Principles and Practice of Endocrinology and Metabolism [3 ed.] 0781717507, 9781469877181, 146987718X

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THIRD EDITION

ASSOCIATE EDITORS IT 13 t* ;i,'; a r E“

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William j. Bremner

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D. Lynn Loriaux

Gary L. Robertson

Eric S. Nylin

Richard H* Snider, jr.

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Wellington Hung

Principles and Practice of

ENDOCRINOLOGY AND METABOLISM

Principles and Practice of

ENDOCRINOLOGY AND

METABOLISM Third Edition

EDITOR

Kenneth L. Becker ASSOCIATE EDITORS

John P. Bilezikian William J. Bremner Wellington Hung C. Ronald Kahn D. Lynn Loriaux Eric S. Nylen Robert W. Rebar Gary L. Robertson Richard H. Snider, Jr. Leonard Wartofsky With 330 Contributors

Lippincott Williams & Wilkins A Wolters Kluwer Company

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© 2001 by LIPPINCOTT WILLIAMS & WILKINS 530 Walnut Street Philadelphia, PA 19106 USA LWW.com All rights reserved. This book is protected by copyright. No part of this book may be reproduced in any form or by any means, including photocopying, or utilized by any information storage and retrieval system without written permission from the copyright owner, except for brief quotations embodied in critical articles and reviews. Materials appearing in this book prepared by individuals as part of their official duties as U.S. gov¬ ernment employees are not covered by the above-mentioned copyright. Printed in the USA

Library of Congress Cataloging-in-Publication Data Principles and practice of endocrinology and metabolism / editor, Kenneth L. Becker ; associate editors, John P. Bilezikian ... [et al.].—3rd ed. p.; cm. Includes bibliographical references and index. ISBN 0-7817-1750-7 1. Endocrinology. 2. Endocrine glands-Diseases. 3. Metabolism-Disorders. I. Becker, Kenneth L. [DNLM: 1. Endocrine Diseases. 2. Metabolic Diseases. WK 100 P957 2000] RC648 .P67 2000 616.4—dc21 00-022095

Care has been taken to confirm the accuracy of the information presented and to describe generally accepted practices. However, the authors, editors, and publisher are not responsible for errors or omissions or for any consequences from application of the information in this book and make no warranty, expressed or implied, with respect to the currency, completeness, or accuracy of the contents of the publication. Application of this information in a particular situation remains the professional responsibility of the practitioner. The authors, editors, and publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accordance with current recommenda¬ tions and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new or infrequently employed drug. Some drugs and medical devices presented in this publication have Food and Drug Administration (FDA) clearance for limited use in restricted research settings. It is the responsibility of health care providers to ascertain the FDA status of each drug or device planned for use in their clinical practice. 10 987654321

EDITORS

EDITOR Kenneth L. Becker, md, PhD Professor of Medicine Professor of Physiology and Experimental Medicine Director of Endocrinology and Metabolism George Washington University School of Medicine and Health Sciences Veterans Affairs Medical Center Washington, DC

ASSOCIATE EDITORS John P. Bilezikian, md Professor of Medicine and Pharmacology Department of Medicine Columbia University College of Physicians and Surgeons New York, New York

William J. Bremner, md, PhD Robert G. Petersdorf Professor and Chairman Department of Medicine University of Washington School of Medicine Seattle, Washington

Wellington Hung, md, PhD Professor Emeritus of Pediatrics Georgetown University School of Medicine Professorial Lecturer in Pediatrics George Washington University School of Medicine and Health Sciences Washington, DC

C. Ronald Kahn, md Mary K. Iacocca Professor of Medicine Harvard Medical School President and Director, Research Division Joslin Diabetes Center Boston, Massachusetts

D. Lynn Loriaux, MD, PhD Professor and Chair Department of Medicine Oregon Health Sciences University School of Medicine Portland, Oregon

Eric S. Nylen, md Associate Professor of Medicine Department of Endocrinology George Washington University School of Medicine and Health Sciences Veterans Affairs Medical Center Washington, DC

Robert W. Rebar, MD Professor Department of Obstetrics and Gynecology University of Cincinnati College of Medicine Chief, Obstetrics and Gynecology University Hospital Cincinnati, Ohio Associate Executive Director American Society for Reproductive Medicine Birmingham, Alabama

Gary L. Robertson, md Professor of Medicine and Neurology Department of Endocrinology Northwestern University Medical School Chicago, Illinois

Richard H. Snider, Jr., PhD Chief Chemist Endocrinology Research Laboratory Veterans Affairs Medical Center Washington, DC

Leonard Wartofsky, md, mph, macp Clinical Professor of Medicine Georgetown University School of Medicine Clinical Professor of Medicine George Washington University School of Medicine and Health Sciences Chair, Department of Medicine Washington Hospital Center Clinical Professor of Medicine Howard University College of Medicine Washington, DC Professor of Medicine and Physiology Uniformed Services University of the Health Sciences F. Edward Hebert School of Medicine Bethesda, Maryland

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CONTRIBUTING AUTHORS

Alaa Abou-Saif, MD Gastroenterology Fellow Department of Medicine Division of Gastroenterology Georgetown University School of Medicine Washington, DC Gary M. Abrams, MD Associate Professor of Clinical Neurology Department of Neurology University of California, San Francisco, School of Medicine San Francisco, California Thomas Aceto, Jr., MD Professor of Pediatrics Chairman Emeritus of Pediatrics Saint Louis University School of Medicine Cardinal Glennon Children's Hospital St. Louis, Missouri Bharat B. Aggarwal, PhD Professor of Medicine and Biochemistry Department of Bioimmunotherapy Chief, Cytokine Research Section University of Texas-Houston Medical School M. D. Anderson Cancer Center Houston, Texas Zalman S. Agus, MD Emeritus Professor of Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania Rexford S. Ahima, MD, PhD Assistant Professor of Medicine Division of Endocrinology, Diabetes and Metabolism University of Pennsylvania School of Medicine Philadelphia, Pennsylvania

K. George M. M. Alberti, MD, DPhil, PRCP, FRCP

Professor of Medicine Department of Diabetes and Metabolism University of Newcastle upon Tyne Faculty of Medicine Newcastle upon Tyne, England Melvin G. Alper, MD Private Practice, Ophthalmology Chevy Chase, Maryland John K. Amory, MD Assistant Professor Department of Medicine University of Washington School of Medicine Veterans Affairs Puget Sound Health Care System Seattle, Washington Neil Aronin, MD Professor of Medicine and Cell Biology Director, Division of Endocrinology and Metabolism University of Massachusetts Medical School Worcester, Massachusetts Louis J. Aronne, MD Clinical Associate Professor of Medicine Weill Medical College of Cornell University New York, New York Gilbert P. August, MD Professor of Pediatrics Department of Endocrinology George Washington University School of Medicine and Health Sciences Children's National Medical Center Washington, DC

Andrew J. Ahmann, MD Assistant Professor of Medicine Director of Adult Diabetes Services Oregon Health Sciences University School of Medicine Portland, Oregon

Lloyd Axelrod, MD Associate Professor of Medicine Harvard Medical School Physician and Chief of the James Howard Means Firm Massachusetts General Hospital Boston, Massachusetts

Abdullah A. Alarifi, MD Consultant Endocrinologist Department of Medicine King Faisal Specialist Hospital and Research Centre Riyadh, Kingdom of Saudi Arabia

Daiva R. Bajorunas, MD Senior Director, Clinical Research Global Project Team Leader, Metabolism Aventis Pharmaceuticals Bridgewater, New Jersey

H. W. Gordon Baker, MD, PhD, FRACP Associate Professor Department of Obstetrics and Gynaecology University of Melbourne School of Medicine Royal Women's Hospital Victoria, Australia James R. Baker, Jr., MD Professor of Medicine Department of Internal MedicineAllergy and Immunology Chief, Division of Allergy University of Michigan Medical School Ann Arbor, Michigan William A. Banks, MD Professor of Internal Medicine Division of Geriatrics Saint Louis University School of Medicine St. Louis, Missouri Robert L. Barbieri, MD Kate Macy Ladd Professor of Obstetrics, Gynecology and Reproductive Biology Harvard Medical School Boston, Massachusetts Marcelo J. Barrionuevo, MD Assistant Professor Department of Obstetrics and Gynecology University of Miami School of Medicine Margate, Florida David S. Baskin, MD, FACS Professor of Neurosurgery and Anesthesiology Baylor College of Medicine Houston, Texas Gerhard Baumann, md Professor of Medicine Northwestern University Medical Center Chicago, Illinois Peter H. Baylis, MD, FRCP, FAMS Professor of Experimental Medicine Dean, Department of Medicine The Medical School University of Newcastle upon Tyne Faculty of Medicine Newcastle upon Tyne, England David V. Becker, md Professor of Radiology and Medicine Division of Nuclear Medicine and Endocrinology Weill Medical College of Cornell University New York Presbyterian Hospital New York, New York

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CONTRIBUTING AUTHORS

Dorothy J. Becker, MB, Bch Professor of Pediatrics University of Pittsburgh School of Medicine Children's Hospital of Pittsburgh Pittsburgh, Pennsylvania

Kenneth L. Becker, MD, PhD Professor of Medicine Professor of Physiology and Experimental Medicine Director of Endocrinology and Metabolism George Washington University School of Medicine and Health Sciences Veterans Affairs Medical Center Washington, DC

Norman H. Bell, MD Distinguished University Professor of Medicine Medical University of South Carolina College of Medicine Charleston, South Carolina

Bankim Bhatt, MD Medical Resident Department of Medicine Georgetown University School of Medicine Washington Hospital Center Washington, DC

John P. Bilezikian, MD Professor of Medicine and Pharmacology Department of Medicine Columbia University College of Physicians and Surgeons New York, New York

Richard E. Blackwell, MD, PhD Professor of Obstetrics and Gynecology University of Alabama School of Medicine Birmingham, Alabama

Vicky A. Blakesley, MD, PhD Director, Department of New Product Evaluation International Division Abbott Laboratories Abbott Park, Illinois Stephen Bloom, ma, md, DSc, FRCPath, FRCP, FMedSci

Professor of Medicine Department of Metabolic Medicine Division of Investigative Science University of London Imperial College School of Medicine London, England

Manfred Blum, MD Professor of Clinical Medicine and Radiology Director, Nuclear Endocrine Laboratory New York University School of Medicine New York, New York

Nanci Bobrow, PhD Assistant Clinical Professor of Pediatrics Cardinal Glennon Children's Hospital Saint Louis University School of Medicine St. Louis, Missouri

Susan Bonner-Weir, PhD Associate Professor of Medicine Harvard Medical School Senior Investigator Joslin Diabetes Center Boston, Massachusetts

Stefan R. Bornstein, MD, PhD Assistant Professor and Research Scholar Pediatric and Reproductive Endocrinology Branch National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland

Eric Bourekas, MD Assistant Professor of Radiology Section of Diagnostic and Interventional Neuroradiology Ohio State University College of Medicine and Public Health Columbus, Ohio

William J. Bremner, MD, PhD Robert G. Petersdorf Professor and Chairman Department of Medicine University of Washington School of Medicine Seattle, Washington

Edward M. Brown, MD Professor of Medicine Endocrine-Hypertension Division Harvard Medical School Brigham and Women's Hospital Boston, Massachusetts Henry B. Burch, MD Associate Professor of Medicine Uniformed Services University of the Health Sciences F. Edward Hebert School of Medicine Bethesda, Maryland Department of Endocrine-Metabolic Service Walter Reed Army Medical Center Washington, DC

Kenneth D. Burman, MD Professor of Medicine Uniformed Services University of the Health Sciences F. Edward Hebert School of Medicine Bethesda, Maryland Clinical Professor Department of Medicine George Washington University School of Medicine and Health Sciences Professor of Medicine Georgetown University School of Medicine Chief, Endocrine Section Washington Hospital Center Washington, DC

Peter H. Byers, MD Professor of Pathology and Medicine University of Washington School of Medicine Seattle, Washington Enrico Carmina, MD Professor Department of Endocrinology University of Palermo Palermo, Italy Visiting Professor Department of Obstetrics and Gynecology Columbia University College of Physicians and Surgeons New York, New York

Thomas O. Carpenter, MD Professor of Pediatrics Yale University School of Medicine Yale-New Haven Hospital New Haven, Connecticut

Bruce R. Carr, MD Professor Paul C. Macdonald Distinguished Chair in Obstetrics and Gynecology Director, Division of Reproductive Endocrinology University of Texas Southwestern Medical Center at Dallas Southwestern Medical School Dallas, Texas Veronica M. Catanese, MD Assistant Professor Department of Medicine and Cell Biology New York University School of Medicine New York, New York

Donald Chakeres, MD Professor of Radiology Ohio State University College of Medicine and Public Health Columbus, Ohio

CONTRIBUTING AUTHORS John R. G. Challis, PhD, Dsc, FlBiol, FRCOG, FRSC

Department of Physiology Medical Sciences Building University of Toronto Faculty of Medicine Toronto, Ontario Canada

Philippe Chanson, md Professor of Medicine Department of Endocrinology University Paris XI Bicetre University Hospital Le Kremlin-Bicetre France

William W. Chin, MD Professor of Medicine Harvard Medical School Boston, Massachusetts Vice President, Lilly Research Laboratories Eli Lilly & Co. Lilly Corporate Center Indianapolis, Indiana

George P. Chrousos, MD Chief, Pediatric and Reproductive Endocrinology Branch National Institutes of Health Bethesda, Maryland

Richard V. Clark, MD, PhD Principal Clinical Research Physician Clinical Pharmacology-Exploratory Department Glaxo Wellcome Research and Development Research Triangle Park, North Carolina

Thomas L. Clemens, MD, PhD Professor of Medicine and Molecular and Cellular Physiology Department of Internal Medicine/ Endocrinology University of Cincinnati College of Medicine Cincinnati, Ohio Fredric L. Coe, MD Professor Departments of Medicine and Physiology University of Chicago Pritzker School of Medicine Chicago, Illinois Joshua L. Cohen, MD Associate Professor of Medicine Department of Endocrinology George Washington University School of Medicine and Health Sciences Washington, DC

Regis Cohen, MD, PhD Praticien Hospitalier Endocrine Staff Physician Avicenne Hospital Bobigny, France University of Leonardo Da Vinci Paris, France Warren E. Cohen, MD Associate Clinical Professor of Pediatrics and Neurology George Washington University School of Medicine and Health Sciences Washington, DC Medical Director, United Cerebral Palsy Nassau County, New York Alessandra Colantoni, md Assistant Professor of Medicine Department of Gastroenterology and Hepatology Loyola University of Chicago Stritch School of Medicine Loyola University Medical Center Maywood, Illinois Richard J. Comi, MD Associate Professor of Medicine Section of Endocrinology and Metabolism Dartmouth Medical School Dartmouth-Hitchcock Medical Center Hanover, New Hampshire Paul E. Cooper, md, frcpc Associate Professor of Neurology Departments of Clinical Neurological Sciences and Medicine University of Western Ontario Faculty of Medicine and Dentistry Health Sciences Addition London, Ontario Canada Dalila B. Corry, MD Associate Clinical Professor of Medicine Department of Medicine University of California, Los Angeles, UCLA School of Medicine Los Angeles, California Chief, Nephrology Olive View Medical Center Sylmar, California Francesco Cosentino, MD, PhD Assistant Professor Department of Experimental Medicine and Pathology University "La Sapienza" Rome, Italy Senior Research Associate Cardiovascular Research Department of Cardiology University Hospital Zurich, Switzerland

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Felicia Cosman, MD Associate Professor of Clinical Medicine Department of Medicine Columbia University College of Physicians and Surgeons New York, New York Helen Hayes Hospital West Haverstraw, New York Brian M. Cox, PhD Professor of Pharmacology and Neuroscience Department of Pharmacology Uniformed Services University of the Health Sciences F. Edward Hebert School of Medicine Bethesda, Maryland Glenn R. Cunningham, MD Associate Chief of Staff Department of Medicine University of Texas-Houston Medical School Veterans Affairs Medical Center Houston, Texas Mary F. Dallman, PhD Professor of Physiology University of California, San Francisco, School of Medicine San Francisco, California Daniel N. Darlington, PhD Associate Professor of Surgery Departments of Surgery and Physiology University of Maryland School of Medicine Baltimore, Maryland Philip Darney, MD, MSc Professor of Obstetrics, Gynecology, and Reproductive Sciences University of California, San Francisco, School of Medicine San Francisco General Hospital San Francisco, California Harish P. G. Dave, MB, ChB, MRCP(UK) Associate Professor of Medicine Department of Hematology George Washington University School of Medicine and Health Sciences Veterans Affairs Medical Center Washington, DC Faith B. Davis, MD Professor of Medicine and Cell Biology and Cancer Research Albany Medical College Staff Physician Stratton Veterans Affairs Medical Center Albany, New York

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CONTRIBUTING AUTHORS

Paul J. Davis, MD Professor of Medicine and Cell Biology and Cancer Research Senior Associate Dean for Clinical Research Albany Medical College Research Physician Wadsworth Center, New York State Department of Health Staff Physician Stratton Veterans Affairs Medical Center Albany, New York

Gerard M. Doherty, MD Associate Professor of Surgery Section of Surgical Oncology and Endocrinology Washington University School of Medicine St. Louis, Missouri

Suzanne M. Jan De Beur, MD Assistant Professor of Medicine Johns Hopkins University School of Medicine Baltimore, Maryland

Marc K. Drezner, MD Professor of Medicine Head, Section of Endocrinology, Diabetes, and Metabolism University of Wisconsin Medical School Madison, Wisconsin

Ralph A. DeFronzo, MD Professor of Medicine Chief, Diabetic Division Member, Nephrology Division University of Texas Medical School at San Antonio University Health Center San Antonio, Texas David M. De Kretser, MD, MBBS, FRACP Professor and Director Monash Institute of Reproduction and Development Monash University Monash Medical Centre, Clayton Clayton, Victoria Australia Nicola De Maria, MD Research Associate Liver Transplant Program Loyola University Medical Center Maywood, Illinois David P. Dempsher, MD, PhD Associate Professor of Pediatrics Cardinal Glennon Children's Hospital Saint Louis University School of Medicine St. Louis, Missouri David W. Dempster, PhD Professor of Clinical Pathology Columbia University College of Physicians and Surgeons New York, New York Director, Regional Bone Center Helen Hayes Hospital West Haverstraw, New York Luca deSimone Nephrology Fellow Beth Israel Medical Center New York, New York

Allan L. Drash, MD Emeritus Professor of Pediatrics University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania

Alan Dubrow, MD Clinical Assistant Professor of Medicine Department of Nephrology Beth Israel Deaconess Medical Center New York, New York D. Robert Dufour, MD Clinical Professor of Pathology George Washington University School of Medicine and Health Sciences Washington, DC Uniformed Services University of the Health Sciences F. Edward Hebert School of Medicine Bethesda, Maryland Chief, Pathology and Laboratory Medicine Service Veterans Affairs Medical Center Washington, DC

William J. Ellis, MD Associate Professor and Clinic Director Department of Urology University of Washington School of Medicine Seattle, Washington Abby Erickson, BA Colorado Center for Bone Research Lakewood, Colorado Gregory F. Erickson, PhD Professor Department of Reproductive Medicine University of California, San Diego, School of Medicine La Jolla, California Eric A. Espiner, MD, FRACP, FRS(NZ) Professor Department of Endocrinology University of Otago Christchurch School of Medicine Christchurch Public Hospital Christchurch, New Zealand Jan Fahrenkrug, MD, DMSci Professor Department of Clinical Chemistry University of Copenhagen Faculty of Health Sciences Bispebjerg Hospital Copenhagen, Denmark Kenneth R. Falchuk, MD Associate Professor of Medicine Harvard Medical School Beth Israel Deaconess Medical Center Boston, Massachusetts

Roberta P. Durschlag, PhD, RD Clinical Assistant Professor Department of Health Sciences Boston University School of Medicine Boston, Massachusetts

Murray J. Favus, MD Professor of Medicine University of Chicago Pritzker School of Medicine Chicago, Illinois

Richard C. Eastman, MD Cygnus, Inc. Redwood City, California

Eva L. Feldman, MD, PhD Professor of Neurology University of Michigan Medical School Ann Arbor, Michigan

George S. Eisenbarth, MD, PhD Professor of Pediatrics, Immunology, and Medicine University of Colorado Health Sciences Center Barbara Davis Center for Childhood Diabetes Denver, Colorado George M. Eliopoulos, MD Associate Professor of Medicine Harvard Medical School Beth Israel Deaconess Medical Center Boston, Massachusetts

Jo-David Fine, MD, mph Professor Department of Dermatology University of North Carolina at Chapel Hill School of Medicine Chapel Hill, North Carolina James D. Finkelstein, MD Senior Clinician Department of Medicine Veterans Affairs Medical Center Washington, DC

CONTRIBUTING AUTHORS Jeffrey S. Flier, MD George C. Reisman Professor of Medicine Harvard Medical School Beth Israel Deaconess Medical Center Boston, Massachusetts Ruth C. Fretts, MD, MPH Assistant Professor Department of Obstetrics and Gynecology Harvard Medical School Beth Israel Deaconess Medical Center Boston, Massachusetts Om P. Ganda, MD Associate Clinical Professor Department of Medicine Harvard Medical School Joslin Diabetes Center Beth Israel Deaconess Medical Center Boston, Massachusetts Luigi Garibaldi Beth Israel Medical Center Newark, New Jersey Gary W. Gibbons, MD Associate Clinical Professor of Surgery Harvard Medical School Director, Quality Improvement Department of Surgery Beth Israel Deaconess Medical Center Boston, Massachusetts John R. Gill, Jr., MD Scientist, Emeritus Hypertension-Endocrine Branch National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland Henry N. Ginsberg, md Professor of Medicine Columbia University College of Physicians and Surgeons New York, New York Joel S. Glaser, MD Professor Departments of Ophthalmology and Neurology University of Miami School of Medicine Bascom Palmer Eye Institute Miami, Florida Department of Ophthalmology Cleveland Clinic of Florida Coral Gables, Florida Allan R. Glass, md Adjunct Professor of Medicine Uniformed Services University of the Health Sciences F. Edward Hebert School of Medicine Bethesda, Maryland

Philip W. Gold, MD Branch Chief Department of Intramural Research Programs National Institute of Mental Health National Institutes of Health Bethesda, Maryland Alisa B. Goldberg, MD Assistant Adjunct Professor Department of Obstetrics, Gynecology and Reproductive Sciences University of California, San Francisco, School of Medicine San Francisco General Hospital San Francisco, California Ira J. Goldberg, MD Professor of Medicine Columbia University College of Physicians and Surgeons New York, New York Stuart L. Goldberg, md Assistant Director, Bone Marrow Transplantation Program Temple University School of Medicine Philadelphia, Pennsylvania Allison B. Goldfine, MD Instructor of Medicine Department of Cellular and Molecular Physiology Harvard University Joslin Diabetes Center Boston, Massachusetts Allan L. Goldstein, PhD Chair, Department of Biochemistry and Molecular Biology George Washington University School of Medicine and Health Sciences Washington, DC David S. Goldstein, MD, PhD Chief, Clinical Neurocardiology Section National Institutes of Health Bethesda, Maryland David Goltzman, MD Professor of Medicine and Physiology McGill University Faculty of Medicine Royal Victoria Hospital Montreal, Quebec Canada Esther A. Gonzalez, MD Assistant Professor Division of Nephrology Saint Louis University School of Medicine St. Louis, Missouri

Michael N. Goodman, PhD Professor of Medicine Department of Internal Medicine University of California, Davis, School of Medicine Sacramento, California

Phillip Gorden, MD Director Emeritus National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland

Frederic D. Gordon, MD Assistant Professor of Medicine Department of Hepatobiliary Surgery and Liver Transplantation Tufts University School of Medicine Lahey Clinic Medical Center Boston, Massachusetts

Daryl K. Granner, MD Joe C. Davis Professor of Biomedical Science Professor of Molecular Physiology, Biophysics, and Internal Medicine Vanderbilt University School of Medicine Director, Vanderbilt Diabetes Center Staff Physician Veterans Affairs Hospital Nashville, Tennessee

Soren Gras, md Senior Registrar Department of Obstetrics and Gynaecology Herlev University Hospital Herlev, Denmark

Douglas A. Greene, MD Executive Vice President Department of Clinical Sciences and Product Development Merck & Co., Inc. Rahway, New Jersey

David A. Gruenewald, MD, FACP Assistant Professor of Medicine University of Washington School of Medicine Veterans Affairs Puget Sound Health Care System Seattle, Washington Joel F. Habener, MD Professor of Medicine Laboratory of Molecular Endocrinology Harvard Medical School Massachusetts General Hospital Boston, Massachusetts

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CONTRIBUTING AUTHORS

Philippe A. Halban, PhD Professor of Medicine Louis-Jeantet Research Laboratories Geneva University Medical Center Geneva, Switzerland Nicholas R. S. Hall, PhD Health and Human Performance Orlando, Florida Allan G. Halline, MD Assistant Professor of Medicine Section of Digestive and Liver Diseases University of Illinois at Chicago College of Medicine Chicago, Illinois Stephen G. Learner, MD Professor of Otolaryngology Department of Otolaryngology Mayo Medical School Rochester, Minnesota Marianne Hatle, MD Resident University of Maryland School of Medicine Baltimore, Maryland Michael J. Hausmann, MD Professor Department of Nephrology Faculty of Health Sciences Ben Gurion University of the Negev Scroka Medical Center of Kupat Holim Beer Sheva, Israel Karin Hehenberger, MD, PhD Research Fellow Joslin Diabetes Center Harvard Medical School Boston, Massachusetts J. Fielding Hejtmancik, MD, PhD Medical Officer National Eye Institute National Institutes of Health Bethesda, Maryland Geoffrey N. Hendy, PhD Professor of Medicine McGill University Faculty of Medicine Royal Victoria Hospital Montreal, Quebec Canada James V. Hennessey, MD Associate Professor of Medicine Division of Endocrinology Brown University School of Medicine Rhode Island Hospital Providence, Rhode Island

Jules Hirsch, MD Professor Emeritus and Physician-in-Chief Emeritus Laboratory of Human Behavior and Metabolism Rockefeller University Rockefeller University Hospital New York, New York

Eva Horvath, PhD Associate Professor of Pathology Department of Laboratory Medicine Division of Pathology University of Toronto Faculty of Medicine St. Michael's Hospital Toronto, Ontario Canada

Angelica Linden Hirschberg, MD, PhD Associate Professor of Obstetrics and Gynecology Karolinska Institute Karolinska Hospital Stockholm, Sweden

Barbara V. Howard, PhD President, MedStar Clinical Research Institute Washington, DC

Max Hirshkowitz, MD Associate Professor Department of Psychiatry Baylor College of Medicine Director, Sleep Center Houston Veterans Affairs Medical Center Houston, Texas Gary D. Hodgen, PhD Professor Department of Obstetrics and Gynecology Eastern Virginia Medical School Chair The Howard and Georgeanna Jones Institute for Reproductive Medicine Norfolk, Virginia

William James Howard, MD Professor of Medicine George Washington University School of Medicine Senior Vice President and Medical Director Washington Hospital Center Washington, DC Ilpo Huhtaniemi, MD, PhD Professor of Physiology University of Turku Faculty of Medicine Turku, Finland Wellington Hung, MD, PhD Professor Emeritus of Pediatrics Georgetown University School of Medicine Professorial Lecturer in Pediatrics George Washington University School of Medicine and Health Sciences Washington, DC

Edward W. Holmes, MD Chairman, Department of Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania

Mehboob A. Hussain, MD Department of Medicine New York University School of Medicine New York, New York

Jens J. Holst, MD Department of Medical Physiology University of Copenhagen Faculty of Health Sciences The Panum Institute Copenhagen, Denmark

Philip M. Iannaccone, MD, PhD George M. Eisenberg Professor Department of Pediatrics Northwestern University Medical School Children's Memorial Institute of Education and Research Chicago, Illinois

Robert N. Hoover, MD, ScD Director, Epidemiology and Biostatistics Program National Cancer Institute National Institutes of Health Bethesda, Maryland Gabriel N. Hortobagyi, MD Professor of Medicine, Chairman Department of Breast and Gynecologic Medical Oncology University of Texas-Houston Medical School M. D. Anderson Cancer Center Houston, Texas

Ivor M. D. Jackson, mb, ChB Professor of Medicine Division of Endocrinology Brown University School of Medicine Rhode Island Hospital Providence, Rhode Island Richard V. Jackson, MBBS, FRACP Associate Professor of Medicine University of Queensland Faculty of Health Sciences Greenslopes Private Hospital Queensland, Australia

CONTRIBUTING AUTHORS Lois Jovanovic, MD Clinical Professor of Medicine University of Southern California School of Medicine Los Angeles, California Director and Chief Scientific Officer Sansum Medical Research Institute Santa Barbara, California William A. Jubiz, MD Director Endocrinology Center Cali, Colombia C. Ronald Kahn, MD Mary K. Iacocca Professor of Medicine Harvard Medical School President and Director, Research Division Joslin Diabetes Center Boston, Massachusetts Cynthia G. Kaplan, MD Associate Professor of Pathology SUNY at Stony Brook School of Medicine Health Sciences Center Stony Brook, New York Edwin L. Kaplan, MD, FACS Professor of Surgery University of Chicago Pritzker School of Medicine Chicago, Illinois Abba J. Kastin, MD Chief of Endocrinology Departments of Medicine and Neuroscience Tulane University School of Medicine Veterans Affairs Medical Center New Orleans, Louisiana Laurence Katznelson, MD Assistant Professor of Medicine Harvard Medical School Massachusetts General Hospital Boston, Massachusetts Harry R. Keiser, MD Scientist Emeritus National Heart, Lung, and Blood Institute Clinical Center National Institutes of Health Bethesda, Maryland Ellie Kelepouris, MD Professor of Medicine Temple University School of Medicine Philadelphia, Pennsylvania Craig M. Kessler, MD Professor of Medicine and Pathology Chief, Division of Hematology-Oncology Georgetown University School of Medicine Lombardy Cancer Center Washington, DC

Parvez Khatri, MD Fellow, Department of Medicine/Nephrology George Washington University School of Medicine and Health Sciences Washington, DC Paul L. Kimmel, MD Professor of Medicine George Washington University School of Medicine and Health Sciences Washington, DC Director, Diabetic Nephropathy Program Division of Kidney, Urologic, and Hematologic Diseases National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland George L. King, MD Professor of Medicine Acting Director of Research Joslin Diabetes Center Harvard Medical School Boston, Massachusetts Anne Klibanski, MD Professor of Medicine Harvard Medical School Chief, Neuroendocrine Unit Massachusetts General Hospital Boston, Massachusetts Mitchel A. Kling, MD Associate Professor of Psychiatry and Medicine University of Maryland School of Medicine Veterans Affairs Medical Center Baltimore, Maryland Mark Korson, MD Associate Professor of Pediatrics Division of Genetics Tufts University School of Medicine New England Medical Center Boston, Massachusetts Kalman Kovacs, MD, PhD Professor of Pathology Department of Laboratory Medicine Division of Pathology University of Toronto Faculty of Medicine Saint Michael's Hospital Toronto, Ontario Canada Andrzej S. Krolewski, MD, PhD Associate Professor of Medicine Chief, Section of Genetics and Epidemiology Harvard Medical School Research Division Joslin Diabetes Center Boston, Massachusetts

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Robert J. Kurman, MD Richard W. TeLinde Distinguished Professor of Gynecologic Pathology Departments of Gynecology, Obstetrics, and Pathology Johns Hopkins University School of Medicine Baltimore, Maryland John C. LaRosa, md, facp President SUNY Downstate Medical Center College of Medicine University Hospital of Brooklyn Brooklyn, New York Robert B. Layzer, MD Professor Emeritus of Neurology University of California, San Francisco, School of Medicine San Francisco, California Jacques LeBlanc, MD Professor Emeritus of Physiology Universite Laval Faculty of Medicine Quebec City, Canada Peter A. Lee, MD, PhD Professor of Pediatrics Pennsylvania State University College of Medicine The Milton S. Hershey Medical Center Hershey, Pennsylvania Z. M. Lei, MD, PhD Assistant Professor of Obstetrics and Gynecology University of Louisville School of Medicine Louisville, Kentucky Hoyle Leigh, MD Professor of Psychiatry University of California, San Francisco, School of Medicine San Francisco, California Derek LeRoith, MD, PhD Chief, Molecular and Cellular Endocrinology Branch National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland Michael A. Levine, MD Professor of Pediatrics, Medicine, and Pathology Director, Pediatric Endocrinology Johns Hopkins University School of Medicine Baltimore, Maryland

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CONTRIBUTING AUTHORS

Jonathan J. Li, PhD Director, Division of Etiology and Prevention of Hormone-Associated Cancers Professor of Pharmacology, Toxicology and Preventive Medicine University of Kansas School of Medicine Kansas Cancer Institute Kansas City, Kansas Sara Antonia Li, MD Associate Director Hormonal Carcinogenesis Laboratory University of Kansas School of Medicine Kansas Cancer Institute Kansas City, Kansas Robert D. Lindeman, MD Professor Emeritus of Medicine Department of Internal Medicine University of New Mexico School of Medicine University of New Mexico Hospital Albuquerque, New Mexico Robert Lindsay, MBChB, PhD, FRCP Professor of Clinical Medicine Columbia University College of Physicians and Surgeons New York, New York Chief of Internal Medicine Helen Hayes Hospital West Haverstraw, New York Timothy O. Lipman, MD Professor of Medicine Georgetown University School of Medicine Chief, Gastroenterology-Hepatology Nutrition Section Veterans Affairs Medical Center Washington, DC Virginia A. Livolsi, MD Professor of Pathology Department of Pathology and Laboratory Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania Rogerio A. Lobo, MD Willard C. Rappleye Professor of Obstetrics and Gynecology Chairman, Department of Obstetrics and Gynecology Columbia University College of Physicians and Surgeons Columbia Presbyterian Medical Center Director, Sloane Hospital for Women New York, New York Rebecca J. Locke Research Assistant Columbia University College of Physicians and Surgeons New York, New York

Christopher J. Logethetis, MD Chairman and Professor Department of Genitourinary Medical Oncology University of Texas-Houston Medical School M. D. Anderson Cancer Center Houston, Texas D. Lynn Loriaux, MD, PhD Professor and Chair Department of Medicine Oregon Health Sciences University School of Medicine Portland, Oregon Harvey S. Luksenburg, MD Assistant Professor of Medicine George Washington University School of Medicine and Health Sciences Washington, DC Thomas F. Liischer, MD Professor and Head of Cardiology Hospital Universitaire de Zurich Zurich, Switzerland Ruth S. MacDonald, RD, PhD Professor of Nutrition Department of Food Science and Human Nutrition University of Missouri-Columbia School of Medicine Columbia, Missouri Michelle Fischmann Magee, MD, MB, BCh, BAO Medical Director, Diabetes Team MedStar Clinical Research Institute Washington Hospital Center Washington, DC Robert W. Mahley, MD, PhD Professor of Pathology and Medicine Director, Gladstone Institute of Cardiovascular Disease University of California, San Francisco, School of Medicine San Francisco, California Christos S. Mantzoros, MD, Dsc Assistant Professor of Medicine Department of Internal Medicine Harvard Medical School Beth Israel Deaconess Medical Center Boston, Massachusetts Eleftheria Maratos-Flier, MD Associate Professor of Medicine Research Division Harvard Medical School Joslin Diabetes Center Boston, Massachusetts Paul Marik, MBBCh, FCP(SA), FRCP(C), FCCM, FCCP Department of Critical Care Mercy Hospital of Pittsburgh Pittsburgh, Pennsylvania

Kevin J. Martin, MB, BCh, FACP Professor of Internal Medicine Department of Nephrology Director, Division of Nephrology -Saint Louis University School of Medicine St. Louis, Missouri William D. Mathers, MD Professor of Ophthalmology Oregon Health Sciences University School of Medicine Casey Eye Institute Portland, Oregon Paul N. Maton, MD, FRCP, FACP, FACG Digestive Disease Specialists Incorporated Digestive Disease Research Institute Oklahoma City, Oklahoma Alvin M. Matsumoto, MD Professor Department of Medicine University of Washington School of Medicine Chief of Gerontology Veterans Affairs Puget Sound Health Care System Seattle, Washington Ernest L. Mazzaferri, MD, MACP Professor Emeritus and Chairman Department of Internal Medicine Ohio State University College of Medicine and Public Health Columbus, Ohio Alan M. McGregor, MA, MD, FRCP Professor of Medicine King's College Guy's, King's and St. Thomas' School of Medicine London, England Karim Meeran, MD, MRCP Senior Lecturer Division of Endocrinology and Metabolism University of London Imperial College School of Medicine Hammersmith Hospital London, England Minesh P. Mehta, MD, MB, ChB Associate Professor and Chairman Department of Human Oncology University of Wisconsin Medical School Madison, Wisconsin James C. Melby, MD Professor of Medicine and Physiology Boston University School of Medicine Boston Medical Center Boston, Massachusetts

CONTRIBUTING AUTHORS Stephen A. Migueles, MD Fellow, Infectious Diseases Laboratory of Immunoregulation National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland Donald L. Miller, MD Professor of Radiology and Nuclear Medicine Uniformed Services University of the Health Sciences F. Edward Hebert School of Medicine Bethesda, Maryland Elizabeth A. Miller Urology Resident University of Washington School of Medicine Seattle, Washington Paul D. Miller, MD Clinical Professor Department of Medicine University of Colorado Health Sciences Center Denver, Colorado Dolly Misra, MD Assistant Clinical Professor of Medicine Division of Endocrinology and Metabolism George Washington University School of Medicine and Health Sciences Washington, DC Diabetes and Endocrine Consultants Waldorf, Maryland Mark E. Molitch, MD Professor of Medicine Center for Endocrinology, Metabolism, and Molecular Medicine Northwestern University Medical School Chicago, Illinois Chulso Moon, MD, PhD Clinical Fellow Department of Medicine University of Texas-Houston Medical School M. D. Anderson Cancer Center Houston, Texas Arshag D. Mooradian, MD Professor of Medicine Director of Endocrinology, Diabetes and Metabolism Saint Louis University School of Medicine St. Louis, Missouri Gregory P. Mueller, PhD Professor of Physiology Uniformed Services University of the Health Sciences F. Edward Hebert School of Medicine Bethesda, Maryland

Beat Muller, MD Department of Internal Medicine Division of Endocrinology University Hospitals Basel, Switzerland Susan E. Myers, MD Assistant Professor of Pediatrics Saint Louis University School of Medicine Cardinal Glenn Children's Hospital St. Louis, Missouri David J. Nashel, MD Professor of Medicine Georgetown University School of Medicine Chief of Medical Service Veterans Affairs Medical Center Washington, DC Adnan Nasir, MD, PhD Department of Dermatology University of North Carolina at Chapel Hill School of Medicine Chapel Hill, North Carolina Jeffrey A. Norton, MD Professor of Surgery Vice Chairman, Department of Surgery University of California, San Francisco, School of Medicine San Francisco Veterans Affairs Medical Center San Francisco, California Robert H. Noth, MD Associate Professor of Medicine Department of Internal Medicine University of California, Davis, School of Medicine Davis, California Veterans Affairs Outpatient Clinic Martinez, California Jennifer A. Nuovo Endocrinologist MedClinic of Sacramento Sacramento, California Eric S. Nylen, MD Associate Professor of Medicine Department of Endocrinology George Washington University School of Medicine and Health Sciences Veterans Affairs Medical Center Washington, DC Donna M. Arab O'Brien, MD Department of Medicine Division of Endocrinology St. Joseph's Health Centre Toronto, Ontario Canada Mary Oehler, MD Staff Radiologist Mount Carmel East Hospital New Albany, Ohio

XV

Robert A. Oppenheim, MD Naperville Eye Associates Naperville, Illinois Jeffrey L. H. O'Riordan Emeritus Professor of Metabolic Medicine University College London, United Kingdom Steven J. Ory, MD Clinical Associate Professor of Obstetrics and Gynecology University of Miami School of Medicine Miami, Florida Harry Ostrer, MD Associate Professor of Pediatrics and Pathology Human Genetics Program New York University School of Medicine New York, New York Weihong Pan, MD, PhD Assistant Professor of Medicine Tulane University School of Medicine New Orleans, Louisiana Yogesh C. Patel, MD, PhD, FACP, FRCP(C), FRACP, FRSC

Professor of Medicine Director, Division of Endocrinology and Metabolism McGill University Faculty of Medicine Royal Victoria Hospital Montreal, Quebec Canada Gary R. Peplinski, MD Surgical Service San Francisco Veterans Affairs Medical Center San Francisco, California Ora Hirsch Pescovitz, MD Professor of Pediatrics, Physiology, and Biophysics Department of Pediatric Endocrinology Indiana University School of Medicine James Whitcomb Riley Hospital for Children Indianapolis, Indiana Kristina C. Pfendler, MD Postdoctoral Scholar Department of Obstetrics and Gynecology University of California, San Francisco, School of Medicine San Francisco, California Joseph J. Pinzone, MD Assistant Professor of Medicine Department of Internal Medicine George Washington University School of Medicine and Health Sciences Washington, DC

XVi

CONTRIBUTING AUTHORS

Mark R. Pittelkow, MD Professor of Dermatology, Biochemistry, and Molecular Biology Mayo Medical School Consultant, Department of Dermatology Mayo Clinic Rochester, Minnesota Stephen R. Plymate, MD Research Professor of Medicine University of Washington School of Medicine Veterans Affairs Puget Sound Health Care System Seattle, Washington Ke-Nan Qin, MD Fellow of Pediatric Endocrinology Department of Pediatrics University of Chicago Pritzker School of Medicine University of Chicago Children's Hospital Chicago, Illinois Ralph Rabkin, MB, Bch, MD Professor of Medicine and Nephrology Department of Medicine Stanford University School of Medicine Stanford, California Veterans Affairs Palo Alto Health Care System Palo Alto, California Miriam T. Rademaker, PhD Professor of Medicine University of Otago Christchurch School of Medicine Christchurch, New Zealand Lawrence G. Raisz, MD Professor of Medicine Department of Endocrinology University of Connecticut School of Medicine University of Connecticut Health Center Farmington, Connecticut Lawrence I. Rand, MD Clinical Assistant Professor of Ophthalmology Harvard Medical School Boston, Massachusetts Ch. V. Rao, PhD Professor and Director Department of Obstetrics and Gynecology University of Louisville School of Medicine Louisville, Kentucky Robert E. Ratner, MD Associate Clinical Professor of Medicine George Washington University School of Medicine and Health Sciences Director, MedStar Clinical Research Institute Washington, DC

Gerald M. Reaven, MD Professor of Medicine Stanford University School of Medicine Stanford, California Robert W. Rebar, MD Professor Department of Obstetrics and Gynecology University of Cincinnati College of Medicine Chief, Obstetrics and Gynecology University Hospital Cincinnati, Ohio Associate Executive Director American Society for Reproductive Medicine Birmingham, Alabama Robert S. Redman, DDS, MSD, PhD Chief, Oral Diagnosis Section, Dental Service Veterans Affairs Medical Center Washington, DC Clinical Associate Professor Department of Oral and Maxillofacial Pathology University of Maryland School of Medicine Baltimore College of Dental Surgery Baltimore, Maryland H. Lester Reed, MD Clinical Professor of Medicine University of Auckland Faculty of Medical and Health Sciences Middlemore Hospital Auckland, New Zealand Domenico C. Regoli, MD Professor Emeritus Department of Pharmacology Universite de Sherbrooke Faculte de Medecine Sherbrooke, Quebec Canada Jens F. Rehfeld, MD, DSc Professor of Clinical Biochemistry University of Copenhagen Faculty of Health Sciences Copenhagen University Hospital Copenhagen, Denmark Robert L. Reid, MD, FRCS(C) Professor Department of Obstetrics and Gynaecology Queen's University School of Medicine Faculty of Health Sciences Kingston General Hospital Kingston, Ontario Canada

Russel J. Reiter, PhD Professor of Neuroendocrinology Department of Cellular and Structural Biology _University of Texas Medical School at San Antonio University Health Center San Antonio, Texas Matthew D. Ringel, MD Assistant Professor of Medicine Uniformed Services University of the Health Sciences F. Edward Hebert School of Medicine Bethesda, Maryland Assistant Clinical Professor of Medicine George Washington University School of Medicine and Health Sciences Section of Endocrinology Washington Hospital Center Washington, DC Antonio Rivera, MD Fellow, Department of Medicine Section of Renal Diseases and Hypertension George Washington University Medical Center Washington, DC Gary L. Robertson, MD Professor of Medicine and Neurology Department of Endocrinology Northwestern University Medical School Chicago, Illinois R. Paul Robertson, MD Professor of Medicine and Pharmacology Scientific Director, Pacific Northwest Research Institute Seattle, Washington Simon P. Robins, PhD, Dsc Head, Skeletal Research Unit Rowett Research Institute Aberdeen, Scotland Alan D. Rogol, MD, PhD Professor of Clinical Pediatrics Department of Pediatrics University of Virginia School of Medicine University of Virginia Medical Center Charlottesville, Virginia Clinical Professor of Internal Medicine Virginia Commonwealth University School of Medicine Richmond, Virginia Prashant K. Rohatgi, MB, MD Professor of Medicine George Washington University School of Medicine and Health Sciences Veterans Affairs Medical Center Washington, DC

CONTRIBUTING AUTHORS Mikael Rorth, MD Professor of Clinical Oncology University of Copenhagen Faculty of Health Sciences Rigshospitalet Copenhagen, Denmark

R. Neil Schimke, MD Professor of Medicine and Pediatrics Chief, Division of Endocrinology and Genetics University of Kansas School of Medicine Kansas City, Kansas

Omega L. Silva, MD Professor Emeritus of Medicine George Washington University School of Medicine and Health Sciences Washington, DC

Robert L. Rosenfield, MD Professor of Pediatrics and Medicine Department of Pediatric Endocrinology University of Chicago Pritzker School of Medicine Chicago, Illinois

James R. Schreiber, MD Elaine and Mitchell Yanow Professor and Head Department of Obstetrics and Gynecology Washington University School of Medicine St. Louis, Missouri

Shonni J. Silverberg, MD Associate Professor of Medicine Columbia University College of Physicians and Surgeons New York, New York

Robert K. Rude, MD Professor of Medicine University of Southern California School of Medicine Los Angeles, California Neil B. Ruderman, MD, DPhil Professor Department of Medicine and Physiology Boston University School of Medicine Boston, Massachusetts James W. Russell, MD Assistant Professor Department of Neurology University of Michigan Medical School Ann Arbor GRECC Ann Arbor, Michigan Lester B. Salans, MD Adjunct Professor The Rockefeller University Clinical Professor of Medicine Mt. Sinai School of Medicine New York, New York Salil D. Sarkar Department of Radiology SUNY Health Sciences Center at Brooklyn College of Medicine Brooklyn, New York David H. Same, MD Associate Professor of Medicine Department of Internal Medicine University of Illinois at Chicago College of Medicine Chicago, Illinois Ernst J. Schaefer, MD Professor of Medicine Lipid Division Tufts University School of Medicine New England Medical Center Boston, Massachusetts Isaac Schiff, MD Joe Vincent Meigs Professor of Gynecology Department of Obstetrics and Gynecology Harvard Medical School Massachusetts General Hospital Boston, Massachusetts

David E. Schteingart, MD Professor of Internal Medicine Division of Endocrinology and Metabolism University of Michigan Medical School Ann Arbor, Michigan Ellen W. Seely, MD Assistant Professor of Medicine Director of Clinical Research Endocrine-Hypertension Division Harvard Medical School Brigham and Women's Hospital Boston, Massachusetts Markus J. Seibel, MD, PD Associate Professor of Medicine Division of Endocrinology and Metabolism University of Heidelberg Medical School Heidelberg, Germany Elizabeth Shane, MD Professor of Medicine Columbia University College of Physicians and Surgeons New York, New York Lawrence E. Shapiro, MD Clinical Professor of Medicine SUNY at Stony Brook School of Medicine Health Sciences Center Stony Brook, New York Director, Division of Endocrinology Winthrop University Hospital Mineola, New York Meeta Sharma, mbbs, md Assistant Director, Diabetes Team Division of Endocrinology Georgetown University School of Medicine MedStar Diabetes Institute Washington Hospital Center Washington, DC R. Michael Siatkowski, MD Associate Professor of Ophthalmology Dean A. McGee Eye Institute Oklahoma City, Oklahoma

Joe Leigh Simpson, MD Ernst W. Bertner Chairman and Professor Department of Obstetrics and Gynecology Baylor College of Medicine Houston, Texas Ethel S. Siris, MD Madeline C. Stabile Professor of Clinical Medicine Department of Medicine Columbia University College of Physicians and Surgeons New York, New York Glen W. Sizemore, MD Professor of Medicine Division of Endocrinology and Metabolism Loyola University of Chicago Stritch School of Medicine Maywood, Illinois Niels E. Skakkebaek, MD Professor of Growth and Reproduction University of Copenhagen Faculty of Health Sciences Rigshospitalet Copenhagen, Denmark Celia D. Sladek, PhD Professor and Acting Chair Department of Physiology and Biophysics Finch University of Health Sciences Chicago Medical School North Chicago, Illinois John R. Sladek, Jr., PhD Professor and Chairman Department of Neuroscience Finch University of Health Sciences Chicago Medical School North Chicago, Illinois Eduardo Slatopolsky, MD Renal Division Washington University School of Medicine St. Louis, Missouri

XViii

CONTRIBUTING AUTHORS

Robert C. Smallridge, MD Professor of Medicine Mayo Medical School Chair, Endocrine Division Mayo Clinic Jacksonville, Florida Robert J. Smith, MD Professor of Medicine Chief of Endocrinology Brown University School of Medicine Director, Hallett Diabetes Center Rhode Island Hospital Providence, Rhode Island Richard H. Snider, Jr., PhD Chief Chemist Endocrinology Research Laboratory Veterans Affairs Medical Center Washington, DC Phyllis W. Speiser, MD Professor of Clinical Pediatrics Department of Pediatrics New York University School of Medicine New York, New York North Shore University Hospital Manhasset, New York Harvey J. Stern, MD, PhD Genetics and IVF Institute Fairfax, Virginia Martin J. Stevens, MD Associate Professor of Internal Medicine University of Michigan Medical School Ann Arbor, Michigan Andrew F. Stewart, MD Professor of Medicine Chief, Division of Endocrinology University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania Elizabeth A. Streeten, MD Clinical Assistant Professor of Medicine Department of Endocrinology, Diabetes, and Obesity University of Maryland School of Medicine Baltimore, Maryland Gordon J. Strewler, MD Professor of Medicine Department of Medical Service Harvard Medical School Boston, Massachusetts Veterans Affairs Boston Healthcare System West Roxbury, Massachusetts Martin I. Surks, MD Professor of Medicine and Pathology Department of Medicine Albert Einstein College of Medicine of Yeshiva University Montefiore Medical Center Bronx, New York

Arthur L. M. Swislocki, MD Associate Professor of Medicine Department of Internal Medicine University of California, Davis, School of Medicine Davis, California Veterans Affairs Outpatient Clinic Martinez, California Shahrad Taheri, MSc, MB, MRCP Wellcome Trust Research Fellow Division of Endocrinology and Metabolism University of London Imperial College School of Medicine Hammersmith Hospital London, England Robert J. Tanenberg, MD, FACP Professor of Medicine Section of Endocrinology and Metabolism Brody School of Medicine East Carolina University School of Medicine Greenville, North Carolina Kamal Thapar, MD Assistant Professor of Neurosurgery University of Toronto Faculty of Medicine Toronto Western Hospital, University Health Toronto, Ontario Canada Ramesh K. Thapar, MD Senior Resident Department of Psychiatry University of Maryland School of Medicine Baltimore, Maryland Michael A. Thomas, MD Associate Professor Department of Clinical Obstetrics and Gynecology University of Cincinnati College of Medicine Cincinnati, Ohio Christopher J. Thompson, MB, ChB, MD, FRCPI

Consultant Physician and Endocrinologist Department of Endocrinology Royal College of Surgeons in Ireland Beaumont Hospital Dublin, Ireland

David J. Torpy, MBBS, PhD, FRACP Senior Lecturer Department of Medicine University of Queensland . Faculty of Health Sciences Brisbane, Australia Carmelita U. Tuazon, MD, mph Professor of Medicine George Washington University School of Medicine and Health Sciences Washington, DC Catherine Tuck, MD Assistant Professor of Medicine Columbia University College of Physicians and Surgeons New York, New York Michael L. Tuck, MD Professor of Medicine University of California, Los Angeles, UCLA School of Medicine Los Angeles, California Veterans Affairs Medical Center, Sepulveda Sepulveda, California Stephen Jon Usala, MD, PhD Clinical Associate Professor Department of Medicine Texas Tech University Health Sciences Center School of Medicine Amarillo, Texas Eve Van Cauter, PhD Professor of Medicine University of Chicago Pritzker School of Medicine Chicago, Illinois Greet H. Van Den Berghe, MD, PhD Associate Professor of Intensive Care Medicine Catholic University of Leuven Leuven, Belgium David H. Van Thiel, MD Director of Transplantation Loyola University of Chicago Stritch School of Medicine Loyola University Medical Center Liver Transplant Office Maywood, Illinois Joseph G. Verbalis, MD Professor of Medicine and Physiology Georgetown University School of Medicine Washington, DC Robert Volpe, MD, FRCP(C), MACP, FRCP (Edin & Lord)

Keith Tornheim, PhD Associate Professor of Biochemistry Boston University School of Medicine Boston, Massachusetts

Professor Emeritus Department of Medicine University of Toronto Faculty of Medicine Toronto, Ontario Canada

CONTRIBUTING AUTHORS Steven G. Waguespack, md Fellow, Adult and Pediatric Endocrinology Departments of Medicine and Pediatrics Division of Endocrinology Indiana University School of Medicine Riley Children's Hospital Indianapolis, Indiana Brian Walsh, MD Director, Menopause Center Department of Obstetrics and Gynecology Harvard Medical School Brigham and Women's Hospital Boston, Massachusetts David O. Walterhouse, MD Assistant Professor of Pediatrics Northwestern University Medical School Children's Memorial Hospital Chicago, Illinois Emily C. Walvoord, MD Senior Fellow Department of Pediatric Endocrinology and Diabetology Indiana University School of Medicine Riley Hospital for Children Indianapolis, Indiana James H. Warram, MD, ScD Senior Investigator Section on Genetics and Epidemiology Research Division Harvard Medical School Joslin Diabetes Center Boston, Massachusetts

Stephen I. Wasserman, md Helen M. Ranney Professor of Medicine Chair, Department of Medicine University of California, San Diego, School of Medicine La Jolla, California Colleen Weber, rn Pediatric Endocrine Nurse Cardinal Glennon Children's Hospital St. Louis, Missouri Anthony Peter Weetman, md, frcp, DSc Professor of Medicine University Department of Clinical Sciences University of Sheffield School of Medicine Northern General Hospital Sheffield, England Gordon C. Weir, MD Professor of Medicine Research Division Harvard Medical School Joslin Diabetes Center Boston, Massachusetts Laura S. Welch, MD Director, Occupational and Environmental Medicine Georgetown University School of Medicine Washington Hospital Center Washington, DC Samuel A. Wells, Jr., MD Professor of Surgery Washington University School of Medicine St. Louis, Missouri

Michelle P. Warren, MD Professor of Obstetrics and Gynecology and Medicine Wyeth Ayerst Professor of Women's Health Columbia University College of Physicians and Surgeons New York, New York

Jon C. White, MD, FACS Director of Surgical Intensive Care Department of Surgery Veterans Affairs Medical Center Associate Professor of Surgery George Washington University School of Medicine and Health Sciences Washington, DC

Leonard Wartofsky, MD, mph, macp Clinical Professor of Medicine Georgetown University School of Medicine Clinical Professor of Medicine George Washington University School of Medicine and Health Sciences Chair, Department of Medicine Washington Hospital Center Clinical Professor of Medicine Howard University College of Medicine Washington, DC Professor of Medicine and Physiology Uniformed Services University of the Health Sciences F. Edward Hebert School of Medicine Bethesda, Maryland

Perrin C. White, MD Professor of Pediatrics University of Texas Southwestern Medical Center at Dallas Southwestern Medical School Dallas, Texas Michael P. Whyte, MD Medical-Scientific Director Department of Metabolic and Molecular Research Professor of Medicine, Pediatrics, and Genetics Division of Bone and Mineral Diseases Washington University School of Medicine Barnes-Jewish Hospital St. Louis, Missouri

xix

Gordon H. Williams, MD Professor of Medicine Harvard Medical School Chair, Endocrine-Hypertension Division Brigham and Women's Hospital Boston, Massachusetts Stephen J. Winters, MD Professor of Medicine Chief, Division of Endocrinology and Metabolism University of Louisville School of Medicine Louisville, Kentucky Joseph I. Wolfsdorf, MB, BCh Associate Professor of Pediatrics Department of Medicine Division of Endocrinology Harvard Medical School Children's Hospital National Medical Center Boston, Massachusetts I-Tien Yeh, MD Associate Professor Department of Pathology University of Texas Medical School at San Antonio University Health Center San Antonio, Texas Paul M. Yen, MD Chief, Molecular Regulation and Neuroendocrinology Clinical Endocrinology Branch National Institute of Diabetes and Digestive and Kidney Disease National Institutes of Health Bethesda, Maryland James E. Zadina, PhD Professor of Medicine Tulane University School of Medicine Director, Neuroscience Laboratory Department of Research Veterans Affairs Medical Center New Orleans, Louisiana Gary P. Zaloga, MA, MD Director of Critical Care Medicine Department of Medicine Georgetown University School of Medicine Washington Hospital Center Washington, DC Charles Zaloudek, md Professor Department of Pathology University of California, San Francisco, School of Medicine San Francisco, California

XX

CONTRIBUTING AUTHORS

Carol Zapalowski, MD, PhD Colorado Center for Bone Research Lakewood, Colorado Thomas R. Ziegler, MD Associate Professor of Medicine Division of Endocrinology/Metabolism Emory University School of Medicine Atlanta, Georgia

Michael Zinger, MD Clinical Instructor Department of Obstetrics and Gynecology Division of Reproductive Endocrinology University of Cincinnati College of Medicine Cincinnati, Ohio

PREFACE

This third edition of Principles and Practice of Endocrinology and Metabolism has been substantially and systematically revised. All of the chapters have been updated, many have been entirely rewritten, and many deal with completely new topics. Furthermore, additional important information and references have been added up until the very date of printing. The new chapters covering topics that did not appear in depth in the prior edition include: Molecular Biology: Present and Future; Pituitary Tumors: Overview of Therapeutic Options; The Incidental Adrenal Mass; Appetite; Pancreas and Islet Transplantation; Syndrome X; Endocrine Effects on Lipids; Compendium of Growth Factors and Cytokines; The Endocrine Blood Cells; The Endocrine Adipocyte; and Endocrine Disorders in Human Immunodeficiency Virus Infection. We would like to welcome the authors of these chapters, and also the new authors who have updated, extensively revised, or have entirely rewritten chapters on topics that appeared in the last edition. A new section has been added to this textbook: Endocrinology of Critical Illness. The six chapters comprising this section address the multiple aspects of this condition in a manner that is unique. Critical illness, which to some extent afflicts the great bulk of humankind at some time in their lives, has enormous hormonal and metabolic dimensions that relate directly to the diagnosis of the illness, influence the response of the host and the consequent evolution of the condition, and play a role in its outcome. The spe¬ cific chapters include Critical Illness and Systemic Inflammation,

Endocrine Markers and Mediators in Critical Illness, The Hypo¬ thalamic-Pituitary-Adrenal Axis in Stress and Critical Illness, Neuroendocrine Response to Acute versus Prolonged Critical Ill¬ ness, Fuel Metabolism and Nutrient Delivery in Critical Illness, and Endocrine Therapeutics in Critical Illness. These subjects are of great importance to every endocrine clinician as well as many who are involved in fundamental endocrine research. Overall, the goal of this textbook is to continue to provide, in a readable, understandable, and well-illustrated format, the clinical and basic information on endocrinology and metabo¬ lism that will be useful to both clinicians and basic scientists. We also wish this book to be a useful source of information for inter¬ nists, house staff, and medical students. We have attempted to cover the field thoroughly and broadly, to include most of the known endocrine and metabolic disorders and hormonal mes¬ senger molecules, to furnish appropriate and current references, and to be of practical benefit to our readers. A complete CD version of this entire textbook is available. It contains approximately 4000 self-assessment questions that have been assembled and edited by Dr. Meeta Sharma. I wish to acknowledge the very helpful library assistance of Joanne Bennett. I am very grateful for the indispensable edito¬ rial and pharmaceutical aid of my Editorial Assistant, Roberta L. Brown, Pharm. D.

Kenneth L. Becker, MD, PhD

xxi

■

PREFACE TO THE FIRST EDITION

Although there are several excellent large textbooks of endo¬ crinology, we have felt the need for a book which would aim at encompassing all aspects of the field, a book which would be disease-oriented, would have practical applicability to the care of the adult and pediatric patient, and could be consulted to obtain a broad range of pathophysiologic, diagnostic, and ther¬ apeutic information. To fulfill this goal we called upon not only eminent special¬ ists in endocrinology but also upon experts in many fields of medicine and science. The first part of the book surveys general aspects of endocrinology. The eight succeeding parts deal with specific fields of endocrinology: The Endocrine Brain and Pitu¬ itary Gland, The Thyroid Gland, Calcium and Bone Metabolism, The Adrenal Glands, Sex Determination and Development, Endocrinology of the Female, Endocrinology of the Male, and Disorders of Fuel Metabolism. Each of these parts contains rele¬ vant anatomic, physiologic, diagnostic, and therapeutic infor¬ mation and, when indicated, pediatric coverage of the topic. Diffuse Hormonal Secretion expounds upon the fact that endocrine function is not confined to anatomically discrete endocrine glands but is also intrinsic to all tissues and organs. This part is divided in two; it first presents a discussion of hor¬ mones which have a diffuse distribution and are not reviewed elsewhere in the book, and subsequently it deals with body con¬ stituents which are important sites of hormonal secretion. Heritable Abnormalities of Endocrinology and Metabolism underlines the importance of genetics in the causation of many endocrine and metabolic abnormalities. Endocrine and meta¬ bolic dysfunction in the young and in the aged is the subject of a separate part, because in both of these age groups hormonal function as well as endocrine disorders differ profoundly from those of individuals in their middle decades. Interrelationships Between Hormones and the Body dis¬ cusses the impact of hormones on the soma and addresses clin¬ ical aspects of the disorders they may engender. Hormones and Cancer examines the phenomenon of hormone-induced neo¬ plasms, elaborating on the fact that all neoplasms secrete hor¬ mones, that several of these hormones can cause additional clinical disorders, and that some neoplasms respond therapeuti¬ cally to hormonal manipulation. The ensuing part, entitled Endocrine and Metabolic Effects of Toxic Agents deals with the sometimes subtle, sometimes pro¬ found influence of four nearly omnipresent agents: medication, alcohol, tobacco, and cannabis; it also addresses the conse¬ quences of environmental toxins on the endocrine system. The last part deals with the therapeutic use of drugs in endocrinol¬ ogy and the proper interpretation of laboratory values. It offers

an extensive table on the clinical use of endocrine-related drugs, a table on reference values, and an outline of the dynamic pro¬ cedures used in endocrinology. The goal of these tabular chap¬ ters is to facilitate the day-to-day evaluation and therapy of the endocrine patient. As a rule, the emphasis of this textbook is on the endocrinol¬ ogy of the human being. Animal data are presented only when contributing to a better understanding of human physiology and pathology. To maximize current relevance, historical infor¬ mation is kept to a minimum. While efforts were made to avoid repetition, the coverage of certain topics may recur when viewed from different standpoints. It is hoped that this will pro¬ vide a wider dimension of the understanding of endocrine and metabolic function and dysfunction. In order not to interrupt continuity, bibliographic references are grouped at the end of each part. Finally, with the interest of the reader in mind, particular attention was given to composing an index as detailed as possible. I wish to thank the associate editors of this text for their skill, their enthusiasm, and their hard work. We all are very grateful for the expertise of our many eminent contributors. During the preparation of the manuscripts, there was considerable inter¬ communication between these contributors and their respective editors concerning both content and presentation. I wish to acknowledge the participation of Richard H. Snider, PhD, and Eric S. Nylen, MD, who have provided outstanding editorial assistance throughout the preparation of the textbook. The field of endocrinology and metabolism is evolving rap¬ idly. New data are being developed continuously, and with this in mind, all contributors were encouraged to add up-to-date information until nearly the date of publication. There are numerous matters upon which there is no current common agreement, and logical arguments can be marshaled to buttress diametrically different viewpoints. This textbook is written by many authors; though most of the beliefs and conclu¬ sions of the contributors tend to reflect those of the editors, no attempt was made to impose a uniformity of pathophysiologic, diagnostic, or therapeutic viewpoints, and the book does not lack for differences of opinion. We hope that the Principles and Practice of Endocrinology and Metabolism will be a relevant sourcebook for those interested in the science and the practice of this fascinating discipline, whether they be clinicians, basic scientists, allied health person¬ nel, or students.

Kenneth L. Becker, md, PhD

XXlll

CONTENTS

PART

I

GENERAL PRINCIPLES OF ENDOCRINOLOGY Kenneth L. Becker, Editor

1 ENDOCRINOLOGY AND THE ENDOCRINE PATIENT 2

4 HORMONAL ACTION

36

DARYL K. GRANNER

KENNETH L. BECKER, ERIC S. NYLEN, and RICHARD H. SNIDER JR

5 FEEDBACK CONTROL IN ENDOCRINE SYSTEMS 50

2 MOLECULAR BIOLOGY: PRESENT AND FUTURE 8

DANIEL N. DARLINGTON and MARY F. DALLMAN

MEHBOOB A. HUSSAIN and JOEL F. HABENER

6 ENDOCRINE RHYTHMS EVE VAN CAUTER

3 BIOSYNTHESIS AND SECRETION OF PEPTIDE HORMONES 24

57

7 GROWTH AND DEVELOPMENT IN THE NORMAL INFANT AND CHILD 68

WILLIAM W. CHIN

GILBERT P. AUGUST

PART II THE ENDOCRINE BRAIN AND PITUITARY GLAND Gary L. Robertson, Editor 8 MORPHOLOGY OF THE ENDOCRINE

16 PITUITARY GONADOTROPINS AND THEIR DISORDERS 170

BRAIN, HYPOTHALAMUS, AND NEUROHYPOPHYSIS 84 JOHN R. SLADEK, JR, and CELIA D. SLADEK

9 PHYSIOLOGY AND PATHOPHYSIOLOGY OF THE ENDOCRINE BRAIN AND HYPOTHALAMUS 90 PAUL E. COOPER

10 PINEAL GLAND

98

WILLIAM J. BREMNER ILPO HUHTANIEMI, and JOHN K. AMORY

17 HYPOPITUITARISM

177

JOSEPH J. PINZONE

18 HYPOTHALAMIC AND PITUITARY DISORDERS IN INFANCY AND CHILDHOOD 192 ALAN D. ROGOL

RUSSEL J. REITER

19 THE OPTIC CHIASM IN ENDOCRINOLOGIC DISORDERS 204

11 MORPHOLOGY OF THE PITUITARY IN HEALTH AND DISEASE 103

R. MICHAEL SIATKOWSKI and JOEL S. GLASER

KAMAL THAPAR KALMAN KOVACS, and EVA HORVATH

20 DIAGNOSTIC IMAGING OF THE SELLAR REGION 219 SECTION

ERIC BOUREKAS, MARY OEHLER, and DONALD CHAKERES

A

ADENOHYPOPHYSIS

21 MEDICAL TREATMENT OF PITUITARY TUMORS AND HYPERSECRETORY STATES 237

12 GROWTH HORMONE AND ITS DISORDERS 129

DAVID H. SARNE

GERHARD BAUMANN

13 PROLACTIN AND ITS DISORDERS

145

LAURENCE KATZNELSON and ANNE KLIBANSKI

14 ADRENOCORTICOTROPIN: PHYSIOLOGY AND CLINICAL ASPECTS 153 DAVID J. TORPY and RICHARD V JACKSON

15 THYROID-STIMULATING HORMONE AND ITS DISORDERS 159 JOSHUA L. COHEN

22 RADIOTHERAPY OF PITUITARYHYPOTHALAMIC TUMORS 243 MINESH P MEHTA

23 NEUROSURGICAL MANAGEMENT OF PITUITARY-HYPOTHALAMIC NEOPLASMS 254 DAVID S. BASKIN

24 PITUITARY TUMORS: OVERVIEW OF THERAPEUTIC OPTIONS 264 PHILIPPE CHANSON XXV

XXVI

CONTENTS

SECTION B NEUROHYPOPHYSIAL SYSTEM 25 PHYSIOLOGY OF VASOPRESSIN, OXYTOCIN, AND THIRST 276 GARY L. ROBERTSON

PART

26 DIABETES INSIPIDUS AND HYPEROSMOLAR SYNDROMES 285 PETER H. BAYLIS and CHRISTOPHER J. THOMPSON 27 INAPPROPRIATE ANTIDIURESIS AND OTHER HYPOOSMOLAR STATES 293 JOSEPH G. VERBALIS

III THE THYROID GLAND Leonard Wartofsky, Editor

28 APPROACH TO THE PATIENT WITH THYROID DISEASE 308 LEONARD WARTOFSKY 29 MORPHOLOGY OF THE THYROID GLAND 311 VIRGINIA A. LIVOLSI 30 THYROID PHYSIOLOGY: SYNTHESIS AND RELEASE, IODINE METABOLISM, BINDING AND TRANSPORT 314 H. LESTER REED 31 THYROID PHYSIOLOGY: HORMONE ACTION, RECEPTORS, AND POSTRECEPTOR EVENTS 321 PAUL M. YEN 32 THYROID HORMONE RESISTANCE SYNDROMES 325 STEPHEN JON USALA 33 THYROID FUNCTION TESTS 329 ROBERT C. SMALLRIDGE 34 THYROID UPTAKE AND IMAGING 336 SAUL D. SARKAR and DAVID V. BECKER 35 THYROID SONOGRAPHY, COMPUTED TOMOGRAPHY, AND MAGNETIC RESONANCE IMAGING 342 MANFRED BLUM 36 ABNORMAL THYROID FUNCTION TEST RESULTS IN EUTHYROID PERSONS 351 HENRY B. BURCH

PART IV

37 ADVERSE EFFECTS OF IODIDE 360 JENNIFER A. NUOVO and LEONARD WARTOFSKY 38 NONTOXIC GOITER 366 PAUL J. DAVIS and FAITH B. DAVIS 39 THE THYROID NODULE 374 LEONARD WARTOFSKY and ANDREW J. AHMANN 40 THYROID CANCER 382 ERNEST L. MAZZAFERRI 41 UNUSUAL THYROID CANCERS 402 MATTHEW D. RINGEL 42 HYPERTHYROIDISM 409 KENNETH D. BURMAN 43 ENDOCRINE OPHTHALMOPATHY 428

44 45

46

47

MELVIN G. ALPER and LEONARD WARTOFSKY SURGERY OF THE THYROID GLAND EDWIN L. KAPLAN HYPOTHYROIDISM 445 LAWRENCE E. SHAPIRO and MARTIN I. SURKS THYROIDITIS 454 IVOR M. D. JACKSON and JAMES V. HENNESSEY THYROID DISORDERS OF INFANCY AND CHILDHOOD 462 WELLINGTON HUNG

CALCIUM AND BONE METABOLISM John P. Bilezikian, Editor

48 MORPHOLOGY OF THE PARATHYROID GLANDS 474 VIRGINIA A. LIVOLSI 49 PHYSIOLOGY OF CALCIUM METABOLISM 478 EDWARD M. BROWN 50 PHYSIOLOGY OF BONE 489 LAWRENCE G. RAISZ

51 PARATHYROID HORMONE 497 DAVID GOLTZMAN and GEOFFREY N. HENDY 52 PARATHYROID HORMONE-RELATED PROTEIN 512 GORDON J. STREWLER

440

CONTENTS

53 CALCITONIN GENE FAMILY OF PEPTIDES 520 KENNETH L. BECKER, BEAT MULLER, ERIC S. NYLEN, REGIS COHEN, OMEGA L. SILVA, JON C. WHITE, and RICHARD H. SNIDER, JR. 54 VITAMIN D 534 THOMAS L. CLEMENS and JEFFREY L. H. O’RI OR DAN 55 BONE QUANTIFICATION AND DYNAMICS OF TURNOVER 541 DAVID W. DEMPSTER and ELIZABETH SHANE 56 MARKERS OF BONE METABOLISM 548 MARKUS J. SEIBEL, SIMON P. ROBINS, and JOHN P. BILEZIKIAN 57 CLINICAL APPLICATION OF BONE MINERAL DENSITY MEASUREMENTS 557 PAUL D. MILLER, ABBY ERICKSON, and CAROL ZAPALOWSKI 58 PRIMARY HYPERPARATHYROIDISM 564 SHONNIJ. SILVER BERG and JOHN P BILEZIKIAN 59 NONPARATHYROID HYPERCALCEMIA 574 ANDREW F. STEWART 60 HYPOPARATHYROIDISM AND OTHER CAUSES OF HYPOCALCEMIA 586 SUZANNE M. JAN DE BEUR, ELIZABETH A. STREETEN, and MICHAEL A. LEVINE

XXVII

61 RENAL OSTEODYSTROPHY 603 KEVIN J. MARTIN, ESTHER A. GONZALEZ, and EDUARDO SLATOPOLSKY 62 SURGERY OF THE PARATHYROID GLANDS 610 GERARD M. DOHERTY and SAMUEL A. WELLS, JR. 63 OSTEOMALACIA AND RICKETS 615 NORMAN H. BELL 64 OSTEOPOROSIS 623 ROBERT LINDSAY and FELICIA COSMAN 65 PAGET DISEASE OF BONE 642 ETHEL S. SIRIS 66 RARE DISORDERS OF SKELETAL FORMATION AND HOMEOSTASIS 651 MICHAEL P. WHYTE 67 DISEASES OF ABNORMAL PHOSPHATE METABOLISM 662 MARC K. DREZNER 68 MAGNESIUM METABOLISM 673 ROBERT K. RUDE 69 NEPHROLITHIASIS 679 MURRAY J. FAVUS and FREDRIC L. COE 70 DISORDERS OF CALCIUM AND BONE METABOLISM IN INFANCY AND CHILDHOOD 688 THOMAS O. CARPENTER

PART V THE ADRENAL GLANDS

D. Lynn Lorianx, Editor 71 MORPHOLOGY OF THE ADRENAL CORTEX AND MEDULLA 698 DONNA M. ARAB O’BRIEN 72 SYNTHESIS AND METABOLISM OF CORTICOSTEROIDS 704 PERRIN C. WHITE 73 CORTICOSTEROID ACTION 714 PERRIN C. WHITE 74 TESTS OF ADRENOCORTICAL FUNCTION 719 D. LYNN LORIAUX 75 CUSHING SYNDROME 723 DAVID E. SCHTEINGART 76 ADRENOCORTICAL INSUFFICIENCY 739 D. LYNN LORIAUX 77 CONGENITAL ADRENAL HYPERPLASIA 743 PHYLLIS W. SPEISER 78 CORTICOSTEROID THERAPY 751 LLOYD AXELROD

79 RENIN-ANGIOTENSIN SYSTEM AND ALDOSTERONE 764 DALILA B. CORRY and MICHAEL L. TUCK 80 HYPERALDOSTERONISM 773 JOHN R. GILL, JR. 81 HYPOALDOSTERONISM 785 JAMES C. MELBY 82 ENDOCRINE ASPECTS OF HYPERTENSION 791 DALILA B. CORRY and MICHAEL L. TUCK 83 ADRENOCORTICAL DISORDERS IN INFANCY AND CHILDHOOD 806 ROBERT L. ROSENFIELD and KE-NAN QIN 84 THE INCIDENTAL ADRENAL MASS 816 D. LYNN LORIAUX 85 PHYSIOLOGY OF THE ADRENAL MEDULLA AND THE SYMPATHETIC NERVOUS SYSTEM 817 DAVID S. GOLDSTEIN

xxviii

CONTENTS

86 PHEOCHROMOCYTOMA AND OTHER DISEASES OF THE SYMPATHETIC NERVOUS SYSTEM 827 HARRY R. KEISER 87 ADRENOMEDULLARY DISORDERS OF INFANCY AND CHILDHOOD 834 WELLINGTON HUNG

88 DIAGNOSTIC IMAGING OF THE ADRENAL GLANDS 837 DONALD L. MILLER 89 SURGERY OF THE ADRENAL GLANDS 843 GARY R. PEPLINSKI and JEFFREY A. NORTON

Robert W. Rebar and Willi, 90 NORMAL AND ABNORMAL SEXUAL DIFFERENTIATION AND DEVELOPMENT 852 JOE LEIGH SIMPSON and ROBERT W. REBAR 885 91 PHYSIOLOGY OF PUBERTY PETER A. LEE

92

893

EMILY C. WALVOORD, STEVEN G. WAGUESPACK, and ORA HIRSCH PESCOVITZ 93 MICROPENIS, HYPOSPADIAS, AND CRYPTORCHIDISM IN INFANCY AND CHILDHOOD 908 WELLINGTON HUNG

PART VII ENDOCRINOLOGY OF THE FEMALE Robert W. Rebar, Editor 94 MORPHOLOGY AND PHYSIOLOGY OF THE OVARY 918 GREGORY F. ERICKSON and JAMES R. SCHREIBER 95 THE NORMAL MENSTRUAL CYCLE AND THE CONTROL OF OVULATION 935 ROBERT W. REBAR, GARY D. HODGEN, and MICHAEL ZINGER 96 DISORDERS OF MENSTRUATION, OVULATION, AND SEXUAL RESPONSE 947 ROBERT W. REBAR 97 OVULATION INDUCTION 967 MICHAEL A. THOMAS 98 ENDOMETRIOSIS 972 ROBERT L. BARBIERI 99 PREMENSTRUAL SYNDROME 977 ROBERT L. REID and RUTH C. FRETTS 100 MENOPAUSE 982 BRIAN WALSH and ISAAC SCHIFF 101 HIRSUTISM, ALOPECIA, AND ACNE 991 ENRICO CARMINA and ROGERIO A. LOBO 102 FUNCTIONING TUMORS AND TUMOR-LIKE CONDITIONS OF THE OVARY 1009 l-TIEN YEH, CHARLES ZALOUDEK, and ROBERT J. KURMAN 103 THE DIFFERENTIAL DIAGNOSIS OF FEMALE INFERTILITY 1015 STEVEN J. ORYand MARCELO J. BARRIONUEVO

104 FEMALE CONTRACEPTION 1022 ALISA B. GOLDBERG and PHILIP DARNEY 105 COMPLICATIONS AND SIDE EFFECTS OF STEROIDAL CONTRACEPTION 1033 ALISA B. GOLDBERG and PHILIP DARNEY 106 MORPHOLOGY OF THE NORMAL BREAST, ITS HORMONAL CONTROL, AND PATHOPHYSIOLOGY 1039 RICHARD E. BLACKWELL 107 CONCEPTION, IMPLANTATION, AND EARLY DEVELOPMENT 1049 PHILIP M. IANNACCONE, DAVID O. WALTERHOUSE, and KRISTINA C. PFENDLER 108 THE MATERNAL-FETAL-PLACENTAL UNIT 1059 BRUCE R. CARR 109 ENDOCRINOLOGY OF PARTURITION JOHN R. G. CHALLIS 110 ENDOCRINE DISEASE IN PREGNANCY 1077 MARK E. MOLITCH 111 TROPHOBLASTIC TISSUE AND ITS ABNORMALITIES 1091 CYNTHIA G. KAPLAN 112 ENDOCRINOLOGY OF TROPHOBLASTIC TISSUE 1096 Z M. LEI and CH. V. RAO

1072

CONTENTS

XXIX

PART VIII ENDOCRINOLOGY OF THE MALE William J. Bremner, Editor 113 MORPHOLOGY AND PHYSIOLOGY OF THE TESTIS 1104 DAVID M. DE KRETSER 114 EVALUATION OF TESTICULAR FUNCTION 1115 STEPHEN J. WINTERS 115 MALE HYPOGONADISM 1125 STEPHEN R. PLYMATE 116 TESTICULAR DYSFUNCTION IN SYSTEMIC DISEASE 1150 H. IN. GORDON BAKER 117 ERECTILE DYSFUNCTION 1159 GLENN R. CUNNINGHAM and MAX HIRSHKOWITZ 118 MALE INFERTILITY 1173 RICHARD V. CLARK

119 CLINICAL USE AND ABUSE OF ANDROGENS AND ANTIANDROGENS 1181 ALVIN M. MATSUMOTO 120 GYNECOMASTIA 1200 ALLAN R. GLASS 121 ENDOCRINE ASPECTS OF BENIGN PROSTATIC HYPERPLASIA 1207 ELIZABETH A. MILLER and WILLIAM J. ELLIS 122 TESTICULAR TUMORS 1212 NIELS E. SKAKKEBAEK and MIKAEL R0RTH 123 MALE CONTRACEPTION 1220 JOHN K. AMORY and WILLIAM J. BREMNER

PART IX DISORDERS OF FUEL METABOLISM C. Ronald Kahn, Editor SECTION A FOOD AND ENERGY 124 PRINCIPLES OF NUTRITIONAL MANAGEMENT 1226 ROBERTA P. DURSCHLAG and ROBERT J. SMITH 125 APPETITE 1233 ANGELICA LINDEN HIRSCHBERG 126 OBESITY 1239 JULES HIRSCH, LESTER B. SALANS, and LOUIS J. ARONNE 127 STARVATION 1247 RUTH S. MACDONALD and ROBERT J. SMITH 128 ANOREXIA NERVOSA AND OTHER EATING DISORDERS 1251 MICHELLE P. WARREN and REBECCA J. LOCKE 129 FUEL HOMEOSTASIS AND INTERMEDIARY METABOLISM OF CARBOHYDRATE, FAT, AND PROTEIN 1257 NEIL B. RUDERMAN, KEITH TORNHEIM, and MICHAEL N. GOODMAN 130 VITAMINS: HORMONAL AND METABOLIC INTERRELATIONSHIPS 1272 ALAA ABOU-SAIF and TIMOTHY O. LIPMAN 131 TRACE MINERALS: HORMONAL AND METABOLIC INTERRELATIONSHIPS 1277 ROBERT D. LINDEMAN

132 EXERCISE: ENDOCRINE AND METABOLIC EFFECTS 1287 JACQUES LEBLANC

SECTION B DIABETES MELLITUS 133 MORPHOLOGY OF THE ENDOCRINE PANCREAS 1292 SUSAN BONNER-WEIR 134 ISLET CELL HORMONES: PRODUCTION AND DEGRADATION 1296 GORDON C. WEIR and PHILIPPE A. HALBAN 135 GLUCOSE HOMEOSTASIS AND INSULIN ACTION 1303 C. RONALD KAHN 136 CLASSIFICATION, DIAGNOSTIC TESTS, AND PATHOGENESIS OF TYPE 1 DIABETES MELLITUS 1307 GEORGE S. EISENBARTH 137 ETIOLOGY AND PATHOGENESIS OF TYPE 2 DIABETES MELLITUS AND RELATED DISORDERS 1315 C. RONALD KAHN 138 NATURAL HISTORY OF DIABETES MELLITUS 1320 ANDRZEJ S. KROLEWSKI and JAMES H. WARRAM

XXX

CONTENTS

139 SECONDARY FORMS OF DIABETES MELLITUS 1327 VERONICA M. CATANESE and C. RONALD KAHN 140 EVALUATION OF METABOLIC CONTROL IN DIABETES 1336 ALLISON B. GOLDFINE 141 DIET AND EXERCISE IN DIABETES 1340 OM P. GANDA 142 ORAL AGENTS FOR THE TREATMENT OF TYPE 2 DIABETES MELLITUS 1344 ALLISON B. GOLDFINE and ELEFTHERIA MARATOS-FLIER 143 INSULIN THERAPY AND ITS COMPLICATIONS 1348 GORDON C. WEIR 144 PANCREAS AND ISLET TRANSPLANTATION 1360 GORDON C. WEIR 145 SYNDROME X 1365 GERALD M. REAVEN 146 SYNDROMES OF EXTREME INSULIN RESISTANCE 1369 JEFFREYS. FLIER and CHRISTOS S. MANTZOROS 147 CARDIOVASCULAR COMPLICATIONS OF DIABETES MELLITUS 1380 KARIN HEHENBERGER and GEORGE L. KING 148 DIABETIC NEUROPATHY 1391 EVA L. FELDMAN, MARTIN J. STEVENS, JAMES W. RUSSELL, and DOUGLAS A. GREENE 149 GASTROINTESTINAL COMPLICATIONS OF DIABETES 1399 FREDERIC D. GORDON and KENNETH R. FALCHUK 150 DIABETIC NEPHROPATHY 1403 RALPH A. DEFRONZO 151 DIABETES AND THE EYE 1418 LAWRENCEI. RAND 152 DIABETES AND INFECTION 1424 GEORGE M. ELIOPOULOS 153 DIABETES AND THE SKIN 1428 ROBERT J. TANENBERG and RICHARD C. EASTMAN 154 THE DIABETIC FOOT 1434 GARY W. GIBBONS

155 DIABETIC ACIDOSIS, HYPEROSMOLAR COMA, AND LACTIC ACIDOSIS 1438 K. GEORGE M. M. ALBERTI 156 DIABETES MELLITUS AND PREGNANCY 1451 LOIS JOVANOVIC 157 DIABETES MELLITUS IN THE INFANT AND CHILD 1459 DOROTHY J. BECKER and ALLAN L. DRASH

SECTION C HYPOGLYCEMIA 158 HYPOGLYCEMIC DISORDERS IN THE ADULT 1469 RICHARD J. COMI and PHILLIP GORDEN 159 LOCALIZATION OF ISLET CELL TUMORS 1477 DONALD L. MILLER 160 SURGERY OF THE ENDOCRINE PANCREAS 1482 JON C. WHITE 161 HYPOGLYCEMIA OF INFANCY AND CHILDHOOD 1488 JOSEPH I. WOLFSDORF and MARK KORSON

SECTION D LIPID METABOLISM 162 BIOCHEMISTRY AND PHYSIOLOGY OF LIPID AND LIPOPROTEIN METABOLISM 1503 ROBERT IN. MAHLEY 163 LIPOPROTEIN DISORDERS 1513 ERNST J. SCHAEFER 164 TREATMENT OF THE HYPERLIPOPROTEINEMIAS 1531 JOHN C. LAROSA 165 ENDOCRINE EFFECTS ON LIPIDS 1538 HENRY N. GINSBERG, IRA J. GOLDBERG, and CATHERINE TUCK 166 LIPID ABNORMALITIES IN DIABETES MELLITUS 1544 ROBERT E. RATNER, BARBARA V HOWARD, and WILLIAM JAMES HOWARD

PART X DIFFUSE HORMONAL SECRETION__ Eric S. Nylen, Editor 167 GENERAL CHARACTERISTICS OF DIFFUSE PEPTIDE HORMONE SYSTEMS 1554 JENS F. R EH FELD

168 ENDOGENOUS OPIOID PEPTIDES BRIAN M. COX and GREGORY R MUELLER

1556

CONTENTS

169 SOMATOSTATIN 1564 YOGESH C. PATEL 170 KININS 1575 DOMENICO C. REGOLI 171 SUBSTANCE P AND THE TACHYKININS 1578 NEIL ARONIN 172 PROSTAGLANDINS, THROMBOXANES, AND LEUKOTRIENES 1581 R. PAUL ROBERTSON 173 GROWTH FACTORS AND CYTOKINES 1588 DEREK LEROITH and VICKY A. BLAKESLEY 174 COMPENDIUM OF GROWTH FACTORS AND CYTOKINES 1601 BHARAT B. AGGARWAL 175 THE DIFFUSE NEUROENDOCRINE SYSTEM 1605 ERIC S. NYLEN and KENNETH L. BECKER 176 THE ENDOCRINE BRAIN 1611 ABBA J. KASTIN, WEIHONG PAN, JAMES E. ZADINA, and WILLIAM A. BANKS 177 THE ENDOCRINE LUNG 1615 KENNETH L. BECKER

178 THE ENDOCRINE HEART 1622 MIRIAM T. RADEMAKER and ERIC A. ESPINER 179 THE ENDOCRINE ENDOTHELIUM 1634 FRANCESCO COSENTINO and THOMAS F. LOSCHER 180 THE ENDOCRINE BLOOD CELLS 1642 HARISH P G. DAVE and BEAT MULLER 181 THE ENDOCRINE MAST CELL 1649 STEPHEN I. WASSERMAN 182 THE ENDOCRINE ENTERIC SYSTEM 1653 JENS J. HOLST 183 THE ENDOCRINE KIDNEY 1666 ALAN DUBROW and LUCA DESIMONE 184 THE ENDOCRINE GENITOURINARY TRACT 1672 JAN FAHRENKRUG and S0REN GRAS 185 THE ENDOCRINE SKIN 1675 MARK R. PITTELKOW 186 THE ENDOCRINE ADIPOCYTE 1686 REXFORD S. AHIMA and JEFFREY S. FLIER

PART XI HERITABLE ABNORMALITIES OF ENDOCRINOLOGY _AND METABOLISM_ Kenneth L. Becker, Editor 187 INHERITANCE PATTERNS OF ENDOCRINOLOGIC AND METABOLIC DISORDERS 1692 R. NEIL SCHIMKE 188 MULTIPLE ENDOCRINE NEOPLASIA 1696 GLEN W. SIZEMORE 189 HERITABLE DISORDERS OF COLLAGEN AND FIBRILLIN 1708 PETER H. BYERS 190 HERITABLE DISEASES OF LYSOSOMAL STORAGE 1719 WARREN E. COHEN

PART

XII

191 HERITABLE DISEASES OF AMINO-ACID METABOLISM 1729 HARVEY J. STERN and JAMES D. FINKELSTEIN 192 HERITABLE DISEASES OF PURINE METABOLISM 1738 EDWARD W. HOLMES and DAVID J. NASH EL

IMMUNOLOGIC BASIS OF ENDOCRINE DISORDERS Leonard Wartofsky, Editor

193 THE ENDOCRINE THYMUS 1748 ALLAN L. GOLDSTEIN and NICHOLAS R. S. HALL 194 IMMUNOGENETICS, THE HUMAN LEUKOCYTE ANTIGEN SYSTEM, AND ENDOCRINE DISEASE 1754 JAMES R. BAKER JR

XXXI

195 T CELLS IN ENDOCRINE DISEASE 1757 ANTHONY PETER WEETMAN 196 B CELLS AND AUTOANTIBODIES IN ENDOCRINE DISEASE 1765 ALAN M. MCGREGOR 197 THE IMMUNE SYSTEM AND ITS ROLE IN ENDOCRINE FUNCTION 1770 ROBERT VOLPE

XXXii

CONTENTS

PART

XIII

ENDOCRINE AND METABOLIC DYSFUNCTION IN THE GROWING CHILD AND IN THE AGED Wellington Hung, Editor

198 SHORT STATURE AND SLOW GROWTH IN THE YOUNG 1784 THOMAS ACETO, JR, DAVID P. DEMPSHER, LUIGI GARIBALDI, SUSAN E. MYERS, NANCI BOBROW, and COLLEEN WEBER

199 ENDOCRINOLOGY AND AGING DAVID A. GRUENEWALD and ALVIN M. MATSUMOTO

1808

PART XIV

INTERRELATIONSHIPS BETWEEN HORMONES _AND THE BODY_ Kenneth L. Becker, Editor

200 CEREBRAL EFFECTS OF ENDOCRINE DISEASE 1834 HOYLE LEIGH 201 PSYCHIATRIC-HORMONAL INTERRELATIONSHIPS 1838 MITCHEL A. KLING, MARIANNE HATLE, RAMESH K. THAPAR, and PHILIP W. GOLD 202 RESPIRATION AND ENDOCRINOLOGY 1846 PRASHANTK. ROHATGI and KENNETH L. BECKER 203 THE CARDIOVASCULAR SYSTEM AND ENDOCRINE DISEASE 1857 ELLEN W. SEELY and GORDON H. WILLIAMS 204 GASTROINTESTINAL MANIFESTATIONS OF ENDOCRINE DISEASE 1865 ALLAN G. HALLINE 205 THE LIVER AND ENDOCRINE FUNCTION 1870 NICOLA DE MARIA, ALESSANDRA COLANTONI, and DAVID H. VAN THIEL 206 EFFECTS OF NONRENAL HORMONES ON THE NORMAL KIDNEY 1885 PAUL L. KIMMEL, ANTONIO RIVERA, and PARVEZ KHATRI 207 RENAL METABOLISM OF HORMONES 1895 RALPH RABKIN and MICHAEL J. HAUSMANN 208 EFFECTS OF ENDOCRINE DISEASE ON THE KIDNEY 1902 ELLIE KELEPOURIS and ZALMAN S. AGUS 209 ENDOCRINE DYSFUNCTION DUE TO RENAL DISEASE 1908 ARSHAG D. MOORADIAN

210 NEUROMUSCULAR MANIFESTATIONS OF ENDOCRINE DISEASE 1912 ROBERT B. LAYZER and GARY M. ABRAMS 211 RHEUMATIC MANIFESTATIONS OF ENDOCRINE DISEASE 1920 DAVID J. NASH EL 212 HEMATOLOGIC ENDOCRINOLOGY 1927 HARVEYS. LUKSENBURG, STUARTL. GOLDBERG, and CRAIG M. KESSLER 213 INFECTIOUS DISEASES AND ENDOCRINOLOGY 1937 CARMELITA U. UJAZON and STEPHEN A. MIGUELES 214 ENDOCRINE DISORDERS IN HUMAN IMMUNODEFICIENCY VIRUS INFECTION 1947 STEPHEN A. MIGUELES and CARMELITA U. TUAZON 215 THE EYE IN ENDOCRINOLOGY 1958 ROBERTA. OPPENHEIM and WILLIAM D. MATHERS 216 OTOLARYNGOLOGY AND ENDOCRINE DISEASE 1977 STEPHEN G. HARNER 217 DENTAL ASPECTS OF ENDOCRINOLOGY 1981 ROBERTS. REDMAN 218 THE SKIN AND ENDOCRINE DISORDERS 1991 JO-DAVID FINE, ADNAN NASIR, and KENNETH L. BECKER

CONTENTS

XXXIII

PART XV HORMONES AND CANCER Kenneth L. Becker, Editor 219 PARANEOPLASTIC ENDOCRINE SYNDROMES 2004 KENNETH L. BECKER and OMEGA L. SILVA 220 ENDOCRINE TUMORS OF THE GASTROINTESTINAL TRACT 2015 SHAHRAD TAHERI, KARIM MEERAN, and STEPHEN BLOOM 221 CARCINOID TUMOR AND THE CARCINOID SYNDROME 2021 PAUL N. MATON 222 HORMONES AND CARCINOGENESIS: LABORATORY STUDIES 2024 JONATHAN J. LI and SARA ANTONIA LI

223 SEX HORMONES AND HUMAN CARCINOGENESIS: EPIDEMIOLOGY 2030 ROBERT N. HOOVER 224 ENDOCRINE TREATMENT OF BREAST CANCER 2039 GABRIEL N. HORTOBAGYI 225 ENDOCRINE ASPECTS OF PROSTATE CANCER 2046 CHULSO MOON and CHRISTOPHER J. LOGETHETIS 226 ENDOCRINE CONSEQUENCES OF CANCER THERAPY 2057 DAIVA R. BAJORUNAS

PART XVI ENDOCRINOLOGY OF CRITICAL ILLNESS Eric S. Nylen, Editor 227 CRITICAL ILLNESS AND SYSTEMIC INFLAMMATION 2068 GARYP ZALOGA, BANKIM BHATT, and PAUL MARIK 228 ENDOCRINE MARKERS AND MEDIATORS IN CRITICAL ILLNESS 2077 ABDULLAH A. ALARIFI, GREET H. VAN DEN BERG HE, RICHARD H. SNIDER, JR, KENNETH L. BECKER, BEAT MULLER, and ERIC S. NYLEN 229 THE HYPOTHALAMIC-PITUITARY-ADRENAL AXIS IN STRESS AND CRITICAL ILLNESS 2087 STEFAN R. BORNSTEIN and GEORGE P CHROUSOS

230 NEUROENDOCRINE RESPONSE TO ACUTE VERSUS PROLONGED CRITICAL ILLNESS 2094 GREET H. VAN DEN BERGHE 231 FUEL METABOLISM AND NUTRIENT DELIVERY IN CRITICAL ILLNESS 2102 THOMAS R. ZIEGLER 232 ENDOCRINE THERAPEUTICS IN CRITICAL ILLNESS 2108 ERIC S. NYLEN, GARYP ZALOGA, KENNETH L. BECKER, KENNETH D. BURMAN, LEONARD WARTOFSKY, BEAT MULLER, JON C. WHITE, and ABDULLAH A. ALARIFI

PART XVII ENDOCRINE AND METABOLIC EFFECTS _OF TOXIC AGENTS_ Kenneth L. Becker, Editor 233 ENDOCRINE-METABOLIC EFFECTS OF ALCOHOL 2124 ROBERT H. NOTH and ARTHUR L. M. SWISLOCKI

234 METABOLIC EFFECTS OF TOBACCO, CANNABIS, AND COCAINE 2131 OMEGA L. SILVA 235 ENVIRONMENTAL FACTORS AND TOXINS AND ENDOCRINE FUNCTION 2134 LAURA S. WELCH

XXXIV

CONTENTS

PART XVIII ENDOCRINE DRUGS AND VALUES Kenneth L. Becker, Editor 236 COMPENDIUM OF ENDOCRINE-RELATED DRUGS 2142 DOLLY MISRA, MICHELLE FISCHMANN MAGEE, and ERIC S. NYLEN 237 REFERENCE VALUES IN ENDOCRINOLOGY 2173 D. ROBERT DUFOUR 238 TECHNIQUES OF LABORATORY TESTING 2226 D. ROBERT DUFOUR 239 EFFECTS OF DRUGS ON ENDOCRINE FUNCTION AND VALUES 2232 MEET A SHARMA

240 DNA DIAGNOSIS OF ENDOCRINE DISEASE 2252 J. FIELDING HEJTMANCIK and HARRY OSTRER 241 DYNAMIC PROCEDURES IN ENDOCRINOLOGY 2260 D. ROBERT DUFOUR and WILLIAM A. JUBIZ

INDEX

2283

PART I

GENERAL PRINCIPLES OF ENDOCRINOLOGY KENNETH L. BECKER,

EDITOR

1. ENDOCRINOLOGY AND THE ENDOCRINE PATIENT.2 2.

MOLECULAR BIOLOGY: PRESENT AND FUTURE.8

3.

BIOSYNTHESIS AND SECRETION OF PEPTIDE HORMONES.24

.

4

HORMONAL ACTION.36

5.

FEEDBACK CONTROL IN ENDOCRINE SYSTEMS .50

6.

ENDOCRINE RHYTHMS.57

7. GROWTH AND DEVELOPMENT IN THE NORMAL INFANT AND CHILD.68

2

PARTI: GENERAL PRINCIPLES OF ENDOCRINOLOGY

CHAPTER 1

ENDOCRINOLOGY AND THE ENDOCRINE PATIENT KENNETH L. BECKER, ERIC S. NYLEN, AND RICHARD H. SNIDER, JR.

DEFINITIONS Endocrinology is the study of communication and control within a living organism by means of chemical messengers that are synthesized in whole or in part by that organism. Metabolism, which is an integral part of the science of endo¬ crinology, is the study of the biochemical control mechanisms that occur within living organisms. The term includes such diverse activities as gene expression; biosynthetic pathways and their enzymatic catalysis; the modification, transformation, and degradation of biologic substances; the biochemical media¬ tion of the actions and interactions of such substances; and the means for obtaining, storing, and mobilizing energy. The chemical messengers of endocrinology are the hormones, endogenous informational molecules that are involved in both intracellular and extracellular communication.

PARACRINE

JUXTACRINE

Synaptic

SOLINOCRINE

Non-synaptic

7 7

l_

_y

ROLE OF THE ENDOCRINE SYSTEM The mammalian organism, including the human, is multicellu¬ lar and highly specialized with regard to sustaining life and reproductive processes. Reproduction requires gametogenesis, fertilization, and implantation. Subsequently, the new intrauter¬ ine conception must undergo cell proliferation, organogenesis, and differentiation into a male or female. After parturition, the newborn must grow and mature sexually, so that the cycle may be repeated. To a considerable extent, the endocrine system influences or controls all of these processes. Hormones partici¬ pate in all physiologic functions, such as muscular activity, res¬ piration, digestion, hematopoiesis, sense organ function, thought, mood, and behavior. The overall purpose of the coordi¬ nating, regulating, integrating, stimulating, suppressing, and modulating effects of the many components of the endocrine system is homeostasis. The maintenance of a healthy optimal internal milieu in the presence of a continuously changing and sometimes threatening external environment is termed allostasis.

HORMONES CHEMICAL CLASSIFICATION Most hormones can be classified into one of several chemical cat¬ egories: amino-acid derivatives (e.g., tryptophan —> serotonin and melatonin; tyrosine —> dopamine, norepinephrine, epinephrine, triiodothyronine, and thyroxine; L-glutamic acid —» y-aminobutyric acid; histidine —> histamine), peptides or polypeptides (e.g., thyrotropin-releasing hormone, insulin, growth hormone, nerve growth factor), steroids (e.g., progesterone, androgens, estrogens, corticosteroids, vitamin D and its metabolites), and fatty acid derivatives (e.g., prostaglandins, leukotrienes, thromboxanes).

SOURCES, CONTROLS, AND FUNCTIONS 'reviously hormones were thought to be synthesized and secreted predominantly by anatomically discrete and circum-

NEUROCRINE

HEMOCRINE

FIGURE 1-1. Different types of hormonal communication detailed in the chapter. The darkened areas on the cell membrane represent recep¬ tors. (H, hormone.) See text for explanations.

scribed glandular structures, called ductless glands (e.g., pitu¬ itary, thyroid, adrenals, gonads). However, many microscopic organoid-like groups of cells and innumerable other cells of the body contain and secrete hormones (see Chap. 175). The classic "glands" of endocrinology have lost their exclu¬ sivity, and although they are important on physiologic and pathologic levels, the widespread secretion of hormones throughout the body by "nonglandular" tissues is of equal importance. Most hormones are known to have multiple sources. Moreover, the physiologic stimuli that release these hormones are often found to differ according to their locale. The response to a secreted hormone is not stereotyped but varies according to the nature and location of the target cells or tissues.

TRANSPORT TYPES OF SECRETORY TRANSPORT Hormones have various means of reaching target cells. In the early decades of the development of the field of endocrinology, hormones were conceived to be substances that traveled to dis¬ tal sites through the blood. This is accomplished by release into the extracellular spaces and subsequent entrance into blood vessels by way of capillary fenestrations. The most appropriate term for such blood-bone communication is hemocrine (Fig. 1-1). Several alternative means of hormonal communication exist, however. Paracrine communication involves the extrusion of hormonal contents into the surrounding interstitial spaces; the hormone then interacts with receptors on nearby cells (see Fig. 1-1 and Chaps. 4 and 175).1 Direct paracrine transfer of cyto¬ plasmic messenger molecules into adjacent cells may occur

Ch. 1: Endocrinology and the Endocrine Patient through specialized gap junctions (i.e., intercrine secretion).2 Unlike hemocrine secretion, in which the hormonal secretion is diluted within the circulatory system, paracrine secretion deliv¬ ers a very high concentration of hormone to its target site. Juxtacrine communication occurs when the messenger molecule does not traverse a fluid phase to reach another cell, but, instead, remains associated with the plasma membrane of the signaling cell while acting directly on an immediately adjacent receptor cell (e.g., intercellular signaling that is adhesion dependent and occurs between endothelial cells and leukocytes and transforming growth factor-a in human endometrium).3'4 Hormones may be secreted and subsequently interact with the same cell that released the substance; this process is auto¬ crine secretion (see Fig. l-l).5 The secreted hormone stimulates, suppresses, or otherwise modulates the activity of the secreting cell. Autocrine secretion is a form of self-regulation of a cell by its own product. When peptide hormones or other neurotransmitters or neu¬ romodulators are produced by neurons, the term neurocrine secretion is used (see Fig. l-l).6 This specialized form of para¬ crine release may be synaptic (i.e., the messenger traverses a structured synaptic space) or nonsynaptic (i.e., the messenger is carried to its local or distal site of action by way of the extracel¬ lular fluid or the blood). Nonsynaptic neurocrine secretion has also been called neurosecretion. An example of neurosecretion is the release of vasopressin and oxytocin into the circulatory sys¬ tem by nervous tissue of the pituitary (see Chap. 25). Several peptides and amines are secreted into the luminal aspect of the gut (e.g., gastrin, somatostatin, luteinizing hormone¬ releasing hormone, calcitonin, secretin, vasoactive intestinal peptide, serotonin, substance P).7 This process may be called solinocrine secretion (see Fig. 1-1), from the Greek word for a hollow tube. Solinocrine secretion also occurs into the bronchi, the urogenital tract, and other ductal structures.8 Commonly, the same hormone can be transported by more than one of these means.9 Extracellular transportation may not always be necessary for hormones to exert their effects. For example, some known hor¬ monal secretions that are transported by one or more of these mechanisms are also found in extremely low concentrations within the cytoplasm of many cells. In such circumstances, these hormones do not appear to be localized to identifiable secretion granules and probably act primarily within the cell. This phenomenon may be called intracrine secretion. As shown in Figure 1-1, the process comprising uptake of a hormone pre¬ cursor Hj and intracellular conversion into H2 (e.g., estrogens) or H3 (e.g., androgens) and subsequent binding and nuclear action is also a form of intracrine communication. OVERLAP OF EXOCRINE AND ENDOCRINE TYPES OF SECRETION Classically, an exocrine gland is a specialized structure that secretes its products at an external or internal surface (e.g., sweat glands, sebaceous glands, salivary glands, oxyntic or gas¬ tric glands, pancreatic exocrine glandular system, prostate gland). An exocrine gland may be unicellular (e.g., mucous or goblet cells of the epithelium of mucous membranes) or multi¬ cellular (e.g., salivary glands). Many multicellular exocrine glands possess a structured histologic organization that is suited to the production and delivery of secretions that are produced in relatively large quantities. A specialized excretory duct or sys¬ tem of ducts usually constitutes an intrinsic part of the gland. Some exocrine glandular cells secrete their substances by means of destruction of the cells themselves (i.e., holocrine secretion); an example is the sebaceous glands. Other exocrine glandular cells secrete their substances by way of the loss of a portion of the apical cytoplasm along with the material being secreted (i.e., apocrine secretion); an example is the apocrine sweat glands. Alternatively, in many forms of exocrine secretion, the secretory cells release their products through the cell membrane, and the

3

cell remains intact (i.e., merocrine secretion); an example is the salivary glands. The constituents of some exocrine glands, par¬ ticularly those opening on the external surface of the body, sometimes function as pheromones, which are chemical sub¬ stances that act on other members of the species.10 Many exocrine glands contain cells of the diffuse neuroen¬ docrine system (see Chap. 175) and neurons; both cell types secrete peptide hormones. Peptide hormones, steroids, and pros¬ taglandins are found in all exocrine secretions (e.g., sweat, saliva, milk, bile, seminal fluid; see Chap. 106).11-14 Although they usually are not directly produced in such glands, thyroid and steroid hormones are found in exocrine secretions as well.15-18 The preferred approach is to view the term "exocrine" as a histologic-anatomic entity and not as a term that is meant to be antithetical to or to contrast with the term "endocrine." Endo¬ crinologists are concerned clinically and experimentally with all means of hormonal communication. The word "endocrine" is best used in a global sense, indicating any and all means of communication by messenger molecules.

TYRANNY OF HORMONE TERMINOLOGY Hormones usually are named at the time of their discovery. Sometimes, the names are based on the locations where they were first found or on their presumed effects. However, with time, other locations and other effects are discovered, and these new locations or effects often are more physiologically relevant than the initial findings. Hormonal names are often overly restric¬ tive, confusing, or misleading. In many instances, such hormonal names have become inap¬ propriate. For example, atrial natriuretic hormone is present in the brain, hypothalamus, pituitary, autonomic ganglia, and lungs as well as atrium, and it has effects other than natriuresis (see Chap. 178). Gastrin-releasing peptide is found in semen, far from the site of gastrin release. Somatostatin, which was found in the hypothalamus and named for its inhibitory effect on growth hormone, occurs in many other locations and has multiple other functions (see Chap. 169). Calcitonin, which ini¬ tially was thought to play an important role in regulating serum calcium and was named accordingly, appears to exert many other effects, and its influence on serum calcium may be quite minor (see Chap. 53). Growth hormone-releasing hor¬ mone and arginine vasopressin are found in the testis, where effects on growth hormone release or on the renal tubular reab¬ sorption of water are most unlikely. Vasoactive intestinal pep¬ tide is found in multiple tissues other than the intestines (see Chap. 182). Insulin, named for the pancreatic islets, is found in the brain and elsewhere.19 Prostaglandins have effects that are far more widespread than those exerted in the secretions of the prostate, from which their name derives (see Chap. 172). The endocrine lexicon also contains substances called hor¬ mones that are not hormones. In the human, melanocyte-stimu¬ lating hormone (MSH) is not a functional hormone, but it comprises amino-acid sequences within the proopiomelanocor¬ tin (POMC) molecule: a-MSH within the adrenocorticotropic hormone (ACTH) moiety, [3-MSH within 5-lipotropin, and 8MSH within the N-terminal fragment of POMC (see Chap. 14). Numerous peptide hormones exist that, because of their effects on DNA synthesis, cell growth, and cell proliferation, have been called growth factors and cytokines (see Chaps. 173 and 174). These substances, which act locally and at a distance, often do not have the sharply delineated target cell selectivity that was attributed to them when they first were discovered. Their terminology also is confusing and often misleading. Aside from occasional readjustments of hormonal nomen¬ clature, no facile solution appears to exist to the quandary of terminology, other than an awareness of the pitfalls into which the terms may lead us.

4

PARTI: GENERAL PRINCIPLES OF ENDOCRINOLOGY

ENDOCRINE SYSTEM INTERACTION WITH ALL BODY SYSTEMS Although speaking in terms of the cardiovascular, respiratory, gastrointestinal, and nervous systems is convenient, the endo¬ crine system anatomically and functionally overlaps with all body systems (see Part X). Extensive overlap is found between the endocrine system and the nervous system (see Chaps. 175 and 176). Hormonal peptides are synthesized in the cell bodies of neurons, are transported along axons to nerve terminals, and are released at the nerve endings. Within these neurons, they coexist with classic neurotransmitters and often are coreleased with them. These substances play a role in neuromodulation or neurosecretion by means of the extracellular fluid. The nerves in which peptide hormones appear to play a role in the transmis¬ sion of information are called peptidergic nerves.20 It is the ample similarity of ultrastructure, histochemistry, and hor¬ monal contents of nerve cells and of many peptide-secreting endocrine cells that has led to the concept of the diffuse neu¬ roendocrine system.

GENETICS AND ENDOCRINOLOGY The rapid application of new discoveries and new techniques in genetics has revolutionized medicine, including the field of endocrinology. DNA probes have been targeted to selected genes, and the chromosomal locations of genes related to many hormones and their receptors have been determined. A complete map of the human genome is gradually emerging.21 This approach has led to new knowledge about hormone bio¬ synthesis and has provided important information concerning species differences and evolution. The elucidation of the chro¬ mosomal loci for genes controlling the biosynthesis of hor¬ mone receptors should provide insights into the physiologic effects of hormones. Clinically, these techniques have poten¬ tial significance as a diagnostic aid in evaluating afflicted patients, a means of identifying asymptomatic heterozygotes, and a method for identification of unborn individuals at risk (i.e., prenatal diagnosis; see Chap. 240). Delineation of pro¬ cesses of genetic expression is revealing the mechanisms of hormonal disease (e.g., obesity22) and also may lead to gene therapy for some forms of endocrine illness or humoral-mediated conditions.23

NORMAL AND ABNORMAL EXPRESSION OR MODULATION OF THE HORMONAL MESSAGE AND ITS METABOLIC EFFECT A sophisticated and faultless machinery is required for appro¬ priate hormonal expression. The hormonal messenger is subject to modifications that may occur anywhere from its initial syn¬ thesis to its final arrival at its target site. Subsequently, the expression of the message at this site (i.e., its action) may also be modified (see Chap. 4). The modulations or alterations of the hormonal message or its final action may be physiologic or pathologic. Table 1-1 summarizes some of the normal and abnormal modulations of a hormonal message and its subse¬ quent metabolic effects. On a physiologic level, the first steps in the genetic ordering of hormonal synthesis, the subsequent posttranslational pro¬ cessing of the hormone, the postsecretory extracellular trans¬ port, the receptor mediation of the hormone and subsequent transduction, and the inactivation and clearance of the hor¬ mone all contribute to expressing, diversifying, focalizing, and specifying the hormonal message and its ultimate action. On a pathologic level, all of these steps are subject to malfunction, causing disease.

Our increased knowledge of endocrine systems has forced us to rethink many traditional concepts. To dispel some com¬ mon misconceptions, listing several "nots" of endocrinology may be worthwhile (Table 1-2).

THE ENDOCRINE PATIENT FREQUENCY OF ENDOCRINE DISORDERS In a survey of the subspecialty problems seen by endocrinolo¬ gists, the six most common, in order of frequency, were found to be diabetes mellitus, thyrotoxicosis, hypothyroidism, non¬ toxic nodular goiter, diseases of the pituitary gland, and dis¬ eases of the adrenal gland. Some conditions seen by endocrinologists are infrequent or rare (e.g., congenital adrenal hyperplasia, pseudohypoparathyroidism), whereas others are relatively common (e.g.. Graves disease, Hashimoto thyroidi¬ tis), and some are among the most prevalent diseases in general practice (e.g., diabetes mellitus, obesity, hyperlipoproteinemia, osteoporosis, Paget disease). The third most common medical problem encountered by general practitioners is diabetes melli¬ tus, and the tenth most frequent problem is obesity.66 Of the total deaths in the United States (i.e., both sexes, all races, and all ages combined), diabetes mellitus is the seventh most common cause. The most common cause of death (heart disease) and the third most common (cerebrovascular acci¬ dents) are greatly influenced by metabolic conditions such as diabetes mellitus and hyperlipemia.67

COST OF ENDOCRINE DISORDERS The frequency and morbidity of endocrine diseases such as osteoporosis, obesity, hypothyroidism, and hyperthyroidism, and the grave consequences of other endocrine disorders such as Cushing syndrome and Addison disease demonstrate that the expense to society is considerable. In the case of diabetes, the health care expenditure is staggering. Approximately 10.3 million people have diabetes in the U.S., and an estimated 5.4 million have undiagnosed diabetes. Direct medical expenses attributed to diabetes total $44.1 billion. The total annual medi¬ cal expenses of people with diabetes average $10,071 per capita, as compared to $2,669 for persons without diabetes.68 Interest¬ ingly, these expenses may be less if the appropriate specialties are involved in the care.60

FACTORS THAT INFLUENCE TEST RESULTS In clinical medicine, hormonal concentrations usually are ascer¬ tained from two of the most easily obtained sources: blood and urine. The diagnosis of an endocrinopathy often depends on the demonstration of increased or decreased levels of these blood or urine constituents. However, several factors must be kept in mind when interpreting a result that appears to be abnormal. These may include age, gender, time of day, exercise, posture, emotional state, hepatic and renal status, presence of other illness, and concomitant drug therapy (see Chaps. 237 and 239). RELIABILITY OF THE LABORATORY DETERMINATION The practice of clinical endocrinology far from a large medical center was previously hindered by the difficulty in obtaining blood and urine tests essential for appropriate diagnosis and follow-up care. However, accurate and rapid analyses now are provided by commercial laboratories. Nevertheless, wherever performed, some tests are unreliable because of methodologic difficulties. Other tests may be difficult to interpret because of a particular susceptibility to alteration by physiologic or pharma¬ cologic factors (e.g., plasma catecholamines; see Chap. 86).

Ch. 1: Endocrinology and the Endocrine Patient

5

TABLE 1-1. Modulation of the Hormone Message and Its Subsequent Physiologic or Pathologic Metabolic Effects Modulation

Explanation

Examples

Gene muta¬ tions

Alteration of one or more nucleotides within the DNA gene sequence may result in a missense gene, a nonsense gene, a gene deletion, or a gene conversion.24'25 The mutation may affect hormone synthesis, enzymatic processing of the hormone, or synthesis of a receptor for a hormone.

Chromosomal deletions

Loss of chromosomal material, with an associated loss of genes.

Alternative gene pro¬ cessing

Alternative splicing of the primary RNA transcript gives rise to multiple messenger RNAs, each encoding a differ¬ ent hormone (see Chap. 3). A similar phenomenon can occur during the synthesis of a hormonal receptor.

Posttranslational pro¬ cessing

Most or all peptide hormones are synthesized in the form of large polypeptide precursors,38 some of which contain more than one functionally distinct hormone (see Chap. 3). Subsequent proteolytic enzymatic processing releases these hormones in their bioactive state.39 Many hormones are transported in the blood in association with protein carrier molecules. These substances facili¬ tate transport and may provide a means of temporary storage of the hormone, protecting it from degradation or retarding its clearance.

Mutant proinsulin syndrome is characterized by a structurally abnormal proinsulin, which results in diminished bioactivity and diabetes.26 A mutation involving the inwardly rectifying potassium (Kir) channels results in the syndrome of persistent hyperinsulinemic hypoglycemia of infancy, in which pancreatic B cells are dysfunctional.27 Some nondia¬ betic Mexican Americans have mutation in the high-affinity sulfonyl urea receptor (SDR2) gene, which regulates insulin secretion. This may be an antecedent to the development of type 2 diabetes in some of these hyperinsulinemic persons.28 Growth hormone resistance (i.e., Laron syn¬ drome) is caused by point mutations in the gene coding for the human growth hormone receptor.29'30 Mutations in the NKCC2 gene (one of the Na+-K+-Cl“ cotransporter isoforms) results in Bartter syndrome (hypokalemic metabolic acidosis, salt wasting, volume depletion, and hypercalciuria).31 Pseudoaldosteronism type 1 (PAH1) is often due to a mutation of the amiloride-sensitive epithelial sodium channel.32 WAGR syndrome (Wilms tumor, aniridia, genitourinary malformations, mental retardation) may have an associated chromosomal deletion involving the gene coding for the (i-subunit of follicle-stimulating hor¬ mone; its deficiency during embryonic development may cause the geni¬ tourinary abnormalities.33 Ovarian dysgenesis (Turner syndrome) is the result of the loss of all or part of an X chromosome (see Chap. 90). CATCH 22 (cardiac defect, abnormal facies, thymic hypoplasia, cleft pal¬ ate, hypocalcemia) is due to a deletion within chromosome 22.34 An alternative exon selection gives rise to the hormone calcitonin or the very differently structured35 hormone calcitonin gene-related peptide (CGRP), a phenomenon that may be altered by a physiologic or patho¬ logic change of the biosynthetic milieu (see Chap. 53).35a Many patients with medullary thyroid cancer have a greater CGRP-to-calcitonin secre¬ tion ratio than do normal persons. Alternative splicing can also produce different forms of receptors (e.g., thyroid receptors).36-37 In the paraneoplastic adrenocorticotropic hormone (ACTH) syndrome in which ACTH is biosynthesized by an extrapituitary tumor (see Chap. 219), much of the hormone that is detectable in the serum is of highmolecular-mass, incompletely processed bioinactive material (see Chaps. 14 and 75). Sex hormone-binding globulin (see Chap. 101) progressively decreases in concentration from infancy to prepuberty, gradually increasing the amount of unbound testosterone and estradiol; this augments the amount of free, tissue-available sex hormones before puberty.40 In famil¬ ial dysalbuminemic hyperthyroxinemia, a variant albumin possesses a high affinity for thyroxine; as a result, these euthyroid persons have a spuriously high total serum thyroxine.41 Some men with idiopathic azoospermia or oligospermia may have rela¬ tively inactive follicle-stimulating hormone isoforms.43

Alterations of transport¬ ing mole¬ cules

Endogenous antihor¬ mones Antibodies to hormones or to their receptors

Receptor or postrecep¬ tor media¬ tion of hormone action

Hormone inactiva¬ tion and clearance

Circulating antihormones antagonize hormone action.42 These substances, which differ slightly in structure from the hormones they antagonize, bind to the appropriate hormonal receptors but lack some or all bioactivity. Although not present normally, antibodies to endogenous hormones may develop. Antibodies also may develop to a hormone receptor.44

Hormones reversibly bind to specific high-affinity protein receptors, leading to intracellular events that culminate in the appropriate cellular response (see Chaps. 4,32, and 72). Altered receptor function or altered transduction (i.e., the biochemical events beyond receptor binding) plays a role in the pathogenesis of several endocrine disorders. Some of these defects are the result of antibodies to receptors (as above) and others result from heritable or acquired (e.g., drug-induced) defects in the receptor or its subsequent transduction. These congenital or acquired conditions may be called target cell resistance disorders. A hormone must be inactivated or removed from its target site so that its effect may terminate. Depending on the hormone, the mechanisms for such termination are hydrolysis by degradative enzymes, oxidation, reduc¬ tion, aromatization, deiodination, conjugation with glucuronide, and other methods.60-64 Depending on the hormone, various tissues or organs are involved in their degradation or clearance from the circulation or from the body (e.g., liver, kidney, muscle, lung).

Antiinsulin antibodies are found in the blood of patients with previously untreated type 1 diabetes.45 In first-degree relatives of patients with type 1 diabetes, the presence of such antibodies may be a predictive marker for the disease.46 Antiinsulin receptor antibodies may produce hypogly¬ cemia through their continuous receptor stimulatory activity47; others can produce severe cellular resistance to both insulin and insulin-like growth factor-I.48 Autoantibodies to the thyrotropin receptor of the thy¬ roid follicular cell appear in Graves disease (see Chap. 42) and may cause the hyperthyroidism.49 Steroid hormone resistance: vitamin D-dependent rickets type 2 (see Chaps. 63 and 70),50 primary glucocorticoid resistance,51 pseudohypoaldosteronism,52 androgen resistance (see Chap. 96).53 Thyroid hormone resistance: pituitary or generalized resistance to thyroid due to a receptor abnormality (see Chap. 32). Peptide hormone resistance: resistant ovary syndrome due to decreased responsivity to gonadotropins (see Chap. 96),54,55 type A syndromes of insulin resistance and acanthosis nigricans (see Chap. 146),56 congenital nephrogenic diabetes insipidus due to resis¬ tance to vasopressin.57-58 Some cases of insulin resistance may be due to abnormalities of the GLUT4 insulin transporter system.59 Ineffective hepatic degradation of endogenous estrogens may result in gynecomastia (see Chap. 120). Renal disease may result in poor degrada¬ tion of the exogenous insulin administered to a type 1 diabetic, resulting in hypoglycemia. A deficiency of the lip-hydroxysteroid dehydrogenase enzyme results in poor clearance of cortisol and a syndrome of mineralocorticoid excess65 (see Chap. 80). The uridine diphospho-glucuronosyltransferase (UGT) family of enzymes conjugate and inactivate steroid hormones in their target tissues, and modifications of their expression may influence hormonal responsivity.65

6

PARTI: GENERAL PRINCIPLES OF ENDOCRINOLOGY

TABLE 1-2. Several “Nots” of Modern Endocrinology 1. Endocrinology is not only the study of internal secretions by ductless glands. It also deals with the secretions of groups of cells, of individual cells, and of the exocrine glands. 2. The secretion of a gland is not unihormonal. There is no gland and prob¬ ably no hormone-secreting cell that secretes only one active substance. 3. Most hormones do not have a single source. With very few exceptions, hormones are produced in more than one location in the body and by more than one cell type. 4. In view of the extensive tissue distribution of most hormones, with some exceptions, the extirpation of any single gland or tissue that pro¬ duces large amounts of a specific hormone usually does not remove that hormone from the body. 5. A hormone is not a substance that acts only at a distal site in the body. Its action often is within the immediate environs, and sometimes it acts on the very cell that secretes the hormone. 6. The term "endocrine" should not be used to connote a means of trans¬ port (i.e., the blood). A hormone is not only blood borne. It also may be borne by extracellular fluid, in lymph, across synapses, or in external secretions, and it may be carried in a functioning state within the con¬ fines of the cell itself. 7. A hormone does not in itself exert a specific action. It depends on arriv¬ ing and interacting with an appropriate receptor that commences the transduction of the hormone message into an action. 8. The receptor-mediated actions of most hormones are not stereotyped. They often differ according to the site-specific characteristics of the receptors and their function at that time. 9. The name of a hormone does not necessarily indicate its exclusive site of production or its predominant physiologic action. 10. The endocrine system is not under the control of a separate and inde¬ pendent nervous system. Instead, the nervous and endocrine systems overlap on both a biochemical and a physiologic level. 11. The effects of hormones are not independent of their concentrations. They vary according to the quantity present at the site of action; an excess may cause effects entirely different from a physiologically suffi¬ cient amount. 12. The effects of hormones are not independent of the age of the individ¬ ual. They vary with the developmental stage of an individual and with his or her age.

on a computed tomography (CT) examination. Intermingled CT-lucent and CT-dense areas are seen on the scan, and such nonhomogeneous areas may be confused with a microade¬ noma.71'72 The increasing use of magnetic resonance imaging (MRI) of the brain may reveal a bona fide asymptomatic microadenoma of the pituitary gland, but extensive endocrine workup often reveals many such lesions to be nonfunctional. They occur in as much as 10% of the normal population 73 Rathke cleft cysts of the anterior sella turcica or the anterior suprasellar cistern often are seen by MRI.74 Although an occa¬ sional patient may have a large and symptomatic lesion,75 most of these lesions are small and asymptomatic. During MRI or CT examination of the brain, the examiner often incidentally encounters a "primary empty sella," an extension of the sub¬ arachnoid space into the sella turcica with a resultant flattening of the pituitary gland in a patient without any pituitary lesion or any prior surgery of that region (see Chap. 11). Although some of these patients may be symptomatic, most have no asso¬ ciated symptoms or hormonal deficit. Another, albeit rare, lesion of the pituitary region seen on MRI is a sellar spine. This asymptomatic anatomic variant is an osseous spine arising in the midline from the dorsum sella that protrudes into the pitu¬ itary fossa; it may be an ossified remnant of the cephalic tip of the notochord.76 MRI or CT scanning of the abdomen may reveal the presence of harmless morphologic variations of the adrenal gland (i.e., incidentalomas) that sometimes leads to unnecessary surgery.77 (See Chap. 84.)

RISKS OF ENDOCRINE TESTING Endocrine testing is not always benign. Many procedures can cause mild to marked side effects.78-81 Other diagnostic maneu¬ vers, particularly angiography, may result in severe illness.82 The expected benefit of any procedure that is contemplated for a patient clearly should be greater than the risk.

COST AND PRACTICABILITY OF ENDOCRINE TESTING Although many tests are sensitive and specific, they all have innate interassay and intraassay variations that may be particu¬ larly misleading when a given value is close to the clinical “medical decision point" (see Chap. 237). Some laboratory dif¬ ferences are due to hormone heterogeneity (e.g., growth hor¬ mone has several isoforms, which bind differently to growth hormone-binding proteins).70 DETERMINATION OF ABNORMAL TEST RESULTS Not uncommonly, the intellectual or commercial enthusiasm engendered by a new diagnostic procedure of presumed importance is found to be unjustified, because the "test" was based on an invalid premise, because too few of ill patients were studied, because normative data to establish reference values were insufficient, or because subsequent studies were not confirmatory (see Chaps. 237 and 241). The increased sophistication of medical testing has made the physician and the patient aware of the presence of "abnormali¬ ties" that may be harmless: physiologic deviations from that which is most common, or pathologic entities that commonly remain asymptomatic. Such findings may cause considerable worry, lead to the expense and risk of further diagnostic proce¬ dures, and even cause needless therapeutic intervention. Some "abnormalities" are the result of methods of imaging. For example, sonography of the thyroid may demonstrate the presence of small nodules within the thyroid gland of a person without any palpable abnormality of that region of the gland; most such microlesions are benign or behave as if they were. Another "abnormality" revealed by imaging is the occa¬ sional heterogeneous appearance of a normal pituitary gland

In addition to being aware of the many factors that influence hormonal values, the limitations of laboratory determinations, and the potential risks of some of these procedures, the endocri¬ nologist must be aware of their expense, particularly because medical costs have increased at an annual rate that is almost twice the rate of overall inflation during the last several years. A hypertensive patient with hypokalemia who is taking neither diuretics nor laxatives should undergo studies of the renin-angiotensin-aldosterone system, and appropriate pharma¬ cologic or dietary manipulations of sodium balance should be instituted (see Chap. 90). But what should be done with the hypertensive patient who is normokalemic? Occasionally, such a person may have an aldosteronoma.83 Should such normo¬ kalemic patients be studied? Similarly, should the approxi¬ mately 25 million hypertensive patients in the United States undergo urinary collections for determinations of catechola¬ mine metabolites to find the rare patient with pheochromocytoma? In the context of the individual physician-patient relationship, the answers to such questions may not be difficult, but they become more controversial when placed within the framework of fiscal guidelines.

CONCLUSION The complexity of the endocrine system presents a profound intellectual challenge. The macrosystem of endocrine glands secretes its hormones under the influence of other gland-based releasing factors or neural influences or both. The very act of secretion alters subsequent secretion by means of feedback con¬ trols (see Chap. 5). Superimposed on this already complex

Ch. 1: Endocrinology and the Endocrine Patient arrangement, the microsystem of dispersed, somewhat inde¬ pendent, but overlapping units throughout the body, as well as the continuous modulation of the receptors for the secreted hormones, allow general or focal actions that are coordinated with other body functions, tempered to the occasion, and appropriate to the needs of the individual. That such a complex system may go awry and that a dysfunction may have a consid¬ erable impact on the patient is not surprising. Because endocrinology and metabolism are broad subjects that incorporate much, if not all, of normal body functions and disease states, they defy easy categorization. However, these enormous complexities, rather than deterring the clinician, researcher, or student, should provide a stimulus to probe deeper into areas difficult to understand and should hasten the eventual application of new developments to patient care.

REFERENCES 1. Hofbauer LC, Khosla S, Dunstan CR, et al. The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption. J Bone Mineral Res 2000; 15:2. 2. Usadel H, Bornstein SR, Ehrhart-Bornstein M, et al. Gap junctions in the adrenal cortex. Horm Metab Res 1993; 25:653. 3. Zimmerman GA, Lorant DE, McIntyre TM, Prescott SM. Juxtacrine inter¬ cellular signaling: another way to do it. Am J Respir Cell Mol Biol 1993; 9:573. 4. Bush MR, Mele JM, Couchman GM, Walmer DK. Evidence of juxtacrine signaling for transforming growth factor alpha in human endometrium. Biol Reprod 1998; 59:1522. 5. Hashimoto K, Higashiyama S, Asada H, et al. Heparin-binding epidermal growth factor-like growth factor is an autocrine growth factor for human keratinocytes. J Biol Chem 1994; 269:20060. 6. Gouin FJ. Morphology, histology and evolution of Myriapoda and insects. 3. The nervous system and the neurocrine systems. Fortschr Zool 1965; 17:189. 7. Uvnas-Wallensten K. Luminal secretion of gut peptides. Clin Gastroenterol 1980; 9:545. 8. Van Minnen J. Production and exocrine secretion of LHRH-like material by the male rat reproductive tract. Peptides 1988; 9:515. 9. Becker KL. The coming of age of a bronchial epithelial cell. Am J Respir Crit Care Med 1994; 149:183. 10. Cohn BA. In search of human skin pheromones. Arch Dermatol 1994; 130:1048. 11. Corps AN, Brown KD, Rees LH, et al. The insulin-like growth factor I con¬ tent in human milk increases between early and full lactation. J Clin Endo¬ crinol Metab 1988; 67:25. 12. Hammami MM, Haq A, Al-Sedairy S. The level of endothelin-like immunoreactivity in seminal fluid correlates positively with semen volume and negatively with plasma gonadotropin levels. Clin Endocrinol 1994; 40:361. 13. Schmidt NA. Salivary cortisol testing in children. Issues Compr Pediatr Nurs 1998; 20:183. 14. Voss HF. Saliva as a fluid for measurement of estriol levels. Am J Obstet Gynecol 1999; 180:S226. 15. Nizankowska B, Abramowicz T, Korezowski R, Rusin J. Triiodothyronine and thyroxine in human, cow's and formula milk. Exp Clin Endocrinol 1988; 91:116. 16. Langer P, Moravec R, Ohradka B, Foldes O. Iodothyronines in human bile. Endocrinol Exp 1988; 22:35. 17. Shieh CC, Chang SC, Tzeng CR, et al. Measurement of testosterone in sem¬ inal plasma, saliva and serum by solid-phase enzyme immunoassay. Andrologia 1987; 19:614. 18. O'Rorke A, Kane MM, Gosling JP, et al. Development and validation of a monoclonal antibody enzyme assay for measuring progesterone in saliva. Clin Chem 1994; 40:454. 19. Hendricke SA, Roth J, Rishi S, Becker KL. Insulin in the nervous system. In: Krieger DT, Bronnstein MJ, Martin J, eds. Brain peptides. New York: John Wiley & Sons, 1983:403. 20. Bean AJ, Zhang X, Hokfelt T. Peptide secretion: what do we know? FASEB J 1994; 8:630. 21. Zabarovsky ER, Allikmets R, Kholodnyuk I, et al. Construction of repre¬ sentative NOTI linking libraries specific for the total human genome and chromosome 3. Genomics 1994; 20:312. 22. Perusse L, Chagnon YC, Weisnagel J, Bouchard C. The human obesity gene map: the 1998 update. Obes Res 1999; 7:111. 23. Moldawer LL, Edwards PD, Minter RM, et al. Application of gene therapy to acute inflammatory disease. Shock 1999; 12:83. 24. Atkinson J, Martin R. Mutations to nonsense codons in human genetic dis¬ ease: implications for gene therapy by nonsense suppressor tRNAs. Nucleic Acids Res 1994; 22:1327. 25. Miller WL. Molecular biology of steroid hormone synthesis. Endocr Rev 1988; 9:295.

7

26. Oohoshi H, Ohgawara H, Nanjo K, et al. Familial hyperproinsulinemia associated with NIDDM. Diabetes Care 1993; 16:1340. 27. Abraham MR, Jahingir A, Alekseev AE, Terzic A. Channelopathies of inwardly rectifying potassium channels. FASEB J 1999; 13:1901. 28. Goksel DL, Fischbach K, Duggirala R, et al. Variant in sulfonylurea recep¬ tor-1 gene is associated with high insulin concentrations in nondiabetic Mexican Americans: SUR-1 gene variant and hyperinsulinemia. Hum Genet 1998; 103:280. 29. Duquesnoy P, Sobrier ML, Duriez B, et al. A single amino acid substitu¬ tion in the exoplasmic domain of the human growth hormone GH receptor confers familial GH resistance (Laron syndrome) with positive GH-binding activity by abolishing receptor homodimerization. EMBO J 1994; 13:1386. 30. Rosenfeld RG, Rosenbloom AL, Guevara-Aguirre J. Growth hormone (GH) insensitivity due to primary GH receptor deficiency. Endocr Rev 1994; 15:369. 31. Haas M, Forbush B III. The Na+-K+-Cl_ cotransporters. J Bioenerg Biomembr 1998; 30:161. 32. Torpy DJ, Chrousos GP. Hyper- and hypoaldosteronism. Vitam Horm 1999; 57:177. 33. Glaser T, Lewis WH, Brung GH, et al. The beta subunit of follicle stimulat¬ ing hormone is deleted in patients with aniridia and Wilms' tumor allow¬ ing a further definition of the WAGR locus. Nature 1986; 321:882. 34. Sergi C, Serpi M, Miiller-Navia J, et al. CATCH 22 syndrome: report of 7 infants with follow-up data and review of the recent advancements in the genetic knowledge of the locus 22qll. Pathologica 1999; 91:166. 35. Stojdl DF, Bell JC. SR protein kinases: the splice of life. Biochem Cell Biol 1999; 77:293. 35a. Franklyn JA, Sheppard MC. Hormonal control of gene expression. Clin Endocrinol (Oxf) 1988; 29:337. 36. Izumo S, Mahdavi V. Thyroid hormone receptor a isoforms generated by alternative splicing differentially activate myosin HC gene transcription. Nature 1988; 334:539. 37. Koenig RJ, Lazar MA, Hodin RA, et al. Inhibition of thyroid hormone action by a non-hormone binding c-erbA protein generated by alternative mRNA splicing. Nature 1989; 337:659. 38. Jung LJ, Kreiner T, Scheller RH. Prohormone structure governs proteolytic processing and sorting in the Golgi complex. Recent Prog Horm Res 1993; 48:415. 39. Seidah NG, Day R, Marcinkiewicz M, Chretien M. Mammalian paired basic amino acid convertases of prohormones and proproteins. Annals N Y Acad Sci 1993; 680:135. 40. Belgorosky A, Rivarola MA. Progressive increase in non sex hormone¬ binding globulin-bound testosterone and estradiol from infancy to late puberty in girls. J Clin Endocrinol Metab 1988; 67:234. 41. Tang KT, Yang HJ, Choo KB, et al. A point mutation in the albumin gene in a Chinese patient with familial dysalbuminemic hyperthyroxinemia. Eur J Endocrinol 1999; 141:374. 42. Dahl KD, Bicsak TA, Hsueh AJW. Naturally occurring antihormones: secre¬ tion of FSH antagonists by women treated with a GnRH analog. Science 1988; 239:72. 43. Wang C, Dahl KD, Leung A, et al. Serum bioactive follicle-stimulating hor¬ mone in men with idiopathic azoospermia and oligospermia. J Clin Endo¬ crinol Metab 1987; 65:629. 44. Bach JF. Antireceptor or antihormone autoimmunity and its relationship with the idiotype network. Adv Nephrol 1987; 16:251. 45. Palmer JP, Asplin CM, Clemons P, et al. Insulin antibodies in insulindependent diabetics before insulin treatment. Science 1983; 222:1337. 46. Yu L, Robles DT, Abiru N, et al. Early expression of antiinsulin autoanti¬ bodies of humans and the NOD mouse: evidence for early determination of subsequent diabetes. Proc Natl Acad Sci U S A 2000; 97:1701. 47. Di Paolo S, Georgino R. Insulin resistance and hypoglycemia in a patient with systemic lupus erythematosus: description of antiinsulin receptor antibodies that enhance insulin binding and inhibit insulin action. J Clin Endocrinol Metab 1991; 73:650. 48. Auclair M, Vigaroux C, Desbois-Mouthon C, et al. Antiinsulin receptor autoantibodies associate with insulin receptor substrate-1 and -2 and cause severe cell resistance to both insulin and insulin-like growth factor-I. J Clin Endocrinol Metab 1999; 84:3197. 49. Chiovato L, Santini F, Vitti P, et al. Appearance of thyroid stimulating anti¬ body and Graves disease after radioiodine therapy for toxic nodular goitre. Clin Endocrinol 1994; 40:803. 50. Liberman UA, Eil C, Marx SJ. Clinical features of hereditary resistance to 1,25-dihydroxyvitamin D (hereditary hypocalcemic vitamin D resistant rickets type II). Adv Exp Med Biol 1986; 196:391. 51. Chrousos GP, Detera-Wadleigh SD, Karl M. Syndromes of glucocorticoid resistance. Ann Intern Med 1993; 119:1113. 52. Zennoro MC, Borensztein P, Seubrier F, et al. The enigma of pseudohypoaldosteronism. Steroids 1994; 59:96. 53. Zoppi S, Wilson CM, Harbison MD, et al. Complete testicular feminization caused by an amino-terminal truncation of the androgen receptor with downstream initiation. J Clin Invest 1993; 91:1105. 54. Talbert LM, Raj MH, Hammond MG, Greer T. Endocrine and immunologic ovary syndrome. Fertil Steril 1984; 42:7411. 55. Fraser IS, Russell P, Greco S, Robertson DM. Resistant ovary syndrome and premature ovarian failure in young women with galactosemia. Clin Reprod Fertil 1986; 4:133.

8

PARTI: GENERAL PRINCIPLES OF ENDOCRINOLOGY 56. Suzuki Y, Hashimoto N, Shimada F, et al. Defects in insulin binding and receptor kinase in cells from a human with type A insulin resistance and from her family. Diabetologia 1991; 34:86. 57. Bichet DG, Razi M, Lonergan M, et al. Hemodynamic and coagulation responses to 1-desamino (8-D-arginine) vasopressin in patients with con¬ genital nephrogenic diabetes insipidus. N Engl J Med 1988; 318:881. 58. Singer I, Forrest JN Jr. Drug-induced states of nephrogenic diabetes insipi¬ dus. Kidney Int 1976; 10:82. 59. Jung Cy, Lee W. Glucose transporters and insulin action: some insights into diabetes management. Arch Pharm Res 1999; 22:329. 60. Visser TJ, Kaptein E, Terpstra OT, Krenning EP. Deiodination of thyroid hormone by human liver. J Clin Endocrinol Metab 1988; 67:17. 61. Bunnett NW. Postsecretory metabolism of peptides. Am Rev Respir Dis 1987; 136:S27. 62. Roupas P, Herington AC. Receptor-mediated endocytosis and degradative processing of growth hormone by rat adipocytes in primary culture. Endo¬ crinology 1987; 120:2158. 63. Benzi L, Ceechetti P, Ciccarone A, et al. Insulin degradation in vitro and in vivo: a comparative study in men. Evidence that immunoprecipitable, par¬ tially rebindable degradation products are released from cells and circulate in blood. Diabetes 1994; 43:297. 64. Yamaguchi T, Fukase M, Kido H, et al. Meprin is predominantly involved in parathyroid hormone degradation by the microvillar membranes of rat kidney. Life Sci 1994; 54:381. 65. Ferrari P, Lovati E, Frey FJ. The role of the II beta-hydroxysteroid dehydro¬ genase type 2 in human hypertension. J Hypertens 2000; 18:241. 66. Hum DW, Belanger A, Levesque E, et al. Characterization of UDP-glucuronyl-transferases active on steroid hormones. J Steroid Biochem Mol Biol 1999; 69:413. 67. National Center for Health Statistics. Monthly Vital Statistics Report 1999; 47(19):1. 68. American Diabetes Association. Economic consequences of diabetes mellitus in the U.S. in 1977. Diabetes Care 1998; 21:296. 69. Levetan CS, Passaro MD, Jablonski KA, Ratner RE. Effect of physician spe¬ cialty on outcomes in diabetic ketoacidosis. Diabetes Care 1999; 22:1790. 70. Baumann G. Growth hormone heterogeneity in human pituitary and plasma. Horm Res 1999; 51(Suppl 1):2. 71. Roppolo HMN, Latchaw RE, Meyer JD, Curtin HD. Normal pituitary gland: 1. Macroscopic anatomy-CT correlation. Am J Neuroradiol 1983; 4:927. 72. Tihansky DP, Crossen J, Markowitz H. Pseudotumor artifact of the dorsum sella in CT scanning. Comput Radiol 1987; 11:241. 73. Hall WA, Luciano MG, Doppman JL, et al. Pituitary magnetic resonance imaging in normal human volunteers: occult adenomas in the general pop¬ ulation. Ann Intern Med 1994; 120:817. 74. Kucharczyk W, Peck WW, Kelly WM, et al. Rathke cleft cysts: CT, MR imaging, and pathologic features. Radiology 1987; 165:491. 75. Hama S, Arita K, Tominago A. Symptomatic Rathke's cleft cyst coexisting with central diabetes insipidus and hypophysitis: case report. Endocr J 1999; 46:187. 76. Fujisawa I, Asato R, Togashi K, et al. MR imaging of the sellar spine. J Com¬ put Assist Tomogr 1988; 12:644. 77. McGrath PC, Sloan DA, Schwartz RW, Kenady DE. Advances in the diag¬ nosis and therapy of adrenal tumors. Curr Opin Oncol 1998; 10:52. 78. Ratzmann GW, Zollner H. Hypomagnesemia and hypokalemia in the insu¬ lin hypoglycemia test. Z Gesamte Inn Med 1985; 40:567. 79. Read RC, Doherty JE. Cardiovascular effects of induced insulin hypoglyce¬ mia in man during the Hollander test. Am J Surg 1972; 104:573. 80. Sobel RJ, Ariad S. Adverse cardiovascular responses to thyrotropin-releasing hormone (200 micrograms) in cardiac patients. Isr J Med Sci 1987; 23:1107. 81. Boice JD Jr. The danger of x-rays—real or apparent. N Engl J Med 1986; 315:828. 82. Hash RB. Intravascular radiographic contrast media: issues for family phy¬ sicians. J Am Board Fam Pract 1999; 12(1):32. 83. Bravo EL, Tarazi RC, Dustan HP, et al. The changing clinical spectrum of primary aldosteronism. Am J Med 1983; 74:641.

CHAPTER

2

MOLECULAR BIOLOGY: PRESENT AND FUTURE MEHBOOB A. HUSSAIN AND JOEL F. HABENER The beginnings of molecular biology as a distinct discipline occurred in the late 1940s and early 1950s with the recognition that pol, nucleotides were the repository of genetic information

in the form of DNA and the transmitters of genetic information in the form of messenger RNA (mRNA), and that transfer RNAs are fundamental for the assembly of amino acids into proteins. Detailed descriptions of the historical developments of this modern era of molecular biology are provided in several books.1-4 These were exciting times, as understanding pro¬ gressed rapidly from the discovery by Avery and Brundage that DNA was a genetic substance; Chargaff established that DNA is composed of four different deoxy ribonucleotides (dATP, dGRP, dTTP, dCTP); Watson and Crick elucidated the double-helical structure of DNA; Jacob and Monod identified mRNA as the intermediary in the transfer of information encoded in DNA to the assembly of amino acids into proteins; Holly discovered transfer RNAs; and Nirenberg et al. discovered the genetic code (i.e., each of the 21 amino acids is specified by a triplet of nucleotides, or codons, within the mRNA to be translated into a protein). In the 1960s, several major discoveries paved the way for the development of recombinant DNA technology and genetic engineering. Two of the major breakthroughs that made this possible were the discoveries of reverse transcriptase5 and restric¬ tion endonucleases;6-8 and techniques for determining the precise sequence of nucleotides in DNA.9-10 Reverse transcriptase, which is found encoded in the RNA of certain tumor viruses, is the means by which the virus makes DNA copies of its RNA templates. It allows molecular biologists to copy mRNA into complementary DNA (cDNA), which is an essential step in the preparation of recombinant DNA for purposes of cloning. Another fundamental discovery was that of restriction endo¬ nucleases, enzymes that cut DNA at specific sequences, typically of 4 to 10 base pairs. The application of specific restriction endo¬ nucleases allows for the cleavage of DNAs at precise locations, a property that is critical for the engineering of DNA segments. A most critical and important discovery was the technologic methodology to determine the sequential order of nucleotides in DNA. Both chemical and enzymatic approaches were devel¬ oped. Currently, the nucleotide sequences of DNAs are deter¬ mined by sophisticated automated instruments using random enzymatic cleavages of DNAs labeled with fluorescent markers. By fortunate coincidence, research into the mechanisms by which bacteria become resistant to certain antibiotics led to the discovery of bacterial plasmids, which are "viruses" that live within bacteria and lend genetic information to the bacteria to ensure their survival. Plasmids faithfully replicate within bacte¬ ria. Importantly, plasmid DNA is relatively simple in structure and is amenable to genetic engineering by excision of DNA sequences and insertion of foreign DNA sequences, which will replicate within bacteria without interference by the host bacte¬ rium. These plasmids have become useful vehicles in which to express and amplify foreign DNA sequences.

CLONING OF GENES Complementary DNA Libraries. The cloning of a particu¬ lar expressed gene begins with the preparation and cloning of cDNAs from mRNAs of a particular cell (Fig. 2-1; Table 2-1) (for a more comprehensive description, see references 11 and 12). The cDNAs are prepared by priming the reverse transcription of mRNAs, using reverse transcriptase and short oligonucleotide fragments of oligodeoxyribothymidine, which preferentially bind to the 3'-polyadenylate, or poly(A), tract that is character¬ istic of cellular mRNAs. Alternatively, random oligonucleotides of different base compositions may be used. Double-stranded DNA is then prepared from the single-stranded cDNA by using DNA polymerase, and the cDNAs are inserted into bacterial plasmids that have been cleaved at a single site with a restric¬ tion endonuclease. To ensure a reasonably high efficiency of insertion of the foreign DNA into the plasmids, cohesive, or "sticky," ends are first prepared by adding short DNA

Ch. 2: Molecular Biology: Present and Future 5'

3'

wv m RN A AA/WWWVN AAA A Reverse transcriptase rdNTP

I

Foreign DNA

Restriction endonuclease pstl i

K

Oligo dT cDNA zWWWWVAAAAA — TTTT

Formation of cohesive ends ccc

1

TTTT

DNA polymerase-1 ^dNTP TTTT

ccc Recombination

9

TABLE 2-1. Approaches for the Selection of Cloned Complementary DNAs (cDNAs) NUCLEIC ACID HYBRIDIZATION Hybridization selection and translation Hybridization arrest and translation Degenerate oligonucleotide probes Direct hybridization to cDNA libraries Polymerase chain reaction (PCR) PROTEIN EXPRESSION FROM CLONED cDNAs Detection by antisera Detection by oligonucleotide containing protein-binding sites (transcrip¬ tion factors) SPECIALIZED APPROACHES Rapid amplification of cDNA ends (RACE) Two-site interaction trap

Nuclease Transfection

O'

^CCCC-

1

Terminal transferase dCTP -CCCCg,

FIGURE 2-1. An approach used in construction and molecular cloning of recombinant DNA. A, Preparation of double-stranded DNA from an mRNA template. The enzyme reverse transcriptase is used to reversetranscribe a single-stranded DNA copy complementary to the mRNA primed with an oligonucleotide of polydeoxythymidylic acid hybrid¬ ized to the poly(A) tract at the 3' end of mRNA. A complementary copy of the DNA strand is then prepared with DNA polymerase. Ends of double-stranded DNA are made flush by cleavage with the enzyme SI nuclease, and homopolymer extensions of deoxycytidine are synthe¬ sized on 3' ends of DNA with the enzyme terminal transferase. Oligo(dC) homopolymer extensions form sticky ends for purposes of insertion of DNA into a linearized bacterial plasmid on which comple¬ mentary oligo(dG) homopolymer extensions have been synthesized. B, Insertion of foreign DNA into a bacterial plasmid for molecular clon¬ ing. A bacterial plasmid, typically pBR322, that has been specifically engineered for purposes of cloning DNA is linearized by cleavage with restriction endonuclease Pst I. Poly(dG) homopolymer extensions are synthesized onto 3' ends of plasmid DNA. Foreign DNA with comple¬ mentary poly(dC) homopolymer extensions is hybridized to and inserted into the plasmid. Recombinant plasmid DNA is transfected into susceptible host strains of bacteria, in which plasmid replicates apart from bacterial chromosomal DNA. Bacteria are then grown on a plate containing tetracycline. Colonies that are resistant to tetracycline are tested for sensitivity to ampicillin. Because native plasmids contain genes encoding resistance to both tetracycline and ampicillin and the gene encoding resistance to ampicillin is inactivated by insertion of a foreign DNA at the Pst I site, bacterial colonies harboring plasmids with DNA inserts are resistant to tetracycline and sensitive to ampicillin. Subsequent screening of tetracycline-resistant, ampicillin-sensitive clones containing specific DNA-inserted sequences is carried out by either DNA hybridization with labeled DNA probes or by other tech¬ niques such as hybridization arrest and cell-free translation. sequences to the ends of the foreign DNA and to the plasmids. Vectors that are commonly used are derivatives of the plasmid pBR322, which was engineered specifically for the purposes of cloning DNA fragments (see Fig. 2-1). Foreign DNA is inserted into a unique site that is prepared by endonuclease cleaving of a desired site within a polylinker, multiple cloning site engi¬ neered into the plasmid. This site is often located within the gene that codes for bacterial (3-galactosidase. The backbone plasmid also carries a gene for resistance to ampicillin or tetra¬ cycline. Thus, bacteria containing the plasmids can be selected by their resistance to ampicillin or tetracycline; those specifi¬ cally containing DNA inserts can be selected by their inability

to express (3-galactosidase and to cleave (3-galactopyranoside (blue-white screening). Hybridization Screening. The recombinant plasmids con¬ taining DNA sequences that are complementary to the specific mRNAs of interest are identified by hybridizing recombinant plasmids to the initial mRNA preparations used in the cloning. The hybrid-selected mRNA is subsequently eluted and trans¬ lated in a cell-free system appropriate for the protein under study. Alternatively, specific Inhibition of the translation of an mRNA can be used to identify the DNA of interest: DNA that is complementary to the mRNA being translated will bind the RNA, thus precluding translation and reducing the amount of the protein being synthesized. The initial techniques of hybridization selection and hybrid¬ ization arrest, in which cell-free translation is used as the assay system, are now supplanted by hybridization of the bacterial colonies with synthetic oligonucleotide probes that are labeled with phosphorus-32 (32P). Mixtures of oligonucleotides in the range of 14 to 17 bases are prepared that are complementary to the nucleotide sequences predicted from the known amino-acid sequences of segments of the protein encoded by mRNA. Because of the degeneracy in the genetic code (there are 61 amino-acid codons and 20 amino acids), mixtures of from 24 to 48 oligonucleotides ordinarily represent all possible sequences complementary to a particular 14- to 17-base region of mRNA. Expression Screening. Later-generation cDNA libraries have been prepared in bacterial phages (X gt-11) or hybrids between plasmids and phages (phagemids), which have been engineered to allow the bacteria infected with the recombinant phages to translate mRNAs expressed from the cDNAs, and thereby to produce the protein products encoded by the cDNAs. The desired sequence of interest can be selected at the protein level by screening the library of bacterial clones with an antiserum directed to the protein. When the desired product is a DNA-binding protein, the library can be screened with a labeled DNA duplex containing copies of the target sequence to which the protein binds. Yeast Two-Site Interaction Trap. The cloning of cDNAs encoding proteins that interact with other known proteins can be accomplished using the yeast two-site interaction trap, which functions much as a bait and fish system. The bait is a cDNA encoding a known protein that is engineered to bind to an enhancer in the promoter of a gene that encodes a factor essen¬ tial for the survival of a yeast cell. The sequences {fish) in the cDNA library are engineered with a strong transcriptional trans¬ activation domain, such as that from the herpes simplex virus and yeast transcription factors VP16 or Gal-4, respectively. The occurrence of protein-protein interactions between the bait and one of the fish activates the expression of the yeast survival

10

PARTI: GENERAL PRINCIPLES OF ENDOCRINOLOGY

gene, which thereby allows for the selection and cloning of the yeast cell that harbors the described cDNA sequence from the cDNA library. Rapid Amplification of Complementary DNA Ends. Most often cDNAs isolated by one or more of the approaches described above lack the complete sequence and are deficient in the 5' ends. The 5' sequences are determined by using the rapid amplification of cDNA ends (RACE) technique.

DNA polymerase, and a second set of oligonucleotide primers is ligated to the extended ends. The extended DNA fragments are then amplified by polymerase chain reaction (see next section), isolated by electrophoresis on agarose gels, and sequenced.

GENOMIC LIBRARIES AND GENE ISOLATION

The development of the polymerase chain reaction PCR, a tech¬ nique for the rapid amplification of specific DNA sequences, constituted a major technological breakthrough.13"16 This proce¬ dure relies on the unique properties of a thermally stable DNA polymerase (Taq polymerase) to allow for sequential annealing of small oligonucleotide primers that bracket a DNA sequence of interest; the result is successive synthesis of the DNA strands. Specific DNA sequences as short as 50 and as long as several thousand base pairs can be amplified over a million-fold in just a few hours by using an automated thermal cycler. The tech¬ nique is so sensitive that DNA (genomic DNA or cDNA reverse-transcribed from RNA) from a single cell can be so amplified. Indeed, a sample containing only a single target DNA molecule can be amplified. The applications of this tech¬ nique are diverse. Not only is it possible to amplify and to clone rare sequences for detailed studies, but also the technique has applications in the fields of medical diagnosis and forensics. Scarce viruses can be detected in a drop of serum or urine or a single white blood cell. Genotyping can be done from a blood or semen stain, saliva, or a single hair. Paradoxically, a major drawback of PCR is its exquisite sensitivity, which leaves open the possibility of false-positive results because of minute con¬ taminations of the samples being tested. Thus, extreme precau¬ tions must be taken to avoid the introduction of contaminants. PCR is carried out using DNA polymerase and oligonucle¬ otide primers complementary to the two 3' borders of the duplex segment to be amplified. The objective of PCR is to copy the sequence of each strand between the regions at which the oligonu¬ cleotide primers anneal. Thus, after the primers are annealed to a denatured DNA containing the segment to be amplified, the primers are extended using DNA polymerase and the four deoxynucleotide triphosphates. Each primer is extended toward the other primer. The result is a double-stranded DNA (which itself is then denatured and annealed again with primer, and the DNA polymerase reaction is repeated). This cycle of steps (denaturation, annealing, and synthesis) may be repeated 60 times. At each cycle, the amount of duplex DNA segment dou¬ bles, because both new and old DNA molecules anneal to the primers and are copied. In principle (and virtually in practice), 2" copies (where n = number of cycles) of the duplex segment bordered by the primers are produced. The heat-stable polymerase isolated from thermophilic bac¬ teria (Thermophilus aquaticus), Taq polymerase, allows multiple cycles to be carried out after a single addition of enzyme. The DNA, an excess of primer molecules, the deoxynucleotide triphosphates, and the polymerase are mixed together at the start. Cycle 1 is initiated by heating to a temperature adequate to assure DNA denaturation, followed by cooling to a tempera¬ ture appropriate for primer annealing to the now-single strands of the template DNA. Thereafter, the temperature is adjusted for DNA synthesis (elongation) to occur. The subsequent cycles are initiated by again heating to the denaturation temperature. Thus, cycling can be automated by using a computer-controlled variable-temperature heating block. In addition to permitting automation, the use of the DNA polymerase of T. aquaticus has another advantage. The enzyme is most active between 70° and 75°C. Base pairing between the oligonucleotide primers and the DNA is more specific at this temperature than at 37°C, the optimal functioning temperature

Southern Blots and Hybridization Screening. The tech¬ niques used in the cloning of genomic DNA are similar to those used for cloning cDNA, except that the genomic sequences are longer than the cDNA sequences and different cloning vectors are required. The common vectors are derivatives of the bacte¬ riophage X that can accommodate DNA fragments of 10 to 20 kilobases (kb). Certain hybrids of bacteriophages and plasmids, called cosmids, can accommodate inserts of DNA of up to 40 to 50 kb. Even larger segments of DNA up to 1 to 2 megabases (Mb) can be cloned and propagated in yeast and are called yeast artificial chromosomes (YACs). In the cloning of genomic DNA, restriction fragments are prepared by partial digestion of unsheared DNA with a restriction endonuclease that cleaves the DNA into many fragments. DNA fragments of proper size are prepared by fractionation on agarose gels and are ligated to the bacteriophage DNA. The fragments of DNA containing the desired sequences can be detected by hybridization of a mem¬ brane blot prepared from the gel with a 32P-labeled cDNA, a Southern blot. The recombinant DNA is mixed with bacterio¬ phage proteins, which results in the production of viable phage particles. The recombinant bacteriophages are grown on agar plates covered with growing bacteria. Then the bacteria are infected by a phage particle, which lyses the bacteria to form visible plaques. Specific phage colonies are transferred by nitro¬ cellulose filters and are hybridized by cDNA probes labeled with 32P, similar to a Southern blot. Libraries of genomic DNA fragments and tissue-specific cDNAs from various animal spe¬ cies cloned in plasmids and bacteriophages are available from a number of commercial laboratories. The development of yeast chromosomal libraries that harbor large segments (several megabases) of chromosomal DNA has markedly accelerated the generation of gene linkage maps. Enhancer Traps. One approach to identifying novel genes imbedded in the genome is to randomly insert a transcriptional reporter gene into chromosomal DNA that has been cleaved into 1- to 2-kb fragments by digestion with a restriction endonuclease. The family of ligated hybrid fragments is then cloned into plas¬ mids that are individually introduced (transfected) into host cell lines (e.g., NIH or BHK fibroblasts). After the transfected cell lines are incubated with the cloned DNA fragments for 1 to 2 days, extracts are prepared from the cells and assayed for expres¬ sion of the transcriptional reporter gene. Typical transcriptional reporter genes used are firefly luciferase, bacterial chloramphenicol acetyl transferase, or bacterial alkaline phosphatase. When, by chance, a transcriptional enhancer is encountered, as determined by the activation of the reporter gene, the particular cloned DNA frag¬ ment is sequenced and searched for transcribed exonic and/or intronic sequences of genes, many of which typically reside 100 to 1000 base pairs from the enhancer sequence. The transcribed sequences of genes usually, but not always, reside 3' (down¬ stream) from enhancer sequences. Rapid Amplification of Genomic DNA Ends. The princi¬ ple of rapid amplification of genomic DNA ends (RAGE) is similar to that of RACE previously described and allows for the identi¬ fication of unknown DNA sequences in genomic DNA. Oligoldeotide primers (amplimers) are annealed to the test genomic ! \T A sample and extended on the genomic DNA template with

GENE AMPLIFICATION BY POLYMERASE CHAIN REACTION

Ch. 2: Molecular Biology: Present and Future of Escherichia coli DNA polymerase. Consequently, the primers are less likely to anneal nonspecifically to unwanted DNA seg¬ ments, especially when the entire genome is present in the tar¬ get DNA.

Transcription assays:

Nucleus Transcription

VARIATIONS OF POLYMERASE CHAIN REACTION

DNA \XXXXXX\ RNA processing

Simple modifications of the PCR conditions can expand the opportunities of the PCR. For example, synthesizing oligonu¬ cleotide primers that recognize domains (motifs) shared by cDNAs and their respective protein products, and choosing less stringent annealing conditions for the primers, permit new sequences of yet unknown DNAs to be generated with PCR, ultimately resulting in the discovery of new cDNAs belonging to the same family. For example, the pancreatic B-cell transcrip¬ tion factor IDX-1 was identified by PCR using oligonucleotide primers that would anneal to sequences shared by the homeodomain transcription factor family. PCR primers can be modified in their sequence and thus are not completely complementary to the template DNA. The amplified PCR product then carries the sequence of the primer and not the original DNA sequence. This strategy can be used to insert mutations site-specifically into known DNA sequences.

11

vPre mRNA mRNA

- Cell-free in vitro - Nuclear run-on - Transfection of promoter-reporters in vivo cell cultures - Transfection of transcription factor vectors - Transgenic in vivo mouse models [3-galactosidase Green fluorescent protein

Cytoplasm mRNA Translation

Messenger RNA assays: - Northern blot hybridization - Solution hybridization RNase protection - RT-PCR Competitor, continuous monitoring

APPROACHES TO THE QUANTITATIVE ASSESSMENT OF GENE EXPRESSION

- In situ hybridization

TRANSCRIPTION ASSAYS protein Nuclear Run-On Assays. Several assays are available that provide an index of relative rates of gene transcription (Fig. 2-2). A simple, straightforward assay is the nuclear run-on assay in which nuclei are isolated from tissue culture cells and nascent RNA chains are allowed to continue to polymerize in the pres¬ ence of radiolabeled deoxyribonucleotides in vitro. This assay has the advantage that it surveys the density of nascent tran¬ scripts made from the endogenous genes of cells and, on aver¬ age, is a good measure of gene transcription rates in response to the existing environmental conditions in which the cultured cells are maintained. Newly synthesized RNA is applied (hybridized) to a nylon membrane on which a cDNA target complementary to the desired RNA has been adsorbed. Radiolabeled RNA hybridized to the cDNA is determined in a radia¬ tion counter. Cell-Free In Vitro Systems. Rates of RNA synthesis can also be determined in broken cell or cell-free lysates to assess the relative strengths of different promoters. To restrict the newly synthesized radiolabeled RNA to a single size and, thus, to enable more ready detection by electrophoresis, a DNA tem¬ plate is used that does not contain guanine bases, called a G-free cassette. RNA synthesis is carried out in the absence of the gua¬ nine nucleotide. After synthesis of a specified length of RNA at the end of which guanine bases are encountered, RNA synthe¬ sis is terminated. Transfection of Promoter-Reporters in In Vivo Cell Culture. Many of the currently used assays of gene transcription employ promoter sequences fused to genes encoding proteins that can be quantitated by bioassays (e.g., bacterial chloramphenicol acetyl transferase, firefly luciferase, alkaline phosphatase, or green fluorescent protein). The hybrid DNAs, so called pro¬ moter-reporter DNAs, are introduced into tissue culture cells by one of several chemical methods (i.e., DNA adsorbed to calcium phosphate precipitates, diethylaminoethyl (DEAE)-dextran incorporated into liposomes, or human artificial chromosomes [Table 2-2]); or physical methods (i.e., electroporation, direct microinjection of DNA, or ballistic injection using a gene gun [Table 2-3]). After introduction of the reporter DNA into the

t

Posttranslational processing

FIGURE 2-2.

Protein expression - Cell-free translation - Pulse and Pulse chase labeling - Western immunoblot - Immunocytochemistry

Approaches to the quantitative assessment of gene

expression. Shown are the various types of assays that can be used to examine regulation of gene expression at various levels. (mRNA, mes¬ senger RNA; RNase, ribonuclease; RT-PCR, reverse transcription poly¬ merase chain reaction.)

cells, the transfected cells are incubated for a specified time under the desired experimental conditions, the cells are har¬ vested, and extracts are prepared for assays of the reporterspecific enzymatic activity. By these transfection methods, cell-type specificity for the expression of gene-promoter sequences can be determined by comparing promoter-reporter efficiencies in cells of different phenotypes. In addition, impor¬ tant transcriptional control sequences in the promoter can be mapped by DNA mutagenesis studies. Transfection of Transcription Factor Expression Vectors. An extension of the promoter-reporter transfection approach is to cotransfect recombinant expression plasmids encoding transcription factors that bind to control sequences in the pro¬ moter DNA and activate transcription of the reporter. By this approach, critical functional components of transcription fac¬ tors and critical bases in DNA control sequences can be exam¬ ined experimentally. Transgenic In Vivo Mouse Models. A method developed for examining specificity of tissue expression and efficiency of expres¬ sion of promoter-reporter genes is their introduction into mice in vivo, using transgenic technology (see the section Genetic Manip¬ ulations in Animals In Vivo). Recombinant promoter-reporter genes are injected into the pronucleus of fertilized mouse ova and implanted into surrogate females. Tire tissues of transgenic neo¬ natal mice are examined for the tissue distribution and relative

12

PART I: GENERAL PRINCIPLES OF ENDOCRINOLOGY TABLE 2-2. Chemical Methods for Introducing Genes into Mammalian Cells Method

Advantages

Disadvantages

DNA-calcium phosphate

Cell death minimal

Good for ex vivo use only

Important in the production of viral vectors

Low transfection efficiency

DNA-DEAEdextran

Simple, inexpensive

Low transgene expression levels

Expression transient or stable

Transient expression only

More reproducible than DNA-calcium morphate

Good for ex vivo only

Can carry large pieces of DNA (chromosomes)

Transient expression only

Can be targeted

Works only in some cell types

Nonimmunogenic Preparations are pyrogen free DNA-lipid-protein (liposomes) Human artificial chromosomes

Can be targeted, transfection rates are better Do not integrate into host chromosomes

Further development required

Large inserts can be accommodated Better transcription control

DEAE, diethylaminoethyl dextran.

strength of the expression of the reporter function. Commonly used reporter functions are the genes encoding either (3-galactosidase or green fluorescent protein.

MESSENGER RNA ASSAYS Northern Blot Hybridization. RNA blotting (Northern blot¬ ting) is analogous to DNA blotting (Southern blotting). RNA is separated according to size by electrophoresis through agarose gels. Generally, the electrophoresis is performed under condi¬ tions that denature the RNA so that the effects of RNA second¬ ary structure on the electrophoretic mobility of the RNAs can be minimized. Alkaline conditions are unsuitable; therefore, agents such as glyoxal, formaldehyde, or urea are used. The size-separated RNA is transferred by blotting to an immobiliz¬ ing membrane without disturbing the RNA distribution along the gel. A labeled DNA is then used as a probe to find the posi¬ tion on the blot of RNA molecules corresponding to the probe. The immobilized RNA is incubated with DNA under condi¬ tions allowing annealing of the DNA to the RNA on the immo¬ bilized matrix. After washing away excess and unspecifically annealed DNA, the matrix is exposed to an x-ray film to detect the position of the probe. RNA blotting allows the estimation of the size of the RNA that is being detected. In addition, the

TABLE 2-3. Physical Methods for Introducing Genes into Mammalian Cells Method

Advantages

Disadvantages

Direct microin¬ jection

High transfection rate

Good for ex vivo use only

Electroporation

High transfection rate

Good for ex vivo use only Excessive cell death

Plasmid injection

Simple

Low transfection rate

Up to 19 kb of DNA can be transferred to muscle Good for use in DNA vac¬ cines Ballistic injection

High transfection rate

Transient transfection

Delivery of precise dosages of DNA

Considerable cell death

Good for use in DNA vac¬ cines

intensity of the band on an x-ray film indicates the abundance of the RNA in the cell or tissue from which the RNA was extracted. Solution Hybridization Ribonuclease Protection. To obtain more precise information on the amount of a specific RNA spe¬ cies in a certain cell or tissue, a single-stranded radioactive probe is generated that is complementary to a portion of the RNA being studied. An excess amount of this single-stranded probe is then mixed in solution with the total RNA of the cells or tissue being investigated. Digestion with ribonuclease of all singlestranded nucleic acids present after hybridization leaves the double-stranded species, consisting of the labeled probe annealed to its complementary RNA, in the solution. The contents of the solution are then size-separated on an electrophoretic gel, which is exposed to an x-ray film. Knowing the amount of input labeled single-stranded probe allows a quantification of the specific RNA present in the total RNA of the cells or tissue. In Situ Hybridization. In situ hybridization with labeled single-stranded probes onto tissues is, in principle, similar to the ribonuclease protection assay. Detection and determina¬ tion of the location of a certain species of RNA within a tissue is possible. Reverse Transcription Polymerase Chain Reaction. Reverse transcription polymerase chain reaction (RT-PCR) can be used to quantitate the abundance of a specific RNA. This method is par¬ ticularly practical when small amounts of tissue or cells are avail¬ able to be analyzed. The RNA is reverse-transcribed to DNA with reverse transcriptase. The cDNA population is then sub¬ jected to PCR amplification with specific primers that recognize the cDNA in question. By choosing the number of PCR cycles within the linear range of product generated after each cycle (i.e., enough primers, nucleotides, and DNA polymerase in the reac¬ tion mixture for none of them to be the limiting factor of the reac¬ tion) and adding to the PCR reaction a defined amount of an artificial DNA template that is also recognized by the primer oli¬ gonucleotides but yields a different-sized product, one can detect differences in abundance of cDNA (and hence RNA in the origi¬ nal sample) among two or more samples. Newer methods allow for an on-line monitoring of each PCR reaction of the product generated. This is achieved by using primer oligonucleotides that can be monitored during the PCR reaction cycles by external optical devices. Such on-line continuous monitoring allows the performance of PCR reactions without prior determination of the number of cycles required to keep the PCR reaction within the linear range of amplification. Continuous PCR monitoring

Ch. 2: Molecular Biology: Present and Future provides immediate information on abundance of a given cDNA species in PCR reactions. Knowledge of the absolute amount of labeled oligonucleotide primer added to the PCR reaction at the start can be used to determine the exact amount of the PCR prod¬ uct generated.

PROTEIN EXPRESSION ASSAYS Cell-Free Translation. A commonly used method to ana¬ lyze proteins encoded by mRNA is to translate the mRNA in cell-free translation systems in vitro. By this method, proteins can be radioactively labeled to a high specific activity. The cellfree translation also provides the primary protein product, such as a proprotein or prohormone, encoded by the mRNA. Pulse and Pulse-Chase Labeling. Studies of protein syn¬ theses can also be carried out in vivo by incubation of cultured cells or tissues with radioactive amino acids (pulse labeling). Posttranslational processing (e.g., enzymatic cleavages of pro¬ hormones) can be assessed by first incubating the cells or tis¬ sues for a short time with radioactive amino acids and then incubating them for an additional period with unlabeled amino acids (pulse-chase labeling). Western Immunoblot. Another approach to the analyses of particular cellular proteins is the Western immunoblot tech¬ nique. Proteins in cell extracts are separated by electrophoresis on polyacrylamide or agarose gels and transferred to a nylon or nitrocellulose membrane, which is then treated with a solution containing specific antibodies to the protein of interest. The anti¬ bodies that are bound to the protein fixed to the membrane are detected by any one of several methods, such as secondary anti¬ bodies tagged with radioisotopes, fluorophores, or enzymes. Immunocytochemistry. A refinement of the Western immunoblot technique is the detection of specific proteins within cells by immunocytochemistry (immunohistochemistry). Cultured cells or tissue sections are fixed on microscope slides and treated with solutions containing specific antibodies. The antibodies that are bound to the proteins within the cells are detected with fluorescently tagged secondary antibodies or by an avidin-biotin complex. Immunocytochemistry is a powerful technique when used for the simultaneous detection of two or even three different proteins with examination by confocal microscopy.

DNA-PROTEIN INTERACTION ASSAYS ELECTROPHORETIC MOBILITY GEL SHIFT AND SOUTHWESTERN BLOTS The binding of proteins such as transcription factors to DNA sequences is commonly done by two approaches: electrophoretic mobility shift assay (EMSA) and Southwestern blotting. Typically, EMSA consists of incubation of protein extracts with a radiola¬ beled DNA sequence or probe. The mixture is then analyzed by electrophoresis on a nondenaturing polyacrylamide gel, fol¬ lowed by autoradiography or autofluorography to evaluate the distribution of the radioactivity or fluorescence in the gel. Inter¬ actions of specific proteins with the DNA probe are manifested by a retardation of the electrophoretic migration of the labeled probe, or band shift. The EMSA technique can be extended to include antibodies to specific proteins in the incubation mix¬ ture. The interaction of a specific antibody with a protein bound to the DNA probe causes a further retardation of migration of the DNA-protein complex, leading to a super shift. PROTEIN-PROTEIN INTERACTION ASSAYS A number of different assays are used to determine and evaluate protein-protein interactions. Two in vitro assays are coimmunopre-

13

cipitation and polyhistidine-tagged glutathione sulfonyl transferase (GST) pull-down. Two in vivo assays are the yeast and mamma¬ lian two-site interaction assays. Coimmunoprecipitation. The commonly used coimmunoprecipitation assay makes use of antisera to specific proteins. In circumstances in which two different proteins, A and B, asso¬ ciate with each other, an antiserum to protein A will immunoprecipitate not only protein A, but also protein B. Likewise, an antiserum to protein B will coimmunoprecipitate proteins B and A. In practice, the proteins under investigation are radiola¬ beled by synthesis in the presence of radioactive amino acids, either in cell-free transcription-translation systems in vitro, or in cell culture systems in vivo. Coimmunoprecipitated proteins are detected by gel electrophoresis and autoradiography. Alter¬ natively, the proteins so immunoprecipitated or coimmunopre¬ cipitated can be assayed by Western immunoblot techniques. Glutathione Sulfonyl Transferase Pull-Down. GST is an enzyme that has a high affinity for its substrate, glutathione. This property of high-affinity interactions has been exploited to develop a cloning vector plasmid encoding GST and containing a polylinker site that allows for the insertion of coding sequences for any protein of interest. Thus, if protein A is believed to interact with protein B, the coding sequence for either protein A or protein B can be inserted into the GST vec¬ tor. The GST-protein A or B fusion protein is synthesized in large amounts by multiplication and expression of the plasmid vector in bacteria. The GST-fusion protein is then incubated with either labeled or unlabeled proteins in extracts of cells or nuclei. Proteins in the extracts bound to protein A or B in the GST-fusion protein are pulled down from the extracts by captur¬ ing the GST on glutathione-agarose beads. Proteins are released from the beads and analyzed by either gel electrophoresis and autoradiography (labeled proteins) or by Western immunoblot (unlabeled proteins). Similar methods using polyhistidine tag in place of GST are also used for pull-down experiments. Far Western Protein Blots. A variation on the Western blotting technique is the Far Western blot. In this technique, a radiolabeled or fluorescence-labeled known protein (instead of an antibody) is applied to a membrane to which proteins from an electrophoretic gel have been transferred. If the known pro¬ tein binds to any one or more proteins on the membrane, it is detected as a labeled band by autoradiograph or autofluorogra¬ phy. Relatively strong protein-protein interactions are required for this approach to succeed. Yeast and Mammalian Cell Two-Site Interaction Traps. The two-site interaction traps are useful for demonstrating proteinprotein interactions in vivo. The principle of the approach is that, when a specific protein-protein interaction occurs, it reconstitutes an active transcription factor which then activates the transcription of a reporter gene. The cells (yeast or mamma¬ lian) are programmed to constitutively express a strong DNAbinding domain, such as Gal-4, fused to the expression sequence of the selected protein, protein A (the bait). The cells are also programmed to express a transcriptional reporter (e.g., CAT or luciferase linked to a promoter) that has binding sites for Gal-4. Thus, protein A anchors to the DNA-binding site of the reporter promoter via the Gal-4 binding domain but does not activate transcription of the reporter gene, and no reporter function is expressed. Protein B, however, is expressed as a fusion protein with a strong transcriptional activator sequence (e.g., the transcriptional transactivation domain of Gal-4 or of VP16). This transcriptional activation domain-protein B fusion protein does not bind DNA, but when, or if, protein B physi¬ cally interacts with (binds to) protein A, a fully active transcrip¬ tion factor is reconstituted, the promoter reporter gene is transcribed, and the reported function is expressed. The yeast two-hybrid system can be used to clone proteins that interact with a bait protein such as protein A. In this

14

PARTI: GENERAL PRINCIPLES OF ENDOCRINOLOGY

instance, a cDNA protein expression library is prepared or obtained that has all of the cDNAs of a given tissue fused to a coding sequence for a transactivation domain (e.g., VP16). Fur¬ ther, the reporter consists of a survival factor essential for the growth of the yeast cell. Thus, when a cDNA encodes a protein B (fish) that interacts with the bait protein A, the yeast cell expresses the survival protein and survives, whereas the other yeast cells die.

KNOCKOUT MICE

TRANSGENIC MICE

1. Transfect DNA into embryonic stem cells

1. Remove fertilized ova

2. Select cells in which homologous recombination occurred at a frequency of

GENETIC MANIPULATIONS IN ANIMALS IN VIVO TRANSGENIC APPROACHES To create transgenic mice, DNA is injected into the male pronu¬ cleus of one-cell mammalian embryos (fertilized ova) that are then allowed to develop by insertion into the reproductive tract of pseudopregnant foster mothers (Fig. 2-3A). The transgenic animals that develop from this procedure contain the foreign DNA integrated into one or more of the host chromosomes at an early stage of embryo development. As a consequence, the foreign DNA is generally transmitted to the germline, and, in a number of instances, the foreign genes are expressed. Because the foreign DNA is injected at the one-cell stage, a good chance exists that the DNA will be distributed among all the progeny cells as development proceeds. This situation provides an opportunity to analyze and compare the qualitative and quanti¬ tative efficiencies of expression of the genes among various organs. The technique is quite efficient; >50% of postinjection ova produce viable offspring, and, of these, -10% efficiently carry the foreign genes. In the transgenic animals, the foreign genes can be passed on and expressed at high levels in subse¬ quent generations of progeny. Transgenic approaches can also be used to prevent the development of the lineage of a particular cell phenotype or to impair the expression of a selected gene. A cell lineage can be ablated by targeting a microinjected DNA containing a subunit of the diphtheria toxin to a particular cell type, using a pro¬ moter sequence specifically expressed in that cell type. The diphtheria toxin subunit inhibits protein synthesis when expressed in a cell, thereby killing the cell. The expression of a particular gene can be impaired by similar cell promoterspecific targeting of a DNA expression vector to a cell that pro¬ duces an antisense mRNA to the mRNA expressed by the gene of interest. The antisense mRNA hybridizes to nuclear tran¬ scripts and processed mRNAs; this results in their degradation by double-stranded RNA-specific nucleases, thereby effec¬ tively attenuating the functional expression of the gene. The efficacy of the impairment of the mRNA can be enhanced by incorporating a ribozyme hammerhead sequence in the expressed antisense mRNA so as physically to cleave the mRNA to which it hybridizes. Another approach to producing a particular gene loss of function is to direct expression of a dominant negative protein (e.g., a receptor made deficient in intracellular signaling by an appropriate mutation, or a mutant transcription factor deficient in transactivation functions but sufficient for DNA binding). These dominant negative proteins compete for the essential functions of the wild-type proteins, resulting in a net loss of function. Another approach, termed targeted transgenesis, combines targeted homologous recombination in embryonic stem (ES) cells with gain-of-function transgenic approaches.11'12-17 This method allows for targeted integration of a single-copy trans¬ gene to a single desired locus in the genome and thereby avoids problems of random and multiple-copy integrations, which may compromise faithful expression of the transgene in the conventional approach.

10'2-10'3

200-500 copies/ova

5'HR

3'HR Cellular ’gk-tk 9ene Knockout vector Recombinatorial replace¬ ment

3. Implant ova in pseudo¬ pregnant surrogate mice

3. Inject cell into blastocoele of 3.5 day blastocyst

Implant blastocyst in pseudopregnant surrogate mice

I 4. Prepare "tail blots'1 hybridization of DNA with 32P-DNA probe

J&.

B

k Chimeric 'Germline offspring offspring

FIGURE 2-3. Approaches for (A) the integration of a foreign gene into the germline of mice, and (B) disruption or knock-out of a specific gene. A, DNA containing a specific foreign gene is microinjected into the male pronucleus of fertilized ova obtained from the oviduct of a mouse. Ova are then implanted into the uterus of pseudopregnant surrogate mothers. Progeny are analyzed for the presence of foreign genes by hybridization with 32P-labeled DNA probe and DNA prepared from a piece of tail from a mouse, which has been immobilized on a nitrocellu¬ lose filter (tail blots). B, To create a knock-out of a gene, pluripotential embryonic stem (ES) cells are used in vitro to introduce an engineered plasmid DNA sequence that will recombine with a homologous gene that is targeted. The recombination excises a portion of the gene in the ES cells, rendering it inactive (no longer expressible). ES cells in which the homologous recombination occurred successfully are selected by a combined positive-negative drug selection. The engineered ES cells are injected into the blastocoele of 3.5-day blastocysts that are then implanted into the uterus of pseudopregnant mice. The offspring are both chimeric and germline for expression of the knock-out gene and must be cross-bred to homozygosity for the genotype of a complete knock-out of the gene that is targeted for disruption.

GENE ABLATION (KNOCK-OUTS) A major advance beyond the gain-of-function transgenic mouse technique has been the development of methods for producing loss of function by targeted disruption or replacement of genes. This approach uses the techniques of homologous recombination in cultured pluripotential ES cells, which are then injected into mouse blastocysts and implanted into the uteri of pseudopreg¬ nant mice (Fig. 2-3B). The targeting vector contains a core replace¬ ment sequence consisting of an expressed-cell lethal-drug

Ch. 2: Molecular Biology: Present and Future

Germ line gene

15

Tissue-specific

Targeting construct \ HSV-tk-neoj Homologous recombination

Homologous recombinant ~fl HSV-tk-neo

Cr e-loxP/ mediated / recom¬ bination , />

b

Type II deletion

\ \ Type I deletion

FIGURE 2-4. Schema of the Cre-loxP approach to conditionally knock out a specifically targeted gene in mice. A, The approach requires the creation of two separate strains of transgenic mice that are crossed to produce double transgenic mice to effect the conditional gene knock¬ outs. One mouse strain is created so as to replace the gene of interest by one that has been flanked by loxP recombination sequences (floxed), using targeted recombinational gene replacement in embryonic stem cells as illustrated in Figure 2-3B. The other mouse strain is a transgenic mouse in which the Cre recombinase enzyme expression vector is tar¬ geted to the tissue of interest using a tissue-specific promoter, such as the proinsulin gene promoter, to target and restrict expression to pan¬ creatic B cells. B, A more detailed depiction of the strategy for prepara¬ tion of the gene replacement by homologous recombination to generate mice with a floxed gene. This approach is similar to that described in Figure 2-3B to create knock-out mice.18

resistance gene (selectable marker) (e.g., neomycin [Pgk-neo]) flanked by sequences homologous to the targeted cellular gene, and a second selectable marker gene (e.g., thymidine kinase [pgktk]). The ES cells are transfected with the gene-specific targeting vector. Cells that take up vector DNA and in which homologous recombination occurs are selected by their resistance to neomy¬ cin (positive selection). To select against random integration, a sus¬ ceptibility to killing by thymidine kinase (negative selection) is used; only homologous recombination in which the thymidine kinase gene has been lost will confer survival benefit. Because the ES cells are injected into multicellular 3.5-day blastocysts, many of the offspring are mosaics, but some are germline het¬ erozygous for the recombined gene. FI generation mice are then bred to homozygosity so as to manifest the phenotype of the gene knock-out. Using this approach of targeted gene disruption, literally thousands of knock-out mice have been created. Many ot these knock-out mice are models for human genetic disorders (e.g., those of endocrine systems such as pancreatic agenesis [homeodomain protein 1DX-1], familial hypocalciuric hypercal¬ cemia [calcium receptor], intrauterine growth retardation [msulin-like growth factor-11 receptor], salt-sensitive hypertension [atrial natriuretic peptide], and obesity [a3-adrenergic receptor]).

B FIGURE 2-5. Diagram showing the approach to reversible conditional expression of a gene in mice, using a tetracycline-inducible gene expres¬ sion system. A, As in the Cre-loxP system (see Fig. 2-4A), the tetracy¬ cline-inducible gene system requires the creation of two independent strains of transgenic mice. One strain of mice targets the expression of a specially engineered transcription factor (rtTA) to the tissue of interest, using a tissue-specific promoter (TSP). B, the rtTA transcription factor consists of a modification of the bacterial tetracycline-responsive repres¬ sor that has been genetically engineered so as to convert it into a tran¬ scriptional transactivator when exposed to tetracycline or one of its analogs. The other mouse strain is one in which a gene of interest is introduced, usually driven by a ubiquitous promoter such as a viral promoter (CMV, RSV) or an actin promoter. The gene of interest could be one encoding an antisense RNA to a messenger RNA of a protein that is to be knocked out. The creation of double transgenic mice then allows for the expression of the gene of interest in a specific tissue under the control of the induced tetracycline. (See text for more detailed description.57) (tet op, tetracycline resistance operon; P, promoter; AS, antisense; TPE, tissue promoter element.)

CONDITIONAL (DEVELOPMENTAL) INTERRUPTION OF GENE EXPRESSION Although targeted transgenesis using chosen site integration and targeted disruption of genes has proven helpful in analy¬ ses of the functions of genes, conditionally to induce expres¬ sion of transgenes or conditionally to inactivate a specific gene is useful. Early on, randomly integrated vectors for the expression of transgenes used the metallothionein promoter that is readily inducible by the administration of heavy metals to transgenic mice. Now techniques have been developed to conditionally inactivate targeted genes in a defined spatial and temporal pattern. Several approaches to achieve condi¬ tional gene inactivation have been developed. Two of these approaches are (a) the Cre recombinase-loxP system (Fig. 24)18 and (b) the tetracycline-inducible transactivator vector (tTA) system (Fig. 2-5).19 Occasionally, both of these systems have been used effectively to knock out (Cre-loxP) or to atten¬ uate (reverse tTA) the expression of specific genes. Both the Cre-loxP and reverse tTA systems require the creation of two independent strains of transgenic mice, which are then crossed to produce double transgenic mice.

16

PARTI: GENERAL PRINCIPLES OF ENDOCRINOLOGY

CRE RECOMBINASE-LOXP SYSTEM The Cre-loxP approach is based on the Cre-loxP recombination system of bacteriophage PI (see Fig. 2-4). This system is capable of mediating loxP site-specific recombination in embryonal stem cells and in transgenic mice. Conditional targeting requires the generation of two mouse strains. One transgenic strain expresses the Cre recombinase under control of a pro¬ moter that is cell-type specific or developmental stage specific. The other strain is prepared by using ES cells to effect a replace¬ ment of the targeted gene with an exact copy that is flanked by loxP sequences required for recombination by the Cre recombi¬ nase. The recombined gene is said to befloxed. The presence of the loxP sites does not interfere with the functional expression of the gene and will be normally expressed in all of its usual tis¬ sues not coexpressing the Cre recombinase. In those tissues in which the Cre is expressed by virtue of its tissue-specific pro¬ moter, the target gene will be deleted by homologous recombi¬ nation. Thus, the Cre-loxP system acts like a timer in which the events that are to take place are predetermined by the prior reprogramming of the genes: the target gene will be ablated during development where and when the promoter chosen to drive the expression of Cre is activated. Thus, a disadvantage of the Cre-loxP system is the lack of control over when the gene knock-out will take place, because it is preprogrammed in the system. Newer genetically engineered Cre derivatives allow for pharmacologic activation of the recombinant event. A potential advantage of the Cre-loxP system is that one can theoretically generate extensive collections of mice expressing the Cre recombinase specifically and individually in many different tis¬ sues so that these mice could be made commercially available to investigators. CONDITIONAL TETRACYCLINE-INDUCIBLE FORWARD AND REVERSE TRANSACTIVATOR VECTOR SYSTEMS The Cre-loxP system leads to the irreversible targeted disrup¬ tion of a particular gene at the time that the promoter encoding the Cre recombinase is activated during development. Having available a system that can be reversibly activated at any time would be desirable. A system that holds promise in this regard is the tetracycline-inducible transactivator vector (forward or reverse tTA), which, in response to tetracycline, switches on a specific gene bearing a promoter containing the tetracyclineresponsive operon (see Fig. 2-5). This system allows any recom¬ binant gene marked by the presence of the tet operon to be turned on or off at will simply by the administration of a potent tetracycline analog to the transgenic mice. The vectors were engineered from the sequences of the E. coli bacterial tetracy¬ cline resistance operon (tet op), in which a repressor sits on the operon, keeping the resistance gene off. When tetracycline binds to the repressor, it is deactivated, falls off of the operon, and turns on the gene. First, the repressor was converted into an activator by fusing the DNA-binding domain to the potent activator sequence (VP16) of the herpes simplex virus. In this system, tetracycline turned off the activator (tet-off) and thereby caused failure of expression of target genes containing the tet operon binding sites for the repressor turned into an activator. This tTA system required the continued presence of tetracycline to keep the gene off and withdrawal of the tetracy¬ cline to turn on the gene, raising problems of long and variable clearance times for the drugs. Turning the gene on by adminis¬ tration of tetracycline (tet-on) would be preferable. Therefore, the tTA vector was reengineered to reverse the action of tetracy¬ cline: in the current vector system, the binding of tetracycline to the reverse tTA enhances its binding to the tet operon. Theoreti¬ cally, as the reverse tTA system now works, any gene can be reversibly turned on by the administration of tetracycline or ne if its more potent analogs in the double transgenic mouse, /hich consists of a cross between a mouse that has the reverse

tTA targeted to express in a specific tissue and a mouse that has a ubiquitously expressed transgene for any gene X under the control of the tet operon. The equivalent of gene knock-outs can be accomplished by constructing gene X in a context to express an antisense RNA containing a ribozyme sequence. When induced by tetracycline, antisense-ribozyme RNA binds to the mRNA expressed by gene X, cleaves it, and thereby function¬ ally inactivates the gene.

PROSPECTS FOR THE FUTURE FOR CONDITIONAL TRANSGENE EXPRESSION The availability of the Cre-loxP and the forward and reverse tTA systems now makes it feasible to combine their key features in the creation of triple transgenic mice so that a targeted recombinational disruption of a gene can be accomplished by the adminis¬ tration of tetracycline. The Cre recombinase could be placed under the control of a tissue-specific promoter containing the tet operon uniquely responsive to the presence of tTA and targeted to a specific tissue by standard pronuclear injection targeted transgenesis. A second transgenic mouse is created with a ubiq¬ uitously expressed promoter during the expression of the reverse tTA. In the third mouse, the gene desired to be deleted would be replaced with an appropriately floxed gene. The latter mouse would be prepared by implantation of recombinantly engineered ES cells into blastocysts. The administration of tetra¬ cycline to the triple transgenic mouse would induce the Cre recombinase in a tissue-specific manner, thus allowing temporal and spatial control of gene knock-outs.

EXPRESSED SEQUENCE TAGS A very informative database of expressed sequence tags (ESTs) is being generated and placed in GenBank. Expressed sequence tags are prepared by random, single-pass sequencing of mRNAs from a repertoire of different tissues, mostly embryonic tissues (e.g., brain, eye, placenta, liver). Currently, the EST database contains -50% of the estimated expressed genes in humans and mammals (70,000-80,000). The EST database will become extremely valu¬ able when the sequences of the human, rat, and mouse genomes are completed.

DNA ARRAYS FOR THE PROFILING OF GENE EXPRESSION Two variants of DNA-array chip design exist.20,21 The first con¬ sists of cDNA (sequences unknown) immobilized to a solid sur¬ face such as glass and exposed to a set of labeled probes of known sequences, either separately or in a mixture of the probes. The second is an array of oligonucleotide probes (sequences known, based on either known genes in GenBank or ESTs) that are synthesized either in situ or by conventional synthesis fol¬ lowed by on-chip immobilization (Fig. 2-6). The array is exposed to labeled sample DNA (unknown sequence) and hybridized, and complementary sequences are determined. In cDNA chips, immobilized targets of single-stranded cDNAs prepared from a specific tissue are hybridized to singlestranded DNA fluorescent probes produced from total mRNAs to evaluate the expression levels of target genes.

OLIGONUCLEOTIDE ARRAYS (GENOMIC AND EXPRESSED SEQUENCE TAGS) The oligonucleotide gene chip (1.28 x 1.28 cm2) consists of a solid-phase template (glass wafer) to which high-density arrays of oligonucleotides (distance between oligonucleotides of 100 A) are attached, with each probe in a predefined posi¬ tion in the array. Each gene EST is represented by 20 pairs of

Ch. 2: Molecular Biology: Present and Future

73 i promoter y PCR

B--=B

STRATEGIES FOR MAPPING GENES ON CHROMOSOMES

in vitro transcription

T7 promoter ^ F

F

17

F

fragmentation x:

GENETIC LINKAGE MAPS AND QUANTITATIVE TRAIT LOCI

io

hybridize & wash ^ p

F

F

1 h FIGURE 2-6. Sample preparation and hybridization for oligonucleotide assay. A complementary DNA (cDNA) is transcribed in vitro to RNA, and then reverse-transcribed to cRNA. This material is fragmented and tagged with a fluorescent tag molecule (F). The fragments are hybrid¬ ized to an array of oligonucleotides representing portions of DNA sequences of interest. After washing, hybridization of the cRNA probe is detected by localization of the fluorescent signals. (PCR, polymerase chain reaction.)

25 base oligonucleotides from different parts of the gene (5’ end, middle, and 3’ end). The specificity of the detection method is controlled by the presence of single-base mismatch probes. Pairs of perfect and single-base mismatch probes corresponding to each target gene are synthesized on adjacent areas on the arrays. This is done to identify and subtract nonspecific background signals. The gene chip is sensitive enough to detect one to five transcripts per cell and is much more sensitive than the Northern blot technique.

COMPLEMENTARY DNA ARRAYS (SPECIFIC TISSUES) Poly (A) mRNA is isolated from cells or tissue of interest, and synthesis of double-stranded cDNA is accomplished by reverse transcription of cDNA, followed by synthesis of doublestranded cDNA using DNA polymerase I. In vitro transcription of double-stranded cDNA to cRNA is accomplished using biotin-16-UTP and biotin-ll-CTP for labeling and a T7 RNA polymerase as enzyme. This cRNA is used for hybridization with the gene chip. The gene chip is stained with R-physoerythrin streptavidin to detect biotin-labeled nucleotides, and different wash cycles are performed. Thereafter the gene chip is scanned digitally and analyzed by special software. (A grid is automati¬ cally placed over the scanned image of the probe array chip to demarcate the probe cells.) After grid alignment, the average intensity of each probe cell is calculated by the software, which then analyzes the patterns and generates a report. The applications of the gene chip include: 1. Simultaneous analysis of temporal changes in gene expression of all known genes and ESTs. 2. Sequencing of DNA. 3. Large-scale detection of mutations and polymorphisms in specific genes (i.e., BRCA1, HIV-1, cystic fibrosis CFTR, (3-thalassemia). 4. Gene mapping by determining the order of overlapping clones. Expensive equipment for generating and analyzing the data using genechips is required. When the cloning of all genes is completed (Human Genome Project), the gene chip will allow monitoring of the expression of all known genes in various situations.

A genetic linkage map shows the relative locations of specific DNA markers along the chromosome.22-27 Any inherited physical or molecular characteristic that differs among individuals and is easily detectable in the laboratory is a potential genetic marker. Markers can be expressed DNA regions or DNA segments that have no known coding function, but whose inheritance pattern can be followed. DNA sequence differences (polymorphisms; i.e., nucleotide differences) are especially useful markers because they are plentiful and easy to characterize precisely. Markers must be polymorphic to be useful in mapping. Alternative DNA polymorphisms exist among individuals, even among members of a single family, so that they are detectable among different families. Polymorphisms are variations in DNA sequence in the genome that occur every 300 to 500 base pairs. Variations within protein-encoding exon sequences can lead to observable pheno¬ typic changes (e.g., differences in eye color, blood type, and dis¬ ease susceptibility). Most variations occur within introns and have little or no effect on the phenotype (unless they alter exonic splicing patterns), yet these polymorphisms in DNA sequence are detectable and can be used as markers. Examples of these types of markers are: (a) restriction fragment length polymorphisms (RPLPs), which reflect sequence variations in DNA sites that are cleaved by specific DNA restriction enzymes; and (b) variable number of tandem repeat sequences (VNTRs), which are short repeated sequences that vary in the number of repeated emits and, therefore, in length. The human genetic linkage map is con¬ structed by observing how frequently any two polymorphic markers are inherited together. Two genetic markers that are in close proximity tend to be passed together from mother to child. During gametogenesis, homologous recombination events take place in the metaphase of the first meiotic step (meiotic recombination crossing-over). This may result in the separation of two markers that originally resided on the same chromosome. The closer the markers are to each other, the more tightly linked they are and the less likely that a recombination event will fall between and separate them. Recombination frequency provides an estimate of the distance between two markers. On the genetic map, distances between markers are measured in terms of centimorgans (cM), named after the American geneti¬ cist Thomas Hunt Morgan. Two markers are said to be 1 cM apart if they are separated by recombination 1% of the time. A genetic distance of 1 cM is roughly equal to a physical distance of 1 million base pairs of DNA (1 Mb). The current resolution of most human genetic map regions is approximately 10 Mb. An inherited disease can be located on the map by following the inheritance of a DNA marker present in affected individu¬ als but absent in unaffected individuals, although the molecu¬ lar basis of a disease or a trait may be unknown. Linkage studies have been used to identify the exact chromosomal loca¬ tion of several important genes associated with diseases, including cystic fibrosis, sickle cell disease, Tay-Sachs disease, fragile X syndrome, and myotonic dystrophy.

RESTRICTION ENZYMES AND CHROMOSOMAL MAPPING The restriction endonucleases, which have been isolated from various bacteria, recognize short DNA sequences and cut DNA molecules at those specific sites. A natural biofunction of restriction endonucleases is to protect bacteria from viral infec¬ tion or foreign DNA by destroying the alien DNA. Some restriction enzymes cut DNA very infrequently, generating a

18

PARTI: GENERAL PRINCIPLES OF ENDOCRINOLOGY

small number of very large fragments, whereas other restriction enzymes cut DNA more frequently, yielding many smaller fragments. Because hundreds of different restriction enzymes have been characterized, DNA can be cut into many different¬ sized fragments.

PHYSICAL MAPS Different types of physical maps vary in their degree of resolu¬ tion. The lowest resolution physical map is the chromosomal (cytogenetic) map, which is based on the distinctive banding pattern observed by light microscopy of stained chromosomes. A cDNA map shows the locations of expressed DNA (exons) on the chromosomal map. The more detailed cosmid contiguous DNA block (contig) map depicts the order of overlapping DNA fragments spanning the genome (see the section High-Resolu¬ tion Physical Mapping). A macrorestriction map describes the order and distance between restriction enzyme cleavage sites. The highest resolution physical map will be the complete elucida¬ tion of the DNA base-pair sequence of each chromosome in the human genome. LOW-RESOLUTION PHYSICAL MAPPING Chromosomal Map. In a chromosomal map, genes or other identifiable DNA fragments are assigned to their respec¬ tive chromosomes, with distances measured in base pairs. These markers can be physically associated with particular bands (identified by cytogenetic staining) primarily by in situ hybridization, a technique that involves tagging the DNA marker with an observable label. The location of the labeled probe can be detected after it binds to its complementary DNA strand in an intact chromosome. As with genetic linkage mapping, chromosomal mapping can be used to locate genetic markers defined by traits observ¬ able only in whole organisms. Because chromosomal maps are based on estimates of physical distance, they are considered to be physical maps. The number of base pairs within a band can only be estimated. Fluorescence In Situ Hybridization.28 29 A fluorescently labeled DNA probe locates a DNA sequence detected on a spe¬ cific chromosome. The fluorescence in situ hybridization (FISH) method allows for the orientation of DNA sequences that lie as close as 2 to 5 Mb. Modifications to the in situ hybridization methods, using chromosomes at a stage in cell division (inter¬ phase) when they are less compact, increase map resolution by an additional 100,000 base pairs. A cDNA map shows the positions of expressed DNA regions (exons) relative to particular chromosomal regions or bands. (Expressed DNA regions are those transcribed into mRNA.) The cDNA is synthesized in the laboratory using the mRNA molecule as the template. This cDNA can be used to map the genomic region of the respective molecule. A cDNA map can provide the chromosomal location for genes whose functions are currently unknown (ESTs). For hunters of disease genes, the map can also suggest a set of candidate genes to test when the approximate location of a disease gene has been mapped by genetic linkage analysis. HIGH-RESOLUTION PHYSICAL MAPPING Two current approaches to high-resolution mapping are termed top-down (producing a macrorestriction map) and bottom-up (resulting in a contig map). With either strategy, the maps rep¬ resent ordered sets of DNA fragments that are generated by cutting genomic DNA with restriction enzymes. The fragments are then amplified by cloning or by PCR methods. Electroohoretic techniques are used to separate the fragments (accordg to size) into different bands, which are visualized by hng or by hybridization with DNA probes of interest. The

use of purified chromosomes, separated either by fluorescenceactivated flow sorting from human cell lines or in hybrid cell lines, allows a single chromosome to be mapped. A number of strategies can be used to reconstruct the origi¬ nal order of the DNA fragments in the genome. Many approaches make use of the ability of single strands of DNA and/or RNA to hybridize to form double-stranded segments. The extent of sequence homology between the two strands can be inferred from the length of the double-stranded segment. Fingerprinting uses restriction enzyme cleavage map data to determine which fragments have a specific sequence (finger¬ print) in common and, therefore, overlap. Another approach uses linking clones as probes for hybridization to chromosomal DNA cut with the same restriction enzyme. In top-down mapping, a single chromosome is cut (using rare-cutter restriction enzymes) into large pieces, which are ordered and subdivided; the smaller pieces are then mapped further. The resulting macrorestriction maps depict the order of and distance between locations at which rare-cutter restriction sites are found in the chromosome. This approach yields maps with more continuity and fewer gaps between fragments than contig maps, but map resolution is lower and the map may not be useful in finding particular genes. In addition, this strategy generally does not produce long stretches of mapped sites. Cur¬ rently, this approach allows DNA pieces to be located in regions measuring -100 kb to 1 Mb. The development of pulsed-field gel (PFG) electrophoretic methods has improved the mapping and cloning of large DNA molecules. Whereas conventional gel electrophoretic methods separate pieces of DNA \\\wvs

clathrin non-clathrin

FIGURE 3-13. The Golgi stack. The Golgi stack consists of numerous membranous compartments, including cis, medial, and trans-Golgi ele¬ ments. These compartments may be differentiated by the presence of spe¬ cific enzymes. Partially processed protein hormones traverse this system by way of intermediate secretory vesicles in a budding-fusion reiterative process. In addition to transport, protein processing occurs. Sorting with routing to ultimate destinations in cellular sites is accomplished in the frans-Golgi network (TGN). Secretory peptides may be sorted to constitu¬ tive or regulated secretory pathways. Constitutive secretory pathways are equivalent to the pathways taken by membrane proteins, whereby non-clathrin-coated membrane segments are used. The regulated secre¬ tory-secretory granule pathway involves a clathrin-coated pit among membrane segments. This is similar to the pathway taken by lysosomal components. (Adapted from Griffiths G, Simons K. Tire trans Golgi net¬ work: sorting at the exit site of the Golgi complex. Science 1986; 234:438.)

32

PART I: GENERAL PRINCIPLES OF ENDOCRINOLOGY

FIGURE 3-14. Proximal and distal glycosylation. The pathway of glycosylation in the rough endoplasmic reticulum (RER) and Golgi is shown. Core carbohydrate moieties are added cotranslationally by way of a dolichol-sugar intermediate (Do/-) to Asn residues in the protein backbone in the RER. Several glycosidases (steps 1-4) remove distal sugars in this compartment. Distal glycosylation occurs by the actions of mannosidases (steps 5-7) and glycosyl transferases (steps 6, 8-1) in the Golgi. Phosphorylation (I, II) of N-acetyl glucosamines in carbohy¬ drate moieties in the cis Golgi occurs in proteins destined for lysosome localization. (From Kornfeld R, Kornfeld S. Assembly of asparaginelinked oligosaccharides. Annu Rev Biochem 1985; 54:631.)

occur, including phosphorylation, acetylation, sulfation, acyla¬ tion, a-amidation of COOH termini, addition of ubiquitin, other modifications, and degradation.46 Another important function of the Golgi stack is the delivery of nascent polypeptides to the appropriate targets within the cell, which occurs in the trans-Golgi region or TGN.47 The pro¬ teins destined for lysosomal sites are targeted to those organelles by way of the mannose-6-phosphate receptor.48 In a similar manner, receptor and secretory proteins are targeted to membrane and secretory granule sites, respectively.49'53 The nearly mature polypeptide emerges from the Golgi stack in the TGN, where transport organelles, known as secretory vesicles or granules, are formed. These vesicles allow the exit of the nearly mature protein hormone from the Golgi stack. Secretory proteins are released from a cell by way of two pathways: the constitutive pathway and the regulated patlnoay.54'55 The constitutive pathway is thought to be mediated by a passive aggregation sorting mechanism whereby peptide hormones form aggregates in the TGN, an action that is facilitated by acidic pH and high calcium concentrations in this compartment. The polarity of the secretory faces of epithelial cells enables proteins that are released in a nonregulated or constitutive manner to be released on the apical surface and regulated release to be per¬ formed at the basolateral surface. Whether such polarity of secre¬ tion exists in endocrine cells is unknown. Constitutive release generally involves the rapid exocytosis of newly synthesized peptides, but regulated secretion involves the classic secretory

granule and signaled degranulation, causing hormone- or factorregulated release of hormones. Secretory peptides must be segre¬ gated into one pathway or the other. Regulated secretion involves the formation of secretory residues and granules com¬ posed of clathrin-containing membrane segments, as found in lysosomes. Proteins destined for regulated secretion must end up in a reservoir known as the secretory granule, where the polypeptide hormones are concentrated and stored. This path¬ way is now considered to operate by active sorting via a signal ligand receptor. Proteins destined for secretion in this manner clearly contain sorting signals in their precursor molecules. For instance, the precursors to proopiomelanocortin (POMC) and proenkephalin have a stretch of aliphatic hydrophobic and acidic amino-acid residues at the N termini that are necessary and suffi¬ cient for efficient sorting into secretory granules. Further, carboxypeptidase E (Cpe) appears to serve as a sorting receptor for these peptide signals as determined by biochemical and genetic approaches. In particular, the Cpefai, which harbors a mutant and ineffective Cpe, is obese, diabetic, and infertile. It has elevated levels of proinsulin in pancreatic B cells and of POMC in the anterior pituitary, and decreased insulin and ACTH release.56 Three types of vesicles are formed in the TGN. One is the secretory vesicle, which is not clathrin coated and mediates non-receptor-dependent transport of membrane proteins and protein to be secreted in the constitutive pathway. The other two are the secretory granule, which is partially clathrin coated and mediates the receptor-dependent transfer of regulated secretory peptides, and the lysosome, which is predominantly clathrin coated and mediates transport of lysosomal enzymes and proteins.57 The secretory vesicle participates in the default, bulk-flow sorting system, but the others require the presence of "sorting patches" or sorting signals based on secondary and tertiary, but not primary, structures.55 Although the secretory granules are derived from immature granules with clathrincoated pits, the precise nature of the receptor-mediated sorting of peptide hormones is unknown. Evidence exists for pH-regulated, receptor-dependent sort¬ ing in the trans-Golgi and TGN. The pH of the compartments decreases as the Golgi stack is traversed from cis to trans regions. Such gradients in pH may participate in the molecular aggregation of polypeptide hormones. Possibly, these aggregates formed in the process of hormone concentration may initiate the budding of secretory granules. Chloroquine, which pre¬ vents Golgi acidification, may inhibit granule formation by pre¬ venting aggregation in neutralized Golgi stacks. Proteolytic processing of protein precursors (i.e., propro¬ teins or polyproteins) to yield smaller bioactive peptides (see Table 3-1) also occurs in acidic Golgi and secretory vesicles 58 Such proteolysis, however, is not required for packaging.

SECRETORY GRANULE Much has been learned about the nature of polypeptide hor¬ mones and secretory granules.55,59'60 The hormones in this organelle are highly concentrated. In particular, a number of polypeptide hormones are condensed in a crystal lattice forma¬ tion to increase the amount of hormone (up to 200-fold) in this organelle. Secretory granules allow cells to store enough hor¬ mone to be released on demand by extracellular signals at a level not possible by de novo synthesis. The f1/2 of stored hor¬ mones may be days, whereas the f1/2 of similar proteins in secretory vesicles may be minutes. The size of secretory granules varies greatly, depending on the nature of the stored hormone. The condensation of hor¬ mone is demonstrated by the presence of electron-opaque or "dense" cores. The granule core is quite stable and is often visi¬ ble even after exocytosis or in vitro enzymatic digestion of the granule membrane. It is osmotically inert yet sensitive to pH levels higher than 7.0 55

Ch. 3: Biosynthesis and Secretion of Peptide Hormones The formation of the secretory granule proceeds in stages, beginning in the trans-Golgi, where the initial hormone concen¬ tration may be observed. This aggregation process603 is facilitated by changes in pH, calcium concentration, and possible presence of other proteins such as secretogranins, chromogranins, and sulfated proteoglycans. Aggregates may form in different regions of the secretory granule. The colocalization of two or more polypeptide hormones in a granule may be observed. Within a cell, the relative distribution of two hormones is constant from granule to granule; however, variability in overall distribution is achieved from cell to cell. The mechanism by which the gonadotrope, a cell that generally produces luteinizing hormone (LH) and follicle-stimulating hormone (FSH), may be regulated to release LH and FSH differentially remains unclear.61

33

TABLE 3-2. Loci of Genetic Regulation of Polypeptide Hormone Synthesis* TRANSCRIPTION

Initiation

TRANSLATION

Protein synthesis

Elongation Termination

POSTTRANSLATION

Protein modification POSTTRANSCRIPTION

Protein maturation

hnRNA processing

Peptide maturation

hnRNA stability

Peptide stability

mRNA

STORAGE

Stability SECRETION

SECRETION POSTSECRETION

Secretory granules release their contents by cytoskeletal pro¬ tein-mediated movement of the granule toward the cellular surface.613 There, secretory granule membranes fuse with the plasma membrane and allow eversion or exocytosis of stored hormone.62 This process of emiocytosis causes secretion of hor¬ mone. The mechanisms involved in stimulus-secretion cou¬ pling are not well known, although responses to cellular signals causing changes in intracellular calcium, ion currents, or intra¬ cellular pH may lead to these events. In the unstimulated cell, a web of actin-associated microfilaments on the cytoplasmic face of the plasma membrane may act as a physical barrier to secretory granule fusion. However, changes in intracellular calcium, ion currents, or intracellular pH may cause differences in actin-binding protein interactions and alterations in the "secretory barrier" and permit exocytosis to occur. Secretion and rapid membrane fusion of multiple secretory granules require an endocytotic pathway to retrieve the extra membranes resulting from exocytosis in the plasma membrane and to return them to the Golgi stack and lysosome.

REGULATION OF POLYPEPTIDE HORMONE SYNTHESIS Regulation of the biosynthesis of polypeptides may occur at any of the biosynthetic levels in the pathway (Table 3-2 and Fig. 3-4). Of major interest is the regulation of peptide hormone syn¬ thesis at the transcriptional level. Studies using gene transfer and structure-function analysis have established that specific DNA elements in the regulatory region of the transcriptional unit are critical for determining transcriptional rates of various structural regions.63,64 In particu¬ lar, hormone-regulatory elements (HREs) have been character¬ ized for glucocorticoid, estrogen, androgen progesterone, vitamin D, mineralocorticoid, retinoic acid, and thyroid hor¬ mone receptors. In each case, a DNA element 8 to 20 nucleotides long may be necessary and sufficient for conferring hormonal regulation to its associated structural region. Several factors, including the steroid and thyroid hormones, interact with nuclear receptor proteins, which interact with DNA elements directly to modulate gene transcription.65-68 For the glucocorti¬ coid receptor, the glucocorticoid ligand binds to the inactive glu¬ cocorticoid receptor in the cytoplasm, present in a complex with heat shock proteins, hsp 90 and hsp 70, and others. The activated receptor-ligand complex interacts as a trans¬ acting factor to bind the DNA element corresponding to the glucocorticoid regulatory element (GRE). Studies have been performed on GREs in genes for mouse mammary tumor virus (MMTV) and murine sarcoma viruses, human metallothionein Ila, tyrosine aminotransferase, tryptophan oxygenase, and growth hormone, and in other genes. The long terminal repeat region of MMTV contains five GREs.64,69 A consensus sequence

*At each step in the pathway of biosynthesis of polypeptide hormones is potential reg¬ ulation by factors and other hormones. At the transcriptional level, the major focus of regu¬ lation is at transcription initiation. However, regulation at elongation and termination steps is also possible. The various steps in heterogeneous nuclear RNA (hnRNA) processing, including splicing and hnRNA stability, may be regulated events. Messenger RNA (mRNA) stability is a major regulated step in determining the steady-state amounts of mRNA that will be translated into protein. Regulation at the translational level has been described and is a possible locus of regulation. Any of the posttranslational steps, including release of secretory granules, may also be focus of regulation. Extracellular processes (postsecretion), including serum protein binding of hormones, activation, and degradation, may also con¬ verge to determine the steady-state level of bioactive polypeptide hormone.

for the putative GRE is shown by the sequence 5'-GGTACANNNTGTTCT-3', in which N = A, C, G, or T. The structures of the steroid and thyroid hormone receptors are better known. These hormone receptors are encoded by genes related to a viral oncogene, v-crM.70,71 The thyroid hor¬ mone receptor is encoded by the protooncogene c-erbA. Each receptor contains a stereotypic structure, including a protein that is ~45 to 60 kDa, with a central DNA-binding domain and a carboxyl-terminal ligand-binding domain. These and other regions mediate frans-activation, dimerization, and nuclear localization. The DNA-binding region consists of multiple cys¬ teine and histidine residues that are critical for the formation of Zn2+ fingers first described in the DNA-binding protein TFllla, a transcription regulatory factor for the 5S ribosomal gene in Xenopus.72 This Zn2+ finger interaction is a common motif for the binding of many eukaryotic proteins to DNA.73-75 The steroid-thyroid hormone receptors represent the first major examples of trans-acting factors well described in mam¬ malian systems. The motif found in prokaryotic systems, par¬ ticularly the interactions of cro and lambda repressor proteins with their target DNA elements in bacteriophage lambda, occurs with a homopolymeric dimer of subunits containing alpha helix-turn-alpha helix structure. The binding generally involves protein dimers; it requires a twofold axis of symmetry in the DNA sequence and involves the major groove of the tar¬ get DNA over several helical turns. Data indicate that the thy¬ roid hormone, retinoic acid, and vitamin D receptors are active only in the heterodimeric state, with other nuclear factors such as retinoid X receptors as their partners. Hormones that act by way of surface membrane receptors may induce the production of second messengers that may directly or indirectly interact with DNA elements within the gene.76-79 HREs may not be restricted to interactions observed with steroid-thyroid hormone receptor complexes, but they may involve other protein-DNA interactions. Advances in the isolation of such frans-acting factors and the identification of cis-acting HREs will probably speed an understanding of the molecular mechanisms of the hormonal regulation of gene expression at the transcriptional level.80 The presence of multiple enhancer elements or HREs in the regulatory regions of genes allows fine tuning of transcriptional

34

PART I: GENERAL PRINCIPLES OF ENDOCRINOLOGY PRE

ACTh

-i—i—r —r 7-MSH A-MSH CLIP

/

PRE

N-TERM

WZft c

N- TERM

ACTH

^

1 1 nri 1

N-TERM

i—I

FIGURE 3-15. Thyroid hormone action. This diagram depicts the mech¬ anism of action of thyroid hormones in the regulation of a thyroid hormone-responsive gene. Thyroxine (T4) or triiodothyronine (T3) enters the cell. T4 is converted to T3 intracellularly in many cells by means of 5'-deiodinase activity. T3 then enters the nucleus, where it binds to the nuclear thyroid hormone receptor, which is encoded by cerbA. This hormone nuclear receptor complex then serves as a trans¬ acting factor for binding to a thyroid hormone regulatory element ('TRE), which may then positively or negatively regulate gene expres¬ sion, with resultant production of RNA and protein derived from the thyroid hormone-regulated gene. (mRNA, messenger RNA.)

efficiency and influences the rate of production of the initial RNA transcript81-85 (Fig. 3-15). Other loci for regulation in this biosynthetic pathway include elongation and termination of transcription86 (see Fig. 3-9). The various steps of RNA maturation, most notably RNA splicing, may also change mRNA levels encoding a particular polypep¬ tide hormone, which ultimately determines the amount of polypeptide produced. The nuclear stability of the hnRNA and transport of the RNA from the nucleus to the cytoplasm also may be regulated. A major determinant of the steady-state levels of mRNA is cytoplasmic mRNA stability. Examples include the estrogen regulation of chicken liver vitellogenin mRNA, prolac¬ tin regulation of breast casein mRNA stabilities, and thyroid hormone control of the TSH (3 subunit.87-89 The interaction of mRNA with the protein synthetic machinery in the process of translation may be regulated. Several examples of translational control have been observed, including glucose regu¬ lation of the translational efficiency of insulin mRNA. Moreover, a number of the posttranslational processing events that occur in the RER and Golgi stack and the control of secretory granule forma¬ tion and release may also be loci for regulation. Even after proteins are released from the secretory cell, the bioactive peptide may be further acted on by degradative pro¬ cesses and proteolytic events that may activate proteins in extracellular steps to determine the bioactivity of a particular polypeptide hormone. A major example involves the cascade of the extracellular enzymatic conversion of the precursors of angiotensin II (see Chap. 79). Another example of postsecretion proteolytic processing of precursor polypeptides involves the conversion of iodinated thyroglobulin to the iodinated thy¬ ronines, thyroxine and triiodothyronine, in the follicular cell of the thyroid. Plasma stability of a polypeptide is a major deter¬ minant of the activity of the hormone in its eventual interaction with target cells.

GENERATION OF DIVERSITY A major example of the generation of diversity is the calcitonin and calcitonin gene-related peptide (CGRP) system. In this system, the C cell of the thyroid expresses a calcitonin-CGRP transcript that initially contains six exons. In the C cell, tissuespecific factors determine the use of the polyadenylation site in cl e fourth exon, but in the brain, transcription through the sixth exon, which encodes CGRP and the alternative polyadenyla¬ tion site present in that exon, provides the alternative splicing

l

1

/9-END

/3-IP*

L&>

6

i

H-LPH

,9-LPH

ACTH

I ip

I

ACTH

I ...1

Anterior and

intermediate

(!) Glycosylation

Rjrtial glycosylation P Phosphorylation

6

0lbasic amin0 acKls 0 Presequonce

1

FIGURE 3-16. Alternative protein processing of the preproopiomelano¬ cortin (POMC) precursor. In the anterior pituitary gland, the single POMC precursor is processed posttranslationally to produce adreno¬ corticotropic hormone (ACTH) and p-lipotropin (P-LPH). However, the intermediate lobe further processes these peptides to a-melanocytestimulating hormone (a-MSH), corticotropin-like intermediate lobe peptide (CLIP), y-lipotropin (y-LPH), and P-endorphin. (From Douglass J, Civielli O, Herbert E. Polyprotein gene expression: generation of diversity of neuroendocrine peptides. Annu Rev Biochem 1984; 53:665.)

and deletion of the fourth exon, which encodes calcitonin. The C cells express mostly calcitonin and not much CGRP; con¬ versely, the hypothalamus produces mostly CGRP but not much calcitonin (see Chap. 53 and Fig. 53-1). Other examples of alternative splicing yielding different polypeptides include the synthesis of the alternate human growth hormone form, sub¬ stance P, substance K, and protooncogenes.90-91 Alternative processing of polypeptides in a posttranslational process is important for the generation of polypeptide diver¬ sity.92-93 A major example of this is the production of ACTH and (3-lipotropin from the POMC precursor (Fig. 3-16). Using the same mRNA transcript, the anterior pituitary gland produces ACTH and (3-lipotropin, and the intermediate lobe of the pitu¬ itary gland performs further alternate proteolytic processing and produces (3-endorphin, corticotropin-like intermediate lobe peptide (CLIP), a-melanocyte-stimulating hormone, and other products (see Chap. 16).

REFERENCES 1. Alberts B, Watson JD, Bray D, et al. Molecular biology of the cell. New York: Garland Publishing, 1994. 2. Matsudaira P, Berk A, Zipursky L, et al. Molecular cell biology. New York: WH Freeman Co, 1999. 3. Lewin BM. Genes VII. New York: Oxford University Press, 1999. 4. Newport JW, Douglass JF. The nucleus: structure, function, and dynamics. Annu Rev Biochem 1987; 56:535. 5. Chin WW. Organization and expression of glycoprotein hormone genes. In: Imura H, ed. The pituitary gland. New York: Raven Press, 1985:164.

Ch. 3: Biosynthesis and Secretion of Peptide Hormones 6. Eipper BA, Mains RE, Glembotski CC. Identification in pituitary tissue of a peptide alpha-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper, and ascorbic acid. Proc Natl Acad Sci U S A1983; 80:5144. 7. Bradbury AF, Finnie MDA, Smyth DG. Mechanism of C-terminal amide formation by pituitary enzymes. Nature 1982; 298:686. 8. Gilbert W. Why genes in pieces? Nature 1978; 271:501. 9. Sudhoff TC, Russell DW, Goldstein JL, et al. Cassette of eight exons shared by genes for LDL receptor and EGF receptor. Science 1985; 228:893. 10. Nevins JR. The pathway of eukaryotic mRNA formation. Annu Rev Biochem 1983; 52:441. 11. Darnell JE Jr. Variety in the level of gene control in eukaryotic cells. Nature 1982; 297:365. 12. Rowley A, Dowell SJ, Diffley FX. Recent developments in the initiation of chromosomal DNA replication: a complex picture emerges. Biochim Biophys Acta 1994; 1217:239. 13. Albright SR, Tjian R. TAFs revisited: more data reveal new twists and con¬ firm old ideas. Gene 2000; 242:1. 14. Zawel L, Reinberg D. Advances in RNA polymerase II transcription. Curr Opin Cell Biol 1992; 4:488. 15. Pugh BF, Tjian R. Diverse transcriptional functions of the multisubunit eukaryotic TFIID complex. J Biol Chem 1992; 267:679. 16. Conaway RC, Conaway JW. General initiation factors for RNA polymerase II. Annu Rev Biochem 1993; 62:161. 17. Busby S, Ebright RH. Promotor structure, promotor recognition, and tran¬ scription activation in prokaryocytes. Cell 1994; 79:743. 18. Ptashne M. Gene regulation by proteins acting nearby and at a distance. Nature 1986; 322:697. 19. Walker MD, Edlund T, Boulet AM, Rutter WJ. Cell-specific expression con¬ trolled by the 5'-flanking region of insulin and chymotrypsin genes. Nature 1983; 306:557. 20. Edlund T, Walker MD, Barr PJ, Rutter WJ. Cell-specific expression of the rat insulin gene: evidence for role of two distinct 5'-flanking elements. Science 1985; 230:912. 21. Platt T. Transcription termination and the regulation of gene expression. Annu Rev Biochem 1986; 55:339. 22. Brawerman G. Determinants of messenger RNA stability. Cell 1987; 48:5. 23. Shatkin AJ. mRNA cap binding proteins: essential factors for initiating translocation. Cell 1985; 40:223. 24. Padgett RA, Grabowski PJ, Konarska MM, et al. Splicing of messenger RNA precursors. Annu Rev Biochem 1986; 55:1119. 25. Sharp PA. Splicing of messenger RNA precursors. Science 1987; 253:766. 26. Keller W. The RNA lariat: a new ring to the splicing of mRNA precursors. Cell 1984; 39:423. 27. Andreadis A, Gallego ME, Nadal-Ginard B. Generation of protein isoform diversity by alternative splicing: mechanistic and biological implications. Annu Rev Cell Biol 1987; 3:207. 28. Weis K. Importins and exportins: how to get in and out of the nucleus. Trends Biol Sci 1998; 23:185. 29. Pelletier J, Sonenberg N. .Insertion mutagenesis to increase secondary structure within the 5'-noncoding region of a eucaryotic mRNA reduces translational efficiency. Cell 1985; 40:515. 30. Darveau A, Pelletier J, Sonenberg A. Differential efficiencies of in vitro translation of mouse c-myc transcript differing in the 5-untranslated region. Proc Natl Acad Sci U S A1985; 82:2315. 31. Shaw G, Kamen R. A conserved AU sequence from the 3'-untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 1986; 46:659. 32. Nielsen DA, Shapiro DJ. Insights into hormonal control of messenger RNA stability. Mol Endocrinol 1990; 4:953. 33. Atwater JA, Wisdom R, Verma IM. Regulated mRNA stability. Annu Rev Genet 1990; 24:519. 34. Kozak M. Compilation and analysis of sequences upstream from the trans¬ lational start site in eukaryotic mRNAs. Nucleic Acids Res 1984; 12:857. 35. Kozak M. Selection of initiation sites by eucaryotic ribosomes: effect of inserting AUG triplets upstream from the coding sequence for preproinsu¬ lin. Nucleic Acids Res 1984; 12:3873. 36. Kozak M. Bifunctional messenger RNAs in eukaryotes. Cell 1986; 47:481. 37. Von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 1986; 14:4683. 38. Gierasch LM. Signal sequences. Biochemistry 1989; 28:1. 39. Walter P, Gilmore R, Blobel G. Protein translocation across the endoplasmic reticulum. Cell 1984; 38:5. 40. Wickner WT, Lodish HF. Multiple mechanisms of protein insertion into and across membranes. Science 1985; 230:400. 41. Wiedmann M, Kurzchalia TV, Hartmann E, Rapoport TA. A signal sequence receptor in the endoplasmic reticulum membrane. Nature 1987; 328:830. 42. Dunphy WG, Rothman JE. Compartmental organization of the Golgi stack. Cell 1985; 42:13. 43. Mellman I, Warren G. The road taken: past and future foundations of mem¬ brane traffic. Cell 2000; 100:99. 44. Griffiths G, Simons K. The trims Golgi network: sorting at the exit site of the Golgi complex. Science 1986; 234:438. 45. Kornfeld R, Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem 1985; 54:631. 46. Dice JF. Molecular determinants of protein half-lives in eukaryotic cells. FASEB J 1987; 1:349. 47. Sanders SL, Schekman R. Polypeptide translocation across the endoplas¬ mic reticulum membrane. J Biol Chem 1992; 267:13791.

35

48. Kornfeld S. Trafficking of lysosomal enzymes in normal and disease states. J Clin Invest 1986; 77:1. 49. Munro S. Pelham HRB. A C-terminal signal prevents secretion of lumenal ER proteins. Cell 1987; 48:899. 50. Munro S, Pelham HRB. An HSP70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell 1986; 46:291. 51. Johnson LM, Bankaitis VA, Emr SD. Distinct sequence determinants direct intracellular sorting and modification for a yeast vacuolar protease. Cell 1987; 48:875. 52. Vails LA, Hunter CP, Rothman JH, Stevens TH. Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptide Y residues in the propeptide. Cell 1987; 48:887. 53. Moore HH, Kelly RB. Re-routing of a secretory protein by fusion with human growth hormone sequences. Nature 1986; 321:443. 54. Kelly RB, Grote E. Protein targeting in the neuron. Annu Rev Neurosci 1993; 16:95. 55. Burgess TL, Kelly RB. Constitutive and regulated secretion of proteins. Annu Rev Cell Biol 1987; 3:243. 56. Loh YP, Snell CR, Cool DR. Receptor-mediated targeting of hormones to secretory granules. Role of carboxypeptidase E. Trends Endocrinol Metab 1997; 8:130. 57. Tooze J, Tooze SA. Clathrin-coated vesicular transport of secretory proteins during the formation of ACTH-containing secretory granules in AtT-20 cells. J Cell Biol 1986; 103:839. 58. Orci L, Ravazzola M, Storch M-J, et al. Proteolytic maturation of insulin is a post-Golgi event which occurs in acidifying clathrin-coated secretory vesi¬ cles. Cell 1987; 49:865. 59. Mellman I, Fuchs R, Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem 1986; 55:663. 60. Hong W, Tang BL. Protein trafficking along the exocytotic pathway. Bioas¬ says 1993; 15:231. 60a. Gerdes HH, Glombik MM. Signal-mediated sorting to the regulated path¬ way of protein secretion. Anat Anz 1999; 181:447. 61. Inoue K, Kurosumi K. Ultrastructural immunocytochemical localization of LH and FSH in the pituitary of the untreated male rat. Cell Tissue Res 1984; 235:77. 61a. Gullberg U, Bengtsson N, Bulow E, et al. Processing and targeting of granule proteins in human neutrophils. J Immunol Methods 1999; 232:201. 62. DeLisle RC, Williams IA. Regulation of membrane fusion in secretory exocytosis. Annu Rev Physiol 1986; 48:225. 63. Thomas G, Thorne BA, Hruby DE. Gene transfer technique to study neu¬ ropeptide processing. Annu Rev Physiol 1988; 50:323. 64. Yamamoto KR. Steroid receptor regulated transcription of specific genes and gene networks. Annu Rev Genet 1985; 19:209. 65. Shupnik MA, Chin WW, Habener JF, Ridgway EC. Transcriptional regula¬ tion of the thyrotropin subunit genes by thyroid hormone. J Biol Chem 1985; 260:2900. 66. Larsen PR, Harney JW, Moore DD. Sequences required for cell type specific thyroid hormone regulation of rat growth hormone promoter activity. I Biol Chem 1986; 261:14373. 67. Wright PA, Crew MD, Spindler SR. Discrete positive and negative thyroid hormone-responsive transcription regulatory elements of the rat growth hormone gene. J Biol Chem 1987; 262:5659. 68. Flug F, Copp RP, Casanova J, et al. Cis-acting elements of the rat growth hormone gene which mediate basal and regulated expression by thyroid hormone. J Biol Chem 1987; 262:6373. 69. Jantzen HM, Strahle U, Gloss B, et al. Cooperativity of glucocorticoid response elements located far upstream of the tyrosine aminotransferase gene. Nature 1987; 49:29. 70. Weinberger C, Thompson CC, Ong ES, et al. The c-erb-A gene encodes a thyroid hormone receptor. Nature 1986; 324:64 1. 71. Green S, Chambon P. A super family of potentially oncogenic hormone receptors. Nature 1986; 324:615. 72. Brown DD. The role of stable complexes that repress and activate eucary¬ otic genes. Cell 1984; 37:359. 73. von Hippel PH, Bear DG, Morgan WD, McSwiggen JA. Protein-nucleic acid interactions in transcription: a molecular analysis. Annu Rev Biochem 1984; 53:389. 74. Harrison SC. A structural taxonomy of DNA-binding domains. Nature 1991; 353:715. 75. Pabo CO. Transcription factors: structural families and principles of DNA recognition. Annu Rev Biochem 1992; 61:1053. 76. Murdoch GH, Franco R, Evans RM, Rosenfeld RG. Polypeptide hormone regulation of gene expression. Thyrotropin-releasing hormone rapidly stimulates both transcription of the prolactin and the phosphorylation of a specific nuclear protein. J Biol Chem 1983; 258:15329. 77. Montminy MR, Sevarino KA, Wagner JA, et al. Identification of a cyclic AMP responsive element within the rat somatostatin gene. Proc Natl Acad Sci U S A 1986; 83:6682. 78. Hunter T, Karin M. The regulation of transcription by phosphorylation. Cell 1992; 70:375. 79. Habener JF. Cyclic AMP response element binding proteins: a cornucopia of transcription factors. Mol Endocrinol 1990; 4:1087. 80. Kadonaga JT, Tjian R. Affinity purification of sequence-specific DNA-bind¬ ing proteins. Proc Natl Acad Sci U S A 1986; 83:5889. 81. Brent R. Repression of transcription in yeast. Cell 1985; 42:3.

36

PARTI: GENERAL PRINCIPLES OF ENDOCRINOLOGY

82. Diamond MI, Miner JN, Yoshinaga SK, Yamamoto KR. Transcriptional fac¬ tor interactions: selectors of positive and negative regulation from a single DNA element. Science 1990; 249:1266. 83. Guarente L. Yeast promoters: positive and negative elements. Cell 1984; 36:799. 84. Jones NC. Negative regulation of enhancers. Nature 1986; 321:202. 85. Maniatis T, Goodboum S, Fischer JA. Regulation of inducible and tissuespecific gene expression. Science 1987; 236:1237. 86. Yanofsky C. Transcription attenuation. J Biol Chem 1988; 263:609. 87. Brock ML, Shapiro DJ. Estrogen stabilizes vitellogenin mRNA against cyto¬ plasmic degradation. Cell 1983; 34:207. 88. Guyette WA, Matusik RJ, Rosen JM. Prolactin-mediated transcriptional and post-transcriptional control of casein gene expression. Cell 1979; 17:1013. 89. Krane IM, Spindel ER, Chin WW. Thyroid hormone decreases the stability and the poly(A) tract length of rat thyrotropin fl-subunit messenger RNA. Mol Endocrinol 1991; 5:469. 90. Koenig RJ, Lazar MA, Hodin RA, et al. Inhibition of thyroid hormone action by a non-hormone binding c-erbA protein generated by alternative mRNA splicing. Nature 1989; 337:659. 91. Chew SL. Alternative splicing of mRNA as a mode of endocrine regulation. Trends Endocrinol Metab 1997; 8:405. 92. Douglass J, Civelli O, Herbert E. Polyprotein gene expression: generation of diversity of neuroendocrine peptides. Annu Rev Biochem 1984; 53:665. 93. Wilson HE, White A. Prohormones: their clinical relevance. Trends Endo¬ crinol Metab 1998; 9:396.

CHAPTER

4

HORMONAL ACTION DARYL K. GRANNER

GENERAL FEATURES OF HORMONE SYSTEMS AND HISTORICAL PERSPECTIVE Multicellular organisms use intercellular communication mech¬ anisms to ensure their survival by coordinating the responses necessary for adjusting to constantly changing external and internal environments. Two systems comprising several highly differentiated tissues have evolved to serve these functions. One is the nervous system, and the other is the endocrine sys¬ tem, which classically has been viewed as using mobile hor¬ monal messages that are secreted from one gland or tissue to act on a distant tissue. There is an exquisite convergence of these regulatory systems. For example, neural regulation of the endocrine system is important; many neurotransmitters resem¬ ble hormones in their synthesis, release, transport, and mecha¬ nism of action; and many hormones are synthesized in the nervous system (see Chap. 175). The focus of this chapter is the endocrine system and how hormones work. The word hormone is derived from a Greek term that means to arouse to activity. Classically defined, a hormone is a sub¬ stance that is synthesized in one organ and transported by the circulatory system to act on another tissue. However, this origi¬ nal description is too restrictive, because hormones can act on adjacent cells (i.e., paracrine action) and on the cell in which they were synthesized (i.e., autocrine action) without entering the circulation. Early studies concentrated on defining the endocrine action of hormones by removing or ablating an organ to localize the site of production. An extract of the tissue was then used to restore the function, and this served as a bioassay for subse¬ quent purification of the hormone and the elucidation of physi¬ ologic and biochemical actions. This classic era of the study of 'mrmonal action was descriptive. During this period, many hormones were discovered, and their major effects were de: ned. Because it was assumed that hormones had a unique

source and a single or predominant action, they were named for the tissue of origin (e.g., thyroid hormone) or for the action (e.g., growth hormone). The next era of investigation of hormonal action was charac¬ terized by the discovery, of many more hormones and by a more detailed analysis of how hormones work. The investiga¬ tion of their functions was aided by methods and ideas previ¬ ously exploited by endocrinologists, including the use of radioisotopes, the concept of turnover, improved means of purifying molecules, and the availability of sophisticated ana¬ lytic machinery. Such studies changed the direction of research in hormonal action from a descriptive (i.e., organ or tissue) to a mechanistic (i.e., molecule or function) approach. Where a mole¬ cule worked was no longer as important as hozo it acted. A sin¬ gle hormone could have hemocrine (i.e., transportation through the blood), paracrine, or autocrine actions but affect the differ¬ ent target cells in a similar way, and some effects could be pro¬ duced by a variety of hormones. For example, naming a single molecule the "growth hormone" was incorrect, because this hormone is but one of several—including the thyroid hor¬ mones, sex hormones, glucocorticoids, insulin, and various growth-promoting polypeptides—that are involved in growth, and growth promotion is only one of the actions of the so-called growth hormone. The principles of hormone synthesis, storage, secretion, transport, metabolism, and feedback control were established during this period. A major contribution was the elaboration of the concept of hormone receptors, and of the properties of spec¬ ificity and selectivity of response, how target cells are defined, how responses are modulated, how signals are transduced from the outside of a cell to its interior, and how hormones can be classified according to their mechanism of action. The techniques of molecular biology and recombinant DNA have been applied to hormonal action with remarkable success. It is now possible to analyze hormonal effects on gene expres¬ sion and to study which few nucleotides of the 3 x 109 in each haploid genome confer the response. Another exciting area is the overlapping spectrum of activity of components of hor¬ monal action systems with nonhormonal proteins. Consider the similar features of the guanosine triphosphate (GTP)-binding proteins involved in the hormone-sensitive adenylate cyclase system with the transforming RAS oncogene family of proteins or with transducin, which is the protein that couples photoacti¬ vation to the visual response.1'2 The homology of plateletderived growth factor (PDGF) gene and the v-sis transforming gene is remarkable, as is the similarity between the insulin and epidermal growth factor receptors, both of which have intrinsic tyrosine kinase activity.3-6 Researchers are exploring the molec¬ ular bases of endocrine diseases, such as pseudohypoparathy¬ roidism, several types of dwarfism. Graves disease, certain types of extreme insulin resistance, testicular feminization, acromegaly, vitamin D resistance, and hereditary nephrogenic diabetes insipidus, to name a few.7-14 This knowledge has chal¬ lenged many of the earlier concepts of hormonal action and endocrine disease.

TARGET CELL CONCEPT There are -200 types of differentiated cells in humans. Only a few produce hormones, but virtually all of the 75 trillion cells in a human body are targets of one or more of the -50 known hor¬ mones. The concept of target cells is undergoing redefinition. It was thought that hormones affected a single cell type, or only a few kinds of cells, and that a hormone elicited a unique bio¬ chemical or physiologic action. For example, it was presumed that thyroid-stimulating hormone (TSH) stimulated thyroid growth and thyroid hormonogenesis; adrenocorticotropic hor¬ mone (ACTH, also called corticotropin) enhanced growth and function of the adrenal cortex; glucagon increased hepatic glu-

Ch. 4: Hormonal Action cose production; and luteinizing hormone (LH) stimulated gonadal steroidogenesis. However, these same hormones also stimulate lipolysis in adipose cells.15 Although the physiologic importance of this effect is unclear, the concept of unique sites of actions of these hormones is untenable. A more relevant exam¬ ple is that of insulin, which effects various responses in different cells and occasionally influences different processes within the same cell. It enhances glucose uptake and oxidation in muscle, lipogenesis in fat, amino acid transport in liver and lympho¬ cytes, and protein synthesis in liver and muscle. These and other examples necessitated a reevaluation of the target cell concept. With the delineation of specific cell-surface and intracellular hormone receptors, the definition of a target has been expanded to include any cell in which the hormone binds to its receptor, whether or not a biochemical or physiologic response has been determined. This definition also is imperfect, but it has heuris¬ tic merit, because it presumes that not all actions of hormones have been elucidated. The response of a target cell is determined by the differenti¬ ated state of the cell, and a cell can have several responses to a single hormone. Cells can respond to a hormone in a hemocrine, paracrine, or autocrine manner. An example is the hormone gastrin-releasing peptide (also called mammalian bombesin). Gastrin-releasing peptide has hemocrine and paracrine actions in the gut but is produced by and stimulates the growth of small cell carcinoma cells of the lung.16 Several factors determine the overall response of a target cell to a hormone. The concentration of a hormone around the tar¬ get cell depends on the rate of synthesis and secretion of the hormone, the proximity of target and source, the associationdissociation constants of the hormone with specific plasma carrier proteins, the rate of conversion of an inactive or suboptimally active form of the hormone into the active form, and the rate of clearance of the hormone from blood by other tissues or by degradation or excretion. The actual response to the hor¬ mone depends on the relative activity and state of occupancy, or both, of the specific hormone receptors on the plasma mem¬ brane or within the cytoplasm or nucleus; the metabolism of the hormone within the target cell; the presence of other factors within the target cell that are necessary for the hormone response; and postreceptor desensitization of the cell. Alter¬ ations of any of these processes can change the hormonal effect on a given target cell and must be considered in addition to the classic feedback loops.

HORMONE RECEPTORS GENERAL FEATURES One of the major challenges in making the hormone-based communication system work is depicted in Figure 4-1. Hor¬ mone concentrations are very low in the extracellular fluid, generally in the range of 10'15 to 10-9 M. This is much lower than that of the many structurally similar molecules (e.g., ster¬ ols, amino acids, peptides) and other molecules that circulate at concentrations in the 10-5 to 10-3 M range. Target cells must identify the various hormones present in small amounts and differentiate a given hormone from the 106- to 109-fold excess of other, often closely related, molecules. This high degree of discrimination is provided by cell-associated recognition mole¬ cules called receptors. Hormones initiate their bioeffects by binding to specific receptors, and because any effective control system must provide a means of stopping a response, hormoneinduced actions usually terminate after the effector dissociates from the receptor. A target cell is defined by its ability to bind a given hormone selectively by means of a receptor, an interaction that is often quantitated using radioactive ligands that mimic hormone binding. Several features of this interaction are important. The

37

"V

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+

O

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150-200 ng/mL) confirm the clinical diagnosis of a prolactin-producing pituitary adenoma. Prolactin-producing adenomas can occur at any age but most often are diagnosed in young women. Based on surgical mate¬ rial, women are more frequently affected than men, but in unse¬ lected adult autopsies, no sex difference can be demonstrated. Since the introduction of bromocriptine therapy, the number of prolactin-producing tumors treated surgically has declined. Patients with prolactin cell adenomas can be effectively treated with various dopaminergic agonists. Depending on the sex of the patient, the treatment abolishes the amenorrhea, stops the galactorrhea, increases the libido, and cures the infertility (see Chap. 13); serum prolactin levels show a marked decline. The tumor regresses, as assessed by various imaging techniques. Local symptoms, such as visual disturbances, often disappear. The effects of treatment are reversible; if drug administration is discontinued, the tumor regrows, serum prolactin levels rise, and clinical symptoms reappear. As a result of bromocriptine medication, the tumor cells themselves decrease in size, indicat¬ ing that tumor regression is mainly a result of a reduction in cell volume. In addition to an overall reduction in cytoplasmic vol¬ ume, there also is a significant reduction in the volume density of the hormone's synthetic and secretory apparatus (rough endoplasmic reticulum and Golgi complex). Overall, this gives a hypercellular appearance to the tumor. The tumoristatic effects of bromocriptine are especially well visualized by electron microscopic examination, in which cells become irregularly shaped, contain heterochromatic nuclei, and maintain a scant cytoplasm with markedly involuted Golgi complexes. The size and volume density of secretory granules remain unaffected. All these ultrastructural changes are fully reversible on discontinua¬ tion of the drug. Prolonged exposure to bromocriptine also may result in fibrotic change within the tumor, an alteration that may adversely complicate surgical extirpation. The presence of cell necrosis after bromocriptine administration also has been reported. Whether bromocriptine is cytotoxic is difficult to assess because foci of necrosis, hemorrhage, and fibrosis may occur in prolactin-producing adenomas without dopaminergic agonist treatment. histopathology. Histologically, prolactin cell adenomas most often are chromophobic or slightly acidophilic, exhibiting distinct prolactin immunostaining in the Golgi complex and secretory granules. On electron microscopic examination, pro¬ lactin cell adenomas can be separated into densely granulated and sparsely granulated variants. Densely granulated prolactin cell adenomas are rare. The adenoma cells resemble nontumor¬ ous resting prolactin cells and are characterized by prominent rough-surfaced endoplasmic reticulum, conspicuous Golgi complexes, and numerous spherical, oval, or irregularly shaped secretory granules measuring up to 700 nm. Sparsely granu¬ lated prolactin cell adenomas are the most frequent tumor type in the human pituitary. The adenoma cells possess a prominent rough-surfaced endoplasmic reticulum, a conspicuous Golgi apparatus, and sparse, spherical, and oval, or irregularly shaped, evenly electron-dense secretory granules measuring 150 to 300 nm (Fig. 11-9). The rough-surfaced endoplasmic reticulum membranes form concentric cytoplasmic whorls, called nebenkerns. Another characteristic ultrastructural feature of prolactin cell adenomas is the presence of misplaced exocytosis—extrusion of secretory granules on the lateral side of the cell, distant from capillaries and intercellular extensions of the basement membrane. Granule extrusion also occurs on the cap¬ illary side of prolactin cells. Prolactin cell adenomas may produce an amyloid-like sub¬ stance and exhibit various degrees of calcification, sometimes so extensive as to be visible with imaging techniques. Amyloid deposition and calcification are characteristic but not pathogno¬ monic; they most frequently occur in prolactin cell adenomas but occasionally may be present in other adenoma types.

120

PART II: THE ENDOCRINE BRAIN AND PITUITARY GLAND

FIGURE 11-9. Sparsely granulated prolactin cell adenoma with promi¬ nent rough-surfaced endoplasmic reticulum membranes (arrows) and misplaced exocytosis (extrusion of secretory granules into the extracel¬ lular space, distant from the basement membrane; arrowheads). x7800

Corticotrope Adenomas. Corticotrope adenomas pro¬ duce ACTH and other peptides of the pro-opiomelanocortin molecule, such as (3-LPH and endorphins. They may secrete hor¬ mones excessively and may be associated with Cushing disease or Nelson syndrome (see Chap. 75). In Cushing disease, the excess ACTH stimulates the adrenal cortex and causes various degrees of hypercortisolism. Historically, bilateral adrenalectomy was undertaken because the ACTH-producing pituitary ade¬ noma either was unrecognized or was unsuccessfully treated. With the development of increasingly precise imaging technol¬ ogy and superior microsurgical techniques, bilateral adrenalec¬ tomy is now a procedure of last resort that is rarely required in the management of Cushing disease. Accordingly, new cases of Nelson syndrome are becoming increasingly uncommon. When bilateral adrenalectomy is necessary to control refractory Cush¬ ing disease, the clinical picture is characteristic. As hypercortiso¬ lism regresses, the tumor frequently behaves in a "dismhibited" fashion, tending to grow more rapidly and to produce large amounts of ACTH and other pro-opiomelanocortin-derived peptides. Patients become hyperpigmented, and the tumor tends to be far more aggressive and invasive than are those corticotropic adenomas that have an intact pituitary-adrenal axis. Further¬ more, only a few patients with Nelson syndrome are cured by surgery and radiotherapy; up to 20% of these patients succumb to uncontrolled local tumor growth. Such aggressive behavior has been ascribed to the loss of negative glucocorticoid feedback resulting from the adrenalectomy; this suggests that corticotrope adenomas, as a whole, are not entirely autonomous, but are sub¬ ject to feedback and modulation of their secretory activity and growth rate by glucocorticoid hormones. Importantly, more than 80% of the adenomas responsible for Cushing disease are microadenomas. Because many are only a few millimeters in diameter, they frequently evade detection, even with the most sophisticated imaging techniques. Given their small size, these tumors present technical difficulties, both to surgeons and to pathologists. From a surgical standpoint, corticotrope microadenomas rarely are situated on the surface of the gland and are exposed only after a thorough dissection of a seemingly normal gland. Most arise in the midline of the pitu¬ itary, within the so-called mucoid wedge, an area in which corticohopes are most numerous. The surgical specimen provided to the pathologist frequently is small and fragmented; as a

FIGURE 11-10. Electron microscopic appearance of densely granulated corticotrope cell adenoma. Note the irregular or dented shape of secre¬ tory granules and bundles of type I filaments (arrows). x9800

result, considerable patience and skill are required to differenti¬ ate adenomatous elements from the normal glandular tissue. Whereas 10% to 15% of microadenomas demonstrate local invasion, fully 60% of macroadenomas are grossly invasive. HlSTOPATHOLOGY. Histologically, corticotrope adenomas usually are basophilic and exhibit various degrees of PAS and lead-hematoxylin positivity. Immunoperoxidase staining reveals ACTH and other fragments of the pro-opiomelanocortin mole¬ cule in the cytoplasm of adenoma cells. On electron microscopic examination, adenomatous corticotropes often resemble nonadenomatous corticotropes; they possess well-developed, rough¬ surfaced endoplasmic reticulum, prominent Golgi apparatuses, and numerous spherical secretory granules that vary in electron density, often line up along the cell membrane, and measure 250 to 700 nm (Fig. 11-10). In corticotrope adenomas removed from patients with Cushing disease, bundles of microfilaments usu¬ ally are present, whereas in adenomas removed from patients with Nelson syndrome, microfilaments are inconspicuous or absent. Otherwise, no ultrastructural differences exist between the corticotrope adenomas of patients with Cushing disease and Nelson syndrome. A few actively secreting corticotrope adenomas are chro¬ mophobic and possess only sparse, fine, PAS-positive cytoplas¬ mic granules. These tumors immunostain for ACTH and related peptides, indicating that they arise in and consist of cor¬ ticotropes. On electron microscopic examination, chromopho¬ bic corticotrope adenoma cells are sparsely granulated and appear less differentiated than their basophilic, densely granu¬ lated counterparts. Chromophobic tumors usually are larger, grow faster, tend to be invasive, and recur more frequently than basophilic adenomas, which generally are small, measuring only a few millimeters in diameter. Silent Corticotrope Adenomas.1’210-22 Silent corticotrope adenomas immunostain for ACTH and related peptides but are not associated with clinical or biochemical evidence of ACTH excess. On light microscopic examination, these tumors are basophilic or chromophobic, show various degrees of PAS posi¬ tivity, and immunostain for ACTH, (1-LPH, and endorphins. On electron microscopic examination, silent corticotrope cell ade¬ nomas are a heterogeneous group. In some cases, the ultrastruc¬ tural features of tumor cells are indistinguishable from those of actively secreting tumors, such as are associated with Cushing

Ch. 11: Morphology of the Pituitary in Health and Disease disease or Nelson syndrome. In other cases, an increase in the size and number of lysosomes, crinophagy (uptake of secretory granules by lysosomes), and marked underdevelopment or involution of the Golgi apparatus suggest a defect in various steps involving hormone synthesis, packaging, or discharge. Two distinct silent corticotrope adenomas, designated silent corticotrope adenomas subtypes I and II, have been identified as spe¬ cific clinicopathologic entities. A third, no longer considered corticotropic in nature, is designated silent subtype III. For the most part, silent subtypes I through III present clinically as large and invasive nonfunctioning sellar masses.1,17 In some instances, hyperprolactinemia is present, sometimes at a level higher than that attributable to the stalk section effect. Accordingly, it has been suggested that some silent subtypes may facil¬ itate prolactin release from nontumorous lactotropes or are themselves capable of prolactin secretion. These findings are particularly applicable to silent subtype III, a tumor frequently seen in women, and therefore often is diagnosed before opera¬ tion as a prolactinoma. The reason that some silent subtype III tumors have been responsible for acromegaly remains a total mystery. A peculiarity of silent subtype tumors relates to their propensity to undergo apoplectic hemorrhage. In the experi¬ ence of the authors, more than 40% of silent subtype adenomas presented in this fashion.22 In all, the silent subtype tumors are enigmatic entities, whose cytogenesis and overall biology war¬ rants further study. Thyrotrope Adenomas. With fewer than 100 cases reported, TSH-secreting adenomas are the least common pituitary tumor phenotype, accounting for only 1% of all pituitary adenomas. Of reported cases, most thyrotrope adenomas have been large, aggressive macroadenomas, both compressive and invasive of surrounding structures.73 Usually, the accompanying clinical history is remarkable for some form of thyroid dysfunction. It once was believed that most arose in the context of long¬ standing primary hypothyroidism, presumably by way of feed¬ back inhibitory loss, induction of thyrotrope hyperplasia, and, later, adenoma formation. Although such a perspective was compatible with earlier experimental studies in which thy¬ roidectomy induced pituitary thyrotrope adenomas in rodents, careful clinicopathologic correlations of human thyrotrope ade¬ nomas indicate an alternate sequence of likely events.10,23 In many patients, the initial manifestations appear to be those of hyperthyroidism and goiter, events wholly compatible with TSH hypersecretion by the tumor. Because secondary (i.e., pitu¬ itary-dependent) hyperthyroidism previously was not a wellrecognized condition, many such patients were incorrectly thought to have primary hyperthyroidism and were subjected to some form of thyroid ablation. This served to ameliorate symptoms, but sometimes was followed later by accelerated tumor growth, optic nerve compression, or recurrence of the hyperthyroid state. Only then was the pituitary correctly identi¬ fied as the site of pathologic involvement. The invasive nature of these tumors appears to be related to two factors, the first of which is the typical diagnostic delay. A more cogent factor, however, is the loss of feedback inhibition. In the same way that end-organ ablation contributes to tumor aggressiveness in the context of Nelson syndrome, similar disinhibiting influences may be operative in the progression of TSH adenomas in the setting of prior thyroidectomy. The routine availability of sen¬ sitive TSH assays coupled with general awareness of the thyro¬ trope adenoma as a potential, though rare, cause of hyperthyroidism should permit more expeditious diagnosis of this tumor type, perhaps while it is still in the microadenoma stage.67 Furthermore, thyrotrope adenomas commonly cosecrete in excess a free a subunit that, if present, may be a helpful diag¬ nostic clue suggesting a pituitary source over a primary thyroid cause of hyperthyroidism (see Chaps. 15 and 42). HlSTOPATHOLOGV. On light microscopic examination, thyro¬ trope adenomas are chromophobic, containing a few small cytoplasmic granules mainly at the cell periphery that stain for

121

PAS, aldehyde fuchsin, and aldehyde thionin. TSH can be dem¬ onstrated immunocytologically in the cytoplasm of the ade¬ noma cells. Occasionally, immunostaining shows only slight or no TSH immunopositivity, suggesting that little hormone is stored in the cytoplasm or that abnormal TSH is produced that is not immunoreactive but may have bioactivity. On electron microscopic examination, thyrotrope adenomas consist of elon¬ gated, angular, or irregular cells with long cytoplasmic pro¬ cesses, scanty rough-surfaced endoplasmic reticulum, an inconspicuous Golgi apparatus, and numerous microtubules. Secretory granules are sparse and spherical, vary slightly in electron density, line up along the cell membrane, and measure 50 to 200 nm. Thyrotrope adenomas often differ in their ultrastructural appearance from nontumorous thyrotropes. In some cases, they consist of highly differentiated thyrotropes with an abundant, slightly dilated, rough-surfaced endoplasmic reticu¬ lum, a conspicuous Golgi apparatus, and varying numbers of secretory granules in the range of 50 to 250 nm. Gonadotrope Adenomas. Gonadotrope adenomas pro¬ duce FSH, FSH and LH, or, rarely, LH alone. They are identified more frequently in older patients. The clinical manifestations of gonadotrope cell adenomas are not clearly defined; patients may show varying degrees of hypogonadism, decreased libido, impotence, and elevated serum FSH/LH concentrations. histopathology. On light microscopic examination, gonado¬ trope adenomas appear chromophobic and may contain sparse PAS-positive cytoplasmic granules. Immunostaining reveals FSH, LH, or both in the cytoplasm of adenoma cells. On elec¬ tron microscopic examination, sexual dichotomy is evident. In men, adenomatous gonadotropes usually are less differentiated and have a few rough-surfaced endoplasmic reticulum mem¬ branes, a moderately developed Golgi apparatus, several microtubules, and sparse, spherical secretory granules that vary slightly in electron density, line up along the cell mem¬ brane, and measure 100 to 300 nm. In women, adenomatous gonadotropes are more differentiated and resemble their non¬ tumorous counterparts. The prominent honeycomb-like Golgi complex consists of several dilated sacculi and vesicles contain¬ ing a few immature secretory granules. Secretory granules are sparse and randomly distributed, vary slightly in electron den¬ sity, and measure 50 to 150 nm. Null Cell Adenomas. Null cell adenomas have no histo¬ logic, immunocytologic, or electron microscopic markers and are not associated clinically and biochemically with any known hormone excess. Although these tumors are endocrinologically inactive and contain no known hormones, they possess the organelles necessary for hormone secretion and have secretory granules.1 It may be that these tumors produce biologically inactive hormone fragments, precursor molecules, or hormones unidentified at present. Together, null cell adenomas and their oncocytic variants (dis¬ cussed later) account for almost one-fourth of all pituitary ade¬ nomas. Both are nonfunctioning sellar masses and are typically seen during middle or old age. They tend to be slow-growing lesions, with some likely growing for years subclinically before manifesting themselves clinically. Despite the regularity with which these tumors are encountered in clinical practice, and the fact that their existence has been known for almost two decades, fundamental questions concerning their causation, cytogenesis, and biology remain unanswered.2,10,74 That these tumors frequently share morphologic similarities with undif¬ ferentiated gonadotrope adenomas has fueled speculation that null cell adenomas may be neoplastic offshoots of gonadotropic lineage. Compelling support for such a notion was provided by the finding that 80% of null cell adenomas and oncocytomas express glycoprotein hormone genes.10 Furthermore, both gonadotropin release and gonadotropin-releasing hormone responsiveness have been demonstrated in null cell adenomas maintained in tissue culture. Alternatively, there is preliminary evidence favoring the existence of nonneoplastic null cells scat-

1 22

PART II: THE ENDOCRINE BRAIN AND PITUITARY GLAND

FIGURE 11-12. Pituitary oncocytoma showing abundance of mito¬ chondria. x7000

FIGURE 11-11. Null cell adenoma. The small adenoma cells contain poorly developed cytoplasmic organelles and sparse, small secretory granules. x9800

tered throughout the normal pituitary.75 It is speculated that such "normal" null cells may be transitional, undifferentiated, or precursor cells that are capable of shifting from hormonally inactive to hormonally active states. Should null cells be vali¬ dated as cellular constituents of the normal pituitary, null cell adenomas could be envisioned as their neoplastic derivatives. HISTOPATHOLOGIC CHARACTERISTICS. On light microscopic examination, null cell adenomas are chromophobic; immunocytologically, they contain no adenohypophysial hormones. In many tumors, however, small groups of adenoma cells or ran¬ domly scattered individual cells immunostain for one or more pituitary hormones, most frequently FSH or the a subunit, less frequently TSH, LH, and prolactin, and occasionally GH or ACTH. Consistent with these findings, in vitro studies reveal that most null cell adenomas produce FSH, TSH, LH, or the a subunit; these hormones can be demonstrated by radioimmunoassay in tumor culture. It is not clear whether this means multidirectional differentiation from an uncommitted precursor, tumor cell heterogeneity, or gradual dedifferentiation. On electron micro¬ scopic examination, null cell adenomas are characterized by closely apposed, polyhedral or irregularly shaped cells with pleo¬ morphic or indented nuclei and poorly developed cytoplasm possessing a few rough endoplasmic reticulum profiles, a moder¬ ately developed Golgi apparatus, and microtubules, which may be abundant in some cells. The secretory granules are sparse and spherical, vary slightly in electron density, line up along the cell membrane, and measure 100 to 200 nm (Fig. 11-11). Oncocytic transformation (i.e., the increase of cytoplasmic volume occupied by mitochondria) is common in null cell adenomas. Because of the lack of endocrine symptoms, some null cell adenomas are recognized only when they enlarge and spread outside the sella, causing local symptoms such as visual distur¬ bances, headache, or injury of cranial nerves. Oncocytomas. Oncocytomas represent the oncocytic vari¬ ant of null cell adenomas. The term oncocytosis is used to describe tumor cells that exhibit intracellular mitochondrial accumulation. Thus, oncocytomas differ from null cell ade¬ nomas only insofar as the cells of the former contain massive numbers of large mitochondria. Both tumors share a similar cli iical profile that is dominated by the neurologic and endocrinologic sequelae of an expansile, nonfunctioning sellar mass.

Like null cell adenomas, oncocytomas are unaccompanied by clinical or biochemical evidence of oversecretion of adenohypo¬ physial hormones, have no morphologic markers that would reveal their cytogenesis, occur most frequently in older men and women, and are rarely diagnosed in patients younger than 40 years. HISTOPATHOLOGY. On light microscopic examination, onco¬ cytomas are chromophobic or acidophilic. The acidophilia is not the result of staining of secretory granules but of the uptake of acid dyes by accumulating mitochondria. Immunostaining of oncocytoma cells fails to demonstrate pituitary hormones. In many cases, however, small groups of adenoma cells or ran¬ domly scattered individual cells immunostain for FSH, the alpha subunit, LH, or TSH, indicating that oncocytoma cells do not lose their potential to produce hormones or that they can differentiate to a hormone-producing cell line. Electron micro¬ scopic examination reveals abundant cytoplasmic mitochon¬ dria (Fig. 11-12), which may be extensive, filling as much as 50% of the cytoplasm (compared with ~8% normally). In some tumors, a transition can be seen between null cell adenoma and oncocytoma. The authors use the term oncocytoma only when abundant mitochondria are evident in practically every ade¬ noma cell. Plurihormonal Adenomas. Plurihormonal adenomas pro¬ duce more than one hormone and can be divided into monomor¬ phous and plurimorphous types. Monomorphous plurihormonal adenomas consist of one morphologically distinct cell type that produces two or more hormones; the cell may differ morpho¬ logically from known adenohypophysial cells. Plurimorphous plurihormonal adenomas are composed of two or more mor¬ phologically distinct cell types, each producing different hor¬ mones; they are similar in ultrastructural appearance to their nontumorous counterparts. Immunocytologic techniques are required to establish the diagnosis. Electron microscopic examination may fail to reveal the cellular origin of the ade¬ noma because ultrastructural features may not be distinct or the tumor may consist of cells not recognized in nontumorous adenohypophyses. In the experience of the authors, 12% of surgically removed pituitary adenomas are plurihormonal. The most frequent hor¬ monal combination produced is GH and prolactin. Three mor¬ phologically distinct adenoma types produce GH and prolactin simultaneously: acidophil stem cell adenoma, mammosomatotrope adenoma, and mixed GH cell-prolactin adenoma. acidophil STEM cell ADENOMAS. Acidophil stem cell ade¬ nomas grow rapidly, often spreading into neighboring tissues.

Ch. 11: Morphology of the Pituitary in Health and Disease They are associated with various degrees of hyperprolactin¬ emia. In some patients, clinical features of acromegaly may be apparent despite normal serum GH levels. Acidophil stem cell adenomas are monomorphous, bihormonal tumors that consist of one cell type, which is assumed to represent the common pro¬ genitor of GH cells and prolactin cells. Immunocytologic tech¬ niques demonstrate both prolactin and GH in the cytoplasm of the same adenoma cells. Immunostaining for GH often is weak or absent. On electron microscopic examination, acidophil stem cell adenomas are composed of closely apposed elongated cells with irregular nuclei and well-developed cytoplasm containing dispersed, short profiles of rough-surfaced endoplasmic reticu¬ lum, an inconspicuous Golgi apparatus, fibrous bodies contain¬ ing microfilaments and smooth-walled tubules, multiple centrioles and cilia, and sparse, irregular secretory granules measuring 100 to 300 nm. Some exocytosis may be evident. Oncocytic change and mitochondrial gigantism occur in most tumors. The correlation between tumor size and blood prolactin concentrations, which is apparent in patients with sparsely granulated prolactin cell adenomas, is often absent in patients with acidophil stem cell adenomas; relatively large tumors may be accompanied by only slight or moderate hyperprolactinemia. MAMMOSOMATOTROPE CELL adenomas. Mammosomatotrope cell adenomas are slowly growing tumors accompanied by ele¬ vated serum GH concentrations, acromegaly, and, in some cases, mild hyperprolactinemia. These monomorphous, bihor¬ monal tumors consist of acidophilic cells. Immunocytologic methods demonstrate GH and prolactin in the cytoplasm of the same adenoma cells. On electron microscopic examination, the adenoma cells appear to be well differentiated and resemble densely granulated GH cells. The secretory granules are often irregular; they may be evenly electron dense or have a mottled appearance, and they measure 200 to 2000 nm. Exocytosis and large extracellular deposits of secretory material are character¬ istic features. MIXED GROWTH HORMONE CELL-PROLACTIN CELL ADENOMAS. Mixed GH cell-prolactin cell adenomas are associated with elevated serum GH levels, acromegaly, hyperprolactinemia, and, occa¬ sionally, galactorrhea, amenorrhea, decreased libido, and impo¬ tence. These bimorphous, bihormonal tumors are composed of two morphologically distinct cell types: densely or sparsely granulated GH cells and prolactin cells. The two cell types form small groups; in several areas, individual cells are interspersed. Immunostaining demonstrates GH and prolactin in the two dif¬ ferent cell populations. Electron microscopic examination shows two morphologically distinct cell types. Every combina¬ tion may occur; most frequently, densely granulated GH cells and sparsely granulated prolactin cells are identified. UNUSUAL PLURIHORMONAL ADENOMAS. Occasional, unusual plurihormonal pituitary adenomas produce bizarre combina¬ tions of two or more hormones, such as GH and TSH; prolactin and TSH; GH, prolactin, and TSH; and, less frequently, GH, pro¬ lactin, and ACTH, or GH, prolactin, FSH/LH, and the a subunit. Such tumors may be monomorphous or plurimorphous. The cell type or types constituting the tumors often cannot be identified, even with detailed electron microscopic investigation. In a few cases, however, two or more ultrastructurally distinct cell types resembling their nontumorous counterparts can be recognized. The hormone content and ultrastructural features of adenomas cannot always be correlated. On light microscopic examination, the unusual plurihor¬ monal adenomas consist of chromophobic, acidophilic, or baso¬ philic cells, or a mixture of cells that stain differently with different histologic techniques. Clinically and biochemically, the secretion of several hormones may be apparent; acromegaly may be accompanied by hyperthyroidism, hypercorticism, or hyperprolactinemia. Some components may be silent. Immu¬ nostaining demonstrates hormones in the cell cytoplasm, but hormone production is not always reflected in hypersecretory symptoms clinically, or in increased serum hormone levels.

123

Plurihormonal adenomas, which are difficult to classify, clearly show that the one cell-one hormone theory, which has dominated pituitary cytophysiology and cytopathology for many years, is oversimplified and requires revision. Beyond the conceptual importance underlying the phenom¬ enon of plurihormonality is a potentially important clinical issue. Although unproven, there is some suggestion that some plurihormonal tumors behave more aggressively than do their monohormonal counterparts.10-75'76 In the case of other endo¬ crine tumors (e.g., pancreatic tumors, medullary carcinoma of the thyroid), plurihormonal tumors are thought to portend a more malignant course than that of monohormonal tumors. Evidence in support of a similar occurrence in the pituitary remains inconclusive. It is known, however, that most plurihor¬ monal pituitary adenomas are macroadenomas at presentation, even in the presence of a hypersecretory syndrome. Further¬ more, -50% of all plurihormonal pituitary adenomas are grossly invasive at the time of diagnosis.76

MALIGNANT PITUITARY LESIONS PRIMARY MALIGNANT NEOPLASMS Primary malignant neoplasms of the hypophysis include carci¬ nomas and sarcomas; they are extremely rare. Primary Adenohypophysial Carcinomas. Primary adeno¬ hypophysial carcinomas, which are derived from anterior pitu¬ itary cells, may secrete GH, prolactin, or ACTH or may not be associated with hormone production. They are rare and were discussed earlier. Electron microscopic and immunocytologic studies fail to distinguish between benign and malignant tumors. Sarcomas. Sarcomas of the adenohypophysis include fibrosarcoma, osteosarcoma, and undifferentiated sarcoma. With few exceptions, virtually all have occurred after radiother¬ apy for either pituitary adenoma, craniopharyngioma, or retin¬ oblastoma. Fibrosarcomas, in particular, are most commonly the consequence of radiotherapy to a pituitary adenoma. Histo¬ logically, these tumors exhibit marked cellular and nuclear pleomorphism, replete with mitotic figures, areas of necrosis, and hemorrhage. In many instances, the sarcomatous component can be seen to be intimately admixed within the substance of a persistent pituitary adenoma. Thus, it has been suggested that radiotherapy induces fibrosarcoma formation by transforming fibroblastic elements within the original adenoma. The latency period for sarcomatous transformation is variable; an average period of 11 years has been reported.10-19 Postirradiation sarco¬ mas are virtually always high-grade malignancies typified by rapid growth, relentless local invasion, and a survival period rarely exceeding a few months. SECONDARY NEOPLASMS Secondary neoplasms of the pituitary most often are found inci¬ dentally at autopsy and are not associated with clinical symp¬ toms or biochemical abnormalities. They occur in 1% to 5% of patients with cancer. The most commonly observed endocrine abnormality is diabetes insipidus (see Chap. 26), which occurs in patients with metastatic carcinoma of the posterior lobe, hypophysial stalk, or hypothalamus. Compression or destruc¬ tion of the production site of hypothalamic releasing and sup¬ pressing hormones or interference with adenohypophysial blood flow (either by blocking transport of hypothalamic hor¬ mones to the adenohypophysis or by inducing ischemia) also may account for the development of endocrine symptoms. Local symptoms may be apparent; however, anterior hypopitu¬ itarism is rare because a substantial part of the adenohypophy¬ sis must be destroyed before a decrease in adenohypophysial hormone secretion becomes manifest clinically and biochemi¬ cally. Hypophysial metastases usually occur at an advanced stage of neoplastic disease, when the malignant process

124

PART II: THE ENDOCRINE BRAIN AND PITUITARY GLAND

involves several organs. Affected patients rarely live long enough to develop anterior hypopituitarism. Only rarely are symptoms of pituitary metastases the first manifestation of a systemic malignancy.77 Hypophysial metastases may come from different primary sources, such as carcinomas of the bronchus, colon, prostate, larynx, or kidney; malignant melanoma; sarcomas; and hema¬ tologic malignancies. In the last of these categories, plasmacy¬ toma is notorious for its periodic presentation in the sellar region. Many plasmacytomas of the sellar region have occurred in the absence of known systemic disease, presenting as seem¬ ingly ordinary pituitary adenomas. Regardless of therapy, most eventually evolve into full-blown multiple myeloma. For women, carcinoma of the breast is the most common primary lesion that metastasizes to the pituitary. In men, carcinoma of the lung is the most culpable primary tumor. Metastases to the posterior lobe are more frequent than to the anterior lobe, presumably because the posterior lobe has a rich direct arterial blood supply. Metastatic tumor deposits usually occur first in the pituitary stalk or posterior lobe and permeate the anterior lobe, either by direct extension from the hypophys¬ ial stalk or posterior lobe, or through the portal circulation, by long or short portal vessels. However, isolated metastases may occur in the anterior lobe, indicating that the secondary tumor in the adenohypophysis is not invariably the result of prior involvement of the posterior lobe and hypophysial stalk; in the genesis of adenohypophysial metastases, routes other than the portal circulation must be considered. Tumor cells may also spread from perihypophysial tissues to the pituitary. In patients with disseminated carcinomatosis, infiltration of long portal vessels by carcinoma cells leads to vascular occlu¬ sion and subsequent ischemia, causing adenohypophysial infarcts. The necrotic foci are not large enough to cause hypopi¬ tuitarism.

CRANIOPHARYNGIOMAS Craniopharyngiomas are histologically benign tumors that are thought to arise from remnants of the Rathke pouch. Such embryonic squamous cell "nests" extend from the tuber cinereum to the pituitary gland, presumably along the track of an incompletely involuted hypophysial-pharyngeal duct. By virtue of their location, craniopharyngiomas simultaneously compromise the function of several intracranial structures and produce numerous clinical effects, including vision loss, anterior and posterior pituitary dysfunction, and increased intracranial pressure. Despite their "benign" nature, craniopharyngiomas can offer considerable resistance to successful treatment. Craniopharyngiomas account for ~3% of intracranial tumors. They primarily affect children, for whom they represent the most common nonglial brain tumor and 10% of all intracranial neoplasms. The age-related incidence of craniopharyngiomas is bimodal, with a major, early peak between 5 and 10 years of age, and a second, smaller peak between 50 and 60 years of age. A slight male preponderance has generally been noted. Topologically, most craniopharyngiomas are suprasellar in location. Those so situated can compress the optic apparatus, cause hydrocephalus by indenting the third ventricle, encroach on hypothalamic structures, distort the infundibulum and pitu¬ itary, and penetrate cerebrospinal fluid spaces to gain access to the middle and posterior cranial fossae. Twenty percent of cra¬ niopharyngiomas originate within the sella, producing sellar enlargement in a fashion similar to that produced by pituitary adenomas. Almost half of all craniopharyngiomas are cystic, 15% are solid, and the remainder are made up of both solid and cystic elements. Although generally well circumscribed, cranio¬ pharyngiomas are not encapsulated lesions and therefore are often tenaciously adherent to basal neural and vascular struc¬ tures. It is this feature of craniopharyngiomas that frequently undermines successful attempts at a safe and curative resection.

FIGURE 11-13. Epithelial component of craniopharyngioma showing tonofilaments (arrows) and absence of secretory granules. x8000

There has been an increasing tendency to view craniopha¬ ryngiomas as being either classically adamantinomatous or papil¬ lary in nature, which is a distinction that some believe to be of clinical, pathologic, and prognostic significance.78 79 Adamantinomatous Craniopharyngioma. Adamantinoma¬ tous craniopharyngioma represents the classic cystic craniopha¬ ryngioma of childhood. It frequently is a bulky, partially calcified tumor that tenaciously insinuates itself around basal brain struc¬ tures. On gross sectioning, it oozes a viscid admixture of shim¬ mering cholesterol crystals and calcific desquamated debris that, in appearance and consistency, often is described as "machinery oil." On light microscopic examination, this variant exhibits an intricate pattern of epithelial growth, including intermixed islands of solid and cystic epithelium within a matrix of variably cellular connective tissue. Nests or cords of columnar and squa¬ mous epithelial cells can be demonstrated. Lymphocytes, mac¬ rophages, clusters of foamy cells, cholesterol crystals, keratin deposits, necrotic debris, and polymorphonuclear leukocytes often are present. Calcification, ranging from areas visible only with a microscope to palpable concretions, is common. Rarely, frank bone formation may be present. Cyst formation is presum¬ ably the result of degeneration of squamous cells, accumulation of keratinous debris, and perivascular stromal degeneration. The results of immunohistochemical studies are conclusively nega¬ tive for adenohypophysial hormones, indicating that craniopha¬ ryngiomas do not originate in adenohypophysial cells and are not capable of hormone production. The results of immunostaining for keratin are positive. Electron microscopic examination shows bundles of tonofilaments, prominent desmosomal attach¬ ments, and an absence of secretory granules (Fig. 11-13). Papillary Craniopharyngioma. The clinical, radiologic, and pathologic profile of papillary craniopharyngioma deviates considerably from that of the conventional adamantinomatous variant.78-79 Accounting for -10% of all craniopharyngiomas, the papillary variant occurs almost exclusively in adults. It usually is suprasellar in location and frequently involves, or arises within, the third ventricle. Most papillary variants are solid or have only a relatively minor cystic component. Papillary craniopharyngio¬ mas are more discretely circumscribed than classic craniopha¬ ryngiomas and lack the calcification and "machinery oil" content so typical of adamantinomatous tumors. Liquid contents, although rarely present, generally are clear. Lacking tenacious adhesions to basal brain structures, the papillary craniopharyn¬ gioma is reputed to be more readily separable from surrounding

Ch. 11: Morphology of the Pituitary in Health and Disease structures. Histologically, it consists of a well-differentiated, albeit less complex, epithelial pattern than that of the adamanti¬ noma tous variant. Prominent, stratified, squamous-lined papil¬ lae, devoid of columnar palisading, microcystic degeneration, calcification, keratinous nodules, and cholesterol clefts, are char¬ acteristic. Whereas the papillary variant is regarded by some as being more amenable to complete and curative resection, not all are in agreement with this view.78-81 Both forms of craniopharyngioma are histologically benign. Mitotic figures and other features of histologic aggressiveness generally are not seen. Despite their histologic benignity, cra¬ niopharyngiomas, particularly the adamantinomatous type, are notorious for their high rate of postoperative recurrence. Even among tumors resected radically, a procedure sometimes accom¬ panied by considerable functional deficit, recurrence rates as high as 25% have been reported. For lesser degrees of resection, recurrence of symptoms is virtually guaranteed, often within 3 years of surgery. Radiotherapy for incompletely excised lesions has proved effective in forestalling recurrence (see Chap. 22). Malignant transformation is exquisitely rare; only a single case of malignant craniopharyngioma has been described. Craniopharyngiomas, both adamantinomatous and papil¬ lary, have been shown to express estrogen receptor mRNA and protein. The significance of this finding remains to be deter¬ mined.82 Rathke Cleft Cysts. Rathke cleft cysts are epithelial cysts apparently derived from remnants of the Rathke pouch. At autopsy, roughly one-fifth of pituitaries contain macroscopic remnants of the Rathke pouch in the form of discontinuous cys¬ tic remnants or microscopic clefts at the interface of the anterior and posterior lobes. Occasionally, these Rathke cleft remnants, as the result of progressive accumulation of colloidal secretions, become sufficiently large and compress surrounding structures. Most cases involving symptoms present as expansile intrasellar masses, occasionally having a suprasellar component. Only exceptionally are they entirely suprasellar in location. Local compressive effects are the basis for presentation in most cases involving symptoms, with headache, hypopituitarism, hyper¬ prolactinemia, visual disturbance, and, rarely, diabetes insipi¬ dus being the principal clinical features.83 Rathke cleft cysts are thin-walled, uniloculate, and filled with fluid, the composition of which ranges from watery to muci¬ nous. The cyst wall frequently is composed of a single layer of cuboidal or columnar, ciliated, or mucin-producing epithelium. Small numbers of adenohypophysial cells also may be evident. Calcification is rare, as is amyloid deposition. Although the epi¬ thelial pattern is considerably less complex than that of cra¬ niopharyngioma, the distinction between these two entities occasionally can be troublesome, emphasizing the importance of generous tissue sampling. Rarely, pituitary adenomas can be found to be admixed with elements of a Rathke cleft cyst. Such biopsy results usually represent the simultaneous sampling of two distinct lesions. Even rarer are more complex lesions, in which Rathke cyst components are intimately associated with adenoma and even squamous metaplasia; such lesions have been termed transitional cell tumors of the pituitary. Whether these are distinct clinicopathologic entities or simply the collision of two distinct lesions remains to be determined. Rathke cleft cysts are definitively treated by drainage and marsupialization of the cyst wall, a procedure that usually results in cure. Recurrences are unusual.

PATHOLOGY OF THE NEUROHYPOPHYSIS AND HYPOTHALAMUS2-4-10-19 Clinically significant diseases of the posterior lobe are rare. Endocrinologically, they can be divided into conditions associ¬ ated with increased or decreased vasopressin secretion. Several abnormalities are unaccompanied by endocrine alterations.

125

INAPPROPRIATE SECRETION OF VASOPRESSIN Inappropriate secretion of vasopressin, or the Schwartz-Bartter syndrome, is the result of vasopressin hypersecretion, either from the posterior pituitary or from extrahypophysial neo¬ plasms (see Chaps. 27 and 219). Renal sodium loss and hyponatremia are characteristic features. Several diseases, including meningitis, myxedema, and cerebral lesions, may be associated with increased vasopressin discharge. Paraneoplas¬ tic ("ectopic") vasopressin secretion may occur in various neo¬ plasms, but is seen mainly in carcinoma of the bronchus. Vasopressin can be extracted from the extrapituitary tumors of patients with vasopressin excess, providing evidence for para¬ neoplastic ("ectopic") production of the hormone.

DIABETES INSIPIDUS Diabetes insipidus is characterized clinically by polyuria and polydipsia (see Chap. 26). In most cases it is caused by vaso¬ pressin deficiency resulting from the destruction of supraoptic and paraventricular nuclei, which is where vasopressin is synthe¬ sized, or by organic damage to the hypophysial stalk or posterior lobe, which is the site of vasopressin discharge. Morphologically, various lesions can be seen in the hypothalamus, especially in the nucleus supraopticus or along the supraopticohypophysial tract. Selective destruction of the posterior lobe results in only moder¬ ate and temporary polyuria and polydipsia. Causes include lesions resulting from head trauma, transection of the hypophys¬ ial stalk, meningoencephalitis, sarcoidosis, granulomas, Langerhans histiocytosis, primary tumors, metastatic carcinomas, lymphomas, and leukemias. Of note, pituitary adenomas, includ¬ ing large and invasive ones, are virtually never accompanied by diabetes insipidus as an initial feature of the tumor. The preop¬ erative presence of diabetes insipidus in association with a sellar region mass argues strongly against a diagnosis of pitu¬ itary adenoma, however suggestive the imaging studies may be. In idiopathic diabetes insipidus, no destructive lesions can be recognized grossly in the hypothalamus, hypophysial stalk, or posterior pituitary. Histologically, nerve cells of the supraoptic and paraventricular nuclei may show a marked reduction in number and size and loss of stainable neurosecretory material. Immunostaining demonstrates an absence of vasopressin in the hypothalamus, hypophysial stalk, and posterior lobe. The renal form of diabetes insipidus (nephrogenic diabetes insipidus) is caused by end-organ failure. Renal tubular cells fail to respond to the antidiuretic effect of vasopressin, resulting in polyuria and polydipsia. No lesions are evident in the hypo¬ thalamus, hypophysial stalk, or posterior lobe. Vasopressin synthesis and release are not impaired.

BASOPHILIC CELL INVASION Basophilic cell invasion of the pituitary is a frequent autopsy finding in older men. It causes no clinical symptoms and cannot be detected by gross examination of the pituitary. Histologi¬ cally, single or small groups of basophilic cells are seen to creep into the posterior lobe. In some cases, large groups of basophilic cells deeply invade the posterior lobe. The cytoplasm of baso¬ philic cells is PAS-positive and contains ACTH and other frag¬ ments of the pro-opiomelanocortin molecule, indicating that these cells are related to corticotropes. However, they differ from the corticotropes located in the anterior lobe: they are smaller and denser, and, except for occasional cases, do not show Crooke hyalinization as a result of cortisol excess. Baso¬ philic cell invasion is not apparent before puberty and cannot be correlated with any clinical endocrine abnormality. From a practical standpoint, the phenomenon of basophil invasion is important only insofar as its presence in a surgical specimen should not be mistaken for a corticotrope adenoma invading the neural lobe of the gland.

126

PART II: THE ENDOCRINE BRAIN AND PITUITARY GLAND

MISCELLANEOUS FINDINGS Squamous cell nests, which are glandular structures resembling salivary glands, and focal mononuclear cell infiltration are com¬ mon incidental findings at autopsy in the posterior lobe and distal end of the hypophysial stalk. Hemorrhages, necroses, and granulomas were reviewed in the discussion on diseases of the anterior lobe. INTERRUPTION OF THE HYPOPHYSIAL STALK Interruption of the hypophysial stalk causes distinct changes along the entire supraopticohypophysial tract. Surgical transec¬ tion or disruption of the hypophysial stalk secondary to head trauma or organic diseases leads to atrophy of the supraoptic and, to a lesser extent, the paraventricular nuclei, as well as the posterior lobe. Various diseases, such as infections, granulo¬ mas, sarcoidosis, and neoplasms, can destroy the supraoptic and paraventricular nuclei and disrupt the hypophysial stalk, thereby impairing the innervation of the posterior lobe. Because the normal functional activity of the posterior lobe depends on the integrity of its nerve supply, diabetes insipidus develops in the absence of innervation. Morphologically, the posterior lobe undergoes marked involution; loss of stainable neurosecretory material and hormone content can be demonstrated. On radio¬ immunoassay, vasopressin concentrations become undetect¬ able; histologically, neurohypophysial tissue is replaced by a fibrous scar. Atrophy of the posterior lobe is noticeable in some cases of anterior hypopituitarism. In postpartum hypopituitar¬ ism, atrophy of the supraoptic and paraventricular nuclei and of the hypophysial stalk and posterior lobe may occur.

NEOPLASMS Neoplasms in the posterior lobe, hypophysial stalk, and hypo¬ thalamus are uncommon. Secondary carcinomas were discussed earlier in relation to the anterior lobe. GRANULAR CELL TUMORS Granular cell tumors (choristomas, or tumorlets) are the most common primary tumors in the posterior lobe and distal part of the hypophysial stalk. They are found in 1% to 8% of unselected adult autopsies, mainly in the elderly. Usually small (1-2 mm or less), they can be detected histologically. Granular cell tumors are slow-growing, histologically benign, sharply demarcated but unencapsulated nodules that usually are not associated with clinical symptoms or biochemical abnormalities and are recog¬ nized incidentally at autopsy. Rare granular cell tumors grow rapidly and become large, causing headaches, vision distur¬ bances, diabetes insipidus, and anterior hypopituitarism.10 Because of increased intracranial pressure or cranial nerve com¬ pression, surgical removal of the tumor may be necessary. Histologically, granular cell tumors consist of loosely apposed, large, spherical, oval, or polygonal cells with eccentric nuclei and abundant, coarsely granular cytoplasm. Numerous large granules that stain strongly with PAS, luxol fast blue, and alcian blue almost fill the entire cytoplasm. On electron microscopic examina¬ tion, the granules correspond to large, membrane-bound, unevenly electron-dense lysosomes. Immunostains reveal an absence of adenohypophysial or neurohypophysial hormones, but may show S-100 protein in the cytoplasm. Granular cell tumors appear to originate in pituicytes, the special glial cells of the posterior lobe. GLIOMAS Other neoplasms are rare in the posterior lobe. Gliomas may originate in pituicytes of the posterior lobe; their histologic fea¬ tures are identical to those of glial tumors deriving from the central nervous system. Most gliomas involving the posterior lobe originate in the hypothalamus or median eminence and

spread downward to the posterior lobe. Diabetes insipidus may develop as a result of massive destruction of the hypothalamus, the hypophysial stalk, or the posterior lobe. Most gliomas that involve the hypothalamus, stalk, and pos¬ terior lobe are low-grade astrocytomas of pilocytic type. Malig¬ nant gliomas arising in these structures are extremely rare, most being a long-term complication of radiotherapy for a sellar region tumor. Postirradiation gliomas occurring in the region generally have been either anaplastic astrocytomas or glioblastomas. The mean latency period is ~10 years.19 They invariably are aggressive lesions, with most patients dying within months of the diagnosis. GANGLIOCYTOMAS (HAMARTOMAS) OF THE SELLAR REGION Lesions composed of neurons can occasionally form symptom¬ atic masses in the sellar region. In fact, constituting this group are a collection of entities, ranging from simple hypothalamic hamartomatous growths on the one hand, to intriguing intra¬ sellar neoplasms composed of both neuronal and adenohypo¬ physial elements. As a group, however, they remain unified by their content of fully differentiated ganglion cells that appear mature and are accompanied by neuropil. The nomenclature surrounding these lesions has been as diverse as it has been confusing, wherein designations such as ganglioneuroma, neuronal hamartoma, gangliocytoma, choristoma, gangliocytoma-pituitary adenoma, pituitary adenomaadenohypophysial choristoma, and a medley of neologisms of one form or another have been inconsistently, interchangeably, and, at times, arbitrarily applied. Understandably, some of this variation in terminology reflects differing views on the pre¬ sumed histiogenetic origins of these lesions, an issue that cur¬ rently is far from settled. It is important to recognize that two topologically distinct variants exist: one arising in the hypothal¬ amus and the second within the sella. Of the former entities that arise in or remain physically attached to the hypothalamus, the term hypothalamic neuronal hamartoma is applied. Of the intrasel¬ lar variants, the majority of which are intimately admixed within the substance of a pituitary adenoma, the terms pituitary adenoma-adenohypophysial neuronal choristoma (PANCH)84 or mixed pituitary adenoma-gangliocytoma are applied.85 Both hypo¬ thalamic and intrasellar variants are discussed separately. Hypothalamic Neuronal Hamartoma. Not uncommonly, a well-defined mass composed of mature central ganglionic tis¬ sue projecting in the leptomeninges from the base of the brain will be encountered. In fact, when carefully sought, minute, macroscopic and nodular foci of ectopic hypothalamic tissue may be found incidentally in -20% of random autopsies. This is usually attached to the ventral hypothalamus, the adjacent pia, or on the surface of the proximal posterior cerebral arteries. Although such hamartomatous nodules are clinically insignifi¬ cant, rarely they may grow to several centimeters in size and compress surrounding structures. Some retain a thick pedicular attachment to the hypothalamus, tuber cinereum, or mammil¬ lary bodies, whereas others form a sessile solitary mass. Most symptomatic examples occur in young males, in whom precocious puberty is the best known manifestation. In some instances, GnRH can be detected immunohistochemically within the neurons of such hamartomas, providing a neuroendocrinologic basis for accelerated sexual maturation. This is not, however, a universal nor a necessary finding. Precocious puberty in immunonegative cases is presumably the result of hypothalamic compression. Rarely, hypothalamic neuronal hamartomas may produce GHRH and clinical acromegaly on a hypothalamic basis, a condition dubbed hypothalamic acromeg¬ aly. In such cases, the pituitary may show either hyperplasia or adenomas of GH-producing cells. In addition to other features of hypothalamic dysfunction (somnolence, hyperphagia, auto¬ nomic disturbances, and diabetes insipidus), these tumors are associated with a peculiar form of epilepsy, one characterized by laughing (gelastic seizures).

Ch. 11: Morphology of the Pituitary in Health and Disease

127

Grossly, the mass in most symptomatic examples will be only 1 to 2 cm in size and often lies behind the pituitary stalk. It is pale, of firm consistency, and has homogeneous cut surfaces. Microscopically, the composition of the tissue resembles that of cerebral gray matter, both qualitatively and quantitatively. The neurons can vary in size, shape, and number, and both unipolar and multipolar forms are represented. Overall, these elements fully resemble mature hypothalamic neurons, although they are often disposed in clusters. Both myelinated and unmyeli¬ nated fibers course among them, forming compact bundles in some areas. Axonal processes may also appear to form illdefined tracts, particularly among examples having pedicular attachment to the hypothalamus. In all examples, the neuronal elements are supported by a normal complement of glial cells of different kinds, although a variable amount of fibrillary glio¬ sis may be found. Immunohistochemical stains reveal a variety of hypotha¬ lamic-releasing peptides that normally reside within the cyto¬ plasm of hypothalamic neurons: immunopositivity for GnRH, somatostatin, GHRH, and CRH may be detectable. In most cases, the immunohistochemical presence of these factors is regarded as a physiologic finding, and should not necessarily imply that these lesions are engaged in pathologic hormone hypersecretion. However, in the appropriate clinical context, such as with precocious puberty or the rare instance of acromeg¬ aly, the trophic effects of these hormones and their pathologic contribution to the endocrinopathy cannot be dismissed. If they are symptomatic and surgically accessible, therapy for hypothalamic neuronal hamartomas is directed primarily at relief of the mass effect. When technically feasible, surgical resection has been successful not only in ameliorating mass effects, but also in regressing secondary sexual characteristics in those experiencing precocious puberty and in improving sei¬ zure control. For lesions less amenable to total resection by vir¬ tue of their deep intrahypothalamic location or other factors limiting surgical accessibility, partial resection may also be of some symptomatic benefit; residual hamartomatous tissue should grow slowly, if at all. Hypothalamic Hamartoblastoma. A special variant of hypothalamic hamartoma is the hypothalamic hamartoblas¬ toma, which occurs in infants and neonates and is associated with other multiple congenital abnormalities. The most fre¬ quent of the co-existing anomalies have included pituitary agenesis; dwarfism; dysmorphic facies; short, broad, or absent olfactory bulbs; hypoplastic thyroid and adrenals; cryptorchid¬ ism; various renal and cardiac malformations; anorectal atresia; syndactyly; and short metacarpals. This lethal syndrome is sometimes referred to as the Pallister-Hall syndrome. The hamartomatous lesion in this condition differs in several subtle respects from the more common hypothalamic hamartoma described earlier. As might be expected from the young age of affected patients, the lesion tends to be more cellular and less differentiated than the typical hypothalamic hamartoma found in older patients, hence the designation hamartoblastoma. A process morphologically intermediate between neoplasm and malformation, the hamartoblastoma is composed of ill-defined clusters of uniform, primitive-appearing, immature neurons unassociated with atypia or mitoses; neuronal differentiation appears incomplete. Intrasellar Adenohypophysial Neuronal Choristoma. The intriguing neoplasm of the sellar region known as an intrasel¬ lar adenohypophysial neuronal choristoma, which, for lack of bet¬ ter designations, has previously been known under the terms intrasellar adenohypophysial neuronal choristoma or the intrasel¬ lar gangliocytoma. It is a composite neoplasm composed of adenohypophysial cells and ganglion-like cells to which the appellation pituitary adenoma-neuronal choristoma (PANCH) has been applied in an attempt to highlight the duality of its composition.84 In a comprehensive review reiterating the composite neuronal and adenohypophysial nature of this

FIGURE 11-14. Adenohypophysial neuronal choristoma. The specimen is from a patient with acromegaly and a large intrasellar and suprasellar mass that was presumed to be an ordinary growth hormone-secreting adenoma. After transsphenoidal resection, the surgical specimen revealed an adenohypophysial neuronal choristoma intermixed within the substance of a growth hormone-secreting pituitary adenoma. Ade¬ noma cells (arrowhead) intermixed with fully differentiated neurons (arrow) are evident in the specimen. These neurons were shown to con¬ tain growth hormone-releasing hormone. Original magnification x400 neoplasm, the authors indicated a preference for the appella¬ tion "mixed adenoma-gangliocytoma."85 This lesion, although sharing some histologic similarities with the hypothalamic hamartoma, differs in several topologic and biologic respects. First and foremost, adenohypophysial neuronal choristomas are intrasellar lesions that have no physi¬ cal attachment to the hypothalamus. Second, they are generally associated with—or more precisely—are intimately admixed with an endocrinologically functioning pituitary adenoma. Accordingly, they are virtually always symptomatic lesions of which the presence is heralded by a hypersecretory state. Finally, the neuronal component of this lesion, like the adenohy¬ pophysial component, are both genuinely neoplastic in nature. The basic lesion consists of islands of ganglion-like cells and accompanying neuropil interspersed within the substance of a pituitary adenoma (Fig. 11-14). The ratio of adenohypo¬ physial cells to neuronal elements can vary considerably, but as a rule, both will be strongly represented. The adenohypo¬ physial cells in virtually all instances are chromophobic and sparsely granulated in nature. They may be disposed in nests, sheets, or otherwise scattered among the neuronal elements. With respect to the latter, the ganglion-like cells are of varying size and number, contain an abundant cytoplasm, complete with peripheral Nissl substance and neuronal processes con¬ taining Herring bodies. The ganglion cells fully resemble nor¬ mal hypothalamic neurons. Of reported cases, most have occurred in the setting of acromegaly, wherein patients have had the clinical, radiologic, and endocrinologic features of a typical GH-producing pitu¬ itary adenoma. On examination of the surgical specimen, a somatotrope adenoma is encountered, invariably of the sparsely granulated type, in which are interspersed varying numbers of neuronal elements, the morphology of which resembles well-differentiated ganglionic cells. Some of these ganglion cells have been found to be immunoreactive for GHRH, whereas others are immunoreactive for somatostatin. Less frequently, the lesion occurs in the setting of Cushing dis¬ ease, wherein the adenoma is of the corticotropic type accom¬ panied by ganglion cells immunoreactive for CRH. Only rarely is the adenohypophysial tumor a prolactinoma or a clinically nonfunctioning-pituitary adenoma. Previously, hypotheses concerning the origin of these lesions centered on the seemingly displaced hypothalamic neurons in

128

PART II: THE ENDOCRINE BRAIN AND PITUITARY GLAND

the pituitary fossa that, as a result of their secretion of trophic factors, might have a paracrine inductive effect on adjacent ade¬ nohypophysial cells, stimulating their growth and eventual neoplastic transformation. According to this view, pituitary ade¬ noma formation is regarded as a secondary, downstream event. Subsequently, however, an alternative hypothesis has been pro¬ posed that features adenohypophysial cells as the instigators of the process, rather than seemingly passive targets of paracrine effect.84 Supported by compelling morphologic evidence, this hypothesis suggests that pituitary adenoma formation is the primary event, and the ganglionic component of the lesion arises as the consequence of neuronal metaplasia of neoplastic adenohypophysial cells. When subjected to careful ultrastructural study, morphologic changes that are indicative of a meta¬ plastic process beginning in adenohypophysial cells that gradually gives way to cells having intermediate and, eventu¬ ally, fully neuronal features can be seen. Whereas metaplastic change is a well-known phenomenon in both normal and neo¬ plastic adenohypophysial cells, as it is in other neuroendocrine neoplasms, actual induction of such metaplastic change has yet to be demonstrated experimentally. A third hypothesis is that both adenohypophysial and neuronal components of this tumor arise from a common, but as yet hypothetical, progenitor cell sequestrated in the sella as an embryonal tissue nest. Because this entity is relatively new, with only small num¬ bers of cases having been collected, and virtually no long-term follow-up data available, it is unclear whether the behavior of these mixed lesions differs significantly from that of the corre¬ sponding pituitary adenoma. The isolated intrasellar gangliocytoma is a lesion unassoci¬ ated with a pituitary adenoma and anatomically distinct from the hypothalamus.85 Although an adenoma was not found in any of seven cases, three examples were associated either with Cushing disease or with acromegaly, a situation not readily explained by a purely gangliocytic tumor. The authors have not encountered such a pure gangliocytic lesion in the sella, particularly in the setting of a hypersecretory state—when an accompanying pituitary adenoma or hyperplasia has always been present.

REFERENCES 1. Hague K, Post KD, Morgello S. Absence of peritumoral Crooke's change is associated with recurrence in surgically treated Cushing's disease. Surg Neurol 2000; 53:77. la. Kovacs K, Horvath E. Tumors of the pituitary gland. In: Hartmann WH, Sobin LH, eds. Atlas of tumor pathology, fascicle 21, series 2. Washington, DC: Armed Forces Institute of Pathology, 1986:1. 2. Horvath E, Scheithauer B, Kovacs K, et al. Regional neuropathology: hypo¬ thalamus and pituitary. In: Graham D, Lantos P, eds. Greenfield's Neuro¬ pathology. London: Arnold, 1997:1007. 3. Lloyd RV, Chander WF, Kovacs K, Ryan N. Ectopic pituitary adenomas with normal anterior pituitary glands. Am J Surg Pathol 1986; 10:546. 4. Thapar K, Kovacs K, Scheithauer B, et al. Classification and pathology of sellar and parasellar tumors. In: Tindall G, Cooper P, Barrow D, eds. The Practice of Neurosurgery. Baltimore: Williams & Wilkins, 1996:1021. 5. Bergland RM, Ray BS, Torack RM. Anatomical variations in the pituitary gland and adjacent structures in 225 human autopsy cases. J Neurosurg 1968; 28:93. 6. Kaufman B, Chamberlin WB Jr. The ubiquitous "empty" sellar turcica. Acta Radiol Diagn (Stockh) 1972; 13:413. 7. Weisberg LA, Housepian EM, Saur DP. Empty sella syndrome as a compli¬ cation of benign intracranial hypertension. J Neurosurg 1975; 43:177. 8. Brismar K, Efendic S. Pituitary function in the empty sella syndrome. Neu¬ roendocrinology 1981; 32:70. 9. McFadzean R, Teasdale G. Pituitary apoplexy. In: Landolt A, Vance M, Reilly P, eds. Pituitary Adenomas. Edinburgh: Churchill Livingstone, 1996:485. 10. Thapar K, Kovacs K. Tumors of the sellar region. In: Bigner DD, McLendon RE, Bruner JM, eds. Russel and Rubinstein's pathology of tumors of the nervous system, 6th ed. Baltimore: Williams & Wilkins, 1998:561. 11. Cardosa ER, Peterson EW. Pituitary apoplexy: a review. Neurosurgery 1984; 14:363. 12. Berger SA, Edberg SC, David G. Infectious disease in the sella turcica. Rev Infect Dis 1986; 8:747.

13. Domingue JN, Wilson CB. Pituitary abscesses: report of seven cases and review of the literature. J Neurosurg 1977; 46:601. 14. Sano T, Kovacs K, Scheithauer BW, et al. Pituitary pathology in acquired immunodeficiency syndrome. Arch Pathol Lab Med 1989; 113:1066. 15. Beressi N, Beressi JP, Cohen R, Modigliani E. Lymphocytic hypophysitis: a review of 145 cases. Ann "Med Interne (Paris) 1999; 150:327. 16. Feigenbaum SL, Martin MC, Wilson CB, Jaffe RB. Lymphocytic adenohypophysitis: a pituitary mass lesion occurring in pregnancy. Proposal for medical treatment. Am J Obstet Gynecol 1991; 164:1549. 17. Lee JH, Laws ER Jr, Guthrie BL, et al. Lymphocytic hypophysitis: occur¬ rence in two men. Neurosurgery 1994; 34:159. 18. Capellan JIL, Olmedo C, Martin JM, et al. Intrasellar mass with hypopitu¬ itarism as a manifestation of sarcoidosis. J Neurosurg 1990; 73:283. 19. Scheithauer BW. The neurohypophysis. In: Kovacs K, Asa SL, eds. Func¬ tional endocrine pathology. Boston: Blackwell, 1991:170. 20. Nishio S, Mizuno J, Barrow DL, et al. Isolated histiocytosis X of the pitu¬ itary gland: case report. Neurosurgery 1987; 21:718. 21. Tampanaru-Sarmesiu A, Stefaneanu L, Thapar K, et al. Transferrin and transferrin receptor in human hypophysis and pituitary adenomas. Am J Pathol 1998; 152:413. 22. Horvath E, Kovacs K. The adenohypophysis. In: Kovacs K, Asa SL, eds. Functional endocrine pathology. Boston: Blackwell, 1991:245. 23. Thapar K, Kovacs K, Laws ER Jr. The pathology and molecular biology of pituitary adenomas. Adv Tech Stand Neurosurg 1995; 22:4. 24. Sano T, Asa SL, Kovacs K. Growth hormone-releasing hormone-producing tumors: clinical, biochemical, and morphological manifestations. Endocr Rev 1988; 9:357. 25. Scheithauer BW, Kovacs K, Randall RV. The pituitary in untreated Addi¬ son's disease: a histologic and immunocytologic study of 18 adenohypo¬ physes. Arch Pathol Lab Med 1983; 107:484. 26. Scheithauer BW, Kovacs K, Randall RV, Ryan N. Pituitary gland in hypothyroidism. Histologic and immunocytologic study. Arch Pathol Lab Med 1985; 109:499. 27. Herman V, Fagan J, Gonsky R, et al. Clonal origin of pituitary adenomas. J Clin Endocrinol Metab 1990; 71:1427. 28. Schulte HM, Oldfield EH, Allolio B, et al. Clonal composition of pituitary adenomas in patients with Cushing's disease: determination by X chromo¬ some inactivation analysis. J Clin Endocrinol Metab 1991; 73:1302. 29. Faglia G, Spada A. The role of the hypothalamus in pituitary neoplasia. Baillieres Clin Endocrinol Metab 1995; 9:225. 30. Asa S, Kovacs K, Stefaneanu L, et al. Pituitary adenomas in mice transgenic for growth hormone-releasing hormone. Endocrinology 1992; 131:2083. 31. Melmed S. Acromegaly. In: Melmed S, ed. The Pituitary. Cambridge: Blackwell Science, 1995:413. 32. Levy A, Lightman S. Growth hormone-releasing hormone transcripts in human pituitary adenomas. J Clin Endocrinol Metab 1992; 74:1474. 33. Rauch C, Li J, Croissandeau G, et al. Characterization and localization of an immunoreactive growth hormone-releasing hormone precursor form in nor¬ mal and tumoral human anterior pituitaries. Endocrinology 1995; 136:2594. 34. Castro M, Brooke J, Bullman A, et al. Synthesis of corticotropin-releasing hor¬ mone (CRH) in mouse corticotropic tumour cells expressing the human proCRH gene, intracellular storage and regulated secretion. J Mol Endocrinol 1991; 7:97. 35. Pagesy P, Li JY, Rentier-Delrue F, et al. Growth hormone and somatostatin gene expression in pituitary adenomas with active acromegaly and mini¬ mal plasma growth hormone elevation. Acta Endocrinol 1990; 122:745. 36. Miller G, Alexander J, Klibanski A. Gonadotropin-releasing hormone mes¬ senger RNA expression in gonadotroph tumors and normal human pitu¬ itary. J Clin Endocrinol Metab 1996; 81:80. 37. Thapar K, Kovacs K, Stefaneanu L, et al. Overexpression of the growth hor¬ mone-releasing hormone gene in acromegaly associated pituitary tumor: an event associated with neoplastic progression and aggressive behavior. Am J Pathol 1997; 151:769. 38. Thapar K, Kovacs K, Muller P. Clinical-pathologic correlations of pituitary tumors. Baillieres Clin Endocrinol Metab 1995; 9:243. 39. Molitch ME. Pathologic hyperprolactinemia. Endocrinol Metab Clin North Am 1992; 21:877. 40. Scheithauer BW, Kovacs K, Randall RV, Ryan N. Effects of estrogen on the human pituitary: a clinicopathologic study. Mayo Clin Proc 1989; 64:1077. 41. Stefaneanu L, Kovacs K, Horvath E, et al. In situ hybridization of estrogen receptor mRNA in human adenohypophysial cells and pituitary ade¬ nomas. J Clin Endocrinol Metab 1994; 78:83. 42. Kovacs K, Stefaneanu L, Ezzat S, et al. Prolactin producing pituitary ade¬ noma in a male to female transsexual patient following protracted estrogen administration: a morphologic study. Arch Pathol Lab Med 1994; 118:562. 43. Vallar L, Spada A, Giannattasio G. Altered Gs and adenylate cyclase activ¬ ity in human GH secreting pituitary tumors. Nature 1987; 330:566. 44. Landis CA, Harsh G, Lyons J, et al. Clinical characteristics of acromegalic patients whose pituitary tumors contain mutant Gs protein. J Clin Endo¬ crinol Metab 1990; 71:1416. 45. Yoshimoto K, Iwahana H, Fukuda A, et al. Rare mutations of the Gs alpha subunit in human endocrine tumors. Mutation detection by polymerase chain reaction-primer introduced restriction analysis. Cancer 1993; 72:1286. 46. Tjordman K, Stem N, Ouaknine G, et al. Activating mutations of the Gs alpha gene in nonfunctioning pituitary tumors. J Clin Endocrinol Metab 1993; 77:765. 47. Williamson E, Daniels M, Foster S, et al. Gs alpha and gi2 alpha mutations in clinically non-functioning pituitary tumors. Clin Endocrinol 1994; 41:815.

Ch. 12: Growth Hormone and Its Disorders 48. Williamson E, Ince P, Harrison D, et al. G-protein mutations in human pitu¬ itary adrenocorticotrophic hormone-secreting adenomas. Eur 1 Clin Invest 1995; 25:128. 49. Karga HJ, Alexander JM, Hedley-White ET, et al. Ras mutations in human pituitary tumors. J Clin Endocrinol Metab 1992; 74:914. 50. Herman V, Drazin NZ, Gonsky R, et al. Molecular screening of pituitary adenomas for gene mutations and rearrangements. 1 Clin Endocrinol Metab 1993; 77:50. 51. Pei L, Melmed S, Scheithauer BW, et al. H-ras mutations in human pitu¬ itary carcinoma metastases. J Clin Endocrinol Metab 1994; 78:842. 52. Bystrom C, Larsson C, Blomberg C, et al. Localization of the MEN1 gene to a small region within chromosome llql3 by deletion mapping tumors. Proc Natl Acad Sci U S A 1990; 87:1968. 53. Chandrasekharappa S, Guru C, Manickam P, et al. Positional cloning of the gene for multiple endocrine neoplasia—type 1. Science 1997; 276:404. 54. Thakkar R, Pook M, Wooding C, et al. Association of somatotrophinomas with loss of alleles on chromosome 11 and with Gsp mutations. J Clin Invest 1993; 91:2815. 55. Boggild M, Jenkinson S, Pistorello M, et al. Molecular genetic studies of sporadic pituitary tumors. J Clin Endocrinol Metab 1994; 78:387. 56. Elarris C, Hollstein M. Clinical implications of the p53 tumor-suppressor gene. N Engl J Med 1993; 329:1318. 57. Thapar K, Scheithauer BW, Kovacs K, et al. p53 expression in pituitary ade¬ nomas and carcinomas: correlation with invasiveness and tumor growth fractions. Neurosurgery 1996; 38:765. 58. Levy A, Hall L, Yeudall WA, et al. p53 gene mutations in pituitary ade¬ nomas: rare events. Clin Endocrinol 1994; 41:809. 59. Buckley N, Bates AS, Broome J, et al. p53 protein accumulates in Cushings adenomas and invasive non-functional adenomas. J Clin Endocrinol Metab 1994; 79:1513. 60. Jacks T, Fazeli A, Schmitt EM, et al. Effects of an Rb mutation in the mouse. Nature 1992; 359:295. 61. Cryns VL, Alexander JP, Klibanski A, Arnold A. The retinoblastoma gene in human pituitary tumors. J Clin Endocrinol Metab 1993; 77:644. 62. Zhu J, Leon S, Beggs A, et al. Human pituitary adenomas show no loss of heterozygosity at the retinoblastoma gene locus. J Clin Endocrinol Metab 1994; 78:922. 63. Pei L, Melmed S, Scheithauer B, et al. Frequent loss of heterozygosity at the retinoblastoma susceptibility gene (RB) locus in aggressive pituitary tumors: evidence for a chromosome 13 tumor suppressor gene other than RB. Cancer Res 1995; 55:1613. 64. Capra E, Rindi G, Pompei-Spina M, et al. Chromosomal abnormalities in a case of pituitary adenoma. Cancer Genet Cytogenet 1993; 681:40. 65. Rock J, Babu V, Drumheller T, et al. Cytogenetic findings in pituitary ade¬ nomas: results of a pilot study. Surg Neurol 1993; 40:224. 66. Alvaro V, Levy L, Deburay C, et al. Invasive human pituitary tumors express a point mutated alpha-protein kinase-C. J Clin Endocrinol Metab 1993; 77:1125.

SECTION

129

67. Klibanski A, Zervas N. Diagnosis and management of hormone-secreting pituitary adenomas. N Engl J Med 1991; 324:822. 68. Scheithauer BW, Kovacs K, Laws ER Jr, Randall RV. Pathology of invasive pituitary adenomas with special reference to functional classification. J Neurosurg 1986; 65:733. 69. Knosp E, Kitz K, Perneczky A. Proliferation activity in pituitary adenomas. Measurement by monoclonal antibody Ki. Neurosurgery 1989; 25:927. 70. Thapar K, Kovacs K, Scheithauer B, et al. Proliferative activity and inva¬ siveness among pituitary adenomas and carcinomas: an analysis using the MIB-1 antibody. Neurosurgery 1996; 38:99. 71. Hsu DW, Hakim F, Biller BMK, et al. Significance of proliferating cell nuclear antigen index in predicting pituitary adenoma recurrence. J Neuro¬ surg 1993; 78:753. 72. Pemicone PJ, Scheithauer BW, Sebo TJ, et al. Pituitary carcinoma. A clinicopathologic study of 15 cases. Cancer 1997; 79:804. 73. Gesundheit N, Petrick PA, Nissim M, et al. Thyrotropin-secreting pituitary adenomas. Clinical and biochemical heterogeneity. Case reports and fol¬ low-up of nine patients. Ann Intern Med 1989; 111:827. 74. Kovacs K, Asa SL, Horvath E, et al. Null cell adenomas of the pituitary: attempts to resolve their cytogenesis. In: Leschago J, Kameya T, eds. Endo¬ crine pathology update. New York: Field & Wood, 1990:17. 75. Kovacs K, Horvath E, Asa SL, et al. Pituitary cells producing more than one hormone. Human pituitary adenomas. Trends Endocr Metab 1989; 1:104. 76. Thapar K, Stefaneanu L, Kovacs K, et al. Plurihormonal pituitary tumors: beyond the one cell-one hormone theory. Endocr Pathol 1993; 4:1. 77. Branch CL, Laws ER Jr. Metastatic tumors of the sella turcica masquer¬ ading as primary pituitary tumors. J Clin Endocrinol Metab 1987; 65:649. 78. Burger P, Scheithauer B. Tumors of the central nervous system. Third series, fascicle 10. Washington, DC: Armed Forces Institute of Pathology, 1994. 79. Crotty R, Scheithauer B, Young W, et al. Papillary craniopharyngioma: a clinicopathological study of 48 cases. J Neurosurg 1995; 83:206. 80. Crotty T, Scheithauer BW, Young WF, Davis D. Papillary craniopharyngio¬ mas: a morphological and clinical study of 46 cases. Endocr Pathol 1992; 3(Suppl):S6(abst). 81. Weiner H, Wishoff J, Rosenberg M, et al. Craniopharyngiomas: a clinicopathologic analysis of factors predictive of recurrence and functional out¬ come. Neurosurgery 1994; 35:1001. 82. Thapar K, Stefaneanu L, Kovacs K, et al. Estrogen receptor gene expression in craniopharyngioma: an in situ hybridization study. Neurosurgery 1994; 35:1012. 83. Brassier G, Morandi X, Tayiar E, et al. Rathke's cleft cysts: surgical-MRI correlation in 16 symptomatic cases. J Neuroradiol 1999; 26:162. 84. Horvath E, Kovacs K, Scheithauer BW, et al. Pituitary adenoma with neu¬ ronal choristoma (PANCH): composite lesion or lineage infidelity? Ultrastruct Pathol 1994; 18:565.

A

ADENOHYPOPHYSIS

CHAPTER

12

GROWTH HORMONE AND ITS DISORDERS

species specificity" refers to the fact that primate GHs are active in lower (evolutionarily earlier) species, but GHs of lower species are inactive in primates, including humans. GH regulation and, in part, GH action also differ among species. This chapter focuses primarily on human GH and its biology.

GROWTH HORMONE GENES GERHARD BAUMANN Growth hormone (GH) is a polypeptide hormone produced by the somatotrope cells in the pituitary gland. It is the master anabolic hormone and possesses numerous bioactivities related to somatic growth, body composition, and intermediary metabolism. Many of the biologic actions of GH are mediated through insulin-like growth factor-I (IGF-I), but GH also has direct effects independent of IGF-I. Unlike most other hormones, GH is species specific, not only in its structure but also partially in its function. Its "one-way

In humans, GH is encoded by two genes on the long arm of chromosome 17.1 They are part of a gene-duplication cluster that also includes the genes for chorionic somatomammotropin (placental lactogen), a protein highly homologous with GH (Fig. 12-1). The GH genes are named GH-N (or GH-1) and GH-V (or GH-2); the former is expressed in the pituitary, the latter in the placenta. GH-V is also called placental GH. The two GH genes are similar in structure; both are composed of five exons and four introns; each spans -1.6 kb.

130

PART II: THE ENDOCRINE BRAIN AND PITUITARY GLAND

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25

30

30

35

40

45

50

55

Minihelix 1 GH-N ■

T

Pituitary

CS-L CS-A GH-V ■■■

Minihelix 3

CS-B

m T_x_I_T

H42

Placental

W46

Helix 3 FIGURE 12-1. Human growth hormone locus on chromosome 17q2224. The top panel shows the organization of the locus with the two GH genes (GH-N and GH-V) and the three chorionic somatomammotropin genes (CS-L, CS-A, CS-B). CS-L is probably a pseudogene. The bottom panel shows the GH-N gene in more detail, with five exons and four introns. GH-N is expressed in the pituitary, the other genes in the locus exclusively in the placenta. (From Parks JS. Molecular biology of growth hormone. Acta Paediatr Scand 1989; 349[Suppl]:127.)

Helix 1

Minihelix 2

GROWTH HORMONE STRUCTURE Human GH is heterogeneous and consists of several molecular variants (Fig. 12-2).2 The principal form is a 191-amino-acid, single-chain protein with a molecular weight of -22,000. It is the most abundant form (~90%-95% of pituitary GH), generally referred to as "GH" or "22K GH." Two disulfide loops are present, and the three-dimensional structure is a twisted bundle of four ahelices (Fig. 12-3).3 Two independent receptor-binding sites are located on opposite surfaces of GH, allowing for ligand-induced receptor dimerization (see later). GH-V (placental GH) has a sim¬ ilar primary structure; it differs from GH-N (22K GH) in 13 of the 191 amino-acid positions (see Fig. 12-2). One important differ¬ ence is the glycosylation consensus site at asparagine 140; GH-V exists as a glycosylated as well as nonglycosylated protein. The second most abundant GH form after 22K GH is an mRNA splice variant that lacks an internal sequence of 15 amino acids. Its molecular weight is -20,000, and hence it is known as "20K GH." It accounts for 5% to 7% of pituitary GH. Other minor vari¬ ants include an Na-acylated and two deamidated variants (see Fig. 12-2). Little is known about the bioactivities or significance of these minor GH forms. In addition to the monomeric forms of

FIGURE 12-2. Primary structure of human growth hormone and its variants. The polypeptide shown is GH-N (22K). Amino-acid substitutions in GH-V are indicated next to the involved residues. The sequence connected by the heavy line (residues 32M6) is deleted in 20K GH. The tree structure at Asn-140 denotes glycosylation in GH-V. The asterisks indicate sites of deamidation, the dot at the amino terminus acylation. (From Baumann G. Growth hormone heterogeneity: genes, isohormones, variants and binding proteins. Endocr Rev 1991; 12:424.)

FIGURE 12-3. Three-dimensional structure of human growth hormone (22K), depicted as a ribbon diagram. The four main a-helices are shown together with three minihelices within the connecting loops. Some resi¬ dues mutated for technical purposes are indicated; they are not relevant in this context. (N, amino terminus; C, carboxy terminus.) (From Ultsch MH, Somers W, Kossiakoff AA, de Vos AM. The crystal structure of affinity-matured human growth hormone at 2 A resolution. J Mol Biol 1994; 236:286.)

GH just described, GH also exists as an oligomeric series of up to at least pentameric GH.4 Both noncovalent and disulfide-linked oligomers occur, and homo- as well as heterooligomers com¬ posed of the various monomeric forms exist in the pituitary and plasma. The biologic significance of GH oligomers is unclear, but they likely act as modulators of overall GH activity because of their different affinities for the GH receptor. The existence of so many molecular forms of GH is one reason for the difficulty with GH measurements and the discrepant results obtained by differ¬ ent assays (see later).

Ch. 12: Growth Hormone and Its Disorders

131

Transmembrane Extracellular

Cytoplasmic

FIGURE 12-4. Schematic representation of the growth hormone recep¬ tor (GHR) complementary DNA {top) and protein (bottom). The GHR is encoded by 10 exons; exons 2-7 encode the extracellular domain, exon 8 the transmembrane domain, and exons 9 and 10 the cytoplasmic domain. The numbers in the upper panel denote the exons; those in the lower panel the amino acids. The transmembrane domain is shaded in black. (Adapted from Kelly PA, Djiane J, Postel-Vinay M-C, Edery M. The prolactin/growth hormone receptor family. Endocr Rev 1991; 12:235.)

GROWTH HORMONE RECEPTOR The GH receptor (GHR) is a 620-amino-acid, -130 kDa, singlechain glycoprotein with a single transmembrane domain, a large extracellular domain containing the GH-binding site, and an intracellular domain involved in GH signaling (Fig. 12-4).5 The extracellular domain also occurs separately as a soluble GHbinding protein (GHBP, see later). The GHR is a member of the cytokine receptor family that also includes the receptors for pro¬ lactin, erythropoietin and other hematopoietic growth factors, many of the interleukins, and others.6 The GHR is encoded by a single gene located on the short arm of chromosome 5. The gene spans at least 87 kb and is divided into 10 exons and 9 introns.7 Exons 2-7 encode the extracellular domain, exon 8 the trans¬ membrane domain, and exons 9 and 10 the intracellular domain. The GHR is expressed ubiquitously, with the liver being the organ most enriched in GHRs. In addition to the full-length GHR, two variants of the GHR are found in humans. A version lacking the 22 amino acids encoded by exon 3 is differentially expressed among tissues8 and/or in different individuals.9 This internal deletion near the amino terminus has no known func¬ tional consequence. The second variant is a GHR truncated at nine amino acids beyond the transmembrane domain, so that it lacks most of the intracellular domain.10 This variant is also expressed ubiquitously. The absence of an intracellular domain renders this variant incapable of signaling and favors prolonged persistence on the cell membrane. The latter may be the reason why this receptor variant contributes substantially to GHBP gen¬ eration (see later). This truncated GHR variant modulates GH action by forming heterodimers with full-length GHRs, thereby sequestering some of the GHRs in a nonfunctional state. GH initiates its action first by binding to the GHR through site 1 on one of its surfaces; this is then followed by binding of a sec¬ ond GHR to site 2 on the other surface of GH.11 This results in a complex containing two GHRs in association with GH (Fig. 12-5). This GH-induced dimerization of the GHR is critical for GHR sig¬ naling and GH action. The functional domains of the GHR are depicted in Figure 12-6. Intracellular signaling is initiated by binding of JAK2 (Janus kinase 2) to a proline-rich region (Box 1) in the proximal intracellular part of the GHR, followed by a JAK2-mediated tyrosine phosphorylation cascade involving JAK2 itself, the GHR, signal transducers and activators of tran¬ scription (Stats) 1,3, and 5, insulin-receptor substrates (IRS) 1 and 2, components of the mitogen-activated protein kinase (MAPK), the protein kinase C, and phosphatidyl inositol-3 kinase path¬ ways, and several other intracellular signaling and adapter pro¬ teins, not all of which are known (Fig. 12-7).12 Gene transcription is then activated through these pathways. Interestingly, as of this writing, the precise pathway responsible for activation of IGF-I gene transcription is not known.

Inactive (antagonist)

Active (agonist)

FIGURE 12-5. Schematic representation of growth hormone (GH)induced dimerization of two growth hormone receptors (GHRs). Bind¬ ing occurs first through site 1 on GH, followed by binding of a second GHR to site 2 on GH. The dimerized GHR (2:1) complex (lower right) is active in transducing the GH signal. At very high (pharmacologic) GH concentrations (lower left), the ratio of GH to GHR is high enough to sat¬ urate GHRs through site 1 binding, with the GHR trapped in an inac¬ tive 1:1 complex. This occurs at GH levels that are not seen in vivo. However, recognition of the potential existence of an inactive 1:1 com¬ plex was important in developing a GH antagonist (see text on treat¬ ment for acromegaly). (hGH, human growth hormone.) (From Fuh G, Cunningham BC, Fukunaga R, et al. Rational design of potent antago¬ nists to the growth hormone receptor. Science 1992; 256:1677.) The GHR binds GH variants with different affinities. The 20K variant as well as the oligomeric GH forms have lower affinity than monomeric 22K GH, but GH-V is equipotent to 22K GH. Little is known about the binding of the other GH variants.

GROWTH HORMONE-BINDING PROTEIN The GHBP is the soluble, extracellular domain of the GHR. In humans and many other species, the GHBP is generated from the GHR by proteolysis; in rodents it is derived from the GHR gene via alternative splicing.13 The GHBP circulates in plasma in nanomolar concentrations, sufficient to complex a substan¬ tial part (-50%) of plasma GH. The serum GHBP level appears to reflect the GHR abundance of the organism, especially in the liver. The biologic significance or importance of the GHBP is not known; it is evolutionarily conserved throughout the verte¬ brates and is generated by different mechanisms in different species, suggesting an important role. The GHBP modulates GH action through a variety of mechanisms. It inhibits GH

132

PART II: THE ENDOCRINE BRAIN AND PITUITARY GLAND Glucose

FIGURE 12-6. Functional domains of the growth hormone receptor (extracellular domain is on top). The proline-rich Box 1 is crucial for JAK2 (Janus kinase 2) binding and initiation of most of the signaling events. (C, extracellular cysteines, only one of which is free [not disulfide linked]-, N, potential N-linked glycosylation sites; Y, intracellular tyro¬ sines, important for phosphorylation and docking of signaling mole¬ cules; WSXWS, tryptophan-serine motif; Stat, signal transducer and activator of transcription; SHC, Src homology containing protein; MAPK, mitogen-activated protein kinase; Spi 2.1, serine protease inhibi¬ tor 2.1; IRS, insulin receptor substrate.) (From Argetsinger LS, Carter-Su C. Mechanisms of signaling by growth hormone receptor. Physiol Rev 1996; 76:1089.) action by competing with the GHR for ligand and by generat¬ ing "unproductive" heterodimers with the GHR at the cell sur¬ face, as opposed to the GHR homodimers necessary for signaling. The GHBP also prolongs the half-life of GH in the cir¬ culation by complexing GH, thereby delaying its elimination. The net effect of these modulatory activities in the intact organ¬ ism is not well understood. In the circulation, GH also binds (with low affinity) to one or more proteins related to a2-macroglobulin.14 This complex accounts for no more than 5% of total GH in serum.

SOMATOTROPE DEVELOPMENT AND GROWTH HORMONE Several pituitary transcription factors are involved in pituitary somatotrope development. Their consideration is important here because mutations in their respective genes lead to abnor¬ malities in the GH axis. The reader is referred to Chapters 8 and 11 for a full discussion of pituitary ontogeny. Developmental genes relevant to GH include the Prop-1, Pit-1 (also known as Poulfl), and GH-releasing hormone receptor (GHRH-R) genes. These genes are expressed sequentially during anterior pitu¬ itary development; each is more restrictive in its impact on dif¬ ferent cell types; and each is dependent on the activity of the preceding one.15 Prop-1 and Pit-1 are POU-homeodomain tran¬ scription factors, the GHRH-R is a seven transmembrane domain receptor signaling through the cyclic adenosine mono¬ phosphate (cAMP) pathway. Prop-1 is important for develop¬ ment of the gonadotrope, somatotrope, lactotrope, and thyroptrope lineages. Pit-1, under the direction of Prop-1, is involved in differentiation of somatotropes, lactotropes, and thyrotropes. The GHRH-R, which is exclusively expressed in

FIGURE 12-7. Intracellular signaling pathways of the growth hormone receptor (GHR) (partial rendition). Principal pathways are the signal transducer and activator of transcription (Stef), protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and insulin receptor substrate (IRS) pathways. The inner ellipse represents the nucleus. Transcriptional elements, their cognate transcription factors, and transactivated genes are shown. (GH, growth hormone; P, phosphorylated sites; JAK2, Janus kinase 2; PC, phosphatidyl choline; PIC, phospholipase C; DAG, diacylglycerol; PI3K, phosphatidyl-inositol-3 kinase.) (From Argetsinger LS, Carter-Su C. Mechanisms of signaling by growth hormone receptor. Physiol Rev 1996; 76:1089.) somatotropes under the direction of Pit-1, is critical for the nor¬ mal expansion of the somatotrope population. The GHRH-R is also necessary for GH synthesis and secretion (see later).

SECRETION NEURAL CONTROL GH secretion is under neural control from the hypothalamus through at least two and possibly three hypophysiotropic factors: GH-releasing hormone (GHRH), somatostatin, and probably ghrelin (see following section). The GHRH neurons are located primarily in the arcuate and ventromedial nuclei, and somatosta¬ tin neurons are located primarily in the anterior periventricular area of the hypothalamus. GHRH and somatostatin release are controlled by a complex and incompletely understood neural network, involving a-adrenergic, dopaminergic, serotoninergic, cholinergic, and histaminergic inputs. In general, a-adrenergic, dopaminergic, serotoninergic, and cholinergic signals stimulate GH secretion. The limbic system plays an important role in GH secretion. A full discussion of the neural pathways regulating GH secretion is beyond the scope of this chapter. From a practical standpoint, it is important to know the physiologic stimuli lead¬ ing to GH secretion, the principal pharmacologic agents used to test GH secretory capacity, and the peripheral feedback control of GH secretion.

GROWTH HORMONE-RELEASING HORMONE AND THE GROWTH HORMONE-RELEASING HORMONE RECEPTOR GHRH is a 40- to 44-amino-acid peptide isolated first from pan¬ creatic tumors that produced it ectopically,16-17 and subse¬ quently from the hypothalamus (Fig. 12-8). Its gene, located on chromosome 20q, encodes a 108-amino-acid precursor from which GHRH is derived by proteolytic cleavage. It is expressed

Ch. 12: Growth Hormone and Its Disorders

GHRH 1-40:

Tyr

Ala

5 '0 15 20 Asp—Ala—lie—Phe—Thr—Asn—Ser—Tyr—Arg—Lys—Val—Leu—Gly—Gin—Leu—Sar—Ala_Arg_

25 30 35 40 Lys—Leu—Uu—Gin—Asp—lie—Met—Ser—Arg-GIn—Gin—Gly—Glu—Ser— Asn-GIn-Glu-Arg-Gly-Als

5 GHRH 1-44:

133

10

15

20

Tyr-Ala-Asp-Ala-lle-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-GIn-Leu-Ser-Ala-Arg35 30 35 40 44 Lys—Leu—Leu—Gin—Asp—lie—Mel—Ser—Arg—Gin—Gin—Gly—Glu—Ser— Asn—Gin—Glu—Arg—Gly—Ala—Arg—Ala—Arg—Leu—NH,

in highest concentration in the hypothalamus, but also in other parts of the brain, in the gut, and in other tissues. The extrahypothalamic role of GHRH is largely unknown; it may act as a sleep inducer. GHRH is released from the median eminence into the pituitary portal system and is the principal stimulatory hypophysiotropic factor promoting GH secretion. (As men¬ tioned earlier, GHRH is also important for the development of somatotrope cells15 and for GH synthesis.18) GHRH, on reach¬ ing the somatotropes, interacts with the GHRH-R, which is a seven transmembrane, Gsa-coupled receptor that signals through the cAMP and Ca2+-channel pathways (Fig. 12-9). Acti¬ vation of these pathways effects GH release from secretory granules as well as GH gene transcription. The GHRH-R is expressed in a variety of tissues, but its biologic role in extrapituitary sites is unknown. GHRH is rapidly inactivated in plasma by an amino peptidase that cleaves the N-terminal dipeptide. Ectopic production of GHRH can occur in carcinoid and pancreatic islet tumors.

SOMATOSTATIN AND SOMATOSTATIN RECEPTORS Somatostatin is a cyclical peptide that exists in two forms: somatostatin-14 and somatostatin-28, the latter being extended at the amino terminus (Fig. 12-10; see Chap. 169). In humans, both somatostatins are encoded by a single gene on the long arm of chromosome 3, and a 92-amino-acid precursor is differ¬ entially processed to the two somatostatin forms, in part in a tissue-specific manner. In the hypothalamus, somatostatin-14 is the predominant form. Somatostatin is widely expressed throughout the central nervous system, the gut, and the pan¬

FIGURE 12-8. Amino-acid sequence of growth hormone-releasing hor¬ mone (GHRH) 1-40 (top) and 1-44 (bottom). GHRH 1-44 has four addi¬ tional amino acids at the C-terminal end of the molecule and is amid a ted.

creas. In extrahypothalamic sites, somatostatin has inhibitory effects on insulin secretion, gut hormone secretion, gut motility, and pancreatic and gastrointestinal exocrine secretions. In the hypothalamic-pituitary system, somatostatin inhibits GH and thyroid-stimulating hormone (TSH) secretion. Five somatosta¬ tin receptor subtypes are known; normal human pituitary expresses subtypes 1, 2, and 5.19 These receptors are members of the seven transmembrane domain, G protein-coupled class. Interaction of somatostatin with its receptors induces coupling to Gj and G0 proteins, which in turn inhibits cAMP production and Ca2+-channel fluxes, thereby blocking release of GH (and other hormones).19

GROWTH HORMONE-RELEASING PEPTIDES AND THE GROWTH HORMONE-RELEASING PEPTIDE RECEPTOR GHRPs are a class of short peptides (5-6 amino acids) that are extremely potent as pharmacologic GH secretagogues. The first prototypes (GHRP-5 and GHRP-6) were described in the early 1980s,20 and many peptide and nonpeptide ana¬ logs have since been synthesized. GHRPs are not entirely specific for GH; they also act to release adrenocorticotropic hormone (ACTH) and prolactin, although the effect on these hormones is relatively modest. The cloning of a specific GHRP receptor in 1996 moved this field from the pharmaco¬ logic to the physiologic realm,21 and a natural ligand, ghrelin, has been identified.213 The GHRP receptor is also a seven transmembrane domain, G protein-coupled receptor that interacts with Gan. It is expressed in the hypothalamus and

®©— NH2 -.0

/T;

00

S ©S$3lu-*Stop

Extracellular

G QOOO

OQO

crY'11\__ OOOO

6 Intracellular

0 COOH —®®®®©0®Svr

*®®0©0® r w®®®®0®00©000®

FIGURE 12-9. Primary structure of the growth hormone-releasing hormone (GHRH) receptor, showing the seven transmembrane helices. The location of a nonsense mutation responsible for familial GHRH-resistant dwarfism is also shown (see text on congenital growth hormone defi¬ ciency). (From Maheshwari HG, Silverman BL, Dupuis J, Baumann G. Phenotype and genetic analysis of a syndrome caused by an inactivating mutation in the growth hormone releasing hor¬ mone receptor: dwarfism of Sindh. J Clin Endo¬ crinol Metab 1998; 83:4065.)

134

PART II: THE ENDOCRINE BRAIN AND PITUITARY GLAND 5

10

14

Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys

Somatostatin-14

Somatostatin-28 5

10

15

20

25

28

Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys

FIGURE 12-10. Amino-acid sequences of somatostatin-14 (top) and somatostatin-28 (bottom). The cyclical nature is indicated by the Cys-Cys bond. to a lesser degree in the pituitary. Ghrelin is expressed in the stomach and hypothalamus; it must be considered as a pot¬ ential candidate for the regulation of GH secretion. Of inter¬ est is that its action on GH secretion is dependent on a functional GHRH system, and that GHRH and GHRP have synergistic actions in vivo. In contrast, the effect of GHRP on ACTH and prolactin release is independent of GHRH. The principal site of action of GHRP on the release of GH, ACTH, and prolactin is the hypothalamus, although for GH a direct, minor effect is also present at the pituitary level. The precise role of the GHRP system in the regulation of GH secretion remains to be determined.

FEEDBACK CONTROL Negative feedback on GH secretion is exerted by IGF-I (a long feedback loop) and by GH itself (a short feedback loop). IGF-I inhibits GH secretion at both the hypothalamic and pituitary levels by influencing GHRH and somatostatin production (hypothalamus) and by interfering with GHRH action (pitu¬ itary). GH inhibits its own secretion by modulating GHRH and somatostatin secretion in the hypothalamus. These feed¬ back effects are superimposed on the neural control men¬ tioned earlier.

OTHER CONTROL MECHANISMS Other important control mechanisms for GH secretion are estrogen- and age-dependent changes. Estrogen has a generally stimulating effect on GH secretion, which results in a distinct sexual dimorphism of GH secretion during the reproductive years. Estrogen can also be used to "prime" a patient to maxi¬ mize response to pharmacologic stimuli. The estrogen effect may in part be mediated by induction of a peripheral GH-resistant state, with lowering of serum IGF-I levels. Pronounced developmental changes occur in GH secretion over the life span. In late fetal and neonatal life, GH secretion is very high and partly unregulated, perhaps in part because of the immaturity of the GHR system and low IGF-I levels. After birth, GH secretion rapidly falls to childhood levels, to be upregulated again during puberty in response to sex steroids. Thereafter, GH secretion declines progressively by -15% per decade, reaching very low levels in old age. This process has been termed "somatopause" and is in part responsible for the body compositional changes associated with aging. Both gen¬ ders are affected by this age-dependent decline. The principal physiologic short-term regulators of GH secre¬ tion are (a) neural endogenous rhythm, (b) sleep, (c) stress, including exercise, and (d) nutritional and metabolic signals. The integrated result of the multiple inputs into the control f GH secretion is a diurnal rhythm of pulsatile secretion that is fa. ly constant in periodicity but varies widely in amplitude. The highest peaks in serum GH are seen during phase IV (slow

wave) sleep, typically 1 to 2 hours after falling asleep. Pulses of smaller amplitude occur throughout the day, on average approximately every 2 hours.22 Many of these pulses are too small to be measured in conventional assays, and perhaps too small to have much biologic activity. Women of reproductive age generally have higher amplitudes as well as higher inter¬ peak GH levels—an effect that has been attributed to estrogen (Fig. 12-11). The extent to which metabolic changes due to inter¬ mittent meals influence GH secretion is unclear; available data suggest that under physiologic circumstances, such effects are minor at best. However, fasting, malnutrition, and obesity have profound effects on GH production (see later). The variability of serum GH levels makes it clear that sampling at single, ran¬ dom time points cannot be used for diagnostic purposes, and that dynamic testing under standardized conditions or diurnal sampling is necessary to arrive at a diagnosis of GH under- or overproduction. There is no known differential secretion or specific stimulus for any of the GH variants. Indeed, they appear to be cosecreted in response to a variety of physiologic or pharmacologic stim¬ uli. However, they have different plasma half-lives, and hence their relative proportions in plasma may differ from that in the pituitary. The average plasma half-life of GH (representing mostly monomeric 22K GH) is -17 minutes.23 The 20K variant and oligomeric forms have longer half-lives. After secretion, GH binds to GHBP in the circulation. This occurs very rapidly, with maximal binding achieved within a few minutes. The amount of GH bound to GHBP varies, depending on the GHBP level in a given person, the GH con¬ centration (which may partially saturate the GHBP), and the time after a secretory pulse. On the average, 40% to 50% of plasma GH is bound to the GHBP.24 The bound fraction has delayed clearance, dampens the oscillations of serum GH, and serves as a circulating GH reservoir. The GHBP level in serum is not influenced by GH pulses; it exhibits no or minimal diur¬ nal variation.

REGULATION OF PLACENTAL GROWTH HORMONE SECRETION GH-V or placental GH is secreted during pregnancy into the maternal (but not fetal) circulation. This process is not regu¬ lated by the factors just described for pituitary GH regulation. The principles regulating GH-V secretion are unknown; it may simply be released constitutively as a function of syncytiotrophoblast mass. Plasma GH-V levels increase progres¬ sively during the second trimester to reach a plateau in the third trimester (Fig. 12-12). Concomitantly, pituitary GH-N levels are suppressed, presumably via negative feedback by GH-V and IGF-I. GH-V binds to GHR with the same affinity as GH-N; its high plasma levels may be responsible for some of the fluid retention and changes in physical features seen in late pregnancy.

Ch. 12: Growth Hormone and Its Disorders

135

Tlme (w**k«) FIGURE 12-12. Plasma levels of the growth hormones GH-N and GHV during pregnancy. Pituitary GH-N is gradually supplanted by pla¬ cental GH-V. (hGH, human growth hormone.) (From Baumann G. Growth hormone heterogeneity: genes, isohormones, variants and binding proteins. Endocr Rev 1991; 12:424; as adapted from Frankenne F, Closset J, Gomez F, et al. The physiology of growth hormones [GHs] in pregnant women and partial characterization of the placental GH variant. J Clin Endocrinol Metab 1988; 66:1171.)

FIGURE 12-11. Characteristic diurnal profiles of serum growth hor¬ mone (GH) levels in a young man (top) and a young woman (bottom). Women of reproductive age have a higher baseline and a greater aver¬ age pulse amplitude than men. The ordinate is logarithmic to empha¬ size the presence of small secretory peaks throughout the day; note that the units for GH are pg/mL. The black bar indicates the period of sleep; the shaded bar shows the 1 ng/mL level that represents the detection limit in many conventional assays; the triangles on top indicate the secretory events as defined by a pulse-detection program. (Adapted from Winer LM, Shaw MA, Baumann G. Basal plasma growth hormone levels in man: new evidence for rhythmicity of growth hormone secre¬ tion. J Clin Endocrinol Metab 1990; 70:1678.)

