Vitamin D - Biochemical, Chemical and Clinical Aspects Related to Calcium Metabolism: Proceedings of the Third Workshop on Vitamin D, Asilomar, Pacific Grove, California, USA, January 1977 [Reprint 2020 ed.] 9783112327203, 9783112327197

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Vitamin D Biochemical, Chemical and Clinical Aspects related to Calcium Metabolism

Vitamin D Biochemical, Chemical and Clinical Aspects Related to Calcium Metabolism Proceedings of the Third Workshop on Vitamin D, Asilomar, Pacific Grove, California, USA, January 1977 Editors A.W. Norman • K. Schaefer • J.W. Coburn • H. F. De Luca D. Fraser • H.G. Grigoleit • D.v. Herrath

W G DE

Walter de Gruyter • Berlin • New York 1977

Editors A.W. Norman, P h . D . , Department of Biochemistry, University of California. Riverside, Ca 9 5 0 2 , U S A K. Schaefer, Priv.-Doz., St. Joseph-Krankenhaus I, Berlin (West), Germany J. W. Coburn, M.D., Nephrology Section, Veterans Administration Wadsworth Hopital Center, Los Angeles, CA 9 0 0 7 3 , U S A H. F. DeLuca, Ph. D., Department of Biochemistry, University of Wisconsin-Madison, Madison WI 5 3 7 0 6 , U S A D. Fräser, M. D., The Hospital for Sick Children, 5 5 5 University Avenue, Toronto 2, Canada H. G. Grigoleit, Dr., Medizinische Abteilung H o e c h s t AG Werk Albert, Wiesbaden, Germany D. v o n Herrath, Dr., St. Joseph-Krankenhaus I, Berlin (West), Germany

CIP-Kurztitelaufnahme

der Deutschen

Bibliothek

Vitamin D: biochem., chem. and clin. aspects related to calcium metabolism; proceedings of the Third Workshop on Vitamin D, Asilomax, Pacific Grove, Calif., USA, January 1977/ed. A. W. Norman. - 1. Aufl. - Berlin, New York: de Gruyter, 1977. ISBN 3-11-006918-0 NE: Norman, Anthony W. [Hrsg.]; Workshop on Vitamin D < 0 3 , 1977, Asilomar, C a l i f >

Library of Congress Cataloging in Publication

Data

Workshop on Vitamin D, 3d, Asilomar Conference Center, Calif., 1977. Vitamin D. Bibliography: p. Includes index. 1. Vitamin D in the body-Congresses. 2. Calcium metabolismCongresses. 3. Bones-Diseases-Congresses. 4. Vitamin DCongresses. I. Norman, Anthony W., 1938 - II. Title. [DNLM: 1. Calcium-Metabolism-Congresses. 2. Calcium metabolism disorders-Congresses. 3. Vitamin D-Congresses. W3 W0512C 1977v/QU173 W926 1977v] QP772. V53W67 1977 612'.399 77-8004 ISBN 3-11006918-0

©Copyright 1977 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Karl Gerike, Berlin. - Binding: Liideritz & Bauer, Buchgewerbe GmbH, Berlin. Printed in Germany.

Foreword

The Third Workshop on Vitamin D was held in the beautiful setting of Asilomar Conference Center, Pacific Grove, California, January 9—13, 1977. At this meeting some 45 invited lectures were presented as well as some 124 "Poster Presentations". The 332 registered delegates represented 20 countries including Argentina, Austria, Australia, Belgium, Canada, Denmark, France, Great Britain, Ireland, Israel, Italy, Japan, The Netherlands, Norway, South Africa, Sweden, Switzerland, the United States of America, West Germany, and Venezuela, as well as representatives from 28 of the 50 states of America.

It has been 26 months since the occurrence of the Second Workshop on Vitamin D in Wiesbaden, West Germany. Clearly there is still an information explosion occurring in the field of calcium and phosphorous metabolism and more specifically vitamin D judging not only by the total number of registered participants but more importantly by the number of submitted communications. It was the view of the Organizing Committee that no attempt should be made to select for verbal presentation only a fraction of the total number of "submitted communications". An important objective of the Workshop was to provide a forum to all scientists interested in vitamin D. Thus on Monday evening at the Workshop sixty "basic contributions" were programmed into a poster session while on Tuesday evening sixty-two presentations related to clinical aspects of vitamin D were programmed. As recently as 10 years ago there were probably only a dozen laboratories in the world which were seriously conducting research relating to vitamin D; it is astounding to consider now the number of participants present at the poster session who were vitally and intimately associated with ongoing investigation into vitamin D and its metabolites. The theme or area of focus of this Third Workshop on Vitamin D was broad in scope and really related to recent developments in our understanding of vitamin D. In the past two years, since the last workshop, important advances have been made in our understanding of the conformation of the vitamin D molecules in solution, as well as to the synthesis of important analogs of various metabolites. In the biochemical and physiological realm new developments have appeared concerning the regulation of the production of the hormonly active vitamin D, 1,25-dihydroxy vitamin D3 as well as in terms of its interaction with the parathyroid gland; and an increased understanding of its molecular mode of action in various target tissues, particularly the intestine. Lastly, there has been a phenomenal increase in the application of this fundamental knowledge into the various clinical aspects of vitamin D, particularly renal osteodystrophy and chronic renal failure, hypoparathyroidism, and possibly osteoporosis. Certainly one of the more vigorous components of the session was the great number of concerned clinicians who attended and participated in the many discussions and debates.

VI

The organizing committee would like to acknowledge the generous financial support of Hoffmann-La Roche, Inc., Nutley, New Jersey; National Institute of Arthritis, Metabolism and Digestive Diseases;The Upjohn Company; the National Dairy Council; Albert-Roussel Pharma GMBH: Diamond Shamrock Corporation; Hoechst Aktiengesellschaft Werk Albert; Merck Sharp and Dohme Research Laboratories, Division of Merck and Company, Inc.; Paul Martini Stiftung; The Procter and Gamble Company; Roche Products Limited, England; Syntex Research; and Ross Laboratories. Without the generous support of these organizations a meeting such as the Third Workshop would have been an impossibility. Of course another important aspect of the Third Workshop on Vitamin D is the publication of the proceedings in an extremely rapid fashion. Through thé cooperation of all authors in providing "camera ready" copies of their manuscripts and through the good efforts of the Organizing Committee as well as the Walter de Gruyter Publishing Company it has been possible to publish the book within six months after the close of the meeting. In view of the rapid pace of developments in the vitamin D world, it is of the utmost importance that new information be rapidly disseminated. The organizing committee is also greatly indebted to the loyalty and dedication of the Conference Secretary, Mrs. Wendy Reid and her colleagues Mrs. Sharon Meacham, Mrs. Billie Luben and Ms. June Bishop for the seemingly unending planning and organization required to successfully present the meeting and this book.

Anthony W. Norman, Riverside Jack W. Coburn, Los Angeles H. F. DeLuca, Madison

D. Fraser, Toronto H. G. Grigoleit, Wiesbaden

K. Schaefer, Berlin-West D. von Herrath, Berlin-West

Contents

List of Participants

XIX

Vitamin D-Chemistry 19-Hydroxy-lOS (19)-Dihydrovitamin D 3 and 25-Hydroxy-24-norvitamin D 3 : Analogs with Anti-Metabolite Activity and Related Studies M. L. Hamond, A. Mourino, P. Blair, W. Wecksler, R. L. Johnson, W. Norman, W. Okamura

1

Synthesis and Separation of C-24 Epimers of some 24-Hydroxylated Vitamin D 3 Analogs N. Ikekawa, M. Morisaki, N. Koizumi, A. Sakamoto

5

The 'Overirradiation Products' of Previtamin D and Tachysterol: Toxisterols H. J. C. Jacobs, F. Boomsma, E. Havinga, A. van der Gen

15

Conformational Analysis of Vitamin D and its Analogs by Ultraviolet, Circular Dichroic, ' H and 1 3 C NMR Spectroscopy E. Berman, N. Friedman, Y. Mazur, M. Sheves

19

I,25-Dihydroxyvitamin D 3 from Solanum Glaucophyllum J. L. Napoli, L. E. Reeve, J. A. Eisman, H. K. Schnoes, H. F. DeLuca

29

Synthesis and Conformational Analysis of Vitamin D Metabolites and Analogs W. H. Okamura, M. L. Hammond, P. Condran, Jr., A. Mourino

33

A Stereoselective Synthesis of l a , 2 4 R , 25-Trihydroxycholecalciferol, a Metabolite of Vitamin D 3 J. J. Partridge, S.-J. Shiuey, E.G. Baggiolini, B. Hennessy, M. R. Uskokovic

47

Crystal Structures and Empirical Force-Field Calculations of Some Steroids of the Vitamin D Series C. Romers, A.J. de Kok, D. H. Faber, C. Altona

57

Approaches to the Synthesis of Vitamin D Metabolites and Analogs W. G. Salmond

61

Conformational Analysis of Vitamin D: NMR and Force Field Calculations R.M.Wing Vitamin D-Like Action of a Steroid from Trisetum Flavescens H. Zucker, O. Kreutzberg, W. A. Rambeck

71 85

Vitamin D-Metabolism and Regulation Vitamin D 3 Metabolism in Immature Japanese Quail: Effects of Ovarian Hormones S. N. Baksi, A. D. Kenny

89

VIII Relationship of Intestinal Calcium and Phosphorus Absorption to Vitamin D Metabolism During Reproductive Activity of Birds A. Bar, A. Cohen, G. Montecuccoli, S. Edelstein, S. Hurwitz

93

On Transcalciferin, the Serum Binding Protein for 25-Hydroxycholecalciferol R. Bouillon, H. van Baelen

97

Ultrastructural Alterations Produced by Vitamin D and its Metabolites on the Chick Parathyroid Gland C. C. Capen, H. L. Henry, A. W. Norman

101

Reduction of Parathyroid Hormone Secretion by 24, 25-DihydroxyCholecalciferol (24, 25-DHCC) A. D. Care, D. W. Pickard, M. Peacock, B. Mawer, C. M. Taylor, J. Redel, A. W. Norman

105

The Influence of Ca 2 + Ions on the Metabolism of 25-Hydroxy Cholecalciferol K. W. Colston, P. J. Butterworth, R. Webdale, I. Maclntyre

109

1,25-Dihydroxyvitamin D 3 : Its Further Metabolism and the Regulation of its Biogenesis H. F. DeLuca, Y. Tanaka, L. Castillo, R. Kumar

113

3

Disappearance from Plasma of Injected H-1,25- (OH) 2 -D 3 in Healthy Humans R. W. Gray, D. R. Wilz, A. E. Caldas, J. Lemann, Jr., H. F. DeLuca

123

Regulation of the Metabolsim o f 2 5 ( O H ) D 3 : l,25(OH) 2 D 3 -Parathyroid Interactions H.L.Henry

125

Conversion of 7-Dehydrocholesterol to Vitamin D 3 in Vivo: Isolation and Identification of Previtamin D 3 from Skin M. F. Holick, J. Frommer, S. McNeill, N. Richtand, J. Henley, J. T. Potts, Jr

135

Assessment of the Effects of 1,25-Dihydroxycholecalciferol (1,25-DHCC) on Parathyroid Secretion Rate in Calves J. G. Hurst, G. P. Mayer

139

Biological Activity of l a , 24-Dihydroxyvitamin D 3 ; A New Synthetic Analog of the Hormonal Form of Vitamin D H. Kawashima, K. Hoshina, S. Ishizuka, Y. Hashimoto, T. Takeshita, S. Ishimoto, T. Noguchi, N. Ikekawa, M. Morisaki, H. Orimo

143

Metabolism of 1,25-Dihydroxyvitamin D 3 : Side Chain Oxidation in Vivo R. Kumar, H. F. DeLuca Calcium and Parathyroid Status in Normal Man Following Administration of 1,25-Dihydroxy-Vitamin D 3 F. Llach, J. W. Coburn, A. S. Brickman, K. Kurokawa, A. W. Norman, J. M. Canterbury, E. Reiss Comparative Aspects of the Biochemistry of the Regulation of Vitamin D Metabolism I. Maclntyre

147

151

155

IX Aspects of the Control of Vitamin D Metabolism in Man E. B. Mawer

165

Metabolism of 25-Hydroxycholecalciferol by Various Lines of Serially Cultured Kidney Cells R. J. Midgett, A. B. Borle

175

Metabolism of 25-Hydroxyvitamin D 3 and la-Hydroxy vitamin D 3 in Experimental Rats with Chronic Renal failure Y. Nishii, K. Kumaki, M. Fukushima, T. Shimizu, M. Ono, H. Ökawa, R. Niki, I. Masunaga, K. Ochi, Y. Töhira, S. Sasaki, T. Suda

179

Modulation of 25-Hydroxyvitamin D 3 Metabolism by la,25-DihydroxyvitaminD 3 J. L. Omdahl

183

Circulating l a , 2 5 - ( O H ) 2 D During Physiological States of Calcium Stress J. Wes Pike, S. Toverud, A. Boass, T. McCain, M. R. Haussler

187

Effect of Glucocorticoids on Vitamin D-Metabolism E. Spanos, I. Maclntyre

191

The Effects of Hypophysectomy on 25-Hydroxyvitamin D 3 Metabolism in the Rat. E. M. Spencer, O. Tobiassen

197

Regulation of the Metabolism of Vitamin D 3 and 25-Hydroxyvitamin D 3 T. Suda, N. Horiuchi, M. Fukushima, Y. Nishii, E. Ogata

201

The Effect of Anticonvulsants on Rat Liver Calciferol 25-hydroxylase S. Sulimovici, M. S. Roginsky

211

Sex Hormonal Control of the Renal Vitamin D Hydroxylases Y. Tanaka, L. Castillo, H. F. DeLuca

215

The Subcellular Localization of 1,25-Dihydroxyvitamin D 3 in the Parathyroid Glands of Chickens W. R. Wecksler, H. L. Henry, W. Norman

219

Vitamin D-Intestinal Action The Dissociation of l , 2 5 ( O H ) 2 D 3 - i n d u c e d CaBPProduction and Alkaline Phosphatase Activity from Calcium Transport by Actinomycin D and Cycloheximide D. D. Bikle, D. T. Zolock, R. L. Morrissey, R. H. Herman

223

Regulation of the Messenger RNA for Calcium Binding Protein by 1,25Dihydroxycholecalciferol A.Charles, J. Martial, D. Zolock, R. Morrissey, D.Bickle, J. Baxter

,227

Cyclic AMP Regulation of the la,25-(OH) 2 D 3 -Mediated Intestinal Calcium Absorptive Mechanism R. A. Corradino

231

A Modified Procedure for the Isolation of Chick Intestinal Calcium Binding Protein E. J. Friedlander, A. W. Norman

241

X Chemical Studies on Bovine Intestinal Calcium Binding Protein and Related Peptides Minor A and Minor B C. S. Fullmer, R. H. Wasserman, D. V . Cohn, J . W. Hamilton

245

The Effect o f Vitamin D Activity on Rat Renal and Intestinal Calcium Binding Proteins. J . E. Harrison, A. J . W. Hitchman, G. B. Gordon, S. A. Hasany

253

Mechanism o f Action o f la,25-Dihydroxyvitamin D 3 at the Intestine and Parathyroids M. R . Hughes, M. R . Haussler

257

1,25-Dihydroxyvitamin D 2 : Characterization, Receptor Affinity and Biological Activity in the Chick P. G. Jones, M. R . Hughes, M. R . Haussler

261

Recent studies on l , 2 5 - ( O H ) 2 D 3 Action in the Intestine E. Lawson, R . Spencer, M. Charman, P. Wilson

265

The Role o f Phosphate in the Intestinal Response to Vitamin D R. Miller, R. Clancy, S. J . Birge

277

Distribution o f Alkaline Phosphatase and Ca-ATPase in Intestinal Epithelial Cell Plasma Membranes: Differential Response to l , 2 5 - ( O H ) 2 D 3 A. K. Mircheff, M. W. Walling, C. H. van Os, E. M. Wright

281

The Relation between the Induction o f Alkaline Phosphatase and 1,25-Dihydroxycholecalciferol Receptor in Chick Embryonic Duodenum S. Moriuchi, S. Yoshizawa, F. Shimura, T. Oku, N. Hosoya

285

Effects o f Dietary Calcium and Phosphorus on the Steady State Levels of some Components o f the Vitamin D Endocrine System E. J . Friedlander, H. L. Henry, A. W. Norman

289

Binding Components for Vitamin D 3 Metabolites in Rat Intestine F . Shimura, S. Moriuchi, N. Hosoya

299

Light and Electron Microscopic Immunoperoxidase Localization of Chick Intestinal Vitamin D-Induced Calcium-Binding Protein A. N. Taylor, J . E. Mcintosh

303

The Effect o f DDT on Vitamin D Metabolism and Calcium Binding Activity in the Chick J . Silver, Z. Alpern

313

Cytoplasmic Binding and Nuclear Uptake of Cholecalciferol Metabolites Inside Rat Duodenal Mucosa Cells A. Ulmann, M. Bachelet, J . F . Cloix, M. Brami, J . L. Funck-Brentano

315

Are the Cytosolic Binding Proteins for 25-Hydroxycholecalciferol Artifacts? H. van Baelen, R. Bouillon

319

XI

Effects of la,25-Dihydroxyvitamin D 3 on Active Intestinal Inorganic Phosphate Absorption M.W. Walling

321

Temporal Patterns of Response of the Intestinal Calcium Absorptive System and Related Parameters to 1,25-Dihydroxycholecalciferol R. H. Wasserman, R. A. Corradino, J. Feher, H. J. Armbrecht

331

Properties of Calcium and Magnesium Stimulated ATP'ase from Human Placenta J. Whitsett, L. I. Kleinman, R. C. Tsang

341

The Effect of l,25(OH) 2 D 3 on Calcium Accumulation, Calcium Transport and Calcium Binding Protein in the Presence and Absence of Cycloheximide D. T. Zolock, R. L. Morrissey, D. D. Bikle

345

Vitamin D-Skeletal Action Effects of Vitamin D and its Metabolites on Bone J. L. Ivey, E. R. Morey, C.-C. Liu, J. I. Rader, D. J. Baylink

349

Bone Cells as well as Bone Remodelling Surfaces in Renal Bone Disorders and Their Changes after Therapy — A Quantitative Analysis G.R.Dellin g

359

3

Localization of H-la,25-Dihydroxycholecalciferol in Rat Bone and Cartilage M. J. Favus, F. H. Wezeman

369

Vitamin D Dependent Bone Disease: Long-Term Responses to Vitamin D Analogs and Effect on Tetracycline Based Bone Dynamics B. Frame, A.M. Parfitt

373

The Dose-reponse Relationship of Tetracycline to the Detectability of Labeled Osteons by Fluorescence Microscopy R. S. Hattner, L.P. Ilnicki, H. C. Hodge

377

Ultrastructural Studies of Bone - Influence of Vitamin D, PTH and Uremia on Bone Cell Ultrastructure and Bone Cell/Bone Matrix Interaction B. Krempien, G. Friedrich, G. Geiger, E. Ritz

381

Multiple Isotope Indicator Studies of Bone Volume Changes in Rachitic and D Treated Rats J. W. K. Lien, M. Kaye

391

Evaluation of a New Method for Studying Resorption by Isolated Bone Cells R. A. Lüben, M. A. Möhler, D. Rosen

395

The Effect of la-Hydroxycholecalciferol on Calcium Balance and Bone Histology in a Randomized Trial B. Lund, R. B. Andersen, M. S. Christensen, T. Friis, L. Hjorth, F. S. J^rgensen, F. Meisen, L. Mosekilde, O. H. S t e n s e n

399

The Effect of Vitamin D Steroles on Bone Histology in Incipient Renal Failure H. H. Malluche, E. Ritz, E. Werner, W. A. Meyer

407

XII

Action of Vitamin D Metabolites and Analogues, Parathyroid Hormone, Calcitonin and Steroid Sex Hormones on Bone in Tissue Culture M. Peacock, G. A. Taylor, A. W. Norman

411

Acid Phosphatase in Bone Cells from Vitamin D Deficient Rats S. Silverton, M. Kaye

415

Vitamin D — Renal Action The Effect of Vitamin D and its Metabolites on the Renal Handling of Phosphate J.-P. Bonjour, H. Fleisch

419

Renal Handling of Calcium in Rats: Influence of Parathyroid Hormone and 1,25-Dihydroxyvitamin D 3 K. Hugi, C. Preston, H. Fleisch, J.-P. Bonjour

433

Adaptive Mechanisms to Phosphate Depletion: The Role of Vitamin D N. Brautbar, M. W. Walling, J. W. Coburn

437

The Effect of Different Solanum Malacoxylon Extracts on Urinary Calcium Excretion in Vitamin D Deficient Rats D. Kraft, D. v. Herrath, K. Schaefer, H. Wagner, E. Ott

441

Quantitative Effect and Pathogenic Mechanism of la-HydroxyCholecalciferol on the Renal Handling of Phosphate S. Madsen, K. 01gaard

443

Failure of Intravenous Calcium to Restore PTH-Induced Phosphaturia in Pseudohypoparathyroidism Type II. A Case Report P. O. Schwüle, D. Vollmar, D. Scholz, H. Schmidt-Gayk

447

Effects of Vitamin D on Renal Tubular Calcium Transport R. A. L. Sutton, C. A. Harris, N. L. M. Wong, J. H. Dirks

451

Vitamin D-Metabolite Assays Assay of Vitamin D 3 (Cholecalciferol) in Plasma Using Gas Chromatography/ Selected Ion Monitoring (GC/SIM) A. A. M. Cruyl, A. P. De Leenheer

455

Development of a Radioimmunoassay for Vitamin D A. Fairney, C. Turner, E. G. Baggiolini, M. R. Uskokovic

459

Radioimmunoassay of Human Serum DBP, and Competitive Binding Protein Radioassay of 24, 25-(OH) 2 D J. G. Haddad, Jr., C. Min, J. Walgate

463

Radioligand Receptor Assay for 1,25-Dihydroxyvitamin D: Biochemical, Physiologic and Clinical Applications M. R. Haussler, M. R. Hughes, J. W. Pike, T. A. McCain

473

XIII

Evolution of Vitamin D Serum Transport Proteins A. W. M. Hay, G. Watson

483

Application of High Pressure Liquid Chromatography for Assay of Vitamin D Metabolites G. Jones

491

Intestinal Cytosol Binders for 1,25-Dihydroxyvitamin D 3 : Use in a Competitive Binding Protein Assay B. E. Kream, J. A. Eisman, H. F. DeLuca

501

Some Problems Associated with the Assay of 25-Hydroxycholecalciferol in Human Serum R. S. Mason, S. Posen

511

Influence of Extraction on Saturation Analysis of 25-Hydroxyvitamin D (25-OH-D) without Prior Chromatography W. Nagel, H. Schmidt-Gayk, M. Zeisner, I. Martiskainen, P. Förster

515

An Assay for 24, 25 and 25, 26 Dihydroxycholecalciferols in sera J. L. H. O'Riordan, R. F. Graham, E. Dolev

519

A "25-Hydroxycholecalciferol-Like Compound" in Milk as Determined by Specific Competitive Binding Radioassays T. W. Osborn, A. W. Norman

523

Specific binding of 25-Hydroxycholecalciferol in Human Mammary cancer M. S. Roginsky, A. Melber

527

Bone Organ Culture Bioassay for Determination of l,25-(OH) 2 D P. H. Stern, T. E. Phillips, S. V. Lucas, A. J. Hamstra, H. F. DeLuca, N.H.Bell

531

The Measurement of 24, 25-Dihydroxycholecalciferol in Human Serum C.M.Taylo r

541

Plasma 25 (OH) Vitamin D levels in Normal Subjects throughout the year and in Patients with Related Diseases C. Velentzas, D. G. Oreopoulos, L. Brandes, D.R. Wilson, W. C. Sturtridge

545

Vitamin D-Nutritional Aspects Cholecalciferol Production P.C.Beadle

549

Biological Activity Evaluation of Chemically Synthesized Vitamin D Metabolites and Analogs A. Boris, J. F. Hurley, T. Trmal

553

Dose-Response of 1,25-Dihydroxycholecalciferol on Serum Calcium and Phosphorus, Urine Electrolytes and Hydroxyproline Excretion in Dairy Cattle C. C. Capen, G. F. Hoffsis, A. W.Norman

-565

XIV

Biochemical Response to 1,25 (OH) 2 Vitamin D Supplementation in Premature Infants: A Prospective Study G. M. Chan, R. C. Tsang, I-Wen Chen, J. J. Steichen, H. F. DeLuca

569

Effects of Vitamin D on Tissue Exchangeable Zinc A. B. Chausmer

573

Parturient Paresis Prophylaxis with 25-Hydroxycholecalciferol F. R. Frank, M. L. Ogilvie, K. T. Koshy, T. J. Kakuk, N. A. Jorgensen

577

Serum and Renal Histologic Changes in the Rat Following Adminstation of Toxic Amounts of 1,25-Dihydroxyvitamin D 3 D. L. Hartenbower, T. M. Stanley, J. W. Coburn, A. W. Norman

587

Elevated 1,25-Dihydroxyvitamin D Levels in Plasma of Dairy Cows in Response to Parturient Hypocalcemia R. L. Horst, J. A. Eisman, B. A. Barton, N. A. Jorgensen, H. F. DeLuca

591

Environmental and Nutritional Influences on Plasma 25-Hydroxyvitamin D Concentration and Calcium Metabolism in Man R. Neer, M. Clark, V. Friedman, R. Belsey, M. Sweeney, J. Buonchristiani, J. Potts, Jr

595

Individual Differences in Susceptibility to Vitamin D-Induced Cardiovascular and Renal Damage: Need to Identify and Characterize its Toxic Metabolites M. S. Seelig, R. G. Mazlen

607

Calcium Absorption and Calcium Balances in Man During Vitamin D Intake H. Spencer, L. Kramer, C. Gatza, D. Osis

611

Potency of Natural and Artificial Ultra-Violet Irradiation as Determinants of Plasma 25-Hydroxyvitamin D in Man T. C. B. Stamp, J. G. Haddad Jr., C. A. Twigg

615

Biological Activity of 24R,25 and 24S,25-Dihydroxycholecalciferol (24R, 25 and 24S,25D, OH 24«-(OH)-Dj H 245-{OH)-D1 H 25-(OH)-D, H la,24/i-(OH)j-D, OH la,24S-(OH),-Dj OH la^S^OH^-Dj OH 24/?,25-(OH)j-Dj H 24S,25-(OH)j-DJ H la,24/?,25-(OH)i-Dj OH 1 a,24S,25-(OH)j-Dj OH

Rj H H OH H H OH H H OH H OH H

Rj H H H OH H H OH H H OH H OH

R. H H H H OH H H OH OH OH OH OH

* Abbreviation used : 24-OH-D3, 24-hydroxyvitamin D 3 ; 24, 25-(OH)2-D3, 24,25-dihydroxyvitamin D 3 ; 1, 24-(OH) 2 ~D 3 , let,24dihydroxyvitamin D 3 ; 1,24,25-(OH)3-D3» la , 24,25-trihydroxyvitamin D3.

6 (1) to determine the absolute configuration at C-24 of biologically produced 24,25- (OH) 2 ~ D 2

and

1

'

2 4

'

2 5

~3

- D

3'

(2) to examine the discrimination in biological activity and metabolic features between 24R and 24S epimers, (3) to have an insight into biological significance of 24hydroxylation in vitamin D^ metabolism and its regulation. It is essential for this project to resolve C-24 epimers at some stage of synthesis and to determine their configuration at C-24.

Those intermediates whose configuration at C-24 was

definitely determined, were converted to vitamin D^ form. We have also been engaged in separating C-24 epimers of vitamin D^ analogs or their appropriate derivatives by chromatographic method. SYNTHESIS Our starting compound was fucosterol (_1) which is widely distributed and almost sole sterol in brown algae.

By simple

procedures fucosterol was converted to 24-oxocholesterol (2^) or desmosterol (3),

from which all of vitamin D^ analogs were

prepared*.*

24,25-(OH) 2 ~P 3 and 1,24,25-(OH) 3 ~P 3 Oxidation of desmosterol benzoate either with m-chloroperbenzoic acid or OsO^ gave C-24 epimeric mixtures of the 24,25-epoxide 4_ and the 24,25-diol

respectivety.

Resolut-

ion of the epoxide was achieved as such by column chromatography on silica gel, while the glycol 1_ was resolved as their 3,24,25-tribenzoate silyl (TMS) ether

or 3,24-dibenzoate 25-trimethyl-

Configurations at C-24 of the 24,25-

epoxides 4a and 4b were determined by acid catalyzed methanolysis followed by application of modified Horeau's (15)

7 24R-OH-D

t' OH

3

Ì A

24R,25-(OH) 2 -D 3

OH

X ^ O M e

T

5a

St

OBz

OBz

i,ii

iii,iv,v

Y ^ C OBz St 6a

4a

mp 173-174 less polar *

mp 164-165' more polar

4b mp 150-152» less polar

CHj

CH 3

A1

CHJ,C»H« =

H

3vX Rol 1

JJ

CHj^X R(/I 1

B1

1

R=CH3orC2Hs

CH3

CH3/C»H1?