REGULATION OF THE GROWTH HORMONE RECEPTOR AND BINDING PROTEIN For reasons of accessibility, little is known about the regula¬ tion of GHRs in human tissues. Therefore, much of the infor¬ mation about GHR regulation is based on (a) animal studies and (b) GHBP measurements in humans, using the GHBP as a surrogate for the GHR. Based on the comparison between direct GHR data in animals and GHBP data in humans, the GHBP level in serum appears to be a reasonable index of GHR abundance in tissues, primarily the liver. The main regulator of GHR/GHBP abundance is ontogeny. In fetal and neonatal life, GHR expression is very low, and serum GHBP levels are correspondingly low. This is a physio-

logic GH-resistant condition, with high GH and low IGF-I lev¬ els. Postnatally a rapid up-regulation of the GHR and GHBP occurs, coincident with the emergence of GH responsivity.25 This process continues throughout childhood, until in the late teens GHBP levels (and presumably GHR levels) reach adult levels. GHBP levels remain constant through adult life until approximately age 60, when a progressive decline ensues that continues until the tenth decade.26 This decline is accompanied by a decline in IGF-I levels and constitutes part of the somatopause. Thus, in old age the changes caused by the decreasing GH secretion are further amplified by the development of GH resistance. Similar changes in GHR expression have been shown in aging animals. Women of reproductive age have slightly higher GHBP levels than men. Estrogens, particularly if given orally, up-regulate serum GHBP, whereas androgens tend to lower GF1BP. Interestingly, no discernible change is seen in GHBP level during puberty in either sex. The effect of GH itself on the GHBP in humans is controversial. The majority of studies find no significant change in GHBP levels in GH deficiency or in response to GH treat¬ ment. However, some reports show an up-regulation, and oth¬ ers a down-regulation of GHBP by GH. On balance, the effect of GH on serum GHBP (and probably, hence, on GHR) in humans is neutral or at least inconsistent. This differs from the case in rodents, in which the GHR (and the GHBP) expression is upregulated by GH. Another important regulator of GHR/GHBP expression is nutritional status, probably, at least in part, medi¬ ated by insulin. A strong positive correlation exists between body mass index and serum GHBP, and IGF-I levels and GH responsivity vary in parallel as a function of adiposity and nutritional status.13

ACTIONS OF GROWTH HORMONE Growth hormone has numerous biologic actions, many occur¬ ring in concert to enhance protein anabolism and tissue accre¬ tion. GH can act directly as well as indirectly through IGF-I, also known as somatomedin C. The mitogenic and proliferative actions of GH are mediated through IGF-I, whereas some of the metabolic actions are direct GH effects. GH has no specific tar¬ get organ; it acts on most if not all tissues through the ubiqui¬ tously expressed GHR.

136

PART II: THE ENDOCRINE BRAIN AND PITUITARY GLAND

INSULIN-LIKE GROWTH FACTORS AND THEIR BINDING PROTEINS The existence of GH-dependent factors that might mediate the action of GH was first suggested by the report that S04 incorporation into growth cartilage chondroitin sulfate was reduced by hypophysectomy, but that exposure of cartilage to GH in vitro could not correct the abnormality. In contrast, serum from animals treated with GH was highly effective in restoring S04 uptake, implicating a GH-dependent "sulfation factor."27 Sulfation factor was later renamed somatomedin, and the "somatomedin hypothesis" was born. It soon became clear that somatomedin was identical to "nonsuppressible insulin-like activity," which led to the characterization of IGF-I and IGF-II. The IGFs are proinsulin-like molecules that are produced in many tissues in response to GH and other regulators. IGF-I production is highly GH-dependent, whereas IGF-II production is less dependent on GH. IGFs act both locally in a paracrine/autocrine fashion and distantly in a hormone-like mode. They have mitogenic and metabolic activities and act through the type I IGF receptor, which is structurally similar to the insulin receptor. Six binding pro¬ teins for IGF (IGFBP) are present in serum and interstitial fluid; they may either enhance or decrease IGF activity.28 In addition, three IGFBP-related proteins bind IGFs with low affinity,29 for a total of nine proteins in the IGFBP family. IGFBP-1 is insulin dependent and has primarily inhibitory activity in vivo. IGFBP-2 is inversely GH dependent; its bio¬ logic role is incompletely understood. IGFBP-3 is the major IGFBP in serum; it is highly GH dependent, and it serves pri¬ marily to retain IGFs in the circulation by forming a 150-kDa ternary complex involving IGF, IGFBP-3, and another GHdependent protein called acid-labile subunit (ALS). This complex is responsible for the high IGF concentrations in serum and serves as a circulating IGF reservoir. Most of the circulating IGF is bound in this complex. IGFBP-4, IGFBP-5, and IGFBP-6 are in part associated with the extracellular matrix and modulate IGF action through restricting or enhancing IGF access to the IGF receptor. IGFBP structure and activity is modulated by proteases that cleave the IGFBPs, thereby decreasing IGF-binding affinity. Protease activity is regulated by a variety of physiologic and patho¬ logic conditions. IGFBPs and their fragments may have activities of their own (such as antianabolic activity) that are independent of their IGF-binding properties. A full descrip¬ tion of the complex IGF-IGFBP system is beyond the scope of this chapter; from a clinical standpoint, an understanding of IGFBP-3 and IGFBP-2 is most important because they can be used diagnostically.

INSULIN-LIKE GROWTH FACTOR RECEPTOR IGF-I and IGF-II bind to and signal through the IGF receptor (also known as type I IGF receptor). IGF-II also binds to the mannose-6-phosphate/type II IGF receptor, but to date no con¬ vincing evidence exists that the lysosomal pathway connected to this receptor is relevant to GH action. The type I IGF receptor is structurally homologous to the insulin receptor; it has a tetrameric structure with two extracellular a-subunits covalently connected to two P-subunits through disulfide bonds. The Psubunits have intrinsic tyrosine kinase activity and signal through a phosphorylation cascade involving IRS-1 and IRS-2, PI3-kinase, MAPK, and other pathways. The IGF receptor dif¬ fers functionally from the insulin receptor in that it promotes primarily mitogenic/proliferative rather than metabolic activi¬ ties. Because both receptors share intracellular pathways, it is presently not clear how this relative specificity of actions is conferred. Nevertheless, some crosstalk occurs between the IG and the insulin systems at high ligand concentrations. The IGF receptor is widely expressed in tissues, with the exception

TABLE 12-1. Bioactivities of Growth Hormone (GH) Direct GH Effects

IGF-mediated Effects

Both (or Unknown)

Lipolysis

Chondrocyte clonal expansion

Insulin antagonism

Amino-acid transport in muscle

Sulfate incorporation into cartilage

Islet cell hyperplasia

Acute hypoglycemic action

DNA and RNA syn¬ thesis

Erythropoiesis

IGF-I production

Phosphorus and Na+ retention in kidney

Nitrogen retention

IGFBP-3 and ALS pro¬ duction

Linear growth in bones

Somatic growth*

Prechondrocyte differen¬ tiation

Enhancement of immune function?

Lactogenesis Somatostatin secretion in hypothalamus IGF-I, insulin-like growth factor-I; IGFBP-3, insulin-like growth factor binding pro¬ tein-3; ALS, acid-labile subunit. "Somatic growth is possible but suboptimal with IGF-I alone. GH assists IGF action through several ancillary effects, such as changing in vivo IGF kinetics through IGFBP-3 and ALS production, synthesizing protein in muscle, differentiating prechondrocytes, etc.

of liver, where it is expressed at very low levels. The IGF sys¬ tem is described in detail in Chapter 173. Because of the widespread expression of the GHR, the IGFs, and the IGF receptor, discerning which of the ultimate biologic actions of GH are direct and which are IGF-mediated has been difficult. Table 12-1 lists the principal GH actions and attempts to assign them to direct and IGF-mediated pathways. In many cases, this assignment is tentative, and both direct and indirect mediation may occur. The principal bioactivities of the GH/IGF system relevant to the intact organism and to clinical medicine are nitrogen retention, protein anabolism, linear growth, lipolysis, insulin antagonism (diabetogenesis), Na+ retention, and negative feed¬ back on GH secretion (short- and long-loop feedback). GH is best viewed as the master postnatal anabolic hormone orches¬ trating a cascade of activities leading to lean body mass accre¬ tion. The exception is adipose tissue, in which GH is largely catabolic. Human GH has lactogenic properties because it can bind to prolactin receptors. This is not a property of animal GHs, and its biologic importance in humans is uncertain. Also unclear is the importance of GH for the immune system; no overt abnormality in immune function is seen in cases of GH deficiency or in GH resistance.

MEASUREMENT OF PLASMA GROWTH HORMONE The measurement of GH in blood is problematic because of the heterogeneous nature of GH. This heterogeneity is one reason for the observation that different assay designs can yield different results for the same blood sample.30 Plasma GH is measured either by conventional polyclonal radioimmunoassay or by a variety of monoclonal immunoradiometric or immunoenzymatic assays. Monoclonal assays frequently yield lower readings than polyclonal assays. This is in part due to the fact that some of the GH variants are not fully reactive in monoclonal assays, but other, poorly understood matrix effects are also involved. The problem of nonreproducibility of results among assays and labo¬ ratories can present a diagnostic dilemma in the classification of a patient as GH deficient or normal. Discrepancies are particu¬ larly notable among monoclonal assays. The need exists for a universal standard and assay design that permits comparison of GH determinations among different laboratories.

Ch. 12: Growth Hormone and Its Disorders TABLE 12-2. Dynamic Tests for Growth Hormone Secretion Stimulatory Insulin hypoglycemia

Dose/Administration 0.05-0.10 U/kg iv

L-dopa

Adults: 500 mg po Children: 250 mg po

Arginine infusion GHRH Clonidine

0.5 g/kg iv over 30 min (30 g maximum) 1Hg/kgiv Adults: 250 gg po Children: 125 gg po

Glucagon

1 mg sc or im

GHRP*

Dose depends on type of GHRP (1 gg/kg iv for GHRP-2 or hexarelin)

GHRH-arginine combined

137

The GHRH-stimulation test cannot be used to distinguish reliably between hypothalamic and pituitary GH deficiency. This is similar to the failure of testing with other hypothalamic releasing hormones (gonadotropin-releasing hormone [GnRH], thyrotropin-releasing hormone [TRH], corticotropin-releasing hormone [CRH]) to clearly differentiate hypothalamic from pituitary disease. Pituitary function tests must be interpreted in the context of other clinical assessments. They can yield misleading results in some conditions not associated with hypothalamic-pituitary disease. Examples are obesity, hypothyroidism, or hypercorticism, in which the GH response to all stimuli tends to be blunted. Conversely, malnutrition, catabolic conditions, or stress may be associated with elevated GH levels that are not normally suppressible.

GHRH-GHRP combined* Inhibitory Glucose-tolerance test

75 or 100 g po

iv, intravenous; po, by mouth; sc, subcutaneous; im, intramuscular; GHRH, growth hormone-releasing hormone; GHRP, growth hormone-releasing peptide. ‘Not yet in standard use. Stimulatory tests can be enhanced by pretreatment with estrogen, pyridostigmine, or propranolol.

DYNAMIC TESTS OF GROWTH HORMONE SECRETION Because of the pulsatile nature of GH secretion, plasma GH lev¬ els vary widely in normal subjects. It follows that a single GH measurement is not diagnostic for an abnormality in the GH axis. Therefore, dynamic testing of the response of plasma GH to stan¬ dardized provocative or inhibitory tests is mandatory for the evaluation of GH disorders. Table 12-2 lists the dynamic tests in clinical use. This topic has been comprehensively reviewed.31 Among the provocative tests, the insulin-tolerance test is consid¬ ered the "gold standard," as it is a potent and reliable stimulus for GH release. Its disadvantage is that induction of hypoglyce¬ mia can be risky, and, therefore, the test must be closely super¬ vised. For the test to be valid, a drop in blood glucose by at least 50% from the starting level must be achieved. GHRH testing has not proven to be as useful as anticipated because the GH response is highly variable, presumably because of differences in prevailing somatostatin tone. Clonidine is used successfully in children, but in adults it is a relatively weak stimulus for GH release. In normal subjects, the highest serum GH levels are seen after combined GHRH-GHRP stimulation. However, the clinical utility of this potent test in the diagnosis of GH deficiency is not yet known. For research purposes, circadian sampling of blood for GH measurements is probably the best procedure to assess sponta¬ neous GH secretion. The frequency of sampling should be at least every 20 minutes for accurate estimation of secretion rate. This can be done over a 24-hour period or overnight, when most of the GH secretion occurs. A variant of circadian sam¬ pling is continuous blood withdrawal by a pump. This yields a pooled "average" GH level but does not permit detection of secretory pulses. These maneuvers are cumbersome and impractical for general diagnostic purposes; they are largely reserved for investigational use. An additional limitation is that the GH secretion rate is quite variable among normal subjects, and the boundary between normal and deficient GH secretion rates is ill defined. Measurement of urinary GH excretion has been advocated as a possible estimate of GH secretion rate. This method is techni¬ cally feasible, but only a small fraction (1300 mm3

% 100 96

Excessive sweating

91

Heel-pad thickness >22 mm

91

Weakness

88

Arthralgias

72

Abnormal glucose tolerance test

68

Malocclusion of teeth

68

New skin tags

58

Serum phosphorus >4.5 mg/dL

48

Carpal tunnel syndrome

44

Hypertension (blood pressure >150/95 mm Hg)

37

Fasting plasma glucose >110 mg/dL

30

Serum testosterone (males) 25 ng/mL

16

8 a.m. serum cortisol 200 ng/mL but no pituitary tumor is visible by appropriate MRI, this possibility should be considered.

SYMPTOMS Hyperprolactinemia may be asymptomatic; an elevated serum value may be a clue to the presence of a pituitary tumor or pituitary stalk impingement.36 However, prolactin elevation from any cause may result in signs and symptoms of hypogonadism in men or women.38'39 A history of osteopenia or osteoporosis may be present.

SIGNS

SIGNS

Signs are often very subtle and nonspecific. Affect may be depressed or labile. Patients may be obese with centrally dis¬ tributed fat. Skin may be thin and dry, and extremities may be cool because of poor venous circulation.34 Parental heights may be used to calculate the predicted height of the patient; this may afford an objective criterion on which to base a diagnosis of short stature in cases of childhood-onset GH deficiency (see Chap. 7).

If hyperprolactinemia results in hypogonadism, the usual signs in men and women may occur (see respective sections of this chapter). Gynecomastia may occur in men. Galactorrhea occurs more commonly in women because estrogen priming of breast tissue facilitates this process.

LABORATORY ASSESSMENT The hypothalamus secretes GH-releasing hormone into the pituitary portal circulation. GH-releasing hormone is trans¬ ported to the pituitary, where it stimulates pulsatile GH secre¬ tion into the peripheral circulation. GH acts at liver and other tissues to induce insulin-like growth factor-I (IGF-I) synthesis and secretion. IGF-I is thought to mediate most of the metabolic and growth-enhancing effects of GH (see Chap. 12). GH levels vary significantly throughout the day, and consid¬ erable disagreement exists about what constitutes laboratory confirmation of GH deficiency in adults. One definition of GH deficiency, in a patient with a compatible history, is a negative response to a standard GH stimulation test. A negative response may be taken to mean a peak serum GH of 1 cm, with the superior margin (solid white arroiv) protruding into the suprasellar cistern without coming in con¬ tact with the optic chiasm (open white arroiv). The carotid arteries (solid black arrows) are displaced slightly laterally in the cavernous sinus regions. There does not appear to be clear-cut involvement of the cav¬ ernous sinus. C, Sagittal Tl-weighted non-contrast-enhanced magnetic resonance image. Again, the small macroadenoma is seen filling the sella and remodeling the sella (solid white arrows). The clivus is slightly remodeled, and the posterior clinoids are not well defined. There is an incidental infarct in the pons (open white arrow).

ism or surgical removal of the adrenals (Nelson syndrome; see later in this chapter and Chap. 75). If the functional status of a suspected adenoma or pituitary mass is in question, venous sampling of the petrosal sinuses can be performed by means of a catheter placed from the femoral vein into the internal jugular vein and then advanced into the greater petrosal veins.6 Analy¬ sis of blood samples can help determine the type of adenoma and the location of a lesion not detected by other imaging modalities. Sampling is also of value to demonstrate that the hormone originated from the gland rather than from an ectopic site. Although inferior petrosal sinus sampling is usually per¬ formed, it has been shown that bilateral, simultaneous cavern¬ ous sinus sampling, using corticotropin-releasing hormone, is as accurate as inferior petrosal sinus sampling in detecting Cush¬ ing disease and is perhaps more accurate in lateralizing the abnormality within the pituitary gland.24 The sella is usually normal in size with microadenomas and CT usually demonstrates no bony expansion, although there may be some asymmetry in the shape of the pituitary gland (see Fig. 20-12). CT, using thin-section coronal images and intra¬ venous contrast, has been used successfully to detect microade¬ nomas. The adenoma is identified as either a hypodense or hyperdense region in the gland after contrast enhancement. Cushing disease adenomas are more difficult to detect by CT,

possibly because of their relative enhancement with respect to the normal gland.25 The recommended modality for examining a pituitary ade¬ noma is MRI, with coronal and sagittal imaging. Detection is best with high-resolution techniques, such as three-dimen¬ sional imaging. The coronal plane is the most sensitive imaging plane, and Tl-weighted spin-echo and three-dimensional imag¬ ing sequences are the best pulse sequences. The use of gadolin¬ ium enhancement is somewhat controversial,26-27 although the vast majority of radiologists believe that contrast is essential in the evaluation of the sella and parasellar regions. Usually, the tumors enhance less-than-normal tissue. Dynamic imaging can be of value in defining the abnormal segment of the gland.13 Of the pituitary macroadenomas, a higher percentage of these are nonfunctioning adenomas. Plain radiographs of the skull may demonstrate bony expansion or erosion of the sella; at times, the masses can be huge, with wide destruction of the skull base (to the extent that the site of origin is not clear). Calcifications are rare. The sensitivity for detecting macroadenomas by CT is higher than for microadenomas; the CT examination should use thinsection coronal and axial imaging with intravenous contrast. Generally, the margins of the macroadenomas are more readily defined by MRI than by CT. Involvement of the optic chiasm, cavernous sinus, sphenoid sinus, orbit, temporal lobes, and

Ch. 20: Diagnostic Imaging of the Sellar Region

231

FIGURE 20-15. Invasive pituitary macroadenoma. This coronal contrast-enhanced 3D gradient echo magnetic resonance image through the sella demonstrates a large irregular aggressive skull-base mass (white arrow) with the pituitary gland enhancing diffusely. The pituitary stalk is displaced to the right. The low-signal-intensity left carotid artery (black arrow) is enveloped by the mass and is displaced interiorly. The mass protrudes into the left suprasellar cistern. It is in contact with the left medial temporal lobe after breaking through the left cavernous sinus. The mass is extending through the left foramen ovale into the masticator space.

carotid arteries can all be seen using MRI. In prolactinomas, MRI is used to evaluate the patient's response to bromocriptine ther¬ apy. A decrease in tumor size can be seen as early as 1 week after the start of therapy. Additionally, MRI can detect posttherapy hemorrhage into macroadenomas and mass effect or inferior herniation of the chiasm as a result of a decrease in the tumor size.28 In macroadenomas, subacute hemorrhage is readily detected by MRI because the breakdown products of hemoglo¬ bin have paramagnetic or diamagnetic effects, depending on their chemical composition. Moreover, MRI is good for evaluat¬ ing invasion into the adjacent cavernous sinus and for docu¬ menting the patency of the carotid arteries (see Fig. 20-15).

INFUNDIBULAR MASSES The thickness of the normal pituitary stalk averages 3.5 mm at the median eminence and 2.8 mm near its midpoint. The normal stalk enhances markedly on CT with contrast and on MRI with gadolin¬ ium. The most common clinical problem associated with disease of the pituitary stalk is diabetes insipidus. When this is present, there usually is absence of the normal hyperintensity of the poste¬ rior pituitary. On Tl-weighted MRI, diabetes insipidus may be found to occur as a result of transection of the pituitary stalk. The differential diagnosis of a thickened stalk includes sar¬ coidosis, tuberculosis, histiocytosis X, and ectopic posterior pituitary as well as germinoma. A thickened stalk can also be due to an extension of a glioma within the hypothalamus. In patients with neurosarcoidosis and tuberculous infiltration of the stalk, the chest radiograph is generally abnormal and may be helpful in the differentiation from histiocytosis X. Clinically, patients with histiocytosis X may have skin lesions, otitis media, or bone lesions in addition to interstitial lung disease.u

FIGURE 20-16. Hypothalamic hamartoma. This young girl presented with precocious puberty. A, Non-contrast-enhanced sagittal Tlweighted image through the midline. A small nodule measuring ~5 mm in dimension (solid white arrow) is seen protruding from the under¬ surface of the hypothalamus into the suprasellar cistern just anterior to the mammillary bodies (small black arrowhead). The pituitary gland and infundibulum are normal (open ivhite arrow). There does not appear to be any deformity of the anterior third ventricle or invasion of the brain. B, Axial proton-density magnetic resonance image. The small hamar¬ toma (straight white arrow) is just anterior to the bifurcation of the basilar artery in the suprasellar cistern. The infundibulum is seen directly ante¬ rior to this (small black arrowhead). Other labeled structures include the supraclinoid carotid arteries (thin black arrows) and the posterior cere¬ bral arteries (curved white arrows).

HYPOTHALAMIC HAMARTOMAS A hamartoma of the tuber cinereum usually presents as preco¬ cious puberty in a young child.29 It is important to differentiate this lesion from a hypothalamic glioma because the prognosis for hamartoma is much more favorable. Imaging is best with MRI thin-section coronal and sagittal planes (Fig. 20-16). The findings are usually characteristic: The mass arises from the

232

PART II: THE ENDOCRINE BRAIN AND PITUITARY GLAND

FIGURE 20-17. Metastatic suprasellar and pituitary ependymoma. A, Coronal contrast-enhanced magnetic resonance imaging scan through the sella. The pituitary gland, stalk, and hypothalamus are all infiltrated by an aggressive irregular mass (white arrows). This is a sec¬ ondary CSF seeding metastasis from a primary ependymoma of the lower thoracic cord. This type of pattern can be seen in sarcoid, histio¬ cytosis X, eosinophilic granuloma, lymphoma, leukemia, and carci¬ noma. It is not uncommon that the sellar metastasis presents before the spinal or other primary tumor site. B, Sagittal Tl-weighted midline tho¬ racolumbar spin-echo image. The primary conus ependymoma (white arrows) is seen as an isointense expansile mass of the conus. Note that there is also thickening of some of the lower lumbar roots consistent with other drop metastases.

undersurface of the hypothalamus and is exophytic. The nodu¬ lar mass (90% of patients. Surgery and radiation therapy are not indicated for this condition. PROLACTIN HYPERSECRETION ASSOCIATED WITH OTHER LESIONS Non-prolactin-secreting adenomas and other central nervous system lesions may increase prolactin levels by interfering with normal hypothalamic inhibition. Bromocriptine, 2.5 to 7.5 mg per day, usually normalizes serum prolactin levels in these patients. The normalization of prolactin is usually associated with the resolution of galactorrhea. Amenorrhea and infertility may also resolve with this mode of therapy; however, when the underlying lesion has disrupted the normal hypothalamic pitu¬ itary axis or has destroyed the gonadotropes, use of bromocrip¬ tine does not restore menses or fertility. DETAILS OF BROMOCRIPTINE THERAPY Bromocriptine (2-Br-a-ergocryptine mesylate) is a semisyn¬ thetic ergot alkaloid. It specifically binds to and stimulates dopamine receptors.

238

PART II: THE ENDOCRINE BRAIN AND PITUITARY GLAND

One-third of an oral dose is absorbed, and peak serum levels are reached 1 to 3 hours after oral administration. It is exten¬ sively metabolized by the liver, with the metabolites being excreted almost entirely by biliary secretion.17 Less than 5% of the drug is excreted in the urine. Maximal suppression of pro¬ lactin occurs 6 to 8 hours after a single dose, and suppression may be maintained for 12 to 14 hours. Treatment with bromocriptine should be initiated with a dose of one-half of a 2.5-mg tablet taken with food just before bedtime, followed by a regimen of 1.25 mg given with food every 8 to 12 hours. Less than 1% of treated patients experience a first-dose phenomenon, characterized by marked faintness or dizziness. This is observed most commonly in elderly patients and in those with a previous history of fainting, peripheral vas¬ cular disease, or use of vasodilators. Increases in dosage should be gradual, no more than 2.5 to 5 mg within a period of a few days to 1 week. The total daily dose is usually divided and administered every 8 to 12 hours. Side effects are usually dose related, with a rapid develop¬ ment of tolerance. Many side effects are potentiated by alcohol, the use of which should be avoided in sensitive patients. To tol¬ erate bromocriptine therapy, some patients may need to begin with a dosage of 0.625 mg per day (one-fourth tablet), thereafter increasing the dosage at 1-week intervals. Nausea is the most common side effect and occurs in up to 25% of treated patients. The nausea is usually mild, may be minimized by administration of the drug with food and by the initial use of low doses, and generally improves with time.18 Constipation is also frequently reported, and some patients experience abdominal cramps. Seven patients receiving high doses of bromocriptine for the treatment of acromegaly were reported to have had major gastrointestinal hemorrhage associ¬ ated with peptic ulcer disease (three of these episodes were fatal).18 However, bromocriptine has not been associated with an increased incidence of peptic ulcer disease. A slight decline in blood pressure is commonly observed in treated patients; however, patients usually remain asymptom¬ atic. Mild orthostatic hypotension has also been noted.18 The decrease in blood pressure is probably related to both a relax¬ ation of vascular smooth muscle and central inhibition of sympa¬ thetic tone. As with the gastrointestinal side effects, symptomatic hypotension usually improves with time. Vascular side effects, including digital vasospasm, livedo reticularis, and erythromelalgia, occur infrequently and are usu¬ ally associated with bromocriptine doses that exceed those used in the treatment of hyperprolactinemia. Significant mental changes, including hallucinations, have been noted, most com¬ monly in elderly patients receiving large doses of bromocrip¬ tine. In two patients, a dose of 5 to 7.5 mg of bromocriptine, administered for treatment of hyperprolactinemia, was reported to have caused psychotic delusions. However, one of these patients had a known history of schizophrenia in remission, and the other was under severe emotional stress. Other side effects of bromocriptine include nasal stuffiness, headache, and fatigue. Women taking bromocriptine should be advised to use mechanical contraception and, if pregnancy is desired or sus¬ pected, to discontinue bromocriptine whenever expected menses are >2 days late. Visual fields should be evaluated regu¬ larly during pregnancy. If evidence of tumor enlargement is found, a choice is made between continued observation, treat¬ ment with bromocriptine, or transsphenoidal surgery, depend¬ ing on the status of the individual patient. In the United States, women are usually advised to discontinue bromocriptine ther¬ apy during pregnancy; in Europe, however, treatment is com¬ monly continued. A review of 1410 pregnancies in 1335 women who received bromocriptine while pregnant revealed that the incidence of spontaneous abortions (11.1%) and congenital .momalies (3.5%) was no higher than that seen in the general population.19 In women not taking other fertility agents, a slightly increased incidence of twin pregnancies (1.8%) was

seen. A retrospective study of 64 children bom to 53 mothers who took bromocriptine while pregnant revealed no evidence of adverse effects on motor or psychological development.20

THERAPY WITH OTHER DOPAMINERGIC AGONISTS Several other dopamine agonists have been developed that may be useful in the treatment of hyperprolactinemia. A parenteral formulation of long-acting bromocriptine has been effective, with intramuscular injections given every 4 weeks. Pergolide is an ergoline derivative that can be given once daily in a dose of 50 to 100 pg.21 Although it is similar to bromocriptine in its effec¬ tiveness and side effects, some patients who do not tolerate bro¬ mocriptine may tolerate pergolide.22 The nonergot dopamine agonist quinagolide (CV 205-502) can be administered in dos¬ ages of 0.1 to 0.5 mg per day, with fewer side effects than bro¬ mocriptine or pergolide. Quinagolide was effective in patients who were unable to tolerate bromocriptine and in some patients who failed to respond adequately to bromocriptine.23 Cabergoline is a long-acting ergoline derivative that can be effective when given weekly or biweekly in doses of 0.5 to 2.0 mg. Its efficacy and side effects profile are similar to or better than those of bromocriptine.24 In several studies, tumor shrink¬ age and normalization of prolactin levels have occurred in patients who could not tolerate bromocriptine or failed to respond adequately.25-27

ADRENOCORTICOTROPIC HORMONE HYPERSECRETION When Cushing syndrome is caused by a pituitary tumor (Cush¬ ing disease), transsphenoidal surgery is the treatment of choice.3'28 Radiation therapy, by comparison, is less often suc¬ cessful and may take 1 to 2 years to be effective3 (see Chap. 22). Drug treatment is generally not used as a primary mode of therapy except in patients who refuse surgery or irradiation. However, drug treatment may be appropriate in severely ill patients with marked hypokalemia, psychiatric disturbances, infection, or poor wound healing or in patients awaiting trans¬ sphenoidal surgery. Medical therapy is also useful in reducing cortisol levels and ameliorating symptoms until pituitary irra¬ diation is fully effective. Finally, drug therapy may be useful in patients in whom surgery and radiation therapy have failed. Patients with Cushing disease who are treated by adrenalec¬ tomy may develop large, ACTH-secreting, pituitary macroade¬ nomas (Nelson syndrome). The response of such lesions to both surgery and irradiation has been disappointing. Agents used in the treatment of ACTH hypersecretion can be divided into two classes—those that act centrally to reduce ACTH release and those that act peripherally to reduce cortisol production or block its effect (Table 21-1; see Chap. 75). Cen¬ trally acting agents are preferred if a drug is to be used for pri¬ mary therapy; moreover, they are the only agents appropriate for the treatment of Nelson syndrome. Peripherally acting drugs are the preferred agents for rapid preoperative treatment of severely ill patients awaiting surgery. When the treatment regimen involves the chronic use of peripherally acting drugs, the resultant reduction in cortisol and in negative feedback may be followed by an increase in ACTH hypersecretion, thereby necessitating increased dosages of the drug.

CENTRALLY ACTING DRUGS BROMOCRIPTINE Unlike the excellent results achieved with bromocriptine ther¬ apy in patients with hyperprolactinemia, long-term administra¬ tion of the drug, even at dosages of 20 to 30 mg per day, effectively reduces ACTH hypersecretion in only a few

Ch. 21: Medical Treatment of Pituitary Tumors and Hypersecretory States

239

TABLE 21-1. Treatment of Adrenocorticotropic Hormone Hypersecretion Metabolism

Excretion

Initial Dose

Maximum Dose

Major Side Effects

Bromocriptine (2.5-mg tablets. 5-mg capsules)

Liver

Liver

1.25 mg bid

60 mg/d

Nausea, hypotension

Cyproheptadine (4-mg tablets)

Liver

Kidney

4 mg bid or tid

32 mg/d

Drowsiness, hyperphagia, weight gain

Valproic acid (250-mg capsules)

Liver

Kidney

250 mg tid

1250 mg/d

Nausea and vomiting, hepatic failure, birth defects

CENTRALLY ACTING AGENTS

PERIPHERALLY ACTING AGENTS Metyrapone (250-mg capsules)

Liver

Kidney

250 mg tid

4 g/d

Gastrointestinal irritation, hirsutism, acne

Mitotane (o,p’-DDD) (500-mg tablets)

Liver*

Liver and kidney

500 mg tid

6 g/d

Nausea and vomiting, diarrhea, ataxia, vertigo, som¬ nolence, depression, pruritus, adrenal necrosis

Aminoglutethimide (250-mg tablets)

Lived

Kidney

250 mg qid

2 g/d

Drowsiness, skin rash, nausea and vomiting, ver¬ tigo, depression, hypothyroidism, birth defects

Trilostane (30-mg and 60-mg capsules)

Liver

Kidney

30 mg qid

480 mg/d

Abdominal pain, nausea and vomiting, diarrhea, spontaneous abortion

Ketoconazole (200-mg tablets)

Liver

Liver (kidney)

400 mg bid

1200 mg/d

Nausea and vomiting, hepatotoxicity

bid, twice a day; lid, three times a day; qid, four times a day. ‘Large amounts of active drug are stored in fat. +Most of the drug is not metabolized; primarily renal excretion.

patients.29 Although a single 2.5-mg dose of bromocriptine reduces ACTH levels in -40% of patients, many of these short¬ term responders fail to improve significantly with long-term treatment. Conversely, some patients who fail to respond to a single dose of bromocriptine demonstrate marked improve¬ ment in symptoms and in ACTH hypersecretion with pro¬ longed therapy.30 Neither the pretreatment ACTH and cortisol levels nor the tumor size can be used to predict accurately the response to therapy. CYPROHEPTADINE The antiserotoninergic effect of cyproheptadine hydrochloride is thought to be the mechanism whereby ACTH secretion is reduced; however, this drug also has anticholinergic, antihistaminic, and antidopaminergic effects. Thirty percent to 50% of patients with Cushing disease achieve an initial clinical remis¬ sion with this agent.31 Usually, when the drug is discontinued, elevated cortisol levels and symptomatic disease promptly return. No clinical features can predict which patients will respond to cyproheptadine. Importantly, many authors report poor efficacy and significant side effects with this drug. Occa¬ sionally, patients with Nelson syndrome have been reported to improve with administration of cyproheptadine. VALPROIC ACID The anticonvulsant agent valproic acid (and its derivatives) is a y-aminobutyric acid transaminase inhibitor that decreases ACTH hypersecretion in some patients with Cushing disease or Nelson syndrome. Reduction of tumor size with valproate sodium has been reported in a single instance A The drug is highly protein bound and has a serum half-life of 6 to 16 hours. Capsules should be swallowed whole and not chewed to avoid local irritation to the mouth and phar¬ ynx. Nausea and vomiting are commonly experienced at the time therapy is initiated. Tolerance to these side effects devel¬ ops rapidly, and symptoms may be reduced by administering the drug with meals. Fatal hepatic failure has occurred in sev¬ eral patients receiving this drug as an anticonvulsant agent. Liver function tests should be performed before the initiation of therapy and at regular intervals during the first year. The drug should not be used in patients with a history of liver disease and should be discontinued if evidence of hepatic dysfunction is found. However, hepatic dysfunction has been known to progress even after discontinuation of the drug. An increased incidence of neural tube defects has been reported

in children whose mothers received this agent during the first trimester of pregnancy.

PERIPHERALLY ACTING DRUGS METYRAPONE Metyrapone reduces the production of cortisol by inhibiting 11P-hydroxylation in the adrenal gland. The dosage is titrated to maintain normal serum cortisol levels (which should be evalu¬ ated at multiple intervals throughout the day) or titrated to keep the 24-hour urine free cortisol level within the physiologic range. The maintenance dosage varies from 250 mg three times a day to 1000 mg four times a day.30-33 The metabolism of metyrapone is accelerated by administration of phenytoin (Dilantin). The most common side effect is gastrointestinal irritation, which can be avoided by administering the drug with food. Despite improvement in serum cortisol levels, some women note worsening of hirsutism and acne during therapy.33 Cost and side effects may be reduced and efficacy enhanced by com¬ bining metyrapone with aminoglutethimide, with 1 g per day of each administered in divided doses. Although the manufac¬ ture of metyrapone tablets has been discontinued, capsules remain available from the manufacturer. MITOTAN E Mitotane (l,l-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane or o,p'-DDD) suppresses the function of the zona fasciculata and zona reticularis of the adrenal cortex. The drug has been known to cause necrosis of the adrenal gland, producing acute adrenal insufficiency. Mitotane is inappropriate for rapid treatment because control of cortisol secretion requires 2 to 4 months of therapy.34 It may be useful in the treatment of patients awaiting the full effect of radiation therapy or in those in whom surgery and irradiation have failed.3 30 AMINOGLUTETHIMIDE Aminoglutethimide reduces cortisol production by inhibiting the conversion of cholesterol to A5-pregnenolone. During short¬ term therapy, serum cortisol levels usually are suppressed to less than one-half of pretreatment values. In some patients, glu¬ cocorticoid insufficiency occurs, necessitating concurrent gluco¬ corticoid replacement therapy. When aminoglutethimide is used to treat patients with Cushing disease, a secondary

240

PART II: THE ENDOCRINE BRAIN AND PITUITARY GLAND

increase in ACTH levels frequently leads to escape from accept¬ able control.30 Few patients have been treated for >3 months. Therapy is begun with administration of one 250-mg tablet every 6 hours. This dosage is then increased by 250 mg per day every 1 to 2 weeks until a total daily dose of 2 g is reached. Significant side effects occur in two-thirds of patients treated with this agent. The most frequent effects of the drug include drowsiness, which occurs in 33% of patients; skin rashes, which affect 16%; and nausea and vomiting, which occur in 13%. Other significant side effects include vertigo and depression. In gen¬ eral, side effects decrease with smaller doses and often improve or disappear after 1 to 2 weeks of continued therapy. Skin rashes may represent allergic or hypersensitivity reactions; if these are severe or persistent, the drug should be discontinued. Interfer¬ ence with thyroid hormone synthesis may produce hypothy¬ roidism. Decreased estrogen synthesis may produce menstrual irregularities and increased hirsutism and acne in some women. Two cases of pseudohermaphroditism were reported in female infants of mothers who took this drug while pregnant. Because aminoglutethimide increases dexamethasone metabo¬ lism, hydrocortisone or cortisone acetate is preferred if glucocorti¬ coid replacement therapy is needed. Inhibition of aldosterone synthesis may produce mineralocorticoid deficiency, presenting with orthostatic or persistent hypotension, which may require therapy with fludrocortisone acetate (Florinef). TRILOSTANE Trilostane is an inhibitor of the 3-(3-hydroxysteroid dehydroge¬ nase: A4,A5-isomerase enzyme system. It is generally less effec¬ tive than the agents described earlier, and results are highly variable.35 Therapy is initiated with 30 mg of trilostane four times a day. This dosage is then increased as required to control serum cortisol and urinary cortisol levels, with an increase every 3 to 4 days until a total dose of 480 mg per day is reached. Significant side effects occur in half of treated patients. Gas¬ trointestinal symptoms are the most common of these, with abdominal pain and discomfort being reported in 16% of patients, diarrhea in 17%, and nausea and vomiting in 5%. Trilostane has been reported to decrease progesterone levels, which has led to cervical dilation and termination of pregnancy in some women. KETOCONAZOLE Ketoconazole is an antimycotic agent that decreases serum cor¬ tisol by inhibiting cholesterol synthesis through blockade of the 14-demethylation of lanosterol. Ketoconazole may also inhibit 11-hydroxylation and may decrease the binding of glucocorti¬ coid to its receptor. This drug has been reported to be effective in the treatment of patients with Cushing disease in whom sur¬ gery and other drug therapy have proved unsuccessful.3,30'36 After oral administration, the drug is rapidly absorbed. An acid pH is required for absorption; therefore, in patients who are also taking antacids or antihistaminic H,-inhibitors, the drug should be administered 2 hours after such therapy. Patients with achlorhydria may need to dissolve the tablets in aqueous hydrochloric acid. In serum, the drug is 99% protein bound. In patients with Cushing disease, therapy is initiated with 400 mg of ketoconazole administered every 12 hours for 1 month; this dosage is then decreased to 400 to 600 mg per day. Urinary cortisol levels were reported to decline significantly within 1 day after onset of therapy. In patients receiving con¬ ventional antifungal doses (200-400 mg per day), the most com¬ mon side effects are nausea and vomiting, occurring in 3%, and abdominal pain, occurring in 1.5%. Hepatotoxicity has been reported to occur in 1 in 10,000 treated patients; this condition usually resolves on discontinuation of the drug. However, one fatal case of hepatic necrosis that progressed despite discontin¬ uation of the drug was reported.

GLUCOCORTICOID RECEPTOR ANTAGONIST Mifepristone (RU 486) is a synthetic steroid agonist antagonist that blocks the binding of glucocorticoids to their receptor. It is under investigation as a potential therapeutic agent in the treat¬ ment of Cushing disease.37

GROWTH HORMONE HYPERSECRETION Transsphenoidal surgery remains the treatment of choice for growth hormone-secreting adenomas (see Chap. 23). The over¬ all rate of cure (defined as serum growth hormone levels of 1000 ng/mL. Hyperprolactinemia secondary to a pituitary adenoma has extragonadal manifestations. Recent rapid weight gain is a fre¬ quent complaint of hyperprolactinemic women and occurs with a frequency that suggests a correlation. Correction of hyperprolactinemia, either by surgery or by medical therapy, has been followed by impressive weight loss in many cases, despite no apparent change in dietary habits. Equally impres¬ sive is the incidence of emotional lability, which is often dra¬ matically reversed after the correction of hyperprolactinemia. Studies demonstrate that the estrogen deficiency secondary to hyperprolactinemia causes bone demineralization, sometimes producing secondary complications.10

The first step in the evaluation of a patient with suspected hyperprolactinemia is to obtain a fasting serum prolactin level. The administration of thyrotropin-releasing hormone (TRH) does not consistently distinguish between functional hyperpro¬ lactinemia and actual prolactinoma11 (see Chap. 13). In men whose basal prolactin values exceed 100 ng/mL, establishing a prolactinoma as the cause of the hyperprolactinemia is not diffi¬ cult. In women, hyperprolactinemia (>200 ng/mL) almost invariably indicates a tumor. Caution must be exercised, because prolactin levels as high as 662 ng/mL have been observed to occur in nonsecreting tumors, presumably due to pronounced pressure on the pituitary stalk, which inhibits the transport of prolactin inhibitory factor to the pituitary gland.12 In the author's experience, the diagnosis of prolactinoma in a patient with basal prolactin levels 600 ng/mL. (For such lesions, the cure rate with surgery, even in the most experienced hands, is only 10%.10) Medical therapy is usually effective for long-term control, with normalization of serum prolactin levels. On the other hand, the presence of vision loss complicates the management of such patients. This is because of the concern that such therapy either may fail or may take too long to produce sufficient reduction of tumor volume to relieve the compression of the vision system, which could result in further irreversible vision damage during the trial of medical treatment. Because vision compromise can reverse after surgical treatment, even when the compression is longstanding, some believe that vision compromise is not a contraindication to a trial of medical therapy. Substantial tumor shrinkage can occur within days, leading to improved vision.13'14 Others,5-6 including the author, believe that surgical intervention is indicated in these patients if they are otherwise healthy, because a risk exists of further permanent vision dam¬ age with the less rapid decompression provided by medical therapy. If medical therapy is selected for patients with vision compromise, careful monitoring of vision is essential.13-14

PROLACTIN-PRODUCING MACROADENOMAS •For macroadenomas, operative removal is recommended if visual compromise is present and if the patient's overall medical condition justifies the small risks of surgical intervention. For macroadenomas without compression of the optic apparatus, surgery may be considered if the tumor is 600 ng/mL, medical therapy is recommended initially. A desire for pregnancy com¬ plicates matters, because pregnant patients with macroade¬ nomas may develop complications related to accelerated tumor growth. Because of this concern, such patients may be candi¬ dates for surgery, even if no visual compromise is present.

PROLACTIN-PRODUCING MICROADENOMAS For microadenomas, opinions differ among surgeons concern¬ ing initial treatment. Some believe that all patients should be treated medically, except for those who develop unacceptable side effects to medical therapy or whose tumors are resistant to dopamine agonists.15 Others believe that surgery should be the initial treatment for healthy patients with microadenomas and that bromocriptine, cabergoline, and irradiation should be reserved for cases of surgical failure or for those in whom the risk of surgery is high.5-6 Surgery does not always cure prolactin-producing microad¬ enomas; in particular, tumors with higher prolactin levels have

a greater likelihood of surgical treatment failure. Serious surgi¬ cal complications can occur, although, in experienced hands, the complication rate is 2 Gy per day are associated with higher complication rates, including injury to the optic nerves or chiasm, and hypopituitarism. Thus, a dose of 50±5 Gy is advisable. If the bulk of the tumor is completely removed surgically, leaving only a minimum residue, and if the patient is young, 45 to 50 Gy is advis¬ able. However, if a large residual lesion is present after surgical resection, if recurrent tumor is found, or if significant suprasellar extension is present, a higher dose (50 to 55 Gy depending on tumor bulk) is recommended.12,21,25 As is the case with surgery, radiother¬ apy must be performed by skilled radiotherapists who have accu¬ mulated considerable experience in pituitary irradiation. The major problem after pituitary irradiation (particularly when used as an adjunct to surgery) is the development of partial hypopi¬ tuitarism or panhypopituitarism. Panhypopituitarism develops in approximately half of patients.13 The variably quoted figures are 30% to 45% for ACTH deficiency, 40% to 50% for gonadotropin deficiency, and 5% to 20% for TSH deficiency.12-14 The prevalence of deficiencies increases with the duration of follow-up: 100% of patients are GH deficient, 96% are gonadotropin deficient, 84% are ACTH deficient, and 49% are TSH deficient after a mean follow-up of 8 years.16 Hypothalamic-pituitary dysfunction may take up to 20 years to develop.20 The sensitivity of the hypothalamus and pitu¬ itary to the effects of radiation is well illustrated by the very fre¬ quent occurrence of endocrine dysfunction that is observed in patients irradiated for nasopharyngeal, extracranial, or primary brain tumors, even though these lesions were anatomically distinct from the hypothalamic-pituitary region.19 Accordingly, prolonged and repeated assessment of pituitary function is mandatory after irradiation therapy. This should permit a precise detection of pitu¬ itary deficiencies and the selection of appropriate replacement ther¬ apy. Nevertheless, one should emphasize that hormonal side effects of irradiation therapy, if diagnosed early, are easily managed. Other disadvantages of radiotherapy include a delayed ther¬ apeutic benefit for patients who have hormonally active tumors, irradiation-induced optic neuropathy, cortical injury, and, rarely, irradiation-induced malignancies (e.g., meningi¬ oma, astrocytoma).15,26 The term "radiosurgery" is applied to high-precision local¬ ized irradiation, given in one session. The "gamma knife" is one of these techniques and uses cobalt sources arranged in a hemi¬ sphere and focused onto a central target. High-precision stereo¬ tactic radiosurgery may also be delivered by adjusted linear accelerators. The aim of radiosurgery is to deliver a high dose of irradiation that is more localized than would be achieved with conventional radiotherapy. However, this is possible only for relatively small adenomas (5 mm from the optic chi¬ asm or optic nerves (due to the risk of irradiation-induced optic neuropathy that causes visual impairment). The long-term results of radiosurgery on hypersecretion, as on subsequent tumor growth, are presently unknown27 (also see Chap. 22).

THERAPEUTIC OPTIONS AND RESULTS BY TYPE OF PITUITARY ADENOMA The recommendations for treatment of the different types of adenomas are summarized in Table 24-2.

GROWTH HORMONE-SECRETING PITUITARY ADENOMAS THERAPEUTIC OPTIONS Surgery and Radiotherapy. Surgery and radiotherapy, as previously summarized, are commonly used in the treatment of acromegaly.

Ch. 24: Pituitary Tumors: Overview of Therapeutic Options

267

TABLE 24-2. Multidisciplinary Treatment Decisions Based on Tumor Type in Pituitary Adenomas Surgery

Radiation Therapy

Chemotherapy

PRL-SECRETING ADENOMAS Microadenoma Macroadenoma

TSS **

or

NR **

DA agonists* DA agonists

GH-SECRETING ADENOMAS Microadenoma

TSS

Macroadenoma

TSS

and/or

Somatostatin ana¬ logs*

and/or

ART*

Mitotane§

and/or

ART§

Mitotane

and

ART

and

ART'1

ACTH-SECRETING ADENOMAS Microadenoma

TSS

Macroadenoma

TSS

and

CLINICALLY NFPA Microadenoma

TSS

Macroadenoma

TSS

PRL, prolactin; TSS, transsphenoidal surgery; DA, dopamine; NR, not recommended; GH, growth hormone; ACTH, adrenocorticotropic hormone; ART, adjuvant radiotherapy; NFPA, nonfunctioning pituitary adenoma. *DA agonists, either as first-line therapy or, in case of persistent hyperprolactinemia, after surgery. '’TSS not recommended as first-line treatment. May be proposed in case of resistance to DA agonists, often followed by radiation therapy. tSomatostatin analogs and/or radiation therapy are proposed when surgery has failed to cure GH hypersecretion. §Mitotane, either alone or in combination with radiation therapy, is proposed when surgery has failed to cure Cushing disease. “•ART is proposed in cases of invasive tumor postoperative remnant.

Medical Treatment. Bromocriptine and other dopamine ago¬ nists are able to improve symptoms of acromegaly in a few patients and to decrease GH secretion.28'29 Somatostatin, the hypothalamic GH-release inhibitory factor and its analogs, SMS 201-995 (octreotide) and BIM 23014 (lanreotide), are able to reduce GH secretion. The native somatosta¬ tin peptide has a half-life that is too short for it to be administered easily. However, octreotide, given subcutaneously three times daily, has been shown to control GH hypersecretion and to decrease tumor volume in a significant proportion of patients with acromegaly with relatively few side effects.30-36 The availability of a long-acting form of octreotide allows oncemonthly intramuscular injections with the same efficacy.37 Another somatostatin analog, lanreotide, when encapsulated in microspheres, has a prolonged release; it has proved to be effec¬ tive in lowering levels of GH and insulin-like growth factor-I (IGF-I), and often in decreasing the tumor mass of acromegalic patients, in a manner comparable to that of octreotide.38-41 The side effects of somatostatin analogs are benign. Digestive prob¬ lems (i.e., abdominal cramps, diarrhea, flatulence) are minor and most often transitory. Cholelithiasis occurs in 10% to 55% of patients, with the incidence related to the duration of the study.30'33'34'36 Generally, it is asymptomatic and is treated con¬ servatively. Despite the reduction in insulin secretion due to the use of somatostatin analogs, glucose-tolerance alterations are of minor significance. Somatostatin analogs are very expensive drugs and need to be given for the remainder of life. Importantly, scintigraphy after administration of labeled octreotide (somatostatin-receptor scintigraphy) allows for the visu¬ alization of pituitary tumors.42 The resulting images are thought to reflect the concentration of the somatostatin recep¬ tors that are present at the surface of the tumor cells. However, scintigraphic findings are poor predictors of long-term results of treatment with somatostatin analog, regardless of the type of pituitary adenoma43 (also see Chap. 169). CRITERIA OF CURE OF ACROMEGALY The results of the various modes of therapy for acromegaly should be analyzed according to stringent criteria. Currently, “cure" (or good control) of acromegaly is defined by plasma GH levels: the mean of sequential sampling or the nadir after oral glucose administration should be 40 mL/kg per 24 hours), while in children the output is greater (>100 mL/kg per 24 hours). Three pathophysiologic conditions result in diabetes insipidus. An absolute or partial deficiency of vasopressin secretion from the neurohypophysis in response to normal osmotic stimulation is termed hypothalamic diabetes insipidus. This disorder is also known as cranial, central, or neurogenic diabetes insipidus. Patients with hypothalamic diabetes insipidus generally have

286

PART II: THE ENDOCRINE BRAIN AND PITUITARY GLAND

normal thirst sensation. Their basic abnormality is insufficient cir¬ culating antidiuretic activity, which is the principal, but not the sole, cause of their polyuria. Diabetes insipidus secondary to decreased renal sensitivity to the antidiuretic effect of vasopressin circulating in normal or high concentrations is usually called nephrogenic diabetes insipidus. Again, these patients rely on normal thirst sensation to regulate water balance. The third mechanism leading to diabetes insipidus is the ingestion of excessive volumes of fluid, which results in suppression of vasopressin release and consequent polyuria. This condition is referred to as dipsogenic diabetes insipidus, sometimes termed primary polydipsia. A decrease in maximal urine-concentrating ability occurs after prolonged periods of polyuria, regardless of the primary cause. The passage of large amounts of dilute urine through the distal nephron removes solute from the renal medullary interstitium, a process known as the washout phenomenon.1'2 The osmotic gradient across the collecting tubular cell, which is essential for the antidiuretic action of vasopressin, is decreased. Thus, any of the three pathophysiologic mechanisms responsi¬ ble for diabetes insipidus may lead to an additional defect that complicates the interpretation of diagnostic tests based on indi¬ rect assessment of the antidiuretic action of vasopressin. In contrast to hypothalamic diabetes insipidus, which is usually the result of a loss of neurosecretory neurons, the chronic hyperosmolar syndromes are frequently the conse¬ quence of a defective thirst mechanism. Thirst osmoreceptors may fail to respond to hypertonicity, which results in hypodipsia. Because the putative thirst osmoreceptors are believed to be in proximity to the osmoreceptors that regulate vasopressin secretion, a defect in osmotically mediated vasopressin release is often associated with hypodipsia. Polyuria is rarely a feature of hyperosmolar syndromes, because many patients secrete small amounts of vasopressin that are sufficient to concentrate urine to some extent, and nonosmotic factors regulating vaso¬ pressin secretion often remain intact. In view of this characteris¬ tic difference between diabetes insipidus and hyperosmolar syndromes, these conditions are discussed separately.

TABLE 26-1. Causes of Diabetes Insipidus HYPOTHALAMIC DIABETES INSIPIDUS Familial Hereditary (usually autosomal dominant) Association of diabetes insipidus with diabetes mellitus, optic atrophy, nerve deafness, atonia of bladder and ureters (DIDMOAD)

Acquired Head trauma Neurosurgery Tumors (craniopharyngioma, germinoma, metastatic deposits in hypo¬ thalamus) Granulomatous disease (tuberculosis, histiocytosis, sarcoidosis, Wegener granulomatosis) Infections (encephalitis, meningitis) Infundibuloneurohypophysitis Vascular disorders (Sheehan syndrome, aneurysms, thrombotic throm¬ bocytopenic purpura) Circulating antibodies to vasopressin (secondary to Pitressin injection) Pregnancy Autoimmunity Idiopathic

NEPHROGENIC DIABETES INSIPIDUS Familial X-linked recessive: V2 receptor gene Autosomal recessive: aquaporin-2 gene

Acquired Chronic renal disease Metabolic disease (hypokalemia, hypercalcemia) Drugs (lithium, demeclocycline) Osmotic diuresis Pregnancy

DIPSOGENIC DIABETES INSIPIDUS Idiopathic Associated with psychosis Sarcoidosis Autoimmune (multiple sclerosis) Drug induced (lithium, tricyclic antidepressants)

DIABETES INSIPIDUS ETIOLOGY In theory, any of a series of defects in the vasopressin neurosecre¬ tory process can be implicated as the cause of hypothalamic dia¬ betes insipidus.3 Abnormalities may arise in the osmoreceptor that controls vasopressin secretion, even when the thirst osmore¬ ceptor is spared. Alternatively, abnormalities may involve the synthesis and packaging of vasopressin (including genetic defects), damage to the vasopressinergic neurons, or disorders of neurohypophyseal hormone release. Enhanced inactivation of vasopressin by circulating degrading enzymes or antibodies is another potential cause of decreased antidiuretic activity. In practice, however, most cases of permanent hypothalamic diabetes insipidus are caused by damage to the hypothalamoneurohypophyseal area. The most common causes of this con¬ dition are listed in Table 26-1.4 FAMILIAL ABNORMALITIES Studies have revealed exciting data regarding a variety of genetic abnormalities found on chromosome 20 in several kin¬ dreds with autosomal dominant hypothalamic diabetes insipi¬ dus. The first report on different families showed single nucleotide substitutions in the region coding for neurophysin (glycine to serine at position 57).5 This mutation is presumed to interfere with the normal vasopressin-neurophysin tetramer complex formation that occurs in the packaging and transport of vasopressin to the neurohypophysis. Two groups investigat¬ ing another extended family discovered a nucleotide substitu-

tion in the signal peptide (alanine to threonine at position -l).6'7 The signal peptide directs the prohormone to the endoplasmic reticulum, where it is cleaved. Both groups speculate that the mutation alters the cleavage mechanism, resulting in abnormal processing of the prohormone. After the description of the first genetic abnormalities causing familial hypothalamic diabetes insipidus, more than 22 different kindreds with unique genetic mistakes have been documented8 (Fig. 26-1). The genetic basis of the DIDMOAD (diabetes insipidus, dia¬ betes mellitus, optic atrophy, deafness), or Wolfram, syndrome is less well understood, although some evidence exists that it is a disorder of mitochondrial DNA.9-10 TRAUMA Closed head trauma or frank damage to the pituitary stalk or hypothalamus as a result of surgical intervention is often the cause of a form of diabetes insipidus that usually presents within 24 hours of injury. In -50% of cases of post-traumatic diabetes insipidus, the condition resolves spontaneously within a few days. Permanent diabetes insipidus develops in another 30% to 40% of these patients, and the remainder exhibit a tripha¬ sic response to injury. In the last group, the onset of polyuria is abrupt and the condition lasts a few days. It is followed by a period of antidiuresis that may last 2 to 14 days before perma¬ nent diabetes insipidus develops. This triple response to injury is believed to be attributable to release of the vasopressin that is stored in granules.11 Recognition of this entity by clinicians

Ch. 26: Diabetes Insipidus and Hyperosmolar Syndromes INTRON

287

INTRON

FIGURE 26-1. Vasopressin gene, vasopressin pre¬ cursor molecule, and vasopressin with its specific neurophysin. Three exons encode for the precursor molecule, which comprises a signal protein, vaso¬ pressin hormone, neurophysin, and a glycoprotein moiety coupled by amino acids. Mutations in the vasopressin gene have been located in all parts of the precursor molecule except vasopressin itself. (Vp, vasopressin.)

should help to prevent inappropriate treatment that would result in hyponatremia during the second of the three phases. TUMORS

Tumors of the anterior pituitary rarely cause diabetes insipidus. In a series of >100 cases of hypothalamic diabetes insipidus, 13% were attributable to tumors, which included glioma, germinoma, and craniopharyngioma.12 In children, a central tumor is a frequent cause of hypothalamic diabetes insipidus, account¬ ing for -25% of cases; in this population, the most common intracranial tumor is germinoma.13 Metastatic deposits in the hypothalamus causing diabetes insipidus usually arise from carcinoma of the breast or bronchus. GRANULOMATOUS DISEASE

Granulomatous disease accounts for only a few cases of diabe¬ tes insipidus in adults (i.e., sarcoidosis, tuberculosis). However, in children with granulomatous disease, histiocytosis X may cause as many as 40% of pediatric cases. IDIOPATHIC

Idiopathic hypothalamic diabetes accounts for -25% of all cases.12 One-third of patients with apparent idiopathic disease have circulating antibodies to the vasopressin-producing cells in the hypothalamus, a finding which suggests an autoimmune origin for the disorder.14 Some patients have an acute lympho¬ cytic infiltration of the infundibulum and neurohypophysis that can be demonstrated on open biopsy and subsequently resolves.15 NEPHROGENIC DIABETES INSIPIDUS

Mild forms of nephrogenic diabetes insipidus are relatively common (see Table 26-1). Mechanisms responsible for renal resistance to the antidiuretic effect of vasopressin may occur at one or more of the many different sites in the chain of biochem¬ ical responses to vasopressin.11,15a Chronic renal disease second¬ ary to numerous conditions, many drugs (e.g., lithium156), prolonged electrolyte disturbances from hypokalemia, and hypercalcemia account for most cases of nephrogenic diabetes insipidus. The inherited forms of nephrogenic diabetes insipi¬ dus are rare, and cause severe polyuria, dehydration, and fail¬ ure to thrive in the young. With the identification of the V2 (antidiuretic) receptor gene on the X chromosome, a variety of substitutions, mutations, or premature stops have been isolated in kindreds with this disorder that cause defects in the trans¬ membrane V2 receptor.16 Studies involving three other families with congenital nephrogenic diabetes insipidus have shown

autosomal inheritance of the disorder, in contrast to the more common X-linked form due to genetic mutations of the V2 receptor gene localized to the Xq28 region of the long arm of the X chromosome. The autosomal form is due to novel genetic mutations of the gene encoded for the vasopressin-sensitive water channel protein, aquapor in-2, which is located in the col¬ lecting tubules.17 DIPSOGENIC DIABETES INSIPIDUS

Dipsogenic diabetes insipidus, also called primary polydipsia or habitual water drinking, is often psychogenic in origin. The course of polyuria in psychotic patients is variable, with fluctu¬ ations in polydipsia and urine volumes occurring over the years. Occasional patients with hypothalamic diabetes insipi¬ dus who are treated with antidiuretic preparations continue to have polydipsia and, consequently, run the risk of developing hyponatremia. Whether these patients continue to drink because of habit or because of a hypothalamic lesion affecting the thirst osmoreceptor is unknown. A few structural abnor¬ malities resulting in increased thirst have been reported. Drugs that cause dryness of the mouth (e.g., thioridazine hydrochlo¬ ride) may increase drinking but do not result in true polydipsia, in contrast to lithium, which can stimulate thirst directly. CLINICAL FEATURES In adults, the major clinical manifestations of diabetes insipidus include the frequent passage of large volumes of dilute urine (often both day and night), excessive thirst, and increased fluid ingestion. Patients with mild degrees of diabetes insipidus may consider their symptoms to be so minimal that they fail to seek medical attention. However, the severity of diabetes insipidus varies widely, with 24-hour urine volumes ranging from 2.5 to 20 L. Even with the most extreme forms of the disorder, patients maintain their water balance as long as thirst appreciation remains intact and adequate volumes of fluid are ingested. The onset of the disease occurs at any time from the neonatal period to old age, and the sex distribution is approximately equal, although one large review of adults with hypothalamic diabetes insipidus reported a slight male preponderance (60%:40%).12 In infants, diabetes insipidus usually presents with evidence of chronic dehydration, unexplained fever, vom¬ iting, neurologic disturbance, and failure to thrive.13 Enuresis, sleep disturbances, and difficulties at school are the most com¬ mon presenting complaints in older children. Usually, no growth retardation or failure to enter puberty occurs. Affected children from families with histories of diabetes insipidus often do not complain; they regard their polydipsia and polyuria as

288

PART II: THE ENDOCRINE BRAIN AND PITUITARY GLAND

the norm. Once hypothalamic diabetes insipidus develops, it rarely goes into remission spontaneously. Patients with hypothalamic diabetes insipidus also may have anterior pituitary dysfunction (see Chap. 17), particularly if their disorder resulted from trauma to or a tumor in the hypothalamo-neurohypophyseal area. Even patients with the idio¬ pathic form of the disease frequently have endocrinologic evidence of anterior pituitary dysfunction, which suggests a more generalized hypothalamic disorder.12 Glucocorticoid defi¬ ciency secondary to impaired corticotropin secretion or to pri¬ mary adrenal disease leads to impairment of the ability of the kidneys to excrete a water load and to dilute urine maximally. At least two mechanisms are responsible for this defect. One involves the distal nephron, which remains partially imperme¬ able to water in the absence of glucocorticoid; the other involves persistent vasopressin secretion, possibly secondary to a reset¬ ting of the osmostat. A similar abnormality of water excretion has been reported with severe thyroid hormone deficiency (see Chap. 45). Thus, impairment of anterior pituitary function can mask hypothalamic diabetes insipidus, which becomes appar¬ ent only when the hypopituitarism is adequately treated. Routine skull radiographs are rarely helpful in patients with hypothalamic diabetes insipidus but nuclear magnetic reso¬ nance imaging (MRI) can be extremely useful. Tl-weighted magnetic resonance imaging of the neurohypophysis produces a characteristic hyperintense signal in healthy persons that dis¬ appears in most patients with hypothalamic diabetes insipi¬ dus.18 The infundibular stalk frequently is thickened in the early phase of the idiopathic form of the disorder, as demon¬ strated by both MRI and computed tomographic scanning.15

DIFFERENTIAL DIAGNOSIS Polyuria may be defined as tire excretion of >2.5 L of urine per 24 hours on two consecutive days, provided that patients are allowed free access to and drink water ad libitum. Once polyuria has been demonstrated, the clinician's first responsibility is to establish the pathophysiologic mechanism—dipsogenic diabetes insipidus, hypothalamic diabetes insipidus, or nephrogenic diabetes insipi¬ dus. Defining the underlying disease process is then important. INDIRECT TESTS Before the development of plasma assays that were capable of detecting low physiologic concentrations of vasopressin, indi¬ rect methods were used to assess the antidiuretic activity of the hormone. The classic diagnostic approach is to measure the responses of urinary osmolality and flow rate to a period of dehydration and, subsequently, to the administration of an exogenous vaso¬ pressin preparation. Various dehydration tests have been described in which changes in plasma and urine osmolalities in patients with polyuria were compared to the responses of healthy persons.19-21 In theory, differentiation between hypo¬ thalamic diabetes insipidus, nephrogenic diabetes insipidus, and dipsogenic diabetes insipidus should be readily possible, but the disorders actually can be diagnosed correctly only in certain circumstances. Frequently, tests yield equivocal results. For example, although patients with primary polydipsia might be anticipated to have significantly lower plasma osmolalities and sodium concentrations than patients with hypothalamic or nephrogenic diabetes insipidus, such a distinction is useful diagnostically in only a few cases.22 The reason for the lack of differentiation is the wide variation in the setpoint of the osmo¬ regulatory mechanisms for thirst and vasopressin secretion. Even after fluid deprivation and the administration of exoge¬ nous vasopressin, urine osmolality frequently fails to attain normal values (Fig. 26-2) because of the secondary nephrogenic diabetes insipidus induced by prolonged polyuria, as explained earlier. A similar ambiguity arises in the interpretation of

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FIGURE 26-2. Urine osmolality is depicted under basal conditions after a period of fluid deprivation (Kydropenia) designed to attain maximum urinary concentration, and after intramuscular injection of 5 U of vaso¬ pressin (Pitressin). The test group included healthy subjects (stippled area) and patients with dipsogenic diabetes insipidus (primary polydip¬ sia, PP), hypothalamic diabetes insipidus (HDI), or nephrogenic diabe¬ tes insipidus (NDI). The bars represent the range of results and the closed circle indicates the mean value for the group. (Adapted from Robertson, GL. Diagnosis of diabetes insipidus. In: Czernichow P, Rob¬ inson AG, eds. Diabetes insipidus in man. Frontiers of hormone research, vol 13. Basel: S Karger, 1985:176.) results from patients with hypothalamic diabetes insipidus. In theory, these patients, who have low circulating concentrations of vasopressin, should demonstrate a substantial increase in urine osmolality to the normal range in response to exogenous vasopressin. In practice, however, they fail to do so (see Fig. 262). Again, the reason for the inadequate urinary response lies in the washout of solute from the renal medullary interstitium. The greater the 24-hour urine volume, the greater is the degree of renal resistance to antidiuretic activity.2 The large overlap in urine osmolality values with these tests is illustrated explicitly in Figure 26-2. The refinement of dehydration tests by calcula¬ tion of free water clearance adds little to their ability to distin¬ guish among the causes of diabetes insipidus. Other means of evaluating the osmoregulatory system using indirect methods to assess antidiuretic activity in an attempt to identify the cause of polyuria (i.e., infusion of hypertonic saline23) also fail to establish an unequivocal diagnosis for the reasons described earlier, as well as because the saline load induces a solute diure¬ sis. Thus, indirect tests of vasopressin function are considerably limited in their ability to establish the cause of polyuria. DIRECT TESTS The introduction of sensitive and specific radioimmunoassays capable of detecting low physiologic concentrations of vaso¬ pressin in plasma not only has clarified and simplified the diagnosis of diabetes insipidus, but also has extended the under¬ standing of its underlying pathophysiologic mechanisms. The measurement of plasma vasopressin, plasma osmolality, and urine osmolality under basal conditions affords little diagnostic discrimination. However, after osmotic stimulation by a period of fluid deprivation, an infusion of hypertonic saline, or both, the estimation of these indices provides a precise diagnosis. PLASMA VASOPRESSIN IN THE DIAGNOSIS OF DIABETES INSIPIDUS Hypothalamic Diabetes Insipidus. Hypothalamic diabe¬ tes insipidus is recognized by the subnormal plasma concentra¬ tions of vasopressin in relation to plasma osmolality that occur in affected persons (Fig. 26-3A). A clear distinction may be made between patients with hypothalamic diabetes insipidus, patients with nephrogenic diabetes insipidus or dipsogenic dia-

Ch. 26: Diabetes Insipidus and Hyperosmolar Syndromes

289

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20

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400

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280

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betes insipidus, and healthy persons by assessing plasma osmolality as it is increased by the infusion of hypertonic saline.24 Many patients with diabetes insipidus have detectable plasma vasopressin, which represents the partial form of the disorder. The ability to secrete vasopressin at high plasma osmolality levels partially explains the ability of some patients to generate a concentrated, if submaximal, urine after fluid dep¬ rivation. A few patients fail to exhibit detectable immunoreactive plasma vasopressin, despite marked increases in plasma osmolality. However, some of these patients still manage to concentrate their urine to some extent, suggesting that their kidneys are particularly sensitive to very low concentrations of vasopressin. Occasionally, patients with hypothalamic diabetes insipidus clearly demonstrate osmotically regulated vaso¬ pressin release (see Fig. 26-3A). In such patients, the theoretic threshold for vasopressin release, obtained from the abscissal intercept of the osmoregulatory line (the function relating plasma, vasopressin to plasma osmolality), is normal, but the slope that defines the sensitivity of osmotically regulated vaso¬ pressin release is significantly reduced. Thus, the osmoreceptor controlling vasopressin secretion is probably intact in this group of patients. Some patients who have been treated with parenteral neuro¬ hypophyseal extract (Pitressin) have developed antibodies to vasopressin. These patients exhibit "vasopressin" in their plasma as a direct result of assay interference, but this can be recognized by laboratory testing simply through detection of plasma-binding activity to synthetic vasopressin. Therefore, screening for vasopressin antibodies in all patients treated with Pitressin is wise to prevent spurious plasma results. After hypertonic saline infusion, patients with primary poly¬ dipsia or nephrogenic diabetes insipidus have plasma vaso¬ pressin and plasma osmolality values that fall within the normal reference range (see Fig. 26-3A). A supranormal plasma vasopressin response to rising plasma osmolality has been demonstrated in a few patients. Whether this response is attrib¬ utable to a state of chronic underhydration is not known. Thus, hypothalamic diabetes insipidus is clearly distinguishable from other forms of diabetes insipidus by relating plasma vaso¬ pressin to plasma osmolality after osmotic stimulation. Nephrogenic Diabetes Insipidus Versus Primary Polydip¬ sia. Analysis of the relationship between plasma vasopressin and urine osmolality after a period of fluid deprivation offers a potential means of differentiating nephrogenic diabetes insipidus from dipsogenic diabetes insipidus (see Fig. 26-3B). Patients with nephrogenic diabetes insipidus have plasma vasopressin concen¬ trations that are inappropriately high in relation to urine osmolal¬ ity. However, prolonged polyuria from any cause can induce renal resistance to vasopressin, with blunting of the maximal uri¬

6

FIGURE 26-3. A, Plasma osmolality and vaso¬ pressin responses to infusion of 5% hypertonic saline solution in representative patients with hypothalamic diabetes insipidus (HDl, •—•), nephrogenic diabetes insipidus (NDI, ■—■ ), and dipsogenic diabetes insipidus (DDI, A—▲ ). B, Plasma vasopressin and urine osmolality responses to a period of dehydration in patients with hypothalamic diabetes insipidus (•), nephro¬ genic diabetes insipidus (■), and dipsogenic diabe¬ tes insipidus (A). The responses of healthy subjects are indicated by the stippled areas. The limit of detection of the vasopressin assay (LD) was 0.3 pmol/L (1 pmol/L = 1.1 pg/mL).

nary concentration in response to vasopressin. This difficulty can be partially overcome by examining the basal values of plasma vasopressin and urine osmolality. Plasma vasopressin tends to be detectable or even elevated in nephrogenic diabetes insipidus, whereas immunoreactive vasopressin is generally undetectable in primary polydipsia2 (see Fig. 26-3B). The administration of exogenous vasopressin after a period of fluid deprivation does not help to discriminate further between the causes of polyuria. Close examination of the data regarding plasma vasopressin and urine osmolality in patients with partial hypothalamic dia¬ betes insipidus reveals an inappropriately high urine osmolal¬ ity in relation to the low plasma vasopressin concentrations (see Fig. 26-3B). This observation confirms the earlier impression that the renal tubule may become extraordinarily sensitive to vasopressin. Because sustained polyuria from any cause induces a state of secondary partial nephrogenic diabetes insip¬ idus, the administration of exogenous vasopressin to patients with partial hypothalamic diabetes insipidus fails to induce maximal urinary osmolality. A systematic study to compare the diagnostic efficacy of indirect tests with direct measurement of osmotically stimu¬ lated vasopressin release has clearly demonstrated that direct plasma vasopressin measurement methods are superior.25 If the clinician does not have ready access to suitable assays for plasma vasopressin measurement, a satisfactory diagnosis may be established using a closely monitored, prolonged thera¬ peutic trial with desmopressin, administered intramuscularly in daily dosages of 1 |ig for as long as 7 days, preferably while patients are hospitalized. Patients with hypothalamic diabetes insipidus who undergo this regimen show an improvement in the degree of polyuria and a reduction in polydipsia. At the end of the trial, a standard water deprivation test, followed by the administration of exogenous vasopressin using indirect assess¬ ment methods, may demonstrate maximal urinary concentra¬ tions that are within the normal reference range. This is because, during the period of the trial, the renal medullary interstitial solute concentration was restored. Patients with pri¬ mary polydipsia experience progressive hyponatremia and gain weight because they continue to drink fluid despite persis¬ tent antidiuresis. Some of these patients run the risk of neuro¬ logic disturbances, particularly seizures, secondary to the development of sudden, profound hyponatremia. Finally, patients with nephrogenic diabetes exhibit little, if any, improvement in thirst or polyuria. Even the most carefully conducted therapeutic trial, how¬ ever, can yield misleading results. For example, a few patients who have had severe hypothalamic diabetes insipidus for many years develop water intoxication when first treated with vasopressin because they initially fail to reduce their water

290

PART II: THE ENDOCRINE BRAIN AND PITUITARY GLAND

intake appropriately, and therefore they appear to have pri¬ mary polydipsia. Thus, with the currently available diagnostic investigations, the measurement of plasma vasopressin levels with plasma and urine osmolalities during osmotic stimulation provides the most reliable method for determining the cause of polyuria.

THIRST IN HYPOTHALAMIC DIABETES INSIPIDUS The thirst mechanism in patients with hypothalamic and neph¬ rogenic diabetes insipidus generally operates normally.26 Such patients rely on an intact thirst appreciation to maintain water balance. In one study that documented the osmolar threshold for the onset of thirst, no significant difference was found between the mean value for a group of 11 patients with hypo¬ thalamic diabetes insipidus and that for 11 healthy persons; however, the range of values was wider in the affected patients. Only a few patients who are treated with vasopressin and who have inappropriate persistent thirst develop episodes of hyponatremia. When thirst appreciation is blunted, hypernatre¬ mia develops.

DIABETES INSIPIDUS AND PREGNANCY Normal human pregnancy is associated with subtle changes in osmoregulation. Plasma osmolality falls by 8 to 10 mOsm/kg due to a lowering of the osmolar thresholds for both thirst and vasopressin release,27 and a small reduction occurs in maximal urinary concentrating ability. Established hypothalamic diabetes insipidus appears to have little effect on fertility, gestation, delivery, and lactation in humans.28 A few studies have demonstrated that the secretion of oxytocin is normal in patients with diabetes insipidus. By contrast, many pregnant patients with hypothalamic diabetes insipidus notice a worsening of their polyuria and polydipsia.29 The mechanisms responsible for this may include depression of the osmolar thirst threshold to levels at which vasopressin secretion is suppressed further; circulating vasopressin that is degraded by the placental enzyme cystine aminopeptidase; and renal resistance to vasopressin, which is increased. Some patients show no change in their polyuria, whereas a few improve. Transient central and nephrogenic diabetes insipidus associ¬ ated with pregnancy has been documented in some patients30'31 and has been found to recur in subsequent pregnancies. Whether this is attributable to an exaggeration of normal physi¬ ologic adaptation to pregnancy or represents a distinct disease entity remains unresolved.

TREATMENT Hypothalamic diabetes insipidus can be treated by the inges¬ tion of adequate volumes of water, with patients relying on the quenching of thirst as the sole indicator of sufficient intake. Some patients who have had severe polyuria since childhood may prefer to manage their symptoms in this manner, thereby avoiding the need for medication. They organize their lives around the inconveniences of frequent micturition and copious drinking. However, good reasons exist why all patients with moderate to severe polyuria should be treated more actively. Prolonged, severe polyuria can result in distention and atonia of the bladder and hydroureter, and, eventually, in hydro¬ nephrosis with consequent renal damage. Susceptibility to potassium deficiency is another potential risk. Furthermore, untreated patients, if deprived of fluid for any reason, are at risk for the development of life-threatening hypernatremia and dehydration. In young children, withholding therapy may lead to failure to thrive. For these reasons, as well as for the relief of symptoms, antidiuretic treatment is advised for patients with 24-hour urine volumes exceeding 4 L.

Hormone replacement therapy using arginine vasopressin, a natural endogenous peptide, is inappropriate for most patients with hypothalamic diabetes, regardless of whether it is admin¬ istered parenterally or intranasally. The peptide has a short half-life and may be associated with significant pressor side effects that render the use of aqueous vasopressin preparations impractical. However, during the last three decades, consider¬ able advances have been made in the development of synthetic vasopressin analogs with various agonist and antagonist activi¬ ties to the pressor (V,) and antidiuretic (V2) receptors. One class of analogs with minimal pressor activity but increased antidi¬ uretic potency and some resistance to degradation in vivo has been developed to treat hypothalamic diabetes insipidus. The current drug of choice is desmopressin.32'33 For adults, it can be administered orally, 50 to 400 gg one to three times daily; intra¬ nasally, 5 to 40 gg once or twice daily; or parenterally, 0.5 to 2.0 gg daily. A wide variation is seen in individual desmopressin requirements for the control of polyuria. For children, the dos¬ age is halved. Desmopressin is not associated with pressor ago¬ nist side effects but carries the potential hazard of dilutional hyponatremia if patients continue to drink inappropriately despite persistent antidiuresis. If desmopressin proves to be too potent, it can be diluted. Alternatively, a shorter-acting preparation—lysine vasopressin— can be administered intranasally. However, because it possesses pressor activity, it may induce vasoconstriction, angina, or renal and intestinal colic when taken in excess. If desmopressin is unavailable, it still may be possible to obtain vasopressin tannate in oil, a crude extract of bovine neurohypophysis containing arginine vasopressin suspended in peanut oil. Before intramus¬ cular injection, this preparation should be warmed and shaken vigorously until the extract is evenly distributed in the oil. A sin¬ gle dose of 5 to 10 IU provides as much as 72 hours of antidiure¬ sis. However, this agent is associated with pressor side effects similar to those of lysine vasopressin, an erratic absorption rate, and the formation of sterile abscesses. Pitressin also has been administered as a nasal insufflation. Now that desmopressin is established as the drug of choice, little need exists to prescribe the partially effective oral agents— chlorpropamide, carbamazepine, clofibrate, or thiazide diuret¬ ics—because all are associated with significant and sometimes dangerous side effects. Rarely, direct treatment of the underlying cause of hypotha¬ lamic diabetes insipidus relieves the symptoms. Documented examples include corticosteroid therapy for hypothalamic sar¬ coidosis, cyclophosphamide therapy for Wegener granulo¬ matosis, and radiotherapy for metastatic disease of the hypothalamus. Effective treatment of nephrogenic diabetes insipidus still poses problems, except for the forms that are drug induced or related to metabolic disorders (see Table 26-1). The latter are fre¬ quently reversible after withdrawal of the drug or correction of the metabolic disturbance. Profound polyuria secondary to the familial forms of this disease is particularly difficult to treat. Restriction of sodium intake, combined with the administration of a thiazide diuretic, reduces urine output by almost 40% in infants. A similar reduction in urine flow may be achieved with the prostaglandin synthetase inhibitor indomethacin when it is administered in dosages of 1.5 to 3.0 mg/kg. The most promis¬ ing results are achieved with the administration of a combined regimen of thiazide, indomethacin, and desmopressin, which reduces diuresis by as much as 80%.

HYPEROSMOLAR SYNDROMES Hyperosmolar or hypematremic syndromes may be defined as plasma osmolality levels and sodium concentrations of >300 mOsm/kg and >145 mEq/L (145 mmol/L), respectively. Although rare, they constitute a major management challenge.

Ch. 26: Diabetes Insipidus and Hyperosmolar Syndromes TABLE 26-2. Specific Causes of Hypodipsic Hypernatremia VASCULAR Anterior communicating artery aneurysm (ligation) Intrahypothalamic hemorrhage Internal carotid artery ligation NEOPLASTIC Primary (craniopharyngioma, pinealoma, meningioma, chromophobe adenoma) Metastatic (lung, breast) GRANULOMATOUS Histiocytosis Sarcoidosis Tuberculosis MISCELLANEOUS Hydrocephalus Head injury Toluene exposure Idiopathic Old age (Adapted from Robertson GL, Aycinena P, Zerbe RL. Neurogenic disorders of osmoregulation. Am J Med 1982; 72:339.)

ETIOLOGY Transient hyperosmolality may occur after the ingestion of large amounts of salt,34 but most hypernatremic states occur after inadequate water intake. This can occur in any healthy individual in whom the combination of excess fluid loss—from skin, gastrointestinal tract, lungs, or kidneys—and inadequate access to water is found. This occurs most commonly in acute illness in which water intake is compromised by vomiting or impaired consciousness and most vividly in patients with dia¬ betes insipidus, before treatment, or when access to water is denied. In other cases, however, hypernatremia reflects a pri¬ mary disorder of thirst deficiency (hypodipsia). A number of conditions are associated with hypodipsia (Table 26-2). One of the more common causes of hypodipsic hypernatremia that the authors have seen is ligation of the ante¬ rior communicating artery, after subarachnoid hemorrhage from a berry aneurysm. Other centers have reported that neo¬ plasms account for 50% of such cases.35 Craniopharyngiomas are particularly associated with hypodipsic diabetes insipidus, sometimes in conjunction with other hypothalamus-related dis¬ orders, such as polyphagia, weight gain, and abnormal ther¬ moregulation. Survivors of diabetic hyperosmolar coma have been shown to have impaired osmoregulated thirst/11 which suggests that hypodipsia contributes to the development of the hypernatremia, which is characteristic of the condition. In almost every case of hypodipsia, associated abnormalities of vasopressin secretion are seen, a finding that reflects the close anatomic proximity of the osmoreceptors for vasopressin secre¬ tion and thirst.

CLINICAL FEATURES In young children and the elderly, hypernatremia may be asso¬ ciated with significant degrees of dehydration.36 Infants are at particular risk, and the mortality is high. In this clinical situa¬ tion, signs are seen of extracellular fluid loss, decreased skin turgor and elasticity, dry and shrunken tongue, tachycardia, and orthostatic hypotension. Affected infants have depressed fontanelles and tachypnea, and their respirations are deep and rapid. Fever is often present, and the temperature may be as high as 40.5°C (105°F). Adults with mild hypernatremia may have no symptoms, but as plasma sodium levels rise above 160 mEq/L, neurologic signs become apparent.36'37 Early symptoms

291

include lethargy, nausea, and tremor, which progress to irrita¬ bility, drowsiness, and confusion. Later features of muscular rigidity, opisthotonus, seizures, and coma reflect generalized cerebral and neuromuscular dysfunction. The most severe neu¬ rologic disturbances are seen at both ends of the age spectrum. The severity of such disturbances is also related to the rate at which hypernatremia develops, as well as to the absolute degree of hyperosmolality. Intracerebral vascular lesions are often the cause of death. In contrast to patients with the life-threatening clinical features of hypernatremic dehydration, patients with long-standing, mod¬ erate hypernatremia (plasma sodium concentrations of 145 to 160 mEq/L) may have few manifestations of the disorder other than lack of thirst. Hypodipsia is the crucial symptom, but it is often overlooked in the clinical setting because patients fail to complain of lack of thirst. However, careful evaluation of these patients reveals that some have no desire to drink any fluid under any circumstances, which suggests a total loss of the thirst osmoreceptor function. Others have only minimal thirst with marked hypertonicity, whereas a third group eventually experiences a normal thirst sensation, but only at high plasma osmolality levels. The key to recognizing subtle differences in thirst apprecia¬ tion rests with a satisfactory measure of thirst. Visual analog scales for measuring thirst during dynamic tests of osmo¬ regulation38-39 have been shown to produce highly reproducible results.40 When these scales are used in evaluating healthy per¬ sons, a linear increase is noted in the degree of thirst and fluid intake with increase in plasma osmolality, and an osmolar threshold for thirst is seen that is a few milliosmoles per kilo¬ gram higher than the osmolar threshold for vasopressin secre¬ tion.39 The application of these techniques to patients with chronic hypernatremia has disclosed numerous disorders of osmoregulation.

OSMOREGULATORY DEFECTS IN CHRONIC HYPERNATREMIA Chronic hypernatremia is characterized by inappropriate lack of thirst despite increased plasma osmolality and mild hypo¬ volemia. Plasma sodium concentrations are typically elevated (150-160 mmol/L) and may reach extremely high concentra¬ tions during intercurrent illnesses (e.g., gastroenteritis) in which body water deficits increase. Although adipsic hyper¬ natremia is uncommon, four distinct patterns of abnormal osmoregulatory function have been described. Type 1 Adipsia. The characteristic abnormalities in type 1 adipsia are subnormal vasopressin levels and thirst responses to osmotic stimulation (Fig. 26-4). The sensitivity of the osmore¬ ceptors is decreased, producing partial diabetes insipidus and relative hypodipsia. Because some capacity remains to secrete vasopressin and experience thirst, such patients are protected from extremes of hypernatremia, as they can produce nearmaximal antidiuresis as plasma osmolality increases. Patients with this type of adipsia usually have normal vasopressin responses to hypotension and hypoglycemia, and show sup¬ pression of vasopressin secretion, with the development of hypotonic diuresis in response to water loading. Type 2 Adipsia. Total ablation of the osmoreceptors pro¬ duces complete diabetes insipidus and absence of thirst in response to hyperosmolality. This is the pattern of osmoregula¬ tory abnormality seen after surgical clipping of aneurysms of the anterior communicating artery,41-42 and despite the com¬ plete absence of osmoregulated thirst and vasopressin release, thirst and vasopressin responses to hypotension and apomorphine are preserved.41-43 Some patients also develop this type of osmoregulatory dysfunction after surgery for large, suprasellar craniopharyngiomas. Interestingly, these patients also have absent baroregulated thirst and vasopressin secretion--pre¬ sumably because the extent of surgical injury is such that both

292

PART II: THE ENDOCRINE BRAIN AND PITUITARY GLAND Patients also have intact nonosmotic release of vasopressin and increased renal sensitivity to vasopressin, so that renal concen¬ trating ability may be reasonably well maintained. Miscellaneous Causes of Adipsia. Osmoregulatory dys¬ function has also been reported in elderly patients, who have diminished thirst in response to hypernatremia.38 Although the defect in thirst appreciation is similar to that in type 1 dysfunc¬ tion, vasopressin responses have variously been reported as being subnormal, normal, or enhanced. Survivors of diabetic hyperosmolar, nonketotic coma have also been reported to have hypodipsia with exaggerated vasopressin secretion.35 In addi¬ tion, a single case has been reported of a young patient who had hypodipsia but a normal osmotically regulated vasopressin release.48 All of these reports lend support to the hypothesis that the osmoreceptors subserving vasopressin release are ana¬ tomically and functionally distinct from those controlling thirst.

TREATMENT

Plasma Osmolality (m Osm/kg) FIGURE 26-4. Thirst and vasopressin responses to osmotic stimulation in adipsic hypernatremia. Type 1: subnormal response of both thirst and vasopressin secretion. Type 2: total lack of response of thirst and vaso¬ pressin secretion. Type 3: reset of osmostat for vasopressin release and thirst to the right of normal. Shaded areas indicate the response ranges in healthy control subjects; the dotted lines are the mean regression lines. (pAVP, plasma arginine vasopressin; LD, limit of detection of the pAVP assay [0.3 pmol/L]).

the osmoreceptors and the paraventricular and supraoptic nuclei are damaged. Patients with complete adipsic diabetes insipidus have no defense against dehydration, and unless they are closely supervised and trained to drink even in the absence of thirst, they can develop profound hypematremic dehydra¬ tion, even in the absence of intercurrent illness. Interest has been shown in the concept that osmoreceptor activity is under bimodal control; that is, a specific stimulus is required to switch off vasopressin secretion in the same way that elevation of plasma osmolality stimulates vasopressin secre¬ tion. Patients with complete osmoreceptor ablation clearly are unable to respond to inhibitory inputs; this has been demon¬ strated in clinical studies in which complete suppression of the secretion of the small quantities of radioimmunoassayable vasopressin or the achievement of maximal free water clear¬ ance during water loading was impossible in a patient with this type of osmoregulatory dysfunction.44 Therefore, in some patients vasopressin secretion may not be entirely suppressed during fluid loads, resulting in significant hyponatremia. Type 3 Adipsia. The osmostats for thirst and vasopressin release may be reset to the right of normal (tvpe 3 in Fig. 26-4), such that vasopressin secretion and thirst do not occur until higher plasma osmolalities are reached. Thereafter, the slope of the osmoregulatory lines are normal. This pattern is found in conjunction with a number of cases of "essential" hypernatre¬ mia, although type 1 defects have also been reported.45-47

Water replacement is the basic therapy for patients with hyper¬ osmolar states associated with dehydration. The oral route is preferred, but if the clinical situation warrants urgent treat¬ ment, the infusion of hypotonic solutions may be necessary. However, overzealous rehydration with hypotonic fluids may result in seizures, neurologic deterioration, coma, and even death secondary to cerebral edema.34'37 Therefore, the decision to treat with hypotonic intravenous fluids should not be made lightly, and rehydration to a euosmolar state should proceed cautiously over at least 72 hours. As plasma osmolality falls, polyuria indicative of hypothalamic diabetes insipidus may develop; this responds to administration of desmopressin. For patients with chronic hyperosmolar syndromes (see Fig. 26-4), longer-term therapy must be considered. Patients with type 3 defects rarely need specific therapy because their osmo¬ regulatory system is essentially intact but operates around a higher than normal plasma osmolality. Patients with type 1 defects (involving partial destruction of the osmoreceptor) should be treated with a regimen of increased water intake (2-4 L every 24 hours). If this leads to persistent polyuria, a small dose of desmopressin can be administered, but plasma osmolality or sodium levels must then be monitored regularlv. Considerable difficulties arise in treating patients who have complete destruction of their osmoreceptors (type 2 defect), because these patients cannot protect themselves from extremes of dehydration and overhydration. Most patients need between 2 and 4 L of fluid per day, but the precise amount varies accord¬ ing to seasonal climatic changes, and the body weight must be monitored daily to provide an index of fluid balance.44 Regular (usually weekly) measurements of plasma osmolality or sodium are needed to ensure that no significant fluctuations occur in body water, and constant supervision is required to make certain the requisite volume of water is consumed. Despite the most vigorous supervision, such patients are extremely vulnerable to swings in plasma osmolality and are particularly prone to severe hypematremic dehvdration.

REFERENCES 1. De Wardener HE, Herxheimer A. The effect of high water intake on the kidneys' ability to concentrate urine in man. J Physiol (Lond) 1957; 139:42. 2. Robertson GL. Diagnosis of diabetes insipidus. In: Czemichow P, Robinson AG, eds. Diabetes insipidus in man. Frontiers of hormone research, vol 13. Basel: S Karger, 1985:176. 3. Maffly RH. Diabetes insipidus. In: Andreoli TE, Grantham JJ, Rector FCJ, eds. Disturbances in body fluid osmolality. Bethesda, MD: American Phys¬ iology Society, 1977:285. 4. Robertson GL. Diabetes insipidus. Endocrinol Metab Clin North Am 1995; 24:549. 5. Ito M, Mori Y, Oiso Y, Saito H. A single base substitution in the coding region for neurophysin II associated with familial central diabetes insipi¬ dus. J Clin Invest 1991; 87:725.

Ch. 27: Inappropriate Antidiuresis and Other Hypoosmolar States 6. Krishnamani MRS, Philips PA III, Copeland KC. Detection of a novel argi¬ nine vasopressin defect by dideoxy fingerprinting. J Clin Endocrinol Metab 1993; 77:596. 7. McLeod JF, Kovacs L, Gaskill MB, et al. Familial neurohypophyseal diabe¬ tes insipidus associated with a signal peptide mutation. J Clin Endocrinol Metab 1993; 77:599A. 8. Heppner C, Kotzka J, Bullmann C, et al. Identification of mutations of the arginine vasopressin-neurophsia II gene in two kindreds with familial cen¬ tral diabetes insipidus. J Clin Endocrinol Metab 1998; 83:693. 9. Rotig A, Cormier V, Chatelain P. Deletion of the mitochondrial DNA in a case of early-onset diabetes mellitus, optic atrophy and deafness. Wolfram syndrome (MIM 222300). J Clin Invest 1993; 91:1095. 10. Barrett TG, Bundey SE. Wolfram (DIDMOAD) syndrome, J Med Genet 1997; 34:838. 11. Verbalis JG, Robinson AG, Moses AM. Postoperative and post-traumatic diabetes insipidus. In: Czernichow P, Robinson AG, eds. Diabetes insipi¬ dus in man. Frontiers of hormone research, vol 13. Basel: S Karger, 1985:247. 12. Moses AM. Clinical and laboratory observations in the adult with diabetes insipidus and related syndromes. In: Czernichow P, Robinson AG, eds. Diabetes insipidus in man. Frontiers of hormone research, vol 13. Basel: S Karger, 1985:156. 13. Baylis PH, Cheetham T. Diabetes insipidus. Arch Dis Child 1998; 79:84. 14. Scherbaum WA, Bottazzo GF. Autoantibodies to vasopressin cells in idio¬ pathic diabetes insipidus: evidence for an autoimmune variant. Lancet 1983; 1:897. 15. Imura H, Nakao K, Shimatsu A, et al. Lymphocytic infundibuloneurohypophysitis as a cause of central diabetes insipidus. N Engl J Med 1993; 329:683. 15a. Knoers NV, Monnens LL. Nephrogenic diabetes insipidus. Semin Nephrol 1999; 19:344. 15b. Bendz H, Aurell M. Drug-induced diabetes insipidus. Drug Saf 1999; 21:449. 16. Bichet DG, Bimbaumer M, Louergan M, et al. Nature and recurrence of AVPR2 mutations in X-linked nephrogenic diabetes insipidus. Am J Hum Genet 1994; 55:278. 17. Hochberg Z, van Lieburg A, Even L, et al. Autosomal recessive nephro¬ genic diabetes insipidus caused by an aquaporin-2 mutation. J Clin Endo¬ crinol Metab 1997; 82:686. 18. Sato N, Ishizaka H, Yagi H, et al. Posterior lobe of the pituitary in diabetes insipidus: dynamic MR imaging. Radiology 1993; 186:357. 19. Dashe AM, Cramm RE, Crist CA, et al. A water deprivation test for the dif¬ ferential diagnosis of polyuria. JAMA 1963; 185:699. 20. Miller MT, Dalakos T, Moses AM, et al. Recognition of partial defects in antidiuretic hormone secretion. Ann Intern Med 1970; 73:721. 21. Baylis PH. Diabetes insipidus. Medicine 1997; 25:9. 22. Robertson GL. The regulation of vasopressin function in health and dis¬ ease. Recent Prog Horm Res 1977; 33:333. 23. Moses A, Streeten D. Differentiation of polyuric states by measurement of responses to changes in plasma osmolality induced by hypertonic saline infusions. Am J Med 1967; 42:368. 24. Baylis PH, Robertson GL. Vasopressin response to hypertonic saline infu¬ sion to assess posterior pituitary function. J R Soc Med 1980; 73:255. 25. Zerbe RL, Robertson GL. A comparison of plasma vasopressin measure¬ ment with a standard indirect test in the differential diagnosis of polyuria. N Engl J Med 1981; 305:1539. 26. Thompson CJ, Baylis PH. Thirst in diabetes insipidus: clinical relevance of quantitative assessment. Q J Med 1987; 65:853. 27. Davison JM, Gilmore EA, Diirr J, et al. Altered osmotic thresholds for vaso¬ pressin secretion and thirst in human pregnancy. Am J Physiol 1984; 246:F105. 28. Amico J. Diabetes insipidus in pregnancy. In: Czernichow P, Robinson AC, eds. Diabetes insipidus in man. Frontiers in hormone research, vol 13. Basel: S Karger, 1985:266. 29. Hime MC, Richardson JA. Diabetes insipidus and pregnancy: case report, incidence and review of the literature. Obstet Gynecol Surv 1978; 33:375. 30. Barron WM, Cohen LH, Ulland LA, et al. Transient vasopressin-resistant diabetes insipidus of pregnancy. N Engl J Med 1984; 310:442. 31. Hughes JM, Barron WM, Vance ML. Recurrent diabetes insipidus associ¬ ated with pregnancy: pathophysiology and therapy. Obstet Gynecol 1989; 73:462. 32. Cobb WE, Spare S, Reichlin S. Diabetes insipidus: management with DDAVP (l-desamino-8-D-arginine vasopressin). Ann Intern Med 1978; 88:183. 33. Williams TDM, Dungar DB, Lyon CC, et al. Antidiuretic effect and pharma¬ cokinetics of oral 1-desamino-8-D-arginine vasopressin. 1. Studies in adults and children. J Clin Endocrinol Metab 1986; 63:129. 34. Ross EJ, Christie SBM. Hypernatremia. Medicine (Baltimore) 1969; 48:441. 35. McKenna K, Morris AM, Azam H, et al. Subnormal osmotically stimulated thirst and exaggerated vasopressin release in human survivors of hyperos¬ molar coma. Diabetologia May 1999; 42:538. 36. Robertson GL, Aycinena P, Zerbe RL. Neurogenic disorders of osmoregula¬ tion. Am J Med 1982; 72:339. 37. Arieff AL, Guisado R. Effects on the central nervous system of hypernatremic and hyponatremic states. Kidney Int 1976; 10:104. 38. Phillips PA, Rolls BJ, Ledingham JGC, et al. Reduced thirst after water dep¬ rivation in healthy elderly men. N Engl J Med 1984; 311:753.

293

39. Thompson CJ, Thompson J, Burd J, Baylis I'll. The osmotic threshold for thirst and vasopressin release are similar in healthy men. Clin Sci 1986; 71:651. 40. Thompson CJ, Selby P, Baylis I’ll. Reproducibility of osmotic and nonosmotic tests of vasopressin secretion in men. Am J Physiol 1991; 260:R533. 41. Pearce SHS, Argent NB, Baylis PH. Chronic hypernatremia due to impaired osmoregulated thirst and vasopressin secretion. Acta Endocrinol (Copenh) 1991; 125:234. 42. Mclver B, Connacher A, Whittle A, et al. Adipsic diabetes insipidus after clipping of anterior communicating artery aneurysm. BMJ 1991; 303:1465. 43. Teelucksingh S, Steer CR, Thompson CJ, et al. Hypothalamic syndrome and central sleep apnea associated with toluene exposure. Q J Med 1991; 286:185. 44. Ball SG, Vaidja B, Baylis PI1. Hypothalamic adipsic syndrome: diagnosis and management. Clin Endocrinol 1997; 47:405. 45. De Rubertis FR, Michelis Ml, Beck N, et al. "Essential" hypernatremia due to ineffective osmotic and intact volume regulation of vasopressin secre¬ tion. J Clin Invest 1971; 50:97. 46. Dunger DB, Seckl JR, Lightman SL. Increased renal sensitivity to vaso¬ pressin in two patients with essential hypernatremia. J Clin Endocrinol Metab 1987; 64:185. 47. Gill G, Baylis PI I, Burn J. A case of "essential" hypernatremia due to reset¬ ting of the osmostat. Clin Endocrinol (Oxf) 1985; 22:545. 48. Hammond DN, Moll GW, Robertson GL, Chelmicks-Schorr E. Hypodipsic hypernatremia with normal osmoregulation of vasopressin. N Engl J Med 1986; 315:433.

CHAPTER

27

INAPPROPRIATE ANTIDIURESIS AND OTHER HYPOOSMOLAR STATES JOSEPH G. VERBALIS

FREQUENCY AND SIGNIFICANCE OF HYPOOSMOLALITY J lypoosmolality of plasma is relatively common in hospitalized patients. The incidence and prevalence of hypoosmolar disor¬ ders depend on the nature of the patient population being stud¬ ied and on the laboratory methods and diagnostic criteria used. Most investigators have used the serum sodium concentration f[Na*i) to determine the clinical incidence of hypoosmolality. When hyponatremia is defined as a serum [Na+] of «_

between half-sites. N refers to nucleotides and arrows indicate direc¬ tions to half-sites on the sense strand.

THYROID-HORMONE RECEPTOR BINDING TO THYROID HORMONE RESPONSE ELEMENTS TRs bind to TREs as monomers, dimers, and heterodimers in vitro.8,9,18 However, the functional roles of these putative com¬ plexes in transcriptional regulation are not well understood. Although TRs can form heterodimers with several other family members on TREs, their major heterodimer partner appears to be the retinoid X receptor (RXR), which bears some amino-acid homology with the retinoic acid receptor and binds 9-cis retinoic acid.8,9,18 At least three major isoforms of RXR exist, so possibly dif¬ ferent TR/RXR-isoform complexes may have differential affinities for TREs and/or abilities to transactivate target genes. TR/RXR heterodimers are likely to be involved in TH-mediated transcriptional regulation. In in vitro experiments, T3 caused rapid dissociation of TR homodimers from TREs (direct repeats and inverted palindrome) but had little effect on the binding of TR/RXR heterodimer to TREs,9 results which suggest that the lat¬ ter complexes may be involved in transcriptional activation. On the other hand, both TR homo- and heterodimers bind to TREs in the absence of ligand and, thus, could be involved in repression of basal transcription by TRs (see the following).

REPRESSION OF BASAL TRANSCRIPTION/COREPRESSORS Unliganded TRs bind to TREs and can repress transcription of positively regulated target genes (Fig. 31-5). Although the physio¬ logic significance of this basal repression is not known, it may influence target-gene expression during early fetal development when the fetal thyroid gland is incapable of producing TH. The basal repression may be due, in part, to interactions with the basal transcription machinery as unliganded TRs can interact with tran¬ scription factor IIB (TFIIB), a key component of the basal transcrip¬ tion complex.19 However, several laboratories have cloned proteins that interact with TR and retinoic acid receptor (RAR) in the absence of their cognate ligands.11,12 These proteins repressed basal transcription by TR and RAR and have been termed corepres¬ sors. One of these corepressors is a 270-kDa protein termed nuclearreceptor corepressor (N-CoR). It has two transferable repression domains and a carboxy-terminal a-helical interaction domain. A truncated version of N-CoR, N-CoRi, which is missing the repres¬ sor region, also has been identified and may represent an alterna¬ tive-splice variant of N-CoR.2'1 This protein blocks basal repression by N-CoR and thus may serve as a natural antagonist for N-CoR. Another corepressor, silencing mediator for RAR and TR (SMRT), is a 168-kDa protein that has some homology with N-CoR.11,12 The hinge region of TR (located between the DNA- and ligand-binding domains) is important for interactions with

0) Q

FIGURE 31-5. Model for repression, derepression, and transcriptional activation by thyroid-hormone receptor (TR). (RXR, retinoid X receptor; T3, triiodothyronine.)

corepressors. Mutations in this region decrease interactions with corepressors and abrogate basal repression, without affect¬ ing transcriptional activation.11,12 Several groups showed that corepressors can complex with another putative corepressor, mSin3, and histone deacetylase.21,22 These findings suggest that local histone deacetylation may play an important role in basal repression by altering the local chromatin structure near the minimal promoter region (where transcription is initiated). In negatively regulated target genes, ligand-independent activation of transcription occurs in the absence of TH.23,24 This activation may determine a "set point" from which liganddependent negative regulation begins. The mechanism of the ligand-independent activation is not known but may also involve TR interaction with corepressors.20,23

TRANSCRIPTIONAL ACTIVATION BY THYROID HORMONE COACTIVATORS Many factors potentially can modulate TH-mediated transcrip¬ tion. These include variability among TR isoforms, TR com¬ plexes, heterodimerization partners, nature of TREs, and TR phosphorylation state.8,23a,23b These factors likely influence interactions of liganded TR with TR-associated nuclear proteins called coactivators. Several such proteins have been identified that interact with liganded TR and enhance TH-mediated tran¬ scriptional activation.11,12 A 160-kDa protein called steroid recep¬ tor coactivator-1 (SRC-1)25 has been identified that interacts with steroid-hormone receptors and TR. SRC-1 mRNA undergoes alternative splicing to generate multiple SRC-1 isoforms, although the functional significance of these SRC-1 isoforms currently is not known.26 Several other 160-kDa proteins also interact with liganded nuclear-hormone receptors and TRs, as well as share partial sequence homology with SRC-1. These findings suggest that a family of coactivators related to SRC-1 may exist.11,12 Some studies have suggested that interaction of coactivators with nuclear-hormone receptors involves the carboxy-terminal AF-2 subregion, although other subregions of the TR ligand-binding domain also may be involved. As seen in Figure 31-6, these putative coactivators have sev¬ eral common features. First, multiple putative nuclear-hormone receptor interaction sites are present, which have a signature LXXLL sequence motif.11,12 Several coactivators also have a poly¬ glutamine region, similar to that found in androgen receptors. In addition, a bHLH motif is seen in the amino-terminal region, suggesting that these coactivators may bind to DNA. Also

324

PART III: THE THYROID GLAND

SRC-1

7

^

bHLH PAS

III

1440

ZE

1

LXXLLs

Q-rich

TIF2/GRIP-1

JI1

1464

1

Histone Deacetylation

mSin3, HDAC

TAF^TFIIE, F, etc. RNA Polymerase II

Ml ZD

1

RAC3/TRAM-1/ACTR/AIB1/pCIP

iv/w/zam

III

i

1424

TATA

TRE

□

FIGURE 31-6. Comparison of the organization and structure of puta¬ tive nuclear-hormone receptor coactivators. (SRC-1, steroid receptor coactivator-1; TIF-2, transcription intermediary factor 2; GRIP-1, gluco¬ corticoid receptor interacting protein; RAC-3, receptor-associated coac¬ tivator 3; TRAM-1, thyroid receptor activator molecule 1; ACTR, activator of thyroid receptor; AlB-1, amplified in breast-1; PCIP, p300/ CBP cointegrator-associated protein.)

+ T►

Histone Acetylation

Ahs /TFIIE, F, etc. phrI-wRNA Polymerase II

located in this region is the PAS (Per/Arnt/Sim) domain, which is shared by a number of hypothalamic genes that regulate circa¬ dian rhythm and may serve as a dimer interface with other cofac¬ tors. Finally, in addition to the SRC-1 family, other cofactors may be associated with liganded TR.11,12,27 The functional roles of these proteins are not well understood. SRC-1 can interact with the cyclic AMP response element binding protein (CREB)-binding protein (CBP), the putative coactivator for cyclic adenosine monophosphate-stimulated transcription as well as the related protein, p300, which interacts with the viral coactivator E1A.11'12 These proteins might serve as integrator molecules for different signaling inputs such as pro¬ tein kinase A- and C-pathway-mediated transcription as well as bridge-liganded TRs with other adapter molecules and/or the basal transcriptional machinery11'12'28 (see Fig. 31-6). In addition, TRs, coactivators, CBP, and the histone acetylase, p300, and CBPassociated factor (PCAF) can form a complex that can remodel local chromatin structure. Indeed, CBP and PCAF as well as SRC-111,12 have intrinsic histone acetylase activity, although the histone substrates are different for these proteins.

MODEL OF THYROID-HORMONE RECEPTOR ACTION On the basis of these findings, a model for the mechanism of basal repression and transcriptional activation has emerged (Fig. 31-7). When ligand is absent, TR homodimers or TR/RXR heterodimers bind to the TRE and complex with corepressor, which, in turn, interacts with mSin3 or a related protein, and histone deacetylase. This complex may keep surrounding histones deacetylated and maintain chromatin near the TRE in a transcriptionally repressed state. When ligand is present, the TR/corepressor complex dissoci¬ ates and is replaced by a coactivator complex that likely contains CBP and the histone acetylase p300/CBP associated factor (PCAF). These changes result in remodeling of chromatin struc¬ ture and nucleosome positioning. The subsequent recruitment of RNA polymerase II to the transcription initiation site within the minimal promoter results in transcriptional activation. This model is probably an oversimplification as other proteins likely exist that form the TR/coactivator complex,29-30 which may interact directly with the basal-transcriptional machinery or other transcription factors. The identities of these proteins and their protein/protein interactions remain to be elucidated. Our understanding of TR action has grown at an accelerating pace and has shed light on both nuclear-hormone receptor action

;
1.5 cm in diameter are more likely to metastasize to distant sites. The pattern of tissue calcitonin staining may differentiate vir¬ ulent from less aggressive tumors, hi one study, patients with primary tumors that showed intense homogeneous calcitonin staining were all clinically well on follow-up examination, whereas patients whose tumors showed patchy localization of calcitonin either developed metastatic disease or died of cancer within 6 months to 5 years of initial surgery.193

DIAGNOSIS Clinical Features. Patients with sporadic disease or previ¬ ously unrecognized familial MTC usually present with one or more painless thyroid nodules in an otherwise normal gland, but the tumor may cause pain, dysphagia, and hoarseness. The dominant sign may be enlarged cervical lymph nodes or, occa¬ sionally, distant metastases, most commonly to the lung, fol¬ lowed in frequency by metastases to the liver, bone (osteolytic or osteoblastic lesions), and brain. In approximately one-half of patients with sporadic MTC, cervical lymph node metastases are present at the time of diagnosis. The thyroid nodule may be cold or normal on radionuclide imaging and is usually solid on echography and malignant on FNA. Radiographs may show dense, irregular calcifications of the primary tumor and cervical nodes, and mediastinal widening that is due to metastases. Rarely, an abnormal phenotype may correctly identify the tumor, or a paraneoplastic syndrome may be the presenting manifestation. Hormonal Features. In addition to calcitonin, MTC may synthesize calcitonin gene-related peptide, L-dopa decarboxy¬ lase, serotonin, prostaglandins, adrenocorticotropin, histaminase, carcinoembryonic antigen, nerve growth factor, and substance P.190 Elevated calcitonin, histaminase, L-dopa decar¬ boxylase, and carcinoembryonic antigen serum levels occur frequently in MTC patients, but not in other thyroid cancers. Approximately 10% of MTC patients have episodes of flushing

397

often induced by alcohol ingestion, calcium infusion, and pentagastrin injection; these episodes may be due to tumor release of prostaglandins and serotonin.190 The only recog¬ nized clinical manifestation of high circulating calcitonin levels is a secretory diarrhea that occurs in ~30% of patients and is usually seen only with advanced tumors. Because of the indo¬ lent course of MTC, the secretion of adrenocorticotropin by the tumor may cause typical Cushing syndrome (see Chaps. 75 and 219). Calcitonin Determination. Assay of plasma calcitonin is helpful in the early diagnosis of C-cell hyperplasia and MTC and has influenced the management of these disorders, partic¬ ularly the MEN2 syndromes. Elevated basal plasma calcitonin levels are found in almost all patients with a palpable MTC and correlate directly with tumor mass.190 Basal calcitonin may not be elevated in patients with small tumors and is almost invariably normal in those with C-cell hyperplasia; however, the plasma calcitonin levels increase to abnormally high levels after stimulation with calcium or pentagastrin. Pentagastrin, 0.5 gg/kg, is given intravenously over 5 seconds, and blood samples are taken at 0, 2, and 5 minutes. Depending on the cal¬ citonin antibody used, this stimulus causes calcitonin levels to rise approximately three- to fivefold with MTC or C-cell hyper¬ plasia.190 Calcium and pentagastrin infused together is more reliable than either used alone because some patients respond to one but not the other. In the combined test, 2 mg /kg of ele¬ mental calcium is infused over 60 seconds, followed by 0.5 |ig/ kg of pentagastrin infused over 5 to 10 seconds, and plasma calcitonin is measured before and 1, 2, 3, 5, and 10 minutes after infusion. Depending on the calcitonin antibody used, the plasma calcitonin level rises approximately five-fold with MTC or C-cell hyperplasia if the basal calcitonin level is mini¬ mally elevated. Diagnosis of familial cases is now possible with genetic screening long before the thyroid tumor is clini¬ cally manifest or calcitonin levels are elevated (see Factors Influencing Prognosis). Hypercalcitoninemia is not absolutely diagnostic of MTC because it occurs in other conditions.190 When the differential diagnosis of a high plasma calcitonin value is between MTC and another malignancy, a higher calcitonin value and a palpa¬ ble thyroid tumor usually can identify patients with MTC. In addition, calcitonin secretion by other cancers is poorly stimu¬ lated by pentagastrin190 (see Chap. 53).

FACTORS INFLUENCING PROGNOSIS MTC is much more aggressive than papillary and follicular car¬ cinoma and has a cancer-specific mortality of -20% at 10 years.182 Also, a significant number of deaths occur from pheochromocytoma.194 The survival rate is substantially worse with sporadic tumors or when metastases are found at the time of diagnosis, with the MEN2B phenotype, and among patients older than 50 years of age at the time of diagnosis. However, the most important prognostic factors are age and tumor stage at the time of diagnosis and the presence of residual disease postopera tively.195,196,1963 Prognosis is best with FMTC and MEN2A. Early detection and treatment has a profound impact on the clinical course of MTC. The 10-year survival rates are nearly similar to those in unaffected subjects when nodal metastases are not present, but fall to -45% with nodal metastases.190 Patients operated on during the first decade of life generally have no evidence of residual disease postoperatively.190 How¬ ever, persistent or recurrent disease is seen in approximately one-third of patients operated on in the second decade and gradually increases in frequency with age until the seventh decade, when approximately two-thirds of patients have per sistent disease after surgery.190 This is largely due to the clinical stage of the disease at the time of surgery.195'196'1963

398

PART III: THE THYROID GLAND

Before 1970, MTC was usually diagnosed in the fifth or sixth decade. With periodic calcitonin screening, affected patients with MEN kindred have been diagnosed at a much earlier age, usually in the second decade or earlier, when they have C-cell hyperplasia or microscopic carcinoma confined to the thyroid.197 Now, with the availability of genetic testing, affected patients can be identified at birth.198 However, the sensitivity of genetic screening for MEN2A offered by diag¬ nostic laboratories that limit RET analysis to exons 10 and 11 is ~83%.199 Genetic testing that includes RET exon 14 results in a more complete and accurate analysis with a sensitivity approaching 95%.199 It is recommended that clinicians con¬ firm the comprehensiveness of a laboratory's genetic screen¬ ing approach for MEN2A to ensure thoroughness of sample analysis.

THERAPY Initial Surgery. Surgery offers the only chance for cure and should be performed as soon as the disease is detected.195-196 Before thyroidectomy, however, pheochromocytoma must be rigorously searched for and excised. The treatment of MTC con¬ fined to the neck is total thyroidectomy because the disease is often multicentric, even in patients with a negative family his¬ tory who are often unsuspected relatives of affected MEN2 kin¬ dred.195-196 Because cervical node metastases occur early and adversely influence survival, all patients with palpable MTC or clinically occult disease that is visible on cut section of the thy¬ roid should undergo routine dissection of lymph nodes in the central neck compartment. The lateral lymph nodes should be dissected when they contain tumor, but radical neck dissection is not recommended unless the jugular vein, accessory nerve, or sternocleidomastoid muscle is invaded by tumor.107 Residual or Recurrent Medullary Thyroid Carcinoma. Residual or recurrent MTC, manifested by elevated calcitonin levels, occurs commonly after primary treatment of the tumor. Reoperation in appropriately selected patients is the only treat¬ ment that consistently and reliably reduces calcitonin levels and may result in excellent local disease control. Although reoperative neck microdissections can normalize calcitonin lev¬ els when metastatic MTC is confined to regional lymph nodes, there is no curative therapy for widely metastatic disease. Improved results are reported with surgical management of ' recurrent MTC, mainly through better preoperative selection of patients and the institution of routine laparoscopic liver exami¬ nation preoperatively, which identifies distant metastases in patients with normal computed tomography and magnetic res¬ onance imaging.200 Patients with widely metastatic MTC often live for years, but many develop symptoms secondary to tumor persistence or progression. Judicious palliative, reoperative resection of discrete, symptomatic lesions provides significant long-term relief of symptoms with minimal operative mortality and morbidity.201 Patients with metastatic MTC causing signifi¬ cant symptoms or physical compromise may respond to pallia¬ tive reoperative resection despite the presence of widespread incurable metastatic disease.201 Inoperable Disease. Patients with inoperable disease are often given palliative treatment, with external radiation for localized disease, or with doxorubicin or other chemotherapy combinations for widespread, life-threatening disease, which is of limited benefit. Radiolabeled metaiodobenzylguanidine and 111 In-octreotide are potentially useful in palliative care. Octre¬ otide does not improve the natural course of advanced stages of MTC.202 Initial reports of the aggressive use of radioimmuno¬ therapy with radiolabeled monoclonal antibodies against carcinoembryonic antigen in patients with far advanced disease appear hopeful. The phase I studies, which show the safety of administering high myeloablative doses of 131I-MN-14 F(ab)2 labeled carcinoembryonic antigen, are encouraging but require confirmation.2023

FOLLOW-UP The efficacy of surgery for MTC is assessed postoperatively by measurement of-plasma calcitonin levels, which may require as long as 6 months to normalize. A normal basal and provoked calcitonin level after surgery indicates cure in most cases. Persistent modest basal calcitonin elevation is often seen after surgery in patients who may remain well for many years, particularly those from a MEN2A kindred who should be observed without further aggressive therapy. When plasma calcitonin levels are extremely high or when diarrhea occurs postoperatively, metastases can be localized by neck palpation, chest radiography, computed tomography, isotope bone (but not liver) scans, liver biopsy, angiography, and venous catheterization with calcitonin measurement. Both 99mTc-dimercaptosuccinic acid and mIn-octreotide studies have similar sensitivity to localized primary MTC; however, these scans do not detect small lymph node involvement (micrometastases) before initial surgery.203 Unfortunately, both scans have no clinical implication for preoperative MTC staging.

FAMILY SCREENING Genetic Screening. Genetic screening should be done in all first-degree relatives of any patient who tests positive for a MEN2 or FMTC mutation. One study found that without genetic testing, even when the mean age at the time of thy¬ roidectomy was ~10 years, a significant number of patients (21%) with MEN2A or MEN2B have persistent or recurrent MTC over a follow-up time of approximately a decade.204 Prophylactic Total Thyroidectomy. In 1993, when muta¬ tions of the RET protooncogene were found to account for hereditary MTC, surgeons gained the opportunity to prophylactically operate on patients at an asymptomatic stage or before the disease became clinically manifest. With this approach, microscopic or grossly evident MTC is often present in the excised thyroid glands, but almost none of the patients have metastasis of their MTC to regional lymph nodes at the time of surgery.198 Almost all patients are biochemically cured with prophylactic total thyroidectomy, which can be performed safely in experienced centers.107 Prophylactic total thyroidectomy done by an experienced surgeon is recommended at age 6 years for patients who test genetically positive for MEN2A.107-205 Thyroidectomy should be done at an even earlier age for children who test positive for MEN2B, because of its aggressive behavior.204-206 Central neck lymph node dissection should be included when calcitonin lev¬ els are elevated or if patients are older than 10 years.107 Surgical Management of Hyperparathyroidism. Surgical management of hyperparathyroidism in those with MEN2A is controversial. Some advocate total parathyroidectomy and heterotopic autotransplantation for all MEN2A patients with hyperparathyroidism.207 Others advocate preservation of the parathyroids, which obviates the potential morbidity associated with total parathyroidectomy and autotransplan¬ tation.208-209 Screening for Pheochromocytoma. Before thyroidectomy is performed, those with MEN2 also should have catechola¬ mine and metanephrine measurements. All patients with MTC require postoperative follow-up of serum calcitonin levels.

REFERENCES 1. Landis SH, Murray T, Bolden S, Wingo PA. Cancer statistics, 1999. CA Can¬ cer] 1999; 49:8. 2. Kosary CL, Ries LAG, Miller BA, et al. SEER cancer statistic review, 19731992: tables and graphs. Bethesda, MD: National Cancer Institute. N1H Pub. No. 96-2789,1995.

Ch. 40: Thyroid Cancer 3. Mazzaferri EL, Jhiang SM. Long-term impact of initial surgical and medical therapy on papillary and follicular thyroid cancer. Am ] Med 1994; 97:418. 4. Hall P, Holm LE. Radiation-associated thyroid cancer: facts and fiction. Acta Oncol 1998; 37:325. 5. Ron E, Lubin JH, Shore RE, et al. Thyroid cancer after exposure to external radiation: a pooled analysis of seven studies. Radiat Res 1995; 141:259. 6. Ron E, Doddy MM, Becker DV, et al. Cancer mortality following treatment for adult hyperthyroidism. JAMA 1998; 280:347. 6a. Franklyn JA, Maisonneuve P, Sheppard M, et al. Cancer incidence and mortality after radioiodine treatment for hyperthyroidism: a populationbased cohort study. Lancet 1999; 353:2111. 7. Jacob P, Goulko G, Heidenreich WF, et al. Thyroid cancer risk to children calculated. Nature 1998; 392:31. 8. National Cancer Institute. Estimated exposures and thyroid doses received by the American people from iodine-131 in fallout following Nevada atmo¬ spheric nuclear bomb tests: a report from the National Cancer Institute. Washington, DC: US Department of Health and Human Services, National Institutes of Health, National Cancer Institute, 1997. 9. Institute of Medicine. Committee on exposure of the American people to I131 from the Nevada atomic-bomb test: implications for public health. Washington, DC: National Academy Press, 1999. 10. Gilbert ES, Tarone R, Bouville A, Ron E. Thyroid cancer rates and 131I doses from Nevada atmospheric nuclear bomb tests. Am J Med 1998; 90:1654. 11. Mazzaferri EL. Thyroid carcinoma: papillary and follicular. In: Mazzaferri EL, Samaan N, eds. Endocrine tumors. Cambridge, England: Blackwell Sci¬ entific Publications Inc., 1993:278. 12. Robbins J, Schneider AB. Radioiodine-induced thyroid cancer: studies in the aftermath of the accident at Chernobyl. Trends 1998; 9:87. 13. Moosa M, Mazzaferri EL. Occult thyroid carcinoma. Cancer J 1997; 10:180. 14. Burgess JR, Duffield A, Wilkinson SJ, et al. Two families with an autosomal dominant inheritance pattern for papillary carcinoma of the thyroid. J Clin Endocrinol Metab 1997; 82:345. 15. Malchoff CD, Sarfarazi M, Tendler B, et al. Familial papillary thyroid carci¬ noma is genetically distinct from familial adenomatous polyposis coli. Thyroid 1999; 9:247. 15a. Lupoli G, Vitale G, Caraglia M, et al. Familial papillary thyroid microcarci¬ noma: a new clinical entity. Lancet 1999; 353:37. 16. Bell B, Mazzaferri EL. Familial adenomatous polyposis (Gardner's syn¬ drome) and thyroid carcinoma: a case report and review of the literature. Dig Dis Sci 1993; 38:185. 17. Cetta F, Olschwang S, Petracci M, et al. Genetic alterations in thyroid carcinoma associated with familial adenomatous polyposis: clinical implications and suggestions for early detection. World J Surg 1998; 22:1231. 18. Burman KD, Ringel MD, Wartofsky L. Unusual types of thyroid neo¬ plasms. Endocrinol Metab Clin North Am 1996; 25:49. 19. Dahia PLM, Marsh DJ, Zheng ZM, et al. Somatic deletions and mutations in the Cowden disease gene, PTEN, in sporadic thyroid tumors. Cancer Res 1997; 57:4710. 20. Stratakis CA, Courcoutsakis NA, Abati A, et al. Thyroid gland abnormali¬ ties in patients with the syndrome of spotty skin pigmentation, myxomas, endocrine overactivity, and schwannomas (Carney complex). J Clin Endo¬

399

34. Schlumberger M, Mancusi F, Baudin E, Pacini F. 131-1 therapy for elevated thyroglobulin levels. Thyroid 1997; 7:273. 35. Yoo KS, Chengazi VU, O'Mara RE. Thyroglossal duct cyst with papillary carcinoma in an 11-year-old girl. J Pediatr Surg 1998; 33:745. 36. LiVolsi VA. Unusual variants of papillary thyroid carcinoma. In: Mazza¬ ferri EL, Kreisberg RA, Bar RS, eds. Advances in endocrinology and metab¬ olism, 6th ed. St. Louis: Mosby-Year Book, Inc., 1995:39. 37. Ain KB. Papillary thyroid carcinoma etiology, assessment, and therapy. Endocrinol Metab Clin North Am 1995; 24:711. 38. Sobrinho-Simoes M, Soares J, Cameiro F. Diffuse follicular variant of papil¬ lary carcinoma of the thyroid: report of eight cases of a distinct aggressive type of tumor. Surg Path 1990; 3:189. 39. Nikiforov Y, Gnepp DR. Pediatric thyroid cancer after the Chernobyl disas¬ ter: pathomorphologic study of 84 cases (1991-1992) from the Republic of Belarus. Cancer 1994; 74:748. 40. Herrera MF, Hay ID, Wu PS, et al. Hurthle cell (oxyphilic) papillary thyroid carcinoma: a variant with more aggressive biologic behavior. World J Surg 1994; 16:669. 41. Katoh R, Harach HR, Williams ED. Solitary, multiple, and familial oxyphil tumours of the thyroid gland. J Pathol 1998; 186:292. 42. Rosai J. Papillary carcinoma. Monogr Pathol 1993; 138. 43. LiVolsi VA. Follicular lesions of the thyroid. In: LiVolsi VA, ed. Surgical pathology of the thyroid. Philadelphia: WB Saunders, 1990:173. 44. Hassoun AAK, Hay ID, Goellner JR, Zimmerman D. Insular thyroid carci¬ noma in adolescents: a potentially lethal endocrine malignancy. Cancer 1997; 79:1044. 45. Schneider AB, Shore-Freedman E, Ryo UY, et al. Radiation-induced tumors of the head and neck following childhood irradiation: prospective studies. Medicine (Baltimore) 1985; 64:1. 46. Mazzaferri EL. Management of a solitary thyroid nodule. N Engl J Med 1993; 328:553. 47. Schneider AB, Bekerman C, Leland J, et al. Thyroid nodules in the follow¬ up of irradiated individuals: comparison of thyroid ultrasound with scan¬ ning and palpation. J Clin Endocrinol Metab 1997; 82:4020. 48. Ezzat S, Sarti DA, Cain DR, Braunstein GD. Thyroid incidentalomas: prev¬ alence by palpation and ultrasonography. Arch Intern Med 1994; 154:1838. 49. Tan GH, Gharib H. Thyroid incidentalomas: management approaches to nonpalpable nodules discovered incidentally on thyroid imaging. Ann Intern Med 1997; 126:226. 50. Fogelfeld L, Wiviott MBT, Shore-Freedman E, et al. Recurrence of thyroid nodules after surgical removal in patients irradiated in childhood for benign conditions. N Engl J Med 1989; 320:835. 51. Dottorini ME, Vignati A, Mazzucchelli L, et al. Differentiated thyroid carci¬ noma in children and adolescents: a 37-year experience in 85 patients. J Nucl Med 1997; 38:669. 52. Harach HR, Williams ED. Childhood thyroid cancer in England and Wales. Br J Cancer 1995; 72:777. 53. Hung W. Well-differentiated thyroid carcinomas in children and adoles¬ cents: a review. Endocrinologist 1994; 4:117. 54. Samuel AM, Rajashekharrao B, Shah DH. Pulmonary metastases in chil¬ dren and adolescents with well-differentiated thyroid cancer. J Nucl Med

crinol Metab 1997; 82:2037. 21. Evans HL. Encapsulated columnar-cell neoplasms of the thyroid, a report of four cases suggesting a favorable prognosis. Am J Surg Pathol 1996;

1998; 39:1531. 55. Zimmerman D, Hay ID, Gough IR, et al. Papillary thyroid carcinoma in children and adults: long-term follow-up of 1039 patients conserva¬ tively treated at one institution during three decades. Surgery 1988;

20:1205. 22. de los Santos ET, Keyhani-Rofagha S, Cunningham JJ, Mazzaferri EL. Cys¬ tic thyroid nodules: the dilemma of malignant lesions. Arch Intern Med

104:1157. 56. Schlumberger M, De Vathaire F, Travagli JP, et al. Differentiated thyroid carcinoma in childhood: long term follow-up of 72 patients. J Clin Endo¬

1990; 150:1422. 23. Baudin E, Travagli JP, Ropers J, et al. Microcarcinoma of the thyroid gland: the Gustave-Roussy Institute experience. Cancer 1998; 83:553. 24. Hedinger C, Williams ED, Sobin LH. The WHO histological classification of thyroid tumors: a commentary on the second edition. Cancer 1989;

crinol Metab 1987; 65:1088. 57. Hay ID. Papillary thyroid carcinoma. Endocrinol Metab Clin North Am 1990; 19:545. 58. Pasieka JL, Thompson NW, McLeod MK, et al. The incidence of bilateral well-differentiated thyroid cancer found at completion thyroidectomy.

63:908. 25. Mazzaferri EL, Young RL, Oertel JE, et al. Papillary thyroid carcmoma: the impact of therapy in 576 patients. Medicine (Baltimore) 1977; 56:171. 26. LiVolsi V, Asa SL. The demise of follicular carcinoma of the thyroid gland. Thyroid 1994; 4:233. 27. LiVolsi VA. Papillary lesions of the thyroid. In: LiVolsi VA, ed. Surgical pathology of the thyroid. Philadelphia: WB Saunders, 1990:136. 28. Noguchi M, Yamada H, Ohta N, et al. Regional lymph node metastases in well-differentiated thyroid carcinoma. Int Surg 1987; 72:100. 29. Sugino K, Ito K Jr, Ozaki O, et al. Papillary microcarcinoma of the thyroid. J Endocrinol Invest 1998; 21:445. 30. Yamashita H, Noguchi S, Murakami N, et al. Extracapsular invasion of lymph node metastasis is an indicator of distant metastasis and poor prognosis in patients with thyroid papillary carcinoma. Cancer 1997, 80:2268. 31. Pacini F, Lari R, Mazzeo S, et al. Diagnostic value of a smgle serum thyroglobulin determination on and off thyroid suppressive therapy in the fol¬ low-up of patients with differentiated thyroid cancer. Clin Endocrinol 1985; 23:405. 32. Pineda JD, Lee T, Ain K, et al. Iodine-131 therapy for thyroid cancer patients with elevated thyroglobulin and negative diagnostic scan. J Clin Endocrinol Metab 1995; 80:1488. 33. Schlumberger M, Arcangioli O, Piekarski JD, et al. Detection and treatment of lung metastases of differentiated thyroid carcinoma in patients with nor¬ mal chest X-rays. J Nucl Med 1988; 29:1790.

World J Surg 1992; 16:711. 59. Shaha AR, Loree TR, Shah JP. Prognostic factors and risk group analysis in follicular carcinoma of the thyroid. Surgery 1995; 118:1131. 60. Sanders LE, Cady B. Differentiated thyroid cancer: reexamination of risk groups and outcome of treatment. Arch Surg 1998; 133:419. 61. Sarda AK, Bal S, Kapur MM. Near-total thyroidectomy for carcinoma of the thyroid. Br J Surg 1989; 76:90. 62. DeGroot LJ, Kaplan EL, McCormick M, Straus FH. Natural history, treat¬ ment, and course of papillary thyroid carcinoma. J Clin Endocrinol Metab 1990; 71:414. 63. Taylor T, Specker B, Robbins J, et al. Outcome after treatment of high-risk papillary and non-Hurthle-cell follicular thyroid carcinoma. Ann Intern Med 1998; 129:622. 64. Mazzaferri EL. Thyroid remnant 131I ablation for papillary and follicular thyroid carcioma. Thyroid 1997; 7:265. 65. Massin JP, Savoie JC, Gamier H, et al. Pulmonary metastases in differenti¬ ated thyroid carcinoma: study of 58 cases with implications for the primary tumor treatment. Cancer 1984; 53:982. 66. Loh KC, Greenspan FS, Gee L, et al. Pathological tumor-node-metastasis (pTNM) staging for papillary and follicular thyroid carcinomas: a retro¬ spective analysis of 700 patients. J Clin Endocrinol Metab 1997; 82:3553. 67. Cady B. Staging in thyroid carcinoma. Cancer 1998; 83:844. 68. Tsang TW, Brierley JD, Simpson WJ, et al. The effects of surgery, radoiodine, and external radiation therapy on the clinical outcome of patients with differentiated thyroid carcinoma. Cancer 1998; 82:375.

400

PART III: THE THYROID GLAND

69. Simpson WJ, McKinney SE, Carruthers JS, et al. Papillary and follicular thyroid cancer: prognostic factors in 1578 patients. Am J Med 1987; 83:479. 70. Kurozumi K, Nakao I, Nishida T, et al. Significance of biologic aggressive¬ ness and proliferating activity in papillary thyroid carcinoma. World J Surg 1998; 22:1237. 71. Sugitani I, Yanagisawa A, Shimizu A, et al. Clinicopathologic and immunohistochemical studies of papillary thyroid microcarcinoma presenting with cervical lymphadenopathy. World J Surg 1998; 22:731. 72. Akslen LA, Varhaug JE. Oncoproteins and tumor progression in papillary thyroid carcinoma: presence of epidermal growth factor receptor, c-erbB-2 protein, estrogen receptor related protein, p21-ras protein, and prolifera¬ tion indicators in relation to tumor recurrences and patient survival. Can¬ cer 1995; 76:1643. 73. Sellers M, Beenken S, Blankenship A, et al. Prognostic significance of cervi¬ cal lymph node metastases in differentiated thyroid cancer. Am J Surg 1992; 164:578. 74. Sisson JC, Giordano TJ, Jamadar DA, et al. 1311 treatment of micronodular pulmonary metastases from papillary thyroid carcinoma. Cancer 1996; 78:2184. 75. Nemec J, Zamrazil V, Pohunkova D, Rohling S. Radioiodide treatment of pulmonary metastases of differentiated thyroid cancer: results and prog¬ nostic factors. Nuklearmedizin 1979; 18:86. 76. Casara D, Rubello D, Saladini G, et al. Different features of pulmonary metastases in differentiated thyroid cancer: natural history and multi¬ variate statistical analysis of prognostic variables. J Nucl Med 1993; 34:1626. 77. Kashima K, Yokoyama S, Noguchi S, et al. Chronic thyroiditis as a favor¬ able prognostic factor in papillary thyroid carcinoma. Thyroid 1998; 8:197. 78. Schaffler A, Palitzsch KD, Seiffarth C, et al. Coexistent thyroiditis is associ¬ ated with lower tumour stage in thyroid carcinoma. Eur J Clin Invest 1998; 28:838. 79. Viswanathan K, Gierlowski TC, Schneider AB. Childhood thyroid cancer: characteristics and long-term outcome in children irradiated for benign conditions of the head and neck. Am J Dis Child 1994; 148:260. 80. Filetti S, Belfiore A, Amir SM, et al. The role of thyroid-stimulating antibod¬ ies of Graves' disease in differentiated thyroid cancer. N Engl J Med 1988; 318:753. 81. Belfiore A, Garofalo MR, Giuffrida D, et al. Increased aggressiveness of thy¬ roid cancer in patients with Graves' disease. J Clin Endocrinol Metab 1990; 70:830. 81a. Mazzaferri EL. Thyroid cancer and Graves' disease: the controversy ten years later. Endocrine Practice 2000; 6:221. 82. Fusco A, Grieco M, Santoro M, et al. A new oncogene in human thyroid papillary carcinomas and their lymph-nodal metastases. Nature 1987; 328:170. 83. Donghi R, Sozzi G, Pierotti MA, et al. The oncogene associated with human papillary thyroid carcinoma (PTC) is assigned to chromosome 10 qll-ql2 in the same region as multiple endocrine neoplasia type 2A (MEN2A). Oncogene 1989; 4:521. 84. Smanik PA, Furminger TL, Mazzaferri EL, Jhiang SM. Breakpoint charac¬ terization of the ret/PTC oncogene in human papillary thyroid carcinoma. Hum Mol Genet 1995; 4:2313. 85. Nikiforov YE, Nikiforova M, Fagin JA. Radiation-induced post-Chernobyl pediatric thyroid carcinomas. Oncogene 1998; 17:1983. 86. Jhiang SM, Sagartz JE, Tong Q, et al. Targeted expression of the ref/PTC 1 oncogene induces papillary thyroid carcinomas. Endocrinology 1996; 137:375. 87. Santoro M, Chiappetta G, Cerrato A, et al. Development of thyroid papil¬ lary carcinomas secondary to tissue-specific expression of the RET/PTC1 oncogene in transgenic mice. Oncogene 1996; 12:1821. 88. American Joint Committee on Cancer. Head and neck tumors: thyroid gland. In: Beahrs OH, Henson DE, Hutter RVP, Myers MH, eds. Manual for staging of cancer, 4th ed. Philadelphia: JB Lippincott, 1992:53. 89. Van De Velde CJH, Hamming JF, Goslings BM, et al. Report of the consensus development conference on the management of differentiated thyroid cancer in the Netherlands. Eur J Cancer Clin Oncol 1988; 24:287. 90. Baldet L, Manderscheid JC, Glinoer D, et al. The management of differenti¬ ated thyroid cancer in Europe in 1988: results of an international survey. Acta Endocrinol (Copenh) 1989; 120:547. 91. Solomon BL, Wartofsky L, Burman KD. Current trends in the management of well differentiated papillary thyroid carcinoma. J Clin 1996; 81:333. 92. Chen H, Zeiger MA, Clark DP, et al. Papillary carcinoma of the thyroid: can operative management be based solely on fine-needle aspiration? J Am Coll Surg 1997; 184:605. 93. Cady B. Our AMES is true: how an old concept still hits the mark: or, risk group assignment points the arrow to rational therapy selection in differ¬ entiated thyroid cancer. Am J Surg 1997; 174:462. 94. Schlumberger MJ. Medical progress: papillary and follicular thyroid carci¬ noma. N Engl J Med 1998; 338:297. 95. Mazzaferri EL. Treating differentiated thyroid carcinoma: where do we draw the line? Mayo Clin Proc 1991; 66:105. 95a. Mazzaferri EL. NCCN thyroid carcinoma practice guidelines. Oncology 1999; 13:391. 96. Newman KD, Black T, Heller G, et al. Differentiated thyroid cancer: deter¬ minants of disease progression in patients A substitution in exon M5, which created a novel splice acceptor site in the maternal allele. The paternal allele is not expressed, although the reasons for this are not known. As in the other example, whereas the mutant recep¬ tor is well expressed in cells, it fails to bind ligand or stimulate cAMP or inositol phosphate production. Evidence has accumulated for the possible existence of other receptors in the PTH/PTHrP system in addition to the two receptors already characterized. A receptor responsive to NH2terminal PTH and PTHrP, which signals through changes in intracellular free calcium rather than cAMP, has been found to exist in keratinocytes and other cells but has not yet been char¬ acterized. A receptor that signals in a similar maimer but is only responsive to PTHrP-(l-34) mediates the release of argininevasopressin from the supraoptic nucleus. Unique biologic roles are predicted for the mid-region of PTHrP (transplacental cal¬ cium transport), the basic region of PTHrP (nuclear import and inhibition of apoptosis), the carboxyl-terminal region of PTHrP (inhibition of bone resorption), and the mid/carboxyl-terminal part of PTH (binding to osteoblast-like cells and modulation of their activity), each of which may act via discrete receptors. Finally, discovery of the PTHR2 suggested the presence of an additional ligand in the PTH family. Thus, the PTHR2, responsive only to PTH and not PTHrP, is expressed mainly in brain, placenta, testis, and pancreas. A novel PTH-like ligand for the PTHR2 has been isolated from the hypothalamus.1353 PARATHYROID HORMONE-INDUCED SIGNAL TRANSDUCTION ADENYLATE CYCLASE Adenylate cyclase stimulation and the subsequent generation of cAMP are believed to be important events in the actions of PTH in the kidney and in the skeleton. Cyclic adenosine mono¬ phosphate mimics phosphaturic and calcium-retaining effects of PTH in the kidney in vivo and in vitro and mediates PTHstimulated renal la-hydroxylase activity.136 Cyclic adenosine monophosphate has also been implicated in the hypercalcemic action of PTH and may simulate PTH-induced bone resorption in vitro. Furthermore, the bone anabolic effect of PTH appears dependent on the capacity to stimulate cAMP. Cyclic AMP produced in target cells for PTH is believed to stimulate cAMP-dependent protein kinase isoenzymes; types I and

506

PART IV: CALCIUM AND BONE METABOLISM

II have different biochemical characteristics and may serve dif¬ ferent functions. The isoenzymes are tetramers consisting of two regulatory and two catalytic subunits that have similar catalytic but different regulatory components, termed RI and RII, respec¬ tively. With binding of cAMP to the regulatory component, the holoenzyme dissociates, releasing the catalytic component that facilitates the transfer of a terminal phosphate group from a nucleotide donor, usually ATP, to an amino acid residue (i.e., serine, threonine, or tyrosine) of the substrate protein. PTHinduced stimulation of the two types of protein kinase has been demonstrated in normal osteoblasts and in a malignant osteo¬ blast line.137 One of the substrates of PKA is the widely expressed cAMP response element binding protein (CREB).40 The phosphorylated CREB bound to the cAMP response element (CRE) in the promoters of responsive genes couples to the basal transcriptional machinery via cointegrators such as CREBbinding protein (CBP). PTH activates several osteoblastic genes, including c-fos,138 by this mechanism. OTHER SECOND MESSENGERS OF PARATHYROID HORMONE ACTION

In addition to cAMP, other second messengers, such as the cal¬ cium ion, have been implicated as being potentially important in PTH action. In some species, transient hypocalcemia, pre¬ sumably caused by calcium entry into bone cells, is the earliest event in the action of PTH on the skeleton in vivo. In vitro stud¬ ies have shown that PTH promotes the uptake of calcium into isolated bone cells, that elevated calcium mimics or potentiates the effects of PTH on the enzymatic activities of isolated bone cells, and that calcium antagonists inhibit and calcium ionophores stimulate bone resorption. PTH stimulates phosphatidylinositol turnover in certain cell types and in renal membranes.139'140 In response to PTH-induced augmentation of phospholipase C action, presumably via Gq stimulation after occupancy of the PTH/PTHrP receptor, increased production of IP3 and diacylglycerol occurs. Increases in cytoplasmic calcium, presumably induced by IP3, have also been demonstrated, as has increased protein kinase C activity. The cellular response to PTH may therefore involve multiple mechanisms of cell signaling, and modulation of one message by another ("cross talk") may affect the final response to the hormone. The relative contributions of adenylate cyclase versus phospholipase C stimulation to the pleiotropic effects of PTH in bone and kidney still remains to be clarified. EFFECTS OF PARATHYROID HORMONE IN TARGET TISSUES BONE

Consistent with its prime function of raising the extracellular fluid calcium concentration, the most appreciated effect of PTH is a catabolic one in bone.141-142 The end result is the breakdown of mineral constituents and bone matrix, as manifested in vivo by the release of calcium and phosphate, by increases in plasma and urinary hydroxyproline, and by other indices of bone resorption. This process appears to be mediated by osteo¬ clastic osteolysis, but the mechanism by which PTH causes osteo¬ clastic stimulation is indirect. Unlike calcitonin, PTH, when administered in vivo, does not bind directly to multinucleated osteoclasts. In vivo studies using autoradiography have revealed PTH binding to mature osteoblasts and to skeletal mononuclear cells in the intertrabecular region of the metaphysis (apparently to preosteoblastic stromal cells).143,144 Consequently, PTH-mediated increases in the number and function of osteoclasts (which are of hematogenous origin) appear to occur indirectly, through effects on cells of the osteoblast series (which are of mesenchymal ori¬ gin).145 The results of these studies have been confirmed in exper-

Osteoclast progenitor

Osteoclast precursor

Pre-osteoclast

Active osteoclast

FIGURE 51-7. Model of the activation of bone resorption and forma¬ tion by parathyroid hormone (PTH) or parathyroid hormone-related protein (PTHrP). PTH of PTHrP bind via their NH2 terminus to the PTH/PTHrP receptor (PTHR) of osteoblastic stromal cells or osteo¬ blasts. Osteoblastic stimulation may lead to bone formation. PTH/ PTHrP binding to the PTHR, most likely on the osteoblastic stromal cell, can also lead to the binding of ODF/OPGL/RANKL to receptors (RANK) on cells of the osteoclast series. This cytokine can then enhance commitment, proliferation, differentiation, and fusion of osteoclast pre¬ cursors to form active bone-resorbing osteoclasts. Alternatively, ODF/ OPGL/RANKL may be bound by OPG, preventing osteoclastic bone resorption.

iments with the cloned PTH/PTHrP receptor; these have confirmed expression of the receptor in osteoblastic cells but not osteoclasts.146 PTH-induced stimulation of multinucleated osteo¬ clasts occurs through the action of PTH-stimulated osteoblastic activity.147 This osteoblastic activity is characterized by release of intermediary factors such as cytokines. One of the most important of these intermediary factors is osteoclast differentiation factor (ODF), also known as osteoprotegerin ligand (OPGL).148 This protein is expressed in bone-lining cells or in osteoblast precursors that support osteoclast recruit¬ ment. ODF/OPGL, which is a member of the tumor necrosis factor (TNF) family, is identical to the molecule TRANCE/ RANKL, which is expressed in T cells and previously was iden¬ tified as a dendritic cell survival factor in vitro. Several bone resorbing factors, such as PTH, PTHrP, PGE2, some of the inter¬ leukins, and l,25(OH)2D3, up-regulate ODF/OPGL gene expres¬ sion in osteoblasts and bone stromal cells (Fig. 51-7). Interaction of ODF/OPGL with its receptor on osteoclast progenitors and osteoclasts then stimulates their recruitment and activation and delays their degradation. The ablation of the ODF/OPGL gene in mice has confirmed it to be a key regulator of osteoclastogenesis as well as of lymphocyte development and lymph-node organogenesis.149 Osteoprotegerin (OPG)/osteoclastogenesis inhibi¬ tory factor (OCIF)150 is a soluble receptor for ODF/OPGL that inhibits recruitment, activation, and survival of osteoclasts and therefore inhibits osteoclastic bone resorption. OPG/OCIF acts as a natural decoy receptor to disrupt the interaction between ODF/OPGL, which is released by osteoblast-related cells, and the ODF/OPGL receptor on osteoclast progenitors. Early and late phases of calcium mobilization have been described after in vivo administration of PTH.151 The early hypercalcemic response occurs from 10 minutes to 3 hours after hormone exposure. This response may be a consequence of increased metabolic activity of preexisting osteoclasts or of other cell types that enhance the transfer to bone of calcium in bone that is already in solution.

Ch. 51: Parathyroid Hormone

A more sustained hypercalcemic response to PTH adminis¬ tration, occurring over approximately 24 hours, appears to depend on new protein synthesis and to involve a quantitative increase in osteoclasts, a change in the structure of the osteo¬ clasts (i.e., increased ruffled borders, which is a zone of the cell believed to be involved in skeletal resorption); an increase in the secretion of lysosomal enzymes, including collagenase and acid hydrolases such as acid phosphatase; and acidification of the extracellular milieu of the osteoclast. In contrast to sustained administration of PTH, which causes bone resorption and hypercalcemia, low and intermit¬ tent doses of PTH-(l-34)—and PTHrP-(l-34) and related ana¬ logs—promote bone formation.152 Furthermore, in clinical studies, daily injections of PTH-(l-34) have been reported to increase hip and spine bone mineral density (BMD). The consequences of the effects of PTH on osteoblast activity are therefore complex. Thus, in addition to using osteoblastic cells to relay signals to the osteoclast lineage to resorb bone, PTH also appears to stimulate osteoblastic cells to enhance new bone formation. This might occur as a result of the ability of PTH to stimulate the production of growth factors, such as IGFI, by osteoblasts. However, the bone-forming activity of PTH may depend more on promoting differentiation of precursor cells into secretory osteoblasts than on an action on mature osteoblasts.153 The precise mechanisms of the anabolic effect of PTH still remain to be clarified, however. The anabolic and catabolic effects of PTH on osteoblasts may therefore represent a combination of direct and indirect effects; effects of different domains of the PTH molecule that signal dif¬ ferently; discrete functions of morphologically similar but func¬ tionally distinct osteoblastic cells; or differences in hormonal effects based on different times of exposure or different hor¬ mone concentrations. KIDNEY

One of the first-described effects of the administration of PTH intravenously was phosphaturia (see Chap. 206). Renal tubular reabsorption of phosphate is an active process, with a limited transport capacity resulting in a maximum rate of tubular reab¬ sorption (Tm P04).154 Because the absolute values of Tm P04 vary considerably among individuals and most of this variation can be explained by differences in glomerular filtration rate (GFR), the Tm P04/GFR ratio has been suggested as a more accurate index of phosphate reabsorption. This index, which represents the sum of the heterogeneous maximum reabsorp¬ tion rates of all individual nephrons, is reduced by increased concentrations of PTH and increased in its absence. The molecular basis of the inhibition of Na+-dependent phosphate transport by PTH was clarified by the cloning of the Na+-phosphate co-transporter, NPT-2.155 Thus, inhibition of phosphate reabsorption by PTH in the renal proximal tubule appears to involve PTH-induced endocytosis of NPT-2 and its subsequent intracellular degradation. Another relatively immediate effect of PTH that contributes to its role in calcium homeostasis is enhanced fractional reab¬ sorption of calcium from the glomerular fluid. However, exces¬ sive circulating concentrations of PTH ultimately are associated with a rise in urinary calcium because of increases in extracellu¬ lar calcium levels and therefore in the filtered load of calcium. The capacity of PTH to produce a mild renal tubular acidosis appears linked to its ability to inhibit the activation of the type 3 Na+-H+ exchanger (NHE3).156 Augmentation of urinary cAMP excretion in response to PTH administration is one of the earliest renal responses and is consis¬ tent with its postulated role as a second messenger for many or most renal responses. Nevertheless, evidence for an important role for inositol phosphates, protein kinase C, and intracellular calcium in the renal actions of PTH is also available; the relative in vivo contributions of the two pathways to the multiple effects of

507

PTH in the kidney still requires clarification. The renal actions of PTH to increase urinary phosphate and cAMP excretion form the basis for the modified Ellsworth-Howard test that is used clini¬ cally to establish renal PTH responsiveness (see Chap. 60). PTH alters renal function because of its interactions with multiple regions of the nephron. Evidence for PTH binding to the primary foot processes of podocytes of renal corpuscles and for the stimulation of adenylate cyclase activity in rat glomeruli can be correlated with reduction of the GFR in rats by PTH as a result of decreasing the ultrafiltration coefficient.144 Evidence for the expression of the cloned PTH/PTHrP receptor in renal glomeruli confirms the previous findings. PTH appears to reach the luminal surface of polar tubular cells by glomerular filtration and the basolateral surface through the peritubular capillary plexus. High-capacity binding sites have been described on the periluminal portion of the earliest part of the proximal tubule, at which, related to the microvillar surface, PTH degradative events appear to occur. Saturable, spe¬ cific, low-capacity binding sites for PTH have been localized after injection in rats to the basolateral surface of the proximal convoluted tubule and pars recta, the thick ascending limb of the loop of Henle, and the distal convoluted tubule and pars arcuata of the collecting duct.144 This pattern of PTH binding and activity correlates well with the expression of the cloned PTH/ PTHrP receptor and with the localization of PTH-stimulated adenylate cyclase activity in rat nephrons, which was demon¬ strated in studies using microdissection of tubules.156 These observations emphasize the diversity of PTH actions on the renal tubule, most of which can be mimicked by the infusion of cAMP onto the luminal aspect of tubular cells.157-158 The use of various techniques, including micropuncture and microperfusion, has localized PTH-induced inhibition of phos¬ phate reabsorption to the proximal convoluted tubule and to the pars recta.158 Inhibition of phosphate reabsorption in the proximal convoluted tubule appears to be accompanied by inhibition of sodium and fluid reabsorption. However, sodium is also reabsorbed more distally. Inhibition of phosphate reab¬ sorption also may occur, although perhaps to a lesser extent, in the distal tubule. The proximal tubule appears to be the major site of action of PTH in stimulating the la-hydroxylase and increasing the production of 1,25-dihydroxyvitamin D. The PTH-induced inhibition of bicarbonate transport occurs in the proximal tubule and the pars recta, and the effect of PTH on this nephron segment may explain the rise in urinary bicarbon¬ ate produced by PTH infusion.158 The important site of PTH action to increase calcium and probably magnesium transport appears to be in the thick ascending limb of the loop of Henle and in the distal convoluted tubule and earliest portion of the cortical collecting tubule. Despite the demonstration of the dual role of PTH in increasing extracellular fluid calcium concentrations through renal and skeletal routes, the relative importance of each is unclear.159 However, contributions of the kidney and skeleton to calcium homeostasis may depend on the concentration of cir¬ culating PTH and the duration of elevated hormonal levels. OTHER TARGET TISSUES

Although the administration of PTH in vivo can enhance intesti¬ nal calcium absorption, this effect appears to be indirect and mediated by the increased production of 1,25-dihydroxyvitamin D.92 Among other reported effects of PTH are direct effects on vas¬ cular tone, stimulation or inhibition of mitosis of various cells in vitro, increased concentrations of calcium in mammary and in sal¬ ivary glands, enhanced hepatic gluconeogenesis, and enhanced lipolysis in isolated fat cells. Tine demonstration of the widespread expression of the PTHrP gene and the equally disseminated expression of the PTH/PTHrP receptor gene suggests that many of the "noncalcemic" actions of PTH may be carried out by PTHrP acting in an autocrine or paracrine manner.160

508

PART IV: CALCIUM AND BONE METABOLISM

MEASUREMENT RADIOIMMUNOASSAYS Radiommunoassays (RIAs) developed for human PTH generally do not use human PTH (hPTH) 1-84 as a tracer because of its scar¬ city and because it lacks a tyrosine residue for convenient radioiodination. Instead, bovine PTH (bPTH) 1-84 is used, possibly contributing to the reduced sensitivity for hPTH. The antisera used in PTH RIAs are generally polyclonal and have been raised against bPTH 1-84 or hPTH 1-84. Nevertheless, populations of antibodies within polyclonal antisera directed against specific epitopes contained in the 1-84 molecule may predominate. Anti¬ body populations recognizing the COOH-terminal region of PTH 1-84 usually are readily obtained after immunization with bPTH 1-84; antibodies recognizing the midregion also occur with high frequency.161 Sufficiently sensitive antisera containing antibodies predominantly interacting with the NH2-terminal region are more unusual but have been successfully raised.162 Attempts to direct antibody specificity have used several strategies. Although the development of monoclonal antibodies to PTH or PTH fragments would seem to be the most direct route to achieve specificity, this approach has not been success¬ ful for various technical and biochemical reasons. Instead, spec¬ ificity has been achieved by using polyclonal antisera with predominant specificity for selected regions, as determined by their reactivity toward synthetic fragments of discrete regions of the molecule or by enhancing the specificity of such antisera by using synthetic fragments of the midregion or COOHterminal end of PTH as tracers. An important advance in the measurement of PTH is the development of immunoradiometric assays (IRMAs) that use antisera directed against the NH2-terminal and midregion or COOH region of the molecule. These assays (i.e., "intact" assays) detect mainly intact PTH 1-84 and appear to be the most sensitive and specific.163 Defining the specificity of an RIA is important because of the complex metabolism of PTH, which results in a multiplicity of circulating molecular forms. The most abundant circulating forms are midregion and COOH fragments because of their longer half-life in the circulation; these become even more pre¬ dominant in renal failure when clearance is further impaired.89-90 The presumed bioactive forms, intact PTH 1-84 and the NH2terminal fragment, are cleared more readily and circulate at lower levels. Midregion and COOH-directed RIAs detect longlived fragments and intact bioactive hormone. These RIAs gener¬ ally do not require the high sensitivity of NH2-directed assays to be useful, because they measure higher absolute levels of PTH. Most material measured by these RIAs is thought to be inac¬ tive, but this has not greatly restricted their clinical usefulness in the diagnosis of primary or secondary hyperparathyroidism. For primary hyperparathyroidism, the midregion and COOHdirected RIAs may be especially useful in providing an index of integrated secretory function of the parathyroid glands.85 How¬ ever, first it should be established that a given midregion or COOH-terminal RIA is clinically useful. Amino-terminal RIAs, which determine the level of short-lived PTH forms, can also be used to detect hypersecretion in primary hyperparathyroidism but may be especially useful in assessing acute changes in PTH secretion in physiologic studies. Two-site IRMAs, which also measure bioactive hormone, are generally more sensitive and can substitute for NH-,-directed assays. For cases of hyperparathyroidism associated with renal fail¬ ure, NH2-directed assays have the theoretical advantage of pro¬ viding a better estimate of circulating bioactive PTH forms and differentiating decreased PTH clearance from hypersecretion. From a practical point of view, two-site IRMAs are preferred to NH,-directed assays. If PTH levels are determined serially dur¬ ing the progression of renal failure, COOH-directed assays also

may provide useful estimates of the severity of the hyperpar¬ athyroidism, and hormone levels determined by COOHdirected assays in renal failure have correlated well with the extent of skeletal resorption.85 It has been suggested that midre¬ gion assays may detect intact PTH more readily than COOHdirected assays and may have a somewhat greater ability to measure a variety of PTH forms than the COOH-directed assays. However, midregion assays are subject to restrictions and advantages similar to those of the COOH-directed assays. Few RIAs, no matter the degree of specificity, completely discriminate between concentrations of immunoreactive PTH found in healthy persons and those detected in hyperparathy¬ roidism, although best results appear to be obtained with twosite IRMAs. It has been suggested that PTH levels should be interpreted in conjunction with prevailing levels of blood cal¬ cium. This assists in the assay analysis; if a given PTH level is measured in a normocalcemic individual and that same PTH level is found in a patient with hypercalcemia, the level in the latter case can be considered inappropriately elevated. Another limitation of most RIAs is the inability to measure values in all healthy individuals. A lower limit of normality generally cannot be established, which makes it difficult to ascertain reduced PTH levels. Sensitive two-site IRMAs often approach a lower limit of normality. PTH RIAs are useful in dif¬ ferentiating hypocalcemia resulting from PTH deficiency (i.e., hypoparathyroidism) from hypocalcemia resulting from other causes. In the former situation, PTH is undetectable or is found in very low concentrations, but in the latter situation, the hypocalcemic stimulus is associated with increased PTH secre¬ tion and elevated levels of PTH (see Chaps. 58 and 61). BIOASSAYS With the unraveling of the complex metabolism of PTH and the resulting heterogeneity of circulating hormone, the care that must be exercised in interpreting the results of PTH RIAs became apparent, and the possibility of using PTH bioassays to supplement the information obtained from RIAs arose. Some PTFI bioassays estimate the biologic effects of the hormone in vivo, and some estimate levels of the hormone based on bio¬ logic effects in vitro. It has been estimated that the normal cir¬ culating levels of bioactive PTH are ~1 pmol/L. IN VIVO BIOASSAYS

Measurements of in vivo phosphaturic effects (e.g., determina¬ tion of Tm P04/GFR) and renal calcium retention (e.g., fractional excretion of calcium) provide estimates of PTH effects in vivo and have been useful as adjunctive studies of PTH. Partly because of their lack of specificity, these assays have not achieved widespread popularity as indices of PTH concentrations in most clinical situations. Conversely, estimates of urinary cAMP excre¬ tion (measured by RIA) have been fairly widely used.164 Several hormones in addition to PTH (e.g., vasopressin) con¬ tribute to urinary cAMP levels, although the PTH-produced component is the major fraction. Greater specificity of urinary cAMP for PTH can be achieved by measuring the fraction of urinary cAMP that is of renal origin. This nephrogenous com¬ ponent can be determined from the clearance of cAMP relative to the GFR, an estimate requiring plasma cAMP measurements, or it can be determined by relating the urinary cAMP to 100 mL of glomerular filtrate. Nephrogenous cAMP is a specific and rather sensitive in vivo bioassay for PTH.164 Nephrogenous cAMP is often elevated in primary hyperparathyroidism and decreased in hypoparathyroidism. There is, however, overlap with the normal range. Most nephrogenous cAMP determina¬ tions accurately reflect circulating PTH levels as determined by RIA, but they do not have the sensitivity or specificity of the best two-site IRMAs.

Ch. 51: Parathyroid Hormone IN VITRO BIOASSAYS

Several attempts have been made to develop clinically useful in vitro bioassays for the measurement of active PTH. The capac¬ ity of PTH to stimulate adenylate cyclase, with the addition of guanyl nucleotides, in purified renal membrane preparations or tumor cell lines and the availability of synthetic antagonistic PTH fragments have imparted useful specificity to such renal membrane bioassay preparations.165-166 Unfortunately, the rela¬ tive insensitivity of this approach has precluded its use for any¬ thing other than experimental purposes. Another approach is the cytochemical PTH bioassay. The most widely used has been a renal cytochemical bioassay (CBA) performed in guinea pig kidney segments maintained in non¬ proliferative organ culture.4-91-167 The major drawbacks of the method are its technical difficulty and low throughput, which have precluded its use for routine clinical assay purposes.

CONCLUSIONS Considerable progress has been made in examining the biosyn¬ thesis of PTH, the regulation of PTH secretion, and the mecha¬ nism of PTH action. These advances include elucidation of the molecular genetics of PTH, discovery of the parathyroid cal¬ cium "sensor," and determination of the structure of the PTH/ PTHrP receptor. Discovery of the ODF/OPGL/RANKL system and the cloning of NPT-2 have greatly contributed to the under¬ standing of the mechanism of PTH action in bone and kidney. The identification of PTHrP has disclosed a new member of the PTH gene family and provided possibilities for understanding the biologic effects of both molecules. This improved compre¬ hension of the biochemistry and physiology of PTH should translate into exciting insights into the pathophysiology of dis¬ ease states in which PTH is implicated.

REFERENCES 1. Kronenberg HM, Igarashi T, Freeman MW, et al. Structure and expression of the human parathyroid hormone gene. Recent Prog Horm Res 1986; 42:641. 2. Cohn DV, Elting J. Biosynthesis, processing and secretion of parathormone and secretory protein-I. Recent Prog Horm Res 1983; 39:181. 3. Walter P, Ibrahimi I, Blobel G. Translocation of proteins across the endoplas¬ mic reticulum. 1. Signal recognition protein (SRP) binds to in vitro-assembled polysomes synthesizing secretory protein. J Cell Biol 1981; 91:545. 4. Goltzman D, Bennett HPJ, Koutsilieris M, et al. Studies of the multiple forms of bioactive parathyroid hormone and parathyroid hormone-like substances. Recent Prog Horm Res 1986; 42:665. 5. Hendy GN, Bennett HPJ, Lazure C, et al. Proparathyroid hormone (ProPTH) is preferentially cleaved to parathyroid hormone (PTH) by the prohormone convertase furin: a mass spectrometric study. J Biol Chem 1995; 270:9517. 6. Canaff L, Bennett HPJ, Hou Y, et al. Proparathyroid hormone processing by the proprotein convertase PC7: comparison with furin and assessment of modulation of parathyroid convertase mRNA levels by calcium and 1,25dihydroxy vitamin D3. Endocrinology 1999; 140:3633. 7. Rabbani SA, Kremer R, Bennett HPJ, Goltzman D. Phosphorylation of parathyroid hormone by human and bovine parathyroid glands. J Biol Chem 1984; 259:2949. 8. Hendy GN, Kronenberg HM, Potts JT Jr, Rich A. Nucleotide sequence of cloned DNAs encoding human preproparathyroid hormone. Proc Natl Acad Sci U S A1981; 78:7365. 9. Vasicek TJ, McDevitt BE, Freeman MW, et al. Nucleotide sequence of genomic DNA encoding human parathyroid hormone. Proc Natl Acad Sci USA 1983; 80:2127. 10. Weaver CA, Gordon DF, Kissil MS, et al. Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone. Gene 1984,28.319. 11. Heinrich G, Kronenberg HM, Potts JT Jr, Habener JF. Gene encoding parathy¬ roid hormone. Nucleotide sequence of the rat gene and deduced amino acid sequence of rat preproparathyroid hormone. J Biol Chem 1984; 259:3320. 12. Russell J, Sherwood LM. Nucleotide sequence of the DNA complementary to avian (chicken) preproparathyroid hormone mRNA and the deduced sequence of the hormone precursor. Mol Endocrinol 1989,3.325. 13. Yasuda T, Banville D, Hendy GN, Goltzman D. Characterization of the human parathyroid hormone-like peptide gene: functional and evolution¬ ary aspects. J Biol Chem 1989; 264:7720.

509

14. Mangin M, Ikeda K, Dreyer BE, Broadus AE. Isolation and characterization of the human parathyroid hormone-like peptide gene. Proc Natl Acad Sci USA 1989; 86:2408. 15. Suva LJ, Mather KA, Gillespie MT, et al. Structure of the 5' flanking region of the gene encoding human parathyroid hormone-related protein (PTHrP). Gene 1989; 77:95. 16. Karaplis AC, Yasuda T, Hendy GN, et al. Gene encoding parathyroid hor¬ mone-like peptide: nucleotide sequence of the rat gene and comparison with the human homologue. Mol Endocrinol 1990; 4:441. 17. Naylor SL, Sakaguchi AY, Shows TB, et al. Human parathyroid hormone gene (PTH) is on the short arm of chromosome 11. Somatic Cell Mol Genet 1983; 9:609. 18. Seldin MF, Mattei M-G, Hendy GN. Localization of mouse parathyroid hor¬ mone-like peptide gene (Pthlh) to distal chromosome 6 using interspecific backcross mice and in situ hybridization. Cytogenet Cell Genet 1992; 60:252. 19. Pausova Z, Morgan K, Fujiwara TM, et al. Molecular characterization of an intragenic minisatellite (VNTR) polymorphism in the human parathyroid hormone-related peptide gene in chromosome region 12pl2.1-pll.2. Genomics 1993; 17:243. 20. Keutmann HT, Sauer MM, Hendy GN, et al. Complete amino acid sequence of human parathyroid hormone. Biochemistry 1978; 17:5723. 21. Motokura T, Bloom T, Kim HG, et al. A novel cyclin encoded by bell-linked candidate oncogene. Nature 1991; 350:512. 22. Chandrasekharappa SC, Guru SC, Manickam P, et al. Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science 1997; 276:404. 23. European Consortium on MEN1. Identification of the multiple endocrine neoplasia type 1 (MEN1) gene. Hum Mol Genet 1997; 6:1177. 24. Guru SC, Goldsmith PK, Bums AL, et al. MENIN, the product of the MEN1 gene, is a nuclear protein. Proc Natl Acad Sci USA 1998; 95:1630. 25. Agarwal SK, Guru SC, Heppner C, et al. Menin interacts with the API transcrip¬ tion factor JunD and represses JunD-activated transcription. Cell 1999; 96:143. 26. Friedman E, Sakaguchi K, Bale AE, et al. Clonality of parathyroid tumors in familial multiple endocrine neoplasia type 1. N Engl J Med 1989; 321:213. 27. Thakker RV, Bouloux P, Wooding C, et al. Association of parathyroid tumors in multiple endocrine neoplasia type 1 with loss of alleles on chro¬ mosome 11. N Engl J Med 1989; 321:218. 28. Arnold A, Horst SA, Gardella TJ, et al. Mutation of the signal peptide¬ encoding region of the preproparathyroid hormone gene in familial iso¬ lated hypoparathyroidism. J Clin Invest 1990; 86:1084. 29. Parkinson DB, Thakker RV. A donor splice site mutation in the parathyroid hormone gene is associated with autosomal recessive hypoparathyroidism. Nature Genet 1992; 1:149. 30. Simpson EL, Mundy GR, D'Souza SM, et al. Absence of parathyroid hor¬ mone messenger RNA in nonparathyroid tumors associated with hyper¬ calcemia. N Engl J Med 1983; 309:325. 31. Nussbaum SR, Gaz RD, Arnold A. Hypercalcemia and ectopic secretion of parathyroid hormone by an ovarian carcinoma with rearrangement of the gene for parathyroid hormone. N Engl J Med 1990; 323:1324. 32. Fischer JA, Oldham SB, Sizemore GW, Arnaud CD. Calcium-regulated parathyroid hormone peptidase. Proc Natl Acad Sci USA 1972; 69:2341. 33. Flueck JA, DiBella FP, Edis AJ, et al. Immunoheterogeneity of parathyroid hormone in venous effluent serum from hyperfunctioning parathyroid glands. J Clin Invest 1977; 60:1367. 34. Hanley DA, Takatsuki K, Sultan JM, et al. Direct release of parathyroid hor¬ mone fragments from functioning bovine parathyroid glands in vitro. J Clin Invest 1978; 62:1247. 35. Mayer GP, Keaton JA, Hurst JG, Habener JF. Effect of plasma calcium con¬ centrations on the relative proportion of hormone and carboxyl fragments in parathyroid venous blood. Endocrinology 1979; 104:1778. 36. Kemper B, Habener JF, Rich A, Potts JT Jr. Parathyroid secretion: discovery of a major calcium-dependent protein. Science 1974; 184:167. 37. Cohn DV, Zangerle R, Fischer-Colbrie R, et al. Similarity of secretory pro¬ tein-I from parathyroid gland to chromogranin A from adrenal medulla. Proc Natl Acad Sci U S A 1982; 79:6056. 38. Bhargava G, Russell J, Sherwood LM. Phosphorylation of parathyroid secretory protein. Proc Natl Acad Sci U S A1983; 80:878. 39. Mouland AJ, Bevan S, White JH, Hendy GN. Human chromogranin A gene; molecular cloning, structural analysis and neuroendocrine cell-specific expression. J Biol Chem 1994; 269:6918. 40. Canaff L, Bevan S, Wheeler D, et al. Analysis of molecular mechanisms controlling neuroendocrine cell specific transcription of the chromogranin A gene. Endocrinology 1998; 139:1184. 41. el-Hajj Fuleihan G, Klerman EB, Brown EN, et al. The parathyroid hor¬ mone circadian rhythm is truly endogenous—a general clinical research center study. J. Clin Endocrinol Metab 1997; 82:281. 42. Samuels MH, Veldhais JD, Kramer P, et al. Episodic secretion of parathy¬ roid hormone in postmenopausal women: assessment by deconvolution analysis and approximate entropy. J Bone Miner Res 1977; 12:616. 43. Mayer GP, Hurst JG. Sigmoidal relationship between parathyroid hormone secretion rate and plasma concentration in calves. Endocrinology 1978; 102:1036. 44. Shoback DM, Thatcher J, Leombruno R, Brown EM. Relationship between parathyroid hormone secretion and cytosolic calcium concentration in dis¬ persed bovine parathyroid cells. Proc Natl Acad Sci U S A1984; 81:3113. 45. Brown EM, Redgrave J, Thatcher J. Effect of the phorbol ester TPA on PTH secretion. Evidence for a role for protein kinase C in the control of PTH release. FEBS Lett 1984; 175:72.

510

PART IV: CALCIUM AND BONE METABOLISM

46. Brown EM, Gambda G, Riccardi D, et al. Cloning and characterization of an extracellular Ca2*-sensing receptor from bovine parathyroid. Nature 1993; 366:575. 47. Poliak MR, Brown EM, Chou Y-HW, et al. Mutations in the human Ca2+sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 1993; 75:1237. 48. Heath H III, Odelberg S, Jackson CE, et al. Clustered inactivating muta¬ tions and benign polymorphisms of the calcium receptor gene in familial benign hypocalciuric hypercalcemia suggest receptor functional domains. J Clin Endocrinol Metab 1996; 81:1312. 49. Cole DEC, Janicic N, Salisbury SR, Hendy, GN. Neonatal severe hyperpara¬ thyroidism, secondary hyperparathyroidism, and familial hypercalciuric hypercalcemia: multiple different phenotypes associated with an inactivat¬ ing Alu insertion mutation of the calcium-sensing receptor gene. Am J Med Genet 1997; 71:202. 50. Poliak MR, Chou Y-HW, Marx SJ, et al. Familial hypocalciuric hypercalce¬ mia and neonatal severe hyperparathyroidism. Effects of mutant gene dos¬ age on phenotype. J Clin Invest 1994; 93:1108. 51. Bai M, Pearce SHS, Kifor O, et al. In vivo and in vitro characterization of neonatal hyperparathyroidism resulting from a de novo, heterozygous mutation in the Ca2+-sensing receptor gene: normal maternal calcium homeostasis as a cause of secondary hyperparathyroidism in familial benign hypocalciuric hypercalcemia. J Clin Invest 1997; 99:88. 52. Pearce SHS, Williamson C, Kifor O, et al. A familial syndrome of hypocal¬ cemia with hypercalciuria due to mutations in the calcium-sensing receptor. N Engl J Med 1996; 335:1115. 53. Cole DEC, Peltekova VD, Rubin LA, et al. A986S polymorphism of the cal¬ cium-sensing receptor and circulating calcium concentrations. Lancet 1999; 353:112. 54. Nemeth EF, Steffey ME, Hammerland LG, et al. Calcimimetics with potent and selective activity on the parathyroid calcium receptor. Proc Natl Acad Sci USA 1998; 95:4040. 55. Silverberg SJ, Bone GH III, Marriott TB, et al. Short-term inhibition of parathyroid hormone secretion by a calcium-receptor agonist in patients with primary hyperparathyroidism. N Engl J Med 1997; 337:1506. 56. Kovacs CS, Ho-Pao CL, Hunzelman JL, et al. Regulation of murine fetalplacental calcium metabolism by the calcium-sensing receptor. J Clin Invest 1998; 101:2812. 57. Brown AJ, Zhong M, Ritter C, et al. Loss of calcium responsiveness in cul¬ tured bovine parathyroid cells is associated with decreased calcium recep¬ tor expression. Biochem Biophys Res Comm 1995; 212:861. 58. Mithal A, Kifor O, Kifor I, et al. The reduced responsiveness of cultured bovine parathyroid cells to extracellular Ca2‘ is associated with marked reduction in the expression of extracellular Ca(2+)-sensing receptor messenger ribonucleic acid and protein. Endocrinology 1995; 136:3087. 59. Russell J, Lettieri D, Sherwood LM. Direct regulation by calcium of cytoplasmic messenger ribonucleic acid coding for pre-proparathyroid hor¬ mone in isolated bovine parathyroid cells. J Clin Invest 1983; 72:1851. 60. Russell J, Sherwood LM. The effects of l,25(OH)2D3 and high calcium on transcription of the pre-proparathyroid hormone gene are direct. Trans Assoc Am Physicians 1987; 100:256. 61. Moallem E, Kilav R, Silver J, Naveh-Many T. RNA-protein binding and post-transcriptional regulation of parathyroid hormone gene expression by calcium and phosphate. J Biol Chem 1998; 273:5253. 62. Brown EM. Four-parameter model of the sigmoidal relationship , between parathyroid hormone release and extracellular calcium con¬ centration in normal and abnormal parathyroid tissue. Endocrinology 1983; 56:572. 63. Kifor O, Moore FD, Wang P, et al. Reduced immunostaining for the extra¬ cellular Ca2+-sensing receptor in primary and secondary hyperparathy¬ roidism. J Clin Endocrinol Metab 1996; 81:1598. 64. Ho C, Conner DA, Pollack MR, et al. A mouse model of human familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Nature Genet 1995; 11:389. 65. Habener JF, Potts JT Jr. Relative effectiveness of magnesium and calcium on the secretion and biosynthesis of parathyroid hormone in vitro. Endocrinology 1976; 98:197. 66. Rude RK, Oldham SB, Sharp CF Jr, Singer FR. Parathyroid hormone secre¬ tion in magnesium deficiency. J Clin Endocrinol Metab 1978; 47:800. 67. Cholst IN, Steinberg SF, Tropper PJ, et al. The influence of hyper¬ magnesemia on serum calcium and parathyroid hormone levels in human subjects. N Engl J Med 1984; 310:1221. 68. Morrissey J, Rothstein M, Mayer G, Slatopolsky E. Suppression of parathy¬ roid hormone by aluminum. Kidney Int 1983; 23:699. 69. Sherwood LM, Mayer GP, Ramberg DF, et al. Regulation of parathyroid hormone secretion: proportional control by calcium, lack of effect of phos¬ phate. Endocrinology 1968; 83:1043. 70. Almaden Y, Canelejo A, Hernandez A, et al. Direct effect of phosphorous on PTH secretion from whole rat parathyroid glands in vitro. J Bone Miner Res 1996; 11:970. 71. Slatopolsky E, Finch J, Denda M, et al. Phosphorous restriction prevents parathyroid gland growth: high phosphorous directly stimulates PTH secretion in vitro. J Clin Invest 1996; 97:2534. 72. Abe M, Sherwood LM. Regulation of parathyroid hormone secretion by adenylate cyclase. Biochem Biophys Res Commun 1972; 48:396. 73. Brown EM, Gardner DG, Windek RA, Aurbach GD. Relationship of intracellular 3',5'-adenosine monophosphate accumulation to parathyroid hor¬

74. 75.

76.

77.

78.

79.

80.

81.

82. 83. 84. 85.

86.

87.

88. 89.

90.

91.

92. 93.

94.

95. 96.

97.

98.

99.

100.

101.

102.

mone release from dispersed bovine parathyroid cells. Endocrinology 1978; 103:2323. Heath H III. Biogenic amines and the secretion of parathyroid hormone and calcitonin. Endocr Rev 1980; 1:319. Epstein S, Heath H III, Bell NH. Lack of influence of isoproterenol, propra¬ nolol and dopamine on immunoreactive parathyroid hormone and calcito¬ nin in normal man. Calcff Tissue Int 1983; 35:32. Fasciotto BH, Gorr S-U, De Franco DJ, et al. Pancreastatin, a presumed product of chromogranin-A (secretory protein-1) processing, inhibits secre¬ tion from porcine parathyroid cells in culture. Endocrinology 1989; 125:1617. Drees BM, Rouse J, Johnson J, Hamilton JW. Bovine parathyroid glands secrete a 26-kDa N-terminal fragment of chromogranin-A which inhibits parathyroid cell secretion. Endocrinology 1991; 129:3381. Silver J, Russell J, Sherwood LM. Regulation by vitamin D metabolites of messenger ribonucleic acid for preproparathyroid hormone in isolated bovine parathyroid cells. Proc Natl Acad Sci U S A1985; 82:4270. Hendy GN, Stotland MS, Grunbaum D, et al. Characteristics of secondary hyperparathyroidism in vitamin D deficient dogs. Am J Physiol 1989; 256:E765. Mackey SL, Heymont JL, Kronenberg HM, Demay MB. Vitamin D receptor binding to the negative human parathyroid hormone vitamin D response element does not require the retinoid x receptor. Molec Endocrinol 1996; 10:298. Kremer R, Bolivar I, Goltzman D, Hendy GN. Influence of calcium and 1,25-dihdroxycholecalciferol on proliferation and proto-oncogene expres¬ sion in primary cultures of bovine parathyroid cells. Endocrinology 1989; 125:935. Fucik RF, Krukeja SC, Hargis GK, et al. Effect of glucocorticoids on function of the parathyroid glands in man. J Clin Endocrinol Metab 1975; 40:152. Silverman R, Yalow RS. Heterogeneity of parathyroid hormone: clinical and physiologic implications. J Clin Invest 1973; 52:1958. Segre GV, Niall HD, Habener JF, Potts JT Jr. Metabolism of parathyroid hor¬ mone: physiological and clinical significance. Am J Med 1974; 56:774. Arnaud CD, Goldsmith RS, Bordier PJ, et al. Influence of immunoheterogeneity of circulating parathyroid hormone on radioimmunoassays of serum in man. Am J Med 1974; 56:785. Reiss E, Canterbury JM. Emerging concepts of the nature of circulating para¬ thyroid hormones: implications for clinical research. Recent Prog Horm Res 1974; 30:391. Neuman WF, Neuman MW, Lane K, et al. The metabolism of labeled para¬ thyroid hormone V. Collected biological studies. Calcif Tissue Res 1975; 18:271. Martin KJ, Hruska KA, Freitag JJ, et al. The peripheral metabolism of par¬ athyroid hormone. N Engl J Med 1979; 301:1092. Segre GV, D'Amour P, Hultman A, Potts JT Jr. Effects of hepatectomy, nephrectomy, and nephrectomy/uremia on the metabolism of parathyroid hormone in the rat. J Clin Invest 1981; 67:439. Papapoulos SE, Hendy GN, Tomlinson S, et al. Clearance of exogenous parathyroid hormone in normal and uraemic man. Clin Endocrinol 1977; 7:211. Goltzman D, Henderson B, Loveridge N. Cytochemical bioassay of para¬ thyroid hormone: characteristics of the assay and analysis of circulating hormonal forms. J Clin Invest 1980; 65:1309. DeLuca HF. Recent advances in the metabolism of vitamin D. Ann Rev Physiol 1981; 43:199. Parsons JA, Rafferty B, Gray D, et al. Pharmacology of parathyroid hor¬ mone and some of its fragments and analogues. In: Talmage RV, Owen M, Parsons JA, eds. Calcium-regulating hormones. Amsterdam: Excerpta Medica, 1975:33. Tregear GW, van Rietschoten J, Greene E, et al. Bovine parathyroid hor¬ mone: minimum chain length of synthetic peptide required for biological activity. Endocrinology 1973; 93:1349. Kemp BE, Moseley JM, Rodda CP, et al. Parathyroid hormone-related pro¬ tein of malignancy: active synthetic fragments. Science 1987; 23:1568. Horiuchi N, Caulfield MP, Fisher JE, et al. Similarity of synthetic peptide from human tumor to parathyroid hormone in vivo and in vitro. Science 1987; 23:1566. Rabbani SA, Mitchell J, Roy DR, et al. Influence of the amino-terminus on in vitro and in vivo biological activity of synthetic parathyroid hormone¬ like peptides of malignancy. Endocrinology 1988; 123:2709. Goltzman D, Peytremann A, Callahan E, et al. Analysis of the requirements for parathyroid hormone action in renal membranes with the use of inhib¬ iting analogues. J Biol Chem 1975; 250:3199. Nussbaum SR, Rosenblatt M, Potts JT Jr. Parathyroid hormone renal recep¬ tor interactions: demonstration of two receptor binding domains. J Biol Chem 1980; 255:10183. Abou-Samra A-B, Uneno S, Jiippner H, et al. Non-homologous sequences of parathyroid hormone and the parathyroid hormone related peptide bind to a common receptor on ROS 17/2.8 cells. Endocrinology 1989; 125:2215. Caulfield MP, McKee RL, Goldman ME, et al. The bovine renal parathyroid hormone (PTH) receptor has equal affinity for two different amino acid sequences: The receptor binding domains of PTH and PTH-related protein are located within the 14-34 region. Endocrinology 1990; 127: 83. Klaus W, Dieckmann T, Wray V, et al. Investigation of the solution structure of the human parathyroid hormone fragment (1-34) by ’H NMR spectros-

Ch. 51: Parathyroid Hormone copy, distance geometry, and molecular dynamics calculations. Biochemis¬ try 1991; 30:6936. 103. Cohen FE, Strewler GJ, Bradley MS, et al. Analogues of parathyroid hor¬ mone modified at positions 3 and 6: Effects on receptor binding and activa¬ tion of adenylyl cyclase in kidney and bone. J Biol Chem 1991; 266:1997. 104. Barden JA, Kemp BE. Stabilized NMR structure of the hypercalcemia of malignancy peptide PTHrP [Ala-26](l-34) amide. Biochim Biophys Acta 1994; 1208:256. 105. Wray V, Federau T, Gronwald W, et al. The structure of human parathyroid hormone from a study of fragments in solution using 1H NMR spectros¬ copy and its biological implications. Biochemistry 1994; 33:1684. 106. Neugebauer W, Barbier J-R, Sung WL, et al. Solution structure and adeny¬ lyl cyclase stimulating activities of C-terminal truncated human parathy¬ roid hormone analogues. Biochemistry 1995; 34:8835. 107. Maretto S, Mammi S, Bissacco E, et al. Mono- and bicyclic analogs of parathy¬ roid hormone-related protein. 2. Conformational analysis of antagonists by CD, NMR, and distance geometry calculations. Biochemistry 1997; 36:3300. 108. Barbier J-R, Neugebauer W, Morley P, et al. Bioactivities and secondary stuctures of constrained analogues of human parathyroid hormone: cyclic lactams of the receptor binding region. J Medicinal Chem 1997; 40:1373. 109. Vickery BH, Avnur Z, Cheng Y, et al. RS-66271, a C-terminally substituted analog of human parathyroid hormone-related protein (1-34), increases tra¬ becular and cortical bone in ovariectomized, osteopenic rats. J Bone Miner Res 1996; 11:1943. 110. Juppner H, Abou-Samra AB, Freeman MW, et al. A G protein-linked recep¬ tor for parathyroid hormone and parathyroid hormone-related peptide. Science 1991; 254:1024. 111. Abou-Samra AB, Juppner H, Force T, et al. Expression cloning of a parathy¬ roid hormone/parathyroid hormone-related peptide receptor from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol triphosphate and increases intracellular free cal¬ cium. Proc Natl Acad Sci U S A 1992; 89:2732. 112. Pausova Z, Bourdon J, Clayton D, et al. Cloning of a parathyroid hor¬ mone/parathyroid hormone-related peptide receptor (PTHR) cDNA from a rat osteosarcoma (UMR 106) cell line: chromosomal assignment of the gene in the human, mouse, and rat genomes. Genomics 1994; 20:20. 113. Schipani E, Harga H, Karaplis AC, et al. Identical complementary deoxyri¬ bonucleic acids encode a human renal and bone parathyroid hormone (PTH)/PTH-related peptide receptor. Endocrinology 1993; 132:2157. 114. Fraher LJ, Hodsman AB, Jonas K, et al. A comparison of the in vivo biochem¬ ical responses to exogenous parathyroid hormone-(l-34) [PTH-(1-34)J and PTH-related peptide-(l-34) in man. J Clin Endocrinol Metab 1992; 75:417. 114a. Fraher LJ, Avram R, Watson PH, et al. Comparison of the biochemical responses to human parathyroid hormone-(l-31)NH2 and hPTH-(l-34) in healthy humans. J Clin Endocrinol Metab 1999; 84:2739. 115. McCuaig KA, Clarke JC, White JH. Molecular cloning of the gene encoding the mouse parathyroid hormone/parathyroid hormone related peptide receptor. Proc Natl Acad Sci USA 1994; 91:5051. 116. Bettoun JD, Minagawa M, Hendy GN, et al. Developmental upregulation of human parathyroid hormone (PTH)/PTH-related peptide receptor gene expression from conserved and human-specific promoters. J Clin Invest 1998; 102:958. 116a. Bettoun JD, Kwan MY, Minagawa M, et al. Methylation patterns of human parathyroid hormone (PTH)/PTH-related peptide receptor gene promoters are established several weeks prior to onset of their function. Biochem Bio¬ phys Res Common 2000; 267:482. 117. Urena P, Kong X-F, Abou-Samra A-B, et al. Parathyroid hormone (PTH)/ PTH-related peptide receptor messenger ribonucleic acids are widely dis¬ tributed in rat tissues. Endocrinology 1993; 133:617. 118. Usdin TB, Gruber C, Bonner TI. Identification and functional expression of a receptor selectively recognizing parathyroid hormone, the PTH2 recep¬ tor. J Biol Chem 1995; 270:15455. 119. Lee CW, Gardella TJ, Abou-Samra AB, et al. Role of the extracellular regions of the PTH/PTHrP receptor in hormone-binding. Endocrinology 1994; 135:1488. 120. Lee CW, Luck MD, Juppner H, et al. Homolog-scanning mutagenesis of the parathyroid hormone (PTH) receptor reveals PTH-(l-34) binding determi¬ nants in the third extracellular loop. Mol Endocrinol 1995; 9:1269. 121. Iida KA, Guo J, Takemura M, et al. Mutations in the second cytoplasmic loop of the rat parathyroid hormone (PTH)/PTH-related protein receptor result in selective loss of PTH-stimulated phospholipase C activity. J Biol Chem 1997; 272:6882. 122. Huang Z, Chen Y, Nissenson RA. The cytoplasmic tail of the G protein-cou¬ pled receptor for parathyroid hormone and parathyroid hormone-related protein contains positive and negative signals for endocytosis. J Biol Chem 1996; 270:151. 123. Mahoney CA, Nissenson RA. Canine renal receptors for parathyroid hor¬ mone: down-regulation in vivo by exogenous parathyroid hormone. J Clin Invest 1983; 72:411. 124. Forte LR, Langeluttig SG, Poelling RE, Thomas ML. Renal parathyroid hor¬ mone receptors in the chick: downregulation in secondary hyperparathy¬ roid animal models. Am J Physiol 1982; 242:E154. 125. Tomlinson S, Hendy GN, Pemberton DM, O'Riordan JLH. Reversible resis¬ tance to the renal action of parathyroid hormone in man. Clin Sci Mol Med 1976; 51:59. 126. Schipani E, Weinstein LS, Bergwitz C, et al. Pseudohypoparathyroidism type lb is not caused by a defect in the coding exons of the human parathy¬

127.

128.

129.

130.

131.

132.

133. 134.

135.

135a. 136.

137.

138.

139.

140.

141. 142.

143.

144.

145.

511

roid hormone (PTH)/PTH-related peptide gene. J Clin Endocrinol Metab 1995; 80:1611. Bettoun JD, Minagawa M, Kwan MY, et al. Cloning and characterization of the promoter regions of the human parathyroid hormone (PTH)/PTHrelated peptide receptor gene: analysis of deoxyribonucleic acid from nor¬ mal subjects and patients with pseudohypoparathyroidism type lb. J Clin Endocrinol Metab 1997; 82:1031. Juppner H, Schipani E, Bastepe M, et al. The gene responsible for pseudo¬ hypoparathyroidism type lb is paternally imprinted and maps in four unrelated kindreds to chromosome 20ql3.3. Proc Natl Acad Sci USA 1998; 95:11798. Yu S, Yu D, Lee E, et al. Variable and tissue-specific hormone resistance in heterotrimeric Gs protein a-subunit (Gsa) knockout mice is due to tissuespecific imprinting of the Gsa gene. Proc Natl Acad Sci USA 1998; 95:8715. Hayward BE, Moran V, Strain L, Bonthron DT. Bidirectional imprinting of a single gene: GNAS1 encodes maternally, paternally, and biallelically derived proteins. Proc Natl Acad Sci USA 1998; 95:15475. Jansen M. Uber atypische Chondrodystrophie (Achondroplasie) und tiber eine noch nicht beschriebene angeborene Wachstumsstorung des Knochensystems: Metaphysare Dysostosis. Orthop Chir 1934; 61:253. Schipani E, Langman CB, Parfitt AM, et al. Constitutively activated recep¬ tors for parathyroid hormone-related peptide in Jansen's metaphyseal chondrodysplasia. N Engl J Med 1996; 335:708. Blomstrand S, Claesson I, Save-Soderbergh J. A case of lethal congenital dwarfism with accelerated skeletal maturation. Pediatr Radiol 1985; 15:141. Zhang P, Jobert A-S, Couvineau A, Silve C. A homozygous inactivating mutation in the parathyroid hormone/parathyroid hormone-related pep¬ tide receptor causing Blomstrand chondrodysplasia. J Clin Endocrinol Metab 1998; 83:3365. Jobert A-S, Zhang P, Couvineau A, et al. Absence of functional receptors for parathyroid hormone and parathyroid hormone-related peptide in Blom¬ strand chondrodysplasia. J Clin Invest 1998; 102:34. Usdin TB, Hoare SR, Wang T, et al. TIP39: a new neuropeptide and PTH2receptor agonist from hypothalamus. Nature Neuroscience 1999; 2:941. Brenza HL, Kimmel-Jehan C, Jehan F, et al. Parathyroid hormone activa¬ tion of the 25-hydroxyvitamin D3-l alpha-hydroxylase gene promoter. Proc Natl Acad Sci U S A1998; 95:1387. Livesey SA, Kemp BE, Re CA, et al. Selective hormonal activation of cyclic AMP-dependent protein-kinase isoenzymes in normal and malignant osteoblasts. J Biol Chem 1982; 257:14983. Pearman AT, Chou WY, Bergman KD, et al. Parathyroid hormone induces c-fos promoter activity in osteoblastic cells through phosphorylated cAMP response element (CRE)-binding protein to the major CRE. J Biol Chem 1996; 271:25715. Stem PH. Cationic agonists and antagonists of bone resorption. In: Cohn DV, Fujita T, Potts JT Jr, Talmage RV, eds. Endocrine control of bone and calcium metabolism. Amsterdam: Excerpta Medica, 1984:109. Hruska KA, Moskowitz D, Esbrit P, et al. Stimulation of inositol triphos¬ phate and diacylglycerol production in renal tubular cells by parathyroid hormone. J Clin Invest 1987; 79:230. Bingham P, Brazell I, Owen M. The effect of parathyroid extract on cellular activity and plasma calcium levels in vivo. J Endocrinol 1969; 45:387. Holtrop ME, Raisz LG, Simmons HA. The effect of parathyroid hormone, colchicine and calcitonin on the ultrastructure and activity of osteoblasts in organ culture. J Cell Biol 1974; 60:346. Rouleau MF, Mitchell J, Goltzman D. In vivo distribution of parathyroid hormone receptors in bone. Evidence that a predominant osseous target cell is not the mature osteoblast. Endocrinology 1988; 123:187. Rouleau MF, Warshawsky H, Goltzman D. Parathyroid hormone binding in vivo to renal, hepatic and skeletal tissues of the rat using a radioauto¬ graphic approach. Endocrinology 1986; 118:919. Rizzoli RE, Somerman M, Murray TM, Aurbach GD. Binding of radioiodinated parathyroid hormone to cloned bone cells. Endocrinology 1983;

113:1832. 146. Amizuka N, Karaplis AC, Henderson JE, et al. Haploinsufficiency of para¬ thyroid hormone-related peptide (PTHrP) results in abnormal postnatal bone development. Dev Biol 1996; 175:166. 147. Takahashi N, Akatsu T, Udagawa N, et al. Osteoblastic cells are involved in osteoclast formation. Endocrinology 1988; 123:2600. 148. Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998; 93:165. 149. Kong Y-Y, Yoshida H, Sarosi L, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 1999; 397:315. 150. Simonet WS, Lacey DL, Dunstan CR, et al. Osteoprotegerin: A novel secreted protein involved in the regulation of bone density. Cell 1997; 89:309. 151. Parsons JA, Potts JT Jr. Physiology and chemistry of parathyroid hormone. In: MacIntyre I, ed. Clinics in endocrinology and metabolism, vol 1. Cal¬ cium metabolism and bone disease. London: WB Saunders, 1972:33. 152. Tam CS, Heersche JNM, Murray TM, Parsons JA. Parathyroid hormone stimulates the bone apposition rate independently of its resorptive action: differential effects of intermittent and continual administration. Endocri¬ nology 1982; 110:506. 153. Corral DA, Amling M, Priemel M, et al. Dissociation between bone resorp¬ tion and bone formation in osteopenic transgenic mice. Proc Natl Acad Sci US A1998; 95:13835.

512

PART IV: CALCIUM AND BONE METABOLISM

154. Bijvoet OLM. Kidney function in calcium and phosphate metabolism. In: Avioli LV, Krane SM, eds. Metabolic bone disease, vol 1. New York: Aca¬ demic Press, 1977:48. 155. Hartmann CM, Hewson AS, Kos CH, et al. Structure of murine and human renal type II Na+-phosphate cotransporter genes (Npt2 and NPT2). Proc Natl Acad Sci U S A1996; 93:7409. 156. Azarani A, Goltzman D, Orlowski J. Structurally diverse N-terminal pep¬ tides of parathyroid hormone (PTH) and PTH-related peptide (PTHRP) inhibit the Na+/H+ exchanger NHE3 isoform by binding to the PTH/ PTHRP receptor type I and activating distinct signalling pathways. J Biol Chem 1996; 271:14931. 157. Morel F. Regulation of kidney functions by hormones: a new approach. Recent Prog Horm Res 1983; 39:271. 158. Agus ZS, Wasserstein A, Goldfarb S. PTH, calcitonin, cyclic nucleotides and the kidney. Annu Rev Physiol 1981; 43:583. 159. Nordin BEC, Peacock M. Role of the kidney in regulation of plasma cal¬ cium. Lancet 1969; 2:1280. 160. Amizuka N, Warshawsky H, Henderson JE, et al. Parathyroid hormonerelated peptide-depleted mice show abnormal epiphyseal cartilage develop¬ ment and altered endochondral bone formation J Cell Biol 1994; 126:1611. 161. Marx SJ, Sharp ME, Krudy A, et al. Radioimmunoassay for the middle region of human parathyroid hormone: studies with a radioiodinated syn¬ thetic peptide. J Clin Endocrinol Metab 1981; 53:76. 162. Papapoulos SE, Manning RM, Hendy GN, et al. Studies of circulating para¬ thyroid hormone in man using a homologous amino-terminal specific immunoradiometric assay. Clin Endocrinol 1980; 13:57. 163. Nussbaum SR, Zahnadnik RJ, Labigne JR, et al. A highly sensitive two-site immunoradiometric assay of parathyrin (PTH) and its clinical utility in evaluating patients with hypercalcemia. Clin Chem 1987; 33:1364. 164. Broadus AE. Nephrogenous cyclic AMP. Recent Prog Horm Res 1981; 37:667. 165. Nissenson RA, Abbott SR, Teitelbaum AP, et al. Endogenous biologically active human parathyroid hormone: measurement by a guanyl nucleotideamplified renal adenylate cyclase assay. J Clin Endocrinol Metab 1981; 52:840. 166. Sato K, Han DC, Ozawa M, et al. A highly sensitive bioassay for PTH using ROS 17/2.8 subclonal cells. Acta Endocrinol (Copenh) 1987; 116:113. 167. Chambers DJ, Dunham J, Zanelli JM, et al. A sensitive bioassay of parathy¬ roid hormone in plasma. Clin Endocrinol 1978; 9:375.

CHAPTER 52

PARATHYROID HORMONERELATED PROTEIN GORDON J. STREWLER

Parathyroid hormone-related protein (PTHrP), sometimes referred to as parathyroid hormone-like protein, is the sister of PTH. Originally identified as the cause of humoral hypercalce¬ mia in malignancy, PTHrP has a distinct set of physiologic func¬ tions that are unrelated to the regulation of systemic calcium homeostasis but rival the actions of PTH in their importance. It has been recognized since Fuller Albright's time that in some ways patients with malignant tumors causing hypercalce¬ mia resemble patients with primary hyperparathyroidism (pHPT). Malignancy-associated hypercalcemia is characterized not only by humorally mediated bone resorption but also by diminished tubule resorption of phosphate with consequent phosphaturia and hypophosphatemia. When it was recognized that the disorder also produced an increase in nephrogenous cyclic adenosine monophosphate (cAMP), which is the compo¬ nent of urinary cAMP secreted into the urine from the renal tubule,1 it became clear that a humoral factor in patients with malignancy-associated hypercalcemia was mimicking PTH at the kidney. Increased nephrogenous cAMP was previously thought to be unique to hyperparathyroidism, reflecting increased secretion of cAMP from the renal tubule, where cAMP is the intracellular second messenger for PTH. Once it was understood that increased cAMP concentrations in kidney or bone cells could be used as a bioassay to detect the humoral factor secreted by malignant tumors, the substance responsible

FIGURE 52-1. Primary structures of the aminoterminal part of parathy¬ roid hormone-related protein and of parathyroid hormone. Compared are the human sequences 1-34. Identical amino acids are highlighted.

for malignancy-associated hypercalcemia was purified and shown to be a protein that was structurally related to PTH.2"4

STRUCTURE AND BIOLOGIC PROPERTIES STRUCTURE AND PROCESSING OF PARATHYROID HORMONE-RELATED PROTEIN AND ITS GENE PTHrP is a protein of 139 to 173 amino acids.5-7 It resembles PTH in primary sequence only at the aminoterminus, where 8 of the first 13 amino acids in the two peptides are identical (Fig. 52-1). Although this is a limited region of homology, it is a criti¬ cal region of both peptides; it is not required for binding but it is necessary for receptors occupied by either hormone to acti¬ vate adenylate cyclase and hence to produce cAMP as a second messenger. Thus, the homology of PTH and PTHrP at the ami¬ noterminal end is responsible for their shared biologic proper¬ ties (see later in this chapter). The gene for PTHrP is located on human chromosome 12. It is considerably more complex than the PTH gene, which is located on chromosome 11, with three promoters and four alter¬ natively spliced exons upstream of the coding sequence.8 The complexity of the promoter region allows for considerable flex¬ ibility in the regulation of PTHrP gene transcription. There is evidence that the promoters are used differentially in different tissues8 and by the viral transactivating protein tax.9 Most of the prepro sequence of PTHrP is encoded on one exon, with the last 2 amino acids of the prepro sequence and 139 amino acids of the mature peptide encoded on a second exon. The splice junction between these two coding exons is precisely conserved between PTHrP and PTH. Downstream of the main coding sequence are two additional exons, which are alternatively spliced to contribute distinct carboxyterminal ends to the isoforms of PTHrP and distinct 3' noncoding sequences. The protein isoforms encoded by these exons are identical through amino acid 139 but comprise 139, 141, or 173 amino acids in toto. This downstream complexity of the PTHrP gene may also be used for regulation. The three transcript fami¬ lies with different 3' untranslated sequences have markedly dif¬ ferent half-lives, ranging from 20 minutes to ~7 hours. In addition, their stability is differentially regulated. Exposure of cells to transforming growth factor-(3 specifically increases the half-life of the most unstable transcript, which encodes the 139 amino acid form of the protein. It is clear from conservation of sequence, gene structure, and chromosomal localization that PTHrP and PTH arose from a common ancestral gene. In addition to the striking resemblance of their aminoterminal sequence and the conservation of the intron-exon boundaries between the two major coding exons, each gene is flanked by duplicated genes for lactate dehydroge¬ nase. It is believed that chromosome 12, on which the PTHrP gene is located, and chromosome 11, where the PTH gene

Ch. 52: Parathyroid Hormone-Related Protein l

37

88-91

102-106

139

—

1 III

PTHrP 36 37

Peptides

PTH-like

94

107

139

Midregion

Osteostatin

Sites of

Cartilage

Placental calcium transport

Bone resorption

Action

Breast

Renal bicarbonate transport?

Brain

Skin

FIGURE 52-2. Top, Structural features of PTHrP. The PTH-homologous domain PTHrP(l-13) is delineated by hatched lines (*), and potential cleavage sites are shown as vertical lines or crosshatched regions (■■). Middle, Peptides known or postulated to be derived from PTHrP. Bottom, Proven or postulated sites of action of individual PTHrP peptides are shown beneath each peptide. resides, arose by an ancient duplication in which the ancestral PTHfPTHrP gene was evidently copied along with its neighbors.

SECRETED FORMS: PEPTIDE HETEROGENEITY The three mature PTHrP peptides predicted by cDNA are 139, 141, and 173 amino acids long. It is not clear whether any of them are secreted as intact proteins, although it seems likely they are. AMINOTERMINAL FRAGMENTS The molecular size of PTHrP containing the bioactive aminoterminal part found in plasma and in tumor extracts ranges anywhere from 6 to 18 kDa,2"4-10 and, apparently, several different aminoterminal fragments of PTHrP are secreted.11 Three different cell types (renal carcinoma cells, PTHrP-transfected RIN-141 cells, and nor¬ mal keratinocytes) use prohormone convertases to cleave PTHrP after arginine-37 to produce an aminoterminal fragment that is probably processed to PTHrP(l-36)12 (Fig- 52-2). This aminotermi¬ nal PTHrP fragment contains the PTH-like region and could account completely for the PTH-like effects of PTHrP, as discussed later. MIDREGIONAL FRAGMENTS The intracellular cleavage step at arginine-37 also produces midregional PTHrP fragments of ~50 to 70 amino acids begin¬ ning with alanine-3812 (see Fig. 52-2). The carboxytermini of these fragments are located at amino acids 94,95, and 101 in the highly basic region PTHrP(88-106).13 Midregional fragments of PTHrP generated intracellularly can apparently enter the regu¬ lated pathway of peptide secretion and become packaged in dense neurosecretory granules, suggesting that they may be under separate secretory control from aminoterminal frag¬ ments.12 Midregional PTHrP fragments of similar size are the predominant circulating forms in the plasma of patients with humoral hypercalcemia of malignancy.14 These midregional PTHrPs play a physiologic role in transplacental calcium trans¬ port (see later), and synthetic midregion peptides also appear to have bioactivities in squamous carcinoma cells. CARBOXYTERMINAL FRAGMENTS The size of the midregional fragments suggests the existence of additional carboxyterminal PTHrP fragments resulting from cleavage at the multibasic amino acid clusters at amino acids 88— 106. Because the most carboxyterminal cleavage site is at amino acid 101, which precedes the lysine-arginine cluster at PTHrP(102-106), it is likely that carboxyterminal peptides such as PTHrP(107-139) are produced and secreted. Peptides with PTHrP(109-138) but not PTHrP(l-36) immunoreactivity have been found in sera of patients with humoral hypercalcemia of malignancy, and a peptide with PTHrP(109-138) immunoreac-

513

tivity circulates as a separate peptide that accumulates in patients with chronic renal failure.10'15-16 The high degree of con¬ servation between human, rat, and chicken PTHrP in the por¬ tion up to amino acid 111 suggests some biologic relevance of such peptides. The very carboxyterminal part of the conserved region, PTHrP(107-lll), was found in several studies to be a potent inhibitor of osteoclastic bone resorption in vitro,17-18 and has been given the name osteostatin, although this result has not been confirmed in all bone resorption assays.19 The correspond¬ ing synthetic peptides (PTHrP[107-lll] and PTHrP[107-139]) induce calcium transients in hippocampal neurons.20 PTHrP is posttranslationally modified in additional ways21; for example, PTHrP secreted by keratinocytes is glycosylated posttranslationally.22 Whether this is also the case with other cell types and with PTHrP forms circulating in plasma is unknown. The findings that cells process PTHrP to multiple peptide fragments, that some of these fragments may be under separate secretory control, and that one or more (in addition to its PTHlike aminoterminus) has its own bioactivity indicate that PTHrP is, like the adrenocorticotropic hormone-melanocytestimulating hormone-endorphin precursor pro-opiomelanocortin, a polyprotein precursor for multiple bioactive peptides.23 The pattern of posttranslational modification may be tissuespecific, and this would add another dimension of specificity to the secreted forms of PTHrP.

IMMUNOASSAYS Because the known PTH-like bioactivity of PTHrP is located within the first 34 amino acids, most immunoassays have been directed against the aminoterminal sequence 1-34 to 1-40 of PTHrP in radioimmunoassays (RIAs)24-26 or against larger frag¬ ments containing aminoterminal PTHrP in immunoradiometric assays.16-27 Because the amounts of aminoterminal PTHrP extractable from normal serum are very low (400 patients with primary hyperparathyroidism for ~30 years indicates that survival, on average, is indistinguish¬ able from the expected longevity from life tables.85 Patients with hypercalcemia in the highest quartile (Ca2+ of 11.2-16.0

Ch. 58: Primary Hyperparathyroidism mg/dL) may have had higher mortality; however, this becomes apparent only after 15 years. These findings are different from those of earlier studies in which an increased risk of death from cancer and cardiovascular events was reported.86'87 These dif¬ ferences may be attributed to the apparently much milder dis¬ ease observed in the Mayo Clinic population, very much like that usually seen in the United States today. Although knowledge of the natural history of primary hyper¬ parathyroidism managed without parathyroid surgery is still incomplete, medical approaches to the management of pri¬ mary hyperparathyroidism should be considered in nonsurgical patients. Patients with acute primary hyperparathyroidism should be treated the same as any patient with severe hyper¬ calcemia88'89 (see Chap. 59). Long-term management of chroni¬ cally elevated, mild hypercalcemia centers on adequate hydration and ambulation. If possible, diuretics should be avoided; in par¬ ticular, thiazide diuretics may worsen the hypercalcemia in some patients. Other diuretics, such as furosemide, may place the patient at risk for dehydration and electrolyte imbalances. Gen¬ eral recommendations for diet are not yet certain, and rationales exist for both low- and high-calcium diets in patients with hyper¬ parathyroidism. Diets high in calcium may suppress levels of endogenous PTH. However, high-calcium diets may lead to greater absorption of calcium because of the elevated levels of l,25(OH),D in some patients.90-91 The recommendation for a lowcalcium diet is based on the notion that less calcium is available for absorption. However, low-calcium diets may predispose patients to further stimulation of endogenous PTH levels. One study showed no effect of dietary calcium on biochemical indices or bone densitometry in patients with primary hyperparathy¬ roidism.92 A normal calcium intake can be followed without adverse effects, except in those patients with elevated 1,25-dihydroxyvitamin D levels. Such patients are advised to be more moderate in their calcium intake to prevent hypercalciuria. Other approaches to the medical management of primary hyperparathyroidism have been considered. Attempts to block PTH secretion with (3-adrenergic inhibitors or H2-receptor antagonists have not been successful.7 Oral phosphate, which has been used for many years in primary hyperparathyroidism, lowers serum calcium by 0.5 to 1.0 mg/dL in most patients. The average dosage is 1 to 2 g daily in divided doses. Phosphate appears to have several mechanisms of action. It may inhibit calcium absorption from the gastrointestinal tract; it prevents calcium mobilization from bone; and it may also impair the production of l,25(OH)2D. The risks of the long-term use of phosphate in the management of patients with primary hyper¬ parathyroidism are unknown. One concern is the possibility of ectopic calcification in soft tissues when the normal solubility product of Ca2+ x P04v is exceeded (normally -40). Phosphate therapy is contraindicated when renal insufficiency or hyper¬ phosphatemia is present. If phosphate is to be used, the serum levels of calcium and phosphate should be monitored at regular intervals. Moreover, the long-term use of phosphate in patients with primary hyperparathyroidism promotes a further increase in PTH.93 The possibility that some of the symptoms and signs of primary hyperparathyroidism are caused by PTH itself and not by hypercalcemia raises questions about further increasing PTH levels in conjunction with phosphate therapy. If sufficient concern exists about lowering the serum calcium level in patients with asymptomatic primary hyperparathyroidism, parathyroid surgery remains the treatment of choice. Estrogen therapy has been proposed as a means of lowering serum calcium, especially because prevalence of primary hyperparathyroidism among women is increased in the post¬ menopausal years. Estrogens have well-known but not clearly understood antagonist actions on PTH-induced bone resorp¬ tion. The serum calcium does tend to fall by -0.5 to 1.0 mg/dL in women receiving estrogens.94-96 However, PTH and phos¬ phorus levels do not change. More studies are required to better delineate the role of estrogen therapy.

571

To date, no available data exist on the role of selective estrogenreceptor modulators (SERMs) in the treatment of primary hyperparathyroidism. Because the antiresorptive actions of these drugs are similar to those of estrogens, SERMs might be pre¬ dicted to lower serum calcium levels in postmenopausal women with primary hyperparathyroidism in a fashion similar to that seen with estrogen. This hypothesis remains to be tested. Calcitonin may have a potential use in treating primary hyperparathyroidism, but no controlled trial has been con¬ ducted to test the efficacy of calcitonin for management of this condition. Data from the use of calcitonin in other states of increased bone turnover indicate that it may never become a useful long-term therapy for primary hyperparathyroidism. The bisphosphonates represent a class of important antiresorbing agents that may emerge as a useful approach to the medical management of primary hyperparathyroidism. Oral etidronate is not useful, and the effect of oral clodronate and alendronate is limited in duration of effect.97 Of the third-gener¬ ation bisphosphonates, risedronate has been shown to have some efficacy in preliminary studies of patients with primary hyperparathyroidism.98 Finally, specifically targeted medical therapy for primary hyperparathyroidism is under active investigation. Calcimimetic agents that target the calcium-sensing receptor on the parathyroid cell99 have shown early promise in animal and in vitro studies.100-101 Data from human investigations are also encouraging. In early studies, one such calcimimetic has been shown to lower serum calcium and PTH levels both in a patient with parathyroid carcinoma and in a group of postmenopausal women with mild primary hyperparathyroidism.102'103

PRIMARY HYPERPARATHYROIDISM DURING PREGNANCY Rarely, primary hyperparathyroidism becomes evident during pregnancy.104 Hyperparathyroidism during pregnancy used to be associated with an increased incidence of fetal death. Perina¬ tal and neonatal complications were also thought to be increased in the hypocalcemic infant whose endogenous PTH production is suppressed by maternal hypercalcemia. Neonatal hypocalcemia and tetany can be the first sign of primary hyper¬ parathyroidism in the mother. Although systematically collected and controlled data are unavailable, experience has indicated that primary hyperpar¬ athyroidism during pregnancy can be managed successfully without resorting to surgery.105-106 The management of primary hyperparathyroidism during pregnancy is controversial.10' Some clinicians advocate surgery during the second trimester to reduce fetal risk of chronic hypercalcemia during the gesta¬ tional period. Others advocate a much more conservative approach.

PARATHYROID CARCINOMA Parathyroid carcinoma is a rare form of primary hyperparathy¬ roidism.108-109 These patients often have hypercalcemia that is more severe than that usually seen in primary hyperparathy¬ roidism. Serum calcium values >14 mg/dL may suggest a par¬ athyroid malignancy. However, parathyroid carcinoma is uncommon, occurring in 4 g of elemental calcium per day in the form of calcium carbonate tablets (e.g., 200-mg tablets) or liquid calcium carbonate antacids. In contrast to the l,25(OH)0D-regulated intestinal absorption of calcium that occurs when dietary calcium intake is normal (~1000 mg per day), ingestion of large amounts of dietary calcium can lead to passive, unregulated, and inappropriate calcium absorption. This latter variety of milk-alkali syndrome is frequently overlooked by physicians for two reasons. First, patients often fail to volunteer the fact that they are using large amounts of calcium carbonate, because they do not consider it a medica¬ tion. Second, physicians fail to specifically ask patients with hypercalcemia if they are using antacids. Hypercalcemia leads to polyuria, polydipsia, mental status changes, dehydration, prerenal reductions in the glomerular fil¬ tration rate; if hypercalcemia, hyperphosphatemia, and dehydra¬ tion persist, nephrocalcinosis and permanent renal impairment may occur. The diagnosis is made by excluding other causes of hypercalcemia and documenting calcium intakes in excess of 4 g per day of elemental calcium. Treatment includes the discontinua¬ tion of the calcium antacid, hydration, and education regarding the large amounts of calcium that can be ingested unintentionally.

In animals, hypophosphatemia leads to osteoclast stimulation, inhibition of mineralization, hypercalciuria, and hypercalce¬ mia. This is true even when animals have been rendered vita¬ min D deficient and surgically hypoparathyroid. In humans, severe hypophosphatemia leads to hypercalciuria, presumably reflecting both increases in osteoclastic activity and hypophos¬ phatemia-induced increases in renal l,25(OH)2D production, with consequent enhanced intestinal calcium absorption. Although hypercalcemia resulting exclusively from hypophos¬ phatemia has not been described in humans, it seems likely that when hypophosphatemia complicates hypercalcemia of any cause, it exacerbates the hypercalcemia. Hypophosphatemia is particularly common among patients with hypercalcemia, regardless of the underlying cause, for the following reasons: Food intake may be reduced due to vomiting or anorexia; dietary phosphorus absorption may be limited by the use of phosphate-binding antacids; hypercalcemia per se is phosphaturic; PTH and PTHrP reduce the renal phosphate threshold; and agents that are used to treat hypercalcemia (calcitonin, loop diuretics, saline infusion) induce phosphaturia.

PARENTERAL NUTRITION Hypercalcemia may develop in two groups of patients receiv¬ ing parenteral hyperalimentation. The first group includes

FAMILIAL HYPOCALCIURIC HYPERCALCEMIA The hypercalcemia that occurs in patients with familial hypocalciuric hypercalcemia, also known as familial benign hypercal¬ cemia (see Chap. 58), has been attributed to diminished renal calcium clearance (excessive tubular reabsorption of calcium) as well as failure of parathyroid gland calcium sensing, both occurring most commonly as a result of inactivating mutations in the calcium-sensing receptor.833

RENAL FAILURE Hypercalcemia has been described occasionally in patients with acute tubular necrosis, especially during the polyuric phase. In one careful study of patients with acute tubular necrosis result¬ ing from rhabdomyolysis, patients initially were hypocalcemic because of severe hyperphosphatemia but later became hypercalcemic with the reversal of oliguria and hyperphos¬ phatemia.84 The hypercalcemia was transient and may have resulted from (a) mobilization of soft-tissue calcium phosphate salts deposited during the early hyperphosphatemic period, and (b) rebound hypercalcemia from the transiently elevated PTH and l,25(OH)2D values that occurred during the early hypocalcemic period. Hypercalcemia often develops in patients with chronic renal failure who are receiving hemodialysis. Hypercalcemia is diffi¬ cult to evaluate in such patients because the usual biochemical

582

PART IV: CALCIUM AND BONE METABOLISM

values [renal phosphate and calcium excretion, serum PTH, serum l,25(OH)2D, and nephrogenous cAMP] are difficult to interpret in this setting. Nevertheless, reductions in dietary cal¬ cium intake and vitamin D supplements may correct the hyper¬ calcemia, a result which suggests that increased intestinal calcium absorption in the face of diminished renal clearance may account for hypercalcemia in some patients. Immobiliza¬ tion of patients receiving hemodialysis may lead to hypercalce¬ mia, presumably through acceleration of the underlying high rates of bone turnover. Tertiary hyperparathyroidism (the evo¬ lution of autonomous parathyroid-dependent hypercalcemia in patients with renal failure and chronic secondary hyperpara¬ thyroidism) may occur. Dialysis against a high-calcium bath also may lead to hypercalcemia. Finally, hypercalcemia may occur as part of the aluminum bone disease that forms an important aspect of renal osteodystrophy.

butions from renal failure, immobilization, total parenteral nutrition, and vitamin D supplementation.

TREATMENT OF HYPERCALCEMIA

Hyperalbuminemia may lead to hypercalcemia because albu¬ min is the major circulating calcium-binding protein. The hypercalcemia is usually mild, and patients in whom the diag¬ nosis is suspected generally are severely dehydrated. Rapid rehydration often corrects the hypercalcemia before careful bio¬ chemical evaluation can be performed. In practice, this form of hypercalcemia can be diagnosed by the determination of a nor¬ mal ionized calcium value in a patient with mildly elevated total serum calcium values, markedly elevated serum albumin levels, and none of the other causes for hypercalcemia listed in Table 59-1. Rarely, patients with multiple myeloma have been described who display elevations in the total serum calcium concentration (sometimes striking elevations) but normal ion¬ ized calcium values.85-86 This factitious hypercalcemia is caused by unusual immunoglobulins with an extremely high binding capacity for calcium. Hypercalciuria, electrocardio¬ graphic changes, and symptoms of hypercalcemia are absent in these unusual patients. Although hypercalcemia caused by a myeloma calcium-binding protein is unusual, it should be con¬ sidered before attributing hypercalcemia in patients with multi¬ ple myeloma to the more common cause, osteolytic bone disease.

Principles of therapy for hypercalcemia should be based on an understanding of its cause, knowledge of its pathophysiology, and careful assessment of the symptom complex in individual patients. The treatment of mild hypercalcemia in a patient with asymptomatic primary hyperparathyroidism (i.e., without nephrolithiasis or apparent skeletal disease) is controversial (see Chap. 58). Similarly, mild hypercalcemia associated with thyrotoxicosis requires no treatment because it resolves sponta¬ neously with treatment of the hyperthyroidism. The hypercal¬ cemia in patients with familial hypocalciuric hypercalcemia almost always is asymptomatic and requires no therapy. Some may argue that severe hypercalcemia in a comatose patient with terminal cancer is best left untreated. The treatment of hypercalcemia in a patient with significant symptomatology may be an urgent problem. The severity of symptoms of hypercalcemia can be related to the absolute cal¬ cium concentration; the rate of development of hypercalcemia (the faster the increase, the greater the symptoms); and the duration of the hypercalcemia. These points, as well as the patient's overall status and prognosis, should be considered before commencing therapy. Therapy is best determined with an understanding of the pathophysiology of a given patient's hypercalcemia. For exam¬ ple, the absorptive hypercalcemia of sarcoidosis is best treated with dietary calcium restriction, whereas the resorptive hyper¬ calcemia of immobilization and malignancy is most appropri¬ ately treated with measures aimed at diminishing osteoclastic activity. Specific treatment of most of the disorders listed in Table 59-1 is self-evident. Therapy for the hypercalcemia associated with primary hyperparathyroidism, renal osteodystrophy, and neo¬ natal disorders is discussed in Chapters 58, 61, and 70, respec¬ tively. Treatment of the underlying disorder responsible for hypercalcemia is an obvious, but frequently overlooked, goal, particularly in patients with malignancy-associated hypercalce¬ mia. In these patients, antitumor therapy should be planned and initiated early in the hospital course, because other mea¬ sures are effective only transiently and may be associated with toxicity. Usually, long-term control of malignancy-associated hypercalcemia can be accomplished only by successful antitu¬ mor therapy.

MANGANESE INTOXICATION

SALINE INFUSION

Hypercalcemia has been reported to occur in patients with manganese intoxication.87-88 Exposure to manganese in doses sufficient to cause intoxication has been reported in miners, in welders, and in people drinking water from manganesecontaminated wells. The mechanisms responsible for the devel¬ opment of hypercalcemia are uncertain. The hypercalcemia may be mild (e.g., 11.5 mg/dL) or severe (e.g., 20 mg/dL).

Renal calcium excretion is enhanced by saline infusion, because of both the increase in glomerular filtration rate and the role of the filtered load of sodium in blocking proximal tubular calcium reabsorption.90 The rate of infusion should be adjusted to the clinical situation. The uncertain cardiovascular status of these patients, many of whom are elderly, dictates caution. Infusion rates of 200 to 400 mL per hour of 0.9% saline are commonly used. The status of hydration should be checked frequently by physical signs (rales, skin turgor, mucous membrane hydration, blood pressure) and by hemodynamic monitoring when appro¬ priate. Aggressive saline infusion may lead to hypokalemia, hypophosphatemia, and hypomagnesemia. These ions should be measured and replaced as necessary.

IDIOPATHIC HYPERCALCEMIA OF INFANCY The complex of disorders known as idiopathic hypercalcemia of infancy is discussed in Chapter 70.

HYPER PROTEIN EM IA

ADVANCED CHRONIC LIVER DISEASE Mild to severe hypercalcemia has been described in one series of 11 patients with advanced chronic liver disease awaiting liver transplantation.89 All the patients had severe jaundice (the mean serum bilirubin value was 28.7 mg/dL), and most had hypoalbuminemia and prolonged prothrombin times and were in mild renal failure (the mean serum creatinine level was 2.8 mg/dL). Hypercalcemia was associated with normal to reduced plasma l,25(OH)2D, 25(OH)D, and PTH concentra¬ tions. Ionized serum calcium values were elevated. The cause of the syndrome appeared to be multifactorial, reflecting contri¬

LOOP DIURETICS Loop diuretics appear to inhibit calcium reabsorption at the level of the thick ascending limb of the Henle loop.90 In theory, use of agents such as furosemide should enhance the calciuresis of a saline infusion. Furosemide in dosages of 40 mg or more.

Ch. 59: Nonparathyroid Hypercalcemia given intravenously or orally, is widely used for this purpose. This drug should not be administered to dehydrated patients until rehydration is complete, because it may further reduce the glomerular filtration rate and, thereby, reduce the filtered load of calcium, decreasing renal calcium clearance even more. The drug is particularly useful if the patient has coexistent conges¬ tive heart failure. Again, careful surveillance of a patient's fluid, electrolyte, and renal status during therapy is critical.

RESTRICTION OF CALCIUM AND VITAMIN D INTAKE Dietary calcium and vitamin D restriction and avoidance of exposure to sunlight and other ultraviolet light sources is appropriate in patients with absorptive causes of hypercalce¬ mia, such as sarcoidosis and other granulomatous diseases dur¬ ing their active stages.90 Dietary calcium intake should be kept at poor matrix formation

Menkes syndrome

Defective cross-linking of collagen

Malnutrition

—

Protein

Poor matrix formation

Copper deficiency

Defective cross-linking of collagen

Hypercortisolism

Generalized decrease in body protein synthesis

Leukemia

(?)

694

PART IV: CALCIUM AND BONE METABOLISM

contain collagen may be affected, as evidenced by joint laxity and thin skin. The sclerae may appear blue because of thinning, allowing the blue pigment of the choroid to be transmitted. Improper formation of dentin may occur, and teeth may be yel¬ low or opalescent and chip easily. Sensorineural or conductive hearing loss may be present. The skeleton is variably affected. Wormian bones (islands of bone with a rich vascular supply) are frequently observed on skull radiographs. Biochemical find¬ ings are nonspecific; however, mild hypercalcemia and hypercalciuria may be present in younger patients. Markers of bone turnover—such as serum alkaline phosphatase activity and osteocalcin as well as urinary excretion of resorptive markers— may also have higher than average values. Serum PTH and vita¬ min D metabolites are nearly always normal. Numerous mutations in genes encoding the a, and a2 chains of type I collagen (bone collagen) have been described.363 Most kindreds are affected with unique mutations, and sporadic mutations are not uncommonly reported. Studies note variable severity associated with the mutations, being dependent on the region of the gene in which the mutation occurs; thus a "regional model" for site of mutation/severity has been pro¬ posed.37 Mutations in introns resulting in abnormal mRNA pro¬ cessing have been described; these result in abnormal ratios of chain production, not abnormal chains, per se. Prenatal diagnosis of osteogenesis imperfecta has been possi¬ ble through genetic studies of chorionic villous samples or through high-resolution fetal ultrasound studies in severe cases. After years of disappointing results with various experimen¬ tal therapies of the disease, trials with intravenous pamidronate have shown dramatic changes in the bone density and fracture rate over the short term.38 The bisphosphonate preparation is given intravenously in 3- to 4-hour daily infusions that are repeated for 3 successive days per cycle. The cycle is repeated at 4-month intervals. Therapy is sufficiently new that long-term effects on the skeleton have not yet been clearly established. Nevertheless, serious consideration for therapy should be undertaken in patients with severe disease. It is less clear whether mild forms of the disease warrant such an intervention. Finally, it seems likely that a number of unidentified muta¬ tions in the collagen gene could be responsible for disorders of childhood osteoporosis that have not classically been diag¬ nosed as osteogenesis imperfecta. Related connective tissue dis¬ orders, particularly the Ehlers-Danlos syndrome and, rarely, Marfan syndrome, may also manifest osteopenia (see Chaps. 66 and 189). IDIOPATHIC JUVENILE OSTEOPOROSIS

ies of bone density in girls with Turner syndrome have indi¬ cated that when bone mineral density is normalized to height or body mass rather than age, bone mass may actually be nor¬ mal. Girls or older women with Turner syndrome may have an increased incidence of fractures. HOMOCYSTINURIA Homocystinuria refers to several disorders of methionine metab¬ olism resulting in increased blood and urinary homocystine levels (see Chap 191). The prototype disorder, caused by cys¬ tathionine (3-synthase deficiency, is commonly manifested by a dislocated lens and a consistent, marked osteoporosis that is most evident in the spine. Some of the skeletal abnormalities resemble those of Marfan syndrome, including kyphosis, scoli¬ osis, pectus excavatum and carinatum, and arachnodactyly. Other features include mental retardation, seizures, malar flush, and vascular thromboses. The detection of homocysteine or homocystine in the urine by the cyanide-nitroprusside reac¬ tion is a useful diagnostic screening procedure. Management has included low methionine diets, large amounts of pyridoxine, and supportive management of complications. LYSINURIC PROTEIN INTOLERANCE Osteopenia is an almost constant feature of lysinuric protein intolerance, which is an autosomal-recessive disorder of aminoacid transport. Episodic vomiting, anorexia, variable hepato¬ megaly, protein aversion, or unexplained osteopenia may bring the child to the physician’s attention. Defective renal, intestinal, and hepatocellular transport of dibasic amino acids (ornithine, lysine, and arginine) cause limited urea cycle activity and epi¬ sodic hyperammonemia. Characteristically, decreased levels of circulating dibasic amino acids and massive urinary excretion of lysine occur. Generalized aminoaciduria also may be present. Very marked elevations in serum lactate dehydrogenase may occur, as well as elevations in serum transaminases. Histomorphometric analysis of bone in one patient confirmed an osteoporotic process. Restricted protein intake probably accounts for the osteoporosis. Citrulline therapy has improved general well-being as well as the osteoporosis. MENKES KINKY HAIR SYNDROME Menkes kinky hair syndrome is an X-linked disorder character¬ ized by defective intestinal absorption and tissue accumulation of copper. Deficient copper-dependent enzymes result in abnor¬ mal hair, neuronal degeneration, and hypothermia. The osteo¬ porosis is caused by defective collagen synthesis, resulting from the defective copper-dependent enzyme lysyl oxidase, which is required for normal cross-linking (also see Chap. 131).

In contrast to osteogenesis imperfecta, idiopathic juvenile osteoporosis usually becomes evident just before puberty. Such individuals may have pain in the weight-bearing skeleton, refusal to walk, or gait abnormalities. Radiographs can show generalized osteopenia, compression fractures of the thora¬ columbar vertebrae, and metaphyseal compression fractures. Marked osteopenia in the metaphyseal region of new bone for¬ mation is characteristic. There are no consistent biochemical changes in this disorder. Increased hydroxyproline excretion and negative calcium bal¬ ance may occur. The circulating PTH level has been reported as elevated relative to the serum calcium level. Serum l,25(OH),D levels may be decreased, normal, or increased. The active dis¬ ease is almost always transient, but residual deformity is not uncommon. Rarely, patients have persistence of active osteoporosis through adolescence and into adulthood (see Chap. 64). Some patients have been shown to respond to bis¬ phosphonate therapy.39

Cushing syndrome, which is caused by an excess of endoge¬ nous glucocorticoids, may present as osteopenia, although other features of hypercortisolism are usually evident. The vita¬ min D metabolites 25-OHD and l,25(OH),D have been pro¬ posed as therapeutic agents for steroid-induced osteoporosis, although use of the latter in adults resulted in a significant inci¬ dence of hypercalcemia. Biphosphate therapy may prove to be useful in this setting.

TURNER SYNDROME

LEUKEMIA

A number of skeletal abnormalities have been reported in Turner syndrome, including osteopenia. However, recent stud¬

Leukemia may produce osteoporosis before abnormal cellular forms are evident in the peripheral circulation.

MALNUTRITION Celiac disease, protein-calorie malnutrition, and copper deficiency may result in osteoporosis. Copper deficiency is thought to cause osteoporosis secondary to defective cross-linking of collagen. HYPERCORTISOLISM

Ch. 70: Disorders of Calcium and Bone Metabolism in Infancy and Childhood OSTEOPENIA OF PREMATURITY Osteopenia of prematurity is common in low-birth-weight infants, in whom it is difficult to provide, ex utero, the mineral requirements necessary for the rapidly growing and develop¬ ing skeleton. Rickets or osteomalacia may be a significant factor in the bone disease of these patients. Human milk fortifiers have been developed to provide adequate mineral to counter this problem but are not without complications (see section on hypercalcemia).

TOWARD A DEFINITIVE DIAGNOSIS In the absence of evidence of primary osteomalacia or other systemic disease, determinations of serum copper, bicarbonate, uric acid, and serum and urine amino-acid levels and, occasion¬ ally, chromosomal analysis should be performed. A search for evidence of hyperadrenalism is undertaken if subtle cushingoid manifestations are present. If the child is severely affected or systemically ill, yet with no features of a specific disorder, a bone marrow aspiration may be necessary and could be per¬ formed during a bone biopsy procedure. Usually, no definitive diagnosis is evident, and one is left to distinguish between idio¬ pathic juvenile osteoporosis and mild osteogenesis imperfecta. Analysis of collagen produced in skin samples obtained at biopsy may confirm a diagnosis of osteogenesis imperfecta. A definitive diagnosis is important in view of the potentially effective therapy with bisphosphonates described earlier.

REFERENCES 1. Hillman LS, Haddad JG. Hypocalcemia and other abnormalities of mineral homeostasis during the neonatal period. In: Heath DA, Marx SJ, eds. Cal¬ cium disorders. Clinical endocrinology, Butterworths international medical reviews. London: Butterworths, 1982; 248. 2. Cole DEC, Carpenter TO, Goltzman D. Calcium homeostasis and disorders of bone and mineral metabolism. In: Collu R, ed. Pediatric endocrinology, comprehensive endocrinology series. New York: Raven Press, 1988; 509. 2a. Walton DM, Thomas DC, Aly HZ, Short BL. Morbid hypocalcemia associ¬ ated with phosphate enema in a six-week-old infant. Pediatrics 2000; 106:E37. 3. Anast CS, David L. Human neonatal hypercalcemia. In: Holick MF, Anast CS, Gray TK, eds. Perinatal calcium and phosphorus metabolism. Amster¬ dam: Elsevier 1983; 363. 4. Marx SJ, Attie MF, Spiegel AM, et al. An association between severe pri¬ mary hyperparathyroidism and familial hypocalciuric hypercalcemia in three kindreds. N Engl J Med 1982; 306:257. 5. Pollack MR, Chou Y-HW, Marx SJ, et al. Familial hypocalciuric hypocalce¬ mia and neonatal severe hyperparathyroidism: effects of mutant gene dos¬ age on phenotype. J Clin Invest 1994; 93:1108. 6. Cooper L, Wertheimer J, Levy R, et al. Severe primary hyperparathyroidism in a neonate who was the product of two related hypercalcemic parents managed by a parathyroidectomy and heterotopic autotransplantation. Pediatrics 1986; 78:263. 7. Harris SS, D'Ercole J. Neonatal hyperparathyroidism: the natural course in the absence of surgical intervention. Pediatrics 1989; 83:53. 8. Ewart AK, Morris CA, Atkinson D, et al. Hemizygosity at the elastin locus in a developmental disorder, Williams syndrome. Nat Genet 1993; 5:11. 9. Taylor AB, Stern PH, Bell NH. Abnormal regulation of circulating 25hydroxyvitamin D in the Williams syndrome. N Engl J Med 1982; 306:972. 10. Goodyer PR, Frank A, Kaplan BS. Observations on the evolution and treat¬ ment of idiopathic infantile hypercalcemia. J Pediatr 1984; 105:771. 11. Garabedian M, Jacqz E, Guillozo H, et al. Elevated plasma 1,25-dihydroxyvitamin D concentrations in infants with hypercalcemia and an elfin facies. N Engl J Med 1985; 312:948. 12. Kruse K, Irle U, Uhlig R. Elevated 1,25-dihydroxyvitamin D serum concen¬ trations in infants with subcutaneous fat necrosis. J Pediatr 1993; 122:460. 13. Clements MR, Johnson L, Fraser DR. A new mechanism for induced vita¬ min D deficiency in children. Nature 1987; 325:62.

695

14. Fu GK, Lin D, Zhang MY, et al. Cloning of human 25-hydroxyvitamin Dlct-hydroxylase and mutations causing vitamin D-dependent rickets type 1. Mol Endocrinol 1997; 11:1961. 15. Balsan S, Garabedian M, Larchet M, et al. Long-term nocturnal calcium infusions can cure rickets and promote normal mineralization in heredi¬ tary resistance to 1,25-dihydroxyvitamin D. J Clin Invest 1986; 77:1661. 16. Carpenter TO. New perspectives on the biology and treatment of X-linked hypophosphatemic rickets. Pediatr Clin North Am 1997; 44:443. 17. Whyte MP, Schranck FW, Armamento-Villareal R. X-linked hypophos¬ phatemia: a search for gender, race, anticipation, or parent of origin effects on disease expression in children. J Clin Endocrinol Metab 1996; 81:4075. 18. Winters RW, Graham JB, Williams TF, et al. A genetic study of familial hypophosphatemia and vitamin D resistant rickets with a review of the lit¬ erature. Medicine 1958; 37:97. 19. The HYP Consortium. A gene (PEX) with homologies to endopeptidases is mutated in patients with X-linked hypophosphatemia. Nat Genet 1995; 11:130. 20. Nesbitt T, Coffman TM, Griffiths R, et al. Cross transplantation of kidneys in normal and Hyp mice: evidence that the Hyp mouse phenotype is unre¬ lated to an intrinsic renal defect. J Clin Invest 1992; 89:1453. 21. Meyer RA Jr, Meyer MH, Gray RW. Parabiosis suggests a humoral factor is involved in X-linked hypophosphatemia in mice. J Bone Miner Res 1989; 4:493. 22. Ecarot B, Glorieux FH, Desbarats M, et al. Effect of 1,25-dihydroxyvitamin D3 treatment on bone formation by transplanted cells from normal and Xlinked hypophosphatemic mice. J Bone Miner Res 1995; 10:424. 23. Carpenter TO, Gundberg CM. Osteocalcin abnormalities in Hyp mice reflect altered genetic expression and are not due to altered clearance, affin¬ ity for mineral, or ambient phosphorus levels. Endocrinology 1996; 137:5213. 24. Glorieux FH, Pierre JM, Pettifor JM, Delvin EE. Bone response to phos¬ phate salts, ergocalciferol, and calcitriol in hypophosphatemic vitamin Dresistant rickets. N Engl J Med 1980; 303:1023. 25. Harrell RM, Lyles KW, Harrelson JM, et al. Healing of bone disease in Xlinked hypophosphatemic rickets/osteomalacia. J Clin Invest 1985; 75:1858. 26. Carpenter TO, Mitnick MA, Ellison A, et al. Nocturnal hyperparathyroid¬ ism: a frequent feature of X-linked hypophosphatemia. J Clin Endocrinol Metab 1994; 78:1383. 27. Rivkees SA, El-Hajj-Fuleihan G, Brown EM, Crawford JD. Tertiary hyper¬ parathyroidism during high phosphate therapy of familial hypophos¬ phatemic rickets. J Clin Endocrinol Metab 1992; 75:1514. 28. Carpenter TO, Keller M, Schwartz D, et al. 24,25 Dihydroxyvitamin D sup¬ plementation corrects hyperparathyroidism and improves skeletal abnor¬ malities in X-linked hypophosphatemic rickets—a clinical research center study. J Clin Endocrinol Metab 1996; 81:2381. 29. Sullivan W, Carpenter TO, Glorieux F, et al. A prospective trial of phos¬ phate and 1,25 dihydroxyvitamin D3 therapy in symptomatic adults with X-linked hypophosphatemic rickets. J Clin Endocrinol Metab 1992; 75:879. 30. Eddy MC, McAlister WH, Whyte MR X-linked hypophosphatemia: nor¬ mal renal function despite medullary nephrocalcinosis 25 years after tran¬ sient vitamin D2-induced renal azotemia. Bone 1997; 21:515. 31. Econs MJ, McEnery PT, Lennon F, Speer MC. Autosomal dominant hypo¬ phosphatemic rickets is linked to chromosome 12pl3. J Clin Invest 1997; 100:2653. 32. O'Neill EM. Linear sebaceous naevus syndrome with oncogenic rickets and diffuse pulmonary angiomatosis. J R Soc Med 1993; 86:177. 33. Konishi K, Nakamura M, Yamakawa H, et al. Case report: hypophos¬ phatemic osteomalacia in von Recklinghausen neurofibromatosis. Am J MedSci 1991; 301(5):322. 34. Hahn SB, Lee SB, Kim DH. Albright's syndrome with hypophosphatemic rickets and hyperthyroidism: a case report. Yonsei Med J1991; 32(2):179. 35. Chen C, Carpenter TO, Steg N, et al. Hypercalciuric hypophosphatemic rickets: mineral balance, bone histomorphometry, and therapeutic implica¬ tions of hypercalciuria. Pediatrics 1989; 84:276. 36. Prockop DJ, Kivirikko KI. Collagens: molecular biology, diseases, and potentials for therapy. Ann Rev Biochem 1995; 64:403. 36a. Nuytinck L, Tutel T, Kayserili H, et al. Glycine to tryptophan substitution in type I collagen in a patient with OI type III: a unique collagen mutation. J Med Genet 2000; 37:371. 37. Wang Q, Orrison BM, Marini JC. Two additional cases of osteogenesis imperfecta with substitutions for glycine in the alpha 2(1) collagen chain. A regional model relating mutation location in phenotype. J Biol Chem 1993; 268(33):25162. 38. Glorieux F, Bishop NJ, Plotkin H, et al. Cyclic administration of pamidronate in children with severe osteogenesis imperfecta. N Engl J Med 1998; 349:947. 39. Shaw NJ, Boivin CM, Crabtree NJ. Intravenous pamidronate in juvenile osteoporosis. Arch Dis Child 2000; 83:143.

.

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.

PART V

THE ADRENAL GLANDS D. LYNN LORIAUX, EDITOR

71. MORPHOLOGY OF THE ADRENAL CORTEX AND MEDULLA.698 72. SYNTHESIS AND METABOLISM OF CORTICOSTEROIDS.704 73. CORTICOSTEROID ACTION.714 74. TESTS OF ADRENOCORTICAL FUNCTION.719 75. CUSHING SYNDROME.723 76. ADRENOCORTICAL INSUFFICIENCY.739 77. CONGENITAL ADRENAL HYPERPLASIA.743 78. CORTICOSTEROID THERAPY.751 79. RENIN-ANGIOTENSIN SYSTEM AND ALDOSTERONE.764 80. HYPERALDOSTERONISM.773 81. HYPOALDOSTERONISM.785 82. ENDOCRINE ASPECTS OF HYPERTENSION.791 83. ADRENOCORTICAL DISORDERS IN INFANCY AND CHILDHOOD.806 84. THE INCIDENTAL ADRENAL MASS.816 85. PHYSIOLOGY OF THE ADRENAL MEDULLA AND THE SYMPATHETIC NERVOUS SYSTEM.817 86. PHEOCHROMOCYTOMA AND OTHER DISEASES OF THE SYMPATHETIC NERVOUS SYSTEM.827 87. ADRENOMEDULLARY DISORDERS OF INFANCY AND CHILDHOOD.834 88. DIAGNOSTIC IMAGING OF THE ADRENAL GLANDS.837 89. SURGERY OF THE ADRENAL GLANDS .... 843

698

PART V: THE ADRENAL GLANDS

CHAPTER

71

MORPHOLOGY OF THE ADRENAL CORTEX AND MEDULLA DONNA M. ARAB O'BRIEN The adrenal glands are paired organs adjacent to the kidneys, containing a cortex and a medulla. These two divisions are best understood as functionally separate endocrine organs. This chapter reviews the embryology, anatomy, and histopathology of the adrenal cortex and medulla, emphasizing those aspects most relevant to clinical endocrinologists.1-5

EMBRYOLOGY The adrenal cortex and medulla arise from separate embryologic tissues (Fig. 71-1). Beginning in the fourth week of gesta¬ tion, cells destined to form the adrenal cortex develop from primitive mesoderm just medial to the urogenital ridge. These cells penetrate the overlying retroperitoneal mesenchyme and, over the next several weeks, form a gradually enlarging adrenal cortex. During the sixth week of gestation, the developing cor¬ tex is penetrated by nerve fibers, along which primitive medul¬ lary cells will eventually migrate. By the eighth week, the cortex has formed two distinct zones: a relatively large and cen¬ trally located fetal zone and a thin rim of definitive cortex, which will later form the adult adrenal cortex. Throughout this period, proliferation is largely confined to the outer definitive cortex, suggesting that the same cells give rise to both the fetal and definitive zones. About this time, the fetal adrenal circulation is established, with several adrenal arteries arising from the descending aorta, and a central vein draining each adrenal gland. Between the second and third month of gestation, the adrenal glands increase in weight from about 5 to 80 mg, are

much larger than the adjacent kidney, and reach their greatest size relative to total fetal weight. Growth of the adrenal cortex after the 20th week of gestation is dependent on pituitary stim¬ ulation (anencephalic fetuses are born with an atrophic adrenal fetal zone). After birth, the fetal zone involutes and the defini¬ tive cortex evolves into the three zones of the adult adrenal cor¬ tex (Fig. 71-2). During fetal development, primitive adrenocortical cells may migrate widely. Accessory adrenocortical rests have been found adjacent to the celiac plexus, in the broad ligament, adja¬ cent to ovarian or spermatic vessels, and around the kidney or uterus. Less common sites include the abdominal organs and, even more rarely, the lung, spinal nerves, or brain. These rests may be responsible for recurrent Cushing disease (see Chap. 75) after bilateral adrenalectomy and, rarely, for neoplastic transformation. Lntratesticular or intraovarian adrenal rests represent a special problem because they may be difficult to distinguish from normal gonadal tissue. Such rests are rarely recognized in normal persons, but they may appear as gonadal enlargement in patients with Nelson syndrome or untreated congenital adrenal hyperplasia (CAH). The adrenal medulla arises from primitive sympathetic ner¬ vous system cells (sympathogonia) derived from the neuroecto¬ derm. These cells differentiate into neuroblasts, which migrate ventrally from the neural crest to form paravertebral and preaortic sympathetic ganglia, and into pheochromoblasts, which form catecholamine-secreting (chromaffin) cells (see Fig. 71-1). In the fetus, chromaffin cells are found not only in the adrenal medulla but also throughout the sympathetic chain. Similarly, neuroblast cell clusters often are present in the fetal adrenal medulla and involute after birth. A rudimentary adre¬ nal medulla may be seen after the sixth week of gestation, but chromaffin cells continue to migrate to this location throughout the third month. After birth, extraadrenal chromaffin tissue generally involutes, although it may persist near the origin of the celiac and mesenteric arteries. The paired organs of Zuckerkandl, located near the inferior mesenteric artery and prominent in the first year of life, represent such extraadrenal catechola¬ mine-secreting tissue. The widespread prenatal occurrence of extraadrenal chromaffin cells accounts for the location of extraadrenal pheochromocytomas later in life.

Neural crest

Intermediate grey

Somite

Ependyn

Aorta

Dorsal root ganglion

Splanchnopleura

Suprarenal cortical primordium

Intermediate grey

Communicating ramus Paravertebral (sympathetic) chain ganglion

Dorsal root ganglion Spinal nerve: dorsal ramus ventral ramus

in*"

Preaortic ganglioi primordium ...

..

" B FIGURE 71-1. Formation of the human adrenal (suprarenal) gland and sympathetic ganglia. A, Migra¬ tion of sympathetic ganglion and chromaffin cells from the neural crest and neural tube. B, The forma¬ tion of the sympathetic ganglia. C, The chromaffin cells of the future suprarenal medulla reaching the provisional cortex. (From Allan FD. Essentials of human embryology, 2nd ed. New York: Oxford Uni¬ versity Press, 1969.)

Cortex BX ) suprarenal Med ullal Preaortic ganglion

c

Medullary primordium 'Enteric ganglia and plexus

Ch. 71: Morphology of the Adrenal Cortex and Medulla

699

T12

Permanent cortex 20% Fetal cortex 75%

Inf. Phrenic

vertebral

Artery

Body

Adrenal Arteries

Medulla 5%

R. Adrenal Vein 5th Fetal Month

8th Fetal Month

Irregular boundary between

First sign of zonation

permanent and fetal cortex

in permanent cortex

Permanent cortex 35%

Renal Artery

Cortex 80% Fetal cortex

Fetal cortex 50%

has disappeared Medulla 15%

L. Adrenal Gland

L. Adrenal Vein

L. Kidney

Renal Vein

20%

Inf. Vena Cava

Newborn Infant All zones of permanent cortex present

4 Years and Older Mature zonation pattern established

FIGURE 71-2. Internal development of the human suprarenal gland. The fetal cortex (cross-hatched) decreases in volume from the fifth fetal month to its complete disappearance in the first year of postnatal life. Zonation of the permanent cortex begins with the zona glomerulosa, indicated in the eighth fetal month; complete zonation is established by the fourth year of life. (From Gray SW. Embryology for surgeons. Phila¬ delphia: WB Saunders, 1972:555; based on data from Sucheston ME, Cannon MS. Development of zonular patterns in the human adrenal gland. J Morphol 1968; 126:477.)

ANATOMY The adrenal glands are paired structures in the retroperitoneal space, lying anteromedial to the upper pole of the kidneys between the level of the 11th thoracic and the first lumbar verte¬ brae (Fig. 71-3). Although often abutting the kidneys, the adrenals in obese patients can be widely separated from the kidneys and other retroperitoneal organs by fat. Normal adult adrenal glands average about 5.0 x 2.5 cm in length and width and are, at most, only about 0.6 cm thick. In cross-section, as seen with computerized imaging (see Chap. 88), the right gland is thickest centrally with two wings of tissue extending at each side, whereas the left gland often has a semi¬ lunar appearance. Each gland has a head, body, and tail. The head lies interiorly, is the largest portion, and contains most of the medulla. The average weight of each gland is between 3 and 5 g, but this may increase by more than 50% during stress. The adrenals are supplied by numerous small arteries aris¬ ing from the celiac, superior mesenteric, inferior phrenic, renal, and iliac arteries and from the aorta. These arteries anas¬ tomose over the surface of the glands, with numerous smaller unbranched arteries descending through the capsule (Fig. 71-4). Blood supplying the adrenal medulla first traverses the cortex through capillary sinusoids. Adrenal blood flow is differen¬ tially regulated in the cortex and the medulla.6 Nitric oxide is important for maintaining high basal levels of blood flow in both zones. Blood flow in the medulla is neurally regulated and correlates with catecholamine secretion. In the cortex, blood flow is not as closely linked to cortical secretory activity, although in many models, increased cortical blood flow is associated with increased adrenal steroid secretion. The adrenal blood empties through a single central vein, which is surrounded by a cuff of normal adrenocortical tissue as it passes through the medulla. The adrenal vein enters the infe-

FIGURE 71-3. Normal adrenal anatomy. Both adrenal glands are sup¬ plied by multiple small arteries. The right adrenal vein empties directly into the vena cava, whereas the left adrenal vein enters the left renal vein. rior vena cava on the right and the renal vein on the left. The acute angle between the right adrenal vein and the vena cava often makes catheterization of this vessel technically difficult.

HISTOPATHOLOGY OF THE ADRENAL CORTEX NORMAL HISTOLOGY The adult adrenal cortex includes the outer 80% of the adrenal glands and a cuff of cortex surrounding the central vein. Three

FIGURE 71-4. The adrenal vasculature showing distinct arteries sup¬ plying medulla and cortex. Note transition of cortical capillaries into veins at the corticomedullary junction (CMJ). (From Breslow MJ. Regu¬ lation of adrenal medullary and cortical blood flow. Am J Physiol 1992; 262:H1317.)

700

PART V: THE ADRENAL GLANDS

FIGURE 71-5. Histologic features of the normal adrenal cortex. Zona glomerulosa cells (G) are distributed focally beneath the capsule (C). Clear cells of the zona fasciculata (F) are arranged in columns between the cortex (or glomerulosa) and the inner compact cells of the zona retic¬ ularis (R). A small area of adrenal medulla can be seen at the lower right (M). (From Neville AM, O'Hare MJ. The human adrenal cortex. Pathol¬ ogy and biology—an integrated approach. New York: Springer-Verlag, 1982:20.)

functional zones are present: glomerulosa, fasciculata, and reticularis (Fig. 71-5). The zona glomerulosa is responsible for aldosterone secretion and constitutes about 5% of the cortex. It is a discontinuous layer of nests of cells beneath the adrenal capsule. These cells are small, with a dense nucleus, a high nuclear/cytoplasmic ratio, and relatively low cytoplasmic lipid content. On electron microscopic examination, there are elon¬ gate mitochondria with abundant tubular cristae, a small amount of smooth endoplasmic reticulum (SER), and few lysosomes and microvilli. Functionally, the zonae fasciculata and reticularis form a unit, with both cell types having the capacity to secrete corti¬ sol and androgens. Histologically, however, they are distinct. The zona fasciculata makes up about 70% of the adrenal cor¬ tex and consists of columns of cells extending from the inner reticularis zone to the glomerulosa (or to the capsule where the glomerulosa is absent). The cells are large, with a low nuclear/cytoplasmic ratio and abundant cytoplasmic lipid. During fixation, the lipid is removed, giving the cells a vacu¬ olated appearance and hence the name clear cells. On electron microscopic examination, the cells form a continuum: from the outer zone, in which the cells have ovoid mitochondria with few internal vesicles, little SER, and few lysosomes, to the inner zone, in which the cells contain spherical mitochon¬ dria with numerous internal vesicles, more SER, and more lysosomes. The zona reticularis comprises the inner 25% of the adre¬ nal cortex. It consists of anastomosing columns of cells that vary widely in size and contain densely granular cytoplasm and sparse lipid (hence, the name compact cells). The nuclear/ cytoplasmic ratio is intermediate to that between cells of the glomerulosa and cells of the fasciculata. Electron microscopic examination reveals ovoid to elongate mitochondria with tubular to vesicular cristae, abundant SER, and numerous lysosomes. In older persons, an increasing amount of lipofuscin may be seen, which imparts a darker color to these cells. The reason for functional zonation of the adrenal cortex has been a matter of considerable debate. Embryologic evidence

FIGURE 71-6. Adrenal nodules. Multiple nodules are shown, separated by fibrovascular connective tissue (CF). Normal cortical architecture is totally disrupted. (Courtesy of Dr. Stephen Boudreau.)

suggests that all cortical cells arise from the same precursor. Although cells from each of the three zones initially have dis¬ tinct steroid secretory patterns in vitro, long-term cultures dem¬ onstrate that the histologic and functional distinctions between these cells disappear.2 The blood supply to the adrenal glands flows inwardly from the capsule. This creates a gradient of increasing steroid hormone concentration from the outer to the inner cortex. Intraadrenal steroid concentrations may modulate the relative activities of the steroidogenic enzymes.7-9 Such hor¬ mone gradients, which likely increase with increasing adrenal size, may explain not only the functional zonation of the adre¬ nal cortex but also the development of the reticularis zone dur¬ ing adrenarche. Electron microscopy has been useful in differentiating among cortical cell types, demonstrating transitional cells between the three zones of the adrenal cortex. It has not been useful, however, in elucidating pathologic changes in the adre¬ nal cortex or in making the difficult distinction between benign and malignant neoplasms.

PATHOLOGY ADRENAL NODULES: THE ADRENAL RESPONSE TO AGING With advancing age, the adrenal glands of most persons begin to show microscopic nodular changes within the cortex. These changes begin as nests of clear cells located peripherally in the cortex or in cortical tissue surrounding the central vein. Larger nodules compress the adjacent cortex and eventually distort the adrenal capsule. They may contain foci of compact cells and areas of fibrosis, hemorrhage, and cyst formation (Fig. 71-6). These "yel¬ low nodules" have been recognized in 3% of normotensive research subjects and may reach 2 to 3 cm in diameter. Their inci¬ dence appears to increase not only with age but also with the presence of vascular damage, as is seen with hypertension and diabetes. Pigmented ("black") nodules seen at autopsy appear to represent a variant of yellow nodules and contain compact (zona reticularis-like) cells with increased amounts of lipofuscin. These nodules are not neoplasms and, although they produce steroids in vitro, are not associated with adrenocortical hyper¬ function. The unaffected cortex remains normal in appearance. Their main significance lies in their incidental detection on radiologic imaging. In the absence of clinical or biochemical evidence of hormonal hypersecretion, small asymptomatic adrenal nodules seen incidentally during abdominal imaging procedures are unlikely to represent clinically significant pathologic entities.10

701

Ch. 71: Morphology of the Adrenal Cortex and Medulla METRIC

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/

,

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FIGURE 71-8. Bilateral nodular hyperplasia with cortical nodules. The adrenal glands from this patient with Cushing disease demonstrate a hyperplastic cortex (dark areas beneath the light-colored capsule) con¬ taining multiple small light-colored nodules (arrows). (Courtesy of Dr. Stephen Boudreau.)

FIGURE 71-7. Effect of stress on the adrenal cortex. Columns of com¬ pact cells have totally replaced the clear cells of the normal zona fasciculata. (From Neville AM, O'Hare MJ. Histopathology of the adrenal cortex. J Clin Endocrinol Metab 1985; 14:791.)

ADRENOCORTICAL HYPERFUNCTION Response to Stress. Adrenal glands examined at autop¬ sies after prolonged illnesses have mean weights of about 6 g and may weigh as much as 9 g. This appears to be the result of prolonged corticotropin (ACTH) stimulation. Histologically, within several hours of ACTH stimulation, clear (fasciculata) cells at the fasciculata-reticularis junction begin to lose their lipid content and take on the light-microscopic and ultrastructural appearance of compact (reticularis) cells (Fig. 71-7). Even¬ tually, this "lipid depletion" may involve the entire fasciculata, and compact cells may extend from the medulla to the glomerulosa or adrenal capsule. Corticotropin-Dependent Cushing Syndrome. Corticotro¬ pin-dependent Cushing syndrome includes both pituitary hypersecretion of ACTH (Cushing disease) and paraneoplastic (ectopic) ACTH secretion from a benign or malignant neo¬ plasm. The pathologic changes in the adrenal glands form a continuum from the mild stress-induced changes to the extreme changes resulting from severe, prolonged ACTH hypersecretion commonly seen with the paraneoplastic ACTH syndrome. Cushing disease accounts for about 60% of endoge¬ nous (noniatrogenic) Cushing syndrome in adults and -35% in children (see Chaps. 14, 75, and 83). The ACTH hypersecretion is generally mild but often prolonged. The adrenal glands are usually enlarged, each weighing 6 to 12 g. The cortex is wid¬ ened, with a broadened zone of compact cells, suggesting a hyperplastic zona reticularis. Some of the reticularis may be replaced by lipid. The clear cells often are larger than normal, with increased lipid content. The zona glomerulosa is normal. Cortical micronodules, consisting of clusters of clear cells, also may be seen in the periphery or around the central vein (Fig. 71-8). The only difference between these micronodules and those seen with aging is that the remainder of the cortex is hyperplastic. With prolonged ACTH stimulation, macronodular hyper¬ plasia may occur. Clinically, this is seen as unilateral or bilateral nodules in bilaterally hyperplastic adrenal glands in patients with long-standing Cushing disease.11 Each nodule-containing gland usually weighs between 12 and 20 g, although much larger glands are possible. The nodules have macroscopic and

microscopic features similar to those of the nodules seen with aging. The remainder of the cortex is hyperplastic. Although adrenal glands with macronodular hyperplasia are not capable of prolonged cortisol secretion without ACTH, there is evi¬ dence that such glands have limited autonomy compared with glands with bilateral hyperplasia that do not have nodules (see Chap. 75). Paraneoplastic ACTH secretion accounts for -15% of endog¬ enous Cushing syndrome in adults. The ACTH levels range from normal to greatly increased. Because of the natural history of the underlying tumor, the duration of disease is often brief (see Chap. 219). The adrenal glands frequently are markedly enlarged, weighing from 12 g to more than 20 g, but nodules usually are not present. Microscopically, columns of hyper¬ trophic compact cells extend from the medulla to the glomeru¬ losa or capsule. Foci of clear cells may be seen capping the compact cell columns. Corticotropin-Independent Cushing Syndrome. The cat¬ egory of ACTH-independent Cushing syndrome includes cortisolsecreting adrenocortical neoplasms, micronodular adrenal disease (which is seen in children and young adults), and druginduced Cushing syndrome (which induces adrenocortical atrophy; see Chap. 75). Benign cortisol-secreting adenomas account for 10% to 15% of cases of endogenous Cushing syn¬ drome. These tumors usually are small (90% identical in amino-acid sequence in their DNA-binding domains. However, the amino-terminal domains of these recep¬ tors are 20 pg/dL, however, prevent the release of ACTH from the normal pituitary gland. Thus, CRH fails to stimulate the release of ACTH in patients with Cushing syndrome if the cause is anything other than a pitu¬ itary tumor that is secreting ACTH. Alternatively, ACTHsecreting pituitary tumors are resistant to the feedback effects of cortisol; they retain the ability to respond to CRH, even at high plasma cortisol concentrations. A plasma ACTH concen¬ tration of >10 pg/mL after CRH administration defines ACTH-dependent hypercortisolism. Values of 10 pg/mL. The untoward effects of CRH are limited to a facial flush and sense of warmth, which occur in -10% of patients. Larger doses can induce hypotension and should not be used.

TESTS FOR ADRENAL HYPOFUNCTION The normal ranges of plasma cortisol and urinary free cortisol overlap the detection limit of most assays. In other words, a value of "zero" can be normal. Thus, reliable tests of adrenal insufficiency evaluate the ability of plasma cortisol to respond to an ACTH stimulus. The cosyntropin (Cortrosyn) test is the most convenient and reliable. This test is performed by admin¬ istering an intravenous bolus of 250 pg of synthetic (31-24 ACTH and measuring the plasma cortisol concentration 45 and 60 minutes later.22 A plasma cortisol concentration of 20 pg/dL is the lower limit of the normal response. Anything less than this suggests impaired adrenal function.23 Localizing the cause of adrenal insufficiency to the adrenal gland itself or to the hypothalamic-pituitary unit can be accomplished efficiently by measuring plasma ACTH. High ACTH concentrations localize the defect to the adrenal gland; normal or low concentrations localize the defect to the hypothalamus or pituitary.24 The RIA for ACTH has been notoriously unreliable, but the commer¬ cially available two-site immunoradiometric assay measure¬ ments are of high quality.25

TESTS FOR CONGENITAL ADRENAL HYPERPLASIA Diagnosis of the various syndromes of congenital adrenal hyperplasia depends on the measurement of the steroid biosyn¬ thetic intermediate before the blocked step (see Chap. 77). All the plasma marker steroids now can be measured by specific RIAs. The appropriate measurements include 17-hydroxyprogesterone (for 21-hydroxylase deficiency), 11-deoxycortisol (for 11-hydroxylase deficiency), 17-hydroxypregnenolone (for 3(3-hydroxysteroid dehydrogenase deficiency), and progester¬ one or pregnenolone (for 17-hydroxylase deficiency).

TESTS FOR LOCALIZATION Four tests are useful in localizing the site of disease in disorders of adrenal function: petrosal sinus sampling, computed tomog¬ raphy (CT), magnetic resonance imaging, and the iodocholesterol scan.

PETROSAL SINUS SAMPLING The venous drainage of the pituitary gland can be sampled in the inferior petrosal sinuses (Fig. 74-7). The finding of an ACTH gra¬ dient between petrosal sinus blood and peripheral blood in a patient with hypercortisolism localizes the site of ACTH produc¬ tion to the sella or parasellar region.26'263 Because the venous drainage of the pituitary gland tends to lateralize (the right half drains into the right inferior petrosal sinus and the left half drains into the left sinus), the site of an ACTH-producing microadenoma usually can be localized within the pituitary gland. The usefulness of the test is increased by sampling during the administration of CRH: the central/peripheral ACTH gradi¬ ents and the right/left gradient are enhanced by this addition. The average gradient in Cushing disease is ~15, with a lower limit of 1.7. The lower limit increases to 3 after CRH administra-

FIGURE 74-7. Anatomy of venous draining of the pituitary gland.

tion, which completely separates this population from those with nonpituitary causes of Cushing syndrome. Current data suggest that the test correctly identifies pituitary disease >95% of the time, which makes it the most powerful single test for differential diagnosis in Cushing syndrome.27 In previously untreated patients, the procedure correctly lateralizes the disease -85% of the time. Untoward effects are largely confined to the catheter¬ ization process. A few patients have had transient signs and symptoms of brainstem dysfunction or other neurological find¬ ings.273 These have not led to permanent disability. Other unto¬ ward effects include bleeding, bruising, and minor infections at the venipuncture site.

COMPUTED TOMOGRAPHY CT has become the procedure of choice in localizing adrenal disease (see Chap. 88). Most adrenal cancers are large, and CT, for practical purposes, identifies all these, as well as nearly all the smaller adrenal adenomas. Other useful findings revealed by CT include adrenal hyperplasia, usually bilateral and sym¬ metric, and an abnormal intraabdominal fat distribution that supports the clinical diagnosis of Cushing syndrome.28-31

MAGNETIC RESONANCE IMAGING Magnetic resonance imaging offers only a single advantage over CT in the differential diagnosis of adrenal disease: the clas¬ sification of adrenal masses into probably benign versus proba¬ bly malignant categories on the basis of the T2-weighted image.32 Normal adrenal glands are dark on Tl-weighted mag¬ netic resonance imaging scans. Benign adenomas remain dark when the scan is shifted to T2-weighting, but malignant lesions become bright. A hyperintense rim on fat-saturated spin-echo magnetic resonance imaging is often seen in adrenal adenomas and may aid in their differentiation from adrenal metastases.33 Adrenal cysts and pheochromocytomas also are bright, but usually present no problem in diagnosis when viewed in the context of other endocrine tests and the ultrasound examina¬ tion for cystic structure (see Chap. 88).

IODOCHOLESTEROL SCAN The iodocholesterol scan is an imaging procedure that depends on the function of the adrenal gland. It is rarely required in the differential diagnosis of hypercortisolism. Circulating choles¬ terol serves as the precursor for most of the steroids produced

Ch. 75: Cushing Syndrome by the adrenal gland. Not surprisingly, therefore, circulating cho¬ lesterol is concentrated in the adrenal glands, lodocholesterol, a y-emitter, can be used to examine this physiologic process. Nor¬ mal and hyperactive glands image bilaterally. Adenomas caus¬ ing Cushing syndrome image, but the contralateral side does not, because the resultant hypercortisolism suppresses ACTH and diminishes the function of the normal gland. Adrenal carci¬ nomas causing Cushing syndrome fail to produce an image because of an inefficient cholesterol-concentrating mechanism. The contralateral normal side also fails to image because ACTH secretion is suppressed. Thus, if both sides are visualized, an ACTH-dependent process is implied. If one side is visualized, an adrenal adenoma is implied. If neither side is seen, adrenal carcinoma or factitious disease is suggested.34-36

REFERENCES 1. Ruder HJ, Guy RL, Lipsett MB. Radioimmunoassay for cortisol in plasma and urine. J Clin Endocrinol Metab 1972; 35:219. 2. Murphy BER Clinical evaluation of urinary cortisol determinations by com¬ petitive protein binding radioassay. J Clin Endocrinol Metab 1968; 28:343. 3. Hsu TH, Bledsoe T. Measurement of urinary free corticoids by competitive protein binding radioassay in hypoadrenal states. J Clin Endocrinol Metab 1970; 30:443. 4. Beardwell CG, Burke CW, Cope CL. Urinary free cortisol measured by competitive protein binding. J Endocrinol 1968; 42:79. 5. Silber RH, Porter CC. The determination of 17-21-dihydroxy, 20-ketosteroids in urine and plasma. J Biol Chem 1954; 210:923. 6. Borushek S, Gold JJ. Commonly used medications that interfere with rou¬ tine endocrine laboratory procedures. Clin Chem 1964; 10:41. 7. Werk EE, MacBee T, Sholiton LJ. Effect of diphenylhydantoin on cortisol metabolism in man. J Clin Invest 1964; 43:1824. 8. Burstein S, Klaiber EL. Phenobarbital-induced increase in 6-hydroxycortisol excretion: clue to its significance in human urine. J Clin Endocrinol Metab 1965; 25:293. 9. Franks RC. Urinary 17-hydroxycorticosteroid and cortisol excretion in childhood. J Clin Endocrinol Metab 1973; 36:702. 10. Krieger DT, Allen W, Rizzo F, Krieger HP. Characterization of the normal temporal pattern of plasma corticosteroid levels. J Clin Endocrinol Metab 1971; 32:266. 11. Doe RP, Zimmerman HH, Flink EB. Significance of the concentration of nonprotein bound plasma cortisol in normal subjects, Cushing s syn¬ drome, pregnancy, and during estrogen therapy. J Clin Endocrinol Metab 12. 13.

14. 15.

16.

1960; 20:1484. Liddle GW, Island DP, Meador CK. Normal and abnormal regulation of corticotropin secretion in man. Recent Prog Horm Res 1962; 18:125. Flack MR, Loriaux DL, Nieman LK. Urinary free cortisol excretion: a better measure of response to the dexamethasone suppression test in Cushing s syndrome? (Abstract). New Orleans: Endocrine Society, 1988:1. Liddle GW, Estep HL, Kendall TW. Clinical application of a new test of pituitary reserve. J Clin Endocrinol Metab 1959; 19:875. Meikle AW, Jubiz W, Hutchings MD, et al. A simplified metyrapone test with determination of plasma 11-deoxycortisol (metyrapone test with plasma S). J Clin Endocrinol Metab 1969; 29:985. Jubiz W, Meikle AW, West CD, Tyler FH. A single dose metyrapone test.

Arch Intern Med 1970; 125:472. 17. Cushman P. Hypothalamic-pituitary-adrenal function in thyroid disorders: effects of metyrapone infusion on plasma corticosteroids. Metabolism 1968; 17:263. 18. Meikle AW, Jubiz W, Matsukura S, et al. Effect of estrogen on the metabolism of metyrapone and release of ACTH. J Clin Endocrinol Metab 1970; 30:259. 19. Jubiz W, Levinson RA, Meikle AW. Absorption and conjugation of metyrapone during diphenylhydantoin therapy: mechanism of abnormal response to oral metyrapone. Endocrinology 1970; 86:328. 20. Chrousos GP, Schulte HM, Oldfield EH, et al. The corticotropin releasing factor stimulation test: an aid in the evaluation of patients with Cushing's syndrome. N Engl J Med 1984; 310:622. 21. Nieman LK, Chrousos GP, Oldfield EH, et al. The corticotropin-releasing hormone stimulation test and the dexamethasone suppression test in the dif¬ ferential diagnosis of Cushing's syndrome. Ann Intern Med 1986; 105:862. 22. Kehlet H, Binder C. Value of an ACTH test in assessing hypothalamic-pituitaryadrenocortical function in glucocorticoid treated patients. BMJ1973; 2:147. 23. Lindholm J, Kehlet H, Blichert-Toft M. Reliability of the 30-minute ACTH test in assessing hypothalamic-pituitary-adrenal function. J Clin Endo¬ crinol Metab 1978; 47:272. 24. Schulte HM, Chrousos GP, Avgerinos P. The CRF test: a possible aid in the evaluation of patients with adrenal insufficiency. J Clin Endocrinol Metab 1984; 58:1064. 25. Slyper AH, Findling JW. Use of a two-site immunoradiometric assay to resolve a factitious elevation of ACTH in primary pigmented nodular adrenocortical disease. J Pediatr Endocrinol 1994; 7:61.

723

26. Oldfield EH, Chrousos GP, Schulte HM, et al. Preoperative localization of ACTH secreting pituitary microadenomas by bilateral and simultaneous inferior petrosal sinus sampling. N Engl J Med 1985; 312:100. 27. Zovickian J, Oldfield EH, Doppman JL, et al. Usefulness of inferior petrosal sinus venous endocrine markers in Cushing's disease. J Neurosurg 1988; 68:205. 27a. Lefournier V, Gatta B, Martinie M, et al. One transient neurological compli¬ cation (sixth nerve palsy) in 166 consecutive interior petrosal sinus sam¬ plings for the etiological diagnosis of Cushing's syndrome. J Clin Endocrinol Metab 1999; 84:3401. 28. Vincent JM, Morrison ID, Armstrong P, Reznek RH. The size of normal adrenal glands on computed tomography. Clin Radiol 1994; 49:453. 29. Ganguly A, Pratt JH, Yune HY, et al. Detection of adrenal tumors by com¬ puterized tomographic scan in endocrine hypertension. Arch Intern Med 1979; 139:590. 30. Kelestimur F, Unlu Y, Ozesmi M, Tolu I. A hormonal and radiological eval¬ uation of adrenal gland in patients with acute or chronic pulmonary tuber¬ culosis. Clin Radiol 1994; 49:453. 31. White FE, White MC, Drury PL, et al. Value of computed tomography of the abdomen and chest in investigation of Cushing's syndrome. BMJ 1982; 284:771. 32. Heinz-Peer G, Honigschabel S, Schneider B, et al. Characterization of adre¬ nal masses using MR imaging with histopathologic correlation. AJR Am J Roentgenol 1999; 173:15. 33. Ichikawa T, Ohtamo K, Uchiyama G, et al. Adrenal adenomas: characteris¬ tic hyperintense rim sign on fat-saturated spin-echo MR images. Radiology 1994; 193:247. 34. Moses DC, Schteingart DS, Sturman MF, et al. Efficacy of radiocholesterol imaging of the adrenal glands in Cushing's syndrome. Surg Gynecol Obstet 1974; 139:201. 35. Chatal JR, Charbonnel B, Le Mevel BP, et al. Uptake of 131I-19-iodocholesterol by an adrenal cortical carcinoma and its metastases. J Clin Endocrinol Metab 1976; 43:248. 36. Beierwaltes WH, Sisson JC, Shapiro B. Diagnosis of adrenal tumors with radionuclide imaging. Spec Top Endocrinol Metab 1984; 6:1.

CHAPTER 75

CUSHING SYNDROME DAVID E. SCHTEINGART Cushing syndrome is the clinical expression of the metabolic effects of persistent, inappropriate hypercortisolism. Since the disorder was first described by Harvey Cushing in 1932, great progress has been made in understanding its pathophysiology and the spectrum of its clinical presentation. The diagnosis can now be precisely established by biochemical means, by noninvasive imaging of the pituitary and adrenal lesions, and by selective identification of the source of corticotropin (ACTH or adrenocorticotropic hormone) in the ACTH-dependent types. The clinical manifestations of Cushing syndrome involve many organ systems and metabolic processes.1 Hypertension, obesity, diabetes, androgen-type hirsutism, and acne are com¬ monly seen in these patients and are prevalent in patients who do not have primary cortisol excess. When present, the following findings increase the suspicion of Cushing syndrome; symptoms and signs of protein catabolism (e.g., ecchymoses, myopathy, osteopenia); truncal obesity; lanugal hirsutism; cutaneous lesions (i.e., wide, purple striae; tinea versicolor; verruca vulgaris); hyperpigmentation; and psychiatric manifestations (i.e., impair¬ ment of affect, cognition, and vegetative functions).2- In some instances, the diagnosis of Cushing syndrome can be strongly suspected on clinical grounds alone, but for most patients, the diagnosis is established by laboratory studies.7

PATHOPHYSIOLOGY Cushing syndrome can be exogenous, resulting from the admin¬ istration of glucocorticoids or ACTH, or endogenous, resulting from a primary increased secretion of cortisol or ACTH. An etiologic classification of Cushing syndrome is given in Figure 75-1.

724

PART V: THE ADRENAL GLANDS tropes; this causes an excessive production of cortisol that is not sufficient to suppress ACTH secretion. In patients with ACTHindependent types of Cushing syndrome, pituitary ACTH secretion is suppressed by excessive cortisol production that originates in adrenocortical tumors (i.e., adenomas or carcino¬ mas) or in autonomous nodular hyperplastic glands.

Iatrogenic EXOGENOUS Factitious ■Pituitary

JCTH-Dependent

150 gg/day

1.0-mg Dexamethasone Suppression Cortisol Normal: 10 pg/dL ACTH Normal: 50 pg/mL

8.0-mg Dexamethasone Suppression Cortisol Pituitary ACTH dependent: 50% of baseline ACTH, corticotropin; IRMA, immunoradiometric assay.

Although the discovery of an adrenocortical adenoma is usu¬ ally made while a patient is being investigated for the possibility of Cushing syndrome, the adenoma occasionally is discovered incidentally during the investigation of abdominal complaints. Silent adrenal masses are found in 2% to 3% of patients who are undergoing CT scans for investigation of abdominal symp¬ toms.343 Although these patients may not have obvious clinical manifestations of Cushing syndrome and their baseline urinary cortisol levels are normal, cortisol secretion is autonomous. Evi¬ dence of autonomous cortisol secretion includes a blunted circa¬ dian rhythm, suppressed ACTH levels, and a lack of response to dexamethasone. Adrenal scintigraphy with iodocholesterol shows increased uptake on the side of the tumor but suppres¬ sion of uptake in the contralateral adrenal gland. Although these patients may have minimal manifestations of Cushing syndrome, these manifestations improve after sur¬ gical removal of the lesion. Patients with primary nodular adrenocortical hyperplasia are clinically indistinguishable from patients with benign adrenocortical adenomas or pituitary ACTH-dependent disease.

DIAGNOSIS STANDARD DIAGNOSTIC EVALUATION A biochemical evaluation of Cushing syndrome is necessary for confirming the clinical diagnosis and for determining the pres¬ ence of the syndrome in patients with an equivocal clinical pre¬ sentation.35 Preliminary testing includes the measurement of urinary free cortisol and of the ability to suppress serum cortisol lev¬ els with a low dose of dexamethasone (Table 75-2). In patients with

729

Cushing syndrome, the urinary free cortisol usually exceeds 90 jig per day. Serum cortisol levels obtained 9 hours after the oral administration of 1 mg of dexamethasone at 23:00 hours fail to decrease below 10 |ig/dL, and plasma ACTH levels (assessed by immunoradiometric assay) fail to decrease to 1.4 gg/dL (38 nmol/L) measured 15 min after the administration of CRH had 100% specificity, sensitivity, and diagnostic accuracy. The test has not been worked out for patients with severe melancholic depression, anorexia nervosa, cortisol resistance syndrome, or recent surgical stress. It also has not been defined for patients

733

with periodic hormonogenesis and intermittent Cushing syn¬ drome. In these patients, measurement of repeated 24-hour urine free cortisol levels may help detect times when an upsurge of ACTH and cortisol secretion occurs. The hypothesis behind the test is that at a dexamethasone dose sufficient to suppress normal cortisol production, patients with pseudoCushing states exhibit low basal plasma cortisol and ACTH lev¬ els and a diminished response to exogenous CRH. By contrast, patients with Cushing disease have higher basal cortisol and ACTH levels after the administration of dexamethasone and a greater peak response after CRH.69

CORTICOTROPIN-RELEASING HORMONE IN THE PATHOPHYSIOLOGY OF PSEUDO-CUSHING SYNDROME CRH secretion may help to differentiate pseudo-Cushing syn¬ drome and true Cushing syndrome. Although CRH secretion may be increased in patients with pseudo-Cushing syndrome, in most patients with ACTH-dependent or ACTH-independent Cushing syndrome, CRH secretion appears to be suppressed. When exogenous CRH is administered to patients with MDD, ambulatory alcoholics, and patients with anorexia nervosa, the ACTH response is diminished, whereas the response is increased in patients with pituitary ACTH-dependent disease. Unfortu¬ nately, a 25% overlap is found between the two groups.65

TREATMENT OF CUSHING DISEASE Optimal treatment of Cushing disease depends on an accurate diagnosis of the underlying pathology. Four approaches are used in the management of pituitary ACTH-dependent Cush¬ ing disease: pituitary surgery, pituitary irradiation, adrenal surgery, and drug therapy.7’70-72

PITUITARY SURGERY The treatment of choice for pituitary ACTH-dependent Cush¬ ing disease is the microsurgical removal of microadenomas or macroadenomas. If a pituitary adenoma or microadenoma can be demonstrated by radiographic techniques, a transsphenoi¬ dal operation of the pituitary gland should be the preferred treatment. If a tumor is not detected by imaging techniques, a transsphenoidal exploration of the pituitary gland is still in order, because the tumor can be found in -90% of patients.73-0 Even with considerable suprasellar extension, the tumor can be resected transsphenoidally. If a tumor invades the dura, total resection may be impossible, but good remission rates of 45% to 75% have been described for these cases. The microsurgical transsphenoidal selective resection of ACTH-secreting pituitary microadenomas is the most common treatment of Cushing syn¬ drome and comes closest to the ideal form of treatment for this condition 76-78 Several reports have described a high cure rate with transsphenoidal surgical treatment of Cushing disease.73-75,79'80 The probability of finding pituitary pathology and of surgically correcting the disease is highest among patients with a typical endocrine testing pattern.81 Typical diagnostic criteria for Cush¬ ing disease consist of elevated basal urinary 17-hydroxycorticosteroids and free cortisol levels, cortisol secretion rates, and mean basal serum cortisol levels; a positive response to metyrapone (i.e., elevated ACTH levels associated with a rise in urinary 17-hydroxycorticosteroids); and abnormal suppression with low-dose dexamethasone but >50% suppression with high-dose dexamethasone. Patients with atypical diagnostic cri¬ teria have elevated basal levels but do not respond as described to metyrapone or to low or high doses of dexamethasone. Pitu¬ itary disease was found in 18 of 19 patients with typical preop¬ erative endocrine test results but in only 6 of 11 patients with atypical test results.

734

PART V: THE ADRENAL GLANDS

If a microadenoma can be identified and totally and dis¬ cretely resected, then the remaining pituitary tissue remains functional, and patients can enjoy remission without loss of endocrine function.82 If a specific adenoma cannot be identified during surgery, the decision must be made as to whether to perform a partial or total hypophysectomy. If preoperative inferior petrosal sinus sampling has been carried out and is clearly lateralizing, an appropriate hemiresection of the hypophysis should be performed. If the endocrine studies strongly indicate a pituitary origin but the petrosal sinus sampling is not lateralizing and the patient does not wish to have children, a total hypophysectomy should be considered, but only after a lengthy preoperative discussion with the patient regarding this possi¬ bility. If the patient wishes to have children, alternative forms of therapy, including medical treatment or a total adrenalectomy, must be considered. Transsphenoidal surgery for Cushing dis¬ ease seems to be a reasonably safe procedure, with a mortality rate of 38°C (100°F), surgical procedures or injuries, and gastroenteritis with associated vomiting and diarrhea. Some studies question the need for increased cortisol during such periods; until this is firmly demonstrated in humans, however, current guidelines should be followed.46 The generally accepted guideline is that the daily dose of glucocorticoid should be doubled during peri¬ ods of minor stress such as low-grade fever, vomiting, and diar¬ rhea. In such circumstances, if the patient is not eating, the corticosteroid must be given parenterally. During periods of major stress, such as intraabdominal surgical procedures or major trauma, the dosage of hydrocortisone should be increased to 200 mg per day. Once the stress has passed, usually by the second postoperative day, the dosage of glucocorticoid is reduced imme¬ diately and directly to the usual daily rate of 12 to 15 mg/m2 (see Chap. 78). The measurement of serum cortisol, serum ACTH, or urinary free cortisol, or of Porter-Silber chromogens is not a reli¬ able guide to the appropriate maintenance dosage of glucocorti¬ coid. Normal serum electrolyte levels, a good appetite, and the patient's feeling of well-being are the best guides to adequate replacement. The appearance of the signs of Cushing syndrome, such as hypertension, weight gain, facial rounding, or supracla¬ vicular puffiness, indicate overtreatment. Overtreatment also may result in diminished bone density.47 The production rate of aldosterone is -100 pg per day at all stages of life in salt-replete humans.48 Fludrocortisone is roughly equipotent with aldosterone but is available only as an oral preparation. In normal persons, mineralocorticoid activity is supplied by both aldosterone and cortisol in roughly equal proportions. Thus, if cortisol is used for replacement, fludrocor¬ tisone, 100 pg per day, will supply the remaining complement of mineralocorticoid activity. If the glucocorticoid preparation used does not have significant mineralocorticoid activity (i.e., prednisone or dexamethasone, which are not recommended for replacement therapy), the dosage of fludrocortisone should be doubled. The serum potassium level is the best guide to the adequacy of mineralocorticoid replacement.

REFERENCES 1. O'Donnell WM. Changing pathogenesis of Addison's disease. Arch Intern Med 1950; 86:266. 2. Loriaux DL. The polyendocrine deficiency syndromes. N Engl J Med 1985; 312:1568. 3. Neufeld M, MacLaren N, Blizzard R. Autoimmune polyglandular syn¬ dromes. Pediatr Ann 1980; 9:154. 4. Vibo R, Aavik E, Peterson P, et al. Autoantibodies to cytochrome P450 enzymes P450 sec, P450 cl 7, and P450 c21 in autoimmune polyglandular disease types I and II and in isolated Addison's disease. J Clin Endocrinol Metab 1994; 78:323. 5. Eisenbarth G, Wilson P, Ward F, Lebovitz HE. HLA type and occurrence of disease in familial polyglandular failure. N Engl J Med 1978; 298:92. 6. Irvine WJ, Barnes EW. Adrenocortical insufficiency. J Clin Endocrinol Metab 1972; 1:549. 7. Crispell KR, Parson W, Hamlin J. Addison's disease associated with histo¬ plasmosis. Am J Med 1956; 20:23. 8. Eberle DE, Evans RB, Johnson RH. Disseminated North American blasto¬ mycosis: occurrence with clinical manifestations of adrenal insufficiency. JAMA 1977; 238:2629.

9. Rawson AJ, Collins LH, Grant JL. Histoplasmosis and torulosis as causes of adrenal insufficiency. Am J Med Sci 1948; 215:363. 10. Forbus WD, Beilerbreurtje AM. Coccidioidomycosis: a study of 95 cases of the disseminated type with special reference to the pathogenesis of the dis¬ ease. Mil Surg 1946; 99:653. 11. Irvine WJ, Toft AD, Feede CM. Addison's disease. In: James VHT, ed. The adrenal gland. New York: Raven Press, 1979:131. 12. O'Connell TX, Aston SJ. Acute adrenal hemorrhage complicating anticoag¬ ulant therapy. Surg Gynecol Obstet 1974; 139:355. 13. Sperling MW, Wolfsen AR, Fisher DA. Congenital adrenal hypoplasia: an isolated defect of organogenesis. J Pediatr 1973; 82:444. 14. Schaumberg H, Powers JW, Raine CS. Adrenoleukodystrophy: a clinical and pathological study of 17 cases. Arch Neurol 1975; 32:577. 15. Griffen JW, Goren E, Schaumberg H, et al. Adrenomyeloneuropathy: a probable variant of adrenoleucodystrophy. Neurology 1977; 27:1107. 16. Blevins LS Jr, Shankroff J, Moser FIW, Ladanson PW. Elevated plasma adrenocorticotropin concentration as evidence of limited adrenocortical reserve in patients with adrenomyeloneuropathy. J Clin Endocrinol Metab 1994; 78:261. 16a. Powers JM, DeCiero DP, Ito M, et al. Adrenomyeloneuropathy: a neuropathologic review featuring its noninflammatory myelopathy. J Neuropathol Exp Neurol 2000; 59:89. 17. Bleasd JF, et al. Acute adrenal insufficiency secondary to heparin-induced thrombocytopenia-thrombosis syndrome. Med J Aust 1952; 157:192. 18. Caron P, Chabannier MH, Cambus JP, et al. Definitive adrenal insufficiency due to bilateral adrenal hemorrhage and primary anti-phospholipid syn¬ drome. J Clin Endocrinol Metab 1998; 83:1437. 19. Sheeler LR, Myers JM, Eversham JJ, Taylor HC. Adrenal insufficiency sec¬ ondary to carcinoma metastatic to the adrenal gland. Cancer 1983; 52:1312. 20. Tapper ML, Rotterdam HZ, Lemer CW, et al. Adrenal necrosis in the acquired immunodeficiency syndrome. Ann Intern Med 1984; 100:239. 21. Greene LW, Cole W, Green JB. Adrenal insufficiency as a complication of the acquired immunodeficiency syndrome. Ann Intern Med 1984; 101:497. 22. Aron DC. Endocrine complications of the acquired immunodeficiency syn¬ drome. Arch Intern Med 1989; 149:330. 23. Danowski TS, Bonessi JV, Sabek G. Probabilities of pituitary-adrenal responsiveness after steroid therapy. Ann Intern Med 1964; 101:11. 24. Plager JE, Cushman P. Suppression of the pituitary ACTH response in man by administration of ACTH or cortisol. J Clin Endocrinol Metab 1962; 22:147. 25. Jasani MK, Boyle TA, Dick WC, et al. Corticosteroid-induced hypothalamopituitary-adrenal axis suppression: prospective study using two regimens of corticosteroid therapy. Ann Rheum Dis 1968; 27:352. 26. Graber AL, Ney RL, Nicholson WE. Natural history of pituitary-adrenal recovery following long term suppression with corticosteroids. J Clin Endocrinol Metab 1965; 25:11. 27. Kehlet H, Binder C. Value of an ACTH test in assessing hypothalamic-pituitaryadrenocortical function in glucocorticoid treated patients. BMJ 1973; 2:147. 28. Banna M. Craniopharyngioma: a review article based on 160 cases. Br J Radiol 1976; 49:206. 29. Hankinson T, Banna M. Pituitary and parapituitary tumors. London: WB Saunders, 1976:51. 30. Vita JA, Silverberg SJ, Goland RS, et al. Clinical clues to the cause of Addi¬ son's disease. Am J Med 1985; 78:461. 31. Stuart CA, Neelon FA, Lebovitz HE. Hypothalamic insufficiency: the cause of hypopituitarism in sarcoidosis. Ann Intern Med 1978; 88:589. 32. Tandon PN, Pathak SN. Tuberculosis of the central nervous system. In: Tropical neurology. London: Oxford University Press, 1973:37. 33. Ortega FJV, Longridge NS. Fracture of the sella turcica. Injury 1975; 6:335. 34. Thom GW. Diagnosis and treatment of adrenal insufficiency. Springfield, IL: Charles C Thomas, 1951. 35. Hertz KC, Gazze LA, Kirkpatrick CH, Katz SI. Autoimmune vitiligo: detec¬ tion of antibodies to melanin producing cells. N Engl J Med 1977; 297:634. 36. Lipsett MB, Pearson OH. Pathophysiology and treatment of adrenal crises. N Engl J Med 1956; 254:511. 37. Jorgensen H. Hypercalcemia in adrenocortical insufficiency. Acta Med Scand 1973; 193:175. 38. Pearson OH, Whitmore WF, West CD. Clinical and metabolic studies of bilateral adrenalectomy for advanced cancer in man. Surgery 1953; 34:543. 39. Lindholm J, Kehlet H, Blichert-Toft M. Reliability of the 30 minute ACTH test in assessing hypothalamic-pituitary-adrenal function. J Clin Endo¬ crinol Metab 1978; 47:272. 40. Kehlet H, Lindholm J, Bjerre P. Value of the 30 min ACTH test in assessing hypothalamic-pituitary-adrenocortical function after pituitary surgery in Cushing's disease. Clin Endocrinol (Oxf) 1984; 20:349. 41. Oelkers W, Diederich S, Bahr V. Diagnosis and therapy surveillance in Addison's disease: rapid adrenocorticotropin (ACTH) test and measure¬ ment of plasma ACTH, renin activity and aldosterone. J Clin Endocrinol Metab 1992; 75:259. 42. Schulte HM, Chrousos GP, Avgerinos P. The corticotropin-releasing hor¬ mone stimulation test: a possible aid in the evaluation of patients with adrenal insufficiency. J Clin Endocrinol Metab 1984; 58:1064. 43. Schultz CL, Haaga JR, Fletcher BD. Magnetic resonance imaging of the adrenal glands: a comparison with computed tomography. Am J Radiol 1984; 143:1235. 44. Gill JR, Bell NH, Barrter FL. Impaired conservation of sodium and potas¬ sium in renal tubular acidosis. Clin Sci 1967; 33:577. 45. Thom GW, Lauler DP. Clinical therapeutics of adrenal disorders. Am J Med 1977; 53:673.

Ch. 77: Congenital Adrenal Hyperplasia 46. Symreng T, Karlberg BE, Kagedal B, Schlidt B. Physiological cortisol substi¬ tution of long term steroid-treated patients undergoing major surgery. Br J Anaesth 1981; 53:949.

743

impair cortisol synthesis do not come under the rubric of CAH and are therefore not discussed here. In addition to a classic form with onset in prenatal life, nonclassic forms of the enzyme deficiencies with onset in childhood or young adult life are also described.

47. Zelissen PM, Croughs RJ, van Rijk PP, Raymakers JA. Effect of glucocorti¬ coid replacement therapy on bone mineral density in patients with Addi¬ son's disease. Ann Intern Med 1994; 120:207. 48. New MI, Seaman MP, Peterson RE. A method for the simultaneous determi¬ nation of the secretion rates of cortisol, 11-deoxycortisol, corticosterone, 11deoxycorticosterone, and aldosterone. J Clin Endocrinol Metab 1969; 29:514.

EMBRYOLOGY

CHAPTER

The fetal gonads remain undifferentiated until about the sev¬ enth week of gestation (see Chap. 90). At that time, in normal 46,XY fetuses, the gene encoding the sex-determining factor of the Y chromosome (i.e., SRY) is transcribed and testicular dif¬ ferentiation begins. SRY initiates a complex cascade of events3 in which other genes are inhibited (e.g., DAX-1, important in ovarian determination) or activated (e.g., SOX9 and SF-1, important in Sertoli cell differentiation and indirectly involved in ovarian inhibition via antimullerian hormone, AMH). Tes¬ tosterone secretion begins by -8 weeks of fetal life. In normal 46,XX fetuses, the absence of high local concentrations of AMH allows differentiation of the ovaries, beginning at -10 weeks' gestation (see Chap. 90). The internal genital duct structures are recognizable by 7 weeks. Mullerian ducts develop into the rostral third of the vagina, the uterus, and fallopian tubes. Wolffian ducts develop into epididymis, vas deferens, seminal vesicles, and ejaculatory ducts under the influence of adequate local quantities of andro¬ gen. Embryonal development of the gonads and internal ducts are generally unaffected by vagaries in sex steroid hormone synthesis. In females, the development of the external genitals is pas¬ sive. The urogenital sinus differentiates into separate urethral and vaginal orifices in normal females, and the labioscrotal folds remain separated by the labia minora, which hood the clitoris. In males, under the influence of high circulating levels of androgen, the urethral and genital orifices fuse to form an elongated penile urethra, and the labioscrotal folds fuse to form the scrotum. The latter changes may also occur in 46,XX fetuses exposed to high androgen levels from fetal adrenal or maternal sources. Conversely, male external genitals may be hypoplastic if a defect exists in testosterone synthesis or action. At -7 weeks of gestation, the adrenal cortex differentiates from mesodermal tissues. Soon after, two adrenal zones form: a small peripheral adult cortex and a large inner fetal cortex. Steroid production by the fetal cortex begins in the latter half of the first trimester. Adrenal mass increases markedly during this time, and peaks at 15 weeks.4 General regulation of adre¬ nal growth early in gestation is incompletely understood and is thought to be only partly attributable to ACTH. Other likely trophic hormones include transforming growth factor-(3

77

CONGENITAL ADRENAL HYPERPLASIA PHYLLIS W. SPEISER

Congenital adrenal hyperplasia (CAH) is a group of inherited diseases caused by defective activity in one of five enzymes that contribute to the synthesis of cortisol from cholesterol in the adrenal cortex (Fig. 77-1). Details of normal adrenal steroido¬ genesis are discussed in detail in Chapter 72. The term adrenal hyperplasia derives from the tendency to glandular enlargement under the influence of adrenocorticotropic hormone (ACTF1) in an effort to compensate for inadequate cortisol synthesis. The alternate term adrenogenital syndrome refers to the common asso¬ ciated finding of ambiguous external genitalia due to incidental deficiency or excess production of adrenal androgens. Each par¬ ticular enzyme deficiency produces characteristic alterations in the ratio of precursor hormone to product hormones. These hor¬ monal imbalances are accompanied by clinically evident abnor¬ malities, including abnormal development of the genitalia and pseudohermaphroditism, disturbances in sodium and potas¬ sium homeostasis, blood pressure dysregulation, and abnormal somatic growth1'2'23 (Table 77-1). The molecular genetic basis for all but one of the enzymatic deficiencies is known; disease-causing mutations have been identified for the genes encoding the respective steroidogenic enzymes. The most prevalent form of CAH is caused by deficiency of the cytochrome P450 enzyme, 21-hydroxylase (>90% of cases), followed by a deficiency of 11 (1-hydroxylase, 17a-hydroxylase/ 17,20-lyase, and 3(l-hydroxysteroid dehydrogenase. No muta¬ tions have been identified in the gene encoding cholesterol desmolase (also termed side-chain cleavage); this apparent enzyme deficiency is instead explained by defective steroidogenic acute regulatory protein. Deficiency of the enzymes that do not

Cholesterol | P450scc P450 cl7

P450C17

DHEA

17-OH-pregnenolone

Pregnenolone —

| 30-HSD

| 3/3-HSD

| 3P-HSD P450 cl7 Progesterone — | P450C21

17-OH-progesterone ■ | P450C21

P450 cl 7

P450 arom Androstenedione

| 170-HSD

| 170-HSD P450 arom

Deoxycorticosterone | P450C11 Corticosterone | P450C11 18-OH-corticosterone | P450C11 Aldosterone

11 -deoxycortisol | P450C11 Cortisol | 110-HSD Cortisone*

Testosterone* —

Estrone*

Estradiol*

FIGURE 77-1. The path¬ ways of corticosteroid syn¬ thesis are diagrammed. Cholesterol is converted in several steps to aldoste¬ rone, cortisol, or sex ster¬ oids. Hormones marked by an asterisk (*) are pro¬ duced largely outside the adrenal cortex. Deficiency of a given enzyme causes accumulation of hormonal precursors and a deficiency of products. (DHEA, dehydroepiandrosterone; HSD, hydroxysteroid dehydro¬ genase.)

744

PART V: THE ADRENAL GLANDS

TABLE 77-1. Clinical, Biochemical, and Genetic Characteristics of Congenital Adrenal Hyperplasia Characteristic

P450c21 Enzyme

P450c21 Enzyme

P450cll Enzyme

P450cl7 Enzyme

3|3-HSD Enzyme

3P-HSD Enzyme

P450scc Enzyme

Nonclassic

Nonclassic

Classic

Deficient phenotype

Classic

Classic

Classic

Classic

Ambiguous genitalia

+ in 46,XX

+ in 46,XX

+ in 46,XY

+ in 46,XY

Addisonian crisis

+ in SW

Rare

Incidence (gen. pop.)

1:14,000

- Puberty in

9

+

Mild in 46,XX

+

1:100

1:100,000

-120

i T i

++ (lethal)

7

cases

Common (?)

Rare

i

nl

i

i often

nl

i

i in cf

T in

Hormones Glucocorticoids

i

nl

i

Mineralocorticoids

i in SW

nl

T

Androgens

TT prenatally

T postnatally

TT prenatally

9

i

Weak androgens Estrogens

Relative defi¬ ciency in 9

Relative defi¬ ciency in 9

Relative deficiency in 9

i

i

i

i

Physiology Blood pressure

i untreated

nl

T often

T often

i

nl

i

Na balance

■1 in SW

nl

T

T

1-

often

nl

K balance

Tin SW

nl

i

+ in SW

T often + if SW

nl

Acidosis

i ± alkalosis

1 T +

± alkalosis

Diagnosis Metabolite

T

Common mutations

++++ 170HP

+ 170HP

DOC, S

DOC, B

DHEA, 17A5Preg

None

SW; del; nt 656 A-*G SV: I172N

V281L

R448H, frame shifts

Term, frame shifts

Term, frame shifts

StAR gene defect

P450c, cytochrome P450; HSD, hydroxysteroid dehydrogenase; +, present; ?, unknown; 1, diminished quantity;

T, increased quantity;

Cf, male; 9, female; SW, salt-water; nl, nor¬

mal; 170HP, 17-hydroxyprogesterone; DOC, deoxycorticosterone; S, 11-deoxycortisol; B, corticosterone; DHEA, dehydroepiandrosterone; 17D5Preg, 17-D5-pregnenolone; del, deletion; nt, nucleotide number; V281L, mutation at valine 281 resulting in leucine substitution; R448H, mutation at arginine 448 resulting in histidine substitution; Term, termination mutation; StAR, steroidogenic acute regulatory protein; SV, simple virilizer; U72N, mutation at isoleucine 172 resulting in asparagine substitution.

(TGF-J3), basic fibroblast growth factor (bFGF), and insulin¬ like growth factor-II (IGF-II).5 Beyond 20 weeks of gestation, adrenal growth and steroidogenesis are almost exclusively responsive to ACTH. In utero administration of glucocorti¬ coids suppresses fetal ACTH and, under most circumstances, inhibits adventitious adrenal steroid production in fetuses affected with a severe virilizing form of CAH (e.g., 21- or 11 (3hydroxylase deficiency).

21-HYDROXYLASE (P450C21) DEFICIENCY

aldosterone production and no salt wasting who have signs of prenatal virilization and markedly increased production of hormonal precursors of 21-hydroxylase (e.g., 17-hydrox¬ yprogesterone) are referred to as simple virilizers. Earlier con¬ fusion regarding the origins of these two classic phenotypes has been resolved to a large extent by the understanding of the molecular genetics of the disease, and allelic variation in the gene encoding active 21-hydroxylase (CYP21) appears to be responsible for most phenotypic variation, as is discussed later. Patients affected with the milder, nonclassic form of 21hydroxylase deficiency may have signs of postnatal androgen excess.6 Except for rare cases showing mild clitoromegaly.

Patients with this commonly diagnosed enzyme deficiency can¬ not adequately synthesize cortisol. Insufficient cortisol synthe¬ sis results in overproduction of adrenal androgens, which are synthesized independently of 21-hydroxylase. Androgens induce somatic growth with inappropriately rapid advance¬ ment of linear growth, early epiphyseal fusion of the long bones, and short stature. Other features include precocious development of sexual hair, apocrine body odor, and penile or clitoral enlargement. Reduced fertility may be observed in both sexes. Clinical features are outlined in Table 77-1. Females affected with the severe classic form of 21hydroxylase deficiency are exposed to excess androgens prenatally and are born with masculinized external genitalia (Fig. 77-2). If the disease goes undiagnosed and the infant is untreated, further virilization ensues (Fig. 77-3). Approxi¬ mately 75% of patients with the classic form cannot synthesize aldosterone efficiently because of impaired 21-hydroxylation of progesterone; these salt-wasting individuals fail to conserve sodium normally and usually come to medical attention in the neonatal period with hyponatremia, hyperkalemia, and hypovolemic shock. These adrenal crises may prove fatal if proper medical care is not delivered. Patients with sufficient

FIGURE 77-2. External genitalia of a 2-month-old female infant with 21-hydroxylase deficiency.

CLINICAL FEATURES

Ch. 77: Congenital Adrenal Hyperplasia

745

TABLE 77-3. Most Common Disease-Causing Mutations in CYP21 Designation

Site

Percentage of Normal Enzyme Activity

GROUP A: NO ENZYME ACTIVITY Deletion

Exons 1 to 8

0

Deletion of 8 base pairs

Exon 3

0

Cluster: Ile-236—>Asn

Exon 6

0

Insert T Phe-306

Exon 7

0

Gln-318—>term

Exon 8

0

Arg-356—>Trp

Exon 8

0

nt 656 A—>G

Intron 2

? amount residual activity

Val-237—>Glu Met-239—>Lys

GROUP B: SEVERELY REDUCED ENZYME ACTIVITY Ile-172—>Asn

Exon 4

2

GROUP C: MODERATELY REDUCED ENZYME ACTIVITY Pro-30—>Leu

Exon 1

30-60

Val-281—>Leu

Exon 7

20-50

?, indeterminate amount of residual activity; term, termination mutation; nt, nucle¬

Urogenital sinus FIGURE 77-3. Variations in the differentiation of the external genitalia in congenital adrenal hyperplasia. In genetic females, adrenal androgen hypersecretion (enzymes 2, 4, and 5) is associated with various degrees of masculinization, leading to apparent male external genitalia. (From Grumbach MM, Ducharme J. The effects of androgens on fetal develop¬ ment: androgen-induced female pseudohermaphroditism. Fertil Steril 1960; 11:157.)

females with the nonclassic disorder are born with normal external genitalia. The syndrome of polycystic ovarian disease has often been confused with nonclassic CAH 21-hydroxylase deficiency in young women with hirsutism, oligomenorrhea, and diminished fertility. Precise clinical distinction between the classic simple virilizing disease and nonclassic disorder is sometimes difficult among males, because the hormonal refer¬ ence standards for diagnosis represent a continuum. Moreover, because males do not manifest ambiguous genitalia as a sign of in utero androgen excess, the only other distinguishing clinical parameters are bone age and somatic growth pattern, which are nonpathognomonic. Phenotypic severity in nonclassic 21hydroxylase deficiency varies greatly, and in some individuals the disease has been detected solely on the basis of hormonal or genetic testing in the course of family studies. Aldosterone synthesis is normal in patients with nonclassic 21-hydroxylase deficiency. Table 77-2 describes features distinguishing saltwasting, simple virilizing, and nonclassic forms of 21-hydroxylase deficiency. Table 77-3 describes specific mutations. Neonatal screening for 21-hydroxylase deficiency measuring 17-hydroxyprogesterone levels in heel-stick blood has been TABLE 77-2. Phenotype in 21-Hydroxylase Deficiency Character¬ istic

Salt Wasting

Age at diag¬ nosis

otide number.

effective in reducing neonatal morbidity and mortality.7 This has been particularly useful in males with salt-wasting disease in whom no obvious phenotypic clue to the diagnosis, such as ambiguous genitalia, is present. The radioimmunoassay of heel-stick blood on filter paper was first used on a wide scale among the Alaskan Yupik Eskimos, one of two geographically isolated and genetically homogeneous groups at high risk for 21-hydroxylase deficiency CAH.8 Subsequently, many other newborn screening programs were developed for 21-hydroxy¬ lase deficiency CAH.9'9a The worldwide incidence of 21hydroxylase deficiency CAH based on newborn screening is 1 in 14,554 live births; approximately 75% of infants in whom the disease is detected in these programs manifest the salt-wasting phenotype.9 According to the Hardy-Weinberg law for popula¬ tions at equilibrium, the heterozygote frequency for all classic 21-hydroxylase gene defects is 1 in 61 persons. A high frequency of nonclassic 21-hydroxylase deficiency has also been discerned.10 This disorder occurs most frequently among Ashkenazi Jews (1 in 27), but it is also common among other ethnic groups, such as Hispanics, residents of the former Yugoslavia, and Italians. Overall, in a mixed white population, the disease is estimated to occur in ~1 in 100 individuals. These disease frequencies were derived indirectly based on response to ACTH stimulation combined with HLA typing. Confirma¬ tion was obtained using the statistical method of commingling distributions.11 Nonclassic 21-hydroxylase deficiency is among the most frequent autosomal recessive disorders in humans. Clinical investigation to diagnose nonclassic 21-hydroxylase deficiency is warranted in any patient showing the signs of androgen excess described previously; particularly high-risk groups include Ashkenazi Jews, children with precocious pubarche, and girls or women with hirsutism and oligomenorrhea.

11 p-HYDROXYLASE (P450C11) DEFICIENCY Simple Virilizing

Nonclassic Form

Infancy

Infancy (females) or childhood (males)

Childhood or adult¬ hood

Aldoster¬ one

Low

Normal

Normal

Virilization

Severe to moder¬ ate

Moderate to severe

None to mild

Mutation

Severe

Moderate (severe + moderate)

Mild (mild to moder¬ ate, mild + severe)

As in the case of 21-hydroxylase deficiency, in patients with llp-hydroxylase deficiency accumulating precursor steroids are channeled into androgen pathways beginning in prenatal life, which causes genital ambiguity in affected newborn females. Male infants show no abnormality of external genitalia. Later signs of androgen excess are observed in both sexes affected with 11 (1-hydroxylase deficiency if the disease is not promptly recognized and treated.12 Patients with llfl-hydroxylase defi¬ ciency account for approximately 5% of all CAH cases. Although in the general population this enzyme defect is found

746

PART V: THE ADRENAL GLANDS

in ~1 in 100,000 live births, the disease frequency is ~1 in 5000 to 7000 among Jews of Moroccan descent.13 No systematic screen¬ ing programs have been initiated to detect forms of CAH other than 21-hydroxylase deficiency. Nonclassic variants of 1113hydroxylase deficiency have also been described.12 Hormonal imbalances differentiate 21-hydroxylase from 11 (3-hydroxylase deficiency. In most cases, classic 21-hydroxy¬ lase deficiency is accompanied by deficient aldosterone synthe¬ sis and limited ability to conserve sodium. In contrast, in patients with 11 (3-hydroxylase deficiency, excessive production of the mineralocorticoid agonist deoxycorticosterone (DOC) or its metabolites results in sodium retention, hypokalemia, vol¬ ume expansion, suppressed plasma renin activity (PRA), and hypertension. Hypertension, however, is not the sine qua non for diagnosis of 11 (3-hydroxylase deficiency and is often absent in young children. Nonclassic cases may show variable eleva¬ tions of DOC and ll(3-deoxycortisol (compound S) and have normal PRA levels.

17a-HYDROXYLASE/17,20-LYASE (P450C17) DEFICIENCY In 17a-hydroxylase/17,20-lyase deficiency, impaired production of glucocorticoids and sex steroids (C19/C18 compounds) causes failure to develop estrogenic sexual characteristics at puberty in genetic females and incomplete development of the external geni¬ tals in genetic males.14-15 Shunting of P450cl7 precursor steroids into the 17-deoxy pathway produces mineralocorticoid excess, with hypokalemic alkalosis and hypertension similar to that in the 11 (3-hydroxylase deficiency. In rare cases, selective 17,20-lyase deficiency is detected. In such patients, cortisol and DOC levels are normal, but adrenal and gonadal C21 to C19 steroid conversion is impaired, so that normal sex steroid production is prevented. More than 120 cases have been reported of severe or com¬ plete 17a-hydroxylase deficiency, mostly in combination with 17,20-lyase deficiency.16 Patients from Canadian-Dutch Mennonite kindreds who share the same genotype have been reported.17 Relatively few genetic females have been detected. Partial deficiency of this enzymatic activity may be found in males with ambiguous genitalia.18 3(3-HYDROXYSTEROID DEHYDROGENASE

DEFICIENCY The enzyme 3(3-hydroxysteroid dehydrogenase (3(3-HSD) is responsible for conversion of A5 to A4 steroids. Deficiency of this enzyme results in inefficient cortisol synthesis; oversecretion of dehydroepiandrosterone (DHEA), which is only weakly andro¬ genic; and oversecretion of pregnenolone, which is ineffective as a mineralocorticoid. Affected individuals typically have cor¬ tisol insufficiency and salt wasting. Genital ambiguity is also part of the syndrome. Although lack of potent androgens pro¬ duces hypospadias in males, high levels of DHEA may cause clitoromegaly without urogenital sinus formation in females.19 In 3(3-HSD and 17a-hydroxylase/17,20-lyase deficiencies, potent androgens are deficient in prenatal life, which predis¬ poses males to gynecomastia. The precise frequency of severe defects in the adrenal 3(3-HSD gene is unknown. A nonclassic form of 3P-HSD deficiency is diagnosed with variable frequency in children with precocious pubarche and females with hirsutism and oligomenorrhea,20-21 but the hormonal profile with ACTH stimulation is less robust a diagnostic tool than that described for 21-hydroxylase deficiency. Some investi¬ gators have suggested that ovarian hyperandrogenism may be confused with 3P-HSD deficiency.22 The physician must also exer¬ cise caution in the interpretation of ACTH stimulation tests per¬ formed in infants younger than 1 year of age, because 3P-HSD is normally deficient in fetal life and relatively inactive in early infancy. Molecular genetic investigation has not revealed muta¬ tions to explain cases of mild 3P-HSD deficiency. No marked vari¬

ations in the ethnic incidence of this defect are known; several classic cases have been identified in consanguineous families.

20,22-DESMOLASE (P450SCC) DEFICIENCY Deficiency of 20,22-desmolase is also called lipoid adrenal hyper¬ plasia or apparent cholesterol desmolase deficiency. This enzyme cata¬ lyzes the initial reaction for all steroid production from cholesterol substrate. Apparent deficiency of side-chain cleav¬ age or cholesterol desmolase is extremely rare, with only ~30 cases reported in the world's literature.23-24 Complete choles¬ terol desmolase deficiency would be expected to produce glo¬ bal adrenocortical insufficiency and death because of marked cortisol deficiency and severe salt wasting. Partial apparent defects in this enzyme result in pseudohermaphroditism in genetic males; lack of secondary sexual characteristics can be expected in genetic females. A single case report describes long¬ term follow-up of a patient diagnosed in the newborn period and successfully treated for 18 years.25 Apparent cholesterol desmolase deficiency seems to occur with less severity and somewhat more frequently among the Japanese. Lipoid adrenal hyperplasia is now known to result from genetic defects in the gene encoding steroidogenic acute regulatory protein, rather than from actual defects in cholesterol desmolase.253

DIAGNOSIS POSTNATAL DIAGNOSIS The diagnosis of 21-hydroxylase deficiency may be confirmed by administering an intravenous bolus of ACTH and measur¬ ing the resultant elevation in blood levels of 17-hydroxyprogesterone.26 Usually, a panel of adrenal hormones is assayed before and after ACTH administration, but the most specific available marker for which testing is commercially available is 17hydroxyprogesterone. Clinicians should be aware that cortisol stimulation is suboptimal after ACTH infusion in patients with severe defects in adrenal steroid synthesis. If for any reason blood testing cannot be used or radioimmunoassays for 17hydroxyprogesterone are unavailable, the examiner can measure 17-ketosteroids or pregnanetriol in a 24-hour urine collection. The latter steroid is the principal direct urinary metabolite of 17-hydroxyprogesterone. Ancillary tests used in the initial evaluation of infants with ambiguous genitalia include karyotype analysis, pelvic and abdominal ultrasonography, and sonogram of the urogenital orifices using radiopaque dyes. Patients with nonclassic 21-hydroxylase deficiency have 17hydroxyprogesterone levels that exceed those seen in heterozy¬ gous carriers of an affected gene, but these levels are lower than those of patients with the classic form of the disorder.26 In the nonstimulated state, these patients may have near-normal serum hormone levels. A serum 17-hydroxyprogesterone level below 200 ng/dL effectively excludes this diagnosis if the sam¬ ple is obtained in the early morning (i.e., by 8:00 a.m.). The diagnosis of 11 (3-hydroxylase deficiency is made by the measurement of elevated basal or ACTH-stimulated DOC and/ or 11-deoxycortisol (i.e., compound S) in the serum or elevated levels of the tetrahydro-compounds (i.e., DOC and/or S) in a 24-hour urine collection.27 Another marker useful in pediatric diagnosis is 6a-hydroxytetrahydro-ll-deoxycortisol, which can be measured by gas chromatography and mass spectrometry of urine.28 As in 21-hydroxylase deficiency, urinary 17-ketoster¬ oids are usually elevated, reflecting increased shunting of 11(3hydroxylase hormonal precursors into the sex steroid pathway. PRA is usually low in older children and is accompanied by low levels of aldosterone. The diagnosis of 17a-hydroxylase/17,20-lyase deficiency is made by a finding of marked elevations of serum DOC and corti-

Ch. 77: Congenital Adrenal Hyperplasia costerone (i.e., compound B) and the metabolites of these two steroids.14 Aldosterone is often low secondary to suppression of renin by excess DOC, as in the case of 11 (3-hydroxylase defi¬ ciency. The 17oc-hydroxylase-deficient patients do not experience adrenal crisis despite inadequate cortisol synthesis. Overproduc¬ tion of corticosterone provides adequate physiologic response to stress. Plasma ACTH levels are less elevated than in other condi¬ tions of impaired cortisol production. Gonadotropin production is extremely elevated in both sexes because of the absence of any sex steroid feedback; the gonads are atrophic. A high ratio of A5 to A4 steroids characterizes the 3(3-HSD defi¬ ciency.29 Serum levels of 17-hydroxypregnenolone and DHEA are elevated before and after ACTH stimulation. Increased excre¬ tion of the A5 metabolites pregnanetriol and 16-pregnanetriol in the urine is also diagnostic for this enzyme disorder.

PRENATAL DIAGNOSIS Prenatal testing for 21-hydroxylase deficiency has been used for two decades in pregnancies known to be at risk.30-31 Hormonal diagnosis is accomplished by finding elevated levels of 17hydroxyprogesterone or 21-deoxycortisol in amniotic fluid.32-33 Genetic diagnosis was first performed by identifying HLA mark¬ ers on fetal cells cultured from the amniotic fluid; the genes encoding HLA antigens are closely linked to CYP21.34'35 Prob¬ lems encountered with these diagnostic techniques included false-negative 17-hydroxyprogesterone levels in non-salt-losing cases and intra-HLA recombination.36 Early amniocentesis and chorionic villus sampling have permitted diagnostic studies to be performed at the end of the first trimester.37-38 DNA obtained from such procedures may be analyzed by molecular genetic techniques, such as allele-specific hybridization with oligonucle¬ otide probes for the normal and mutant alleles of CYP21.39-40 In pregnancies known to have a 25% risk for 21-hydroxylase deficiency, "blind" prenatal treatment of the fetus may be initi¬ ated by administering dexamethasone to the mother beginning in the first trimester.41^3 Deferral of therapy until a molecular genetic diagnosis is known could hamper the ability to prevent genital ambiguity.44 Although prenatal treatment usually ame¬ liorates virilization of affected females, results of prenatal treat¬ ment have not been completely successful in this regard.45-46 Failure to produce normal female genitalia in 20% to 25%47 of affected girls through the use of prenatal dexamethasone ther¬ apy has been attributed to cessation of therapy in midgestation, to noncompliance, or to suboptimal dosing. Some treatment failures had no ready explanation 48 No fetus treated with low-dose dexamethasone has been born with a congenital malformation specifically attributable to dexamethasone therapy.49 The incidence of fetal deaths in treated pregnancies does not exceed that for the general popu¬ lation. Complications observed in a rodent model of in utero exposure to high-dose glucocorticoids included cleft palate, placental degeneration, intrauterine growth retardation, and unexplained fetal death.50 Other concerns for human fetal expo¬ sure have been raised based on observations of relatively late sequelae involving neurologic and vascular changes in lower mammals.51 Although at present no data exist to suggest that such sequelae occur in humans, the oldest prenatally treated children from CAH studies are only now reaching adolescence, and more long-term follow-up is required. The incidence of maternal complications has varied among investigations. Serious side effects, such as overt Cushing syn¬ drome, massive weight gain, and hypertension, have been reported in -1% of all treated pregnant women. Caution must be exercised in recommending prenatal ther¬ apy with dexamethasone, and women must be fully informed of these potential risks and nonuniformity of beneficial outcome to the affected female fetus. Despite these caveats, many parents of affected girls still choose prenatal medical treatment because of the severe psychological impact of ambiguous genitalia.

747

A similar diagnostic and therapeutic approach is effective in cases of 11 (3-hydroxylase deficiency, in which affected female fetuses are also at risk for prenatal virilization.

TREATMENT Patients with simple virilizing or salt-wasting classic 21hydroxylase deficiency or those with 11 (3-hydroxylase, 17ahydroxylase/17,20-lyase, and 3(3-HSD deficiencies, as well as select symptomatic patients with nonclassic forms of these dis¬ eases, are treated with daily oral hydrocortisone or similar drugs. Treatment with glucocorticoids suppresses excessive secretion of ACTH, correcting the adrenal hormone imbalance. Patients with the salt-wasting form of CAH require additional supplementation with mineralocorticoids (e.g., fludrocortisone acetate [Florinef], 50-200 gg per day) and sodium chloride sup¬ plements (1 to 2 g/10 kg body weight). Older children and adults with simple virilizing disease who are treated ade¬ quately with glucocorticoids usually do not have a clinically apparent deficiency of aldosterone, nor is renin markedly ele¬ vated. Many pediatric endocrinologists empirically treat all CAH patients with fludrocortisone and sodium chloride despite the lack of signs of salt wasting. Prudence dictates fol¬ lowing PRA in all patients as an index of the need for mineralocorticoid and salt supplements. Caution is advised to avoid development of hypertension consequent to excessive or unnecessary treatment with the latter regimen. Glucocorticoid treatment also leads to reduction of mineralocorticoid hormones in 11 (3-hydroxylase and 17a-hydroxylase deficiency, with amelioration of hypertension. In cases of long¬ standing hypertension, adjunctive antihypertensive drugs may be required to completely normalize blood pressure. The usual mode of treatment for CAH in childhood is with two to three divided daily doses of hydrocortisone totaling 10 to 20 mg/m2 per day (average dosage -15 mg/m2 per day). Even this relatively low dosage may be supraphysiologic, because healthy children and adolescents secrete an average of ~7 gg/m2 of cortisol daily.52-53 Experience indicates that oncedaily doses of hydrocortisone, because of its relatively rapid metabolism, is therapeutically suboptimal over the long term. Treatment efficacy should be monitored with frequent mea¬ surements of serum 17-hydroxyprogesterone (good control is indicated by a level of Thr) in the CYP17 (P45017a) gene lead to ambiguous external genitalia in a male patient with partial combined 17ahydroxylase deficiency. J Clin Endocrinol Metab 1992; 74:667. 19. Bongiovanni AM. The adrenogenital syndrome with deficiency of 3(3hydroxysteroid dehydrogenase. J Clin Invest 1962; 41:2086. 20. Bongiovanni AM. Acquired adrenal hyperplasia: with special reference to 3(i-hydroxysteroid dehydrogenase. Fertil Steril 1981; 35:599. 21. Zerah M, Schram P, New MI. The diagnosis and treatment of nonclassical 3|3-HSD deficiency. Endocrinologist 1991; 1:75. 22. Ehrmann DA, Rosenfield RL. Hirsutism—beyond the steroidogenic block. N Engl J Med 1990; 323:909. 23. Prader A, Gurtner HP. Das Syndrom des Pseudohermaphroditismus masculinus bei kongenitaler Nebennierenrinden-Hyperplasia ohne Androgentiber, produktion (adrenaler Pseudoherm masc). Helv Pediatr Acta 1955; 10:397. 24. Prader A, Siebenmann RE. Nebenniereninsuffizienz bei kongenitaler Lipoid-hyperplasie der Nebennieren. Helv Pediatr Acta 1957; 12:569. 25. Hauffa BP, Miller WL, Grumbach MM, et al. Congenital adrenal hyperpla¬ sia due to deficient cholesterol side-chain cleavage activity (20,22-desmolase) in a patient treated for 18 years. Clin Endocrinol 1985; 23:481. 25a. Stocco DM. The role of the StAR protein in steroidogenesis: challenges for the future. J Endocrinol 2000; 164(3):247. 26. New MI, Lorenzen F, Lemer AJ, et al. Genotyping steroid 21-hydroxylase deficiency: hormonal reference data. J Clin Endocrinol Metab 1983; 57:320. 27. Eberlein WR, Bongiovanni AM. Plasma and urinary corticosteroids in the hypertensive form of congenital adrenal hyperplasia. J Biol Chem 1956; 223:85. 28. Hughes IA, Arisaka O, Perry LA, et al. Early diagnosis of 11-beta-hydroxy¬ lase deficiency in two siblings confirmed by analysis of a novel steroid metabolite in newborn urine. Acta Endocrinol (Copenh) 1986; 111:349. 29. Bongiovanni AM. Urinary excretion of pregnanetriol and pregnenetriol in two forms of congenital adrenal hyperplasia. J Clin Invest 1971; 60:2751. 30. Jeffcoate TNA, Fleigner JRH, Russell SH, et al. Diagnosis of the adrenogen¬ ital syndrome before birth. Lancet 1965; 2:553. 31. Merkatz IR, New MI, Seaman MP. Prenatal diagnosis of adrenogenital syn¬ drome by amniocentesis. J Pediatr 1969; 75:977. 32. Frasier SD, Thomeycroft IH, Weiss BA, Horton R. Elevated amniotic fluid concentration of 17a-hydroxyprogesterone in congenital adrenal hyperpla¬ sia. J Pediatr 1975; 86:310. 33. Gueux B, Fiet J, Couillin P, et al. Prenatal diagnosis of 21-hydroxylase defi¬ ciency congenital adrenal hyperplasia by simultaneous radioimmunoassay of 21-deoxycortisol and 17-hydroxyprogesterone in amniotic fluid. J Clin Endocrinol Metab 1988; 66:534. 34. Couillin P, Nicolas H, Boue J, Boue A. HLA typing of amniotic-fluid cells applied to prenatal diagnosis of congenital adrenal hyperplasia. Lancet 1979; 1:1076. 35. Pollack MS, Levine LS, Pang S, et al. Prenatal diagnosis of congenital adrenal hyperplasia (21-hydroxylase deficiency) by HLA typing. Lancet 1979; 1:1107.

36. Pang S, Pollack MS, Loo M, et al. Pitfalls of prenatal diagnosis of 21hydroxylase deficiency congenital adrenal hyperplasia. J Clin Endocrinol Metab 1985; 61:89. 37. Odink RJH, Boue A, Jansen M. The value of chorion villus sampling in early detection of 21-hydroxylase deficiency. Pediatr Res 1988; 23:131A. 38. Shulman DI, Mueller OT, Gallardo LA, et al. Treatment of congenital adre¬ nal hyperplasia in utero. Pediatr Res 1989; 25:93A. 39. Owerbach D, Draznin MB, Carpenter RJ, Greenberg F. Prenatal diagnosis of 21-hydroxylase deficiency congenital adrenal hyperplasia using the polymerase chain reaction. Hum Genet 1992; 89:109. 40. Speiser PW, White PC, Dupont J, et al. Prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency by allele-specific hybridization and Southern blot. Hum Genet 1994; 93:424. 41. David M, Forest MG. Prenatal treatment of congenital adrenal hyperplasia resulting from 21-hydroxylase deficiency. J Pediatr 1984; 105:799. 42. Evans MI, Chrousos GP, Mann DW, et al. Pharmacologic suppression of the fetal adrenal gland in utero. JAMA 1985; 253:1015. 43. Forest MG, Betuel H, David M. Prenatal treatment of congenital adrenal hyperplasia with 21-hydroxylase deficiency: a multicenter study. Ann Endocrinol (Paris) 1987; 48:31. 44. Speiser PW, LaForgia N, Kato K, et al. First trimester prenatal treatment and molecular genetic diagnosis of congenital adrenal hyperplasia (21hydroxylase deficiency). J Clin Endocrinol Metab 1990; 70:838. 45. Migeon CJ. Comments about the need for prenatal treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. (Editorial). J Clin Endocrinol Metab 1990; 70:836. 46. Prenatal treatment of congenital adrenal hyperplasia. (Editorial). Lancet 1990; 510. 47. Forest MG, Morel Y, David M. Prenatal diagnosis and treatment of congen¬ ital adrenal hyperplasia. Horm Res 1997; 48(suppl 2): 22. 48. Pang S, Pollack MS, Marshall RN, Immken L. Prenatal treatment of con¬ genital adrenal hyperplasia due to 21-hydroxylase deficiency. N Engl J Med 1990; 322:111. 49. Lajic S, Wedell A, Bui TH, Ritzen EM, Holst M. Long-term somatic follow¬ up of prenatally treated children with congenital adrenal hyperplasia. J Clin Endocrinol Metab 1998; 83:3872. 50. Goldman AS, Sharpior BH, Katsumata M. Human foetal palatal corticoid receptors and teratogens for cleft palate. Nature 1978; 272:464. 51. Seckl JR, Miller WL. How safe is long-term prenatal glucocorticoid treat¬ ment? JAMA 1997; 277:1077. 52. New MI, Seaman MP. Secretion rates of cortisol and aldosterone in various forms of congenital adrenal hyperplasia. J Clin Endocrinol 1970; 30:361. 53. Linder BL, Esteban NV, Yergey AL, et al. Cortisol production rate in child¬ hood and adolescence. J Pediatr 1990; 117:892. 54. Golden MP, Lippe BM, Kaplan SA, et al. Management of congenital adre¬ nal hyperplasia using serum dehydroepiandrosterone sulfate and 17hydroxyprogesterone concentrations. Pediatrics 1978; 61:867. 55. Cutler GB Jr, Laue L. Seminars in medicine of the Beth Israel Hospital, Bos¬ ton: congenital adrenal hyperplasia due to 21-hydroxylase deficiency. N Engl J Med 1990; 323:1806. 56. Merke DP, Keil MF, Jones JV, et al. Flutamide, testolactone, and reduced hydrocortisone dose maintain normal growth velocity and bone matura¬ tion despite elevated androgen levels in children with congenital adrenal hyperplasia. J Clin Endocrinol Metab 2000; 85(3):1114. 57. Van Wyk JJ, Gunther DF, Ritzen EM, et al. The use of adrenalectomy as a treatment for congenital adrenal hyperplasia. J Clin Endocrinol Metab 1996; 81:3180. 58. Spritzer P, Billaud L, Thalabard J-C, et al. Cyproterone acetate versus hydrocortisone treatment in late-onset adrenal hyperplasia. J Clin Endo¬ crinol Metab 1990; 70:642. 59. Donahoe PK, Gustafson ML. Early one-stage surgical reconstruction of the extremely high vagina in patients with congenital adrenal hyperplasia. J Pediatr Surg 1994; 29:352. 60. Mulaikal RM, Migeon CJ, Rock JA. Fertility rates in female patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. N Engl J Med 1987; 316:178. 61. Kuhnle U, Bollinger M, Schwarz HP, Knorr D. Partnership and sexuality in adult female patients with congenital adrenal hyperplasia. First results of a cross-sec¬ tional quality-of-life evaluation. J Steroid Biochem Mol Biol 1993; 45:123. 62. Knudson AG Jr. Mixed adrenal disease of infancy. J Pediatr 1951; 39:408. 63. Bentinck RC, Hinman F Sr, Lisser H, et al. The familial congenital adrenal syndrome: report of two cases and review of the literature. Postgrad Med 1952; 11:301. 64. Childs B, Grumbach MM, Van Wyk JJ. Virilizing adrenal hyperplasia: a genetic and hormonal study. J Clin Invest 1956; 35:213. 65. Dupont B, Oberfield SE, Smithwick EM, et al. Close genetic linkage between HLA and congenital adrenal hyperplasia (21-hydroxylase defi¬ ciency). Lancet 1977; 2:1309. 66. White PC, New MI. Genetic basis of endocrine disease, 2: congenital adre¬ nal hyperplasia due to 21-hydroxylase deficiency. J Clin Endocrinol Metab 1992; 74:6. 67. White PC, Grossberger D, Onufer BJ, et al. Two genes encoding steroid 21hydroxylase are located near the genes encoding the fourth component of complement in man. Proc Natl Acad Sci U S A 1985; 82:1089. 68. Carroll MC, Campbell RD, Porter RR. The mapping of 21-hydroxylase genes adjacent to complement component C4 genes in HLA, the major his¬ tocompatibility complex in man. Proc Natl Acad Sci U S A 1985; 82:521. 69. White PC, Vitek A, Dupont B, New Ml. Characterization of frequent dele-

Ch. 78: Corticosteroid Therapy tions causing steroid 21-hydroxylase deficiency. Proc Natl Acad Sci U S A 1988; 85:4436. 70. Owerbach D, Crawford YM, Draznin MB. Direct analysis of CYP21B genes in 21-hydroxylase deficiency using polymerase chain reaction amplifica¬ tion. Mol Endocrinol 1990; 4:125. 71. Mornet E, Crete P, Kuttenn F, et al. Distribution of deletions and seven point mutations on CYP21B genes in three clinical forms of steroid 21hydroxylase deficiency. Am J Hum Genet 1991; 48:79. 72. Higashi Y, Hiromasa T, Tanae A, et al. Effects of individual mutations in the P-450(C21) pseudogene on the P-450(C21) activity and their distribution in the patient genomes of congenital steroid 21-hydroxylase deficiency. J Biochem 1991; 109:638. 73. Speiser PW, Dupont J, Zhu D, et al. Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase defi¬ ciency. ] Clin Invest 1992; 90:584. 74. Wedell A, Thilen A, Ritzen EM, et al. Mutational spectrum of the steroid 21hydroxylase gene in Sweden: implications for genetic diagnosis and associa¬ tion with disease manifestation. J Clin Endocrinol Metab 1994; 78:1145. 75. Higashi Y, Tanae A, Inoue H, et al. Aberrant splicing and missense muta¬ tions cause steroid 21-hydroxylase [P-450(C21)j deficiency in humans: pos¬ sible gene conversion products. Proc Natl Acad Sci U S A 1988; 85:7486. 76. Tusie-Luna MT, Traktman P, White PC. Determination of functional effects of mutations in the steroid 21-hydroxylase gene (CYP21) using recombi¬ nant vaccinia virus. J Biol Chem 1990; 265:20916. 77. Tusie-Luna MT, Speiser PW, Dumic M, et al. A mutation (Pro-30 to Leu) in CYP21 represents a potential nonclassic steroid 21-hydroxylase deficiency allele. Mol Endocrinol 1991; 5:685. 78. Higashi Y, Hiromasa T, Tanae A, et al. Effects of individual mutations in the P-450(C21) pseudogene on the P-450(C21) activity and their distribution in the patient genomes of congenital steroid 21-hydroxylase deficiency. J Biochem (Tokyo) 1991; 109:638. 79. Dickerman Z, Grant DR, Faiman C, Winter JSD. Intraadrenal steroid con¬ centrations in man: zonal differences and developmental changes. J Clin Endocrinol Metab 1984; 59:1031. 80. Nikoshkov A, Lajic S, Vlamis-Gardikas A, et al. Naturally occurring mutants of human steroid 21-hydroxylase (P450c21) pinpoint residues important for enzyme activity and stability. J Biol Chem 1998; 273:6163. 81. Mornet E, Dupont J, Vitek A, White PC. Characterization of two genes encoding human steroid 11 (3-hydroxylase (P-450 11(3). J Biol Chem 1989; 264:20961. 82. Chua SC, Szabo P, Vitek A, et al. Cloning of cDNA encoding steroid 11(3hydroxylase (P450cll). Proc Natl Acad Sci U S A1987; 84:7193. 83. Kawamoto T, Mitsuuchi Y, Toda K, et al. Cloning of cDNA and genomic DNA for human cytochrome P-450-ufS . FEBS Lett 1990; 269:345. 84. Kawamoto T, Mitsuuchi Y, Ohnishi T, et al. Cloning and expression of a cDNA for human cytochrome P-450aldos as related to primary aldoste¬ ronism. Biochem Biophys Res Commun 1990; 173:309. 85. Cumow KM, Tusie-Luna MT, Pascoe L, et al. The product of the CYP11B2 gene is required for aldosterone biosynthesis in the human adrenal cortex. Mol Endocrinol 1991; 5:1513. 86. White PC, Pascoe L, Cumow KM, et al. Molecular biology of ll|3-hydroxylase and ll(3-hydroxysteroid dehydrogenase enzymes. J Steroid Biochem Mol Biol 1992; 43:827. 87. Cumow KM, Slutsker L, Vitek J, et al. Mutations in the CYP11B1 gene caus¬ ing congenital adrenal hyperplasia and hypertension cluster in exons 6, 7, and 8. Proc Natl Acad Sci U S A 1993; 90:4552. 88. White PC, Dupont J, New MI, et al. A mutation in CYP11B1 (Arg-448->His) associated with steroid 11 (3-hydroxylase deficiency in Jews of Moroccan origin. J Clin Invest 1991; 87:1664. 89. Naiki Y, Kawamoto T, Mitsuuchi Y, et al. A nonsense mutation (TGG116TAG[Stop() in CYP11B1 causes steroid 11 (3-hydroxylase defi¬ ciency. J Clin Endocrinol Metab 1993; 77:1677. 90. Helmberg A, Ausserer B, Kofler R. Frame shift by insertion of 2 basepairs in codon 394 of CYP11B1 causes congenital adrenal hyperplasia due to ster¬ oid 11 beta-hydroxylase deficiency. J Clin Endocrinol Metab 1992; 75:1278. 91. Nakagawa Y, Yamada M, Ogawa H, Igarashi Y. Missense mutation in CYP11B1 (CGA[Arg-384] —>GG A[GlyJ) causes steroid 11 beta-hydroxylase deficiency. Eur J Endocrinol 1995; 132:286. 92. Yang LX, Toda K, Miyahara K, et al. Classic steroid 11 beta-hydroxylase deficiency caused by a C—>G transversion in exon 7 of CYP11B1. Biochem Biophys Res Commun 1995; 216:723. 93. Geley S, Kapelari K, Johrer K, et al. CYP11B1 mutations causing congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency. J Clin Endo¬ crinol Metab 1996; 81:2896. 94. Merke DP, Tajima T, Chhabra A, et al. Novel CYP11B1 mutations in con¬ genital adrenal hyperplasia due to steroid 11 beta-hydroxylase deficiency. J Clin Endocrinol Metab 1998; 83:270. 95. Joehrer K, Geley S, Strasser-Wozak EM, et al. CYP11B1 mutations causing non-classic adrenal hyperplasia due to 11 beta-hydroxylase deficiency. Hum Mol Genet 1997; 6:1829. 96. Matteson KJ, Picado-Leonard J, Chung B-C, et al. Assignment of the gene for adrenal P450cl7 (steroid 17a-hydroxylase/17,20-lyase) to human chro¬ mosome 10. J Clin Endocrinol Metab 1986; 63:789. 97. Chung B-C, Picado-Leonard J, Haniu M, et al. Cytochrome P450cl7 (ster¬ oid 17a-hydroxylase/17,20-lyase): cloning of human adrenal and testis cDNAs indicates the same gene is expressed in both tissues. Proc Natl Acad Sci U S A 1987; 84:407. 98. Suzuki Y, Nagashima T, Nomura Y, et al. A. A new compound heterozygous

751

mutation (W17X, 436 + 5G-*T) in the cytochrome P450cl7 gene causes 17 alphahydroxylase/17,20-lyase deficiency. J Clin Endocrinol Metab 1998; 83:199. 99. Lachance Y, Luu-The V, Labrie C, et al. Characterization of human 3(3hydroxysteroid dehydrogenase/A5-A4 isomerase gene and its expression in mammalian cells. J Biol Chem 1990; 265:20469. 100. Lorence MC, Corbin CJ, Kamimura N, et al. Structural analysis of the gene encoding human 3(3-hydroxysteroid dehydrogenase /A5_>4-isomerase. Mol Endocrinol 1990; 4:1850. 101. Lachance Y, Luu-The V, Verreault H, et al. Structure of the human type II 3(3-hydroxysteroid dehydrogenase/A5-A4 isomerase (3J3-HSD) gene: adre¬ nal and gonadal specificity. DNA Cell Biol 1991; 10:701. 102. Berube D, Luu-The V, Lachance Y, et al. Assignment of the human 3(3-HSD gene to the pl3 band of chromosome 1. Cytogenet Cell Genet 1989; 52:199. 102a. McCartin S, Russell AJ, Fisher RA, et al. Phenotypic variability and origins of mutations, in the gene encoding 3(3-hydroxysteroid dehydrogenase type II. Mol Endocrinol 2000; 24:75. 103. Rheaume E, Simard J, Morel Y, et al. Congenital adrenal hyperplasia due to point mutations in the type II 3P-hydroxysteroid dehydrogenase gene. Nature 1992; 1:239. 104. Chang YT, Kappy MS, Iwamoto K, et al. Mutations in the type II 3 betahydroxysteroid dehydrogenase (3 beta-HSD) gene in a patient with classic salt-wasting 3 beta-HSD deficiency congenital adrenal hyperplasia. Pediatr Res 1993; 34:698. 105. Simard J, Rheaume E, Sanchez R, et al. Molecular basis of congenital adre¬ nal hyperplasia due to 3(3-hydroxysteroid dehydrogenase deficiency. Mol Endocrinol 1993; 7:716. 106. Sakkal-Alkaddour H, Zhang L, Yang X, et al. Studies of 3 beta-hydroxysteroid dehydrogenase genes in infants and children manifesting premature pubarche and increased adrenocorticotropin-stimulated delta 5-steroid lev¬ els. J Clin Endocrinol Metab 1996; 81:3961. 107. Chung BC, Matteson KJ, Voutilainen R, et al. Human cholesterol side-chain cleavage enzyme, P450scc: cDNA cloning, assignment of the gene to chromo¬ some 15, and expression in the placenta. Proc Natl Acad Sci U S A1986; 83:8962. 108. Lin D, Gitelman SE, Saenger P, Miller WL. Normal genes for the cholesterol side chain cleavage enzyme, P450ssc, in congenital lipoid adrenal hyper¬ plasia. J Clin Invest 1991; 88:1955. 109. Miller WL. Congenital lipoid adrenal hyperplasia: the human gene knockout for the steroidogenic acute regulatory protein. J Mol Endocrinol 1997; 19:227. 110. Ulick S, Wang JZ, Morton DH. The biochemical phenotypes of two inborn errors in the biosynthesis of aldosterone. J Clin Endocrinol Metab 1992; 74:1415. 111. Harada N, Ogawa H, Shozu M, Yamada K. Genetic studies to characterize the origin of the mutation in placental aromatase deficiency. Am J Hum Genet 1992; 51:666. 112. Ito Y, Fisher CR, Conte FA, et al. Molecular basis of aromatase deficiency in an adult female with sexual infantilism and polycystic ovaries. Proc Natl Acad Sci U S A1993; 90:11673. 113. Simpson ER, Zhao Y, Agarwal VR, et al. Aromatase expression in health and disease. Recent Prog Horm Res 1997; 52:185. 114. Eckstein B, Cohen S, Farkas A, Rosier A. The nature of the defect in familial male pseudohermaphroditism in Arabs of Gaza. J Clin Endocrinol Metab 1989; 68:477. 115. Rosier A, Belanger A, Labrie F. Mechanisms of androgen production in male pseudohermaphroditism due to 17 beta-hydroxysteroid dehydroge¬ nase deficiency. J Clin Endocrinol Metab 1992; 75:773. 116. Castro-Magana M, Angulo M, Uy J. Male hypogonadism with gynecomas¬ tia caused by late-onset deficiency of testicular 17-ketosteroid reductase. N Engl J Med 1993; 328:1297. 117. Pang S, Softness B, Sweeney WJ, New MI. Hirsutism, polycystic ovarian disease, and ovarian 17-ketosteroid reductase deficiency. N Engl J Med 1987; 316:1295. 118. Andersson S, Moghrabi N. Physiology and molecular genetics of 17 betahydroxysteroid dehydrogenases. Steroids 1997; 62:143. 119. Moghrabi N, Hughes IA, Dunaif A, Andersson S. Deleterious missense mutations and silent polymorphism in the human 17beta-hydroxysteroid dehydrogenase 3 gene (HSD17B3). J Clin Endocrinol Metab 1998; 83:2855. 120. Nyckoff JA, Seely EW, Hurwitz S, et al. Glucocorticoid-remediable aldos¬ teronism and pregnancy. Hypertension 2000; 35:668.

CHAPTER 78

CORTICOSTEROID THERAPY LLOYD AXELROD This chapter examines the risks associated with the use of glu¬ cocorticoids and of mineralocorticoids for various illnesses, and provides guidelines for the administration of these commonly prescribed substances.

752

PART V: THE ADRENAL GLANDS

FIGURE 78-1. The structures of commonly used glucocorticoids. In the depiction of cortisol, the 21 carbon atoms of the glucocor¬ ticoid skeleton are indicated by numbers and the four rings are designated by letters. The arrows indicate the structural differ¬ ences between cortisol and each of the other molecules. (From Axelrod L. Glucocorticoid ther¬ apy. Medicine [Baltimore] 1976; 55:39, and Axelrod L. Glucocor¬ ticoids. In: Kelley WN, Harris ED Jr, Ruddy S, Sledge CB, eds. Textbook of rheumatology, 4th ed. Philadelphia: WB Saunders, 1993:779.)

i' CH,OH

CHjOH

*>C=0

C=0

CHjOH

I

I C=0

PREDNISOLONE ch2oh

CHjOH

I

STRUCTURE OF COMMONLY USED GLUCOCORTICOIDS Figure 78-1 indicates the structures of several commonly used glucocorticoids.1'2 Cortisol (hydrocortisone) is the principal circu¬ lating glucocorticoid in humans. Glucocorticoid activity requires a hydroxyl group at carbon 11 of the steroid molecule. Cortisone and prednisone are 11keto compounds. Consequently, they lack glucocorticoid activ¬ ity until they are converted in vivo to cortisol and prednisolone, the corresponding 11-hydroxyl compounds.3-4 This conversion occurs predominantly in the liver. Thus, topical application of cortisone is ineffective in the treatment of dermatologic dis¬ eases that respond to topical application of cortisol.4 Similarly, the antiinflammatory action of cortisone delivered by intraarticular injection is minimal compared with the effect of cortisol administered in the same manner.3 Cortisone and prednisone are used only for systemic therapy. All glucocorticoid prepara¬ tions marketed for topical or local use are 11-hydroxyl com¬ pounds, which obviates the need for biotransformation.

PHARMACODYNAMICS

CHjOH

I

c=o

GLUCOCORTICOIDS

METHYL PREDNISOLONE

I

c=o

c=o

PREDNISONE

DEXAMETHASONE

circulation is in the range of 80 to 115 minutes.1 The T1/2s of other commonly used agents are cortisone, 0.5 hours; pred¬ nisone, 3.4 to 3.8 hours; prednisolone, 2.1 to 3.5 hours; methylprednisolone, 1.3 to 3.1 hours; and dexamethasone 1.8 to 4.7 hours.1-7-8 Prednisolone and dexamethasone have comparable circulating T1/2s, but dexamethasone is clearly more potent. Similarly, the correlation between the circulating T1/2 of a glu¬ cocorticoid and its duration of action is poor. The many actions of glucocorticoids do not have an equal duration, and the dura¬ tion of action may be a function of the dose. The duration of ACTH suppression is not simply a function of the level of antiinflammatory activity, because variations in the duration of ACTH suppression are achieved by doses of glucocorticoids with comparable antiinflammatory activity. The duration of ACTH suppression produced by an individual glu¬ cocorticoid, however, probably is dose related.5 TABLE 78-1. Commonly Used Glucocorticoids

Duration of Action*

Gluco¬ corticoid Potency+

Equivalent Glucocorticoid Dose (mg)

Mineralo¬ corticoid Activity

SHORT-ACTING

HALF-LIFE, POTENCY, AND DURATION OF ACTION The important differences among the systemically used gluco¬ corticoid compounds are duration of action, relative glucocorti¬ coid potency, and relative mineralocorticoid potency (Table 78-1).1-2 The commonly used glucocorticoids are classified as short-acting, intermediate-acting, and long-acting on the basis of the duration of corticotropin (ACTH) suppression after a single dose, equivalent in antiinflammatory activity to 50 mg of pred¬ nisone (Table 78-1).3 The relative potencies of the glucocor¬ ticoids correlate with their affinities for the intracellular glucocorticoid receptor.6 The observed potency of a glucocorti¬ coid, however, is determined not only by the intrinsic biologic potency, but also by the duration of action.6-7 Consequently, the relative potency of two glucocorticoids varies as a function of the time interval between the administration of the two steroids and the determination of the potency. In particular, failure to account for the duration of action may lead to a marked under¬ estimation of the potency of dexamethasone.7 The correlation between the circulating half-life (T1/2) of a glu¬ cocorticoid and its potency is weak. The T1/2 of cortisol in the

Cortisol (hydrocortisone)

1

20

Yes*

Cortisone

0.8

25

Yes*

Prednisone

4

5

No

Prednisolone

4

5

No

Methylprednisolone

5

4

No

5

4

No

Betamethasone

25

0.60

No

Dexamethasone

30

0.75

No

INTERMEDIATE-ACTING Triamcinolone LONG-ACTING

The classification by duration of action is based on Harter JG. Corticosteroids. NY State J Med 1966;66:827. The values given for glucocorticoid potency are relative. Cortisol is arbitrarily assigned a value of 1. •Mineralocorticoid effects are dose related. At doses close to or within the basal physiologic range for glucocorticoid activity, no such effect may be detectable. (Data from Axelrod L. Glucocorticoid therapy. Medicine [Baltimore] 1976;55:39; Axelrod L. Adrenal corticosteroids. In: Miller RR, Greenblatt DJ, eds. Handbook of drug therapy. New York: Elsevier North-Holland, 1979:809; and Axelrod L. Glucocorticoids. In: Kelley WN, Harris ED Jr, Ruddy S, Sledge CB, eds. Textbook of rheumatology, 4th ed. Philadelphia: WB Saunders, 1993:779.)

Ch. 78: Corticosteroid Therapy In short, the slight differences in the circulating T1/2s of the glu¬ cocorticoids contrast with their marked differences in potency and duration of ACTH suppression. Thus, the duration of action of a glucocorticoid is not determined by its presence in the circulation. This is consistent with the mechanism of action of steroid hor¬ mones. A steroid molecule binds to a specific intracellular receptor protein (see Chap. 4). This steroid-receptor complex modifies the process of transcription by which RNA is transcribed from the DNA template. This process alters the rate of synthesis of specific proteins. The steroid thereby modifies the phenotypic expression of the genetic information. TTius, the glucocorticoid continues to act inside the cell after it has disappeared from the circulation. More¬ over, the events initiated by the glucocorticoid may continue to occur, or a product of these events (such as a specific protein) may be present after the disappearance of the glucocorticoid.

753

either prednisone or prednisolone.12 This is further complicated by the lower percentage of plasma prednisolone that is bound to protein in patients with active liver disease; the unbound fraction is inversely related to the serum albumin concentra¬ tion. An increased frequency of prednisone side effects is observed at low serum albumin levels.12 Both these findings may reflect impaired hepatic function. Because the impairment of conversion of prednisone to prednisolone is quantitatively small in the presence of liver disease and is offset by a decreased rate of clearance of prednisolone, and because of the marked variability in plasma prednisolone levels after the administration of either corticosteroid, there is no clear man¬ date to use prednisolone rather than prednisone in patients with active liver disease or cirrhosis.8 If prednisone or pred¬ nisolone is used, however, a somewhat lower than usual dose should be given if the serum albumin level is low.8

BIOAVAILABILITY, ABSORPTION, AND BIOTRANSFORMATION Normally, a person's plasma cortisol level is much lower after the oral administration of cortisone than after an equal dose of cortisol.9 Consequently, although oral cortisone may be ade¬ quate replacement therapy in chronic adrenal insufficiency, the oral form of this agent should not be used when larger, phar¬ macologic effects are sought. Comparable plasma prednisolone levels are achieved in normal persons after equivalent oral doses of prednisone and prednisolone.8,10 After the administra¬ tion of either of these corticosteroids, however, there is wide variation in individual prednisolone concentrations, which may reflect variability in absorption.8 In contrast to the marked rises that follow the intramuscular injection of hydrocortisone, plasma cortisol levels rise little or not at all after an intramuscular injection of cortisone acetate. When it is given intramuscularly, cortisone acetate does not pro¬ vide adequate plasma cortisol levels and offers no advantage over hydrocortisone delivered by the same route. The explana¬ tion for the failure of intramuscular cortisone acetate to provide adequate plasma cortisol levels is unknown. It may reflect poor absorption from the site of injection. Alternatively, intramuscular cortisone acetate, which reaches the liver through the systemic circulation, may be metabolically inactivated before it can be converted to cortisol in the liver, in contrast to oral cortisone ace¬ tate, which reaches the liver through the portal circulation. PLASMA TRANSPORT PROTEINS In normal humans, circadian fluctuations occur in the capacity of corticosteroid-binding globulin (transcortin) to bind cortisol and prednisolone. Patients who have been treated with prednisone for a prolonged period have no diurnal variation in the binding capacity of corticosteroid-binding globulin for cortisol or pred¬ nisolone, and both capacities are reduced in comparison with normal persons. Thus, long-term glucocorticoid therapy not only alters the endogenous secretion of steroids, but also affects the transport of some glucocorticoids in the circulation. This may explain why the disappearance of prednisolone is more rapid in those persons who have previously received glucocorticoids.

GLUCOCORTICOID THERAPY IN THE PRESENCE OF LIVER DISEASE Plasma cortisol levels are normal in patients with hepatic disease. Although the clearance of cortisol is reduced in patients with cir¬ rhosis, the hypothalamic-pituitary-adrenal (HPA) homeostatic mechanism remains intact. Consequently, the decreased rate of metabolism is accompanied by decreased synthesis of cortisol (see Chap. 205). The conversion of prednisone to prednisolone is impaired in patients with active liver disease.11 This is largely offset by a decreased rate of elimination of prednisolone from the plasma in these patients.11 In patients with liver disease, the plasma availability of prednisolone is quite variable after oral doses of

GLUCOCORTICOID THERAPY AND THE NEPHROTIC SYNDROME When hypoalbuminemia is caused by the nephrotic syndrome, the fraction of prednisolone that is protein bound is decreased. The unbound fraction is inversely related to the serum albumin concentration. The unbound prednisolone concentration remains normal, however.13,14 Because the pharmacologic effect is deter¬ mined by the unbound concentration, altered prednisolone kinetics do not explain the increased frequency of predniso¬ lone-related side effects in these patients.

GLUCOCORTICOID THERAPY AND HYPERTHYROIDISM The bioavailability of prednisolone after an oral dose of pred¬ nisone is reduced in patients with hyperthyroidism because of decreased absorption of prednisone and increased hepatic clearance of prednisolone.15

GLUCOCORTICOIDS DURING PREGNANCY Glucocorticoid therapy is well tolerated in pregnancy.16 Gluco¬ corticoids cross the placenta, but there is no compelling evi¬ dence that this produces clinically significant HPA suppression or Cushing syndrome in neonates,16 although subnormal responsiveness to exogenous ACTH may occur. Similarly, there is no evidence that glucocorticoids increase the incidence of congenital defects in humans.16 Glucocorticoids do appear to decrease the birth weight of full-term infants; the long-term consequences of this are unknown. Because the concentrations of prednisone and prednisolone in breast milk are low, the administration of these drugs to the mother of a nursing infant is unlikely to produce deleterious effects in the infant.

GLUCOCORTICOID THERAPY AND AGE The clearance of prednisolone and methylprednisolone decreases with age.17,18 Despite the higher prednisolone levels seen in elderly subjects compared with young subjects after com¬ parable doses, endogenous plasma cortisol levels are suppressed to a lesser extent in the elderly.17 These findings may be associ¬ ated with an increased incidence of side effects and suggest the need to use smaller doses in the elderly than in young patients.

DRUG INTERACTIONS The concomitant use of medications can alter the effectiveness of glucocorticoids; the reverse also is true.19 EFFECTS OF OTHER MEDICATIONS ON GLUCOCORTICOIDS The metabolism of glucocorticoids is accelerated by substances that induce hepatic microsomal enzyme activity, such as pheny-

754

PART V: THE ADRENAL GLANDS

toin, barbiturates, and rifampin. The administration of these medi¬ cations can increase the corticosteroid requirements of patients with adrenal insufficiency or lead to deterioration in the condi¬ tions of patients whose underlying disorders are well controlled by glucocorticoid therapy. These substances should be avoided in patients receiving corticosteroids. Diazepam does not alter the metabolism of glucocorticoids and is preferable to barbiturates in this setting. If drugs that induce hepatic microsomal enzyme activ¬ ity must be used in patients taking corticosteroids, an increase in the required dose of corticosteroids should be anticipated. Conversely, ketoconazole increases the bioavailability of large doses of prednisolone (0.8 mg/kg) because of inhibition of hepatic microsomal enzyme activity.20 Oral contraceptive use decreases the clearance of prednisone and increases its bioavailability.21 The bioavailability of prednisone is decreased by antacids in doses comparable to those used clinically.22 The bioavailability of prednisolone is not impaired by sucralfate, H2-receptor blockade, or cholestyramine.

TABLE 78-2. Considerations before the Use of Glucocorticoids as Pharmacologic Agents 1. How serious is the underlying disorder? 2. How long will therapy berequired? 3. What is the anticipated effective corticosteroid dose? 4. Is the patient predisposed to any of the potential hazards of glucocorti¬ coid therapy? Diabetes mellitus Osteoporosis Peptic ulcer, gastritis, or esophagitis Tuberculosis or other chronic infections Hypertension and cardiovascular disease Psychological difficulties 5. Which glucocorticoid preparation should be used? 6. Have other modes of therapy been used to minimize the glucocorticoid dosage and to minimize the side effects of glucocorticoid therapy? 7. Is an alternate-day regimen indicated?

EFFECTS OF GLUCOCORTICOIDS ON OTHER MEDICATIONS

The concurrent administration of a glucocorticoid and a salicy¬ late may reduce the serum salicylate level. Conversely, reduc¬ tion of the corticosteroid dose during the administration of a fixed dose of salicylate may lead to a higher and possibly toxic serum salicylate level. This interaction may reflect the induc¬ tion of salicylate metabolism by glucocorticoids.23 Glucocorticoids may increase the required dose of insulin or oral hypoglycemic agents, antihypertensive drugs, or glaucoma medications. They also may alter the required dose of sedativehypnotic or antidepressant therapy. Digitalis toxicity can result from hypokalemia caused by glucocorticoids, as from hypo¬ kalemia of any cause. Glucocorticoids can reverse the neuro¬ muscular blockade induced by pancuronium.

CONSIDERATIONS BEFORE INITIATING THE USE OF GLUCOCORTICOIDS AS PHARMACOLOGIC AGENTS Cushing syndrome (see Chap. 75) is a life-threatening disorder. The 5-year mortality was higher than 50% at the beginning of the era of glucocorticoid and ACTH therapy.24 Infection and cardio¬ vascular complications were frequent causes of death. High-dose exogenous glucocorticoid therapy is similarly hazardous. Table 78-2 summarizes the important questions to consider before initiating glucocorticoid therapy.25 These questions enable the physician to assess the potential risks that must be weighed against the possible benefits of treatment. The more severe the underlying disorder, the more readily can systemic glucocorti¬ coid therapy be justified. Thus, corticosteroids are commonly used in patients with severe forms of systemic lupus erythemato¬ sus, sarcoidosis, active vasculitis, asthma, chronic active hepatitis, transplantation rejection, pemphigus, or diseases of comparable severity. Generally, systemic corticosteroids should not be administered to patients with mild rheumatoid arthritis or mild bronchial asthma; such patients should receive more conserva¬ tive therapy first. Although these patients may experience symp¬ tomatic relief from glucocorticoids, it may prove difficult to withdraw the drugs. Consequently, they may unnecessarily experience Cushing syndrome and HPA suppression. DURATION OF THERAPY

The anticipated duration of glucocorticoid therapy is another critical issue. The use of glucocorticoids for 1 to 2 weeks for a condition such as poison ivy or allergic rhinitis is unlikely to be associated with serious side effects in the absence of a contraindi¬ cation. An exception to this rule is a corticosteroid-induced psy¬ chosis. This complication may occur after only a few days of high-dose glucocorticoid therapy, even in patients with no previ¬ ous history of psychiatric disease (see Chap. 201 ).26-27 Because the risk of so many complications is related to the dose and duration

(Modified from Thorn GW. Clinical considerations in the use of corticosteroids. N Engl J Med 1966; 274:775.)

of therapy, the smallest possible dose should be prescribed for the shortest possible period. If hypoalbuminemia is present, the dose should be reduced. If long-term treatment is indicated, the use of an alternate-day schedule should be considered. LOCAL USE

A local corticosteroid preparation should be used whenever pos¬ sible because systemic effects are minimal when these substances are administered correctly. Examples include topical therapy in dermatologic disorders, corticosteroid aerosols in bronchial asthma and allergic rhinitis, and corticosteroid enemas in ulcer¬ ative proctitis. Systemic absorption of inhaled glucocorticoids leading to Cushing syndrome and HPA suppression is a rare occurrence when these agents are administered correctly at pre¬ scribed doses.28 29 The intraarticular injection of corticosteroids may be of value in carefully selected patients if strict aseptic tech¬ niques are used and if frequent injections are avoided. SELECTING A SYSTEMIC PREPARATION

Agents with little or no mineralocorticoid activity should be used when a glucocorticoid is prescribed for pharmacologic purposes. If the dosage is to be tapered over a few days, a long-acting agent may be impractical. For alternate-day therapy, a short-acting agent that generally does not cause sodium retention (e.g., pred¬ nisone, prednisolone, or methylprednisolone) should be used. There is no indication for glucocorticoid conjugates designed to achieve a prolonged duration of action (several days or several weeks) after a single intramuscular injection. The bioavailability of such preparations cannot be regulated precisely, the duration of action cannot be estimated reliably, and it is not possible to taper the dosage rapidly in the event of an adverse reaction such as a corticosteroid-induced psychosis. The use of such preparations may cause HPA suppression more frequently than do comparable doses of the same glucocorticoid given orally. The use of supple¬ mental medications to minimize the systemic corticosteroid dose and to reduce the side effects of systemic glucocorticoids should always be considered. In asthma, for example, treatment should include inhaled glucocorticoids and bronchodilators, such as 0adrenergic agonists and theophylline, and may include cromolyn.

EFFECTS OF EXOGENOUS GLUCOCORTICOIDS ANTIINFLAMMATORY AND IMMUNOSUPPRESSIVE EFFECTS

Endogenous glucocorticoids protect the organism from damage caused by its own defense reactions and the products of these reac-

Ch. 78: Corticosteroid Therapy tions during stress.30,303 Consequently, the use of glucocorticoids as antiinflammatory and immunosuppressive agents represents an application of the physiologic effects of glucocorticoids to the treat¬ ment of disease.30 Glucocorticoids have many effects on inflamma¬ tory and immune responses, which are described in this section. Glucocorticoids inhibit synthesis of almost all known cytok¬ ines and of several cell surface molecules required for immune function.31-33 When an immune stimulus such as tumor necrosis factor binds to its receptor, nuclear factor kappa B (NF-kB) moves to the nucleus, where it activates many immunoregulatory genes. This activation of NF-kB involves the degradation of its cytoplas¬ mic inhibitor IkBoc and the translocation of NF-kB to the nucleus. Glucocorticoids are potent inhibitors of NF-kB activation. This inhibition is mediated by the induction of the IkBoc inhibitory pro¬ tein, which traps activated NF-kB in inactive cytoplasmic com¬ plexes.31-33 This reduction in NF-kB activity appears to explain the ability of glucocorticoids to inhibit the production of cytokines and cell surface molecules and to suppress the immune response. Influence on Blood Cells and on the Microvasculature. Glucocorticoid effects on inflammatory and immune phenom¬ ena include effects on leukocyte movement, leukocyte function, and humoral factors (Table 78-3). In general, glucocorticoids have a greater effect on leukocyte traffic than on function, and more effect on cellular than on humoral processes.34,35 Gluco¬ corticoids alter the traffic of all the major leukocyte populations in the circulation (see Chap. 212). Probably the most important antiinflammatory effect of glu¬ cocorticoids is the ability to inhibit the recruitment of neutrophils and monocyte-macrophages to an inflammatory site.35 Cortico¬ steroids modify the increased capillary and membrane perme¬ ability that occurs in an area of inflammation. By decreasing the dilation of the microvasculature and the increased capillary per¬ meability that occur during an inflammatory response, the exu¬ dation of fluid and the formation of edema may be reduced, and the migration of leukocytes may be impaired.2,35,36 The decrease in the accumulation of inflammatory cells is also related to decreased adherence of inflammatory cells to the vascular endo¬ thelium. It is not possible to determine the relative contributions of the direct vascular effect, the effect on inflammatory cell adherence to the vascular wall, and the effect on chemotaxis to the reduction in inflammation caused by glucocorticoids. Glucocorticoids have multiple effects on leukocyte func¬ tion.35 Corticosteroids suppress cutaneous delayed hypersensi¬ tivity responses. Monocyte-macrophage traffic and function are sensitive to glucocorticoids (see Table 78-3). Glucocorticoids in divided daily doses depress the bactericidal activity of mono¬ cytes. The sensitivity of monocytes to glucocorticoids may explain the effectiveness of these agents in many granuloma¬ tous diseases because the monocyte is the principal cell involved in granuloma formation.35 Although neutrophil traffic is sensitive to glucocorticoids, neutrophil function appears to be relatively resistant to these agents.35 Whereas most in vivo studies of neutrophil phagocytosis have found no evidence for impairment of phagocytosis or bacterial killing,35 other studies suggest that glucocorticoids induce a generalized phagocytic defect affecting both granulocytes and monocytes. Glucocorticoid therapy retards the disappearance of sensi¬ tized erythrocytes, platelets, and artificial particles from the cir¬ culation.35 This may account for the efficacy of glucocorticoids in the treatment of idiopathic thrombocytopenic purpura and autoimmune hemolytic anemia. Influence on Arachidonic Acid Derivatives. Glucocorticoids inhibit prostaglandin (PG) and leukotriene synthesis by inhibiting the release of arachidonic acid from phospholipids/' This inhibi¬ tion of arachidonic acid release appears to be mediated by the induction of lipocortins, a family of related proteins that inhibit phospholipase A2, which is an enzyme that liberates arachidonic acid from phospholipids (see Chap. 172) 38,39 This mechanism is distinct from the mechanism of action of the nonsteroidal antiin¬ flammatory agents, such as salicylates and indomethacin, which

755

TABLE 78-3. Effects of Glucocorticoids on Inflammatory and Immune Responses in Humans EFFECTS ON LEUKOCYTE MOVEMENT Lymphocytes Circulating lymphocytopenia 4-6 hours after drug administration, sec¬ ondary to redistribution of cells to other lymphoid compartments Depletion of recirculating lymphocytes Selective depletion of T lymphocytes more than B lymphocytes Monocyte-Macrophages Circulating monocytopenia 4-6 hours after drug administration, proba¬ bly secondary to redistribution Inhibition of accumulation of monocyte-macrophages at inflammatory sites Neutrophils Circulating neutrophilia Accelerated release of neutrophils from the bone marrow Blockade of accumulation of neutrophils at inflammatory sites Eosinophils Circulating eosinopenia, probably secondary to redistribution Decreased migration of eosinophils into immediate hypersensitivity skin test sites

EFFECTS ON LEUKOCYTE FUNCTION Lymphocytes Suppression of delayed hypersensitivity skin testing by inhibition of recruitment of monocyte-macrophages Suppression of lymphocyte proliferation to antigens more easily than proliferation to mitogens Suppression of mixed leukocyte reaction proliferation Suppression of T lymphocyte-mediated cytotoxicity (at high concentra¬ tions in vitro) No effect on antibody-dependent cell-mediated cytotoxicity Suppression of spontaneous (natural) cytotoxicity Regulatory effects on helper and suppressor cell populations Monocyte-Macrophages Suppression of cutaneous delayed hypersensitivity by inhibition of lymphokine effect on the macrophage Blockade of Fc receptor binding and function Depression of bactericidal activity Possible decrease in monocyte chemotaxis Neutrophils Possibly no effect on phagocytic and bactericidal capability (controversial) Increase in antibody-dependent cellular cytotoxicity Probable decrease in lysosomal release but little effect on lysosomal membrane stabilization at pharmacologic concentrations Inhibition of chemotaxis only by suprapharmacologic concentrations

EFFECTS ON HUMORAL FACTORS Mild decrease in immunoglobulin levels Decreased reticuloendothelial clearance of antibody-coated cells Decreased synthesis of prostaglandins and leukotrienes Inhibition of plasminogen activator release Potentiation of the actions of catecholamines Antagonism of histamine-induced vasodilation (Adapted from Parrillo JE, Fauci AS. Mechanisms of glucocorticoid action on immune processes. Annu Rev Pharmacol Toxicol 1979;19:179.)

inhibit the cyclooxygenase that converts arachidonic acid to the cyclic endoperoxide intermediates in the PG synthetic pathway; in some tissues, glucocorticoids inhibit cyclooxygenase activity. Thus, the glucocorticoids and the nonsteroidal antiinflammatory agents exert their antiinflammatory effects at two distinct but adja¬ cent loci in the synthetic pathway of arachidonic acid metabolism. Glucocorticoids and nonsteroidal antiinflammatory agents have different spectra of antiinflammatory effects. Some of the thera¬ peutic effects of corticosteroids that are not produced by the non¬ steroidal agents may be related to the inhibition of leukotriene formation.37

756

PART V: THE ADRENAL GLANDS

TABLE 78-4. Adverse Reactions to Glucocorticoids OPHTHALMIC Posterior subcapsular cataracts, increased intraocular pressure and glau¬ coma, exophthalmos CARDIOVASCULAR Hypertension Congestive heart failure in predisposed patients GASTROINTESTINAL Peptic ulcer disease, pancreatitis ENDOCRINE-METABOLIC Truncal obesity, moon facies, supraclavicular fat deposition, posterior cer¬ vical fat deposition (buffalo hump), mediastinal widening (lipomato¬ sis), hepatomegaly caused by fatty liver Acne, hirsutism or virilism, erectile dysfunction, menstrual irregularities Suppression of growth in children Hyperglycemia; diabetic ketoacidosis; hyperosmolar, nonketotic diabetic coma; hyperlipoproteinemia Negative balance of nitrogen, potassium, and calcium Sodium retention, hypokalemia, metabolic alkalosis Secondary adrenal insufficiency MUSCULOSKELETAL Myopathy Osteoporosis, vertebral compression fractures, spontaneous fractures Aseptic necrosis of femoral and humeral heads and other bones NEUROPSYCHIATRIC Convulsions Benign intracranial hypertension (pseudotumor cerebri) Alterations in mood or personality Psychosis DERMATOLOGIC Facial erythema, thin fragile skin, petechiae and ecchymoses, violaceous striae, impaired wound healing IMMUNE, INFECTIOUS Suppression of delayed hypersensitivity Neutrophilia, monocytopenia, lymphocytopenia, decreased inflammatory responses Susceptibility to infections (Data from Axelrod L. Adrenal corticosteroids. In: Miller RR, Greenblatt DJ, eds. Handbook of drug therapy. New York: Elsevier North-Holland, 1979:809; and Axelrod L. Glucocorticoids. In: Kelley WN, Harris ED Jr, Ruddy S, Sledge CB, eds. Textbook of rheumatology, 4th ed. Philadelphia: WB Saunders, 1993:779.)

SIDE EFFECTS The side effects of glucocorticoids include the diverse manifesta¬ tions of Cushing syndrome and HPA suppression (Table 78-4).40 Iatrogenic Cushing syndrome differs from endogenous Cushing syndrome in several respects: hypertension, acne, menstrual dis¬ turbances, male erectile dysfunction, hirsutism or virilism, striae, purpura, and plethora are more common in endogenous Cushing syndrome; benign intracranial hypertension, glaucoma, posterior subcapsular cataract, pancreatitis, and aseptic necrosis of bone are virtually unique to iatrogenic Cushing syndrome; and obesity, psychiatric symptoms, and poor wound healing have nearly equal frequency in both.40-41 These differences may be explained as follows. When Cushing syndrome is caused by exogenous glu¬ cocorticoids, ACTH secretion is suppressed. In spontaneous, ACTH-dependent Cushing syndrome, the elevated ACTH output causes bilateral adrenal hyperplasia. In the former circumstance, the secretion of adrenocortical androgens and mineralocorticoids is not increased. Conversely, when ACTH output is elevated, the secretion of adrenal androgens and mineralocorticoids may be increased.1 The augmented secretion of adrenal androgens may account for the higher prevalence of virilism, acne, and menstrual irregularities in the endogenous form of Cushing syndrome, and the enhanced production of mineralocorticoids may explain the higher prevalence of hypertension.1

Some of the complications that are virtually unique to iatro¬ genic Cushing syndrome arise after the prolonged use of large doses of glucocorticoids. Examples are benign intracranial hypertension, posterior subcapsular cataract, and aseptic necro¬ sis of bone.1 Although the association of glucocorticoid therapy and pep¬ tic ulcer disease is controversial,42^7 glucocorticoids appear to increase the risk of peptic ulcer disease and also gastrointestinal hemorrhage (see Chap. 204).45-46 The magnitude of the associa¬ tion between glucocorticoid therapy and these complications is small and is related to the total dose and duration of therapy.42-45 The risk of peptic ulcer disease and related gastrointestinal problems is increased by the concurrent use of glucocorticoids and nonsteroidal antiinflammatory drugs.48-49 Glucocorticoid therapy, especially daily therapy, may sup¬ press the immune response to skin tests for tuberculosis. When possible, tuberculin skin testing is advisable before the initia¬ tion of glucocorticoid therapy. Routine isoniazid prophylaxis probably is not indicated for corticosteroid-treated patients, even for those with positive tuberculin skin test results.50 At similar doses, some patients respond to and experience side effects of glucocorticoids more readily than do others. Varia¬ tions in responsiveness to glucocorticoids may be a consequence of drug interactions or of variations in the severity of the under¬ lying disease. Alterations in bioavailability probably do not account for variations in the therapeutic response to glucocorti¬ coids. In patients who experience side effects, the metabolic clearance rate of prednisolone and the volume of distribution are lower10-51 and the circulating T1/2 is longer51 than in those who do not experience side effects. Impaired renal function may contrib¬ ute to a decrease in the clearance of prednisolone and an increase in the prevalence of Cushingoid features.52 Patients who have a Cushingoid habitus while taking prednisone have higher endogenous plasma cortisol levels than do those without this complication, perhaps because of resistance of the HPA axis to suppression by exogenous glucocorticoids.53 Variations in the effectiveness of corticosteroids may be the result of altered cellular responsiveness to the drugs.54-57 In patients with primary open-angle glaucoma, exogenous gluco¬ corticoids produce a more pronounced rise of intraocular pressure54; a greater suppression of the 8:00 a.m. plasma corti¬ sol level when dexamethasone, 0.25 mg, is administered the previous evening at 11:00 p.m.56; and greater suppression of phytohemagglutinin-induced lymphocyte transformation55-57 than in normal persons. Primary open-angle glaucoma is rela¬ tively common. These findings suggest that a distinct subpopu¬ lation of patients are hyperresponsive to glucocorticoids and that this sensitivity is genetically determined (see Chap. 215). PREVENTION OF SIDE EFFECTS Increasingly, the issues of concern to physicians and patients with respect to glucocorticoid therapy are not only HPA suppres¬ sion but long-term complications such as glucocorticoid-induced osteoporosis and Pneumocystis carinii pneumonia. Of course, the risk of many complications can be reduced by the use of the blu¬ est possible dose of a glucocorticoid for the shortest possible period, by the use of regional or topical rather than systemic steroids, and by the use of alternate-day corticosteroid therapy. In addition, pharmaco¬ logic interventions to prevent specific complications such as bone disease and P. carinii pneumonia are now widely used. Osteoporosis. The majority of patients who receive long¬ term glucocorticoid therapy will develop low bone mineral density. By some estimates, more than one-fourth of these patients will sustain osteoporotic fractures.58 The prevalence of vertebral fractures in asthmatic patients on glucocorticoid ther¬ apy for at least a year is 11%.58 Patients with rheumatoid arthri¬ tis who are treated with glucocorticoids have an increased incidence of fractures of the hips, ribs, spine, legs, ankles, and feet.58 Skeletal wasting occurs most rapidly during the first year

Ch. 78: Corticosteroid Therapy of therapy. Trabecular bone is affected more than cortical bone. The effects on the skeleton are related to the cumulative dose and duration of treatment.58 Alternate-day glucocorticoid ther¬ apy does not reduce the risk of osteopenia. Inhaled steroids have been associated with bone loss. The pathogenesis of glucocorticoid-induced osteoporosis involves several different mechanisms.58 Glucocorticoids decrease intestinal absorption of calcium and phosphate by vitamin Dindependent mechanisms. Urinary calcium excretion is increased, possibly as a result of direct effects on renal tubular calcium reabsorption. These changes may lead to secondary hyperparathyroidism in at least some patients. Glucocorticoids reduce sex hormone production. This may be a direct effect by decreasing gonadal hormone release. It may also be indirect by reducing ACTH secretion and adrenal androgen production. Also, inhibition of luteinizing hormone secretion can result in decreased estrogen and testosterone production by the gonads. Glucocorticoids also have an inhibitory effect on the prolifera¬ tion of osteoblasts, attachment of osteoblasts to matrix, and the synthesis of type I collagen and noncollagenous proteins by osteoblasts. The evaluation of a patient should emphasize medical risk factors for osteoporosis, including inadequate dietary calcium and vitamin D intake, alcohol consumption, smoking, meno¬ pause, and any history of infertility or impotence suggesting hypogonadism in males. Attention should also be devoted to the possible presence of thyrotoxicosis, overtreatment with thyroid medication, renal osteodystrophy, multiple myeloma, osteomala¬ cia, or primary hyperparathyroidism. When appropriate, labora¬ tory studies should be ordered for evaluation of these disorders. When glucocorticoid therapy will be administered for more than a few months, it is reasonable to obtain a baseline measurement of bone mineral density using dual energy x-ray absorptiometry. In general, all patients should receive calcium and vitamin D supplementation to correct any nutritional deficiency. Calcium therapy alone is associated with rapid rates of spinal bone loss and offers only partial protection from this loss. There is no evi¬ dence that the combination of calcium and vitamin D com¬ pletely prevents bone loss caused by glucocorticoids.59 Calcitriol and bisphosphonates, specifically alendronate and etidronate, are effective in the prevention of bone loss.60-63 If calcitriol is used, careful follow-up determinations of serum levels is necessary. Hypogonadotropic men should receive tes¬ tosterone therapy; hormone replacement therapy should be considered for postmenopausal women. Patients should be educated about the risks and the consequences of osteoporosis and the factors in their own lives that may contribute. Because glucocorticoids also affect muscle mass and function, patients should be advised about exercises for maintaining muscle strength. Pneumocystis carinii Pneumonia. Glucocorticoids pre¬ dispose patients to infections of many varieties. Until recently, prophylaxis against infections for patients treated with gluco¬ corticoids was limited to patients receiving transplantation of organs, who also receive other forms of immunosuppression. Currently, prophylaxis for patients with other disorders who are treated with glucocorticoids is being used, particularly for P. carinii pneumonia.64'65 In a series of 116 patients without acquired immunodefi¬ ciency syndrome (AIDS) who experienced a first episode of P. carinii pneumonia between 1985 and 1991, 105 (90.5%) had received glucocorticoids within 1 month before the diagnosis of P. carinii pneumonia was established.64 The median daily dose was equivalent to 30 mg prednisone; 25% of the patients had received as little as 16 mg daily. The median duration of gluco¬ corticoid therapy was 12 weeks before the development of the pneumonia. In 25% of the patients, P. carinii pneumonia devel¬ oped after 8 weeks or less of glucocorticoid therapy. However, the attack rate in patients with primary or metastatic central nervous system tumors who received glucocorticoid therapy

757

was 1.3% and may be lower in other conditions.65 Also, prophy¬ lactic therapy may produce side effects. Some physicians recommend prophylaxis (e.g., with trimethoprim-sulfa, one double-strength tablet a day) for patients with impaired immune competence conferred by che¬ motherapy, transplantation, or an inflammatory disorder who have received prednisone 20 mg or more per day for more than 1 month. No controlled studies with such prophylaxis in steroid-treated patients are available. Among patients undergo¬ ing bone marrow or organ transplantation at the Mayo Clinic from 1989 to 1995, no cases of P. carinii pneumonia were detected in those who received adequate chemoprophylaxis. WITHDRAWAL FROM GLUCOCORTICOIDS

The symptoms associated with glucocorticoid withdrawal include anorexia, myalgia, nausea, emesis, lethargy, headache, fever, desquamation, arthralgia, weight loss, and postural hypotension. Many of these symptoms can occur with normal plasma glucocor¬ ticoid levels and in patients with normal responsiveness to con¬ ventional tests of the HPA system.66-67 These patients may have abnormal responses to a more sensitive test using 1 pg of a-1-24 ACTH rather than the conventional 250-pg dose.68-69 Because glu¬ cocorticoids inhibit PG production and because many of the fea¬ tures of the corticosteroid withdrawal syndrome can be produced by PGs such as PGE2 and PGI2, this syndrome may be caused by a sudden increase in PG production after the withdrawal of exoge¬ nous corticosteroids. The corticosteroid withdrawal syndrome may contribute to psychologic dependence on glucocorticoid treatment and to difficulties in withdrawing such therapy.

SUPPRESSION OF THE HYPOTHALAMICPITUITARY-ADRENAL SYSTEM DEVELOPMENT OF HYPOTHALAMIC-PITUITARY-ADRENAL SUPPRESSION

Few well-documented cases of acute adrenocortical insuffi¬ ciency have been reported after prolonged glucocorticoid ther¬ apy and none have been reported after ACTH therapy.1 After the introduction of ACTH and glucocorticoids into clinical practice in the late 1940s, patients were described in whom shock was attributed to adrenocortical insufficiency induced by these agents, but biochemical evidence of adrenocortical insuf¬ ficiency was not available to substantiate the diagnosis.1 Prolonged hypotension, or even an apparent response of hypotension to intravenous hydrocortisone, is not a reliable means of assessing adrenocortical function. It must be demon¬ strated simultaneously that the plasma cortisol level is lower than the values found in normal persons experiencing, a compa¬ rable degree of stress. When testing for plasma cortisol levels became available in the early 1960s, three cases were described in which these criteria were met. The paucity of reports may reflect the fact that acute adrenocortical insufficiency after glu¬ cocorticoid therapy is uncommon in properly treated patients, and that physicians may be reluctant to report such events. The minimal duration of glucocorticoid therapy that can produce HPA suppression must be ascertained from studies of adrenocortical weight and adrenocortical responsiveness to provocative tests.1-2 Any patient who has received a glucocorti¬ coid in dosages equivalent to 20 to 30 mg per day of prednisone for more than 5 days should be suspected of having HPA sup¬ pression.1-2 If the dosages are closer to but above the physio¬ logic range, 1 month is probably the minimal interval.1-2 The stress of general anesthesia and surgery is not hazard¬ ous to patients who have received only replacement doses (no more than 25 mg hydrocortisone, 5 mg prednisone, 4 mg triam¬ cinolone, or 0.75 mg dexamethasone), provided the corticoster¬ oid is given early in the day. If doses of this size are given late in the day, suppression may occur as a result of inhibition of the diurnal surge of ACTH release.

758

PART V: THE ADRENAL GLANDS

TABLE 78-5. Assessment of Hypothalamic-Pituitary-Adrenal (HPA) Function in Patients Treated with Glucocorticoids METHOD Withhold exogenous corticosteroids for 24 hr Give cosyntropin [synthetic cxl-24 ACTH] 250 gg as intravenous bolus or intramuscular injection Obtain plasma cortisol level 30 or 60 min after administration of ACTH Performance of the test in the morning is customary but not essential

INTERPRETATION Normal response: Plasma cortisol level >18 pg/dL at 30 or 60 minutes after ACTH administration Note: Traditional recommendations also specify an increment above baseline of 7 gg/dL at 30 minutes or 11 pg/dL at 60 minutes and a doubling of the baseline value at 60 minutes. These end-points are valid in normal, unstressed subjects but are frequently misleading in ill patients with a normal HPA axis, in whom stress may raise the baseline plasma cortisol level by an increase in endogenous ACTH levels. (From Axelrod L. Glucocorticoids. In: Kelley WN, Harris ED Jr, Ruddy S, Sledge CB, eds. Textbook of rheumatology, 4th ed. Philadelphia: WB Saunders, 1993:779.)

ASSESSMENT OF HYPOTHALAMIC-PITUITARY-ADRENAL FUNCTION

logic range. These supraphysiologic levels may produce a normal plasma cortisol level in patients with partial adrenocor¬ tical insufficiency. Nevertheless, the low-dose short ACTH test has not yet replaced the conventional-dose short ACTH test. The lower limit of the normal range for the low-dose ACTH test has not yet been defined.69 Also, there are no commercially avail¬ able preparations of ACTH available for direct use in the lowdose short ACTH test. The injection for the low-dose short ACTH test must be prepared by dilution, which is a source of inconvenience and possible error. Insulin-induced hypoglycemia may be hazardous (especially in patients with cardiac or neuro¬ logic disease), and the symptoms may be uncomfortable. This procedure is more time-consuming and more costly than the ACTH test because more cortisol values must be determined. The measurement of plasma cortisol levels before and after the administration of corticotropin-releasing hormone also has been recommended.71 This test also is longer and more expen¬ sive than the ACTH test and has not been compared to a physi¬ ologic stress such as anesthesia and surgery. It offers no clear advantage over the ACTH test. CORTICOTROPIN AND THE HYPOTHALAMIC-PITUITARYADRENAL SYSTEM

When HPA suppression is suspected, the physician may wish to assess the integrity of the HPA system. A test of HPA reserve is indicated only when the result will modify therapy. In practice, this applies to patients who may need an increase in the cortico¬ steroid dosage to cover a stressful event (such as general anesthe¬ sia and surgery) and to patients in whom withdrawal of glucocorticoid therapy is contemplated. In the latter group, a test of the HPA axis usually is indicated only when the glucocorti¬ coid dosage has been reduced to replacement levels, for example, 5 mg prednisone daily (or an equivalent dosage of another gluco¬ corticoid). In stable patients receiving prolonged glucocorticoid therapeutic regimens, frequent tests of HPA reserve function are not indicated. For example, it is not necessary to test before each reduction in dosage during tapering of the steroid regimen. The responsiveness of the HPA system may change as corticosteroid therapy continues, and repeated testing is costly. The short ACTH test is a useful guide to the presence or absence of HPA suppression in patients treated with glucocorti¬ coids (Table 78-5). Although this test assesses directly only the adrenocortical response to ACTH, it is an effective measure of the integrity of the HPA axis. Because hypothalamic-pituitary

Pharmacologic doses of ACTH cause elevated cortisol secretory rates and increased plasma cortisol levels. The elevated plasma cortisol levels might be expected to suppress ACTH release. Actually, there is no evidence of clinically significant hypotha¬ lamic-pituitary suppression in patients who have received ACTH therapy.1 The failure of ACTH to suppress HPA function is not explained by the dose of ACTH used, the frequency of injection, the time of administration, or the plasma cortisol pat¬ tern after ACTH administration. Alternatively, it is possible that the hyperplastic and overactive adrenal cortex that results from ACTH therapy compensates for hypothalamic or pituitary sup¬ pression. Although threshold adrenocortical sensitivity to ACTH is not changed in patients who have received daily ACTH therapy, there may be altered adrenocortical responsive¬ ness to ACTH in the physiologic range. Moreover, the normal response of the plasma cortisol level in patients treated with ACTH may be preserved, at least in part, because ACTH treat¬ ment reduces the rate of ACTH secretion but not the total amount secreted, whereas glucocorticoids reduce both the rate of secretion and the total amount secreted.72

function returns before adrenocortical function during recovery from HPA suppression, a normal adrenocortical response to ACTH in this setting implies that hypothalamic-pituitary function also is normal.

RECOVERY FROM HYPOTHALAMIC-PITUITARY-ADRENAL SUPPRESSION

This rationale is supported by direct observation. Thus, the maximal response of the plasma cortisol level to ACTH corre¬ sponds to the maximal plasma cortisol level observed during the induction of general anesthesia and surgery in patients who have received glucocorticoid therapy.1-2 A normal response to ACTH before surgery is unlikely to be followed by markedly impaired secretion of cortisol during anesthesia and surgery in corticosteroid-treated patients. An abnormal response to ACTH is a necessary but not a sufficient condition for the diagnosis of adrenal insufficiency in glucocorticoid-treated patients who undergo surgery; some patients with an abnormal response to ACTH tolerate surgery without glucocorticoid treatment.70 Moreover, hypotension in the operative or postoperative period in patients who have been treated previously with glucocorti¬ coid therapy is often a result of other causes, such as volume depletion and reactions to anesthetic medication. The hypoten¬ sion often responds to treatment of these factors. Other tests of HPA function generally are not indicated. The low-dose (1 pg) short ACTH test is more sensitive than the conventional-dose ACTH test in patients who have been treated with glucocorticoids.68-683 The conventional dose of ACTH used in the short ACTH test (and other ACTH tests) pro¬ duces circulating ACTH levels that are far above the physio¬

During the recovery from HPA suppression, hypothalamicpituitary function returns before adrenocortical function.1-2-73 Twelve months must elapse after the withdrawal of large doses of glucocorticoids given for a prolonged period before HPA function, including responsiveness to stress, returns to nor¬ mal.1'2-7? Conversely, recovery from HPA suppression that has been induced by a brief course of corticosteroids (i.e., 25 mg prednisone twice daily for 5 days) occurs within 5 days.74 Patients with mild suppression of the HPA axis (i.e., normal basal plasma and urine corticosteroid levels but diminished responses to ACTH and insulin-induced hypoglycemia) resume normal HPA function more rapidly than do those with severe depression of the HPA axis (i.e., low basal plasma and urine corticosteroid levels and diminished responses to ACTH and insulin-induced hypoglycemia). The time course of recov¬ ery correlates with the total duration of previous glucocorticoid therapy and the total previous corticosteroid dose. Neverthe¬ less, in an individual patient, it is not possible to predict the duration of recovery from a course of glucocorticoid therapy at supraphysiologic doses lasting more than a few weeks. Conse¬ quently, persistence of HPA suppression should be suspected for 12 months after such treatment. The recovery interval after suppression of the contralateral adrenal cortex by the products

Ch. 78: Corticosteroid Therapy of an adrenocortical tumor may exceed 12 months. The recov¬ ery from HPA suppression that is induced by exogenous gluco¬ corticoids may be more rapid in children than in adults.

759

-1 CLINICALLY APPARENT J DISEASE ACTIVITY

WITHDRAWAL OF PATIENTS FROM GLUCOCORTICOID THERAPY RISKS OF WITHDRAWAL The decision to discontinue glucocorticoid therapy provokes apprehension among physicians. Tire deleterious consequences of such an action include precipitation of adrenocortical insuffi¬ ciency, development of the corticosteroid withdrawal syndrome, or exacerbation of the underlying disease. Adrenocortical insuffi¬ ciency after the withdrawal of glucocorticoids is justly feared. The likelihood of precipitating the underlying disease depends on the activity and natural history of the illness in question. When there is any possibility that the underlying illness will flare up, the glu¬ cocorticoid should be withdrawn gradually, over an interval of weeks to months, with frequent reassessment of the patient. ■TREATMENT OF PATIENTS WITH HYPOTHALAMIC-PITUITARYADRENAL SUPPRESSION No proven means exists for hastening a return to normal HPA function once inhibition has resulted from glucocorticoid therapy. The use of ACTH does not prevent or reverse the development of glucocorticoid-induced adrenal insufficiency. Conversion to an alternate-day schedule permits but does not accelerate recovery. In children, alternate-day glucocorticoid therapy actually may delay recovery. The recovery from corticosteroid-induced adrenal insuffi¬ ciency is time dependent and spontaneous. The rate of recovery is determined not only by the doses given when the corticoster¬ oids are being tapered, but also by the doses administered dur¬ ing the initial phase of treatment, before tapering is commenced. During the course of recovery, small doses of hydrocortisone (10-20 mg) or prednisone (2.5-5.0 mg) given in the morning may alleviate the withdrawal symptoms. Recovery of HPA function still occurs when small doses of glucocorticoids are administered in the morning. The possibility cannot be excluded, however, that small doses of glucocorticoids given in the morning retard the rate of recovery from HPA suppression.

ALTERNATE-DAY GLUCOCORTICOID THERAPY Alternate-day glucocorticoid therapy is defined as the adminis¬ tration of a short-acting glucocorticoid with no appreciable mineralocorticoid effect (i.e., prednisone, prednisolone, or methylprednisolone) once every 48 hours in the morning, at about 8:00 a.m. The purpose of this approach is to minimize the adverse effects of glucocorticoids while retaining the therapeu¬ tic benefits. The original basis for this schedule was the hypothe¬ sis that the antiinflammatory effects of glucocorticoids persist longer than do the undesirable metabolic effects.75"77 This hypothesis is not supported by observations of the duration of corticosteroid effects. A second hypothesis emphasizes that intermittent rather than continuous administration produces a cyclic, although not diurnal, pattern of glucocorticoid levels in the circulation and within the target cells that simulates the nor¬ mal diurnal cycle.34 This may prevent the development of Cushing syndrome and HPA suppression while providing ther¬ apeutic benefit. Because the full expression of a disease fre¬ quently occurs only when the level of inflammatory activity is elevated over a protracted period, the intermittent administra¬ tion of a glucocorticoid may be sufficient to shorten the interval during which the disorder develops without interruption and thereby to prevent the level of disease activity from becoming apparent clinically (Fig- 78-2).34 The duration of action of the glucocorticoid is important here. The selection of prednisone, prednisolone, and methylprednisolone as the agents of choice

TIME (DAYS)

FIGURE 78-2. The effect of glucocorticoid administration on the activ¬ ity of the underlying disease. A divided daily dosage schedule may be necessary initially in some disorders. When the disease is controlled, or from the start of therapy in certain diseases, altemate-day therapy may be effective. (From Fauci AS, Dale DC, Balow JE. Glucocorticosteroid therapy: mechanisms of action and clinical considerations. Ann Intern Med 1976; 84:304.)

for alternate-day therapy and of 48 hours as the appropriate interval between doses has an empiric basis. It has been found that intervals of 36, 24, and 12 hours were accompanied by adrenal suppression, and that an interval of 72 hours was thera¬ peutically ineffective when prednisone (and, occasionally, tri¬ amcinolone) was used.77 An interval of 48 hours is optimal. ALTERNATE-DAY GLUCOCORTICOID THERAPY AND MANIFESTATIONS OF CUSHING SYNDROME An alternate-day regimen can prevent or ameliorate the mani¬ festations of Cushing syndrome.1'2 The susceptibility to infec¬ tions that characterizes Cushing syndrome may be alleviated. Patients have been described in whom refractory infections appeared to clear after conversion from daily to alternate-day regimens. In addition, there is a low frequency of infections in patients receiving alternate-day therapy. Children treated with alternate-day steroid therapy regain or retain tonsillar and peripheral lymphoid tissue. The available information strongly suggests that alternate-day regimens are associated with a lower incidence of infections than are daily regimens, but it does not firmly establish this point. Host defense mechanisms have been studied in patients receiving alternate-day therapy. Patients maintained on such schedules who have been studied on the days they do not take the medication have normal blood neutrophil and monocyte counts, normal cutaneous inflammatory responses, and normal neutrophil T1/2s. Patients receiving daily therapy, however, demonstrate neutrophilia, monocytopenia, decreased cutane¬ ous neutrophil and monocyte inflammatory responses, and pro¬ longation of the neutrophil T1/2. Patients studied on the days they do not receive treatment do not have the lymphocytopenia observed in patients who receive daily therapy. Monocyte cellu¬ lar function is normal in patients receiving alternate-day treat¬ ment at 4 hours and at 24 hours after a dose. Intermittently normal leukocyte kinetics, preservation of delayed hypersensi¬ tivity, and preservation of monocyte cellular function may explain the apparently reduced susceptibility to infection of patients receiving alternate-day therapy.78-80 EFFECTS OF ALTERNATE-DAY GLUCOCORTICOID THERAPY ON HYPOTHALAMIC-PITUITARY-ADRENAL RESPONSIVENESS Patients receiving alternate-day glucocorticoid therapy may have some suppression of basal corticosteroid levels, but they have normal or nearly normal responsiveness to provocative

760

PART V: THE ADRENAL GLANDS

tests such as the corticotropin-releasing hormone stimulation test, the ACTH stimulation test, insulin-induced hypoglycemia, and the metyrapone test.1'2-81 They have less suppression of HPA function than do patients receiving daily therapy. EFFECTS OF ALTERNATE-DAY THERAPY ON THE UNDERLYING DISEASE Alternate-day glucocorticoid therapy is as effective, or nearly as effective, in controlling diverse disorders as daily therapy in divided doses.1'2 This approach has provided apparent benefit in patients with the following disorders: childhood nephrotic syn¬ drome, adult nephrotic syndrome, membranous nephropathy, renal transplantation, mesangiocapillary glomerulonephritis, lupus nephritis, ulcerative colitis, rheumatoid arthritis, acute rheumatic fever, myasthenia gravis, Duchenne muscular dystro¬ phy, dermatomyositis, idiopathic polyneuropathy, asthma, Sjogren syndrome, sarcoidosis, alopecia areata and other chronic dermatoses, and pemphigus vulgaris. Prospective, controlled studies demonstrate the efficacy of alternate-day therapy in membranous nephropathy and renal transplantation. The role of alternate-day therapy in giant cell arteritis is controversial.82-84 USE OF ALTERNATE-DAY THERAPY Because alternate-day therapy can prevent or ameliorate the mani¬ festations of Cushing syndrome, can avert or permit recovery from HPA suppression, and is as effective (or nearly as effective) as contin¬ uous therapy, patients for whom long-term glucocorticoid administration is indicated should be placed on such programs whenever possible. Nevertheless, physicians sometimes are reluctant to use alternate-day schedules, often because of an unsuccessful experience. Many efforts fail because of lack of familiarity with the indications for and use of such therapy. The benefits of alternate-day glucocorticoid therapy are demonstrable only when corticosteroids are used for a pro¬ longed period. There is no reason to use an alternate-day sched¬ ule when the anticipated duration of therapy is less than several weeks. Alternate-day therapy may not be necessary or appropriate during the initial stages of therapy or during exacerbation of the underlying disease. Nevertheless, patients with many chronic disorders have been treated with an alternate-day reg¬ imen as initial therapy with apparent benefit.1'2 In patients with rheumatoid arthritis, it appears to be easier to establish treatment with alternate-day corticosteroids than to convert from daily therapy. Physicians treating recipients of renal transplants initially use daily therapy and then convert to an alternate-day schedule. Alternate-day therapy may be hazardous in the presence of adrenocortical insufficiency of any cause because patients are unprotected against glucocorticoid insufficiency during the last 12 hours of the 48-hour cycle. In patients who have been taking glucocorticoids for more than a brief period, or in those who may have adrenal insufficiency on another basis, the adequacy of HPA function should be determined before the initiation of an alternate-day program. It may be possible to surmount this obstacle by giving a small dose of a short-acting glucocorticoid (i.e., 10 mg hydrocortisone) in the afternoon of the second day; this approach has not been studied systematically. Alternate-day glucocorticoid therapy may fail to prevent or ameliorate the manifestations of Cushing syndrome or HPA suppression if a short-acting glucocorticoid is not used, or if it is used incorrectly. For example, the use of prednisone four times a day on alternate days may be less successful than the use of the same total dose once every 48 hours. An abrupt alteration from daily to alternate-day therapy should be avoided. First, the prolonged use of daily-dose gluco¬ corticoids may have caused HPA suppression. In addition, patients with normal HPA function may experience withdrawal symptoms and have an exacerbation of the underlying disease.

No program of conversion from continuous therapy to alter¬ nate-day therapy has been shown to be optimal. One approach is to reduce the frequency of drug administration until the total dose for each day is given in the morning, and then to increase the dosage gradually on the first day of each 2-day period and to decrease the dosage on the second day. Another approach is to double the dosage on the first day of each 2-day cycle, to give this as a single morning dose if possible, and then to taper the dosage gradually on the second day.85 It is not clear how often changes in dosage should be made with any approach. This depends on many variables, including the underlying disease involved, the duration of previous glucocorticoid therapy, the personality of the patient, and the physician's ability to use adjunctive therapy. Nonetheless, the conversion should be made as quickly as the patient can tolerate it. If adrenal insuffi¬ ciency, the corticosteroid withdrawal syndrome, or an exacer¬ bation of the underlying disease develops, the previously effective regimen should be reinstituted and then tapered more gradually. Occasionally, it is necessary to resume full daily dos¬ ages temporarily. An absolute change of dosage represents a larger percentage change in dosage at small total daily doses than at large total daily doses. Changes in the dosage should be about 10 mg prednisone (or equivalent) at total daily doses of more than 30 mg, 5 mg at total doses of more than 20 mg, and 2.5 mg at lower doses. The interval between changes in the dos¬ age may be as short as 1 day or as long as many weeks. Optimal results from alternate-day glucocorticoid therapy may not be achieved because of failure to use supplemental therapy for the underlying disorder. Conservative (nongluco¬ corticoid) therapy often is used until a glucocorticoid is initi¬ ated, at which time these less toxic therapeutic measures are ignored. Adjunctive therapeutic measures may facilitate the use of the lowest possible corticosteroid dose. With alternate-day therapy, these measures especially should be used during the end of the second day, when symptoms may be prominent. Supplemental therapy may be especially helpful in disorders in which patients are likely to experience symptoms of the disease on the day off therapy, such as asthma and rheumatoid arthri¬ tis. In illnesses in which disabling symptoms are less likely to appear on the alternate day, such as the childhood nephrotic syndrome, less difficulty may be encountered. Alternate-day therapy may fail because of failure to inform patients about the purposes of this regimen. Because glucocor¬ ticoids may induce euphoria, patients may be reluctant to accept modification of a schedule of frequent doses. A careful explanation about the risks of glucocorticoid excess, attuned to patients' intellectual and emotional ability to comprehend, enhances the prospects of success.

DAILY SINGLE-DOSE GLUCOCORTICOID THERAPY Sometimes, alternate-day therapy fails because patients experi¬ ence symptoms of the underlying disease during the last few hours of the second day. In these situations, single-dose gluco¬ corticoid therapy may be of value.1'2 This regimen appears to be as effective as divided daily doses in controlling such underly¬ ing diseases as rheumatoid arthritis, systemic lupus erythema¬ tosus, polyarteritis, and proctocolitis. In giant cell arteritis, a daily dose in the morning is nearly as effective as daily therapy in divided doses.82 Daily single-dose therapy reduces the likeli¬ hood that HPA suppression will develop. The manifestations of Cushing syndrome, however, probably are not prevented or ameliorated by a daily single-dose regimen.

GLUCOCORTICOIDS OR CORTICOTROPIN? Disorders that respond to glucocorticoid therapy also respond to ACTH therapy if the adrenal cortex is normal. There is no evidence, however, that ACTH is superior to glucocorticoids for the treatment of any disorder when comparable doses are

Ch. 78: Corticosteroid Therapy used.1'2'86 Hydrocortisone and ACTH, given intravenously in pharmacologically equivalent doses (determined by plasma cortisol levels and urinary corticosteroid excretion rates), are equally effective in the treatment of inflammatory bowel dis¬ ease.87 Similarly, there is no apparent difference in the effective¬ ness of prednisone and ACTH for the treatment of infantile spasms.88 Because ACTH does not appear to offer any thera¬ peutic advantage, glucocorticoids are preferable for therapeutic purposes: they can be administered orally, the dose can be regu¬ lated precisely, their effectiveness does not depend on adreno¬ cortical responsiveness (an important consideration in patients who have been treated with glucocorticoids), and they produce a lower frequency of certain side effects such as acne, hyperten¬ sion, and increased pigmentation.1'2 If alternate-day therapy cannot be used, ACTH might appear to be preferable because it does not suppress the HPA axis. This benefit usually is out¬ weighed by the advantages of glucocorticoids and by the fact that daily injections of ACTH are not superior to single daily doses of short-acting glucocorticoids; in both cases, HPA sup¬ pression is unlikely to result, but Cushing syndrome is not pre¬ vented. In life-threatening situations, glucocorticoids are indicated because maximal blood levels are obtained immedi¬ ately after intravenous administration, whereas with ACTH infusion, the plasma cortisol level rises to a plateau over several hours. The principal indication for ACTH continues to be the assessment of adrenocortical function.

DOSAGE ANTIINFLAMMATORY OR IMMUNOSUPPRESSIVE THERAPY The glucocorticoid dosage required for antiinflammatory or immunosuppressive therapy is variable, and depends on the disease under treatment. In general, the dosage ranges from just above that needed for long-term replacement therapy up to 60 to 80 mg prednisone or its equivalent daily. Although much larger dosages sometimes are recommended for diseases such as asthma, systemic lupus erythematosus, and cerebral edema, controlled studies have not established the need for such large amounts of medication. The role of massive doses of cortico¬ steroids in asthma is controversial.89'90 Most studies report no advantage of high-dose therapy (e.g., more than 60-80 mg prednisone per day). Many physicians use intravenous pulse therapy (e.g., 1 g per day of methylprednisolone intravenously for 3 consecutive days) for severe manifestations of systemic lupus erythematosus, rapidly progressive glomerulonephritis, or other entities. There are no controlled studies that compare the results of pulse therapy with 60 to 80 mg per day of pred¬ nisone, however. Thus, the superiority of pulse therapy has not been demonstrated.91'92 When alternate-day therapy is used, the dosage is variable and depends on the disease under treatment. It may range from just above that needed for long-term replacement therapy to 150 mg prednisone every other day. PERIOPERATIVE MANAGEMENT Traditional doses of glucocorticoids recommended for periop¬ erative coverage in patients treated with steroids (e.g., 100 mg hydrocortisone intravenously every 8 hours or 20 mg methyl¬ prednisolone intravenously every 8 hours on the day of sur¬ gery, with a gradual taper over subsequent days) are arbitrary and have no empirical basis.70 A study in cynomolgus monkeys explored the doses required to prevent postoperative hypoten¬ sion.93 Bilateral adrenalectomies were performed in the experi¬ mental animals, and replacement doses of glucocorticoids were given for 4 months. The animals were then divided into three groups, given normal, one-tenth normal, or 10 times the normal replacement doses of glucocorticoids. A cholecystectomy was performed on each animal under these conditions. The animals that received one-tenth normal replacement doses had an

761

increased mortality rate, decreased peripheral vascular resis¬ tance, and hypotension. The group that received normal replacement doses of glucocorticoids had no more hypotension or postoperative complications than did the group receiving 10 times the replacement dose. A double-blind study in patients provided similar results.94 The investigators studied patients who had taken at least 7.5 mg prednisone a day for several months and had an abnormal response to an ACTH test. All patients received their usual daily dose of prednisone on the day of surgery. One group of 12 patients received perioperative injections of saline. The other group of 6 patients received hydrocortisone in the saline. There was no significant difference in outcome between the groups in this small study. It appears that patients with secondary adrenal insufficiency resulting from glucocorticoid therapy do not experience hypotension or tachycardia when given only their usual daily dose of steroids for surgical procedures such as joint replacements and abdomi¬ nal operations. Based on an analysis of the literature, an interdisciplinary group suggests the use of variable doses, depending on the magnitude of the surgical stress 70 For minor surgical stress (e.g., an inguinal herniorrhaphy), the glucocorticoid target dose would be 25 mg hydrocortisone or equivalent. For moderate sur¬ gical stress (e.g., a lower extremity revascularization or total joint replacement), the target would be 50 to 75 mg hydrocortisone or equivalent. This might constitute continuation of the patient's usual dose of prednisone (i.e., 10 mg a day) and 50 mg hydro¬ cortisone intravenously intraoperatively. For major surgical stress (e.g., esophagogastrectomy or cardiopulmonary bypass), the patient might receive his or her usual steroid dose (e.g., 40 mg prednisone or the parental equivalent preoperatively within 2 hours of surgery) and 50 mg hydrocortisone intravenously every 8 hours after the initial dose for the first 48 to 72 hours. Corticosteroid therapy should not be tapered inadvertently to a dosage below that known to control the underlying disease. In patients with primary adrenocortical insufficiency, hydro¬ cortisone has the advantage of having mineralocorticoid as well as glucocorticoid activity at high dosages. At dosages less than 100 mg per day, it is necessary to use a mineralocorticoid agent in addition to hydrocortisone; fludrocortisone can be used when patients can take oral medications. Parenteral mineralo¬ corticoid preparations such as desoxycorticosterone acetate are no longer available commercially in the United States. DRUG INTERACTIONS If patients also must take a hepatic microsomal enzyme inducer, the metabolism of the glucocorticoid will be acceler¬ ated and larger daily dosages may be needed. The treatment of adrenocortical insufficiency is considered in Chapter 76.

MINERALOCORTICOIDS PHARMACOLOGY Mineralocorticoids are 21-carbon steroids characterized by their effects on fluid and electrolyte balance. They promote renal sodium reabsorption and potassium excretion (see Chap. 79). Mineralocorticoid deficiency (see Chap. 81) causes hyponatre¬ mia, volume depletion, hypotension, hyperkalemia, and a hyperchloremic metabolic acidosis. Mineralocorticoid excess (see Chap. 80) is associated with the retention of sodium and water, hypertension, potassium depletion, hypokalemia, and a metabolic alkalosis. The excessive secretion or administration of a mineralocorticoid causes sodium retention, with consequent fluid retention and weight gain. Patients retain several hundred milliequivalents of sodium and gain several kilograms of weight. If mineralocorticoid excess persists, mineralocorticoid escape occurs; further sodium retention and weight gain do not

762

PART V: THE ADRENAL GLANDS CH;OH

CH2OH

C=0

c=o

FLUDROCORTISONE (9a- FLUOROHYDROCORTISONE)

FIGURE 78-3. Structure of the mineralocorticoid fludrocortisone. The arrow indicates the structural difference between this synthetic steroid and cortisol.

occur. During this escape phenomenon, urinary sodium excre¬ tion increases until patients come into balance, and urinary sodium excretion again reflects sodium intake. Thus, patients with mineralocorticoid excess often do not retain sufficient fluid for peripheral edema to develop (although it occasionally does) unless there is another cause for edema such as hypoalbuminemia or right ventricular failure. Therefore, the absence of edema does not exclude the possibility of mineralocorticoid excess. Aldosterone is the principal mineralocorticoid in humans. Desoxycorticosterone, corticosterone, and cortisol (hydrocorti¬ sone) also are secreted in amounts sufficient to cause salt reten¬ tion in certain pathologic situations. AGENTS USED CLINICALLY The agents with mineralocorticoid action that are used clini¬ cally are hydrocortisone and fludrocortisone (9a-fluorohydrocortisone, Florinef [Apothecon, a Bristol-Meyers Squibb Co., Princeton, NJ]; Fig. 78-3). When hydrocortisone is given in large dosages (e.g., 100 mg per day or more), a mineralocorticoid effect may be anticipated. Aldosterone is not used clinically, although it is a potent mineralocorticoid and is essentially devoid of glucocorticoid effect. It would be of limited value because of its brief duration of action. Fludrocortisone is available only for oral therapy. The presence of the fluorine atom in tire 9a position enhances the mineralocorti¬ coid potency of hydrocortisone. The enhanced mineralocorticoid potency of 9a-fluorohydrocortisone is explained by impaired renal conversion of this molecule to 9a-fluorocortisone by 11-hydroxysteroid dehydrogenase, in contrast to the rapid conversion of corti¬ sol to cortisone.95 At recommended dosages, fludrocortisone is an effective mineralocorticoid, but it is essentially free of glucocorti¬ coid activity. Its duration of action is ~12 to 24 hours.96-99 If patients also need large dosages of a glucocorticoid, hydrocortisone can be used alone. A dosage of 100 mg per day or more provides mineralocorticoid activity.96-98 Experimen¬ tally, it was found that fludrocortisone, 1 mg per day orally, is equivalent in mineralocorticoid activity to aldosterone, 1 mg per day in four divided intramuscular doses.98-99 No mineralocorticoid by itself can increase the sodium stores of sodium-depleted patients. The effectiveness of a min¬ eralocorticoid hormone depends on substrate availability; patients with mineralocorticoid deficiency not only need the hormone, they also need salt and water. Both hormonal therapy and proper fluid and electrolyte administration are necessary to achieve an optimal clinical response.

DRUG INTERACTIONS Mineralocorticoid activity is antagonized by spironolactone; the latter has no effect in patients with hypoaldosteronism. Amiloride (Midamor), a potassium-sparing diuretic agent that acts even in the absence of aldosterone, can reduce the effects of a mineralocorticoid on sodium and potassium balance.

Anything that promotes salt loss, such as a diuretic medica¬ tion, impairs mineralocorticoid efficacy. For patients who ordi¬ narily need a diuretic (e.g., for the treatment of hypertension or fluid retention), the desired effect may be achieved by modify¬ ing the dosage of mineralocorticoid, the intake of salt, or both. Medications that alter sweating or promote vomiting or diar¬ rhea may affect salt balance and therefore change the effective¬ ness of a mineralocorticoid.

INDICATIONS Mineralocorticoid therapy is indicated for primary adrenocorti¬ cal insufficiency; isolated hypoaldosteronism; salt-losing forms of congenital adrenal hyperplasia; and chronic orthostatic hypotension caused by autonomic insufficiency (multiple sys¬ tems atrophy [e.g., the Shy-Drager syndrome], idiopathic orthostatic hypotension, and diabetic autonomic neuropathy).

DOSAGE The usual dosage of fludrocortisone is 0.1 mg daily, but may range from 0.1 mg on alternate days to 0.2 mg daily. Generally, the starting dosage is 0.1 mg daily, with adjustments made according to clinical response. Orthostatic vital signs are of great value in assessing the adequacy of mineralocorticoid replacement therapy. A marked rise in the heart rate or a fall in blood pressure on standing may precede other manifestations of mineralocorticoid deficiency.

REFERENCES 1. Axelrod L. Glucocorticoid therapy. Medicine (Baltimore) 1976; 55:39. 2. Axelrod L. Glucocorticoids. In: Kelley WN, Harris ED Jr, Ruddy S, Sledge CB, eds. Textbook of rheumatology, 4th ed. Philadelphia: WB Saunders, 1993. 3. Hollander JL, Brown EM Jr, Jessar RA, Brown CY. Hydrocortisone and cor¬ tisone injected into arthritic joints. JAMA 1951; 147:1629. 4. Robinson RCV, Robinson HM Jr. Topical treatment of dermatoses with ste¬ roids. South Med J 1956; 49:260. 5. Harter JG. Corticosteroids: their physiologic use in allergic disease. NY State J Med 1966; 66:827. 6. Ballard PL, Carter JP, Graham BS, Baxter JD. A radioreceptor assay for eval¬ uation of the plasma glucocorticoid activity of natural and synthetic ste¬ roids in man. J Clin Endocrinol Metab 1975; 41:290. 7. Meikle AW, Tyler FH. Potency and duration of action of glucocorticoids: effects of hydrocortisone, prednisone and dexamethasone on human pitu¬ itary-adrenal function. Am J Med 1977; 63:200. 8. Pickup ME. Clinical pharmacokinetics of prednisone and prednisolone. Clin Pharmacokinet 1979; 4:111. 9. Jenkins JS, Sampson PA. Conversion of cortisone to cortisol and pred¬ nisone to prednisolone. BMJ 1967; 2:205. 10. Gambertoglio JG, Amend WJC Jr, Benet LZ. Pharmacokinetics and bio¬ availability of prednisone and prednisolone in healthy volunteers and patients: a review. J Pharmacokinet Biopharm 1980; 8:1. 11. Davis M, Williams R, Chakraborty J, et al. Prednisone or prednisolone for the treatment of chronic active hepatitis? A comparison of plasma avail¬ ability. Br J Clin Pharmacol 1978; 5:501. 12. Lewis GP, Jusko WJ, Burke CW, et al. Prednisone side effects and serumprotein levels, a collaborative study. Lancet 1971; 2:778. 13. Frey FJ, Frey BM. Altered prednisolone kinetics in patients with the neph¬ rotic syndrome. Nephron 1982; 32:45. 14. Gatti G, Perucca E, Frigo GM, et al. Pharmacokinetics of prednisone and its metabolite prednisolone in children with nephrotic syndrome during the active phase and in remission. Br J Clin Pharmacol 1984; 17:423. 15. Frey FJ, Horber FF, Frey BM. Altered metabolism and decreased efficacy of prednisolone and prednisone in patients with hyperthyroidism. Clin Phar¬ macol Ther 1988; 44:510. 16. Schatz M, Patterson R, Zeitz S, et al. Corticosteroid therapy for the preg¬ nant asthmatic patient. JAMA 1975; 233:804. 17. Stuck AE, Frey BM, Frey FJ. Kinetics of prednisolone and endogenous cor¬ tisol suppression in the elderly. Clin Pharmacol Ther 1988; 43:354. 18. Tornatore KM, Logue G, Venuto RC, Davis PJ. Pharmacokinetics of methylprednisolone in elderly and young healthy males. J Am Geriatr Soc 1994; 42:1118. 19. Jubiz W, Meikle AW. Alterations of glucocorticoid actions by other drugs and disease states. Drugs 1979; 18:113. 20. Ziircher RM, Frey BM, Frey FJ. Impact of ketoconazole on the metabolism of prednisolone. Clin Pharmacol Ther 1989; 45:366.

Ch. 78: Corticosteroid Therapy 21. Legler UF, Benet LZ. Marked alterations in dose-dependent prednisolone kinet¬ ics in women taking oral contraceptives. Clin Pharmacol Ther 1986; 39:425. 22. Uribe M, Casian C, Rojas S, et al. Decreased bioavailability of prednisone due to antacids in patients with chronic active liver disease and in healthy volunteers. Gastroenterology 1981; 80:661. 23. Graham GG, Champion GD, Day RO, Pauli PD. Patterns of plasma concen¬ trations and urinary excretion of salicylate in rheumatoid arthritis. Clin Pharmacol Ther 1977; 22:410. 24. Plotz CM, Knowlton AI, Ragan C. The natural history of Cushing's syn¬ drome. Am J Med 1952; 13:597. 25. Thom GW. Clinical considerations in the use of corticosteroids. N Engl J Med 1966; 274:775. 26. Boston Collaborative Drug Surveillance Program. Acute adverse reactions to prednisone in relation to dosage. Clin Pharmacol Ther 1972; 13:694. 27. Perry PJ, Tsuang MT, Hwang MH. Prednisolone psychosis: clinical obser¬ vations. Drug Intell Clin Pharm 1984; 18:603. 28. Hollman GA, Allen DB. Overt glucocorticoid excess due to inhaled corti¬ costeroid therapy. Pediatrics 1988; 81:452. 29. Stead RJ, Cooke NJ. Adverse effects of inhaled corticosteroids. BMJ 1989; 298:403. 30. Munck A, Guyre PM. Glucocorticoid physiology and homeostasis in rela¬ tion to anti-inflammatory actions. In: Schleimer RP, Claman HN, Oronsky A, eds. Anti-inflammatory steroid action: basic and clinical aspects. San Diego: Academic Press, 1989. 30a. Sapolsky RM, Romero LM, Munck AU. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev 2000; 27:55. 31. Scheinman RI, Cogswell PC, Lofquist AK, Baldwin AS Jr. Role of transcrip¬ tional activation of IicBa in mediation of immunosuppression by glucocor¬ ticoids. Science 1995; 270:283. 32. Auphan N, DiDonato JA, Rosette C, et al. Immunosuppression by gluco¬ corticoids: inhibition of NF-kB activity through induction of IkB synthesis. Science 1995; 270:286. 33. Marx J. How the glucocorticoids suppress immunity. Science 1995; 270:232. 34. Fauci AS, Dale DC, Balow JE. Glucocorticosteroid therapy: mechanisms of action and clinical considerations. Ann Intern Med 1976; 84:304. 35. Parrillo JE, Fauci AS. Mechanisms of glucocorticoid action on immune pro¬ cesses. Annu Rev Pharmacol Toxicol 1979; 19:179. 36. Cupps TR, Fauci AS. Corticosteroid-mediated immunoregulation in man. Immunol Rev 1982; 65:133. 37. Samuelsson B. Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science 1983; 220:568. 38. DiRosa M, Flower RJ, Hirata F, et al. Anti-phospholipase proteins. Prosta¬ glandins 1984; 28:441. 39. Parente L, DiRosa M, Flower RJ, et al. Relationship between the anti-phos¬ pholipase and anti-inflammatory effects of glucocorticoid-induced pro¬ teins. Eur J Pharmacol 1984; 99:233. 40. Axelrod L. Side effects of glucocorticoid therapy. In: Schleimer RP, Claman H, Oronsky A, eds. Antiinflammatory steroid action: basic and clinical aspects. San Diego: Academic Press, 1989. 41. Ragan C. Corticotropin, cortisone and related steroids in clinical medicine: practical considerations. Bull NY Acad Med 1953; 29:355. 42. Conn HO, Blitzer BL. Nonassociation of adrenocorticosteroid therapy and peptic ulcer. N Engl J Med 1976; 294:473. 43. Langman MJS, Cooke AR. Gastric and duodenal ulcer and their associated diseases. Lancet 1976; 1:680. 44. Jick H, Porter J. Drug-induced gastrointestinal bleeding. Lancet 1978; 2:87. 45. Messer J, Reitman D, Sacks HS, et al. Association of adrenocorticosteroid therapy and peptic-ulcer disease. N Engl J Med 1983; 309:21. 46. Spiro HM. Is the steroid ulcer a myth? N Engl J Med 1983; 309:45. 47. Conn HO, Poynard T. Adrenocorticosteroid administration and peptic ulcer: a critical analysis. J Chronic Dis 1985; 38:457. 48. Piper JM, Ray WA, Daugherty JR, Griffin MR. Corticosteroid use and pep¬ tic ulcer disease: role of nonsteroidal anti-inflammatory drugs. Ann Intern Med 1991; 114:735. 49. Gabriel SE, Jaakkimainen L, Bombardier C. Risk for serious gastrointestinal complications related to use of nonsteroidal anti-inflammatory drugs. A meta-analysis. Ann Intern Med 1991; 115:787. 50. Schatz M, Patterson R, Kloner R, Falk J. The prevalence of tuberculosis and positive tuberculin skin tests in a steroid-treated asthmatic population. Ann Intern Med 1976; 84:261. 51. Kozower M, Veatch L, Kaplan MM. Decreased clearance of prednisolone, a factor in the development of corticosteroid side effects. J Clin Endocrinol Metab 1974; 38:407. 52. Bergrem H, Jervell J, Flatmark A. Prednisolone pharmacokinetics in Cushing¬ oid and non-cushingoid kidney transplant patients. Kidney Int 1985; 27:459. 53. Frey FJ, Amend WJC Jr, Lozada F, et al. Endogenous hydrocortisone, a pos¬ sible factor contributing to the genesis of Cushingoid habitus in patients on prednisone. J Clin Endocrinol Metab 1981; 53:1076. 54. Becker B. Intraocular pressure response to topical corticosteroids. Invest Ophthalmol 1965; 4:198. 55. Bigger JF, Palmberg PF, Becker B. Increased cellular sensitivity to glucocor¬ ticoids in primary open angle glaucoma. Invest Ophthalmol 1972; 11:832. 56. Becker B, Podos SM, Asseff CF, Cooper DG. Plasma cortisol suppression in glaucoma. Am J Ophthalmol 1973; 75:73. 57. Becker B, Shin DH, Palmberg PF, Waltman SR. HLA antigens and cortico¬ steroid response. Science 1976; 194:1427.

763

58. American College of Rheumatology Task Force on Osteoporosis Guide¬ lines. Recommendations for the prevention and treatment of glucocorti¬ coid-induced osteoporosis. Arthritis Rheum 1996; 39:1791. 59. Sambrook PN. Calcium and vitamin D therapy in corticosteroid bone loss: what is the evidence? J Rheumatol 1996; 23:963. 60. Sambrook P, Birmingham J, Kelly P, et al. Prevention of corticosteroid osteoporosis. A comparison of calcium, calcitriol, and calcitonin. N Engl J Med 1993; 328:1747. 61. Adachi JD, Bensen WG, Brown J, et al. Intermittent etidronate therapy to prevent corticosteroid-induced osteoporosis. N Engl J Med 1997; 337:382. 62. Reid IR. Preventing glucocorticoid-induced osteoporosis. N Engl J Med 1997; 337:420. 63. Saag KG, Emkey R, Schnitzer TJ, et al. Alendronate for the prevention and treatment of glucocorticoid-induced osteoporosis. N Engl J Med 1998; 339:292. 64. Yale SH, Limper AH. Pneumocystis carinii pneumonia in patients without acquired immunodeficiency syndrome: associated illnesses and prior corti¬ costeroid therapy. Mayo Clin Proc 1996; 71:5. 65. Sepkowitz KA. Pneumocystis carinii pneumonia without acquired immuno¬ deficiency syndrome: who should receive prophylaxis? Mayo Clin Proc 1996:71:102. 66. Amatruda TT Jr, Hollingsworth DR, D'Esopo ND, et al. A study of the mecha¬ nism of the steroid withdrawal syndrome: evidence for integrity of the hypo¬ thalamic-pituitary-adrenal system. J Clin Endocrinol Metab 1960; 20:339. 67. Amatruda TT Jr, Hurst MM, D'Esopo ND. Certain endocrine and meta¬ bolic facets of the steroid withdrawal syndrome. J Clin Endocrinol Metab 1965; 25:1207. 68. Dickstein G, Shechner C, Nicholson WE, et al. Adrenocorticotropin stimu¬ lation test: effects of basal cortisol level, time of day, and suggested new sensitive low-dose test. J Clin Endocrinol Metab 1991; 72:773. 68a. Henzen C, Suter A, Lerch E, et al. Suppression and recovery of adrenal response after short-term, high-dose glucocorticoid treatment. Lancet 2000; 355:542. 69. Streeten DHP. Shortcomings in the low-dose (1 pg) ACTH test for the diag¬ nosis of ACTH deficiency states. J Clin Endocrinol Metab 1999; 84:835. 70. Salem M, Tainsh RE Jr, Bromberg J, et al. Perioperative glucocorticoid cov¬ erage. A reassessment 42 years after emergence of a problem. Ann Surg 1994; 219:416. 71. Schlaghecke R, Kornely E, Santen RT, Ridderskamp P. The effect of long¬ term glucocorticoid therapy on pituitary-adrenal responses to exogenous corticotropin-releasing hormone. N Engl J Med 1992; 326:226. 72. Daly JR, Fletcher MR, Glass D, et al. Comparison of effects of long-term corticotrophin and corticosteroid treatment on responses of plasma growth hormone, ACTH, and corticosteroid to hypoglycaemia. BMJ 1974; 2:521. 73. Graber AL, Ney RL, Nicholson WE, et al. Natural history of pituitary-adre¬ nal recovery following long-term suppression with corticosteroids. J Clin Endocrinol Metab 1965; 25:11. 74. Streck WF, Lockwood DH. Pituitary adrenal recovery following short-term suppression with corticosteroids. Am J Med 1979; 66:910. 75. Haugen HN, Reddy WJ, Harter JG. Intermittent steroid therapy in bron¬ chial asthma. Nord Med 1960; 63:15. 76. Reichling GH, Kligman AM. Alternate-day corticosteroid therapy. Arch Dermatol 1961; 83:980. 77. Harter JG, Reddy WJ, Thom GW. Studies on an intermittent corticosteroid dosage regimen. N Engl J Med 1963; 269:591. 78. MacGregor RR, Sheagren JN, Lipsett MB, Wolff SM. Altemate-day pred¬ nisone therapy: evaluation of delayed hypersensitivity responses, control of disease and steroid side effects. N Engl J Med 1969; 280:1427. 79. Dale DC, Fauci AS, Wolff SM. Altemate-day prednisone: leukocyte kinetics and susceptibility to infections. N Engl J Med 1974; 291:1154. 80. Fauci AS, Dale DC. Altemate-day therapy and human lymphocyte sub¬ populations. J Clin Invest 1975; 55:22. 81. Schurmeyer TH, Tsokos GC, Avgerinos PC, et al. Pituitary-adrenal respon¬ siveness to corticotropin-releasing hormone in patients receiving chronic, alternate day glucocorticoid therapy. J Clin Endocrinol Metab 1985; 61:22. 82. Hunder GG, Sheps SG, Allen GL, Joyce JW. Daily and alternate day corti¬ costeroid regimens in treatment of giant cell arteritis: comparison in a pro¬ spective study. Ann Intern Med 1975; 82:613. 83. Abruzzo JL. Altemate-day prednisone therapy. Ann Intern Med 1975; 82:714. 84. Bengtsson B-A, Malmvall B-E. An alternate-day corticosteroid regimen m maintenance therapy of giant cell arteritis. Acta Med Scand 1981; 209:347. 85. Fauci AS. Altemate-day corticosteroid therapy. Am J Med 1978; 64:729. 86. Allander E. ACTH or corticosteroids? A critical review of results and possibili¬ ties in the treatment of severe chronic disease. Acta Rheum Scand 1969; 15:277. 87. Kaplan HP, Portnoy B, Binder HJ, et al. A controlled evaluation of intrave¬ nous adrenocorticotropic hormone and hydrocortisone in the treatment of acute colitis. Gastroenterology 1975; 69:91. 88. Hrachovy RA, Frost JD Jr, Kellaway P, Zion TE. Double-blind study of ACTH vs prednisone therapy in infantile spasms. J Pediatr 1983; 103:641. 89. Editorial. Steroids in acute severe asthma. Lancet 1992; 340:1384. 90. McFadden ER Jr. Dosages of corticosteroids in asthma. Am Rev Respir Dis 1993; 147:1306. 91. Elenbaas J. Steroid pulse therapy in systemic lupus erythematosus. Drug Intell Clin Pharm 1983; 17:342. 92. Kurki P, ed. High dose intravenous corticosteroid therapy of systemic lupus erythematosus and primary crescenteric rapidly progressive glomer¬ ulonephritis. Proceedings of a symposium. Scand J Rheumatol 1984; Suppl 54:1.

764

PART V: THE ADRENAL GLANDS

93. Udelsman R, Ramp J, Gallucci WT, et al. Adaptation during surgical stress: a reevaluation of the role of glucocorticoids. J Clin Invest 1986; 77:1377. 94. Glowniak JV, Loriaux DL. A double-blind study of perioperative steroid requirements in secondary adrenal insufficiency. Surgery 1997; 121:123. 95. Oelkers W, Buchen S, Diederich S, et al. Impaired renal 11 (3-oxidation of 9afluorocortisol: an explanation for its mineralocorticoid potency. J Clin Endocrinol Metab 1994; 78:928. 96. Goldfein A, Laidlaw JC, Haydar NA, et al. Fluorohydrocortisone and chlorohydrocortisone, highly potent derivatives of compound F. N Engl J Med 1955; 252:415. 97. Renold AE, Haydar NA, Reddy WJ, et al. Biological effects of fluorinated derivatives of hydrocortisone and progesterone in man. Ann NY Acad Sci 1955; 61:582. 98. Thom GW, Renold AE, Morse WI, et al. Highly potent adrenal cortical ster¬ oids; structure and biological activity. Ann Intern Med 1955; 43:979. 99. Thom GW, Sheppard RH, Morse WI, et al. Comparative action of aldoster¬ one and 9-alpha-fluorohydrocortisone in man. Ann NY Acad Sci 1955; 61:609.

CHAPTER 79

RENIN-ANGIOTENSIN SYSTEM AND ALDOSTERONE DALI LA B. CORRY AND MICHAEL L. TUCK

THE RENIN-ANGIOTENSIN SYSTEM ANGIOTENSI NOGEN Angiotensinogen (AGT), also termed renin substrate, is the pre¬ cursor for the angiotensin peptides, which include angiotensin (A)-I, II, III, and IV, and Aj_7. Levels of this peptide, which can be rate-limiting for the renin-angiotensin system, are produced mainly in the liver, where its precursor preproangiotensinogen is synthesized and glycosylated in the hepatocytes; nonetheless, there is evidence of production in other tissues as well (e.g., the heart, blood vessels, kidney, and adipocytes). In the circulation, AGT, with a half-life of 16 hours, is cleaved by renin (-10%) and/or other enzymes to release A-I (Fig. 79-1). In many tissues not expressing renin, AGT can be cleaved by enzymes other than renin (e.g., cathepsin G, tonin, and chymase). The extent to which AGT is glycosylated may influence the kinetics of renin in the circulation. Indeed, this has been hypothesized in a proposed, separate, brain-AGT system. There is one copy of the AGT gene in the mammalian genome, which is -11,800 base pairs (bp) in length.1'2 This gene

has 5 exons that encode for the protein, separated by 4 introns. Exon 1 codes for the 5'-nontranslated region, whereas exon 2 contains the signal peptide and coding regions. AGT is secreted constitutively from hepatocytes; however, several factors (e.g., glucocorticoids, estrogens, thyroid hormone, insulin, and A-II) may exert a positive feedback.3-4 RELATIONSHIP TO HYPERTENSION A high molecular-weight form of AGT, which is released during pregnancy, may play a role in pregnancy-induced hypertension. In adipose tissue, there is a form of AGT that is increased by insu¬ lin and decreased by 3-adrenergic blockade and that possibly contributes to obesity-related hypertension. Moreover, AGT may play a role in certain other forms of hypertension, as observed in glucocorticoid excess states (e.g., Cushing syndrome) and thy¬ roid disorders.1-4 Interestingly, antisense nucleotide sequences that have been used to block AGT mRNA can reduce blood pressure. In spontaneously hypertensive rats, central nervous system (CNS) administration of antisense sequences against the mRNA encoding AGT lowers blood pressure.5 Furthermore, rats made transgenic with human AGT develop hypertension because AGT is expressed in the blood vessel wall.6-7 Thus, there is ample documentation for a role of AGT in experimental hypertension. Further documentation for a role of AGT in hypertension is as follows: Epidemiologic studies have shown a relationship between plasma AGT and human hypertension.8-9 Polymor¬ phisms of the AGT gene have been linked to familial hyperten¬ sion, renal disease, and cardiovascular risk factors.9 Increased levels of AGT are associated with essential hypertension, and an M235T polymorphism in the AGT gene is associated with nephropathy in type 2 diabetics.10 The AGT M235T molecular variant—threonine substituted for methionine at amino acid 235—is associated by linkage analysis with essential hyperten¬ sion, especially in whites.11-12 Subjects bearing the 235 allele have higher levels of AGT (i.e., a 20% increase in homozygous subjects [TT] and a 10% increase in heterozygous subjects [MT]) compared with homozygous (MM) individuals. This linkage of AGT variants to hypertension is population-dependent (i.e., strong in whites, weak in Mexican Americans13 and the Chinese14). AGT mutations probably play a significant but modest role in blood pressure variation. All of these findings suggest that the renin-AGT reaction kinetics are increased by AGT variants, leading to more A-II production, which in turn may increase vascular resistance and growth. In turn, vascular injury induces AGT gene expression in the vascular media and neointima, suggesting that the renin-angiotensin system partic¬ ipates in myointimal proliferation.15 Finally, gene knock-out mice for AGT develop hypotension with polyuria when chal¬ lenged with a high-salt diet.16

RENIN SYNTHESIS AND RELEASE NON-ACE PATHWAYS

ACE PATHWAYS Angiotensinogen ensinogen ■

Tissue Plasminogen Activator

C I

^

Angiotensin I

Cathepsin G

Chymase CAGE

Angiotensin II

j Renin Ar jace

c,

„ . Bradykinin

■ Inactivated

\

AT, Receptor

ATj Receptor

t

Vasodilation Vasoconstriction Antiproliferative

'V

Cell growth (proliferative)

*—*

Sodium and Sympathetic water retention activation

FIGURE 79-1. Angiotensinogen and angiotensin I and II pathways and their role in vascular and fluid homeostasis via angiotensin II receptors (AT] and AT2). (CAGE, chymotrypsin-like angiotensin-generating enzyme.)

Renin, an aspartyl proteolytic enzyme, catalyzes the rate-limiting cleavage of AGT (between the leucine at position 10 and the valine at position 11) to form the decapeptide A-I, which is fur¬ ther converted by angiotensin-converting enzyme I (ACE-I) to the octapeptide, A-II. It is the plasma renin activity that is accepted as an index of the renin-angiotensin system, because it is difficult to measure other components (e.g., A-II). Renin synthesis begins with the formation of preprorenin in the juxtaglomerular (JG) cells of the kidney.17-18 This precursor is transported into the rough endoplasmic reticulum (Fig. 79-2), where it is cleaved to form a 23-amino-acid inactive form (pro¬ renin), which is passed through the Golgi apparatus, glycosy¬ lated, and deposited in lysosomal granules.18 Here it is cleaved by cathepsin B to form renin, which can be secreted in response to various stimuli.18 Basally, there is a low rate of renin release, and this is increased several-fold in response to stimuli.

Ch. 79: Renin-Angiotensin System and Aldosterone

765

Cytoplasm

FIGURE 79-2. Renin synthesis, activation, and release. Prorenin, which is not regulated by the factors that control renin release (i.e., pressure and volume changes), is released into the circulation at levels several-fold higher than those of bioactive renin.19 Whereas the role of circulating prorenin is unknown, it is found in tissue sites such as the adrenal glands, pituitary, and submandibular glands as well as in the kidney, and in spontaneously hypertensive rats, which are strokeprone, prorenin may raise blood pressure.20 Several local factors, which are under genetic control, also contribute to further enhance the renin response to salt restric¬ tion and to other stimuli. The vascular tissue-derived JG cells are situated in the afferent arteriole near the glomerulus and the mac¬ ula densa. With salt depletion, there is a recruitment of new cells facilitating a continued renin release.21 Additionally, the expres¬ sion of the renin gene can be increased by sodium restriction.22 Regulation of renin release is multifactorial (i.e., by renal baroreceptors, JG cells, the macula densa, renal nerves, and humoral factors). The renal baroreceptor is an intrarenal vascular recep¬ tor in the afferent arteriole that stimulates renin secretion in response to reduced renal perfusion pressure. It is attenuated when renal perfusion is elevated (Fig. 79-3). This is the most powerful mechanism for renin release; it is exemplified in the Goldblatt models of hypertension, in which a unilateral stenotic lesion of the renal artery causes hypoperfusion. The hypoperfu¬ sion induces increases in renin, and, hence, results in a renindependent form of hypertension. The macula densa is a modified group of cells in the distal tubules near the end of the loop of Henle and adjacent to the

T Aldosterone, Volume

Negative feedback Bradykinin Substance P

afferent arteriole and the JG cells. These cells sense distal tubu¬ lar Na+ delivery through Cl" and act through the Na+,K+,C1" cotransporter pathway. Diuretics, which stimulate the cotrans¬ porter pathway, enhance renin release. Other factors (e.g., ade¬ nosine, prostaglandin E2, nitric oxide, and the Pj-adrenergic system) also may mediate the macula densa renin-response pathway.17'23 The renal nerves (i.e., the Pradrenergic system) are stimu¬ lated through mechanoreceptors in the heart, pressor receptors in the aorta, and chemoreceptors in the vagal nerve. Central sympathetic stimulation increases renin secretion and is the major acute pathway for stress and posture; thus, upright pos¬ ture increases plasma renin activity two- to four-fold. Cyclic adenosine monophosphate (cAMP) is the main intra¬ cellular signal pathway for renin release, which is induced by P,-adrenergic agonists and by prostaglandins.24 Increased intra¬ cellular calcium provides another signal pathway; renin secre¬ tion is suppressed after rises in cytosolic calcium. Electrical depolarization of the JG cells (by increased A-II) opens calcium channels, and the high intracellular calcium inhibits renin release. Hyperpolarization of the JG cells with a resultant decrease of intracellular calcium has the opposite effect.24 Other inhibitors of renin release include adenosine Aj, a-adrenergic agonists, thromboxane, and endothelin.24 Atrial natriuretic pep¬ tides (ANPs) and nitric oxide inhibit renin release through stim¬ ulation of cyclic guanosine monophosphate (cGMP) and guanylate cyclase. Phospholipase C-regulated changes in intra¬ cellular calcium also affect renin secretion.24

w

Inactive fragments

@ Angiotensin \ \

non-Renin

non-ACE

\\

Angiotensin II

t

X

Negative feedback

FIGURE 79-3. Control of renin release by negative blood pressure

feedback.

766

PART V: THE ADRENAL GLANDS

Angiotensinogen

Kinogen

i

1

Renin

Kallikrein

Angiotensin I

Bradykinin

(Inactive)

(Active)

iOcOi

Angiotensin II

(Aldosterone secretion Vasoconstriction)

Nitric oxide Prostaglandin

Bradykinin,^ (Inactive)

FIGURE 79-4. Metabolism of vasoactive peptides by angiotensin-Iconverting enzyme (ACE).

Mice and rats that are transgenic for the renin message TGR(mREN2)27 (a model representing a precisely defined defect for the renin-angiotensin system) demonstrate that the renin gene is an important candidate gene for hypertension.25 Thus, TGR(mREN2)27 rats, harboring the murine REN-2 gene, develop severe hypertension despite low levels of renin. The high expression of the transgene in several tissues should cause high tissue levels of renin and A-II. In this model, the high blood pressure and suppressed plasma renin activity in the face of an increased tissue renin expression suggests that an extrarenal renin-angiotensin system can mediate the hypertension.

ANGIOTENSIN l-CONVERTING ENZYME Angiotensin I-converting enzyme (ACE) converts the inactive decapeptide angiotensin I to the active octapeptide angiotensin II (Fig. 79-4). The enzyme is not specific for angiotensin because it cleaves bradykinin, luteinizing hormone-releasing hormone, the enkephalins, and substance P.26-27 It is a more efficient kininase than it is a converting enzyme.27 ACE activates the vaso¬ constrictor A-II and metabolizes the vasodilator bradykinin (see Fig. 79-4). ACE inhibitors block angiotensin formation and bradykinin degradation. The beneficial effects of ACE inhibi¬ tors in the heart and kidney may be a result of both A-II reduc¬ tion and bradykinin accumulation. ACE is a metalloenzyme that requires zinc as a cofactor and also needs chloride ions to cleave most substrates.28 Human A'CE has a molecular mass of 150 kDa to 180 kDa, of which 146.6 kDa is protein and the remainder is carbohydrate.28 Most ACE is bound to plasma membranes of endothelial cells within vascular tissue. ACE is inserted into the membrane by a 17amino acid hydrophobic region near the carboxyl terminus (Cterminus hydrophobic anchor peptide). ACE can be released from the plasma membrane by proteolytic cleavage; therefore, some soluble ACE can be detected in blood, urine, edema fluid, amniotic fluid, cerebrospinal fluid, lymph, seminal plasma, and prostate.28'29 The highest concentration of ACE is seen in the blood ves¬ sels of the lung, retina, and brain.29 The human kidney also con¬ tains a very large amount of this enzyme.28 The brush border of the proximal tubules (where A-II has major functions in electro¬ lyte transport) is rich in ACE. It is also abundant in the choroid plexus, the placenta, and the epithelial lining of the small intes¬ tine, and seems to be highly concentrated in some areas of the brain (e.g., the subfornical organ, area postrema, substantia nigra, and locus coeruleus). The primary structure of ACE consists of two active centers that are located within two homologous domains.29’30 Most tissues have both domains, but testicular tissue has only one domain, per¬ haps representing an early form of the enzyme that has not been duplicated.30 In endothelial cells, ACE contains two functional sites (requiring two zinc ions) and two inhibitor-binding sites.

ACE has in vitro activity for a broad range of substrates besides A-I (e.g., N-acetyl-seryl-aspartyl-lysyl-proline [AcSDKP], which is a regulatory factor for hematopoiesis).26'27'31 In vivo, plasma levels of Ac-SDKP increase sharply after oral administration of an ACE inhibitor.31 ACE inhibition, which is the treatment of choice'for the erythrocytosis that occurs after renal transplantation, lowers the erythrocyte count indepen¬ dently of any effect on erythropoietin. Thus, ACE appears to have a role in the regulation of hematopoiesis. The human ACE gene displays an insertion (I)/deletion (D) polymorphism, which accounts for at least half of the variability in serum ACE levels.32 One such I/D polymorphism, which has been found in the noncoding region, corresponds to the presence or absence of a 287-bp sequence in intron 16.32 Individuals who are homozygous for the insertion polymorphism (II) have lower levels of circulating ACE than do individuals with the deletion (DD) genotype.33 The D allele may be associated with an increased risk for coronary heart disease and diabetic nephropa¬ thy. Individuals with diabetic nephropathy who are DD homozy¬ gous may respond less well to ACE inhibitors with respect to reducing blood pressure and proteinuria.33 These individuals are also more salt sensitive, such that on a high salt diet, ACE inhibi¬ tion is less effective. Furthermore, a deletion variant of the ACE D allele is associated with an increased risk of diabetic nephropathy and hastened progression of IgA nephropathy.34 This deletion in ACE does not appear to affect outcome of renal transplants.35 A link between the D allele and plasminogen activator inhibitor-1 levels in diabetes and microangiopathy suggests that an adverse consequence of thrombotic mechanisms can be activated with ACE polymorphism.36 Other A-II-forming serine proteinases such as chymase have been identified in the human cardiac left ventricle and in vascu¬ lar, renal, and other tissues.37 These enzymes serve as alterna¬ tive routes in the conversion of A-I to A-II.37 These non-ACE enzymes may explain the incomplete blockade of A-II forma¬ tion during ACE-inhibition therapy.

ANGIOTENSIN METABOLISM Of the several active angiotensins, A-II is the most important. The enzymes that are most active in degrading angiotensin are the aminopeptidases (Fig. 79-5). Glutamyl aminopeptidase cleaves A-II to form A-III (A2_8), and arginyl aminopeptidase cleaves A-III to A-IV (A3_g).38 Although Aj_g does not have major pressor activity, it does increase cGMP levels and may have central effects.38 The amino-terminal heptapeptide, A,_7 [des-phe8], which is formed directly from A-I by several tissue endopeptidases or by ACE,39 can stimulate the release of argi¬ nine vasopressin (AVP), vasodilator prostaglandins, bradyki¬ nin, and nitric oxide.39-40 Most angiotensins have a very short duration of action and undergo enzymatic degradation and endocytosis. The AT, receptor can mediate intracellular trans¬ port of A-II and the ligand-receptor can also be internalized. The acute inhibition of ACE decreases levels of A-II, but chronic inhibition therapy may not suppress A-II, although it does raise levels of A|_7 (a competitive inhibitor of A-II); this phenomenon possibly explains the long-term antihypertensive effects of ACE-I.39'40

TISSUE RENIN-ANGIOTENSIN SYSTEM Local renin-angiotensin systems in multiple tissues can pro¬ duce A-II independently of the circulating renin-angiotensin system (Table 79-1). Their function of these systems is probably to permit regional modulation of blood flow and also to assist in the action of growth factors. Most contractile cells (e.g., vas¬ cular smooth muscle, heart cells, mesangial cells, and sperm tails) have a well-defined local renin-angiotensin system. A-II is

Ch. 79: Renin-Angiotensin System and Aldosterone

767

TABLE 79-1. Differences in Circulating and Tissue Renin-Angiotensin System Control and Action CIRCULATING

TISSUE

Control

Short-term effect due to rapid feedback

Long-term effects

Kidney

Na+ and water reabsorption

Intraglomerular pressure

Heart

Chronotropic effect

Myocyte cell growth

Vessels

Vasoconstriction

Arrythmogenic

Angiotensin IV FIGURE 79-5. Enzymes that convert angiotensin I into other angio¬ tensin peptide products.

formed in these cells either by renin or through other proteases such as chymase, cathepsin, or tonin 41-43 Kidney Renin-Angiotensin System. In addition to being the major site for release of systemic renin, the kidney has a local renin-angiotensin system that is involved in renal growth.44 ACE-I administration during pregnancy causes severe fetal renal abnormalities. The tubular epithelial cells con¬ tain ACE, and local A-II is found in proximal tubules and mesangial cells45 The intrarenal A-II constricts afferent and efferent arterioles and directly increases Na+ reabsorption. Locally, A-II is also involved in renal pathologic conditions (e.g., the development of nephrosclerosis). Cardiac Renin-Angiotensin System. Evidence exists for a role for an intracardiac renin-angiotensin system in both nor¬ mal and failing hearts.41'42 There is a direct expression of AGT and the A-II receptor (AT,) in cardiomyocytes. The exact source of the renin in cardiac tissue is controversial, because levels of mRNA are low.43 The heart can also express A-I, which may be converted by intracardiac ACE to A-II.43 Probably, cardiac renin-angiotensin system activity is regulated by the amount of AGT, ACE, or renin-binding protein in the cardiomyocytes. Consequently, local A-II can regulate vascular tone, contractil¬ ity, growth, and cardiac hypertrophy. ' Vascular Renin-Angiotensin System. Vascular tissue has an active tissue renin-angiotensin system. Both ACE mRNA and product are found in the endothelium, and also, to a lesser degree, in vascular smooth muscle cells.43 AGT, which is also expressed in cultured vascular smooth muscle cells, is stimu¬ lated by insulin and insulin-like growth factor-I (IGF-I).46 The specific origin of the renin in the vasculature is more controver¬ sial, because the mRNA is low. Nonetheless, in situ conversion of A-I to A-II does occur in the vasculature; thus, there is an active ACE, which probably has a regulatory role.43 Insulin and IGF-I-induced vascular growth and hypertrophy are mediated in part by a local renin-angiotensin system and attenuated by ACE-I and AT,-receptor blockers.46 Ovarian Renin-Angiotensin System. The ovarian cells pro¬ duce high concentrations of both renin and prorenin, and all components of the renin-angiotensin system are found in follicu¬ lar fluid.47 The granulosa and theca cells also contain renin and angiotensin. Because prorenin is secreted throughout pregnancy,

Vascular cell growth

it may have functional significance during this time. A possible role for A-II as a reproductive hormone is being investigated.48 Testicular Renin-Angiotensin System. A-I, -II, and -III are found in the testicular tissue, and AT, receptors are present in Leydig cells.48,49 The structure of testicular ACE is unique; it has only one of the two binding domains. Perhaps, testicular ACE and A-II participate in spermatogenesis and may play a role in male fertility.49 Adipose Renin-Angiotensin System. Adipose tissue is a rich source for AGT.50 Locally formed A-II from adipose tissue may cause vasoconstriction and hypertension, especially as body fat content increases. Skin Renin-Angiotensin System. The human skin fibro¬ blast expresses A-II and AGT mRNA. Local injury elevates local A-II concentrations, and it then participates in wound healing through growth factors. Adrenal Renin-Angiotensin System. Some of the highest levels of A-II are found in the adrenal cortex, within the adrenal zona glomerulosa and fasciculata.51 Local A-II is thought to play an important role in aldosterone and corticosteroid pro¬ duction. Renin is known to be produced in adrenal tissue and acts independently of plasma renin. Brain Renin-Angiotensin System. The blood-brain bar¬ rier prevents any blood-borne components of the renin-angio¬ tensin system from entering the brain; thus, this organ has a local renin-angiotensin system. The expression of renin mRNA is low in the brain, but some cells (e.g., glial cells) have marked expression of AGT.52 However, the expression and production of ACE is widespread throughout the brain.52 Moreover, AT, receptors are in the hypothalamus and AT2 receptors are in the locus coeruleus and in the inferior olivary region. In addition, receptors for A-IV and A,_7 are also found in the brain."2 Direct injection of A-II into the brain causes changes in drinking behavior and elevations of blood pressure. The anterior pituitary gland has high levels of renin and A-II (located mainly in the gonadotropes); thus, the renin-angio¬ tensin system may contribute to the regulation of estrogen pro¬ duction in females and testosterone production in males. Interestingly, A-II inhibits prolactin release. Importantly, the human brain (especially the pineal gland) is rich in chymase, which can convert A-I to A-II.53

ANGIOTENSIN RECEPTORS A-II binds and acts on its receptors at the cell surface. Several recep¬ tor subtypes have been found, including the ATV AT2, and AT,.54-56 AT, Receptor Subtype. Almost all actions of A-II are mediated through the AT, receptor, including its effects on vasoconstriction and aldosterone production and cell growth (Fig. 79-6). The AT, receptor is part of the superfamily of peptide hormone receptors with seven membrane-spanning regions linked to G proteins (Fig. 79-7). AT, activates phospholipase C, which in turn hydrolyzes phosphoinositide to inositol triphos¬ phate (IP3) and diacylglycerol.54 These changes increase the intracellular calcium levels that in turn activate protein kinases. Also, AT, receptors appear to lower levels of intracellular

768

PART V: THE ADRENAL GLANDS

Angiotensin

*

«n

AT-i Receptor

TABLE 79-2. Actions of the Angiotensin AT, and AT2 Receptors

N

FIGURE 79-6. Three major pathways of the angiotensin II [ATX) receptor.

cAMP, an effect that is most evident in the adrenal gland on the control of aldosterone secretion.54 Although the AT, receptor contains no intrinsic protein kinase activity, numerous studies indicate that A-II activates both nonreceptor-type and receptor-type tyrosine kinases.56 Thus, A-II binding to the AT, receptor stimulates a large vari¬ ety of regulated cellular events, including activation of phos¬ pholipases, second messenger generation, and protein phosphorylation. The enzymes that may be coupled to the AT, receptor include adenylate cyclase and phospholipases C, D, and A.54 AT2 Receptor Subtype. Table 79-2 contrasts the actions of the A-II AT, and AT2 receptor subtypes. Attention has been directed to the AT2 receptor subtype, which is found in fetal tis¬ sue, where it is responsible for the growth and remodeling of organs.57 It is likely that the actions of the AT, receptor may antagonize those of the AT, receptor (Table 79-2). Using the gene transfer approach, it has been shown that the AT2 receptor partic¬ ipates in vascular smooth muscle growth through apoptosis.57 AT2 receptors have been identified in the adult brain, in the adre¬

AT, RECEPTOR

at2 receptor

Vasoconstriction

Vasodilation

Growth

Growth inhibition

Anti-apoptotic

Pro-apoptotic

Pro-fibrotic

Fibrosis (?)

Pro-thrombotic

Thrombosis (?)

Pro-oxidant

Anti-oxidant (?)

nal medulla, in the gastrointestinal tract, and in other tissues. AT, receptors also facilitate intestinal sodium reabsorption. It has been demonstrated that an A-II receptor antagonist can correct endothelial dysfunction in human essential hypertension.

ACTIONS OF ANGIOTENSIN II Renal Sodium and Water Retention. The best known function of A-II is to support the circulation under conditions of reduced intravascular volume. However, there are >50 known physiologic actions. These include vasoconstriction, salt reten¬ tion through aldosterone, increased thirst and secretion of antidiuretic hormone resulting in retention of water, increased cardiac output, and stimulation of sympathetic nervous system activity (Table 79-3). Also, there are direct effects of A-II on early proximal tubule Na+ reabsorption that are mediated in part through the Na+, H+ antiporter; this accounts for 40% to 50% of sodium and water reabsorption.58 In AGT gene-null mutant mice, chronic volume depletion causes a sharp decrease in glomerular filtration rate (GFR) and a marked concentration defect, which is insensitive to AVP.59 Furthermore, the reninangiotensin system is essential for two fundamental homeostatic functions in the kidney, stabilizing the GFR and maintaining urine diluting and concentrating capacity.

TABLE 79-3. Angiotensin II Actions in Various Target Tissues

FIGURE 79-7. Model of angiotensin II (AT,) receptor. (From Hunyady L, Balia T, Catt KJ. The ligand binding sites of the angiotensin ATI receptor. Trends Pharmacol Sci 1996; 17:135.)

Action

Target Tissue

Vasoconstriction

Vascular smooth muscle

Hypertophy/hyperplasia

Vascular smooth muscle

Aldosterone secretion

Adrenal glomerulosa

Contractility, hypertrophy

Myocardium

Embryogenesis

Kidney

Sodium reabsorption

Proximal tubule of kidney

Contraction

Mesangium of kidney

Inhibition of renin release

Juxtaglomerular cells

Increased prostaglandins, nitric oxide, endothelin

Vascular endothelium

Extracellular matrix synthesis

Vascular connective tissue

Platelet aggregation

Platelets

Monocyte adhesion

Vessel wall

Prolactin inhibition

Anterior pituitary

ADH inhibition, thirst

Posterior pituitary

Norepinephrine release

Sympathetic neurons

Catecholamine release

Adrenal medulla

Pressor control

Brain

Baroreceptor control

Brain

ADH control

Brain

Salt/water absorption

Intestine (AT2)

Angiotensinogen synthesis

Liver

Glycogenolysis

Liver

Decreased apoptosis

Tissues

Ch. 79: Renin-Angiotensin System and Aldosterone Systemic Vasoconstriction. A-II produces arteriolar vaso¬ constriction and elevates vascular resistance and blood pressure. The signal transduction for this A-II-mediated vasoconstriction includes an increase of intracellular calcium and formation of pro¬ tein kinase C.60 The facilitated release of norepinephrine61 and endothelin-I62 may also contribute to the vasoconstriction that is induced by A-II. A-II stimulates the release of arachidonic acid products in the vasculature (e.g., lipoxygenase products and P450 epoxides), which have vasoconstrictive properties.63 Regulation of Gomerular Filtration Rate. A-II regulates the GFR and renal blood flow by constricting the efferent and afferent glomerular arterioles, and the interlobular artery.64 A-II differentially increases efferent resistance to levels that are two to three times those of the afferent resistance; this elevates glo¬ merular capillary pressure to maintain the single nephron GFR. In AGT gene-null mutant mice, chronic volume depletion causes a sharp decrease in GFR and a marked concentration defect, which is insensitive to AVP.59 These physiologic changes are paralleled by changes in renal structure at the glomerular level as well as at the renal papilla and underscore the key role of angiotensins in the development of the mammalian kidney. As mentioned previously, A-II can also stimulate prostaglan¬ dins (e.g., thromboxane) that mediate renal hemodynamics.65 A-II controls the GFR by constricting the mesangium and alter¬ ing tubuloglomerular feedback. In sodium-replete women, A-II infusion elicits a blunted renal microcirculatory response com¬ pared with the response in sodium-replete men; this demon¬ strates that sex steroids modulate the effects of A-II.66 A-II also has nonhemodynamic effects on the kidney (e.g., stimulation of cytokines in diabetic nephropathy).67 Cardiac Effects. The cardiac effects of the tissue angio¬ tensin system are multiple.68 A-II is involved in cardiac remod¬ eling and, through the ATj receptor, promotes extracellular production of matrix as well as a fibrous tissue response and increasing collagen levels. In addition, the vasoconstriction caused by A-II and oxidative stress further contribute to ischemia and left ventricular hypertrophy with a resultant greater stiffness of the myocardial wall.'14 Elevations in plasma renin activity are associated with increased risk for myocardial infarction.70 This risk may be related to renin-angiotensinsystem-induced changes in the fibrinolytic system, because A-II inhibits fibrinolysis (thereby promoting clot formation) and stimulates excess production of plasminogen activator inhibitor.71 Also, the ACE-induced breakdown of bradykinin leads to lower tissue type plasminogen activator levels and decreases nitric oxide concentrations. A low-salt diet increases plasminogen activator inhibitor activity by increasing the renin-angiotensin system and raising the aldosterone levels.7These findings help to explain why blockade of the reninangiotensin system by ACE-I or by AT1 receptor antagonists protect against thrombotic cardiac events and reduce the risk of recurrent myocardial infarctions. Brain Effects. There are several actions of the angio¬ tensins in the brain, an organ in which all three receptor subtypes exist.52 In addition, A-II exerts a central pressor activity and causes blunting of the baroreceptor; it also stimulates dipsogenic behavior and the synthesis of AVP. Other Effects. Other actions of A-II include platelet adhe¬ sion, glycogenolysis, and inhibition of prolactin release. A-II also has interactions with prostaglandins, nitric oxide, and endothelin. More diverse effects include contraction of the uterus, blockade of the effects of glucagon, stimulation of cate¬ cholamines, stimulation of endothelial cell growth, stimulation of ovulation, and regulation of steroidogenesis. Moreover, A-II selectively stimulates transforming growth factor-(3 and stimu¬ lates mRNA in the heart and kidneys (thereby contributing to cardiac and renal fibrosis), and it suppresses clusterin, a glyco¬ protein that is expressed in response to tissue injury.73 Functional analysis of A-II and other components of the reninangiotensin system can now be achieved by transgenic and gene¬

769

targeting technology.74,75 The direct transfection of the human renin gene using gene-transfer techniques in hepatocytes in vivo elevates blood pressure. In vivo transfection of antisense oligodeoxynucleotides for AGT decreases plasma AGT levels and blood pressure in spontaneously hypertensive rats.74 Transfection of antisense to AGT or AT4 receptors decreases blood pressure in spontaneously hypertensive rats.76 Vascular injury is associated with abundant expression of AGT and ACE. This process of vas¬ cular hypertrophy can be duplicated by transfecting ACE vector locally into intact rat carotid arteries 74 In vivo transfer of antisense ACE oligodeoxynucleotides inhibits neointimal formation in balloon-injured rat carotid arteries.74 Antisense technology has been used to examine the role of the renin-angiotensin system in the central nervous system (see earlier).76 Conceivably, a gene therapy for hypertension could use an antisense inhibition with adeno-associated viral vector delivery to target the A-II type 1 receptor messenger ribonucleic acid.76 Prolonged reductions in blood pressure in hypertension may be achieved with a single dose of adeno-associated viral vectors to deliver antisense oligodeoxynucleotides to inhibit ATj receptors.76

ALDOSTERONE Aldosterone is the most potent of several mineralocorticoid steroid hormones that act on the epithelium, especially in the kidney, to produce Na+ reabsorption and K+ excretion.77 Other steroids that have weaker mineralocorticoid effects include deoxycorticosterone (DOC), 18-oxycortisol, 19-nor-deoxycorticosterone (19-nor-DOC), and 18-hydroxy deoxycorticosterone (18-OH-DOC).

SYNTHESIS The adrenal gland is partitioned into different zones, which con¬ tain specific enzymes that produce either glucocorticoids (i.e., cortisol), mineralocorticoids (i.e., aldosterone), or weak adrenal androgens.78,79 Aldosterone is synthesized in the outer region of the adrenal gland (zona glomerulosa). Cortisol is synthesized in the inner regions in the zona fasciculata and reticularis. The same adrenal enzymes share most of the early synthetic steps of these steroids, but they differ markedly in their terminal enzymes. The zona glomerulosa is unique in lacking 17a-hydroxylase, the essential enzyme for cortisol formation. However, the zona glom¬ erulosa is rich in the enzyme aldosterone synthase, which is required for the terminal steps in aldosterone synthesis.78-80 Aldosterone synthase is a single multifunctional P450 enzyme on the inner mitochondrial membrane that converts corticosterone to aldosterone. It catalyzes three successive hydroxylation steps: (a) conversion of deoxycorticosterone to corticosterone; (b) addi¬ tion of a hydroxyl group to corticosterone (18-OH-corticosterone); and (c) conversion of 18-OH-corticosterone to an aldehyde, with consequent formation of aldosterone. Two cytochrome P450 enzymes have been found, including llp-hydroxylase and aldo¬ sterone synthase.78 Their genes are located in close proximity (40 kb) on chromosome 8 (8q21-q22) and display 95% homology. In the familial disorder glucocorticoid-remediable hyperaldosteronism, there is hypertension and variable hypokalemia as a result of the formation of a chimeric gene that makes aldosterone synthesis ACTH-dependent78 (see Chap. 80).

MINERALOCORTICOID RECEPTORS The mineralocorticoid receptor (steroid receptor type 1 [SRI]) binds both aldosterone and cortisol with equal affinity, whereas the cortisol receptor (steroid receptor type 2 [SR2]) binds only cor¬ tisol with high affinity.81,82 Both receptors are part of the steroid/ vitamin D/retinoic acid superfamily of transcription regulators. Both receptors have high amino acid homology and may overlap the

770

PART V: THE ADRENAL GLANDS

actions of progesterone and androgen receptors. In humans, there are two isoforms of the SRI; the a and |3 isoforms.83'84 The SRls are found mainly on epithelial cells of the renal tubule, the parotid gland, and the colon. SRls have been found in vascular smooth muscle, in the heart, and in areas of the brain. These receptors can undergo down-regulation, as seen with sodium loading in which the (1 isoform expression is decreased.84 In contrast, SR2s are found in most cells of the body. Glucocorticoids and mineralocorticoids bind to SRI with equal affinity, and glucocorticoid levels are 1000-fold higher than those of mineralocorticoids; thus, one would expect the receptor to be saturated with cortisol. However, specificity is conferred by the enzyme llfi-hydroxysteroid dehydrogenase (HSD),78’79-85 which oxidizes cortisol to inactive corticoster¬ one, thus allowing aldosterone to bind to its receptor. Thus, coexpression of 11(3-HSD and SRI in tissues appears to be a mechanism by which specificity is conferred to aldosterone action. There are multiple forms of this enzyme in the mam¬ malian kidney,86 including a high density form in the princi¬ pal and intercalated cells.87 In the syndrome of apparent mineralocorticoid excess, the renal isoform for 11(1-HSD9 is missing, thus allowing the xaormally high concentrations of cortisol in the kidney to bind to SRI.78 This effect creates an excess Na+ reabsorption with resultant hypertension and hypokalemia (see Chap. 80). The active ingredient of licorice, glycyrrhetinic acid, also inhibits 11(3-HSD^, causing an acquired syndrome of mineralocorticoid excess in humans. However, several questions remain regarding mineral corticoid receptors.873

ALDOSTERONE ACTION The major site of aldosterone action is on the distal nephron, where it increases Na+ reabsorption and K+ and H+ secretion. Aldosterone initiates its action by diffusing across the plasma membrane of the cell and binding avidly to specific receptors, which are located mostly in the cytoplasm.82 In the nucleus, the aldosterone-receptor complex acts on chromatin to increase mRNA and ribosomal RNA transcription.87-88 The binding of steroid to the carboxyl terminal of the receptor causes release of heat shock proteins, after which the receptor changes confor¬ mation to allow dimerization and binding to the regulatory end of specific target genes (hormone-responsive elements).82 Mineralocorticoids affect electrolyte balance by inducing and activating proteins in epithelial cells (e.g., aldosteroneinduced protein may act as a regulatory unit for the luminal membrane Na+ channel to allow luminal Na+ to enter the cell).89,90 Another aldosterone-induced protein may be a subunit of the Na+, K+ pump. This strengthens the argument that aldo¬ sterone not only has an indirect effect through ionic changes but also has a direct action on the Na+, K+ pump.88 The major sites for aldosterone action are in the connecting segment and in the collecting tubules.78 Aldosterone promotes NaCl reabsorption and K+ secretion in the connecting segments and in the principal cells in the cortical collecting tubules. In addition, it may act on Na+ in the papillary (inner medullary) collecting tubule and is thought to increase ionic transport by increasing the number of open Na+ and K+ channels in the luminal membrane.89'90 Aldosterone also stimulates the Na+, K+ pump in the basolateral membrane. Na+ then diffuses into the tubular cells from which the Na+ is removed by the Na+, K+ pump, thus creating an electrogenic negative potential differ¬ ence within the lumen. K+ is also secreted from the cell as Clmoves in to maintain electroneutrality. Another mechanism for K+ efflux is through the Na+, K+ pump. It is believed that aldo¬ sterone mediates Na+ channel movement by the methylation of channel proteins that, in turn, activate silent or inoperative Na+ channels.90 Because amiloride blocks luminal Na+ permeability, it is likely that increasing luminal permeability is one of the pri¬ mary actions of aldosterone.91 Intracellular calcium changes

may serve as second messenger for this response by increasing the number of open Na+ channels.92 The intercalated cells in the renal cortex and the tubular cells of the outer medullary region seem to be the sites for aldoster¬ one action on H+ secretion.93 These cells do not activate Na+ reabsorption; therefore, aldosterone-mediated H+ secretion and Na+ reabsorption occur in different regions of the kidney. How¬ ever, there is a modest Na+/H+ exchange process mediated by aldosterone in the principal cells. Although aldosterone acts almost exclusively on the kidney, there are other epithelial sites (e.g., the colon, sweat and salivary glands) where the hormone reduces the Na+ and raises the K+ content of secretions.78 In very end-stage renal failure, the colonic secretion of K+ can become an important route of elimination. Aldosterone may be implicated in the fibrosis of left ventric¬ ular hypertrophy and congestive heart failure.94-97 In fact, the heart could be capable of de novo synthesis. The hormone may have effects on vascular remodeling and collagen formation as well as modifying endothelial cell function, and the cardiac angiotensin AT, receptor may be one of its targets.94-97 A local or tissue aldosterone, which has been found in vascular smooth muscle, may contribute to A-II-induced vascular hypertro¬ phy.96 Thus, hypertension, cardiac fibrosis, and hypertrophy by nonepithelial cells may, in part, be consequences of high aldos¬ terone levels, in either the primary or the secondary forms of hyperaldosteronism.95'96 Evidence suggests that many of the effects of aldosterone on blood pressure and the resultant pathologic changes could be mediated through specific miner¬ alocorticoid receptors in the AV3V region of the brain and in the heart.98 Some of these nonepithelial effects are blocked either by aldosterone-receptor antagonists (e.g., spironolactone) or by selective aldosterone-receptor agonists (e.g., eplerenone). In the Randomized Aldactone Evaluation Study (RALES) trial, the addition of low-dose spironolactone (25 mg) to standard ther¬ apy for congestive heart failure further reduced total mortality and/or the hospitalization rate for heart failure by 31%." Hyperaldosteronism occurs in renal failure despite normal renin levels. Furthermore, aldosterone appears to contribute to the progression of renal disease by causing fibrosis and glomer¬ ular damage, as seen in the rat remnant kidney model.100 More details of aldosterone action may be seen in a review.1003

CONTROL OF ALDOSTERONE SECRETION The Renin-Angiotensin System. A-II is the major stimula¬ tor of aldosterone secretion. The proximal site of action of A-II is in enhancing the conversion of cholesterol to pregnenolone, but it also can modulate the distal regulatory site influencing the con¬ version of corticosterone to aldosterone by acting on aldosterone synthase.80,101'102 In general, the renin-angiotensin system is the major mediator of the volume-induced changes in aldosterone. Thus, volume and/or salt depletion will stimulate renin release, producing more A-II to act on the adrenal gland. The resulting Na+ retention then restores the volume to normal and shuts off renin release. The converse effect is seen with volume overload or with a high-salt diet, in which the renin-angiotensin system and aldosterone secretion are suppressed, allowing excess Na+ to be excreted. With volume expansion, there also is release of the ANPs and of dopamine, which, as inhibitors of aldosterone secre¬ tion, may act in concert with a low A-II level to limit the release of this mineralocorticoid. Plasma Potassium Concentration. There is a direct rela¬ tionship between increases in plasma K+ and increases in aldos¬ terone secretion.103 Studies of K+ infusion in humans show that a 0.1- to 0.2-mEq/L change in plasma K+ can alter aldosterone levels, suggesting that the zona glomerulosa is exquisitely sen¬ sitive to K\104 In the feedback cycle, an increasing plasma K+ level stimulates aldosterone, which in turn increases K+ secre¬ tion and restores it to normal. This mechanism protects against excessive K+ in the body; it represents the single pathway for

Ch. 79: Renin-Angiotensin System and Aldosterone

handling K+ overload. K+ has a direct effect on the zona glomerulosa cells to stimulate aldosterone synthesis, thereby activat¬ ing the conversion of cholesterol to pregnenolone.105 Although both A-II and K+ act on the zona glomerulosa through different and independent effects, their cumulative action can be syner¬ gistic.106 Thus, A-II is more effective in releasing aldosterone when the subject is on a high K+ diet. This effect may be a result of adrenal renin, because in vitro studies show that K+ enhances adrenal renin and adrenal A-II. Both young and elderly subjects have similar basal serum potassium levels and similar responses to a K+ infusion; however, during a K+ infusion, hyperkalemia is more pronounced in elderly subjects because of a blunted adrenal response.107 Corticotropin. As with cortisol release, ACTH, acting through adenylate cyclase, acutely stimulates aldosterone secretion in a dose-dependent manner; however, this effect is not sustained.108 Thus, ACTH is not considered an important chronic regulator of aldosterone. ACTH initially stimulates and then suppresses the aldosterone-synthase gene.108 ACTH also directs steroid biosynthesis through the enzyme 17a-hydroxylase, which diverts synthesis away from the mineralocorticoid pathway and into the glucocorticoid and androgen pathways. Other Factors. The plasma Na+ concentration is a weak regulator of aldosterone; hyponatremia increases aldosterone levels, and hypernatremia possibly has the opposite effect.109 In humans, extreme changes in plasma Na+ are required to alter aldosterone secretion independently from more powerful stim¬ uli (e.g., A-II and K+). Hyponatremia usually involves water retention, which (by suppression of A-II) overrides any direct action of Na+ on adrenal gland aldosterone synthesis.110 There is a modest effect of systemic acidosis on adrenal aldosterone release; this increases the excretion of acid and reduces sys¬ temic acidemia.111 The effect of aldosterone is thought to be through the H+-ATPase pumps on the luminal membrane of the collecting tubules. Aldosterone escape occurs when, after several days of adminis¬ tration of this hormone, there is a loss of normal sodium- and fluid-retaining capacity, and a spontaneous diuresis occurs. This escape mechanism, which is caused by increases in ANPs and by a rise in renal perfusion pressure that limits Na+ retention, is observed in primary hyperaldosteronism and perhaps in the inherited forms of mineralocorticoid hypertension, conditions in which edema is not seen, despite a volume retention state.112

REFERENCES 1. Corvol P, Jeunemaitre X. Molecular genetics of human hypertension: role of angiotensinogen. Endocr Rev 1997; 18(5):662. 2. Kim HS, Krege JH, Kluckman KD, et al. Genetic control of blood pressure and the angiotensinogen locus. Proc Natl Acad Sci USA 1995; 92:2735. 3. Brasier AR, Li J. Mechanisms for inducible control of angiotensinogen gene transcription. Hypertension 1996; 27:465. 4. Klett CP, Printz MP, Bader M, et al. Angiotensinogen messenger RNA sta¬ bilization by angiotensin II. J Hypertens 1996; 14(Suppl 1):S25. 5. Wielbo D, Sernia C, Gyurko R, Phillips MJ. Antisense inhibition of hyper¬ tension in the spontaneously hypertensive rat. Hypertension 1995; 25:314. 6. Naftilan AJ, Zuo WM, Ingelfinger J, et al. Localization and differential reg¬ ulation of angiotensinogen mRNA expression in the vessel wall. J Clin Invest 1991; 88:1300. 7. Schunkert H, Ingelfinger JR, Hirsch AT, et al. Evidence for tissue specific activation of renal angiotensinogen mRNA expression in chronic stable experimental heart failure. J Clin Invest 1992; 90:1523. 8. lnoue I, Nakajima T, Williams CS, et al. A nucleotide substitution in the promoter of human angiotensinogen is associated with essential hyperten¬ sion. J Clin Invest 1997; 99:1786. 9. Corvol P, Persu A, Gimenez-Roqueplo AP, Jeunemaitre X. Seven lessons from two candidate genes in human essential hypertension: angiotensino¬ gen and epithelial sodium channel. Hypertension 1999; 33:1324. 10. Freire MBS, Ji L, Onuma T, et al. Gender-specific association of M235T polymorphism in angiotensinogen gene and diabetic nephropathy in NIDDM. Hypertension 1998; 31:896. 11. Kunz R, Kreutze R, Beige J, et al. Association between the angiotensinogen 235T variant and essential hypertension in whites: a systematic review and methodological appraisal. Hypertension 1997; 30:1331.

771

12. Staessen JA, Kuznetsova T, Wang JG, et al. M235T angiotensinogen poly¬ morphism and cardiovascular renal risk. J Hypertens 1999; 17:9. 13. Atwood LD, Kammerer CM, Samollow PB. Linkage of essential hyperten¬ sion to the angiotensinogen locus in Mexican Americans. Hypertension 1997; 30:326. 14. Niu T, Xu X, Rogus J, et al. Angiotensinogen gene and hypertension in Chi¬ nese. J Clin Invest 1998; 101:188. 15. Rakugi H, Jacob HJ, Krieger JE, et al. Vascular injury induces angiotensino¬ gen gene expression in the media and neointima. Circulation 1993; 87:283. 16. Umemura S, Kihara M, Sumida Y, et al. Endocrinological abnormalities in angiotensinogen-gene knockout mice: studies of hormonal responses to dietary salt loading. J Hypertens 1998; 16:285. 17. Navar LG, Inscho EW, Majid SA, et al. Paracrine regulation of the renal microcirculation. Physiol Rev 1996; 76:425. 18. Skott O, Jensen BL. Cellular and intrarenal control of renin secretion. Clin Sci 1993; 84:1. 19. Toffelmire EB, Slater K, Corvol P, et al. Response of prorenin and active renin to chronic and acute alterations of renin secretion in normal humans. Studies using a direct immunoradiometric assay. J Clin Invest 1989; 83:679. 20. Hosoi M, Kim S, Takada T, et al. Effects of prorenin on blood pressure and plasma renin concentrations in stroke-prone spontaneously hypertensive rats. Am J Physiol 1992; 262:E234. 21. Gomez RA, Chevalier RL, Everett AD. Recruitment of renin gene-express¬ ing cells in adult rat kidney. Am J Physiol 1990; 259:F660. 22. Sigmund CD, Gross KW. Structure, expression, and regulation of the murine ren genes. Hypertension 1991; 18:446. 23. Kopp U, DiBona GF. Interaction of renal (11-adrenoreceptors and prosta¬ glandins in reflex renin release. Am J Physiol 1983; 244:F418. 24. Wagner C, Jensen BL, Kramer BK, Kurtz A. Control of the renal renin sys¬ tem by local factors. Kidney Int 1998; 54(Suppl 67):S78. 25. Lee MA, Bohm M, Paul M, et al. Physiologic characterization of the hyper¬ tensive transgenic rat TGR(mREN2)27. Am J Physiol 1996; 270(Endocrinol Metab 33):E919. 26. Erdos EG, Skidgel RA. Metabolism of bradykinin by peptidases in health and disease. In: Farmer SG, ed. The kinin system: handbook of immunopharmacology. London: Academic Press, 1997:112. 27. Linz W, Wiemer G, Gohlke P, et al. Contribution of the kinins to the cardio¬ vascular actions of angiotensin-converting enzyme inhibitors. Pharmacol Rev 1995; 47:25. 28. Ramchandran R, Kasturi S, Douglas J, et al. Metalloprotease-mediated cleavage secretion of pulmonary ACE by vascular endothelial and kidney epithelial cells. Am J Physiol 1996; 27LH744. 29. Soubrier F, Wei L, Hubert C, et al. Molecular biology of the angiotensin Iconverting enzyme, II: structure function: gene polymorphism and clinical implications. J Hypertens 1993; 11:599. 30. Wei I, Clauser F, Athenc-Gelas F, Corval P. The two homologous domains of human angiotensin I-converting enzyme interact differently with com¬ petitive inhibitors. J Biol Chem 1992; 267:13398. 31. Azizi M, Rousseau A, Ezan E, et al. Acute angiotensin-converting enzyme inhibition increases the plasma level of the natural stem cell regulator Nacetylseryl-lysyl-proline. J Clin Invest 1996; 97:839. 32. Rigat B, Hubert C, Alhenc-Gelas F, et al. An insertion/deletion polymor¬ phism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest 1990; 86:1343. 33. Van der Kliej FGH, Schmidt A, Navis GJ, et al. Angiotensin converting enzyme insertion / deletion polymorphism and short-term renal response to ACE inhibition: role of sodium status. Kidney Int 1998; 53(Suppl 63):S23. 34. Marre M, Bernadet P, Gallois Y, et al. Relationship between angiotensin I converting enzyme gene polymorphism, plasma levels, and diabetic retinal and renal complications. Diabetes 1994; 43:384. 35. Biege J, Scherer S, Weber A, et al. Angiotensin-converting enzyme geno¬ type and renal allograft survival. J Am Soc Nephrol 1997; 8:1319. 36. Kimura H, Geiyo F, Suzuki S, et al. Polymorphisms of angiotensin convert¬ ing enzyme and plasminogen activator inhibitor-1 genes in diabetes and microangiopathy. Kidney Int 1998; 54:1659. 37. Urata H, Nishimura H, Ganten D. Chymase-dependent angiotensin II forming system in humans. Am J Hypertens 1996; 9:277. 38. Harding JW, Wright JW, Swanson GN, et al. AT4 receptors: specificity and distribution. Kidney Int 1994; 46:1510. 39. Chappel MC, Pirro NT, Sykes A, Ferrario CM. Metabolism of angiotensin (1-7) by angiotensin converting enzyme. Hypertension 1998; 31:362. 40. Ferrario CM, Chappel MC, Tallant EA, et al. Counterregulatory actions of angiotensin (1-7). Hypertension 1997; 30:535. 41. Unger T, Gohlke P. Tissue renin-angiotensin systems in the heart and vas¬ culature: possible involvement in the cardiovascular actions of converting enzyme inhibitors. Am J Cardiol 1990; 65:31. 42. Dostal DE, Baker KM. Evidence for a role of an intracardiac renin-angiotensin system in normal and failing hearts. Trends Cardiovasc Med 1993; 3:67. 43. Martin P, Wagner J, Dzau VJ. Gene expression of the renin-angiotensin sys¬ tem in human tissues: quantitative analysis by polymerase chain reaction. J Clin Invest 1993; 91:2058. 44. Wang DH, Du Y, Zhao H, et al. Regulation of angiotensin I receptor and its gene expression: role in renal growth. J Am Soc Nephrol 1997; 8:193. 45. Yanagawa N. Potential role of local luminal angiotensin II in proximal tubule sodium transport. Kidney Int Suppl 1991; 32:S33. 46. Kamide K, Hori M, Tuck ML, et al. Insulin-mediated growth in aortic smooth muscle and the vascular renin-angiotensin system. Hypertension 1998; 32:482.

772

PART V: THE ADRENAL GLANDS

47. Nemeth G, Pepperell JR, Yamada Y, et al. The basis and evidence of a role for the ovarian renin-angiotensin system in health and disease. J Soc Gynecol Investig 1994; 1:118. 48. Speth RC, Daubert DL, Grove KL. Angiotensin II: a reproductive hormone too? Regul Pept 1999; 79(1):25. 49. Hageman JR, Moyer JS, Bachman ES, et al. Angiotensin-converting enzyme and male fertility. Proc Natl Acad Sci U S A1998; 95:2552. 50. Jones BH, Standbridge MK, Taylor JW, et al. Angiotensinogen gene expres¬ sion in adipose tissue, analysis of obese models and hormonal and nutritional control. Am J Physiol 1997; 273(Regulatory Integrative Comp Physiol 42):236. 51. Kifor I, Moore TJ, Fallo F, et al. Potassium-stimulated angiotensin release from superfused adrenal capsules and enzymatically digested cells of the zona glomerulosa. Endocrinology 1991; 129:823. 52. Phillips MI, Speakman EA, Kimura B. Levels of angiotensin and molecular biology of the tissue renin angiotensin system. Regul Pept 1993; 43:1. 53. Baltatu O, Nishimura H, Hoffman S, et al. High levels of human chymase expression in the pineal and pituitary gland. Brain Res 1997; 759:269. 54. Griendling KK, Lassegue B, Alexander RW. The vascular angiotensin (ATI) receptor. Thromb Haemost 1993; 70(1):188. 55. Coffman TM. Gene targeting in physiological investigations: studies of the renin-angiotensin system. Am J Physiol 1998; 274 (Renal Physiol):F999. 56. Sadoshima J. Versatility of the angiotensin II type 1 receptor. Circ Res 1998; 82:1352. 56a. Schiffrin EL, Park JB, Intengan HD, Touyz RM. Correction of arterial struc¬ ture and endothelial dysfunction in human essential hypertension by the angiotensin receptor antagonist losartan. Circulation 2000; 101(14):1653. 57. Horiuchi M, Akishita M, Dzau VJ. Recent progress in angiotensin II type 2 receptor research in the cardiovascular system. Hypertension 1999; 33:613. 58. Cogan MG. Angiotensin II: a powerful controller of sodium transport in the early proximal tubule. Hypertension 1990; 15:451. 59. Okubo S, Numura F, Matsusaka T, et al. Angiotensinogen gene null-mutant lacks homeostatic regulation of glomerular filtration and tubular reabsorp¬ tion. Kidney Int 1998; 53:617. 60. Scholz H, Kurtz A. Role of protein kinase C in renal vasoconstriction caused by angiotensin II. Am J Physiol 1990; 259:C421. 61. Clemson B, Gaul L, Gubin SS, et al. Prejunctional angiotensin II receptors. Facilitation of norepinephrine release in the human forearm. J Clin Invest 1994; 93:684. 62. Moreau P, d'Uscio LV, Shaw S, et al. Angiotensin II increases tissue endothelin and induces vascular hypertrophy: reversal by ET(A)-receptor antagonist. Circulation 1997; 96:1593. 63. Golub MS, Hori MT, Tuck ML. Arachidonic acid metabolites in the vascutature. In: Sowers JR, ed. Endocrinology of the vasculature. Totowa, NJ: Humana Press, 1996:357. 64. Ichikawa I, Harris RC. Angiotensin actions in the kidney: renewed insight into the old hormone. Kidney Int 1991; 40:583. 65. Wilcox CS, Welch WJ, Snellen H. Thromboxane mediates renal hemody¬ namic response to infused angiotensin II. Kidney Int 1991; 40:1090. 66. Miller JA, Anacata LA, Cattran DC. Impact of gender on the response to angiotensin II. Kidney Int 1999; 55:278. 67. Wolf G, Ziyadeh FN. The role of angiotensin II in diabetic nephropathy: emphasis on nonhemodynamic mechanisms. Am J Kidney Dis 1997; 29:153. 68. Dzau VJ, Re R. Tissue angiotensin system in cardiovascular medicine: a paradigm shift. Circulation 1994; 89:493. 69. Gibbons GH. The pathophysiology of hypertension. The importance of angiotensin II in cardiovascular remodeling. Am J Hypertens 1998; 11:177S. ’ 70. Alderman MH, Madhaven S, Ooi WL, et al. Association of renin-sodium profile with risk of myocardial infarction in patients with hypertension. N Engl J Med 1991; 324:1098. 71. Vaughan DE. The renin-angiotensin system and fibrinolysis. Am J Cardiol 1997; 79(5A):12. 72. Brown NJ, Agirbasli MA, Williams GH, et al. Effect of activation and inhi¬ bition of the renin-angiotensin system on plasma PAI-1. Hypertension 1998; 32:965. 73. Hwan K, Thornhill B, Wolstenholme T, Chevalier RL. Tissue-specific regu¬ lation of growth factors and clusterin by angiotensin II. Am J Hypertens 1998; 11:715. 74. Morishita R, Ogihara T. In vivo evaluation of in vitro hypothesis using "gain or loss" of function: functional analysis of the renin-angiotensin sys¬ tem. Am J Hypertens 1998; 11:507. 75. Tomita N, Morishita R, Higaki J, et al. Transient decrease in high blood pressure by in vitro transfer of antisense oligodeoxynucleotides against angiotensinogen. Hypertension 1995; 26:131. 76. Phillips MI. Antisense inhibition and adeno-associated viral vector deliv¬ ery for reducing hypertension. Hypertension 1997; 29:177. 77. Marver D, Kokko JP. Renal target sites and the mechanism of action of aldosterone. Min Elect Metab 1983; 9:1. 78. White PC. Mechanisms of disease. Disorders of aldosterone biosynthesis and action. N Engl J Med 1994; 331:250. 79. Connell JM, Fraser R. Adrenal cortical synthesis and hypertension. J Hyper¬ tens 1991; 9:97. 80. Holland OB, Carr B. Modulation of aldosterone synthase messenger ribo¬ nucleic acid levels by dietary sodium and potassium and by adrenocorticotropin. Endocrinology 1993; 132:2666. 81. Arriza JL, Weinberger C, Cerelli C, et al. Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. Science 1987; 237:268.

82. Funder JW. Mineralocorticoids, glucocorticoids, receptors and response elements. Science 1993; 259:1132. 83. Bastl CP, Hayslett. The cellular action of aldsterone in target epithelia. Kid¬ ney Int 1992; 42:250. 84. Zennaro M-C, Farman N, Bonvalet J-P, et al. Tissue-specific expression of alpha and beta messenger ribonucleic acid isoforms of the human mineralocorticoid receptor in normal and pathological states. J Clin Endocrinol Metab 1997; 82:1345. 85. Funder JW. Enzymes and the regulation of sodium balance. Kidney Int 1992; 37(Suppl):S114. 86. Kenouch S, Coutry N, Farman N, Bonvalet J-P. Multiple patterns of 11(1hydroxysteroid dehydrogenase catalytic activity along the mammalian nephron. Kidney Int 1992; 42:56. 87. Naray-Fejes-Toth A, Rusvai E, Fejes-Toth G. Mineralocorticoid receptors and ll|3-hydroxysteroid dehydrogenase activity in renal principal and intercalated cells. Am J Physiol 1994; 266:F76. 87a. Funder JW. Aldosterone and mineralocorticoid receptors: orphan ques¬ tions. Kidney Int 2000; 57(4):1358. 88. Horisberger JD, Rossier BC. Aldosterone regulation of gene transcription leading to control of ion transport. Hypertension 1992; 19:221. 89. Szerlip H, Palevsky P, Cox M, Blazer-Yost B. Relationship of the aldoster¬ one-induced protein GP70 to the conductive channel. J Am Soc Nephrol 1991; 2:1108. 90. Schafer JA, Hawk CT. Regulation of Na+ channels in the cortical collecting duct by AVP and mineralocorticoids. Kidney Int 1992; 41:255. 91. Hayhurst RA, O'Neil RG. Time-dependent actions of aldosterone and amiloride-blockable sodium channels in A6 epithelia. Am J Physiol 1988; 254:F689. 92. Petzel D, Ganz MB, Nestler EJ, et al. Correlates of aldosterone-induced increases in Ca?+ and Isc suggest that Ca?+ is the second messenger for stimulation of apical membrane conductance. J Clin Invest 1992; 89:150. 93. Hays SR. Mineratocorticoid modulation of apical and basolateral mem¬ brane HVOH /HCO3- transport processes in rabbit inner strip of outer medullary collecting duct. J Clin Invest 1992; 90:180. 94. Brilla CG, Weber KT. Mineralocorticoid excess, dietary medium and myo¬ cardial fibrosis. J Lab Clin Med 1992; 120:893. 95. Young M, Fullerton M, Dilley P, Funder J. Mineralocorticoids, hypertension and cardiac fibrosis. J Clin Invest 1994; 93:2578. 95a. Falkenstein E, Christ M, Feuring M, Wehling M. Specific nongenomic actions of aldosterone. Kidney Int 2000; 57(4):1390. 96. Hakeyama H, Miramuri I, Fujita T, et al. Vascular aldosterone, biosynthesis and a link to angiotensin Il-induced hypertrophy of vascular smooth mus¬ cle. J Biol Chem 1994; 269:24316. 97. Robert V, Heymes C, Silvestre J-B, et al. Angiotensin ATI receptor subtype as a cardiac target of aldosterone; role in aldosterone-salt-induced fibrosis. Hypertension 1999; 33:981. 98. Gomez Sanchez EP. What is the role of the central nervous system in min¬ eralocorticoid hypertension? Am J Hypertension 1991; 4:374. 99. The RALES Investigators. Effectiveness of spironolactone added to an angiotensin converting enzyme inhibitor and a loop diuretic for severe chronic congestive heart failure (The Randomized Aldactone Evaluation Study [RALES]). Am J Cardiol 1996; 78:902. 100. Ibrahim HN, Rosenberg ME, Greene EL, et al. Aldosterone is a major factor in the progression of renal disease. Kidney Int 1997; 52(Suppl 63):S115. 100a. Farman N, Verrey F. Forefronts in nephrology news in aldosterone action. Kidney Int 2000; 57:1239. 101. Brann DW, Hendry LB, Mahesh VB. Emerging diversities in the mechanism of action of steroid hormones. J Steroid Biochem Molec Biol 1995; 52:113. 102. Adler GK, Chen R, Menachery Al, et al. Sodium restriction increases aldosterone synthesis by increased late pathway, not early pathway, messenger ribonucleic acid levels and enzyme activity in normal rats. Endocrinology 1993; 133:2235. 103. Young DB. Quantitative analysis of aldosterone's role in potassium regula¬ tion. Am J Physiol 1988; 255:F811. 104. Himathongan T, Dluhy R, Williams GH. Potassium-aldosterone-renin interrelationships. J Clin Endocrinol Metab 1975; 41:153. 105. Kifor I, Moore TJ, Fallo F, et al. Potassium-stimulated angiotensin release from superfused adrenal capsules and enzymatically digested cells of the zona glomerulosa. Endocrinology 1991; 129:823. 106. Young DB, Smith MJ Jr, Jackson TE, Scott RE. Multiplicative interaction between angiotensin II and K concentration in stimulation of aldosterone. Am J Physiol 1984; 247:E328. 107. Mulkerrin E, Epstein FH, Clark BA. Aldosterone responses to hyperkale¬ mia in healthy elderly humans. J Am Soc Nephrol 1995; 6:1459. 108. Braley LM, Adler GK, Mortensen RM, et al. Dose effect of adrenocorticotropin on aldosterone and cortisol biosynthesis in cultured bovine adrenal glomerulosa cells. In vitro correlate of hyperreninemic hypoaldosterone. Endocrinology 1992; 131:187. 109. Merrill DC, Ebert TJ, Skelton MM, Cowley AW Jr. Effect of plasma sodium on aldosterone during angiotensin II stimulation in normal humans. Hypertension 1989; 14:164. 110. Taylor RE, Glass GT, Radke KJ, Schneider EG. Specificity of effect of osmo¬ lality on aldosterone secretion. Am J Physiol 1987; 252:E118. 111. Jones GV, Wall BM, Williams HH, et al. Modulation of plasma aldosterone by physiologic changes in hydrogen ion concentration. Am J Physiol 1992; 262:R269. 112. White PC. Inherited forms of mineralocorticoid hypertension. Hyperten¬ sion 1996; 28:927.

Ch. 80:

Hyperaldosteronism

773

Hypokalemia with Alkalosis

CHAPTER

80

/

HYPERALDOSTERONISM

\

Hypertension

Normal Blood Pressure

JOHN R. GILL, JR.

DISORDERS OF MINERALOCORTICOID EXCESS Mineralocorticoid excess, whether primary or secondary, and disorders that mimic it are commonly manifested clinically as hypokalemia, which usually but not always is accompanied by alkalosis. Thus, a useful strategy for diagnosing hypokalemia is to approach the evaluation as a differential diagnosis of disor¬ ders of mineralocorticoid excess. Clinical features that accom¬ pany hypokalemia and may be used to determine the specific disorders that need to be considered are illustrated in Table 80-1 and in the flow chart in Figure 80-1. This basic framework is expanded in the course of this review to include the disorders appropriate to each of the four listed patterns of abnormalities of renin and aldosterone. Ideally, evaluation of the renin and aldosterone systems should be done in the absence of medica¬ tion during a standardized sodium intake (e.g., 100 mEq per day of sodium as sodium chloride). If this is not possible, then at least the use of medications that affect the production of renin and aldosterone (i.e., diuretics and angiotensin convert¬ ing enzyme inhibitors) should be discontinued and the blood pressure should be controlled with a calcium channel blocking agent. The level of sodium intake immediately preceding the

Low PRA High ALDO

Low PRA Low ALDO

High PRA High ALDO

High PRA High ALDO

FIGURE 80-1. Flow chart for evaluation of patients with hypokalemic alkalosis. {PRA, plasma renin activity; ALDO, aldosterone.) (See Table 80-1 for continuation of evaluation.)

sampling of blood for plasma renin activity and plasma aldo¬ sterone should be estimated by collecting a 24-hour aliquot of urine for the determination of sodium excretion. The blood for the plasma renin activity and plasma aldosterone evaluations should be drawn after overnight bedrest (in the hospital) or after 30 minutes of bedrest (in the clinic), and then drawn again after 2 hours of standing and walking.

PHYSIOLOGY OF ALDOSTERONE SYNTHESIS The zona glomerulosa cells of the adrenal cortex synthesize aldosterone from corticosterone in a two-step, mixed function oxi¬ dation reaction that is catalyzed by the enzyme corticosterone

TABLE 80-1. Disorders Characterized by Hypokalemia and Alkalosis HYPERALDOSTERONISM (SO-CALLED "PRIMARY")

Hypertension

Clinical Features

High plasma renin activity

Hypokalemia Alkalosis

High aldosterone levels

Disorders

Hypertension

Renal artery stenosis

Low plasma renin activity

Renin-secreting tumor

High aldosterone levels

Disorders Aldosterone-producing adenoma Idiopathic hyperaldosteronism Primary adrenal hyperplasia Dexamethasone-suppressible hyperaldosteronism Adrenocortical carcinoma

SYNDROMES OF REAL OR APPARENT MINERALOCORTICOID EXCESS NOT CAUSED BY ALDOSTERONE Clinical Features Hypokalemia Alkalosis Hypertension

Malignant hypertension Chronic renal disease

SECONDARY HYPERALDOSTERONISM AND NORMAL BLOOD PRESSURE Clinical Features Hypokalemia Alkalosis Normal blood pressure High plasma renin activity High aldosterone levels

Disorders Renal disorders Renal tubular acidosis

Low plasma renin activity

Nephritis

Low aldosterone levels

Cystinosis

Disorders 11 (1-Hydroxylase deficiency 17-Hydroxylase deficiency Liddle syndrome 11 (3-Hydroxysteroid dehydrogenase deficiency Licorice ingestion

SECONDARY HYPERALDOSTERONISM AND HYPERTENSION Clinical Features Hypokalemia Alkalosis

Bartter syndrome Magnesium-losing tubulopathy Calcium-losing tubulopathy Hepatic cirrhosis Cardiac failure Gastrointestinal disorders Covert vomiting Covert laxative abuse Familial chloride diarrhea Covert diuretic abuse

774

PART V: THE ADRENAL GLANDS

the renin-angiotensin system and its control of aldosterone secretion is presented in Chapters 79 and 183; ANH is discussed in Chapter 178.

M-Enz i

0 HO

OH CH

R

O? M-Enz STEP 2 CORTICOSTERONE

OH

18-HYDROXY

ALDOSTERONE

CORTICOSTERONE FIGURE 80-2. Proposed mechanism and structure of intermediates in the conversion of corticosterone to aldosterone. (From Ulick S. Diagno¬ sis and nomenclature of the disorders of the terminal portion of the aldosterone biosynthetic pathway. J Clin Endocrinol Metab 1976; 43:92.)

methyl oxidase1 (Fig. 80-2). Normally, the adrenal glands secrete 60 to 200 pg aldosterone each day, depending on the state of sodium chloride and water balance of the body; the steroid circulates in the blood either free or loosely bound to albumin. Although some of the circulating aldosterone may be excreted in the urine in the free form, most of it is metabolized by the liver to tetrahydroaldosterone and by the kidneys to aldosterone-18-glucuronide. Measurement of the excretion of the 18-glucuronide metabolite has been used as an index of aldosterone production; values obtained by this method range from 1 to 15 pg per day in normal persons receiving an unrestricted sodium chloride intake.

SECRETION Aldosterone biogenesis is regulated primarily by the renin-angio¬ tensin system, with potassium, atrial natriuretic hormone (ANH), and dopamine also making important contributions. Corticotropin (ACTH) also may stimulate aldosterone production, but its effects are self-limited except in certain disease states. In addi¬ tion, the factors that determine aldosterone secretion have direct effects on the kidney and vasculature and, along with aldoster¬ one, contribute to the maintenance of the volume of extracellu¬ lar fluid, the concentration of extracellular potassium, and, ultimately, blood pressure. Thus, when the intake of sodium chloride is restricted and blood volume is contracted, an increase in the formation of angiotensin II stimulates the secre¬ tion of aldosterone. Angiotensin II and aldosterone, in turn, both increase the reabsorption of sodium by the renal tubule to cur¬ tail sodium loss and restore extracellular volume. Conversely, when sodium chloride intake is increased and blood volume is expanded, the formation of angiotensin II decreases, ANH is secreted by the heart, and the formation of dopamine by the adrenal glands and kidneys increases. ANH and dopamine inhibit aldosterone biogenesis; they also increase sodium excre¬ tion through direct effects on the kidney. Potassium exerts dif¬ ferential effects on aldosterone biosynthesis. Hyperkalemia, secondary to potassium administration or retention, stimulates aldosterone secretion, whereas hypokalemia, secondary to potassium depletion, inhibits it. A more detailed discussion of

ACTIONS Aldosterone exerts its biologic actions by crossing the plasma membrane of target cells and binding to its receptor in the cyto¬ sol.2 The aldosterone-receptor complex then migrates to the nucleus, where it binds, thereby initiating transcription, trans¬ lation, and synthesis of proteins responsible for expression of the action of aldosterone on the functions of that target cell (see Chap. 4). Collecting tubule cells of the kidney and epithelial cells of other transporting tissues (i.e., sweat, salivary gland, and intestine) are target tissues in which the physiologic effects of aldosterone are most discernible, although effects on vascular3 and other tissues may occur. In epithelial cells for which the physiologic actions of aldosterone have been exten¬ sively characterized, the steroid facilitates passage of sodium across the apical membrane into the cell and accelerates extru¬ sion of sodium and uptake of potassium by Na+,K+-adenosine triphosphatase located in the basolateral membrane.4 In the renal collecting tubule, these actions of aldosterone increase the reabsorption of sodium and the secretion of potassium, leading to retention of sodium by the body and loss of potassium (see Chap. 206). As the foregoing discussion indicates, the renin-angiotensinaldosterone systems regulate the volume of extracellular fluid and contribute to the maintenance of the extracellular potas¬ sium concentration. As a result of disease, the production of aldosterone may become supernormal and cease to be responsive to the physiologic stimuli that regulate it. The overproduc¬ tion of aldosterone may result from overactivity of the reninangiotensin system (secondary hyperaldosteronism) or from other abnormalities (primary hyperaldosteronism). The consequences of continual overproduction of aldosterone, or hyperaldoster¬ onism, depend in part on the disorder with which it is associ¬ ated, and may include excessive sodium chloride and water retention, potassium loss, alkalosis, and hypertension.

“PRIMARY ALDOSTERONISM” Autonomous overproduction of aldosterone occurs in ~2% of patients with hypertension and was recognized initially as being associated with either a unilateral adenoma (aldosterone-producing adenoma, or APA) or bilateral hyperplasia of the zona glomerulosa. Because patients with bilateral adrenal disease tended to have a clinical presentation similar to that of patients with an APA (hypertension and hypokalemia) and, like the patients with unilateral disease, they showed suppression of the renin-angio¬ tensin system, they also were assumed to have a primary adrenal disorder. Subsequent observations have not proved this to be true; they suggest, instead, that bilateral hyperplasia of the zona glomerulosa probably is not a primary adrenal disorder in most cases and may have more than one cause. Unilateral adrenalec¬ tomy usually cures or ameliorates the hypertension in patients with an APA, whereas subtotal or total adrenalectomy, with few exceptions, has little effect on the blood pressure of patients with bilateral hyperplasia. In the case of those few patients who have primary adrenal hyperplasia, unilateral adrenalectomy may be curative.5 In some patients with bilateral hyperplasia, the hyper¬ aldosteronism was dependent on ACTH (so-called glucocorticoidremediable hyperaldosteronism). The hypothesis that a trophic hormone, possibly of pituitary origin, may be responsible for bilateral hyperplasia not caused by ACTH has received consider¬ able support, although it has not been definitively proved. As a result of advances in knowledge, the inclusive designation of "primary hyperaldosteronism" for those patients with hyperal¬ dosteronism associated with both adenoma and bilateral hyper-

Ch. 80: Hyperaldosteronism

775

ALDOSTERONE-PRODUCING ADENOMAS

and water retention may contribute to the increase in blood pressure that occurs, the precise mechanisms responsible for an increase in peripheral resistance and the associated hyperten¬ sion are unclear. Receptors for aldosterone have been found in blood vessels and in the brain, and the effects of aldosterone on ion transport in these tissues may be important mediators of the increase in blood pressure. Preliminary studies in dogs indi¬ cate that an amount of aldosterone that is too small to exert an effect when infused peripherally increases blood pressure when it is infused into the third ventricle of the brain.13

CLINICAL FEATURES AND PATHOPHYSIOLOGY

DIAGNOSIS AND DIFFERENTIAL DIAGNOSIS

Aldosterone-producing adrenocortical adenomas usually are small (0.5-2.5 cm), unilateral, solitary, and associated with hypoplastic zona glomerulosa. Occasionally, however, these lesions may be multiple and associated with hyperplasia of the zona glomerulosa. This benign tumor of the adrenal cortex occurs most frequently in the third through the fifth decades of life but also has been observed in prepubertal children6 and older persons (range, 13-66 years). Aldosterone-producing ade¬ nomas occur equally in men and women and do not appear to have a racial predisposition; they tend to develop more fre¬ quently in the left adrenal gland than in the right, but this dif¬ ference is small. (The occurrence of primary aldosteronism resulting from APA in two or more members of the same family has been reported,7 as well as the familial occurrence of idiopathic aldo¬ steronism. In one of the families, one member had bilateral nodular hyperplasia and another had APA. This familial form of primary aldosteronism was labeled familial hyperaldoster¬ onism type II to distinguish it from glucocorticoid-remediable hyperaidosteronism. The clinical features of familial hyperal¬ dosteronism type II do not differ from those of sporadic patients with APA or idiopathic hyperaidosteronism.) The symptoms reported most frequently by patients with an APA are headache, easy fatigability, and weakness, although high blood pressure may be the only symptom in many cases. Hypokalemia, suppressed plasma renin activity, and high plasma and urinary aldosterone values are the findings usually associated with APA, but they may not always be demonstrable.8 Aldosterone production by an APA is autonomous and is affected only minimally by angiotensin II because of the decreased formation of this peptide as well as the decreased responsiveness of the adenoma. An APA is unresponsive to the infusion of dopamine, although endogenous dopamine may exert tonic inhibitory effects.9 ANH, levels of which appear to be elevated in patients with this disorder,10 does not inhibit aldosterone biogenesis by APA cells in vitro, as it does in nor¬ mal glomerulosa cells.11 Therefore, this hormone may have lit¬ tle effect on aldosterone secretion by an APA in vivo. In contrast, changes in serum potassium or ACTH levels may exert important regulatory effects on aldosterone secretion in patients with this tumor: aldosterone production may be blunted by the potassium loss and the hypokalemia that it induces. The circadian rhythm of aldosterone in patients with APA tends to mirror that of ACTH. As a consequence of sustained overproduction of aldoster¬ one, retention of sodium chloride and water occurs, but this usually is limited; "escape" occurs when extracellular fluid has been expanded by -500 mL.12 Initially, the renal excretion of potassium increases. As hypokalemia develops, potassium excretion decreases to reflect intake of this cation. Hydrogen ion excretion, which increases principally because of an increase in urinary ammonium, is responsible for the development and maintenance of metabolic alkalosis. Urinary calcium and mag¬ nesium levels are high; presumably, this is a reflection of the effects of expansion of extracellular fluid on the tubular reab¬ sorption of these ions. The urinary concentrating ability is impaired because of potassium depletion. Although sodium

Patients with hypertension who have borderline or low serum potassium concentrations, low stimulated plasma renin activity (14 ng/dL) should be evaluated further for auton¬ omous overproduction of aldosterone. The evaluation should be done before antihypertensive treatment is instituted or after it has been discontinued for 2 weeks. Determining aldosterone suppressibility, by combining a high sodium intake and treatment with a mineralocorticoid such as 9-afluorohydrocortisone for 3 or more days, or by intra¬ venous saline infusion, is a useful first step. Of these two inter¬ ventions, the infusion of 2 L normal saline over 4 hours with the measurement of plasma aldosterone levels before and at the conclusion of the infusion is simpler and may be readily adapted to the evaluation of outpatients.14 At the conclusion of the infusion, the plasma aldosterone level normally is 5 ng/dL or less; in patients with hyperaidosteronism, it usually exceeds 10 ng/dL. Determination of the plasma aldosterone to plasma renin activity ratio also has been used to detect aldosteronism. Single deter¬ minations of this ratio tend to be of limited use because values from patients with hyperaidosteronism frequently are the same as those from patients with normal adrenal function. A solution to the problem of overlap that appears to improve specificity considerably is to collect blood samples every 30 minutes for 6 hours, so that an integrated plasma aldosterone/plasma renin activ¬ ity ratio can be determined.15 A somewhat simpler but probably equally effective alternative is to collect blood 90 minutes after the administration of 25 to 50 mg captopril, which is a convert¬ ing enzyme inhibitor, for determination of the plasma aldos¬ terone/plasma renin activity ratio16 (Fig. 80-3). Both procedures are safe and do not require dietary preparation or hospitalization. It has been suggested that all patients with hypertension undergo captopril screening tests,17 because determination of the serum potassium concentration and plasma renin activity may be inadequate measures for screen¬ ing the hypertensive population for hyperaidosteronism.8 The normogram in Figure 80-3 indicates the degree of separation of patients with aldosterone excess from those with essential hypertension.16 Subsequent evaluation of the captopril test indicates a false-negative rate of 6.3% and a false-positive rate of 0.6% in patients with primary hyperaidosteronism.18 The problem of identifying those patients with hyperaidosteronism who harbor an adenoma has been greatly facilitated by techno¬ logic advances. Computed tomography (CT) appears to have the capacity to discern an adrenal mass in -80% of patients with an aldosterone-producing adenoma.19,20 Magnetic resonance imag¬ ing also has been used to evaluate the adrenal glands in patients with hyperaidosteronism; apart from confirming the findings of CT, however, it does not appear to offer any advan¬ tage over CT. These new techniques for imaging adrenal masses have replaced the more cumbersome and time-consuming pro¬ cedure of [131I]iodocholesterol imaging in most centers (see Chap. 88). A limitation of all the imaging techniques, however, is that an adenoma may be poorly visualized or not detected at all. These problems result from the small size of some ade¬ nomas, which may be less than 1 cm in diameter. The problem

plasia has tended to be replaced by the more specific designations of aldosterone-producing adenoma, idiopathic hyperaldosteronism, pri¬ mary adrenal hyperplasia, and glucocorticoid-remediable hyper¬ aldosteronism. Rarely, adrenocortical carcinoma may overproduce aldosterone. These five disorders present as hypokalemic alkalo¬ sis, hypertension, low or suppressed plasma renin activity, and high aldosterone concentrations (see Table 80-1).

776

PART V: THE ADRENAL GLANDS

ADENOMA

A°

HYPERPLASIA

APA o

500IHA A 400-

ALDO/PRA RATIO

300o 200-

o

A

180HB ng/dL

1 00-

m -'-1—

—i-1-1

50

100

150

—i

200

1-1—

250

ALDO (ng/dL) FIGURE 80-3. Values for the plasma aldosterone (ALDO)/plasma renin activity (PRA) ratio plotted as a function of plasma aldosterone for nor¬ mal and hypertensive control subjects (hatched area), patients with aldos¬ terone-producing adenoma (APA), and patients with idiopathic hyperaldosteronism (IHA) 2 hours after the administration of 25 mg captopril. The plasma aldosterone value decreased to 50 ng/dL in a patient with hyperaldosteronism suggests that the diagnosis is more likely to be an aldosterone-producing adenoma than bilateral hyper¬ plasia, even though the adrenal glands may appear normal (Fig. 80-4). If the plasma aldosterone level were to show a "par¬ adoxic" fall paralleling the circadian fall in plasma cortisol dur¬ ing 2 hours of ambulation after early morning sampling, this also would suggest that the diagnosis is more likely to be an aldosterone-producing adenoma than bilateral hyperplasia.22 So many patients with an adenoma, like those with bilateral hyperplasia, show a rise rather than a fall in the plasma aldos¬ terone level that the usefulness of the procedure is limited.8 The most reliable means of assessing adrenal glands in patients with hyperaldosteronism is bilateral catheterization of adrenal veins to sample for corticosteroids 23 The aldosterone/ cortisol ratio in samples of venous blood from both adrenal glands and a peripheral site, before and after ACTH stimula¬ tion, provides a good comparative characterization of adrenal function and an extremely reliable means of distinguishing between bilateral and unilateral overproduction of aldosterone. Table 80-2 shows the results of adrenal vein sampling in a patient with an aldosterone-producing adenoma. Determina¬ tion of the cortisol level provides important confirmation of roentgenographic evidence that the site of sampling is actually an adrenal vein; it also is used to correct values of adrenal venous aldosterone for dilution by blood of extraadrenal origin. In the illustrated patient, the findings show the importance of stimulation with ACTH to ensure that an adenoma is function¬ ing, rather than quiescent, at the time of sampling and also to demonstrate suppression of aldosterone production by the con¬ tralateral gland. Although adrenal vein sampling is an invasive procedure, it is associated with minimal morbidity. When used as just described, it has an accuracy >90% in the lateralization of an adenoma, making the distinction between an APA and those disorders associated with bilateral overproduction of

ALDO ng/dL

FIGURE 80-4. Plasma 18-hydroxycorticosterone (180HB) and plasma aldosterone (ALDO) concentrations at bedrest and after 4 hours of ambulation in patients with aldosterone-producing adenomas (ade¬ noma) or idiopathic hyperaldosteronism (hyperplasia). Note the wide separation in resting values for 180HB between patients with adenoma and patients with hyperplasia and the different responses of the two groups to ambulation. (From Biglieri EG, Schambelan M. The signifi¬ cance of elevated levels of plasma 18-hydroxycorticosterone in patients with primary aldosteronism. J Clin Endocrinol Metab 1979; 49:87.)

TABLE 80-2. Adrenal Venous Sampling

Location

Aldosterone (ng/dL)

Cortisol (gg/dL)

Aldosterone/ Cortisol Ratio

PATIENT WITH ALDOSTERONE-PRODUCING ADENOMA Basal

Inferior vena cava

17

13

1.3

Left adrenal vein

106

20

5.3

Right adrenal vein

110

571

0.2

After Corticotropin Inferior vena cava Left adrenal vein Right adrenal vein

91

25

3.6

143,000

2230

64.1

290

2440 PATIENT WITH IDIOPATHIC HYPERALDOSTERONISM

0.1

Basal

Inferior vena cava

18

9

2.0

Left adrenal vein

432

21

20.6

Right adrenal vein

300

18

16.7

After Corticotropin Inferior vena cava

36

14

2.6

Left adrenal vein

15,200

1700

8.9

Right adrenal vein

10,400

1230

8.6

Ch. 80: Hyperaldosteronism

aldosterone. The observations that APA may be associated with other abnormalities seen on CT scans such as ipsilateral and contralateral nonfunctioning nodules and thickening of an adrenal limb indicate the important role of adrenal vein sam¬ pling in reaching a correct diagnosis.20 Only occasionally will a patient with idiopathic aldosteronism have a gradient between the two adrenal glands with an aldosterone/cortisol ratio that approaches 4 to 1, the lower limit of the range of the ratio of APA to normal adrenal.24

TREATMENT The preferred therapy for patients with an aldosterone-produc¬ ing adenoma is operative removal of the adrenal gland contain¬ ing the tumor.25 Adenomectomy alone is not sufficient.253 This can be done laparoscopically with minimal morbidity26 (see Chap. 89). The cure rate (correction of hypertension as well as aldosteronism) ranges from 60% to 75%. Because the prevalence of a family history of hypertension in patients with an aldosteroneproducing adenoma may range from 40% to 60%, this, along with the duration of the hypertension, may account for the per¬ sistence of an elevated blood pressure despite the correction of hyperaldosteronism. Most of the patients who remain hyperten¬ sive after the correction of hyperaldosteronism become more responsive to antihypertensive medication. Spironolactone is probably the single most effective drug for treating patients with an APA. It acts as a competitive inhibitor, competing with aldosterone for its cytosol receptor, thereby antagonizing the action of the mineralocorticoid in target tis¬ sue. Spironolactone also is both an antiandrogen and a pro¬ gestagen, and this explains many of its distressing side effects;2,4 decreased libido, mastodynia, and gynecomastia may occur in 50% or more of men, and menometrorrhagia and mastodynia may occur in an equally large number of women taking the drug.27 The problem of side effects may preclude the long-term use of spironolactone, particularly in younger men and women, and is more likely to occur when the dosage exceeds 100 mg per day. The usual dosage of spironolactone ranges from 50 mg once daily to 100 mg twice daily. Drugs such as amiloride and triamterene also may oppose the effect of aldosterone on the renal tubule, but these agents act by inhibiting sodium reabsorption and potassium secretion through a direct effect on the tubule cell, not by competing with aldosterone for its receptor. This may explain why triamterene and amiloride tend to be less effective than spironolactone as antihypertensive agents in patients with hyperaldosteronism. Calcium channel blocking drugs, such as nifedipine, also may be useful therapeutic agents in patients with hyperaldo¬ steronism. In addition to its antihypertensive action, nifedipine may decrease aldosterone production.28 Thus, the combined use of nifedipine and a potassium-sparing diuretic may serve as an alternative to spironolactone in the medical treatment of patients with an APA.

IDIOPATHIC HYPERALDOSTERONISM CLINICAL FEATURES AND PATHOPHYSIOLOGY Idiopathic bilateral adrenocortical hyperplasia of the zona glomerulosa (idiopathic hyperaldosteronism) is characterized by fea¬ tures similar to those associated with APAs, although they are less pronounced in many patients.29 The reported prevalence of idiopathic hyperaldosteronism has ranged from a high of 70% of those patients with primary hyperaldosteronism14 to a low of 8%.20 The true prevalence probably is somewhere between these two extremes and may be ~30%. Although the etiology of idiopathic aldosteronism has not been established, the belief that a circulating stimulatory factor is responsible for hyperfunction of the zona glomerulosa is

777

generally accepted. Extracts of urine from normal persons con¬ tain a protein fraction that selectively stimulates the production of aldosterone and produces hypertension when injected into rats.30 Subsequent studies of one of these extracts have shown that (a) it is a peptide of pituitary origin, (b) it is increased in patients with idiopathic hyperaldosteronism but not in those with an APA, and (c) it is not suppressed by dexamethasone.31 The specific peptide has not been identified. In other studies, plasma concentrations of y-melanotropin32 and of (5endorphin33 were increased in patients with idiopathic hyper¬ aldosteronism but not in those with an aldosterone-producing adenoma. These two peptides are fragments of a larger pep¬ tide, pro-opiomelanocortin, which is formed in the pituitary; they may stimulate the secretion of aldosterone or increase the sensitivity of the adrenal gland to angiotensin II, or both. The report of an enlargement of the intermediate lobe of the pitu¬ itary in a patient with idiopathic hyperaldosteronism,34 together with the observations that central serotoninergic stim¬ ulation of aldosterone secretion may occur35 and that cypro¬ heptadine, which is an inhibitor of central serotonin release, may decrease plasma aldosterone in patients with idiopathic hyperaldosteronism,36 is consistent with a possible role for the pituitary in the pathogenesis of idiopathic hyperaldosteronism. It should be noted, however, that a pathogenetic schema for idiopathic hyperaldosteronism has to account for the hyperten¬ sion as well as the hyperaldosteronism because adrenalectomy rarely lowers blood pressure. Therefore, a putative aldoster¬ one-stimulating factor of pituitary or other origin, or changes proximal to its release, must be responsible for the hyperten¬ sion in idiopathic hyperaldosteronism, unless the hypertension is a separate process. The effects of the putative aldosteronestimulating factors on blood pressure are not known. As in an APA, the overproduction of aldosterone in idiopathic hyperal¬ dosteronism causes increased sodium reabsorption and potas¬ sium secretion by the renal tubule, expansion of the volume of extracellular fluid, suppression of plasma renin activity, and hypokalemia.

DIAGNOSIS AND DIFFERENTIAL DIAGNOSIS Although the features that characterize prolonged aldosterone excess may be as prominent in patients with idiopathic hyperal¬ dosteronism as in patients with an APA, they usually are less pronounced and more subtle.29 (It has been suggested that approximately half the patients with hypertension and sup¬ pressed plasma renin activity who show a decrease in plasma aldosterone to values between 5 and 10 ng/dL in response to saline infusion may have idiopathic hyperaldosteronism, even though baseline plasma aldosterone and serum potassium val¬ ues may be normal.14 When these diagnostic criteria are used, it may be difficult to distinguish between idiopathic hyperaldo¬ steronism and low-renin essential hypertension. Such a sharp distinction between idiopathic hyperaldosteronism and lowrenin essential hypertension may not be made with certainty until the pathogenesis of both disorders is more clearly delin¬ eated.) More important distinctions are those between idio¬ pathic hyperaldosteronism and an APA, and between idiopathic hyperaldosteronism and primary bilateral adrenal hyperplasia, because therapy for these disorders is different. The response to saline infusion may be helpful because the aldosterone/cortisol ratio increases in an APA but remains unchanged or decreases, with a value of 2.2 or less, in idiopathic hyperaldosteronism37 (Fig. 80-5). A value for plasma 18-hydroxycorticosterone less than 50 ng/dL at 8:00 a.m. after overnight bedrest is the usual finding in idiopathic hyperaldosteronism.8'21 In patients with an APA, as well as in most patients with primary hyperplasia, val¬ ues for 18-hydroxycorticosterone are >50 ng/ dL and may exceed 100 ng/dL.5'21,38 An increase in plasma aldosterone levels associated with a decrease in plasma cortisol levels after 2 hours of ambulation in the morning favors a diagnosis of idiopathic

778

PART V: THE ADRENAL GLANDS PATIENT 1 F 32 Yrs

DEX 2 mg/d

Mean 115|— BP mm Hg 100^Serum K* meq/L

4.5r3.01—

•—•

20 UP PRA 10 ng/ml/hr

Urine ALDO Mg/d

Urine K+ meq/d

40 20

0 60

Intake

30

0 160 Urine NA+ meq/L

120

Intake

80 40

0 BW Kg FIGURE 80-5. The plasma aldosterone/plasma cortisol ratio before 0control) and after the administration of 1250 mL of a normal saline infu¬ sion (SI) over 2 hours from 8 a.m. to 10 a.m. Note consistent increases and higher values for the ratio in patients with aldosterone-producing adenoma (APA) than in those with idiopathic hyperaldosteronism (IHA). (From Arteaga E, Klein R, Biglieri EG. Use of the saline infusion test to diagnose the cause of primary aldosteronism. Am J Med 1985; 79:722.)

hyperaldosteronism but also may occur in association with an APA.22 An increase in plasma aldosterone levels is more difficult to interpret than is a paradoxic decrease. Because of the number of false-positive and false-negative results, the test, as originally described, is not sufficiently dependable to be a reliable tool.8 A refinement in the interpretation of the postural stimulation test (i.e., correction of the percentage increase in aldosterone by sub¬ traction of the percentage increase in cortisol with standing) and acceptance of an increase in levels of plasma aldosterone with standing of 0.85 in women). It is visceral or subcutaneous truncal fat rather than intraperitoneal fat that is high risk; this type of fat and its distribution are also predictors of a low HDL2 choles¬ terol,108 insulin resistance,109 and diabetes.110 Intraabdominal fat is detected more accurately by CT scan. Obese hypertensive patients have a unique hemodynamic profile: Blood volume and cardiac output are increased, but systemic vascular resistance is normal to low.107 There are dif¬ ferences between normotensive and hypertensive obese sub¬ jects: Normotensive obese subjects have high cardiac output but a systemic vascular resistance that is below the levels found in normotensive lean subjects. Hypertensive obese subjects have a higher systemic vascular resistance than do normoten¬ sive obese subjects.107 In obesity, norepinephrine and insulin levels are high.,lb Because insulin increases norepinephrine release, it is possible that the caloric excess causes hyperinsulinemia and stimulation of the sympathetic nervous system and BP.111 Techniques to assess sympathetic nervous system activity in humans (plasma and urinary catecholamine levels, norepinephrine spillover, and microneurography) have provided important data on obe¬ sity and its relation to hypertension. Some studies have found that plasma norepinephrine levels are variably high in obesity. During weight loss there is an even more consistent relation¬ ship between reductions in norepinephrine and BP.112 Regional norepinephrine spillover in obesity is high in the kidney and could lead to sodium retention.113 Using microneurography techniques (which determine muscle sympathetic outflow), marked elevations were found in obese normotensive young men,114 but it is unclear if sympathetic activity is higher in hypertensive obese subjects. Thus, human obesity is character¬ ized by abnormalities in sympathetic cardiovascular control, but its role in hypertension is still undetermined. Other variables may alter sympathetic nervous system activ¬ ity and hypertension in obesity. Sleep apnea syndrome occurs in a substantial subgroup of obese patients, and activation of

797

the sympathetic nervous system by repeated bouts of hypoxia could affect BP. The muscle sympathetic activity has been found to be high only in those obese subjects who have obstruc¬ tive sleep apnea; when these patients are factored out, there are no demonstrable abnormalities in sympathetic nervous system activity in obesity.115 Leptin produced in adipose tissue acts on central receptors to decrease appetite and increase energy expenditure. Like insulin, leptin has cardiovascular properties that include increasing sympathetic nervous system activity, altering sodium excretion, and possibly affecting BP.116 Also, there is a reported association of a polymorphism in the P3adrenergic receptor gene with features of the insulin resistance syndrome.117 The hypotensive response to weight loss is accompanied by reductions in plasma volume, insulin, norepinephrine, and renin.106-112 Often a successful BP response is attained before ideal body weight is achieved. Interestingly, sodium balance does not alter the effect of weight loss on BP.106 Based on the results of multiple weight loss studies, it can be estimated that for every kilogram of weight loss there is a 0.5 to 1.0 mm Hg fall in mean BP. Reversal of obesity is difficult to achieve, and anti¬ hypertensive agents are often needed to control the hyperten¬ sion.118 HYPERCALCEMIA AND HYPERTENSION Estimates of hypertension in primary hyperparathyroidism have ranged from 50% to 70%, and as many as 35% of patients with chronic hypercalcemia of other etiologies are hyper¬ tensive119 (see Chaps. 58 and 59). Hypertension is also seen in the syndrome of parathyroid hormone (PTH) resistance (pseudohypoparathyroidism) and in secondary hyperparathy¬ roidism due to severe renal failure. Hypertension in primary hyperparathyroidism is sometimes associated with renal insuf¬ ficiency, but other factors, such as hypercalcemia; PTH and its analog, PTH-related protein; and phosphorus and magnesium deficiency contribute to the hypertension. Calcium is a major mediator of vascular contractility; calcium infusion can increase BP120 and potentiate A-II and norepinephrine. The positive rela¬ tionship between serum calcium and BP in hypertensive sub¬ jects suggests a cause-effect relationship.120 PTH may be involved in raising BP, but on acute administra¬ tion it acts as a vasodilator and induces a fall in BP. However, more chronic exposure to PTH may raise BP. Approximately half of patients with pseudohypoparathyroidism have hyper¬ tension, but their serum calcium is low and their PTH is high.121 Perhaps the high PTH sustains the hypertension, as normaliza¬ tion of calcium has no effect on BP.121 1,25-dihydroxycholecalciferol is high in primary hyperparathyroidism and can exert mild effects on calcium mobilization in blood vessels. PRA is high in hyperparathyroidism, suggesting that the renin-angiotensin system participates in raising the BP122; in some, parathyroid surgery normalizes PRA, aldosterone, and the BP.123 However, in renal insufficiency, hypertension persists or is only partially corrected.123 Hypertension, per se, is not an indication for parathyroid surgery in patients with primary hyperparathyroidism. Despite the heightened renin-angio¬ tensin system in primary hyperparathyroidism, the BP response to ACEI is limited, and there is no information on the best anti¬ hypertensive agent in this disorder. Of note, hypocalcemia also can raise BP,124 and, in such cir¬ cumstances, calcium supplementation may show beneficial effects on systolic BP in patients with essential hypertension.125 In studies of normocalcemic patients with essential hyperten¬ sion, calcium supplementation produces a mild, but statistically significant, reduction in BP.125 These studies have been disputed, but it appears that a subgroup of hypertensive patients do have BP sensitivity to calcium. An ionic hypothesis of cardiovascular dis¬ ease is based on the findings of a negative correlation between ionic (free) calcium and magnesium, and BP.126

798

PART V: THE ADRENAL GLANDS

ACROMEGALY AND HYPERTENSION Hypertension is very common in acromegaly. In the largest published series, hypertension was found in up to 51% of 500 patients with acromegaly127 (see Chap. 12). Human growth hor¬ mone promotes sodium reabsorption, and the extracellular fluid volume is increased in acromegaly; this is accompanied by suppressed renin levels.128'129 Also, there is evidence for overac¬ tivity of the sympathetic nervous system in acromegaly. An increase of left ventricular mass is frequently found in active disease, accompanied by an increased stroke volume and a decreased afterload. Thus, increased cardiac output in acro¬ megaly could be responsible for the rise in BP.130 Moreover, some have postulated a specific cardiomyopathy of acromegaly that could accompany the hypertension. The successful treat¬ ment of acromegaly often normalizes or improves BP and is beneficial to the other cardiac abnormalities, especially if the disease is treated early.

ENDOCRINE SYSTEMS IN ESSENTIAL HYPERTENSION In essential hypertension, there is evidence for the participation of several hormone systems, e.g., the renin-angiotensin system, serum aldosterone, and the sympathetic nervous system. Also, natriuretic hormones (e.g., the atrial natriuretic peptides [ANPs]), ouabain-like factors, endogenous vasodilator sub¬ stances (e.g., the eicosanoids [prostaglandins, lipoxygenases, P450 epoxides]), and the kallikrein-kinin system have been implicated in hypertension. Many of these systems act in an autocrine-paracrine fashion; new additions to this list include endothelial factors such as nitric oxide and endothelin. How¬ ever, the clinical use of measurements of these systems in the evaluation of hypertensive subjects is limited to special study situations. On the other hand, therapy with agonists or antago¬ nists of these systems may prove to be promising. RENIN-ANGIOTENSIN SYSTEM

THYROID DISEASE AND HYPERTENSION Thyroid hormone affects all regulatory aspects of the cardiovas¬ cular system.131-134 Thyroid hormone excess increases heart rate, cardiac output, stroke volume, and systolic BP; however, it decreases peripheral vascular resistance and diastolic BP. Car¬ diovascular changes with thyroid hormone are attributed to increased adrenergic nervous system activity through the (3adrenergic receptor.133'134 Because thyroid hormones are struc¬ turally related to catecholamines, they may be taken up and released at the neural synapse.133'134 Thyroid hormone stimu¬ lates the renin-angiotensin system mainly through increasing the hepatic production of angiotensinogen (renin substrate).134 Hypothyroidism and Hypertension. The hypertension, which occurs in some patients with hypothyroidism, mainly involves elevations in diastolic BP; the incidence of the hypertension is reported to be 15% to 30%, but reports have varied widely from 0% up to 50% of subjects.135 Overall, hypothyroidism may account for 1% to 3% of patients who are diagnosed as having essential hypertension. Hypothyroidism is common in older individuals in whom the independent incidence of hyperten¬ sion is high. However, comparison studies have shown an increased prevalence of hypertension in hypothyroid individu¬ als even when age is accounted for.135 Sympathetic nervous sys¬ tem activity is increased in hypothyroidism, as documented by high circulating levels of norepinephrine.131-134 This enhanced sympathetic nervous system activity is predominantly a-adrenergic in origin and may raise total peripheral resistance and decrease cardiac output—hemodynamic findings that have been verified in hypertensive, hypothyroid patients. In general, PRA in hypertensive, hypothyroid patients is low.135 In about 30% of these patients, thyroid hormone replacement alone will reduce BP, although most of the patients will require antihyper¬ tensive therapy. Hyperthyroidism and Hypertension. Approximately 30% of subjects with thyrotoxicosis will have elevated BP that is predominantly an elevation in systolic BP.131-132'134 The high systolic pressure and pulse pressure are best explained by increased cardiac output. These cardiac adaptations to thyroid hormone excess are mediated through the adrenergic nervous system; (3-adrenergic blockade reduces the cardiac output and pulse pressure.133 Nevertheless, circulating catecholamine lev¬ els are normal to reduced in hyperthyroidism.133 In hyper¬ thyroid rats, there is an increased density of (5-adrenergic receptors in the cardiovascular tissue, indicating that thyroid hormone may increase adrenergic activity through these receptors.133 Although PRA levels are elevated in hyperthy¬ roidism, the increased renin-angiotensin system activity is not thought to contribute to the hypertension.131'132 Successful treatment of hyperthyroidism, especially in younger subjects, usually corrects the hemodynamic abnormalities and reduces the systolic BP (see Chap. 42).

In hypertension, the renin-angiotensin system activity, almost always measured as PRA, varies widely. There probably is a continuum of levels of PRA in hypertension; however, some authors, who believe that there are subsets, profile hypertensive subjects as having low, normal, and high PRA.136-137 Procedures to test for the release of PRA include sodium restriction, upright posture, diuretic administration, or correlation of PRA with 24hour sodium excretion. There is an inverse relationship between PRA and sodium excretion; the concept of renin profil¬ ing in hypertension may enable a more definitive classification of hypertension. In support of the renin-profiling procedure is the increased cardiovascular risk associated with high circulat¬ ing levels of PRA.137 There also may be differences in BP response in renin subgroups. Low-Renin Hypertension. Approximately 30% of subjects with essential hypertension have low PRA levels, and a higher incidence is found in certain ethnic groups such as AfricanAmericans.136 Normally, low PRA is also thought to be associ¬ ated with older age, but PRA may not be suppressed in the hypertensive individuals who survive to an older age.138 In patients with low-renin hypertension, there is an increased inci¬ dence of salt sensitivity, and these persons are at lower risk for myocardial infarction.137 In addition, individuals with low-renin hypertension have a superior antihypertensive response to diuretics and calcium-channel blockers.139 However, the magni¬ tude of difference of BP response to thiazide diuretics between renin groups is small.140 The mechanism for low renin in essen¬ tial hypertension is probably multifactorial, including mineralocorticoid excess, alterations in the tissue renin-angiotensin system, disordered calcium metabolism, and/or low sympa¬ thetic function. The best example of low-renin hypertension is primary aldosteronism in which volume expansion suppresses PRA levels. Other genetic disorders of mineralocorticoid excess (including glucocorticoid remediable aldosteronism,141 the apparent mineralocorticoid excess syndrome,142 and Liddle syndrome143) exhibit a suppressed PRA accompanied by hyper¬ tension and hypokalemia. Initial studies of the mechanisms for low-renin hypertension focused on aldosterone and other mineralocorticoids. Several candidate steroids (18-hydroxycorticos¬ terone, 18-hydroxy-DOC, DOC, and 19-norDOC) have turned out not to be the cause.144 In addition, hypokalemia rarely occurs in low-renin essential hypertension, and aldosterone lev¬ els are normal. The exact cause of low-renin essential hyperten¬ sion remains unknown; however, these subjects might have abnormal calcium and magnesium metabolism; they display a positive relationship between diastolic BP and cytosolic calcium and an inverse relationship between systolic and diastolic BP and intracellular free magnesium.145 High-Renin Hypertension. Approximately 10% to 20% of patients with essential hypertension have elevated levels of PRA.136 It is uncertain if these moderate increments represent a

Ch. 82: Endocrine Aspects of Hypertension

Normotensives

Hypertensives

FIGURE 82-3. Changes in paraaminohippuric acid (PAH) clearance (renal blood flow) in normotensive and essential hypertensive subjects on high-sodium diet in response to angiotensin II. Nonmodulators fail to change renal blood flow with high salt and angiotensin II levels. (From Shoback D, Williams GH, Moore TJ, et al. Defect in the sodiummodulated tissue responsiveness to angiotensin II in essential hyper¬ tension. J Clin Invest 1983; 72:2115.)

distinct pathophysiologic abnormality, but these individuals are at greater risk for cardiovascular disease.137 One explana¬ tion for elevated PRA in the hypertensive subject is nephron heterogeneity, with discordant renin secretion and sodium excretion; this causes a hypertensive vasoconstriction-volume relationship.146 The renin dependency of the BP is supported by showing a greater hypotensive response to blockade of the renin-angiotensin system (ACEI, A-II-receptor blockers) in high-renin subjects. However, interpretation of these findings is difficult, as the antihypertensive responses related to ACEI cor¬ relate only minimally with baseline PRA.147 Specific secondary causes of high-renin hypertension include renovascular hyper¬ tension, malignant hypertension, and renin-producing tumors. Normal-Renin Hypertension. Normal PRA values are found in -60% of subjects with hypertension.136 An abnormal tissue response to A-II has been reported in subjects with normalrenin hypertension (termed nonmodulating essential hyperten¬ sion).148 The nonmodulation trait may be an intermediate phenotype that is inherited and is associated with a single nucleotide polymorphism (SNP) in the coding region of the angiotensinogen gene at codon 235.149 The phenotype occurs twice as frequently in men.149 Up to 85% of subjects have a fam¬ ily history of hypertension.149 In these subjects, the aldosterone response to A-II infusion is subnormal on a low-sodium diet, and the renal blood flow response to A-II is subnormal on a high-sodium intake (Fig. 82-3). Thus, sodium fails to normally modulate the tissue response to A-II in both the adrenal glands and the kidneys. Because administration of an ACE inhibitor completely corrects the abnormal responses, tissue A-II defects may account for nonmodulation.149 Nonmodulation could par¬ tially explain the impaired renal sodium handling and salt sen¬ sitivity that are found in hypertension. The diagnosis of nonmodulation is difficult, requiring sodium balance and A-II infusion. However, several tests, such as angiotensinogen gene expression and the red blood cell sodium, lithium (Na+,Li+) countertransport assay could be markers for nonmodulation.149 Tissue Renin-Angiotensin System. Many of the con¬ clusions from profiling of circulating PRA must now be interpreted in light of the presence of a tissue (local) reninangiotensin system in blood vessels, heart, kidney, adrenal glands, and other target organs.150 It is possible that much of the cause of essential hypertension resides in the tissue renin-angiotensin system. For example, in the kidney, A-II levels can be 1000-fold higher than in the circulation, especially in certain areas such as

799

the glomerular and peritubular sites.151 In transgenic rats in which a mouse renin gene is inserted, there is hypertension, but circulating levels of renin and A-II and renal renin content are low at the same time that vascular A-II is high.152 Treatment with ACEI and removal of the adrenals corrects the BP, suggest¬ ing a tissue mediation of the hypertension in what was other¬ wise a low-renin state.152 Genetics of the Renin-Angiotensin System. Genetic aspects of the renin-angiotensin system in hypertension are being intensely studied. Polymorphisms of the angiotensinogen gene on chromosome 1 have been linked to essential hypertension, and there is a 33% excess of shared angiotensinogen alleles in male sibling pairs with diastolic hypertension.153 This mutation might account for 6% of hypertension in young white subjects. One mutation, the T235 variant of the angiotensinogen gene, is associ¬ ated with elevated levels of angiotensinogen. Another mutation, involving an A for G substitution in the 6 position of the promoter region, occurs on the angiotensinogen gene; in a large study, sub¬ jects with the AA genotype had better BP responses to sodium and weight reduction.154 Thus, genotyping for angiotensinogen might potentially help to predict BP responses to therapy. ACE gene polymorphism has also been noted in hypertension and vascular disease. The alleles found for ACE include insertion (I) and deletion (D); the DD genotype is associated with coronary heart disease,155 carotid artery disease,156 and atherosclerosis. These studies show that polymorphism of ACE is strongly asso¬ ciated with cardiovascular complications. Insulin resistance and angina pectoris also relate to converting-enzyme gene polymor¬ phism.157 Other studies have shown that the DD allele is associ¬ ated with higher ACE levels and the II genotype with lower ACE levels in the renal disease of type 1 diabetic patients.158 The dia¬ betic group has less reduction in BP and proteinuria on treatment with an ACEI if the subjects are on a high-salt diet.159 SYMPATHETIC NERVOUS SYSTEM IN HYPERTENSION Increased sympathetic nervous system activity (see Chap. 85) occurs in essential hypertension.160 In studies measuring plasma norepinephrine, young hypertensive subjects had higher values than did normotensive controls.161 Hypertensive subjects also have increased renal and cardiac norepinephrine spillover, indicating regional sympathetic excess.162 In studies using microneurography to measure regional sympathetic nerve activity, the recorded activity in skeletal muscle is high in essential hypertension.163 Inhibitors of sympathetic activity lower BP proportionately to the baseline level of plasma norepi¬ nephrine. Stress, isometric exercise, and tilt table maneuvers all produce exaggerated norepinephrine responses in hyperten¬ sion, and hyperactive BP responses occur with norepinephrine infusion. Thus, the combination of exaggerated BP responses and high norepinephrine levels could provide a substantial neurogenic stimulus for hypertension. Certain subgroups may be more prone to develop neurogenic hypertension, including young hypertensive subjects, very obese individuals, and those defined as having a hyperdynamic circulation.160 There may be two phases of sympathetic nervous system activity participation in the evolution of hypertension. In the early phase there is high cardiac output that is initiated by an increase in sympathetic nervous system activity.160 This early hyperdynamic phase of hypertension then converts to a more long-term phase of chronic BP elevation in which cardiac output returns toward normal and systemic vascular resistance rises.164 With time high sympathetic nervous system activity and BP also induce structural and hypertrophic changes in blood vessels that further sustain the hypertension.165 In humans, there is sup¬ port for the concept of an inappropriately high sympathetic ner¬ vous system activity, both early and late, in the course of essential hypertension, implying that factprs that would nor¬ mally or physiologically suppress the sympathetic nervous sys¬ tem are not operative.164 In studies to support this notion, drugs

800

PART V: THE ADRENAL GLANDS

that lower BP cause an excessive response of norepinephrine, suggesting an unmasking of sympathetic nervous system responses. There may be a central disinhibition failure due to abnormal central function in the cells of the rostral ventricular medulla in neurogenic forms of hypertension.166 Thus, values considered in the normal range in a person with established hypertension may be abnormal or inappropriately high. Much of the neurogenic contribution to hypertension may be at a regional or organ-specific level. Increased organ-specific radiotracer spillover has been demonstrated in the heart and kidneys of essential hypertensive subjects.162 Similar regional increases in sympathetic burst activity or firing rates are found in borderline and established hypertensive individuals.163 A mutually reinforcing effect of the sympathetic nervous system and renin-angiotensin system may sustain high BP, since adren¬ ergic receptors mediate renin release and A-II can activate the sympathetic nervous system.167 Both of these systems also have parallel growth-promoting properties that probably mediate the degenerative changes in chronic hypertension. Dopamine is an indispensable catecholamine product that not only acts as a neurotransmitter in neuronal tissues but also has autocrine and paracrine functions in nonneuronal tissues. When stimulating specific receptors located in the blood vessels or along the nephron, dopamine can regulate renal hemody¬ namics as well as sodium and water transport, and in the adre¬ nal gland inhibits aldosterone secretion. In humans with essential hypertension, the response to dopamine is impaired at the receptor level. Renal dopamine modulates sodium excre¬ tion during sodium loading, mediated through the renal dopamine D3-receptor (as shown in mice with inactive D3receptors).168 Both BP and the renin-angiotensin system are higher in these mice. The D3-receptor may be an important reg¬ ulator of renal renin release and BP.168 A dopamine agonist (e.g., fenoldopam and the powerful dopamine D2-receptor agonist carmoxirole) can lower BP in very severe hypertension and has been used in hypertensive crises.169 Interestingly, a reduced uri¬ nary free dopamine response to salt loading is observed in essential hypertension, indicating a deficiency of renal dopa¬ mine that could blunt normal natriuretic mechanisms.170 SODIUM AND HYPERTENSION A positive relationship between sodium intake and hyperten¬ sion is documented in epidemiologic studies,171 but increased salt intake does not uniformly raise BP in all persons. There may be individual differences in BP sensitivity to salt.171'172 Some hypertensive subjects may be salt sensitive (defined as an increase in mean BP of >10 mm Hg in response to a high-salt diet). Salt-sensitive hypertension is more common in older sub¬ jects, in African-Americans, and in diabetic subjects.171'172 Fac¬ tors that control sodium (e.g., membrane transport systems, ANPs, and sodium pump inhibitors) are abnormal in hyperten¬ sion. Other factors that regulate sodium (e.g., aldosterone, dopamine, prostaglandins, and bradykinin) also play a role in the relationship of salt to BP. MEMBRANE SODIUM TRANSPORT A major function of most cells is to pump in an outward direc¬ tion Na+ in exchange for extracellular K+ by the action of the Na+,K+-adenosine triphosphatase (ATPase) pump. Tire pump then establishes a gradient of sodium across cell membranes that can serve as energy for other passive ion transport systems. Red and white blood cells from subjects with essential hypertension show a high Na+ content, a fording that matches the observations of high Na+ content in blood vessels and other target tissue from hypertensive animal models.173,174 Disturbances in several mem¬ brane ionic transport pathways are described in human hyper¬ tension.173,174 Measurement of these transport systems in humans uses peripheral cells (erythrocytes, leukocytes, platelets). The transport pathways are discussed in the following sections.

Na+,K+ Pump. The Na+,K+-ATPase or Na+,K+pump medi¬ ates the active transport of Na+ and K+, and is central for acute and chronic regulation of cell ions. Factors such as insulin, cate¬ cholamines, and P2-agonists increase intracellular uptake of K+ through the pump. Compounds such as digitalis increase cell Na+ by direct inhibition of the pump; the activity can be mea¬ sured by ATPase enzyme activity, ion fluxes, ouabain binding, and so forth. In skeletal muscle, the activity of the pump is increased by training, thyroid hormones, and glucocorticoids. The pump is down-regulated in hypothyroidism, cardiac fail¬ ure, muscle disease, K+ deficiency, and some forms of hyperten¬ sion. Insulin stimulates K+ uptake through the pump in 3T3 fibroblasts by means of phosphatidylinositol 3-kinase and pro¬ tein kinase C.175 Several studies found a defective pump activity in human and animal models of hypertension that could lead to a high intracellular Na+.176 This defect could produce hypertension by partial membrane depolarization and activation of voltagedependent Ca2+ channels (with increased influx of Ca2+), thus promoting blood vessel contraction. Another proposal is that high intracellular Na+ could activate the Na+,Ca2+ exchanger that would increase intracellular Ca2+.176 Other studies find high pump activity in blood vessels, especially in animal meta¬ bolic models such as the fructose-fed rat.177 Na+,Li+ Countertransport. An elevated erythrocyte Na+,Li+ countertransport has been noted in numerous studies of subjects with essential hypertension.173,174 Tire Na+,Li+-countertransport system is genetically transmitted by a single gene determinant and may be a candidate marker for genetic hypertension. Na+,Li+countertransport kinetics are abnormal in relatives of hyperten¬ sive patients who have high countertransport.178 One question has been the usefulness of this assay as a predictor of future hypertension. In a prospective study of normotensive middleaged men, those who eventually developed hypertension had high Na+,Li+ countertransport, suggesting the assay as a predictor of subsequent hypertension.179 Enhanced erythrocyte Na+,Li+ countertransport is found in hypertensive patients who have hyperlipidemia.180 High coun¬ tertransport is also a marker for metabolic correlates [higher fasting blood glucose, greater glucose, and insulin responses to an oral glucose tolerance test (OGGT)] in hypertensive sub¬ jects.181 In nonmodulating essential hypertension, in people who fail to change renal blood flow in response to high salt intake, countertransport is high, and the assay may be a marker for nonmodulation and salt-sensitive hypertension.182 The pathophysiologic consequences of an abnormal counter¬ transport are more difficult to understand. The system is quan¬ titatively a minor transport pathway, and its functional significance remains to be determined. The Na+,Li+-countertransport system is also a risk marker for hypertension and renal complications in diabetes mellitus. A high countertransport appears to be an inherited-risk marker for diabetic nephropathy. The assay may identify those type 2 diabetics who will develop microalbuminuria.183 The major kinetic abnormality of the Na+,Li+ countertransporter in diabe¬ tes with renal disease is a raised affinity for extracellular sodium.184 Studies show a membrane defect in countertrans¬ port in diabetic nephropathy that explains the abnormal kinet¬ ics, including an increased Rmax and I7max/Km(So) ratio, which reflects higher ion association for the transport system.185 The abnormal kinetic parameters can be completely corrected by thiol group alkylation with N-ethylmaleimide. Similar to the findings in essential hypertension, high countertransport val¬ ues are correlated with insulin resistance and BP in patients with type 2 diabetes.186 Na+,H+ Exchange. Elevated Na+,H+-exchanger activity is found in various cell types from patients with essential hyper¬ tension and is a recognized intermediate phenotype for this dis¬ order.187 The phenotype of an increased maximal transport capacity is preserved in Epstein-Barr virus immortalized lym-

Ch. 82: Endocrine Aspects of Hypertension phoblasts from hypertensive patients, suggesting strong genetic control.188-189 Both lymphocytes190 and erythrocytes191 from patients with essential hypertension show an enhanced Na+,H+-exchanger activity. Higher Na+,H+ exchanger in periph¬ eral cells is also noted in normotensive subjects with a family history of hypertension.187 Very similar findings for elevated Na+,H+ exchanger are noted in vascular myocytes from the spontaneously hypertensive rat.192-193 There is a family of Na+,H+-exchanger isoforms of which the most studied in hypertension is the NHE-1 isoform. The NHE-1 isoform and, in some tissues, the NHE-3 isoform have been reported to be ele¬ vated in human and experimental hypertension in several cells, including erythrocytes, platelets, and fibroblasts.187 However, the gene for the NHE-1 isoform of the exchanger is not the cause of hypertension as shown in genetic linkage studies.191 Although this transporter is involved in growth regulation, the enhanced proliferation pattern seen in vascular tissue in hyper¬ tension is mostly independent of ion transport.194 The mechanism of Na+,H+ abnormality in hypertension is unclear. The physiologic role of this transport system is to respond to intracellular acid loads, and intracellular pH does appear to be slightly lower in hypertension.192 Exchanger abun¬ dance is identical in hypertensive versus normotensive subjects, indicating that the increased activity in the hypertensive group is due to elevated turnover of the exchanger.188-195 Na+,H+ exchanger phosphorylation is elevated in quiescent cells.188 Its dysfunction could represent a defect in signal transduction as it responds to several agonists including A-II.188 It has been further noted that A-II stimulates p90rsk in vascular smooth muscle cells, leading to the hypothesis of a potential Na+,H+-exchanger kinase.195 Meta¬ bolic acidosis, high salt intake, and circulating hormones (e.g., insulin) also regulate Na+,H+ exchange.187 Although increased Na+,H+ exchange is high in the erythrocytes of patients with pri¬ mary aldosteronism, aldosterone directly added to cells in vitro does not affect this system.196 ACEI corrects the Na+,H+-exchanger overactivity in essential hypertension.197 The addition of chelerythrine, a blocker of pro¬ tein kinase C, also reduces Na+,H+-exchanger activity in spon¬ taneously hypertensive rats.198 The addition of an amiloride derivative, which inhibits the Na+,H+ exchanger, increases BP in spontaneously hypertensive rats.199 The Na+,H+ exchanger also exhibits altered kinetics in dia¬ betic nephropathy.200-201 This phenotype persists in immortal¬ ized lymphoblasts201 and in skin fibroblasts202 from diabetic subjects with nephropathy. Similarly to hypertension, the mechanism in diabetes is due to posttranslational factors.201-202 Na+,K+,(2CI-) Cotransport. This transport pathway is inhibited by loop diuretics (furosemide, bumetanide). Initial studies found low erythrocyte cotransport in essential hyperten¬ sion, but later reports noted high activity, probably representing a different hypertensive population.203 Ethnic and geographic variations in cotransport may make it a poor marker for essential hypertension. In a metaanalysis from the literature, it was found that essential hypertension, family history of hypertension, gen¬ der, and antihypertensive medications were the main determi¬ nants of cotransport.204 Indirect evidence has shown a circulating cotransport inhibitor of unknown nature.205 In summary, various membrane cation-transport defects are thought to contribute to the high cell sodium content and increased Na+ reabsorption seen in hypertension. In addition, there is evidence for increased passive inward Na+ leak in hyper¬ tension. These abnormal membrane ion transport pathways sug¬ gest a more complex picture for sodium handling than can be explained by a circulating pump inhibitor, natriuretic hormones, and many of the other factors that control sodium homeostasis. SODIUM PUMP INHIBITORS Enhanced sodium excretion in response to volume expansion is associated with increased blood levels of a factor(s) that inhibits the Na+,K+-ATPase pump.206 Reduced Na+ excretion causes vol¬

801

ume expansion and signals a circulating pump inhibitor to block sodium reabsorption and correct volume overload. This pump inhibitor, however, could act on other tissue Na+,K+ATPase pump sites. For example, in the blood vessels, there would be an increase of intracellular Na+ and Ca2+ and a reset¬ ting of vascular tone.206 Increased intracellular Na+ and Ca2+ and reduced Na+,K+-ATPase pump activity are found in experi¬ mental and human hypertension. The isolation and structural identification of a circulating compound that inhibits the Na+,K+-ATPase pump has been difficult. One group has identi¬ fied and characterized a ouabain-like compound from human plasma and adrenal glands that inhibits Na+,K+-ATPase pump activity.207 The physiologic effects of this endogenous ouabain include stimulation of vascular contraction and control of intra¬ cellular calcium stores.207 The endogenous ouabain is a novel ster¬ oid counterpart that is isomeric with the plant glycoside ouabain.207 The primary source of endogenous ouabain is in the adrenal zona glomerulosa where ACTH and A-II AT2 receptors stimulate its release. The signal pathway for endogenous oua¬ bain release is different than that for aldosterone. A binding site for endogenous ouabain has been found on the sodium pump. It mediates long-term BP control through neuronal pathways, yielding a slow pressor effect that could contribute to the induction of hypertension. ENDOGENOUS NATRIURETIC PEPTIDES There is a family of endogenous natriuretic peptides, including ANP, brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP).208-209 ANP, which was first isolated from the heart, has numerous actions, including stimulation of natriuresis and vasodilation, and inhibition of the renin-angiotensin system, aldosterone, the sympathetic nervous system, and the endothelin system.208 ANP is synthesized in atrial myocytes, implying an endocrine function in the heart (see Chap. 178). ANP and BNP are released by atrial stretch (intravascular vol¬ ume, atrial pressure). BNP has properties similar to those of ANP, but is synthesized and stored in the central nervous sys¬ tem and in atrial cells.208 In hypertension, the levels of BNP may be greater than the levels of ANP; thus, it is debated which peptide is more important in guarding against BP elevations. CNP is produced in the endothelium and is a potent vasoactive peptide that dilates both veins and arteries, but it has no natri¬ uretic properties. CNP is not a circulating peptide and acts in a paracrine fashion. Much of the function of these ANPs is determined by their receptors. Two of these receptors, ANPR-A and ANPR-B, are linked to guanyl cyclases. ANP and BNP bind to A receptors in the endothelium of blood vessels, whereas CNP binds to B receptors.208 The third is the ANPR-C receptor, which is a clear¬ ance receptor and plays an active role in determining availabil¬ ity of the peptides by controlling their rate of removal from the circulation.208 In essential hypertension, the levels of ANP and BNP are elevated, but it has been argued that these values are actually relatively low for the level of BP.208 The concept of a natriuretic peptide deficiency state in essential hypertension is appealing, but unproved. The most promising therapeutic approach to raise natriuretic peptide levels and lower BP is through the reg¬ ulation of the ANPR-C clearance receptors and use of neutral endopeptidase inhibition (ompatrilat) to potentiate their bioac¬ tion.210 These agents also have ACE inhibitor properties and accumulate kinins.210a Cardiac natriuretic peptide levels may also be predictors of mortality in heart failure.211 KININS IN HYPERTENSION The products of the kallikrein-kinin system, the kinins, are natriuretic and diuretic and have vasodilating properties, through stimulation of nitric oxide and prostaglandins212-213 (see Chap. 170). Kinins are produced from kininogens through cleavage by the enzyme kallikrein to form the two main kinins.

802

PART V: THE ADRENAL GLANDS

bradykinin and lysyl-bradykinin (kallidin). Kinins act as para¬ crine and autocrine hormones through two receptors, the B,and B2-receptors. The B2-receptors, which belong to the family linked to G proteins, are the ones that mediate most known reg¬ ulating effects on blood flow to meet metabolic demands. Renal kinins play a role in the regulation of the microcirculation and of water and sodium metabolism; the natriuretic effect is medi¬ ated in part by prostaglandins. Neutral endopeptidase inhibi¬ tors are natriuretic through the blocking of kinin hydrolysis leading to their accumulation. ACE is one of the main pepti¬ dases that hydrolyze kinins; ACE inhibitors block the break¬ down of kinins. Tissue and urinary kinins increase with ACEI application. Although kinin accumulation potentiates the hypotensive action of ACE inhibitors, the administration of a kinin antagonist only blocks the acute but not chronic hypoten¬ sive effect of ACEI. Kinin release after ACEI may mediate the cardioprotection of ACEI during ischemic episodes and in the prevention of remodeling.214'215 A reduction in the kallikrein-kinin system has been found in animal models of genetic hypertension; in children, it is a good marker of genetic hypertension.212'213 A restriction fragment length polymorphism for the kallikrein gene family is linked to BP in the spontaneously hypertensive rat.212 The administration of kinin inhibitors raises BP, whereas in animals, which are transgenic for kinin expression, there is a prolonged hypoten¬ sive response. The bradykinin B2-receptor gene knockout mouse also develops hypertension on a high-salt diet. NITRIC OXIDE IN HYPERTENSION Nitric oxide (NO) is formed from endogenous arginine by nitric oxide synthase (NOS) or from exogenous nitrovasodilators such as nitroglycerin (see Chap. 179). Cells and tissues oxidize via a five-electron oxidation of the guanido nitrogen of arginine to form citrulline and NO.216 This reaction is catalyzed by the enzyme NOS, which exists in three isoforms (neuronal NOS, inducible NOS, and endothelial eNOS).216-217 Studies have found eNOS to be membrane associated in the Golgi apparatus, in plasmalemmal membranes, and in caveolae that contain several key signal-transduction complexes.218 The enzymes are dependent on several cofactors, including reduced nicotinamide adenine dinu¬ cleotide phosphate (NADP), flavin, adenine dinucleotide, and tetrahydrobiopterin; they use heme as the prosthetic group. In addition, intracellular calcium and calmodulin can activate NOS and NO production, but there are also several calcium-indepen¬ dent pathways.219 The actions of agents such as acetylcholine, his¬ tamine, thrombin, and insulin are mediated through NO. Certain cytokines and endotoxins also activate NO in vascular smooth muscle, liver, and peripheral cells. Shear stress induces NO pro¬ duction through activation of heat-shock proteins such as HSP90.220 The major signal-transduction pathway for NO is through guanylate cyclase, forming cyclic guanosine monophosphate (GMP).221 NO binds to the heme-containing enzyme guanylate cyclase to convert guanosine triphosphate (GTP) to cGMP. This reaction activates protein kinase G with phosphorylation of numerous proteins that mediate vascular relaxation and have antigrowth effects. Numerous hormones and drugs regulate NO through effects on NOS activity and at other regulatory sites.222 These factors can also act on the NO-cGMP complex to alter signal transduc¬ tion. Several agents such as phosphodiesterase inhibitors, scav¬ enger agents (hemoglobin), agents that alter cellular calcium, NOS-substrate inhibitors, and hormone and receptor antago¬ nists will alter NO. The NO-cGMP complex mediates smooth muscle vasodila¬ tation in blood vessels and other hollow organs. The system is also operative in platelets, nerves, macrophages, and other tar¬ get organs. NO contributes to the regulation of insulin secretion and action, platelet function, neurotransmission, memory, penile erection, cytotoxicity induced by macrophages, and

atherogenesis.222 NO can operate through cGMP-independent pathways having molecular effects on iron-containing enzymes, free radicals, proteins, and DNA synthesis. The role of NO in hypertensive vascular disease is complex.223 With the preponderance of data showing that NO is crucial in vascular homeostasis, it should be a major contributor to hyper¬ tension. A logical hypothesis is that decreased eNOS expression should be seen in hypertension. There have been eNOS polymor¬ phisms reported in essential hypertension such as a calcium repeat polymorphism in intron 13,224 and a G to T substitution in exon 7 of the eNOS gene resulting in a Glu298Asp conversion.225 However, these altered genes seem to have a weak effect on phe¬ notype or cardiovascular risk. A depressed response of blood flow to acetylcholine, which is eNOS sensitive, is taken as an indicator of endothelial dysfunction and decreased bioactive NO. Attenuated vasodilator responses are seen in hypertension and coronary heart disease.223 Paradoxically, NO responses can be increased in both genetic and primary hypertension, and the response may vary in different vascular beds.226 In the spontane¬ ous hypertensive rat, eNOS is up-regulated in the aorta but nor¬ mal in other vascular beds. Oxidative stress may be another important determinant of endothelial function. Increased pro¬ duction of superoxide radicals (O,-) may account for the variable findings of NO activity in hypertension, as overproduction of the NO scavenger 02~ (rather than a change in eNOS production) may be important in endothelial dysfunction. An increased vas¬ cular 02~ has been associated with hypercholesterolemia and ani¬ mal models of hypertension.226 In summary, reduced production or bioavailability of NO should promote cellular events in blood vessels that promote vasoconstriction, inhibit vasodilatation, and activate structural damage in vessels. Blockade of NO synthase with inhibitors will raise BP in humans and animal models.223 Administration of arginine will cause a mild reduction in BP both in humans and in animal models.223 ENDOTHELIN IN HYPERTENSION Endothelin-1 (ET-1) is a potent 21-amino acid vasoconstrictor peptide produced by the endothelium. There are three mamma¬ lian endothelins (i.e., ET-1, ET-2, and ET-3), which were first found in the vascular endothelium but exist in many other cells.227 They regulate cardiovascular function and other noncardiovascular actions such as airway smooth muscles, the digestive tract, endocrine glands, the renal system, and the ner¬ vous system. Endothelial cells produce preproendothelin, from which is derived proendothelin, which is converted to big endothelin by endothelin-converting enzyme, a neutral endopeptidase.227 Big endothelin (39 amino acids) then is processed into endothelin (22 amino acids). Vascular endothelial cells (VEC) release pre¬ dominantly ET-1 in response to low shear stress, A-II, vaso¬ pressin, catecholamines, and transforming growth factor-[3. ET-1 acts on the ETA and ETB receptors to induce contraction, proliferation, and cell hypertrophy.227 Through the ETB receptor, ET-1 may release NO and prostacyclin, thus having the capacity to be both a vasoconstrictor and a vasodilator. This dual activity depends on the receptor predominance in any vascular bed. Cor¬ onary arteries lack ETB receptors, so ET-1 is a vasoconstrictor, whereas in cardiomyocytes ETB receptors predominate. In the kidney, predominantly ETA receptors are found in blood vessels and mesangial cells. In the distal tubule, the ETA receptor regu¬ lates sodium excretion. The endothelin system plays an important role in develop¬ ment. Several models of gene disruption produce malforma¬ tions in the upper airways and aortic arch and abnormalities in pigment and megacolon. These models do not exhibit major changes in BR The endothelin system seems to be activated mostly in severe salt-dependent hypertension such as the deoxycorticosterone

Ch. 82: Endocrine Aspects of Hypertension acetate, salt-sensitive rat and the Dahl salt-sensitive rat. These models overexpress ET-1 in blood vessels. Endothelin antagonist therapy reverses hypertrophic remodeling in small blood ves¬ sels.228 In human hypertension, administration of endothelinreceptor antagonists has a mild BP-lowering effect, but the effect of a combined ETA/ETB antagonist is equal to that of an ACE inhibitor.229-230 Endothelins may be involved in renal failureinduced hypertension, in cyclosporine-induced hypertension, in erythropoietin-induced hypertension, in pheochromocytomainduced hypertension, and in pregnancy-induced hyperten¬ sion.229 In general, although the exact role of endothelins in hypertension is not yet clear, the receptor antagonists may have a promising future in the control and prevention of cardiovascular disease.229

REFERENCES 1. Hypertension Detection and Follow-Up Program Cooperative Group. The hypertension detection and follow-up program. Circ Res 1977; 40(Suppl 1):106. 2. The Sixth Report of the Joint National Committee on Detection, Evaluation and Treatment of High Blood Pressure (JNC VI). Arch Intern Med 1997; 157:2413. 3. Working Group on Renovascular Hypertension. Detection, evaluation and treatment. Arch Intern Med 1987; 147:820. 4. Mann SJ, Pickering TG. Detection of renovascular hypertension: state of the art. Ann Intern Med 1992; 117:845. 5. Connolly JO, Higgens RM, Walters HL, et al. Presentation, clinical features and outcome in different patterns of atherosclerotic renovascular disease. Q J Med 1994; 87:413. 6. Canzanello VJ, Textor SC. Noninvasive diagnosis of renovascular disease. Mayo Clin Proc 1994; 69:1172. 7. Ram CVS. Renovascular hypertension. Curr Opin Nephrol Hypertens 1997; 6:575. 8. McGrath BP, Clarke K. Renal artery stenosis: current diagnosis and treat¬ ment. Med J Aust 1993; 158:343. 9. Rimmer JM, Gennari FJ. Atherosclerotic renal vascular disease and pro¬ gressive renal failure. Ann Intern Med 1993; 118:712. 10. Choudhri AH, Cleland JG, Rowlands PL. Unsuspected renal artery steno¬ sis in peripheral vascular disease. Br Med J 1990; 301:1197. 11. Pickering TG, Devereux RB, James GD. Recurrent pulmonary edema in hypertension due to bilateral renal artery stenosis: treatment by angio¬ plasty or surgical revascularization. Lancet 1988; 2:551. 12. Svetkey LP, Kadir S, Dunnick NR, et al. Similar prevalence of renovascular hypertension in selected blacks and whites. Hypertension 1991; 17:678. 13. Pohl MA. Renal artery stenosis, renal vascular hypertension and ischemic nephropathy. In: Schrier RW, Gottschalk CW, eds. Disease of the kidney, 6th ed. Boston: Little Brown and Company, 1997:1367. 14. Martinez-Maldonado M. Pathophysiology of renovascular hypertension. Hypertension 1991; 17:707. 15. Mitchell KD, Braam B, Navar LG. Hypertensinogenic mechanisms medi¬ ated by renal actions of renin-angiotensin system. Hypertension 1992; 19(Suppl 1):I-18-1. 16. Navar LG. The kidney in blood pressure regulation and development of hypertension. Med Clin North Am 1997; 8:1165. 17. Mitchell KD, Navar LG. Intrarenal actions of angiotensin II in pathogenesis of experimental hypertension. In: Laragh JH, Brenner BM, eds. Hyperten¬ sion: pathophysiology, diagnosis, and management. New York: Raven Press, 1995:1437. 18. Guan S, Fox MJ, Mitchell KD, Navar LG. Angiotensin and angiotensin-converting enzyme tissue levels in two-kidney, one-clip hypertensive rats. Hypertension 1992; 20:763. 19. Romero JC, Feldstein AE, Rodriguez-Porcel MG, et al. New insights into the pathophysiology of renovascular hypertension. Mayo Clin Proc 1997 72:251. 20. Corry DB, Tuck ML. Secondary aldosteronism. Endocrinol Metab Clin North Am 1995; 24:511. 21. Thornbury JR, Stanley JC, Fryback DG. Hypertensive urogram: a nondiscriminatory test for renovascular hypertension. AJR 1982; 138:43. 22. Wilcox CS. Use of angiotensin-converting-enzyme inhibitors for diagnos¬ ing renovascular hypertension. Kidney Int 1993; 44:1379. 23. Elliot WJ, Martin WB, Murphy MB. Comparison of two noninvasive tests for renovascular hypertension. Arch Intern Med 1993; 153:755. 24. Postma CT, Van Der Steen PH, Hoefnagels WH, et al. The captopril test in the detection of renovascular disease in hypertensive patients. Arch Intern Med 1990; 150:625. 25. Roubidoux MA, Dunnick NR, Klotman PE, et al. Renal vein renins: inabil¬ ity to predict response to revascularization in patients with hypertension. Radiology 1991; 178:819. 26. Nally JV, Black HR. State of the art review: captopril renography patho¬ physiological considerations and clinical observations. Semin Nucl Med 1992; 22:85.

803

27. Mann SJ, Pickering TG, Sos TA, et al. Captopril renography in the diagno¬ sis of renal artery stenosis: accuracy and limitations. Am J Med 1991; 90:30. 28. Halpem EJ, Deane CR, Needleman L, et al. Normal renal artery spectral Doppler wave form. A closer look. Radiology 1995; 196:667. 29. Olin JW, Piedmonte MR, Young JR, et al. The utility of duplex ultrasound scanning of the renal arteries for diagnosing significant renal artery steno¬ sis. Ann Intern Med 1995; 196:667. 30. Rene PC, Oliva VL, Bui BT, et al. Renal artery stenosis: evaluation of Doppler US after inhibition of angiotensin converting enzyme with captopril. Radiol¬ ogy 1995; 196:675. 31. Riehl J, Schmitt H, Bongatz, et al. Renal artery stenosis: Evaluation with colour duplex ultrasonography. Nephrol Dial Transplant 1997; 12:1608. 32. Debatin JF, Spritzer CE, Grist TM, et al. Imaging of the renal arteries: value of MR angiography. AJR 1991; 157:981. 33. Postma CT, Joosten FB, Rosenbusch G, Thien T. Magnetic resonance angiography has a high reliability in the detection of renal artery stenosis. Am J Hypertens 1997; 10:957. 34. Rieumont MJ, Kaufman JA, Geller SC, et al. Evaluation of renal artery stenosis with dynamic gadolinium-enhanced MR angiography. AJR 1997; 169:39. 35. DeHaan MW, Kouwenhoven M, Thelissen GRP, et al. Renovascular disease in patients with hypertension: detection with systolic and diastolic gating in three-dimensional, phase-contrast MR angiography. Radiology 1996; 198:449. 36. Olbricht CJ, Arlet IP. Magnetic resonance angiography—the procedure of choice to diagnose renal artery stenosis? Nephrol Dial Transplant 1998; 13:1620. 37. Olbricht CJ, Paul K, Prokop M, et al. Minimally invasive diagnosis of renal artery stenosis by spiral computed tomography angiography. Kidney Int 1995; 48:1332. 38. Weibull H, Bergqvist D, Bergentz SE, et al. Percutaneous transluminal angioplasty versus reconstruction of atherosclerotic renal artery stenosis. Prospective randomized study. J Vase Surg 1993; 18:841. 39. Lawrie GM, Morris GC, Glaeser DH, DeBakey MI. Renovascular recon¬ struction: factors affecting long-term prognosis in 919 patients followed up to 31 years. Am J Cardiol 1989; 63:1085. 40. Hansen KJ, Starr SM, Sands E, et al. Contemporary surgical management of renovascular disease. J Vase Surg 1992; 16:310. 41. Textor SC. Revascularization in atherosclerotic renal artery disease. Kidney Dis 1998; 53:799. 42. Caps MT, Zierler RE, Polissar NL, et al. Risk of atrophy in kidneys with atherosclerotic renal artery stenosis. Kidney Int 1998; 53:735. 43. Greco BA, Breyer JA. Atherosclerotic ischemic renal disease. Am J Kidney Dis 1997; 29:167. 44. Derkx FH, Schalecamp MA. Renal artery stenosis and hypertension. Lancet 1994; 344:237. 45. Ramsey LE, Weller PC. Blood pressure response to percutaneous angio¬ plasty. an overview of published series. Br Med J 1990; 300:569. 46. Bonelli FS, McKusick MA, Textor SC. Renal artery angioplasty: technical results and clinical outcome in 320 patients. Mayo Clin Proc 1995; 70:104. 47. Plouin PF, Chatelleir G, Dame B, et al. Blood pressure outcome of angio¬ plasty in atherosclerotic renal artery stenosis. A randomized trial. Hyper¬ tension 1998; 31:823. 48. Van de Ven PJ, Beutler JJ, Kaatee R. Transluminal vascular stent for ostial atherosclerotic renal artery stenosis. Lancet 1995; 346:672. 49. Blum U, Krumme B, Flugel P, et al. Treatment of ostial-artery stenosis with vascular endoprosthesis after unsuccessful balloon angioplasty. N Engl J Med 1997; 336:459. 50. Shannon HM, Gillespie IN, Moss JG. Salvage of the solitary kidney by insertion of a renal artery stent. AJR 1998; 171:459. 51. Hollenberg NK. The treatment of renovascular hypertension: surgery, angioplasty, and medical therapy with converting enzyme inhibitors. Am J Kidney Dis 1987; 10(Suppl 1):52.' 52. Pohl MA, Novick AC. Natural history of atherosclerotic and fibrous renal artery disease: clinical implications. Am J Kidney Dis 1985; 5(4):A120. 53. Dean RH. Comparison of medical and surgical treatment of renovascular hypertension. Nephron 1986; 44(Suppl 1):101. 54. Stradness DE Jr. Natural history of renal artery stenosis. Am J Kidney Dis 1994; 24:630. 55. Marshall FI, Hagen S, Mahaffy RG, et al. Percutaneous transluminal angio¬ plasty for theromatous renal artery stenosis—blood pressure response and discriminant analysis of outcome predictors. Q J Med 1990; 75:483. 56. Corvol P, Pinet F, Galen FX, et al. Seven lessons from seven renin-secreting tumors. Kidney Int 1988; 34(Suppl 25):S38. 57. Mimran A. Renin-secreting tumors. In: Swales J, ed. Textbook of hyperten¬ sion. Oxford, UK: Blackwell Science, 1994:858. 58. Bruneval P, Fournier JG, Soubrier F, et al. Detection and localization of renin messenger mRNA in human pathologic tissues using in situ hybrid¬ ization. Am J Pathol 1988; 131:320. 59. Anderson PW, Macaulay L, Do YS, et al. Extrarenal renin-secreting tumors: insights into hypertension and ovarian renin production. Medicine 1990; 68:257. 60. Misiani R, Sonzogni A, Poletti EM, et al. Hyponatremic hypertensive syn¬ drome and massive proteinuria in a patient with renin-producing leiomyo¬ sarcoma. Am J Kidney Dis 1994; 24:83. 61. Aurell M, Rudin A, Tisell LE, et al. Captopril effect on hypertension in patients with renin-producing tumor. Lancet 1979; 2:149.

804

PART V: THE ADRENAL GLANDS

62. Kawai M, Sahashi K, Yamase H, et al. Renin producing adrenal tumor: report of a case. Surg Today 1998; 28:974. 63. Carey RM, Wang ZQ, Siragy HM. Role of the angiotensin type 2 receptor in the regulation of blood pressure and renal function. Hypertension 2000; 35(1 Pt 2):155. 64. Lifton RP, Dluhy RG, Powers M, et al. Glucocorticoid remediable hyperal¬ dosteronism: chimeric gene from cross-over between 11 [3-hydroxylase and aldosterone synthase genes. Nature 1992; 355:262. 65. Snyder PM, Price MP, McDonald FJ, et al. Mechanism by which Liddle's syndrome mutations increase activity of a human epithelial Na+ channel. Cell 1995; 83:969. 66. White PC, Speiser PW. Steroid 11 (1-hydroxylase deficiency and related dis¬ orders. Endocrinol Metab Clin North Am 1994; 23:325. 66a. Farman N, Verrey F. Forefronts in nephrology news in aldosterone action. Kidney Int 2000; 57:1239. 67. Malchoff DM, Brufsky A, Reardon G, et al. A mutation of the glucocorti¬ coid receptor in primary cortisol resistance. J Clin Invest 1993; 91:1918. 68. Norbiato G, Bevilacqua M, Vago T, et al. Glucocorticoid resistance and the immune function in the immunodeficiency syndrome. Ann NY Acad Sci 1998; 840:835. 69. Gomez-Sanchez CE. Cushing's syndrome and hypertension. Hypertension 1986; 8:256. 70. Whitworth JA. Studies on the mechanisms of glucocorticoid hypertension in humans. Blood Pressure 1994; 3:24. 71. Danese RD, Aron DC. Cushing's syndrome and hypertension. In: Bravo EL, ed. Endocrine hypertension. Endocrinol Metab Clin North Am 1994; 23:299. 72. Parks LL, Turney MK, Gaitan D, Kovacs WJ. Expression of 11 [i-hydroxysteroid dehydrogenase type 2 in an ACTH-producing small cell lung can¬ cer. J Steroid Biochem Mol Biol 1998; 67(4):341. 73. Sowers JR, Epstein M. Diabetes mellitus and associated hypertension, vascu¬ lar disease and nephropathy. An update. Hypertension 1995; 26(Pt 1): 869. 74. Corry DB, Tuck ML. Insulin and glucoregulatory hormones: implications for antihypertensive therapy. In: Epstein M, ed. Calcium antagonists in clinical medicine. Philadelphia: Hanley and Belfus, 1998:217. 75. Mogensen CE. The kidney and hypertension. Boston: Kluwer Academic Publishers, 1998:1. 76. Chaturvedi N, Sjolie A-K, Stephenson JM, et al. Effect of lisinopril on pro¬ gression of retinopathy in normotensive people with type 1 diabetes. Lan¬ cet 1998; 351:28. 77. Groop LC, Kankuri M, Schalin-Jantti, et al. Association between polymor¬ phism of the glycogen synthase gene and non-insulin-dependent diabetes. Kidney Int 1993; 328:10. 78. Weidmann P, Boehlen LM, de Courten M. Pathogenesis and treatment of hypertension associated with diabetes mellitus. Am Heart J 1993; 125:1498. 79. Stem N, Tuck ML. Pathogenesis of hypertension in diabetes. In LeRoith D, Taylor S, Olefsky J, eds. Diabetes mellitus: a fundamental and clinical text. Philadelphia: Lippincott-Raven Publishers, 1996:780. 80. Nosandi R, Sambataro M, Thomaseth K, et al. Role of hyperglycemia and insulin resistance in determining sodium retention in non-insulin-depen¬ dent diabetes. Kidney Int 1993; 44:139. 81. Tuck ML, Corry DB, Trujillo A. Salt-sensitive blood pressure and exagger¬ ated vascular reactivity in the hypertension of diabetes mellitus. Am J Med 1990; 88:210. 82. Randeree HA, Omar HA, Mortala AA, Seedat MA. Effect of insulin on blood pressure in NIDDM patients with secondary failure. Diabetes Care 1992; 15:1258. 83. Trujillo A, Egenna P, Barrett J, Tuck M. Renin regulation in type II diabetes mellitus: influence of dietary sodium. Hypertension 1989; 13:200. 84. Hseuh WA. Effect of renin-angiotensin system in the vascular disease of type II diabetes mellitus. Am J Med 1992; 92(Suppl):13S 85. Tomiyama H, Kushiro T, Abeta A, et al. Kinins contribute to the improve¬ ment of insulin sensitivity during treatment with angiotensin converting enzyme inhibitor. Hypertension 1994; 23:450. 86. Steinberg HO, Brechtel G, Johnson A, et al. Insulin-mediated skeletal mus¬ cle vasodilation is nitric oxide dependent. A novel mechanism for insulin resistance. J Clin Invest 1995; 96:786 87. O'Driscoll G, Green D, Rankin J, et al. Improvement in endothelial function by angiotensin converting enzyme in insulin-dependent diabetes mellitus. J Clin Invest 1997; 100:678. 88. Reaven GM, Lithell H, Landsberg L. Hypertension and associated meta¬ bolic abnormalities—the role of insulin resistance and the sympathoadre¬ nal system. N Engl J Med 1996; 334:374. 89. Muller DC, Elahi D, Pratley RE, et al. An epidemiological test of the hyperinsulinemia-hypertension hypothesis. J Clin Endocrinol Metab 1993; 76:544. 90. Lithell HO. Hyperinsulinemia, insulin resistance and the treatment of hypertension. Am J Hypertens 1996; 9:154. 91. He J, Klag MJ, Caballero B, et al. Plasma insulin levels and incidence of hyper¬ tension in African Americans and whites. Arch Intern Med 1999; 159:498. 92. Tchemof A, Prud'homme D, Despres J-P, et al. Relation of steroid hor¬ mones to glucose tolerance and plasma insulin levels in men. Diabetes Care 1995; 18:292. 93. Mantzorose CS, Georgiadis El, Young P, et al. Relative androgenicity, blood pressure levels, and cardiovascular risk factors in young healthy women. Am J Hypertens 1995; 8:606. 94. Masuo K, Mikami H, Ogihara T, Tuck ML. Differences in insulin and sym¬ pathetic responses to glucose ingestion due to family history of hyperten¬ sion. Am J Hypertens 1996; 9:739.

95. Hausberg M, Sinkey CA, Mark AL, et al. Sympathetic nerve activity and insulin sensitivity in normotensive offspring of hypertensive parents. Am J Hypertens 1998; 11:1312. 96. Wu DA, Bu X, Warden CH, et al. Quantitative trait locus mapping of human blood pressure to a genetic region at or near the lipoprotein lipase gene locus on chromosome 8q22. J Clin Invest 1996; 97:2111. 97. Steinberg HO, Chaker H, Learning R, et al. Obesity/insulin resistance is associated with endothelial dysfunction. Implications for the syndrome of insulin resistance. J Clin Invest 1996; 97:2601. 98. Zeng G, Quon MJ. Insulin-stimulated production of nitric oxide is inhibited by wotmannin. Direct measurement in vascular endothelial cells. J Clin Invest 1996; 98:894. 99. Nickenig G, Roling J, Strehlow K, et al. Insulin induces upregulation of vascular ATI receptor gene expression by postranscriptional mechanisms. Circulation 1998; 98:2453. 100. Kamide K, Hori MT, Zhu J-H, Tuck ML. Insulin-mediated growth in aortic smooth muscle and the vascular renin-angiotensin system. Hypertension 1998; 32:482. 101. Hall JE, Coleman TG, Mizelle HL, Smith MJ. Chronic hyperinsulinemia and blood pressure regulation. Am J Physiol 1990; 258:F722. 102. Heise T, Magnusson K, Heinemann L, Sawicki PT. Insulin resistance and the effect of insulin on blood pressure in essential hypertension. Hyperten¬ sion 1998; 32:243. 103. Kaplan NM. Obesity, insulin and hypertension. Cardiovasc Risk Factors 1994; 4:133. 104. Peiris AN, Sothman MS, Hoffman RG, et al. Adiposity, fat distribution and cardiovascular risk. Ann Intern Med 1989; 110:867. 105. Tuck ML. Obesity, the sympathetic nervous system, and essential hyper¬ tension. Hypertension 1992; 19(Suppl l):I-67. 106. Reisin E. Sodium and obesity in the pathogenesis of hypertension. Am J Hypertens 1990; 3:164. 107. Schmieder RE, Messerli FH. Does obesity influence early target organ dam¬ age in hypertensive patients? Circulation 1993; 87:1482. 108. Ostlund RE, Staten M, Kohrt WM, et al. The ratio of waist-to-hip circumfer¬ ence, plasma insulin level, and glucose intolerance as independent predictors of the HDL2 cholesterol level in older adults. N Engl J Med 1990; 322:229. 109. Abate N, Garg A, Peshock RM, et al. Relationship of generalized and regional adiposity to insulin sensitivity in man. J Clin Invest 1995; 96:88. 110. Chan JM, Rimm EB, Colditz GA, et al. Obesity, fat distribution and weight gain as risk factors for clinical diabetes in men. Diabetes Care 1994; 17:961. 111. Landsberg L. Hyperinsulinemia: possible role in obesity-induced hyper¬ tension. Hypertension 1992; 19(Suppl 1):I-61. 112. Maxwell MH, Heber D, Waks AU, Tuck ML. Role of insulin and norepi¬ nephrine in the hypertension of obesity. Am J Hypertens 1994; 7:402. 113. Vaz M, Jennings G, Turner A, et al. Regional sympathetic and oxygen con¬ sumption in obese normotensive human subjects. Circulation 1997; 6:3423. 114. Grassi G, Seravalle G, Cattaneo BM, et al. Sympathetic activation in obese normotensive subjects. Hypertension 1995; 25[Pt I]:560. 115. Narkiewiez K, van de Borne PJ, Cooley RL, et al. Sympathetic activity in obese subjects with and without obstructive sleep apnea. Circulation 1998; 98:772. 116. Haynes WG, Morgan DA, Walsh SA, et al. Receptor-mediated regional sympathetic nerve activation by leptin. J Clin Invest 1997; 100:270 117. Wilden E, Lehto M, Kanninen T, et al. Association of a polymorphism in the (33-ad renergic receptor gene with features of the insulin resistance syn¬ drome in Finns. N Engl J Med 1995; 333:348. 118. Reisin E, Mathew R, Falkner B, et al. Lisinopril versus hydrochlorothiazide in obese hypertensive patients. A multicenter placebo-controlled trial. Hypertension 1997; 30[Pt I]:140. 119. Lind L, Hvarfner A, Palmer M, et al. Hypertension in primary hyperpar¬ athyroidism in relation to histopathology. Eur J Surg 1991; 157;457. 120. Brickman AS, Nyby MD, vonHungen K, Tuck ML. Calciotropic hormones, platelet calcium and blood pressure in essential hypertension. Hyperten¬ sion 1990; 16:515. 121. Brickman AS, Stern N, Sowers JR. Hypertension in pseudohypoparathy¬ roidism. Am J Med 1988; 85:785. 122. Gennari C, Nami R, Gonelli A. Hypertension and primary hyperparathy¬ roidism: the role of adrenergic and renin-angiotensin-aldosterone systems. Miner Electrolyte Metab 1995; 21:77. 123. Sancho JJ, Rouco J, Riera-Vidal R, Sitges-Serra A. Long-term effects of para¬ thyroidectomy for primary hyperparathyroidism on arterial hypertension. World J Surg 1992; 16:732. 124. Zawada E Jr, Brickman AS, Maxwell MH, Tuck ML. Hypertension associ¬ ated with hyperparathyroidism is not responsive to angiotensin blockade. J Clin Endocrinol Metab 1980; 50:912. 125. Sowers JR, Standley PR, Tuck ML, Ram JL. Calcium and calcium regulatory hormones in hypertension. In: Laragh JH, Brenner BM, eds. Hypertension: pathophysiology, diagnosis and management, 2nd ed. New York: Raven Press, 1995:1155. 126. Resnick L. The cellular ionic basis of hypertension and allied clinical condi¬ tions. Prog Cardiovasc Dis 1999; 42(1):1. 127. Ezzat S, Forster MJ, Berchtold P, et al. Acromegaly: clinical and biochemical features in 500 patients. Medicine 1994; 73:233. 128. Kratz C, Benker G, Weber F, et al. Acromegaly and hypertension: preva¬ lence and relationship to the renin-angiotensin-aldosterone system. Klin Wochenschr 1990; 68:583. 129. Connell JMC, Davies DL. Endocrine hypertension: thyroid disease and

Ch. 82: Endocrine Aspects of Hypertension

130.

131.

132. 133. 134. 135.

136.

137.

138.

139.

140.

141.

142.

143.

144.

145.

146.

147. 148.

149.

150.

acromegaly. In: Swales JD, ed. Textbook of hypertension. Oxford, UK: Blackwell Science, 1994:959. Lopez-Velasco R, Escobar-Morreale HF, Vega B, et al. Cardiac involvement in acromegaly: specific myocardiopathy or consequence of systemic hyper¬ tension? J Clin Endocrinol Metab 1997; 82:1047. Akpununu BE, Mulrow PJ, Hoffman EA. Secondary hypertension: evalua¬ tion and treatment: thyrotoxicosis and hypertension. Dis Mon 1996; 42:689 and 42:696. Saito I, Saruta T. Hypertension and thyroid disorders. Endocrinol Metab Clin North Am 1994; 23:379. Levy GS, Klein I. Catecholamine-thyroid hormone interactions and cardio¬ vascular manifestations of hyperthyroidism. Am J Med 1990; 88:642. Klein I, Ojama K. Cardiovascular manifestations of endocrine disease. ] Clin Endocrinol Metab 1992; 75:339. Streeten DHP, Anderson GH, Howland T, et al. Effects of thyroid function on blood pressure: recognition of hypothyroid hypertension. Hypertension 1988; 11:78. Laragh JH, Sealey JE. The renin-angiotensin-aldosterone system and the renal regulation of sodium and potassium and blood pressure homeosta¬ sis. In: Windhager EE, ed. Handbook of renal physiology. New York: Oxford University Press, 1992:1409. Alderman MH, Madhavan S, Ooi WL, et al. Association of the reninsodium profile with risk of myocardial infarction in patients with hyper¬ tension. N Engl J Med 1991; 324:1098. Trenkwalder P, James GD, Laragh JH, Sealey JE. Plasma renin activity and plasma prorenin are not suppressed in hypertensives surviving to old age. Am J Hypertens 1996; 9:621. Blaufox MD, Lee HB, Davis B, et al. Renin predicts the blood pressure response to nonpharmacologic and pharmacologic therapy. JAMA 1992; 267:1221. Wyndham RN, Gimenez L, Walker WG, et al. Influence of renin levels on the treatment of essential hypertension with thiazide diuretics. Arch Intern Med 1987; 147:1021. Rich GM, Ulick S, Cook S, et al. Glucocorticoid-remediable aldosteronism in a large kindred: clinical spectrum and diagnosis using a characteristic biochemical phenotype. Ann Intern Med 1992; 116:813. Soro A, Ingram MC, Tonolo G, et al. Evidence of coexisting changes in 11(3dehydrogenase and 5(3-reductase activity in subjects with untreated essen¬ tial hypertension. Hypertension 1995; 25:67. Snyder PM, Price MP, McDonald FJ, et al. Mechanism by which Liddle's syndrome mutations increase activity of a human epithelial Na+ channel. Cell 1995; 83:969. Griffing GT, Dale SL, Holbrook MM, Melby JC. The regulation of urinary free 19-nordeoxycorticosterone and its relation to systemic arterial blood pressure in normotensive and hypertensive subjects. J Clin Endocrinol Metab 1983; 56:99. Resnick LM, Muller FB, Laragh JH. Calcium-regulating hormones in essen¬ tial hypertension. Relation to plasma renin activity and sodium metabo¬ lism. Ann Intern Med 1986; 105:649. Sealey JE, Blumenfeld JD, Bell GM, et al. On the renal basis for essential hypertension: nephron heterogeneity with discordant renin secretion and sodium excretion causing a hypertensive vasoconstriction-volume rela¬ tionship. J Hypertens 1988; 6:763. Williams GH. Converting-enzyme inhibitors in the treatment of hyperten¬ sion. N Engl J Med 1988; 319:1517. Redgrave J, Rabinowe S, Hollenberg NK, Williams GH. Correction of abnormal renal blood flow response to angiotensin II by converting enzyme inhibitor. J Clin Invest 1985; 75:1285. Williams GH, Fisher ND, Hunt SC, et al. Effects of gender and genotype on the phenotypic expression of nonmodulating essential hypertension. Kid¬ ney Int 2000; 57(4):1404. Johnston Cl, Franz Volhard Lecture. Renin-angiotensin system: a dual tis¬ sue and hormonal system for cardiovascular control. J Hypertens 1992;

10(Suppl 7):S13. 151. Seikaly MG, Arant BS, Seney FD. Endogenous angiotensin concentration in specific intrarenal fluid compartments of the rat. J Clin Invest 1990; 86:1352. 152. Bachman S, Peters J, Engler E, et al. Transgenic rats carrying the mouse renin gene-morphologic characterization of a low-renin hypertension model. Kidney Int 1992; 41:24. 153. Jeunemaitre X, Soubrier F, Kotelevtsev YV, et al. Molecular basis of human hypertension: role of angiotensinogen. Cell 1992; 71:169. 154. Hunt SC, Cook NR, Oberman A, et al. Angiotensinogen genotype, sodium retention, weight loss and prevention of hypertension: trials of hyperten¬ sion prevention. Hypertension 1998; 32:393. 155. Lindpianter K, Pfeffer MA, Kreutz R, et al. A prospective evaluation of an angiotensin-converting enzyme gene polymorphism and the risk of ischemic heart disease. N Engl J Med 1995; 332:706. 156. Kauma H, Piavansalo M, Savolainen MJ, et al. Association between angio¬ tensin converting enzyme gene polymorphism and carotid atherosclerosis. J Hypertens 1996; 14:1183. 157. Takezako T, Saku K, Zhang B, et al. Angiotensin I converting enzyme gene polymorphism and insulin resistance in patients with angina pectoris. Am J Hypertens 1999; 12:291. 158. Marre M, Bemadet P, Gallois Y, et al. Relationships between angiotensin I converting enzyme gene polymorphism, plasma levels, and diabetic retinal and renal complications. Diabetes 1994; 43:384.

805

159. Fujisawa T, Ikegami H, Kawaguchi Y, et al. Meta-analysis of association of insertion/deletion polymorphism of angiotensin I-converting gene with diabetic nephropathy and retinopathy. Diabetologia 1998; 41:47. 160. Julius S. Changing role of the autonomic nervous system in human hyper¬ tension. J Hypertens 1990; 9(Suppl 7):S59. 161. Goldstein DS, Kopin IJ. The autonomic nervous system and catechola¬ mines in normal blood pressure control and in hypertension. In: Laragh JH, Brenner BM, eds. Hypertension: Pathophysiology, Diagnosis and Man¬ agement. New York: Raven Press, 1990:711 . 162. Esler M, Jennings G, Lambert G, et al. Overflow of catecholamine neu¬ rotransmitters to the circulation: source, fate, and function. Physiol Rev 1990; 70:963. 163. Mark AL. The sympathetic nervous system in hypertension: a potential long-term regulator of arterial pressure. J Hypertens 1996; 14(Suppl):S159. 164. Izzo JL. Sympathoadrenal activity, catecholamines and tire pathogenesis of vasculopathic hypertensive target-organ damage. Am J Hypertens 1989; 2:3055. 165. Brooks VL, Osborn JW. Hormonal-sympathetic interactions in the long¬ term of arterial pressure: an hypothesis. Am J Physiol 1995; 268:R1343. 166. McCarty R, Gold P. Catecholamine, stress and disease: a psychobiologic perspective. Psycosom Med 1996; 58:590. 167. Ichihara A, Inscho EW, Imig JD, et al. Role of renal nerves in afferent arteriolar reactivity in angiotensin-induced hypertension. Hypertension 1997; 29:442. 168. Ascio LP, Ladiness C, Fuchs S, et al. Distribution of the dopamine D3 recep¬ tor gene produces renin-dependent hypertension. J Clin Invest 1998; 102:493. 169. Pa J, Eisner GM. Renal dopamine receptors in health and hypertension. Pharmacol Ther 1998; 80:149. 170. Gill JR, Grossman E, Goldstein DS. High urinary dopa excretion and low urinary dopamine: dopa ratio in salt-sensitive hypertension. Hypertension 1991; 18:614. 171. Weinberger MH. Salt sensitivity of blood pressure in humans. Hyperten¬ sion 1996; 27:481. 172. Lijnen P. Alterations in sodium metabolism as an etiological model for hypertension. Cardiovasc Drugs 1995; 9:377. 173. Corry DB, Tuck ML. Altered sodium and potassium metabolism in the pathogenesis of hypertension. In: Maxwell MH, Kleeman C, Narrins R, eds. Disorders of fluid and electrolyte metabolism, 5th ed. New York: McGraw-Hill, 1993:1245. 174. Bobick A, Neylon CB, Little PJ. Disturbances of vascular smooth muscle cation transport and the pathogenesis of hypertension. In: Swales JD, ed. Textbook of hypertension. Oxford, UK: Blackwell Science, 1994:175. 175. Sweeney G, Somwar R, Ramlal T, et al. Insulin stimulation of K+ uptake in 3T3-L1 fibroblasts involves phosphatidylinositol 3-kinase and protein kinase C-zeta. Diabetologia 1998; 41:1199. 176. Blaustein MP. Physiologic effects of endogenous ouabain: control of intra¬ cellular calcium stores and cell responsiveness. Am J Physiol 1993; 264:0367. 177. Berger ME, Ormsby BL, Bunnag P, et al. Increased functional Na(+)-K(+) pump activity in the vasculature of fructose-fed hyperinsulinemic and hypertensive rats. Hypertens Rev 1998; 21(2):73. 178. Rutherford PA, Thomas TH, Wilkinson R. Na-Li countertransport kinetics in the relatives of hypertensive patients with abnormal Na-Li countertrans¬ port activity. Biochem Mol Med 1997; 62(1):106. 179. Strazzulo P, Siani A, Cappuccio FP, et al. Red blood cell sodium-lithium countertransport and risk of future hypertension: the Olivetti Prospective Heart Study. Hypertension 1998; 31(6):1284. 180. Van Norren K, Thien T, Berden JH, et al. Relevance of erythrocyte Na+/Li+ countertransport measurement in essential hypertension, hyperlipidemia and diabetic nephropathy: a critical review. Eur J Clin Invest 1998; 28(5):339. 181. Ragone E, Strazullo P, Siani A, et al. Ethnic differences in red blood cell sodium/lithium countertransport and metabolic correlates of hyperten¬ sion: an international collaborative study. Am J Hypertens 1998; 11(8; Pt 1): 935. 182. Sanchez RA, Gimenez MI, Migliorini M, et al. Erythrocyte sodium-lithium countertransport in non-modulating offspring and essential hypertensive individuals: response to enalapril. Hypertension 1997; 30:99. 183. Monciotti CG, Semplicini A, Morocutti A, et al. Elevated sodium-lithium countertransport activity in erythrocytes is predictive of the development of microalbuminuria in IDDM. Diabetologia 1997; 40(6):654. 184. Carr SJ, Moore D, Sikand K, Norman RI. Raised affinity for extracellular sodium of the sodium-lithium countertransporter is associated with a fam¬ ily history of hypertension and uraemia in patients with renal disease. Clin Sci 1997; 92(5):497. 185. Jones SC, Thomas TH, Marshall SM. Thiol group modulation of sodiumlithium countertransport kinetics in diabetic nephropathy. Diabetologia 1997; 40:1079. 186. Giordano M, Castellino P, Solini A, et al. Na+/Li+ and Na+/H+ counter¬ transport activity in hypertensive non-insulin-dependent diabetic patients: role of insulin resistance and antihypertensive treatment. Metabolism 1997; 46:1326. 187. Siffert W, Dusing R. Sodium-proton exchange and primary hypertension: an update. Hypertension 1995; 26:649. 188. Ng LL, Sweeney FP, Siczkowski M, Davies JE, et al. Na(+)-H(+) antiporter phenotype, abundance, and phosphorylation of immortalized lympho¬ blasts from humans with hypertension. Hypertension 1995; 25:971. 189. Rosskopf D, Schroder KJ, Siffert W. Role of sodium-hydrogen exchange in the proliferation of immortalised lymphoblasts from patients with essen¬ tial hypertension and normotensive subjects. Cardiovasc Res 1995; 29:254.

806

PART V: THE ADRENAL GLANDS

190. Garciandia A, Lopez R, Tisaire J, et al. Enhanced Na(+)-H(+) exchanger activity and NHE-1 mRNA expression in lymphocytes from patients with essential hypertension. Hypertension 1995; 25(3):356. 191. Diez J, Alonso A, Garciandia A, et al. Association of increased erythrocyte Na*/H‘ exchanger with renal Na+ retention in patients with essential hypertension. Am ] Hypertens 1995; 8(2):124. 192. LaPointe MS, Ye M, Moe OW, et al. Na+/H+ antiporter (NHE-1 isoform) in cultured vascular smooth muscle from the spontaneously hypertensive rat. Kidney Int 1995; 47(1):78. 193. Siczkowski M, Davies JE, Ng LL. Na(+)-H(+) exchanger isoform 1 phospho¬ rylation in normal Wistar-Kyoto and spontaneously hypertensive rats. Circ Res 1995; 76:825. 194. Siffert W, Rosskopf D, Moritz A, et al. Enhanced G protein activation in immortalized lymphoblasts from patients with essential hypertension. ] Clin Invest 1995; 96:759. 195. Takahashi E, Abe J, Berk BC. Angiotensin II stimulates p90rsk in vascular smooth muscle cells. A potential Na(+)-K(+) exchanger kinase. Circ Res 1997; 81 (2):268. 196. Koren W, Postnov IY, Postnov YV. Increased Na(+)-H(+) exchange in red blood cells of patients with primary aldosteronism. Hypertension 1997; 29(2):587. 197. Fortuno A, Tisaire J, Lopez R, et al. Angiotensin converting enzyme inhibi¬ tion corrects Na+,H+ exchanger overactivity in essential hypertension. Am J Hypertens 1997; 10(1):84. 198. Gende OA. Chelerythrine inhibits Na(+)-H(+) exchange in platelets from spontaneously hypertensive rats. Hypertension 1996; 28(6):1013. 199. Chen YF, Yang RH, Meng QC, et al. Sodium-proton (Na+/H+) exchange inhibition increases blood pressure in spontaneously hypertensive rats. Am J Med Sci 1994; 308:145. 200. Trevisan R, Viberti G. Sodium-hydrogen antiporter: its possible role in the genesis of diabetic nephropathy. Nephrol Dialysis Transplant 1997; 12(4):643. 201. Ng LL, Davies JE, Siczkowski M, et al. Abnormal Na+/H+ antiporter phe¬ notype and turnover of immortalized lymphoblasts from type 1 diabetic patients with nephropathy. J Clin Invest 1994; 93(6):2750. 202. Siczkowski M, Davies JE, Sweeney FP, et al. Na+/K+ exchanger isoform-1 abundance in skin fibroblasts of type I diabetic patients with nephropathy. Metabolism 1995; 44:791. 203. Tuck ML, Corry DB, Maxwell M, et al. Erythrocyte Na+,K+ cotransport and Na+,K+ pump in black and Caucasian hypertensive patients: a kinetic anal¬ ysis. Hypertension 1987; 10:204. 204. Tepper T, Sluiter WJ, Huisman RM, deZeeuw D. Co-transport measure¬ ment in essential hypertension: useful diagnostic tool or failure? A meta¬ analysis of 17 years of literature. 205. Pares I, de la Sierra A, Coca MM, et al. Detection of a circulating inhibitor of the Na(+)-K(*J-Cl(~) cotransport system in plasma and urine after high salt intake. Am J Hypertens 1995; 8:965. 206. Blaustein MP. The physiological effects of endogenous ouabain: control of cell responsiveness. Am J Physiol 1993; 264:0367. 207. Hamlyn JM, Hamilton BP, Manunta P. Endogenous ouabain, sodium balance and blood pressure: a review and an hypothesis. J Hypertens 1996; 14:151. 208. Wilkens MR, Redondo J, Brown LA. The natriuretic peptide family. Lancet 1997; 349:1307 209. Roller KJ, Goeddel DV. Molecular biology of the natriuretic peptides and their receptors. Circulation 1992; 86:1081. 210. Burnett JC Jr. Vasopeptidase inhibition: a new concept in blood pressure management. J Hypertens 1999; 17(Suppl 1):S37. 210a. Schriger JA, Grantham JA, Kullo IJ, et al. Vascular actions of brain natri¬ uretic peptide: modulation by atherosclerosis and neutral endopeptidase inhibition. J Am Coll Cardiol 2000; 35:796. 211. Lainchbury JG, Espinar EA, Frampton CM, et al. Cardiac natriuretic pep¬ tides as predictors of mortality. J Intern Med 1997; 241:257. 212. Bhoola KD, Figueroa CD, Worthy K. Bioregulation of kinins: kallikreins, kininogens, and kinases. Pharmacol Rev 1992; 44:1. 213. Carretero OA, Scili AG. The kallikrein-kinin system. In: Fozzard HA, ed. The heart and cardiovascular system. New York: Scientific Foundations, Raven Press, 1991:1851. 214. Liu LH, Yang XP, Sharov VG, et al. Effects of angiotensin-converting enzyme inhibitors and angiotensin II type 1 receptor antagonists in rats with heart fail¬ ure: role of kinins and angiotensin type 2 receptor. J Clin Invest 1997; 99:1926. 215. Yang XP, Liu YH, Scicli GM, et al. Role of kinins in the cardioprotective effect of preconditioning: study of myocardial ischemia/reperfusion injury in B2 kinin receptor knockout mice and kininogen-deficient rats. Hypertension 1997; 30:735. 216. Dattilo JB, Makhoul RG. The role of nitric oxide in vascular biology and pathobiology. Ann Vase Surg 1997; 11:307. 217. Ignarro L, Murad F. Nitric oxide biochemistry, molecular biology, and ther¬ apeutic implications. In: JT August, MW Anders, F Murad, ed. Advances in Pharmacology. New York: Academic Press, 1995. 218. Garcia-Cardena G, Martasek P, Masters BS, et al. Dissecting the interaction between nitric oxide synthase (NOS) and caveolin: functional significance of the NOS caveolin binding domain in vivo. J Biol Chem 1997; 272:25437. 219. Busse R, Fleming I. Nitric oxide, nitric oxide synthase and hypertensive vascular disease. Curr Hypertension Rep 1999; 1:88. 220. Garcia-Cardena G, Fan R, Shah V, et al. Dynamic activation of endothelial nitric oxide by Hsp90. Nature 1998; 292:821. 221. Murad F. The 1996 Albert Lasker Medical Research Awards. Signal transduction using nitric oxide and cyclic guanosine monophosphate. JAMA 1996; 276:1189.

222. Haller H. Endothelial function: general considerations. Drugs 1997; 53(Suppl 1):1. 223. Ferro CJ, Webb DJ. Endothelial dysfunction and hypertension. Drugs 1997; 53 (Suppl 1):30. 224. Nakayama T, Soma M, Takahashi Y, et al. Association analysis of CA repeat polymorphism of the endothelial nitric oxide synthase gene with essential hypertension in Japanese. Clin Genet 1997; 51:26. 225. Miyamoto Y, Saito Y, Kajiyama N, et al. Endothelial nitric oxide synthase gene expression is positively associated with essential hypertension. Hypertension 1998; 32:3. 226. Bauersachs J, Bouloumie A, Mulsch A, et al. Vasodilator dysfunction in aged spontaneously hypertensive rats; changes in NO synthase III and sol¬ uble guanylyl expression and in superoxide anion production. Cardiovasc Res 1998; 37:772. 227. Luscher TF, Oemar BS, Boulanger CM, Hahn AWA. Molecular and cellular biology of endothelin and its receptors, Pts 1 and 2. J Hypertens 1993; 11:7 and 11:121. 228. Schiffrin EL, Intengan HD, Thibault G, Touyz RM. Clinical significance of endothelin in cardiovascular disease. Curr Opin Cardiol 1997; 12:354. 229. Moreau P, Rabelink TJ. Endothelin and its antagonists in hypertension: can we foresee the future? Curr Hypertens Rep 1999; 1:69. 230. Krum H, Viskoper RV, Lacourciere Y, et al. The effects of an endothelin receptor antagonist, bosantin, on blood pressure in patients with essential hypertension. N Engl J Med 1998; 338:784.

CHAPTER

83

ADRENOCORTICAL DISORDERS IN INFANCY AND CHILDHOOD ROBERT L. ROSENFIELD AND KE-NAN QIN

GENERAL PRINCIPLES Along with the well-known perturbations of fluid, electrolyte, and glucose homeostasis, adrenocortical diseases that occur in children also cause disturbed growth. The proper management of children with these disorders requires careful documentation of height and weight at regular intervals. Physicians who are relatively unfamiliar with pediatric patients may assume that the fluid and electrolyte requirements of infants and children are similar to those of adults. Actually, infants are hypermetabolic, as compared to adults, because of the relatively large size of their high-energy-consuming organs (i.e., the brain, heart, liver, and kidneys) as compared to their somatic size. Water requirements change in proportion to caloric requirements. One milliliter of water is required for each kilocalorie of energy expenditure. Water and calorie require¬ ments are approximately constant throughout life relative to surface area (1500 keal/m2 per day). Surface area can be calcu¬ lated as the square root of (cm x kg/3600). Normal daily sodium and potassium maintenance require¬ ments are ~2 mEq/dL and 1 mEq/dL water, respectively. When calculating electrolyte replacement, it must be remembered that the exchangeable fluid compartment is relatively larger in chil¬ dren than in adults, ranging from 60% of body weight in small children to 40% in adults. Because of the rapid rate of turnover of children's body fluids, the electrolyte concentrations of parenteral fluids should be distributed evenly throughout the day to prevent shifts in the tonicity of body fluids. Infants and children are intolerant of prolonged fasting because of their high metabolic rate and functionally immature gluconeogenic enzyme systems. When fasted, the normal young child will become hypoglycemic within as few as 20 hours. To prevent glycogenolysis, infants and young children need 6 to 8 mg/kg per minute of glucose.1

807

Ch. 83: Adrenocortical Disorders in Infancy and Childhood TABLE 83-1. Typical Normal Plasma Values in Infancy and Childhood for Renin-Aldosterone Axis Age Term, 15 yr, ambulatory >15 yr, supine

PRA (ng/mL/h) Range

Aldosterone (ng/dL) Range

performance IQ Cognitive deficits (e.g., space-form blindness) Immature personality, probably secondary to short stature

CRANIOFACIAL Premature fusion of spheno-occipital and other sutures, producing brachycephaly Triangular facies Low-set ears Abnormal pinnae Retruded mandible Epicanthal folds (25%)+ Ptosis High-arched palate (36%) "Double" eyelashes Abnormal dentition Visual anomalies, usually strabismus (22%) Auditory deficits: sensorineural or secondary to middle ear infections "Wooly" hair

NECK Pterygium coli (46%) Short broad neck (74%) Low nuchal hair line (71%)

CHEST Rectangular contour (i.e., shield chest; 53%) Apparent widely spaced nipples

FIGURE 90-15. A, Appearance of one of original seven cases of Turner syndrome reported in 1938. B, Same patient, photographed in 1972, almost 35 years after publication of the original case report. The karyo¬ type was documented to be 45,X. The patient had received little estro¬ gen in intervening years and experienced severe osteoporosis. (Courtesy of Dr. R. Rebar and Dr. S. S. C. Yen, University of California, San Diego.)

Inverted nipples Tapered lateral ends of clavicle

CARDIOVASCULAR Coarctation of aorta or ventricular septal defect (10-16%) Aortic stenosis Pulmonic stenosis (rare)

RENAL (38%) Horseshoe kidneys Unilateral renal aplasia Duplicated ureters

NONGONADAL FEATURES OF TURNER SYNDROME. The com¬ mon anomalies of Turner syndrome include epicanthal folds, high arched palate, low nuchal hairline, webbed neck, shield¬ like chest, coarctation of the aorta, ventricular septal defect, renal anomalies, pigmented nevi, lymphedema, hypoplastic nails, and cubitus valgus (Table 90-3). Inverted nipples and double eyelashes may be present as well. No feature is pathog¬ nomonic, but in aggregate, they form a spectrum of anomalies more likely to exist in 45,X individuals than in normal 46,XX individuals. These anomalies are the Turner stigmata, the pres¬ ence of which suggests the coexistence of gonadal dysgenesis. Individuals with a 45,X karyotype have low birth weights (adjusted mean, 2851.1 ± 65.1 g).66 Total body length at birth is sometimes less than normal, but often it is normal. Height velocity before puberty generally lies in the 10th to the 15th percentile, and the mean heights of 45,X adults (16 years or older) range from 141 to 146 cm (55.5-57.5 in).72"75 In untreated patients, the epiphyses remain open; additional growth occurs when sex steroids are administered. Despite the diminished final height of such patients, their adult stature tends to corre¬ late with parental height.75 (Also see ref. 75a.) That not all patients with gonadal dysgenesis are short indi¬ cates that sex steroid deficiency is not the cause. For example, normal stature is characteristic of individuals with 46,XX gonadal dysgenesis. Growth hormone (GH) levels have long been considered essentially normal in individuals with gonadal dysgenesis.76-77 Cellular resistance to GH, however, has been sug¬ gested. This relative resistance may be overcome by treatment with exogenous GH at higher doses than those used for classic GH deficiency.78 Anti-GH antibodies have been detected, and GH reserve may be decreased.79'80 More evidence suggests that abnormalities in GH or insulin-like growth factor-I (IGF-I), which fall below the normal range after 8 years of age, are sec¬ ondary to the lack of gonadal activation and estrogen secretion.81

GASTROINTESTINAL Telangiectasias

SKIN AND LYMPHATICS Pigmented nevi (63%) Capillary hemangiomas Lymphedema (39%) secondary to hypoplasia of superficial vessels (especially neonatal)

NAILS Hypoplasia or malformation (66%)

SKELETAL Cubitus valgus (54%) Radial tilt of articular surface of trochlear eminence of the humerus Clinodactyly V Short metacarpals, usually fourth finger (48%) Decreased carpal arch (mean angle, 117 degrees) Deformities of medial tibial condyle Hypoplastic or fused (rarely) cervical vertebrae Kyphosis Scoliosis Square lumbar vertebral bodies

DERMATOGLYPHICS Increased total digital ridge count (mean, 166.1 ± 8.6) Increased distance between palmar triradii a and b Distal axial triradius in position t1

PELVIC ALTERATIONS Android pelvic inlet Small iliac wings Narrow sacrosciatic notches Narrow pubic arch *A list of somatic anomalies frequently associated with a 45,X chromosomal com¬ plement. Many other anomalies have been reported in 45,X individuals, frequencies are given in parentheses. (Data from Simpson JL. Ovarian determinants through phenotypic-karyotypic deductions: progress and pitfalls. In: Rosenfield R, Grumbach M, eds. Turner syndrome. New York: Marcel Dekker, 1990:65; Simpson JL. Disorders of sexual differentiation: eti¬ ology and clinical delineation. New York: Academic Press, 1976; and Simpson JL. Gonadal dysgenesis and abnormalities of the human sex chromosomes: current status of phenotypic-karyotypic correlations. Birth Defects 1975; 11[4]:23.)

864

PART VI: SEX DETERMINATION AND DEVELOPMENT

Short stature may be common in gonadal dysgenesis because the epiphyses are structurally abnormal. This hypothe¬ sis is compatible with observations that decreased growth occurs in the long bones, teeth, and skull.82-83 One aspect of Turner syndrome may be a skeletal dysplasia. Most 45,X patients have normal intelligence, but any given 45, X patient has a slightly higher probability of being retarded than a 46,XX individual. The frequency of overt retardation is 11% to 17%.72 Biases of ascertainment dictate that this preva¬ lence probably represents the maximum risk. Performance IQ is definitely lower than verbal IQ, however; 45,X individuals have an unusual cognitive defect characterized by an inability to appreciate the shapes and relations of objects with respect to one another (i.e., space-form blindness).84-86 The patients usu¬ ally appear socially immature, probably in part because they are short and sexually immature.87 METABOLIC ALTERATIONS. Diabetes mellitus, thyroid dis¬ ease, and essential hypertension often are present in individuals with 45,X karyotypes. Abnormal oral glucose-tolerance tests may occur in as many as 40% of these individuals.88 Both autoim¬ mune thyroiditis and Graves disease are observed with increased frequency in patients with Turner stigmata. Approximately onethird of adult 45,X patients have essential hypertension, which also may occur in young 45,X girls. Therapy for any metabolic alterations is not unique, although reduction or careful monitor¬ ing of exogenous estrogen therapy may be necessary. Abnormalities of the X Chromosome. Several different abnormalities of the X chromosome have been associated with gonadal dysgenesis. The cytologic origin of these defects was considered earlier. DELETION OF THE SHORT ARM OF CHROMOSOME X. A ter¬ minal deletion of the X short arm [del(Xp)] may or may not cause gonadal dysgenesis, short stature, and other features of the Turner stigmata. The phenotype depends on the amount of Xp that is deficient. Spontaneous menstruation, albeit usually leading to sec¬ ondary amenorrhea, has occurred in almost 40% of reported 46, X,del(X)(pll) individuals (see Fig. 90-7). Almost all 46,X,del(X)(p21) individuals menstruate, but only approximately one-half become pregnant.7-34-89'90 These data indicate that ovarian tissue persists more often in del(Xp) individuals than in 45,X indi¬ viduals and that complete ovarian failure (with primary amenor¬ rhea) occurs only if the proximal and the terminal portions of Xp are deleted. However, the mean adult heights are 140 cm (55 in) for 46,X,del(X)(pll) and 146.5 cm (57.5 in) for 46,X,del(X)(p21) persons89-91 (see Fig. 90-8). Inasmuch as 46,X,del(X)(p21) individu¬ als are short but have normal ovarian function, determinants for ovarian maintenance and for stature must be located in different regions of Xp; statural determinants are more distal.35-36-91 No evi¬ dence yet exists that any given X-specific probe bears a special relation to X-ovarian or X-structural determinants. ISOCHROMOSOME FOR THE X LONG ARM (1 [XQ]). Almost all 46,X,i(Xq) patients have streak gonads, short stature, and features of the Turner stigmata. In addition to having a dupli¬ cation of Xq,i(Xq), these individuals differ from del(X)(pll) persons because the terminal portion and almost all of Xp is deleted. The better gonadal function in del(X)(pll) than in i(Xq) individuals is consistent with the location of ovarian determinants at several different sites on Xp. A locus on Xp may be deleted in i(Xq) karyotype but be retained near the cen¬ tromere in 46,X,del(X)(pll) cases. Because duplication of Xq (i.e., 46,X,i[Xq]) does not compensate for deficiency of Xp, the gonadal determinants on Xq and Xp must have different func¬ tions. Whether duplication of Xq per se produces abnormali¬ ties is unknown. DELETION OF THE LONG ARM OF CHROMOSOME X (DEL[XQ]). Most patients with a deletion of the X long arm have streak gonads and never menstruate. This is especially true of individuals with del(X)(ql3). However, deletions of dis¬ tal Xq are more likely to be associated with premature ovarian

failure than with primary amenorrhea. The Xq appears to con¬ tain more than one region that is required for normal ovarian function. Perhaps several Xq loci can affect ovarian function in additive fashion. The only clue to the nature of any of these products is the suggestion that the diaphanous (DM) gene, localized to Xq25, plays a role; however, other genes in distal Xq are also pivotal to ovarian development. Originally, deletions of Xq were not thought to result in short stature, but later tabulations show a definitely decreased mean height of persons with del(X)(ql3).36-72-89-92 Whether the short stature reflects specific loci or vicissitudes of X-inactivation is unclear.93 Mosaicism MOSAICISM INVOLVING ONLY X CHROMOSOMES. The 45,X/ 46,XX individuals have fewer anomalies than 45,X individuals. In one survey, 12% of 45,X/46,XX individuals menstruated, compared with only 3% of 45,X individuals.72 In that survey, the mean adult height was greater in 45,X/46,XX persons than in 45,X individuals. More mosaic patients (25%) than nonmo¬ saic patients (5%) reach adult heights greater than 152 cm (60 in). Somatic anomalies are less likely to occur in 45,X/46,XX than in 45,X patients. MOSAICISM WITH A Y CHROMOSOME. Individuals with a 45,X cell line and at least one line containing a Y chromosome manifest a variety of phenotypes, ranging from almost normal males with cryptorchidism or penile hypospadias to females indistinguishable from those with the 45,X Turner syndrome. The different phenotypes presumably reflect different tissue dis¬ tributions of the various cell lines, although this assumption remains unproven. At any rate, 45,X/46,XY individuals may show unambiguous female external genitalia, ambiguous exter¬ nal genitalia, or almost normal male external genitalia. Some 45,X/46,XY individuals with female external genitalia have the Turner stigmata and are clinically indistinguishable from 45,X individuals. Others, however, are female but of nor¬ mal stature and without somatic anomalies. As in other types of gonadal dysgenesis, the external genitalia, vagina, and miillerian derivatives remain unstimulated because of deficient sex steroids. Breasts fail to develop, and little pubic or axillary hair grows. In fact, breast development in a 45,X/46,XY individual should lead one to suspect an estrogen-secreting tumor, most commonly a gonadoblastoma or dysgerminoma. The streak gonads of 45,X/46,XY individuals usually are indistinguishable histologically from the streak gonads of indi¬ viduals with 45,X gonadal dysgenesis. However, gonadoblastomas or dysgerminomas develop in 15% to 20% of 45,X/46,XY individuals.55-56-94 Such neoplasms may arise as early as the first two decades of life. Gonadoblastomas occur almost exclusively in 46,XY or 45,X/46,XY individuals and usually are benign. However, they may be associated with dysgerminomas or other germ-cell tumors that are malignant. The gonads of 45,X/46,XY individuals should be extirpated regardless of the patient's age. Because of the risk of neoplasia, 45,X/46,XY gonadal dysgene¬ sis should be differentiated from forms of gonadal dysgenesis lacking a Y chromosome. When the polymerase chain reaction for SRY was used, unrecognized Y-chromosome material was found in 1 of 40 patients with Turner syndrome.95 Because the detection of SRY sequences in patients with gonadal dysgenesis is correlated with the presence of Y-chromosomal DNA and carries the risk of tumor development, the application of this technique in search of SRY may be justified in all individuals with this disorder. Individuals may show one streak gonad and one dysgenetic testis. The terms asymmetric gonadal dysgenesis or mixed gonadal dysgenesis are often applied to such individuals. They usually have ambiguous external genitalia. Many investigators believe that the phenotype of asymmetric gonadal dysgenesis is almost always associated with 45,X/46,XY mosaicism, with ostensibly nonmosaic cases reflecting merely an inability to analyze appro¬ priate tissues. Most 45,X/46,XY individuals with ambiguous

Ch. 90: Normal and Abnormal Sexual Differentiation and Development

FIGURE 90-16. A case of 46,XX gonadal dysgenesis: a 19-year-old woman with primary amenorrhea. Notice the normal stature and diminished breast development. Circulating gonadotropin levels were markedly elevated.

external genitalia have mullerian derivatives (e.g., a uterus). The presence of a uterus is helpful diagnostically, because a uterus is absent in most genetic forms of male pseudohermaphroditism. If an individual has ambiguous external genitalia, bilateral tes¬ tes, and a uterus, one may reasonably infer the presence of 45,X/ 46,XY mosaicism, whether or not both lines can be demon¬ strated cytogenetically. Occasionally, the uterus is rudimentary, or a fallopian tube fails to develop ipsilateral to a testis. The 45,X/46,XY mosaicism has less commonly been detected in individuals with almost normal male external geni¬ talia. In some individuals, hypospadias is present, but the sexof-rearing is unequivocally male. The 45,X/47,XXY and 45,X/46,XY/47,XXY complements exist, albeit much less often than 45,X/46,XY. These comple¬ ments are associated with the same phenotypic spectrum as 45,X/46,XY. Of particular interest is one family in which two and possibly three sibs had 45,X/46,XY/47,XYY mosaicism.95 The parents were second cousins, suggesting recessive factors. Gonadal Dysgenesis in 46,XX Individuals. The first indi¬ vidual with gonadal dysgenesis and an apparently normal female (46,XX) complement was reported in I960.12 By 1971, a survey of 61 such individuals led to the conclusion that the dis¬ order was inherited in an autosomal recessive fashion.1' A study of all XX gonadal dysgenesis cases in Finland confirmed this conclusion.97 The external genitalia and the streak gonads (see Fig. 90-14) in XX gonadal dysgenesis are indistinguishable from those in gonadal dysgenesis secondary to a sex chromosomal abnormal¬ ity. Likewise, the endocrine profiles do not differ, but individu¬ als with XX gonadal dysgenesis usually have normal stature (Fig. 90-16). Several pathogenic mechanisms can be postulated, but firm data have not been gathered. Phenocopies for XX gonadal dysgenesis also are well recognized (see Chap. 96). Both XX gonadal dysgenesis and neurosensory deafness have occurred in multiple sibs in several families; the occur¬ rence of deaf but fertile male sibs confirms autosomal inheri¬ tance.98 The coexistence of gonadal and auditory anomalies probably indicates a syndrome distinct from XX gonadal dys¬

865

genesis without deafness (i.e., genetic heterogeneity). Further evidence for genetic heterogeneity can be cited. In several other families, unique patterns of somatic anomalies indicate the existence of mutant genes distinct from those already dis¬ cussed. These include: XX gonadal dysgenesis and myopathy; XX gonadal dysgenesis and cerebellar ataxia; XX gonadal dys¬ genesis and metabolic acidosis; XX gonadal dysgenesis, micro¬ cephaly, and arachnodactyly.99-101 Follicle-Stimulating Hormone-Receptor Mutations. At least one form of XX gonadal dysgenesis is now known to be caused by a mutation of the FSH receptor (FSHR). Affected women present with primary or secondary amenorrhea and elevated serum FSH levels indicative of premature ovarian failure97-102 (see Chap. 96). The mutation originally was identified in a large number of sporadic and familial cases in Finland. Most cases were found in north central Finland, a sparsely populated part of the country. The overall frequency of the disorder in Finland was 1 per 8300 females, a relatively high incidence attributed to a founder effect. The segregation ratio of 0.23 for female sibs was consistent with autosomal recessive inheritance, as was the consanguinity rate of 12%. Sib-pair analysis using polymorphic DNA markers was used to localize the gene to a specific region of chromosome arm 2p, a region known to contain genes for both FSHR and the LH receptor (LHR). One specific mutation (C566T:alanine to valine) in exon 7 was observed in six multiplex families.102-103 On transvaginal ultrasonography, most of these patients have demonstrable ovarian follicles, raising the possibility of resid¬ ual receptor activity.103 The C566T mutation was not found in all Finnish XX gonadal dysgenesis patients and is rarely detected in 46,XX women with ovarian failure who reside outside Finland.104-105 Gonadal Dysgenesis in 46,XY Individuals. Gonadal dys¬ genesis also can occur in 46,XY individuals. In XY gonadal dysgenesis, affected individuals are phenotypic females who show sexual infantilism and bilateral streak gonads (Fig. 9017). The gonads may undergo neoplastic transformation (2030% prevalence)56 (Fig. 90-18). At least one form of XY gonadal dysgenesis results from an X-linked recessive or male-limited autosomal dominant gene.106-107 Sporadic cases may result from deletion or point mutations within SRY on the Y short arm.14-108-109 Further evidence for genetic heterogeneity lies in the exis¬ tence of at least three syndromes having XY gonadal dysgenesis as one of their componen ts: XY gonadal dysgenesis and long-limbed camptomelic dwarfism, XY gonadal dysgenesis and ectodermal defects, and the genitopalatocardiac syndrome.36-no-m Rudimentary Ovary Syndrome and Unilateral Streak Gonad Syndrome. The rudimentary ovary syndrome is a poorly defined entity of unknown cause said to be characterized by decreased numbers of follicles. This "syndrome" is heteroge¬ neous, not a single entity. Many cases have been associated with sex chromosomal abnormalities, particularly 45,X/46,XX mosaicism. Similar statements also apply to individuals with the unilateral streak ovary syndrome. For example, a unilat¬ eral streak gonad and a contralateral polycystic ovary have been observed in a 46,XX/46,X,i(Xq) individual who became pregnant.113 EVALUATION AND TREATMENT OF GONADAL DYSGENESIS.

When Turner stigmata are present, the diagnosis of gonadal dysgenesis usually is made early in childhood. The index of suspicion should be high for any infant with lymphedema of the hands and feet at birth, especially because the somatic anomalies are not very obvious in neonates (see Fig. 90-9). Other children present for evaluation of sexual infantilism, and still others with a male sex-of-rearing may not virilize at the expected age of puberty. Short stature is another com¬ mon reason that evaluation is sought. The measurement of circulating gonadotropin concentra¬ tions and the determination of the karyotype can establish the

866

PART VI: SEX DETERMINATION AND DEVELOPMENT

FIGURE 90-17. A and B, Two examples of 46,XY gonadal dysgenesis. Both 16-year-old individuals pre¬ sented with primary amenorrhea and markedly ele¬ vated concentrations of circulating gonadotropins. Both patients had dysgerminoma of an ovary; the patient in B also had a large gonadoblastoma of the contralateral ovary. Breast development as in B is extremely rare and no doubt secondary to hormone production by the patient's gonadal neoplasm. (B, Photograph reproduced from Villanueva AL, Benirschke K, Campbell J, et al. Complete development of secondary sexual characteris¬ tics in a case of 46,XY gonadal dysgenesia. Obstet Gynecol 1984; 64:68S.) diagnosis. Longitudinal studies have documented elevated gonadotropin levels at all ages in gonadal dysgenesis, indicat¬ ing absence of appropriate feedback inhibition of the hypotha¬ lamic-pituitary unit by the dysgenetic gonads even in childhood.114-115 Chromosomal studies are indicated to eliminate the possi¬ bility of a Y chromosome. The use of the polymerase chain reaction to detect sequences of SRY may well be warranted. If the phenotype and karyotype are compatible, the only tissue needed is blood for lymphocyte culture. If the phenotype and karyotype are incompatible (e.g., tall "45,X" subjects), skin or gonadal fibroblasts also should be cultured to detect any mosa¬ icism. If a Y chromosome is identified, surgical extirpation of the dysgenetic gonads is indicated to prevent neoplasms. Streak gonads usually can be removed by laparoscopy. In appropriate cases in which disseminated malignancies do not involve the gonads, the uterus may be left in situ for donor in vitro fertilization or embryo transfer. The evaluation of other commonly involved organ systems should include a careful physical examination, with special attention to the cardiovas¬ cular system, and should include thyroid function tests (including antibodies), fasting blood glucose level, renal func¬ tion tests, and an intravenous urogram or a renal ultrasono¬ graphic scan. The treatment of individuals with short stature has received significant attention. A multicenter, prospective, randomized trial of administration of GH, alone and in combination with oxandrolone, was initiated in 1983.116-117 Data on 62 girls, who have received 3 to 6 years of treatment, have been published. Given an average height of 143 cm for untreated girls in the United States, the mean height of 151.9 cm in the 30 girls whose therapy was terminated represented a net increase of 8.1 cm (therapy was terminated because the subjects had met study criteria for cessation of treatment, including bone age >14 years and a growth velocity of 27. As with the much better studied androgen-receptor gene (see earlier), DNA-binding (exons 2 and 3) and estrogen-binding domains (exons 4-8) are found. Although it is not technically male pseudohermaphroditism, a mutation in the estrogen receptor has been reported in a 28year-old genetic male with normal male sexual develop¬ ment.274,275 Incomplete epiphyseal closure led to tall stature. Endogenous serum gonadotropin and estrogen levels were ele¬ vated, and neither decreased after exogenous estrogen adminis¬ tration. The molecular basis proved to be a homozygous transition in exon 2 that resulted in a premature stop codon.274,275 5oc-REDUCTASE DEFICIENCY (PSEUDOVAGINAL PERINEOSCRO¬ TAL HYPOSPADIAS). Some genetic males have ambiguous external genitalia at birth but otherwise develop like normal males. At puberty, they undergo virilization with phallic enlargement, increased facial hair, muscular hypertrophy, voice deepening, and no breast development (see Chap. 115). The external genitalia consist of a phallus that resembles a clitoris more than a penis, a perineal urethral orifice, and, usually, a

878

PART VI: SEX DETERMINATION AND DEVELOPMENT

FIGURE 90-25. A, External genitalia of one of three 46,XY sibs with pseudovaginal perineoscrotal hypospadias (PPSH) phenotype who have deficiencies of 5a-reductase. The clitoris measures 5 to 6 cm long. Bilateral testes were present, one of which is visible (arroiv). B, When phallus is elevated, the pseudovaginal opening is evident. (From Opitz JM, Simpson JL, Sarto CE, et al. Pseudovaginal perineoscrotal hypospa¬ dias. Clin Genet 1972; 3:1.)

separate blindly ending perineal orifice that resembles a vagina (i.e., pseudovagina; Fig. 90-25). The testes are relatively normal in size but may be undescended, and they secrete normal amounts of testosterone. Subjects have erections with ejacula¬ tion from the perineal urethra. This abnormality, inherited in autosomal recessive fashion, results from a deficiency of 5a-reductase.276~286 The enzyme 5areductase converts testosterone to DHT, the androgen active within cells. That intracellular 5a-reductase deficiency results in characteristic genital abnormalities is consistent with embryologic findings that virilization of the external genitalia during embryogenesis requires DHT, but wolffian differentiation requires only testosterone. Two 5a-reductase (SRD5) genes exist. The type I gene (SRD5A1) is located on chromosome 5, and the type II gene (SRD5A2) on chromosome band 2p23. Only type II is expressed in gonads; thus, of the two isoforms, type II is deficient in male pseudohermaphroditism. Consisting of five exons,283 the SRD5A2 gene has been shown to have undergone deletions far less often284 than point mutations.285 Different ethnic groups have different mutations (founder effect), scattered among the five exons. The molecular etiology can be exploited for prenatal diagnosis and genetic counseling in kindreds, but usually only after one affected case has been identified. In adolescent subjects, the diagnosis is made most easily by finding an elevated ratio of plasma testosterone to DHT after hCG stimulation. Circulating LH levels are increased despite the presence of normal to high levels of testosterone, suggesting a feedback role for DHT in the regulation of LH secretion. FSH levels also tend to be elevated, perhaps because of the testicular damage induced by cryptorchidism. The deficiency in 5a-reductase also is present in cultured fibroblasts, in fibroblast homogenates, and in tissue homoge¬ nates. Because levels of 5a-reductase normally are highest in genital tissue, most investigators prefer to assay cells derived from genital tissue (e.g., foreskin). Unfortunately, a great vari¬ ability exists in 5a-reductase activity, with near overlap between controls and patients clearly deficient for the enzyme.287 Several hormonal studies may be needed to confirm the diagnosis. Measurement of 5a and 5(i urinary metabolites constitutes one approach. The ratio of plasma testosterone to DHT can be measured after hCG stimulation or after testoster¬ one stimulation.287-288 The diagnosis can be made on the basis of

an elevated ratio of urinary tetrahydrocortisol to 5a-tetrahydrocortisol.288 Gender identity changes from female to male in untreated individuals have been reported, leading to the use of the descriptive term "penis at 12" ("guevedoces") syndrome to describe this disorder.278 These changes in gender identity underscore the importance of androgens in affecting behav¬ ior and suggest that DHT is more important than testoster¬ one in utero (see Chap. 115). Two individuals with documented type II 5a-reductase deficiency had normal sperm concentrations, suggesting that dihydrotestosterone does not play a major role in spermatoge¬ nesis.289 However, low levels of DHT may be sufficient for normal spermatogenesis. INFERTILE MALES WITH ANDROGEN RESISTANCE. Some phe¬ notypic males with infertility have been said to have partial androgen resistance.290 Gynecomastia may exist. The suggestion has been made that a significant proportion of male infertility may be due to this cause. This view is plausible, but whether the disorder constitutes one defect or several remains unclear. Affected males typically have azoospermia or oligospermia and normal or increased circulating levels of LH and testosterone. The family history is unremarkable, somewhat surprisingly in view of the inheritance patterns of other syndromes of andro¬ gen insensitivity and casts doubt on the primary role of andro¬ gen resistance as the cause of infertility. Histologically, the testes show maturation arrest of Sertoli cells only. Decreased or abnor¬ mal androgen receptors have been identified. Potential Teratogenic Forms. At least four potential ter¬ atogens have been identified, but fortunately none has been reported as causing a case of female pseudohermaphroditism in humans. Although not approved for this indication, the four drugs have been used to treat women with hirsutism. Cyproterone acts by blocking uptake of androgens by receptors and may have some effect on 5a-reductase as well291; thus, mater¬ nal ingestion of high doses during embryogenesis should result in female external genitalia in 46,XY fetuses. Spironolac¬ tone is an aldosterone antagonist that blocks androgen action by inhibiting cytochrome P450-linked enzymes involved in steroidogenesis and increases the clearance of testoster¬ one.292-293 Finasteride inhibits 5a-reductase, and thus should be capable of producing the form of pseudohermaphroditism bearing that designation (5a-reductase deficiency.)294 Flutamide is a nonsteroidal compound that inhibits the androgen receptor295 and may reduce the synthesis of androgens or increase their metabolism at high doses.296 Other Disorders HYPOSPADIAS WITHOUT OTHER DEFECTS. In hypospadias, the external urinary meatus terminates on the ventral aspect of the penis, proximal to its usual site at the tip of the glans penis. Hypospadias can be classified according to the site of the ure¬ thral meatus; along the glans penis, on the penile shaft, at the penoscrotal junction, or on the perineum (see Fig. 93-3). Some¬ times, testicular hypoplasia coexists; however, more often tes¬ ticular volume is normal (see Chap. 93). Multiple affected sibs and affected individuals in several gen¬ erations have been reported to have uncomplicated hypospa¬ dias.7 After the birth of one affected child, the recurrence risk for subsequent male progeny is 6% to 10%.297 These risks are higher than those usually associated with multifactorial or polygenic traits, suggesting genetic heterogeneity with the existence of unappreciated autosomal recessive forms. Hypospadias some¬ times is only one of several components of multiple malforma¬ tion patterns that are known to be inherited in this fashion. The presence of other anomalies should therefore be excluded before parents are quoted recurrence risks of 6% to 10%. GENITAL AMBIGUITY WITH MULTIPLE MALFORMATION PAT¬ TERNS. Genital ambiguity may occur in individuals with mul¬ tiple malformation patterns, as tabulated elsewhere.7 Previously unrecognized syndromes continue to be reported.32-298

Ch. 90: Normal and Abnormal Sexual Differentiation and Development

879

MICROPHALLUS. As measured from the pubic ramus to the tip of the glans, the mean stretched penile length in the term infant is 3.5 cm. The 3rd and 97th percentiles are 2.8 and 4.2 cm, respectively.299 In premature infants, the mean stretched phallic length at 28 and 34 weeks is 2.2 and 2.8 cm, respectively. The mean width at term is 1.1 cm; the 3rd and 97th percentiles are 0.9 and 1.3 cm, respectively; and at 28 and 34 weeks the mean widths are 0.8 and 0.9 cm, respectively. If the phallus in a term infant is + +

++-> + + + +

+ + ++

+

+ —> + +

+ + -> + + + +

+++ +

+

+

+

+ (T with priming)

+ —» + +

++-» + + + +

++++

+

+

++-> + + + +

++++

+ -» + +

++-» + + +

+ + + —> + + + +

++++

++++

+++

++ -> +

+

FSH response to GnRH Circulating gonadotropins Mean

0 -> + +

+++

Episodic Nocturnal Gonadal response to gonadotropins Circulating gonadal steroids Circulating adrenal androgens Feedback sensitivity to sex steroids

0 —> + + +

++ +

0 -> + + + +

+++

0 —> + + + +

+ + + + —> +

0-> + +,+

—>

+

+ -> + + +

—>

++

Positive feedback to estrogen +

Males -> +

Females

+

LH, luteinizing hormone; GnRH, gonadotropin-releasing hormone; FSH, follicle-stimulating hormone.

genital differentiation. Nevertheless, gonadotropin stimulation may be required for normal ovarian development with oocyte and primordial follicle production. Whether estrogen is required for sex-specific differentiation of the female hypothal¬ amus and pituitary is unclear. Observations in humans and ani¬ mal experiments suggest that gonadal steroids do affect differentiation of the CNS-hypothalamus-pituitary in the fetus and the timing of puberty.5

NEONATAL AND CHILDHOOD PERIOD Hormonal dynamics during the neonatal period reveal consid¬ erable HPG maturation.1 At birth, gonadotropin and sex steroid levels are high, but these levels decline during the first few days. Gonadotropin levels begin to rise again before 1 week of life, suggesting a negative feedback response to the decline in circulating levels of sex steroids originating from the placenta. During the next several weeks, plasma levels of LH and FSH are higher than during the rest of childhood in both sexes (female levels, particularly of FSH, are greater than male lev¬ els). Levels of testosterone in males and, less dramatically, lev¬ els of estradiol in females are also higher for children at this age than for older children, a finding that suggests pituitary gona¬ dotropin stimulation of gonadal steroidogenesis. These data indicate that gonadotropin secretion, gonadal response with steroid production, and negative feedback mechanisms are all functional at this age. Circulating levels of gonadotropins and sex steroids peak at 2 to 3 months, after which they begin to drop to the low levels that persist for several years. Inhibiti B, the biologically active inhibin among males that appears to be produced primarily by Sertoli cells, also rises to peak levels at ~3 months. Concentrations gradually fall over the next year or more; thus, the profile is more extended than for LH, FSH, or testosterone. Inhibin B may play a crucial role in seminiferous tubule development at this age.6 7 The fall in these hormone levels cannot be explained simply by an adjustment of the negative feedback mechanism, because a similar pattern with a drop in gonadotropin levels occurs among agonadal children. This phenomenon suggests that gonadotro¬ pin secretion is controlled by CNS differentiation and that sex steroid feedback is not an obligatory element in the decreased secretion during childhood. Although gonadotropin levels are lowest during midchildhood, levels usually are not only measur¬ able and not completely suppressed but also indicate episodic release and diurnal (sleep-enhanced) variation.2 Furthermore, gender differences persist, with female FSH levels being greater than male levels. Sex steroids, including estradiol,8 and also inhibin may be detectable at very low levels in some children.

PUBERTY GONADOTROPIN-RELEASING HORMONE AND GONADOTROPINS As noted, the HPG axis is active beginning early in fetal life. Potentially, the gonad is capable of adult hormonal and germinal function throughout childhood and will respond if stimulated with pubertal or adult levels of gonadotropins. The pituitary in the prepubertal child can also secrete gonadotropin whenever stimulated with GnRH. The pituitary is capable of adult func¬ tion; thus, the restraint of pubertal maturation lies at the level of GnRH secretion or higher CNS loci. Because the pituitary and gonad are ready to respond, the controlling level is expressed by the magnitude and frequency of GnRH stimulation. The increasing mean levels of gonadotropins that precede the onset and continue during the progression of puberty result from increased secretion of gonadotropins in an episodic fash¬ ion, reflective of episodic GnRH secretion. This change in GnRH stimulation initiates the increased secretion of gonado¬ tropins of puberty. Prepubertal children already have an epi¬ sodic pattern of gonadotropin release, with infrequent pulses of low amplitude and greater secretion occurring during sleep. As pubertal maturation approaches, the episodic pattern becomes more regular and pulses attain a greater amplitude. This enhanced episodic pattern of puberty initially becomes appar¬ ent during sleep, already the time of increased activity during childhood.1-2'9-13 Just before the clinical onset of puberty and during early puberty, episodic pulses and mean levels are dis¬ tinctly higher during sleep than during wakefulness. These changes are more dramatic for LH than FSH, with the relative rise of LH being much greater than that of FSH during puberty. In the mature individual the LH episodic release occurs approximately every 90 minutes, with peak levels persisting for 20 ng/mL) on initial testing, the measurement should be repeated, because prolactin levels are increased by a number of nonspecific stimuli, including stress, sleep, and food ingestion. If thyroid function is normal and pro¬ lactin levels are elevated, further evaluation is warranted to rule but a pituitary tumor and other causes (see Chap. 13). Basal prolactin concentrations should be determined in all amenorrheic women, not just in those with galactorrhea, because prolactin levels are elevated in more than one-third of all amenorrheic women.8 Increased serum TSH levels (generally >5 pU/mL utilizing sensitive assays) with or without increased levels of prolactin indicate primary hypothyroidism (see Chaps. 15 and 45). The increased secretion of thyrotropin-releasing hormone (TRH) in this disorder stimulates increased secretion of prolactin and TSH in some affected women. High serum FSH levels (>30 mlU/mL in most laboratories) imply ovarian failure. Chromosomal evaluation is indicated in all women with increased serum FSH levels who are younger than 30 years of age when the amenorrhea begins, because a number of karyotypic abnormalities have been identified in such women. Gonadectomy is indicated in any such individual who has a portion of a Y chromosome because of the malignant potential of the gonads.9 If prolactin, TSH, and FSH levels are normal or low, further evaluation is based on the clinical presentation. Circulating thy¬ roid hormone levels should be determined if there is any sug¬ gestion of thyroid dysfunction. Serum total testosterone levels should be determined whether or not the patient is hirsute; not all hyperandrogenic women are hirsute because of relative insensitivity of the hair follicles to androgens in some women. Although slightly increased levels of serum testosterone and perhaps of dehydroepiandrosterone sulfate (DHEAS) suggest polycystic ovarian syndrome (PCO), androgen levels occa¬ sionally are not elevated in PCO, because of alterations in the metabolic clearance rates of androgens and in sex-hormonebinding-globulin (SHBG) concentrations.10 Circulating levels of luteinizing hormone (LH) may also aid in differentiating PCO from hypothalamic-pituitary dysfunction or failure. LH levels often are increased in PCO such that the ratio of LH to FSH is increased, but this too is not always so.11 However, LH and FSH levels are normal or slightly reduced in women with hypotha¬ lamic-pituitary dysfunction.12 There is some overlap between women with PCO-like disor¬ ders and those with hypothalamic-pituitary dysfunction. In an effort not to miss a serious cause of amenorrhea, some radiographic assessment of the region of the sella turcica is indicated in all amenorrheic women in whom LH and FSH levels are low (generally 50 pg/mL or if the LH level is significantly greater than the FSH level (in terms of mlU/mL) in any sample, the probability of viable oocytes is considerable. Irregular uterine bleeding, as an indication of estrogen stimulation, also provides good evidence of remaining functional ovarian follicles. It is not uncommon to

952

PART VII: ENDOCRINOLOGY OF THE FEMALE

TABLE 96-3. Causes of Chronic Anovulation

mimic the normal menstrual cycle, with oocyte donation for embryo transfer, may provide the greatest possibility for preg¬ nancy in women desiring pregnancy.43-44

CHRONIC ANOVULATION OF CENTRAL ORIGIN HYPOTHALAMIC CHRONIC ANOVULATION (FUNCTIONAL)

CHRONIC ANOVULATION

Anorexia nervosa and bulimia Amenorrhea associated with simple weight loss, diet, malnutrition Exercise-associated amenorrhea Psychogenic hypothalamic amenorrhea Pseudocyesis Due to systemic illness FORMS OF ISOLATED GONADOTROPIN DEFICIENCY (INCLUDING KALLMANN SYNDROME) HYPOPITUITARISM Idiopathic After hypothalamic-pituitary damage Neoplasms "Empty sella" syndrome After surgery After irradiation After trauma After infection After infarction HYPERPROLACTINEMIC CHRONIC ANOVULATION (GALACTOR¬ RHEA-AMENORRHEA) OF MULTIPLE CAUSES

CHRONIC ANOVULATION CAUSED BY INAPPROPRIATE FEED¬ BACK (i.e., POLYCYSTIC OVARY SYNDROME) EXCESSIVE EXTRAGLANDULAR ESTROGEN PRODUCTION (i.e., OBESITY) ABNORMAL BUFFERING INVOLVING SEX HORMONE-BINDING GLOBULIN (INCLUDING LIVER DISEASE) FUNCTIONAL ANDROGEN EXCESS OF ADRENAL OR OVARIAN ORIGIN NEOPLASMS PRODUCING ANDROGENS OR ESTROGENS NEOPLASMS PRODUCING CHORIONIC GONADOTROPIN

CHRONIC ANOVULATION CAUSED BY OTHER ENDOCRINE AND METABOLIC DISORDERS ADRENAL HYPERFUNCTION Cushing syndrome Congenital adrenal hyperplasia (female pseudohermaphroditism) THYROID DYSFUNCTION Hyperthyroidism Hypothyroidism (Modified from Rebar RW. Chronic anovulation. In Serra GB, ed. The ovary. New York: Raven Press, 1983:217.)

identify women with intermittent menstruation, hypoestrogenism, and hypergonadotropinism. Because a number of preg¬ nancies have occurred after biopsy of ovaries devoid of oocytes, ovarian biopsy cannot be recommended for affected women. Even in women with intermittent ovarian failure, estrogen replacement is appropriate to prevent the accelerated bone loss that occurs in affected women.42 The estrogen should always be given sequentially with a progestin to prevent endometrial hyperplasia (see Chap. 100). Because women with ovarian fail¬ ure may conceive while on estrogen therapy (including com¬ bined oral contraceptive agents), affected women should be counseled appropriately and cautioned to have a pregnancy test if withdrawal bleeding does not occur or if signs and symp¬ toms develop that are suggestive of pregnancy. Despite these considerations, probably no other contraceptive agent is required for those women who do not wish pregnancy but who are sexually active, because pregnancy occurs in far less than 10%.13 Although rare pregnancies in women with premature ovarian failure have occurred after ovulation induction with human menopausal and chorionic gonadotropins, the low like¬ lihood should lead the physician to discourage patients from selecting such therapy. Hormone replacement treatment to

Chronic anovulation may be viewed as a steady state in which the monthly rhythms associated with ovulation are not func¬ tional. Although amenorrhea is common, irregular menses and oligomenorrhea may occur as well. Chronic anovulation fur¬ ther implies that viable oocytes remain in the ovary and that ovulation can be induced with appropriate therapy. Chronic anovulation is the most common endocrine cause of oligomenorrhea or amenorrhea in women of reproductive age (Table 96-3). Appropriate management requires determination of the cause of the anovulation. However, anovulation can be interrupted transiently by nonspecific induction of ovulation in most affected women. CHRONIC ANOVULATION OF CENTRAL ORIGIN Hypothalamic Chronic Anovulation. Hypothalamic chronic anovulation may be defined as anovulation in which dysfunc¬ tion of hypothalamic signals to the pituitary gland causes fail¬ ure to ovulate. It remains unclear whether the primary abnormality is always present within the hypothalamus or sometimes occurs as a result of altered inputs to the hypothala¬ mus. The term is used to refer to women who may be affected with suprahypothalamic or hypothalamic chronic anovulation. Although isolated gonadotropin deficiency frequently is caused by hypothalamic dysfunction, it is preferable to consider such individuals separately. However, it may be virtually impossible to differentiate partial forms of isolated gonadotropin defi¬ ciency from hypothalamic chronic anovulation. Some reports have documented an increased incidence of amenorrhea in women who exercise strenuously, diet exces¬ sively, or are exposed to severe emotional or physical stresses of any kind45-47 (see Chap. 128). Such amenorrheic persons fall into this group of women considered as having hypothalamic chronic anovulation, which is sometimes called functional amen¬ orrhea. The diagnosis of hypothalamic chronic anovulation is suggested by the abrupt cessation of menses in women younger than 30 years of age who have no clinically evident anatomic abnormalities of the hypothalamic-pituitary-ovarian axis or any other endocrine abnormalities. The term hypotha¬ lamic amenorrhea was first proposed by Klinefelter and col¬ leagues in 1943 for anovulation in which hypothalamic dysfunction is thought to interfere with the pituitary secretion of gonadotropin.48 Although hypothalamic chronic anovulation is a common cause of oligomenorrhea and amenorrhea, relatively little is known about its pathophysiologic basis. The diversity of women with hypothalamic chronic anovulation indicates that this is a heterogeneous group of disorders with similar mani¬ festations. Compared with a matched control population, young women with secondary amenorrhea are more likely to be unmarried, to engage in intellectual occupations, to have had stressful life events, to use sedative and hypnotic drugs, to be underweight, and to have a history of previous menstrual irregularities.45 Although it has been suggested that the per¬ centage of body fat controls the maintenance of normal men¬ strual cycles, it is more likely that diet, exercise, stress, body composition, and other unrecognized nutritional and environ¬ mental factors contribute in various proportions to amenor¬ rhea (Fig. 96-3). Hormonally, basal circulating concentrations of pituitary (i.e., LH, FSH, TSH, prolactin, growth hormone), ovarian (i.e., estrogens, androgens), and adrenal hormones (i.e., dehydroepiandrosterone, DHEAS, cortisol) typically are within the normal range for women of reproductive age.49 However, mean serum gonadotropin, gonadal steroid, and DHEAS levels often are

Ch. 96: Disorders of Menstruation, Ovulation, and Sexual Response

FIGURE 96-3. Schematic representation of postulated associations among various forms of hypothalamic chronic anovulation and com¬ mon linked factors. These disorders appear to be closely interrelated. (Reprinted from Rebar RW. The reproductive age: chronic anovulation. In: Serra BG, ed. The ovary. New York: Raven, 1983:217.)

slightly decreased, and circulating and urinary cortisol levels are generally increased compared with those in normal women in the early follicular phase of the menstrual cycle.47-50 Despite low levels of circulating estrogen, affected women rarely have symptoms related to estrogen deficiency. Typically, the pulsatile secretion of gonadotropin is diminished, but these individuals respond normally to exogenous gonadotropin-releasing hor¬ mone (GnRH; Fig. 96-4). ANOREXIA NERVOSA. Anorexia nervosa may represent the severest form of functional hypothalamic chronic anovulation, or it may have one or more distinct pathophysiologic bases. The constellation of amenorrhea often preceding the weight loss, a distorted and bizarre attitude toward eating, food, or weight, extreme inanition, and a disordered body image makes the diagnosis of anorexia nervosa obvious in almost all cases51-54 (see Chap. 128). Demographically, 90% to 95% of anorectic women are white and come from middle- and upper-income families.

PSEUDOCYESIS HYPOGONADAL (n = 6)

NORMAL CYCLE

953

Peripheral gonadotropin and gonadal steroid levels gener¬ ally are lower than in the early follicular phase of the menstrual cycle.55 As patients undergo therapy, gain weight, and improve psychologically, sequential studies of the ultradian gonadotro¬ pin rhythms show progressive gonadotropin changes parallel¬ ing those normally seen during puberty. Initially, there is a nocturnal rise in gonadotropins, followed by an increase in mean basal gonadotropin levels throughout the 24-hour period.56-58 The responses of severely ill anorectics to GnRH are also similar to those observed in prepubertal children and become adult-like with recovery or with treatment with pulsa¬ tile GnRH.59-60 Because the metabolism of estradiol and test¬ osterone is also abnormal, normalizing with weight gain, some of the gonadotropin changes may be secondary to peripheral alterations in steroids.61 Several abnormalities indicate hypothalamic dysfunction, including mild diabetes insipidus and abnormal thermoregula¬ tory responses to heat and cold.54 Affected individuals have altered body images as well.62 Still other central and peripheral abnormalities exist. There is evidence of chemical hypothyroidism, with affected patients having decreased body temperature, bradycardia, low serum triiodothyronine (T3) levels, and increased reverse T3 concentrations.63-65 Circulating cortisol levels also are ele¬ vated, but the circadian cortisol rhythm is normal.66 The increased cortisol seems to be caused by the reduced meta¬ bolic clearance of cortisol as a result of the reduced affinity constant for corticosteroid binding globulin (CBG) present in such patients.67 Moreover, like women with endogenous depression, anorectics suppress significantly less after dexamethasone administration than do normal subjects.68 Anorectics also have reduced ACTH responses to exogenous corticotropin¬ releasing hormone (CRH), suggesting normal negative pituitary feedback by the increased circulating cortisol.69 Although rigorous studies have not been performed of women with bulimia, presumably such individuals have endo¬ crine disturbances similar to those of women with anorexia nervosa. SIMPLE WEIGHT LOSS AND AMENORRHEA. Societal attitudes encourage dieting and pursuit of thinness, particularly in young women. Several reproductive problems, including hypo¬ thalamic chronic anovulation, have been associated with simple weight loss. Affected women are distinctly different from anorectics in that they do not fulfill the psychiatric criteria for

“HYPOTHALAMIC" Hypogonadotropinism

FIGURE 96-4. Basal concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and their pul¬

HOURS

satile patterns during the early follicular phase of the nor¬ mal menstrual cycle are compared with exaggerated patterns in subjects without ovarian feedback (hypogonadal), in patients with pseudocyesis, and in the absence of pulsatile fluctuations observed in various forms of hypo¬ thalamic hypogonadotropism. (IGD, isolated gonadotropin deficiency.) (Reprinted from Yen SSC, Lakely BL, Wang CF. The operating characteristics of the hypothalamic-pituitary system during the menstrual cycle and observations of bio¬ logical action of somatostatin. Recent Prog Horm Res 1975; 31:321.)

954

PART VII: ENDOCRINOLOGY OF THE FEMALE

FIGURE 96-5. Some factors apparently involved in the pathophysiol¬ ogy of exercise-associated amenorrhea. (Reprinted from Rebar RW. Effect of exercise on reproductive function in females. In: Givens JR, ed. The hypothalamus in health and disease. Chicago: Year Book Medical Publishers, 1984:245.)

anorexia.52 The cessation of menses does not occur before sig¬ nificant weight loss in such women, although this sequence is common in anorectics. The few studies that have been con¬ ducted in amenorrheic women with simple weight loss suggest that the abnormalities are similar to those observed in anorec¬ tics, but are more minor and more easily reversible with weight gain.70 Although it has been suggested that the amenorrhea in these women is secondary to metabolic defects resulting from undernutrition, the possibility of separate central defects has not been excluded.70 The importance of normal body weight to normal reproductive function is evident in studies of a tribe of desert-dwelling hunter-gatherers in Botswana.71 The weights of the women vary markedly with the season, being greatest in the summer, and the peak incidence of parturition follows exactly 9 months after the attainment of maximal weight. EXERCISE-ASSOCIATED AMENORRHEA. Regular endurance training in women is associated with at least three distinct disor¬ ders of reproductive function: delayed menarche, luteal dysfunc¬ tion, and amenorrhea.72'73 The American College of Sports Medicine has coined the term the "female athletic triad" to describe the three disorders recognized as sometimes occurring together in female athletes: disordered eating, amenorrhea, and osteoporosis.74-743 Activities associated with an increased fre¬ quency of reproductive dysfunction include those favoring a slimmer, lower-body-weight physique such as middle and long distance running, ballet dancing, and gymnastics. Swimmers and bicyclists appear to have lower rates of amenorrhea despite comparable training intensities. The cause of these disorders remains to be established and may involve many factors. Dietary changes, the hormonal effects of acute and chronic exercise, alter¬ ations in hormone metabolism because of the increased lean to fat ratio, and the psychological and physical "stress" of exercise itself may all contribute and may vary in importance in different individuals (Fig. 96-5; see Chaps. 128 and 132). Menstrual dysfunction was induced in untrained women who underwent a program of strenuous aerobic exercise (run¬ ning 4-10 miles per day) combined with caloric restriction.75 The spectrum of abnormalities in these women included luteal phase dysfunction, loss of the midcycle LH surge, prolonged menstrual cycles, altered patterns of gonadotropin secretion, and amenorrhea. Subsequent studies have indicated that luteal phase defects can occur soon after endurance training is begun in the majority of untrained women.76 However, in contrast to these findings, others observed that a progressive exercise program of moderate intensity did not affect the reproductive system of gynecologically mature (mean age, 31.4 years), untrained, eumenorrheic women.77 It was suggested that rela¬ tively young gynecologic age or an earlier age of training onset in particular adversely affects menstrual cyclicity.

Many amenorrheic athletes welcome the onset of amenor¬ rhea. However, significant osteopenia, usually affecting trabec¬ ular bone, has been reported in these women.78-80 It appears that the loss in bone density secondary to hypoestrogenism nul¬ lifies the beneficial effects of weight-bearing exercise in strengthening and remodeling bone.79'81 Such women are at risk for stress fractures, particularly in the weight-bearing lower extremities, and bone density may remain below those of eumenorrheic athletes even after resumption of menses.82 Stress is generally acknowledged to play a role in the cause of this form of amenorrhea, even though it remains difficult to define the term stress. Amenorrheic runners subjectively associ¬ ate greater stress with running than do runners with regular menses.83 However, no increase in amenorrhea was observed in a competitive group of young classical musicians, who presum¬ ably were experiencing similar stress, compared with a group of young ballet dancers, in whom the incidence of amenorrhea was quite high.84 Basal levels of circulating cortisol and urinary free cortisol excretion, which are indicative of increased stress, are increased in eumenorrheic and amenorrheic runners.85 Because levels of CBG, the disappearance rate of cortisol from the circulation, and the response of cortisol to adrenocorticotropin (ACTH) were not altered in the women runners com¬ pared with sedentary control subjects, secretion of ACTH and possibly of CRH must be increased in women who run. Abnormalities of the hypothalamic-pituitary-adrenal axis also are indicated by the observations that serum ACTH and cortisol responses to exogenous CRH are blunted, as are the responses to meals.86 The observation that amenorrheic runners also have subtle abnormalities in hypothalamic-pituitary-adrenal function pro¬ vides support for the concept that exercise-associated amenor¬ rhea is similar to other forms of hypothalamic amenorrhea.87 PSYCHOGENIC HYPOTHALAMIC AMENORRHEA. Amenorrhea may occur in women with a definite history of psychological and socioenvironmental trauma.45'78-80 The incidence of amenorrhea is quite high among depressed women, and it is difficult to differ¬ entiate the effects of lifestyle and nutritional status from variables such as stress. Studies of individuals in whom a definite psycho¬ logical traumatic event preceded the onset of amenorrhea have revealed low to normal basal levels of serum gonadotropins with normal responses to GnRH, prolonged suppression of gonado¬ tropins in response to estradiol, and failure of a positive feedback response to estradiol.78-97 Increased basal levels of cortisol and decreased levels of DHEAS also have been noticed in women with psychogenic amenorrhea compared with eumenorrheic women.46 The mean levels of circulating cortisol are increased in such women largely because of an increase in the amplitude of the pulses of cortisol.98 Moreover, studies of depressed women have revealed abnormal circadian rhythms of cortisol and early "escape" from dexamethasone suppression 99-101 The mechanism by which emotional states or stressful expe¬ riences cause psychogenic amenorrhea is not yet established. Evidence suggests, however, that a cascade of neuroendocrine events that may begin with limbic system responses to psychic stimuli impairs hypothalamic-pituitary activity.102 It has been suggested that increased amounts of hypothalamic (5-endorphin is important in inhibiting gonadotropin secretion.102 Psychological studies have found several social and psycho¬ logical correlates of psychogenic amenorrhea: a history of pre¬ vious pregnancy losses, including spontaneous abortion103-105; stressful life events within the 6-month period preceding the amenorrhea106-108; and poor social support or separation from significant family members during childhood and adoles¬ cence.101-108'109 Many women with psychogenic amenorrhea report stressful events associated with psychosexual problems and socioenvironmental stresses during the teenage years.89 Women with psychogenic amenorrhea also tend to have nega¬ tive attitudes toward sexually related body parts, more partnerrelated sexual problems, and greater fear of or aversion to

Ch. 96: Disorders of Menstruation, Ovulation, and Sexual Response menstruation than do eumenorrheic women.107 Distortions of body image and confusion about basic bodily functions, espe¬ cially sexuality and reproduction, are common.104 DIMINISHED GONADOTROPIN-RELEASING HORMONE AND LUTEINIZING HORMONE SECRETION IN ALL FORMS. The vari¬ ous forms of hypothalamic chronic anovulation associated with altered lifestyles have several features in common. Altered GnRH and LH secretion seems to be the common result from altered hypothalamic input. It remains unclear if these disorders form a single disorder or several closely related disorders. More¬ over, similar forms of amenorrhea are sometimes seen in women with severe systemic illnesses or with hypothalamic damage from tumors, infection, irradiation, trauma, or other causes. TREATMENT. The treatment of patients with hypothalamic chronic anovulation is controversial. Psychological therapy and support or a change in lifestyle may cause cyclic ovulation and menses to resume. However, ovulation does not always resume, even after the lifestyle is altered. The treatment of affected women in whom menses do not resume and who do not desire pregnancy is difficult. Most physicians now advocate the use of exogenous sex steroids to prevent osteoporosis. Ther¬ apy consisting of oral conjugated estrogens (0.625-1.250 mg), ethinyl estradiol (20 gg), micronized estradiol-17(l (1-2 mg), or estrone sulfate (0.625-2.500 mg) or of transdermal estradiol-17f) (0.05-0.10 mg) continuously with oral medroxyprogesterone acetate (5-10 mg) or oral micronized progesterone (200 mg) added for 12 to 14 days each month is appropriate. Sexually active women can be treated with oral contraceptive agents. These women appear to be particularly sensitive to the unde¬ sired side effects of sex steroid therapy, and close contact with the physician may be required until the appropriate dosage is established. If sex steroid therapy is provided, patients must be informed that the amenorrhea may still be present after therapy is discontinued. Some physicians believe that only periodic observation of affected women is indicated, with barrier methods of contracep¬ tion recommended for fertility control. Contraception is neces¬ sary for sexually active women with hypothalamic chronic anovulation because spontaneous ovulation may resume at any time (before menstrual bleeding) in these mildly affected indi¬ viduals. Women who refuse sex steroid therapy should be encouraged to have spinal bone density evaluated at intervals to document that bone loss is not accelerated. Adequate calcium ingestion should be encouraged in all affected women. For women who desire pregnancy but who do not ovulate spontaneously, clomiphene citrate (50-100 mg per day for 5 days beginning on the third to fifth day of withdrawal bleed¬ ing) can be used. Treatment with human menopausal and chorionic gonadotropins (hMG-hCG) or with pulsatile GnRH may be effective in women who do not ovulate in response to clomiphene. Because the underlying defect in hypothalamic amenorrhea is decreased endogenous GnRH secretion, admin¬ istration of pulsatile GnRH to induce ovulation can be viewed as physiologic; it offers the additional advantages of decreased need for ultrasonographic and serum estradiol monitoring, a decreased risk of multiple pregnancies, and a virtual absence of ovarian hyperstimulation. A starting intravenous dose of GnRH of 5 gg every 90 minutes is effective.110 After ovulation is detected by urinary LH testing or ultrasound, the corpus luteum can be supported by continuation of pulsatile GnRH or by hCG (1500 IU every 3 days for four doses). Ovulation rates of 90% and conception rates of 30% per ovulatory cycle can be expected.111 Isolated Gonadotropin Deficiency. As originally described in 1944, Kallmann syndrome consisted of the triad of anosmia, hypogonadism, and color blindness in men.112 Women may be affected as well, and other midline defects may be associ¬ ated.113-116 Because autopsy studies have shown partial or com¬ plete agenesis of the olfactory bulb, the term olfactogenital dysplasia has also been used to describe the syndrome.117

955

Because isolated gonadotropin deficiency may also occur in the absence of anosmia, the syndrome is considered to be quite het¬ erogeneous. Data indicate that the defect is a failure of GnRH neurons to form completely in the medial olfactory placode of the develop¬ ing nose or the failure of GnRH neurons to migrate from the olfactory bulb to the medial basal hypothalamus during embryogenesis.118 In some patients, structural defects of the olfactory bulbs can be seen on magnetic resonance imaging.116 It appears likely that this disorder forms a structural continuum with other midline defects, with septo-optic dysplasia repre¬ senting the severest disorder. Clinically, affected individuals typically present with sexual infantilism and an eunuchoidal habitus, but moderate breast development may also occur. Primary amenorrhea is the rule. The ovaries usually are small and appear immature, with folli¬ cles rarely developed beyond the primordial stage.120 These immature follicles respond readily to exogenous gonadotropin with ovulation and pregnancy, and exogenous pulsatile GnRH can also be used to induce ovulation.121122 Replacement therapy with estrogen and progestin should be given to affected women who do not desire pregnancy. Circulating LH and FSH levels generally are quite low. The response to exogenous GnRH is variable, sometimes being diminished and sometimes normal in magnitude, but rarely may be absent.123'124 Although the primary defect in most indi¬ viduals appears to be hypothalamic, with reduced GnRH syn¬ thesis or secretion, a primary pituitary defect may occasionally be present. In addition, partial gonadotropin deficiency may be more frequent than has been appreciated (see Chap. 115). Hyperprolactinemic Chronic Anovulation. Approximately 15% of amenorrheic women have increased circulating concen¬ trations of prolactin, but prolactin levels are increased in more than 75% of patients with galactorrhea and amenorrhea.8 Radiologic evidence of a pituitary tumor is present in -50% of hyperprolactinemic women, and primary hypothyroidism must always be considered. Individuals with galactorrheaamenorrhea (i.e., hyperprolactinemic chronic anovulation) frequently complain of symptoms of estrogen deficiency, including hot flushes and dyspareunia. However, estrogen secretion may be essentially normal.125 It is not clear if it is the hyperprolactinemia or the "hypoestrogenism" that causes the accelerated bone loss seen in such individuals.126 Signs of androgen excess are observed in some women with hyperpro¬ lactinemia; androgen excess may rarely result in PCO. In hyper¬ prolactinemic women, serum gonadotropin and estradiol levels are low or normal. Most hyperprolactinemic women have disordered reproduc¬ tive function, and it appears that the effects on gonadotropin secretion are primarily hypothalamic. The mechanism by which hypothalamic GnRH secretion is disrupted is unknown but may involve an inhibitory effect of tuberoinfundibular dopaminergic neurons.125,127 It has been proposed that increased hypothalamic dopamine is present in hyperprolactinemic women with pituitary tumors but is ineffective in reducing pro¬ lactin secretion by adenomatous lactotropes. The dopamine can, however, reduce pulsatile LH secretion and produce acy¬ clic gonadotropin secretion through a direct effect on hypotha¬ lamic GnRH secretion (see Chap. 13). It has been suggested that mild nocturnal hyperprolactine¬ mia may be present in some women with regular menses and unexplained infertility.128 Galactorrhea in women with unex¬ plained infertility may reflect increased bioavailable prolactin and may be treated appropriately with bromocriptine.126 Bro¬ mocriptine or cabergoline therapy may also be indicated in normoprolactinemic women with amenorrhea and increased prolactin responses to provocative stimuli.130 Hypopituitarism. Hypopituitarism may be obvious on cursory inspection or it may be quite subtle (see Chaps. 12 to 18). The clinical presentation depends on the age at onset, the

956

PART VII: ENDOCRINOLOGY OF THE FEMALE tered hypothalamic hormones have failed to localize the cause to the hypothalamus or the pituitary gland in affected patients. Radiographic evaluation of the sella turcica is indicated in any individual with suspected hypopituitarism. The ovaries appear immature and unstimulated, but because oocytes still are present, ovulation can be induced with exogenous gonado¬ tropins when pregnancy is desired. Exogenous pulsatile GnRFl may also be used to induce ovulation if the disorder is hypotha¬ lamic. Moreover, oocytes may undergo some development, and even ovarian cysts may appear in the absence of significant gonadotropic stimulation (see Chap. 94). When pregnancy is not desired, maintenance therapy with cyclic estrogen and progestin is indicated to prevent signs and symptoms of estro¬ gen deficiency (see Chap. 100). CHRONIC ANOVULATION RESULTING FROM INAPPROPRIATE FEEDBACK IN POLYCYSTIC OVARY SYNDROME

FIGURE 96-6. Hypopituitarism in a 28-year-old woman with a cra¬ niopharyngioma diagnosed at age 16 years. She had received total replacement therapy since the time of diagnosis. Breast development has not advanced beyond Tanner stage 3, little pubic hair is present, and the body habitus is not that of a mature adult. The deep pigmentation of the areolae occurred during therapy several years earlier with fluoxymesterone in an attempt to induce pubic and axillary hair growth.

cause, and the woman's nutritional status (Fig. 96-6). Loss of axillary and pubic hair and atrophy of the external genitalia should lead the physician to suspect hypopituitarism in a pre¬ viously menstruating young woman who develops amenor¬ rhea. In such cases, a history of past obstetric hemorrhage suggesting postpartum pituitary necrosis (i.e., Sheehan syn¬ drome) should be sought.131 Failure to develop secondary sex¬ ual characteristics or to progress in development once puberty begins must always prompt a workup for hypopituitarism (see Chap. 18). Individuals with pituitary insufficiency often complain of weakness, easy fatigability, lack of libido, and cold intolerance. Short stature may occur in individuals developing hypopituitar¬ ism during childhood. Symptoms of diabetes insipidus may be observed if the posterior pituitary gland is involved. On physi¬ cal examination, the skin is generally thin, smooth, cool, and pale (i.e., "alabaster skin") with fine wrinkling about the eyes; the pulse is slow and thready; and the blood pressure is low. An evaluation of thyroid and adrenal function is of para¬ mount importance in these individuals. Thyroid replacement therapy must be instituted and the patient must be euthyroid before adrenal testing is initiated (see Chaps. 14,15,18, and 74). Serum gonadotropin and gonadal steroid levels typically are low in hypopituitarism. Responses to exogenously adminis¬

Heterogeneous Disorder. In 1935, Stein and Leventhal focused attention on a common disorder in which amenorrhea, hirsutism, and obesity were frequently associated.132 With the development of radioimmunoassays for measuring reproduc¬ tive hormones, it became clear that women with what is called PCO shared several distinctive biochemical features. Compared with eumenorrheic women in the early follicular phase of the menstrual cycle, affected women typically have elevated serum LH levels and low to normal FSF1 levels11 (Fig. 96-7). Virtually all serum androgens are moderately increased, and estrone lev¬ els are generally greater than estradiol levels133 (Fig. 96-8). Ovarian inhibin physiology is normal.134 Many women with the biochemical features of PCO have small or even morphologically normal ovaries and are not over¬ weight or hirsute. Not all women with PCO have the characteris¬ tic features. Moreover, excess androgen from any source or increased conversion of androgens to estrogens can lead to the constellation of findings observed in PCO.10 Included are such disorders as Cushing syndrome, congenital adrenal hyperplasia, virilizing tumors of ovarian or adrenal origin, hyperthyroidism and hypothyroidism, obesity, and type 2 diabetes.1343 In all of these disorders, the ovaries may be morphologically polycystic. Although no clinical and biochemical criteria describe the syndrome strictly, a conference convened by the National Insti¬ tutes of Health135 developed diagnostic criteria for PCO: 1. Clinical evidence of hyperandrogenism (e.g., hirsutism, acne, androgenetic alopecia) and/or hyperandrogemia (e.g., elevated total or free testosterone). 2. Oligoovulation (i.e., cycle duration >35 days or 50 mlU/mL, then the diagnosis of menopause is likely. The perimenopause begins with the onset of symptoms of hypoestrogenism and ends with the final menses. During this time, it is not uncommon for menstrual cycles to become more variable, lasting anywhere from 20 to 60 days. After age 40 years, it is more common for the follicular phase to be shortened. Anovulatory cycles may be interspersed with ovulatory cycles, and anovulatory bleeding may occur, indicated by variation and unpredictability in the amount of flow and in the duration and timing of the bleeding. Luteal phase defects also become more common as menopause is approached. Periods of amenorrhea with elevated plasma FSH levels suggesting menopause can occur, only to be followed a few months later by an ovulatory cycle. The decline in estrogen production by the perimenopausal ovarian follicle lessens the negative feedback on the hypothala¬ mus and pituitary, causing FSH levels to rise. Moreover, the folli¬ cles themselves are less sensitive to FSH stimulation.

PREMATURE MENOPAUSE Premature ovarian failure is the onset of menopause before age 35 years, and is diagnosed by finding elevated plasma FSH levels. The reported incidence of this condition among women with amenorrhea varies from 4% to 10%. Women who pass through menopause at a younger age have the typical symptoms of estrogen deficiency, including vasomotor flushes and genital atrophy. However, it may not be permanent, and some women may ovulate again.6'63 the causes of premature ovarian failure are many (see Chaps. 96 and 102). Genetic abnormalities, usually associated with dele¬ tions of the long arm of the X chromosome or mosaicism, may lead to depletion of oogonia.7-8 Autoimmune disorders, either alone or in association with other autoimmune endocrine disorders such as Addison disease, may lead to ovarian failure. Radiation or

984

PART VII: ENDOCRINOLOGY OF THE FEMALE

TABLE 100-1. Representative Reproductive Hormone Levels in Women* Premenopausal Hormone

Postmenopausal

Plasma Level

Production Rate

Plasma Level

Production Rate

ANDROSTENEDIONE

150 ng/dL

2.7 mg/d

90 ng/dL

16 mg/d

0

TESTOSTERONE

35 ng/dL

200 pg/d

25 ng/dL

150 pg/d

>90

DEHYDROEPIANDROSTERONE

4-5 ng/mL

1.8 ng/mL

DEHYDROEPIANDROSTERONE SULFATE

1500 ng/mL

300 ng/mL

ESTRONE

40-200 pg/mL

80-4000 pg/d

35 pg/mL

55 pg/d

0

ESTRADIOL

40-350 pg/mL

50-500 pg/d

13 pg/mL

12 pg/d

50

LUTEINIZING HORMONE

10^0 mlU/mL

70 mlU/mL

FOLLICLE-STIMULATING HORMONE

10-40 mlU/mL

80 mlU/mL

PROLACTIN

10 ng/mL

8 ng/mL

% Tightly Bound

0 0

‘Also see Chapter 237. (From Korenman SG. Menopausal endocrinology and management. Arch Intern Med 1982; 142:1131. Copyright 1982, American Medical Association, reproduced by permission of the American Medical Association.)

chemotherapy can cause ovarian failure. There is evidence that chemotherapy administered before puberty and in the absence of radiation therapy does not affect the ovaries.9 In a condition known as Savage syndrome, the ovarian follicles are resistant to the gonadotropins. The plasma gonadotropin levels are elevated. This disorder could be caused by a gonadotropin receptor or postreceptor defect within the ovary. Galactosemia is associated with primary ovarian failure; in this case, it is thought that an abnormal metabolite interferes with postreceptor activity. Another form of premature ovarian failure is surgical menopause. In all of these cases, hormone-replacement therapy should be considered. Women with premature ovarian failure are vul¬ nerable at an early age to develop genital atrophy, osteoporosis, vasomotor symptoms, and probably heart disease.

URETHRA The urethra has the same biologic origin as the vagina (the uro¬ genital sinus) and also undergoes postmenopausal atrophy. Some women experience problems with dysuria and frequency in the absence of infection; estrogen treatment is effective in providing relief. UTERUS With estrogen depletion, the postmenopausal uterus becomes smaller and firmer. Vascularity is reduced, and uterine leiomyo¬ mas decrease in size. Inner migration of the squamocolumnar junction of the cervix occurs; thus, it may become more difficult to diagnose cervical cancer. The fallopian tubes show deciliation and decreased secretion.

GENITAL ATROPHY CARDIOVASCULAR DISEASE VULVA The postmenopausal vulva has little subcutaneous fat and elastic tissue, resulting in a narrower opening, and sparser and coarser pubic hair than the premenopausal vulva. The labia majora shrink more than the labia minora, so that the relative propor¬ tions of the prepubertal organ are restored. The Bartholin glands secrete less fluids for lubrication, and vaginal dryness is often a problem. Moreover, the vulva may become more pruritic. VAGINA The postmenopausal vaginal mucosa loses its papillae, the rugae flatten, and the vaginal walls become smoother and thin¬ ner. These changes often cause dyspareunia, burning, and occasional bleeding through breaks in the vaginal wall. For women who have ceased having intercourse, the vagina may become stenotic.10 With decreasing estrogen production, the vaginal glycogen content diminishes and the pH is increased, leading to inhibi¬ tion of lactobacilli, which in turn permits other organisms to grow, including streptococci, staphylococci, diphtheroids, and coliforms. These bacteria are often responsible for vaginal dis¬ charge and vaginal infections after menopause. In such cases, antibiotics and other preparations provide temporary relief but are not curative. Estrogen treatment is more effective in provid¬ ing long-term resolution of vaginal dryness and dyspareunia. Some women passing through the menopause experience decreased intensity and duration of sexual response. However, many, if not most, postmenopausal women continue to be sexu¬ ally active. If dryness is a problem, estrogens may be very help¬ ful.11 Nonhormonal vaginal lubricants (such as Replens, Columbia Pharmaceuticals) may also be useful.

Cardiovascular disease (CVD) is the leading cause of death among women in industrialized countries: >50% of postmeno¬ pausal women will die of CVD. Estrogens have been hypothe¬ sized to protect against atherosclerosis, because the incidence of CVD is quite low before the menopause. Premenopausal women have approximately one-fifth the CVD mortality of men, but after the menopause their mortality exponentially rises to approach that of men.12 One explanation is that the estrogen of a premenopausal woman confers protection, which is lost at menopause; this is supported by the observation that women who undergo a premature surgical menopause (i.e., bilateral oophorectomy) and who do not use postmenopausal estrogens have twice as much CVD as age-matched premenopausal con¬ trols. If they use postmenopausal estrogens, however, their inci¬ dence of CVD is the same as that of premenopausal women of the same age.13 Premature natural menopause, in contrast, has not been found to increase the risk of CVD when subjects are controlled for age, smoking, and estrogen use.13 Most epidemiologic studies have found that postmeno¬ pausal estrogen users have a lower incidence of CVD compared with nonusers. The Nurses' Health Study,14 the largest cohort study, which followed 121,000 women for as long as 18 years, identified 425 cases of fatal myocardial infarction (MI). The adjusted relative risk of death from coronary heart disease was significantly reduced to 0.47 (95% Cl, 0.32-0.69) for current hor¬ mone use but unchanged at 0.99 (95% Cl, 0.75-1.30) for past use. The greatest decrease was seen in women who had at least one risk factor for heart disease (current tobacco use, hypercho¬ lesterolemia, hypertension, diabetes, parenteral history of pre¬ mature MI, obesity). Substantially less benefit was seen in women with no risk factors. Concomitant progestin use did not appear to detract from this benefit.

Ch. 100: Menopause Additional evidence for the benefit of estrogens was provided by a prospective study of more than 8000 postmenopausal women living in a moderately affluent retirement community in southern California.15 After 7 years of observation, >1400 of these women had died. The investigators found that the women who had ever taken postmenopausal estrogens had 20% less all-cause mortality compared with women who did not (relative risk [RR] of death, 0.80; 95% Cl, 0.70-0.87). The greatest reductions in mortality were seen with current use and with long durations of use: current use for more than 15 years was associated with a 40% reduction in mortality rates. This reduction in mortality rates was not depen¬ dent on the dosage of estrogen used: both high (i.e., >1.25 mg daily) and low (i.e., 10% of women in the United States, is a much more serious disease than endometrial cancer. Besides being common, breast cancer has a high mortality rate. This dis¬ ease is frequently disfiguring and emotionally very disturbing. Whether estrogens actually cause breast cancer is presently unknown. Although some studies have found no increased risk of breast cancer.69 Other large studies found excess risk among long-term users.70 This observation was confirmed by the Nurses' Health Study, which analyzed 1935 cases of breast cancer prospectively seen during 725,000 person-years of observation.71 They found that the risk of death due to breast cancer in women who had taken estrogen for five or more years was increased 45%. Other studies are in agreement.713 This conclusion is consis¬ tent with available animal data suggesting that breast cancer can be induced with high-dose estrogens (see Chap. 222). Also, estro¬ gens can maintain breast tumor growth in tissue culture. They also found that the addition of a progestin to estrogen treatment did not influence the increased risk of breast cancer seen with long-term estrogen use.71 Interestingly, hormone replacement therapy reduces the sensitivity of mammography.716

THERAPEUTIC ASPECTS Theoretically, the ideal estrogen to administer should be the one the woman's own ovaries produced in the premenopausal years, namely, estradiol. Estradiol taken orally is converted to estrone in the gut and liver. However, estradiol given vaginally, by injection, or transdermally is absorbed rapidly; because it bypasses the liver, it appears in the plasma predominantly as estradiol. Estra¬ diol remains biologically potent because it can suppress gonado¬ tropins when given by any of the above routes.72 The transdermal approach has the advantage of delivering constant physiologic levels of estradiol73 Because the liver is bypassed, it may be con¬ sidered for women at risk for phlebitis or hypertension. The most common form of estrogen-replacement therapy uses conjugated equine estrogens prescribed orally. The dose that is effective for osteoporosis and flushes is 0.3 to 0.625 mg daily. Estropipate (piperazine estrone sulfate) (1.25 mg per day) or micronized estradiol (Estrace) (0.5 mg) may also be used. Oral or injectable estrogens with prolonged half-lives generally should not be used. Transdermal estradiol is applied to the skin twice weekly. It is designed to deliver 0.05 to 0.10 mg per day of estradiol, which achieves a blood level in the range of the nor¬ mal early follicular phase of the menstrual cycle. The drug avoids the first-pass hepatic metabolism of oral preparations; there is no stimulation of renin substrate, and no increase in sex hormone-binding globulin, corticosteroid-binding globulin, or thyroxine-binding globulin 74 Oral estrogens do increase the lev¬ els of these globulins by their effect on the liver, but any long¬ term adverse reactions of these increases are unknown. On the other hand, with oral estrogen administration the liver produces more HDL-cholesterol24 and clears more LDL-cholesterol23 from the circulation, which is presumably a benefit. The standard regimen adds a progestin75 such as medroxy¬ progesterone acetate, 5 mg daily, from the 1st to the 13th days of the month to reduce the risk of endometrial cancer. A woman with a uterus will have a 90% chance of experiencing with¬ drawal bleeding. It has been shown that with continuous use of estrogens and progestins, this annoying side effect can be mini¬ mized.76 However, irregular and unpredictable bleeding can occur in the first several months of continuous combined ther¬ apy and results in high dropout rates. The long-term safety of this regimen needs to be established. There are reports of endometrial cancer developing years later in women treated in this fashion.77 Endometrial biopsies need not be performed before estrogen therapy is begun unless irregular bleeding has occurred. Biop¬ sies need only be performed during hormone treatment if with-

Ch. 100: Menopause drawal bleeding occurs before day 10 or after day 20 of monthly cyclic progestin therapy78 or after 6 months of continu¬ ous progestin therapy. Vaginal probe ultrasonography may reduce the number of biopsies required; endometrial cancer is highly unlikely if endometrial thickness is 84 The effect of raloxifene on cognitive functioning and on the incidence of Alzheimer's disease is unknown.

CONCLUSION Each patient has to be informed about the possible benefits and risks of the currently available therapies, including alternatives. Only in this way can she share in making the decision about the treatment she is prescribed.