32

X/HJ J'" 0R

JL,

,C«H"

CH3?8H1' V s

C H 3

CH3/C8h17 J

CH^»"" V s

^

Y]

D1

83

E1

C2

Tfi

H

CHj-yik

CH 3

C1

A3

Jh

C H

»•••f/à

CH 3

CH3.C8H17

TH

CH 3 ,C8H17

CH 3 C 8 H 1 7

(TXy

CHJ A2

CH3/C8Hl7

CH3/C«H"

D2

R1

D3

Fig. 1. Overirradiation products from previtamin D/tachysterol : Toxisterols A1

A2

A3

B1

B2

B3

CI

C2

D1

EtOH

2

5

at the t a r g e t tissue

in

This

straight-phase and

1,25-(OH)2D^•

(0.125 ml) c o l l e c t e d s h o w e d that

precisely coincided with authentic This d e m o n s t r a t e d the p r e s e n c e of

(intestine)

tracer

1,25-(OH)2D

in v i t a m i n D - d e f i c i e n t rats w h e n

d o s i n g w a s p r o d u c i n g its c a l c e m i c

the

S.g.

effects. 3

T h e s e r u m e x t r a c t s w e r e e x t r a c t e d , t r e a t e d w i t h tracer 1 , 2 5 - ( O H ) 2 ~ [ 2 3 , 2 4 D 3 , and c h r o m a t o g r a p h e d o n S e p h a d e x L H - 2 0 as d e s c r i b e d e l s e w h e r e

(5, 6).

(Skellysolve B / C H C l 3 / M e 0 H ,

T h e c o m b i n e d 1,25-(0H),,D 2 and

HJ-

9:1:1)

l,25-(OH)2D3

f r a c t i o n s w e r e c h r o m a t o g r a p h e d o n the s e m i - p r e p a r a t i v e H P L C s y s t e m as w e r e the i n t e s t i n a l e x t r a c t s . no 1 , 2 5 - ( 0 H ) 2 D 2

R a d i o r e c e p t o r a s s a y of the f r a c t i o n s

in d o s e d or c o n t r o l a n i m a l s .

H o w e v e r , s e r u m f r o m S.g.

d o s e d a n i m a l s c o n t a i n e d 300 p g / m l of 1 , 2 5 - ( 0 H ) 2 D 3 t i o n of 20 p g / m l i n the c o n t r o l

demonstrated

c o m p a r e d to a

concentra-

animals.

T h e p r e c i s i o n a n d r e s o l v i n g p o w e r of the c h r o m a t o g r a p h y c o n s i d e r e d w i t h s e n s i t i v i t y a n d s p e c i f i c i t y of the r a d i o r e c e p t o r a s s a y leave no d o u b t is a s o u r c e of 1 , 2 5 - ( O H ) 2 D 3 .

S i n c e the a c t i v e p r i n c i p l e as

f r o m S_.j>. is w a t e r s o l u b l e , l , 2 5 - ( O H ) 2 D

the

that

extracted

in the a n i m a l m u s t r e s u l t

from

m e t a b o l i s m of a h y d r o p h y l i c c o n j u g a t e of this h o r m o n e p r e s e n t in the p l a n t .

31 In addition, no results to date rule out the presence of a la-hydroxyvitamin D^ conjugate in the plant.

Nevertheless, calcemic disease subsequent to

S^j*. ingestion must result from high tissue concentrations of 1,25-(OH) ^D.^. It is unlikely that

extracts will replace synthetic l ^ S - i O H ^ D ^

therapeutically, however. REFERENCES 1.

Wasserman, R. H.

2.

Holick, M. F., Schnoes, H. K., DeLuca, H. F., Suda, T., and Cousins, R. J.

(1971)

(1975)

Nutr. Rev. 33, 1-5.

Biochemistry 10, 2799-2804.

3.

Holick, M. F., and DeLuca, H. F.

(1971)

4.

Jones, G., and DeLuca, H. F.

5.

Eisman, J. A., Hamstra, A. H., Kream, B. E., and DeLuca, H. F.

(1975)

J. Lipid Res. 12, 460-465.

J. Lipid Res. 16, 448-453. (1976)

Science 193, 1021-1023. 6.

Eisman, J. A., Hamstra, A. H., Kream, B. E., and DeLuca, H. F.

(1976)

Arch. Biochem. Biophys. 176, 235-243. 7.

The radioreceptor assay is equally sensitive to l,25-(OH)2D2 or l,25-(OH) 2 D 3 -

Besides 1,24(R),25-(OH) 3 D, which is about 1/3 as active

as l,25-(OH)2D, the assay is insensitive to all other known D metabolites with 1,000 times or greater molar excesses necessary to reduce 3 specific l,25-(OH) 2 -[ HjD^ binding to the protein. Eisman, J. A., Kream, B. E., and DeLuca, H. F., submitted for publication. ACKNOWLEDGEMENT Supported by program-project grant AM-14881 from the National Institutes of Health and Contract EY-76-S-02-1668 from the U. S. Energy Research and Development Administration.

SYNTHESIS AND CONFORMATIONAL ANALYSIS OF VITAMIN D METABOLITES AND ANALOGS

W i l l i a m H.

Okamura, M i l t o n

and A n t o n i o

of

California

92502,

1A f o r

lites)

is

the

of

biologically

the pathway i n much t h e

hormones

exemplified

haustive

reviews

order

evaluate

to

University

structures

This metabolite

function

Condran,

Jr.

of

California,

Riverside,

USA

the most

D3 t h r o u g h to

Chemistry,

la,25-dihydroxyvitamin

Figure known.

Patrick

Mourino

Department

The s t e r o i d

L . Hammond,

is

active

by t h o s e

have appeared the

given

D^ a n d

its

form of 2 and

the

on t h e s e structural

metabo-

it

from is

classical

in F i g u r e

(see

calciferol

biologically

in F i g u r e

same way a s

further

[la,25-(OH)2D3]

vitamin

produced

shown

D3

3.

subjects

vitamin

believed steroid Several

(1-3) .

requirements

exIn

neces-

FIGURE 1 ( f o l l o w i n g p a g e ) . STRUCTURES OF VITAMIN D3, ITS METABOLITES AND ANALOGS. A b b r e v i a t i o n s : v i t a m i n D3 [ D3] ; 2 5 - h y d r o x y v i t a m i n D3 [25-OHD3]; l a , 2 5 - d i h y d r o x y v i t a m i n D3 [ l a , 2 5 - ( O H ) 2 D 3 ] ; 2 4 R , 2 5 - d i h y d r o x y v i t a m i n D3 [24R, 25-(OH) 2 D 3 ]; 2 5 ^ , 2 6 - d i h y d r o x y v i t a m i n D3 [ 2 5 £ , 2 6 - ( O H ) 2 D 3 ] ; l a , 2 4 R , 2 5 - t r i h y d r o x y v i t a m i n D3 [la,24R,25-(OH)3D 3 ]; l a - h y d r o x y v i t a m i n D3 [ i a - 0 H D 3 ] ; 3 - d e o x y - i a - h y d r o x y v i t a m i n D3 [ 3-d-ia-OHD3]; 3 - d e o x y - l a , 2 5 - d i h y d r o x y v i t a m i n D3 [ 3 - d - i a , 2 5 - ( O H ) 2 ° 3 J > 3 - d e o x y - 3 a - m e t h y l - i a - h y d r o x y v i t a m i n D3 [ 3 - d - 3 a - M e - i a OHD3]; 3 - d e o x y - 3 3 - m e t h y l - l a - h y d r o x y v i t a m i n D3 [3-d-33-Me-ia-OHD 3 ]; 3 - d e o x y 3 , 3 - d ime t h y 1 - l a -hyd r o x y v i t amln D3 [ 3 - d - 3 , 3 - ( M e ) 2 - l a - O H D 3 ] ; l a - h y d r o x y - e p i v i t a m i n D3 [ l a - 0 H - e p i - D 3 ] ; 2 0 , 2 1 , 2 2 , 2 3 , 2 4 , 2 5 , 2 6 , 2 7 - o c t a n o r v i t a m i n D3 [ o c t a n o r - D 3 ] ; v i t a m i n D 2 [D 2 ]; 1 0 S , 1 9 - d i h y d r o v i t a m i n D3 [DHV3-II or t h e lOS-a i s o m e r ] ; 1 0 R , 1 9 - d i h y d r o v i t a m i n D3 [DHV3-III or t h e lOR-a i s o m e r ] ; 19h y d r o x y - l O S , 1 9 - d i h y d r o v i t a m i n D3 [19-OHDHV 3 -Il]; 1 9 - h y d r o x y - 1 0 R , 1 9 - d i h y d r o v i t a m i n D3 [I9-OHDHV3-III]; 2 4 S , 2 5 - d i h y d r o x y v i t a m i n D3 [24S,25-(OH) 2 D 3 ]; 24-homo-25-hydroxyvitamin D3 [24-homo-25-OHD3]; 2 4 - n o r - 2 5 - h y d r o x y v i t a m i n D3 [24-nor-25-OHD 3 ]; 2 3 , 2 4 - d i n o r - 2 5 - h y d r o x y v i t a m i n D3 [ 2 3 , 2 4 - d i n o r - 2 5 - O H D 3 ] ; 2 2 , 2 3 , 2 4 - t r i n o r - 2 5 - h y d r o x y v i t a m i n D3 [ 2 2 , 2 3 - 2 4 - t r i n o r - 2 5 - O H D 3 ] ; 2 0 , 2 1 , 2 2 , 2 3 , 2 4 - p e n t a n o r - 2 5 - h y d r o x y v i t a m i n D3 [ 2 0 , 2 1 , 2 2 , 2 3 , 2 4 - p e n t a n o r - 2 5 - O H D 3 ] ; 5Ev i t a m i n D3 [5E-D3 or 5 , 6 - t r a n s - D 3 ] ; 2 5 - h y d r o x y - 5 E - v i t a m i n D3 [25-OH-5E-D 3 ]; d i h y d r o t a c h y s t e r o l 3 [DHT3 or t h e lOS-b i s o m e r ; a l s o 1 0 S , 1 9 - d i h y d r o - 5 E - D 3 I ; 2 5 - h y d r o x y d i h y d r o t a c h y s t e r o l 3 [25-OHDHT3]; 1 0 R , 1 9 - d i h y d r o - 5 E - v i t a m i n D3 [DHV3-IV, t h e lOR-b isomer o r 1 0 R , 1 9 - d i h y d r o - 5 E - D 3 ] ; 1 9 - h y d r o x y d i h y d r o t a c h y s t e r o l 3 [19-OHDHT3]; 1 9 - h y d r o x y d i h y d r o v i t a m i n D3-IV [I9-OHDHV3-IV]; d i h y d r o t a c h y s t e r o l 2 [DHT 2 ]; d i h y d r o v i t a m i n D 2 -IV [DHV 2 -IV]; i s o v i t a m i n D3 [iso-D3]; i s o t a c h y s t e r o l 3 [ i s o - T 3 l ; t a c h y s t e r o l 3 [T3]•

A. D 3 ANO METABOLITES

D3

B

5Z (5,6-cis)

l9-0HDHV,-ffl

25-OHDJ

la,25-(OH) 2 D 3

24R, 25-(OH) 2 D 3

2 5 i ,26-(OH) 2 D 3

lcl,24R,25-(OH) 3 D 3

ANALOGS

24-nor-

24S.25-(OH),D, 25-OHD3

25-OHO3

23,24-dinor25-OHD3

22,23,24tnnor25-OHD 3

C

5 E ( 5 , 6 - t r a n s ) ANALOGS

25-0H0HT

I9-OHDHV3-IE

DHT2

0HV2-nz:

T

3

3

DHV3-ET

ISO-D3

I9-0HDHT

¡SO-T3

3

35

VITAMIN

D

METABOLISM STEROID

HORMONES

HO H 13/3)

la,25-(0H) 2 -D 3 HORMONALLY ACTIVE FORM

OH

THE I a - O H APPEARS PARTICULARLY CRITICAL BIOLOGICAL ACTIVITY.

Ecdytonc

FOR

Figure 3

Figure 2

sary for optimal or minimal vitamin D activity, we have directed our attention to the synthesis, structural analysis and biological evaluation of analogs of vitamin D^ metabolites.

It is of interest to design and prepare:

a more in-

expensively synthesized mimic of la,25-(OH) ^D^; an analog with more selective biological action than the metabolites; and a substance with anti-metabolite properties (4).

Through

a cooperative effort with several colleagues at the University of California at Riverside, which include the research groups of Professors Richard M. Wing and Anthony W. Norman, we have studied numerous analogs possessing interesting structural and biological properties

(3).

Figures IB and lc depict vari-

ous analogs of the 5Z and 5E series, respectively, which have been synthesized or designed in these and other laboratories. Particularly significant are those analogs possessing la-OH groups in the 5Z series (Figure IB) or pseudo-la-OH groups (OH groups in approximately the same topology as the la-OH of la,25-(OH)in

the 5E series (Figure 1C).

This hydroxyl

36 or its pseudo-equivalent is of unusual importance in imparting significant biological activity to analogs (2,5). Recently our studies on the hydroboration and reduction reactions of vitamin D have led to the isolation and stereochemical characterization of dihydrotachysterol^

(DHT^) and a host

of related 10,19-dihydrovitamin D 3 's (DHV3-II, -III, -IV, their 19-OH counterparts OH forms) (3,6-7).

and more recently some of their 25-

The results obtained for the pseudo-la-OH

analogs DHT^ (lOS-b) and its C 10 -methyl epimer DHV 3 ~IV (lOR-b) are of greatest interest in the context of the ensuing discussion.

It was established for the first time that their con-

figurations are 10S and 10R, respectively (Figure 4).

As we

earlier determined by ^"H-NMR spectroscopy for the natural hormone la,25-(OH) 2 D 3

(7-8), the A-rings of DHT 3 and DHV 3 ~IV are

also partitioned between two chair like conformations 4).

Of the two A-ring chair forms

(Figure

(diequatorial-la-OH/10S-

CH 3 and diaxial-la-OH/10S-CH3), lOS-b (DHT3) exists mainly (~90%) as the diequatorial con former.

For the corresponding

two chair forms (equatorial-la-OH/axial-10R-CH3 and axial-la0H/equatorial-10R-CH3) of lOR-b (DHV3~IV), they are present in nearly equal amounts.

It is emphasized that these conclu-

sions were reached by analysis of ^H-NMR data obtained in solution (deutero-chloroform as solvent) at 24°C.

It is

known that the equilibrium population ratio will vary somewhat depending upon solvent and temperature (9); for cyclohexanol, Anet (10) has shown that in water (polar) versus carbon disulfide (nonpolar), the % equatorial-OH conformation varies between 90% and 75%, respectively.

Boat conformations, or the

more likely twist boat conformations (Figure 5), are also possible for cyclohexane but they are considered higher energy intermediates in the rapid partial rotation between the more stable degenerate chair forms of cyclohexane.

At any given

instant at room temperature, there exists greater than 10,000 chair cyclohexanes for each twist boat form and the various conformational forms are in rapid equilibrium with one an-

37

C ID) 5£ - VITAMIN

f7

D3

(5,6-TRANS-VITAMIN D3) CYCLOHEXANE CONFORMATIONS

i

J J

J

CHAIR I

IN SOLUTION

CHAIR n

>99.99%

95%

*kcal/mole CH, y

H—

HjCOH AGAG 0 AG 0

(CH3) (0H) (I-I)

I

i

- 1.8

-0.8

-2.6 (~99%I)

CH,

I

"OH

OH

•OH

AG° (CHJ) AG» ( O H ) AG" ( I Y - M )

H,C-

N

-1.8

+ 0.8

-1.0 (~85%IY)

75

TABLE I Relationship between Percentage of More Stable Isomer at Equilibrium, Equilibrium Constant K, and Standard Free-energy Difference AG° at 25°C. and 60°C. for an Equilibrium of Isomers: A £ B. % More Stable Isomer

AG

K

AG25 Kcal./mole

60 Kcal./mole

50

1.00

0.00

0.00

60

1.50

0.24

0.27

70

2.33

0.50

0.56

85

5.67

1.03

1.14

2.00

2.20

97

32.0

Once again refering to Figure 2 one notes maxima in the energy profile.

These maxima represent the barriers to con-

version of one equivalent staggered form to another, and the heights of the barriers determine whether the rate of the conversion will be slow (large barrier) or fast (low barrier). There is virtually no barrier at all for the interconversion of five membered rings.

The pathways for interconversion of

six membered rings are very sensitive to the degree of unsaturation.

Two common cases are shown in figures 4 and 5

which illustrate that as six membered rings are flattened (eg by inclusion of unsaturation) interconversion between forms becomes more facile.

Generally speaking the rates of

conformational interconversion for five and six membered 4 -1 rings are extremely rapid (> 10 sec ) at room temperature. Therefore a change in environment may in many instances result in an instantaneous change in conformational populations for the more flexible molecules. Molecules which undergo rapid structural interconversions of any type are called fluxional.

An important concept in

dealing with fluxional molecules is limiting structure. limiting structures we mean the instantaneous structures which are involved in the dynamic equilibrium process, _ie

By

76 Figure 4: Interconversion pathways for cyclohexane. Two pathways are available, one which transverses mirror symmetry (C5) forms and another which transverses twofold (C2) forms (Figure 7) .

half c h a i r

twistboat

Figure 5: Interconversion pathway for cyclohexene.

23

fluxional process.

For example, the limiting structures for

cyclohexane are the two chair forms given in Figure 3.

Flux-

ional molecules have properties which are a weighted average of the properties of the limiting structures. All analytical methods for determining the characteristics of fluxional molecules require knowledge of some observable property of the various limiting structures.

Computations of

the best average over the contributing limiting structures are then used to predict the structural composition (12). Limiting structures are often simulated by using substituents

77 which produce a large conformational bias vide supra since many molecules remain fluxional at the lowest temperatures compatible with experiment. can be quenched

If the interconversion process

(eg by lowering the temperature) one can de-

rive very detailed information concerning the interconversion pathway from a variable temperature study of the system. In view of the fact that a) small changes in AG0 produce shifts in population that become more amplified as the initial populations approach equality (Table I), and b) that the strengths of binding of steroids to macromolecules

(eg recep-

tor protein) may be 5-6 Kcal (or several multiples of this value if several hydrogen bonds are formed) one must be aware of the possibility that steroid conformations may adapt to the requirements of the macromolecule.

Thus while correlation of

steroid conformation with biological activity serves as a powerful guide to the synthesis of analogues, complete understanding of the topological requirements of steroid hormones will only come from structural studies of the integrated systems.

Nuclear Magnetic Resonance (13) Generally individual torsion angles can be estimated from the magnitudes of the spectral splittings (14) (Spin-Spin coupling constants) or other spectral parameters, but it is unusual indeed to be able to measure all of the angles required to rigorously define the structure.

Thus the total structure

must be inferred from a few structural parameters.

The

accuracy of the torsion angles measured is perhaps a factor of three less than those from x-ray structures (ie ±3 degrees as against ±1 degree).

The measurements can, however, be

made under a wide variety of conditions, including those in which the steroid is absorbed on a protein receptor.

78 When several conformers are in rapid equilibrium with one another an accurate measurement of their relative concentrations can be made from the time averaged spectrum of the mixture if spectral parameters for the limiting structures can be determined or are known (15). If the fluxional process is -1 4 slow (< 10 sec ) each conformer will contribute its own spectrum and the structures can be accessed as above, with the relative concentrations coming from a direct measurement of signal intensities. Thus one can observe conformational changes and, except for the rare case in which the number of conformations exceed the number of observations, define the conformational populations in some detail.

An example analysis based on vicinal coupling

constants is given for vitamin D, in Figure 6.

Figure 6: Analysis of the low field resonances of vitamin D 0

HO (a)

|/3

57%

43% 3j3a

4/3

=

7.6 Hz

Jaa ~ II Hz Jee ~ 3 Hz

(observed)

79 Molecular Mechanics (16) The concept of partitioning the energy of a molecule among various individual structural features (limiting structures) leads naturally to formulation of numerical methods for the calculation of the optimal structure of a conformer by simultaneous minimization of those contributions.

This approach is

empirical and therefore gives acceptable results only when suitably calibrated with model compounds of the same type for which one wishes to make predictions.

Steroids, fortunately,

constitute a class which is very well calibrated.

This ap-

proach perhaps has its most useful application in predicting structures and conformational populations for new yet-to-besynthesized putative analogues.

The results (17) for 5,6

trans D^ are typical of this approach (Figure 7). CRITICAL ANALYSIS OF CONFORMATIONAL RESULTS Structural results for vitamin D are available from four independent crystal structure analyses, Hodgkin (2), Knobler (3), Woolfson (6), and Dahl (7).

These have been compared with

each other as well as with the molecular mechanics structure determined by Rego (17) using a least squares molecular superposition technique (18).

It is quite apparent (Table II)

that the molecular mechanics derived structure gives a very adequate representation of vitamin D conformation. TABLE II Comparison of vitamin D structures A)

3 3 .OH equatorial H

H

W

17 a

3.8

5.3

6.2

8.0

D

R 14

23

.20

.30 .14

3.8

80 B)

3 3 OH axial

g

K

W

D

R

K

—

.63

.27

.29

W

17.3

—

.44

.43 .31

D

7.5 11.9

—

R

7.9 11.5

8.3

The upper right triangle gives the root mean square deviations between atoms in A. ^The lower left triangle gives residuals R = 100 vE AX "IT"

—

Although there is much better agreement between the 36 OH equatorial A ring conformers than for the cases with 3S OH axial, generally the comparisons between structures are very unsatisfactory.

Furthermore, correspondence between

(except for the rigid C rings)

subunits

is also less than might be ex-

pected based on the quality of the structures.

Clearly

crystal packing forces easily distort the flexible regions of these fluxional molecules. Conformational equilibria

for the vitamin D's have been mea-

sured by several NMR methods and are compared to each other as well as molecular mechanics estimates

in Table III.

Table III Summary of A ring Conformational J . VIC

LIS

57 (4) 3

Equilibria

c

MM

i A values

57(2.)

56

72

80

69(5)

76(3)

51

78

80

DHT

88 (6)

89 (2)

81

99

100

DHV IV

50(5)

42(6)

53

—

DHV II

0(6)

6(3)

7

0

100

92 (3) 100

100

15

Molecules/Methods D

3 5,6t-D 3

DHV III

94 (6)

13

15

a

V a l u e s in the parens represent the standard deviation last significant figure.

The molecular mechanics calculation does a less job of predicting equilibria than structures, the trends are reproduced culations

satisfactory

none-the-less

sufficiently well to use these cal-

in a predictive fashion.

free energy approach

in the

In contrast the additive

(A values) illustrated

very badly for this series of molecules.

in Figure 2 fails

81

e:a 3ß-OH ratio of

71:29.

82 SUMMARY Experimental and computational methods exist for rapidly and accurately determining the conformations and conformational equilibria of the vitamin D's.

Results are available for a

large selection of analogues, now new analogues are being characterized routinely, and we are capable at this time of predicting structural characteristics before synthesis. Structure-function correlations are being established and refined as new bioassay data become available. The spectral characteristics of the vitamin D's especially 13 the C resonances will ultimately be used to study the interaction of vitamin D with receptor and carrier proteins. REFERENCES 1.

Fieser, L. F. and Fieser, M. , "Steroids," Reinhold, New York, N.Y., Chapter 4 (1959).

2.

Hodgkin, D. C., Rimmer, B. M., Dunitz, J. D., and Trueblood, K. N., J. Chem. Soc. 4945-4955 (1963).

3.

Knobler, C., Romers, C., Braum, P. B., and Hornstra, J., Acta. Crystallogr. B28, 2097-2103 (1972).

4.

Wing, R. M., Okamura, W. H., Rego, A., Pirio, M. R., and Norman, A. W., J. Amer. Chem. Soc. 97, 4980-4985 (1975).

5.

LaMar, G. N. and Budd, D. L., J. Amer. Chem. Soc. 96, 7317-7324 (1974).

6.

Hull, S. E., Leban, I., Main, P., White, P. S., and Woolfson, M. M., Acta. Crystallogr. in press (1976).

7.

Trinh-Toan, Deluca, H. F., and Dahl, L. F., J. Org. Chem. in press (1976).

8.

Bucort, R., "Topics in Stereochemistry" _ed. Eliei, E. L. and Allinger, N. L., Interscience, John Wiley, New York, N.Y. 8, 159-224 (1974).

9.

Altona, C., Geise, H. J. and Romers, C., Tetrahedron, 24, 13-32 (1968).

83 10.

Duax, W. L., Weeks, C. M. and Rohrer, D. C., Topics in Stereochemistry, 9, 271-383, Allinger and Eliel, Editors (1976).

11.

Eliel, E. L., Allinger, N. L., Angyal, S. J., and Morrison, G. A., "Conformational Analysis," Interscience, New York, N.Y., pp 44, 436-442 (1965).

12.

In the case of only two limiting forms, the equilibrium K = (P]_-P) / (P-P2) requires the knowledge of P]^ and P2 based on measurements on conformers 1 and 2 as well as the observed value of the property, P, for the mixture.

13.

Jackman, L. M. and Sternhell, S., Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry, Pergamon Press, London (1969).

14.

The magnitude of splitting of a proton resonance via coupling to another proton separated by 3 bonds is related to the torsion angle about the intermediate bond is related to the torsion angle about the intermediate bond (Figure 6). The functional dependence of the splitting is known as the Karplus Rule. Examples of the use of the Karplus Rule are given in Reference 13, pp 281298.

15.

Garbish, Jr., E. W., Hawkins, B. L. and Mackay, K. D., in "Conformational Analysis: Scope and Present Limitations" ed. Chiurdoglu, G., Academic Press, New York, N.Y., pp 93-109 (1971).

16.

Engler, E. M., Androse, V. D., and Schleyer, P. von R., J. Amer. Chem. Soc. _95 , 8005-8025 (1973).

17.

Rego, A., private communication of unpublished (1975) .

18.

Nyberg, S. C., Acta Cryst. B30, 251-252

results

(1974).

VITAMIN D-LIKE ACTION OF A STEROID FROM TRISETUM FLAVESCENS

H. Zucker, 0. Kreutzberg and Vi.A. Rambeck Institut für Physiologie, Physiologische Chemie und Ernährungsphysiologie, Fachbereich Tiermedizin, Universität München, Veterinärstraße 13, D-8000 M ü n c h e n 22 Trisetum flavescens, a common meadow and pasture grass, was identified by Dirksen et al. in 1972

(1) as the cause of a

chronic calcification disease occurring in cattle in Upper Bavaria. Similar symptoms were produced in sheep and rabbits fed Trisetum flavescens

(2). The same phenomena were

reported

from South- and Northamerica where the calcinosis is associated with the ingestion of Solanum malacoxylon

(3) and Cestrum

diurnum. For S. malacoxylon it has b e e n shown that the water soluble calcinogenic factor is a 1,25-(OH^2^3-glycoside

(4).

The present studies were designed to concentrate, isolate and study the unknown calcinogenic compound of Trisetum

flaves-

cens. A new bioassay procedure was developed for this purpose. Growing rats were fed a low calcium diet free of vitamin D for periods of 3 weeks, then changed to a low phosphorus, high calcium diet ( C a / P ~ 2 0 ) . This change produces an immediate fall in serum phosphate from 8 to 9 mg/100 ml to about 3 mg/100 ml. V i t a m i n D or the various test materials were fed or given by stomach tube at the time of the dietary change. Serum phosphate is determined on the 3rd day after administration of the test materials. There is a rise in serum phosphate, which is closely related to the amount of vitamin D or vitamin D-like activity

(Table 1).

To isolate and purify the active principle (see Fig. 1) 15 g of ground lyophilized Trisetum flavescens were extracted in

86 diethyl ether. The supernatant was saponified and the non saponifiable part was applied to a 20x2 cm column packed with 60 g A^O-^. The fraction eluted with 9% diethyl ether in light petroleum contained all the biological activity. Further purification at a preparative scale was accomplished by highpressure liquid chromatography using first a small-particle silica column (p-Porasil). Solvent systems were 5 to 10 % isopropanol in hexane. Peaks detected with a UV monitor and a refractometer were collected and checked for biological activity. The active fraction was brought to dryness, redissolved in acetonitril, filtered, and the filtrate further purified by a reversed phase HPLC column (ju-Bondapak) . Fig. 1.

Isolation and purification of Trisetum factor Lyophilized ground T. flavescens |

Diethyl ether extraction

Unsoluble (discard)

Soluble extract

|

Saponification

Saponifiable

(discard)

Non saponifiable |

A ^ O ^ column chromatography

Active eluate |

HPLC silica column

Active fraction | Unsoluble

1 (discard)

Dissolved in CHjCH

Soluble |

HPLC reversed phase

Purified extract Table 1.

Rise in serum phosphate. % over control

400 I.U.Vit.D 3 /kg feed

461

5 g S.malacoxylon/kg feed 511

90 g T.flavescens/kg feed 451 Purified extract of 90 g T.flavescens/kg feed

34%

This purified extract was cochromatographed with vitamin D^ and its metabolites (see Fig.2).Though the chemical structure

87 is still unknown, the highly purified biologically active material seems not to be identical with l ^ S C O H ^ D ^ . This conclusion is drawn not only from the strikingly different chromatographic behaviour on the two HPLC colums, but also from analysis via direct probe mass spectrometry. The results indicate that the calcinogenic substance in T. flavescens is a highly active vitamin D-like compound not identical with the active principle in S. malacoxylon. Fig. 2. Elution profile of a mixture of vit.D^, its metabolites and of the purified extract of T. flavescens

2)

14 Repletion with vitamin D„ diminishes the amount of C0„ formed after a 14 14 dose of 25-0H-[26,27- C]D 3 but not after a dose of 1,25-(OH) 2 ~[26,27CJD 3 3)

in all rats fed the various dietary levels of calcium or phosphorus in their diets, except rats fed a low calcium, normal phosphorus diet. In this 14 } group of rats, the amount of C0„ 2 formed after a dose of 25-OH-[26,2714C]D 3 was unchanged in the vitamin D-deficient or repleted state. 14 4)

The production of

14 C0 2 from 1,25-(OH) 2 ~[26,27-

C]D 3 occurs in germ

free animals thus showing that the process is not bacterial and occurs in some organ of the animal (34.98% + 1.8% controls vs. 33.76% + 0.43% experimental animals at 24 hr) . 5)

Administration of actinomycin D 3-4 hr prior to dosing the animals with 14 14 1,25-(0H) 2 ~[26,27- C]D 3 reduces the amount of C0 2 formed at the early

time points (8 hr 13.03 + 1.9% dose experimental vs. 17.8 + 1.83% controls). 14 However, by 12 hr the cummulative amount of C0 2 formed in both groups of animals was equal.