REFERENCES 1. Statistical abstract of the United States: 1981, 102nd ed. Washington, DC: US Bureau of the Census, 1981. 2. U.S. Department of Commerce, Special Studies. 65+ in the United States. P23-190; 1996:2. 3. Grodin JN, Siiteri PK, MacDonald PC. Source of estrogen production in postmenopausal women. J Clin Endocrinol Metab 1973; 36:207. 4. Longcope C, Jaffee W, Griffing G. Production rates of androgens and estro¬ gens in postmenopausal women. Maturitas 1981; 3:215. 5. MacDonald PC, Edman CD, Hemsell DL, et al. Effect of obesity on conver¬ sion of plasma androstenedione to estrone in postmenopausal women with and without endometrial cancer. Am J Obstet Gynecol 1978; 130:448. 6. Rebar RW, Erickson GF, Yen SSC. Idiopathic premature ovarian failure: clinical and endocrine characteristics. Fertil Steril 1982; 37:35. 6a. Kalantaridou SN, Nelson LM. Premature ovarian failure is not premature menopause. Ann NY Acad Sci 2000; 900:393. 7. Coulam CB. Premature gonadal failure. Fertil Steril 1982; 38:645. 8. Krauss CM, Turksoy RN, Atkins L, et al. Familial premature ovarian failure due to an interstitial deletion of the long arm of the X chromosome. N Engl J Med 1987; 317:125. 9. Stillman RJ, Schinfeld JS, Schiff I, et al. Ovarian failure in long-term survi¬ vors of childhood malignancy. Am ] Obstet Gynecol 1981; 139:62. 10. Schiff I, Wilson E. Clinical aspects of aging of the female reproductive sys¬ tem. In: Schneider E, ed. Aging of the reproductive system. New York: Raven Press, 1978:9. 11. Morrell MJ, Dixen JM, Carter CS, Davidson JM. The influence of age and cycling status on sexual arousability in women. Am J Obstet Gynecol 1984; 148:66. 12. Lemer DJ, Kannel WB. Patterns of coronary disease morbidity and mortality: a 26-year follow-up of the Framingham population. Am Heart J 1986; 111(2):383. 13. Colditz GA, Willett WC, Stampfer MJ, et al. Menopause and the risk of cor¬ onary heart disease in women. N Engl J Med 1987; 316:1105. 14. Grodstein F, Stampfer MJ, Colditz GA, et al. Postmenopausal hormone therapy and mortality, N Engl J Med 1997; 336:1769. 15. Henderson BE, Paganini-Hill A, Ross RK. Decreased mortality in users of estrogen replacement therapy. Arch Intern Med 1991; 151:75. 16. Tire Coronary Drug Project Research Group. Findings leading to discontin¬ uation of the 2.5 mg/day estrogen group. JAMA 1973; 226:652. 17. Sullivan JM, Vander Zwagg R, Hughes FP, et al. Estrogen replacement and coronary artery disease. Arch Intern Med 1990; 150:2557. 18. Sullivan JM, Vander Zwagg R, Lemp GF, et al. Postmenopausal estrogen use and coronary atherosclerosis. Ann Intern Med 1988; 108:358. 19. Stamler J, Katz LN, Pick R, et al. Effects of long-term estrogen therapy on serum cholesterol-lipid-lipoprotein levels and mortality in middle aged men with previous myocardial infarction. Circulation 1980; 22:658. 20. DeVogt HJ, Smith PH, Davone-Macaluso M, et al. Cardiovascular side effects of diethylstilbestrol, cyproterone acetate, and medroxyprogesterone acetate used for treatment of prostatic cancer. J Urol 1986; 135:303. 21. Gordon T, Castelli WP, Hjortland MC, et al. High density lipoprotein as a protective factor against coronary heart disease. Am J Med 1977; 62:707. 22. Matthews KA, Meilahn E, Kuller LH, et al. Menopause and risk factors for coronary heart disease. N Engl J Med 1989; 321:641. 23. Walsh BW, Schiff I, Rosner B, et al. Effects of postmenopausal estrogen replacement on the concentrations and metabolism of plasma lipoproteins. N Engl J Med 1991; 325:1196. 24. Walsh BW, Li H, Sacks FM. Effects of postmenopausal hormone replace¬ ment with oral and transdermal estrogen on high-density lipoprotein metabolism. J Lipid Res 1994; 35:2083. 25. Sacks FM, McPherson R, Walsh BW. Effect of postmenopausal estrogen replacement on plasma lipoprotein (a) concentrations. Arch Intern Med 1994; 154:1106. 26. Sack MN, Rader DJ, Cannon RO. Oestrogen and inhibition of oxidation of low-density lipoproteins in postmenopausal women. Lancet 1994; 343:269. 27. Wagner JD, Clarkson TB, St. Clair RW, et al. Estrogen and progesterone replacement therapy reduces low density lipoprotein accumulation in the coronary arteries of surgically postmenopausal cynomolgus monkeys. J Clin Invest 1991; 88:1995. 28. Williams JK, Adams MR, Herrington DM, Clarkson TB. Short-term admin¬ istration of estrogen and vascular responses of atherosclerotic coronary arteries. J Am Coll Cardiol 1992; 20:452. 29. Steinleitner A, Stanzyk FZ, Levin JH, et al. Decreased in vitro production of 6-keto prostaglandin by uterine arteries from postmenopausal women. Am J Obstet Gynecol 1989; 161:1677. 30. McGill HC. Sex steroid hormone receptors in the cardiovascular system. Postgrad Med 1989; (April):64. 31. Hyashi T, Fukuto JM, Ignarro LJ, Chaudhuri G. Basal release of nitric oxide from aortic rings is greater in female rabbits than in male rabbits: implica¬ tions for atherosclerosis. Proc Natl Acad Sci U S A 1992; 89:11259. 32. Hulley S, Grady D, Bush T, et al. Randomized trial of estrogen plus proges¬ tin for secondary prevention of coronary heart disease in postmenopausal women. JAMA 1998; 280:605. 33. Heaney RP. Estrogens in postmenopausal osteoporosis. Clin Obstet Gynecol 1976; 19:791. 34. World Health Organization. Research on menopause in the 1990's. Geneva, Switzerland, WHO, Series 1996; 866:40. 35. Gallagher JC, Nordin BEC. Calcium metabolism in the menopause. In:

Ch. 101: Hirsutism, Alopecia, and Acne Curry AS, Hewitt JV, eds. Biochemistry of women: current concepts. Cleve¬ land: CRC Press, 1974:145. 36. Lindsay R, Herrington BS. Estrogens in osteoporosis. Semin Reprod Endo¬ crinol 1983; 1:55. 37. Weiss NS, Ure CL, Ballard JH, et al. Decreased risk of fractures of the hip and lower forearm with postmenopausal use of estrogen. N Engl J Med 1980; 303:1195. 38. Paganini-Hill A, Ross RK, Gerkins VR, et al. Menopausal estrogen therapy and hip fractures. Ann Intern Med 1981; 95:28. 39. Felson DT, Zhang Y, Hannan MT, et al. The effect of postmenopausal estro¬ gen therapy on bone density in elderly women. N Engl J Med 1993; 329:1141. 40. Gallagher JC, Kable WT, Goldgar D. Effect of progestin therapy on cortical and trabecular bone: comparison with estrogen. Am J Med 1991; 90:171. 41. Eriksen EF, Colvard DS, Berg NJ, et al. Evidence of estrogen receptors in normal human osteoblast-like cells. Science 1988; 241:84. 42. Komm BS, Teipening CM, Benz DJ, et al. Estrogen binding, receptor mRNA, and biologic response in osteoblast-like osteosarcoma cells. Science 1988; 241:81. 43. Kaplan FS, Fallon MD, Boden SD, et al. Estrogen receptors in bone in a patient with polyostotic fibrous dysplasia (McCune-Albright syndrome). N Engl J Med 1988; 319:421. 44. Gray TK, Flynn TC, Gray KM, Nabell LM. 17|3-Estradiol acts directly on the clonal osteoblastic cell line UMR106. Proc Natl Acad Sci U S A1987; 84:6267. 45 Chapuy MC, et al. Vitamin D, and calcium to prevent hip fractures in elderly women. N Engl J Med 1992; 327:1637. 46. Liberman UA, Weiss SR, Broil J, et al. Effect of oral alendronate on bone mineral density and the incidence of fractures in postmenopausal osteoporosis. N Engl J Med 1995; 333:1437. 47. Black DM, Cummings SR, Karpf DB, et al. Randomised trial of effect of alendronate on risk of fracure in women with existing vertebral fractures: Fracture Intervention Trial Research Group. Lancet 1996; 348:1535. 48. deGroen PC, Lubbe DF, Hirsh LJ, et al. Esophagitis associated with the use of alendronate. N Engl J Med 1996; 335:1016. 49. Hopper JL, Seeman E. The bone density of female twins discordant for tobacco use. N Engl J Med 1994; 330:387. 50. Barrett-Connor E, Chang JC, Edelstein SL. Coffee-associated osteoporosis offset by daily milk consumption. JAMA 1994; 271:280. 51. Christiansen C, Riis BJ, Rodbro P. Prediction of rapid bone loss in post¬ menopausal women. Lancet 1987; 1(8542):1105. 52. Tulandi T, Lai S. Menopausal hot flash. Obstet Gynecol Surv 1985; 40:553. 53. Tataryn IV, Lomax P, Meldrum DR, et al. Objective techniques for the assessment of postmenopausal hot flashes. Obstet Gynecol 1981; 57:340. 54. Cignarelli M, Cicinelli E, Corso M, et al. Biophysical and endocrine-meta¬ bolic changes during menopausal hot flashes: increase in plasma free fatty acid and norepinephrine levels. Gynecol Obstet Invest 1989; 27:34. 55. Ravnikar V, Elkind-Hirsch K, Schiff I, et al. Vasomotor flushes and the release of peripheral immunoreactive luteinizing hormone-releasing hor¬ mone in postmenopausal women. Fertil Steril 1984; 41:881. 56. Meldrum DR, Erlik Y, Lu JK, Judd HL. Objectively recorded hot flushes in patients with pituitary insufficiency. J Clin Endocrinol Metab 1981; 52:684. 57. Simpkins JW, Katovich MJ, Song IC. Similarities between morphine with¬ drawal in the rat and the menopausal hot flush. Life Sci 1983; 32:1957. 58. Coope J. Double-blind crossover study of estrogen replacement therapy. In: Campbell S, ed. The management of the menopause and postmenopausal years. Baltimore: University Park Press, 1976:159. 59. Schiff I, Tulchinsky D, Cramer D, Ryan KJ. Oral medroxyprogesterone treatment of postmenopausal symptoms. JAMA 1980; 244:1443. 60. Thompson J, Oswald I. Effect of estrogen on the sleep, mood, and anxiety of menopausal women. BMJ 1977; 2:1317. 61. Schiff I, Regestein Q, Tulchinsky D, Ryan KJ. Effects of estrogens on sleep and psychological state of hypogonadal women. JAMA 1979; 242:2405. 62. Erlik Y, Tartaryn I, Meldrum D, et al. Association of waking episodes with menopausal hot flushes. JAMA 1981; 345:1741. 63. Campbell S. Double-blind psychometric studies on the effects of natural estrogens on postmenopausal women. In: Campbell S, ed. The manage¬ ment of the menopausal and postmenopausal years. Baltimore: University Park Press, 1976:149. 64. Klaiber El, Broverman DM, Vogel W, et al. Effects of estrogen therapy on plasma MAO activity and EEG during responses of depressed women. Am J Psychiatry 1972; 128:1492. 64a. Mulnard RA, Cotman CW, Kawas C, et al. Estrogen replacement therapy for treatment of mild to moderate Alzheimer disease: a randomized con¬ trolled trial. JAMA 2000; 283:10007. 65. Smith DC, Prentice R, Thompson DJ, Herrmann WL. Association of exoge¬ nous estrogens and endometrial cancer. N Engl J Med 1975; 293:1164. 66. Ziel HK, Finkle WD. Increased risk of endometrial carcinoma among users of conjugated estrogens. N Engl J Med 1975; 293:1167. 67. Chu J, Schweid Al, Weiss NS. Survival among women with endometrial cancer: a comparison of estrogen users and nonusers. Am J Obstet Gynecol 1982; 143:569. 68. Gambrell RD Jr. Clinical use of progestins in the menopausal patient. J Reprod Med 1982; 27(Suppl):531. 69. Sourander L, Rajala T, Raiha I, et al. Cardiovascular and cancer morbidity and mortality and sudden cardiac death in postmenopausal women on oestrogen replacement therapy (ERT). Lancet 1998; 352:1965. 70. Brinton LA, Hoover R, Fraumeni JF. Menopausal oestrogens and breast cancer risk: an expanded case-control study. Br J Cancer 1986; 54:825. 71. Colditz GA, Hankinson SE, Hunter DJ, et al. The use of estrogens and

991

progestins and the risk of breast cancer in postmenopausal women. N Engl J Med 1995; 332:1589. 71a. Willet WC, Colditz G, Stampfer M. Postmenopausal estrogens—opposed, unopposed, or none of the above. JAMA 2000; 283:534. 71b. Kavanaugh AM, Mitchell AM, Giles GG. Hormone replacement therapy and accuracy of mammographic screening. Lancet 2000; 355:270. 72. Schiff I, Tulchinsky D, Ryan KJ. Vaginal absorption of estrone and estra¬ diol-1713. Fertil Steril 1977; 28:1063. 73. Padwick ML, Endacott J, Whitehead MB. Efficacy, acceptability, and meta¬ bolic effects of transdermal estradiol in the management of postmeno¬ pausal women. Am J Obstet Gynecol 1985; 152:1085. 74. Haas S, Walsh B, Evans S, et al. The effect of transdermal estradiol on hormone and metabolic dynamics over a six-week period. Obstet Gynecol 1988; 71:671. 75. Whitehead MI, Siddle N, Lane G. The pharmacology of progestogens. In: Mishell DR, ed. Menopause, physiology and pharmacology. Chicago: Year Book Medical Publishers, 1987:326. 76. Archer DF, Pickar JH, Bottiglioni F. Bleeding patterns in postmenopausal women taking continuous combined or sequential regimens of conjugated estrogens with medroxyprogesterone acetate. Obstet Gynecol 1994; 83:686. 77. Leather AT, Savvas M, Studd JWW. Endometrial histology and bleeding patterns after 8 years of continuous combined estrogen and progestin ther¬ apy in postmenopausal women. Obstet Gynecol 1991; 78:1008. 78. Padwick ML, Pryse-Davies J, Whitehead MI. A simple method for deter¬ mining the optimal dosage of progestin in postmenopausal women receiv¬ ing estrogens. N Engl J Med 1986; 315:930. 79. Smith-Bindman R, Kerlikowske K, Feldstein V, et al. Endovaginal ultra¬ sound to exclude endometrial cancer and other endometrial abnormalities. JAMA 1998; 280:1510. 80. Ettinger B, Selby J, Citron JT, et al. Cyclic hormone replacement therapy using quarterly progestin. Obstet Gynecol 1994; 83:693. 81. Grey AB, Stapleton JP, Evans MC, Reid IR. The effect of the anti-estrogen tamoxifen on cardiovascular risk factors in normal postmenopausal women. J Clin Endocrinol Metab 1995; 80:3191. 81a. Guzzo JA. Selective estrogen receptor modulators—a new age of estrogens in cardiovascular disease? Clin Cardiol 2000; 23:15. 82. Delmas PD, Bjamason NH, Mitlak BH, et al. The effects of raloxifene on bone mineral density, serum cholesterol, and uterine endometrium. N Engl J Med 1997; 337:1641. 83. Eli Lilly and Company. Evista® (raloxifene hydrochloride) tablets prescrib¬ ing information. Indianapolis, IN; 1997, Dec 10. 84. Walsh BW, Kuller LH, Wild RA, et al. Effects of raloxifene on serum lipids and coagulation factors in healthy postmenopausal women. JAMA 1998; 279:1445. 85. Sacks FM, Pfeffer MA, Moye LA, et al. The effect of pravistatin on coronary events after myocardial infarction in patients with average cholesterol lev¬ els. Cholesterol and Recurrent Events Trial investigators. N Engl J Med 1996; 335(14):1001. 86. Kannel WB, Wolf PA, Castelli WP, d'Augustino RB. Fibrinogen and risk of cardiovascular disease. JAMA 1987; 258:1183. 87. Mannucci PM, Bettega D, Chantarangkul V, et al. Effect of tamoxifen on measurements of hemostasis in healthy postmenopausal women. Arch Intern Med 1996; 156:1806. 88. Shewmon DA, Stock JL, Rosen CJ, et al. Tamoxifen and estrogen lower cir¬ culating lipoprotein(a) concentrations in healthy postmenopausal women. Arterioscler Thromb 1994; 14:1586. 89. McDonald CC, Stewart HJ, for the Scottish Breast Cancer Committee. Fatal myo¬ cardial infarction in the Scottish adjuvant tamoxifen trial. BMJ 1991; 303:435. 90. Rutqvist LE, Mattsson A, for the Stockholm Breast Cancer Study Group. Cardiac and thomboembolic morbidity among postmenopausal women with early stage breast cancer in a randomized trial of adjuvant tamoxifen. J Natl Cancer Inst 1993; 85:1398. 91. Data on file, Lilly Research Laboratories, Indianapolis, IN. 92. Hulley S, Grady D, Bush T, et al. Randomized trial of estrogen plus proges¬ tin for secondary prevention of coronary heart disease in postmenopausal women. JAMA 1998; 280:605. 93. Cummings SR, Eckert S, Krueger KA, et al. The effect of raloxifene on risk of breast cancer in postmenopausal women. JAMA 1999; 281:2189.

CHAPTER 101_

HIRSUTISM, ALOPECIA, AND ACNE ENRICO CARMINA AND ROGERIO A. LOBO The effects of androgen on female skin include acne, hirsut¬ ism, and alopecia. Although derangements of androgen pro¬ duction and metabolism do not explain all such cases of dermatopathology, they often are of central importance in

992

PART VII: ENDOCRINOLOGY OF THE FEMALE OVARIES

ADRENALS OM

(T>

Testosterone

0

d4- Androstenedione (Adione)

d4-Androstenedione

G

61 G hoGa 0

Dehydroepiandrosterone

Dehydroepiandrosterone

,DHEA>

O

cr

Sulfate (DHEAS)

G

SO,H OH

d5-Androstenediol (d5-diOl)

d5-Androstenediol

HO\/

Commonly used abbreviations are in parentheses.

producing these abnormalities. To fully understand this interaction, the clinician must be familiar with androgen production in women and with the factors that modulate androgen action.

ANDROGEN PRODUCTION IN WOMEN Androgen production in women can be discussed in terms of three separate sources of production: the ovaries and the adre¬ nal glands, which are glandular sources, and the peripheral com¬ partment, which comprises all extrasplanchnic and nonglandular areas of androgen production. The peripheral compartment includes many tissues, the largest of which is the skin. The peripheral compartment modulates androgens produced by the ovaries and the adrenals. The ovary directly secretes testosterone, A4-androstenedione (Adione), and, to a lesser degree, dehydroepiandrosterone (DHEA) (Fig. 101-1). The adrenal gland normally does not secrete testosterone but does secrete Adione in amounts equal to that produced by the ovary in the follicular phase.1 The adrenal gland almost exclusively secretes dehydroepiandros¬ terone sulfate (DHEAS). DHEAS is secreted systemically in larger quantities than is any other androgen, and its serum concentration correlates significantly with urinary 17-ketosteroid excretion, but it appears to be a more specific marker of adrenal androgen production.2 Another specific marker of adrenal androgen secretion is ll|3-hydroxyandrostenedione. Under normal circumstances, the adrenal, but not the ovary, has the ability to 11-hydroxylate. The sensitivity of 11(3hydroxyandrostenedione as an adrenal marker is similar to that of DHEAS, but these two steroids are not correlated and probably reflect different aspects of adrenal androgen produc¬ tion.1 DHEA and Adione are also secreted in greater quantities by the adrenal than by the ovary.4

FIGURE 101-1. Androgens secreted by the normal ovaries and adrenals.

DIFFERENTIATION OF OVARIAN FROM ADRENAL ANDROGEN SECRETION Investigators have used various stimulation and suppression protocols to determine the source of androgen production in normal and hyperandrogenic women. Selective catheterization of adrenal and ovarian veins also has been attempted. How¬ ever, none of these techniques adequately shows the contribu¬ tion of these glands to the total androgen pool. Selective venous catheterization data have confirmed that almost all exoge¬ nously administered agents (e.g., dexamethasone, human chorionic gonadotropin [hCG], adrenocorticotropin [ACTH]) affect the ovary and the adrenal.5 Although these catheteriza¬ tion techniques may be useful in the detection of androgenproducing neoplasms, they have no place in the evaluation of other hyperandrogenic conditions. The only probe that has the potential to differentiate ovarian from adrenal androgen secretion is the gonadotropin-releasing hormone (GnRH) agonist (see Chap. 16).6,7 By down-regulating the gonadotrope and the ovary, ovarian androgens (i.e., tes¬ tosterone and Adione) can be differentiated from adrenal andro¬ gens (i.e., DHEA, DHEAS) in normal women and those with polycystic ovary syndrome (PCOS) (Fig. 101-2). Because Adione can be secreted equally by the ovary and adrenal, serum tes¬ tosterone is the better ovarian marker. Although only one-third of testosterone production (0.25-0.35 mg per day) is secreted directly by the ovary, the remaining two-thirds is derived in near-equal proportions from ovarian and adrenal precursors. Normally, two-thirds of circulating testosterone may originate from the ovary. These concepts, supported by data using a GnRH agonist, allow the conclusion that serum testosterone is the primary marker of ovarian androgen production, and DHEAS and llfi-hydroxyandrostenedione are the best serum markers of adrenal androgen production. However, DHEAS testing is more readily available, and the levels are not subject to diurnal variation.

Ch. 101: Hirsutism, Alopecia, and Acne |

FIGURE 101-2. Mean (± standard error) androgen and cortisol levels with gonadotropin-releasing hormone agonist (GnRH-a) treatment (100 pg per day) in women with polycystic ovary (PCO) syn¬ drome and normal ovulatory women compared with oophorectomized women (shaded bars). (DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate.) (From Chang RJ. Ovarian steroid secretion in polycystic ovarian dis¬ ease. Semin Reprod Endocrinol 1984; 2:244.) Unlike in normal ovulatory women, in women with hyperandrogenic disorders such as PCOS, the ovary may contribute to the circulating pool of DHEAS and lip-hydroxyandrostenedione.8-9 Research using a GnRH agonist has shown that the ovary may be responsible for -20% of circulating DHEAS.10 The effort to differ¬ entiate ovarian from adrenal androgen secretion is also con¬ founded by the fact that, in most hyperandrogenic conditions, both adrenal and ovarian androgen secretion are increased. In PCOS, 50% to 70% of patients present with adrenal hyperandrogenism,9 whereas in nonclassic 21-hydroxylase deficiency, 50% to 80% present with evidence of ovarian hyperandrogenism.11

Gn RH-o, lOCVq/doy

Weeks

Weeks

Logically, DHT should serve as the primary marker of peripheral androgen production; however, because of rapid cel¬ lular turnover of DHT and its great affinity for sex hormone¬ binding globulin (SHBG), serum DHT does not reflect the stepup phenomenon in peripheral tissues in terms of androgenicity.

17/3-Hydroxyandrogens OH

SERUM MARKERS FOR ANDROGENICITY IN WOMEN Testosterone exerts significant androgenic activity, whereas DHEAS is relatively inert as an androgen; however, 20 mg per day of DHEAS is produced, compared with only 3 mg per day of Adione and 8 mg per day of DHEA. These weak adrenal androgens exert their effects largely after conversion to more potent andro¬ gens. Testosterone, A5-androstenediol, dihydrotestosterone (DHT), and 3cx-androstanediol (3a-diol) are classified as 17[f-hydroxyandrogens and are the most potent circulating androgens; they are formed largely from other precursor androgens (Fig. 101-3). To exert effects in the periphery, including skin and genitalia, even the more potent androgens such as testosterone must be converted to DHT. Androgen action in peripheral tissues requires receptor occupancy by DHT, which necessitates signifi¬ cant 5a-reductase activity (5a-RA) within the cell.12 A more distal metabolite of DHT, 3a-diol, is also produced exclusively in peripheral tissues and is one of the 17(3-hydroxyandrogens known to exert some biologic effect. Most of its activity, however, is linked to DHT production. In the peripheral compartment, other enzymatic activities, such as 17-ketoreductase and aromatase activities, are largely responsible for producing less potent androgens (e.g., Adione from testosterone) and estrogens. A balance appears to exist between the "step up" of androgen action when DHT is formed and the "step down" when less potent androgens and estrogens are formed. An understanding of the relative control of these enzymatic processes is key to an appreciation of the effects of androgen on skin (Fig. 101-4). Two isoenzymes for 5a-reductase have been identified (types 1 and 2), and the genes are encoded on chromosomes 5 and 2, respectively.13'14 Although some overlap between the localization of these enzymes within the body is probable, type 1 is found predominantly in the liver and nonsexual skin, and type 2 is the predominant 5ot-reductase in the genitalia and prostate in men.

993

I

H

FIGURE 101-3. Serum markers for androgenicity in women.

994

PART VII: ENDOCRINOLOGY OF THE FEMALE

17 KSreductase TESTOSTERONE-ANDROSTENEDIONE

5a- reductase

5a- reductase

17 KSreductase \ ♦ DIHYDROTESTOSTERONE-- ANDROSTANEDIONE

3 KS- reductase

3 KS- reductase

I

1

glucuronyl-transferase

glucuronyl-transferase

I

I

17 KSreductase 3a- ANDROSTANEDIOL-- ANDROSTERONE

/

sulfuryltransferase

3a- ANDROSTANEDIOL GLUCURONIDE

3a- ANDROSTANEDIOL SULFATE

!S(^y

sulfuryltransferase

ANDROSTERONE GLUCURONIDE

3a- ANDROSTANEDIOL SULFATE

FIGURE 101-4. Peripheral metabolism of testosterone and androstenedione. (KS, ketosteroid.) Investigators have focused on more distal metabolites of DHT. The most studied are 3a-diol and its glucuronide, 3a-androstanediol glucuronide (3a-diol G)15 (see Fig. 101-4). Although 3a-diol and 3a-diol G are exclusively produced in peripheral tissues, 3a-diol G serves as a better marker of androgen action

because, once formed, no reconversion to DHT takes place. An excellent correlation is found between serum 3a-diol G level and the manifestation of androgenicity.12'16 A good correlation is also seen between androgenicity and the level of skin 5aRA16 (Fig. 101-5). Although some investigators have argued that serum 3a-diol G may not exclusively reflect peripheral or skin activity and that it is largely derived from adrenal andro¬ gens,17 data confirm the earlier observations. DHT produces much higher serum levels of 3a-diol G when it is applied to the skin than when it is delivered intravenously.18 Serum 3a-diol G also shows excellent concordance with the improvement in hir¬ sutism scores, which occurs with GnRH agonist treatment.19 Serum concentrations of androgen metabolites are also strongly influenced by the circulating levels of androgens.20 In hirsute women, 3a-diol G levels depend both on levels of more potent androgens (e.g., testosterone and androstenedione) and on peripheral 5a-RA.18'20 Although serum 3a-diol G levels best reflect the presence and the severity of hirsutism, measurement of other androgen metabolites may be useful clinically. Assay of one of these metabolites, androsterone glucuronide, may be particularly helpful in patients with acne.21

MODULATORS OF ANDROGEN ACTION No correlation is found between androgen levels and the degree or severity of hirsutism. However, the modulation of androgen status by 5a-RA and the androgen receptor content in tissues are pivotal. Androgen receptor content appears to be less important than 5a-RA levels. Receptor concentrations alone do not explain differences in clinical androgenicity,22 although some studies have suggested that polymorphisms of the androgen receptor may be implicated in the pathogenesis of some forms of alopecia and hirsutism23 and may influence the peripheral expression of hyperandrogenism.24 The most important modulator of peripheral androgen action is 5a-reductase, (type 1 and 2)13'14 (Table 101-1). The skin expres¬ sion of 5a-reductase is highly influenced by racial and ethnic factors, as well as inheritance. Moreover, skin production of 5areductase is also regulated by local factors (e.g., transforming growth factor-[l [TGF-(3] and insulin-like growth factor-I [IGF-I] and its binding proteins) and circulating factors (e.g., circulating IGF-I and androgens)25-27 In blood, tire primary modulator of the androgen signal is the transport protein SHBG, also known as testosterone-estradiol¬ binding protein (see Chap. 114). All conditions that decrease SHBG binding increase unbound concentrations of the active 17(3hydroxyandrogens, augmenting their effect. The percentage of unbound estradiol (normally 35% is SHBG bound) also increases. Although several factors regulate the liver production of SHBG, probably the most important factor is insulin, which reduces SHBG blood concentrations and, thus, increases unbound testosterone.28 This effect may be important in some conditions in which insulin levels are increased (e.g., obesity and PCOS) and explains why weight reduction may be important to the treatment of hirsutism. TABLE 101-1. Characteristics of Two 5a-Reductase Isoenzymes

FIGURE 101-5. In vivo percentage conversion of testosterone (T) to dihydrotestosterone (DHT) by 5a-reductase in a genital skin preparation and relation to the clinical evaluation of hirsutism (Ferriman-Gallwey score). (From Serafini P, Lobo RA. Increased 5a-reductase activity in idiopathic hirsutism. Fertil Steril 1985; 43:74.)

5a-Reductase Type 1

5a-Reductase Type 2

Structure

292 amino acids

254 amino acids

Molecular weight

29,000

29,000

PH Gene

Basic

Acid

Short arm of chromosome 8

Short arm of chromosome 2

Localization

Adult female genital skin, adult skin

Prostate, liver, male geni¬ tal skin

Sensitivity to finasteride

+

++

Ch. 101: Hirsutism, Alopecia, and Acne

995

Vellus Hair

Terminal Hair

FIGURE 101-6. Changes occurring after several generations of hair cycles that result in the transition from terminal to vellus hair. During puberty, under the influence of androgens, some of the hair changes from vellus to terminal, especially in the axillary and pubic regions. However, after puberty, for uncertain reasons, an increase in the normally low level of 5a-reductase activity in the scalp may induce a transition from terminal to vellus hair, leading eventually to alopecia. (Modified from Montagna W, Parakkal PF. The structure and function of skin, 3rd ed. New York: Academic Press, 1974:250.)

The measurement of non-SHBG-bound or "free'' testos¬ terone has been advocated in the routine evaluation of androgen excess to more accurately detect subtle forms of hyperandrogenism.29 However, the correlation of total and non-SHBG-bound testosterone is excellent and frequently can be predicted.30 The assay of unbound testosterone gener¬ ally is not necessary and should be used only in patients who have signs of androgen excess (i.e., hirsutism, acne, or alope¬ cia) in the presence of normal levels of total testosterone and DHEAS. In an evaluation of 588 hirsute women, an increase of non-SHBG testosterone was found, with normal levels of total testosterone and DHEAS, in only 12 patients (1%).31 The normal serum levels for these sex steroids in women are as follows: testosterone, 20 to 70 ng/dL (some laboratories report val¬ ues up to 100 ng/dL); dialyzable free testosterone, 1 to 8 pg/mL (free, by dialysis); non-SHBG-bound testosterone, 1 to 10 ng/dL (dialyzable free plus loosely bound); androstenedione, 20 to 250 ng/dL; DHEA, 130 to 980 ng/dL; DHEAS, 0.5 to 2.8 pg/mL (some laboratories report values to 3.3 pg/mL); ll(3-hydroxyandrostenedione, 15 to 200 ng/dL; As-androstenediol, 20 to 80 ng/dL; DHT, 5 to 30 ng/dL; 3a-diol, 0.5 to 6.5 ng/dL; and 3a-dial G, 60 to 300 ng/dL (range has been extended with refinement in assay).

PILOSEBACEOUS UNIT An understanding of the clinical conditions associated with hyperandrogenism requires some background information about the pilosebaceous unit (PSU), the common structure in skin that gives rise to hair and sebaceous glands. PSUs and hair are distributed over virtually the entire body except the palms and soles. If the sebaceous component of the PSU is prominent, the hair is merely vellus—soft, fine, and unpigmented hair that may remain unrecognized (Fig. 101-6). If the pilary component is prominent, the terminal hair is differentiated from vellus hair by its darker color, greater length, and coarseness. Before puberty, the predominant body hair is vellus. During puberty, some of the vellus hair normally is transformed into terminal hair, particularly in the pubic and axillary regions. After puberty, terminal hair undergoes normal cyclic changes, the control of which is only partly understood.

The characteristics and distribution of body hair differ greatly among women and are strongly influenced by ethnic and racial factors (i.e., Native Americans, Asians, fair-skinned whites, and some blacks have less hair). Body hair is also influenced by imme¬ diate genetic factors; family members frequently have similar hair characteristics. Elderly women often have increased facial hair, which may be associated with a diminution in pubic and axillary hair. Body hair is more noticeable in women with dark hair. The attitude toward body hair varies among different socie¬ ties and individuals. In some societies, a relatively large amount of body hair in women is admired; in others, it is con¬ sidered unattractive. Similarly, the psychology and immediate environment of a woman may alter markedly her attitude toward a degree of body hair that most women would not con¬ sider excessive, but which she finds alarming or intolerable.

GROWTH PHASES OF HAIR Anagen is the growth phase of hair; the length of each phase of growth varies according to body site. This process is somewhat influenced by the hormonal environment. A primary feature of anagen hair is its pigmentation and medullary component (Fig. 101-7). After the growth phase, the transitional catagen phase ensues, in which the club-shaped bulb moves distally, eventu¬ ally releasing the dermal papilla and becoming inactive. A rest¬ ing stage, telogen, follows until the hair is shed and active follicular growth begins again.

HORMONAL CONTROL ANDROGENS Although the usual factors that control the transition between phases of hair growth remain elusive, androgens are probably the most important factors in determining the type and distribu¬ tion of hair over the human body. Androgens may convert hair follicles into terminal hair and concomitantly prolong the anagen phase of hair growth.32 Therefore, not only do androgens alter the type of hair, but they also increase its length and oiliness (because of their effects on sebaceous glands). As previously noted, most of this effect is determined by DHT, which is formed by the peripheral conversion of testosterone, via the action of 5a-

996

PART VII: ENDOCRINOLOGY OF THE FEMALE

Sebaceous gland

/

Sebaceous gland Cortex

\ Arrector pili muscle

Medulla

Club

Cortex Cortex

Cuticle Outer root sheath Cuticle Huxley’s layer Henle’s layer Collagen

Inner root sheath

Club

Germ

\t Autophagic vacuole

Basal lamina Basal lamina — Fibroblast • » __Collagen •.t’;, _Macrophage

•"/

Germ

Dermal papilla Dermal papilla

.Dermal papilla Bulb

Anagen

Telogen

Catagen

FIGURE 101-7. Changes in the hair follicle during growth (anagen), regression (catagen), and rest (telo¬ gen). (Adapted from Montagna W, Parakkal PF. The structure and function of skin, 3rd ed. New York: Academic Press, 1974:187.) reductase. The two isoenzymes have different locations within the PSU; type 1 is localized mostly in the distal portions of the seba¬ ceous gland, in the dermal papilla and in the outer root sheaths, whereas type 2 is found mostly in the dermal papilla.33 Other enzymes, mostly 17(3-hyd roxysteroid dehydrogenase type 2 (17(3-HSD2), 17p-hydroxysteroid dehydrogenase type 1 (17(3-HSD1), and 3(3-hydroxysteroid dehydrogenase (3J3-HSD) are also highly expressed within the PSU and are localized in the outer root sheath and sebaceous gland but not in the dermal papilla.34 Thus, these two compartments may have the capacity to convert relatively weak androgens, such as DHEA, into tes¬ tosterone. Finally, data are conflicting with respect to the pres¬ ence of cytochrome P450 aromatase, which converts androgens to estrogens and may be viewed as providing a protective mechanism against the effects of androgens.33-34 One should keep in mind that different body areas may express different concentrations of these enzymes. Patients with adrenal androgen excess have lower 5a-RA in pubic skin than do patients with testosterone excess.35 However, in the scalp, cir¬ culating DHEAS levels rather than testosterone levels correlate positively with the proliferation index of hair bulb cells.36 OTHER FACTORS Other factors that influence the PSU include growth hormone, insulin-like growth factors, adrenal steroids, and a-melanocytestimulating hormone.37-38 Adrenal steroids alone appear to be sufficient for the normal production of hair in the axilla and pubis. Hair in these areas appears even in the absence of 5aRA, but it is unlikely to be present in cases of androgen receptor deficiency (i.e., testicular feminization; see Chap. 90). This sug¬ gests that low levels of androgens are sufficient to stimulate some hair growth in pubic and axillary regions. However, some mechanism for androgen action is required, possibly one that does not need DHT or requires only a low level of DHT for receptor activation. The scalp does not require androgen for hair growth. Paradoxically, an increase in the normally low

level of 5a-RA in the scalp may cause a transition from terminal to vellus hair (see Fig. 101-6) and the development of alopecia. Various growth factors and cytokines may affect hair growth.38-40 These factors generally act on cells of the dermal papilla as well as on follicular stem cells. They include fibro¬ blast growth factor (FGF) and platelet-derived growth factor (PDGF) that potentiate the growth of dermal papilla cells, mostly by increasing the production of stromolysin, a metalloprotease that accelerates the growth of the PSU.39 Other local factors such as TGF-p, IGF-I, interleukin-1, and epidermal growth factor (EGF) inhibit or attenuate the growth of the PSU.40

ACNE HORMONAL CONTROL OF SEBUM PSUs, which are primarily sebaceous, are influenced by many fac¬ tors, and once they are stimulated, their actions may lead to oily skin and acne lesions. The density of PSUs is greatest on the face and scalp (400-800 glands/cm2) and lowest on the extremities (50 glands/cm2).41 Androgen is unequivocally linked to stimulation of sebum production.42 Androgen stimulates sebaceous gland cell division and intracellular lipid synthesis.42 In animal studies that have examined the effects of testosterone and antiandrogens, estradiol has been shown to be an extremely potent inhibitor of sebum production. However, estradiol appears to have little or no effect on cell division. Corticosteroids have a stimulatory effect on sebaceous glands, and those progestins that have androgenic properties act at physiologic levels to stimulate sebaceous activity.

PREVALENCE AND SCORING Acne is present to some degree in almost all individuals but occurs most commonly during puberty, when it is found in as

Ch. 101: Hirsutism, Alopecia, and Acne

many as 50% of adolescent girls and 85% of boys. After adoles¬ cence, acne disappears in most subjects but persists in some individuals. In other subjects, acne appears during adult life. Patients with persistent acne during adulthood (and patients with severe adolescent acne) can benefit from a systemic treat¬ ment (Fig. 101-8). Acne may be graded according to several methods. An easy method is to score acne on a scale of 0 to 3.43 In the modified method, the absence of lesions is scored as 0. Grade 1 (mild) lesions are characterized by comedones, with either no or only a few inflammatory papules or pustules. Grade 2 (moderate) lesions are characterized by numerous inflammatory lesions but with rare cystic activity. Grade 3 (severe) lesions are the worst lesions and are characterized by the presence of innumer¬ able inflammatory papules or pustules as well as cysts. For research purposes, use of the method that scores the severity of lesions from 0 to 9 is preferable.44

PATHOGENESIS Acne is a multifactorial disease in which androgens have a cen¬ tral role. Four processes determine the appearance of the acneic skin lesions45: (a) excessive keratinization of the infra-infundibu¬ lum and cohesion of horny cell masses that lead to retention of hyperkeratosis; (b) increased sebum production; (c) bacterial colonization; and (d) inflammation (Fig. 101-9). In this process, androgens appear to be key in the process of hyperkeratosis and the increased sebum production. Bacteria (e.g., Propionibacterium acnes) are implicated in the inflamma¬ tory process.46 Obstruction to sebum excretion is a characteristic feature of comedone formation. Bacteria, specifically anaerobes such as Corynebacterium species in the deeper tissues and aer¬ obes such as Staphylococcus epidermidis at the surface, are responsible for the breakdown of sebum by lipases, resulting in the liberation of irritant fatty acids. In acne, the colonization correlates with sebum excretion, and sebaceous lipids are believed to be essential etiologic factors in P. acnes colonization of human skin.46 Rupture of the cyst wall and expulsion of the irritant fatty acids into the dermis causes inflammatory and cystic lesions. Excessive bacterial counts in skin and surface irritants on the skin, such as cosmetic oils, also contribute to this process; diet is probably less important. A genetic predisposition is seen to the development of acne.

997

FIGURE 101-9. Electron photomicrograph of comedo formation begin¬ ning in infra-infundibulum. Large numbers of Propionibacterium acnes are present in the pilosebaceous canal. Keratohyalin is prominent. Cells of horny layer remain intact and begin to adhere to each other. Many cells contain lipid droplets not seen in normal infundibulum. (From Knutson DD. Comedo formation: ultrastructure. In: Frank SB, ed. Acne: update for the practitioner. New York: Yorke Medical Books, 1979:81.)

Whether this results from familial hyperandrogenemia during puberty, end organ (i.e., PSU) sensitivity, or both is uncertain.

HYPERANDROGENISM AND ACNE Several investigators have demonstrated a link between hyperandrogenism and acne; some studies have shown that most patients with acne have elevated serum androgens, reflecting increased ovarian and/or adrenal production.4 46 Although some studies have reported specific correlations of acne with lev¬ els of serum free testosterone49 or serum DFIEAS,50 the authors and others21'51-52 have not observed any particular androgen pro¬ file in hyperandrogenic patients with acne. Also, no correlation exists between the degree of hyperandrogenism and the severity of acne. This suggests that peripheral factors are important in determining the appearance of acneic lesions in hyperandro¬ genic patients. These factors may include changes in peripheral androgen metabolism, alterations of some local factors, or both. Consistent with the first hypothesis, a particular profile of andro¬ gen metabolites has been shown to be present in hyperandro¬ genic acne patients characterized by relatively higher levels of androsterone glucuronide.21 Indeed, although all studied andro¬ gen metabolites (including 3a-diol G) are increased in hyperan¬ drogenic acne patients (because of the increase of androgen substrate), only androsterone glucuronide is higher in hyperan¬ drogenic patients with acne as compared to hyperandrogenic

998

PART VII: ENDOCRINOLOGY OF THE FEMALE

2500-1

THERAPY

200

Group

Group

FIGURE 101-10. Mean serum levels of androsterone sulfate (A sulfate) and androsterone glucuronide (A glucuronide) in normal controls (group 1), hirsute hyperandrogenic patients with acne but no hirsutism (group 2), hirsute hyperandrogenic patients with acne (group 3), and hirsute hyperandrogenic women without acne (group 4). (*+ p 27. If this is the classic clinical presentation of PCOS, many patients exhibit different clinical features. In fact.

FIGURE 101-17. Serum levels of immunoreactive luteinizing hormone (LH), ratio of LH to follicle-stimulating hormone (FSH), and biologi¬ cally active LH in control subjects (C), women with chronic anovulation (CA), and women with polycystic ovary syndrome (PCO). Closed circles for women with PCO indicate values exceeding 3 standard deviations of mean control levels. (From Lobo RA, Kletzky OA, Campeau JD, diZerega GS. Elevated bioactive luteinizing hormone in women with the polycystic ovary syndrome [PCO]. Fertil Steril 1983; 39:674.)

PCOS may be diagnosed in many hyperandrogenic women with normal menses because of chronic anovulation or because of the presence of sonographic and hormonal features typical of the syndrome. The authors have calculated that -20% of eumenorrheic hyperandrogenic women have chronic anovula¬ tion despite reporting "normal" menses, and another 50% have other features of PCOS.85 Many women with PCOS also have normal body weight, and in the authors' experience obesity is present in only 44% of patients with classic PCOS (hyperandro¬ genism and chronic anovulation).78 The characteristic biochemical disturbance is inappropri¬ ate gonadotropin secretion with high serum luteinizing hor¬ mone (LH) levels, an exaggerated response of LH to GnRH, and normal or slightly low follicle-stimulating hormone (FSH) levels.86 This usually yields elevated serum LH/FSH ratios. Abnormally high LH/FSH ratios may occur in only 75% of patients, but serum bioactive LH is elevated in most patients87 (Fig. 101-17). Mild elevations in serum prolactin levels (between 22 and 40 ng/mL) may be found in a few patients (12% in the authors' experience).88 The elevations in serum prolactin, coupled with GnRH and LH secretory abnormalities, have suggested a relative dopamine defi¬ ciency in this syndrome, but only suggestive data for some patients support this hypothesis. These abnormalities more probably are linked to increases in serum estrone and serum unbound estradiol.89 PCOS is a disorder characterized by a chronic hyperestrogenic state, and it may contribute to the inappropriate gonadotropin secretion.86 Ovarian hyperandrogenism is a cardinal feature of PCOS. The ovaries produce increased amounts of testosterone, androstenedione, and DHEA, but elevations of serum tes¬ tosterone are the most frequently encountered findings. The androgen excess in PCOS is milder than that observed in tumors or in ovarian hyperthecosis, and circulating levels of testosterone generally do not exceed 150 ng/dL. This may be demonstrated using a GnRH-agonist test. Interestingly, the most consistent alteration is the increase of serum 17hydroxyprogesterone (17-OHP) that may help to distinguish

Ch. 101: Hirsutism, Alopecia, and Acne women with PCOS from women with other forms of hyperandrogenism.90 That this syndrome has been considered to be ovarian does not exclude the presence of adrenal androgen secretory distur¬ bances. In this regard, as many as 50% of patients with docu¬ mented PCOS have increased adrenal androgen production.9-91 In a prospective study in several countries, the prevalence of adrenal androgen excess in PCOS was 50% to 60%.92 Several researchers have suggested that adrenal hyperandrogenism may be central to the pathogenesis of this syndrome. The best evidence, however, suggests that in most patients with PCOS, adrenal hyperandrogenism is secondary to the hormonal status of the syndrome.93 Insulin resistance and ovarian steroid pro¬ duction (coupled with elevations in unbound estradiol) seem to be the main contributors to the development of adrenal hyper¬ androgenism in PCOS.94 Mild hyperinsulinemia and insulin resistance are charac¬ teristic features of PCOS that are worsened by obesity but also are present in -50% of normal-weight women with the syndrome.95 Excess circulating insulin influences the clinical presentation of PCOS in several major ways78: (a) direct stim¬ ulation of ovarian androgen secretion; (b) reduction of insulin¬ like growth factor-binding protein-1 production and, as a consequence, increase in bioavailable IGF-I activity; (c) influ¬ ence on gonadotropin and adrenal androgen secretion; and (d) contribution to abnormalities in lipids and lipoproteins and to alterations in glucose tolerance. The molecular mechanisms of insulin resistance in PCOS appear to be different from those found in other syndromes of insulin resistance. In many patients the mechanism consists of an abnormality of insulin receptor signaling and phosphoryla¬ tion,96 which is probably genetically transmitted.95 This sug¬ gests that insulin resistance may play a central role in the pathogenesis of the syndrome. Pathogenesis of Hirsutism and Acne in Polycystic Ovary Syndrome. Hirsutism and acne are common in PCOS but cannot be explained solely on the basis of elevated blood androgen levels. Serum androgen levels are similar in all patients with PCOS, whether or not hirsutism is present.12 The response of the peripheral compartment appears to determine the manifestation of hirsutism. In one study, serum 3a-diol G levels were normal in nonhirsute patients with PCOS but were increased in the group with hirsutism (see Fig. 101-16). Genital skin 5a-RA is elevated to a similar degree in patients with PCOS and idiopathic hirsutism who have similar Ferriman-Gallwey scores. This further reinforces the concept that the immediate tissue environment of the PSU rather than the androgen substrate delivered to it is more pivotal in the expression of androgenicity. Some patients have responded to weight reduction by a resumption of ovulation and decreased androgen secretion. The treatment of PCOS is discussed more extensively in Chapter 96. Central to the pathophysiology of PCOS is insulin resistance, which occurs in most women with PCOS and may or may not be associated with the finding of acanthosis nigri¬ cans. Although excessive body weight probably accentuates this abnormality, it may be found in women of normal weight. In studies in the United States, Italy, and Japan, insulin resis¬ tance was equally prevalent (-70%), even in the leaner Japa¬ nese women.92 As discussed, hyperinsulinemia has many ramifications in the pathophysiology of PCOS, which are beyond the scope of this review. However, an important association is found between hyperinsulinemia and ovarian hyperandrogenism. Insulin and IGF-I stimulate ovarian androgen production and decrease SHBG, leading to elevated levels of free testosterone. Moreover, evidence suggests that IGF-I may play a role in the control of skin 5a-RA. Evidence also suggests that insulin con¬ tributes to the hyperandrogenism by augmenting ovarian androgen production.

1003

STROMAL HYPERTHECOSIS Hyperthecosis is an ovarian disorder that overlaps with true PCOS and ovarian tumors. The history is more compatible with that of PCOS, but onset is not sudden. Serum testoster¬ one levels often exceed 150 to 200 ng/dL, however, and DHEAS and gonadotropin levels are usually normal. The dis¬ ease is explained by luteinized thecal cells in the ovarian stroma that produce the excessive serum testosterone levels. The process is bilateral and usually diagnosed histologically only after oophorectomy. The ovaries are fleshy and do not resemble those seen in classic PCOS.97 Patients with stromal hyperthecosis often respond poorly to suppression therapy, except for the use of a GnRH agonist, which is highly effective if therapy is prolonged. As these patients become older, and particularly for those who experience progression from hir¬ sutism to frank virilization, bilateral oophorectomy often becomes the treatment of choice. NONCLASSIC 21-HYDROXYLASE DEFICIENCY Nonclassic 21-hydroxylase deficiency (NC-21-OH) represents an attenuated form of classic congenital adrenal 21-hydroxylase deficiency and, as in the classic form, has an autosomal reces¬ sive mode of transmission.98-100 The prevalence of NC-21-OH varies greatly according to ethnicity100 and is very low in patients of Northern European ancestry, although it is relatively common in Southern Europe (-3% prevalence among hyperandrogenic women in Southern Italy98) and in some populations such as Ashkenazi Jews. In most cases, the clinical presentation is identical to that seen in PCOS,98-101 and patients present with signs of hyperandrogenism during puberty or at menarche. Because of this later presentation, the disorder is also referred to as late-onset 21-hydroxylase deficiency. Most patients (70%) have menstrual irregularities and chronic anovulation; the androgen pattern is one of increased levels of androstenedione, testosterone, and unbound testosterone. DHEAS levels are often normal, because the adrenal gland produces mainly A4-androgens.101 Up to 100% of patients with CAH have sonographic evidence of polycystic ovaries. The diagnosis of NC-21-OH requires an elevation of early morning (8 a.m.) serum 17-OHP. Because serum 17-OHP rises during the luteal phase of the cycle, the blood sample should be obtained during the follicular phase in women with a men¬ strual factor. Basal levels of >10 ng/mL make the diagnosis clear, whereas in patients with 17-OHP levels from 3 to 10 ng/ mL, the confirmation of the diagnosis requires an ACTH stim¬ ulation test. Importantly, very few patients with normal 17OHP levels have an exaggerated response to ACTH. In some patients, a molecular study of the CYP21 gene helps to estab¬ lish the diagnosis.102 In the absence of clinical and androgen data characteristic of this disorder, a 17-OHP assay should be performed in all hyperandrogenic women. Most NC-21-OH patients must be considered to be affected by a mixed form of hyperandro¬ genism (both adrenal and ovarian).101'103 This is probably a consequence of the effect of increased levels of estrogen (spe¬ cifically unbound estradiol) on gonadotropin secretion.104 Because of the increase of ovarian androgen secretion, gluco¬ corticoid therapy may be relatively ineffective in many patients with NC-21-OH, and appropriate treatment of hirsut¬ ism may necessitate the administration of antiandrogens or GnRH-agonists.103/105 OTHER LATE-ONSET ADRENAL ENZYMATIC DEFICIENCIES Two other late-onset enzymatic deficiencies, 11-hydroxylase (11-OH) deficiency and 3|J-ol-dehydrogenase-isomerase (3(3ol) deficiency, result in hyperandrogenism during adult life. However, late-onset 11-OH deficiency is very uncommon and

1004

PART VII: ENDOCRINOLOGY OF THE FEMALE

few patients have been described.106 In contrast, late-onset 3fiol deficiency is relatively common (-1-2% of all hyperandrogenic women), and these patients present with high levels of DHEAS but with normal or even low levels of testosterone.107 The diagnosis requires an ACTH stimulation test with com¬ parison of the rises in A4 and A5 steroid precursors. In many patients, the defect may be sufficiently mild that the diagnosis is considered to be PCOS. Unlike for late-onset 21-OH defi¬ ciency, molecular studies have not shown any alteration of the type I or type II 3(3-ol gene in patients with late-onset 3(3-ol deficiency.108'109 This has reinforced the notion that this disor¬ der may be functional and represents an example of func¬ tional adrenal hyperandrogenism.

TREATMENT OF HIRSUTISM The evaluation of androgen excess should focus on establishing the cause and source of the hyperandrogenism. For treatment of hirsutism, the levels of serum testosterone and DHEAS must be known. Levels of these two hormones can serve as guides for suppression of ovarian or adrenal androgen production. This approach does not require a specific diagnosis for the more func¬ tional states, such as idiopathic hirsutism and PCOS, but instead focuses on the sources of production. A specific diagnosis such as congenital adrenal hyperplasia or a tumor warrants more spe¬ cific treatment (i.e., corticosteroids or surgery, respectively). Although measurements of serum 3a-diol G levels can con¬ firm a peripheral source of hyperandrogenism, this evaluation is not essential. Normal testosterone and DHEAS levels imply a peripheral abnormality. ORAL CONTRACEPTIVES If testosterone is elevated, ovarian hyperandrogenism is best suppressed with oral contraceptives. The combination of progestin and synthetic estrogen decreases LH-dependent ovarian androgen production. The estrogen also increases the production of SHBG, so that the lowered androgen (e.g., test¬ osterone) is more avidly bound, yielding much lower serum non-SHBG-bound testosterone levels. Progestins alone are less effective than combination oral contraceptives. Certain oral contraceptives decrease adrenal androgen production by at least 30% and are helpful in cases of mild adrenal hyperfunc¬ tion (DHEAS levels 30 cm in diameter (median, 9 cm). Small neoplasms are solid, encapsulated by a thin fibrous layer, and located in the ovarian cortex, whereas larger tumors may replace the ovary and become focally cystic. Virilizing tumors tend to be thin-walled, cystic tumors. Blood is often present within the cysts and explains the presentation of hemoperitoneum secondary to rupture that occurs in 10% of cases.

FIGURE 102-3. Granulosa tumor, microfollicular pattern. x200

Granulosa tumors display many histologic patterns, including microfollicular (the most characteristic, showing many Call-Exner bodies; Fig. 102-3), macrofollicular, trabecular, insular, solid or dif¬ fuse, and juvenile. The pathologist must be aware of these pat¬ terns so that poorly differentiated carcinomas are not misdiagnosed as diffuse granulosa tumors. This relatively com¬ mon error accounts for many of the examples of poor-prognosis granulosa tumors reported in the literature. Alpha-inhibin is expressed in granulosa cell tumors and other sex cord stromal tumors, but not in most carcinomas.15 Immunohistochemical staining for this peptide may aid in the differential diagnosis. The histologic appearance of granulosa cell tumors does not correlate with the prognosis in most studies. The neoplastic cells resemble normal granulosa cells, being small, round to ovoid, and uniform. The nuclei have a characteristic longitudinal groove (a "coffee bean" appearance), and the cytoplasm is pale with poorly defined borders. Occasional cells are luteinized. Reactive thecal cells occur in -25% of granulosa tumors. Anti-mullerian hormont (AMH) is a useful serum marker for these tumors.153 The juvenile granulosa tumor, which accounts for 85% of granulosa cell tumors occurring before puberty, has distinctive features: the cells are larger, more pleomorphic, and often luteinized. Nuclear grooves are rare, and mitoses may be numerous.16 Despite these ominous-appearing features, the prognosis is excellent. Ultrastructural, immunocytochemical, and in vitro incubation studies have confirmed that the granulosa cells within these neo¬ plasms can produce progesterone and testosterone in addition to estrogens, and receptors for these hormones may be detected as well.17,18 These findings are in accord with the occasional viriliz¬ ing and progestational activity displayed by these tumors. Granulosa tumors are regarded as having low malignant potential, with a 5-year survival rate of >90%. However, these tumors recur in 10% to 33% of patients, and late recurrences (as much as 25 years after primary tumor resection) are characteris¬ tic. The prognosis is worse in patients with tumors >5 cm in diameter, bilateral tumors, rupture, and spread beyond the ovary.19 Unilateral salpingo-oophorectomy is adequate treat¬ ment for premenopausal women with stage 1A neoplasms, whereas postmenopausal women are treated by total abdomi¬ nal hysterectomy and bilateral salpingo-oophorectomy.

THECOMAS Thecomas comprise 2% to 3% of all ovarian neoplasms and are found primarily in perimenopausal and postmenopausal women.

1012

PART VII: ENDOCRINOLOGY OF THE FEMALE

FIGURE 102-4. Thecoma. Spindle cells are in interlacing fascicles. Clear cytoplasm contains lipid. x200

Patients generally present with abnormal uterine bleeding and an abdominal mass. Most functional thecomas are estrinizing, but a few are virilizing.20 Endometrial hyperplasia and carcinoma occur in association with these tumors but not as often as with granulosa tumors. Grossly, these are solid, smooth, white to yellow tumors with occasional cysts and calcified areas. The tumors are bilat¬ eral in 5% of patients. Microscopically, thecomas have interlac¬ ing whorls of spindle cells, many containing abundant lipid (Fig. 102-4). Fibrocollagenous tissue makes up various propor¬ tions of these tumors, and if significant numbers of fibroblasts are present, the tumor may be designated a fibrothecoma. These tumors are regarded as benign, and excision results in cure. A few cases of "malignant thecomas" have been said to show both clinical and pathologic features of malignancy, namely, inva¬ sion or metastases, atypical nuclei, and an increased mitotic rate.

SERTOLI-LEYDIG TUMORS Sertoli-Leydig cell tumors, also known as androblastomas, usu¬ ally occur in women in the third or fourth decade of life. Less than 50% of the tumors are associated with androgenizing signs, including hirsutism and virilization (temporal balding, deepening of the voice, development of male body configura¬ tion, and clitoral enlargement) secondary to secretion of tes¬ tosterone.20-21 A few patients have estrinizing signs attributable to peripheral aromatization of androgens. The usual presenta¬ tion is oligomenorrhea or amenorrhea. Defeminization, defined as regression of female secondary sex characteristics (amenor¬ rhea, atrophy of endometrium and vaginal mucosa, decreased breast size), occurs initially and is followed by masculinization. The serum testosterone level is elevated, but because these tumors produce little or no androstenedione and dehydroepiandrosterone, urinary 17-ketosteroids are in the normal range. Masculinizing adrenal tumors produce high levels of andro¬ stenedione and dehydroepiandrosterone and small amounts of testosterone. Consequently, the urinary 17-ketosteroids are ele¬ vated in patients with masculinizing adrenal tumors. Sertoli-Leydig cell tumors are unilateral in >95% of patients. The cut surface is homogeneous and gray-pink to yelloworange, with occasional cysts, areas of hemorrhage, or necrosis. Well-differentiated tumors are usually 40 mJU/mL are consistent with ovar¬ ian failure. A serum FSH determination obtained on the second or third day of the menstrual cycle reflects ovarian reserve. Patients with an abnormal serum FSH level, >15 mlU/mL, on cycle day 2 or 3 have a poor prognosis for success with in vitro fertilization and probably with other treatment modalities as well.22

FIGURE 103-1. Endometrial biopsy is representative of day 27 of the menstrual cycle, with coalescent decidua and infiltration of leukocytes consistent with normal late luteal function. (Hematoxylin and eosin; xlOO)

tional method of evaluating luteal function17 (Figs. 103-1 and 103-2). Although some subjectivity exists in the interpretation, the test is less expensive than multiple progesterone determina¬ tions. However, it is invasive and may require repetition. More¬ over, as documented in the original publication, an error in interpretation occurs in 20% to 25% of specimens. The specific¬ ity of endometrial biopsy and effectiveness of available treat¬ ment remain controversial. Additional techniques for evaluating luteal function have been described. Prolactin production in endometrial explant culture is depressed in patients with luteal deficiency. Tissue obtained by endometrial biopsy in patients with luteal defi¬ ciency produces less prolactin, which is directly correlated with the amount of decidualization.18 Luteal function has been

ASSESSMENT OF UTERINE CAVITY AND FALLOPIAN TUBES Hysterosalpingography is the accepted standard for evaluating the uterine cavity and tubal lumen (Fig. 103-3). Water-soluble or oil-soluble radiopaque contrast material is injected through an endocervical cannula; the flow of contrast is followed with fluo¬ roscopy; and radiographs are taken as a permanent record. Some researchers claim a therapeutic effect, with enhanced fer¬ tility 6 to 12 months after hysterosalpingography, but this bene¬ fit has not been documented in a controlled clinical trial.23 Although the procedure is generally safe, the possible compli¬ cations include uterine perforation, postexamination hemor¬ rhage, hypersensitivity reactions to the iodine in the contrast material, exacerbation of pelvic inflammatory disease, granu¬ loma formation, venous and lymphatic intravasation, and pain. The uterine cavity also may be evaluated with hysteroscopy and ultrasonography using saline or Albunex (albumin micro¬ spheres) as a contrast medium. These techniques may be used when hysterosalpingography is contraindicated or for further study of a suspected abnormality.

ASSESSMENT OF THE MALE FACTOR Semen analysis remains the most accurate and widely used test to study male fertility. A small population of subfertile men with normal semen analyses has been identified, and new tests are being developed to assess the most critical element of sperm function: the ability to fertilize an ovum. Assessment of sperm morphology using the strict criteria of Kruger is more predic¬ tive of unsuspected male factor infertility; abnormalities are more often associated with failed in vitro fertilization and intrauterine insemination.24-25 The presence of antisperm anti¬ bodies as well as tests of the 24-hour survival of the sperm are important predictors of male fertility potential. Sperm binding to the zona pellucida can be evaluated with hemizona assay; failure to bind is predictive of poor success with in vitro fertili¬ zation and intrauterine inseminations.2-26

EVALUATION OF THE CERVICAL FACTOR

FIGURE 103-2. Endometrial biopsy, representative of day 24 of the menstrual cycle, shows periarteriolar and subcapsular pseudodecidual changes with stromal edema. If this specimen had been obtained the day before menstruation, it would be consistent with luteal-phase defi¬ ciency. However, to establish the diagnosis of luteal deficiency, the biopsy findings must be confirmed in two sequential cycles. (Hematox¬ ylin and eosin; xlOO)

Assessment of the cervical factor in infertility remains one of the most controversial areas of investigation. The postcoital test (Sims-Huhner test) traditionally is used. This test has undergone modification since its original description, and considerable variation is seen in its performance and interpretation. It usu¬ ally is performed at midcycle, as close to the time of ovulation as possible, within 8 hours of intercourse. A sample of cervical mucus is removed from the endocervix, with care taken to avoid vaginal contamination; the sample is placed on a slide for

1020

PART VII: ENDOCRINOLOGY OF THE FEMALE

FIGURE 103-3. Hysterosalpingograms. A, Normal hysterosalpingogram. (C, uterine cornu; io, internal os of cervix; eo, external os of cervix.) B, T-shaped cavity characteristic of intrauterine diethylstilbestrol expo¬ sure. C, Endometrial polyp in the right cornu (arrow). D, Nonpatent hydrosalpinx, left oviduct (arrow).

evaluation of its estrogen effect (gauged by the volume, cellularity, spinnbarkeit, and ferning pattern of the mucus) to con¬ firm the appropriateness of the timing of the test and is examined for the presence of motile sperm. Some women exhibit mucus receptiveness toward the sperm for only 1 day, with the mucus being relatively impermeable to the sperm at other times. A poor test result most often is due to performance of the test at a suboptimal time in the cycle. After the mucus has been evaluated, sperm are identified. Considerable debate continues over the criteria for the mini¬ mum number of motile sperm present in a normal postcoital test: estimates range from 1 to >20 per high-power field. One review cited little difference in fertility rates when 1 to 5 motile sperm were seen per high-power field compared with 11 to 20.27 Most sperm should demonstrate good forward motility; immobile sperm and rotary motion are abnormal.28 Tests with the latter results should be repeated; often the sperm are found to be normal on reexamination. Less frequently, these patterns are present in patients with sperm immobilizing or sperm agglutinating antibodies. Postcoital tests often are misleading. One study of fertile couples found abnormal postcoital tests (less than one sperm per high-power field) in 20%.29 In another study, six of eight women with abnormal postcoital tests (i.e., persistently show¬ ing no sperm) had motile and presumably normal sperm recov¬ ered from the peritoneal cavity at laparoscopy.30 In another study involving 355 infertile couples without severe defects in ovulation or seminal or tubal function, no correlation was found between the results of the postcoital test and subsequent pregnancy, with a minimum follow-up of 18 months.31 The overall poor positive and negative predictive value of the post¬ coital test severely limits its clinical utility. Although abnormal postcoital test results should be viewed with some skepticism, a normal study is helpful. It confirms that coital technique is appropriate and is associated with a more favorable prognosis. Sperm antibody testing may be indicated when the sperm are nonmotile or demonstrate shaking movement without for¬ ward progression and when significant sperm agglutination has been observed in multiple semen specimens after infection has been excluded. Sperm agglutinating and immobilizing antibod¬ ies have been identified, and a diverse literature exists regarding their role in infertility. An association appears to exist between the presence of serum agglutinating antibodies and infertility in men, but a similar association does not appear to exist in women.32 Sperm immobilizing antibodies in the serum and cer¬ vical mucus of infertile women also may be a reliable marker, because they are rarely detected in fertile controls.33,34 An immunobead-binding assay may provide a semiquantitative means of assessing and localizing antibodies on the sperm.35,36 The value and current role of sperm antibody testing remain controversial, because no specific antigen associated with immunologic infer¬

tility has been identified. Antibody-mediated infertility proba¬ bly occurs in 3 hours requires the use of a backup method for 2 days while continuing pill-taking. The efficacy of minipills is excellent among women older than age 40 and lactating women. In women older than 40 years, decreased fecundity contributes to the efficacy of the minipill, whereas in lactating women, the prolactin-induced suppression of ovulation contributes to its efficacy. Another rea¬ son that the minipill is an excellent method of birth control in lactating women is that it does not decrease milk volume and has no negative impact on infant growth or development.36-38 Side Effects. The most common side effect associated with the minipill is irregular uterine bleeding. Women using the minipill may have irregular bleeding, spotting, or amenor¬ rhea. Other side effects include acne and the development of functional ovarian follicular cysts. PROGESTIN-ONLY IMPLANTABLE CONTRACEPTIVES Norplant, a progestin-only implantable contraceptive, was first introduced in Chile in 1972 and in the United States in 1990. The Norplant system is comprised of six silastic rods, each 34mm long, filled with levonorgestrel. The semipermeable silastic rods allow for a slow release of levonorgestrel at an initial rate of 80 gg per day (equivalent to the amount in a progestin-only pill). After ~9 to 12 months of use, the rods release levonor¬ gestrel at -30 |ig per day. They maintain excellent contraceptive efficacy (99.7% per year) and are FDA approved for 5 years of continuous use. In the near future, Implanon, a one-rod/3-year implant system containing 3-keto-desogestrel, and Norplant II, a two-rod/3-year implant system containing levonorgestrel, will be released. Mechanism of Action. The principal mechanism by which progestin-only implants exert their contraceptive efficacy is by altering the cervical mucus and making it impenetrable to sperm. The continuous low levels of progestin also serve to suppress LH and prevent ovulation. Compared to cervical mucus changes, the prevention of ovulation is less reliable, and, as progestin levels decline over time, more ovulatory cycles are noted. With Norplant, in the first 2 years of use only 10% to 20% of cycles appear to be ovulatory, whereas in the fifth year of use, -45% of cycles appear to be ovulatory. However, even

cycles that appear to be ovulatory are often not completely nor¬ mal cycles. Women who have regular menstrual cycles on Nor¬ plant have been shown to have subnormal levels of LH, FSH, and progesterone.39-40 Progestin-only implants also induce changes in the endome¬ trial lining. These changes are likely responsible for the irregular uterine bleeding associated with implant use. With Norplant, the endometrial lining has been found to be hypotrophic with an increased microvascular density of capillaries that appear to be particularly fragile.41 The exact mechanism of these changes is unclear. Study has shown that postfertilization prevention of implantation on an unfavorable endometrial lining is not a mechanism for the contraceptive action of Norplant.42 Use of Implants. Contraceptive implants are inserted subdermally, in the upper inner arm, under local anesthesia using the provided trocar. Insertion is a simple procedure that usually takes 5 to 10 minutes. Special care must be taken to place the implants in the correct plane. Placement of all six implants in the same subdermal plane allows easy removal. Because the silastic rods are not biodegradable, after 5 years of use, or at the woman's request, the implants must be removed. Under local anesthesia, a small incision is made, and the implants can be removed either with finger pressure or with a pair of small hemostatic clamps. Removal of the implants can take from 5 to 60 minutes. The major advantage of one- and two-rod implant systems is faster and easier insertion and removal. Implant sys¬ tems using biodegradable capsules are in development. Absolute contraindications to progestin-only implants include undiagnosed vaginal bleeding, suspected or confirmed pregnancy, active liver disease or tumors, active thromboem¬ bolic disorders, and known or suspected breast cancer. Relative contraindications include severe acne, depression, vascular migraine headaches, and the concomitant use of medications that increase the hepatic metabolism of progestins—and, hence, decrease the efficacy of implants—such as phenytoin, carbamazepine, phenobarbital, and rifampin. Progestin-only implants may be very well suited for women with hypertension, diabetes, a history of cardiovascular disease (such as stroke, myocardial infarction, or prior deep venous thrombosis), gallbladder disease, hypercholesterolemia, or hyper¬ triglyceridemia. Implants are also appropriate contraception for heavy smokers, including women older than age 35 years. Side Effects. The most common side effect of progestinonly implants, and the most commonly cited reason for removal of Norplant, is irregular menstrual bleeding. In the first year of use, 68% of women using Norplant report menstrual problems.43 Of the women with these problems, 23% report increased bleed¬ ing, 16% report decreased bleeding or amenorrhea, and 29% report irregular bleeding or spotting.43 Over time, bleeding pat¬ terns tend to improve, and many women report regular men¬ strual cycles. The Norplant 5-year cumulative removal rate for bleeding problems is 17.5% 43 The cause of the irregular men¬ strual bleeding is not entirely clear. Under the influence of progestin-only contraception, the endometrial lining becomes hypotrophic, with an increased microvascular density of fragile capillaries.41 These fragile capillaries may be especially prone to bleeding. Varying levels of estrogen, produced by partially stim¬ ulated follicles, may also contribute to the irregular bleeding associated with progestin-only implants.39 Other side effects occurring in 10% to 16% of Norplant users in the first year of use include headache, acne, weight gain, leukorrhea, pelvic pain, vaginal fungal infections, genital pruritus, and reaction at the implantation site.43 The incidence of these side effects, with the exception of genital pruritus, decreases in later years. Of these side effects, only weight gain, mood changes, and headache lead to removal rates of >1%.43 After 5 years of use, 59% of U.S. women with implants gained weight. The mean 5-year weight gain for these women was 5.2 kg.43 Another complication of implant use is difficulty in remov¬ ing the rods. In a large U.S. study, 8% of Norplant removals

Ch. 104: Female Contraception were classified as difficult, and 3% were associated with adverse effects (including multiple incisions and a reaction to the local anesthetic).43 The amount of difficulty encountered and the time required for removal are related to the provider's skill and experience. New systems with fewer implant rods should minimize difficulty with insertion and removal. No adverse effects on fertility are seen after removal of the rods. Within 24 hours after removal, fertility returns to base¬ line.40 No consistent effects on blood pressure, lipoproteins, or coagulation have been noted. Also, because hypoestrogenemia does not occur, bone density is not affected. PROGESTIN-ONLY INJECTABLE CONTRACEPTIVES

1029

TABLE 104-6. Known and Potential Noncontraceptive Benefits of Depot Medroxyprogesterone Acetate KNOWN Decreased incidence of sickling events in women with sickle cell disease Rise in seizure threshold and improvement in seizure control in women with seizure disorders Decreased incidence of endometrial cancer Decreased menstrual volume and anemia Increased volume of breast milk in lactating women

POTENTIAL Decreased incidence of pelvic inflammatory disease

DMPA (Depo-Provera) is an injectable progestin-only contracep¬ tive. It was first introduced in the mid-1960s and, since then, has been used extensively around the world. In 1992 it was approved by the FDA for use in the United States. It is an easy-to-use, pri¬ vate, and very effective (99.7%) method of birth control. DMPA is given as an intramuscular injection every 3 months. Peak serum hormone levels occur shortly after injec¬ tion and then progressively fall, yet remain in the effective con¬ traceptive range, over the next 3 months. Mechanism of Action. As do other progestin-only contra¬ ceptives, DMPA exerts its contraceptive effects by thickening the cervical mucus, making it impenetrable to sperm. However, compared with Norplant, which provides continuous low lev¬ els of levonorgestrel, DMPA provides much higher levels of progestin. These high levels of progestin are effective at inhibit¬ ing the LH surge, and as a result DMPA is effective at inhibiting ovulation. Despite the fact that DMPA is more effective at inhibiting ovulation than Norplant, both have similar contra¬ ceptive efficacy. Although DMPA effectively inhibits the LH surge, it does not completely suppress FSH and, thus, stimulated follicles continue to make estrogen. Estrogen levels in DMPA users have been found to be approximately at the level found in the early follicular phase in a normal menstrual cycle.44 Use of Depot Medroxyprogesterone Acetate. DMPA is an aqueous solution of suspended crystals, given in a dose of 150 mg intramuscularly, either in the deltoid or gluteus maximus muscle. The first injection should be given within 5 days of the menstrual period to ensure the patient is not pregnant at that time. The next injection should be given 12 to 13 weeks after the first injection. If these guidelines are followed, ovulation is inhibited from the onset of use. If the patient is not within 5 days of the onset of her menstrual period, or is beyond 13 weeks after her last injection, a pregnancy test is indicated. If a sensitive pregnancy test is negative and no episodes of unprotected intercourse have occurred in the prior 2 weeks, the injection can be given. If any doubt exists as to whether unprotected intercourse has occurred within the 2 weeks before the negative pregnancy test, a backup method of birth control should be used, and a second pregnancy test given 2 weeks later. If the sensitive pregnancy test is still negative, the injection may be given. In these situations, a backup method of birth control should be used for the first 2 weeks of DMPA use, as ovulation may not be inhibited during this time. DMPA is contraindicated in women who are pregnant and those who have undiagnosed vaginal bleeding. Relative con¬ traindications include active liver disease, breast cancer, severe depression, severe cardiovascular disease, and a desire to con¬ ceive within 1 year. SIDE EFFECTS. The most common side effect associated with DMPA use is irregular menstrual bleeding.44"46 Within the first year of use, ~70% of women using DMPA have irregular bleeding.44 With continued use, a majority of women become amenorrheic. After 5 years of use, -80% of women are amenorrheic.47 The irregular bleeding associated with DMPA use is usually not excessive in quantity, but can be of increased frequency or duration. Although average hemoglobin levels rise in women

Decrease in symptoms associated with endometriosis Decreased incidence of ectopic pregnancy Halt in growth of uterine leiomyomas

using DMPA, frequent and prolonged bleeding are the most common reasons for discontinuation of DMPA.47 As many as 25% of new DMPA users discontinue this contraceptive in the first year of use due to frequent or prolonged bleeding.48 Other side effects reported with DMPA use include weight gain, depression, decreased libido, headaches, dizziness, abdominal pain, anxiety, and delayed return to fertility. Sys¬ temic levels of the drug may persist for 9 months after injection, and long infertile periods of up to 18 months may occur.49 The increased risk of breast cancer found in beagle dogs treated with DMPA has not been observed in humans using DMPA. A multinational study of breast cancer risk in women using DMPA has shown no increased risk50 (see Chap. 105). In cross-sectional studies, DMPA use has been associated with decreased bone mineral density.51-53 This loss in bone min¬ eral density has been shown to be reversible with discontinua¬ tion of DMPA.54 The long-term effect of a temporary loss of bone mineral density on osteoporosis and fractures later in life is unknown (see Chap. 105). NONCONTRACEPTIVE BENEFITS OF DEPOT MEDROXYPROGES¬ TERONE ACETATE. DMPA has been shown to have many non¬

contraceptive benefits (Table 104-6). Although DMPA is FDA approved only for use as a contraceptive and in the treatment of metastatic endometrial cancer, it has been shown to raise the seizure threshold and to improve seizure control in some women with seizure disorders, to decrease the incidence of sickling events in women with sickle cell disease, and to decrease the incidence of endometrial cancer. DMPA is also safe for use in lactating women. In contrast to COCs, DMPA causes an increased volume of breast milk in lactating women.

POSTCOITAL “EMERGENCY” CONTRACEPTIVES Postcoital contraception is an "emergency" aid that can be pro¬ vided for women who have experienced a single unprotected or inadequately protected act of intercourse within the previous 72 hours. The mechanism of action of postcoital contraception is unclear. Studies have shown that emergency contraceptive pills (ECs) both alter the endometrium and delay ovulation.55-58 Postcoital contraception also may prevent fertilization. Various regimens containing either an estrogen-progestin combination (combined ECs) or a progestin alone (progestin ECs) have been used with considerable success (Table 104-7). The first dose of a combined EC regimen should be given within 72 hours of unprotected intercourse, and the second dose should be given 12 hours later. The reported failure (pregnancy) rate is -2%. Women having unprotected intercourse during the second or third week of their menstrual cycle have an 8% chance of con¬ ceiving that cycle. Thus, postcoital contraception may decrease the risk of conception from 8% to 2%, a 75% decrease in risk.59-60 The principal side effects of combined ECs are nausea and vom¬ iting. The prophylactic use of an antiemetic 1 hour before each dose

1030

PART VII: ENDOCRINOLOGY OF THE FEMALE

TABLE 104-7. Oral Contraceptives Used for Emergency Contraception* Number of Doses

continue to use this method through the first year and -60% continue through the second year. The incidence of complica¬ tions differs with the device used, but overall morbidity is low. In the United States, the Cu-T380A and Progestasert are the only IUDs available.

Brand Name

Preparation

Tablets Per Dose and Color

Ovral

EE 50 pg + NG 0.50 mg

2 white

2

Preven

EE 50 pg + LNG 0.25 mg

2 light blue

2

USE OF THE INTRAUTERINE DEVICE

Lo/Ovral

EE 30 pg + NG 0.30 mg

4 white

2

Levlen

EE 30 pg + LNG 0.15 mg

4 light orange

2

Nordette

EE 30 pg + LNG 0.15 mg

4 light orange

2

Tri-Levlen

EE 30 pg + LNG 0.05, 0.075,0.125 mg

4 yellow

2

Triphasil

EE 30 pg + LNG 0.05, 0.075,0.125 mg

4 yellow

2

Alesse

EE 20 pg + LNG 0.10 mg

5 pink

2

NG 0.075 mg

20 yellow

2

The IUD is a safe, effective method of contraception for appro¬ priate candidates. Women who are at low risk for STIs and who are without menstrual dysfunction or anatomic distortion of the uterine cavity are ideally suited to IUD use. Because the IUD provides no protection against STIs, and may increase the severity of existing infections, a thorough screening of women for STI risk factors is mandatory before IUD insertion. Absolute contraindications to IUD insertion include a history of current, recent, or recurrent pelvic inflammatory disease, acute cervicitis, acute vaginitis, intrauterine pregnancy, allergy to copper (with copper IUDs), and immunosuppression. The relative con¬ traindications to IUD use include multiple sexual partners, nulligravid condition, menometrorrhagia, hypermenorrhea, severe dysmenorrhea, uterine abnormalities distorting the cavity, anti¬ coagulation therapy or a bleeding disorder, and valvular heart disease. Diabetes mellitus is not a contraindication to IUD use. Women with type 1 or type 2 diabetes mellitus (with no other contraindications to IUD use) may safely use this method of contraception.65-66 The IUD should be inserted during the last days of men¬ strual flow to ensure that the woman is not pregnant, and because the cervix is partially open at that time, allowing for easier insertion. An IUD may also be placed immediately after a first-trimester abortion without increased rates of expulsion or infection.67-68 In the postpartum period, or after a secondtrimester abortion, IUD insertion should be delayed 4 to 8 weeks because of the higher risk of IUD expulsion if performed sooner. Aside from an increased risk of expulsion, IUD inser¬ tion immediately postdelivery is not associated with an increased risk of complications.69

Ovrette

EE, ethinyl estradiol; NG, norgestrel; LNG levonorgestrel. •Combined pills: begin within 72 hours of unprotected intercourse; doses 12 hours apart. Progestin-only pills: begin within 48 hours of unprotected intercourse; doses 12 hours apart.

can significantly reduce these symptoms.60 Progestin ECs cause less nausea and vomiting. The only absolute contraindication to the use of postcoital contraception is confirmed or suspected ongoing pregnancy. Postcoital contraception with combined or progestin ECs does not terminate an ongoing pregnancy. No data are available on the safety of combined ECs in women with contraindications to estrogen. The short duration of use make significant complications unlikely; however, the progestinonly regimen has similar efficacy and should be considered for these women. In a woman planning to use an IUD for contraception, a cop¬ per IUD inserted within 5 days of unprotected intercourse is also a very effective method of emergency contraception (fail¬ ure rates of 2000 years. In addition to providing contra¬ ception, most barrier methods offer some protection against STIs. CONDOM The use of a male or female condom, if made of latex, protects against the transmission of bacteria and viruses, including human immunodeficiency virus (HIV).74 Between 1982 and 1995, condom use rose from 12% to 20% in the United States.1 This rise likely reflects efforts to prevent STIs, specifically HIV. Many couples use a condom both for contraception and to pre¬ vent STIs. Others use condoms for disease prevention in addi¬ tion to another, more effective contraception method.

1031

TABLE 104-8. Failure Rates of Different Techniques of Tubal Sterilization Type of Procedure

Number of Failures per 100 Women

Laparotomy (modified Pomeroy technique) Postpartum

0.75

Interval

2.0

Laparoscopy Unipolar electrocautery

0.75

Silastic bands

1.8

Bipolar electrocautery

2.5

Hulka clips

3.7

(From Petersen HB, Xia Z, Hughes JM, et al. The risk of pregnancy after tubal steril¬ ization: findings from the U.S. Collaborative Review of Sterilization. Am J Obstet Gynecol 1996; 174:1161.)

DIAPHRAGM The diaphragm is a round latex barrier that is placed in the vagina before intercourse. The spring-like edges of the diaphragm allow it to collapse to enable placement in the vagina. Once it is properly positioned in front of the cervix, the spring opens and keeps the diaphragm in place. The three basic types of diaphragms are the arcing spring, the coil spring, and the flat spring. Diaphragms come in sizes between 50 and 105 mm in diameter and must be individually fitted for each woman. They must be refitted after childbirth. Various clinical studies indicate typical use effectiveness rates ranging from 2 to 25 pregnancies per 100 woman-years. This broad range of contraceptive effectiveness is attributable to differences in the degree of motivation of the woman and to experience with the method. The principal contraindications to diaphragm use are ana¬ tomic factors causing poor fit and allergies to latex or spermi¬ cide. The use of a diaphragm plus spermicide provides prophylaxis against many STIs and has been associated with a decrease in cervical dysplasia (likely due to decreased spread of human papilloma virus). The principal complication of diaphragm use is recurrent cystitis, probably due to partial urethral blockage. If, despite adequate antibiotic treatment, the frequency of cystitis increases, another method of contraception should be considered. A serious cause of concern among diaphragm users is the reporting of several nonfatal cases of toxic shock syndrome. In all of these cases, however, the patients had left the diaphragm in place for long periods of time. Women should be carefully instructed never to leave the diaphragm in the vagina longer than 24 hours. SPERMICIDAL PREPARATIONS A great variety of spermicidal preparations are available as foams, creams, jellies, films, or suppositories. All spermicides con¬ tain a surfactant, which is responsible for the contraceptive effect. Surfactants have long-chain alkyl groups that easily penetrate the lipoprotein membrane of spermatozoa, increasing the permeabil¬ ity of the cell and leading to irreversible loss of motility. The vagina absorbs some of the spermicidal chemicals. No human studies have reported deleterious effects resulting from the absorption of surfactants. A double-blind, placebocontrolled trial has colposcopically evaluated the local effects of the spermicide nonoxynol-9 on the vagina and cervix and found no increase in epithelial disruption.75 Two studies have suggested a greater risk of congenital birth defects in the off¬ spring of women using vaginal spermicides,76'77 but other stud¬ ies have not shown any association between spermicide exposure and congenital malformations. Not only do spermicidal preparations provide a contracep¬ tive benefit, but the incidence of cervical gonorrhea, vaginal candidiasis, trichomoniasis, and genital infection with herpes

simplex virus are all decreased by these chemical agents. Clini¬ cal trials to assess the efficacy of nonoxynol-9 in preventing HIV transmission are in progress.75

FEMALE STERILIZATION Sterilization has become the most common method of fertility regulation in the United States. It is an elective procedure that offers women permanent, nonreversible contraception. Female sterilization is performed by ligating, excising, cauterizing, banding, or clipping portions of both fallopian tubes. The majority of these procedures are performed either laparoscopically or via laparotomy. Failure rates for the most commonly used techniques of tubal sterilization are listed in Table 104-8.78 The larger the amount of tube destroyed, the poorer the poten¬ tial for a surgical reversal, should the woman desire to have this latter procedure later. Although pregnancy after tubal sterilization is not common, when pregnancies do occur after sterilization, they are almost as likely to be ectopic as intrauterine pregnancies. The U.S. Col¬ laborative Review of Sterilization found a 10-year cumulative probability of pregnancy of 18.5 per 1000 procedures (for all types of sterilization procedures combined). They found a 10year cumulative probability of ectopic pregnancy of 7.3 per 1000 procedures. Women at highest risk for ectopic pregnancy after tubal sterilization are those sterilized under age 30 using the bipolar electrocautery technique.79 In the United States, deaths attributable to female sterilization are rare, with a case fatality rate of -1.5 per 100,000 procedures.80 This is markedly lower than the maternal mortality rate associ¬ ated with childbearing, which is ~10 per 100,000 live births. Deaths associated with female sterilization have resulted from complications of general anesthesia as well as from infection and hemorrhage. Although major complications are infrequent, one study foimd that 1.7% of laparoscopic sterilizations are compli¬ cated by penetrating injuries, injuries to major abdominal and pelvic vessels, and bowel burns.81 Complication rates are proba¬ bly lower among clinicians with more laparoscopy experience.

REFERENCES 1. Piccinino LJ, Mosher WD. Trends in contraceptive use in the United States: 1982-1995. Fam Plann Perspect 1998; 30:4. la. Westhoff C, Davis A. Tubal sterilization: focus on the U.S. experience. Fertil Steril 2000; 73:913. 2. Trussell J, Kowal D. The essentials of contraception. In: Hatcher RA, et al. Contraceptive technology, 17th ed. New York: Ardent Media, 1998:216. 3. Collins DC. Sex hormone receptor binding, progestin selectivity, and the new oral contraceptives. AmJ Obstet Gynecol 1994; 170:1508. 4. Goldzieher JW. Selected aspects of the pharmacokinetics and metabolism of ethinyl estrogens and their clinical implications. Am J Obstet Gynecol 1990; 163:318.

1032

PART VII: ENDOCRINOLOGY OF THE FEMALE

5. Mishell DR, Damey PD, Burkman RT, Sulak PJ. Practice guidelines for OC

38. World Health Organization, Special Programme of Research, Development, and Research Training in Human Reproduction, Task Force for Epidemiolog¬

selection: update. Dialogues Contracept 1997; 5(4):7.

ical Research on Reproductive Health. Progestogen-only contraceptives dur¬

6. Bagwell MA, Coker AL, Thompson SJ, et al. Primary infertility and oral

ing lactation. II. Infant development. Contraception 1994; 50:55.

contraceptive steroid use. Fertil Steril 1995; 63:1161. 7. Carpenter S, Neinstein LS. Weight gain in adolescent and young adult oral

39. Faundes A, Brache V, Tejada AS, et al. Ovulatory dysfunction during con¬ tinuous administration of low-dose levonorgestrel by subdermal implants.

contraceptive users. J Adolesc Health Care 1986; 7:342. and the risk of deep venous thromboembolic disease. Am J Epidemiol

Fertil Steril 1991; 56:27. 40. Walker DM, Damey PD. Implantable contraception. In: Sciarra JJ, ed. Gyne¬

1991; 133:32. 9. World Health Organization Collaborative Study of Cardiovascular Disease

41. Fraser IS, Hickey M, Song J. A comparison of mechanisms underlying dis¬

and Steroid Hormone Contraception. Ischaemic stroke and combined oral

turbances of bleeding caused by spontaneous dysfunctional uterine bleed¬

8. Gerstman BB, Piper JM, Tomita DK, et al. Oral contraceptive estrogen dose

cology and obstetrics. Philadelphia: Lippincott-Raven Publishers, 1998.

ing or hormonal contraception. Hum Reprod 1996; 11 (Suppl 2):165.

contraceptives: results of an international, multicentre, case-control study. Lancet 1996; 348:498. 10. World Health Organization Collaborative Study of Cardiovascular Disease and Steroid Hormone Contraception. Venous thromboembolic disease and

42. Segal SJ, Alvarez-Sanchez F, Brache V, et al. Norplant implants: the mecha¬ nism of contraceptive action. Fertil Steril 1991; 56:273. 43. Sivin I, Mishell DR, Damey PD, et al. Levonorgestrel capsule implants in the United States: a 5-year study. Obstet Gynecol 1998; 92:337.

combined oral contraceptives: results of international multicentre casecontrol study. Lancet 1995; 346:1575.

44. Speroff L, Damey PD. Injectable contraception. In: A clinical guide for con¬ traception, 2nd ed. Baltimore: Williams & Wilkins, 1996.

11. Pettiti DB, Sidney S, Bernstein A, et al. Stroke in users of low-dose oral con¬ traceptives. N Engl J Med 1996; 335:8.

45.

ology and management. Dialogues Contracept 1998; 5(5):1.

12. Sidney S, Pettiti DM, Quesenberry CP Jr, et al. Myocardial infarction in users of low-dose oral contraceptives. Obstet Gynecol 1996; 88:939.

46. Grimes DA, Wallach M. Injectable contraception. In: Modern contracep¬ tion: updates from the contraception report. Totowa, NJ: Emron, 1997.

13. Beral V, Hermon C, Kay C, et al. Mortality associated with oral contracep¬ tive use: 25 year follow up of cohort of 46,000 women from Royal College

47. Gardener JM, Mishell DR Jr. Analysis of bleeding patterns and resumption of fertility following discontinuation of a long-acting injectable contracep¬

of General Practitioners' oral contraception study. BMJ 1999; 318:96. 14. Croft P, Hannaford PC. Risk factors for acute myocardial infarction in women: evidence from the Royal College of General Practitioners' oral

tive. Fertil Steril 1970; 21:286. 48. World Health Organization. Clinical evaluation of the therapeutic effec¬ tiveness of ethinyl oestradiol and oestrone sulphate on prolonged bleeding

contraception study. BMJ 1989; 298:165.

in women using depot medroxyprogesterone acetate for contraception.

15. Vandenbroucke JP, Koster T, Briet E, et al. Increased risk of venous throm¬

Hum Reprod 1996; ll(Suppl 2):1.

bosis in oral-contraceptive users who are carriers of factor V Leiden muta¬ tion. Lancet 1994; 344:1453.

49. Kaunitz AM. Injectable depot medroxyprogesterone acetate contraception: an update for clinicians. Int J Fertil 1998; 43(2):73.

16. Schwingl PJ, Ory HW, Visness CM. Estimates of the risk of cardiovascular death attributable to low-dose oral contraceptives in the United States. Am

50. World Health Organization Collaborative Study of Neoplasia and Steroid Contraceptives. Breast cancer and depot-medroxyprogesterone acetate: a

I Obstet Gynecol 1999; 180:241.

multinational study. Lancet 1991; 338:833.

17. Burkman RT Jr. Benefits and risks of oral contraceptives: a reassessment. J Reprod Med 1991; 36(Suppl):217.

51. Cundy T, Evans M, Roberts H, et al. Bone density in women receiving depot medroxyprogesterone acetate for contraception. BMJ 1991; 303:13.

18. Larsson G, Milsom I, Lindstedt G, Rybo G. The influence of a low-dose combined oral contraceptive on menstrual blood loss and iron status. Con¬

52. Cromer BA, Blair JM, Mahan JD, et al. A prospective comparison of bone density in adolescent girls receiving depot medroxyprogesterone acetate

traception 1992; 46:327.

(Depo-Provera), levonorgestrel (Norplant) or oral contraceptives. J Pediatr

19. Wolner-Hanssen P, Eschenbach DA, Paavonen J, Kiviat N, et al. Decreased

1996; 129:671.

risk of symptomatic chlamydial pelvic inflammatory disease associated with oral contraceptive use. JAMA 1990; 263:54.

Darney PD, Klaisle CM. Contraception-associated menstrual problems: eti¬

53. Scholes D, Lacroix AZ, Ott SM, et al. Bone mineral density in women using

20. Barbone F, Austin H, Louv WC, Alexander WJ. A follow-up study of meth¬

depot-medroxyprogesterone acetate for contraception. Obstet Gynecol

ods of contraception, sexual activity, and rates of trichomoniasis, candidia¬

1999; 93:233. 54. Cundy T, Cornish J, Evans MC, et al. Recovery of bone density in women

sis, and bacterial vaginosis. Am J Obstet Gynecol 1990; 163:510.

who stop using medroxyprogesterone acetate. BMJ 1994; 308:247.

21. Brinton LA, Vessey MP, Flavel R, et al. Risk factors for benign breast dis¬ ease. Am J Epidemiol 1981; 113:203.

55. Swahn ML, Westlund P, Johannisson E, Bygdeman M. Effect of post-coital contraceptive methods on the endometrium and the menstrual cycle. Acta

22. Schlesselman JJ. Risk of endometrial cancer in relation to use of combined

Obstet Gynecol Scand 1996; 75:738.

oral contraceptives. A practitioner's guide to meta-analysis. Hum Reprod 1997; 12:1851. 23. Sherman ME, Sturgeon S, Brinton LA, et al. Risk factors and hormone lev¬ els in patients with serous and endometriod uterine carcinomas. Mod

56. Ling WY, Robichaud A, Zayid I, et al. Mode of action of DL-norgestrel and ethi¬ nyl estradiol combination in postcoital contraception. Fertil Steril 1979; 32:297. 57. Rowlands S, Kubba AA, Guillebaud J, Bounds W. A possible mechanism of action of danazol and an ethinyl estradiol/norgestrel combination used as

Pathol 1997; 10:963. 24. Mol BW, Ankum WM, Bossuyt PM, Van der Veen F. Contraception and the risk of ectopic pregnancy: a meta-analysis. Contraception 1995; 52:337.

postcoital contraceptive agents. Contraception 1986; 33:539. 58.

Ling WY, Wrixon W, Acorn T, et al. Mode of action of dl-norgestrel and

25. Hankinson SE, Colditz GA, Hunter DJ, et al. A quantitative assessment of

ethinyl estradiol combination in postcoital contraception. III. Effect of pre¬

oral contraceptive use and ovarian cancer. Obstet Gynecol 1992; 80:708.

ovulatory administration following the luteinizing hormone surge on ovarian steroidogenesis. Fertil Steril 1983; 40:631.

26. Lanes SF, Birmann B, Walker AM, Singer S. Oral contraceptive type and functional ovarian cysts. Am J Obstet Gynecol 1992; 166:956.

59. Trussell J, Ellertson C, Stewart F. The effectiveness of the Yuzpe regimen of postcoital contraception. Fam Plann Perspect 1996; 28(2):58.

27. Grimes DA, Godwin AJ, Rubin A, et al. Ovulation and follicular develop¬ ment associated with three low-dose oral contraceptives: a randomized

60.

cians and Gynecologists, December 1996 (no. 3).

28. Redmond GP, Olson WH, Lippman JS, et al. Norgestimate and ethinyl estradiol in the treatment of acne vulgaris: a randomized, placebo-

61. Trussell J, Ellertson C. Efficacy of emergency contraception. Fertil Control 62.

Rev 1995; 4(2):8. Lockwood CJ, Krikin G, Papp C, et al. Biological mechanisms underlying

62a.

Baird DT. Clinical uses of antiprogestagens. J Soc Gynecol Investig 2000;

controlled trial. Obstet Gynecol 1997; 89:615. 29. Kleerekoper M, Brienza RS, Schultz LR, Johnson CC. Oral contraceptive

RU486 clinical effects: inhibition. J Clin Endocrinol Metab 1994; 79:786.

use may protect against low bone mass. Arch Intern Med 1991; 151:1971. 30. Ness RB, Keder LM, Soper DE, et al. Oral contraception and the recogni¬ tion of endometritis. Am J Obstet Gynecol 1997; 176:580. 31. Cramer DW, Goldman M, Schiff I, et al. The relationship of tubal infertility

7(1 Suppl):S49. 63. Glasier A, Thong KJ, Dewar M, et al. Mifepristone (RU486) compared with high-dose estrogen and progestogen for emergency postcoital contracep¬

to barrier method and oral contraceptive use. JAMA 1987; 257:2446.

tion. N Engl J Med 1992; 237:1041.

32. Speroff L, Glass RH, Kase NG. Clinical gynecologic endocrinology and infertility, 5th ed. Baltimore: Williams & Wilkins, 1994:60.

63a. Harvey SM, Beckman LJ, Sherman C, Petitti D. Women's experience and sat¬ isfaction with emergency contraception. Fam Plann Perspective 1999; 31:237.

33. Speroff L, Damey PD. Oral contraception. In: A clinical guide for contra¬ ception, 2nd ed. Baltimore: Williams & Wilkins, 1996.

American College of Obstetricians and Gynecologists. Practice patterns. Emergency oral contraception. Washington: American College of Obstetri¬

controlled trial. Obstet Gynecol 1994; 83:29.

63b. Hewitt G, Cromer B. Update on adolescent contraception. Obstet Gynecol Clin North Am 2000; 27:143.

34. Damey PD. The androgenicity of progestins. Am J Med 1995; 98(Suppl 1 A):104. 35. World Health Organization Committee on Contraceptive Research for

64. Braken MB. Oral contraceptives and congenital malformations in off¬

Human Reproduction. A multicentered phase III comparative clinical trial

spring; a review and meta-analysis of the prospective studies. Obstet

of Mesigna, Cyclofem and Injectable No. 1 given monthly by intramuscu¬

Gynecol 1990; 76:552. 65. Kjos SL, Ballagh SA, La Cour M, et al. The copper T380A intrauterine device

lar injection to Chinese women. Contraception 1995; 51:167.

in women with type II diabetes mellitus. Obstet Gynecol 1994; 84:1006.

36. World Health Organization, Special Programme of Research, Develop¬ ment, and Research Training in Human Reproduction, Task Force on Oral Contraceptives. Effects of hormonal contraceptives on milk volume and infant growth. Contraception 1984; 30:505. 37. World Health Organization, Special Programme of Research, Development, and Research Training in Human Reproduction, Task Force for Epidemio¬ logical Research on Reproductive Health. Progestogen-only contraceptives during lactation. I. Infant growth. Contraception 1994; 50:35.

66.

Kimmerle R, Weiss R, Berger M, Kurz K. Effectiveness, safety and accept¬ ability of a copper intrauterine device (Cu Safe 300) in type I diabetic women. Diabetes Care 1993; 16:1227.

67. Speroff L, Damey PD. Intrauterine contraception. In: A clinical guide for contraception, 2nd ed. Baltimore: Williams & Wilkins, 1996. 68. Querido L, Retting E, Haspels AA. IUD insertion following induced abor¬ tion. Contraception 1985; 31:603.

Ch. 105: Complications and Side Effects of Steroidal Contraception 69. Chi I-C, Farr G. Postpartum IUD contraception—a review of an interna¬ tional experience. Adv Contracept 1989; 5:127. 70. Lee NC. The intrauterine device and pelvic inflammatory disease revisited: new results from the Women's Health Study. Obstet Gynecol 1988; 72:1. 71. Kessel E. Pelvic inflammatory disease with intrauterine device use: a reas¬ sessment. Fertil Steril 1989; 51:1. 72. Cramer DW. Tubal infertility and intrauterine device. N Engl J Med 1985312:941. 73.

Daling JR. Primary tubal infertility in relation to use of intrauterine device. N Engl J Med 1985; 312:937.

74. Laga M, Alary M, Nzila N, et al. Condom promotion, sexually transmitted disease treatment, and declining incidence of HIV-1 infection in female Zairian sex workers. Lancet 1994; 1:246. 75. Jick H. Vaginal 1245:1329.

spermicides

and

congenital

disorders.

JAMA

1981;

76. Cordero JF, Layde PM. Vaginal spermicides, chromosomal abnormalities and limb reduction defects. Fam Plann Perspect 1983; 15:16. 77. Van Damme L, Niruthisard S, Atisook R, et al. Safety evaluation of nonox-

1033

curves being equal. The metabolic degradation path of the two estrogens is identical, with the principal urinary metabolite being ethinyl estradiol glucuronide. The progestins used in hormonal contraceptives are absorbed rapidly, with peak concentrations reached in ~1 hour after a pill is taken. The acetate compounds attain peak concen¬ trations somewhat later because they must be deacetylated in the gastrointestinal tract before the progestin can be absorbed. All progestins are hydroxylated and conjugated in the liver before excretion primarily in the urine. Drugs that accelerate hepatic metabolism of steroids (rifampin, barbiturates, phenytoin, carbamazepine, fluconazole) can decrease the serum con¬ centrations of low-dose hormonal contraceptives, such as the minipills or Norplant, and decrease efficacy.

ynol-9 gel in women at low risk of HIV infection. AIDS 1998; 12:433. 78.

Petersen HB, Xia Z, Hughes JM, et al. The risk of pregnancy after tubal ster¬ ilization: findings from the U.S. Collaborative Review of Sterilization. Am J Obstet Gynecol 1996; 174:1161.

79. Petersen HB, Xia Z, Hughes JM, et al. The risk of ectopic pregnancy after tubal sterilization. N Engl J Med 1997; 336:762. 80. Escobedo LG, Petersen HB, Grubb GS, Franks AL. Case-fatality rates for tubal sterilization in U.S. hospitals, 1979 to 1980. Am J Obstet Gynecol 1989; 160:147. 81. DeStefano F. Complications of interval laparoscopic tubal sterilization. Obstet Gynecol 1983; 61:163.

CHAPTER

1 05

COMPLICATIONS AND SIDE EFFECTS OF STEROIDAL CONTRACEPTION ALISA B. GOLDBERG AND PHILIP DARNEY

All steroidal contraceptives are composed either of a progestin alone or a combination of an estrogen and a progestin. The side effects or complications observed with the use of hormonal contraception can be attributed either to the estrogen or proges¬ tin component. Understanding which effects are estrogen related and which are progestin related can help clinicians indi¬ vidualize hormonal contraceptive use for their patients.

PHARMACOLOGY Hormonal contraceptives are formulated from synthetic steroid 19-nortestosterone derivatives, which include norethindrone, norethindrone acetate, norethynodrel, ethynodiol diacetate, norgestrel, levonorgestrel (LN), desogestrel, gestodene, norgestimate, and dienogest. Exceptions are the injectable contraceptives (Depo-Provera, Lunelle), which use the progesterone derivative, depot medroxyprogesterone acetate (MPA). One of these progestins is combined with various dosages of an estrogen, either ethinyl estradiol or ethinyl estradiol-3-methyl ether (mestranol) in oral tablets and in some long-acting methods (injectables, vaginal rings, patches). The presence of a C17 ethinyl group on all synthetic estrogens and progestins enhances the oral activities of these agents by slowing their rapid hydroxylation and conjugation in the hepatic portal system.1-2 Ethinyl estradiol and mestranol are fairly well absorbed; -60% of the oral dose is recovered in the urine. In the liver, mestranol is demethylated to ethinyl estradiol. After oral administration, concentrations of both estrogens peak at 1 to 2 hours, with the areas under the plasma concentration time

BIOLOGIC POTENCIES OF CONTRACEPTIVE STEROIDS Various animal tissue responses (e.g., rat ventral prostate) have been used to assess the biopotency of contraceptive steroids. Much scientific criticism has been directed at these test systems, particularly at the extrapolation of dog and rodent data to humans. Specific steroid-receptor binding assays are now used. These allow in vitro comparisons of the androgenic, progestogenic, and estrogenic properties of sex steroids. In vivo, how¬ ever, these effects are modulated by endogenous sex steroids and their binding globulins, notably sex hormone-binding globulin (SHBG). Progestins differ in their bioactivities. This variation in bio¬ activity among different progestins is in large part due to modifications in the steroid structure that result in different receptor-binding affinities and different rates of metabolism. These factors require that different doses of each different progestin be used to achieve contraceptive efficacy. The admin¬ istration of estrogen together with a progestin in combined con¬ traceptives allows for use of a lower dose of progestin. The dose of progestin required for contraceptive efficacy in combined contraceptives is affected by the amount of estrogen adminis¬ tered. Because differences among progestin potencies are com¬ pensated for by dose adjustment, scales that attempt to correlate steroid dose with clinical effect are not useful.3

METABOLIC EFFECTS Pharmacologic doses of contraceptive hormones have wide¬ spread metabolic effects, but many of these are merely alter¬ ations in laboratory values without clinical significance. Nevertheless, some laboratory test alterations may reflect clini¬ cally significant metabolic changes. For example, changes in coagulation factors may predispose certain women to intravas¬ cular clotting, and changes in renin and angiotensin may affect blood pressure in a few users. Many of the metabolic alterations associated with steroid contraceptive use are attributable to the estrogenic component of the combination pill. These effects would not be expected with the use of progestin-only contra¬ ception. In contrast, the metabolic alterations caused by progestins, which would be expected in progestin-only contra¬ ceptive users, may be altered by the concomitant use of estro¬ gen.4 For example, the estrogen in combined oral contraceptives (COCs) raises triglycerides and high-density lipoprotein (HDL), whereas most progestins have the opposite effects.

CARBOHYDRATE METABOLISM Combined Oral Contraception. Early studies with highdose COCs showed impairment of glucose tolerance and increased insulin resistance. However, multiple studies of lowdose COCs have not shown a clinically significant impact of COCs on carbohydrate metabolism.5-6 Even women with a his¬ tory of gestational diabetes have not been found to be at addi-

1034

PART VII: ENDOCRINOLOGY OF THE FEMALE

tional risk of developing diabetes due to COC use/ 8 The small increase in insulin resistance seen with low-dose COC use may alter glucose metabolism in some women with overt diabetes mellitus; however, this effect has not been consistent among individual patients. Also, the use of COCs has not been found to increase the risk of development of nephropathy or retinopathy in patients with type 1 diabetes mellitus.9 Current consensus opinion is that healthy diabetic women with no end-organ com¬ plications of diabetes mellitus may safely use low-dose COC. The changes observed in carbohydrate metabolism with oral contraceptive use (increased insulin resistance and decreased glucose tolerance) are believed to be attributable almost entirely to the progestin component of combination pills, and are dose related. Ethinyl estradiol administered alone, even in high doses, does not cause glucose tolerance deterioration or abnormal insulin responses. Progestin-Only Contraceptives. Progestin-only oral con¬ traceptives, minipills or "POPs," may decrease carbohydrate tolerance in healthy women, but this effect is generally not clin¬ ically significant.10 Because they do not adversely affect breast milk volume or quality, POPs are often used by lactating women. Those containing norethindrone have been found to increase the risk of developing diabetes among high-risk breast-feeding women (obese Latinas with a history of gesta¬ tional diabetes; relative risk [RR] = 2.87, 95% confidence inter¬ val [Cl] = 1.57-5.27).8 The same study found no increase in the rate of development of diabetes on the basis of breast-feeding alone.8 The predominantly progestogenic state of lactation combined with POPs may be enough to cause significant glu¬ cose intolerance in women at high risk. It remains unknown whether POPs will have a similar diabetogenic effect on highrisk, nonlactating women. One study of non-breast-feeding, type 1 diabetic women using POPs containing lynestrenol found no change in insulin requirements.11 LN implant contraceptives (e.g., Norplant, Jadelle) have been shown to have no clinically significant effect on carbohy¬ drate metabolism in healthy women. In one study, 100 healthy women had a glucose-loading test before Norplant insertion, and then annually over 5 years of its use. The investigators found no significant changes in mean fasting serum glucose levels or in 2-hour postprandial serum glucose levels. The 1hour postprandial glucose levels were elevated from baseline in years 1 and 2 of Norplant use, but not in years 3 through 5. These elevations were not above the normal range.12 There is no evidence that LN implants increase the risk of developing dia¬ betes among lactating or nonlactating women at high risk, as POPs containing norethindrone may. Since depot MPA acetate injection (DMPA, Depo-Provera) results in higher serum concentrations of progestin at the beginning of each 3-month injection cycle, its effects on carbo¬ hydrate metabolism might differ from those of lower-dose POPs or continuous-release progestin implant systems. Studies have shown no deterioration in glucose tolerance in nondia¬ betic women using DMPA for contraception for short dura¬ tions; however, after 4 to 5 years of continuous use, some studies show an increase in glucose intolerance.11 Whether this is due to DMPA itself, or to associated weight gain, is unclear. DMPA is not contraindicated in diabetic women, and often is an excellent method of contraception for women with vascular disease; however, changes in glucose metabolism may occur.

LIPID METABOLISM Combined Oral Contraceptives. All estrogen-containing contraceptive pills increase serum triglyceride levels by an aver¬ age of -50%. The progestin-only pill has not been associated with such changes. The increased triglyceride levels are attributable mainly to an increase in very low-density lipoproteins (LDLs; see Chap. 162). Most low-dose COCs do not cause significant increases in mean serum cholesterol levels; however, high-dose

estrogen-progestin formulations can decrease HDL while increasing LDL.10 Although estrogens increase HDL levels and progestins decrease HDL levels, COCs may have various effects because of endogenous factors that modulate these effects. COCs containing the less androgenic progestins (desogestrel, dienogest, gestodene, and norgestimate) moderately elevate triglycerides (as does the estrogen component of all COCs), as well as total cholesterol, but the increase in total cho¬ lesterol is all in the HDL fraction. LDL levels fall, so that except for women with very high triglycerides (>450 mg/dL), less androgenic COCs can improve the lipoprotein profile.14"16 Whether this effect has consequences for cardiovascular health is undetermined. High-dose COCs can decrease HDL and increase LDL; however, they have not been associated with arteriosclerotic disease. In fact, the estrogen component of highdose COCs protects against plaque deposition despite the adverse lipid effects of high doses of progestins. Progestin-Only Contraceptives. Low-dose, sustainedrelease contraceptives (e.g., Norplant) do not perturb lipopro¬ tein metabolism. A study of more than 20 LN implant users followed for 5 years showed that modest changes in the choles¬ terol/HDL ratio were accounted for by weight gain and aging. The low serum concentrations of LN (0.3-0.5 ng/mL) had no persistent effect on lipoprotein metabolism (Population Coun¬ cil, data on file). Other smaller, shorter-term studies also failed to show significant lipoprotein effects of LN implants.121" With LN implant use, triglyceride levels fall slightly because no estrogen is administered, and endogenous estradiol production is modestly suppressed. The effect of DMPA on lipid metabolism is not clear. Some studies have suggested that DMPA has a negative impact on lipids, because it has been associated with decreased HDL cho¬ lesterol and increased total and LDL cholesterol levels.18 Other studies have not found DMPA to be associated with these nega¬ tive changes in lipids.19,20 Epidemiologic studies have not asso¬ ciated DMPA with cardiovascular disease.21

CARDIOVASCULAR EFFECTS THROMBOEMBOLIC DISEASE Combined Oral Contraceptives. Early epidemiologic studies22-24 indicated a four-fold to eight-fold increase in the risk of venous thromboembolism (VTE) among oral contraceptive users. As the estrogen content of COCs declined, reported risks of VTE fell to approximately three-fold, but the increased risk of VTE remains the greatest health threat that COCs pose.25,26,26a A large World Health Organization (WHO) international multicenter hospital-based case-control study found an increased risk of VTE with low-dose COC use (Europe: odds ratio [OR] = 4.24, 95% Cl 3.07-5.87; developing countries: OR = 3.02, 95% Cl 2.28^.00) 27 Further analysis of the data from this study,28 as well as others,26 has suggested an additional two-fold increase in VTE risk among users of COCs containing desogestrel and gestodene, compared to users of COCs contain¬ ing LN. The additional risk of VTE observed with the use of desogestrel- or gestodene-containing COCs is best explained by selection and prescribing bias.26 Women at highest risk of VTE, new starters, and women who have had complications on COCs in the past are most likely to have received COCs containing desogestrel or gestodene. In contrast, women who had been using COCs without complication for years comprise a popula¬ tion at low risk of complications, and were more likely to remain on older formulations. Whether selection and prescribing bias completely account for this observed effect is unknown. Genetic predisposition also plays a role in modifying the risk of VTE associated with oral contraceptive use. The factor V Leiden mutation is a point mutation that results in resistance to the anticoagulant effects of activated protein C. The factor V

Ch. 105: Complications and Side Effects of Steroidal Contraception Leiden mutation is estimated to affect 4.4% of Europeans and 0.6% of Asians, and is extremely rare (or nonexistent) in populations from Africa and Southeast Asia.29 Women with the factor V Leiden mutation have an eight-fold increased risk of VTE (RR = 7.9, 95% Cl 3.2-19.4), compared to women without the mutation.30 When a woman with the factor V Leiden mutation uses COCs, her base¬ line risk of VTE increases four-fold, and her overall risk of VTE is 35 times greater (RR = 34.7,95% Cl 7.8-154) than for women with¬ out the mutation who are not using COCs.30 Screening the general population for the factor V Leiden mutation is currently not rec¬ ommended; however, women who are known to have the muta¬ tion or a strong family history of thromboembolic disease should avoid COCs. Other thrombophilic disorders, such as protein C or S deficiency, also increase the risk of VTE with COC use. The increased risk of VTE with COCs is largely due to the estrogen component. Progestin-only contraceptives do not appear to confer an increased risk of VTE.21 Therefore, POPs, implants, and injectables are often reasonable contraceptive choices for women at high risk of VTE.

MYOCARDIAL INFARCTION Combined Oral Contraceptives. Several early epidemio¬ logic studies of COCs containing 50 |ig estrogen showed an increased risk of myocardial infarction (MI).31 Much of the observed increased risk of MI among COC users was actually due to the increased incidence of smoking and hypertension among COC users in the past. More recent studies of low-dose COCs have not revealed an increased risk of MI among nonhypertensive COC users who do not smoke.32'33 Similarly, current or past use of COCs has not been associated with an increased risk of mortality from MI.34 Among women who smoke and use COCs, the RR of MI has been reported to range from 3.5 (1.3-9.5) for those who smoke 15 ciga¬ rettes per day32 This risk is modified by age. For women younger than age 35 years, the overall risk of dying from all cardiovascular diseases due to COC use and smoking is exceedingly small (3.3 per 100,000 for smokers vs. 0.65 per 100,000 for nonsmokers).35 Therefore, younger women who smoke can use COCs with little risk of cardiovascular complications. For women older than age 35 years using COCs, the risk of cardiovascular death among smok¬ ers is 29.4 per 100,000 compared to 6.21 per 100,000 for nonsmok¬ ers. This level of risk is unacceptably high, and COC use is contraindicated in women older than age 35 years who smoke.35 Progestin-Only Contraceptives. There does not appear to be any increased risk of MI with the use of progestin-only methods compared to nonhormonal methods (pills: RR = 0.98 [0.16-5.97]; injectables: RR = 0.66 [0.07-6.00]).21

CEREBROVASCULAR ACCIDENTS Combined Oral Contraceptives. The occurrence of stroke in young women is rare, with an estimated incidence of 11.3 cases per 100,000 woman-years of observation.36 In the absence of smoking and hypertension, low-dose COC users do not have an increased risk of either hemorrhagic or thrombotic stroke.36-39 Both smoking and hypertension appear to have a synergistic effect when combined with COC use to increase the risk of stroke.36'38'39 In WHO studies,38'39 smoking COC users had a seven-fold increased risk for ischemic stroke (RR = 7.2, 95% Cl 3.23-16.1) and a three-fold increased risk for hemorrhagic stroke (RJR = 3.1, 95% Cl 1.65-5.83) when compared to non¬ smoking, non-COC users. In the same studies, hypertensive women using COCs had a ten-fold increased risk for ischemic stroke (RR = 10.7,95% Cl 2.04-56.6) and for hemorrhagic stroke (RR = 10.3, 95% Cl 3.27-32.3) as compared to nonhypertensive, non-COC users. In these studies, the risk of stroke among COC users with a history of hypertension was double the risk for hypertensive women not using COCs; however, approximately half the participants were using high-dose COCs.38'39

1035

Progestin-Only Contraception. There is no increased risk of stroke among nonsmoking, nonhypertensive women using progestin-only contraception.21 Smokers have a baseline increased risk of stroke, but this risk is not altered with progestin-only con¬ traceptive use.21 It is unclear whether progestin-only contraception increases the risk of stroke among hypertensive women; limited data suggest that this might be the case.21

HYPERTENSION Combined Oral Contraceptives. High-dose COCs have been found to induce hypertension in ~5% of users.40 Low-dose COCs also can induce hypertension, although the risk is much lower. One study estimated that low-dose COCs induce hyper¬ tension in 0.42% of users (41.5 cases per 10,000 person-years).40 The effect of COCs on blood pressure appears to be short-lived and reverses within 3 months after discontinuation of pills. The pathophysiologic mechanism for the oral contraceptiveinduced hypertensive effect involves the renin-angiotensin sys¬ tem. In most women, the COC-induced increase in angiotensinogen is physiologically compensated for by a decrease in renin secretion, and normal blood pressure is maintained. Some women, however, do not make the necessary physiologic adjustments, and hypertension results. There are no good pre¬ dictors of which patients will develop hypertension on COCs; therefore, all patients should have their blood pressure checked annually while on COCs. A history of pregnancy-induced hypertension is not associated with an increased risk of COCinduced hypertension.41 It is likely that both the estrogen and progestin components of COCs can alter blood pressure. Progestin-Only Contraceptives. Although past studies of high-dose COCs suggested that the progestin component was primarily responsible for the observed elevations in blood pres¬ sure, multiple studies of currently used POPs, implants, and injectables have not revealed an increased incidence of hyper¬ tension with these methods of contraception. Hypertensive women may use progestin-only contraception,42 although their blood pressure should be closely followed, and if uncontrolla¬ ble elevations result the method should be discontinued and nonhormonal methods considered.

EFFECTS ON THE BREASTS BENIGN BREAST CHANGES Combined Oral Contraceptives. The estrogen compo¬ nent of COCs can cause breast fullness and tenderness. Although the fullness often remains as long as COCs are taken, the tenderness usually resolves after a few months of use. If the tenderness persists, one should consider switching the patient to a COC containing a lower dose of estrogen. The incidence of benign breast disease (fibrocystic changes and fibroadenomas) is decreased among women who have used high-dose COCs for more than 2 years.43 Women using high-dose COCs for more than 6 years have an approximately 50% reduction in benign breast disease. Low-dose COCs may have an attenuated benefit. The quantity and quality of milk produced during lactation have been found to be decreased with COC use.44 Similarly, women using COCs have been found to breast-feed for shorter lengths of time postpartum. Both estrogens and progestins are secreted in the milk, and although these steroids decrease the levels of both proteins and fats in human milk, no adverse effects on the growth or development of infants have been iden¬ tified.45 Although COC use during lactation is not harmful to infants, it is not recommended because it makes breast-feeding more difficult. Progestin-Only Contraception. LN implants (Norplant) have been associated with breast tenderness in 11% of users in a

1036

PART VII: ENDOCRINOLOGY OF THE FEMALE

large U.S. study.46 The tenderness may be due to temporarily elevated follicular estradiol levels in women with lower LN serum concentrations and usually resolves over time. After excluding pregnancy as a possible cause of the mastalgia, reas¬ surance is usually all that is necessary. POPs and DMPA are rarely associated with breast complaints.

BREAST CANCER Combined Oral Contraceptives. For many years a great deal of controversy surrounded the issue of breast cancer risk in COC users. Some epidemiologic studies have shown no increased risk of breast cancer with COC use,47^19 whereas oth¬ ers have shown an increased risk of breast cancer in women using COCs before the age of 25 years.50-53 In 1996, a metaanalysis was performed using the data from 54 epidemiologic studies conducted in 25 countries.54 This study analyzed data for >53,000 women with breast cancer and >100,000 controls. They found that women who are currently using COCs have a small increase (RR = 1.24, Cl 1.15-1.33) in the risk of breast cancer as compared to nonusers. This risk remains slightly elevated within the first 10 years after discon¬ tinuation of use (1-4 years after stopping: RR = 1.16, Cl 1.081.23; 5-9 years after stopping: RR = 1.07, Cl 1.02-1.13). Beyond 10 years after discontinuation of use, there is no increased risk of breast cancer (10 or more years after stopping: RR = 1.01, Cl 0.96-1.05). They found no significant effect of duration of use, age at first use, or the type of hormone contained in the COC on breast cancer risk. In this study, although women currently using COCs were at increased risk of a new diagnosis of breast cancer, their tumors were significantly more likely to be confined to the breast at diagnosis than were those of nonusers (RR = 0.89 for spread to lymph nodes at diagnosis; RR = 0.70 for distant spread at diag¬ nosis). Whether these effects are due to earlier diagnosis in cur¬ rent or recent COC users or due to a pathophysiologic effect of COCs on breast cancer remains unclear. The risk of breast can¬ cer may be greater for younger than for older women.543 (See also Chaps. 222 and 223.) Progestin-Only Contraceptives. DMPA was initially found to cause malignant mammary tumors in beagle dogs. This find¬ ing created great fear as to whether DMPA would have the same effect in humans. Two large case-controlled studies, and one pooled analysis of these studies, have addressed this con¬ cern and have found no increased risk of breast cancer in everusers of DMPA as compared to never-users.55-57 The pooled analysis did show a slightly increased risk of breast cancer in DMPA users within the first 5 years of use (pooled analysis: RR = 2.0, Cl 1.5-2.8).57 Whether this is due to increased surveillance in DMPA users or stimulation of preexisting tumors is unclear. Two of the studies showed no increase in breast cancer risk with increasing duration of use.55,57 The third study showed an increase in breast cancer risk with >6 years of DMPA use only in those women who began using DMPA before age 25 years (RR = 4.2, Cl 1.1-16.2).56 Based on a few studies, POPs do not appear to increase breast cancer risk.50'58 To date, there are no epidemiologic stud¬ ies evaluating the effect of LN implants on breast cancer risk.

EFFECTS ON THE REPRODUCTIVE TRACT

Progestin-Only Contraceptives. An increased incidence of follicular cysts has been noted with LN implant (Norplant) use. The continuous low level of LN allows follicles to develop, and follicular cysts often result.603 These cysts only need to be evaluated sonographically or laparoscopically if they become large and painful. Sinjilar to LN implants, the low dose of progestin in POPs allows follicles to develop and follicular cysts to form. However, one large cohort study evaluating ovar¬ ian cysts in COC users also looked at progestin-only pill users, and found no cases of follicular cysts in 219 person-months of observation.59 In contrast to POPs and implants, DMPA effec¬ tively suppresses follicular development and ovulation; thus, follicular cysts are rare among DMPA users. OVARIAN CANCER Combined Oral Contraceptives. Women using COCs have a markedly reduced risk of ovarian cancer, with increasing duration of use increasing the protective effect.61-63 A 10% decrease in risk is noted after 1 year of COC use, and a ~50% decrease in risk is achieved after 5 years of use.62 This protec¬ tive effect has been found to extend to women at highest risk of ovarian cancer, including women with BRCA-1 and BRCA-2 mutations64 (see also Chap. 223). Progestin-Only Contraception. DMPA is probably asso¬ ciated with a slightly decreased risk of epithelial ovarian cancer, given that it effectively inhibits ovulation; however, studies have been unable to detect a difference in ovarian cancer risk largely because of the high parity of DMPA users.65 There are no epidemiologic data evaluating the effects of POPs or implants on ovarian cancer risk.

ENDOMETRIAL EFFECTS MENSTRUAL CHANGES In the majority of COC users, menses are regular, and become shorter in duration, lighter in flow, and associated with less dysmenorrhea. Progestin-only contraceptives are usually asso¬ ciated with disruption of the menstrual cycle, but no overall increase in menstrual blood loss. Menstrual irregularity is one of the leading causes of dissatisfaction with progestin-only methods. (See Chap. 104 for a complete discussion of menstrual changes with hormonal contraceptives.) ENDOMETRIAL CANCER Combined Oral Contraceptives. Multiple studies indi¬ cate that the risk of endometrial cancer is reduced among users of COCs.66'67 Longer duration of use is associated with increased protection against endometrial cancer. A metaanaly¬ sis found that women using COCs for 4 years had a 56% decreased risk of endometrial cancer; after 8 years of use, a woman's risk was reduced by 67%, and after 12 years of use, risk was reduced by 72%.68 Although the greatest risk reduction is observed for women who are currently using COCs, even 20 years after discontinuation, the risk of endometrial cancer is -50% lower in ever-users than never-users (see also Chap. 223). Progestin-Only Contraceptives. DMPA use has been associated with a decreased risk of endometrial cancer.69 Although epidemiologic studies have not yet shown a decreased risk of endometrial cancer with POPs or LN implant use, given the protective effect of progestin on the endometrium, these agents may decrease endometrial cancer risk as well.

OVARIAN EFFECTS CERVICAL EFFECTS OVARIAN CYSTS Combined Oral Contraceptives. High-dose COCs have been shown to decrease the incidence of functional ovarian cysts.59 Low-dose COCs have not demonstrated a similar effect, although an attenuated effect may exist.60

CERVICAL CANCER Combined Oral Contraceptives. The relation of oral con¬ traceptive use to cervical dysplasia, carcinoma in situ, and inva¬ sive squamous cell cervical cancer is unclear, since some studies

Ch. 105: Complications and Side Effects of Steroidal Contraception

show positive relations between cervical dysplasia and con¬ tinuing oral contraceptive use, whereas other studies report no relation.70-71 Cervical dysplasia, carcinoma in situ, and squa¬ mous cell carcinoma are thought to be at least partly of viral origin (human papilloma virus). Consequently, the confound¬ ing factors of coitus at early age, multiple sexual partners, sexu¬ ally transmittable diseases, and oral contraceptive use are difficult to decipher. Similarly, women using COCs are subject to increased surveillance as compared to nonusers. Increased surveillance results in increased detection of cervical squamous cell disease, and although the association between COC use and cervical squamous cell disease may be real, it may reflect screening bias or confounding (see Chap. 223). Nonetheless, annual Papanicolaou (Pap) smear screening should be recom¬ mended for all women taking COCs, and women at highest risk because of multiple sexual partners or a history of sexually transmitted diseases should be screened twice a year. There is convincing evidence suggesting a relationship between COC use and adenocarcinoma of the cervix.72 Data from the Surveillance, Epidemiology and End Results (SEER) tumor registry for Los Angeles found ever-users of COCs to have an RR of 2.1 of adenocarcinoma of the cervix, compared to never-users (Cl 1.1-3.8). With COC use of >12 years' duration, this rose to a RR of 4.4 for ever-users (Cl 1.8-10.8). This associa¬ tion may be mediated directly via estrogen or progestin recep¬ tors on endocervical cells, or may be explained by the increased incidence of ectropion in oral contraceptive users. Ectropion results in exposure of endocervical cells to the vagina, which may result in increased exposure to carcinogens. Progestin-Only Contraceptives. DMPA does not appear to have an independent effect on cervical cancer or dysplasia. One study found an increased risk of dysplasia among DMPA users; however, this increased risk was attributable to known risk factors for cervical dysplasia among DMPA users.73 A WHO case-control study found a slightly higher risk of carci¬ noma in situ in DMPA users.74 Although this may be a real find¬ ing, it may reflect confounding or screening biases. No epidemiologic data yet exist evaluating the effects of POPs and implants on cervical cancer risk. When these data become avail¬ able, it is likely that similar issues of confounding and screen¬ ing bias will make interpretation difficult.

EFFECTS ON THE GASTROINTESTINAL TRACT LIVER AND BILIARY TREE EFFECTS Combined Oral Contraception. COCs have a variety of effects on the liver. Estrogen influences the hepatic synthesis of DNA, RNA, enzymes, plasma proteins, lipids, and lipopro¬ teins. It also influences the hepatic metabolism of carbohy¬ drates and intracellular enzyme activity. Progestins have less, if any, effect on the liver. Although the liver is affected in a variety of ways by COCs, many of these changes have proved to be of no clinical significance, and others remain incompletely under¬ stood. Virtually all phase II and phase III studies of COCs have evaluated the effects of COCs on liver function tests, and have shown no effect.75 Some studies have shown an increased inci¬ dence of gallbladder disease and gallstones among current COC users.76 Other studies77 and a metaanalysis78 of the effect of COCs on biliary disease showed that COCs initially did increase the risk of biliary disease (RR = 1.36, 95% Cl 1.15— 1.62),78 but over time the risk returns to baseline. This suggests that, under the influence of COCs, susceptible (or asymptom¬ atic) women become symptomatic from biliary disease shortly after starting the pill, whereas nonsusceptible women do not develop biliary disease over time with continued use. The mechanism by which estrogen has its cholestatic effects79 is incompletely understood. Under the influence of estrogen, bile becomes increasingly saturated with choles-

1037

terol.80 This effect is probably secondary to elevated cholesterol concentrations in the bowel caused by altered cholesterol and lipid metabolism dependent on estrogen dose. Given these effects, women with active hepatitis, jaundice, or cholestasis should avoid COCs. Women with a past history of hepatitis can safely be given COCs. Progestin-Only Contraceptives. POPs, implants, and injectables do not alter liver function tests.81'82 Women with a past history of liver disease may safely use these methods. Whether women with active hepatitis or cirrhosis should use progestinonly methods is controversial, and the decision should be strongly influenced by the likelihood of pregnancy with other methods. Clearly, nonhormonal methods are safest for these women; however, progestin-only methods are less likely to have an adverse effect on their liver disease than either COCs or pregnancy.83 It is unlikely that progestin-only methods increase the risk of gallbladder disease.83

LIVER TUMORS Combined Oral Contraceptives. The use of COCs has been associated with an increased risk of hepatocellular ade¬ noma, and risk may increase with longer duration of use.84'85 Although hepatocellular adenomas are benign tumors, they may rupture, causing hemorrhage and death. Several case-control studies have shown an increased inci¬ dence of hepatocellular carcinoma among COC users,86 whereas other similar studies have not demonstrated the same effect.85 A large population-based study evaluated trends in the inci¬ dence of primary liver cancer and concomitant oral contracep¬ tive use in three countries, and found no association of oral contraceptive use with primary liver cancer.87 Progestin-Only Contraceptives. The WHO found no association between DMPA use and liver cancer.69 There are no data linking POPs or implants to benign or malignant liver tumors.

EFFECTS ON BONE MINERAL DENSITY Estrogen-replacement ther¬ apy has been proven to prevent bone loss and fractures in post¬ menopausal women. Similarly, women with a history of COC use are less likely to have low bone mineral density later in life (RR = 0.35, 95% Cl 0.2-0.5).88 The effect of COCs on increasing bone mineral density appears to be related to duration of use.88 As the first large population of COC users pass into menopause, future epidemiologic studies will determine whether women with a history of COC use have fewer fractures than women who never used COCs. It also remains to be seen if a past history of COC use is protective against osteoporosis in the presence and absence of postmenopausal estrogen-replacement therapy. Progestin-Only Contraceptives. In cross-sectional studies, DMPA use has been associated with a decrease in bone mineral density.89"913 This effect appears to be greater with a longer dura¬ tion of use and among young women (ages 18-21).91 This decrease in bone mineral density is likely the result of decreased endogenous estrogen secretion due to the suppression of follicu¬ lar development by DMPA. With discontinuation of DMPA, bone mineral density recovers92; however, the long-term effects of a temporary loss in bone mineral density remain unknown. Since POPs and implants do not completely suppress follicu¬ lar development, endogenous estrogen secretion remains within tire normal premenopausal range93; it is unlikely that progestinonly pills or implants adversely affect bone mineral density. Different progestins also might directly affect bone mineral density differently, hr vitro, osteoblasts have been found to have both estrogen and progesterone receptors.94 hr other cell lines, the nortestosterone derivatives (i.e., norethindrone, norgestrel) have been shown to stimulate the growth of estrogen-receptor-positive Combined Oral Contraceptives.

1038

PART VII: ENDOCRINOLOGY OF THE FEMALE

cells, whereas MPA did not.95 Perhaps the nortestosterone group of progestins has an estrogenic effect on bone that MPA does not.

COMMON MINOR SIDE EFFECTS Other common side effects of hormonal contraception include weight gain,96 nausea, headaches, skin changes, and changes in mood and libido. Although these side effects are usually not dangerous, they often limit the acceptability of a contraceptive method. These side effects are discussed in Chapter 104.

REFERENCES

•

1. Goldzieher JW. Selected aspects of the pharmacokinetics and metabolism of ethinyl estrogens and their clinical implications. Am J Obstet Gynecol 1990; 163:318. 2. Collins DC. Sex hormone receptor binding, progestin selectivity, and the new oral contraceptives. Am J Obstet Gynecol 1994; 170:1508. 3. Upmalis D, Phillip A. Receptor binding and in vivo activities of the new progestins. J Soc Obstet Gynecol Can 1991; 13(Suppl):35. 4. Krauss RM, Burkman RT Jr. The metabolic impact of oral contraceptives. Am J Obstet Gynecol 1992; 167:1177. 5. Rimm EB, Manson JE, Stampfer MJ, et al. Oral contraceptive use and the risk of type 2 (non-insulin dependent) diabetes mellitus in a large prospec¬ tive study of women. Diabetologia 1992; 35:967. 6. Duffy TJ, Ray R. Oral contraceptive use: prospective follow-up of women with suspected glucose intolerance. Contraception 1984; 30:197. 7. Kjos SL, Shoupe D, Douyan S, et al. Effect of low-dose oral contraceptives on carbohydrate and lipid metabolism in women with recent gestational diabetes: results of a controlled, randomized, prospective study. Am J Obstet Gynecol 1990; 163:1822. 8. Kjos SL, Peters RK, Xiang A, et al. Contraception and the risk of type 2 dia¬ betes mellitus in Latina women with prior gestational diabetes mellitus. JAMA 1998; 280:533. 9. Garg SK, Chase HP, Marshall G, et al. Oral contraceptives and renal and retinal complications in young women with insulin-dependent diabetes mellitus. JAMA 1994; 271:1099. 10. Godsland IF, Crook D, Simpson R, et al. The effects of different formula¬ tions of oral contraceptive agents on lipid and carbohydrate metabolism. N Engl J Med 1990; 323:1375. 11. Radberg T, Gustafson A, Skryten A, Karlsson K. Oral contraception in dia¬ betic women: a cross-over study on serum and high density lipoprotein (HDL) lipids and diabetes control during progestogen and combined estro¬ gen/progestogen contraception. Horm Metab Res 1982; 14:61. 12. Singh K, Viegas OAC, Loke D, Ratnam SS. Effect of Norplant implants on liver, lipid and carbohydrate metabolism. Contraception 1992; 45:141. 13. Liew DF, Ng CS, Yong YM, Ratnam SS. Long-term effects of Depo-Provera on carbohydrate and lipid metabolism. Contraception 1985; 31:51. 14. Speroff L, De Cherney A. Evaluation of a new generation of oral contracep¬ tives. Obstet Gynecol 1993; 81:1034. 15. Kloosterboer HJ, Vonk-Noordegraaf CA, Turpijn EW. Selectivity in progesterone and androgen receptor binding of progestins in oral contraceptives. Contraception 1988; 38:325. 16. Petersen KR, Skouby SO, Pedersen RG. Desogestrel and gestadene in oral contraceptives: 12 months assessment of carbohydrate and lipoprotein metabolism. Obstet Gynecol 1991; 78:666. 17. Viegas OAC, Singh K, Liew D, et al. The effects of Norplant on clinical chemistry in Singaporean acceptors after 1 year of use: metabolic changes. Contraception 1988; 38:79. 18. World Health Organization. A multicentre comparative study of serum lip¬ ids and apolipoproteins in long-term users of DMPA and a control group of IUD users. Contraception 1993; 47:177. 19. Mainwaring R, Hales HA, Stevenson K, et al. Metabolic parameter, bleed¬ ing and weight changes in U.S. women using progestin only contracep¬ tives. Contraception 1995; 51:149. 20. Garza-Flores J, De la Cruz DL, Valles de Bourges V, et al. Long-term effects of depot medroxyprogesterone acetate on lipoprotein metabolism. Contra¬ ception 1991; 44:61. 21. World Health Organization Collaborative Study of Cardiovascular Disease and Steroid Hormone Contraception. Cardiovascular disease and use of oral and injectable progestogen-only contraceptives and combined inject¬ able contraceptives. Contraception 1998; 57:315. 22. Royal College of General Practitioners. Oral contraceptives and health: interim report. New York: Pitman, 1974. 23. Collaborative Group for the Study of Stroke in Young Women. Oral contra¬ ception and increased risk of cerebral ischemia or thrombosis. N Engl J Med 1973; 288:871. 24. Collaborative Group for the Study of Stroke in Young Women. Oral contracep¬ tives and stroke in young women: associated risk factors. JAMA 1975; 231:718. 25. Gerstman BB, Piper JM, Tomita DK, et al. Oral contraceptive estrogen dose and the risk of deep venous thromboembolic disease. Am J Epidemiol 1991; 133:32. 26. Lewis MA, Heinemann LAJ, MacRae KD, et al. The increased risk of

venous thromboembolism and the use of third generation progestagens: role of bias in observational research. Contraception 1996; 54:5. 26a. Editorial. Oral contraceptives and cardiovascular risk. Drug Ther Bull 2000; 38:1. 27. World Health Organization Collaborative Study of Cardiovascular Disease and Steroid Hormone Contraception. Venous thromboembolic disease and combined oral contraceptives: results of international multicentre case-con¬ trol study. Lancet 1995; 346:1575. 28. World Health Organization Collaborative Study of Cardiovascular Disease and Steroid Hormone Contraception. Effect of different progestagens in low oestrogen oral contraceptives on venous thromboembolic disease. Lancet 1995; 346:1582. 29. Rees DC, Cox M, Clegg JB. World distribution of factor V Leiden. Lancet 1995; 346:1133. 30. Vandenbrouke JP, Koster T, Briet E, et al. Increased risk of venous thrombo¬ sis in oral-contraceptive users who are carriers of factor V Leiden mutation. Lancet 1994; 344:1453. 31. Pettiti DB, Sidney S, Quesenberry CP. Oral contraceptive use and myocar¬ dial infarction. Contraception 1998; 57:143. 32. Croft P, Hannaford PC. Risk factors for acute myocardial infarction in women: evidence from the Royal College of General Practitioners' oral contraception study. Br Med J 1989; 298:165. 33. Sidney S, Pettiti DB, Quesenberry CP, et al. Myocardial infarction in users of low-dose oral contraceptives. Obstet Gynecol 1996; 88:939. 34. Beral V, Hermon C, Kay C, et al. Mortality associated with oral contracep¬ tive use: 25 year follow up of cohort of 46,000 women from Royal College of General Practitioners' oral contraception study. Br Med J 1999; 318:96. 35. Schwingl PJ, Ory HW, Visness CM. Estimates of the risk of cardiovascular death attributable to low-dose oral contraceptives in the United States. Am J Obstet Gynecol 1999; 180:241. 36. Pettiti DB, Sidney S, Bernstein A, et al. Stroke in users of low-dose oral con¬ traceptives. N Engl J Med 1996; 335:8. 37. Stampfer MJ, Willett WC, Colditz GA. A prospective study of past use of oral contraceptive agents and risk of cardiovascular diseases. N Engl J Med 1988; 319:1313. 38. World Health Organization Collaborative Study of Cardiovascular Disease and Steroid Hormone Contraception. Ischaemic stroke and combined oral contraceptives: results of an international, multicentre, case-control study. Lancet 1996; 348:498. 39. World Health Organization Collaborative Study of Cardiovascular Disease and Steroid Hormone Contraception. Haemorrhagic stroke, overall stroke risk, and combined oral contraceptives: results of an international, multi¬ centre, case-control study. Lancet 1996; 348:505. 40. Chasan-Taber L, Willett WC, Manson JE, et al. Prospective study of oral contraceptives and hypertension among women in the United States. Cir¬ culation 1996; 94:483. 41. Pritchard JA, Pritchard SA. Blood pressure response to estrogen-progestin oral contraception after pregnancy-induced hypertension. Am J Obstet Gynecol 1977; 733. 42. Speroff L, Damey PD. Implant contraception: Norplant. In: A clinical guide for contraception, 2nd ed. Baltimore: Williams & Wilkins, 1996. 43. Brinton LA, Vessey MP, Flavel R, Yeates D. Risk factors for benign breast disease. Am J Epidemiol 1981; 113:203. 44. World Health Organization Task Force on Oral Contraceptives. Effects of hormonal contraceptives on milk volume and infant growth. Contracep¬ tion 1984; 30:505. 45. Nillson S, Mellbin T, Hofvander Y, et al. Long-term follow-up of children breast-fed by mothers using oral contraceptives. Contraception 1986; 34:443. 46. Sivin I, Mishell DR, Damey P, et al. Levonorgestrel capsule implants in the United States: a 5-year study. Obstet Gynecol 1998; 92:337. 47. Centers for Disease Control Cancer and Steroid Hormone Study. Long¬ term oral contraceptive use and risk of breast cancer. JAMA 1983; 249:1591. 48. World Health Organization Collaborative Study of Neoplasia and Steroid Contraceptives. Breast cancer and combined oral contraceptives: results from a multinational study. Br J Cancer 1990; 61:110. 49. Huggins GR, Zucker PK. Oral contraceptives and neoplasia: 1987 update. Fertil Steril 1987; 47:733. 50. United Kingdom National Case-Control Study Group. Oral contraceptive use and breast cancer risk in young women. Lancet 1989; 1:1973. 51. Stadel BV, Schlesselman JJ, Murray PA. Oral contraceptives and breast can¬ cer. Lancet 1989; 1:1257. 52. Vessey MP, McPherson K, Villard-Mackintosh L. Oral contraceptives and breast cancer: latest findings in a large cohort study. Br J Cancer 1989; 59:613. 53. White E, Malone KE, Weiss NS, Daling JR. Breast cancer among U.S. women in relation to oral contraceptive use. J NCI 1994; 86:505. 54. Collaborative Group on Hormonal Factors in Breast Cancer. Breast cancer and hormonal contraceptives: collaborative reanalysis of individual data on 53,297 women with breast cancer and 100,239 women without breast cancer from 54 epidemiological studies. Lancet 1996; 347:1713. 54a. Pathak DR, Osuch JR, He J. Breast carcinoma etiology: current knowledge and new insights into the effects of reproductive and hormonal risk factors in black and white populations. Cancer 2000; 88(5 Suppl):1230. 55. World Health Organization Collaborative Study of Neoplasia and Steroid Contraceptives. Breast cancer and depot-medroxyprogesterone acetate: a multinational study. Lancet 1991; 338:833. 56. Paul C, Skegg DCG, Spears GFC. Depot medroxyprogesterone (DepoProvera) and risk of breast cancer. Br Med J 1989; 299: 759.

Ch. 106: Morphology of the Normal Breast, Its Hormonal Control, and Pathophysiology 57. Skegg DCG, Noonan EA, Paul C, et al. Depot medroxyprogesterone acetate and breast cancer. JAMA 1995; 273:799. 58. The Cancer and Steroid Hormone Study of the Centers for Disease Control and the National Institute of Child Health and Human Development. Oral contraceptive use and the risk of breast cancer. N Engl J Med 1986; 315:405. 59. Lanes SF, Birmann B, Walker AM, Singer S. Oral contraceptive type and functional ovarian cysts. Am J Obstet Gynecol 1992; 166:956. 60. Grimes DA, Godwin AJ, Rubin A, et al. Ovulation and follicular develop¬ ment associated with three low-dose oral contraceptives: a randomized controlled trial. Obstet Gynecol 1994; 83:29. 60a. Alvarez-Sanchez F, Brache V, Montes de Oca V, et al. Prevalence of enlarged ovarian follicles among users of levonorgestrel subdermal contra¬ ceptive implants (Norplant). Am J Obstet Gynecol 2000; 182:535. 61. Centers for Disease Control Cancer and Steroid Hormone Study. Oral con¬ traceptive use and risk of ovarian cancer. JAMA 1983; 249:1596. 62. Hankinson SE, Colditz GA, Hunter DJ. A quantitative assessment of oral contraceptive use and risk of ovarian cancer. Obstet Gynecol 1992; 80:708. 63. Cancer and Steroid Hormone Study of the Centers for Disease Control and the National Institutes of Child Health and Human Development. The reduction in risk of ovarian cancer associated with oral contraceptive use. N Engl J Med 1987; 316:650. 64. Narod SA, Risch H, Moslehi R, et al. Oral contraceptives and the risk of hereditary ovarian cancer. N Engl J Med 1998; 339:424. 65. World Health Organization Collaborative Study of Neoplasia and Steroid Contraceptives. Depot-medroxyprogesterone acetate (DMPA) and risk of epithelial ovarian cancer. Int J Cancer 1991; 49:191. 66. Centers for Disease Control Cancer and Steroid Hormone Study. Oral con¬ traceptive use and risk of endometrial cancer. JAMA 1983; 249:1600. 67. Cancer and Steroid Hormone Study of the Centers for Disease Control and the National Institutes of Child Health and Human Development. Combination oral contraceptive use and the risk of endometrial cancer. JAMA 1987; 257:796. 68. Schlesselman JJ. Risk of endometrial cancer in relation to use of combined oral contraceptives. A practitioner's guide to meta-analysis. Hum Reprod 1997; 12:1851. 69. World Health Organization Collaborative Study of Neoplasia and Steroid Contraceptives. Depot-medroxyprogesterone acetate (DMPA) and risk of endometrial cancer. Int J Cancer 1991; 49:186. 70. Brinton LA. Oral contraceptives and cervical neoplasia. Contraception 1991; 43:581. 71. World Health Organization Collaborative Study of Neoplasia and Steroid Contraceptives. Combined oral contraceptives and risk of cervical carci¬ noma in situ. Int J Epidemiol 1995; 24:19. 72. Ursin G, Peters RK, Henderson BE, et al. Oral contraceptive use and adeno¬ carcinoma of the cervix. Lancet 1994; 344:1390. 73. The New Zealand Contraception and Health Study Group. History of long-term use of depot-medroxyprogesterone acetate in patients with cer¬ vical dysplasia; case-control analysis nested in a cohort study. Contracep¬ tion 1994; 50:443. 74. Thomas DB, Ye Z, Ray RM, and the World Health Organization Collabora¬ tive Study of Neoplasia and Steroid Contraception. Cervical carcinoma in situ and use of depot-medroxyprogesterone acetate (DMPA). Contracep¬ tion 1995; 51:25. 75. Goldzieher JW. Effects on other tissues. In: Fraser IS, ed. Estrogens and progestagens in clinical practice. London: Churchill Livingstone, 1998. 76. Grodstein F, Colditz GA, Hunter DJ, et al. A prospective study of symp¬ tomatic gallstones in women: relation with oral contraceptives and other risk factors. Obstet Gynecol 1994; 84:207. 77. Royal College of General Practitioners' Oral Contraception Study Oral contraceptives and gallbladder disease. Lancet 1982; 2:957. 78. Thijs C, Knipschild P. Oral contraceptives and the risk of gallbladder dis¬ ease: a meta-analysis. Am J Pub Health 1993; 83:113. 79. Sillem MH, Teichmann AT. The liver. In: Goldheizer J, Fotherby K (eds). Phar¬ macology of the contraceptive steroids. New York: Raven Press, 1994:247. 80. Bennion LJ, Ginsberg RL, Gamick MB, Bennett PH. Effects of oral contracep¬ tives on the gallbladder bile of normal women. N Engl J Med 1976; 294:189. 81. Korba VD, Paulson SR. Five years of fertility control with microdose norgestrel: an updated clinical review. J Reprod Med 1974; 13:71. 82. Population Council. Norplant levonorgestrel implants: a summary of sci¬ entific data. New York: The Population Council, 1990. 83. McCann MF, Potter LS. Progestin-only oral contraception: a comprehen¬ sive review. Contraception 1994; 50(Suppl 1):S96. 84. Palmer JR, Rosenberg L, Kaufman DW, et al. Oral contraceptive use and liver cancer. Am J Epidemiol 1989; 130:878. 85. World Health Organization Collaborative Study of Neoplasia and Steroid Contraceptives. Combined oral contraceptives and liver cancer. Int J Can¬ cer 1989; 43:254. 86. Prentice RL. Epidemiologic data on exogenous hormones and hepatocellu¬ lar carcinoma and selected other cancers. Prev Med 1991; 20:38. 87. Waetjen LE, Grimes DA. Oral contraceptives and primary liver cancer: temporal trends in three countries. Obstet Gynecol 1996; 88:945. 88. Kleerekoper M, Brienza RS, Schultz LR, Johnson CC. Oral contraceptive use may protect against low bone mass. Arch Intern Med 1991; 151:1971. 89. Cundy T, Evans M, Roberts H, et al. Bone density in women receiving depot medroxyprogesterone acetate for contraception. Br Med J 1991; 303:13. 90. Cromer BA, Blair JM, Mahan JD, et al. A prospective comparison of bone den¬ sity in adolescent girls receiving depot medroxyprogesterone acetate (DepoProvera), levonorgestrel (Norplant®) or oral contraceptives. J Pediatr 1996; 129:671.

1039

91. Scholes D, Lacroix AZ, Ott SM, et al. Bone mineral density in women using depot-medroxyprogesterone acetate for contraception. Obstet Gynecol 1999; 93:233. 91a. Petiti DB, Piaggio G, Mehta S, et al. for the WHO Study of Hormonal Con¬ traception and Bone Health. Steroid hormone contraception and bone min¬ eral density: a cross-sectional study in an international population. Obstet Gynecol 2000; 95:736. 92. Cundy T, Cornish J, Evans MC, et al. Recovery of bone density in women who stop using medroxyprogesterone acetate. BMJ 1994; 308:247. 93. Faundes A, Brache V, Tejada AS, et al. Ovulatory dysfunction during con¬ tinuous administration of low-dose levonorgestrel by subdermal implants. Fertil Steril 1991; 56:273. 94. Eriksen EF, Colvard DS, Berg NJ, et al. Evidence of estrogen receptors in normal human osteoblast-like cells. Science 1988; 241:84. 95. Jordan VC, Jeng MH, Catherino WH, Parker CJ. The estrogenic activity of synthetic progestins used in oral contraceptives. Cancer 1993; 71(Suppl):1501. 96. Gluntz S, Gluntz JC, Campbell-Heider N, Schaff E. Norplant use among urban minority women in the United States. Contraception 2000; 61:83.

CHAPTER

106

MORPHOLOGY OF THE NORMAL BREAST, ITS HORMONAL CONTROL, AND PATHOPHYSIOLOGY RICHARD E. BLACKWELL

MORPHOLOGY AND HORMONAL CONTROL COMPARATIVE ANATOMY OF LACTATION The constituents of milk products differ widely among species, undoubtedly reflecting differences in the nutritional require¬ ments of the neonate and the environmental restrictions on the mother. The mammary gland is unique in the animal kingdom in that only 4200 species of mammals possess this organ. Most of these mammals (95%) belong to the subclass Eutheria; the remainder belong either to the subclass Monotremata, which contains the primitive egg-laying mammals such as the duck¬ bill platypus, or to the Metatheria, which contains the single¬ order Marsupilia (i.e., kangaroos).1

HISTORY OF THE HORMONAL CONTROL OF THE BREAST Haller, in 1765, was the first to conclude that milk was derived from blood. The relation of blood and milk production was investigated by Sir Astley Cooper, who first described the early physiologic occurrence of milk letdown and lactogenesis. In the 1930s, it was shown by means of pressure monitors that milk secretion and ejection are separate events.2 In 1928, prolactin was extracted and demonstrated to be different from other known pituitary hormones.3 In the 1940s, it was pro¬ posed that during pregnancy, estrogen and progesterone pro¬ mote full mammary growth while progesterone inhibits estrogen stimulation of prolactin secretion, and that at parturi¬ tion, an increase in circulating prolactin and cortisol accompa¬ nied by a fall in estrogen and progesterone trigger lactation.4 Although incorrect in some aspects, this hypothesis endured for more than 20 years. It began to be challenged with the dis¬ covery that mammary growth occurs in the absence of steroid hormones in adrenalectomized and gonadectomized rats that are recipients of pituitary mammotrophic tumor xenografts secreting prolactin, growth hormone, and adrenocorticotropic

1 040

PART

VII: ENDOCRINOLOGY OF THE FEMALE

FIGURE 106-1. Anatomy of the breast (sagittal view).

hormone.5 Subsequent studies showed that estrogen stimu¬ lates the secretion of prolactin.6 Partially inhibiting the response, progesterone suppresses prolactin secretion below baseline. It has been proposed that elevated progesterone lev¬ els during pregnancy prevent the secretion of milk and that the withdrawal of this hormone after parturition is in part responsible for lactogenesis.7

ANATOMY OF THE MAMMARY GLAND The mammary gland lies on the pectoralis fascia and muscula¬ ture of the chest wall over the upper anterior rib cage (Fig. 106-1). It is surrounded by a layer of fat and encased in skin. The tissue extends into the axilla, forming the tail of Spence. The mammary gland consists of 12 to 20 glandular lobes or lobules that are connected by a ductal system. The ducts are surrounded by connective and periductal tissues, which are under hormonal control. The lactiferous ducts enlarge as they approach the nipple, which is pigmented and surrounded by the areola. The ductal tissue is lined by epithelial cells. The indi¬ vidual functional unit of the breast is the alveolar cell, which is surrounded by the hormonally responsive myoepithelial cells. Milk is produced at the surface of the alveolar cells and is ejected by the contraction of the myoepithelial cells under the influence of oxytocin. Fibrous septa run from the lobules into the superficial fascia. The suspensory ligaments of Cooper per¬ mit mobility of the breast. The principal blood supply of the breast comes from the lat¬ eral thoracic and internal thoracic arteries, although compo¬ nents have been identified from the anterior intercostal vessels. The breast is innervated chiefly by the intercostal nerves carry¬ ing both sensory and autonomic fibers. The nipple and areola are innervated by the interior ramus of the fourth intercostal nerve. Seventy-five percent of the lymphatic drainage involves axillary pathways through the pectoral and apical axillary nodes. Drainage also occurs through parasternal routes.

EMBRYOLOGY AND HISTOLOGY OF THE MAMMARY GLAND The mammary gland can be identified 6 weeks after fertiliza¬ tion; it is derived from ectoderm. At 20 weeks' gestation, the 16 to 24 primitive lactiferous ducts invade the mesoderm. These ectodermal projections continue to branch and grow deeper into the tissue. Canalization occurs near term. Importantly, although the central lactiferous duct is present at birth, the gland does not differentiate until it receives the appropriate hormonal signals.

By the time an embryo is 7 mm in length, the mammary tis¬ sue has thickened to form a ridge (known as the mammary crest or milk line) extending along the ventrolateral body wall from the axillary to the inguinal region on each side. The caudal epi¬ thelium regresses, and the crest in the thoracic region thickens further to form a primordial mammary bud by the time the embryo is 10 to 12 mm in length. These embryologic origins account for the occasional development of supernumerary nip¬ ples and accessory breast tissue. Although mammary tissue remains relatively unresponsive until pregnancy, it is responsive to systemic hormone adminis¬ tration during fetal life. In the third trimester, when fetal prolac¬ tin levels increase, terminal differentiation of ductal cells occurs. This hormonal milieu accounts for the witch's milk expressible from the nipples of some normal newborn girls. After birth, these cells revert slowly to a more primitive state.8 The glands remain quiescent until the establishment of ovula¬ tory menstrual cycles, at which time breast development pro¬ ceeds in the manner described by Marshall and Tanner9 (see Chap. 91). Although the hormonal regulation of mammogenesis is unclear, estrogen in vivo appears to bring about ductal proliferation, although it has little ability to stimulate lobuloalveolar development.10 In vitro, however, estrogens do not pro¬ mote mammary growth. It has been suggested that various epidermal growth factors participate in this process.11 When progesterone is administered in vivo, lobuloalveolar develop¬ ment occurs.12 However, the administration of estrogen and progesterone to hypophysectomized animals fails to promote mammogenesis.13 These data strongly suggest that hormones other than estrogens and progestogens play a role in mammo¬ genesis. For instance, if the pituitary and adrenal glands are removed from oophorectomized rats, the addition of estrogen plus corticoids and growth hormone restores duct growth simi¬ lar to that seen in puberty.14

NONPREGNANT (INACTIVE) MAMMARY GLAND Before pregnancy, breast lobules consist of ducts lined with epi¬ thelium and embedded in connective tissue. The preponder¬ ance of the tissues in the gland are of the connective and adipose types. There is a scant contribution from glandular parenchyma, and a few bud-like sacculations arise from the ducts. The entire gland consists predominantly of the lactifer¬ ous ducts. The breast does undergo cyclic changes associated with normal ovulation, and the premenstrual breast engorge¬ ment noted by most women is probably secondary to tissue edema and hyperemia. Epithelial proliferation is also detectable during the menstrual cycle.15

MAMMARY DEVELOPMENT IN PREGNANCY After conception, the mammary gland undergoes remarkable development. Lobuloalveolar elements differentiate during the first trimester. Both in vitro and in vivo, it is possible to induce mammary development with either placental lactogen or pro¬ lactin in the absence of steroid hormones.16 Although both pla¬ cental lactogen and prolactin increase throughout pregnancy, data suggest that either of these hormones can stimulate com¬ plete mammogenesis. The role of estrogen in mammogenesis appears to be secondary, since lactation has been reported in pregnancies of women with placental sulfatase deficiency.1' Progesterone, although stimulating lobuloalveolar develop¬ ment, also appears to antagonize the terminal effects induced by prolactin, at least in vitro. Cortisol, which potentiates the action of prolactin on mammary differentiation, apparently is unnecessary for either ductal or alveolar proliferation.18 Insulin and other growth factors also stimulate mammogene¬ sis.19 For example, insulin is required for the survival of postna¬ tal mammary tissue in vitro. It is also possible that insulin-like molecules such as the insulin growth factors participate in this

Ch. 106: Morphology of the Normal Breast, Its Hormonal Control, and Pathophysiology

1041

TABLE 106-1. Hormone Regulation of the Breast

TABLE 106-2. Some Bioactive Substances in Milk of Humans and Other Mammals

GROWTH

PITUITARY HORMONES

Ducts

Prolactin

Growth hormone

Growth hormone

Estrogen

Thyroid-stimulating hormone

Cortisol

Follicle-stimulating hormone

Alveoli Estrogen Progesterone

Adrenocorticotropic hormone Oxytocin

HYPOTHALAMIC HORMONES

Cortisol

Thyroid-releasing hormone

Prolactin

Somatostatin

Growth hormone

LACTOGENESIS

Gonadotropin-releasing hormone

GROWTH FACTORS

Prolactin

Insulin-like growth factors

Insulin

Insulin-like growth factor-binding proteins

Insulin-like growth factor-I

Nerve growth factor

Cortisol

Epidermal growth factor Transforming growth factor-a Transforming growth factor-(3

process. However, studies suggest that epidermal growth factor is involved in mammogenesis and, together with glucocorti¬ coids, facilitates the accumulation of type IV collagen, a compo¬ nent of the basal lamina, on which epithelial cells are supported (Table 106-1).

Growth inhibitors macrophage-derived and macrophage-activating factor Relaxin Platelet-derived growth factor

THYROID AND PARATHYROID HORMONES Thyroxine Triiodothyronine

CONTROL OF LACTATION

Reverse triiodothyronine Calcitonin

Although milk letdown occurs fairly abruptly between the sec¬ ond and fourth postpartum days in the human, the transition from colostrum production to mature milk secretion is gradual. This process may take up to a month and seems to coincide with a fall in plasma progesterone and a rise in prolactin levels. Twelve weeks before parturition, changes in milk composi¬ tion begin20: increased production of lactose, proteins, and immunoglobulins and decreased sodium and chloride content. There is an increase in blood flow and in oxygen and glucose uptake in the breasts. There is also a marked increase in the amount of citrate at about the time of parturition. The composi¬ tion of milk remains fairly stable until term, which is best exem¬ plified by the stable production of a-lactalbumin, a milk-specific protein. At parturition, there is a marked fall in placental lacto¬ gen production, and progesterone levels reach nonpregnant lev¬ els within several days.21 Plasma estrogen falls to basal levels in 5 days, whereas prolactin decreases over 14 days.22 A fall in the progesterone level seems to be the most important event in the establishment of lactogenesis. Exogenous progesterone prevents lactose and lipid synthesis after ovariectomy in pregnant rats and in ewes.23 Furthermore, progesterone administration inhibits casein and a-lactalbumin synthesis in vitro.24 The major proteins of human milk are a-lactalbumin (30% of the total protein content), lactoferrin (10-20%), casein (40%), and immunoglobulin A (IgA; 10%). Milk also contains many sub¬ stances that are potentially capable of exerting biologic effects. Their physiologic role is, as yet, largely unexplored25 (Table 106-2).

Parathyroid hormone Parathyroid hormone-related protein

STEROID HORMONES Estradiol Estriol Progesterone Testosterone Corticosterone Vitamin D

GASTROINTESTINAL PEPTIDES Vasoactive intestinal peptide Bombesin Cholecystokinin Gastrin Gastric inhibitory peptide Pancreatic peptide YY Substance P Neurotensin

OTHERS Prostaglandin E Prostaglandin F2a Cyclic adenosine monophosphate Cyclic guanosine monophosphate Delta sleep-inducing peptide Transferrin Lactoferrin Casomorphin

MAINTENANCE OF LACTATION In the human, lactation is maintained by the interaction of numerous hormones. After removal of either the pituitary or the adrenal glands from a number of animal species, milk pro¬ duction is terminated rapidly.26 The species dictates the type of replacement therapy required to reinstitute milk production. For instance, in rabbits and sheep, prolactin is effective alone, whereas in ruminants, milk secretion is restored by the addition of corticosteroids, thyroxine, growth hormone, and prolactin. In humans, prolactin appears to be a key hormone in the mainte¬ nance of lactation, since the administration of bromocriptine

Erythropoietin (Modified from Grosvenor CE, Picciano MF, Baumrucker CR. Hormones and growth factors in milk. Endocr Rev 1993; 14:710.)

blocks lactogenesis.27 The role of thyroid hormones in lactation is unclear. Thyroidectomy inhibits lactation, and replacement therapy with thyroxine increases milk yield. It has been sug¬ gested that growth hormone and thyroxine synergize to alter milk yield, and triiodothyronine acts directly on mouse mam¬ mary tissue in vitro to increase its sensitivity to prolactin.28

1042

PART VII: ENDOCRINOLOGY OF THE FEMALE

Despite species differences, in humans, prolactin levels reach a peak before delivery and subsequently rebound after the initiation of lactation. This phenomenon can be inhibited by progesterone. Despite the importance of declining progesterone levels in initiating this event, lactation fails to occur with inade¬ quate prolactin production. Prolactin production becomes attenuated over time, with the most dynamic period being 8 to 41 days postpartum. By the 63rd day, the prolactin response will be attenuated by a factor of 5, and this is maintained to ~194 days postpartum.29-32 SUCKLING AND MILK EJECTION The integrated baseline level of prolactin is elevated in lactating women.33 Suckling or manipulation of the breasts leads to eleva¬ tion in prolactin within 40 minutes.34 In rats, this response can be mimicked by electrical stimulation of the mammary nerves. Both growth hormone and cortisol are also increased. The response appears to be greatest in the immediate postpartum period and is attenuated over 6 months. If lidocaine is applied to the nipple, thus blocking nerve conduction, the rise in prolactin levels is abolished.35 If two infants suckle simultaneously, the rise in pro¬ lactin is amplified. Along with prolactin release, suckling increases the secretion of oxytocin. After the application of a stimulus, there is an 8- to 12-hour delay before milk secretion is fully stimulated. This response seems to be correlated with the frequency and duration of vigorous suckling. There is no correla¬ tion between the amount of prolactin released and the milk yield. Suckling of the breasts increases intramammary pressure bilaterally, secondary to contraction of the myoepithelial cells in response to the octapeptide oxytocin. This contraction follows the application of stimulation to the nipple, which activates sensory receptors transmitting impulses to the spinal cord and hypothalamus. Oxytocin-producing neurons are located both in the paraventricular and supraoptic nuclei (see Chap. 25). It is estimated that ~2 ng oxytocin is released per 2- to 4-second pulse interval.36 The synthesis and release of oxytocin are rapid, because 90 minutes after injection of a radioactive amino acid into cerebrospinal fluid, radiolabeled oxytocin is released by exocytosis, and electrical pulse activity has been measured in oxytocic neurons 5 to 15 seconds before milk ejection. The response may be conditioned, since the cry of an infant or vari¬ ous other perceptions associated with nursing can trigger activ¬ ity in the central pathways. Thus, both oxytocin and prolactin are released in response to suckling, but the patterns of release clearly are different.37 When nursing women are allowed to hold their infants but not to breast-feed, serum prolactin concentrations do not increase, despite the occurrence of the milk letdown reflex; prolactin lev¬ els rise only with nursing. The increase in prolactin with nursing is apparently sufficient to maintain lactogenesis and an ade¬ quate milk supply for the next feeding. This accounts for the ability of "wet nurses" to continue to breast-feed infants for years—even after the menopause—once lactation is established. RESOLUTION OF LACTATION Postpartum lactation can be maintained over an extended period of time by discontinuing suckling. Nevertheless, prolac¬ tin levels decrease progressively over a number of weeks despite breast-feeding. The physiologic hyperprolactinemia is achieved by altering the endogenous secretory rate of each pro¬ lactin pulse. No alteration occurs in the number of bursts of prolactin or its half-life. A large group of Australian women breast-feeding for extended periods of time demonstrated a mean of 322 days of anovulation and 289 days of amenorrhea. Fewer than 20% of the women ovulated or had menstruated by 6 months postpartum. Ovulation was delayed to a maximum of 750 days and menstruation to 698 days.38 During pregnancy, luteinizing hormone (LH) and folliclestimulating hormone (FSH) secretion are inhibited through

hypothalamic mechanisms. The exogenous opioid tone is increased during the postpartum period, and the administra¬ tion of exogenous gonadotropin-releasing hormone (GnRH) pulses restores gonadotropin secretion. All of this suggests a central blockade of folliculogenesis secondary to hyperpro¬ lactinemia.39

BREAST FUNCTION AND AGING In the reproductive-aged woman, glandular tissue makes up -20% of the breast volume. The remainder of the breast is com¬ posed of connective and adipose tissue. Breast volume changes throughout the menstrual cycle by -20% secondarily to vascu¬ lar and lymphatic congestion. Adding to the increased volume of the breast is increased mitotic activity in nonglandular tissue. Breast engorgement and change in volume result in some ele¬ ment of mastalgia in most women, and this combined with an increase in tactile sensitivity of the breast results in the premen¬ strual tenderness found in most women.40'41 With the advent of menopause and a decrease in secretion of the gonadotropins estrogen and progesterone, involution of both glandular and ductal components of the breast occurs. Without replacement estrogen therapy there is a decrease in the number and size of glandular elements and both ducts and lob¬ ules become atrophic. Over time, the volume of the breast is primarily replaced with both adipose and stromal tissues, and as with most tissues there is a loss of both contour and struc¬ ture, which makes the aging breast more amenable to surveil¬ lance with mammography.42'43

PATHOPHYSIOLOGY Any disorder of the breast is viewed by the patient with alarm. Although, with the exception of carcinoma, disorders of the breast are not life threatening, any deviation from nor¬ mal size and appearance must be thoroughly evaluated. Because the development of the breasts is hormone dependent and breast disorders either may have a hormonal etiology or may be misconstrued as having a hormonal cause, the endo¬ crinologist should be familiar with the pathophysiology of these organs.

DEVELOPMENTAL ANOMALIES It was not until 1969 that a system for classifying breast devel¬ opment was established by Marshall and Tanner9 (see Chap. 91). In addition to its obvious use in evaluating the adequacy of breast development, this classification can be used to determine the presence of pathology. CONGENITAL ANOMALIES Congenital anomalies of the breast itself are uncommon; how¬ ever, one frequently sees anomalies of development. Even so, amastia, congenital absence of the breast; athelia, congenital absence of the nipple; polymastia, multiple breasts; polythelia, multiple nipples; or some combination, occur in 1% to 2% of the population and may have a familial tendency.44 If treatment is deemed necessary, surgical augmentation or excision is recom¬ mended (Fig. 106-2). Young patients may present with the problem of premature thelarche (see Chap. 92). Many would define this condition as breast development beginning before age 8. Affected individu¬ als may have either bilateral or unilateral development. The disorder may be differentiated from precocious puberty by the finding of prepubertal serum levels of gonadotropin and estro¬ gen. Precocious thelarche is self-limited and demands no ther¬ apy other than assurance, once complete or incomplete isosexual precocity has been ruled out.

Ch. 106: Morphology of the Normal Breast, Its Hormonal Control, and Pathophysiology

1043

FIGURE 106-2. Patient with Poland syndrome (aplasia of the pectoralis muscles, rib deformities, webbed fingers, and radial nerve aplasia, often associated with unilateral amastia) after partial reconstruction. The areola and nipple remain to be reconstructed.

BREAST ASYMMETRY Breast asymmetry is fairly common (Fig. 106-3) and presumably is secondary to a difference in end-organ sensitivity to estrogen and progesterone. Occasionally, full symmetry is obtained in adolescents by the administration of an oral contraceptive agent, although either augmentation or reduction mammoplasty may be required if severe asymmetry does not resolve. Most patients with breast asymmetry do not require therapy other than reas¬ surance that this is simply a variation of normal. HYPOPLASIA OF THE BREASTS Perhaps one of the most common disorders of the breast involves hypoplasia. These individuals may simply have small

FIGURE 106-3. Normal mammogram showing breast asymmetry. Con¬ temporary mammography uses a low kilovoltage-high milliamperage technique; the dose generally ranges from 20 to 30 kVp. Limitation of breast motion with a compression device allows decreased milliamper¬ age and increased magnification to produce a more uniform image den¬ sity. The breast image varies with the age of the patient. Virginal breasts are small and have near-consistent fibroglandular tissue. Breasts of a reproductive-aged individual, as shown in this figure, vary between well-developed fibroglandular and adipose tissues. Ducts and fibrous tissue are difficult to differentiate and are often found together. A wide variation is noted in the postmenopausal period, but there generally is increased fat content, making trabeculae, subareolar ducts, and veins easily visible. The atrophic breast shows a ground-glass homogeneity with prominent residual trabeculae.

FIGURE 106-4. Breast hypoplasia (top) and same patient after augmen¬ tation mammoplasty (bottom).

breasts secondary to a transient delay in puberty or may have a genetic tendency toward hypoplasia, with other siblings having similar problems. Such breasts show a physiologic response to pregnancy, and lactation can follow. Not uncommonly, because of social pressure to have "normal-sized" breasts, augmenta¬ tion is often sought by affected individuals (Fig. 106-4). Such augmentation will not interfere with lactation or breast-feeding but does increase the difficulty of self-examination and surveil¬ lance for breast malignancies. Breast hypoplasia is sometimes found in patients with severe anorexia nervosa and other vari¬ ants of psychogenic amenorrhea associated with decreased body weight or extremes of exercise (see Chap. 128); such indi¬ viduals have an altered fat/lean mass body ratio, which gener¬ ally renders them hypogonadotropic and hypoestrogenic. The removal of estrogen-progesterone stimulation leads to breast atrophy. Reconstructive therapy is contraindicated in this group; correction of nutritional requirements is the therapy of choice, although this must often be accompanied by psycho¬ therapy in patients with emotionally related weight loss. Breast hypoplasia also occurs in the female pseudohermaph¬ roditism of congenital adrenal hyperplasia (see Chap. 77) and in Turner syndrome (see Chap. 90). The early institution of cor¬ ticosteroid therapy will greatly benefit the former patient; the latter should be treated at the appropriate time with cyclical estrogen and progesterone. BREAST HYPERTROPHY Breast hypertrophy or macromastia is encountered commonly in both adolescents and adults. The breasts may be either symmet¬ ric or asymmetric. The patient frequently presents seeking advice on reduction mammoplasty (Fig. 106-5), perhaps because of chest wall pain secondary to the weight of the breasts, difficulty in finding clothes that fit the upper body, and difficulty with her self-image. Frequently, young women are under intense sexual pressure and are often embarrassed by

1044

PART VII: ENDOCRINOLOGY OF THE FEMALE

FIGURE 106-5. Patient before {top left and right) and after {bottom left and right) reduction mammoplasty. peers during gymnasium classes or when wearing swimming suits. As an alternative to surgical correction, danazol has been tried.45 Unfortunately, this drug has many side effects and defi¬ nitely is not acceptable for long-term therapy. NIPPLE INVERSION Nipple inversion is common but rarely presents as a complaint to the clinician. Cosmetic repair can be performed, but breast¬ feeding is difficult after such procedures.

GALACTORRHEA Galactorrhea, the inappropriate production and secretion of milk, may be intermittent or continuous, bilateral or unilateral, free flowing or expressible. By definition, fat droplets must be present on microscopic examination for a breast secretion to be considered milk and as evidence of galactorrhea. Galactorrhea is frequently associated with hyperprolactinemia (see Chap. 13),46 which should be sought by repeatedly measuring serum prolac¬ tin levels, remembering that prolactin is a stress-related hormone whose secretion may be increased by breast examination and stimulation, acute exercise, food intake (particularly protein), and sleep.47 Although the differential diagnosis of hyperpro¬ lactinemia is extensive, the common causes of this condition are prolactinoma, primary hypothyroidism, and drug intake. Galact¬ orrhea should be evaluated by the measurement of multiple serum prolactin levels, thyroxine, and thyroid-stimulating hor¬ mone and by radiographic or magnetic resonance imaging stud¬ ies of the pituitary. The prolactin level at which radiographic surveillance is begun is debated; however, computed tomogra¬ phy or magnetic resonance imaging should be done if basal pro¬ lactin levels exceed 100 ng/mL. Galactorrhea and its treatment are considered in more detail in Chapters 13,21, 22, and 23.

MASTODYNIA Mastodynia, painful engorgement of the breasts, is usually cyclic, becoming worse before menstruation.48 Although most women describe mastodynia at some times, they require no therapy. However, some patients require cyclic analgesics or nonsteroidal antiinflammatory drugs. Occasionally mastodynia is a complaint of women experiencing the premenstrual syn¬ drome; some affected patients will sporadically obtain some

relief with nonspecific therapy, as discussed in Chapter 99. Mastodynia may also be treated effectively with danazol, but the side effects of the drug mandate its use only in severe cases. In addition, a second generation of drugs, the GnRH ana¬ logs, have been used to induce hypogonadotropism and hypoestrogenism, thus treating disorders such as endometrio¬ sis, fibroids, hirsutism, and premenstrual syndrome. Treatment with these agents, either on a daily or monthly basis, will result in profound hypogonadism, breast atrophy, and relief of masto¬ dynia. These drugs are not approved by the Food and Drug Administration for this purpose, and therapy beyond 6 months results in reversible bone demineralization. To compensate for this loss in other disorders, estrogen "add-back" therapy, cotreatment with progestogens, and the use of variable-dose estrogen-progestogen overlapping protocols have been used to counter this and other side effects.

BREAST INFECTIONS Breast infections are often confused with galactorrhea but require therapy with appropriate systemic antibiotics. Patients present with unilateral or bilateral breast drainage, which, when examined by microscopy, fails to show fat globules. Gram stain frequently will reveal Staphylococcus, Streptococcus, or Escherichia coli. If the discharge has a greenish tint. Pseudomonas should be suspected. If the discharge is accompanied by abscess forma¬ tion, drainage as well as antibiotics should be used. The galactocele, or retention cyst, which usually occurs after cessation of lactation, is caused by duct obstruction and can masquerade as mastitis. These lesions usually lie below the are¬ ola and are often tender to palpation. Such cysts occasionally can be emptied by properly placed pressure; however, drainage frequently must be carried out. Untreated galactoceles may be sites of future sepsis and can calcify and become confused with malignant lesions radiologically. The drainage from a galacto¬ cele may range from milky to clear to yellow-green purulentappearing material; however, these lesions are usually sterile.

MAMMARY DYSPLASIAS Mammary dysplasia is perhaps the most common lesion of the female breast (Fig. 106-6). Historically, mammary dysplasias have carried the label fibrocystic disease, chronic lobular hyper-

Ch. 106: Morphology of the Normal Breast, Its Hormonal Control, and Pathophysiology

1045

FIGURE 106-7. Light mammograph of tissue sample showing scleros¬ ing adenosis.

on mammography. Approximately half the patients have a his¬ tory of trauma. Excisional biopsy is the treatment of choice. At biopsy, one may note hemorrhage into the fatty tissue. FIGURE 106-6. Composite mammogram showing bilateral fibrocystic disease. Note multiple large cysts in body of breast {arrows). plasia, cystic hyperplasia, or chronic cystic mastitis.49 The term cystic mastitis should be discarded, since inflammation is not present in this disorder. Mammary dysplasia may be unilateral or bilateral and most frequently occurs in the upper outer quadrants. The disorder tends to be exacerbated in the premenstrual period. Patients usually complain of pain or lumps in the breasts. The breasts may be tender in many locations; axillary adenopathy is gener¬ ally not found. Palpable breast lumps are usually cystic and tense; they shrink after menstruation. The natural history of the disease varies; however, it tends to resolve at menopause. Mammary dysplasia may be accompanied by a nipple dis¬ charge, which may be clear or bloody, in up to 15% of patients. The disorder may be confused with carcinoma, and a Papanico¬ laou smear of the discharge, mammography, and perhaps nee¬ dle aspiration may be necessary to rule out a malignancy. At the time of aspiration, one may evacuate a cyst filled with a dirty gray-green fluid. The management of this problem includes fre¬ quent breast examination, periodic mammography, use of a brassiere with good support, perhaps avoiding methylxanthines and chocolate, and, in extreme cases, the use of danazol therapy in daily doses of 100 to 800 mg in two divided doses. Although bromocriptine suppresses prolactin secretion, it has not been effective in the management of fibrocystic disease. It does decrease cyclic mastodynia, however. Likewise, GnRH analogs can be used in extreme cases. The dose of leuprolide acetate used to induce hypogonadism is 3.75 mg given intra¬ muscularly every month.

TUMORS OFTEN CONFUSED WITH BREAST CARCINOMA

SCLEROSING ADENOSIS Breast adenosis may be confused with carcinoma, particularly if sclerosing adenosis is present. This latter condition is character¬ ized by the proliferation of ductal tissue, producing a palpable lesion. These lesions are common in younger women, especially in the third and fourth decades of life; they are rarely seen postmenopausally. Grossly, breast carcinoma is often firm and gritty to palpation, whereas adenosis is usually rubbery. Microscopi¬ cally, one sees a nodule or whorl pattern. In sclerosing adenoma¬ tous adenosis, one also sees circumscription of the lesion, central attenuation of ductal caliber, an organoid arrangement of the ducts, mild epithelial tissue surrounding the ducts, and absence of intraductal epithelial bridging (Fig. 106-7). INTRADUCTAL PAPILLOMA Intraductal papilloma is a benign lesion of the lactiferous duct walls that occurs centrally beneath the areola in 75% of cases.51 Such lesions present as pain or bloody discharge. They are soft, small masses that are difficult to palpate. Indeed, if the patient presents with a small palpable mass associated with a bloody nipple discharge, there is a 75% chance that an intraductal pap¬ illoma will be found. If no mass can be palpated, Paget disease of the nipple or a carcinoma must be considered. Intraductal papilloma is not premalignant and is best managed by excision of the duct by wedge resection. Although intraductal papillo¬ mas generally occur in women in the late childbearing years, they may present in the adolescent. In these younger patients, such lesions are generally found at the periphery of the breast; multiple ducts may be involved, and cystic dilation is noted. These lesions have been called "Swiss cheese disease" or juve¬ nile papillomatosis. The treatment of juvenile papillomatosis involves excisional biopsy (Fig. 106-8). FIBROADENOMA

A few lesions, such as fat necrosis, adenosis (especially the sclerosing type), intraductal papilloma (including juvenile papillomatosis), and fibroadenoma (including cystosarcoma phylloides), are often confused with carcinoma of the breast.50 FAT NECROSIS Fat necrosis may present as a hard lump that may be tender; it rarely enlarges. Skin retraction may be seen, along with irregu¬ larity of the edges; fine, stippled calcifications may be present

One of the most common benign neoplasms of the adolescent and adult breast is the fibroadenoma52’53 (Fig. 106-9). These tumors may be small, firm nodules or large, rapidly growing masses that are multiple 20% of the time. They are more com¬ mon in black than in white women. They may be painful. The fibroadenoma may be hormonally responsive; rapid growth occurs during pregnancy and lactation. These tumors are best treated by excisional biopsy. Rare variants of the fibroadenoma (cystosarcoma phylloides, also known as the giant fibroade-

1 046

PART VII: ENDOCRINOLOGY OF THE FEMALE TABLE 106-3. Breast Self-Examination 1. Begin at 20 years 2. Carry out exams at least once per month 3. Assess the following: a. Symmetry b. Size c. Lumps d. Skin discoloration, dimpling, puckering e. Nipple discharge f. Pain g. Nipple inversion

FIGURE 106-8. Light micrograph of tissue sample showing intraductal papilloma.

noma) have been described. Although these tumors are gener¬ ally benign, a few have true sarcomatous potential.

ASSESSMENT OF BREAST DISEASE SELF-EXAMINATION Breast self-examination is still one of the most important meth¬ ods for the diagnosis of diseases of the breast, either benign or malignant. A poll conducted for the American Cancer Society found that the physician plays a pivotal role in encouraging patients to practice breast self-examination.53 It was noted that when patients received personal instruction from their physi¬ cians, 92% continued to practice breast self-examination regu¬ larly. Once taught self-examination, many patients can detect nodules in their own breasts before they are palpable by a skilled physician. Care must be taken to convince each patient that her breasts are not homogeneous but rather contain various struc¬ tures and different degrees of nodularity, thickening, and small lumps. The texture of the breast changes throughout a woman's life and during the menstrual cycle (Table 106-3). It is suggested that breast self-examination be practiced each month, preferably just after the menstrual period.54 The exami¬ nation should consist of inspection of the size, shape, and skin color and for puckering, dimpling, retraction of any of the sur¬ face, and any nipple discharge. The patient should look at her breasts by placing her hands on her hips and flexing the shoul¬ ders forward, then raising the hands behind her head. Breasts may be asymmetric in size, but asymmetry in movement is an indication of pathology. Next, palpation of the breast should be carried out in both the sitting and supine positions. Each quad¬ rant should be palpated systematically, including the nipple and areolar area. Special attention should be paid to the upper

outer quadrant and the axilla, because this is the most frequent site of breast carcinoma. Examination can be carried out with the fingertips or with a rotary motion as suggested by the American Cancer Society. An annual physical examination also should be performed by a physician skilled in the diagnosis of breast disease. There are different variations and techniques of breast examination, but a consistent systematic examination is central to all. MAMMOGRAPHY Breast imaging dates back to 1913. Mammography has been refined subsequently such that improved image, clarity, and contrast have increased its accuracy to well more than 90%.55-59 Despite widespread publicity urging the use of mammogra¬ phy and mass screening, breast cancer still strikes many women and is a common cause of death.60 Although most recommenda¬ tions relating to breast screening are aimed at women aged 35 or older, the incidence of breast cancer in women younger than 35 is not zero. An interdisciplinary task force in the United States has made the following recommendations61: First, mammography should be a part of clinical examination for breast disease and does not substitute for any part. Second, the mainstay of detection of breast disease remains self-examination and physician consul¬ tation. Third, women are candidates for mammography at any age if they have masses or nipple discharge, masses felt by the patient but not confirmed by a physician,62 previous surgical alteration of the breast by augmentation procedures or implants, contralateral disease, previous breast cancer, history of breast can¬ cer in a mother or sister, first pregnancy after age 30, and abnor¬ mal patterns in baseline mammography suggestive of increased risk63 (Table 106-4). Women older than age 50 should receive regu¬ lar breast examinations, including mammography, as determined by the physician. Baseline mammography should be performed on all women at some time between the ages of 35 and 50. BREAST IMAGING WITHOUT RADIATION Because of concerns about the hazards of multiple radiation exposures for mammography, several other modes have been introduced in the field of breast imaging.

TABLE 106-4. Principal Breast Cancer Risk Factors REPRODUCTIVE EXPERIENCE Risk increases with increased age at which a woman bears her first fullterm child OVARIAN ACTIVITY 70% risk reduction in women who undergo oophorectomy before age 35 BENIGN BREAST DISEASE Four-fold increase in risk in women with history of mammary dysplasia FAMILIAL TENDENCY Two-fold to three-fold increased risk if female relative has breast cancer

FIGURE 106-9. Mammogram showing fibroadenoma (arrow).

Ch. 106: Morphology of the Normal Breast, Its Hormonal Control, and Pathophysiology

1047

patients who had severe chronic inflammatory reactions around implanted Silastic ventriculoperitoneal shunt tubing.68 A study evaluating the sera of 79 women with breast implants who expe¬ rienced a wide variety of problems found that half had antibody levels >2 standard deviations above the control group without implants.69 Subsequent studies have been contradictory. Access to silicone implants has been restricted, and the Food and Drug Administration approved a protocol to evaluate silicone implants in women whose saline-filled implants are considered medically unsatisfactory.70 Augmentation mammoplasty creates a second problem, that is, the effect of capsular contracture on the quality of mammog¬ raphy. Moderate contracture has been predicted to result in a 50% reduction in the quality of visualization.71-72 These factors need to be considered in advising patients about augmentation mammoplasty.

BREAST CANCER ETIOPATHOLOGY

FIGURE 106-10. Breast sonogram showing fibrocystic disease. Multi¬ ple cysts are outlined by the small white dots.

Thermography. Thermography maps focal variations in skin temperature by various techniques.64 Invasive breast can¬ cers produce higher skin temperature, and thermography is accurate for detecting advanced disease. However, it is ineffec¬ tive in the diagnosis of nonpalpable cancers, detecting only approximately one-half the cases that can be discovered by mammography. Thus it is not an acceptable modality for popu¬ lation screening. Ultrasound Mammography. Breast ultrasound mammog¬ raphy can produce images in conjunction with immersion of the glands in a water bath65 (Fig. 106-10). However, it has poor reso¬ lution; it will not image structures smaller than 1 mm or identify microcalcification. Ultrasonography seems to be most success¬ fully applied to the diagnosis of breast disease in younger patients and is thought to be complementary to mammography.

IMPORTANCE OF EARLY DIAGNOSIS OF BREAST ANOMALIES AND DISEASES The endocrinologist should encourage early diagnosis of con¬ genital and acquired breast disorders. Fear of breast disease and subsequent surgical mutilation often causes the patient to defer evaluation, often worsening the outcome. In particular, the mor¬ tality from breast cancer remains high, in part because of such delays.66 It thus should be emphasized that both benign and malignant diseases of the breast are diagnosable at the most treatable stage by self-examination, early physician consulta¬ tion, radiographic study, and, sometimes, other methods, and that the treatment of developmental anomalies and of benign and malignant breast disease may be hormonal as well as surgi¬ cal (see Chap. 224). Moreover, even when surgery is mandated, early diagnosis plus available plastic surgery procedures can produce both cure and aesthetically satisfactory results.

COMPLICATIONS OF BREAST AUGMENTATION Major developments in breast augmentation such as silicone implants, mucocutaneous flaps, and autogenous tissue transfers have occurred within the past 20 years. When silicone implants were first introduced in 1964,67 they were thought to be biocom¬ patible products. However, it has been postulated that biomateri¬ als such as silicone might behave like other immunogenic substances. Antibodies to silicone were described in sera of two

Despite the investments that have been made in the diagnosis and treatment of breast cancer over the last two decades, only modest headway has been made in managing this disease. Cur¬ rently, women in the United States have a 1 in 8 risk, which is twice that found in 1940. In one study, at age 25, a woman had a 1 in 19,608 risk of developing breast cancer; by age 40 this had increased to 1 in 217; by age 70,1 in 14; and by age 85,1 in 9.73 Family history seems to play a major role in the development of breast cancer, with a two- to three-fold increased risk in the inci¬ dence of the disease being found in women who have female rela¬ tives with the disease. For instance, the patient with an affected mother or sister has a 2.3 relative risk and an affected aunt 1.5 rela¬ tive risk, and a 14% incidence when both mother and sister are affected. Hereditary forms of breast cancer make up ~8% of the disease population, and those women who have a strong family history tend to develop the disease at a younger age.74 In this respect, perhaps the most exciting event to have occurred in breast cancer research is the identification of genes predisposing to breast cancer. BRCA-1 and BRCA-2 together account for approximately two-thirds of familial breast cancer or roughly 5% of all cases.75-78 It also appears that BRCA-1 is associated with the predisposition of ovarian cancer. BRCA-1 is located on a locus on chromosome 17Q, and an analysis of 200 families has shown that BRCA-1 is responsible for multiple cases of breast cancer in -33% of families but more than 80% of families in which there is both breast cancer and epithelial ovar¬ ian cancer. Women who inherit the BRCA genes have a 60% risk of acquiring breast cancer by age 50, and a 90% overall lifetime risk. BRCA-2 lies within a 6-centimorgan interval on chromo¬ some 13Q12.13 centered on D13S260. The loss of this gene may also result in elimination of suppressor function. The discovery of these genes presents the possibility for genetic testing, which remains controversial at present. Cigarettes, coffee, alcohol, and diet may play a role in the development of breast cancer. Tobacco-related cancers appear in the lung, esophagus, oral cavity, pancreas, kidney, bladder, and breast. Therefore, smoking remains the chief preventable cause of death and illness in the United States. It is responsible for -70% of all deaths; however, although smoking decreased from 40% in 1965 to 29% in 1987, more than 5 million Ameri¬ cans continue to smoke. The incidence of smoking in women has risen at an alarming rate, and this parallels the increase in lung cancer found in women. Further, there has been an abrupt increase in smoking in girls aged 11 through 17.79 Methylxanthine-containing compounds have been impli¬ cated as a causative factor in the development of fibrocystic dis¬ ease of the breast and cancer. The Boston Collaborative Drug Surveillance Program showed an increased risk in women who drank between one and three cups of coffee or tea per day.80

1 048

PART VII: ENDOCRINOLOGY OF THE FEMALE

Several studies have evaluated the role of alcohol and its association with an increased risk of breast cancer. Women who consume more than three drinks per day have been reported to have a 40% increase in the risk of breast cancer.81-82 Dietary fat intake has been thought to be linked to breast cancer, but this relationship is controversial.823 It was noted that postmenopausal women in the United States are at a much higher risk for breast cancer than are Asian women. This does not appear to be a geographic phenomenon as movement of Asian women to either the Hawaiian Islands or Pacific Coast seems to eradicate the difference in incidence. The suppression, however, usually requires one to two generations to demon¬ strate significance. Other populations with high fat intake but relatively low risk of cancer such as seen in Greece or Spain use monounsaturated fats composed primarily of oleic acid. Like¬ wise, fish oil which is rich in omega-3 fatty acids has been asso¬ ciated with a lower incidence of breast cancer in countries ranging from Greenland to Japan.83 Steroid hormones are thought to affect the expression of breast cancer. For instance, a woman who has a child at the age of 18 has approximately one-third the risk of a woman who delivers after age 35. However, pregnancy must occur before age 30 to be protective, but, in fact, a woman who gives birth after age 35 appears to be at greater risk than a woman who has never been pregnant. There is also a 70% reduction of risk in the incidence of breast cancer in women who undergo oophorectomy before the age of 35. There also appears to be a small increased risk in patients who experience early menarche as well as late meno¬ pause. It has been suggested that tire endocrine milieu influences the susceptibility of the breast to environmental carcinogens.84 This is the so-called estrogen window hypothesis, which suggests that an unopposed estrogen stimulation at certain periods of life favors tumor induction. The longer the unopposed estrogen stimulation acts on the breast, the greater is the risk factor. Per¬ haps pregnancy, a high progesterone state, closes the window, because progesterone is known to down-regulate the estrogen receptors in the endometrium and is protective against the devel¬ opment of endometrial cancer. Although the data appear to be inconclusive at present, one might speculate that a similar mech¬ anism may be achieved at the level of the breast. Various chemical agents have been implicated in a decrease or increase in breast cancer. Estrogens of all types and their analogs may stimulate tumorigenesis. Progestogens, while regulating estrogen expression, can induce significant mitosis of both epi¬ thelial and stromal components.85 Historically, birth control pills have been evaluated using a variety of different study designs, and many, but not all reports have shown no increased risk of breast cancer.86-88 GnRH analogs decrease estrogen production and therefore are thought to be protective against breast cancer. Tamoxifen is a weak estrogen agonist that antagonizes the bio¬ logic effect of 17{}-estradiol. It is now used for the treatment of breast cancer in both menopausal and perimenopausal women, and current data suggest that this drug may in fact retard the expression of breast cancer. Raloxifene, a selective estrogen recep¬ tor modulator (SERM), used in hormonal replacement therapy, has also been shown to reduce the incidence of breast cancer and is given as a hormonal replacement therapy in menopause.883 POSTOPERATIVE REHABILITATION OF THE PATIENT WITH BREAST CANCER Chapter 224 discusses the current therapy of breast cancer. Thirty years ago, radical mastectomy was considered by many surgeons to be the treatment of choice for resectable breast can¬ cer. Reconstructive options were few, and required 3 to 4 stage procedures to create an adequate breast replacement. Usually, women were required to wear external breast prostheses. This resulted in surgical patients feeling disfigured, having a poor body image, lower self-esteem, and diminished feelings of sex¬ ual attractiveness and of femininity. Now, reconstructive tech¬

niques can be carried out immediately, or can be delayed. There has been a trend toward immediate reconstruction, as this tends to reduce the degree of psychological morbidity experienced by the patient, and the reconstructed breast is integrated into the body image. Further, the integrity of the soft tissue that envelops the breast is intact at the time of surgery; there is no fibrosis or contraction of the tissue, and a plastic surgeon can be involved in the surgery and the reconstruction to give the best cosmetic result, whether implant or autologous tissue is used.89-92 Following reconstruction, breast cancer patients are usually examined every 3 months for the first 5 years, at every 6 months for the next 5 years, and yearly thereafter. A metastatic survey including a complete blood cell count, blood chemistry, chest x-ray, and mammogram should be performed routinely in patients with stage I or II disease.

REFERENCES 1. Mepham T. Physiological aspects of lactation. In: McPhan T, ed. Biochemis¬ try of lactation. New York: Elsevier, 1983:3. 2. Peterson LV. Lactation. Physiol Rev 1944; 24:340. 3. Riddle O, Bates R, Dykshorn S. The preparation, identification and assay of prolactin-A hormone of the anterior pituitary. Am J Physiol 1933; 105:191. 4. Meites J, Turner C. Studies concerning the mechanism controlling the initi¬ ation of lactation at parturition: II. Why lactation is not initiated during pregnancy. Endocrinology 1942; 30:719. 5. Clifton K, Furth J. Ducto-alveolar growth in mammary glands of adrenogonadectomized male rats bearing mammotropic pituitary tumors. Endocri¬ nology 1960; 66:893. 6. Chen C, Meites J. Effects of estrogen and progesterone on serum and pitu¬ itary levels in ovariectomized rats. Endocrinology 1970; 86:503. 7. Kuhn N. Progesterone withdrawal as the lactogenic trigger in the rat. J Endocrinol 1969; 44:39. 8. McKiernan J, Coyne J, Canglone S. Histology of breast development in early life. Arch Dis Child 1988; 63:136. 9. Marshall W, Tanner J. Variations in pattern of pubertal changes in girls. Arch Dis Child 1969; 44:291. 10. Cowie AT. Backward glances. In: Yokoyama A, Mizuno H, Nagasawa II, eds. Physiology of mammary glands. Baltimore: University Park Press, 1978:43. 11. Tonelli G, Sorof S. Epidermal growth factor: requirement for development of cultured mammary glands. Nature 1980; 285:250. 12. Ichinose R, Nandi S. Influence of hormones on lobulo-alveolar differentia¬ tion of mouse mammary glands in vitro. J Endocrinol 1966; 35:331. 13. Cowie A, Tindal ], Yokoyama A. The induction of mammary growth in the hypophysectomized goat. ] Endocrinol 1966; 34:184. 14. Lyons WR. Hormonal synergism in mammary growth. Proc R Soc Biol 1958; 149:303. 15. Going JJ, Anderson T], Battersby S, et al. Proliferative and secretory activity in human breast during natural and artificial menstrual cycles. Am J Pathol 1988; 130:193. 16. Talwalker P, Meites T. Mammary lobulo-alveolar growth induced by ante¬ rior pituitary hormones in adreno-ovariectomized-hypophysectomized rats. Proc Soc Exp Biol Med 1961; 107:880. 17. France J, Seddon R, Liggins G. A study of a pregnancy with low estrogen production due to placental sulfatase deficiency. J Clin Endocrinol Metab 1973; 36:19. 18. Topper Y, Freeman C. Multiple hormone interactions in the developmental biology of the mammary gland. Physiol Rev 1980; 60:1049. 19. Elias J. Effect of insulin and cortisol on organ cultures of adult mouse mammary gland. Proc Soc Exp Biol Med 1959; 101:500. 20. Fleet I, Goode ], Hamon M, et al. Secretory activity of goat mammary glands during pregnancy and the onset of lactation. J Physiol 1975; 251:763. 21. Weiss G, Facog E, O'Byme E, et al. Secretion of progesterone and relaxin by the human corpeus luteum at midpregnancy and at term. Obstet Gynecol 1977; 50:679. 22. Martin R, Glass M, Wilson G, Woods K. Human oc-lactalbumin and hor¬ monal factors in pregnancy and lactation. Clin Endocrinol (Oxf) 1980; 13:223. 23. Hartmann P, Trevethan P, Shelton ]. Progesterone and oestrogen and the initiation of lactation in ewes, j Endocrinol 1973; 59:249. 24. Bruce J, Ramirez V. Site of action of the inhibitory effect of estrogen upon lactation. Neuroendocrinology 1978; 6:19. 25. Grosvenor CE, Picciano MF, Baumrucker CR. Hormones and growth fac¬ tors in milk. Endocr Rev 1993; 14:710. 26. Hearn J. Pituitary inhibition of pregnancy. Nature 1973; 241:207. 27. Brun del Re R, del Pozo E, deGrandi P, et al. Prolactin inhibition and sup¬ pression of puerperal lactation by a Br-ergocriptine (CB 154): a comparison with estrogen. Obstet Gynecol 1973; 41:884. 28. Vonderhaar BK. Studies on the mechanism by which thyroid hormones enhance a-lactalbumin activity in explants from mouse mammary glands. Endocrinology 1977; 150:1423.

Ch. 107: Conception, Implantation, and Early Development 29. Riggs LA, Yen SSC. Multiphasic prolactin secretion during parturition in human subjects. Am J Obstet Gynecol 1977; 128:215. 30. Kuhn NJ. Lactogenesis. The search for trigger mechanisms in different spe¬ cies. Symp Zool Soc Lond 1977; 41:165. 31. Noel GL, Suh HK, Frantz AG. Prolactin release during nursing and breast stimulation in postpartum and non-postpartum subjects. J Clin Encocrinol Metab 1974; 38:413. 32. Nunley WL, Urban RT, Kitchin JD, et al. Dynamics of pulsatile prolactin release during the postpartum lactational period. J Clin Endocrinol Metab 1991; 72:287. 33. Gross B, Eastman C, Bowen C, McEldruff A. Integrated concentration of pro¬ lactin in breast-feeding mothers. Aust NZ J Obstet Gynaecol 1979; 19:150. 34. Howie P, McNeilly A, McArdle T, et al. The relationship between sucklinginduced prolactin response and lactogenesis. J Clin Endocrinol Metab 1980; 50:670. 35. Tyson J. Nursing and prolactin secretion: principal determinants in the mediation of puerperal infertility. In: Crosignani P, Robyn C, eds. Prolactin and human reproduction. New York: Academic Press, 1977:97. 36. Lincoln O, Wakerley J. Electrophysiological evidence for the activation of supraoptic neuronics during the release of oxytocin. J Physiol (Lond) 1974; 242:533. 37. Brandts C, Rozenberg S, Meuris S. Advances in physiology of human lacta¬ tion. In: Angeli A, Bradlow H, Dogliotti L, eds. Endocrinology of the breast. Ann NY Acad Sci 1986; 464:66. 38. Lewis PR, Brown JB, Renfree MB, et al. The resumption of ovulation and menstruation in a well-nourished population of women breast feeding for an extended period of time. Fertil Steril 1991; 55:529. 39. Matsuzaki T, Azuma K, Irabara M, et al. Mechanism of anovulation in hyperprolactinemic amenorrhea determined by pulsatile gonadotropin¬ releasing hormone injection combined with human chorionic gonadotro¬ pin. Fertil Steril 1994; 62:2254. 40. Milligan D, Drife JO, Short RV. Changes in breast volume during normal menstrual cycle and after oral contraceptives. Br Med J 1975; 4:494. 41. Robinson JE, Short RV. Changes in breast sensitivity at puberty, during the menstrual cycle, and at parturition. Br Med J 1977; 1:1188. 42. Cowie AT, Forsyth JA, Hart JC. Hormonal control of lactation. Berlin: Springer-Verlag, 1980. 43. Andolina V, Lille S, Wilson KM, eds. Mammographic imaging: a practical guide. Philadelphia: Lippincott, 1992. 44. Pellegrini J, Wagner R. Polythelia and associated conditions. Am Fam Phy¬ sician 1983; 28:192. 45. Aksu MF, Tzingounis VA, Greenblatt RB. Treatment of benign breast dis¬ ease with danazol: a follow-up report. J Reprod Med 1978; 31:181. 46. Blackwell RE. Diagnosis and treatment of hyperprolactinemic syndromes. In: Wynn RM, ed. Obstetrics and gynecology annual 1985. Norwalk, CT: Appleton-Century-Crofts, 1985:305. 47. Blackwell RE. Diagnosis and management of prolactinomas. Fertil Steril 1985; 43:5. 48. Pilnik S. Clinical diagnosis of benign breast disease. J Reprod Med 1979; 22:277. 49. Love S, Gelman R, Silen W. Fibrocystic "disease" of the breast: a nondis¬ ease? N Engl J Med 1982; 307:1010. 50. Oberman HA. Benign breast lesions confused with carcinoma. In: McDiuitt RW, Oberman HA, Ozzello L, Kaufman N, eds. International Academy of Pathology monograph: the breast. Baltimore: Williams & Wilkins, 1984:1. 51. Haagensen C, Stout A, Phillips J. Neoplasms of the breast: I. Benign intra¬ ductal papilloma. Am J Surg 1951; 133:18. 52. Hertel B, Zaloudek C, Kempson R. Breast adenomas. Cancer 1976; 37:2891. 53. Egan R. Breast imaging, 3rd ed. Baltimore: University Park Press, 1984:5. 54. Wilson RW. The breast. In: Sabiston D, ed. Davis-Christopher textbook of surgery, 10th ed. Philadelphia: WB Saunders, 1972:573. 55. Colman M, Mattheiem W. Imaging techniques in breast cancer: workshop report. Eur J Cancer Clin Oncol 1988; 24:69. 56. Maisey MN. Imaging techniques in breast cancer: what is new? What is useful? A review. Eur J Cancer Clin Oncol 1988; 24:61. 57. Bassett LW, Gold RH. The evolution of mammography. AJR Am J Roent¬ genol 1988; 150:493. 58. Salomon A. Beitrage zur Pathologie und Klinik des Mammarkarzinome. Arch Klin Chir 1913; 101:573. 59. Egan R. Mammography. Springfield, IL: Charles C Thomas Publisher, 1964:1. 60. Pietsch J. Breast disorders. In: Lavery J, Sanfilippo J, eds. Pediatric and ado¬ lescent obstetrics and gynecology. New York: Springer-Verlag, 1985:103. 61. Executive Board of the American Academy of Obstetrics and Gynecology. ACOG statement of policy mammography statement. Chicago: American College of Obstetrics and Gynecology, 1979:1. 62. Edeiken S. Mammography and palpable cancer of the breast. Cancer 1988; 61:263. 63. Solin LJ, Legoretta A, Schultz DJ, et al. The importance of mammographic screening relative to the treatment of women with carcinoma of the breast. Arch Intern Med 1994; 154:745. 64. Gauterie M, Gross C. Breast thermography and cancer risk prediction. Cancer 1980; 45:51. 65. Wild J. Review of the ultrasonic examination of the breast. In: Jellins J, Kobayashi T, eds. Ultrasonic examination of the breast. New York: John Wiley and Sons, 1983:21. 66. Carlson RW, Stockdale FE. The clinical biology of breast cancer. Annu Rev Med 1988; 39:453. 67. Cronin TD, Gerow F. Augmentation mammoplasty: a new "natural feel"

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prosthesis. In: Transactions of the Third International Congress of Plastic Surgeons. Amsterdam: Excerpta Medica, 1964. 68. Goldblum RM, Pelley RP, O'Donell AA, et al. Antibodies to silicone elas¬ tomers and reactions to ventriculoperitoneal shunts. Lancet 1992; 340:510. 69. Heggers JP, Goldblum RM, Pyron MT, et al. Immunologic responses to sili¬ cone implants: fact or fiction? Plast Surg Forum 1990; 8:13. 70. Randall T. First clinical study of breast implants launched. JAMA 1992; 268:1822. 71. Douglas KP, Bluth El, Sauter ER, et al. Roentgenographic evaluation of the augmented breast. South Med J 1991; 64:49. 72. Handel N, Silverstein MJ, Gamagami P. Factors affecting mammographic visu¬ alization of the breast after augmentation mammaplasty. JAMA 1992; 268:1913. 73. Davis DL, Dinse GE, Hoel DG. Decreasing cardiovascular disease and increasing cancer among whites in the United States from 1973 through 1978. JAMA 1994;271:431. 74. Colton T, Greenberg ER, Noller K, et al. Breast cancer in mothers pre¬ scribed diethylstilbestrol in pregnancy. JAMA 1993; 269:2096. 75. Futreal PA, Liu Q, Shattuck-Eldens D, et al. BRCA1 mutations in primary breast and ovarian carcinomas. Science 1994; 266:120. 76. Miki Y, Swensen J, Shattuck-Eldens D, et al. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 1994; 266:66. 77. Nowak R. Breast cancer gene offers surprises. Science 1994; 265:1796. 78. Wooster R, Neuhausen SL, Mangion J, et al. Localization of a breast cancer susceptibility gene, BRCA2 to chromosome 13ql2-13. Science 1996; 265:2088. 79. Rosenhert L, Schwingi PA. Breast cancer and cigarette smoking. N Engl J Med 1984; 310:92. 80. Welsch CW. Caffeine and the development of a normal and neoplastic mammary gland. Proc Soc Exp Biol 1994; 207:1. 81. Schotzkin A, Jones DY, Hoover RN, et al. Alcohol consumption and breast cancer in the epidemiologic followup study of the first national health and nutrition examination survey. N Engl J Med 1987; 316:1169. 82. Willett WC, Colditz G, Stampler MJ, et al. A prospective study of alcohol intake and risk of breast cancer. Am J Epidemiol 1986; 124:540. 82a. Velie E, Kulldorff M, Schaiver C, et al. Dietary fat, fat sib types, and breast cancer in postmenopausal women: a prospective cohort sudy. J Natl Can¬ cer Inst 2000; 92:833. 83. Bland KI. Risk factors as an indicator for breast cancer screening in asymp¬ tomatic patients. Maturitas 1987; 9:135. 84. Korenmann SC. Estrogen window hypothesis of the etiology of breast can¬ cer. Lancet 1980; 1:700. 85. Li JJ, Li SA. Estrogen carcinogenesis in hamster tissues: a critical review. Endocrinol Rev Monograph 1. Endocrine Aspects of Cancer 1993; 1:86. 86. Ramcharan S, Pellegrin FA, Ray RM, Hau J-P. The Walnut Creek Contra¬ ceptive Drug Study: a prospective study of the side-effects of oral contra¬ ceptives. J Reprod Med 1980; 25:366. 87. Royal College of General Practitioners Oral Contraceptive Study. Further analysis of mortality in oral contraceptive users. Lancet 1981; 1:541. 88a. Minton SE. Chemoprevention of breast cancer in the older patient. Hematol Oncol Clin North Am 2000; 14:113. 88. Kay CR, Hannaford PC. Breast cancer and the pill—a further report from the Royal College of General Practitioners Oral Contraceptive Study. Br J Cancer 1988; 58:675. 89. Lewis FM, Bloom JR. Psychosocial adjustment to breast cancer: a review of selected literature. Int J Psychiatry Med 1978; 9:1. 90. Stevens LA, McGrath MH, Druss RG, et al. The psychological impact of immediate breast reconstruction for women with early breast cancer. Plast Reconsti Surg 1984; 73:619. 91. Rowland JH, Holland JC, Chaglassian T, et al. Psychological response to breast reconstruction. Psychosomatics 1993; 34:241. 92. Moran SL, Herceg S, Kurtelawicz K, Serletti JM. TRAM flap breast recon¬ struction with expanders and implants. AORN J 2000; 71:354.

CHAPTER

107

CONCEPTION, IMPLANTATION, AND EARLY DEVELOPMENT PHILIP M. IANNACCONE, DAVID O. WALTERHOUSE, AND KRISTINA C. PFENDLER The hormonal milieu plays an essential role in the production of parental germ cells, the biology of the reproductive process, and the subsequent creation, development, and survival of the offspring. To understand fully the impact of hormones on the adult, the child, the newborn, and the as-yet-unborn, the endo-

1050

PART VII: ENDOCRINOLOGY OF THE FEMALE

crinologist must be aware of the processes of conception, implantation, and early fetal development. Human loss through fetal wastage is significant. There are -600,000 clinically apparent spontaneous abortions per year in the United States, and the 3 million live births probably repre¬ sent 10 million conceptions.1 Fetal loss can occur at any of the major steps in development. The probability of pregnancy in any given menstrual cycle under optimal conditions is ~30%.2"4 The probability of successful fertilization may be as high as 85%, but 25% to 35% of conceptuses do not implant, and as many as 30% fail shortly after implantation. Undoubtedly, this loss represents a reproductive strategy. The rate-limiting fea¬ ture of reproduction in mammals is the childbearing period. Therefore, if there is a possibility of loss of the individual dur¬ ing or immediately after the pregnancy, it is in the best interests of the species to eliminate the pregnancy as soon as possible and to make the mother available for another pregnancy. Thus, several critical steps of increasing sophistication of cellular coordination are required for the conceptus to enter midgesta¬ tion and the organogenesis phase of development.

Second meiotic division

First meiotic division

12 Anaphase

£• nj O

3 •3 (T3

14 Pronuclear egg

OOCYTE MATURATION Oocyte maturation in mammals proceeds from the development of a differentiated gonadal ridge in the fetus. For example, at approximately day 11 in the female mouse, primordial germ cells have located within the genital ridge, and ovarian development ensues.5 hi humans, primordial germ cells arise in the yolk sac in the fourth week of fetal life and begin migration caudally toward the genital ridge during the fifth week. The first meiotic division occurs in the fetal ovary, and the oocyte becomes arrested at the diplotene stage. A germinal vesicle (nucleus) then forms. The first meiotic division is completed in the adult ovary, and the onset of this process is heralded by germinal vesicle breakdown. After telophase I, the first polar body of the oocyte is formed. The second meiotic division begins before ovulation, and the mature oocyte is fertilizable at the anaphase II stage. Fertiliza¬ tion occurs during anaphase II, and the completion of telophase II finds the zygote with two polar bodies and two pronuclei (Fig. 107-1). The oocyte and follicular maturation are discussed in Chapter 94. Maturation of the oocyte and ovulation are regulated by hor¬ mone levels, notably those of follicle-stimulating hormone (FSH). The extruded oocyte and its closely adherent cumulus adherens (follicular cells; corona radiata) are collected by the fim¬ briated end of the oviduct. The adherent cells communicate with one another through a complex network of intercellular bridges that extends from the innermost cells through the zona pellucida to the perivitelline space and into the oocyte6 (Fig. 107-2). These cells may have important nutritional functions for the oocyte and may control events in maturation or fertilization.7 The cumulus cells can bind tightly to the epithelial cells of the tube and may help initiate tubal transport. Transport of the egg to fertilization sites at the distal end of the oviduct and transport of the fertilized ovum to the uterus appear to be the concerted effort of the ciliary movement of the epithelium and muscular contractions of the myosalpinx. These contractions are not peri¬ staltic. The sperm at this time are moving in the opposite direc¬ tion and, although the cilia beat in the direction of the uterus, the muscular contractions of the oviduct do not give direction to moving particles within it. Particles can be propelled in either direction in the fallopian tubes of most species.8 The role of tubal secretions in the development of the early embryo has not been elucidated. These secretions do not have a demonstrable effect on the sperm because capacitation, which permits the acrosome reaction, can occur in chemically defined media.9 Because human oocytes can undergo spontaneous matura¬ tion in vitro, there appears to be an active inhibition of oocyte

FIGURE 107-1. Diagram of oocyte maturation. Completion of the prophase of first meiotic division occurs in the fetal ovary of most ani¬ mals. At zygotene (stage 2), homologous maternal and paternal chro¬ mosomes commence pairing; at pachytene (stage 3), pairing has occurred throughout their lengths, and they form bivalents. Each homologue separates longitudinally to create two sister chromatids; thus, each bivalent forms a tetrad. It is during this stage that crossing over occurs, causing an interchange of genetic material between the paternal and maternal chromatids. At the diplotene stage (stage 4), the chromosomes commence their separation; they remain connected at their points of interchange (chiasmata). The germinal vesicle appears after the first meiotic arrest after the diplotene stage. The dictyate stage is a quiescent period, which may last for many years. In the adult ovary, the first meiotic division is completed. Ovulation occurs after extrusion of the first polar body (stage 11), and the second meiotic division (stages 12-14) is completed after sperm penetration. The zona pellucida is shown as a stippled ring (see Chap. 87). (From Tsafiri A. Oocyte matu¬ ration in mammals. In: Jones RE, ed. The vertebrate ovary. New York: Plenum Publishing, 1978:410.) maturation within the follicle. Meiosis is prevented by a matu¬ ration inhibitor produced by the granulosa cells of the follicle.10 Meiosis is resumed within the follicle after a surge of luteiniz¬ ing hormone. If oocytes are removed from cumulus cells, matu¬ ration inhibitors are ineffective. Moreover, receptors for luteinizing hormone have been demonstrated on cumulus cells but not on denuded oocytes. Therefore, the cumulus cells are important mediators of both maturation inhibition and resumption of meiosis, directed by the preovulatory luteinizing hormone surge. Interestingly, in humans, oocyte maturation in vitro, as judged by germinal vesicle breakdown, is not necessary for sperm penetration, because penetration can be demonstrated at the first meiotic division. After sperm penetration of the mature oocyte, the sperm head swells and a pronucleus forms, with the sperm midpiece remaining visible. In immature oocytes, the sperm penetrates and swells but no pronucleus is formed. Thus, fertilization competence in humans is achieved only in fully mature oocytes at the time of the second meiotic division.

SPERM CAPACITATION AND FERTILIZATION The relatively thick and rigid structure that invests the mam¬ malian egg, called the zona pellucida, has necessitated some

Ch. 107: Conception, Implantation, and Early Development

FIGURE 107-2. A, Photomicrograph of unfertilized, mature ovum with associated corona radiata cells. Coronal cells close to the ovum send processes through the zona pellucida. These processes (e.g., arrow) are evident as granules in the perivitelline space. B, Higher magnification in phase contrast shows these connecting processes of the corona radi¬ ata cells more clearly (arroiu). (From Shettles LB. Ovum humanum. Munich: Urban und Schwarzenberg, 1960; 42:52.) changes in the physiology of fertilization, particularly with respect to the sperm. Mammalian sperm require the occurrence of two events before they can fertilize an oocyte. The first, known as capaci¬ tation, is the process by which sperm become competent for fertilization, an act they are not able to accomplish before an appropriate, species-dependent incubation time within the female reproductive tract milieu or similar in vitro medium.11-13 During this time, the sperm not only mature but also attain a state of hyperactivated motility that is necessary for them to move through the length of the female reproductive tract and to generate the force necessary to pierce through the cumulus oophorus and the zona pellucida of the oocyte. In addition, certain incompletely defined factors known as decapacitation factors must be removed from the sperm before they become competent for fertilization. Presumably, these factors are macromolecules that are blocking certain receptor sites neces¬ sary for this functional change to occur, and there is evidence that removal of these factors increases the response of the sperm to extracellular Ca2+.n

1051

Once the sperm are capacitated, the acrosome reaction can begin, and it is through this process that the sperm can ulti¬ mately fuse with the oocyte. The morphology of the sperm head is such that an inner acrosomal membrane is immediately adjacent to the nuclear membrane of the cell, whereas an outer acrosomal membrane and the plasma membrane act as the lim¬ iting membrane of the acrosome.14 The acrosome itself contains proteases, such as acrosin, and other enzymes necessary for the sperm to navigate the interstices of the corona radiata. The outer acrosomal membrane possesses specific molecules for attachment to the zona before penetration of the egg, including a receptor that binds to a glycoprotein named ZP3 of the zona pellucida of the oocyte and a galactosyltransferase that recognizes N-acetylglucosamine residues.15-18 This morphol¬ ogy necessitates some interesting adaptations during the fusion of the sperm to the oocyte. Because the surface molecules neces¬ sary for attachment to the zona must be retained, the outer membrane must remain intact after the release of enzymes. The spermatozoon joins with the egg by membrane fusion of a mid¬ portion membrane, the equatorial region of the sperm head. The acrosome reaction, then, seems designed to create the structural alterations required for these various constraints to be overcome. First, the sperm-limiting membrane changes to allow influx of calcium, presumably along an electrochemical gradient. Immediately thereafter, the acrosomal membrane becomes fenestrated, appearing to allow the acrosomal con¬ tents to be released while leaving the acrosomal membrane, with its putative zona attachment elements, largely intact. The equatorial portion of the membrane is left intact for fusion with the oolemma, the limiting membrane of the unfertilized egg. Once fusion has occurred, the sperm head swells rapidly and forms the male pronucleus, leaving the sperm midpiece visible within the fertilized egg.19-22 Numerous cations play distinctive roles in these processes of capacitation and acrosomal exocytosis.22a Moreover, it is thought that the female reproductive tract is instrumental in regulating these processes by forming gradients of the cations at different positions along its length, as well as allowing their concentra¬ tions to change during certain times of the menstrual cycle.10 Ca2+, one of the most studied of these cations, is necessary for achieving the hyperactivated motility and the fertilizing ability associated with capacitation, in addition to being required for the acrosome reaction. It has been postulated that the binding of sperm to ZP3 of the zona pellucida triggers a G-protein path¬ way that ultimately leads to the release of bound Ca2+,15 and that this Ca2+ stimulates adenylate cyclase to produce cyclic adenosine monophosphate, which in turn activates cyclic ade¬ nosine monophosphate-dependent protein kinases that alter the sperm function during these prefertilization events.11 Na+ also has been shown to be critical for capacitation and the acrosome reaction, although much higher concentrations of Na+ are required for the latter process. Finally, K+ plays a cru¬ cial role in these events, albeit in a more regulatory capacity. High levels of K+ do not inhibit capacitation, but they do sup¬ press the fertilizing potential of the sperm. Before ovulation, Ca2+ and Na+ concentrations in the female reproductive tract are sufficient for capacitation, but the K+ concentration is too high to permit either the acrosome reaction to proceed or fertili¬ zation to occur. Follicular fluids released during ovulation, however, are thought to cause a substantial decrease in K+ con¬ centration, as well as an added increase in Na+ concentration, which result in the fulfillment of fertilizing potential. In addi¬ tion to the increased potential for fertilization during ovulation that is regulated by the concentrations of these ions, concentra¬ tions also seem to vary along the length of the female reproduc¬ tive tract to help ensure that sperm proceed through capacitation and the acrosome reaction at the proper time and place to optimize fertilization.10-11 Immediately after fertilization, the maternal genome is acti¬ vated and forms the female pronucleus. The sperm nucleus re-

1052

PART VII: ENDOCRINOLOGY OF THE FEMALE

forms and is evident morphologically as the male pronucleus. As the cells enter mitosis, the nuclear membranes of the pronuclei break down, and the chromosomes comigrate to the poles of the cell, where they are packaged as a unit in the nuclei of the prog¬ eny blastomeres. Thus, at the first cleavage, there is a symmetric division of the fertilized egg, and the two blastomeres have fused nuclei containing the maternal and the paternal genomes. It is clear that genetic information from both the mother and the father is an absolute requirement for normal development. When the maternal or paternal pronucleus is removed from fertilized mouse eggs and the egg is manipulated such that it contains either two maternal or two paternal pronuclei, devel¬ opment cannot proceed past midgestation. Bipaternal concep¬ tions form only placenta, while bimaternal conceptions form disorganized embryonal tissue.23'24 As a result of these types of experiments, it has become evident that the same gene derived from the mother may be functionally different when derived from the father, leading to the concept of imprinting,25 Imprint¬ ing refers to a situation in which a gene is "marked" or "imprinted" during either female or male gametogenesis so that it is not expressed and, consequently, either the remaining paternal or maternal allele is exclusively expressed.26 The mechanism of imprinting remains uncertain, and it is not clear if each imprinted gene is imprinted using the same mechanism. Whatever the mechanism, the imprint must be maintained in somatic tissues during specific periods of devel¬ opment but must be reversible in the germline. Most imprinted genes are methylated in a parental-specific manner in the germ¬ line, and DNA methylation appears to be the most likely mech¬ anism of imprinting.27-28 In fact, mice deficient for DNA methyltransferase, an enzyme that helps maintain methylation stability, lose monoallelic expression of several imprinted genes and die in the early postimplantation period, probably because of instability of primary imprints.29 Methylation may alter chro¬ matin structure and modulate binding of transcriptional regu¬ latory proteins to imprinted genes. The list of imprinted genes is expanding and includes Wilms tumor 1 (WT1), insulin, insulin¬ like growth factor-U (IGF-II), insulin-like growth factor-ll receptor (IGF-IIR), H19, X-inactive specific transcript (Xist), and others.28 The extent of monoallelic expression varies for different imprinted genes during development. These discoveries are of great importance to medicine because aberrant imprinting has been demonstrated in the set¬ ting of human syndromes and cancer. Biallelic expression of an imprinted gene results in overexpression of the gene product compared with monoallelic expression as seen with imprinting. Expression from two alleles may occur by loss of the imprint or by deletion of the imprinted allele, with reduplication of the expressed allele resulting in uniparental disomy. Paternal uni¬ parental disomy for the IGF-II locus has been described in Beckwith-Wiedemann syndrome (BWS).30 BWS is an over¬ growth disorder characterized by gigantism, macroglossia, and visceromegaly. Because IGF-II is maternally imprinted, redupli¬ cation of the paternal allele results in a double dose of IGF-II expression. Since IGF-II functions as a fetal growth factor, this may be in part responsible for the overgrowth phenotype. Loss of the maternal IGF-II imprint or paternal uniparental disomy, again resulting in a double dose of IGF-II expression, has also been described in Wilms tumor cells.31 Here the excess growth factor may contribute to tumorigenesis. Finally, inheritance of a paternal deletion of chromosome region 15qll—13 is associated with Prader-Willi syndrome, characterized by obesity, hypogo¬ nadism, and mental retardation, whereas inheritance of this same deletion on the maternal chromosome is associated with Angelman syndrome, characterized by ataxic movements, inappropriate laughter, mental retardation, and hyperactivity.32 In both cases, the allele located in this region on the one normal chromosome 15 is not capable of sustaining normal develop¬ ment, suggesting that imprinting occurs on a chromosome of particular parental descent. Clearly, imprinting plays a crucial

PREIMPLANTATION

IMPLANTATION

DAY 5-7

DAY 7-8

Embryonic

Zona Pellucida

Primitive Ectoderm

Inner Cell Mass Trophectoderm

Primitive Endoderm

Blastocoele

Perivitelline Space Abembryonic

POSTIMPLANTATION DAY 10-11

DAY 13-15 Primitive Streak

with maternal blood lakes

_

.

Endometrium

FIGURE 107-3. Early stages of mammalian development. The preim¬ plantation stages shown are blastocysts with and without their zonae pellucidae. The embryonic end of the embryo contains the inner cell mass, which will form the fetus. Trophectodermal cells are fated to form the extraembryonic tissues, including the placenta. The primitive endo¬ derm forms at the time of implantation and eventually will produce yolk sac structures. The primitive ectoderm will form the definitive ectoderm, endoderm, and mesoderm following the stages shown in the lower half of the diagram (postimplantation). The proamnion forms within the substance of the primitive ectoderm, and the trophoblast begins to differentiate into definitive placental structures (cytotrophoblast and syntrophoblast). By day 13, the primitive ectoderm has formed a single layer of columnar cells, and the craniocaudal groove (primitive streak) begins to form. Mesoderm differentiates from the primitive ectoderm at the point of the primitive streak in most primates.

role in determining nonequivalence of the maternal and pater¬ nal genomes and is necessary for normal development.

PREIMPLANTATION DEVELOPMENT The preimplantation period of development in mammals, the time from conception to nidation (implantation), has variable lengths in the various species. In humans, the preimplantation period lasts for ~7 days; in the mouse, it is 4 days, whereas in the rat, it is 5 days. The fertilized egg (Figs. 107-3 and 107-4) is morphologically similar to the mature unfertilized egg. The embryo at this stage is 100 pm in diameter, is associated with two polar bodies remaining from meiotic division, and is sur¬ rounded by the amorphous zona pellucida. The zona pellucida, which is composed primarily of three complex glycoproteins known as ZPI, ZP2, and ZP3, is important to early develop¬ ment for several, largely mechanical, reasons. First, there is evi¬ dence suggesting that certain glycoproteins of the zona pellucida may play a role in the recognition of the egg by the sperm. Competitive inhibition assays have shown that by incu¬ bating mouse sperm with ZP3 before fertilization, binding of the sperm to the zona pellucida is inhibited, thus suggesting that this glycoprotein is responsible for the recognition and binding of the sperm to the zona pellucida.15 Second, the zona responds nearly instantaneously to sperm penetration and ren¬ ders the egg impervious to additional penetration. Third, the zona provides a constraint to cleavage and ensures that as the

Ch. 107: Conception, Implantation, and Early Development

FIGURE 107-4. Photomicrographs (Hoffmann modulation contrast) of living preimplantation mouse embryos. A, One-cell pronuclear-stage egg ~12 hours after fertilization. A prominent pronucleus (arrow) and a polar body are evident. B, Two-cell cleavage stage. One of the two polar bodies is evident. C, Four-cell cleavage stage. One of the two polar bod¬ ies is evident. D, Eight-cell cleavage stage. E, Compacted 16-cell cleavage stage. F, Early blastocyst stage. A nascent blastocoele is evident. G, Mid¬ blastocyst stage. A well-formed blastocoele is evident in each embryo. Individual trophectodermal cells can be distinguished. The inner cell mass is apparent as an amorphous mass of cells. H, Expanded late blas¬ tocyst stage embryo. Inner cell mass is evident at lower right pole of the embryo. Individual trophectodermal cells are also evident. The embryos are surrounded by zonae pellucidae. The outside diameter of the embryos remains ~100 pm until the expanded blastocyst stage.

blastomeres divide, they remain together and in the proper ori¬ entation. Finally, the zona prevents the naturally sticky cleavagestage embryo from adhering to the wall of the oviduct as it progresses to the uterus. The mammalian oviduct also plays an important role in these early stages of preimplantation development. Not only does it provide a route through which the embryo is trans¬ ported from the ovary to its site of implantation in the uterus, but it also provides a crucial timing mechanism for a process known as cleavage division.33-35 At this stage of development, the embryo divides symmetrically and reductively such that a geo¬ metric increase in the number of cells (blastomeres) occurs without an actual increase in the overall size of the embryo.

1053

These divisions occur entirely in the oviduct while the embryo is being propelled through its length as a result of ciliary action and muscular contractions in the oviduct wall. The primary function of development at this stage is to provide additional cells and membrane. Beginning after the first division in the mouse embryo and after the second division in the human embryo, a critical tran¬ sition occurs in the genetic control of development.36 Before this time, the embryo contains a host of maternally derived mRNAs, ribosomes, and macromolecules that are sufficient to drive transcription and translation through the first (or second, as in the human embryo) cleavage division.37 Further develop¬ ment, however, is dependent on the activation of embryonic control of transcription and the subsequent degradation of maternal mRNAs and proteins.37 In the mouse, one population of polypeptides exhibits at least a two-fold decrease in abun¬ dance during the two-cell stage, whereas another population of polypeptides exhibits a similar increase in abundance.38 Although this change most likely reflects the degradation of maternal mRNA and the appearance of new embryonic mRNA, it is possible that this transition is not complete and that some maternal products still may persist for a time after this transition.37 Once the embryonic genome has been activated, two impor¬ tant morphogenetic events occur in the embryo during the pre¬ implantation period. The first, known as compaction, occurs late in the eight-cell stage when individual blastomeres condense and their boundaries become less prominent, thus forming a cellular mass known as a morula (see Fig. 107-4F). This process results in several profound changes in the embryo (Fig. 107-5). During this time, several new gene products are expressed that contribute to many of the morphologic manifestations of com¬ paction. Included in this group are E-cadherin, gap junction proteins, tight junction proteins, growth factors, and compo¬ nents of the cytoskeleton.39 E-cadherin (which originally was referred to as uvomorulin in the morula-stage mouse and later was identified as E-cadherin) acts as a Ca2+-dependent cell adhesion molecule that binds adjacent blastomeres together and appears to facilitate the formation of junctional complexes, which include both gap junctions and tight junctions. Gap junc¬ tions form between all cells of the compaction-stage embryo and are constructed from a family of proteins known as the connexins, the structure of which creates channels between cells that allow for communication between blastomeres. During compaction, these gap junctions migrate from central regions of intercellular contact to peripheral locations of contact where tight junctions also form, thus creating junctional complexes between lateral surfaces of the outer blastomeres.37,39'40 The tight junctions within these complexes serve a dual purpose. First, tight junctions play a critical role in the second preim¬ plantation morphogenetic process known as cavitation. Second, they contribute to the polarization of the outer blastomeres by separating an apical region, where microvilli will form, from a basolateral region, to which the nuclei will migrate. By the 16cell stage, these outer polar blastomeres form the trophectoderm, a cell lineage leading to the formation of extraembryonic tissues such as the placenta, whereas the inner apolar blas¬ tomeres form the inner cell mass, which ultimately will develop into the embryo proper.41,42 This is the first stage of commit¬ ment of cells to a particular fate. Before this time, each blastomere in the two-cell, four-cell, and early eight-cell embryo is totipotent and, therefore, has the potential to develop into a complete organism when it is isolated from the remaining embryo. Once the 16-cell stage of development has been reached, however, the embryo has sufficient cells to form an inside and an outside, and thus establishes the conditions nec¬ essary for the first step of embryonic commitment. It is at this point that some of the embryo's cells lose their totipotency. Although the details of the mechanism by which the mor¬ phologic change in cellular contact associated with compaction

1054

PART VII: ENDOCRINOLOGY OF THE FEMALE

■ 8-cell morula

• • • • C

o

o ro

a

E

o

o

I

cell adhesion (i.e. E-cadherin) microvilli -*vl,/ basal nuclei -* • polarization of outer blastomeres

(crosssection)

• continued polarization of outer blastomeres • formation of tight junctions gap junctions

• 16-cell post-compacted morula • 2 cell lineages distinguishable: trophectoderm inner cell mass (crosssection)

c o re

• ion gradient established • osmotic uptake of water • formation of blastocoele

>

re O

detected by the blastomeres that causes a G protein to activate phospholipase C, which in turn cleaves phosphatidylinositol 4,5-bisphosphate. This results in the formation of two products: Ins (1,4,5)P3, which causes an increase in intracellular calcium, and 1,2-diacylglycerol, which activates protein kinase C. The activated protein kinase C is then available to phosphorylate proteins involved in nuclear migration and cell adhesion.43 It remains to be seen, however, what triggers this pathway, if indeed the phosphatidylinositol cycle functions to activate pro¬ tein kinase C during compaction. The second major morphogenetic event to occur in preim¬ plantation development is known as cavitation. This process begins several days after conception (3 days in the mouse, 4 in the rat, and 6 in the human) and culminates in the formation of the blastocyst (see Fig. 107-4F through H). At least two factors are known to be critical to the proper execution of this event. First, the tight junctions that form between plasma membranes of the outer blastomeres not only provide an apical/basolateral polarization of the cells, but also prevent paracellular leakage of fluid from the nascent blastocoele. Second, the cell adhesion properties of E-cadherin, which is located in the basolateral regions of the plasma membrane, are crucial in restricting the distribution of Na+/K+-adenosine triphosphatases to this region as well. With these two factors in place, the polar distri¬ bution of Na+/K+-adenosine triphosphatases to this basolateral location causes a Na+ gradient to be established within the inte¬ rior of the embryo, and subsequently osmotic uptake of water occurs such that it accumulates in the extracellular space of the nascent blastocoele. Because the tight junctions prevent this fluid from leaking out, it accumulates until the blastocoelic cav¬ ity is fully expanded.37'39 At this stage, the two cell types are easily distinguishable: The inner cell mass cells are located internally at the embryonic pole of the embryo, whereas the trophectodermal cells, which are extremely large, owing to acytokinetic cell division, surround both the inner cell mass cells and the blastocoele.

OVIDUCT TRANSPORT FIGURE 107-5. Compaction and cavitation of the preimplantation mouse embryo. During the late eight-cell stage, embryos begin a mor¬ phogenetic process known as compaction, which ultimately results in the polarization of the outer blastomeres and the establishment of two cell lineages. During this process, individual blastomeres become less evi¬ dent as the cell adhesion molecule E-cadherin functions to bind adjacent cells to one another. Simultaneously, microvilli form on the apical sur¬ faces of the outer blastomeres, the nuclei migrate basolaterally, gap junctions form between all adjacent blastomeres, and tight junctions form between outer blastomeres, thus separating apical and basolateral regions. These changes result in the formation of two cell lineages by the 16-cell stage; the outer polar cells will form the trophectoderm, whereas the inner apolar cells will form the inner cell mass. The second morphogenetic event, cavitation, begins as soon as the two cell lineages are established and results in the formation of a blastocoelic cavity. The basolateral location of E-cadherin aids in restricting the distribution of Na+/K+-adenosine triphosphatases to this region, thus causing a Na+ gradient to form within Ere embryo. Water flows into the embryo osmotically, and the presence of the tight junctions in the outer blas¬ tomeres prevents this fluid from leaking out. Thus, a blastocoelic cavity forms, and the embryo is now known as a blastocyst.

can induce all of these varied events are incompletely deci¬ phered, evidence suggests that protein kinase C and subse¬ quent phosphorylation of proteins may be involved.43-45 Early activation of protein kinase C not only can trigger premature compaction through its effect on E-cadherin, but also can induce the migration of blastomere nuclei to a basolateral posi¬ tion.43-44 If protein kinase C functions in the signal pathway leading to events of compaction much as it does in other signal pathways, it is possible that some type of surface signal is

The role of oviduct transport in the maturation of the mamma¬ lian embryo is poorly understood. It is reasonable to assume that oviduct fluids have a central role in the nourishment of the embryo and in gas exchange; however, the fluids also may con¬ tain substances that control or somehow enhance the develop¬ ment of the cleavage-stage embryo. The mammalian embryo can survive and progress in various artificial media. The embryo completes its passage through the oviduct at the early blastocyst stage and is propelled into the uterus. In the mouse, this occurs at day 3 of gestation, and in humans, at day 5 to 6. The blastocyst continues to develop in the uterus for another 24 to 48 hours, during which time it greatly expands its blastocoelic cavity until the inner cell mass is little more than a plaque of cells on the embryonic pole. Then, the embryo loses its zona pellucida. Despite attempts to isolate the factors involved in this process, little is known about the loss of the zona. In vitro, the zona can be removed by enzymatic digestion, mechanical disruption,16-17 or an acid milieu.46 In rabbits in vivo, the egg vestments are removed enzymatically at the implantation site and not while the blastocyst is free in the uterus.

X CHROMOSOME INACTIVATION In eutherian females (placental mammals, i.e., other than monotremes and marsupials), one of the two X chromosomes is inactivated early in embryonic development, thus providing a mechanism for genetic dosage compensation.47 In eutherians, this inactivation begins in the trophectoderm in the early blasto¬ cyst stage and is characterized by a preferential paternal X chro-

Ch. 107: Conception, Implantation, and Early Development mosome inactivation. This also is true of X chromosome inactivation that subsequently occurs in the primitive endoderm during the midblastocyst stage. During the late blastocyst stage, however, X chromosome inactivation occurs randomly in the inner cell mass, with no paternal or maternal preference, thus resulting in mosaic females composed of a mixture of cells that have either a maternally or paternally active X chromosome. In somatic cells, this inactivation becomes fixed such that all descendants from a particular cell maintain the same inactivated X chromosome. In the germline, however, this inactivation must be reversed at the time of meiosis so that each X chromosome has an equal chance of contributing to the gametes.48 In marsupials, this pattern of X chromosome inactivation is different in that it is always the paternal X chromosome that is inactivated. This may not necessarily be a functional difference, however, because the marsupial blastocyst has no inner cell mass. The coincidence of the timing of X chromosome inactiva¬ tion and cell commitment to either trophectoderm or inner cell mass lineages in eutherians strongly suggests that these two processes are linked in some meaningful way. Perhaps the pref¬ erential X chromosome inactivation may be part of a system that is necessary to prevent rejection of the conceptus. Alterna¬ tively, it also has been suggested that preferential X chromo¬ some inactivation may prevent the accumulation of genes necessary to the proper development of extraembryonic mem¬ branes on the paternal X chromosome. This would adversely affect the development of boys because they do not possess a paternally derived X chromosome.48-49 X chromosome inactivation was first proposed as a mecha¬ nism of gene dosage control by Lyon.50-51 The best evidence of it exists in women heterozygous at the glucose-6-phosphate dehydrogenase (G6PD) locus of the X chromosome. G6PD is a dimeric dimorphic enzyme; that is, there are two distinguish¬ able allelic forms of the enzyme (isoenzymes) and the enzyme is composed of two subunits that must combine to form a holoenzyme. In heterozygous women, the two isoenzymes can combine to form heteropolymeric forms, which are distinguish¬ able from the other two subunits. When X chromosome inacti¬ vation occurs in women heterozygous at the G6PD locus, two populations of cells are created: one with the paternal allele active and the other with the maternal allele active. The two isoenzymes can be distinguished by electrophoresis. No het¬ eropolymeric form is present, however, indicating that the two alleles were not active simultaneously in the same cells at the time of sampling.52 Insights have been made into the multistep mechanism con¬ trolling X inactivation in mammals. First, the number of X chro¬ mosomes is counted by an as yet unknown process, tallying up the number of X inactivation centers (Xic). Second, a single X chromosome is chosen to remain active, and inactivation of any additional X chromosomes is initiated by expression of the Xist from the Xic. Finally, this inactivation spreads over the length of each inactive chromosome. Xist is only expressed on the inactive X in somatic cells of females, in male germ cells during spermatogenesis, and on the imprinted paternal X chromosome of the trophectoderm and primitive endoderm of the blastocyst. It does not encode a pro¬ tein but instead remains as an RNA moiety that stays bound to the X chromosome undergoing inactivation. Furthermore, the expression of Xist coincides with the imprinted X inactivation that occurs in the trophectoderm and primitive endoderm of the blastocyst, which is then turned off before X inactivation in the embryonic lineage. DNA methylation of Xist correlates with its activity; it is unmethylated where it is expressed on an inac¬ tive X chromosome and methylated as an inactive allele on an active X chromosome. Xist can operate from multiple promoters, resulting in pro¬ duction of either stable or unstable RNA, suggesting one mech¬ anism whereby Xist can be developmentally regulated. Stable Xist forms as a result of activation of one promoter on the

1055

imprinted paternal X chromosome of the trophectoderm. Alter¬ natively, unstable Xist RNA results from activation of a differ¬ ent promoter when the imprint is erased before random X inactivation of the somatic cells.53-54 Questions that remain to be answered about X inactivation are how X/'cs are counted and how an inactivation signal can propagate throughout the entire length of the X chromosome and yet let certain genes escape this signal.

IMPLANTATION The embryo now undergoes implantation, which begins with attachment of the late blastocyst to the uterine tissue at a nida¬ tion site. The selection of this site is tightly regulated, because it usually occurs in a predictable manner, but little else is known. Implantation can be classified on the basis of the usual position of the site in the uterus and, hence, may be noninvasive and central, noninvasive and eccentric, or interstitial as in humans (Fig. 107-6). In humans, the embryo attaches to the lin¬ ing of the uterine fundus, with the embryonic pole usually attaching to the antimesometrial lining. The endometrial cells of the uterus have microvilli on their luminal surfaces that begin to interdigitate with the microvilli of the trophectodermal cells. Pinocytosis (the cellular process of active engulfing of liquid) in the endometrial epithelial cells increases at this time and is thought to enhance or at least stabilize attachment, perhaps by removing uterine fluids from the attachment site. This pinocytosis is stimulated by progestins and inhibited by estrogens.21 Actual cell fusion between the embryonic trophec¬ toderm and the uterine epithelium does not occur in most spe¬ cies. The presence of the blastocyst in the uterus undoubtedly provides some signal to the uterus and to the ovary to main¬ tain the pregnancy.55 The blastocyst is capable of producing human chorionic gonadotropin, which supports the corpus luteum, and the luteal phase of human conception cycles maintains higher progesterone levels from day 3 through day 8 than in nonconception cycles.56-58 Implantation may be enhanced by proteases. These pro¬ teolytic enzymes are thought to have two functions: to cause the removal of the zona pellucida, which must precede the attach¬ ment of the embryo to the uterine lining, and to aid the embryo's invasion of the endometrial lining. The cells of the human trophoblast frankly invade uterine tissue as implanta¬ tion proceeds. Early theories of the role of such enzymes sug¬ gested that they were necessary to digest maternal tissues; however, their actual role, if any, beyond the removal of the zona may be far more subtle. For example, such enzymes may act on the invasion process through limited proteolysis (e.g., blastolemmase) by beginning a cascade of activation of other enzymes.34 Implantation has at least three phases. Tire first is attachment, in which specific receptor sites may be responsible for binding of either the embryonic pole or the abembryonic pole, depend¬ ing on the species, to the endometrial epithelium. The second phase is invasion. In humans, the trophectodermal cells invade through the basement membrane of the uterine epithelium to establish a nidation site in the stroma of the endometrium (see Fig. 107-6). The ability of the human embryo to invade tissue may explain the high frequency of ectopic pregnancies in women relative to other mammalian species. The third phase is the endometrial response to the implanted embryo. In a few eutherian species (including humans, other primates, and murine rodents), the uterine stromal cells undergo a specific reaction called decidualization. The name derives from the fact that these cells occasionally are shed at term. The stromal cells in the immediate area of the embryo become large, eosinophilic, and transcriptionally active. The cells of the decidual swelling may be important in the support of the pregnancy (e.g., by the pro¬ duction of luteotropin, which supports the corpus luteum); in

1056

PART VII: ENDOCRINOLOGY OF THE FEMALE Cytotrophoblast Blostocelo Embryoblost (inner cell mass) Cytotrophoblast

Syncytiotrophoblas'

Uterine mucosal epithelium

Uterine gland

Connective tissue cells in process of decidual transformation Spiral artery

Dilated vein

A Epithelium

Cytotrophoblast Syncytiotrophoblast Mesenchyme Heuser's membrane Primitive yolk sac Entoderm Ectoderm

Glands

Ammotic cavity Amnion Lacunae of syncytiotrophoblast

Maternol mucosa

Myometrium

FIGURE 107-6. Diagram of human implantation site. A, Trophoblast invasion of uterine epithelium at the time of attachment. B, Nidation site is completed with the embryo in its interstitial position. There is a single layer of abembryonic trophectodermal cells in contact with the uterine lumen. Primitive entoderm and primitive ectoderm are distinguishable. (From TuchmannDuplessis H, David G, Haegel P. Illustrated human embryology, vol I: embryogenesis. New York: Springer-Verlag, 1972.)

the prevention of immune rejection of the implanted embryo; or in some other, unknown capacity.59 Among mammals, there is a great variation in the specific details of development at this stage. Although many attempts at generalizations across species have been made, by and large, they are either not helpful or are actually incorrect. For exam¬ ple, much of what is kmown concerning reproductive endocri¬ nology is derived from experiments in mouse and rat. These animals, like other diapause mammals (i.e., those that can delay implantation while keeping the embryo alive), express an estro¬ gen surge, which seems to be necessary for the progesteroneprimed uterus to accept the initiation of implantation. This is not true of humans. In the rabbit, the zona pellucida is removed

B

at the site of implantation, and the blastocyst is invested with additional coverings that must be removed enzymatically. In the mouse, the blastocyst can exist free in the uterine cavity without its zona pellucida. Virtually nothing is known about the removal of the zona in humans. One important reason for these variations is that there are several successful solutions to problems of early development in viviparous animals, and many of the specific details of reproductive strategy do not allow for clear winners or clear losers. While the molecular control of implantation is not fully understood, many factors are necessary for proper implanta¬ tion of the embryo. Most of the factors identified to date are produced or released by the uterus (many in response to estro-

Ch. 107: Conception, Implantation, and Early Development gen or progesterone), but it is becoming evident that embry¬ onic factors are important as well. COX-1, leukemia inhibitory factor (LIF), HB-EGF, and amphiregulin are expressed by the uterine epithelium at the time of implantation, while adhesion molecules such as lectins, carbohydrate moieties, and heparin sulfate proteoglycan interact between the surfaces of the blastocyst and endometrium. Interleukin (IL)-la and IL-1(3 are released by the blastocyst and adhere to the endometrial epithelial ftyintegrin subunit, while trophoblast giant cells produce proteinases such as gelatinases A and B and urokinasetype plasminogen activator (uPA) that mediate the invasion of the decidua.60-64 hi diapause mammals such as the mouse or rat, there are uter¬ ine inhibitory factors that can prevent implantation. The blasto¬ cyst-stage embryo can overcome the inhibition by a process of activation, which occurs in response to the prenidation estrogen surge in these animals. This process does not occur in humans.

POSTIMPLANTATION DEVELOPMENT Development after implantation is rapid and complex. The embryo must establish both its placental compartment and its definitive fetal structures in a short time. The polar or embry¬ onic trophectoderm (that overlying the inner cell mass) devel¬ ops into an ectoplacental cone in the mouse, whereas in most primates, the trophoblast differentiates into syncytiotrophoblast and cytotrophoblast, the latter having a high mitotic rate. Rapid division produces a syncytial trophoblast surrounding the pri¬ mate embryo, although the mural trophectoderm (that facing the uterine cavity) remains a single layer of cells. Lacunar spaces form within the syntrophoblast, which eventually becomes contiguous with the maternal capillary circulation, into which the chorionic villi will grow. The ectoplacental cone of the mouse and rat undergoes similar development, and the resulting placental structure is hemochorial, as in humans. The major placental classification among mammalian orders is derived from the number of tissue layers that separate the fetal and maternal circulations. There are six such potential barriers to exchange. FFumans, like many other primates and murine species, have a hemochorial placenta in which three fetal tissues (endothelium, connective tissue, and chorionic epithelium) are bathed in maternal blood.65 As the process of placentation proceeds, definitive embry¬ onic structures are developing. Immediately after implantation, a layer of cells appears at the blastocoele margin on the side of the inner cell mass. This layer is called the endoderm. The remaining cells of the inner cell mass are now called the epiblast or the primitive ectoderm. The endoderm proliferates rapidly and eventually surrounds the blastocoele. The epiblast cells (embryonic ectoderm) are now arranged in a columnar manner. Cells contiguous with the epiblast, called amnioblasts, appear; spaces between the amnioblasts develop (the proamnion) and eventually form the amnionic cavity. Although it is a matter of some debate, it seems possible that the amnioblast cells are the source of the amniotic fluid, which cushions and thereby pro¬ tects the developing embryo. Apoptosis also plays a critical role in cavitation of the early embryo (not to be confused with cavi¬ tation of the blastocyst). A signal from the primitive endoderm acts over a short distance to induce apoptosis of the inner ecto¬ derm cells, while survival of the outer ectodermal cells is medi¬ ated by interaction with the adjacent basement membrane that separates the ectoderm from the endoderm.66 By approximately 7 days in the mouse and 13 days in humans, all three germ layers are present. The primitive ecto¬ derm (epiblast) gives rise to definitive ectoderm, definitive endoderm, and mesoderm, which appears between the primi¬ tive endoderm and the primitive ectoderm. The primitive endo¬ derm gives rise to several extraembryonic tissues (see Fig. 107-3). The first indication of craniocaudal axis and bilateral symmetry

1057

in the embryo appears as a longitudinal depression in the columnar embryonic ectoderm. This depression is called the primitive streak and, in most primates, it seems to be the site of origin of mesodermal cells (see Fig. 107-4). Most of the available information concerning cell lineage in the early embryo is derived from experiments performed in the mouse, and it is not clear whether these principal features of the fates of early cells are applicable to human development. It may be some time before this can be determined because, at present, the only way this information can be obtained is by experimental manipu¬ lation and disruption of the embryo. A case in point is a series of experiments that defined the ultimate fates of areas of the egg cylinder-stage embryo of the mouse (day 7). This work required microsurgical removal of some structures from the embryo with development in culture, or transplantation of radioactively labeled structures to unlabeled embryos, after the development of the combined structure. Early postimplantation stages are responsible for establish¬ ing the structures that ultimately allow organogenesis to pro¬ ceed.33'35 An understanding of the molecular biology of the control of differentiation of the definitive structures will have far-reaching implications for many gestational diseases and certainly for human cancer.67 Correct fetal development requires the coordinated expression of thousands of genes. The correct temporal and spatial expression of these genes could not occur without the intervention of some relatively small set of supervisor genes that can orchestrate the process. Such genes are being found based on information from diverse ani¬ mal studies.68

SPONTANEOUS ABORTION The most common manifestation of the failure of embryonic and fetal development is spontaneous abortion: the failure of con¬ ception to produce a live birth. Spontaneous abortion, then, is either the disruption of pregnancy once it can be recognized or the expulsion of a nonviable fetus. Precise clinical definitions are much more difficult. Most often, these definitions must invoke low birth weight, because below certain weights, the fetus is unlikely to survive. Other definitions include loss of pregnancy before 20 or 28 weeks of gestation. Accurate esti¬ mates of the incidence of spontaneous abortion, therefore, are difficult to obtain. The frequency of clinically evident spontane¬ ous abortion is ~15% of pregnancies. Undoubtedly, the risk is much higher in women with a previous spontaneous abortion, with the risk as high as 46% after three consecutive abortions. However, if the abortus is karyotypically abnormal, the risk of consecutive abortion is substantially lower.69-70 The association of prior spontaneous abortion with subsequent poor pregnancy outcome has been well documented, even when all other risk factors have been controlled. The effects of specific risk factors seem to be much stronger than the history. Early pregnancy losses are occult. Early abortion has many causes and must not be considered a single disease entity. One of the principal observations in human embryos that fail to cleave normally is the presence of structural abnormalities of chromo¬ somes. In a large series of fetal deaths, the karyotypes of the off¬ spring were compared with the morphology of the conception products.71 More than half of the small or unformed fetuses had chromosomal abnormalities, whereas only 6% of fetuses of nor¬ mal size with or without malformations had chromosomal aberrations (see Chap. 90). Intrauterine death may occur in asso¬ ciation with chromosomal abnormalities that also can be seen in live births. These deaths result from the failure of embryonic development, not the gross anomalies frequently associated in live offspring with the deviant karyotype. There may exist a con¬ tinuum of anomalies in the offspring into which spontaneous abortion fits, from failure of fetal development through to birth with malformations. Nonchromosomal causes of pregnancy loss

1058

PART

VII: ENDOCRINOLOGY OF THE FEMALE

include maternal metabolic disturbances such as endometrial growth factor disturbances or hyperglycemia.72'73 One potential source of disruption of pregnancy is exposure of the woman to toxic substances. Of particular concern are exposures in the early periods of pregnancy. Few data are avail¬ able, however, in some measure because of the traditional view that preimplantation development is refractory to toxic insult. However, the general presumption that early-stage embryos are either killed or left unaffected to implant and develop nor¬ mally is an oversimplification. For example, the blastocyst is sensitive to cyclophosphamide, heavy metals, and trypan blue. Such exposures decrease cell numbers in the early embryo and can lead to vascular anomalies in midgestation when exposure occurs at the blastocyst stage. Exposure to toxic substances can be environmental, such as in the workplace, or self-inflicted, such as maternal smoking.733 Maternal smoking is important because of the numerous per¬ sons involved, and has been implicated by association in a wide array of pregnancy complications involving both the mother and the offspring. These complications include low birth weight, spontaneous abortion, sudden infant death syndrome, placenta previa, excessive maternal bleeding, and perinatal mortality.74-76 Many laboratories have been investigating possi¬ ble reasons for the adverse role of maternal smoking in preg¬ nancy outcome (see Chap. 234), and several conclusions have emerged. First, the embryo can be affected directly by chemical exposure; there need not be any intervening maternal role. Nevertheless, injury to maternal pregnancy support systems, such as the corpus luteum, may occur, or maternal tissues may activate deleterious compounds in tobacco smoke. Second, the embryo is at risk for adverse effects much sooner than was pre¬ viously suspected. The blastocyst-stage embryo is sensitive to compounds such as those in cigarette smoke with respect to implantation, decidual response, gross dysmorphogenesis, live birth rate, and perinatal mortality. These events can be mani¬ fested long after the exposure to the chemicals.77-81 Because mothers are unaware of early pregnancy, these data may require a reevaluation of the advice given to women who are considering pregnancy: It is becoming clear that one should not wait for evidence of the pregnancy to refine the potential envi¬ ronment of the developing embryo.82

PERSPECTIVES The study of the progression of embryonic tissue is the study of evolution, organization, differentiation, and molecular control. It has attracted the attention of endocrinologists, biologists, cli¬ nicians, and amateur naturalists for centuries. There can be no doubt that detailed investigation of the issues surrounding reproductive strategies of species both related and unrelated to humans will yield abundant insight that will help alleviate human ailments as diverse as birth defects and cancer.

REFERENCES 1. Fabro S. Reproductive toxicology: state of the art. Am J Ind Med 1983; 4:391. 2. Roberts CJ, Lowe CR. Where have all the conceptions gone? Lancet 1975; 1:498. 3. Hertig AT. The overall problem in man. In: Benirschke K, ed. Comparative aspects of reproductive failure. Berlin: Springer-Verlag, 1967:11. 4. King CR, Pernoll ML, Prescott G. Reproductive wastage. Obstet Gynecol Annu 1982; 11:59. 5. Newbold RR, Carter DB, Harris SE, et al. Molecular differentiation of the mouse genital tract: altered protein synthesis following prenatal exposure to diethylstilbestrol. Biol Reprod 1984; 30:459. 6. Shettles LB. Ovum humanum. Munich: Urban und Schwarzenberg, 1960:79. 7. Racowsky C, Satterlie RA. Metabolic, fluorescent dye and electrical cou¬ pling between hamster oocytes and cumulus cells during meiotic matura¬ tion in vivo and in vitro. Dev Biol 1985; 108:191.

8. Jansen RP. Endocrine response in the fallopian tube. Endocr Rev 1984; 5:525. 9. Chang MC. The meaning of sperm capacitation: a historical perspective. J Androl 1984; 5:45. 10. O'Neill C, Quinn P. Inhibitory influence of uterine secretions on mouse blastocysts decreases at the time of blastocyst activation. J Reprod Fertil 1983; 68:269. 11. Fraser LR, Umar G, Sayed S. Na'-requiring mechanisms modulate capaci¬ tation and acrosomal "exocytosis in mouse spermatozoa. J Reprod Fertil 1993; 97:539. 12. Fraser LR. Requirements for successful mammalian sperm capacitation and fertilization. Arch Pathol Lab Med 1992; 116:345. 13. Spungin B, Levinshal T, Rubinstein S, Breitbart H. A cell free system reveals that capacitation is a prerequisite for membrane fusion during the acrosome reaction. FEBS Lett 1992; 311:155. 14. Oura C, Toshimori K. Ultrastructural studies on the fertilization of mam¬ malian gametes. Int Rev Cytol 1990; 122:105. 15. Gilbert SF. Developmental biology, 3rd ed. Sunderland, MA: Sinauer Asso¬ ciates Inc, 1991:33. 16. Bleil JD, Wassarman PM. Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking. Proc Natl Acad Sci U S A 1990; 87:5563. 17. Shur BD, Hall NG. A role for mouse sperm surface galactosyltransferase in sperm binding to the egg zona peUucida. J Cell Biol 1982; 95:574. 18. Shur BD, Neely CA. Plasma membrane association purification and partial characterization of mouse sperm (3 1,4-galactosyltransferase. J Biol Chem 1988; 268:17706. 19. Bedford JM. Significance of the need for sperm capacitation before fertiliza¬ tion in eutherian mammals. Biol Reprod 1983; 28:108. 20. Hinrichsen-Kohane AC, Hinrichsen MJ, Schill WB. Molecular events lead¬ ing to fertilization—a review. Andrologia 1984; 16:321. 21. Hendrickx AG. Disorders of fertilization, transport, and implantation. Prog Clin Biol Res 1984; 160:211. 22. Farooqui AA. Biochemistry of sperm capacitation. Int J Biochem 1983; 15:463. 22a. Espinosaal F, Lopez-Gonzaleza T, Munoz-Garaya C, et al. Dual regulation of the T-Type Ca (2+) current by serum albumin and beta-estradiol in mammalian spermatogenic cells. FEBS Lett 2000; 475:251. 23. Barton SC, Surani MAH, Norris ML. Role of paternal and maternal genomes in mouse development. Nature 1994; 311:374. 24. Spindle A, Sturm KS, Flannery M, et al. Defective chorioallantoic fusion in mid-gestation lethality of parthenogenone-tetraploid chimeras. Dev Dyn 1996; 173:447. 25. Sapienza C, Peterson AC, Rossant J, et al. Degree of methylation of trans¬ genes is dependent on gamete of origin. Nature 1987; 328:251. 26. Pedersen RA, Sturm KS, Rappolee DA, Werb Z. Effects of imprinting on early development of mouse embryos. In: Bauister BD, ed. Preimplantation embryo development. New York: Springer-Verlag, 1993:212. 27. Surani MA. Imprinting and the initiation of gene silencing in the germ line. Cell 1998; 93:309. 28. Barlow DP. Gametic imprinting in mammals. Science 1995; 270:1610. 29. Li E, Beard C, Jaenisch R. Role for DNA methylation in genomic imprint¬ ing. Nature 1993; 366:362. 30. Junien C. Beckwith-Wiedemann syndrome, tumorigenesis and imprinting. Curr Opin Genet Dev 1992; 2:431. 31. Okawa O, Eccles MR, Szeto J, et al. Relaxation of insulin-like growth factor II gene imprinting implicated in Wilms' tumour. Nature 1993; 362:749. 32. Cassiday SB, Schwartz S. Prader-Willi and Angelman syndromes: disor¬ ders of genomic imprinting. Medicine 1998; 77:140. 33. McLaren A. Early mammalian development. Prog Clin Biol Res 1985; 163A:29. 34. Denker HW. Basic aspects of ovoimplantation. Obstet Gynecol Annu 1983; 12:15. 35. Swartz WJ. Early mammalian embryonic development. Am J Ind Med 1983; 4:51. 36. Telford NA, Watson AJ, Schultz GA. Transition from maternal to embry¬ onic control in early mammalian development: a comparison of several species. Mol Reprod Dev 1990; 26:90. 37. Watson AJ, Kidder GM, Schultz GA. How to make a blastocyst. Biochem Cell Biol 1992; 70:849. 38. Latham KE, Garrels JI, Chang C, Solter D. Quantitative analysis of protein synthesis in mouse embryos 1. Extensive reprogramming at the one- and two-cell stages. Development 1991; 112:921. 39. Watson AJ. The cell biology of blastocyst development. Mol Reprod Dev1992; 33:492. 40. Becker DL, Leclerc-David C, Warner A. The relationship of gap junctions and compaction in the preimplantation mouse embryo. In: Stern C, Ing¬ ham P, eds. Gastrulation (Dev Suppl). Cambridge: The Company of Biolo¬ gists Limited, 1992:113. 41. Rossant J, Papaioannou VE. The biology of embryogenesis. In: Sherman MI, ed. Concepts in mammalian embryogenesis. Cambridge, MA: MIT Press, 1977:36. 42. Sutherland AE, Calarco-Gillam PG. Analysis of compaction in the preim¬ plantation mouse embryo. Dev Biol 1983; 100:328. 43. Winkel GK, Ferguson JE, Takeichi M, Nuccitelli R. Activation of protein kinase C triggers premature compaction in the four-cell stage mouse embryo. Dev Biol 1990; 138:1. 44. Ohsugi M, Ohsawa T, Yamamura H. Involvement of protein kinase C in nuclear migration during compaction and the mechanism of the migration: analyses in two-cell mouse embryos. Dev Biol 1993; 156:146.

Ch. 108: The Maternal-Fetal-Placental Unit 45. Bloom T, McConnell J. Changes in protein phosphorylation associated with compaction of the mouse preimplantation embryo. Mol Reprod Dev 1990; 26:199. 46. Nicolson GL, Yanagimachi R, Yanagimachi H. Ultrastructural localization of lectin binding site on the zonae pellucidae and plasma membranes of mammalian eggs. ] Cell Biol 1975; 66:263. 47. Migeon BR. X-chromosome inactivation: molecular mechanisms and genetic consequences. Trends Genet 1994; 10:230. 48. Gartler SM, Dyer KA, Goldman MA. Mammalian X-chromosome inactiva¬ tion. Mol Genet Med 1992; 2:121. 49. Solter D. Differential imprinting and expression of maternal and paternal genomes. Annu Rev Genet 1988; 88:127. 50. Lyon MF. Mechanisms and evolutionary origins of variable X-chromosome activity in mammals. Proc R Soc Lond [Biol] 1974; 187:243. 51. Chapman VM, Shows TB. Somatic cell genetic evidence for X-chromosome linkage of three enzymes in the mouse. Nature 1976; 259:665. 52. Migeon BR. Glucose-6-phosphate dehydrogenase as a probe for the study of X-chromosome inactivation in human females. Curr Top Biol Med Res 1983; 9:189. 53. Goto T, Monk M. Regulation of X-chromosome inactivation in develop¬ ment in mice and humans. Microbiol Mol Biol Rev 1998; 62:362. 54. Johnston CM, Nesterova TB, Formstone EJ, et al. Developmentally regulated Xist promoter switch mediates initiation of X inactivation. Cell 1998; 94:809. 55. Kennedy TG. Embryonic signals and the initiation of blastocyst implanta¬ tion. Aust J Biol Sci 1983; 36:531. 56. Casper RF, Wilson E, Collins JA, et al. Enhancement of human implanta¬ tion by exogenous chorionic gonadotropin. (Letter). Lancet 1983; 2:1191. 57. Kusuda M, Nakamura G, Matsukuma K, et al. Corpus luteum insufficiency as a cause of nidatory failure. Acta Obstet Gynecol Scand 1983; 62:199. 58. Buster JE. Gestational changes in steroid hormone biosynthesis, secretion, metabolism, and action. Clin Perinatol 1983; 10:527. 59. Bazer FW, Roberts RM. Biochemical aspects of conceptus-endometrial interactions. J Exp Zool 1983; 228:373. 60. Rinkenberger JL, Cross JC, Werb Z. Molecular genetics of implantation in the mouse. Dev Genet 1997; 21:6. 61. Smith SE, French MM, Julian J, et al. Expression of heparan sulfate pro¬ teoglycan (Perlecan) in the mouse blastocyst is regulated during normal and delayed implantation. Dev Biol 1997; 184:38. 62. Cullinan EB, Abbondanzo SJ, Anderson PS, et al. Leukemia inhibitory fac¬ tor (LIF) and LIF receptor expression in human endometrium suggests a potential autocrine/paracrine function in regulating embryo implantation. Proc Natl Acad Sci U S A1996; 93:3115. 63. Das SK, Wang X-N, Paria BC, et al. Lleparin-binding EGF-like growth fac¬ tor gene is induced in the mouse uterus temporally by the blastocyst solely at the site of its apposition: a possible ligand for interaction with blastocyst EGF-receptor in implantation. Development 1994; 120:1071. 64. Schultz GA, Edwards DR. Biology and genetics of implantation. Dev Genet 1997; 21:1. 65. Benirschke K. Placentation. J Exp Zool 1983; 228:385. 66. Coucouvanis E, Martin GR. Signals for death and survival: a two-step mechanism for cavitation in the vertebrate embryo. Cell 1995; 83:279 67. Sanford JP, Chapman VM, Rossant J. DNA methylation in extraembryonic lineages of mammals. Trends Genet 1985; 1:89. 68. Utset MF, Awqulewitsch A, Ruddle FH, McGinnis W. Region-specific expression of two mouse homeo box genes. Science 1987; 235:1379. 69. Huisjes HJ. Spontaneous abortion. New York: Churchill Livingstone, 1984:205. 70. Wilcox AJ. Surveillance of pregnancy loss in human populations. Am J Ind Med 1983; 4:285. 71. Byrne J, Warburton D, Kline J, et al. Morphology of early fetal deaths and their chromosomal characteristics. Teratology 1985; 32:297. 72. Freinkel N, Lewis NJ, Akazawa S, et al. The honeybee syndrome: implica¬ tions of the teratogenicity of mannose in rat-embryo culture. N Engl J Med 1984; 310:223. 73. Giudice LC. Growth factors and growth modulators in human uterine endometrium: their potential relevance to reproductive medicine. Fertil Steril 1994; 61:1. 73a. Zenzes MT. Smoking and reproduction: gene damage to human gametes and embryos. Hum Reprod Update 2000; 6:122. 74. Tovares R, Ramos P, Palminha J, et al. Transplacental exposure to genotoxins. Evaluation in hemoglobin of hydroxyethylvaline adduct levels in smoking and non-smoking mothers and their newborns. Carcinogenesis 1994; 15:1271. 75. Jacobson JL, Jacoboson SW, Sokol RJ, et al. Effects of alcohol use, smoking, and illicit drug use on fetal growth in black infants. J Pediatr 1994; 124:757. 76. Naeye RL. Common environmental influences on the fetus. Monogr Pathol 1981; 22:52. 77. Iannaccone PM, Tsao TY, Stols L. Effects on mouse blastocysts of in vitro exposure to methylnitrosourea and 3-methylcholanthrene. Cancer Res 1982; 42:864. 78. Iannaccone PM. Long-term effects of exposure to methylnitrosourea on blastocysts following transfer to surrogate female mice. Cancer Res 1984; 44:2785. 79. Iannaccone PM, Fahl WE, Stols L. Reproductive toxicity associated with endometrial cell mediated metabolism of benzo[a]pyrene: a combined in vitro, in vivo approach. Carcinogenesis 1984; 5:1437.

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80. Bossert NL, Iannaccone PM. Midgestational abnormalities associated with in vitro preimplantation N-methyl-N-nitrosourea exposure with subse¬ quent transfer to surrogate mothers. Proc Natl Acad Sci U S A1985; 82:8757. 81. Dwivedi RS, Iannaccone PM. Effects of environmental chemicals on early development. In: Korach K, ed. Reproductive and developmental toxicol¬ ogy. New York: Marcel Dekker Inc, 1998:11. 82. Iannaccone PM, Bossert NL, Connelly CS. Disruption of embryonic and fetal development due to preimplantation chemical insults: a critical review. Am J Obstet Gynecol 1987; 157:476.

CHAPTER

108

THE MATERNAL-FETALPLACENTAL UNIT BRUCE R. CARR

The hormonal changes and maternal adaptations of human preg¬ nancy are among the most remarkable phenomena in nature. During pregnancy, the placenta, which is supplied with precur¬ sor hormones from the maternal-fetal unit, synthesizes large quantities of steroid hormones as well as various protein and peptide hormones and secretes these products into the fetal and maternal circulations. Near the end of pregnancy, a woman is exposed daily to ~100 mg estrogen, 250 mg progesterone, and large quantities of mineralocorticoids and glucocorticosteroids. The mother, and to a lesser extent the fetus, are also exposed to large quantities of human placental lactogen (hPL), human chori¬ onic gonadotropin (hCG), prolactin, relaxin, and prostaglandins and to smaller amounts of proopiomelanocortin (POMC)derived peptides such as adrenocorticotropic hormone (ACTH) and endorphin, gonadotropin-releasing hormone (GnRH), thyroid-stimulating hormone (TSH), corticotropin-releasing hor¬ mone (CRH), somatostatin, and other hormones. Implantation, the maintenance of pregnancy, parturition, and finally lactation are dependent on a complex interaction of hormones in the maternal-fetal-placental unit. Moreover, there exists a complex regulation for the secretion of steroid hor¬ mones by means of protein and peptide hormones also pro¬ duced within the placenta. In this chapter the discussion is focused on the hormones secreted by the placenta, the endocri¬ nology of the fetus and the mother, the effect of various endo¬ crine diseases on the maternal-fetal unit, and the use of endocrine tests to assess fetal well-being.

PLACENTAL COMPARTMENT In mammals, especially humans, the placenta has evolved into a complex structure that delivers nutrients to the fetus, pro¬ duces numerous steroid and protein hormones, and removes metabolites from the fetus to the maternal compartment. The structure of the placenta is discussed in Chapter 111.

PROGESTERONE The principal source of progesterone during pregnancy is the placenta, although the corpus luteum is the major source dur¬ ing the first 6 to 8 weeks of gestation,1 when progesterone is essential for the development of a secretory endometrium to receive and implant a blastocyst. Apparently, the developing trophoblast takes over as the principal source of progesterone by 8 weeks, since removal of the corpus luteum before this time, but not after, leads to abortion.2 After 8 weeks, the corpus luteum contributes only a fraction of the progesterone secreted.

1 060

PART VII: ENDOCRINOLOGY OF THE FEMALE

50

findings not only have provided new insights into the biochemi¬ cal basis for placental progesterone formation, but they have also provided clues to other aspects of maternal-placental physi¬ ology. For example, the rate of progesterone secretion may depend on the number of LDL receptors on the trophoblast and may be independent of placental blood flow: (a) Cholesterol side-chain cleavage by "the placental mitochondria is in a highly activated state, perhaps meaning that the placenta is under con¬ stant trophic stimulation and that hCG and GnRH produced by the placenta are the trophic substances; (b) cholesterol synthesis from acetate is limited, as discussed earlier; (c) the fetus does not contribute precursors for placental biosynthesis; and (d) the lev¬ els of maternal LDL are not rate-limiting for the placental prod¬ ucts of cholesterol or progesterone.5 A functioning fetal circulation is unimportant for the regula¬ tion of progesterone levels in the maternal unit. In fact, fetal death, ligation of the umbilical cord, or anencephaly—which all are associated with a decrease in estrogen production—have no significant effect on progesterone levels in the maternal com¬ partment.6'7 The physiologic role of the large quantity of progesterone includes binding to receptors in uterine smooth muscle, thereby inhibiting contractility and leading to myometrial quiescence. Progesterone also inhibits prostaglandin formation, which is critical in human parturition8 (see Chap. 109). Progesterone is essential for the maintenance of pregnancy in all mammals, possibly because of its ability to inhibit the T-lymphocyte cellmediated responses involved in graft rejection. The high local levels of progesterone can block cellular immune response to foreign antigens such as a fetus, creating immunologic privi¬ lege for the pregnant uterus.9

C ESTRIOL

ESTROGEN iso

iso

20 50 WNkt Gntotioo

FIGURE 108-1. Range (mean ± 1 standard deviation) of progesterone (A), estradiol-17(3 (B), and estriol (C) in plasma of normal pregnant women as a function of a week of gestation. (Courtesy of Dr. C. Richard Parker, Jr.)

The placenta of a term pregnancy produces -250 mg progester¬ one each day. Maternal progesterone plasma levels rise from 25 ng/mL during the late luteal phase to 40 ng/mL near the end of the first trimester to 150 ng/mL at term (Fig. 108-1/4). Most progesterone (90%) secreted by the placenta enters the maternal compartment. Although the placenta produces large amounts of progester¬ one, it normally has very limited capacity to synthesize precur¬ sor cholesterol from acetate. Radiolabeled acetate is only slowly incorporated into cholesterol in placental trophoblasts, and the activity of the rate-limiting enzyme of cholesterol biosynthesis— HMG-CoA reductase—in placental microsomes is low. There¬ fore, maternal cholesterol, in the form of low-density lipoprotein (LDL) cholesterol, is the principal substrate for the biosynthesis of progesterone.3'4 LDL cholesterol attaches to its receptor on the trophoblast and is taken up and degraded to free cholesterol, which then is converted to progesterone and secreted. These

During human pregnancy, the rate of estrogen production and the levels of estrogen in plasma increase markedly (Fig. 108-1B and C), and the levels of urinary estriol increase 1000-fold.10 In fact, it has been estimated that during a pregnancy, a woman produces more estrogen than a normal ovulatory woman could produce in 150 years!5 The corpus luteum of pregnancy is the principal source of estrogen during the first few weeks; subse¬ quently, nearly all of the estrogen is formed by the trophoblast of the placenta. The mechanism by which estrogen is synthesized by the pla¬ centa is unique (Fig. 108-2). The placenta cannot convert progesterone to estrogens because of a deficiency of 17ahydroxylase (CYP17). Thus, it must rely on androgens pro¬ duced in the maternal and fetal adrenal glands. Estradiol-17(3 and estrone are synthesized by the placenta by conversion of dehydroepiandrosterone sulfate (DHEAS) that reaches it from both the maternal and the fetal blood. Near term, 40% of the estradiol-17(3 and estrone is formed from maternal DHEAS and 60% of the estradiol-17(3 and estrone arises from fetal DHEAS precursor.11 The placenta metabolizes DHEAS to estrogens through placental sulfatase, A4'5-isomerase and 3(3hydroxysteroid dehydrogenase, and aromatase enzyme complex. Estriol is synthesized by the placenta from 16ahydroxydehydroepiandrosterone sulfate (16a-OHDS) formed in the fetal liver from circulating DHEAS. At least 90% of uri¬ nary estriol ultimately is derived from the fetal adrenal gland,11 which secretes steroid hormones at a high rate, sometimes up to 100 mg per day, mostly as DHEAS. The principal precursor for this DHEAS is LDL cholesterol circulating in fetal blood. A minor source is formation from pregnenolone secreted by the placenta. Only 20% of fetal cholesterol is derived from the maternal compartment, and because amniotic fluid cholesterol levels are negligible, the principal source of cholesterol appears to be the fetus itself. The fetal liver synthesizes cholesterol at a high rate and may supply sufficient cholesterol to the adrenals to maintain steroidogenesis.12

Ch. 108: The Maternal-Fetal-Placental Unit

MOTHER

PLACENTA

CH0LES7ER0L(LDL)

•pregnenolone

DHEAS

Adrenal

I

16 a OHDS^ Liver

1061

FETUS CHOLESTEROL (LDL)

*r DHEAS"*-

1. Sulfatase 2.3 p OHSDH 3. Aromatase EiE2

l. 16 a OHDSCholl 1. Sulfatase 2.3 p OHSDH 3. Aromatase

FIGURE 108-3. Mean concentration of chorionic gonadotropin (hCG) and placental lactogen (hPL) in sera of women throughout normal preg¬ nancy. (From Pritchard JA, MacDonald PC, Gant NF. Williams obstet¬ rics, 17th ed. Norwalk, CT: Appleton-Century-Crofts, 1985:121.)

Fetal Liver

FIGURE 108-2. Sources of estrogen biosynthesis in the matemal-fetalplacental unit. (LDL, low-density lipoprotein; chol, cholesterol; OHDS, hydroxydehydroepiandrosterone sulfate; OHSDH, hydroxysteroid dehy¬ drogenase; C2 pool, carbon-carbon unit; DHEAS, dehydroepiandrosterone sulfate; E2, estrone; E„ estradiol-17p7; E3, estriol.) (From Carr BR, Gant NE. The endocrinology of pregnancy-induced hypertension. Clin Perinatal 1983; 10:737.) Estetrol is a unique estrogen, the 15a-hydroxy derivative of estriol, which is derived exclusively from fetal precursors and fetal metabolism. Although the measurement of estetrol in pregnant women had been proposed as an aid in monitoring a fetus at risk for intrauterine death, it is not superior to the mea¬ surement of urinary estriol.13 Several disorders lead to low urinary excretion of estriol by the mother. A particularly interesting one is placental sulfatase deficiency,14 also known as the steroid sulfatase deficiency syn¬ drome, an X-linked metabolic disorder characterized during fetal life by decreased maternal estriol production secondary to this deficient enzymatic activity (see Fig. 108-2), which ren¬ ders the placenta unable to cleave the sulfate moiety from DHEAS. Placental sulfatase deficiency is associated with pro¬ longed gestation and difficulty in cervical dilatation at term, often necessitating cesarean section. Steroid sulfatase defi¬ ciency is thought to occur in 1 of every 2000 to 6000 neonates. The male offspring are, of course, sulfatase deficient, manifest clinically by ichthyosis during the first few months of life. The genetic locus for steroid sulfatase deficiency is on the distal short arm of the X chromosome.15 Most of the estrogen secreted by the placenta is destined for the maternal compartment, as is true for progesterone: 90% of the estradiol-17(3 and estriol enters the maternal compartment. Interestingly, estrone is the estrogen preferentially secreted into the fetal compartment.16 The physiologic role of the large quan¬ tity of estrogen produced by the placenta is not completely understood. It may regulate or fine-tune the events leading to parturition, because pregnancies are often prolonged when estrogen levels in maternal blood and urine are low, as in pla¬ cental sulfatase deficiency or when the fetus is anencephalic. Estrogen stimulates phospholipid synthesis and turnover, increases incorporation of arachidonic acid into phospholipids, stimulates prostaglandin synthesis, and increases the number of lysosomes in the uterine endometrium.8 Estrogens increase

uterine blood flow and may also play a role in fetal organ matu¬ ration and development.17

HUMAN CHORIONIC GONADOTROPIN The hCG secreted by the syncytiotrophoblast of the placenta is released into both the fetal and maternal circulation. This hor¬ mone is a glycoprotein with a molecular mass of -38,000 daltons that consists of two noncovalently linked subunits: a and |3.18 It has been used extensively as a pregnancy test and can be detected in the serum as early as 6 to 8 days after ovulation. Plasma levels rise rapidly during normal pregnancy, with a doubling in concentration every 2 to 3 days,19 reaching a peak between 60 and 90 days of gestation (Fig. 108-3). Thereafter, the maternal concentration declines and plateaus from -120 days until delivery.20 The levels of hCG are higher in multiple preg¬ nancies, in pregnancies associated with Rh isoimmunization, and in diabetic women. Levels also are higher in pregnancies associated with hydatidiform moles or in women with chorio¬ carcinoma (see Chap. 112). There is some evidence that the rate of secretion of hCG is regulated by a paracrine mechanism involving the release of GnRH by the cytotrophoblast.21 Fetal concentrations of hCG reach a peak at 11 to 14 weeks' gestation, thereafter falling pro¬ gressively until delivery. The most accepted theory regarding the role of hCG in preg¬ nancy is the maintenance of the early corpus luteum to ensure continued progesterone and, possibly, relaxin secretion by the ovary until this function is taken over by the growing trophoblast. Likewise, some investigators have demonstrated that hCG promotes steroidogenesis (progesterone) by the trophoblast.21 Others have suggested a role for hCG in promoting early growth and androgen secretion by the developing fetal zone of the human adrenal gland.22 It is more likely that a primary role for hCG is to regulate the development as well as the secretion of testosterone by the fetal testes. Male sexual differentiation occurs at an early but critical time when hCG is present in fetal serum. At this time, fetal hCG levels are higher—before the vasculariza¬ tion of the fetal pituitary, when fetal plasma luteinizing hormone (LH) levels are low.23 Another role may be to create immunologic privilege to the developing trophoblast.24 Finally, tire excess thy¬ rotropic activity during the clinical development of hyperthy¬ roidism observed in some women with neoplastic trophoblastic disease is secondary to excessive hCG secretion. hCG and TSH have similar structures, and purified hCG inhibits binding to

1062

PART VII: ENDOCRINOLOGY OF THE FEMALE

thyroid membranes and stimulates adenylate cyclase in thyroid tissues25 (see Chaps. 15 and 112). HUMAN PLACENTAL LACTOGEN Placental lactogen is a single-chain polypeptide of 191 aminoacid residues with a molecular mass of -22,000 daltons.26 The hormone has both lactogenic and growth hormone (GH)-like activity and is also referred to as chorionic growth hormone or chorionic somatomammotropin. However, hPL exhibits princi¬ pally lactogenic activity, having only 3% or less of the growthstimulating activity of human GH. The amino-acid sequences of hPL and GH are similar,27 and their genes are close together on chromosome 17: It has been proposed that the two hor¬ mones evolved from a similar ancestral polypeptide (see Chaps. 12 and 13). The nucleotide sequence for hPL has been reported, and the gene has been cloned.28 hPL is secreted by the syncytiotrophoblast and can be detected in serum by radioimmunoassay as early as the third week after ovulation.26 The serum level of the hormone contin¬ ues to rise with advancing gestational age and appears to plateau at term (see Fig. 108-3), the concentration closely following and being correlated with increasing placental weight.21’ The serum half-life of hPL is short. For example, although the serum level of hPL before delivery is the highest of all the protein hormones secreted by the placenta, hPL cannot be detected after the first postpartum day. The time sequence and peak of hPL secretion are significantly different from those of hCG (see Fig. 108-3), which suggests a different regulation for each hormone. This is interesting, because both are secreted by the syncytiotrophoblast rather than by the cytotrophoblast. Moreover, hPL secretion is not limited to the trophoblast, since immunoreactive hPL has been detected in patients with various malignant tumors includ¬ ing lymphomas, hepatomas, and bronchogenic carcinomas. Interestingly, hPL appears to be secreted primarily into the maternal circulation; only low levels are found in cord blood of neonates. Thus, most of the physiologic roles proposed for hPL have centered on its sites of action in maternal tissues. It has been suggested that hPL has a significant effect on maternal glucose, thereby providing adequate and continued nourishment for the developing fetus.27 It has been proposed that hPL exerts meta¬ bolic effects in pregnancy similar to those of GH, including stim¬ ulation of lipolysis, thus increasing the circulating free fatty acids available for maternal and fetal nutrition; inhibition of glucose uptake in the mother, yielding increased maternal insulin levels; development of maternal insulin resistance; and inhibition of gluconeogenesis, which favors transportation of glucose and protein to the fetus.30 A few cases of deficient hPL in maternal serum have been described in otherwise normal pregnancies, however, raising issue with this proposed role of hPL.31 HUMAN GROWTH HORMONE VARIANT A "true" placental GH has been shown to be produced by the syncytiotrophoblast of the placenta and secreted in parallel with hPL.32-35 This GH variant is now recognized to be the product of the hGH-V gene34 and differs from the major 22-kDa GH in 13 amino-acid residues.36 A glycosylated variant of this GH form has also been described in an in vitro system,37 but it is not known if this form circulates. Because concentrations of the placental GH variant in maternal plasma correlate with plasma levels of insulin-like growth factor-I (IGF-I), it has been suggested that placental GH is involved in the control of serum IGF-I levels in normal pregnant women.38 OTHER PLACENTAL PEPTIDE HORMONES In addition to hCG and hPL, several other placental hormones that are similar or closely related to hypothalamic, pituitary, or other hormones in their biologic and immunologic activity have

been described (e.g., POMC, human chorionic follicle-stimulating hormone [FSH], human chorionic gonadotropin-releasing hor¬ mone [hCGnRH], human chorionic thyrotropin [hCT]-releasing hormone, human chorionic corticotropin-releasing hormone, relaxin, somatostatin, gastrin, and vasoactive intestinal peptide). Information regarding these hormones is limited.21 The regula¬ tion of their secretion is poorly understood, although it appears that classic negative feedback inhibition does not exist. Further¬ more, their function and significance are speculative. Most of these hormones do not cross the placenta and are believed to enter primarily the maternal compartment. HUMAN CHORIONIC PROOPIOMELANOCORTIN PEPTIDES

Considerable evidence exists for a chorionic corticotropin or ACTH produced and secreted by placental tissue. Along with ACTH, other products that are processed from a similar 31-kDa POMC peptide are found in placental tissue, including (3-endorphin, (3-lipotropin, and a-melanocyte-stimulating hormone.21-39-40 HUMAN CHORIONIC THYROTROPIN

A substance with TSH-like activity has been identified pre¬ sumptively in placental tissue. However, the structure of this "hCT" is not identical to that of human pituitary TSH,41 and its physiologic role is unclear. The increased thyroid activity observed in some women with gestational trophoblastic dis¬ ease is believed to be secondary to the action of excessive hCG secretion and not to hCT. HUMAN CHORIONIC GONADOTROPIN-RELEASING HORMONE AND OTHER HORMONES

A substance with bioimmunoreactivity similar to that of hypothalamic GnRH has been localized to and shown to be synthesized by the cytotrophoblast layer of the placenta. It has been proposed that hCG secretion by the syncytiotrophoblast is regulated in part by hCGnRH.21 Similarly, substances simi¬ lar to thyrotropin-releasing hormone (TRH), somatostatin, and CRH are also synthesized by the trophoblast. CRH mRNA has been localized in the placenta, principally in the cytotrophoblast.42 CRH levels increase in maternal plasma and amniotic fluid throughout pregnancy, but the role for this increase is unclear.43 The FSH-suppressing hormone follistatin has been found in human placenta. Inhibin and activin are secreted by the placenta, and maternal levels increase near term.44 (See Chap. 112 for a discussion of these and other pla¬ cental hormones.)

FETAL MEMBRANES AND DECIDUA Fetal membranes consisting of amnion and chorion were origi¬ nally thought to be inactive endocrinologically. The amnion is a thin structure (0.02-0.5 mm) and contains no blood vessels or nerves. However, the fetal membranes play important roles during pregnancy in the transport and metabolism of hor¬ mones and in the events that lead eventually to parturition.45 Thus, although fetal membranes apparently do not synthesize hormones de novo, they have extensive enzymatic capabilities for regulating steroid hormone metabolism. Some of these enzymes are 5a-reductase, 3(3-hydroxysteroid dehydrogenase, A^-isomerase, 20a-hydroxysteroid oxidoreductase, 17(l-hydroxysteroid dehydrogenase, aromatase, and sulfatase. Also, fetal membranes contain large quantities of arachidonic acid, the obligate precursor of prostaglandins. Furthermore, they contain phospholipase A2 and other enzymes that stimulate the release of arachidonic acid from glycerophospholipids in the amnion or chorion.8 The decidua is a complex structure of specialized endome¬ trial stromal cells that proliferate in response to progesterone secreted during the luteal phase of the menstrual cycle and later

Ch. 108: The Maternal-Fetal-Placental Unit in response to hormones secreted by the developing trophoblast. Evidence suggests that the decidua is also a rich source of enzymatic activity and secretion of hormones. The decidua may be important in fetal homeostasis and in the maintenance of pregnancy, since the decidua appears to communicate directly with the fetus via transport through the fetal mem¬ branes and into the amniotic fluid as well as directly into the myometrium by simple diffusion. The hormones and enzymatic activities localized to the decidua include prolactin, relaxin, prostaglandins, and lahydroxylase. The concentration of prolactin in amniotic fluid is extremely high compared with that in fetal or maternal plasma; it arises from the decidua.46 Prolactin is secreted by decidual cells in culture but not by trophoblast or placental membranes. The prolactin secreted by the decidua is immunologically, structurally, and biologically similar to that from pituitary sources.47 However, the regulation of decidual prolactin forma¬ tion and secretion is more complex. Bromocriptine treatment of pregnant women reduces maternal and fetal plasma levels but not amniotic fluid levels of prolactin. Prolactin secretion by decidual cells or tissues is not affected by treatment with dopamine, dopaminergic agonists, or TRH. The function of decidual prolactin remains speculative. Because most of the prolactin synthesized and secreted by the decidua reaches amniotic fluid, a regulatory role in amniotic fluid osmolality and homeostasis has been proposed.48 Relaxin is a peptide consisting of two chains (A and B) of 22 and 31 amino acids covalently linked.49 Relaxin is secreted by the corpus luteum, decidua, and basal plate and septa of the placenta.50 The greatest source appears to be the corpus luteum of pregnancy, and it is thought to be regulated by hCG. That the decidua and placenta can synthesize relaxin is intriguing because of the proximity of the pregnant uterus. This is relevant because relaxin is believed to play a role along with progester¬ one in reducing uterine activity as well as in the softening of pelvic tissues and cervix before parturition (see Chap. 94).50

FETAL COMPARTMENT The understanding and elucidation of the human fetal endocrine system have required the development of assays for minute quantities of hormone. The regulation of the fetal endocrine sys¬ tem, like that of the placenta, is not completely independent, since synthesis relies to some extent on precursor hormones secreted directly by the placenta or obtained from the maternal unit. As the fetus develops, its endocrine system becomes more independent in preparation for extrauterine existence.

HYPOTHALAMIC-PITUITARY AXIS The fetal hypothalamus begins differentiation from the fore¬ brain during the first few weeks of fetal life, and by 12 weeks, hypothalamic development is well advanced. Most of the hypothalamic-releasing hormones, including GnRH, TRH, dopamine, norepinephrine, and somatostatin, and their respec¬ tive hypothalamic nuclei have been identified as early as 6 to 8 weeks of fetal life.23 The neurohypophysis is detected first at 5 weeks, and by 14 weeks, the supraoptic and paraventricular nuclei are fully developed.23-51 Rathke pouch appears in the human fetus at 4 weeks. The premature anterior pituitary cells that develop from the cells lining Rathke pouch can secrete GH, prolactin, FSH, LH, and ACTH in vitro as early as 7 weeks of fetal life.52 Evidence sug¬ gests that the intermediate lobe of the pituitary is a significant source of POMC hormones.53-54 The hypothalamic-pituitary portal system is the functional link between the hypothalamus and the anterior pituitary. Vas¬ cularization of the anterior pituitary begins by 13 weeks of fetal life, but a functioning intact portal system is absent cm til 18 to

1063

Weeks Gestation

FIGURE 108-4. Ontogeny of pituitary hormones in human fetal serum. (Prl, prolactin; TSH, thyroid-stimulating hormone; ACTH, adrenocorti¬ cotropic hormone; GH, growth hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone.) (From Parker CR Jr. The endocri¬ nology of pregnancy. In: Carr BR, Blackwell RE, eds. Textbook of repro¬ ductive medicine. Norwalk, CT: Appleton & Lange, 1993:17.) 20 weeks.55 However, there is indirect evidence that hypotha¬ lamic secretion of releasing hormones influences anterior pitu¬ itary function before this time by simple diffusion, given their proximity in early fetal development. There is also evidence that fetal adrenal feedback is operative at the hypothalamicpituitary axis as early as week 14 of fetal life. Elevated levels of fetal androgens are detected at this time in amniotic fluid in fetuses affected with congenital adrenal hyperplasia secondary to 21-hydroxylase deficiency.56

GROWTH HORMONE, PROLACTIN, VASOPRESSIN, AND OXYTOCIN GH is detected in the fetal pituitary as early as 12 weeks' gesta¬ tion, and fetal pituitary GH concentrations increase until 25 to 30 weeks' gestation, thereafter remaining constant until term. Fetal plasma GH levels peak at 20 weeks and then fall rapidly until birth57 (Fig. 108-4). However, fetal plasma concentrations of GH always exceed maternal concentrations, which are sup¬ pressed, possibly by the high circulating levels of hPL. The regulation of GH release in the fetus appears to be more complex than in the adult. To explain the high levels of plasma GH at midgestation and a fall thereafter, unrestrained release of growth hormone-releasing hormone (GHRH) leading to exces¬ sive release of GH at midgestation has been postulated.23 Thereafter, as the hypothalamus matures, somatostatin may increase arid GHRH levels decline, reducing GH release. The role of GH in the fetus is also unclear. There is considerable evi¬ dence that GH is not essential to somatic growth in primates.58 For example, in neonates with pituitary agenesis, congenital hypothalamic hypopituitarism, or familial GH deficiency, birth size and length are usually normal. However, the somatomedins, in particular IGF-I and IGF-II, increase in fetal plasma, and IGF-I and IGF-II levels correlate better than do GH levels with fetal growth.59 Although GH is an important trophic hor¬ mone for somatomedin production in the fetus, somatomedin regulation may be independent of GH, depending instead on other factors. Prolactin, hPL, and insulin also stimulate somatomedin production.23 Prolactin is present in lactotropes by 19 weeks of life. The prolactin content of the fetal pituitary increases throughout ges¬ tation,60 whereas plasma levels increase slowly until 30 weeks' gestation, when the levels rise sharply until term and remain elevated until the third month of postnatal life (see Fig. 108-4). In humans, TRH and dopamine as well as estrogens appear to affect fetal prolactin secretion. Bromocriptine both lowers maternal prolactin levels and crosses the placenta to inhibit

1064

PART VII: ENDOCRINOLOGY OF THE FEMALE

fetal prolactin release and lower prolactin levels in fetal blood.61 It has been suggested that prolactin in the fetus influences adre¬ nal growth, lung maturation, and amniotic fluid volume. Arginine vasopressin (AVP) and oxytocin are found in hypo¬ thalamic nuclei and in the neurohypophysis during early fetal development.52 However, there are relatively few studies on the regulation and secretion of these hormones. The levels of AVP are high in fetal plasma and cord blood at delivery. The principal stimulus to AVP release appears to be fetal hypoxia, although acidosis, hypercarbia, and hypotension also play a role.62 The ele¬ vated AVP levels in fetal blood may lead to increased blood pres¬ sure, vasoconstriction, and the passage of meconium by the fetus. Oxytocin levels in the fetus are not affected by hypoxia but appear to increase during labor and delivery (see Chap. 109).

Birth

THYROID GLAND The placenta apparently is relatively impermeable to TSH and thyroid hormone, so that the fetal hypothalamic-pituitarythyroid axis develops and functions independently of the maternal system. Although thyroxine (T4) may cross the pla¬ centa to a slight degree, human thyroid-stimulating immuno¬ globulins (TSI) as well as iodine and antithyroid drugs given to women with hyperthyroidism pass through the placenta and may affect fetal thyroid function.63 TRH is detectable in hypothalamic nuclei, and TSH is found in pituitary tissues by 10 to 12 weeks of fetal life.64 High concen¬ trations of TRH are detected in fetal blood, and the source is thought to be the fetal pancreas. However, this source of TRH appears to have little effect on the pituitary release of TSH, and the function of pancreatic TRH is unknown.65 The fetal thyroid has developed sufficiently by the end of the first trimester that it can concentrate iodine and synthesize iodothyronines. The levels of TSH and thyroid hormone are rel¬ atively low in fetal blood until midgestation. At 24 to 28 weeks' gestation, serum TSH concentrations rise abruptly to a peak but decrease slightly thereafter until delivery.66 In response to the surge of TSH, T4 levels rise progressively after midgestation until term (Fig. 108-5). During this time, both thyroid respon¬ siveness to TRH and pituitary TRH content increase. At birth, there is an abrupt release of TSH, T4, and triiodothyronine (T3), and the levels of these hormones fall during the first few weeks after birth.66 The relative hyperthyroid state of the fetus is believed to be necessary to prepare it for the thermoregulatory adjustments of extrauterine life. The abrupt changes of TSH and T4 that occur at birth are believed to be stimulated by the cooling associated with delivery.67 Finally, 3,3’,5'-triiodo-Lthyronine (reverse T3) levels are high during early fetal life, begin to fall at midgestation, and continue to fall after birth (see Chap. 47). The difference between the formation of T3 and reverse T3 is thought to be related to maturation of peripheral iodothyronine metabolism (see Chap. 30).

GONADS Bioactive and immunoreactive GnRH has been detected in the fetal hypothalamus by 9 to 12 weeks of life. The amount increases with fetal age, with the maximum noted between 22 and 25 weeks in females and between 34 to 38 weeks in males.23 The dominant gonadotropin fraction in the fetal pituitary is the a subunit. However, the fetal pituitary in vitro is capable of secreting intact LH by 5 to 7 weeks.53 The pituitary content of LH increases from 10 weeks to 24 weeks and then falls slowly near term. The content of LH is higher in females than in males, a difference thought to be secondary to a greater negative feed¬ back in response to higher concentrations of plasma testoster¬ one in fetal male plasma.68 The FSH content of the fetal pituitary increases until midgestation, then falls until term. The FSH content is higher in female than in male fetuses because of greater negative feedback in the latter. The plasma concentra-

FIGURE 108-5. Maturation of serum thyroid-stimulating hormone (TSH), thyroxine (T4), and triiodothyronine (T3) during the last half of gestation and early neonatal life. The increase in thyrotropin-releasing hormone (TRH) effect or content is also illustrated. (From Fisher DA. Maternal-fetal neurohypophyseal system. Clin Perinatol 1983; 10:615.)

tion of FSH rises slowly to a peak near week 25 and then falls to low levels by term (see Fig. 108-4). The FSH levels parallel the pituitary content of FSH with respect to sexual dimorphism, being higher in females than in males. The pattern of LH levels in fetal plasma parallels that of FSH. The fall in gonadotropin pituitary content and plasma concentration after midgestation is thought to be attributable to the maturation of the hypothala¬ mus.23 The hypothalamus also becomes more sensitive to sex steroids circulating in fetal blood that originate in the placenta. The differentiation of the bipotential fetal gonad into a testis or an ovary is discussed in Chapter 94. In the male, testosterone secretion begins soon after differentiation of the gonad into a tes¬ tis and the formation of Leydig cells at 7 weeks of fetal life. Maxi¬ mal levels are observed at ~15 weeks and decrease thereafter.69 This early secretion of testosterone is important in regulating sex¬ ual differentiation. It is believed that hCG is the primary stimu¬ lus to the early development and growth of Leydig cells and the subsequent peak of testosterone. The pattern of hCG, the concen¬ tration of testicular hCG receptors, and the pattern of plasma tes¬ tosterone are related closely.70 Thus, it appears that sexual differentiation of the male does not rely solely on fetal pituitary gonadotropins. However, fetal LH and FSH are still required for complete differentiation of the fetal ovary and testis. For exam¬ ple, anencephalic fetuses with low levels of circulating LH and FSH have appropriate secretion of testosterone at 15 to 20 weeks secondary to adequate levels of hCG, but they have a decreased number of Leydig cells, exhibit hypoplastic external genitalia, and often have undescended testes.71 Likewise, male fetuses with congenital hypopituitarism often have an associated micro-

Ch. 108: The Maternal-Fetal-Placental Unit

penis. These observations suggest that beginning about midges¬ tation fetal pituitary gonadotropins affect testosterone secretion from the testes. The regulation of testosterone secretion also appears to depend on fetal cholesterol, principally LDL choles¬ terol, to maintain maximal rates of testosterone secretion. Fetal testicular LDL receptors and rates of de novo synthesis of choles¬ terol also parallel the secretion of hCG and testosterone.72 The fetal ovary is involved primarily with the formation of follicles and germ cells. Although follicular development appears to be relatively independent of gonadotropins, the anencephalic female fetus has small ovaries and a decreased number of ovarian follicles. However, the fetal ovaries do not contain hCG receptors, at least by 20 weeks of gestation. The ovaries appear to be relatively inactive with respect to steroido¬ genesis during fetal life, but they can aromatize androgens to estrogens in vitro as early as 8 weeks of life.73

ADRENAL GLAND The human fetal adrenal glands secrete large quantities of ster¬ oid hormones (up to 200 mg per day near term).74 This rate of steroidogenesis may be five times that observed in the adrenal glands of adults at rest. The principal steroids are C-19 steroids (mainly DHEAS), which serve as substrate for estrogen biosyn¬ thesis in the placenta. It was recognized early that the human fetal adrenal gland con¬ tains a unique fetal zone that accounts for the rapid growth of the gland and that this zone disappears during the first few weeks after birth.12 The fetal zone differs histologically and biochemi¬ cally from the neocortex (also known as the definitive or adult zone). The uniqueness of a transient fetal zone has been reported in cer¬ tain higher primates and some other species, but only humans possess tire extremely large fetal zone that involutes after birth. The cells of the adrenal cortex arise from coelomic epithelium. Those cells comprising the fetal zone can be identified in the 8- to 10-mm embryo and before the appearance of the cells of the neo¬ cortex (14-mm embryo).75 Growth is most rapid during the last 6 weeks of fetal life. By 28 weeks' gestation, the adrenal gland may be as large as the fetal kidney and may be equal to the size of the adult adrenal by term (Fig. 108-6). The fetal zone accounts for the largest percentage of growth; after birth, the gland shrinks sec¬ ondary to involution and necrosis of fetal zone cells. Histologically, the central portion of the adrenal contains the fetal zone cells, which are eosinophilic cells with pale-staining nuclei that at term make up 80% to 85% of the volume of the gland. The neocortex is the outer rim of cells containing a small quantity of cytoplasm and dark-staining nuclei. The neocortex

1065

is thought to originate the zona glomerulosa, zona fasciculata, and zona reticularis after birth.12 In vitro studies utilizing fetal adrenal tissues or cells, in vivo perfusion studies of previable fetuses, and cord blood measure¬ ments of steroid hormones demonstrate that the fetal zone can secrete the full complement of steroid hormones secreted by the adult adrenal cortex. However, the fetal zone has a reduced capacity to secrete C-21 steroids because of the low activity of 3(3-hydroxysteroid dehydrogenase and A4'5-isomerase com¬ plex,76 probably secondary to estrogen or other factors produced by the placenta that may inhibit enzyme action. Thus, the princi¬ pal steroids secreted by the fetal zone cells are A5-sulfoconjugates, namely, DHEAS and pregnenolone sulfate.77-78 In contrast, the principal secretory product of the neocortex is cortisol. Elec¬ tron microscopic investigations suggest fetal zone activity as early as the seventh week and indicate that it is the most ste¬ roidogenic zone throughout gestation. The neocortex cells exhibit little steroidogenic activity until the third trimester.79 During gestation, DHEAS levels in fetal plasma rise, peak¬ ing between 34 and 40 weeks.80 This pattern coincides with the marked increase in fetal adrenal growth. After birth, DHEAS levels decline, paralleling the regression of the fetal zone. Corti¬ sol plasma levels also increase during fetal life, but there is little evidence after 25 weeks' gestation of a sharp rise like that of DHEAS. Moreover, a significant portion of the circulating corti¬ sol in fetal plasma arises from placental transfer from the maternal compartment.81 REGULATION OF FETAL ADRENAL GROWTH ACTH stimulates steroidogenesis in vitro,12 and there is clinical evidence that ACTH is the principal trophic hormone of the fetal adrenal gland in vivo. For example, in anencephalic fetuses, the plasma levels of ACTH are very low, and the fetal zone is markedly atrophic. Maternal glucocorticosteroid ther¬ apy suppresses fetal adrenal steroidogenesis by suppressing fetal ACTH secretion.82 Further evidence that ACTH regulates steroidogenesis early in fetal life is provided by the observation of elevated levels of 17a-hydroxyprogesterone in the amniotic fluid of fetuses with congenital adrenal hyperplasia secondary to the absence of 21-hydroxylase. Despite these observations, other ACTH-related peptides (e.g., fetal pituitary or placental POMC derivatives) have been proposed as trophic hormones for the fetal zone, but the evidence is weak.12 Other hormones or growth factors, including prolactin, hCG, GH, hPL, and epi¬ dermal and fibroblast growth factor, have no consistent signifi¬ cant effect on steroidogenesis or adenylate cyclase activity in cultures of fetal zone organs or monolayer cells or of membrane preparations. However, a role of these or other hormones in promoting growth of adrenal cells is possible.83-84 After birth, the adrenal gland shrinks by more than 50% sec¬ ondary to regression of fetal zone cells. This suggests that a trophic substance other than ACTH is withdrawn from the maternal or placental compartment or that the secretion rates of some other trophic hormone are altered to initiate regression of this fetal zone. FETAL ADRENAL STEROIDOGENESIS

Trimesters

Months

Years

FIGURE 108-6. Size of adrenal gland and its component parts during fetal life, infancy, and childhood. (From Carr BR, Simpson ER. Lipopro¬ tein utilization and cholesterol synthesis by the human fetal adrenal gland. Endocr Rev 1981; 2:306.)

The availability of precursor substrates may assist ACTH in regu¬ lating the rate of steroid hormone production by the fetal zone. Circulating pregnenolone and progesterone have long been sug¬ gested as the principal precursors of fetal adrenal steroidogenesis, but a number of factors make tiffs unlikely. For example, in view of the fetal adrenal blood flow and the levels of unconjugated pregnenolone in fetal plasma, 5

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H+-H-W

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r = -0 92

p100 lb overweight or >200% of ideal body weight), respiratory impairment, which may be a major source of morbidity, takes three forms. The first is directly related to increased chest wall and abdominal fat, and consists of a reduction in lung and chest wall compliance. In an ambulatory setting, these patients do not retain carbon dioxide, but they may be slightly hypoxic, especially when supine.143 After abdominal surgery, such as a cesarean section, massively obese patients have a further reduction in arterial partial pressure of oxygen (Pa02), with the nadir appearing on the second postoperative day.143 A much smaller group of mas¬ sively obese patients have hypoventilation, hypercapnia.

hypoxia, somnolence, and markedly reduced lung compli¬ ance—a constellation of findings that has been referred to as the pickwickian syndrome. Finally, some patients with obesity develop upper airway obstruction when asleep (or sedated) and have hypoxia and even apnea. These concerns have important implications for obese women who become pregnant. Several series reporting data in such women have been published. In women in the top fifth percentile for weight (>90 kg at term), the incidence of hyper¬ tension and gestational diabetes was increased two- to eight¬ fold.144-146 Slight increases also were noted in the rates of other complications, including thrombophlebitis, preeclampsia, uri¬ nary tract infection, and infection of an episiotomy or other wound.144-146 Although an increased rate of respiratory compli¬ cations related to the influence of the expanding uterine con¬ tents on an already impaired respiratory apparatus would be expected, this was not found in these series.144-146 The rate of cesarean section was increased in these women in one series,146 but not in the other two.144-145 Thus, obese women should be screened periodically for gestational diabetes and monitored carefully for the development of hypertension, urinary tract infection, and preeclampsia. For obese women with compli¬ cated deliveries who may remain at bed rest for extended peri¬ ods, prophylactic subcutaneous heparin may be indicated. In massively obese women, care also must be taken to prevent res¬ piratory compromise. Fetal complication rates also are increased by maternal obe¬ sity. Birth weights are higher, and this is not due to the associ¬ ated gestational diabetes.144-146 Follow-up shows that, at 12 months, the infants of obese mothers are significantly more obese than the infants of nonobese mothers.145 Increased shoul¬ der dystocia, meconium, and late deceleration during labor also are noted.146 Although one series found an increase in perinatal mortality due to obesity,146 this was not found in the other two series.144,145 Estimates are that 40,000 to 80,000 additional calories are required during pregnancy.147 The normal, nonobese woman usually gains little during the first trimester, 0.36 kg per week between weeks 13 and 18, 0.45 kg per week between weeks 18 and 28, and 0.36 kg per week between week 28 and term.147 Of the recommended 9- to 13.5-kg weight gain during pregnancy, -3.6 kg is actually maternal fat.147 Many obese women resist diets that cause them to gain the recommended amount of weight during pregnancy, and 10% to 40% gain •»■*.

»*:

%

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INVASIVE MOLE The invasive mole is the most common form of persistent tro¬ phoblastic disease. It is usually diagnosed through persistent hCG elevation. This lesion is rarely seen as a pathologic speci¬ men unless complications necessitate hysterectomy (Fig. 111-8). In such cases, histology shows villi, typical of a classic mole, extending into the uterine wall. The diagnosis cannot usually be made on evacuated material. Most invasive moles are ade¬ quately treated by chemotherapy.3-4'6

CHORIOCARCINOMA Choriocarcinoma is a true malignant neoplasm of trophoblastic cells. Approximately half of cases follow molar pregnancies. The remainder occur after spontaneous abortions and ectopic or normal pregnancies. Grossly, choriocarcinoma is an extremely hemorrhagic lesion in both primary and metastatic sites. Microscopically, it is a haphazard mixture of cytotropho¬ blast and syncytiotrophoblast (Fig. 111-9). The syncytiotropho¬ blast may be extensively vacuolated. In general, anaplasia is not striking, although it may be present. No molar villi are seen, and their presence precludes the diagnosis. Such patients usu¬ ally present with bleeding after a molar or other pregnancy. Treatment with multiagent chemotherapy is usually curative, and later normal pregnancy is possible.3-4-6

PLACENTAL SITE TROPHOBLASTIC TUMOR Placental site trophoblastic tumor is an avillous lesion com¬ posed of uninucleate intermediate trophoblastic cells that invade myometrium and blood vessels. The cells secrete pre¬ dominantly human placental lactogen and a small amount of hCG. This tumor tends to present with amenorrhea and a uter¬ ine mass. It may perforate the uterus. Ten percent to 15% of cases behave in a malignant fashion. Treatment is surgical because response to chemotherapy is poor.3-4-8

. _ «•*

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teau or rise in (3-subunit human chorionic gonadotropin (hCG) levels indicates persistent disease requiring chemother¬ apy.4'6'7 The incidence of such disease is 10% to 20% after a complete mole but 5% or less after a partial mole. Choriocarci¬ noma is extremely rare after a partial mole. Most patients with complete and partial moles later have normal pregnancies.6

a • * '

* *$• « a ... • m*.' %r * \»

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FIGURE 111-9. Choriocarcinoma is characterized by irregularly inter¬ lacing aggregates of syncytiotrophoblast (S) and cytotrophoblast (C). Villus structures are not found. (Hematoxylin and eosin; x400)

PLACENTAL SITE LESIONS As described previously, the placental site is largely com¬ posed of intermediate trophoblastic cells. These cells are involved in several benign placental site processes. The exag¬ gerated placental site reaction (syncytial endometritis) con¬ tains multinucleate as well as uninucleate intermediate cells. Although large masses of cells may be present, the underly¬ ing architecture is retained. Placental site nodules and plaques show hyalinized material surrounding the interme¬ diate cells. These are remnants of former gestations and may be found years later.3-10

REFERENCES 1. Jirasek JE. Atlas of human prenatal morphogenesis. Boston: Martinus Nijhoff, 1983:23. 2. Popek EJ. Normal anatomy and histology of the placenta. In: Lewis SH, Perrin E, eds. Pathology of the placenta, 2nd ed. Philadelphia: Churchill Livingstone, 1999. 3. Benirschke K, Kaufmann P. Pathology of the human placenta, 3rd ed. New York: Springer-Verlag, 1990. 4. Silverberg SG, Kurman RJ. Tumors of the uterine corpus and gestational trophoblastic disease. Washington: Armed Forces Institute of Pathology, 1992. 5. Wells M, Bulmer JN. The human placental bed: histology, immunohistology, and pathology. Histopathology 1988; 13:483. 6. Szulman AE. Trophoblastic diseases: complete and partial hydatidiform mole. In: Lewis SH, Perrin E, eds. Pathology of the placenta, 2nd ed. Phila¬ delphia: Churchill Livingstone, 1999. 7. Lage JM, Wolf NF. Gestational trophoblastic disease: new approaches to diagnosis. Clin Lab Med 1995;15:631. 8. Gillespie AM, Kumar S, Hancock BW. Treatment of persistent trophoblastic disease later than 6 months after diagnosis of molar pregnancy. Br ] Cancer 2000; 82:1393. 9. Carr DH. Cytogenetics of human reproductive wastage. In: Kalter H, ed. Issues and reviews in teratology. New York: Plenum, 1983. 10. Baergen RN. Trophoblastic lesions of the placental site. Gen Diagn Pathol 1997; 143:143.

1 096

PART VII: ENDOCRINOLOGY OF THE FEMALE

CHAPTER

1 1 2

TABLE 112-1. Regulatory Molecules Produced by Human Trophoblast Tissue Steroid hormones

Estrogens, progesterone Human chorionic gonadotropin (hCG), human placen¬ tal lactogen (HPL), prolactin (PRL), growth hormone (GH), corticotropin, thyrotropin (TSH), parathyroid hormone (PTH), calcitonin (CT), relaxin, leptin, renin, inhibins, follistatin, activins, leptin

ENDOCRINOLOGY OF TROPHOBLASTIC TISSUE

Protein hormones

Z. M. LEI AND CH. V. RAO

Neuropeptides

Gonadotropin-releasing hormone (GnRH), thyrotro¬ pin-releasing hormone (TRH), growth hormone¬ releasing hormone (GHRH), somatostatin, corti¬ cotropin-releasing hormone (CRH), CRH-binding protein, oxytocin, neuropeptide Y, opioids

Growth factors/ cytokines

Epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF)a and -0, platelet-derived growth factor (PDGF), placental growth factor, vascular endothelial growth factor (VEGF), insulin, insulin-like growth factor (IGF)-I and -II, IGF-binding pro¬ teins (IGFBPs), macrophage colony-stimulating factor (MCSF), erythropoietin, stem cell factor Interleukins (IL-1, IL-2, IL-6, IL-8, IL-10, IL-13), leu¬ kemia inhibitory factor (LIF), tumor necrosis fac¬ tor (TNF)-a, interferon-a, -p, and -y

Eicosanoids

Prostaglandins, thromboxane A2, leukotrienes, and 5-, 12-, and 15-hydroxyeicosatetraenoic acids

The placenta develops from trophectoderm of the blastocyst through the processes of cell proliferation and differentiation. It consists of an outer layer of trophoblasts and an inner connec¬ tive tissue core with macrophages (Hofbauer cells), fibroblasts, and blood vessels branched out from the umbilical vessels. The trophoblast layer and connective tissue core communicate with each other through the molecules made by each of them. The trophoblast cell layer contains outer syncytiotrophoblasts and inner mononuclear cytotrophoblasts and extravillous mononuclear trophoblasts that invade the uterus to establish a vascular con¬ nection with the maternal circulation.1 Signals of invasion may come from syncytiotrophoblasts through human chorionic gonadotropin (hCG).2-5 Villus and extravillus cytotrophoblasts can proliferate and are relatively inactive in the synthesis of many placental hormones. Upon appropriate stimulation, vil¬ lus cytotrophoblasts can undergo aggregation and fusion of their plasma membranes to become syncytiotrophoblasts, which are terminally differentiated and serve as the primary source of many placental hormones and other regulatory agents. The placenta deters the noxious agents and yet allows nutrients to enter the fetal circulation and fetal metabolic waste to empty into the maternal circulation, while preventing mater¬ nal immune system cells from attacking the fetus. Structural features, and the ability to produce a wide array of regulatory molecules, allow the placenta to grow, to differentiate, and to perform numerous diverse functions, including possible com¬ munications with the brain.6-9

TROPHOBLAST HORMONES The placenta produces a wide variety of regulatory hormones. In fact, it is difficult to find anything not made by placenta that is produced elsewhere in the body. Many regulatory molecules produced by human trophoblastic tissue are summarized in fable 112-1 7'8-1(M6 These hormones have diverse functions in the fetoplacental unit and in maternal and fetal tissues. Interest¬ ingly, some have similar functions, suggesting there might be a hierarchy. Having more than one regulatory hormone may allow for more control points in the regulation. Control of cytotrophoblastic differentiation is one example of multiple regulatory agents having a similar function with one playing a central role. Like hCG, epidermal growth factor (EGF), trans¬ forming growth factor-a (TGF-a), and leukemia inhibitory fac¬ tor (LIF) promote differentiation and also increase the synthesis of hCG.17-21 Inhibiting hCG-receptor synthesis by treatment with 21 mer phosphorothioate antisense oligodeoxynucleotide prevents not only hCG action, but also the actions of EGF, TGFa, and LIF in promoting differentiation of cytotrophoblasts.--^3

HUMAN CHORIONIC GONADOTROPIN hCG is the signature hormone of the placenta. It is produced in large amounts, has pervasive actions, and has a profile of an early rise followed by a decrease to low steady-state levels. Although tro¬ phoblast is the major source, a wide variety of normal tissues, including anterior pituitary, can make hCG. Nontrophoblastic hCG is not glycosylated and its levels are very low in the circu-

lation due to rapid clearance. Some nontrophoblastic tissues may not even release hCG, so that it will serve as a local ligand for hCG/luteinizing hormone (LH) receptors.24'25 This chapter focuses on trophoblastic hCG and some of its newly discov¬ ered actions in the fetoplacental unit and elsewhere in the body that are necessary for completion of a successful fullterm pregnancy.

STRUCTURE AND REGULATION OF HUMAN CHORIONIC GONADOTROPIN SYNTHESIS hCG is a heterodimeric glycoprotein hormone consisting of noncovalently bound a and (3 subunits.26 The a subunit is iden¬ tical to that of others in the glycoprotein hormone family, which consists of LH, follicle-stimulating hormone (FSH), and thyroidstimulating hormone (TSH) (see Chaps. 15 and 16). The (3 sub¬ unit, on the other hand, is hormone specific and, while similar, is not identical to the (3 subunit of LH.26 The primary difference is that the hCG-(3 subunit contains 30 additional amino acids at the carboxyterminus with four O-linked oligosaccharide chains, enabling intact hCG to survive in the circulation longer than does LH and also establishing a basis for developing highly specific immunoassays. The (3-hCG assay measures not only the (3 subunit but also intact hCG. The circulatory half-life of intact hCG, 24 to 36 hours, is much longer than that for LH (2-3 hours), for free hCG-a (10-15 minutes), and for free hCG-(3 (35-45 minutes). Thus, not only the amount of carbohydrate on the protein backbone but also the conformation of the native hormone plays an important role in the clearance from the circulation.11-26-27 The ot subunit of hCG contains 92 amino acids with 10 half¬ cystine residues, forming five intrachain disulfide linkages. The (3 subunit of hCG contains 145 amino acids with 12 half-cystines that form six conserved disulfide bridges.27 The folding pattern of the a and (3 subunits, with three disulfide bonds, forms a dis¬ tinct seatbelt-like motif that is found in a family of cystine-knot growth factors, which include nerve growth factor, TGF-p, and platelet-derived growth factor-p.28 A single gene on chromosome 6q21.1-23 encodes the a sub¬ unit of hCG.29 It contains five introns, four exons, and a consen¬ sus TATA box located 30 base pairs (bp) upstream from a single transcription initiation site. The P subunit of hCG is encoded by a cluster of six genes spanning more than 52 kbp on chromo-

Ch. 112: Endocrinology of Trophoblastic Tissue TABLE 112-2. Trans-Acting Factors and C/s-Acting Elements in the Transcription of Human Chorionic Gonadotropin Subunit Genes Trmis-Acting

Cis-Acting

Subunit

1097

TABLE 112-3. Regulators of Human Chorionic Gonadotropin Biosynthesis Regulator

Effect

Interaction

ACTIVATION

P-Adrenergic agonists

Stimulation

cAMP

Stimulation

Ap-2

URE

a & (3

Direct binding

DHEA

Stimulation

CREB

CRE(s)

a& p

Direct binding

EGF & TGF-a

Stimulation

CAT-binding proteins

CAT box

a

Direct binding

Glucocorticoids

Stimulation

JREB

JRE

a

Direct binding

GnRH

Stimulation

GATAB

GATA

a

Direct binding

IL-1 & -6

Stimulation

TSEB(s)

TSE

a

Direct binding

Insulin

Stimulation

SF-1

GSE

a

Direct binding

LIF

Stimulation

LH-2

PGBE

a

Direct binding

MCSF

Stimulation

Retinoic acid

Stimulation

INHIBITION c-Jun

CRE(s)

a & (3

Direct binding

Thyroid hormone

Stimulation

Oct-3/4

Octamer

a& p

Direct binding to the p promoter

Activin

Potentiation of GnRH effect

Indirect action on the a promoter

hCG

Biphasic effect

Phorbol ester

Synergistic effect with cAMP

Glucocorticoid receptor

GRE

a

Indirect action

Dopamine

Inhibition

T, receptor

TRE

a

Indirect action

GnRH antagonists

Inhibition

Inhibin

Inhibition

Prolactin

Inhibition

AP-2, activator protein-2; URE, upstream regulatory element; CREB, cyclic ade¬ nosine monophosphate (cAMP) response element-binding protein; CRE(s), cAMP response elements; JREB, junctional regulatory element-binding protein; /RE, junc¬ tional regulatory element; GATAB, GATA-binding protein; TSEB(s), trophoblastspecific element-binding proteins; TSE, trophoblast-specific element; SF-1, steroidogenic factor-1; GSE, gonadotrope-specific element; PGBE, pituitary glycoprotein hormone basal element; GRE, glucocorticoid response element; TRE, thyroid hormone response element.

Progesterone

Inhibition

Mifepristone

Inhibition

TGF-P

Inhibition

TSH

Inhibition

DHEA, dehydroepiandrosterone. See Table 112-1 for explanations of the peptide abbreviations.

some 19ql3.3.30'31 The genes are a closely spaced array of tan¬ dem inverted copies. The hCG-(3 subunit genes 5, 3, and 8 contain a natural promoter and have high transcriptional activ¬ ity. The 5'-flanking region of other hCG-(J subunit genes contains several gaps and deletions that make them transcriptionally less active; sensitive methods are required to detect their expression.32 Analysis of sequence divergence in the coding and noncod¬ ing regions of hCG-fS subunit genes suggests that multiple copies are probably derived from duplication of the ancestral LH-(3 subunit gene.33 During this duplication, deletion of a single base resulted in read-through of the translation stop codon. Thus, a part of the 3'-untranslated region became the coding sequence for the additional 30 amino acids in the carboxyterminus of the hCG-p subunit.34 Unlike the gonadotropin-a subunit gene, which contains a single promoter site, the evolution of the hCG-(3 subunit gene introduced a new pro¬ moter and a number of c/s-acting elements that confer cellspecific transcription.35 The a subunit gene is transcriptionally more active than are the hCG-P subunit genes. However, the hCG-P subunit is rate limiting in the synthesis of intact hCG.27'36 The trans-acting factors and ds-acting elements that are involved in transcriptional activation or inhibition of the a and p subunit genes of hCG are summarized in Table 112-2 35-38 These confer the specificity of regulation by agents that modu¬ late the hCG synthesis. The regulation of hCG synthesis also involves posttranscriptional mechanisms such as the stability of the hCG-subunit mRNA and protein and posttranslational modifications.30-41 The placenta primarily secretes intact hCG and, to a small extent, the free a subunit, nicked hCG, and the P-core frag¬ ment.26'27 The control mechanisms for their release are probably different and are not well understood. Intact hCG is bioactive by virtue of its ability to bind to hCG/LH receptors. The free subunits, on the other hand, are bioinactive because they can¬ not bind to hCG/LH receptors. It is difficult to reconcile the few reports42-44 claiming that free subunits are also bioactive because no separate receptors for subunits have been identi¬

fied. The functional relevance of nicked hCG and P-core frag¬ ment is unknown. A number of agents produced by the placenta can regulate the synthesis of hCG. Some of them inhibit, whereas others stimulate; depending on the concentration, hCG can do both. Many of the regulatory agents that control hCG synthesis are listed in Table 112-3.2'8'15'36'41 hCG synthesis can be controlled at two different levels: (a) by increasing the number of syncytiotrophoblasts that produce large amounts of hCG45 (these are formed from cytotrophoblasts, which produce very little hCG) and (b) by regulating the expression of hCG-subunit genes in syncytiotrophoblasts. The self-regulation of hCG synthesis is a new concept, which has developed from the discovery that cyto- and syncytiotropho¬ blasts contain hCG/LH receptors and that exogenous, as well as endogenous, hCG can promote the differentiation of cytotrophoblasts and the expression of hCG-subunit genes by both transcriptional and posttranscriptional mechanisms. The effects of hCG are concentration dependent. Lower concentrations stimulate and higher concentrations inhibit the differentiation of cytotrophoblasts as well as the expression of hCG-subunit genes.17-40 Although hCG/LH receptors are present in the firsttrimester placenta, they are not functional in self-regulation of hCG biosynthesis. Self-regulation can potentially explain the pregnancy profile of hCG. Its rapid increase to peak levels is probably due to lack of feedback inhibition by hCG during the first trimester. Once peak levels are reached, feedback inhibition results in a drop in hCG levels. When they decrease, they can never fall to zero because low levels stimulate synthesis. When they begin to rise, they can never return to previous high levels because high con¬ centrations inhibit synthesis. Thus, low steady-state levels are maintained throughout the second and third trimester of preg¬ nancy.2 This profile is quite different from that of other placen¬ tal hormones (i.e., human placental lactogen and the steroid hormones; see Chaps. 108 and 109), which progressively increase throughout pregnancy.

1 098

PART VII: ENDOCRINOLOGY OF THE FEMALE

TABLE 112-4. Human Chorionic Gonadotropin/Luteinizing Hormone Receptor Distribution

TABLE 112-5. Actions of Human Chorionic Gonadotropin during Pregnancy Support luteal synthesis of progesterone through 6-9 weeks

Ovary

Inhibit cyclic release of luteinizing hormone

Placenta Fetal membranes

Induce behavioral changes, i.e., nausea and vomiting, craving, drowsiness, and decreased physical activity, etc.

Decidua

Promote early embryonic growth and development

T cells, monocytes, and macrophages

Promote implantation

Umbilical cord

Maintain myometrial quiescence

Uterus

Increase uterine blood flow

Oviduct

Regulate placental steroid and eicosanoid biosynthesis

Urinary bladder

Increase invasion of extravillous trophoblasts

Skin

Prevent maternal T-cell activation through up-regulation of 2,3-indoleamine dioxygenase in syncytiotrophoblasts

Adrenal cortex-zona reticularis Brain Neural retina Breast

Regulate the differentiation of cytotrophoblasts Regulate the expression of human chorionic gonadotropin subunit genes Facilitate nutrient and metabolite exchange between maternal and fetal cir¬ culation through its actions on umbilical vessels Promote weakening of fetal membranes and softening of cervix toward the end of pregnancy through increased prostaglandin production

The self-regulation of hCG biosynthesis is probably not an allor-none phenomenon. Its onset may be gradual, potentially explaining the decrease in doubling times as hCG levels rapidly increase during the first trimester. The presence or absence and the strength of self-regulation may explain individual variations in hCG levels, variations in the same woman during different pregnancies, and the high hCG levels in preeclampsia and Down syndrome as compared to those found in normal pregnancy.7-9'46

SHIFT IN THE CURRENT PARADIGM FOR HUMAN CHORIONIC GONADOTROPIN ACTIONS For a long time, it was believed that the only function of hCG was to rescue and maintain the corpus luteum from regression in a fertile cycle until the placenta could start producing ade¬ quate amounts of progesterone. Once this luteoplacental shift was completed, hCG was considered a vestigial hormone. The persistence of hCG throughout pregnancy should have aroused suspicions that it might have other functions. However, this possibility was not considered since it was not known that hCG has pervasive actions throughout the body,9-46-47 as evidenced by the low levels of functional hCG/LH receptors found in sev¬ eral nongonadal tissues9-46^19 (Table 112-4). The receptors in nongonadal tissues, as in gonadal tissues, have been demonstrated by mRNA, protein, and hormone¬ binding studies.9-46-49 The existence of these receptors indicates that hCG can regulate nongonadal tissue functions. Indeed, studies have substantiated this possibility. The actions of hCG from early embryonic growth and development to the end of pregnancy are summarized in Table 112-5.9'46'47'49 The absence of cyclic LH release during pregnancy could be due to hCG reaching gonadotropin-releasing hormone (GnRH) neurons in hypothalamus to inhibit its synthesis.6'50-51 Many pregnancy-associated behaviors can be induced in the rat model by injecting hCG into the peripheral circulation or into brain ventricles.52-53 This implies that peripheral hCG is trans¬ ported into cerebrospinal fluid by receptors in choroid plexus to act on brain areas that are associated with behavioral regula¬ tion.6'52-53 Locally made, peripherally and/or blastocystderived hCG/LH may promote early embryonic growth, development, and implantation.54'55 hCG derived from pla¬ centa may maintain myometrial quiescence,56-57 increase the uterine blood flow,58-59 and induce numerous other changes in the fetoplacental unit.60-61 hCG levels are very low in the fetal circulation, suggesting that hCG secretion is directed into the maternal circulation and is prevented from entering into the fetus. The reason for this could be that the hCG surge may interfere with growth and developmental programs in the fetus. The low fetal hCG levels

are derived primarily from the fetal kidney and liver62 Fetal hCG is structurally and functionally similar to, but not identi¬ cal to, the maternal hormone. Fetal hCG may control fetal adrenal androgen synthesis, gonadal steroid production,62-63 and brain growth and differentiation,64-66 and may relax the umbilical vessels, keeping the umbilical cords from becoming too rigid.60 hCG reportedly has antiviral properties, which may pro¬ tect the fetus from certain viral infections.67 Although this may depend on the stage of pregnancy, the viral load, and the type of virus, studies on a human immunodeficiency virus (HIV) transgenic mouse model may support these observa¬ tions.68 In this model, heterozygous mothers bear homozy¬ gous and heterozygous litters that cannot be differentiated until ~3 days after birth. Then, homozygous pups begin to show severe signs of wasting syndrome and 100% of them die in a few weeks. If these pups have been treated with hCG, the disease progression slows and the animals survive. Thus, transgenic HIV mice fetuses are protected in utero, and this protection can be extended by hCG administration after birth. Although the mechanisms have not been worked out, a num¬ ber of different ones could be involved in this protective action of hCG.68-69 The influence of hCG on myometrium should diminish toward the end of pregnancy, so that active labor can begin. In fact, the myometrial hCG/LH receptors appear to decrease as labor approaches.70 hCG acts on the fetal membranes to increase the synthesis of prostaglandins, possibly weakening them by collagen breakdown.61 Since fetal membranes lie in the cervical canal during active labor, any prostaglandins pro¬ duced by the fetal membranes will have an opportunity to cause cervical softening through collagenolysis. Thus, the waning influence of hCG on the myometrium and its increas¬ ing influence on the fetal membranes and the cervix are proba¬ bly meant for smooth progression of labor and delivery These opposing influences of hCG on different tissues at the same time may occur through interactions with other agents present within the fetomaternal environment. Table 112-5 does not include some of the predictable changes during pregnancy. For example, the presence of hCG/ LH receptors predicts pregnancy-associated skin changes.71 The presence of receptors in the urinary bladder and the possi¬ ble smooth muscle-relaxing activity of hCG suggest that fre¬ quent urination during early pregnancy could be caused by hCG.72 Changes in visual processing during pregnancy may occur through the actions of hCG/LH receptors contained in

Ch. 112: Endocrinology of Trophoblastic Tissue the neural retina.73 The synthesis of maternal and fetal adrenal androgens may, in part, be controlled by hCG.62 Since fetal adrenal androgens are precursors of estriol synthesis in the placenta, and urinary estriol reflects fetal well-being, ultimate fetal and placental well-being could be determined by actions of hCG.

PRODUCTION AND ACTION OF HUMAN CHORIONIC GONADOTROPIN IN TUMOR TISSUES hCG belongs to a family of embryonically related marker pro¬ teins that include carcinoembryonic antigen and a-fetoprotein.74 Thus, a wide variety of cancers and cancer cells contain intact hCG and/or one of its subunits; to date, >70 different cancer cell lines have been shown to contain them.25'75-78-783 The presence of hCG and/or one of its subunits in cancer cells is probably due to synthesis rather than sequestration. The regu¬ latory mechanisms involved in the expression of hCG-subunit genes in cancer cells is not known. Ectopic production of hCG is considered a recapitulation of the embryonic state, as is cancer. The expression of hCG and/or one of its subunits increases in advanced cancers, suggesting that they might be involved in the progression of the disease. In fact, contraceptive hCG vaccine is now being tested, especially against cancers of the colon and pancreas.79-81 Like other hormones, hCG acts via binding to its receptors. A demonstration of hCG/LH receptors in cancers of nongonadal tissues has reinforced a belief that hCG may indeed play a role. In fact, studies suggest that hCG may have dual roles in cancers. It promotes some cancers (endometrial cancer,82-86 chorio¬ carcinomas4'5 [see Chap. Ill], and lung cancer87-88), whereas it inhibits others (prostate cancer89-92 and breast cancer93-96). Some controversies on whether hCG prevents or promotes cancers could be due to whether they produce intact hCG or just its (3 subunit, which may have a stimulatory effect, probably due to the formation of homodimers.97-99 When intact hCG or LH promotes cancer, its presence in can¬ cer tissues can be expected to be associated with a poor progno¬ sis. When these hormones protect against cancer, their presence indicates a good prognosis. In the latter case, injection of the hormone into the lesion may slow the cancer progression. Gestational trophoblastic neoplasms (GTNs) contain high hCG/LH receptor levels, which further increase in more malignant phenotypes such as choriocarcinomas.100 These high receptor levels are due to a loss of self-regulation of hCG bio¬ synthesis. This may explain how GTNs can produce much higher levels of hCG than do normal trophoblasts.101 The high hCG levels produced by choriocarcinomas may promote their growth, development, and metastasis in the host body.5 hCG-producing tumors (see Chaps. 120 and 219) in young boys can cause precocious puberty by virtue of constant stim¬ ulation of the testis to produce testosterone102 (see Chap. 92). Such tumors in young girls usually do not have obvious adverse effects unless they are associated with the ovaries. The reason for this gender difference is that both LH and FSH are required for ovarian estradiol synthesis, whereas LH alone is capable of stimulating testicular synthesis of tes¬ tosterone. This gender difference is also seen when there is an activating LH receptor mutation.103 hCG-producing tumors in some men can cause gynecomastia, possibly due to direct actions of hCG on the breast. hCG-producing tumors in women cause disruption of the menstrual cycle and dysfunc¬ tional uterine bleeding.

POTENTIAL THERAPEUTIC USES OF HUMAN CHORIONIC GONADOTROPIN There are several potential therapeutic uses of hCG. Since hCG has pervasive actions during pregnancy, some unexplained pregnancy losses could be due to aberrant or inadequate

1099

actions of hCG; this may be corrected by the administration of hCG. hCG levels progressively decrease during threatened abortion, but whether this is a cause or consequence is not known. Administration of hCG also may help in some of these cases. hCG treatment may work by increasing the placental endocrine activity, by preventing immunologic mechanisms that promote fetal rejection, by increasing uterine blood flow, by decreasing uterine activity, and so forth.49 This treatment may not work if infection, anatomic defects, fetal anomalies, and so forth, are responsible for these conditions. The ability of hCG to maintain myometrial quiescence sug¬ gests it may be used in the treatment of preterm labor and delivery, unless it is caused by infection, premature rupture of membranes, and so forth. In fact, administration of hCG has a tocolytic effect in a mouse preterm-labor model.104 If it is proved that hCG works in women, it would be the most natural means of preventing preterm labor. The rationale for giving hCG when women already have it in their circulation is that perhaps their levels are not adequate, and increasing levels by giving exogenous hormone might delay events that lead to pre¬ term labor and delivery. Epidemiologic data, the rat breast cancer model studies, and the anticancer effects of hCG in human breast cancer cells suggest that the decreased incidence of breast cancer in women who complete a full-term pregnancy before 20 years of age could be due to hCG.105-108 This hormone may act on breasts to promote nonreversible differentiation of proliferation-compe¬ tent epithelial cells into secretory cells in terminal end buds. Coincidentally, this differentiation, which is a physiologic phenomenon to prepare the breast for lactation, also makes the cells less susceptible to carcinogenic transformation.109 Additional mechanisms, such as inhibition of cell growth and invasion,94-95-110 increase of apoptosis,111'112 and the cell's abil¬ ity to repair DNA damage, also may play roles in the protec¬ tive actions of hCG in the breast.113,114 Increased inhibin and insulin-like growth factor (IGF)-binding proteins and/or decreased IGF-I and its receptors may mediate hCG actions in breast cancer cells.109'115'116 These findings do not necessarily mean that every woman who completes a full-term pregnancy at a young age will never get breast cancer. Several other factors, such as family history, radiation, environment, and so forth, contribute to the develop¬ ment of this disease; therefore, pregnancy may not be able to overcome some or all of these factors. Potential uses of hCG in the treatment of HIV infections and Kaposi sarcomas are controversial.117,118 Nonetheless, these treatment strategies may have some merit because of the reported antiviral properties of hCG as well as its ability to act on cells of the immune system and numerous other target tis¬ sues throughout the body.46,49,67,119-121

SUMMARY AND PERSPECTIVES Trophoblastic tissue is a transient and unique endocrine organ that is capable of producing a vast array of bioactive sub¬ stances. Among them, hCG is the best known and perhaps most important. hCG is not just a gonadal-regulating hor¬ mone as once was believed. It is a pluripotent regulatory mol¬ ecule with the actions of a classic hormone, a growth factor, and a cytokine. It plays a pivotal role in the regulation of the functions of the fetoplacental unit and of a number of other tissues during pregnancy. Although the evolutionary signifi¬ cance of the broad spectrum of hCG actions is unknown, it could have evolved to orchestrate numerous functions during pregnancy in women. LH may fulfill some of the roles of hCG in other species. These far-ranging actions are not unique to hCG, as prolactin is another example of a hormone having multiple targets in the body. hCG research has helped to explain many unknown, and to rationalize several known, effects of hCG.

1100

PART VII: ENDOCRINOLOGY OF THE FEMALE

REFERENCES 33. 1. Bemischke K, Kaufmann P. Pathology of the human placenta, 3rd ed. New York: Springer-Verlag. 1995. 2. Rodway MR, Rao ChV. A novel perspective on the role of human chorionic gonadotropin during pregnancy and in gestational trophoblastic disease. Early Pregnancy Biol Med 1995; 1(3):176. 3. El-Hendy KA, Subramanian MG, Diamond MP, Yelian FD. hCG modulates MMP-9 activity in first trimester trophoblast cells. J Soc Gynecol Invest 1998; 5:118A. 4. Zygmunt M, Hahn D, Munstedt K, et al. Invasion of cytotrophoblastic JEG3 cells is stimulated by hCG in vitro. Placenta 1998; 19(8):587. 5. Lei ZM, Taylor DD, Gercel-Taylor C, Rao ChV. Human chorionic gonado¬ tropin promotes tumorigenesis of choriocarcinoma JAR cells. Trophoblast Res 1999; 13:147. 6. Lei ZM, Rao ChV, Kornyei JL, et al. Novel expression of human chorionic gonadotropin/luteinizing hormone receptor gene in brain. Endocrinology 1993; 132(5):2262. 7. Kliman HJ. Placental hormones. Endocrinol Pregnancy 1994; 5(4):591. 8. Petraglia F, Florio P, Nappi C, Genazzani AR. Peptide signaling in human placenta and membranes: autocrine, paracrine, and endocrine mecha¬ nisms. Endocr Rev 1996; 17(2):156. 9. Rao ChV. Potential novel roles of luteinizing hormone and human chori¬ onic gonadotropin during early pregnancy in women. Early Pregnancy Biol Med 1997; 3(1):1. 10. Walsh SW. Prostaglandins in pregnancy. In: Sciarra JJ, ed. Gynecology and obstetrics. Philadelphia: JB Lippincott Co, 1992. 11. Reyes FI. Protein hormones of the placenta. In: Sciarra JJ, ed. Gynecology and obstetrics. Philadelphia: JB Lippincott Co, 1992. 12. Guilbert L, Robertson SA, Wegmann TG. The trophoblast as an integral component of a macrophage-cytokine network. Immunol Cell Biol 1993; 71 (Pt 1):49. 13. Senaris R, Garcia-Caballero T, Casabiell X, et al. Synthesis of leptin in human placenta. Endocrinology 1997; 138(10):4501. 14. Conrad KP, Benyo DF. Placental cytokines and the pathogenesis of pre¬ eclampsia. Am J Reprod Immunol 1997; 37(3):240. 15. Petraglia F, Santuz M, Florio P, et al. Paracrine regulation of human placenta: control of hormonogenesis. J Reprod Immunol 1998; 39(1— 2):221. 16. He Y, Smith SK, Day KA, et al. Alternative splicing of vascular endothelial growth factor (VEGF)-Rl (FLT-1) pre-mRNA is important for the regula¬ tion of VEGF activity. Mol Endocrinol 1999; 13(4):537. 17. Shi QJ, Lei ZM, Rao ChV, Lin J. Novel role of human chorionic gonadotro¬ pin in differentiation of human cytotrophoblasts. Endocrinology 1993; 132(3):1387. 18. Sawai K, Azuma C, Koyama M, et al. Leukemia inhibitory factor (LIF) enhances trophoblast differentiation mediated by human chorionic gonad¬ otropin (hCG). Biochem Biophys Res Commun 1995; 211(1):137. 19. Sawai K, Matsuzaki N, Kameda T, et al. Leukemia inhibitory factor pro¬ duced at the fetomatemal interface stimulates chorionic gonadotropin pro¬ duction: its possible implication during pregnancy, including implantation period. J Clin Endocrinol Metab 1995; 80(4):1449. 20. Mochizuki M, Maruo T, Matsuo H, et al. Biology of human trophoblast. Int J Gynaecol Obstet 1998; 60(Suppl 1):S21. 21. Morrish DW, Dakour J, Li H. Functional regulation of human trophoblast differentiation. J Reprod Immunol 1998; 39(1-2):179. ■ 22. Yang M, Lei ZM, Rao ChV. Mechanism of leukemia inhibitory factor induced differentiation of human cytotrophoblasts into syncytiotrophoblasts. In: The Endocrine Society Annual Meeting, San Diego, CA; 1999. Abstract Pl-6. 23. Yang M, Lei ZM, Rao ChV. How does epidermal growth factor promote the differentiation of human cytotrophoblasts into syncytiotrophoblasts. J Soc Gynecol Invest 1999; 6(Suppl 1):Abstract 31. 24. Lei ZM, Toth P, Rao ChV, Pridham D. Novel coexpression of human chori¬ onic gonadotropin (hCG)/human luteinizing hormone receptors and their ligand hCG in human fallopian tubes. J Clin Endocrinol Metab 1993; 77:863. 25. Acevedo HF, Hartsock RJ, Maroon JC. Detection of membrane-associated human chorionic gonadotropin and its subunits on human cultured cancer cells of the nervous system. Cancer Detect Prev 1997; 21(4):295. 26. Pierce JG, Parsons TF. Glycoprotein hormones: structure and function. Annu Rev Biochem 1981: 50:465. 27. lies RK, Chard T. Molecular insights into the structure and function of human chorionic gonadotrophin. J Mol Endocrinol 1993; 10(3):217. 28. Lapthom AJ, Harris DC, Littlejohn A, et al. Crystal structure of human chorionic gonadotropin. Nature 1994; 369(6480):455. 29. Fiddes JC, Goodman HM. The gene encoding the common alpha sub¬ unit of the four human glycoprotein hormones. J Mol Appl Genet 1981; 1(1):3. 30. Boorstein WR, Vamvakopoulos NC, Fiddes JC. Human chorionic gonado¬ tropin beta-subunit is encoded by at least eight genes arranged in tandem and inverted pairs. Nature 1982; 300(5891):419. 31. Policastro P, Ovitt CE, Hoshina M, et al. The beta subunit of human chori¬ onic gonadotropin is encoded by multiple genes. J Biol Chem 1983; 258(19):11492. 32. Bo M, Boime I. Identification of the transcriptionally active genes of the

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35.

36. 37.

38.

39.

40.

41. 42.

43.

44.