This suggests that the enzyme(s) required for this

process are rapidly turned over. 14 6)

The process occurs in chickens but the amount of 14

C0 2 formed after the 14

injection of 25-OH-[26,27- C]D 3 or 1,25-(OH) 2 ~[26,27- C]D 3 is 5% and 12% respectively, obviously less than in rats. The results outlined above demonstrate unequivocally a side chain, oxidation pathway of vitamin D metabolism. in the pathway is 1,25-(OH) 2 D 3 .

It is clear that the immediate precursor 14 The rates of

C0 2 formation are maximal

at the time that 1,25-(0H) 2 D 3 is acting upon the gut and bone.

It is

possible that the pathway may in some way be involved in the mechanism of action of 1,25-(OH) 2 D 3 .

It is equally possible that it is involved in

the degradation of 1,25-(0H) 2 D 3 in either case demonstrating its fundamental importance.

lk9 REFERENCES 1.

D e L u c a , H. F., and S c h n o e s , H. K.

(1976)

Ann. Rev. B i o c h e m . 4J5, 6 3 1 -

666. 2.

F r o l i k , C. A . , a n d D e L u c a , H. F.

(1972)

J. C l i n . I n v e s t . 51,

2900-

(1973)

J. C l i n . I n v e s t . 52,

543-548.

2906. 3.

F r o l i k , C. A . , and D e L u c a , H. F.

4.

S u d a , T . , D e L u c a , H. F., and H a l l i c k , R. B. 43,

5.

Biochem.

G r a y , R. W . , O m d a h l , J. L . , G h a z a r i a n , J. G . , and D e L u c a , H . F.

10,

(1972)

7528-7532.

O m d a h l , J., H o l i c k , M . , S u d a , T., T a n a k a , Y . , and D e L u c a , H . F. Biochemistry

7.

Anal.

139-146.

J. Biol. Chem. 247, 6.

(1971)

(1971)

2935-2940.

S u d a , T., D e L u c a , H. F., a n d T a n a k a , Y.

(1970)

J. N u t r . 100,

1049-

1052. 8.

J e f f a y , H . , and A l v a r e z , J.

(1961)

A n a l . Chem. 33,

612-615.

ACKNOWLEDGEMENT S u p p o r t e d by p r o g r a m - p r o j e c t AM-05331-02

g r a n t A M - 1 4 8 8 1 and a p o s t d o c t o r a l

from the N a t i o n a l I n s t i t u t e s of H e a l t h .

fellowship

CALCIUM TRATION

A N D P A R A T H Y R O I D S T A T U S IN N O R M A L OF 1 , 2 5 - D I H Y D R O X Y - V I T A M I N D 3

MAN

FOLLOWING

ADMINIS-

F. L l a c h, J . W . C o b u r n , A . S . B r i c k m a n , K. K u r o k a w a , A . W . N o r m a n , J . M . C a n t e r b u r y and E. R e i s s D e p t s . of M e d . , VA W a d s w o r t h H o s p . C t r . , C e d a r s - S i n a i M e d . C t r . , U C L A Sch. of M e d . , L o s A n g e l e s , C A , U n i v . of M i a m i , M i a m i , FL and D e p t . of B i o c h e m . , UC R i v e r s i d e , R i v e r s i d e , CA. The by

possibility l,25(OH)

Chertow the

et

D

ments,

al.

found

iPTH

hormone they

from to

serum

iPTH the

found

evaluate and

3 on

s e rum

Ca .

Eight

normal

1.0 ml

Ca,

on of

received

the

ug

of

7 am

and

hourly

to

mental

Days

Urine

slices.

Ca

that

volunteers day

obtained cyclic

values

on

the

obtained

s u b j ec t . RESULTS

at

the

Day)

, ., orally

blood

of

time

was

of

undero n

AMP

and

to

effect

the

level

on

2 successive

subjects

of

of

days:

ingested

second

day

they

Day).

Be-

.

were

iPTH

day the

release

of

Control

at

para-

l^SCOH^D^

the

by

experi-

(Experimental

C a , P and

on

the

study

,^

by v o i d i n g

AMP,

on

both

Ca, P,

experimental same

the

samples

on

raised-

early

a inhibitory

and

where-

been

4 hours

cyclic

studied

vehicle

intervals

of

those

were

3

measurement

be

has

in v i £ r o

of

independent

(Control

2

and

might

l,25(OH)„D„

for m e a s u r e m e n t obtained

P,

there

secretion

'

were

present

urinary

half-hourly

samples

In

inhibited

The

exists

immunoreactive

and

7 pm, venous

for

(1).

l^SCOH^D^

propandiolethanol 2.7

rat

serum

effects

first

6

tween

the

given

short-term

iPTH

male

in

reduced

loop

secretion

the

hypothesis

l,25(OH)2

7 am

that

PTH

l^SiOH^D^

route,

(iPTH)

feed-back

inhibits

that

parathyroid

taken

test

a negative

itself,

intraperitoneal

thyroid

at

that

and

collected and

Experi^

creatinine,

2-hourly

intervals

creatinine. were

compared

control

at

day

for

The with each

152 For

the

entire

rum

Ca,

P or

Ca

of

0.2

giving

to

the

decreased

iPTH.

there

0.4 m g / d l

33

were

However, were

l,25(OH)2D3 by

negative

group

to

_n

50%

correlation

4

in

no

significant

significant observed

p a t i e n t s

these

between

A t

patients

serum

increments

7 to

.

iPTH

CASE

changes

12 h o u r s 7

_10

and

a

in

serum

s e r u m

1 P T H

significant

Ca.

(Figure

1).

Relation

between

rum

and

iPTH

Ca

representative

80

of

the

ing 00

70

60 33 H O. •H S ^ PS Cd in

r -0.84

r

9.5

SERUM 2°3

when

serum At

increment not

circles

sent

values

played

ingestion trol

serum

in

small of

end

the

of

the

iPTH

Experimental

the

4 other

increments

3 of

in

l^SCOH^D^

these

patients;

w

The

values

number

at

are

11

after

in iPTH

and

12

l,25(OH)

rose.

experimental

in

and

in

hours

actually

the

circles

levels

(mg/dl)

iPTH

the

in

change

Ca

Day

ca.

reprein

parentheses

10

in-

closed

Day.

9.0

show-

serum

Open

Control 50

case

significant in

sein a

patients

crement

w

se-

after

h r s >

with

in

serum

ith

n o

day,

there

patients.

however,

iPTH

7 to

changes

was

Serum

3 of

small

Ca

them

10 h o u r s

observed

a

did

dis-

after

on

the

the con-

day .

DISCUSSION The

present

ificant mal

man.

2^2

on

ments

data

effect Thus,

indicate

within

in

serum

a few

urinary

secret:

i

on

iPTH

that

Ca

hours

after

excretion

PTH. were

l^SiOH^D^

seen

On in

the

its

c a n

exert

ingestion

is a u g m e n t e d contrary,

several

a

by

small

subjects.

signin

nor-

l,25(OH) increSuch

a

153 response who

is

consistent

found

no

the

goat

from

2D^(2). iPTH

decrease

with or

even

parathyroid

Also,

Oldham

in r a c h i t i c

administration

et

gland

substantially. an

immediate

(OH^D^ The

in

a vitamin

reasons

of

Chertow

It

is

for et

al.

of

These

stores

the

or

amount

Finally, for of

the

it

of

(1) and that

may

the

quantities sterol

al.

of

given,

et

a bolus

present

Ca

al,

in

serum

intravenous , .

had

(4)

,

increased

reported

injection

of

and

factors

species our

are

might

of

state

of

1,25

results

clear. the

other

of v i t a m i n in

the

D

tissues,

administration.

differences

results

not

and

present

route

the

modify

glands

previous

its

between

study,

l^SiOH^D^

the

l,25(OH)

decrease

serum

al.

secretion

with

after

et

dog.

the

between

(1) in

the

parathyroid

that

iPTH

,

discrepancies

the

include

is p o s s i b l e

et

after

a number on

no

hours

Canterbury

apparent

discrepancies

Chertow

PTH

in

Care

,

D replete

ljZSCOH^D^

tissues.

the

the

impossible

action

of

of

perfused

found

3, w h e n

addition,

release

being

10-12

l,25(OH)„D„

In

increase

(3)

until

I

observations

an

al.

puppies

of

the

in m a n

may

account

and

those

rat.

REFERENCES 1.

Chertow, B.S., Baylink, D.J., Wergedal, J.E., Norman,A.W.: D e c r e a s e in s e r u m i m m u n o r e a c t i v e p a r a t h y r o i d h o r m o n e in r a t s and in p a r a t h y r o i d h o r m o n e s e c r e t i o n in v i t r o by 1,25-dihydroxycholecalciferol. J Clin Invest 56,668-678 (1975) .

2.

Care, A.D., Bates, R.F.L, Pickard, D.W., Peacock, M., T o m l i s o n , S., O ' R i o r d a n , J.C.H., M a w e r , E.B., Taylor, C . M . , D e L U C A , H . F . , N o r m a n , A . W . ; T h e e f f e c t s of v i t a m i n D m e t a b o l i t e s and t h e i r a n a l o g u e s on the s e c r e t i o n of parathyroid hormone. P r o c 1 1 t h E u r o p e a n Symp C a l c T i s s u e (In P r e s s ) .

3.

Oldham, S.B., Smith, R., Hartenbower, D.L., Henry, G.H.: E f f e c t s of l a 2 5 - d i h y d r o x y - v i t a m i n D (la25 ( O H ) 2 D ) on serum c a l c i u m ( S ^ ) , i m m u n o r e a c t i v e p a r a t h y r o i d h o r m o n e ( i P T H ) and i n t e s t i n a l and p a r a t h y r o i d c a l c i u m b i n d i n g proteins (ICaBPaPCaBP). Fed P r o c 3 5 , 534 ( 1 9 7 6 ) .

4.

C a n t e r b u r y , J.M., B r i c k e r , N.A., Levy, G.S., K o z l o v i s k i s , P.L. R u i z , E., Zull, J . E . , R e i s s , E.: M e t a b o l i s m of bovine parathyroid hormone: i m m u n o l o g i c a l and b i o l o g i c a l c h a r a c t e r i s t i c of f r a g m e n t s generated by liver p e r f u s i o n . J. Clin Invest 55, 12 4 5 - 5 2 ( 1 9 7 5 )

COMPARATIVE ASPECTS OF THE BIOCHEMISTRY OF THE REGULATION OF VITAMIN D METABOLISM

u

M a c I n t y r e

Endocrine Unit, Royal Postgraduate Medical School, Ducane Road, London W12 OHS, England. The studies described here are mainly the result of a joint collaborative effort by Professor M. Haussler, J.W. Pike, A.M. Goldner, and T.A. McCain from the departments of Biochemistry and Physiology at the University of Arizona and by Professor I. MacIntyre, E. Spanos, K. Colston and I.M.A. Evans from the Endocrine Unit, Royal Postgraduate Medical School, Ducane Road, London W12 OHS, England. Calcitonin and vitamin D are both present in fish and both are active. Thus 1,25(0H)2D3 markedly stimulates bone resorption with a consequent elevation in plasma phosphate; calcitonin opposes these effects These actions are similar to those present in mammals

(1).

: 1,25(0H)2D3

enhances resorption of bone but also increases calcium and phosphorus absorption from the gut; calcitonin, as in fish, opposes the action of 1,25(OH)2D3 on bone and in general acts to conserve the skeleton.

Despite

the controversies of recent years, a number of factors have become accepted as physiological by the kidney.

regulators of the production of 1,25(0H)2D3

These are : 1,25(0H)2D3 itself, dietary phosphate and

calcium; and parathyroid hormone (2,1).

The latter agent probably

acts by changing phosphate metabolism and can have different effects depending on the electrolyte status. But none of these regulators is adequate to account for the changes in vitamin D metabolism which are likely to accompany the major physiological

changes in calcium and vitamin D metabolism during growth,

pregnancy and lactation.

We present evidence here that such changes

in the production of 1,25(OH)2D3 are most probably mediated by the integrated action of prolactin, placental

lactogen and the steroid

hormones. Prolactin Prolactin has long been known to have a marked effect in elevating calcium.

plasma

This action probably depends on the hormones recently

discovered marked action on the renal

la-hydroxylase

(3).

Physiological

doses of prolactin markedly stimulate the renal enzyme in chicks

(Fig.l)

156 Fig. 1

CONTROL

PROLACTIN

TREATED

10,000 -I 1000 la,25(OH)?D3

§ [ 500

Elution volume (ml)

and this effect is accompanied by a striking elevation of plasma (4) (Fig.2). 40

30 Fig.2

•o CJ1

20-

a

40

31 O

40

CSJ

10-

0

J

Control

20ug

lOOug

Prolactin I day

1,25(0H)2D

157 Further, elevated levels of 1,25(0H)2D are found during egg laying in chicks (4) (Fig.3) when plasma prolactin levels are known to be elevated. Fig.3 20 h 10

CD

e o CNJ I

CNJ

d> on

15-

10

5 -

•

12

0 J Hens

•

24

Nonlaying

Laying

Young

Recent studies (5) strongly suggest that t h i s action of prolactin i s also of major importance in mammals and that i t may be responsible, at least in part, for the enhanced absorption of calcium during pregnancy and lactation.

But i t i s possible that (in contradistinction to the young

chick) that the prolactin receptors concerned with i t s action on vitamin D in mammals are not present until maturity. Steroids Kenny (6,7) has shown that la-hydroxylase i s increased during the reproductive period in birds and DeLuca (8) has recently produced evidence that this may be due to oestrogen although no studies of plasma levels of 1,25(0H)2D were reported.

Further, the Wisconsin results suggested that

addition of testosterone was necessary before an effect could be seen in immature birds.

158

Our own results are somewhat different but give strong support to Kenny's results.

We find that oestrogen alone i s highly effective in young

male chicks and that a combination of testosterone and progesterone also has some effect (Figs. 4 and 5). Fig. 4

400 -i

1

300 -

CD -w

O

I

Q-

200

| 24,25 P T H

Placental lactogen Oestradiol

Acknowledgements This research was supported in part by grants from the Wellcome Trust and Medical Research Council and NIH Grant AM 15781.

162

LEGENDS TO FIGURES Figure 1:

Effect of 50 yg of prolactin on kidney enzyme activities after one hour.

Figure 2:

LH-20 columns; 15 day male chicks.

Effect on daily prolactin for 5 days on plasma la-25(0HD). Forty 15-day old male chicks per group.

Figure 3:

Serum 1,25(0H)2D levels in the laying hen.

Figure 4:

Renal enzyme activities 24 hours after diethyl stilboestrol Male Chicks; weight 80-104 g; diet 1% calcium, 0.54% P, 20 IU D/day.

Figure 5:

The effect of 5 mg steroids on renal enzymes at 24 hours. T, testosterone; P, progesterone; DES, diethyl stilboestrol Other conditions as in Figure 4.

Figure 6:

Urinary oestradiol in human pregnancy (from Brown, 1956).

Figure 7:

Summary of the concept of vitamin D regulation.

163 REFERENCES 1.

Maclntyre, I., Colston, K.W., Evans, I.M.A., Lopez, E., MacAuley, S.J., Piegnoux-Deville, J., Spanos, E. and Szelke, M. : Regulation of Vitamin D : An Evolutionary View. Clinical Endocrinology, 5, Suppl., 85S-95S (1976).

2.

Haussler, M.R., Baylink, D.J., Hughes, M.R., Brumbaugh, P.F., Wergedal, J.E., Shen, F.H., Nielsen, R.L., Counts, S.J., Bursac, K.M. and McCain, T.A. : The assay of la,25-dihydroxyvitamin D3 : Physiologic and Pathologic Modulation of Circulating Hormone Levels. Clinical Endocrinology, 5, Suppl., 151S-165S (1976).

3.

Spanos, E., Colston, K.W., Evans, I.M.A., Galante, L.S., MacAuley, S.J. and Maclntyre, I. : Effect of Prolactin on Vitamin D Metabolism. Molecular and Cellular Endocrinology, 5, 163-167 (1976).

4.

Spanos, E., Pike, J.W., Haussler, M.R., Colston, K.W., Evans, I.M.A., Goldner, A.M., McCain, T.A. and Maclntyre, I. : Circulating la,25Dihydroxyvitamin D in the Chick : Enhancement by Injection of Prolactin and During Egg Laying. Life Sciences, 19 (11), 1751-1756 (1976).

5.

Haussler, M.R. and Maclntyre, I. (unpublished).

6.

Kenney, A.D. : Vitamin D metabolism : Physiological

Regulation

in egg laying Japanese quail. American J. Physiology, 230, 1609-1915 (1976). 7.

Kenney, A.D., Lamb, J., David, N.R. and Losty, T.A. : Regulation of Vitamin D metabolism in egg laying Birds. Federation Proceedings, 33, 679 (1974).

8.

Tanaka, Y., Castillo, L. and DeLuca, H.F. : Control of renal vitamin D hydroxylases in birds by sex hormones.

Proceedings of the

National Academy of Science, 73, 2701-2705 (1976).

164 9.

Nicoll, C.S., Meites, J. and Blackwell, C. : Estrogen Stimulation of Prolactin Production by Rat Adenohypophysis In Vitro. Endocrinology, 70, 272-277 (1962).

10.

Meites, J. : International Symposium on Human Prolactin, 105-118 (1973) Excerpta Medica, Amsterdam.

11.

Brown, J.B. : Urinary Secretion of Oestrogens during pregnancy, lactation and re-establishment of menstruation. Lancet, i, 704-707 (1956).

12.

Spanos, E., Colston, K.k1. and Maclntyre, I. (Unpublished).

13.

Schaefer, K., von Herrath, D., Koch, H-V, and Opitz, A. : Effect of Cortisone on Vitamin D Metabolism. of Medical Sciences,

14.

Israel Journal

7, 533-534 (1971).

Lukert, B.P., Stanbury, S.W. and Mawer, E.B. : Vitamin D and Intestinal Transport of Calcium : Endocrinology, 93, 718-722 (1973).

Effects of Prednisolone.

A S P E C T S OF THE CONTROL OF V I T A M I N

E.

Barbara

The p u r p o s e o f many o f been

the s e a r c h

aetiology

University

the

for

o f some o f

implicated. amin

metabolic

the d i s e a s e

subjects

states

of metabolism in

and

from f a i l u r e

in h e a l t h y

the d i h y d r o x y 1 a t e d

25-OH-D s y n t h e s i s duction of less

delineation

hydroxy1 a t i o n

is

are

(D3)

by d i s t r i b u t i o n Uptake of

D3

seems

t o be

of

to reach

inhibition

patterns

of

vitof

abnormalities.

occur

the a p p r o p r i a t e

the v a r i o u s

enzyme

organs

systems

steps.

metabolite in

the

D i n man may t h e o r e t i c a l l y

is

controlled;

in c o m p a r i s o n w i t h

the

internal less

but

the

la,25"(OH)2~D,

probably

is

environment. likely

than

(25-OH-D)*

regulation

finely and

D

of

controlled apparently

Defects

much

in

those at a

pro-

25"

later

D metabolism.

is

D 3 TO 2 5 - H Y D R O X V V I T A M I N

i n t o body

into

D3

IN CONTROL

removed f r o m s e r u m by e x c r e t i o n ,

the

-

tissues

liver

25 0H-D3

from the

-

The

'vitamin

D1

D3

is

not p o s s i b 1 e .

is

and by h e p a t i c

from serum

synthesised

term

the normal

humans o f b o t h 2 5 _ h y d r o x y v i t a m i n

therefore

CONVERSION OF V I T A M I N D3

of

recognition

the v i t a m i n

total

coarse

to changes

of vitamin

Vitamin

to the

metabolites

the hormonal

sensitive

stage

or

of

the h y d r o x y l a t i o n

The p r o d u c t i o n

the v i t a m i n

in man the

h a v e been c o n c e r n e d w i t h m e t a b o l i s m o f

although

to p a r t i a l

D metabolism

that might e x p l a i n

in which

in the m e t a b o l i s m o f v i t a m i n

a t many p o i n t s ,

England.

of vitamin

abnormalities

m e t a b o l i s m must be a p r e r e q u i s i t e

Interference

of Manchester,

recorded s t u d i e s

Fewer s t u d i e s

in h e a l t h y

involved

IN MAN

Mawer

Department of M e d i c i n e ,

has

D METABOLISM

is

rapid,

via

SUBJECTS

the b i l e

and 1

faeces,

25"hydroxy 1 a t i o n . > > 3 as

is

the

2

release of

newly

liver.

u s e d when d i s t i n c t i o n

between v i t a m i n s

D2 and

166 The r a t e o f c o n v e r s i o n o f an i n t r a v e n o u s 25-OH-D3

p u l s e dose o f

radioactive

D3 to

i s markedly a f f e c t e d by the s t a t e o f v i t a m i n D n u t r i t i o n o f

individual

subject.

1

There

is e v i d e n c e

(see below)

that t h i s

e f f e c t e d by the c o n c e n t r a t i o n o f D r a t h e r than by a f e e d - b a c k i n v o l v i n g 25~0H-D; but s i n c e parent v i t a m i n ,

there

the n u t r i t i o n a l

assayed concentration of 25 0H-D 25-OH-D l e v e l

probably

reflects

control

in serum.

the

in terms o f

In most c i r c u m s t a n c e s

accurately

is

mechanism

i s no ready method o f m e a s u r i n g

s t a t u s must be a s s e s s e d

-

the

the

the

the c o n c e n t r a t i o n o f D but

the

r e l a t i o n s h i p may not always be p r e d i c t a b l e .

The f o r m a t i o n o f D3

l a b e l l e d 25-OH-D3 2k h a f t e r

bears a s t r o n g negative

25-OH-D in serum, F i g . subjects with

1.

relationship The r e g r e s s i o n

in whom the 25-OH-D l e v e l

levels

i n j e c t i o n o f a p u l s e dose o f

to the a s s a y e d c o n c e n t r a t i o n

i s based on data from 30 c o n t r o l

ranged from 2~k0

above 30 ng/ml had r e c e i v e d o r a l

ng/ml; those

D3 ( 1 . 5 mg)

received

other subjects ceased. within

subjects

shown in F i g .

labelled D3 shortly

(open c i r c l e s )

1.

25-0H-D

the 95% c o n f i d e n c e

after extensive s o l a r exposure,

r e c e i v e d D3 3 months a f t e r

l i m i t s of

irradiation

two s u b j e c t s , however, formed i n a p p r o p r i a t e l y

Q

x o

rise

it follows

labelled

o f D was l a r g e ;

25-OH-D3

the

immedlatter

of

S i n c e the serum h a l f -

than t h a t o f the 25-OH-D to w h i c h

that s i m i l a r

two had

that

high concentrations

l a b e l l e d 25-OH-D3 f o r t h e i r a s s a y e d 25-OH-D v a l u e s .

1

irradiation

the r e g r e s s i o n , s u g g e s t i n g

the serum pool

l i f e o f v i t a m i n D i s much s h o r t e r

(closed

Two s u b j e c t s

The former s u b j e c t s produced c o n c e n t r a t i o n s o f

iately after solar

gives

earlier.

the 2 5 - h y d r o x y 1 a t i o n of the l a b e l l e d p u l s e o f D3 i s p r o v i d e d by

data from k a d d i t i o n a l circles)

subjects

10 days

E v i d e n c e t h a t some f a c t o r o t h e r than the serum c o n c e n t r a t i o n o f controls

of

it

l e v e l s o f 25-OH-D may be a s s o c i a t e d

100

iA IN

W < >-

F i g . 1. R e l a t i o n s h i p between s y n t h e s i s o f 25-OH-D3 and a s s a y e d concent r a t i o n of 25-OH-D3(r = -0.91) The c o n c e n t r a t i o n of l a b e l l e d 2 5 - 0 H D 3 was measured 2k h a f t e r i n j e c t i o n o f label led D 3 .


jmol/hr) and PTH (1 U/hr) cAMP (0.9>jmol/hr) dbcAMP (1 ^imol/hr)

e

/0 of total

13.6 4; 0.2 2.9 + U. 2 12.5 + 4.7 + +

0.4 0.7 1.5 0.7 0.7

13. 2+ 10.7

1.7 1.0

12.8

recovered

radioactivity

in p l a s m a

Relative ratio

100 21 31 92 35 94 97 79

All chemicals were constantly infused into D-deficient rats immediately after TPTX. 24 hr later, the rats were given [ H]-25-OH-D^, and plasma was collected 6 hr thereafter. hours in vivo (11,12).

Thus we constantly infused cAMP, dbcAMP or theophy-

lline into D-deficient TPTX rats for a considerable period of time.

As

shown in Fig 5, 2 ^imol/hr of cAMP constantly infused into the TPTX rats starting 18 hr after the surgery restored 1Q(,25-(OH)^-D^ synthesis to the level of D-deficient sham rats within 6 hr.

Seven and a half U/hr of PTH

also restored 10/,25-(OH) ^-D^ synthesis to the same level within 12 hrTo evaluate further whether cAMP plays the role of second messenger in the PTH action on renal 10/-hydroxylase, the effect of theophylline was examined.

Neither theophylline nor a submaximal dose of 1 U/hr PTH alone re-

stored the 10l-hydroxylation reaction (Table 4).

When 0.5^imol/hr of theo-

phylline was constantly infused along with a submaximal dose of PTH, 101,25(OHJ^-DJ synthesis increased to the maximal level obtained by PTH. sion of 1 ^imol/hr of dbcAMP also restored the 101,25- (OH) 2

-D

Infu-

3 synthesis to

a level of 79% of that of D-deficient sham rats (Table 4). These results, together with previous reports (15-19), apparently satisfy Sutherland's four criteria for implicating cAMP as a second messenger of a given hormone action.

First, renal adenylate cyclase activity is markedly

enhanced by PTH in D-deficient rats and chicks (15,16).

Second, when PTH

is administered to D-deficient rats, the renal accumulation of cAMP is stimulated within min (18,19), and this increment precedes the PTH effect

210 of the stimulation of renal biosynthesis of 1 0 1 , 2 5 - ( O H ) T h i r d r

the

constant infusion of cAMP into D-deficient TPTX rats restores 101,25-(OH) production quantitatively to a level similar to that obtained by PTH

D

infusion (Fig 5 and Table 4).

Fourth, the iri vivo conversion of 25-OH-D^

to 101,25-(OH)2-D3 is restored to control levels when theophylline and a submaximal dose of PTH are infused together (Table 4).

The mode of cAMP

action and its interaction with Ca are currently under investigation. REFERENCES 1.,2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Bhattacharyya, M. H., and DeLuca, H. F. J. Biol. Chem. 248, 2969-2973 and 2974-2977 (1973) Rojanasathit, S., and Haddad. J. G. Biochim. Biophys. Acta 421, 12-21 (1976) Miller, L. L., Bly, C. G., Watson, M. L., and Bale, W. F. J. Exptl. Med. 9j4, 431-453 (1951) Fukushima, M., Suzuki, Y., Tohira, Y. , Matsunaga, I., Ochi, K., Nagano, H., Nishii, Y., and Suda, T. Biochem. Biophys. Res. Commun. 66, 632-638 (1975) Fukushima, M., Suzuki, Y., Tohira, Y., Nishii, Y., Suzuki, M., Sasaki, S. , and Suda, T. FEBS Letters 65_, 211-214 (1976) Henry, H. L., and Norman, A. W. Biochem. Biophys. Res. Commun. 62, 781-788 (1975) Brumbaugh, P. F., Hughes, M. R., and Haussler, M. R. Proc. Nat. Acad. Sci. USA 72_, 4871-4875 (1975) Oldham, S. B., Fisher, J. S., Shen, L. H., and Arnaud, C. D. Biochemistry 13_, 4790-4796 (1974) Chertow, B. S., Baylink, D. J., Wergedal, J. E., Su, M. H. H. , and Norman, A. W. J. Clin. Invest. 56, 668-678 (1975) Horiuchi, N., Suda, T., Sasaki, S., Takahashi, H., Shimazawa, E., and Ogata, E. Biochem. Biophys. Res. Commun. in press. Garabedian, M., Holick, M. F., DeLuca, H. F., and Boyle, I. T. Proc. Nat. Acad. Sci. USA 69, 1673-1676 (1972) Suda, T., Horiuchi, N., Sasaki, S., Ogata, E., Ezawa, I., Nagata, N. , and Kimura, S. Biochem. Biophys. Res. Commun. 54_, 512-518 (1973) Horiuchi, N., Suda, T., Sasaki, S., Ogata, E., Ezawa, I., Sano, Y., and Shimazawa, E. Arch. Biochem. Biophys. 171, 540-548 (1975) Forte, L. R., Nickols, G. A., and Anast, C. S. J. Clin. Invest. 5J_, 559-568 (1976) Larkins, R. G., MacAuley, S. J., Rapoport, A., Martin, T. J., Tulloch, B. R. , Byfield, P. G. H., Matthews, E. W., and Maclntyre, I. Clin. Sci. Mol. Med. 46_, 569-582 (1974) Rasmussen, H. , Wong, M., Bikle, D., and Goodman, D. B. P. J. Clin. Invest, SI, 2502-2504 (1972) Nagata, N. , and Rasmussen, H. Biochemistry 1_, 3728-3733 (1968) Kakuta, S., Suda, T., Sasaki, S., Kimura, N., and Nagata, N. Endocrinology 97, 1288-1293 (1975) Ohara, K., Yoshida, M. , Ohno, J., Suda, T., Sasaki, S. , Wakabayashi, K. , Nishii, Y. , and Abe, E. Bone Metabolism 189-191 (1976) in Japanese.