45. 46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

chorionic gonadotropin beta gene cluster in vivo. J Biol Chem 1992; 267(5):3179. Talmadge K, Vamvakopoulos NC, Fiddes JC. Evolution of the genes for the beta subunits of human chorionic gonadotropin and luteinizing hormone. Nature 1984; 307(5946):37. Fiddes JC, Goodman HM. The cDNA for the beta-subunit of human chori¬ onic gonadotropin suggests evolution of a gene by readthrough into the 3'untranslated region. Nature 1980; 286(5774):684. Albanese C, Colin IM, Crowley WF, et al. The gonadotropin genes: evolu¬ tion of distinct mechanisms for hormonal control. Recent Prog Horm Res 1996; 51:23. Jameson JL, Hollenberg AN. Regulation of chorionic gonadotropin gene expression. Endocr Rev 1993; 14(2):203. Liu L, Roberts RM. Silencing of the gene for the beta subunit of human chorionic gonadotropin by the embryonic transcription factor Oct-3/4. J Biol Chem 1996; 271(28):16683. Liu L, Leaman D, Villalta M, Roberts RM. Silencing of the gene for the alpha-subunit of human chorionic gonadotropin by the embryonic tran¬ scription factor Oct-3/4. Mol Endocrinol 1997; 11(11):1651. Fuh VL, Burrin JM, Jameson JL. Cyclic AMP (cAMP) effects on chorionic gonadotropin gene transcription and mRNA stability: labile proteins medi¬ ate basal expression whereas stable proteins mediate cAMP stimulation. Mol Endocrinol 1989; 3(7):1148. Licht P, Cao H, Lei ZM, et al. Novel self-regulation of human chorionic gonadotropin biosynthesis in term pregnancy human placenta. Endocri¬ nology 1993; 133(6):3014. Merz WE. Biosynthesis of human chorionic gonadotropin: a review. Eur J Endocrinol 1996; 135(3):269. Blithe DL, Richards RG, Skarulis MC. Free alpha molecules from preg¬ nancy stimulate secretion of prolactin from human decidual cells: a novel function for free alpha in pregnancy. Endocrinology 1991; 129(4):2257. Moy E, Kimzey LM, Nelson LM, Blithe DL. Glycoprotein hormone alphasubunit functions synergistically with progesterone to stimulate differenti¬ ation of cultured human endometrial stromal cells to decidualized cells: a novel role for free alpha-subunit in reproduction. Endocrinology 1996; 137(4):1332. Wolkersdorfer GW, Bomstein SR, Hilbers U, et al. The presence of chori¬ onic gonadotrophin beta subunit in normal cyclic human endometrium. Mol Hum Reprod 1998; 4(2):179. Ringler GE, Strauss JF. In vitro systems for the study of human placental endocrine function. Endocr Rev 1990; 11(1):105. Rao ChV. The beginning of a new era in reproductive biology and medi¬ cine: expression of low levels of functional luteinizing hormone/human chorionic gonadotropin receptors in nongonadal tissues. J Physiol Pharma¬ col 1996; 47(Suppl 2):41. Rao ChV. Novel concepts in neuroendocrine regulation of reproductive tract functions. In: Bazer FW, ed. The endocrinology of pregnancy. Totowa, NJ: Humana Press, 1998:125. Ziecik AJ, Derecka-Reszka K, Rzucidlo SJ. Extragonadal gonadotropin receptors, their distribution and function. J Physiol Pharmacol 1992; 43(4 Suppl 1):33. Rao ChV. A paradigm shift on the targets of luteinizing hormone/human chorionic gonadotropin actions in the body. J Bellevue Obstet Gynecol Soc 1999; 15:26. Lei ZM, Rao ChV. Signaling and transacting factors in the transcriptional inhibition of gonadotropin releasing hormone gene by human chorionic gonadotropin in immortalized hypothalamic GT1-7 neurons. Mol Cell Endocrinol 1995; 109(2):151. Lei ZM, Rao ChV. Cfs-acting elements and fraus-acting proteins in the tran¬ scriptional inhibition of gonadotropin-releasing hormone gene by human chorionic gonadotropin in immortalized hypothalamic GT1-7 neurons. J Biol Chem 1997; 272(22):14365. Toth P, Lukacs H, Hiatt ES, et al. Administration of human chorionic gonad¬ otropin affects sleep-wake phases and other associated behaviors in cycling female rats. Brain Res 1994; 654(2):181. Lukacs H, Hiatt ES, Lei ZM, Rao ChV. Peripheral and intracerebroventricular administration of human chorionic gonadotropin alters several hippocampus-associated behaviors in cycling female rats. Horm Behav 1995; 29(1):42. Han SW, Lei ZM, Rao ChV. Up-regulation of cyclooxygenase-2 gene expression by chorionic gonadotropin during the differentiation of human endometrial stromal cells into decidua. Endocrinology 1996; 137(5):1791. Han SW, Lei ZM, Rao ChV. Treatment of human endometrial stromal cells with chorionic gonadotropin promotes their morphological and functional differentiation into decidua [In Process Citation). Mol Cell Endocrinol 1999; 147(1—2):7. Ambrus G, Rao ChV. Novel regulation of pregnant human myometrial smooth muscle cell gap junctions by human chorionic gonadotropin. Endocrinology 1994; 135(6):2772. Eta E, Ambrus G, Rao ChV. Direct regulation of human myometrial con¬ tractions by human chorionic gonadotropin. J Clin Endocrinol Metab 1994; 79(6):1582. Toth P, Li X, Rao ChV, et al. Expression of functional human chorionic gonadotropin/human luteinizing hormone receptor gene in human uter¬ ine arteries. J Clin Endocrinol Metab 1994; 79(1):307.

Ch. 112: Endocrinology of Trophoblastic Tissue 59. Toth P, Gimes G, Rao ChV. hCG treatment in early gestation: its impact on uterine blood flow and pregnancy outcome. In: 16th World Con¬ gress on Fertility and Sterility and 54th Annual Meeting of the Ameri¬ can Society for Reproductive Medicine; 1998, San Francisco, CA; Abstract S46. 60. Rao ChV, Li X, Toth P, et al. Novel expression of functional human chori¬ onic gonadotropin/luteinizing hormone receptor gene in human umbilical cords. J Clin Endocrinol Metab 1993; 77(6):1706. 61. Toth P, Li X, Lei ZM, Rao ChV. Expression of human chorionic gonadotro¬ pin (hCG)/luteinizing hormone receptors and regulation of the cyclooxygenase-1 gene by exogenous hCG in human fetal membranes. J Clin Endocrinol Metab 1996; 81(3):1283. 62. McGregor WG, Kuhn RW, Jaffe RB. Biologically active chorionic gonado¬ tropin: synthesis by the human fetus. Science 1983; 220(4594):306. 63. Dobozy O, Brindak O, Csaba G. Influence of pituitary hormones (hCG, TSH, Pr, GH) on testosterone level and on the functional activity of the Leydig cell in rat fetuses. Acta Physiol Hung 1988; 72(2):159. 64. Al-Hader AA, Lei ZM, Rao ChV. Novel expression of functional luteinizing hormone/chorionic gonadotropin receptors in cultured glial cells from neonatal rat brains. Biol Reprod 1997; 56(2):501. 65. Al-Hader AA, Lei ZM, Rao ChV. Neurons from fetal rat brains contain functional luteinizing hormone/chorionic gonadotropin receptors. Biol Reprod 1997; 56(5):1071. 66. Al-Hader AA, Tao YX, Lei ZM, Rao ChV. Fetal rat brains contain luteiniz¬ ing hormone/human chorionic gonadotropin receptors. Early Pregnancy Biol Med 1997; 3(4):323. 67. Harris PJ. Human chorionic gonadotropin hormone is antiviral. Med Hypotheses 1996; 47(2):71. 68. De SK, Wohlenberg CR, Marinos NJ, et al. Human chorionic gonadotropin hormone prevents wasting syndrome and death in HIV-1 transgenic mice. J Clin Invest 1997; 99(7):1484. 69. Shapira A, Bao S, Lei ZM, et al. Treatment of homozygous HIV-1 transgenic mouse pups with human chorionic gonadotropin (hCG) upregulates the skin hCG/luteinizing hormone receptor levels. In the program of The Endocrine Society Annual Meeting, 1998, Abstract P2-149. 70. Zuo J, Lei ZM, Rao ChV. Human myometrial chorionic gonadotropin/ luteinizing hormone receptors in preterm and term deliveries. J Clin Endo¬ crinol Metab 1994; 79(3):907. 71. Bird J, Li X, Lei ZM, et al. Luteinizing hormone and human chorionic gonadotropin decrease type 2 5 alpha-reductase and androgen receptor protein levels in women's skin. J Clin Endocrinol Metab 1998; 83(5):1776. 72. Tao YX, Heit M, Lei ZM, Rao ChV. The urinary bladder of a woman is a novel site of luteinizing hormone-human chorionic gonadotropin receptor gene expression. Am J Obstet Gynecol 1998; 179(4):1026. 73. Thompson DA, Othman MI, Lei ZM, et al. Localization of receptors for luteinizing hormone/chorionic gonadotropin in neural retina. Life Sci 1998; 63(12):1057. 74. Jacobs EL, Haskell CM. Clinical use of tumor markers in oncology. Curr Probl Cancer 1991; 15(6):299. 75. Acevedo HF, Krichevsky A, Campbell-Acevedo EA, et al. Expression of membrane-associated human chorionic gonadotropin, its subunits, and fragments by cultured human cancer cells. Cancer 1992; 69 (7):1829. 76. Acevedo HF, Krichevsky A, Campbell-Acevedo EA, et al. Flow cytometry method for the analysis of membrane-associated human chorionic gonad¬ otropin, its subunits, and fragments on human cancer cells. Cancer 1992; 69(7):1818. 77. Acevedo HF, Tong JY, Hartsock RJ. Human chorionic gonadotropin-beta subunit gene expression in cultured human fetal and cancer cells of differ¬ ent types and origins [see comments]. Cancer 1995; 76(8):1467. 78. Lazar V, Diez SG, Laurent A, et al. Expression of human chorionic gonado¬ tropin beta subunit genes in superficial and invasive bladder carcinomas. Cancer Res 1995; 55(17):3735. 78a. Fujikawa K, Matsui Y, Oka H, et al. Prognosis of primary testicular semi¬ noma: a report of 57 new cases. Cancer Res 2000; 60:2152. 79. Triozzi PL, Martin EW, Gochnour D, Aldrich W. Phase lb trial of a synthetic beta human chorionic gonadotropin vaccine in patients with metastatic cancer. Ann N Y Acad Sci 1993; 690:358. 80. Triozzi PL, Stevens VC, Aldrich W, et al. Effects of a beta-human chorionic gonadotropin subunit immunogen administered in aqueous solution with a novel nonionic block copolymer adjuvant in patients with advanced can¬ cer. Clin Cancer Res 1997; 3(12 Pt 1):2355. 81. Triozzi PL, Stevens VC. Human chorionic gonadotropin as a target for can¬ cer vaccines. Oncol Rep 1999; 6(1):7. 82. Lin J, Lei ZM, Lojun S, et al. Increased expression of luteinizing hormone/ human chorionic gonadotropin receptor gene in human endometrial carci¬ nomas. J Clin Endocrinol Metab 1994; 79(5):1483. 83. Bax CR, Chatzaki E, Davies S, Gallagher CJ. Elucidating the role of gonado¬ tropins in endometrial cancer cell growth. Biochem Soc Trans 1996; 24:443S. 84. Konishi I, Koshiyama M, Mandai M, et al. Increased expression of LH/ hCG receptors in endometrial hyperplasia and carcinoma in anovulatory women. Gynecol Oncol 1997; 65(2):273. 85. Nagamani M, Cao HA. Specific binding and proliferative effects of lutein¬ izing hormone in human endometrial cancer cell lines. J Soc Gynecol Invest 1997; 4:132 A. 86. Han SW, Zhou XL, Lei ZM, Rao ChV. Role of luteinizing hormone in human endometrial carcinoma. In: The Endocrine Society Annual Meeting; 1999, Abstract Pl-593.

1101

87. Rivera RT, Pasion SG, Wong DT, et al. Loss of tumorigenic potential by human lung tumor cells in the presence of antisense RNA specific to the ectopically synthesized alpha subunit of human chorionic gonadotropin J Cell Biol 1989; 108(6):2423. 88. Kumar S, Talwar GP, Biswas DK. Necrosis and inhibition of growth of human lung tumor by anti-alpha human chorionic gonadotropin antibody. J Natl Cancer Inst 1992; 84(1):42. 89. Tao YX, Bao S, Ackermann DM, et al. Expression of luteinizing hormone/ human chorionic gonadotropin receptor gene in benign prostatic hyperpla¬ sia and in prostate carcinoma in humans. Biol Reprod 1997; 56(1):67. 90. Bao S, Lei ZM, Rao ChV. The presence of functional luteinizing hormone/ chorionic gonadotropin receptors in human prostate cell lines. In: The Endocrine Society Annual Meeting; 1997, Abstract P3^403. 91. Dimhofer S, Berger C, Hermann M, et al. Coexpression of gonadotropic hormones and their corresponding FSH- and LH/CG-receptors in the human prostate. Prostate 1998; 35(3):212. 92. Lei ZM, Rao ChV. Direct luteinizing hormone regulation of male reproduc¬ tive tract In: Coutinho EMSP, ed. Reproductive medicine: a millennium review. The proceedings of the 10th World Congress on Human Reproduc¬ tion; 2000. London: Parthenon Publishing. 2000, In press. 93. Russo IH, Russo J. Chorionic gonadotropin: a tumoristatic and preventive agent in breast cancer. In: Teicher BA, ed. Drug resistance in oncology. New York: Marcel Dekker Inc, 1993:537. 94. Lojun S, Bao S, Lei ZM, Rao ChV. Presence of functional luteinizing hor¬ mone/ chorionic gonadotropin (hCG) receptors in human breast cell lines: implications supporting the premise that hCG protects women against breast cancer. Biol Reprod 1997; 57(5):1202. 95. Li X, Lei ZM, Rao ChV. The actions of human chorionic gonadotropin in MCF-7 cells support the premise that it may protect woman against breast cancer. In: The Endocrine Society Annual Meeting, 1999. Abstract P3. 96. Lei ZM, Rao ChV. Protective role of human chorionic gonadotropin and luteinizing hormone against breast cancer. In: Bamea ER, Jaunaiux JE, Schwartz PE, Schofield PN, eds. Cancer and pregnancy. London: SpringerVerlag, 2000. 97. Gillott DJ, lies RK, Chard T. The effects of beta-human chorionic gonado¬ trophin on the in vitro growth of bladder cancer cell lines. Br J Cancer 1996; 73(3):323. 98. Bieche I, Lazar V, Nogues C, et al. Prognostic value of chorionic gonadotro¬ pin beta gene transcripts in human breast carcinoma. Clin Cancer Res 1998; 4(3):671. 99. Butler SA, Laidler P, Porter JR, et al. The beta-subunit of human chorionic gonadotrophin exists as a homodimer [In Process Citation]. J Mol Endo¬ crinol 1999; 22(2):185. 100. Lei ZM, Rao ChV, Ackerman DM, Day TG. The expression of human chorionic gonadotropin/human luteinizing hormone receptors in human gestational trophoblastic neoplasms. J Clin Endocrinol Metab 1992; 74(6):1236. 101. Licht P, Cao H, Zuo J, et al. Lack of self-regulation of human chorionic gonadotropin biosynthesis in human choriocarcinoma cells. J Clin Endo¬ crinol Metab 1994; 78(5):1188. 102. Perilongo G, Rigon F, Murgia A. Oncologic causes of precocious puberty. Pediatr Hematol Oncol 1989; 6(4):331. 103. Themmen AP, Martens JW, Brunner HG. Activating and inactivating muta¬ tions in LH receptors. Mol Cell Endocrinol 1998; 145(1-2):137. 104. Kurtzman JT, Spinnato JA, Zimmerman MJ, et al. Human chorionic gonad¬ otropin exhibits potent inhibition of preterm delivery in a small animal model. Am J Obstet Gynecol 1999; 181:853. 105. Hildreth NG, Shore RE, Dvoretsky PM. The risk of breast cancer after irra¬ diation of the thymus in infancy N Engl J Med 1989; 321(19):1281. 106. Tokunaga M, Land CE, Tokuoka S. Follow-up studies of breast cancer inci¬ dence among atomic bomb survivors. J Radiat Res (Tokyo) 1991; 32 (Suppl):201. 107. MacMahon B, Cole P, Lin TM, et al. Age at first birth and breast cancer risk. Bull WHO 1970; 43(2):209. 108. Trapido EJ. Age at first birth, parity, and breast cancer risk. Cancer 1983; 51(5):946. 109. Russo J, Russo IH. Hormonally induced differentiation: a novel approach to breast cancer prevention. J Cell Biochem Suppl 1995;22:58. 110. Alvarado MV, Alvarado NE, Russo J, Russo IH. Human chorionic gonad¬ otropin inhibits proliferation and induces expression of inhibin in human breast epithelial cells in vitro. In Vitro Cell Dev Biol Anim 1994; 30A(1):4. 111. Srivastava P, Russo J, Russo IH. Chorionic gonadotropin inhibits rat mam¬ mary carcinogenesis through activation of programmed cell death. Car¬ cinogenesis 1997; 18(9):1799. 112. Srivastava P, Russo J, Mgbonyebi OP, Russo IH. Growth inhibition and activation of apoptotic gene expression by human chorionic gonadotropin in human breast epithelial cells. Anticancer Res 1998; 18(6A):4003. 113. Huang Y, Bove B, Wu Y, et al. Microsatellite instability during the immor¬ talization and transformation of human breast epithelial cells in vitro. Mol Carcinog 1999; 24(2):118. 114. Russo J, Yang X, Hu YF, et al. Biological and molecular basis of human breast cancer. Front Biosci 1998; 3:D944. 115. Alvarado MV, Russo J, Russo IH. Immunolocalization of inhibin in the mammary gland of rats treated with hCG. J Histochem Cytochem 1993; 41(1):29.

1102

PART VII: ENDOCRINOLOGY OF THE FEMALE

116. Huynh H. In vivo regulation of the insulin-like growth factor system of mitogens by human chorionic gonadotropin. Int J Oncol 1998; 13(3):571. 117. Bourinbaiar AS, Nagorny R. Inhibitory effect of human chorionic gonado¬ tropin (hCG) on HIV-1 transmission from lymphocytes to trophoblasts. FEBS Lett 1992; 309(1):82. 118. Darzynkiewicz Z. The butler did it: search for killer(s) of Kaposi's sarcoma cells in preparations of human chorionic gonadotropin. ] Natl Cancer Inst 1999; 91(2):104.

119. Lin J, Lojun S, Lei ZM, et al. Lymphocytes from pregnant women express human chorionic gonadotropin/luteinizing hormone receptor gene. Mol Cell Endocrinol 1995; 111:R13. 120. Zhang YM, Lei ZM, Rao ChV. Human macrophages contain luteinizing hormone and chorionic gonadotropin receptors. J Soc Gynecol Invest 1998; 5(Suppl l):Abstract 212. 121. Zhang YM, Lei ZM, Rao ChV. Functional importance of human monocyte luteinizing hormone and chorionic gonadotropin receptors. J Soc Gynecol Invest 1999;6(Suppl l):Abstract 46.

PART VIII

ENDOCRINOLOGY OF THE MALE WILLIAM J. BREMNER, EDITOR

113. MORPHOLOGY AND PHYSIOLOGY OF THE TESTIS.1104 114. EVALUATION OF TESTICULAR FUNCTION.1115 115. MALE HYPOGONADISM.1125 116. TESTICULAR DYSFUNCTION IN SYSTEMIC DISEASE.1150 117. ERECTILE DYSFUNCTION .1159 118. MALE INFERTILITY.1173 119. CLINICAL USE AND ABUSE OF ANDROGENS AND ANTIANDROGENS .1181 120. GYNECOMASTIA.1200

121. ENDOCRINE ASPECTS OF BENIGN PROSTATIC HYPERPLASIA.1207 122. TESTICULAR TUMORS.1212 123. MALE CONTRACEPTION .1220

1104

PART VIII: ENDOCRINOLOGY OF THE MALE

CHAPTER

113

MORPHOLOGY AND PHYSIOLOGY OF THE TESTIS DAVID M. DE KRETSER

STRUCTURAL ORGANIZATION OF THE TESTIS In most mammalian species, the testis is located within the scrotum, having descended from an intraabdominal position during fetal development1 (see Chap. 93). The intrascrotal posi¬ tion allows the testis to function at a lower temperature than is found within the abdomen. This is a requirement for normal spermatogenesis in many mammals including humans, although in some, such as the elephant, the testes do not descend and spermatogenesis is unaffected by the higher tem¬ perature within the abdomen. Because of testicular descent, the vascular supply originates relatively proximally, from the aorta near the origin of the renal arteries. The venous drainage, com¬ mencing as an anastomotic plexus of veins (the pampiniform plexus that surrounds the testicular artery), terminates in the renal vein on the left and in the inferior vena cava on the right.

FIGURE 113-1. A, The arrangement of the seminiferous tubules, rete testis, efferent ducts, and epididymis is illustrated. B, The structure of the rete testis is detailed. (From de Kretser DM, Temple-Smith PD, Kerr JB, et al. Anatomical and functional aspects of the male reproductive organs. In: Bandhauer K, Frick J, eds. Handbuch der Urologie, vol XVI. Berlin: Springer-Verlag, 1982:1.)

This arrangement of vessels acts as a countercurrent mecha¬ nism to maintain lower testicular temperature; the cooler venous blood surrounds the testicular artery, decreasing its temperature as it approaches the testis. The remnant of the peritoneal sac, the processus vaginalis, surrounds the testis on its anterior and lateral sides as the tunica vaginalis. The outer dense connective tissue covering, the tunica albuginea, sends septa, which run posteriorly toward the medi¬ astinum of the testis and divide the testis into a series of lobules. Within these lobules lie the seminiferous tubules, which are coiled, extending as loops from the region of the mediastinum of the testis, from which they drain by straight tubules into an anastomotic network of ducts, the rete testis2 (Fig. 113-1). The products of the tubules drain from the rete testis by a series of 15 to 20 ducts, the ductuli efferentes, which in humans constitute part of the head of the epididymis. In turn, the ductuli drain into the duct of the epididymis, whose coils form the remainder of the head, body, and tail of the epididymis. The connective tissue surrounding the seminiferous tubules contains vascular and lymphatic vessels and the Leydig cells, which are responsible for the androgenic output of the testis.

SPERMATOGENESIS The sequence of cytologic changes that produce spermatozoa from spermatogonia is called spermatogenesis. It can be subdi-

Ch. 113: Morphology and Physiology of the Testis

1105

that retains its stem cell function.5,6 In the human male, three types of spermatogonia are recognized classically: the A dark, thought to represent the most primitive; the A pale; and the B spermatogonia. They lie adjacent to the basement membrane of the tubule, interspersed with the basal aspects of the Sertoli cells. During cell division, the spermatogonia do not complete cytokinesis and remain linked by intercellular bridges.7 Popula¬ tions of spermatogonia that are injected into tubules devoid of germ cells will partially restore spermatogenesis.8 These studies will provide the experimental basis for further evaluation of the nature and control of the stem cells in the testis.

MEIOSIS

FIGURE 113-2. Light micrograph of a section from a normal human testis illustrates spermatogonia (SG), primary spermatocytes (SC), spermatids (SD), Sertoli cell nuclei (arrows), and Leydig cells (L). Stages of the human seminiferous cycle are denoted where identifiable (STG). x950

vided into three major phases: the replication of stem cells, meiosis, and spermiogenesis.

Responding to unknown signals, groups of type B spermatogo¬ nia begin meiosis, which involves two cell divisions. The sper¬ matogonia lose their contact with the basement membrane of the tubule and are then called primary spermatocytes.9 During the prophase of the first division (see Chap. 90), the primary sper¬ matocytes, which have already replicated their DNA and con¬ tain twice the diploid content, undergo a series of characteristic nuclear changes consistent with the appearance and pairing of homologous chromosomes (Fig. 113-3). During the leptotene stage, the chromosomes appear as single, randomly coiled threads, which thicken and commence pairing during zygotene. During pachytene, the chromosomes appear as condensed, closely paired structures, which begin to repel each other during diplotene. Diakinesis is associated with further repulsion of the pairs of chromosomes, which each consist of a pair of daughter chromatids. During the metaphase of this first division, each member of the pair of homologous chromosomes moves to each daughter cell, reducing the number of chromosomes to the dip¬ loid number; however, because each chromosome is composed of two chromatids, the DNA content of a diploid cell is retained. Incomplete cytokinesis causes the formation of a pair of joined secondary spermatocytes from each primary spermatocyte. During the second division, the 23 chromosomes, each com¬ prising a pair of chromatids, attach to the spindle, and the chro¬ matids separate. This yields a cell, a spermatid, containing the haploid DNA and chromosomal complement.

REPLICATION OF STEM CELLS SPERMIOGENESIS The replication of stem cells commences in fetal life with the migration of the primordial germ cells into the mesenchyme of the gonadal ridge. There is evidence that this migration is con¬ trolled by stem cell factor and its receptor c-kit.3 After a period of prenatal mitotic division, the gonocytes, which populate the seminiferous cords at birth, remain quiescent until immediately before puberty, when they divide by mitosis to form the spermatogonial population.4 The types of spermatogonia (Fig. 113-2) that can be characterized cytologically vary with each species. However, a feature common to all is the division of the stem cell population to provide two pools of cells, one that moves through the subsequent steps of spermatogenesis and the other

INTERPHASE

Cell division stops after the formation of the spermatids. How¬ ever, a dramatic metamorphosis, spermiogenesis, transforms a conventional cell into a highly specialized cell with the capabil¬ ity of flagellar-derived motility (Figs. 113-4 and 113-5). Little is known of the mechanisms by which these dramatic cytologic changes are controlled. The developmental phases are termed the Sax, Sb j, Sb2, Sc2, Sdx, and Sd2 stages according to Clermont, but many of the details can be determined only by electron microscopy.9-11 With the increasing use of spermatids extracted directly from the testis by biopsy, the specific features that char¬ acterize each of the above stages become crucial in identifying FIGURE 113-3. This diagram illustrates the process by which homologous chromosomes pair in the first meiotic division involving the primary sperma¬ tocytes. In tire leptotene phase, the chromosomes are represented by unpaired fine "threads." These pair during zygotene and thicken during pachytene, even¬ tually repelling each other during diplotene. The pairing process involves the formation of a tripar¬ tite structure, synaptinemal complex, which can be identified under the electron microscope. During the pairing, exchange of genetic material occurs between the maternal and paternal chromosomes in a process called crossing over. (From de Kretser DM, Kerr JB. The cytology of the testes. In: Knobil E, Neill JD, eds. The physiology of reproduction, 2nd ed. New York: Raven Press, 1994.)

1106

PART VIII: ENDOCRINOLOGY OF THE MALE

FIGURE 113-4. Initial cytologic changes during spermiogenesis are shown. See Figure 113-7 for explanation of Sa, Sty, and Sb2. (From de Kretser DM. The light and electron microscope anatomy of the normal human testis. In: Santen RJ, Swerdloff RS, eds. Male sexual dysfunction: diagnosis and management of hypogonadism, infertility and impo¬ tence. New York: Marcel Dekker Inc, 1986:3.)

the nature of the cell type injected into the cytoplasm of the oocyte to achieve fertilization. The changes can be subdivided into nuclear, acrosome formation, flagellar development, redistribu¬ tion of cytoplasm, and spermiation. NUCLEAR CHANGES During the Sa, and Sb, stages, the nucleus, which ultimately forms the head of the sperm, remains centrally placed, but it is subsequently displaced peripherally, coming into apposition with the cell membrane, separated only by the acrosomal cap. There is also a progressive decrease in nuclear volume associ¬ ated with chromatin condensation, causing the development of resistance by the DNA to degradation by the enzyme DNAase. ACROSOME FORMATION During the Sa, and Sb, stages, the Golgi complex of the sperma¬ tid produces several large vacuoles, which are applied to one pole of the nucleus. These vacuoles form a cap-like structure, the acrosome, which contains substances for penetration of the ovum during fertilization.113 Sperm that do not contain an acrosome cannot fertilize the ovum; because of the globular shape of the sperm head, the condition is known asglobozoospermia. The Golgi complex subsequently migrates to the abacrosomal pole of the spermatid and is lost in the residual cytoplasm. FLAGELLAR DEVELOPMENT The initial development of the tail occurs from the pair of centrioles located near the Golgi complex of the Sa, spermatid. The

Redundant nuclear envelope

FIGURE 113-5. Subsequent cytologic changes during spermiogenesis. (See also Fig. 113-7.) (From de Kretser DM. The light and electron micro¬ scope anatomy of the normal human testis. In: Santen RJ, Swerdloff RS, eds. Male sexual dysfunction: diagnosis and management of hypogo¬ nadism, infertility and impotence. New York: Marcel Dekker Lnc, 1986:3.)

microtubular structure, comprising nine peripheral doublets surrounding a pair of single microtubules, grows out from the distal centriole and forms the axoneme or core of the tail. The developing axoneme lodges in a facet at the abacrosomal pole of the nucleus by a complex articulation called the neck of the spermatid. Initially, the axoneme distal to the neck is sur¬ rounded by the cell membrane, but several specializations develop. Immediately adjacent to the outer nine doublets, a set of nine electron-dense fibers develop, which are connected cranially to the neck but distally taper to disappear eventually. A pair of these dense fibers, which lie diametrically opposite to each other, persist distally and become surrounded by a collec¬ tion of microtubules. These microtubules form the precursors for solid electron-dense fibers, or ribs, which run transversely around the axoneme, joining the pair of longitudinal, dense fibers. The region of the tail surrounded by these ribs is the principal piece. The region between the principal piece and the neck is the last segment to become organized. Some of the genes, which encode the proteins comprising the outer dense fibers and the fibrous sheath, have been identified; these will provide the basis for future studies on the formation and function of the components.12-15 Relatively late in spermiogenesis, between the Sd, and Sd2 stages, the mitochondria that lie in the peripheral regions of the cell coalesce to form several helical arrays around the axoneme, forming the midpiece of the sperm. The axoneme is identical to the core structure of cilia throughout the plant and animal kingdoms. The two centrally placed, single microtubules appear to be connected to the nine

Ch. 113: Morphology and Physiology of the Testis Outer fiber (doublet)

STAGE 1

STAGE 2

STAGE 3

STAGE 4

1107

* Sb,

ijP

\

FIGURE 113-6. The structure of a typical axoneme of a sperm tail.

I Sb2T*-»' *## * •

" m *r

«

/*

,»

'

%

m

peripheral doublets by a series of radial spokes (Fig. 113-6). One of each pair of doublets, termed subfiber A, is smaller and more electron dense; it sends a pair of hook-like extensions, or dynein arms, toward the adjacent doublet. They represent con¬ densations of the protein, dynein, which has adenosine triphos¬ phatase activity and is vital to the generation of flagellar motility; the absence of dynein arms is associated with immo¬ bility of sperm and cilia.16'17 REDISTRIBUTION OF CYTOPLASM Associated with the eccentric placement of the nucleus is a sig¬ nificant repositioning of the cytoplasm and organelles. This probably is caused by a palisade of microtubules, the manchette, which extends as a cylindrical collection from the head, in the region where the acrosome ends, to the caudal pole of the sper¬ matid. Ultimately, most of this cytoplasm is shed by the sper¬ matid and effectively appears to be pulled off by processes of Sertoli cell cytoplasm, which invaginate into pouches develop¬ ing in the spermatid cytoplasm.10 This cytoplasmic remnant, the residual body, is phagocytosed and degraded by the adjacent Sertoli cells. SPERMIATION The release of spermatids at the Sd2 stage occurs in association with the loss of the residual cytoplasm.

FIGURE 113-7. The light microscopic features of the stages of the human seminiferous cycle are illustrated. (L, leptotene primary sperma¬ tocyte; M, spermatocytes in meiosis; P, pachytene primary spermato¬ cyte; Z, zygotene primary spermatocyte; Sav Sbv Sb2, Sc, Sdv spermatids at different steps of spermiogenesis; Ser, Sertoli cell nucleus.) (From de Kretser DM, Temple-Smith PD, Kerr JB, et al. Ana¬ tomical and functional aspects of the male reproductive organs. In: Bandhauer K, Frick J, eds. Handbuch der Urologie, vol XVI. Berlin: Springer-Verlag, 1982:1.)

SPERMATOGENIC CYCLE Careful and extensive cytologic studies have demonstrated a char¬ acteristic sequence of cell associations through which the seminif¬ erous epithelium passes and that constitute the cycle of the seminiferous epithelium.6 In the rat, this consists of 14 cell associa¬ tions, each of which may extend over several millimeters of tubule. Flowever, in the human, six cell associations or stages have been identified, and each may occupy only 25% to 33% of a cross section of a seminiferous tubule9 (Fig. 113-7). Studies in which dividing cells are labeled by tritiated thymidine have shown that the time taken for the daughter cells of spermatogonial divisions to mature to Sd2 spermatids released from the seminiferous tubule is 64 ± 6 days or 4.5 cycles of the human seminiferous cycle.18 The time for spermatogenesis is unique for each species and repre¬ sents a biologic constant. The germ cells either pass through these stages at a specified speed or degenerate. It is uncertain whether these time constraints are an innate function of each cell or whether they are imposed by other con¬

trolling factors. Coordination of the spermatogenic process may be achieved by the unusual organization of the seminiferous epithelium, wherein the germ cells remain joined by intercellu¬ lar bridges.7 Additionally, coordination may be achieved by the Sertoli cells, whose arborizing branches maintain contact with many germ cell stages around the radial axis of the seminifer¬ ous tubules. SERTOLI CELLS Sertoli Cell Cytology. The general features of the Sertoli cell are similar in most mammalian species.19-20 The cells extend from the basement membrane of the tubule to the luminal sur¬ face of the epithelium, and, although the base of the cell is clearly identifiable, the central portions cannot be distinguished by light microscopy. This results from the formation of many thin cytoplasmic prolongations that extend in an arborizing network around the germ cells undergoing spermatogenesis.

1108

PART VIII: ENDOCRINOLOGY OF THE MALE

Electron microscopy has demonstrated that these processes are always surrounded by the cell membrane of the Sertoli cell. In the Sertoli cell, the cytoplasmic organelles exhibit some degree of polarity. The basal aspects, which abut the basement membrane of the tubule, interspersed between spermatogonia, often show collections of mitochondria. The nucleus is pleo¬ morphic, is aligned perpendicular to the basal lamina, and con¬ tains a prominent nucleolus. The nucleolus is a feature of the postpubertal Sertoli cell, probably related to the follicle-stimu¬ lating hormone (FSH)-induced maturation of the protein syn¬ thetic capacity of the mature Sertoli cell.21-22 In the perinuclear region, small collections of rough endo¬ plasmic cisternae and Golgi membranes can be seen. Significant numbers of lipid inclusions, often surrounded by smooth endo¬ plasmic reticulum, are found adjacent to the nucleus; in some species, these inclusions exhibit significant changes in number with the stages of the seminiferous cycle. Lysosomes, lipofuscin pigment, and residual bodies, which are the phagocytosed cytoplasmic remnants of the spermatids, may be seen deep within the Sertoli cell cytoplasm. The shape of the Sertoli cell is probably maintained by the cytoskeleton, consisting of a peri¬ nuclear array of fine filaments and microtubules that often extend into the smaller processes of cytoplasm surroimding the germ cells. Inter-Sertoli Cell Junctions. One of the characteristic features of the Sertoli cell is the specialized inter-Sertoli cell junction, which occurs where adjacent Sertoli cells abut. These usually commence at a level in the epithelium just luminal to the basal row of the spermatogonia and extend centrally. This unique cell junction consists of small occluding junctions, rep¬ resenting points of fusion of the cell membranes that obliterate the intercellular space. Adjacent to these points of occlusion, smooth-membraned cisternae run parallel to the cell membrane and demarcate a narrow band of cytoplasm containing bundles of fine filaments.23 The effect of these complexes is to prevent transport by way of the intercellular space to the centrally placed germ cells.23 The inter-Sertoli cell junctions divide the seminiferous epithelium into two compartments: a basal one containing the spermatogonia and preleptotene primary sper¬ matocytes and an adluminal one containing the subsequent stages of germ cell maturation. The cell junctions represent the site of the blood-testis barrier; by preventing intercellular transport, they create a highly selective permeability barrier based on transport systems within the Sertoli cell.24 These cell junctions and the blood-testis barrier are absent in the imma¬ ture testis but develop during pubertal maturation.21'25 The resultant development of the cell junctions and barrier coincides with the onset of meiosis and seminiferous fluid secretion.22'26 These cell junctions are not permanent structures since they must disassemble and re-form basally as preleptotene sperma¬ tocytes lose their attachment to the basement membrane and move centrally. Sertoli Cell Function. There has been a great expansion in the knowledge of Sertoli cell function 27 Because of the intimate relationship of the Sertoli cells to germ cells, the function of these cells is probably crucial for the successful completion of spermatogenesis. The concept that the number of Sertoli cells determines the spermatogenic output of the testis has gained considerable support, and clearly, the Sertoli cell is critically important for the transport of substances into the seminiferous tubule. Numerous examples indicate that the Sertoli cells are essential for the metabolic activities of the germ cells: For exam¬ ple, the germ cells, which are unable to metabolize glucose, are provided with lactate by the Sertoli cells 28 Key roles of the Ser¬ toli cells are discussed later. Sertoli Cell Replication. Studies in the rat demonstrate that the Sertoli cell population divides by mitosis in fetal and postnatal life until day 15 but remains stable thereafter.29 Neo¬ natal hypothyroidism in the rat can extend the period of Sertoli cell replication, and in adult life the increased numbers of Ser¬

toli cells lead to a marked increase in sperm output.30 These data strongly suggest that the number of Sertoli cells in the tes¬ tis is a major factor in controlling the spermatogenic potential of the testis. Use of this model has also shown that the func¬ tional maturation of the Sertoli cells is delayed during hypothyroidism31 and that there is a marked delay in the pro¬ cess of spermatogenesis.32 The control of Sertoli cell prolifera¬ tion involves FSH, thyroid hormones, and growth factors such as activin A.33 Seminiferous Tubule Fluid Production. The Sertoli cells secrete a fluid, with characteristics distinct from plasma, into the lumen of the seminiferous tubule.34 This secretion com¬ mences during sexual maturation, after the formation of the blood-testis barrier; it is dependent on FSH for its produc¬ tion.35'36 In animals, the production of seminiferous tubule fluid can be measured by unilateral efferent duct ligation; the increasing difference in weight, with time, between the ligated and unligated sides provides this index.35 Under such condi¬ tions, fluid production is maintained for 24 hours, after which a pressure atrophy of the seminiferous epithelium occurs and fluid production decreases, eventually ceasing altogether.35-36 Androgen-Binding Protein. The discovery that the Ser¬ toli cell produces an androgen-binding protein (ABP) that is capable of binding dihydro testosterone and testosterone with high affinity has provided a biochemical marker of Sertoli cell function.27-37 Purification and characterization have revealed that, in the rat, ABP is a protein with a molecular mass of 85,000 daltons, for which a radioimmunoassay has been developed.38 The amount of ABP secreted by the Sertoli cells varies among species; it is absent in some, and in others, such as humans, it is uncertain whether it is produced in the testis, because there is contamination of testis tissue with blood, which contains an ABP formed in the liver. ABP and the sex steroid-binding glob¬ ulin (SSBG) found in some species are very similar, and repre¬ sent the same protein produced in the liver and testis under different regulatory mechanisms.39-40 ABP/SSBG acts as a hor¬ mone or growth factor, as evidenced by several studies that have identified high-affinity binding sites.41-42 Furthermore, there are multiple alternate RNA transcripts of ABP in different tissues, and one of these encodes a nuclear targeting signal.43-44 The secretion of ABP occurs principally into the lumen of the seminiferous tubules (Fig. 113-8), but it also passes across the basal aspect of the Sertoli cells into interstitial fluid and blood.38 ABP may provide a mechanism for ensuring a large store of tes¬ tosterone within the tubule, stabilizing fluctuations in testoster¬ one secretion by the Leydig cells. It may also provide a mechanism for ensuring a high concentration of testosterone for the caput epididymis, because ABP and its bound testoster¬ one are transported into the epididymis, where they are absorbed by the principal cells. Secretion of Other Plasma Proteins. The Sertoli cell may produce a number of other proteins found in plasma, perhaps circumventing the presence of the blood-testis barrier and ensuring adequate local concentrations. Albumin, transferrin, and plasminogen activator are such proteins secreted by the Sertoli cell.45-17 Their specific functions are unclear. Plasmino¬ gen activator has been proposed as a mechanism for enabling the discrete disruption of the inter-Sertoli cell junctions to allow germ cells to pass through from the basal to the adlumi¬ nal compartments. Transferrin production by the Sertoli cells facilitates the transport of iron across the blood-testis barrier and enables the provision of adequate amounts of iron for the metabolism of germ cells.47 Inhibins. The Sertoli cell secretes inhibin (see Fig. 113-8), which increases in the testis after efferent duct ligation.48"50 Inhibin, originally isolated from follicular fluid, is a disulfidelinked dimer of two dissimilar subunits termed a and (IT1 Two forms exist that share a common a subunit but distinct P sub¬ units: These forms are inhibin A (aPA) and inhibin B (aPB).51 Both forms suppress FSH, whereas dimers of the P subunits—

Ch. 113: Morphology and Physiology of the Testis

FIGURE 113-8. A diagram of the testis shows the relationship of Sertoli cells to germ cells. Factors controlling the testicular compartments are outlined. Evidence for the secretion of estradiol (E2) and gonadotropin¬ releasing hormone (GnRH) by the adult Sertoli cell is tentative, but the hormones have been suggested as local modulators of Leydig cell func¬ tion. (T, testosterone; ABP, androgen binding protein; FSH, follicle-stim¬ ulating hormone; LH, luteinizing hormone.)

termed activins (activin A [PAPA], activin AB [PAPB], activin B [PbPbD—stimulate FSH. The genes coding the subunits pro¬ duce larger precursor molecules that dimerize and are proteolytically cleaved to form the mature substances.52'53 The subunits of inhibin show significant homology to transforming growth factor-P, antimullerian hormone (AMH), and numerous other proteins, such as the protein coded for by the decapentaplegic gene complex of Drosophila.52*54 The Sertoli cells secrete inhibin B as the principal protein feedback on FSH secretion, and there is a good correlation between sperm output and inhibin B levels in the circula¬ tion.55-56 Inhibin B levels are inversely correlated to FSH concen¬ trations; therefore, there have been suggestions that inhibin B is a useful marker for Sertoli cell function.57 Experiments using recombinant inhibin A indicate that, although both testosterone and inhibin modulate FSH, inhibin can normalize FSH levels in castrated rams in the absence of testosterone.58-59 Antimullerian Hormone. Antimullerian hormone is pro¬ duced by the Sertoli cell during fetal and early postnatal life.60-603 It has been purified and shown to be a glycoprotein. In fetal life it directs the regression of the mullerian ducts during male sexual differentiation (see Chap. 90). Steroidogenic Function. The presence of smooth endo¬ plasmic reticulum, lipid droplets, and mitochondria with tubu¬ lar cristae in the cytoplasm of the Sertoli cells led to the proposal that the Sertoli cells could be the site of steroid biosynthesis.61 By separating seminiferous tubules from interstitial tissue, it was demonstrated that the tubules do not have the capacity to metabolize cholesterol to products more distant in the ste¬ roidogenic pathway. However, if provided with substrates such as progesterone, the seminiferous tubules have the capacity to metabolize these materials to androgens.62 Unique metabolites of C-19 steroids have been identified in Sertoli cells, and the pat¬ tern of metabolism can be altered by FSH treatment. Despite the uncertainty about the steroidogenic capacity of the seminiferous tubules and Sertoli cells, cultures of immature rat Sertoli cells have the ability to metabolize androgens, with the enzyme aro-

1109

matase, to estradiol.63 However, in the rat, this activity decreases rapidly with age and is not detectable 20 days after birth. Evi¬ dence suggests that estradiol production by the testis after this time occurs within the Leydig cells.64 In the immature rat, the activity of aromatase is FSH inducible, and this reaction has been used as a basis for an in vitro bioassay for FSH.65 Age-Dependent Changes in Sertoli Cell Function. Before 20 days, the Sertoli cells can respond dramatically to FSH by an increase in cyclic adenosine monophosphate (AMP) and in pro¬ tein synthesis, together with the stimulation of the enzyme aro¬ matase. Additionally, the immature Sertoli cells produce AMH in fetal life and for a short period after birth. These functions disappear ~20 days after birth in the rat and are replaced by increases in ABP secretion, the onset of fluid production, and an increase in inhibin secretion.66 The mechanisms causing these changes in Sertoli cell function are unknown, but some may be related to the onset of pubertal secretion of FSH by the pituitary. Stage-Dependent Changes in Sertoli Cell Function. The seminiferous tubule undergoes a sequence of cytologic changes, causing the formation of specific cell associations, which have been identified as the seminiferous cycle. In association with these changes in the germ cell complement of the seminiferous tubule, there are distinct cytologic changes that have been identi¬ fied within the Sertoli cell, particularly in the rat, in which there is a very well-defined seminiferous cycle. In this species, a num¬ ber of biochemical parameters vary according to the stage of the seminiferous cycle.67 Thus, FSH-receptor levels, ABP production, and cyclic AMP production change according to the stage of the seminiferous cycle in response to FSH stimulation.9 The nature of the products from germ cells that modulate Sertoli cell function is still unknown. The concept that these may arise from late sper¬ matids, possibly through the phagocytosis of residual bodies, has been reviewed.68 CONTROL OF THE SEMINIFEROUS TUBULE Hormonal Control. The function of the testis depends on the secretion of the gonadotropic hormones, FSH and luteiniz¬ ing hormone (LH), by a functional hypothalamic-pituitary unit (see Chap. 16). Moreover, the action of LH on spermatogenesis is mediated through the secretion of testosterone by the Leydig cells. However, there is considerable controversy concerning the relative roles of FSH and testosterone in the control of semi¬ niferous tubule function. Initiation of Spermatogenesis. The role of testosterone in this process is not questioned, and current data indicate that constitutive-activating mutations in the LH receptor cause pre¬ cocious puberty, while inactivating mutations result in familial testosterone resistance and male pseudohermaphroditism.69-70 The earlier view that, in humans and other mammalian species, both FSH and LH are required for the initiation of spermatoge¬ nesis during pubertal maturation71-73 has been challenged by three studies in mice. First, testosterone alone has been shown to induce spermatogenesis in the hpg mouse, which lacks the capacity to secrete gonadotropin-releasing hormone and, thus, cannot produce both FSH and LH.74 Secondly, inactivation of the gene encoding the (3 subunit of FSH did not prevent the onset of full spermatogenesis during pubertal maturation in these mice.75 The third study of several males with inactivating mutations of the FSH receptor found that the males were able to complete spermatogenesis, but in most, the testicular vol¬ umes and sperm counts were very significantly impaired.76 In all these studies it was noted that the testes were smaller and that the sperm output was lower than normal. The possi¬ bility that this results from an impairment of Sertoli cell multi¬ plication (normally stimulated by FSH) is being explored. The view that both FSH and LH were required originated from studies of patients with hypogonadotropic hypogonadism during the induction of spermatogenic activity using FSH and LH. Some patients respond to LH or human chorionic gonado-

1110

PART VIII: ENDOCRINOLOGY OF THE MALE

tropin (hCG) alone, but most require the action of both gonado¬ tropic hormones.77 There are conflicting data for the rat, suggesting that LH, through the action of testosterone, may play a more dominant role during pubertal maturation; how¬ ever, the rat is a very poor model for studies of sexual matura¬ tion because it does not have a prepubertal period. The spermatogenic process in the rat occupies 48 to 50 days, and mature spermatozoa can be seen in the rat 45 to 50 days after birth. Consequently, changes closely related to the time of birth in the rat may be involved in the initiation of spermatogenesis. Maintenance of Spermatogenesis. Given the results of the studies in the hpg mouse and those in genetically modified mice in which the |3 subunit of FSH is not produced, the con¬ troversy regarding the role of FSH in the maintenance of spermatogenesis after its establishment at puberty is still unre¬ solved. In the rat, the observations that testosterone alone, given immediately after hypophysectomy, could maintain sper¬ matogenesis without FSH have received some support from experiments in humans using an alternative design. In these studies, the suppression of FSH and LH secretion by the administration of contraceptive doses of testosterone caused azoospermia or severe oligospermia. These changes could be reversed by the administration of hCG or LH, which presum¬ ably act by stimulating Leydig cells to increase the intratesticular levels of testosterone.77-78 However, when researchers used the same design to suppress spermatogenesis, highly purified FSH was also able to restore the sperm count, presumably with¬ out altering intratesticular levels of testosterone.79 Several stud¬ ies have shown that the levels of testosterone normally found within the testis are not required to maintain spermatogenesis, which can proceed successfully at concentrations -10% of nor¬ mal.80-81 Nonetheless, these levels still represent twice the nor¬ mal circulating concentrations, in turn raising questions as to why the testicular androgen receptor requires significantly greater stimulation than other androgen-dependent tissues, for example, prostate. Both in rats and in humans, if regression of the spermatoge¬ nic epithelium has occurred after hypophysectomy, testoster¬ one alone is insufficient to restore spermatogenesis.80-82 However, this view has been challenged by studies in stalksectioned monkeys in whom testosterone treatment alone reversed the testicular regression that had occurred, although testicular volumes returned to only 60% of presurgical levels.83 The claim that no biochemical action of FSH was found in the adult rat testis84 has now been shown to be erroneous since the sensitivity to FSH stimulation varies with the stage of the seminiferous cycle within the seminiferous tubule.67 These studies demonstrate that an FSH-induced effect can be obtained in the adult testis provided an FSH-sensitive phase of the seminiferous cycle is selected. Positive evidence for the role of FSH in the spermatogenic process includes the fact that receptors for FSH have been identified on the Sertoli cell and on spermatogonia.85 The action of FSH is mediated by the cyclic AMP-protein kinase system and stimulates protein synthesis by the Sertoli cell. A number of proteins, such as ABP, aromatase, plasminogen activator, RNA polymerase, inhibin, and proteoglycan, are responsive to FSH or cyclic AMP.49-66-84 Stud¬ ies in primates and in normal men have shown that FSH is important in maintaining the transition of type A to type B spermatogonia.86-87 Testosterone is clearly important in maintaining the seminif¬ erous epithelium. This action of testosterone is mediated through androgen receptors found within the Sertoli cell and on peritubular and Leydig cells.88-90 Further evidence for the role of androgens in the stimulation of Sertoli cell function was obtained from Sertoli cells in culture, in which RNA poly¬ merase and ABP production could be stimulated indepen¬ dently of any action of FSH.91-92 After hypophysectomy, testosterone alone can maintain fluid production and the secre¬ tion of inhibin by the rat testis.36-93 Unfortunately, the rat proves

FIGURE 113-9. The relationship between germ cells and Sertoli cells in the rat is shown. In the presence of low intratesticular testosterone levels, spermatids at stages VII to VIII of the cycle are shed from the epithelium. (From McLachlan RI, Wreford NG, Robertson DM, et al. Hormonal con¬ trol of spermatogenesis. Trends Endocrinol Metab 1995; 6:95.)

to be a difficult model in which to explore the effect of high doses of testosterone, since these stimulate FSH secretion.94 Morphometric studies have shown that spermiogenesis is exquisitely sensitive to testosterone,81-94 and further data sug¬ gest that withdrawal of testosterone disrupts the conversion of step 7 to 8 because of premature sloughing of round spermatids into the epididymis.95-96 It is likely that this cell loss is due to disruption of the ectoplasmic specializations between sperma¬ tids and Sertoli cells (Fig. 113-9). Further, the studies in the hpg model indicate that testosterone is important in facilitating the survival of primary spermatocytes.74 The relative role of testosterone and dihydrotestosterone in spermatogenesis has been perplexing, given that the concentra¬ tions of intratesticular testosterone are significantly greater. However, studies with 5a-reductase inhibitors have shown that dihydrotestosterone is a significant stimulator of spermatogen¬ esis when intratesticular testosterone concentrations decline.96 It is recognized that the testis secretes estradiol, arising from the conversion of androgens by the enzyme aromatase. Evi¬ dence for an effect of estradiol on spermatogenesis has emerged from gene-targeted disruption of the P450 aromatase gene, which demonstrated that the initially fertile mice progressively became infertile as a result of decreases in spermatid numbers, increased apoptosis, and abnormal acrosome development."7-99 More direct evidence of the actions of estradiol was obtained from gene knock-out of the estradiol a receptor: These mice were infertile due to the actions of estradiol on fluid reabsorp¬ tion in the epithelium of the efferent ductules.100 The resultant back pressure resulted in loss of germ cells from the seminifer¬ ous epithelium. However, estradiol can still act on the seminif¬ erous epithelium in these mice, since a functional estradiol (i receptor remains in the testis.101 Initial reports of the knock-out of this gene indicate that the mice are fertile at 6 weeks of age.102 In summary, while recent studies shed some doubt as to the critical importance of FSH in enabling spermatogenesis to pro¬ ceed to completion, others have defined specific points at which FSH appears to be very important in maintaining a nor¬ mal throughput of germ cells. These points appear to be in its action on Sertoli cell mitosis and in facilitating the formation and survival of type B spermatogonia. Nonhormonal Control. There are numerous steps that must be successfully completed before the testis can success¬ fully produce a normal sperm output. These involve molecular mechanisms that require key regulators that are not hormones. As these molecular controllers are identified, usually through experiments that involve the exploration of the function of a protein through gene-targeted disruption, additional regulators of spermatogenesis emerge. It is not possible to consider these

Ch. 113: Morphology and Physiology of the Testis proteins exhaustively in this chapter, but a few examples are given that illustrate these developments. For successful sperm production, the development and dif¬ ferentiation of the testis must proceed normally. Any mutation or rearrangement in genes, which is crucial for normal testis development, will impair sperm output or testicular develop¬ ment. Mutations in key domains of the androgen receptor can disrupt hormone binding and result in testicular feminization, but data indicate that there are mutations that occur in other regions of the gene encoding the receptor that do not interfere with sexual differentiation but can impair spermatogenesis. These include expansions of the CAG repeat sequence in the amino-terminal region of the protein.103 The deletion of genes on the long arm of the Y chromosome has demonstrated the presence of testis-specific genes that are essential to enable normal sperm production. One of these, DAZ (deleted in azoospermia), encodes an apparent RNAbinding protein and exhibits homology to the boule gene in Drosophila, mutations that cause sterility in flies. Mutations or deletions of the DAZ genes, for which there are multiple copies in humans, result in severe disruption of spermatogenesis, without any effect on sexual differentiation.104-105 Numerous studies in mice have shown that mutations in the gene encoding stem cell factor or its receptor c-kit result in testes devoid of germ cells because of disruption of the migra¬ tion of the primordial germ cells and their transformation into spermatogonia.3-106 Later stages of spermatogenesis can show disruption by interference in molecular mechanisms. For instance, targeted disruption of the gene encoding heat shock protein 70-2, a molecular chaperone, results in the arrest of spermatogenesis at the primary spermatocyte stage since this protein appears to be crucial to enabling the completion of meiosis.107 There are numerous studies to indicate that apoptotic mech¬ anisms are important regulatory pathways in the testis, espe¬ cially following hormonal modulation.108 Evidence supporting this view has emerged from studies of targeted disruption of the gene encoding bcl-w, a cell survival molecule; the first wave of spermatogenesis in mice almost progressed to completion but collapsed, resulting in infertility and ultimately in a Sertoli cell-only phenotype.109 Whether there are hormonal mecha¬ nisms that regulate this and other proteins, which control cell survival, remains to be established. INTERTUBULAR TISSUE The seminiferous tubules are supported by a loose connective tissue, which is supplied by a rich vascular network. It is bounded by the basement membrane of the seminiferous tubule, which is surrounded by a varying number of layers of contractile myoid cells interspersed with collagen fibers and a basement membrane-type material that is applied to some of the layers of the myoid cells. The myoid cells are modified smooth muscle cells that cause the contraction of the seminifer¬ ous tubules. The general organization of the intertubular tissue varies among species, based on the number of Leydig cells, which are responsible for androgen secretion, the arrangement of the lymphatics, and the extent of the connective tissue.110 The intertubular tissue contains varying numbers of Leydig cells, fibroblasts, macrophages, mast cells, and small unmyelinated nerve fibers. LEYDIG CELLS The Leydig cells are derived from the mesenchyme of the gonadal ridge; two generations of Leydig cells are developed. In fetal life, the differentiation of mesenchyme into Leydig cells induces the secretion of androgens that generate the sexual dif¬ ferentiation of the external genitalia. These fetal Leydig cells degenerate shortly after birth, and the prepubertal period is characterized by the absence of Leydig cells from the intertubu¬

1111

lar tissue. Associated with the pubertal secretion of gonadotro¬ pins, adult Leydig cells redifferentiate from connective tissue precursors111 within the intertubular tissue. In other species, such as the rat, in which the interval from birth to sexual matu¬ ration is short, some overlap occurs between the fetal and adult generations. The testosterone is secreted into the intertubular tissue, where it is absorbed by blood vessels, lymphatics, and the seminiferous tubules. Leydig Cell Cytology. Leydig cells form small collections around blood vessels and have a variable appearance by light microscopy, usually attributed to the lipid inclusions, which cause a variable vacuolation (see Fig. 113-2). The Leydig cells are characterized by an ovoid nucleus exhibiting a conspicuous nucleolus. The cytoplasm contains a large amount of smooth endoplasmic reticulum in the form of interconnected tubules. Mitochondria contain both lamellar and tubular cristae, the lat¬ ter being typical of steroid-secreting cells. The content of lipid, lysosomes, and lipofuscin pigment is variable. The human Ley¬ dig cells are characterized by the presence of crystalloid inclu¬ sions, the crystals of Reinke, the functional significance of which is unknown. The amount of smooth endoplasmic reticulum and mitochondria declines after hypophysectomy and increases after LH or hCG stimulation.112 Leydig Cell Function. The Leydig cells secrete testoster¬ one and are able to synthesize cholesterol from acetate, with the cholesterol acting as a substrate for steroidogenesis. The amount of cholesterol obtained by lipoprotein uptake relative to synthesis from acetate varies among species. The enzymatic steps involved in the synthesis of testosterone from cholesterol are outlined in Figure 113-10. Besides the secretion of testoster¬ one, the Leydig cells secrete estradiol, contributing 20% to 30% of the total circulating estradiol; the remainder is derived from the peripheral aromatization of androgenic substrates. There is a subcellular localization of the enzymes involved in steroido¬ genesis, with the conversion of cholesterol to pregnenolone localized in mitochondria; the remaining steps of steroid bio¬ synthesis in the testis depend on enzymes located within the smooth endoplasmic reticulum of the Leydig cell. Several studies have expanded the understanding of the mechanisms underlying cholesterol transport into the mito¬ chondria, a step that is critical in enabling cleavage of the side chain of pregnenolone. The isolation and characterization of steroidogenic acute regulatory protein (StAR)m and the cloning of the gene encoding this protein demonstrated that it had a cru¬ cial role in the transport of cholesterol into mitochondria. Muta¬ tions in this gene or its targeted disruption profoundly interfered with steroid hormone biosynthesis in all steroidsecreting endocrine tissues; in humans, mutations were shown to be responsible for the condition of lipoid congenital adrenal hyperplasia.114-115 Influence of Luteinizing Hormone. The Leydig cell con¬ tains receptors for LH on its cell membrane; this hormone con¬ trols testosterone secretion by means of cyclic AMP.116 The principal enzyme controlled by LH is the side-chain cleavage enzyme involved in the conversion of cholesterol to preg¬ nenolone. Besides the immediate events initiated through the phosphorylation of proteins that induce testosterone secretion, LH is trophic to the Leydig cell and stimulates the incorpora¬ tion of labeled amino acids into specific proteins. This trophic activity causes a hypertrophy of the Leydig cells and probably increases the number of Leydig cells. The stimulation of Leydig cells with large doses of LH or hCG rapidly reduces the number of their receptors, a phenome¬ non termed down-regulation.m'n7 Although these changes decrease testosterone secretion 24 to 48 hours after an injection, repeated stimulation does not yield the same results. Addition¬ ally, a single injection of hCG is followed by a prolonged steroidogenic response characterized by two phases of tes¬ tosterone secretion, one initially occurring over the first 6 to 18 hours and the second occurring 48 to 72 hours later.117 The

1112

PART VIII: ENDOCRINOLOGY OF THE MALE Acetate

17 cx - hydroxypregnenolone

17cx-hydroxyprogesterone

I7cx-hydroxy. 20cx dihydro¬ progesterone

O

FIGURE 113-10. These are the biochemical path¬ ways in the synthesis of testosterone.

results indicate that hCG can be administered at 6- to 7-day intervals due to the prolonged steroidogenic response. The nadir between the two phases of testosterone secretion is due to the block in testosterone production induced by the large injec¬ tion of hCG, and recovery from the inhibition allows restimula¬ tion of testosterone secretion by the existing plasma hCG, because of the long half-life of hCG. INFLUENCE OF OTHER FACTORS. There is increasing evi¬ dence, principally from rat studies, that other factors may be involved in the stimulation of testosterone secretion by Leydig cells. This concept originated from observations that damage to the seminiferous tubules by a number of experimental agents caused changes within the Leydig cells,118 which included Ley¬ dig cell hypertrophy, partial loss of LH receptors, and a hyper¬ responsiveness of the Leydig cells to hCG stimulation in vitro. If unilateral damage to the testis was induced, such as after uni¬ lateral cryptorchidism, the Leydig cell changes were present exclusively in the damaged testis, thereby ruling out circulating humoral factors, such as LH, participating in these changes. The resultant hypothesis suggested that the seminiferous tubules somehow modulate the Leydig cells (see Fig. 113-8). Reasoning that any factor passing from the seminiferous tubules to the Leydig cells must pass across the lymphatic sinusoidal system in the rat, investigators have demonstrated a proteinaceous factor that was not LH and that stimulated ster¬ oidogenesis.119 This substance stimulates testosterone more than the maximum levels generated by LH or hCG. Also, a number of studies have suggested that the macrophages present in both the testis and ovary may secrete substances capable of stimulating steroidogenesis, thereby increasing the potential for local control of Leydig cells.118 This view has been substantiated by the

impaired development and function of Leydig cells in mice wherein the gene encoding CSF-1 has been disrupted.120 The rela¬ tive importance of these local factors and of LH in the physiology of testosterone secretion requires clarification. The possibility that these changes may be explained by modulation of StAR or the peripheral benzodiazepine receptor121 requires further work.

INTERCOMPARTMENTAL MODULATION IN THE TESTIS Throughout this chapter, the seminiferous tubules and intertu¬ bular tissue have been considered as independent entities. However, there is increasing evidence that this approach is unwarranted. It is well documented that testosterone is required for the process of spermatogenesis, indicating that the Leydig cells at a local level are able to influence the seminifer¬ ous tubules.122 Additionally, the seminiferous tubules are involved in the modulation of Leydig cell function. There is also evidence that the Leydig cells exhibit changes in size according to the stage of the seminiferous cycle in the tubules immediately adjacent to them.123 Furthermore, the Sertoli cells show marked changes in function in association with spermatogenic damage and the stage of the seminiferous cycle.124 All of the agents used to induce experimental damage to sper¬ matogenesis decrease the parameters of Sertoli cell function, such as seminiferous tubule fluid production, ABP production, and inhibin production. These changes occur despite a rela¬ tively well-maintained morphology of the Sertoli cell; they indi¬ cate the importance of obtaining sensitive biochemical indices of Sertoli cell function.

Ch. 113: Morphology and Physiology of the Testis The testis should be considered as a functional unit and not as individual compartments with few functional interrelation¬ ships. Consideration of these factors may soon provide expla¬ nations about why spermatogenesis in certain infertile men does not proceed to completion despite a well-maintained stem cell population within the seminiferous epithelium.

IMMUNOLOGIC CONTROL IN THE TESTIS There is increasing interest in the concept that the testis is an immunologically privileged site; this is based on observations that grafts in the testis survive for prolonged periods.125'126 The maintenance of this environment may be crucial in preventing the formation of autoantibodies to sperm components. Atten¬ tion has been drawn to the large population of macrophages in the intertubular tissue of the testis that have an impaired capac¬ ity to respond to an inflammatory stimulus by the secretion of proinflammatory cytokines. The reason for this observation is still unclear but may involve the production of unidentified substances by the Leydig and Sertoli cells. These cells have the capacity to produce cytokines such as interleukin (IL)-l and IL6 in response to inflammatory stimuli.127

REFERENCES 1. Wartenberg H. Differentiation and development of the testes. In: Burger HG, de Kretser DM, eds. The testis. New York: Raven Press, 1981:39. 2. de Kretser DM, Temple-Smith PD, Kerr JB. Anatomical and functional aspects of the male reproductive organs. In: Bandhauer K, Frick J, eds. Handbuch der Urologie, vol XVI. Berlin: Springer-Verlag, 1982:1. 3. Marziali G, Lazzaro D, Sorrentino V. Binding of germ cells to mutant SId Sertoli cells is defective and is rescued by expression of the transmembrane form of the c-kit ligand. Dev Biol 1993; 157:182. 4. Muller J, Skakkebaek NE. Quantification of germ cells and seminiferous tubules by stereological examination of testicles from 50 boys who suffered from sudden death. Int J Androl 1983; 6:143. 5. Huckins C. The spermatogonial stem cell population in adult rats: I. Their morphology, proliferation and maturation. Anat Rec 1971; 169:533. 6. Clermont Y. Kinetics of spermatogenesis in mammals: seminiferous epithe¬ lium cycle and spermatogonial renewal. Physiol Rev 1972; 52:198. 7. Dym M, Fawcett DW. Further observations on the numbers of spermatogo¬ nia, spermatocytes and spermatids connected by bridges in the mamma¬ lian testis. Biol Reprod 1971; 4:195. 8. Brinster RL, Zimmerman JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci U S A1994; 11298. 9. Clermont Y. The cycle of the seminiferous epithelium in man. Am J Anat 1963; 112:35. 10. de Kretser DM. Ultrastructural features of human spermiogenesis. Z Zellforsch 1969; 98:477. 11. Holstein AF, Roosen-Runge EC. Atlas of human spermatogenesis. Berlin: Grosse Verlag, 1981. 11a. Abou-Haila A, Tulsiani DR. Mammalian sperm acrosome: formation, con¬ tents, and function. Arch Biochem Biophys 2000; 379:173. 12 Fulcher KD, Mori C, Welch JE, et al. Characterization of FSC-1 complemen¬ tary deoxyribonucleic acid for a mouse sperm fibrous sheath component. Biol Reprod 1995; 52: 41. 13. Schalles U, Shao X, van der Hoorn FA, Oko R. Developmental expression of the 84 kDa ODF sperm protein: localization to both the cortex and medulla of outer dense fibers and to the connecting piece. Develop Biol 1998; 199:250. 14. Burfeind P, Hoyer-Fender S. Sequence and developmental expression of a mRNA encoding a putative protein of rat sperm outer dense fibers. Develop Biol 1991; 148:195. 15. O'Bryan MK, Loveland KL, Herzfield D, et al. Identification of a rat testis specific gene encoding a potential rat outer dense fibre protein. Mol Reprod Develop 1998; 50:313. 16. Gibbons IRL. Mechanisms of flagellar motility. In: Afzelius BA, ed. The func¬ tional anatomy of the spermatozoon. Oxford: Pergamon Press, 1975:127. 17. Afzelius BA, Eliasson R, Johnsen O, Lindholmer C. Lack of dynein arms in immotile human spermatozoa. J Cell Biol 1975; 66:225. 18. Heller CG, Clermont Y. Kinetics of the germinal epithelium in man. Recent Prog Horm Res 1964; 20:545. 19. Fawcett DW. Ultrastructure and function of the Sertoli cell. In: Hamilton DW, Greep RO, eds. Handbook of physiology, section 7, vol 5. Baltimore: Williams & Wilkins, 1975:21. 20. de Kretser DM, Kerr JB. The cytology of the testis, hr: Knobil E, Neill JD, eds. Physiology of reproduction. New York: Raven Press, 1994:1177. 21. de Kretser DM, Burger HG. Ultrastructural studies of the human Sertoli cell in normal men and males with hypogonadotropic hypogonadism

1113

before and after gonadotropic treatment. In: Saxena BB, Beling CG, Gandy HM, eds. Gonadotropins. New York: Wiley Interscience, 1972:640. 22. Means AR, Fakunding JL, Huckins C, et al. Follicle stimulating hormone, the Sertoli cell and spermatogenesis. Recent Prog Horm Res 1976; 32:477. 23. Dym M, Fawcett DW. The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol Reprod 1970; 3:308. 24. Setchell BP, Waites GMH. The blood-testis barrier. In: Hamilton DW, Greep RO, eds. Handbook of physiology, section 7, vol 5. Baltimore: Williams & Wilkins, 1975:143. 25. Flickinger CJ. The postnatal development of the Sertoli cells of the mouse Z Zellforsch 1967; 78:92. 26. Gilula NB, Fawcett DW, Aoki A. The Sertoli cell occluding junctions and gap junctions in mature and developing mammalian testis. Dev Biol 1976; 50:142. 27. Russell LD. Form, dimensions and cytology of mammalian Sertoli cell. In: Russell LD, Griswold MD, eds. The Sertoli cell. Vienna, IL: Cache River Press, 1993:1. 28. Jutte NHPM, Jansen R, Grootegoed AJ, et al. Regulation of survival of rat pachytene spermatocytes by lactate supply from Sertoli cells. J Reprod Fertil 1982; 65:431. 29. Orth JM, Gunsalus GL, Lamperti AA. Evidence from Sertoli cell—depleted rats indicates that spermatid number in adults depends on numbers of Ser¬ toli cells. Endocrinology 1988; 122:787. 30. Cooke PS, Hess RA, Porcelli J, Meisami E. Increased sperm production in adult rats after transient neonatal hypothyroidism. Endocrinology 1991; 129:244. 31. Bunick KD, Kirby J, Hess RA, Cooke PS. Developmental expression of tes¬ tis messenger ribonucleic acids in the rat following propylthiouracilinduced neonatal hypothyroydism. Biol Reprod 1994; 51:706. 32. Simorangkir DR, Wreford NG, de Kretser DM. Impaired germ cell devel¬ opment in the testis of immature rats with neonatal hypothyroidism J Androl 1997; 18:186. 33. Boitani C, Stefanini M, Fragale A, Morena AR. Activin stimulates Sertoli cell proliferation in a defined period of rat testis development. Endocrinol¬ ogy 1995; 136:5438. 34. Setchell BP. Do Sertoli cells secrete fluid into the seminiferous tubules? J Reprod Fertil 1969; 19:391. 35. Jegou B, Le Gac F, de Kretser DM. Seminiferous tubule fluid and interstitial fluid production: I. Effects of age and hormonal regulators in immature rats. Biol Reprod 1982; 27:590. 36. Jegou B, Le Gac F, Irby D, de Kretser DM. Studies on seminiferous tubule fluid production in the adult rat: effect of hypophysectomy and treatment with FSH, LH, and testosterone. Int J Androl 1983; 6:249. 37. French FS, Ritzen EM. A high-affinity androgen binding protein (ABP) in rat testis: evidence for secretion into efferent duct fluid and absorption by epididymis. Endocrinology 1973; 93:88. 38. Gunsalus GL, Musto NA, Bardin CW. Immunoassay of androgen binding protein in blood: a new approach for study of the seminiferous tubule. Sci¬ ence 1978; 200:65. 39. Hansson V, Ritzen EM, French FS, et al. Testicular androgen-binding pro¬ tein (ABP): comparison of ABP in rabbit testis and epididymis with a simi¬ lar androgen-binding protein (TeBG) in rabbit serum. Mol Cell Endocrinol 1975; 3:1. 40. Joseph DR, Hall SH, French FS. Rat androgen binding protein: evidence for identical subunits and amino acid sequence homology with human sex hormone binding globulin. Proc Natl Acad Sci U S A1987; 84:337. 41. Hryb DJ, Khan MS, Romus NA, Rosner W. The solubilization and partial characterization of the sex hormone—binding globulin receptor from human prostate. J Biol Chem 1989; 264:5378. 42. Porto CS, Abreu LC, Gunsalus GL, Bardin CW. Binding of sex hormonebinding globulin (SHBG) to testicular membranes and solubilized recep¬ tors. Mol Cell Endocrinol 1992; 89:33. 43. Joseph BR, Becchis M, Fenstermacher DA, Petrusz P. The alternate Nterminal sequence of rat androgen binding protein/sex hormone binding globulin contains a nuclear targetting signal. Endocrinology 1996; 137:1138. 44. Joseph DR, Wang YM, Sullivan PS. Characterization and sex hormone reg¬ ulation of multiple alternate androgen-binding protein/sex hormone¬ binding globulin RNA transcript in rat brain. Endocr J 1994; 2:749. 45. Wright WW, Musto NA, Mather JP, Bardin CW. Sertoli cells secrete both testis-specific and serum proteins. Proc Natl Acad Sci U S A1981; 78:7565. 46. Lacroix M, Smith FE, Fritz IB. Secretion of plasminogen activator by Sertoli cell enriched cultures. Mol Cell Endocrinol 1977; 9:227. 47. Huggenvik J, Sylvester SR, Griswold MD. Control of transferrin in RNA synthesis in Sertoli cells. Ann NY Acad Sci 1984; 438:1. 48. Steinberger A, Steinberger E. Secretion of an FSH-inhibiting factor by cul¬ tured Sertoli cells. Endocrinology 1976; 99:918. 49. Le Gac F, de Kretser DM. Inhibin production by Sertoli cells. Mol Cell Endocrinol 1982; 28:487. 50. Au CL, Robertson DM, de Kretser DM. An in vivo method for estimating inhibin production by adult rat testes. J Reprod Fertil 1985; 71:259. 51. Robertson DM, Foulds LM, Leversha L, et al. Isolation of inhibin from bovine follicular fluid. Biochem Biophys Res Commun 1985; 126:220. 52. Mason AJ, Hayflick JS, Ling N, et al. Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor. Nature 1985; 318:659. 53. Forage RG, Ring JM, Brown RW, et al. Cloning and sequence analysis of cDNA species coding for the two subunits of inhibin from bovine follicular fluid. Proc Natl Acad Sci U S A1986; 83:3091.

1114

PART VIII: ENDOCRINOLOGY OF THE MALE

54. Massague J. The TGF-JS family of growth and differentiation factors. Cell 1987; 49:437. 55. Jensen TK, Andersson AM, Hjollund NHI, et al. Inhibin B as a serum marker of spermatogenesis: correlation to differences in sperm concentra¬ tion and follicle stimulating hormone levels. A study of 349 Danish men. J Clin Endocrinol Metab 1997; 82:4059. 56. Anderson RA, Wallace EM, Groome NP, et al. Physiological relationships between inhibin B, follicle stimulating hormone secretion and spermatoge¬ nesis in normal men and response to gonadotrophin suppression by exoge¬ nous testosterone. Hum Reprod 1997; 12:746. 57. Anawalt BD, Bebb RA, Matsumoto AM, et al. Serum inhibin B levels reflect Sertoli cell function in normal men and men with testicular dysfunction. J Clin Endocrinol Metab 1996; 81:3341. 58. Tilbrook AJ, de Kretser DM, Clarke IJ. Human recombinant inhibin A sup¬ presses plasma follicle stimulating hormone to intact levels but has no effect on luteinizing hormone in castrated rams. Biol Reprod 1993; 49:779. 59. Tilbrook AJ, de Kretser DM, Clarke IJ. Human recombinant A and test¬ osterone act directly at the pituitary to suppress plasma concentrations of FSH in castrated rams. J Endocr 1993; 138:181. 60. Josso N, Picard J, Tran D. The antimullerian hormone. Recent Prog Horm Res 1977; 33:117. 60a. de Santa Barbara P, Moniot B, Poulat K, Berta P. Expression and subcellular localization of SF-1, Sox 9, WT1, and AMH proteins during early human testicular development. Dev Dyn 2000; 217:293. 61. Brokelmann J. Fine structure of germ cells and Sertoli cells during the cycle of the seminiferous epithelium in the rat. Z Zellforsch 1963; 59:820. 62. Christensen AK, Mason NR. Comparative ability of seminiferous tubules and interstitial tissue of rat testes to synthesize androgen from progesterone-4-15C in vitro. Endocrinology 1965; 76:646. 63. Dorrington JH, Armstrong DT. Follicle stimulating hormone stimulates estradiol-17|3 synthesis in cultured Sertoli cells. Proc Natl Acad Sci U S A 1975; 72:2677. 64. Tsai-Morris CH, Aquilano DR, Dufau ML. Cellular localization of rat testic¬ ular aromatase activity during development. Endocrinology 1985; 116:38. 65. Van Damme MP, Robertson DM, Marana R, et al. A sensitive and specific in vitro bioassay method for the measurement of follicle stimulating hormone activity. Acta Endocrinol (Copenh) 1979; 91:224. 66. Hodgson Y, Robertson DM, de Kretser DM. The regulation of testicular function. In: Greep RO, ed. International review of physiology, vol 27, reproductive physiology IV. Baltimore: University Park Press, 1983:275. 67. Parvinen M. Regulation of the seminiferous epithelium. Endocr Rev 1982; 3:404. 68. Jegou B, Syed V, Sourdaine P, et al. The dialogue between late spermatids and Sertoli cells in vertebrates: a century of research. In: Nieschlag E, Habernicht UF, eds. Spermatogenesis, fertilization, contraception. Berlin: Springer-Verlag, 1992:57. 69. Toledo SP. Leydig cell hypoplasia leading to two different phenotypes: male pseudohermaphroditism and primary hypogonadism not associated with this. Clin Endocrinol 1992; 36:521. 70. Latronico AC, Anasti J, Arnhold IJ, et al. Brief report: testicular and ovarian resistance to luteinizing hormone caused by inactivating mutations of the luteinizing hormone-receptor gene. N Engl J Med 1996; 334:507. 71. Steinberger E, Root A, Ficher M, Smith KD. The role of androgens in the initiation of spermatogenesis in man. J Clin Endocrinol Metab 1973; 37:746. 72. Burger HG, Baker HWG. Therapeutic considerations and results of gona¬ dotropin treatment in male hypogonadotropic hypogonadism. Ann N Y Acad Sci 1984; 438:447. 73. Paulsen CA. The effect of human menopausal gonadotropin on spermato¬ genesis in hypogonadotropic hypogonadism. In: Gual C, ed. Proceedings of the sixth Pan-American Congress in Endocrinology: International Con¬ gress Series 112. Amsterdam: Excerpta Medica, 1966:398. 74. Singh J, O'Neill C, Handlesman DJ. Induction of spermatogenesis by andro¬ gens in gonadotropin deficient (hpg) mice. Endocrinology 1995; 136:5311. 75. Kumar TR, Wang Y, Lu N, Matzuk M. Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nature Genet 1997; 15:201. 76. Tapanainen JS, Aittomaki K, Min J, et al. Men homozygous for an inactivat¬ ing mutation of the follicle stimulating hormone (FSH) receptor gene present variable suppression of spermatogenesis and fertility. Nature Genet 1997; 15:205. 77. Bremner WJ, Matsumoto AM, Paulsen CA. Gonadotropin control of sper¬ matogenesis in man: studies of gonadotropin administration in spontane¬ ous and experimentally induced hypogonadotropic states. Ann N Y Acad Sci 1984; 438:465. 78. Matsumoto AM, Paulsen CA, Bremner WJ. Stimulation of sperm produc¬ tion by human luteinizing hormone in gonadotrophin-suppressed normal men. J Clin Endocrinol Metab 1984; 55:882. 79. Matsumoto AM, Karpas AE, Bremner WJ. Chronic human chorionic gona¬ dotropin administration in normal men: evidence that follicle stimulating hormone is necessary for the maintenance of quantitatively normal sper¬ matogenesis in man. J Clin Endocrinol Metab 1986; 62:1184. 80. Cunningham GR, Huckins C. Persistence of complete spermatogenesis in the presence of low intra-testicular concentration of testosterone. Endocri¬ nology 1979; 105:177. 81. Sun YT, Wreford NG, Robertson DM, de Kretser DM. Quantitative cytological studies of spermatogenesis in intact and hypophysectomised rats: identi¬ fication of androgen-dependent stages. Endocrinology 1990; 127:1215.

82. Macleod J, Pazianos A, Ray B. The restoration of human spermatogenesis and of the reproductive tract with urinary gonadotropins following hypophysectomy. Fertil Steril 1966; 17:7. 83. Marshall GR, Wickings EJ, Liidecke DK, Nieschlag E. Stimulation of sper¬ matogenesis in stalk-sectioned rhesus monkeys by testosterone alone. J Clin Endocrinol Metab 1983; 57:152. 84. Means AR, Dedman JR, Tash JS, et al. Regulation of the testis Sertoli cell by follicle stimulating hormone. Annu Rev Physiol 1980; 42:59. 85. Orth J, Christensen AK. Autoradiographic localization of specifically bound 125I-labelled follicle stimulating hormone on spermatogonia of the rat testis. Endocrinology 1978; 103:1944. 86. Zhengwei Y, Wreford NG, Royce P, et al. Stereological evaluation of human spermatogenesis following suppression by testosterone treatment: hetero¬ geneous pattern of spermatogenic impairment. J Clin Endocrinol Metab 1998; 83:1284. 87. Zhengwei Y, Wreford NG, Schlatt S, et al. GnRH antagonist-induced gona¬ dotropin withdrawal acutely and specifically impairs spermatogonial development in the adult macaque (Macaca fascicularis). J Reprod Fertil 1998; 112:139. 88. Grootegoed JA, Peters MJ, Mulder E, et al. Absence of nuclear androgen recep¬ tor in isolated germinal cells of rat testes. Mol Cell Endocrinol 1977; 9:159. 89. Sanborn BM, Steinberger A, Tcholakian RK, Steinberger E. Direct measure¬ ment of androgen receptors in cultured Sertoli cells. Steroids 1977; 29:493. 90. Bremner WJ, Millar MR, Sharpe RM. Immunohistochemical localization of androgen receptors in the rat testis: evidence of a stage-dependent expres¬ sion and regulation by androgens. Endocrinology 1994; 135:1227. 91. Lamb DJ, Tsai YH, Steinberger A, Sanborn BM. Sertoli cell nuclear tran¬ scriptional activity: stimulation by follicle stimulating hormone and tes¬ tosterone in vivo. Endocrinology 1981; 108:1020. 92. Louis BG, Fritz IB. FSH and testosterone independently increase the pro¬ duction of ABP by Sertoli cells in culture. Endocrinology 1979; 104:454. 93. Au CL, Irby DC, Robertson DM, de Kretser DM. Effects of testosterone on testicular inhibin and fluid production in intact and hypophysectomized adult rats. J Reprod Fertil 1986; 76:257. 94. Sun YT, Irby DC, Robertson DM, de Kretser DM. The effects of exoge¬ nously administered testosterone on spermatogenesis in intact and hypo¬ physectomised rats. Endocrinology 1989; 125:1000. 95. O'Donnell L, McLachlan RI, Wreford NG, et al. Testosterone withdrawal promotes stage-specific detachment of round spermatids from the rat sem¬ iniferous epithelium. Biol Reprod 1996; 55:895. 96. O'Donnell L, Stanton PG, Wreford NG, et al. Inhibition of 5a reductase activity impairs T-dependent restoration of spermiogenesis in rats. Endo¬ crinology 1996; 137:2703. 97. Janulis L, Bahr JM, Hess RA, et al. Rat testicular germ cells and epididymal sperm contain active P450 aromatase. J Androl 1998; 19:65. 98. Carreau S, Bilinska B, Levallet J. Male germ cells: a new source of estrogens in the mammalian testis. Ann Endocrinol (Paris) 1998; 59:79. 99. Robertson DM, O'Donnell L, Jones ME, et al. Impairment of spermatogene¬ sis in mice lacking a functional aromatase (cyy 19) gene. Proc Natl Acad Sci USA 1999; 96:7986. 100. Hess RA, Bunick D, Lee KH, et al. A role for oestrogens in the male repro¬ ductive tract. Nature 1997; 390:509. 101. Saunders PTK, Maguire SM, Gaughan J, Millar MR. Expression of oestro¬ gen receptor beta (ER beta) in multiple cell types including some germ cells, in the rat testis. J Endocrinol 1997; 156:R13. 102. Krege JG, Hodgin JB, Couse JF, et al. Generation and reproductive pheno¬ types of mice lacking estrogen receptor beta. Proc Natl Acad Sci U S A 1998; 95:15677. 103. Yong EL, Wang Q, Tut TG, et al. Male infertility and the androgen receptor: molecular, clinical and therapeutic aspects. Reprod Med Rev 1997; 6:113. 104. Reijo R, Lee TY, Salo P, et al. Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nature Genet 1995; 10:383. 105. Eberhart CG, Maines JZ, Wasserman SA. Meiotic cell cycle requirement for a fly homologue of human deleted in azoospermia. Nature 1996; 381:783. 106. Loveland KL, Schlatt S. Stem cell factor and c-kit in the mammalian testis: les¬ sons originating from mother nature's gene knockouts. J Endocr 1997; 153:337. 107. Dix DJ, Allen JW, Collins BW, et al. Targetted disruption of Hsp 70-2 results in failed meiosis, germ cell apoptosis and male infertility. Proc Natl Acad Sci U S A 1996; 93:3264. 108. Sinha Hikim AP, Swerdloff RS. Hormonal and genetic control of germ cell apoptosis in the testis. Rev Reprod 1999; 4:38. 109. Print CG, Loveland K, Gibson L, et al. Apoptosis regulator Bcl-w is essen¬ tial for spermatogenesis but is otherwise dispensable. Proc Natl Acad Sci USA 1998; 95:12424. 110. Fawcett DW, Neaves WB, Flores MN. Comparative observations on inter¬ tubular tissue of the mammalian testis. Biol Reprod 1973; 9:500. 111. Lording DW, de Kretser DM. Comparative ultrastructural and histochemical studies of the interstitial cells of the rat testis during fetal and postnatal development. J Reprod Fertil 1972; 29:261. 112. Ewing LL, Zirkin B. Leydig cell structure and steroidogenic activity. Recent Prog Horm Res 1983; 39:599. 113. Clark BJ, Wells J, King SR, Stocco DM. The purification, cloning, and expression of a novel LH-induced mitochondrial protein in MA-10 mouse Leydig tumor cells: characterization of the steroidogenic acute regulatory protein (StAR). J Biol Chem 1994; 269:28314. 114. Bose HS, Pescovitz OH, Miller WL. Spontaneous feminization in a 46XX

Ch. 114: Evaluation of Testicular Function

115. 116. 117.

118. 119.

female patient with congenital lipoid adrenal hyperplasia due to a homozygous frameshift mutation in the acute steroid regulatory protein. J Clin Endocrinol Metab 1997; 82:1511. Fujieda K, Tajima T, Nakae J, et al. Spontaneous puberty in 46XX subjects with congenital lipoid adrenal hyperplasia. J Clin Invest 1997; 99:1265. Catt KJ, Harwood JP, Aguilera G, Dufau ML. Hormonal regulation of pep¬ tide receptors and target cell responses. Nature 1979; 280:109. Padron RS, Wischusen J, Hudson B, et al. Prolonged biphasic response of plasma testosterone to single intramuscular injections of human chorionic gonadotropin. J Clin Endocrinol Metab 1980; 50:1100. de Kretser DM. Sertoli cell-Leydig cell interaction in the regulation of tes¬ ticular function. Int J Androl 1982; 5(Suppl):ll. Sharpe RM, Cooper I. Intratesticular secretion of a factor(s) with major stimulating effects on Leydig cell testosterone secretion in vitro. Mol Cell Endocrinol 1984; 37:159.

120. Cohen PE, Hardy MP, Pollard JW. Colony-stimulating factor-1 plays a major role in the development of reproductive function in male mice. Mol Endocrinol 1997; 11:1636. 121. Papadopoulos V. Peripheral-type benzodiazepine/diazepam binding inhib¬ itor receptor: biological role in steroidogenic cell function. Endocrinol Rev 1993;14:222. 122. McLachlan RI, Wreford NG, de Kretser DM, Robertson DM. The effects of testosterone on spermatogenic cell populations in the adult rat. Biol Reprod 1994;51:945. 123. Bergh A. Local differences in Leydig cell morphology in the adult rat testis: evidence for a local control of Leydig cells by adjacent seminiferous tubules. Int J Androl 1982; 5:325. 124. Rich KA, de Kretser DM. Spermatogenesis and the Sertoli cell. In: de Kret¬ ser DM, Burger HG, Hudson B, eds. The pituitary and testis: clinical and experimental studies. Berlin: Springer-Verlag, 1983:85. 125. Head JR, Billingham RE. Immune privilege in the testis. II: evaluation of potential local factors. Transplantation 1985; 40:269. 126. Hedger MP. Testicular leucocytes: what are they doing? Rev Reprod 1997; 2:38. 127. Cucicini C, Kercret H, Touzlain AM, et al. Vectorial production of interleu¬ kin 1 and interleukin 6 by rat Sertoli cells cultured in a dual culture com¬ partment system. Endocrinology 1997; 138:2863.

CHAPTER

114

EVALUATION OF TESTICULAR FUNCTION STEPHEN J. WINTERS

Male hypogonadism signifies impaired production of testoster¬ one by Leydig cells, or deficient spermatogenesis, but in most clinical disorders both compartments of the testis are abnormal. This is not surprising because extensive biochemical communi¬ cation occurs between the Leydig cells and the seminiferous tubules (see Chap. 113). Hypogonadism due to pathology intrinsic to the testis (primary testicular failure) or to deficient gonadotropin drive to the testis (hypogonadotropic hypogo¬ nadism) may produce similar clinical features, and the tests used to diagnose most forms of hypogonadism overlap. There¬ fore, a general clinical and laboratory approach to evaluating testicular function will be presented.

CLINICAL EVALUATION

1115

TABLE 114-1. Symptoms and Signs of Hypogonadism in Adult Men Symptoms

Signs

Decreased libido

Soft smooth skin

Asthenia

Decreased beard, axillary, and pubic hair

Erectile dysfunction

Decreased muscle mass and strength

Infertility

Small testes

Osteopenia/ fractures

Gynecomastia Small prostate

A complete physical examination is needed to evaluate hypogonadal males. Prepubertal Leydig cell insufficiency causes eunuchoidism, including a juvenile voice; childlike facies; scant facial, pubic, and body hair; smooth skin; poorly developed skel¬ etal musculature; fat accumulation in the hips, buttocks, and lower abdomen; a small prostate; and failure of epiphyseal clo¬ sure of the long bones, resulting in an arm span that exceeds the height by >6 cm and disproportionately long legs. The testis size is readily measured by approximating its length and width with a ruler or with a commercially available series of ovoids (see Chap. 93). The testes generally reach adult size by 18 years of age. The median testis length among normal men is 5 cm, equiv¬ alent to 25 mL in volume.1 The left testis is often slightly smaller than the right. Although testes that are 4 cm long may be normal, many men with hypospermatogenesis will have testes of this size. There are genetic differences in testis size, with smaller tes¬ tes found among Asian men. Testis size declines with aging.2 Var¬ icocele (see Chap. 118), a distention of the pampiniform plexus within the scrotum due to dysfunctional valves within the sper¬ matic vein, is a common finding in infertile men, and is associ¬ ated with a reduction in testis size beginning in adolescence, particularly on the left.3-4 Usually, the venous distention is visible or palpable in the upright posture, increases if the patient per¬ forms a Valsalva maneuver, and disappears as soon as the patient is recumbent. Color Doppler ultrasonography can be used to confirm the presence of a varicocele.5 The skin of hypogonadal men may be soft and smooth. Mus¬ cle mass may decline, and fat mass may increase. Gynecomastia is a frequent finding in hypogonadal teenagers and in adults. Examination of the male breast can be difficult, however, because the distinction between fat and breast tissue is often inexact. Galactorrhea, on the other hand, is a rare finding in males (see Chap. 13). Physical changes regress slowly in sexu¬ ally mature men who acquire Leydig cell dysfunction. If some of the physical changes of eunuchoidism in previously normal men are present, androgen deficiency is both severe and long standing. Therefore, testosterone deficiency is often present in men with limited physical findings. Reduced visual acuity or a visual field disturbance suggests a mass lesion in the hypothal¬ amus-pituitary (see Chap. 19). The digital rectal examination is useful in assessing sphincter tone in men with erectile dysfunc¬ tion, and for estimating prostate size.

LABORATORY EVALUATION TESTOSTERONE

Patients with impaired testicular function present variably, depending on the age at onset of their disease. Hypogonadism in the fetus results in genital ambiguity (see Chap. 90). When the disturbance begins in childhood, puberty is delayed or does not occur (see Chap. 92). Adult men with hypogonadism often present with a decrease in libido and energy, or with infertility, among other symptoms (Table 114-1), and although these symptoms are sensitive indicators of androgen deficiency, they are nonspecific. For example, a reduced libido in men is also a characteristic of depression as well as of performance anxiety.

Physiologic Aspects of Testosterone Testing. Testoster¬ one is both a paracrine regulator of spermatogenesis (see Chap. 113) and a hemocrine hormone. The testicular content of testosterone is ~50 pg (1 pg/g testis), whereas the blood production rate is -5000 pg per day, indicating that only a small portion of the testosterone produced each day is stored in the testes. The testosterone precursor steroids, including pregnenolone, 17-OH pregnenolone, dehydroepiandrosterone (DHEA), progesterone, 17a-hydroxyprogesterone, and

1116

PART VIII: ENDOCRINOLOGY OF THE MALE

17-Ketosteroids

FIGURE 114-1. Metabolic pathways for testoster¬ one. Enzymes are (A) aromatase, (B) 5a-reductase, (C) 17p-hydroxysteroid dehydrogenase, (D) vari¬ ous hydroxylases and transferases, and (E) 3ahydroxysteroid dehydrogenase. The percentages represent the average percent bioconversion in nor¬ mal men to these active and inactive metabolities.

HO

HO' Androstan - 3a, 17(3-diol (3a-diol)

H

glucuronides and sulfates

i 3a-diol-glucuronide

androstenedione, are also secreted by the testis, and the rela¬ tive concentrations of these steroids in spermatic vein blood are proportional to their testicular concentrations.6 The release of precursor steroids into the circulation may indicate that they are unnecessary by-products in the orderly biotransformation of pregnenolone to testosterone, because none is known to have a physiologic action in the male. Androstenedione is of special interest because it is used as a performance-enhancing drug. Androstenedione is biocon¬ verted to testosterone and to estrone, but there is little pub¬ lished information on the endocrine profile following androstenedione administration. A single sample of blood, generally drawn in the morning, can be used to measure testosterone. The usual normal range in morning samples is 3 to 10 ng/mL (10-40 nmol/L). There is a diurnal variation in testosterone in adult men, with highest lev¬ els in the early morning, followed by a progressive fall through¬ out the day, reaching the lowest levels in the evening and during the first few hours of sleep. Peak and nadir values dif¬ fer by -15%, although more pronounced differences are some¬ times observed.7 The diurnal rhythm is blunted with aging8 and in men with testicular failure.9 The metabolic clearance of testosterone is not thought to vary throughout the day, so that the diurnal testosterone rhythm is presumed to result from a day-night difference in testosterone production. Because there are no parallel changes in serum LH levels, however, the origin as well as physiologic significance of the diurnal testosterone rhythm remains uncertain. It is important to measure testosterone in the morning, because reference ranges are based on morning values. Frequent sampling of spermatic venous blood reveals testosterone secretory pulses at a frequency of -1 pulse per hour,6 but because of this rapid frequency and relatively low pulse amplitude only small fluctuations occur in peripheral plasma. Testosterone secretory bursts are more readily defined in the peripheral blood when pulse frequency is low.7 There is a good correlation between the plasma testosterone level at first sampling and the mean of multiple samples taken over 1 year, so that one morning sample is reasonably representative,10 although abnormal and borderline values should be confirmed. There is a prominent sleep-related increase in serum tes¬ tosterone in pubertal boys, with abrupt rises from female to adult male levels.11 This difference can be used clinically to evaluate boys with delayed puberty (see Chap. 92), because the rise to a higher testosterone value in the morning may precede pubertal testis growth and indicates that puberty has begun.12

Men with hypogonadism and hyperprolactinemia have an exaggerated diurnal testosterone rhythm, leading to very low levels in the afternoon and evening, which can explain clinical hypogonadism despite a normal morning total testosterone level,9 as sometimes occurs in men with a prolactinoma. The testosterone level tends to rise during intense exercise because of hemoconcentration13 and to decline 12 to 24 hours later because gonadotropin-releasing hormone (GnRH) secretion is reduced.14 Testosterone levels are also reduced in acute and chronic illness15 and with fasting for at least 48 hours. These factors can confound an evaluation of testicular function. Testosterone Metabolism. The metabolism of testosterone is shown in Figure 114-1. Testosterone is metabolized into two biologically important products, dihydrotestosterone (DHT) and estradiol, by the enzymes 5a-reductase and aromatase, respec¬ tively. Most of the metabolism of testosterone occurs in the liver, however, via 3a- and 3(3-hydroxysteroid dehydrogenase, 5a- and 5(l-steroid reductase, and oxidation of the D-ring to the 17-ketosteroids androsterone (3a-hydroxy-5a-androstane-17-one) and etiocholanolone (3a-hydroxy-5p-androstane-17-one), which are then excreted in the urine. Most of the 10 to 25 mg per day of ketosteroids in the urine of men is of adrenal origin, however, so that the urinary 17-ketosteroid excretion is not a test of testicular func¬ tion. Testosterone metabolites are also conjugated to sulfuric and glucuronic acids at the 3- or 17-position and excreted in the urine and bile. A small fraction (2%) of the circulating testosterone is excreted unchanged in the urine.

SEX HORMONE-BINDING GLOBULIN Of the circulating testosterone in normal men,

c 400

w 200 o a> 100

1117

TABLE 114-2. Conditions with Abnormal Sex Hormone-Binding Globulin Concentrations Increased

Decreased

Aging

Hyperinsulinemia

Androgen deficiency

Obesity

Estrogen treatment

Androgen treatment

Thyrotoxicosis

Hypothyroidism

Alcoholic cirrhosis

Hypercortisolism

Hepatitis

Nephrotic syndrome

Growth hormone deficiency

Acromegaly Familial

Young Adult

Obese

Elderly

FIGURE 114-2. Distribution of testosterone in blood plasma. The free fraction represents 1% to 4% of the total testosterone (T). The albuminbound testosterone represents a loosely bound complex of albumin and testosterone that dissociates readily. Together the free and albuminbound testosterone have been termed bioavailable testosterone. Sex hormone-binding globulin (SHBG) has a high affinity for testosterone and dihydrotestosterone, and its role in androgen action remains con¬ troversial. When the level of SHBG is reduced, as in obesity, the total testosterone is low, but the bioavailable and free testosterone levels are generally normal. In older men, the mean total testosterone level declines, but because SHBG increases with aging, the decrease in total testosterone is less than the decrease in free and bioavailable testoster¬ one. (CBG, cortisol-binding globulin.) controversial. The finding of membrane-binding sites for androgen-binding protein in the epididymis and for SHBG in testis, prostate, and other tissues and the activation of the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway, which is a coactivator of androgen receptors (ARs), suggest that these binding proteins could play a direct role in androgen action. However, according to the free hormone hypothesis, which states that only free steroid enters target cells, SHBG functions solely as a reservoir for testosterone.

FREE OR BIOAVAILABLE TESTOSTERONE For most clinical applications the measurement of the total tes¬ tosterone level is entirely satisfactory. However, when circulat-

FIGURE 114-3. Relationship between the level of sex hormone-binding globulin (SHBG) and total testosterone in plasma from normal-weight and obese men. (Plasma samples were kindly provided by Drs. Bret Goodpaster and David Kelley of the University of Pittsburgh; repub¬ lished with the permission of Clinical Chemistry.)

ing SHBG levels are altered (Table 114-2), this change may be reflected as an increase or a decrease in the measured serum total testosterone concentration. For example, a low testoster¬ one level in an obese man may be misinterpreted to suggest androgen deficiency (see Fig. 114-2). Because there is consider¬ able evidence that the non-SHBG-bound portion of circulating testosterone represents the biologically active fraction, many methods for determining non-SHBG-bound or free testosterone have been developed.20 METHODOLOGY Total Testosterone. Total testosterone can be measured directly in serum by immunoassay, or after extraction with organic solvents with or without further chromatographic separation. Commercial kits for the direct assay of testosterone in unex¬ tracted serum or plasma using an iodine-125 (125I)-labeled tracer are technically easy to use, precise, and sufficiently accu¬ rate for most purposes. When SHBG levels are low, however (see later), the result for testosterone may be overestimated, whereas high SHBG levels may lead to underestimation of the actual testosterone value. This artifact appears to result from differences between the SHBG levels in the assay standards and samples, with nonlabeled testosterone binding to both SHBG and to the tes¬ tosterone antiserum, whereas the 125I-testosterone tracer binds primarily to the antiserum.21 Direct assays also tend to overesti¬ mate the true values of testosterone at low concentrations. To discriminate among low levels more accurately, or to control for the effects of abnormal SHBG concentrations, immunoassays using organic solvent extraction and chro¬ matographic separation may be needed, but are more costly and difficult to perform. Fully automated immunoassay analyzers have been introduced into clinical laboratories for competitive and two-site assays.22 These electrochemiluminescence immunoassays (ECLIAs) use nonradiolabeled detection methods, and have very short incu¬ bation times. Thus, they are attractive for clinical laboratories. The precision and accuracy are quite acceptable for samples from adult men, with results similar to those of radioimmu¬ noassays (RIAs). At low levels, however, ECLIAs may produce values that are 50% to 100% higher than those of RIAs. Lipemia may also produce inaccurate high values. Equilibrium Dialysis Assay. The equilibrium dialysis assay is used to calculate the percent free testosterone. The diffusion across a semipermeable membrane of tracer amounts of 3Htestosterone added to the sample is measured. The free testosterone concentration is then calculated from the product of the total tes¬ tosterone level and the percentage of tracer crossing the dialysis mem¬ brane (percent free testosterone), the latter is usually 1% to 4%, with a free testosterone level commonly ranging from 4 to 20 ng/dL. This assay is complex, with potential errors due to tem¬ perature, sample dilution, and tracer impurities. (Testosterone can also be measured by RIA in the dialysate, avoiding the use of 3H-testosterone, but a very sensitive immunoassay is needed.) Centrifugal ultrafiltration is a variation of equilibrium

1118

PART VIII: ENDOCRINOLOGY OF THE MALE

FIGURE 114-4. Relation between the free testosterone (T) level calculated from the plasma levels of total testosterone and sex hormone-binding globulin (SHBG) and the non-SHBG testosterone among normal thin and overweight men, determined by ammonium sulfate precipitation. dialysis in which high-speed centrifugation is used to separate bound from free hormone across a dialysis membrane. Calculated Free Testosterone. Another approach for cor¬ recting the total testosterone value for variations in SHBG con¬ centrations is to calculate the free testosterone level from the levels of total testosterone and SHBG, using binding constants for SHBG and albumin.23 This value appears to correlate quite well with the value obtained by equilibrium dialysis. Free Testosterone Index. The "free testosterone index" has been calculated as the ratio: total testosterone/SHBG using units of nmol/L. Although the calculation is easy to perform and is believed to be valid in women, it appears to be less useful in men, because most of the SHBG in men is bound to testosterone.24 Analog Kits. The free testosterone level has been deter¬ mined directly using solid-phase free testosterone RIA analog kits, which use an 125I-labeled testosterone analog as the tracer. This assay is based on the selective binding of the analog tracer to the testosterone antiserum, but not to SHBG, and is a popular, high-precision, single-step, nonextraction method. Although there is a strong positive correlation between free testosterone levels measured by these analog kits and by equilibrium dialy¬ sis assay, the kits produce substantially (75%) lower values.233 Moreover, the level of SHBG is a positive predictor of the free testosterone level as measured by analog methods. The percent¬ age of free testosterone (determined by the analog method) does not decrease as SHBG increases, and this free testosterone level is almost perfectly positively correlated with the total tes¬ tosterone. Thus, both the total testosterone and the free tes¬ tosterone (determined by the analog method) appear to provide essentially the same clinical information.25 Non-Sex Hormone-Binding Globulin Assay (Bioavailable Testosterone). The concept of the non-SHBG—testosterone assay (sometimes referred to as bioavailable testosterone) is that the testosterone bound to albumin, because of its low-affinity binding, is as readily available to target tissues as is free tes¬ tosterone.26 The most widely used technique to measure nonSHBG-bound testosterone involves the precipitation of tracer amounts of 3H-testosterone bound to SHBG using ammonium sulfate. The non-SHBG testosterone is calculated by multiplying the total testosterone level times the percent of3H-testosterone remaining in the supernatant after precipitation. Figure 114-2 shows that the decline in bioavailable testosterone as men grow older is sub¬ stantially greater than the fall in total testosterone, whereas the bioavailable testosterone level is normal in obesity although SHBG is reduced. On the other hand, SHBG increases as men grow older.27 Figure 114-4 illustrates the excellent correlation

between the non-SHBG testosterone (i.e., bioavailable testoster¬ one) and the free testosterone calculated from the level of SHBG and total testosterone in normal men, suggesting that the mea¬ sures provide equally useful information. Salivary Testosterone. Testosterone concentrations have also been measured in saliva in which the testosterone level is 2% to 3% of the concentration in serum. This approach is useful in field studies. Because the salivary gland basement mem¬ brane excludes proteins, salivary testosterone correlates with free testosterone. Differences between salivary and free tes¬ tosterone levels have been explained by enzymes in salivary glands or saliva that convert androstenedione to testosterone, or that metabolize testosterone further, and by meal-related changes in saliva production. Sex Hormone-Binding Globulin. Several two-site immunoradiometric assays (IRMAs) for SHBG, which use rabbit and mouse polyclonal antibodies, are available.28 The normal range for men is 10 to 50 nmol/L, children 45 to 90 nmol/L, and women 30 to 90 nmol/L. SHBG can also be measured indirectly using radioligand binding assays based on the specific binding of 3Htestosterone or 3H-DHT by serum.29 The range of normal values for men approximates 0.3 to 1.2 pg bound DHT/dL, indicating that most of the circulating SHBG in plasma in men is complexed to androgens. Aging and various medical disorders alter plasma levels of SHBG (see Table 114-2). Measurement of SHBG in plasma is useful in interpreting testosterone concentrations, and as a marker for insulin resistance and cardiovascular risk.30

LUTEINIZING HORMONE AND FOLLICLE-STIMULATING HORMONE The pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH)—like thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG)—are heterodimeric glycoproteins composed of a common a subunit and a hormone-specific P subunit.31 LH activates Leydig cells, whereas FSH stimulates Sertoli cells. Gonadotropin synthesis is up-regulated by GnRH, and suppressed by gonadal steroids. FSH-P mRNA and thereby FSH secretion are also regulated at the pituitary level by testicular inhibin, and perhaps by pituitary activin and follistatin.32 LH is secreted into the circulation in normal adult men in discrete pulses approximately once every 1 to 2 hours throughout the day and night.33 This mode of secre¬ tion results from the pulsatile release of GnRH into the portal blood by the anterior hypothalamus (see Chap. 19). To estimate mean serum LH levels for a given patient, three blood samples can be drawn at 20-minute intervals. These can be assayed indi¬ vidually, or equal aliquots of each sample can be pooled, and the pool assayed. FSH levels are more constant in peripheral blood,34 presumably because the FSH response to GnRFl is of lesser magnitude, and because the clearance of FSH from blood is slow, as compared to LH. Highly sensitive and specific two-site immunoassays for LH and FSH are now widely available. Specificity results from the use of two distinct antibodies, which bind to separate sites on the protein. Often, one antibody is to the a subunit, and the other antibody is to the (3 subunit. One antibody, which is present in excess, is immobilized to facilitate separation of bound from free label; it is known as the capture antibody. The other antibody, which is coupled to a detectable label, is the detection antibody. The signal increases in proportion to the level of hormone, rather than decreasing in proportion to the level of hormone, as in RIAs. The detection antibody is radiolabeled with 125I in IRMAs, whereas ion-chelated antibodies are used in immunofluorometric assays (IFMAs). Delayed addition of an enhancement solution dissociates the metal from the antibody. The metal then binds to other constituents of the enhancement solution, forming a highly fluorescent chelate. Because the low molecular weight metal chelates to an antibody without alter¬ ing its binding affinity, IFMAs tend to be more sensitive than

Ch. 114: Evaluation of Testicular Function are IRMAs. In addition, with IFMAs there are no radioactive materials to dispose of, and the shelf life of the reagents is longer. However, reagents for IFMAs tend to be more expen¬ sive, and the interassay variation tends to be greater than for IRMAs. Enzyme-linked immunosorbent assays (ELISAs) for gonadotropins are also available. These assays are also noniso¬ topic, but are generally less sensitive than are IRMAs or IFMAs because of the coupling of the macromolecular enzyme to the antibody, and they have a higher nonspecific binding. Two-site assays using monoclonal antibodies may be too specific. For example, an LH-(I sequence variant, which appears to represent a polymorphism, was identified when a specific monoclonal antiserum to the LH-a/p dimer was unable to recognize LH in the sera and urine of healthy Finnish men and women.35 LH levels as measured by two-site immunoassays are detectable in all adult male serum samples and in most samples from normal prepubertal boys, especially nighttime samples, whereas LH levels are often below the normal range in gona¬ dotropin-deficient patients.36 Nevertheless, the diagnosis of gonadotropin deficiency should not be made based on the serum LH level alone. Instead, the serum testosterone level should also be low. In contrast to their barely detectable LH lev¬ els, serum FSH levels in patients with GnRH deficiency are readily measurable,37 consistent with paracrine stimulation of FSH-P mRNA and FSH secretion by activin, as occurs in cul¬ tured rat pituitary cells. The finding of elevated LH and FSH concentrations in serum indicates primary testicular failure. Serum LH levels rise when testosterone production falls because the negative feed¬ back effects of testosterone on GnRH secretion are reduced. Ele¬ vated serum FSH concentrations, which generally indicate a disturbance of seminiferous tubule function, are now known to result from deficient secretion of testicular inhibin B as well as sex steroids.38 LH levels are rarely increased in men without a concomitant increase in FSH concentrations because seminifer¬ ous tubules are more readily damaged than are Leydig cells, but elevated FSH and normal LH levels are often found in severely oligospermic men.39 Pituitary tumors may produce FSH,40 and rarely dimeric LH.41

DIHYDROTESTOSTERONE The concentration of DHT in the circulation of adult men is -10% that of testosterone. Of the circulating DHT, -25% is secreted by the testis, and the remainder arises from the biocon¬ version of testosterone in liver, kidney, muscle, prostate, and skin.42 The DHT concentration in prostate is 5- to 10-fold greater than that in the peripheral blood.43 There are two isoen¬ zymes that convert testosterone to DHT, 5a-reductase types 1 and 2. The type 1 enzyme is found in the liver and skin, and per¬ haps in other tissues. The type 2 enzyme predominates in geni¬ tal and male accessory gland tissues in which 5a-reduction is a prerequisite for normal androgen-mediated function.44 Unlike testosterone, DHT levels in plasma do not decrease as men grow older 45 Because serum DHT levels are usually normal in men with testicular dysfunction, the measurement of DHT is not recommended for routine clinical purposes. Moreover, anti¬ sera to DHT may cross-react with testosterone, making the complete separation of these two steroids difficult. Only a small fraction of the DHT produced in target tissues reenters plasma; rather it is metabolized by 3a reduction to 5aandrostane 3(3-17(3-diol (3a-diol), which reenters plasma and is further metabolized by glucuronide conjugation and by other pathways.46 3a-Diol-G is present in significantly reduced con¬ centrations in plasma from hypogonadal and elderly men, and in patients with androgen resistance and 5a-reductase defi¬ ciency. Plasma 3a-diol-G levels are increased in men with severe acne or dense chest hair,47 and in hirsute women. Plasma 3a-diol-G is derived from ketosteroids secreted by the adrenals as well as from DHT, however, so that interpretation

1119

of plasma 3a-diol-G levels is complex. Therefore, its measure¬ ment remains a research tool.

PROLACTIN The measurement of prolactin in serum is of great importance in the evaluation of men with sexual dysfunction, impaired libido, or delayed adolescence, because these symptoms are common in men with prolactin-producing pituitary tumors48 (see Chap. 13). Slight elevations of prolactin, which become more pronounced after stimulation with dopamine antagonists or thyrotropin-releasing hormone (TRH), may be found in men with primary testicular failure,49 perhaps because of androgen deficiency and unchanged or increased estrogen production, because prolactin (PRL) gene expression is stimulated by estra¬ diol. On the other hand, men with complete GnRH deficiency, with very low circulating levels of testosterone and estradiol, have low prolactin levels that normalize after treatment with hCG or testosterone.50 Prolactin levels are rarely increased in otherwise healthy men with infertility.51

ESTROGENS Most of the estrogens in the circulation in normal adult men are derived from the bioconversion of testosterone to estradiol, and androstenedione to estrone, by the aromatase enzyme complex in fat, muscle, kidney, and liver. Thus, plasma estro¬ gen concentrations in men are determined by both testicular and adrenal substrate production, and by the aromatase enzyme activity in several tissues. The normal range is gener¬ ally 6 WBCs per high-power microscopic field in expressed pros¬ tatic secretions suggests infection. Seminal plasma pH may be elevated. Cytokines produced by WBCs in response to infection could damage sperm cell membrane integrity and impair fertil¬ ization. Infection with Chlamydia trachomatis is now the most common sexually transmitted disease, and is known to cause symptomatic pelvic inflammatory disease in women. Chlamy¬ dia urethritis in men has been proposed to produce chronic prostatitis and seminal vesicle infection. Detection of chlamy¬ dia in a first-void urine sample or in semen can be accom¬ plished by specific PCR or ligase chain reaction assays. C. trachomatis seems to be rare in the semen and urine of asymp¬ tomatic infertile men, however.106

Ch. 114: Evaluation of Testicular Function BIOCHEMICAL ANALYSIS OF SEMEN There is a tremendous array of seminal plasma constituents, each of which presumably plays a role in maintaining the proper milieu for fertilization.107 After ejaculation, human semen coagulates because of the formation of a dense fibrous network. Proteolytic enzymes of prostatic origin lyse the fibers in 10 to 30 minutes. Sperm can then be separated from seminal plasma by gentle centrifugation. Because of cell breakage, how¬ ever, intracellular constituents invariably are present in seminal plasma, and specific seminal plasma constituents may be reduced or absent because they bind to the sperm surface and are removed. The protein content of seminal plasma ranges from 3.5 to 5.0 g/dL. Some of these proteins are identical to that of blood plasma, including transferrin, insulin-like growth factor (IGF)-I and -II, and inhibin; others are specific for semen such as sperm ndherins, which are glycoproteins that are thought to play a role in sperm binding to the zona pellucida. The prostate contrib¬ utes protective redox enzymes such as superoxide dismutase as well as a number of peptidases such as prostate-specific anti¬ gen, which, although a marker for prostate cancer, plays a physiologic role in semen liquefaction. Carbohydrates are present in semen, both free and associated with proteins. Fruc¬ tose is the principal sugar of seminal plasma. Bilateral agenesis or complete obstruction of the seminal vesicles results in ejacu¬ lates that are nearly free of fructose. Androgen deficiency impairs the function of the accessory organs, with a reduction in the concentrations of many substances normally present in semen. Among the steroid hormones, testosterone, several of its precursor steroids, DHT, and 17(3-estradiol are present in semen.108 Seminal plasma contains many trace elements, with calcium and zinc being the most abundant.109 Carnitine, acetylcarnitine, glycerylphosphorylcholine, and citric acid are among the products of the human prostate. Carnitine is also present in sperm, and treatment with oral carnitine has been proposed to improve sperm motility. High levels of the cytokine interleukin-6 (IL-6) in semen have been proposed as a marker for infection of the male acces¬ sory glands, although no relationship has been shown between seminal plasma IL-6 levels and sperm parameters.110 Prosta¬ glandins of both the E and F series are produced by the testis and throughout the excurrent duct system, and presumably regulate ejaculatory function. There is substantial concern that environmental pollutants damage the male reproductive sys¬ tem. An estimation of internal dosing can be made by measur¬ ing chemicals in serum or semen. Blood lead levels have been a better indicator of seminiferous tubule dysfunction than is the level of lead in semen,111 whereas the concentration of alumi¬ num in spermatozoa may be a more reliable biomarker of alu¬ minum toxicity.112

STUDY OF TESTICULAR TISSUE Over the past 30 years, physical examination of the testes and measurement of plasma testosterone, LH, and FSH levels have replaced testicular biopsy for distinguishing gonadotropin defi¬ ciency from primary testicular failure. The use of routine testic¬ ular biopsy in men with unexplained infertility has also declined because the finding of damaged seminiferous tubular epithelium with incomplete spermatogenesis has provided lit¬ tle insight into the pathogenesis of male infertility, and the ulti¬ mate therapeutic impact of the biopsy results has been limited. However, testicular biopsy is performed in azoospermic men with normal plasma FSH levels in an effort to identify genital tract obstruction, which can be successfully treated by micro¬ surgery. In many centers, fine-needle aspiration biopsy of the testis has replaced open biopsy.113 In addition, transrectal ultrasonography and transurethral vasography can be used to localize the site of an obstruction.114 In men with marked

1123

hypospermatogenesis, testicular sperm aspiration (TESA) and epididymal sperm aspiration (PESA) are used to obtain sperm for intracytoplasmic sperm injection (ICSI) when few or no sperm are present in the ejaculate.115

GENETIC STUDIES Cytogenetic studies are helpful in clarifying the cause of pri¬ mary testicular failure, and for genetic counseling in men plan¬ ning ICSI because mutations can be passed on to the progeny. Standard chromosomal analyses were abnormal in 13.7% of azoospermic men and 4.6% of oligospermic men.116 Klinefelter syndrome (47,XXY) and its variants (e.g., 46,XY/47,XXY) are the most common cause of azoospermia and are detected by peripheral blood leukocyte karyotyping with banding proce¬ dures, and no longer by the examination of buccal mucosal cells for condensed chromatin (Barr body). Genes on the long arm of the Y chromosome are required for spermatogenesis. This region of the Y is known as the AZF region because it contains genes related to azoospermia. Using the PCR to analyze DNA from peripheral blood leukocytes, small deletions of AZF genes, which escape detection under the microscope, can be identified in 15% to 30% of men with azoospermia.117 The absence of these deletions in the fathers of infertile men indicates that they represent de novo mutations, and provides good evidence that they relate to male infertility. Because, with ICSI, Y microdeletions will be passed on to sons, screening tests for Y microdeletions have been recommended. Congenital bilateral absence of the vas deferens accounts for 3.5% to 8.0% of cases of azoospermia, and represents a mild form of cystic fibrosis in -70% of cases.118 This autosomal reces¬ sive disorder results from mutations involving the cystic fibro¬ sis transmembrane conductase regulator gene, which codes for a membrane protein that functions as an ion channel, and appears to play a role in the development of the epididymis, seminal vesicles, and vas deferens. Testing for this mutation should be performed in men with obstructive azoospermia. Computed tomographic (CT) scans of the paranasal sinuses, chest radiography, and pulmonary function tests should also be obtained, and affected men should be cautioned not to smoke cigarettes.

REFERENCES 1. Takihara H, Sakatoku J, Fujii M, et at. Significance of testicular size mea¬ surement. In: Andrology. I. A new orchiometer and its clinical application. Fertil Steril 1983; 39:836. 2. Stearns EL, MacDonnell JA, Kaufman BJ, et al. Decline of testicular func¬ tion with age: hormonal and clinical correlates. Am J Med 1974; 57:761. 3. Sawczun IS, Hensle TW, Burbie KA, Nagler HM. Varicoceles: effect on tes¬ ticular volume in prepubertal and pubertal males. Urology 1993; 41:466. 4. Haans LCF, Laven JSE, Mali WPThM, et al. Testis volumes, semen quality, and hormonal patterns in adolescents with and without a varicocele. Fertil Steril 1991; 56:731. 5. Chiou RK, Anderson JC, Wobig RK, et al. Color Doppler ultrasound criteria to diagnose varicoceles: correlation of a new scoring system with physical examination. Urology 1997; 50:953. 6. Winters SJ, Takahashi J, Troen R Secretion of testosterone and its delta-4 precursor steroids into spermatic vein blood in men with varicocele-associ¬ ated infertility. J Clin Endocrinol Metab 1999; 84:997. 7. Spratt DI, O'Dea L St L, Schoenfeld D, et al. Neuroendocrine-gonadal axis in men: frequent sampling of LH, FSH and testosterone. Am J Physiol 1988; 254:E658. 8. Tenover JS, Matsumoto AM, Clifton DK, Bremner WJ. Age-related alter¬ ations in the circadian rhythms of pulsatile luteinizing hormone and tes¬ tosterone secretion in healthy men. j Gerontol 1988; 43:M163. 9. Winters SJ. Diurnal rhythm of testosterone and luteinizing hormone in hypogonadal men. J Androl 1991; 12:185. 10. Vermeulen A, Verdonck G. Representativeness of a single point plasma tes¬ tosterone level for the long term hormonal milieu in men. j Clin Endocrinol Metab 1992; 74:939. 11. Boyar RN, Rosenfeld RS, Kapen S, et al. Human puberty: simultaneous augmented secretion of luteinizing hormone and testosterone during sleep. J Clin Invest 1974; 54:609.

1124

PART VIII: ENDOCRINOLOGY OF THE MALE

12. Wu F, Brown DC, Butler GE, et al. Early morning plasma testosterone is an accurate predictor of imminent pubertal development in prepubertal boys. J Clin Endocrinol Metab 1993; 76:26. 13. Zmuda JM, Thompson PD, Winters SJ. Exercise increases serum testoster¬ one and sex hormone-binding globulin levels in older men. Metabolism 1996; 45:935. 14. Kujala H, Alem M, Huhtaniemi IT. Gonadotrophin-releasing hormone and human chorionic gonadotropin tests reveal that both hypothalamic and testicular endocrine functions are suppressed during acute prolonged exer¬ cise. Clin Endocrinol 1990; 33:219. 15. Turner HE, Wass JAH. Gonadal function in men with chronic illness. Clin Endocrinol 1997; 47:379. 16. Dunn JF, Nisula BC, Rodbard D. Transport of steroid hormones: binding of 21 endogenous steroids to both testosterone-binding globulin and corticosteroid¬ binding globulin in human plasma. J Clin Endocrinol Metab 1981; 53:58. 17. Petra PH. The plasma sex steroid binding protein (SBP or SHBG). A critical review of recent developments on the structure, molecular biology and function. J Steroid Biochem Molec Biol 1991; 40:735. 18. Terasaki T, Nowlin DM, Pardridge WM. Differential binding of testosterone and estradiol to isoforms of sex hormone-binding globulin: selective alter¬ ation of estradiol binding in cirrhosis. J Clin Endocrinol Metab 1988; 67:639. 19. Joseph DR. Structure, function, and regulation of androgen-binding pro¬ tein/sex hormone-binding globulin. Vitam Horm 1994; 49:197. 20. Wheeler MJ. The determination of bioavailable testosterone. Ann Clin Bio¬ chem 1995; 32:345. 21. Masters AM, Hahnel R. Investigation of sex-hormone binding globulin interference in direct radioimmunoassays for testosterone and estradiol. Clin Chem 1989; 35:979. 22. Wheeler MJ, D'Souza A, Matadeen J, et al. Ciba Coming ACS:180 testoster¬ one assay evaluated. Clin Chem 1996; 42:1445. 23. Sodergard R, Backstrom T, Shanbhag V, Carstensen H. Calculation of free and bound fractions of testosterone and estradiol 17-fS to human plasma proteins at body temperature. J Steroid Biochem 1982; 16:810. 24. Kapoor P, Luttrell BM, Williams D. The free androgen index is not valid for adult males. J Steroid Biochem 1993; 45:325. 24a. Rosner W. Errors in the measurement of plasma free testosterone. J Clin Endrocrinol Metab 1997; 82:2014. 25. Winters SJ, Kelley DE, Goodpaster B. The analog free testosterone assay: are the results in men clinically useful? Clin Chem 1998; 44:2178. 26. Manni A, Pardridge WM, Cefalu W, et al. Bioavailability of albumin-bound testosterone. J Clin Endocrinol Metab 1985; 61:705. 27. Vermeulen A, Kaufman JM, Giagulli. Influence of some biological indexes on sex hormone-binding globulin and androgen levels in aging or obese males. J Clin Endocrinol Metab 1996; 81:1821. 28. Cox C, Caulier C, Havelange G, et al. Two-sites immunoradiometric assay using monoclonal antibodies for the determination of serum human sex hormone binding globulin. J Immunoassay 1992; 13:355. 29. Nisula BC, Loriaux DL, Wilson YA. Solid phase method for measurement of the binding capacity of testosterone-estradiol binding globulin in human serum. Steroids 1979; 31:681. 30. Pugeat M, Crave JC, Toumaire J, Forest MG. Clinical utility of sex hor¬ mone-binding globulin measurement. Horm Res 1996; 45:148. 31. Chin WW, Boime I, eds. Glycoprotein hormones. Norwell, MA: Serono Symposia, 1990. 32. Shupnik MA. Gonadotropin gene modulation by steroids and gonadotro¬ pin-releasing hormone. Biol Reprod 1996; 54:279. 33. Nankin HR, Troen P. Repetitive luteinizing hormone elevations in serum in normal men. J Clin Endocrinol Metab 1971; 33:558. 34. Veldhuis JD, King JC, Urban RJ, et al. Operating characteristics of the male hypothalamo-pituitary-gonadal axis: pulsatile release of testosterone and follicle-stimulating hormone and their temporal coupling with luteinizing hormone. J Clin Endocrinol Metab 1987; 65:929. 35. Haavisto A-M, Pettersson K, Bergendahl M, et al. Occurrence and biologi¬ cal properties of a common genetic variant of luteinizing hormone. J Clin Endocrinol Metab 1995; 80:1257. 36. Wu FCW, Butler GE, Kelnar CJH, et al. Patterns of pulsatile luteinizing hor¬ mone and follicle-stimulating hormone secretion in prepubertal (midchildhood) boys and girls and patients with idiopathic hypogonadotropic hypogonadism (Kallmann's syndrome): a study using an ultrasensitive timeresolved immunofluorometric assay. J Clin Endocrinol Metab 1991; 72:1229. 37. Odink RJ, Schoemaker J, Schoute E, et al. Predictive value of serum folliclestimulating hormone levels in the differentiation between hypogonadotro¬ pic hypogonadism and constitutional delay of puberty. Horm Res 1998; 49:279. 38. Anawalt BD, Bebb RA, Matsumoto AM, et al. Serum inhibin B levels reflect Sertoli cell function in normal men and men with testicular dysfunction. J Clin Endocrinol Metab 1996; 81:3341. 39. Rosen SW, Weintraub BD. Monotropic increase of serum FSH correlated with low sperm count in young men with idiopathic oligospermia and aspermia. J Clin Endocrinol Metab 1972; 32:410. 40. Snyder PJ. Gonadotroph cell adenomas of the pituitary. Endocr Rev 1985; 6:552. 41. Vos P, Croughs RJM, Thijssen JHH, et al. Response of luteinizing hormone secreting pituitary adenoma to a long-acting somatostatin analog. Acta Endocrinol 1988; 118:587. 42. Ito T, Horton R. The source of plasma dihydrotestosterone in man. J Clin Invest 1971; 50:1621.

43. McConnell JD, Wilson JD, George FW, et al. Finasteride, an inhibitor of 5areductase, suppresses prostatic dihydrotestosterone in men with benign prostatic hyperplasia. J Clin Endocrinol Metab 1992; 74:505. 44. Thigpen AE, Silver RI, Guileyardo JM, et al. Tissue distribution and ontog¬ eny of steroid 5a-reductase isozyme expression. J Clin Invest 1993; 92:903. 45. Belanger A, Candas B, Dupont A, et al. Changes in serum concentrations of conjugated and unconjugated steroids in 40- to 80-year old men. J Clin Endocrinol Metab 1994; 79:1086. 46. Horton R. Dihydrotestosterone is a peripheral paracrine hormone. J Androl 1992; 13:23. 47. Lookingbill DP, Egan N, Santen RJ, Demers LM. Correlation of serum 3aandrostenediol glucuronide with acne and chest hair density in men. J Clin Endocrinol Metab 1988; 67:986. 48. Perryman RL, Thorner MO. The effects of hyperprolactinemia on sexual and reproductive function in men. J Androl 1981; 2:233. 49. Spitz IM, LeRoith D, Livshin J, et al. Exaggerated prolactin response to TRH and metoclopramide in primary testicular failure. Fertil Steril 1980; 34:573. 50. Winters SJ, Johnsonbaugh RE, Sherins RJ. The response of prolactin to chlorpromazine stimulation in men with hypogonadotropic hypogo¬ nadism and early pubertal boys: relationship to sex steroid exposure. Clin Endocrinol (Oxf) 1982; 10:321. 51. Sigman M, Jarow JP. Endocrine evaluation of infertile men. Urology 1997; 50:659. 52. Nagel SC, vom Saal FS, Welshons WV. The effective free fraction of estra¬ diol and xenoestrogens in human serum measured by whole cell uptake assays: physiology of delivery modifies estrogenic activity. Proc Soc Exp Biol Med 1998; 217:300. 53. Khosla S, Melton LJ, Atkinson EJ, et al. Relation of serum sex steroid levels and bone turnover markers with bone mineral density in men and women: a key role for bioavailable estrogens. J Clin Endocrinol Metab 1998; 83:2266. 54. Stanik S, Dornfeld LP, Maxwell MH, et al. The effect of weight loss on reproductive hormones in obese men. J Clin Endocrinol Metab 1981; 53:828. 55. Schioler V, Thode J. Six direct radioimmunoassays of estradiol evaluated. Clin Chem 1988; 34:949. 56. Winters SJ, Troen P. Pulsatile secretion of immunoreactive a-subunit in man. J Clin Endocrinol Metab 1985; 60:344. 57. Winters SJ, Troen P. a-Subunit secretion in men with idiopathic hypogona¬ dotropic hypogonadism. J Clin Endocrinol Metab 1988; 66:338. 58. Pralong FP, Pavlou SN, Waldstreicher J, et al. Defective regulation of glyco¬ protein free a-subunit in males with isolated gonadotropin-releasing hor¬ mone deficiency—a clinical research center study. J Clin Endocrinol Metab 1995; 80:3682. 59. Kourides IA, Weintraub BD, Ridgway EC, Maloof F. Pituitary secretion of free alpha and beta subunit of human thyrotropin in patients with thyroid disorders. J Clin Endocrinol Metab 1975; 40:872. 60. Blackman MR, Weintraub BD, Kourides IA, et al. Discordant elevation of the common a-subunit of the glycoprotein hormones compared to (3subunits in serum of uremic patients. J Clin Endocrinol Metab 1981; 53:39. 61. Somjen D, Tordjman K, Kohen F, et al. Combined beta FSH and beta LH response to TRH in patients with clinically non-functioning pituitary ade¬ nomas. Clin Endocrinol 1997; 46:555. 62. Fein HG, Rosen SW, Weintraub BD. Increased glycosylation of serum human chorionic gonadotropin and subunits from eutopic and ectopic sources: comparison with placental and urinary forms. J Clin Endocrinol Metab 1980; 50:1111. 63. Sailer B, Clara R, Spottl G, et al. Testicular cancer secretes intact human choriogonadotropin (hCG) and its free (3-subunit: evidence that hCG (+hCG-(3) assays are the most reliable in diagnosis and follow-up. Clin Chem 1990; 36:234. 64. Burger HG. Inhibin in the male: progress at last. Endocrinology 1997; 138:1361. 65. Hayes FJ, Hall JE, Boepple PA, Crowley WF Jr. Differential control of gona¬ dotropin secretion in the human: endocrine role of inhibin. J Clin Endo¬ crinol Metab 1998; 83:1835. 65a. Anderson RA, Sharpe RM. Regulation of inhibin production in the human male and its clinical applications. Int J Androl 2000; 23:136. 66. Illingworth PJ, Groome NP, Byrd W, et al. Inhibin-B: a likely candidate for the physiologically important form of inhibin in men. J Clin Endocrinol Metab 1996; 81:1321. 67. Robertson DM, Cahir N, Findlay JK, et al. The biological and immunologi¬ cal characterization of inhibin A and B forms in human follicular fluid and plasma. J Clin Endocrinol Metab 1997; 82:889. 68. Groome NP, Illingworth PJ, O'Brien M, et al. Quantification of inhibin proaC-containing forms in human serum by a new ultrasensitive two-site enzyme-linked immunosorbent assay. J Clin Endocrinol Metab 1995; 80:2926. 69. Andersson AM, Toppari J, Haavisto AM, et al. Longitudinal reproductive hormone profiles in infants: peak of inhibin B levels in infant boys exceeds levels in adult men. J Clin Endocrinol Metab 1998; 83:675. 70. Nachtigall LB, Boepple PA, Seminara SB, et al. Inhibin B secretion in males with gonadotropin releasing hormone (GnRH) deficiency before and dur¬ ing long-term GnRH replacement: relationship to spontaneous puberty, testicular volume, and prior treatment—a clinical research center study. J Clin Endocrinol Metab 1996; 81:3520.

Ch. 115: Male Hypogonadism 71. Wallace EM, Groome NP, Riley SC, et al. Effects of chemotherapy-induced testicular damage on inhibin, gonadotropin and testosterone secretion: a prospective longitudinal study. J Clin Endocrinol Metab 1997; 82:3111. 72. Petersen P, Andersson A-M, Rorth M, et al. Undetectable inhibin B serum levels in men after testicular irradiation. J Clin Endocrinol Metab 1999; 84:213. 73. Pierik FH, Vreeburg JTM, Stijnen T, et al. Serum inhibin B as a marker of spermatogenesis. J Clin Endocrinol Metab 1998; 83:3110. 74. Petersen PM, Skakkebaek NE, Vistisen K, et al. Semen quality and repro¬ ductive hormones before orchiectomy in men with testicular cancer. J Clin Oncol 1999; 17:941. 74a. Hiort O, Holterhus PM. The molecular basis of male sexual differentiation. Eur J Endocrinol 2000; 142:101. 75. Lee MM, Donahoe PK, Silverman BL, et al. Measurements of serum miillerian inhibiting substance in the evaluation of children with nonpalpable gonads. N Engl J Med 1997; 336:1480. 76. Pierce JG, Parsons TF. Glycoprotein hormones: structure and function. Annu Rev Biochem 1982; 50:465. 77. van Damme M-P, Robertson DM, Diczfalusy E. An improved in vitro bio¬ assay method for measuring luteinizing hormone (LH) activity using mouse Leydig cell preparations. Acta Endocrinol 1974; 77:655. 78. Dahl KD, Stone MP. FSH isoforms, radioimmunoassays, bioassays, and their significance. J Androl 1992; 13:11. 79. Jaakkola T, Ding Y-Q, Kellokumpu-Lehtinen P, et al. The ratios of serum bioactive/immunoreactive luteinizing hormone and follicle-stimulating hormone in various clinical conditions with increased and decreased gona¬ dotropin secretion: reevaluation by a highly sensitive immunometric assay. J Clin Endocrinol Metab 1990; 70:1496. 80. Christin-Maitre S, Bouchard P. Bioassays of gonadotropins based on cloned receptors. Mol Cell Endocrinol 1996; 125:151. 81. Beato M, Truss M, Chavez S. Control of transcription by steroid hormones. Ann NY Acad Sci 1996; 784:93. 82. Deslypere JP, Young M, Wilson JD, McPhaul MJ. Testosterone and 5 alphadihydrotestosterone interact differently with the androgen receptor to enhance transcription of the MMTV-CAT reporter gene. Mol Cell Endo¬ crinol 1992; 88:15. 83. Quigley CA, de Beilis A, Marschke E, et al. Androgen receptor defects: his¬ torical, clinical and molecular perspectives. Endocr Rev 1995; 16:27. 84. Aiman J, Griffin JE. The frequency of androgen receptor deficiency in infer¬ tile men. J Clin Endocrinol Metab 1982; 54:725. 85. Wang Q, Ghadessy FJ, Yong EL. Analysis of the transactivation domain of the androgen receptor in patients with male infertility. Clin Genet 1998; 54:185. 86. MacLean HE, Wame GL, Zajac JD. Spinal and bulbar muscular atrophy: androgen receptor dysfunction caused by a trinucleotide repeat expansion. J Neurol Sci 1996; 135:149. 87. Tut TG, Ghadessy FJ, Trifiro MA, et al. Long polyglutamine tracts in the androgen receptor are associated with reduced trans-activation, impaired sperm production, and male infertility. J Clin Endocrinol Metab 1997; 82:3777. 88. Kantoff P, Giovannucci E, Brown M. The androgen receptor CAG repeat polymorphism and its relationship to prostate cancer. Biochim Biophys Acta 1998; 1378:C1. 89. Forest MG. Pattern of the response of testosterone and its precursors to human chorionic gonadotropin stimulation in relation to age in infants and children. J Clin Endocrinol Metab 1979; 49:132. 90. Aynsley-Green A, Zachmann M, Illig R, et al. Congenital bilateral anorchia in childhood: a clinical, endocrine and therapeutic evaluation of twentyone cases. Clin Endocrinol (Oxf) 1976; 5:381. 91. Cisek LJ, Peters CA, Atala A, et al. Current findings in diagnostic laparo¬ scopic evaluation of the nonpalpable testis. J Urol 1998; 160:1145. 92. Lee PA, Danish RK, Mazur T, Migeon CJ. Micropenis III. Primary hypogo¬ nadism, partial androgen insensitivity syndrome, and idiopathic disorders. Johns Hopkins Med J 1980; 147:175. 93. de Kretser DM, Burger HG, Hudson B, Keogh EJ. The hCG stimulation test in men with testicular disorders. Clin Endocrinol (Oxf) 1975; 4:591. 94. Vermeulen A. Decreased androgen levels and obesity in men. Ann Med 1996; 28:135. 95. Korenman SG, Morley JE, Mooradian AD, et al. Secondary hypogonadism in older men: its relation to impotence. J Clin Endocrinol Metab 1990; 71:963. 96. Tenover JS, Matsumoto AM, Plymate SR, Bremner WJ. The effects of aging in normal men on bioavailable testosterone and luteinizing hormone secre¬ tion: response to clomiphene citrate. J Clin Endocrinol Metab 1987; 65:1118. 97. Harman SM, Tsitouras PD, Costa PT, et al. Evaluation of pituitary gonado¬ tropic function in men: value of luteinizing hormone-releasing hormone response versus basal luteinizing hormone level for discrimination of diag¬ nosis. J Clin Endocrinol Metab 1982; 54:196. 98. Hudson RW. The endocrinology of varicoceles. Fertil Steril 1988; 49:199. 99. Ghai K, Cara JF, Rosenfield RL. Gonadotropin releasing hormone agonist (nafarelin) test to differentiate gonadotropin deficiency from constitution¬ ally delayed puberty in teen-age boys—a clinical research center study. J Clin Endocrinol Metab 1995; 80:2980. 100. World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical-mucus interaction, 3rd ed. Cam¬ bridge, England: Cambridge University Press, 1992.

1125

101. Zuckerman Z, Rodriguez-Rigau L, Smith KD, Steinberger E. Frequency distribution of sperm counts in fertile and infertile males. Fertil Steril 1977; 28:1310. 102. Bonde JPE, Ernst E, Jensen EK, et al. Relation between semen quality and fertility: a population-based study of 430 first-pregnancy planners. Lancet 1998; 352:1172. 103. Critser JK, Nodes EE. Bioassays of sperm function. Semin Reprod Endo¬ crinol 1993; 11:1. 103a. Carrell DT. Semen analysis at the turn of the century: an evaluation of potential uses of new sperm function assays. Arch Androl 2000; 44:65. 104. Adeghe JH. Male subfertility due to sperm antibodies: a clinical overview. Obstet Gynecol Surv 1993; 48:1. 105. Clarke GN, Bourne J, Baker HWG. Intracytoplasmic sperm injections for treating infertility associated with sperm immunity. Fertil Steril 1997; 68:112. 106. Fujisawa M, Nakano Y, Matsui T, et al. Chlamydia trachomatis detected by ligase chain reaction in the semen of asymptomatic patients without pyospermia or pyuria. Arch Androl 1999; 42:41. 107. Mann T, Lutwak-Mann C. Male reproductive function and semen. New York: Springer-Verlag, 1981. 108. Garcia Diez LC, Gonzalez Buitrago IM, Corrales IJ, et al. Hormone levels in serum and seminal plasma of men with different types of azoospermia. J Reprod Fertil 1983; 67:208. 109. Abou-Shakra FR, Ward NI, Everard DM. The role of trace elements in male infertility. Fertil Steril 1989; 52:307. 110. Matalliotakis I, Kirakou D, Fragouli I, et al. Interleukin-6 in seminal plasma of fertile and infertile men. Arch Androl 1998; 41:43. 111. Alexander BH, Checkoway H, Faustman EM, et al. Contrasting associa¬ tions of blood and semen lead concentrations with semen quality among lead smelter workers. Am J Industr Med 1998; 34:464. 112. Hovatta O, Venalainen ER, Kuusimaki L, et al. Aluminum, lead, and cad¬ mium concentrations in seminal plasma and spermatozoa, and semen quality in Finnish men. Hum Reprod 1998; 13:115. 113. Dajani YF, Kilani Z. Role of testicular fine needle aspiration in the diagno¬ sis of azoospermia. Int J Androl 1998; 21:295. 114. Kuligowska E, Baker CE, Oates RD. Male infertility: role of transrectal US in diagnosis and management. Radiology 1992; 185:353. 115. Belker AM, Sherins RJ, Dennison-Lagos L, et al. Percutaneous testicular sperm aspiration: a convenient and effective office procedure to retrieve sperm for in vitro fertilization with intracytoplasmic sperm injection. J Urol 1998; 160:2058. 116. Van Assche E, Bonduelle M, Toumaye H, et al. Cytogenetics of infertile men. Hum Reprod 1996; ll(Suppl 4):1. 117. Roberts KP. Y-chromosome deletions and male infertility. State of the art and clinical implications. J Androl 1998; 19:255. 118. Durieu I, Bey-Omar F, Rollet J, et al. Diagnostic criteria for cystic fibrosis in men with congenital absence of the vas deferens. Medicine 1995; 74:42.

CHAPTER

115

MALE HYPOGONADISM STEPHEN R. PLYMATE Male hypogonadism may be defined as a failure of the testes to produce testosterone, spermatozoa, or both (Table 115-1). This may be caused by a failure of the testes or of the anterior pitu¬ itary. Hypogonadism may also occur if a testicular product is unable to exert an effect, as in the androgen-resistance syn¬ dromes (Table 115-2).

CLINICAL CHARACTERISTICS OF HYPOGONADISM The clinical presentation of hypogonadism depends on whether the onset was in utero, prepubertal, or postpubertal. If hypogo¬ nadism is present because of a defect that occurred in utero, the individual will have ambiguous genitalia (see Chaps. 77 and 90). The clinical pictures of testicular androgen failure of prepu¬ bertal and postpubertal onset are presented in Table 115-2. Although the findings on physical examination may be nor¬ mal, a problem with seminiferous tubule function may manifest

1126

PART VIII: ENDOCRINOLOGY OF THE MALE

TABLE 115-1. Classification of Male Hypogonadism

TABLE 115-2. Manifestations of Testicular Androgen Failure

PRIMARY HYPOGONADISM

PREPUBERTAL TESTICULAR FAILURE

Klinefelter syndrome

Testes, 50 ng/mL. Com¬ puterized tomography of the sella is the current standard for demonstrating the tumor mass and should be performed in men with elevated serum prolactin levels. Although magnetic resonance imaging (MRI) of the sella may be useful, instru¬ ments of stinfectious complication of epididymitis or urethritis with subelinical spread up the vas and can also be due to congenital strictures anywhere along the outflow tract.11 This should be suspected in patients with azoospermia and can be documented bv a vasogram and a testicular biopsy confirming adequate spermatogenesis. Young syndrome is a rare condition, charac¬ terized by chronic sinopulmonary infections and obstructive azoospermia, in which thickened mucous secretions lead to blockage of the bronchioles and epididymis.69 Cystic fibrosis can be associated with congenital absence of the vas deferens and azoospermia or epididymal obstruction.70 1 lowever, vasectomy is the most common cause of obstructive azoospermia.

TABLE 118-5. Medications Associated with Impaired Testicular Function ANTIANDROGENS Spironolactone Cyproterone Cimetidine Flutamide ANDROGEN SYNTHESIS INHIBITORS Ketoconazole Leuprolide ANTINEOPLASTIC DRUGS Cyclophosphamide Cisplatin Melphalan Chlorambucil Nitrosoureas Busulfan Procarbazine ANTIHYPERTENSIVES/CARDIOVASCULAR AGENTS a-Methyldopa Digoxin Calcium-channel blockers Reserpine Amiodarone

IMMUNOLOGIC FACTORS

PSYCHOACTIVE AGENTS Tricyclic antidepressants

Immunologic factors have been associated with infertility.71,713 Sperm antibodies have been reported in infertile men and their partners, although the incidence is low.72 These also can be found in fertile couples, and the association with infertility has not been shown to be directly causative. However, spermspecific antigens can be present in semen and may be related to sperm agglutination or poor sperm motility despite normal sperm counts. Therapy with glucocorticoids has been used with some success, although well-controlled studies are not available ' However, these drugs can cause significant side effects as glucose intolerance, hypertension, fluid retention, myopathy, and aseptic necrosis of the hip, and should be used with caution (see Chap, 78). 1CS1 seems to offer the best thera¬ peutic approach in difficult cases.74

INFECTIONS Infections in the male reproductive tract can cause infertility (see Chap. 213).743 Clinically apparent infections of the testis, epididymis, or prostate can lead to reduced sperm count or motility l pididvmitis is considered secondary to urethritis or cystitis, and the ipsilateral testis usually is involved. Causative organisms typically are Neisseria yonorrhoeae and Chlamydia tra¬ chomatis in sexually active men younger than 35 years of age and colitorm bacteria in men older than 35 years of age.11 A variety of other organisms have been associated with epididvmitis, including Mycobacterium tuberculosis, Ureaplasma ureaU/ticum, herpes simplex virus, and mumps virus. Mumps orchitis virtually always occurs in postpubertal males in -33% of cases of mumps parotitis.12 Mumps orchitis can cause pro¬ gressive. severe, irreversible tubular damage and presents with soft testes. oligospermia, and elevated gonadotropins. Prostati¬ tis can take se\ eral forms: acute or chronic, bacterial or nonbacterial, s\ mptomatic or asymptomatic. The presence of excess leukocv tes in the semen suggests prostatitis (>1 leukocyte/100 sperm), ' I he physical examination often reveals a soft, tender, enlarged prostate gland. Culture is best obtained using a twoor four-glass urine collection with prostatic massage.76 The most common infectious organism is Escherichia coli. Ureaplasm infections have been implicated in infertility, and treatment w ith do\\ c\ cline improves sperm function or fertility, although a controlled studv did not show an effect.77

Amphetamines Narcotics Major tranquilizers Minor tranquilizers OTHER DRUGS Anabolic steroids Phenytoin Ethanol Penicillamine Isoniazid

DRUGS, TOXINS, AND RADIATION Several drugs, chemicals, and radiation can cause damage to the seminiferous epithelium (Table 118-5). Many of the anticancer drugs cause direct damage to the germ cells17 (see Chap. 226). Particularly toxic are the alkylating agents, including cyclophos¬ phamide, nitrosoureas, chlorambucil, melphalan, busulfan, and cisplatin, and other agents such as procarbazine, doxorubicin, and cytosine arabinoside. Antimetabolites, such as methotrexate, 5-fluorouracil, and 6-mercaptopurine, do not appear to affect tes¬ ticular function significantly. Alcohol can suppress Leydig cells, germ cells, and gonadotropin release by the pituitary, leading to a spectrum of hypogonadism13 (see Chaps. 205 and 233). Crude marijuana is a potential suppressant of testicular function, although available data are not definitive14 (see Chap. 234). Radi¬ ation at doses as low as 15 rad can transiently decrease sperm output, and doses >200 rad can produce prolonged azoosper¬ mia.1 ’ Although only a few drugs, such as sulfa drugs and cime¬ tidine, have been shown to cause testicular injury, the chronic use of any drug may be a factor in infertility.78,79

MANAGEMENT OF MALE INFERTILITY The primary management decision is whether there is an iden¬ tifiable and treatable cause for the infertility. The causes of tes¬ ticular dysfunction are listed in Table 118-6. The failure to identify a known factor leads to the unsatisfy¬ ing diagnosis of idiopathic infertility. This category probably

1179

Ch. 118: Male Infertility TABLE 118-6. Causes of Male Infertility USUALLY IRREVERSIBLE Chromosomal abnormality

T

20

Sperm 1Q (millions)

P.T.

Absent vas deferens Young syndrome Nonmotile sperm

Basal

Drug

Placebo

Mumps orchitis Cryptorchidism Drugs/toxins Epididymal dysfunction

POTENTIALLY REVERSIBLE Vas or epididymal occlusion

X

10 Sperm (millions)

Retrograde ejaculation

T n

^

Basal

Prostatitis, epididymitis

£ Drug

w.s.

Pi i 1Placebo 1 ft

Gonadotropin deficiency Varicocele Drugs/toxins Heat or irradiation Sexual dysfunction Immunologic dysfunction Systemic illness

includes various factors, not yet identified, that produce a sim¬ ilar clinical picture of low sperm count, poor sperm motility, and infertility. Numerous agents have been proposed for the treatment of idiopathic infertility. An evaluation of their efficacy is difficult because of the scarcity of well-conducted therapeutic trials that exclude spousal factors and improper coital techniques and include adequate basal evaluation with three to six semen sam¬ ples collected during the 4 to 6 months before therapy, plus an adequate number of semen samples during and after therapy to document any effect. The treatment period should be at least 3 months to span one germ-cell cycle. Moreover, a double-blind placebo control is necessary because spontaneous pregnancies may occur that are unrelated to treatment.80 A review using metaanalyses of randomized controlled trials concluded that there is no demonstrable efficacy of medical treatments for idio¬ pathic male infertility.81 Despite controversial efficacy, three hormonal approaches to therapy are often empirically employed: exogenous gonadotro¬ pins, antiestrogens, and aromatase inhibitors, which reduce conversion of androgens to estrogens.82-873 Gonadotropins have been used to provide an additional stimulus to the germinal epithelium and Leydig cells by increasing intratesticular tes¬ tosterone with hCG or by stimulating germ-cell maturation with hMG or rFSH. Some studies indicate a positive response to exogenous gonadotropins, but this has not been confirmed in several carefully studied series.82'83 Clomiphene is an antiestro¬ gen that increases gonadotropin release. Controlled studies with clomiphene at a dosage between 25 and 50 mg per day for 3 to 6 months have reported both negative and positive responses, and the present data must be regarded as discourag¬ ing.84'85 An aromatase inhibitor, testolactone, has been used, based on the possibility that excess estrogen may impair the germinal epithelium. An initial study without a control group reported success, but a subsequent study with placebo controls and patient crossover showed no effect on sperm count or fer¬ tility.86'87 Random, sustained increases in sperm count occur during both placebo and drug therapy (Fig. 118-3), illustrating the importance of controls in therapeutic trials. Therapies designed to isolate more functional sperm, such as sperm washing for intrauterine insemination or IVF, are proving to be more useful in treating male factor infertility.88 Microinjection of sperm into ooplasm, ICSI, is performed at a

Sperm (millions)

Sperm (millions)

FIGURE 118-3. Changes in mean sperm output over time. Mean total sperm output is plotted for 4-month intervals in individual patients on therapeutic protocol for oligospermic infertility. Means are based on the minimum of four samples from each treatment period: basal, drug, or placebo. Note that marked increases in output occurred while the patient was on either drug or placebo therapy. (Based on data from Clark RV, Sherins RJ. Use of semen analysis in the evaluation of the infertile couple. In: Santen RJ, Swerdloff RS, eds. Male reproductive dysfunction. New York: Marcel Dekker Inc, 1986:253.) number of centers and appears to be effective without increased risk of fetal abnormalities or loss.64-89'90'91 Such spe¬ cialized reproductive techniques currently offer the greatest chance for success in treating idiopathic male infertility.

REFERENCES 1. Forti G, Krausz C. Evaluation and treatment of the infertile couple. J Clin Endocrinol Metab 1998; 83:4177. 2. Franken DR, Acosta AA, Kruger TF, et al. The hemizona assay: its role in identi¬ fying male factor infertility in assisted reproduction. Fertil Steril 1993; 59:1075. 3. Barratt CLR, St. John JC. Diagnostic tools in male infertility. Hum Reprod 1998; 13(Suppl 1):51. 4. Clark RV, Sherins RJ. Use of semen analysis in the evaluation of the infer¬ tile couple. In: Santen RJ, Swerdloff RS, eds. Male reproductive dysfunc¬ tion. New York: Marcel Dekker Inc, 1986:253. 5. Bostofte E, Bagger P, Michael A, Stakemann G. Fertility prognosis for infertile men: results of follow-up study of semen analysis in infertile men from two populations evaluated by the Cox regression model. Fertil Steril 1990; 54:1100. 6. Collins JA, Burrows EA, Willan AR. Prognosis for live birth among untreated infertile couples. Fertil Steril 1995; 64:22 7. Clark RV. Clinical andrology: history and physical examination. Endo¬ crinol Metab Clin North Am 1994; 23:699. 8. Irvine DS. Epidemiology and aetiology of male infertility. Hum Reprod 1998; 13(Suppl 1):33.

1180

PART VIII: ENDOCRINOLOGY OF THE MALE

9. Whitehead ED, Leiter E. Genital abnormalities and abnormal semen analy¬ ses in male patients exposed to diethylstilbestrol in utero. J Urol 1981; 125:47, 10. Thomas AJ Jr. Ejaculatory dysfunction. Fertil Steril 1983; 39:445. 11. Berger RE, Elolmes KK. Infection and male infertility. In: Santen RJ, Swerdloff RS, eds. Male reproductive dysfunction. New York: Marcel Dekker Inc 1986:407. 12. Beard CM, Benson RC, Kelalis PP, et al. The incidence of mumps orchitis in Rochester, Minnesota, 1935 to 1974. Mayo Clin Proc 1977; 52:3. 13. Van Thiel DH, Lester R, Sherins RJ. Hypogonadism in liver disease: evi¬ dence for a double defect. Gastroenterology 1974; 67:1188. 14. Hembree WC, Zeidenberg P, Nahas G. Marihuana effects on human gonadal function. In: Nahas G, Poton WDM, Idanpaan-Heittila J, eds. Marihuana: chemistry, biochemistry, and cellular effects. New York: Springer-Verlag, 1976:521. 15. Clifton DK, Bremner WJ. The effect of testicular X-irradiation on spermato¬ genesis in man. J Androl 1983; 4:387. 16. Tas S, Lauwerys R, Lison D. Occupational hazards for the male reproduc¬ tive system. Crit Rev Toxicol 1996; 26:26. 17. Tielemans E, Burdorf A, te Velde ER, et al. Occupationally related exposures and reduced semen quality: a case control study. Fertil Steril 1999; 71:690. 17a. De Celis R, Feria-Kelasco A, Gonzalez-Unzaga M, et al. Semen quality of workers occupationally exposed to hydrocarbons. Fertil Steril 2000; 73:221. 18. Sherins RJ. Adverse effects of treatment: gonadal dysfunction. In: DeVita VT Jr, Heilman S, Rosenberg SA, eds. Cancer: principles and practice of oncology, 4th ed. Philadelphia: JB Lippincott Co, 1993:2395. 19. Winters JSD, Faiman C. Pituitary-gonadal relations in male children and adolescents. Pediatr Res 1972; 6:126. 20. Lenz S, Giwercman A, Elsborg A, et al. Ultrasonic testicular texture and size in 444 men from the general population: correlated to semen quality. Eur Urol 1993; 24:231. 21. World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction, 3rd ed. Cam¬ bridge, UK: Cambridge University Press, 1992. 22. Davis RO, Katz DF. Operational standards for computer-aided sperm anal¬ ysis instruments. J Androl 1993; 14:385. 23. Vantman D, Zinaman M, Koukoulis G, et al. Computer-assisted semen analysis: evaluation of method and assessment of the influence of sperm concentration on linear velocity determination. Fertil Steril 1988; 49:510. 24. Baker HWG, Burger HC, de Kretser DM, et al. Factors affecting the vari¬ ability of semen analysis results in infertile men. Int J Androl 1981; 4:609. 25. Tielemans E, Heederik D, Burdorf A. Intraindividual variability and redun¬ dancy of semen parameters. Epidemiology 1997; 8:99. 26. Tyler JPP, Crockett NG, Driscoll L. Studies of human seminal parameters with frequent ejaculation. I. Clinical characteristics. Clin Reprod Fertil 1982; 1:273. 27. Cooper TG, Keck C, Oberdieck U, Nieschlag E. Effects of multiple ejacula¬ tions after extended periods of sexual abstinence on total, motile, and nor¬ mal sperm numbers from healthy normal and oligospermic men. Hum Reprod 1993; 8:1251. 28. Sherins RJ, Brightwell D, Steruthal PM. Longitudinal analysis of semen of fertile and infertile men. In: Troen P, Nankin HR, eds. The testis in normal and infertile men. New York: Raven Press, 1977:473. 29. Barfield A, Melo J, Coutinho E, et al. Pregnancies associated with sperm concentrations below 10 million/mL in clinical studies of a potential male contraceptive method, monthly depot medroxyprogesterone acetate and testosterone esters. Contraception 1979; 20:121. 30. Burris AS, Clark RV, Vantman DJ, et al. A low sperm concentration does not preclude fertility in men with isolated hypogonadotropic hypogo¬ nadism after gonadotropin therapy. Fertil Steril 1988; 50:343. 31. Ombelet W, Bosmans E, Janssen M, et al. Semen parameters in a fertile ver¬ sus subfertile population: a need for change in the interpretation of semen testing. Hum Reprod 1997; 12:987. 32. Burris AS, Rodbard HW, Winters SJ, Sherins, RJ. Gonadotropin therapy in men with isolated hypogonadotropic hypogonadism: the response to human chorionic gonadotropin is predicted by initial testicular size. J Clin Endocrinol Metab 1988; 66:1144. 33. Anawalt BD, Bebb RA, Matsumoto AM, et al. Serum inhibin B levels reflect Sertoli cell function in normal men and men with testicular dysfunction. J Clin Endocrinol Metab 1996; 81:3341. 34. Bouchard P, Lagoguey M, Brailly S, Schaisan G. Gonadotropin-releasing hormone pulsatile administration restores luteinizing hormone pulsatility and normal testosterone levels in males with hyperprolactinemia. J Clin Endocrinol Metab 1985; 60:258. 35. Silber SJ, Rodriguez-Rigau LJ. Quantitative analysis of testicle biopsy: determination of partial obstruction and prediction of sperm count after surgery for obstruction. Fertil Steril 1981; 36:480. 36. Toumaye H, Liu J, Nagy PZ, et al. Correlation between testicular histology and outcome after intracytoplasmic sperm injection using testicular sper¬ matozoa. Hum Reprod 1997; 11:127. 37. Whitcomb RW, Crowley WF. Diagnosis and treatment of isolated gonado¬ tropin-releasing hormone deficiency in men. J Clin Endocrinol Metab 1990; 70:3. 38. Nachtigall LB, Boepple PA, Pralong FP, Crowley WF. Adult-onset idio¬ pathic hypogonadotropic hypogonadism, a treatable form of male infertil¬ ity. N Engl J Med 1997; 336:410. 39. Simoni M, Gromoll J, Hoppner W, et al. Mutational analysis of FSH recep¬ tor in normal and infertile men: identification and characterization of two discrete FSH receptor isoforms. J Clin Endocrinol Metab 1999; 84:751.

40. Aiman J, Griffin JE. The frequency of androgen receptor deficiency in infer¬ tile men. J Clin Endocrinol Metab 1982; 54:725. 41. Eil C, Gamblin GT, Hodge JW, et al. Whole cell and nuclear androgen uptake in skin fibroblasts from infertile men. J Androl 1985; 6:365. 42. Tut TG, Ghadessy FJ, Trifiro MA, et al. Long polyglutamine tracts in the andro¬ gen receptor are associated with reduced trans-activation, impaired sperm productions, and male infertility. J Clin Endocrinol Metab 1997; 82:3777. 43. Wang Q, Ghadessy FJrYong EL. Analysis of the trans-activation domain of the androgen receptor in patients with male infertility. Clin Genet 1998; 54:185. 44. Kidd GS, Glass AR, Vigersky RA. The hypothalamic-pituitary testicular axis in thyrotoxicosis. J Clin Endocrinol Metab 1979; 48:798. 44a. Donnelly P, White C. Testicular dysfunction in men with primary hypothy¬ roidism; reversal of hypogonadotropic hypogonadism with replacement thyroxine. Clin Endocrinol 2000; 52:197. 45. Luton JP, Thieblot P, Valcke JC, et al. Reversible gonadotropin deficiency in male Cushings disease. J Clin Endocrinol Metab 1977; 45:488. 46. Paulsen CA, Gorden DL, Carpenter RW, et al. Klinefelters syndrome and its variants: a hormonal and chromosomal study. Recent Prog Horm Res 1968; 24:321. 47. Wang C, Baker HWG, Burger HW, et al. Hormonal studies in Klinefelters syndrome. Clin Endocrinol (Oxf) 1975; 4:399. 48. Santen RJ, de Kretser DM, Paulsen CA, Vorhees J. Gonadotropins and tes¬ tosterone in the XYY syndrome. Lancet 1970; 2:371. 49. Skakkebaek NE, Zeuthen E, Nielsen J, Yde H. Abnormal spermatogenesis in XYY males: a report on four cases ascertained through a population study. Fertil Steril 1973; 24:390. 50. Hasen J, Boyar RM, Shapiro LR. Gonadal function in trisomy 21. Horm Res 1980; 12:345. 51. Afzelius BA. A human syndrome caused by immotile cilia. Science 1976' 193:317. 52. Gagnon C, Sherins RJ, Phillips DM, Bardin CW. Deficiency of protein car¬ boxyl methylase in immotile spermatozoa in infertile men. N Engl J Med 1982; 306:821. 53. Najmabadi H, Huang V, Yen P, et al. Substantial prevalence of microdele¬ tions of the Y-chromosome in infertile men with idiopathic azoospermia and oligozoospermia detected using a sequence-tagged site-based map¬ ping strategy. J Clin Endocrinol Metab 1996; 81:1347. 54. Pryor JL, Kent-First M, Muallem A, et al. Microdeletions in the Y chromo¬ some of infertile men. N Engl J Med 1997; 336:534. 55. Kim ED, Bischoff FZ, Lipshultz LI, Lamb DJ. Genetic concerns for the subfertile male in the era of ICSI. Prenat Diagn 1998; 18:1349. 56. Lipshultz LI, Caminos-Torres R, Greenspan C. Testicular function after uni¬ lateral orchiopexy. N Engl J Med 1976; 295:15. 57. Alpert PF, Klein RS. Spermatogenesis in the unilateral cryptorchid testis after orchiopexy. J Urol 1983; 129:301. 58. Gorelick JI, Goldstein M. Loss of fertility in men with varicocele Fertil Steril 1993; 59:613. 59. Fariss BL, Fenner DK, Plymate SR, et al. Seminal characteristics in the pres¬ ence of a varicocele as compared with those of expectant fathers and pre¬ vasectomy men. Fertil Steril 1981; 35:325. 60. Nagao RR, Plymate SR, Berger RE, et al. Comparison of gonadal function between fertile and infertile men with varicoceles. Fertil Steril 1986; 46:930. 61. Hudson RW. The endocrinology of varicoceles. Fertil Steril 1988; 49:199. 62. Rodriguez-Rigau LJ, Smith KD, Steinberger E. Varicocele and the morphol¬ ogy of spermatozoa. Fertil Steril 1981; 35:54. 63. Nieschlag E, Hertle L, Fischedick A, Behre HM. Treatment of varicocele: counsel¬ ling as effective as occlusion of the vena spermatica. Hum Reprod 1995; 10:347. 64. Dubin L, Amelar RD. Varicocelectomy: 986 cases in a twelve-year study. Urology 1977; 10:446. 65. Madgar I, Weissenberg R, Lunenfield B, et al. Controlled trial of high sper¬ matic vein ligation for varicocele in infertile men. Fertil Steril 1995; 63:120. 66. Proctor KG, Howards SS. The effect of sympathomimetic drugs on postlymphadenectomy aspermia. J Urol 1983; 129:837. 67. Urry RL, Middleton RG, McGavin S. A simple and effective technique for increasing pregnancy rates in couples with retrograde ejaculation. Fertil Steril 1986; 46:1124. 68. Scammell GE, Stedronska-Clark J, Edmonds DK, Hendry WF. Retrograde ejaculation: successful treatment with artificial insemination. Br J Obstet Gynaecol 1989; 63:198. 69. Handelsman DJ, Conway AJ, Boylan LM, Turtle JR. Young's syndrome: obstructive azoospermia and chronic sinopulmonarv infections. N Engl J Med 1984; 310:3. 70. van der Ven K, Messer L, van der Ven H, et al. Cystic fibrosis mutation screen¬ ing in healthy men with reduced sperm quality. Hum Reprod 1996; 11:513. 71. Marshburn PB, Kutteh WH. The role of antisperm antibodies in infertility Fertil Steril 1994; 61:799. 71a. Dickman AB, Norton EJ, Westbrook VA, et al. Anti-sperm antibodies from infertile patients and their cognate sperm antigens: a review. Am J Reprod Immunol 2000; 43:134. 72. Clarke GN, Eliott PJ, Smaila C. Detection of sperm antibodies in semen using the immunobead test: a survey of 813 consecutive patients. Am J Reprod Immunol Microbiol 1985; 7:118. 73. Bals-Pratsch M, Doren M, Karbowski B, et al. Cyclic corticosteroid immu¬ nosuppression is unsuccessful in the treatment of sperm-antibody-related male infertility: a controlled study. Hum Reprod 1992; 7:99. 74. Clarke GN, Bourne H, Baker HW. Intracytoplasmic sperm injection for treat¬ ing infertility associated with sperm autoimmunity. Fertil Steril 1997; 68:112.

Ch. 119: Clinical Use and Abuse of Androgens and Antiandrogens 74a. Diemer T, Ludwig M, Huwe P, et al. Influence of urogenital infection on sperm function. Curr Opin Urol 2000; 10:39. 75. Branigan EF, Muller CH. Efficacy of treatment and recurrence rate of leukocytospermia in infertile men with prostatitis. Fertil Steril 1994; 62:580. 76. Berger RE, Karp LE, Williamson RA, et al. The relationship of pyospermia and seminal fluid bacteriology to sperm function as reflected in the sperm penetration assay. Fertil Steril 1982; 37:557. 77. Toth A, Lesser ML, Brooks C, Labriola D. Subsequent pregnancies among 161 couples treated for T-mycoplasma genital tract infection. N Engl J Med 1983; 308:505. 78. Toth A. Reversible toxic effect of salicylazosulfapyridine on semen quality. Fertil Steril 1979; 31:538. 79. Van Thiel DH, Gavaler JS, Smith WJ. Hypothalamic-pituitary-gonadal dys¬ function in men using cimetidine. N Engl J Med 1979; 300:1012. 80. Collins JA, Wrixon W, Janes LB, Wilson EH. Treatment-independent preg¬ nancy among infertile couples. N Engl J Med 1983; 309:1201. 81. O'Donovan PA, Vandekerchove P, Lilford RJ, Hughes E. Treatment of male infertility: is it effective? Review and meta-analyses of published random¬ ized controlled trials. Hum Reprod 1993; 8:1209. 82. Schill WB, Jungst D, Unterburger P, Braun S. Combined hMG/hCG treat¬ ment in subfertile men with idiopathic normagonadotropic oligospermia. IntJAndrol 1982; 5:467. 83. Knuth UA, Honigl W, Bals-Pratsch M, et al. Treatment of severe oligospermia with human chorionic gonadotropin/human menopausal gonadotropin: a placebo-controlled, double blind trial. J Clin Endocrinol Metab 1987; 65:1081. 84. Newton R, Schinfeld JS, Schiff I. Clomiphene treatment of infertile men: failure of response with idiopathic oligospermia. Fertil Steril 1980; 34:399. 85. Sokol RZ, Steiner B, Bastillo M, et al. Controlled comparison of the efficacy of clomiphene citrate in male infertility. Fertil Steril 1988; 49:865. 86. Vigersky RA, Glass AR. Effects of testolactone on the pituitary-testicular axis in oligospermic men. J Clin Endocrinol Metab 1981; 52:897. 87. Clark RV, Sherins RJ. Treatment of men with idiopathic oligospermic infertil¬ ity using the aromatase inhibitor, testolactone: results of a double blinded, randomized placebo controlled trial with crossover. J Androl 1989; 10:240. 87a. Vandekerekhove P, Lilford R, Vail A, Hughes E. Clomiphene or tamoxifen for idiopathic oligolashenospermia. Cochrane Database Syst Rev 2000; 2:CD000151. 88. Schlegel PN, Girardi SK. In vitro fertilization for male factor infertility. J Clin Endocrinol Metab 1997; 82:709. 89. Palermo G, Joris H, Devroey P, Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992; 340; 17. 90. Nagy Z, Liu J, Verheyen G, et al. The results of intracytoplasmic sperm injection are not related to any of the three basic sperm parameters. Hum Reprod 1995; 10:1123. 91. Givens CR. Intracytoplasmic sperm injection: what are the risks? Obstet Gynecol Surv 2000; 55;58. 92. Tarlatzis BC, Bili H. Intracytoplasmic sperm injection. Survey of world results. Ann N Y Acad Sci 2000; 900:336.

CHAPTER

119

CLINICAL USE AND ABUSE OF ANDROGENS AND ANTIANDROGENS ALVIN M. MATSUMOTO

ANDROGENS: PAST AND PRESENT The popular belief that failure of testicular function was respon¬ sible for symptoms of old age in men stimulated early attempts to isolate an active testicular substance that would rejuvenate aging men. In 1889, Brown-Sequard reported that an extract he prepared from dog and guinea pig testes and administered to himself resulted in increased vigor, strength, intellectual capacity, and sexual potency.1 Because of his stature in the scientific com¬ munity, many people in the public and medical profession began using testicular extracts, but others castigated Brown-Sequard. Although his aqueous extract probably was devoid of steroid hormone and bioactivity, the controversy concerning his "elixir of life" stimulated further studies of internal secretions of glands, from which evolved the science of modem endocrinology. Soon after the discovery of androgenic substances in the urine, urinary extracts were reported to increase the frequency

1181

of erections and improve well-being in hypogonadal men. Because of their limited availability and weak activity, however, urinary androgens were not widely used. In the mid-1930s, tes¬ tosterone was isolated and identified as the major androgenic principle of the testes. Soon thereafter, the chemical structure of testosterone was elucidated, and the hormone was synthesized and came to be used clinically in the treatment of male hypogo¬ nadal states. Because of the short duration of action of testoste¬ rone, numerous analogs and derivatives of testosterone with greatly prolonged action were developed. Today, the principal clinical use of androgens remains the treatment of androgen deficiency resulting from hypogonadism and delayed puberty.2-4 Androgens are also used to stimulate erythropoiesis in hypoproliferative anemias related to renal or bone marrow failure; to treat micropenis and microphallus (see Chap. 93); to treat hereditary angioneurotic edema; as adjuvant hormonal therapy for female breast cancer (see Chap. 224); top¬ ically to treat vulvar lichen sclerosus; and to stimulate bone for¬ mation in the treatment of osteoporosis.2^1 Preliminary studies have suggested a number of potential uses of androgens for treatment of clinical syndromes associated with wasting syndromes (e.g., acquired immunodeficiency syn¬ drome [AIDS] and cancer); chronic illnesses, such as renal failure and chronic obstructive pulmonary disease (COPD); long-term use of certain medications (e.g., glucocorticoid therapy); autoim¬ mune rheumatologic diseases (e.g., rheumatoid arthritis); and aging. Evidence is also emerging that androgens may be useful in treating reduced libido and in increasing bone mass in post¬ menopausal women. Finally, interest is renewed in investigating the use of androgens to promote protein anabolism in catabolic states (e.g., after major trauma or surgery, bums, space travel, and spinal cord injury). Although the short- and long-term bene¬ fits and risks of androgen therapy in these conditions are not fully established, the potential for expanded clinical uses of androgens has stimulated interest in the development of new androgens and alternative androgen formulations, and has once again sparked both public and physician enthusiasm for the use of testosterone as an agent to prevent frailty in aging men. Although androgens have been used to treat children with short stature, their value for this purpose is dubious, and such a use is inappropriate. Extremely high dosages of androgenic, ana¬ bolic steroids are increasingly consumed by athletes to improve performance and by adolescents to enhance appearance. Whether androgens improve athletic performance is uncertain, and the high dosages are often associated with severe side effects. Despite condemnation by the medical community and by athletic organizations, this form of androgen abuse remains widespread. The potential for androgen abuse has resulted in reclassification of androgen and androgenic anabolic steroid preparations as Schedule III controlled substances by the U.S. Food and Drug Administration (FDA). With the enactment of the Dietary Supplement Health and Education Act of 1994, however, preparations containing the androgens dehydroepiandrosterone (DHEA) and A4-androstenedione (androstenedione), both of which may be converted to testosterone in the body, became available over the counter as "dietary supplements" free of FDA regulation. Despite the lack of evidence for their benefits or risks, DHEA and androstenedione are being abused by athletes to enhance strength and performance, and by men and women in the hope of "preventing the aging process." Antiandrogens are compounds that bind to the androgen receptor and competitively inhibit androgen binding, thereby antagonizing androgen action at target organs. Clinically, they are useful in the treatment of androgen-dependent malignan¬ cies (e.g., prostate and male breast cancer) and conditions such as hirsutism and acne (see Chaps. 101 and 225).

PHARMACOLOGY OF ANDROGEN PREPARATIONS Because of the short duration of testosterone action, pharmaco¬ logic strategies have been developed to achieve more sustained

1182

PART VIII: ENDOCRINOLOGY OF THE MALE

blood levels of testosterone and thereby prolong its androgenic action. The development of novel methods of testosterone admin¬ istration and chemically modified analogs of the native testoste¬ rone molecule are the two major pharmacologic strategies used to prolong androgen action.2 In general, the chemical modifications of testosterone involve esterification of the 17(3-hydroxyl group or alkylation of the 17a-position of the D ring, with or without other modifications of the ring structure of native testosterone (Table 119-1). Mesterolone and dihydrotestosterone (DHT) are androgen preparations that have A-ring modifications without any alter¬ ations at the 17-position of the D ring (see Table 119-1).

TESTOSTERONE Orally administered testosterone is rapidly absorbed from the gastrointestinal tract into the portal blood and accumulated in the liver. Because it is efficiently degraded by the liver, very lit¬ tle testosterone reaches the systemic circulation, and sustained blood levels are very difficult to maintain. No oral forms of unmodified testosterone are available in the United States. A microparticulate form of testosterone has been administered to a small number of hypogonadal men in Europe and has been shown to achieve therapeutic blood levels of testosterone in some patients.5 Absorption of this preparation is erratic, how¬ ever, and very large dosages (200-400 mg per day) taken sev¬ eral times a day are required to maintain adequate serum testosterone levels. Furthermore, the long-term toxicity to the liver of this large burden of testosterone is unknown. Intramus¬ cular injections of aqueous native testosterone are also rapidly absorbed from the injection site and promptly degraded by the liver. Therefore, unmodified testosterone administered either orally or parenterally is impractical for achieving sustained physiologic testosterone levels in blood and androgenic effects on target organs. 7RANSDERMAL TESTOSTERONE DELIVERY Currently, three transdermal testosterone patches are approved for use in androgen replacement therapy for hypogonadal men (see Table 119-1): a scrotal matrix patch (Testoderm), the first to be marketed; a nonscrotal, permeation-enhanced, reservoir patch (.Androderm or Andropatch [United Kingdom]); and a nonscrotal reservoir patch that does not contain permeation enhancers (Testoderm TTS).6 Because a normal adult man pro¬ duces ~7 mg per day of testosterone (i.e., up to 100-fold the daily production of estradiol), testosterone patches are larger and require more frequent (daily) application than do estrogen patches. The scrotal Testoderm patch consists of an ethylene-vinyl acetate copolymer containing testosterone within this matrix.7-8 When applied to scrotal skin, the 40 cm2 or 60 cm2 patches deliver 4 or 6 mg of testosterone over 24 hours, respectively. The unusual superficial vascularity of scrotal skin permits 5- to 40-fold greater testosterone absorption from this site than from other skin sites. Because of initial problems with adhesion of these patches to scrotal skin, thin, lightly adhesive strips were incorporated onto the patch. Daily application in the morning on the scrotum of hypogonadal men produces physiologic lev¬ els of testosterone that mimic the normal circadian rhythm of testosterone in young men, with peak testosterone levels reached within 2 to 4 hours. Because these patches do not con¬ tain permeation enhancers, they are generally well tolerated, with only occasional itching (~7%) and moderate skin irritation (-5%). The use of this patch, however, does require an adequatesize scrotum that is clean, dry, and shaven for optimal adhe¬ sion and effectiveness; this limits its acceptability and clinical utility. Also, supraphysiologic concentrations of DHT are pro¬ duced, as a result of the high 5a-reductase activity in scrotal skin. The physiologic significance of high circulating DHT lev¬ els on androgen-responsive target organs (e.g., prostate) is not

known, but careful monitoring for adverse androgenic effects is recommended. The nonscrotal Androderm transdermal system consists of an adhesive patch with a central reservoir containing testoste¬ rone and permeation enhancers in an alcohol-based gel that are delivered to skin through a microporous polyethylene mem¬ brane.9-11 Permeation -enhancers facilitate testosterone absorp¬ tion through nonscrotal sites (e.g., back, abdomen, thighs, or upper arm). In hypogonadal men, nightly application of a single 5 mg (44 cm2) or two 2.5 mg (37 cm2) patches on nonscrotal skin also maintains serum testosterone concentrations in the normal physiologic range with a circadian rhythm similar to that of young men. The Androderm patch causes more local skin irrita¬ tion (-30% of users) than do scrotal Testoderm patches (~5%).12 Unlike scrotal patches, they may cause allergic contact dermati¬ tis (-12%) and, uncommonly, a burn-like blister reaction, which is usually associated with placement of the patch over bony prominences. The severity and incidence of skin irritation is reduced by coapplication of triamcinolone acetonide (0.1%) cream under the drug reservoir. However, skin irritation limits the acceptability and use of this nonscrotal patch. Unlike with the scrotal testosterone patch, serum DHT levels remain within the physiologic range during Androderm use. Another nonscrotal transdermal testosterone delivery sys¬ tem, the Testoderm TTS patch, is available for androgen replacement therapy.13-15 It is composed of a relatively large (72 cm2) lightly adhesive patch with a reservoir (60 cm2 contact area) containing testosterone in an alcohol-based gel that does not contain permeation enhancers. Nightly application of the Testoderm TTS patch on nonscrotal skin (arms, upper buttocks, or torso) in hypogonadal men results in physiologic serum tes¬ tosterone and DHT levels with pharmacokinetics similar to that of the Androderm patch. Early clinical experience in hypogo¬ nadal men suggests that this patch is associated with less skin irritation but is also less adherent to skin than the permeationenhanced Androderm patch. Currently available testosterone patches are also being tested for treatment of other patient populations (e.g., adoles¬ cent boys with hypogonadism and men and women with mus¬ cle wasting associated with AIDS).16-18 Because of problems with skin adherence and irritation associated with available transdermal testosterone patches, other matrix and reservoir patches are being developed and tested for androgen therapy (e.g., in hypogonadal men and postmenopausal women).19-20 A transdermal 1% hydroalcoholic gel preparation of tesosterone (AndroGel) has been approved and become available for androgen replacement therapy in hypogonadal men. In initial studies in hypogonadal men, the application of 50 to 100 mg per day of AndroGel divided over four different sites increased serum testosterone levels and maintained them in the physio¬ logic range for 180 days.21 Unlike testosterone creams or oint¬ ments, the testosterone gel dried rapidly, left no residue and, more importantly, did not cause skin irritation. A caution with testosterone gel is the transfer of androgens and induction of androgenic effects in female and childhood contacts. AndroGel also produces a disproportionate increase in serum DHT levels. Long-term studies have demonstrated continued efficacy (improved sexual function, mood, body composition, and bone mineral density) and safety without significant skin irritation.21a-21b AndroGel offers an easy, acceptable, rapidly reversible, and titratable method for delivering androgen therapy for a vari¬ ety of disorders in men, and perhaps in women and children. TESTOSTERONE IMPLANTS Subcutaneous surgical implantation of fused pellets or capsules of unmodified testosterone has been used to achieve a prolonged, sustained release of physiologic levels of testosterone for 4 to 6 months for androgen replacement therapy of hypogonadal men.22'2. This form of testosterone administration is used rarely

Ch.

119: Clinical Use and Abuse of Androgens and Antiandrogens

1183

TABLE 119-1. Common Androgen Preparations TESTOSTERONE

HO

H

I. Modalities currently available: Treatment Modality

Trade Name

Route

Transdermal testosterone

Testoderm (Alza)

Scrotal patch

Androderm (Watson)

Nonscrotal patch

Testoderm TTS (Alza)

Nonscrotal patch

AndroGel (Unimed)

Hydroalcoholic gel

Testosterone pellets (Organon)*

SC implant

Testosterone pellets

Testopel pellets (Bartor)

II. Modalities being developed (not currently available): Testosterone microspheres+ (testosterone microencapsulated in biodegradable matrix [85:15, DL-lactide: glycolide copolymer])

— (BioTek)

IM

Testosterone-cyclodextrin complex+ (testosterone complexed with hydroxypropyl-(3-cyclodextrin)

— (Bio-Technology)

SL

Transbuccal testosterone+

—(Watson; Columbia)

Buccal

TESTOSTERONE DERIVATIVES I. 17/3-hydroxyl esterification (with or zvithout other ring modifications)

R = COCH2CH3

Testosterone propionate

(Generic)

IM

Testosterone cypionate (cyclopentylpropionate)

Depo-Testosterone (Pharmacia Upjohn)! (Generic)

IM

Testosterone enanthate (heptanoate)

Delatestryl (Bio-Technology) (Generic)

IM

R = CO(CH2)5CH3

Testosterone undecanoate’1

Restandol, Andriol (Organon)

PO

R = CO(CH2)5CH=CH2

Nandrolone phenpropionate

Durabolin (Organon) Hybolin Improved (Hyrex) (Generic)

IM

Deca-Durabolin (Organon) (Generic)

IM

R = COCH2CH2 —

r = coch2ch2-

)

Position 19: removal of —CH3 Nandrolone decanoate

Hybolin Decanoate (Hyrex) Testosterone buciclate*+ (butylcyclohexylcarboxylate, 20 Aet-1)

R = CO(CH2)4CH3 Position 19: removal of—CH,

IM R__ CO=^

\—(CH2)3CH

(continued)

1184

PART VIII: ENDOCRINOLOGY OF THE MALE

TABLE 119-1. (continued) TESTOSTERONE DERIVATIVES II. 17a-alkylation (with or without other ring modifications)*

Methyltestosterone

Android (ICN)

PO

X = CH3

Testred (ICN) Oreton Methyl (Schering) Virilon (Star) Fluoxymesterone

Oreton Methyl Buccal

Buccal

Halotestin (Pharmacia Upjohn)

PO

(Generic)

X = CH3 Position 9:-F Position 11: —OH

Oxandrolone

Oxandrin (Bio-Technology)

PO

X = CH3 Position 2: occupied by oxygen Position 5:-H

Oxymetholone

Anadrol-50 (Unimed)

PO

X = CH3 Position 2: =CHOH Position 5:-H

Stanozolol

Winstrol (Sanofi)

PO

X = CH3 Position 5:-H Position 2, 3:

Danazol

Danocrine (Sanofi)

X = CH = CH

Danazol (Barr) Position 2, 3: TESTOSTERONE DERIVATIVES III. Ring Modification Without Position 17 Modifications Mesterolone*

Mestoranum (Schering)

PO

Position 1:-CH3

Percutaneous

Position 5:-H

Pro-viron Dihydrotestosterone^

Position 5:-H

Andractim (Besins-Iscovesco) Gelovit

SC, subcutaneous; SL, sublingual; IM. intramuscular;-, alpha substitution; —, beta substitution; ’Not available in the United States. ’Being developed; not currently available.

) (shaded circles), position of modifications.

t°ther trade names for testosterone cypionate include: Dep Andro (Forest); Duratest (Hauck); Depotest (Hyrex); Testred Cvpionate (ICN); Andro Cyp (Keene)- T-Cypionate (Legere); Andronate (Pasadena); Virilon IM (Star). r v mother trade names for testosterone enanthate include: Testro LA (CO Truxton); Andro LA (Forest); Durathate (Hauck); Everone (Hvrex); Testrin (Pasadena); Andropositorv (Rugby). ■Also being developed in oil for IM injections. r J ° J •Because of limited clinical usefulness, potential hepatotoxidty, and possibility for abuse, many oral 17cc-alkvlated androgens have been withdrawn from the market in the United States and in some countries in Europe. Some preparations such as methandrostenolone (Dianabol, Nerobol, Danabol, Metanabol), norethandrolone (Nilevar), methenolone (Primobolan), bolasterone (Tes-10), formebolone (Esiclene, Hubemol), and oxymesterone (Oranabol), ethylestrenol (Maxibolin), and methandriol (Stenediol) remain available in some foreign countries. These preparations are not listed in the table. nAlso being developed as a 0.7% hydroalcoholic gel formulation in the United States.

in the United States and is not acceptable to many hypogonadal men because of the large number and/or size of the implants, the need for a minor surgical procedure using a large trocar, extrusion of the pellets (8-9% of cases), and, although it is small, the risk of bleeding (2-3%) and infection (800 mg per day) it is a potent inhibitor of gonadal and adrenal steroidogen¬ esis, inhibiting both 17a-hydroxylase and 17,20-desmolase activity188-189 (see Chaps. 21 and 75). In a dosage of 400 mg every 8 hours, ketoconazole has been used with some success as a second-line agent to treat metastatic prostate cancer (see Chap. 225).190,191 After an initial suppression to castrate levels, testoste¬ rone levels rise moderately, concomitant with the activation of feedback mechanisms, resulting in increased luteinizing hor¬ mone secretion. Combining ketoconazole with an antigonadotropic agent, such as a GnRH analog, may help to prevent this secondary rise in serum testosterone levels. Because adrenal ster¬ oid synthesis is inhibited, glucocorticoid replacement is needed in conjunction with the high dosages of ketoconazole used for the treatment of prostate cancer. Ketoconazole has also been used in lower dosages to treat hirsutism and, combined with a GnRH agonist, to treat male-limited precocious puberty.192,193 The potential idiosyncratic hepatotoxicity of ketoconazole, however, although uncommon, limits its use in benign conditions.

20. 21.

21a.

21b.

22. 23. 24.

25. 26.

27.

28.

REFERENCES 29. 1. Wilson JD. Charles-Edouard Brown-Sequard and the centennial of endocri¬ nology. J Clin Endocrinol Metab 1990; 71:1403. 2. Bagatell C], Bremner WJ. Androgens in men—uses and abuses. N Engl J Med 1996; 334:707. 3. Bhasin S, Bremner WJ. Emerging issues in androgen replacement therapy. J Clin Endocrinol Metab 1997; 82:3. 4. Matsumoto AM. Androgen treatment of male hypogonadism. In: Bagdade JD, ed. 1998 Yearbook of endocrinology. St. Louis: Mosby, 1998:xix. 5. Daggett PR, Wheeler MJ, Nabarro JDN. Oral testosterone, a reappraisal. Horm Res 1978; 9:121. 6. Amory JK, Matsumoto AM. The therapeutic potential of testosterone patches. Exp Opin Invest Drugs 1998; 7:1977. 7. Findlay JC, Place VA, Snyder PJ. Treatment of primary hypogonadism in men with the transdermal administration of testosterone. J Clin Endocrinol Metab 1989; 68:369. 8. Place VA, Atkinson L, Prather DA, et al. Transdermal testosterone replace¬ ment through genital skin. In: Nieschlag E, Behre HM, eds. Testosterone: action, deficiency, substitution. Berlin: Springer-Verlag, 1990:165. 9. Meikle AW, Mazer NA, Moeilmer JF, et al. Enhanced transdermal delivery of testosterone across nonscrotal skin produces physiological concentra¬ tions of testosterone and its metabolites in hypogonadal men. J Clin Endo¬ crinol Metab 1992; 74:623. 10. Arver S, Dobs AS, Meikle AW, et al. Long-term efficacy and safety of a per¬ meation-enhanced testosterone transdermal system in hypogonadal men. Clin Endocrinol (Oxf) 1997; 47:727. 11. Parker S, Armitage M. Experience with transdermal testosterone replace¬ ment therapy for hypogonadal men. Clin Endocrinol (Oxf) 1999; 50:57. 12. Jordan WP Jr. Allergy and topical irritation associated with transdermal testosterone administration: a comparison of scrotal and nonscrotal transdermal systems. Am J Contact Derm 1997; 8:108. 13. Yu A, Gupta SK, Hwang SS, et al. Transdermal testosterone administration in hypogonadal men: comparison of pharmacokinetics at different sites of application and at the first and fifth days of application. J Clin Pharmacol 1997; 37:1129. 14. Yu A, Gupta SK, Hwang SS, et al. Testosterone pharmacokinetics after application of an investigational transdermal system in hypogonadal men. J Clin Pharmacol 1997; 37:1139. 15. Jordan Jr WP, Atkinson LE, Lai C. Comparison of the skin irritation poten¬ tial of two testosterone transdermal systems: an investigational system and a marketed product. Clin Ther 1998; 20:80. 16. De Sanctis V, Vullo C, Urso L, et al. Clinical experience with Androderm® testosterone transdermal system in hypogonadal adolescents and young men with (i-thalassemia major. J Pediatr Endocrinol Metab 1998; 11:891. 17. Miller K, Corcoran C, Armstrong C, et al. Transdermal testosterone admin¬ istration in women with acquired immunodeficiency syndrome wasting: a pilot study. J Clin Endocrinol Metab 1998; 83:2717. 18. Bhasin S, Storer TW, Asbel-Sethi N, et al. Effects of testosterone replace¬ ment with a nongenital, transdermal system, Androderm, in human immunodeficiency virus-infected men with low testosterone levels. J Clin Endocrinol Metab 1998; 83:3155. 19. Rolf C, Gottschalk I, Behre HM, et al. Pharmacokinetics of new testosterone

30. 31. 32. 33.

34.

35. 36.

37.

38. 39. 40.

41.

42.

43.

44.

45.

46. 47. 48.

1197

transdermal therapeutic systems in gonadotropin-releasing hormone antag¬ onist-suppressed normal men. Exp Clin Endocrinol Diabetes 1999; 107:63. Buckler HM, Robertson WR, Wu FCW. Which androgen replacement for women? J Clin Endocrinol Metab 1998; 83:3920. Swerdloff RS, Wang C, Cunningham G, et al. Long-term pharmacokinetics of transdermal testosterone gel versus testosterone patch in hypogonadal men [abstract]. In: Proceedings of The Endocrine Society 82nd Annual Meeting, Toronto, Ontario, Canada, June 21-24, 2000; 2347. Wang C, Swerdloff RS, Iranmanesh A, et al. Transdermal testosterone gel improves sexual function, mood, muscle strength, and body composition parameters in hypogonadal men. J Clin Endocrinol Metab 2000; in press. Wang C, Swerdloff RS, Iranmanesh A, et al. Effects of transdermal tes¬ tosterone gel on bone turnover markers and bone mineral density in hypogonadal men [abstract]. In: Proceedings of The Endocrine Society 82nd Annual Meeting, Toronto, Ontario, Canada, June 21-24, 2000; 2348. Conway AJ, Boylan LM, Howe C, et al. Randomized clinical trial of testoste¬ rone replacement therapy in hypogonadal men. Int J Androl 1988; 11:247. Handelsman DJ, Conway AJ, Boylan LM. Pharmacokinetics and pharmaco¬ dynamics of testosterone pellets in man. J Clin Endocrinol Metab 1990; 71:216. Jockenhovel F, Vogel E, Kreutzer M, et al. Pharmacokinetics and pharmaco¬ dynamics of subcutaneous testosterone implants in hypogonadal men. Clin Endocrinol (Oxf) 1996; 45:61. Handelsman DJ, Mackey M-A, Howe C, et al. An analysis of testosterone implants for androgen replacement therapy. Clin Endocrinol (Oxf) 1997; 47:311. Stuenkel CA, Dudley RE, Yen SSC. Sublingual administration of testosteronehydroxypropyl-p-cyclodextrin inclusion complex simulates episodic andro¬ gen release in hypogonadal men. J Clin Endocrinol Metab 1991; 72:1054. Salehian B, Wang C, Alexander G, et al. Pharmacokinetics, bioefficacy, and safety of sublingual testosterone cyclodextrin in hypogonadal men: com¬ parison to testosterone enanthate—a clinical research center study. J Clin Endocrinol Metab 1995; 80:3567. Wang C, Eyre DR, Clark R, et al. Sublingual testosterone replacement improves muscle mass and strength, decreases bone resorption, and increases bone formation markers in hypogonadal men—a clinical research center study. J Clin Endocrinol Metab 1996; 81:3654. Dobs AS, Hoover DR, Chen M-C, Allen R. Pharmacokinetic characteristics, efficacy, and safety of buccal testosterone in hypogonadal males: a pilot study. J Clin Endocrinol Metab 1998; 83:33. Ben-Galim E, Hillman RE, Weldon VV. Topically applied testosterone and phallic growth. Am J Dis Child 1980; 134:296. Klugo RC, Cerny JC. Response of micropenis to topical testosterone and gonadotropin. J Urol 1978; 119:667. Bracco GL, Carli P, Sonni L, et al. Clinical and histologic effects of topical treat¬ ments of vulval lichen sclerosus. A critical evaluation. J Reprod Med 1993; 38:37. Bomstein J, Heifetz S, Kellner Y, et al. Clobetasol dipropionate 0.05% ver¬ sus testosterone propionate 2% topical application for severe vulvar lichen sclerosus. Am J Obstet Cynecol 1998; 178:80. Bhasin S, Swerdloff RS, Steiner B, et al. A biodegradable testosterone micro¬ capsule formulation provides uniform eugonadal levels of testosterone for 10-11 weeks in hypogonadal men. J Clin Endocrinol Metab 1992; 74:75. Snyder PJ, Lawrence DA. Treatment of male hypogonadism with testoste¬ rone enanthate. J Clin Endocrinol Metab 1980; 51:1335. Skakkebaek NE, Bancroft J, Davidson DW, Warner P. Androgen replace¬ ment with oral testosterone undecanoate in hypogonadal men: a double¬ blind controlled study. Clin Endocrinol (Oxf) 1981; 14:49. Butler GE, Sellar RF, Walker RF, et al. Oral testosterone undecanoate in the management of delayed puberty in boys: pharmacokinetics and effects on sexual maturation and growth. J Clin Endocrinol Metab 1992; 75:37. Gooren LJG. A ten year safety study of the oral androgen testosterone undecanoate. J Androl 1994; 15:212. Zhang G-Y, Gu Y-Q, Wang X-H, et al. A pharmacokinetic study of injectable testosterone undecanoate in hypogonadal men. J Androl 1998; 19:761. Behre HM, Abshagen K, Oettel M, et al. Intramuscular injection of tes¬ tosterone undecanoate for the treatment of male hypogonadism: phase I studies. Eur J Endocrinol 1999; 140:414. Behre HM, Nieschlag E. Testosterone buciclate (20 Aet-1) in hypogonadal men: pharmacokinetics and pharmacodynamics of the new long-acting androgen ester. J Clin Endocrinol Metab 1992; 75:1204. Minto CF, Howe C, Wishart S, et al. Pharmacokinetics and pharmacody¬ namics of nandrolone esters in oil vehicle: effects of ester, injection site and injection volume. J Pharmacol Exp Ther 1997; 281:93. Luisi M, Franchi F. Double blind group comparative study of testosterone undecanoate and mesterolone in hypogonadal male patients. J Endocrinol Invest 1980; 3:305. Schaison G, Nahoul G, Couzinet B. Percutaneous dihydrotestosterone sys¬ tem. In: Nieschlag E, Behre HM, eds. Testosterone: action, deficiency and substitution. Berlin: Springer Verlag, 1990:155. Mendonca BB, Inacio M, Costa EM, et al. Male pseudohermaphroditism due to steroid 5alpha-reductase 2 deficiency. Diagnosis, psychological eval¬ uation, and management. Medicine 1996; 75:64. Choi SK, Han SW, Kim DH, de Lignieres B. Transdermal dihydrotestosterone therapy and its effects on patients with microphallus. J Urol 1993; 150:657. de Lignieres B. Transdermal dihydrotestosterone treatment of "andropause." Ann Med 1993; 25:235. Wang C, Iranmanesh A, Berman N, et al. Comparative pharmacokinetics of three doses of percutaneous dihydrotestosterone gel in healthy elderly men—a clinical research center study. J Clin Endocrinol Metab 1998; 83:2749.

1198

PART VIII: ENDOCRINOLOGY OF THE MALE

49. Swerdloff RS, Wang C. Dihydrotestosterone: a rationale for its use as a nonaromatizable androgen replacement therapeutic agent. Baillieres Clin Endocrinol Metab 1998; 12:501. 50. Cummings DE, Kumar N, Bardin CW, et al. Prostate-sparing effects in pri¬ mates of the potent androgen 7oc-methyl-19-nortestosterone: a potential alternative to testosterone for androgen replacement and male contracep¬ tion. J Clin Endocrinol Metab 1998; 83:4212. 51. Anderson RA, Martin CW, Kung AW, et al. 7a-methyl-19-nortestosterone maintains sexual behavior and mood in hypogonadal men. J Clin Endo¬ crinol Metab 1999; 84:3356. 52. Edwards JP, West SJ, Pooley CLF, et al. New nonsteroidal androgen recep¬ tor modulators based on 4-(trifluoromethyl)-2(lH)-pyrrolidino[3,2-g]quinolone. Bioorg Med Chem Lett 1998; 8:745. 53. Matsumoto AM. The testis. In: Bennett JC, Plum F, eds. Cecil textbook of medicine, 20th ed. Philadelphia: WB Saunders, 1996:1325. 54. Burstein S, Grumbach MM, Kaplan SL. Early determination of androgen responsiveness is important in the management of microphallus. Lancet 1979; 2:983. 55. Tsur H, Shafir R, Shachar J, Eshkol A. Microphallic hypospadias: testoste¬ rone therapy prior to surgical repair. Br J Plast Surg 1983; 36:398. 56. Matsumoto AM. Hormonal therapy of male hypogonadism. Endocrinol Metab Clin North Am 1994; 23:857. 57. Davidson JM, Kwan M, Greenleaf W. Hormonal replacement and sexuality in men. Clin Endocrinol Metab 1982; 11:599. 58. Burris AS, Banks SM, Carter CS, et al. A long-term prospective study of the physiologic and behavioral effects of hormone replacement in untreated hypogonadal men. J Androl 1992; 13:297. 59. Bagatell C], Heiman JR, Rivier JE, Bremner WJ. Effects of endogenous tes¬ tosterone and estradiol on sexual behavior in normal young men. J Clin Endocrinol Metab 1994; 78:711. 60. Wang C, Alexander G, Berman N, et al. Testosterone replacement therapy improves mood in hypogonadal men—a clinical research center study. J Clin Endocrinol Metab 1996; 81:3578. 61. Janowsky JL, Oviatt SK, Orwoll ES. Testosterone influences spatial cogni¬ tion in older men. Behav Neurosci 1994; 108:325. 62. Alexander GM, Swerdloff RS, Wang C, et al. Androgen-behavior correla¬ tions in hypogonadal and eugonadal men. II. Cognitive abilities. Horm Behav 1998; 33:85. 63. Leibenluft E, Schmidt PJ, Turner EH, et al. Effects of leuprolide-induced hypogonadism and testosterone replacement on sleep, melatonin, and pro¬ lactin secretion in men. J Clin Endocrinol Metab 199-7; 82:3203. 64. Katznelson L, Finkelstein JS, Schoenfeld DA, et al. Increase in bone density and lean body mass during testosterone administration in men with acquired hypogonadism. J Clin Endocrinol Metab 1996; 81:4358. 65. Bhasin S, Storer TW, Berman N, et al. Testosterone replacement increases fat-free mass and muscle size in hypogonadal men. J Clin Endocrinol Metab 1997; 82:407. 66. Behre HM, Kliesch S, Leifke E, et al. Long-term effect of testosterone ther¬ apy on bone mineral density in hypogonadal men. J Clin Endocrinol Metab 1997; 82:2386. 67. Marin P, Arver S. Androgens and abdominal obesity. Baillieres Clin Endo¬ crinol Metab 1998; 12:441. 68. Bagatell CJ, Knopp RH, Vale WW, et al. Physiologic testosterone levels in normal men suppress high-density lipoprotein cholesterol levels. Ann Intern Med 1992; 116:967. 69. Zgliczynski S, Ossowski M, Slowinska-Srzednicka J, et al. Effect of tes¬ tosterone replacement therapy on lipids and lipoproteins in hypogonadal and elderly men. Atherosclerosis 1996; 121:35. 70. Bagatell CJ, Bremner WJ. Androgen and progestogen effects on plasma lip¬ ids. Prog Cardiovasc Dis 1995; 38:255. 71. English KM, Steeds R, Jones TH, Channer KS. Testosterone and coronary heart disease: is there a link? QJM 1997; 90:787. 72. Shahidi NT. Androgens and erythropoiesis. N Engl J Med 1973; 289:72. 73. Ammus SS. The role of androgens in the treatment of hematologic disor¬ ders. Adv Intern Med 1989; 34:191. 74. Jockenhovel F, Vogel E, Reinhardt W, Reinwein D. Effects of various modes of androgen substitution therapy on erythropoiesis. Eur J Med Res 1997; 2:293. 75. Kulin HE. Delayed puberty in boys. Curr Ther Endocrinol Metab 1997; 6:346. 76. Houchin LD, Rogol AD. Androgen replacement in children with constitu¬ tional delay of puberty: the case for aggressive therapy. Baillieres Clin Endocrinol Metab 1998; 12:427. 77. Aynsley-Green A, Zachman M, Prader A. Interrelation of the therapeutic effects of growth hormone and testosterone on growth in hypopituitarism. J Pediatr 1976; 89:992. 78. Parker MW, Johanson AJ, Rogol AD, et al. Effect of testosterone on somatomedin C concentrations in prepubertal boys. J Clin Endocrinol Metab 1984; 58:87. 79. Veldhuis JD, Metzger DL, Martha PM Jr, et al. Estrogen and testosterone, but not non-aromatizable androgen, direct network integration of the hypothalamo-somatotrope (growth hormone)-insulin-like growth factor I axis in the human: evidence from pubertal pathophysiology and sex-steroid hormone replacement. J Clin Endocrinol Metab 1997; 82:3414. 80. Hendler ED, Goffinet JA, Ross S, et al. Controlled study of androgen ther¬ apy in anemia of patients on maintenance hemodialysis. N Engl J Med 1974; 291:1046. 81. Neff MS, G ildberg J, Slifkin RF, et al. A comparison of androgens for ane¬ mia in patients on hemodialysis. N Engl J Med 1981; 304:871.

82. Teruel JL, Aguilera A, Marcen R, et al. Androgen therapy for chronic renal fail¬ ure. Indications in the erythropoietin era. Scand J Urol Nephrol 1996; 30:403. 83. Teruel JL, Marcen R, Navarro-Antolin J, et al. Androgen versus erythropoi¬ etin for the treatment of anemia in hemodialyzed patients: a prospective study. J Am Soc Nephrol 1996; 7:140. 84. Ballal SH, Domoto DT, Polack DC, et al. Androgens potentiate the effects of erythropoietin in the treatment of anemia of end-stage renal disease. Am J Kidney Dis 1991; 17:29. 85. Gaughan WJ, Liss KA, Dunn SR, et al. A 6-month study of low-dose recom¬ binant human erythropoietin alone and in combination with androgens for the treatment of anemia in chronic hemodialysis patients. Am J Kidney Dis 1997; 30:495. 86. Van Hengstrum M, Steenbergen J, Haanen C. Clinical course in 28 unse¬ lected patients with aplastic anemia treated with anabolic steroids. Br J Haematol 1979; 41:323. 87. Branda RF, Amsden TW, Jacob HS. Randomized study of nandrolone ther¬ apy for anemias due to bone marrow failure. Arch Intern Med 1977; 137:65. 88. Camitta BM, Thomas ED, Nathan DG, et al. A prospective study of andro¬ gens and bone marrow transplantation for treatment of severe aplastic ane¬ mia. Blood 1979; 53:504. 89. Harrington WJ Sr, Kolodny L, Horstman LL, et al. Danazol therapy for par¬ oxysmal nocturnal hemoglobinuria. Am J Hematol 1997; 54:149. 90. Ahn YS. Efficacy of danazol in hematologic disorders. Acta Hematol 1990; 84:122. 91. Guthrie RD, Smith DW, Graham CB. Testosterone treatment of micropenis during early childhood. J Pediatr 1973; 83:247. 92. Gearhart JP, Jeffs RD. The use of parenteral testosterone therapy in genital reconstructive surgery. J Urol 1987; 138:1077. 93. Price P, Wass JAH, Griffin JE, et al. High-dose androgen therapy in male pseudohermaphroditism due to 5 a-reductase deficiency and disorders of the androgen receptor. J Clin Invest 1984; 74:1496. 94. Frank MM, Gelfand JA, Atkinson JP. Hereditary angioedema: the clinical syndrome and its management. Ann Intern Med 1976; 84:580. 95. Barbosa J, Seal US, Doe RP. Effects of anabolic steroids on haptoglobin, orosomucoid, plasminogen, fibrinogen, ceruloplasmin al antitrypsin, 0glucuronidase, and total serum proteins. J Clin Endocrinol Metab 1971; 33:388. 96. Madanes AE, Farber M. Danazol. Ann Intern Med 1982; 96:625. 97. Zurlo JJ, Frank MM. The long-term safety of danazol in women with hereditary angioedema. Fertil Steril 1990; 54:64. 98. Allegra JC. Rationale approaches to the hormonal treatment of breast can¬ cer. Semin Oncol 1983; 10(Suppl 4):25. 99. Katznelson L. Therapeutic role of androgens in the treatment of osteoporo¬ sis in men. Baillieres Clin Endocrinol Metab 1998; 12:453. 100. Finkelstein JS, Klibanski A, Neer RM, et al. Increases in bone density dur¬ ing treatment of men with idiopathic hypogonadotropic hypogonadism. J Clin Endocrinol Metab 1989; 69:776. 101. Finkelstein JS, Neer RM, Biller BM, et al. Osteopenia in men with a history of delayed puberty. N Engl J Med 1992; 326:600. 102. Anderson FH, Francis RH, Faulkner K. Androgen supplementation in eugonadal men with osteoporosis—effects of six months' treatment on bone mineral density and cardiovascular risk factors. Bone 1996; 18:171. 103. Reid IR, Wattie DJ, Evans MC, Stapleton JP. Testosterone therapy in gluco¬ corticoid-treated men. Arch Intern Med 1996; 156:1173. 104. Savvas M, Studd JW, Norman S, et al. Increase in bone mass after one year of percutaneous oestradiol and testosterone implants in post-menopausal women who have previously received long-term oral oestrogens. Br J Obstet Gynaecol 1992; 99:757. 105. Davis SR, McCloud P, Strauss BJ, Burger H. Testosterone enhances estradiol's effects on post-menopausal bone density and sexuality. Maturitas 1995; 21:227. 106. Watts NB, Notelovitz M, Timmons MC, et al. Comparison of oral estrogens and estrogens plus androgen on bone mineral density, menopausal symp¬ toms, and lipid-liporotein profiles in surgical menopause. Obstet Gynecol 1995; 85:529. 107. Barrett-Connor E. Efficacy and safety of estrogen/androgen therapy. Menopausal symptoms, bone, and cardiovascular parameters. J Reprod Med 1998; 43(Suppl 8):746. 108. Need AG, Horowitz M, Bridges A, et al. Effects of nandrolone decanoate and antiresorptine therapy on vertebral density in osteoporotic post-menopausal women. Arch Intern Med 1989; 149:57. 109. Passeri M, Pedrazzoni M, Pioli G, et al. Effects of nandrolone decanoate on bone mass in established osteoporosis. Maturitas 1993; 17:211. 110. Studd J, Arnala I, Kicovic PM, et al. A randomized study of tibolone on bone mineral density in osteoporotic post-menopausal women with previ¬ ous fractures. Obstet Gynecol 1998; 92:574. 111. Chesnut CH, Ivey JL, Gruber HE, et al. Stanozolol in post-menopausal osteoporosis: therapeutic efficacy and possible mechanisms of action. Metabolism 1983; 32:571. 112. Davis SR, Burger HG. The rationale for physiological testosterone replace¬ ment in women. Baillieres Clin Endocrinol Metab 1998; 12:391. 113. Burger HG, Hailes J, Menelaus M, et al. The management of persistent menopausal symptoms with oestradiol-testosterone implants: clinical, lipid and hormonal results. Maturitas 1984; 6:351. 114. Sherwin BB, Gelfand MM, Brender W. Androgen enhances sexual motiva¬ tion in females: a prospective, crossover study of sex steroid administration in the surgical menopause. Psychosom Med 1985; 47:339. 115. Tenover JS. Androgen administration to aging men. Endocrinol Metab Clin North Am 1994; 23:877.

Ch. 119: Clinical Use and Abuse of Androgens and Antiandrogens 116. Bhasin S, Bagatell CJ, Bremner WJ, et al. Issues in testosterone replacement in older men. J Clin Endocrinol Metab 1998; 83:3435. 117. Tenover JS. Effects of testosterone supplementation in the aging male. J Clin Endocrinol Metab 1992; 75:536. 118. Sih R, Morley JE, Kaiser FE, et al. Testosterone replacement in older hypogonadal men: a 12-month randomized controlled trial. J Clin Endocrinol Metab 1997; 82:1661. 119. Hajjar RR, Kaiser FE, Morley JE. Outcomes of long-term testosterone replacement in older hypogonadal males: a retrospective analysis. J Clin Endocrinol Metab 1997; 82:3793. 119a. Snyder PJ, Peachey H, Hannoush P, et al. Effect of testosterone treatment on bone mineral density in men over 65 years of age. J Clin Endocrinol Metab 1999; 84:1966. 119b. Snyder PJ, Peachey H, Hannoush P, et al. Effect of testosterone treatment on body composition and muscle strength in men over 65 years of age. J Clin Endocrinol Metab 1999; 84:2647. 120. Bhasin S, Storer TW, Berman N, et al. The effects of supraphysiologic doses of testosterone on muscle size and strength in normal men. N Engl J Med 1996; 335:1. 121. Bross R, Casaburi R, Storer TW, Bhasin S. Androgen effects on body com¬ position and muscle function: implications for the use of androgens as ana¬ bolic agents in sarcopenic states. Baillieres Clin Endocrinol Metab 1998; 12:365. 122. Coodley GO, Coodley MK. A trial of testosterone therapy for HIV-associ¬ ated weight loss. AIDS 1997; 11:1347. 123. Grinspoon S, Corcoran C, Askari H, et al. Effects of androgens in men with AIDS wasting syndrome. A randomized, double-blind, placebo-controlled trial. Ann Intern Med 1998; 129:18. 124. Rabkin JG, Wagner GJ, Rabkin R. Testosterone therapy for human immu¬ nodeficiency virus-positive men with and without hypogonadism. J Clin Psychopharmacol 1999; 19:19. 125. Sattler FR, Jaque SV, Schroder ET, et al. Effects of pharmacological doses of nandrolone decanoate and progressive resistance training in immunodeficient patients infected with human immunodeficiency virus. J Clin Endo¬ crinol Metab 1999; 84:1268. 126. Strawford A, Barbieri T, Van Loan M, et al. Resistance exercise and supraphysiological androgen therapy in eugonadal men with HIV-related weight loss. JAMA 1999; 281:1282. 127. Corcoran C, Grinspoon S. Treatments for wasting in patients with the acquired immunodeficiency syndrome. N Engl J Med 1999; 340:1740. 127a. Bhasin S, Storer TW, Javanbakht M, et al. Testosterone replacement and resistance exercise in HIV-infected men with weight loss and low testoste¬ rone levels. JAMA 2000; 283:763. 128. Johansen KL, Mulligan K, Schambelan M. Anabolic effects of nandrolone decanoate in patients receiving dialysis. A randomized controlled trial. JAMA 1999; 281:1275. 129. Schols AM, Soeters PB, Mostert R, et al. Physiological effects of nutritional support and anabolic steroids in patients with chronic obstruction pulmo¬ nary disease. A placebo-controlled randomized trial. Am J Respir Crit Care Med 1995; 152:1268. 130. Ferreira IM, Verreschil T, Nery LE, et al. The influence of 6 months of oral anabolic steroids on body mass and respiratory muscles in undernour¬ ished COPD patients. Chest 1998; 114:19. 131. Demling RH, DeSanti L. Oxandrolone, an anabolic steroid, significantly increases the rate of weight gain in the recovery phase after major burns. J Trauma 1997; 43:47. 132. Zachwieja JJ, Smith SR, Lovejoy JC, et al. Testosterone administration pre¬ serves protein balance but not muscle strength during 28 days of bed rest. J Clin Endocrinol Metab 1999; 84:207. 133. Amory JK, Bremner WJ. The use of testosterone as a male contraceptive. Baillieres Clin Endocrinol Metab 1998; 12:471. 134. Lesser MA. Testosterone propionate therapy in one hundred cases of angina pectoris. J Clin Endocrinol 1946; 6:549. 135. Jaffe MD. Effect of testosterone cypionate on postexercise ST segment depression. Br Heart J 1977; 39:1217. 136. Rosano GMC, Leonardo F, Pagnotta P, et al. Acute anti-ischemic effect of testosterone in men with coronary artery disease. Circulation 1999; 99:1666. 137. Webb CM, Adamson DL, de Zeigler D, Collins P. Effect of acute testoster¬ one on myocardial ischemia in men with coronary artery disease. Am J Cardiol 1999; 83:437. 137a. Webb CM, McNeill JG, Hayward CS, et al. Effects of testosterone on coro¬ nary vasomotor regulation in men with coronary artery disease. Circula¬ tion 1999; 100:1690. 138. Wilson JD. Androgen abuse by athletes. Endocr Rev 1988; 9:181. 139. Yesalis CE, Bahrke MS. Anabolic-androgenic steroids. Current issues. Sports Med 1995; 19:326. 140. American Academy of Pediatrics. Committee on Sports Medicine and Fitness. Adolescents and anabolic steroids: a subject review. Pediatrics 1997; 99:904. 141. Haupt HA, Rovere GD. Anabolic steroids: a review of the literature. Am J Sports Med 1984; 12:469. 142. Wu FC. Endocrine aspects of anabolic steroids. Clin Chem 1997; 43:1289. 143. Sullivan ML, Martinez CM, Gennis P, Gallagher EJ. The cardiac toxicity of anabolic steroids. Prog Cardiovasc Dis 1998; 41:1. 144. Porcerelli JH, Sandler BA. Anabolic-androgenic steroid abuse and psycho¬ pathology. Psychiatr Clin North Am 1998; 21:829. 145. Webb OL, Laskarzewski PM, Glueck CJ. Severe depression of high-density lipoprotein cholesterol levels in weight lifters and body builders by self-

1199

administered exogenous testosterone and anabolic-androgenic steroids. Metabolism 1984; 33:971. 146. Thompson PD, Cullinane EM, Sady SP, et al. Contrasting effects of tes¬ tosterone and stanozolol on serum lipoprotein levels. JAMA 1989; 261:1165. 147. Glazer G. Atherogenic effects of anabolic steroids on serum lipid levels. Arch Intern Med 1991; 151:1925. 148. Labrie F, Belanger A, Luu-The V, et al. DHEA and the intracrine formation of androgens and estrogens in peripheral tissues: its role during aging. Steroids 1998; 63:322. 149. Nippoldt TB, Nair KS. Is there a case for DHEA replacement? Baillieres Clin Endocrinol Metab 1998; 12:507. 150. Morales AJ, Haubrich RH, Hwang JY, et al. The effect of six months of treatment with a 100 mg daily dose of dehydroepiandrosterone (DHEA) on circulating sex steroids, body composition and muscle strength in ageadvanced men and women. Clin Endocrinol (Oxf) 1998; 49:421. 151. Gordon CM, Grace E, Jean Emans S, et al. Changes in bone turnover mark¬ ers and menstrual function after short-term oral DHEA in young women with anorexia nervosa. J Bone Miner Res 1999; 14:136. 152. Flynn MA, Weaver-Osterholtz D, Sharpe-Timms KL, et al. Dehydroepiandros¬ terone replacement in aging humans. J Clin Endocrinol Metab 1999; 84:1527. 152a. Baulieu EE, Thomas G, Legrain S, et al. Dehydroepiandrosterone (DHEA), DHEA sulfate, and aging: contribution of the DHEAge Study to a sociobio¬ medical issue. Proc Natl Acad Sci U S A 2000; 97:4279. 152b. Arlt W, Callies F, van Vlijmen JC, et al. Dehydroepiandrosterone replace¬ ment in women with adrenal insufficiency. N Engl J Med 1999; 341:1013. 153. King DS, Sharp RL, Vukovich MD, et al. Effect of oral androstenedione on serum testosterone and adaptations to resistance training in young men. A randomized controlled trial. JAMA 1999; 281:2020. 153a. Leder BZ, Longcope C, Catlin DH, et al. Oral androstenedione administra¬ tion and serum testosterone concentrations in young men. JAMA 2000; 283:779. 154. Bettman HK, Goldman HS, Abramowicz M, Sobel EH. Oxandrolone treat¬ ment of short stature: effect on predicted mature height. J Pediatr 1971; 79:1018. 155. Binder D, Grauer ML, Wehner AV, et al. Outcome in tall stature. Final height and psychological aspects in 220 patients with and without treat¬ ment. Eur J Pediatr 1997; 156:905. 156. Rolf C, Nieschlag E. Potential adverse effects of long-term testosterone therapy. Baillieres Clin Endocrinol Metab 1998; 12:521. 157. Wang C, Alexander G, Berman N, et al. Testosterone replacement therapy improves mood in hypogonadal men—a clinical research center study. J Clin Endocrinol Metab 1996; 81:3578. 158. Tricker R, Casaburi R, Storer TW, et al. The effects of supraphysiological doses of testosterone on angry behavior in healthy eugonadal men—a clin¬ ical research center study. J Clin Endocrinol Metab 1996; 81:3754. 159. Behre HM, Bohmeyer J, Nieschlag E. Prostate volume in testosteronetreated and untreated hypogonadal men in comparison to age-matched normal controls. Clin Endocrinol (Oxf) 1994; 40:341. 160. Jackson JA, Waxman J, Spiekerman AM. Prostatic complications of tes¬ tosterone replacement therapy. Arch Intern Med 1989; 149:2365. 161. Matsumoto AM, Sandblom RE, Schoene RB, et al. Testosterone replace¬ ment in hypogonadal men: effects on obstructive sleep apnoea, respiratory drives, and sleep. Clin Endocrinol (Oxf) 1985; 22:713. 162. Schneider BK, Pickett CK, Zwillich CW, et al. Influence of testosterone on breathing during sleep. J Appl Physiol 1986; 61:618. 163. Matsumoto AM. Effects of chronic testosterone administration in normal men: safety and efficacy of high dosage testosterone and parallel dosedependent suppression of luteinizing hormone, follicle-stimulating hor¬ mone, and sperm production. J Clin Endocrinol Metab 1990; 70:282. 164. Ishak KG, Zimmerman HJ. Hepatotoxic effects of anabolic/androgenic ster¬ oids. Semin Liver Dis 1987; 7:230. 165. Geller J. Megestrol acetate plus low-dose estrogen in the management of advanced prostatic carcinoma. Urol Clin North Am 1991; 18:83. 166. Ottery FD, Walsh D, Strawford A. Pharmacologic management of anorexia/cachexia. Semin Oncol 1998; 25(Suppl 6):35. 167. Loprinzi CL, Michalak JC, Quella SK, et al. Megestrol acetate for the pre¬ vention of hot flashes. N Engl J Med 1994; 331:347. 168. Grinspoon S, Corcoran C, Lee K, et al. Loss of lean body and muscle mass with androgen levels in hypogonadal men with acquired immunodefi¬ ciency syndrome and wasting. J Clin Endocrinol Metab 1996; 81:4051. 169. Subramanian S, Goker H, Kanji A, Sweeney H. Clinical adrenal insufficiency in patients receiving megestrol therapy. Arch Intern Med 1997; 157:1008. 170. Mann M, Roller E, Murgo A, et al. Glucocorticoidlike activity of megestrol. A summary of Food and Drug Administration experience and a review of the literature. Arch Intern Med 1997; 157:1651. 171. Neumann F. The antiandrogen cyproterone acetate: discovery, chemistry, basic pharmacology, clinical use and tool in basic research. Exp Clin Endo¬ crinol (Oxf) 1994; 102:1. 172. Barradell LB, Faulds D. Cyproterone acetate. A review of its pharmacology and therapeutic efficacy in prostate cancer. Drugs Aging 1994; 5:59. 173. Lopez M. Cyproterone acetate in the treatment of metastatic cancer of the male breast. Cancer 1985; 55:2334. 174. Shaw JC. Antiandrogen and hormonal treatment of acne. Dermatol Clin 1996; 14:803. 175. Barth JH, Cherry CA, Wojnarowska F, et al. Cyproterone acetate for severe hirsutism: results of a double-blind dose-ranging study. Clin Endocrinol (Oxf) 1991; 35:5.

1200 176

177. 178.

179.

180. 181. 182. 183. 184. 185. 186.

187. 188. 189.

190. 191. 192.

193.

PART VIII: ENDOCRINOLOGY OF THE MALE

Meriggiola MC, Bremner WJ, Paulsen CA, et al. A combined regimen of cyproterone acetate and testosterone enanthate as a potentially highly effective male contraceptive. J Clin Endocrinol Metab 1996; 81:3018. Goktas S, Crawford ED. Optimal hormonal therapy for advanced prostate carcinoma. Semin Oncol 1999; 26:162. Eisenberger MA, Blumenstein BA, Crawford ED, et al. Bilateral orchidectomy with of without flutamide for metastatic prostate cancer. N Engl J Med 1998; 339:1036. Cusan L, Dupont A, Gomez J-L, et al. Comparison of flutamide and spironolactone in the treatment of hirsutism: a randomized controlled trial. Fertil Steril 1994; 61:281. Muderris II, Bayram F. Clinical efficacy of lower dose flutamide 125 mg/ day in the treatment of hirsutism. J Endocrinol Invest 1999; 22:165. Goa KL, Spencer CM. Bicalutamide in advanced prostate cancer. A review. Drugs Aging 1998; 12:401. Dole EJ, Holdsworth MT. Nilutamide: an antiandrogen for the treatment of prostate cancer. Ann Pharmacother 1997; 31:65. Givens J. Treatment of hirsutism with spironolactone. Fertil Steril 1985; 43:841. Jeffcoate W. The treatment of women with hirsutism. Clin Endocrinol (Oxf) 1993; 39:143. Pittaway DE, Maxson WS, Wentz AC. Spironolactone in combination drug therapy for unresponsive hirsutism. Fertil Steril 1985; 43:878. Werber Leschek E, Jones J, Barnes KM, et al. Six-year results of spironolac¬ tone and testolactone treatment of familial male-limited precocious puberty with addition of deslorelin after central puberty onset. J Clin Endocrinol Metab 1999; 84:175. Lissak A, Sorpkin Y, Calderon I, et al. Treatment of hirsutism with cimetidine: a prospective randomized controlled trial. Fertil Steril 1989; 51:247. Pont A, Graybill JR, Craven PC, et al. High-dose ketoconazole therapy and adrenal and testicular function in humans. Arch Intern Med 1984; 144:2150. Rajfer J, Sikka SC, Rivera F, Handelsman DJ. Mechanism of inhibition of human testicular steroidogenesis by oral ketoconazole. J Clin Endocrinol Metab 1986; 63:1193. Small EJ, Vogelzong NJ. Second-line hormonal therapy for advanced pros¬ tate cancer: a shifting paradigm. J Clin Oncol 1997; 15:382. Bok RA, Small EJ. The treatment of advanced prostate cancer with ketocon¬ azole: safety issues. Drug Saf 1999; 20:451. Venturoli S, Marescalchi O, Colombo FM, et al. A prospective randomized trial comparing low dose flutamide, finasteride, ketoconazole, and cypro¬ terone acetate-estrogen regimens in the treatment of hirsutism. J Clin Endocrinol Metab 1999; 84:1304. Holland FJ, Kirsch SE, Selby R. Gonadotropin-independent precocious puberty ("testotoxicosis"): influence of maturational status on response to ketoconazole. J Clin Endocrinol Metab 1987; 64:328.

TABLE 120-1. Causes of Gynecomastia PHYSIOLOGIC Neonatal Pubertal Senescent

NEOPLASMS Steroid-producing (adrenal, testis) Human chorionic gonadotropin-producing (especially testis and lung)

DRUGS (see Table 120-2)

CONGENITAL DISORDERS Klinefelter syndrome and variants Anorchia (vanishing testis syndrome) Cryptorchidism Defects in enzymes of testosterone biosynthesis Defects in androgen action True hermaphroditism

ACQUIRED TESTICULAR DAMAGE Surgery Trauma Irradiation Infection (mumps and other viruses, leprosy)

ENVIRONMENTAL Phytoestrogens Hepatotoxic agents

SYSTEMIC DISORDERS Renal failure Hepatitis Cirrhosis Thyrotoxicosis

MISCELLANEOUS Spinal cord injury Myotonic dystrophy Refeeding after starvation Increased aromatization of androgens Psychological stress Human immunodeficiency virus infection Chest wall trauma

CHAPTER

1 20

GYNECOMASTIA ALLAN R. GLASS

GENERAL CONSIDERATIONS Gynecomastia (enlargement of the male breast secondary to an increase in glandular tissue and stroma) can be a vexing clinical problem.1-3 Often, it is a benign finding, but sometimes it is an important clue to disease elsewhere4 (Table 120-1). Thus, the condition cannot be dismissed as a simple cosmetic defect, although, in a substantial number of cases, no underlying cause is ever found. On physical examination, it is necessary to distinguish between pseudogynecomastia, which is breast enlargement caused by increased adipose tissue, and true gynecomastia (Fig. 120-1). In the latter condition, there is enlargement of the mammae. The glandular tissue can be palpated as radially arranged cords, usu¬ ally around the nipple, and may form a discrete button-like mass or merge gradually with the surrounding adipose tissue. The are¬ olae may be enlarged and convex, and the small areolar glands (glands of Montgomery) often are prominent. Frequently, the mammary papilla or nipple is enlarged and protuberant (Fig. 120-2). Breast tenderness (mastodynia) may be present, particu-

FIGURE 120-1. Gynecomastia in 30-year-old man who had unilateral testicular atrophy after postpubertal mumps orchitis.

Ch. 120: Gynecomastia

1201

HORMONAL CONTEXT OF GYNECOMASTIA

larly if the onset is recent. Although commonly bilateral and sym¬ metric, gynecomastia of any cause may be unilateral or markedly asymmetric, either initially or throughout its course (Fig. 120-3). Histologically, there may be hyperplasia of the epithelial or stro¬ mal cells or an increase in fibrous tissue. Tire appearance tends to correlate with the duration of the condition (Fig. 120-4). Thus, gynecomastia of more recent onset tends to show cellular hyper¬ plasia that, over time, progresses to increased fibrous tissue with¬ out increased cellularity, including new induction of type VI collagen.5-7 This increase in fibrous tissue explains why long¬ standing gynecomastia is so difficult to treat by nonsurgical means. Because so many conditions can cause gynecomastia, it is not unusual to encounter the condition clinically. In addition, several studies have reported a high prevalence of gynecomas¬ tia in the normal population (>40% of young, healthy men8) and in hospitalized patients.1 Often, gynecomastia represents the quiescent residual enlargement attributable to a condition that is no longer present.

Given the long-recognized role of estrogens in stimulating mam¬ mary growth, it is not surprising that high circulating estrogen lev¬ els of exogenous or endogenous origin can be associated with gynecomastia. More recently, however, gynecomastia has been noted in disorders with deficient androgen production even when estrogen production is normal, leading to the concept that gyneco¬ mastia often may be related to increases in the circulating estro¬ gen/androgen ratio rather than to absolute increases in estrogen per se. Finally, the occurrence of gynecomastia in conditions in which the androgen sensitivity of peripheral target tissues is reduced, even without significant changes in circulating andro¬ gens or estrogens, has refined this concept to suggest that gyneco¬ mastia is related to the resultant estrogen/androgen ratio effect at the breast. Although experimental evidence is inconclusive, this has proved to be a useful framework for evaluating gynecomastia. In men, the principal circulating androgenic activity resides in testosterone, which originates almost exclusively from testic¬ ular secretion. By contrast, the major estrogenic effect in men is related to circulating estradiol and estrone, most of which origi¬ nate from the conversion of circulating androgenic precursors to estrogens by peripheral tissues such as fat. Because a sub¬ stantial fraction of these androgen precursors is of adrenal ori¬ gin, an endocrine dysfunction limited to the testis can cause significant reductions in circulating androgens with less effect on circulating estrogens, and the resulting increase in the estro¬ gen/androgen ratio can lead to gynecomastia. This general scheme is thought to apply in a variety of testicular disorders. Primary increases in estrogen production also have second¬ ary effects that can further elevate the estrogen/androgen ratio and enhance gynecomastia: stimulation of sex hormone-binding globulin (SHBG) levels or suppression of androgen production by direct inhibition of testicular biosynthetic enzymes or by inhibition of luteinizing hormone (LH). Increased activity of the enzyme aromatase, which converts androgens to estrogens, has been noted in tissues from some patients with gynecomastia,9 suggesting that local production of estrogen within the breast may also play a contributory role. In addition to the role of androgens and estrogens, the role of gonadotropins in relation to gynecomastia needs to be con¬ sidered. LH and human chorionic gonadotropin (hCG) increase

FIGURE 120-3. Unilateral gynecomastia secondary to spironolactone therapy in 38-year-old man.

FIGURE 120-4. Histologic appearance of gynecomastia of relatively acute onset. There is epithelial proliferation of the ducts and loose peri¬ ductal fibroblastic tissue. There also is a mild inflammatory infiltrate. (Courtesy of Dr. Nirmal Saini.)

FIGURE 120-2. Gynecomastia in 36-year-old man with renal failure. Note enlarged and convex areolae and protuberant nipples.

1202

PART VIII: ENDOCRINOLOGY OF THE MALE

the secretion of estradiol from Leydig cells in the testis; there¬ fore, gynecomastia is particularly common in forms of testicu¬ lar dysfunction in which circulating gonadotropin levels are

TABLE 120-2. Drugs Associated with Gynecomastia*

elevated (primary testicular failure). Conversely, gynecomastia is less common when testicular dysfunction is accompanied by low circulating gonadotropin levels, as may occur in some pitu¬ itary disorders. Also, in the latter condition, coexistent impair¬ ment of adrenocorticotropic hormone (ACTH) release may decrease the adrenal output of the androgens that serve as pre¬ cursors for circulating estrogens, thus decreasing further the estrogen/androgen ratio. Hyperprolactinemia, which is implicated strongly in galactor¬ rhea (milky breast discharge), rarely is a cause of gynecomastia. However, disorders causing increases in the circulating estro¬ gen/androgen ratio may result in both hyperprolactinemia and gynecomastia as independent secondary effects. In addition, a patient with hyperprolactinemia may have gynecomastia from secondary hypogonadism consequent to the prolactin excess or from another cause totally unrelated to the hyperprolactinemia. Galactorrhea appears more likely to occur in a hyperprolactine¬ mic man who happens to have concurrent gynecomastia.

Angiotensin-converting enzyme inhibitors (captopril, enalapril) Antiandrogens (flutamide, cyproterone, zanoterone, finasteride, spironolac¬ tone) Antihypertensives (methyldopa, reserpine) Antiinfectives (minocycline, indinavir, ketoconazole, ethionamide, metro¬ nidazole, isoniazid [INH]) Arthritis drugs (auranofin, sulindac) Calcium-channel blockers (verapamil, nifedipine, diltiazem, amlodipine) Cancer chemotherapy drugs (alkylating agents, methotrexate) Cardiac drugs (digitalis, amiodarone) Central nervous system (CNS)-acting drugs (diazepam, phenytoin, phenothiazines, tricyclic antidepressants) Diuretics (spironolactone) Drugs of abuse (marijuana, heroin, methadone, amphetamines, anabolic steroids) Gastric motility enhancers (metoclopramide, domperidone) Miscellaneous (clomiphene, penicillamine, etretinate, theophylline) Peptide hormones (gonadotropins, growth hormone) Steroids (estrogens, aromatizable androgens, anabolic steroids) Ulcer drugs (cimetidine, omeprazole [rare])

CAUSES OF GYNECOMASTIA

'Some of these drug associations are clearly evident and others are anecdotal.

PHYSIOLOGIC GYNECOMASTIA Physiologic gynecomastia can be seen at the extremes of life. Neo¬ natal gynecomastia usually is transient, reflecting the effect of the high level of estrogens in the maternal-fetal unit. The gynecomas¬ tia seen in senescence may be related to several factors. Older people may be more likely to develop (or to have developed) some of the various disorders associated with gynecomastia. Fur¬ ther, aging seems to be associated with a progressive primary testicular dysfunction, including low-normal or low serum tes¬ tosterone levels, increases in serum LH and follicle-stimulating hormone (FSH), and normal or increased serum estrogen lev¬ els.10-11 The resulting increase in the circulating estrogen/andro¬ gen ratio then could account for gynecomastia. Some controversy revolves around whether this progressive loss of testicular func¬ tion is related solely to aging per se or also reflects underlying dis¬ eases to which older people become susceptible12 (see Chap. 199). In addition, aging is associated with accumulation of adipose tis¬ sue, which is an important source of the aromatase enzyme that converts circulating androgen precursors to estrogens. Physiologic gynecomastia is most common during sexual maturation; such pubertal gynecomastia can affect as many as two-thirds of normal adolescents. It usually lasts for only a few months to a few years but sometimes persists into adulthood. A wide variety of alterations in androgens, estrogens, or estrogen/ androgen ratios have been described in subjects with pubertal gynecomastia,13-16 but their etiology and causative relation to the breast enlargement remain largely speculative. Persistent puber¬ tal gynecomastia also has been called essential gynecomastia. In some cases, the breasts become female-appearing in size and conformation, and these otherwise hormonally normal adults have been said to have persistent pubertal macromastia.

TUMORS Tumors of the steroid-producing organs (adrenal, testis) are an uncommon cause of gynecomastia but one that must be ruled out in a male with breast enlargement (see also Chap. 219). Feminiz¬ ing adrenal tumors usually are malignant. Commonly, such tumors are large. Many are palpable, and they are easily seen by computed tomography or magnetic resonance imaging. Biochem¬ ically, they produce large amounts of various steroid precursors that can be detected as elevated plasma dehydroepiandrosterone sulfate (DHEAS) and serve as the substrate for peripheral conver¬ sion to estrogens. Serum estradiol levels are high.

In contrast to feminizing adrenal tumors, estrogen-producing tumors of the testis often are small and can be benign. If not pal¬ pable, they may be imaged by testicular ultrasound. Although estrogen-producing testicular tumors are usually of Leydig-cell origin, there is an association between gynecomastia, feminizing Sertoli cell tumors, and the Peutz-Jeghers syndrome.17 The gynecomastia associated with some tumors has been related to high levels of aromatase activity (which converts androgens to estrogens) within the tumor itself; this phenome¬ non has been noted in some testicular tumors18-19 as well as in hepatocellular carcinoma.20 Clinically, one should suspect an estrogen-producing neo¬ plasm if gynecomastia is of rapid onset, if serum estrogen levels are very high, or if a mass is found in the abdomen or testis. Often, the autonomous estrogen production has suppressed the pituitary, leading to low serum LH and FSH levels and, conse¬ quently, to secondary hypogonadism and testicular atrophy. More commonly, tumors lead to gynecomastia through the paraneoplastic production of gonadotropin, particularly hCG. This hormone and LH in high levels tend to stimulate testicular estrogen production disproportionate to androgen production, leading to increases in the circulating estrogen/androgen ratio. Testicular tumors are the most common neoplasm with clini¬ cally evident hCG production (7% of all testicular tumors are associated with gynecomastia21), but many other tumors can produce this hormone22 (see Chaps. 122 and 219). Serum estro¬ gen levels tend to be high, and serum hCG-p virtually always is elevated. Occasionally, the source of the ectopic hCG produc¬ tion is not evident, in which case catheterization of veins drain¬ ing various organs to determine the source of the hormone can be helpful. In such cases, one should pay particular attention to the possibility of an occult testicular tumor.23

DRUGS Medications commonly are implicated in gynecomastia24 (Table 120-2). For some drugs, studies have revealed the mechanism by which the estrogen/androgen ratio effect at the breast is increased. For exogenous estrogens, as in the treatment of prostate cancer, the mechanism of gynecomastia is self-evident. Other exposures to exogenous estrogen are not so apparent, however, such as estrogen-containing creams used by a spouse or mother or designed to treat baldness, foods prepared from estrogen-treated

Ch. 120: Gynecomastia animals, or occupational exposure during the manufacture of estrogen-containing medications.243 Crude marijuana extracts, but not the purified active ingredient tetrahydrocannabinol, interact with estrogen receptors, suggesting that marijuana-associated gynecomastia may reflect the presence of plant estrogens.25 Digi¬ talis preparations also may have estrogen-like effects. Other drugs may produce gynecomastia by interfering with androgen production or action, thus increasing the estrogen/ androgen ratio effect at the breast.253 The antifungal agent ketoconazole is a potent inhibitor of testosterone biosynthesis and can cause gynecomastia.26 However, because of its pharmacokinetics, the incidence of gynecomastia is low when the drug is given in a once-a-day dosage because this regimen allows the serum testoster¬ one levels to normalize before each succeeding dose. Traditional antiandrogens (cyproterone, flutamide), as well as the H^-blocker cimetidine, appear to cause gynecomastia by blocking androgen receptors at the breast rather than by decreasing androgen produc¬ tion; they cause an increased effective estrogen/androgen ratio in the breast tissue. This side effect of cimetidine is most common when high doses are used to treat Zollinger-Ellison syndrome and is seen much less frequently with other antiulcer drugs, such as ran¬ itidine or omeprazole. The blockade of androgen action at the breast by a commercial insecticide has been implicated in an epi¬ demic of gynecomastia in Haitian immigrants.27 The aldosterone antagonist spironolactone, frequently used as a diuretic, may block androgen receptors as well as interfere with androgen production, thereby leading to an increased effective estrogen/androgen ratio at the breast28 Here, too, the breast enlargement is seen most com¬ monly at high dosages, as in the treatment of primary hyperaldos¬ teronism. Finasteride, which is used in the treatment of prostate disorders and which inhibits 5-a reductase and reduces intracellu¬ lar levels of active androgen (dihydrotestosterone) in target tissues, has also been associated with gynecomastia29 (see Chap. 115). A wide variety of neurotransmitter agonists, antagonists, or modulators that are used to treat hypertension or psychiatric disorders have been associated with gynecomastia, but the nature and mechanism of this connection are largely unex¬ plored. Also, such agents commonly are associated with hyper¬ prolactinemia, which may cause secondary hypogonadism. An increasingly frequent cause of drug-related gynecomastia is cancer chemotherapeutic agents. Gynecomastia associated with such medications may be increasing in prevalence as their spec¬ trum of use is extended to nonmalignant conditions (e.g., gyneco¬ mastia following methotrexate treatment of rheumatoid arthritis). It has been known for many years that such drugs, particularly alkylating agents, are highly toxic to the spermatogenic epithe¬ lium, causing primary testicular failure with azoospermia, small testicles, and high serum LH and FSH levels.30 Serum testosterone levels, however, usually are normal or low normal, but in some cases of chemotherapy-related gynecomastia, serum estrogen lev¬ els have been elevated.31 A reasonable, but improved, theory is that chemotherapeutic agents produce compensated Leydig cell failure, with normal or low-normal serum testosterone levels maintained only with the stimulus of high serum LH. This increased serum LH then causes a relative increase in testicular estrogen output, leading to an increase in the circulating estro¬ gen/ androgen ratio and then to gynecomastia. Such chemother¬ apy-related gynecomastia can occur during or after cancer treatment, often resolves spontaneously, and has no prognostic significance with regard to the effectiveness of chemotherapy or recurrence of tumor. If the chemotherapy is given for a tumor that produces hCG, a chemotherapy-induced gynecomastia then must be differentiated from tumor recurrence by means of the measure¬ ment of serum hCG-(3, which does not cross-react with LH.

CONGENITAL DISORDERS Any congenital or acquired defect of androgen production or of androgen action at target tissues can cause gynecomastia. The most common congenital disorder associated with gynecomastia

1203

is Klinefelter syndrome, in which the abnormal karyotype (usu¬ ally 47,XXY) is associated with primary testicular failure (see Chap. 115). Serum free testosterone levels are low normal or frankly low, whereas serum estradiol levels are normal or ele¬ vated.32 The resulting increase in the circulating estrogen/andro¬ gen ratio leads to gynecomastia, which is seen in more than half of these patients. Moreover, the incidence of breast cancer is markedly increased in men with Klinefelter syndrome.33 Another congenital disorder leading to gynecomastia is anorchia (vanishing testis syndrome), in which no testes can be located. In this condition, the presence of the testis during fetal development may be inferred from the male phenotype of the external genitalia; the reasons for the subsequent testicular dis¬ appearance are unknown. In two other genetic disorders associated with the develop¬ ment of hypogonadism, namely myotonic dystrophy and sickle cell anemia, gynecomastia is uncommon (/, cup canned kidney beans

2 oranges >/2 cup canned kidney beans

OH CH,-CH»CHCOOH

TABLE 124-2. Representative Food Choices for Low-, Medium-, and High-Fiber Diets

I

NH,

NH,

1229

CH,—CHCOOH

CH,CH,—CHCOOH

I

NH,

NH,

NH,

Tryptophan HC= CCH,—CHCOOH

I

I

N

I

H

I

NH

NH,

Arginine

Lysine

NH, CH,CH,CH,CH,«CHCOOH

I

NH,

I

HN —C= NH

I

CH,CH,CH,—CHCOOH

I

NH,

NON-ESSENTIAL Serine

Alanine

Glycine

OH CH,COOH

CH,—CHCOOH

I

I

I

CH,—CHCOOH

I

NH,

NH,

NH,

Cysteine

Tyrosine

SH

NH,

I

CH,—CHCOOH

I

CH,—CHCOOH

HO30 with no concomitant obesity-related risk factors or diseases, and for patients with a BMI of >27 with concomitant obesity-related risk factors or dis¬ eases. Weight loss drugs should never be used without concom¬ itant lifestyle modifications. Continual assessment of drug therapy for efficacy and safety is necessary. If the drug is effica¬ cious in helping the patient lose and/or maintain weight loss and there are no serious adverse effects, it can be continued. If not, it should be discontinued."2 Two medications have been approved for long-term use by the FDA: sibutramine (Meridia), a norepinephrine and seroto¬ nin reuptake inhibitor that enhances satiety,40 and orlistat (Xenical), an inhibitor of pancreatic lipase.41 Thyroid hormone, diuretics, and digitalis should be used in the treatment of obesity only when specific indications exist (e.g., hypothyroidism, edema, hypertension, or congestive heart fail¬ ure), and never for the achievement of weight reduction. Diabetes and hypertension occurring in obesity should be treated aggres¬ sively. Human chorionic gonadotropin and growth hormone have no place in the treatment of obesity. SURGICAL TECHNIQUES Surgical techniques, such as the vertical-banded gastroplasty and Roux en Y gastric bypass, have been used for the treatment of morbid obesity.42 An NIH Consensus Conference on Gastrointes¬ tinal Surgery for Severe Obesity had evaluated the need for this treatment.43 Surgery was only recommended for cooperative patients who have been at least twice their ideal body weight for at least 5 years, have failed all other treatment modalities, have no psychiatric, alcohol, or drug abuse problems, and have no medical contraindications to surgery. Substantial improvement in comorbid conditions has been reported; however, short- and long-term complications of surgery are common, and the risk/ benefit ratio must be carefully examined for each patient. Refer¬ ral of suitable patients to a medical center specializing in this type of procedure is recommended. Of note, laparoscopic tech¬ niques to accomplish stomach restriction are under investigation. However, their safety and efficacy await evaluation.

CONCLUSION The treatment of obesity is unsatisfactory and frequently unsuccessful. Nevertheless, the evidence is overwhelming that obesity is a significant risk factor for many illnesses. The physi¬ cian and the obese patient must recognize that nothing short of a change in lifestyle (nutritional change, increase in physical activity, diminution of stressful life situations, etc.) is needed for treatment. No pill, formula, or unusual dietary manipula¬ tion is available to provide certain success. It may not be possi¬ ble to correct all of the obesity; however, even some weight loss may ameliorate concurrent diabetes, hypertension, or other complicating diseases. The recent rapid growth of biomedical

science and increasing attention to the understanding of the basics of energy metabolism may eventually provide further insights and better therapies for this unfortunate situation.

REFERENCES

'

1. Bray GA, Bouchard C, James WP. Handbook of obesity. New York: Marcel Dekker Inc, 1995. 2. Clinical guidelines on the identification, evaluation, and treatment of over¬ weight and obesity in adults—the evidence report. Obes Res 1998; 6(Suppl 2):51S. 3. NIH Technology Assessment Conference Panel. Methods for voluntary weight loss and control. Ann Intern Med 1993; 119:764. 4. Brozek J, Henschel A, eds. Techniques for measuring body composition: proceedings of a conference. Quartermaster Research and Engineering Center, January 22-23, 1959. Washington, DC: National Academy of Sciences-National Research Council, 1961. 5. Anonymous. Proceedings of a panel on the clinical uses of whole-body counting, Vienna, June 28-July 2, 1965. Vienna: International Atomic Energy Agency, 1966. 6. Wing RR, Marcus MD, Epstein LH, et al. Long-term effects of modest weight loss in type II diabetic patients. Arch Intern Med 1987; 147:1749. 7. Eliahou HE, Iana A, Gaon T. Body weight reduction necessary to attain normotension in the overweight hypertensive patient. Int J Obese 1981; 5(Suppl 1):157. 8. Kalkhoff RK, Hartz AH, Rupley D, et al. Relationship of body fat distribu¬ tion to blood pressure, carbohydrate tolerance and plasma lipids in healthy obese women. J Lab Clin Med 1983; 102:61. 9. Bjomtorp P. Regional obesity. In: Bjomtorp P, Brodoff B, eds. Obesity. Phila¬ delphia: JB Lippincott Co, 1992; 579-586. 10. Pouliot MC, Despres JP, Lemieux S, et al. Waist circumference and abdomi¬ nal sagittal diameter: best simple anthropometric indexes of abdominal visceral adipose tissue accumulation and related cardiovascular risk in men and women. Am J Cardiol 1994; 73(7):460. 11. Van Baeyer HC. Maxwell's dream. New York: Random House Inc, 1998. 12. Kleiber M. The fire of life: an introduction to animal energetics. New York: Robert E Krieger Publishing Co, 1975. 13. Newburgh LH, Johnston MW. Endogenous obesity—a misconception. Ann Intern Med 1993; 3:815. 14. Leibel RL, Rosenbaum M, Hirsch J. Changes in energy expenditure result¬ ing from altered body weight. N Engl J Med 1995; 332:621. 15. Rosenbaum M, Leibel RL, Hirsch J. Obesity. Medical progress. N Engl J Med 1997; 337:396. 16. Hirsch J, Leibel RL. The genetics of obesity. Hosp Pract 1998; 33(3):55. 17. Ristow M, Muller-Wieland D, Pfeffer A, et al. Obesity associated with a mutation in a genetic regulator of adipocyte differentiation. N Engl J Med 1998; 339:953. 18. Ricquier D, Fleury C, LaRose M, et al. Contributions of studies on uncou¬ pling proteins to research on metabolic disease. J Intern Med 1999; 245:637. 19. Freake HC. A genetic mutation in PPARy is associated with enhanced fat cell differentiation: implications for human obesity. Nutr Rev 1999; 1:154. 20. Salans LB, Cushman SW, Weismann RE. Studies of human adipose tissue: adipose cell size and number in nonobese and obese patients. J Clin Invest 1973; 52:929 21. Hirsch J, Batchelor BR. Adipose tissue cellularity in human obesity. Clin Endocrinol Metab 1976; 5(2):299. 22. Nimrod A, Ryan KJ. Aromatization of androgens by human abdominal and breast fat tissue. J Clin Endocrinol Metab 1975; 40:367. 23. Salans LB, Knittle JL, Hirsch J. Obesity, glucose intolerance and diabetes mellitus. In: Ellenberg M, Rifkin H, eds. Diabetes mellitus: theory and prac¬ tice. New York: Medical Examination Publishing, 1983:469. 24. Boden G. Role of fatty acids in the pathogenesis of insulin resistance and NIDDM. Diabetes 1990; 39:226. 25. Rebrin K, Steil GM, Getty L, Bergman RN. Free fatty acids as a link in the regu¬ lation of hepatic glucose output by peripheral insulin. Diabetes 1995; 44:1038. 26. Paolissa G, Gambardeira A, Amato L, et al. Effect of long-term fatty acid infusion on insulin secretion in healthy subjects. Diabetologia 1995; 38:1295. 27. Segal KR, Landt M, Klein S. Relationship between insulin sensitivity and plasma leptin concentration in lean and obese man. Diabetes 1996; 45:988. 28. Auwerx J, Staels B. Leptin. Lancet 1998; 351:737. 29. Hotamisligil GS, Spiegelman BM. Tumor necrosis factor, as key component in the obesity-diabetes link. Diabetes 1994; 43:1271. 30. Frittitta L, Youngren JF, Sbraccia P, et al. Increased adipose tissue PC-1 pro¬ tein content, but not tumor necrosis factor-gene expression, is associated with a reduction of both whole body insulin sensitivity and insulin recep¬ tor tyrosine-kinase activity. Diabetologia 1997; 40:282. 31. Pan XR, Li GW, Hu YH, et al. Effects of diet and exercise in preventing NIDDM in people with impaired glucose tolerance: the Da Qing IGT and Diabetes Study. Diabetes Care 1997; 20:537. 32. Drenick E. The prognosis of conventional treatment in severe obesity. In: Bjomtorp P, Cairella M, Howard A, eds. Recent advances in obesity research III. London: John Libbey, 1981:80. 33. National Task Force on the Prevention and Treatment of Obesity. Very low calorie diets. JAMA 1993; 270:967.

Ch. 127: Starvation 34. Thomas PR. Weighing the options—criteria for evaluating weight manage¬ ment programs. Washington, DC: National Academy Press, 1995. 35. Brownell KD, Greenwood MRC, Stellar E, Shrager EC. The effects of repeated cycles of weight loss and regain in rats. Physiol Behav 1986; 38:459. 36. Reed GW, Hill JO. Weight cycling: a critical review of the animal literature. Obes Res 1993; 1:392. 37. Wing RR. Weight cycling in humans: a critical review of the literature. Ann Behav Med 1992; 14:113. 38. Lissner L, Odell PA, D'Agostino RB, et al. Variability of body weight and health outcomes in the Framingham population. N Engl J Med 1991; 324:1839 39. Weintraub M. Long-term weight control: the National Heart, Lung, and Blood Institute funded multimodal intervention study. Clin Pharmacol Ther 1992; 51:581. 40. Fanghanel G, Cortinas L, Sanchez-Reyes L, Berber A. A Clinical trial of the use of sibotramine for the treatment of patients suffering essential obesity. Int J Obes Related Metab Disord 2000; 24:144. 41. Heymsfield SB, Segal KR, Hauptman J, et al. Effects of weight loss with orlistat on glucose tolerance and progression to type 2 diabetes in obese adults. Arch Intern Med 2000; 160:1321. 42. Balsiger BM, Murr MM, Poggio JL, Sarr MG. Bariatric surgery. Surgery for weight control in patients with morbid obesity. Med Clin North Am 2000; 84:477. 43. Gastrointestinal surgery for severe obesity: Proceedings of an NIH Consen¬ sus Development Conference, March 25-27, 1991. Am J Clin Nutr 1992; 55(Suppl 2):615S.

CHAPTER

127

STARVATION RUTH S. MACDONALD AND ROBERT J. SMITH Protein-energy malnutrition is a major public health problem in many parts of the world. Severe protein-energy malnutrition is characterized by cessation of growth in children, body wasting, mental apathy, and loss of pigmentation of the hair and skin.1 When both dietary energy sources and protein are deficient, the syndrome called marasmus develops, characterized by general¬ ized emaciation, an absence of subcutaneous fat, muscle wasting, and wrinkled and dry skin (Fig. 127-1, left). A severe deficiency of protein, with either low or adequate energy intake, causes the syndrome called kwashiorkor, characterized by emaciated limbs as evidence of a loss of lean body mass but a swollen abdomen sec-

1247

ondary to edema and hepatomegaly2 (see Fig. 127-1, right). Fea¬ tures of marasmus and kwashiorkor often occur simultaneously or at different times in one person. Both conditions are associated with reduced resistance to disease and infection, and with dimin¬ ished capacity for recovery from concurrent illnesses.1'3'4 Periods of partial or total starvation and resulting malnutri¬ tion often occur during the course of catabolic illnesses or after injury. During starvation in otherwise normal persons, there is a coordinated adaptive response within different tissues such that nutrient stores (glycogen, triglycerides, and protein) are used efficiently. These metabolic changes occur in response to alterations in nutrient availability and in the levels of several hormones. In treating patients with both malnutrition and an additional illness, it is important to understand these normal nutrient and hormonal control mechanisms and the possible alterations in the normal adaptive responses.

METABOLIC RESPONSE TO STARVATION The metabolic adaptation to starvation can be divided into four temporal phases: the postabsorptive period (5-6 hours after a meal), early starvation (1-7 days), intermediate starvation (1-3 weeks), and prolonged starvation (more than 3 weeks). During the first three phases, a series of metabolic changes occurs pro¬ gressively, until a more stable, near steady state is reached dur¬ ing prolonged starvation.5 Metabolic fuel use and production by liver, muscle, adipose tissue, kidney, and brain during the four phases of starvation are summarized in Table 127-1. During the postabsorptive period and early in starvation, glucose is readily available from hepatic stores of glycogen and is consumed as a principal fuel by the brain, muscle, kidney, and other tissues. As hepatic glycogen stores become depleted during the first 24 hours of fasting, plasma levels of glucose decrease modestly, gluconeogenesis becomes a progressively more important source of glucose, and alternative substrates begin to replace glucose as a metabolic

TABLE 127-1. Adaptations in Metabolic Fuel Use and Production during Progressive Starvation Postabsorptive, 5-6 hr Glucose is primary fuel in tissues such as skeletal muscle, kidney, and brain. Primary source of glucose is hepatic glycogenolysis. Early starvation, 1-7 d Fatty acids assume increasing importance as fuels for muscle and kidney. Ketone bodies partially replace glucose as a fuel for the brain. Primary source of fatty acids is adipose tissue triglyceride. Ketone bodies are synthesized in the liver from fatty acid precursors. Hepatic glycogen stores become depleted and gluconeogenesis from amino acids and glycerol becomes the primary source of glucose. Intermediate starvation, 1-3 wk Fatty acids and ketone bodies predominate as fuels for muscle and kidney. Ketone bodies become more important and glucose less important as fuels for brain. Adipose tissue lipolysis and hepatic ketogenesis increase. Glucose is derived from both hepatic and renal gluconeogenesis. Prolonged starvation, >3 wk Fatty acids and ketone bodies remain the predominant fuels for muscle and kidney. Ketone body utilization further increases and glucose utilization decreases in brain. Adipose tissue triglyceride becomes the primary fuel source. Hepatic gluconeogenesis decreases as muscle proteolysis and the release

FIGURE 127-1.

Typical appearance of children with advanced protein-

energy malnutrition.

Left,

Marasmus.

Right,

Kwashiorkor. (Photograph

from Kivu region, Republic of the Congo, by R. J. Smith.)

of amino acids from muscle decreases. Renal gluconeogenesis from glutamine increases.

1248

PART IX: DISORDERS OF FUEL METABOLISM

fuel.6 It has been estimated that 200 mg/dL)

OR Fasting plasma glucose concentration of >126 mg/dL on more than one occasion

OR Glucose concentration at 2 hours of >200 mg/dL during an oral glucosetolerance test One or more of these diagnostic criteria must be met on a subsequent day for diagnosis of diabetes.

GESTATIONAL DIABETES Two or more of the following plasma glucose concentrations after fasting and a 100-g oral glucose dose: Fasting >105 mg/dL 1 hour >190 mg/dL 2 hour >165 mg/dL 3 hour >145 mg/dL

IMPAIRED GLUCOSE TOLERANCE Fasting >110 mg/dL and 140 mg/dL and 800 patients with type 1A diabetes, the author and his associates have typed five who have had DQB1*0602, and one 0602 patient had the polyendocrine autoimmune type I syndrome with its coexistent mucocutaneous candidiasis. The manner in which this DQ molecule protects against type 1A diabetes is unknown. The highest-risk DQ alleles associated with type 1A dia¬ betes are DQA1*0501/DQB1*0201 associated with DR3, and DQA1*0301/DQB1*0302 associated with DR4. Persons in the general population heterozygous for these two alleles have a risk of diabetes similar to that of an offspring of a parent with type 1 diabetes (-1:16). Such persons make up -2% of the U.S. population, but account for 40% of patients with type 1A diabetes. A research program to identify these per¬ sons at birth is under way. Subsequent studies will define the timing and sequence of the appearance of autoantibodies, with the long-term goal of predicting type 1A diabetes in the general population. The fact that the incidence of diabetes in HLA-identical sib¬ lings (16%) is less than that in identical twins (40-50%) strongly suggests that another gene outside the MHC contributes to dia¬ betes susceptibility. This is similar to the genetics of diabetes susceptibility of BB rats and NOD mice. When either of these animals is crossed with normal-strain animals, only offspring inheriting a "diabetogenic" MHC gene and other autosomal genes develop diabetes. Multiple other genes influence the sus¬ ceptibility of NOD mice, and a gene on chromosome 4 influ¬ ences susceptibility associated with a T-cell immunodeficiency of BB rats (see Table 136-3). Too many false-positive results occur with HLA typing (3040% of the general population express DR3 or DR4) for this study to aid in clinical decision making. Moreover, HLA typing is relatively expensive and can never indicate a risk for diabetes greater than that of DR3/4 HLA-identical siblings (25-40%). (Within a family, nondiabetic siblings who are HLA-identical by serologic typing to a sibling with type 1A diabetes are usually [>99% of the time] identical at all HLA loci; nevertheless, their risk of developing diabetes is only ~17%.) The imprecision of HLA typing for predicting diabetes even within families proba¬ bly results in part from the inability to identify another genetic linkage group for type 1A diabetes. Even when such a linkage group is discovered, genetic prediction of the risk of type 1A diabetes cannot exceed the concordance rate of identical twins (50%). In addition to alleles within the MHC on chromosome 6, alleles of the insulin gene on chromosome 11 contribute to dia¬ betes susceptibility.29 Approximately 90% of persons with type

1311

1A diabetes are homozygous for a common allele compared with 60% of the general population. Some controversy exists regarding whether this insulin-gene polymorphism shows "imprinting" (i.e., a differential influence on diabetes suscepti¬ bility depending on whether it is inherited from the father or the mother). These insulin alleles differ not in their coding sequence but in the 5' region of the gene. Thus, differential reg¬ ulation of expression of insulin, particularly for expression in the thymus, may have an important influence on diabetes risk. In addition to genes within the MHC and insulin alleles, other genes probably influence diabetes risk. With molecular and computational tools provided by the Genome Project, and shared national repositories of cell lines and DNA from families with multiple affected members, the search for these additional genes is under way and has identified multiple loci that may be associated with diabetes risk. Environmental factors may not be necessary for the triggering of type 1A diabetes. As in many cancers, somatic mutations may randomly trigger disease expression. Testing of this hypothesis will likely depend on the localization of major susceptibility genes. GENETIC AND ENVIRONMENTAL FACTORS The lack of 100% concordance for type 1A diabetes in identical twins has been used to argue that environmental factors must contribute to the development of this disease.30-31 One environ¬ mental factor known to increase the incidence of type 1A diabe¬ tes is congenital rubella. After prenatal infection with this virus, as many as 20% of children later develop diabetes. As in those who spontaneously develop type 1A diabetes, individuals with congential rubella who later develop diabetes express HLA alleles DR3 and DR4.32 These children often also have thyroidi¬ tis and other immunologic disorders (e.g., agammaglobuline¬ mia) in association with an abnormal T-lymphocyte phenotype that differs from that in both normal persons and usual patients with type 1A diabetes. No epidemiologically defined environmental factors other than congenital rubella have been clearly associated with type 1A diabetes. Viral infections, in particular those that occur close to the time of onset of overt diabetes, are known to precipitate hyperglycemia (secondary to insulin resistance associated with infection), but they are unlikely to play a primary pathogenic role. Although coxsackievirus B4 has been isolated from the pancreas of a child with recent-onset diabetes,33 the pancreas had multiple pseudoatrophic islets (islets with no B cells but abundant A and D cells) with no inflammation, indicating chronic B-cell destruction preceding the viral infection. Any search for environmental factors that may trigger autoimmu¬ nity, such as drugs, unknown viruses, or dietary components (e.g., milk proteins), must focus on factors that act months to years before the onset of diabetes rather than on acutely diabe¬ togenic factors. ISLET CELL ANTIBODIES AND OTHER IMMUNOLOGIC MARKERS Approximately 5% of first-degree relatives of patients with type 1A diabetes also develop diabetes. Immunologic and endocrinologic assays capable of identifying those relatives most likely to develop diabetes and predicting approximately when overt diabetes will occur include the immunofluorescence assay for cytoplasmic islet cell antibody, the results of which are positive in 70% to 80% of patients with new-onset type 1A diabetes. Some assays for islet cell antibodies34-36 and a few radioimmu¬ noassays for antiinsulin autoantibodies37,38 have the requisite specificity to identify persons at high risk. Examples are the complement-fixation tests for cytoplasmic islet cell antibodies, variants of the standard cytoplasmic islet cell antibody assays, fluid-phase antiinsulin autoantibody assays, and autoantibod¬ ies to a 64-kDa islet protein (predominantly antibodies to glutamic acid decarboxylase [GAD]) and antibodies to a mole¬ cule termed ICA512(IA-2).38 Autoantibody assays using defined

1312

PART IX: DISORDERS OF FUEL METABOLISM

% Not Diabetic

FIGURE 136-2. Progression to type 1A diabetes of first-degree relatives of patients with diabetes based on the number of defined antiislet autoantibodies (Ab; of GAD65 [glutamic acid decarboxylase], ICA512, and insulin). One relative (of -500) lacking autoantibodies progressed to diabetes. (From Verge CF, Gianani R, Kawasaki E, et al. Prediction of type 1 diabetes in first-degree relatives using a combination of insulin, GAD and ICA512bdc/IA-2 autoantibodies. Diabetes 1996; 45:926.)

islet autoantigens (GAD65, ICA512, or insulin) have improved so much (Fig. 136-2) that for most clinical settings the difficultto-standardize cytoplasmic islet cell antibody assay should be abandoned. The presence of antiislet cell antibodies can precede the development of overt diabetes by more than a decade34'39 (Fig. 136-3). HLA typing of antibody-positive relatives and even antibody-positive "normal" persons indicates that they have the same HLA distribution as do patients with type 1A diabe¬ tes, and within 7 years, -50% develop overt diabetes. The appearance of autoantibodies to human insulin also can precede by years the development of type 1A diabetes.40 Anti¬ insulin antibodies are present in both cytoplasmic antibody¬ positive and antibody-negative persons who develop diabetes; these antibodies provided the first radioimmunoassay aid for

FIGURE 136-3. Development of insulin autoantibodies and cytoplas¬ mic islet cell antibodies (ICA) in overt diabetes. (Adapted from Soeldner JS, Tuttleman M, Srikanta S, et al. Insulin dependent diabetes mellitus and initiation of autoimmunity: islet cell autoantibodies, insu¬ lin autoantibodies and beta cell failure. N Engl J Med 1985; 313:893.)

predicting type 1A diabetes. Antiinsulin autoantibodies are found in -60% of persons who develop diabetes. When this test is combined with assays for cytoplasmic islet cell antibodies, 90% of patients with new-onset type 1A diabetes are found to have evidence of autoimmune disease. In addition to the presence of cytoplasmic islet cell antibod¬ ies and insulin autoantibodies, many immunologic abnormali¬ ties are present in patients with type 1A diabetes and their relatives.41 Many of these abnormalities (e.g., presence of antithyroglobulin and microsomal antibodies, antibodies to singlestranded DNA, antibodies to the surface of rat islet cells and a rat insulinoma cell line) are inherited independently of the HLA susceptibility to type 1A diabetes and are present in as many as 30% of first-degree relatives. Such abnormalities pro¬ vide relatively little prognostic information but appear to be related to the autoimmune background of type 1A diabetes. A major advance in the past several years has been the bio¬ chemical characterization of a series of islet autoantigens, including insulin, GAD (a major component of the 64-kDa autoantigen),42 carboxypeptidase H,43 a milk-related islet pro¬ tein (ICA69),44 ICA512,45 and ganglioside GM2-1.46 Biochemical assays are now available that use recombinant human proteins to measure antibodies to insulin, GAD, and ICA512. When just these three assays are used, >98% of patients with new-onset type 1A diabetes and prediabetes express at least one antibody, and >80% express two or more. Specificity and sensitivity are much higher with these biochemical assays than with cytoplas¬ mic islet cell antibody testing. In contrast to cytoplasmic islet cell antibody testing, with its inherent problems of reproduc¬ ibility, biochemical determination of autoantibodies is remark¬ ably stable in the prediabetic phase. International workshops to standardize insulin and GAD radioassays are under way. Such assays should rapidly replace standard cytoplasmic islet cell antibody testing in both the diagnosis and prediction of type 1A diabetes. FIRST-PHASE INSULIN SECRETION AS AN INDEX OF EARLY TYPE 1 DIABETES MELLITUS Approximately 3% of nondiabetic relatives of patients with type 1A diabetes have positive results on screening assays for islet cell autoantibodies. When such antibodies are detected, intravenous glucose-tolerance testing can be used to assess first-phase insulin release as a measure of subclinical B-cell dys¬ function (Fig. 136-4). The loss of first-phase insulin secretion, as well as its rate of fall, aids in predicting the time of onset of overt diabetes.47,48 At the time of initial detection of islet cell antibodies, one of four patients has first-phase insulin secretion below the first percentile of normal persons. Almost all persons who develop type 1A diabetes lose first-phase insulin secretion before they develop overt diabetes. For patients with initially normal insulin release, intravenous glucose-tolerance testing is performed again in 3 to 6 months and, depending on its stabil¬ ity, at subsequent 3- to 12-month intervals. Immunologically and endocrinologically, persons with abnormal results may be alerted to the risk of type 1A diabetes and advised concerning routine home monitoring for glucosuria or capillary blood glu¬ cose determination. To aid in predicting the time of onset of type 1A diabetes among antibody-positive relatives of persons with type 1A dia¬ betes, the following mathematic formula has been developed: years to diabetes = -0.12 + 1.35 x loge (insulin secretion) -0.59 loge (insulin autoantibody concentration). This simple formula appears to account for -50% of the variance in the time of dia¬ betes onset.48 Immunologic assays also are being used to aid in the classifi¬ cation of patients with diabetes. Insulin dependence is a physi¬ ologic state that can evolve slowly, even in type 1A diabetes, from a stage in which hyperglycemia is controlled with diet or oral medication to a stage in which death occurs in the absence

Ch. 136: Classification, Diagnostic Tests, and Pathogenesis of Type 1 Diabetes Mellitus

1313

IMPLICATIONS FOR PREVENTIVE THERAPY

FIGURE 136-4. Loss of first-phase insulin secretion in a prediabetic twin with islet cell antibody. The y-axis gives insulin concentrations at the times indicated after the intravenous injection of glucose. Initial phase of insulin release (1 and 3 minutes) is progressively lost. (DM, diabetes mellitus; F, fasting.) (Adapted from Srikanta S, Ganda OP, Eisenbarth GS, Soeldner JS. Islet cell antibodies and beta cell function in monozygotic triplets and twins initially discordant for type I diabetes. N Engl J Med 1983; 308:322.)

of insulin therapy (Fig. 136-5). Antiislet antibodies are found in as many as 10% of patients with classic type 2 diabetes at the time of diagnosis, and over the ensuing 5 years, many of these patients become insulin dependent.35 Epidemiologic data from Japan, Pittsburgh, the Netherlands, and Poland indicate that 1 in 200 children die of ketoacidosis at the time of diagnosis of their diabetes. Such deaths probably could be prevented by early detection and treatment of diabetes.

(? Precipitating event)

The newer immunologic knowledge concerning type 1A diabe¬ tes and the success of a wide variety of immunotherapies in preventing the diabetes of BB rats and NOD mice have led to trials of immunotherapy in patients with recent-onset type 1A diabetes, and to a few trials in persons at high risk for the development of type 1A diabetes. These trials indicate that lim¬ iting B-cell destruction is possible. Toxic side effects of the most powerful (and most effective) drugs are a serious problem, however. For example, cyclosporine A is nephrotoxic at the dosages that appear to be required to induce remissions of type 1A diabetes,49-50 and azathioprine may be associated with epi¬ thelial malignancies. Prednisone given after the onset of type 1A diabetes is ineffective, and a series of other therapies, such as plasmapheresis and treatment with antithymocyte globulin or monoclonal antibody T12, produce no long-term benefit. A series of new agents that are not significantly immuno¬ suppressive yet are able to limit B-cell destruction in animal models of type 1A diabetes are being studied. All immunologic attempts to limit B-cell destruction are considered investiga¬ tional and should be used only under the oversight of a human investigation committee.51 Trials investigating the prevention of type 1A diabetes and the amelioration of further B-cell loss after diabetes onset are concentrating on nonimmunosuppressive therapies. Such tri¬ als include administration of the vitamin nicotinamide,52 which modestly delays diabetes onset in NOD mice; vaccina¬ tion with bacille Calmette-Guerin53; administration of oral insulin54; and therapy with parenteral insulin.55 Nicotinamide may have an effect by limiting free radical damage to B cells. In the NOD mouse model, a single injection of bacille Cal¬ mette-Guerin (BCG) prevents diabetes but not insulitis. Such therapy may limit B-cell destruction by altering cytokines pro¬ duced by infiltrating T cells. A completed German trial of nic¬ otinamide found no benefit, but a larger European trial is continuing. In randomized trials, BCG had no beneficial effect. Oral administration of insulin delays or prevents type 1 diabetes in NOD mice. Its effect is likely due to the generation of T cells (by peptides of insulin present in the intestinal mucosa) that suppress inflammation. The most dramatic pre¬ vention of diabetes in both the NOD mouse and the BB rat has been obtained with parenteral administration of insulin. Such therapy prevents not only diabetes, but also infiltration of islets by T cells and destruction of B cells. A small pilot trial51 of intravenous insulin and low-dose subcutaneous insulin for the prevention of diabetes in high-risk relatives of patients with diabetes suggests that such therapy may delay the onset of type 1 diabetes, and a large U.S. prevention trial (DPT-1 [Diabetes Prevention Trial-1]) is under way.

AUTOIMMUNE POLYGLANDULAR FAILURE

FIGURE 136-5. Stages in the development of type 1 diabetes, begin¬ ning with genetic predisposition and ending with insulin-dependent diabetes with essentially complete B-cell destruction. (Adapted from Eisenbarth GS. Type I diabetes mellitus: a chronic autoimmune disease. N Engl J Med 1986; 314:1360.)

Approximately 20% of patients with type 1A diabetes develop other organ-specific autoimmune diseases (see Chap. 197), such as celiac disease. Graves disease, hypothyroidism, Addison disease, and pernicious anemia.56'57 Some patients develop multiple disorders as a part of two inherited polyendocrine autoimmune syndromes (type I and type II). The type I syn¬ drome usually has its onset in infancy, with hypoparathyroid¬ ism, mucocutaneous candidiasis, and, somewhat later, Addison disease and other organ-specific disorders. Fifteen percent of these children develop type 1A diabetes. This disorder is inher¬ ited in an autosomal recessive manner with no FILA association due to mutations of a gene (AIRE) located on chromosome 21. This mutated gene codes for a DNA-binding protein that is expressed in the thymus. The type II polyendocrine autoim¬ mune syndrome (Addison disease, type 1 diabetes [50% of patients], Graves disease, hypothyroidism, myasthenia gravis, and other organ-specific diseases) is strongly FILA-associated

1314

PART IX: DISORDERS OF FUEL METABOLISM

and has an onset from late childhood to middle age. In these families, a high prevalence of undiagnosed organ-specific auto¬ immune disease is seen, and, at a minimum, thyroid function tests should be performed in the first-degree relatives of these patients. Biochemical evaluation for adrenal insufficiency and pernicious anemia should be performed if any suggestive symptom or sign is present (e.g., decreasing insulin require¬ ments can herald the development of Addison disease in a patient with type 1 diabetes before electrolyte abnormalities or hyperpigmentation develop). Excellent autoantibody tests can facilitate the detection of Addison disease (21-hydroxylase autoantibodies) or celiac disease (transglutaminase autoanti¬ bodies) in patients with type 1A diabetes. As many as 1 in 200 patients with type 1A diabetes develop Addison disease, and 1 in 20 develop celiac disease.

REFERENCES 1. Report of the Expert Committee on the Diagnosis and Classification of Dia¬ betes Mellitus. Diabetes Care 1997; 20:1183. 2. World Health Organization Expert Committee on Diabetes Mellitus. Sec¬ ond report. Geneva: World Health Organization, 1980:646. Technical report series 1980. 3. Marble A, Ferguson BD. Diagnosis and classification of diabetes mellitus and the non-diabetic melliturias. In: Marble A, Krall LP, Bradley RF, et al., eds. Joslin's diabetes mellitus, 12th ed. Philadelphia: Lea & Febiger, 1985:332. 4. Polonsky KS, Stuns J, Bell GI. Non-insulin-dependent diabetes mellitus: a genetically programmed failure of the beta cell to compensate for insulin resistance. N Engl J Med 1996; 334:777. 5. Nelson RL. Oral glucose tolerance test: indications and limitations. Mayo Clin Proc 1988; 63:263. 6. Ganda OP, Srikanta S, Brink SJ, et al. Differential sensitivity to beta cell secretagogues in "early" type I diabetes. Diabetes 1984; 33:516. 7. Bergman RN, Finegood DJ, Ader M. Assessment of insulin sensitivity in vivo. Endocr Rev 1985; 6:45. 8. Fajans SS, Conn JW. An approach to the prediction of diabetes mellitus by modification of the glucose tolerance test with cortisone. Diabetes 1954; 3:296. 9. Koenig RJ, Cerami A. Hemoglobin Alc and diabetes mellitus. Annu Rev Med 1980; 31:29. 10. Starkman HS, Soeldner JS, Gleason RE. Oral glucose tolerance—relation¬ ship with hemoglobin Alc. Diabetes Res Clin Pract 1987; 6:343. 11. Garg SK, Potts RO, Ackerman NR, et al. Correlation of fingerstick blood glucose measurements with Gluco Watch biographer glucose results in young subjects with type 1 diabetes. Diabetes Care 1999; 22:1708. 12. Agner E, Thorsteinssen B, Eriksen M. Impaired glucose tolerance and dia¬ betes mellitus in elderly subjects. Diabetes Care 1982; 5:600. 13. Gepts W. The pathology of the pancreas in human diabetes. In: Adreani D, Federlin KF, DiMario U, Heding LG, eds. Immunology in diabetes. Lon¬ don: Kimpton Publishers, 1984:21. 14. Gepts W. Islet cell morphology in type I and type II diabetes. In: Irvine WJ, ed. Immunology in diabetes. Edinburgh: Teviot Scientific Publications, 1982:255. 15. Eisenbarth GS. Type I diabetes mellitus: a chronic autoimmune disease. N Engl ] Med 1986; 314:1360. 16. Makino S, Harada M, Kishimoto Y, Hayashi Y. Absence of insulitis and overt diabetes in athymic nude mice with NOD genetic background. Jikken Dobitsu Exp Anim 1986; 35:495. 17. Inoue H, Tanizawa Y, Wasson J, et al. A gene encoding a transmembrane protein is mutated in patients with diabetes mellitus and optic atrophy (Wolfram syndrome). Nat Genet 1998; 20:143. 18. Mordes JP, Greiner DL, Rossini AA. Animal models of autoimmune diabe¬ tes mellitus. In LeRoith D, Taylor SI, Olefsky JM, eds. Diabetes mellitus. Philadelphia: Lippincott-Raven Publishers, 1996:349. 19. Feingold KR, Lee TH, Chug MY, et al. Muscle capillary basement mem¬ brane width in patients with Vacor-induced diabetes mellitus. J Clin Invest 1986; 78:102. 20. Jackson RA, Buse JB, Rifai R, et al. Two genes required for diabetes in BB rats. J Exp Med 1984; 159:1629. 21. Nepom GT, Kwok WW. Perspectives in diabetes: molecular basis for HLADQ associations with IDDM. Diabetes 1998; 47:1177. 22. Todd JA. From genome to aetiology in a multifactorial disease, type 1 dia¬ betes. Bioessays 1999; 21:164. 23. Bellgrau D, Eisenbarth GS. Immunobiology of autoimmunity. In: Eisen¬ barth GS, ed. Molecular mechanisms of endocrine and organ specific autoimmunity. Austin: RG Landes Company, 1991:1. 24. Redondo MJ, Kawasaki E, Mulgrew CL, et al. DR and DQ associated pro¬ tection from type 1 diabetes: comparison of DRBl‘1401 and DQAl‘0102DQBl‘0602. J Clin Endocrinol Metab 2000; In press. 25. Nepom GT. Immunogenetics and IDDM. Diabetes Rev 1993; 1:93. 26. Kawasaki E, Noble J, Erlich H, et al. Transmission of DQ haplotypes to patients with type 1 diabetes. Diabetes 1998; 47:1971.

27. Erlich HA, Griffith RL, Bugawan TL, et al. Implication of specific DQB1 alleles in genetic susceptibility and resistance by identification of IDDM siblings with novel HLA-DQB1 allele and unusual DR2 and DR1 haplo¬ types. Diabetes 1991; 40:478. 28. Greenbaum CJ, Cuthbertson D, Eisenbarth GS, et al. Islet cell antibody pos¬ itive relatives with HLA-DQA1‘0102, DQB 1*0602: Identification by the Diabetes Prevention Trial-1. J Clin Endocrinol Metab 2000; 85:1255. 29. Bennett ST, Lucassen AM, Gough SCL, et al. Susceptibility to human type I diabetes at IDDM2 is determined by tandem repeat variation at the insulin gene minisatellite locus. Nat Genet 1995; 9:284. 30. Barnett AH, Eff C, Leslie RDG, Pyke DA. Diabetes in identical twins: a study of 200 pairs. Diabetologia 1981; 20:87. 31. Blom L, Dahlquist G, Nystriim L, et al. The Swedish childhood diabetes study—social and perinatal determinants for diabetes in childhood. Diabe¬ tologia 1989; 32:7. 32. Menser MA, Forrest JM, Brensby RD. Rubella infection and diabetes melli¬ tus. Lancet 1981; 1:57. 33. Yoon JW, London WT, Curfman BL, et al. Coxsackie virus B4 produces transient diabetes in non-human primates. Diabetes 1986; 35:712. 34. Bingley PJ, Gale EAM. Current status and future prospect for prediction of IDDM. In: Palmer JP, ed. Prediction, prevention, and genetic counseling in IDDM. Chichester, England: John Wiley, 1996:227. 35. Turner R, Stratton I, Horton V, et al. UKPDS 25: autoantibodies to islet-cell cytoplasm and glutamic acid decarboxylase for prediction of insulin requirement in type 2 diabetes. UK Prospective Diabetes Study Group. Lancet 1997; 30:1288. 36. Gorsuch AN, Spencer KM, Lister J, et al. Evidence for a long prediabetic period in type I (insulin-dependent) diabetes mellitus. Lancet 1981; 2:1363. 37. Palmer JP, Asplin CM, Clemons P, et al. Insulin antibodies in insulindependent diabetics before insulin treatment. Science 1983; 222:1337. 38. Vardi P, Tuttleman M, Grinbergs M, et al. Consistency of anti-islet autoim¬ munity in "pre-type I diabetics" and genetically susceptible subjects: evi¬ dence from an ultrasensitive competitive insulin autoantibody (CIAA) radioimmunoassay. Diabetes 1986; 35(Suppl 1):86A. 39. Gale EAM. Islet cell autoantibodies: a family story. Eur J Endocrinol 1996; 135:643. 40. Kuglin B, Bertrams J, Linke C, et al. Prevalence of cytoplasmic islet cell antibodies and insulin auto-antibodies is increased in subjects with geneti¬ cally defined high risk for insulin-dependent diabetes mellitus. Klin Wochenschr 1989; 67:66. 41. Radetti G, Paganini C, Gentili L, et al. Frequency of Hashimoto's thyroidi¬ tis in children with type 1 diabetes mellitus. Acta Diabetol 1995; 32:121. 42. Baekkeskov S, Aanstoot H, Christgau S, et al. Identification of the 64K autoantigen in insulin dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase. Nature 1990; 347:151. 43. Castano L, Russo E, Zhou L, et al. Identification and cloning of a granule autoantigen (carboxypeptidase H) associated with type I diabetes. J Clin Endocrinol Metab 1991; 73:1197. 44. Pietropaolo M, Castano L, Babu S, et al. Islet cell autoantigen 69 kDa (ICA69): molecular cloning and characterization of a novel diabetes associ¬ ated autoantigen. J Clin Invest 1993; 92:359. 45. Verge CF, Stenger D, Bonifacio E, et al. Combined use of autoantibodies (IA-2ab, Gadab, IAA, ICA) in type 1 diabetes: combinatorial islet autoanti¬ body workshop. Diabetes 1998; 47:1857. 46. Nayak RC, Omar MAK, Rabizadeh A, et al. "Cytoplasmic" islet cell anti¬ bodies: evidence that the target antigen is asialoglycoconjugate. Diabetes 1985; 34:617. 47. Srikanta S, Ganda OP, Soeldner JS, Eisenbarth GS. First-degree relatives of patients with type I diabetes: islet cell antibodies and abnormal insulin secretion. N Engl J Med 1985; 313:461. 48. Eisenbarth GS, Gianani R, Yu L, et al. Dual parameter model for prediction of type 1 diabetes mellitus. Proc Assoc Am Physicians 1998; 110:126. 49. Stiller CR, Dupre J, Gent M, et al. Effect of cyclosporine immunosup¬ pression in insulin-dependent mellitus of recent onset. Science 1984; 223:1362. 50. Feutren G, Asson G, Karsenty G, et al. Cyclosporine increases the rate and length of remissions in insulin-dependent diabetes of recent onset: results of a multi-center trial. Lancet 1986; 2:119. 51. Gottlieb PA, Eisenbarth GS. Diagnosis and treatment of pre-insulin depen¬ dent diabetes (IDDM). Annu Rev Med 1998; 49:391. 52. Lampeter EF, Klinghammer A, Scherbaum WA, et al. The Deutsche Nicotin¬ amide Intervention Study: an attempt to prevent type 1 diabetes. DENIS Group. Diabetes 1998; 47:980. 53. Sadelain MWJ, Qin H-Y, Lauzon J, Singh B. Prevention of type I diabetes in NOD mice by adjuvant immunotherapy. Diabetes 1990; 39:583. 54. Zhang JZ, Davidson L, Eisenbarth GS, Weiner HL. Suppression of diabetes in nonobese diabetic mice by oral administration of porcine insulin. Proc Natl Acad Sci U S A1991; 88:10252. 55. Keller RJ, Eisenbarth GS, Jackson RA. Insulin prophylaxis in individuals at high risk of type I diabetes. Lancet 1993; 341:927. 56. Nuefeld M, Maclaren N, Blizzard RM. Two types of autoimmune Addi¬ son's disease associated with different polyglandular autoimmune (PGA) syndromes. Medicine (Baltimore) 1981; 60:355. 57. Verge C, Eisenbarth GS. Autoimmune polyendocrine syndromes. In: Wil¬ son JD, Foster DW, eds. Williams textbook of endocrinology, 9th ed. Phila¬ delphia: WB Saunders, 1998:1651.

Ch. 137: Etiology and Pathogenesis of Type 2 Diabetes Mellitus and Related Disorders

CHAPTER

1315

137

ETIOLOGY AND PATHOGENESIS OF TYPE 2 DIABETES MELLITUS AND RELATED DISORDERS C. RONALD KAHN

NON-INSULIN-DEPENDENT (TYPE 2) DIABETES MELLITUS Type 2 diabetes mellitus is by far the most prevalent endocrine disease. It is estimated to affect >15 million people in the United States, approximately one-third of whom are undiagnosed.1,2 The prevalence increases with age, and >9% of those older than 65 years have the disease. The prevalence is higher in Mexican Americans, blacks, and Native Americans, reaching as high as 50% among adult Pima Indians. Development of type 2 diabe¬ tes is strongly influenced by genetic factors and environmental factors, including obesity, decreased physical activity, and a low level of physical fitness.3

_i_i_i_i_

100

200

300

400

MEAN GLUCOSE ( mg/dL) 2 HOURS AFTER GLUCOSE LOAD

FIGURE 137-1. Relation between insulin secretion and glucose level 2 hours after a glucose load. Data were obtained for Pima Indians with various degrees of glucose intolerance. Similar results have been observed in whites. (Data from Savage PJ, Dippe SE, Bennett PH, et al. Hyperinsulinemia and hypoinsulinemia: insulin responses to oral car¬ bohydrate over a wide spectrum of glucose tolerance. Diabetes 1975; 24:262.)

PATHOGENESIS Although type 2 or non-insulin-dependent diabetes mellitus is the more common form of the disease, the exact nature of its pathogenesis remains controversial, and in contrast to type 1 diabetes, in which immunologic markers confirm the pathogen¬ esis of the disease, no specific diagnostic tests are available for type 2 diabetes. Type 2 diabetes has strong genetic influences and occurs in identical twins with almost total concordance4,5; however, defining the exact genes involved has posed a great challenge. In type 2 diabetes, B-cell mass is relatively well preserved, but insulin secretion in response to specific secretagogues such as glucose is reduced, and clear evidence exists for resistance to insulin action in the peripheral tissues.6-9 The precise genetic defects have been identified in some of the minor forms of type 2 diabetes, including those in rare patients with genetic syn¬ dromes of insulin resistance (see Chap. 146) and in patients with maturity-onset diabetes of youth (MODY). For the common form of type 2 diabetes, controversy contin¬ ues over whether the decreased insulin secretion or insulin resistance is the principal factor in the pathogenesis of the dis¬ ease, and which occurs first in the longitudinal development of the syndrome. Uncertainty also exists about the extent of the heterogeneity and the identity of the primary lesion.

INSULIN SYNTHESIS AND SECRETION Even before the introduction of radioimmunoassays, morpho¬ logic studies suggested that the pancreas of a patient with type 2 diabetes has at least 50% (and sometimes up to 100%) of the normal B-cell mass, whereas that of a patient with type 1 diabe¬ tes of more than a few years' duration has virtually no B cells.6 This finding is consistent with the data on extractable insulin as measured in bioassay and radioimmunoassay. Immunoassay of plasma insulin and of C peptide has confirmed the presence of functioning B cells in patients with this disease, but the extent of function varies considerably depending on the type of stimu¬ lus used, the body weight of the patient, and the stage of dis¬ ease.8-10 In long-standing disease, islets do produce amyloid deposits, which are partly comprised of a second hormone

(termed islet amyloid polypeptide or amylin), which is secreted by B cells.10 Although some studies have implicated this peptide in the abnormal B-cell function of type 2 diabetes, this hypothesis remains unproven, and it seems more likely that the amyloid deposits are a marker of chronic insulin hypersecretion. In most individuals with type 2 diabetes, basal insulin levels are normal or elevated, and the degree of elevation correlates with the degree of obesity.11 Elevated insulin levels also occur in thin individuals with type 2 diabetes. Although a few patients have been identified with a defect in the conversion of proinsu¬ lin to insulin or mutant insulin molecules, in most patients with type 2 diabetes, the proportion of proinsulin to insulin is nor¬ mal or only slightly increased, and the insulin and proinsulin have normal receptor binding and bioactivity. Although basal insulin levels usually are elevated in patients with type 2 diabetes, the insulin secretory responses to oral glucose differ considerably, depending on the extent of glucose intolerance. In patients with normal fasting glucose lev¬ els and 2-hour postprandial levels of 200 mg/dL.7,8 In contrast to the relative preservation of insulin response to meals and to oral glucose, a loss of acute-phase (first-phase) insulin release in response to intravenous glucose occurs in vir¬ tually all patients with significant fasting hyperglycemia.9 Acute insulin release in response to (1-adrenergic stimuli, amino acids, and other insulin secretagogues in these same individu¬ als often is normal, suggesting a specific defect in glucorecognition rather than a general defect in B-cell function.12,13 This pattern of response resembles that seen in patients in the early, preclinical phase of type 1 diabetes. In studies of perfused pan¬ creases from animal models of type 2 diabetes, although firstphase secretion may be lost, glucose maintains its ability to potentiate arginine-induced insulin secretion.14 The preserva-

1316

PART IX: DISORDERS OF FUEL METABOLISM

tion of response to an oral glucose load probably is a reflection of the importance in this response of the potentiation of the glu¬ cose effect by gastrointestinal hormones. In humans with type 2 diabetes, however, the glucose potentiation is also blunted.12 The lost first-phase response to glucose can be restored, at least partially, by a-adrenergic blockade, opiate-receptor blockade, inhibitors of prostaglandin synthesis, reduction of plasma glu¬ cose by dietary restriction, use of oral hypoglycemic agents, or even administration of insulin.15 The fact that this lesion is functional and at least partially reversible makes it potentially amenable to therapeutic manipulation. A fragment of glucagon¬ like peptide-1 (GLP-1) may potentiate glucose-induced insulin secretion in persons with type 2 diabetes; this offers a possible new avenue for therapy that is currently being explored in clin¬ ical trials.16

INSULIN RESISTANCE Virtually all patients with type 2 diabetes have some degree of insulin resistance.17 Conditions associated with the develop¬ ment of insulin resistance, especially obesity and advancing age, greatly increase the risk of type 2 diabetes. Insulin resis¬ tance correlates with certain patterns of obesity and is greater in individuals with central obesity than in those with more gener¬ alized obesity.18 At any given body weight, a high waist-to-hip ratio correlates with insulin resistance and increased risk of type 2 diabetes. Insulin resistance and hyperinsulinemia are also associated with hypertension and hypertriglyceridemia, deceased high-density lipoprotein cholesterol, and increased risk of atherosclerosis and cardiovascular disease.19,20 The asso¬ ciation of insulin resistance with these features in the absence of clinical diabetes has been referred to as the metabolic syndrome or syndrome X (see Chap. 145).19 In cases of type 2 diabetes and syndrome X, insulin resistance is suggested by the elevated insulin levels and the fact that the patient develops glucose intolerance with circulating insulin levels well above those seen in the type 1 diabetic. The simplest test of insulin sensitivity is the measurement of the fall in plasma glucose in response to a given dose of exogenous insulin (i.e., an insulin-tolerance test). In normal individuals, glucose usually falls by >50% in response to a dose of 0.05 to 0.1 U per kilogram of body weight. In type 2 diabetics, this response may be markedly blunted, and as much as 0.3 U/kg may be required to produce a 50% fall in glucose. Because the variability of endogenous insulin secretion and the counterregulatory hormones released during hypoglycemia may modify the response to the insulin test, more sophisticated measures of insulin resistance have been developed in which these factors are minimized. These tests include the measure¬ ment of steady-state glucose during simultaneous insulin and glucose infusion in which pancreatic insulin is suppressed with propranolol and epinephrine or somatostatin or the use of the euglycemic insulin clamp technique.21 In the latter, doseresponse curves for the effect of insulin on glucose disposal in normal humans can be constructed (Fig. 137-2). When this test is used, the nature of the insulin resistance can be dissected into changes in median effective dose (ED50) on dose-response curves (i.e., changes in insulin sensitivity) and changes in maxi¬ mal insulin effect (i.e., changes in insulin responsiveness).21,22 In type 2 diabetes, decreased sensitivity in insulin action to decreased splanchnic glucose output (i.e., an effect primarily on the liver) and decreased sensitivity and decreased responsive¬ ness of insulin action to increased glucose utilization (i.e., effects primarily on muscle and fat tissue) are seen.21,23 The resistance to insulin that occurs in the patient with type 2 diabetes mellitus could result from defects at several levels of insulin action (see Chap. 135). To produce a signal at the target cell, insulin must bind to its receptor, generate a transmem¬ brane signal by activation of the insulin-receptor kinase, and initiate a complex network of intracellular signals that ulti-

/NSUL/N (fiU/mL) FIGURE 137-2. Glucose disposal during a euglycemic clamp in normal patients and in those with type 2 diabetes. Predicted level of insulin resistance, assuming the only defect to be that of insulin binding, was compared with the observed data. The observed data indicate more severe insulin resistance and thus suggest the presence of a postbinding defect as well. (Redrawn from data of Scarlett JA, Gray RS, Griffin J, et al. Insulin treatment reverses the insulin resistance of type II diabetes mellitus. Diabetes Care 1982; 5:353.) mately culminate in the activation and inhibition of the differ¬ ent cellular processes responsible for the physiologic effects of insulin. Studies of tissues taken from animal models of type 2 diabetes, as well as biopsies of tissues from humans with the disease, have revealed multiple alterations, including defects at the insulin receptor (e.g., binding and kinase activation) and at several of the postreceptor steps involved in insulin action. Decreased insulin-receptor binding has been described in obese and thin individuals with type 2 diabetes23,24 (Fig. 137-3). This decrease in binding is attributable to a decrease in receptor

FIGURE 137-3. Defect in insulin-receptor binding in diabetes and obe¬ sity. The negative relation between receptor concentration and plasma insulin concentration is illustrated. Data are plotted as insulin binding versus plasma insulin concentration. Shaded areas indicate the normal range. Data on the left are from thin and obese patients with type 2 dia¬ betes and data on the right are from obese individuals with and without diabetes. (Data for diabetic patients from Kahn CR. Insulin action, diabetogenes, and the cause of type II diabetes [Banting lecture]. Diabetes 1994; 43[8]:1066; and from Ferrannini E, Mari A. How to measure insu¬ lin sensitivity. J Hypertens 1998; 16[7]:895. Data for obese patients from Caro JF, Sinha MK, Raju SM, et al. Insulin receptor kinase in human skeletal muscle from obese subjects with and without non-insulindependent diabetes. ] Clin Invest 1987; 79:1330.)

Ch. 137: Etiology and Pathogenesis of Type 2 Diabetes Mellitus and Related Disorders T3

Liver

Plasma Glucose

Dependent Glucose Utilization

Controls FIGURE 137-4. Defect in insulin-receptor kinase activity in type 2 dia¬ betes, in which the activity of this enzyme in liver is decreased by -50%, even when expressed per receptor. (N/DDM, non-insulin-dependent diabetes mellitus.) (Redrawn from Caro JF, Ittoop O, Pories WJ, et al. Studies in the mechanism of insulin resistance in the liver from humans with non-insulin-dependent diabetes. J Clin Invest 1986; 78:249.)

number, with no changes in receptor affinity, and is thought to be secondary to down-regulation of the receptor by the elevated basal endogenous insulin level.16-24 Similar decreases in insulin bind¬ ing are observed in patients with impaired glucose tolerance and in some obese individuals with normal glucose tolerance. These findings indicate that the decrease in insulin receptors alone probably does not entirely account for the insulin resistance. Because "spare" receptors for insulin action are present in many tissues, a decrease in receptors would be expected to produce only a shift in the dose-response curve, with no changes in maxi¬ mal response (i.e., decreased sensitivity).22 As noted previously, euglycemic clamp studies have indicated that both decreased sensitivity and decreased responsiveness to insulin are present, indicating postbinding defects in insulin action.23 These include defects in activation of the insulin-receptor kinase and phosphorylation of intracellular substrates.23-23-28 Sequence variations have been identified in several of the pro¬ teins involved in insulin signaling, and in the case of insulinreceptor substrate-1 (IRS-1), these occur with increased frequency in patients with type 2 diabetes.29 Most studies have indicated that a defect in glucose transport is also present due to a defect in glucose transporter translocation30-31 (Fig. 137-4). Finally, a defect also is present in glycogen synthesis, which forms the major com¬ ponent of nonoxidative glucose metabolism.6-31 Which of these defects is primary and whether other defects are present in the intracellular steps of insulin action remain unknown; however, some of these alterations can be found in normoglycemic off¬ spring of parents with type 2 diabetes.31 Studies in both animal models and cell culture have demon¬ strated that a number of defects in insulin signaling may be sec¬ ondary to different metabolic abnormalities present in the type 2 diabetic patient. For example, prolonged exposure to high insulin levels produces postbinding desensitization and recep¬ tor down-regulation, suggesting a role for increased basal insu¬ lin levels in these defects.32 The increased levels of tumor necrosis factor-a (TNF-a) and free fatty acids (FFAs), which are also found in obesity, produce insulin resistance by inhibiting the insulin-receptor kinase and by altering postreceptor metab¬ olism, respectively.33-35 Hyperglycemia, both directly and through the production of intermediates like glucosamine, can also contribute to increasing levels of insulin resistance.36 Thus, like the defects in insulin secretion, the defects in insulin action are largely reversible when the diabetes is treated and the meta¬ bolic abnormalities are corrected. This is true whether the treat¬

1317

Brain

Fat & Muscle

FIGURE 137-5. Schema for pathogenesis of type 2 diabetes, illustrating insulin resistance to glucose uptake at muscle and fat and failure of insulin to suppress hepatic glucose output, coupled with a defect in glucose sensing at the B cell.

ment involves diet, oral hypoglycemic agents, or intensive insulin treatment.36-38 A schematic diagram illustrating the var¬ ious factors involved in the pathogenesis of type 2 diabetes is shown in Figure 137-5.

EVENTS IN THE DEVELOPMENT OF TYPE 2 DIABETES Like type 1 diabetes, type 2 diabetes is preceded by a long pre¬ diabetic phase in which glucose-tolerance tests are normal, but insulin resistance is present17-39'40 (Fig. 137-6). Many patients also pass through a stage of impaired glucose tolerance in which basal and stimulated insulin levels are increased, find¬ ings which further suggest that insulin resistance precedes the functional insulin deficiency. At this stage, decreases in insulinreceptor binding and insulin action in muscle and fat can be detected. Fasting hyperglycemia suggests unsuppressed gluco-

INITIATION FACTORS

PROGRESSION FACTORS

Insulin Resistance Genes

Obesity

©

Insulin Secretion Genes

B-cell Toxins

©

B-cell Capacity Genes

Diet/Environmental toxins ©

Obesity Genes

Activity/Age _

©

Type II > Diabetes Mellitus

Failing Insulin Secretion Glucose Desensitization of Beta Cell Increased Insulin Secretion Decreased Insulin and Glucose Sensitivity FIGURE 137-6. Model of the progressive pathogenesis of type 2 diabe¬ tes mellitus.

1318

PART IX: DISORDERS OF FUEL METABOLISM

neogenesis and indicates further resistance to insulin action at the liver. These patients also have a significant functional defect in insulin secretion, especially in glucose recognition.12-13

insulin response to intravenous glucose.51 These changes are similar to those observed in type 2 diabetes and may reflect an unmasking of the diabetic state by the hormonal milieu of preg¬ nancy, particularly the increased levels of placental lactogen.

GENETICS OF TYPE 2 DIABETES MATURITY-ONSET DIABETES OF YOUTH Although type 2 diabetes has a very strong genetic influence, the genes leading to development of this disease are poorly understood. In most patients, type 2 diabetes probably is poly¬ genic in nature, and the polygenes involved may be different among different families or population groups. However, a small number of patients show a monogenic form of type 2 dia¬ betes. These include patients with maturity-onset diabetes of youth (MODY; described below), a few families in whom genetic defects in the insulin molecule lead to generation of a mutant insulin or a failure to process proinsulin and hyperproinsulinemia,41-42 patients with defects in mitochondrial DNA, and patients with genetic defects in the insulin receptor. In each of these cases, the resulting syndrome usually has clinical fea¬ tures that are distinct from typical type 2 diabetes. Genetic defects in the insulin receptor have been described in at least 100 individuals, virtually all with different mutations in the gene. Most of these patients have syndromes of severe insulin resistance (e.g., leprechaunism, Rabson-Mendenhall syndrome, or the type A syndrome of insulin resistance and acanthosis nigri¬ cans).43-44 These patients are discussed in Chapter 146. A variant of type 2 diabetes associated with a point mutation in the gene encoding the transfer RNA for leucine has been described. This gene is present in mitochondrial DNA rather than in nuclear DNA. Because mitochondrial DNA is inherited almost exclusively from the mother, this form of diabetes is characterized by a maternal inheritance pattern 45,46 This condi¬ tion of diabetes is also associated with nerve deafness and some somatic defects, and may represent as much as 0.8% of type 2 diabetes in some populations.

RELATED CONDITIONS IMPAIRED GLUCOSE TOLERANCE Impaired glucose tolerance (IGT) is present in 7% to 11% of the population, depending on the diagnostic criteria used and the group studied. Recommendations by the American Diabetes Association Expert Committee on the Diagnosis and Classifica¬ tion of Diabetes Mellitus suggested that this diagnosis be based simply on a fasting glucose level of 110 to 125 mg/dL.47 Many of the characteristics of this population, other than the level of hyperglycemia, are similar to those of patients with type 2 dia¬ betes, suggesting that IGT may be an intermediate step in the development of overt type 2 diabetes.47-48 IGT may also be a component of syndrome X. The presence of IGT significantly increases the risk of subsequent development of type 2 diabe¬ tes, although in many individuals, IGT is transient or does not progress. Researchers at the National Institutes of Health are about to undertake a major study to determine if the progres¬ sion of IGT to type 2 diabetes can be prevented by changes in lifestyle or pharmacologic intervention. GESTATIONAL DIABETES MELLITUS Gestational diabetes mellitus is defined as the development of dia¬ betes during pregnancy in a woman with no previous history of disease (see Chap. 156).49-52 Gestational diabetes may occur in 2% to 5% of all pregnancies. Although some of these cases repre¬ sent the coincidence of detection of type 1 or type 2 diabetes with pregnancy, in most patients with true gestational diabetes, the hyperglycemia disappears after delivery. These women have an increased risk of developing type 2 diabetes, which is usually estimated at 2% to 3% per year of follow-up 49,50 Pathophysiologically, women with gestational diabetes have insulin resis¬ tance, as measured in a euglycemic clamp, and decreased early

From a genetic perspective, the best-characterized subset of patients with type 2 diabetes are those with MODY.53~59 As implied by the name, this form is characterized by an early age of onset and a strong family history, with affected members in at least three generations, suggesting an autosomal dominant mode of inheritance. Although the typical patient in these kin¬ dreds is first found to be diabetic in the teens or 20s, some may be diagnosed as early as 5 years of age. As in other forms of type 2 diabetes, the level of hyperglycemia increases gradually, allowing treatment with diet or sulfonylureas, although in the latter stages of disease some patients require insulin for control of blood glucose. These patients develop the typical chronic complications of diabetes mellitus, including retinopathy, nephropathy, and neuropathy. Pathophysiologically, the disor¬ der is heterogeneous, but in many families, the major defect appears to be relatively low insulin secretion. Genetic studies have revealed at least five subtypes of MODY. These have been designated MODY1 through MODY5, based on the order in which the genetic loci were identified. All five forms of MODY thus far identified are due to genetic defects impairing B cell function. The most common form of MODY in the United States and in most European countries is MODY3, which is present in 25% to 50% of families that meet the clinical criteria for MODY. This form is due to genetic defects in the nuclear transcrip¬ tion factor hepatic nuclear factor-la (HNF-la), a gene that plays a major role in control of insulin synthesis and B-cell function.53-54 From 10% to 40% of the kindreds have MODY2, in which the molecular abnormalities are genetic defects in the enzyme glucokinase.55'56 Glucokinase is the major form of hexokinase, the enzyme responsible for phosphorylation of glucose to glucose-6-phosphate in the liver and in islets of Langerhans. In the latter site, the enzyme is one of the rate-limiting steps in glucose sensing by tire B cell. In comparison with other forms of type 2 diabetes, MODY2 is usually characterized by relatively mild degrees of hyperglycemia and glucose intolerance. MODY1, MODY4, and MODY5 are also due to defects in the transcription factors HNF-4a, PDX-1, and HNF-1 (3, respectively. Each of these affects only a few families. In all forms of MODY, the gene involved may have a wide variety of mutations, including point mutations, nonsense mutations lead¬ ing to premature termination, and splicing defects. This makes screening for these defects more difficult in individual patients. Almost all patients are heterozygous for the mutation, with one normal allele and one mutant allele. The mechanism by which the mutant enzyme becomes dominant over the normal enzyme is poorly understood. The genetic defect in 15% to 30% of families has not yet been determined.57-59

NONDIABETIC MELLITURIA The presence of some form of sugar in the urine is not necessar¬ ily diagnostic of diabetes. At one time, the forms of nondiabetic mellituria were confused with diabetes, but this occurs rarely now that the diagnosis of diabetes is based primarily on blood glucose measurement. Moreover, urinary glucose is determined with more specific glucose oxidase detection systems (e.g., TesTape, Clinistix, Diastix, Chemstrip uG). Nonetheless, sugar in the urine may be due to diseases other than diabetes.

RENAL GLUCOSURIA The most commonly recognized nondiabetic mellituria is renal glucosuria.60 This disorder accounted for as many as 1 of 500

Ch. 137: Etiology and Pathogenesis of Type 2 Diabetes Mellitus and Related Disorders patients referred for evaluation of diabetes when the diagnosis depended primarily on urine glucose measurements. The diag¬ nosis of renal glycosuria requires the detection of glucosuria with simultaneous normal plasma glucose on timed urine sam¬ ples taken during an oral glucose-tolerance test. Renal gluco¬ suria usually is not associated with other urinary tubular absorptive defects (e.g., aminoaciduria of the Fanconi syn¬ drome should be excluded) and appears to be benign. Some patients with renal glucosuria have mistakenly been treated for decades with insulin. With increased reliance on measurements of serum and capillary glucose and on hemoglobin A1C, such mistaken diagnoses should be extremely rare. Normally, glucose is actively reabsorbed at the proximal renal tubule after filtration at a concentration equal to that in plasma. In normal individuals, the reabsorptive capacity of the proximal tubule exceeds the filtered load of glucose, and little glucosuria occurs with a plasma glucose concentration of 10 times as many (i.e.. Ill) would have developed type 2 diabetes by this age. In many situations, however, the time of onset of the diabe¬ tes cannot be determined. All one can do is count individuals with diabetes (preexisting as well as recent cases) in the popula¬ tion at a specific point in time and express this number as a pro¬ portion or prevalence. Figure 138-1 shows the prevalence of diabetes in a random sample of adults in the U.S. population from 1988 to 1994/ Whereas Table 138-1 shows the incidence rates of diagnosed diabetes. Figure 138-1 shows the prevalence of diagnosed and undiagnosed diabetes together, and the prev-

30

o c a>

15% achieve a good response. The rate of secondary failure in patients who have an initial response to treatment with sulfonylureas ranges from 5% to 10% each year; after 10 years, only 50% have satisfactory con¬ trol.4 The causes of secondary failure include B-cell exhaustion and dietary noncompliance. Numerous drugs (e.g., niacin, thi¬ azide diuretics, (3-blockers, corticosteroids) reduce insulin sen¬ sitivity and, thus, decrease the clinical efficacy of sulfonylureas, and their use may lead to deteriorating glycemic control. Potential alternatives to these drugs that do not worsen insulin resistance should be considered if continued sulfonylurea ther¬ apy is desirable.

MEGLITINIDES Meglitinides are oral hypoglycemic agents derived from ben¬ zoic acid and chemically unrelated to the sulfonylurea agents. Repaglinide, the first meglitinide to receive Food and Drug Administration approval, lowers blood glucose by stimulating the release of insulin from the pancreas. Thus, it requires func¬ tioning B cells and is ineffective in type 1 diabetes. Repaglinide, which is rapidly absorbed after oral administration, reaches peak plasma levels within 1 hour and is rapidly eliminated by

1 346

PART IX: DISORDERS OF FUEL METABOLISM

oxidation and conjugation with glucuronic acid (with a half-life of -1 hr). The cytochrome P450 enzyme system, specifically 3A4, is involved in further metabolism; however, metabolites do not contribute to the glucose-lowering effect of the drug. Tablets are available in 0.5-, 1-, and 2-mg strengths. Recom¬ mended dosage is from 0.5 to 4 mg taken with meals 2 to 4 times daily, with a maximum dosage of 16 mg per day. This dosage may need adjustment in liver disease. Patients who skip (or add) extra meals should skip (or add) a dose for that meal. Repaglinide is effective with metformin; however, it should not be used in conjunction with the sulfonylureas. Stimulation of insulin secretion occurs by processes both similar to and distinct from those of the sulfonylureas. Like sul¬ fonylureas, repaglinide binds to a site on the B-cell membrane; however, the affinity of the binding sites appears different for meglitinide and glipizide.28 Binding closes ATP-dependent K+ channels. The consequent K+ channel blockade, in turn, leads to depolarization of the B cell by the opening of the Ca2+ channels, resulting in calcium influx which induces insulin secretion. The ion-channel binding is highly tissue specific; low-affinity bind¬ ing is seen in cardiac and skeletal muscle tissues.

BIGUANIDES Three derivatives of guanidine—phenformin, metformin, and buformin—have glucose-lowering effects.29 Of these, only met¬ formin, which is available in the United States, is used clinically, because phenformin and buformin use is associated with the development of lactic acidosis.30 This also has been observed with metformin use, but almost all affected patients had impaired renal function. When metformin is used appropriately, the incidence of lactic acidosis is rare. In 56,000 patient-years of use in Canada, no cases of lactic acidosis were reported.31 Metformin is partially absorbed from the gastrointestinal tract and has a bioavailability of 50% to 60%. The drug is stable, does not bind to plasma proteins, and is not metabolized. It is excreted in the urine and has a plasma half-life of 1.7 to 4.5 hours. Of a given dose, 90% is cleared within 12 hours.32 Metformin does not have a hypoglycemic effect, but acts as an "antihyperglycemic."33It lowers blood glucose levels in hypergly¬ cemic patients with type 2 diabetes but has no effect on glucose levels in normal subjects. The mechanism of action remains unclear. Metformin reduces absorption of glucose from the gas¬ trointestinal tract, probably due to inhibition of glucose uptake at the mucosal surface.34 Metformin also inhibits hepatic glucose production35 and increases insulin-stimulated glucose uptake at the periphery36'37 by stimulating the GLUT4 glucose transporter.38 Unlike therapy with insulin or sulfonylureas, therapy with metformin does not lead to weight gain and may be associated with modest weight loss because of a slight anorectic effect of the drug.39 In addition, patients with hypertriglyceridemia may expe¬ rience reductions in triglyceride levels of as much as 50%.40'41 Starting dosages of metformin range from 1.0 to 2.0 g per day in divided doses of 500 mg or 850 mg. The maximum dosage is 3 g, usually given in three divided doses. Primary failure rates of 5% to 20% have been reported. Secondary failure rates range from 5% to 10% per year. Metformin has been used successfully in combination with sulfonylureas, especially in patients older than 60 years.42,43 Contraindications to metformin therapy include impaired creatinine clearance, liver disease, heart fail¬ ure, chronic obstructive lung disease, and alcohol abuse. Side effects are mainly gastrointestinal; abdominal discomfort, diar¬ rhea, and nausea and vomiting have been reported.

CARBOHYDRASE INHIBITORS The group of agents known as carbohydrase inhibitors includes acarbose44; miglitol is still in clinical trials. The mech¬

anism of action of both drugs is inhibition of the a-glucosidases in the intestinal brush border, leading to a delay in carbohydrate absorption. Clinical studies with these agents have demonstrated a reduction in postprandial glucose eleva¬ tions in both types 1 and 2 diabetes mellitus.45-4" Doses of the carbohydrase inhibitors must be titrated to balance the decreased glycemic response against malabsorption and other gastrointestinal side effects. For both agents, the therapeutic dose is between 50 and 100 mg; higher doses lead to abdomi¬ nal distention, flatulence, malabsorption, and diarrhea. Both these agents have also been used in combination with sulfonylureas.50,51 The use of these agents has generally been associated with only modest improvements in glycosylated hemoglobin measurements but may moderate the weight gain seen with the sulfonylureas.

THIAZOLI DIN EDI ONES In contrast to sulfonylureas and meglitinides, which are oral hypoglycemic agents, thiazolidinediones act to enhance insulin sensitivity. These agents have been studied extensively in obese, insulin-resistant rodent models, including kk, db/db, and db/db mice, and the Zucker rat. Two drugs in this category are currently marketed in the United States: rosiglitazone and pioglitazone. Rosiglitazone is dosed at 2 or 4 mg twice daily or 8 mg once daily, and studies show that effectiveness is decreased little with once daily dosing. This product may not have a favor¬ able effect on lipid profiles: total cholesterol and low-density lipoproteins (LDL) are slightly increased. Triglycerides are unaffected, although high-density lipoproteins (HDL) may be increased. In premarketing studies, rosiglitazone was not associated with drug-induced hepatotoxicity or elevation of the liver enzyme alanine transaminase. Although rare reports of hepatic toxicity have appeared, one occurring two weeks after commencement of therapy,52 these cases are not clearly attributable to the use of this agent. The drug has been approved for type 2 diabetes as monotherapy and in combi¬ nation with sulfonylureas or with metformin, but not with insulin. Pioglitazone is the most recent thiazolidinedione to be mar¬ keted. The initial dosage is 15 to 30 mg once daily, without relationship to meals. The maximum dose is 45 mg. The lipid effects may be beneficial: triglycerides are decreased, HDL is increased; total cholesterol and LDL are unaffected.53 In vitro, the drug inhibits the growth of cultured vascular smooth muscle cells (VSMC). In vivo studies of stroke-prone sponta¬ neously hypertensive rats, which are subjected to endothelial injury, reveal a protective effect against both acute and chronic vascular injury via inhibition of VSMC prolifera¬ tion.54 In patients, pioglitazone did not increase hepatic enzymes compared with controls, and, to date, hepatotoxicity has not been encountered. Pioglitazone is approved in type 2 diabetes as monotherapy and in combination with sulfonyl¬ ureas, metformin, or insulin, but not for triple combination therapy. All of the thiazolidinediones are associated with a statisti¬ cally increased incidence of edema and weight gain when com¬ pared with placebo. When edema occurs, it tends to be mild or moderate. The thiazolidinediones should not be used in patients with congestive heart failure. The drugs are associated with an increase of plasma fluid volume and may result in mild, dilutional-related decreases in hemoglobin, hematocrit, and white cell count. In some studies, the weight gain was thought to correlate with the improvement in hyperglycemia. Also, weight gain is a known occurrence in many longitudinal studies of patients with type 2 diabetes in general. With rosiglitazone or pioglitazone, therapy should not be commenced if the plasma aspartate transaminase exceeds 2.5

Ch. 142: Oral Agents for the Treatment of Type 2 Diabetes Mellitus times the upper limit of normal. Liver enzymes should be mea¬ sured before therapy, then perhaps every 2 months for a year, and periodically thereafter. Women taking these drugs who have polycystic disease or who are taking oral contraceptives may ovulate; hence, alternative forms of contraception should be suggested. Oral doses of thiazolidinediones lower blood glucose levels and decrease insulin levels. An increase in the insulin content of pancreatic islets is also noted.55'56 No hypoglycemia is seen when the drugs are administered to nondiabetic animals. The mechanism of action of the thiazolidinediones is not completely understood. Initial steps include binding to a member of the nuclear receptor peroxisome proliferator-activated receptor (PPAR) family to regulate the transcription of a number of insu¬ lin-responsive genes.57 They may also stimulate uncoupling proteins, although less evidence exists for this. Binding is spe¬ cific to the PPAR-y isoform that is present mainly in adipose tis¬ sue. However, because thiazolidinediones influence metabolism in liver, pancreas, and skeletal muscle—if the drugs work solely through tills receptor—either all of these tissues must contain sufficient levels of this PPAR isoform, or in vivo and in vitro iso¬ form binding may differ. PPARs exist as heterodimers with another nuclear receptor, the retinoid X receptor (RXR). In addi¬ tion, the drug effects are dependent on the presence of insulin, perhaps through separate insulin-regulated transcription fac¬ tors. Nuclear-receptor binding is linked to an increased rate of the transcription of PPAR-y responsive genes, including glucose transporters, critical for the control of glucose metabolism. In addition, some studies suggest improved vascular function through increased expression of plasminogen activator inhibi¬ tor type 1 (PAI-1) in endothelial cells58 and inhibition of angio¬ genesis.59 The mechanisms of action of oral agents are compared in Table 142-2.

OTHER STRATEGIES FOR DRUG THERAPY Several other strategies for treating type 2 diabetes are under investigation. New approaches include products that antago¬ nize glucagon action to suppress hepatic glucose production (e.g., glucagon analogs that antagonize the action of glucagon and glucagon-receptor antagonists60,61) and oxidation inhibitors that might interdict the actions of fatty acids in stimulating hepatic gluconeogenesis and in attenuating glucose disposal in muscle, as well as in diminishing the rates of ketone, choles¬ terol, and triglyceride synthesis.62 Another strategy involves modulators of the RXR, which interact with the PPAR nuclear receptors to improve insulin sensitivity. In addition, peptides with insulinotropic effects, such as glucagon-like peptides, are under investigation.623 New methods to target comorbid condi¬ tions that worsen insulin resistance are also under study, such as P3-receptor agonists for the treatment of obesity and neu¬ ropeptides to suppress appetite. The ideal therapy for type 2 diabetes probably will not be achieved until the fundamental pathogenesis of the disease is better understood, and then ther¬ apies likely will be tailored to specific molecular defects. Weight loss to improve insulin sensitivity and glucose con¬ trol as well as lipid abnormalities should also be strongly con¬ sidered in obese patients with type 2 diabetes.63,64 Currently, approved agents for weight loss are limited. Sibutramine acts to suppress appetite by inhibiting reuptake of serotonin, norepi¬ nephrine, and dopamine.65-66 It is available in 5-, 10-, and 15-mg tablets that are administered once daily and is metabolized into active metabolites in the liver by the cytochrome P450 enzymes. It is contraindicated in patients with coronary artery disease (because it may increase the heart rate and blood pressure) and in those requiring monoamine oxidase inhibitors. Orlistat, a lipase inhibitor, prevents the gastrointestinal absorption of -30% of dietary fat and may lower the glucose level, the choles¬ terol level, and blood pressure.67 It is given at a dosage of 120

1347

TABLE 142-2. Mechanisms of Action of Oral Agents Used to Treat Type 2 Diabetes Mellitus Class

Example

Action

Sulfonylureas

Glyburide

Insulinotropic, increases circu¬ lating insulin

Meglitinides

Repaglinide

Insulinotropic, increases circu¬ lating insulin

Biguanides

Metformin

Increases insulin-stimulated glucose uptake, reduces hepatic glucose production

Carbohydrase inhibitors

Acarbose

Delays carbohydrate absorption

Thiazolidinediones

Pioglitazone

Increases insulin sensitivity

mg three times daily with meals. The drug may cause gas¬ trointestinal discomfort. One should emphasize that weight loss is modest with both of these agents, and as both agents are new, no data exist on long-term safety or efficacy for either com¬ pound. Finally, the role of gastric bypass surgery to manage weight and glucose intolerance should be considered in the morbidly obese diabetic patient, with attention to the benefits as well as the limitations and risks of this procedure.68

REFERENCES 1. United Kingdom Prospective Diabetes Study (UKPDS) Group. Intensive blood-glucose control with sulphonylureas or insulin compared with con¬ ventional treatment and risk of complications in patients with type 2 diabe¬ tes (UKPDS 33). Lancet 1998; 352:837. 2. Loubatieres A. The hypoglycemic sulfonamides: history and development of the problem from 1942-1945. Arm N Y Acad Sci 1957; 71:4. 3. Melander A, Bitzen P-O, Faber O, Groop L. Sulphonylurea antidiabetic drugs: an update of their clinical pharmacology and rational therapeutic use. Drugs 1989; 37:58. 4. Skillman TG, Feldman JM. The pharmacology of sulfonylureas. Am J Med 1981; 70:361. 5. Gerich JE. Oral hypoglycemic agents. N Engl J Med 1989; 321:1231. 6. Kahn CR, Schecte Y. Oral hypoglycemic agents. In: Gilman AG, Rail TW, Nies AS, Taylor P, eds. The pharmacologic basis of therapeutics. New York: McGraw-Hill, 1993:1485. 7. Carlson RF, Isley WL, Ogrine FG, Klobucar TR. Efficacy and safety of refor¬ mulated, micronized glyburide tablets in patients with non-insulin-dependent diabetes mellitus: a multicenter, double blind, randomized trial. Clin Ther 1993; 15:788. 8. Groop L, Luzi L, Melander A, et al. Different effects of glyburide and glip¬ izide on insulin secretion and hepatic glucose production in normal and NIDDM subjects. Diabetes 1987; 36:1320. 9. Shmid-Antomarchi H, DeWeille J, Fosset M, Lazdunski M. The receptor for the antidiabetic sulfonylureas controls the activity of the ATP-modulated K+ in insulin secreting cells. J Biol Chem 1987; 262:15840. 10. Siconolfi-Baez L, Banerji MA, Lebovitz HE. Characterization and signifi¬ cance of sulfonylurea receptors. Diabetes Care 1990; 13:2. 11. Boyd AE III. Sulfonylurea receptors, ion channels and fruit flies. Diabetes 1988; 37:847. 12. Feldman JM, Lebovitz HE. Endocrine and metabolic effects of glibenclamide. Diabetes 1971; 20:745. 13. Kolterman OG, Olefsky JM. The impact of sulfonylurea treatment upon the mechanisms responsible for the insulin resistance in type II diabetes. Dia¬ betes Care 1984; 7:81. 14. Birkeland KI, Furuseth K, Melander A, et al. Long-term randomized pla¬ cebo-controlled double-blind therapeutic comparison of glipizide and gly¬ buride: glycemic control and insulin secretion during 15 months. Diabetes Care 1994; 17:43. 15. Groop L, Groop P-H, Stenman S, et al. Comparison of pharmacokinetics, metabolic effects and mechanisms of action of glyburide and glipizide dur¬ ing long term treatment. Diabetes Care 1987; 10:671. 16. Tsalikian E, Dunphy TW, Bohannon NV, et al. The effect of chronic oral antidiabetic therapy on insulin and glucagon responses to a meal. Diabetes 1977; 26:314. 17. Pfeifer MA, Beard JC, Halter JB, et al. Suppression of glucagon secretion during a tolbutamide infusion in normal and noninsulin-dependent dia¬ betic subjects. J Clin Endocrinol Metab 1983; 56:586. 18. Pek S, Fajans SS, Floyd JC Jr, et al. Failure of sulfonylureas to suppress plasma glucagon in man. Diabetes 1972; 21:216. 19. Efendic S, Enzmann F, Nylen A, et al. Effect of glucose/sulfonylurea inter¬ action on release of insulin, glucagon and somatostatin from isolated per¬ fused rat pancreas. Proc Natl Acad Sci U S A1979; 76:5901.

1348

PART IX: DISORDERS OF FUEL METABOLISM

20. Beck-Nielsen H, Hother-Nielsen O, Pedersen O. Mechanism of action of sulphonylureas with special reference to the extrapancreatic effect: an overview. Diabet Med 1988; 5:613. 21. Rossetti L, Smith D, Shulman GI, et al. Correction of hyperglycemia with phlorizin normalizes tissue sensitivity to insulin in diabetic rats. J Clin Invest 1987; 79:1510. 22. Olefsky JM, Reaven GM. Effects of sulfonylurea therapy on insulin binding in mononuclear leukocytes of diabetic patients. Am ] Med 1976; 60:89. 23. Beck-Nielsen H, Pedersen O, Lindskov HO. Increased insulin sensitivity and cellular insulin binding in obese diabetics following treatment with glibenclamide. Acta Endocrinol (Copenh) 1979; 90:451. 24. Grunberger G, RuanJ, Gorder P. Sulfonylureas do not affect insulin binding or glycemic control in insulin-dependent diabetics. Diabetes 1982; 31:890. 25. Bernhard H. Long-term observations on oral hypoglycemic agents in dia¬ betes. The effect of carbutamide and tolbutamide. Diabetes 1965; 14:59. 26. Balodimos MC, Camerini-Davalos RA, Marble A. Nine years' experience with tolbutamide in the treatment of diabetes. Metabolism 1966; 11:957. 27. Lebovitz HE. Clinical utility of oral hypoglycemic agents in the manage¬ ment of patients with noninsulin-dependent diabetes meOitus. Am ] Med 1983; 75: 94. 28. Fuhlendorff J, Rorsman P, Kofod H, et al. Stimulation of insulin release by repaglinide and glibenclamide involves both common and distinct pro¬ cesses. Diabetes 1998; 47:345. 29. Bailey CJ. Biguanides and NIDDM. Diabetes Care 1992; 15:755. 30. Williams RH, Palmer JP. Farewell to phenformin for treating diabetes mellitus. Ann Intern Med 1975; 83:567. 31. Lucis OJ. The status of metformin in Canada. Can Med Assoc J 1983; 128:24. 32. Hermann LS. Metformin: a review of its pharmacologic properties and therapeutic use. Diabetes Metab Rev 1979; 5:233. 33. Bailey CJ. Metformin revisited: its actions and indications for use. Diabet Med 1988; 5:315. 34. Caspary WF, Creutzfeldt W. Analysis of the inhibitory effect of biguanides on glucose absorption: inhibition of sugar transport. Diabetologia 1971; 7:379. 35. Inzucchi SE, Maggs DG, Spollett GR, et al. Efficacy and metabolic effects of met¬ formin and troglitazone in type II diabetes mellitus. N Engl J Med 1998; 338:867. 36. Rossetti L, DeFronzo RA, Gherzi R, et al. Effect of metformin treatment on insulin action in diabetic rats: in vivo and in vitro correlations. Metabolism 1990; 39:425. 37. Schernthaner G. Improvement in insulin action is an important part of the antidiabetic effect of metformin. Horm Metab Res Suppl 1985; 15:116. 38. Klip A, Leiter LA. Cellular mechanisms of action of metformin. Diabetes Care 1990; 13:696. 39. Clarke BF, Duncan LJP. Comparison of chlorpropamide and metformin treatment on weight and blood glucose response of uncontrolled obese dia¬ betics. Lancet 1968; 1:123. 40. Wu MS, Johnston P, Sheu WH, et al. Effect of metformin on carbohydrate and lipoprotein metabolism in NIDDM patients. Diabetes Care 1990; 13:1. 41. Schneider J, Erren T, Zofel P, Kaffarnik H. Metformin induced changes in serum lipids, lipoproteins and apoproteins in non-insulin dependent dia¬ betes mellitus. Atherosclerosis 1990; 82:97. 42. Clarke BF, Duncan LJP. Biguanide treatment in the management of insulin independent (maturity-onset) diabetes: clinical experience with metformin. Res Clin Forums 1979; 1:52. 43. Nattrass M, Hinks L, Smythe P. Metabolic effects of combined sulphonylurea and metformin therapy in maturity-onset diabetics. Horm Metab Res 1979; 11:332. .44. Lindstrom J, Tuomilehto J, Spenglert M. Acarbose treatment does not change the habitual diet of patients with type 2 diabetes mellitus. Diabet Med 2000; 17:20. 45. Vierhapper H, Bratusch-Marrain A, Waldhause W. a-Glucosidase hydro¬ lase inhibition in diabetes. Lancet 1978; 2:1386. 46. Hanefeld M, Fischer S, Schulze J. Therapeutic potentials of acarbose as first-line drug in NIDDM insufficiently treated with diet alone. Diabetes Care 1991; 14:732. 47. Walton RJ, Sherif IT, Noy GA, Alberti KGGM. Improved metabolic profiles in insulin-treated diabetic patients given an a-glucosidehydrolase inhibi¬ tor. BMJ 1979; 1:220. 48. Dimitriadis G, Hatziagelaki E, Ladas S, et al. Effects of prolonged admin¬ istration of two new a-glucosidase inhibitors on blood glucose control, insulin requirements and breath hydrogen excretion in patients with insulin-dependent diabetes mellitus. Eur J Clin Invest 1988; 18:33. 49. Arends J, Wilms BH. Smoothening effect of a new a-glucosidase inhibitor, BAY m 1099, on blood glucose profiles of sulfonylurea-treated type II dia¬ betic patients. Horm Metab Res 1986; 18:761. 50. Reaven GM, Lardinois CK, Greenfield MS, et al. Effect of acarbose on car¬ bohydrate and lipid metabolism in NIDDM patients poorly controlled by sulfonylureas. Diabetes Care 1990; 13:32. 51. Johnston PS, Coniff FR, Hoogwerf BJ, et al. Effects of the carbohydrase inhibitor miglitol in sulfonylurea-treated NIDDM patients. Diabetes Care 1994; 17:20. 52. Al-Salman J, Arjomand H, Kemp DG, Mittal M. Hepatocellular injury in a patient receiving rosiglitazone. Ann Intern Med 2000; 132:121. 53. Yamasaki Y, Kawamori R, Wasada T, et al. Pioglitazone (AD-4833) amelio¬ rates insulin resistance in patients with NIDDM. AD-4833 glucose clamp study group, Japan. Tohoku J Exp Med 1997; 183:173. 54. Yoshimoto T, Naruse M, Shizume H, et al. Vasculo-protective effect of insu¬ lin sensitizing agent pioglitazone in neointimal thickening and hyperten¬ sive vascular hypertrophy. Atherosclerosis 1999; 145:333.

55. Colca JR, Wyse BM, Sawada G, et al. Ciglitazone, a hypoglycemic agent: early effects on the pancreatic islets of ob/ob mice. Metabolism 1988; 37:276. 56. Fujiwara T, Wada M, Fukuda K, et al. Characterization of CS-045, a new oral antidiabetic agent, II. Effects on glycemic control and pancreatic islet structure at a late stage of the diabetic syndrome in c57BL/KsJ-db/db mice. Metabolism 1991;40:1213. 57. Krebs EG. Historical perspectives on protein phosphorylation and a classi¬ fication system for protein kinases. Philos Trans R Soc Lond Biol Sci 1983; 302:3. 58. Krebs EG. The enzymology of control by phosphorylation. In: Boyer PD, Krebs EG, eds. The enzymes, 3rd ed. Orlando, FL: Academic Press, 1986:3. 59. Xin X, Yang S, Kowalski J, Gerritsen ME. Peroxisome proliferator-activated receptor gamma ligands are potent inhibitors of angiogenesis in vitro and in vivo. J Biol Chem 1999; 274:9116. 60. Unson XG, MacDonald D, Ray K, et al. Position 9 replacement analogs of glucagon uncouple biological activity and receptor binding. J Biol Chem 1991; 266:2763. 61. Madsen P, Brand CL, Holst JJ, Knudsen B. Advances in non-peptide gluca¬ gon receptor antagonists. Curr Pharm Des 1999; 5:683. 62. Foley JE. Rationale and application of fatty acid oxidation inhibitors in treatment of diabetes mellitus. Diabetes Care 1992; 15:773. 62a. Vella A, Shah P, Basu R, et al. Effect of glucagon-like peptide 1 (7-36) amide on glucose effectiveness and insulin action in people with type 2 diabetes. Diabetes 2000; 49:611. 63. Kelley DE, Goodpaster B, Wing RR, Sinoneau JA. Skeletal muscle fatty acid metabolism in association with insulin resistance, obesity, and weight loss. Am J Physiol 1999; 277:EU30. 64. Purnell JQ, Kahn SE, Albers JJ, et al. Effect of weight loss with reduction of intra-abdominal fat on lipid metabolism in older men. J Clin Endocrinol Metab 2000; 85:977. 65. Lean ME. Sibutramine—a review of clinical efficacy. Int J Obes 1997; 21:S30. 66. Cuellar GE, Ruiz AM, Monsalve MC, et al. A Six-month treatment of obe¬ sity with sibutramine 15 mg; a double-blind, placebo-controlled mono¬ center clinical trial in a Hispanic population. Obes Res 2000; 8:71. 67. Rossner S, Sjostrom L, Noack R, et al. Weight loss, weight maintenance, and improved cardiovascular risk factors after 2 years treatment with orlistat for obesity. Obes Res 2000; 8:49. 68. Sjostrom CD, Lissner L, Wedel H, Sjostrom L. Reduction in incidence of diabe¬ tes, hypertension and lipid disturbances after intentional weight loss induced by bariatric surgery: the SOS intervention study. Obes Res 1999; 7:477.

CHAPTER

143

INSULIN THERAPY AND ITS COMPLICATIONS GORDON C. WEIR Insulin is the mainstay of therapy for all patients with type 1 diabetes and for many of those with type 2 diabetes. Subcutane¬ ous insulin therapy attempts to mimic normal physiologic insu¬ lin secretion and regulation of fuel metabolism.1 It is subject to many variables that can be controlled only partially in every¬ day life. Insulin therapy differs from physiologic insulin secre¬ tion in at least two major ways. First, insulin secretion in normal persons is an exquisitely regulated process that rapidly responds to changes in the circulating concentrations of glu¬ cose, amino acids, other fuels, gut hormones, and the auto¬ nomic nervous system. Second, insulin normally is secreted into the portal circulation, where it acts on hepatic metabolic processes before entering the peripheral circulation. With injec¬ tion of insulin, a constant problem exists of matching fuel flux with appropriate insulin levels. Nonetheless, considerable suc¬ cess can be achieved with this imperfect therapy.

INSULIN PURITY AND SOURCES The insulin market in most nations is now dominated by human insulin preparations; pork insulin and beef-pork mix¬ tures may soon be completely discontinued by the major

Ch. 143: Insulin Therapy and Its Complications

1349

TABLE 143-1. Characteristics of the Most Widely Available Insulins in the United States Action Profile (Hours)* Type and Preparation

Constituents

Onset

Peak

Duration

Special Considerations

For intravenous, intraperitoneal, and pump use

SHORT-ACTING Regular (human)

Solution of unmodified zinc insulin crystals

0.5-1.0

2-3

6-8

Lispro

Analog

0.2

0.5-1.5

2-3

U500

Concentrated, unmodified

1-3

6-12

12-18

This is a purified pork preparation For use only in cases of insulin resistance with requirements >200 U per day

INTERMEDIATE-ACTING NPH (human)

Protamine zinc, phosphate buffer

1.5

5-8

18-24

Lente (human)

Amorphous, acetate buffer

2.5

6-12

18-28

Amorphous and crystalline mix

3-4

9-15

22-26

NPH 70%, regular 30% NPH 50%, regular 50%

0.5-1.0 0.5-1.0

3-8 3-8

18-24 18-24

Premixing with delay before injection may result in loss of regular insulin action

LONG-ACTING Ultralente (human) MIXTURES N PH/regular

Biphasic action, not suitable when frequent dose adjustments are required

NPH, neutral protamine Hagedom insulin. "Onset, peak, and duration of action of insulins are approximations, because they vary according to injection technique, site, presence of insulin antibodies, and other variables that affect insulin pharmacokinetics.

manufacturers. Beef insulin is the most immunogenic of these, whereas pork insulin is only slightly more immuno¬ genic than human insulin.1'2 This is consistent with the fact that beef insulin differs from human insulin by three amino acids, whereas pork insulin differs from human insulin by only one amino acid. Previously, insulin preparations con¬ tained trace amounts of proinsulin-like intermediates, desaminoinsulin, glucagon, pancreatic polypeptide, somatostatin, and other contaminants of islet and exocrine tissue. Antibod¬ ies to many of these substances were found in the plasma of patients treated with these older insulin preparations. Mod¬ ern purified insulins are much "cleaner" than those used 20 years ago, and the contaminants are usually present at levels of 24 hours, causing early morning hypoglycemia; for some patients, this may be due to circulating antibodies that were generated by previous use of less purified insulins. Care must be taken to ensure that "frosting" of NPH (precipitation on the walls of the vial that leads to loss of insulin potency) does not occur.17 This phenomenon can be due to freez¬ ing or overheating of the vials. Patients should be taught to rou¬ tinely examine the vials carefully before injection.

LONG-ACTING INSULINS Manufacture of the first long-acting insulin preparation, prota¬ mine zinc insulin, has been discontinued. The only currently available long-acting preparation is human Ultralente. It acts like a slow NPH, with an onset of action at 3 to 4 hours, a broad peak at 9 to 15 hours, and a duration that may not reach 24 hours.4-18 Ultralente is usually given twice a day, most com¬ monly before breakfast and before supper.

model.19 The distribution space has been estimated to approxi¬ mate the extracellular space, which is -16% of body weight. The apparent distribution space is increased in the presence of antibodies19 but otherwise appears to be similar in normal per¬ sons and patients with insulin-treated diabetes.20 The liver is the major -site of insulin clearance, accounting for -50% of the total; the kidney accounts for -30%, and skeletal mus¬ cle accounts for most of the rest.22 The metabolic clearance rate of insulin is 700 to 800 mL/m2 per minute.21 At supraphysiologic concentrations of insulin, hepatic clearance may become saturated, but this does not seem to occur in the kidney.23 Reduced insulin clearance has been found in obese persons.22 Renal function also is an important determinant of circulating insulin levels. Insulin doses usually must be lowered as renal function deteriorates. Insulin pharmacokinetics are complicated further by the many factors that alter insulin absorption from the subcutaneous site.24 These may cause much more variability in insulin action than is generally appreciated, both from one individual to another and from day to day in a given individual.25 Circulating insulin antibodies have important effects on insulin pharmacoki¬ netics, delaying the onset of action of rapid-acting insulin and increasing the duration of action of both short- and longer-acting insulins.26 Today, they rarely cause insulin resistance. Factors that tend to produce relatively increased insulin absorption include low doses of insulin, dilute insulin solution, increased subcutaneous blood flow (exercise, massage, heat), local tissue injury, abdominal injection, and intramuscular injection. Factors that tend to produce relatively decreased insu¬ lin absorption include high doses of insulin, concentrated insulin solution, decreased subcutaneous blood flow (shock, cold, standing), lipohypertrophy, intradermal injection, and injection into limbs (at rest). The insulin absorption rate is inversely pro¬ portional to the volume and concentration of the injected insu¬ lin.24 Thus, even U500 regular insulin, when given in large volumes, may have a duration of action similar to that of smaller volumes of intermediate-acting insulin.

SITE OF INJECTION Insulin conventionally is injected into the subcutaneous tissues of the abdomen, buttock, anterior thigh, and dorsal arm. In unusual circumstances, insulin also may be injected intramus¬ cularly to achieve more rapid uptake. Prolonged intramuscular insulin therapy, besides being painful, causes tissue scarring. Insulin is absorbed more rapidly from the abdominal subcuta¬ neous tissue than from the thigh or upper limb. If the limb is exercised, absorption is more rapid than expected because of increased blood flow. Traditionally, rotation of insulin injection sites has been advocated to prevent lipohypertrophy or lipoatrophy. The increased purity of insulin preparations has greatly reduced the incidence of lipoatrophy, but hypertrophy still occurs if insulin is repetitively injected into the same site. The injection patterns should be consistent; for example, abdominal sites might be used in the morning and thigh sites at night.

SUBCUTANEOUS BLOOD FLOW With upright posture, subcutaneous blood flow diminishes considerably in the lower limbs (and to a lesser extent in the abdominal wall), decreasing the absorption rate of insulin.27 Conversely, massage, increased ambient temperature (includ¬ ing hot baths and showers), and exercise increase the rate of absorption. Insulin reactions seem to occur more often during heat waves.

INSULIN PHARMACOKINETICS MIXTURE OF INSULINS After intravenous injection, the half-life of circulating insulin appears to be 5 to 10 minutes in normal persons and patients with diabetes who do not have insulin antibodies.19 The disap¬ pearance curve is explained best by a multiexponential

When regular (and probably lispro) insulin is mixed in the same syringe with Lente insulin, some of the rapid-acting com¬ ponent can be lost in just a few minutes, so the mixture must be

Ch. 143: Insulin Therapy and Its Complications injected immediately.28 The problem is potentially even more severe if regular insulin is mixed with Ultralente insulin.4'29'30 No difficulty is found when regular or lispro insulin is mixed with NPH insulin.11'24 Premixed insulin preparations are widely used. A mixture of 70% NPH and 30% regular insulin is the most common, but a mixture of 50% NPH and 50% regular insulin is also available. Mixtures of lispro insulin and interme¬ diate-acting insulin are under study.31

INDICATIONS FOR INSULIN THERAPY Virtually all patients with type 1 diabetes should be treated with insulin. Although some patients with very early type 1 diabetes can be kept in control with sulfonylureas, the theoreti¬ cal potential of insulin to slow the process of autoimmune destruction32 makes the use of insulin advisable. Also, patients with gestational diabetes who have a fasting glucose level of >120 mg/dL should be treated with insulin. Insulin is indicated in patients with type 2 diabetes who fail to respond to an ade¬ quate trial of diet and oral hypoglycemic agents.1'33

DIABETES CONTROL AND COMPLICATIONS TRIAL The 9.5-year Diabetes Control and Complications Trial (DCCT) was carried out in 29 centers and included 1441 patients with type 1 diabetes.34 The study had two arms, a group with no complica¬ tions and a group with "very mild to moderate" nonproliferative retinopathy. A mean hemoglobin Alc of ~7% was achieved in the intensive therapy group compared with a level of ~9% in the stan¬ dard therapy group. The efficacy of intensive treatment was dra¬ matic. In the primary prevention group, the risk of developing retinopathy was reduced by 76%, and in the secondary interven¬ tion cohort, the risk of progression was reduced by 54%. The risk of developing microalbuminuria was reduced by 34% and 56%, respectively. The risk of developing neuropathy was reduced by 57% and 69%, respectively. A tendency was seen for a reduction in the number of major cardiovascular and peripheral vascular events that had borderline statistical significance. Questions remain about the existence of a threshold for glycemic control that must be reached before benefits occur. The data suggest a contin¬ uum of beneficial effect such that any improvement in glycemic control should be expected to help. This is an important and prac¬ tical concept for those patients who are not able make a full com¬ mitment to a DCCT-style regimen. With regard to adverse effects, the incidence of severe hypoglycemia (reactions needing assistance) was increased three-fold in the intensive therapy group. Intensive therapy also was associated with weight gain, which was 4.6 kg more than in the conventional group after 5 years. Although concern exists regarding how many patients will be able to follow this difficult regimen, the DCCT sets a new standard of care for persons with type 1 diabetes. Caution must be used in applying a DCCT level of care to children younger than 13 years, persons with hypogly¬ cemic unawareness, and patients with advanced complications.

UNITED KINGDOM PROSPECTIVE DIABETES STUDY Much debate existed about whether the DCCT results could be extrapolated to patients with type 2 diabetes. This question has been largely answered by the United Kingdom Prospective Dia¬ betes Study (UKPDS).35'36 In this study, 5102 patients were fol¬ lowed for an average of 10 years. Treatment was with insulin, sulfonylureas, or metformin. Although the statistical power for the group receiving insulin was insufficient to allow separate

1351

analysis, evaluation of all groups revealed that lowering of the hemoglobin Alc from 7.9% to 7.0% produced a 25% reduction in microvascular complications. Moreover, a continuous relation¬ ship was found between risk of microvascular complications and glycemia, and no evidence was seen for a threshold for the devel¬ opment of complications above a hemoglobin Alc of 6.2%. These data support the hypothesis that hyperglycemia causes or is the major contributor to these complications. These results were not surprising because of the similarity in the progression and character of the complications of type 1 and type 2 diabetes.37 Various arguments have been raised about whether insulin treatment, by raising plasma insulin concentrations, might worsen macrovascular disease.38 The UKPDS did not unequivocally answer this question, but for the combined treatment groups, a 16% reduction was seen in combined fatal or nonfatal myocardial infarctions and sudden death (p = 0.052). For the group receiving insulin, no evidence was found of an increase in macrovascular disease or death rate, even though this group showed higher fast¬ ing plasma insulin levels than were found in the other treatment groups. More weight gain occurred in the patients treated with insulin and sulfonylureas than in those treated with metformin. The results of the UKPDS are very helpful for countering the anxiety about the potential cardiovascular risks of insulin therapy.