The Effect of Anticonvulsants on .{at Liver Calciferol 25-hydroxylase

S. Sulimovici and M.S. Roginsky Nassau County Medical Center, East Meadow, New York, and State University of New York, Stony Brook INTRODUCTION Anticonvulsant osteomalacia has been attributed to an alteration in Vitamin D metabolism (l) resulting in low serum levels of 25-OH-Vitamin D(2).

The

most accepted explanation for this finding has been the observed accelerated rate of conversion of both cholecalciferol (D^) and 25-hydroxycholecalciferol (25-OHL^) to more polar inactive metabolites (3,^).

Induction

of liver microsomal cytochrome ri+c,q by the anticonvulsant (AC) drugs is considered to be the mechanism for these changes (4).

The primary, if not

the only source of calciferol 25-hydroxylase, the enzyme involved in the initial hydroxylation of D^ to its active metabolite 25-OHD^, is the liver (5).

The effect of AC on this enzyme has not been studied.

The present

work has been designed to investigate the effect of prolonged administration of AG to Vitamin D-deficient rats on liver calciferol 25-hydroxylase activity. MATERIALS AND METHODS Male albino rats (Holtzman Company, Madison, Wisconsin) were used in the present experiments.

The rats were fed on a low Vitamin D diet (Nutrition-

al Biochemicals, Cleveland, Ohio) and water supplemented ad libitum.

Phé-

nobarbital (?) or diphenylhydantoin (DPH) 75 mg/kg body weight dissolved in 0.2 ml normal saline was injected intraperitoneally into the rats once daily for 25 days.

Control animals received saline only.

Calciferol 25-

hydroxylase activity was assayed using both liver homogenate and microsomal fractions.

An aliquot of the liver homogenate corresponding to 1 g

wet tissue was incubated in the presence of 0.1 M I^HPO^ buffer (pH 7 A ) MgSO/f. 5.0 mM, KCl 0.1 M, nicotinamide 0.16 M, ATP 20 mM, NADP~r OA

mM,

glucose-6-phosphate 22 mM, glucose-6-phosphate dehydrogenase 2.5 units and

212 0.2 pCi (3H) D^ (sp. act. 12,3 Ci/mmol) dissolved in a small amount of ethanol.

The total volume was 3 ml and the incubations were carried out

in air for two hours at 37° C.

The incubation period was terminated by

the addition of 0.2 ml acetic acid and freezing. ed and separated as previously reported (6,7).

The sterols were extractFor the estimation of en-

zymic activity in the microsomes it was essential to preincubate the liver homogenate at 37° C f°r

minutes prior to differential centrifugation.

The isolation of microsomes was carried out as described by 3hattacharyya & DeLuca (8).

The incubation media for mxcrosomes was identical to that

used for liver homogenate with the addition of succinate (10 mM).

The en-

zymic activity for liver homogenate and microsomal fractions was expressed as pmoles 25-OHD^ formed per g liver tissue and per 100 mg microsomal protein respectively.

In both cases this was calculated from the data ob-

tained from the conversion of added (3H) D^ to (3H) 25-OHD^. RESULTS AND DISCUSSION As shown in Table 1 both DPH and P had a significant inhibitory effect on calciferol 25-hydroxylase activity of both liver homogenate and microsomal fractions.

In neither control nor treated groups of animals was any other

metabolite than 25-OHD^ found. ed in the AG treated animals.

Total liver microsomal protein was increasDespite this increase, enzymic activity

calculated on the basis of total microsomal protein was significantly less in the AG treated as compared to control animals confirming the overall inhibition of the liver calciferol 25-hydroxylase enzyme.

SUMMARY DPH and P administered in vivo for 25 days to Vitamin D-deficient rats inhibit liver calciferol 25-hydroxylase activity.

The inhibition was demon-

strated in both liver homogenate and microsomal fractions.

These results

suggest that an inhibition of the enzyme system which converts D^ to 25OHD^ may explain the low plasma levels of circulating 25-OHD^ found in AG treated patients.

213 Table 1, Effect of in vivo administration of anticonvulsant drugs on rat liver calciferol 25-hydroxylase activity, (mean - S.E.M.)

Exp. 1 Control DPH P Exp. 2 Control DPH P Exp. 3 Control DPH P Exp. 4 Control DPH P * **

Total homogenate 25-OHD^ pmoles/g liver

Microsomes 25-OHDo pmoles/lOO mg protein

2.6 - 0.06 (4) 2.3 J 0.08 (4)* 1.5 - 0.08 (4)**

3.8 ± 0.3 (4J 2.4 - 0.2 (4)** 1.2 - 0.1 (4)**

6.1

5.2 I 0.20 (4) 2.9 + 0.20 (4)** 2.0 - 0.15 (4)**

10.2 I 0.3 (4) 2.6 - 0.3 (4)** 1.5 - 0.2 (4)**

8.5 3.3 3.2

5.5 t 0.30 (4) 3.0 - 0.15 (5)** 3.5 - 0.15 (4)**

>.0 -T 0.3 _ CO 3.9 + 0.3 (4)** 1.5 - 0.2 (4)**

6.9 5.3 3.7

4.7 - 0.10 (4) 2.6 - 0.15 (4)** 1.7 1 0.10 (4)**

6.6 - 0.4 (4) 3.7 r 0.3 GO** 0.9 - 0.1 (4)**

9.9 7.0 2.7

25-OHD, pmoles/total microsomal protein 9.5 6.1

statistically non significant p2D3--induction of CaBP

in organ- cultured chick duodenum* CaBP 2+

(Ca ), mM

(yg/mg total protein)

0

0 2,• 30±

0.3125 0.625

0

.21+

3.,26 ± • 09 3,.61 + 0 .20 0

1.25 2.5 5

3..1*7 ± 3..08 ±

* Values are x + S.E.; 5 duodena/group. bly involves cAMP and calcium.

.31 0 .11

0

Culture period: 2h hr.

Of course, the control of la,25-(OH) 9 D, 2+

biosynthesis by calcium regulating hormones and, possibly, by Ca

and

phosphate ions per se, plays a central though indirect role in determining intestinal responsiveness to vitamin D-like sterols (22). As a consequence of these observations, it was desirable to determine what effect alteration of the endogenous cAMP concentration of the cultured duodenum would have on CaBP induction. employed.

Several experimental treatments were

Diphenylhydantoin, known to interfere with calcium absortion in

vivo (23), inhibited vitamin D induction of CaBP, reduced calcium uptake and cAMP concentration in the organ-cultured duodenum (2b).

Aspirin, and

several other agents, similarly inhibited these vitamin D-mediated responses. 3-Isobutyl-l-methylxanthine, a potent phosphodiesterase inhibitor, was used to stimulate endogenous cAMP concentration in the presence of high 2+ and low Ca

in the medium.

In Table 3, vitamin D induction of CaBP and

stimulation of cAMP concentration can be seen to be dependent on ambient calcium concentration.

IBMX elevated endogenous cAMP concentration essen2+

tially independent of ambient Ca

concentration.

However, in the pre-

sence of increased cAMP, vitamin D-induction of CaBP was reduced in the high calcium medium and increased in the low calcium medium. dibutyryl cAMP produced quite similar results.)

(Exogenous

These data support the

hypothesis that vitamin D-induction can be regulated locally by cAMP and calcium.

A possible mechanism has been postulated (3): in essence, cAMP

237 Table 3-

Effect of 3-isobutyl-l-methylxanthine (IBMX) and C a 2 + on vitamin

D, activity in organ-cultured chick duodenum. IBMX (Ca

High

Low

) , mM

(0.1 mM)

°3 (26 uM)

(1..25

-

-

(1.• 25

-

+

• 25

+

-

(1,• 25

+

+

(0,.31

-

-

(0..31

-

+

(0..31

+

-

(0.• 31

+

+

(1_

Culture period: i+8 hr.

CaBP (yg per 100 mg; duodenum)

cAMP (pmoles/mg DNA)

0

116 + 2

IT. 1 + 0 . 9

170 + 3

0 10. 1+ ±

239 + 5 0

.8

302 + 11

0

109 + k

12. 5 + 0 .7

1U0 + 2

0 18. 3 ±

223 + 5 0

313 + 9

.7

may alter calcium concentration at some critical intracellular site (25) and, consequently, modulate genetic expression including the amount of CaBP induced.

It is clear that neither exogenous cAMP nor cAMP stimulated en-

dogenously serves as a "second messenger" of vitamin D^ action - there is no CaBP induction in the absence of vitamin D 3 even though cAMP may be elevated by other means. Cyclic AMP may function, generally, via activation of cAMP-dependent protein kinases (26) which in turn phosphorylate enzymes, membrane or nuclear proteins, etc. Table k.

(A specific endogenous substrate for one hormonally-

la,25-(OH) D -stimulated, cAMP-dependent protein kinase activity

in chick intestine. Protein kinase activity ratio -cAMP/+cAMP Treatment Rachitic chicks

-lq,25-(0H) 2 D 3

+la,25-(OH) 2 P 3

0.h6 + 0.02

0.35 + 0.02**

0.h2

0.28

(1 ug la,25-(0H) 2 D 3 , P.O., 36 hr) Embryonic chick duodenum in culture (65 nM la,25-(OH) 2 D 3 , 2k

hr)*

* Protein kinase fractionated through the (NH ) SO step of the procedure of Kuo and Greengard (28). ** Significantly lower than minus la, 25-(OH) 2 D 3 control; P < 0 . 0 1 .

238 stimulated, cAMP-dependent protein kinase has

now "been unequivocally dem-

onstrated, and there is an intriguing recent possibility

(27)).

Cyclic AMP-dependent protein kinase activity has been detected in rachitic chicks treated with la,25-(0H) 2 D 3 and in embryonic chick duodena incubated in the presence of la,25-(0H) 2 D 3

(Table U).

There is as yet no information

concerning the possible endogenous substrate(s) for this kinase.

However,

on the basis of the known nuclear site of action of la,25-(OH)^Dj-increased DNA template activity (1*0 and RNA synthesis (15-18) - and the fact that such functions can be stimulated by hormone-responsive,

cAMP-dependent

protein kinases in certain target tissues (29,30) suggests that such a substrate may be found in the intestinal cell nucleus.

There is also evidence

that cAMP-activated protein kinase catalytic subunits are translocated from a cytosolic locus to the nucleus in hormone stimulated tissues (30). In summary, it is likely that cAMP and calcium are involved in the regulation of lct,25-(0H) 2 D 3 activity in the intestinal cell.

The mechanism is

unknown but phosphorylation (and/or dephosphorylation) of critical endogenous substrate(s) involving a hormone-stimulated, cAMP-dependent protein kinase is a clear possibility. ACKNOWLEDGEMENTS Supported by NIH Grants AM-15355 and AM-0U652 and Research Career Development Award AM-00115-

Thanks are due Karen Bucher Ni and Eileen M. Frelier

for their excellent technical assistance and Dr. Milan Uskokovic of Hoffmann-LaRoche for his generous gifts of synthetic la,25-(0H) 2 D 3 . REFERENCES 1.

Neville, E. , Holdsworth, E. S.: A "second messenger" for vitamin D. FEBS Letters 2, 313-316, ( 1 9 6 9 ) .

2.

Corradino, R. A.: Embryonic chick intestine in organ culture: Interaction of adenylate cyclase system and vitamin D 3 -mediated calcium absorptive mechanism. Endocrinology 9h_, l60T-l6lU (197*0.

3.

Corradino, R. A.: Involvement of cyclic AMP in the vitamin D mediated intestinal calcium absorptive mechanism. In Calcium Regulating Hormones (Talmage, R. V. , Owen, M. , Parsons, J. A., eds.) pp. 3*+6361, Excerpta Medica, Amsterdam, 1975-

239 U.

Walling, M. W., Brasitus, T. A., Kimberg, D. V. : Elevation of cyclic AMP levels and adenylate cyclase activity in duodenal mucosa from vitamin D-deficient rats by la,25-dihydroxycholecalciferol (la,25-(OH) 2 D ). Endocrine Res. Commun. 3, 83-91 (1976).

5.

Corradino, R. A., Ebel, J. G., Craig, P. H., Taylor, A. N., Wasserman, R. H.: Calcium absorption and the vitamin D-dependent calcium-binding protein. I. Inhibition by dietary strontium. Calc. Tiss. Res. 7, 81-92 (1971).

6.

Omdahl, J. L., DeLuca, H. F.: Rachitogenic activity of dietary strontium. I. Inhibition of intestinal calcium absorption and 1,25dihydroxycholecalciferol synthesis. J. Biol. Chem. 2^7, 5520-5526 (1972).

J.

Wasserman, R. H., Corradino, R. A., Krook, L., Hughes, M. R., Haussler, M. R.: Studies on the la,25-dihydroxycholecalciferol-like activity in a calcinogenic plant, Cestrum diurnum, in the chick. J. Nutr. 106, i+57—i+65 (1976).

8.

Corradino, R. A.: Maintenance of embryonic chick duodenum in large scale organ culture. In Tissue Culture Association Manual: Techniques , Methods and Procedures for Cell, Tissue and Organ Culture (Evans, V. J., Perry, V. P., Vincent, M. M. , eds. ) pp. 71-71*, Tissue Culture Association, Rockville, Md., 1975-

9-

Corradino, R. A.: Embryonic chick intestine in organ culture. A unique system for the study of the intestinal calcium absorptive mechanism. J. Cell Biol. 5 8 , 6U-78 (1973).

10.

Corbin, J. D., Reimann, E. M.: Assay of cyclic AMP-dependent protein kinases. Methods Enzymol. 38, 287-290 (1970).

11.

Brumbaugh, P. F., Haussler, D. H., Bressler, R., Haussler, M. R.: Radioreceptor assay for la,25-dihydroxyvitamin D^. Science 183, 1089-1091 (197U).

12.

Birge, S. J., Alpers, D. H.: Stimulation of intestinal mucosal proliferation by vitamin D. Gastroenterology 6h_, 977-982 (1973).

13.

Whitfield, J. F., MacManus, J.P., Rixon, R. H., Boynton, A. L., Youdale, T., Swierenga, S.: The positive control of cell proliferation by the interplay of calcium ions and cyclic nucleotides. In Vitro 12, 1-18 (1976).

1^.

Zerwekh, J. E., Lindell, T. J., Haussler, M. R.: Increased intestinal chromatin template activity. Influence of la,25-dihydroxyvitamin D, and hormone-receptor complexes. J. Biol. Chem. 251, 2388-239*+ (1976).

15-

Corradino, R. A.: 1,25-dihydroxycholecalciferol: inhibition of action in organ cultured intestine by actinomycin D and a-amanitin. Nature 2^3, Ul-1+3 (1973).

16.

Tsai, H. C., Midgett, R. J., Norman, A. W.: Studies on calciferol metabolism, tissue and subcellular localization and action of vitamin D 3 . Arch. Biochem. Biophys. 157, 339-3^7 (1973).

17.

Tsai, H. C., Norman, A. W.: Studies on the mode of action of calciferol VI: Effect of 1,25-dihydroxyvitamin D 3 on RNA synthesis in the intestinal mucosa. Biochem.Biophys.Res . Commun. 622-627 (1973).

2kO 18.

Z e r w e k h , J. E . , H a u s s l e r , M. R . , L i n d e l l , T. J.: Rapid enhancement o f c h i c k i n t e s t i n a l D N A - d e p e n d e n t R N A p o l y m e r a s e II a c t i v i t y by la,25d i h y d r o x y v i t a m i n D , in v i v o . P r o c . N a t . A c a d . Sci. 2337-23^+1 (197U).

19.

E m t a g e , J. S . , L a w s o n , D. E. M . , K o d i c e k , E.: T h e r e s p o n s e of the s m a l l i n t e s t i n e to v i t a m i n D. Isolation and properties of chick intestinal polyribosomes. B i o c h e m . J. l U o , 2 3 9 - 2 ^ 7 ( 1 9 7 U ) .

20.

C o r r a d i n o , R. A . , F u l l m e r , C. S . , W a s s e r m a n , R. H . : Embryonic chick i n t e s t i n e in o r g a n c u l t u r e : S t i m u l a t i o n o f c a l c i u m t r a n s p o r t by exogeneous vitamin D-induced calcium-binding protein. Arch. Biochem. B i o p h y s . 1 7 U , 7 3 8 - 7 ^ 3 (1976).

21.

C o r r a d i n o , R. A.: E m b r y o n i c c h i c k i n t e s t i n e in o r g a n c u l t u r e : r e s p o n s e to v i t a m i n D a n d its m e t a b o l i t e s . S c i e n c e 1 7 9 , ¿+02-^+05 (1973).

22.

D e L u c a , H. F . , S c h n o e s , H. K.: v i t a m i n D. A n n . Rev. B i o c h e m .

23.

V i l l a r e a l e , M . , G o u l d , L. V . , W a s s e r m a n , R. H . , B a r , A . , B e r g s t r o m , W. H.: Diphenylhydantoin: E f f e c t s o n c a l c i u m m e t a b o l i s m in t h e chick. S c i e n c e 1 8 3 , 6 7 1 - 6 7 3 (197 1 *).

2k.

C o r r a d i n o , R. A . : Diphenylhydantoin: Direct inhibition of the vitam i n D j - m e d i a t e d c a l c i u m a b s o r p t i v e m e c h a n i s m in o r g a n c u l t u r e d duodenum. B i o c h e m . P h a r m a c o l . 25., 863-86*+ (1976).

25.

B o r l e , A. B.: R e g u l a t i o n o f the m i t o c h o n d r i a l c o n t r o l o f c e l l u l a r c a l c i u m h o m e o s t a s i s a n d c a l c i u m t r a n s p o r t by p h o s p h a t e , p a r a t h y r o i d hormone, calcitonin, vitamin D and cyclic AMP. In C a l c i u m R e g u l a t i n g H o r m o n e s ( T a l m a g e , R. V. , O w e n , M . , P a r s o n s , J. A . , eds.) pp. 2 1 7 - 2 2 8 , E x c e r p t a M e d i c a , A m s t e r d a m , 1975-

26.

C o r b i n , J. D . , K e e l y , S. L . , S o d e r l i n g , T. R . , P a r k , C. R. Hormonal r e g u l a t i o n of a d e n o s i n e 3 ' , 5 ' - m o n o p h o s p h a t e - d e p e n d e n t p r o t e i n k i n a s e . A d v . C y c l i c N u c l e o t i d e Res. 2 6 5 - 2 7 9 (1975).

27.

K i r c h b e r g e r , M. A . , Chu, G.: Correlation between protein kinasem e d i a t e d s t i m u l a t i o n of c a l c i u m t r a n s p o r t b y c a r d i a c s a r c o p l a s m i c r e t i c u l u m a n d p h o s p h o r y l a t i o n of a 2 2 , 0 0 0 d a l t o n p r o t e i n . Biochim. B i o p h y s . A c t a U l 9 , 5 5 9 - 5 6 2 (1976).

28.

K u o , J. F . , G r e e n g a r d , P.: Cyclic nucleotide-dependent kinases. VI. J. B i o l . Chem. 2^+5, 2^93-2^+98 (1970).

29.

C h u a n g , D. M . , H o l l e n b e c k , R . , C o s t a , E.: Enhanced template activity i n c h r o m a t i n f r o m a d r e n a l m e d u l l a a f t e r p h o s p h o r y l a t i o n o f chromosomal proteins. Science 193, 60-62 ( 1 9 7 6 ) .

30.

J u n g m a n n , R. A . , Lee, S . , D e A n g e l o , A. B . : T r a n s l o c a t i o n of c y t o plasmic protein kinase and cyclic adenosine monophosphate-binding p r o t e i n to i n t r a c e l l u l a r a c c e p t o r sites. Adv. C y c l i c N u c l e o t i d e Res. 5., 2 8 1 - 3 0 6 (1975).

M e t a b o l i s m a n d m e c h a n i s m o f a c t i o n of 6 3 1 - 6 6 6 (1976).

protein

A MODIFIED PROCEDURE FOR THE ISOLATION OF CHICK INTESTINAL CALCIUM BINDING PROTEIN

Friedlander, E.J. and Norman, A.W. Department of Biochemistry University of California, Riverside, CA

Intestinal

calcium

binding

proteins

92502

(CaBP) have been

and purified from several species of animals (1-5).

identified

The method utilized

in our laboratory to obtain chick intestinal CaBP in an electrophoretically homogenous state will be described in this report.

The procedure

is a modification of that originally devised by Wasserman et al (1), wherein the protein was purified from the soluable fractions of intestinal mucosal homogenates by the following 3-step process:

1) (NH^)'^

S0^ fractionation, 2) gel filtration on Sephadex G-100 and 3) preparative acrylamide disc gel electrophoresis. The crude supernatant starting material consists of the high speed supernatants

from

intestinal mucosal

homogenates.

This material

is

first subjected to (NH^)SO^ fractionation. As in the original procedure, solid (NH^)^ S0^ is slowly added till 75% saturation at 4 C. then centrifuged the original employed.

to remove the precipitated protein.

procedure,

a second

This is allowed

(NH^^SO^

to stand

This is

However, unlike

cut to 99% saturation

for 12 hours at 4 C.

is

Following

centrifugation at 100,000 x g for 1 hour, the supernatant is disgarded, and the pellets resuspended in 8-12 ml of Tris buffer.

By contrast, in

the published procedure the 75% (NH^^SO^ cut is subjected to two rounds of dialysis which must be followed by concentration molecular

sieve unit to obtain a suitable volume

in a pressurized

for gel

filtration

chromatography. The proteins from the 75-99% (NH^^SO^ cut were then subjected to molecular seive chromatography on Sephadex G-100. was

nearly

identical

to

that of the originally

The elution profile published

procedure,

thus indicating that the extra (NH^^SO^ cut did not markedly alter the amounts of protein carried through to this part of the isolation procedure.

In comparison with the original procedure, the yield of CaBP and

reduction in total protein is similar.

2k2 In

the

published

procedure,

the

calcium

binding

peak

from

the

S e p h a d e x G - 1 0 0 c o l u m n was p o o l e d , c o n c e n t r a t e d , and r u n o n a p r e p a r a t i v e disc

gel

CaBP

to e l e c t r o p h o r e t i c

ion

electrophoresis

exchange

step.

apparatus

to

homogeneity.

The

CaBP

was

It was applied

c o l u m n and e l u t e d w i t h a N a C l g r a d i e n t the

results

that

two

of

of

this

the

chromatography

three

major

effectuate

the

decided to

a

purification

of

to s u b s t i t u t e

an

DEAE

(A50)

in this b u f f e r .

step.

protein

It was

peaks

Sephadex

Figure 1A shows

interesting

contained

to

calcium

binding

a c t i v i t y . P r e v i o u s to this, there h a d b e e n n o r e p o r t s that c h i c k tinal m u c o s a c o n t a i n e d tained

subunits.

demonstrating

two c a l c i u m b i n d i n g

In v i e w

of

that p o r c i n e

the

results

proteins,

of

note

intes-

or that C a B P

Hitchman

con-

and H a r r i s o n

i n t e s t i n a l CaBP has a r e d u c e d a n i o n i c

charge

u p o n b i n d i n g c a l c i u m , the n o t i o n w a s e n t e r t a i n e d that p e r h a p s these protein

peaks r e p r e s e n t

in r e s p e c t to b o u n d

the same

identical

this,

and also

to h o m o g e n e i t y ,

the CaBP p e a k from a n o t h e r

through

Sephadex

the

ion-exchanger with

protein, but differing

G-100

stage

was

1 mM C a C ^ .

of these c h r o m a t o g r a p h y

step u t i l i z i n g E D T A

in the b u f f e r

binding

peak

dient peat

chromatography

CaBP eluted binding

of

calcium binding

this

in

the

in the peak

of

Figure

A

strates a marked decrease trophoretically

elutes.

Thus

The

chick

CaBP

was

obtained

chromatography

o n l y one

calcium

same part of the 1A.

containing

intestinal

following

CaCl^,

calcium

CaBP

these

of

the

demonElec-

two

ion-

steps.

a modification

stages

gra-

Upon re-

Thus c h i c k i n t e s t i n a l CaBP has b e e n p u r i f i e d to h o m o g e n e i t y ing

the

IB and

in a n i o n i c c h a r g e u p o n b i n d i n g c a l c i u m .

homogenous

CaBP

on

Figures

in F i g u r e

system

first

in the e a r l i e r part of the g r a d i e n t w h e r e the first

peak,

exchange

peak

steps.

system demonstrated

chromatographed

as the late e l u t i n g

to finally p u r i f y

chromatographed

1 m M E D T A and t h e n w i t h

This

only

identical preparation worked

1C s h o w the r e s u l t s

peak.

two

calcium.

I n an e f f o r t to d e m o n s t r a t e

to

(2)

the

of

the

purification

originally procedure

published reported

of the c h a n g e in a n i o n i c c h a r g e u p o n b i n d i n g

procedure. here-in

takes

The

utilizfinal

advantage

calcium.

REFERENCES 1.

Wasserman, R.H., Corradina, 243, 3 9 8 7 - 3 9 8 6

(1968).

R.A., and T a y l o r , A.N.

J. Biol.

Chem.

2^3 Figure 1A. Separation of proteins from the pooled Ca binding peak of a Sephadex G100 column on DEAE(A50) Sephadex. Protein was applied in .0137M Tris, 0.07M NaCl, pH 7.4. Elution for the first 100 ml was with this buffer following which the NaCl in the buffer was increased to 0.4M in a linear gradient. The gradient was monitored by conductivity measurements. CaBP was assayed by the Chelex-100 procedure and binding activity expressed as CaPr/CaR. B) Separation of + the proteins from the pooled Ca binding peak of a Sephadex G-100 column on DEAE (A-50) Sephadex with EDTA. Conditions are exactly as described above, only ImM EDTA is included in the buffer system. C) Chromatography of CaBP from B) on DEAE(A50) Sephadex with CaCl^. Conditions are exactly as described above, only ImM CaCl^ is included in the buffer system.

Figure I A .

10

DEAE

20

30

Figure B.

10 Figure C.

40

DEAE

20

30

DEAE

SEPHADEX

50

60

70

SEPHADEX

with

40

50

SEPHADEX

60 with

80

90

EDTA

70

80

CoClj

0.45 CoPr CaR

0.3

0.1

/ //

j 1 \\JL

0.2 " 0.30 0--0--o

fl i]V' I» >

0.15

1

—•-»— 1 2—0—/7 » 20i 30' 4•0 50i 10 FRACTION

2.

90

Hitchman, A.J., Kerr, M.K., and Harrison, J.E.

60

i

70

80

!•

»0

NO.-4.7ml

Arch. Biochem•

Biophys. 155, 221-222 (1973). 3.

Hitchman, A.J. and Harrison, J.E.

4.

Harmeyer, M. and DeLuca, H.F.

Can. J. Biochem. 50, 758-765 (1972).

Arch. Biochem. Biophys. 133, 247-251

(1969). 5.

Fullmer, C.S., and Wasserman, R.H. 317, 172-180 (1973).

Biochem. Biophys. Acta.

Chemical Studies on Bovine Intestinal Calcium Binding and R e l a t e d P e p t i d e s M i n o r A and M i n o r B

Protein

Curtis S. F u l l m e r , R o b e r t H. W a s s e r m a n , D a v i d V. C o h n , and J a m e s W . Hamilton C o r n e l l U n i v e r s i t y , Ithaca, N.Y. 14853 and V e t e r a n s A d m i n i s t r a t i o n H o s p i t a l , K a n s a s C i t y , Mo. 6 4 1 2 8 We r e c e n t l y i n i t i a t e d studies o n s t r u c t u r a l c o m p a r i s o n s b e t w e e n the v i t a m i n D - d e p e n d e n t c a l c i u m b i n d i n g p r o t e i n s of v a r i o u s s p e c i e s a n d

sources.

D u r i n g the e a r l y stages of this w o r k , w h i c h w a s f o c u s e d o n b o v i n e

CaBP,

we r e p e a t e d l y o b s e r v e d a p p a r e n t l y c o n t r a d i c t o r y d a t a w h e n w e a t t e m p t e d p r e p a r e t r y p t i c d i g e s t s of the p r o t e i n for p e p t i d e m a p p i n g .

Some

pre-

p a r a t i o n s of C a B P w e r e r e a d i l y d i g e s t e d b y the t r y p s i n w h i l e o t h e r 1 p a r a t i o n s s e e m e d to r e s i s t d i g e s t i o n preparations

.

W e s o o n r e c o g n i z e d that

to

pre-

those

that w e r e e a s i l y d i g e s t e d h a d b e e n d i a l y z e d in o r d e r

to

r e m o v e p r o t e i n - b o u n d c a l c i u m , w h e r e a s , those that r e s i s t e d d i g e s t i o n h a d not been dialyzed.

Figure 1 illustrates

this e f f e c t of d i a l y s i s

and

shows h o w a d d e d c a l c i u m a f f e c t s the d i g e s t i o n of C a B P b y t r y p s i n . the d i a l y z e d p r e p a r a t i o n , several b a n d s r e p r e s e n t i n g

tryptic

of CaBP are s e e n f o l l o w i n g p o l y a c r y l a m i d e e l e c t r o p h o r e s i s

peptides

(gel 3).

the d i g e s t i o n of the d i a l y z e d C a B P was c o n d u c t e d w i t h a d d e d

When

calcium

(gel 4), we found p r i m a r i l y two n e w s p e c i e s o n the g e l s , b o t h m i g r a t i n g t h a n the n a t i v e p r o t e i n .

With

faster-

T h e y are h e r e i n r e f e r r e d to as

c a l c i u m - t r y p s i n I and c a l c i u m - t r y p s i n

II.

T h e s e r e s u l t s s u g g e s t that

CaBP

u n d e r g o e s a c o n f o r m a t i o n a l c h a n g e w h e n it b i n d s c a l c i u m such that m o s t of those p e p t i d e b o n d s that are n o r m a l l y a t t a c k e d by t r y p s i n ( - l y s y l - x ) are somehow

shielded.