SETTING GOALS OF THERAPY Although normalization of all aspects of metabolism is the ideal in the treatment of type 1 and type 2 diabetes, this goal rarely is attainable with current forms of therapy. Nevertheless, near¬ normal glycemia can be achieved in selected patients and, ide¬ ally, should be the goal of therapy for all, with recognition that this goal must be modified for many individuals. The American Diabetes Association (ADA) has set forth goals of obtaining preprandial glucose values of between 80 and 120 mg/dL and bedtime glucose levels of 100 to 140 mg/dL.39 The goal for hemoglobin Alc is 255 A, pat¬ tern A) or smaller LDL particles (diameter of 150 mL, and recurrent urinary tract infec¬ tions. Management of diabetic cystopathy emphasizes the facil¬ itation of bladder emptying and the treatment of urinary incontinence. The Crede method and intraabdominal straining (Valsalva technique) increase intravesicular pressure and may allow for passive voiding in milder cases. Medications, includ¬ ing cholinergic stimulants (e.g., bethanechol chloride), which facilitate bladder contractions, and a-adrenergic agonists (e.g., phenoxybenzamine, prazosin, and terazosin), which relax the internal sphincter, may help facilitate bladder emptying. In advanced conditions, chronic catheterization may be necessary. If incontinence develops because of an incomplete sphincter, placement of an artificial urinary sphincter bladder, bladder neck reconstruction, or a fascial sling bladder neck suspension may be of benefit.1-3 Neuropathic sexual dysfunction affects 50% of diabetic men, producing impotence or retrograde ejaculation. Erectile impotence must be distinguished from psychogenic impotence and organic impotence of other causes by a detailed sexual history and appropriate laboratory tests. Yohimbine, an a2-adrenergic blocker, may help increase penile rigidity by increasing blood flow to the corpus of the penis. Other vasoac¬ tive medications, including phentolamine, papaverine, or pros¬ taglandins, may be injected directly into the corpora cavernosa. Several mechanical devices are available that help produce erections by creating a vacuum to draw blood into the penis. If these treatments are ineffective, a penile prosthesis may be effi¬ cacious.1-3 Retrograde ejaculation, caused by the uncoordinated closure of the internal vesicle sphincter and relaxation of the external vesicle sphincter, is diagnosed by an absent ejaculate plus the recovery of live, mobile sperm in the postcoital urine (see Chaps. 117 and 118). Anatomic, nondiabetic causes of retro¬ grade ejaculation must be excluded. Artificial insemination of sperm recovered from fresh urine samples may circumvent the resulting infertility.1-3 The blunted epinephrine response to hypogly¬ cemia produces hypoglycemic unawareness that greatly compli¬ cates intensive insulin treatment; it is ascribed to autonomic neuropathy of the adrenal medulla, although it may occur in patients without apparent neuropathy. Gastrointestinal Neuropathy. Gastrointestinal neuropa¬ thy, which can involve the entire gastrointestinal tract, probably explains the high frequency of complaints involving this sys¬ tem in diabetics1-3 (see Chap. 149). Esophageal motility disorders, although usually asymptomatic, are common in long-standing diabetes.1-3 Diabetic gastropathy diminishes gastric acid secre¬ tion and motility, explaining both the reduced frequency of duodenal ulceration in diabetics and the presence of anorexia.

1398

PART IX: DISORDERS OF FUEL METABOLISM

nausea, vomiting, postprandial fullness, and early satiety in the absence of demonstrable intrinsic lesions. Delayed gastric emptying may complicate diabetic control because of retarded postprandial caloric absorption. Gastric emptying may be improved by erythromycin, which increases gastric motility through simulating the action of motilin; by bethanechol, which increases gastric contractibility through stimulation of muscar¬ inic receptors; or by metoclopramide, which inhibits central and peripheral dopaminergic pathways and allow endogenous parasympathetic tone to act uninhibited.1'3 In refractory cases of gastroparesis, surgical procedures including jejunostomy, pyloroplasty, or partial gastrectomy may be beneficial. Diabetic diarrhea may reflect intestinal hypermotility from decreased sympathetic inhibition, hypomotility with consequent bacterial overgrowth, bile salt malabsorption, diabetic "sprue" (steator¬ rhea with mucosal histology resembling gluten sensitivity), and pancreatic insufficiency.1'3 Cardiovascular Autonomic Neuropathy. Cardiovascular autonomic neuropathy (see Chap. 147) produces exercise intoler¬ ance and orthostatic hypotension, the former from reduced augmen¬ tation of cardiac output and impaired constriction of the visceral vascular bed and the latter from an impaired sympathetically mediated compensatory increase in peripheral vascular resis¬ tance.1'3 Initial treatment of symptomatic orthostatic hypotension includes the use of waist-high compressive stockings, keeping the head of the bed elevated at night, and use of salt supplements, all of which act to increase blood volume and cardiac filling. If nonpharmacologic measures are ineffective, medications including fludrocortisone or short-acting pressor agents (e.g., ephedrine, midodrine, yohimbine, ergotamine, clonidine, and phenylephrine hydrochloride [used as an atomized nasal spray]) may be of bene¬ fit.1-3 Some studies suggest that erythropoietin may increase standing blood pressure in diabetic patients with orthostatic hypotension. Octreotide has also been used with some success. Cardiac denervation is marked by an invariant pulse of 80 to 90 beats per minute that is unresponsive to stress, exercise, or sleep; it is associated with painless myocardial infarction, coronary artery spasm, and sudden cardiac death.1-3

PATHOGENESIS AND THERAPEUTIC IMPLICATIONS OF DIABETIC NEUROPATHY A pathogenetic role for the metabolic alterations resulting from ir\sulin deficiency is now generally accepted for diabetic neu¬ ropathy; however, the nature, extent, and importance of that role remain controversial.6 There is an array of interrelated met¬ abolic alterations in diabetic nerves resulting from elevated ambient glucose concentrations (Fig. 148-8), several of which appear to interact, in a self-reinforcing or synergistic fashion, with a negative impact on nerve function. These changes include (a) increased activity of the polyol pathway leading to accumulation of sorbitol and fructose and imbalances in nicotinamide adenine dinucleotide phosphate (NADP)/ NADPH,17-19 (b) formation of reactive oxygen species via glu¬ cose autooxidation,20'203 (c) nonenzymatic glycation of proteins resulting in production of "advanced glycation end products" (AGEs),18-19'193 and (d) inappropriate activation of protein kinase C.18-19 One unifying mode of injury among these differ¬ ent metabolic impairments lies in the production and decreased scavenging of reactive oxygen species, thereby promoting cel¬ lular oxidative stress and mitochondrial dysfunction that can lead to programmed cell death of nervous system tissues.12'17-24

POLYOL PATHWAY The polyol pathway refers to the enzymatic conversion of glucose to sorbitol and then to fructose. Because this reaction is dependent on the concentration of glucose, sorbitol and fructose are formed

FIGURE 148-8. Increased glucose is thought to initiate a cascade of putatively cytotoxic metabolic events through autooxidation, glycation, and formation of advanced glycation end products (AGEs), and through increased sorbitol pathway activity. Autooxidation and/or AGEs are thought to promote the generation of toxic free radicals, including a variety of reactive oxygen species (ROS). Sorbitol production produces compensatory depletion of other organic osmolytes such as myoinositol and taurine, and endogenous antioxidant, with resulting attenuation of oxidative defense. Sorbitol accumulation and/or reciprocal osmolyte depletion also produce osmotic stress, which may damage mitochon¬ dria and stimulate protein kinase C (PKC). PKC may also be activated by shifts in triose phosphates toward diacylglycerol production as a result of increased sorbitol pathway activity. Both ROS and PKC activa¬ tion have been implicated in microvascular dysfunction in peripheral nerve and retina, producing tissue ischemia. Ischemia impairs mito¬ chondrial function and generates additional ROS. Mitochondrial dys¬ function and ROS further impair oxidative defense mechanisms. Neurotrophic support through nerve growth factor (NGF) and perhaps other neurotrophins that regulate oxidative defense mechanisms may be impaired by ROS. Mitochondrial damage is thought to lead to the release of cytochromes, activation of caspases, and induction of apopto¬ sis or programmed cell death (PCD).

in high concentrations in many tissues of the diabetic, including nerves. Conversion of glucose to sorbitol is dependent on aldose reductase, while sorbitol is converted to fructose via sorbitol dehy¬ drogenase. Both reactions change the oxidation/reduction state of the cell, decreasing NADPH/NADP+ and NADH/NAD+ ratios, respectively. This alters the normal redox potential of the cell and not only decreases the ability of the cell to detoxify reactive oxygen species but also promotes nerve ischemia and free radical injury of mitochondria.17-20'23'24 Mitochondrial dysfunction further impairs the cell's ability to function in the presence of unchecked reactive oxygen species by decreasing the available adenosine triphos¬ phate (ATP), which is needed for the de novo synthesis of free rad¬ ical scavengers.25 Left unchecked, reactive oxygen species produce (a) lipid, DNA, and protein peroxidation17-20; (b) further ischemia and reduced nerve blood flow21-24; and (c) cellular apoptosis.12'26 These collective metabolic and vascular impairments result in peripheral nervous system injury and clinical neuropathy. Animal studies have revealed that indicators of oxidative stress parallel neuropathy, and antioxidants that block formation of reactive oxy¬ gen species prevent neuropathy.6 In addition, insulin and aldose reductase inhibitors (which block the accumulation of sorbitol) prevent formation of reactive oxygen species and experimental diabetic neuropathy.17-24

AUTOOXIDATION OF GLUCOSE AND ADVANCED GLYCATED END PRODUCT FORMATION During diabetes, trace amounts of free transition metals (e.g., iron and copper) promote autooxidation of glucose, leading to further formation of reactive oxygen species.17-19,23 In experi¬ mental diabetes, metal-chelating agents preserve both nerve function and blood flow. Reactive oxygen species also may link

Ch. 149: Gastrointestinal Complications of Diabetes autooxidation and AGE formation. High ambient glucose results in glycation of proteins and oxidation by reactive oxy¬ gen species, with the final end products known as AGEs. Reac¬ tive oxygen species enhance AGE formation, which, in turn, further accelerates reactive oxygen species (ROS) formation (a process known as autooxidative glycosylation). AGE accumula¬ tion correlates with endothelial dysfunction, impaired nerve blood flow, and ischemia.17_19-23

NERVE GROWTH FACTORS Once diabetic neuropathy becomes clinically evident, structural and functional abnormalities within the peripheral nervous sys¬ tem presumably are widespread and of long standing. To what degree these structural abnormalities are reversible is unclear; however, the studies described earlier have shown greater improvement after treatment in patients with early neuropathy. This suggests that interventions should be introduced as early as possible and should be geared toward slowing the progres¬ sion of neuropathy rather than reversing the clinical signs and symptoms. Studies evaluating other neurotropic growth factors, which induce axonal sprouting in injured neurons, and may be a means to overcome some of the structural damage in diabetic neuropathy, are either planned or are under way.27-

REFERENCES 1. Greene DA, Feldman EL, Stevens MJ, et al. Diabetic neuropathy. In: Porte D Jr, Sherwin R, eds. Diabetes mellitus. East Norwalk, CT: Appleton & Lange, 1997:1009. 2. DCCT Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulindependent diabetes mellitus. N Engl J Med 1993; 329:977. 3. Feldman EL, Stevens JJ, Greene DA. Clinical management of diabetic neu¬ ropathy. In: Veves A, ed. Clinical management of diabetic neuropathy. Totowa: Humana Press, 1998:89. 4. Salpeter MM, Spanton S, Holley K, Podleski TR. Brain extract causes ace¬ tylcholine receptor redistribution which mimics some early events at developing neuromuscular junctions. J Cell Biol 1982; 93:417. 5. Stevens MJ, Feldman EL, Thomas T, Greene DA. Pathogenesis of diabetic neuropathy. In: Veves A, ed. Clinical management of diabetic neuropathy. Totowa: Humana Press, 1998:13. 6. Feldman EL, Russell JW, Sullivan KA, Golovoy D. New insights into the pathogenesis of diabetic neuropathy. Curr Opin Neurol 1999; 12:553. 7. Pirart J. Diabetes mellitus and its degenerative complications; a prospective study of 4,400 patients observed between 1947 and 1973. Diabetes Care 1978; 1:168. 8. Fedele D, Comi G, Coscelli C, et al. A multicenter study on the prevalence of diabetic neuropathy in Italy. Diabetes Care 1997; 20:836. 9. Young MJ, Boulton AJM, Macleod AF, et al. A multicentre study of the prevalence of diabetic peripheral neuropathy in the United Kingdom hos¬ pital clinic population. Diabetologia 1993; 36:150. 10. Dyck PJ, Kratz KM, Karnes JL, et al. The prevalence by staged severity of various types of diabetic neuropathy, retinopathy, and nephropathy in a population-based cohort: the Rochester Diabetic Neuropathy Study. Neu¬ rology 1993; 43:817. 11. Maser RE, Steenkiste AR, Dorman JS, et al. Epidemiological correlates of diabetic neuropathy. Report from Pittsburgh Epidemiology of Diabetes Complications Study. Diabetes 1989; 38:1456. 12. Russell JW, Sullivan KA, Windebank AJ, et al. Neurons undergo apoptosis in animal and cell culture models of diabetes. Neurobiol Dis 1999; 6:347. 13. Stevens MJ, Feldman EL, Greene DA. Diabetic peripheral neuropathy. In: DeFronzo RA, ed. Current therapy of diabetes mellitus. St. Louis: MosbyYear Book, 1997:160. 14. Greene DA, Feldman EL, Stevens MJ. Neuropathy in the diabetic foot: new concepts in etiology and treatment. In: Levin M, O'Neal L, eds. The dia¬ betic foot. St. Louis: Mosby, 1993:135. 15. Pfeifer MA, Ross DR, Schrage JP, et al. A highly successful and novel model for treatment of chronic painful diabetic peripheral neuropathy. Diabetes Care 1993; 16:1103. 16. The Capsaicin Study Group. Treatment of painful diabetic neuropathy with topical capsaicin: a multicenter, double-blind, vehicle controlled study. Arch Intern Med 1991; 151:2225. 17. Feldman EL, Stevens MJ, Greene DA. Pathogenesis of diabetic neuropathy. Clin Neurosci 1997; 4:365. 18. Greene DA, Stevens MJ, Obrosova I, Feldman EL. Glucose-induced oxida¬ tive stress and programmed cell death in diabetic neuropathy. Eur J Phar¬ macol 1999; 375:217. 19. Greene DA, Obrosova IG, Stevens MJ, Feldman EL. Pathways of glucosemediated oxidative stress in diabetic neuropathy. In: Packer L, Tritschler

19a. 20. 20a.

21.

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HJ, King GL, Azzi A, eds. Antioxidants in diabetes management. New York: Marcel Dekker, Inc., 2000: 111. Rahbar S, Hadler JL. A new rapid method to detect inhibition of Amador; product generated by delta-gluonolactone. Clin Chim Acta 1999; 287:123. van Dam PS, Bravenboer B. Oxidative stress and antioxidant treatment in diabetic neuropathy. Neurosci Res Commun 1997; 21:41. Stevens MJ, Obrosova I, Cao X, et al. Effects of DL-alpha-lipoic acid on peripheral nerve conduction, blood flow, energy metabolism, and oxida¬ tive stress in experimental diabetic neuropathy. Diabetes 2000; 49:1006. Low PA, Nickander KK, Tritschler HJ. The roles of oxidative stress and antioxidant treatment in experimental diabetic neuropathy. Diabetes 1997; 46(Suppl 2):S38.

22. Tomlinson DR. Future prevention and treatment of diabetic neuropathy. Diabetes Metab 1998; 24(Suppl 3):79. 23. Zochodne DW. Diabetic neuropathies: features and mechanisms. Brain Pathol 1999; 9:369. 24. Cameron NE, Cotter MA. Metabolic and vascular factors in the pathogene¬ sis of diabetic neuropathy. Diabetes 1997; 46(Suppl 2):S31. 25. Heiden MG, Chandel NS, Schumacker PT, Thompson CB. Bcl-xL prevents cell death following growth factor withdrawal by facilitating mitochon¬ drial ATP/ADP exchange. Mol Cell 1999; 3:159. 26. Anderson KM, Seed T, Ou D, Harris JE. Free radicals and reactive oxygen species in programmed cell death [In Process Citation], Med Hypotheses 1999; 52:451. 27. Feldman EL, Windebank AJ. Growth factors and peripheral neuropathy. In: Dyck PJ, Thomas PK, eds. Diabetic neuropathy. Philadelphia: WB Saun¬ ders, 1998:377.

CHAPTER

149

GASTROINTESTINAL COMPLICATIONS OF DIABETES FREDERIC D. GORDON AND KENNETH R. FALCHUK Diabetes mellitus can affect many organs in the gastrointestinal tract. The metabolic derangements caused by diabetes can have a significant impact on the function of the digestive and hepato¬ biliary systems. Rundles, in 1945, was the first to draw attention to the effects of diabetes on the gut.1 Subsequently, others have confirmed the observation of altered motility in diabetics with autonomic neuropathy. Dysphagia, nausea, vomiting, diarrhea, constipation, and fecal incontinence are common complaints among these patients. Differentiating the various causes of these symptoms can be difficult and should not necessarily be attributed to diabetes alone. Recent developments have led to a better understanding of the pathophysiology of diabetic gastroenteropathy. These advances have broadened the therapeutic options. In this chapter the focus is on the pathophysiology, clinical features, diagnosis, and treatment of the common gas¬ trointestinal complications of diabetes.

PATHOGENESIS Despite recent scientific advances, the pathogenetic mecha¬ nisms causing diabetic gastrointestinal symptoms remain poorly understood. The most widely accepted theory is that an autonomic visceral neuropathy plays an important role in the development of symptoms. This is supported by the observa¬ tion that diabetics with impaired gastric emptying have the same clinical constellation and radiographic findings as postvagotomy patients.2 The delay in gastric emptying may result from impaired myogenic contraction and altered cholin¬ ergic pathways. Nitric oxide may play a critical role in mediat¬ ing gastric wall tone and compliance. In diabetic animal models, nitric oxide synthase activity is decreased, resulting in

1400

PART IX: DISORDERS OF FUEL METABOLISM

gastrointestinal motor dysfunction.3 In addition, electron microscopic studies have demonstrated a decreased density of unmyelinated axons in the vagus nerve of diabetics with gastroparesis.4 Furthermore, additional manifestations of auto¬ nomic neuropathy such as orthostatic hypotension, urinary bladder dysfunction, and impotence are often present in these patients. Other mechanisms have also been proposed. Hyper¬ glycemia and electrolyte imbalance (e.g., hyperkalemia, hypokalemia) may affect nerve function and delay gut motil¬ ity.5 Diabetics may also have altered production of various hor¬ mones such as motilin, pancreatic polypeptide, somatostatin, vasoactive intestinal peptide, calcitonin gene-related peptide, glucagon, and gastrin, which are known to influence gas¬ trointestinal tract motility, absorption, and secretion.6'7 Finally, diabetic microangiopathy may play a causal role in the devel¬ opment of gastrointestinal tract complications.8 Surprisingly, none of the abnormalities caused by these proposed mecha¬ nisms correlate with the duration of diabetes, the severity of the neuropathy, the degree of blood sugar control, the presence of other diabetic complications, or the age or sex of the patient.9

ESOPHAGUS Although diabetics rarely complain of symptoms referable to the esophagus, the most typical symptoms are heartburn, and less frequently dysphagia and chest pain. Esophageal motor dysfunction in diabetics has been extensively studied using various techniques, including barium studies, nuclear radiographic methods, and manometry. Delayed esophageal transit and emptying have been demonstrated in up to 35% of both type 1 and type 2 diabetics.10-12 Esophageal manometry can reveal multiple aberrant motor patterns, including reduced or absent peristalsis, tertiary contractions, and decreased lower esophageal sphincter pressures.13 The correlation between investigative studies and clinical symptoms is inconsistent. Diabetics with dysphagia warrant complete investigation to rule out gastroesophageal reflux, benign or malignant esoph¬ ageal strictures, and infectious esophagitis. Endoscopy is the preferred method of evaluation. Biopsy samples can be obtained and therapeutic maneuvers can be performed. Gas¬ troesophageal reflux disease can be treated with H2-receptor antagonists or proton pump inhibitors alone or in combina¬ tion with a prokinetic agent. If esophageal biopsy samples document candidal esophagitis, antifungal therapy can be instituted.

STOMACH The most common gastric disorder of diabetes is gastroparesis, which is manifested by postprandial fullness, vague epigastric pain, nausea, vomiting, heartburn, and anorexia. The onset of symptoms is usually insidious, but symptoms may arise acutely, especially in association with ketoacidosis. There is a strong correlation between the onset of gastroparesis and the development of other diabetic complications, such as periph¬ eral neuropathy, retinopathy, and nephropathy. It is important to exclude other causes of impaired gastric emptying such as gastric outlet obstruction, peptic ulcer disease, side effects of pharmacologic agents, and uremia. Barium studies in patients with gastroparesis can demon¬ strate an elongated stomach with sluggish, ineffective, or absent peristalsis. At times, solid residue can be found in the stomach of these patients (Fig. 149-1). Because gastric emptying of liquids is usually normal, barium contrast studies are insen¬ sitive in assessing gastric dysmotility. By using solid, nondigestible radiopaque markers, the gastric emptying of nondigestible solids is found to be more prolonged than the

FIGURE 149-1.

Barium upper gastrointestinal radiograph in a patient

with diabetic gastroparesis. The stomach is elongated and filled with food debris.

emptying of digestible solids.14 Results of radioisotope gastricemptying studies are more accurate because both liquid- and solid-phase motility are assessed.15 Because of the impaired gastric emptying, these patients are predisposed to the forma¬ tion of bezoars, consisting of nondigestible foods. Gastroparesis leads to the irregular emptying of solids into the small intestine, resulting in erratic serum glucose control that may, in turn, worsen gastric emptying. To break this cycle, frequent small meals and close monitoring of the serum glucose concentration are advised. Should symptoms persist, various antiemetic and proki¬ netic agents can be used (Table 149-1). Metoclopramide is an antidopaminergic drug that inhibits dopamine-induced gas¬ tric smooth muscle relaxation.16 It also has cholinergic activity that probably acts by increasing the release of acetylcholine from postganglionic neurons or by enhancing acetylcholine receptor sensitivity in the gastric wall.1 In addition to its cho¬ linergic effects, metoclopramide binds to medullary chemoreceptors in the brain, preventing emesis. As a result of its ability to penetrate the blood-brain barrier, side effects such

TABLE 149-1. Prokinetic Agents Commonly Used for the Treatment of Diabetic Gastroparesis: Their Mechanisms of Action and Side Effects Drug

Mechanisms of Action

Side Effects

Metoclopramide

Dopamine antagonist

Hyperprolactinemia

Cholinergic agonist

Diarrhea, cramps, tremor Tardive dyskinesia

Domperidone

Central antiemetic effect

Anxiety, depression

Dopamine antagonist

Hyperprolactinemia

Central antiemetic effect

Erythromycin

Motilin receptor agonist

Diarrhea, cramps, nausea

Ch. 149: Gastrointestinal Complications of Diabetes as sedation, tremulousness, headache, galactorrhea, and amenorrhea due to hyperprolactinemia can occur. Several studies have shown that while metoclopramide is effective with short-term use, tachyphylaxis may develop.18 Domperidone is a potent dopamine antagonist lacking cholinergic activity. It enhances gastric emptying and has central anti¬ emetic properties. Side effects of domperidone are infrequent because of its limited penetration of the blood-brain barrier, although hyperprolactinemia can occur. It is beneficial for long-term treatment and has an excellent safety profile.19-20 Erythromycin is another drug that is effective in relieving symptoms of gastroparesis. It is a macrolide antibiotic that has prokinetic properties attributed to its ability to bind to motilin receptors in the antrum and duodenum. Numerous studies have shown improvement in gastric emptying.21-22 Gastroparesis can be a difficult disorder to manage; symp¬ toms may persist despite aggressive medical treatment. Patients who have failed prokinetic therapy may benefit from a percuta¬ neous endoscopic gastrostomy and jejunostomy that allows gastric decompression and adequate nutritional support. This nonpharmacologic approach is well tolerated 23 Gastric pacing has been shown to improve gastric emptying. This technique entrains and improves gastric slow-wave activ¬ ity and enhances slow-wave amplitude. This leads to a normal¬ ization of gastric dysmotility 24

1401

CELIAC DISEASE Celiac disease has been reported to occur in diabetics with greater frequency than in the general population.29-30 The histo¬ compatibility antigens HLA-DR3 and HLA-DQB1>I'0201 have been linked to celiac disease and type 1 diabetes, suggesting a genetic predisposition to both conditions. The clinical manifes¬ tations of celiac disease and diabetic diarrhea are similar, although celiac disease may be distinguished by the absence of peripheral neuropathy, the onset of diarrhea before the initial diagnosis of diabetes, and steatorrhea, which is rare in diabetic diarrhea. In these patients, biopsy of the small bowel is essen¬ tial. A presumptive diagnosis of celiac disease can be made if the biopsy shows flattened villi and increased cellularity in the lamina propria. A gluten-free diet will improve both the symp¬ toms and histology and thus confirm the diagnosis. Serum immunoglobulin A (IgA) antiendomysial and IgA antigliadin antibodies may also be useful in diagnosing and monitoring patients with celiac disease. Several studies have shown 80% to 100% sensitivity and specificity of these antibodies for celiac disease, although their use cannot substitute for a histologic diagnosis.31-32 With successful treatment, titers of these antibod¬ ies decrease or even disappear and can, therefore, be used to monitor compliance with a gluten-free diet.

LARGE INTESTINE SMALL INTESTINE The incidence of diarrhea in the diabetic population is -22%, occurring primarily in patients with poorly controlled diabe¬ tes.25 The clinical features are nonspecific, consisting of chronic intermittent diarrhea alternating with normal bowel move¬ ments or constipation. Despite the chronicity, weight loss is distinctly uncommon and should elicit a search for other causes of diarrhea. The etiology of diarrhea in diabetes is multifactorial. Auto¬ nomic dysfunction plays a role in diabetic diarrhea. Specifically, a2-adrenergic receptors in enterocytes of the small and large intestines stimulate absorption of sodium chloride and inhibit bicarbonate secretion, resulting in a net influx of fluids and ions into the gut.26 A second cause may be bacterial overgrowth. Up to 4% of diabetics have achlorhydria,27 and many have delayed gastric and small bowel transit. These features alone or in com¬ bination can allow overgrowth of Escherichia coli, enterococci, and staphylococci. These bacteria deconjugate bile salts, pre¬ venting micelle formation and resulting in the development of fat malabsorption. There is also evidence that pancreatic insuf¬ ficiency is a contributing factor.28 The evaluation of diabetics with diarrhea should be directed by their clinical presentation. Stool output should be quantified to separate true diarrhea from fecal incontinence. Stool cultures for enteric pathogens and 72-hour fecal fat studies should be performed. Patients with documented steatorrhea need to be evaluated for exocrine pancreatic insufficiency, bacterial over¬ growth, or a primary malabsorptive disorder such as celiac dis¬ ease. Pancreatic insufficiency can be diagnosed by a secretin/ cholecystokinin stimulation test or a clinical response to a trial of pancreatic enzyme replacement. Bacterial overgrowth can be diagnosed by various methods, including breath tests and quantitative small bowel bacterial cultures. Alternatively, a response to a 2-week trial of antibiotics, such as tetracycline, can assist in the evaluation of this condition. Malabsorptive processes often require biopsy of the small bowel for diagnosis. Patients without steatorrhea or evidence of enteric infection fall into the category of "idiopathic diabetic diarrhea" and may benefit from a trial of clonidine, conventional antidiarrheal agents (loperamide, diphenoxylate, codeine), anticholinergic agents, or psyllium.

The most common gastrointestinal complaint among diabetics is chronic constipation, occurring in 20% to 60% of patients.24-33 The pathogenesis is poorly understood, although neurogenic dysfunction is thought to be the primary cause. Diminished or delayed postprandial myoelectric colonic activity is observed in diabetics without constipation, and absent activity is observed in those with constipation.34 Pharmacologic challenge with neostigmine or metoclopramide induces a return of the myo¬ electric response. The evaluation of chronic constipation in the diabetic should be guided by the patient's age, associated symptoms, and fam¬ ily history. In patients with rectal bleeding, occult blood in the stool, age older than 40, or a family history of colon cancer, malignancy should be ruled out by appropriate studies such as flexible sigmoidoscopy, barium enema, or colonoscopy. Treat¬ ment of constipation should include dietary fiber, stool soften¬ ers, and rarely laxatives. Fecal incontinence is common and often coincides with the onset of diarrhea.343 There is a strong association of incontinence with autonomic neuropathy. The pathogenesis is complex and incompletely understood. Initially it was thought that dimin¬ ished rectal sensation with intact anal sphincter function allowed incontinent passage of stool.35 Later, anorectal manometry was used to demonstrate that fecal incontinence is caused by aberrant sphincter function; however, it is unclear whether autonomic neuropathy or abnormal intrinsic sphincter smooth muscle underlies the manometric findings.36 Therapy for fecal inconti¬ nence is difficult but responds to the control of diarrhea. If this fails, biofeedback and sphincter tone improving techniques can be used, although the results are not always successful.

GALLBLADDER DISEASE The management of gallstones in diabetics is a controversial subject because of the vast number of studies and the oppos¬ ing conclusions reached. In autopsy studies, diabetics have approximately twice the incidence of gallstones as compared with the nondiabetic population.363-37 Although no specific cause has been determined, factors that may predispose to cholelithiasis include impaired gallbladder emptying, altered

1402

PART IX: DISORDERS OF FUEL METABOLISM

glucose metabolism, hyperinsulinemia, and supersaturation of gallbladder bile levels with decreased bile acid concentra¬ tions.38 Other studies, however, indicate that comorbid condi¬ tions, such as obesity and hyperlipidemia, are confounding variables and that diabetics are not at increased risk of gall¬ stone formation.39 Asymptomatic gallstones in the general population rarely lead to life-threatening complications.40 It is not clear whether this applies to diabetic patients. Several studies in diabetics have shown that when gallstones become symptomatic, morbidity and mortality are increased, prima¬ rily due to infectious complications.41 Previously, emergency cholecystectomy in the diabetic population was reported to have a five- to ten-fold increase in mortality when compared with the nondiabetic population.42 It has been demonstrated that diabetes itself does not increase morbidity and mortality, but rather its associated conditions (e.g., renal failure, vascu¬ lar disease) are responsible.43 Additionally, complication rates have decreased significantly because of the early detection of gallstones and cholecystitis by ultrasonography, the more effective use of antibiotics, and improved intraoperative hemodynamic monitoring.44 Because diabetics often have comorbid conditions, patients with biliary colic or cholecystitis should be advised to undergo cholecystectomy as soon as possible. In the past, it has been rec¬ ommended that diabetics with asymptomatic gallstones also have surgery to reduce the chance of potential morbidity in the future. This concept has been carefully reevaluated in the past 10 years. Expectant management of diabetics with asymptom¬ atic gallstones has been shown to be safer and less costly than prophylactic cholecystectomy.44-45 The introduction of laparo¬ scopic cholecystectomy, while potentially less invasive than conventional surgery, should not influence the decision to oper¬ ate on a patient with asymptomatic cholelithiasis until this con¬ cept is studied in more detail.

LIVER DISEASE The incidence of liver dysfunction in diabetics ranges from 28% to 39%.46 Viral hepatitis is seen in diabetics two to four times more frequently than in the general population 47 Previously, this was thought to be due to needle sticks; however, the associ¬ ation remains constant even in the era of disposable needles. More likely, it is caused by increased nosocomial exposures or diminished resistance to infection.48 Many of the medications used to treat diabetes and its com¬ plications can cause liver function test abnormalities. These drugs include the oral hypoglycemic sulfonylureas, many anti¬ hypertensive agents, and antibiotics. Both transaminase eleva¬ tions and cholestatic jaundice can be seen as side effects of medication. Rarely does the side effect manifest clinically, and stopping the offending agent usually leads to a clinical and bio¬ chemical resolution. Increased glycogen deposition is the most common hepatic disorder found in diabetics. Excessive glycogen stores have been documented in up to 80% of persons with type 1 and 2 diabetes and is thought to be stimulated by exogenous hyperinsulinemia.47 A second liver lesion associated with diabetes is nonalco¬ holic steatohepatitis (NASH).49 This condition must be distin¬ guished from hepatic steatosis, a noninflammatory process, which occurs in up to 50% of type 2 diabetics. The diagnosis of NASH requires a liver biopsy showing moderate to gross macrovesicular steatosis in association with inflammation. Additionally, there must be negligible alcohol consumption and absence of active viral infection. The mechanism of fat accumulation in type 1 diabetics is thought to be due to chronic hyperglycemia coupled with inadequate insulin levels that stimulate the release of fatty acids from adipose tissue. These fatty acids are then transported to the liver, where they

are deposited into the hepatocyte. Fat accumulates because of increased hepatocellular triglyceride production and dimin¬ ished secretion in the form of very low-density lipoprotein. In type 2 diabetes the mechanism of fat deposition is different and probably accounts for the increased incidence of NASH when compared with type 1 diabetes. In the type 2 diabetic population, obesity rather than hyperglycemia and hypoinsulinemia seems to be the critical factor. Intake of excessive dietary fats and carbohydrates leads to high levels of free fatty acids that are stored in hepatocytes.46 Accumulation of fatty acids may be responsible for hepatic inflammation because they are highly reactive and can damage biomembranes. Physical examination of the diabetic patient with NASH can be normal or reveal an enlarged, tender liver. The alkaline phosphatase value is typically normal or slightly elevated. Serum transaminase levels can be slightly elevated, but more significant elevations should prompt an evaluation of other etiologies of hepatitis (e.g., infections, drugs). Radiologic eval¬ uation may be normal or show evidence of fatty infiltration of the liver. The diagnosis of NASH, however, can be confirmed only by liver biopsy. the course of NASH is typically indolent, but nearly 50% of patients develop hepatic fibrosis and up to 15% will develop cirrhosis. There is no proven therapy for NASH although grad¬ ual and sustained weight loss is recommended.-41

REFERENCES 1. Rundles RW. Diabetic neuropathy. Medicine 1945; 24:111. 2. Zitomer BR, Gramm HF, Kozak GP. Gastric neuropathy in diabetes mellitus: radiologic observations. Metabolism 1968; 17:199. 3. Takahashi T, Nakamura K, Itoh H, et al. Impaired nitric oxide synthase in the gastric myenteric plexus of spontaneously diabetic rats. Gastroenterol¬ ogy 1997; 113:1535. 4. Guy R, Dawson J, Garrett ], et al. Diabetic gastroparesis from autonomicneuropathy: surgical considerations and changes in vagus nerve morphol¬ ogy. J Neurol Neurosurg Psychiatry 1984; 47:686. 5. DeBoer SY, Masclee A AM, LamersCBHW. Effect of hyperglycemia on gas¬ trointestinal and gallbladder motility. Scand J Gastroenterol 1992; 27(Suppl 194):13. 6. Hilsted J. Pathophysiology in diabetic autonomic neuropathy: cardiovas¬ cular, hormonal, and metabolic studies. Diabetes 1982; 31:730. 7. Belai A, Lincoln, J, Bumstock G. Lack of vasoactive intestinal polypeptide and calcitonin gene-related peptide during electrical stimulation of enteric nerves in streptozotocin diabetic rats. Gastroenterology 1987; 93:1034. 8. Liberski S, Koch K, Atnip R, et al. Ischemic gastroparesis: resolution after revascularization. Gastroenterology 1990; 99:252. 9. Keshavarzian A, Iber F, Nasrallah S. Radionuclide esophageal emptying and manometric studies in diabetes mellitus. Am J Gastroenterol 1987; 82:625. 10. Westin L, Lilja B. Oesophageal scintigraphy in patients with diabetes melli¬ tus. Scand J Gastroenterol 1986; 21:1200. 11. Borgstrom P, Olsson R, Sundkvist G, et al. Pharyngeal and oesophageal function in patients with diabetes mellitus and swallowing complaints. Br J Radiol 1988; 61:817. 12. Horowitz M, Harding P, Maddox A, et. al. Gastric and oesophageal emptying in patients with type 2 diabetes mellitus. Diabetologia 1989; 32:151. 13. Mandelstam P, Lieber A. Esophageal dysfunction in diabetic neuropathy gastroenteropathv. JAMA 1967; 201:88.14.Feldman M, Smith H, Simon T. Gastric emptying of solid radiopaque markers: studies in healthy subjects and diabetic patients. Gastroenterology 1984; 87:895. 15. Siegel JA, Urbain JL, Adler LP, et al. Biphasic nature of gastric emptying. Gut 1988; 29:85. 16. Peringer E, Jenner P, Donaldson JM, et al. Metoclopramide and dopamine receptor blockade. Neuropharmacology 1976; 15:463. 17. Hay AM, Man WK. Effect of metoclopramide on guinea pig stomach: criti¬ cal dependence on intrinsic stores of acetylcholine. Gastroenterology 1979; 76:492. 18. Schade RR, Dugas MC, Lhostkv DM, et al. Effect of metoclopramide on gastric liquid emptying in patients with diabetic gastroparesis. Dig Dis Sci 1985; 30:10. 19. Soykan I, Sarosiek I, McCallum RW. The effect of oral domperidone ther¬ apy on gastrointestinal symptoms, gastric emptying, and quality of liver in patients with gastroparesis. Am J Gastroenterol 1997; 92(6):976. 20. Silvers D, Kipnes M, Broadstone V, et al. Domperidone in the management of symptoms of diabetic gastroparesis. Clin Ther 1998; 20:438.

Ch. 150: Diabetic Nephropathy 21. Altomare DF, Rubini D, Pilot M-A, et al. Oral erythromycin improves gas¬ trointestinal motility and transit after subtotal but not total gastrectomy for cancer. Br J Surg 1997; 84:1017. 22. Janssens J, Peeters TL, Vantrappen G, et al. Erythromycin improves delayed gastric emptying in diabetic gastroparesis. N Engl J Med 1990; 322:1028. 23. Kim CH, Nelson DK. Venting percutaneous gastrostomy in the treat¬ ment of refractory idiopathic gastroparesis. Gastrointest Endosc 1998; 47(1):67. 24. McCallum RW, Chen JDZ, Lin Z, et al. Gastric pacing improves emptying and symptoms in patients with gastroparesis. Gastroenterology 1998; 114:456. 25. Feldman M, Schiller L. Disorders of gastrointestinal motility associated with diabetes mellitus. Ann Intern Med 1983; 98:378. 26. Ogbonnaya K, Arem R. Diabetic diarrhea: pathophysiology, diagnosis, and management. Arch Intern Med 1990; 150:262. 27. Ungar B, Stocks AE, Martin FIR, et al. Intrinsic-factor antibody, parietal-cell antibody, and latent pernicious anemia in diabetes mellitus. Lancet 1968; 2:415. 28. Newihi H, Dooley C, Saad C, et al. Impaired exocrine pancreatic function in diabetics with diarrhea and peripheral neuropathy. Dig Dis Sci 1988; 33:705. 29. Walsh CH, Cooper BT, Wright AD, et al. Diabetes mellitus and celiac dis¬ ease: a clinical study. Q J Med 1978; 47:89. 30. Rensch MJ, Merenich JA, Lieberman M, et al. Gluten-sensitive enteropathy in patients with insulin-dependent diabetes mellitus. Ann Intern Med 1996; 124(6):564. 31. Corroccio A, Iacono G, Montalto G, et al. Immunologic and absorptive tests in celiac disease: can they replace intestinal biopsy? Scand J Gastroenterol 1993; 28:673. 32. McMillan SA, Haughton DJ, Biggart JD, et al. Predictive value for coeliac disease of antibody to gliadin, endomysium, and jejunum in patients attending for jejunal biopsy. Br Med J 1991; 303:1163. 33. Goyal RK, Spiro HM. Gastrointestinal manifestations of diabetes mellitus. Med Clin North Am 1971; 55:1031. 34. Battle WM, Snaper WJ Jr, Alavi A, et al. Colonic dysfunction in diabetes mellitus. Gastroenterology 1980; 79:1217. 34a. Folnaczny C, Riepl R, Tschop M, Landgraf R. Gastrointestinal involvement in patients with diabetes mellitus: part I. Epidemiology, pathophysiology, clinical findings. Z Gastroenterol 1999; 37:803. 35. Katz LA, Kaufman HJ, Spiro HM. Anal sphincter pressure characteristics. Gastroenterology 1967; 52:513. 36. Schiller LR, Santa Ana C A, Schmulen AC, et al. Pathogenesis of fecal incon¬ tinence in diabetes mellitus. N Engl J Med 1982; 307:1666. 36a. Ruhl CE, Everhart JE. Association of diabetes, serum insulin and C-peptide with gallbladder disease. Hepatology 2000; 31:299. 37. Lieber MM. The incidence of gallstones and their correlation with other diseases. Ann Surg 1952; 135:394. 38. DeSantis A, Attili AF, Corradini SG, et al. Gallstones and diabetes: a case-control study in a free-living population sample. Hepatology 1997;25:787. 39. Persson GE, Thulin AJG. Prevalence of gallstone disease in patients with diabetes mellitus: a case-control study. Eur J Surg 1991; 157:579. 40. Gracie WA, Ransohoff DF. The natural history of silent gallstones. N Engl J Med 1982; 307:798. 41. Hickman MS, Schwesinger WH, Page CP. Acute cholecystitis in the dia¬ betic. Arch Surg 1988; 123:409. 42. Pellegrini C. Asymptomatic gallstones: does diabetes mellitus make a dif¬ ference? Gastroenterology 1986; 91:245. 43. Haff RC, Butcher HR, Ballinger WF. Factors influencing morbidity in bil¬ iary tract operations. Surg Gynecol Obstet 1971; 132:195. 44. Friedman LS, Roberts MS, Brett AS, et al. Management of asymptomatic gallstones in the diabetic patient. Ann Intern Med 1988; 109:913. 45. Ransohoff DF, Gracie MD, Wolfenson LB, et al. Prophylactic cholecystec¬ tomy or expectant management for silent gallstones. Ann Intern Med 1983; 99:199. 46. Falchuk KR, Fiske S, Haggitt R, et al. Pericentral hepatic fibrosis and intra¬ cellular hyalin in diabetes mellitus. Gastroenterology 1980; 78:535. 47. Stone B, Van Thiel D. Diabetes mellitus and liver disease. Semin Liver Dis 1985; 5:8. 48. Khuri KG, Shamma MH, Abourizk N. Hepatitis B virus markers in diabe¬ tes mellitus. Diabetes Care 1985; 8:250. 49. Luyckx FH, Lefebvre PJ, Scheen AJ. Non-alcoholic steatohepatitis: associa¬ tion with obesity and insulin resistance, and influence of weight loss. Dia¬ betes Metab 2000; 26:98. 50. Sheth SG, Gordon FD, Chopra S. Nonalcoholic steatohepatitis. Ann Intern Med 1997; 126(2):137.

CHAPTER

1403

1 50

DIABETIC NEPHROPATHY RALPH A. DEFRONZO

Of the ~1 million people with type 1 (previously called insulindependent) diabetes mellitus in the United States, 30% to 40% eventually develop end-stage renal failure,1 and there is little evidence that the incidence of diabetic nephropathy in type 1 diabetics has changed within the last decade.2 Type 2 (previ¬ ously called non-insulin-dependent) diabetes mellitus is much more common, affecting some 15 million people.3 Among type 2 diabetic patients, the incidence of renal disease is lower (~510%).4'5 However, the incidence of nephropathy in type 2 dia¬ betics varies considerably among ethnic groups, being three to four times higher in blacks and Hispanics and seven times higher in Native Americans when compared to whites.6-8 Nev¬ ertheless, in absolute terms, more type 2 than type 1 diabetic patients eventually progress to renal insufficiency. The magni¬ tude of the problem can be readily appreciated when it is real¬ ized that every third person who is started on dialysis is diabetic,9 and that the Medicare payment for diabetics is -$51,000 per year per patient.10

DIABETIC NEPHROPATHY Since the original description of diabetic nephropathy by Kimmelstiel and Wilson11 in 1936, numerous studies have reported the renal histologic changes and the clinical course, which is characterized by hypertension, edema, heavy albuminuria, and varying degrees of renal insufficiency.5 Three major histopatho¬ logic alterations have been described in the diabetic kidney: glomerulosclerosis, vascular involvement, and tubulointersti¬ tial disease.12-14

GLOMERULOSCLEROSIS Glomerular involvement is the most characteristic feature of diabetic nephropathy and includes three distinctive lesions: dif¬ fuse intercapillary glomerulosclerosis, nodular glomeruloscle¬ rosis, and glomerular basement membrane (GBM) thickening (Fig. 150-1). DIFFUSE AND NODULAR LESIONS Diffuse intercapillary glomerulosclerosis, the most frequent histologic abnormality, is characterized by increased, periodic acid-Schiff-positive, eosinophilic material within the mesan¬ gial region (see Fig. 150-1A). The process is diffuse, involving the entire glomerulus, and generalized, affecting all glomeruli throughout the kidney. The earliest change is a widening of the mesangial matrix. With time, this mesangial material expands and coalesces, encroaching on adjacent capillary lumina. As this process progresses, entire glomeruli may become hyalinized. There is no increase in mesangial cell number, but mesangial cell volume is increased.15 In 40% to 50% of patients, the increase in mesangial matrix forms large acellular nodules at the center of peripheral glomerular lob¬ ules (see Fig. 150-1B). This nodular lesion is invariably associ¬ ated with the diffuse lesion and is pathognomonic of diabetes mellitus. The nodular lesion correlates poorly with the sever¬ ity of clinical renal disease; the best predictor of clinical renal disease is the diffuse lesion.13-15 Immunofluorescent staining reveals diffuse linear deposi¬ tion of immunoglobulin G (IgG) along the GBM. Although this

1 404

PART IX: DISORDERS OF FUEL METABOLISM Biochemical analysis of the normal GBM has demonstrated significant differences in the collagen and protein content com¬ pared with basement membranes from nonrenal tissues.16 These differences in chemical composition account for the high glom¬ erular filtration coefficient, which permits a water flux that is 50 to 100 times greater than,in other capillaries, yet totally restricts the passage of serum proteins. The ability to restrict proteins selectively also is related to the strong negative charge provided by heparin sulfate and sialic acid residues within the GBM. A number of biochemical alterations in the composition of the GBM occur in diabetic nephropathy.16'17 Moreover, studies in alloxan diabetic rats have demonstrated increased activity of glucosyltransferase, the enzyme involved in the assembly of hydroxylysine-linked carbohydrate units. The restoration of normoglycemia with insulin normalized the activity of this enzyme. These results suggest that the accelerated synthesis of hydroxylysine-glucose-galactose subunits contributes to the GBM thickening; other investigators have been unable to dem¬ onstrate a significant alteration in amino acid or glucosylgalactose content in diabetic GBM. A polyantigenic expansion, involving all of the intrinsic components of the GBM and mesangium, has been demonstrated.18 This suggests an overall increase in the synthetic rate of normal basement membrane or a decrease in the rate of degradation. In diabetics with advanced nephropathy, the sialic acid and heparin sulfate pro¬ teoglycan content of the GBM is uniformly diminished. This decrease in anionic charge contributes to the increased clear¬ ance of negatively charged macromolecules, such as albumin.1'1 In vitro studies using isolated glomeruli have shown that glu¬ cose stimulates its own incorporation into capillary basement membrane in a dose-dependent fashion. Excessive glycosylation of the GBM may render collagen more resistant to degrada¬ tion and contribute to the GBM thickening.

VASCULAR INVOLVEMENT

FIGURE 150-1. A, Diffuse diabetic glomerulosclerosis with marked mesangial matrix hyperplasia (thick arrows) and thickened glomerular basement membranes (thin arrows). No hypercellularity is evident. B, Nod¬ ules (thick arrow) usually are observed in the peripheral capillary loops and rarely are seen without the diffuse lesion. Note the prominent arteriolosclerosis in the efferent and afferent arterioles. C, Interstitial fibrosis, tubular atrophy (thick arrow), and thickening of the tubular basement membranes. (From DeFronzo RA. Diabetes and the kidney: an update. In: Olefsky JM, Sherwin RS, eds. Diabetes mellitus: management and complications. New York: Churchill Livingstone, 1985:161.) linear pattern of immunoglobulin deposition is reminiscent of anti-GBM nephritis, eluted IgG has no affinity for the GBM and most likely represents nonspecific trapping of filtered proteins. BASEMENT MEMBRANE THICKENING

Thickening of the GBM is an early and characteristic change of diabetic glomerulosclerosis (see Fig. 150-1A). Basement mem¬ brane thickening is not limited to the glomerulus but can be observed in capillaries throughout the body, including muscle, skin, and retina. The GBM is a collagen-like protein that is synthesized by visceral epithelium of the glomerulus.12 The integrity of the GBM is maintained by the continuous addition of new material from the epithelial aspect and by the simultaneous removal from the endothelial side by mesangial cells. The half-life of the GBM is -100 days. Both increased epithelial synthesis and impaired removal by mesangial cells contribute to the base¬ ment membrane thickening.

Accelerated renal arteriosclerosis and arteriolosclerosis are more characteristic of the diabetic than the nondiabetic kidney (see Fig. 150-1B). In the larger arteries, atheromatous changes are often advanced and may contribute to renal failure by caus¬ ing ischemic parenchymal atrophy. In the smaller renal arteri¬ oles, hyaline thickening involves the afferent and efferent vessels. Although arteriosclerosis and arteriolosclerosis may be extensive, neither process correlates with the severity of glom¬ erular change.

TUBULOINTERSTITIAL DISEASE Although not usually appreciated, tubulointerstitial changes (see Fig. 150-1C) are common in the diabetic kidney, and in advanced cases, there is marked tubular atrophy, thickening of the tubular basement membrane, and interstitial fibrosis.14-20 21 Such changes are typically observed with renal ischemia, but in the diabetic, the tubular changes correlate poorly with the degree of vascular involvement and may be seen in their absence. The involved tubules often show thickening of the tubular basement membrane, and immunofluorescent studies have demonstrated deposition of IgG along the tubular base¬ ment membrane. The interstitial area surrounding the involved tubules is fibrotic and may contain a cellular infiltrate of lym¬ phocytes and plasma cells. These changes progress with increasing duration of the diabetes.13-14 The interstitial reaction does not imply infection, such as pyelo¬ nephritis, although asymptomatic bacteriuria and pyelonephritis are twice as common in diabetics, particularly women, than in nondiabetics.22 This propensity to urinary tract infection probably results from various factors, including impaired renal blood flow, bladder dysfunction, interstitial scarring, impaired polymorpho¬ nuclear leukocyte function, defective leukocyte chemotaxis, and glucosuria, which enhance bacterial growth. It has been difficult

1405

Ch. 150: Diabetic Nephropathy TABLE 150-1. Metabolic and Genetic Theories of Diabetic Nephropathy

-3

0

Time (years)

3

15

-,-

1

.

25 j

120

150

150

GFR(mUmin)

120

60

2.0

>10

15

10

10

Serum Urea Nitrogen (mg/dL)

15

>30

80% of diabetic patients have evidence of retinopathy, but as many as 30% to 50% have no laboratory evidence of renal disease.

DIABETIC NEUROPATHY The association between diabetic nephropathy and neuropathy is much less impressive than between diabetic nephropathy and retinopathy. In patients who have had diabetes for 20 years, approximately one-half have some evidence of neuro¬ pathic involvement.87-90-92 In diabetics with end-stage renal fail¬ ure, the incidence of neuropathy varies from 50% to 90%, depending on how carefully one looks for evidence of diabetic neuropathic involvement. Peripheral neuropathy is more com¬ mon than autonomic neuropathy. In one study, symptomatic diabetic gastroparesis was observed in -50% of diabetic patients treated with dialysis.93 However, the specificity of these neuropathologic abnormalities, especially those relating to peripheral neuropathy, must be questioned because dialysis and transplantation often lead to reversal of the abnormali¬ ties,94 suggesting a uremic etiology. As with retinopathy, uremia appears to exacerbate the progression of diabetic neuropathy. When diabetic nephropathy is first diagnosed, fewer than half of patients have clinically evident diabetic neuropathy.

HYPERTENSION The incidence of hypertension, as well as the relationship between hypertension and renal disease, are very different in type 1 and type 2 diabetes mellitus. In newly diagnosed type 2 diabetics, -50% to 60% have hypertension, whereas 300-500 mg per day or elevated serum creatinine). c. Uremia is associated with insulin resistance and increased insulin requirements.

d. With advanced uremia (GFR 1000 pg/mL, although tumors may occur in patients whose glucagon levels are 500 patients who otherwise faced a major amputation. If successful revascularization is accomplished, more conser¬ vative reconstructive foot surgery, distal amputations, or revi¬ sions can be carried out to achieve healing and limb salvage.17 Gradual and progressive weight-bearing under careful surveil¬ lance is mandatory and often requires special orthotics and/or shoes to keep these high-risk areas healed and pressure free.

CONCLUSION The fear of gangrene or amputation is one of the overwhelming concerns of diabetic patients who experience the many compli¬ cations of their disease. Neuropathy, ischemia, and an altered host defense mechanism make these patients particularly prone to developing foot ulcers, which often become infected. Occa¬ sionally, it is only the complications of odor, hyperglycemia, or systemic symptoms that bring the patient to the hospital with a septic foot. Preventing limb-threatening ulcers or infections begins with patient education and understanding. Early recognition of any foot problem and its prompt treatment are essential. Treating serious limb-threatening conditions requires considerable expe¬ rience. Diabetics generally have greater risk factors (usually car¬ diac), and diabetic arteries require the maximum skill and experience of the operating surgeon. A team approach is the most cost-effective method to salvage the diabetic foot.18 In the past decade, the amputation rate at all levels of the diabetic lower extremity has been reduced by utilizing the concepts out¬ lined in this chapter.19 An aggressive approach to limb salvage is less expensive than resorting to major amputation, and the ben¬ efits to the patient and society are unquestionably superior.20

REFERENCES 1. US Department of Health and Human Services. Healthy People 2000— national health promotion and disease prevention objectives. Washington, DC: US Government Printing Office, 1991:73. 2. LoGerfo FW, Coffman JD. Vascular and microvascular disease in the dia¬ betic foot: implications for foot care. N Engl J Med 1984; 311:1615. 3. Berceli SA, Chan AK, Pomposelli FB Jr. Efficacy of dorsal pedal artery bypass in limb salvage for ischemic heel ulcers. J Vase Surg 1999; 30:499. 4. King TA, DePalma RG, Rhodes RS. Diabetes mellitus and atherosclerosis involvement of the profunda femoris artery. Surg Gynecol Obstet 1984; 159:553. 5. Gibbons GW, Wheelock FC Jr. Problems in the noninvasive evaluation of the peripheral circulation in the diabetic. Prac Cardiol 1982; 8:115.

6. Gibbons GW, Freeman DV. Diabetic foot infections. In: Howard RJ, Simmons RL, eds. Surgical infectious diseases. Norwalk, CT: Appleton & Lange, 1988:585. 7. Gibbons GW. Diabetic foot sepsis. In: Brewster D, ed. Common problems in vascular surgery. Chicago: Year Book, 1989:412. 8. Delbridge L, Appleberg M, Reeve TS. Factors associated with development of foot lesions in the diabetic. Surgery 1983; 93:78. 8a. Benotmane A, Mohammedi F, Ayed F, et al. Diabetic foot lesions: etiologic and prognostic factors. Diabetes Metab 2000; 26:113. 9. Gibbons GW, Wheelock FC Jr. Cutaneous ulcers of the diabetic foot. In: Ernest CB, Stanley JC, eds. Current therapy in vascular surgery. Philadel¬ phia: BC Decker, 1987:233. 10. Gibbons GW. Diabetic foot sepsis. Semin Vase Surg 1992; 5(4):1. 11. Sinha S, Frykberg RG, Kozak GP. Neuroarthropathy in the diabetic foot. In: Kozak G, ed. Clinical diabetes mellitus. Philadelphia: WB Saunders, 1983:415. 12. Gibbons GW, Eliopoulos GM. Infection of the diabetic foot. In: Kozak GP, Hoar CS, Rowbotham JL, et al., eds. Management of the diabetic foot prob¬ lem. Joslin Clinic and New England Deaconess Hospital. Philadelphia: WB Saunders, 1984:97. 13. Gibbons GW, Freeman DV. Vascular evaluation and treatment of the dia¬ betic. Clin Podiatr Med Surg 1987; 4:337. 13a. Feinglass J, Kaushik S, Handel D, et al. Peripheral bypass surgery and amputation: northern Illinois demographics. Arch Surg 2000; 135:75. 14. Hoar CS, Campbell DR. Aorto-iliac reconstruction. In: Kozak GP, Hoar CS, Row¬ botham JL, et al., eds. Management of the diabetic foot problem. Joslin Clinic and New England Deaconess Hospital. Philadelphia: WB Saunders, 1984:159. 15. Wheelock FC, Gibbons GW. Arterial reconstruction: femoral, popliteal, tibial. In: Kozak GP, Hoar CS, Rowbotham JL, et al., eds. Management of the diabetic foot problem. Joslin Clinic and New England Deaconess Hospital. Philadelphia: WB Saunders, 1984:173. 15a. Akbari CM, Pomposelli FB Jr, Gibbons GW, et al. Lower extremity revascu¬ larization in diabetes: late observations. Arch Surg 2000; 135:452. 16. Pomposelli FB, Jepson SJ, Gibbons GW, et al. A flexible approach to infrapopliteal vein grafts in patients with diabetes mellitus. Arch Surg 1991; 126:724. 17. LoGerfo FW, Gibbons GW, Pomposelli FB, et al. Trends in the care of the diabetic foot. Arch Surg 1992; 127:617. 18. Caputo GM, Cavanagh PR, Ulbrecht JS, et al. Current concepts: assessment and management of foot disease in patients with diabetes. N Engl J Med 1994; 13:854. 19. Gibbons GW, Maracaccio EJ, Burgess AM, et al. Improved quality of dia¬ betic foot care, 1984 vs. 1990: reduced length of stay and costs, insufficient reimbursement. Arch Surg 1993; 1283:576. 20. Gibbons GW, Burgess AM, Guadagnoli E, et al. Return to well-being and function after infrainguinal revascularization. J Vase Surg 1995;21:35.

CHAPTER 155

DIABETIC ACIDOSIS, HYPEROSMOLAR COMA, AND LACTIC ACIDOSIS K. GEORGE M. M. ALBERTI The diabetic acidoses and comas remain a significant cause of mortality and morbidity, much of it unnecessary. Many of the problems encountered could be avoided by the education of patients, health care professionals, and physicians in appropriate preventive measures and by the use of systematic, logical ther¬ apy. Several reviews are recommended for further reading.1-9

DIABETIC KETOACIDOSIS Diabetic ketoacidosis (DKA) may be defined as a state of uncontrolled diabetes mellitus in which there is hyperglycemia (usually >300 mg/dL or 16.7 mmol/L) with a significant lower¬ ing of arterial blood pH (5 mmol/L). The cutoff between DKA and hyperosmolar hyperglycemic nonketotic coma (HONK) is somewhat arbitrary, although hyperglycemia tends to be much more severe in the latter, with ketone body levels lower.

Ch. 155: Diabetic Acidosis, Hyperosmolar Coma, and Lactic Acidosis

1439

EPIDEMIOLOGY There are few good data available on the incidence of DKA. In a survey in Rhode Island, DKA accounted for 1.6% of all admis¬ sions to the hospital, with previously undiagnosed diabetes accounting for 20% of these. The annual incidence was 14 per 100,000 people.10 ha Denmark an annual incidence of 4.5% was reported, with highest risk ha female adolescents.11 The best data have been compiled by the National Diabetes Group ha the United States, who report an annual incidence of three to eight episodes per 1000 diabetic patients, with 20% to 30% occurring in new diabetics.12 Higher figures have been reported by the Cen¬ ters for Disease Control and Prevention13 at 26 to 38 per 100,000 population and an annual incidence of 1.0% to 1.5% of all diabet¬ ics in the period 1980 to 1987. Rates were three-fold higher in black men than in white men. There is increasing recognition of DKA ha type 2 diabetic patients, particularly nonwhites such as American Indians,14 Japanese,15 blacks,16 and Asian Indians.17 Mortality for established DKA is relatively high, accounting for 10% of all diabetes-related deaths in the United States between 1970 and 1978.18 Rates were highest in xaonwlaites and in the older than 65-year-old age group (59% of all DKA deaths). In most published series, mortality lies between 4% and 10%, although 0% has been reported in one large series in the United States.19 A report from Birmingham, United King¬ dom, showed a rate of 3.9% in 929 episodes over a 21-year period, with 50% of identified causes in nearly all series. This may occur in established type 1 diabetics or in previously undiagnosed patients, who form 20% to 30% of the total admis¬ sions. Infections may be minor, such as urinary tract infection, skin lesions, or bacterial throat infections, or more severe. Infec¬ tion causes a marked increase in secretion of cortisol and gluca¬ gon. Established type 1 diabetics with infections often diminish their food intake and may mistakenly decrease their insulin doses, whereas these should be increased based on the results of home blood glucose monitoring. Urine ketones should also be checked routinely in ill type 1 diabetics with increased insu¬ lin doses if ketones are more than trace positive. Other precipitating factors include omission of insulin doses in known type 1 diabetics, a well-known phenomenon in many "brittle" diabetics and in any young type 1 diabetic with recurrent episodes of DKA. In a large study in Scotland, young people admitted in DKA were less likely to have taken their prescribed doses of insulin,26 and in New Zealand, 61% of DKA patients were found to have made errors in insulin self-administration.27 Children with DKA have a greater risk of associated psychopathology28 and are more likely to come from disadvantaged backgrounds.29 Children presenting for the first time in DKA tend to be those with the lowest Cpeptide (i.e., insulin) reserves.30 Another cause of "pure" insu¬ lin deficiency is the malfunction of insulin infusion devices, with very high occurrence rates in some centers, in particular because of catheter failure, with a rate of 0.14 per patient-year in one large series.31 Other established precipitating factors include cerebrovas¬ cular accidents, acute myocardial infarction, and trauma. Each of these is accompanied by increased secretion of catechol¬ amines, glucagon, and cortisol, with predictable metabolic con¬ sequences if insulin doses are not increased. Rarer causes include Fourier gangrene (sudden, severe gangrene of the scro¬ tum), infusion of (3-sympathomimetic agents, pheochromocytoma, and treatment with the antipsychotic drugs clozapine and olanzapine.32

SIGNS AND SYMPTOMS The classic clinical presentation of severe DKA includes Kussmaul respiration (i.e., deep, sighing hyperventilation), dehy¬ dration, hypotension, tachycardia, warm skin, normal or low temperature, and altered state of consciousness. Only -10% are totally unconscious, and even this figure has diminished recently. Often, the breath smells of acetone, and there may be oliguria in the later stages. Preceding symptoms include nausea and vomiting, thirst, and polyuria in nearly all cases, and, less commonly, leg cramps and abdominal pain. There may also be no bowel sounds, with gastric stasis and pooling of fluid. Occasionally, the patient may present with an acute abdomen (pain and rigidity). If this occurs in young patients, virtually all cases resolve with conservative treatment: in older patients, there may be intraabdominal disease.33 It is sensible to treat the metabolic disturbance first. If there is no resolution or there is worsening of the abdominal state during

1442

PART IX: DISORDERS OF FUEL METABOLISM

TABLE 155-1. Signs and Symptoms of Diabetic Ketoacidosis Symptoms, Signs

Cause

Polyuria, polydipsia Anorexia, fatigue

Osmotic diuresis ?

Nausea, vomiting

?Ketosis

Weight loss

Protein, fat catabolism

Abdominal pain

?K+ depletion, fluid pooling

Leg cramps

?K+ depletion

Dehydration

Osmotic diuresis

Hypotension

Dehydration, acidemia

Tachycardia

Dehydration, acidemia

Hyperventilation

Acidemia

Gastric stasis

?K* depletion

Hypothermia

Peripheral vasodilation, acidemia

Impaired consciousness

Hyperosmolality

the first 3 to 4 hours of treatment, diagnostic reevaluation should be considered. Nearly all the signs and symptoms of DKA can be ascribed to different aspects of the metabolic disturbance (Table 155-1). Thus, the dehydration, polyuria, and thirst are secondary to the osmotic diuresis. The hypotension and tachycardia are caused by the fluid loss and the acidemia. Acidemia also causes the hyperventilation. The inappropriately low body temperature and warm skin are the result of the vasodilatation caused by the acidemia. The vomiting and nausea are probably the conse¬ quence of hyperketonemia, and the leg cramps and gastric sta¬ sis may be secondary to intracellular potassium depletion. The impaired consciousness correlates only with plasma osmolality, implying that intracellular fluid loss from cerebral cells is involved, although impaired cerebral circulation caused by hypotension may also contribute. Hypovolemia and hypoten¬ sion cause prerenal failure with consequent oliguria.

DIAGNOSIS In most cases, the rapid diagnosis of DKA should be possible at the bedside (Table 155-2). The clinical history usually is helpful. Clinical examination and bedside measurement of blood glucose and plasma ketones (using a test strip) should complete the diagnosis. Emergency room staff should be instructed in test-strip glucose measurement. The condition of "euglycemic" ketoacidosis should also be noted, in which blood glucose levels are not very elevated despite severe ketoacidosis,34 although if this is defined as a blood glucose level of 150-155 mmol/L). In this case, half-normal (0.45%) saline should be used, but infused more slowly. When saline is used, plasma sodium levels inevitably rise, partly because the infused fluid has a higher sodium content than the extracellular fluid in DKA patients, partly because water without sodium will be moving into cells, and partly because glucose levels will be falling. This rise in sodium levels is helpful, however, in that it prevents plasma osmolality from falling too quickly. This may be beneficial in preventing cerebral edema. Hyperchlore¬ mia almost invariably develops late in treatment, but there is little evidence to suggest that it is harmful. Most DKA patients who are hypotensive on presentation respond with a rise in BP to the first 1 to 2 L saline. If systolic BP remains below 90 mm Hg, 1 to 2 U blood or plasma expanders should be given. If this fails, 100 mg hydrocortisone sodium succinate may be given iv, with an appropriate increase in sub¬ sequent insulin therapy. Once blood glucose levels have fallen to 250 mg/dL (13.8 mmol/L), 10% glucose is substituted for saline. If this occurs before the patient is adequately rehydrated, saline should be continued simultaneously. The importance of adequate early rehydration cannot be over¬ emphasized. Simple rehydration alone lowers blood glucose lev¬ els by improving the renal excretion of glucose, with hemodilution accounting for as much as a 23% fall in blood glucose. Tissue per¬ fusion is also improved, allowing the small amounts of insulin present to begin to act. Rehydration, even without insulin, decreases counterregulatory hormone secretion. INSULIN

Before 1973, large doses of insulin (i.e., hundreds of units) were routinely used in DKA. Then it was shown that relatively small amounts of insulin given intramuscularly (im) or as a continu¬ ous iv infusion were just as effective in lowering blood glucose,

1 444

PART IX: DISORDERS OF FUEL METABOLISM

and had several advantages.46-47 Among the advantages of the low-dose regimens are decreased problems with hypokalemia during therapy, a lower occurrence of late hypoglycemia, and a more predictable response to therapy. There is also an adequate but somewhat slower rate of fall of blood glucose levels, which is less likely to cause osmotic disequilibrium. Insulin Resistance. The major concern in the use of low doses of insulin has been insulin resistance. It has been shown that fractional glucose turnover and the rate of fall of blood glu¬ cose levels in DKA patients are decreased up to ten-fold com¬ pared with nonketotic, well-controlled diabetics. Similarly, in animals and humans, insulin binding by adipocytes is decreased, as is postreceptor insulin action. This is accompa¬ nied, in ketoacidotic rats, by a marked diminution in total body insulin responsiveness. These changes correlate with the degree of acidemia, which is most severe when the pH is 150 pU/mL, well above those found in normal persons. Simi¬ larly, with standard im regimens, levels of >80 pU/mL are engendered. These levels are sufficient, even in the face of insu¬ lin resistance, to inhibit lipolysis, thereby cutting off the supply of substrate for ketogenesis, restraining hepatic gluconeogenesis and helping to decrease glucagon levels. There is little impact on peripheral glucose uptake initially, but there are ade¬ quate circulating fuels, and ketone body use steadily increases; the insulin influence on potassium transport into cells is also submaximal, which may be an advantage. Intravenous Insulin Regimens. Several insulin regimens have been proposed, with doses ranging from 2 to 10 U per hour as a continuous infusion. Good results have been reported with 1.4 to 1.6 U per hour after an initial small bolus.19 The author's routine is to give 6 U per hour in saline, using an infu¬ sion pump and a separate line (see Appendix 1). In children, a dosage of 0.1 U/kg per hour is used. Because insulin has a cir¬ culating half-life of 4 to 5 minutes and a biologic half-life of no more than 30 minutes, it is critical that the insulin be given con¬ tinuously. If the infusion stops for any reason, the effects will rapidly disappear. Insulin adsorbs to plastic and glass; there¬ fore, some physicians recommend making the insulin solution in polygeline or albumin or drawing back 1 mL of the patient's blood into the syringe. In practice, adsorption is not a problem. Blood glucose levels should be checked after the first 2 hours. If there has not been a significant fall (50-100 mg/dL or 2.8-5.7 mmol/L), then the infusion pump and line, the rehydra¬ tion scheme, and the BP should be checked, and the insulin infusion rate should be doubled if these are satisfactory. This should be repeated every 2 hours until blood glucose levels are falling satisfactorily. When blood glucose levels have fallen to 250 gg/dL and 10% dextrose has been substituted for saline (100 mL per hour), the insulin dose should be decreased to 4 U per hour, and subsequently modified according to hourly bed¬ side blood glucose readings. Intramuscular Regimen. Hourly im insulin provides an alternative to continuous iv insulin and is particularly useful in centers where reliable infusion pumps are not available or where nursing care is inadequate. In this case, a loading dose of insulin should be given as either 20 U im or 10 U im plus 10 U iv in hvpotensive or very dehydrated patients. Thereafter, 5 to 6 U should be given hourly as deep im injections. In children, a loading dose of 0.25 U/kg is given, followed by 0.1 U/kg hourly. Adequate rehydration is critical for im insulin to be

effective. If, after 2 hours, there is not a significant response, the im regimen should be substituted with continuous iv insulin, having first checked that rehydration is progressing satisfacto¬ rily. When blood glucose reaches 250 mg/dL and iv glucose is commenced, the insulin dose should be decreased to 5 to 6 U every 2 hours (see Appendix 1). POTASSIUM More iatrogenic deaths during the treatment of DKA have been caused by changes in plasma potassium than by any other fac¬ tor. There is usually a large deficit of intracellular total body potassium (3-12 mmol/kg body weight). Despite this, as many as 33% of DKA patients may have elevated plasma K+ levels initially. This loss of intracellular potassium into the extracellu¬ lar space, which occurs in all cases, has been attributed to the acidemia, intracellular volume depletion, and lack of insulin. The role of acidemia has been questioned, however. Additional factors include a direct effect of hyperglycemia and hyperglucagonemia. In a careful analysis, glucose, pH (negatively), and the anion gap were independent, significant determinants of plasma K+ on presentation.48 Once treatment commences, plasma K+ levels inevitably fall, except in those presenting with the sick-cell syndrome. The fall is the result of intracellular vol¬ ume repletion, hemodilution, reversal of the acidemia, loss of K+ in the urine as urine flow is reestablished, and a direct effect of insulin on intracellular K+ transport. Thus, hypokalemia is inevitable unless potassium is replaced. There are arguments about when potassium replacement should begin. Some recommend waiting until plasma potas¬ sium levels are known, levels are low normal or low, and urine flow has been reestablished; probably, this is too late. The author's practice is to start cautious replacement at 20 mmol KC1 per hour in the saline infusion from the time of the first dose of insulin, then to modify the amount infused according to subsequent plasma values (see Appendix 1). It has been sug¬ gested that potassium should be given as phosphate or half as phosphate and half as chloride. However, phosphate require¬ ments are very different from those for potassium, so it is prob¬ ably sensible to replace them separately, if at all, and to use KC1. Electrocardiographic monitoring is an invaluable guide to rapid changes in plasma potassium, and all patients should be monitored at least in the early stages of therapy. For as long as iv therapy is continued, iv potassium replacement should be continued. Thereafter, oral potassium replacement should be continued for several days, because much of the potassium administered iv will be lost in the urine, and the total body deficit will be only partly replenished. If alkali is given, addi¬ tional potassium should be given (20 mmol/100 mmol sodium bicarbonate). OTHER ELECTROLYTES There is a deficiency of magnesium, calcium, and phosphate, as well as of sodium and potassium in DKA patients. It is argu¬ able, however, whether these need to be replaced immediately. Most debate has concerned phosphate. During treatment of DKA, plasma phosphate levels fall, sometimes to undetectable levels. Red cell 2,3-diphosphoglycerate levels are also very low and take 4 to 48 hours to return to normal. This may cause impaired oxygen delivery to tissues when the acidemia is corrected. It has been argued that the low phosphate levels impede recovery of 2,3-diphosphoglycerate. Several trials of phosphate replacement have been carried out. None of the more recent trials has shown benefit, and in all cases, biochemical hypocalcemia was found in the treated group.49 It is possible that phosphate changes are less with the use of low-dose insulin than they were previously. It is not the author's practice to replace phosphate. Similarly, although magnesium levels are low during therapy, no convincing evi¬ dence shows that replacement is beneficial.

Ch. 155: Diabetic Acidosis, Hyperosmolar Coma, and Lactic Acidosis TABLE 155-4. General Therapy for Diabetic Ketoacidosis Search for and treat any precipitating factors Nasogastric suction Catheterization

1445

TABLE 155-5. Monitoring Therapy for Diabetic Ketoacidosis Parameter

Action

CLINICAL Pulse, BP

Every half hour for 4 h, every hour for 4 h, then every 2-4 h

Intravenous furosemide for oliguria Whole blood or plasma expanders for hypotension

Temperature

At 0, 2,4, 6 h, then every 6 h

Central venous pressure monitoring

Urine flow

Hourly for 6 h, then every 4 h with fluid balance chart

Electrocardiographic monitoring

Conscious state

Every hour

Antibiotics

CVP

Every hour in those with CVP line

Low-dose heparinization

ALKALI There is still no universal agreement about correcting the aci¬ demia of severe DKA. The acidemia has certain pathophysio¬ logic consequences, including negative inotropism, peripheral vasodilatation, central nervous system depression, and insulin resistance. On the other hand, vigorous alkalinization has dele¬ terious consequences, including hypokalemia, a paradoxical fall in cerebrospinal fluid pH, impaired oxyhemoglobin dissoci¬ ation, and rebound alkalosis.50 Human data suggest that bicar¬ bonate either has no benefit or, indeed, may slow clearance of ketones and metabolic normalization.51-53 Despite this, it is usually considered advisable to give mod¬ erate amounts of bicarbonate when the pH is 6 mmol/L. Maintain K* between 4 and 5 mmol/L. If K+ 7.0, give 50 mmol containing 10 mmol KC1. 5. Continued treatment. A. Monitor glucose with test strip at bedside hourly and in laboratory at hours 2 and 6, then every 4 hours. B. Monitor K+ in laboratory at hours 2 and 6, then every 6 hours. C. Laboratory measurements of creatinine and electro¬ lytes at hours 3, 6, and 24. D. Frequent monitoring of pulse, BP, temperature (see Table 155-5). E. When blood glucose 120% of ideal body weight.19 The screening test consists of oral administration of 50 g of glucose and a plasma glucose determination at 1 hour, and it may be given regardless of the time of the last meal. If the plasma glucose concentration 1 hour after the oral load is 120 mg/dL (6.7 mmol/ L), then insulin should be prescribed immediately.

Eye examination Physical examination

5.3

Two or more of the venous plasma concentrations must be met or exceeded for a positive diagnosis. ^ormoglycemia = fasting glucose level of 7.8 mmol/L), a glucose-tolerance test is indicated. In the summary of the Fourth International Gestational Dia¬ betes Mellitus Workshop-Conference,15 the recommendations for the diagnostic oral glucose-tolerance test allow the clinician to choose between a 100-g glucose drink20 and a 75-g glucose drink.21 Table 156-2 describes these tests. Either glucose load should be administered after a minimum of 8 hours of fasting. A fasting plasma glucose blood sample should be drawn no later than 9:00 a.m. In the case of the 100-g load, plasma glucose levels should be obtained 1,2, and 3 hours after the glucose load. In the case of the 75-g glucose load, testing is really necessary only at the 0-, 1-, and 2-hour time points. The glucose level should be determined on venous plasma using the hexokinase method, and not on capillary blood using glucose oxidase-impregnated test strips, which are less accurate for this purpose. Once the diagnosis of gestational diabetes is made, however, these strips become the mainstay of treatment strategies.

DIAGNOSTIC STRATEGIES The diagnostic criteria for gestational diabetes are based on the oral glucose-tolerance test (see Table 156-2).20 These criteria cor¬ rectly identify women at risk for a stillbirth. They do not identify women at risk of delivering a macrosomic infant. Other tests and cutoffs need to be used to identify the macrosomic fetus.22 Blood glucose values at 4 and 5 hours after glucose load have no diagnostic significance. Measurement of glycosylated hemoglo¬ bin levels, which is a test of long-term plasma glucose control in type 1 and type 2 diabetes mellitus, is not sensitive enough to diagnose gestational diabetes.19 Use of a single diagnostic test, rather than the two-step method used in most of the United States, greatly simplifies the procedure. A single 75-g glucose load21 is all that is necessary; gestational impaired glucose tolerance is defined as a 2-hour postload glucose concentration of >140 mg/dL (7.8 mmol/L). This constitutes a diagnosis of gestational diabetes and warrants treatment.

1454

PART IX: DISORDERS OF FUEL METABOLISM

TABLE 156-3. Diet Calculation for Women 80% to 120% of Ideal Body Weight % of Daily Carbo¬ hydrate Allowed

Time

Meal

Fraction (kcal/24 hours)

8:00 a.m.

Breakfast

2/18

10

10:30 a.m.

Snack

1/18

5

12:00 noon

Lunch

5/18

30

3:00 p.m.

Snack

2/18

10

5:00 p.m.

Dinner

5/18

30

8:00 p.m.

Snack

2/18

5

11:00 p.m.

Snack

1/18

10

TREATMENT OF GESTATIONAL DIABETES The goal of management of gestational diabetes is to maintain normoglycemia. Most pregnant women never exceed 120 mg/ dL of plasma glucose, even at 1 hour after a meal, despite the ingestion of large quantities of carbohydrate. If the peak post¬ prandial glucose value is >120 mg/dL, the risk of macrosomia rises exponentially.23 Because a nutritious meal for the mother and her unborn child necessitates at least a 40 mg/dL increase of plasma glucose, if the woman's fasting glucose level is much greater than 90 mg/dL (whole-blood capillary glucose), she will be unable to maintain her postprandial levels at 120 mg/dL, 2 to 4 U of regular insulin is given subcutaneously each hour until the blood glucose level is 70 to 80 mg/dL and is titrated to the target infu¬ sion rate of 2.55 mg/kg per minute as active labor is achieved. In the case of an elective cesarean section, the bedtime dose of NPH insulin is repeated at 8 a.m. on the day of surgery and every 8

DECREASED MATERNAL INSULIN REQUIREMENTS Maternal insulin requirements usually drop precipitously post¬ partum; therefore, women with gestational diabetes may not require further insulin. On the other hand, women with pregestational diabetes require the resumption of insulin therapy, but these requirements may be decreased for 48 to 96 hours postpartum. Insulin require¬ ments should be recalculated at 0.6 U/kg based on the postpar¬ tum weight and should be started when the 1-hour postprandial plasma glucose value is >150 mg/dL or the fasting glucose level is >100 mg/dL. The postpartum caloric requirements are 25 kcal/kg per day, based on postpartum weight. For women who wish to breast-feed, the calculation is 27 kcal/kg per day and insulin requirements are 0.6 U/kg per day. The insulin require¬ ment during the night drops dramatically during lactation, owing to siphoning of glucose into the breast milk. Thus, the majority of the insulin requirement is needed during the daytime to cover the increased caloric needs of breast-feeding. Normogly¬ cemia should especially be prescribed for nursing diabetic women, because hyperglycemia elevates milk glucose levels.57 POSTPARTUM COURSE OF GESTATIONAL DIABETES Almost 98% of all women with gestational diabetes revert to normoglycemia postpartum. A glucose-tolerance test should be performed 6 weeks postpartum, or when breast-feeding is stopped, to ensure that the diabetes has disappeared. Women should be warned that the probability that diabetes will recur in each subsequent pregnancy is -90%. Therefore, women should be screened during each pregnancy. Moreover, if a woman remains overweight, she has a 60% chance of manifesting overt diabetes within 20 years. Therefore, the postpartum period is an important time in which to initiate the process of weight loss. The second goal of management of pregnancies complicated by gestational diabetes is to prevent obesity-induced diabetes in the future.

NEONATAL CARE If the blood glucose concentration is normalized throughout pregnancy in a woman with diabetes, no evidence exists that excess attention need be paid to her offspring. If the normal blood glucose level has not been documented throughout preg¬ nancy, however, the wise course is to monitor the neonate in an intensive care situation for at least 24 hours postpartum. Blood glucose levels should be monitored hourly for 6 hours. If the neonate shows no signs of respiratory distress, hypocalcemia, or hyperbilirubinemia at 24 hours after delivery, he or she can be safely discharged to the normal nursery.58

PERINATAL OUTCOME With the advent of tools and techniques to maintain normogly¬ cemia before, during, and between all pregnancies complicated by diabetes, infants of diabetic mothers now have the same chances of good health as infants born to nondiabetic women. Animal and human studies clearly implicate glucose as the teratogen.583 In a Boston study, hyperglycemia in the first trimes¬ ter was associated with a 23% incidence of major malformations; in an East Germany study, the malformation rate associated with elevated glycohemoglobin during the first trimester was

1458

PART IX: DISORDERS OF FUEL METABOLISM

15.8%.41-42 In the latter study, when the glucose level was normal¬ ized before conception, the malformation rate dropped to 1.6%. These studies and others emphasize the need for preconceptional programs to achieve and maintain normoglycemia.44'59 The morbidity and subsequent development of the infant of the diabetic mother is associated with hyperglycemia.60 Neona¬ tal macrosomia, hyperinsulinism, and hypoglycemia improve after maternal glucose is normalized. In one study series, all 53 infants born to 52 women with type 1 diabetes who maintained normoglycemia were normal.58 Therefore, the goal for all pregnancies complicated by diabe¬ tes is to achieve and maintain normoglycemia.

REFERENCES 1. White P. Pregnancy and diabetes. In: Marble A, White P, Bradley RF, Krall LP, eds. Joslin's diabetes mellitus, 11th ed. Philadelphia: Lea & Febiger, 1971:50. 2. Pedersen J. The pregnant diabetic and her newborn: problems and manage¬ ment. Baltimore: Williams & Wilkins, 1967. 3. Jovanovic L, Peterson CM, Fuhrmann K, eds. Diabetes and pregnancy: ter¬ atology, toxicology and treatment. Philadelphia: Praeger, 1985. 4. Pedersen J, Pedersen LM. Diabetes mellitus and pregnancy: the hyperglyce¬ mia, hyperinsulinemia theory and the weight of the newborn baby. In: Rod¬ riguez RR, Vallance-Owen ], eds. Proceedings of the 7th Congress of the International Diabetes Federation. Amsterdam: Excerpta Medica, 1971:678. 4a. Butte NF. Carbohydrate and lipid metabolism in pregnancy: normal compared with gestational diabetes mellitus. Am J Clin Nutr 2000; 71(5 Suppl):1256S. 5. Herrera E, Knopp RH, Freinkel N. Plasma fuels, insulin liver composition, gluconeogenesis and nitrogen metabolism during late gestation in the fed and fasted rat. J Clin Invest 1969; 48:2260. 6. Gillmer MDG, Beard RW, Brooke FM, Oakley NW. Carbohydrate metabo¬ lism in pregnancy. II. Diurnal plasma glucose profile in normal and diabetic women. BMJ 1975; 3:399. 7. Felig P. Maternal and fetal fuel homeostasis in human pregnancy. Am J Clin Nutr 1973; 26:998. 8. Bleicher SG, Sullivan JB, Freinkel N. Carbohydrate metabolism in preg¬ nancy: the interrelationships among glucose, insulin and free fatty acids in late pregnancy and postpartum. N Engl J Med 1964; 271:866. 9. Freinkel N. Effects of the conceptus on maternal metabolism during preg¬ nancy. In: Leibel BS, Wrenshall GA, eds. On the nature and treatment of dia¬ betes. Amsterdam: Excerpta Medica, 1965:679. 10. Klopper A. The assessment of placental function in clinical practice. In: Klopper A, Diczfalusy E, eds. Foetus and placenta. Oxford: Blackwell Scien¬ tific Publications, 1969:471. 11. Josimovich JB. Placental lactogenic hormone. In: Endocrinology of preg¬ nancy. New York: Harper & Row, 1971:184. 12. Doe RP, Dickinson P, Zinneman HH, Seal US. Elevated non-protein-bound cortisol in pregnancy, during estrogen administration and in carcinoma of the prostate. J Clin Endocrinol 1983; 29:757. 13. Freinkel N, Goodner CJ. Carbohydrate metabolism in pregnancy: the . metabolism of insulin by human placental tissue. J Clin Invest 1960; 39:116. 14. Hadden DR. Geographic, ethnic and racial variations in the incidence of gestational diabetes mellitus. Diabetes 1985; 34:8. 15. Metzger BE, Coustan DR, and the Organizing Committee. Summary and recommendations of the Fourth International Workshop-Conference on Gestational Diabetes Mellitus. Diabetes Care 1998; 21:B161. 16. Moses RG, Moses J, Davis WS. Gestational diabetes: do lean young Cauca¬ sian women need to be tested? Diabetes Care 1998; 21:1803. 17. Pettitt DJ. Gestational diabetes mellitus. Who to test. How to test. Diabetes Care 1998; 21:1789. 18. Sweet success guidelines for care. State program guide. Sacramento: Cali¬ fornia Diabetes & Pregnancy Program, Maternal & Child Health Branch, California Department of Health Services, 1998. 19. Jovanovic L, Peterson CM. Screening for gestational diabetes: optimal tim¬ ing and criteria for retesting diabetes. Diabetes 1985; 34:21. 20. O'Sullivan JB, Mahan CB. Criteria for the oral glucose tolerance test in preg¬ nancy. Diabetes 1964; 13:278. 21. Moses RG, Moses M, Russell KG, Schier GM. The 75-g glucose tolerance test in pregnancy. A reference range determined on a low-risk population and related to selected pregnancy outcomes. Diabetes Care 1998; 21:1807. 22. Jovanovic-Peterson L, Crues J, Durak E, Peterson CM. Magnetic resonance imaging in pregnancies complicated by gestational diabetes predicts infant birth weight ratio and neonatal morbidity. Am J Perinatal 1994; 10:432. 23. Jovanovic-Peterson L, Peterson CM, Reed G, et al. Postprandial blood glu¬ cose levels predict birthweight: the Diabetes in Early Pregnancy Study. Am J Obstet Gynecol 1991; 164:103. 24. Jovanovic-Peterson L, Peterson CM. Dietary manipulation as a primary treatment strategy for pregnancies complicated by diabetes. J Am Coll Nutr 1990; 9:320. 25. Peterson CM, Jovanovic-Peterson L. Percentage of carbohydrate and glycemia response to breakfast, lunch, and dinner in women with gestational dia¬ betes. Diabetes 1991; 40(Suppl 2): 172.

26. Jovanovic-Peterson L, Peterson CM. Nutritional management of the obese gestational diabetic woman. (Guest editorial). J Am Coll Nutr 1992; 11:246. 27. Jovanovic-Peterson L, Peterson CM. Sweet success, but an acid aftertaste? (Editorial). N Engl J Med 1991; 325:959. 28. Jovanovic-Peterson L, Durak EP, Peterson CM. Randomized trial of diet versus diet plus cardiovascular conditioning on glucose levels in gesta¬ tional diabetes. Am J Obstet Gynecol 1989; 161:415. 29. Jovanovic-Peterson L, Peterson CM. Is exercise safe or useful for gestational diabetic women? Diabetes 1991; 40(Suppl 2):179. 30. Jovanovic L, Peterson CM, Saxena BB, et al. Feasibility of maintaining normal glucose profiles in insulin-dependent pregnant women. Am J Med 1980; 68:105. 31. Jovanovic L, Peterson CM. Is pregnancy contraindicated in women with diabetes mellitus. Diabetic Nephrop 1984; 3:36. 32. Jovanovic-Peterson L, Peterson CM. Diabetic retinopathy. In: Coustan DR, guest ed; Pitkin RM, Scott JR, eds. Clinical obstetrics and gynecology: dia¬ betes and pregnancy. Philadelphia: JB Lippincott, 1991:516. 33. Fuhrmann K. Outcome of normoglycemic diabetic pregnancies in Karlsburg. In: Jovanovic L, Peterson CM, Fuhrmann K, eds. Diabetes and pregnancy: ter¬ atology, toxicology and treatment. Philadelphia: Praeger, 1985:168. 34. Mestman JH. Autoimmune diseases associated with diabetic pregnancies. In: Jovanovic L, Peterson CM, Fuhrmann K, eds. Diabetes and pregnancy: teratology, toxicology and treatment. Philadelphia: Praeger, 1985:32. 35. Jovanovic-Peterson L, Peterson CM. De novo hypothyroidism in pregnan¬ cies complicated by type I diabetes, subclinical hypothyroidism, and pro¬ teinuria: a new syndrome. Am J Obstet Gynecol 1988; 159:442. 36. Chew EY, Mills JL, Metzger BE, et al. Metabolic control and progression of retinopathy. The Diabetes in Early Pregnancy Study. National Institute of Child Health and Human Development Diabetes in Early Pregnancy Study. Diabetes Care 1995; 18:631. 37. Jovanovic L, Peterson CM, eds. Contemporary issues in nutrition: diabetes mellitus. New York: Alan R Liss, 1985. 38. Committee of Nutrition. Nutrition in maternal health care. Chicago: Amer¬ ican College of Obstetricians and Gynecologists, 1974. 39. Jovanovic L, Metzger BE, Knopp RH, et al. The Diabetes in Early Pregnancy Study: beta-hydroxybutyrate levels in type 1 diabetic pregnancy compared with normal pregnancy. National Institute of Child Health and Human Devel¬ opment Diabetes in Early Pregnancy Study Group. Diabetes Care 1998; 21:1978. 40. Jovanovic L, Peterson CM. The clinical utility of glycosylated hemoglobin. Am J Med 1981; 70:331. 41. Miller E, Hare JW, Clogerty JP, et al. Elevated maternal hemoglobin Ale in early pregnancy and major congenital anomalies in infants of diabetic mothers. N Engl J Med 1981; 304:1331. 42. Fuhrmann K, Ruher H, Semmler K, et al. Prevention of congenital malforma¬ tions in infants of insulin dependent diabetic mothers. Diabetes Care 1983; 6:219. 43. Mills JL, Simpson JL, Driscoll SG, et al. Incidence of spontaneous abortion among normal women and insulin dependent diabetic women whose preg¬ nancies were identified within 21 days of conception. National Institutes of Child Health and Human Development Diabetes in Early Pregnancy Study. N Engl J Med 1988; 319:1617. 44. Mills JL, Knopp RH, Simpson JL, et al. Lack of relation of increased malfor¬ mation roles in infants of diabetic mothers to glycemic control during organogenesis. N Engl J Med 1988; 318:671. 45. Kitzmiller JL, Gavin LA, Gin GD, et al. Preconceptional care of diabetes: gly¬ cemic control prevents congenital anomalies. JAMA 1991; 265:731. 46. Mills JL, Baker L, Goldman A. Malformations in infants of diabetic mothers occur before the seventh gestational week: implications for treatment. Dia¬ betes 1979; 23:292. 47. Jovanovic L, Mills JL, Peterson CM. The rationale for the use of human insulin in pregnancy. In: Jovanovic L, Peterson CM, Fuhrmann K, eds. Diabetes mel¬ litus: teratology, toxicology and treatment. Philadelphia: Praeger, 1985:157. 48. Jovanovic-Peterson L, Kitzmiller JL, Peterson CM. Randomized trial of human versus animal species insulin in pregnancies complicated by diabe¬ tes. Am J Obstet Gynecol 1992; 167:1325. 49. Jovanovic L, Ilic S, Pettitt DJ, et al. Metabolic and immunologic effects of insu¬ lin lispro in gestational diabetes. Diabetes Care 1999; 22(9):1422. 50. Gabbe SG, Lowenson RI, Wu PY, Guerra G. Current patterns of neonatal mor¬ bidity and mortality in infants of diabetic mothers. Diabetes Care 1978; 1:335. 51. Driscoll SG, Benirshke K, Curtis GW. Neonatal deaths among infants of dia¬ betic mothers. Am J Dis Child 1961; 100:818. 52. Kenny JD, Adams JM, Corbet AJ, Rudolph AJ. The role of acidosis at birth in the development of hyaline membrane disease. Pediatrics 1976; 58:181. 53. Buchanan TA, Kjos SL, Montoro MN, et al. Use of fetal ultrasound to select metabolic therapy for pregnancies complicated by mild gestational diabe¬ tes. Diabetes Care 1994; 17:275. 54. Freeman RK. Obstetric management of the diabetic patients. Contemp Obstet Gynecol 1976; 1:51. 55. Gurson CT, Etili L, Soyak S. Relation between endogenous lipoprotein lipase activity, free fatty acids, and glucose in plasma of women in labor and of their newborns. Arch Dis Child 1968; 43:679. 56. Jovanovic L, Peterson CM. Glucose and insulin requirements during labor in insulin-dependent pregnant diabetic women. Am J Med 1983; 75:607. 57. Jovanovic-Peterson L, Fuhrmann K, Hedden K, Walker L, Peterson CM. Maternal milk and plasma glucose and insulin levels: studies in normal and diabetic subjects. Am J Nutr 1989; 8:125. 58. Jovanovic L, Druzin M, Peterson CM. Effect of euglycemia on the outcome of pregnancy in insulin-dependent diabetic women as compared with nor¬ mal control subjects. Am J Med 1981; 71:921.

Ch. 157: Diabetes Mellitus in the Infant and Child 58a. Suhonen L, Hiilesmaa V, Teramo K. Glycaemic control during early preg¬ nancy and fetal malformations in women with type 1 diabetes mellitus. Diabetologia 2000; 43:79. 59. Jovanovic L, Peterson CM, Fuhrmann K, eds. Diabetes in pregnancy: tera¬ tology, toxicology and treatment. Philadelphia: Praeger, 1985. 60. Petersen M, Pedersen SA, Greisen G, et al. Early growth delay in diabetic pregnancy: relation to psychomotor development at age 4. BMJ 1988; 296:598.

CHAPTER

157

DIABETES MELLITUS IN THE INFANT AND CHILD DOROTHY J. BECKER AND ALLAN L. DRASH Diabetes mellitus can present very different medical and psy¬ chosocial issues in the child from those found in the adult. Even among childhood cases, manifestations, goals of therapy, clini¬ cal course, and susceptibility to a variety of acute, intermediate, and chronic complications vary with age.

MAGNITUDE OF THE PROBLEM Insulin-dependent diabetes mellitus (IDDM) is one of the most common serious chronic diseases of childhood. The majority of cases are classified as type 1 diabetes, which usually is due to autoimmune destruction of the B cells of the islets of Langerhans. In the United States, an apparent increase is occurring in the frequency of "atypical type 1 diabetes," which is seen mostly in black adolescents but also in Hispanics and whites. Such patients typically are obese with acanthosis nigricans, and present with episodes of ketosis or ketoacidosis; although they initially require insulin therapy, they later become non-insulin dependent.1 Type 1 diabetes in the neonate may be transient, in which case it is often related to intrauterine growth retardation. Cases of permanent neonatal diabetes that is nonautoimmune and usually familial have also been described, with some patients showing agenesis of the pancreas or islet cells. Interestingly, classic type 2 (non-insulin dependent) diabetes is being seen with increasing frequency by pediatricians. It occurs primarily in obese black and Hispanic patients and is seen more com¬ monly in girls than in boys.2 Another form of nonautoimmune type 1 diabetes being recognized more often in childhood is maturity-onset diabetes of youth (MODY). MODY patients eventu¬ ally require insulin; often they are initially treated with oral agents, and usually they are not obese. Their insulin-secretory deficits are due to genetic mutations of the glucokinase gene and of a number of transcription factors (MODY 1-5).3 Secondary dia¬ betes is being seen with increasing frequency in patients with cystic fibrosis, who are living longer, and also in patients receiv¬ ing immunosuppressive agents for organ transplantation, which now is more common.

TYPE 1 DIABETES Among children, type 1 diabetes remains the most prevalent form of the disease in the United States. This disorder occurs with an annual incidence of 17 to 18 cases per 100,000 persons younger than 19 years.4 The prevalence is increasing because the incidence is rising and mortality rates are decreasing. One of the

1459

more intriguing aspects of the study of the epidemiology of type 1 diabetes is the remarkable variation in prevalence and inci¬ dence in various parts of the world. Incidence figures are lowest in Asia; Japan, Korea, and China report approximately one case per 100,000 population each year. Rates are highest in the Scan¬ dinavian countries, especially Finland, and in Sardinia, with incidence rates approaching 40 cases per 100,000 children and adolescents per year.5 The explanation for these geographic dif¬ ferences is still unclear. Although the frequency of genetic sus¬ ceptibility haplotypes probably plays a major role among the different races, other populations have very similar gene fre¬ quencies. Thus, geographic variations and the rising incidence of type 1 diabetes around the world point to the role of yet-tobe-determined environmental factors in precipitating insulin deficiency.5-8 Approximately 10% of the 10,000,000 Americans with known diabetes have type 1 diabetes. Although a large proportion of these patients (probably >50%), acquire the disease before the age of 20, recognition is increasing that type 1 diabetes may present in adulthood, either with a typical acute onset, or with an indolent course, and is often misdiagnosed as type 2 diabetes. In childhood, the mean age of onset is ~8 years. A peak is seen in adolescence, which occurs somewhat earlier in girls than in boys. A rise in incidence has been reported among children younger than 5 years.9 No significant difference is seen in sex distribution of diabetes during childhood.10 Mortality is higher in diabetic children and adolescents than in age- and sex-matched nondiabetic American children.11-12 On the other hand, although no recent analyses have been pub¬ lished of mortality due to diabetes in childhood, the general impression is that these rates are decreasing. Death occurring at the onset, however, often related to a missed diagnosis, contin¬ ues to occur. Death related to the chronic complications of type 1 diabetes is almost never seen in adolescents today. The single most important diabetes-related cause of death during childhood is diabetic ketoacidosis (DKA).13 This occurs both at presentation and later during the course of type 1 diabetes and accounts for >40% of pediatric deaths. In the authors' experience, -40% of patients with newly diagnosed type 1 diabetes present in DKA. The mor¬ tality due to DKA at the authors' institution over the past 30 years is 1 and a cholesterol content of

8 24 16 E

FIGURE 158-2. Physiology and symptoms of hypoglycemia. Plasma levels of glucose, insulin, and counterregulatory hormones and total symp¬ tom score (i.e., adrenergic and neuroglycopenic) during glucose clamp-induced hypoglycemia (filled circles) and euglycemia (open circles) in 10 nonobese, nondiabetic individuals. Total symptom score and hormonal responses rise when glucose is below 66 mg/dL (after 165-180 minutes); the exception was cortisol secretion, which required a lower plasma glucose level to become activated. (From Mitrakou A, Ryan C, Veneman T, et al. Hier¬ archy of glycemic thresholds for counterregulatory hormone secretion, symptoms and cerebral dys¬ function. Am ] Physiol 1991; 260:E67.)

o>

8

0 450

350

E

CT> 250

0

60

120

180

MINUTES

240

300

360

0

60

120

180

MINUTES

240

300

360

150

Ch. 158: Hypoglycemic Disorders in the Adult TABLE 158-2. Differential Diagnosis of Acute Hypoglycemia MEDICATION OR TOXIN INDUCED Excessive insulin effect Insulin Sulfonylureas Rodenticides (e.g., Vacor) Diffuse hepatic dysfunction Ethanol Nonselective (1-blocking agents Other medications

FASTING HYPOGLYCEMIA Excessive insulin effects Insulinoma Surreptitious insulin injection Surreptitious sulfonylurea ingestion Insulin-receptor autoantibodies Antiidiotypic antibodies to antiinsulin antibodies Humoral tumor-associated hypoglycemia Diffuse hepatic dysfunction Congestive heart failure Septic shock syndrome Combined endocrine deficiency states Limitation of substrate for gluconeogenesis Uremia Excessive glucose consumption Large sarcomas (?)

POSTPRANDIAL HYPOGLYCEMIA Excessive insulin effect After gastric surgery Reactive hypoglycemia Hepatic dysfunction Hypoglycin ingestion (i.e., Jamaican vomiting illness)

tions (see Table 158-2). Preprandial recurrent symptoms are more suggestive of pathologic hypoglycemia, which requires elabo¬ rate evaluation and invasive intervention, than are postprandial symptoms (i.e., within 4 to 5 hours of eating). A dietary history, including snacking and recent changes in eating patterns, is important, because many patients with true hypoglycemic disor¬ ders change their dietary patterns to prevent hypoglycemic epi¬ sodes. The longest regular fast of the day, usually bedtime to breakfast, should be identified and scrutinized for symptoms. The use of insulin, sulfonylureas, alcohol, or other medications should be considered as the possible cause of hypoglycemia, par¬ ticularly in the setting of poor or erratic nutrition.

CAUSES OF HYPOGLYCEMIA INSULIN-INDUCED HYPOGLYCEMIA IN THE TREATMENT OF DIABETES The most common cause of hypoglycemia in adults is insulin treatment in the management of diabetes.13'14 The emphasis on aggressive attempts to maintain plasma glucose values in the normal range has increased the risk of episodes of hypoglyce¬ mia. The administration of a depot of subcutaneous insulin that is then slowly released into the circulation is an inflexible deliv¬ ery of insulin, independent of dietary modifications. Erratic dietary and exercise patterns, which are typical of many individ¬ uals, can lead to hypoglycemic episodes. In the management of the diabetic patient with frequent episodes of hypoglycemia, meals, exercise, and the dose and duration of action of the insu¬ lin must be considered.13-14

1471

Patients with diabetes may be less aware of hypoglycemia because of the use of other medications such as jl-blockers, a change in the usual symptom complex after switching from ani¬ mal to human insulins, or autonomic neuropathy and loss of the catecholamine-mediated symptoms. In virtually all patients with type 1 diabetes, the glucagon response to hypoglycemia is lost after 2 to 3 years of diabetes, rendering them more suscepti¬ ble to prolonged episodes of hypoglycemia. Concern exists that patients who switch from animal to human insulins become insensitive to hypoglycemia. The loss of hypoglycemic symp¬ toms after initiation of human insulin therapy has not been ver¬ ified in randomized controlled trials, but patients should be counseled that the subjective warning signs of hypoglycemia may change when they switch from animal to human insulins because of improved glycemic control or more gradual diminu¬ tion in blood glucose under a new insulin regimen.15-16 Another common manifestation of hypoglycemia in insulintreated diabetics is the "Somogyi effect," in which fasting glu¬ cose concentrations seem to increase in response to increasing doses of insulin (see Chap. 143). This is the result of episodes of marked hypoglycemia at hours when patients are not testing their glucose concentrations (2:00 to 3:00 a.m.). It is followed by secretion of counterregulatory hormones, which in turn pro¬ duces high levels of serum glucose at a later time when patients are testing their glucose concentrations (6:00 to 8:00 a.m.). The treatment of acute episodes of hypoglycemia, insulininduced or otherwise, is the administration of a nutrient and monitoring of plasma glucose over the next 1 to 2 hours. In epi¬ sodes of severe hypoglycemia (i.e., confusion, loss of conscious¬ ness, seizures), intravenous glucose (50 mL of 50% glucose) should be administered to reverse the situation rapidly. In milder episodes, a small amount (15 g) of simple carbohydrates fol¬ lowed by nutrients with slower gastrointestinal absorption (e.g., whole milk, cheese) may be used. The response to administered nutrients should be immediate. Prolonged or delayed resolution of symptoms (>10 minutes) suggests inadequate treatment or another cause of the symptoms. Prolonged or repeated hypogly¬ cemia can cause permanent neurologic injury.17

FASTING HYPOGLYCEMIA EVALUATION The evaluation of fasting hypoglycemia (Fig. 158-3) is essentially the workup for possible insulinoma.18-18a The investigation of insulinoma entails invasive testing, and the treatment is primarily surgical. Fasting hypoglycemia and an inappropriate hyperinsulinemia at the time of hypoglycemia must be documented before any further tests are performed. The single most useful test is the in-hospital monitored fast lasting for up to 48 hours. The insulin that is released by insulinomas usually is not suppressed by declining plasma glucose levels. Fasting individuals with insulinomas become hypoglycemic because the fasting plasma glucose level depends on the hepatic output of glucose. In con¬ trast, normal individuals rarely develop plasma glucose concen¬ trations 8% of all the pancre¬ atic tumors evaluated.87 Because the prognosis and treatment of nonductal tumors is different from that of the more commonly encountered ductal neoplasms, a histopathologic diagnosis should be established for all pancreatic masses before a treat¬ ment plan is formulated. Nonfunctional tumors are predominantly malignant (90%)88 and are found most often in the head of the pancreas. Despite their size and location, 40% are resectable at the time of discovery.15 Because they do not produce debilitating syndromes related to the elaboration of humoral products, the risks and benefits of resective surgery should be weighed carefully. If a formal pancreatic resection can extirpate the entire tumor, most surgeons would agree that this is the preferred approach.89 Although some sur¬ geons advocate tumor debulking,90 others question the advisabil¬ ity of any resection short of curative extirpation in patients without Immorally related disease. Biliary bypass, gastrointesti¬ nal bypass, and chemical splanchnicectomy are used to relieve symptoms created by the mass effects of the tumor in patients with adenocarcinoma of the pancreas. These also are appropriate operations in patients with nonfunctional endocrine tumors of the pancreas, who often have symptoms related to local disease. Non¬ operative percutaneous or endoscopic biliary bypass also can be helpful in selected cases when the risks of surgery are prohibitive. Unlike patients with adenocarcinoma of the pancreas, patients with nonfunctional endocrine tumors camhave long-term sur¬ vival, and this should be taken into account when considering palliative procedures. Specifically, if an operation is performed for biliary obstruction, a concomitant gastrointestinal bypass should be considered, because enteric obstruction becomes more likely with prolonged survival. Long-term survival after gastroje¬ junostomy also makes peristomal jejunal ulceration more likely. For this reason, vagotomy should be performed or appropriate H2-receptor-blocker prophylaxis initiated. Elevated plasma levels of a-fetoprotein have been measured in metastatic nonfunctioning endocrine tumors of the pan¬ creas.91 Alpha-fetoprotein is, most likely, a tumor marker for all metastatic islet cell malignancies and may be used to track the progress of metastatic disease. This feature would make it par¬ ticularly helpful in following nonfunctioning tumors that do not elaborate other measurable hormones or peptide markers.

Ch. 160: Surgery of the Endocrine Pancreas

SURGICAL ASPECTS OF PANCREATIC ENDOCRINE TUMORS EXOCRINE TUMORS VERSUS ENDOCRINE TUMORS Adenocarcinoma of the pancreas is the fifth leading cause of cancer death in the United States. Approximately 20,000 new cases are diagnosed each year, and 5-year survival is ~2% regardless of therapy.92 By comparison, 200 to 1000 endocrine tumors of the pancreas are found in the United States each year,29-93 and 5-year survival after surgery is nearly 100% for benign tumors and >40% for malignant tumors.94 Another advantage of operating on endocrine tumors is the significant symptomatic relief from hormonal syndromes that can be obtained by curative resection or tumor cytoreduction.

SURGICAL PROCEDURES Like ductal tumors, islet cell tumors of the pancreas may be solid or cystic.95 The treatment of cystic endocrine tumors, whether benign, malignant, functional, or nonfunctional, is similar to that of solid endocrine tumors. Operations performed for endo¬ crine tumors of the pancreas include enucleation, segmental resection, distal pancreatectomy, pancreaticoduodenectomy, total and near-total pancreatectomy, tumor cytoreduction, bypass procedures, and surgery on other involved organs such as the stomach, duodenum, and liver (see Table 160-2). Multiple cases of laparoscopic (minimally invasive) resections of islet cell tumors of the distal pancreas have been reported.96 Some centers have performed total hepatectomy with orthotopic liver transplantation for patients with metastases confined to the liver.97 One unusual feature of endocrine tumors of the pancreas not shared by nonendocrine tumors is the response to tumor debulking. With some of these tumors, surgical reduction of the size of the lesion alone can significantly improve long-term survival.98 In contrast, surgery for adenocarcinoma of the pancreas is lim¬ ited mainly to total resection by pancreaticoduodenectomy or palliation with bypass.

INTRAOPERATIVE LOCALIZATION Tumor localization is important and sometimes difficult for endocrine neoplasms, which can be small but clinically symp¬ tomatic. On occasion, tumors cannot be localized before surgery and must be found at surgery. Surgeons performing these oper¬ ations should be familiar with intraoperative ultrasonography, duodenotomy, intraarterial methylene blue administration, and other intraoperative localizing techniques, as well as with the indications for biopsy or blind resection. Any physician evaluating patients with pancreatic masses must understand the possibility and significance of finding an endocrine tumor. Also, any surgeon operating on patients with pancreatic endocrine tumors must be thoroughly familiar with the evaluation and localization of these lesions, with the intraop¬ erative decision-making process, and with the wide range of ablative procedures used for these unusual neoplasms.

REFERENCES 1. Warshaw AL, Swanson RS. What's new in general surgery: pancreatic can¬ cer in 1988. Ann Surg 1988; 208:541. 2. Gold EB. Epidemiology of and risk factors for pancreatic cancer. Surg Clin North Am 1995; 75:819. 3. National Institutes of Health, National Cancer Institute. SEER Cancer Sta¬ tistics Review, 1973-1990. Bethesda, MD: National Institutes of Health, 1993. NIH publication 93-2789.

1487

4. De Jong SA, Pickleman J, Rainsford K. Nocturnal tumors of the pancreas. Arch Surg 1993; 128:730. 5. Yeo CJ, Wang BH, Anthone GJ, Cameron JL. Surgical experience with pan¬ creatic islet-cell tumors. Arch Surg 1993; 128:1143. 6. Capella C, Heitz PH, Hofler H, et al. Revised classification of neuroendo¬ crine tumors of the lung, pancreas and gut. Virchows Arch 1995; 425:547. 7. Bottger TH, Seidl C, Seifert JK, et al. Value of quantitative DNA analysis in endocrine tumors of the pancreas. Oncology 1997; 54:318. 8. Grant CS. Surgical management of malignant islet cell tumors. World J Surg 1993; 17:498. 9. Delcore R, Friesen SR. Gastrointestinal neuroendocrine tumors. J Am Coll Surg 1994; 178:187. 10. Strodel WE, Vinik Al, Lloyd RV, et al. Pancreatic polypeptide-producing tumors. Silent lesions of the pancreas? Arch Surg 1984; 119:508. 11. Brunt LM, Mazoujian G, O'Dorisio TM, Wells SA. Stimulation of vasoactive intestinal peptide and neurotensin secretion by pentagastrin in a patient with VIPoma syndrome. Surgery 1994; 115:362. 12. McCleod MK, Vinik AL Calcitonin immunoreactivity and hypercalcitoninemia in two patients with sporadic, nonfamilial, gastroenteropancreatic neu¬ roendocrine tumors. Surgery 1992; 111:484. 13. Perkins PL, McLeod MK, Jin L, et al. Analysis of gastrinomas by immunohistochemistry and in situ hybridization histochemistry. Diagn Mol Pathol 1992; 1:155. 14. Bieligk S, Jaffe BM. Islet cell tumors of the pancreas. Surg Clin North Am 1995; 75:1025. 15. Modlin IM, Lewis JJ, Ahlman H, et al. Management of unresectable malig¬ nant endocrine tumors of the pancreas. Surg Gynecol Obstet 1993; 176:507. 16. Hammond PJ, Jackson JA, Bloom SR. Localization of pancreatic endocrine tumors. Clin Endocrinol (Oxf) 1994; 40:3. 17. Vinik Al, Delbridge L, Moattari R, et al. Transhepatic portal vein catheteriza¬ tion for localization of insulinomas: a ten year experience. Surgery 1991; 109:1. 18. Van Eijck CH, Brunning HA, Reubi JC, et al. Use of isotope-labeled somato¬ statin analogs for visualization of islet-cell tumors. World J Surg 1993; 17:444. 19. Van Eijck CH, Lamberts SW, Lemaire LC, et al. The use of somatostatin receptor scintigraphy in the differential diagnosis of pancreatic duct cancers and islet cell tumors. Ann Surg 1996; 224:119. 20. Doppman JL, Miller DL, Chang R, et al. Intraarterial calcium stimulation test for detection of insulinomas. World J Surg 1993; 17:439. 21. Ariyama J, Suyama M, Satoh K, Wakabayashi K. Endoscopic ultrasound and intraductal ultrasound in the diagnosis of small pancreatic tumors. Abdom Imaging 1998; 23:380. 22. Bottger TC, Junginger T. Is preoperative radiographic localization of islet cell tumors in patients with insulinomas necessary? World J Surg 1993; 17:427. 23. Norton JA, Cromack DT, Shawker TH, et al. Intraoperative ultrasono¬ graphic localization of islet cell tumors. Ann Surg 1988; 207:160. 24. Joffe SN. Pancreatic islet cell tumor. In: Cameron JL, ed. Current surgical therapy, 2nd ed. St. Louis: CV Mosby, 1986:285. 25. Udelsman R, Yeo CJ, Hruban RH, et al. Pancreaticoduodenectomy for selected pancreatic endocrine tumors. Surg Gynecol Obstet 1993; 177:269. 26. Menegaux F, Schmitt G, Mercadier M, Chigot JP. Pancreatic insulinomas. Am J Surg 1993; 165:243. 27. Von Eyben FE, Grodum E, Gjessing HJ, et al. Metabolic remission with octreotide in patients with insulinoma. J Intern Med 1994; 235:245. 28. Buchanan KD. Effects of somatostatin on neuroendocrine tumors of the gas¬ trointestinal system. Recent Results Cancer Res 1993; 129:45. 29. Lo CY, Lam KY, Fan ST. Surgical strategy for insulinomas in multiple endo¬ crine neoplasia type I. Am J Surg 1998; 175:305. 29a. Tomassetti P, Migliori M, Corinaldesi R, Gullo L. Treatment of gastroentero¬ pancreatic neuroendocrine tumors with octreotide LAR. Aliment Pharma¬ col Ther 2000; 14:557. 30. Friesen SR. Tumors of the endocrine pancreas. N Engl J Med 1982; 306:580. 31. Shepard JJ, Challis DR, Davies PF, et al. Multiple endocrine neoplasia, type 1. Arch Surg 1993; 128:1133. 32. Cherner JA, Sawyers JL. Benefit of resection of metastatic gastrinoma in multiple endocrine neoplasia type 1. Gastroenterology 1992; 102:109. 33. Zollinger RM, Ellison EH. Primary peptic ulcerations of the jejunum associ¬ ated with islet cell tumors of the pancreas. Ann Surg 1955; 142:709. 34. Stabile BE, Morrow DJ, Passaro E. The gastrinoma triangle: operative impli¬ cations. Am J Surg 1984; 147:25. 35. Sawicki MP, Howard TJ, Dalton M, et al. The dichotomous distribution of gastrinomas. Arch Surg 1990; 125:1584. 36. Howard TJ, Sawicki MP, Stabile BE, et al. Biologic behavior of sporadic gas¬ trinoma located to the right and left of the superior mesenteric artery. Am J Surg 1993; 165:101. 37. Imamura M, Takahashi K. Use of selective arterial secretin injection test to guide surgery in patients with Zollinger Ellison syndrome. World J Surg 1993; 17:433. 37a. Jensen RT, Gibril F. Somatostatin receptor scintography in gastrinomas. Ital J Gastroenterol Hepatol 1999; 31(Suppl 2):S179. 38. Kvols L, Brown M, O'Connor L, et al. Evaluation of radiolabeled somato¬ statin analog (e.g., 123I-octreotide) in the detection and localization of carci¬ noid and islet cell tumors. Radiology 1993; 187:129. 39. Thompson NW, Pasieka J, Fukuuchi A. Duodenal gastrinomas, duodenot¬ omy, and duodenal exploration in the surgical management of ZollingerEllison syndrome. World J Surg 1993; 17:455. 40. Ko TC, Flisak M, Prinz RA. Selective intra-arterial methylene blue injection: a novel method of localizing gastrinoma. Gastroenterology 1992; 102:1062.

1 488

PART IX: DISORDERS OF FUEL METABOLISM

41. Harmon j VV, Norton J A, Collin MJ, et al. Removal of gastrinomas for control of Zollinger-Ellison syndrome. Ann Surg 1984; 200:396. 42. Norton JA, Doppman JL, Jensen RT. Curative resection in Zollinger-Ellison syndrome: results of a 10-year prospective study. Ann Surg 1992; 215:8. 43. Howard T, Zinner M, Stabile B, et al. Gastrinoma excision for cure. Ann Surg 1990; 211:9. 44. Delcore R, Friesen SR. Role of pancreatoduodenectomy in the management of primary duodenal wall gastrinomas in patients with Zollinger-Ellison syndrome. Surgery 1992; 112:1016. 45. Orloff SL, Debas HT. Advances in the management of patients with Zollinger-Ellison syndrome. Surg Clin North Am 1995; 75:511. 46. Phan GQ, Yeo CJ, Cameron JL, et al. Pancreaticoduodenectomy for selected periampullary neuroendocrine tumors: fifty patients. Surgery 1997; 122:989. 47. Richardson CT, Feldman M, McClelland RN, et al. Effect of vagotomy in Zollinger-Ellison syndrome. Gastroenterology 1979; 77:682. 48. Zollinger RM, Ellison EC, Fabri PJ, et al. Primary peptic ulcerations of the jejunum associated with islet cell tumors: twenty five year evaluation. Ann Surg 1980; 192:422. 49. Farley DR, van Heerden JA, Grant CS. The Zollinger-Ellison syndrome: a collective surgical experience. Ann Surg 1992; 215:561. 50. Sheppard B, Norton J, Doppman J, et al. Management of islet cell tumors in patients with multiple endocrine neoplasia: a prospective study. Surgery 1989; 106:1108. 51. Delcore R, Friesen SR. Zollinger-Ellison syndrome. Arch Surg 1991; 126:556. 52. Fishbeyn VA, Norton JA, Benya RV, et al. Assessment and prediction of long-term cure in patients with the Zollinger-Ellison syndrome: the best approach. Ann Intern Med 1993; 119:199. 53. Pisegna JR, Norton JA, Slimak GG, et al. Effects of curative gastrinoma resection on gastric secretory function and antisecretory drug requirement in the Zollinger-Ellison syndrome. Gastroenterology 1992; 102:767. 54. Arnold R, Neuhaus C, Benning R, et al. Somatostatin analog Sandostatin and inhibition of tumor growth in patients with metastatic endocrine gastroenteropancreatic tumors. World J Surg 1993; 17:511. 54a. Sato T, Konishi K, Kimura H, et al. Strategy for pancreatic endocrine tumors. Hepatogastroenterology 2000; 47:537. 55. Soga J, Yakuwa Y. Glucagonomas/diabetico-dermatogenic syndrome (DDS): a statistical evaluation of 407 reported cases. J Hepatobiliary Pancreat Surg 1998; 5:312. 56. Becker SW, Kahn D, Rothman S. Cutaneous manifestations of internal malignant tumors. Arch Dermatol Syph 1942; 45:1069. 57. McGavran MH, Unger RH, Recant L, et al. A glucagon-secreting alpha-cell carcinoma of the pancreas. N Engl J Med 1966; 274:1408. 58. Higgins GA, Recant L, Fischman AB. The glucagonoma syndrome: surgi¬ cally curable diabetes. Am J Surg 1979; 137:142. 59. Norton JA, Kahn CR, Shiebinger R, et al. Amino acid deficiency and the skin rash associated with glucagonoma. Ann Intern Med 1979; 91:213. 60. Ingemansson S, Holst J, Larsson LI, Lunderquist A. Localization of glu¬ cagonomas by catheterization of the pancreatic veins and with glucagon assay. Surg Gynecol Obstet 1977; 145:509. 61. Park S, O'Dorisio M, O'Dorisio T. Vasoactive intestinal polypeptide-secret¬ ing tumours: biology and therapy. Baillieres Clin Gastroenterol 1996; 10:673. 62. Jaffe BM. Surgery for gut hormone-producing tumors. Am J Med 1987; 82:68. 63. Cesani F, Ernst R, Walser E, Villanueva-Meyer J. Tc-99m sestamibi imaging of a pancreatic VIPoma and parathyroid adenoma in a patient with multiple type I endocrine neoplasia. Clin Nucl Med 1994; 19:532. 64. Nagomey DM, Bloom SR, Polak JM, Blumgart LH. Resolution of recurrent Vemer. Morrison syndrome by resection of metastatic vipoma. Surgery 1983; 93:348. 65. Kraenzlin ME, Ch'ng JLC, Wood SM, et al. Long-term treatment of a VIPoma with somatostatin analogue resulting in remission of symptoms and possible shrinkage of metastases. Gastroenterology 1985; 88:185. 66. Mathoulin-Portier MP, Payan MJ, Monges G, et al. Pancreatic and duodenal somatostatinoma. Two clinico-pathologic entities. Ann Pathol 1996; 16:299. 67. Larsson LI, Holst JJ, Kuhl C, et al. Pancreatic somatostatinoma: clinical fea¬ tures and physiologic implications. Lancet 1977; 1:666. 68. Sakazaki S, Umeyama K, Nakagawa H, et al. Pancreatic somatostatinoma. Am J Surg 1983; 146:674. 69. Kelly TR. Pancreatic somatostatinoma. Am J Surg 1983; 146:671. 70. Krejs GJ, Orci L, Conlon JM, et al. Somatostatinoma syndrome: biochemical, morphologic and clinical features. N Engl J Med 1979; 301:285. 71. Kaneko H, Yanaihara N, Ito S, et al. Somatostatinoma of the duodenum. Cancer 1979; 44:2273. 72. O'Brien TD, Chejfec G, Prinz RA. Clinical features of duodenal somatostatinomas. Surgery 1993; 114:1144. 73. Angeletti S, Corleto VD, Schillaci O, et al. Use of the somatostatin analogue octreotide to localise and manage somatostatin-producing tumours. Gut 1998; 42:792. 74. Adrian T, Uttenthal L, Williams S, et al. Secretion of pancreatic polypeptide in patients with pancreatic endocrine tumors. N Engl J Med 1986; 315:287. 75. Mozell E, Stenzell P, Woltering E, et al. Functional endocrine tumors of the pancreas: clinical presentation, diagnosis, and treatment. Curr Probl Surg 1990; 27:303. 76. Miraliakbari BA, Asa L, Boudreau SF. Parathyroid hormone-like peptide in pancreatic endocrine carcinoma and adenocarcinoma associated with hypercalcemia. Hum Pathol 1992; 23:884. 77. Tarver DS, Birch SJ. Case report: life-threatening hypercalcemia secondary to pancreatic tumor secreting parathyroid hormone-related protein—suc¬ cessful control by hepatic arterial embolization. Clin Radiol 1992; 46:204.

78. Mitlak BH, Hutchinson JS, Kaufman SD, Nussbaum SR. Parathyroid hor¬ mone-related peptide mediates hypercalcemia in an islet cell tumor of the pancreas. Horm Metab Res 1991; 23:344. 79. Price DE, Absalom SR, Davidson K, et al. A case of multiple endocrine neo¬ plasia: hyperparathyroidism, insulinoma, GRF-oma, hypercalcitoninemia and intractable peptic ulceration. Clin Endocrinol (Oxf) 1992; 37:187. 80. Gullo L, De Giorgio R, D'Errico A, et al. Pancreatic exocrine carcinoma pro¬ ducing adrenocorticotropic hormone. Pancreas 1992; 7:172. 81. Tsuchihashi T, Yamaguchi K, Abe K, et al. Production of immunoreactive corticotropin-releasing hormone in various neuroendocrine tumors. Jpn J Clin Oncol 1992; 22:232. 82. Amikura K, Alexander HR, Norton JA, et al. Role of surgery in management of adrenocorticotropic hormone-producing islet cell tumors of the pan¬ creas. Surgery 1995; 118:1125. 83. Venkatesh S, Ordonez NG, Ajani J, et al. Islet cell carcinoma of the pancreas. Cancer 1990; 65:354. 84. Buetow PC, Miller DL, Parrino TV, Buck JL. Islet cell tumors of the pancreas: clinical, radiologic, and pathologic correlation in diagnosis and localization. Radiographics 1997; 17:453. 85. White TJ, Edney JA, Thompson JS, et al. Is there a prognostic difference between functional and nonfunctional islet cell tumors? Am J Surg 1994:168:627. 86. Heitz PU, Kasper M, Polak JM, et al. Pancreatic endocrine tumors. Hum Pathol 1982; 13:263. 87. De Jong SA, Pickleman J, Rainsford K. Nonductal tumors of the pancreas. The importance of laparotomy. Arch Surg 1993; 128:730. 88. Kent RB, van Heerden JA, Weiland LH. Nonfunctioning islet cell tumors. Ann Surg 1981; 193:185. 89. Evans DB, Skibber JM, Lee JE, et al. Nonfunctioning islet cell carcinoma of the pancreas. Surgery 1993; 114:1175. 90. Eckhauser FE, Cheung PS, Vinik Al, et al. Nonfunctioning malignant neu¬ roendocrine tumors of the pancreas. Surgery 1986; 100:978. 91. Lesur G, Bergemer AM, Turner L, et al. Increases in alpha-fetoprotein in pancreatic endocrine tumors with hepatic metastases. Gastroenterol Clin Biol 1996; 20:204. 92. Gordis L, Gold EB. Epidemiology of pancreatic cancer. World J Surg 1984; 8:808. 93. Brennan MF, MacDonald JS. The endocrine pancreas. In: DeVita V, Heilman S, Rosenberg SA, eds. Principles and practice of oncology, 2nd ed. Philadel¬ phia: JB Lippincott, 1985:1206. 94. Thompson GB, van Heerden JA, Grant CS, et al. Islet cell carcinoma of the pancreas: a twenty-year experience. Surgery 1988; 104:1011. 95. Schwartz RW, Munfakh NA, Zweng T, et al. Nonfunctioning cystic neu¬ roendocrine neoplasms of the pancreas. Surgery 1994; 115:645. 96. Vezakis A, Davides D, Larvin M, McMahan MJ. Laparoscopic surgery com¬ bined with preservation of the spleen for distal pancreatic tumors. Surg Endosc 1999; 13:26. 97. Dousset B, Houssin D, Soubrane O, et al. Metastatic endocrine tumors: is there a place for liver transplantation? Liver Transpl Surg 1995; 1:111. 98. Danforth DN, Gorden P, Brennan MF. Metastatic insulin-secreting carci¬ noma of the pancreas: clinical course and the role of surgery. Surgery 1984; 96:1027. 99. Debas HT, Mulvihill SJ. Neuroendocrine gut neoplasms: important lessons from uncommon tumors. Arch Surg 1994; 129:965. 100. Buchanan KD, Johnston CF, O'Hare MMT, et al. Neuroendocrine tumors: a European view. Am J Surg 1986; 81:14.

CHAPTER 1 61

HYPOGLYCEMIA OF INFANCY AND CHILDHOOD JOSEPH I. WOLFSDORF AND MARK KORSON Glucose is the predominant metabolic fuel utilized by the brain. Because the brain cannot synthesize glucose or store more than a few minutes' supply as glycogen, survival of the brain requires a continuous supply of glucose.1 Recurrent hypoglycemia dur¬ ing the period of rapid brain growth and differentiation in infancy can result in long-term neurologic sequelae and psycho¬ motor retardation. Therefore, prevention of hypoglycemia and expeditious diagnosis and vigorous treatment when it occurs are essential to prevent the potentially devastating conse¬ quences of hypoglycemia on the brain.

Ch. 161: Hypoglycemia of Infancy and Childhood

1489

FIGURE 161-1. Transition from the fed to the fasted state. A, Between meals: Glucose is derived from hepatic glycogenolysis; free fatty acids are an important fuel for muscle. B, Overnight fast (postabsorptive state): Liver glycogen becomes depleted; gluconeogenesis becomes the principal source of glucose. Hepatic ketone production increases, providing an alternative fuel for brain and muscle. C, Prolonged fasting: Fatty acids and ketones are the principal metabolic substrates. Brain utilization of ketones increases. Glucose is derived from gluconeogenesis.

INCREASED SUSCEPTIBILITY OF THE INFANT AND CHILD TO HYPOGLYCEMIA Hypoglycemia is most common in the newborn period. During infancy and childhood, it occurs most frequently when nighttime feeding is discontinued and when intercurrent illness interrupts the normal feeding pattern, causing periods of relative starvation. Basal energy needs during infancy are high. A full-term newborn baby, for example, has a ratio of surface area to body mass that is more than twice that of an average adult, necessitating a high rate of energy expenditure to maintain body temperature. Also, the infant brain is large relative to body mass and its energy require¬ ment is mainly derived from the oxidation of circulating glucose. To meet the high demand for glucose, the rate of glucose produc¬ tion in infants and young children is two to three times that of older children and mature adults.2 Although the demand for glu¬ cose is high, the activity of several liver enzymes involved in energy production is low in the newborn compared to that of older children and adults. Consequently, until feeding is well established, maintenance of glucose homeostasis in the newborn period is more precarious than it is later in childhood. In the postabsorptive state, the rate of glucose turnover in adults is ~2 mg/kg per minute (8-10 g per hour), whereas the average basal (4-6 hours after feeding) rate of glucose turnover is 6 mg/kg per minute in newborns, approximately three times the adult rate. During prolonged fasting, infants and children cannot sustain this high rate of glucose production. Normal children, 18 months to 9 years of age, fasted for 24 hours, have a mean blood glucose concentration of 52 ± 14 (standard deviation [SD]) mg/dL. Indeed, 22% have blood glucose concentrations 3.3 mmol/L) without intravenous infusions of glucose or glucagon, a feeding schedule appropri¬ ate for the age of the infant, and a fasting tolerance of 6 to 8 hours in the newborn infant, 12 hours in infants up to 1 year of age, or 16 hours or more in older children. Prompt effective treatment is necessary to minimize the risk of long-term adverse neurologic sequelae 42 Initially, this requires a glucose infusion at two- to four-fold (average 14.5 ±1.7 mg/kg per minute43) the basal rate of glucose production, and occasionally reaches 25 mg/kg per minute. Placement of a central venous line is usually necessary to be able to infuse hypertonic glucose solutions. Treatment with oral diazoxide, which opens normal KATP channels and thereby suppresses insulin secretion, should be given a trial (15-25 mg/ kg per day in 3 doses at 8-hour intervals). Its effect may be potentiated by the addition of a thiazide diuretic. Diazoxide is ineffective in infants whose hyperinsulinism is caused by muta¬ tions of the Katp channel. A long-acting somatostatin analog (octreotide) may be successful in maintaining normoglycemia in up to 50% of cases of congenital hyperinsulinism. Octreotide inhibits insulin secretion by decreasing the influx of calcium ions into B cells and through a direct effect on secretory gran¬ ules. The starting dose is 5 pg/kg every 6 to 8 hours. If glucose is not maintained (>60 mg/dL), the dosage of octreotide is increased up to a maximum of 40 to 60 pg/kg per day, divided into three to six doses. Because of the marked variability of response to octreotide, the therapeutic regimen has to be adapted for each individual patient, and its effects closely mon-

1 494

PART IX: DISORDERS OF FUEL METABOLISM

itored.44 Many infants fail to respond to medical therapy and require a 95% subtotal pancreatectomy to restore normoglycemia 45453

HORMONE DEFICIENCY ADRENOCORTICOTROPIC HORMONE/CORTISOL DEFICIENCY Cortisol limits glucose utilization in several tissues, including skel¬ etal muscle, by directly opposing the action of insulin and, second¬ arily, by promoting lipolysis. It stimulates protein breakdown and increases release of gluconeogenic precursors from muscle and fat. Cortisol stimulates hepatic gluconeogenesis and glycogen synthe¬ sis and exerts permissive influences on the gluconeogenic and gly¬ cogenolytic effects of glucagon and epinephrine. By all these effects, cortisol tends to raise plasma glucose concentrations. Adrenocortical insufficiency should be considered in the dif¬ ferential diagnosis of patients who present with hypoglycemia and ketosis. In infancy, adrenocortical insufficiency may be sec¬ ondary to congenital adrenal hyperplasia or congenital adrenal hypoplasia. In older children, adrenocortical insufficiency is more likely to be caused by Addison disease. Adrenocortico¬ tropic hormone (ACTH) deficiency or panhypopituitarism can present with hypoglycemia in infancy or in later childhood. Diagnosis. A serum cortisol concentration 40 million per¬ sons) in the United States who are 20 years of age or older fall into the high-risk blood cholesterol classification, and another 54 million people have borderline-high blood cholesterol levels.4 Approximately 20% of males and 5% of females have low HDL cholesterol levels, and fewer than 5% of men and women have elevated triglyceride levels. All patients who are screened should receive information about an NCEP or American Heart Association step 1 diet and CHD risk factors. According to the NCEP Adult Treatment Panel guidelines, patients who have desirable total cholesterol and normal HDL cholesterol values should have their values checked again within 5 years.3-4-52 If the patient has a borderlinehigh value, information about other CHD risk factors should be obtained3-4 (Table 163-1). If the patient has a cholesterol value in the borderline-risk category and a normal HDL cholesterol level, in the absence of CHD (i.e., prior myocardial infarction or angina) or two or more CHD risk factors (see Table 163-1), dietary information should be provided and the cholesterol value checked within the next year. If the patient has a borderline-high value and a history of CHD or two or more CHD risk factors, or the patient has a highrisk total cholesterol value or has a low HDL cholesterol value, LDL cholesterol levels should be assessed so that an appropriate treatment regimen can be determined.3-4 LDL cholesterol is rou¬ tinely calculated after measuring serum total cholesterol, tri¬ glyceride, and HDL cholesterol after an overnight fast. The normal ranges for total cholesterol, triglyceride, very-low-density lipoprotein (VLDL) cholesterol, LDL cholesterol, and HDL cho¬ lesterol are provided in Table 163-1, and options for measuring LDL cholesterol are discussed later. Another issue is whether apoliprotein (apo) A-I, apo B, Lp(a), or LDL size should be measured for assessing CHD risk. In pro¬ spective studies, only Lp(a) among these parameters has been shown to be an independent risk factor, after smoking, blood pressure, diabetes, LDL cholesterol, and HDL cholesterol were taken into account.55-115 In the author's view, direct measure¬ ments of LDL cholesterol, HDL cholesterol, remnant lipoprotein cholesterol, and Lp(a) cholesterol will become the method of choice for lipoprotein assessment in the future. A remnant lipo¬ protein cholesterol >10 mg/dL confers a 100% increased risk in women, and a substantially increased risk in men.70-74 Similarly, an Lp(a) cholesterol >10 mg/dL confers a 100% increased CHD risk, especially in men.98 An Lp(a) cholesterol and remnant lipo¬ protein cholesterol should be part of CHD risk assess¬ ment.74-98-112-115 Measurement of other parameters cannot be recommended at this time.

TOTAL CHOLESTEROL AND HIGH-DENSITY LIPOPROTEIN CHOLESTEROL

NATIONAL CHOLESTEROL EDUCATION PROGRAM GUIDELINES

The classification system begins with the measurement of total cholesterol and HDL cholesterol levels for screening the general population in the fasting or nonfasting state. In the author's view, it is not unreasonable to get a screening triglyceride value at that time. Accurate fingerstick methods are available for cho¬ lesterol and HDL cholesterol screening in the office setting.33 An accurate home cholesterol test that can be self-administered by

The NCEP Adult Treatment Panel has developed guidelines for the diagnosis and treatment of individuals older than 20 years of age with elevated blood cholesterol levels associated with an increase in LDL cholesterol levels.3-4 The goals of therapy and the particular level of LDL cholesterol requiring the initiation of diet and drug therapy depend on the presence or absence of CHD or two or more CHD risk factors (Table 163-2). The pres-

generally develop premature CHD in the middle decades of life.5-7'50

ANIMAL MODEL EVIDENCE Animal models have demonstrated a direct relationship between LDL cholesterol and atherosclerosis. Animals consum¬ ing diets high in saturated fat and cholesterol develop LDL cho¬ lesterol elevation and atherosclerosis.51 Such diets also increase HDL cholesterol, an effect that may be compensatory. These hypercholesterolemic animals develop intimal lesions that progress from fatty streaks to ulcerated plaques, resembling those of human atherosclerosis. In laboratory trials, severe ath¬ erosclerosis in monkeys regresses when blood cholesterol is lowered through diet or drug therapy. Such studies support a causal relationship between LDL cholesterol and atherosclerosis and suggest reversibility of the process with the reduction of the serum LDL cholesterol level.51 The combined findings of these studies support the concept that lowering total and LDL cholesterol levels can reduce the incidence of CHD events and the death rate due to myocardial infarction.13-48 Moreover, the pooled analysis of clinical trial findings suggests that intervention is as effective in preventing recurrent myocardial infarction and mortality in patients expe¬ riencing a recurrent attack as it is in primary prevention. The complete set of evidence strongly supports the concept that reducing total and LDL cholesterol levels can reduce CHD risk in younger and older men, in women, and in individuals with moderate elevations of cholesterol.4 It is important to recognize the magnitude of CHD reduction associated with lowering serum cholesterol levels. For persons with serum cholesterol levels initially in the range of 250 to 300 mg/dL (6.5-7.8 mmol/L), each 1% reduction in serum choles¬ terol level yields approximately a 1% to 2% reduction in CHD rates.9 A 30% reduction in the LDL cholesterol level will reduce CHD risk by as much as 50%.13-47 Moreover, studies indicate that aggressive lipid modification can result in stabilization of existing coronary atherosclerosis and some degree of regres¬ sion.19-38-47

PATIENT EVALUATION

1515

Ch. 163: Lipoprotein Disorders TABLE 163-1. Normal Values for Plasma Lipid and Lipoprotein Cholesterol Concentrations* Plasma Cholesterol (mg/dL)

Plasma Triglyceride (mg/dL)

VLDL Cholesterol (mg/dL)

HDL Cholesterol (mg/dL)

LDL Cholesterol (mg/dL)

HDL/Cholesterol Ratio

Percentiles 160 mg/dL or 4.1 mmol/L) must be ruled out. These include hypothyroidism, obstructive liver disease, and nephrotic syn¬ drome. LDL cholesterol decision points for initiating diet and drug therapy are given in Table 163-3. The NCEP guidelines

TABLE 163-2. National Cholesterol Education Program Major Coronary Heart Disease Risk Factors in Addition to Low-Density Lipoprotein Cholesterol High-density lipoprotein (HDL) cholesterol 60 mg/dL (1.6 mmol/L)

have been accepted by all major U.S. medical organizations, including the American College of Physicians, the American Heart Association, and the American Medical Association.4 Guidelines for the general population and children and adoles¬ cents have also been developed.116/117 TABLE 163-3. National Cholesterol Education Program Adult Treatment Panel II Treatment Guidelines Low-Density Lipoprotein Cholesterol Values (mg/dL [mmol/L]) Therapy

^>13ol3A)

>160 (4.2)

>190 (5.0)

DIET*

Yes, ifCHD is present

Yes, if 2 or more CHD risk factors are present

Yes

DRUGS (AFTER DIET*)

Yes, ifCHD is present

Yes, if 2 or more CHD risk factors are present

Yes

CHD, coronary heart disease. ’The goal of diet therapy is reading the initiation value, and the goal of drug ther¬ apy is 30 mg/dL or 0.8 mmol/L below the initiation value. CHD risk factors are listed in Table 163-2. All CHD patients should be placed on a National Cholesterol Education Program step 2 diet.

1516

PART IX: DISORDERS OF FUEL METABOLISM

The recommendation that LDL cholesterol values be used as the primary criterion for treatment decisions for patients with elevated cholesterol levels makes accurate measurement a national public health imperative as reviewed by the NCEP Lab¬ oratory Standardization Panel.118 If a patient has an LDL cholesterol level of 160 mg/dL (4.1 mmol/L), it represents approximately the 75th percentile for mid¬ dle-aged Americans (see Table 163-1). It is important to confirm any abnormalities by repeat determinations. Hospitalization or acute illness can affect lipid values, and lipid determinations should gen¬ erally be carried out in the free-living state.119 An elevated or borderline-high triglyceride level (200 mg/dL or 2.3 mmol/L) has not clearly been shown to be an independent risk factor for premature heart disease. However, an elevated triglyceride level is inversely associated with a low level of HDL cholesterol, which has been shown to be a significant risk factor for CHD. Common secondary causes of elevated LDL cholesterol and triglyceride values and of decreased HDL cholesterol include diabetes, hypothyroidism, obstructive liver disease, kidney disease, excess alcohol intake, and the use of corticosteroids, anabolic steroids, estrogens, P-blocking agents, and thiazide diuretics.4 If possible, these factors should be screened for and treated before diet or drug therapy for lipid disor¬ ders is initiated. Screening should include an evaluation of glucose, albumin, liver transaminases, alkaline phosphatase, creatinine, and thyroid-stimulating hormone, and the patient should be asked about alcohol intake and the use of [1-blockers, estrogens, cortico¬ steroids, anabolic steroids, and thiazides.

LOW-DENSITY LIPOPROTEIN CHOLESTEROL MEASUREMENT Unlike total cholesterol quantitation, there is no consensusapproved and validated reference method for the direct mea¬ surement of LDL cholesterol. The accurate measurement of LDL cholesterol depends on the separation of LDL particles in serum from other lipoproteins: chylomicrons, VLDL, and HDL. Tradi¬ tionally, LDL has been defined as all lipoproteins within the density range of 1.019 to 1.063 g/mL. However, in common practice, the definition has been broadened to include interme¬ diate-density lipoprotein (IDL; 1.006-1.019 g/mL). Using this definition, LDL is composed of LDL + IDL + Lp(a). This defini¬ tion serves as the basis for the cut-points defined by the NCEP Adult Treatment Panel. The options for measuring LDL choles¬ terol include ultracentrifugation, the Friedewald calculation for estimating LDL cholesterol levels, and a direct method for mea¬ suring LDL cholesterol that uses immunoseparation of lipopro¬ teins by their respective apolipoprotein content. Ultracentrifugation involves the separation of lipoproteins based on their density differences after an 18-hour spin at 109,000 x g. The VLDL and chylomicrons float to the top and are sepa¬ rated using a tube slicing technique from the 1.006-g/mL infranatant (i.e., "1.006 bottom"). This infranatant fraction contains LDL and HDL. A heparin-manganese precipitation reagent is added to the 1.006 bottom to precipitate LDL, leaving HDL in the superna¬ tant. The cholesterol concentrations of the 1.006 g/mL of infrana¬ tant and the HDL cholesterol supernatant are measured using the Abell-Kendall cholesterol reference method: LDL cholesterol = infranatant cholesterol - HDL cholesterol. This procedure has been adopted by the Centers for Disease Control and Prevention and the Reference Network Laborato¬ ries for Standardizations as a means of directly measuring LDL cholesterol in the research setting and serves as the standard.118 However, ultracentrifugation is poorly suited to the routine, clinical laboratory for several reasons. It requires cumbersome procedures; it is extremely labor intensive and technique depen¬ dent; it requires expensive instrumentation; and although it is the accepted reference method, it is an indirect measurement.

Most clinical laboratories use the equation known as the Friedewald formula119 to estimate a patient's LDL cholesterol con¬ centration: estimation of LDL cholesterol = total cholesterol HDL cholesterol - VLDL cholesterol. The estimation of VLDL cholesterol equals the triglyceride level divided by five.119 The Friedewald formula estimates the LDL cholesterol con¬ centration by subtracting the cholesterol associated with the other classes of lipoproteins from total cholesterol. This involves three independent lipid analyses, each contributing a potential source of error. It also involves a potentially inaccurate estimate of VLDL cholesterol. Because no direct VLDL cholesterol assay is available, it is calculated from the triglyceride value divided by a factor of five. This divisor can also add error to all LDL cho¬ lesterol estimates, but it is especially inappropriate for individu¬ als with elevated triglyceride levels. Clinical laboratories use automated enzymatic analyses for cholesterol and triglyceride within serum or plasma, and HDL cholesterol is measured after precipitation of other lipoproteins in serum or plasma with hep¬ arin manganese chloride, dextran magnesium sulfate, or phosphotungstic acid.118 On-line direct assays of HDL cholesterol are now available. The drawbacks of using the Friedewald formula for determining levels of LDL cholesterol are that it is estimated by calculation; it requires multiple assays and multiple steps, each adding a potential source of error; it is increasingly inaccu¬ rate as triglyceride levels increase; it requires that patients fast for 12 to 14 hours before specimen collection to avoid a triglyc¬ eride bias; and it is not standardized.118'119 Moreover, LDL cho¬ lesterol concentrations cannot be reported for individuals with elevated triglyceride levels (>400 mg/dL or 4.5 mmol/L) or in the nonfasting state.119'120 It has been reported that the formula becomes increasingly inaccurate in calculating true LDL choles¬ terol levels at borderline triglyceride levels (200^400 mg/dL or 2.3-4.5 mmol/L).112-115 The inadequacies of the methods for measuring LDL choles¬ terol necessitated the development of a direct method by which clinical laboratories may accurately and practically assess LDL cholesterol concentrations in patient samples. In 1990, the Labo¬ ratory Standardization Panel of the NCEP recommended the development of a direct LDL cholesterol measurement method.118 The direct method for measuring serum or plasma LDL cholesterol concentration that was introduced was suitable for routine use in the clinical laboratory. This immunoseparation technology uses affinity-purified goat polyclonal antisera to human apo A-I and apo E, which are coated on latex particles; this facilitates the removal of chylomicrons, VLDL, and HDL in nonfasting or fasting specimens. After incubation and centrifu¬ gation, LDL cholesterol remains in the filtrate solution. The LDL cholesterol concentration is obtained by performing an enzy¬ matic cholesterol assay on the filtrate solution. The direct LDL cholesterol immunoseparation method allows for the direct quantitation of LDL cholesterol from one measure¬ ment, the use of fasting and nonfasting samples, and an LDL cholesterol measurement regardless of elevated triglyceride lev¬ els. When the direct LDL cholesterol assay was carried out on serum obtained from 115 subjects, who were fasting or nonfast¬ ing and were normal or hyperlipidemic, and was compared with those obtained by ultracentrifugation analysis, the correla¬ tion was 0.97, with a small negative bias of 2.9%. Subjects with LDL cholesterol levels >160 mg/dL, as obtained by ultracentrif¬ ugation, were correctly classified 93.8% of the time. In a similar study carried out on serum obtained from 177 subjects with nor¬ mal or elevated lipid levels, the correlation between the direct LDL cholesterol and the value obtained by ultracentrifugation was 0.98, with between-run and within-run coefficients of vari¬ ation 200 mg/dL or 2.3 mmol/L) and normal LDL cholesterol levels, there are no clear medication guidelines.3,4 However, diet and exercise are encouraged, as well as the elimination of secondary causes of elevated triglycerides, such as lack of exercise, obesity, diabetes, alcohol, estrogens, and (1-blockers. If the patient has a fasting

1518

PART IX: DISORDERS OF FUEL METABOLISM

triglyceride level >1000 mg/dL (11.3 mmol/L) while on a restricted diet, medication to reduce the risk of pancreatitis is recommended. However, before taking this step, the physician should make sure that these patients are not taking estrogens, thiazides, or (1-blockers; are not using alcohol; or do not have uncontrolled diabetes mellitus. Caloric and fat restriction (10 mg/dL) and decreased HDLC (240 mg/dL as “high blood cholesterol." An HDL cholesterol level

CARBOXYPEPTIDASE E

1 GR EXTENDED CCK PROHORMONE CONVERTASE |

PEPTIDYLGLY

III’

I

oAMIDATING

MONOXYGENASE

——G,V

COMPLEX

2. G EXTENDED CCK

F-

3. BIOACTIVE CCKs

-—-- COHN?

CCK 83

-COHN?

CCK 58

f-

--

CONH?

CCK -33

|-COHN?

CCK 22

COHN?

CCK-8

FIGURE 167-1. Schematic illustration of the posttranslational process¬ ing of preprocholecystokinin in cerebral neurons and the I cells of the small intestine. (CCK, cholecystokinin; G, glycine; GR, glycine-arginine.) co- and posttranslational processing comprises two sorts of covalent modifications: cleavage of peptide bonds (often at the carboxyl end of single or double basic amino-acid residues),8-10 and amino-acid derivations (acetylations, amidations, glycosylations, methylations, sulfations, and the like). The degree of processing varies considerably. Sometimes the preprohormones are processed through a few steps to a single active form, as for insulin. Other preprohormones undergo multiple modifications to yield several bioactive forms, as illustrated for cholecystoki¬ nin in Figure 167-1. Because prohormone maturation is governed by the activity of processing enzymes, much effort has been devoted to charac¬ terization of these enzymes. So far, a number of essential enzymes have been identified. Of central significance is a fam¬ ily of subtilisin-related endoproteases, which cleave the dibasic Arg-Arg and Lys-Arg sites. These prohormone convertases are all structurally related, calcium-dependent, serine proteases. The enzyme family contains nine or more dibasic-specific endoproteases, but prohormone convertases 1 (PCI/3) and 2 (PC2) appear most important for prohormone processing in mammals.8'9 Also, an aspartyl endoprotease that cleaves prohor¬ mones at monobasic sites has been identified.10 Because the bio¬ logic activity of half of the diffuse peptide hormone systems depends on a-amidation of the C-terminal acid group, much interest has accompanied the identification of the amidation enzyme complex. Carboxyamidation requires two sequentially acting enzymes that use glycine as the amide donor. The first enzyme is a copper- and ascorbate-dependent peptidyl-glycine a-hydroxylating monooxygenase (PHM), derived from the Nterminal part of the amidation enzyme precursor. The second enzyme is a separate peptidyl-a-hydroxyglycine a-amidating lyase (PAL), derived from the remaining intragranular region of the same amidation enzyme precursor.11'12 Although proteolysis at dibasic or monobasic sites and carboxyamidation are funda¬ mental in the maturation of most prohormones, the vast num¬

1555

ber of different posttranslational modifications indicates that more processing enzymes are to be found. The full extent to which defective processing enzymes can explain endocrine dis¬ eases remains to be shown; however, fat /fat mice experience obesity and slow-onset diabetes mellitus due to carboxypeptidase-E deficiency,13 and mutations in the gene encoding PCI may be associated with obsesity, mild diabetes mellitus, and hypogonadotropic hypogonadism in humans.14 Although endocrine cells or neurons may contain several bio¬ active products of the preprohormone, these cells frequently release only a few active forms in significant amounts. The released forms may then undergo postsecretory enzymatic modi¬ fications in the blood or, alternatively, at the target site. One such enzyme, neutral endopeptidase, which belongs to the neprilysin family,15 is anchored in the plasma membrane and actively cleaves peptides in the immediate vicinity. Poorly and/or inap¬ propriately processed prohormones in the synthesizing tissue are, accordingly, intermediate precursors or degradation frag¬ ments. Their clinical significance relates to hypersecretion by endocrine tumors, which often release incompletely processed intermediates of variable bioactivity; sometimes, a tumor secretes normal amounts of the principal mature peptide but also releases into the blood increased amounts of intermediate products. Molecular heterogeneity of a peptide system may not be attributable only to multiple steps in the posttranslational phase of its biosynthesis. Differences in posttranscriptional pro¬ cessing (alternative splicing) may also produce different molecular forms of the same hormone, as illustrated by the 22- and 20kDa forms of human growth hormone.6 Finally, heterogeneity may result when multiple genes encode the same peptide hor¬ mone with only a few amino-acid substitutions. These substitu¬ tions may have no effect on bioactivity, as, for example, in the two forms of insulin that occur in the rat. On the other hand, they may have considerable pathogenetic implications, as in the case of the mutant insulins found in some families with dia¬ betes mellitus. Regardless of whether the molecular heteroge¬ neity is caused by multiple posttranslational or postsecretory modifications, by multiple genes or gene transcripts, the phe¬ nomenon may merit considerable biochemical investigation and sophisticated diagnostic assays before the clinical implica¬ tions can be dealt with appropriately.

WIDESPREAD EXPRESSION All cells in the body (except haploid sex cells) have all of the genes for all hormonal peptides. In principle, therefore, all cells could conceivably synthesize all hormones. Endocrine cells and neurons are highly differentiated, however, so at one time the assumption was that one endocrine cell type could express the gene for only one peptide system. The names assigned to endo¬ crine cells and neurons reflected this unambiguous concept: the secretin cell, the insulin cell, the oxytocin neuron, among others. Studies have revealed a system for regulating gene activation that is considerably more complex, however. Most hormonal peptides are synthesized in several different cell types in differ¬ ent regions of the body (see Chap. 175). This "diffuse" synthesis varies greatly at different ontogenic and phylogenic stages. Thus, in adult mammals, somatostatin is synthesized in and released from endocrine cells in the thyroid (C cells), the pan¬ creas (D cells), endocrine and paracrine cells in the upper gut, and various neurons in both the central and peripheral nervous systems. Cholecystokinin is synthesized in large amounts in all regions of the brain, in various pituitary neurons, in peripheral neurons in the distal part of the gut, as well as in endocrine cells in the gut (I cells), adrenal medulla, and pituitary.5-16 The phenomenon of widespread expression casts doubt on the concept that hormone production by tumors is really "ectopic." Perhaps hormone-secreting tumors always originate from cells that have been tailored to synthesize the hormones

1 556

PART X: DIFFUSE HORMONAL SECRETION

that are subsequently released from that particular tumor. Such synthesis may occur at a low level, or it may follow a cell-specific type of processing. Therefore, this may not be detected until the transformation of the cells has increased the rate of synthesis to an extent sufficient to cause symptoms (see Chap. 219).

CELL-SPECIFIC PROCESSING To some extent, widespread peptide gene expression is differen¬ tiated. Thus, although a gene for a hormone may be expressed at the translational level in different cell types, the processing of the prohormone may vary in different cells. For instance, the single gastrin gene is expressed in three different endocrine cells in mammals: the antral G cells, the pituitary corticotropes, and the endocrine cells dispersed in the exocrine pancreas.5-16 The posttranslational processing in these cells differs greatly. The antral cells contain a mixture of large and small gastrins in tyrosine-Osulfated, as well as nonsulfated, forms. The end product in the pituitary is exclusively nonsulfated large gastrins, whereas the pancreas synthesizes only completely sulfated gastrin-17. Similar cell specificity of the posttranslational processing has been found for cholecystokinin, glucagon, somatostatin, the opioid peptides, the tachykinins, and other peptide systems. Such cell specificity occurs not only at the posttranslational level, however. For example, the calcitonin gene and other hor¬ mone genes can be processed at the posttranscriptional level to completely different mRNAs through alternative splicing.17 Thus, whereas the calcitonin gene in the thyroid C cells is expressed as the well-known hormone calcitonin, the same gene in hypothalamic and other neurons is expressed as a struc¬ turally unrelated peptide, calcitonin gene-related peptide (see Chaps. 3 and 53). Cell-specific processing, therefore, economizes with genes, so that a single gene may be expressed in a number of different pep¬ tides with different bioactivities. That the differentiation may occur during both the posttranscriptional and posttranslational phases only emphasizes the versatility of gene expression mecha¬ nisms. Again, the transformed cells in hormone-secreting tumors do not respect the normal cell-specific differentiation, and the hormone genes may be expressed as many kinds of peptides. The diagnosis and, perhaps, the therapeutic control of such tumors may depend on an understanding of these possible variations in both posttranscriptional and posttranslational processing.

sophisticated manner, this process may appear to be chaotic in the case of hormone-secreting tumors.

REFERENCES 1. Bayliss WM, Starling EH. On the causation of the so-called "peripheral reflex secretion" of the pancreas. Proc R Soc Lond 1902; 69:352. 2. Starling EH. The chemical correlation of the functions of the body: the chemi¬ cal control of the functions of the body. Croonian Lecture I. Lancet 1905; 2:338. 3. Krieger D. Brain peptides: what, where and why? Science 1983; 222:975. 4. Larsson LI, Goltermann N, de Magistris L, et al. Somatostatin cell pro¬ cesses as pathway for paracrine secretion. Science 1979; 205:1395. 4a. Carlevaro MF, Cermelli S, Cancedda R, et al. Vascular endothelial growth factor (VEGF) in cartilage neovascularization and chondrocyte differentiation: auto¬ paracrine role during endochondral bone formation. J Cell Sci 2000; 113:59. 5. Rehfeld JF. The new biology of gastrointestinal hormones. Physiol Rev 1998; 78:1087. 6. Seeburg PH. The human growth hormone locus: the genes and their prod¬ ucts. In: Hakanson R, Thorell J, eds. Biogenetics of neurohormonal pep¬ tides. London: Academic Press, 1985:83. 7. Fradkin JE, Eastman RC, Lesniak MA, Roth J. Specificity spillover at the hor¬ mone receptor: exploring its role in human disease. N Engl J Med 1989; 320:640. 8. Steiner DF. The proprotein convertases. Curr Opin Chem Biol 1998; 2:31. 9. Nakayama K. Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. Biochem ] 1997; 327:625. 10. Bourbonnais Y, Ash J, Daigle M, Thomas DY. Isolation and characterization of S. cerevisiae mutants defective in somatostatin expression: cloning and functional role of a yeast gene encoding an aspartyl protease in precursor processing at monobasic cleavage sites. EMBO J 1993; 12:285. 11. Mains RE, Milgram SL, Keutmann HT, Eipper BA. The NH2 terminal pro¬ region of peptidylglycine a-amidating monooxygenase facilitates the secretion of soluble proteins. Mol Endocrinol 1995; 9:3. 12. Eipper BA, Stoffers DA, Mains RE. The biosynthesis of neuropeptides: pep¬ tide y-amidation. Ann Rev Neurosci 1992; 15:57. 13. Naggert JK, Fricker LD, Varlamov O, et al. Hyperinsulinemia inf at/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity. Nat Genet 1995; 10:135. 14. Jackson RS, Creemers JWM, Ihagi S, et al. Obesity and impaired prohor¬ mone processing associated with mutations in the human prohormone convertase 1 gene. Nat Genet 1997; 16:303. 15. Turner AJ, Brown CD, Carson JA, Barnes K. The neprilysin family in health and disease. Adv Exp Med Biol 2000; 477:229. 16. Rehfeld JF, Solinge WW. The tumor biology of gastrin and cholecystokinin. Adv Cancer Res 1994; 63:295. 17. Amara SG, Jonas V, Rosenfeld MG, et al. Alternative RNA processing in calcitonin gene expression. Nature 1982; 298:240. 18. Cuello AC, ed. Co-transmission. London: Macmillan, 1985. 19. Rehfeld JF. Accumulation of nonamidated products of preprogastrin and preprocholecystokinin in pituitary corticotrophs: evidence for posttransla¬ tional control of cell differentiation. J Biol Chem 1986; 261:5841.

COSYNTHESIS A final characteristic of the diffuse peptide hormonal endocrine system is that many cells known to synthesize a given peptide hormone or transmitter also synthesize other hormones or trans¬ mitters.18 These may be amines, but often they are genetically and structurally unrelated peptide systems. When assayed using the immunocytochemical methods that predominate (with the inaccuracies inherent in these techniques at the molecular level), this cosynthesis may appear to be haphazard. However, more indepth examinations using more accurate biochemical methods have indicated that some order exists to peptide cosynthesis. For instance, endocrine cells and neurons that produce peptides belonging to the opioid family (products of the proopiomelano¬ cortin, proenkephalin, and prodynorphin genes) often contain peptides belonging to the gastrin-cholecystokinin family. When adrenocorticotropic hormone or opioid peptides, or both, are the main products of the cell, however (as, for example, with cortico¬ tropes and melanotropes), the gastrin-cholecystokinin peptide concentration is low and the peptide processing is directed toward inactive molecular forms.19 Conversely, antral gastrin cells contain low amounts of opioid peptides that occur in forms that differ from the known active peptides. Although the cosyn¬ thesis of peptides in normal cells may seem to be regulated in a

CHAPTER

1 68

ENDOGENOUS OPIOID PEPTIDES BRIAN M. COX AND GREGORY P. MUELLER

CHEMISTRY Three families of endogenous opioid peptides have been identi¬ fied: endorphins, enkephalins, and dynorphins (Figs. 168-1 and 168-2). The simplest of the opioid peptides—methionine and leucine enkephalin (ME and LE, respectively)—were isolated from extracts of porcine brain.1 Subsequently, the ME sequence was recognized to be contained in the 31-amino-acid C-terminal fragment of a previously isolated pituitary peptide, P-lipotropin ((5-LPH). This fragment was shown to have a high affinity for opioid receptors, and it became known as p-endorphin (PEND). Another pituitary opioid, dynorphin A (DYN A), is a C-

Ch. 168: Endogenous Opioid Peptides Enkephalin family 1 5 ME Tyr-Gly-Gly-Phe-Met LE Tyr-Gly-Gly-Phe-Leu

ME-RF ME-RGL

1557

1 5 Tyr-Gly-Gly-Phe-Met-Arg-Phe Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-NH2

(3-endorphin family 1 5 10 15 20 25 30 B-END Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-GIn-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-lle-lle-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu a-END B-END,.,, y-END B-END,.17 Dynorphin family 15 10 15 DYN A Tyr-Gly-Gly-Phe-Leu-Arg-Arg-lle-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-GIn DYN B Tyr-Gly-Gly-Phe-Leu-Arg-Arg-GIn-Phe-Lys-Val-Val-Thr a-neo-END Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-Lys

Endomorphins/Exomorphin* 1 5 Endo-1 Tyr-Pro-Trp-Gly-NH2 B-caso morph in Tyr-Pro-Phe-Pro-Gly-Pro-lle

Endo-2 gluten exorphin A5

1 5 Tyr-Pro-Phe-Phe-NH2 Gly-Tyr-Tyr-Pro-Thr

Nociceptin/orphanin FQ 15 10 15 N/OFQ Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-GIn FIGURE 168-1. Structure of major endogenous opioids and exorphins. (ME, [Met5]enkephalin; ME-RF, [Met5]enkephalyl-Arg-Phe; LE, leucine enkephalin; ME-RGL, [Met5]enkephalyl-Arg-Gly-Leu; END, endorphin; DYN, dynorphin; neo-END, neoendorphin; Endo, endomorphin; N/OFQ, nociceptin/orphanin FQ.) terminal-extended LE.2 At least three different genes direct the synthesis of endogenous opioid peptides: one for the enkepha¬ lins, one for (3-END, and one for dynorphins (see later). These genes are expressed in cells of the nervous, endocrine, and immune systems. Related peptides are found in the hydrolysis products of casein and wheat gluten (see Fig. 168-1). The first four amino acids of the enkephalin sequence (TyrGly-Gly-Phe) represent the minimum sequence for activation of opioid receptors, although this tetrapeptide has weak activity and does not occur naturally in significant concentrations. Cterminal extensions of this peptide, such as Met5 or Leu5 in the enkephalins, Met5-Glu31 in (3-END, and Leu5-Gln17 in DYN A, increase affinity at one or more of the various types of opioid receptor. They provide some specificity in the actions of each peptide. The Tyr1 residue must have a free a-amino group for opioid activity. Thus, N-terminal-extended peptides, as found in precursors, have a negligible affinity for opioid receptors. Many analogs of the naturally occurring peptides have been synthesized. Some of these have increased resistance to degra¬ dation by peptidases and increased specificity for particular opioid-receptor types. After the cloning of three major types of opioid receptor, another mRNA coding for a related receptor-like protein was identified by homology cloning. This receptor, designated ORL-1 (opioid receptor-/ike clone l)3 has high sequence homology with the other opioid receptors but, when expressed, does not bind any of the peptides noted earlier or most opi¬ ate drugs. Screening of brain extracts for peptides that would bind to the expressed ORL-1 receptor yielded a novel peptide with high sequence homology to DYN A, with the important exception that the Tyr1 of DYN A was replaced by a Phe1 residue. This novel peptide was called nociceptin4 and orphanin FQ5 by the two groups that independently isolated the peptide; here it is identified as N/OFQ, because no con¬ sensus name for this peptide yet exists. Subsequent studies have shown that N/OFQ and ORL-1 are normally expressed in the nervous system and elsewhere. N/OFQ has no effect

at the three major types of opioid receptor. Similarly, the enkephalins, dynorphin and B-endorphin do not interact with ORL-1 receptors. The high sequence homology of N/OFQ to DYN A, and of ORL-1 to the K-type opioid receptor, indicate that the N/OFQ and its receptor, ORL-1, have evolved from or in paralled with the other endogenous opioid systems. Functional studies also suggest that N/OFQ is involved in regulation of function in many systems regulated in part by the classic opioid peptides, although the effects of N/OFQ often differ from those of the other opioids.6 N/OFQ may also serve other functions not served by enkephalins or other opioids.

ANATOMIC DISTRIBUTION AS A REFLECTION OF BIOLOGIC FUNCTION The anatomic distribution of endogenous opioid peptides pro¬ vides an important insight into their biologic functions/ The presence of high concentrations of (3-END in the pituitary gland and of enkephalins in the adrenal medulla, and their release into blood, together point to potential endocrine functions of these peptides. In the central nervous system (CNS), endor¬ phin, enkephalin, and dynorphin peptides exist in neuronal systems of arborizing axons and terminal networks with numerous varicosities that resemble classic monoamine neu¬ rons. In vitro studies that have demonstrated the presence of brain opioid receptors and have shown that the release of the peptides is calcium dependent have also provided additional evidence for the role of endogenous opioid peptides as brain neurotransmitters. These peptides are also produced by dis¬ crete, specialized cells in several peripheral organs and, in some cases, are localized in peripheral neurons (e.g., ME in the myen¬ teric plexuses of the gastrointestinal tract). In other instances, such as endorphin peptides in pancreatic D cells, peptides derived from opioid peptide genes in testicular Leydig cells/ and enkephalins in some gastrin-containing cells of the stom-

1558

PART X: DIFFUSE HORMONAL SECRETION 1--p-LPH--1

PREPRO-OPIOMELANOCORTIN |- ACTH —-II- y-LPH -II|r-MSH \ 1

s'8nal

!

A

1

26

|(XMSH|

i-1 A

CLIP —|

1-II

▲ 132

A

77

|— p-MSH -|F p-END (1-27) -| 1-l_ ■1 A A j

l A

▲▲

—|

p-END

173

215

235

265

PREPROENKEPHALIN |— peptide F —| ME ME

ME

AAA

▲ A

■

I 1

ME-RGL

24

97 105

MEt-NHp ■h

A

135

182

PREPRODYNORPHIN

A

LE

ME-RF

ES

m A

► ►

Signal 1

► ►

|

peptide Eh

206

226

|-DYN-32—| a-neo-END 1-1 p-neo-END

h DYN-A HFLeumorphinH DYN-A( 1-8) DYN B

| Signal 1

1 AAA

1

257 263

20

174

A A

209

A A

228

1

256

PREPRONOCICEPTIN/ORPHANIN FQ Nociceptin/Orphanin FQ | Signal 1

1

1

1

^ A

A

20

130

A

146

AA

:

FIGURE 168-2. Comparison of the structures of preproopiomelanocortin, preproenkephalin, preprodynorphin, and prepronociceptin. The N terminus of each peptide is to the left. The positions of paired basic amino-acid residues, signals for cleavage by processing enzymes, are indicated by the black trian¬ gles. The horizontal bars indicate the positions of identified peptides from each precursor. (LPH, lipotropin; ACTH, adrenocorticotropic hormone; END, endorphin; MSH, melanocyte-stimulating hormone; CLIP, corticotropin-like intermediate lobe peptide; ME, [Met5]enkephalin; ME-RGL, [Met']enkephalylArg-Gly-Leu; Met-NH2, metorphamide; LE, [Leu5]enkephalin; ME-RF, [Mebs]enkephalyl-Arg-Phe; neoEND, neoendorphin; DYN, dynorphin.) Numbers refer to amino-acid positions in the precursor peptides.

ach, endogenous opioid peptides are produced by peripheral endocrine cells and presumably have a role in paracrine regula¬ tion. Nociceptin is most highly expressed in the brain and spi¬ nal cord and only weakly in the ovary.7 Anatomical and functional evidence for roles of endogenous opioid peptides is steadily growing. Cells of the immune system express both opi¬ oid peptides and receptors and may, thus, coordinate immune functions via opioid signaling as discussed in greater detail later.

SYNTHESIS AND PROCESSING SYNTHESIS The genes that encode for the endorphin, enkephalin, dynor¬ phin, and N/OFQ precursor proteins have been elucidated, and the mechanisms through which their expression is regulated is under active investigation.8-10 The large, inactive precursors are synthesized on membrane-bound ribosomes and undergo a series of posttranslational modifications en route through the

Golgi apparatus and during maturation in secretory granules. Paired, dibasic residues (Arg-Lys, Lys-Arg, Lys-Lys, or ArgArg) are recognition sites for the action of the trypsin-like pro¬ protein convertase (PC) enzymes that tleave the precursors. These calcium-dependent enzymes constitute a family of five endoproteinases that are expressed in a tissue-specific manner and process a wide variety of secretory precursor proteins, including those of the endogenous opioid peptides.11 Carboxypeptidase E (CPE) trims the C terminals of the peptide interme¬ diates, after which a-amidation may ensue, as occurs for metorphamide. Gene disruption studies and investigation of naturally occurring mutations have clearly demonstrated the essential roles of the PCs and CPE in mediating the formation of peptide messengers. For example, loss of either proprotein con¬ vertase 1 (PCI) or CPE by mutation in humans results in defec¬ tive prohormone processing, elevated plasma concentrations of unprocessed prohormone, hypogonadism, hvpocortisolism, and obesity.12-13 Mutational and knock-out analyses of the amidating enzyme, peptidylglycine a-amidating monooxygenase (PAM)14 have yet to be conducted. Other posttranslational modifications include glycosylation, phosphorylation, and tyrosine sulfation.

Ch. 168: Endogenous Opioid Peptides Both propiomelanocortin (POMC) and proenkephalin A are subject to glycosylation on asparagine residues, and POMC is phosphorylated as well. Although these modifications to the precursors do not show up in the final opioid peptide products, they are necessary for guiding their movement in the secretory pathway.15-16 N-acetylation of (3-END occurs in the pituitary neurointermediate lobe and to a small extent in the brain. Although this modification completely eliminates opioid activ¬ ity, it generates yet another class of chemical messengers having possible actions on immune cells and other targets. With the cloning of the posttranslational processing enzymes (PCI, PC2, PC3, PC4, PC5, CPE, and PAM), the molecular mechanisms that coordinate the expression of opioid peptide precursors with the activity of their respective processing enzymes can now be investigated. Findings indicate that processing enzymes are themselves regulated and may, therefore, represent therapeutic targets for drug actions.15-17

PATTERNS OF PRECURSOR PROCESSING PROOPIOMELANOCORTIN The pattern of precursor processing differs among various cell types.11 In corticotropes of the anterior pituitary, POMC is pro¬ cessed primarily to adrenocorticotropic hormone (ACTH), (3LPH, and y-endorphin (y-END). In the melanotropes of the intermediate lobe, these products are processed further to yield primarily a-melanocyte-stimulating hormone (a-MSH), (3LPH, and shortened and acetylated forms of endorphin that are inactive at opioid receptors. The processing of POMC in the brain resembles a hybrid of the two patterns that occur in the pituitary gland. POMC-ergic neurons mainly express (3-END1_31 and smaller amounts of the shortened and acetylated forms of (3-END, ACTH, and a-MSH. Tissue-specific processing of POMC is rigidly sustained under extreme conditions. For example, intermediate pituitary tumors induced by a POMCsimian virus large T-antigen transgene are faithful in their pro¬ cessing of POMC according to the pattern that exists in normal melanotropes.18 PROENKEPHALIN, PRODYNORPHIN, AND PRONOCICEPTIN Regional differences in the processing of proenkephalin and prodynorphin also exist. The adrenal glands of some species (e.g., bovine) are especially rich in enkephalins and provide a model for studying proenkephalin processing; the posterior pituitary is used in the study of prodynorphin processing. Proenkephalin contains six copies of ME and one of LE; how¬ ever, as predicted by the positioning of paired dibasic amino acids, two of the ME sequences occur in two C-terminalextended forms—ME-Arg-Gly-Leu and ME-Arg-Phe—which are stored and secreted intact. In adrenal glands, ME and LE account for only 20% of the total enkephalin peptides. The remainder consist of the larger, ME-containing peptides: pep¬ tide F, peptide I, peptide E, and peptide B.13 This is in marked contrast to the brain, in which the immunoreactive enkephalins are largely ME, LE, ME-Arg-Phe, and ME-Arg-Gly-Leu. The gene for preprodynorphin encodes for three sequences of LE, multiple forms of dynorphin, and two neoendorphins, but not ME or (3-END. Three LE sequences in prodynorphin form the N termini of a-neoendorphin, DYN A, and dynorphin B (DYN B). In some tissues, these peptides may be processed further to aneoendorphin, DYN Aj_g, or LE. In the brain, levels of LE and DYN Aj_g are considerably higher than those of longer pro¬ dynorphin products. Pronociceptin contains only one copy of N/OFQ in addition to at least one other peptide whose sequence is conserved across rodent and human species.4'5'10 Although potential roles of N/OFQ in pain perception and anx¬ iety are being revealed by genetic studies in mice lacking the nociceptin receptor,19-20 functions of other products of pronoci¬ ceptin remain to be determined.

1559

RECEPTORS FOR ENDOGENOUS OPIOIDS Endogenous opioids serve as neuronal and endocrine cell regu¬ lators by interacting with opioid receptors. Four major types of opioid receptors are found, which have been given the follow¬ ing designations: |i, with preferential affinity for morphine and related drugs; 8, with preferential affinity for ME and LE; k, with preferential affinity for DYN A and related peptides; and ORL-1, highly selective for N/OFQ. The mRNAs for each type of opioid receptor have been cloned and their amino-acid sequences determined.21-22 Opioid-receptor mRNAs have been detected in brain and several peripheral tissues. The size of mRNA forms expressed in different tissues is variable.21 Each receptor is com¬ posed of a single protein chain with seven putative transmem¬ brane domains, placing these receptors in the large group of plasma membrane receptors whose effects are transduced by interactions with guanine nucleotide binding proteins (G pro¬ teins). Overall homology in the amino-acid sequences of |i. 8, k, and ORL-1 receptors is greater than that between opioid recep¬ tors and other G protein-coupled receptors.22 The structural features necessary for binding or activation of each type of opioid receptor have been extensively studied, and subtypes of each of the major forms have been pro¬ posed.23-24 The molecular basis for this apparent heterogeneity remains uncertain, however; the reported heterogeneity in mRNAs for each receptor type does not fully explain the func¬ tional evidence for receptor subtypes, raising the possibility that additional genes for the subtypes await identification. Receptors responding specifically to (3-END (termed e recep¬ tors) have also been proposed,25 but these have not yet been cloned. The nature of proposed receptors in immune cells (mainly lymphocytes) with selectivity for (3-END, (3-LPH, and extended enkephalin-containing products of proenkephalin is also unknown.

OPIOID RECEPTOR-EFFECTOR SYSTEMS Opioid receptors are expressed in the plasma membranes of selected neuronal, endocrine, and immune system cells. Opioidreceptor protein can be observed in intracellular membrane fractions and presumably reflects newly synthesized receptor protein moving to the plasma membrane or internalized recep¬ tors destined for degradation or recycling back to the plasma membrane. Plasma membrane-bound opioid receptors regu¬ late G proteins of the Gt or G0 type. Agonist activation of p, 8, k, or ORL-1 receptors facilitates the dissociation of guanosine diphosphate from the a subunits of the receptor-associated G protein. This allows guanosine triphosphate (GTP) to bind to the a subunit, triggering dissociation of the a subunit from the (3 and y subunits. The liberated a subunit can now interact with effector proteins; as the effector is activated, the G protein a subunit hydrolyzes bound GTP to guanosine diphosphate (GDP). The Py subunit complex may also play a role in opioidreceptor signal transduction. After hydrolysis of GDP, reassoci¬ ation of the a and Py subunits occurs. Thus, GTP is essential for opioid-receptor activation of effector systems.26 Cellular effec¬ tor systems regulated by opioid receptors are listed in Table 168-1. Opening of channels results in membrane hyperpolar¬ ization; closing of N-type Ca2+ channels reduces neurotrans¬ mitter release.26-27 Regulation of adenylate cyclases, activation of L-type Ca2+ channels, or activation of mitogen-activated pro¬ tein kinase may be involved in opioid-mediated regulation of gene expression and cell proliferation in some cell types 28

LONG-TERM OPIOID TREATMENT: TOLERANCE AND DEPENDENCE Long-term treatment with opiate drugs or opioid peptides may cause a state of tolerance; the opioid becomes less effec-

1560

PART X: DIFFUSE HORMONAL SECRETION

TABLE 168-1. Opioid Receptor Transduction Systems Effector System

Opioid-Receptor Type

Effects of Opioid-Receptor Activation

G-Protein Transducer

Adenylate cyclase (type V)52

g, 5, k, and ORL-1

Inhibition

ai

Adenylate cyclase (type II)52

M M

Activation

Py

Phospholipase C53

Activation

a, via Ca2+ influx; possibly Py

MAPK28

g, 8, ORL-1

Activation

Probably Py

K+ channels26

|i, 8. k, and ORL-1

Channel opening; in many cell types

Both a and Py

Ca2+ channels (N-type)26

g, 8, k, and ORL-1

Channel closing; in neurons

Both a and Py

Ca2+ channels (L-type)53

g, 8. K

Channel opening; in neurons and nonneuronal cells

ai, other?

ORL-1, opioid receptor-like clone 1; MAPK, mitogen-activated protein kinase.

tive in inducing a biologic response. In some systems, a state of dependence on the presence of opioid can also arise. In these systems, normal function requires the continued pres¬ ence of opioid agonist. Dependence probably develops as a result of a compensatory increase in the activity of enzymes (e.g., adenylate cyclase) and other regulatory proteins whose activity is depressed during the acute phase of opioid action. Such enhanced activity may result from an increased rate of synthesis of the effector protein to compensate for the level of inhibition induced by the presence of opioid. If the opiate drug is removed, either by discontinuation of administration or by competition for the receptor sites by an antagonist opi¬ ate (e.g., naloxone), the increased level of effector protein results in a hyperactivity of the opioid-regulated pathway, which is manifest as the opiate withdrawal syndrome.26 Enhanced activity of opioid-regulated effector systems also contributes to the tolerance to opioid action that occurs during long-term opiate drug treatment, but other factors may also play a part in tolerance. Long-term opioid treatment reduces the extent of coupling between opioid receptors and the G proteins, thereby mediating their effects and making receptor activation less effective. High concentrations of opioids may also induce a reduction in the number of receptors available to be activated. These adaptive changes in opioid effect, result¬ ing from long-term opioid exposure, affect the activity of endogenous opioid peptides as well as opiate drugs. Receptor

sensitivity may recover quickly after discontinuation of opiate drug exposure, but changes in effector system activity, which may involve altered expression of certain proteins, can persist for days or even weeks.26-27

DISTRIBUTION OF RECEPTORS Opioid receptors of all types are distributed widely throughout the CNS and peripheral autonomic nervous system. Some cor¬ relation is seen between the distribution of receptors and that of endogenous opioid peptides. The correlation in distribution is not perfect, however, because the peptides are often synthe¬ sized in neural cell somata for secretion from axon terminals at a distant site. Considerable differences exist among species in the distribution of both endogenous opioids and their receptors (Table 168-2).

RELATIONSHIP BETWEEN PHYSIOLOGIC FUNCTION AND RECEPTOR TYPE IN THE NERVOUS SYSTEM Endogenous opioids play a modulatory role in the regulation of neural function. Complete blockade of opioid receptors with a nonselective antagonist like naltrexone does not produce a cata¬ strophic disruption of function. A unique association of one endogenous opioid and one opioid-receptor type with a spe¬ cific neural system also seldom is found.

TABLE 168-2. Properties of Receptors for Endogenous Opioids Receptor Type

|i Receptors

8 Receptors

k Receptors

ORL-1 Receptors

SELECTIVE ENDOGENOUS OPIOIDS

Endomorphins 1 and 2; (1casomorphin (low affinity)

Unknown

DYN A; DYN B; aneo-END

N/OFQ

ENDOGENOUS OPIOIDS WITH HIGH, BUT NONSELECTIVE, AFFINITY

P-END; ME; LE; metorphamide; DYN A,_g

P-END; ME; LE; DYN A,_g

DYN A] 8; p-neoEND

None

SELECTIVE OPIATE DRUGS

Morphine

Ethylketocyclazocine*; U69593+

None

ANTAGONISTS (NONSELECTIVE DRUGS APPEAR IN PARENTHESES)

Naloxonet; naltrexone*

ICI 1748641 (nalox¬ onet)

Nor-binaltorphimine (naloxonet)

None

REGULATION OF AGONIST AFFINITY BY Na+ AND GUANOSINE TRIPHOSPHATE

Yes

Yes

Yes (weak)

Yes

STRUCTURES WITH HIGH RECEPTOR CONCENTRATIONS IN CENTRAL NERVOUS SYSTEM

Thalamus; pons-medulla; dorsal horn (spinal cord)

Limbic system; stri¬ atum; globus pallidus

Cortex; cerebellum (some species); dorsal hom (spi¬ nal cord)

Hypothalamus; amygdala; anterior olfactory nucleus; periaqueduc¬ tal gray; brainstem

PERIPHERAL TISSUES

Myenteric plexus (gas¬ trointestinal tract)

Vas deferens (mouse, hamster)

Myenteric plexus; vas deferens (rabbit); placenta (human)

Gastrointestinal tract; vas deferens; liver; spleen

ORL-1, opioid receptor-like clone 1; DYN, dynorphin; N/OFQ, nociceptin/orphanin FQ; neo-END, neoendorphin; END, endorphin; ME, methionine enkephalin; LE, leucine enkephalin. ‘Ethylketocyclazocine shows K activity in pharmacologic assays and is an antagonist at g receptors. *U69593 is a selective agonist at K receptors. The structure is (5a, 7a, 8b)-(-)-N-Met-N-(7-[l-pyrroIidinyl]-l-oxaspiron[4,5j-dec-8-yl)benzenacetamide. •Naloxone and naltrexone have preferential affinity for |i receptors, but at high concentrations they antagonize agonist effects at 6, K, and e receptors. 1ICI 174864 is a selective antagonist at 8 receptors. Its structure is N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (Aib is a-amino isobutyric acid).

Ch. 168: Endogenous Opioid Peptides The roles of specific receptor types or specific opioid pep¬ tide gene products have been evaluated by examining changes in phenotype associated with specific gene deletions (Table 168-3). These studies confirm previous work indicat¬ ing that natural opiates (such as morphine) and synthetic analog drugs (such as methadone or fentanyl) produce anal¬ gesia exclusively through g receptors, the receptor type for which they have highest affinity. Receptors of the g type are located at critical points in the major afferent sensory path¬ way from periphery to brain (e.g., in the dorsal horn of spi¬ nal cord and in several thalamic nuclei), as well as at critical points in descending pain control pathways (e.g., periaque¬ ductal gray matter, ventral medulla), which serve to modu¬ late activity in the afferent pain pathways. Endogenous opioid peptides and receptors of the 8. k, and ORL-1 types are also present at several of these sites, suggesting that endogenous opioids and each of the major opioid-receptor types probably play a physiologic role in the regulation of pain sensitivity.29-30 Endogenous opioids also play a role in neural reinforce¬ ment pathways and motor control mechanisms. Dopamine release in the nigrostriatal, mesolimbic, and mesocortical sys¬ tems is activated (indirectly) by g and 8 agonists, and inhib¬ ited by k agonists acting directly on the dopamine neurons. Cells containing y-aminobutyric acid (GABA) in the regions innervated by these dopamine-containing neurons also carry g and 8 receptors. These systems, in which endogenous opi¬ oids are also present in relatively high concentration, are pri¬ mary locations for opiate-induced reinforcement and modulation of behaviors. Hypothalamic [3-END acts on g receptors to decrease the pulsatile release of gonadotropin-releasing hormone, appar¬ ently by inhibiting nitric oxide-expressing interneurons involved in the activation and release of the hormone.31'32 In the case of stress,33 this mechanism is initiated through the local release of corticotropin-releasing hormone. Endogenous opioids also exert a reciprocal inhibitory tone over the secre¬ tion of corticotropin-releasing hormone; however, this regula¬ tion is complex, involving more than one opioid peptide and receptor subtype.34 Intracerebroventricular administration of N/OFQ exerts potent anxiogenic effects.19 Because this peptide and its recep¬ tor, ORL-1, are present in relatively high concentrations in the amygdala, N/OFQ likely plays a physiologic role in anxiety and fear responses. Mice with deletion of the ORL-1 gene sur¬ prisingly showed enhanced learning abilities relative to con¬ trols (see Table 168-3), suggesting that the anxiogenic actions of endogenous N/OFQ might lead to some impairment of learn¬ ing in complex environments.20

1561

HEMOCRINE AND PARACRINE FUNCTIONS PITUITARY PROOPIOMELANOCORTIN SYNTHESIS AND CONCENTRATIONS Beta-END is cosynthesized and secreted with ACTH and (3LPH from the anterior pituitary. The intermediate lobe, present in most mammals but not in adult humans, contains higher concentrations of total endorphins (together with a-MSH), but 90% of this material consists of shorter, acetylated forms of (3-END that are not active on conventional opioid receptors. In the anterior and intermediate lobes of pituitary, (3-END synthe¬ sis is regulated by different mechanisms (Fig. 168-3).35 Normal circulating levels of peptides derived from POMC are as follows: (3-END, 5 to 20 fmol/mL; (3-LPH, 10 to 35 fmol/ mL. In chronic pain, these values are 230 fmol/mL and 440 fmol/mL, respectively. After adrenalectomy, the levels increase markedly ((3-END, 1700 fmol/mL; (3-LPH, 7600 fmol/mL. PHYSIOLOGIC TARGETS Physiologic targets for the POMC-derived peptides of the pituitary gland include the adrenal gland, melanophores, and, possibly, peripheral nerves carrying pain signals, cells of the gastrointestinal tract, and lymphocytes. Pituitary (3-END and other pituitary peptides may exert some actions directly on the CNS, but firm evidence to support this hypothesis is lack¬ ing. Some evidence suggests that, in addition to ACTH, (3-LPH may also act on the adrenal cortex, enhancing secretion of aldosterone. Primary afferent nociceptor neurons (PAN) respond with increasing firing frequency to intense mechani¬ cal, thermal, or chemical stimuli. These changes in firing rate, which ultimately result in the interneuronal release of sub¬ stance P and the perception of pain, are modulated by endo¬ genous opioid peptides. The p receptors located on peripheral terminals of PAN decrease firing rate through a G proteincoupled mechanism. This response underlies the physiologic means for alteration of the perception of pain by circulating opioid peptides. These peripheral opioid receptors also respond to endogenous opioids secreted by immune cells infiltrating inflamed tissues,36 in addition to those released by the pituitary and adrenal glands. The 8 and k receptor subtypes, present on central terminals of PAN, also reduce the release of substance P from PAN. In contrast, very low doses of N/OFQ, acting at ORL-1 receptors, facilitate release of sub¬ stance P from the peripheral terminals of PAN. Second-order nociresponsive neurons in the dorsal horn express all four opioid-receptor subtypes, allowing endogenous and exoge¬ nous opioids to control directly the flow of information in

TABLE 168-3. Phenotypic Consequences of Deletions of Selected Opioid Peptide or Receptor Genes in Mice Gene Deletion

Phenotype—Physiology

Phenotype—Pharmacology

g Receptor54

Unchanged nociceptive threshold

Loss of morphine analgesia, respiratory depression, and behavioral rein¬

Unchanged levels of 6 and K receptors

Impairment of immune function

forcement (reward)

ORL-1 receptor20

Increased cell proliferation during hematopoiesis

Unchanged analgesic effects of K-receptor agonists

Reduced sperm counts and impaired mating function in males

Reduced analgesic and respiratory depressant effects of some 5-receptor agonists

Unchanged nociceptive threshold and locomotor activity

Loss of inhibition of locomotor activity by N/OFQ

Impaired adaptation to auditory stimuli Facilitation of long-term potentiation and memory

Proenkeph¬ alin55

Unchanged levels of (3-endorphin and dynorphin Unchanged stress-induced analgesic responses Increased anxiety and aggressiveness Hypersensitivity to noxious stimuli

ORL-1, opioid receptor-like clone 1; N/OFQ, nociceptin/orphanin FQ.

(Not reported)

1562

PART X: DIFFUSE HORMONAL SECRETION

ENKEPHALIN AND DYNORPHIN IN THE PARS NERVOSA OF THE PITUITARY Immunocytochemical studies have shown that ME, LE, DYN A, and DYN B are all present in the magnocellular neuronal perikaryon in the supraoptic and paraventricular nuclei of the hypothalamus,42-43 and that they are cosequestered with vaso¬ pressin and oxytocin in the same neurosecretory vesicles of the pars nervosa. These peptides are probably coreleased in response to appropriate stimuli. Parallel changes in the levels of vasopressin and dynorphins in the pars nervosa after dehydra¬ tion or hemorrhage have been reported. The functions of opioids in the pars nervosa are only par¬ tially understood. The K-type receptors are found in this struc¬ ture. Because the molar ratio of vasopressin to DYN A in the rat pars nervosa is >500:1, the primary target of released dynorphin probably is within the pituitary itself. A paracrine role for secretory regulation of the neurohypophysial peptides seems likely, because opioid-induced modulation of vaso¬ pressin and oxytocin secretion from isolated neural lobes of rat pituitary has been reported. Also K-selective agonists induce a profound diuresis,44 whereas the opiate antagonist naloxone potentiates the stimulated release of vasopressin and oxytocin. Thus, evidence exists for opioid regulation of neurohypophys¬ ial peptide secretion. ENDOGENOUS OPIOIDS IN THE ADRENAL MEDULLA

FIGURE 168-3. Pathway for secretion and regulation of pituitary opioid peptides. Brain norepinephrine (NE) and serotonin (5-HT) neurons evoke the release of corticotropin-releasing hormone (CRH) into hypophysial portal blood. In the anterior lobe, CRH stimulates corticotropes to release adrenocorticotropic hormone (ACTH), 13endorphin (/3-END), and P-lipotropin (fi-LPH) into the general circu¬ lation. ACTH enhances the secretion of adrenal cortisol, which, in turn, acts at the level of the hypothalamus to inhibit CRH release and at the level of the pituitary to inhibit CRH action. Release of P-END and melanotrope peptides from the intermediate lobe is stimulated directly by circulating NE and epinephrine (EPI) via a P-adrenergic receptor (/?). By contrast, hypothalamic dopamine (DA) neurons innervate and tonically inhibit melanotrope secretion via a dopamine 2 (D2)-receptor mechanism. (a-MSH, oc-melanocyte-stimulating hor¬ mone; PNS, peripheral nervous system; CNS, central nervous system; Gl, gastrointestinal.)

ascending pain pathways.37 Neuronal release of opioid pep¬ tides also exerts control over nociception at sites within the central nervous system. Lymphocytes express the conventional opiate receptors as well as receptors for both ACTH and P-END.38-40 Speculation is that pituitary ACTH and P-END play a role in marshaling immune responses. The P-END-binding sites recognize the middle and C-terminal regions of P-END. This is in marked contrast to the classic opioid receptors, which require the Nterminal regions of opiate peptides. Lymphocytes express the POMC gene and store y-END. Thus, to consider that lympho¬ cytes themselves may be targets for the endorphin peptides they elaborate is not unreasonable. Endorphin peptides alter several functions of the immune system, including increasing T-cell blastogenesis, natural killer cell cytotoxicity, and tumor resistance. Evidence that the lymphokine interleukin-1 acti¬ vates the release of POMC peptides from the pituitary gland indicates that the interaction between the neural and endocrine systems is bidirectional.39'41 The mRNA for p-type opioid recep¬ tors is also expressed in lymphocytes, and long-term morphine treatment is known to suppress immune system function. Immune cell death induced by Fas (a receptor on the cell sur¬ face that triggers cell death by apoptosis when activated by its ligand, FasL) is promoted by morphine treatment.41

Proenkephalin products are present in relatively high concen¬ trations in the adrenal medulla of most species. In some species, including humans, opioids derived from prodynorphin and POMC are also present. The enkephalins are stored together with catecholamines in secretory vesicles of adrenal chromaffin cells and are secreted into adrenal venous blood after treat¬ ments that stimulate catecholamine secretion.45 Studies have also revealed the presence of opioid receptors in adrenal medulla, a finding that suggests a local mechanism for control¬ ling the release of adrenal catecholamines. Enkephalin-containing peptides are secreted into the circulation after splanchnic nerve stimulation to exert effects in other tissues. Although pentapeptide enkephalins are hydrolyzed very rapidly in circulating blood, a role for enkephalins secreted from the adrenal medulla in modulating the function of circulating cells of the immune system is likely. Finally, the colocalization of enkephalins with catecholamines, as well as the nature of some of the potential target sites for adrenal enkephalins, suggest that they may have a role that is complementary to that of the catecholamines in the physiologic response to stress. ENDOGENOUS OPIOIDS AND THE GASTROINTESTINAL TRACT Both enkephalins and dynorphins are present in the neurons that innervate the gastrointestinal tract.46-47 Enkephalins and dynorphins are present in many myenteric plexus neurons and a few neurons of the submucous plexus throughout the gas¬ trointestinal tract. These neurons innervate sphincters, circular muscle, and longitudinal muscle of the stomach and intestine. The release of dynorphin-like material into the vascular perfu¬ sate of guinea pig intestine during peristalsis has been reported. The presence of |i, 8, and k opioid receptors through¬ out the digestive tract48 suggests that endogenous opioid pep¬ tides act locally in this system. Gastrointestinal motility is substantially modified by opi¬ oids, but the effects often vary widely among species. In humans, opioids increase the tone of sphincters and the fre¬ quency of segmenting, nonpropulsive contractions of circular muscle. This action has been ascribed to the increased local release of serotonin by opioids. A reduction in nitric oxidedependent neurotransmission may also be involved.49 Concom-

Ch. 168: Endogenous Opioid Peptides itantly, the peristaltic reflex is depressed, and the contractions of longitudinal muscle are decreased as a result of a reduction in acetylcholine secretion. Enkephalins reduce secretions, both in the stomach and in the intestine. Thus, the propulsion of gas¬ trointestinal contents is decreased, and water content is reduced. Opioids, therefore, provide symptomatic relief of diar¬ rhea. These actions are all reversed by naloxone. Hydrolysis of the milk protein (3-casein yields a peptide-(3-casomorphin that has some activity at jt receptors.50 In breast-fed neonates, this peptide may be produced locally in sufficient concentrations to reduce gastrointestinal activity.51

REFERENCES 1. Hughes J, Smith TW, Kosterlitz HW, et al. Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature 1975; 258:577. 2. Cox BM. Endogenous opioid peptides: a guide to structures and terminol¬ ogy. Life Sci 1982; 31:1645. 3. Mollereau C, Parmentier M, Mailleux P, et al. ORL-1, a novel member of the opioid receptor family—cloning, functional expression and localiza¬ tion. FEBS Lett 1994; 341:33. 4. Meunier JC, Mollereau C, Toll L, et al. Isolation and structure of the endog¬ enous agonist of opioid receptor-like ORL-1 receptor. Nature 1995; 377:532. 5. Reinscheid RK, Nothacker H-P, Bourson A, et al. Orphanin FQ: a neu¬ ropeptide that activates an opioidlike G protein-coupled receptor. Science 1995; 270:792. 6. Calo G, Guerrini R, Rizzi A, et al. Pharmacology of nociceptin and its receptor: a novel therapeutic target. Br J Pharmacol 2000; 129:1261. 7. Douglass J, Cox BM, Quinn B, et al. Expression of the prodynorphin gene in male and female mammalian reproductive tissues. Endocrinology 1987; 120:707. 8. Herbert E, Seasholtz A, Comb M, et al. Study of the regulation of expres¬ sion of neuropeptide genes by gene transfer methods. In: Meltzer HY, ed. Psychopharmacology: the third generation of progress. New York: Raven Press 1987:373. 9. Leslie FM, Chen Y, Winzer-Serhan UH. Opioid receptor and peptide mRNA expression in proliferative zones of fetal rat central nervous system. Can J Physiol Pharmacol 1998; 76:284. 10. Mollereau C, Simons MJ, Soularue P, et al. Structure, tissue distribution, and chromosomal localization of the prenociceptin gene. Proc Natl Acad Sci USA1996; 93:8666. 11. Steiner DF. The proprotein convertases. Curr Opin Chem Biol 1998; 2:31. 12. Jackson RS, Creemers JW, Ohagi S, et al. Obesity and impaired prohor¬ mone processing associated with mutations in the human prohormone convertase 1 gene. Nat Genet 1997; 16:218. 13. Asteria C. Genetic alterations of enzymes involved in prohormone process¬ ing as common molecular mechanisms of human and rodent obesity. Eur J Endocrinol 1998; 139:150. 14. Prigge ST, Kolhekar AS, Eipper BA, et al. Amidation of bioactive peptides: the structure of peptidylglycine alpha-hydroxylating monooxygenase. Sci¬ ence 1997; 278:1300. 15. Bennett HPJ, Bradbury AF, Huttner WB, Smyth DG. Processing of propep¬ tides: glycosylation, phosphorylation, sulfation, acetylation and amidation. In: Loh YP, ed. Mechanisms of intracellular trafficking and processing of proproteins. Boca Raton, FL: CRC Press, 1993:251. 16. Dickerson IM, Not'd G. Tissue-specific peptide processing. In: Fricker LD, ed. Peptide biosynthesis and processing. Boca Raton, FL: CRC Press 1991:71. 17. Eipper BA, Bloomquist BT, Husten, et al. Peptidylglycine a-amidating monooxygenase and other processing enzymes in the neurointermediate pituitary. Ann N Y Acad Sci 1993; 689:147. 18. Low MJ, Liu B, Hammer GD, et al. Post-translational processing of proopi¬ omelanocortin (POMC) in mouse pituitary melanotrope tumors induced by a POMC-simian virus 40 large T antigen transgene. J Biol Chem 1993; 268:24967. 19. Jenck G, Moreau JL, Martin JR, et al. Orphanin FQ acts as an anxiolytic to attenuate behavioral responses to stress. Proc Natl Acad Sci U S A 1997; 94:14854. 20. Manabe T, Noda Y, Mamiya T, et al. Facilitation of long-term potentiation and memory in mice lacking nociceptin receptors. Nature 1998; 394:577. 21. Evans CF, Keith DE Jr, Morrison H, et al. Cloning of a delta opioid receptor by functional expression. Science 1992; 258:1952. 22. Blake AD, Bot G, Reisine T. Structure-function analysis of the cloned opiate receptors: peptide and small molecule interactions. Chem Biol 1996; 3:967. 23. Jiang Q, Takemori AE, Sultana M, et al. Differential antagonism of opioid delta antinociception by [D-Ala2, Leu5, Cys6]enkephalin and naltrindole 5'isothiocyanate: evidence for delta receptor subtypes. J Pharmacol Exp Ther 1991; 257:1069. 24. Kim K-W, Cox BM. Inhibition of norepinephrine release from rat cortex slices by opioids: differences among agonists in sensitivities to antagonists suggest receptor heterogeneity. J Pharmacol Exp Ther 1993; 267:1153.

1563

25. Schulz R, Wiister M, Herz A. Pharmacological characterization of the eopiate receptors. J Pharmacol Exp Ther 1981; 216:604. 26. Cox BM. Opioid receptor-G protein interactions: acute and chronic effects of opioids. In: Herz A, ed. Handbook of experimental pharmacology, vol 104/1. Opioids I. Berlin: Springer-Verlag, 1993:145. 27. Nestler EJ, Hope BT, Widnell KL. Drug addiction: a model for the molecu¬ lar basis of neural plasticity. Neuron 1993; 11:995. 28. Ignatova EG, Belcheva MM, Bohn LM, et al. Requirement of receptor inter¬ nalization for opioid stimulation of mitogen-activated kinase: biochemical and immunofluorescence confocal microscopic evidence. Neuroscience 1999; 19:56. 29. Fields HL. Brainstem mechanisms of pain modulation: anatomy and phys¬ iology. In: Hertz A, ed. Handbook of experimental pharmacology, Vol 104/ II. Opioids II. Berlin: Springer-Verlag, 1993:3. 30. Aicher SA, Punnoose A, and Goldberg A. Mu-opioid receptors often colo¬ calize with the substance P receptor (NK1) in the trigeminal dorsal horn. J Neurosci 2000; 20:4325. 31. Ferin M. Neuropeptides, the stress response, and the hypothalamic-pitu¬ itary-gonadal axis in the female rhesus monkey. Ann N Y Acad Sci 1993; 697:106. 32. Faletti AG, Mastronardi CA, Lomnuczi A, et al. (3-Endorphin blocks luteinizing hormone releasing hormone release by inhibiting the nitricoxidergic pathway controlling its release. Proc Natl Acad Sci U S A 1999; 96:1722. 33. Rivest S, Plotsky PM, Rivier C. CRF alters the infundibular LHRH secre¬ tory system from the medial preoptic area of female rats: possible involve¬ ment of opioid receptors. Neuroendocrinology 1993; 57:236. 34. Pechnick RN. Effects of opioids on the hypothalamic-pituitary-adrenal axis. Annu Rev Pharmacol Toxicol 1993; 32:353. 35. Vale W, Rivier J, Guillemin R, Rivier C. Effects of purified CRF and other substances on the secretion of beta-endorphin-like immunoreactivities by cultured anterior or neurointermediate pituitary cells. In: Collu R, Barbeau A, Ducharme JR, Rochefort J-G, eds. Central nervous system effects of hypothalamic hormones and other peptides. New York: Raven Press, 1979:163. 36. Schafer M, Carter L, Stein C. Interleukin 1(1 and corticotrophin-releasing factor inhibit pain by releasing opioids from immune cells in inflamed tis¬ sue. Proc Natl Acad Sci U S A1994; 91:4219. 37. Levine JD, Fields HL, Basbaum AL Peptides and the primary afferent noci¬ ceptor. J Neurosci 1993; 13:2273. 38. Peterson PK, Molitor TW, Chao CC. The opioid-cytokine connection. J Neuroimmunol 1998: 83:63. 39. Sapolsky R, Rivier C, Yamamoto G, et al. Interleukin-1 stimulates the secretion of hypothalamic corticotropin-releasing factor. Science 1987; 238:522. 40. Blalock JE. Proopiomelamoncortin and the immune-neuroendocrine con¬ nection. Ann NY Acad Sci 1999: 885:161. 41. Yin D, Mufson RA, Wang R, Shi Y. Fas-mediated cell death promoted by opioids. Nature 1999; 397:218. 42. Martin R, Geis R, Holl R, et al. Co-existence of unrelated peptides in oxyto¬ cin and vasopressin terminals of rat neurohypophyses: immunoreactive methionine5-enkephalin-, leucine5-enkephalin-, and cholecystokinin-like substances. Neuroscience 1983; 8:213. 43. Eriksson M, Ceccatelli S, Uvnas-Moberg K, et al. Expression of Fos-related antigens, oxytocin, dynorphin and galanin in the paraventricular and supraoptic nuclei of lactating rats. Neuroendocrinology 1996; 63:356. 44. Leander JD. A kappa opioid effect: increased urination in the rat. J Pharma¬ col Exp Ther 1983; 224:89. 45. Chaminade M, Foutz AS, Rossier J. Co-release of enkephalins and precur¬ sors with catecholamines from perfused cat adrenal gland in situ. J Physiol 1984; 353:157. 46. Yuferov VP, Culpepper-Morgan JA, La Forge KS, et al. Regional quantita¬ tion of preprodynorphin mRNA in guinea pig gastrointestinal tract. Neurochem Res 1998; 23:505. 47. Kromer W. Endogenous and exogenous opioids in the control of gas¬ trointestinal motility and secretion. Pharmacol Rev 1989; 40:121. 48. Wittert G, Hope P, Pyle D. Tissue distribution of opioid receptor gene expression in the rat. Biochem Biophys Res Commun 1996; 218:877. 49. Bayguinov O, Sanders KM. Regulation of neural responses in the canine pyloric sphincter by opioids. Br J Pharmacol 1993; 108:1024. 50. Brand V, Teschemacher H, Blasig J, et al. Opioid acdvities of beta-casomorphins. Life Sci 1981; 28:1903. 51. Froetschel MA. Bioacdve peptides in digesta that regulate gastrointestinal function and intake. J Anim Sci 1996; 74:2500. 52. Avidor-Reiss T, Nevo I, Saya D, et al. Opiate-induced adenylyl cyclase superactivadon is isozyme-specific. J Biol Chem 1997; 272:5040. 53. Smart D, Hirst RA, Hirota K, et al. The effects of recombinant rat p-opioid receptor acdvadon in CHO cells on phospholipase C, [Ca2+]i and adenylyl cyclase. Br J Pharmacol 1997; 120:1165. 54. Matthes HWD, Maldonaldo R, Simonin F, et al. Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the popioid-receptor gene. Nature 1996; 383:819. 55. Konig M, Zimmer AM, Steiner H, et al. Pain responses, anxiety and aggres¬ sion in mice deficient in pre-proenkephalin. Nature 1996; 383:535.

1564

PART X: DIFFUSE HORMONAL SECRETION

CHAPTER

169

SOMATOSTATIN YOGESH C. PATEL Somatostatin (SST) was first found in the mammalian hypothal¬ amus as a tetradecapeptide (SST-14), which inhibited the release of growth hormone (GH). It has since come to be known as a multifunctional hormone that is also produced throughout the central nervous system and in most peripheral organs. Somatostatin acts on a diverse array of endocrine, exocrine, neuronal, and immune cell targets to inhibit secretion, to modu¬ late neurotransmission, and to regulate cell growth. These actions are mediated by a family of G protein-coupled recep¬ tors with five distinct subtypes (termed SSTR1 through SSTR5). SST is best regarded as an endogenous inhibitory regulator of the secretory and proliferative responses of many different tar¬ get cells. In addition, the peptide may be of importance in the pathophysiology of several diseases such as neoplasia, inflam¬ mation, Alzheimer and Huntington diseases, and acquired immunodeficiency syndrome (AIDS), and has found a number of clinical applications in the diagnosis and treatment of neu¬ roendocrine tumors and various gastrointestinal disorders.1-8

SOMATOSTATIN GENES AND GENE PRODUCTS Like other protein hormones, somatostatin is synthesized as part of a large precursor protein (proSST) that is processed to generate two bioactive forms, SST-14 and SST-28 (Fig. 169-1). In humans only one somatostatin gene is found, located on the long arm of chromosome 3, which encodes for both SST-14 and SST-28, whereas lower vertebrates (e.g., fish) have two somato¬ statin genes that separately encode for SST-14 or SST-28.2-4'8 The

SST-28

Se r-Ala -As n -Se r-As n-Pro -Ala -Me t-Ala -Pro -Arg Glu-Arg-Lys-Ala-GLy-Cys-Lys-Asn-Phe-Phe—--|-rp Cys-Ser-Thr-Phe-Thr —Lys

SST-14

Ala-Gly-Cys-Lys-Asn-Phe-Phe— jrD l

.

Cys-Ser-Thr-Phe-Thr— Lys

CST-17

Asp-Arg-Met-Pro-Cys-Arg-Asn-Phe-Phe— -|-rp Lys-Cys-Ser-Ser-Phe-Thr— Lys

SMS 201-995 octreotide

Dphe-Cys-Phe — DTrp Thr(ol)-Cys-Thr —*-ys

BIM23014 lanreotide

DpNal-Cys-Tyr-— DTrp | . Thr-Cys-Val — y

FIGURE 169-1. Structure of naturally occurring somatostatin peptides. (SST-28, somatostatin-28; SST-14, somatostatin-14; CST-17, human cortistatin-17.) Amino-acid residues necessary for bioactivity are shown in bold. Octreotide and lanreotide are synthetic somatostatin analogs.

FIGURE 169-2. Schematic depiction of the rat somatostatin gene and its regulatory domains. The mRNA coding region consists of two exons of 238 and 367 base pairs (bp) separated by an intron of 621 bp. Located upstream (i.e., 5' end) from the start site of mRNA transcription (arrow) are the regulatory elements TATA, cyclic adenosine monophosphate response element (CRE), atypical glucocorticoid response element (aGRE), and somatostatin promoter silence element (SMS-PS). Tissuespecific elements (TSE) consisting of TATA motifs that operate in con¬ cert with CRE to provide high-level constitutive activity are shown. (3'UT, 3' untranslated region; SST-14, somatostatin-14; SST-28, somato¬ statin-28.) transcriptional unit of the rat SST gene consists of exons of 238 and 367 base pairs (bp) separated by an intron of 621 bp (Fig. 169-2). The 5'-upstream region contains a number of regulatory elements for tissue-specific and extracellular signals, including a cyclic adenosine monophosphate (cAMP) response element (CRE) and two nonconsensus glucocorticoid response elements (GREs).2-4 The SST-14 sequence has been totally conserved throughout vertebrate evolution, whereas the amino-acid struc¬ ture of SST-28 has changed -30% during evolution from fish to humans.2-4-8 A novel second SST-like gene, cortistatin (CST), which has been described in humans, yields two cleavage prod¬ ucts, CST-17 and CST-29, which are comparable to SST-14 and SST-28 (see Fig. 169-1).9 The CST peptides interact with all five SSTRs, but unlike somatostatin, expression of cortistatin is restricted to the cerebral cortex and its biofunction(s) remains unknown.3-9

ANATOMIC DISTRIBUTION OF SOMATOSTATIN CELLS Somatostatin-producing cells occur in high densities through¬ out the central and peripheral nervous systems, and in the endocrine pancreas and gut. They occur in smaller numbers in the thyroid, adrenal medulla, testes, prostate, submandibular gland, kidneys, and placenta1-8-10 (Table 169-1). The typical mor¬ phologic appearance of an SST cell is that of a neuron with mul¬ tiple branching processes, or of a secretory cell, often with short cytoplasmic extensions (D cells). In the brain, the highest con¬ centrations of SST are found in the hypothalamus, neocortex, and basal ganglia, throughout the limbic system, and at all lev¬ els of the major sensory systems.1-8-10-11 The approximate rela¬ tive amounts of SST in the major regions of the brain are as follows: cerebral cortex, 49%; spinal cord, 30%; brainstem, 12%; hypothalamus, 7%; olfactory lobe, 1%; an*! cerebellum, 1%.” SST cells in the pancreas are almost exclusively islet D cells and account for 2% to 3% of the total adult islet cell popula¬ tion.12 Gut SST cells are of two types: D cells in the mucosa and neurons that are intrinsic to the submucous and myenteric plexuses.13 In the thyroid, SST coexists with calcitonin in a sub¬ population of C cells.1 In addition to these typical SST-producing neuroendocrine cells, which secrete large amounts of the pep¬ tide from storage pools, inflammatory and immune cells also produce SST, usually in small amounts on activation.14-15 In the rat, the gut accounts for -65% of total body SST, whereas lesser amounts occur in the brain (25%), the pancreas (5%), and the remaining organs (5%). The relative proportions of SST-14 and SST-28 synthesized and secreted vary considerably in different tissues.4 SST-14 is the predominant form in the brain, pancreas, upper gut, and enteric neurons, whereas SST-28 is an important constituent of brain and is the predominant molecular form in the intestinal mucosa.

Ch. 169: Somatostatin TABLE 169-1. Localization of Somatostatin Body Region

Type of Cells

MAJOR SITES Nervous system

Neurons

Locale

Hypothalamus Cerebral cortex Limbic system Basal ganglia Major sensory systems Spinal cord Dorsal root ganglia Autonomic ganglia

Pancreas Gut MINOR SITES Adrenal Placenta Reproductive organs Submandibular gland Thyroid Urinary system

D cells

Islets

D cells

Mucosal glands

Neurons

Submucous and myenteric plexuses

—

Scattered medullary cells

—

Cytotrophoblasts in chorionic villi

—

Testis, epididymis, prostate

D cells

Scattered ductal cells

C cells

Scattered parafollicular cells (coex¬ isting with calcitonin) Scattered cells in renal glomerulus and collecting ducts

1565

quately investigated.4-8 Glucocorticoids exert a dose-dependent biphasic effect on SST secretion; low doses are stimulatory and high doses are inhibitory.4 Insulin stimulates hypothalamic SST release but has an inhibitory effect on the release of SST from islet and gut4 Finally, members of the growth factor-cytokine family such as GH, insulin-like growth factor-I (IGF-I), interleukin-1 (IL1), tumor necrosis factor-a (TNF-a), and interleukin-6 (IL-6) are capable of stimulating SST secretion from brain cells.2 Many of the agents that influence SST secretion also regulate SST gene expression.2-4-8 Steady-state SST mRNA levels are stimu¬ lated by growth factors and cytokines (e.g., GH, IGF-I, IGF-II, ILI, TNF-a, IL-6, interferon-y, and interleukin-10), glucocorticoids, testosterone, estradiol, and N-methyl-D-aspartate-receptor ago¬ nists; and are inhibited by insulin, leptin, and transforming growth factor-(i (TGF-[I). Among the intracellular mediators known to modulate SST gene expression are Ca2+, cAMP, cyclic guanosine monophosphate (cGMP), and nitric oxide (NO).2-4-8 Activation of the adenylate cyclase-cAMP pathway plays an important role in the stimulation of SST secretion and gene transcription.20 Cyclic AMP-dependent transcriptional enhance¬ ment is mediated by the nuclear protein cAMP response element¬ binding protein (CREB), which binds to the cAMP response element on the SST gene.20 Ca2+-dependent induction of the SST gene occurs through phosphorylation of CREB by the Ca2+dependent protein kinase I and protein kinase II. GH, IGF-I, IGFII, and glucocorticoids have all been shown to induce the SST gene by direct interaction with its promoter.4 The molecular mechanisms underlying the effects of estrogens, testosterone, cGMP, and NO on SST mRNA levels remain to be determined 4

SOMATOSTATIN IN THE PLASMA AND OTHER BODY FLUIDS ACTIONS OF SOMATOSTATIN Both SST-14 and SST-28 are released readily from tissues and are detected in blood.1'4-816 The main source of circulating SST is the gastrointestinal tract.17 Circulating SST is inactivated rapidly by the Uver and kidneys. The plasma half-life of SST-14 is 2 to 3 minutes, whereas that of SST-28 is slightly longer.4 Fasting plasma concen¬ trations of SST-like immunoreactivity (SST-LI) range from 5 to 18 pmol/L. These levels double in response to the ingestion of a mixed meal4 The bioactive circulating forms consist of SST-14, desAla1 SST-14 (a postsecretory conversion product of SST-14), and SST-28.16 With few exceptions, fluctuations in peripheral plasma levels of SST-LI are small. The main clinical utility of plasma mea¬ surements is in the diagnosis of SST-producing tumors, which are associated with marked hypersomatostatinemia. SST is secreted into the cerebrospinal fluid (CSF), probably from all parts of the brain.18-19 It is stable in this medium and attains a concentration that is approximately twice that in the general circulation. Significant amounts are also excreted in the urine (4-6 pmol/L). Semen contains high levels of SST-LI, 200fold greater than those in plasma. Amniotic fluid is rich in SSTLI originating from the fetus.

REGULATION OF SOMATOSTATIN SECRETION AND GENE EXPRESSION Because SST cells are so widely distributed and interact with many different body systems, the fact that the secretion of SST can be influenced by a broad array of secretagogues, ranging from ions and nutrients to neuropeptides, neurotransmitters, classic hormones, and growth factors, is not surprising.1-2-4-8 Gluca¬ gon, GH-releasing hormone (GHRH), neurotensin, corticotropin¬ releasing hormone (CRH), calcitonin gene-related peptide (CGRP), and bombesin are potent stimulators of SST release, whereas opioids and y-aminobutyric acid (GABA) generally inhibit SST secretion.1-4-8 Of the various hormones studied, thy¬ roid hormones enhance SST secretion from the hypothalamus; their effect on secretion from other tissues has not been ade¬

SST not only has wide anatomic distribution, but also acts on multiple targets, including the brain, pituitary, endocrine and exocrine pancreas, gut, kidney, adrenal, thyroid, and immune cells1-2-7-8 (Fig. 169-3). Its actions include inhibition of virtually every known endocrine and exocrine secretion, and of various neurotransmitters; behavioral and autonomic effects if centrally administered; and effects on gastrointestinal and biliary motility, vascular smooth muscle tone, and intestinal absorption of nutri¬ ents and ions. SST also blocks the release of growth factors (e.g., IGF-I, epidermal growth factor [EGF], and platelet-derived growth factor [PDGF]) and cytokines (e.g., IL-6, interferon-y), and inhibits the proliferation of lymphocytes and of inflamma¬ tory, intestinal mucosal, and cartilage and bone precursor cells.2 All of these diverse effects of SST can be explained by its inhibi¬ tion of two key cellular processes, secretion and cell proliferation.

SOMATOSTATIN RECEPTORS, RECEPTOR SUBTYPES, AND SIGNAL TRANSDUCTION Somatostatin acts through high-affinity plasma membrane receptors that are pharmacologically heterogeneous and feature several different isoforms.2-3-5-6-21 Molecular cloning has revealed a family of five structurally related SSTR subtype genes that encode for seven transmembrane domain, G protein-coupled receptor proteins that display distinct agonist-binding profiles for natural and synthetic SST peptides2-3-21 (Table 169-2). Recep¬ tor types 1 through 4 bind SST-14 and SST-28 approximately equally, whereas the type 5 receptor displays relative selectivity for binding of SST-28.2-3-21 Four of the genes (the exception is SSTR2) appear to have no introns. Each of the receptor genes is located on a separate chromosome. The mRNA for individual human SSTR subtypes is widely expressed in brain, pituitary, pancreatic islets, stomach, jejunum, colon, lung, kidney, and liver, with a characteristic tissue-specific pattern for each receptor.2-3-21'22 Typically, more than one subtype occurs in a given tar-

1566

PART X: DIFFUSE HORMONAL SECRETION

Excitation ol many neurons General arousal Hyperkinesia Rigidity Catalepsy Autonomic effects

TRH I CRH I Norepinephrine I Somatostatin ) GHI TSH I ACTH ) Insulin I Glucagon) Somatostatin) Enzymes) Bicarbonates t

SOMATOSTATIN

& Aldosterone) Catecholamine)

Renin ) Water absorption)

TABLE 169-2. Characteristics of Cloned Human Somatostatin Receptor (hSSTR), Types 1-5 hSSTRl

hSSTR2

hSSTR3

hSSTR4

hSSTR5

Amino adds

391

369

418

388

363

Chromosomal location

14

17

22

20

16

-

Agonist binding’ SST-14

++

++

++

++

++

SST-28

++

++

++

++

+++

Octreotide

-

++

+

-

++

Lanreotide

-

++

+

-

++

Yes

Yes

Yes

Yes

Yes

Adenylate cyclase activity

4

4

4

4

4

Tyrosine phos¬ phatase activity

T

t

T

T

T

MAPK activity

T

4

4T

T

4

Brain

Yes

Yes

Yes

Yes

Pituitary

Yes

Yes

Yes

Islet

Yes

Yes

Yes

Yes

Yes

Stomach

Yes

Yes

Yes

Yes

Yes

Liver

Yes

G-protein coupling Effector coupling

Gastrin * Secretin) CCK * VIP) Motilin) Neurotensin) Gastric acid) Pepsin) Blood flow) Motility) Nutrient, ion absorption) Mucosal proliferation)

FIGURE 169-3. Principal actions of somatostatin. Somatostatin inhibits the release of dopamine from the midbrain and of norepinephrine, thyrotropin-releasing hormone (TRH), corticotropin-releasing hormone (CRH), and endogenous somatostatin from the hypothalamus. It also inhibits both the basal and the stimulated secretion of growth hormone (GH), thyroid-stimulating hormone (TSH), and islet hormones. It has no effect on luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, or adrenocorticotropic hormone (ACTH) in normal subjects. It does, however, suppress elevated ACTH levels in Addison disease and in ACTH-producing tumors. In addition, it inhibits the basal and the TRHstimulated release of prolactin in vitro and diminishes elevated prolactin levels in acromegaly. In the gastrointestinal tract, somatostatin inhibits the release of virtually every gut hormone that has been tested. It has a gener¬ alized inhibitory effect on gut exocrine secretion (gastric acid, pepsin, bile, colonic fluid) and suppresses motor activity generally, through inhibition of gastric emptying, gallbladder contraction, and small intestine segmen¬ tation. Somatostatin, however, stimulates migrating motor complex activ¬ ity. The effects of somatostatin on the thyroid include inhibition of the TSH-stimulated release of thyroxine (T4) and triiodothyronine (T3). The adrenal effects consist of the inhibition of angiotensin II-stimulated aldos¬ terone secretion and the inhibition of acetylcholine-stimulated medullary catecholamine secretion. In the kidneys, somatostatin inhibits the release of renin stimulated by hypovolemia and inhibits antidiuretic hormone (ADH)-mediated water absorption. (CCK, cholecystokinin; VIP, vasoac¬ tive intestinal peptide.) (Modified from Patel YC. General aspects of the biology and function of somatostatin. In: Weil C, Muller EE, Thomer MO, eds. Somatostatin. Basic and clinical aspects of neuroscience series, vol 4. Berlin: Springer-Verlag, 1992:1.)

get tissue (e.g., SSTR1 through SSTR5 in the brain, stomach, pancreatic islets, and aorta; SSTR1, SSTR2, SSTR3, and SSTR5 in the pituitary). SSTR2 is the most abundantly expressed subtype, in terms of both the number of tissues that express this receptor and the level of expression. It is preferentially expressed by islet A cells and immune cells. SSTR1 and SSTR5 are the main subtypes expressed by islet B cells.23 SSTR2 and SSTR5 are the prin¬ cipal subtypes found in somatotropes. SST receptors elicit their cellular responses through G proteinlinked modulation of multiple second messenger systems (Fig. 169-4), including (a) receptor coupling to adenylate cyclase, (b) receptor coupling to K+ channels, (c) receptor coupling to Ca2+ channels, (d) receptor coupling to exocytotic vesicles, (e) receptor

Tissue distribution+

Lungs Kidneys Placenta

Yes Yes

Yes Yes

Yes Yes

t, increased; 4, decreased; SST-14, somatostatin-14; SST-28, somatostatin-28; MAPK, mitogen-activated protein kinase. ’Binding potency shown is based on the concentration required for half maximal inhibition of binding (IC50) value for each agonist: -, IC50 150-1000 nM; +, IC^ 10-20 nM; ++, ICjq 1—10 nM; +++, IC-q IGF-II > insulin

IGF-II receptor

IGF-II » IGF-I > insulin

Insulin receptor

Insulin » IGF-II > IGF-I

Hybrid receptor

IGF-I » insulin

IGF, insulin-like growth factor.

1 590

PART X: DIFFUSE HORMONAL SECRETION

brane (3 subunits by disulfide bonds that are formed after pro¬ teolytic cleavage of the proreceptor into a and (3 subunits in the Golgi apparatus. The (3 subunit consists of a short extracellular domain, a transmembrane region, a cytoplasmic juxtamembrane region in which tyrosine-containing motifs important for receptor internalization and interaction with endogenous sub¬ strates occur, a tyrosine kinase domain that includes an adeno¬ sine triphosphate (ATP)-binding motif, a cluster of tyrosine residues subject to autophosphorylation, and a C-terminal domain containing several tyrosine residues that are also autophosphorylated following activation of the receptor. The tyro¬ sine kinase domain is the most highly conserved (~87%), while the C-terminal domain is the least conserved between the IGF-I and insulin receptors. The IGF-I and insulin receptor are mem¬ bers of a receptor tyrosine kinase family that includes the epi¬ dermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors, but they are atypical in that they occur in a heterotetrameric structure and are considered to be "pre¬ dimerized," unlike the other receptors of this family that require ligand binding for dimerization and activation (Fig. 173-16). The IGF-II/mannose-6-phosphate receptor is unrelated struc¬ turally to the IGF-I and insulin receptors, because it contains a long extracellular domain consisting of numerous repeats of a short amino-acid sequence, a transmembrane domain, and a short cyto¬ plasmic tail.9 The extracellular domain contains binding sites for IGF-II and mannose-6-phosphate, and ligand binding at one site influences ligand binding at the other. This receptor is important in the uptake and intracellular trafficking of mannose-6-phosphate-containing lysosomal enzymes and may play a role in the clearance of IGF-II from the circulation. The cytoplasmic tail lacks any obvious catalytic activity or classic binding sites for known endogenous substrates and—in the absence of any convincing evi¬ dence to the contrary—has generally been considered to lack sig¬ naling properties. Proteolytic cleavage of the extracellular domain releases a circulating form of the receptor, which may then act as a binding protein for IGF-II. Since the IGF-I receptor is considered to mediate the actions of the IGFs, functional aspects of this receptor are dealt with in more detail in the following section. INSULIN-LIKE GROWTH FACTOR-1 RECEPTOR The IGF-I receptor gene is expressed ubiquitously in most tis¬ sues.10 The gene contains 21 exons, and two mRNA species can be found in human tissues, one of ~11 kilobases (kb) and a second of ~7 kb.11 The mRNA contains an ~l-kb 5' untranslated (UT) region, a coding region of ~5 kb, and a 3' UT region of ~5 kb. Both the 5' UT and 5' flanking regions of the gene are enriched in GC sequences, and the expression is regulated by SP1 and WT1 transcription fac¬ tors that bind specifically to these GC-rich boxes in the promoter region.12 This region is also responsible for the regulation of the IGF-I receptor gene by growth factors.13-14 EGF, PDGF, and bFGF up-regulate gene expression, whereas IGF-I down-regulates its own receptor at the transcriptional level.15-16 The 3' UT region most likely is involved in the stability of the mRNA. The sequence of events initiated by ligand binding to receptor tyrosine kinases has been elucidated for a number of these recep¬ tors.17 In the case of the IGF-I receptor, ligand binding to the a subunit alters the conformation of the extracellular and transmem¬ brane portions of the receptor, leading to a change in the cytoplas¬ mic domain, which in turn results in activation of the tyrosine kinase and phosphorylation of a cluster of three tyrosine residues in the kinase domain itself.18-19 Subsequently, tyrosines in the juxtamembrane and C-terminal domains are phosphorylated along with other substrates.20 The insulin receptor substrate (IRS) family of proteins, of which four have been characterized together with the protein She, represent the most well studied of the substrates involved in IGF-I and insulin-receptor signal transduction.21 Their primary site of interaction is the phosphorylated tyrosine at resi¬ due 960 within the juxtamembrane domain of the IGF-I receptor. IGF-I-receptor activation results in tyrosine phosphorylation of

IRS (1-4) and She.22-24 Phosphorylated tyrosine residues on the IRS molecules are found in consensus sequences for the binding of Src homology domain (SFI2) domains of several proteins of the signal transduction pathways.25 These include the p85 subunit of phosphoinositide-3'kinase (PI3’K), growth factor receptor bound protein-2 (Grb-2), the tyrosine phosphatase Syp, Crk adapter pro¬ teins, and Nek26 (Fig. 173-2). PI3'K activation leads to pathways involved in preventing apoptosis (via protein kinase B/Akt), sig¬ naling to focal adhesion sites and even mitogenesis.27 Grb-2 is an adapter protein that interacts via its SH2 domain with both tyrosine phosphorylated IRS and She.28 Bound Grb-2 then inter¬ acts via its SH3 domain with the mammalian homologue of sonof-sevenless (mSOS), which contains the appropriate proline-rich region required for SH3 domain interaction.29-30 This interaction recruits mSOS to the plasma membrane where mSOS, a guanine nucleotide exchange factor, activates p21ras by facilitating the dis¬ placement of guanine diphosphate (GDP) from the guanine nucle¬ otide-binding site of p21ras and its replacement with guanine triphosphate (GTP). GTP-p21ras interacts with raf proteins and thereby activates the mitogen-activated protein (MAP) kinase/Erk pathway. The final result is the transport of active molecules into the nucleus and enhancement of specific gene transcription, lead¬ ing to increased mitogenic signals and cellular proliferation. These major pathways are the ones that have been most clearly charac¬ terized for IGF-I-receptor signaling. Additionally, a host of other substrates and pathways have been delineated. The Crk family of adapter proteins, CrkI and II and CrkL, while lacking catalytic activity, play important roles in the signal trans¬ duction pathways of growth factor receptors including the IGF-I receptor.31-32 IGF-I-receptor activation results in tyrosine phospho¬ rylation of both CrkH and CrkL, which probably occurs by the association of these molecules with members of the IRS family of proteins. Crkll is involved in the differentiated function of the IGFI receptor, whereas CrkL results in the more transforming pheno¬ type 33-35 Examples of recently described substrates involved in IGF-I receptor-signaling cascades include the vav protooncogene in hematopoietic cells, two isoforms of the 14-3-3 family of pro¬ teins that interact directly with the IGF-I receptor, protein kinase C isoforms (i.e., PKC a), the G protein G(3y, focal adhesion kinase, Janus kinase 1 (JAK1), signal transducers and activators of tran¬ scription (Stat) 3, and suppressor of cytokine signaling (SOCS-2). IGF-I-receptor activation also results in enhanced expression of a large number of genes. These include z>ascular endothelial growth /actor (VEGF), elastin, myogenin, cyclin Dl, c-myc, c-fos, c-jun, tubulin, and neurofilament. The wide array of genes affected by IGF-I-receptor signaling attests to the pleiotropic responses to this receptor, which range from cell-cycle progression and cellular pro¬ liferation to differentiated functions in specialized tissues.

INSULIN-LIKE GROWTH FACTOR-BINDING PROTEINS A third, yet important, component of the IGF system is the fam¬ ily of IGF-binding proteins (IGFBPs).36-37 IGFBPs-1 through -6 are encoded by a gene family and are characterized by cysteinerich N and C termini with less-conserved central regions.38 IGFBPs are produced by a variety of biologic tissues and are found in serum and other biologic fluids.39 Some of the general characteristics are shown in Table 173-3.40 REGULATION The production of IGFBPs is regulated by several factors. All of the IGFBPs are developmentally regulated from fetal levels through the aging process. IGFBP-1 gene expression and circulat¬ ing levels are regulated by nutritional status, insulin, and gluco¬ corticoids. GH, on the other hand, increases the levels of both IGFBP-3 and the acid-labile subunit (ALS), both of which form the major serum complex that binds the majority of circulating IGFs. The effect of GH on IGFBP-3 is probably mediated by IGF-I

Ch. 173: Growth Factors and Cytokines

1591

IGF-IR

+ # iiiMiiiiMiMMiiiimiiiiiiiiiMiiiiiMiiiiiiiiiiiiiiiiiiiiimmiiiii. IMIIIIIIIII|Mi»

40-

20-

# 5

0J

FIGURE 178-7. Venous plasma atrial natriuretic peptide (ANP) levels (filled circles) and concurrent brain natriuretic peptide (BNP) levels (open circles) in normal subjects and patients with circulatory disorders (mean ± standard error of the mean; n = number of subjects in each group). All samples were assayed in the same laboratory using previ¬ ously published techniques. Hypertensive patients showed no evidence of significant end-organ disease. Patients with acute (uncomplicated) myocardial infarction (MI) had blood drawn within 24 hours of admission. Heart failure was of recent onset. New York Heart Associa¬ tion functional class II-IV. In patients with chronic renal failure, blood was drawn before and after acute volume depletion by ultrafiltration. (Data for hypertensive patients from Richards AM, Crozier IG, Espiner EA, et al. Plasma brain natriuretic peptide and endopeptidase 24.11 inhibition in hypertension. Hypertension 1993; 22:231; and from Pidgeon BG, Richards AM, Nicholls MG, et al. Differing metabolism and bioactivity of atrial and brain natriuretic peptides in essential hypertension. Hypertension 1996; 27:906. Data for patients with acute MI from Foy SG, Crozier IG, Richards AM, et al. Neurohormonal changes after acute myocardial infarction: relationships with haemo¬ dynamic indices and effects of ACE inhibition. Eur Heart J 1995; 16:770. Data for normal patients and those with heart failure from Yandle TG, Richards AM, Gilbert A, et al. Assay of brain natriuretic pep¬ tide [BNP] in human plasma: evidence for high molecular weight BNP as a major plasma component in heart failure. J Clin Endocrinol Metab 1993 76:832. Data for patients with chronic renal failure from Corboy JC, Walker RJ, Simmonds MB, et al. Plasma natriuretic peptides and cardiac volume during acute changes in intravascular volume in hae¬ modialysis patients. Clin Sci [Colch] 1994 87[6]:679.)

and BNP levels occur in hypertension (see Fig. 178-7) and corre¬ late with left ventricular hypertrophy.101 Consistent with the effect of atrial or ventricular pacing in experimental heart fail¬ ure, a variety of tachyarrhythmias may markedly increase plasma hormone levels (ANP > BNP in humans).142 Rapid supraventricular tachycardia may initiate a polyuric syndrome with natriuresis, as described 20 years before the discovery of the natriuretic peptides.143 The possibility that increases in cir¬ culating natriuretic peptides may underlie syndromes of cere¬ bral salt wasting144 has received support from studies of patients with subarachnoid hemorrhage.145 Although one study reports a uniquely increased secretion of BNP (ten-fold the basal level, with ANP unchanged),145 comparable studies in the authors' laboratory show that both ANP and BNP are similarly increased after the acute insult. Further work is required to determine the mechanism of this increase and its significance in the pathogenesis of hyponatremic syndromes accompanying acute brain injury. Thus, altered levels of circulating hormones, ANP and BNP, are largely secondary to changes in the intracardiac pressure and/or altered myocardial work. Few, if any, reports have been published of primary disorders of natriuretic peptide secretion or action that resulted in disease, with the possible exception of cases of

familial open-angle glaucoma.146 The findings in transgenic ani¬ mal studies111,133 134 suggest that, conceivably, polymorphism in the NPR-A or other genes may contribute to some forms of essential hypertension147-1473 or predispose to the onset of early heart failure after myocardial injury in humans. Excessive pro¬ duction of ANP has been reported148; however, proof of a gradi¬ ent across the tumor and restoration of normal levels after resection of the tumor has yet to be shown. Even if plasma hor¬ mone assays have yet to prove themselves in the diagnosis of primary disorders of blood pressure or volume status, a large body of evidence now supports their use as markers of cardiac function. For example, raised levels of ANP or BNP, or their Nterminal peptides (which have slower clearance rates and, hence, higher plasma concentrations) are indicators of symp¬ tomless left ventricular dysfunction149-150 and predict subsequent deterioration in hemodynamic function or the development of frank heart failure.151-152 In patients presenting with acute dysp¬ nea, raised plasma concentrations of BNP give strong support to a diagnosis of cardiac failure, as opposed to primary lung disease,153 whereas in patients with essential hypertension, elevated levels of circulating natriuretic peptides may reflect increased left ventricular mass.154 The prognosis for patients who experience acute myocardial infarction,152-155-156 or those with established heart failure,157 is also predicted by levels of the cardiac natriuretic peptides. BNP in particular (due to its predominantly ventricular origin, more rapid induction, and slower clearance rate), and its N-terminal fragment (NTBNP),158 may be especially useful in clinical diagnosis and in determining prognosis, as these hormones correlate better with severity and indicators of cardiac dysfunction than do ANP and NT-ANP (Fig. 178-8).25-44-156 Thus, a relatively cheap and rapid blood test may ulti¬ mately be a guide for the introduction or intensification of cardiac treatment. Further, patients with possible ventricular dysfunc¬ tion may be screened using plasma BNP assays, permitting selection of patients for more detailed (and costly) investiga-

FIGURE 178-8. Kaplan-Meier survival curves for 121 patients with myocardial infarction. Patients were divided into two subgroups with early postinfarction plasma natriuretic peptide concentrations (atrial natriuretic peptide [ANP] and its N-terminal fragment [N-ANP], brain natriuretic peptide [BNP] and its N-terminal fragment [N-BNP]) that were above (solid line) and below (dashed line) the group median (med). Venous blood was drawn between 24 and 96 hours after the onset of symptoms. Inclusion criteria were age younger than 80 years of age, absence of cardiogenic shock, and survival for at least 24 hours after myocardial infarction, p Values refer to significance values for the differ¬ ences between the two groups. (From Richards AM, Nicholls MG, Yandle TG, et al. Plasma N-terminal pro-brain natriuretic peptide and adrenomedullin: new neurohormonal predictors of left ventricular function and prognosis after myocardial infarction. Circulation 1998; 97:1921.)

Ch. 178: The Endocrine Heart tions, such as echocardiography. Indeed, the levels of cardiac peptides appear to be superior to time-honored indicators (such as ejection fraction) in detecting heart failure based on diastolic dys¬ function, which comprises one-third of all clinical heart failure.159 Further, a study has shown that the use of plasma NT-proBNP measurements to guide treatment for heart failure (compared to standardized clinical assessment) both reduced total cardio¬ vascular events and delayed the time of the first event.1593 The same hormone measurements may provide prognostic insights after myocardial infarction'155'156 and in heart failure10 that are inde¬ pendent of ejection fraction. How useful the cardiac natriuretic peptides become in everyday clinical decision making involv¬ ing patients at risk for heart disease remains to be seen. Again, whether the measurement of natriuretic peptide levels will eventually help clinicians to assess volume status in noncar¬ diac disorders is unclear. Plasma ANP (which increases) is more responsive to salt loading than plasma renin activity (which decreases),160 a finding that has led to the use of ANP assays in assessing the adequacy of mineralocorticoid replacement in patients with Addison disease.161 Similarly, the possibility exists that, along with aldosterone and renin, measurement of plasma natriuretic peptides (e.g., NT-BNP) may prove to be a useful marker of the hyperexpanded state associated with primary hyperaldosteronism162

THERAPEUTIC IMPLICATIONS Due to the vasodilator, natriuretic/diuretic, renin-angiotensinaldosterone-inhibitory and antimitogenic actions of these pep¬ tides, any maneuver that increases their circulating and tissue levels is likely to be helpful in the treatment of a variety of car¬ diovascular and volume-overload disorders. As mentioned previously, the short-term intravenous administration of exog¬ enous ANP and BNP in various doses has been shown to have beneficial hemodynamic and renal effects in patients with hypertension or heart failure.71'104'163 Natriuretic peptide administration may also be a beneficial adjunctive component of reperfusion therapy for patients with acute myocardial infarction, as ANP is reported to inhibit reperfusion-induced ventricular arrhythmias and preserve ATP content in the ischemic myocardium.164 The infusion of ANP has been shown to reduce anginal symptoms,165 whereas BNP is reported to suppress hyperventilation-induced attacks in patients with variant angina by reducing coronary artery spasm.166 Further¬ more, ANP gene therapy, by direct intraluminal delivery via the affected vessel, is already a prospect.167 In patients with acute renal failure, large doses of ANP significantly improve glomerular function and reduce the need for dialysis.168 Although the peptidic nature (requiring intravenous adminis¬ tration) and relatively short half-lives of ANP and BNP in plasma limit their clinical application, the routine infusion of ANP is currently approved in Japanese hospitals for patients with fluid-overload states. An alternative approach is to enhance levels of the endogenous peptides through inhibition of their enzymatic breakdown by NEP 24.11. This approach appears to have a number of advantages, including availability of oral inhibitors and a relatively pro¬ longed effect in enhancing circulating levels of the cardiac natriuretic peptides (ANP more than BNP in humans). The ben¬ eficial hemodynamic, hormonal, and renal effects of short-term NEP inhibition in hypertension and heart failure are well docu¬ mented and mimic those of exogenous ANP and BNP.43'169'170 A major advantage of NEP inhibition (compared with exogenous natriuretic peptide administration) is the increased protection of the peptides from degradation within the kidney, thus increasing the local hormone concentration and enhancing natriuresis. Indeed, NEP administration in heart failure is asso¬ ciated with an increased natriuresis and diuresis170 in contrast with the blunted renal response observed with exogenous ANP infusion alone.87 Long-term use of NEP inhibitors has also been

1631

shown to reduce intraocular pressure and may have a place in the management of glaucoma.171 Another potential approach increases endogenous natriuretic peptide levels by blocking the clearance receptor, NPR-C.78 The blockade of both degradative pathways, as shown in sheep with experimental heart failure, induces greater than additive increments in plasma ANP, BNP, and cGMP concentrations and enhances the beneficial hemody¬ namic and renal effects (compared with blocking enzyme or receptor alone).78 The long-term use of compounds that poten¬ tiate circulating levels of the natriuretic peptides and their actions may also promote the antiproliferative effects of these peptides and may impede the detrimental consequences of car¬ diac and vascular smooth muscle hypertrophy and endothelial proliferation.172

FUTURE PROSPECTS AND CONCLUSIONS A wealth of information exists on the molecular biology, phys¬ iology, and pathophysiology of the cardiac natriuretic pep¬ tides. They have a well-established role in pressure and volume homeostasis. A number of questions remain unan¬ swered, however. Do ANP and BNP simply duplicate one another's roles, or do they have distinct functions? Differences listed in Table 178-1 suggest that distinct functions are highly probable. If so, do specific receptors exist for either ANP or BNP? Do the amino-terminal forms (NT-ANP, NT-BNP) per¬ form any role in health or disease? The picture is complicated by the identification of multiple natriuretic hormones—some circulating, others largely confined to tissues—with the poten¬ tial to interact, as observed in other vasoactive hormonal sys¬ tems such as the renin-angiotensin system. Untangling the individual roles of these hormones will be difficult and will require specific antagonists and/or use of transgenic models. Results from BNP knock-out, disruption of NPR-B and NPR-C receptors, or combinations of these are awaited with interest. Finally, how hormones of the natriuretic peptide system inter¬ act with other local vasoactive (nonangiotensin) systems—

TABLE 178-1. Comparative Physiology of Atrial Natriuretic Peptide (ANP) and Brain Natriuretic Peptide (BNP) in Humans

Major site of synthesis

ANP

BNP

Cardiac atrium

Cardiac ventricle

Cardiac mRNA content: Normal

Ventricle « atrium

Ventricle > atrium

Heart failure

Ventricle > atrium

Ventricle » atrium

Induction

Slow

Rapid

mRNA turnover

Slow

Rapid

Major stimulus to syn¬ thesis

Atrial transmural pressure

Ventricular wall tension

Major type of secretion

Regulated

Constitutive

Major storage form in atria

Prohormone

Mature hormone (BNP32)

Major plasma form

Mature hormone (ANP-28)

(ANP1-126)

Prohormone (BNP1-108)

Normal plasma levels

5-20 pmol/L

0.6-9.0 pmol/L

Plasma half-life

3 min

22 min

C-receptor affinity

High

Lower than ANP

Affinity for NEP

High

Lower than ANP

Bioreceptor type

NPR-A (other?)

Dissimilar bioactivity

NPR-A (other?) Less cGMP generation? Less inhibition of aldos¬ terone?

NEP, neutral endopeptidase; NPR-A, natriuretic peptide receptor A; GMP, guanosine monophosphate.

1 632

PART X: DIFFUSE HORMONAL SECRETION

such as nitric oxide, endothelin, and adrenomedullin—is largely unexplored.

REFERENCES 1. Kisch B. Electron microscopy of the atrium of the heart. Exp Med Surg 1956; 14:99. 2. Henry JP, Gauer ON, Reeves JL. Evidence on the atrial location of receptors influencing urine flow. Circ Res 1956; 5:85. 3. De Bold AJ, Borenstein HB, Veress AT, Sonnenberg H. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extracts in rats. Life Sci 1981; 28:89. 4. Kangawa H, Marsuo H. Purification and complete amino acid sequence of alpha human atrial natriuretic polypeptide (alpha-h ANP). Biochem Biophys Res Commun 1984; 18:131. 5. Sudoh T, Kangawa K, Minamino N, Matsuo H. A new natriuretic peptide in porcine brain. Nature 1988; 332:78. 6. Sudoh T, Minamino N, Kangawa K, Matsuo H. C-type natriuretic peptide (CNP): a new member of natriuretic peptide family identified in porcine brain. Biochem Biophys Res Commun 1990; 168:863. 7. Espiner EA, Richards AM, Yandle TG, Nicholls MG. Natriuretic hormones. Endocrinol Metab Clin North Am 1995; 24:481. 8. Hama N, Itoh H, Shirakami G, et al. Detection of C-type natriuretic peptide in human circulation and marked increase of plasma CNP levels in septic shock patients. Biochem Biophys Res Commun 1994; 198:1177. 9. Totsune K, Takahashi K, Murakami O, et al. Elevated plasma C-type natri¬ uretic peptide concentrations in patients with chronic renal failure. Clin Sci 1994; 87:319. 10. Wei CM, Heublein DM, Perrella MA, et al. Natriuretic peptide system in human heart failure. Circulation 1993; 88:1004. 11. Itoh H, Suga S, Ogawa Y, et al. Cellular and molecular aspects of C-type natriuretic peptide (CNP). In: Samson WK, Levin ER, eds. Contemporary endocrinology: natriuretic peptides in health and disease. Totowa, NJ: Humana Press, 1997:Chapter 7. 12. Lindpaintner K, Ganten D. The cardiac renin angiotensin system. An appraisal of present experimental and clinical evidence. Circ Res 1991; 68:905. 13. Colucci WS. Myocardial endothelin. Does it play a role in myocardial fail¬ ure? Circulation 1996; 93:1069. 14. Jougasaki M, Wei CM, Heublein DM, et al. Immunohistochemical localiza¬ tion of adrenomedullin in canine heart and aorta. Peptides 1995; 16:773. 15. Bui TD, Shallal A, Malik AN, et al. Parathyroid hormone related peptide gene expression in human fetal and adult heart. Cardiovasc Res 1993; 27:1204. 16. Tamura N, Ogawa Y, Yasoda A, et al. Two cardiac natriuretic peptide genes (atrial natriuretic peptide and brain natriuretic peptide) are organized in tan¬ dem in the mouse and human genomes. J Mol Cell Cardiol 1996; 28:1811. 17. Yandle TG. Biochemistry of natriuretic peptides. J Intern Med 1994; 235:561. 18. Klein RM, Kelly KB, Meriskoliversidge EM. A clathrin-coated vesiclemediated pathway in atrial natriuretic peptide (ANP) secretion. J Mol Cell Cardiol 1993; 25:437. 19. Bloch KD, Seidman JG, Naftilan JD, et al. Neonatal atria and ventricles secrete atrial natriuretic factor via tissue-specific secretory pathways. Cell 1986; 47:695. 20. Irons CE, Sei CA, Glembotski CC. Regulated secretion of atrial natriuretic factor from cultured ventricular myocytes. Am J Physiol 1993; 264:H282. 21. Misono KS, Grammer RT, Fukumi H, Lnagami T. Rat atrial natriuretic fac¬ tor: isolation, structure and biological activities of four major peptides. Bio¬ chem Biophys Res Commun 1984; 123:444. 22. Suga S, Nakao K, Hosoda M, et al. Receptor selectivity of natriuretic pep¬ tide family, atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide. Endocrinology 1992; 130:229. 23. Kambayashi Y, Nakao K, Kumura H, et al. Biological characterization of human brain natriuretic peptide (BNP) and rat BNP: species-specific actions of BNP. Biochem Biophys Res Commun 1990; 173:599. 24. Hunt PJ, Yandle TG, Nicholls MG, et al. The amino-terminal portion of pro¬ brain natriuretic peptide (Pro-BNP) circulates in human plasma. Biochem Biophys Res Commun 1995; 214:1175. 25. Mukoyama M, Nakao K, Hosoda K, et al. Brain natriuretic peptide as a novel cardiac hormone in humans. Evidence for an exquisite dual natri¬ uretic peptide system, atrial natriuretic peptide and brain natriuretic pep¬ tide. J Clin Invest 1991; 87:1402. 26. Ogawa Y, Nakao K, Mukoyama M, et al. Natriuretic peptides as cardiac hormones in normotensive and spontaneously hypertensive rats. The ven¬ tricle is a major site of synthesis and secretion of brain natriuretic peptide. Circ Res 1991; 69:491. 27. Ruskoaho H. Atrial natriuretic peptide: synthesis, release, and metabolism. Pharmacol Rev 1992; 44:479. 28. Forssmann W-G. Urodilatin (ularitide, INN): a renal natriuretic peptide. Molecular biology and clinical importance. Nephron 1995; 69:211. 29. Nakamura S, Naruse M, Naruse K, et al. Atrial natriuretic peptide and brain natriuretic peptide coexist in the secretory granules of human cardiac myocytes. Am J Hypertens 1991; 4:909. 30. Hosoda K, Nakao K, Mukoyama M, et al. Expression of brain natriuretic peptide gene in human heart. Hypertension 1991; 17:1152.

31. Tanaka M, Hiroe M, Nishikawa T, et al. Cellular localization and structural characterization of natriuretic peptide-expressing ventricular myocytes from patients with dilated cardiomyopathy. J Histochem Cytochem 1994; 42:1207. 32. Ledsome JR, Wilson N, Courneya CA, Rankin AJ. Release of atrial natri¬ uretic peptide by atrial distention. Can J Physiol Pharmacol 1985; 63:739. 33. Leskinen H, Vuolteenaho O, Toth M, Ruskoaho H. Atrial natriuretic pep¬ tide (ANP) inhibits its own secretion via ANPA receptors: altered effect in experimental hypertension. Endocrinology 1997; 138:1893. 34. Leskinen H, Vuolteenaho O, Toth M, Ruskoaho H. Combined inhibition of endothelin and angiotensin receptors blocks volume load induced cardiac hormone release. Circ Res 1997; 80:114. 35. Cameron VA, Aitken GD, Ellmers LJ, et al. The sites of gene expression of atrial, brain and C-type natriuretic peptides in mouse fetal development: temporal changes in embryos and placentas. Endocrinology 1996; 137:817. 36. Yandle TG, Richards AM, Gilbert A, et al. Assay of brain natriuretic peptide (BNP) in human plasma: evidence for high molecular weight BNP as a major plasma component in heart failure. J Clin Endocrinol Metab 1993; 6:832. 37. Buckley MG, Sethi D, Markandu ND, et al. Plasma concentrations and com¬ parisons of brain natriuretic peptide and atrial natriuretic peptide in normal subjects, cardiac transplant recipients and patients with dialysis-indepen¬ dent or dialysis-dependent chronic renal failure. Clin Sci 1992; 83:437. 38. Lang CC, Coutie WJ, Khong TK, et al. Dietary sodium loading increases plasma brain natriuretic peptide levels in man. J Hypertens 1991; 9:779. 39. Thibault G, Doubell AF, Garcia R, et al. Endothelin-stimulated secretion of natriuretic peptides by rat atrial myocytes is mediated by endothelin A receptors. Circ Res 1994; 74:460. 40. Toth M, Vuorinen KH, Vuolteenaho O, et al. Hypoxia stimulates release of ANP and BNP from perfused rat ventricular myocardium. Am J Physiol 1994; 266:H1572. 41. Kinnunen P, Vuolteenaho O, Ruskoaho H. Mechanisms of atrial and brain natriuretic peptide release from rat ventricular myocardium: effect of stretching. Endocrinology 1993; 132:1961. 42. Nicholls MG. The natriuretic peptides in heart failure. J Intern Med 1994; 235:515. 43. Richards AM, Crozier IG, Espiner EA, et al. Plasma brain natriuretic pep¬ tide and endopeptidase 24.11 inhibition in hypertension. Hypertension 1993; 22:231. 44. Richards AM, Crozier IG, Yandle TG, et al. Brain natriuretic factor: regional plasma concentrations and correlations with haemodynamic state in car¬ diac disease. Br Heart J 1993; 69:414. 45. Nicholson S, Richards M, Espiner E, et al. Atrial and brain natriuretic pep¬ tide response to exercise in patients with ischaemic heart disease. Clin Exp Pharmacol Physiol 1993; 20:535. 46. Lang CC, Choy AMJ, Turner K, et al. The effect of intravenous saline load¬ ing on plasma levels of brain natriuretic peptide in man. J Hypertens 1993; 11:737. 47. Morita E, Yasue H, Yoshimura M, et al. Increased plasma levels of brain natriuretic peptide in patients with acute myocardial infarction. Circula¬ tion 1993; 88:82. 48. Yoshibayashi M, Kamiya T, Saito Y, Matsuo H. Increased plasma levels of brain natriuretic peptide in hypertrophic cardiomyopathy. N Engl J Med 1993; 329:433. 49. Shimoike H, Iwai N, Kinoshita M. Differential regulation of natriuretic peptide genes in infarcted rat hearts. Clin Exp Pharmacol Physiol 1997; 24:23. 50. Yoshimura M, Yasue H, Okumura K, et al. Different secretion patterns of, atrial natriuretic peptide and brain natriuretic peptide in patients with con¬ gestive heart failure. Circulation 1993; 87:464. 51. Yasue H, Yoshimura M, Sumida H, et al. Localisation and mechanism of secretion of B-type natriuretic peptide in comparison with those of A-type natriuretic peptide in normal subjects and patients with heart failure. Cir¬ culation 1994; 90:195. 52. Hanford DS, Thuerauf DJ, Murray SF, Glembotski CC. Brain natriuretic peptide is induced by al-adrenergic agonists as a primary response gene in cultured rat cardiac myocytes. J Biol Chem 1994; 269:26227. 53. Liang F, Gardener DG. Autocrine/paracrine determinants of strain-acti¬ vated brain natriuretic peptide gene expression in cultured cardiac myo¬ cytes. J Biol Chem 1998; 273:14612. 54. Maack T, Nikonova LN, Friedman O, Cohen D. Functional properties and dynamics of natriuretic factor receptors. Proc Soc Exp Biol Med 1996; 213:109. 55. Kishi Y, Ashikaga T, Watanabe R, Numano F. Atrial natriuretic peptide reduces cyclic AMP by activating cyclic GMP-stimulated phosphodi¬ esterase in vascular endothelial cells. J Cardiovasc Pharmacol 1994; 24:351. 56. Anand-Srivastava MB, Sehl PD, Lowe DG. Cytoplasmic domain of natri¬ uretic peptide receptor-C inhibits adenylyl cyclase. Involvement of a per¬ tussis toxin-sensitive G protein. J Biol Chem 1996; 271:19324. 57. Levin ER. Natriuretic peptide C-receptor: more than a clearance receptor. Am J Physiol 1993; 264:E483. 58. Hutchinson HG, Trindade PT, Cunanan DB, et al. Mechanisms of natriuretic-peptide-induced growth inhibition of vascular smooth muscle cells. Cardiovasc Res 1997; 35:158. 59. Engel AM, Schoenfeld JR, Lowe DG. A single residue determines the dis¬ tinct pharmacology of rat and human natriuretic peptide receptor-C. J Biol Chem 1994; 269:17005.

Ch. 178: The Endocrine Heart 60. Nakao K, Ogawa Y, Suga S, Imura H. Molecular biology and biochemistry of the natriuretic peptide system. II: Natriuretic peptide receptors. J Hypertens 1992; 10:1111. 61. Yamamoto T, Feng L, Mizuno T, et al. Expression of mRNA for natriuretic peptide receptor subtypes in bovine kidney. Am J Physiol 1994; 267:F318. 62. Yoshimoto T, Naruse M, Naruse K, et al. Differential gene expression of vascular natriuretic peptide receptor subtype in artery and vein. Biochem Biophys Res Commun 1995; 216:535. 63. Kato J, Lanier-Smith KL, Currie MG. Cyclic GMP down-regulates atrial natriuretic peptide receptors on cultured vascular endothelial cells. J Biol Chem 1991; 266:14681. 64. Yoshimoto T, Naruse M, Naruse K, et al. Angiotensin II-dependent downregulation of vascular natriuretic peptide type C receptor gene expression in hypertensive rats. Endocrinology 1996; 137:1102. 65. Jaiswal RK. Endothelin inhibits the atrial natriuretic factor stimulated cGMP production by activating the protein kinase C in rat aortic smooth muscle cells. Biochem Biophys Res Commun 1992; 182:395. 66. Yoshimoto I, Naruse K, Shionoya K, et al. Angiotensin converting enzyme inhibitor normalizes vascular natriuretic peptide type A receptor gene expression via bradykinin-dependent mechanism in hypertensive rats. Biochem Biophys Res Commun 1996; 218:50. 67. Kollenda MC, Vollmar AM, McEnroe GA, Gerbes AL. Dehydration increases the density of C receptors for ANF on rat glomerular membranes. Am ] Physiol 1990; 256:R1084. 68. Suga S, Nakao K, Kishimoto I, et al. Phenotype-related alteration in expression of natriuretic peptide receptor in aortic smooth muscle cells. Circ Res 1992; 71:34. 69. Pilo A, Iervasi G, Clerico A, et al. Circulatory models in metabolic studies of rapidly renewed hormones: application to ANP kinetics. Am J Physiol 1998; 274:E560. 70. Florkowski CM, Richards AM, Espiner EA, et al. Renal, endocrine, and hemodynamic interactions of atrial and brain natriuretic peptides in nor¬ mal man. Am J Physiol 1994; 266:R1244. 71. Yoshimura M, Yasue H, Morita E, et al. Hemodynamic, renal, and hor¬ monal responses to brain natriuretic peptide in patients with congestive heart failure. Circulation 1991; 84:1581. 72. Iervasi G, Clerico A, Pilo A, et al. Evidence that atrial natriuretic peptide tissue extraction is not changed by large increases in its plasma levels induced by pacing in humans. J Clin Endocrinol Metab 1997; 82:884. 73. Deschodt-Lanckman M, Micheaux F, De Prez E, et al. Increased serum lev¬ els of endopeptidase 24.11 ("enkephalinase") in patients with end-stage renal failure. Life Sci 1989; 45:133. 74. Richards AM, Wittert G, Espiner EA, et al. EC 24.11 inhibition in man alters clearance of atrial natriuretic peptide. J Clin Endocrinol Metab 1991; 72:1317. 75. Richards M, Espiner E, Frampton C, et al. Inhibition of endopeptidase EC 24.11 in humans—renal and endocrine effects. Hypertension 1990; 16:269. 76. Schwartz J-C, Gros C, Lecomte J-M, Bralet J. Enkephalinase (EC 3.4.24.11) inhibitors: protection of endogenous ANF against inactivation and poten¬ tial therapeutic applications. Life Sci 1990; 47:1279. 77. Yandle T, Richards AM, Smith MW, et al. Assay of endopeptidase.il (EC3.4.24.11) activity in human plasma. Application to in vivo studies of endopeptidase inhibitors. Clin Chem 1992; 38:1785. 78. Rademaker MT, Charles CJ, Kosoglou T, et al. Clearance receptors and endopeptidase: equal role in natriuretic peptide metabolism in heart fail¬ ure. Am J Physiol 1997; 273:H2372. 79. Anand-Srivastava MB, Trachte GJ. Atrial natriuretic factor receptors and signal transduction mechanisms. Pharmacol Reviews 1993; 45:455. 80. Vesely DL, Douglass MA, Dietz JR, et al. Three peptides from the atrial natri¬ uretic factor prohormone amino terminus lower blood pressure and produce diuresis, natriuresis, and/or kaliuresis in humans. Circulation 1994; 90:1129. 81. Weir ML, Honrath U, Flynn TG, Sonnenberg H. Lack of biological activity or specific binding of amino-terminal pro-ANP segments in the rat. Regul Pept 1994; 53:111. 82. Richards AM, McDonald D, Fitzpatrick MA, et al. Atrial natriuretic hor¬ mone has biological effects in man at physiological plasma concentrations. J Clin Endocrinol Metab 1988; 67:1134. 83. Maack T. Role of atrial natriuretic factor in volume control. Kidney Int 1996; 49:1732. 84. Hunt P, Espiner EA, Nicholls MG, et al. Differing biological effects of equimolar atrial and brain natriuretic peptide infusions in normal man. J Clin Endocrinol Metab 1996; 81:3871. 85. Hildebrandt DA, Mizelle HL, Brands MW, et al. Intrarenal natriuretic peptide infusion lowers arterial pressure chronically. Am J Physiol 1990; 259:R585. 86. Lohmeier TE, Shin Y, Reinhart GA, Hester RL. Angiotensin and ANP secre¬ tion during chronically controlled increments in atrial pressure. Am J Phys¬ iol 1994; 266:R989. 87. Cody RJ, Atlas SA, Laragh JH, et al. Atrial natriuretic factor in normal sub¬ jects and heart failure patients. Plasma levels and renal, hormonal and hemodynamic responses to peptide infusion. J Clin Invest 1986; 78:1362. 88. Lee EY, Humphreys MH. Phosphodiesterase activity as a mediator of renal resistance to ANP in pathological salt retention. Am J Physiol 1996; 271 :F3. 89. Lohmeier TE, Mizelle HL, Reinhart GA, et al. Atrial natriuretic peptide and sodium homeostasis in compensated heart failure. Am J Physiol 1996; 27LR1353. 90. Richards AM, Rao G, Espiner EA, Yandle TG. Interaction of angiotensin converting enzyme and atrial natriuretic factor. Hypertension 1989; 13:193. 91. Jensen KT, Eiskjaer H, Carstens J, Pederson EB. Renal effects of brain natri¬ uretic peptide in patients with congestive heart failure. Clin Sci 1999; 96:5.

1633

92. Winquist RJ, Faison EP, Nutt RF. Vasodilator profile of synthetic atrial natriuretic factor. Eur J Pharmacol 1984; 102:169. 93. Richards AM. Atrial natriuretic factor administration to humans: 1984. J Cardiovasc Pharmacol 1989; 13:S69. 94. Shen Y-T, Graham RM, Vatner SF. Effects of atrial natriuretic factor on blood flow distribution and vascular resistance in conscious dogs. Am J Physiol 1991; 260:H1893. 95. Woods RL. Vasoconstrictor actions of atrial natriuretic peptide in the splanchnic circulation of anesthetized dogs. Am J Physiol 1998; 275:R1822. 96. Floras JS. Sympathoinhibitory effects of atrial natriuretic factor in normal humans. Circulation 1990; 81:1860. 97. Volpe M. Atrial natriuretic peptide and baroreflex control of circulation. Am J Hypertens 1992; 5:488. 98. Charles CJ, Espiner EA, Richards AM. Cardiovascular actions of ANF: con¬ tributions of renal, neurohumoral and hemodynamic factors in sheep. Am J Physiol 1993; 264:R533. 99. Charles CJ, Espiner EA, Richards AM, et al. Chronic infusions of brain natriuretic peptide in conscious sheep: bioactivity at low physiological lev¬ els. Clin Sci 1998; 95:701. 100. Melo LG, Veress AT, Ackermann U, Sonnenberg H. Chronic regulation of arterial blood pressure by ANP: role of endogenous vasoactive endothelial factors. Am J Physiol 1998; 275:H1826. 101. Richards AM. The natriuretic peptides and hypertension. J Intern Med 1994; 235:543. 102. Atlas SA, Maack T. Atrial natriuretic factor. In: Windhager EE, ed. Handbook of physiology: renal physiology. New York: Oxford University Press, 1992:1577. 103. Janssen MT, de Zeeuw D, van der Hem GK, de Jong PE. Antihypertensive effect of a 5-day infusion of atrial natriuretic peptide in man. Hypertension 1989; 13:640. 104. Marcus LS, Hart D, Packer M, et al. Hemodynamic and renal excretory effects of human brain natriuretic peptide infusion in patients with conges¬ tive heart failure. Circulation 1996; 94:3184. 105. Appel RG. Growth-regulatory properties of atrial natriuretic factor. Am J Physiol 1992; 262:F911. 106. Wu CF, Bishopric NH, Pratt RE. Atrial natriuretic peptide induces apopto¬ sis in neonate rat cardiac myocytes. J Biol Chem 1997; 272:14860. 107. Fujisaki H, Ito H, Hirata Y, et al. Natriuretic peptides inhibit angiotensin IIinduced proliferation of rat cardiac fibroblasts by blocking endothelin-1 gene expression. J Clin Invest 1995; 96:1059. 108. Suga S, Nakao K, Itoh H, et al. Endothelial production of C-type natriuretic peptide and its marked augmentation by transforming growth factor [i Possible existence of "vascular natriuretic peptide system." J Clin Invest 1992; 90:1145. 109. Suga S, Itoh H, Komatsu Y, et al. Endothelial production of C-type natriuretic peptide—evidence for cytokine regulation. (Abstract). Circulation 1993; 88:1-621. 110. Furuya M, Aiska K, Miyazaki T, et al. C-type natriuretic peptide inhibits intimal thickening after vascular injury. Biochem Biophys Res Commun 1993; 193:248. 111. Oliver PM, Fox JE, Kim R, et al. Hypertension, hypertrophy, and sudden death in mice lacking natriuretic peptide receptor A. Proc Natl Acad Sci USA 1997; 94:14730. 112. Florkowski CM, Richards AM, Espiner EA, et al. Low-dose brain natri¬ uretic peptide infusion in normal men and the influence of endopeptidase inhibition. Clin Sci 1997; 92:255. 113. Kurtz A, Bruna RD, Pfeilschifter J, et al. Atrial natriuretic peptide inhibits renin release from juxtaglomerular cells by a cGMP-mediated process. Proc Natl Acad Sci U S A1986; 83:4769. 114. Cherradi N, Brandenburger Y, Rossier MF, et al. Atrial natriuretic peptide inhibits calcium-induced steroidogenic acute regulatory protein gene tran¬ scription in adrenal glomerulosa cells. Mol Endocrinol 1998; 12:962. 115. Beland B, Tuchelt H, Bahr V, Oelkers W. The role of atrial natriuretic factor[a-human A NF(996)J in the hormonal and renal adaptation to sodium deficiency. J Clin Endocrinol Metab 1994; 79:183. 116. Lichardus B, Veress AT, Field LJ, Sonnenberg H. Blood pressure regulation in ANF-transgenic mice: role of angiotensin and vasopressin. Physiol Res 1994; 43:145. 117. Kanamori T, Wada A, Tsutamoto T, Kinoshita M. Possible regulation of renin release by ANP in dogs with heart failure. Am J Physiol 1995; 269:H2281. 118. Khurana ML, Pandey KN. Receptor-mediated stimulatory effect of atrial natriuretic factor, brain natriuretic peptide, and C-type natriuretic peptide on testosterone production in purified mouse Leydig cells: activation of cholesterol side-chain cleavage enzyme. Endocrinology 1993; 133:2141. 119. Johnson KM, Hughes FM, Fong YY, et al. Effects of atrial natriuretic pep¬ tide on rat ovarian granulosa cell steroidogenesis in vitro. Am J Reprod Immunol 1994; 31:163. 120. Hagiwara H, Inoue A, Yamaguchi A, et al. cGMP produced in response to ANP and CNP regulates proliferation and differentiation of osteoblastic cells. Am J Physiol 1996; 270:0311. 121. Suda M, Ogawa Y, Tanaka K, et al. Skeletal overgrowth in transgenic mice that overexpresses brain natriuretic peptide. Proc Natl Acad Sci U S A1998; 95:2337. 122. Imura H, Nakao K, Itoh H. The natriuretic peptide system in the brain: implications in the central control of cardiovascular and neuroendocrine functions. Frontiers Neuroendocrinol 1992; 13:217. 123. Goetz KL. Evidence that atriopeptin is not a physiological regulator of sodium excretion. Hypertension 1990; 15:9. 124. Westenfelder C, Birth FM, Baranowski RL, et al. Volume homeostasis in calves with artificial atria and ventricles. Am J Physiol 1990; 258:F1005.

1634

PART X: DIFFUSE HORMONAL SECRETION

125. Lee ME, Miller WL, Edwards BS, Burnett JC. Role of endogenous atrial natriuretic factor in acute congestive heart failure. J Clin Invest 1989; 84:1962. 126. Hirata Y, Matsuoka H, Suzuki E, et al. Role of endogenous atrial natriuretic peptide in DOCA-salt hypertensive rats. Effects of a novel antagonist for atrial natriuretic peptide receptor. Circulation 1993; 87:554. 127. Yamamoto K, Burnett JC, Redfield MM. Effect of endogenous natriuretic peptide system on ventricular and coronary function in failing heart. Am J Physiol 1997; 273:H2406. 128. Wada A, Tsutamoto T, Matsuda Y, Kinoshita M. Cardiorenal and neurohumoral effects of endogenous atrial natriuretic peptide in dogs with severe congestive heart failure using a specific antagonist for guanylate cyclasecoupled receptors. Circulation 1994; 9:2232. 129. Wada A, Tsutamoto T, Maeda Y, et al. Endogenous atrial natriuretic peptide inhibits endothelin-1 secretion in dogs with severe congestive heart failure. Am J Physiol 1996; 270:H1819. 130. Itoh H, Nakao K, Mukoyama M, et al. Chronic blockade of endogenous atrial natriuretic polypeptide (ANP) by monoclonal antibody against ANP acceler¬ ates the development of hypertension in spontaneously hypertensive and deoxycorticosterone acetate-salt-hypertensive rats. J Clin Invest 1989; 84:145. 131. Barbee RW, Perry BD, Re RN, et al. Hemodynamics in transgenic mice with overexpression of atrial natriuretic factor. Circ Res 1994; 74:747. 132. Ogawa Y, Itoh H, Tamura N, et al. Molecular cloning of the cDNA and gene that encode mouse brain natriuretic peptide gene and generation of transgenic mice that overexpress the brain natriuretic peptide gene. J Clin Invest 1994; 93:1911. 132a. Matsukawa N, Grzesik WJ, Takahashi N, et al. The natriuretic peptide clearance receptor locally modulates the physiological effects of the natri¬ uretic peptide system. Proc Nat Acad Sci U S A 1999; 96:7403. 133. John SW, Krege JH, Oliver PM, et al. Genetic decreases in atrial natriuretic peptide and salt-sensitive hypertension. Science 1995; 267:679. 134. Oliver PM, John SW, Purdy KE, et al. Natriuretic peptide receptor 1 expres¬ sion influences blood pressures of mice in a dose-dependent manner. Proc Natl Acad Sci U S A 1998; 95:2547. 135. Crozier IG, Nicholls MG, Ikram H, et al. Atrial natriuretic peptide in humans: production and clearance by various tissues. Hypertension 1986; 8:11-11. 136. Nishigaki K, Tomita M, Kagawa K, et al. Marked expression of plasma brain natriuretic peptide is a special feature of hypertrophic obstructive cardiomyopathy. J Am Coll Cardiol 1996; 28:1234. 137. Ando K, Hirata Y, Emori T, et al. Circulating forms of atrial natriuretic pep¬ tide in patients with congestive heart failure. J Clin Endocrinol Metab 1990; 70:1603. 138. Shemin D, Dworkin LD. Sodium balance in renal failure. Curr Opin Neph¬ rol Hypertens 1997; 6:128. 139. Nagaya N, Nishikimi T, Okano Y, et al. Plasma brain natriuretic peptide levels increase in proportion to the extent of right ventricular dysfunction in pulmonary hypertension. J Am Coll Cardiol 1998; 31:202. 140. Mitaka C, Hirata Y, Nagura T, et al. Increased plasma concentrations of brain natriuretic peptide in patients with acute lung injury. J Crit Care 1997; 12:66. 141. Wong F, Blendis F. Pathophysiology of sodium retention and ascites forma¬ tion in cirrhosis: role of atrial natriuretic factor. Semin Liver Dis 1994; 14:59. 142. La Villa G, Padeletti L, Lazzeri C, et al. Plasma levels of natriuretic peptides during ventricular pacing in patients with a dual chamber pacemaker. Pac¬ ing Clin Electrophysiol 1994; 17:953. 143. Wood P. Polyuria in paroxysmal tachycardiac and paroxysmal atrial flutter and fibrillation. Br Heart J 1963; 25:273. 144. Cort JH. Cerebral salt wasting. Lancet 1954; i:752. 145. Berendes E, Walter M, Cullen P, et al. Secretion of brain natriuretic peptide in patients with aneurysmal subarachnoid haemorrhage. Lancet 1997; 349:245. 146. Tunny TJ, Richardson KA, Clark CV, Gordon RD. The atrial natriuretic peptide gene in patients with familial primary open-angle glaucoma. Biochem Biophys Res Commun 1996; 223:221. 147. Rutledge DR, Sun Y, Ross EA. Polymorphisms within the atrial natriuretic peptide gene in essential hypertension. J Hypertens 1995; 13:953. 147a. Sarzani R, Dessi-Fulgheri P, Salvi F, et al. A novel promoter variant of the natriuretic peptide clearance receptor gene is associated with lower atrial natriuretic peptide and higher blood pressure in obese hypertensives. J Hypertens 1999; 17:1301. 148. Shimizu K, Nakano S, Nakano Y, et al. Ectopic atrial natriuretic peptide production in small cell lung cancer with the syndrome of inappropriate antidiuretic hormone secretion. Cancer 1991; 68:2284. 149. Francis GS, Benedict C, Johnstone DE, et al. Comparison of neuroendocrine activation in patients with left ventricular dysfunction with and without congestive heart failure: a substudy of the Studies of Left Ventricular Dys¬ function (SOLVD). Circulation 1990; 82:1724. 150. Lerman A, Gibbons RJ, Rodeheffer RJ, et al. Circulating N-terminal atrial natriuretic peptide as a marker for symptomless left-ventricular dysfunc¬ tion. Lancet 1993; 341:1105. 151. Foy SG, Crozier IG, Richards AM, et al. Neurohormonal changes after acute myocardial infarction: relationships with haemodynamic indices and effects of ACE inhibition. Eur Heart J 1995; 16:770. 152. Rouleau JL, Packer M, Moye L, et al. Prognostic value of neurohumoral activation in patients with acute myocardial infarction: effect of captopril. J Am Coll Cardiol 1994; 24:583. 153. Davis M, Espiner E, Richards G, et al. Plasma brain natriuretic peptide in assessment of acute dyspnoea. Lancet 1994; 343:440.

154. Takeda T, Kohno M. Brain natriuretic peptide in hypertension. Hypertens Res 1995; 18:259. 155. Omland T, Aakvaag A, Bonarjee WS, et al. Plasma brain natriuretic pep¬ tide as an indicator of left ventricular systolic function and long-term sur¬ vival after acute myocardial infarction: comparison with plasma atrial natriuretic peptide and N-terminal proatrial natriuretic peptide. Circula¬ tion 1996; 93:1963. 156. Richards AM, Nicholls MG, Yandle TG, et al. Plasma N-terminal pro-brain natriuretic peptide and adrenomedullin: new neurohormonal predictors of left ventricular function and prognosis after myocardial infarction. Circula¬ tion 1998; 97:1921. 157. Swedberg K, Eneroth P, Kjekshus J, Wilhelmsen L. Hormones regulating cardiovascular function in patients with severe congestive heart failure and their relation to mortality. Circulation 1990; 82:1730. 158. Hunt PJ, Richards AM, Nicholls MG, et al. Immunoreactive amino-termi¬ nal pro-brain natriuretic peptide (NT-proBNP): a new marker of cardiac impairment. Clin Endocrinol 1997; 47:287. 159. Lainchbury JG, Nicholls MG, Espiner EA, et al. Cardiac hormones. Auck¬ land, New Zealand: The National Heart Foundation of New Zealand, 1997. Technical report no. 72. 159a. Troughton RW, Frampton CM, Yandle TG, et al. Treatment of heart failure guided by plasma aminoterminal brain natriuretic peptide (N-BNP) con¬ centrations. Lancet 2000; 355:1126. 160. Espiner EA, Nicholls MG. Human atrial natriuretic peptide. Clin Endo¬ crinol 1987; 26:637. 161. Cohen N, Gilbert R, Wirth A, et al. Atrial natriuretic peptide and plasma renin levels in assessment of mineralcorticoid replacement in Addison's disease. J Clin Endocrinol Metab 1996; 81:1411. 162. Naruse M, Takeyama Y, Tanabe A, et al. Atrial and brain natriuretic pep¬ tides in cardiovascular diseases. Hypertension 1994; 23:1-231. 163. Pidgeon BG, Richards AM, Nicholls MG, et al. Differing metabolism and bioactivity of atrial and brain natriuretic peptides in essential hyperten¬ sion. Hypertension 1996; 27:906. 164. Takata Y, Hirayama Y, Kiyomi S, et al. The beneficial effects of atrial natri¬ uretic peptide on arrhythmias and myocardial high-energy phosphates after reperfusion. Cardiovasc Res 1996; 32:286. 165. Lai CP, Egashira K, Tashiro H, et al. Beneficial effects of atrial natriuretic peptide on exercise induced myocardial ischaemia in patients with stable angina pectoris. Circulation 1993; 87:144. 166. Kato H, Yasue H, Yoshimura M, et al. Suppression of hyperventilationinduced attacks with infusion of B-type (brain) natriuretic peptide in patients with variant angina. Am Heart J 1994; 128:1098. 167. Isner JM, Feldman LJ. Gene therapy for arterial disease. Lancet 1994; 344:1653. 168. Rahman SN, Kim GE, Mathew AS, et al. Effects of atrial natriuretic peptide in clinical acute renal failure. Kidney Int 1994; 45:1731. 169. Kimmelstiel CD, Perrone R, Kilcoyne L, et al. Effects of renal neutral endopeptidase inhibition on sodium excretion, renal hemodynamics and neuro-hormonal activation in patients with congestive heart failure. Cardi¬ ology 1996; 87:46. 170. Rademaker MT, Charles CJ, Espiner EA, et al. Neutral endopeptidase inhi¬ bition: augmented atrial and brain natriuretic peptide, haemodynamic and natriuretic responses in ovine heart failure. Clin Sci 1996; 91:283. 171. Wolfensberger TJ, Singer DRJ, Freegard T, et al. Evidence for a new role for natriuretic peptides: control of intraocular pressure. Br J Ophthalmol 1994; 78:446. 172. Thompson JS, Sheedy W, Morice A. Effects of the neutral endopeptidase inhibitor SCH 41495, on the cardiovascular remodelling secondary to chronic hypoxia in rats. Clin Sci 1994; 87:109.

CHAPTER

179

THE ENDOCRINE ENDOTHELIUM FRANCESCO COSENTINO AND THOMAS F. LOSCHER In addition to the cells of the "classic" endocrine glands that secrete hormones, several other cells, such as the vascular endothelium, are capable of generating substances that can cir¬ culate and affect neighboring smooth muscle cells and blood cells. Hence, the endothelium acts as an important regulatory organ within the circulation by generating vasoactive sub¬ stances that act in a paracrine, autocrine, solinocrine, and, under certain conditions, hemocrine fashion (see Chap. 1).

Ch. 179: The Endocrine Endothelium TABLE 179-1. Angiogenic and Antiangiogenic Factors Angiogenic Factors

TABLE 179-2. Cardiovascular Diseases Associated with Elevated Endothelin Levels Antiangiogenic Factors

Acute myocardial infarction

Vascular endothelial growth factor (VEGF)

Angiostatin

Atherosclerosis

Basic fibroblast growth factor (bFGF)

Endostatin

Cardiogenic shock

Epidermal growth factor (EGF) Transforming growth factor-P (TGF-P) Angiopoietin-1 Angiogenin

Transforming growth factor-P (TGF-P)

Cerebral/myocardial vasospasm Congestive heart failure

Angiopoietin-2

Diabetes

Interferon-y

Endotoxic shock

Platelet factor-4 fragment

Hypertension

Lymphotoxin

Pulmonary hypertension

Thrombospondin

Raynaud phenomenon

Critically located as a barrier between smooth muscle cells and the blood, the endothelium plays a pivotal functional role in maintaining the homeostasis of the normal vessel by generat¬ ing substances that modulate vascular tone as well as growth, coagulation, platelet function, and the release of circulating hormones.1 Furthermore, the endothelium is a target organ in cardiovascular disease (Tables 179-1 and 179-2). Endothelial cells produce and release a variety of vasoactive substances1 (Fig. 179-1): (a) endothelium-derived relaxing fac¬ tors (EDRFs), such as nitric oxide (NO), endothelium-derived hyperpolarizing factor, and prostacyclin (prostaglandin I2 [PGI2]), as well as other prostaglandins; and (b) endotheliumderived contracting factors, including cyclooxygenase-derived contracting factors, endothelin, and angiotensin II.

Stretch

•{cyclooxygenase

ENDOTHELIUM-DERIVED RELAXING FACTORS ENDOTHELIUM-DERIVED NITRIC OXIDE In the presence of endothelium, acetylcholine induces relaxation. This relaxation cannot be prevented by the use of inhibitors of cyclooxygenase (which blocks PGI2 production), suggesting that a different EDRF must be involved.2 Endothelium-dependent relaxations have been demonstrated in large (conduit) arteries and in resistance vessels of most mammalian species, including humans.1-3'4 The release of EDRF can be demonstrated under basal conditions: in response to mechanical forces such as shear

AA

Synthasel:

ATP-sensitive :K* Channels cGMPjlj cAMP

CONTRACTI ON

1635

RELAXATION

FIGURE 179-1. Endothelium-derived vasoactive substances. The endothelium releases relaxing factors {right) and contracting factors (left). The relaxing factors include nitric oxide (NO), prostacyclin (PG/2), and endothelium-derived hyperpolarizing factor (EDHF). NO and PGI2 cause not only relaxation but also inhibi¬ tion (O) of platelet function. The contracting factors include the local vascular renin-angiotensin system, endothelin (ET), and cyclooxygenase-derived contracting factors such as thromboxane A, (TXA2) and pros¬ taglandin H2 (PGH2). In addition, the cyclooxygenase pathway is a source of oxygen-derived free radicals (Of ). Circles represent receptors. (ATI, angiotensin I; TGF-f5v transforming growth factor-p,; Thr, thrombin; All, angiotensin II; Ach, acetylcholine; AA, arachidonic acid; ADP, adenosine diphosphate; 5-HT, serotonin; BK, bradykinin; T, thrombin receptor; A, angiotensin receptor; M, muscarinic receptor; P, phosphate; ETB, endothelin-receptor subtype; S3, serotonergic receptor; S2, bradykinin receptor; ATG, angiotensinogen; ACE, angiotensin-converting enzyme; ECE, endothelin-converting enzyme; ET-1, endothelin-1; L-Arg, L-arginine; NOS, nitric oxide synthase; ATII, angiotensin II; ATV angiotensin subtype 1 receptor; ETA, endo¬ thelin-receptor subtype; TX, thromboxane receptor; cGMP, cyclic guanosine monophosphate; cAMP, cyclic adenosine monophosphate; ATP, adenosine triphosphate.) (From Liischer TF, Boulanger CM, Dohi Y, Yang Z. Endothelium-derived contracting factors. Hypertension 1992; 19:117.)

1 636

PART X: DIFFUSE HORMONAL SECRETION

stress (exerted by the circulating blood)5'6 and after activation of receptor-operated mechanisms by acetylcholine, neurotransmit¬ ters, various local and circulating hormones, and substances derived from platelets and the coagulation system3-4 (see Fig. 179-1). The EDRF is a diffusible substance with a half-life of a few seconds that has been identified as NO.7-8 Relaxation of smooth muscle cells by endothelium-derived NO (EDNO) is associated with activation of soluble guanylate cyclase (GC) and an increase in intracellular cyclic 3'5’-guanosine monophosphate (cGMP) in vascular smooth muscle9 (see Fig. 179-1). An inhibitor of soluble GC, methylene blue, prevents the production of cGMP and inhibits endothelium-dependent relaxations. Soluble GC is also present in platelets and is acti¬ vated by EDNO10 (see Fig. 179-1). Increased levels of cGMP in platelets are associated with reduced adhesion and aggrega¬ tion. Therefore, EDNO causes both vasodilatation and platelet deactivation and, thereby, represents an important antispastic and antithrombotic feature of the endothelium. EDNO is formed from L-arginine by oxidation of its guanidino-nitrogen terminal11 (see Fig. 179-1) by NO synthase, which has been cloned.12 NO synthase is primarily a cytosolic enzyme requiring calmodulin, Ca2+, (3-nicotinamide-adenine dinucleotide hydrogen phosphate (NADPH), and tetrahydrobiopterin, and has similarities with cytochrome P450 enzymes.13 Several isoforms of the enzyme occur in endothelial cells, as well as in platelets, mac¬ rophages, vascular smooth muscle cells, and the brain. Analogs of L-arginine, such as NG-monomethyl L-arginine (LNMMA), or L-nitroarginine methyl ester (L-NAME), inhibit endothelium-dependent relaxations to serotonin in porcine coro¬ nary arteries, an effect that is restored by L-arginine but not by D-arginine.14 In quiescent arteries, L-NMMA causes endotheliumdependent contractions.15 In intact organs, L-NAME markedly decreases local blood flow.16 When infused in rabbits, L-arginine methyl ester induces long-lasting increases in blood pressure that are reversed by L-arginine.17 This demonstrates that the vascula¬ ture is in a constant state of vasodilation because of the continu¬ ous basal release of NO from the endothelium. Of particular pathophysiologic interest is the discovery of an endogenous inhibitor of the L-arginine-NO pathway known as asymmetric dimethyl-arginine,18 which is also produced by cultured endothe¬ lial cells. This indicates that endogenously produced substances can regulate the activity of this pathway both locally and systemically (it is also detected in plasma). Hence, increased production or elimination of this endogenous inhibitor can profoundly affect the function of the cardiovascular system.19

ENDOTHELIUM-DERIVED HYPERPOLARIZING FACTORS In the porcine coronary circulation, L-NMMA inhibits relax¬ ations to serotonin but only slightly inhibits those to bradykinin.12'14 Other inhibitors of the action of EDNO, such as hemoglobin and methylene blue, as well as inhibitors of cyclooxygenase, also are ineffective. Thus, endothelial cells appear to release a relaxing factor distinct from NO and PGI2. Acetylcholine causes not only endothelium-dependent relax¬ ation but also endothelium-dependent hyperpolarization of vascular smooth muscle 20 An endothelium-dependent hyperpolarizing factor (see Fig. 179-1) distinct from NO could explain these responses, although NO also has been shown to have hyperpolarizing properties under certain conditions. The endothelium-derived hyperpolarizing factor appears to acti¬ vate adenosine triphosphate-sensitive K+ channels,19 but its chemical nature remains elusive.

PROSTACYCLIN Endothelial cells are an important source of PGI2, which is syn¬ thesized within the vasculature in response to shear stress, hypoxia, and several mediators also leading to the formation of EDNO22 (see Fig. 179-1 and Chap. 172). Prostacyclin causes

relaxation by increasing cyclic 3',5’-adenosine monophosphate (cAMP) in smooth muscle and platelets,23 where it also inhibits platelet aggregation, particularly together with NO.

ENDOTHELIUM-DERIVED CONTRACTING FACTORS CYCLOOXYGENASE-DEPENDENT ENDOTHELIUMDERIVED CONTRACTING FACTOR Exogenous arachidonic acid can evoke endothelium-dependent contractions that can be prevented by indomethacin (an inhibitor of cyclooxygenase), suggesting that cyclooxygenase products, besides PGI„ can produce vasoconstriction.1-24 In the human saphenous vein, acetylcholine and histamine evoke endotheliumdependent contractions; in the presence of indomethacin, how¬ ever, endothelium-dependent relaxations are unmasked.25 The products of cyclooxygenase-mediated contractions are throm¬ boxane A2 (TXA2), in the case of acetylcholine, and endoperoxides (prostaglandin H2 [PGH2]), in the case of histamine.25 TXA2 and endoperoxide activate both vascular smooth muscle and platelets, thereby counteracting the protective effects of NO and PGL, in the blood vessel wall (see Fig. 179-1). The cyclooxygenase pathway is also the source of superoxide anions that can mediate endothelium-dependent contractions either by enhancing the breakdown of NO or by directly affect¬ ing vascular smooth muscle.1'26 Thus, the cyclooxygenase path¬ way produces various potentially contracting factors. Their release appears particularly prominent in veins and in the cere¬ bral and ophthalmic circulation (see Chap. 172). The use of more specific inhibitors than indomethacin (such as the TXA2/PGH2 receptor antagonists and the superoxide anion scavengers) is allowing their importance in vascular function to be character¬ ized. Such a selective pharmacologic approach has emphasized the important role of superoxide anions in the balance between endothelium-dependent contractions and relaxations.27

ENDOTHELIN Endothelial cells produce the 21-amino-acid peptide endothelin28 (Fig. 179-2). Of the three peptides endothelin-1, endothelin-2, and endothelin-3, endothelial cells appear to produce exclusively endothelin-1. The translation of mRNA generates preproendothelin, which is converted to big endothelin. Conversion of the latter to endoc thelin-1 by endothelin-converting enzyme (ECE) is necessary for the development of full vascular activity.28 The expression of mRNA and the release of the peptide are stimulated by thrombin, transforming growth factor-(3, interleukin-1, epinephrine, angio¬ tensin II, arginine vasopressin, calcium ionophore, and phorbol ester (Fig. 179-3).28-31 In addition, hypoxia stimulates the release of endothelin in isolated vessels.32 Endothelin-1 is a potent vasoconstrictor both in vitro and in vivo.28-33 In the human heart, eye, and forearm, endothelin causes vasodilation at lower concentrations and marked con-

Endothelin-1

CSCSSLMDKECVYFCHLDIIW

Endothelin-2 Endothelin-3 FIGURE 179-2. The structures of the three 21-amino-acid human endothelins. Complete sequence homologies between the three pep¬ tides are underlined. Amino adds are designated by standard one-letter abbreviations (see Chap. 94, Fig. 94-23 for the key to these amino-acid abbreviations).

Ch. 179: The Endocrine Endothelium All AVP Thr TGFb

Platelets

ET-1

1637

Monocyte

BK Endothelium

^PDGF

|3. Proliferation!

TGFP

t t

Internal elastic lamina

2. Migration!

pdgf

|l • Proliferation!

Vascular Smooth muscle

FIGURE 179-3. The vascular endothelin system and its interactions with the L-arginine-nitric oxide pathway. (All, angiotensin II; AVP, arginine vasopressin; Thr, thrombin; TGF-fi, transforming growth factor^; ET-1, endothelin-1; BK, bradykinin; ECE, endothelin converting enzyme; ETB, endothelin-receptor subtype; cNOS, constitutive nitric oxide synthase; NO, nitric oxide; ETA, endothelin-receptor subtype; PLC, phospholipase C; cGMP, cyclic guanosine monophosphate; sGC, soluble guanylate cyclase.)

tractions at higher concentrations.1'16-34 In the heart, this may lead to ischemia, arrhythmias, and death. Circulating levels of endothelin-1 are low,28 suggesting that little of the peptide is formed under physiologic conditions because of the absence of stimuli or the presence of potent inhibitory mechanisms. Alternatively, it may be released prefer¬ entially toward smooth muscle cells.23-28 Possible pathways involved in regulating the mechanism of endothelin production are (a) a cGMP-dependent pathway,30-35 (b) a cAMP-dependent pathway,36 and (c) a pathway involving an inhibitory factor produced by vascular smooth muscle cells.37 Furthermore, endothelin can release NO and prostacyclin from endothelial cells, possibly representing a negative-feedback mechanism.38 EDNO also modulates the actions of endothelin at the level of vascular smooth muscle. The contractions in response to endo¬ thelin are enhanced after endothelial removal, indicating that basal production of EDNO reduces its response.33 Stimulation of the formation of EDNO by acetylcholine reverses endothelininduced contractions in most blood vessels, although this mechanism appears to be less potent in veins.33 Three distinct endothelin receptors have been cloned: the ETa, ETb, and ETC receptors.39-41 Endothelial cells express ETB receptors linked to the formation of NO and PGI2, possibly explaining the transient vasodilator effects of endothelin when it is infused into intact organs or organisms. In vascular smooth muscle, ETa and, partly, ETB receptors mediate contraction and proliferation (see Fig. 179-3). ETB receptors bind endothelin-1 and endothelin-3 equally, whereas ETA receptors preferentially bind endothelin-1. Several endothelin antagonists lower blood pressure, suggesting that endothelin may contribute to blood pressure regulation.42

ANGIOTENSINS Angiotensin II is a vasoactive octapeptide formed from its inac¬ tive decapeptide precursor, angiotensin I, by the action of a dipeptidyl carboxypeptidase, angiotensin-converting enzyme (ACE), which also is present on endothelial cells43 (see Fig. 179-1 and Chap. 79). Possible local angiotensin II synthesis in the vas¬ cular wall is of special interest in view of the multiple vascular actions of angiotensin II. Angiotensin II not only exerts a direct vasoconstrictor effect but also enhances sympathetic noradrenergic transmission,44

FIGURE 179-4. Endothelium-derived vasoactive factors and vascular growth. The endothelium produces growth inhibitors such as heparin (HP), heparan sulfate (HS), and nitric oxide (EDNO). It releases endo¬ thelium-derived growth promoters such as platelet-derived growth fac¬ tor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor-P (TGF-fi), and endothelin (ET). At sites of damaged endothe¬ lium, the production of EDNO and prostacyclin (PGI2) is diminished, favoring monocyte adhesion and platelet aggregation^ Growth factors are released by these cells as well as the endothelium and lead to prolif¬ eration as well as migration of vascular smooth muscle cells into the intima. (Thr, thrombin; All, angiotensin II; AI, angiotensin I; O,-, oxy¬ gen-derived free radicals; NO, nitric oxide.) (From Liischer TF, Noll G. In: Braunwald E, ed. Heart disease. Philadelphia: WB Saunders, 1996.)

exhibits mitogenic and trophic actions in the vasculature,45 and induces endothelin synthesis.31 Hence, vascular or endothelial ACE activity may play an important role in regulating normal vascular function.

REGULATION OF VASCULAR STRUCTURE AND ANGIOGENESIS VASCULAR STRUCTURE The endothelium produces several factors that regulate the proliferation and migration of underlying smooth muscle cells46-47 (Fig. 179-4). Denudation of endothelial cells is fol¬ lowed by platelet adhesion and aggregation, resulting in the release of platelet-derived growth factor (PDGF) and other mitogens. These events lead to the migration and proliferation of vascular smooth muscle cells and strongly suggest that the endothelium normally has a net inhibitory influence on these responses (see Fig. 179-4). The endothelium synthesizes sub¬ stances, including heparan sulfate, NO, and prostaglandins, that inhibit the growth of smooth muscle cells; this may explain why vascular structure normally remains stable46-49 (see Fig. 179-4). Under certain conditions, the endothelium can generate substances such as PDGF, basic fibroblast growth fac¬ tor, insulin-like growth factor-I, colony-stimulating factor I, endothelin-1, transforming growth factor-p, interleukin-1, and tumor necrosis factor-a that can either induce proliferation by themselves or stimulate growth factor gene expression in smooth muscle cells.46-47'50 In addition, endothelial dysfunction is associated with adhesion of circulating blood cells, such as platelets and monocytes, which also are an important source of growth factors. Endothelial dysfunction in certain disease states could markedly alter the effects of endothelial cells on the behavior of smooth muscle cells and contribute to changes in vascular structure. Structural abnormalities of the media of large conduit and resistance arteries are involved in the pathophysiology of hypertension. In large conduit arteries, intimal thickening and atherosclerosis are important consequences of hypertension

1 638

PART X: DIFFUSE HORMONAL SECRETION ET

and other cardiovascular risk factors, which are responsible for cardiovascular complications such as myocardial infarction and stroke (see later). Hypertensive resistance arteries exhibit an increased media/lumen ratio, which primarily involves the migration and rearrangement of vascular smooth muscle cells within the media (i.e., remodeling). This contributes to the increase in peripheral vascular resistance in hypertension. Although not observed, an imbalance in the production of endogenous inhibitors of migration and proliferation, and of promoters of these responses by endothelial cells could par¬ tially explain these structural vascular changes occurring in hypertension.

ANGIOGENESIS Normal organ growth and development as well as the mainte¬ nance of homeostasis rely on precise control of the blood sup¬ ply by the circulatory system. This system delivers oxygen and nutrients to each organ. Indeed, during the development of the fetus the circulatory system is the earliest organ system to develop. Once the rudimentary system of the early embryo has been formed, further growth of blood vessels (angiogenesis) occurs by proliferation of existing vascular endothelial cells in response to factors secreted by surrounding tissues. Quiescent endothelium responds to vascular endothelial growth factor or basic fibroblast growth factor by entering the cell cycle. Angio¬ genesis, similar to all tissue growth, is under multiple positive and negative regulatory controls. The fact that a growing tumor secretes factors that induce blood vessel growth in order to sup¬ port its own growth and survival is well known. Consequently, angiogenesis is an attractive target for antican¬ cer therapies. This therapeutic approach has been shown to be promising by the discovery of angiostatin51 and endostatin,52 strong and specific inhibitors of the proliferation of endothelial cells. Sequence analysis of these two polypeptides revealed that they are proteolytic fragments of plasminogen and collagen XVIII, respectively. They can inhibit angiogenesis in both in vivo and in vitro assay systems and block the growth of metastases as well as of several primary tumors. The first step toward using such inhibitors for human therapy, however, is to determine the most effective delivery system (i.e., systemic injection of puri¬ fied or recombinant protein, or the gene delivery system). Cur¬ rently, antiangiogenic therapy is a promising form of cancer therapy.53 As shown in Table 179-1, however, a growing number of factors are known to regulate angiogenesis, underscoring the need for more extensive studies to better characterize the basic molecular mechanisms of blood vessel formation.

ENDOCRINE EFFECTS OF ENDOTHELIAL MEDIATORS The endothelium-derived mediators can affect the production of circulating hormones and, at least in certain disease states, increased production of these substances allows them to act as humoral factors (Fig. 179-5).

RENIN-ANGIOTENSIN SYSTEM In isolated renal tissue, NO, released either from isolated canine blood vessels or from cultured porcine endothelial cells, inhib¬ its renin production.54 Considering that NO is released in response to shear stress, this mechanism could regulate renin secretion in response to changes in local hemodynamics and, hence, act as an intrarenal baroreceptor.1 Endothelin inhibits renin production in vitro55 in isolated glomeruli of the rat55 but augments renin production markedly in vivo because of the pronounced renal vasoconstriction.56 Angiotensin II, however, which is the final product of the renin-angiotensin system.

FIGURE 179-5. Endocrine actions of endothelium-derived mediators. Nitric oxide (NO) and endothelin (ET) can affect various endocrine reg¬ ulators of the cardiovascular system, such as the renin-angiotensin sys¬ tem, atrial natriuretic peptide/factor (ANF), the hypophysis, and the adrenal glands. (ACTH, adrenocorticotropic hormone; AVP, arginine vasopressin; NE, norepinephrine; E, epinephrine; All, angiotensin II; ACE, angiotensin-converting enzyme; Al, angiotensin I; PGH2, prosta¬ glandin H2; PG12, prostacyclin.) stimulates endothelin production in endothelial cells in cul¬ ture,29 as well as in intact blood vessels.31

ATRIAL NATRIURETIC PEPTIDE In the atrium of the rat, removal of the endocardium augments the basal release of atrial natriuretic peptide57 (ANH; see Chap. 178). A similar effect can be obtained with inhibitors of EDNO, suggesting that the endothelium acts as an inhibitor of the myo¬ cardial production of ANH. Endothelin-1, however, is a potent secretagogue for ANH in cultured rat atrial myocytes.58 ANH released from myocytes can increase cGMP in the endothelium and, in turn, inhibit the release of endothelin-1 and, possibly, of NO.35

PITUITARY HORMONES Endothelin is produced by neuronal cells and also is found in human cerebrospinal fluid, findings which suggest that it acts as an important mediator in the central nervous system. In humans, intravenous administration of endothelin-1 increases basal plasma concentrations of corticotropin, whereas levels of prolactin, thyrotropin, luteinizing hormone, follicle-stimulating hormone, and growth hormone remain unchanged.Stimu¬ lated serum concentrations of luteinizing hormone and folliclestimulating hormone tend to be higher in the presence of endothelin infusion, however, whereas the peptide exerts a sup¬ pressive action on stimulated plasma concentrations of prolac¬ tin and growth hormone. In addition, endothelin-1 reduces the antidiuretic effects of arginine vasopressin in vivo.

CATECHOLAMINES The addition of endothelin-1 to primary cultures of bovine adrenal chromaffin cells augments the efflux of norepinephrine and epinephrine.60 In contrast, endothelin inhibits adrenergic neurotransmission in the guinea pig femoral artery.61 In adrenocortical glomerulosa cells, endothelin-1 stimulates aldos¬ terone release.62

Ch. 179: The Endocrine Endothelium

ENDOTHELIUM DYSFUNCTION IN HYPERTENSION Abnormal vascular tone and growth are important in the patho¬ physiology of hypertension and atherosclerosis.1 The endothe¬ lium is critically involved in this process because several endothelium-derived vasoactive factors influence these features in an autocrine or paracrine fashion. In hypertension, certain mor¬ phologic and functional alterations of the endothelium occur.1 Endothelial cells of hypertensive vessels have an increased vol¬ ume and bulge into the lumen, and the subintimal space exhibits structural changes, with increased fibrin and cell deposition. Fur¬ thermore, the interaction of platelets and monocytes with the endothelium is greater than in normotensive control subjects.

Genetic Hypertension

Ach All

± *

ADP ATP Thr 5-HT

W

*

ADP ATP Thr 5-HT Ach

i H I ndothellum

inactive

ENDOTHELIUM-DEPENDENT RELAXATION Endothelium-dependent relaxation in response to acetylcholine is reduced in the aortic, cerebral, and peripheral microcircula¬ tions of most experimental models of hypertension.63-65 Simi¬ larly, the vasodilator effects of acetylcholine in the human forearm of hypertensive subjects have been found to be blunted in most studies.66-71 In the coronary circulation of the spontane¬ ously hypertensive rat (SHR), little endothelial dysfunction occurs.72 In the human coronary circulation, however, endothe¬ lium-dependent responses are impaired in epicardial vessels and microvessels in patients with hypertension, particularly in the presence of left ventricular hypertrophy.73 The response to the direct vasodilator sodium nitroprusside remains preserved, and the impaired responses to acetylcholine must be related to alterations in endothelial function. In experimental animals, the degree of impairment of endo¬ thelium-dependent responses is positively correlated with the level of blood pressure and seems to increase as a function of the severity and duration of hypertension.74 This suggests that most of the endothelial dysfunction in hypertension is a conse¬ quence rather than a cause of the high blood pressure. As in perfused mesenteric resistance arteries of the SHR, endothe¬ lium-dependent relaxation is reduced by intraluminal but not by extraluminal application of acetylcholine.75 The mechanisms responsible for impaired endothelium-dependent responses in hypertension (Fig. 179-6) include (a) decreased release or increased inactivation of EDNO; (b) decreased release of other endothelium-derived vasodilator substances, such as endo¬ thelium-dependent hyperpolarizing factor or PGI2; (c) impaired diffusion of these substances from the endothelium to the vascular smooth muscle cells; (d) decreased responsiveness of the vascular smooth muscle cells to vasodilator substances; and (e) augmented release of endothelium-derived contracting factors.

FORMATION OF NITRIC OXIDE Although endothelium-dependent relaxation is either dimin¬ ished or normal in spontaneous hypertension, the production of NO seems to be increased. The release of NO from isolated coro¬ nary vessels is augmented in the SHR.76 The activity of constitu¬ tive nitric oxide synthase (cNOS) is also enhanced in the SHR.77 These data suggest that, in the rat, blood pressure per se is a stim¬ ulus for NOS activation. This interpretation is reinforced by the fact that cNOS activity is normal in the prehypertensive 4-week old SHR.77 In spite of an increased activity of the L-arginine path¬ way, however, the bioavailability of NO probably is diminished due to its increased inactivation.75 Nevertheless, NO production and inactivation might be heterogeneously affected in different forms of hypertension. Indeed, in Dahl salt-sensitive rats, endo¬ thelium-dependent relaxation is impaired, without an involve¬ ment of the cyclooxygenase-dependent pathway (see later). This suggests that the decreased NO production could contribute to the pathogenesis of this form of hypertension (see Fig. 179-6).

1639

Vascular Smooth Muscle

Contraction

Relaxation

Salt-induced Hypertension

FIGURE 179-6. Heterogeneity of endothelial dysfunction in spontane¬ ous and salt-induced hypertension. Although the L-arginine-nitric oxide (NO) pathway is overactive in the former, NO seems to be inacti¬ vated by oxygen-derived free radicals (Of) in the latter. In addition, a vasoconstrictor prostanoid (prostaglandin H2 [PGH2]) is formed (top panel). In contrast, in the Dahl rats, a deficient production of NO is most likely (bottom panel). Circles represent receptors. (ADP, adenosine diphosphate; Ach, acetylcholine; All, angiotensin II; ATP, adenosine triphosphate; Thr, thrombin; 5-HT, serotonin; M, muscarinic receptor; T, thrombin receptor; P2, purinergic receptor; Sj, serotonergic receptor; COX-1, cyclooxygenase-1; L-Arg, L-arginine; NOS, nitric oxide syn¬ thase; Tx, thromboxane receptor; cGMP, cyclic guanosine monophos¬ phate; ET-1, endothelin-1; ETA, endothelin-receptor subtype; ETg, endothelin-receptor subtype.) (From Moreau P, Nava E, Takase H, Luscher TF. Handbook of hypertension, vol 17. Pathophysiology of hyper¬ tension. Zanchetti A, Mancia G, eds. Amsterdam: Elsevier Science, 1997.) Pharmacologic experiments in humans have provided indirect evidence for a diminished basal and stimulated NO production. Most studies have found a reduced endothelium-dependent vasodilation in patients with primary or secondary hyperten¬ sion.69 In patients with hypertension, the endothelium-dependent vasodilation in response to acetylcholine is improved after treatment with a cyclooxygenase inhibitor.69 Because inhibition of cyclooxygenase-derived contracting factors does not fully normalize endothelium-dependent vasodilation in hyperten¬ sive subjects, however, an additional defect that involves the L-arginine-NO pathway is implicated. In patients with essential hypertension, treatment with L-arginine does not affect the

1 640

PART X: DIFFUSE HORMONAL SECRETION

response to acetylcholine,78 suggesting that this defect involves either the uptake of the precursor of NO or another pathway (i.e., endothelium-dependent hyperpolarizing factor; see earlier).

native 5-HT oxLDL LDL

SIN-1

5-HT TxA,

ENDOTHELIUM-DEPENDENT CONTRACTIONS Although the assumption is commonly made that impaired endothelium-dependent relaxations are primarily related to reduced activity of NO, they may also be caused by increased pro¬ duction of endothelium-derived contracting factors (EDCF)63 (see Fig. 179-6). In the SHR, the reduced response to acetylcholine in the aorta is related to the production of PGHr In the circulation of the human forearm, impaired vasodilation to acetylcholine is improved (although not normalized) by pretreatment with indomethacin (a cyclooxygenase inhibitor) in patients with essen¬ tial hypertension,78 suggesting that increased production of PGH2 or another cyclooxygenase-derived contracting factor contributes to impaired endothelium-dependent vascular regulation in human hypertension. A similar observation has been made in a new model in which hypertension was produced by long¬ term administration of L-NAME; in these animals, increased endothelium-dependent contractions mediated by TXA2/PGH2 were seen in the aorta.79 This finding does not seem to be limited to the use of acetylcholine, because adenosine diphosphateadenosine triphosphate and serotonin also triggered an enhanced production of EDCF in the cerebral and coronary microcirculation of the SHR. Platelet-derived substances may, therefore, produce more contraction when aggregating in damaged vessels of hyper¬ tensive animals or individuals via a cyclooxygenase-dependent pathway, thus contributing to the complications of hypertension (i.e., stroke, myocardial infarction).64

ENDOTHELIN Circulating levels of endothelin typically are not increased in experimental hypertensive models or humans with hyperten¬ sion.28 This suggests that at least the luminal release of the pep¬ tide into the circulation is unaltered except in the presence of vascular disease (i.e., atherosclerosis) or renal failure. Because more than twice as much endothelin is released abluminally,23 however, measurement of circulating endothelin levels may not be appropriate to determine local vascular endothelin produc¬ tion. In mesenteric resistance arteries of DOCA (desoxycorticosterone acetate)-salt hypertensive rats, but not in SHRs, increased production of endothelin occurs even in the presence of normal circulating levels of the peptide (see Fig. 179-6). In contrast to the direct contractile responses to endothelin, the potentiating properties of low and threshold concentrations of endothelin are increased with aging and hypertension,28-31 indicating that this indirect amplifying effect of endothelin could contribute to increased vascular contractility as pressure rises and the blood vessel wall ages. The use of endothelin-receptor antagonists, which are becoming increasingly available, will help to determine the role of endothelin in health and disease. In hypertension the picture is still unclear. In DOCA-salt hypertensive rats, endothelin seems to be implicated in the maintenance of hypertension and in vascular hypertrophy.80 In SHRs, however, the effect does not seem consistent, as two studies reported conflicting effects of BQ-123, a selective ETA-receptor antagonist.81-82 Studies of human hypertension using the first approved antagonists are elucidat¬ ing the contribution of endothelin in hypertension.83

ENDOTHELIUM-DEPENDENT RESPONSES IN ATHEROSCLEROSIS HYPERLIPIDEMIA Morphologically, the endothelium remains intact in preatherogenesis to the early stage of atherogenesis.85 Functional alter¬

r'NO (EDRF,) Relaxation EDRF2 ■-r..-.-.—

TContraction |

cGMPt

Vascular smooth muscle cell FIGURE 179-7. Schematic representation of the effects of low-density lipoproteins (LDL) in the blood vessel wall. Most likely, oxidation of LDL is an important step in the dysfunction of the endothelium in hyperlipidemia and atherosclerosis. Oxidized low-density lipoproteins (OX-LDL) may interact with the intracellular availability of L-arginine (L-Arg) and the G protein (G,) of the serotonergic receptor (S,), and they may also inactivate nitric oxide (NO). In addition, OX-LDL can increase the endothelial production of endothelin-1 by protein kinase C. (5-HT, serotonin; BK, bradykinin; SIN-1, molsidomine [nitric acid donor]; TXA2, thromboxane A2; EDRFv endothelium-derived relaxing factor 1; cGMP, cyclic guanosine monophosphate; S2, 5-HT2 [5-hydroxytryptamine] serotonergic receptor.)

ations occur, however, possibly due to the presence of oxidized low-density lipoproteins (OX-LDLs).86 In isolated porcine coronary arteries, endothelium-dependent relaxation to platelets, serotonin, and thrombin is inhibited by OX-LDLs.87-88 In contrast, relaxation to the NO-donor linsidornine is maintained, excluding reduced responsiveness of smooth muscle to EDNO. This inhibition is specific for OX-LDLs because it is not induced by comparable concentrations of native LDL.87 The inhibitor of NO production, L-NMMA, exerts an inhibitory effect on endothelium-dependent relaxation similar to that of the modified lipoproteins, suggesting that OX-LDLs inter¬ fere with the L-arginine pathway. The activity of NO synthase appears to remain unaffected, because L-arginine evokes full relaxation in vessels treated with OX-LDLs. Pretreatment of iso¬ lated vessels with L-arginine improves the reduced endotheliumdependent responses to serotonin.89 Thus, OX-LDLs may interact with the intracellular signal transduction mechanisms (e.g., the function of Gs proteins)90 or the availability of L-arginine87 (Fig. 179-7). Similarly, in hypercholesterolemic pigs, in vivo inhibition of endothelium-dependent relaxation in response to serotonin occurs in coronary arteries exposed to OX-LDLs.91 Furthermore, in humans with hypercholesterolemia, L-arginine infusion aug¬ ments the blunted increase in local blood flow in response to ace¬ tylcholine.92 In addition to their effect on the L-arginine pathway, OX-LDLs inactivate NO and cause endothelium-dependent,89 as well as endothelium-independent, contractions.93 OX-LDLs induce endothelin-1 mRNA expression and endothelin-1 release94 (see Fig. 179-7). Threshold and low con¬ centrations of endothelin potentiate contractions induced by serotonin in the human coronary artery. Similarly, endothelin-1 potentiates norepinephrine- and serotonin-induced contrac¬ tions in the human internal mammary artery. Thus, even small increases in local endothelin levels may be important.95

ATHEROSCLEROSIS Atherosclerosis is associated with severe morphologic changes of the intima of large arteries (i.e., intimal thickening, prolifera¬ tion of smooth muscle cells, accumulation of lipid-containing macrophages).85 Endothelial denudation does not occur, how¬ ever, except at late stages. In porcine coronary arteries, established atherosclerosis severely impairs endothelium-dependent relaxation to seroto¬ nin and also reduces endothelium-dependent relaxation to

Ch. 179: The Endocrine Endothelium

bradykinin in the presence of hypercholesterolemia.91 Endothe¬ lium-independent relaxation to nitrovasodilators remains pre¬ served, however, except in severely atherosclerotic arteries. Similarly, in atherosclerotic human coronary arteries, endothe¬ lium-dependent relaxation to substance P, bradykinin, aggre¬ gating platelets, and calcium ionophores is attenuated,96 and in vivo acetylcholine causes paradoxical vasoconstriction. In vivo, the activity of the L-arginine/NO pathway is a bal¬ ance between the synthesis and the breakdown of NO.97 Con¬ troversy exists regarding the mechanism responsible for the marked impairment or loss of endothelium-dependent relax¬ ation in atherosclerosis. EDRF release as measured by bioassay experiments in porcine coronary arteries with hypercholester¬ olemia and atherosclerosis have shown that the release of bioac¬ tive NO is reduced.91 Direct measurements of NO in the rabbit aorta, however, suggest increased formation of NO with con¬ comitant massive breakdown of the endogenous nitrovasodilator (to the bioinactive nitrite and nitrate).98 This observation suggests that increased formation of superoxide radicals and other products in the endothelium inactivates NO, possibly as a result of decreased activity of superoxide dismutase in the ath¬ erosclerotic blood vessel wall. Increased circulating levels of endothelin are associated with human atherosclerosis,99 and the increase in endothelin levels correlates positively with the degree of atherosclerotic disease and the number of vascular beds involved. The increased endo¬ thelin production is derived not only from endothelial cells of ath¬ erosclerotic blood vessels, but also from vascular smooth muscle cells migrating into the intima. Increased local levels of endothe¬ lin may contribute to the known vasoconstrictor responses of atherosclerotic blood vessels and, because of the proliferative properties of endothelin,50 to the atherosclerotic process itself.

DIABETES Micro- and macrovascular damage are hallmark abnormalities associated with diabetes (see Chap. 147). Endothelial dysfunction, including abnormal expression of NO and endothelin, is noted in type 1 and type 2 diabetes mellitus and in the earliest stages, includ¬ ing in impaired glucose tolerance, and in first-degree relatives.100-101

REFERENCES 1. Luscher TF, Vanhoutte PM. The endothelium: modulator of cardiovascular function. Boca Raton, FL: CRC Press, 1990:1. 2. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980; 299:373. 3. Yang Z, Stulz P, von Segesser L, et al. Different interactions of platelets with arterial and venous coronary bypass vessels. Lancet 1991; 337:939. 4. Luscher TF, Diederich D, Siebenmann R, et al. Difference between endothe¬ lium-dependent relaxations in arterial and in venous coronary bypass grafts. N Engl J Med 1991; 319:462. 5. Rubanyi GM, Romero JC, Vanhoutte PM. Flow-induced release of endothe¬ lium-derived relaxing factor. Am J Physiol 1986; 250:H1145. 6. Pohl U, Floltz J, Busse R, Bassenge E. Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension 1986; 8:37. 7. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the bio¬ logical activity of endothelium-derived relaxing factor. Nature 1987; 327:524. 8. Feelisch M, te Poel M, Zamora R, et al. Understanding the controversy over the identity of EDRF. Nature 1994; 368:62. 9. Rapoport RM, Draznin MB, Murad F. Endothelium-dependent relaxation in rat aorta may be mediated through cyclic GMP-dependent protein phos¬ phorylation. Nature 1983; 306:174. 10. Radomski MW, Palmer RMJ, Moncada S. Comparative pharmacology of endothelium-derived relaxing factor, nitric oxide and prostacyclin in plate¬ lets. Br J Pharmacol 1987; 92:181. 11. Palmer RMJ, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 1988; 333:664. 12. Bredt DS, Hwang PM, Glatt CE, et al. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature 1991; 351:714. 13. Cosentino F, Luscher TF. Tetrahydrobiopterin and endothelial function. Eur Heart J 1998; suppl G:G3. 14. Richard V, Tschudi MR, Luscher TF. Differential activation of the endothe¬ lial L-arginine pathway by bradykinin, serotonin and clonidine in porcine coronary arteries. Am J Physiol 1990; 259:H-1433.

1641

15. Tschudi M, Richard V, Biihler FR, Luscher TF. Importance of endotheliumderived nitric oxide in intramyocardial porcine coronary arteries. Am J Physiol 1990; 260:H13. 16. Meyer P, Flammer J, Luscher TF. Endothelium-dependent regulation of the ophthalmic microcirculation in the perfused porcine eye. Role of nitric oxide and endothelins. Invest Ophthalmol Vis Sci 1993; 34:3614. 17. Rees DD, Palmer RMJ, Moncada S. The role of endothelium-derived nitric oxide in the regulation of blood pressure. Proc Natl Acad Sci U S A 198986:3375. 18. Vallance P, Leone A, Colver A, et al. Accumulation of an endogenous inhib¬ itor of nitric acid synthesis in chronic renal failure. Lancet 1992; 339:572. 19. Boger RH, Bode-Boger SM, Szuba A, et al. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction. Its role in hyper¬ cholesterolemia. Circulation 1998; 98:1842. 20. Feletou M, Vanhoutte PM. Endothelium-dependent hyperpolarization of canine coronary smooth muscle. Br J Pharmacol 1988; 93:515. 21. Standen NB, Quayle JM, Davies NW, et al. Hyperpolarizing vasodilators activate ATP-sensitive K+-channels in arterial smooth muscle. Science 1989; 245:177. 22. Moncada S, Vane JR. Pharmacology and endogenous roles of prostaglan¬ din endoperoxides, thromboxane A2 and prostacyclin. Pharmacol Rev 1979; 30:293. 23. Wagner O, Christ G, Wojta J, et al. Polar secretion of endothelin-1 by cul¬ tured endothelial cells. J Biomed Chem 1992; 267:16066. 24. De Mey JG, Claeys M, Vanhoutte PM. Endothelium-dependent inhibitory effects of acetylcholine, adenosine diphosphate, thrombin and arachidonic acid in the canine femoral artery. J Pharmacol Exp Ther 1982; 222:166. 25. Yang Z, von Segesser L, Bauer E, et al. Differential activation of the endo¬ thelial L-arginine and cyclooxygenase pathway in the human internal mammary artery and saphenous vein. Circ Res 1991; 68:52. 26. Katusic ZS, Vanhoutte PM. Superoxide anion is an endothelium-derived contracting factor. Am J Physiol 1989; 257:H33. 27. Cosentino F, Katusic ZS. Role of superoxide anion in mediation of endothe¬ lium-dependent contractions. Hypertension 1994; 23:229. 28. Luscher TF, Boulanger CM, Dohi Y, Yang Z. Endothelium-derived contract¬ ing factors. (Brief review). Hypertension 1992; 19:117. 29. Kohno M, Yasunari K, Yokokawa K, et al. Inhibition by atrial and brain natri¬ uretic peptide of endothelin. A secretion after stimulation with angiotensin II and thrombin of cultured human endothelial cells. J Clin Invest 1991; 87:1999. 30. Boulanger C, Luscher TF. Release of endothelin from the porcine aorta: inhibition by endothelium-derived nitric oxide. J Clin Invest 1990; 85:587. 31. Dohi Y, Hahn AWA, Boulanger CM, et al. Endothelin stimulated by angio¬ tensin II augments vascular contractility of hypertensive resistance arter¬ ies. Hypertension 1992; 19:131. 32. Kourembanas S, Marsden PA, McQullan LP, Faller DV. Hypoxia induces endothelin gene expression and secretion in cultured human endothelium. J Clin Invest 1991; 88:1054. 33. Luscher TF, Yang Z, Tschudi M, et al. Interaction between endothelin-1 and endothelium-derived relaxing factor in human arteries and veins. Circ Res 1990; 66:1088. 34. Kiowski W, Luscher TF, Linder L, Buhler FR. Endothelin-l-induced vasocon¬ striction in man: reversal by calcium channel blockade but not by nitrova¬ sodilators or endothelium-derived relaxing factor. Circulation 1991; 83:469. 35. Saijonmaa O, Ristimaki A, Fyhrquist F. Atrial natriuretic peptide, nitroglyc¬ erine, and nitroprusside reduce basal and stimulated endothelin produc¬ tion from cultured endothelial cells. Biochem Biophys Res Common 1990; 173:514. 36. Yokokawa K, Kohno M, Yasunari K, et al. Endothelin-3 regulates endothelin-1 production in cultured human endothelial cells. Hypertension 1991; 18:304. 37. Stewart DJ, Langleben D, Cemacek P, Cianflone K. Endothelin release is inhibited by coculture of endothelial cells with cells of vascular media. Am J Physiol 1990; 259:H1928. 38. Warner TD, Mitchell JA, de Nucci G, Vane JR. Endothelin-1 and endothe¬ lin-3 release EDRF from isolated perfused arterial vessels of the rat and rabbit. J Cardiovasc Pharmacol 1989; 13(Suppl 5):85. 39. Arai H, Hori S, Aramori I, et al. Cloning and expression of a cDNA encod¬ ing an endothelin receptor. Nature 1990; 348:730. 40. Sakurai T, Yanagisawa M, Takuwa Y. Cloning of a cDNA encoding a non¬ isopeptide-selective subtype of the endothelin receptor. Nature 1990; 348:732. 41. Emori T, Hirata Y, Marumo F. Specific receptors for endothelin-3 in cul¬ tured bovine endothelial cells and its cellular mechanism of action. FEBS Lett 1990; 263:261. 42. Luscher TF. Do we need endothelin antagonists? Cardiovasc Res 1993; 27:2089. 43. Shai S-Y, Fishel RS, Martin BM, et al. Bovine angiotensin converting enzyme cDNA cloning and regulation. Increased expression during endo¬ thelial cell growth arrest. Circ Res 1992; 70:1274. 44. Severs WB, Daniels-Severs AE. Effects of angiotensin on the central ner¬ vous system. Pharmacol Rev 1973; 25:415. 45. Dubey RK, Roy A, Overbeck HW. Culture of renal arteriolar smooth mus¬ cle cells: mitogenic responses to Ang II. Circ Res 1992; 71:1143. 46. Luscher TF, Tanner FC. Endothelial regulation of vascular tone and growth. Am J Hypertens 1993; 6:283S. 47. Dzau VJ, Gibbons GH. Vascular remodelling: mechanisms and implica¬ tions. J Cardiovasc Pharmacol 1993; 21(Suppl I):S1. 48. Garg UC, Hassid A. Nitric-oxide generating vasodilators and 8-bromocyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular SMCs. J Clin Invest 1989; 83:1774.

1 642

PART X: DIFFUSE HORMONAL SECRETION

49. Dubey RK, Ganten D, Luscher TF. Enhanced migration of smooth muscle cells from Ren-2 transgenic rats in response to angiotensin II: inhibition by nitric oxide. Hypertension 1993; 22:412. 50. Hirata Y, Takagi Y, Fukuda Y, Marumo F. Endothelin is a potent mitogen for rat vascular smooth muscle cells. Atherosclerosis 1989; 78:225. 51. O'Reilly MS, Holmgren L, Shing Y et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by Lewis lung carci¬ noma. Cell 1994; 79:315. 52. O'Reilly MS, Boehm T, Shin Y, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 1997; 88:277. 53. Harris AL. Are angiostatin and endostatin cures for cancer? Lancet 1998; 351:1598. 54. Vidal-Ragout MJ, Romero JC, Vanhoutte PM. Endothelium-derived relax¬ ing factor inhibits renin release. Eur J Pharmacol 1988; 149:401. 55. Matsumura Y, Nakase K, Ikegawa R, et al. The endothelium-derived vasocon¬ strictor peptide endothelin inhibits renin release in vitro. Life Sci 1989; 44:149. 56. Miller WL, Redfield MM, Burnett JC Jr. Integrated cardiac, renal, and endo¬ crine actions of endothelin. J Clin Invest 1989; 83:317. 57. Lorenz RR, Sanchez-Ferrer CF, Burnett JC, Vanhoutte PM. Influence of endocardial derived factor(s) on the release of atrial natriuretic factor. (Abstract). FASEB J 1988; 2:1293. 58. Fukuda Y, Hirata Y, Yoshimi H, et al. Endothelin is a potent secretagogue for atrial natriuretic peptide in cultured rat atrial myocytes. Biochem Biophys Res Commun 1988; 155:167. 59. Vierhapper H, Wagner O, Nowotny P, Waldhausl W. Effect of endothelin-1 in man. Circulation 1990; 81:1415. 60. Boarder MR, Marriott DB. Characterization of endothelin-1 stimulation of catecholamine release from adrenal chromaffin cells. J Cardiovasc Pharma¬ col 1989; 13(Suppl 5):223. 61. Wiklundin NP, Oehlen A, Cederqvist B. Inhibition of adrenergic neuroef¬ fector transmission by endothelin in the guinea-pig femoral artery. Acta Physiol Scand 1988; 134:311. 62. Gomez-Sanchez CE, Foecking MF, Chiou S. Endothelin binding to cultured calf adrenal zona glomerulosa cells and stimulation of aldosterone secre¬ tion. J Clin Invest 1989; 84:1032. 63. Luscher TF, Vanhoutte PM. Endothelium-dependent contractions to acetyl¬ choline in the aorta of the spontaneously hypertensive rat. Hypertension 1986; 8:344. 64. Luscher TF, Vanhoutte PM. Endothelium-dependent responses to aggre¬ gating platelets and serotonin in spontaneously hypertensive rats. Hyper¬ tension 1986; 8(Suppl II):55. 65. Mayhan WG, Faraci FM, Heistad DD. Impairment of endothelium-dependent responses of cerebral arterioles in chronic hypertension. Am J Physiol 1987; 253:H1435. 66. Linder L, Kiowski W, Biihler FR, Luscher TF. Indirect evidence for release of endothelium-derived relaxing factor in human forearm circulation in vivo: blunted response in essential hypertension. Circulation 1990; 81:1762. 67. Panza JA, Quyyumi AA, Brush JE Jr, Epstein SE. Abnormal vascular endo¬ thelium-dependent vascular relaxation in patients with essential hyperten¬ sion. N Engl J Med 1990; 323:22. 68. Creager MA, Roddy M-A, Coleman SM, Dzau VJ. The effect of ACE inhibition on endothelium-dependent vasodilation in hypertension. J Vase Res 1992; 29:97. 69. Taddei S, Virdis A, Mattei P, Salvetti A. Vasodilation to acetylcholine in primary and secondary forms of human hypertension. Hypertension 1993; 21:929. 70. Luscher TF. The endothelium and cardiovascular disease—a complex rela¬ tion. N Engl J Med 1994; 330:1081. 71. Cockcroft J, Chowienczyk PJ, Benjamin N, Ritter JM. Preserved endothe¬ lium-dependent vasodilation in patients with essential hypertension. N Engl J Med 1994; 330:1036. 72. Tschudi MR, Criscione L, Luscher TF. Effect of aging and hypertension on endothelial function of rat coronary arteries. J Hypertens 1991; 9(Suppl 6):164. 73. Treasure CB, Manoukian SV, Klein JL, et al. Epicardial coronary artery responses to acetylcholine are impaired in hypertensive patients. Circ Res 1992; 71:776. 74. Luscher TF, Vanhoutte PM, Raij L. Antihypertensive therapy normalizes endothelium-dependent relaxations in salt-induced hypertension of the rat. Hypertension 1987; 9(Suppl III):193. 75. Dohi Y, Thiel M, Biihler FR, Luscher TF. Activation of the endothelial Larginine pathway in pressurized mesenteric resistance arteries: effect of age and hypertension. Hypertension 1990; 15:170. 76. Kelm M, Feelisch M, Krebber T, et al. The role of nitric oxide in the regula¬ tion of coronary vascular resistance in arterial hypertension: comparison of normotensive and spontaneously hypertensive rats. J Cardiovasc Pharma¬ col 1992; 20:183. 77. Nava E, Noll G, Luscher TF. Increased activity of constitutive nitric oxide synthase in cardiac endothelium in spontaneous hypertension. Circulation 1995; 91:2310. 78. Panza JA, Casino PR, Badar DM, Quyyumi AA. Effect of increased avail¬ ability of endothelium-derived nitric oxide on endothelium-dependent vascular relaxation in normals and in patients with essential hypertension. Circulation 1993; 87:1475. 79. Kung CF, Moreau P, Takase H, Luscher TF. L-NAME-induced hypertension impairs endothelial function in rat aorta: reversal by trandolapril and verapamil. Hypertension 1995; 26(5):744. 80. Li JS, Lariviere R, Schiffrin EL. Effect of a nonselective endothelin antago¬ nist on vascular remodeling in deoxycorticosterone acetate salt hyperten¬

81. 82.

83.

84. 85. 86.

87.

88.

89.

90.

91.

92.

sive rats: evidence for a role of endothelin in vascular hypertrophy. Hypertension 1994; 24:183. Bank N, Aynedijan HS, Khan GA. Mechanism of vasoconstriction induced by chronic inhibition of nitric oxide in rats. Hypertension 1994; 24:322. Nishikibe M, Ikada M, Tsuchida S, et al. Antihypertensive effect of a newly synthesized endothelin antagonist, BQ-123, in genetic hypertension mod¬ els. J Hypertens 1992; 10(Suppl 4):53. Krum H, Viskoper RJ, Lacourciere Y, et al. The effect of an endothelinreceptor antagonist,-bosentan, on blood pressure in patients with essential hypertension. N Engl J Med 1998; 338:784. Hayzer PJ, Cicila G, Cockerham C, et al. Endothelin A and B receptors are downregulated in the hearts of hypertensive rats. Am J Med Sci 1994; 307:222. Ross R. The pathogenesis of atherosclerosis—an update. N Engl J Med 1986; 314:488. Yla-Herttuala S, Palinski W, Rosenfeld ME, et al. Evidence for the presence of oxidatively modified low-density lipoproteins in atherosclerotic lesions of rabbit and man. J Clin Invest 1989; 84:1086. Tanner FC, Noll G, Boulanger CM, Luscher TF. Oxidized low-density lipo¬ proteins inhibit relaxations of porcine coronary arteries: role of scavenger receptor and endothelium-derived nitric oxide. Circulation 1991; 83:2012. Kugiyama K, Kerns SA, Morrisett JD, et al. Impairment of endotheliumdependent arterial relaxation by lysolecithin in modified low-density lipo¬ proteins. Nature 1990; 344:160. Simon BC, Cunningham LD, Cohen RA. Oxidized low density lipoproteins cause contraction and inhibit endothelium-dependent relaxation in the pig coronary artery. J Clin Invest 1990; 86:75. Flavahan NA. Atherosclerosis or lipoprotein-induced endothelial dysfunc¬ tion: potential mechanisms underlying reduction in dysfunction in EDRF/ nitric oxide activity. Circulation 1992; 85:1927. Shimokawa H, Vanhoutte PM. Impaired endothelium-dependent relaxation to aggregating platelets and related vasoactive substances in porcine coronary arteries in hypercholesterolemia and atherosclerosis. Circ Res 1989; 64:900. Creager MA, Gallagher SH, Girerd XJ, et al. L-arginine improves endothe¬ lium-dependent vasodilation in hypercholesterolemic humans. J Clin

Invest 1992; 90:1248. 93. Galle J, Bassenge E, Busse R. Oxidized low-density lipoproteins potentiate vasoconstrictions to various agonists by direct interaction with vascular smooth muscle. Circ Res 1990; 66:1287. 94. Boulanger CM, Tanner FC, Hahn AWA, et al. Oxidized low-density lipo¬ proteins induce mRNA expression and release of endothelin from human and porcine endothelium. Circ Res 1992; 70:1191. 95. Yang Z, Richard V, von Segesser L, et al. Threshold concentrations of endothelin-1 potentiate contractions to norepinephrine and serotonin in human arteries: a new mechanism of vasospasm? Circulation 1990; 82:188. 96. Forstermann U, Mtigge A, Alheid U, et al. Selective attenuation of endothe¬ lium-mediated vasodilation in atherosclerotic human coronary arteries. Circ Res 1988; 62:185. 97. Wever RMF, Luscher TF, Cosentino F, Rabelink TJ. Atherosclerosis and the two faces of endothelial nitric oxide synthase. Circulation 1998; 97:108. 98. Minor RL, Myers RR Jr, Guerra R Jr, et al. Diet-induced atherosclerosis increases the release of nitrogen oxides from rabbit aorta. J Clin Invest 1990; 86:2109. 99. Lerman A, Edwards BS, Hallett JW, et al. Circulating and tissue endothelin immunoreactivity in advanced atherosclerosis. N Engl J Med 1991; 325:997. 100. De Vriese AS, Verbeuren TJ, Van de Voorde J, et al. Endothelial dysfunction in diabetes. Br J Pharmacol 2000; 130:963. 101. Balletshoffer BM, Rittig K, Enderle MD, et al. Endothelial dysfunction is detectable in young normotensive first-degree relatives of subjects witji type 2 DM associated with insulin resistance. Circ 2000; 101:1780.

CHAPTER

180

THE ENDOCRINE BLOOD CELLS HARISH P. G. DAVE AND BEAT MOLLER

FLOATING ENDOCRINE SYSTEM Blood cells play a critical role in tissue oxygenation as well as in hemostasis and immune function. Besides these well-estab¬ lished and accepted roles of blood cells, recognition is emerging of the ability of these cells to elaborate a variety of substances (e.g., cytokines, regulatory peptides, and glycoproteins) that

Ch. 180: The Endocrine Blood Cells TABLE 180-1. Hormones Produced by Blood Cells

LYMPHOCYTES IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-14, G-CSF, GM-CSF, M-CSF, interferons, MIP-1, RANTES, TNF, LIF, NGF, CGRP, MIF, ACTH, TSH, GH and IGF-I, PTHrP, VIP, prolactin, vasopressin, enkephalin, preprotachykinin, GnRH, CRH, TRH

MONOCYTES/MACROPHAGES/DENDRITIC CELLS IL-1, IL-5, IL-6, IL-8, IL-10, IL-12, G-CSF, GM-CSF, M-CSF, interferons, TNF, LIF, MIF, TGF, FGF, MIP, NGF, VEGF, ACTH, preprotachykinin, proglucagon, prosomatostatin, propancreatic polypeptide, preproinsu¬ lin, VIP, TSH, ?GnRH

NEUTROPHILS IL-1, IL-3, IL-6, IL-8, G-CSF, GM-CSF, M-CSF, interferons

EOSINOPHILS IL-1, IL-3, IL-5, IL-6, GM-CSF, TNF, MIP, TGF

BASOPHILS/MAST CELLS IL-1, IL-3, IL-4, IL-5, IL-6, IL-8, GM-CSF, MIP, TNF, LIF

PLATELETS IL-1, PDGF, TGF, EGF, RANTES See text for abbreviation nomenclature.

have autocrine, paracrine, juxtacrine, and hemocrine effects. They also secrete classic hormones of the endocrine system. With this plurality of hormonal effects, blood cells constitute a floating endocrine system.

ENDOCRINE BLOOD CELLS Blood cells consist of lymphocytes, neutrophils, monocytes, eosinophils, and basophils—all subsets of leukocytes (white cells)—as well as thrombocytes (platelets) and erythrocytes (red cells).1 All of these cell types, except for the mature cir¬ culating erythrocytes, produce hormones. The red cell loses its nucleus before emerging from the bone marrow and does not directly elaborate hormones. The hormones produced by these various blood cell types are summarized in Table 180-1.

MONOCYTES Monocytes are bone marrow-derived cells that represent from 1% to 6% of leukocytes in the adult. After a short stay in the cir¬ culation, they enter the tissues and are recognizable there as macrophages, which are longer-lived cells. Tissue macrophages may also replenish themselves by cell division depending on local factors and are not wholly dependent on the blood mono¬ cyte pool. They have three main functions2: as phagocytic cells, they engulf microbes and other foreign particles, and, together with monocytes and polymorphonuclear granulocytes, form the phagocytic system of the body; as antigen-presenting cells, they take up, process, and present antigen to T and B lympho¬ cytes; finally, as immunomodulators, they produce and release various cytokines (also called monokines). Macrophages produce an array of secretory chemicals that are important regulators of inflammation. The lymphocyte acti¬ vation that occurs from antigen presentation results in the secre¬ tion of additional factors that further activate the macrophage. The secretory products include polypeptide hormones, cyto¬ kines, inhibitors of cytokines, bioactive oligopeptides and lipids, sterol hormones, reactive oxygen and its intermediaries, and reactive nitrogen intermediaries.3 Macrophages are strategically positioned to provide a defense against organisms entering the body via the respira¬

1643

tory tract (alveolar macrophages), gastrointestinal tract (Kupffer cells of the liver and peritoneal macrophages), blood¬ stream (splenic macrophages), skin (Langerhans cells), and lymphatics (lymph node macrophages). In addition, they are present in all other organs and tissues in a large variety of phe¬ notypes, extending from microglial cells of the brain, synovial A cells of the synovial cavity of joints, and mesangial phago¬ cytes in the kidney, to the multinucleated osteoclasts of the bone. Kupffer cells in the liver contribute up to 90% of all the macrophages in the body and up to 15% of all cells in the liver. The phagocytic tissue macrophages, together with endothelial cells, constitute what was previously called the "reticuloendo¬ thelial system" (RES). Through phagocytosis, tissue macrophages play an impor¬ tant role in the clearing of microbes and the removal of dam¬ aged or effete tissue cells or of extracellular matrix. Specific receptors on the cell surface of macrophages mediate the phagocytic function: receptors for the Fc portion of immuno¬ globulin G antibody recognize antibody-coated microbes; CR1 and CR3 receptors recognize complement; and CD40 receptors recognize lipopolysaccharide found in the cell wall of gram¬ negative bacteria, among others. Specialized macrophages, also known as interdigitating dendritic cells, are typical antigen-presenting cells. These cells are able to initiate a specific immune response against a microbial antigen. Dendritic cells ingest a microbial or tumor antigen and process it. After migrating into second¬ ary lymphoid tissue, they present the processed antigen (on the surface in association with the major histocompatibility complex [MHC] class II) to other cells of the immune system (i.e., T cells).4 Dendritic cells are highly mobile cells, and the sequential migration of these cells into and out of tissues is accompanied by phenotypical as well as functional changes that are instrumental to their role as sentinels of the immune system.Dendritic cells can be of macrophage or lympho¬ cyte origin. The cytokine expression pattern and behavior differ between dendritic cells that are morphologically and immunophenotypically related to macrophages, between those that are classified as immature or mature dendritic cells, and also between those that are related to B or to T lymphocytes. A detailed list of the cytokines that these cells produce is likely to be incomplete, because newer subsets are in the process of being defined and tested under various experimental conditions. At a minimum, most dendritic cells produce a number of interleukins (i.e., IL-ip, IL-6, IL10, IL-12, and IL-18) and tumor necrosis factor-a (TNF-a).8-13 Determining the precise cellular origin of interferons has been difficult due to the rapid apoptosis of these cells; none¬ theless, evidence indicates that the dendritic cells are the source of these cytokines, which play an important role in the antiviral immune response. Intriguing evidence also exists for the expression of preproinsulin by murine thymic dendritic cells. Dendritic cells also produce macrophage inflammatory protein-ly (MIP-ly), macrophage inflamma¬ tory protein-la (MIP-la), and macrophage inflammatory protein-2 (MIP-2).8 Nitric oxide, a potent regulator of dis¬ parate cellular processes, is also elaborated by dendritic cells.14 Dendritic cell-derived nitric oxide promotes apoptosis15 of autoreactive T cells and may therefore play a role in autoimmunity. Cytokines. Macrophages produce and secrete a wide array of substances (termed cytokines or monokines) ranging in molecular mass from 32 Da (superoxide anion) to almost 500,000 kDa (fibronectin), and ranging in bioactivity from induction of cell growth to cell death.16 TNF-a and several interleukins (e.g., IL-1 and IL-6) are the classic mediators of these cells; however, the nitric oxide that is also produced by macrophages may differentially affect vascular tone in differ¬ ent vascular beds. Macrophage migration inhibitory factor

1644

PART X: DIFFUSE HORMONAL SECRETION

(M1F) is released by macrophages and T cells in response to glucocorticoids as well as to various proinflammatory stim¬ uli. Once secreted, the MIF “overrides" the immunosuppres¬ sive effects of glucocorticoids on macrophages and T cells. MIF is also produced by many other cell types, which in turn can trap and also respond to these cytokines via cognate receptors.2 Cytokines are acutely synthesized and secreted in response to stimuli, unlike most classic hormones, which are preformed and stored within cell granules before a suitable stimulus is received. Simi¬ lar to most classic hormones, most cytokines (with the notable exceptions of IL-1 and TNF-a) are synthesized in the form of precursors and have an amino-terminal leader sequence direct¬ ing their transport to the Golgi apparatus in preparation for sub¬ sequent secretion. Most of the monokines have potent and diverse systemic effects in addition to their effects on immune function. The effects of different cytokines are often pleiotropic and overlapping; they can be synergistic or antagonistic, depending on the experimental system. A subset of monokines with known interactions with the endocrine system is listed in Table 180-2. Reports of the production of classic hormones by macro¬ phages (detected at the mRNA or protein level) have also appeared. These include preprotachykinin in human mononu¬ clear phagocytes and lymphocytes, as well as preproinsulin, proglucagon, propancreatic polypeptide, and prosomatostatin within the human and murine thymus.17 Hormone expression is enriched in the antigen-presenting cell population, which is presumed to be a mixture of macrophages and dendritic cells. Autoimmunity and self-reactivity are prevented by the dele¬ tion of lymphocyte clones reactive to self-antigens. The close association of hormone-producing cells and dendritic cells in the thymus could lead to the development of a self-reactive lymphocyte clone that is subsequently deleted, thereby reduc¬ ing the likelihood that hormones and other proteins expressed at low levels will incite an autoimmune reaction. Thus, the presence of these pancreatic hormones (i.e., preproinsulin, proglucagon, propancreatic polypeptide, and prosomatosta¬ tin) at low levels in the thymus may be critical in inducing central tolerance to proteins of restricted expression. This would suggest that other self-antigens will also be found in the thymus. Indeed, the presence of albumin, insulin, gluca¬ gon, thyroid peroxidase, glutamic acid peroxidase, thyroglobulin, myelin basic protein, and retinal S antigen has been demonstrated by reverse transcription polymerase chain reac¬ tion in the human thymus at ages from 8 days to 13 years.18 (This study did not exclude the presence of some of these sub¬ stances in thymic epithelial cells.) Vasoactive intestinal pep¬ tide (VIP) and VIP-1 receptor have been detected in rat macrophages.19 Monocytes. Although monocytes are less well studied, they have a pattern of cytokine production similar to that of macrophages. Besides the various cytokines, these cells also synthesize prostaglandins and leukotrienes. Transforming growth factor-p (TGF-(3) is an autocrine hormone for these cells, and its production is further stimulated by 1,25 dihydroxyvitamin D3 and retinoids.20 Interestingly, monocytes in culture can be stimulated by thyrotropin-releasing hormone (TRH) to release thyroid-stimulating hormone (TSH), an effect that can be totally blocked by adding triiodothyronine to the cultured cells.21 Thus, these cells exhibit the same type of control as the hypothalamic-pituitary-thyroid axis. They also display receptors for various peptide hormones and, as with lymphocytes, undergo an increase in receptor number on activation. Activation of monocytes by growth hormone (GH) results in an increase in interferon-y (IFN-y) secretion,22 which, in turn, has additional effects on the surrounding immune cells.