In terms of their m i g r a t i o n , these two b a n d s r e s e m b l e d two i d e n t i f i e d p e p t i d e s c a l l e d M i n o r A and M i n o r B that w e r e

previously

generated

246 spontaneously when CaBP was stored for various periods of time in the

2 freezer

.

It seemed possible that calcium-trypsin I and II were, in

fact, identical to Minor A and Minor B.

Fig. 1

2

In order to better understand

3

4

the relationship of calcium to the con-

formation of CaBP and the possible interrelationships of these generated peptide bands to the native peptide, we have examined more closely phase of CaBP chemistry.

this

Figure 2 shows that the peptides generated

native protein by calcium-trypsin exhibited

from

immunological activity to

anti-CaBP antisera and were fully able to bind calcium (as was previously found for Minor A and B).

Thus, it appeared that these

calcium-trypsin

peptides must be structurally similar to the native CaBP.

Native CaBP has a blocked amino terminus.

The calcium-tryptic peptides I

and II and Minor A and B, in contrast, were unblocked and hence, susceptible to direct sequence analysis by Edman degradation.

For this reason,

we initiated structural studies on both of these peptides.

Our results

2k7 h a v e p r o v e d v a l u a b l e o n two c o u n t s :

F i r s t , our d a t a s h o w that there

are

3 e r r o r s in o u r p r e v i o u s l y p u b l i s h e d s e q u e n c e of n a t i v e C a B P

.

Second,

ex-

a m i n a t i o n of the a u t o c o n v e r s i o n of n a t i v e C a B P to M i n o r A a n d B in the p r e s e n c e of c a l c i u m h a s r e s u l t e d in i n t e r e s t i n g version process

f i n d i n g s a b o u t the

con-

itself.

We h a v e n o t y e t c o m p l e t e d the s t r u c t u r e of the e n t i r e C a B P m o l e c u l e h a v e d a t a o n m u c h of its amino t e r m i n a l r e g i o n . The r e s u l t s of one a n a l y s i s of M i n o r A and M i n o r B are s h o w n in F i g u r e

lysine f r o m c a l c i u m trypsin II. a p p e a r to be i d e n t i c a l supporting

generated

is the a b s e n c e of the

Moreover,

initial

the s e q u e n c e s of M i n o r A and B

to the c a l c i u m - t r y p s i n g e n e r a t e d p e p t i d e s ,

the r a t i o n a l e

such

3.

As far as we h a v e s e q u e n c e d through the two c a l c i u m - t r y p s i n f r a g m e n t s , the o n l y d i f f e r e n c e o b s e r v e d

but

that c a l c i u m - t r y p s i n

I a n d M i n o r A , and

t r y p s i n II and M i n o r B are i d e n t i c a l . The p o s s i b i l i t y e x i s t s that e n c e s o c c u r at the C - t e r m i n a l e n d of the two p a i r s of p e p t i d e s .

thus, calciumdifferIf this

is the c a s e , h o w e v e r , such d i f f e r e n c e s m u s t be m i n i m a l . 120l

1

100

Fig. 2. A s s a y of c a l c i u m b i n d i n g and i m m u n o r e a c t i v i t y f o l l o w i n g i n c u b a t i o n of CaBP w i t h t r y p s i n for 4 h o u r s .

1

1

—

80

g o o 60 F z O £ 40 • ? £ | - 20£ £ >>

o120 •

o iooH)i—. * O 1« ss z 80 5 ' z 5

1«

2 (25-(0H)D2) and to test its effects jji vitro by competitive protein binding assay and in vivo, by measuring stimulation of calcium absorption.

These results would then

hopefully negate or confirm any of the possible explanations listed above for the cause of discrimination against vitamin D^ by the chick. Following incubation of 25-(0H)D2 with kidney homogenates from rachitic chicks, the presumed l,25-(0H)gD2 product was purified by silicic acid and Sephadex LH-20 chromatography.

A 1x35 cm Celite column with a solvent

system of 10% ethyl acetate in hexane and 4-5% water in ethanol was used as a final column to isolate the 1,25-(0H)2D2 product and to resolve it from the tritiated marker 1,25-dihydroxyvitamin D^ (1,25-(OH^D^)•

An ultra-

violet spectrum of the putative 1,25-(011)^02 revealed a qualitative resemblance to the vitamin D-sterols.

The generated 1,25-(0H)2Dg metabolite

was identified by direct probe mass spectrometry according to the procedure of Haussler et al. (11) and was in accord with the mass spectrum reported by Jones et al. (10).

Radioreceptor assays of 1,25-(0H)2D2 were

performed by competitive binding utilizing a modification of the chick receptor assay system as originally described by Brumbaugh ejt a^-. (12,13). Absorption of calcium was determined by a modification of the procedure of Coates and Holdsworth (14).

o in Figure 1. Competitive binding curves f o r 1,25-(0H) D and 1,25-(QH) 2 D .

10 15 20 25 30 HOURS Figure 2. Time course of stimulation of calcium absorption by 1,25-(0H) 2 D 2 in the chick.

The f i r s t parameter examined f o r 1,25-(0H)^D^ was i t s a b i l i t y t o bind t o the chick i n t e s t i n a l receptor system f o r the 1,25-(0H) 2 D^ hormone ( 1 5 ) . Figure 1 i l l u s t r a t e s that l,25-(OH) 2 D 2 competes with an e f f i c a c y virtuallyi d e n t i c a l t o that of the natural l ^ ^ - ^ O E ) ^ ^ s t e r o l . have been reported by Eisman e t a l .

(l6).

Similar r e s u l t s

1,25-(0H) 2 D 2 was next b i o -

assayed by measuring calcium absorption at the appropriate time a f t e r o r a l administration of the s t e r o l dissolved in 1,2-propanediol.

Figure 2 shows

that the k i n e t i c s of action of 1,25-(0H) 2 D 2 are similar t o those already reported f o r the natural 1,25-(0H) 2 D^ hormone (17) with a peak of

activity

at 10 hours and a rapid e f f e c t at 5 hours plus a submaximal sustained a c t i v i t y between 15 and 30 hours.

A f u r t h e r quantitative comparison of

the e f f e c t s of 1,25-(0H) 2 D 2 and 1,25-(0H) 2 D

on calcium absorption 9 hours

a f t e r dosing with 3 1 2 and 625 pmoles revealed that 1,25-(0H) o D„ was 1 / 3

to

3/k as a c t i v e as 1,25-(0H) 2 D . Thus, based upon these r e s u l t s , we conclude that 1,25-(0H) 2 D 2 can be generated i n r a c h i t i c chick kidney homogenates and that the p r o p e r t i e s of t h i s s t e r o l are similar t o those of the more e x t e n s i v e l y studied natural hormone.

1,25-(OH) CD

in the i n t e s t i n e ,

i s not discriminated against at the molecular

level

since i t binds t o the cytosol-nuclear receptor system

with an a f f i n i t y equal t o that of l,25-(OK)^> .

This f i n d i n g tends t o

263 eliminate target organ discrimination against the active l,25-(OH^Dg-form as an explanation for the lack of efficacy of vitamin D^ in the chick. Since Jones et al. (9) have shown that 1,25-( OH^D,-, is produced, in vitro, by chick kidney mitochondria at a rate identical to that of 1,25inefficient metabolism of vitamin D^ to its active form is also an unlikely possibility.

However, 1,25-(0H) D

is only l/3 to 3/U as active as

in vivo, although it does rapidly stimulate calcium absorption.

These data imply that l,25-(0H)2Dg may be transported in the blood

less effectively than l^J-COH^D^ or may be degraded more quickly than the D^-form. REFERENCES 1.

Steenbock, H., Kletzien, S. W. F., and Halpin, J. G. (1932) J. Biol. Chem. 97, 2U9-26U 2. Chen, P. S., and Bosmann, H. B. (I96U) J. Nutr. 83, 133-139 3. Hibberd, K. A., and Norman, A. W. (19697 Biochem. Pharmacol. 18, 23^7-2355 k. Drescher, D., DeLuca, H. F., and Imrie, M. H. (1969) Arch. Biochem. Biophys. 130, 657-661 5. Imrie, M. H., Neville, P. F., Snellgrove, A. W., and DeLuca, H. F. (1967) Arch. Biochem. Biophys. 120, 525-532 6. Haussler, M. R., and Norman, A. W. (1969) Proc. Natl. Acad. Sei. U.S.A. 62, 155-162 7. Belsey, R. E., DeLuca, H. F., and Potts, J. T., Jr. (197*0 Nature 2^7, 208-209 8. Jones, G., Schnoes, H. K., and DeLuca, H. F. (1975) Biochemistry 1*1, 1250-1256 9. Jones, G., Schnoes, H. K., and DeLuca, H. F. (1976) J. Biol. Chem. 251, 21+-28

10.

Jones, G., Baxter, L. A., DeLuca, H. F., and Schnoes, H. K. Biochemistry 15, 713-716 11. Haussler, M. R., Wasserman, R. H., McCain, T. M., Peterlik, M., Bursac, K. M., and Hughes, M. R. (1976) Life Sei. l8, 10^9-1056 12. Brumbaugh, P. F., Haussler, D. H., Bressler, R., and Haussler, M. R. (197*0 Science 183, 1080-1091 13. Brumbaugh, P. F., Haussler, D. H., Bursac, K. M., and Haussler, M. R. (197*0 Biochemistry 13, ¿+091-4097 1*K Coates, M. E., and Holdsworth, E. S. (1961) Br. J. Nutr. 15, 131-l*+7 15. Brumbaugh, P. F., and Haussler, M. R. (1975) J. Biol. Chem. 250, 1588-159U 16. Eisman, J. A., Hamstra, A. J., Kream, B. E., and DeLuca, H. F. (1976) Arch. Biochem. Biophys. 176, 235-2*13 17. Haussler, M. R., Boyce, D. W., Littledike, E. T., and Rasmussen, H. (1971) Proc. Natl. Acad. Sei. U.S.A. 68, 177-181

Recent studies on 1,25-(0H) 9 D

Action in the Intestine

E.Lawson, R.Spencer, M.Charman, P.Wilson MRC Nutrition Unit, Milton Road, Cambridge, U.K. Our understanding of the mechanism by which Ca is absorbed and of the part played by vitamin D in regulating this process, has improved significantly since the discovery of 1,25-(011)203. There is now convincing evidence from a number of laboratories that the kidney hormone 1, 2 5 - ( O H ) D ^

5

is the active form of

vitamin D in the intestine of animals.

1,25-(OH) Q D ^

j

is more

potent and acts more rapidly than any other known substance; it is active in anephric rats whereas vitamin D^ and its other metabolites are not.

It is the major form of vitamin D

found in the intestine of vitamin D-deficient animals after physiological doses of the vitamin and at times w h e n Ca absorption is proceeding maximally. There is no evidence that 1,25-(OH)„D is metabolised in the intestine to other active ^ i forms. The intracellular distribution of l,25-(OH) D in the 2 J intestine is that expected of a steroid hormone in its target tissue.

Furthermore it is the only hormone which

directly

regulates Ca absorption, the effect of parathyroid hormone

on

this process being mediated through its control of 1,25-(OH) 2 D

j

synthesis.

At least part of the mechanism of action of l,25-(OH) D 2 i involves the stimulation of intestinal protein synethesis,in particular of a protein called calcium-binding protein

(CaBP).

The finding that actinomycin D and puromycin inhibit l,25-(OH)„D stimulated intestinal Ca absorption is consistent ^ 3 with this view. (For reviews see refs. 1-4). In a series of experiments we have attempted to study the biosynethesis of CaBP in detail not only to understand how 1,23-(0H)^^^

controls its synthesis but also to obtain some

indication of the part CaBP plays in Ca absorption and to what extent its presence is obligatory.

266

Fig. 1. CaBP concentration in chick intestine after l,25-(OH) D (125 ng) or D (12.5 Ug) a n d CaBP mRNA activity after 1,25-fOH) D mRNA activity is % of maximum response.

It is of interest to compare the amounts of CaBP by the intestines of rachitic chicks dosed with

synthesised either

vitamin D or l,25-(OH) D (Fig. l). As might be expected the 2 J synthesis of CaBP is increased more rapidly in response to 1,25-(0H) D than to vitamin D . And considering the 2 j j differences in the amounts of steroids administered to the birds it is not surprising that the amount of CaBP produced by 1,25-(OH) D is very much less than that produced by 2 J vitamin D^ . Nevertheless, the maximal rate of CaBP synthesis which was reached in response to 125

n

S of 1,25-(OH)_D is 2 j similar to that found after a physiological dose (12.5 us) of

the vitamin.

Consequently, this amount of l,25-(OH) D must 2 j have saturated for a time at least all the sites in the intestine able to respond to the hormone.

The much larger

amounts of CaBP produced by vitamin D^ would appear to be due to the continued production of 1,25-(OH) 2 D J over an extended period thereby maintaining the increased CaBP synthetic activity.

Note also that after a pulse dose of l,25-(OH) 2 D^

the concentration of CaBP reached a maximum value after

267

Fig. 2. CaBP synthesising capacity of intestinal polysomes from D-dosed and deficient birds. /3/-I-CaBP antiserum was incubated with isolated polysomes which were then analysed on sucrose gradients. Only polysomes from dosed birds (o,*; bind the antiserum. -1 in polysomes of deficient birds.

268 24-48 h and then declined quite rapidly so as to be undetectable by 96 h. dose of l,25-(OH)_D ^

j

The intestinal mRNA activity after a reached a maximum after 12 h and then

declined to almost 90% of its maximum level at 24 h.

This

relationship between CaBP synthesis and its level in the intestine shows that CaBP once formed is a stable molecule and suggests that its presence in this tissue is related to the turnover time of the mucosal cells.

This suggestion may

explain the presence of CaBP3 in kidneys and brain of vitamin D-deficient animals, both tissues having a slow cell turnover. The increased synthesis of CaBP by chick intestine response to l,25-(OH)_D ^

j

in

can of course be observed in vitro

with a polysome fraction obtained from such intestines and incubated with the factors necessary for protein to continue"'.

Since this system for in vitro

synthesis

protein

synthesis can be designed so that only the polysomes

come

from the chick intestine, the effect of l,25-(OH) D

must be

on some component of this cell organelle.

An alternative

method of demonstrating the same phenomenon is shown in Fig. 2.

This figure shows that intestinal polysomes

from

vitamin D dosed but not deficient chicks can bind CaBP antiserum.

RNA has been extracted from intestinal polysomes

of vitamin D and 1,25-(OH) ^ D j -treated birds and shown to contain mRNA activity for CaBP. The translation of the messenger extracts has been effected by a rabbit reticulocyte 6 7 lysate and an extract of wheat germ . In both cases the CaBP in the newly synthesised protein was recognised by its immunological and electrophoretic

characteristics.

The properties of the CaBP messenger RNA have also been 7

studied .

The most remarkable

feature of this material is

its size which, with a molecular weight of 700,000 and a sedimentation constant of approximately l8S, is about three times that expected if this mRNA were required to code only for a protein of the size of chick intestinal CaBP.

As with

269

Fig. 3. Gel electrophoresis of [ H ] polypeptides synthesised by the wheat germ system primed with intestinal polysomal RNA. A. Proteins synthesised by the cell-free system with RNA from deficient chicks (•) and chicks dosed with 1,25-(0H)2D 12 h before killing (o) after precipitation with CaBP antiserum. B. Ratio of immunoprecipitable radioactive proteins from dosed and deficient RNA in A.

Fig. 4. Time course of changes in CaBP levels and in Ca absorption after D3 (A) and l , 2 5 - ( O H ) 2 D 3 (B). CaBP was detected by immuno-electrophoresis.

270 most other mRNAs that for CaBP also has a poly A tract which can be recognised by its ability to bind to poly U-Sepharose. This poly A tract, however, only accounts for part of the excess nucleotide sequences and even although all mRNAs are much larger than necessary

(after allowing for the poly A

tract) the CaBP messenger is particularly large.

For example

the mRNA for chick ovalbumin (M.W. 43,000) is apparently the same size as that for chick intestinal CaBP.

One

explanation

for the large size of the mRNA for CaBP could be that the messenger coded for a precursor molecule for CaBP which was cleaved to the 27,000 molecular weight CaBP following

its

release from the polysomes.

series

However, in an extensive

of experiments in which the products of polysomal RNA

from

dosed birds were compared to the products of polysomal RNA from vitamin D-deficient birds no evidence has been obtained for the existence of a precursor to CaBP (Fig. 3). In the early studies the changes in CaBP levels and in Ca absorption after a dose of vitamin D were consistent with an g obligatory role for CaBP in Ca absorption .

Thus CaBP was

detected immunologically in the intestine of vitamin Dtreated birds about two hours before Ca absorption was increased (Fig. 4A).

first

The CaBP levels and Ca absorption as

measured by an in vivo technique then increased rapidly and remained at these raised rates for some considerable

time.

More recently changes in Ca absorption in response to l,25-(OH) D

have been measured using the everted intestinal 9 sac technique . The results showed that Ca absorption was detectable before CaBP (Fig, 4B).

Furthermore, although the

intestinal polysomes of l,25-(OH) ^ D 3 treated birds can be shown to be synthesising CaBP, the activity is very low and only increases w h e n Ca absorption is about 50% of the maximum (and at the time that CaBP can be detected by Immunoelectrophoresis).

At later time intervals there was also no

correlation between Ca absorption and CaBP levels.

The

absorption of calcium declines quite rapidly after reaching

271

Gel

Slice

Fig. 5. Slices of intestinal tissue from D^ dosed and deficient chicks were incubated for 2 h in presence of 3H-leucine and l^C-leucine respectively. The pooled cytoplasm was electrophoresed. Only one area of the gel had a raxsed 3 H / 1 4 C ratio corresponding to position of CaBP.

86000

86000

45000

45000

treated for 6 h with 1 , 25-( O H ^ D ^ .

272 its maximum so that it is proceeding at the same rate as in untreated birds at the time (48 h ) w h e n CaBP levels are at, or close to, their maximum.

It appears therefore that

addition to CaBP the absorption of calcium requires factors which are also 1,25-(0H) 2 D^ dependent.

in

other

These

factors

should be recognisable by their relatively short half-life the intestinal cell.

in

Following a dose of l,25-(OH) D the ^ 3

level of the hormone in the intestine after reaching

its

maximum declines rapidly, and Ca absorption and mRNA for CaBP decline at about the same rate.

Consequently these

additional

factors will be recognisable by having a similar turnover t ime. Among the possibilities as a factor involved in Ca absorption is obviously cAMP since the intestinal levels of this substance have been shown to be affected by 1,25-(OH) D

. 3 However, attempts have recently been made by us to identify other proteins which could be involved in the absorption process.

calcium

The procedure adopted involves

incubating

slices of intestine from steroid treated birds and from 3 vitamin D-deficient birds in the presence of H-leucine and 14 C-leucine respectively.

All birds were rachitic at the

beginning of the experiment.

After 2 h the incubation was

stopped, cell fractions prepared, the proteins

solubilised

usually by SDS treatment and the proteins fractionated by gel electrophoresis. The distribution of 3H and 14C along the 3 gel was recorded. Any increase in H relative to C indicates the presence of a protein synthesised in response to the steroid.

Fig. 5 shows that CaBP is the only protein

in the cytoplasm synthesised in response to Vitamin D^. Irrespective of whether the birds were dosed with vitamin D or 1,25-(OH) 9 D and at whatever time interval after dosing 3 that the analyses were made, CaBP is the only cytoplasmic protein whose synthesis is increased by these

steroids.

Brush border proteins were also analysed similarly.

In this

cell fraction at least three proteins were detected whose

273

Fig. 7 . Changes in the rate of synthesis of membranous proteins of MW 45,000 and 86,000 and also in Ca transport in response to 1,25-(OH) D^. A , 45,000 protein; •, 86,000 protein; o, Ca transport. Bars show duration of incubat ion.

27^ synthesis is stimulated by l,25-(OH) D 2 j

These membrane

proteins are synthesised at different rates according to the time interval after the hormone was administered to the birds. For 4-6 h there is an increase in the synthesis of two relatively small proteins and at later time intervals the synthesis of a much larger molecule is increased. molecular weights of these proteins appear to be

The 45,000,

86,000 and one greater than 150,000. The results in Fig. 6 illustrate the changes in ratio along the gel that are found after analysing the brush border proteins from intestines of chicks dosed 6 h previously with l,25-(OH) D . ^ J

The two peaks shown are the two

smaller

proteins, the larger one not being seen in this gel system. It is possible by appropriate analysis of the radioactivity on the gel to establish the amounts of the two proteins formed.

Fig. 7 shows the changes in the level of the two

proteins formed at various time intervals after l,25-(OH) 2 D J . Also shown are the changes in Ca absorption after l,25-(OH) 2 D The mechanism by which l,25-(OH) D stimulates the synthesis 2 j of these proteins is unclear. The intracellular distribution of the hormone and the finding that actinomycin D inhibits the increased synthesis of these membranous proteins that the hormone

is acting on a transcriptional

suggests

event.

However, the time scale of their appearance is very rapid for the synthesis of membranous proteins.

Only

further

experiments will establish whether these proteins are involved in Ca absorption but their rapid appearance

in

response to l,25-(OH) 2 D J suggests that they have some role to play in the function of this hormone.

275 References 1.

Omdahl, (1973).

J.L.,

2.

Norman,

A.W. : Vitam.

3.

Wasserman, R . H . , C o r r a d i n o , R . A . , A . N . : Vitam. & Horm. 32_, 299-325

4.

Lawson, (1974).

5.

Emtage, J . S . , Lawson, 239-247 (197^).

D.E.M.,

Kodicek,

E.:

Biochem.J.l40

6.

Emtage, J . S . , Lawson, 100-101 (1973).

D.E.M.,

Kodicek,

E.:

Nature 2 46,

7.

Spencer, R . , Charman, M., Emtage, J . S . , Europ.J.Biochem. i n press. (1976).

8.

E m t a g e , J . S . , Lawson, D.E.M., K o d i c e k , E.: 339-346 (1974).

9.

Spencer, R . , Charman, M., W i l s o n , Nature 263, I6I-I63 (1976).

10.

DeLuca, H . F . :

D.E.M.,

Physiol.Revs.

J.S.:

Fullmer, (1974).

Vitam.

327-372

(1974).

& Horm. 32_, 326-384

Emtage,

¿3,

C.S.,

Taylor,

& Horm. 32,

277-298

P.W.,

Lawson,

D.E.M.:

Biochem.J.l44,

Lawson,

D.E.M.:

C o r r a d i n o , R . A . : In C a l c i u m - r e g u l a t i n g hormones (R.V.Talmage, M.Owen, J . A . P a r s o n s , E d s . ) pp. 346-361, E x c e r p t a Medica, Amsterdam (1975).

The Role of Phosphate in the Intestinal Response to Vitamin D

R. Miller, R. Clancy, S.J. Birge Department of Medicine, The Jewish Hospital of St. Louis and W a s h i n g t o n University, St. Louis, Missouri 63110. Recent investigations have led to the recognition that the restoration of phosphate homeostasis in rachitic animals is the consequence of the direct action of vitamin D o n a variety of tissues, including the intestine, bone, kidney, and the mobilization of phosphate from other soft tissue stores. In addition, vitamin D is essential for the m a i n tenance of normal growth. In the vitamin D deficient animal 25-hydroxycholecalciferol (25HCC) stimulates the p r o l i f e r a tion of the intestinal m u c o s a (1) and accelerates the accumulation of phosphate and the synthesis of protein in muscle (2). The identification in essentially all tissues of a cytoplasmic protein w h i c h specifically binds 25HCC w i t h high affinity (3) suggests that vitamin D and specifically 25HCC may influence cellular metabolism at a non-specific or basic level w h i c h results in the maintenance of normal tissue growth and function. In order to investigate the direct action of 25HCC, as well as 1,25HCC on the intestine, an organ culture preparation of explants from vitamin D deficient chick ileum was established similar to that described by Kagnoff et al. (4). In this preparation, 1,25HCC, at a concentration of 50 pg/ml, and 25HCC, at a concentration of 20 ng/ml, stimulated the accumulation of phosphate by the explants. This response was o b served as early as 30 min. after the addition of the calciferol and w a s inhibited by m e t a b o l i c inhibitors and cycloheximide. The stimulation of phosphate accumulation preceded by 30 min. stimulation of 4 5 c a uptake and by 150 min. stimulation of ^H-thymidine incorporation into DNA (Figure 1). To examine the role of increased intracellular phosphate in m e d i a t i n g the response to 1,25HCC, the extracellular p h o s phate concentration was increased from 0.5 m M to 3.0 mM. The synthesis of DNA and uptake of 45ca were stimulated by the high phosphate in the absence of the sterol. (Table I). Reduction of the extracellular phosphate to 0.05 m M blunted the response to 1,25HCC (Figure 2). These observations suggest that both 25HCC and 1,25HCC act directly on the intestinal m u c o s a to stimulate the accumulation of phosphate. The stimulation of calcium uptake and DNA synthesis is dependent in p a r t on the availability of phosphate. It is postulated that in the vitamin D deficient intestine intracellular phosphate concentrations are deficient and that the restoration of intracellular phosphate concentrations towards normal may play an important, if n o t primary, role in the ultimate ex-

278 pression of the cells response to the vitamin D sterols. Table I - Influence of Iodoacetamide (IA) on 1,25HCC-Induced and Phosphate-Induced Stimulation of 4 5 C a Uptake Culture Conditions PO^ Cone . 1,25HCC 0.5 0.5 0. 5 0.5 3. 0 3.0

45

IA

C a Uptake/mg Protein %Control No. Paired Obs.

_

mM mM mM mM mM mM

+

-

+ +

-

+

-

-

-

+

EFFECT OF

l,25(OH)2D

100 113 167 115 170 132

ON

24 13 13 11 11 45

Co

P-Value .05 . 001 .05 . 005 . 05

32 AND.^PQ, UPTAKE d

O oc i— 50 Z

o u §

o

25 H-TdR

o

z < X

u 1

2 3 4 5 HOURS OF I N C U B A T I O N

6

Figure 1 - Explants from vitamin D deficient chicks were incubated in the presence of 60 pg/ml of 1,25HCC or in the absence of the sterol (control). Closed circles indicate the accumulation of 32p-phosphate in 16 min. by the explants. The closed triangles indicate the uptake of 45ca in 4 min. by the explants. The open triangles indicate the cumulative incorporation of 3n-thymidine (^H-TdR) into DNA. The vertical bars indicate the S.E.M. The data are expressed as dpm/mg protein as a percent change from control plotted as a function of total duration of incubation.

279

INFLUENCE

OF P0 4 O N EXPLANT RESPONSE TO 1,25HCC

KWWN

0.05

KWXNN

0.5

l\V-

3.0

CONC. P04 (mM) Figure 2 - Explants from vitamin D deficient chicks were incubated at various phosphate concentration for a total duration of incubation of 3 hrs. in the presence (closed bars) and absence (open bars) of 1,25HCC.

References: 1.

Birge, S.J. and Alpers, D.H. Stimulation of intestinal mucosal proliferation by vitamin D. Gastroenterology 64: 977-982 (1973).

2.

Birge, S.J. and Haddad, J.G. 25-Hydroxycholecalciferol stimulation of muscle metabolism. J. Clin. Invest. 56: 1100-1107 (1975).

3.

Haddad, J.G. and Birge, S.J. Widespread, specific binding of 25-hydroxycholecalciferol in rat tissue. Biol. Chem. 250:299-303 (1975).

4.

J.

Kagnoff, M.F., Donaldson, R.M. and Trier, J.S. Organ culture of rabbit small intestine : Prolonged in vitro steady state protein synthesis and secretion and secretory IgA secretion. Gastroenterology 63:541-551 (1972).

Distribution of Alkaline Phosphatase and Ca-ATPase in Intestinal Epithelial Cell Plasma Membranes:

Differential Response to 1,25-(OH) D.

A.K. Mircheff, M.W. Walling, C.H. van Os, and E.M. Wright Departments of Physiology and Medicine, UCLA School of Medicine and Nephrology Unit, VA Wadsworth Hospital Center, Los Angeles, Ca. 90024, USA

INTRODUCTION

The small intestine absorbs Ca v i a a mechanism w h i c h is stimulated by l^S-iOH^D^.

Analysis of the electrochemical potential of Ca in the

intestinal epithelium indicates that flux of Ca into cells m a y be passive, while extrusion must be active.

A n asymmetrical distribution of active

Ca extrusion, with a higher rate on the basal lateral membranes (BLM) than on the brush border membranes (BBM), would generate net Ca absorption. ATPases have been implicated in active ion transport in numerous systems, and Ca-ATPase activity has been demonstrated in both BBM and BLM.

We

report here a preliminary evaluation of the role of this enzyme in intestinal Ca transport.

METHODS

Male Holtzman rats were raised from weaning under vitamin D-deficient conditions (-D) for six weeks. verified the - D state.

Severe hypocalcemia and hypophosphatemia

Repleted rats (+1,25) received 405 ng. IP doses

of l ^ S - i O H ^ D ^ 48 and 24 hr. before sacrifice.

An analytical procedure

(1) was used to isolate plasma membranes from proximal duodenum, m i d jejunum, and terminal ileum. previously

(1).

Enzyme activities were assayed as described

Unidirectional Ca fluxes were measured under short-

circuited conditions

(2).

282 RESULTS AND DISCUSSION If Ca-ATPase is involved in active Ca-transport, the segmental distributions of Ca-ATPase and transport should be similar. is demonstrated in Table I.

That this is the case

Since it has been suggested that Ca-ATPase is

identical to alkaline phosphatase (AP) (e.g., ref. 3), the segmental distribution of AP is also included, and the distribution of Na,K-ATPase is presented for comparison.

The gradients of Ca-ATPase and AP activities

and Ca absorption were similar, decreasing from duodenum to ileum, and much greater than the gradient of Na,K-ATPase activity. Table I Comparison of segmental distributions of Ca-absorption and enzyme activities in +1,25-intestine Duodenum

Jejunum

Ileum

13.2

1.1

0.9

Ca-ATPase

3,200

700

200

Alkaline phosphatase

81,700

7,600

800

Na,K-ATPase

1,900

900

1,200

Net Ca absorption

All activities are given in units of nmole/mg protein-hr. Ca absorption was converted from units of nmole/cm 2 hr. with factors of 6.8, 5.9, and 3.5 mg/cir.2 measured in duodenum, jejunum and ileum, respectively.

Since duodenum is the site of the greatest rate of Ca absorption, analytical experiments were performed to determine the total BLM and BBM contents of Ca-ATPase and AP in -D and +1,25 duodenum; the results are summarized in Table II.

BLM contain twice as much Ca-ATPase as BBM.

Such an asymmetrical distribution is predicted to generate net Ca absorption; furthermore, the excess BLM capacity for Ca-stimulated ATP hydrolysis is about 100-fold greater than the observed net rate of transport. Both Ca-ATPase and Ca absorption increased in response to 1,25-(OH)^D^, and the relative increases were roughly similar. The data in Table II indicate dissociation of Ca-ATPase from AP in two respects:

(a) the AP distribution favored BBM over BLM, and (b) 1,25-

me

(OH)2D2~ di ate d increases of the two activities were not parallel. There arises, therefore, question about the relationship between Ca-ATPase

283 and AP.

Sucrase and Na,K-ATPase activities also increased in response to

l^S-COH^D^;

this result calls for further work on the nature of 1,25-

(OH)2D2-mediated changes in plasma membrane enzyme activities.

Table II C a absorption and specific activities of plasma m e m b r a n e localized enzymes in homogenates of duodenal m u c o s a

Ca Absorption

-D

+1,25

Ca--ATPase BLM BBM

Alkaline Phosphatase Bl.M BBM

Na,K ATPase BI.M-marker

Sucrase BBM-marker

4.8

I II

700 1,400

400 500

15,400 24,800

29,100 28,400

1,100 1,500

980 1,000

13.2

I 11

2,000 2,300

1,200 1,000

20,300 51,200

46,000 78,300

1,300 2,700

1 ,200 1,600

BLM and Ei'.M contents of C.i ATPase aru! alkaline phosphatase were calculated from analytical Isolation data. - D and 4],25 animals were from the same groups and were processed simultaneously in experiments I a n d II.

ACKNOWLEDGMENT

This work was supported by USPHS grants NS 09666, AM 19567, and AM 14750 and VA Medical Research Funds.

REFERENCES

1.

Mircheff, A.K., Wright, E.M.:

Analytical isolation of intestinal

epithelial cell plasma membranes.

J. Memb. Biol. 2^8: 309-333

2.

Active secretion of calcium by adult

Walling, M.W., Kinberg, D.V.:

rat ileum and jejunum in vitro. 3.

Am. J. Physiol. 225: 415-422

Haussler, M.R., Nagode, L.A., Rasmussen, H.:

brush border alkaline phosphatase by v i t a m i n ATPase.

Nature 228: 1199-1200

(1970).

(1976).

(1973).

Induction of intestinal

D and identity with Ca-

The Relation between the Induction of Alkaline Phosphatase and 1,25-Dihydroxycholecalciferol Receptor in Chick Embryonic Duodenum

S. Moriuchi, S. Yoshizawa, F. Shimura, T. Oku and N. Hosoya Department of Nutrition,

School of Health Sciences,

University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, Japan Alkaline phosphatase (al-Pase) in chick embryonic duodenum is induced at the time of hatching (1).

D^ is known to

induce al-Pase in vitamin D deficient animals (2).

However,

it is not known that D^ is required for the upsurge of intestinal al-Pase during embryonic development.

Therefore,

the effect of D^ metabolites on the induction of al-Pase was observed using organ culture system of chick embryonic duodenum in relation to the development of l ^ S - t O H ^ D ^ receptor in chick embryonic duodenum in ovo. One day old White Leghorn fertilized eggs were obtained from Kazusa Hachery (Chiba, Japan) and incubated at 37.5°C 60% relative humidity.

with

Duodena from various stages of

embryos were used for organ culture and for the assay of 1,25-(OH)2D^ binding activity. Duodena from 14-. 18- and 20-day embryos were cultured in the presence or absence of 62.5nM LX-OHD^.

After 48 hours,

al-Pase activity was assayed in the duodenal homogenate. 1 -OHD^ stimulated al-Pase activity significantly in cultured duodena from 2 0-day embryos, however, there was not effect in 14- and 18-day embryo.

On the other hand, maltase activity

of cultured 20-day embryo was not affected by l^-OHD^ (3). Furthermore, the effect of various D^ metabolites on al-Pase activity of cultured 20-day embryo was examined. l,25-(OH)^D2 produced a graded increase in the duodenal al-Pase in

286 culture at concentration of 0.625 to 62.5 nM. to 1 , 2 5 - ( O H )

a n d

1 -0HD

In contrast

3 / 25-OHD3 and D 3 did not give any

significant responce at the effective level of 1,25-(OH) and l^-OHD^ , although a small response to 25-OHD3 was observed at 625 nM (4).

The effect of l,25-(OH) 2 D 3 and

lcx-OHD3 was abolished by the addition of actinomycin D in culture medium.

This result suggests that the induction of

al-Pase during organ culture involves the process of protein synthesis. On the other hand, development of cytosol l,25-(OH)2D3 receptor in_ ovo was observed in chick embryonic duodena using sucrose density gradient ultracentrifugation techniques and the embryonic l,25-(OH)2D3 receptor was compared with the binding profile for l,25-(OH)2D3 of the cytosol of vitamin D deficient chick duodenum. The binding profile for 1,25-(OH)2D2

the cytosol of vitamin D deficient chick

duodenum on the sucrose density gradient revealed 3 binding components, and the sedimentation constant was estimated as 2.5, 3.5 and 5.5 S respectively.

In good agreement with

the results of other investigators (5), the 3.5 S binding component has high affinity and low capacity for l,25-(OH)2D3 and is thought to be l,25-(OH)2D3 receptor.

In the course

of chick development, the 3.5 S binding component was not detected in 13-day embryonic duodenum, it appeared on 15th day of incubation and then gradually increased to the level of vitamin D deficient chick on 19th day of incubation.

The

5.5 S binding component v/as specific for 25-OHD3 and it was found even in 13-day embryo, but it did not show any significant change during development.

The 2.5 S component

was not specific either l ^ S - i O H ^ D ^ or 25-OHD 3>

However,

it was a main binding component in early stages of development (6) . These results suggest that 1, 25-(OH) „D., is one of essential

287 factors in the induction of al-Pase and the action of l ^ S - i O H ^ D ^ would require the presence of l,25-(OH) 2 D 3 receptor.

The inability to induce al-Pase in 14-, and 18-

day embryo could be ascribed to the absence or low level of 1,25-(OH)2^2 receptor. References 1. Moog, F. ; The functional differentiation of the small intestine I. The accummulation of alkaline phosphomonoesterase in the duodenum of the mouse, J. Exp. Zool., 115, 109-120 (1951) 2. Norman, A. W. , Mircheff, A. K., Adams, T. H., and Spielvogel, A. : Studies on the mechanism of action of calciferol III. Vitamin D-mediated increase of intestinal brush border alkaline phosphatase activity, Biochim. Biophys. Acta, 215, 348-359 (1970) 3. Yoshizawa, S., Sugisaki, N., Moriuchi, S., and Hosoya, N.: The effect of l>-hydroxyvitamin D^ and cortisone on the development of chick duodenal alkaline phosphatase in organ culture, J. Nutr. Sci. Vitaminol., 22^, 21-28 (1976) 4. Yoshizawa, S., and Moriuchi, S. : 1,25-Dihydroxycholecalciferol and induction of alkaline phosphatase in organ culture, J. Nutr. Sci. Vitaminol., 22_, 263-265 (1976) 5. Brumbaugh, P. F., and Haussler, M. R. : lex , 2 5-Dihydroxycholecalciferol receptors in intestine II. Temperaturedependent transfer of the hormone to chromatin via a specific cytosol receptor, J. Biol. Chem., 249, 12581262 (1974) 6. Oku, T., Shimura, F., Moriuchi, S., and Hosoya, N. ; Development of 1,25-dihydroxycholecalciferol receptor in the duodenal cytosol of chick embryo, Endocrinol. Jap., 23, 375-381 (1976)

EFFECTS OF DIETARY CALCIUM AND PHOSPHORUS ON THE STEADY STATE LEVELS OF SOME COMPONENTS OF THE VITAMIN D ENDOCRINE SYSTEM

E. J. Friedlander, H. L. Henry, and A. W. Norman Department of Biochemistry University of California, Riverside, CA 92506 USA

The intestinal absorption of calcium is known to be strictly dependent upon the availability of vitamin D, particularly its hormonally active

metabolite

1,25-dihydroxyvitamin

D^

[1,25-(OH) 2 ~D 3 ]

(1).

An

interesting aspect of this process is that intestinal calcium absorption is adaptively regulated by the organism such that there is an increased efficiency

of absorption under conditions where the calcium demand of

the organism

is high

(i.e.,

conditions

of

low dietary

calcium)

(2).

Much evidence has accumulated to suggest that adaptation by an organism to the varying calcium demands involves the endocrine mediated regulation of production of 1,25-(OH)2~D2 by the kidney.

This steroid then

localizes in the intestinal mucosa (3) and stimulates calcium absorption (4).

This presumably occurs via stimulation of RNA and protein syn-

thesis (5), including the induction of a calcium binding protein (CaBP) (6-8). The process of lot-hydroxylatin of 25-hydroxyvitamin D^ which occurs exclusively

in the kidney,

is believed to be the primary

site of these regulatory processes (9-12). vitamin D^

intake,

there

is a highly

^S-OH-D^),

Under conditions of normal

significant

inverse

correlation

between the 25-hydroxyvitamin D^-l -hydroxylase (1-hydroxylase)

activ-

ity and serum calcium levels (9).

The regulation of the 1-hydroxylase

has

through

been proposed

to be mediated

levels of parathyroid hormone. demonstrated of

levels

of

in the

circulating

For example, Hughes ej: a_l. have recently

that parathyroidectomy

circulating

changes

in rats abolished the enhancement

1,25-(OH)2~D2

produced

in response

to

low

calcium diets (13). Augmentation of intestinal calcium absorption may also occur under conditions of adequate or normal dietary calcium if the dietary phorus is very low (14). are not yet clear.

phos-

Possible mechanisms involved in this phenomena

It is known that the administration of exogenous

290 1,25-(OH)2~D2 eliminates the adaptation of intestinal calcium transport to low calcium diets, while intestinal

calcium

it does not eliminate the stimulation of

transport

to

low phosphorus

diets

(15).

Thus

dietary phosphorus deprivation may stimulate calcium transport through an as yet unrecognized mechanism. This report quantitatively evaluates the steady state relationships existent

in

endocrine calcium

the

and phosphorus.

calcium

state

level

levels (16),it

between

various

components

of

of of

and

phosphorus depletion was

intestinal

CaBP

the

intestinal

calcium

parallel

represents

D

in dietary

In particular, the role of the 1-hydroxylase

of

CaBP

vitamin

the

system under conditions of adaptation to changes

under

measure

chick

the

a good

was

investigated.

utilized

in

absorption

these

The

steady

studies

response.

as

Since

a

the

levels

of

intestinal

calcium

absorption

indicator

of

intestinal calcium

absorption

activity. The basic approach utilized in all these studies involved raising chickens that were previously rachitic on diets of varying calcium and phosphorus content and administering either vitamin D^ or 1,25-(OH) daily over a two week period.

Since the regulation of the production

of 1,25-(OH)2~D2 is an important control point in the adaptation to a low

calcium

or

phosphorus

status,

it was

of

interest

to bypass

the

endogenous output of 1 ^ S - C O H ^ - D ^ and supply this steroid exogenously. Figure

1 shows the comparative effects of either daily vitamin D^ or

1,25-(OH)2~D3 administration on the steady state level of CaBP in birds raised under various calcium and phosphorus dietary conditions.

In the

presence of vitamin D^, CaBP was significantly elevated under both low calcium

and

low phosphorus

dietary

conditions.

Fig. 1. Differential production of intestinal CaBP following daily administration of vitamin D or l«',25(OH)2D3. Groups of 6 to 8 chicks, 14 days old, were raised on the indicated experimental diet for an additional two weeks over which time they received 1.6 nmoles/day of vitamin D- (upper panel) or 1.0 nmoles/ day o f l,25-(OH) „-D-, (lower panel). Analyses of the d i e t s i n d i c a t e d that t h e p e r c e n t calcium was L=0.05%, N=1.2%, H=2.4%; while dietary phosphorus was L=0.05%, N = 0 . 7 % , H=l.l%. Intestinal CaBP was quantitated by the method of radial immunodiffusion.

However, when

X ,

Dietary Dietary

Ca N P L

L N

1,25-

Vitomin D s (1.6 nmolaa/day

N N

H N

(OH^-D^

was

presented

under

similar

dietary

conditions

the

r e s p o n s e to the low c a l c i u m c o n t e n t of the diet w a s e n t i r e l y whereas

that d u e to low p h o s p h o r u s was still

adaptive

eliminated,

present.

Since the level of CaBP c a n be s i g n i f i c a n t l y

e l e v a t e d by low d i e -

tary p h o s p h o r u s c o n d i t i o n s , e v e n in the p r e s e n c e of h i g h levels of (OH^D^, lase

then

plays

the

any

levels?

In

between

the

question

arises

significant

an

effort

role

to

gain

1-hydroxylase

and

as

to w h e t h e r

in a d a p t a t i o n

the

to

an understanding the

adaptation

of

this

enzyme

was

measured

in c h i c k e n s

renal

1-hydroxy-

changing

phosphorus

of

the

response

C a B P to c h a n g i n g c a l c i u m and p h o s p h o r u s c o n d i t i o n s

relationships of

intestinal

the s p e c i f i c

supplemented

with

2 shows the h i g h l y

serum

calcium

levels

these

birds.

As

corresponding

and

shown

significant

specific in

Figure

to c h i c k s w i t h

serum

e x c l u d e d , the slope and s t a t i s t i c a l tion were to

increased.

extremely

elevation general

in

equivalent mg/100

values

the

specific nature

excluding to

activity of

1-hydroxylase

above

levels ml

of

11.5 mg/

calcium who

is

levels

a

a

of

above serum

in

values

100 m l

are

correla-

from 11.5 m g / 1 0 0 m l striking

1-hydroxylase.

values had

fall

there

the

circulating

chicks

the

1-hydroxylase

s i g n i f i c a n c e of the inverse

5 mg/100

serum

excluding

calciums

serum calcium of

when

the

between

Due

serum 11.5

18

fold

to

the

calcium

and

mg/100

phosphorus

ml

below

is 4

ml.

When phorus

low

reciprocal

phosphorus,

As

of

D^

conditions.

inverse c o r r e l a t i o n

activity 2B,

activity

vitamin

a n d r a i s e d u n d e r a v a r i e t y of d i e t a r y c a l c i u m and p h o s p h o r u s Figure

1,25-

the

levels,

same two

types

of

analysis

significant

trends

is r e p e a t e d w i t h c a n be o b s e r v e d

F i g . 2. R e l a t i o n s h i p b e t w e e n s p e c i f i c a c t i v i t y o f the 1 - h y d r o x y l a s e a n d s e r u m c a l c i u m levels. T h e d i e t a r y g r o u p s u t i l i z e d in this and all s u b s e q u e n t e x p e r i m e n t s w e r e (1) 0.7% P h o s p h o r u s 0 . 2 % C a l c i u m ; (2) 0.1% P h o s p h o r u s , 2.2% C a l c i u m ; (3) 0.7% P h o s p h o r u s , 2.2% C a l c i u m ; (4) 1.2% P h o s p h o r u s , 2.2% C a l c i u m . (A) I n c l u d e s all 1 - h y d r o x y l a s e v a l u e s for all s e r u m c a l c i u m determinations; (B) Excludes 1 -hydroxylase v a l u e s for s e r u m c a l c i u m s above 11.5 m g / 1 0 0 m l or p h o s p h o r u s b e l o w 4 m g / 1 0 0 m l .

the s e r u m in the

6.0

80

phos-

relation-

100

12.0

140

Serum Ca (mg/IOOml)

I 2t-

1.0-

0.80.6-

04 0.2100

292 F i g . 3. R e l a t i o n s h i p b e t w e e n s p e c i f i c a c t i v i t y of the 1 - h y d r o x y l a s e and serum phosphorus l e v e l s . (A) Includes 1 - h y d r o x y l a s e f o r a l l serum phosphorus v a l u e s (B) excludes 1 - h y d r o x y l a s e values f o r serum phosphorus values below 4 mg/100 ml; (C) excludes 1 - h y d r o x y l a s e values f o r serum phosphorus values above 8 mg/100 ml.

• n

21 0.690 2-D2 i s o l a t e d from the i n t e s t i n e i s

D^.

paral-

296 leled by increasing steady state levels of CaBP.

Furthermore, the low

phosphorus dietary mineral status which was responsible for an elevated steady

state

level

of

intestinal

CaBP, 3

increased localization of intestinal that

low phosphorus n

1,25-(OH)2~D2 The

i-

results

correlation

and

dietary

the

presented

evaluation

of

vitamin D endocrine system. capability amount

of

tissue

and

of

the kidney

via

the

to produce

unknown first

corresponding

level

the

of

of

mechanism.

quantitative steps of the

in concert the enzymatic

1,25-(OH)^-D^

in

an

It thus appears

some of the key component

We have measured

with

the localization

some

represent

1,25-(OH)localizing the

associated

stimulates

mucosa

here

also

H-l,25-(0H)2~D2.

conditions

intestinal

is

intestinal

induction

of

as well

as

mucosal CaBP,

the

target

all

as

a

function of changes in dietary calcium and phosphorus. We have been able to demonstrate that upon inducing a high calcium demand in the organism, there is a striking elevation of the 1-hydroxylase which inducible increase

in turn

levels in

the

of

is

correlated

intestinal 3

amount

of

inate the adaptation of CaBP H-D3

CaBP.

Furthermore,

increase there

isolated

Also, exogenous

B

Exp.

6 0 r—

« e

o

k_ Q.

in

the

is a 5 fold

from

the

1,25-(0H)2-D^

intes-

can elim-

levels to varying calcium dietary

60

condi-

3 H - I , 2 5 ( 0 H ) 2 D 3 Exp. n= 5 r = 0.99 p =

CT> E

:30 o> Q. m o o

a striking

H-l,25-(0H)2~D2

tinal mucosa of these birds.

3

with

Q_

m 6.4 SerumCa l, 25- (OH) 2Dy> D3^lo Si

©

m

'

g |

- r-

^ Hi?

Figure 1. Light microscopic localization of CaBP. Cryostat cut, acetone fixed section of duodenum from vitamin D 3 dosed (500 I.U., 72 Hr.) chick reacted with the complete immunoperoxidase enzyme bridge. Intense locali z a t i o n of DAB reaction product is present continuously along the absorptive surface of the villi and in the goblet cells. A fainter reaction is present in the lamina propria area which was also noted in the immunological control (Fig. 2). Figure 2. Immunological control employing the same tissue and fixation protocol as in Fig. 1. Non-immune serum replaced the specific antiserum. Figure 3. Light microscopic localization of CaBP. The same immunological protocol was used as in Fig. 1 on tissue from the same vitamin D3 treatment group. The tissue was fixed with ethanol instead of acetone. The higher power view illustrates the intense DAB reaction product in goblet cells and at the absorptive surface of two adjacent villi (separated by the clear luminal space). Figure 4. Light microscopic localization of CaBP employing noncoagulant fixatives (formaldehyde and glutaraldehyde). The DAB reaction product is present intermittently along the absorptive surface of the villi, in the goblet cells and in strands of material (incompletely fixed mucus?) connecting groups of goblet cells. The dark staining nuclei were also present in immunological controls (sections not shown).

308 cells when anti-CaBP was the primary antiserum in the immuno-enzyme bridge.

Figure 6 illustrates the distribution when non-immune serum was

used, i.e., an immunological

control.

A comparison of Figs. 5 & 6 allows

a qualitative localization of CaBP in the mucigen granules of the goblet cells since that is the major difference in deposition of DAB between the two treatments.

At higher magnification

(Fig. 7), the typical

pentagon

shape of the DAB due to the underlying PAP complex (12) is seen in high concentrations over the mucigen granules of the goblet cell.

This is con-

sistent with the light microscope findings and provides a slightly finer resolution concerning its site within goblet cells.

Heavy deposits of DAB were also noted over spherical granules located between the goblet cell theca and the goblet cell nuclei which are more basally displaced than the absorptive cell nuclei

(Figs. 8 & 9).

presumably represent condensing vacuoles in transit from the Golgi to the goblet cell theca.

These complex

Such a localization in condensing vacuoles is

consistent with the concept that CaBP is synthesized de novo at the RER*, is transferred to the Golgi where it is packaged for subsequent storage in the theca, and finally is secreted from the goblet cell.

The relative-

ly high background or non-specific binding observed in these studies may have prevented a localization over the Golgi or RER since none was observed.

Occasionally thin sections were examined in which mucus had been retained in the gut lumen adjacent to the microvillar border.

The DAB reaction

product was as concentrated over the secreted mucus as it was over the mucigen granules in the goblet cells (Fig. 10).

Therefore, there is lim-

ited evidence showing the secretion of CaBP from the goblet cells into the intestinal

lumen with the mucus.

Because of the relatively high non-specific cfistribution of DAB noted in this study, a localization of CaBP in other regions in which the concentration was less than that in mucigen granules or condensing vacuoles would most likely be obscured.

Therefore, it is not possible to eliminate

other sites of lower concentrations of CaBP.

Whether or not the con-

densing vacuoles, the goblet cell mucigen granules and secreted mucus

309

Figure 5. E.M. immunoperoxidase localization of CaBP. Goblet cell from the duodenum of a vitamin D3 treated (500 I.U., 72 Hr.) chick illustrating the high concentration of DAB reaction product over the mucigen granules. The random distribution of DAB over other parts of the section is not different from that seen in the immunological control (Fig. 6), therefore, can not be distinguished from non-specific binding. X 13,000. Figure 6. Immunological control. Duodenal goblet cell from same treatment group as Fig. 5. Non-specific antiserum replaced anti-CaBP in the enzyme bridge, therefore, non-specific binding only. X 9,000. Figure 7. DAB reaction product localized over individual mucigen granules in a goblet cell from a chick treated as in Fig. 5. At this magnification the characteristic pentagonal shape of the DAB reaction product due to the PAP complex can be seen. X 38,000.

310

Figure 8. DAB reaction product localized over the mucigen granules in the theca of a goblet cell and in the underlying condensing vacuoles. Duodenal tissue from same treatment group as that in Fig. 5. X 6,000. Figure 9. Higher magnification of a field similar to Fig. 8 showing the discrete localization of DAB reaction product over two condensing vacuoles (arrows) and the basal portion of the theca (GC). X 13,000. Figure 10. This field illustrates an infrequently observed condition in which recently secreted mucus (M) is retained in the lumen (L) near the microvillar border (MV). Although random deposits of DAB are present over the entire field as in the immuno-controls, a higher concentration is present over the mucigen granules and the mucus in the lumen. X 5,000.

311 represent the only sites of CaBP, they certainly represent the major sites. It also suggests that the light microscopic CaBP localization at the absorptive cell surface probably represents secreted mucus trapped along the villi.

The preparative technique employed for light microscopy would fa-

vor retention of luminal substances, whereas those for electron microscopy would favor loss of materials not firmly attached to the epithelial cells (see METHODS section). If CaBP is a significant factor in the vitamin D-mediated process of Ca absorption as many studies have suggested, and if the only sites of CaBP in the intestine are those demonstrated in this study, and since there is no evidence to support the concept that goblet cells are absorptive in nature, then one must conclude that the CaBP present in the secreted mucus is the functional CaBP.

Unfortunately, the process of collecting and pro-

cessing intestinal tissue for electron microscopic observation disrupts whatever spatial relationships there might have been between the mucus and the underlying absorptive cells.

Therefore, it is difficult to speculate

how the CaBP in the mucus might function in Ca absorption.

Its normal

presence there, however, might explain the success of the reconstitution experiments of Corradino et.al. (13) in which purified CaBP when added in vitro to embryonic intestine elevated the Ca transport capacity of that tissue.

If on the other hand, other undetected lower concentration sites

of CaBP actually represent the functionally significant CaBP, then from the standpoint of cell economy, the question remains:

What purpose is

served by the relatively massive production of a specific-protein (3% of the soluble proteins) by vitamin D stimulation with the subsequent secretion of that protein into the intestinal

lumen?

ACKNOWLEDGEMENTS We acknowledge the excellent technical assistance of Helen Irvin for cutting the ultrathin sections and of M. Brindak and C.S. Fullmer in the preparation and purification of the primary antisera. We also thank S.S. Spicer for his personal assistance with the immunoperoxidase technique, and W.L. Davis, J.H. Martin and J.L. Matthews for their valuable advice. The research was supported by NIH grant AM-04652 (to R.H. Wasserman) and by a research grant from The Upjohn Company.

312 REFERENCES 1. Taylor, A. N., Wasserman, R. H.: Immunofluorescent l o c a l i z a t i o n of vitamin D-dependent CaBP. J. Histochem. Cytochem. 18^ 107-115 (1970). 2. Taylor, A. N., Wasserman, R. H.: C o r r e l a t i o n s between the vitamin Dinduced CaBP and the i n t e s t i n a l absorption of Ca. Fed. Proc. 28, 18341838 (1969). 3. Wasserman, R. H., Corradino, R. A.: Vitamin D, Ca, and protein synt h e s i s . Vitamin Horm. 31_, 43-103 (1973). 4. Wasserman, R. H., Corradino, R. A . , Fullmer, C. S . , Taylor, A. N.: Some aspects of vitamin D a c t i o n ; Ca absorption and the vitamin D-dependent CaBP. Vitamin Horm. 32, 299-324 (1974). 5. Emtage, J. S . , Lawson, D. E. M., Kodicek, E.: The response of the small i n t e s t i n e to vitamin D; c o r r e l a t i o n between CaBP production and i n creased Ca absorption. Biochem. J. 144, 339-346 (1974). 6. Helmke, K., F e d e r l i n , K., P i a z o l o , P., Stroder, J . , Jeschke, R., Franz, H. E.: L o c a l i z a t i o n of CaBP in i n t e s t i n a l t i s s u e by immunofluorescence in normal, vitamin D - d e f i c i e n t and uraemic subjects. Gut 1_5, 875879 (1974). 7. P i a z o l o , P., Hotz, J . , Helmke, K., Franz, H. E., Schleyer, M.: CaBP in the duodenal mucosa of uremic patients and normal subjects. Kidney I n t . 8. 110-118 (1975). 8. M o r r i s s e y , R. L . , Bucci, T., Empson, R. N., Lufkin, E. G.: CaBP, i t s c e l l u l a r l o c a l i z a t i o n in jejunum, kidney and pancreas. Proc. Soc. Exptl. B i o l . Med. H 9 , 56-60 (1975). 9. Arnold, B. M., Kovacs, K., Murray, T. M.: C e l l u l a r l o c a l i z a t i o n of i n t e s t i n a l CaBP in pig duodenum. Digestion 1_4, 77-84 (1976). 10. L i p p i e l l o , L . , Wasserman, R. H.: Fluorescent antibody l o c a l i z a t i o n of the vitamin D-dependent CaBP in the oviduct of the l a y i n g hen. J. H i s t o chem. Cytochem. 23, 111-116 (1975). 11. Wasserman, R. H., Taylor A. N.: I n t e s t i n a l absorption of phosphate in the chick. J. Nutr. 103, 586-599 (1973). 12. Sternberger, L. A . , Hardy, P. H., C u c u l i s , J. J . , Meyer, H. G.: The unlabeled antibody enzyme method of immunohistochemistry. J. Histochem. Cytochem. 18, 315-333 (1970). 13. Corradino, R. A . , Fullmer, C. S . , Wasserman, R. H.: Embryonic chick i n t e s t i n e in organ c u l t u r e : stimulation of Ca transport by exogenous vitamin D-induced CaBP. Arch. Biochem. Biophys. V74, 738-743 (1976).