LYMPHOCYTES Lymphocytes are mononuclear cells that represent 20% to 50% of leukocytes. They are further characterized by a cluster of dif¬ ferentiation (CD) antigens. Broadly, they can be categorized as B, T, and natural killer (NK) lymphocytes, although clearly, the CD expression pattern can lead to much more refined subtyp¬ ing. The majority of lymphocytes are long-lived cells that play a critical role in the humoral and cellular immune response; they are the repositories of immune memory. Like the macrophages and dendritic cells, the lymphocytes are metabolically active cells. They produce a multitude of inter¬ leukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-14), colony-stimulating factors (granulocyte colony-stimulating fac¬ tor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF], and macrophage colony-stimulating factor [M-CSF]), interferons (IFN-a, IFN-y), and other factors such as MIP-1, RANTES (regulated on activation, normal T-expressed and secreted), TNF-a, leukemia inhibitory factor (LIF), and nerve growth factor (NGF).1'23 They are also well endowed with recep¬ tors for many of the interleukins and interferons, so that the cells can be affected in both an autocrine and paracrine fashion. In addition, lymphocytes express receptors for many bioactive pep¬ tides such as adrenocorticotropic hormone (ACTH), calcitonin, endorphins, enkephalins, vasopressin, oxytocin, thyrotropin, GH, somatostatin, substance P, and VIP.3 In general, the density of peptide hormone receptors increases markedly after activation of the cells, implying a role for classic hormones as well as the various interleukins in lymphocyte regulation. Just as hormone receptors have been identified on these cells, so has the ability to produce some of their cognate hormones. Concanavalin A-stimulated human lymphocyte cultures pro¬ duce detectable levels of prolactin, luteinizing hormone (LH) and follicle-stimulating hormone (FSH).24 Prolactin production, which can also follow T-cell stimulation of B cells,25 in turn, enhances NK cell function, activates the interferon-regulated factor-1 (IRF-1) transcription factor, and interacts with or gener¬ ates IL-2 and IFN-y.26 The suggestion has also been made that lymphocytes may have an endocrine role in infertility: lower lev¬ els of these hormones are produced by the lymphocytes of infer¬ tile women. Unstimulated lymphocytes secrete GH.27 However, unlike the situation with TSH in monocytes, this GH secretion is not subject to the same regulatory mechanisms as GH produc¬ tion by pituitary cells. Incubation of lymphocytes in media con¬ taining growth hormone-releasing hormone (GHRH) and somatostatin has no effect on GH release.28 Acromegaly has been reported in a patient with a non-Hodgkin lymphoma whose tumor cells secreted GH.28a In addition, pituitary GH deficiency has not been associated with immunodeficiency in humans. Lymphocytes also express the proopiomelanocortin (POMC) gene, leading to the detection of (J-endorphin, enkephalin, and ACTH in these cells.29'30 Human immunodeficiency virus (HIV) infection of lymphocytes leads to the production of ACTH, which has the same bioactivity as ACTH produced by the pitu¬ itary.31 The mechanism for this is unclear, but it may reflect part of the pleiotropic effects of TAT protein produced by the virus. The fratts-activation of the enkephalin gene by HTLV-I TAX protein, which is closely analogous to TAT, has been demon¬ strated 32 This ACTH production may lead to further immuno¬ suppression in HIV-infected individuals. Macrophage MIF is released by macrophages and T cells in response to glucocorticoids as well as in response to stimula¬ tion by various proinflammatory stimuli.33 MIF has also been isolated from anterior pituitary cells.33 Calcitonin gene-related peptide (CGRP), vasopressin, and VIP have been detected in certain subsets of lymphocytes, but their roles in lymphocyte development and immune function are unclear.19'34'35

Ch. 180: The Endocrine Blood Cells

1645

TABLE 180-2. Some Cytokines/Monokines Released by Blood Cells and Their Relationship to the Endocrine System* Cytokine_Major Immune Functions_Endocrine Effects and Clinical Implications INTERLEUKINS (ILs) IL-1 (a and P form)

Prototypic "multifunctional" monokine; affects nearly every cell type, often in concert with other cytokines; mediates acute-phase inflammatory response; cofactor for immune and endothelial cell proliferation and acti¬ vation; enhances production of tumor necrosis factor (TNF), IL-1, inter¬ feron (IFN), and colony-stimulating factors.

Stimulates hypothalamic-pituitary-adrenocortical (HPA) axis, including corticotropin-releasing hormone secre¬ tion; inhibits gonadotropin-releasing hormone secretion; inhibits thyroid cell function.

IL-1 receptor antagonist (IL-lra)

Binds to IL-1 receptor; fails to transduce signal, thereby antagonizing effects of IL-1.

Antagonizes IL-1 effects.

IL-6

Mediates inflammatory response by stimulating the production of acutephase proteins in hepatocytes; activates hematopoietic progenitor cells and shortens their G0 period; induces growth and/or differentiation of T cells, B cells, hepatocytes, keratinocytes, and nerve cells; enhances B-cell differentiation to immunoglobulin-secreting plasma cells; induces matu¬ ration of megakaryocytes, thereby increasing the number of platelets.

Stimulates HPA axis, gonadotropin, vasopressin, and growth hormone secretion. Suppresses thyroid axis and serum lipid levels; regulates osteoclast proliferation and recruitment. Raised levels found in Paget disease, estro¬ gen deficiency, and hyperthyroidism.

IL-10

Produced by antigen-presenting cells to modulate T-helper cell immune response (TH1 and TH2); potent immunosuppressant of macrophage function by down-regulation of major histocompatibility complex (MHC) class II expression, and cytokine synthesis; enhances B-cell growth and secretion of immunoglobulin; cofactor for mast cell growth.

Glucocorticoids favor the development of a TH2 immune response, possibly by stimulation of IL-10 and suppres¬ sion of IL-12 production in antigen-presenting cells.

IL-12

Initiates cell-mediated immunity by inducing the differentiation of TH1 cells from uncommitted T cells; stimulates the growth and functional activity of T cells and natural killer cells, induces IFN-y production.

Injection of IL-12 in cancer patients increased the serum lev¬ els of cortisol, prolactin, and estradiol. IL-12 is expressed in cultured thyroid cells, especially after stimulation with thyroid-stimulating hormone, IL-1, or IFN-y.

Together with IL-1 is the principal mediator of tissue destruction in many immune-inflammatory diseases. Important mediator of inflammation; vital in keeping infections localized and for the maturation and function of the immune system; induces fever, endothelial cell activation, and angiogenesis; cofactor for macrophage activation and for B-cell and Tcell proliferation; enhances expression of adhesion molecules on leuko¬ cytes; induces catabolic state; membrane form mediates cytotoxicity.

Stimulates secretion of luteinizing hormone, prolactin, and adrenocorticotropic hormone from the pituitary; media¬ tor in the pathogenesis of autoimmune type 1 diabetes. TNF-a plays a role in the state of insulin resistance asso¬ ciated with obesity and type 2 diabetes. Thiazolidinediones specifically block TNF-a-induced insulin resistance, contributing to their antidiabetic action.

TUMOR NECROSIS FACTORS (TNFs) TNF-a (and -p)

INTERFERON (IFN)

Initially characterized for their ability to "interfere" with viral replication.

EFN-a

Interferes with viral replication; increases expression of MHC class I; enhances natural killer cell function.

Directly stimulates adrenal glucocorticoid production; par¬ ticipates in the regulation of various endocrine systems; modulates temperature, glucose sensitivity, feeding pat¬ tern, and opiate activity.

EFN-y

Produced mainly by T cells; same biologic functions as IFN-a; in addition, increases MHC class II expression; activates macrophages; inhibits IgE production; inhibits proliferation of TH2 cells.

Possible pathophysiologic role in the autoimmune insulitis of type 1 diabetes.

CHEMOKINES

Chemotactic cytokines.

a-Family (e.g., IL-8, mac¬ rophage inflamma¬ tory protein [MCPJ-2)

Chemoattractants for neutrophil granulocytes.

IL-8 is secreted from primary follicular thyroid cell cultures, indicating a possible link with autoimmune thyroiditis.

P-Family (e.g., monocyte chemoattractant pro¬ tein [MCP]-1, MIP-la and MIP-lp)

Chemoattractants and activators for lymphocytes, monocytes, eosinophil granulocytes, and basophil granulocytes; stimulate production of other inflammatory mediators such as IL-1, TNF-a, and histamine.

MCP-1 expressed in thyroid cells with possible link to autoimmune thyroiditis; correlation between prolactin and MIP-la in patients with rheumatoid arthritis.

Stimulates growth of mononuclear phagocytes (M-CSF) and granulocytes (G-CSF) and enhances their function.

M-CSF, together with transforming growth factor-p, is important for the growth and differentiation of the periimplantation embryo. GM-CSF increases serum levels of cortisol and growth hormone in cancer patients.

Macrophage migration inhibitory factor (MIF)

Released from macrophages and T lymphocytes that have been stimulated by glucocorticoids; inhibits random migration of macrophages.

Glucocorticoid counterregulator; overcomes the inhibitory effects of glucocorticoids on cytokine production.

1,25-Dihydroxyvitamin

Behaves as a paracrine factor in the immune system; produced by monocytes and macrophages; has potent actions on immune cells; pathophysiologic roles in sarcoidosis and autoimmune diseases such as type 1 diabetes.

Important mediator in bone metabolism and calcium homeostasis.

Calcitonin precursors (CTpr)

Reliable sepsis marker; novel mediator and potential therapeutic target in sepsis; increases cytokine production in peripheral blood mononuclear cells. Stimulated macrophages are, at least in part, responsible for the CTpr production in parenchymatous tissues such as liver, lung, kidney, spleen, and others.

Mature calcitonin, which is not elevated in sepsis, plays a role in calcium homeostasis and bone metabolism. Endo¬ crine functions of calcitonin precursors not yet known.

Adrenomedullin (ADM)

Inhibits secretion of cytokine-induced neutrophil chemoattractant, a mem¬ ber of the IL-8 family, from activated rat alveolar macrophages in vitro. Endometrial macrophages of women receiving tamoxifen strongly express ADM, and the angiogenesis capabilities of ADM might play a role in tamoxifen-induced endometrial hyperplasia.

Discovered in extracts of human pheochromocytoma but also produced in many other tissues. Very potent vasodi¬ lator and has potential role in septic shock. Elevated in patients with primary hypertension, and in patients with Graves disease.

COLONY-STIMULAT¬ ING FACTORS Macrophage colonystimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), granu¬ locyte-macrophage colony-stimulating factor (GM-CSF)

"HORMONE-LIKE" SUBSTANCES

d3

’Most of these substances are produced by a variety of cell types and have many additional effects not mentioned in this table. The complexity of the system is necessarily over¬ simplified for didactic purposes.

1 646

PART X: DIFFUSE HORMONAL SECRETION

NEUTROPHILS Polymorphonuclear neutrophils (PMNs) are phagocytic cells for which the primary function is the ingestion and destruction of invading microorganisms. Biochemically, they are highly active cells. The primary granules contain digestive and hydro¬ lytic enzymes that are used in the phagosomes. The secondary and tertiary granules, which are released by exocytosis in response to external stimuli, are crucial to mobilizing mediators of inflammation. Neutrophils are subject to the effects of G-CSF and GM-CSF during their maturation and secrete certain interleukins (e.g., IL-1, IL-3, IL-6, IL-8, IL-10).1'3'36 TNF-a and its receptors are also pro¬ duced by neutrophils. Soluble TNF-receptor production increases whereas a concomitant decrease occurs in the cell-surface recep¬ tor.37 Thus, neutrophils can decrease the inflammatory response by altering the cellular effect of TNF-a via modulation of levels of both cell-surface and soluble TNF receptors. Lactoferrin released from these granules can inhibit the release of GM-CSF activity from macrophages, thereby attenuating granulopoiesis. Proenkephalin has been demonstrated in neutrophils, as well as in lymphocytes and neuronal cells. Prolactin receptors are present on neutrophils, although no evidence exists of pro¬ lactin production per se by these cells. Less is known about neutrophils than about monocytes, macrophages, and lymphocytes because of the difficulty of iso¬ lating them without activating them and thereby causing changes in the granule content and composition. Isolating mRNA from these enzyme-rich cells is difficult, further hinder¬ ing molecular studies. Neutrophils are likely to have as rich an array of hormone receptors as do the mononuclear cells and to be influenced by the neuro-immuno-endocrine axis.

EOSINOPHILS Eosinophils are relatively uncommon polymorphonuclear leu¬ kocytes. They are notable for their strikingly large eosinophilic granules. The number of these cells is low in the nonallergic adult, usually 0.1% residual activity) of HPRT are associated with early (teenage)-onset gout and uric acid crystal formation (infancy), and, occasionally, acute renal insufficiency.10 Neurologic symptoms either are absent or mild. Rarely, mothers of HPRT-deficient male children exhibit mild degrees of purine overproduction and lateonset gout, indicating that this disorder can be inherited as an incomplete X-linked dominant trait in unusual situations. HPRT deficiency can be diagnosed readily by assay of erythrocyte lysates. A deficiency of this enzyme has two important consequences for purine synthesis: First, PRPP accumulates because it is not used in this salvage reaction, and second, purine nucleotide formation through salvage mecha¬ nisms is reduced (see Fig. 192-1). Consequently, there is increased activity of the first enzyme in the de novo pathway of purine synthesis (see Fig. 192-3). (HPRT deficiency is also discussed in the section Disorders of Purine Salvage.)

1742

PART XI: HERITABLE ABNORMALITIES OF ENDOCRINOLOGY AND METABOLISM centrations of hypoxanthine and xanthine in the urine. As a conse¬ quence, there is heightened risk of xanthine calculi formation. Sonography has been successfully used to detect these xanthinecontaining calculi.11 In patients with normal HPRT activity, the accumulation of hypoxanthine as a result of XO inhibition leads to increased purine base salvage and decreased de novo purine syn¬ thesis. This effect reduces the total amount of purine (hypoxan¬ thine, xanthine, and uric acid) produced each day. XO inhibition also is beneficial because it replaces uric acid as the end product of purine metabolism with the more soluble purine base, hypoxan¬ thine. The latter effect reduces urate and uric acid concentrations in serum and urine, respectively, thereby decreasing crystal forma¬ tion in all patients with gout, even those with HPRT deficiency. In patients with normal HPRT activity, there is the added effect of decreased purine production. Although there still is no satisfac¬ tory treatment for the neurologic symptoms associated with HPRT deficiency, the production of a mouse model for Lesch-Nyhan syndrome12 and the prospect of germline gene modification13 raise the potential for somatic gene therapy and possibly even future treatment and prevention of this devastating disorder.

DISORDERS OF PURINE SALVAGE HYPOXANTHINE-GUANINE PHOSPHOR I BOS YLTRANSFE RASE DEFICIENCY

FIGURE 192-5. Lesch-Nyhan syndrome. Choreoathetosis of upper extremities and scissoring of lower extremities are typical of the spastic¬ ity observed in this disorder.

MANAGEMENT OF PURINE OVERPRODUCTION The treatment of purine overproduction states in patients with a primary derangement in purine synthesis, as in PRPP synthetase overactivity or HPRT deficiency, or in patients with a secondary increase in purine synthesis due to a primary defect in purine catabolism, is aimed at reducing uric acid formation. This is accomplished through the use of allopurinol, a drug that inhibits XO activity (Fig. 192-6; see Fig. 192-1). In the setting of LeschNyhan syndrome, the use of allopurinol results in increased con-

Hypoxanthine

Allopurinol

Xanthine

Uric acid

Oxypurinol

FIGURE 192-6. Substrates and inhibitors of xanthine oxidase.

HPRT deficiency is a disorder of both purine base salvage and purine synthesis. Failure to reuse hypoxanthine (and guanine) contributes to the excessive rate of uric acid production observed in patients with this enzyme defect. In normal per¬ sons, a large portion of the hypoxanthine produced each day through purine nucleotide catabolism is salvaged by the HPRT reaction. Loss of this salvage mechanism contributes to the excess production of uric acid characteristic of this disorder. Sev¬ eral mutations have been described.13®

ADENINE PHOSPHOR IBOSYL TRANSFERASE DEFICIENCY Reutilization of the purine base, adenine, is catalyzed by APRT (see Fig. 192-1). This enzyme is the product of a gene different from that for HPRT. The salvage of adenine is quantitatively much less significant than that of hypoxanthine because humai\ cells have a limited capacity to produce adenine. Thus, APRT deficiency has no discernible effect on purine nucleotide synthe¬ sis, and patients with this enzyme defect do not exhibit increased rates of purine synthesis and uric acid production. When adenine is not phosphoribosylated by APRT, this purine base becomes available for oxidation by XO (see Fig. 192-1). The affinity of APRT for adenine is considerably greater than that of XO, and in normal individuals, essentially all of the adenine is metabolized through the salvage pathway. Symptoms in patients with APRT deficiency are the conse¬ quence of failure to "scavenge" or reuse the small amount of adenine produced or ingested each day.14 Oxidation of adenine to 2,8-dihydroxyadenine leads to the formation of an extremely insoluble purine base, and crystals of 2,8-dihydroxyadenine form in the urine, causing renal calculi.15 Complete deficiency of APRT is inherited as an autosomal recessive trait and can be diagnosed readily by assay of erythrocyte lysates. Recurrent radiolucent renal calculi may form in children and young adults with this enzyme defect. Renal insufficiency has developed in some patients. Occasionally, the stones have been misdiagnosed as uric acid calculi because of the similar physical properties of 2,8-dihydroxyadenine and uric acid. The treatment of this disor-

Ch. 192: Heritable Diseases of Purine Metabolism der depends on early recognition of the type of stone being formed and institution of appropriate dietary (low purine) and drug therapy (allopurinol to inhibit XO). Partial deficiency of APRT appears to be a common genetic polymorphism. Gener¬ ally it is of no clinical significance but has been associated with urolithiasis.15 Using gene-targeting, a mouse model of APRT deficiency has been generated. The kidney disease that ensues resembles that seen in humans and offers new avenues of research into this disorder.16

DISORDERS OF PURINE CATABOLISM Disorders in one portion of the purine pathway frequently lead to secondary changes in other parts of this pathway, as well as producing derangements in other metabolic pathways. This is best illustrated by the group of disorders categorized as defects in purine catabolism. Inherited defects in other pathways may alter purine catabolism, secondarily, and the resultant derange¬ ments in purine metabolism contribute to the symptoms observed in these patients.

1743

PURINE NUCLEOSIDE PHOSPHORYLASE DEFICIENCY Purine PNP catalyzes the reaction in which inosine, deoxy¬ inosine, guanosine, and deoxyguanosine undergo phosphorolysis to purine bases (see Fig. 192-1). PNP deficiency is characterized by recurrent infections with nonbacterial organ¬ isms, reflecting the primary defect in cellular immunity.21 Labora¬ tory tests reveal lymphopenia and a diminished number of T cells. Although there are normal numbers of B cells, their function may be significantly impaired. A confirmation of the diagnosis is obtained by the assay of erythrocyte lysates or other cell extracts. The deficiency of PNP, which is inherited as an autosomal recessive trait, leads to the accumulation of several purine nucle¬ osides, the most important of which is deoxyguanosine. Thus, deoxyguanosine triphosphate concentrations become elevated in T lymphocytes, leading to the inhibition of ribonucleotide reduc¬ tase, a reduction in DNA synthesis, and the death of T cells. The prognosis in PNP deficiency usually is better than in ADA defi¬ ciency; the replacement of PNP activity by bone marrow trans¬ plantation or red blood cell infusion has been less successful.

XANTHINE OXIDASE DEFICIENCY ADENOSINE DEAMINASE DEFICIENCY Adenosine deaminase deaminates both adenosine and deoxyadenosine to form inosine and deoxyinosine, respectively (see Fig. 192-1). Deficiency of this enzyme activity, which is inherited as an autosomal recessive disorder, leads to profound lym¬ phopenia.17 Patients having this enzyme defect have reduced numbers of T and B cells, decreased immunoglobulin levels, and diminution in cellular and humoral immune responses. ADA deficiency accounts for a significant number of patients with severe combined immunodeficiency (SCID). These patients experience recurrent infections, and without therapy, they often die within the first few years of life. More recently, ADA defi¬ ciency has been recognized as a cause of lymphopenia and immunodeficiency in adults.18 ADA deficiency can be diagnosed by assay of any easily obtained tissue, such as erythrocytes. The deficiency leads to the accumulation of both adenosine and deoxyadenosine. The buildup of these naturally occurring purine nucleosides, par¬ ticularly deoxyadenosine, may alter lymphocyte growth and function through derangements in purine, nucleic acid, or transmethylation reactions. The accumulation of deoxy-ATP may inhibit ribonucleotide reductase, leading to a decrease in DNA synthesis and lymphocyte replication. An accumulation of S-adenosylhomocysteine may inhibit transmethylation reactions. It is unclear why a generalized deficiency of ADA has such profound effects on lymphocytes while most other organs function normally, but the susceptibility of lympho¬ cytes to ADA deficiency may be explained partly by other fea¬ tures of purine metabolism in lymphocytes that lead to the accumulation of deoxy-ATP or other purine intermediates in these cells. The removal of deoxyadenosine and adenosine by replace¬ ment of ADA activity through red blood cell transfusion or bone marrow transplantation has restored immune function in some of these patients. Several SCID patients have been treated with injections of polyethylene glycol-modified bovine ADA, result¬ ing in significant clinical improvement.19 The successful cloning of the gene responsible for ADA deficiency has opened new ave¬ nues of therapeutic intervention for this disorder. In one of the first uses of gene transfer therapy, patients with SCID have been given infusions of genetically corrected T cells, resulting in improved immune status.20

XO catalyzes the last reactions in the purine catabolic pathway, that is, oxidation of hypoxanthine to xanthine and of xanthine to uric acid (see Fig. 192-1). This disorder is suspected in individu¬ als with persistently low (15 years discordant) to their diabetic twinmates.100No immu¬ nosuppressive therapy was provided because the recipients were monozygotic twins. At first the diabetic state cleared, but within 4 months, lymphocytic infiltration appeared in the grafts, with B-cell destruction. Thus, without any apparent anti¬ genic stimulation other than the presence of the normal islets, the immune system still was abnormal and capable of reestab¬ lishing the disease.

IMMUNOTHERAPY Interestingly, a fourth twin in the foregoing study, who also had been transplanted with a pancreatic segment, was treated prophylactically with azathioprine after the transplantation, and the diabetic state did not recur.99 This raises the issue of immunotherapy in the treatment of type 1 diabetes, before total destruction of the B cells has occurred. Because B-cell destruction does proceed at a subclinical level for years before the onset of overt diabetes, it clearly would be of importance to predict which person is going to develop diabetes and to com¬ mence therapy before B-cell destruction is complete. This may soon be possible with new means of detecting antibodies or Tlymphocyte response to islet cell antigens, such as GAD-65.92 It is well known that cyclosporine, an immunosuppressive agent, when given to newly diagnosed diabetic patients, will suppress the diabetic process, despite the considerable B-cell loss that has already occurred.101 Azathioprine has also proved

1 778

PART XII: IMMUNOLOGIC BASIS OF ENDOCRINE DISORDERS

similarly useful.102-103 Indeed, insulin administration itself, given before the development of frank type 1 diabetes, might prevent or delay the onset by "resting" the B cells and reducing the presentation of their antigens.102-104 Immunotherapy with other models (e.g., T-cell vaccination,105-106 and oral vaccina¬ tion with myelin basic protein107) is being investigated. Vacci¬ nation with GAD-65 to susceptible, but not yet diabetic, mice has prevented the development of type 1 diabetes.108-109 This is an exciting development in the quest for a means to prevent this very serious malady.

INSULIN RESISTANCE DUE TO INSULIN-RECEPTOR ANTIBODIES Brief mention should be made of this rare entity, which is not genetically related to type 1 diabetes or the other organ-specific endocrinopathies. It is termed type B insulin resistance and may be associated with profound hyperglycemia and acanthosis nig¬ ricans, although occasionally hypoglycemia may be noted when the insulin-receptor antibodies manifest an agonist, rather than antagonist, effect on insulin action.110 However, insulin-receptor antibodies may be seen in occasional type 1 diabetic patients.111

AUTOIMMUNE DISEASES OF ADRENALS, GONADS, PARATHYROIDS, AND PITUITARY ADDISON DISEASE The original description by Addison112 of 11 examples of this disorder included cases now recognized as idiopathic (now known to be autoimmune) adrenal atrophy, as well as tubercu¬ losis of the adrenal gland and metastatic carcinoma. Although tuberculosis once accounted for most patients with Addison dis¬ ease (see Chap. 213), autoimmune adrenalitis has become the most common form of this condition in Western countries. There is ample evidence supporting an autoimmune basis for this disease.1-2-18-113 The evidence derives from the histology of the disorder, the finding of autoantibodies against the adrenal cortex in many patients with this condition, the association with other organ-specific autoimmune disease, study of HLA anti¬ gens, genetic studies, and experimental observations.1-2-18'113'113a In the human disease, both adrenal glands are found to be very small, and difficult to locate at autopsy. The capsule is gen¬ erally thickened and the cortex is usually completely destroyed. The remaining adrenocortical cells may be single or in small clusters. A mononuclear cell infiltration is invariable, with lym¬ phocytes, plasma cells, macrophages, and, occasionally, germi¬ nal centers. The few remaining parenchymal cells are surrounded by the heaviest infiltration of lymphocytes, and a variable amount of fibrosis is evident.1-2-18-113 HUMORAL IMMUNITY Antiadrenal antibodies are detectable in approximately twothirds of patients with autoimmune Addison disease.1-2-18-113 The means of detection have included the complement fixation test and immunofluorescence, but with identification of the actual antigens (21-hydroxylase in the adult disease and 17hydroxylase in the type I childhood Addison disease),18-113 Western blotting has been utilized. The adrenal antibodies tend to be more common in those patients with a short duration of disease, and in those who develop the disorder at an early age. The titers of adrenal antibodies are much lower than for thyroid or gastric antibodies in patients with AITD or pernicious ane¬ mia, respectively, but they may persist for many years after ade¬

quate medical therapy. Such antibodies are found very rarely in the control population and are also quite rare in first-degree rel¬ atives of patients with Addison disease (providing that these relatives do not have idiopathic hypoparathyroidism). In "idio¬ pathic" hypoparathyroidism, adrenal antibodies occur in 25% to 30% of patients. In patients with Addison disease who have antiadrenal anti¬ bodies, there also may be antibodies that react with ovary, testis, and steroid-producing cells in the placenta. This cross-reacting antibody may be associated with primary ovarian failure. Although these are IgG antibodies and, therefore, can cross the placenta, there is no evidence that they cause damage to the fetal adrenal glands. In patients with autoimmune Addison disease, there is a high prevalence of antibodies to other organ antigens, includ¬ ing not only steroid-producing cells, but also parathyroid, thy¬ roid, islet cell antigens, or gastric antigens. There is also a higher prevalence of other overt organ-specific autoimmune diseases associated with these same antibodies (Table 197-6). Thus, in patients with autoimmune adrenal disease, careful consideration should be given to the probability that there will be other organ-specific autoimmune diseases, either in an overt or occult (serologic) form. GENETIC STUDIES Autoimmune Addison disease tends to be familial. It generally is considered to be an autosomal recessive characteristic, although the inheritance has not been completely settled. There is an increased incidence of HLA-B8 and DR3 in whites, similar to that seen with GD. This is true, however, only with patients who do not have generalized candidiasis and hypoparathyroidism.

OVARIAN FAILURE Approximately 25% of women with autoimmune Addison dis¬ ease have premature menopause or amenorrhea.1-2-113 Most of these have circulating antibodies against steroid-secreting cells. Such antibodies are almost never detected in patients with amen¬ orrhea that is not associated with Addison disease. The question

TABLE 197-6. Associated Organ-Specific Autoimmune Diseases in Patients with Autoimmune Adrenalitis (Addison Disease)

Associated Disorders Primary ovarian failure Thyroid disease

Middlesex Hospital Series

Edinburgh Series

No.

No.

%

%

25

8

51

18

(56)

(46)

Primary thyrotoxicosis

20

(19) 7

21

(16) 7

Primary myxedema

33

11

20

7

3

1

5

2

Insulin-dependent diabetes mellitus

45

15

27

9

Idiopathic parathyroid deficiency

12

4

16

5.5

7

2

(12)

(4)

12 *

4 *

Number of patients affected

118'

40

106

37

Total number of patients

294

Goitrous autoimmune thyroiditis

Pernicious anemia Positive prolactin-cell antibodies (?subclinical hypophysitis)

289

'Not tested. 'Discrepancy in numbers due to polyendocrine cases. (From Doniach D, Bottazzo GF. Polyendocrine immune disease. In: Franklin EC, ed. Clinical immunology update. New York: Elsevier-North Holland, 1981:96; and Irvine WJ. Polyendocrine immune disease. In: Besser GM, ed. Advanced medicine, vol 13. Tun¬ bridge Wells, England: Pittman, 1977:115.)

Ch. 197: The Immune System and Its Role in Endocrine Function arises whether autoimmune gonadal failure is a closely associ¬ ated, but separate, organ-specific autoimmune disease, or whether it results from cross-reactive antigens shared by gonads and adrenals. Certainly, some steroid cell antibodies are cross¬ reactive between adrenal, gonadal, and placental antigens. This would also explain the finding of antiovarian antibodies in some males with autoimmune adrenal failure. Sensitized T lympho¬ cytes may be similarly cross-reactive.1'2 In some instances, how¬ ever, premature menopause, which is only occasionally of proven autoimmune etiology, may not be related to Addison disease. Histologic features of the ovaries of patients with amenorrhea associated with autoimmune Addison disease will show lympho¬ cytic infiltration and fibrous tissue, similar to that seen in AITD. Autoimmune testicular failure associated with Addison disease is uncommon; it generally is associated with polyendocrine autoim¬ mune failure related to candidiasis and hypoparathyroidism.

HYPOPARATHYROIDISM Autoimmune hypoparathyroidism occurs mostly in children and adolescents, and is often associated with mucocutaneous candidiasis (type I polyendocrine autoimmune disease). Thus, it frequently is associated with Addison disease and other organspecific autoimmune (see Chaps. 60 and 70) diseases.1 The pathologic picture is characterized by lymphocytic infil¬ tration and atrophy. Antiparathyroid antibodies and evidence for cell-mediated immunity have been demonstrated.1

HYPOPHYSITIS There are an increasing number of cases of autoimmune hypophysitis being reported, all in women between the third and eighth decade.2-114 In many of these patients, the diagnosis was made at autopsy. A conspicuous feature of this condition has been its association with pregnancy and the postpartum state. In many of the reported cases, the disease was detected after deliv¬ ery, with the longest interval after gestation being 14 months. Possibly, cases that have been diagnosed as postpartum Shee¬ han syndrome may instead be examples of postpartum autoim¬ mune pituitary disease, developing insidiously during and after pregnancy. Another prominent feature of autoimmune lympho¬ cytic hypophysitis has been its association with other organspecific autoimmune disorders, such as HT, adrenalitis, and pernicious anemia (see Chaps. 11 and 17).

AUTOIMMUNE POLYENDOCRINE DISEASE Theoretically, any patient with one expressed autoimmune endocrine disease showing serologic reactivity with another organ should be considered as potentially belonging to the polyendocrinopathies.115'1153 Although many of these target organs are indeed endocrine glands, other nonendocrine organ-specific autoimmune diseases that are associated in increased frequency include pernicious anemia, myasthenia gravis, Sjogren disease, vitiligo, alopecia areata, chronic active hepatitis, idiopathic thrombocytopenic purpura, and rheumatoid arthritis.116-119 Those patients with overt autoimmune diseases of these organs would belong to the categories depicted in Table 197-7.115 Of the categories listed in Table 197-7, only categories II and III are associated with definite HLA genes.115 Category I, which seems to be the most severe form of autoimmune polyglandular endocrine failure, does not have any particular HLA type and generally occurs in children. Indeed, the inheritance has now been assigned to chromosome 21q22.3 (i.e., not part of the HLA system).120 Possibly, in this condition, the putative suppressor T-

1779

TABLE 197-7. Classification of Polyendocrine Autoimmune Disease I. Candidiasis, hypoparathyroidism, Addison disease (2 or 3 present) II. Addison disease and thyroid autoimmune disease and/or type 1 diabe¬

tes mellitus III. a. Thyroid autoimmune disease and type 1 diabetes mellitus

b. Thyroid autoimmune disease and pernicious anemia c. Thyroid autoimmune disease and vitiligo and/or alopecia and/or other organ-specific autoimmune diseases not falling into the above categories (From Neufeld M, Blizzard RM. Polyglandular autoimmune disease. In: Pinchera A, ed. Autoimmune aspects of endocrine disorders. New York: Academic Press, 1980:357.)

lymphocyte defect is more severe and more nonspecific than that seen in the other entities. There are various other, rarer, polyendocrine autoimmune syndromes, such as central diabetes insipidus, autoimmune enteropathy, autoimmunity to gut hormone-secreting cells, and autoimmunity directed against specific prolactin cells in the anterior pituitary.115 The observation that all of the disorders listed in Table 197-1 have a close association with one another and often are associated with specific HLA genes suggests a very similar pathogenesis for all these autoimmune entities. It may well be that each disease has separate genes, and each a separate organ-specific defect in immunoregulation. Probably, the defect is an organ-specific abnormality in regulatory T-lymphocyte function, which is spe¬ cific for each disease. The fact that some persons develop two or more diseases may relate to the inheritance of more than one closely related gene, or sets of genes. Nonetheless, much remains to be learned before these disorders are fully understood.117'119

REFERENCES 1. Volpe R. The autoimmune endocrinopathies. Contemporary Endocrinology Series. Totowa, NJ: Humana Press, 1999:1. 2. Volpe R. Autoimmune diseases of the endocrine system. Boca Raton, FL: CRC Press, 1990:1. 3. Davies TF. Autoimmune endocrine disease. New York: John Wiley and Sons, 1983:1. 4. Plotnikoff N, Murgo A, Faith R, Wybran J. Stress and immunity. Boca Raton, FL: CRC Press, 1991:1. 5. Nepom GT, Erlich H. MHC class II molecules and autoimmunity. Ann Rev Immunol 1990; 90:493. 6. Nagataki S, Yamashita S, Tamai H. Immunogenetics of autoimmune endo¬ crine disease. In: Volpe R, ed. Autoimmune diseases of the endocrine sys¬ tem. Boca Raton, FL: CRC Press, 1990:51. 7. Wachtel SS, Koo GC, Boyce EA. Evolutionary conservation of HY male anti¬ gen. Nature 1975; 254:270. 8. Kovacs W, Olsen N. Sex and autoimmune disease. In: Volpe R, ed. The autoimmune endocrinopathies. Contemporary Endocrinology Series. Totowa, NJ: Humana Press, 1999:183. 9. Svejgaard A, Platz P, Ryder LR. HLA and endocrine disease. In: Volpe R, ed. Autoimmunity in endocrine disease. New York: Marcel Dekker Inc, 1985:93. 10. Tomer Y, Steinberg D, Davies TF. Immunogenetics of autoimmune endo¬ crine diseases. In: Volpe R, ed. The autoimmune endocrinopathies. Contem¬ porary Endocrinology Series. Totowa, NJ: Humana Press, 1999:57. 11. Tomer Y, Davies TF. Infection, thyroid disease and autoimmunity. Endocr Rev 1993; 14:107. 12. Martin A, Davies TF. T cells in human autoimmune thyroid disease: emerg¬ ing data show lack of need to invoke suppressor T cell problems. Thyroid 1992; 2:247. 13. Volpe R. A perspective on human autoimmune thyroid disease: is there an abnormality of the target cell which predisposes to the disorder? Autoim¬ munity 1992; 12:3. 14. Volpe R. Suppressor T lymphocyte dysfunction is important in the patho¬ genesis of autoimmune thyroid disease. Thyroid 1993; 3:345. 15. Volpe R. Immunoregulation in autoimmune thyroid disease. Thyroid 1994; 4:157. 16. Iwatani Y. Normal mechanisms for tolerance. In: Volpe R, ed. The autoim¬ mune endocrinopathies. Contemporary Endocrinology Series. Totowa, NJ: Humana Press, 1999:1. 17. Ishimura H, Kuchroo V, Abramson-Leeman S, Dorf ME. Comparison between helper and suppressor cell induction. Immunol Rev 1988; 106:93.

1 780

PART XII: IMMUNOLOGIC BASIS OF ENDOCRINE DISORDERS

!8. Drexhage HA. Autoimmune adrenocortical failure. In: Volpe R, ed. The autoimmune endocrinopathies. Contemporary Endocrinology Series. Totowa, NJ: Humana Press, 1999:309. 19. Murakami M, Miyashita K, Monden T, et al. Evidence that a soluble form of TSH receptor is present in the peripheral blood of patients with Graves' dis¬ ease. In: Nagataki S, Mori T, Torizuka K, eds. 80 Years of Hashimoto disease. Amsterdam: Elsevier, 1993:683. 20. Volpe R. Immunology of autoimmune thyroid disease. In: Volpe R, ed. The autoimmune endocrinopathies. Contemporary Endocrinology Series. Totowa, NJ: Humana Press, 1999:217. 21. Weetman AP, McGregor AM. Autoimmune thyroid disease: further devel¬ opments in our understanding. Endocr Rev 1994; 15:788. 22. Burman KD, Baker JR. Immune mechanisms in Graves' disease. Endocr Rev 1985; 6:183. 23. Hashimoto H. Zue Kenntnis der Iymphomatosen Veranderung der Schilddruse (Struma lymphomatosa). Arch Klin Chir 1912; 97:219. 24. McGregor AM, Weetman AP, Ratanachaiyavong S, et al. Iodine: an influence on the development of autoimmune thyroid disease? In: Hall R, Kobberling J, eds. Thyroid disorders associated with iodine deficiency and excess. New York: Raven Press, 1985:209. 25. Sundick RS, Bagchi N, Brown TR. Mechanisms by which iodine induces autoimmunity. In: Drexhage HA, de Vijlder JJM, Wiersinga WM, eds. The thyroid gland, environment and autoimmunity. Amsterdam: Elsevier Sci¬ ence, 1990:13. 26. Helfand M, Redfern CC. Screening for thyroid disease: an update. Ann Intern Med 1998; 129:144. 27. Dayan CM. The natural history of autoimmune thyroiditis: how normal is autoimmunity? Proc R Coll Physicians Edinb 1996; 26:419. 28. Roitt IM, Doniach D, Campbell RN, Hudson RV. Autoantibodies in Hashi¬ moto's disease (lymphadenoid goitre). Lancet 1956; 2:820. 29. Rose NR, Witebsky E. Studies on organ specificity: V. Changes in the thyroid glands of rabbits following active immunization with rabbit thyroid extracts. J Immunol 1956; 76:417. 30. Adams DD, Purves HD. Abnormal responses in the assay of thyrotrophin. Univ Otago Med School Proc 1956; 34:11. 31. McKenzie JM. Humoral factors in the pathogenesis of Graves' disease. Physiol Rev 1968; 48:252. 32. Nagayama Y, Wadsworth HL, Russo D, et al. Binding domains of stimula¬ tory and inhibitory thyrotropin (TSH) receptor autoantibodies determined with chimeric TSH-lutropin/chorionic gonadotropic receptors. J Clin Invest 1991; 88:336. 33. Wolf M, Misaki T, Bech K, et al. Immunoglobulins of patients recovering from Yersinia enterocolilica infections exhibit Graves'-like activity in human thyroid membranes. Thyroid 1991; 1:315. 34. Nordyke RA, Gilbert FI, Miyamoto LA, Fleury KA. The superiority of antimicrosomal over antithyroglobulin antibodies for detecting Hashimoto's thyroiditis. Arch Intern Med 1993; 153:862. 35. Haubruck H, Mauch L, Cook NJ, et al. Expression of recombinant thyroid peroxidase by the bacillovirus system and its use in ELISA screening for diagnosis of autoimmune thyroid disease. Autoimmunity 1993; 15:275. 36. Bagnasco M, Pesce G. Autoimmune thyroid disease: immunological model and clinical problem. Fundam Clin Immunol 1996; 4:7. 37. Resetkova E, Nishikawa M, Mukuta T, et al. Homing of 51Cr-labelled human peripheral lymphocytes to Graves' disease tissue xenografted into SCID mice. Thyroid 1995; 5:293. 38. Davies TF, Concepcion ES, Ben-Nun A, et al. T cell receptor V gene use in AITD: direct assessment by thyroid aspiration. J Clin Endocrinol Metab 1993; 76:660. 39. Volpe R, Row W. Role of antigen-specific suppressor T lymphocytes in the pathogenesis of autoimmune thyroid disease. In: Walfish PG, Wall JR, Volpe R, eds. Autoimmunity and the thyroid. Orlando, FL: Academic Press, 1985:79. 40. Gerstein H, Rastogi B, Iwatani Y, et al. The decrease in nonspecific suppres¬ sor T lymphocytes in female hyperthyroid patients is secondary to the hyperthyroidism. Clin Invest Med 1987; 10:337. 41. Grubeck-Loebenstein B, Derfler K, Kassal H, et al. Immunological features of non-immunogenic hyperthyroidism. J Clin Endocrinol Metab 1984; 60:150. 42. Okita N, Row W, Volpe R. Suppressor T lymphocyte deficiency in Graves' disease and Hashimoto's thyroiditis. J Clin Endocrinol Metab 1981; 52:528. 43. Topliss DJ, Okita N, Lewis M, et al. Allosuppressor T lymphocytes abolish migration inhibition factor production in autoimmune thyroid disease: evi¬ dence from radiosensitivity experiments. Clin Endocrinol 1981; 15:335. 44. Topliss DJ, How J, Lewis M, et al. Evidence for cell-mediated immunity and specific suppressor T lymphocyte dysfunction in Graves' disease and dia¬ betes mellitus. J Clin Endocrinol Metab 1983; 57:700. 45. Vento S, Hegarty JE, Bottazzo GF, et al. Antigen-specific suppressor cell function in autoimmune chronic active hepatitis. Lancet 1984; 1:1200. 46. Vento S, O'Brien CJ, Cundy T, et al. Cellular immunity and specific defects of T-cell suppression in patients with autoimmune thyroid disorders. In: Pinchera A, Ingbar SF1, McKenzie JM, Fenzi GF, eds. Thyroid autoimmunity. New York: Plenum Press, 1987:304. 4/. Noma I, Yata J, Shishiba Y, Inatsuki B. In vitro detection of antithyroglobu¬ lin antibody forming cells from the lymphocytes of chronic thyroiditis patients and analysis of their regulation. Clin Exp Immunol 1982; 49:465. 48. Mori H, Hamada N, DeGroot LJ. Studies in thyroglobulin-specific suppres¬ sor T cell function in autoimmune thyroid disease. J Clin Endocrinol Metab 1985; 61:306.

49. Tao TW, Gatenby PA, Leu SL, et al. Helper and suppressor activities of lym¬ phocyte subsets on antithyroglobulin production in vitro. J Clin Endocrinol Metab 1985; 61:520. 50. Benveniste P, Row VV, Volpe R. Studies of the immunoregulation of thyroid autoantibody production in man. Clin Exp Immunol 1985; 61:274. 51. I i taka M, Aquayo J, Iwatani Y, et al. Studies of the effect of suppressor T lym¬ phocytes on the induction of antithyroid microsomal antibody-secreting cells in autoimmune thyroid disease. J Clin Endocrinol Metab 1988; 66:708. 52. Iitaka M, Aquayo f, Iwatani Y, et al. In vitro induction of antithyroid microsomal antibody secreting cells in peripheral blood mononuclear cells from normal subjects. J Clin Endocrinol Metab 1988; 69:749. 53. Yoshikawa N, Morita T, Resetkova E, et al. Reduced antigen-specific activa¬ tion of suppressor T lymphocytes in autoimmune thyroid disease. J Endo¬ crinol Invest 1993; 16:609. 54. Mukuta T, Yoshikawa N, Resetkova E, et al. Activation of T cell subsets by synthetic TSH receptor peptides and recombinant glutamate decarboxylase in autoimmune thyroid disease and insulin-dependent diabetes mellitus. J Clin Endocrinol Metab 1995; 80:1264. 55. Weetman AP, Volkman DJ, Burman KD, et al. The in vitro regulation of human thyrocyte HLA-DR antigen expression. J Clin Endocrinol Metab 1985; 61:817. 56. Arreaza G, Yoshikawa N, Resetkova E, et al. Expression of intercellular adhesion molecule-1 on human thyroid cells before and after xenografting in nude and severe combined immunodeficient mice. J Clin Endocrinol Metab 1995; 80:3224. 57. Yanagawa T, Mangklabruks A, Chang YB, et al. Human histocompatibility leukocyte antigen-DQA I‘0501 allele associated with genetic susceptibility to Graves’ disease in a Caucasian population. J Clin Endocrinol Metab 1993; 76:1569. 58. McLachlan S. The genetic basis of autoimmune thyroid disease: time to focus on chromosomal loci other than the major histocompatibility complex (HLA in man). J Clin Endocrinol Metab 1993; 77:605A. 59. How J, Topliss DJ, Strakosch C, et al. T lymphocyte sensitization and sup¬ pressor T lymphocyte defect in patients long after treatment for Graves' dis¬ ease. Clin Endocrinol 1983; 18:61. 60. Weetman AP, McGregor AM, Hall R. Evidence for an effect of antithyroid drugs on the natural history of Graves' disease. Clin Endocrinol 1984; 21:163. 61. Ratanachaiyawong S, McGregor AM. Immunosuppressive effects of anti¬ thyroid drugs. Clin Endocrinol Metab 1985; 14:449. 62. Volpe R. Evidence that the immunosuppressive effects of antithyroid drugs are mediated through actions on the thyroid cell: a review. Thyroid 1994; 4:217. 63. Totterman TH, Karlsson FA, Bengtsson M, Mendel-Hartvig I. Induction of circulating activated suppressor-like T cells by methimazole therapy for Graves' disease. N Engl J Med 1987; 316:15. 64. Volpe R. Immunoregulation in autoimmune thyroid disease. N Engl J Med 1987; 316:44. 65. Eisenbarth GS. Autoimmune [) cell insufficiency-diabetes mellitus type 1. Triangle 1984; 23:111. 66. Rossini AA, Mordes JP, Like AA. Immunology of insulin dependent diabe¬ tes mellitus. Annu Rev Immunol 1985; 3:289. 67. Bach JF. Autoimmunity and type I diabetes. Trends Endocr Metab 1997; 8:71. 68. Yoon JW, Austin M, Onodera T, Notkins AL. Virus induced diabetes melli¬ tus: isolation of a virus from the pancreas of a child with diabetic ketoacido¬ sis. N Engl J Med 1979; 300:1173. 69. Gepts W. The pathology of the pancreas in human diabetes. In: Andreani A, DiMario U, Federlin KF, Heding LG, eds. Immunology and diabetes. Lon- ■» don: Kimpton, 1984: 21. 69a. Varela-Calvino R, Sojarbi G, Arif S, Peakman M. T-cell reactivity to the P2C nonstructural protein of a diabetogenic strain of Coxsakie virus B4. Virology 2000; 274:56. 70. Menser MA, Forrest JM, Bransby JM. Rubella infection and diabetes melli¬ tus. Lancet 1987; 1:57. 71. Karjalainen J, Martin JM, Knip M, et al. A bovine albumin peptide as a pos¬ sible trigger of insulin dependent diabetes mellitus. N Engl J Med 1992; 327:302. 72. Atkinson MA, Bowman MA, Kao KJ, et al. Lack of immune responsiveness to bovine serum albumin in insulin-dependent diabetes. N Engl J Med 1993; 329:1853. 73. Todd JA, Bell JI, McDevitt HO. HLA-DQbt’1'’ gene contributes to susceptibility and resistance to insulin dependent diabetes mellitus. Nature 1987; 329:599.’ 74. Nepom GT. Immunogenetics and IDDM. Diab Rev 1993; 1:93. 75. Atkinson MA, MacLaren NK. Islet cell autoantigens in insulin dependent diabetes mellitus. J Clin Invest 1993; 92:1608. 76. Harrison LC. Islet cell antigens in insulin-dependent diabetes: Pandora's box revisited. Immunol Today 1992; 13:348. 77. Chase HP, Voss MA, Butler-Simon N, et al. Diagnosis of pre-type 1 diabetes. J Pediatr 1987; 111:807. 78. Ziegler AG, Ziegler R, Vardi P, et al. Life table analysis of progression to dia¬ betes of anti-insulin autoantibody-positive relatives of individuals with type 1 diabetes. Diabetes 1989; 38:1320. 79. Dean BM, McNally JM, Bonifacio E, et al. Comparison of insulin autoanti¬ bodies in diabetes-related and healthy populations bv precise displacement ELISA. Diabetes 1989; 38:12751. 80. Sachs JA, Cudworth AG, Jaraquemade D, et al. Type I diabetes and the HLA-D locus. Diabetologia 1980; 18:41.

Ch. 197: The Immune System and Its Role in Endocrine Function 81. Schatz DA, Winter WE, Maclaren NK. Immunology of diabetes mellitus. In: Volpe R, ed. Autoimmune diseases of the endocrine system. Boca Raton, FL: CRC Press, 1990:241. 82. Tarn AC, Thomas JN, Dean BM, et al. Predicting insulin dependent diabetes. Lancet 1988; 1:845. 83. McCulloch DK, Klaff LJ, Kahn SE, et al. Non-progression of subclinical beta cell dysfunction among first degree relatives of IDDM patients. Five year follow-up of the Seattle family study. Diabetes 1990; 39:549. 84. Bonifacio E, Bingley P, Shattock M, et al. Quantification of islet cell antibod¬ ies and prediction of insulin dependent diabetes. Lancet 1990; 335:147. 85. Riley WJ, Maclaren NK, Krischer J, et al. A prospective study of the devel¬ opment of diabetes in relatives of patients with insulin dependent diabetes. N Engl J Med 1990; 323:1167. 86. Peig M, Gomis R, Ercilla G, et al. Correlation between residual (5 cell func¬ tion and islet cell antibodies in newly diagnosed type I diabetes: follow-up study. Diabetes 1989; 38:1396. 87. Spencer KM, Tam A, Dean BM, et al. Fluctuating islet cell autoimmunity in unaffected relatives of patients with insulin dependent diabetes. Lancet 1984; 1:764. 88. Lernmark A, Baekkescov S. Islet cell antibodies—theoretical and practical implications. Diabetologia 1981; 24:431. 89. Brogren CH, Lernmark A. Islet cell antibodies in diabetes. J Clin Endocrinol Metab 1982; 11:409. 90. Nerup J, Mandrup-Poulsen T, Molvig J. The HLA-IDDM association: impli¬ cations for the etiology and pathogenesis of IDDM. Diabetes Metab Rev 1987; 3:779. 91. Baekkeskov S, Aanstoot HJ, Fu Q, et al. The glutamate decarboxylase and 38 KD autoantigens in type I diabetes: aspects of structure and epitope recog¬ nition. Autoimmunity 1993; 15:24. 92. Harrison LC, Honeyman MC, DeAizpurua HJ, et al. Inverse relation between humoral and cellular immunity to glutamic acid decarboxylase in subjects at risk of insulin dependent diabetes. Lancet 1993; 341:1365. 93. Bottazzo GF, Dean BM, McNally JM, et al. In situ characterization of autoim¬ mune phenomena and expression of HLA molecules in the pancreas in dia¬ betic insulitis. N Engl J Med 1985; 313:353. 94. Nerup J, Andersen OO, Bendixen G, et al. Anti-pancreatic cellular hyper¬ sensitivity in diabetes mellitus. Diabetes 1971; 20:424. 95. Boitard C, Debray-Sachs M, Pouplard A, et al. Lymphocytes from diabetics suppress insulin release in vitro. Diabetologia 1981; 21:41. 96. Boitard C, Yasunami R, Dardenne M, Bach JF. T cell mediated inhibition of the transfer of autoimmune diabetes in NOD mice. J Exp Med 1989; 169:1669. 97. Fairchild RS, Kyner JL, Abdou NI. Suppressor cell dysfunction in insulin dependent diabetes. Diabetes 1980; 29:52A. 98. Lohmann D, Krug J, Lampeter EF, et al. Defect of suppressor cell activity and cell mediated anti-beta cell cytotoxicity in type I diabetes mellitus. Dia¬ betologia 1986;29:421. 99. Boitard C, Timsit J, Larger E, et al. Pathogenesis of IDDM: immune regula¬ tion and induction of immune tolerance in the NOD mouse. Autoimmunity 1993; 15(Suppl):12. 100. Sutherland DER, Sibley R, Xu XZ, et al. Twin to twin pancreas transplanta¬ tion reversal and re-enactment of the pathogenesis of type I diabetes. Trans Assoc Am Physicians 1984; 97:80. 101. Stiller CR, Dupre J. Immune interventional studies in type I diabetes melli¬ tus: summary of the London (Canada) and Canadian-European experience. In: Eisenbarth GS, ed. Immunotherapy of diabetes and selected autoim¬ mune diseases. Boca Raton, FL: CRC Press, 1989:73.

1781

102. Harrison LC, Colman PG, Dean B, et al. Increase in remission rate in newly diagnosed type I diabetic subjects treated with azathioprine. Diabetes 1985; 34:1306. 103. Silverstein J, Maclaren N, Riley W, et al. Immunosuppression with azathio¬ prine and prednisone in recent onset insulin dependent diabetes mellitus. N Engl J Med 1988; 319:599. 104. Muir A, Luchetta R, Song HY, et al. Insulin immunization protects NOD mice from diabetes. Autoimmunity 1993; 15:58A. 105. Boitard C. The differentiation of the immune system towards anti-islet autoimmunity: clinical prospects. Diabetologia 1992; 35:1101. 106. Formby B, Shao T. T cell vaccination against autoimmune diabetes in nonobese diabetic mice. Ann Clin Lab Sci 1993; 23:137. 107. Weiner HL. Treatment of autoimmune disease by oral tolerance to autoan¬ tigens. Autoimmunity 1993; 15:6. 108. Kaufman DL, Clare-Saizier M, Tian J, et al. Spontaneous loss of T-cell toler¬ ance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature 1993; 366:69. 109. Tisch R, Yang XD, Singer S, et al. Immune response to glutamic acid decar¬ boxylase correlates with insulitis in non-obese diabetic mice. Nature 1993; 366:72. 110. Bloise W, Wajchenberg BL, Moncada VY, et al. Atypical antiinsulin receptor antibodies in a patient with type B insulin resistance and scleroderma. J Clin Endocrinol Metab 1989; 68:227. 111. Maron R, Elias D, Dejong BM, et al. Autoantibodies to the insulin receptor in juvenile onset insulin dependent diabetes. Nature 1983; 303:817. 112. Addison T. On the constitutional and local effects of disease of the suprare¬ nal glands. In: Wilks S, Daldy T, eds. A collection of the published writings of the late Thomas Addison, M.D., physician to Guy's Hospital. London: New Sydenham Society, 1868:209. 113. Muir A, Schatz DH, MacLaren NK. Autoimmune Addison's disease. Springer Semin Immunopathol 1993; 14:275. 113a. de Carmo Silva R, Kater SA, Laureti S, et al. Autoantibodies against recom¬ binant human steroidogenic enzymes 21-hydroxylase, side-chain cleavage and 17a-hydroxylase in Addison's disease and autoimmune polyendocrine syndrome Type III. Europ J Endocrinol 2000; 142:187. 114. Josse R. Autoimmune hypophysitis. In: Volpe R, ed. Autoimmune diseases of the endocrine system. Boca Raton, FL: CRC Press, 1990:331. 115. Bottazzo GF, Doniach D. Polyendocrine autoimmunity: an extended con¬ cept. In: Volpe R, ed. Autoimmunity and endocrine disease. New York: Mar¬ cel Dekker Inc, 1985:375. 115a. Ekwall O, Hedstrand H, Haavik J, et al. Pteridin-dependent hydroxylases as autoantigens in autoimmune polyendocrine syndrome type 1. J Clin Endo¬ crinol Metab 2000; 85:2944. 116. Green ST, Ng JP, Chan-Lam D. Insulin dependent diabetes mellitus, myas¬ thenia gravis, pernicious anemia, autoimmune thyroiditis and autoimmune adrenahtis in a single patient. Scott Med J 1988; 33:213. 117. Neufeld M, Blizzard RM. Polyglandular autoimmune disease. In: Pinchero A, ed. Autoimmune aspects of endocrine disorders. New York: Academic Press, 1980:357. 118. Irvine WJ. Autoimmunity in endocrine disease. In: Besser GM, ed. Advanced medicine, vol 13. Tunbridge Wells, England: Pittman, 1977;115. 119. Eisenbarth G, Maes M. Autoimmune polyendocrine syndromes. In: Volpe R, ed. The autoimmune endocrinopathies. Contemporary Endocrinology Series. Totowa, NJ: Humana Press, 1999:349. 120. Aaltonen J, Bjorses P, Sandkuijl L, et al. An autosomal locus causing autoim¬ mune disease: autosomal polyglandular disease type I assigned to chromo¬ some 21. Nat Genet 1994; 8:83.

■

'

PART XIII

ENDOCRINE AND METABOLIC DYSFUNCTION IN THE GROWING CHILD AND IN THE AGED WELLINGTON HUNG,

EDITOR

198. SHORT STATURE AND SLOW GROWTH IN THE YOUNG.1784 199. ENDOCRINOLOGY AND AGING.1808

1 784

PART XIII: ENDOCRINE AND METABOLIC DYSFUNCTION IN THE GROWING CHILD AND IN THE AGED

CHAPTER

1 98

SHORT STATURE AND SLOW GROWTH IN THE YOUNG THOMAS ACETO, JR., DAVID P. DEMPSHER, LUIGI GARIBALDI, SUSAN E. MYERS, NANCI BOBROW, AND COLLEEN WEBER

Those infants who decelerate in their linear growth do so during the second to sixth months and achieve a new, lower growth channel by an average age of 13 months (Fig. 198-2). By the age of 18 months to 2 years, the shifts in growth rate usually cease, and growth proceeds along the same percentile.

INTRAUTERINE GROWTH RESTRICTION/PREMATURITY INTRAUTERINE GROWTH RESTRICTION

NORMAL LINEAR GROWTH, POSTNATAL After birth, many infants shift from a growth rate that is deter¬ mined by maternal factors to one that is increasingly related to the infant's own genetic background, as reflected by midparental size. Thus, for approximately two-thirds of normal infants, the linear growth rate shifts percentiles during the first 18 months of life. The number of infants shifting upward in growth rate and the number shifting downward are approximately equal. Normal full-term infants who are relatively small at birth but whose genetic background dictates a larger size accelerate toward the new growth rate soon after birth and achieve a new channel of growth by an average age of 13 months (Fig. 198-1).

FIGURE 198-1. The average linear growth of 18 middle-class normal infants who had been below the 10th percentile for length at full-term birth but who achieved the 50th percentile or better by the age of 2 years, at which time their stature correlated well with that of their par¬ ents. The catch-up in linear growth began soon after birth, and a new channel of growth had been achieved by 4 to 18 months. (From Smith DW, Truog W, Rogers JE, et al. Shifting linear growth during infancy: illustration of genetic factors in growth from fetal life through infancy. J Pediatr 1976; 89:225.)

Intrauterine growth restriction occurs in association with envi¬ ronmental, maternal, placental, and fetal factors.1'13 Whether different types of intrauterine growth restriction exist or whether the condition should be diagnosed solely on the basis of birth weight that is low for gestational age is unclear.2'3 Neo¬ nates with body weights of 60% of cases, and virtually all the remaining patients have maternal disomy of 15q

RUSSELL-SILVER77 84

Sporadic

Short stature of prenatal onset (intrauterine growth retardation) and continuing postnatally; mildly to moderately short stature in childhood; triangular face with down-turned comers of the mouth, asymmetry of the extremi¬ ties; clinodactyly of the fifth finger

ULLRICHNOONAN77'85

Sporadic or autosomal dominant

Moderately short stature; epicanthal folds, ptosis, low-set ears, webbed neck; shield chest; mental retardation; pul¬ monic stenosis, septal defects; small penis, cryptorchidism, occasional hypogonadism; occasional lymphedema of the dorsum of hands and feet; improperly called Turner-like syndrome

WILLIAMS77'86

Sporadic

Prenatal and postnatal growth deficiency; mildly to moderately short stature in childhood; short palpebral fis¬ sures, depressed nasal bridge, anteverted nares, prominent lips with open mouth; mental retardation (average IQ: 50-60) with friendly personality; supravalvular aortic stenosis or other congenital heart disease or arterial anomaly; occasional hypercalcemia in early infancy ("idiopathic hypercalcemia of infancy")

also should be ~2 SD below the mean (i.e., 152 cm [60 in] for the mother and 164 cm [64.5 in] for the father). Further evaluation is unnecessary; bone age is within normal limits. If the patient's height in standard deviations is significantly less than that of his parents (e.g., 4 SD below the mean), normal genetic short stature is not the cause and further evaluation is indicated. If the patient's height and a parent's height are >2 SD below the mean, both may have abnormal short stature and should be studied for a genetically controlled disease such as congenital GHD or skel¬ etal dysplasia (e.g., hypochondroplasia, multiple epiphyseal dysplasia syndrome). THERAPY AND PROGNOSIS No therapy is available for normal genetic short stature. Whether drug therapy for these normal children will ever be indicated is uncertain, given the difficulty of evaluating the long-term effects of drugs such as GH in healthy youngsters with normal short stature. Most parents are reassured by the knowledge that their nor¬ mally short child is healthy and will grow to adulthood like them¬ selves as a normally short and functional individual. Affected teenage boys should be encouraged to engage in sports that they enjoy and that are safe (wrestling, baseball, swimming, tennis, ice skating, sailing) and to avoid sports that pose a potential risk (tackle football). They should be reassured that normally short girls are delighted to date normally short young men. Those par¬ ents, usually fathers, who find the diagnosis difficult to accept should be reassured repeatedly. Therapy for abnormal genetic short stature depends on the underlying disease.

SLOW GROWTH WITH OR WITHOUT SHORT STATURE DEPRIVATION SYNDROME Deprivation, caloric or emotional, slows weight gain and, even¬ tually, linear growth. The type I deprivation syndrome has more

of a nutritional component and the type II syndrome has more of a psychosocial component.98-99 Type I deprivation syndrome is seen in infants and young children. For various reasons, these patients have not received enough food or, in some cases, enough attention. The parent or caregiver may be disorganized, inadequately trained, mis¬ guided, overwhelmed, or disturbed. This condition also has been described in the second decade of life.100 Type II depriva¬ tion syndrome, the childhood variety, affects children older than 3 years and, occasionally, teenagers. Parents or caregivers, who frequently are alcoholics, abuse these children emotionally. Although the disorder occurs more often in the lower socioeco¬ nomic classes, the authors have documented the deprivation syndrome in the upper classes. Boys are affected most com¬ monly.101 At the initial evaluation, hypopituitarism, including' GHD, often is present. Without intervention, the prognosis for normal growth and development is guarded to dismal for patients with both types of deprivation syndrome. PRESENTING MANIFESTATIONS Infants with type I deprivation syndrome have slowing of growth, a scrawny appearance, and a relatively alert demeanor, although some look dejected. Kwashiorkor (see Chap. 127) rarely occurs in the United States. Eight patients, aged 14 to 27 months, were found to have consumed excessive amounts of fruit juice with resultant failure to thrive (abnormally low weight)102; how¬ ever, these patients recovered after nutritional intervention. Six¬ teen extensively studied patients with nutritional dwarfing (aged 10 to 16 years) were found to have inappropriate eating habits and subnormal weight gain (accompanied by a propor¬ tionate decline in growth velocity), but no signs of emaciation. Parents reported that these children became satiated early dur¬ ing the course of a meal.100 Children with type II deprivation syndrome, those with "psychosocial dwarfism," generally are withdrawn, grow extremely slowly, and have delayed sexual maturation. Most

Ch. 198: Short Stature and Slow Growth in the Young

1803

have an appropriate weight for their height, and some resemble patients with celiac dwarfism, with protuberant abdomens and wasted buttocks. Eventually, a history emerges of polydipsia, polyphagia, stealing of food, eating from garbage cans, and drinking from toilet bowls. Developmentally, patients in both groups perform suboptimally. ESTABLISHMENT OF THE DIAGNOSIS The gold standard for establishing the diagnosis of deprivation syndrome is the observation of accelerated weight gain in infants, and accelerated growth as well as weight gain in older children, when patients have new caretakers (e.g., hospitals or foster homes). Feeding should be increased gradually to the rec¬ ommended number of kilocalories per kilogram of the ideal body weight for the patient's age. In infants, definitive weight gain occurs in ~2 weeks; in older children, acceleration of growth and weight gain can take several months. Laboratory studies are of little help in establishing the diagnosis. The bone age is retarded, particularly in patients with psychosocial dwarfism. Infants whose linear growth rates are slowing (e.g., dropping from the 50th to the 20th percentile from the ages of 6 to 12 months, respectively) present diagnostic difficulties. Such infants may be perfectly normal and may simply be experienc¬ ing a shift from an intrauterine growth rate influenced by mater¬ nal factors to an extrauterine growth rate dictated by their own genetic backgrounds (i.e., midparental height). These normal infants establish their permanent normal growth rate (e.g., along the 20th percentile) by the age of 18 to 24 months. THERAPY AND PROGNOSIS When the caretaker is disturbed, a new caretaker must be located. For some parents of malnourished infants, education in feeding is helpful. If a biologic parent is a psychologically dis¬ turbed caretaker, psychotherapy is essential. If the parent refuses psychotherapy or the infant does not improve rapidly, the physician must use every means necessary to place the child in a new permanent home. With intervention, the long-term overall prognosis for chil¬ dren with type I deprivation syndrome is generally good. For children with type II deprivation syndrome, the long-term prog¬ nosis for growth and sexual maturation is favorable, and intel¬ lectual ability improves to some extent (Fig. 198-23). However, both intellectual function and emotional development are likely to be permanently compromised.103

ATYPICAL CROHN DISEASE104 Crohn disease, a chronic inflammatory disease of the bowel of unknown etiology but with a strong genetic component, often interferes with growth and sexual maturation, probably as a result of chronic undemutrition, and secondarily, of low serum concen¬ trations of IGF-I.105 Several factors contribute to the nutritional problems, including increased nutrient losses and malabsorption. PRESENTING MANIFESTATIONS Growth failure can herald Crohn disease. The weight often is more compromised than the height, and puberty is delayed but the patient looks well. Perianal fistulas are common. On ques¬ tioning, patients may describe intermittent attacks of abdominal pain and diarrhea. CONFIRMATION OF THE DIAGNOSIS Barium contrast radiographs of the small and large bowels often are characterized by an irregular mucosa or a cobblestone-like

FIGURE 198-23. Growth curve of a girl with deprivation syndrome. Her growth as a young child was severely stunted, during which time she was being severely mistreated. She was removed from her home and responded remarkably to kindness and attention.

pattern, a thickened bowel, and the presence of enteric fistulas. The segmental distribution of the lesions frequently is diagnos¬ tic. Biopsy samples of the rectal mucosa obtained by colonos¬ copy show typical granulomas. The erythrocyte sedimentation rate usually is elevated, the bone age is retarded, and the hemo¬ globin and serum albumin levels occasionally are depressed. THERAPY AND PROGNOSIS Control of the disease and provision of adequate nutrition are the prerequisites for growth, but an optimal method of accom¬ plishing these goals has not been identified. Growth may accel¬ erate with initial daily glucocorticoid therapy followed by alternate-day therapy in cases of stable disease. Calories have been administered by central or peripheral intravenous hyperalimentation, elemental diets, and special¬ ized formulas.106 When the underlying disease is controlled, good nutrition alone, regardless of the method used to deliver it, stimulates growth. When disease activity cannot be stabi¬ lized and growth cannot be achieved with medical and nutri¬ tional support, surgical intervention should be considered. For growth to occur, resection must be performed before late puberty, all actively diseased bowel must be resected, and a prolonged disease-free postoperative period must be achieved. Ongoing nutritional therapy may augment the accelerated growth rate.107 The effects of various treatment plans on growth rate and final adult height require evaluation in large cooperative studies.

1804

PART XIII: ENDOCRINE AND METABOLIC DYSFUNCTION IN THE GROWING CHILD AND IN THE AGED suggests the presence of adrenocortical hyperplasia caused by an abnormality of the hypothalamic-pituitary area, whereas fail¬ ure to suppress is strongly suggestive of an adrenocortical tumor. Hypochloremic hypokalemic metabolic alkalosis can be present, and—in those cases caused by pituitary adenomas— late evening serum ACTH levels may be elevated. In a study of a short boy with periodic Cushing syndrome, the CRH test was found to be helpful. ACTH and cortisol concentrations were undetectable both in the basal state and after stimulation with CRH. As expected, the patient had bilateral micronodular adre¬ nal hyperplasia at surgery. In searching for a pituitary lesion, radiographs of the sella turcica, computed tomography of the pituitary area, MRI, petro¬ sal sinus sampling, and, eventually, direct transsphenoidal visu¬ alization of the pituitary are helpful. For demonstrating the presence of an adrenal tumor, a radiograph of the abdomen can be useful to look for calcification of certain areas indicative of such a tumor. To reveal the tumor itself, ultrasonography, com¬ puted tomography, MRI, and radioactive iodocholesterol uptake can be used (see Chap. 88). In almost 300 patients, when plasma was sampled from the inferior petrosal sinuses with the conjunctive use of CRH, patients with Cushing disease could be distinguished from those with ectopic ACTH secretion.114 THERAPY AND PROGNOSIS

FIGURE 198-24. Seventeen-year-old boy with Cushing syndrome caused by bilateral adrenal hyperplasia. Note moon facies, buffalo hump, and obesity, especially of the trunk.

CUSHING DISEASE OR SYNDROME Patients with Cushing syndrome secrete excess cortisol and other adrenocortical hormones, usually continuously but some¬ times periodically.108 The underlying disease can be caused by an ACTH-secreting microadenoma of the pituitary (basophilic or mixed basophilic chromophobic, with a resultant bilateral adrenal hyperplasia) or by an adrenal tumor. Rarely, it is caused by a tumor secreting corticotropin-releasing hormone (CRH) or by an ectopic ACTH-producing tumor.109 The natural history of Cushing disease (adrenal hyperplasia) is unknown, but rare cases of spontaneous remission have been reported.110-111 Because many of the adrenal tumors are malignant, the mortal¬ ity rate is high (see Chaps. 75 and 83). After the age of 7 years, the most common underlying problem is an ACTH-producing pituitary adenoma.112-113 PRESENTING MANIFESTATIONS Although Cushing disease is rare in the young, infants as well as teenagers can be affected. Usually, help is sought for the increas¬ ingly abnormal appearance—moon facies, obesity (especially of the trunk and face), purplish striae, hypertension, acne, emo¬ tional lability, and virilization (Fig. 198-24). A review of growth data invariably reveals a pathologically slow growth rate over months or years. The slowing of growth occasionally precedes the abnormal appearance by several months or years. CONFIRMATION OF THE DIAGNOSIS The diagnosis of Cushing disease depends on the demonstra¬ tion of pathologically elevated cortisol secretion—that is, ele¬ vated urinary free cortisol levels that cannot be suppressed with low doses of dexamethasone (see Chap. 77). The response to administration of larger doses of dexamethasone helps to local¬ ize the lesion. Suppression with high doses of dexamethasone

Transsphenoidal microsurgery is the treatment of choice for patients with adrenal hyperplasia from a demonstrated pitu¬ itary tumor115 (see Chap. 23). Results are excellent when the tumor is visualized and removed at surgery. Many of these patients become permanently glucocorticoid deficient. The former approach, bilateral adrenalectomy, rarely is indicated. Long-term remission has been reported with pituitary irradia¬ tion (see Chap. 22). Ketoconazole can facilitate regression of the stigmata of Cushing disease.116 Surgical excision is the treatment of choice for demonstrable adrenal tumors (see Chap. 89). Well-localized adenomas have a good prognosis. However, microscopic examination does not always distinguish benign from malignant lesions. The results of chemotherapy for malignant tumors are disappointing. After successful therapy, the signs and symptoms of Cush¬ ing syndrome disappear, and many children grow in an accel¬ erated fashion. As a group, they achieve a reasonable adult height.112

IATROGENIC EFFECTS ON GROWTH STIMULANT MEDICATION Certain neurostimulant drugs, especially methylphenidate but also pemoline and methamphetamine, can inhibit weight gain and growth before puberty but probably not after puberty. Desipramine does not inhibit the linear growth of children.117-120 PRESENTING MANIFESTATIONS Patients with attention deficit disorders who have been treated with neurostimulant drugs can have moderate slowing of growth and weight gain or even weight loss. Generally, these patients have received "high-normal" dosages (e.g., >1 mg /kg per day) of methylphenidate hydrochloride for many months. Anorexia is common and dose dependent, but abates with continued therapy. The results of physical examination usually are normal. ESTABLISHMENT OF THE DIAGNOSIS The diagnosis is clinical and can be confirmed only when linear growth accelerates after cessation of the medication. Discontinu-

Ch. 198: Short Stature and Slow Growth in the Young ance of therapy during summer vacations seems to result in accelerated growth, but catch-up is incomplete. If linear growth does not increase during a drug "holiday," patients should be evaluated for other causes of slow growth. THERAPY AND PROGNOSIS A controlled study involving 124 preadolescent boys with atten¬ tion deficit/hyperactivity disorder (ADHD) has been reported.120 Small but significant differences in height were found between children with and without ADHD. The height deficits were evident in early, but not late, adolescence, however, and were not related to the use of psychotropic medications. These data suggest that ADHD may be associated with tempo¬ rary deficits in height gain through midadolescence that fre¬ quently normalize by late adolescence. This effect appears to be mediated by ADHD and not by its treatment. Thus, treatment with hGH is not indicated for this disorder.120 No definitive therapy is available, except for discontinuing drug therapy, decreasing a large dosage, or substituting desipramine. Thus, judicious prescribing of these drugs is important. During withdrawal, some children become tempo¬ rarily hyperactive or depressed.

PSYCHOSOCIAL MANAGEMENT OF SHORT STATURE Current research builds on two decades of interest in psychoso¬ cial adjustment, personality and behavior factors, cognitive development, and school achievement, plus success in adult¬ hood of children with diagnoses of GHD, constitutional delay of growth,121-126 and Turner syndrome.127-130 Interest is increasing in the effect of GH therapy on the cognitive and behavioral func¬ tioning of short children, regardless of whether they demon¬ strate GHD.121-131-134 Societal bias toward tall stature from childhood through adulthood, even into the workplace, pre¬ sents a challenge to the short individual in aspects of self¬ esteem, achievement, and acceptance. Therefore, their medical therapy is only one dimension of the care necessary to meet the multifaceted and variable needs of this population. The overall goal for short children is to enable them to become financially self-supporting adults who hold jobs com¬ mensurate with their intellectual and educational ability, who function independently in their social environment, and who are satisfied with their lives. To maximize the possibility of suc¬ cess, physicians must interact with their patients in age-appropriate ways, being especially sensitive to the physical environment, to the use of language, and to the power and influ¬ ence of the therapeutic relationship. The multidisciplinary team approach, including psychological intervention, skilled and compassionate nursing, and educational and vocational coun¬ seling, is most efficacious. Achieving realistic treatment expecta¬ tions is essential. In their interactions with short children, adults tend to infantilize, to overprotect, and to lower behavioral expectations. Some children respond by acting in a manner appropriate for their "height age" rather than their chronologic age. Short chil¬ dren may show a tendency to withdraw from their peer group and to continue this social isolation through adolescence and into adulthood. Boys, especially those with delayed onset or absence of puberty, may avoid interaction with male peers and form nonromantic liaisons with girls or younger children. Fur¬ ther social isolation, including rejection of dating and heterosex¬ ual interaction, and poor school achievement are additional risks. Accompanying these withdrawal syndromes are lack of assertiveness and ambition, anxiety, low self-esteem, and

1805

dependency. GH therapy may add to the sense of well-being and overall health, thus leading to better psychological adjust¬ ment.131'134 Children of all ages must be allowed assertively and politely to correct an adult who mistakes their age or remarks about their height in a derogatory manner. The support of the entire family, including siblings and parents, plays a crucial role in helping them to be emotionally expressive, competent, selfreliant, and independent. Listening to parents, assessing their emotional capacity, and empowering and teaching them to meet the stresses of raising a child with a diagnosis of short stature are essential. Special issues relate to the language specific to the diagnosis of short stature. Sensitize parents to avoid the many "short"-related pejoratives in our language, such as calling someone "short¬ sighted" or referring to being "short-changed." Gender inequal¬ ity creates a greater burden for males, because short females may be viewed as "cute" or "petite." Teaching parents to help their children to respond to teasing by bullies can be invaluable to the child who is not spontaneous in rebuffing a put-down with snappy repartee. Advise parents to encourage the develop¬ ment of hobbies, skills, and special talents that can help foster the high self-esteem that will cause peers to look up to their child. Specific suggestions include playing a musical instru¬ ment, singing, developing computer proficiency, debating, and engaging in arts and crafts such as painting, drawing, handi¬ crafts, and sewing. Children must wear age-appropriate clothing, and teens should be allowed to feel that peer group fashion trends are permitted. Creating a physical environment at home that fos¬ ters self-sufficiency and inclusion in family routines, including chores, should be stressed. Specific suggestions include mak¬ ing stepstools available, relaxing rules about climbing on counters, rearranging usual kitchen configurations, providing light switch pull cords, and lowering closet rods. Whether it be blocks added to bike pedals or devices to make driving acces¬ sible to the teen, these aids are necessary to foster a sense of self-worth and peer group inclusion. Celebrations of the ritual rites of passage, developmental milestones, and birthdays, particularly entrance into the teenage years, are pivotal times to enhance self-esteem. Because short children tend to be at a physical disadvan¬ tage in body contact sports, encourage more personally selfcompetitive sports such as bettering one's own track record, rock climbing, tennis, golf, fishing, swimming, diving, skiing, martial arts, gymnastics, and wrestling in appropriate weight classes. Encourage individual problem solving; however, if, for example, the teen is self-conscious about changing clothes or showering in front of peers, altering of rigid school rules that may be changed only by the intervention of a parent or physi¬ cian can save pain and humiliation. Allowing the individual to make his or her own choices and decisions fosters a sense of self-reliance. Parents must be encouraged to be advocates for their short children at school, working closely with school personnel to pre¬ empt problems. Transition years when children move from ele¬ mentary to middle and to high school may be times of increased stress when they have to redefine themselves in the hierarchy of their peer group. Special attention may be needed to insure that school achievement remains at the level of intellectual capacity. Relocating to a new community may increase stress and chal¬ lenge academic and social adjustment. As a group, short children have normal intellectual function. A few patients with GHD caused by tumors or by cranial irradi¬ ation may experience varying degrees of intellectual impair¬ ment. Some girls with Turner syndrome are at greater risk for visuospatial perception deficits that cause difficulties with

1806

PART XIII: ENDOCRINE AND METABOLIC DYSFUNCTION IN THE GROWING CHILD AND IN THE AGED

directional sense and map and graph reading, as well as some inability to use a computer mouse. Learning environments should maximize the child's skills while removing distractions, anxiety, or overload. Remediation education should be insti¬ tuted if specific math deficits are diagnosed. Vocational plan¬ ning should be consistent with each individual's pattern of strengths and weaknesses. The best means for part-time employment for teens and per¬ manent employment for young adults may be through a friend or a nonjudgmental person who has had experience with people of short stature. Networks of such employers should be culti¬ vated as a resource for this patient population. Role models in the community make ideal individuals to open doors and smooth the way for the short individual. The formal support groups that have been established-such as Human Growth Foundation (www.hgfound.org). Magic Foundation for Children's Growth (www.magicfoundation.org) and Little People of America (www.lpaonline.org)—are impor¬ tant conduits for peer group interaction as well as information and family networking. The groups are an effective advocate¬ lobbying group in the health care and political arenas.

REFERENCES 1. Cowett RM, Stern L. The intrauterine growth retarded infant: etiology, pre¬ natal diagnosis, neonatal management, and long-term follow-up. In: Lifshitz F, ed. Pediatric endocrinology. New York: Marcel Dekker, 1985:109. la. Kramer MS, Seguin L, Lydon J, Galet L. Socio-economic disparities in preg¬ nancy outcome: why do the poor fare so poorly? Paediatr Perinat Epidemiol 2000; 14:194. 2. Miller HC. Intrauterine growth retardation: past, present and future. Growth Genet Horm 1992; 8(3):5. 3. Warshaw JB. Intrauterine growth restriction revisited. Growth Genet Horm 1992; 8(1 ):5. 4. Cruise MO. A longitudinal study of the growth of low birth weight infants. I. Velocity and distance growth, birth to 3 years. Pediatrics 1973; 51:620. 5. Kitchen WH, Doyle LW, Ford GW. Very low birthweight and growth in infants with atypical fetal growth patterns. Am J Dis Child 1977; 131:1078. 6. Ross G, Lipper EG, Auld PAM. Growth achievement of very low birth weight premature children at school age. J Pediatr 1990; 117:307. 7. Holmes GE, Miller HC, Khatab H, et al. Postnatal somatic growth in infants with atypical fetal growth patterns. Am J Dis Child 1977; 131:1078. 8. Herman-Giddens ME, Slora E], Wasserman RC, et al. Secondary sexual char¬ acteristics and menses in young girls seen in office practice: a study from the pediatric research in office settings network. Pediatrics 1997; 99:505. 9. Palmert MR, Malvin HV, Boepple PA. Unsustained or slowly progressive puberty in young girls: initial presentation and long-term follow-up of 20 untreated patients. J Clin Endocrinol Metab 1999; 84:415. 10. Klein KO. Precocious puberty: who has it? Who should be treated? J Clin Endocrinol Metab 1999; 84:411. 10a. Lebrethon MC, Bourguignon JP. Management of central isosexual precocity: diagnosis, treatment, outcome. Curr Opin Pediatr 2000; 12:394. 11. Feuillam PP, Jones JV, Barnes K, et al. Reproductive axis after discontinua¬ tion of gonadotropin-releasing hormone analog. Treatment of girls with precocious puberty. Long-term follow-up comparing girls with hypotha¬ lamic hamartoma to those with idiopathic precocious puberty. J Clin Endo¬ crinol Metab 1999; 84:44. 12. Butler GE, Sellar RE, Walker RF, et al. Oral testosterone undecaenoate in the management of delayed puberty in boys: pharmacokinetics and effects on sexual maturation and growth. J Clin Endocrinol Metab 1992; 75:37. 13. Wing-yee T, Boyukgelwiz A, Hindmarsh PC, et al. Long-term outcome of oxandrolone treatment in boys with constitutional delay of growth and puberty. J Pediatr 1990; 117:588. 14. LaFranchi S, Hanna CE, Mandel SH. Constitutional delay of growth: expected versus final adult height. Pediatrics 1991; 87:82. 15. Finkelstein JS, Neer RM, Biller BMK, et al. Osteopenia in men with a history of delayed puberty. N Engl J Med 1992; 326:600. 16. Wallis M. The molecular bases of growth hormone deficiency. Mol Aspects Med 1988; 10:429. 17. De Boer H, Blok G-J, Van der Veen E. Clinical aspects of growth hormone deficiency in adults. Endocr Rev 1995; 16:63. 18. Blum WF, Albertsson-Wikland K, Rosberg S, Rauke MB. Serum levels of IGF-I and IGFBP-3 reflect spontaneous growth hormone secretion. J Clin Endocrinol Metab 1993; 76:1612. 19. Frasier SD. A review of growth hormone stimulation tests in children. Pedi¬ atrics 1974; 53:929. 20. Lawson W. The diagnosis and treatment of endocrine disorders in child¬ hood and adolescence. Springfield, IL: Charles C Thomas, 1950.

21. American Academy of Pediatrics Committee on Drugs and Committee on Bioethics. Considerations related to the use of recombinant human growth hormone in children. Pediatrics 1997; 99:122. 22. Spillotis BE, August GP, Hung W, et al. Growth hormone neurosecretory dysfunction: a treatable cause of short stature. JAMA 1984; 251:2223. 23. Daughaday WH. Recognition of growth hormone secretory disorders. (Edi¬ torial). JAMA 1984; 251:2251. 24. Moore WV, Donaldson DL, Hollowell JG, et al. Growth hormone secretory profiles: variation on'consecutive nights. J Pediatr 1989; 115:51. 25. Frasier SD. A review of growth hormone stimulation tests in children. Pedi¬ atrics 1974; 53:929. 26. Rose SR, Ross JL, Uriarte M, et al. The advantage of measuring stimulated as compared with spontaneous growth hormone levels in the diagnosis of growth hormone deficiency. N Engl J Med 1988; 319:201. 27. Donaldson DL, Pan F, Hollowell JG, et al. Reliability of stimulated and spontaneous growth hormone (GH) levels for identifying the child with low GH secretion. J Clin Endocrinol Metab 1991; 72:647. 28. Loche S, Cambiaso P, Setzu S, et al. Final height after growth hormone ther¬ apy in non-growth-hormone-deficient children with short stature. J Pediatr 1994; 125:196. 29. Zadik Z, Mira U, Landau H. Final height after growth hormone therapy in peripubertal boys with a subnormal integrated concentration of growth hormone. Horm Res 1992; 37:150. 30. Frohman LA, Aceto T Jr, MacGillivray MH. Studies of growth hormone secretion in children: normal hypopituitary and constitutionally delayed. J Clin Endocrinol Metab 1967; 27:1409. 31. Comblath M, Parker ML, Reisner SH, et al. Secretion and metabolism of growth hormone in premature and full term infants. J Clin Endocrinol Metab 1965; 25:209. 32. Martha PM Jr, Gorman KM, Blizzard RM, et al. Endogenous growth hor¬ mone secretion and clearance rates in normal boys, as determined by decon¬ volution analysis: relationship to age, pubertal status, and body mass. J Clin Endocrinol Metab 1992; 74:336. 33. Celniker AC, Chen AB, Wert RM Jr, Sherman BM. Variability in the quantity of circulating growth hormone using commercial immunoassays. J Clin Endocrinol Metab 1989; 68:469. 34. Granada ML, Sanmarti A, Lucas A, et al. Assay-dependent results of immunoassayable spontaneous 25-h growth hormone secretion in short children. Acta Paediatr Scand Suppl 1990; 370:63. 35. Baumann G. Growth hormone heterogeneity: genes, isohormones, variants, and binding proteins. Endocr Rev 1991; 12:424. 36. Chatelain P, Bouillat B, Cohen R, et al. Assay of growth hormone levels in human plasma using commercial kits: analysis of some factors influencing the results. Acta Paediatr Scand Suppl 1990; 370:56. 37. Rosenfeld RG, Albertsson-Wikland K, Cassorla F, et al. Diagnostic contro¬ versy: the diagnosis of childhood growth hormone deficiency revisited. J Clin Endocrinol Metab 1995; 80:1532. 38. Rosenfeld RG, Gargosky SE. Assays for insulin-like growth factors and their binding proteins: practicalities and pitfalls. J Pediatr 1996; 128:S52. 39. Werther GA. Growth hormone measurements versus auxologic treatment decisions: the Australian experience. J Pediatr 1996; 128:S47. 40. Strasburger CJ, Wu Z, Pflaum CD, Dressendorfer RA. Immunofunctional assay of human growth hormone in serum: a possible consensus for hGH measurement. J Clin Endocrinol Metab 1996; 81:2613. 41. L'Hermite-Balerjaux M, Copinschi G, Cauter EV. Growth hormone assays early to latest test generations compared. Clin Chem 1996; 42:1978. 42. Kaplan SA. Growth and growth hormone: disorders of the anterior pitu¬ itary. In: Kaplan SA, ed. Clinical pediatric and adolescent endocrinology, Philadelphia: WB Saunders, 1982:20. 43. Burstein S. Growth disorders after cranial irradiation in childhood. J Clin Endocrinol Metab 1994; 78:1280. 44. Ogilvy-Stuart AL, Clayton PE, Shalet SM. Cranial irradiation and early puberty. J Clin Endocrinol Metab 1994; 78:1282. 45. Blatt J, Bercu BB, Gillin JC, et al. Reduced pulsatile growth hormone secre¬ tion in children after therapy for acute lymphoblastic leukemia. J Pediatr 1984; 104:182. 46. Aceto T Jr, Frasier SD, Hayles AB, et al. Collaborative study of the effects of human growth hormone deficiency. I. First year of therapy. J Clin Endo¬ crinol Metab 1972; 35:483. 47. Arslanian SA, Becker DJ, Lee PA, et al. Growth hormone therapy and tumor recurrence findings in children with brain neoplasms and hypopituitarism. Am J Dis Child 1985; 139:347. 48. Truilizi F, Scotti G, Di Natale B, et al. Evidence of a congenital midline brain anomaly in pituitary dwarfs: a magnetic resonance imaging study in 101 patients. Pediatrics 1994; 93:409. 49. Cacciari E, Zucchini S, Ambrosetto P, et al. Empty sella in children and ado¬ lescents with possible hypothalamic-pituitary disorders. J Clin Endocrinol Metab 1994; 78:767. 50. Baroncelli GI, Bertelloni S, Ceccarelli C, Saggese G. Measurement of volu¬ metric bone mineral density accurately determines degree of lumbar under¬ mineralization in children with growth hormone deficiency. J Clin Endocrinol Metab 1998; 83:3150. 51. Frasier SD, Foley TP Jr. Creutzfeldt-Jakob disease in recipients of pituitary hormones. J Clin Endocrinol Metab 1994; 78:1277. 52. Aceto T Jr, Sotos J, Garibaldi L, et al. Response to increasing doses of growth hormone (GH) in classic GH deficiency. Pediatr Res 1992; 31:71 A. 53. Blethen SL, Compton P, Lippe B, et al. Factors predicting the response to

Ch. 198: Short Stature and Slow Growth in the Young

54.

55.

56. 57.

58.

59. 60. 61. 62. 63.

64.

65.

65a.

66. 67.

68.

68a.

69.

70. 71. 72.

73.

74. 75. 75a.

76.

77. 78. 79.

80. 81. 82.

growth hormone (GH) therapy in prepubertal children with GH deficiency. J Clin Endocrinol Metab 1993; 76:574. Hedin L, Olsson B, Diczfalusy M, et al. Intranasal administration of human growth hormone (hGH) in combination with membrane permeation enhancer in patients with GH deficiency: a pharmacokinetic study. J Clin Endocrinol Metab 1993; 76:962. Dhawan J, Pan LC, Pavlath GK, et al. Systemic delivery of human growth hormone by injection of genetically engineered myoblasts. Science 1991; 254:1509. Barr E, Leiden JM. Systemic delivery of recombinant proteins by genetically modified myoblasts. Science 1991; 254:1507. Allen DB, Julius R, Breen TJ, Attie KM. Treatment of glucocorticoid-induced growth suppression with growth hormone. J Clin Endocrinol Metab 1998; 83:2824. Hopwood NJ, Hintz RL, Gertner JM, et al. Growth response of children with non-growth-hormone deficiency and marked short stature during three years of growth hormone therapy. J Pediatr 1993; 123:215. Allen DB, Frasier SD, Foley TP Jr, Pescowitz OH. Growth hormone for children with Down syndrome. (Editorial). J Pediatr 1993; 123:742. Wilson DM, Lee PD, Morris AH, et al. Growth hormone therapy in hypophosphatemic rickets. Am J Dis Child 1991; 145:1165. Rotenstein D, Reigel DH, Flom LL. Growth hormone accelerates growth of short children with neural tube defects. J Pediatr 1989; 115:417. Angulo M, Castro-Magana C, Uy J. Pituitary evaluation and growth hor¬ mone treatment in Prader-Willi syndrome. J Pediatr Endocrinol 1991; 4:167. Finkelstein BS, Silvers JB, Marrero U, et al. Insurance coverage, physician recommendations, and access to emerging treatments. Growth hormone therapy for childhood short stature. JAMA 1998; 279(9):663. Saenger PH, Attie KM, DiMartino-Nardi J, et al. Metabolic consequences of 5-year growth hormone (GH) therapy in children treated with GH for idio¬ pathic short stature. Genentech Collaborative Study Group. J Clin Endocrinol Metab 1998; 83:3115. Rosenfeld RG, Frane J, Attie KM. Six-year results of a randomized prospecfive trial of human growth hormone and oxandrolone in Turner syndrome. J Pediatr 1992; 121:49. Chernausek SD, Attie KM, Cara JF, et al. Growth hormone therapy of Turner syndrome: the impact of age of estrogen replacement on final height. J Clin Endocrinol Metab 2000; 85:2439. Holm VA, Cassidy SB, Butler MG, et al. Prader-Willi syndrome: consensus diagnostic criteria. Pediatrics 1993; 91:398. Angulo M, Castro-Magana M, Mazur B, et al. Growth hormone secretion and effects of growth hormone therapy on growth velocity and weight gain in children with Prader-Willi syndrome. J Pediatr Endocrinol Metab 1996; 3:393. Ritzen ME, Lindgren AC, Hagenas L, Blichfeldt S, et al. Growth hormone treatment of children with Prader-Willi syndrome affects linear growth and body composition favourably. Acta Paediatr 1998; 87:28. Myers SE, Carrel AL, Whitman BY, Allen DB. Sustained benefit after 2 years of growth hormone upon body composition, fat utilization, physical agility, and growth in Prader-Willi syndrome. J Pediatr 2000; 137:42. Haffner D, Schaefer F, Nissel R, et al. Effect of growth hormone treatment on the adult height of children with chronic renal failure. N Engl J Med 2000; 343:923. Hintz RL. Untoward events in patients treated with growth hormone in the USA. Horm Res 1992; 38(Suppl 1):44. Blethen SL. Pseudotumor cerebri: the national cooperative growth study. Contribution proceedings. 1993:14. Kohn B, Julius JR, Blethen SL. Combined use of growth hormone and gona¬ dotropin-releasing hormone analogues: the national cooperative growth hormone study experience. German Study Group for Growth Hormone Treatment in Chronic Renal Failure. Pediatrics 1999; 104:1014. Laron Z, Pertzeland A, Mannheimer S. Genetic pituitary dwarfism with high serum concentrations of growth hormone: a new inborn error of metabolism? J Med Sci 1966; 2:152. Rosenfeld RG, Rosenbloom Al, Guevara-Aguirre J. Growth hormone (GH) insensitivity due to primary GH receptor deficiency. Endocr Rev 1994; 15:369. Clemmons DR, Underwood LE. Uses of human insulin-like growth factor-I in clinical conditions. J Clin Endocrinol Metab 1994; 79:4. Laron Z. The essential role of IGF-I: Lessons from the long-term study and treatment of children and adults with Laron syndrome. J Clin Endocrinol Metab 1999; 84:4397. Juul A, Kastrup KW, Pedersen SA, et al. Growth hormone (GH) provocative retesting of 108 young adults with childhood-onset GH deficiency and the diagnostic value of insulin-like growth factor I (IGF-I) and IGF-binding protein-3. J Clin Endocrinol Metab 1997; 82:1195. Rosen T, Bengtsson BA. Premature mortality due to cardiovascular disease in hypopituitarism. Lancet 1990; 336:285. Sonksen PH, Weissberger AJ. Growth hormone deficiency in adults. Growth Genet Horm 1998; 14:41. Baum HB, Katznelson L, Sherman JC, et al. Effects of physiological growth hormone (GH) therapy on cognition and quality of life in patients with adult-onset GH deficiency. J Clin Endocrinol Metab 1998; 83:3184. Zimmerman D, Saenger PH, Gharib H. AACE clinical practice for growth hormone use in adults. Endocr Pract 1998; 4:165. Aleman A, Verharr HJJ, DeHaan EHF, et al. Insulin-like growth factor-I and cognitive function in healthy older men. J Clin Endocrinol Metab 1999; 84:471. Pfeiffer M, Verhomer R, Zizek B, et al. Growth hormone treatment reverses

83.

84.

85.

85a. 86. 87. 88. 89.

90. 91. 92. 93. 94. 95. 96.

97.

98.

99.

100. 101. 102. 103.

104.

105. 106.

107. 108.

109.

110. 111. 112. 113. 114.

115.

116. 117.

1807

early atherosclerotic changes in GH-deficient adults. J Clin Endocrinol Metab 1999; 84:453. Klein RZ, Arnold MB, Bigos ST, et al. Correlation of cognitive test scores and adequacy of treatment in adolescents with congenital hypothyroidism. J Pediatr 1994; 124:383. Sekadde CB, Slaunwhite WR, Aceto T Jr. Rapid radioimmunoassay of tri¬ iodothyronine in clinical radioassay procedures: a compendium. In: Besch PK, ed. American Association of Clinical Chemists, 1975:292. Jones KL. Achondroplasia syndrome. In: Jones KL, ed. Smith's recognizable patterns of human malformation, 5th ed. Philadelphia: WB Saunders, 1997:298. Hunter I, Greene SA, MacDonald TM, Morris AD. Prevalence and aetiology of hypothyroidism in the young. Arch Dis Child 2000; 83:207. Sekadde CB, Slaunwhite WR Jr, Aceto T Jr, Murray K. Administration of thyroxine once a week. J Clin Endocrinol Metab 1975; 39:759. Pantsiotou S, Stanhope R, Uruena M, et al. Growth prognosis and growth after menarche in primary hypothyroidism. Arch Dis Child 1991; 66:838. Man EB, Jones WS. Thyroid function in human pregnancy, V. Am J Obstet Gynecol 1969; 104:898. Haddow JE, Klein RZ, Mitchell ML, et al. Maternal thyroid deficiency dur¬ ing pregnancy and subsequent neuropsychological development of the child. N Engl J Med 1999; 341:549. Beighton P, Giedion ZA, Gorlin R, et al. International classification of osteochondrodysplasias. Am J Med Genet 1992; 44:223. Hall BD. Approach to skeletal dysplasia. Pediatr Clin North Am 1992; 39:279. Shapiro F. Epiphyseal disorders. N Engl J Med 1987; 317:1702. Jones KL. Osteochondrodysplasias. In: Jones KL, ed. Smith's recognizable patterns of human malformation, 5th ed. Philadelphia: WB Saunders, 1997. Taybi H, Lachman RS. Radiology of syndromes, metabolic disorders, and skeletal dysplasias, 3rd ed. Chicago: Year Book, 1990:671. Saleh M, Burton M. Leg lengthening: patient selection and management in achondroplasia. Orthop Clin North Am 1991; 22:589. Lavini F, Renzi-Brivio L, de Bastianai G. Psychologic, vascular, and physio¬ logic aspects of lower limb lengthening in achondroplastics. Clin Orthop 1990; 250:138. Jones KL. Hypochondroplasia syndrome. In: Jones KL, ed. Smith's recog¬ nizable patterns of human malformation, 5th ed. Philadelphia: WB Saun¬ ders, 1997:304. Powell GF, Brasel JA, Blizzard RM. Emotional deprivation and growth retardation simulating idiopathic hypopituitarism. I. Clinical evaluation of the syndrome. N Engl J Med 1967; 276:1271. Powell GF, Brasel JA, Raid S, et al. Emodonal deprivation and growth retar¬ dation simuladng idiopathic hypopituitarism. II. Endocrine evaluation of the syndrome. N Engl J Med 1967; 276:1279. Sandberg DE, Smith MM, Fomari V, et al. Nutritional dwarfing: is it a con¬ sequence of disturbed psychosocial functioning? Pediatrics 1991; 88:926. Rudolf MCJ, Hochberg Z. Annotadon. Are boys more vulnerable to psycho¬ social growth retardation? Dev Med Child Neurol 1990; 32:1022. Smith MM, Lifshitz F. Excess fruit juice consumption as a contributing fac¬ tor in nonorganic failure to thrive. Pediatrics 1994; 93:438. Money J, Annecillo C, Kelly JF. Growth of intelligence: failure and catch-up associated respectively with abuse and rescue in the syndrome of abuse dwarfism. Psychoneuroendocrinology 1983; 8:309. Rosenthal SR, Snyder JD, Hendricks KM, Walker WA. Growth failure and inflammatory bowel disease. Approach to treatment of a complicated ado¬ lescent problem. Pediatrics 1983; 72:481. Kirschner BS. Growth and development in chronic inflammatory bowel dis¬ ease. Acta Paediatr Scand Suppl 1990; 366:98. Polk DB, Hattner JAT, Kemer JA Jr. Improved growth and disease activity after intermittent administration of a defined formula diet in children with Crohn's disease. J Parenter Enteral Nutr 1992; 16:499. Lipson AB, Savage MO, Davies PSW, et al. Acceleration of linear growth fol¬ lowing intestinal resection for Crohn's disease. Eur J Pediatr 1990; 149:687. Muguruza MTG, Chrousos GP. Periodic Cushing syndrome in a short boy: usefulness of the ovine corticotropin releasing hormone test. J Pediatr 1989; 115:270. Preeyasombat C, Sikikulchayanonta V, Mahaclok Elert-Wattana P, et al. Cushing's syndrome caused by Ewing's sarcoma secreting corticotropin releasing factor-like peptide. Am J Dis Child 1992; 146:1103. Putnam TI, Aceto T Jr, Abbassi V, Kenny FM. Cushing's disease with a spon¬ taneous remission. Pediatrics 1972; 50:477. Kammer H, Barton M. Spontaneous remission of Cushing's disease: a case report and review of the literature. Am J Med 1979; 67:519. McArthur RG, Cloutier MD, Hayles AB, Sprague RF. Cushing's disease in children: findings in 13 cases. Mayo Clin Proc 1972; 47:318. Thomas CG, Smith AT, Griffith JM, Askin FB. Hyperadrenalism in child¬ hood and adolescence. Ann Surg 1984; 199:538. Oldfield EG, Doppman JL, Nieman LK, et al. Petrosal sinus sampling with and without corticotropin-releasing hormone for the differential diagnosis of Cushing's syndrome. N Engl J Med 1991; 325:897. Tyrell JB, Brooks RM, Fitzgerald PA, et al. Cushing's disease: selective trans¬ sphenoidal resection of pituitary microadenomas. N Engl J Med 1978; 298:753. Sonino N, Boscaro M, Merola G, Mantero F. Prolonged treatment of Cushing disease by ketoconazole. J Clin Endocrinol Metab 1985; 61:718. Roche AF, Lipman RS, Overall JE, Hung W. The effects of stimulant medica¬ tion on the growth of hyperkinetic children. Pediatrics 1979; 63:847.

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PART XIII: ENDOCRINE AND METABOLIC DYSFUNCTION IN THE GROWING CHILD AND IN THE AGED

118. Dickinson LC, Lee J, Ringdahl IC, et al. Impaired growth in hyperkinetic children receiving pemoline. J Pediatr 1979; 94:538. 119. Vincent J, Varley CK, Leger P. Effects of methylphenidate on early adoles¬ cent growth. Am J Psychiatry 1990; 147:501. 120. Spencer T, Biederman J, Wilens T. Growth deficits in children with attention deficit hyperactivity disorder. Pediatrics 1998; 102:501. 121. Sandberg D. Short stature in middle childhood: a survey of psychosocial functioning in a clinic-referred sample. In: Stabler B, Underwood L, eds. Growth, stature, and adaptation. Chapel Hill, NC: University of North Carolina Press, 1994:19. 122. Stabler B, Underwood L, eds. Growth, stature, and adaptation. Chapel Hill, NC: University of North Carolina Press, 1994. 123. Zimet G. Psychosocial functioning of adults who were short as children. In: Stabler B, Underwood L, eds. Growth, stature, and adaptation. Chapel Hill, NC: University of North Carolina Press, 1994:73. 124. Sartorio A, Conti A, Molinari E, et al. Growth, growth hormone and cogni¬ tive functions. Horm Res 1996; 45:23. 125. Stabler B, Clopper RR, Siegel PT, et al. Links between growth hormone defi¬ ciency, adaptation and social phobia. Horm Res 1996; 45:30. 126. Burman P, Deijen JB. Quality of life and cognitive function in patients with pituitary insufficiency. Psychother Psychosom 1998; 67:154. 127. Lagrou K, Xhouret-Heinrichs D, Heinrichs C, et al. Age-related perception of stature, acceptance of therapy, and psychosocial functioning in human growth hormone-treated girls with Turner's syndrome. J Clin Endocrinol Metab 1998; 83:1494. 128. McCauley E. Self-concept and behavioral profiles in Turner Syndrome. In: Stabler B, Underwood L, eds. Growth, stature, and adaptation. Chapel Hill, NC: University of North Carolina Press, 1994:181. 129. Skuse D. Psychosocial functioning in the Turner Syndrome: a national sur¬ vey. In: Stabler B, Underwood L, eds. Growth, stature, and adaptation. Chapel Hill, NC: University of North Carolina Press, 1994:151. 130. Rovet J. School outcome in Turner Syndrome. In: Stabler B, Underwood L, eds. Growth, stature, and adaptation. Chapel Hill, NC: University of North Carolina Press, 1994:165. 131. Cuttler L, Silvers JB, Singh J, et al. Short stature and growth hormone ther¬ apy. A national study of physician recommendation patterns. JAMA 1996; 276:531. 132. Hintz RL. Growth hormone treatment of idiopathic short stature. Horm Res 1996; 46:208. 133. Rao JK, Julius JR, Breen TJ, Blethen SL. Response to growth hormone in attention deficit hyperactivity disorder: effects of methylphenidate and pemoline therapy. Pediatrics 1998; 102(Suppl):497. 134. Stabler B, Siegel PT, Clopper RR, et al. Behavior change after growth hor¬ mone treatment of children with short stature. J Pediatr 1998; 133:366.

CHAPTER

1 99

ENDOCRINOLOGY AND AGING DAVID A. GRUENEWALD AND ALVIN M. MATSUMOTO The number of elderly people is growing faster than the popu¬ lation at large. The number of Americans older than age 65 years is expected to increase from 35 million in 2000 to 78 million in 2050. Furthermore, the number of the "oldest old," those older than age 85, is expected to increase from 4 million in 2000 to almost 18 million in 2050.1 Thus, the latter, most frail group of elderly people with the greatest burden of age-associated diseases is the group that is growing the most rapidly.13 Because endocrine diseases such as osteoporosis, type 2 diabetes mellitus, and hypothyroidism are extremely common in older people, adult endocrinologists will see an increasing proportion of elderly patients in their practices in the future. With aging, changes occur in many parameters of endocrine function, such as decreases in growth hormone (GH) and gonadal steroid levels, and increases in cholesterol levels and adiposity. Many of these changes predispose to morbidity and mortality in later life; for example, ovarian failure affects bone mass and frac¬ ture risk. The effects of other age-related alterations (e.g., declin¬

ing GH and testosterone levels) are of uncertain significance. Furthermore, the clinical presentation, diagnosis, treatment, and prognosis of certain endocrine diseases are altered with aging, greatly increasing the clinical challenges of evaluation and man¬ agement in elderly patients. In turn, endocrine diseases in geri¬ atric patients often have profound effects on functional status and quality of life, and these issues are often much more impor¬ tant to patients than the underlying diseases per se. Ultimately, the primary goal of medical management in elderly people is not to eliminate disease, but rather to help the older person achieve the highest possible level of functioning and quality of life.

PRINCIPLES OF GERIATRIC ENDOCRINOLOGY IMPAIRED HOMEOSTASIS Aging is characterized by a decline in functional reserve of major body organs, leading to impaired ability to restore equilibrium after environmental stresses. This age-related impairment of homeostatic regulation is evident in many endocrine functions but may become clinically evident only during acute or signifi¬ cant long-term stress. For example, fasting blood glucose levels exhibit very little change with normal aging, but after challenge with a glucose load, glucose levels increase more in healthy elderly people than in young adults. The function of endocrine systems may be maintained through homeostatic mechanisms and/or changes in hormone metabolism that offset the loss of function. For example, pituitary luteinizing hormone (LH) secre¬ tion and serum LH levels are increased in many elderly men and testosterone metabolism is decreased, thus compensating for a reduction in testicular testosterone secretion. In some cases, how¬ ever, these changes are insufficient to maintain normal function with aging even under basal conditions. One example is aldos¬ terone production, which declines disproportionately to its clear¬ ance rate with aging, a situation that leads to age-related decreases in basal plasma aldosterone levels. Several principles of geriatric endocrinology illustrate the complexity and challenge of evaluating frail older patients with endocrine disease. These include the atypical presentations of illness; the presence of multiple coexisting medical problems; the large number of symptoms, signs, and abnormal laboratory 4 findings often present in individual elderly patients; underre¬ porting of symptoms; and problems in the cognitive, psychiat¬ ric, social, economic, and functional domains. Failure to appreciate these challenges and to appropriately assess older patients with these issues in mind may result in missed or incor¬ rect diagnoses, inappropriate treatments, and poor functional outcomes.

NONSPECIFIC AND ATYPICAL PRESENTATION Endocrinopathies commonly present in elderly people with nonspecific, muted, or atypical symptoms and signs. For exam¬ ple, hypothyroidism and hyperthyroidism may present simi¬ larly in older adults with nonspecific symptoms, including weight loss, fatigue, weakness, constipation, and depression. The presentation of endocrine disease in geriatric patients may also be atypical compared with that in younger patients (e.g., apathy, depression, and psychomotor retardation may be asso¬ ciated with hyperthyroidism, and marked hyperglycemia and hyperosmolarity without ketoacidosis may be present in elderly patients with type 2 diabetes). In some patients, regardless of the cause of the illness, its manifestations may occur in the most

Ch. 199: Endocrinology and Aging compromised body system. Thus, in an older patient with underlying gait and balance abnormalities, falling may be the primary symptom of diseases as diverse as pneumonia, myocar¬ dial infarction, uncontrolled diabetes mellitus, or hypothyroid¬ ism. Illnesses may present in the guise of other disabling geriatric syndromes such as delirium, urinary incontinence, and dementia. Endocrine disorders may produce or be associated with any or all of these syndromes, so endocrinologists must have a basic understanding of these disorders. Several excellent reviews of these geriatric syndromes are available.2-4

DIFFICULTIES IN LABORATORY EVALUATION In addition to the atypical or nonspecific presentations of endo¬ crine disease described earlier, with aging, it is increasingly com¬ mon for illnesses such as hypothyroidism to present without any symptoms. The presence of disease may be appreciated only on routine laboratory screening, as in the case of asymptomatic hypercalcemia secondary to hyperparathyroidism. Furthermore, the presence of multiple medical problems and the use of multiple medications may confound the evaluation of older patients. For example, decreased serum thyroxine (T4) and triiodothyronine (T3) levels and alterations in the levels of serum thyroidstimulating hormone may occur in elderly patients who are systemically ill or are taking certain medications (e.g., glucocorti¬ coids, dopamine) but are euthyroid (euthyroid sick syndromes), giving a misleading impression of an endocrine abnormality. The evaluation of the older patient is further complicated by the fact that normal ranges for endocrine laboratory tests are usually established in healthy young subjects and may not reflect normal values in healthy elderly people. Moreover, nor¬ mative data for older populations are often confounded by the inclusion of subjects with age-associated diseases. Finally, most studies of aging and endocrine function in humans are crosssectional rather than longitudinal and, therefore, may not accu¬ rately predict age-related changes within a given individual. Indeed, variability among individuals is a hallmark of aging.

GERIATRIC ASSESSMENT AND TREATMENT The onset of functional decline may be an important, and some¬ times the only, clue to the development of an acute illness or exacerbation of a chronic disease in geriatric patients. Accord¬ ingly, a structured geriatric assessment should be a part of the clinical evaluation, especially in frail elderly patients. Func¬ tional assessment can detect impairments in physical function, cognition, emotional status, sensory capabilities, and activities of daily living that are not detected by standard clinical exami¬ nations,5 and these impairments are often much more important to patients than the underlying diseases that give rise to them. Such an assessment can also help to determine the response to treatment and to predict the patient's ultimate degree of disabil¬ ity. A useful approach to general outpatient screening of older patients for functional disability has been suggested.6 Patients with evidence of functional impairment on screening examina¬ tion may benefit from a comprehensive functional assessment by an interdisciplinary care team. However, comprehensive evaluation is time consuming and expensive; therefore, it should be targeted to the most appropriate patients: frail or ill elderly people with a real or anticipated functional decline (including patients on the verge of requiring institutionaliza¬ tion), those with inadequate primary medical care, and those with poor economic and social support systems.7 Treatment decisions involving geriatric patients with endo¬ crine disease must consider age-associated factors such as alter¬ ations in clearance rate and target-organ effects, coexisting

1809

medical illnesses, and the medications taken by the patient. Older patients consume a disproportionate share of medications compared with the population at large. Moreover, drug toxicities are more frequent and severe in the elderly than in young patients receiving the same drug regimen.3 Dysfunction in mul¬ tiple organ systems together with cognitive and visual impair¬ ment further predispose older patients to adverse drug effects. As a result, older people are at high risk for the development of medication side effects and drug interactions secondary to polypharmacy. To minimize these risks, dosage levels for hor¬ mone replacement and medications must be adjusted for changes in clearance rate with aging, and patients should receive the lowest dosage of medication needed to achieve the therapeutic effect. New medications should be initiated using low doses and increased very gradually as needed. Finally, the medication regimen should be reviewed periodically, and med¬ ications no longer needed should be discontinued.

HYPOTHALAMUS AND PITUITARY GLAND HYPOTHALAMUS Studies directly assessing the effects of aging on parameters of hypothalamic neuroendocrine function in humans have not been performed. Some of these effects, however, can be inferred by assessing age-related alterations in circadian and ultradian rhythms (e.g., pulsatile release) of pituitary hormones and by determining pituitary hormonal responsiveness to administra¬ tion of hypothalamic releasing hormones or to agents that either block end-organ feedback (e.g., clomiphene and metyrapone) or stimulate hypothalamic-pituitary hormonal secretion (e.g., stimulation of antidiuretic hormone [ADH] secretion by hyper¬ tonic saline administration or stimulation of GH secretion by insulin-induced hypoglycemia). Age-related blunting of the circadian rhythm of LH pulse fre¬ quency has been observed in healthy elderly men, suggesting altered regulation of the gonadotropin-releasing hormone (GnRH) pulse generator with aging.8 Furthermore, LH pulse fre¬ quency is relatively decreased despite reduced testosterone lev¬ els in some healthy elderly men as compared with young men, implying decreased GnRH pulse frequency in these older men.q Interestingly, despite this impairment in baseline LH pulse fre¬ quency with aging, fasting decreases LH pulse frequency in healthy young men but not in older men, indicating altered reproductive axis regulation in response to a fasting stress.10 Finally, administration of naloxone, an opioid antagonist, does not increase LH pulse frequency in healthy older men as it does in young men, suggesting altered hypothalamic opioid regula¬ tion of LH secretion.11 In contrast to the findings for the repro¬ ductive axis, adrenocorticotropic hormone (ACTH) pulse frequency, cortisol levels, and ACTH response to corticotropin¬ releasing hormone (CRH) stimulation are unchanged in healthy elderly compared with young men, suggesting that hypotha¬ lamic regulation of pituitary/adrenocortical function may be relatively unimpaired by aging.12 Hypothalamic-pituitary feedback sensitivity to some endorgan hormones is altered with aging. For example, most stud¬ ies have found increased feedback sensitivity to testosterone with aging,13'14 whereas glucocorticoid feedback sensitivity is decreased with aging.15

POSTERIOR PITUITARY: ANTIDIURETIC HORMONE The bulk of evidence suggests an increase in basal ADH levels with aging.16 Furthermore, aging is associated with an increased

1810

PART XIII: ENDOCRINE AND METABOLIC DYSFUNCTION IN THE GROWING CHILD AND IN THE AGED

ADH responsiveness to osmotic stimuli such as hypertonic saline infusion, and pharmacologic inhibition of ADH secretion (e.g., with ethanol infusion) is impaired in elderly subjects compared with young adults.17 Taken together with the age-associated decline in glomerular filtration rate, the increased prevalence of conditions such as congestive heart failure and hypothyroidism, and the use of sulfonylurea or diuretic medication, these changes in ADH secretion predispose elderly people to the development of hyponatremia by impairing free water clearance. Elderly people are also at increased risk for dehydration and hypernatremia. Although ADH secretory capacity is unim¬ paired with aging, the renal response to ADH is blunted, possi¬ bly due to chronic exposure to elevated levels, resulting in decreased maximal urinary concentrating capacity.16 Further¬ more, baroreceptor responsiveness to ADH declines with aging, such that release in response to hypotension or hypovolemia is decreased and the risk of volume depletion is higher. Other fac¬ tors predisposing older adults to water depletion include the impairment in thirst responses to dehydration18 and the com¬ mon occurrence of states that limit access to free water (e.g., altered mental status, immobility, and surgery).

ANTERIOR PITUITARY GROWTH HORMONE The GH axis undergoes significant alterations in many healthy elderly people (see Chap. 12). In adult men, GH secretion declines progressively after age 40, and by age 70 to 80 approxi¬ mately half of all men have no significant GH secretion over a 24hour period. Levels of plasma somatomedin C (insulin-like growth factor-I [IGF-I]) show a corresponding decline, such that by age 70 to 80, -40% of subjects exhibit plasma IGF-I levels sim¬ ilar to those found in GH-deficient children (see Chaps. 12 and 173). These low IGF-I levels in octogenarians correlate with an absence of significant nocturnal GH pulses.19 Circulating levels of IGF-binding protein-3 (IGFBP-3) also decrease with aging, but whether this affects the bioactivity of IGF-I in older adults is unclear.20 Based on animal studies, this dramatic decrease in GH secre¬ tion with aging is thought to be due primarily to decreased hypothalamic secretion of GH-releasing hormone (GHRH) and increased hypothalamic somatostatin production, rather than to an age-related decrease in pituitary GHRH responsiveness.19'21 Furthermore, the ability of exogenous IGF-I to suppress serum GH levels decreases with advancing age in humans, suggesting that declining GH secretion with aging is not due to increased sensitivity to IGF-I negative feedback.22 In line with these obser¬ vations, normal GH and IGF-I levels can be achieved in GHdeficient elderly subjects with GHRH administration.19 Of note, the magnitude of the increase in pulsatile GH secretion induced by fasting was found to be similar in elderly and young adult human subjects, although the absolute levels of GH secretion of older subjects were -50% lower in both fed and fasted condi¬ tions.23 These findings suggest that age-related hyposomatotropism may be partially reversible by lifestyle modifications such as changes in diet or exercise. The GH response to various secretagogues (e.g., insulin, arginine, and L-dopa) and to GHRH is normal or reduced with aging. As with the thyrotropin-releasing hormone (TRH) stimu¬ lation test, a normal GH response may be useful in verifying intact pituitary (somatotrope) function. The insulin-tolerance test is considered the definitive test to diagnose GH deficiency in adults,24 but in elderly people this test is associated with increased risk due to the high prevalence of ischemic heart dis¬ ease in this population. The arginine-stimulation test25 and the combined administration of arginine and GHRH24 have been

proposed as alternative methods for the assessment of GH secre¬ tion in older patients. Many age-associated changes in body composition, such as increased adiposity and decreased muscle and bone mass, are similar to those associated with GH deficiency in younger patients.21-26 This observation has led to the hypothesis that decreased GH secretion with aging contributes to alterations in body composition (including diminished muscle and bone mass) and increased frailty in older adults, and to the suggestion that GH supplementation might be clinically useful in preventing or reversing these age-related changes. As noted earlier, compared with young adults, many healthy older adults are deficient in GH and IGF-I, and frail elderly people living in nursing homes have even lower IGF-I levels than healthy elderly subjects.27 Whether young adult or age-adjusted reference ranges are the most appropriate standards to use for older adults is unclear, however, and definitive criteria for clinically significant GH defi¬ ciency in elderly people have not been established. Short-term GH replacement in older men with low plasma IGF-I levels was found to increase lean body mass by 9% and reduce fat mass by 14%.28 These beneficial effects were sustained in a similar study over 1 year of follow-up and regressed partially after cessation of treatment, results which suggest that hyposomatotropism contributes to age-related alterations in body com¬ position.29 "Physiologic" replacement in elderly people, however, may require a lower dosage than in young adults.30 Side effects such as carpal tunnel syndrome and gynecomastia were common in older subjects with plasma IGF-I levels exceeding 1.0 U/mL during treatment,19 even though normal IGF-I levels in young adults may be as high as 1.5 U/mL. Moreover, whether GH sup¬ plementation can achieve meaningful improvements in func¬ tional status or quality of life in elderly people is unclear. In healthy older men with low IGF-I levels but intact functional capacity, GH replacement increased lean body mass and reduced adiposity but did not yield any discernible improvement in func¬ tional capacity.31 GH supplementation, however, possibly could improve functioning in frail GH- and IGF-I-deficient elderly peo¬ ple with preexisting functional deficits. Other abnormalities of GH secretion, including GH defi¬ ciency in adults with hypothalamic-pituitary disease, are cov¬ ered in Chapter 12. PROLACTIN No clinically significant changes in basal prolactin levels occur ' with aging. The amplitude of nocturnal pulsatile prolactin secre¬ tion, however, is lower in elderly than in young men; this may be due to age-related alterations in dopaminergic regulation of prolactin secretion.32 Furthermore, several medications com¬ monly used by elderly patients, including phenothiazines, metoclopramide, and cimetidine, inhibit dopamine secretion and sometimes cause elevated prolactin levels. Hypothyroidism increases hypothalamic TRH release, which in turn stimulates prolactin secretion. When hyperprolactinemia does occur, its clinical manifestations are usually subtle and often unrecog¬ nized. Prolactin excess has antigonadotropic effects; therefore, hyperprolactinemia causes secondary hypogonadism and may contribute to sexual dysfunction and bone loss. Less common manifestations of hyperprolactinemia in older people include gynecomastia and, rarely, galactorrhea. ADRENOCORTICOTROPIC HORMONE No significant age-related changes occur in basal ACTH and cortisol levels, or in cortisol responses to exogenous ACTH stim¬ ulation. However, evidence obtained using stimuli such as metyrapone, insulin-induced hypoglycemia, or ovine CRH,

Ch. 199: Endocrinology and Aging with and without vasopressin, indicates that cortisol and ACTH responses to stimuli at or above the level of the anterior pituitary are increased or prolonged with aging.33'34 Furthermore, the sen¬ sitivity of the hypothalamic-pituitary-adrenal (HPA) axis to glucocorticoid negative feedback is decreased with aging (see discussion of adrenal cortex physiology later). THYROID-STIMULATING HORMONE Conflicting data have been reported on the effect of aging on thyroid-stimulating hormone (TSH) levels. Some studies have found unchanged or slightly increased TSH levels in normal elderly people, with elevated TSH occurring more commonly in females. However, TSH was found to decrease with aging in healthy subjects who were carefully selected to exclude subclinical primary hypothyroidism.35-36 In fact, primary hypothyroid¬ ism is very common in older adults, with 3% of older men and 7% of elderly women having TSH levels >10 iiU/mL.37 In other cases, TSH levels were suppressed by concurrent glucocorticoid use, and by fasting and stress, which were associated with severe nonthyroidal illnesses. Thyroid responsiveness to TSH administration is preserved with normal aging, but the TSH response to TRH is diminished or even absent in healthy elderly people, particularly in men. Therefore, an abnormal TRH-stimulation test cannot be used to support a diagnosis of hyperthy¬ roidism in elderly people. A normal TSH response to TRH may be useful in ruling out hyperthyroidism, but this test is rarely needed with the widespread availability of highly sensitive TSH assays. Finally, in young adults, TSH secretion exhibits a circa¬ dian variation, with the highest levels of TSH released during the night. This nocturnal TSH peak is blunted with aging, how¬ ever, suggesting hypothalamic dysfunction.38 GONADOTROPINS In the early perimenopausal phase of the menopausal transition, the number of ovarian follicles gradually declines, leading to a reduction in inhibin-B production.39 The decrease in negative feedback at the pituitary due to reduced inhibin-B levels is thought to be the primary stimulus leading to an increase of the serum follicle-stimulating hormone (FSFI) levels that sustain inhibin-A and estradiol production until late in the menopausal transition.39 Ultimately, inhibin A and estradiol levels decline and FSH levels increase substantially, marking the progression to late perimenopausal status—although LH levels do not increase during this period. After menopause, FSH levels are increased to a greater extent than are LH levels, although by 15 years postmenopause, LH levels fall to below premenopausal levels. Postmenopausal women exhibit an exaggerated gona¬ dotropin response to GnRH, due to loss of negative feedback from ovarian hormones. Furthermore, older postmenopausal women exhibit lower basal 24-hour mean LH levels and greater suppression of LH and FSH secretion by estradiol than younger women with premature ovarian failure, suggesting hypotha¬ lamic-pituitary alterations in aging women.40 Aging men exhibit higher basal LH and FSH levels than younger men, but gonadotropin levels often remain within the normal range. Testosterone levels are decreased in many healthy older men with elevated gonadotropin levels, implying primary testicular failure. Furthermore, decreased LH pulse frequency (an indicator of hypothalamic GnRH pulse generator activity) is evident in some healthy elderly men despite reduced testosterone levels.10-41 Evidence of altered pituitary function is also seen with aging, with slightly impaired LH responses to GnRH administration in elderly men compared with young men.42 Decreased testosterone levels together with inappropriately normal (i.e., not elevated) gonadotropin levels

1811

is a common finding in both healthy and systemically ill elderly men, suggesting secondary hypogonadism. Additional clinical and hormonal evidence of pituitary dysfunction is needed in these cases to justify imaging studies to rule out pituitary tumors.

PITUITARY ADENOMAS AND THE EMPTY SELLA SYNDROME The incidence of pituitary tumors is not markedly altered with aging.43 Autopsy studies reveal that pituitary "incidentalomas" are common, occurring in 13% to 27% of subjects.44 Most func¬ tioning adenomas are microscopic prolactin-producing tumors (see Chap. 13). In contrast, nonfunctioning adenomas are the most common type of pituitary tumors diagnosed during life in older adults, comprising 61% to 73% of cases.44 Many of these apparently nonfunctioning adenomas, however, actually pro¬ duce quantities of gonadotropins (especially FSH) or the a subunit of these glycoprotein hormones. Nonsecreting tumors and tumors that secrete LH, FSH, or a subunit are usually large at the time they are diagnosed, because few or no symptoms of hormonal hypersecretion occur. These tumors typically present with a mass effect, including visual field abnormalities and headaches, as incidental findings on imaging studies, or with manifestations of panhypopituitarism. Vision changes are the most common presentation of pituitary adenoma in elderly patients,45 and for pituitary tumors to present with symptoms of hormonal overproduction (e.g., acromegaly or Cushing disease) is uncommon. As iir younger adults, management of large pituitary tumors usually involves transsphenoidal decompression and debulking, along with assessment of anterior pituitary hormone func¬ tion and replacement of hormone deficiencies. Prolactinomas are managed medically with dopamine agonists, such as bro¬ mocriptine, pergolide, or cabergoline. Both transsphenoidal pituitary surgery and radiotherapy appear to be effective and relatively well tolerated by older patients who are appropriate candidates. The clinician may find it appropriate, however, to manage elderly patients with normal endocrine status and no visual field defects who are asymptomatic or at high surgical risk with only serial magnetic resonance imaging (MRI) and visual field assessment.44 Panhypopituitarism is difficult to diagnose in older patients because the symptoms are nonspecific and are difficult to distin¬ guish from other common age-related symptoms. Among the presentations of hypopituitarism reported in case series of elderly patients are postural hypotension, recurrent falls, hyponatremia, weakness, weight loss, immobility, drowsiness and confusion, and urinary incontinence.44 Elderly patients with panhypopituitarism require replacement of thyroid hormone and cortisol; the indications for replacement of estrogen, tes¬ tosterone, and GH in these patients have not been as clearly established.44 With the widespread use of brain-imaging techniques such as computerized tomography (CT) and MRI, both pituitary masses and the empty sella syndrome are being identified with greater frequency. An MRI study of healthy young and elderly subjects reported that pituitary height and volume tends to decrease with aging, and empty sella was observed in 19% of elderly subjects but in none of the young subjects. No relation¬ ship was seen between pituitary volume and anterior pituitary hormone levels,46 however, confirming other reports that clini¬ cally apparent pituitary dysfunction is uncommon in empty sella syn¬ drome.47 This study also found that the posterior pituitary bright signal on T1 -weighted MRI, which is thought to reflect stored ADH-neurophysin complex, was not detected in 29% of healthy

1812

PART XIII: ENDOCRINE AND METABOLIC DYSFUNCTION IN THE GROWING CHILD AND IN THE AGED

elderly subjects, whereas it was detected in all young adult sub¬ jects.46 None of the subjects had clinical manifestations of diabe¬ tes insipidus, but plasma osmolarity and ADH levels were higher in the elderly subjects. Thus, the absence of the posterior pituitary bright signal on Tl-weighted MRI appears to reflect a phys¬ iologic depletion of ADH neurosecretory granules rather than a patho¬ logic occurrence. Based on the foregoing, the functional significance of an incidental finding of empty sella or altered posterior pituitary bright signal on MRI in apparently healthy elderly patients is unclear. In such patients, conservative man¬ agement is appropriate, although visual field testing, and mea¬ surement of serum anterior pituitary hormone levels in patients with empty sella, are indicated to rule out subclinical pituitary dysfunction and suprasellar involvement.

TABLE 199-1. Alterations in Thyroid Physiology and Hormones with Aging T4 production 4 T4 clearance 1 T4 to T3 conversion i T3 clearance 1 Serum total and free T4 20 pU/mL, and high titers of thyroid antimicrosoma 1 antibodies are associated with an increased likelihood of even¬ tual thyroid failure.54 Treatment with L-thyroxine may be beneficial for some elderly patients with subclinical hypothyroidism, with improvements in myocardial contractility and psychometric testing. The results of randomized trials of treatment for subclinical hypothyroidism have been mixed, however.54 In those who are not started on Lthyroxine, the TSH level can be followed and thyroid replace¬ ment initiated if TSH levels progressively increase. Alternatively, L-thyroxine can be initiated at low dosages and carefully titrated to achieve normal serum TSH levels. Some physicians advise starting L-thyroxine treatment in all patients with serum TSH levels of >10 mU / L, as well as in those with both a borderline ele¬ vated TSH of between 5 and 10 mU/L and an abnormal elevation of thyroid antimicrosomal antibodies.36 Potential adverse effects such as exacerbation of myocardial ischemia and the patient's ability to comply with treatment should also be taken into consideration, however. Replacement should be with L-thyroxine in a reliable preparation of accurate dosage and predictable bioavailability. Thyroid hormone requirements decrease with age, due to a reduction in clearance rate; replacement dosages average 110 pg per day in elderly patients compared with 130 pg per day in younger patients.60 With advancing age or declining nutritional status, a reduction in T4 dosage may be necessary over time. In most older patients, the initial replacement dosage is 25 pg per day, increasing by 25 pg every 4 weeks until serum TSH lev¬ els have normalized. In those with known cardiac disease, treat¬ ment is usually begun even more gradually, at an initial dosage of 12.5 to 25 pg per day, with increments every 4 weeks as toler¬ ated. However, severely hypothyroid patients may require ini¬ tial replacement doses of 50 to 100 pg orally, or up to 400 pg intravenously for myxedema stupor and coma, even with evi¬ dence of concomitant cardiac disease. In these markedly hypothyroid patients, the clinician is advised to test for the pos¬ sibility of coexistent adrenal insufficiency and to protect against precipitation of adrenal crisis during thyroid replacement by giving stress dosages of glucocorticoid replacement (e.g., 200 mg hydrocortisone) before T4 administration. Although restoring the euthyroid state without exacerbating angina is not always possible, treatment of hypothyroidism should not be withheld based on the fear of an adverse effect of thyroid replacement. Treatment of patients with coexisting ischemic cardiac disease is individualized to reduce or eliminate symptoms of hypothyroidism without inducing intolerable anginal symptoms. Obtaining a baseline level of creatine phosphokinase (CPK), which can be elevated in some cases of hypothyroidism, in case the patient develops ischemic cardiac symptoms during initiation of treatment, may be useful. Other areas of concern during thyroid-replacement therapy in older people include effects on dosages of other medications and avoidance of thyroid overreplacement. As hypothyroidism is corrected, the clearance rate of other medications may be affected, such that adjustments in dosage are required. Exces¬ sive T4 replacement should be avoided, because it may result in decreased bone density and exacerbation of underlying heart

1815

disease, especially in high-risk osteopenic or cardiac patients. Finally, in patients with secondary hypothyroidism due to pitu¬ itary or hypothalamic disease, serum TSH levels cannot be used either to diagnose or to monitor thyroid hormone replacement as in those with primary hypothyroidism. Normalization of total or free T4 levels should be the goal in these patients.

NODULAR THYROID DISEASE AND THYROID CANCER With aging, the thyroid gland becomes increasingly nodular, and the incidence of multinodular goiter increases. Estimates are that 90% of women older than age 70 and 60% of men older than age 80 have thyroid nodules, most of which are nonpalpable.36 Older patients with multinodular goiter are susceptible to iodine-induced thyrotoxicosis (e.g., after radiocontrast or amiodarone administration). Multinodular goiters frequently have functionally autonomous areas, and attempts to suppress the growth of these goiters with thyroid hormone may lead to iatro¬ genic hyperthyroidism. In elderly patients with nontoxic nodu¬ lar goiter, surgical management is usually reserved for those with significant compressive symptoms, for those for whom suspicion of malignancy is high, or occasionally for patients with cosmetic concerns. After age 50, the frequency of clinically evident solitary thy¬ roid nodules decreases. However, even well-differentiated pap¬ illary and follicular carcinomas are more aggressive and are associated with increased mortality in elderly patients, so a care¬ ful evaluation is indicated when any new solitary nodule is noted or when the size of an existing nodule increases. Other clinical manifestations suggesting thyroid carcinoma include dysphagia, hoarseness, pain, adherence of the thyroid to adja¬ cent structures, cervical lymphadenopathy, and a hard consis¬ tency of the thyroid. The incidence of both anaplastic thyroid carcinoma and thyroid lymphoma is higher in elderly than in younger adults.36

DISORDERS OF PARATHYROID GLANDS AND CALCIUM METABOLISM AGING AND REGULATION OF SERUM CALCIUM With aging, calcium homeostasis is preserved at the expense of elevated parathyroid hormone (PTH) levels and consequent reduction in bone mass. PTH protects against hypocalcemia by stimulating renal 1,25-dihydroxyvitamin D [l,25(OH)2D] produc¬ tion, promoting calcium conservation by the kidney, and increas¬ ing bone resorption. With aging, however, PTH is less effective in stimulating l,25(OH)2D production, apparently due to decreased renal la-hydroxylase activity. The resulting decrease in l,25(OH)2D levels, together with impaired intestinal responsive¬ ness to R25(OH)2D action, in turn contribute to a decrease in intestinal absorption of dietary calcium that is evident by age 50 to 60 years.69 Furthermore, decreased calcium absorption is exac¬ erbated in some elderly individuals by decreased gastric acid secretion and lactase deficiency, which may result in avoidance of dairy products, ha turn, a mild decrease in serum calcium levels and a reduction in renal clearance of PTH lead to a 30% increase in serum PTH levels between ages 30 and 80.70 Serum calcium lev¬ els are normalized by the increased PTH levels, but bone resorp¬ tion is increased relative to bone formation, resulting in loss of bone mass and increased fracture risk. Ultimately, these patho¬ physiologic changes result in type II osteoporosis and an increased risk of fractures in many elderly adults (see later).

1816

PART XIII: ENDOCRINE AND METABOLIC DYSFUNCTION IN THE GROWING CHILD AND IN THE AGED

VITAMIN D DEFICIENCY Dietary calcium intake is grossly inadequate in most older indi¬ viduals.71 As a consequence of age-related renal PTH resistance and intestinal l,25(OH)?D resistance, elderly people are unable to substantially increase intestinal absorption of calcium in response to a low-calcium diet and are, therefore, more depen¬ dent than young adults on adequate dietary calcium inges¬ tion.72-73 Age-related abnormalities in PTH secretion and bone turnover are not inevitable, however. Older women treated with 2400 mg per day of calcium for 3 years reversed these alter¬ ations,74 and even 1000 mg per day significantly lowers PTH lev¬ els.75 Furthermore, vitamin D deficiency commonly coexists with calcium deficiency in the elderly. In one study of elderly subjects performed in the United States, hypovitaminosis D was found in 38% of nursing home residents and 54% of homebound people residing in the community.76 Vitamin D deficiency appears to be equally common in medical inpatients in the United States, occurring in 57% of unselected patients on a general medical ward, including substantial numbers of patients without appar¬ ent risk factors for vitamin D deficiency and those with vitamin D intakes above the recommended daily amount.77 Vitamin D supplementation in postmenopausal women decreases PTH lev¬ els and bone turnover, and increases bone mineral density at the femoral neck.78 Combined calcium and vitamin D supplementa¬ tion reduces fracture rates in elderly women.79 Based on these and other data, the current consensus is that an elemental cal¬ cium intake of at least 1000 to 1500 mg per day (and possibly more in some) is desirable in older adults, and adequate vitamin D intake (400 to 800IU per day) must also be ensured.80 The opti¬ mal amount of calcium needed to preserve bone mineral density in older women, however, is still unknown.

PAGET DISEASE OF BONE Paget disease of bone (see Chap. 65) rarely occurs before the age of 35 years. Its prevalence increases with aging, and it affects 2% to 5% of individuals >50 years of age, although significant geo¬ graphic variation is found.81 Commonly affected sites include the pelvis, spine, femur, and skull. Most people with Paget dis¬ ease are asymptomatic, and the disorder is usually identified incidentally when radiographs are obtained for an unrelated indication, or an otherwise unexplained elevation in serum alkaline phosphatase is noted. In symptomatic patients, pain is the most common presenting symptom, usually localized to the affected bones. In one-third of cases, however, the pain is due to secondary osteoarthritic changes, often in the hips, knees, and vertebrae. Bony deformities occur in -15% of patients at the time of diagnosis, usually involving the long bones of the lower extremities and often presenting as a bowing of the affected extremity. With involvement of the skull, compression of the eighth cranial nerve may result in sensorineural hearing loss. In patients with Paget disease of the hip joint, the results of total hip arthroplasty are comparable to those of hip arthroplasties performed in patients unaffected by Paget disease.82 The bisphosphonates are the current treatment of choice and are effec¬ tive in suppressing the accelerated bone turnover and bone remodeling that is characteristic of this disease.

fracture a hip are at a 10% to 20% increased risk of mortality over the following year84 and have increased morbidity, includ¬ ing institutionalization and impairment in mobility and func¬ tional status. Two clinical syndromes of osteoporosis have been proposed on epidemiologic and biochemical grounds.85 Type I ("postmeno¬ pausal") osteoporosis is thought to occur in women typically between 51 and 75 years old and is characterized by an acceler¬ ated rate of bone loss, mainly in trabecular bone, with distal radius and vertebral fractures. The loss of the direct restraining effects of estrogen on bone-cell function is thought to be the most important mediator of this accelerated bone loss, leading to increased sensitivity of bone to PTH and increased calcium release from bone.86 PTH levels are slightly decreased at steady state in these patients. Type II ("senile") osteoporosis is character¬ ized by a late, slow phase of bone loss occurring in both men and women older than 70 years. This condition is associated with progressive secondary hyperparathyroidism and loss of both tra¬ becular and cortical bone with vertebral and hip fractures. The proposal was originally made that type II osteoporosis is due pri¬ marily to decreased serum l,25(OH)2D levels and calcium mal¬ absorption, resulting in a secondary increase in PTH levels and bone resorption. Subsequently, however, the proposal has been put forward that both the secondary hyperparathyroidism and the decreased bone formation characterizing this slow phase of bone loss are manifestations of underlying estrogen deficiency in both elderly men and elderly women, with loss of estrogen action resulting in net calcium wasting and in an increase in the level of dietary calcium intake required to maintain bone homeostasis.86 Aging men have decreased circulating levels of both bioavailable estrogen and testosterone. Bioavailable estrogen levels appear to be a better correlate of bone mass than testosterone levels, sup¬ porting the hypothesis that estrogen deficiency is an important cause of bone loss in elderly men.86 As in younger patients, secondary causes of osteoporosis, osteomalacia, and primary hyperparathyroidism must be con¬ sidered in the assessment of elderly osteopenic patients (Table 199-4). Osteomalacia due to vitamin D deficiency is relatively TABLE 199-4. Causes of Osteopenia OSTEOPOROSIS Primary

Type I Type II Secondary

Excessive alcohol intake Smoking Glucocorticoid excess Anticonvulsant use (associated with both osteoporosis and osteomalacia) Hyperthyroidism Hyperprolactinemia Hypogonadism in men Malignancy, especially multiple myeloma Long-term heparin use Immobilization Rheumatoid arthritis Malnutrition Chronic liver disease (biliary cirrhosis)

OSTEOPOROSIS

OSTEOMALACIA Vitamin D deficiency

Osteoporosis is a major cause of morbidity and mortality in older people. Estimates are that 1.5 million fractures annually are a direct result of osteoporosis. By the age of 90 years, -32% of women and 17% of men have fractured a hip.83 Patients who

Hypophosphatemia Miscellaneous (e.g., renal tubular acidosis) PRIMARY HYPERPARATHYROIDISM

Ch. 199: Endocrinology and Aging common in elderly people, particularly in those with a history of gastrectomy, chronic renal failure, malabsorption, or anti¬ convulsant use. Screening for osteomalacia is warranted in frail elderly people, including measurement of serum calcium and phosphate levels, which are decreased in vitamin D defi¬ ciency, and alkaline phosphatase level, which is increased. Findings of decreased 24-hour urinary calcium and serum 25hydroxyvitamin D levels and increased PTH levels help to con¬ firm the diagnosis.

MANAGEMENT OF OSTEOPOROSIS The management of osteoporosis and osteomalacia is discussed in detail in Chapters 64 and 63, respectively. The following points should be emphasized in the care of frail older patients with osteoporosis. First, older people at risk for falling should be identified. Important risk factors for falls in elderly adults include cogni¬ tive impairment, abnormalities of gait and balance, use of mul¬ tiple medications, use of psychoactive medications, nocturia, disabilities of the lower extremities, and environmental factors such as household clutter. Second, a performance-oriented assessment of gait and balance should be undertaken. Direct observation of gait and balance is probably more useful than the standard neuromuscular examination in identifying patients with increased fall risk and treatable mobility problems.87-89 Assessment should include gait parameters (such as initiation of gait, step height, step length, step symmetry, path deviation, and trunk stability) and balance parameters (including ability to rise from a chair, immediate and sustained standing balance [with eyes open and closed], stability after sternal nudge, turn¬ ing balance, and stability while looking upward or bending down). Management of patients at risk for falling may include treatment of underlying conditions contributing to falls (e.g., Parkinson disease, postural hypotension, foot disorders); mini¬ mization of medications increasing the risk of falling; gait retraining and strengthening; provision of assistive devices (e.g., a cane or walker); and environmental alterations (e.g., elimination of obstacles and clutter, provision of proper lighting and handrails).88 Secondary causes of osteoporosis, such as alco¬ holism, glucocorticoid excess, hypogonadism in men, hyperthy¬ roidism, and multiple myeloma, should be sought and treated. For prophylaxis and for treatment of established osteoporo¬ sis, most women (and men) should ingest at least 1 to 1.5 g of elemental calcium per day beginning in the perimenopausal period, unless a contraindication such as a history of nephroli¬ thiasis or hypercalciuria is present. Calcium carbonate is an inexpensive formulation that is acceptable for most patients, although calcium citrate is better absorbed by patients with achlorhydria. This should be accompanied by a daily multivita¬ min containing 400 to 800 IU of vitamin D. Intake of >1000 IU per day may cause increased bone resorption, although if bio¬ chemical evidence of osteomalacia is found, higher doses of vitamin D may be needed. Also, exercise is important.893 Aside from calcium and vitamin D supplementation, estrogenreplacement therapy has been considered by some to be the treatment of first choice for established postmenopausal osteoporosis, based on long-term experience, well-documented effectiveness in preventing bone loss, and the potential for other benefits aside from its effects on bone. However, relatively few data are available on the effects of estrogen use on rates of fracture (especially hip fracture) in postmenopausal women.40 Estrogens act primarily to prevent bone resorption, but the ability to restore lost bone mass may be minimal. Furthermore, evidence exists that the beneficial effects of estrogen replacement are more marked in women who begin treatment within five years after

1817

menopause.90 Therefore, maximum benefit from estrogen ther¬ apy is obtained by beginning use as soon as possible after meno¬ pause. Estrogens also play an important role in the treatment of type II osteoporosis,91 however, and other studies have reported similar benefits from estrogen use on bone density, both in women older than age 70 and in younger women.83 The optimal duration of estrogen use has not been established, but because cessation of estrogen treatment leads to resumption of bone loss, continuing treatment until age 65 to 70 or longer in those with¬ out contraindications to their use may be reasonable. Alterna¬ tively, the suggestion has been made that starting estrogen use at age 65 may provide almost as much protection against osteoporotic fractures as starting at menopause, and may reduce the risks of long-term estrogen therapy (see Chaps. 100 and 223 for discussions of the risks).92'93 The selection of estrogen replacement regimens for elderly women is discussed later in the Menopause section. The bisphosphonates alendronate and etidronate have been demonstrated to decrease the fracture rate in women with post¬ menopausal osteoporosis and are effective alternatives to estro¬ gen therapy, especially for women who are concerned about the potential adverse effects of estrogens. Continuous high-dose etidronate may lead to impaired bone mineralization; therefore, etidronate must be given intermittently at a lower dosage (e.g., 400 mg per day for 2 weeks every 3 months).94 An additive ben¬ efit of combined intermittent etidronate and cyclical estrogen and progesterone replacement on hip and spine bone mineral density was demonstrated over a 4-year period in postmeno¬ pausal women with established osteoporosis.95 Low-dose alen¬ dronate (5 mg per day) prevents bone loss in postmenopausal women without established osteoporosis.96-97 This approach is appropriate for those women who are at high risk for future fractures and who are unable or unwilling to take estrogen.98 The efficacy and safety of other bisphosphonates (e.g., risedronate) in the prevention and treatment of osteoporosis are cur¬ rently under investigation. Calcitonin may be less effective in the prevention of bone loss in osteoporotic patients than estrogens or bisphosphonates, and its long-term efficacy in fracture prevention has not been as well documented. Raloxifene, a selective estrogen-receptor modula¬ tor, has been shown to increase bone density in postmenopausal women,99 although to a smaller extent than estrogen replace¬ ment, and data regarding reduction in fractures are limited.100 Although raloxifene does not appear to stimulate the endo¬ metrium in these women, it does decrease total and low-density lipoprotein (LDL) cholesterol levels. Unlike estrogens, however, raloxifene does not increase high-density lipoprotein (HDL) lev¬ els, and raloxifene is associated with an increased incidence of hot flushes, leg cramps, and thromboembolic events. Further¬ more, no long-term data are available to assess the effects of ral¬ oxifene on the incidence of coronary heart disease (CHD) events, cognitive function, or the incidence of breast, ovarian, and uterine cancers.100 At present, recommendation of other potential osteoporosis treatments such as fluoride, androgenic steroids, and parathyroid hormone injections is premature until studies are available that document improvements in osteo¬ porotic fracture rates without significant adverse effects.

HYPERCALCEMIA PRIMARY HYPERPARATHYROIDISM Primary hyperparathyroidism occurs most commonly in adults between 45 and 60 years of age, although it may develop at any age. The condition occurs more often in women than in men by

1818

PART XIII: ENDOCRINE AND METABOLIC DYSFUNCTION IN THE GROWING CHILD AND IN THE AGED

a ratio of nearly 3:1. The annual incidence of the disease is ~1 per 1000.101 The diagnosis is often suspected based on a finding of elevated serum calcium levels on routine laboratory testing; indeed, asymptomatic hyperparathyroidism is by far the most common presentation.102 Compared with younger patients, however, elderly people with primary hyperparathyroidism more often present with neuropsychiatric and neuromuscular symptoms, and with osteoporosis associated with fractures.103 Other common complaints in older patients include altered mental status, fatigue, depression, weakness, personality change, memory loss, anorexia, and constipation. The approach to the diagnosis and treatment of elderly patients with primary hyperparathyroidism is similar to that of younger patients (see Chap. 58).102 Serum calcium level is high and phosphate level is often low to low normal, whereas alka¬ line phosphatase level is often high normal or mildly increased. Assays for intact PTH are the diagnostic tests of choice, and the presence of primary hyperparathyroidism is established by a high-normal or elevated PTH level in the setting of hypercalce¬ mia. (Nonparathyroid causes of hypercalcemia are associated with undetectable or clearly decreased PTH levels. The most common cause of hypercalcemia in hospitalized patients is a malignancy producing a PTH-related protein [PTHrP] that can be measured in reference laboratories.) The skeletal effects of primary hyperparathyroidism are selective, with a reduction in cortical bone but relative protection against cancellous bone loss. Accordingly, bone densitometry is an important part of the evaluation of these patients, and measurements at the forearm and hip are the best indicators of cortical bone density (the latter is the most important site because of its significance as a risk fac¬ tor for hip fracture). Surgery is the only definitive treatment and is indicated in surgical candidates with markedly elevated serum calcium lev¬ els (>12 mg/dL), overt manifestations of primary hyperparathy¬ roidism (e.g., nephrolithiasis), marked hypercalciuria, and markedly reduced cortical bone density. For the patients who are managed conservatively, checking serum calcium levels every 6 months and monitoring 24-hour urine calcium excre¬ tion, creatinine clearance, and bone densitometry annually is appropriate. These patients should be instructed to avoid thia¬ zide diuretics and to avoid dehydration, but avoidance of dairy products is unnecessary. Most patients followed expectantly remain stable over time. Medical therapy for hyperparathyroid¬ ism may include administration of estrogens in women, oral phosphate in patients with low serum phosphate levels, and bisphosphonates.

HYPERCALCEMIA OF MALIGNANCY In most patients with malignancy-related hypercalcemia, an obvious neoplasm is evident on examination and routine diag¬ nostic evaluation. Diagnostic possibilities include humoral hypercalcemia of malignancy, in which a PTHrP is produced by a cancer, usually of squamous cell type (e.g., lung, head, and neck), that occurs commonly in the elderly. In addition, multiple myeloma and some lymphatic tumors secrete osteoclast¬ activating factors, many of which are cytokines (e.g., lymphotoxin, interleukin-1, tumor necrosis factor). Treatment of hypercalcemia of malignancy includes volume repletion followed by immedi¬ ate forced diuresis with saline infusion and furosemide. Parenteral bisphosphonates (e.g., pamidronate), calcitonin, or mithramycin should also be given, and the underlying malig¬ nancy should be treated if possible. Glucocorticoid therapy is reserved primarily for myeloma and lymphatic tumors. In elderly patients with advanced malignancies, short life expect¬ ancy, and poor functional status, not treating the hypercalcemia

may be appropriate and may provide a more comfortable mode of exit for these terminally ill patients.

ADRENAL CORTEX PHYSIOLOGY Decreased cortisol production is offset by decreased cortisol clearance, resulting in unchanged basal serum cortisol levels with aging. Urinary free cortisol levels are the same in elderly persons as in young adult subjects.104 Stimulation of cortisol secretion by exogenous ACTH is unaltered with aging.105 Fur¬ thermore, cortisol and ACTH responses to metyrapone, insulininduced hypoglycemia, ovine CRH, and perioperative stress are normal or slightly prolonged in elderly subjects,106-107 indi¬ cating an intact HPA axis responsiveness to stimulation with aging. In addition, ACTH pulse frequency is similar in healthy young and healthy elderly men, suggesting that baseline hypo¬ thalamic regulation of glucocorticoid function is intact with aging.12 Clear evidence now exists, however, that feedback sen¬ sitivity to glucocorticoids decreases with aging.15-108 Although the clinical implications of this decreased responsiveness to glucocorticoid feedback inhibition are uncertain, some have hypothesized that decreased negative feedback results in pro¬ longed glucocorticoid exposure; this, in turn, damages hippo¬ campal neurons regulating glucocorticoid secretion, leading to additional glucocorticoid hypersecretion and further damage to mechanisms regulating glucocorticoid feedback inhibi¬ tion.34-109 This process may be involved in mediating a "gluco¬ corticoid cascade" of neurodegeneration in Alzheimer disease and, to a lesser extent, in the normal aging brain.110 Thus, although the issue is still controversial, age-related glucocorti¬ coid dysregulation may be a potentially modifiable risk factor for Alzheimer disease.

LABORATORY DIAGNOSIS OF ADRENOCORTICAL DISEASE IN OLDER PATIENTS Adrenal hyperfunction and hypofunction are less common in elderly than in middle-aged adults. However, manifestations that are associated with either adrenal hyperfunction (e.g., hypertension, obesity, and diabetes mellitus) or adrenal insuffi¬ ciency (e.g., orthostatic hypotension and weight loss) occur more commonly in older than in young adults. Therefore, adre¬ nal disease must be considered in the evaluation of elderly patients with these manifestations, and patients with suggestive findings on physical examination or laboratory screening should undergo further assessment. In addition, benign adrenal masses are common incidental findings observed during imag¬ ing procedures of the abdomen. Benign adenomas usually range in size from 1 to 6 cm and weigh 10 to 20 g, whereas malignant tumors generally weigh >100 g.106-111 Incidentally detected adre¬ nal masses of 6 cm should probably be removed in patients who are appropriate candidates for surgery, although the natu¬ ral history of incidentally discovered adrenal lesions in the eld¬ erly is unknown. Several factors may interfere with testing of HPA axis func¬ tion in the elderly. First, the excretion of steroids commonly mea¬ sured in urine is decreased with renal impairment, and measurements are unreliable if the creatinine clearance is 5% of bone mass compared to baseline. Calcium supplementation with 1500 mg per day of ele¬ mental calcium, together with vitamin D, 800 IU per day, should be ensured to minimize bone loss caused by a corticosteroid-

1819

induced decrease in intestinal calcium absorption and an increase in urinary calcium losses. The exogenous administration of corticosteroids induces suppression of gonadotropins and sex steroids; therefore, sex hormone-replacement therapy should be considered unless contraindicated. For postmenopausal women, appropriate hormone-replacement regimens are the same as for women not receiving corticosteroids. Men with low testosterone levels should also be given hormone replacement, either with tes¬ tosterone enanthate or testosterone cypionate, 100 to 200 mg intramuscularly every 2 weeks, or with daily transdermal appli¬ cation of a testosterone patch. The bisphosphonate alendronate (5 or 10 mg per day) or etidronate (administered cyclically, 400 mg per day for 14 days every 3 months) are effective in the pri¬ mary and secondary prevention of bone loss in patients receiv¬ ing glucocorticoid therapy.114-116 Whether bisphosphonate treatment reduces fracture risk in patients with established corticosteroid-related osteoporosis is not yet clear, however. Calcitonin is also effective in the prevention and treatment of glucocorticoid-induced bone loss.113'117 The restriction of dietary sodium and the administration of thiazide diuretics are useful to decrease corticosteroid-induced hypercalciuria, although the effects of these interventions on bone density have not been fully investigated.

HYPOADRENOCORTICISM Hypoadrenocorticism is covered in detail in Chapter 76. As in younger adults, iatrogenic adrenal failure secondary to long¬ term glucocorticoid administration is the most common cause of hypoadrenocorticism in elderly people. Only very uncommonly does autoimmune adrenocortical insufficiency present initially in an elderly patient. Some nonautoimmune causes of adrenal insufficiency occur more commonly in older adults, however, including tuberculosis, adrenal hemorrhage in patients taking anticoagulants, and metastatic involvement of the adrenals.106 Some elderly patients with chronic adrenal insufficiency (e.g., secondary to hypopituitarism) present with nonspecific symp¬ toms of "failure to thrive"—such as weight loss, anorexia, weak¬ ness, and decreased functional status. Moreover, one-third of older patients with adrenal insufficiency do not have hyperkale¬ mia at initial presentation. Of note, compared with adrenal insufficiency in younger adults, adrenal insufficiency in the elderly has historically been more often fatal and more com¬ monly diagnosed only at autopsy.118 Therefore, a high index of suspicion is required to detect this treatable, life-threatening problem in many older patients. Recovery of HPA axis responsiveness after cessation of glu¬ cocorticoid therapy is variable and in some individuals may not be complete even after several months. A number of factors may put older adults at higher risk to develop iatrogenic adre¬ nal insufficiency. Elderly people who are on complicated med¬ ication regimens or who are cognitively impaired may become confused about their medications or forget to take them. High medication costs or medication-related side effects may cause older patients to discontinue their medicines abruptly without consulting their physicians. In addition, as noted earlier, the clinical manifestations of adrenocortical insufficiency are often nonspecific. When adrenocortical insufficiency due to cessa¬ tion of long-term glucocorticoid therapy is suspected in an older person, an appropriate course is to perform the ACTHstimulation test and institute therapy. As with younger adults, older people with persistent adrenocortical insufficiency should be given glucocorticoid replacement and coverage for major surgery and other stressful events until HPA axis func¬ tion has recovered.

1820

PART XIII: ENDOCRINE AND METABOLIC DYSFUNCTION IN THE GROWING CHILD AND IN THE AGED

ALDOSTERONE AND RENIN Aldosterone secretion and clearance rates decrease with aging. In contrast to cortisol levels, however, basal plasma aldosterone levels are not maintained at normal levels in elderly subjects but decline by -30% in healthy octogenarians compared with younger adults.119 In response to dietary sodium restriction, aldosterone secretion increases three-fold in the young but only two-fold in older adults. These changes in aldosterone secretion with aging are thought to be due to corresponding reductions in plasma renin activity, which is decreased in the basal state and in response to sodium restriction or upright posture.120 Further¬ more, conversion of inactive to active renin is thought to be impaired with aging.121 Age-related increases in atrial natri¬ uretic hormone (ANH) secretion also contribute to the agerelated decrease in aldosterone secretion by directly inhibiting aldosterone release and by inhibiting renal renin secretion, plasma renin activity, and angiotensin II levels. In addition, renal responsiveness to ANH is increased with aging, suggest¬ ing that ANH is an important contributor to age-related renal sodium losses.16 Declining aldosterone levels with aging predispose older peo¬ ple to renal salt wasting. Several other factors place elderly people at higher risk for volume depletion and dehydration, including decreased thirst sensation, increased ANH levels and renal ANH sensitivity, decreased renal ADH responsiveness, and possibly decreased renal-tubular responsiveness to aldosterone.16 In addi¬ tion, age-related hypoaldosteronism is characterized by a decreased aldosterone response to hyperkalemia, which may con¬ tribute to an increased susceptibility to hyperkalemia if other potassium regulatory systems fail.122 Accordingly, elderly patients with hyporeninemic hypoaldosteronism are at higher risk of becoming hyperkalemic during treatment with potassium¬ sparing diuretics, (3-blocking agents, or nonsteroidal antiinflam¬ matory drugs. Older diabetic patients with renal insufficiency are particularly susceptible to this complication.

ADRENAL ANDROGENS Adrenal androgen secretion declines progressively beginning in the third decade, with plasma levels of the principal adrenal androgen, dehydroepiandrosterone (DHEA), declining to just 10% to 20% of young adult levels by the eighth and ninth decades.123-124 This age-related decrease in DHEA levels is due to a reduction in adrenal DHEA secretion rather than to an increase in DHEA metabolism. Furthermore, the DHEA response to adrenal stimulation by ACTH is markedly decreased in older people. However, levels of DHEA and its sulfate, DHEAS, exhibit marked interindividual variability at all ages. Epidemiologic data suggest that DHEA levels may play an important role in the deterioration of a variety of physiologic functions with aging. For example, low DHEA levels have been reported in states of poor health (e.g., after surgery or accidents) or in the setting of immunologic dysregulation (e.g., active rheu¬ matoid arthritis or acquired immunodeficiency syndrome).125 Other studies have reported positive correlations between plasma DHEA levels and vigor and longevity, and inverse cor¬ relations with cancers and cardiovascular disease.126-127 Further¬ more, high-functioning community-dwelling elderly subjects have higher levels of DHEAS than low-functioning subjects,128 such that DHEAS levels are linked to functional status. As a result of these associations, considerable interest has been generated in the potential therapeutic effects of DHEA administration in older adults. Data from randomized con¬ trolled trials in which DHEA was administered in dosages of 50 to 100 mg per day over a 6- to 12-month period have shown sub¬

jective improvements in physical and psychological well-being, increased serum IGF-I levels and, at higher dosages, increased lean body mass and muscle strength at the knee.129 Levels of cir¬ culating lipids, glucose, and insulin as well as bone density were comparable in treated and placebo groups. Concerns remain, however, regarding the potential for androgenization in women, gynecomastfa in men, possible adverse effects on lipo¬ protein metabolism at supraphysiologic doses, and potential hepatotoxicity.130-131 Furthermore, DHEA is metabolizable to estrogens and to androgens, including testosterone and dihy¬ drotestosterone,125'129 and its effects on the risk of breast cancer in women and of prostate cancer in men have not been deter¬ mined. Thus, although these data are potentially promising, the long-term safety and efficacy of DHEA treatment have not been established.

CATECHOLAMINES Norepinephrine (NE) is the principal neurotransmitter released by sympathetic postganglionic neurons. After release, most NE is taken up again into the axon terminals, whereas only a small fraction is released into the circulation. Substantial evidence indicates that sympathetic nervous sys¬ tem (SNS) activity is increased with aging in humans. Basal plasma NE levels, and the NE secretory response to various stimuli such as upright posture and exercise, are increased with aging.132-133 In contrast, circulating epinephrine levels and epi¬ nephrine responses to various stimuli exhibit little change with aging. Although sympathetic tone is increased with aging, phys¬ iologic responsiveness to both a- and (3-adrenergic receptormediated stimulation appears to decrease with aging.134 Decreased catecholamine responsiveness is thought to be due to changes at both the receptor and the postreceptor level. The clinical effects of this age-related increase in sympathetic tone may include the development of hypertension. A number of studies have reported a correlation between hypertension and increased plasma NE levels in elderly people.135 Body fat content is independently associated with both aging and plasma NE levels, however, suggesting that obesity may contribute to hypertension by increasing sympathetic tone, independent of the effects of aging per se. Certain diseases commonly associated with aging may give rise to autonomic insufficiency with orthostatic hypotension, including Parkinson disease, multiple system atrophy, and dia-^ betes mellitus. Moreover, certain drugs that interfere with SNS function may also cause orthostatic hypotension; these include antihypertensive agents such as clonidine and a-methyldopa and psychoactive drugs such as phenothiazines and tricyclic antidepressants. Other factors such as volume depletion, pro¬ longed bed rest, and venous insufficiency may also cause or exacerbate postural hypotension. The management of ortho¬ static hypotension includes the treatment of hypovolemia, dis¬ continuation of medications that may exacerbate postural hypotension, and instructing the patient to sit with legs dan¬ gling for several minutes before getting out of bed, to elevate the head of the bed at night, and to use elastic support stockings and an abdominal binder to promote venous return. Useful medica¬ tions include caffeine, fludrocortisone to expand plasma vol¬ ume, and midodrine, a sympathomimetic amine. Postprandial hypotension is a common disorder involving SNS dysfunction in elderly people. It possibly results from inad¬ equate SNS compensation for pooling of blood in the splanchnic vessels after a meal, impaired baroreceptor reflex function, impaired peripheral vasocontriction, release of vasoactive gas¬ trointestinal peptides, and/or inadequate postprandial increases in cardiac output.136 This condition is especially common in

Ch. 199: Endocrinology and Aging elderly hypertensive patients. Postprandial hypotension may be an important cause of syncope in elderly patients and should be considered in the evaluation of older people with unexplained syncope.137 The management of this condition should include the avoidance of dehydration, discontinuation of unnecessary drugs that could exacerbate postprandial hypotension, consumption of frequent small meals, avoidance of alcohol, and avoidance of strenuous exercise within 2 hours after meals. Caffeine use has not been shown to be helpful in this condition.

1821

TABLE 199-5. Manifestations of Estrogen Deficiency in Menopausal Women Primarily in the menopausal period

Vasomotor instability—"hot flushes" Spreading sensation of heat Palpitations Diaphoresis Sleep disturbances Irregular menstrual bleeding and eventual cessation of menses Neuropsychiatric symptoms probably related to low estrogen state

FEMALE REPRODUCTIVE AND ENDOCRINE FUNCTION: MENOPAUSE The mean age for menopause in women, 51 years, has not changed significantly over the last century. Because life expec¬ tancy for women has increased markedly over the same period, however, most women can expect to spend more than one-third of their lives in the postmenopausal state. Accordingly, clini¬ cians caring for perimenopausal and postmenopausal women must work closely with them to consider the potential impact of menopausal changes on future health and functional status. Failure of ovarian end-organ function occurs by the fifth or sixth decade, with cessation of ovarian follicular development, estradiol secretion, and menstruation, and unresponsiveness to gonadotropin stimulation. Premenopausally, estradiol (E2) and estrone (Et) are secreted by maturing ovarian follicles and are produced by aromatization of ovarian and adrenal androgenic precursors in peripheral tissues. An elevation in serum FSH lev¬ els heralds the onset of menopause and is the most sensitive clinically available indicator of cessation of ovarian follicle development.138 As noted in the discussion of gonadotropins earlier, levels of inhibin B appear to decline slightly before FSH levels increase,39 but an inhibin-B assay is not yet clinically avail¬ able. After menopause, circulating E2 and E1 are derived almost entirely from aromatization of adrenal androstenedione, and E1 levels are higher than those of E2. FSH levels increase to a greater extent than LH levels. Obese postmenopausal women exhibit increased production of adrenal androgenic precursors and increased peripheral aromatization of androgens to estrogens, especially to E17 which is associated with a decreased risk of osteoporosis. The manifestations of estrogen deficiency experienced by menopausal women are listed in Table 199-5. Vasomotor symp¬ toms occur in three out of four women during the perimeno¬ pausal period139 and may be associated with other symptoms such as palpitations, faintness, fatigue, and vertigo. These symptoms, along with atrophy of the sexual tissues, are relieved by estrogen replacement. Clonidine, methyldopa, or medroxy¬ progesterone may occasionally be effective for treating hot flushes in women who are unable to take estrogens. As discussed earlier, long-term treatment with estrogens in postmenopausal women may prevent or delay the occurrence of osteoporosis and fractures. Furthermore, the incidence of coro¬ nary artery disease rises sharply in postmenopausal women, and some evidence from observational studies has suggested that estrogen replacement in postmenopausal women decreases the risk of cardiovascular disease by up to 50% compared with those not receiving estrogens.83-140 This apparent cardioprotective effect of estrogens has been attributed to favorable effects on lipid metabolism, including decreased serum LDL and increased HDL cholesterol levels, and to other effects of estrogens on clot¬ ting mechanisms, glucose regulation, blood vessels, and myocar¬ dial tissue.141 Cardiac catheterization studies have reported that women receiving estrogen replacement develop less severe coro¬ nary atherosclerosis, and that the greatest benefit of estrogens on

Decreased concentration Irritability Anxiety Depressed mood Decreased libido (probably related to low testosterone levels) Alterations in lipid metabolism Increased LDL cholesterol Slight decline in HDL cholesterol Primarily postmenopausal

Urogenital atrophy Vulvar pruritus Dyspareunia Urinary complaints, including dysuria and stress incontinence Increased risk of pelvic prolapse due to loss of supporting structures Osteoporosis Increased risk of cardiovascular disease (?) Cognitive dysfunction Possible decreased short-term memory Possible increased risk of Alzheimer disease LDL, low-density lipoprotein; HDL, high-density lipoprotein.

coronary disease is realized by women with more severe athero¬ sclerosis at the time of the initial catheterization.142-143 The cardiovascular benefits of estrogen-replacement therapy have been called into question, however, by the results of the Heart and Estrogen/Progestin Replacement Study (HERS)—a randomized, placebo-controlled trial of hormone replacement for secondary prevention of CHD in postmenopausal women— which found no reduction in the rate of coronary events over a period of >4 years, with a tendency toward a higher coronary event rate in the treatment group in the first year and fewer events in years 4 and 5.144 Furthermore, thromboembolic events were increased within the first year of therapy. Based on these data, insufficient evidence exists at this time to support the initia¬ tion of hormone-replacement therapy specifically to prevent coronary events in women with established CHD. Estrogen replacement possibly may be beneficial in the primary preven¬ tion of coronary events; however, data from randomized, con¬ trolled trials addressing this issue will not be available until the results of studies such as the Women's Health Initiative and the Women's International Study of Long Duration Oestrogen after Menopause (WISDOM) are completed.145 Furthermore, given the trend toward a cardiovascular benefit after several years of treatment in the HERS study, continuing therapy for this indica¬ tion in women who are already receiving replacement may be appropriate. Nevertheless, even long-term therapy may be asso¬ ciated with some risks. One large prospective observational study reported that overall mortality was reduced by 37% in women currently taking hormone replacement, but the magni¬ tude of this apparent benefit decreased to 20% in those receiving therapy for >10 years.146 Some, but not all, evidence suggests that estrogen use may improve cognitive function in postmenopausal women with

1822

PART XIII: ENDOCRINE AND METABOLIC DYSFUNCTION IN THE GROWING CHILD AND IN THE AGED

TABLE 199-6. Contraindications to Postmenopausal Hormone Replacement Contraindications

Known or suspected estrogen-dependent neoplasm Breast carcinoma Endometrial carcinoma Melanoma Vaginal bleeding of unknown origin Active liver disease or impairment in liver function Active or recent thrombophlebitis or thromboembolic disease Relative contraindications

History of endometrial hyperplasia Hypertension associated with estrogen use Uterine fibroids Active pancreatitis Familial hypertriglyceridemia Migraine headaches Endometriosis Gallbladder disease

Alzheimer disease (AD). Retrospective studies have found an inverse correlation between the dosage and duration of estrogenreplacement therapy and the incidence of AD,147-148 and estro¬ gen administration in small open trials appeared to improve cognition and mood in women with AD.149'150 Moreover, some aspects of cognitive function were found to improve with transdermal estrogen replacement in a small double-blind, placebocontrolled trial of postmenopausal women with AD.151 Larger prospective studies are needed, however, to document any alleged therapeutic role for estrogens in the treatment of AD. Established risks associated with estrogen replacement include a nearly six-fold increase in the incidence of endometrial cancer in those receiving unopposed estrogen therapy. Coad¬ ministration of a progestin protects against this complication, however. The lifetime risk of developing breast cancer appears to be 10% to 30% higher in postmenopausal women receiving estrogen-replacement therapy for >10 years, and this risk must be weighed against the potential benefits of hormone replace¬ ment.141 Hypercoagulability is a potential side effect of estrogen therapy. Estrogen use may also predispose to cholelithiasis and hypertriglyceridemia. Table 199-6 summarizes the contraindica¬ tions to estrogen-replacement therapy. In summary, further clarification of the risks and benefits of postmenopausal hormone-replacement therapy must await the completion of additional prospective, randomized, controlled trials such as the Women's Health Initiative. For the time being, the beneficial effects on bone density and urogenital atrophy, and—in the immediate postmenopausal period—the ameliora¬ tion of vasomotor symptoms such as hot flushes, are the pri¬ mary indications for hormone-replacement therapy in some postmenopausal women. An estrogen-progestin combination is appropriate for women with an intact uterus to decrease the risk of endome¬ trial cancer, although inclusion of a progestin may partially attenuate some of the favorable effects of estrogens on lipids, and the effects of progestins on cardiovascular risk are uncer¬ tain. Estrogens should be used alone in women who have had a hysterectomy. Many older women may prefer continuous rather than cyclic hormone administration, because objection¬ able resumption of menses often occurs with cyclic therapy. An appropriate continuous combined hormonal regimen for many of these women is 0.625 mg daily of an oral conjugated estrogen, together with 2.5 mg daily of medroxyprogesterone acetate. Potential disadvantages to this approach include the

possibility of irregular bleeding and the relative lack of infor¬ mation on the effects of continuous progesterone on reduction in endometrial cancer risk and serum lipid levels. Further¬ more, some women may experience weight gain and depres¬ sion with daily progesterone administration. Patients should be informed that spotting often occurs during the first few months on this regimen, but that most women become amenorrheic after a year of treatment. For women who find a cyclic regimen suitable, 0.625 mg of conjugated estrogen may be used daily, with addition of a 10- to 14-day course of medroxy¬ progesterone acetate, 5 to 10 mg daily, every 3 or 4 months to induce withdrawal bleeding. This approach has the disad¬ vantage of a greater likelihood of inducing menses, but menses will occur at a predictable time, and more is known about the effects of cyclic hormone regimens on uterine can¬ cer risk and lipids. With either approach, very gradual intro¬ duction of conjugated estrogen may reduce unpleasant symptoms such as breast swelling, beginning with 0.3 mg every other day, advancing to 0.3 mg per day after 1 month and 0.625 mg per day the following month. Alternatives to conjugated estrogens include oral, transdermal, and vaginal estradiol preparations. Alternatives to estrogen replacement are available for women who are unable or unwilling to take estrogens—specifi¬ cally, the selective estrogen-receptor modulators such as ralox¬ ifene. As noted earlier, raloxifene has beneficial effects on bone mass and serum lipid levels. The increase in bone mass, how¬ ever, may be less with raloxifene than with estrogen therapy, and although the reduction in total and LDL cholesterol levels is similar with estrogens and raloxifene, the latter does not increase HDL cholesterol levels.100 The effects of selective estrogen-receptor modulators on cardiovascular mortality and mortality due to all causes are unknown. In contrast to estrogens, raloxifene commonly pro¬ duces hot flushes and is, therefore, not indicated for the treat¬ ment of vasomotor symptoms of menopause. Raloxifene has estrogen-antagonist effects on breast and uterine tissues, and does not cause endometrial hyperplasia. Tamoxifen, another estrogen antagonist, reduces the risk of invasive breast cancer in women who are at increased risk for breast cancer or who have a history of breast carcinoma in situ.152 If raloxifene is shown to have similar effects, it may be particularly useful in women with risk factors for breast cancer or a history of the disease.

MALE REPRODUCTIVE AND ENDOCRINE FUNCTION HORMONAL CHANGES The age-related changes in reproductive function in men are less dramatic than those that occur in aging women. In aging men, reproductive changes occur gradually, exhibit considerable vari¬ ation among individuals, and usually do not result in severe hypogonadism. A modest degree of primary testicular failure is evident in many healthy elderly men, as evidenced by decreases in daily sperm production, diminished total and free testoste¬ rone levels, and reduced testosterone responses to exogenous gonadotropin administration, together with increased serum gonadotropin levels.42'153 Some medical illnesses and malnutri¬ tion may further impair testicular function, as can some medica¬ tions (Table 199-7).153a In more frail older men, testicular failure is extremely common. For example, among male nursing home residents, 45% of individuals have been reported to exhibit tes¬ tosterone levels within the hypogonadal range.154 Some men,

Ch. 199: Endocrinology and Aging TABLE 199-7. Medications Associated with Decreased Serum Testosterone Levels and Hypogonadism Opiates Ethanol Cytotoxic drugs Cimetidine Ketoconazole Glucocorticoids Phenytoin Spironolactone

however, maintain serum testosterone levels within the normal range even after age 80.42'153 In addition to primary testicular failure, subtle age-related changes occur in hypothalamicpituitary control of testicular function, including decreased gonadotropin responsiveness to exogenous GnRH administra¬ tion42 and decreased LH pulse frequency in some healthy aging men.41 Furthermore, many healthy older men have inappropri¬ ately normal gonadotropin levels (i.e., not elevated above the normal range) in the presence of low testosterone levels, sug¬ gesting secondary testicular failure. A small number of aging men exhibit more obvious testic¬ ular failure, with total testosterone levels clearly below the lower limits of the normal range and clear manifestations of androgen deficiency (e.g., decreased libido and potency, osteoporosis, gynecomastia, and hot flushes). In the absence of contraindications, androgen replacement is indicated for these older men, as for young men. More often, however, the clini¬ cian is faced with an older patient with slightly decreased serum testosterone levels (e.g., 2.5 to 3.0 ng/mL) and nonspe¬ cific symptoms that may include impotence, loss of libido, muscle weakness, or osteopenia, and whether these men should be treated is unclear. In young hypogonadal men, androgens are important for the maintenance of normal bone and muscle mass, sexual drive, and erectile function.42 The hypothesis has been raised that age-related androgen defi¬ ciency may contribute to declining muscle and bone mass and other concomitants of aging, but few randomized, controlled studies have been performed to determine whether androgen supplementation in older men is beneficial and whether the benefits outweigh the risks. Two reports involving a small number of older hypogonadal men found that testosterone administration for 3 months increased lean body mass, reduced biochemical indices of bone turnover, and increased libido, without adverse effects on lipid metabolism or symp¬ toms of prostatism.155'156 Another study of testosterone replacement over 12 months in older men with low testoste¬ rone levels found increases in grip strength but failed to dem¬ onstrate changes in body composition.157 Several of the subjects were withdrawn from treatment, however, due to ele¬ vations in hematocrit. Finally, visceral fat mass and fasting blood glucose levels declined in middle-aged men receiving testosterone.158 Although some placebo-controlled studies have shown improvements in some measures of strength with testosterone therapy, no data are yet available to show whether testosterone improves functional performance or quality of life in elderly men.159 At the moment, larger and longer term studies are needed to determine the risks and benefits of androgen-replacement therapy before this approach can be recommended for aging men with slightly reduced testos¬ terone levels. Androgen-replacement therapy is discussed in detail in Chapter 119.

1823

SEXUAL ACTIVITY AND ERECTILE DYSFUNCTION In general, sexual activity and libido decline with aging, although some healthy elderly men exhibit stable or increased sexual desire with aging.160'161 Important determinants of sexual behavior in elderly men include perceived health status and the level of sexual activity during the younger years. Kinsey estimated the prevalence of impotence to be 55% by the age of 75 years.162 In contrast to the earlier view that most impotence is psychogenic, erectile dysfunction is now thought to have an organic basis in the large majority of cases. Furthermore, the prevalence of organic causes increases with advancing age.163 Important contributors to impotence in older men include arte¬ rial and venous abnormalities, neuropathies, use of medications, and coexisting medical illnesses. The most common cause of erectile dysfunction in older men is vascular disease, with half of all men older than age 50 exhibiting evidence of impaired penile blood flow. Venous insufficiency (failure to occlude venous out¬ flow) may occur due to leakage, arteriovenous malformations, or increased shunting between the corpora cavernosa and the glans. Although overt hypogonadism is present in 126 mg/dL is thought to be an equally potent predictor of complications in elderly people and in younger adults. Furthermore, estimates are that one-third of elderly patients with diabetes remained undiagnosed using the threshold of 140 mg/dL.182 Diabetes screening in asymptomatic, undiagnosed adults is recommended every 3 years for all individuals older than age 45, or more often in those who are obese, have a first-degree rel¬ ative with diabetes, are members of a high-risk ethnic population (e.g., black, Hispanic, or Native American), are hypertensive, have low HDL cholesterol and/or high triglyceride levels, or have a history of impaired glucose tolerance or impaired fasting glucose levels.189 However, reductions in disease-specific out¬ comes as a result of screening asymptomatic elderly people for diabetes have not yet been demonstrated.190 Furthermore, rou¬ tine screening and yearly monitoring of fasting glucose levels have been recommended for nursing home residents.57 This may be appropriate in some nursing home residents, but the physician must consider whether the patient or surrogate deci¬ sion maker desires testing and treatment, whether the results would affect treatment decisions, and whether treatment would improve functional status and quality of life.

MANAGEMENT GLYCEMIC CONTROL Increasing evidence indicates that, as in type 1 diabetes, the degree of hyperglycemia is a major determinant of morbidity in type 2 diabetes. The Diabetes Control and Complications Trial (DCCT) reported a reduction in microvascular and neurologic complica¬ tion rates in young individuals with type 1 diabetes who were treated with intensive insulin therapy,191 raising the question of whether intensive intervention might similarly benefit patients with type 2 diabetes. The Wisconsin Epidemiologic Study of Dia¬ betic Retinopathy found a relationship between the degree of hyperglycemia and the incidence and progression of microvas¬ cular and macrovascular complications in individuals with both type 1 and type 2 diabetes, but did not indicate whether interven¬ tion would be equally beneficial in the two disorders.192-193 A small intervention trial of intensive insulin therapy versus con¬ ventional insulin therapy in patients with type 2 diabetes, how¬ ever, demonstrated significant reductions in the development or progression of diabetic retinopathy, nephropathy, and neuropa¬ thy, as well as of macrovascular events in the intensively treated group.194 Furthermore, the United Kingdom Prospective Diabe¬ tes Study of middle-aged type 2 diabetic patients reported that, over a 10-year period, intensive treatment with either oral hypoglycemic agents or insulin significantly reduced diabetesrelated complications, especially microvascular events, although intensively treated patients experienced more hypoglycemic epi¬ sodes.195 These findings indicate that, in both type 1 and type 2 diabetes, optimizing glucose control is important in minimizing morbidity from this disease.

The management of diabetes in older patients must be indi¬ vidualized. Despite the foregoing observations, tight glycemic control is not an appropriate goal in many older patients with diabetes. As demonstrated in the DCCT and other trials, tight control involves an increased risk of hypoglycemia, even in young patients who are closely monitored. In elderly diabetic patients, this risk may be further increased by patients' tendency to err by up to 20% in their dosing of insulin.196 Moreover, a vari¬ ety of other coexisting medical, social, cognitive, economic, and functional problems may increase the risk of hypoglycemia or make accurate blood glucose monitoring difficult in frail older patients (see Table 199-9). Taken together, these problems often make efforts at rigorous glycemic control either unsafe or tech¬ nically not feasible. A major goal in all patients is to treat hyperglycemia to relieve symptoms while avoiding hypoglycemia. In many elderly diabetic patients, controlling glucose levels more rigorously in an attempt to minimize long-term complications such as mac¬ rovascular and microvascular disease is also appropriate, but the potential for benefit must be balanced with the risks of hypoglycemia and any practical constraints on more aggressive glucose control. The degree of glycemic control and other treat¬ ment goals should be agreeable to the patient, the physician, and the home caregivers assisting with diabetes care. A team approach is ideal for the care of older diabetic patients with complex medical, social, and functional issues. Available resources will determine the exact composition of the team, but one team member should be identified as a diabetes educator in charge of assessment, education, and follow-up.197 Careful attention should be given to other risk factors for cardiovascular disease, such as systolic and diastolic hypertension, smoking, and hyperlipidemia; good foot care is essential. The modalities available for the treatment of diabetes in elderly patients include diet, exercise, and the use of oral hypoglycemic agents and/or insulin, as in younger patients. Treatment of type 2 diabetes in older patients often begins with a trial of dietary therapy. No single approach can be uni¬ formly recommended, however, as the following consider¬ ations demonstrate. DIET AND EXERCISE The effects of dietary intervention in older diabetics have not been well studied, and in practice, marked changes in dietary habits may be difficult to achieve. Dietary alterations may' adversely affect the patient's quality of life, and this must be considered along with the potential benefits of treatment. In contrast to younger patients with type 2 diabetes, many frail eld¬ erly diabetic patients are not overweight. In these patients, ade¬ quate nutrition and even weight gain are advisable. In older diabetic patients who are >20% above their ideal body weight, the goal of dietary therapy is long-term moderate weight reduc¬ tion (not >5% to 10% reduction in body weight). Appropriate dietary prescriptions for these patients might include restriction of dietary fat to 25% to 30% of total calories, and moderate total calorie restriction (250 to 500 kcal per day reduction). In contrast, most patients who undertake a very low calorie diet eventually regain weight lost initially.198 The diet prescription should be simple and easy for the patient to understand, and should take the patient's food prefer¬ ences and habits into consideration. Functional limitations affecting the elderly person's ability to eat, shop for food, and prepare meals should be assessed. Family members and others involved in meal preparation should be instructed in dietary recommendations and should be involved in helping the patient to comply. As with younger patients with type 2 diabetes, an intensive team approach to dietary therapy, together with regu-

Ch. 199: Endocrinology and Aging lar exercise as indicated (see later) and active family involve¬ ment, is most likely to be successful. Few data are available to indicate whether the benefits of reg¬ ular exercise observed in younger patients with type 2 diabetes occur in older patients. Exercise training in some older type 2 diabetic patients may achieve modest improvements in glucose tolerance, although these benefits appear to be transitory if exer¬ cise is discontinued.199 Exercise alone has not been shown to markedly improve glucose levels in patients with diabetes, but it may have significant benefits on cardiovascular function, hypertension, and lipid levels. Accordingly, recommending an exercise program for many older diabetic patients, with specific precautions, seems reasonable. Diabetic patients are at increased risk for silent myocardial ischemia, and those with proliferative retinopathy are susceptible to retinal detachments and vitreous hemorrhage; therefore, an exercise treadmill test and a careful retinal examination are advisable before an exercise program is initiated. Exercise should be aerobic and should be undertaken gradually under close supervision. Proper foot care, adequate glucose control before initiation of an exercise program, and avoidance of hypoglycemia are also essential. USE OF ORAL HYPOGLYCEMIC AGENTS Oral agents are usually the next step in the treatment of type 2 dia¬ betes that is not well controlled by diet and exercise, as in younger patients. Oral agents are particularly useful in treating obese or normal-weight elderly patients with visual problems, arthritis, or memory deficits, in whom insulin administration may be prob¬ lematic. Sulfonylureas are traditionally the oral agents of first choice, although data from a long-term randomized, controlled trial of glucose-lowering agents in patients with type 2 diabetes suggested that metformin may be superior to other available agents in decreasing the incidence of diabetes-related complica¬ tions in obese patients.200 Advantages of using the sulfonylureas in older diabetic patients include their efficacy in lowering glucose levels, the simplicity of dosing regimens, long experience with these agents, and their relative safety. Underweight older diabetic patients often do not respond to sulfonylureas because they are relatively insulin deficient. Some elderly people are very sensitive to the hypoglycemic effects of these medications, however. There¬ fore, treatment of patients with mild to moderate hyperglycemia should begin with very low dosages (e.g., glipizide, 2.5-5.0 mg, or glyburide, 1.25-2.5 mg, each morning), with small incremental increases every 1 to 2 weeks if needed. Elderly diabetic patients with more marked hyperglycemia in the range of 300 to 400 mg/ dL are unlikely to be adequately controlled with sulfonylurea therapy and may be better managed with insulin therapy. Elderly people are at higher risk than younger patients for prolonged hypoglycemia due to the use of oral sulfonylureas.201 Chlorpropamide is not recommended for use in the elderly due to its very long half-life, which may increase the risk of pro¬ longed hypoglycemia. In addition, chlorpropamide also may cause hyponatremia due to inappropriate ADH secretion. Second-generation sulfonylureas (glipizide, glyburide) are pref¬ erable because they are nonionically bound to albumin in the circulation. As a result, these agents are not displaced from albu¬ min by other anionic drugs such as warfarin and salicylates, and drug interactions are less likely to occur. Either of these agents is acceptable for initial use in most older patients with type 2 dia¬ betes, and the reported incidence of hypoglycemia is similar in patients taking glyburide and in those taking glipizide.202 In general, oral hypoglycemic agents should not be prescribed for patients with severe renal or hepatic failure, which may lead to drug accumulation and toxicity. Several nonsulfonylurea oral hypoglycemic agents are now available. Metformin, a biguanide, is thought to act primarily by

1827

suppressing hepatic glucose production. In a 10-year, random¬ ized, controlled trial comparing metformin treatment with sul¬ fonylurea and insulin therapy in obese middle-aged patients with type 2 diabetes, metformin-treated subjects experienced fewer diabetes-related complications and lower overall mortal¬ ity, as well as less weight gain and fewer hypoglycemic epi¬ sodes, than did the other treatment groups 200 Gastrointestinal discomfort is relatively common with metformin, although the appetite reduction and mild weight loss associated with its use may be potentially beneficial in some obese elderly patients. Severe lactic acidosis has been reported with metformin; there¬ fore, the drug should be avoided in patients at risk for decreased tissue perfusion, for example, those with acute illnesses or con¬ gestive heart failure. Hepatic and renal insufficiency are other contraindications to its use. Other available oral hypoglycemic agents include acarbose, an a-glucosidase inhibitor, and troglitazone, a thiazolidinedione. Acarbose inhibits the breakdown of ingested carbohydrates in the gastrointestinal tract, thus preventing their absorption. Diarrhea is a common side effect and potentially may be therapeutic in older people with constipation. Little information is available, however, regarding the tolerability and effectiveness of this agent in the older diabetic patient. Troglitazone, now off the market, acts by increasing insulin sensitivity. It had generally been well tolerated in clinical trials that had included older patients, but its disadvantages included its expense and the occurrence of severe hepatotoxicity and death in rare instances.182 Monitoring of liver enzymes is mandatory for patients receiving drugs in this class, and they should not be used in patients with coexisting liver dis¬ ease. Repaglinide is a nonsulfonylurea insulin secretagogue developed for the management of type 2 diabetes; available data suggest that the risk of hypoglycemic episodes may be lower with repaglinide than with sulfonylurea drugs such as glyburide.203 However, its role in the treatment of elderly patients with type 2 diabetes has not been determined. INSULIN USE Insulin is indicated for older insulinopenic patients and for patients with type 2 diabetes whose blood glucose cannot be adequately controlled with diet, exercise, and oral hypoglyce¬ mic agents. In these patients, the best approach is to begin with a low insulin dosage and to increase slowly as needed, while ensuring that the patient is never hypoglycemic. As with younger patients receiving insulin, prerequisites for safe insulin therapy include accurate home blood glucose monitoring and record keeping, and a stable pattern of food intake and activity throughout the day. In older patients who are unable to adjust their food intake regimen, the insulin regimen may have to be adjusted instead. Patients with visual or manual dexterity prob¬ lems may require devices such as syringe magnifiers or dose gauges to help draw up the correct amount of insulin, or syringes with premeasured insulin doses.204 Lispro insulin, an insulin analog that more closely mimics the action of endogenous postprandial insulin release than regular insulin, may be useful in the management of diabetes in some elderly patients (see Chap. 143). Lispro insulin has a rapid onset of action and a short duration of action, and should be given within 15 minutes of meal consumption. It may be especially useful in elderly patients with erratic food intake patterns, because it can also be given immediately after a meal. Lispro insulin is also useful in older diabetic patients with impaired renal function, who are at increased risk of hypoglycemia due to the prolonged duration of action of regular insulin 204 In frail elderly patients with complex medical, cognitive, social, or functional problems (see Table 199-9), the insulin regi¬ men should be kept as simple as possible to reduce medication

1 828

PART XIII: ENDOCRINE AND METABOLIC DYSFUNCTION IN THE GROWING CHILD AND IN THE AGED

errors and to improve compliance. Many elderly patients requir¬ ing insulin can be adequately managed with a single daily dose of intermediate-acting insulin in the morning. The dawn phenom¬ enon is markedly reduced or absent in normal elderly people,205 so for many elderly diabetic patients, giving a single dose in the evening may be inappropriate and could induce prolonged and unrecognized early morning hypoglycemia.

LIPID LEVELS In parallel with body weight, average total plasma cholesterol levels increase during early adulthood but level off beyond approximately age 50 in men and age 60 in women. In senes¬ cence, cholesterol and triglyceride levels may decline together with body weight, although all of these remain stable in some healthy elderly people. One longitudinal study reported declin¬ ing HDL and LDL cholesterol levels with aging in healthy older men and women,206 although women continue to have higher HDL levels, on average, than men of the same age. HDL cholesterol levels are inversely associated with risk of coronary artery disease in older adults of both sexes,207'208 whereas a high LDL level appears to be an important risk factor for coronary disease in older women.209 Unlike in middle-aged adults, however, elevated total cholesterol levels do not strongly predict coronary events in elderly people, causing considerable controversy regarding the benefits of cholesterol screening in the older population. The presence of comorbidity or debilita¬ tion and their association with low cholesterol levels appear to account for this reduction in predictive capacity of cholesterol levels for coronary events in older people 210 Extremes of choles¬ terol levels are associated with an increased mortality risk, with the lowest mortality risk in those with intermediate cholesterol levels.211 These findings are consistent with the notion that a low cholesterol level in an older person may be a marker for frailty or serious underlying illnesses. Indeed, low total cholesterol lev¬ els are associated with early demise in nursing home residents and hospitalized patients.212 On the other hand, long-standing or lifelong hypocholesterolemia is associated with a lower risk of cardiovascular disease.213 Taken together, these observations suggest that judicious use of cholesterol screening may be indi¬ cated in more robust older adults. Lowering of elevated cholesterol levels in middle-aged adults has been shown to reduce morbidity and mortality from CHD in randomized, controlled trials, not only in those with established atherosclerotic disease214 but also in hypercholesterolemic men without a history of myocardial infarction.215 Fur¬ thermore, lipid-lowering therapy decreased the number of coronary events in middle-aged subjects without hypercholes¬ terolemia,216 and reduced the coronary event rate in middleaged subjects with average total cholesterol levels who did not have clinically evident CHD.217 Taken together, these data indi¬ cate an important role for cholesterol screening and lowering in both primary and secondary prevention of CHD events in middle-aged patients. No trials have been performed in an exclusively elderly pop¬ ulation to determine whether cholesterol reduction decreases the occurrence of CHD in these patients. Secondary prevention trials (e.g., Scandinavian Simvastatin Survival Study [4S] and Cholesterol and Recurrent Events [CARE] trials), however, have shown benefits of cholesterol lowering in reducing CHD mortal¬ ity in the subgroup of older subjects up to 75 years of age.218-219 Moreover, in a trial of the use of a statin drug for primary pre¬ vention of CHD, the subset of older subjects up to 73 years old benefited from a reduction in CHD event rates.217 Data are not yet available to indicate whether the benefits of cholesterol low¬

ering in primary and secondary CHD prevention apply equally to people older than age 75. Many physicians have been reluc¬ tant to treat hypercholesterolemia aggressively in older patients because of concerns about efficacy, cost, side effects, decreasing strength of total cholesterol levels as a relative risk factor for CHD with aging, and the possibility of minimal benefit in patients with limited longevity. Because of the markedly increased prevalence of CHD with aging, however, the attribut¬ able risk, or amount of coronary artery disease risk due to hypercholesterolemia, is greater in the elderly population as a whole. Furthermore, lipid-lowering agents are effective in reducing lipid levels in the elderly, and no evidence is found to indicate that such therapy is less effective in preventing CHDrelated morbidity and mortality in the elderly. In addition, angiographic studies in younger adults have shown regression of coronary artery plaques with lipid-lowering therapy, indicat¬ ing the potential to alter the natural history of the atherosclerotic lesion.220 Finally, stratification of data by age of subject in both primary and secondary prevention trials shows significant reduction in CHD events in both older and middle-aged adults.215-216'218 Studies of the cost effectiveness of the use of statin drugs to lower cholesterol in those with and without preexisting CHD have yielded varying estimates of the cost of such treat¬ ment per year of life saved, but in general their cost effectiveness is greatest in patients with the highest risk of CHD.221-222 Treat¬ ment of all patients without regard to CHD risk would be pro¬ hibitively expensive. Based on these potential benefits, aggressive management of hypercholesterolemia may be appropriate in selected elderly patients, but the decision to treat should be made after carefully weighing individual factors, including overall health, patient motivation, the impact of atherosclerotic disease on quality of life, and the potential risks and benefits of therapy. Given the decreasing association between cholesterol levels and CHD with advancing age, whether or not lipid-lowering agents are helpful for primary prevention of CHD in the elderly is unclear. Lipid¬ lowering strategies for secondary prevention may be beneficial in patients with established atherosclerotic disease and a good life expectancy, whereas extremely old patients and those with marked functional limitations due to comorbid illnesses such as congestive heart failure, dementia, malignancies, and chronic renal or lung disease are unlikely to derive much benefit from lipid-lowering therapy. In all elderly patients, regardless of whether they are hypercholesterolemic, modification of other CHD risk factors including hypertension, smoking, and diabetes mellitus is the highest therapeutic priority, because treatment of these conditions has clearly been shown to benefit the elderly.223 According to the National Cholesterol Education Program guidelines, the approach to treatment initiation and the goals of treatment are similar in elderly and younger adults, and are based on both LDL cholesterol levels and CHD risk status.214 As for young adults, older patients with one or more risk factors besides age (smoking, family history of CHD, diabetes, hyper¬ tension, HDL level of 130 mg/dL, and drug treatment for LDL levels of >160 mg/dL. The presence of CHD, however, lowers these treatment initiation levels to >100 and >130 mg/dL, respectively.214 Although these treatment guidelines have sug¬ gested a goal of 50% of the gland is destroyed. Some reported cases have been discovered only at autopsy.3 The clinical presentation may vary, but usually is related to the symptoms of a mass lesion (i.e., headache and vision disturbance). The endocrine dysfunction may manifest as amenorrhea, polydipsia, polyuria, or panhypopituitarism. Radiologic findings of a pituitary abscess reveal changes con¬ sistent with an expanding mass in the sella turcica. The pressure of an expanding mass may lead to erosion of the walls of the sella turcica, with depression of the floor into the sphenoid sinus and upward rotation and osteoporosis of the anterior and poste¬ rior clinoids.8-10 With computed tomographic (CT) scans and magnetic resonance imaging (MRI), suprasellar extension may be seen. Although these techniques are useful in defining the anatomic location and characteristics of an abscess of the hypo¬ thalamic-pituitary region, it may be difficult to distinguish such an abscess from tumor.9-10 Even before the development of MRI, it had been suggested that the presence of diabetes insipidus (DI) could help to differentiate pituitary abscess from an ade¬ noma. DI occurs in only 10% of patients with adenomas as com¬ pared to almost half of patients with an abscess.2 Treatment of a pituitary abscess usually consists of the administration of antibiotics combined with surgical drainage, using the transsphenoidal approach with drainage into the sphenoid sinus. Fungal infections of the pituitary caused by Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Sporothrix schenckii, Candida species, and Aspergillus species have been described. They usually are associated with disseminated dis¬ eases and immunosuppression.

Parasitic infections of the pituitary are extremely rare. Pitu¬ itary involvement has been described in patients with dissemi¬ nated toxoplasmosis. An intrasellar hydatid cyst also has been reported. Amebic brain abscess and malaria have been associ¬ ated with pituitary necrosis. African trypanosomiasis (SS) is an anthropozoonosis transmitted by the tsetse fly. Infection with Trypanosoma brucei in humans is associated with adynamia, leth¬ argy, anorexia, and more specifically, amenorrhea/infertility in women and loss of libido/impotence in men. Evidence suggests that experimental infection with T. brucei species causes polyglandular endocrine failure by local inflammation of the pituitary, thyroid, adrenal, and gonadal glands. In a study of Ugandan patients with untreated SS, there was a high preva¬ lence of hypogonadism (85%), hypothyroidism (50%), and adre¬ nal insufficiency (27%). Pituitary function tests suggested an unusual, combined central (hypothalamic/pituitary) and peripheral defect in hormone secretion. The presence of hypo¬ pituitarism correlated with high cytokine concentrations (i.e., tumor necrosis factor-a [TNF-a], interleukin-6 [IL-6]), and direct parasitic infiltration of the endocrine glands, is involved in the pathogenesis of SS-associated endocrine dysfunction.11 Viral infections may alter hypothalamic-pituitary function or cause actual necrosis of the gland. All of the herpesviruses, as well as influenza, measles, mumps, poliomyelitis, Coxsackie B, rabies, St. Louis encephalitis, rubella, and epidemic hemor¬ rhagic fever, have been implicated.3

THE THYROID INFECTIONS OF THE THYROID Infections of the thyroid gland are uncommon. Clinically, they may be divided into two categories: acute suppurative thyroiditis, usually caused by bacterial, fungal, mycobacterial, or parasitic infections, and subacute thyroiditis, more commonly attributed to a viral etiology12 (see Chap. 46). Of 224 reported cases of acute suppurative thyroiditis, 68% were bacterial, 9% were mycobacterial, 5% were parasitic, and 3% were the result of syphilitic gummatous infections.13 Acute bacterial thyroiditis may occur in patients of any age; it is more common in women and in persons with preexisting thyroid disease. The thyroid infection often is preceded by infec¬ tion elsewhere in the body, usually the upper respiratory tract, throat, or head and neck. Clinical manifestations include ante¬ rior neck pain, tenderness, and dysphagia; the area may be warm and erythematous. Unilateral vocal cord paralysis has been reported as a complication of acute suppurative thyroidi¬ tis.14 Often, there is fever and concurrent pharyngitis. Other than the leukocytosis (frequently more than 10,000 cells, usually neu¬ trophilia), laboratory data, including thyroid function tests, usually are normal. The most frequent bacterial isolates include S. aureus, S. pyogenes, S. pneumoniae, and Enterobacter species.12-13 Other bacteria—Actinomyces, Salmonella, Klebsiella pneumoniae, and Brucella—have been isolated.15 In rare circumstances, some multisystemic infections (e.g., Lyme disease) can be superim¬ posed on severe primary hypothyroidism such that if the thyroi¬ dal symptoms are sufficiently advanced and pronounced, the nonspecific symptoms of Borrelia infections are easily over¬ looked.16 Fine-needle aspiration, with or without ultrasono¬ graphic guidance, may aid in the diagnosis.17 The treatment of acute bacterial thyroiditis consists of rest, local heat, and appro¬ priate antibiotics. If an abscess occurs as a complication, surgical drainage should be performed (Fig. 213-1). A role for Yersinia enterocolitica has been proposed in patients with Graves disease, nontoxic goiter, and other thyroid disor¬ ders on the basis of the presence of antibodies in their sera. In the United States and Israel, where serotype 0:3 is not commonly

Ch. 213: Infectious Diseases and Endocrinology

1939

may require surgery as well as antifungal therapy (e.g., flucona¬ zole or amphotericin B).

FIGURE 213-1. A 25-year-old man with thyroid abscess caused by bac¬ terial infection after attempted intravenous heroin injection. At first, it was thought that he had subacute granulomatous thyroiditis. Usually, immediate incision and drainage are necessary for this life-threatening condition. Note the initial pustule. (G, goiter; L, larynx; P, pustule.)

isolated, 0:3 antibody titers greater than 1:8 were reported in 52% of patients with thyroid disorders—a finding that suggests the occurrence of a cross-reaction rather than a causal relation in these patients. Cross-reactivity may occur at the level of the receptor for thyroid-stimulating hormone (TSH).18 Helicobacter pylori, a newly recognized pathogen, has a mark¬ edly increased prevalence in patients with autoimmune atro¬ phic gastritis (87.5%). Anti-H. pylori immunoglobulin G (IgG) levels and results of breath tests are also higher in patients with Graves disease and Hashimoto thyroiditis. A positive linear regression has been found between the levels of microsomal autoantibodies and anti-H. pylori IgG in patients with atrophic thyroiditis. Such a relationship suggests that the H. pylori anti¬ gen may be involved in the development of autoimmune atro¬ phic thyroiditis or that autoimmune function in atrophic thyroiditis may increase the likelihood of H. pylori infection.19 The incidence of tuberculosis (TB) of the thyroid gland is dif¬ ficult to assess. Previous reports were based mainly on the pres¬ ence of granulomas on tissue biopsy without cultures; such findings easily could represent sarcoidosis, syphilis, or granulo¬ matous thyroiditis. In one review of bacteriologically proven TB of the thyroid, most cases were associated with coexistent dis¬ seminated infection. Compared with patients who had nontuberculous bacterial thyroiditis, fever, pain, and tenderness were less prominent and symptoms had been present longer.13 Rarely, TB of the thyroid may present as an isolated area of thyroiditis in the form of a nodular lesion.20 On rare occasions, atypical mycobacterium (e.g., Mycobacterium avium intracellulare) has been reported as a cause of suppurative thyroiditis.21 Of 31 reported cases of thyroiditis caused by fungi, Aspergil¬ lus infections were most common, accounting for >80% of cases, with C. immitis, Candida species, and Allescheria boydii implicated in the remaining 15% of cases.13 Aspergillosis of the thyroid usu¬ ally is a manifestation of disseminated disease and is seen in immunocompromised hosts. The diagnosis most often is made at autopsy. The pathogenesis probably is by hematogenous spread to the thyroid, with subsequent formation of focal abscesses, and with patchy hemorrhagic lesions from vascular invasion by the fungus. Therapy for disseminated infection usu¬ ally consists of high doses of amphotericin B. Coccidioidomyco¬ sis of the thyroid is very rarely recognized antemortem.22 Cases usually occur as part of a fatal disseminated infection. Treatment

The authors treated a patient with underlying sickle cell dis¬ ease and sarcoidosis who was receiving low-dose corticosteroid therapy and had an abscess of the thyroid caused by Cryptococ¬ cus neoformans. The infection failed to clear with amphotericin B therapy but subsequently responded to fluconazole. Parasitic infections of the thyroid are extremely rare. A few cases of thyroidal echinococcal infection have been reported, which usually present as a chronic, slow-growing mass, with signs and symptoms of compression. The initial diagnosis at the time of surgery generally is a nontoxic goiter. Cysticercosis of the thyroid also has been reported.13 Severe hypothyroidism was reported in a patient with chronic intestinal giardiasis due to isolated levothyroxine malabsorption. Treatment with met¬ ronidazole resulted in complete elimination of the parasites and recovery of regular intestinal thyroid hormone absorption.23 Syphilitic gumma of the thyroid gland is extremely rare. This infection mimics a slow-growing tumor; the most common symptoms are those of local compression.13 Thyroiditis is a rare manifestation of disseminated disease with the protozoan Pneumocystis carinii. One patient, who was chemically euthyroid at the time of diagnosis, became hypothy¬ roid during therapy and required the use of thyroid hormone replacement.24 Infectious agents have been implicated in the pathogenesis of autoimmune thyroid disease.25 Classic autoimmune thyroid dis¬ ease (i.e.. Graves disease and Hashimoto thyroiditis) has been shown to be associated with a variety of infectious agents (e.g., V. enterocolitica and retroviruses), while infection of the thyroid gland (e.g., subacute thyroiditis and congenital rubella) has been shown to be associated with autoimmune thyroid phenomena.25 Subacute thyroiditis is a remitting inflammatory disease of the thyroid gland that may be caused by a viral infection; mumps, measles, influenza, Coxsackie virus, St. Louis encepha¬ litis, cytomegalovirus (CMV), and infectious mononucleosis all have been reported.26'27 However, it has been difficult to culture virus or to demonstrate viral inclusion bodies. Therefore, in most cases, the actual role of viral agents in the etiology of thy¬ roiditis is merely a supposition. EUTHYROID SICK SYNDROME The euthyroid sick syndrome refers to an alteration of thyroid function that occurs in patients with various nonthyroid ill¬ nesses, such as chronic renal failure, liver disease, stress, drug use, starvation, surgery, fever, and infection (see Chap. 36). Of the infections involved, sepsis probably is implicated most com¬ monly, but pyelonephritis, pulmonary infections, viral hepatitis, other viral infections, typhoid fever, brucellosis, and malaria also have been reported.28-30 The euthyroid sick syndrome has been estimated to occur in -50% of all patients in the medical intensive care unit setting, many of whom have severe infections. The prognosis corre¬ lates with the level of serum thyroxine (T4). Of a large number of patients studied with nonthyroid illness, 84% with a T4 level 5 pg/dL died.31 As patients recover, there may be a transient, slight rise in the serum TSH level.3 Subsequently, when the serum triiodothyronine (T3) level begins to rise, the TSH level normalizes. Patients with the euthyroid sick syndrome are clinically euthyroid, but have a low serum T3 level and a high reverse T3 level. The serum total T4 level may be low, high, or normal, and the TSH level often is normal. The depression of serum T3 is thought to result from an impaired peripheral conversion of T4 to T3, which, in turn, may cause a relative increase of T4 or an

1940

PART XIV: INTERRELATIONSHIPS BETWEEN HORMONES AND THE BODY

increased reverse T3. A decreased total T4 level may occur because of a reduction of thyroid-binding globulin in patients who are malnourished. Moreover, a thyroid hormone-binding inhibitor has been described that may interfere with thyroid¬ binding globulin binding of T4, such that the total T4 level is decreased but the free T4 level remains normal. Infection may cause the euthyroid sick syndrome; among those who already have the syndrome for other reasons, the inci¬ dence of complicating infection also is high. It has been sug¬ gested that thyroid hormone-binding inhibitor may be similar in structure to a phagocytosis inhibitor, and that small amounts of thyroid hormone-binding inhibitor can inhibit the phagocy¬ tosis of E. coli in some tissues.

DISORDERS OF CALCIUM AND PHOSPHATE IN RELATION TO INFECTIOUS DISEASES HYPOCALCEMIA AND THE TOXIC SHOCK SYNDROME Hypocalcemia is a common finding in patients with toxic shock syndrome. The hypocalcemia has been thought to result from concurrent hypoalbuminemia. The severity of the hypocalcemia often is correlated with the inflammatory response.313 However, studies have indicated that the hypocalcemia often occured in conjunction with elevated concentrations of serum immunoreactive calcitonin,32 which now has been found to be due to increased procalcitonin and other calcitonin precursors (Chap. 53). Often, hypophosphatemia is present.323 HYPOCALCEMIA DURING SEPSIS AND OTHER INFECTIONS A low ionized calcium level is common during bacterial sepsis and has been implicated as a prognostic factor. In a study of 60 critically ill patients with bacterial sepsis, 20% had ionic hypo¬ calcemia.33 A significant mortality was observed among patients with hypocalcemia (up to 50%), compared with patients who had normal calcium levels (29%). Interestingly, only patients with gram-negative sepsis had hypocalcemia. The two most common organisms isolated from blood cultures were E. coli and Pseudomonas aeruginosa. Serum calcium concentrations normal¬ ized in each of those patients with sepsis who survived. Hypo¬ calcemia in the patients with sepsis resulted from an efflux of calcium from the vascular space that was not met by a concom¬ itant influx. The cause of defective calcium influx appeared to be multifactorial and resulted from acquired parathyroid gland insufficiency, renal la-hydroxylase insufficiency, dietary vita¬ min D deficiency, and acquired tissue resistance to calciferol. The role that calcium plays in sepsis is not clear. Patients with sepsis often have decreased plasma ionized calcium lev¬ els. It appears that calcium replacement would be beneficial for normal cardiovascular and cellular function. However, in experimental models of septic shock, in hypocalcemic ani¬ mals, calcium replacement resulted in increased mortality.34'35 Studies on animal models of sepsis demonstrated increased intracellular free calcium levels, which may contribute to cel¬ lular injury.35 The administration of exogenous calcium may act to increase the intracellular flux of calcium, leading to det¬ rimental effects on the cell. The mechanism for the increase in intracellular calcium during sepsis is not known. Mediators of septic shock such as bacterial endotoxin or tumor necrosis fac¬ tor do not alter lymphocyte free intracellular calcium levels in vitro; however, incubation with lysophosphatidylcholine (an endogenous membrane lipid) significantly increases intracel¬ lular free calcium levels.36 Additional animal studies, which investigated the blockade of calcium influx with calciumchannel blockers, led to improved survival.37'38 Dantrolene, which is thought to reduce intracellular calcium by decreas¬

ing calcium release from the sarcoplasmic reticulum, also may have beneficial effects on cellular function during sepsis.39 The question of whether to replace calcium in patients with hypocalcemia remains unanswered. No human trials have been performed. A temporary decrease in the serum calcium level also has been described in patients with hypoparathyroidism who are treated with cholecalciferol during febrile illness.40 HYPERCALCEMIA IN GRANULOMATOUS AND VIRAL INFECTIONS Hypercalcemia can be associated with chronic granulomatous processes and chronic infection. Increased production of dihydroxyvitamin D by activated macrophages has been shown to be the cause in most cases. Hypercalcemia occasionally occurs in patients with TB.41 Often, normocalcemia is present at the time of hospital admission, and hypercalcemia develops after patients are in the hospital, are eating, and are taking multivita¬ mins containing vitamin D. The increased serum calcium responds to a corticosteroid suppression test. Usually, serum calcium levels normalize spontaneously. Many of these patients have hyperglobulinemia. Their serum phosphate concentra¬ tions generally are normal. It has been postulated that the tuber¬ culous lymph nodes of these patients contain the lahydroxylase enzyme, which induces an increased production of 1,25-dihydroxyvitamin D3, leading to the hypercalcemia. Coccidioidomycosis and, occasionally, other fungal infec¬ tions (i.e., histoplasmosis) are associated with hypercalcemia. Hypercalcemia was reported in a patient with Nocardia asteroides pericarditis correlated with the duration of infection. The hypercalcemia resolved after treatment of N. asteroides with sulfisoxazole.42 Adult T-cell leukemia (ATL) caused by human T-cell leuke¬ mia virus (HTLV)-I infection is associated with hypercalcemia in >66% of patients. A parathyroid hormone-related protein, which is implicated in the hypercalcemia of ATL, is transactivated by the HTLV-I and HTLV-II tax proteins.43 HYPOPHOSPHATEMIA Hypophosphatemia occurs in approximately one-third of all patients with Legionella pneumophila pneumonia. In one study, 10 of 18 patients had serum phosphate levels of 1000 mg/ dL. Significant reductions in total cholesterol and total triglycer¬ ides in patients treated with gemfibrozil, 600 mg twice a day, and/or atorvastatin (starting at 10 mg per day) have been noted.63 Caution is advised when using a fibrate in combination with the statin drugs due to an increased risk of myositis. If treatment with a lipid-lowering agent becomes necessary, a statin that is not exclusively metabolized by the cytochrome P450 3A4 isoform (e.g., pravastatin or fluvastatin) may minimize the potential for drug interactions with the Pis. Modification of other reversible cardiac risk factors such as hypertension and tobacco use should be included in risk-reduction counseling. The hyperglycemia observed in patients with HAFR is gener¬ ally nonketotic and responsive to treatment with sulfonylureas or insulin. Most patients do not require discontinuation of Plcontaining therapy. Given the strong likelihood that insulin resistance is pathophysiologic, metformin might prove to be effi¬ cacious. Preliminary data indicate that relatively low dosages of this agent relieve hyperinsulinemia in HIV-infected patients with fat redistribution and abnormal glucose homostasis.69a Guidelines

have not been established regarding glucose monitoring in PI recipients. Serum concentrations of the oral hypoglycemic agents glipizide, glyburide, or tolbutamide may be affected by coadmin¬ istration with Pis by influencing cytochrome P-450 metabolism.

THE ENDOCRINE PANCREAS Pancreatic abnormalities have been identified at autopsy in up to 50% of patients with AIDS70 (see Chap. 133). Nonspecific inflam¬ mation is the predominant finding. Infiltration by CMV, HSV, M. tuberculosis, M. avium complex, C. neoformans, Aspergillus spe¬ cies, T. gondii, P. carinii, Cryptosporidium parvum, Microsporidia, lymphoma, and Kaposi sarcoma usually occurs with dissemi¬ nated disease, but rarely results in clinically significant endocrine dysfunction.71 Hypoglycemia due to pancreatic destruction by Kaposi sarcoma has been reported70 (see Chap. 158). Studies conducted in the pre-HAART era revealed that insulinrequiring nonoxidative glucose disposal, hepatic glucose pro¬ duction, insulin clearance, and peripheral insulin sensitivity are enhanced in symptomatic HIV-infected individuals compared to seronegative controls.72 These findings differ from those of sepsis, in which insulin resistance and hyperglycemia are com¬ mon.73 Spontaneous insulin-requiring diabetes mellitus has been diagnosed in a few patients without apparent opportunis¬ tic infection or autoimmunity and has raised the possibility of direct HIV-induced islet cell dysfunction.70-70® Impaired glucose tolerance associated with HAART and syndrome(s) of abnormal fat redistribution has received considerable attention. The pre¬ sumed mechanism is insulin resistance. Drugs (see Table 214-2). Several medications used in the treatment of HIV disease are toxic to the pancreas. Acute pancre¬ atitis is the main clinical manifestation, but glucose homeostasis may also be perturbed. Pentamidine, an agent used in the treat¬ ment and prophylaxis of PCP, causes hypoglycemia in -25% of recipients because of insulin release from acutely damaged B cells.70 This adverse effect has appeared weeks after ceasing ther¬ apy due to the long tissue half-life.74 In some patients, progres¬ sive B-cell destruction may ensue, ultimately leading to insulin deficiency and frank diabetes mellitus. Risk factors associated with significant pentamidine-induced B-cell cytotoxicity include high dosage, prolonged duration of therapy, prior pentamidine treatment, intravenous administration, and renal insufficiency.70 Although much less frequent, dysglycemia has occurred with ' aerosolized pentamidine.70 Serum glucose concentrations should be monitored during intravenous pentamidine therapy, and patients should be advised of the warning symptoms of hypogly¬ cemia and diabetes. Symptomatic hypoglycemia should prompt glucose administration and discontinuation of the drug. Individ¬ uals experiencing hyperglycemia typically have reduced serum C-peptide concentrations and require insulin administration. Stimulation of B-cell insulin secretion by the sulfonamide component of trimethoprim-sulfamethoxazole is the presumed mechanism for the rare association of this agent with hypogly¬ cemia.71 Interferon-a, megestrol acetate, and didanosine have been reported to result in hyperglycemia and frank diabetes in patients with AIDS.70

ELECTROLYTE AND MINERAL METABOLISM The same principles guiding the evaluation and treatment of electrolyte disorders in seronegative patients should be applied to HIV-infected individuals. Attempts should be made to iden¬ tify and eliminate any precipitating factors with special atten¬ tion directed to recently administered medications.

Ch. 214: Endocrine Disorders in Human Immunodeficiency Virus Infection Hyponatremia. Hyponatremia is the most common electro¬ lyte disturbance in HIV-infected patients, occurring in up to 50% of hospitalized patients and in -20% of ambulatory patients.7 Hypovolemic hyponatremia results from excessive cutaneous, renal, or gastrointestinal fluid losses. Renal-salt wasting has been associated with pentamidine and amphotericin B.7 Excessive hypotonic solution administration should be avoided, as it may further dilute the serum sodium concentrations. As adrenocortical deficiency is causal in a minority of patients, exclusion of this entity should be considered, based on the associated findings (e.g., hyperkalemia, metabolic acidosis) and/or refractoriness to fluid replacement. Sulfonamide-induced interstitial nephritis produc¬ ing hyporeninemic hypoaldosteronism has been described in recipients of trimethoprim-sulfamethoxazole. Furthermore, trime¬ thoprim inhibits sodium channels in the distal nephron, mimick¬ ing the action of a potassium-sparing diuretic75 (see Table 214-2). Euvolemic hyponatremia may be a manifestation of SIADH. Pri¬ mary polydipsia in the setting of dementia has also been reported. Hypernatremia. Hypernatremia results from dehydration when free water deficits exceed sodium wasting. Increased insensi¬ ble losses associated with fever and impaired urinary concentrating capacity related to certain medications (e.g., amphotericin B) have been etiologic. Central (neurogenic) DI occurs rarely in HTV-infected patients with CNS disease8 and nephrogenic DI has occurred with foscamet administration for CMV retinitis9 (see Table 214-2). Hypercalcemia. Hypercalcemia is caused by neoplastic, infectious, or granulomatous processes in HIV-infected patients and results mostly from the extrarenal conversion of 25hydroxyvitamin D to 1,25-dihydroxyvitamin D by la-hydroxylase occurring within immune cells39 (see Chap. 59). Besides lymphoma, raised serum calcium levels have complicated infec¬ tions with C. neoformans, disseminated CMV, M. avium complex, and P. carinii in HIV-infected individuals.10-39 The hypercalcemia appearing in patients coinfected with human T-cell lymphotropic virus type 1, a retrovirus linked to T-cell leukemia and lym¬ phoma, has been attributed to viral-induced production of IL-2 and/or parathyroid hormone (PTH)-related peptide.39 Hypocalcemia. As in seronegative individuals, hypocalce¬ mia has been described in HIV-infected patients with sepsis, hypoalbuminemia, and impaired vitamin D metabolism due to renal insufficiency or malabsorption. Elevated circulating free fatty acids may reduce serum calcium levels by enhancement of calcium binding to albumin. Relative hypoparathyroidism determined by reductions of PTH at baseline and following ethylenediaminetetraacetic acid (EDTA)-induced hypocalcemia has been demonstrated in patients with AIDS. These findings correlated with the degree of hypocalcemia. Destructive infiltra¬ tion by opportunistic pathogens (e.g., P. carinii or CMV) or direct invasion by HIV due to expression of CD4-like surface mole¬ cules on parenchymal cells could be pathogenic. Frank hypopara¬ thyroidism in a patient presenting with muscle cramps, tetany, and undetectable serum PTH levels has also been described. The significance of modest reductions in serum calcium concentra¬ tions, which are associated with advanced HIV infection, increased circulating TNF-a levels, and reduced concentrations of 1,25-dihydroxyvitamin D in some patients, is unknown.76 Adverse medication effects account for a majority of cases of hypocalcemia in HIV-infected patients. While foscarnet com¬ plexes with ionized calcium in a dose-dependent manner, renal tubular damage may also be operative in some cases given the association with hypomagnesemia and hypokalemia.77 Since total calcium levels may not be altered, the ionized fraction should be monitored during treatment in those who develop compatible symptoms. Pentamidine-induced hypocalcemia has been reported and may be profound if concurrently adminis¬ tered with foscamet.78 Ketoconazole can interfere with vitamin D metabolism and exacerbate hypocalcemia. Marked hypo¬

1957

magnesemia resulting from amphotericin B therapy potentiates hypocalcemia through inhibition of PTH release as well as induction of peripheral PTH resistance (see Table 214-2). Hyperkalemia. HIV-related causes of hyperkalemia include primary adrenal insufficiency, hyporeninemic hypoaldoster¬ onism, and adverse effect of medications.39 Pentamidine induces hyperkalemia by interfering with distal sodium trans¬ port (see Table 214-2). Mechanisms responsible for raised serum potassium concentrations with trimethoprim-sulfamethoxazole therapy have been discussed. Hypokalemia. Malnutrition, gastrointestinal fluid losses, foscarnet administration, and amphotericin B-related renal tubular acidosis have been associated with hypokalemia in HIVinfected patients.

REFERENCES 1. Mosca L, Costanzi G, Antonacci C, et al. Hypophyseal pathology in AIDS. Histol Histopathol 1992; 7:291. 2. Milligan SA, Katz MS, Craven PC, et al. Toxoplasmosis presenting as pan¬ hypopituitarism in a patient with the acquired immune deficiency syn¬ drome. Am J Med 1984; 77:760. 3. Sullivan WM, Kelley GG, O'Connor PG, et al. Hypopituitarism associated with a hypothalamic CMV infection in a patient with AIDS. (Letter). Am J Med 1992; 92:221. 4. Schwartz LJ, St. Louis Y, Wu R, et al. Endocrine function in children with human immunodeficiency virus infection. Am J Dis Child 1991; 145:330. 5. Geffner ME, Yeh DY, Landaw EM, et al. In vitro insulin-like growth factor1, growth hormone, and insulin resistance occurs in symptomatic human immunodeficiency virus-l-infected children. Pediatr Res 1993; 34:66. 6. Frost RA, Fuhrer J, Steigbigel R, et al. Wasting in the acquired immune defi¬ ciency syndrome is associated with multiple defects in the serum insulinlike growth factor system. Clin Endocrinol (Oxf) 1996; 44:501. 7. Tang WW, Kaptein EM, Feinstein El, Massry SG. Hyponatremia in hospital¬ ized patients with the acquired immunodeficiency syndrome (AIDS) and the AIDS-related complex. Am J Med 1993; 94:169. 8. Keuneke C, Anders HJ, Schlondorff D. Adipsic hypernatremia in two patients with AIDS and cytomegalovirus encephalitis. Am J Kidney Dis 1999; 33:379. 9. Navarro JF, Quereda C, Gallego N, et al. Nephrogenic diabetes insipidus and renal tubular acidosis secondary to foscamet therapy. Am J Kidney Dis 1996; 27:431. 10. Sellmeyer DE, Grunfeld C. Endocrine and metabolic disturbances in human immunodeficiency virus infection and the acquired immune deficiency syn¬ drome. Endocr Rev 1996; 17:518. 11. Del Monte P, Bemasconi D, De Conca V, et al. Endocrine evaluation in patients treated with interferon-alpha for chronic hepatitis C. Horm Res 1995; 44:105. 12. Glasgow BJ, Steinsapir KD, Anders K, Layfield LJ. Adrenal pathology in the acquired immune deficiency syndrome. Am J Clin Pathol 1985; 84:594. 13. Arabi Y, Fairfax MR, Szuba MJ, et al. Adrenal insufficiency, recurrent bacte¬ remia, and disseminated abscesses caused by Nocardia asteroides in a patient with acquired immunodeficiency syndrome. Diagn Microbiol Infect Dis 1996; 24:47. 14. Membreno L, Irony I, Dere W, et al. Adrenocortical function in acquired immunodeficiency syndrome. J Clin Endocrinol Metab 1987; 65:482. 15. Villette JM, Bourin P, Doinel C, et al. Circadian variations in plasma levels of hypophyseal, adrenocortical and testicular hormones in men infected with human immunodeficiency virus. J Clin Endocrinol Metab 1990; 70:572. 16. Norbiato G, Bevilacqua M, Vago T, et al. Cortisol resistance in acquired immunodeficiency syndrome. J Clin Endocrinol Metab 1992; 74:608. 17. Azar ST, Melby JC. Hypothalamic-pituitary-adrenal function in non-AIDS patients with advanced HIV infection. Am J Med Sci 1993; 305:321. 18. Chrousos GP. The hypothalamic-pituitary-adrenal axis and immune-mediated inflammation. N Engl J Med 1995; 332:1351. 19. Findling JW, Buggy BP, Gilson IH, et al. Longitudinal evaluation of adreno¬ cortical function in patients infected with the human immunodeficiency virus. J Clin Endocrinol Metab 1994; 79:1091. 20. Mulder JW, Frissen PH, Krijnen P, et al. Dehydroepiandrosterone as predic¬ tor for progression to AIDS in asymptomatic human immunodeficiency virus-infected men. J Infect Dis 1992; 165:413. 21. Clerici M, Trabattoni D, Piconi S, et al. A possible role for the cortisol/anti¬ cortisols imbalance in the progression of human immunodeficiency virus. Psychoneuroendocrinology 1997; 22:S2 7. 22. Steer KA, Kurtz AB, Honour JW. Megestrol-induced Cushing's syndrome. Clin Endocrinol (Oxf) 1995; 42:91. 23. Gallant JE, Chaisson RE, Moore RD. The effect of adjunctive corticosteroids for the treatment of Pneumocystis carinii pneumonia on mortality and subse¬ quent complications. Chest 1998; 114:1258. 24. Heufelder AE, Hofbauer LC. Human immunodeficiency virus infection and the thyroid gland. Eur J Endocrinol 1996; 134:669. 25. Martin-Davila P, Quereda C, Rodriguez H, et al. Thyroid abscess due to Rhodococcus equi in a patient infected with the human immunodeficiency virus. Eur J Clin Microbiol Infect Dis 1998; 17:55.

1 958

PART XIV: INTERRELATIONSHIPS BETWEEN HORMONES AND THE BODY

26. Mollison LC, Mijch A, McBride G, Dwyer B. Hypothyroidism due to destruction of the thyroid by Kaposi's sarcoma. Rev Infect Dis 1991; 13:826. 27. LoPresti JS, Fried JC, Spencer CA, Nicoloff JT. Unique alterations of thyroid hormone indices in the acquired immunodeficiency syndrome (AIDS). Ann Intern Med 1989; 110:970. 28. Hommes MJ, Romijn JA, Endert E, et al. Hypothyroid-like regulation of the pituitary-thyroid axis in stable human immunodeficiency virus infection. Metabolism 1993; 42:556. 29. Raffi F, Brisseau JM, Planchon B, et al. Endocrine function in 98 HIV-infected patients: a prospective study. AIDS 1991; 5:729. 30. Grunfeld C, Feingold KR. Metabolic disturbances and wasting in the acquired immunodeficiency syndrome. N Engl J Med 1992; 327:329. 31. van der Poll T, Romijn JA, Wiersinga WM, Sauerwein HP. Tumor necrosis factor: a putative mediator of the sick euthyroid syndrome in man. J Clin Endocrinol Metab 1990; 71:1567. 32. Ozawa M, Sato K, Han DC, et al. Effects of tumor necrosis factor-alpha/cachectin on thyroid hormone metabolism in mice. Endocrinology 1988; 123:1461. 33. Sato K, Satoh T, Shizume K, et al. Inhibition of 125I organification and thyroid hor¬ mone release by interleukin-1, tumor necrosis factor-alpha, and interferon-gamma in human thyrocytes in suspension culture. J Clin Endocrinol Metab 1990; 70:1735. 34. Rahman A, Esmaili A, Saatcioglu F. A unique thyroid hormone response ele¬ ment in the human immunodeficiency virus type 1 long terminal repeat that overlaps the Spl binding sites. J Biol Chem 1995; 270:31059. 35. Gilquin J, Viard JP, Jubault V, et al. Delayed occurrence of Graves' disease after immune restoration with HAART. Highly active antiretroviral ther¬ apy. Lancet 1998; 352:1907. 36. Sumida S, Miller K, Vogel S, et al. Hypothyroidism is associated with IL-2 therapy in a randomized controlled trial of IL-2 for the treatment of HIV infection. Abstracts of the 36th Annual Meeting of the Infectious Diseases Society of America, Denver, 1998. 37. De Paepe ME, Waxman M. Testicular atrophy in AIDS: a study of 57 autopsy cases. Hum Pathol 1989; 20:210. 38. Zhang H, Domadula G, Beumont M, et al. Human immunodeficiency virus type 1 in the semen of men receiving highly active antiretroviral therapy. N Engl J Med 1998; 339:1803. 39. Hofbauer LC, Heufelder AE. Endocrine implications of human immunode¬ ficiency virus infection. Medicine (Baltimore) 1996; 75:262. 40. Grinspoon S, Corcoran C, Lee K, et al. Loss of lean body and muscle mass correlates with androgen levels in hypogonadal men with acquired immu¬ nodeficiency syndrome and wasting. J Clin Endocrinol Metab 1996; 81:4051. 41. Merenich JA, McDermott MT, Asp AA, et al. Evidence of endocrine involve¬ ment early in the course of human immunodeficiency virus infection. J Clin Endocrinol Metab 1990; 70:566. 42. Ellerbrock TV, Wright TC, Bush TJ, et al. Characteristics of menstruation in women infected with human immunodeficiency virus. Obstet Gynecol 1996; 87:1030. 43. Grinspoon S, Corcoran C, Miller K, et al. Body composition and endocrine function in women with acquired immunodeficiency syndrome wasting. J Clin Endocrinol Metab 1997; 82:1332. 43a. Miller K, Corcoran C, Armstrong C, et al. Transdermal testosterone admin¬ istration in women with acquired immunodeficiency syndrome wasting: a pilot study. J Clin Endocrinol Metab 1998; 83:2717. 44. Martin ME, Benassayag C, Amiel C, et al. Alterations in the concentrations and binding properties of sex steroid binding protein and corticosteroid¬ binding globulin in HIV+ patients. J Endocrinol Invest 1992; 15:597. 44a. Bhasin S, Storer TW, Javanbakht M, et al. Testosterone replacement and resistance exercise in HIV-infected men with weight loss and low testoste¬ rone levels. JAMA 2000; 283:763. 45. Grunfeld C, Pang M, Doerrler W, et al. Lipids, lipoproteins, triglyceride clear¬ ance, and cytokines in human immunodeficiency virus infection and the acquired immunodeficiency syndrome. J Clin Endocrinol Metab 1992; 74:1045. 46. Hellerstein MK, Grunfeld C, Wu K, et al. Increased de novo hepatic lipogenesis in human immunodeficiency virus infection. J Clin Endocrinol Metab 1993; 76:559. 47. Feingold KR, Krauss RM, Pang M, et al. The hypertriglyceridemia of acquired immunodeficiency syndrome is associated with an increased prevalence of low density lipoprotein subclass pattern B. J Clin Endocrinol Metab 1993; 76:1423. 48. Strawford A, Hellerstein M. The etiology of wasting in the human immuno¬ deficiency virus and acquired immunodeficiency syndrome. Semin Oncol 1998; 25:76. 49. Dobs AS, Few WL 3rd, Blackman MR, et al. Serum hormones in men with human immunodeficiency virus-associated wasting. J Clin Endocrinol Metab 1996; 81:4108. 50. Grinspoon S, Corcoran C, Askari H, et al. Effects of androgen administra¬ tion in men with the AIDS wasting syndrome. A randomized, double-blind, placebo-controlled trial. Ann Intern Med 1998; 129:18. 50a. Corcoran C, Grinspoon S. Treatments for wasting in patients with the acquired immunodeficiency syndrome. N Engl J Med 1999; 340:1740. 51. Oster MH, Enders SR, Samuels SJ, et al. Megestrol acetate in patients with AIDS and cachexia. Ann Intern Med 1994; 121:400. 52. Strawford A, Barbieri T, Van Loan M, et al. Resistance exercise and supraphysiologic androgen therapy in eugonadal men with HIV-related weight loss. A randomized controlled trial. JAMA 1999; 281 (14):1282. 53. Schambelan M, Mulligan K, Grunfeld C, et al. Recombinant human growth hormone in patients with HIV-associated wasting. A randomized, placebocontrolled trial. Serostim Study Group. Ann Intern Med 1996; 125:873. 54. Reyes-Teran G, Sierra-Madero JG, Martinez del Cerro V, et al. Effects of tha¬

55.

56. 57. 58.

59. 60. 61. 62.

63. 64. 65.

65a. 66.

67. 68.

69. 69a.

70. 70a.

71. 72. 73. 74.

75. 76.

77. 78.

lidomide on HIV-associated wasting syndrome: a randomized, double¬ blind, placebo-controlled clinical trial. AIDS 1996; 10:1501. Carpenter CC, Fischl MA, Hammer SM, et al. Antiretroviral therapy for HIV infection in 1997. Updated recommendations of the International AIDS Society-USA panel. JAMA 1997; 277:1962. Miller KK, Daly PA, Sentochnik D, et al. Pseudo-Cushing's syndrome in human immunodeficiency virus-infected patients. Clin Infect Dis 1998; 27:68. Miller KD, Jones E, Yanovski JA, et al. Visceral abdominal-fat accumulation associated with use of indinavir. Lancet 1998; 351:871. Carr A, Samaras K, Burton S, et al. A syndrome of peripheral lipodystrophy, hyperlipidaemia and insulin resistance in patients receiving HIV protease inhibitors. AIDS 1998; 12:F51. Danesh J, Collins R, Peto R. Chronic infections and coronary heart disease: is there a link? Lancet 1997; 350:430. Henry K, Melroe H, Huebsch J, et al. Severe premature coronary artery dis¬ ease with protease inhibitors. Lancet 1998; 351:1328. Lo JC, Mulligan K, Tai VW, et al. "Buffalo hump" in men with HIV-1 infec¬ tion. Lancet 1998; 351:867. Walli R, Herfort O, Michl GM, et al. Treatment with protease inhibitors asso¬ ciated with peripheral insulin resistance and impaired oral glucose toler¬ ance in HIV-1-infected patients. AIDS 1998; 12:F167. Henry K, Melroe H, Huebesch J, et al. Atorvastatin and gemfibrozil for protease-inhibitor-related lipid abnormalities. (Letter). Lancet 1998; 352:1031. Martinez E, Gatell J. Metabolic abnormalities and use of HIV-1 protease inhibitors. Lancet 1998; 352:821. Carr A, Samaras K, Chisholm DJ, Cooper DA. Pathogenesis of HIV-1-protease inhibitor-associated peripheral lipodystrophy, hyperlipidaemia, and insulin resistance. Lancet 1998; 351:1881. Murata H, Hruz PW, Mueckler M. The mechanism of insulin resistance caused by HIV protein inhibitor therapy. J Biol Chem 2000; 275(27):20251. Hirsch MS, Klibanski A. What price progress? Pseudo-Cushing's syndrome associated with antiretroviral therapy in patients with human immunodefi¬ ciency virus infection. Clin Infect Dis 1998; 27:73. Kino T, Gragerov A, Kopp JB, et al. The HIV-1 virion-associated protein vpr is a coactivator of the human glucocorticoid receptor. J Exp Med 1999; 189:51. Yanovski JA, Miller KD, Kino T, et al. Endocrine and metabolic evaluation of human immunodeficiency virus-infected patients with evidence of protease inhibitor-associated lipodystrophy. J Clin Endocrinol Metab 1999; 84:1925. Saint-Marc T, Touraine JL. "Buffalo hump" in HIV-1 infection. (Letter). Lan¬ cet 1998; 352:319. Hadigan C, Corcoran C, Basgoz N, et al. Metformin in the treatment of HIV lipodystrophy syndrome: a randomized controlled trial. JAMA 2000; 284(4):472. Brivet FG, Naveau SH, Lemaigre GF, Dormont J. Pancreatic lesions in HIVinfected patients. Baillieres Clin Endocrinol Metab 1994; 8:859. Hadigan C, Corcoran C, Stanley T, et al. Fasting hyperinsulinemia in human immunodeficiency virus-infected men: relationship to body composition, gonadal function, and protease inhibitor use. J Clin Endocrinol Metab 2000; 85:35. Cappell MS, Hassan T. Pancreatic disease in AIDS—a review. J Clin Gastro¬ enterol 1993; 17:254. Hommes MJ, Romijn J A, Endert E, etal. Insulin sensitivity and insulin clearance in human immunodeficiency virus-infected men. Metabolism 1991; 40:651. Grunfeld C, Feingold KR. The metabolic effects of tumor necrosis factor and other cytokines. Biotherapy 1991; 3:143. Waskin H, Stehr-Green JK, Helmick CG, Sattler FR. Risk factors for hypoglycemia associated with pentamidine therapy for Pneumocystis pneu¬ monia. JAMA 1988; 260:345. Hsu I, Wordell CJ. Hyperkalemia and high-dose trimethoprim/sul¬ famethoxazole. Ann Pharmacother 1995; 29:427. Haug CJ, Aukrust P, Haug E, et al. Severe deficiency of 1,25-dihydroxyvitamin D3 in human immunodeficiency virus infection: association with immunological hyperactivity and only minor changes in calcium homeosta¬ sis. J Clin Endocrinol Metab 1998; 83:3832. Gearhart MO, Sorg TB. Foscamet-induced severe hypomagnesemia and other electrolyte disorders. Ann Pharmacother 1993; 27:285. Youle MS, Clarbour J, Gazzard B, Chanas A. Severe hypocalcaemia in AIDS patients treated with foscarnet and pentamidine. Lancet 1988; 1:1455.

CHAPTER

21 5

THE EYE IN ENDOCRINOLOGY ROBERT A. OPPENHEIM AND WILLIAM D. MATHERS The eye is frequently a window into the systemic conditions of the body. There is no better illustration of this than the interrela¬ tionship between the eye and the endocrine system. Many changes, subtle and gross, are manifested in the eye by normal

Ch. 215: The Eye in Endocrinology

1959

fluctuations in hormones, exogenous hormones, endocrine dis¬ orders, and metabolic abnormalities.

OCULAR EFFECTS OF NORMAL HORMONAL FLUCTUATION MENSTRUAL CYCLE Studies of the serum levels of luteinizing hormone, follicle-stim¬ ulating hormone, estriol, progesterone, and testosterone throughout the menstrual cycle, combined with determinations of intraocular pressure, anterior chamber depth, corneal thick¬ ness, outflow facility, and tear production, established no statis¬ tically valid correlation between any of these physiologic measurements and the hormonal fluctuations.1'2

PREGNANCY Both physiologic and pathologic ocular changes occur during pregnancy. In addition, there are several preexisting ocular con¬ ditions that are either exacerbated or ameliorated by pregnancy.3 PHYSIOLOGIC OCULAR CHANGES DURING PREGNANCY Visual Acuity. Blurred vision due to a change in refraction commonly occurs during pregnancy and usually resolves after delivery. The index of refraction of the cornea may change due to changes in corneal thickness. Lids. Chloasma, the increased pigmentation of the cheeks that often occurs in pregnancy, can also involve the eyelids. It is caused by increased melanocyte stimulation and resolves after delivery.4 Ptosis, or droopiness of the upper lid, is a well-docu¬ mented complication of lumbar anesthesia during childbirth.5 Ptosis associated with miosis and anhidrosis (i.e., Horner syn¬ drome) has also been noted to occur with the use of lumbar anes¬ thesia at the time of delivery.6 Conjunctiva. Conjunctival vessels undergo a progres¬ sively decreased flow of blood during pregnancy that is mani¬ fested by a decrease in the number of visible conjunctival capillaries.7 Subconjunctival hemorrhages have also been observed during pregnancy and resolve with no sequelae. Cornea. Corneal sensitivity decreases after the 31st week of pregnancy with a return to normal by 6 to 8 weeks postpar¬ tum.8 A slight increase in corneal thickness, perhaps due to edema, also occurs and is a possible cause of new-onset contact lens intolerance during pregnancy. Intraocular Pressure. A reduction in intraocular pressure usually occurs in pregnancy, probably as a result of an increase in outflow facility, which is especially evident during the last trimester.9 PATHOLOGIC OCULAR CHANGES DURING PREGNANCY Preeclamptic-eclamptic hypertensive retinopathy can occur after the 20th week of pregnancy. Findings on fundus examination include narrowing of the arterioles, flame-shaped hemorrhages, cotton¬ wool spots, retinal edema, and disc swelling. There is a correla¬ tion between the severity of the retinopathy and the risk that the mother will have permanent renal damage,10 as well as the risk that the fetus will die.11 Ten percent of patients with eclampsia and 1% to 2% of patients with severe preeclampsia have exudative retinal detachment, which usually resolves within weeks after delivery.12'13 Occasionally, residual retinal pigment epithelial (RPE) alterations may cause decreased visual acuity. Indocyanine green (ICG) angiography suggests that damage to the choroidal vasculature compromises the overlying RPE, leading to subretinal exudation.14 Transient cortical blindness15 and acute ischemic optic neuropathy16 have also been associated with toxemia.

FIGURE 215-1. Fundus of a 26-year-old woman who had a 15-year his¬ tory of juvenile-onset diabetes mellitus before pregnancy. Her prolifera¬ tive diabetic retinopathy (PDR) quickly progressed during pregnancy. Typical changes of PDR seen here are neovascularization of the retina (thin small arrow), neovascularization of the optic disc (large arrow), cotton-wool spots (arrowhead), retinal hemorrhage (arrow outline), and irregular venous dilation (short fat arrow). In central serous choroidopathy (CSC), dysfunction of the RPE allows serous fluid to leak under the retina, causing elevation of the macula. Subretinal exudates are seen in a high proportion of patients during pregnancy, as compared to the incidence of CSC in nonpregnant patients.17 The condition usually resolves spon¬ taneously after delivery.18 Pseudotumor cerebri (idiopathic intracranial hypertension) is a condition in which there is increased intracranial pressure with papilledema in the presence of normal results on head imaging scan and normal cerebrospinal fluid composition. Symptoms may include headache, visual obscurations, and visual field loss. It may be associated with pregnancy; however, nonpreg¬ nant pseudotumor cerebri patients are typically overweight women of childbearing age.3,19 EFFECTS OF PREGNANCY ON PREEXISTING OCULAR CONDITIONS Two ocular conditions are known to improve with pregnancy: glaucoma, by virtue of decreased intraocular pressure (see earlier), and intraocular inflammation.20 Both sarcoid uveitis and VogtKoyanagi-Harada syndrome have been noted to improve during pregnaray and to rebound after delivery.20'21 It is postulated that this is due to the increased cortisol in the bloodstream during preg¬ nancy and to the subsequent reduction that occurs after childbirth. The possibility of exacerbation of diabetic retinopathy during pregnancy is an extremely important clinical issue. The presence of diabetic retinopathy before pregnancy and the degree of its severity determine the course of this condition during preg¬ nancy. It is therefore recommended that women with diabetes undertake childbearing at a young age, before retinopathy has developed.22 Of women with diabetes who do not have retinop¬ athy before pregnancy, only -10% develop mild retinopathy that usually regresses after delivery.3 Background diabetic retinopa¬ thy tends to progress transiently during pregnancy.23'24 These patients should undergo dilated fundus examination every tri¬ mester. The real concern is for women who have proliferative diabetic retinopathy before pregnancy. Approximately 50% of these patients experience vision-threatening progression of their retinopathy during pregnancy3 (Fig. 215-1). Therefore, it is rec-

1960

PART XIV: INTERRELATIONSHIPS BETWEEN HORMONES AND THE BODY

ommended that patients with proliferative retinopathy undergo panretinal photocoagulation before conceiving. Dilated fundus examination should be performed once a month during the preg¬ nancy. Without close supervision and treatment, pregnancy in a woman with proliferative diabetic retinopathy can lead to irrep¬ arable loss of sight. Although the pathogenesis is unclear, one hypothesis suggests that there is an absence of autoregulation, which leads to increased retinal blood flow in the presence of a hyperdynamic circulation during pregnancy.25 Graves disease, which is common in young women, can be aggra¬ vated by pregnancy. It also may present during pregnancy. Because many of the symptoms of Graves disease, such as heat intolerance, tachycardia, and emotional fragility, are also seen in pregnancy, the ocular signs of exophthalmos and lagophthalmos may be impor¬ tant clues that a pregnant patient also has Graves disease.3 Uveal melanomas have been noted to grow during preg¬ nancy.26 Studies suggest that women with uveal melanoma are not at increased risk for metastases and that women with poste¬ rior uveal melanoma have a similar 5-year survival compared with nonpregnant women.27-28 Just as the normal pituitary is known to enlarge in pregnant women, so do pituitary adenomas.3 Pituitary adenomas that previ¬ ously caused no symptoms may do so for the first time during pregnancy. Affected women have headache, decreased visual acu¬ ity, and visual field disturbances such as bitemporal hemianopsia. The symptoms usually resolve after delivery with spontaneous regression of the tumor. Bromocriptine, which has been shown to produce regression of the tumor, appears not to be detrimental to the developing fetus. Pituitary adenoma enlargement during preg¬ nancy also can be caused by pituitary apoplexy, which can threaten the mother's life. A woman with a known pituitary adenoma who develops a headache or a new visual field defect should undergo magnetic resonance imaging and, possibly, lumbar puncture to rule out subarachnoid hemorrhage from the tumor. Meningiomas, which typically grow in a chronic and insidious manner, may demonstrate an accelerated course producing rel¬ atively acute vision loss during pregnancy. This accelerated growth pattern is probably hormone related. Many cases of meningioma have presented during pregnancy.29

The most common hormonal preparations used in the treatment of eye diseases are corticosteroids, such as prednisolone, dexamethasone, fluorometholone, and rimexolone. Along with anti¬ biotics, topical steroids and oral prednisone play a central role in the medical armamentarium of ophthalmologists. Their tremen¬ dous usefulness stems from both their antiinflammatory and immunosuppressive effects. ANTIINFLAMMATORY EFFECTS Inflammation in the eye can cause scarring and opacification of ocular structures that can lead to loss of vision. Corticosteroids are frequently used to diminish inflammation after eye surgery such as cataract extraction, refractive surgery, and corneal trans¬ plantation. In addition, these agents are used to treat most forms of uveitis involving either the anterior or posterior segment. Although topical application is effective for anterior segment inflammation, uveitis involving structures posterior to the cili¬ ary body requires periocular injection or systemic administra¬ tion. Corticosteroids are also a mainstay of therapy for other forms of ocular inflammation, such as scleritis, optic neuritis, temporal arteritis, and cystoid macular edema. IMMUNOLOGIC SUPPRESSION Topical and systemic corticosteroids are used to control harmful immunologic activity. They are essential after corneal transplant surgery to prevent and, when relevant, reverse rejection of the graft. Ocular infections, such as bacterial keratitis and endoph¬ thalmitis, are also treated with topical corticosteroids once the eye infection is believed to be sterile after the initiation of antibi¬ otic treatment. Finally, there is a wide range of ocular immuno¬ logic conditions that resolve in response to corticosteroid treatment. One example is the type IV immune response that causes phlyctenular keratitis, an allergic response that involves the limbus and mimics a corneal ulcer. When herpes simplex antigens remain in the corneal stroma after the infection resolves, a protracted immunologic response, mediated by both T and B cells, can cause pain and clouding of the cornea. Corti¬ costeroids are the only drugs used routinely to suppress this del¬ eterious response.

MENOPAUSE SIDE EFFECTS AND COMPLICATIONS In clinical practice, the most commonly recognized effect of meno¬ pause on the eye is the occurrence of dry eyes due to lacrimal insuf¬ ficiency. This traditionally has been associated with the decrease in estrogen levels that occurs after menopause. However, treatment with exogenous estrogen does not modify this dryness. In addition, lacrimal insufficiency also occurs with increased frequency during pregnancy and the use of estrogen-containing birth control pills, when estrogen and prolactin levels are elevated. This has led some investigators to speculate that the hormonal mediator of the lacri¬ mal gland is androgen, rather than estrogen.30 Androgen levels are decreased during the high-estrogen states of pregnancy and oral contraceptive use. When ovarian function declines during meno¬ pause, both estrogens and androgens decrease. Studies also sug¬ gest that prolactin, in addition to androgens, is involved in regulating exocrine secretion of the lacrimal gland.31

OCULAR EFFECTS OF EXOGENOUS HORMONES CORTICOSTEROIDS Hormones have many effects on the eye when they are used as therapeutic agents for both eye disease and systemic diseases.

Topical and systemic corticosteroid therapy can be associated » with multiple ocular complications. The risk is directly related to increased length of treatment, and all patients started on topical corticosteroid therapy must receive close follow-up to monitor for complications. The most common side effects are glaucoma and posterior subcapsular cataracts. Twenty-five percent of patients who receive topical corticosteroids have an increase in intraocular pressure. Some preparations, such as fluorometholone and rimexolone, are less likely to increase intraocular pressure. It is common for patients with systemic conditions such as sarcoidosis, rheumatoid arthritis, or lupus, who have been treated long-term with systemic corticoste¬ roids, to have advanced cataracts necessitating extraction. Microbial infections can be tragically exacerbated by the use of corticosteroids. Bacterial and fungal keratitis can be made worse if topical corticosteroids are initiated prematurely. The use of topical corticosteroids in the presence of actively repli¬ cating herpes simplex virus in the corneal epithelium may cause rapid acceleration of the infection, as well as corneal per¬ foration. Finally, topical steroids can accelerate corneal melts by increasing collagenase activity, although topical steroids may be helpful if there is associated corneal stromal infiltra¬ tion. Systemic corticosteroid use or withdrawal may be associ¬ ated with pseudotumor cerebri.

Ch. 215: The Eye in Endocrinology

OVARIAN STEROIDS HARMFUL EFFECTS OF ORAL CONTRACEPTIVES Oral contraceptives have no known therapeutic benefits in the treatment of eye disease; however, they have been implicated in the pathogenesis of many types of eye disease.32 Numerous case reports and studies have indicated that these drugs can affect nearly every part of the eye.33 Oral contraceptive use has been associated with edema of the eyelids and conjunctiva, subcon¬ junctival hemorrhage, conjunctivitis, and allergic reactions. Other studies have described vascular complications, such as central retinal vascular occlusions, retinal periphlebitis, and retinal hemorrhages.34-36'363 In addition, macular edema, central serous chorioretinopathy, and RPE disturbances have been reported.37 Many cases of optic neuritis and retrobulbar neuritis have been associated with oral contraceptives. These agents are well-known causes of pseudotumor cerebri, which manifests itself as papilledema and can easily be confused with optic neuritis. Finally, many subjective visual disturbances can be attrib¬ uted to oral contraceptives.38 Photophobia, glare, transient blur¬ ring of vision, difficulty in focusing, and other similar symptoms frequently occur in patients taking these agents. The role of oral contraceptive-induced migraine in these events remains obscure, but undoubtedly plays a part (see Chap. 105). NONSTEROIDAL ANTIESTROGENS Tamoxifen is an antiestrogen that prevents estrogen stimulation of breast cancer cells. It is the treatment of choice in women with estrogen receptor-positive advanced breast cancer. Ocular complications include corneal opacities (cornea verticillata; Fig. 215-2), optic nerve toxicity, and retinopathy. Retinal abnormali¬ ties include bilateral macular edema and yellow-white dots in the paramacular and foveal areas. Most ocular changes are reversible after the withdrawal of tamoxifen.39 Clomiphene is a competitive antagonist of estrogen receptors that is used to stimulate ovulation in the treatment of female infertility. Ocular side effects include blurred vision and scintil¬ lating scotomas that are dose-related and reversible on discon¬ tinuation of the drug. Changes in retinal cell function have been reported. Visual abnormalities are considered a contraindication to continuing use of this medication.

1961

OCULAR EFFECTS OF ENDOCRINE DISORDERS Two common endocrine disorders have frequent and often severe involvement of the eyes: diabetes mellitus and Graves disease. So extensive is the eye involvement in these conditions that an entire chapter in this text is devoted to each of them (see Chaps. 43 and 151). Patients should be referred to an ophthalmologist at the time diabetes is diagnosed. The ophthalmologist will perform a base¬ line and thereafter yearly eye examination to monitor not only the development of diabetic retinopathy and macular edema, but also primary open-angle glaucoma (which occurs with increased fre¬ quency m patients with diabetes). Careful monitoring and timely treatment of these two conditions can reduce potential loss of vision. Diabetes can also contribute to the development of many other eye conditions, including cranial nerve abnormalities (such as isolated third, fourth, or sixth cranial nerve palsies) as well as acute disc edema and mucormycosis (a rare but life-threatening orbital infection). Finally, patients with diabetes are at increased risk for cataracts (posterior subcapsular cataracts) and often expe¬ rience sudden shifts in refraction due to acute hyperglycemia. Thyroid ophthalmopathy is clinically apparent in -50% of patients with Graves disease.40 Clinical signs range from asymp¬ tomatic exophthalmos to vision-threatening optic neuropathy. Intensive medical or surgical treatment is required in only -4% of cases.40 Most patients with Graves disease who have no readily apparent eye findings can be demonstrated to have more subtle eye changes when examined with orbital ultrasound or magnetic resonance imaging. Any patient with Graves disease who complains of eye discomfort or decreased vision should promptly undergo a complete eye examination. With the exception of diabetes mellitus and Graves disease, all other endocrine disorders that involve the eyes are less common and, in most cases, less pervasive. However, all aspects of the eye and ocular adnexa can be affected by endocrine conditions. Knowledge of these associations and observation of these ocular findings can serve as important clues to what may be an otherwise diagnostically elusive condition. For example, the presence of a subluxated lens can confirm the diagnosis of Marfan syndrome, and a Kayser-Fleischer ring can help in the diagnosis of Wilson disease. A description of ocular signs that are associated with endo¬ crine conditions is provided here, beginning from the ocular adnexa and proceeding from the front to the back of the eye. The endocrine and metabolic disorders that are associated with each of these eye signs are summarized in Table 215-1. It should be noted that the differential diagnosis of these eye signs includes many ocular and systemic conditions that are not included here.

ORBIT

FIGURE 215-2. Whorl-like opacities (arrow) seen in the corneal epithelium as seen in patients with Fabry disease or with tamoxifen administration.

Endocrine and metabolic conditions are often associated with changes in the location of the globe in relationship to the bony cavity that contains it. The most common cause of both unilat¬ eral and bilateral exophthalmos, or protrusion of the eye, is thy¬ roid ophthalmopathy. Hypothyroidism and several other disorders are also associated with enophthalmos, or recession of the globe within the orbit. Changes in the distance between the two orbits can also be affected by endocrine conditions. Hypertelo¬ rism is increased separation between the bony orbits, and hypotelorism is decreased separation between the orbits. Changes in the size and shape of the osteal bones and their cavities are also associated with systemic disease. Endocrine and metabolic conditions are associated with deep-set eyes, a promi¬ nent supraorbital ridge, and hypertrophy of the orbital bones. Addi¬ tional associations are osteolysis of the bony orbit, erosion of the lateral wall of the optic canal, and enlargement of the superior orbital fissure.

1 962

PART XIV: INTERRELATIONSHIPS BETWEEN HORMONES AND THE BODY

TABLE 215-1. Ocular Conditions and Their Endocrine and Metabolic Associations ORBIT Exophthalmos

Brown tumor of hyperparathy¬ roidism Cretinism (hypothyroidism) Cushing syndrome Graves disease Hunter syndrome (MPS II-H) Hurler syndrome (MPS I-H) Hypophosphatasia (phosphoethanolaminuria) Obesity Pseudotumor cerebri Scheie syndrome (MPS I-S) Thyroid disorder Enophthalmos

Cretinism (hypothyroidism) Maple syrup urine disease (branched-chain ketoaciduria) Morquio syndrome (MPS IV) Hypertelorism

Cretinism (hypothyroidism) Ehlers-Danlos syndrome Hunter syndrome (MPS Il-H) Hurler syndrome (MPS I-H) Infantile hypercalcemia with sup¬ ravalvular aortic stenosis (Wil¬ liams syndrome) Maple syrup urine disease Marfan syndrome Morquio syndrome (MPS IV) Hypotelorism

Maternal phenylketonuria fetal defects Turner syndrome Deep-Set Eyes

Lowe syndrome (oculocerebrore¬ nal syndrome) Marfan syndrome Prominent Supraorbital Ridge

Hurler syndrome (MPS I-H) Marfan syndrome Hypertrophy of Orbital Bones

Acromegaly Osteolysis of Bony Orbit

Hyperparathyroidism Erosion of Optic Canal (Lateral Wall)

Craniopharyngioma Pituitary tumor Enlargement of Superior Orbital Fissure

Pituitary neoplasm Extraocular Muscle Enlargement on Computed Tomography (CT)

Acromegaly Graves disease Orbital Bruit (Bilateral)

Hyperthyroidism Ptosis

Abetalipoproteinemia Addison disease Corticosteroid ptosis (prolonged use of topical corticosteroids) LIDS Ptosis

Cretinism Eclampsia and preeclampsia

Ehlers-Danlos syndrome Graves disease Hunter syndrome Hurler syndrome (MPS I-H) Hyperparathyroidism Hyperthyroidism Hypocalcemia Hypoparathyroidism Laurence-Moon-Biedl syndrome Maple syrup urine disease Morquio syndrome Normal pregnancy Obesity (floppy eyelid syndrome) Homer Syndrome

Pituitary tumor Lid Retraction

Graves disease Dalrymple sign—widening of palpebral fissure Stellwag sign—retraction of upper lid associated with infrequent or incomplete blinking Lid Lag

Excess intake of thyroid hormone Graves disease Ectropion

Congenital ectropion Lowe syndrome Facial Palsy

Diabetes mellitus (Willis syndrome) Blepharospasm

Addison disease Hypocalcemia (in hypoparathy¬ roidism) Infrequent Blinking

Thyrotoxicosis (Stellwag sign) Including exophthalmic ophthal¬ moplegia Lid Edema

Angioneurotic edema caused by corticosteroids Hyperthyroidism Stasis, including premenstrual edema Mongoloid Obliquity

Laurence-Moon-Biedl syndrome Prader-Willi syndrome Epicanthus

Abetalipoproteinemia Ehlers-Danlos syndrome Hurler syndrome Infantile hypercalcemia Laurence-Moon-Biedl syndrome Lowe syndrome Turner syndrome Hyperpigmentation

Adrenocorticotropic hormone (ACTH) therapy ACTH-secreting pituitary tumors Addison disease Estrogen therapy Gaucher disease (cerebroside lipi¬ dosis) Hemochromatosis Melasma Niemann-Pick disease (essential lipid histiocytosis)

Porphyria cutanea tarda Pregnancy Wilson disease Hypopigmentation

Corticosteroids Fanconi syndrome Homocystinuria Hyperthyroidism (Graves disease) Hypopituitarism Phenylketonuria Vitiligo (in autoimmune adrenal or thyroid disease) Xanthelasma

Diabetes mellitus Hyperlipemia Thickened Eyelids

Acromegaly Congenital hypothyroidism Trichomegaly (Long Lashes)

Isolated adrenal malfunction and ovarian atrophy Madarosis (Loss of Lashes)

Ehlers-Danlos syndrome Hyperthyroidism Hypocalcemia Hypoparathyroidism Hypothyroidism Pituitary insufficiency Pituitary necrosis (SimmondsSheehan syndrome) Poliosis

Albinism Wemer syndrome (progeria of adults) Coarse Eyebrows

Congenital hypothyroidism (cretin¬ ism) CPD syndrome (chorioretinopathy and pituitary dysfunction) Hunter syndrome (MPS II) Hurler syndrome (MPS I) Sanfilippo syndrome (MPS III) Hertogh Sign (Loss of Outer Third of Eyebroiv)

Hypogonadism Hypothyroidism LACRIMAL SYSTEM Dry Eye

Multiple mucosal neuromas Pheochromocytoma Bloody Tears

Vicarious menstruation with ectopic tissue Excessive Tears

Morquio syndrome (MPS IV) Thyrotoxicosis EXTRAOCULAR MUSCLES Strabismus

Arylsulfatase A deficiency syndrome Diabetes mellitus Ehlers-Danlos disease Gangliosidosis Infantile (GM,) Juvenile (GM2) Gaucher disease Gout Graves disease

Homocystinuria Hurler disease (mucopolysaccha¬ ridosis type I) Hutchinson syndrome (adrenal cortex neuroblastoma with orbital metastasis) Hypocalcemia Infantile hypercalcemia with sup¬ ravalvular aortic stenosis (Wil¬ liams syndrome) Laurence-Moon-Biedl syndrome Lowe syndrome Marfan syndrome Maple syrup urine disease Prader-Willi syndrome (hypoto¬ nia-obesity syndrome) Pseudohypoparathyroidism Tay-Sachs disease (familial amau¬ rotic idiocy) Cyclic Strabismus

Graves disease Vertical Nystagmus

Diabetes mellitus Nystagmus

Abetalipoproteinemia Chediak-Higashi syndrome (anom¬ alous leukocytic inclusions with constitutional stigmata) Eclampsia and preeclampsia Gangliosidosis (generalized gan¬ gliosidosis, infantile) Hermansky-Pudlak syndrome (oculocutaneous albinism and hemorrhagic diathesis; MPS I-H) Hurler syndrome Hypervitaminosis D and other forms of hypercalcemia Hypothyroidism (cretinism) Laurence-Moon-Biedl syndrome (retinal pigmentosa-polydactyly-adiposogenital syn¬ drome) Lowe disease (oculocerebrorenal syndrome) Marfan syndrome Obesity (cerebral-ocular-skeletal anomalies syndrome) Ocular albinism (Nettleship-Falls and Forsius-Eriksson types-Xlinked) Prader-Willi syndrome (hypoto¬ nia-obesity syndrome) Pseudohypoparathyroidism Tay-Sachs disease Werner syndrome (progeria of adults) Wilson disease (hepatolenticular degeneration) Pendular Nystagmus

Albinism Laurence-Moon-Biedl syndrome (retinal pigmentosa-polydactyly-adiposogenital syn¬ drome) Periodic Alternating Nystagmus

Diabetes mellitus Ocular Bobbing

Encephalopathy of GM2 gangli¬ osidoses (continued)

1963

Ch. 215: The Eye in Endocrinology TABLE 215-1. Ocular Conditions and Their Endocrine and Metabolic Associations (Continued) Paralysis of Third Cranial Nerve Cavernous sinus syndrome Pituitary adenoma (lateral extension) Diabetes (usually pupil-sparing) Paralysis of Fourth Cranial Nerve Intracranial

Soft Globe Diabetic coma

Corneal Opacity, Diffuse Cystinosis

SCLERA

Fabry disease

Episcleritis

GMj gangliosidosis type I deficiency

Addison disease Scleritis

Hurler syndrome Maroteaux-Lamy syndrome (MPS VI)

Cretinism (hypothyroidism)

Morquio syndrome

Craniopharyngioma

Gout

Mucopolysaccharidosis

Diabetes

Porphyria cutanea tarda

Multiple sulfatase deficiency

Paralysis of Sixth Cranial Nerve

Staphyloma of Sclera

Cretinism

Ehlers-Danlos syndrome

Pseudo-Hurler polydystrophy (mucolipidosis III)

Diabetes mellitus

Hyperparathyroidism

Scheie syndrome

Gaucher disease (cerebroside lipidosis)

Marfan syndrome associated with myopia

Sialidosis, Goldberg type

Massive pituitary adenoma External Ophthalmoplegia Abetalipoproteinemia

Porphyria cutanea tarda Blue Sclera

Comeal Opacification in Infancy Corneal lipidosis Lowe syndrome

Diabetes mellitus (Willis disease)

Ehler-Danlos syndrome

Refsum syndrome (phytanic acid a-hydroxylase deficiency)

Hallermann-Streiff syndrome (bird-faced dwarf)

Generalized gangliosidosis (GM, gangliosidosis I and II)

Hypophosphatasia (phosphoethanolaminuria)

Lipomucopolysaccharidosis

Internuclear Ophthalmoplegia Fabry disease (a-galactocerebrosidase deficiency) Painful Ophthalmoplegia Diabetic ophthalmoplegia Double Elevator Palsy Graves disease Transient Ophthalmoplegia Wilson disease Poor Convergence Graves disease (Mobius sign) Paralysis of third nerve (see earlier) Divergence Paralysis Diabetes mellitus (vascular disease) CONJUNCTIVA Congestion of Conjunctiva Gout Hyperparathyroidism Hypoparathyroidism Hypothyroidism Chronic Mucopurulent Conjunctivitis Gout Conjunctival Aneurysms, Varicosities, Tortuosities, and Telangiectasias

Lowe syndrome Marfan syndrome Osteogenesis imperfecta Phenylketonuria Pseudohypoparathyroidism Pseudoxanthoma elasticum Scleral thinning secondary to scleritis Werner syndrome (adult progeria) Dilated Episcleral Vessels Endocrine exophthalmos of rapid development Scleral Rigidity Decreased scleral rigidity Graves disease

Mucolipidosis

Pseudo-Hurler polydystrophy Mucopolysaccharidoses Hurler syndrome Maroteaux-Lamy syndrome

Interstitial Keratitis Incontinentia pigmenti Corneoscleral Keratitis Gout Marginal Corneal Ulcers Gout Pigmentation of Cornea Metallic pigmentation Wilson disease (KayserFleischer ring) Yellow discoloration Tangier disease Anterior Embryotoxon Alport syndrome (hereditary nephritis-deafness syn¬ drome) Familial hypercholesterolemia Staphyloma of Cornea Occurs in advanced keratoconus (see earlier) Trigger Mechanisms for Recurrent Herpes Simplex Keratitis Menses Comeal Disease Associated with Lenticular (Lens) Problems

Morquio syndrome

Addison disease

Scheie syndrome

Apert syndrome

Riley-Day syndrome

Cataracts

Von Gierke disease

Corneal ulcers

Comeal Opacity, Localized Fucosidosis Cornea Verticillata

Ehlers-Danlos syndrome Hypoparathyroidism Keratic moniliasis

Fabry disease

Keratoconjunctivitis

Tamoxifen administration

Lowe syndrome

Comeal Crystals

Marfan syndrome

Increased scleral rigidity

Cystinosis syndrome

Mucolipidosis IV

Diabetes mellitus

Gout

Pseudohypoparathyroidism

Hyperthyroidism

Refsum syndrome

CORNEA Microcomea Ehlers-Danlos syndrome Laurence-Moon-Biedl syndrome Marfan syndrome Megalocomea

Cornea Plana Marfan syndrome Keratoconus Apert syndrome (acrocephalosyndactylia) Ehlers-Danlos syndrome

Sanfilippo syndrome (MPS III) Scheie syndrome Werner syndrome Comeal Disease Associated zvith Retinal Problems Apert syndrome

Diabetes

Lowe syndrome

Fabry disease (diffuse angiokeratosis)

Marfan syndrome

Laurence-Moon-Biedl syndrome

Cystinosis

Osteogenesis imperfecta

Marfan syndrome

Ehlers-Danlos syndrome

Pseudoxanthoma elasticum

Fabry disease

Conjunctival Xerosis Sjogren syndrome (keratoconjunc¬ tivitis sicca), seen in association with autoimmune thyroid disease Vitamin A deficiency (seen in pregnancy) Subconjunctival Hemorrhage Diabetes (due to fragility of vessel walls) Ehlers-Danlos syndrome Conjunctival Hyperpigtnenta tion Addison disease

Scheie syndrome (MPS I-S) Hyperplastic Corneal Nerves Multiple endocrine neoplasia type 2B Increased Visibility of Comeal Nerves Refsum syndrome Comeal Anesthesia Diabetes mellitus Metachromatic leukodystrophy (arylsulfatase-A deficiency) Nephropathic cystinosis Band-Shaped Keratopathy

GLOBE

Gout

Microphthalmia

Hypercalcemia

Colobomatous microphthalmia Laurence-Moon-Biedl syndrome Noncolobomatous microphthalmia Lowe syndrome Buphthalmos Hurler syndrome Lowe syndrome

Hyperparathyroidism Hypophosphatasia Paget disease Renal failure, associated with Fanconi syndrome (cystinosis) Sclerocomea Hurler syndrome

Punctate Keratitis

Hunter syndrome

CRST syndrome (Calcinosis, Raynaud phenomenon, Sclerodactyly, Telangiectasia)

Hurler-Scheie syndrome

Diabetes mellitus

Hyperparathyroidism

Graves disease

Idiopathic hypercalcemia

Hypothyroidism

Marfan syndrome

Riley-Day syndrome

Mucolipidosis IV

Filamentary Keratitis Diabetes mellitus Keratitis Sicca Autoimmune thyroid disease Diabetes mellitus Anterior Comeal Mosaic Endocrine exophthalmos Comeal Dermoids Incontinentia pigmenti Pannus Hypothyroidism

Hurler syndrome Hyperlipoproteinemia

Porphyria cutanea tarda Refsum syndrome Scheie syndrome Von Gierke disease (glycogen storage disease type I) Werner syndrome INTRAOCULAR PRESSURE Hypotony Diabetes mellitus Homocystinuria Morquio syndrome

(continued)

1964

PART XIV: INTERRELATIONSHIPS BETWEEN HORMONES AND THE BODY

TABLE 215-1. Ocular Conditions and Their Endocrine and Metabolic Associations (Continued) Glaucoma Acromegaly Cretinism

Heterochromia Incontinentia pigmenti Iris Atrophy

Albinism

Diabetic retinopathy

Diabetes mellitus

Toxemia retinopathy of preg¬ nancy

Cushing syndrome

Diabetes mellitus

Fabry disease

Diabetes mellitus

Homocystinuria

Galactokinase deficiency

Hurler syndrome

Galactose-transferase deficiency

Ehlers-Danlos syndrome Homocystinuria Hunter syndrome Hurler syndrome

LENS Anterior Subcapsular Cataracts Addison disease

Hyperthyroidism

Albinism

Lowe syndrome

Diabetes mellitus

Marfan syndrome

Hypothyroidism

Multiple endocrine neoplasia type 1

Tyrosinosis

Scheie syndrome

Wilson disease

Sulfite oxidase deficiency Elevated Intraocular Pressure in Upgaze Graves ophthalmopathy Glaucoma Associated with Shal¬ low Anterior Chamber Cystinosis Conditions Simulating Congenital Glaucoma Cystinosis Familial lipidosis Glycogen storage disease type I Hurler disease Incontinentia pigmenti Maroteaux-Lamy disease Morquio syndrome

Werner syndrome Nuclear Cataracts Maple syrup urine disease Von Gierke disease (glycogen stor¬ age disease type I) Lamellar Cataracts

Punctate Cataracts Cretinism Galactokinase deficiency

Morquio syndrome Refsum disease Homer syndrome Afferent Pupillary Defect Diabetic retinopathy IRIS Aniridia Homocystinuria syndrome Iris Coloboma Laurence-Moon-Biedl syndrome Marfan syndrome Rubeosis Iridis Diabetes mellitus

Diabetes mellitus

Marfan syndrome

Fabry disease

Prader-Willi syndrome (hypogenital dystrophy with diabetic ten¬ dency) Refsum disease

Lowe syndrome

Tyrosinosis

Pseudohypoparathyroidism

Wilson disease

Posterior Subcapsular Cataract

Zellweger syndrome (cerebrohepatorenal syndrome) Spasm of Accommodation Diabetes mellitus Paresis of Accommodation

Parafoveal Telangiectasia Diabetes mellitus Aneurysmal Dilation of Retinal Vessels Diabetes mellitus Neovascularization of the Retina Diabetes mellitus Ehlers-Danlos syndrome Generalized Arterial Narrowing Adrenal tumor hyperaldosteronism Cushing disease Hunter syndrome Jansky-Bielschowsky disease (gangliosidosis GM2 type III)

Incontinentia pigmenti

After pregnancy

Pheochromocytoma

Laurence-Moon-Biedl syndrome

Diabetes mellitus

Sanfilippo syndrome

Lactation VITREOUS

Tay-Sachs disease (amaurotic familial idiocy)

Vitreous Hemorrhage

Toxemia of pregnancy

Werner syndrome

Marfan syndrome

Maple syrup urine disease

Hypercalcemia

Refsum disease

Lowe syndrome

Mannosidosis

Diabetes mellitus Macular Edema

Passow syndrome (congenital syringomyelia)

Mucopolysaccharidoses

Diabetes mellitus

Lowe syndrome

Diabetic retinopathy Hard Exudates

Marfan syndrome

Marfan syndrome

Argyll Robertson pupil

Laurence-Moon-Biedl syndrome

Osteogenesis imperfecta congenita

Hypoparathyroidism

Miosis

Incontinentia pigmenti

Angioid streaks

Mannosidosis

Hyphema

Diabetic ophthalmoplegia (usually spares pupillary fibers)

Hypophosphatasia

Hypophosphatasia

Fabry disease

Fixated Pupil

Hypoparathyroidism

Incontinentia pigmenti Macular Hemorrhage in Young Adults

Hunter syndrome

ANTERIOR CHAMBER

Lowe syndrome

Hypoglycemia (in infants)

Diabetes mellitus

Hurler syndrome

Diabetes mellitus

Mydriasis

Hypercalcemia

Retinovitreal Hemorrhage in Young Adults

Multiple sulfatase deficiency

Scheie disease

PUPIL

Homocystinuria

Diabetes mellitus Porphyria cutanea tarda

Morquio syndrome

Abetalipoproteinemia

Iris Processes

Hagberg-Santavuori syndrome (hereditary encephalopathy, microcephaly, optic atrophy)

Dilated Retinal Veins and Retinal Hemorrhages

Galactokinase deficiency

Riley-Day syndrome

Diabetes mellitus

Cotton-wool spots

Cretinism

Iridescent Crystalline Deposits in Lens Cretinism Hypocalcemia Posterior Lenticonus Lowe syndrome Microphakia or Spherophakia Homocystinuria Hyperlysinemia Lowe syndrome Marfan syndrome Dislocated Letts Ehlers-Danlos syndrome Homocystinuria Hyperlysinemia Marfan syndrome Pseudoxanthoma elasticum Sulfite oxidase deficiency Lenticular Disease Associated with Corneal Problems Diabetes mellitus Fabry disease Wilson disease Syndromes and Diseases Associated with Cataracts

Diabetes mellitus RETINA Anatomic Classification of Macular Diseases Nerve fiber-ganglion cell layers Goldberg disease (fl-galactosidase deficiency)

Zellweger syndrome (cerebrohepatorenal syndrome) Retinitis or Pseudoretinitis Pig¬ mentosa Abetalipoproteinemia Cystinosis Haltia-Santavuori syndrome (neu¬ ronal ceroid lipofuscinosis)

Jansky-Bielschowsky disease (gangliosidosis GM2 type III)

Hunter syndrome (MPSII)

Mucolipidoses

Hurler syndrome (MPS I)

Sphingolipidoses

Hypophosphatasia

Vogt-Spielmeyer disease (heredi¬ tary cerebroretinal degenera¬ tion, mental retardation)

Infantile phytanic acid storage disease

Outer plexiform layer Cystoid macular degeneration Diabetes mellitus Lipid deposits in macula sec¬ ondary to vascular disease in retina Diabetes mellitus Bruch membrane Angioid streaks

Kearns-Sayre syndrome (external ocular muscle myopathy, blepharoptosis, mental defi¬ ciency, heart block, and occa¬ sional hypothyroidism) Laurence-Moon-Biedl syndrome Mucolipidosis IV Multiple sulfatase deficiency Pseudohypoparathyroidism Refsum disease

Acromegaly

Sanfilippo syndrome (MPS III)

Abetalipoproteinemia

Ehlers-Danlos syndrome

Scheie syndrome (MPS I-S)

Addison syndrome

Pseudoxanthoma elasticum

Stock-Spielmeyer-Vogt syndrome

(continued)

»

Ch. 215: The Eye in Endocrinology

1965

TABLE 215-1. Ocular Conditions and Their Endocrine and Metabolic Associations (Continued) Retinal "Sea Fans" Diabetes mellitus Incontinentia pigmenti Retinal Vascular Tumors and Angi¬ omatosis Retinal Syndromes Pheochromocytoma Retinal Vascular Tortuosity Fabry disease Maroteaux-Lamy syndrome (MPS type VI) Lipemia Retinalis

Tay-Sachs disease (gangliosidosis GM2 type I) White or Yellow Flat Macular Lesion and Pigmentary Change Diffuse leukoencephalopathy Gaucher disease Macular Pucker Diabetes mellitus Pigmented Fundus Lesions Abetalipoproteinemia Incontinentia pigmenti

Ischemic Infarcts of Choroid (Elschnig Spots) Toxemia of pregnancy Choroidal Ischemia Toxemia of pregnancy Choroidal Folds Graves disease Choroidal Hemorrhage

Hypophosphatasia Hypopituitarism Infantile type neuronal ceroid lipofuscinosis Laurence-Moon-Biedl syndrome Maple syrup urine disease Maroteaux-Lamy disease Metachromatic leukodystrophy

Diabetes mellitus

Morquio syndrome

Ehlers-Danlos syndrome

Mucolipidosis IV

Choroidal Detachment

Niemann-Pick disease

Diabetes mellitus with hyper¬ lipemia

Laurence-Moon-Biedl syndrome

Diabetes mellitus

Passow syndrome

Lignac-Fanconi syndrome

Toxemia of pregnancy

Pituitary gigantism

Hypothyroidism, untreated

Refsum disease

Idiopathic hypercalcemia Primary hyperlipoproteinemia

Pale Fundus Lesions Generalized pallor

Progressive lipodystrophy

Albinism

Secondary hyperlipoproteinemia

Fabry disease

Central Retinal Vein Occlusion Diabetes mellitus Cholesterol Emboli of Retina Diabetes mellitus Retinal Artery Occlusion Fabry disease Homocystinuria Cherry-Red Spot in Macula (3-Galactosidase deficiency Hurler syndrome Lipomucopolysaccharidoses Multiple sulfatase deficiency Sphingolipidoses Farber syndrome (congenital soft tissue nodules, muscle hypotonia, mental retar¬ dation with ceramide dep¬ osition) Gangliosidosis GM type 2 Gaucher disease Goldberg syndrome Infantile metachromatic dys¬ trophy Niemann-Pick disease

Gaucher disease

Ceroid lipofuscinosis Stock-Spielmeyer-Vogt syndrome Pigmentary Changes in Macula Abetalipoproteinemia

Cystinosis

Riley-Day syndrome Sanfilippo disease Stock-Spielmeyer-Vogt syn¬ drome Tay-Sachs disease

Lipidoses

Von Gierke disease (glycogen storage disease type I)

Congenital

Endocrine exophthalmos

Gangliosidosis GM2 type III

Pseudotumor cerebri

Kufs disease (inherited pro¬ gressive mental deterio¬ ration with ceroidlipofuscin deposition systemically)

Addison disease

Stock-Spielmeyer-Vogt syn¬ drome Tay-Sachs disease Localized pale areas Hypercholesterolemia Incontinentia pigmenti Laurence-Moon-Beidl syn¬ drome Lignac-Fanconi syndrome Refsum syndrome Retinal Detachment Apert syndrome Ehlers-Danlos syndrome Homocystinuria Incontinentia pigmenti Norrie syndrome (X-linked blind¬ ness, deafness, and mental retardation)

Bull's Eye Macular Lesion

Conditions Simulating Posterior Uveitis in Children

Refsum disease

OPTIC NERVE

Stock-Spielmeyer-Vogt syn¬ drome

Albinism

Homocystinuria

Porphyria cutanea tarda

Papilledema

Marfan syndrome

Macular Hypoplasia

Glaucoma

Hyperlipemia

Sandhoff disease (ganglioside storage disorder)

Tay-Sachs disease

Syndromes Associated with Uveitis

Toxemia of pregnancy Warburg syndrome (recessive hydrocephalus, cryptorchid¬ ism, micropenis, and eye abnor¬ malities) Peripheral Retinal Degeneration Cystinosis Crystalline Retinopathy Cystinosis

Vogt-Spielmeyer disease

CHOROID

Gangliosidosis GM2 type 3

Angioid Streaks

Zollinger-Ellison syndrome Optic Nerve Hypoplasia

Diabetes mellitus

Albinism

Fabry disease

Children of diabetic mothers

Hunter syndrome

Osteogenesis imperfecta

Hyperparathyroidism

Pituitary abnormalities

Hypocalcemia

Diabetes insipidus

Hypoparathyroidism

Growth retardation

Idiopathic hypercalcemia Maroteaux-Lamy syndrome Menarche Menses

Neonatal hypoglycemia Neovascularization of Optic Disc Incontinentia pigmenti achromians

Mucolipidosis III

VISUAL DISTURBANCE

Obesity

Acquired Myopia

Oral contraceptives

Albinism

Pregnancy

Diabetes mellitus

Pseudohypoparathyroidism

Hypothyroidism

Systemic corticosteroids Optic Neuritis

Toxemia of pregnancy Myopia

Diabetes mellitus

Ehlers-Danlos syndrome

Hyperthyroidism

Homocystinuria

Hypoparathyroidism

Laurence-Moon-Biedl syndrome

Hypothyroidism

Marfan syndrome

Ischemic Optic Neuropathy Diabetes mellitus Optic Nerve Drusen Diabetes mellitus

Scheie syndrome Acquired Hyperopia Diabetes mellitus Cortical Blindness

Pituitary tumor

Galactosemia

Pseudoxanthoma elasticum

Pompe disease (glycogen storage disease type II)

Optic Atrophy Abetalipoproteinemia Albinism

Tay-Sachs disease Blindness in Childhood

Cretinism

Laurence-Moon-Biedl syndrome

Cushing syndrome

Marfan syndrome

Diabetes mellitus

Acromegaly

Metachromatic leukodystrophy

Hunter disease (MPS II)

Galactosylceramide lipidosis

Hurler syndrome (MPS I-H)

Diffuse lipomatoses

Niemann-Pick disease

Gangliosidosis GM, type 2

Refsum disease (phytanic acid storage disease)

Ehlers-Danlos syndrome

Gangliosidosis GM2 type 3 Homocystinuria

Sandhoff disease (ganglioside storage disorder with GM, accumulation)

Sanfilippo syndrome (MPS III)

Pseudoxanthoma elasticum Choroidal Neovascularization

Hunter syndrome

Night Blindness

Scheie syndrome (MPS I-S)

Angioid streaks

Hurler syndrome

Refsum syndrome

Tangier disease

Osteogenesis imperfecta

Hyperparathyroidism

Retinitis pigmentosa

(Adapted from Roy FH, ed. Ocular differential diagnosis, 5th ed. Philadelphia: Lea & Febiger, 1993.)

1 966

PART XIV: INTERRELATIONSHIPS BETWEEN HORMONES AND THE BODY

Besides the globe, the orbit contains nerves, fat, blood vessels, and the extraocular muscles. Several endocrine conditions, such as thyroid ophthalmopathy and acromegaly, cause enlargement of the extraocular muscles that is seen on orbital imaging. Another orbital sign that requires a special test for detection is an orbital bruit. This is the audible flow of blood that can be heard with a stethoscope held over the orbit. It is associated with hyperthyroidism. LIDS Endocrine and metabolic disorders associated with abnormali¬ ties of the eyelids are particularly common. In addition, of all ocular abnormalities, those of the lids are among the most easily detected by the internist because they are signs that can be noted on general inspection without the use of specialized ophthalmic equipment such as the slit lamp or indirect ophthalmoscope. Many of the changes of the lids are only cosmetically impor¬ tant. More serious, mostly because they threaten the health of the underlying cornea, are abnormalities that change the rela¬ tionship of the lids to the globe and abnormalities in a patient's blink. Ptosis, or droopiness of the upper lid, if severe, can impair a person's visual field. Horner syndrome, which is caused by paralysis of the sympathetic nerve supply, is manifested by a droopy eyelid and a pupil that is smaller than the pupil of the fellow eye. The disparity in pupil size is greater in dim illumina¬ tion. One cause of Horner syndrome is a pituitary tumor. Lid retraction is present when the lower eyelid exposes sclera, or the white of the eye, beneath the inferior limbus or when the upper eyelid is at or above the superior limbus. The limbus is the sclerocorneal junction. In lid lag, the eyelids briefly lag behind when the patient looks down. Both lid retraction and lid lag are com¬ mon signs in patients with Graves disease. In ectropion, the lid margin turns out from the globe. Abnormalities in blinking that are related to endocrine condi¬ tions include facial palsy, blepharospasm, and infrequent blinking. In facial palsy, which can be caused by diabetes mellitus, the patient cannot close the lids on the involved side because of paral¬ ysis of the facial muscles supplied by the seventh cranial nerve. The inability to blink leads to corneal exposure and, if not treated, can cause serious corneal damage. Blepharospasm is involuntary twitching, blinking, or closure of the eyelids. It is associated with Addison disease (see Chap. 76) and hypoparathyroidism. Mongoloid obliquity is the term used when the temporal corner of the eye is higher than the nasal corner. Epicanthus refers to a congenital fold of skin that overlies the inner canthus of the eye. It simulates the appearance of the eye turning in. Changes in the pigmentation of the lid skin have multiple endocrine associa¬ tions and include both hyperpigmentation and hypopigmentation. Growths on the lids include xanthelasma (or xanthoma), which are yellowish fatty deposits on the eyelid (Fig. 215-3). These are frequently seen in lipid disorders (see Chap. 163). Alterations in the texture, amount, growth pattern, and color of the eyebrows and lashes are also manifestations of endocrine disorders. These include trichomegaly (long lashes), madarosis (loss of lashes), and poliosis (whitening of the lashes or brows). In some conditions, especially several of the mucopolysacchari¬ doses, coarse eyebrows are noted. The Hertogh sign refers to the lack of the outer third of the eyebrows and can occur in patients with hypothyroidism (Fig. 215-4).

FIGURE 215-3. Palpebral xanthoma (xanthelasma) of the eyelid [arrow) in a patient with mild familial hypercholesterolemia.

Strabismus refers to misalignment or dissociation of the eyes (i.e., when the two eyes are not looking in the same direction). The most common examples of this are esotropia (the eye turned in) and exotropia (the eye turned out). Cyclic strabismus is a man¬ ifest strabismus that recurs regularly. Nystagmus refers to involuntary repetitive oscillating move¬ ments of the eyes in the horizontal, vertical, or rotary directions. In pendular nystagmus, the oscillations are smooth and equal in velocity in both directions. In periodic alternating nystagmus, there are rhythmic jerk-type movements in one direction for 60 to 90 seconds and then in the reverse direction for 60 to 90 seconds. Ocular bobbing is present when both eyes move together spontaneously by intermittently dropping downward a few millimeters and then returning to the straight-ahead position. Several cranial nerves are devoted to innervating the extraoc¬ ular muscles, and lesions anywhere along these nerves can have an effect on ocular movement. Paralysis of the third cranial nerve is manifested by (a) droopiness of the eyelid; (b) limitation of the ability to look up, down, and in; and (c) if the pupil is involved, a dilated, unreactive pupil (Fig. 215-5). Paralysis of the fourth cra¬ nial nerve causes superior oblique muscle palsy that results in limitation of the eye's ability to look down and in. Paralysis of the sixth cranial nerve causes a palsy of the lateral rectus muscle ' resulting in limitation of temporal gaze in that eye. Paralysis of any of these three nerves can be caused by diabetic neuropathy.

EXTRAOCULAR MUSCLES The extraocular muscles are the muscles associated with move¬ ments of the globe. Thus, pathology involving these muscles or their nerve supply is manifested by abnormalities in eye move¬ ment, including involuntary movement, weakened movement, and paralysis.

FIGURE 215-4. Loss of eyelashes and outer third of eyebrows in a patient with previously diagnosed hypothyroidism. With thyroid hor¬ mone therapy, the patient currently is euthyroid. The eyelashes did not return.

Ch. 215: The Eye in Endocrinology

1967

FIGURE 215-5. A 22-year-old woman with Cushing syndrome secondary to an adrenocorticotropic hor¬ mone-producing pituitary tumor. There is a third cra¬ nial nerve palsy on the right side due to tumor compression, as manifested by ptosis (A), a dilated pupil (B), and loss of the right eye's ability to look up, down, and medially (C). In the central picture (C), the patient is looking straight ahead. The remaining pic¬ tures are located in a position that corresponds to the direction of the patient's gaze. For example, in the upper left photograph, the patient is looking up and to the right. Note the inability of the right eye to look up.

External ophthalmoplegia, or generalized paralysis of the extraocular muscles, is manifested by a droopy eyelid and inability to move the eye. Internuclear ophthalmoplegia refers to a situation in which the ability to move one eye inward is decreased or lost, with concomitant nystagmus of the fellow eye when it tries to look outward. Double elevator palsy is characterized by limitation of upward gaze in the involved eye and a turning downward of that eye when looking straight ahead. Poor convergence refers to the inability to move the eyes inward to fixate on a close object. Divergence paralysis is an esodeviation, that is, a latent or manifest turning in of the eye that is greater at distance than at near.

CONJUNCTIVA The conjunctiva is the transparent, vascularized membrane that covers the inner surface of the eyelids and the globe adjacent to the cornea. Congestion of the conjunctiva refers to the injection of the conjunctival vessels, giving the appearance of a "red eye." Chronic mucopurulent conjunctivitis refers to chronic hypere¬ mia of the conjunctiva that is accompanied by a mucopurulent discharge.

GLOBE Congenital systemic conditions can affect the size of the globe. Microphthalmos refers to a small globe, whereas buphthalmos refers to a large globe.

SCLERA The sclera is the white, avascular supportive wall of the eye. The episclera is the vascularized connective tissue located between the sclera and the overlying conjunctiva. Episcleritis is a sectoral, although sometimes diffuse, erythema of the eyes resulting from engorgement of the large vessels that can be seen beneath the conjunctiva. These vessels blanch with the application of topical epinephrine. Episcleritis is accompa¬ nied by ocular discomfort as opposed to severe pain, which occurs in scleritis. Scleritis, or inflammation of the sclera, is manifested by engorgement of the large vessels beneath the conjunctiva that do not blanch with the application of topical phenylephrine. A staphyloma of the sclera refers to a localized area of thinned sclera that bulges outward. In blue sclera, the sclera has a blue coloration.

CORNEA The cornea is one of the parts of the eye most commonly involved by systemic disease. Endocrine and metabolic abnor¬ malities can affect the size, shape, and clarity of this normally transparent structure. Microcornea refers to a cornea that is 14 mm in diam¬ eter. The term hyperplastic corneal nerves refers to an overgrowth of corneal nerves 70 dB. Speech reception thresh¬ old is the sound intensity at which the subject begins to hear words. Speech discrimination is the ability to understand a list of unrelated words. Sensorineural hearing loss is the most common type of loss. Treat¬ ment usually involves the use of hearing aids and aural rehabilitation. (ANSI, American National Standards Institute.)

neural hearing loss as well (Fig. 216-1). Deafness is associated • with diabetes insipidus in the DIDMOAD syndrome1 (diabetes insipidus, diabetes wellitus, optic Atrophy, and deafness). In olfactory-genital dysplasia (Kallmann syndrome), anosmia is the primary feature (see Chap. 115).la Craniopharyngiomas (see Chap. 11) are congenital cysts that arise in the basisphenoid and may erode into the pituitary and hypothalamus. This tumor usually appears as a cystic mass in the sphenoid and nasophar¬ ynx; it may be detected on the basis of clinical signs or on an imaging study of the sinuses. The treatment options include transoral and transnasal resections.

PITUITARY GLAND In acromegaly, the tongue is increased in size. Also, most affected patients have a change in voice that consists of decreased pitch and a huskier sound. Occasionally, the recurrent laryngeal nerve is stretched, which causes vocal cord paralysis. If the condition does not improve spontaneously, the voice change can be treated by thyroplasty. A patient may develop diabetes insipidus as a result of trauma to the skull base secondary to an operation or a disease process that is otolaryngologic in origin. Large pituitary tumors can infiltrate the sinuses.

FIGURE 216-2. Audiogram demonstrates normal hearing in the left ear and conductive hearing loss in the right ear. Notice that the air con¬ duction levels in the right ear are separated from the bone conduction markers. The speech reception threshold corresponds to the air conduc¬ tion level. Speech discrimination scores are normal in both ears. This type of hearing loss usually is associated with some malfunction in the ossicular chain, tympanic membrane, or external ear canal. Surgical correction usually is possible. (ANSI, American National Standards Institute.)

The major interest of the otolaryngologist in pituitary disease has been patient management. The transseptal-transsphenoidal surgical approach is primarily a team procedure in which the rhinologist provides access for the neurosurgeon.2-3 This type of cooperative effort has markedly improved the management and outcome of these patients (see Chap. 23).

THYROID GLAND With hyperthyroidism (thyrotoxicosis, Graves disease), a patient may have a goiter and Graves ophthalmopathy. Often, the oto¬ laryngologist is involved in the surgical management of the oph¬ thalmopathy. Transantral orbital decompression is the most widely used surgical procedure for this condition.4 This procedure allows removal of the inferior and medial orbital walls to provide space for the excess tissue in the sinuses. No external incisions are neces¬ sary, and complications are minimal. Endoscopic decompression has been described and appears successful (see Chap. 43).5 A number of pertinent findings are associated with myxe¬ dema (hypothyroidism). A conductive hearing loss secondary to serous otitis media may be present (Fig. 216-2). Also, a sensori¬ neural hearing loss may be present. The conductive loss usually

Ch. 216: Otolaryngology and Endocrine Disease resolves with treatment, but the sensorineural loss generally persists. Generalized mucosal edema produces nasal obstruc¬ tion, thickened tongue, facial edema, hoarseness, and slowed speech. Although diagnosis is rarely a problem, if doubt arises, biopsy of the nasal mucosa can be performed and will reveal an increase in acid mucopolysaccharide content. Carcinoma of the thyroid may present as a mass in the gland, a neck mass of unknown cause, or a cause of vocal cord paralysis. Vocal cord paralysis secondary to recurrent laryngeal nerve involve¬ ment or surgical trauma often responds to thyroplasty, arytenoidectomy, or other appropriate therapy.6-7 When the tumor is under control, the customary practice is to wait 6 months before instituting therapy. If the prognosis is poor, however, and the patient is having trouble with aspiration or a weak voice, the therapy is performed immediately. Acute bacterial thyroiditis (see Chaps. 46 and 213) may be associated with a large neck mass, hoarseness, vocal cord paralysis, or a compromised airway. In such cases, tracheostomy or vocal cord injection with fat may be indicated. An infant with congenital hypothyroidism (cretinism) usually presents with a severe sensorineural hearing loss (see Fig. 216-1), a broad flat nose, and a high-pitched cry8 (see Chap. 47). Correc¬ tion of the hypothyroidism improves the voice, but the hearing loss remains. In Pendred syndrome9—a rare congenital syndrome associated with bilateral sensorineural hearing loss and a euthy¬ roid goiter—the hearing loss involves high-frequency sound pri¬ marily, is of varying severity, and is nonreversible. The goiter results from a defect in the organification of thyroid hormone. Thyroid function tests are normal, and the diagnosis is confirmed using a perchlorate washout test. No treatment is available, but genetic counseling may be appropriate.10 Another nontreatable syndrome—Hollander syndrome—is characterized by progres¬ sive sensorineural hearing loss and euthyroid goiter.

1979

duces nerve irritability, which causes laryngeal stridor, laryngospasm, and a positive Chvostek sign (see Chap. 60). Frequently, this hypoparathyroidism is a sequela of a surgical procedure in the neck. Severe hypocalcemia and hypo¬ magnesemia can cause bilateral vocal cord paralysis.14 Hypophosphatasia, as well as hyperphosphatasia, has been reported to occur in infants found to have an associated hearing loss.15

METABOLIC BONE DISEASE Several metabolic diseases of bone produce significant otolaryn¬ gologic findings, one of which is Paget disease of the bone (see Chap. 65). This is a disease process that has truly protean mani¬ festations that are revealed in middle and old age. Skull changes are relatively common, as is involvement of the temporal bone. Clinically, a significant number of these patients have hearing loss. Classically, this loss begins as a mixed hearing loss and then becomes purely sensorineural (Fig. 216-3). Moreover, these

Hertz 125

250

500

1000

2000

4000

8000

CALCIUM AND PHOSPHATE METABOLISM Patients with hyperparathyroidism may have hearing loss, dys¬ phagia, fasciculations of the tongue, tumors of the facial bones, and lesions of the oral mucosa. The hearing loss is sensorineural and nonreversible (see Fig. 216-1). The lesions seen in the facial bones are called brown tumors (osteitis fibrosa cystica). These lesions are benign, are most often located in the maxilla, and need not be excised unless they cause functional or cosmetic problems. The nodular lesion of the oral mucosa, called epulis, requires no therapy. Hyperparathyroidism is an important com¬ ponent of multiple endocrine neoplasia (MEN) (see Chap. 188). In MEN type 1, the findings may include those of hyperparathy¬ roidism, pituitary tumor, and pancreatic tumor. In MEN type 2A, hyperparathyroidism, pheochromocytoma, and medullary thyroid carcinoma (occasionally presenting as a neck mass) are found. In MEN type 2B, the findings include medullary thyroid cancer, pheochromocytoma, and neuromas involving the mucosa lining the lips, oral cavity, nose, larynx, and eyes; hyper¬ parathyroidism is rare. These neuromas are histologically benign, but their presence should alert the clinician to the possi¬ bility of a MEN syndrome. In any patient suspected of having MEN type 2A or 2B, the clinician must screen for pheochro¬ mocytoma preoperatively. If a secreting pheochromocytoma is present, anesthesia and surgery are exceedingly risky.11 Hypercalcemia that is unrelated to parathyroid malfunction may be seen with malignant lesions involving the head and neck region and with sarcoidosis (see Chap. 59). Careful head and neck examination should reveal any primary cancer. Sarcoidosis causes many characteristic findings in the head and neck region.12-13 Granular lesions of the nasal mucosa, ulcers of the larynx, neck masses, and swelling of the salivary glands are commonly seen. Hypoparathyroidism with hypocalcemia pro¬

Left ear

X

3

Speech Audiometry Type of speech signal Speech reception threshold

75

50

dB I_—— dB

Most comfortable level

R_dB I_dB

Discomfort level

R_dB L_dB

Speech discrimination scores

30

Right-% at

dB sensation level

Right_% at_dB sensation level

84

Left —Z——% at_dB sensation level Left_% at-dB sensation level

FIGURE 216-3. Audiogram reflects the type of hearing loss that is often seen with Paget disease of the bone. In the left ear, a mixed hearing loss is present: part of the loss is sensorineural arid part is conductive. Notice the separation, in the low frequencies, between air conduction and bone conduction levels on the left side. The speech reception threshold corre¬ sponds to the air conduction level, but speech discrimination remains fairly good at this point. The hearing level in the right ear is fairly typical of end-stage Paget disease of the temporal bone. This is a fairly severe sensorineural hearing loss with poor speech discrimination. In such cases, the mixed hearing loss might occur relatively early in the course of the disease, whereas the sensorineural hearing loss might occur rela¬ tively late. (ANSI, American National Standards Institute.)

1 980

PART XIV: INTERRELATIONSHIPS BETWEEN HORMONES AND THE BODY

PREGNANCY The most common otolaryngologic abnormality related to preg¬ nancy is a severe vasomotor rhinitis. This usually arises in the second or third trimester. No predisposing factors have been identified. Treatment with decongestants is of limited benefit. Another unusual finding is hoarseness, which arises from vascu¬ lar engorgement of the true and false vocal cords. An extremely dry throat—laryngitis sicca gravidarum—occasionally is seen. All of these conditions resolve after delivery. Finally, the incidence of facial paralysis is three times greater in pregnant women than in nonpregnant women; the cause for this is unknown, and treat¬ ment usually is limited to careful observation.23

DIABETES MELLITUS

FIGURE 216-4.

Radiograph showing fibrous dysplasia of the left max¬

illa. Severe cystic involvement (arrow), overall expansion of the bone, and extreme thinning of the cortex (arrowhead) are evident. Notice the asymmetry of the orbits, as well as the gross deformity of the left orbit.

patients can have tinnitus and vertigo, which seem to be related to the disease.16 Calcitonin therapy may halt the progress of the involvement.17 Osteogenesis imperfecta is a genetically induced disease with varying levels of involvement18 (see Chaps. 66, 70, and 189). Affected patients may have a conductive hearing loss sec¬ ondary to involvement of the ossicular chain. Clinically and his¬ tologically, the condition is identical to otosclerosis. Surgical treatment and use of hearing aids may be necessary. At opera¬ tion, if the incus is involved, performing a stapedectomy may not be possible. In fibrous dysplasia (see Chap. 66), the maxilla is the bone most commonly involved in the head (Fig. 216-4). Mass lesions can form anywhere, leading to cosmetic and functional prob¬ lems.19-20 These lesions can be debulked or removed, but they have a tendency to recur. Malignant transformation is rare but is more likely if the patient has been irradiated.21 Osteopetrosis (Albers-Schonberg disease) may have oto¬ logic complications as well. The involvement varies greatly from patient to patient, but the temporal bone is commonly affected.22 Temporal bone disease causes sensorineural and conductive hearing losses. Usually, these are not treatable except with a hearing aid. Moreover, facial paralysis may be seen; this tends to be recurrent, and decompression often is advised. Dental caries occurs frequently and is severe. This condition may cause osteomyelitis of the mandible, which is difficult to control.

ADRENAL CORTEX Patients with adrenal insufficiency (Addison disease) may present with sunken eyes, dry tongue, and hyperpigmentation of the skin and tongue (see Chap. 76). Endolymphatic hydrops (Meniere syndrome), dysosmia, and dysgeusia all have been reported to occur with adrenal insufficiency. Adrenocortical hyperactivity (Cushing disease) causes moon facies and promi¬ nent supraclavicular fat pads.

Sensorineural hearing loss has been reported to appear earlier and is more severe in diabetic individuals than in the normal population. When diabetic patients are compared with a general population of the same age and sex, however, no difference in hearing levels is found.24-25 Other conditions that have been thought to be more prevalent in diabetic persons are Meniere syndrome, facial nerve palsy (Bell palsy), and vocal cord paral¬ ysis, but none of these has been conclusively shown to be more common in these patients than in the population as a whole. Infections are a great problem in diabetic individuals. Two infectious processes are unique and are reviewed in some detail. The first is malignant (necrotizing) external otitis, a disease that affects elderly diabetic patients.26-27 It is a unilateral process that begins as a routine external otitis. Despite therapy, it evolves from a soft tissue infection into an osteomyelitis of the temporal bone and eventually involves the base of the skull. Pseudomonas aeruginosa is the major pathogen. The cardinal symptoms are severe otalgia and otorrhea. Granulation tissue is present at the anterior junction of the bony and the cartilaginous external auditory canal. The clinical appearance is deceptively mild, and the course of the disease is prolonged. Once the bone is involved, cranial nerve deficits develop. The facial nerve is the one most commonly affected, but cranial nerves VI through XII can be involved. If more than one cranial nerve is affected, the prognosis is poor. The treatment must be aggressive and multi¬ faceted. Local treatment consists of placing an antibiotic-soaked wick in the ear canal. Systemic therapy uses an aminoglycoside and one of the synthetic penicillins. Prolonged therapy with a cephalosporin or fluoroquinolone may be effective.28 Treatment must be continued for at least 4 weeks, and probably for 6 weeks. Surgical management is limited to debridement of the temporal bone, usually a radical mastoidectomy. Controlling the diabetes concurrently and monitoring renal function is impera¬ tive. Treatment must be continued until all clinical evidence of the disease disappears, the erythrocyte sedimentation rate nor¬ malizes, the gallium bone scan improves, and radiographic evi¬ dence of resolution is noted.29 At one time, the mortality rate associated with this disease was 50%, but it is now around 10% (see Chap. 152). The second infection of concern is mucormycosis. The two forms seen in the head and neck region are the rhino-orbitalcerebral form and the otic form.30-33-33® The nasal form presents with blindness, ophthalmoplegia, proptosis, facial swelling, pal¬ atal ulcer, or disorders of consciousness. Examination reveals brick red or black areas within the nasal cavity. Biopsy confirms the clinical impression of mucormycosis. The otic presentation usually is accompanied by otorrhea, followed by facial paralysis and then altered sensorium. Treatment must be prompt and aggressive. The diabetes must be controlled, because ketoacido-

Ch. 217: Dental Aspects of Endocrinology sis and dehydration are invariably present at the onset of the disease. Systemic therapy is initiated with amphotericin B. Sur¬ gical debridement is more important in mucormycosis than in malignant otitis externa. Surgical management involves the removal of all necrotic tissue and the establishment of drainage from areas of infection. If the facial or optic nerve is involved, decompression is indicated. The mortality rate associated with this condition is 40%.

HYPOGLYCEMIA Reactive postprandial hypoglycemia34 has been associated with Meniere syndrome, fluctuating hearing loss, and episodic ver¬ tigo.3^ Although some authors believe that hypoglycemia plays a major role in the evolution of these symptom complexes, the objective data are not impressive. The subjective nature of the symptoms and the tendency for remission to occur make scien¬ tific evaluation difficult.

LIPID METABOLISM Hyperlipidemia causes characteristic xanthomas of the face and may be associated with sensorineural hearing loss.36 The view once was that patients with Meniere syndrome, fluctuating hearing loss, episodic imbalance, and premature hearing loss often had hyperlipidemia.37 Most of those studies had flaws in their design, and hyperlipidemia probably is not a significant factor in these conditions. In abetalipoproteinemia, which is a rare recessive disorder, the presenting features include ataxia, acanthocytosis, and sensorineural hearing loss.38

REFERENCES 1. Ito H, Takamoto T, Nitta M, et al. DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy and deafness) syndrome associated with myocardial disease. Jpn Heart J 1988; 29:371. la. Jenkin A, Renner D, Hahn F, Larsen J. A case of primary amenorrhea, diabe¬ tes and anosmia. Gynecol Endocrinol 2000; 14:65. 2. Laws ER Jr, Thapar K. Surgical management of pituitary adenomas. Baillieres Clin Endocrinol Metab 1995; 9:391. 3. Carrau RL, Jho HD, Ko Y. Transnasal-transsphenoidal endoscopic surgery of the pituitary gland. Laryngoscope 1996; 106:914. 4. Hamer SG. Orbital decompression techniques. In: Gorman CA, Waller RR, Dyer JA, eds. The eye and orbit in thyroid disease. New York: Raven Press, 1984:221. 5. Metson R, Shore JW, Gliklich RE, Dallow RL. Endoscopic orbital decom¬ pression under local anesthesia. Otolaryngol Head Neck Surg 1995; 113:661. 6. Bauer CA, Valentino J, Hoffman HT. Long-term result of vocal cord aug¬ mentation with autogenous fat. Ann Otol Rhinol Laryngol 1995; 104:871. 7. Netterville JL, Aly A, Ossoff RH. Evaluation and treatment of complications of thyroid and parathyroid surgery. Otolaryngol Clin North Am 1990; 23:529. 8. Chaouki ML, Maoui R, Benmiloud M. Comparative study of neurological and myxoedematous cretinism associated with severe iodine deficiency. Clin Endocrinol (Oxf) 1988; 28:399. 9. Friis J, Johnsen T, Feldt-Rasmussen U, et al. Thyroid function in patients with Pendred's syndrome. J Endocrinol Invest 1988; 11:97. 10. Maisel RH, Brown DR, Ritter FN. Endocrinology. In: Paparella MM, Shumrick DA, eds. Otolaryngology, vol 1. Philadelphia: WB Saunders, 1980:779. 11. Werbel SS, Ober KP. Pheochromocytoma. Update on diagnosis, localization, and management. Med Clin North Am 1995; 79:131. 12. Krespi YP, Kuriloff DB, Aner M. Sarcoidosis of the sinonasal tract: a new staging system. Otolaryngol Head Neck Surg 1995; 112:221. 13. Benjamin B, Dalton C, Croxson G. Laryngoscopic diagnosis of laryngeal sar¬ coid. Ann Otol Rhinol Laryngol 1995; 104:529. 14. Lye WC, Leong SO. Bilateral vocal cord paralysis secondary to treatment of severe hypophosphatemia in a continuous ambulatory peritoneal dialysis patient. Am J Kidney Dis 1994; 23:127. 15. Schuknecht HF. Pathology of the ear. Cambridge, MA: Harvard University Press, 1974:172. 16. Hamer SG, Rose DE, Facer GW. Paget's disease and hearing loss. Otolaryn¬ gology 1978; 86:869. 17. El Samma M, Linthicum FH Jr, House HP, House JW. Calcitonin as treat¬ ment for hearing loss in Paget's disease. Am J Otolaryngol 1986; 7:241.

1981

18. Bergstrom L. Osteogenesis imperfecta: otologic and maxillofacial aspects. Laryngoscope 1977; 87(Suppl):l. 19. Feldman MD, Rao VM, Lowry LD, Kelly M. Fibrous dysplasia of the para¬ nasal sinuses. Otolaryngol Head Neck Surg 1986; 95:222. 20. Megerian CA, Sofferman RA, McKenna MJ, et al. Fibrous dysplasia of the temporal bone: ten new cases demonstrating the spectrum of otologic sequelae. Am J Otol 1995; 16:408. 21. Sofferman RA. Cysts and bone dyscrasias of the paranasal sinuses. In: English GM, ed. Otolaryngology, vol 2. Philadelphia: Harper & Row, 1985:13. 22. Stocks RM, Wang WC, Thompson JW, et al. Malignant infantile osteopetro¬ sis: otolaryngological complications and management. Arch Otolaryngol Head Neck Surg 1998; 124:689. 23. Hilsinger RL Jr, Adour KK. Idiopathic facial paralysis, pregnancy, and the menstrual cycle. Ann Otol Rhinol Laryngol 1975; 84:433. 24. Hamer SG. Hearing in adult-onset diabetes mellitus. Otolaryngol Head Neck Surg 1981; 89:322. 25. Duck SW, Prazma J, Bennett PS, Pillsbury HC. Interaction between hyper¬ tension and diabetes mellitus in the pathogenesis of sensorineural hearing loss. Laryngoscope 1997; 107:1596. 26. Chandler JR. Malignant external otitis and osteomyelitis of the base of the skull. Am J Otol 1989; 10:108. 27. Slattery WH 3rd, Brackmann DE. Skull base osteomyelitis. Malignant exter¬ nal otitis. Otolaryngol Clin North Am 1996; 29:795. 28. Levenson MJ, Parisier SC, Dolitsky J, Bindra G. Ciprofloxacin: drug of choice in the treatment of malignant external otitis (MEO). Laryngoscope 1991; 101:821. 29. Grandis JR, Curtin HD, Yu VL. Necrotizing (malignant) external otitis: pro¬ spective comparison of CT and MR imaging in diagnosis and follow-up. Radiology 1995; 196:499. 30. Vessely MB, Zitsch RP 3rd, Estrem SA, Renner G. Atypical presentations of mucormycosis in the head and neck. Otolaryngol Head Neck Surg 1996; 115:573. 31. Sugar AM. Mucormycosis. (Review). Clin Infect Dis 1992; l(Suppl 14):S126. 32. Harril WC, Stewart MG, Lee AG, Cernoch P. Chronic rhinocerebral mucor¬ mycosis. Laryngoscope 1996; 106:1292. 33. Gussen R, Canalis RF. Mucormycosis of the temporal bone. Ann Otol Rhinol Laryngol 1982; 91:27. 33a. Ferguson BJ. Mucormycosis of the nose and paranasal sinuses. Otolaryngol Clin North Am 2000; 33:349. 34. Betteridge DJ. Reactive hypoglycemia. BMJ 1987; 295:286. 35. de Vincentiis I, Ralli G. New pathogenetic and therapeutic aspects of Meniere's disease. Adv Otorhinolaryngol 1987; 37:97. 36. Pulec JL, Pulec MB, Mendoza I. Progressive sensorineural hearing loss, sub¬ jective tinnitus and vertigo caused by elevated blood lipids. Ear Nose Throat J 1997; 76:716. 37. Spencer JT Jr. Hyperlipoproteinemia and inner ear disease. Otolaryngol Clin North Am 1975; 8:483. 38. Liston S, Meyerhoff WH. Metabolic hearing loss. In: English GM, ed. Oto¬ laryngology, vol 1. Philadelphia: Harper & Row, 1985:9.

CHAPTER

217

DENTAL ASPECTS OF ENDOCRINOLOGY ROBERT S. REDMAN Most of the major circulating hormones are important in the nor¬ mal growth and development of the orofacial region, including the teeth, and most of them also participate in the maintenance of the health and integrity of these structures. Consequently, hormonal abnormalities commonly have dental and oral mani¬ festations. Many of these oral signs and symptoms, and the endocrinopathies and other disorders with which they may be associated, are summarized in Tables 217-1 and 217-2.

ONTOGENY OF THE OROFACIAL STRUCTURES When one considers the potential for hormonal effects on the development of mature oral structures, two phenomena must be examined. First, with regard to tooth development, the various

1 982

PART XIV: INTERRELATIONSHIPS BETWEEN HORMONES AND THE BODY

TABLE 217-1. Dental and Orofacial Abnormalities That May Be Associated with Specific Metabolic or Endocrine Disorders Dental or Oral Abnormality

Metabolic or Endocrine Disorders

TEETH Enamel hypoplasia, hypocalcifkation

Cretinism; juvenile myxedema; rickets; hypophophatasia; pseudohypophosphatasia; hypopara¬ thyroidism, including APECS; pseudohypoparathyroidism; fluorosis

To be distinguished from: amelogenesis imperfecta; results of local trauma or severe systemic illness in infancy or early childhood Dental hypoplasia, hypocalcifkation

Cretinism; juvenile myxedema; rickets; familial hypophosphatemia; hypophosphatasia and pseudohypophosphatasia; hypoparathyroidism and APECS

Short roots, occluded pulp chambers

Cretinism; myxedema

To be distinguished from: dentinogenesis imperfecta; results of trauma or illness in early childhood To be distinguished from: regional odontodysplasia Short roots, enlarged pulp chambers

Pseudohypoparathyroidism

Dental defects leading to periapical infection

Vitamin D-resistant rickets (familial hypophosphatemia)

Delayed eruption

Cretinism; myxedema; hypopituitarism; rickets

Precocious eruption

Hyperthyroidism; congenital adrenal hyperplasia; precocious puberty

To be distinguished from: dentinogenesis imperfecta; dentinal dysplasia

BONES OF THE MAXILLA AND MANDIBLE Hypocalcifkation (developmental)

Cretinism; juvenile myxedema; rickets; hypophosphatasia; pseudohypophosphatasia; familial hypophosphatemia

Hypocalcifkation (postdevelopmental)

Decreased estrogen (postmenopause); hyperparathyroidism; vitamin D deficiency; long-term inadequate dietary calcium or high dietary phosphate/calcium ratio

Loss of lamina dura

Hyperparathyroidism; vitamin D deficiency (osteomalacia and rickets); renal dialysis

Central (intraosseous) giant cell lesions

Hyperparathyroidism; renal dialysis

Underdevelopment; relative maxillary prognathism

Pituitary dwarfism; cretinism; juvenile myxedema

To be distinguished from: Down syndrome Postpubertal enlargement

Acromegaly (greater degree of enlargement in the mandible than in the maxilla)

To be distinguished from: Paget disease of bone (in which the enlargement is usually greater in the maxilla) ORAL MUCOSA Macroglossia (secondary to edema)

Hypothyroidism at any age

To be distinguished from: cellulitis; lymphangioma; amyloidosis; relative or absolute hyperplasia (true macroglossia), as in Down syndrome and acromegaly Amyloidosis ((32-microglobulin)

Renal dialysis (ABM2 amyloidosis)

To be distinguished from: all other types of amyloidosis Glossodynia; metallic taste

Diabetes; decreased estrogen (postmenopause); also occurs in conjunction with pernicious or iron deficiency anemia and ingestion of bismuth and lead

Candidiasis Acute pseudomembranous type (tongue, buccal mucosa, gingiva)

Greater incidence in diabetic patients than in normal population

Chronic hyperplastic type (tongue, buccal mucosa, and especially the labial angular commissures)

Hypoparathyroidism, APECS; hyperadrenocorticism; also occurs in immune deficiencies, includ¬ ing AIDS x

If lesions are resistant to antifungal therapy, APECS, hyperadrenocorticism (including corticoster¬ oid therapy) and hypoparathyroidism should be considered. Also occurs in immune deficien¬ cies, both congenital and acquired (AIDS).

To be distinguished from: Angular cheilosis associated with closed bite or nutritional deficiencies; premalignant dyskeratosis; carcinoma; also occurs in immune deficiencies, including AIDS Atrophic type (especially focal on tongue and hard palate)

Patchy melanin pigmentation (buccal, labial, and gingi¬ val mucosa)

Greater prevalence in diabetic patients than in general population To be distinguished from: median rhomboid glossitis; benign migratory glossitis; glossitis secondary to anemia or vitamin B deficiency, also occurs in immune deficiencies, including AIDS Hypoadrenocorticism

To be distinguished from: amalgam tattoo; bismuth or lead deposits; pigmentations associated with African or Mediterranean ancestry; nevus; melanoma; Peutz-Jeghers syndrome

Aphthae; herpes labialis

Frequently associated with menses

Hyperplastic gingivitis; pyogenic granuloma

Increased female sex hormones, as occur in pregnancy, hypergonadism, use of oral contraceptives, or estrogen therapy; diabetes

To be distinguished from: hyperplastic gingivitis due to vitamin C deficiency and medications such as phenytoin (Dilantin), cyclosporine, and nifedipine Periodontitis that is progressive despite treatment

Poorly controlled diabetes; hyper- and hypothyroidism; hyperparathyroidism; hyperadrenocorti¬ cism; renal dialysis; also occurs in immune deficiencies

SALIVARY GLANDS Xerostomia

Diabetes

To be distinguished from: postirradiation atrophy and fibrosis of salivary glands; Sjogren syndrome; and the effects of medications such as diuretics and antipsychotics Bilateral, generalized enlargement, especially the parotid glands

Diabetes

To be distinguished from: alcoholism; chronic undernutrition; Sjogren syndrome; iodine therapy (iodine mumps); bismuth ingestion; sarcoidosis

APECS, autoimmune polyendocrinopathy candidiasis syndrome; AIDS, acquired immunodeficiency syndrome.

Ch. 217: TABLE 217-2. Reported Effects of Various Endocrine Conditions on the Sense of Taste* Abnormality/Disorder

Effect

HYPOGONADOTROPIC HYPOGONADISM (KALL¬ MANN SYNDROME)

Decreased sense of taste (largely attribut¬ able to loss of sense of smell); defects are irreversible.

HYPOTHYROIDISM

Distortion of taste (dysgeusia), as well as hyposmia; decreased taste acuity, espe¬ cially to bitter stimuli. Defects are revers¬ ible with thyroid hormone therapy.

HYPERCALCEMIA

Bitter taste in mouth; reversible with nor¬ malization of serum calcium levels

PSEUDOHYPOPARATHY¬ ROIDISM

Impaired taste for sour and bitter (as well as impaired olfaction)

ADDISON DISEASE

Lowered threshold for the sensation of saltiness; reversible with corticoster¬ oid therapy

GONADAL DYSGENESIS

Variable disorders of gustatory (as well as olfactory) functions

DIABETES MELLITUS

Blunted sensation for sweetness and generalized decreased taste acuity for saltiness and bitterness; metallic taste

Dental Aspects of Endocrinology

1983

MAXILLA

Taste (gustation) is an extremely adaptive chemical sense. The thousands of taste buds are located mostly on the dorsal surface of the tongue but also are located on pala¬ tal, pharyngeal, and buccal mucosae. Taste buds consist of supporting cells and gusta¬ tory cells. The latter cells, which respond to dissolved substances, are supplied afferently by the facial nerve (anterior two-thirds of the tongue), glossopharyngeal nerve (posterior one-third of the tongue), and vagus nerve (throat); the impulses eventu¬ ally arrive in the parietal cortex, where they are intermixed with sensations of touch, temperature, and smell.

stages of odontogenesis occur at different ages for each pair of teeth. The first primary teeth are initiated at ~6 weeks after con¬ ception, whereas the last secondary teeth (the third molars) finish root formation at -20 years after birth.1 Thus, significant devia¬ tions from the normal chronology of tooth eruption (Fig. 217-1) can be an important sign of endocrinopathy, as well as of meta¬ bolic or nutritional problems. Furthermore, any part of a tooth that has completely calcified undergoes no significant further developmental changes in shape or composition. Thus, the duration, as well as the severity, of hormonal changes determine which parts of which teeth are affected. Once the teeth have fully formed and erupted, they can be altered only by destructive pro¬ cesses, such as periodontitis and caries, and their capacity for repair is limited. Therefore, abnormalities of shape or mineral¬ ization that are restricted to certain teeth also may serve as a permanent record, indicating when and for how long an endocrinopathy has been a factor (Fig. 217-2). Second, the max¬ illa, mandible, and most of the facial bones grow by intramembranous ossification, except for epiphyseal plate-like growth in a rim of hyaline cartilage on the head of each mandibular condyle. This cartilage persists in the adult.2 Thus, with appro¬ priate hormonal stimulation, during adulthood, the facial bones and both jaws can enlarge by accretion, but the mandible also can elongate from the condyles.

PITUITARY GLAND Experiments in which the incisors continuously erupt have indi¬ cated the relative importance of the pituitary and several of its target endocrine gland hormones in the production of dental and alveolar bone abnormalities in hypopituitarism.3 In hypophysectomized rats, the rate of eruption progressively slows, then ceases, and the incisors become reduced in size and mis¬ shapen. Amelogenesis, morphogenesis, and rate of eruption are largely restored by thyroxine administration, whereas dentino-

FIGURE 217-1. Chronology of eruption of the permanent, or second¬ ary, teeth. Eruption time, as depicted in this diagram, is defined as the time when the tooth first pierces the gingiva and becomes visible in the oral cavity. The numbers within the teeth designate the mean eruption times in years and months; example, the maxillary central incisors (next to the midline) erupt at the age of 7 years, 5 months in boys, and 7 years, 2 months in girls. The third molars ("wisdom teeth") contain no numbers because their eruption times are extremely variable and are, therefore, of little use as a sign of metabolic or hormonal disturbance. Of the deciduous teeth (not shown), the mandibular incisors usually erupt first, beginning at -6 months after birth, followed by the first molars, canines, and second molars. Notice that when the deciduous molars are shed, they are replaced by the permanent premolars. All of the deciduous teeth usually have erupted by the age of 2 years. Like the permanent teeth, the deciduous teeth tend to erupt earlier in girls than in boys. (Modified from Sinclair D. Human growth after birth. London: Oxford University Press, 1978:103.) genesis and alveolar bone growth nearly normalize with growth hormone supplementation. Decreased levels of adrenocortical hormones also may participate in the abnormal morphogenesis of the incisors.

HYPOPITUITARISM In pituitary dwarfism (growth hormone deficiency), eruption of both primary and secondary dentitions is delayed, and shed¬ ding of the primary teeth is delayed.3"6 The crowns of the teeth reportedly are smaller than normal, although some researchers have suggested that the crowns appear smaller only because

1984

PART XIV: INTERRELATIONSHIPS BETWEEN HORMONES AND THE BODY

FIGURE 217-3. The maxillary anterior teeth of a 52-year-old man with acromegaly are pictured. Notice the wide spaces between the teeth, as well as their anterior inclination. FIGURE 217-2. Severe hypoplasia of enamel and dentin. The parents of this 7-year-old girl were unsure of the nature of her illness in early childhood. The limitation of the dental defect to the incisal third of the secondary teeth, of which only the central incisors (c) have erupted, would be compatible with the onset of an endocrinopathy, such as hypothyroidism, shortly after birth and its subsequent diagnosis and appropriate treatment at 14 to 16 months of age. (Courtesy of Douglas J. Sanders, DDS.)

they are incompletely erupted.3 Also, the roots of the teeth are noticeably stunted.3-6 The overall growth of the jaws is retarded, with the maxilla being less affected than the mandible.3 In this condition, the alveolar (tooth-supporting) regions of both jaws grow at a disproportionately reduced rate. Consequently, the dental arches are too small to accommodate all of the teeth, caus¬ ing crowding and malocclusion.3'5'6 Hypofunction of the sali¬ vary glands also may occur, leading to increased dental caries and periodontitis.6 Adult-onset panhypopituitarism has no specific effects on the teeth, but characteristic orofacial changes include thinning of the mucosa of the lips and an immobile facial expression.3

HYPERPITUITARISM Growth hormone excess that occurs before puberty (gigantism) causes progressive, symmetric enlargement of the jaws, tongue, and teeth,3-6 and the eruption of the teeth is accelerated 3 Orofa¬ cial features of acromegaly emerge when the hyperpituitarism continues past, or begins after, 8 to 10 years of age.3-5 The jaws and facial bones enlarge disproportionately in relation to most of the bones of the skull because resumption of osseous growth is more vigorous in the intramembranous bones. Moreover, endochondral ossification resumes in the hyaline cartilage of the heads of the mandibular condyles, and the mandibular angles become flattened. This causes progressively more severe man¬ dibular prognathism. The palatal arch is flattened, and pan¬ oramic dental radiographs may demonstrate enlarged maxillary sinuses. The periosteum of the jaws may become ossified at points of attachment of the muscles and tendons. The crowns of the teeth are not enlarged, but often excessive deposition of cementum on the roots (hypercementosis) occurs. The tongue may become so large that indentations form where it encroaches on the teeth. Partly from this pressure, and partly from the enlargement of the jaws, the teeth become spaced and out¬ wardly tipped (Fig. 217-3). The nose and lips also are enlarged, adding to the general coarsening of the facial features (see Chap. 12).

Enlargement of the bones of the face and skull, and spacing and hypercementosis of the teeth can occur in osteitis deformans (Paget disease of bone).3-4 In contrast to that associated with acromegaly, however, the enlargement in this disease is limited to the bones. The lips may become thinner because of stretching, and the maxilla tends to enlarge disproportionately to the man¬ dible, producing an anterior open bite and a maxillary prog¬ nathism. Also, the jaws and skull bones are especially inclined to exhibit radiolucency (osteoclastic stage) and fuzzy (cotton¬ wool) radiodensity (osteoblastic stage).

THYROID GLAND The serum concentration of thyroid hormone is low in neonatal rats and mice but increases to adult levels between 2 and 3 weeks of age. When this rise is prevented by thyroidectomy, the weaning process, tooth eruption, and maturation of the salivary glands are all retarded but not prevented.7-9 Rodent parathyroid glands are enclosed in the poles of the thyroid gland, and surgi¬ cal removal of the thyroid eliminates both glands. Similar devel¬ opmental retardation occurs when hypothyroidism is induced by propylthiouracil, however, and is prevented with timely' replacement of thyroxine.7 This indicates that the effects are attributable to the lack of thyroxine and not to any disturbance of parathyroid hormone. Furthermore, the administration of thyroxine to suckling mice and rats induces precocious develop¬ ment of salivary glands8 as well as of the teeth.3 Also, in mature rats and mice, deficiency of thyroxine causes up to 50% reduc¬ tions in salivary flow and of gland stores of amylase, protease, and other salivary proteins.9 These findings suggest that decreased salivary function, as well as the previously observed enamel hypocalcification, may contribute to the increased den¬ tal caries that occur in juvenile hypothyroidism. Of note, the major salivary glands actively concentrate iodine from body fluids.9 This does not complicate radioiodine uptake or scanning tests of the thyroid because of the relatively small amounts used, the lesser uptake by the salivary glands, and the anatomic separation of these organs and the thyroid gland. If radioiodine is used to destroy thyroid tissue in a patient with thyroid hyperplasia, adenoma, or carcinoma, however, the sali¬ vary glands also may be seriously damaged.10'11 The resulting xerostomia may be permanent, and rampant caries and peri¬ odontal destruction follow unless long-term, specific dental care is instituted in a timely manner. Appropriate care includes scru-

Ch. 217: Dental Aspects of Endocrinology pulous oral hygiene, frequent professional dental cleanings, use of saliva substitutes for oral moistness and comfort, and the daily topical application of a fluoride gel onto the teeth.

HYPOTHYROIDISM Cretinism is characterized by maxillary prognathism because the underdevelopment of the maxilla is less severe than that of the mandible.3'6 Radiographic examination often reveals hypocalcification of the jaws, and sometimes abnormal devel¬ opment of the sinuses or nonunion of the mandibular sym¬ physis is seen. The characteristic facies consists of a concave nasal bridge and flared alae nasi; stiff facial expression, thick¬ ened lips and enlarged tongue, owing to doughy, nonpitting edema; and a mouth held partly open because of the lack of room for the tongue inside the underdeveloped mandible.3-6 The somewhat similar facies in Down syndrome arises from a disproportionately underdeveloped maxilla and a relative macroglossia that is not associated with edema.3 In both juve¬ nile myxedema and cretinism, development of the teeth is retarded, frequently with hypocalcification, enamel hypopla¬ sia (see Fig. 217-2), persistence of large pulp chambers, and open apical foramina.5 Eruption of both dentitions and shed¬ ding of the primary dentition are generally (but erratically) delayed.3-6 This, and the underdevelopment of the jaws, causes a malocclusion that may be severe and also may be complicated by spreading and flaring of the teeth secondary to pressure from the enlarged tongue. Hypothyroidism at any age seems to predispose affected patients to excessive dental caries, as well as to accelerated alve¬ olar bone loss in both dentulous areas (periodontitis) and eden¬ tulous ridges (atrophy).5-6 The increased caries and periodontitis are related to hyposalivation and to the drying effects of mouth breathing caused by the enlarged, protruding tongue.6 Adults with myxedema also develop thickened lips and a swollen tongue (from edema). Pressure from the latter, in conjunction with an exacerbation of periodontitis, may cause spreading and splaying of the teeth.3-5 Generally, the earlier that childhood hypothyroidism is treated, the greater is the success in prevent¬ ing or reversing orofacial maldevelopment, except for the affected parts of dentin and enamel that have completed all phases of development. Hypothyroid patients often are unable to tolerate prolonged dental procedures. Also, they usually have an exaggerated response to premedication with narcotics or barbiturates.5

HYPERTHYROIDISM In children, hyperthyroidism accelerates the development of the teeth and jaws, but maldevelopment is unusual.3-5 Malocclusion results occasionally when shedding of primary teeth and erup¬ tion of secondary teeth are disproportionately precocious to jaw growth. Usually, the teeth are normal in terms of size, morphol¬ ogy, and calcification. Periodontitis may begin at an unusually early age, and both caries and periodontitis reportedly are exac¬ erbated in hyperthyroidism at any age. In severe hyperthyroid¬ ism, rapid bone demineralization, manifested radiographically as osteoporosis of the jaws and loss of alveolar bone in both den¬ tulous and edentulous areas. People with hyperthyroidism are likely to be poor dental patients because they are unable to hold still for dental proce¬ dures and are likely to develop cardiac arrhythmias.3-4-6 The anx¬ iety, stress, and trauma associated with dental treatment thus may precipitate a medical emergency in the dental office. In par¬ ticular, the use of epinephrine and other vasoconstrictors in local anesthetics is contraindicated.6

1985

CALCIUM AND PHOSPHORUS METABOLISM Hypoplasia of enamel and dentin, marked chronologic devia¬ tions in eruption and exfoliation of teeth, loss of radiodensity of the jawbones (especially the lamina dura), and the presence of giant cell lesions in the jaws may alert the dentist to previously undiagnosed disorders of calcium and phosphorus metabolism. Abnormalities of mineral metabolism that produce oral signs and symptoms include disturbances of particular nutritional factors (calcium, fluoride, vitamin D) or hormones (parathyroid hormones, adrenal corticosteroids), and renal function.

NUTRITIONAL FACTORS Calcium deficiency may be an important factor in osteoporosis, which may accentuate alveolar bone loss in edentulous ridges.3 Fluoride deficiency has only one overt oral effect: a greatly increased susceptibility to dental caries. It also may contribute to osteoporosis when calcium intake is inadequate. Dental fluoro¬ sis, or mottled (hypoplastic) enamel, occurs with increasing fre¬ quency and severity when fluoride concentrations exceed 1.0 ppm in the drinking water if it is ingested while tooth develop¬ ment and calcification are in progress3 (see Chap. 131). When the water supply contains 5 mEq/hour in patients with previous gastric surgery. The secretin provocative test is usually carried out in patients with borderline gastrin levels and equivocal acid-secretion results. In normal individuals, the administration of secretin suppresses gastrin secretion, but in gastrinoma patients a paradoxical rise in gastrin levels occurs in response to secretin. (The fasted patient is given 2 U/kg of secretin intravenously, and blood is collected for gastrin measurement at 2, 5,10, and 20 minutes; in the pres¬ ence of gastrinoma, gastrin levels increase by at least 50% from baseline.) Up to 87% of patients with gastrinoma demonstrate a positive response to secretin. If the diagnosis of gastrinoma is confirmed biochemically, tumor localization is carried out to identify the small proportion of patients (9%) with localized disease, because these persons may benefit from curative surgery. Radiolabeled octreotide scanning provides the most useful information regarding the primary tumor and any metastases (see Chap. 169).5-6 As with other neuroendocrine tumors, the detection of radiolabeled octreotide binding also identifies patients who may respond best to therapy with somatostatin analogs. Magnetic resonance imag¬ ing (MRI) provides information regarding hepatic metastases but is not helpful in localizing the primary tumor. Small tumors may be detected with endoscopic ultrasonography and/or selective arterial angiography in combination with computed tomographic (CT) scanning. On occasion, intraarterial secretin injection at the time of angiography may help localize the gastrinoma (see Chap. 159). Surgical exploration, often in conjunction with intraopera¬ tive ultrasonography, duodenal transillumination, and duodenotomy, may be necessary to localize any solitary tumor not detected by the above imaging techniques. Fortunately, symptomatic control can be achieved, without many side effects, with acid antisecretory drugs (particularly H+-K+ ATPase inhibitors) at dosages titrated to the patient's response. The aim of therapy is to reduce gastric acid secretion to 400 mEq per day. In the early stages, this is usually intermittent, but with progressive tumor growth, it becomes continuous and can be life-threatening. The stool is otherwise normal and does not contain mucus or blood. Steator¬ rhea is not a feature. The severe potassium loss can result in tem¬ porary quadriplegia and is frequently accompanied by a metabolic acidosis due to bicarbonate loss in the stool. The aver¬ age duration of symptoms before diagnosis is -3 years. Other features of the VIPoma syndrome are hypercalcemia, glucose intol¬ erance, and mild diabetes mellitus. In up to 20% of patients, flush¬ ing of the head and trunk may occur in association with a patchy erythematous rash. The diagnosis of VIPoma depends on the demonstration of secretory diarrhea associated with elevated fasting plasma VIP levels (usually >200 pg/mL). On the whole, a stool volume of 70% of the patients have metastatic disease (usually in the liver and regional lymph nodes). The most characteristic feature of the glucagonoma syndrome is the presence of a necrolytic migratory erythematous rash (Fig. 220-2).14 The rash usually starts in the groin and perineum and then gradually migrates to the distal extremities. Typically, the ini¬ tial lesions are erythematous macules, which become raised and bullous. Then the lesions break down and heal, often leaving a residual area of hyperpigmentation. The rash is intensely painful and pruritic, and secondary bacterial and fungal infections are common. The underlying cause of the rash is unknown, but several factors such as direct action of glucagon on the skin, amino-acid and fatty acid deficiency, and zinc deficiency have been implicated

2018

PART XV: HORMONES AND CANCER extent of metastatic disease.153 Endoscopic ultrasonography is sensitive in the detection of pancreatic primary tumors, but lim¬ ited penetration reduces the detection of distant spread. Localized solitary tumors may require multiple techniques for localization. Localized, solitary glucagonomas should be surgically excised to aim for a cure, whereas palliative measures are used for meta¬ static disease. The glucagonoma rash responds to oral and topical zinc, and somatostatin analogs. Longer acting somatostatin ana¬ logs and longer lasting preparations of octreotide are likely to be increasingly used.16-17 With time, the tumor may become less responsive to such therapy, and increasing doses and/or surgery or induced embolization to reduce tumor bulk may be required. Control of cachexia is extremely difficult and may require dietary intervention such as consumption of a high-protein diet. Glucose intolerance may require insulin therapy. Aspirin has been advo¬ cated for prevention of thrombotic episodes, and anticoagulant therapy is used for patients with proven thrombosis. Psychiatric symptoms, such as psychosis or depression, require appropriate psychiatric assessment and treatment. The available palliative measures directed at the tumor and its metastases include surgi¬ cal debulking of the tumor, hepatic tumor embolization, and cyto¬ toxic chemotherapy. Glucagonomas are relatively insensitive to chemotherapeutic agents, but occasionally a patient may benefit from this treatment modality.

SOMATOSTATINOMA FIGURE 220-2. Truncal rash in a patient with a glucagonoma. (From Bloom SR, Polak JM. Glucagonoma syndrome. Am J Med 1987; 82[Suppl 5B]:25.)

in its etiology. The rash is commonly associated with mucosal involvement, which results in stomatitis, cheilitis, and glossitis. Cachexia is a common feature of glucagonoma and may mislead the physician into believing that a more aggressive tumor, such as pancreatic carcinoma, is responsible for the patient's symptoms. As glucagon opposes the effects of insulin on blood glucose homeostasis, impaired glucose tolerance, which usually results in mild diabetes, is also a manifestation of the glucagonoma syndrome. Other manifestations of glucagonoma include normocytic normo¬ chromic anemia, dystrophy of the nails, diarrhea, a tendency to venous thrombosis and pulmonary embolism, and neuropsychiatric symptoms. • Paraneoplastic syndromes, such as optic atrophy, have also been reported in association with glucagonoma. The diagnosis of glucagonoma depends greatly on clinical sus¬ picion. Once the diagnosis is considered, measurement of a highly elevated plasma glucagon value after an overnight fast is confir¬ matory. Plasma glucagon levels may be increased in patients with prolonged fasting, renal and hepatic failure, diabetic ketoacidosis, or therapy with oral contraceptives or danazol, and in those with trauma, bums, sepsis, or Cushing syndrome. These conditions are easily distinguishable from glucagonoma, however, and rarely result in markedly raised glucagon levels. A rare condition associ¬ ated with elevated glucagon levels is familial hyperglucagonemia,15 Cosecretion of a second gut hormone is common, and one-fifth of glucagonoma patients also have elevated gastrin levels, which may cause acid hypersecretion. Elevated plasma insulin, pancre¬ atic polypeptide, VIP, and urinary 5-hydroxyindoleacetic acid lev¬ els have all been observed with glucagonoma. Secretion of other cleavage products of preproglucagon by these tumors may result in gastrointestinal mucosal hypertrophy. The majority of glucagonomas are large and metastatic at presentation. Tumor localization can be achieved with ultra¬ sonography, CT, or visceral angiography (see Chap. 159). Soma¬ tostatin-receptor scintigraphy is most useful for determining the

Somatostatinomas18 are extremely rare tumors with an annual incidence of ~1 in 40 million. They occur mostly in the pancreas but can also arise in the duodenum. Duodenal somatostatinomas may be associated with neurofibromatosis type 1 (von Reckling¬ hausen disease) and pheochromocytoma.19 Duodenal soma¬ tostatinomas usually present early with obstructive symptoms and only rarely result in the tumor syndrome. Pancreatic tumors present late, with hepatic metastases, and often with the tumor syndrome characterized by the triad of cholelithiasis, diabetes, and steatorrhea. Hypoglycemia may occasionally occur and is likely to be caused by larger molecular forms of somatostatin.20-21 Other features include anemia, hypochlorhydria, postprandial fullness, and weight loss. Occasionally, somatostatinomas may secrete adreno¬ corticotropic hormone (ACTH), resulting in Cushing syndrome. The diagnosis of somatostatinoma is confirmed by detection of highly elevated plasma somatostatin levels. Tumor localization ' uses the same techniques as described for other endocrine tumors of the gut. Treatment is mainly surgical. The palliative measures used are similar to those used for other gastrointestinal endocrine tumors.

PPoma Many types of pancreatic endocrine tumors also secrete pancre¬ atic polypeptide (PP).22-24 Elevated plasma PP levels can, there¬ fore, be used as a marker of gastrointestinal endocrine tumors, particularly VIPomas (see Chap 182). Rare tumors produce only PP, but this does not result in any unique clinical or biochemical features. Histologically, the tumors are usually composed of mixed cell types, one of which can be immunocytochemically identified as producing PP.

NEUROTENSINOMA Neurotensin is a 13-amino-acid peptide released from N cells in the small intestine. Neurotensin immunoreactivity has also been

Ch. 220: Endocrine Tumors of the Gastrointestinal Tract detected in enteric neurons. The hormone is produced by a small number of pancreatic endocrine tumors, which usually also pro¬ duce VIP.2:i When infused in humans, neurotensin causes increased watery secretions from the small intestine and frequent defecation.26 Neurotensin may therefore contribute to the clinical features of the VIPoma syndrome. When neurotensin is the sole product of an endocrine tumor, however, no clinical features occur. (Presumably, escape occurs from long-continued eleva¬ tions of plasma neurotensin.) Neurotensin is also detected in nonendocrine tumors in the gastrointestinal tract, suggesting that it may have growth regulatory functions in the pancreas and colon.