T H E E F F E C T O F D D T ON VITAMIN D M E T A B O L I S M AND C A L C I U M BINDING A C T I V I T Y IN T H E CHICK

J u s t i n S i l v e r and Zvi A l p e r n lipid R e s e a r c h Laboratory, Hadassah University Hospital, P. O. Box 499, J e r u s a l e m , I s r a e l C h r o n i c DDT e x p o s u r e c a u s e s t h i n a v i a n e g g s h e l l s w h i c h m a y b e a f a c t o r in t h e d e c r e a s e in p o p u l a t i o n of c e r t a i n a v i a n s p e c i e s ( l ) . T h e m e c h a n i s m of t h i s DDT e f f e c t i s not c l e a r . C h i c k s fed DDT h a v e b e e n s h o w n to h a v e a r e d u c e d i n t e s t i n a l a b s o r p t i o n of c a l c i u m with no a l t e r a t i o n in v i t a m i n D m e t a b o l i s m (2). We fed DDT t o c h i c k s and s t u d i e d t h e i r m e t a b o l i s m of 3 H - v i t a m i n D 3 and the l e v e l s of i n t e s t i n a l c a l c i u m b i n d i n g activity (Ca.B.A.). METHODS One d a y old White L e g h o r n c h i c k s w e r e g i v e n n o r m a l f e e d f o r 1 week, and t h e n divided i n t o e x p e r i m e n t a l g r o u p s . T h e c o n t r o l g r o u p w a s a l lowed t o f e e d f r e e l y . A s e c o n d c o n t r o l g r o u p ( s t a r v e d c o n t r o l s ) w a s a l l o w e d only l i m i t e d a c c e s s t o food in o r d e r to l i m i t t h e i r w e i g h t g a i n a s o c c u r s in the DDT g r o u p . DDT t e c h n i c a l g r a d e (79% p , p ' DDT and 20% o , p ' DDT) w a s m i x e d i n t o the f e e d to a c o n c e n t r a t i o n of 400 p p m , and fed t o a t h i r d g r o u p of c h i c k s f o r 3 w e e k s . E a c h c h i c k r e c e i v e d 0.02 n m o l f l , 2 - 3 H l c h o l e c a l c i f e r o l in 0.1 m l e t h a n o l b y i n t r a p e r i t o n e a l i n j e c t i o n 24 h b e f o r e b e i n g k i l l e d . P l a s m a w a s e x t r a c t e d and c h r o m a t o g r a p h e d on S e p h a d e x LH20 c o l u m n s (3). I n t e s t i n a l m u c o s a c a l c i u m b i n d i n g a c t i v i t y w a s m e a s u r e d u s i n g the C h e l e x r e s i n a s s a y (4). RESULTS The DDT fed c h i c k s w e r e s i g n i f i c a n t l y l i g h t e r t h a n the c o n t r o l c h i c k s (173 ± 9 g : 241 ± 18 g; p < 0.01) but n o t d i f f e r e n t in w e i g h t f r o m the s t a r v e d c h i c k s (197 ± 1 1 g). In s p i t e of t h i s r e d u c t i o n in body w e i g h t the l i v e r w e i g h t / b o d y w e i g h t r a t i o w a s h i g h e s t in t h e DDT f e d g r o u p t h a n in t h e c o n t r o l and s t a r v e d c o n t r o l s (5 ± 0.1, 3 ± 0.2, 3 ± 0 . 2 ; p < 0.01). T h e DDT c h i c k s ' l i v e r s a p p e a r e d m u c h p a l e r t h a n the c o n t r o l s ' l i v e r s and w e r e p r e s u m a b l y m o r e h e a v i l y i n f i l t r a t e d with f a t . 24 H o u r s a f t e r an i n t r a p e r i t o n e a l i n j e c t i o n of 3 H - c h o l e c a l c i f e r o l the a m o u n t s of p l a s m a r a d i o a c t i v i t y c h r o m a t o g r a p h i n g a s v i t a m i n D, and i t s m e t a b o l i t e s , w e r e not s i g n i f i c a n t l y d i f f e r e n t a m o n g s t the 3 g r o u p s . E x a m i n a t i o n of the C a . B . A . of d u o d e n a l m u c o s a s u p e r n a t a n t s h o w e d t h a t the DDT f e d c h i c k s had s i g n i f i c a n t l y l e s s C a . B . A . t h a n t h e f r e e f e e d i n g c o n t r o l s (4.2 ± 0.3 X 1 0 ' 4 c p m : 8.1 ± 1 . 2 X 10" 4 c p m ; p < 0.01), but t h e C a . B . A . in both t h e

31^

DDT fed and s t a r v e d g r o u p s (3.8 ± 0.5 X 10

4

cpm) was s i m i l a r .

DISCUSSION T h e s e r e s u l t s c o n f i r m t h a t the i m p a i r m e n t of c a l c i u m a b s o r p t i o n a s s o c i a t e d with DDT f e e d i n g i s not due t o a n e f f e c t on v i t a m i n D m e t a b o l i s m (2), but m a y w e l l be r e l a t e d t o t h e c o n s i d e r a b l y l o w e r i n t e s t i n a l C a . B . A . T h i s m a y r e f l e c t a d i r e c t e f f e c t on i n t e s t i n a l c a l c i u m b i n d i n g p r o t e i n p r o d u c tion. H o w e v e r , the c o m p a r a b l e r e d u c t i o n of C a . B . A . in t h e s t a r v e d c h i c k s s u g g e s t s t h a t the r e d u c e d C a . B . A . with D D T f e e d i n g m a y s i m p l y r e s u l t f r o m r e d u c e d food i n t a k e . T h e i m p o r t a n c e of n o r m a l food i n t a k e in t h i s r e g a r d i s f u r t h e r s u p p o r t e d b y t h e o b s e r v a t i o n in r a t s (5) t h a t e x p e r i m e n t a l p r o t e i n m a l n u t r i t i o n r e d u c e s both c a l c i u m a b s o r p t i o n and i n t e s t i n a l c a l c i u m b i n d i n g p r o t e i n without a f f e c t i n g the m e t a b o l i s m of 3 H - v i t a m i n D 3 . We t h e r e f o r e s u g g e s t t h a t DDT i n d u c e d a n o r e x i a i m p a i r s i n t e s t i n a l c a l c i u m b i n d i n g p r o t e i n and s u b s e q u e n t c a l c i u m a b s o r p t i o n . T h i s w o r k w a s s u p p o r t e d b y g r a n t s f r o m t h e M i n i s t r y of H e a l t h and J o i n t R e s e a r c h F u n d of t h e H e b r e w U n i v e r s i t y and H a d a s s a h . REFERENCES 1.

Cooke, A. S.: (1973) E n v i r o n . P o l l u t . , 4, 8 5 - 1 5 2 .

2.

Nowicki, H. G., Wong, R. G., M y r t l e , J. F . , and N o r m a n , A. W . : (1972) J. A g r . F o o d . C h e m , 20, 3 7 6 - 3 8 0 .

3.

S i l v e r , J . , N e a l e , G., and T h o m p s o n , G. R . : (1974) Clin. Sci. and Mol. M e d . , 46 4 3 3 - 4 4 8 .

4.

W a s s e r m a n , R. H. and T a y l o r , A. N . : (1966) S c i e n c e 152, 7 9 1 - 7 9 3 .

5.

Kalk, W. J . and P i m s t o n e , B. L . : (1974) B r . J. N u t r .

32, 5 6 9 - 5 7 8 .

CYTOPLASMIC BINDING ÄND NUCLEAR UPTAKE OF CHOLECALCIFEROL METABOLITES INSIDE RAT DUODENAL MUCOSA CELLS.

A. UIMANN, M. BACHELET, J.F. CLOIX, M. BRAMI and J.L. FUNCK-BRENTANO. I.N.S.E.R.M. U.90 - Hôpital Necker - 161, rue de Sèvres - 75730 - Paris Cedex 15 - FRANCE. Inside chick intestine, 1 a,25-dihydroxycholecalciferol (1,25-(OH), the active metabolite of vitamin D^ (1) is thought to act in a similar way than steroid hormones do (2). Unlike for the chick, few data has been presented concerning its cellular mechanism of action inside rat intestine (3). We have previously demonstrated that both 1,25-(OH)^D^ and its hepatic precursor 25-hydroxycholecalciferol (25-(OH)D^) bind with a high affinity (1.2 to 2 nM) to cytoplasmic macromolecules from rat duodenal mucosa cells (4). Further, we suggested that each of these sterols bind to distinct sites. Additional data is presented herein concerning their cytoplasmic binding and nuclear uptake. MATERIALS AND METHODS 3 H-25-(OH)D3 (7.1 to 9.7 Ci/mmole) was purshased from Arrersham. 25-(OH)D3 and 5-6-Trans-cholecalciferol (5-6-Trans-D3) were given by Roussel Laboratories. 1,25-(OH) 311(1 24,25-dihydroxycholecalciferol (24,25-(OH)2D3) were kindly supplied by Dr Uskokovic (Hoffman-La Roche Laboratories, Nutley, N.J., U.S.A.) and 1 -hydroxycholecalciferol (la-OH)D, by Leo 3 Laboratories (Aarhus, Denmark). Biologically prepared H-l,25-(OH)^D^ (7.1 to 9.3 Ci/mmole) was generously given by Dr. M. Garabedian (Hôpital des Enfants Malades, Paris). The cytosol was prepared by high-speed centrifugation at 4° of duodenal mucosa homogenates from male weanling rachitic Wistar rats. Each cytosol fraction was incubated with sterols dissolved in 1 % (final concentration) of ethanol. Free and bound sterols were separated using a dextran-

316 coated charcoal suspension. To measure sterol nuclear uptake, duodenal mucosa cells were incubated at 37° for 1 hour with sterols in buffer A (TrisHCl : 10 mM, Sucrose : 250 mM, MgCl 2 : 1 mM, pH 7.4) . They were then extensively washed at 4° with the same buffer without sterol and homogenized. The homogenate was centrifuged at 800 x g for 10 min. The resulting crude nuclear pellet was rinsed and purified across a 2 M sucrose solution. The purified nuclear pellet was then mixed with buffer A containing Triton X-100 (1 %) and then rinsed with Triton-free buffer A. Finally, radioactivity and DNA content of these nuclei were measured. RESULTS Sterol Cytosol Binding : Aliquots of cytosol were incubated at 4° with 10 nM of either 3 or

H-1,25- (OH)

3 H-25-(OH)D_.

either alone (control binding) or in the presence of

large amounts of various unlabelled sterols. Table I shows the amount of tritiated sterol bound after a 3-hour incubation. Table I : Influence of various conpetitors on radioactive sterol (10 nM) binding.

Unlabelled sterol

Radioactive sterol binding (percent of control) 3

None

H-25-(OH)D 3

3

H-1,25-(OH)^

100

100

l,25-(OH) 2 D 3 (1 jjM)

35

24

25- (OH) D^ (1>IM)

23

23

24,25-(OH) 2 D 3 (1jjM)

23

23

la-(OH)D 3 (1 ;JM)

100

57

5-6-Trans-D 3 (2 ;jM)

100

84

D 3 (1 JJM)

100

100

3 Sterols without the 25-hydroxy1 group have no effect on H-25-(OH)D^ cytoplasmic binding. Conversely, 1 -(OH)D-. and 5-6-Trans-D-, have a low but 3 definite affinity for H-l,25-(OH)„D-, binders. In order to study sterol

317

binding dissociation, cytosol fractions were incubated at 0° with 10 nM of either 3H-1,25-(OH)2D3 or 3H-25-(OH)D3. After incubation, unlabelled 1,25-(OH)or 25-(OH)D^ (10 }M) were added respectively and radioactive binding measured after various times at 0° or 20°. At 20°, binding of both 3 3 H-1,25-(OH)and H-25-(OH)D^ is reversible within a few minutes. However 3H-l,25-(OH)2D3 dissociates faster than 3H-25-(OH)D3 does. At 0°, 3 H-1,25-(OH)„ binding remains reversible within 45 min. On the contrary, at 0°, H-25-(OH)D3 binding is reversible only if incubation time is below 1 hour. Over a 1-hour incubation, binding becomes non-reversible. Sterol Nuclear Uptake : 3 Duodenal mucosa fragments were incubated with H-25-(OH)D3 (5 nM) for 1 hour at 37°. Radioactivity was measured in purified nuclei as described in "Materials and3Methods". 0.78 + 0.41 pmole/mg DNA (mean + S.E.M. of 5 experiments) of H-25- (OH) D_. were found in association with nuclei (total 3 nuclear uptake). Similar experiments were carried out with H-25-(OH)D3 (5 nM) plus cold 25-(OH)D3 (0.5 ;JM) : 3H-25-(OH)D3 nuclear uptake was then 0.83 + 0.19 pmole/mg DMA (mean + S.E.M. of 5 experiments). Thus, no saturable 25-(OH)D3 nuclear uptake was demonstrated. When similiar experiments were carried out with 3H-1,25-(OH)2D3 (8 nM) , a 1.42 + 0.24 prole/mg DNA (mean + S.E.M. of 3 experiments) total nuclear uptake was observed. The presence of cold 1,25-(OH)~D, (0.8 JJM) in incubation media decreased 3 H-1,25-(OH)2D3 nuclear uptake to 0.75 + 0.13 pmole/mg DNA (mean + S.E.M. of 3 experiments). In other experiments 3 duodenal mucosa fragments were incubated with increasing amounts of H-l,25-(OH)2D3 either alone (total uptake) or in the presence of a 100-fold relative excess of unlabelled 1,25-(OH)2D3 ("non specific"uptake). 3H-1,25-(OH)2D3 "specific nuclear uptake" (total minus "non specific" uptake) appeared to be 3 saturable (maximum "specific" nuclear uptake : 1.4 pirole/mg DNA). This H-l,25-(OH)2D3 nuclear uptake was specific as incubations in the presence of cold 25- (OH) D 3 ( 8 jjM) resulted in no decrease of 3H-1,25-(OH) 2 D 3 nuclear uptake. Incubations in the presehae of la3 (OH) - (0.8 -_pM) resulted in a slight but non significant decrease of H-1,25-(OH)9D-. nuclear uptake.

318

DISCUSSION Experiments performed on cytosol confirm our previous report (4) that both 25-(OH)D^ and 1,25-(OH)^D^ bind to distinct macromolecular sites. It appears from competition experiments that binding to the ^H-25-(OH)D^-labelled sites requires the presence of a 25-hydroxy1 group as neither 1 a -(OH)D^ nor 5-6-Trans-D3 have any affinity for these sites. There results are in agreement with recently published work on 25-(OH)D^ cytoplasmic binding sites by Lawson et al. (5). Dissociation studies bring evi3 dence that these sites are distinct from H-1,25-(OH)„D^-labelled sites. 3 In addition H-25-(OH)D,-binding dissociation studies at 0° suggest that 3 H-25-(OH)D^-labelled sites are able of kinetic modification in the presence of 25-(OH)D^ itself. A 25-(OH)D^ covalent binding is unlikely since binding is reversible at 20°. As to sterol nuclear association, a specific and saturable nuclear uptake was demonstrated for 1,25-(OH)^D^, but not for 25- (OH) D^ - That is in agreement with experiments performed in vivo by Chen and De Luca (3). However, the physiological significance of 1,25-(OH)2^2 nuclear uptake and that of 25-(OH)D3 and l,25-(OH)2D3 cytosolic binding still remains unclear and requires further investigations. ACKNOWLEDGMENTS This work was supported by a grant of the I.N.S.E.R.M. REFERENCES 1. FRASER, D.R. and KODICEK, E. (1970) Nature 228, 764-766. 2. BRUMBAUGH, P.F. and HAUSSLER, M.R. (1975) J. Biol. Chem. 250, 15881594. 3. CHEN, T.C. and DE LUCA, H.F. (1973) J. Biol. Chem. 248, 4890-4895. 4. ULMANN, A., BRAMI, M., PEZANT, E., GARABEDIAN, M. and FUNCK-BRENTANO, J.L. (1977) Acta Endocrinol., in the press. 5. LAWSON, D.E.M., CHARMAN, M., WILSON, P.W. and EDELSTEIN, S. (1976) Biochim. Biophys. Acta 437, 403-415.

ARE THE CYTOSOLIC BINDING PROTEINS FOR

25-HYDROXYCHOLECALCIFEROL

ARTIFACTS?

H. Van Baelen and R. Bouillon Laboratorium voor Experimentele Geneeskunde, Rega Instituut, Minderbroedersstraat

10, B-3000 Leuven, Belgium.

Several authors (1-8) have shown recently that most nucleated tissues of man and rat contain a 5-6 S macromolecule responsible for the specific binding of 25-hydroxycholecalciferol

(250HD^)•

In our opinion this cytosolic binding protein is related to the serum 250HD2 _ binding protein (transcalciferin) which sediments at 4.1 S. conclusion is based on the following

This

observations:

I. A 250HD^ - binding protein, sedimenting at 5.8 S, can be formed

by incu-

bating appropriate amounts of cytosol and diluted rat or human plasma. This binding protein could not be formed with cytosol heated at 60°C.

2. The cytosolic binding protein can be transformed binding protein by heating cytosol at 60°C.

into a 4.! S 250HD^-

Incubation of heated cyto-

sol with untreated cytosol results in the formation of a 5.8 S binding protein.

3. A complete immunological

identity

is found between serum transcalcifer-

in and a component of human kidney cytosol using monospecific man transcalciferin

anti-hu-

antiserum.

The 5.8 S cytosolic binding protein is undetectable in cytosol of extensively washed rat kidney cells but detectable after addition of diluted serum.

These results strongly suggest that the cytosolic binding protein,

found previously widely distributed in different species, is largely an artifact due to plasma contamination during the preparation of cellular homogenates.

320 F u r t h e r studies have s h o w n that the c y t o s o l i c c o m p o n e n t , r e s p o n s i b l e the t r a n s f o r m a t i o n of s e r u m t r a n s c a l c i f e r i n , labile.

is n o t d i a l y s a b l e and h e a t

Since c y t o s o l , treated w i t h t r y p s i n , does n o t i n t e r a c t

transcalciferin,

the c y t o s o l i c c o m p o n e n t is p r o b a b l y a p r o t e i n .

sucrose gradients

for

with In

linear

the c y t o s o l i c p r o t e i n s e d i m e n t s a r o u n d 4 S.

A s far as the m o l e c u l a r b a s i s of the t r a n s f o r m a t i o n of s e r u m f e r i n is c o n c e r n e d

two m o d e l s are e s p e c i a l l y

transcalci-

attractive:

1. The 5.8 S c y t o s o l i c b i n d i n g p r o t e i n is the r e s u l t of a d i m e r i z a t i o n of s e r u m t r a n s c a l c i f e r i n , p r o m o t e d by the 4 S c y t o s o l i c

protein.

2. The 5.8 S c y t o s o l i c b i n d i n g p r o t e i n is a c o m p l e x f o r m e d b e t w e e n t r a n s c a l c i f e r i n and the 4 S c y t o s o l i c

serum

protein.

REFERENCES

1. H a d d a d , J . G . , and B i r g e , S.J.

(1971) B i o c h e m . B i o p h y s . Res. C o m m u n .

45,

829-834 2. H a d d a d , J . G . , and B i r g e , S.J.

(1975) J. Biol. C h e m . 2 5 0 ,

3. H a d d a d , J . G . , W a l g a t e , J., M i n , C . , and H a h n , T.J. p h y s . A c t a 444, 4. E d e l s t e i n , S.

(1976) B i o c h i m .

Bio-

921-925

(1974) B i o c h e m .

Soc. Spec. P u b l . _3> 43-54

5. B o u i l l o n , R . , V a n K e r k h o v e , P . , and De M o o r , P. R e s e a r c h suppl.

299-303

to vol. 2J_,

(1976) C a l c i f i e d

172-175

6. L a w s o n , D . E . M . , C h a r m a n , M . , W i l s o n , P . W . , and E d e l s t e i n , S. B i o c h i m . B i o p h y s . A c t a 437,

Tissue

(1976)

403-415

7. K r e a m , B . E . , R e y n o l d s , R . D . , K n u t s o n , J . C . , E i s m a n , J . A . , and D e L u c a , H.F.

(1976) A r c h . B i o c h e m . B i o p h y s . J_76, 779-787

8. O k u , T . , O o i z u m i , K . , H o s o y a , N. 9-25

(1974) J. N u t r . Sci. V i t a m i n o l .

20,

EFFECTS OF lct,25-DIHYDROXYVITAMIN D 3 ON ACTIVE INTESTINAL

INORGANIC

PHOSPHATE ABSORPTION

Marl in W. Walling VA Wadsworth Hospital Center and Schools of Dentistry and Medicine, University of California, Los Angeles, California Since the 1930's it has been clear that vitamin D increases the percentage of phosphorus that is absorbed from the diet (1-3).

However, early inves-

tigators concluded that the effects of vitamin D on phosphorus

balance

were an indirect result of its marked effects on calcium absorption

(3,4).

In addition, studies on the kinetics of inorganic phosphate (Pi) absorption indicated that this molecule crossed the intestinal epithelium by passive diffusion

(5,6).

However, there is recent evidence for carrier-

mediated Pi absorption in chick intestine (7).

In 1961, the extensive

everted gut sac studies by Harrison and Harrison convincingly

demonstrated

that vitamin D increased inorganic phosphate (Pi) absorption by rat small intestine (8).

Nevertheless, these workers also found an apparent link of

Pi transport to Ca because the removal of Ca abolished concentrative Pi absorption as well as the effect of vitamin D (8). The interpretation of the movement of ions across intestinal epithelia is complicated by the existence of an electrical

potential difference (PD) of

about 5mV which can produce net diffusional movement (the blood side is electropositive relative to the gut lumen).

Evaluation of rat intestinal

Pi transport according to thermodynamic criteria led Asano (9) and Noble and Matty (10) to the conclusion that Pi movement was not an active process because the observed flux ratios were not different from those predicted for passive diffusion by the Ussing equation (11) (at pH 7.4 Pi valence is -1.6, if the PD is 5mV and there is no concentration gradient the passive ratio is 1.35).

However, the data of the Harrisons'

(8) are

highly suggestive of active transport because the Pi concentration appear to exceed those that could be produced by the PD.

ratios

The first rigor-

ous demonstration of active Pi absorption by the intestine was provided by Helbock et al in 1966 (12) when they measured unidirectional across rat duodenum in vitro with electrochemical

Pi fluxes

gradients eliminated by

322 the method of Ussing and Zerahn (13).

More recently, Walling and Kimberg

employed a similar protocol and demonstrated that 1,25 dihydroxyvitamin D3 (1 ^ ( O H ^ D g )

stimulates active Pi absorption across rat duodenum (14).

The data subsequently presented will provide numerous examples of active Pi absorption by rat small

intestine and will conclude with a model

de-

scribing the effects of 1,25(0H)2D3 on this tissue. EXPERIMENTAL

PROCEDURES

The data which are presented herein were obtained employing a modification of the in vitro technique of Ussing and Zerahn (13) (see references 14-16 for details).

Unless specified otherwise, a modified Krebs-Ringer, HCO3-

buffer, pH 7.4 was employed, containing llmM D-glucose, 1.25 mM Ca and 2.4 mM Pi.

Because experiments were conducted in the absence of electrochemi-

cal gradients, a significant net flux requires an active transport process. All animals referred to as vitamin D-deficient were male Holtzman rats raised from weaning on a non-rachitogenic vitamin D-free diet in a room devoid of UV light for a minimum of 6 weeks.

Chemically synthesized vita-

min D-compounds were provided by Hoffman-LaRoche, Nutley, N.J. 40

UJ I2D3 have demonstrated that Ca transport is stimulated before the appearance of CaBP (3,4).

Furthermore, we have observed that Ca transport is induced

by 1,25(OH>2D3 even when CaBP synthesis is blocked by cycloheximide (Cyclo) (5).

We report here that the appearance of CaBP correlates inversely with the

retention of calcium by the mucosa after 1,25(01-1 >203 administration.

Therefore,

we propose that CaBP may be essential for the regulation of Ca accumulation by the intestinal absorptive cell.

One-day-old chicks were fed a vitamin D-deficient diet containing 0.43% Ca and 0.3% phosphorus for 2-1/2 weeks.

These chicks were then given 62.5 pmol of

1,25(OH)2D3 im in 100 nI of propylene glycol (PG).

Cyclo in doses of 20 /ug

in 100 MI PG was injected ip starting one hour before 1,25(OH)2D3 administration, at 4-hour intervals for the first 12 hours, and then at subsequent 6-hour intervals. PG (100 pi) was given to the control groups in place of 1,25(OH>2D3 or Cyclo. Ca transport and Ca accumulation were measured in vivo at various times after 1,25(OH>2D3 administration by the ligated duodenal loop technique.

After the

chicks were anesthetized, 0.25 ml of modified Trowells T8 medium containing 3 mM Ca, 2.5 ¡ i d

45

Ca, and 5 fiC\

3

H-polyethylene glycol (PEG) was injected

into the proximal half of the duodenum.

After 15 minutes, blood was collected,

3^6 the duodenal loop was removed, luminal contents were recovered, and the tissue was solubilized.

^H-PEG was used to correct for the amount of extracellular

in the tissue digest.

The cytosol from the mucosal homogenate of the distal

half of the duodenum was used to determine CaBP content by radial diffusion.

^Ca

immuno-

We repeated the experiment with 50 /¿9 doses of Cyclo and also ex-

amined the number of granules within the mitochondria of intestinal absorptive cells fixed in

osmium-pyroantimoniate.

Ca transport as measured by serum ^ C a

was stimulated by

presence and absence of Cyclo within 4 hours.

1,25(OH>2D3 in the

Ca accumulation in the mucosa

of the 1,25(OH>2D3 treated chicks was stimulated at 4 hours, peaked at 6 hours, and then decreased to control level (0 hr) by 24 hours while Ca accumulation the 1,25(OH)2D3 + Cyclo treated chicks continued to rise (Figure 1). chicks given Cyclo without control.

in

Rachitic

1,25(OH)2D3 showed no significant difference f r o m the

CaBP was first detected in the 1,25(OH>2D3 treated animals at 8 hours.

CaBP synthesis was completely blocked in chicks receiving Cyclo except for the 24 hour 1,25(OH>2D3 + Cyclo treated animals where CaBP was extensively

inhibited.

When we repeated the experiment using a higher dosage of Cyclo (50 jug) and an

Figure 1.

The effect of

CaBP

1,25(OH)2D3 on Ca accumulation by chick intestine in the absence ( — 0 — )

and in the

presence of cyclo and of cyclo alone

( — • — ) (—-A-—).

Closed symbols indicate a significant difference (P2D3 is indicated.

8

12 TIME ( h o u r i )

16

oral administration of 12D3, the results were similar except for the following:

no CaBP was detected in any chick receiving Cyclo; Ca transport and Ca

accumulation were stimulated by 3 hours; and CaBP was detected at 6 hours.

The

preliminary results on mitochondrial mineralization in the 18 hour treated groups indicated that the mitochondria of cells near the tip of the intestinal villus for chicks receiving 1,25(OH>2D3 plus Cyclo contained five times as many granules as those from either the control (0 hr) or 1,25(OH)2D3 treated animals.

The stimulation of intestinal Ca accumulation by 1,25(OH)2D3 does not require CaBP and in fact is decreased as CaBP is synthesized.

This decrease of Ca accu-

mulation does not occur if CaBP synthesis is blocked by Cyclo.

This conclusion

is supported by our preliminary studies on mitochondrial mineralization.

We pro-

pose that CaBP may be essential to the intestinal absorptive cell's ability to regulate intracellular calcium content.

References 1.

Emtage, J.S., Lawson, D.E.M., and Kodicek, E.:

Vitamin D-induced synthesis

of mRNA for calcium binding protein; Nature, 246, 100-101 (1973). 2.

Ebel, J.G., Taylor, A.N., and Wasserman, R.H.:

Vitamin D-induced calcium

binding protein of intestinal mucosa (Relation of vitamin D dose level and lag period); Am. J. Clin. Nutr. 22, 431-436 (1969). 3.

Morrissey, R.L., Zolock, D.T., Bikle, D.D., Empson, R.N., and Bucci, T.J.:

Intestinal response to 1a,25-dihydroxycholecalciferol; (Unpublished results presented at FASEB, 1975). 4.

Spencer, R., Charman, M., Wilson, P., and Lawson, E.:

Vitamin D-stimulated

intestinal calcium absorption may not involve calcium binding protein directly; Nature, 263, 161-163 (1976). 5.

Bikle, D.D., Zolock, D.T., Morrissey, R.L., and Herman, R.H.:

The disso-

ciation of 1,25(OH>2D3-induced CaBP production and alkaline phosphatase activity from calcium transport by actinomycin D and cycloheximide; (Published in these Proceedings).

EFFECTS OF VITAMIN D AND ITS METABOLITES ON BONE

J . L . I v e y , E.R. Morey, C.-C. L i u , J . I . Rader, and D.J. Baylink American Lake Veterans Administration H o s p i t a l , Tacoma, WA 98493, and Department of Medicine, U n i v e r s i t y of Washington, S e a t t l e , WA

98195,

and NASA-Ames Research Center, Moffett F i e l d , CA 94035, USA. Effects of vitamin D (+D) or i t s deficiency (-D) on bone could be manifested by changes in c e l l number, cell a c t i v i t y , or the quantity and quali t y of the mineral and matrix deposited.

We propose to examine each of

these and assess the mechanisms involved in the action of vitamin D on bone. The change in bone most frequently associated with -D i s impaired bone mineralization.

The processes involved in m i n e r a l i z a t i o n of bone include:

production of osteoid by o s t e o b l a s t s , maturation of the osteoid, tion of m i n e r a l i z a t i o n , and m i n e r a l i z a t i o n . these processes appears to be decreased (1).

initia-

In -D the rate of each of In a number of c o n d i t i o n s ,

i n c l u d i n g -D, i n i t i a t i o n of m i n e r a l i z a t i o n ( I ) occurs without delay when osteoid i s mature, as evidenced by l o s s of a c i d i c proteoglycans Thus, in -D, the delay in I maturation (R

om

(2).

i s due to the decrease in the rate of osteoid

).

The effect of -D on the rate of m i n e r a l i z a t i o n has been determined using an electron microprobe to measure the concentration of Ca and P in minera l i z i n g bone (3).

The concentration of Ca in bone as a function of time

(based on the distance from the m i n e r a l i z i n g front and the rate of bone formation) i s shown in Figure 1.

In each group, the shape of the curve

of bone [P] as a function of time was s i m i l a r to that shown in Figure 1 for bone [Ca].