2019

levels; the highest levels of chromogranin A are seen in meta¬ static disease.32 Small gastrinomas can result in high plasma chromogranin A levels, because gastrin causes hyperplasia of enterochromaffin-like cells that also secrete chromogranin A. Severe renal failure can result in elevated plasma chromogranin A levels comparable to levels seen with neuroendocrine tumors. Chromogranin A is most useful as a marker of nonfunctioning tumors. The peptide GAWK,33 the product of chromogranin B, is also a useful marker for neuroendocrine tumors, as are secretoneurins.33a The role of the above tumor markers remains to be defined, but at present they are not routinely used in the diagno¬ sis and follow-up of patients.

ENTEROGLUCAGONOMA SOMATOSTATIN THERAPY In the intestinal mucosa, proglucagon-derived peptides are syn¬ thesized and released by L cells of the terminal ileum and colon.27 Proglucagon undergoes tissue-specific posttranslational processing in the pancreas and intestine. In the pancreas, the end products of proglucagon processing are glucagon, glicentinrelated pancreatic polypeptide (GRPP), and a large major frag¬ ment. In the intestine, the products are glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), and glicentin (or enteroglucagon). Interest in these peptides as intestinal growth factors began with the observation that intestinal villous hyper¬ trophy occurred in a patient with a tumor that was secreting proglucagon-derived peptides. Resection of the tumor resulted in the normalization of the previously increased blood levels of these peptides and regression of intestinal villous hypertrophy (see Chap. 160).28 Further evidence was provided by the fact that, after small bowel resection, proglucagon messenger RNA expression was increased in the remnant intestine. Other studies have reported that overexpression of the glucagon gene or exog¬ enously administered proglucagon-derived peptides in rodents are associated with bowel growth and regeneration.29 Although initially the belief was that enteroglucagon was the growth fac¬ tor released by the tumor described (hence, the name "enteroglucagonoma"), GLP-2, a 33-amino-acid peptide, appears to be the mediator of epithelial cell proliferation in the gut.30

OTHER ENDOCRINE TUMORS OF THE GUT Gastrointestinal endocrine tumors may secrete PTHrP, resulting in hypercalcemia. The hypercalcemia can be controlled by regular infusion of bisphosphonates, hepatic tumor embolization, and surgical debulking. Tumors have been described that secrete growth hormone-releasing hormone (GHRH), causing acro¬ megaly, and ACTH, causing Cushing syndrome. These may occur either alone or in association with other gastrointestinal hormone tumor syndromes. Numerous other peptides have been detected either in the plasma or in tissue from endocrine tumors of the gut. These include neuropeptide Y (NPY), neuromedin-B, calcitonin gene-related peptide (CGRP), motilin, and bombesin, but these are not associated with specific clinical syndromes.

TUMOR MARKERS No generally useful tumor marker exists for gastrointestinal endocrine tumors. Some interest has been shown in the use of chromogranins and neuron-specific enolase (NSE) as markers. Histochemically, chromogranins and NSE have been used for some time in the immunocytochemical identification of neu¬ roendocrine tumors.31 In neuroendocrine tumors, a correlation is seen between tumor burden and circulating chromogranin A

Somatostatin analogs are commonly used in the treatment of gut endocrine tumors. Somatostatin has widespread inhibitory effects in the gastrointestinal tract.34 It is used to reduce the secretion of peptides from endocrine tumors and also has anti¬ neoplastic actions.35 Somatostatin has a half-life of 50% of the liver parenchyma is replaced by tumor, embolization may precip¬ itate fulminant hepatic failure. Other contraindications to tumor embolization include blood coagulation disorders, intercurrent infection, and end-stage disease. Embolization can result in the release of numerous vasoactive peptides from the tumor and.

2020

PART XV: HORMONES AND CANCER

FIGURE 220-3. A, Hepatic arteriogram showing three large hepatic metastases in the right lobe. B, Hepatic arteriogram of the same patient after hepatic artery embolization, which completely occluded the arte¬ rial supply to the secondary tumors seen in A.

therefore, a hypotensive crisis. Patients are prepared for the proce¬ dure by prehydration. An octreotide infusion is used to block the effects of the peptides. Intravenous aprotinin and prophylactic broad-spectrum antibiotics are also administered to minimize the risks of the procedure. The procedure may result in fever, malaise, nausea, vomiting, abdominal pain, and paralytic ileus. Fever necessitates appropriate microbiologic investigations; in such cases, the development of a hepatic abscess should be excluded by abdominal ultrasonographic examination. Fortunately, complica¬ tions of tumor embolization are rare in the hands of an experi¬ enced operator. Chemoembolization, using agents such as doxorubicin and iopamidol, has been advocated in the treatment of metastatic neuroendocrine tumors. Morbidity is reported to be less than for cytotoxic chemotherapy or for embolization alone.

REFERENCES 1. Zollinger RM, Ellison EH. Primary peptic ulceration of the jejunum associ¬ ated with islet cell tumors of the pancreas. Ann Surg 1955; 142:709. 2. Polak JM, Bloom SR. Review: The enterochromaffin-like cell, intragastric acidity and the trophic effects of plasma gastrin. Aliment Pharmacol Ther 1988; 2:291.

3. Walsh JH. Gastrin. In: Walsh JH, Dockray GJ, eds. Gut peptides. New York: Raven Press, 1994:75. 3a. Goebel SU, Heppner C, Bums AL, et al. Genotype/phenotype correlation of multiple endocrine neoplasia type 1 gene mutations in sporadic gastrino¬ mas. J Clin Endocrinol Metab 2000; 85:116. 4. Jensen RT. Gastrointestinal endocrine tumours. Gastrinoma. Bailliere's Clin Gastroenterol 1996; 10(4):673. 5. Modlin 1M, Tang LEf. Approaches to the diagnosis of gut neuroendocrine tumors: the last word (today). Gastroenterology 1997; 112:583. 6. Hammond PJ, Jackson JA, Bloom SR. Localization of pancreatic endocrine tumors. Clin Endocrinol 1994; 40:3. 7. Jensen RT. Management of the Zollinger-Ellison syndrome in patients with multiple endocrine neoplasia type 1. J Intern Med 1998; 243:477. 8. Vemer JV, Morrison AB. Islet cell tumor and a syndrome of refractory diar¬ rhea and hypokalemia. Am J Med 1958; 25:374. 9. Park SK, O'Dorisio MS, O'Dorisio TM. Gastrointestinal endocrine tumours. Vasoactive intestinal polypeptide-secreting tumours: biology and therapy. Baillieres Clin Gastroenterol 1996; 10(4):673. 10. Bloom SR, Polak JM, Pearce AGE. Vasoactive intestinal peptide and waterydiarrhoea syndrome. Lancet 1973; 2:14. 11. Dockray G. Vasoactive intestinal polypeptide and related peptides. In: Walsh JH, Dockray GJ, eds. Gut peptides. New York: Raven Press, 1994:447. 12. O'Dorisio TM. VIP and watery diarrhea. In Bloom SR, ed. Gut hormones. Edinburgh: Churchill Livingstone, 1978:581. 13. Frankton S, Bloom SR. Gastrointestinal endocrine tumours. Glucagonomas. Baillieres Clin Gastroenterol 1996; 10(4):673. 14. Bloom SR, Polak JM. Glucagonoma syndrome. Am J Med 1987; 82:25. 15. Boden G, Owen OE. Familial hyperglucagonemia: an autosomal dominant disorder. N Engl J Med 1977; 296:534. 15a. Johnson DS, Coel MN, Bornemann M. Current imaging and possible thera¬ peutic management of glucagonoma tumors. Clin Nuclear Med 2000; 25:120. 16. Arnold R, Frank M. Gastrointestinal endocrine tumours. Gastrointestinal endocrine tumours: medical management. Baillieres Clin Gastroenterol 1996; 10(4):737. 17. Trautman ME, Neuhaus C, Lenze H, et al. The role of somatostatin analogs in the treatment of endocrine gastrointestinal tumors. Horm Metab Res 1995; 27:24. 18. Kreijs GJ, Orci L, Conlon JM, et al. Somatostatinoma syndrome. N Engl J Med 1979; 301:285. 19. Griffiths DF, Williams GT, Williams ED. Duodenal carcinoid tumors, phaeochromocytoma and neurofibromatosis: islet cell tumor, phaeochromocytoma and the von Hippel-Lindau complex: two distinctive neuroendocrine syndromes. Q J Med 1987; 64:769. 20. Penman E, Lowry PJ, Wass JAH, et al. Molecular forms of somatostatin in normal subjects and patients with pancreatic somatostatinomas. Clin Endo¬ crinol (Oxf) 1980; 12:611. 21. Bloom SR, Polak JM. Somatostatin. BMJ 1987; 295:288. 22. Vinik Al, Strodel WE, Eckhauser FE, et al. Somatostatinomas, PPomas, neurotensinomas. Semin Oncol 1987; 14:263. 23. Adrian TE, Lettenthal LO, Williams SJ, Bloom SR. Secretion of pancreatic polypeptide in patients with pancreatic endocrine tumors. N Engl J Med 1986; 315:287. 24. Polak JM, Bloom SR, Adrian TE, et al. Pancreatic polypeptide in insulino¬ mas, gastrinomas, VIPomas and glucagonomas. Lancet 1976; 1:328. 25. Blackburn AM, Bryant MG, Adrian TE, Bloom SR. Pancreatic tumors pro¬ ducing neurotensin. J Clin Endocrinol Metab 1981; 52:820. 26. Calam J, Unwin R, Peart WS. Neurotensin stimulates defaecation. Lancet 1983; 1:737. 27. Holst JJ. Enteroglucagon. Ann Rev Physiol 1997; 59:257. 28. Bloom SR. An enteroglucagon tumor. Gut 1972; 13(7):520. 29. Drucker DJ, Erlich, P, Asa SL, et al. Induction of intestinal epithelial prolifera¬ tion by glucagon-like peptide 2. Proc Natl Acad Sci U S A1996; 93 (15):7911. 30. Hussain MA. A biological function for glucagon-like peptide 2. Eur J Endocr 1998; 139:265. 31. Bishop AE, Polak JM. Gastrointestinal endocrine tumours. Pathology. Baillieres Clin Gastroenterol 1996; 10(4):555. 32. Nobels FRE, Kwekkeboom DJ, Bouillon R, et al. Chromogranin A: its clinical value as marker of neuroendocrine tumors. Eur J Clin Invest 1998; 28:431. 33. Sekiya K, Ghatei MA, Salahuddin MJ, et al. Production of GAWK (Chromogranin-B 420-493)-like immunoreactivity by endocrine tumors and its possible diagnostic value. J Clin Invest 1989; 83:1834. 33a. Ischia R, Gasser RW, Fischer-Collorie R, et al. Levels and molecular proper¬ ties of secretoneurin-immunoreactivity in the serum and urine of control and neuroendocrine tumor patients. J Clin Endocrinol Metab 2000; 85:355. 34. Chiba T, Yamada T. Gut somatostatin. In: Walsh JH, Dockray GJ, eds. Gut Peptides. New York: Raven Press, 1994:123. 35. Pollack MN, Schally AV. Mechanisms of antineoplastic action of somato¬ statin analogs. Proc Soc Exp Biol Med 1998; 217:143. 36. Lamberts SWJ, Van der Lely A-J, De Herder WW, et al. Octreotide. N Engl J Med 1996; 334(4):246. 37. Patel YC, Greenwood MT, Panetta R, et al. The somatostatin receptor family. Life Sci 1995; 57(13):1249. 38. De Herder WW, Hofland LJ, Van der Lely AJ, Lamberts SW. Gastrointesti¬ nal endocrine tumours. Peptide receptors in gut endocrine tumors. Bailli&res Clin Gastroenterol 1996; 10(4):571. 39. Virgolini I. Mark Forster Award Lecture. Receptor nuclear medicine: vasointestinal peptide and somatostatin receptor scintigraphy for diagnosis and treatment of tumour patients. Eur J Clin Invest 1997; 27 (10):793.

Ch. 221: Carcinoid Tumor and the Carcinoid Syndrome

CHAPTER

221

CARCINOID TUMOR AND THE CARCINOID SYNDROME PAUL N. MATON

Carcinoids are found in 1% of autopsies, but clinical data sug¬ gest an incidence of 2 cases per 100,000 per year.1 Although the incidence of subclinical gastric carcinoids has probably been underestimated,2'3 most carcinoids remain localized and are not clinically significant. The major interest in these tumors relates to the few that produce 5-hydroxytryptamine and other sub¬ stances and cause flushing, diarrhea, heart disease, and asthma—the carcinoid syndrome.3'4

CELL OF ORIGIN Carcinoids are neuroendocrine tumors that usually arise from enterochromaffin (EC) cells, which are found scattered throughout the body but occur principally in the submucosa of the intestine and main bronchi.3'4 The EC cell population is het¬ erogeneous, which may explain the variety of features associ¬ ated with carcinoid tumors (Table 221-1). Some EC cells are argentaffinic, whereas others are argyrophilic (see Chap. 175). Furthermore, some EC cells contain peptides, such as substance P, enkephalins, or motilin.3 Most gastric carcinoids arise not from EC cells but from enterochromaffin-like (ECL) cells, which may be important in the production of histamine.2,5

PATHOLOGY Carcinoid tumors are benign or of low-grade malignancy. Some authors, however, have included more aggressive "atypical" car¬ cinoids and even highly malignant neuroendocrine carcinomas in accounts of carcinoids.6 The relative distribution of carcinoid tumors, their propensity to metastasize, and their ability to cause the carcinoid syndrome are given in Table 221-2. One analysis of 8000 carcinoids1 showed appendiceal carcinoids to be the most common. Overall, 75% of carcinoids occur in the gut, 24% in the lungs, and only 1% in other sites. Eighty percent of small intesti¬ nal carcinoids (which may be multiple) occur within 60 cm (2 ft) of the ileocecal valve, and 85% of bronchial carcinoids are in the main bronchi. The primary tumors tend to remain small and to

2021

extend outward, away from the lumen. They then spread to local lymph nodes. A marked fibrotic reaction may occur, which, with midgut carcinoids, may distort the gut and mesentery, and some¬ times cause intestinal obstruction or vascular occlusion. Further spread occurs to the peritoneum or liver, and distant metastases may occur at almost any site; such distant lesions may include osteolytic and osteoblastic bone metastases.3-7,8 Most gastric ECL cell carcinoids are under the influence of gastrin. In patients with hypergastrinemia due to gastric atro¬ phy (with or without pernicious anemia) or Zollinger-Ellison syndrome with multiple endocrine neoplasia type 1 (MEN1), a generalized hypertrophy of ECL cells is present that in some cases leads to the formation of multiple carcinoids.2,5 The many small polyps (sometimes numbering in the hundreds) that are seen initially depend on gastrin but may become autonomous. Metastases to lymph nodes are rare6 but are more common in sporadic (non-hypergastrinemia-associated) ECL cell carci¬ noids (see Table 221-2). Carcinoids may contain and secrete various peptides and amines, and some carcinoids, particularly those of the foregut, can produce other clinical syndromes with or without the carci¬ noid syndrome. Carcinoids that secrete insulin, growth hor¬ mone, corticotropin, (3-melanocyte-stimulating hormone, gastrin, calcitonin, substance P, growth hormone-releasing hor¬ mone, and bombesin-like peptides have been described.3 Many carcinoids secrete chromogranin, a peptide common to many neuroendocrine tumors.9 Carcinoid tumors of the stomach can occur in MEN1. The MEN1 gene, a tumor suppressor gene, is on chromosome 11 (llql3). In MEN1 patients gastric carcinoids (like parathyroid and islet cell tumors) exhibit loss of heterozygosity at llql3 with deletion of the wild-type allele.10 This developmental mecha¬ nism is also true for lung carcinoids (and possibly for sporadic gut carcinoids,10,11 but not for thymic carcinoids12), irrespective of MEN 1.13,14 The carcinoid syndrome is much rarer than carcinoid tumors: the estimated incidence is approximately three cases per million population per year. The syndrome occurs only when vasoactive substances reach the systemic circulation; therefore, most gut carcinoids cause the syndrome only when hepatic metastases are present. Even then, the carcinoid syndrome occurs only in a few cases (see Table 221-2), and the histamine-type syndrome caused by gastric carcinoids has been described in less than ten cases (all carcinoids were sporadic ECL cell carcinoids). Ovarian and bron¬ chial tumors, which drain directly into the systemic circulation, can cause the carcinoid syndrome without metastases; however, with bronchial carcinoids, metastases are usually present. Rarely, medullary carcinoma of the thyroid and small cell tumors of the lung cause the syndrome.

TABLE 221-1. Characteristics and Embryologic Derivation of Carcinoid Tumors Site of Tumor Foregut

Midgut

Hindgut

HISTOLOGIC FEATURES

Trabecular

Solid mass of cells

Mixed

APPEARANCE OF CYTOPLASMIC GRANULES (ELECTRON MICROSCOPY)

Variable density, 180 pm diameter

Uniformly dense, 230 pm diameter

Variable density, 190 pm diameter

Positive

Positive

Positive

Argyrophilic or negative

Argentaffin

Negative

5-HTP, peptides; histamine (gastric)

5-HT

None

Unusual

Common

STAINING: NSE SILVER TUMOR PRODUCTS METASTASES TO BONE AND SKIN

Common

NSE, neuron-specific enolase; 5-HTP, 5-hydroxytryptophaii; 5-HT, 5-hydroxytryptamine.

2022

PART XV: HORMONES AND CANCER

TABLE 221-2. Ability of Carcinoid Tumors to Metastasize and Produce the Carcinoid Syndrome

CARCINOID SYNDROME

No. of Cases With Metastases

With Carcinoid Syndrome

2

—

—

Total FOREGUT Esophagus Stomach* With gastric atrophy

197

17

—

With ZES

21

12

—

Sporadic1,

32

17

9

Duodenum

115

23

4

5

1

1

18

6

1

5

—

—

Pancreas Gallbladder Bile duct Ampulla

7

1

—

Larynx

4

2

—

Bronchus

>500t

Thymus

74

>1004 19

66 —

MIDGUT Jejunum Ileum

56

20

91

1013

355

—

44

8

6

1687

34

6

89

53

5

4

—

—

34

2

17

2

—

1

33

8

1

573

18

1

Meckel diverticulum Appendix Colon Liver Ovary Testis Cervix

HINDGUT Rectum

ZES, Zollinger-Ellison syndrome. ’From Rindi G, Luinetto O, Cornaggia M, et al. Three subtypes of gastric argyrophil carcinoid and the gastric neuroendocrine carcinoma: a clinical pathologic study. Gastro¬ enterology 1993; 104:994. +Nof associated with hypergastrinemia. ♦Only approximate data are available.

CLINICAL FEATURES CARCINOIDS WITHOUT SYSTEMIC FEATURES Carcinoids without systemic features occur most commonly in the appendix and small intestine1 (see Table 221-2) and usually are found incidentally at surgery.3-4'7'8 Small intestinal carcinoids are usually symptomless but, occasionally, cause intestinal obstruc¬ tion or vascular occlusion. Ileal tumors generally are not demon¬ strated by simple radiology; therefore, barium infusion studies or angiography is required.15 Duodenal and gastric carcinoids most often are found incidentally at endoscopy. Colonic, rectal, and esophageal carcinoids also may be found incidentally or may cause obstruction.16 Bronchial carcinoids may be discovered as a coin lesion on chest radiographs or may be seen at bronchoscopy. They also may present with cough, wheeze, hemoptysis, or seg¬ mental obstruction and infection. Mediastinal and ovarian carci¬ noids appear as masses.17 Most carcinoids occur as an isolated disease, but associations are seen between foregut carcinoids and MEN1; between gastric carcinoids and hypergastrinemia, whether due to achlorhydria or to Zollinger-Ellison syndrome, especially as part of MEN118; between ampullary carcinoids and von Reck¬ linghausen disease3; and between renal carcinoids and horseshoe kidney.19 The diagnosis of all carcinoids without systemic features depends on the histologic structure and staining.

The carcinoid syndrome3-4-7'8 is characterized by flushing, diar¬ rhea, and heart disease, although the relative importance of the symptoms varies in different patients, reflecting differences in tumor origin, bulk, tumor products, and length of history. The primary tumor may have been removed many years before the development of the syndrome, or it may never have become clinically evident. Most patients with the carcinoid syndrome, however, have an ileal tumor, with evident hepatic metastases at the time of presentation (see Table 221-2). Carcinoid flushing is erythematous, and principally affects the upper part of the body. Some patients are unaware of the flushing, whereas others are distressed by it. Flushes may be brief or prolonged. They often are spontaneous, but they may be precipitated by alcohol, certain foods, abdominal palpation, or anxiety. Several patterns of flushing have been described,7 but only two are clinically distinctive. Gastric carcinoids may pro¬ duce a bright red, geographic flush, often precipitated by food consumption.20 Bronchial carcinoids may cause severe pro¬ longed flushes with salivation, lacrimation, sweating, facial edema, palpitations, diarrhea, and hypotension. After many months of flushing, a fixed, facial telangiectasia, edema, and cyanotic plethora may occur. Diarrhea occurs in most cases, although it is less evident with gastric carcinoids. In some patients, diarrhea is related to episodes of flushing; in others, the two seem independent. Watery diarrhea is more frequent than malabsorption and is due to increased motility and possibly intestinal secretion, but intermittent intesti¬ nal obstruction, cholorrheic diarrhea after previous intestinal resection for tumor, or vascular insufficiency or lymphatic obstruction may occur in some patients. Abdominal pain may be due to these abnormalities or to necrosis of hepatic metastases. Heart disease occurs in -30% of patients. Insidious right heart failure often worsens during periods of flushing, and, in such cases, tricuspid regurgitation or stenosis is typical; less commonly, pulmonary stenosis may occur. The left side of the heart also may be involved, usually in association with bron¬ chial carcinoids. The heart disease is caused by a unique form of fibrosis that involves the endocardium and valves.21-213 Fibrosis in other sites can cause constrictive pericarditis, retroperitoneal fibrosis, pleural thickening,22 and Peyronie disease. Wheezing occurs in 10% of patients and can be the presenting, feature. A pellagra-like syndrome may occur, and confusional states have been described with foregut carcinoids. Rarely, patients experience arthralgia or a myopathy.23

PATHOGENESIS OF THE CARCINOID SYNDROME The flushing is poorly understood but may be due to the release of kinins in some patients.24-25 Carcinoids contain kallikrein, an enzyme that, when released into the circulation spontaneously or stimulated by alcohol or catecholamines, acts on plasma kininogens to generate bradykinin3-7 (see Chap. 170). In gastric carcinoids, the distinctive flush is mediated by histamine (see Chap. 181). The diarrhea is caused largely by 5-hydroxytryptamine (serotonin) through its effects on gut motility. The 5-hydroxytryptamine also contributes to the asthma and is probably implicated in the cardiac fibrosis. The diversion of tryptophan to the tumor for 5-hydroxytryptamine synthesis can lead to reduced protein synthesis, with hypoalbuminemia, and to nicotinic acid deficiency, with pellagra.26 The prostaglandins27 and many gut peptides28 probably are not mediators of the flushing or diarrhea in most patients. The

Ch. 221: Carcinoid Tumor and the Carcinoid Syndrome role of other peptides such as substance P or other tachykinins (neurokinin A, neuropeptide K) has yet to be fully evaluated.25

DIAGNOSIS OF THE CARCINOID SYNDROME Once the carcinoid syndrome has been considered, confirmation of the diagnosis is often not difficult and rests on clinical features, measurement of the principal 5-hydroxytryptamine metabolite in urine (5-hydroxyindoleacetic acid [5-HIAA]) and, occasion¬ ally, the provocation of flushing with epinephrine.3'4,7'8 In a patient who flushes, who has an enlarged liver, and in whom un¬ itary 5-HIAA excretion is >30 mg per day (normal is 75% for tumors from all sites. In patients with distant metastases, the 5-year survival was 30% or less.32 One analysis indicates an overall 5-year survival of 50%, with 45% of carci¬ noids having metastasized at the time of diagnosis.1 In the larg¬ est reported series of patients with the carcinoid syndrome, the median survival from first flush was 3 years but survival ranged up to 17 years.8 The median survival in patients with heart dis¬ ease was 14 months, and in patients with a large tumor burden (5-HIAA level of >150 mg per day), it was 11 months.

TREATMENT CONTROL OF THE TUMOR Except in the case of gastric ECL cell carcinoids that are of low malignancy and can be observed for some years, surgery should be considered in all patients because the resection of local dis¬ ease can result in cure of carcinoid tumors, and in cure of the car¬ cinoid syndrome due to some bronchial and ovarian tumors.33 Interestingly, the appropriate surgery for multiple gastric carci¬ noids in patients with gastric atrophy is not removal of the tumors, but removal of the gastric antrum, leading to normaliza¬

2023

tion of plasma gastrin and tumor regression.5 Resection of iso¬ lated hepatic metastases detected by computed tomographic scan or somatostatin analog scintigraphy34-36 also may be mark¬ edly beneficial in selected cases7,8; however, in the presence of extensive metastases, partial hepatic resection is not warranted, nor is removal of the primary tumor unless it is causing local problems. Hepatic transplantation has been beneficial on occa¬ sion.37 Chemotherapy for carcinoid tumors has been disappoint¬ ing, with responses occurring in a minority and lasting only a mean of 7 months.8 Single agents have produced responses in up to 30%, streptozocin being the most effective agent. Various combinations of streptozocin with 5-fluorouracil, cyclophospha¬ mide, and doxorubicin have produced response rates of up to 35%.8 Given the variation in tumor growth, questionable effi¬ cacy, and undoubted toxicity of chemotherapy, as well as the availability of other symptomatic therapy, chemotherapy should be reserved for advanced tumors that are actively grow¬ ing. Administration of interferon-a, 3 to 6 million IU per day subcutaneously, reduces tumor size in -15% of cases and stabi¬ lizes tumor size in another 30% to 40%.8,38^41 Octreotide acetate, used mainly for its effect on symptoms (see later), reduces tumor bulk in -5% and stabilizes tumor size in another 20% 42-44 Radiotherapy is useful only for symptomatic therapy of bone and skin metastases. Hepatic artery occlusion leads to selective necrosis of hepatic metastases. Surgical ligation of the hepatic artery has been used to necrose the hepatic tumor in the carcinoid syndrome,8 but percutaneous arterial embolization is less traumatic, more selec¬ tive, and can be repeated.45-47 Embolization, alone or in combi¬ nation with chemotherapy,8,48 can produce a striking relief of symptoms and reduction in urinary 5-HIAA levels, even in patients with symptoms that are resistant to other modes of therapy. Complete remissions of up to 30 months have been reported. Second remissions may follow repeat embolizations, and survival may be prolonged.

CONTROL OF SYMPTOMS Many patients have considerable hepatic tumor, yet they remain well, apart from occasional flushing or diarrhea.3,7 They should be advised to avoid precipitants of flushing and to supplement their diet with nicotinamide. Heart failure can be treated with diuretics, asthma with albuterol (salbutamol) (which does not precipitate flushing), and diarrhea with loperamide. If patients require further therapy, various other agents may be tried. For diarrhea, cyproheptadine, 4 to 8 mg every 6 hours, is the best oral agent49 In the rare patient with carcinoid syndrome due to a gas¬ tric carcinoid, a combination of diphenhydramine 50 mg every 6 hours, together with an H2 antagonist (e.g., cimetidine 300 mg every 6 hours), has proved effective for flushes.50 For most patients, however, the most effective agent for both diarrhea and flushes is the long-acting somatostatin analog octreotide acetate (100-500 gg every 8-12 hours or the long-acting formulation every 24 hours subcutaneously), which has produced responses in >80% of patients.43,44,51 Another somatostatin analog, lanreotide, is also effective. Interferon-a, used principally for its effect on the tumor, reduces flushing and diarrhea in -50% of patients8,38-40 (see Chap. 169). If drugs fail to control symptoms, hepatic embolization should be considered. Progressive cardiac disease can be halted only by removal of the tumor and cure of the carcinoid syndrome, but the occasional, carefully selected patient may benefit from tricuspid valve replacement.7,52 Anesthetics, surgery, chemotherapy, and hepatic artery occlu¬ sion can precipitate extremely severe flushing with hypoten¬ sion—a carcinoid crisis. The risk of developing such a crisis can be reduced by appropriate premedication, careful monitoring, the

2024

PART XV: HORMONES AND CANCER

judicious use of anesthetic drugs and techniques, and avoidance of flush-provoking agents, such as catecholamines.45'46'53 Should a crisis occur, hypotension should be treated with octreotide acetate 100 jig intravenously, which should be available whenever patients with the carcinoid syndrome undergo procedures.54 If octreotide acetate is not available, intravenous methoxamine, 3 to 5 mg, can be used. Other pressor agents should be avoided.

REFERENCES 1. Modlin IM, Sandor A. An analysis of 8305 cases of carcinoid tumors. Cancer 1997; 79:813. 2. Rindi G, Luinetto O, Cornaggia M, et al. Three subtypes of gastric argyrophii carcinoid and the gastric neuroendocrine carcinoma: a clinical patho¬ logic study. Gastroenterology 1993; 104:994. 3. Maton PN, Hodgson HJF. Carcinoid tumours and the carcinoid syndrome. In: Bouchier IAD, Allan RN, Hodgson HJF, Keighly MRB, eds. Textbook of gastroenterology. London: Bailliere-Tindall, 1984:620. 4. Feldman JM, Zakin D, Dannenberg AJ. Carcinoid tumors and syndrome. Semin Oncol 1987; 14:237. 5. Maton PN, Dayal Y. Clinical implications of hypergastrinemia. In: Zakim DH, Dannenberg AJ, eds. Peptic ulcer disease and other acid-related disor¬ ders. New York: Academic Research Associates, 1991:213. 6. Bordi C, Falchetti A, Azzoni C, et al. Aggressive forms of gastric neuroendocrine tumors in multiple endocrine neoplasia type 1. Am J Surg Pathol 1997; 21:1075. 7. Grahame-Smith DG. The carcinoid syndrome. London: William Heinemann, 1972. 8. Moertel CG. An odyssey in the land of small tumors. J Clin Oncol 1987; 5:1503. 8a. Nuttall KL, Pingree SS. The incidence of elevations in urine 5-hydroxyindoleacetic acid. Ann Clin Lab Sci 1998; 28:167. 9. Nobels FR, Kwekkeboom DJ, Coopmans W. Chromogranin A as a serum marker for neuroendocrine neoplasia: comparison with neuron-specific enolase and the alpha-subunit of glycoprotein hormones. J Clin Endocrinol Metab 1997; 82:2622. 10. Debelenko LV, Emmert-Buck MR, Zhuang Z, et al. The multiple endocrine neoplasia type 1 gene locus is involved in the pathogenesis of type II gastric carcinoids. Gastroenterology 1997; 113:773. 11. Jakobovitz O, Nass D, De Marco L, et al. Carcinoid tumors frequently dis¬ play genetic abnormalities involving chromosome 11. J Clin Endocrinol Metab 1996; 81:164. 12. Teh BT. Thymic carcinoids in multiple endocrine neoplasia type 1. J Intern Med 1998; 243:501. 13. Dong Q, Debelenko LV, Chandrasekharappa SC, et al. Loss of heterozygos¬ ity at llql3: analysis of pituitary tumors, lung carcinoids, lipomas, and other uncommon tumors in subjects with familial multiple endocrine neo¬ plasia type 1. J Clin Endocrinol Metab 1997; 82:1416. 14. Walch AK, Zitzelsberger HF, Aubele MM, et al. Typical and atypical carci¬ noid tumors of the lung are characterized by llq deletions as detected by comparative genomic hybridization. Am J Pathol 1998; 153:1089. 15. Jeffree MA, Barter SJ, Hemingway AP, Nolan DJ. Primary carcinoid tumors of the ileum: the radiological appearances. Clin Radiol 1984; 35:451. 16. Spread C, Berkel H, Jewell L, et al. Colon carcinoid tumors: a populationbased study. Dis Colon Rectum 1994;37:482. 17. Wang DY, Chang D-B, Kuo S-H, et al. Carcinoid tumors of the thymus. Tho¬ rax 1994; 49:357. 18. Hakanson R, Sundler F, eds. Mechanisms for the development of gastric carcinoids: proceedings of an international symposium. Digestion 1986; 35(Suppl 1):1. 19. Krishnan B, Truong LD, Saleh G, et al. Horseshoe kidney is associated with an increased relative risk of primary renal carcinoid tumor. J Urol 1997; 157:2059. 20. Roberts LJ, Marney SR, Oates JA. Blockade of the flush associated with met¬ astatic gastric carcinoid by combined H, and H2 receptor antagonists: evi¬ dence for an important role of H2 receptors in human vasculature. N Engl J Med 1979; 300:236. 21. Wikowske MA, Hartman LC, Mullaney CJ, et al. Progressive carcinoid heart disease after resection of primary ovarian carcinoid. Cancer 1994; 73:1889. 21a. Sakai D, Mukakami M, Kasazoe K, Tsutsumi Y. Ileal carcinoid tumor com¬ plicating carcinoid heart disease and secondary retroperitoneal fibrosis. Pathol Int 2000; 50:404. 22. Moss SF, Lehner PJ, Gilbey SG, et al. Pleural involvement in the carcinoid syndrome. Q J Med 1993; 86:49. 23. Lederman RJ, Bukowski RM, Nickelson P. Carcinoid myopathy. Cleve Clin J Med 1987; 54:299. 24. Lucas KJ, Feldman JM. Flushing in the carcinoid syndrome and plasma kallikrein. Cancer 1986; 58:2290. 25. Grahame-Smith DG. What is the cause of the carcinoid flush? Gut 1987; 28:1413. 26. Swain CP, Tavill AS, Neale G. Studies of tryptophan and albumin metabo¬ lism in a patient with carcinoid syndrome, pellagra and hypoproteinemia. Gastroenterology 1976; 74:484. 27. Metz SA, McRae JR, Robertson PR. Prostaglandins as mediators of parane¬ oplastic syndromes: review and update. Metabolism 1981; 30:299. 28. Long RG, Peters JR, Bloom SR, et al. Somatostatin, gastrointestinal peptides and the carcinoid syndrome. Gut 1981; 22:549.

29. Wilkin JK. Flushing reactions: consequences and mechanisms. Ann Intern Med 1981; 95:468. 30. Aldrich LB, Moattari R, Vinik AL Distinguishing features of idiopathic flushing and carcinoid syndrome. Arch Intern Med 1988; 148:2614. 31. Young DS, Pestaner LC, Gibberman V. Effects of drugs on clinical laboratory tests. Clin Chem 1975; 21:398D. 32. Godwin JD. Carcinoid tumors: an analysis of 2837 cases. Cancer 1975; 36:560. 33. Norton JA. Neuroendocrine tumors of the pancreas and duodenum. Curr Probl Surg 1994; 31:77. 34. Kwekkeboom DJ, Krenning EP, Bakker WH, et al. Somatostatin analog scin¬ tigraphy in carcinoid tumors. Eur J Nucl Med 1993; 20:283. 35. Kwekkeboom DJ, Krenning EP. Somatostatin receptor scintigraphy in patients with carcinoid tumors. World J Surg 1996; 20:157. 36. Kisker O, Weinel RJ, Geks J, et al. Value of somatostatin receptor scintigra¬ phy for preoperative localization of carcinoids. World J Surg 1996; 20:162. 37. Le-Treut YP, Delpero JR, Dousset B, et al. Results of liver transplantation in the treatment of metastatic neuroendocrine tumors. A 31-case French mul¬ ticentric report. Ann Surg 1997; 225:355. 38. Oberg K, Eriksson B. Role of interferons in the management of carcinoid tumors. Br J Hematol 1991; 79(Suppl 1):74. 39. Janson ET, Oberg K. Long-term management of the carcinoid syndrome: treatment with octreotide alone and in combination with alpha interferon. Acta Oncol 1993; 32:225. 40. Oberg K, Norheim I, Lind E, et al. Treatment of malignant carcinoid tumors with human leukocyte interferon. Cancer Treat Rep 1986; 70:1296. 41. Oberg K. Advances in chemotherapy and biotherapy of endocrine tumors. Curr Opin Oncol 1998; 10:58. 42. Arnold R, Benning R, Neuhaus C, et al. Gastroenteropancreatic endocrine tumors: effect of Sandostatin on tumor growth. The German Sandostatin Study Group. Metabolism 1992; 41(Suppl 2):116. 43. Kvols LK, Moertel CG, O'Connell MJ, et al. Treatment of the malignant car¬ cinoid syndrome. N Engl J Med 1986; 315:663. 44. Gorden P, Comi RJ, Maton PN, Go VLW. Somatostatin and somatostatin analogue (SMS 201-995) in treatment of hormone-secreting tumors of the pituitary and gastrointestinal tract and non-neoplastic diseases of the gut. Ann Intern Med 1989; 110:35. 45. Maton PN, Camilleri M, Griffin G, et al. The role of hepatic arterial emboli¬ sation in the carcinoid syndrome. BMJ 1983; 287:932. 46. Martensson H, Norbin A, Bengmark S, et al. Embolisation of the liver in the management of metastatic carcinoid tumors. J Surg Oncol 1984; 27:152. 47. Ruszdiewski P, Malka D. Hepatic arterial chemoembolization in the manage¬ ment of advanced digestive endocrine tumors. Digestion 2000; 62(Suppl 1):79. 48. Drougas JG, Anthony LB, Blain TK, et al. Hepatic artery chemoembolization for management of patients with advanced metastatic carcinoid tumors. Am J Surg 1998; 175:408. 49. Moertel CG, Kvols LK, Rubin J. A study of cyproheptadine in the treatment of metastatic carcinoid tumor and the malignant carcinoid syndrome. Can¬ cer 1991; 67:33. 50. Oates JA. The carcinoid syndrome. N Engl J Med 1986; 315:702. 51. Saslow SB, O'Brien MD, Camilleri M, et al. Octreotide inhibition of flushing and colonic motor dysfunction in carcinoid syndrome. Am J Gastroenterol 1997; 92:2250. 52. Codd JE, Prozda J, Merjavy J. Palliation of carcinoid heart disease. Arch Surg 1987; 122:1076. 53. Tornebrandt K, Nobin A, Ericsson M, Thompson D. Circulation, respiration and serotonin levels in carcinoid patients during neuroleptic anaesthesia. % Anaesthesia 1983; 38:957. 54. Marsh HM, Martin JK Jr, Kvols LK, Moertel CG. Carcinoid crisis during anesthesia: successful treatment with somatostatin analogue. Anesthesiol¬ ogy 1987; 66:89.

CHAPTER

222

HORMONES AND CARCINOGENESIS: LABORATORY STUDIES JONATHAN J. LI AND SARA ANTONIA LI The resurgence and rapid growth of the field of hormonal car¬ cinogenesis—the role of hormones in the etiology and growth of cancer—are due in large part to growing concerns regarding two of the most common human cancers, breast and prostate.1-4 Although other hormone-associated cancers occur at lower fre-

Ch. 222: Hormones and Carcinogenesis: Laboratory Studies

2025

TABLE 222-1. Animal Models in Hormonal Carcinogenesis Hormone

Species

Organ Site

Incidence (%)

17p-E2, DES, E,

Hamster

Kidney

90-100

7, 39,43,44

EE

Hamster

Liver

25-35

45

References

EE + ANF

Hamster

Liver

100

46

17P-E2/DES + PRL

Mouse

Testis

30-70

37,38

17P-E2, Ev DES

Rat

Mammary gland

70-100

9,14,16,17

17P-E2, DES

Mouse

Cervix/uterus

20-60

28-29

DES

Monkey

Uterus

70

33

DES, 17P-E2

Rat

Pituitary

15-85

34-36

MPA

Mouse

Mammary gland

60

20

17P-E2 + T/DES

Hamster

Ductus deferens

100

31,63

17P-E, /DES + P

Rat

Mammary gland

100

19

17P-E2 + T/T

Rat

Prostate

20-100

11,47,48

27/J-E,, 17p-estradiol; DES, diethylstilbestrol; £,, estrone; EE, ethynylestradiol; ANF, a-naphthoflavone; PRL, prolactin; MPA, medroxyprogesterone acetate; T, testosterone; P, progesterone.

quencies, they are also of clinical importance; these include endometrial, ovarian, testicular, cervicovaginal, pituitary, thy¬ roid, and sex hormone-related hepatic neoplasms.5-7 That these cancers cannot be attributable to any specific exogenous physi¬ cal, environmental, or dietary factor is becoming increasingly clear. Despite the long history of hormonal carcinogenesis research, the precise mechanism whereby hormones affect neo¬ plastic transformation remains elusive. A better understanding of the effect of hormones, both ovarian/testicular and pituitary, on normal cellular processes of growth and differentiation is needed to ascertain more precisely their involvement in neo¬ plastic development. Nevertheless, after intensive study,8 some of the cellular and molecular alterations elicited by hormones during tumorigenesis are beginning to be revealed. That hormones can induce neoplasms in experimental ani¬ mals has been known for >60 years.9 Moreover, with a few nota¬ ble exceptions, for nearly every human neoplasm with a hormonal association, a corresponding animal tumor model can be induced by hormones alone. Of the various hormonal agents, sex hormones, particularly estrogens93 and to a lesser extent progesterone and prolactin (PRL) in women, and androgens in men, have been associated with tumor induction.

GENERAL CONSIDERATIONS Hormones can affect neoplastic processes by acting either as the sole etiologic agent or in conjunction with physical agents (i.e., ionizing radiation) or nonhormonal chemical carcinogens.8 A number of general mechanisms exist whereby hormones may modify a target tissue during one or more phases of the events initiated by carcinogenic events, such as viral infection, chemical

exposure, or exposure to ionizing radiation. For example, hor¬ mones may be involved in (a) promotional or carcinogenic effects, (b) alterations of the host immune system, (c) activation of viruses, and (d) modification of hormone receptors or alter¬ ation of metabolic rates affecting carcinogen activation. The pri¬ mary concern of this chapter, however, is the induction of tumors, benign and malignant, by hormones, either endogenously produced or exogenously administered. The concept that a given hormone acts specifically on individual target organs or tissues is somewhat misleading, because most organs or tissues are differentially sensitive to various hormones acting alone, or in concert with or opposition to, other hormones. The general characteristics of hormonal carcinogenesis are (a) tissue, strain, and species spec¬ ificity; (b) long induction period; (c) sustained and prolonged hormone exposure; and (d) cellular proliferation. Sex hormones have been implicated in the induction and growth of a wide variety of experimental tumors as summarized in Table 222-1. A common characteristic during endocrine-induced tumorigene¬ sis appears to be a prolonged and severe derangement in normal homeostasis and regulatory relationships as a result of chronic hormone exposure. Although the minimum oncogenic dose for a given sex hor¬ mone to elicit a high incidence of tumors at any tissue site is not precisely known, clearly the conditions required to induce a high tumor yield do not require particularly high concentrations of hormones, either at the serum or tissue level (Table 222-2). For example, the serum level of 17(3-estradiol (17(3-E2) in a female ACI rat in estrus is 75 to 80 pg/mL,10 and the sustained onco¬ genic dose required to elicit a high incidence of mammary tumors is only twice estrous levels. Such levels approach those found during pregnancy in this species. In the male Syrian ham¬ ster, continuous administration of exogenous 17[L-E2 induces

TABLE 222-2. Sex Hormone Levels Required for Experimental Hormonal Carcinogenesis Serum (ng/mL)

Tissue (pg/mg protein)

Reference

Kidney

1.9-2.7

4.5-5.4

11

Mammary gland

0.18-0.21

ND

10

0.045

0.10*

12

2.3

0.90+

Organ

Hormone

Species

17p-E2

Hamster

17P-E2

Rat Rat

Prostate

17P-E2 Testosterone

J7/3-E2,17[5-estradiol; ND, not determined. *pg/g tissue. fng/g tissue.

2026

PART XV: HORMONES AND CANCER

renal tumors, and the serum estrogen levels are approximately seven-fold higher than the mean estrous levels found in normal untreated females (see Table 222-2). The 17|3-E2 levels in the male kidney are equal to or slightly lower than those found in the uterus during estrus, however, because this organ site has only modest ability to concentrate estrogens.11 Similarly, even lower levels of estrogen and androgen are required to induce a high incidence of prostate carcinomas in the male Noble rat.12

EXPERIMENTAL ANIMAL MODELS Numerous murine and one primate species develop tumors in response to sex hormone exposure alone. Although some of these hormone-induced tumor models either may involve viral mediation or may arise in conjunction with the stimulation of other pertinent hormonal factors, most are considered to be the result of the direct carcinogenic action of the hormonal agents themselves. The induction of tumors by hormones characteristi¬ cally occurs in hormone-dependent target tissues. The Syrian hamster kidney model is included in this group because it is essentially an estrogen-responsive target organ.7'11

MAMMARY GLAND More than 65 years ago, Lacassagne9 first demonstrated that long-term estrone (E,) administration induced a high frequency of mammary cancer in male mice. Subsequent studies showed that numerous other mouse strains were also susceptible to the carcinogenic action of estrogens at this tissue site.13 Whereas, in the past, the suggestion was that estrogens were direct carcino¬ gens, the belief now is that, in mice, sex hormones alone cannot effect a high incidence of mammary tumors in the absence of a mouse mammary tumor virus (MMTV) or chemical carcinogen exposure. The lack of an established viral association in rats, however, suggests that mammary tumor induction by female sex hormones in susceptible strains may result from direct hor¬ monal action. High incidences (54-100%) of mammary tumors have been elicited with natural and synthetic estrogens, includ¬ ing Ej, 17(3-E2, ethynylestradiol (EE), and diethylstilbestrol (DES), in both male and female Noble, Wistar, Long-Evans, and ACI rats after 5.0 to 10.0 months of continuous estrogen treat¬ ment (Fig. 222-1).6'10'14 As in the mouse, genetic factors also • clearly play a significant role in the induction of mammary tumors by estrogens in the rat. Despite the marked differences in incidence between the sexes in humans, the lack of a sex dif¬ ference in the ability of estrogens to induce mammary tumors in the rat may actually be analogous to the situation seen in humans. For instance, the strongest risk factor for breast cancer in men is known to be Klinefelter syndrome, a condition result¬ ing from inheritance of an extra X chromosome and character¬ ized by testicular dysfunction and gynecomastia.15 This finding clearly indicates that breast cancer in men develops under con¬ ditions favoring excessive endogenous estrogen levels. Perti¬ nent distinctions can be made between mammary tumors in the rat induced by estrogen and by a chemical carcinogen (e.g., dimethylbenzanthracene [DMBA], N-nitrosomethylurea [NMU]). Estrogen-induced primary mammary neoplasms exhibit a mod¬ est but distinct frequency of metastases (-15%) to other tissue sites, including lymph nodes, liver, and lung,16-17 whereas rat mammary tumors induced by chemical carcinogens do not exhibit any significant metastatic potential.18 Approximately 85% of the mammary tumors originating in rats are estrogen dependent, similar to breast cancer in postmenopausal women, whereas most (-90%) of the mouse mammary tumors are estro¬ gen independent.18 Also, PRL plays a permissive, if not essen-

FIGURE 222-1. Mammary gland carcinomas (arrowheads) induced after continuous administration of 17P-estradiol for 6 months to a female ACI rat. Hormone pellets (20-mg pellet containing 4 mg of 17|}-estradiol) were renewed every 4 months to maintain constant estrogen levels. Serum estradiol concentrations were 165 to 170 pg/mL throughout the treatment period.

tial, role in the induction of mammary tumors by estrogen in most rat strains.14 Finally, combined estrogen and progesterone treatment has been shown to induce a higher incidence of mam¬ mary tumors in Wistar-WAG rats than estrogen exposure' alone.19 Of interest, however, medroxyprogesterone acetate (MPA, Provera) is capable of inducing mammary tumors in mice in the absence of added estrogen.20 The biosynthesis of the estrogens Ej and 17P-E, from their androgen precursors, androstenedione and testosterone, is cata¬ lyzed by aromatase, a microsomal cytochrome P450-dependent enzyme. In postmenopausal women with breast cancer, aro¬ matase activity in the peripheral tissues is a major endogenous source of estrogen for tumor growth.21-22 The dynamics of androgen and estrogen production and metabolism, particu¬ larly the percentage of peripheral aromatization in nonhuman primates (cynomolgus, rhesus, and baboons), closely resembles that in humans.23 This is not found in murine species. Primate species have been used as models of human peripheral aromati¬ zation to test the therapeutic effects of steroidal and nonsteroi¬ dal aromatase inhibitors in vivo.24

OVARY Presently, an animal model for hormonally induced epithelial ovarian tumors does not exist. Nevertheless, granulosa cell tumors of the ovary develop in 25% to 50% of BALB/c mice

Ch. 222: Hormones and Carcinogenesis: Laboratory Studies when they are implanted with progesterone pellets.25 In one study, 19-norprogesterone was more effective than progesterone in inducing these neoplasms. The contraceptive agents norethindrone and norethynodrel elicited a 52% incidence of ova¬ rian tumor. Castrated rats with intrasplenic ovarian transplants that resulted in constant high levels of gonadotropins showed a high frequency of tumors in the transplanted ovaries.26 The resulting ovarian tumors were thecal granulosa cell tumors, however, and were not of epithelial origin. In these rats, the long-term excess of gonadotropins is believed to be largely responsible for promoting ovarian tumor development.

UTERUS Despite the well-established association between estrogen and endometrial cancer in women,4 an animal model of similar hor¬ monally induced cancer at this site is lacking. Although endome¬ trial tumors are produced with a high incidence in rabbits after estrogen treatment, these adenocarcinomas are preceded by cys¬ tic hyperplasia, which occurs spontaneously with high frequency (75%) in aging animals.27 Endometrial carcinomas in the uterine horns have also been induced with either DES or 17(3-E2 in mice.28 Uterine carcinomas were observed in 90% of mice receiving DES for 5 days neonatally 29 The induction of these uterine tumors was age and dose dependent. In addition, these uterine tumors were estrogen dependent because they partially regressed after ova¬ riectomy and, when transplanted into nude mice, required estro¬ gen for continued growth. The involvement of a MMTV in mice uterine tumor development remains a possibility. Similarly, estro¬ gen-dependent uterine tumors can be induced in hamsters when DES is administered to newborn animals.30 Filially, a high inci¬ dence of uterine leiomyosarcomas has been induced in hamsters after combined estrogen and androgen treatment.31 Interestingly, the addition of progesterone to this combined treatment inhibited the induction of these uterine smooth muscle cell tumors. Partic¬ ularly relevant is the induction of uterine mesotheliomas in a non¬ human primate species (squirrel monkey) after prolonged treatment with either DES or estradiol benzoate.32

CERVIX-VAGINA Cervical or vaginal squamous cell carcinomas occur after pro¬ longed estrogen administration in C3H, C57, and BC mouse strains.33 Moreover, no spontaneous occurrence of such tumors has been reported. Generally, the belief is that these tumorigenic effects are produced by the direct carcinogenic action of estro¬ gens. Also, prolonged testosterone administration causes cervi¬ cal tumors in female hybrid mice.

PITUITARY GLAND Some strains of mice and rats are highly susceptible to the induc¬ tion of pituitary tumors by estrogens, whereas other strains are largely resistant.34'33 Males appear to be more susceptible to estrogen-induced pituitary tumors than females. Once pituitary tumors develop in mice, they do not regress after estrogen treat¬ ment ceases. Histologically, these tumors are described as chro¬ mophobe adenomas. The predominant secretion of these tumors is PRL, and growth-promoting properties as well as adrenocorticotropin-like effects have been reported. These pitu¬ itary tumors can be induced either by natural steroidal estrogens or by synthetic steroidal and stilbene estrogens. Intermediatelobe pituitary adenomas also have been induced in rats and hamsters after prolonged estrogen treatment.36 Present evidence indicates that these pituitary tumors are also induced by direct hormonal action.

2027

TESTES The induction of testicular tumors in mice with estrogens has been studied extensively. Initially, malignant tumors of the interstitial cells were reported to develop in the A1 strain of mice receiving 17(J-E2.37 Since then, similar testicular tumors have been induced with high incidence in other mouse strains, including BALB/c, ABj, and ACrg, but not in several other strains.38 As a consequence of estrogen treatment of susceptible mice, alterations in androgen biosynthetic enzyme systems, transient induction of DNA synthesis, and a greater nuclear estrogen content in Leydig cells may contribute to their neoplas¬ tic transformation.38 Apparently, the pituitary plays a permis¬ sive role in estrogen-induced testicular tumors, because hypophysectomy prevents the appearance of these tumors.

KIDNEY The most extensively studied experimental model in hormonal carcinogenesis is the estrogen-induced renal carcinoma of the Syrian hamster. Long-term exposure of either castrated or intact male hamsters (but not female hamsters) to either steroidal or stilbene estrogens results in essentially 100% incidence of multi¬ ple bilateral renal neoplasms.39 Because the reproductive and urogenital tracts of the Syrian hamster arise from the same embryonic germinal ridge, the kidney of this species appears to have carried over genes that are expressed and responsive to estrogens. Complete chemoprevention of renal tumorigenesis can be effected by administering the estrogen concomitantly with androgen, progesterone, antiestrogens, or EE.40-41 Evidence strongly indicates that the estrogen-induced renal tumor arises primarily from undifferentiated committed epithelial stem cells in the interstitium.42 Not all estrogens are equally active in inducing these renal tumors.43 With the exception of EE, which elicits only a 10% renal tumor incidence, potent estrogens (17(3E2, DES, hexestrol, and lip-methoxyethinylestradiol [Moxestrol]) exhibit high incidences of renal neoplasms compared with weak estrogens (estriol, 4-hydroxyestrone). Moreover, estrogens that possess low or negligible estrogenic activity (17aE2, (3-dienestrol, 2-hydroxy-estradiol) do not induce kidney tumors. The lack of strong carcinogenic activity of EE in the hamster kidney, despite its known potent estrogenic activity, may be the result of a differential effect of EE on the proliferation of a subset of renal tubule cells, rather than on the stem cells residing in the interstitium.41 One of the most unusual features of the Syrian hamster kidney is its ability to behave as an estrogen-responsive and estrogendependent organ. Estrogen treatment elevates the level of estro¬ gen receptors and induces progesterone receptors in the kidney. These effects are characteristic of estrogen action in target tis¬ sues. A comprehensive model for estrogen carcinogenicity in the hamster kidney is proposed (Fig. 222-2). Briefly, estrogens induce proliferation of preexisting estrogen-sensitive interstitial cells, as well as reparative proliferation secondary to cellular damage. The proliferation of the interstitial cells leads to aneuploidy and chromosomal instability, resulting in gene overexpres¬ sion, amplification, and suppression (specifically, protooncogene, and suppressor gene expression) and eventually leads to tumor formation via a multistep process.44

LIVER A few hepatic tumors have been induced in mice, rats, and ham¬ sters by various synthetic estrogens and progestins.45 A 20% to 30% incidence of liver tumor has been reported in hamsters after long-term administration of EE. However, in the presence of

2028

PART XV: HORMONES AND CANCER required for preneoplasia, and estrogen is required for full tumor development.52

PERINATAL EFFECTS Perinatal effects of estrogens have been studied extensively in the mouse.53 When these animals receive prenatal and neonatal exposure to DES or 17[}-E2, cervicovaginal adenosis and adeno¬ carcinomas occur in females, and testicular lesions occur in the rete testis of the males. The mechanism for these transplacental and perinatal effects remains to be elucidated.

HORMONES AS COCARCINOGENS OR PROMOTERS

FIGURE 222-2. Multistep model for estrogen-induced carcinogenesis in the Syrian hamster kidney. (E, estrogen; ER, estrogen receptor; E2F1, transcription factor E2F1; WT1, suppressor gene Wilm tumor 1.)

0.3% a-naphthoflavone (ANF) in the diet, or in a 20-mg pellet form, EE administration induced an 80% to 100% incidence of hepatocellular carcinomas in castrated male hamsters.46 Because ANF is not known to behave as a carcinogen or to possess sub¬ stantial mutagenic activity, the belief is that it modifies the metabolism of synthetic estrogens, thus enhancing their carcino¬ genicity by increasing the amount of the parent hormone. A cocarcinogenic role for ANF cannot be ruled out, however, in the induction of these hamster liver tumors.46

PROSTATE Long-term exposure of either Noble or Lobund Wistar rats to testosterone results in prostatic carcinomas.47 Tumor incidence was 50% when testosterone treatment was applied for 13 months and then Et was substituted for 6 months. Maximum tumor yields were obtained when testosterone plus 17(3-E2 was given for 19 months, with the tumor incidence approaching 90%.48 The resultant tumor nodules attained only microscopic proportions, however, somewhat limiting the usefulness of this model. Similar simultaneous exposure to testosterone plus 17(3-E2 for 4 months resulted in consistent dysplastic lesions in the dorsolateral lobe of the prostate in Noble rats.49 When tes¬ tosterone was replaced by dihydrotestosterone, the active androgen in many species, prostatic tumors were not seen.50 These data suggest that 17P-E9 may be involved in the etiology of these prostatic neoplasms, because testosterone is known to enhance proliferative activity at this organ site. Current evidence suggests that estrogen, acting on the androgensupported prostate, induces cell proliferation through a receptormediated process.51

DUCTUS DEFERENS AND SCENT GLAND As with tumors in the Noble rat prostate model, other hormoneinduced tumors also require the presence of two hormones, both estrogen and androgen. Examples are a leiomyosarcoma induced in the hamster ductus deferens31 and an unknown type of epithelial tumor induced in the scent gland after long-term coadministration of these gonadal hormones.52 Although, pres¬ ently, the relationship between these hormones in inducing these tumors is not well understood, the scent gland tumor is a particularly interesting model system because androgen is

Finally, hormones can act as cocarcinogens or promoters in con¬ junction with either physical carcinogens (e.g., ionizing radia¬ tion) or chemical carcinogens (DMBA, diethylnitrosamine [DEN], N-nitrosobutylurea [NBU]) at different organ sites. For example, either DES or EE plus x-ray treatments yields a high incidence of mammary tumors in female ACI rats, a rat strain that is relatively insensitive to radiation treatment alone.54 These same hormones are capable of promoting mammary tumors in other rat strains exposed to DMBA and DEN/NBU.55

IN VITRO CELL CULTURE MODELS Hormonal effects on in vitro cell transformation and mutagenic assays have important implications regarding the role of these substances in oncogenic processes. Hormonal agents have yielded some negative results in numerous in vitro tests, includ¬ ing lack of gene mutations in the Salmonella typhimurium assay. Positive findings in other in vitro cell assay systems are signifi¬ cant, however, and strongly suggest the possibility that hor¬ mones may possess epigenotoxic characteristics that could affect in vivo malignant cell transformation.56

SYRIAN HAMSTER EMBRYO CELL SYSTEM One of the most intensively studied in vitro assays is the Syrian hamster embryo (SHE) cells in culture.57 In this assay system, DES and some of its metabolites induce morphologic and neo-> plastic transformation of SHE cells. However, no detectable gene mutations at two genetic loci were found. In the presence of a rat postmitochondrial supernatant fraction, DES also induced unscheduled DNA synthesis.

BALB/C 3T3 CELL SYSTEM Another in vitro cell system that has been studied in consider¬ able detail is the BALB/c 3T3 cell system.58 In this system, 17P-E2, DES, and Ej induce a statistically significant cell trans¬ formation frequency. The natural steroidal estrogens require three- to five-fold higher concentrations than DES to induce an equivalent transformation frequency.

OTHER CELL SYSTEMS In other systems studied,59 DES induced gene mutations in mouse lymphoma cells in the presence of a rat liver postmito¬ chondrial supernatant, and unscheduled DNA synthesis in HeLa cells in the presence of the same postmitochondrial super¬ natant fraction. Sister chromatid exchanges have been induced in human fibroblasts and lymphocytes in culture by DES but not by 17p-E2.

Ch. 222: Hormones and Carcinogenesis: Laboratory Studies The major drawbacks of the cells used in most short-term systems include the fact that many are not primarily of epithelial origin, they are not considered target cells for sex hormones, and in many instances they are neoplastic.

METABOLISM AND COVALENT BINDING STUDIES Investigations in both animal models and short-term in vitro cell culture assays provide indirect evidence for the bioactivation of sex hormones as a pertinent aspect of hormone-induced tumor cell transformation. In this regard, estrogens have been more extensively studied. Based on numerous reports, no doubt exists that estrogens can form reactive species capable of covalent binding to cellular macromolecules.60 Whether such reactive intermediates have any involvement in initiating oncogenesis in whole animal systems remains controversial, however, because of the high microsomal protein and hormone concentrations required to demonstrate their formation.61

GROWTH FACTOR AND ONCOGENE INVOLVEMENT An analysis of carcinogenesis, especially hormonal carcinogen¬ esis, must include consideration of the possible role of growth factors and oncogenes in these processes.62 This is especially pertinent because many growth factors produced by normal cells are involved, singly or in combination with other mitogens, in the proliferation of specific target cells, both normal and neo¬ plastic. Growth factors such as insulin-like growth factors (IGF) and transforming growth factors (TGF) can be produced by tar¬ get cells. Also, the likelihood is that transformed cells may both synthesize and respond to growth factors and, consequently, proliferate independently through autocrine secretion. Thus, growth factors, which are basically peptide hormones, may be involved in the regulation of growth of both normal and neo¬ plastic endocrine tissues. In vitro studies with serum-free media have clearly shown the proliferative effects of epidermal growth factor (EGF) and TGF-a on endocrine target cells. Oncogenes, including cellular protooncogenes, are thought to play an important role in carcinogenesis in animals and humans, perhaps through their proliferative functions. Onco¬ genes could participate in carcinogenesis in several ways. Some of them possibly may be coding for growth factors or their receptors. The one gene of simian sarcoma virus (sis) is almost identical to a gene coding for a precursor of one polypeptide chain of platelet-derived growth factor (PDGF).63 The expres¬ sion of the c-sis protooncogene is known to be under androgenic control in a ductus deferens smooth muscle tumor cell line (DDTMF-2). Moreover, these cells synthesize and secrete a PDGF-like growth factor that is implicated in the autocrine reg¬ ulation of DDT:MF-2 cell proliferation.64 In addition, studies of the amino-acid sequence of immunoaffinity-purified EGF recep¬ tor have shown that the v-erb-B oncogene of avian erythroblas¬ tosis virus may encode for a truncated receptor lacking the external ligand-binding domain for EGF. These findings provide direct evidence that oncogenes may contribute to malignant cell transformation by inappropriate production of growth factors or through expression of uncontrolled growth factor receptor functions, causing unregulated cell proliferation. Although little is known about the expression of oncogenes by endocrine target cells, prolonged hormonal stimulation, a prerequisite for hormonal carcinogenesis, may cause inappro¬ priate gene overexpression, amplification, and suppression. For estrogens, immediate estrogen response genes (e.g., c-myc, c-fos, c-jun) may be overexpressed.7 Moreover, cell-cycle genes as well

2029

as their regulatory genes (e.g., pl6, p21, p27) may be deregulated and frequently overexpressed. In conclusion, many chemical and physical agents are known to be involved in carcinogenesis in animals and humans. These agents have been classified into various categories, such as initi¬ ators, promoters, cocarcinogens, and others. Hormones proba¬ bly possess one or more of these characteristics, depending on the experimental model system in question. A unique and fun¬ damental feature of carcinogenesis resulting from hormonal imbalance is the consistent finding that transformation usually follows a discrete pathway from normal cell hyperplasia to hormone-responsive and hormone-dependent neoplasia to hormone-independent neoplasia (i.e., autonomous tumors). Both hormone-induced and chemical carcinogen-induced tumors require a long latent period. Unlike hormonally induced cancers, however, cancers that are induced by chemical carcino¬ gens in endocrine glands or in their target tissues usually do not depend on hormones for their growth. An exception to this gen¬ eral rule is the chemical carcinogen-induced mammary cancer in rats. What the nature of hormonal involvement is and whether hormones have a direct or indirect influence in one or more parts of the sequence of events leading to carcinogenesis remain to be elucidated. However, hormone-mediated genomic instability may be a key element common to a number of differ¬ ent hormonally induced model systems at various organ sites.

REFERENCES 1. Colditz GA, Stampfer MJ, Willett WC, et al. Prospective study of estrogen replacement therapy and risk of breast cancer in postmenopausal women. JAMA 1990; 264:2648. 2. Toniolo PG. Endogenous estrogens and breast cancer risk: the case for pro¬ spective cohort studies. Environ Health Perspect 1997; 105:587. 3. Ross RK, Bernstein L, Lobo RA, et al. 5-Alpha-reductase activity and risk of prostate cancer among Japanese and U.S. white and black males. Lancet 1992; 339:887. 4. Colditz GA, Hankinson SE, Hunter DJ, et al. The use of estrogens and progestins and the risk of breast cancer in postmenopausal women. N Engl J Med 1995; 332:1589. 5. Hertz R. An appraisal of the concepts of endocrine influence on etiology, pathogenesis, and control of abnormal and neoplastic growth. Cancer Res 1957; 17:423. 6. Nandi S. Role of hormones in mammary neoplasia. Cancer Res 1978; 38:4046. 7. Hou X, Li JJ, Chen WB, et al. Estrogen-induced protooncogene and suppres¬ sor gene expression in the hamster kidney: significance for estrogen carcino¬ genesis. Cancer Res 1996; 56:2616. 8. Li JJ, Li SA. The effect of hormones on tumor induction. I. Brief overview of the endocrine system. II. Hormonal carcinogenesis. III. Effect of hormones on carcinogenesis by non-hormonal chemical agents. In: Arcos JC, Argus MF, Woo WT, eds. Chemical induction of cancer. Boston: Birkhauser, 1996:397. 9. Lacassagne A. Apparition de cancers de la mamelle chez la souris male, soumise a des injections de folliculine. Compt Rend Acad Sci 1932; 195:630. 9a. Lippert TH, Seeger H, Mueck AO. The impact of endogenous estradiol metabolites on carcinogenesis. Steroids 2000; 65:357. 10. Shull JD, Spady TJ, Snyder M, et al. Ovary-intact, but not ovariectomized female ACI rats treated with 17fS-estradiol rapidly develop mammary carci¬ nomas. Carcinogenesis 1997; 18:1595. 11. Li SA, Xue Y, Xie Q, et al. Serum and tissue levels of estradiol during estro¬ gen-induced renal tumorigenesis in the Syrian hamster. J Steroid Biochem Mol Biol 1994; 48:283. 12. Leav I, Ho S-M, Ofner P, et al. Biochemical alterations in sex hormone induced hyperplasia and dysplasia of the dorsolateral prostates of Noble rats. J Natl Cancer Inst 1988; 80:1045. 13. Rudali G, Coezy E, Frederic F, et al. Susceptibility of mice of different strains to the mammary carcinogenic action of natural and synthetic oestrogens. Prev Europ Etudes Clin Biol 1971; 16:425. 14. Blankenstein MA, Broerse JJ, van Zwieten MJ, et al. Prolactin concentration in plasma and susceptibility to mammary tumors in female rats from different strains treated chronically with estradiol 17[i. Breast Cancer Res Treat 1984; 4:137. 15. Thomas DB, Jimenez LM, McTieman A, et al. Breast cancer in men: risk fac¬ tors with hormonal implications. Am J Epidemiol 1992; 135:734. 16. Dunning WF, Curtis MR, Segaloff A. Strain differences in response to diethylstilbestrol and the induction of mammary gland and bladder cancer in the rat. Cancer Res 1947; 7:511 17. Cutts JH, Noble RL. Estrone-induced mammary tumors in the rat. I. Induc¬ tion and behavior of tumors. Cancer Res 1964; 24:1116.

2030

PART XV: HORMONES AND CANCER

18. Nandi S, Yang J, Guzman R. Hormones and the cellular origin of mammary cancer: a unifying hypothesis. In: Li JJ, Li SA, Gustafsson JA, et al., eds. Hor¬ monal carcinogenesis, vol II. New York: Springer-Verlag, 1996:11. 19. Hannouche N, Samperez S, Riviere MR, Jouan P. Estrogen and progesterone receptors in mammary tumors induced in rats by simultaneous administra¬ tion of 17(J-estradiol and progesterone. J Steroid Biochem 1982; 17:415. 20. Lanari C, Molinolo AA, Dosne Pasqualini C. Induction of mammary adeno¬ carcinomas by medroxyprogesterone acetate in BALB/c female mice. Can¬ cer Lett 1986; 33:215. 21. Varela, RM, Dao TL. Estrogen synthesis and estradiol binding by human mammary tumors. Cancer Res 1978; 38:2429. 22. Longcope C, Femino A, Johnston ON. Androgen and estrogen dynamics in the female baboon (Papio anubis). J Steroid Biochem 1988; 31:195. 23. Brodie AMH, Hammond JO, Ghosh M, et al. Effect of treatment with aromatase inhibitor 4-hydroxyandrostenedione on the nonhuman primate menstrual cycle. Cancer Res 1989; 49:4780. 24. Dukes M, Edwards PN, Large M, et al. The preclinical pharmacology of "Arimidex" (Anastrozole; ZD1033)—a potent, selective aromatase inhibi¬ tor. J Steroid Biochem Mol Biol 1996; 58:439. 25. Lipschutz A, Iglesias R, Pamosevick V, et al. Ovarian tumors and other ova¬ rian changes induced in mice by two 19-nor contraceptives. Br J Cancer 1967; 21:153. 26. Biskind GR, Kordan B, Biskind MS. Ovary transplanted to spleen in rats: the effect of unilateral castration, pregnancy, and subsequent castration. Cancer Res 1950; 10:309. 27. Griffiths CT. Effects of progestins, estrogens, and castration on induced endometrial cancer in rabbits. Surg Forum 1963; 14:399. 28. Highman B, Greeman DL, Norvell MJ, et al. Neoplastic and preneoplastic lesions induced in female C3H mice by diets containing diethylstilbestrol or 17(3-estradiol. J Environ Path Toxicol 1980; 4:81. 29. Newbold RR, Bulock BC, McLachlan JA. Uterine adenocarcinomas in mice following developmental treatment with estrogens: a model for hormonal carcinogenesis. Cancer Res 1991; 50:7677. 30. Leavitt WW, Evans RW, Hendry WJ III. Etiology of DES-induced uterine tumors in Syrian hamster. In: Leavitt WW, ed. Hormones and cancer. New York: Plenum Publishing, 1982:63. 31. Kirkman H, Algard FT. Characteristics of an androgen / estrogen-induced uter¬ ine smooth muscle cell tumor of the Syrian hamster. Cancer Res 1970; 30:794. 32. McClure HM, Graham CE. Malignant uterine mesotheliomas in squirrel monkeys following diethylstilbestrol administration. Lab Animal Sci 1973; 23:493. 33. Bischoff F. Carcinogenic effects of steroids. Adv Lipid Res 1969; 7:165. 34. Banerjee SK, Zoubine MH, Sarkar DK, et al. 2-Methoxy estradiol blocks estrogeninduced rat pituitary tumor growth and tumor angiogenesis: possible role of vascular endothelial growth factor. Anticancer Res 2000; 20:2641. 35. Nakagawa K, Ohara T, Tashiro K. Pituitary hormones and prolactin-releas¬ ing activity in rats with primary estrogen-induced pituitary tumors. Endo¬ crinology 1980; 106:1033. 36. Koneff AA, Simpson ME, Evans HM. Effect of chronic administration of diethylstilbestrol on the pituitary and other endocrine organs of hamsters. Anat Rec 1946; 94:169. 37. Bonser GM, Robison JM. The effects of prolonged estrogen administration upon male mice of various strains: development of testicular tumors in the strong A strain. J Pathol Bacteriol 1940; 51:9. 38. Sato B, Spomer W, Huseby RA, Samuels LT. The testicular estrogen receptor system in two strains of mice differing in susceptibility to estrogen-induced Leydig cell tumors. Endocrinology 1979; 104:822. 39. Kirkman H. Estrogen-induced tumors of the kidney. III. Growth character¬ istics in the Syrian hamster. Natl Cancer Inst Monogr 1959; 1:1. 40. Li JJ, Cuthbertson TL, Li SA. Inhibition of estrogen carcinogenesis in the Syr¬ ian golden hamster kidney by antiestrogens. J Natl Cancer Inst 1980; 64:795. 41. Li JJ, Hou X, Bentel JM, et al. Prevention of estrogen carcinogenesis in the hamster kidney by ethynylestradiol: some unique properties of a synthetic estrogen. Carcinogenesis 1998; 19:471. 42. Oberley TD, Gonzalez A, Lauchner LJ, et al. Characterization of early lesions in estrogen-induced renal tumivs in the Syrian hamster. Cancer Res 1991; 51:1922. 43. Li JJ, Li SA, Klicka JK, et al. Relative carcinogenic activity of various syn¬ thetic and natural estrogens in the hamster kidney. Cancer Res 1983; 43:5200. 44. Li SA, Hou X, Li JJ. Estrogen carcinogenesis: a sequential, epi-genotoxic multi-stage process. In: Li JJ, Li SA, Gustafsson JA, et al., eds. Hormonal car¬ cinogenesis, vol II. New York: Springer-Verlag, 1996:200. 45. Li JJ, Kirkman H, Li SA. Synthetic estrogens and liver cancer: Risk analysis of animal and human data. In: Li JJ, Nandi S, Li SA, eds. Hormonal carcino¬ genesis, New York: Springer-Verlag, 1992:217. 46. Li JJ, Li SA. High incidence of hepatocellular carcinoma after synthetic estrogen administration in Syrian hamsters fed a-naphthoflavone: a new tumor model. J Natl Cancer Inst 1984; 73:543. 47. Drago JR. The induction of Nb rat prostatic carcinomas. Anticancer Res 1984; 4:255. 48. Ofner P, Bosland MC, Vena RL. Differential effects of diethylstilbestrol and estradiol-17p in combination with testosterone on rat prostate lobes. Toxicol Appl Pharmacol 1992; 112:300. 49. Bruchovsky N, Lesser B. Control of proliferative growth in androgen responsive organs and neoplasms. Adv Sex Horm Res 1976; 2:1.

50. Pollard M, Snyder DL, Lochert PH. Dihydrotestosterone does not induce prostate adenocarcinoma in L-W rats. Prostate 1987; 10:325. 51. Ho S-M, Yu M, Leav I, Viccione T. The conjoint action of androgens and estrogens in the induction of proliferative lesions in the rat prostate. In: Li JJ, Nandi S, Li SA, eds. Hormonal carcinogenesis. New York: SpringerVerlag, 1992:18. 52. Kirkman, H, Algard FT. Androgen-estrogen-induced tumors I. The flank organ (scent glandj-chaetepithelioma of the Syrian hamster. Cancer Res 1964; 24:1569. 53. Bern HA, Talamantes FJ. Neonatal mouse models and their relation to dis¬ ease in the human female. In: Herbst SL, Bern HA, eds. Developmental effects of diethylstilbestrol (DES) in pregnancy. New York: Thieme-Stratton, 1981:129. 54. Holtzman S, Stone JP, Shellabarger CJ. Synergism of estrogens and x-rays in mammary carcinogenesis in female ACI rats. J Natl Cancer Inst 1981; 67:455. 55. Russo IH, Russo J. Mammary gland neoplasia in long-term rodent studies. Environ Health Perspect 1996; 104:938. 56. Li JJ. Perspectives in hormonal carcinogenesis: animal models to human disease. In: Huff J, Boyd J, Barrett JC, eds. Cellular and molecular mecha¬ nisms in hormonal carcinogenesis: environmental influences. Philadelphia: Wiley-Liss 1996:447. 57. Tsutsui T, Degen GH, Schiffmann D, et al. Dependence on exogenous meta¬ bolic activation for induction of unscheduled DNA synthesis in Syrian ham¬ ster embryo cells by diethylstilbestrol and related compounds. Cancer Res 1984; 44:184. 58. Friedrich U, Thomale J, Nass G. Induction of malignant transformation by various chemicals in BALB/3T3 clone A31-1-1 cells and biological charac¬ terization of some transformants. Mutation Res 1985; 152:113. 59. Rao PN, Engelberg J. Structural specificity of estrogens in the induction of mitotic chromatid non-disjunction in HeLa cells. Exp Cell Res 1967; 48:71. 60. Tsibris JCM, McGuire PM. Microsomal activation and binding to nucleic acids and proteins. Biochem Biophys Res Commun 1977; 78:411. 61. Beleh MA, Lin YC, Brueggemeier RW. Estrogen metabolism in microsomal, cell, and tissue preparations from kidney and liver from Syrian hamster. J Steroid Biochem Mol Biol 1995; 52:479. 62. Schavorovsky OG, Rozados VR, Gervasoni SI, Matar P. Inhibition of ras onco¬ gene: a novel approach to antineoplastic therapy. J Biomed Sci 2000; 7:292. 63. Waterfield MD. Oncogenes may encode a growth factor or part of the recep¬ tor for a growth factor. Br J Cancer 1984; 50:242. 64. Smith RG, Nag A, Syms AJ, Norris JS. Steroid regulation of receptor concen¬ tration and oncogene expression. J Steroid Biochem 1986; 24:51.

CHAPTER

223

SEX HORMONES AND HUMAN CARCINOGENESIS: EPIDEMIOLOGY ROBERT N. HOOVER Because of the central role that the hormonal milieu plays in var¬ ious carcinogenic processes, clinical endocrinologists must be aware of malignancies to which their patients may be predis¬ posed, either because of the nature of their illness or because of the nature of the hormonal therapy being instituted.

CARCINOGENESIS AND ENDOGENOUS SEX HORMONE STATUS Endogenous hormone status has long been thought to be an important factor in the etiology of a number of human malig¬ nancies. This belief has been based on animal carcinogenesis studies (see Chap. 222), the responsiveness of a number of tumors to hormonal manipulation (see Chaps. 224 and 225), the relationship of risk of certain tumors to a variety of reproductive and other factors thought to influence hormonal status, and the simple fact that some organs depend on hormonal status for their normal function.1 Speculation about a causal role for hor-

2031

Ch. 223: Sex Hormones and Human Carcinogenesis: Epidemiology mones has focused on malignancies of the female breast and the reproductive tract. Some evidence for hormonal carcinogenesis has been observed for a variety of other tumors, however, including prostate, liver, testis, thyroid, and gallbladder cancers, and malignant melanoma. Despite these long-standing suspi¬ cions, little success has been achieved in identifying the specific hormonal factors that might be responsible for these tumors, with the possible exception of endometrial cancer.

CARCINOGENESIS AND EXOGENOUS SEX HORMONE THERAPY Within the last 50 years, a new element in the area of hormonal influences on cancer risks has been added, that of exogenous sex hormone exposure. Pharmacologic levels of estrogens, progestins, androgens, and pituitary trophic hormones, alone or in combina¬ tion, have been administered to large segments of the population for various reasons. These large-scale "natural experiments" have provided more specific insights into the relationship between hormonal factors and several different malignancies.2 Moreover, enthusiasm has grown for the widespread treatment of relatively healthy segments of the population (e.g., women receiving oral contraceptive agents or menopausal replacement therapy). Con¬ siderable interest has arisen in the use of estrogens for post¬ menopausal prevention of osteoporosis and osteoporotic fractures3 (see Chaps. 64 and 100). Some evidence supports the long-suspected potential of menopausal estrogens to prevent clinical coronary heart disease.4 In addition, within the general population, a substantial increase has been seen in the use of dietary supplements, many of which have significant hormonal activity (e.g., androstenedione, melatonin). Because of this enthu¬ siasm on the part of physicians and the public, appropriate eval¬ uations of the carcinogenic consequences of these exposures have become important to public health, as well as to understanding the biology of the tumors involved.

ENDOMETRIAL CANCER ENDOGENOUS FACTORS IN ENDOMETRIAL CANCER The cancer for which the evidence for both an endogenous and an exogenous hormonal cause is best established is endometrial cancer. Various factors related to endogenous hormone production have been associated with endometrial cancer.5 Medical conditions related to increased risk include functional (estrogen-secreting) ovarian tumors, the polycystic ovary syndrome, diabetes mellitus, and hypertension. Reproductive factors, including nullipar¬ ity and a late natural menopause, also have consistently been found to be related to increased risk. Some dietary factors also seem to influence risk. Obesity is a risk factor and a vegetarian diet is a possible protective factor.6 Age, a determinant of levels of most endogenous hormones, also influences endometrial cancer risk in a unique manner. Endometrial cancer rates are extremely low in women younger than 45 years of age, rise precipitously among women in their late 40s and throughout their 50s (much more dramatically than for other tumors), and then decline in women approximately age 60 and older (Fig. 223-1).

EXOGENOUS SEX HORMONES AND ENDOMETRIAL CANCER Exposure to exogenous hormones also has been linked to endometrial cancer.5

FIGURE 223-1.

Age-specific incidence rates for breast and uterine cor¬

pus cancers among white women during 1986 through 1990. (Data from the Surveillance, Epidemiology and End Results Program.)

ESTROGENS AND ENDOMETRIAL CANCER Estrogen-replacement therapy of 2 years or longer for menopausal women is associated with an excess relative risk of endometrial can¬ cer. Table 223-1 shows estimated relative risks (i.e., the risk of the disease among those exposed to estrogen therapy compared with the risk among those not exposed).7-14 The relative risk among users compared with nonusers ranges from two-fold to eight-fold. It increases even further with long duration of use and with high average daily doses. Thus far, every type of estrogen that has been investigated has shown this relationship, including conjugated equine estrogens, ethinyl estradiol, and diethylstilbestrol (DES). The highest risk occurs among current users. The risk declines with each year after cessation of use, although apparently some residual excess risk is present even 10 years after cessation. The risk is highest for early-stage malignancies, but a two-fold to three¬ fold excess risk is seen for the advanced stages of disease as well. EFFECT OF ESTROGEN AND PROGESTERONE IN SEQUENCE A profound trend has been seen away from unopposed estrogen treatment of menopausal symptoms and toward treatment with

TABLE 223-1. Relative Risks* of Endometrial Cancer Associated with Menopausal Estrogen Use from Selected Case-Control Studies Over¬ all RR

RR among LongTerm Users+

Health plan

7.6

13.9

Retirement community

5.6

8.8

Gray9

Private practice

3.1

11.6

Pike et al.]0

Community

2.1

24.2

Green et al.11

General population

3.7

16.3

Hulka et al.12

Gynecology patients

1.8

4.1

Shapiro et al.13

Hospital patients

3.9

6.0

Brinton et al.14

Community

3.0

6.0

Reference

Source of Controls

Ziel and Finkle7 Mack et al.8

RR, relative risk. *Risk of cancer relative to a risk of 1.0 for women who never used menopausal estrogens. ^Definition of long-term varied from >5 to >15 years.

2032

PART XV: HORMONES AND CANCER

a sequence of an estrogen that is then combined with a proges¬ tin. Substantial evidence15 indicates that such cyclic treatment reduces the frequency of hyperplasia and atypical hyperplasia associated with unopposed estrogen treatment. Although the epidemiologic data concerning endometrial cancer risk are still developing, certain patterns are emerging. The risk of endome¬ trial cancer is lower among women using the combined regimen than among women using estrogen alone.10,16 Evidence implies that, at least in the short term, the risk is related to the number of days that a progestin is used with estrogen in a monthly cycle. Those using the progestin for >10 days per month, including those using the combined regimen continuously, have a risk similar to that of women not using any hormone-replacement therapies. Those using progestins for 75% in 70% to 80% of cases and reduced tumor masses in many patients with advanced prostate cancer.86-87 Toxicity can be severe, however; therefore, patients should be considered for suramin therapy only in the context of clinical trials. Encouraging results have been obtained with combinations of estramustine and either vinblastine, etoposide, or paclitaxel. Preclinical data suggest that these combinations may exert their effects through inhibition of microtubule function.86

2056

PART XV: HORMONES AND CANCER

REFERENCES 1. Parker SL, Tong T, Bolden S, et al. Cancer Statistics 1996. CA Cancer J Clin 1996; 46:5. 2. Welsch CW. Host factors affecting the growth of carcinogen-induced rat mammary carcinomas: a review and tribute to Charles Brenton Huggins. Cancer Res 1985; 45:3415. 3. Holund B. Latent prostatic cancer in consecutive autopsy series. Scand J Urol Nephrol 1980; 14:29. 4. Catalona WJ, ed. Epidemiology and etiology. In: Prostate cancer. New York: Grune & Stratton, 1984:1. 4a. Ozen M, Pathak S. Genetic alterations in human prostate cancer: a review of current literature. Anticancer Res 2000; 20:1905. 5. Viola MV, Fromowitz F, Oravez S, et al. Expression of ras oncogene p21 in prostate cancer. N Engl J Med 1986; 314:133. 6. Gao X, Honn KV, Grignon D, et al. Frequent loss of expression and loss of heterozygosity of the putative tumor suppressor gene DCC in prostate car¬ cinomas. Cancer Res 1993; 53:2723. 7. Gleason DF. Histologic grading and clinical staging of carcinoma of the prostate. In: Tannenbaum M, ed. Urologic pathology: the prostate. Philadel¬ phia: Lea & Febiger, 1977:171. 8. Gleason DF, Mellinger GT, Veterans Administration Cooperative Urological Research Group. Prediction of prognosis for prostatic adenocarcinoma by combined histological grading and clinical staging. J Urol 1974; 111:58. 9. Catalona WJ, ed. Pathology. In: Prostate cancer. New York: Grune & Strat¬ ton, 1984:15. 10. Diamond DA, Berry SJ, Umbricht C, et al. Computerized image analysis of nuclear shape as a prognostic factor for prostatic cancer. Prostate 1982; 3:321. 11. Nativ O, Winkler HZ, Raz Y, et al. Stage C prostatic adenocarcinoma: flow cytometric nuclear DNA ploidy analysis. Mayo Clin Proc 1989; 64:911. 12. Winkler HZ, Rainwater LM, Myers RP, et al. Stage D1 prostatic adenocarci¬ noma: significance of nuclear DNA ploidy patterns studied by flow cytom¬ etry. Mayo Clin Proc 1988; 63:103. 13

Bonkhoff H, Stein U, Welter C, et al. Differential expression of the pS2 pro¬ tein in the human prostate and prostate cancer: association with malignant changes and neuroendocrine differentiation. Hum Pathol 1995; 26:824. 14. Carter HB. Current status of PSA in the management of prostate cancer Adv Surg 1994; 27:81. 14a. Lavoipierre AM. Ultrasound of prostate and testicles. World J Surg 2000' 24:198. 15. Donohue RE, Mani JH, Whitesel JA, et al. Pelvic lymph node dissection: guide to patient management in clinically locally confined adenocarcinoma of prostate. Urology 1982; 20:559. 16. Bonkhoff H, Remberger K. Differential pathways and histologenetic aspects of normal and abnormal prostatic growth: a stem cell model. Prostate 1996; 28:98. 17. Garabedian EM, Hunphrey PA, Gordon JI. A transgenic mouse model of metastatic prostate cancer originating from neuroendocrine cells. Proc Natl Acad Sci U S A 1995; 26:15382. 18. Krijen JL, Bogdanowicz JF, Seldenrijk C A, et al. The prognostic value of neu¬ roendocrine differentiation in adenocarcinoma of the prostate in relation to progression of disease after endocrine therapy. J Urol 1997; 158:171. 19. Isaacs JT, Coffey DS. Adaptation versus selection as the mechanism respon¬ sible for the relapse of prostatic cancer to androgen ablation therapy as studied in the Dunning R3327H adenocarcinoma. Cancer Res 1981; 41:5070. 20. Partin AW, Coffey DS. Benign and malignant prostatic neoplasms: human studies. Recent Prog Horm Res 1994; 49:293. 21. The Leuprolide Study Group. Leuprolide vs diethylstilbestrol for metastatic prostatic cancer. N Engl J Med 1984; 311:1281. 22. Parmar H, Phillips RH, Lightman SL, et al. Randomized controlled study of orchidectomy vs. long-acting D-Trp-6-LHRH microcapsules in advanced prostatic cancer. Lancet 1985; 2:1201. 23. Fowler JE Jr, Whitmore WF Jr. The response of metastatic adenocarcinoma of the prostate to exogenous testosterone. J Urol 1981; 126:372. 24. Visakorpi T, Kallioniemi AH, Syvanen AC, et al. Genetic changes in primary and recurrent prostate cancer by comparative genomic hybridization. Can¬ cer Res 1995; 55:342. 25. Kibel AS, Schutte M, Kern SE, et al. Identification of 12p as a region of fre¬ quent deletion in advanced prostate cancer. Cancer Res 1998; 58:5652. 26. Meyers FJ, Gumerlocj PH, Chi SG, et al. Very frequent p53 mutations in met¬ astatic prostate carcinoma and in matched primary tumors. Cancer 1998; 83:2534. 27. Ueda T, Ichikawa T, Tamaru J, et al. Expression of KA1 protein in benign prostatic hyperplasia and prostate cancer. Am J Pathol 1996; 149:1435. 28. Lu J, Danielsen M. Differential regulation of androgen and glucocorticoid receptors by retinoblastoma protein. J Biol Chem 1998; 273:31528. 29. Sementchenko VI, Schweinfest CW, Papas TS, et al. ETS2 function is required to maintain the transformed state of human prostate cancer cells. Oncogene 1998; 17:2883. 30. Wolf RM, Schneider SL, Pontes JE, et al. Estrogen and progestin receptors in human prostatic carcinoma. Cancer 1985; 55:2477. 31. Glick JH, Wein A, Padavic K, et al. Phase II trial of tamoxifen in metastatic carcinoma of the prostate. Cancer 1982; 49:1367. 32. Klugo RC, Farrah RN, Cerny JC. Bilateral orchiectomy for carcinoma of prostate: response to serum testosterone and clinical response to subse¬ quent estrogen therapy. Urology 1981; 17:49.

33. Cohen P, Peehl DM, Stamey TA, et al. Elevated levels of insulin-like growth factor-binding protein-2 in the serum of prostate cancer patients. J Clin Endocrinol Metab 1993; 76:1031. 34. Santen RJ. The testis. In: Felig P, Baxter J, Broadus A, Frohman L, eds. Endo¬ crinology and metabolism, 2nd ed. New York: McGraw-Hill, 1986:821. 35. Sanford EJ, Paulsen DF, Rohner TJ, et al. The effects of castration on adrenal testosterone secretion in men with prostatic carcinoma. J Urol 1977; 118:1019. 36. Harper ME, Pike A, Peeling WB, Griffiths K. Steroids of adrenal origin metabolized by human prostatic tissue both in vivo and in vitro. J Endo¬ crinol 1984; 60:117. 37. Geller J. Rationale for blockade of adrenal as well as testicular androgens in the treatment of advanced prostate cancer. Semin Oncol 1985; 12(Suppl 1):28. 38. Connolly JG, Mobbs EG. Clinical applications and value of receptor levels in treatment of prostate cancer. Prostate 1984; 5:477. 39. Trachtenberg J, Walsh PC. Correlation of prostatic nuclear androgen-receptor content with duration of response and survival following hormonal ther¬ apy in advanced prostatic cancer. J Urol 1982; 127:466. 40. Ghanadian R, Auf G, Williams G, et al. Predicting the response of prostatic carcinoma to endocrine therapy. Lancet 1981; 2:1418. 41. Byar DP. The Veterans Administration Cooperative Urological Group's studies of cancer of the prostate. Cancer 1973; 32:1126. 42. Byar DP, Corle DK. Hormone therapy for prostate cancer: results of the Vet¬ erans Administration Cooperative Urological Research Group studies. In: National Cancer Institute Monographs, no 7. Washington: US Government Printing Office, 1988:165. 43. Zincke H, Bergstralh EJ, Larson-Keller JJ, et al. Stage D1 prostate cancer treated by radical prostatectomy and adjuvant hormonal treatment. Cancer 1992; 70(Suppl):311. 44. Catalona WJ, ed. Endocrine therapy. In: Prostate cancer. New York: Grune & Stratton, 1984:145. 45. Beck PH, McAninch JW, Goebel JL, Stutzman RE. Plasma testosterone in patients receiving diethylstilbestrol. Urology 1978; 11:157. 46. Glashan RW, Robertson MRG. Cardiovascular complications in the treat¬ ment of prostatic carcinoma. Br J Urol 1981; 53:624. 47. Santen RJ, Manni A, Harvey H. Gonadotropin releasing hormone (GnRH) analogs for the treatment of breast and prostatic carcinoma. Breast Cancer Res Treat 1986:129. 48. Warner B, Worgul TJ, Drago J, et al. Effects of very high-dose D-leucine-6gonadotropin-releasing hormone proethylamide on the hypothalamic-pitu¬ itary testicular axis in patients with prostatic cancer. J Clin Invest 1983; 71:1842. 49. Santen RJ, Demers LM, Max DT, et al. Long-term effects of administration of a gonadotropin-releasing hormone superagonist analog in men with prostatic carcinoma. J Clin Endocrinol Metab 1984; 58:397. 50. Evans RM, Doelle GC, Lindner J, et al. A luteinizing hormone releasing hor¬ mone agonist decreases biologic activity and modifies chromatographic behavior of luteinizing hormone in man. J Clin Invest 1984; 73:262. 51. Santen RJ, English HF, Warner BA. GnRH superagonist treatment of pros¬ tate cancer: hormonal effects with and without an androgen biosynthesis inhibitor. In: Labrie F, Belanger A, DuPont A, eds. LHRH and its analogues: basic and clinical aspects. Amsterdam: Excerpta Medica, 1984:336. 52. Ahmed SR, Grant J, Shalet SM, et al. Preliminary report on use of depot for¬ mulation of LHRH analog, ICI 118630 (Zoladex) in patients with prostatic cancer. BMJ 1985; 289:185. 53. Debruyne FM, Denis L, Lunglmayer G, et al. Long-term therapy with a ' depot luteinizing hormone-releasing hormone analogue (Zoladex) in patients with advanced prostatic carcinoma. J Urol 1988; 140:775. 54. Ahmann FR, Citrin DL, deHaan HA, et al. Zoladex: a sustained-release, monthly luteinizing hormone-releasing analogue for the treatment of advanced prostate cancer. J Clin Oncol 1987; 5:912. 55. Kruger H, Mohring K, Dorsam J, Vecsei P. The slow release form of leupro¬ lide (TAP 144 SR) in the treatment of prostatic carcinoma: a hormonal pro¬ file. In: Proceedings of International Symposium on Endocrine Therapy, Monaco, November 19-21,1988. Abstract A16. 56. Denis L, Mahler D. Ketoconazole in the treatment of prostate cancer. In: International Symposium on Hormonal Manipulation of Cancer: peptides, growth factors and new antisteroidal agents, June 4-6,1986. 57. Sogani PC, Whitmore WF Jr. Experience with flutamide in previously untreated patients with advanced prostatic cancer. J Urol 1979; 122:640. 58. Smith RB, Walsh PC, Goodwin WE. Cyproterone acetate in the treatment of advanced carcinoma of the prostate. J Urol 1973; 110:106. 59. Beurton D, Grail J, Davody P, Cukier J. Treatment of prostatic cancer with cyproterone acetate as monotherapy. Prog Clin Biol Res 1987; 243A:369. 60. Visakorpi T, Hyytinen E, Kovisto P, et al. In vivo amplification of androgen receptor gene and progression of human prostate cancer. Nat Genet 1995; 9:401. 61. Koivisto P, Kolmer M, Visakorpi T, et al. Androgen receptor gene and hor¬ monal therapy failure of prostate cancer. Am J Pathol 1998; 152:1. 62. Brendler H. Adrenalectomy and hypophysectomy for prostatic cancer. Urology 1973; 2:99. 63. Worgul TJ, Santen RJ, Samojlik E, et al. Clinical and biochemical effect of aminoglutethimide in the treatment of advanced prostatic carcinoma. J Urol 1983; 129:51. 64. Santen RJ, English H, Rohner T, et al. Androgen depletion/repletion in com¬ bination with chemotherapy: strategy for secondary treatment of metastatic

Ch. 226: Endocrine Consequences of Cancer Therapy

65. 66.

67. 68. 69. 70. 71.

72.

73.

74. 75.

76. 77.

78.

79.

80.

81.

82.

83. 84. 85. 86.

87.

prostatic cancer. In: Schroeder FH, Richards B. eds. EORTC Genitourinary Group monograph 2 (part A): Therapeutic principles in metastatic prostatic cancer. New York: Alan R Liss, 1985:359. Sogani PC, Ray B, Whitmore WF Jr. Advanced prostatic carcinoma: flutamide therapy after conventional endocrine treatment. Urology 1975; 6:164. Trump DL, Havlin KH, Messing EM, et al. High-dose ketoconazole in advanced hormone-refractory prostate cancer: endocrinologic and clinical effects. J Clin Oncol 1989; 7:1093. Susan LP, Roth RB, Adkins WC. Regression of prostatic cancer metastasis by high doses of diethylstilbestrol diphosphate. Urology 1976; 7:598. Benson RC, Wear JB, Gill GM. Treatment of Stage D hormone resistant car¬ cinoma of the prostate with estramustine phosphate. J Urol 1979; 121:452. Eisenberger MA. Chemotherapy for prostate carcinoma. National Cancer Institute Monographs, no 7. Washington, DC: US Government Printing Office, 1988:151. Klotz L. Hormone therapy for patients with prostate carcinoma. Cancer 2000; 88(12 Suppl):3009. Crawford CD, Eisenberger MA, McLeod DG, et al. A controlled trial of leuprolide with and without flutamide in prostatic carcinoma. N Engl J Med 1989; 321:419. Brisset JM, Bertagna C, Fist J, et al. Total androgen blockade vs. orchiectomy in stage D prostate cancer. Monograph Series of European Organization, Research and Treatment, 1987:17. Eisenberger M, Crawford Ed, McCleod D, et al. A comparison of leuprolide and flutamide vs. leuprolide alone in newly diagnosed stage D2 prostate cancer. (Abstract). Proc Am Soc Clin Oncol 1992; 11:201. Denis L, Mettlin C. Conclusions. Cancer 1990; 66(Suppl):1086. Beland G, Elhilai M, Fradet Y, et al. A controlled trial of castration with and without nilutamide in metastatic prostatic carcinoma. Cancer 1990; 60(Suppl):1074. Crombie C, Raghaven D, Page J, et al. Phase II study of megestrol acetate for metastatic carcinoma of the prostate. Br J Urol 1987; 59:443. Loeng SA, Beckley S, Brandy MF, et al. Comparison of estramustine phos¬ phate, methotrexate and ds-platinum in patients with advanced, hormone refractory prostate cancer. J Urol 1983; 129:1001. Pienta KJ, Reidman B, Hussian M. Phase II evaluation of oral estramustine and oral etoposide in hormone-refractory adenocarcinomas of prostate. J Clin Oncol 1994; 12:2005. English HF, Heitjan DF, Lancaster, et al. Beneficial effects of androgenprimed chemotherapy in the Dunning R3327 G model of prostatic cancer. Cancer Res 1991; 51:1760. Manni A, Bartholomew M, Caplan R, et al. Androgen priming and chemo¬ therapy in advanced prostate cancer. Evaluation of determinants of clinical outcome. J Clin Oncol 1988; 6:1456. Seifter EJ, Bunn PA, Cohen MH, et al. A trial of combination chemotherapy followed by hormonal therapy for previously untreated metastatic carci¬ noma of prostate. J Clin Oncol 1986; 4:1365. Osborne CK, Blumenstein B, Crawford ED, et al. Combined versus sequen¬ tial chemo-endocrine therapy in advanced prostate cancer: final results of a randomized Southwest Oncology Group study. J Clin Oncol 1990; 8:1675. Feldman D, Zhao X-Y, Krishan AV. Editorial mini-review: vitamin D and prostate cancer. Endocrinology 2000; 141:5. Clarke NW, Holbrook IB, McClure J, et al. Osteoclast inhibition by pamidronate in metastatic prostate cancer: a preliminary study. Br J Cancer 1991; 63:420. Small EJ, Meyer M, Marshall ME, et al. Suramin therapy for patients with symptomatic hormone-refractive prostate cancer. J Clin Oncol 2000; 18:1440. Myers C, Cooper M, Stein C, et al. Suramin: a novel growth factor antago¬ nist with activity in hormone-refractory metastatic prostate cancer. J Clin Oncol 1992; 10:881. Eisenberger MA, Reyno LM, Jodrell DI, et al. Suramin, an active drug for prostate cancer: interim observations in a Phase I trial. J Natl Cancer Inst 1993; 85:611.

CHAPTER

226

ENDOCRINE CONSEQUENCES OF CANCER THERAPY DAIVA R. BAJORUNAS Survival statistics for cancer have improved dramatically dur¬ ing the past three decades. Overall, with aggressive multimodal¬ ity therapy, nearly 60% of newly diagnosed cancer patients can expect to survive beyond 5 years after diagnosis. Clinical cancer research efforts, therefore, increasingly focus on longer-term medical, psychologic, and economic effects of such treatment.

2057

Endocrine system dysfunction as a consequence of chemother¬ apy, radiotherapy, and immunotherapy for malignancies is recog¬ nized with increasing frequency.1 Prominent among acute effects are disordered glucose and mineral metabolism, hyperlipidemia, and cytokine-induced autoimmune thyroiditis (Tables 226-1 and 226-2). Long-term adverse effects of chemotherapy and radiother¬ apy on hypothalamic-pituitary, thyroid, parathyroid, and gonadal function continue to be well described in the literature, and the multifactorial nature of osteopenia in cancer survivors is being defined. These late sequelae remain the most clinically rel¬ evant endocrine complications of cancer therapy; they have prompted vigorous efforts at early detection and treatment as well as a search for measures to reduce or prevent such morbidity.

THERAPY-INDUCED HYPOTHALAMICPITUITARY GLAND DYSFUNCTION Iatrogenic hypothalamic-pituitary dysfunction commonly occurs after cranial irradiation for leukemia and tumors of the head and neck region, or with total body irradiation (TBI) in bone marrow transplantation (BMT). In patients receiving incidental hypotha¬ lamic-pituitary axis (HPA) irradiation in doses of up to 70 Gy, the risk of endocrine deficiencies may exceed 90%.2 Growth hormone (GH) secretion is the first to fail after cranial radiation, and, in chil¬ dren, GH deficiency and premature sexual development are the most common therapy-induced neuroendocrine problems.2-3 In adults, hyperprolactinemia is commonly observed, and complete or partial gonadotropin deficiency, tertiary hypothyroidism, or decreased thyrotropin (thyroid-stimulating hormone, TSH) reserve occur progressively in descending order of frequency. Whereas clinically significant adrenal insufficiency is relatively uncommon, subtle abnormalities in adrenal function may be present in up to one-third of patients. More than one-half of irradiated patients have manifest dysfunction of multiple endocrine axes. Inexplica¬ bly, diabetes insipidus does not occur after external radiation, even in patients with therapy-induced panhypopituitarism. The available data indicate that the hypothalamus is more commonly the site of damage than is the pituitary.2 Hypotha¬ lamic blood flow is reduced after cranial irradiation,4 and incongruent GH secretory dynamics (normal responses to provocative stimuli, subnormal spontaneous secretion) and dose-dependent alteration in circadian/ultradian GH rhythms and pulsatility5 suggest neurosecretory or regulatory dysfunction. Anterior pitu¬ itary responses to testing with hypothalamic peptides are charac¬ teristic of pathologically documented hypothalamic disease, and satisfactory end-organ responses to long-term treatment with gonadotropin- or GH-releasing hormone (GnRH, GHRH) have been described.6-7 Data8 suggest that a hierarchy of sensitivity to radiation damage exists, with the most vulnerable being extrahypothalamic neurotransmitter control of GH. Both the total dose of cranial radiation and the fraction sizes determine the frequency of neuroendocrine dysfunction. GH deficiency and precocious puberty have been noted after con¬ ventional fractionated HPA irradiation with doses of >18 Gy, and after total doses as low as 9 to 10 Gy when given in a single dose, as in TBI for BMT.3 A significant inverse relationship is seen between the dose of radiation and the stimulated peak GH responsiveness. Hypothalamic-pituitary radiation doses of >24 Gy place patients at high risk of developing GH deficiency.5-9 The central nervous system is more sensitive to radiation at an early age; for any given dose of irradiation, the incidence of GH deficiency is lower in adults than in children, and the very young child who receives craniospinal irradiation is most at risk of extreme short stature. In adults, hyperprolactinemia or defi¬ ciencies of gonadotropins, TSH, or adrenocorticotropic hor¬ mone (ACTH) are very uncommon at doses under 40 to 50 Gy."

2058

PART XV: HORMONES AND CANCER

TABLE 226-1. Effects of Chemotherapy on Endocrine Function

TABLE 226-2. Endocrine Effects of Immune Modulators in Cancer Therapy

Chemotherapeutic Agents

Immune Modulators

Endocrine Effect

Interleukin-2

Thyroid dysfunction, ± hyperthyroidism or hypo¬

Endocrine Function

HYPOTHALAMUS-PITUITARY Busulfan; 6-mercaptopurine

Secondary adrenal insufficiency*

L-Asparaginase'

4 TSH release

Vincristine (animal data)

1 GH release

Cyclophosphamide; vincristine; vin¬ blastine; melphalan; cisplatin

Inappropriate vasopressin secretion (SIADH)

thyroidism (± LAK), on an autoimmune basis in some; acute painless thyroiditis; autoimmune hyperglycemia, with insulin and/or islet cell autoantibodies (± LAK); dyslipidemia, with 4- TC, 4- HDL-C, 4 LDL-C, T remnant lipoproteins, T TG (± interferon-a, isotretinoin); 4 testosterone, nl LH, 4 DHEA; T cortisol, T (3-endorphin, 4 melatonin

THYROID L-Asparaginase

4 TBG levels

Interleukin-4

5-Fluorouracil; mitotane

T TBG levels

Interleukin-6

4 TSH, 4 T3, 4 or nl T4, T rT,

BVP (bleomycin, vinblastine, cisplatin) chemotherapy

4 Thyroid hormone clearance (? via 4 deiodinase activity)

Interferons

a, y. Thyroid dysfunction, ± hyperthyroidism, or

Vinblastine (animal data)

4 Thyroidal hormone secretion

131I-containing radiopharmaceuticals; aminoglutethimide; vincristine, carmustine (or lomustine), procarba¬ zine; mechlorethamine, vinblastine, procarbazine*1'

T TSH; 4 T,, T4

Cisplatin, vinblastine; busulfan, cyclophosphamide

T TSH; normal T,, T4

Dactinomycin

T Radiosensitization T Calcitonin levels*

Multidrug chemotherapy

hypothyroidism, on an autoimmune basis in some; autoimmune diabetes; T TG

a: Insulin allergy; 4 TC, 4 HDL-C, 4 LDL-C (3: T Cortisol, T ACTH, T prolactin, T GH, t urinary free cortisol

T- Hyperglycemia with insulin resistance; T cortisol; T ACTH, T GH; 4 osteoclast formation (animal

PARATHYROID Vinblastine, L-asparaginase (animal data); multidrug chemotherapy for acute leukemia and breast cancer*

4 Bone resorption, 4 calcium (animal data)

4- PTH secretion

PANCREAS Vincristine; L-asparaginase; streptozocin; plicamycin; mitomycin C, 5-fluorouracil

4 Insulin secretion

Cyclophosphamide

Autoimmune diabetes

Dacarbazine, mitomycin, doxorubicin, cisplatin, GM-CSF1

T Insulin secretion, 4 insulin

L-Asparaginase+

T Glucagon secretion

action

ADRENAL 5-Fluorouracil; 5-fluorodeoxyuridine

-l In vitro steroidogenesis

Mitotane; aminoglutethimide

4 Cortisol, ± 4- aldosterone

RENAL Cisplatin; multidrug chemotherapy for ALL, AML+

4.1,25-dihydroxyvitamin D, 4. Ca, 4. Mg

Ifosfamide; streptozocin

Nephrogenic DI

BONE Chlorambucil

T Bone mineral density*

Plicamycin; dactinomycin; cisplatin,* estramustine

4- Bone resorption

ATRA

T Ca, T bone resorption

Methotrexate; doxorubicin (animal data); multidrag chemotherapy for ALL;+ cisplatin, doxorubicin, cyclo¬ phosphamide

Osteopenia

Dactinomycin

T Radiosensitization

Ifosfamide

Rickets

METABOLIC DISORDERS Multidrag chemotherapy for leukemias, lymphomas and (rarely) solid tumors

Tumor lysis syndrome, with T P, 4- Ca, T K, T uric acid

Mitotane; isotretinoin; etretinate; Lasparaginase; tamoxifen; cisplatin*; 5-fluorouracil

T Cholesterol

Isotretinoin; etretinate; acitretin;

T Triglycerides

ATRA; L-asparaginase; tamoxifen; 5-fluorouracil (animal data) 4, decreased; T. increased; TSH, thyroid-stimulating hormone; GH, growth hor¬ mone; SIADH, syndrome of inappropriate secretion of antidiuretic hormone; TBG, thy¬ roxine-binding globulin; T3, triiodothyronine; T4, thyroxine; PTH, parathyroid hormone; GM-CSF, granulocyte-macrophage colony-stimulating factor; ALL, acute lymphoblastic leukemia; AML, acute myeloblastic leukemia; DI, diabetes insipidus; ATRA, all-fnws ret¬ inoic acid. •Requires further substantiation. 'Concomitant corticosteroid administration.

data) Tumor necrosis factor

T Cortisol, T ACTH; exacerbation of hypothyroid¬ ism; autoimmune thyroid dysfunction (with interleukin-2); 4 HDL-C, T TG; T glucagon

Cyclosporine A

T TC, T LDL-C

4. decreased; T. increased; L4K, lymphokine-activated killer cells; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TG, triglycerides; nl, normal; LH, luteinizing hormone; DHEA, dehydroepiandrosterone; TSH, thyroid-stimulating hormone; T3, triiodothyronine; T4, thyroxine; rTv reverse triiodo¬ thyronine; ACTH, adrenocorticotropic hormone; GH, growth hormone.

Slowed growth velocity that is inappropriate for a child's age and stage of puberty is a very common, but not universal, out¬ come of GH deficiency in this setting. Age at irradiation is corre¬ lated not only with final height, with the youngest at irradiation having the worst growth prognosis, but also with the age at onset of puberty.10 The majority of subjects who experience premature sexual maturation also have GH deficiency, and early puberty contributes to their poor growth.3'10 An inverse relationship exists between time since therapy and stimulated peak GH responsiveness11 (Fig. 226-1). In contrast to findings in patients with isolated idiopathic GH deficiency, young adults with radia¬ tion-induced GH dysfunction rarely revert to normal GH status.12 Little direct evidence exists that cytotoxic drugs impair ante-' rior pituitary function (see Table 226-1). Growth deceleration in children has been seen to occur during antileukemic multimo¬ dality therapy, however, with some degree of “catch-up" growth occurring after completion of chemotherapy.13 Moreover, final height as well as growth velocity after craniospinal irradiation for brain tumors is more profoundly affected in children who have received adjuvant chemotherapy than in those receiving craniospinal irradiation alone, suggesting potentiation of radia¬ tion-induced growth failure by the chemotherapy.10 Cancer patients receiving chemotherapy regimens that include short¬ term, high-dose courses of corticosteroids are at risk for the development of adrenal suppression, which does not necessar¬ ily correlate with either the corticosteroid dosage or the duration of therapy. Impaired release of other pituitary hormones in response to provocative stimulation also can occur in such patients and is not inconsistent with the well-known multiple effects of corticosteroids on hypothalamic-pituitary function.

PREVENTION AND TREATMENT Routine hypothalamic-pituitary shielding during cranial radia¬ tion, dose targets of 10 Gy) and dactinomycin than in those receiving radiation alone.20

Information has been conflicting regarding the role of the iodine load associated with the use of radiographic contrast agents (especially the ethiodized oil used in lymphangiography) in pre¬ disposing such patients to radiation injury of the thyroid. More recently, a time-adjusted multivariate analysis of data for patients with Hodgkin disease, which accounted for other potentially important variables, found lymphangiography to be the only variable that significantly influenced the development of hypothyroidism.29 Other cancer populations at risk for primary thyroid dysfunc¬ tion after cancer therapy are being identified. In children and ado¬ lescents treated for acute lymphoblastic leukemia with cranial or craniospinal irradiation, subtle primary hypothyroidism is rela¬ tively common, with significantly elevated mean nadir diurnal TSH and mean peak nocturnal TSH levels reported.30 Among patients receiving single-fraction TBI for BMT in childhood, 73% develop overt (15%) or subclinical (58%) hypothyroidism within a mean follow-up period of 3.2 years; fractionating the irradiation results only in transiently elevated TSH levels in 25%.31 Spinal axis irradiation for central nervous system malignancies results in hypothyroidism in 20% to 68% of children.19 Irradiation-induced hypothyroidism shows a dose depen¬ dency, with the prevalence and severity of thyroid dysfunction lower in patients receiving 30 Gy. The differences between curves 1 and 2 (p = .0001), curves 2 and 3 (p = .0083), and curves 1 and 3 (p 9.5 g/m2 were independent significant determinants of recovery/0 At one time, a prepubertal age in a patient undergo¬ ing chemotherapy was thought to be protective. Long-term studies, however, indicate a high incidence of germinal cell damage in boys treated before adulthood for Hodgkin disease.71 In an assessment of fertility after chemotherapy for testicular germ cell cancers, the prechemotherapy sperm count had the strongest predictive value for recovery.72 Several reports, how¬ ever, have shown decreased potential fertility and testicular his¬ tologic abnormalities in pretherapy semen analysis specimens and testicular biopsy material in patients with malignant dis¬ ease, especially those with Hodgkin disease and those with tes¬ ticular carcinoma. These findings raise important questions about the impact of the underlying disease per se on the degree of infertility seen after therapy. In addition to impairment of steroidogenesis and sperm pro¬ duction, chemotherapy-induced increased aneuploid frequency and an increase in chromosomal abnormalities have been dem¬ onstrated in patients treated for various malignancies.73 Data concerning the outcome of pregnancies, however, have not shown any increase in genetically mediated birth defects, altered sex ratios, or birth weight effects in the offspring of can¬ cer survivors,69 possibly as a result of selection bias against genetically abnormal sperm. PREVENTION AND TREATMENT In a murine model, the use of a GnRH analog has provided impressive testicular protection from histologically detectable cyclophosphamide-induced damage. These results indicate that inhibition of the pituitary-gonadal axis might reduce the rate of spermatogenesis and render the testis less susceptible to the effects of chemotherapy. However, subsequent clinical and experimental trials with such analogs, with or without testoste¬ rone, in patients undergoing cancer therapy have shown no ben¬ eficial effect. As animal data suggest that hormonal treatment may enhance the recovery of spermatogenesis from surviving stem cells rather than protect the cells from damage during cyto¬ toxic or radiation insult, continuing suppressive therapy in patients for a fixed time after completion of irradiation or che¬ motherapy may prove more successful.69 Semen cryopreservation with artificial insemination has become standard practice and should be offered to all men before cytotoxic cancer therapy. Long-term experience with this practice has suggested that a prefreeze sperm motility of >15% predicts a postthaw motility of >10%.74 Newer methods of assisted reproductive techniques, such as intracytoplasmic sperm injection, permit conception even in cases of severe oligoasthenospermia; the success rate with this technique in cancer patients is ~20% per cycle.73 A future consideration is stem cell autoimplantation after freeze storing before the start of steriliz¬ ing therapy, a technique demonstrated with donor stem cells in animals.76 For men with uncommon therapy-related hypogo¬ nadism, treatment with transdermal or parenteral testosterone preparations, given in physiologic replacement doses, can result in improvement in mood, physical strength, libido, and potency.

EFFECTS OF THERAPY ON BONE Abnormal bone mineral density is an increasingly recognized long-term consequence of cancer therapy, described in both males and females, adults and children. Altered mineral metab¬ olism has been noted in children with acute leukemia at diagno-

Ch. 226: Endocrine Consequences of Cancer Therapy sis, probably secondary to the leukemic process, which becomes more prevalent with treatment.77 Various metabolic abnormali¬ ties have been described in such patients (see Table 226-1). The most obvious contributor to the therapy-related skeletal mor¬ bidity seen in young leukemic patients is corticosteroid therapy; however, long-term untreated GH deficiency may also partici¬ pate in the pathogenesis of this bone loss. Osteoporosis is com¬ mon among patients undergoing marrow transplantation. Among the mechanisms invoked are the baseline disease, the use of immunosuppressive drugs, and, in women, estrogen defi¬ ciency. Hypogonadism in women is well established as a cause of osteoporosis, and after treatment for Hodgkin disease, young, prematurely menopausal women have been shown to have reductions in bone mass comparable to that seen in normal post¬ menopausal women several decades older but equally estrogen deficient.78 In such patients, however, additional adverse effects of chemotherapy have been suggested.78-79 The pretherapy lean body mass is the most important predictor of subsequent bone mineral loss in patients undergoing anticancer chemotherapy.

REFERENCES 1. Yeung SJ, Chiu AC, Vassilopoulou-Sellin R, Gagel RF. The endocrine effects of nonhormonal antineoplastic therapy. Endocr Rev 1998; 19:144. 2. Constine LS, Woolf PD, Cann D, et al. Hypothalamic-pituitary dysfunction after radiation for brain tumors. N Engl ] Med 1993; 328:87. 3. Sklar CA, Constine LS. Chronic neuroendocrinological sequelae of radia¬ tion therapy. Int J Radiat Oncol Biol Phys 1995; 31:1113. 4. Chieng PU, Huang TS, Chang CC, et al. Reduced hypothalamic blood flow after radiation treatment of nasopharyngeal cancer: SPECT studies in 34 patients. Am J Neuroradiol 1991; 12:661. 5. Blatt J, Lee P, Suttner J, Finegold D. Pulsatile growth hormone secretion in children with acute lymphoblastic leukemia after 1800 cGy cranial radia¬ tion. Int J Radiat Oncol Biol Phys 1988; 15:1001. 6. Hall JE, Martin KA, Whitney HA, et al. Potential for fertility with replace¬ ment of hypothalamic gonadotropin-releasing hormone in long term female survivors of cranial tumors. J Clin Endocrinol Metab 1994: 79:1166. 7. Ogilvy-Stuart AL, Stirling HF, Kelnar CJH, et al. Treatment of radiationinduced growth hormone deficiency with growth hormone-releasing hor¬ mone. Clin Endocrinol 1997; 46:571. 8. Jorgensen EV, Schwartz ID, Hvizdala E, et al. Neurotransmitter control of growth hormone secretion in children after cranial radiation therapy. J Pediatr Endocrinol 1993; 6:131. 9. Shalet SM, Crowne EC, Didi MA, et al. Irradiation-induced growth failure. Baillieres Clin Endocrinol Metab 1992; 6:513. 10. Ogilvy-Stuart AL, Shalet SM. Growth and puberty after growth hormone treatment after irradiation for brain tumours. Arch Dis Child 1995; 73:141. 11. Brennan BMD, Rahim A, Mackie EM, et al. Growth hormone status in adults treated for acute lymphoblastic leukaemia in childhood. Clin Endocrinol 1998; 48:777. 12. Nicolson A, Toogood AA, Rahim A, Shalet SM. The prevalence of severe growth hormone deficiency in adults who received growth hormone replacement in childhood. Clin Endocrinol 1996; 44:311. 13. Schriock EA, Schell MJ, Carter M, et al. Abnormal growth patterns and adult short stature in 115 long-term survivors of childhood leukemia. J Clin Oncol 1991; 9:400. 14. Talvensaari KK, Lanning M, Paakko E, et al. Pituitary size assessed with magnetic resonance imaging as a measure of growth hormone secretion in long term survivors of childhood cancer. J Clin Endocrinol Metab 1994; 79:1122. 15. Carroll PV, Christ ER, Bengtsson BA, et al. Growth hormone deficiency in adulthood and the effects of growth hormone replacement: a review. J Clin Endocrinol Metab 1998; 83:382. 16. DeGroot LJ. Effects of irradiation on the thyroid gland. Endocrinol Metab Clin North Am 1993; 22:607. 17. Healy JC, Shafford EA, Reznek RH, et al. Sonographic abnormalities of the thyroid gland following radiotherapy in survivors of childhood Hodgkin's disease. Br J Radiol 1996; 69:617. 18. Ron E, Lubin JH, Shore RE, et al. Thyroid cancer after exposure to external radiation: a pooled analysis of seven studies. Radiat Res 1995; 141:259. 19. Hancock SL, McDougall IR, Constine LS. Thyroid abnormalities after ther¬ apeutic external radiation. Int J Radiat Oncol Biol Phys 1995; 31:1165. 20. Tucker MA, Morris Jones PH, Boice Jr JD, et al. Therapeutic radiation at a young age is linked to secondary thyroid cancer. Cancer Res 1991; 51:2885. 21. Fogelfeld L, Bauer TK, Schneider, et al. p53 Gene mutations in radiationinduced thyroid cancer. J Clin Endocrinol Metab 1996; 81:3039. 22. Nikiforov YE, Rowland JM, Bove KE, et al. Distinct pattern of ret oncogene rearrangements in morphological variants of radiation-induced and spo¬ radic thyroid papillary carcinomas in children. Cancer Res 1997; 57:1690.

2065

23. Leprat F, Alapetite C, Rosselli F, et al. Impaired DNA repair as assessed by the "Comet" assay in patients with thyroid tumors after a history of radia¬ tion therapy: a preliminary study. Int J Radiat Oncol Biol Phys 1998; 40:1019. 24. Becker DV, Robbins J, Beebe GW, et al. Childhood thyroid cancer following the Chernobyl accident. Endocrinol Metab Clin North Am 1996; 25:197. 25. Likhtarev I, Kairo 1, Tronko ND, et al. Thyroid cancer risk to children calcu¬ lated. Nature 1998; 392:31. 26. Ron E, Morin Doody M, Becker DV, et al. Cancer mortality following treat¬ ment for adult hyperthyroidism. JAMA 1998; 280:347. 27. Hancock SL, Cox RS, McDougall IR. Thyroid diseases after treatment of Hodgkin's disease. N Engl J Med 1991; 325:599. 28. Constine LS. What else don't we know about the late effects of radiation in patients treated for head and neck cancer? Int J Radiat Oncol Biol Phys 1995; 31:427. 29. Fein DA, Hanlon AL, Corn BW, et al. The influence of lymphangiography on the development of hypothyroidism in patients irradiated for Hodgkin's disease. Int J Radiat Oncol Biol Phys 1996; 36:13. 30. Pasqualini T, McCalla J, Berg S, et al. Subtle primary hypothyroidism in patients treated for acute lymphoblastic leukemia. Acta Endocrinol 1991; 124:375. 31. Thomas BC, Stanhope R, Plowman PN, Leiper AD. Endocrine function fol¬ lowing single fraction and fractionated total body irradiation for bone mar¬ row transplantation in childhood. Acta Endocrinol 1993; 128:508. 32. Wasnich RD, Grumet FC, Payne RO, Kriss JP. Graves' ophthalmopathy fol¬ lowing external neck irradiation for nonthyroidal neoplastic disease. J Clin Endocrinol Metab 1973; 37:703. 33. Petersen M, Keeling CV, McDougall IR. Hyperthyroidism with low radioio¬ dine uptake after head and neck irradiation for Hodgkin's disease. J Nucl Med 1989; 30:255. 34. Krouse RS, Royal RE, Heywood G, et al. Thyroid dysfunction in 281 patients with metastatic melanoma or renal carcinoma treated with interleukin-2 alone. J Immunother Emphasis Tumor Immunol 1995; 18:272. 35. Cohen J, Gierlowski TC, Schneider AB. A prospective study of hyperpara¬ thyroidism in individuals exposed to radiation in childhood. JAMA 1990; 264:581. 36. Redman JR, Bajorunas DR. Therapy-related thyroid and parathyroid dys¬ function in patients with Hodgkin's disease. In: Lacher MJ, Redman JR, eds. Hodgkin's disease: the consequences of survival. Philadelphia: Lea & Febiger, 1989:222. 37. Glazebrook GA. Effect of decicurie doses of radioactive iodine 131 on para¬ thyroid function. Am J Surg 1987; 154:368. 38. Faddy MJ, Gosden RG, Gougeon A, et al. Accelerated disappearance of ova¬ rian follicles in mid-life: implications for forecasting menopause. Hum Reprod 1992; 7:1342. 39. Wallace WHB, Shalet SM, Hendry JH, et al. Ovarian failure following abdominal irradiation in childhood: the radiosensitivity of the human oocyte. Br J Radiol 1989; 62:995. 40. Lushbaugh CC, Ricks RC. Some cytokinetic and histopathologic consider¬ ations of irradiated male and female gonadal tissues. Front Radiat Ther Oncol 1972; 6:228. 41. Stillman RJ, Schinfeld JS, Schiff I, et al. Ovarian failure in long-term survi¬ vors of childhood malignancy. Am J Obstet Gynecol 1981; 139:62. 42. Doll R, Smith PG. The long-term effects of x-irradiation in patients treated for metropathia haemorrhagica. Br J Radiol 1968; 41:362. 43. Raymond JP, Izembart M, Marliac V, et al. Temporary ovarian failure in thy¬ roid cancer patients after thyroid remnant ablation with radioactive iodine. J Clin Endocrinol Metab 1989; 69:186. 44. Madsen BL, Giudice L, Donaldson SS. Radiation-induced premature meno¬ pause: a misconception. Int J Radiat Oncol Biol Phys 1995; 32:1461. 45. Thomas PRM, Winstanly D, Peckham MJ, et al. Reproductive and endocrine function in patients with Hodgkin's disease: effects of oophoropexy and irradiation. Br J Cancer 1976; 33:226. 46. Hadar H, Loven D, Herskovitz P, et al. An evaluation of lateral and medial transposition of the ovaries out of radiation fields. Cancer 1994; 74:774. 47. Byrne J, Fears TR, Gail MH, et al. Early menopause in long-term survivors of cancer during adolescence. Am J Obstet Gynecol 1992; 166:788. 48. Himelstein-Braw R, Peters H, Faber M. Influence of irradiation and chemo¬ therapy on the ovaries of children with abdominal tumours. Br J Cancer 1977; 36:269. 49. Chatterjee R, Mills W, Katz M, et al. Prospective study of pituitary-gonadal function to evaluate short-term effects of ablative chemotherapy or total body irradiation with autologous or allogenic marrow transplantation in post-menarcheal female patients. Bone Marrow Transplant 1994; 13:511. 50. Spinelli S, Chiodi S, Bacigalupo A, et al. Ovarian recovery after total body irradiation and allogeneic bone marrow transplantation: long-term follow up of 79 females. Bone Marrow Transplant 1994; 14:373. 51. Gradishar WJ, Schilsky RL. Ovarian function following radiation and che¬ motherapy for cancer. Semin Oncol 1989; 16:425. 52. Bines J, Oleske DM, Cobleigh MA. Ovarian function in premenopausal women treated with adjuvant chemotherapy for breast cancer. J Clin Oncol 1996; 14:1718. 53. Quigley C, Cowell C, Jimenez M, et al. Normal or early development of puberty despite gonadal damage in children treated for acute lymphoblas¬ tic leukemia. N Engl J Med 1989; 321:143. 54. Sy Ortin TT, Shostak CA, Donaldson SS. Gonadal status and reproductive function following treatment for Hodgkin's disease in childhood: The Stan¬ ford experience. Int J Radiat Oncol Biol Phys 1990; 19:873.

2066

PART XV: HORMONES AND CANCER

55. Lalos O, Lalos A. Urinary, climacteric and sexual symptoms one year after treatment of endometrial and cervical cancer. Eur J Gynaec Oncol 1996; 17:128. 56. Sorosky JI, Sood AK, Buekers TE. The use of chemotherapeutic agents dur¬ ing pregnancy. Obstet Gynecol Clin North Am 1997; 24:591. 57. Mensley ML, Reichman BS. Fertility and pregnancy after adjuvant chemo¬ therapy for breast cancer. Crit Rev Oncol Hematol 1998; 28:121. 58. Critchley HO, Wallace WH, Shalet SM, et al. Abdominal irradiation in childhood: the potential for pregnancy. Br J Obstet Gynaecol 1992; 99:392. 59. Sanders J E, Hawley J, Levy W, et al. Pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total-body irradiation and bone marrow transplantation. Blood 1996; 87:3045. 60. Sauer MV, Paulson RJ, Ary BA, Lobo RA. Three hundred cycles of oocyte donation at the University of Southern California: assessing the effect of age and infertility diagnosis on pregnancy and implantation rates. J Assist Reprod Genet 1994; 11:92. 61. Blumenfeld Z, Avivi I, Linn S, et al. Prevention of irreversible chemotherapyinduced ovarian damage in young women with lymphoma by a gonado¬ trophin-releasing hormone agonist in parallel to chemotherapy. Hum Reprod 1996; 11:1620. 62. Donnez J, Bassil S. Indications for cryopreservation of ovarian tissue. Hum Reprod Update 1998; 4:248. 63. Rowley MJ, Leach DR, Warner GA, Heller CG. Effect of graded doses of ion¬ izing radiation on the human testis. Radiat Res 1974; 59:665. 63a. Beumer TL, Roepers-Gajadien HL, Gademan IS, et al. Apoptosis regulation in the testis: involvement of Bcl-2 family members. Mol Reprod Dev 2000' 56:353. 64. Ogilvy-Stuart AL, Shalet SM. Effect of radiation on the human reproductive system. Environ Health Perspect 1993; 101(Suppl 2):109. 65. Centola GM, Keller JW, Henzler M, Rubin P. Effect of low-dose testicular irradiation on sperm count and fertility in patients with testicular seminoma. J Androl 1994; 15:608.

66. Handelsman DJ, Turtle JR. Testicular damage after radioactive iodine (I131) therapy for thyroid cancer. Clin Endocrinol (Oxf) 1983; 18:465. 67. Rivkees SA, Crawford JD. The relationship of gonadal activity and chemo¬ therapy-induced gonadal damage. JAMA 1988; 259:2123. 68. Meikle AW, Cardoso de Sousa JC, Ward JH, et al. Reduction of testosterone synthesis after high dose interleukin-2 therapy of metastatic cancer. J Clin Endocrinol Metab 1991; 73:931. 69. Howell S, Shalet S. Gonadal damage from chemotherapy and radiotherapy. Endocrinol Metab Clin North Am 1998; 27:927. 70. Pryzant RM, Meistrich ML, Wilson G, et al. Long-term reduction in sperm count after chemotherapy with and without radiation therapy for nonHodgkin's lymphomas. J Clin Oncol 1993; 11:239. 71. Heikens J, Behrendt H, Adriaanse R, Berghout A. Irreversible gonadal dam¬ age in male survivors of pediatric Hodgkin's disease. Cancer 1996; 78:2020. 72. Lampe H, Horwich A, Norman A, et al. Fertility after chemotherapy for tes¬ ticular germ cell cancers. J Clin Oncol 1997; 15:239. 73. Robbins WA. Cytogenetic damage measured in human sperm following cancer chemotherapy. Mutation Res 1996; 355:235. 74. Padron OF, Sharma RK, Thomas Jr AJ, Agarwal A. Effects of cancer on sper¬ matozoa quality after cryopreservation: a 12-year experience. Fertil Steril 1997; 67:326. Costabile RA, Spevak M. Cancer and male factor infertility. Oncology 199812:557. 76. Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci U S A 1994; 91:11298. 77 Halton JM, Atkinson SA, Fraher L, et al. Altered mineral metabolism and bone mass in children during treatment for acute lymphoblastic leukemia. J Bone Miner Res 1996; 11:1774. 78. Redman JR, Bajorunas DR, Wong G, et al. Bone mineralization in women following successful treatment of Hodgkin's disease. Am J Med 1988; 85:65. 79. Douchi T, Kosha S, Kan R, et al. Predictors of bone mineral loss in patients with ovarian cancer treated with anticancer agents. Obstet Gynecol 1997; 90:12.

PART XVI

ENDOCRINOLOGY OF CRITICAL ILLNESS ERIC S. NYLEN,

EDITOR

227. CRITICAL ILLNESS AND SYSTEMIC INFLAMMATION. 2068 228. ENDOCRINE MARKERS AND MEDIATORS IN CRITICAL ILLNESS. 2077 229. THE HYPOTHALAMIC-PITUITARYADRENAL AXIS IN STRESS AND CRITICAL ILLNESS. 2087 230. NEUROENDOCRINE RESPONSE TO ACUTE VERSUS PROLONGED CRITICAL ILLNESS. 2094 231. FUEL METABOLISM AND NUTRIENT DELIVERY IN CRITICAL ILLNESS.2102 232. ENDOCRINE THERAPEUTICS IN CRITICAL ILLNESS.2108

2068

PART XVI: ENDOCRINOLOGY OF CRITICAL ILLNESS

CHAPTER

227

CRITICAL ILLNESS AND SYSTEMIC INFLAMMATION GARY P. ZALOGA, BANKIM BHATT, AND PAUL MARIK Endocrinology is predominantly the study of homeostatic systems; thus, the fact that endocrine perturbations occur as a consequence of critical illness has been recognized since the seminal works of Bernard, Camion, Cuthbertson, Selye, and others is not surpris¬ ing. The notion of a general adaptation syndrome has been further refined by placing it within the context of a complex of inflamma¬ tory, immune, and endocrine mediators. Importantly, the full expression of this response involves mutual and reciprocal inter¬ action among these compartments. Central to understanding the organism's response and recovery from critical illness has been the unveiling of the role of inflammatory mediators and the embracing of the concept of systemic inflammation. Critical illness is an acute medical condition that is immediately or imminently life-threatening. One or more organ systems deterio¬ rate to such an extent that they can no longer support the inde¬ pendent functioning of the patient (e.g., cardiac failure, renal failure, respiratory failure). Common to many forms of critical illness are activation of systemic inflammatory cascades and decreased perfusion of one or more organ systems. Critical illness dif¬ fers from terminal illness in that it is potentially reversible. Criti¬ cally ill patients are usually managed in an intensive care unit where they receive temporary physiologic support concurrent with the management of their acute medical condition. Shock, trauma, burns, systemic infections, and organ ischemia are com¬ mon examples of critical illnesses.

IMMUNE SYSTEM AND INFLAMMATORY MEDIATORS A primary function of the immune system is the removal of foreign antigens (e.g., invading organisms, malignant cells, and necrotic tissue), a role that is vital to the survival of the host. Cytokines are • soluble proteins that activate and regulate T and B lymphocytes and mediate many of the manifestations of the inflammatory response. They are produced by a wide variety of hematopoietic and nonhematopoietic cells (see Chap. 173). Like hormones, they mediate communication between cells in the body by hemocrine, autocrine, and paracrine mechanisms. They also circulate in the blood, thereby producing systemic effects distant from their sites of synthesis via distinct and specific receptors. Although the view once was that endocrine glands did not synthesize or respond directly to cytokines, evidence now demonstrates that not only do they respond directly to cytokines but they also syn¬ thesize and secrete these mediators. Cytokines differ from classic hormones in their redundancy1 (different cytokines have similar functions). In addition, cytokines are pleiotropic in that they are able to act on many different cell types. The cytokines are conveniently divided into three groups: immunoregulatory cytokines that are involved in the activation, growth, and differentiation of lymphocytes, monocytes, and leukocytes (i.e., interleukin-2 [IL-2], interleukin-3 [IL-3], interleukin-4 [IL-4]); proinflammatory cytokines that are produced predominantly by mononuclear phagocytes in response to infectious agents (i.e., interleukin-l(3 [IL-1[3], tumor necrosis factor-a [TNF-a], interleukin-6 [1L-6]); and antiinflammatory cytokines (i.e., IL-4, IL-6, interleukin-10 [IL-10], interleukin-13

[IL-13], and transforming growth factor-|3 [TGF-J3]). Some cyto¬ kines (e.g., IL-4 and IL-6) have overlapping actions. Although monocytes, macrophages, and CD4-TH (helper) cells are the most important sources of cytokines, they are also pro¬ duced by many other cells, including glial cells, Kupffer cells, keratinocytes, bone marrow stromal cells, mast cells, eosinophils, fibroblasts, endothelial cells, gut mucosal cells, mesangial cells, and endocrine glands. Monocytes and macrophages are the prin¬ cipal sources of the proinflammatory cytokines. CD4-TH cells develop into two distinct subsets of cells, TH1 and TH2 cells.2 TH1 cells (T helper cell subtype 1) secrete IL-2, TNF, and interferon-y (IFN-y), and are the principal effectors of cell-mediated immunity against intracellular microbes. TH2 cells (T helper cell subtype 2), on the other hand, secrete IL-4, IL-5, IL-10, and IL-13, which largely inhibit macrophage function.2'3 A number of factors play a role in driving native CD4-TH cells toward TH1 or TH2 cells, includ¬ ing antigen-presenting cells, hormones, and cytokines.2 Glucocor¬ ticoids enhance TH2 activity and synergize with IL-4, whereas dehydroepiandrosterone and IFN-y enhance TH1 activity.4-5 Cytokine receptors are predominantly integral plasma mem¬ brane glycoproteins with three distinct domains: (a) a recognition domain protruding from the plasma membrane that confers specificity with regards to ligand binding; (b) a hydrophobic region spanning the plasma lipid bilayer; and (c) the cytoplasmic domain, located on the inner surface of the plasma membrane, which has intrinsic enzyme activity. All the cytokine receptors are associated with one or more members of the Janus kinases, which couple ligand binding to tyrosine phosphorylation of var¬ ious known signaling proteins and transcription factors termed the signal transducers and activators of transcription (Stats).6

SYSTEMIC INFLAMMATION The idea that cytokines play pivotal roles in the pathogenesis of sepsis is'based on several lines of evidence: (a) intravenous administration of cytokines (i.e., TNF-a, IL-1) induces a sepsis¬ like syndrome in animals and humans; (b) inhibition of the effects of some cytokines (i.e., TNF-a, IL-1) by administration of neutralizing antibodies, soluble cytokine receptors, or receptor antagonists attenuates sepsis in animals; (c) administration of antiinflammatory cytokines such as IL-10 mitigates severe sep¬ sis in animals; and (d) plasma levels of cytokines (i.e., TNF-a, IL1, IL-6, IL-8, and macrophage-migration inhibitory factor [MIF]) ' increase in models of sepsis as well as in human sepsis, and the levels generally reflect the severity of sepsis. Further evidence that cytokines are essential for the manifestations of sepsis is derived from knock-out models. TNF-deficient mice are resis¬ tant to the lethality of lipopolysaccharide (LPS).7 Transgenic TNF-receptor (TNF-R55) knock-out mice do not display hemo¬ dynamic or systemic immune alterations to intravenous LPS.8'9 Transgenic mice lacking the IL-1 converting enzyme are resis¬ tant to the effects of LPS.10 Mice lacking IL-1 receptor antagonist (IL-lra) are more susceptible than controls to lethal endotoxemia, and IL-lra overproducers are protected from the lethal effects of endotoxemia.11 In patients with sepsis, TNF-a is the first proinflammatory cytokine released, followed by IL-1 and IL-6.12'13 TNF-a and IL1 (the most important proinflammatory cytokines) are closely related biologically, act synergistically, and are largely responsi¬ ble for the clinical manifestations of sepsis.12-18 IL-6 is not a prox¬ imal inflammatory cytokine; it does not cause shock in mice or primates.19 Nuclear factor-KB (NF-kB), which plays a critical role in the transcriptional induction of proinflammatory mediators,20'20a is activated by endotoxin, viruses, oxidants, TNF-a, and platelet-activating factor (PAF).21'22 In addition to activating a proinflammatory cytokine cascade, inflammatory stimuli acti-

Ch. 227: Critical Illness and Systemic Inflammation

Extracellular insult

Proinflammatory cytokines

Chemokines

TABLE 227-1. Criteria for Systemic Inflammation (Systemic Inflammatory Response Syndrome)

Adhesion molecules

Two or more of the following:

Chemokines

Temperature

>38°C or 90 beats/min

Respiratory rate

>20 breaths/min

White blood cell count

>12.0 x 109/L, 0.1 immature forms (bands)

Proinflammatory cytokines Adhesion molecules

2069

Glucocorticoids Anti-inflammatory cytokines

FIGURE 227-1. Balance of proinflammatory and antiinflammatory forces in systemic inflammation. (Modified from McKay LI, Cidlowski ]A. Molecular control of immune/inflammatory responses: interactions between nuclear factor-kappa B and steroid signaling pathways. Endocr Rev 1999; 20:435.)

vate the production of specific cytokine-neutralizing molecules, which include cytokine receptors and cytokine-receptor antago¬ nists. Circulating soluble cytokine receptors result from pro¬ teolytic cleavage of the extracellular binding domain of the receptors.23 Soluble cytokine receptors from both of the TNF receptors (p75 and p55), the IL-1 receptor, and the IL-6 receptor have been identified. The release of surface receptors may down-regulate membrane cytokine receptor responsiveness, with circulating receptors acting as a buffer for the free cyto¬ kines in the circulation. Additional factors, such as suppressors of cytokine signaling, (i.e., SOCS 1-7), modulate the response via effects on Janus kinase (JAK)-Stat kinase activity. After the release of IL-1 and TNF-a, antiinflammatory cytokines (i.e., IL-4, IL-10, IL-13, and TGF-P) are released into the circulation. The production of antiinflammatory cytokines is associated with a switch from TH1 to TH2 activation. The antiinflammatory cytokines suppress the expression of the genes for IL-1 and TNF-a and inhibit antigen presentation by monocytes as well as T- and B-lymphocyte function. IL-lra is released into the circulation, and binds to and neutralizes IL-1. The role of the antiinflammatory cytokines is to limit the inflammatory response (Fig. 227-1). If the compensatory antiinflam¬ matory response is excessive, it manifests clinically as anergy, immune depression, and an increased susceptibility to infection, known as the compensatory antiinflammatory response syndrome (CARS).24'25 In most healthy persons, the body is able to achieve a balance between proinflammatory and antiinflammatory mediators, and homeostasis is restored. In some persons, however, this balance is upset, which results in systemic inflammatory response syndrome (SIRS) (Table 227-1) arid can progress to multisystem organ dysfunc¬ tion (MODS).24'25-253 The systemic inflammatory response is seen in association with a large number of clinical conditions. Besides infec¬ tious insults that may produce SIRS (characterized by fever, tachy¬ cardia, tachypnea, and leukocytosis), noninfectious causes include pancreatitis, ischemia, trauma and tissue injury, hypovolemic shock, drugs, and immune-mediated injuries. Although a similar pathogenesis and pathophysiology are assumed to underlie the various clinical entities that comprise SIRS, the nature of the precip¬ itating insult as well as other factors such as genetic polymorphism, sex, age, race, and nutritional status likely affect the production of inflammatory mediators and their interactions. The properties and bioeffects of the relevant cytokines are summarized in Table 227-2.

TUMOR NECROSIS FACTOR TNF-a is produced by mononuclear cells, whereas TNF-(J (lymphotoxin) is produced by T lymphocytes. TNF-a shares 28% amino-acid homology with TNF-(3. Both cytokines bind to the same receptors, but their synthesis is differentially regulated. Active TNF-a consists of a trimer of three identical polypeptide

chains with molecular masses of 17 kDa each. The 17-kDa sub¬ units are released from a 26-kDa membrane-associated precur¬ sor protein by proteolytic cleavage. The enzyme that processes precursor TNF-a is a microsomal metalloprotease, TNF-aconverting enzyme (TACE).26-28 Nitric oxide plays a regulatory role in the activation of metal-dependent proteases.29 Synthetic inhibitors of metalloproteases prevent the processing of the TNF-a precursor with decreased production of TNF-a, as well as release of the 55-kDa and 75-kDa TNF receptors. They do not affect the release of other cytokines.30-33 Although many factors lead to the release of TNF-a, the most important and well studied is endotoxin (i.e., LPS). Endotoxin released from bacteria binds with a 60-kDa protein (LPS-binding protein or LBP), normally present in the blood. The LPS-LPB complex, in turn, binds to a 55-kDa cell-surface receptor on monocytes (the CD-14 molecule), activating the monocyte.34'35 Teichoic acid and peptidoglycan from gram-positive bacteria activate monocytes and macrophages and induce the produc¬ tion of TNF-a by a CD-14-independent pathway.36 Exposure of macrophages to endotoxin results in a three-fold increase in the transcriptional rate of the TNF-a gene, which is mediated through the induction of the transcription factor NF-kB, a heterodimeric protein that is normally found in the cytoplasm bound to its 37-kDa inhibitor (IkB) until a stimulatory signal is sensed at the cell surface. Several signal-transduction pathways may be involved in NF-kB activation, but all act by means of protein kinases that phosphorylate and degrade IkB. The production of TNF-a is tightly controlled. Transcription of TNF-a genes is regulated by nuclear transcription factors (e.g., NF-kB) and suppressed by a variety of repressors. Further¬ more, the messenger (mRNA) transcripts of TNF-a have short half-lives. Differences in the regulatory sequences of the TNF-a gene may influence the response to TNF-a after microbial chal¬ lenge. In patients with severe sepsis, genomic polymorphism within the TNF-a locus is associated with TNF-a production and patient outcome.37 Various agents are known to modulate the biosynthesis of TNF-a by macrophages (e.g., norepineph¬ rine, PAF, granulocyte-macrophage colony-stimulating factor, C5a, engagement of CDllb/CD18, and nitric oxide may enhance the synthesis of TNF-a by macrophages). Agents that increase intracellular cyclic adenosine monophosphate (e.g., (3agonists, prostaglandin E2, and phosphodiesterase inhibitors) decrease TNF-a mRNA in response to LPS.38 Glucocorticoids decrease the production of the proinflammatory cytokines and mediators, probably by activating glucocorticoid receptors that bind to activated NF-kB and prevent gene transcription.39 In addition, IL-4, IL-10, IL-13, and TGF-P decrease TNF-a synthe¬ sis. The activity of TNF-a in biologic fluids is controlled by a number of mechanisms. Proteinases released by activated neu¬ trophils can inactivate TNF-a, and the increased circulating sol¬ uble TNF receptors (sTNF-R) in sepsis bind 90% of the free TNF-a. Although sTNF-Rs significantly attenuate TNF-a activity, this inhibition is unlikely to be sufficient to neutralize the toxic activ¬ ities of TNF in sepsis, because even at the highest concentrations of sTNF-R, 10% of TNF-a is still active.

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PART XVI: ENDOCRINOLOGY OF CRITICAL ILLNESS

TABLE 227-2. Cytokine Actions Cytokine

Source

Major Actions

IL-2

Activated T cells

T & B cell growth-and differentiation; immunoglobulin secretion by B cells; NK cell growth and activity; production of IFN-y and TNF-P

IL-4

TH2 cells

T & B cell growth and differentiation

IL-5

T cells; mast cells, eosinophils

Differentiation of B cells and eosinophils; chemotaxis

IL-7

Bone marrow stromal cells

Pre-B and pre-T cell growth and maturation

IL-11

Bone marrow stromal cells

Megakaryocyte colony formation, myelopoiesis, erythropoiesis, lymphopoiesis

IFN-y

Activated T cells

Antiviral activity; activates monocytes, macrophages, neutrophils; NK cells; T-cell cytotoxicity

IL-ip

Macrophages; T & B cells; endothelial cells

Activates T, B, NK cells, neutrophils; induces proinflammatory cytokines, coagulation, fibrinolysis; mediates acute-phase response; down-regulates IL-lra; increases ACTH, endorphins, vasopressin, somatostatin release

TNF-a

Macrophages, activated T & B cells

See Table 227-3

IL-6

Many cells including macrophages, activated T & B cells, endothelial cells, smooth muscle cells, fibroblasts, mast cells, intestinal cells

Activates T & B cells; acute-phase proteins; activates phospho¬ lipase A,

IL-8

Monocytes, macrophages, neutrophils, endothelial cells

Neutrophil and basophil chemotaxis; neutrophil activation

MCP-1, MCP-2, MCP-3

Monocytes, macrophages, fibroblasts, B cells, endothe¬ lial cells

Chemotactic for monocytes; release of lysosomal enzymes and superoxide anion; stimulates eosinophils and basophils

MIP-1

T & B cells

Chemotactic for monocytes; expression of [5-1 integrins

RANTES

T cells, platelets, renal epithelium, mesangial cells

Chemotactic for monocytes, CD4 cells, eosinophils, basophils

IL-lra

Monocytes, macrophages

Inhibits IL-la and IL-lp

IL-4

Th2 cells

Induces IL-lra, IL-10; inhibits antigen presentation; inhibits produc¬ tion of IL-1, TNF, IL-8

IL-6

Decreases TNF production; induces IL-lra and sTNF-R55

IL-10

Many cells, including macrophages, activated T & B cells, endothelial cells, smooth muscle cells, mast cells Activated T & B cells

IL-13

Th2 cells

B cell growth and differentiation; promotes IL-lra production; inhibits IL-1, IL-6, IL-8, IL-10, TNF, IFN-a production

IMMUNOREGULATORY

PROINFLAMMATORY

ANTIINFLAMMATORY

Inhibits IFN, IL-1, IL-6, and TNF production; induces IL-lra

IL, interleukin; NK, natural killer; TNF, tumor necrosis factor; TH2, helper T lymphocyte type 2; 1FN, interferon; ACTH, adrenocorticotropic hormone; MCP, monocvte chemotactic pro-

tem; MU, macrophage inflammatory protein; RANTES, regulation on activation, normal T-expressed and secreted; IL-lra; interleukin-1 receptor antagonist.

TNF-a release into the circulation during endotoxemia occurs in a stereotypical pattern. Within minutes after intravenous endotoxin administration, a burst of TNF-a occurs that peaks in 90 to 120 minutes. TNF-a is then cleared quickly, with circulating - levels becoming undetectable within 4 to 6 hours.40-41 Repeat administration of endotoxin is followed by a markedly attenu¬ ated or absent secondary release of TNF-a. TNF-a down-regula¬ tion occurs independent of IL-1, because IL-1 production is not attenuated after a second endotoxin dose.42-43 The serum half-life of recombinant TNF-a is short (i.e., 20 to 40 minutes).41-44'45 These data suggest that most patients with acute-onset sepsis have a peak TNF-a level very early in the course of the illness. Two different TNF receptors (TNF-Rs), which have been cloned and characterized in most cells and tissues, have molec¬ ular masses of 55 kDa (TNF-R55) and 75 kDa (TNF-R75). Similar to other receptors, TNF-Rs have an extracellular domain, a sin¬ gle hydrophobic transmembrane region, and an intracellular domain. A TNF-a trimer has three binding sites for TNF-R, each located at the interface between two subunits. Thus, one TNF trimer molecule can cross-link up to three TNF-Rs. In vitro stud¬ ies indicate that the cytotoxic and most inflammatory effects are triggered by TNF-R55, whereas the proliferative effects are trig¬ gered by TNF-R75.46 Circulating TNF-a is principally responsible for the release of its receptors into the circulation.3-23-47-49 The levels of soluble TNF receptors may, thus, reflect the TNF-a levels.50 Soluble TNF-R concentrations show a dose-dependent increase to peak

concentrations within 2 hours; however, in contrast to TNF-a levels, TNF-R concentrations remain elevated for 48 hours. Fur¬ thermore, animals pretreated with antibody to TNF-a have reduced circulating levels of TNF-R. TNF-a and IL-1 have many overlapping actions and syner-' gisms. Binding of TNF-a and IL-1 to their cellular receptors induces activation and generation of a number of secondary messengers mediated by G proteins, adenylate cyclase, phos¬ pholipase A2 and C, and oxygen free radicals. In addition, a number of genes are transcribed, including those for intracellular adhesion molecule-1 (ICAM-l) and endothelial-leukocyte adhesion molecule (ELAM); the clotting and fibrinolytic proteins, tissue factor, urokinase-type plasminogen activator, and plasminogen activator inhibitor-1; the proinflammatory cytokines IL-4, IL-6, and IL-8; the antiinflammatory cytokines IL-4, IL-10, and IL1RA; phospholipase A2; inducible nitric oxide synthetase; and cyclooxygenase. The biologic and clinical effects of TNF-a are summarized in Tables 227-3 and 227-4. INTERLEUKIN-ip Interleukin-1 shares many functional and biologic characteris¬ tics with TNF-a. The IL-1 family of cytokines consists of three peptides, IL-la, IL-1 P, and IL-1RA, that are encoded by three distinct genes. IL-la and IL-1 [3 activate the same receptors and, therefore, have similar biologic properties. IL-1 (3, the predomi¬ nant form of this mediator, is produced by activated mononu-

Ch. 227: Critical Illness and Systemic Inflammation TABLE 227-3. Biologic Effects of Tumor Necrosis Factor a

TABLE 227-4. Clinical Effects of Tumor Necrosis Factor a

ACTIVATES MANY CELLS INCLUDING:

INCREASED VASCULAR PERMEABILITY

Macrophages, lymphocytes, neutrophils, eosinophils

MYOCARDIAL DEPRESSION

Fibroblasts, osteoclasts, chondrocytes

HYPOTENSION

Endothelial cells, neural cells

COAGULATION

EXPRESSION OF ADHESION MOLECULES ICAM-1, ELAM, VCAM-1 ACTIVATES CYCLOOXYGENASE, PHOSPHOLIPASE A2, NITRIC OXIDE SYNTHETASE

2071

FIBRINOLYSIS PULMONARY HYPERTENSION MICROTHROMBI CATABOLISM

Production of PAF, PGE„ PGI2, NO ACTIVATES COMPLEMENT CYTOKINES AND HEMATOPOIETIC FACTORS Proinflammatory cytokines: IL-1, IL-6, IL-8, MCP-1 Antiinflammatory cytokines: IL-4, IL-10, IL-lra PDGF, IL-2 COAGULATION SYSTEM Activates contact system Increases urokinase-type plasminogen activator Increases plasminogen activator inhibitor Down-regulates thrombomodulin VASCULAR SYSTEM Endothelin-1 NEUTROPHILS Expression of surface adhesion molecules, C3B receptors, L-selectin Superoxide production Phagocytosis, enzyme release ICAM-1, intracellular adhesion molecule-1; ELAM, endothelial-leukocyte adhesion molecule; PAF, platelet-activating factor; PCE2, prostaglandin E2; PGJ,, prostaglandin I2; NO, nitric oxide; IL, interleukin; IL-lra; interleukin-1 receptor antagonist; MCP, mono¬ cyte chemotactic protein; PDGF, platelet-derived growth factor.

clear cells. The mature 17-kDa form is released from a 31-kDa precursor via proteolytic cleavage by a cysteine proteinase, the IL-1 (3-converting enzyme, or caspase-1.51 As with TNF, two IL-1 receptors have been isolated: IL1-RI and IL1-RII. IL1-RI is found on most cells of the body, whereas IL1-RII is restricted to B cells, neutrophils, and bone marrow cells. IL1-RI mediates signaling of cells by IL-1, whereas IL1-RII competes with IL1-RI for IL-1, acting as a decoy receptor. Under physiologic conditions, only a few hundred IL-IRs are found per cell, but under inflammatory conditions receptor levels increase to 20,000 per cell. IL-la and IL-1 (3 mimic many of the bioactivities of TNF-a. Infusion of either form into humans causes fever, hemodynamic abnormalities, anorexia, malaise, arthralgia, headache, and neutrophilia (i.e., signs of sepsis). The activity of IL-1 in biologic fluids is regulated by the production and release of IL-lra, which is a pure competitive antagonist of IL-1(3.

INTERLEUKIN-6 IL-6 is produced not only by immune cells but also by many nonimmune cells (e.g., osteoblasts, keratinocytes, and intestinal epithelial cells). It is involved in inflammation and the regula¬ tion of endocrine and metabolic function. It is secreted during stress of diverse origins, probably through (3-adrenergic receptor mechanisms, and is a major mediator of the stress-response.52 IL-6 exerts its effects by binding to specific receptors (IL-6Rs) that are structurally related to receptors for IL-2, IL-3, IL-5, and IL-7. The IL-6R, which has a very short intracytoplasmic compo¬ nent, is associated with a second membrane protein (termed gpl30) that is responsible for signal transduction. Evidence is found for IL-6 antagonists in vivo. Unlike TNF-a and IL-1, IL-6 does not cause the septic response when injected into animals or into transgenic mouse models.53-59

IL-6 stimulates growth of some cells and inhibits that of oth¬ ers. It is a growth and differentiation factor for T and B lympho¬ cytes and is responsible for the stimulation of acute-phase proteins, fever, bone resorption, and thrombopoietic activity. Although once thought to be a proinflammatory cytokine, IL-6 also has antiinflammatory properties. IL-6 reduces TNF-a pro¬ duction and causes the induction of circulating IL-lra and sTNF-R55.60'61 It is a potent inhibitor of matrix metalloproteinases that are responsible for the release of active TNF-a.62 IL-6 increases the liver production of acute-phase proteins, which are believed to attenuate the effects of proinflammatory media¬ tors.63 IL-6 also has proinflammatory effects and induces the expression of phospholipase A2, which plays a central role in inflammation by producing potent lipid mediators (e.g., leukotrienes, prostaglandins, and PAF).64-65 A number of studies in diverse groups of patients with sepsis have demonstrated a strong association between IL-6 levels and outcome.66-73 In addition, IL-6 levels appear to correlate with the severity of sepsis (see Chapter 228, Fig. 228-3). IL-6 levels in patients with sepsis may reflect effects of IL-1 and TNF-a.74

INTERLEUKIN-8 A salient feature of acute and chronic inflammation is the infil¬ tration of the affected tissues by polymorphonuclear and mono¬ nuclear cells. This recruitment of inflammatory cells is directed mainly by a number of structurally related cytokines, the chemokines. IL-8 precursor protein is synthesized as a single 99amino-acid peptide chain. The 79-amino-acid mature form is proteolytically cleaved at the amino-terminus to yield various forms with slightly different biologic properties. The predomi¬ nant form of IL-8 consists of 72 amino acids. Two different types of IL-8 receptors are found, IL-8Ra and IL-8R(3, which are mem¬ bers of a superfamily of receptors coupled to guanine nucle¬ otide-binding proteins. IL-8 is produced by a variety of cells, including monocytes, macrophages, neutrophils, and endothe¬ lial cells. TNF-a, IL-1, and endotoxin release IL-8, which has remarkable specificity for neutrophils and basophils. It has chemoattractant activity and is able to induce degranulation, to elicit a respiratory burst, and to activate arachidonate-5-lipoxygenase in neutrophils.

INTERLEUKIN-10 IL-10 (a peptide of 160 amino acids and a molecular mass of 16.5 kDa), like IL-4 and IL-13, is produced by TH2 cells. Its receptor has a molecular mass of 90 to 100 kDa and has structural homol¬ ogy with the IFN-y receptor. IL-10 is a potent inhibitor of the synthesis and release of proinflammatory cytokines (i.e., TNFa, IL-1, IL-6, IL-8), probably by inhibition of transcription of the genes for these cytokines.75-77 IL-10 inhibits antigen-stimulated T-cell proliferation by decreasing the surface expression of major histocompatibility complex II molecules.7* It also inhibits

2072

PART XVI: ENDOCRINOLOGY OF CRITICAL ILLNESS

tissue factor expression and induction of procoagulant activity on monocytes.

culating cytokines also enter the brain through the organum vasculosum (no blood-brain barrier) and alter hypothalamic function.

INTERLEUKIN-4 AND INTERLEUKIN-13

Immune stimulation (i.e., inflammation) increases the activ¬ ity of the HPA axis, a phenomenon directly and indirectly medi¬ ated through cytokines.81 Corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) are released by cytokines and play important roles in the release of ACTH. CRH and AVP release are also modulated by catecholamines, prostaglandins, and nitric oxide, mediators released by cytokines. In addition, endotoxemia and tissue inflammation up-regulate brain levels of TNF-a, IL-1 (3, and IL-6, providing additional mechanisms whereby inflammation stimulates the brain. IL-1, IL-2, IL-6, IL-8, and TNF stimulate hypothalamic release of CRH.82 These cytokines activate the HPA axis inde¬ pendently and in combination; their effects are synergistic.83-88 They are effective when produced by the brain (paracrine) or from the circulation. In humans, IL-6 increases serum ACTH and cortisol levels beyond those achieved by CRH. This effect is mediated through the combined release of CRH and AVP from the hypothalamus.89-90 In addition, IL-1 stimulates expres¬ sion of proopiomelanocortin (POMC), the precursor for ACTH. IFN-y has an inhibitory effect on the release of CRH,80 and TGF selectively blocks acetylcholine-stimulated CRH release from the hypothalamus.91 Immunoneutralization of CRH blocks cytokine stimulation of ACTH release and glucocorticoid secre¬ tion, suggesting that the major effects of cytokines on the HPA axis are through CRH.

1L-4 and IL-13 are synthesized by TH2 cells and macrophages, as well as certain CD8 T cells, mast cells, and B cells. IL-4 promotes differentiation of CD4-TH cells into TH2 cells. IL-4 inhibits TH1 cells and decreases antibody-dependent cell-mediated cytotox¬ icity. IL-13 decreases production of IL-1, IL-8, macrophage inflammatory peptide-1, and nitric oxide, and increases produc¬ tion of IL-lra and IL-1RII.

INFLAMMATORY MEDIATORS AND ENDOCRINE ACTION Neuroendocrine changes are an important and essential compo¬ nent of the response to critical illness, as the immune and neu¬ roendocrine systems share numerous regulatory factors. This system of shared regulators allows one system to modulate functions of the other (see Fig. 227-2). Most endocrine cells are affected by cytokines, and major alterations in hormonal balance accom¬ pany the acute-phase response. The reciprocal interactions between the immune, endocrine, and nervous systems are extremely complex. For example, in addition to cytokine receptors, immune cells contain receptors for classic endocrine hormones (e.g., corticosteroids, insulin, prolactin, growth hormone [GH], estradiol, testosterone, (3-adrenergic agonists, acetylcholine, endorphins, enkephalins, substance P, somatostatin, and vaso¬ active intestinal peptide [VIP]1). In addition, immune cells (i.e., leukocytes) synthesize classic hormones (e.g., adrenocortico¬ tropic hormone [ACTH], (3-endorphin, GH, prolactin, VIP, and substance P) (see Chap. 180). On the other hand, classic endo¬ crine organs contain receptors for cytokines and other immune-derived products as well as for hormones. In particu¬ lar, interleukin receptors (i.e., IL-6, IL-1) have been identified on pituitary, adrenal, thyroid, pancreas, testicular, and ovarian tissues. These tissues also synthesize various cytokines. Inter¬ estingly, stimulation of immune or neuroendocrine cells with specific cytokines or hormones often alters responses to other hormones or cytokines. Evidence is increasing that cytokines contribute to the patho¬ genesis of immune-mediated target cell damage, leading to func¬ tional insufficiency of various endocrine glands (i.e., hypophysitis, thyroiditis, parathyroiditis, adrenalitis, insulitis) (see Chaps. 46, 60, 76,137). Cytokines affect endocrine glands at many different levels. In particular, they affect the release of hormones from the hypo¬ thalamus and the pituitary. They also act on the pituitary as paracrine and/or autocrine regulators, modulating hormone secretion and cell growth. The major cytokines that affect the hypothalamic-pituitary axis are IL-1, IL-2, IL-6, TNF, and IFN. The predominant effects of these cytokines are stimulation of the hypothalamic-pituitary-adrenal (HPA) axis (see Chap. 229), suppression of the hypothalamic-pituitary-gonadal (HPG) axis and hypothalamic-pituitary-thyroid (HPT) axis, and release of GH (see Chap. 230).

IL-1 and TNF mediate their hypothalamic stimulatory effects via generation of second messengers (i.e., prostaglandins, nor¬ epinephrine, nitric oxide). The mechanism by which IL-2 acti¬ vates the release of CRH is activation of IL-2 receptors on cholinergic interneurons near cell bodies of CRH neurons in the paraventricular nucleus.92 The release of acetylcholine from these interneurons stimulates muscarinic-type receptors that, in turn, stimulate the release of CRH.92 This release of CRH can be blocked by atropine.92 IL-1 (3, IL-2, IL-6, and IFN-a enhance the release of AVP in hypothalamic cells, whereas TGF-(3 selectively blocks acetylcho¬ line release of AVP.91 Cytokines also affect other hypothalamic hormones such as gonadotropin-releasing hormone (GnRH), growth hormone-releasing hormone (GHRH), and thyrotropin¬ releasing hormone (TRH). IL-1 indirectly inhibits the release of GnRH from the hypothalamus via CRH, AVP, norepinephrine,' prostaglandins, excitatory amino acids, and endorphins.93-97 IL1 and TNF-a stimulate the release of somatostatin.98-100 Both IL1 and TNF inhibit the release of TRH, and IL-1 also stimulates the release of dopamine and somatostatin, which, in turn, strongly inhibit the synthesis of TRH.93-94 Somatostatin inhibits release of GH by the pituitary.79 On the other hand, IL-6 enhances GH secretion, whereas IL-1 and TNFa both stimulate and inhibit GH release. IL-1 is a weak stimula¬ tor of GHRH release from the hypothalamus. IFN-y blocks GHRH-dependent release of GH. Although cytokines have inhibitory effects on hypothalamic signals for GH secretion, GH levels are elevated during inflammatory diseases, the acutephase response, and experimental endotoxemia. Perhaps the IL6 effects on the pituitary predominate.

HYPOTHALAMUS

PITUITARY GLAND

Numerous cytokines are expressed in the central nervous sys¬ tem (CNS).79 IL-1, IL-6, and TNF-a are synthesized in the hypothalamus80 and alter anterior pituitary function via the por¬ tal circulation. In particular, IL-1 is abundantly expressed in the paraventricular nucleus, the arcuate nucleus, and the median eminence.80 Although cytokines are produced in the brain, cir¬

The pituitary gland expresses cytokines and their receptors101'102 (e.g., IL-6 is produced in anterior pituitary cells1). Interestingly, this production of IL-6 is induced by LPS, TNF, IL-1 [3, IFN-y, and prostaglandin E2 and is inhibited by glucocorticoids. IL-2 recep¬ tor transcripts and protein products are colocalized in ACTH-, prolactin-, and GH-producing cells. IL-2 and IL-6 are involved

Ch. 227: Critical Illness and Systemic Inflammation in the autocrine/paracrine regulation of normal and tumor anterior pituitary hormone-producing cell growth.101 IL-ip has been localized to cytoplasmic granules in anterior pituitary cells and colocalizes with TSH. IL-1 regulates the growth of normal pituitary cells, whereas IL-lra, which blocks this action, is expressed in pituitary adenomas (mainly GH and ACTH). In ACTH-producing cells, IL-1 enhances glucocorticoid feedback, stimulating glucocorticoid response-element transcriptional activity. Constitutive production of TNF-a mRNA also occurs in the pituitary.1 Cytokines act as inter/autocellular factors that regulate not only the function but also the growth of anterior pituitary cells.101 In the pituitary, IL-1, IL-2, IL-6, TNF-a, and IFN-y stimulate the release of ACTH.80 IL-1 also stimulates the expression of the POMC gene,100 the common precursor of ACTH, endorphins, and a-melanocyte-stimulating hormone. In addition to ACTH, the cytokines affect other anterior pitu¬ itary hormones (e.g., luteinizing hormone [LH], follicle-stimu¬ lating hormone [FSH], GH, and TSH). IL-1 stimulates LH via IL-6, whereas it inhibits FSH release.103-104 IL-6 causes increased release of GH from the pituitary, whereas IL-1 and TNF-a have divergent effects on GH secretion.93-94-105-106 IFN-y interferes with GHRH-dependent release of GH.94 The release of TSH is enhanced directly by IL-1 and IL-6103-107 and indirectly by IL-1. The prolactin receptor is a member of a larger family, known as the cytokine class 1 receptor superfamily, which cur¬ rently has > 20 different members. Prolactin receptors are widely distributed throughout the body and have diverse actions, including immune regulation.108 Prolactin promotes antibody production and macrophage function. IL-1 stimu¬ lates dopamine release and inhibits VIP and TRH secretion in the hypothalamus, resulting in a pronounced inhibition of pro¬ lactin secretion. On the other hand, IL-1 stimulates the release of IL-6 from the folliculostellate cells of the anterior pituitary, thereby stimulating the release of prolactin from lactotropes.109 IL-1, TNF-a, and IFN-y have both stimulatory and inhibitory effects on TRH/VIP-stimulated prolactin secretion.93-94-110-116 The net effect of cytokines is inhibition of prolactin secretion, which during the acute-phase response may help down-regulate the immune response to inflammation. Hormones of the posterior pituitary are also affected by the cytokines. IL-1 stimulates the secretion of AVP via norepi¬ nephrine and angiotensin II.94 IL-1 also stimulates the secre¬ tion of several neuropeptides that augment the release of oxytocin.117

2073

secretion is inhibited.135 These effects are associated with an increase or reduction in preproinsulin gene transcription/ translation, protein biosynthesis, oxygen consumption, and oxidative metabolism. With prolonged IL-1 incubation, pan¬ creatic B cells are destroyed via apoptosis.136 The cytotoxic effects of IL-1 may contribute to the pathogenesis of autoim¬ mune diabetes mellitus. In vitro, low concentrations of IL-6 augment glucose-stimu¬ lated insulin release.137 IL-6, TNF-a, and IFN-y at high concen¬ trations inhibit glucose-stimulated insulin release.138-139 In combination with TNF-a, TNF-(3, IFN-y, and IL-1, IL-6 synergizes to inhibit the release of insulin and causes the destruction of pancreatic B cells.140-141 In general, low levels of cytokines stimulate insulin secretion, whereas high concentrations of proinflammatory cytokines inhibit insulin secretion. The elevated blood glucose concentra¬ tions seen in critically ill patients (i.e., stress hyperglycemia) result from a combination of insulin resistance and impaired insulin secre¬ tion induced by proinflammatory cytokines. In addition to effects on insulin, interleukins alter the secre¬ tion of glucagon. IL-1 causes increased secretion of glucagon from islets.142 Prolonged incubation of islets with IL-1 causes functional inhibition of glucagon secretion and A-cell toxicity.135-136 Hyperglucagonemia seen in patients with inflamma¬ tion and infection probably results partially from elevated levels of IL-1 and TNF-a. Glucagon assists with substrate mobilization during the acute-phase response. THYROID IL-1 and TNF inhibit TRH release. IL-1 also stimulates release of somatostatin and dopamine, which serve to inhibit TRH synthe¬ sis.143-144 In contrast, IL-1 and IL-6 stimulate TSH release by the pituitary.143 However, IL-1, TNF, IL-6, and IFN-y reduce expres¬ sion of TSH receptors143 and thus inhibit the thyroidal axis. The effect of these cytokines on TRH release predominates over the pituitary effects and is believed to contribute to low TSH con¬ centrations during the acute-phase response. The predominant effects of cytokines on the HPT axis are inhibitory.143-145-147 Release of cytokines from immune and nonimmune cells during illness and inflammatory states contributes to the euthyroid sick syndrome. In addition, thyrocyte-produced cytokines and cytokines produced by intrathyroidal immune cells (i.e., lym¬ phocytes, monocytes) may modulate thyroid function, growth, and response to immune attack. ADRENAL GLAND

PANCREAS Cytokines have multiple effects on carbohydrate, lipid, and pro¬ tein metabolism. IL-1 enhances uptake of glucose by renal tubular cells,118 adipose cells,119 synoviocytes,120 and fibroblasts.121-122 The combination of IFN-y and TNF-a increases uptake of glucose in fibroblasts.122 In animals without endogenous insulin production, IL-1 causes a decrease in blood glucose by increasing the activity of hepatic 5-hydroxytryptamine.123 In addition, IL-1 decreases lipid uptake from the intestine and into peripheral tissues.124 It also stimulates proteolysis.125 IFN-y causes insulin resistance, increased insulin clearance, impaired glucose tolerance, and increased counterregulatory hormone responses.126 TNF-a causes hyperglycemia and hypertriglyceridemia by increasing liver tri¬ glyceride production.127 Long-term infusion of TNF-a causes insulin resistance,128 whereas neutralization of TNF-a improves insulin resistance.129-130 Insulin secretion from the pancreas is directly and pro¬ foundly affected by cytokines.131-133 At low concentrations of IL-1, glucose-stimulated insulin secretion is increased.134 At higher concentrations of IL-1, glucose-stimulated insulin

IL-1 immunoreactivity is found in noradrenergic chromaffin cells in the adrenal gland.1 Adrenal production of IL-la is decreased by systemic administration of cholinergic agonists. On the other hand, endotoxin increases production of adrenal IL-la. Immunoreactive IL-1 is also present in cultured sympa¬ thetic ganglia. The release of IL-6, which is synthesized by adre¬ nal zona glomerulosa cells, is stimulated by IL-1 (a and P), ACTH, endotoxin, prostaglandin E2, and angiotensin. Fetal, but not adult, adrenal glands express TNF-a. Cytokines activate the HPA axis79-81 and increase glucocorti¬ coid secretion. At the level of the adrenal cortex, IL-1, IL-2, IL-6, TNF-a, and IFN-a stimulate glucocorticoid secretion in isolated adrenal glands and hypophysectomized animals.7" IL-6 produc¬ tion by the adrenal gland further contributes to glucocorticoid secretion. Interestingly, IL-6 inhibits hepatic synthesis of corticosteroid¬ binding globulin, increasing free cortisol concentrations (and cortisol bioavailability). In addition to directly stimulating adre¬ nal glucocorticoid secretion, TNF-a also inhibits the gluco¬ corticoid response to ACTH via a nitric oxide-dependent

2074

PART XVI: ENDOCRINOLOGY OF CRITICAL ILLNESS

mechanism.79 TNF-(I also inhibits glucocorticoid production. Prolonged overproduction of TNF may contribute to adrenal insufficiency in the chronically critically ill. Glucocorticoids produced by the adrenal gland decrease CRH and ACTH release via a negative-feedback mechanism. They also decrease production of cytokines by mononuclear cells, down-regulating the immune system and decreasing the inflammatory response. GONADS The testes and ovaries synthesize cytokines and contain recep¬ tors for cytokines.1 These cytokines play important roles in the regulation of gonadal function and may play a role in inflamma¬ tory destruction of these glands. IL-1 inhibits GnRH secretion from the hypothalamus.79 On the other hand, IL-6 stimulates pituitary synthesis of LH. IL-1 can stimulate LH release via IL-6. IL-1 inhibits FSH release, whereas Sertoli cells produce IL-1 and express TNF receptors. TNF-a blocks gonadotropin-stimulated inhibin synthesis, decreasing negative feedback on FSH secretion. Leydig cells also make IL-1 and express TNF receptors. IL-1, IL-2, and TNFa inhibit testosterone synthesis. Testosterone, in turn, inhibits IL-1 secretion from mononuclear cells. Overall, the effects of cytokines on the hypothalamic-pituitary-testosterone axis are complex but may contribute to hypogonadotropic hypogonadism during illness. An intraovarian cytokine system of agonists, receptors, and antagonists also exists.79 Theca cells express IL-1R, TNF-a, and TGF-P; granulosa cells express IL-1, IL-1R, IL-6, TNF-a, and TGF-P; and oocytes express TNF-a and TGF-(3. IL-1 stim¬ ulates progesterone secretion from theca cells, whereas TNF-a has both stimulatory and inhibitory effects. Theca-cell andro¬ gen synthesis is inhibited by TNF-a. In granulosa cells, IL-1 and TNF-a interfere with sex hormone production; TNF-a inhibits estrogen synthesis, whereas IL-1, IL-6, and TNF-a inhibit progesterone synthesis. Sex steroids also inhibit cyto¬ kine production.79 Estrogens inhibit IL-1, IL-6, and TNF-a pro¬ duction, and progesterone and androgens inhibit IL-1 synthesis by mononuclear cells. Overall, cytokines (especially IL-1) participate in follicle growth and maturation, luteal growth, and luteolysis. The inhibitory effects on gonadotropinstimulated sex steroid production by cytokines may contribute to hypogonadotropic hypogonadism, anovulation, oligomen¬ orrhea and amenorrhea, and infertility during inflammatory processes.

SUMMARY The acute-phase response, a generalized host reaction to a variety of pathologic processes, is characterized by activation of the immune and neuroendocrine systems (Fig. 227-2). Like classic hormones, cyto¬ kines have autocrine, paracrine, and hemocrine effects. Most endocrine cells produce cytokines, contain cytokine receptors, and are affected by cytokines. Cytokines are responsible for many of the neuroendocrine alterations that accompany the acute-phase response and systemic inflammation. These changes include increased levels of epinephrine, cortisol, aldosterone, insulin, glucagon, GH, prolactin, and AVP, and reduced levels of thyroid hormones and gonadal steroids. Not only do cytokines affect endocrine cells, but endocrine hormones affect immune¬ cell function and cytokine secretion. The result is an integrated immunoendocrine network. Improved understanding of the actions of cytokines on endocrine cells and the effects of endocrine hor¬ mones on immune cells may lead to new therapeutic approaches to both inflammatory and endocrine diseases.

FIGURE 227-2. Reciprocal interactions of immune-neuro-endocrine systems in critical illness.

REFERENCES 1. Besedovsky HO, Del Rey A. Immune-neuroendocrine interactions: facts and hypotheses. Endocr Rev 1996; 17:64. 2. Romagnani S. Biology of human TH1 and TH2 cells. ] Clin Immunol 1995; 15:121. 3. Curfs JH, Meis JF, Hoogkamp-Korstanje JA. A primer on cytokines: sources, receptors, effects, and inducers. Clin Microbiol Rev 1997; 10:742. 4. Daynes RA, Meikle AW, Araneo BA. Locally active steroid hormones may facilitate compartmentalization of immunity by regulating the types of lymphokines produced by helper T cells. Res Immunol 1991; 142:40. 5. Rook GAW, Hernandez-Pando R, Lightman SL. Hormones, peripherally activated prohormones and regulation of the Thl/Th2 balance. Immunol Today 1994; 15:301. 6. Ihle JN. Cytokine receptor signaling. Nature 1995; 377:591. 7. Marino MW, Dunn A, Grail D, et al. Characterization of tumor necrosis factor-deficient mice. Proc Natl Acad Sci U S A 1997; 94:8093. 8. Peschon JJ, Torrance DS, Stocking KL, et al. TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation. J Immunol 1998; 160:943. 9. Pfeffer K, Matsuyama T, Kundig T. Mice deficient for the 55-kd tumor necro¬ sis factor receptor are resistant to endotoxic shock, yet succumb to L. mono¬ cytogenes infection. Cell 1993; 73:457. 10. Li P, Allen H, Banerjee S, et al. Mice deficient in IL-1 (1-converting enzyme are defective in production of mature IL-1 (3 and resistant to endotoxic shock. Cell 1995; 80:401. 11. Hirsch E, Irikura VM, Paul SM, Hirsh D. Functions of interleukin 1 receptor antagonist in gene knockout and overproducing mice. Proc Natl Acad Sci USA 1996; 93:11008. 12. Hack CE, Aarden LA, Thijs LG. Role of cytokines in sepsis. Adv Immunol 1997; 66:101. 13. Thijs LG, Hack CE. Time course of cytokine levels in sepsis. Intensive Care Med 1995; 21(Suppl 2):S258. 14. Okusawa S, Gelfand JA, Ikejima T. Interleukin 1 induces a shock like state in rabbits: synergism with tumor necrosis factor and the effect of cyclooxy¬ genase inhibition. J Clin Invest 1988; 81:1162. ' 15. Fong Y, Tracey KJ, Moldawer LL, et al. Antibodies to cachectin/tumor necrosis factor reduce interleukin 1(3 and interleukin 6 appearance during lethal bacteremia. J Exp Med 1989; 170:1627. 16. Tracey KJ, Fong Y, Hesse DG, et al. Anti-cachectin/TNF monoclonal anti¬ bodies prevent septic shock during lethal bacteraemia. Nature 1987; 330:662. 17. Tracey KJ, Beutler B, Lowry SF, et al. Shock and tissue injury induced by recombinant human cachectin. Science 1986; 234:470. 18. Kumar A, Thota V, Dee L, et al. Tumor necrosis factor alpha and interleukin 1 (3 are responsible for in vitro myocardial cell depression induced by human septic shock serum. J Exp Med 1996; 183:949. 19. Dinarello CA, Cannon JG, Mancilla J. Interleukin-6 as an endogenous pyro¬ gen: induction of prostaglandin E, in brain but not peripheral blood mono¬ nuclear cells. Brain Res 1991; 562:199. 20. Barnes PJ, Karin M. Nuclear factor-tcB—a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 1997; 336:1066. 20a. Paterson RL, Galley HF, Phillon TK, Webster NR. Increased nuclear factor kappa B activation in critically ill patients who die. Crit Care Med 2000; 28:1047. 21. Carter AB, Monick MM, Hunninghake GW. Lipopolysaccharide-induced NF-kappaB activation and cytokine release in human alveolar macrophages is PKC-independent and TK- and PC-PLC dependent. Am J Respir Cell Mol Biol 1998; 18:384. 22. Blackwell TS, Christman JW. The role of nuclear factor-kappa B in cytokine gene regulation. Am J Respir Cell Mol Biol 1997; 17:3. 23. Lantz M, Gullberg U, Nilsson E, Olsson 1. Characterization in vitro of a human tumor necrosis factor-binding protein. A soluble form of a tumor necrosis factor receptor. J Clin Invest 1990; 86:1396. 24. Bone RC. Sir Isaac Newton, sepsis, SIRS and CARS. Crit Care Med 1996; 24:1125.

Ch. 227: Critical Illness and Systemic Inflammation 25. Bone RC, Grodzin CJ, Balk RA. Sepsis: a new hypothesis for pathogenesis of the disease process. Chest 1997; 112:235. 25a. Papathanassoglou ED, Moynihan TA, Ackerman MH. Does programmed cell death (apoptosis) play a role in the development of multiple organ dys¬ function in critically ill patients? Crit Care Med 2000; 28:537. 26. Moss ML, Jin SL, Becherer JD, et al. Structural features and biochemical properties of TNF-alpha converting enzyme (TACE). J Neuroimmunol 1997; 72:127. 27. Moss ML, Jin SL, Milla ME, et al. Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-a. Nature 1997; 385:733. 28. Black RA, Rauch CT, Kozlosky CJ, et al. A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature 1997; 385:729. 29. Murrell GA, Jang D, Williams RJ. Nitric oxide activates metalloprotease enzymes in articular cartilage. Biochem Biophys Res Commun 1995; 206:15. 30. Solomon KA, Covington MB, DeCicco CP, Newton RC. The fate of pro-TNFalpha following inhibition of metalloprotease-dependent processing to sol¬ uble TNF-alpha in human monocytes. J Immunol 1997; 159:4524. 31. Gallea-Robache S, Morand V, Millet S, et al. A metalloproteinase inhibitor blocks the shedding of soluble cytokine receptors and processing of trans¬ membrane cytokine precursors in human monocytic cells. Cytokine 1997; 9:340. 32. Solorzano CC, Ksontini R, Pruitt JH, et al. A matrix metalloproteinase inhib¬ itor prevents processing of tumor necrosis factor alpha (TNF alpha) and abrogates endotoxin-induced lethality. Shock 1997; 7:427. 33. Gearing AJ, Beckett P, Christodoulou M, et al. Matrix metalloproteinases and processing of pro-TNF-alpha. J Leukoc Biol 1995; 57:774. 34. Wright SD, Ramos RA, Tobias PS. CD 14, a receptor for complexes of LPS and LPS binding protein. Science 1990; 249:1431. 35. Gallay P, Barras C, Tobias PS, et al. Lipopolysaccharide (LPS)-binding pro¬ tein in human serum determines the tumor necrosis factor response of monocytes to LPS. J Infect Dis 1994; 170:1319. 36. Heumann D, Barras C, Severin A, et al. Gram-positive cell walls stimulate synthesis of tumor necrosis factor alpha and interleukin-6 by human mono¬ cytes. Infect Immun 1994; 62:2715. 37. Stuber F, Petersen M, Bokelmann F, Schade U. A genomic polymorphism within the tumor necrosis factor locus influences plasma tumor necrosis factor-alpha concentrations and outcome of patients with severe sepsis [see comments]. Crit Care Med 1996; 24:381. 38. Trinchieri G. Regulation of tumor necrosis factor production by monocytemacrophages and lymphocytes. Immunol Res 1991; 10:8. 39. Van der Poll T, Buller HR, ten Cate H, et al. Activation of coagulation after administration of tumor necrosis factor to normal subjects. N Engl J Med 1990; 322:1622. 40. Hesse DG, Tracey KJ, Fong Y, et al. Cytokine appearance in human endotoxemia and primate bacteremia. Surg Gynecol Obstet 1988; 166:147. 41. Beutler BA, Milsark IW, Cerami A. Cachectin/tumor necrosis factor: pro¬ duction, distribution, and metabolic fate in vivo. J Immunol 1985; 135:3972. 42. Zuckerman SH, Evans GF, Butler LD. Endotoxin tolerance: independent regulation of interleukin-1 and tumor necrosis factor expression. Infect Immun 1991; 59:2774. 43. Salkowski CA, Vogel SN. Lipopolysaccharide increases glucocorticoid recep¬ tor expression in murine macrophages. A possible mechanism for glucocorti¬ coid-mediated suppression of endotoxicity. J Immunol 1992; 149:4041. 44. Waage A. Production and clearance of tumor necrosis factor in rats exposed to endotoxin and dexamethasone. Clin Immunol Immunopathol 1987; 45:348. 45. Palladino MAJ, Shalaby MR, Kramer SM, et al. Characterization of the anti¬ tumor activities of human tumor necrosis factor-alpha and the comparison with other cytokines: induction of tumor-specific immunity. J Immunol 1987; 138:4023. 46. Friers W. Tumor necrosis factor: characterization at the molecular, cellular and in vivo levels. FEBS Letters 1991; 285:199. 47. Lantz M, Malik S, Slevin ML, Olsson I. Infusion of tumor necrosis factor (TNF) causes an increase in circulating TNF-binding protein in humans. Cytokine 1990; 2:402. 48. Kern WV, Engel A, Kern P. Soluble tumor necrosis factor receptors in febrile neutropenic cancer patients. Infection 1995; 23:64. 49. Van Zee KJ, Kohno T, Fischer E, et al. Tumor necrosis factor soluble recep¬ tors circulate during experimental and clinical inflammation and can pro¬ tect against excessive tumor necrosis factor alpha in vitro and in vivo. Proc Natl Acad Sci U S A1992; 89:4845. 50. Redl H, Schlag G, Adolf GR, et al. Tumor necrosis factor (TNF)-dependent shedding of the p55 TNF receptor in a baboon model of bacteremia. Infect Immun 1995; 63:297. 51. Cerretti DP, Kozlosky CJ, Mosley B, et al. Molecular cloning of interleukin beta converting enzyme. Science 1992; 256:97. 52. Papanicolaou DA, Wilder RL, Manolagas SC, Chrousos GP. The pathophys¬ iologic roles of interleukin-6 in human disease. Ann Intern Med 1998; 128:127. 53. Blackwell TS, Christman JW. Sepsis and cytokines: current status. Br J Anaesth 1996; 77:110. 54. Dinarello CA. Interleukin-1 and interleukin-1 antagonism. Blood 1991; 77:1627. 55. Wewers MD, Rinehart JJ, She Z-W. Tumor necrosis factor infusions in humans' prime neutrophils for hypochlorous acid production. Am J Physiol Lung Cell Mol Physiol 1990; 259:L276.

2075

56. Pfeffer K, Matsuyama T, Kundig T. Mice deficient for the 55-kd tumor necro¬ sis factor receptor are resistant to endotoxic shock, yet succumb to L. mono¬ cytogenes infection. Cell 1993; 73:457. 57. Smith JW, Urba WJ, Curti BD. The toxic and hematologic effects of interleu¬ kin-1 alpha administered in a phase I trial of patients with advanced hema¬ tologic malignancies. J Clin Oncol 1992; 10:1140. 58. Woodroofe C, Muller W, Ruther U. Long-term consequences of interleukin6 overexpression in transgenic mice. DNA Cell Biol 1992; 11:587. 59. Preiser JC, Schmartz D, van der Linden P, et al. Interleukin-6 administration has no acute hemodynamic or hematologic effects in the dog. Cytokine 1991; 3:1. 60. Tilg H, Trehu E, Atkins MB, et al. Interleukin-6 (IL-6) as an anti-inflamma¬ tory cytokine: induction of circulating IL-1 receptor antagonist and soluble tumor necrosis factor receptor p55. Blood 1994; 83:113. 61. Mizuhara H, O'Neill E, Seki N, et al. T cell activation-associated hepatic injury: mediation by tumor necrosis factors and protection by interleukin 6. J Exp Med 1994; 179:1529. 62. Shingu M, Miyauchi S, Nagai Y, et al. The role of IL-4 and IL-6 in IL-1dependent cartilage matrix degradation. Br J Rheumatol 1995; 34:101. 63. Alcorn JM, Fierer J, Chojkier M. The acute-phase response protects mice from D-galactosamine sensitization to endotoxin and tumor necrosis factoralpha. Hepatology 1992; 15:122. 64. Crowl RM, Stoller TJ, Conroy RR, Stoner CR. Induction of phospholipase A2 gene expression in human hepatoma cells by mediators of the acute phase response. J Biol Chem 1991; 266:2647. 65. Shrikant P, Weber E, Jilling T, Benveniste EN. Intercellular adhesion molecule-1 gene expression by glial cells. Differential mechanisms of inhibition by IL-10 and IL-6. J Immunol 1995; 155:1489. 66. Casey LC, Balk R, Bone RC. Plasma cytokine and endotoxin levels correlate with survival in patients with sepsis syndrome. Ann Intern Med 1993; 119:771. 67. Presterl E, Staudinger T, Pettermann M, et al. Cytokine profile and correla¬ tion to the APACHE III and MPM II scores in patients with sepsis. Am J Respir Crit Care Med 1997; 156:825. 68. Helfgott DC, Tatter SB, Santhanam RH, et al. Multiple forms of IFN-beta/ IL-6 in serum and body fluids during acute bacterial infection. J Immunol 1989; 142:948. 69. Calandra T, Gerain J, Heumann D, et al. High circulating levels of interleu¬ kin-6 in patients with septic shock; evolution during sepsis, prognostic value and interplay with other cytokines. Am J Med 1991; 91:23. 70. Meduri GU, Headley S, Kohler G, et al. Persistent elevation of inflammatory cytokines predicts a poor outcome in ARDS. Plasma IL-1 beta and IL-6 lev¬ els are consistent and efficient predictors of outcome over time. Chest 1995; 107:1062. 71. Antonelli M, Raponi GM, Martino P, et al. High IL-6 serum levels are asso¬ ciated with septic shock and mortality in septic patients with severe leu¬ kopenia due to hematological malignancies. Scand J Infect Dis 1995; 27:381. 72. Damas P, Canivet J-L, De Groote D, et al. Sepsis and serum cytokine concen¬ trations. Crit Care Med 1997; 25:405. 73. Damas P, Ledoux D, Nys M, et al. Cytokine serum level during severe sepsis in human IL-6 as a marker of severity. Ann Surg 1992; 215:356. 74. Dinarello CA. Proinflammatory and antiinflammatory cytokines as media¬ tors in the pathogenesis of septic shock. Chest 1997; 112:321S. 75. Lalani I, Bhol K, Ahmed AR. Interleukin-10: biology role in inflammation and autoimmunity. Ann Allergy Asthma Immunol 1997; 79:469. 76. Berg DJ, Kuhn R, Rajewsky K, et al. Interleukin-10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance. J Clin Invest 1995; 96:2339. 77. Howard M, Muchamuel T, Andrade A, Menon S. Interleukin-10 protects mice from lethal endotoxemia. J Exp Med 1993; 177:1205. 78. de Waal Malefyt R, Haanen J, Spits H. IL-10 and viral IL-10 strongly reduced antigen specific T cell proliferation by diminishing the antigen presenting capacity of monocytes via down regulation of class major histocompatibil¬ ity complex expression. J Exp Med 1991; 174:915. 79. Mandrup-Poulsen T, Nerup J, Reimers JI, et al. Cytokines and the endocrine system. I. The immunoendocrine network. Eur J Endocrinol 1995; 133:660. 80. Mandrup-Poulsen T, Nerup J, Reimers JI, et al. Cytokines and the endocrine system. I. The immunoendocrine network. Eur J Endocrinol 1995; 133:660. 81. Turnbull AV, Lee S, Rivier C. Mechanisms of hypothalamic-pituitary-adre¬ nal axis stimulation by immune signals in the adult rat. Ann N Y Acad Sci 1998; 840:434. 82. Chrousos GP. The hypothalamic-pituitary-adrenal axis and immune-mediated inflammation. N Engl J Med 1995; 332:1351. 83. Imura H, Fukata J, Mori T. Cytokines and endocrine function: an interaction between the immune and neuroendocrine systems. Clin Endocrinol 1991; 35:107. 84. Bemardini R, Kamilaris TC, Calogero AE, et al. Interactions between tumor necrosis factor-a, hypothalamic corticotropin-releasing hormone and adrenocorticotropin secretion in the rat. Endocrinology 1990; 126:2876. 85. Sapolsky R, Rivier C, Yamamoto G, et al. Interleukin-1 stimulates the secretion of hypothalamic corticotropin-releasing factor. Science 1987; 238:522. 86. Naitoh Y, Fukata J, Tominaga T, et al. Interleukin-6 stimulates the secretion of adrenocorticotropic hormone in conscious, free-moving rats. Biochem Biophys Res Commun 1988; 155:1459.

2076

PART XVI: ENDOCRINOLOGY OF CRITICAL ILLNESS

87. Perlstein RS, Mougey EH, Jackson WE, Neta R. Interleukin-1 and interleukin-6 act synergistically to stimulate the release of adrenocorticotropic hor¬ mone in vivo. Lymphokine Cytokine Res 1991; 10:141. 88. Perlstein RS, Whitnall MH, Abrams JS, et al. Synergistic roles of interleukin6, interleukin-1, and tumor necrosis factor in adrenocorticotropin response to bacterial lipopolysaccharide in vivo. Endocrinology 1993; 132:946. 89. Mastorakos G, Chrousos GP, Weber JS. Recombinant interleukin-6 activates the hypothalamic-pituitary-adrenal axis in humans. J Clin Endocrinol Metab 1993; 77:1690. 90. Mastorakos G, Weber JS, Magiakou MA, et al. Hypothalamic-pituitaryadrenal axis activation and stimulation of systemic vasopressin secretion by recombinant interleukin-6 in humans: potential implications for the syn¬ drome of inappropriate vasopressin secretion. J Clin Endocrinol Metab 1994; 79:934. 91. Raber J, Sorg O, Horn TF, et al. Inflammatory cytokines: putative regulators of neuronal and neuro-endocrine function. Brain Res Brain Res Rev 1998; 26:320. 92. McCann SM, Lyson K, Karanth S, et al. Mechanism of action of cytokines to induce the pattern of pituitary hormone secretion in infection. Ann N Y AcadSci 1995; 771:386. 93. Scarborough DE. Cytokine modulation of pituitary hormone secretion. Ann N Y Acad Sci 1990; 594:169. 94. Jones TH, Kennedy RL. Cytokines and hypothalamic-pituitary function. Cytokine 1993; 5:531. 95. Rivest S, Lee S, Attardi B, Rivier C. The chronic intracerebroventricular infu¬ sion of interleukin-1 p alters the activity of the hypothalamic-pituitarygonadal axis of cycling rats. I. Effect of LHRH and gonadotropin biosynthesis and secretion. Endocrinology 1993; 133:2424. 96. Bonavera JJ, Kalra SP, Kalra PS. Evidence that luteinizing hormone suppres¬ sion in response to inhibitory neuropeptides P-endorphin, interleukin-1 p, and neuropeptide K may involve excitatory amino acids. Endocrinology 1993; 133:178. 97. Rettori V, Gimeno MF, Karara A, et al. Interleukin la inhibits prostaglandin E2 release to suppress pulsatile release of luteinizing hormone but not folli¬ cle-stimulating hormone. Proc Natl Acad Sci U S A1991; 88:2763. 98. Scarborough DE. Somatostatin regulation by cytokines. Metabolism 1990; 39:108. 99. Scarborough DE, Lee SL, Dinarello CA, Reichlin S. Interleukin-1 p stimu¬ lates somatostatin biosynthesis in primary cultures of fetal rat brain. Endo¬ crinology 1989; 124:549. 100. Brown SL, Smith LR, Blalock JE. Interleukin 1 and interleukin 2 enhance proopiomelanocortin gene expression in pituitary cells. J Immunol 1987; 139:3181. 101. Arzt E, Paez Pereda M, Costas M, et al. Cytokine expression and molecular mechanisms of their auto/paracrine regulation of anterior pituitary func¬ tion and growth. Ann N Y Acad Sci 1998; 840:525. 102. Arzt E, Stalla GK. Cytokines: autocrine and paracrine roles in the anterior pituitary. Neuroimmunomodulation 1996; 3:28. 103. Spangelo BL, Judd AM, Isakson PC, MacLeod RM. Interleukin-6 stimulates anterior pituitary hormone release in vitro. Endocrinology 1989; 125:575. 104. Murata T, Ying SY. Effects of interleukin-1 beta on secretion of follicle stim¬ ulating hormone (FSH) and luteinizing hormone (LH) by cultured rat ante¬ rior pituitary cells. Life Sci 1991; 49:447. 105. Walton PE, Cronin MJ. Tumor necrosis factor-a inhibits growth hormone secretion from cultured anterior pituitary cells. Endocrinology 1989; 125:925. 106. Nash AD, Brandon MR, Bello PA. Effects of tumor necrosis factor-alpha on growth hormone and interleukin 6 mRNA in ovine pituitary cells. Mol Cell Endocrinol 1992; 84:R31. 107. Bemton EW, Beach JE, Holaday JW, et al. Release of multiple hormones by a direct action of interleukin-1 on pituitary cells. Science 1987; 238:519. 108. Bole-Feysot C, Goffin V, Edery M, et al. Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr Rev 1998; 19:225. 109. Yamaguchi M, Matsuzaki N, Hirota K, et al. Interleukin 6 possibly induced by interleukin ip in the pituitary gland stimulates the release of gonadotro¬ pins and prolactin. Acta Endocrinol 1990; 122:201. 110. Schettini G, Florio T, Meucci O, et al. Interleukin-l-p modulation of prolac¬ tin secretion from rat anterior pituitary cells: involvement of adenylate cyclase activity and calcium mobilization. Endocrinology 1990; 126:435. 111. Schettini G, Landolfi E, Grimaldi M, et al. Interleukin 1 beta inhibition of TRH stimulated prolactin secretion and phosphoinositide metabolism. Biochem Biophys Res Commun 1989; 165:496. 112. Yamaguchi M, Koike K, Yoshimoto Y, et al. Effect of TNF-a on prolactin secretion from rat anterior pituitary and dopamine release from the hypo¬ thalamus: comparison with the effect of interleukin ip. Endocrinol Jpn 1991; 38:357. 113. Koike K, Hirota K, Ohmichi M, et al. Tumor necrosis factor-a increases release of arachidonate and prolactin from rat anterior pituitary cells. Endo¬ crinology 1991; 128:2791. 114. Walton PE, Cronin MJ. Tumor necrosis factor-a and interferon-y reduce pro¬ lactin release in vitro. Am J Physiol 1990; 259:E672. 115. Koike K, Masumoto N, Kasahara K, et al. Tumor necrosis factor-a stimulates prolactin release from anterior pituitary cells: a possible involvement of intracellular calcium mobilization. Endocrinology 1991; 128:2785. 116. Yamaguchi M, Koike K, Matsuzaki N, et al. The interferon family stimulates the secretions of prolactin and interleukin-6 by the pituitary gland in vitro. J Endocrinol Invest 1991; 14:457.

117. Naito Y, Fukata J, Shindo K, et al. Effects of interleukins on plasma arginine vasopressin and oxytocin levels in conscious, freely moving rats. Biochem Biophys Res Commun 1991; 174:1189. 118. Kohan DE, Schreiner GF. Interleukin-1 modulation of renal epithelial glu¬ cose and amino acid transport. Am J Physiol 1988; 254:F879. 119. Garcia-Welsh A, Schneiderman JS, Baly DL. Interleukin-1 stimulates glu¬ cose transport in rat adipose cells. Evidence for receptor discrimination between IL-ip and It-la. FEBS Lett 1990; 269:421. 120. Hernvann A, Aussel C, Cynober Let al. IL-ip, a strong mediator for glucose uptake by rheumatoid and non-rheumatoid cultured human synoviocytes. FEBS Lett 1992; 303:77. 121. Bird TA, Davies A, Baldwin SA, Saklavala J. Interleukin 1 stimulates hexose transport in fibroblasts by increasing the expression of glucose transporters. J Biol Chem 1990; 265:13578. 122. Taylor DJ, Faragher EB, Evanson JM. Inflammatory cytokines stimulate glu¬ cose uptake and glycolysis but reduce glucose oxidation in human dermal fibroblasts in vitro. Circul Shock 1992; 37:105. 123. Endo Y, Suzuki R, Kumagai K. Interleukin 1-like factors can accumulate 5hydroxytryptamine in the liver of mice and can induce hypoglycaemia. Bio¬ chem Biophys Acta 1985; 840:37. 124. Argiles JM, Lopez-Soriano F, Evans RD, Williamson DH. Interleukin-1 and lipid metabolism in the rat. Biochem J 1989; 259:673. 125. Zamir O, Hasselgren P-O, Von Allmen D, Fischer JE. In vivo administration of interleukin-la induces muscle proteolysis in normal and adrenalectomized rats. Metabolism 1993; 42:204. 126. Koivisto VA, Pelkonen R, Cantell K. Effect of interferon on glucose tolerance and insulin sensitivity. Diabetes 1989; 38:641. 127. Feingold KR, Soued M, Staprans I, et al. Effect of tumor necrosis factor (TNF) on lipid metabolism in the diabetic rat. Evidence that inhibition of adipose tissue lipoprotein lipase activity is not required for TNF-induced hyperlipidemia. J Clin Invest 1989; 83:1116. 128. Lang CH, Dobrescu C, Bagby GJ. Tumor necrosis factor impairs insulin action on peripheral glucose disposal and hepatic glucose output. Endocri¬ nology 1992; 130:43. 129. Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor a: direct role in obesity-linked insulin resistance. Science 1993; 259:87. 130. Hofmann C, Lorenz K, Braithwaite SS, et al. Altered gene expression for tumor necrosis factor a and its receptors during drug and dietary modula¬ tion of insulin resistance. Endocrinology 1994; 134:264. 131. Mandrup-Poulsen T, Helquist D, Molvig J, et al. Cytokines as immune effec¬ tor molecules in autoimmune endocrine disease with special reference to insulin-dependent diabetes mellitus. Autoimmunity 1989; 4:191. 132. Sandler S, Eizirik DL, Svensson C, et al. Biochemical and molecular actions of interleukin-1 on pancreatic (I cells. Autoimmunity 1991; 10:241. 133. Purrello F, Buscema M. Effects of interleukin-1 P on insulin secretion by pan¬ creatic beta-cells. Diab Nutr Metab 1993; 6:295. 134. Palmer JP, Helquist S, Spinas GA, et al. Interaction of P-cell activity and IL1 concentration and exposure time in isolated rat islets of Langerhans. Dia¬ betes 1989; 38:1211. 135. Mandrup-Poulsen T, Bendtzen K, Nerup J, et al. Affinity-purified human interleukin 1 is cytotoxic to isolated islets of Langerhans. Diabetologia 1986; 39:63. 136. Mandrup-Poulsen T, Egeberg J, Nerup J, et al. Ultrastructural studies of time-course and cellular specificity of interleukin 1 mediated islet cytotox¬ icity. Acta Path Microbiol Immunol Scand 1987; 95:55. x 137. Buschard K, Aaen K, Horn T, et al. Interleukin 6: a functional and structural in vitro modulator of beta cells from islets of Langerhans. Autoimmunity 1990; 5:185. 138. Southern C, Schultser D, Greene IC. Inhibition of insulin secretion from rat islets of Langerhans by interleukin-6. Biochem J 1990; 272:243. 139. Bendtzen K, Mandrup-Poulsen T, Nerup J, et al. Cytotoxicity of human pl7 interleukin-1 for pancreatic islets of Langerhans. Science 1986; 232:1545. 140. Mandrup-Poulsen T, Bendtzen K, Dinarello CA, Nerup J. Human tumor necrosis factor potentiates human interleukin-1 mediated rat pancreatic beta-cell cytotoxicity. J Immunol 1987; 139:4077. 141. Rabinovitch A, Sumoski W, Rajotte RV, Warnock GL. Cytotoxic effects of cytokines on human pancreatic islet cells in monolayer culture. J Clin Endo¬ crinol Metab 1990; 71:152. 142. Helqvist S, Zumsteg UW, Spinas GA, et al. Repetitive exposure of pancreatic islets to interleukin-1 (L An in vitro model of pre-diabetes? Autoimmunity 1991; 10:311. 143. Mandrup-Poulsen T, Nerup J, Reimers JI, et al. Cytokines and the endocrine system. II. Roles in substrate metabolism, modulation of thyroidal and pan¬ creatic endocrine cell functions and autoimmune endocrine diseases. Eur J Endocrinol 1996; 134:21. 144. Jones TH, Kennedy RL. Cytokines and hypothalamic-pituitary function. Cytokines 1993; 5:531. 145. Dubuis JM, Dayer JM, Siegrist-Kaiser CA, Burger AG. Human recombinant interleukin-lB decreases plasma thyroid hormone and thyroid stimulating hormone levels in rats. Endocrinology 1988; 123:2175. 146. Van der Poll T, Romijn JA, Wiersinga WM, Sauerwein HP. Tumor necrosis factor: a putative mediator of the sick-euthyroid syndrome in man. J Clin Endocrinol Metab 1990; 71:1567. 147. Bartalena L, Grasso L, Brogioni S, Martino E. Interleukin-6 effects on the pituitary-thyroid axis in the rat. Eur J Endocrinol 1994; 131:302.

Ch. 228: Endocrine Markers and Mediators in Critical Illness

CHAPTER

228

ENDOCRINE MARKERS AND MEDIATORS IN CRITICAL ILLNESS ABDULLAH A. ALAR I FI, GREET H. VAN DEN BERGHE, RICHARD H. SNIDER, JR., KENNETH L. BECKER, BEAT MOLLER, AND ERIC S. NYLEN Inflammation is the response to an injury (e.g., infection, trauma, heat, cold, locally toxic chemicals, etc.). It is characterized by vasodilatation with increased blood flow, increased capillary permeability resulting in passage of macromolecules and fluid into the interstitial space, chemoattraction of hematopoietic cel¬ lular elements to the site of injury, stimulation of neutrophilic phagocytosis, and activation of monocytes. The inflammatory response is mediated by a multitude of humoral substances including several cytokines, which emanate from cells of the immune system as well as many other cells throughout the body. These have a particularly key role in stimulating, perpet¬ uating, and/or modulating the inflammatory response. Some of the cytokines are proinflammatory, some are antiinflammatory, and some manifest either of these functions, depending on their local or systemic concentration, and the coexisting chemical or hor¬ monal milieu. Stress can be defined as any condition that threatens to dis¬ rupt the equilibrium of bodily functions (i.e., homeostasis). Stress is a somewhat broader and less specific term than inflammation and usually includes in its definition the response of the immune, neural, and endocrine systems to a concurrent pertur¬ bation or injury. Previously, the concept of stress had focused primarily on the hypothalamic-pituitary-adrenal (HPA) axis, the catecholamines, and glucose and insulin metabolism. The histopathologic changes, physiologic alterations, and hormonal and cytokine responses observed in many studies, however, have documented a substantial overlap between the concepts of "inflammation" and "stress." The appellation critical illness is best applied to a lifethreatening condition that merits intensive medical and/or surgical intervention. The patient with critical illness is always under stress and, not uncommonly, manifests some or all of the physi¬ ologic and biologic characteristics of inflammation. Generally, different stressors evoke a multihormonal and selective response, the magnitude of which frequently correlates with the degree and type of stress. Although the endocrine changes during stress are well documented, their mechanisms, their extent, their timing, their duration, the prognostic value of these changes, and whether they are adaptive or maladaptive (i.e., harmful) are much less well understood. In the short term, stress-induced changes in endocrine function are generally adaptive: they promote optimal intravascular volume, perfu¬ sion pressure, and substrate availability. Nonetheless, if the stress is prolonged or if the homeostatic response is inadequate, the endocrine response can contribute to a worsened clinical sta¬ tus and, hence, to the potential need for therapeutic interven¬ tions (see Chaps. 230 and 232). Much of our current understanding of the endocrine response to stress originated with Bernard's concept of the "internal environment." Cannon coined the term homeostasis to describe the complete bioresponse necessary to maintain a steady state and documented the role of the sympathoadrenal system. Selye described the enlargement of the adrenal cortex in

2077

response to diverse noxious agents (later termed the general adaptation syndrome); this shifted attention to the HPA axis. Later, the role of glucocorticoids in stress was redefined as the modula¬ tion of other adaptive responses (especially those involving proinflam¬ matory cytokines), thereby protecting the host from the effects of "overreaction."'1 Further clarifying the endocrine response to stress has been the emerging recognition of the cytokines as piv¬ otal mediators and the endocrine endothelium as an essential site of activation in critical illness (see Chap. 227). Also, the expression of heat-shock protein in stress tolerance at the cellu¬ lar level, its involvement in mediating the immune response to stress, and its potential protective effect appear to provide another fundamental component. Finally, as discussed in Chap¬ ter 227, a hallmark of the stress response is a multidirectional immunoneuroendocrine interaction (see Chap 227, Fig 227-2) A3

GLUCOCORTICOIDS The most accepted explanation for the increased requirement of circulatory glucocorticoids in response to stress is that, although they exert a permissive action during periods of no stress (allow¬ ing other hormones and factors to maintain homeostasis4), they exert a regulatory role during stress.1 These regulatory effects are essential in preventing immunotoxicity.1 The secretion of glucocorticoids is part of a closed, feedback-loop system that is ideal for providing physiologic levels at all times, while also allowing for an emergency override via the central nervous sys¬ tem (CNS) (see Chap. 229). This is the single most important stress-response system, without which survival is unlikely.5 Critical illness induces an increase in the serum cortisol con¬ centration,6 the extent of which is determined by the type and severity of stress. Elevated cortisol levels can block the HPA response to minor stress but do not alter the response to major stress. Generally, the HPA responds in a graded fashion to increasing stress.7 In surgery, the degree of activation depends on the type and extent of surgery and anesthesia.8 Reversal of general anesthesia is also a potent stimulator of adrenocortico¬ tropic hormone (ACTH) secretion.9 (Interruption of the neural connections from the operative site can block the rise in ACTH and cortisol.10) Typically, the serum cortisol increase is in response to corticotropin-releasing hormone (CRH) and ACTH stimulation. These increases are associated with loss of circadian rhythm, resistance to dexamethasone suppression, and hyperre¬ sponsiveness to CRH stimulation.9 Both ACTH and CRH levels decrease to below presurgical levels by the first postoperative day. In contrast, elevations in ACTH and CRH due to sepsis may last for several days; the cortisol levels remain high, gradually returning to normal.11 Suppressed ACTH levels indicate the res¬ toration of normal feedback sensitivity, whereas high cortisol secretion, in the presence of low ACTH levels, indicates either an increased adrenal sensitivity to ACTH, cortisol resistance, or the presence of alternative pathway(s) for cortisol secretion (see Chaps. 229 and 232). Thus, clinical assessment of adrenal func¬ tion based on the cortisol concentration can be misleading (see Chap. 232). Another change that occurs in critical illness is the shift of adrenal steroid production from mineralocorticoid and androgens to cortisol.12 Associations between the severity of injury and the degree of HPA activation have been reported.13 For example, the Glasgow Coma Score is significantly correlated with cortisol concentra¬ tions in patients who have experienced both head and systemic injuries.14 Furthermore, serum cortisol concentrations typically correlate inversely with critical illness outcome.13,14 Glucocorticoids stimulate gluconeogenesis in the liver, kid¬ ney, and skeletal muscle; inhibit glucose uptake; and facilitate the synthesis and release of catecholamines (see Chap. 73). They

2078

PART XVI: ENDOCRINOLOGY OF CRITICAL ILLNESS

also inhibit the secretion and action of insulin and stimulate the secretion of glucagon, causing protein wasting and inhibition of protein synthesis in skeletal muscle. Thus, although the impor¬ tance of glucocorticoids to survival is well recognized, just how these effects influence the adaptation to stress is less clear. The hypercortisolism elicited by critical illnesses can be interpreted as an attempt by the body to mute its own inflammatory cascade and thus protect itself against possible endogenous overreac¬ tion.1 Moreover, the acute cortisol-induced metabolic effects provide immediately available energy and postpone anabolism. Nonetheless, the benefit of prolonged hypercortisolism is ques¬ tionable, as it may lead to immune suppression, stress hypergly¬ cemia, myopathy, a catabolic state, and impaired healing. At the cellular level, glucocorticoids act via ligand binding to a superfamily of nuclear receptors (see Chap. 73). As transcrip¬ tional modifiers (regulators), they affect the expression of a vari¬ ety of genes, thereby serving as an important antiinflammatory force that is counteracted by proinflammatory cytokines and transcriptional factors (e.g., nuclear factor-KB) (see Chap. 227, Fig. 227-1).15 Despite a half-century of extensive research into the actions of glucocorticoids, their multifaceted actions have yet to be fully unraveled.16

CATECHOLAMINES The control center of the stress response is located in the cerebral cortex, limbic system, hypothalamus, and brainstem (see Chap. 229). Various noxious stressors cause increased activity of the sympathetic nervous system (SNS) and adrenal medulla. This results in secretion of epinephrine and norepinephrine from the adrenal medulla and norepinephrine from the sympathetic nerve terminals, respectively (norepinephrine comprises -20% of adrenal medullary catecholamine secretion). Although most norepinephrine is taken up by presynaptic neurons, some "overflow" into the plasma occurs, serving as an indicator of sympathetic neuronal activity. Increasingly, this stress response appears to show specificity (i.e., the degree of the response and the ratio of epinephrine to norepinephrine secretion depend on the specific type of stress). The catecholaminergic response, which is rapid and transient, can be attenuated by epidural analgesia.17 The severity of the stres¬ sor determines the degree of the catecholamine response18; how¬ ever, the catecholamine concentration is not predictive of the final outcome in critically ill patients.19 With prolonged stress, marked compensatory changes occur in the adrenal medulla, including increased activity of the enzymes involved in catecholamine bio¬ synthesis and elevated tissue concentrations of catecholamines. These changes in enzymatic activity are regulated to varying degrees by glucocorticoids, ACTH, and neuronal activity.20 The released catecholamines trigger metabolic and hemody¬ namic changes characteristic of the "flight or fight" response, which range from increased blood pressure to increased glycogenolysis. Catecholamines have direct effects on the metabolism of lip¬ ids, proteins, and carbohydrates, and on regulatory hormones (e.g., insulin and glucagon).21 They augment hepatic glucose output by stimulating both gluconeogenesis and glycogenolysis, increase lipolysis, inhibit insulin secretion, impair tissue sen¬ sitivity to insulin, and increase glucagon and growth hormone secretion.21'22 As with the HPA axis, multidirectional effects are also found between the SNS and cytokines. Infusion of tumor necrosis factor¬ ed (TNF-a) or interleukin-1 (IL-1) causes a rapid and marked increase of catecholamines.23 Norepinephrine inhibits lipopolysaccharide (LPS)-induced TNF-a and IL-6 release in human whole blood; this effect is blocked by (3 antagonism, suggesting

that norepinephrine may modulate ongoing cytokine production in acute sepsis.24

THYROID HORMONES The changes in thyroid function during acute critical illness are well known25 (see Chaps. 36 and 232). During a mild illness, tri¬ iodothyronine (T3) production is rapidly decreased by inhibition of conversion of thyroxine (T4) to T3, with a reciprocal increase in reverse T3 (rT3; loiv T3 syndrome). With an increase in severity of illness, T, decreases (low T4 syndrome). The serum thyroidstimulating hormone (thyrotropin, TSH) concentration also may be low or normal. The nocturnal rise in serum TSH concentra¬ tions may be blunted, but usually the serum TSH response to thyrotropin-releasing hormone (TRH) is normal.26 Among patients in intensive care units (ICUs), up to 70% have low T3 syndrome and 22% have low T4 syndrome.27 Transient eleva¬ tions in serum TSH concentrations precede the eventual normal¬ ization of serum T4 concentrations during recovery from the critical illness.28 These changes occur within hours. Declines in T4 and T3 levels are apparent within a few hours of injury29 and become maximal within 4 days.30 Reverse T3 peaks at 12 hours after injury and returns to normal by 2 weeks.31 The magnitude of thyroid function changes in patients with critical illness varies with the severity of the illness. The degree of T3 suppression with concomitantly low TSH correlates posi¬ tively with the severity and duration of disease, and correlates negatively with outcome.32 Decreased T4 concentration corre¬ lates with increased mortality; total T4 levels of Q.

normal

*

20 0

growth

hormone

n = 14 p< 0.0001

i

thyrotropin

prolactin

n=14 p< 0.0001

n = 14 p< o.oooi

1

B FIGURE 230-3. A, In patients with prolonged critical illness, in the presence of low-normal nocturnal serum concentrations (growth hormone [GH]: 1.5 ± 0.24 |ig/L; thyroid-stimulating hormone [TSH]: 1.25 ± 0.42 mlU/L; prolactin [PRL]: 9.4 ± 0.9 gg/L), the fraction of hormone released in a pulsatile fashion was consistently reduced for all three hormones (GH: 51 ± 6% vs. normal mean 99%; TSH: 32 ± 6% vs. normal 65%; and PRL: 16 ± 3% vs. normal 48%). Time series were obtained between 9 p.m. and 6 a.m. (Values are mean ± standard error of the mean.) B, The reduced nocturnal pulsatile GH and TSH production, calcu¬ lated with deconvolution analysis as the amount of hormone released in a pulsatile fashion per liter of distribution volume (Lv) over 9 hours, correlated positively with, respectively, low circulating insulin-like growth factor-I (IGF-I; 106 ± 11 |ig/L) and low triiodothyronine (T3; 0.64 ± 0.06 nmol/L). (Adapted from Van den Berghe G, de Zegher F, Veldhuis JD, et al. The somatotropic axis in critical illness: effect of contin¬ uous GHRH and GHRP-2 infusion. J Clin Endocrinol Metab 1997; 82:590; and from Van den Berghe G, de Zegher F, Veldhuis JD, et al. Thyrotropin and prolactin release in prolonged critical illness: dynamics of spontaneous secretion and effects of growth hormone secretagogues. Clin Endocrinol 1997; 47:599.)

substances released as a consequence of the inflammatory response to disease or trauma (e.g., tumor necrosis factor-a [TNF-a] and other interleukins) are also candidates to play a role in the pathogenesis of altered GH-secretory control. The cir¬ culating levels of these cytokines, however, are often low to undetectable in the chronic phase of critical illness. Alterna¬ tively, adaptive mechanisms within the central nervous system, possibly mediated by endogenous dopamine or serotonin, may modulate regulation of GH secretion during chronic stress. As the spontaneous GH secretion is suppressed, which is in con¬ trast to the pronounced responses to GH secretagogues, an altered SRIH secretion alone is unlikely to explain the complete picture. The observed GH-releasing effect of GHRP infusion indicates at least some availability of endogenous GHRH to maintain the synthesis of GH during critical illness. Therefore, a lack of GHRP in combination with a reduced SRIH tone would provide a suitable explanation for the findings in prolonged crit¬ ical illness.17'19'46 Infusing GHRP in this condition could accord¬ ingly be interpreted as a hypothalamic replacement therapy. From a therapeutic perspective, this provides a pathophysio¬ logic basis for exploring the safety and efficacy of GH-secretagogue administration as a strategy to counter the wasting syndrome and, consequently, to accelerate the process of recov¬ ery from prolonged critical illness. In summary, the acute stress-regulated changes within the somatotropic axis appear to consist primarily of an activated GH secretion and a peripheral shift toward its direct effects, whereas the chronic phase is mainly characterized by a relative hyposomatotro-

pism with a hypothalamic component. When a renewed acute phase, such as an intercurrent infection or an urgent surgical interven¬ tion, complicates the chronic phase, protease activity reappears in serum, and blood levels ofIGFBP-3 and IGF-I drop?4 In other words, repetitive episodes of GH resistance may appear on a back¬ ground of relative hyposomatotropism, thus forming mixed con¬ ditions that may be difficult to interpret and may explain some of the apparent paradoxes on this issue. Infusion of GH secreta¬ gogues precisely in the chronic phase of critical illness amplifies GH secretion, which is followed by a rise in the somatomedins. Supplying a hypothalamic releasing factor allows for feedback inhibition and for peripheral adjustment of metabolic pathways according to the needs determined by the disease. Thus, infusing GH secretagogues may be a safer strategy than the administra¬ tion of human recombinant GH and/or IGF-I for reversing the wasting syndrome of prolonged critical illness. THYROID AXIS Critical illness is characterized by multiple and complex alter¬ ations in the thyroid axis (see Chaps. 30-33).9'48 Again, a dual presentation appears to exist: Mainly, changes occur in periph¬ eral metabolism, binding, and receptor occupancy of thyroid hormones during acute illness and/or starvation, and a low activity state of primarily neuroendocrine origin occurs in pro¬ longed critical illness (see Fig. 230-3). Mixed forms are again possible and may further complicate the difficult interpretation of thyroid function tests in this setting.

2098

PART XVI: ENDOCRINOLOGY OF CRITICAL ILLNESS

350

T Placebo

GHRH

Placebo □ n=10

300

young: n=2 old: n=8

250 _i

3

200 21 h

I O

150

GHRH + GHRP-2

GHRP-2

100

50

..

0 0

-f

t

I

f

20

40

60

120

Minutes

21 h

06 h

B

IGF-I:

r2 = 0 67

ALS:

r2 = 0 76

IGFBP-3: r2 = 057

Pulsatile GH (pg/Lv over 9 h)

FIGURE 230-4. The somatotropes appear to readily release large amounts of growth hormone (GH) on stimulation with GH secretagogues. Shown are the serum peak GH responses to placebo, growth hormone¬ releasing hormone (GHRH; 1 pg/kg intravenously [IV]), GH-releasing peptide-2 (GHRP-2; 1 pg/kg IV), and GHRH plus GHRP-2 (1 + 1 pg/kg IV) obtained in 11 younger critically ill patients (age 28 ± 2.6 years, open circles) and 29 older critically ill patients (age 66 ± 2 years, squares). Lines interlink mean values; shaded area indicates mean ± standard error of the mean. (Reproduced from Van den Berghe G, de Zegher F, Bowers CY, et al. Pituitary responsiveness to growth hormone [GH] releasing hormone, GH-releasing peptide-2 and thyrotropin releasing hormone in critical ill¬ ness. Clin Endocrinol 1996; 45:341.) Acute illness or trauma induces alterations in thyroid function within hours. Although serum levels of thyrotropin (thyroid-stim¬ ulating hormone, TSH) usually remain normal, circulating tri¬ iodothyronine (T3) levels drop rapidly (partly due to decreased conversion of thyroxine [T4] to T3 by 5'-deiodinase).49 An increased turnover of thyroid hormones may also be involved.50 The magni¬ tude of the T3 drop within 24 hours reflects the severity of ill¬ ness.51-52 Serum reverse T3 (rT3) levels increase, partly due to reduced rT3 degradation.48 In animal models, hepatic nuclear T3 receptors appear to decrease in number and in occupancy.53-54 The

FIGURE 230-5. A, Nightly serum growth hormone (GH) profiles in the prolonged phase of illness, illustrating the effects of continuous infusion of placebo, growth hormone-releasing hormone (GHRH; 1 pg/kg per hour), GH-releasing peptide-2 (GHRP-2; 1 pg/kg per hour), or GHRH plus GHRP-2 (1 + 1 pg/kg per hour). Age range of the patients was 62 to 85 years; duration of illness was from 13 to 4& days. Infusions were started 12 hours before the beginning of the respective profiles. B, Exponential regression lines have been reported between pulsatile GH secretion and the changes in circulat¬ ing insulin-like growth factor-I (IGF-I), acid-labile subunit (ALS), and insulin-like growth factor-binding protein-3 (IGFBP-3) obtained with 45-hour infusion of either placebo, GHRP-2, or GHRH plus GHRP-2. They indicate that the parameters of GH responsiveness increase in proportion to GH secretion up to a certain point, beyond which a further increase of GH secretion has apparently little or no additional effect. In the chronic, nonthriving phase of critical ill¬ ness, GH sensitivity is clearly present; in contrast, the acute phase of illness is thought to be primarily a condition of GH resistance. (A, Adapted from Van den Berghe G, de Zegher F, Veldhuis JD, et al. The somatotropic axis in critical illness: effect of continuous GHRH and GHRP-2 infusion. J Clin Endocrinol Metab 1997; 82:590; and from Van den Berghe G, de Zegher F, Baxter RC, et al. On the neu¬ roendocrinology of prolonged critical illness: effect of continuous thyrotropin-releasing hormone infusion and its combination with growth hormone-secretagogues. J Clin Endocrinol Metab 1998; 83:309. B, Adapted from Van den Berghe G, de Zegher F, Baxter RC, et al. On the neuroendocrinology of prolonged critical illness: effect of continuous thyrotropin-releasing hormone infusion and its com¬ bination with growth hormone-secretagogues. J Clin Endocrinol Metab 1998; 83:309.)

Ch. 230: Neuroendocrine Response to Acute versus Prolonged Critical Illness absence of a TSH elevation in the face of low T3 feedback suggests that a degree of suppression or setpoint alteration also occurs at the hypothalamic-pituitary level.55-57 Experimental data suggest that enhanced nuclear T3-receptor occupancy within the thyrotropes as well as a reduced expression of the prothyrotropin (TRH) messen¬ ger RNA in the paraventricular nucleus could be involved.56-57 The cytokines TNF-cx, IL-1, and IL-6 have been investigated as putative mediators of the acute low T3 syndrome.57-60 Although these cyto¬ kines are capable of mimicking the acute stress-induced alterations in thyroid status, cytokine antagonism in sick mice failed to restore normal thyroid function.61 Endogenous thyroid hormone analogs resulting from alternative deamination and decarboxylation, such as triiodothyroacetic and tetraiodothyroacetic acid, may also par¬ ticipate in the pathogenesis of the low T3 syndrome by blunting the TSH response to low thyroid hormone feedback and by competing with active thyroid hormone for binding to transport proteins.62-63 Finally, low concentrations of binding proteins and inhibition of hormone binding, transport, and metabolism by elevated levels of free fatty acids and bilirubin have been proposed as factors contrib¬ uting to the low T3 syndrome at the tissue level.64 Teleologically, the acute changes in the thyroid axis that occur during starvation have been interpreted as an attempt to reduce energy expenditure65 and, thus, represent an appropriate response that does not warrant intervention. Whether this is also applicable to other acute stress conditions is conceivable but unproven. Alterations in the thyroid axis during the prolonged phase of ICU-dependent critical illness appear to be different. Essen¬ tially, in the presence of mean nocturnal serum TSH concentra¬ tions at the low limit of the normal range, pulsatile TSH secretion is substantially diminished and positively related to low serum levels of T318-19 (see Fig. 230-3). The normally occur¬ ring nocturnal TSH surge was consistently found to be absent, independent of concomitant sleep. The suggestion has been made that tire low TSH levels of the low T3 syndrome reflect an adaptive pituitary suppression in response to thyroid hormone levels that may be perceived as relatively high for the catabolic condition. According to this hypothesis, one would expect a negative or no correlation between circulating T3 and pitu¬ itary TSH secretion. The finding of a positive correlation between reduced pulsatile TSH secretion and serum T3 does not support this hypothesis. The alternative concept of a true central hypothyroidism is more plausible. The observation of a normal mean number of TSH bursts and the absence of the nightly TSH surge both support this concept. The exact mechanisms underlying central hypothyroidism accompanying prolonged critical illness are still unclear. As TRH is thought to determine the TSH setpoint for feedback inhibition by peripheral thyroid hormone levels, a secretory deficiency of hypothalamic TRH or decreased responsiveness to TRH may be involved. The observation that hypothalamic TRH gene expression is positively related to serum T3 after death from prolonged critical illness66 and the finding that an increase in serum TSH is a marker of the onset of recovery from severe illness55 support the hypothesis of hypothalamic TRH deficiency. Endogenous dopamine could also play a role, because dopamine has been found to blunt TSH, PRL, and GH secretory patterns in a similar fashion during critical ill¬ ness, and to decrease pituitary responsiveness to TRH.16 Likewise, alterations in somatostatin or cortisol secretion could be involved.67 Endogenous thyroid hormone analogs resulting from alternative deamination and decarboxylation, such as triiodothyroacetic acid and tetraiodothyroacetic acid, may also participate in a decreased pituitary responsiveness to TRH.62-68-69 Finally, intrapituitary or cir¬ culating cytokines may modulate pituitary hormone release.60-70 The normal TSH pulse frequency during critical illness suggests that the still unidentified pacemakers governing pulsatile hormone release by the thyrotropes are not altered. Uncertainty remains as to whether the low serum and tissue T3 concentrations71 are involved

2099

in several problems specifically associated with prolonged critical illness, such as diminished cognitive status with lethargy72; somno¬ lence or depression; ileus and gallbladder dysfunction; pleural and pericardial effusions; glucose intolerance and insulin resistance; hyponatremia; normocytic normochromic anemia; and deficient clearance of triglycerides. The hypothesis of a low T3 syndrome of neuroendocrine origin during prolonged critical illness has been further explored by investigating the effect of TRH administration.19 The thyroid axis of patients in the ICU for long periods can indeed be reactivated by a continuous infusion of TRH, producing TSH secretion in addition to increases in circulating thyroid hormones (Fig. 230-6). Interestingly, coinfusion of TRH and GH-secretagogues has been

A

Placebo

TRH

TRH + GHRP-2

7 6

3 5

E. 4 i 3

10 days postinjury) (see Chap. 230).4 In the acute period, the secretory activity of the anterior pituitary is generally augmented, with increased blood levels of adreno¬ corticotropic hormone (ACTH), prolactin, luteinizing hormone, and GH.11 In contrast, plasma thyroid-stimulating hormone (TSH) levels are usually within normal limits; however, they may be decreased, possibly due to increased endogenous corti¬ sol and dopamine release or their exogenous administration. In

patients requiring prolonged care, the secretory activity of these anterior pituitary hormones is often suppressed, and plasma levels are reduced toward or below the normal range.4,11 Excep¬ tions to this pattern are blood TSH levels, which occasionally increase during recovery, and levels of arginine vasopressin (AVP), which are often elevated acutely and may remain so dur¬ ing prolonged critical illness.12,13 Impaired anterior pituitary hormone secretion results in a propor¬ tionate decrease of the respective peripheral target organ hormones over time, except for cortisol, of which the circulating levels remain high despite decreased ACTH concentrations,n HYPOTHALAMIC-PITUITARY-ADRENAL AXIS In contrast to the general observation of increased plasma corti¬ sol levels, adrenal hypofunction is not uncommon in some groups of septic and critically ill patients.14 Subnormal plasma cortisol levels in response to standard stimulation tests, with or without blunted aldosterone responses, appear to predict increased mor¬ bidity and mortality in ICU patients.14 Glucocorticoids increase peripheral amino-acid efflux from skeletal muscle, facilitating gluconeogenesis by liver and kidney.1,2 In adipose tissue, cortisol stimulates lipolysis and inhibits glucose» uptake, resulting in elevated plasma free fatty acids, triglycerides and glycerol.1,2 In contrast to the increase in cortisol synthesis and secretion, critical illness is associated with a dissociated, reduced production of the androgens dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) by the adrenal glands11,15 (Table 2312). Blood androstenedione concentrations have been reported to be decreased in bum patients, but they are elevated in critically ill nonbum patients.15 DHEA and DHEAS concentrations are consis¬ tently decreased after severe illnesses or burns, and the DHEA response to ACTH administration is blunted.15 THYROID AXIS Within hours of acute illness or trauma, circulating levels of triiodothyronine (T3) decline, due to inhibited peripheral con¬ version of thyroxine (T4) to T3 and/or increased turnover of thyroid hormones in the hypermetabolic phase of illness4,16 (see Chaps. 228 and 232). Changes in thyroid hormone levels in ICU patients mirror the severity and duration of illness and correlate negatively with outcome variables.7 Central hypothyroidism has been hypothesized to contribute to the catabolic state of prolonged critical illness, because normal concentrations of plasma and tissue T3 are necessary for pro-

Ch. 231: Fuel Metabolism and Nutrient Delivery in Critical Illness TABLE 231-2. Endocrine Responses in Acute versus Prolonged Critical Illness* Acute (Initial 7-10 Days)

Prolonged (>10 Days)

T i

Aldosterone

Tt i T

Glucagon

TT

T

Insulin

NL

Catecholamines

TT

Triiodothyronine (total)

a

Thyroxine (total)

NL

IGF-I

i

IGFBP-3

i

Testosterone

i

F F F

Cytokines (TNF, IL-1, IL-6)

T

NL

Cortisol Adrenal androgens

NL

to i

to F

T

T F

to T

NL

to F

T, increased blood levels; 1, decreased blood levels; NL, generally normal blood lev¬ els; IGF, insulin-like growth factor; IGFBP, IGF-binding protein; TNF, tumor necrosis fac¬ tor; IL, interleukin. ‘Responses vary as a function of the nature, timing and severity of illness, underly¬ ing nutritional status, drug administration, and other factors. Alterations in growth hor¬ mone and thyroid-stimulating hormone pulse amplitude and/or pulse number also may occur.

tein synthesis, lipolysis, fuel utilization by muscle, and GH secretion.1'2'17 Thyroid hormones may induce protein catabo¬ lism when levels are increased, however; thus, the low T3 syn¬ drome may be an adaptive response to conserve body protein, as in simple malnutrition.1 PITUITARY-GONADAL AXIS Serum testosterone levels in men decrease markedly in a number of catabolic states, including malnutrition and criti¬ cal illness.4'18 Acute injury results in Leydig cell suppression, as evidenced by low serum testosterone concentrations in association with elevated plasma luteinizing hormone (LH) concentrations but normal levels of follicle-stimulating hor¬ mone (FSH) and inhibin in plasma.4'19 In women, prolonged critical illness is associated with a decrease in both FSH and LH levels.20 In men, prolonged critical illness and hypoandrogenemia is associated with low LH concentrations.4 Adminis¬ tration of dopamine and long-term administration of opioids result in additional suppression of gonadotrope function.18 The magnitude of the decrease in plasma testosterone corre¬ lates with the severity of the insult in prolonged coma after trauma.21 Recovery of gonadal function may take weeks, even months, after recovery from critical illness. Thus, prolonged hypogonadism, especially in men, may delay anabolism and clinical recovery. GROWTH HORMONE-INSULIN-LIKE GROWTH FACTOR AXIS Malnutrition, exogenous and endogenous glucocorticoids, somatostatin, and drugs (e.g., dopamine) can attenuate pulsatile GH secretion and result in low concentrations of IGF-I. In criti¬ cally ill patients the rise in GH concentrations coupled with low levels of IGF-I may serve to shift metabolic responses to those directly induced by GH, including lipolysis and insulin antago¬ nism, and to concomitantly attenuate protein-sparing anabolic effects mediated by IGF-I.5'6 A summary of peripheral endocrine response patterns for major body hormones in acute versus prolonged critical illness is given in Table 231-2 (also see Chap. 230). The onset of clinical recovery is characterized by restored sensitivity of the anterior pitu¬ itary to reduced feedback controlA11-13

2105

NUTRITIONAL SUPPORT IN CRITICAL ILLNESS GENERAL CONCEPTS The primary focus of nutritional support in critically ill patients is to supply adequate energy, protein, and micronutrients (e.g., vitamins, minerals, electrolytes) to facilitate organ function, wound and tissue healing, and, thus, patient recovery. Protein is critical for organ structure and function, and a significant ero¬ sion of lean tissue is associated with a worsened clinical out¬ come, including diminished immune function, increased rates of infection, decreased wound healing, skeletal muscle dysfunc¬ tion, and delayed convalescence.9'10 This relationship is espe¬ cially apparent in those with preexisting malnutrition before the onset of severe catabolic stress. Direct cause-and-effect relation¬ ships between nutritional status and patient outcomes are diffi¬ cult to prove, however, because malnutrition reflects, in part, the severity or nature of the underlying illness.22 Nonetheless, a rea¬ sonable assumption is that the provision of nutritional support in critically ill adult and pediatric patients is beneficial if feeding is not possible for 5 to 7 days.23 The goal of nutritional support is to maintain organ structure and function and to support body metabolism and protein syn¬ thesis. Thus, major objectives of nutritional and metabolic sup¬ port in malnourished or catabolic patients are to detect and correct preexisting depletion of macro- and micronutrients, attenuate progressive protein wasting, optimize fluid and elec¬ trolyte status, and, thus, reduce morbidity related to malnutri¬ tion.10-23 Parenteral and enteral nutritional support is adjunctive to primary therapies (e.g., fluid resuscitation, oxygen delivery, abscess drainage, and provision of antibiotics). Use of parenteral nutrition (PN) and tube feedings has become a standard of care in most ICUs throughout the world. Few objective data from properly designed, randomized, controlled studies are available to determine the true efficacy of, and the indications for, nutri¬ tional support in critical illness.24 Nonetheless, several clear indications exist for the use of PN and tube feedings in critically ill patients (Table 231-3). NUTRITIONAL ASSESSMENT Nutritional assessment in the hospital setting involves an inte¬ gration of several factors, including medical/surgical history, type and severity of the acute underlying illness, physical exam¬ ination findings, weight loss history, prior dietary intake pat¬ tern, evaluation of current organ function, and fluid status and

TABLE 231-3. Some Clinical Indications for Specialized Enteral or Parenteral Nutrition (PN) Support in Critically III Patients Food intake not possible for >5-7 days due to underlying illness Severe catabolic stress (e.g., burns, trauma, sepsis) Major gastrointestinal operations (PN) Medical illness associated with prolonged gastrointestinal dysfunction (diar¬ rhea, nausea/vomiting) and/or illness in which oral food intake is con¬ traindicated: Bone marrow transplantation Inflammatory bowel disease Pancreatitis High-output enterocutaneous fistula Ileus or bowel obstruction Short bowel syndrome Preexisting moderate to severe protein-energy malnutrition and inability to maintain adequate enteral feeding to promote anabolism (PN)

2106

PART XVI: ENDOCRINOLOGY OF CRITICAL ILLNESS

TABLE 231-4. Components of Comprehensive Nutritional Assessment in Hospitalized Patients PHYSICAL EXAMINATION Skeletal muscle and fat wasting Skin lesions suggestive of micronutrient deficiency Fluid status

BODY WEIGHT PATTERN Current body weight as percentage of ideal body weight

BEE for females (kcal/24 hours) = 655.1 + (9.6 x kg body weight) + (1.8 x height in cm) - (4.7 x age in years) The BEE is then multiplied by factors to account for activity (1.2 to 1.3 times BEE, unless the patient is sedated), and occa¬ sionally for illness severity, to arrive at the energy prescription. The estimated maintenance energy requirement is -1.3 times BEE.23 24 In obese subjects (defined as >20% above ideal body weight), the adjusted body weight should be used in the calcula¬ tion of energy and protein needs:

Usual body weight Percentage weight loss over past several weeks and months

DIETARY INTAKE PATTERN Food intake pattern Previous intravenous or enteral nutritional support Use of nutritional supplements

INTESTINAL TRACT FUNCTION Gastrointestinal symptoms (nausea, emesis, diarrhea, steatorrhea) Delayed gastric emptying, gastroparesis, ileus, obstruction, intraabdomi¬ nal infection, perforation, hemorrhage

SELECTED SERUM OR PLASMA BIOCHEMICAL INDICES Standard organ function indices (liver and renal function tests), blood glucose Electrolytes, including calcium, magnesium, and phosphorus Triglycerides Vitamins and mineral levels (e.g., serum zinc, vitamin C), if suggested by physical examination, by dietary history, or by underlying illness Serum proteins (e.g., albumin, prealbumin) are not helpful in the intensive care unit setting (values markedly influenced by degree of stress and by fluid status)

selected biochemical values (Table 231-4). Circulating concen¬ trations of proteins (e.g., albumin, prealbumin, IGF-I, transfer¬ rin, and retinol-binding protein) generally are not useful as indices of the underlying nutritional status in ICU patients. Plasma levels of these proteins are markedly affected by nonnutritional factors (e.g., fluid status, decreased hepatic synthesis and increased metabolic clearance associated with inflammation or infection).22-23 Because of the long turnover of albumin (18-21 days), levels in blood remain low despite adequate feeding. The basic principle in considering PN therapy is that the patient must be unable to achieve adequate energy, protein, and micronutrient intake via the enteral route with oral food, liquid supplements, or tube feedings. Compared with PN, enteral feeding is much less expensive, maintains intestinal mucosal structure and function, is safer in terms of mechanical and met¬ abolic complications, and is associated with reduced rates of nosocomial infections.23'24 Thus, the enteral route of feeding should be used whenever possible. PROVISION OF ENERGY AND PROTEIN Studies on nutrient utilization efficiency in severely catabolic patients suggest that lower amounts of total energy (calories) and protein should be administered than have been routinely given in the past.22'23 Excessive dietary calories and protein loads (hyperalimentation) may induce metabolic complications, including carbon dioxide overproduction, azotemia, hypergly¬ cemia, electrolyte alterations, and hepatic dysfunction.24 Energy intake should be advanced slowly over several days after the initiation of specialized feeding to provide maintenance energy intake. Energy requirements may be estimated using standard equations (e.g., the Harris-Benedict equation) that incorporate the patient's age, sex, weight, and height to determine basal energy expenditure (BEE): BEE for males (kcal/24 hours) = 66.5 + (13.8 x kg body weight) + (5.0 x height in cm) - (6.8 x age in years)

Adjusted body weight = (current weight - ideal body weight [from standard tables or equations] x 0.25) + ideal body weight The estimated dry weight should be used to determine energy and protein needs (see later) in settings of peripheral or central edema due to fluid overload or the capillary leak syndrome. Energy expenditure varies considerably from day to day in critically ill ICU patients. In light of the complications related to overfeeding outlined earlier, energy provision in the range of 25 to 30 kcal/kg per day is generally safe for most ICU patients and for stable patients without severe malnutrition.23-24 In clinically stable non-ICU patients with severe malnutrition who require nutritional repletion, 35 to 40 kcal/kg per day may be provided, with careful monitoring of serum chemistries, as outlined later. Carbon dioxide overproduction, as evidenced by an indirect calorimetry-derived respiratory quotient of >1.0 (the ratio of carbon dioxide production to oxygen consumption) was not uncommon in the past, when ICU patients routinely received excessive energy doses. This complication is unusual, however, with current standards of nutritional care in ICU settings. Dextrose in PN or tube feeds should be given at a dosage not to exceed 5 mg/kg per minute (-500 g per day for a 70-kg per¬ son).23 Catabolic patients are unable to efficiently oxidize larger carbohydrate loads, which may induce hyperglycemia, hepatic steatosis, and/or excessive carbon dioxide production over time. Dextrose should provide 70% to 80% of nonprotein energy, unless the patient is hyperglycemic. In this case, the dextrose load should be reduced and/or regular insulin should be pro¬ vided in parenteral feeding or as a separate insulin drip to main¬ tain blood glucose between 100 and 150 mg/dL.25 Intravenous lipid emulsions are used to provide essential linoleic and linolenic fatty acids or as an energy source, and are generally infused over a 24-hour period in patients requiring PN. The maximal recommended rate of fat emulsion infusion is» -1.0 g/kg per day.23 ICU patients generally clear intravenous fat emulsions well from plasma; however, large doses of fat emul¬ sion have been associated with impaired reticuloendothelial function and possibly immune suppression. Serum triglycerides should be monitored serially to assess the clearance of the intra¬ venous fat emulsion. The triglyceride levels should be main¬ tained at 2.0 g/kg per day are not efficiently used for protein synthesis, and the excess may be oxidized, contributing to azotemia. In most cata¬ bolic patients requiring specialized feeding, a generally recom¬ mended protein dose is 1.5 g/kg per day in individuals with normal renal function.9-23 The administered protein dosage should be adjusted downward as a function of the degree of azotemia and hyperbilirubinemia. This strategy takes into account the relative inability of ICU patients to efficiently use exogenous nutrients and the knowledge that most protein and lean tissue repletion occurs over a period of several weeks to months during convalescence. Adequate nonprotein energy is essential to allow amino acids to be effectively used for protein

Ch. 231: Fuel Metabolism and Nutrient Delivery in Critical Illness TABLE 231-5. Guidelines for Protein Administration in Hospitalized Patients Condition

Protein Intake Goal’ (g/kg/day)

Malnourished, clinically stable

1.5-2.0

TABLE 231-6. Guidelines for Electrolyte and Micronutrient Administration in Parenteral Nutrient (PN) Solutions* Element

Peripheral PN

Central PN

Mild to moderate catabolic stress

1.2-1.5

Potassium (mEq/L)

20^0

40-60

Critically ill

1.2-1.5

Sodium (mEq/L)

30-75

50-75

Encephalopathy

0.6

Phosphorus (mmol/L)

5-8

10-15

Hepatic failure

0.6-1.0

Calcium (mEq/L)

5

5

Renal failure, not dialyzed

0.6-0.8

Magnesium (mEq/L)

5-8

15

Renal failure, dialyzed

1.2

’Protein load is adjusted proportional to hepatic and renal function indices.

synthesis. The nonprotein calorie/nitrogen ratio used in most centers ranges from 100:1 to 150:1 (nitrogen = protein/6.25). Highly catabolic patients are given protein loads at the lower end of this range, assuming near-normal renal and hepatic func¬ tion.9'23 To diminish the risk of phlebitis, peripheral vein PN solu¬ tions provide low concentrations of dextrose (480 mg/d are not recommended. ACTIONS AND SIDE EFFECTS: Trilostane reversibly inhibits 3B-hydroxysteroid dehydrogenase, causing decreased production of cortisol and aldosterone. Contraindicated in severe hepatic or renal disease. Side effects include abdominal discomfort or pain, diarrhea, oral or nasal burning, headache, myalgia, arthralgia and skin rash. May prevent maximal adrenal response in a stressed patient or when used in combination with other adrenal steroid inhibitors. May also cause orthostatic hypotension by its suppression of aldosterone production._

Drug|Trade Names and Preparations ADRENOCORTICOTROPIC HORMONE (ACTH) INDICATIONS: Assessment of adrenocortical function. Disorders that respond to glucocorticoid therapy also respond to ACTH if the adrenal cortex is intact [see Chap. 78], Powder: ACTH Acthar Corticotropin

Repository: 40 U/vial ACTH-80 25 or 40 U/vial H.P. Acthar Gel 40 U/vial INDICATIONS: Limited use in testing adrenocortical function and disorders responsive to corticosteroids.

I. CORTICOTROPIN

80 U/mL 40 or 80 U/mL

DOSE: Use as much as 80 U as a single injection for test of adrenal responsiveness. ACTIONS AND SIDE EFFECTS: Prepared from animal pituitaries. Higher risk of allergic reactions than for the synthetic analog of ACTH, cosyntropin (below). May be used as a Rx agent. If used long-term, patients should be monitored for side effects of hypercortisolism. These preparations are all for IM or SC injection. "ACTH" and Acthar also may be given IV for Dx purposes. The others all are repository gels and are not for IV use. II. COSYNTROPIN ‘^^^^^^T^ortrosyt^"*"'**^"^^^5^t^'per vial INDICATIONS: (1) The short ACTFI stimulation test is used as a screening test of adrenocortical function in the assessment of hypothalamicpituitary-adrenal function in patients treated with glucocorticoids. (2) The long ACTH stimulation test is performed in the evaluation of adrenal insufficiency when the response to the short test is abnormal. It helps to delineate whether the source is primary or central. It is the preferred preparation for Dx testing of adrenocortical function. Given IM or IV. DOSE: 1 pg or 250 gg bolus push. See Chapters 74 and 241 for use in ACTH stimulation tests. ACTIONS AND SIDE EFFECTS: A synthetic corticotropin analog of the first 24 amino acids of the corticotropin molecule, which possesses full bioactivity. Not used as a Rx agent [see Chaps. 78 and 241 ].

2145

Trade Names and Preparations

_ ANDROGENS 1. TESTOSTERONE A. TRANSDERMAL SYSTEM

mg/d 4&6 5 6

Testoderm Testoderm TTS Testoderm + adhesive

mg/d 2.5 & 5 50,75 & 100

Androderm AndroGel 1%

INDICATIONS: Replacement therapy for conditions associated with a deficiency or lack of endogenous testosterone (i.e., congenital and acquired primary and hypogonadotropic hypogonadism). DOSE: Testoderm should be applied to dry shaven scrotal skin every 22-24 h. Total testosterone measurement should be determined after 3-4 wk of Rx 2-4 h after application. Testoderm TTS may be applied to skin of arm, back, or upper buttocks every 24 h. Serum testosterone concentrations should be measured 2-4 h after application. Androderm may be applied to skin of back, abdomen, upper arms, or thighs at bedtime. Serum testosterone concentrations should be measured the morning after application. AndroGel 1% (i.e., 5G delivers 50 mg testosterone) is applied once daily to clean dry intact skin of shoulders, upper arms, or abdomen. Do not apply gel to genitals. Avoid showering or swimming for about 5-6 h. Check testosterone level after 14 days. _SIDE EFFECTS: See discussion below.

~b7 testosterone SUBCUTANEOUS IMPLANT T estopel INDICATIONS: As for the transdermal system (see above)

75 mg pellets

DOSE: In androgen-deficient males, use 150-450 mg SC q 3-6 mo. Adjust dose as follows: implant two 75 mg pellets for each 25 mg testosterone propionate required weekly. SIDE EFFECTS: See discussion below. II. TESTOSTERONE (In Aqueous Suspension)

Testosterone (Aq) Histerone 100

mg/mL 25, 50 & 100 100

Testamone Testandro

mg/mL 100 100

mg/mL

III. TESTOSTERONE DERIVATIVES A. 17P-Hydroxyl Esterification 1. NANDROLONE DECANOATE IN OIL 2. NANDROLONE PHENPROPIONATE IN OIL 3. TESTOSTERONE CYPIONATE IN OIL 4. TESTOSTERONE ENANTHATE IN OIL 5. TESTOSTERONE PROPIONATE IN OIL B. 17a-Alkylation 1. FLUOXYMESTERONE 2. METHYLTESTOSTERONE

Nandrolone decanoate D

50, 100 & 200

Nandrolone phenpropionate

25 & 50

Testosterone cypionatelOO & 200 depAndro 100 100 depAndro 200 200 Testosterone enanthate 100 & 200 Andro L.A. 200 200

Testosterone propionate

(tabs) Fluoxymesterone (tabs) Methyltestosterone Android-10 Android-25 Oreton Methyl Oxandrin

Androlone-D Deca-Durabolin

200 50, 100 & 200

Durabolin Hybolin Improved

25 & 50 50

Depo-T estosterone Depotest 100 Depotest 200 Andropository-200 Delatestryl

100 & 200 100 200 200 200

Hybolin Decanoate Neo-Durabolic

50 & 100 50 & 200

Nandrobolic

25

Duratest-100 Duratest-200

100 200

Durathate-200 Everone

200 200

100

mg/tab 10

(tabs) Halotestin

mg/tab 2, 5 & 10 mg/tab 10 10

(tabs)

mg/tab (All contain tartrazine)

mg/tab (buccal tabs) (caps) mg/tab 10 & 25 Methyltestosterone Testred 10 10 Oreton Methyl Virilon 10 25 10 3. OXANDROLONE 2.5 4. OXYMETHOLONE Anadrol-50 50 5. STANOZOLOL Winstrol 2 INDICATIONS: See Chapter 119 for clinical uses and abuses of androgens. Also see Chapter 92, use in delayed puberty; Chapter 18, pediatric dose in pituitary hormonal deficiency; Chapter 17, replacement dose in adult hypopituitarism; Chapter 224, use in breast Ca. The aqueous testosterone preparations and 1713-Hydroxyl esterification derivatives are for use by IM injections. 1713-Hydroxyl derivatives are in oil and are long-acting. 17aAlkylation derivatives are po preparations. Bioavailability of po 17a-Alkylation derivatives is lessened by passage through the hepatic circulation. Also, they have potential hepatotoxicities. The so-called anabolic steroids are related to testosterone; they have a high anabolic/low androgenic ratio, and have been prescribed for weight gain in underweight, recently ill patients; for osteoporosis; for reversal of nitrogen loss during corticosteroid Rx; for certain varieties of anemia; and for hereditary angioedema. These agents include nandrolone phenpropionate, nandrolone decanoate, oxandrolone. oxymetholone, and stanozolol. DOSE: See Chapters in Indications. ACTIONS AND SIDE EFFECTS: Side effects include virilization in the female, hepatotoxicity, prostatic hypertrophy, inhibition of spermatogenesis, gynecomastia, acne, baldness, hirsutism, polycythemia, sleep apnea, hyperlipidemia, edema, and/or hypercalcemia with malignancy or immobilization. Periodic evaluation of LFT, PSA, lipids, and C'BC is recommended. Their use has been much abused [see Chap. 119], Rule out pregnancy if considering use of any androgen in a woman.

2146

IV. DANAZOL

I Danocrine

50, 100 & 200 mg/cap

INDICATIONS: (1) May be used to induce medical "pseudomenopause" in endometriosis; (2) fibrocystic disease of breast if severe pain and tenderness; (3) prophylactically in hereditary angioedema. Unlabeled uses: premenstrual syndrome [see Chap. 99], precocious puberty, gynecomastia, menorrhagia, idiopathic immune and lupus-associated thrombocytopenias, autoimmune hemolytic anemia. DOSE: Dosage must be individualized. In females, start only during menses or with negative pregnancy test. For mild endometriosis, initially use 200-400 mg/d in 2 divided doses. Severe cases may require up to 800 mg/d in 2 divided doses. Dosage is adjusted according to response [see Chap. 98], Rx is continued for 3-6 mo; may extend to 9 mo. In fibrocystic breast disease, dosage is 100-400 mg/d in 2 divided doses. In hereditary angioedema, initial dosage is 200 mg bid to tid. ACTIONS AND SIDE EFFECTS: Synthetic androgen derivative of 17a-ethinyltestosterone. Side effects include: androgenization, hypoestrogenicity and hepatic dysfunction. Exclude Ca of the breast before Rx. Long-term experience limited. Androgenic effects may not reverse when drug is stopped [see Chap. 98]._

Drug_Trade Names and Preparations ANTIANDROGENS I. BICALUTAMIDE II. FLUTAMIDE_

Casodex

50 mg tab

Eulexin

125 mg cap

INDICATIONS: Treatment in combination with GnRH analogs for stage D: prostate Ca [see Chap. 225]. The flutamide GnRH combination is also indicated in the treatment of stage B2 to C prostate Ca in conjunction with radiation therapy. Unlabeled uses include hirsutism and prostatic hyperplasia. DOSE: Casodex 50 mg po qd. Eulexin 250 mg po tid q8h. ACTIONS AND SIDE EFFECTS: All agents are nonsteroidals with antiandrogenic properties. Contraindicated in pregnant women. Common side gynecomastia, nausea, and impotence. Rarely causes reversible elevation in L.FT and jaundice. 7ir’*NTLUTAM7DE~"*"^'**~^ INDICATIONS: Treatment in combination with surgical castration for treatment of stage D2 metatstatic prostatic Ca. DOSE: 300 mg po qd for 30 days, then 150 mg po qd. ACTIONS AND SIDE EFFECTS: As above. Impaired adaptation to darkness. Rarely, interstitial pneumonitis. Baseline chest x-ray should be obtained.___

ttttnXste^e--w^b--r^b— INDICATIONS: Proscar for benign prostatic hyperplasia. Propecia for androgenetic alopecia. Unlabeled use for adjuvant monotherapy following radical prostatectomy. DOSE: Proscar 5 mg po qd. May take 6-12 months to see response. Propecia 1 mg po qd. Generally takes 3 or more months to see benefit. ACTIONS AND SIDE EFFECTS: A competitive and specific inhibitor of type II 5a-reductase reducing conversion of testosterone to dihydrotestosterone. Contraindicated in women who are or may potentially be pregnant, and women should not handle crushed or broken tablets. PSA levels drop in patients with benign prostatic hyperplasia or prostate Ca, but does not indicate a beneficial effect._

Trade Names and Preparations

Drug ANTITHYROID DRUGS

I. Thionamide Derivatives A. METHIMAZOLE B. PROPYLTHIOURACIL INDICATIONS: Used in medical management of Graves disease or toxic nodular goiter [see Chap. 42], Propylthiouracil may be preferred to methimazole in treatment of (1) severe hyperthyroidism including thyroid storm, because it blocks peripheral conversion of T4 to T3; (2) hyperthyroidism in pregnancy, because less placental transfer and no teratogenic effects reported. Dosage here should be adjusted to keep T4 in uppernormal range; (3) lactation (only if patient insists, with scrupulous monitoring of infant's thyroid function. PTU crosses into breast milk less than methimazole). Frequently possible to maintain patients on once-daily dosing with methimazole. DOSE: See Chapters in Indications. ACTIONS AND SIDE EFFECTS: Use has been associated with aplasia cutis in the fetus, but the significance of this finding has been questioned. Agranulocytosis, skin rash, jaundice, abnormal LFT. II. Iodide-Containing Compounds A. IODIDES

Soln: Potassium iodide Pima SSKI Strong Iodine soln: (Lugol's) Tabs: Thyro-Block Injection: Sodium Iodide

1 g potassium iodide/mL 325 mg potassium/5 mL syrup 1 g potassium iodide/mL 5% iodine + 10% potassium iodide soln 130 mg potassium iodide tabs

10% sodium iodide for injection in 10 mL amps

2147

INDICATIONS: (1) Thyroid storm > 1 h after 6-adrenergic blocker and thionamide Rx instituted; (2) postradioactive iodine Rx to control hyperthyroidism, while awaiting the therapeutic radioactive effect. Iodide started about 5 d after radioactive iodine administration; (3) preparation of hyperthyroid patient for surgery: Iodide given for about 10 d before; (4) radiation exposure emergencies to block thyroidal uptake of iodine. DOSE: In hyperthyroidism, dosage generally is 5 drops of soln po tid or I tab tid [see Chapter 42 for adult doses. Chapter 47 for pediatric doses]. In radiation exposure, the recommended dosages are: age 1 yr=l tab or 6 drops po/d. In thyroid storm, the parenteral dosage of sodium iodide is 2 g/d. ACTIONS AND SIDE EFFECTS: Considered "non-routine" agents in Rx of hyperthyroidism. See Chapters 37, 42 and 47 for specifics regarding uses, mechanisms of action and adverse reactions. Administration in multinodular goiter may lead to an increase in thyroid hormone levels and thyrotoxicosis. Thyro-Block tabs are available only to state and federal agencies. Caution: test for idiosyncrasy before giving iodide parenterally. B. Cholecystographic Dyes 1. IOCETAMIC ACID Cholebrine 62% iodine as 750 mg tabs 2. IOPANOIC ACID Telepaque 66.7% iodine as 500 mg tabs 3. IPODATE SODIUM Bilivist 61.4% iodine as 500 mg caps Oragrafin Sodium 61.4% iodine as 500 mg caps (contains tartrazine) 4. IPODATE CALCIUM Oragrafm Calcium 61.7% iodine as 3 g/pack oral suspension 5. TYROPANOATE SODIUM Bilopaque 57.4% iodine as 750 mg caps INDICATIONS: May be used for short-term early Rx of hyperthyroidism or as an adjunct to thionamides. DOSE: 500-1000 mg po/d. _ACTIONS ANDSJDE JEFFECTS: lopanoic acid_may not be effective as long-term Rx in Graves disease; Ti reduction may not be sustained. C. Radioactive Iodine Iodotope 8-100 mCi/cap; 7.05 mCi/rnL oral soln SODIUM 1,11_Sodium Iodide lnl Therapeutic 0.75-100 mCi; cap; 3.5-150 mCi/vial soln_ INDICATIONS: Sodium 1 11 is indicated for: (I) Hyperthyroidism [Graves, toxic solitary or multiple nodular goiter]; (2) papillary and follicular Ca of the thyroid. DOSE: 1 ' I dose calculated by gland size and radioactive iodine uptake [see Chap. 42]. Toxic multinodular goiter may be relatively resistant to its effects. The usual dose of1311 in thyroid Ca is 50 mCi to ablate the thyroid bed and 100-150 mCi as a Rx dose. ACTIONS AND SIDE EFFECTS: Half-life of1 11 is 8.06 d. B-Radiation causes 90% of the local effect and y, 10% [see Chaps. 40, 42 and 47],_

Drug

Trade Names and Preparations

AROMATASE INHIBITORS Arimidex

ANASTROZOLE

mg/tab

INDICATIONS: Treatment in advanced breast Ca and in postmenopausal women with disease progression following antiestrogen Rx. DOSE: 1 mg po qd. ACTIONS AND SIDE EFFECTS: A nonsteroidal competitive inhibitor of aromatase enzyme that prevents conversion of androgens to estrogens. -Generally well-tolerated. May cause diarrhea, nausea, hot flashes, headache and edema. II. LETROZOLE

Femara

2.5 mg tabs

INDICATIONS: Same as anastrozole. DOSE: 2.5 mg qd. SIDE EFFECTS: Same as anastrozole. III. TESTOLACTONE

Teslac

50 mg tabs

INDICATIONS: Adjuvant Rx for palliative treatment of advanced or disseminated breast Ca in postmenopausal women, when hormonal Rx is indicated. May also be used in premenopausal women after ovarian function has been terminated. Unlabeled use in congenital adrenal hyperplasia and gynecomastia. DOSE: 250 mg po qid. SIDE EFFECTS: A synthetic antineoplastic agent that inhibits steroid aromatase activity. The inhibition may be noncompetitive and irreversible. Occasionally, rash, paresthesia, increased blood pressure, acne, edema, glossitis, anorexia, nausea, and vomiting._

Drug

Trade Names and Preparations

BISPHOSPHONATES I. ALENDRONATE SODIUM

1 Fosamax

5, 10 & 40 mg tabs

INDICATIONS: (1) Prevention and treatment of osteoporosis, including the osteoporosis associated with glucocorticoids. (2) Symptomatic Paget disease of the bone. DOSE: Prevention of osteoporosis: 5 mg tab po qd with 8 ounces of water at least 30 min before food, beverage, or other medications. Patients should avoid lying down for at least 30 min after ingestion. Treatment of osteoporosis (men and women): 10 mg tab po qd. Treatment of Paget disease of bone: 40 mg tab po qd for 6 months. Retreatment in patients with relapse may be considered after a 6-month drug-free period. Periodic alkaline phosphatase measurement is recommended. Adequate calcium and vitamin D should be ensured.

2148

ACTIONS AND SIDE EFFECTS: Contraindicated in patients with hypocalcemia or hypersensitivity to any components. Not recommended for _^iit>£^iLWith_creatinine>clearanc^2-4 wk causes down-regulation of pituitary gonadotropin receptors and a fall in androgens in males to castrate levels and of estrogens in premenopausal females to postmenopausal levels. Side effects when used in Rx of prostate Ca include a flare of bone pain in the first 4-10 d, which may be attenuated by the prior and concomitant use of

lT7^FlRmN7cETATE-INDICATIONS: Rx of endometriosis and central precocious puberty. DOSE: Dosage for endometriosis is 400 pg/d. Dosage for central precocious puberty is 1600 pg/d up to 1800 pg/d. ACTIONS AND SIDE EFFECTS: Rhinitis and hypoestrogenemia.

Trade Names and Preparations

-dj.:hs. GONADOTROPIN RELEASING HORMONE-ANTAGONIST GAMRELIX ACETATE

Antagon

250 pg/5 mL prefilled syringe

INDICATIONS: Inhibition of premature LH surges during controlled ovarian hyperstimulation. DOSE: 0.25 mg daily SC. ACTIONS AND SIDE EFFECTS: Synthetic decapeptide generated by amino acid substitution of native GnRH. Competitively blocks GnRH receptors. Pregnancy must be excluded. Should not be used in patients with hypersensitivity to GnRH.

Drug

Trade Names and Preparations

GONADOTROPINS I. CHORIONIC GONADOTROPIN, HUMAN (hCG)

Chorionic gonadotrop in A.P.L. Chorex-5 & 10 Choron 10 Gonic Pregnyl Profasi

5-10-20,000 U.S.P. units with 10 mL diluent, for IM use only 5-10-20,000 U.S.P. units with 10 mL diluent, for IM use only 5-10,000 U.S.P. units with 10 & 25 mL diluent, for IM use only 10,000 U.S.P. units with 10 mL diluent, for IM use only 10,000 U.S.P. units with 10 mL diluent, for IM use only 10,000 U.S.P. units with 10 mL diluent, for IM use only 5-10,000 U.S.P. units with 10 mL diluent, for IM use only INDICATIONS: Rx of infertility: (1) In females: (a) in the induction of ovulation in combination with menotropins; (b) for Rx of luteal phase defect; (2) in males (a) in selected cases of hypogonadotropic hypogonadism; (b) prepubertal cryptorchidism not due to anatomical obstruction.

DOSE: See Chapter 97 for protocol for ovulation induction xAvk. After 18-24 mo, hMG may be added.

One regimen for Rx of male hypogonadotropic infertility is 2000 IU of hCG IM 2-3

ACTIONS AND SIDE EFFECTS: hCG is a placental hormone extracted from the urine of pregnant women. Biologically, hCG mimics the action of LH. Headaches, irritability, gynecomastia, and precocious puberty. II. FOLLITROPINS A. FOLLINOTROPIN ALPHA B. FOLLINOTROPIN BETA C. UROFOLLINOTROPIN

Gonal-F Follistim Fertinex

75 & 150 IU 75 IU 75 & 150 IU/mL

INDICATIONS: (1) Ovulation induction in anovulatory infertile women; (2) Follicle stimulation in ovulatory women undergoing “Assisted Reproductive Technologies.” DOSE: See protocol for ovulation induction. ACTIONS AND SIDE EFFECTS: Urofollinotropin is highly purified FSH extracted from the urine of postmenopausal women. Follinotropin alfa and beta are human FSH preparations of recombinant DNA origin. Follinotropins stimulate ovarian follicular growth. Treatment with hCG may be needed in the absence of an endogenous LH surge. Ovarian hyperstimulation syndrome, multiple pregnancies, ovarian enlargement, thromboembolic events, and acute respiratory distress syndrome.

2158

III. MENOTROPINS (hMG)

Pergonal

75 IU each of FSH and LH activity per 2 mL amp of hMG 150 1U each of FSH and LH activity per 2 mL amp of hMG

INDICATIONS: (1) Induction of ovulation in selected patients who do not respond to clomiphene, or do not produce endogenous gonadotropins; (2) with hCG in Rx of hypogonadotropic or idiopathic male infertility when no response to clomiphene. DOSE: See Chapter 97 for protocol for ovulation induction using hMG-hCG. In male infertility, the dosage of hMG is 25-75 IU 3 x/wk with hCG injections 2 x/wk. ACTIONS AND SIDE EFFECTS: hMG is a preparation of human menopausal gonadotropins extracted from the urine of postmenopausal women. Side effects include hyperstimulation syndrome and gynecomastia.

Drug

Trade Names and Preparations

GROWTH HORMONE (GH) SOMATROPIN

Genotropin Humatrope Norditropin Nutropin Nutropin AQ Saizen Serostim

1.5, 5.8 & 13.8 mg/mL (5 & 12 mg cartridge may be used with pen). 5 mg (~ 13 IU) per vial 4 & 8 mg per vial (also available in cartridge for the Nordipen) 5 & 10 mg (~ 13 & 26 IU) per vial 10 mg (-30 IU) 5 mg per vial 5,6 mg per vial

SOMATREM

Protropin 5 & 10 mg (~ 13 & 26 IU) per vial INDICATIONS: (I) Rx of pediatric GH deficiency (GHD); (2) adult GHD; (3) pediatric chronic renal insufficiency; (4) Turner syndrome; (5) cachexia treatment of AIDS. Somatropin and somatrem are synthetic preparations of GH. Somatropin is identical to human pituitary GH, while somatrem has an additional methionine. DOSE: General guidelines for indication #1 are 0.18-0.3 mg/kg total weekly dose divided into 3-7 injections/wk SC or 1M. For indication #2. 0.375 mg/kg total weekly dose divided into 3-7 injections/wk SC or IM. For indication #3, 0.35 mg/kg total weekly dose divided into qd SC injections. (In hemodialysis, administer hs or 3-4 h post dialysis. With chronic cycling peritoneal dialysis give q morning. In chronic ambulatory dialysis, give q hs.) For indication #4, 0.002-0.006 mg/kg (Nutropin AQ only) SC qd. Starting dose is titrated q 6 wk to maximum of 0.0125 mg/kg/d. For indication #5, 0.1 mg/kg (Serostim only) SC hs. ACTIONS AND SIDE EFFECTS: In children: injection site reactions, lipoatrophy, headache, hematuria, hypothyroidism, mild hyperglycemia. A small number of patients may develop antibodies to protein that may interfere with growth response. Relationship between leukemia and GH Rx is uncertain. Benign intracranial hypertension may occur soon after initiation of Rx. A dose reduction or discontinuation resolves signs and symptoms. Contraindicated with any evidence of neoplastic activity. Discontinue if any evidence of tumor growth. Do not use with fused epiphyses. Concomitant glucocorticoid Rx may inhibit the growth promoting effects of GFI. Baseline and periodic thyroid function studies, fundoscopic exams, and bone age should be monitored. Patients should be observed for glucose intolerance. Use with caution in patients with diabetes. Patients with endocrine disorders have a higher incidence of slipped capital femoral epiphyses. Patients with chronic renal insufficiency should have baseline X-rays of the hip to monitor for renal osteodystrophy and avascular necrosis of the femoral head. Patients with Turner syndrome should be evaluated carefully for otitis media and other ear disorders while on GH Rx. In adults: Patients with childhood GHD should be reevaluated before replacement with GH. Patients should be monitored with 1GF-I levels q 6 wk while titrating dose. Side effects include edema, arthralgias, carpal tunnel syndrome, rarely malignant transformation of nevi. gynecomastia, and pancreatitis.

Drug

1

Trade Names and Preparations

GROWTH HORMONE RELEASING HORMONE (GHRH) I. SERMORELIN ACETATE

Geref

0.5 & 1 mg per vial

Geref Diagnostic

50 pg per vial

INDICATIONS: (1) Treatment of idiopathic GH deficiency (GHD) in children with growth failure; (2) Dx aid for GHD. DOSE: For Dx GH testing: 1 (tg/kg IV. For Rx, 30 pg/kg SC/d hs. All children should be prepubescent. Geref Dx test should be performed in all children. Those who do not achieve a peak GH level > 2 ng/mL should be excluded from receiving Geref Rx. Treatment response is best when initiated at a bone age < 7.5 years for females and < 8 years for males. Relative GH response to stimulation is not predictive of growth response. Height shoud be assessed at least q 6 months. Treatment should be discontinued when the epiphyses are fused. Patients who fail to respond should be reevaluated, and a change to Rx with GH should be considered. Baseline & periodic thyroid function and bone age should be monitored. Concomitant glucocorticoid Rx may inhibit growth promoting effects. ACTIONS AND SIDE EFFECTS: Sermorelin is the acetate salt of an amidated synthetic 29 amino acid peptide (GHRH |.2q) that corresponds to the amino terminal segment of human GHRH, which is composed of 44 amino acids. It is the shortest fragment of GHRH known to possess full bioactivity. Brief local injection reactions, rarely headache and flushing, dysphagia, dizziness, hyperactivity, somnolence, and urticaria. Side effects

Trade Names and Preparations

Drug HYPERGLYCEMIC AGENTS I. DIAZOXIDE A. Oral Preparation

Proglycem

50 mg/cap; 50 mg/mL oral suspn

2159

DOSE: Dosage must be individualized. Adults and children: 3-8 mg/kg/d in 2-3 equal doses q 8-12 h. Infants and neonates: 8-15 mg/kg/d in 2-3 equal doses q 8-l_2 h. B. Parenteral Preparation Hyperstat IV 300 mg in 20 mL amp Diazoxide Injection 15 mg/mL vial in 10 mL or 20 mL vials J)OSE^^didts^^^Tigdhito^£eripheraWeim^ayi^ej|epeated^t^212£iiiiiiiiil£l^!^ INDICATIONS: (1) Hypoglycemia caused by hyperinsulinism; (2) hypertensive crisis. Oral preparations may be used for persistent hypoglycemia caused by hyperinsulinism in adults with islet cell neoplasia and in infants and children with leucine sensitivity, islet cell hyperplasia, nesidioblastosis, and islet cell neoplasms. Parenteral preparations may be used for the emergency lowering of blood .pressure. Insulin or tolbutamide will reverse the hyperglycemic effects. When given po, onset of action is /2” (peach/wh.),' ’ (pink/wh,), “2” (green/wh.) & "‘3” ^ellowAG^ INDICATIONS: Thyroid preparations are used for (1) Rx of hypothyroidism and (2) TSH suppression. Natural thyroid products are derived from beef or pork sources. Desiccated thyroid and thyroglobulin are thought by many to be obsolete. Their standardization is inexact and their shelf-lives are limited. Levothyroxine is the synthetic sodium salt of the L isomer of T-i. Its fi/2 is about 1 wk. A steady-state serum level is reached after about 4 wk on a given dose. FDA standardization procedures recently have been improved. Bioequivalence may vary between brands, and it is suggested that they not be interchanged. See Chapters 45 and 199 for initiation guidelines for adults and Chapter 47 for pediatric doses. 0.1 mg of levothyroxine = approximately I gr thyroid USP. Liothyronine sodium is the synthetic sodium salt of the L isomer of Tj. Ti is rapidly metabolized and excreted. Its tV2 is 1-2 d. Serum levels fluctuate widely. Postabsorptive T3 peaks could be dangerous in the elderly and in subjects with coronary artery disease. Liothyronine is not recommended for routine thyroid hormone replacement Rx. T 3 is used (1) to prepare patients for scanning for presence of metastatic thyroid Ca in order to allow for a relatively short period between discontinuation of thyroid hormone Rx and scanning; (2) may be used in the performance of a T3 suppression test, where it must be used with caution because it may precipitate angina pectoris or myocardial infarction; (3) myxedema coma [use injectable form q 4 hr at a rate of 65 pg/d. The initial dose ranges from 25 to 50 pg. In patients with known cardiovascular disease, start with 10 to 20 pg. Liotrix is a mixture of synthetic T-i and T3 in a 4:1 ratio. Generally, there is no clinical advantage to its use over that of T-i alone. All strengths of Euthroid, except “2," contain tartrazine. 60 mg of Liotrix = approximately 1 gr thyroid USP. The reference strength of Euthroid “I" = 60 pg T-i + 15 pg T3. This provides a so-called “thyroid equivalent” of 60 mg. Reference strength of Thyrolar “1” = 50 pg T4 + 12.5 pg T3. This provides a "thyroid equivalent" of 60 mg. ACTIONS AND SIDE EFFECTS: Side effects of thyroid hormone Rx generally reflect hyperthyroidism secondary to overdosage. hypersensitivity reactions may occur to vehicles in tablets.

Drug

|

Rarely,

Trade Names and Preparations

THYROTROPIN (TSH) THYROGEN Thytropar 1.1 mg vials & 10 mL diluent (10 IU of thyrotropic activity/vial) INDICATIONS: Adjunctive diagnostic tool for serum thyroglobulin testing with or without radioiodine imaging in the follow-up of patients with thyroid cancer [see Chap. 40], DOSE: 0.9 mg IM q 24 h for 2 doses or q 72 h for 3 doses. Radioiodine administration should be given 23 h following the final thyrogen dose. Scanning is typically done 48 h after radioiodine administration and 72 h after final thyrogen dose. ACTIONS AND SIDE EFFECTS: Purified recombinant form of human TSH produced by recombinant DNA technology. Side effects include nausea, weakness, dizziness, headache, and vomiting with overdosage.

Drug

|

Trade Names and Preparations

THYROTROPIN RELEASING HORMONE (TRH, Protirelin) Thypinone Relefact TRH

500 pg/mL in 1 mL 500 pg/mL in 1 mL amps

Thyrel TRH

500 pg/mL in 1 mL amps

INDICATIONS: Dx agent in the assessment of (1) thyroid function; (2) pituitary or hypothalamic dysfunction; (3) adequacy of thyrotropin suppression in patients on thyroid hormone Rx [see Chap. 241], DOSE: In the TRH stimulation test, protirelin is administered as a 500 pg bolus in adults. In children aged 6-16 years, 7 pg/kg, up to 500 pg. ACTIONS AND SIDE EFFECTS:

2170

Protirelin is a synthetic tripeptide thyrotropin releasing hormone.

It causes release of thyroid stimulating

hormone and prolactin from the anterior pituitary. In acromegaly, about 2/3 of patients will have a paradoxical rise of growth hormone levels in response to protirelin. Side effects, usually minor, occur in about 50% of patients and include transient hypo- or hypertension, nausea, flushing, urge to urinate, abdominal discomfort, a metallic taste in the mouth, and headache. Occasionally a patient may experience chest or throat tightness. Rarely, seizures may occur in persons with predisposing central nervous system disease and amaurosis has occurred transiently in patients with pituitary tumors.

Drug

Trade Names and Preparations

V ASOPRESSIN DERIVATIV ES ” DESMOPRESSIN ACETATE

DDAVP DDAVP DDAVP DDAVP Stimate

0.1 and 0.2 mg tabs nasal spray pump (Nasal soln of 0.1 mg/mL) rhinal tube delivery system (Nasal soln of 0.1 mg/mL) injection 4-15 pg/mL 0.15 mg/mL nasal soln

DOSE: In Rx of neurogenic diabetes insipidus, dose of DDAVP in adults is 0.5-1 mL/d (2-4 pig) IV or SC in 2 divided doses; 0.1-0.4 mL/d (10-40 mg) intranasally in 2-3 doses (100 pig = 100 IU AVP), or 0.05 mg po bid stalling dose (0.1-1.2 mg/d). Enuresis: 2 puffs (0.2 mL; 20 pig) intranasally q hs (10-40 pig range); or 0.2 mg po q hs starting (0.2-0.6 mg range). II. LYPRESSIN

| Diapid

0.185 mg/mL in 8 mL bottles of nasal spray (1 spray = approx 2 posterior pituitary pressor U)

DOSE^^T^^^sua^dos^^or^dult^^n^^lMldren^s.^^r^.^^^y^^^l^^^^hjTostril^q^d^i^Nn^dditiona^^ed^^^^ose^Tia^^e^jequire^^o^reven^nocmH^.

III. VASOPRESSIN DOSE: Pitressin synthetic, 5-10 U, usually yields a full physiologic response in adults. In diabetes insipidus, the dosage is 5-10 U bid to tid IM or SC as necded:jjrjj2trana^ by spray or by dropper. INDICATIONS: (1) Central diabetes insipidus (intranasal, oral, parenteral); (2) primary nocturnal enuresis (intranasal); (3) congenital bleeding disorders (intranasal or parenteral). Unlabeled uses include (1) management of bleeding esophageal varices; (2) congenital and acquired bleeding disorders. ACTIONS AND SIDE EFFECTS: These agents are derivatives of vasopressin, a posterior pituitary hormone. They have pressor and antidiuretic hormone activity. See Chapter 26 for their use in diabetes insipidus. Desmopressin acetate is 1-deamino-8-D-arginine vasopressin. It is a synthetic analog of arginine vasopressin. A single dose has an antidiuretic effect that lasts 8-20 h. By injection, its antidiuretic effect is about 10 times as potent as by the nasal route. DDAVP presently is the drug of choice in Rx of diabetes insipidus. Lypressin is 8-lysine vasopressin, which has antidiuretic activity and vasopressor effects. The peak of antidiuretic effect is 30-60 min. Its duration of action is 3-8 h. It is useful in patients who become unresponsive or have reactions to other preparations. May induce vasoconstriction if taken in excess. Pitressin is synthetic 8-arginine vasopressin. The duration of action of vasopressin is 2-8 h, and of vasopressin tannate is 24-96 h. Fluid and electrolyte status should be monitored closely to avoid fluid overload and hyponatremia. Use with caution in patients with atherosclerosis or hypertension. Side effects include hypersensitivity reactions, tremor, vertigo, abdominal cramps, bronchoconstriction, and anaphylaxis.

Trade Names and Preparations

Drug V ITAMIN D I. CALCIFEDIOL (25|OH]DQ

Calderol

20 & 50 (ig/cap

INDICATIONS: (1) Renal osteodystrophy in the presence of hypocalcemia, hyperparathyroidism, osteomalacia, or proximal myopathy [see Chap. 61]; (2) hypoparathyroidism [see Chap. 60]; and (3) osteoporosis. (Calcifediol is 25-hydroxycholecalciferol or 25(OH)D3. The time to optimal Rx effect is 2-4 wk. Serum calcium levels must be monitored closely and patients should be warned of the symptoms of hypercalcemia. For a discussion of individual agents, see Chapter 63.) ^^OSE^^or^ena^steodystro£hy^20to2002i^o/d^ForjTy£oparathyroidism^0-200jr£po/d^ome^ive^?^oii^lteniat£da^ II. CALCITRIOL (l,25|OHl2D3)

Oral. Rocaltrol

0.25 & 0.5 pg/cap

Injection: _Calcijex_1 & 2 pg/mL for IV injection_ INDICATIONS: (1) Renal failure in the presence of hypocalcemia, osteodystrophy, tertiary hyperparathyroidism, osteomalacia, or proximal myopathy [see Chap. 61]; (2) hypoparathyroidism [see Chap. 60]; (3) pseudohypoparathyroidism; (4) osteomalacia; (5) tumor-induced osteomalacia; (6) X-linked hypophosphatemic rickets [see Chaps. 63 and 70]; (7) vitamin D-dependent rickets type I [see Chaps. 63 and 70]; and (8) vitamin Ddependent rickets type II [see Chap. 70]. Calcitriol is 1,25-dihydroxycholecalciferol [l,25(OH)2D.?]. The time to optimal Rx effect for this preparation is 1-3 d. Because of its short time to onset and offset of action, calcitriol is the preparation of choice in Rx of hypocalcemia in pregnancy. It is commonly used in the Rx of renal osteodystrophy in the presence of the indications listed here. In the Rx of osteomalacia, if hypophosphatemia is present, phosphate also is added to the Rx regimen. In vitamin D-dependent rickets type II, parenteral calcium Rx may be necessary to heal the bone disease. Calcium levels should be checked at least twice weekly until the dose of calcitriol is established. DOSE: Starting po dosage is 0.25 pg/d. In dialysis patients, increase by 0.25 pg as needed at 4-8 wk intervals up to 1 pg/d. In hypoparathyroidism, increase at 2-4 wk intervals to 0.5-2 pg/d in adults; to 0.25-0.75 pg/d in children 1-5 yr. Other doses: 0.5-2 pg/d in X-linked hypophosphatemic rickets; 0.5-3 pg po/d in vit. D-dependent rickets type I. Also see Chapter 70; doses as high as 15-20 pg/d may be required in vitamin D-dependent rickets type II. III. CHOLECALCIFEROL Delta-D 400 IU tabs (Vitamin Dj)_Vitamin Dj_1000 IU tabs___ INDICATIONS: (1) Dietary vitamin D supplementation and (2) Rx of vitamin D deficiency. (1 mg = 40,000 IU vitamin D activity.)

2171

DOSE: 400-1000 IU po/d. IV. DIHYDROTACHYSTEROL (DHT)

DHT

0.125, 0.2 & 0.4 mg tabs; 0.2 mg/mL Intensol soln (20% ethanol); 0.2 mg/5 mL oral soln (4% ethanol) Hytakerol 0.125 mg caps; 0.25 mg/mL in oil INDICATIONS: (1) Hypoparathyroidism and tetany [see Chap. 60], Unlabeled use: Renal osteodystrophy in the presence of hypocalcemia, secondary hyperparathyroidism, osteomalacia, or proximal myopathy. DHT is a synthetic reduction product of tachysterol and is very similar to vitamin Dv It is hydroxylated in the liver, but does not require renal activation. Its time to optimal Rx effect is 1-2 wk. It has only weak antirachitic activity [1/450 that of vitamin D], 1 mg of DHT is equivalent to about 3 mg [120,000 IU] of vitamin p2- Monitor serum calcium. DOSE: In renal osteodystrophy. DHT dose is 0.20-2.0 mg po/d. In hypoparathyroidism, maintenance dose is 0.2-1 mg po/d.

V. ERGOCALCIFEROL (Vitamin D2)

Drops: Calciferol Drops Drisdol Vitamin D Oral Drops

8,000 IU/mL in 60 mL dropper 8,000 IU/mL in 60 mL dropper bottle 8,000 IU/mL in 60 mL dropper bottle

Caps or Tabs: Vitamin D Calciferol Drisdol (Many over-the-counter

50,000 IU/cap 50,000 IU/tab 50,000 IU/cap (with tartrazine) small-dosage forms are available, e.g., 400, 800 & 1000 IU/cap or tab)

Injection, IM: Calciferol in oil 500,000 IU/mL INDICATIONS: (1) Hypoparathyroidism [see Chap. 60]; (2) pseudohypoparathyroidism; (3) malabsorption of vitamin D; (4) vitamin D-resistant rickets; (5) vitamin D-deficient rickets [see Chap. 63]; and (6) vitamin D-dependent rickets type II [see Chaps. 63 and 70]. Ergocalciferol is vitamin D2. 1.25 mg provides 50,000 IU of vitamin D activity. Its time to optimal Rx effect is 4-6 wk. This is the least expensive vitamin D preparation available. 400 lU/d of vitamin D: satisfies daily allowances for most age groups. Monitor serum calcium. Vitamin D-dependent rickets type II may respond to Rx with vitamin D:. For malabsorption, ergocalciferol may be given IM. DOSE: In hypoparathyroidism in adults, dosage is 1.25-5 mg (50,000-200,000 IU) po/d [see Chap. 60], In vit. D-resistant rickets: 12,000 to 500,000 IU/d po in adults; 1000-16,000 IU/d po in children. If compliance is a problem, 600,000 IU may be given po or IM for 1 dose [also see Chap. 70]. In vit, D-deficient rickets: 5,000-10,000 IU/d po until bone is healed. The dose then is reduced to 400 IU po/d. Vi. PARICALCITOL (19-NOR-la, 25-DIHYDROXY VITAMIN D)_ Zemplar_5 pg/mL 1 & 2 mL vials_ INDICATIONS: Prevention and Rx of secondary hyperparathyroidism associated with chronic renal failure. DOSE: 0.04-0.1 (ig/kg IV with dialysis no more frequently than qod. Adjust by 2-4 pg every 2-4 wk as needed. ACTIONS AND SIDE EFFECTS: Vitamin D products may cause hypercalcemia and the calcium-phosphate product should not exceed 70. Early symptoms include weakness, headache, somnolence, nausea, vomiting. Late symptoms include polyuria, polydipsia, irritability, generalized vascular calcification.

2172

Ch. 237: Reference Values in Endocrinology

CHAPTER

237

REFERENCE VALUES IN ENDOCRINOLOGY D. ROBERT DUFOUR Laboratory tests are widely used by endocrinologists in the diagnosis and monitoring of disease. On receiving the results of such tests, the first thing physicians do is check to see whether the results are "normal" by comparing them with a list of values prepared by the laboratory, which commonly are referred to as "normal ranges." Textbooks often provide such lists. Although comparing a test result to a published standard may seem sim¬ ple, many considerations may influence the interpretation. This chapter, instead of simply providing a list of normal values, dis¬ cusses the factors that may affect the interpretation of test results as well as common situations that may change test results. Table 237-1, at the end of the chapter, lists common factors that may influence the results of each of these tests.

REFERENCE RANGES: DO THEY MEAN “NORMAL”? DEFINITIONS OF NORMAL Although one review documented seven possible meanings of the word normal, to most endocrinologists and their patients, normal is synonymous with absence of disease.1 By corollary, abnormal indicates the presence of disease, which has caused a change from the normal, healthy state. A second common mean¬ ing of normal, which is derived from statistics, is often used by scientists: a bell-shaped frequency distribution in which 95% of all results are within 2 standard deviations (SD) of the average value. Although these two definitions do not necessarily describe the same thing, they frequently are used interchangeably in medi¬ cine, particularly in the interpretation of laboratory results.2,3

REFERENCE VALUES All clinical pathology laboratories publish a list of reference val¬ ues. Until recently, these were called tables of normal values, with the laboratory using the term normal in the sense of the second definition given earlier. Reference value now is widely used to indi¬ cate that the value provides a reference, something to be used for comparison with a test result, rather than a declaration of values expected in normal, healthy persons.4 This subtle but important difference is the source of much confusion among physicians seeking to interpret the results of laboratory tests. Less commonly, reference values may be established for disease, such as expected values of glycated hemoglobin in persons with varying degrees of glycemic control. Because the principles and assumptions used in establishing such reference values are similar, this latter type of reference range is not discussed further here. ESTABLISHMENT OF A REFERENCE RANGE Reference ranges can be established in several ways. A sample of suitable size is needed to guarantee the statistical validity of results. Usually, 100 to 200 persons constitute a sample large enough to be valid but small enough to be workable. For many difficult or expensive procedures, published reference ranges may be based on results from fewer than 20 persons.

2173

Representative Sampling. The first assumption is that the sample is representative of the population that ultimately will be tested. Most volunteers are hospital employees, blood donors, or medical students. None of these groups is necessarily representative of the population that eventually will be exam¬ ined? This is especially a problem in inner city and charity hos¬ pitals, in which the usual volunteers are not likely to be from the same population as the patients. If the sample is not representa¬ tive, the reference range may or may not be valid, but its validity cannot be tested. Absence of Disease. A second assumption is that no per¬ sons in the sample group have the diseases for which the testing is being done. This is important for disorders with a high preva¬ lence of asymptomatic disease. An example is the current wide¬ spread effort to screen the U.S. population for risk factors for coronary artery disease. Autopsy studies show a high incidence of atherosclerosis in persons without symptoms.6 Serum choles¬ terol is thought to be related to the development of atheroscle¬ rotic plaques.7,8 To develop a reference range for serum cholesterol levels to separate persons without atherosclerosis from those with atherosclerosis, a sample of persons without ath¬ erosclerosis would have to be selected. This is not commonly done. Until the late 1980s, most laboratories used reference ranges derived from "normal" volunteers, many of whom actu¬ ally had atherosclerosis. This led to extremely wide reference ranges in which virtually no one except those with familial hypercholesterolemia had abnormal results.9 Currently, "refer¬ ence" values for cholesterol are based on the risk of development of coronary artery disease, rather than the selection of "typical" values.10 (Other common disorders that may affect laboratory tests are diabetes mellitus, hypertension, and alcohol abuse.) Conditions of Testing. Although the method used for obtaining samples is not commonly considered, differences in patient preparation and the technique used for venipuncture can affect test results.11 Fasting increases bilirubin and uric acid levels, whereas glucose, triglycerides, and insulin levels increase after meals. A number of constituents show diurnal variation, including cortisol, osteocalcin, and urinary excretion of collagen fragments (N-telopeptide, deoxypyridinoline). If all volunteers are requested to come in after an overnight fast (a common instruction given by laboratories), then reference ranges will be valid only for comparing samples obtained under those condi¬ tions.5 In most clinical settings, patients are seen at other times of the day for blood drawing, and they may have results outside the reference range solely for that reason. In an elegant series of experiments, the effect of various forms of prevenipuncture position on test results was evalu¬ ated.12 Drawing blood after the person stood and walked for 30 minutes before sitting down increased the apparent concentra¬ tions of proteins and protein-bound molecules (which affect many of the tests related to endocrine evaluation) by 3% to 5% over those obtained after the same person had been sitting for 30 minutes. If the person had been supine for 30 minutes before venipuncture, a 5% to 10% decrease was seen in the same con¬ stituents. Because the first condition describes the common pro¬ cedure for obtaining blood from outpatients, and the second describes the usual method of obtaining blood from inpatients, different interpretations of results could easily be made. In vir¬ tually all laboratories, reference ranges are derived using sam¬ ples obtained from ambulatory persons, so that some results are 8% to 15% higher than expected for supine inpatients. STATISTIC INTERPRETATION Mean and Standard Deviation. The typical approach in evaluating the data is to consider the central 95% of all values as the reference range. In most laboratories, this is determined by

21 74

PART XVIII: ENDOCRINE DRUGS AND VALUES

TEST RESULTS

FIGURE 237-1. This skewed distribution of test results is typical of the pattern observed for many serum constituents. Using the mean ± 2 standard deviations (SD) as reference limits would falsely classify results between 0 and 5 as normal and results between 40 and 45 as abnormal.

calculating the mean and SD and defining the reference limits as the mean ± 2 SD. Several problems are associated with this seemingly simple statistical procedure. The assumption is that the data are graphically distributed in a bell-shaped fashion, that is, as the Gaussian or normal distribution. For many param¬ eters, distribution of results is not symmetric but is skewed to one side or the other.13 Often, the degree of skewing of the data is not enough to invalidate the assumption that values within 2 SD of the mean include 95% of all data points. Some statistical tests demonstrate whether the degree of skewing is too great for this assumption to be used.14 Many laboratories neither inspect the data for skewing nor use these statistical tests to determine whether skewing is too great; they simply define the reference range as the mean ± 2 SD. Figure 237-1 illustrates the conse¬ quences of this approach. If values are skewed toward higher concentrations, >5% of the sample have results above the upper reference limit, and the converse is true for values skewed to lower concentrations. Even if valid sampling techniques are used, such statistical errors lead to incorrect reference limits. Evaluation of the sensitivity of several statistical techniques to differences in data distributions has shown that some are virtu¬ ally unaffected by skewing of results.15 Limitations of Gaussian Statistics. An even more impor¬ tant problem is associated with this method of defining limits of a reference range: Gaussian statistics describe the distribution of repeated measurements of the same parameter.16 For example, if 100 different scales were used to measure one person's weight, the chance would be 95% that the actual weight would lie within 2 SD of the average value for all weight measurements. When results obtained from populations are described, no valid statis¬ tical reason exists for selecting these limits as defining an expected range of normality. Once limits are set to include only the central 95% of all values as normal, then 5% of results from the sample are considered to be outside the reference limits, even though all persons in the sample originally were consid¬ ered normal. Thus, if reference limits are used as a guide to what is normal in the medical sense, then 5% of results from the sam¬ ple must indicate disease. This same condition holds true for each test performed. If each test assays an independent variable, and results for each test are distributed randomly in the popula¬ tion, then the likelihood that n tests are within the reference

range is (0.95)". The likelihood (p) that all independent test val¬ ues are within the reference ranges (assuming that reference lim¬ its include 95% of all values) is as follows for different values of n: n = l,p = .95; n = 2,p = .90; n = 6,p = .74; n = 10, p = .60; n = 12, p = .54; n = 15, p = .46; n = 20,p = .36; n = 24, p = .29. As n becomes larger and approaches the number of tests commonly included in screening profiles by many laboratories, most patients should have at least one result outside the reference limits solely because of the chance distribution of results. Because of this statistical fact, results outside reference values are frequently produced, even for apparently healthy persons. The author has been using results of medical students' labora¬ tory tests to illustrate this phenomenon to the students for sev¬ eral years. When samples were taken from 597 healthy medical students, and reference values were defined as within 2 SD of the average, only 65% of students had all “normal" test results; 27% had one “abnormal" test result; and 8% had more than one "abnormal" test result. When the central 95% of the distribution was used as the reference values (because several tests showed statistically significant skewness), the results were similar (64% had all "normal" results, 27% had one “abnormal" result, and 9% had two or more "abnormal" results), although in -15% of students, the two methods disagreed on the number of "abnor¬ mal" results. Several publications have highlighted this statisti¬ cal phenomenon and have used it to suggest that widespread screening programs are unlikely to be beneficial, because most “abnormal" results are probably the result of random statistical occurrences.17'18 The statistical assumptions of independence become more complicated, however, because the results of many commonly measured tests change in parallel, rather than independently (i.e., sets of compounds such as serum electro¬ lytes; calcium, phosphate, and magnesium; blood urea nitrogen and creatinine; and triiodothyronine [T3], thyroxine [T4], and thyroid-stimulating hormone [TSH]). Another assumption limiting the use of reference values is that results are randomly distributed in the population, and that an individual is no more likely to have any one result than another. Many studies have shown that each individual main¬ tains concentrations of many critical parameters within an extremely narrow range and, thus, results would not be ran¬ domly distributed in the population.19-21 The ratio of average variation within an individual to the variation within a popula¬ tion has been termed the index of individuality. An index below 1.4 suggests that standard reference ranges will be insensitive' markers of significant changes in the condition of a person. As an example, the range of cholesterol values seen in the medical students (central 95% of values) is from 126 to 252 mg/dL. In contrast, the average day-to-day variation in cholesterol level in a person in relation to his or her average blood cholesterol level is -6% (1 SD). Thus, significant changes in serum cholesterol can occur without producing an "abnormal" result. Other common tests with small individual variation in results include calcium, alkaline phosphatase, and thyroid function tests. IMPLICATIONS OF A REFERENCE RANGE The usefulness of reference ranges depends on the techniques used to establish them. Even if all of the influences have been considered, it does not follow that a result within the reference range indicates health or that a result outside the reference range indicates disease. Because the reference range is a descriptive statistic of the sample (and, if valid sampling techniques were used, of the population), then results outside the reference range indicate only that an individual is not from the same population. This could be due to disease, to population differences, or to physiologic variations in the individual. Similarly, a result within the reference range does not guarantee health, because

Ch. 237: Reference Values in Endocrinology some diseases may have a high enough prevalence in the popu¬ lation that the test cannot separate healthy and diseased individ¬ uals. These observations have led to several alternatives to reference ranges.

2175

greater specificity and sensitivity. The original results for sensi¬ tivity and specificity are unlikely to remain valid when the same test is used for patients with fewer manifestations of disease.25 RELATIVE OPERATING CHARACTERISTIC CURVES

MEDICAL DECISION LEVELS Medical decision levels are cutoff points that reliably indicate disease or the need for additional action.22 The limits for these values usually are wider than those for reference ranges, so that finding a result beyond these limits as a result of chance alone would be uncommon. Because they indicate the need for medical intervention, less reason exists to consider qualifying factors in interpreting results. Tables of medical decision lev¬ els are found in several articles, and one book gives medical decision levels for 137 tests.23 Decision levels may prove use¬ ful for analytes that are commonly subject to minor, physio¬ logic variations. SENSITIVITY AND SPECIFICITY Sensitivity refers to the ability to detect disease, expressed as per¬ centage of patients with disease having an abnormal test result. Specificity refers to the ability to exclude disease, expressed as percentage of persons without disease having a normal test result. One limitation of medical decision levels is that, although they are highly specific for disease, they tend to be less sensitive and are of less use in screening persons for early stages of a dis¬ ease process that produces gradual changes in test results. Fig¬ ure 237-2 shows the effect of changing the reference limits of a test to improve either sensitivity or specificity: Improving the sensitivity and specificity of a given test simultaneously is impossible. For most disease-test combinations, sensitivity and specificity are unknown.24 In addition, the use of sensitivity and specificity has been criticized, because values usually are derived by applying the test to patients with known disease, which was confirmed by some other technique of equal or

Relative operating characteristic (ROC) curves are a graphic depiction of sensitivity and specificity over a continuous range of cutoff points or decision levels.26 Typically, sensitivity is plotted in an ascending fashion on the Y-axis and specificity is plotted in a descending fashion on the X-axis. Such graphs are useful for selecting optimal decision points to use in diagnos¬ ing disease and for comparing the performance of diagnostic tests. The point closest to the upper left corner of the graph (perfect performance) correctly classifies the greatest percent¬ age of patients. For comparison of two or more tests, the curve closer to the upper left corner performs better in clinical usage. Decision points can be selected to give high sensitivity or high specificity, depending on the use of the test. When medical decision levels are used, the specificity is increased to virtually 100%, meaning that all abnormal results indicate disease. Fig¬ ure 237-3 illustrates a typical ROC curve for a diagnostic test. Several possible cutoff points are indicated, showing why med¬ ical decision levels have a lower sensitivity than other possible decision levels. INTERLABORATORY DIFFERENCES Because laboratories use many different techniques for measur¬ ing most analytes, different cutoff values may be needed for dif¬ ferent laboratories. Thus, some time will be required before enough universally applicable formulas are available for diag¬ nosis of disease by laboratory tests alone.

Glucose Concentration (mg/dL) FIGURE 237-2. The distribution of fasting glucose levels in patients with normal glucose tolerance (solid line) and those with "diabetic" glu¬ cose tolerance (dotted line) illustrates the impossibility of improving both sensitivity and specificity simultaneously. Use of a fasting glucose level of 140 mg/dL (solid bar) to predict abnormal glucose tolerance excludes all persons with normal glucose tolerance (specificity 100%), but it misses 40% of those with abnormal glucose tolerance (sensitivity 60%). With use of a fasting glucose level of 115 mg/dL (open bar), sensi¬ tivity is improved to 100%, but specificity falls to 75%. With any value between these two points, specificity improves and sensitivity falls. Moving below 115 or above 140 mg/dL lowers specificity or sensitivity, respectively, without improving the other component.

FIGURE 237-3. This relative operating characteristic curve illustrates the results obtained in several screening programs for primary hyper¬ parathyroidism. Point A represents a serum calcium level of 10.0 mg/ dL; all patients with primary hyperparathyroidism continuously have serum calcium levels above this value, but -30% of normal persons have one or more values above this. Point B represents an upper refer¬ ence limit of 10.5 mg/dL. Approximately 90% of serum calcium values from patients with primary hyperparathyroidism fall above this level; 5% to 8% of values from normal persons are above it, usually due to hemoconcentration. Point C is the medical decision level of 11.0 mg/dL recommended by Statland.26 Only 70% of values from patients with pri¬ mary hyperparathyroidism are above this level, but it reliably indicates an elevated serum calcium value of clinical significance.

21 76

PART XVIII: ENDOCRINE DRUGS AND VALUES

INDIVIDUAL REFERENCE RANGES Another approach is the use of individual reference ranges.4'27 The concentrations of many analytes are closely regulated in most individuals, so that the variation within a person is much smaller than the variation between persons.19-21 With the feasibility of performing widespread population screening for different dis¬ ease states and the common practice of doing "profiles" of labora¬ tory tests as part of periodic physical examinations, a database exists for determining a person's typical concentrations of several analytes. If the results of laboratory tests are available on comput¬ ers, this analysis is easier to perform. One way in which these ranges can be useful is in diagnosing myocardial infarction, hi one common approach, standard reference ranges are used and serum isoenzyme determinations of creatine kinase are obtained only if the enzyme levels exceed the reference limit. In several studies, many patients with apparent myocardial infarction had extremely low baseline values for serum creatine kinase and showed acute rises in the level of this enzyme that did not exceed the upper reference limit.28'29 Thus, comparing a patient's test results to his or her own values improves the sensitivity of crea¬ tine kinase measurements for diagnosing myocardial infarction.30 The use of individual reference ranges should better define the expected limits of normal physiologic changes and provide the ultimate in sensitivity for early detection of disease. As dis¬ cussed earlier, intraindividual variation is generally much less than the variation seen in the population.31 The major impedi¬ ments are the extremely high cost of screening the population using every conceivable test and the enormous amount of data storage capacity required. Another possible problem is the lack of acceptable reference ranges for allowable changes in an indi¬ vidual. Although the studies cited provide approximate refer¬ ence ranges for intraindividual variation, they are not all inclusive, and a wide difference is seen in the values given. Moreover, data suggest that intraindividual variation is greater in persons with certain disease processes.32 A range that may detect early changes in healthy persons may be a false-positive indicator of impending complications in persons with disorders such as diabetes. Despite these problems, the use of individual reference ranges probably will increase.

COMMON CAUSES OF “ABNORMAL” ENDOCRINE TEST RESULTS Although reference ranges often are used to define "normal," results outside reference limits are commonly seen when no evi¬ dence of an underlying disease process is present. In this section, some frequent causes of variation in laboratory test results are described. A more complete listing is available in Table 237-1 at the end of this chapter.

PHYSIOLOGIC INFLUENCES ON ENDOCRINE TEST RESULTS

DIURNAL AND PULSATILE RHYTHMS Diurnal variation is a well-known phenomenon for cortisol and adrenocorticotropic hormone, but smaller degrees of diurnal vari¬ ation also are found for other hormones, including prolactin, TSH, and testosterone.35-37 Marked diurnal variation in serum iron concentrations is a common finding, and many hormones, such as gonadotropins, growth hormone, and prolactin, are released in a pulsatile fashion, such that interpreting a single test result is difficult.38 Even for substances measured by common hematology and chemistry tests, a significant diurnal variation is seen.383 For measurement of most pituitary hormones, pooling several serum samples and assaying the pooled specimen pro¬ vides a more accurate indication of average hormone concentra¬ tion and pituitary function. For most other tests, consistently sampling in the early morning yields more reproducible results. PHYSICAL AND EMOTIONAL STRESS Exercise leads to the release of catecholamines, prolactin, and muscle-specific enzymes into the circulation.39 Even mild exer¬ cise can cause a marked increase in the frequency of menstrual irregularities,393 probably due to hormonal changes. Stress leads to catecholamine release; prolonged stress can cause marked changes in the concentrations of various blood lipids and hor¬ mones.40-42 An entire section of this book (Part XVI) is devoted to the endocrine changes seen in acute illness. AGE The values of many serum constituents change markedly in con¬ centration during a person's lifetime. Although it is accepted that values may differ in children (see Chaps. 7,18,47,70,83,87, 90-92, 157, 161, and 198), changes also occur at other times in life. For example, testosterone and renin decrease with increas¬ ing age in adults, and alkaline phosphatase and parathyroid hormone (PTH) levels continue to increase in persons older than 50 years. (Also see Chap. 199.) RACE For many commonly measured parameters, values in persons of African ancestry are different from those in persons of European ancestry; differences in other races have not been evaluated as carefully. For example, blacks tend to have higher values for high-density lipoprotein cholesterol and PTH, but lower values for vitamin D metabolites and renin. SEX Obvious differences are found between the sexes in levels of sex hormones and prolactin, but women and men often have differ¬ ent concentrations of many of the analytes commonly mea¬ sured. Serum levels of free T4 and copper are higher in women, but lower values for renin, aldosterone, and most blood lipids also are the rule. MENSTRUATION AND PREGNANCY

The effects of normal physiologic changes in a patient are not often considered in interpreting laboratory findings, yet they are commonly the explanation for an unexpected test result. DIET Occasionally, dietary factors are responsible for unexpected changes, either due to the ingestion of food (e.g., transient alkalo¬ sis, intracellular shifts in phosphate, release of intestinal alkaline phosphatase into the circulation, elevated levels of hormones) or due to the chemical substances in food that cause physiologic changes (e.g., caffeine-induced catecholamine release).33-54

During the normal menstrual cycle, in addition to the obvious changes in levels of estrogens and progesterone, levels of vaso¬ pressin, prolactin, and PTH increase. The onset of menstruation causes a fall in serum sodium and phosphate levels, and a rise in renin and aldosterone concentrations. Pregnancy induces some unexpected changes: Levels of PTH, calcitonin, cortisol, and aldos¬ terone increase, and fasting levels of glucose and glycohemoglobin (hemoglobin Alc) fall. The effects of pregnancy may last long after the baby is delivered. Studies have shown lower levels of prolactin and dehydroepiandrosterone in women who have borne children than in those who have never been pregnant.

Ch. 237: Reference Values in Endocrinology HEIGHT AND WEIGHT A person's height and weight are related to the concentrations of several substances. In children, a positive correlation is seen between height and serum alkaline phosphatase levels. Weight is much more closely associated with the concentrations of sev¬ eral parameters, especially in obese persons, who have higher serum concentrations of cortisol, insulin, and glucagon, but lower than normal levels of gonadotropins and sex hormone¬ binding globulin. Weight loss in obese persons causes changes, such as a fall in renin and aldosterone levels (see Chap. 126). ALTITUDE Changes in the concentrations of many analytes are found in persons living at altitudes above 5000 ft. Although some of these changes are transient, occurring only during the process of accli¬ matization, others appear to persist. Persons living at these heights tend to have higher levels of erythropoietin and lower levels of renin, angiotensin II, and aldosterone.

EFFECTS OF DRUGS ON ENDOCRINE TEST RESULTS Drugs affect laboratory tests by two mechanisms.43 The first is a direct pharmacologic effect of the drug, such as hypokalemia due to diuretic therapy. Although most endocrinologists are familiar with this particular effect, many unfamiliar drug effects occur. For example, anticonvulsants increase levels of alkaline phosphatase (and related enzymes such as y-glutamyltransferase), prolactin, and vasopressin, but decrease total and free T4 and 25-hydroxyvitamin D levels, as well as urinary excretion of corticosteroid metabolites.44 A second type of drug interference results from cross-reac¬ tion in the assay. Two common examples are the cross-reaction of phenothiazines with some assays for urinary metanephrine, and the cross-reaction of certain cephalosporins with many assays for creatinine.45-46 Usually, information on the medica¬ tions being taken by each patient tested is not given to the labo¬ ratory; even if this information were available, screening all test requests manually to detect possible drug-test interferences would be virtually impossible. With the increasing use of com¬ puters, however, it should be possible to construct a program to review the pharmacy record for each patient and search for drug interferences when abnormal results are encountered. Several comprehensive lists of drug effects on laboratory tests are avail¬ able,47-48 and a compendium of drug effects on endocrine labora¬ tory tests is provided in Chapter 239.

HEMOCONCENTRATION Hemoconcentration causes an increase in proteins (e.g., albu¬ min) and, consequently, in the level of protein-bound substances in the blood. The common causes of hemoconcentration are dehydration, postural differences, and the use of tourniquets during blood collection, but evaporation of serum after collec¬ tion may produce the same effect. Ambulatory patients have a mild degree of relative hemoconcentration as a result of the shift of extracellular fluid from intravascular to extravascular loca¬ tions, caused by the increased hydrostatic pressure in the upright position.12 This causes an increase of 3% to 5% in the concentration of proteins and protein-bound substances (e.g., some hormones, lipids, and ions such as Mg, Ca, and Fe). A sim¬ ilar hemoconcentrating effect is produced by leaving on a tour¬ niquet for as little as 40 seconds while drawing blood.12 The suggested approach is to use a tourniquet only after a vein has been located and the skin has been prepared; longer use can cause 5% to 10% hemoconcentration. Fist clenching during

21 77

blood collection, with a tourniquet applied, leads to leakage of muscle contents, especially potassium, which can increase by 1 to 1.5 mmol/L in as little as 1 minute. The combined effects of posture and tourniquet use may cause a 10% to 20% increase in the apparent concentration of proteins. In one study, 15% of all persons evaluated for hypercal¬ cemia eventually were found to have normal serum calcium lev¬ els; hypercalcemia was artifactual as a result of concentration of serum proteins.49 In the author's laboratory, 5% to 10% of per¬ sons with "hypercalcemia" have serum albumin levels of >4.5 g/dL on initial study; usually, their calcium levels are within the reference range if their blood was collected (without prolonged use of a tourniquet) after they sat for 30 minutes. In one year, two patients were admitted for the evaluation of "hypercalce¬ mia" that was found to be caused by hemoconcentration.

CHANGES AFTER COLLECTION OF SPECIMENS Difficulties occasionally arise from changes that occur in the test tube during or after specimen collection. Some of the more com¬ mon specimen-related problems include hemolysis, which increases the apparent concentration of all abundant substances within cells, such as potassium, phosphate, magnesium, and enzymes; and delay in transport of the specimen to the labora¬ tory, which allows utilization and exhaustion of glucose, after which the cells leak their intracellular contents.50-51 Less com¬ monly, extremely high white blood cell or platelet counts cause difficulties; the former increases the rate of glucose use, and the latter leads to the release of potassium during coagulation.52-53 In rare instances, an interaction occurs between a substance in the collection tube and the patient's blood that causes artifactual abnormalities, such as an increase in potassium induced by hep¬ arin in patients with extremely high lymphocyte counts.54 Renin levels increase with storage in ice water, as a result of the conver¬ sion of prorenin to renin.55

CLERICAL AND ANALYTIC ERRORS A final source of unexpected test results may be the laboratory itself if the results reported are not matched to the correct patient. Although all laboratories strive to minimize errors, such mistakes do occur. Clerical errors in specimen identification, which can occur at any time from collection to final reporting, are the single most common cause (25%) of erroneous results.56 Less commonly, actual errors in performing the test cause incorrect results. Explainable causes of abnormal laboratory test results always should be considered. These factors are likely if the result is only minimally outside the reference limits; if evidence exists of hemoconcentration, such as a serum albumin level above the reference limit; if patients are receiving multiple med¬ ications; or if results from the current specimen differ markedly from previous results for a patient who is clinically stable. In any of these circumstances, the best approach is to contact the labo¬ ratory and submit a new specimen. Many reference laboratories perform repeated testing at no charge or at a reduced rate to maintain good customer relationships and to document possible causes of unexpected results.

USE OF LABORATORY TESTS IN DIAGNOSIS Although the major purpose of this chapter is to familiarize the endocrinologist with the techniques used in laboratories that can influence the interpretation of laboratory tests, it would not be complete without a brief discussion of the uses of laboratory tests in endocrine diagnosis.

2178

PART XVIII: ENDOCRINE DRUGS AND VALUES

Sensitivity = Likelihood ot ‘ abnormal'' result in presence of disease

True Positive Results (TP) Total Persons With Disease

Specificity = Likelihood ot "normal" result in absence of disease

True Negative Results (TN) Total Persons Without Disease

High Cortisol Cushing's

23.75

Normal Cortisol

Total

1.25

25

Predictive Value (TP) = True Positives (Total) = Sensitivity* Prevalence

Obese

3 75

71.25

27.5

72.5

Calcium

Calcium

>10.5

s10.5

23.75 27 5

88%

75

|FP] = False Positives (Total) = (1 - Specificity) x (1 - Prevalence) Predictive Value of Positive = Likelihood that a positive result indicates the presence of disease _(Sensitivity) (Prevalence)_ (Sensitivity x Prevalence)+ |(1 - Specificity) x (1 - Prevalence))

_113_ "

Hyperpara¬ thyroid

0.9

0.1

A

1

Predictive Value = QJ = 3.8% 25.9

[TP1 + IFP]

FIGURE 237-4. Bayes' theorem. The predictive value of a positive result answers the question: What is the likelihood that a positive result indicates the presence of disease? For any disease, increasing sensitivity or specificity of the tests used increases the predictive value. The major determinant of predictive value is disease prevalence. If the prevalence is low, then the true-positive results (TP) become small compared with false-positive results (FP), because TP = sensitivity x prevalence. The equation then reduces to TP/FP, or TP/[(1 - specificity) x (1 - preva¬ lence)]. Because (1 - prevalence) is ~1, the predictive value is [sensitiv¬ ity /([l - specificity] x prevalence)] x prevalence.

Normal

Hypothyroid

25

975

25.9

975.1

High TSH

Normal TSH

0.9

0.1

1000 B

1

Predictive Value = — = 8.3% 109 Euthyroid

10

3990

4000

109

“ABNORMAL” TEST RESULTS—BAYES’ THEOREM Not all endocrine results that fall outside the reference range indicate disease. When such results occur, the physician must answer this question: What is the likelihood that this result indi¬ cates a particular disease in this patient? Several statistical mod¬ els could be used to answer the question. The most widely publicized is Bayes' theorem. Bayes' theorem answers the question directly, giving a numeric probability that the test result indicates a given disease. The principles of Bayes' theorem are given in Figure 237-4. The information needed to answer the question includes the fre¬ quency with which abnormal results are found in persons with the disease (sensitivity), the frequency with which normal results are found in persons without the disease (specificity), and the frequency of the disease in the population. The answer is called the predictive value of a positive result. Figure 237-5 illus¬ trates the importance of disease prevalence on the predictive value. The percentage of abnormal results that indicate disease decreases as the frequency of the disease decreases. Since the introduction of Bayes' theorem into medical use, it has found increasing application as a model for the diagnostic process, and several reviews highlighting it have appeared in the internal medicine literature.1'57'58 The weaknesses of Bayes' theo¬ rem for medical decision making have not been emphasized. The first limitation is its use of a single decision level for predicting the presence of disease. For example, in the evaluation of hypercalce¬ mia, a serum calcium level of 10.6 mg/dL would be treated the same as a calcium level of 12.1 mg/dL or a calcium level of 16.5 mg/dL. Few practicing endocrinologists would view such levels this way. A second obstacle is the difficulty of using Bayes' theo¬ rem for more than one variable. Although applications for multi¬ ple variables do exist, they are not commonly used, and they further compound the first limitation by looking at only a single decision level for each variable studied. Furthermore, equal emphasis is placed on each result. Physicians are well aware, however, that certain findings are virtually pathognomonic, whereas others are extremely nonspecific. A further drawback lies not in the theorem itself, but in the way it has been used. In screening for rare diseases (e.g., neo¬ natal hypothyroidism) or even in screening for relatively corn-

FIGURE 237-5. Examples of the use of Bayes' theorem. A, In obese patients with abdominal striae and centripetal obesity, the frequency of Cushing syndrome is -25%. In testing 100 patients using urinary free cortisol levels, the sensitivity and specificity are each -95%. The predic¬ tive value of elevated urinary free cortisol levels in this setting is 86%; thus, the certainty of diagnosis is increased from 25% to the higher fig¬ ure by using this one test. B, In screening for primary hyperparathy¬ roidism, -90% of all serum calcium values in affected persons are above 10.5 mg/dL. Because this is the upper reference limit, 2.5% of normal persons have a serum calcium value above this figure by chance alone. In screening studies, the prevalence of hyperparathyroidism is -1:1000. The likelihood that a serum calcium value above 10.5 mg/dL represents primary hyperparathyroidism, given only the result of the calcium test, is 3.5%. C, Neonatal hypothyroidism screening programs sometimes use measurements of thyroid-stimulating hormone (TSH) in cord blood for the initial test. Because -10% of cases are due to hypothalamic-pitu¬ itary insufficiency, the sensitivity of such testing is 90%. The cutoff value is >3 standard deviations above the mean value for normal neo¬ nates; this excludes 99.75% of normal infants. Because the prevalence of hypothyroidism is -1:4000 newborns, the predictive value of an ele¬ vated TSH level is -8%, but most affected infants have an elevated TSH level. Thus, when 4000 infants are screened, only 11 infants need further studies to find the one infant with hypothyroidism.

mon endocrine disorders (e.g., primary hyperparathyroidism), most abnormal restilts from screening tests do not indicate the presence of disease. This has led many to criticize the screening of asymptomatic persons as worthless, because most abnormal results are false-positives.17/18 The purpose of screening tests is not to make diagnoses, however, but to identify persons who are at high risk for having a disease. For example, as shown in Figure 237-5, only -8% of infants with an elevated level of TSH have congenital hypothyroidism. What the screening program does, however, is virtually rule out hypothyroidism in 99.75% of the screened newborns. The task of finding affected infants is now much simpler because further tests can be performed only on the few infants with elevated TSH levels. Without screening, discovering infants with hypothyroidism would be like finding a needle in a haystack; screening puts the needle into a pincushion. Bayes' theorem is an inadequate model for the diagnostic process. Although its popularization has focused attention on the inherent uncertainty in the interpretation of any data, other mathematical models and artificial intelligence systems (e.g.,

C

Ch. 237: Internist) provide much closer approximations to the probabil¬ ity of diagnoses based on data obtained.59

REFERENCE VALUES IN ENDOCRINOLOGY Reference values should be used as a relative means of compar¬ ison, not as an absolute declaration of health or disease. Table 237-1 provides a context for the endocrinologist to interpret results for an individual patient. Reference values are given first in the conventional units used in the United States. The conversion factor to transform these results to SI units and the reference range in SI units also are given. SI refers to the Systeme International, which is an attempt to create a universally accepted scientific nomenclature. This sys¬ tem is used widely in Europe and in other parts of the world. The SI recommends expressing concentration in moles (or fractions thereof) of a substance per liter, rather than in weight per volume. Young outlined the rationale for making this conversion. In sum¬ mary, the major advantage of SI units is that they make interrela¬ tions between substances much easier to understand.651 A good example is in the formula for calculated osmolality, in which glu¬ cose is divided by 18 and blood urea nitrogen by 2.8 to convert from weight units to moles (osmolality is related to moles of a substance in solution). In this example, expressing concentration in moles makes the calculation easy and eliminates the need for remembering the appropriate factors for conversion. Other advantages of using SI units include a better understanding of relationships between a compound and its target or receptor, and universality in the reporting system, which should improve the international usefulness of scientific studies. The recommendation has been made that peptide hormone concentrations still be expressed in standard mass nomencla¬ ture. This decision is difficult to understand, because hormones with active forms of different molecular weights cannot be expressed accurately using such mass nomenclature. Hormone concentrations are reported in mass units; however, because all immunoassays detect molar concentrations of a substance, hor¬ mone immunoassays make use of some conversion factor. For the many hormones with multiple circulating forms, the conver¬ sion factor may be either an average molecular weight of all forms present, a weighted average, or the molecular weight of the most abundant or most physiologically important form (i.e., the immunoassay standard). Because the exact conversion fac¬ tors may vary, Table 237-1 gives the conversion factor only for hormones with a single predominant circulating form.

Reference Values in Endocrinology

2179

The major disadvantage of conversion to SI units is that phy¬ sicians in the United States would be required to learn a new set of reference ranges for all commonly ordered tests. Canada adopted SI units without major difficulty. In the late 1980s, sev¬ eral journals announced their intention to convert to SI units for all published scientific articles; however, most have returned to the use of conventional units. The reference ranges given in Table 237-1 are those used in the author's laboratory or in the reference laboratories the author uses, employing the method listed first under "Methodologic Considerations." Reference ranges are for serum or plasma unless otherwise specified. For some tests, reference ranges published in the literature are included, with the refer¬ ence number in superscript. Some of these reference ranges are based on results from fewer than 20 persons. These ranges may not be applicable to other methods or laboratories, and readers are urged to utilize reference ranges published by the laboratory they most commonly use. For tests in which reference ranges often differ by an order of magnitude (because of the lack of an acceptable standard or differences in reactivity of antibodies), the reference range carries the notation "method dependent." For each test, the attempt has been made to define the common intraindividual, physiologic, drug-related, and methodologic fac¬ tors influencing the results for a particular test. For each test, these comments apply to the specimen type (e.g., urine or serum) listed first, for the method given in the "Methodologic Considerations" column unless otherwise specified. Intraindividual variation includes day-to-day variation, within-day variation, and changes occurring during the menstrual cycle. Physiologic changes include commonly encountered alterations in expected physiol¬ ogy, such as exercise, obesity, pregnancy, and the effects of age, sex, and race. The effects of renal insufficiency and alcohol or tobacco use on test results also are given when indicated. This is not a comprehensive list of reference ranges for children; instead, the magnitude and direction of such changes is given. For more information on pediatric reference ranges, the reader should con¬ sult pertinent chapters of this book. In addition, two textbooks devoted to pediatric chemistry include endocrine values.652'653 The column for drug effects includes only pharmacologic effects of drugs; cross-reactions are listed under "Methodologic Consid¬ erations," as are any special collection or handling requirements. Several general references and a publication on drug effects are the sources of much of the data in this table.47-48-652-659 In addition, specific references for each individual test are listed under the test name. (see References on page 2216)

Abbreviations = angiotensin-converting enzyme = ACTH adrenocorticotropic hormone

hCG

= human chorionic gonadotropin

HDL

= high-density lipoprotein

AMP

5-HIAA

ACE

ANH

= adenosine monophosphate = atrial natriuretic hormone

cAMP = cyclic adenosine monophosphate CEA = carcinoembryonic antigen

PTHrP = parathyroid hormone-related protein = red blood cell RBC = radioimmunoassay RIA

= decreased

HVA

= diethylstilbestrol

IGF-I

= insulin-like growth factor-I

IGFBP-3 IV

= insulin-like growth factor-binding protein-3 = increased = intravenously

GAD GFR

INC

= females = follicle-stimulating hormone

LATS

= long-acting thyroid stimulator

LDL

= glutamic acid decarboxylase = glomerular filtration rate

M

= low-density lipoprotein = luteinizing hormone = males

= growth hormone GnRH = gonadotropin-releasing hormone GH

Program

HPLC

DEC

FSH

National Cholesterol Education

NSAID = nonsteroidal antiinflammatory drug

DES

F

—

= 5-hydroxyindoleacetic acid = human immunodeficiency virus HIV HMG-CoA = 3-hydroxy-3-methylglutaryl coenzyme A = high-performance liquid chromatography = homovanillic acid

DHEA = dehydroepiandrosterone EDTA = ethylenediaminetetraacetic acid ELISA = enzyme-linked immunosorbent assay

NCEP

LH MAO N/A

= monoamine oxidase = not applicable

PSA PTH

= prostate-specific antigen = parathyroid hormone

t3

= sex hormone-binding globulin = Systeme international d'Unites = triiodothyronine

U TBG

= thyroxine = thyroxine-binding globulin

THC TSH

= tetrahydrocannabinol = thyroid-stimulating hormone (thyrotropin)

VMA

= vanillylmandelic acid

SHBG SI

m 2180

PART XVIII: ENDOCRINE DRUGS AND VALUES

TABLE 237-1. Reference Values in Endocrinology Test Name

Reference Range (Conventional Units)

Conversion Factor

Reference Range (SI Units)

a SUBUNIT94-96

0-1.0 ng/mL

1

0-1.0 pg/L

0.02-0.2 mmol/L

1

0.02-0.2 mmol/L

Probably similar to ketones.

0-0.5 U/L

1

0-0.5 U/L

Highest in p.m. with nadir in a.m. Within-day variation 25-50%. Day-today variation 50-100%.

9-52 pg/mL

0.22

2-11.5 pmol/L

INC: Highest shortly before waking, released in episodic spikes during the day, lowest in early sleep.

27.7

Supine: 50-250 pmol/L Upright: 80-970 pmol/L

2.77

High salt: 0-13.9 nmol/d Normal salt: 6.4-86 nmol/d Low salt: 47-122 nmol/d

Intraindividual Variation

ACE. See ANGIOTENSIN¬ CONVERTING ENZYME ACETO ACETATE60-61'660 (see also KETONES) ACETONE. See KETONES ACID PHOSPHATASE62 64

ACTH. See ADRENOCORTICO¬ TROPIC HORMONE 3',5'-ADENOSINE MONOPHOS¬ PHATE. See CYCLIC ADEN¬ OSINE MONOPHOSPHATE ADH. See VASOPRESSIN ADRENOCORTICOTROPIC HORMONE65-71

ALBUMINURIA, MINIMAL. See MICROALBUMIN, URINE ALDOSTERONE72-77 661

Serum: Supine: 2-9 ng/dL Upright: 3-35 ng/dL Urine: High salt: 0-5 pg/d Normal salt: 2.3-31 gg/d Low salt: 17-44 pg/d

While supine, decreases during day. Upright, increases progressively and remains elevated for 6 h. Day-to-day variation 40%. In women, higher in late luteal phase.

ALKALINE PHOSPHATASE78-82

30-130 U/L

1

30-130 U/L

No diurnal variation. Day-to-day variation 5-10%. Higher in fall than in spring.

ALKALINE PHOSPHATASE BONE ISOENZYME83-”-662

M: 5.9-22.9 gg/L F: 3.9-15.1 gg/L Postmenopausal: 6.4-24.4 M-g/L

1

M: 5.9-22.9 pg/L F: 3.9-15.1 pg/L Postmenopausal: 6.4-24.4 Mg/L

Day-to-day variation 10%. Higher in fall than in spring.

ALKALINE PHOSPHATASE ISOENZYMES83-”'662

Bone: 12-84 U/L Liver: 13-92 U/L Intestine: 0-14 U/L Other: trace or less

ALUMINUM (Al)97-100'174

0-6 pg/L

AMP, CYCLIC. See CYCLIC ADENOSINE MONOPHOS¬ PHATE

Bone: 12-84 U/L Liver: 13-92 U/L Intestine: 0-14 U/L Other: trace or less

0.0371

7.0 at room temperature. Other chemical assays detect other isoenzymes of acid phosphatase from bone, platelet, and RBC; these assays may show falsely high values in hemolyzed specimens, and higher values in serum than in plasma.

INC: stress, pregnancy, exercise, hypoglycemia, hemo¬ concentration DEC: weight loss, breast feeding In neonates, values 2x normal during first 24 h of life.

INC: insulin, desipramine, erythro¬ poietin, interferon-(3, ketoconazole, L-dopa, mifepristone (RU-486), vasopressin DEC: glucocorticoids, clonidine

Immunometric assay: Detects intact hormone, may not detect small fragments of ACTH in ectopic ACTH pro¬ duction, but usually reports increased levels.

INC: pregnancy, high temperature, prolonged fast, obesity DEC: high altitude, ethanol, weight loss (in obesity), severe acute illness. Highest in neonates, adult levels by 3 mo, significantly lower over age 60. Lower in blacks than in whites.

INC: volume-depleting agents, lith¬ ium, ethanol (acutely), spironolac¬ tone, verapamil DEC: licorice, heparin, propranolol, ACE inhibitors, NSAIDs, raniti¬ dine. Nifedipine decreases the response to the upright position.

RIA: A variety of steroids interfere in urine assays. |iadrenergic antagonists and calcium-channel blockers increase aldosterone/renin ratio but rarely cause sig¬ nificant elevation of aldosterone. Urine requires boric acid as a preservative.

INC: pregnancy, periods of bone growth, after meals, hemoconcentration, renal failure Higher in blacks than in whites and higher in men than in women (until after menopause). Higher in persons of blood groups O and B, especially after meals. Higher in children, rises during puberty and then falls to adult levels by age 20. Stable until age 50, then rises in both sexes. In adults, directly related to body mass, inversely related to height. In infants, transient marked elevations may occur after acute illness.

INC: drugs causing cholestasis, lith¬ ium, anticonvulsants (especially phenytoin) DEC: oral contraceptives, clofibrate, tamoxifen, glucocorticoids

Spectrophotometric: Many anticoagulants (EDTA, fluo¬ ride, oxalate) inhibit the enzyme reaction and cause falsely decreased results. The method is zinc depen¬ dent; patients with zinc deficiency show falsely low values. Transiently low after blood transfusion, prob¬ ably due to zinc chelation. Valproic acid causes slight overestimation in some methods, whereas methylxanthines inhibit alkaline phosphatase activity.

INC: renal failure, lactation Higher in children, increases at puberty, declines to reach adult levels by age 20. Higher in men than in women until after menopause, when values become higher in women. Increases throughout pregnancy. Increases with age older than 50 yr.

INC: lithium, phenytoin DEC: clofibrate

Immunoassay: Approximately 3% cross-reactivity with liver isoenzyme. Does not agree well with results of immunoinhibition assays in course of treatment of metabolic bone disease.

Bone: Higher in children, increase at puberty, decline to reach adult levels by age 20 yr. Higher in men than in women. Both bone and liver isoenzymes increase with age >50 yr. Bone isoenzyme increases throughout pregnancy and remains elevated during lactation.

Bone: INC: lithium, phenytoin Bone: DEC: clofibrate Liver: INC: anticonvulsants (espe¬ cially phenytoin), drugs causing cholestasis Liver: DEC: oral contraceptives

High-resolution electrophoresis: Resolves bone, liver, intestinal, placental isoenzymes. Standard electro¬ phoresis resolves all isoenzymes except bone and liver. Heat fractionation is poorly reproducible and relatively inaccurate in most laboratories.

INC: hemoconcentration Reflects recent exposure; returns to normal within months if only single exposure. Serum correlates poorly with total body aluminum.

INC: aluminum-containing antacids, sucralfate (in renal failure)

Atomic absorption: Contamination from glass, needles, and heparin a significant problem.

(continued)

2182

PART XVIII: ENDOCRINE DRUGS AND VALUES

TABLE 237-1. Reference Values in Endocrinology (Continued) Test Name

Reference Range (Conventional Units)

Conversion Factor

ANDROSTANEDIOL GLUCURONIDE101-105

M: 270-1500 ng/dL F: 60-300 ng/dL

ANDROSTENEDIONE103-111

Reference Range (SI Units)

Intraindividual Variation

0.021

M: 5.6-31.2 nmol/L F: 1.2-6.2 nmol/L

Higher in a m. than in p.m. In women, lower in follicular than in luteal phase.

60-300 ng/dL

0.0349

2.1-10.5 nmol/L

Highest in a.m., lowest in p.m. In women, highest at midcycle, 40% lower at onset of menses. Approximately 3050% day-to-day variation.

ANGIOTENSIN I76112

50%. In women, higher in luteal than in follic¬ ular phase. Higher in spring than in autumn.

___j 11.4

Urine: 0-456 pmol/d Serum: 11.4—27.4 gmol/L

Higher in summer than in winter. Dayto-day variation 40% (urine).

0-3.2 mU/mL

1

0-3.2 U/L

In women, increases to peak near time of ovulation, nadir in late luteal phase. Episodic release occurs near term preg¬ nancy, uncertain if release is episodic at other times due to lack of sensitivity of assays.

26-300 pg/mL

0.24

6-72pmol/L

Marked episodic fluctuation, average 200% within-day variation.

17-OXOGENIC STEROIDS. See 17-KETOGENIC STEROIDS 17-OXOSTEROIDS. See 17KETOSTEROIDS OXYTOCIN438-440

P. See PHOSPHATE PANCREATIC POLYPEPTIDE441-447

PANCREOZYMIN. See CHOLECYSTOKININ

Ch. 237: Reference Values in Endocrinology

2203

Physiologic Changes

Drug Effects

Methodologic Considerations

Less subject to dietary cross-reactants than VMA.

INC: amphetamines, caffeine, hydra¬ zines, MAO inhibitors, prochlor¬ perazine DEC: L-dopa

Spectrophotometric after chromatographic purification: a-Methyldopa and methylglucamine (radiopaque dye) cross-react to give false elevations in all assays; Padrenergic blockers, imipramine, phenothiazines, phenacetin, and oxytetracycline cross-react in some assays; propranolol inhibits color formation. Acetami¬ nophen interferes with plasma normetanephrine assay.

INC: acute illness, exercise, ethanol, smoking, stress, hyper¬ tension, high-fat meal, urinary tract bleeding or infection DEC: high-fiber diet Low in children, increases during puberty to peak in mid¬ teens, then declines to adult levels by age 30. Higher in blacks than in whites, lower in females than in males.

INC: lithium DEC: heparin

Immunoassay: No interferences reported. Stable on stor¬ age at room temperature or in refrigerator up to 7 days. Dipsticks are less precise and accurate than quantitative immunoassays.

INC: renal failure; after meals, especially lipid containing

DEC: morphine

RIA: Must be frozen immediately. Different molecular weight forms may react differently. (Also see Chap. 182.) Atomic absorption: Elevated cadmium, gold may cause slight underestimation.

INC: tissue necrosis (myocardial infarct, stroke, burns), renal failure, ethanol Higher in women than in men.

INC: dehydration, exercise, ethanol DEC: at high altitudes, pregnancy

INC: steroids causing salt retention, mannitol DEC: vincristine, cyclophosphamide, diuretics

Freezing point depression: When vapor pressure depression osmometers are used, volatile substances such as ketones, ethanol, isopropanol, and methanol do not increase osmolality, causing absence of "osmotic gap."

INC: renal failure, weight lifting DEC: ethanol, acute illness, pregnancy Highest in children, with peak values occurring during periods of bone growth; falls to adult levels by age 20 yr. In adults, increases throughout life. Higher in men than in women, higher in whites than in blacks. Decreases in early pregnancy, returns to normal by term; increased during lactation.

INC: calcitriol, omeprazole, phenytoin DEC: glucocorticoids, warfarin, estro¬ gen, oral contraceptives, tamoxifen, thiazides

RIA: Significantly lower if blood collected with anticoag¬ ulants, particularly oxalate and EDTA. RIA measures both active and inactive forms; active form is decreased by treatment with warfarin, so that RIA is falsely elevated.

INC: malabsorptive disorders, renal failure (serum), liver failure, ingestion of oxalate-containing foods (spinach, tomatoes, strawberries, rhubarb) DEC: renal failure (urine) Ratio of oxalate to creatinine highest in infancy, falls throughout childhood.

INC: vitamin C, especially in renal failure, methoxyflurane DEC: pyridoxine

Spectrophotometric: Wide interlaboratory variation; methods are poorly reproducible, with most methods overestimating actual concentration. Oxalate concen¬ tration rises rapidly at room temperature, especially in serum; specimens must be frozen or, for urine, col¬ lected in acid.

INC: pregnancy, hemoconcentration DEC: stress

INC: oral contraceptives

RIA: No interferences reported.

INC: after meals, acute stress, renal failure, especially with dialysis Higher in men than in women, higher in children than in adults. Lowest values in persons 40 yr.

Probably similar to testosterone

Immunoassay after ammonium sulfate precipitation: No interferences reported. Measures testosterone not bound to SHBG, as ammonium sulfate precipitates SHBG-bound testosterone.

Same as for testosterone, although hemoconcentration does not affect results.

INC: barbiturates, clomiphene, dana¬ zol DEC: androgens, antiepileptics, DES, digoxin, spironolactone, phenothiazines, THC, ketoconazole

Equilibrium dialysis: Results are generally similar to those calculated from total testosterone and SHBG levels. Variation in incubation temperature significantly affects results obtained. Analog immunoassays are widely used, but results are significantly lower and are affected by changes in the level of SHBG; factors affect¬ ing SHBG (see earlier) produce erroneous results.

(continued)

2210

PART XVIII: ENDOCRINE DRUGS AND VALUES

TABLE 237-1. Reference Values in Endocrinology (Continued) Reference Range (Conventional Units)

Conversion Factor

Reference Range (SI Units)

Intraindividual Variation

18 pg/dL >18 pg/dL Two to 5 times basal values

basal and each day of test

RENIN-ANGIOTENSIN-ALDOSTERONE AXIS

STIMULATORY 1. Sodium restriction109-113

10-20 mmol sodium diet until urine Na 5 ng/mL/h >25 ng/dL >100 pg/d

‘The methodology, interpretation, and utility of some of these procedures are disputed; for further information and clinical recommendations, see the indicated chapters.

Response greater in p.m. than in a.m.; day-to-day variation 20-30%

Ch. 241: Dynamic Procedures in Endocrinology

2271

Physiologic Factors

Drug Effects

Methodologic Considerations

Special Considerations/Interpretation

INC: depression, bipolar disorder.

INC (cortisol, ACTH): phenytoin.

Because ACTH is extremely labile in vitro, proper speci¬ men collection and handling is vital (see Table 237-1). Results from different assays are not directly comparable; many cortisol assays have poor reproducibility at such low values.

Absent response is seen in normal individuals and pseudo-Cushing syndrome. Most patients with psy¬ chiatric disorders, particularly affective disorders, will fail to show suppression of response to CRH despite administration of dexamethasone. Some have suggested use of dexamethasone 0.5 mg q6h for 2 days before administration of CRH.

DEC: acute illness, obesity, preg¬ nancy, acute stress (for overnight

INC: amphetamines, carbamazepine, non¬ steroidal antiinflam¬ matory agents,

Cortisol assay is important in interpreting results of over¬ night testing because some assays may be imprecise at low levels (near the 5 (ig/dL decision level). Urine 17-OH steroids or free cortisol may be inaccurate in patients with renal failure.

The overnight low-dose test shows lack of suppression in 98% of cases of Cushing syndrome, regardless of cause. The low-dose test has better specificity for Cushing syndrome than the overnight test but is nor¬ mal in a significant minority of patients with Cushing syndrome. The high-dose tests show suppression with Cushing disease, but usually not with other causes of Cushing syndrome; occasional patients with Cushing disease require higher doses of dexamethasone to show a fall in cortisol production. With the 1 mg overnight test, lack of suppression is com¬ mon in affective psychiatric disorders, alcohol abuse, and acute illness. Lack of suppression in low-dose tests is common in patients with diabetes mellitus, but is not related to diabetic control. Some have sug¬ gested using larger doses of dexamethasone (32 mg/ day) to improve ability to differentiate Cushing dis¬ ease from other causes of Cushing syndrome. Poor dexamethasone absorption has been reported in patients with chronic renal failure. Ingestion of dex¬ amethasone should be verified; in doubtful cases, plasma dexamethasone concentration is useful. (See Chaps. 74, 78, and 201.)

If assays that show cross-reactiv¬ ity with prednisone and corti¬ sone are used (competitive protein binding and some

With 250 gg, diminished or no response is seen in patients with primary adrenal insufficiency or severe second¬ ary adrenal insufficiency, and in patients treated with high-dose glucocorticoids in whom normal pituitaryadrenal responsiveness has not returned. Low-dose ACTH shows diminished response in patients receiv¬ ing even low-dose long-term corticosteroids and, in one review, in 95% of patients with secondary adrenal insufficiency. It agrees better with results of insulininduced hypoglycemia or metyrapone procedures in such cases than does the higher-dose test in some stud¬ ies, although others suggest it is less sensitive than tests evaluating hypothalamic or pituitary response. Response to synthetic ACTH persists for at least 3-4 weeks following onset of secondary adrenal insuffi¬ ciency; these tests should not be used in the first month

and 2-d low-dose tests). In renal failure and alcohol abuse, lack of suppression is common with low-dose overnight and 2-d tests. Nonsuppression is more common in children and in those >age 65 yr, especially if cortisol is measured at 4 p.m. as is rec¬ ommended in psychiatric uses of the test.

Response is similar in newborn infants, children, adults, and the elderly.

benzodiazepines. DEC: phenytoin, barbi¬ turates, estrogens (high doses), rifampicin.

INC: estrogens, theophylline. DEC: calcium-channel blockers, glucocorti¬ coids, ketoconazole, megestrol acetate.

immunoassays), a high basal value with no response to infusion may be seen in patients receiving the medica¬ tions.

after pituitary surgery. Patients with Cushing disease often hyperrespond, and those with adrenal carcinoma or ectopic ACTH production usually do not. As many as 50% of patients with adrenal adenomas show a response to ACTH. In normal individuals, the response decreases as basal serum cortisol increases. (See Chaps. 74 and 78.)

Response decreases with increas¬ ing age.

Drugs that alter aldoster¬ one excretion also affect dynamic responses (see Table 237-1).

This degree of sodium restriction often is not achieved in prac¬ tice, particularly in children. Appropriate sample collection is essential, because renin activity increases if specimens are not kept at room tempera¬ ture until plasma is frozen; aldosterone is labile (see Table 237-1 for additional informa¬ tion on collection).

In persons with aldosteronoma, typically no change occurs in aldosterone, whereas an increase may be seen in persons with hyperplasia of the zona glomerulosa. In primary or secondary hypoaldosteronism, a blunted aldosterone response is seen; renin increases only in primary hypoaldosteronism. Hypokalemia and hypopituitarism blunt the response to volume depletion; in hypopituitarism, cortisol alone does not correct the response, but cor¬ tisol plus thyroxine does. (See Chaps. 79-81, and 183.)

(continued)

2272

PART XVIII: ENDOCRINE DRUGS AND VALUES

TABLE 241-1. Dynamic Endocrine Procedures* (Continued) How Performed

Substance Measured

Expected Response

80 mg furosemide PO; have patient stand upright for 2 h, starting 2 h after diuretic administration

Plasma renin activity at 4 h Plasma aldosterone at 4 h

>5 ng/mL/h >25 ng/dL

1. Sodium loading109"113

Oral: 120 mmol Na diet until

Plasma renin activity Urinary aldosterone Plasma renin activity at 4 h Plasma aldosterone, basal and at 4 h