I t i s apparent that the rate of Ca deposition in bone i s

s i g n i f i c a n t l y reduced in -D. [Ca] in -D was s t i l l

In a d d i t i o n , the s l i g h t decrease in bone

apparent at 200 h, suggesting that the maximum extent

350

i

6

1

§ i

\th 8e

I a

^

75-

I" w e t w t Control 1 H r 2. 15 0.92 (1.3)* (4.6) 0.24 (1.•1) Repleted 1.65 0.20 0.23 Control Repleted

6 Hr

1. 74 1.53

(1.1)

24 H r 1. 18 Control (5.6) Repleted 0.21 * R a t i o of C o n t r o l to R e p l e t e d

1. 72 0. 73

(2.4)

0.57 0.60

(0. 9)

0. 30 0.19

(1.6)

0. 23 0.16

(1..4)

Discussion The d i m i n i s h e d l o c a l i z a t i o n of 3 H - l a , 2 5 ( O H ) 2 D 3 in b o n e from rats p r e t r e a t ed w i t h l a , 2 5 ( O H ) 2 D 3 s u g g e s t s that b o n e c o n t a i n s a finite n u m b e r of s p e c i fic b i n d i n g s i t e s for the h o r m o n e . The i n c o r p o r a t i o n of the m e t a b o l i t e at the t a r g e t site is r a p i d in b o t h r a c h i t i c and r e p l e t e d g r o u p s as e v i d e n c ed by the d i s p l a c e m e n t of 3 H - l a , 2 5 ( O H ) 2 D 3 from b i n d i n g sites by u n l a b e l e d l a , 2 5 ( O H ) 2 D 3 in the e a r l i e s t time group. The l a b e l e d m e t a b o l i t e b i n d s s p e c i f i c i a l l y to b o n e cell p o p u l a t i o n s as i n d i c a t e d by the a u t o r a d i o g r a p h ic images. This s t u d y , h o w e v e r , d i d n o t e v a l u a t e the q u a n t i t a t i v e s e l e c tivity of the h o r m o n e for c e r t a i n types of b one c e l l s u n d e r these e x perimental conditions. L a b e l e d m e t a b o l i t e w a s o b s e r v e d p r i m a r i l y in o s t e o c y t e s w i t h l e s s e r a c c u m u l a t i o n in o s b e o b l a s t s . Osteoclasts contained l a b e l e d m e t a b o l i t e b u t the r a t i o s of l a b e l e d m a t e r i a l in such cell p o p u lations was not assessed. The r e s u l t s c l e a r l y d e m o n s t r a t e the p r o b a b l e c e l l u l a r s i t e s of a c t i o n 3 H - l a , 2 5 ( O H ) 2 D 3 in r a c h i t i c rat b o n e , a n d its lack of i n c o r p o r a t i o n into r a c h i t i c e p i p h y s e a l c a r t i l a g e . M a n i p u l a t i o n of a v a i l a b l e b i n d i n g s i t e s using u n l a b e l e d l a , 2 5 ( O H ) 2 D 3 i n d i c a t e s the p r e s e n ce of a finite n u m b e r of sites u p o n the d e v e l o p m e n t of v i t a m i n D d e f i ciency .

371

A u t o r a d i o g r a p h s s h o w i n g i n c o r p o r a t i o n of -^H-la, 25 ( O H ) 2 D 3 in v i t a m i n D - d e ficient c o n t r o l (Fig 1-3, 1 hr, 6 h r a n d 24 hr) and l a , 2 5 ( O H ) 2 D 3 r e p l e t e d rats (Fig 4-6, 1 hr, 6 hr a n d 24 hr). References 1. Tanaka, Y. a n d H. F. D e L u c a , Bone m i n e r a l m o b i l i z a t i o n a c t i v i t y of 1 , 2 5 - d i h y d r o x y c h o l e c a l c i f e r o l , a m e t a b o l i t e of v i t a m i n D, A r c h B i o c h e m B i o p h y s 1 4 6 : 5 7 4 - 5 7 8 , 1971. 2. Raisz, L. G., C. L. T r u m m e l , M. F. H o l i c k and H. F. D e L u c a , 1 , 2 5 - D i h y d r o x y c h o l e c a l c i f e r o l : a p o t e n t s t i m u l a t o r of b o n e r e s o r p t i o n in tissue c u l ture, S c i e n c e 1 7 5 : 7 6 8 - 7 6 9 , 1972. 3. M e y e r , W. L. and A. S. K u n i n , The i n d u c t i v e e f f e c t of r i c k e t s on g l y c o l y t i c e n z y m e s of rat e p i p h y s e a l c a r t i l a g e a n d its r e v e r s a l by v i t a m i n D a n d p h o s p h a t e , A r c h B i o c h e m B i o p h y s 1 2 9 : 4 3 8 - 4 4 6 , 1969. 4. W e z e m a n , F. H., 2 5 - H y d r o x y v i t a m i n D 3 ¡ A u t o r a d i o g r a p h i c e v i d e n c e of s i t e s of a c t i o n in e p i p h y s e a l c a r t i l a g e a n d b o n e , S c i e n c e 1 9 4 : 1 0 6 9 - 1 0 7 1 , 1976. 5. W e z e m a n , F. H., M. J. Favus, W. A. R e y n o l d s a n d K. E. K u e t t n e r , I n c o r p o r a t i o n of 3 H - 2 5 H y d r o x y c h o l e c a l c i f e r o l by fetal b o n e a n d e p i p h y s e a l c a r tilage a f t e r p l a c e n t a l t r a n s f e r , Ant Rec. 1977. P r o c e e d i n g s of the 9 0 t h M e e t i n g , A m e r i c a n A s s o c i a t i o n of A n a t o m i s t s . 6. Frolik, C. A. and H. F. D e L u c a , M e t a b o l i s m of 1 , 2 5 - d i h y d r o x y c h o l e c a l c i ferol in the rat, J. C l i n Invest 5 1 : 2 9 0 0 - 2 9 0 6 , 1972.

V I T A M I N D DEPENDENT B O N E DISEASE: LONG-TERM RESPONSES TO V I T A M I N D A N A LOGS AND EFFECT O N TETRACYCLINE BASED B O N E DYNAMICS

B o y Frame, M.D., and A.M. Parfitt, M.D. H e n r y F o r d Hospital, Detroit, MI 48202 Two sisters aged 25 and 17 years w i t h vitamin D dependent bone disease have b e e n observed since infancy.

The diagnosis rests on the early onset

of severe clinicfil and radiographic riclets in the absence of dietary deficiency of v i t a m i n D, hypomagnesemia, intestinal malabsorption or renal failure; autosomal recessive mode of inheritance; hypocalcemia and hypophosphatemia in the untreated state (Table l); secondary hyperparathyroidism; and the need for continued pharmacologic doses of vitamin D 0 (2.5 - 5.0mg/d), DHT (l.4 - 1.8mg/d), and 25KCC (240 - 320/|g/d) to prevent biochemical relapse and maintain bone healing.

B y contrast, doses

of 1CTHCC m u c h closer to physiological (l.2 - 1.8/Jg/d) were adequate.

The

m e a n plasma values during treatment for at least one year with the three latter compounds are shown in Table 2. Normal plasma levels of 25HCC were found during treatment w i t h DHT and normal 1,25DHCC levels during treatment w i t h lOHCC (by courtesy of M. Haussler).

These findings exclude a defect in 25 hydroxylation.

Table 1

Pre-treatment biochemical data M.C.

K.C. 8 mo.

Age of onset Serum Ca (mg/lOOml)

6.0 - 6.5

7.0 - 7.9

Serum P

1.6 - 3.0

3.6 - 4.3

22 - 30

29 - 43

(mg/lOOml)

Alk P'tase (BU) B U N (mg/lOOir!) Table 2

K.C.

M.C.

l6 mo.

7 - 1 0

9 - 1'»

Biochemical data during treatment

fJsA

C

1400

9.67

25HCC

260

lOHCC

mg/l00mf

/LlEn/ml^S 40 )

3.69(7)

45.o(l)

10.29

3.56(12)

28.0(3)

1.2

9.95

3.00(10)

46.5(2)

1600

9.I3

2.16(10)

80.5(2)

25HCC

320

9.57

3.37(10)

25.0(2)

1QHCC

1.7

9.77

3.07(11)

38.5(4)

DHT

DHT

37^ Unexplained were the o b s e r v a t i o n s t h a t PTH l e v e l s

i n hoth p a t i e n t s

i n c r e a s e d d u r i n g treatment with DI!T and 1CCTICC, and normal a d m i n i s t r a t i o n of 25IICC.

were

only d u r i n g the

I n M.C. t h i s occmrred even though the plasma

calcium was h i g h e r on 1CTFTCC than on 25HCC. I n c l u d i n g the two p a t i e n t s

of t h i s r e p o r t ,

the response to treatment with

1,2I5T)ITCC or 1GITCC has been determined in 10 p a t i e n t s w i t h v i t a m i n D d e pendency ( l - ' t ) , was 1.

and the mean d a i l y dose r e q u i r e d f o r h e a l i n g of

The minimum d a i l y dose of

vitamin D d e f i c i e n c y r i c k e t s Withdrawal

rickets

1, 2TDHC0 and IflTTCC needed to

i s not known, b u t i s not more than 0.5j^g

of vitamin D 9 and phosphate a t age 17 in M.C. and 9 y e a r s

K.C. was f o l l o w e d a f t e r 13 to 18 months by moderate biochemical r e c u r r e n c e of

overt r i c k e t s

in K.C.

and changes i n c o r t i r a l

m o d e l l i n g data obtained from t e t r a c y c l i n e V.'hile on treatment, rate

o s t e o i d seam number,

(MATt), the percentage of

of r e s o r p t i o n spaces,

seam width,

seams t a k i n g a t e t r a c y c l i n e

sigma^ ( l i f e - s p a n of the o s t e o i d

i n seam number,

and bone f o r m a t i o n r a t e ,

seam width,

bone

mineral

(3)» in

relapse,

labelled rib biopsies

f o r m a t i o n r a t e were w i t h i n the normal range, increase

cure

re(Table

3).

appositional

label,

number

seam) and bone

riff treatment t h e r e was an

number of

resorption

and a r e d u c t i o n i n MAR.

spaces,

sietna^

These o b s e r v a t i o n s

are

c o n s i s t e n t with the bone e f f e c t s of decreased vitamin P and i n c r e a s e d PTTl. K.C.

M.C.

on

off

on

off

Ca mg/lOOml

9.8

8.8

9.2

8.8

P mg/lOOml

4. 2

3.1

3.7

2.4

14.2

30.9

6.3

11.7

3.3

5.5

2.1

4.7

P'tase

(BU)

Seams/mm^

11.1

Seam width^im MAR fjm/(1 t

70

Label

Ties. Spaces Sigma^ Vf

1.03

(days)

tfyear

/mm^

2.09 94 21

23.2 0.64 78 2.38 12^ 34

6.« 1.05 95 0.55 73 25

24.9 0.77 88 ] .10 98 56

Age comparable normal valxies

1.4 10.6 1.3 85 0.55 55 21

+ + + + + +

+

1.3 4.2 0.43 5 0.49 36 15

T a b l e 3 - T e t r a c y c l i n ^ - b a s e d c o r t i c a l - b o n e remodelling data from l e f t eleventh r i b i n K.C. and M.C. on and o f f treatment with e r g o c a l c i f e r o i (Vitamin P 0 ) and phosphate

375 On thr> assumption that a steady state hart been attained, the degree of uncoupling between matrix and mineral appositions! rates necessary to produce the observed increases in seam width can be estimated at 0.2f)|jm/«l in K.C. and 0.29jum/d in M.C., with estimated values for matrix appositional rate of 0.8'^jm/d in K.C. and l.O^fm/d In M.C.

This is a much greater

degree of uncoupling than vo observe in adults with osteomalacia of various etiologies.

Whether this is simply due to age or is peculiar to

vitamin D dependent bone disease is unknown.

However, both this morpho-

logic difference and the difference in PTII suppressibility might reflect a difference in vitamin !) profile between vitamin D deficiency in which all metabolites are low, and vitamin D dependency in which there is selective loss of 1 ,hvdroxylated metabolites only with normal levels of 25HCC and 2'i, 25DHCC.

A similar difference in metabolite profile might

account for the recurrence of florid radiographic rickets in K.C. at a time when the usiial bone dynamic abnormalities of osteomalacia were present to only minimal degree. Conclusions:

1) In vitamin D dependency the mean dose of ICfHCC or

1,25DIICC required for adequate treatment of vitamin D dependency is l.ltyfg/day, which is more than twice that needt-d for the treatment of vitamin D deficiency rickets. 2) Tetracycline based bone remodelling dynamics in vitamin D dependency indicates an \mcoupling between matrix synthesis and mineral appositional rate which is greater than that observed in adults with osteomalacia. 3) It seems unlikely that an absence of renal 25HCC 1 hydroxylase will explain all features of vitamin D dependent bone disease. REFERENCES 1. Fraser, D., Sang V.Tiay Kooh, Kind, H.P., Rollick, M.F., Tanaka, Y., DeLuca, H.F. (1973) N Engl J Med 289:817-822 2. Reade, T.M., Scriver, C.R., Glorieux, F.H., Nogrady, B., Delvin, E., Poirier, R., Holick, M.F., DeLuca, H.F. (1975) Ped Res 9:593-59^ 3. Balsan, S. and Garabedian, M. (1975) Pe 10" M PTH and 10 _6 M 250HD 3> d i s s o c i a t i o n constant for 1,25(0H) 2 D 3 was increased from 10 5

6x10 M ethinyl oestradiol the maximal response.

-10

The _8

M to 10 M by

in the culture medium but there was no change in

C a l c i t o n i n at a concentration of 6xlO _ 1 1 M 9

inhibits

50% of the bone resorption induced by both 10~ M 1,25(0H)2D 3 and 10 _7 M PTH.

413 Table 2. Inhibition of bone resorption by calcitonin (CT), ethinyl oestradiol (EO), progesterone(Pr) and androstenedione (An), induced by a maximal dose of 1,25(0H)2D3, PTH and 25OHD3. Resorbing hormone: Maximal dose

Inhibitin g hormone: 50? inhibitory-c ose CT EO Pr An

1,25(0H)2D3

10"9M

6xlO_11M

6X10"5M

6xl0-5

PTH

10"7M

6xlO-11M

6X10"5M

6x10"5

250HD3

10"6M

6X10"5M

-

6xl0"5 -

-

-

2 4 R , 2 5 ( 0 H ) 2 D 3 did not inhibit the action of 1 , 2 5 ( 0 H ) 2 D 3 on bone; no dose of 2 4 R , 2 5 ( 0 H ) 2 D 3 was found to alter either the maximal

response of the

dissociation constant of 1,25(OH)2^3 for bone. Conclusions 1. A hydroxyl

group at the la and 25 position bestows bone resorbing

properties on the vitamin D molecule. both hydroxyls are present.

Maximum potency is achieved when

The maximal

response for 2 5 0 H D 3 is greater

than that for laOHD 3 . 2. Calcitonin, at the same concentration, inhibits the action of PTH and 1,25(0H)2D3. 3. The steroid sex hormones, at the same concentrations, inhibit the action of 1 , 2 5 ( 0 H ) 2 D 3 , 2 5 0 H D 3 and PTH. 4. The natural metabolite 2 4 R , 2 5 ( 0 H ) 2 D 3 resorbs bone and has no inhibitory action on

1,25(0H)2D3.

References 1. Reynolds JJ, Holick MF and DeLuca HF: The role of vitamin D metabolites in bone resorption. Calcif. Tiss. Res. 25.» 333-339

(1974).

2. Peacock M, Taylor GA and Redel J: The action of two metabolites of vitamin D 3 : 25,26 dihydroxycholecalciferol

and 24,25

ciferol on bone resorption. Febs Lett.

248-250

dihydroxycholecal-

(1976).

3. Webster LA, Atkins D and Peacock M: A bioassay for parathyroid

hormone

using whole mouse calvaria in tissue culture. J. Endocr. 62_, 631-637

(1974).

4. Atkins D and Peacock M: A comparison of the effects of the calcitonins, steroid hormones and thyroid hormones on the response of bone to parathyroid hormone in tissue culture. J. Endocr. 64, 573-583

(1975).

Acid Phosphatase in Bone Cells from Vitamin D Deficient Rats

S. Silverton and M. Kaye Montreal General Hospital Research Institute, 1650 Cedar Avenue, Montreal, Quebec, Canada H3G 1A4 The action of vitamin D metabolites on bone cells is more difficult to assess than similar actions of peptide hormones.

As D metabolites,

being lipid soluble are probably transported intracellularly, assessment of their effects is most easily made at the end product: a presumed change is activation or synthesis of an enzyme or enzyme system in the target cell.

Our experiments have attempted to look at one D-specific

response in bone cells, acid phosphatase activity, which we have quantitated in individual osteoclasts and osteoblasts obtained from the femurs of D-deficient and control rats. Methods Weanling male Sprague-Dawley rats were maintained on a semi-synthetic Vitamin D deficient diet containing 0.8% Ca, and 0.7% P for six weeks. Control animals were given the same diet with added Vitamin D2 (80 I.U./ lOOg).

At sacrifice, bone smears from the metaphyses of the femurs

were prepared on microscope slides and immediately quick-frozen.

Lyo-

philized slides were incubated for acid phosphatase activity using the Barka and Anderson diazo dye method with Napthol AS-TR phosphate as substrate and pararosanilin as coupler.

Osteoclasts and osteoblasts from

each animal were analyzed for the optical density of dye product using a computer-linked microdensitometer.

Osteoclasts were identified as

acid phosphatase positive multinucleate cells.

Osteoblasts were distin-

guished from other mononucleate cells by comparison with calvaria smears which had also been incubated for acid phosphatase. acid phosphatase positive cells was also quantitated.

A third group of These cells were

irregular mononuclear cells with centrally located nuclei.

Results from

different cell types were compared for statistical significance using Student's "t" test.

kl6 Results The optical density values from all groups are shown in Figure 1. Osteoclasts and osteoblasts from D-replete animals showed significantly higher levels of acid phosphatase activity than the same cells from D-deficient animals.

Other acid phosphatase positive cells showed no

significant differences in activity whether the cells were from D-deficient or control animals.

0.1200

10.004 6

£0.0154 0.0074 +

0.1000 i0.0063

0.0800 *£0.0032

0.0600-

significant difference p-c.01

*"not significant at p30% at a dilution of 1:1,000 sixty-eight days after immunisation. Further studies of this particular serum have been performed. Serum dilution curves in the presence of increasing amounts of cholecalciferol showed significant displacement of the binding to tritium labelled cholecalciferol. This displacement was not seen when the pre-immunisation serum was used. Studies of the post immunisation serum at a dilution of 1:2,000 showed significant cross reaction with cholecalciferol, 25 hydroxycholecalciferol, 25 hydroxyvitamin D 2 , 24,25 dihydroxycholecalciferol and 1,25 (OH^D^ and, to a much lesser extent, with la hydroxycholecalciferol, 1, 24,25 trihydroxycholecalciferol and vitamin D^• This serum did not significantly cross react with cholesterol, 7-dehydrocholesterol and dihydrotachysterol.

RADIOIMMUNOASSAY SYSTEM FOR CHOLECALCIFEROL The antiserum prepared above was used at a dilution of 1:2,500 to produce a sensitive and reproducible standard curve for cholecalciferol. Preliminary studies on human serum show that following extraction with chloroform/ methanol, cholecalciferol can be separated satisfactorily from its more polar metabolites by paper chromatography (4) and the extracts eluted off the paper in a suitable form for use in the assay.

461 PREPARATION OF ANTISERUM TO 1,25-DIHYDROXYCHOLECALCIFEROL 1.

Preparation of 1,25-Dihydroxycholecalciferol - Bovine Serum Albumin Conjugate l,25-(OH)2Do 25-hemisuccinate was prepared from 1,25-(OH)2"/-dehydrocholesterol 25-hemisuccinate by photolysis and thermal rearrangement. This conjugate was prepared in the manner described above for the cholecalciferol-protein conjugate. The incorporation of 1,25-(OH)2D3 into the conjugate was assessed as before. The maximum value obtained was 19 moles of 1,25-(OH)2D3 per mole of bovine serum albumin.

2.

Immunisation of Rabbits Eighteen rabbits were immunised using 200 yg and 400 yg of conjugate per animal as before. They were bled at weekly intervals for 3 weeks after immunisation as for the cholecalciferol series of rabbits. The sera obtained were tested for binding to tritiated 1,25 (OH)2D3 which was prepared from tritiated 25 hydroxycholecalciferol using a vitamin D deficient chick kidney homogenate as described by Norman et al (5).

3.

Results Four weeks after immunisation sera from 3 rabbits shows a small rise in the titre of binding to tritiated 1,25-(OH)2D3 when compared to the pre-immunisation sera for each particular rabbit. The serum from one rabbit has been studied further and significant displacement of binding to the tracer by l,25-(OH)2l>3 can be demonstrated .

ACKNOWLEDGEMENT S We are grateful to the National Kidney Research Fund for financial support and to Professor V. K. T. James for expert advice. REFERENCES 1.

Erlanger, B. F., Borek, F., Beiser, S. M. and Lieberman, S. (1958) J. Biol. Chem. 234 1090

2.

Vaitukaitis, J., Robbins, J. B., Nieschlag, E. and Ross, G. T. (1971) J. Clin. Endocr. Metab. 33 988 McLaughlin, M., Fairney, A., Lester, E., Raggatt, P. R., Brown, D. J. and Wills, M. R. (1974) Lancet 1 536

3. 4. 5.

Bikle, D. D. and Rasmussen, H. (1974) Biochem. Biophys. Acta 362 425 Norman, A. W., Midgett, R. J., Myrtle, J. F. and Nowicki, H. G. (1971) Biochem, Biophys. Res. Comm. 42

1082

Radioimmunoassay of Human Serum DBP, and Competitive Binding Protein Radioassay of 24,25-(OH)~D

John G. Haddad, Jr., Chong Min, and Jean Walgate Washington University School of Medicine, Department of Medicine, Division of Endocrinology, the Jewish Hospital of St. Louis, St. Louis, Missouri 63110. Human serum DBP has been isolated by ion-exchange chromatography, gel filtration and preparative gel electrophoresis. DBP binds 25-OHD3, D 3 and l,25-(OH)2D3 mole per mole, but binds 25-OHD3 with higher affinity. A radioimmunoassay for human DBP has been developed, and indicates normal sera to contain 7-9 x 10~6m DBP. Serum DBP is higher during pregnancy and estrogen therapy, and unchanged during vitamin D deficiency or excess. No correlation between serum 25-OHD and DBP concentrations was observed. Since total serum antiricketic sterol concentration is unlikely to exceed 2 x DBP is normally < 3% saturated. This accounts for the usefulness of normal serum as a binding protein source in competitive protein binding radioassays. Since 24,25-(OH)-Do is equipotent to 25-OHD3 in the competitive displacement of 3H25-OHD3 from rat serum DBP, an assay for 24,25-(OH)2D3 has been developed. Lipid extracts of serum are chromatographed on 1 x 15 cm columns of Sephadex LH-20 slurried in CHC13:n-hexane (65:35, v/v) to isolate 24, 25-(OH)2D by batch elution. Since the assay tracer can be 3H 25-OHD 3 7 only small amounts of 3H24,25-(OH)2D3 are biosynthesized in order to monitor the recovery of the extraction and chromatographic steps. 24,25-(OH)2D concentrations are 1-3 x 10"^M in normal human serum. Since sera from anephric patients contain normal levels of 24,25-(OH)2D, significant extra-renal 24-hydroxylase activity is present in these subjects. A.

Radioimmunoassay of Human Serum DBP

Vitamin D and its metabolites have been identified to associate with a protein of 1 8 y r .

No. o f Patients old)

Plasma 1,25¡ - ( O H ) 9 D (ng/dl ± S . D . ) 3-3

+

0.6

k

6.k

+

0.9

Pregnant ( 3 8 w k s . )

5

7-3

+

1.9

Hypoparathyroidism

11

2.8

+

0.9

8

2.9

+

l.k

26

5 A

+

2.1

3

l.k

+

0.5

A n t i c o n v u l s a n t drug t h e r a p y

25

k.i

+

3.6

F a m i l i a l hypophosphatemic

11

3-3

+

l.k

5

3.k

+

o.k

15

3-k

+

1.2

3.6

+

1.3

5-0

+

1.6

3-6

+

1.5

Normal (5 - 10 y r .

58

old)

Pseudohypoparathyroidism Primary h y p e r p a r a t h y r o i d i s m Nutritional

Juvenile (10-15

osteomalacia rickets

osteoporosis yr.

old)

Postmenopausal

osteoporosis

Sarcoidosis Idiopathic

k hypercalciuria

Hypervitaminosis D

In conclusion,

of

ko 5

application of radioligand receptor assay for

l,25-(OH)2D

has led t o new i n s i g h t s i n t o t h e b i o c h e m i s t r y and p h y s i o l o g y o f v i t a m i n D and has p e r m i t t e d a d d i t i o n a l c o n c e p t u a l understanding o f m e t a b o l i c bone d i s e a s e s and t h e i r r e l a t i o n s h i p t o v i t a m i n D metabolism. ACKNOWLEDGMENTS This r e s e a r c h was supported i n p a r t by NIH Grant AM-15781. REFERENCES 1. 2. 3.

Brumbaugh, P. F . , and H a u s s l e r , M. R. ( 1 9 7 5 ) J . B i o l . Chem. 2 5 0 , 1588-159^ Brumbaugh, P. F . , H a u s s l e r , D. H., B u r s a c , K. M., and H a u s s l e r , M. R. (197*0 B i o c h e m i s t r y 13, ^091-1+097 Hughes, M. R . , B a y l i n k , D. J . , J o n e s , P. G., and H a u s s l e r , M. R. (1976) J . C l i n . I n v e s t . 58, 61-70

482 k. 5. 6. 7. 8. 9. 10. 11. 12. 13. 1k. 15. 16. 17. 18. 19. 20. 21. 22.

Haussler, M. R. (1977) in Clinical and Nutritional Aspects of Vitamin D (Norman, A. W., ed.) pp. 000-000, Marcel Dekker, Inc., New York Eisman, J. A., Hamstra, A. J., Kream, B. E., and DeLuca, H. F. (1976) Arch. Biochem. Biophys. 1 7 6 , 235-2^3 Brumbaugh, P. F., and Haussier, M. R. (197*0 J. Biol. Chem. 2^9, 1251-1257 Haussier, M. R. and Brumbaugh, P. F. ( 1 9 7 6 ) in Hormone-Receptor Interaction: Molecular Aspects (Levey, G. S., ed.) pp. 301-332, Marcel Dekker, Inc., New York Procsal, D. A., Okamura, W. H., and Norman, A. W. (1975) J. Biol.

Chem. 250, 8382-8388

Zerwekh, J. E., Brumbaugh, P. F., Haussler, D. H., Cork, D. J., and Haussier, M. R. (197*0 Biochemistry 13, *+097-Ul02 Wasserman, R. H. (1975) Nutr. Rev. 33, 1-5 Peterlik, M., Bursac, K., Haussier, M. R., Hughes, M. R., and Wasserman, R. H. (1976) Biochem. Biophys. Res. Commun. 70, 797-80*4Haussier, M. R., Wasserman, R. H., McCain, T. A., Peterlik, M., Bursac, K. M., and Hughes, M. R. (1976) Life Sei. l8, 10*19-1056 Wasserman, R. H., Henion, J. D., Haussier, M. R., and McCain, T. A. (1976) Science 19U, 853-855 Hughes, M. R., Brumbaugh, P. F., Haussler, M. R., Wergedal, J. E., and Baylink, D. J. (1975) Science 190, 5 7 8 - 5 8 0 Spanos, E., Pike, J. W., Haussier, M. R., Colston, K. W., Evans, I. M. A., Goldner, A. M., McCain, T. A., and Maclntyre, I. (1976) Life Sei. 19, 1751-1756 Pike, J. W., Toverud, S., Boass, A., McCain, T., and Haussier, M. R. (1977) Proc. Third Workshop on Vitamin D, Pacific Grove (Asilomar), California Haussler, M. R., Baylink, D. J., Hughes, M. R., Brumbaugh, P. F., Wergedal, J. E., Shen, F. H., Nielsen, R. L., Counts, S. J., Bursac, K. M., and McCain, T. A. ( 1 9 7 6 ) Clin. Endocrinol. 5, 151s-l65s Hughes, M. R., Baylink, D. J., Gonnerman, W. A., Toverud, S. U., Ramp, W. K., and Haussier, M. R. (1977) Endocrinology, in press Kaplan, R. A., Haussier, M. R., Deftos, L. J., Bone, H., and Pak, C. Y. C. (1977) J- Clin. Invest., in press DeLuca, H. F. (197*0 Fed. Proc. 33, 2211-2219 Shen, F., Baylink, D. J., Nielson, R., Hughes, M. R., and Haussier, M. R. (1975) Clinical Research 23, U23 Abstr Jubiz, W., Haussier, M. R., McCain, T. A., and Tolman, K. G. (1977) J. Clin. Endocrinol. Metabol., in press

Evolution of vitamin D serum transport proteins

Alastair W.M. Hay and. Graham Watson Nuffield Institute of Comparative Medicine, Zoological Society of London, Regent's Park, London, NW1 URY Vitamin D and its metabolites are transported in blood by serum proteins, the nature of which vary according to the species of vertebrate.

Lipo-

proteins transport 25-hydroxycholecalciferol in cartilaginous fish (l) and amphibia (1,2) whereas in bony fish, reptiles, birds and mammals the secosteroid is carried on specific globulins or albumin (1-7). However, it is not only the nature of the transport proteins which vary, the properties of these proteins are also variable.

In the chick, a

species which utilises vitamin D^ as an anti-rachitic agent far less efficiently than vitamin D^ (8,9) the serum transport proteins bind 25-0HD^ more efficiently than 25-OH-Dg (10).

By contrast, species such as

humans, cattle, Old World primates and rats which use vitamins D^ and D^ with similar efficiency (10-12) have transport proteins which exhibit equivalence of binding for 25-0Ii-D2 and 25-OH-D3 (10,13). In order to test the general hypothesis that the degree of affinity of transport proteins for the various forms of vitamin D and its metabolites might determine their efficiency as anti-rachitic agents(10), a survey was made of 63 vertebrate species. A competitive protein binding assay (lU) was used to test the affinities of the serum proteins of 1+ fish, 7 reptiles, 18 birds and 32 mammals for 25-0H-D2 and 25-OH--D

(15).

Fish, reptile, bird and monotreme serum

proteins bound 25-0H-D2 considerably less efficiently than 25-0^0^; the results for the Green tree python (Chondropython viridis) are shown in Fig. 1.

Over two thirds of the mammalian proteins exhibited equivalence

of binding for the two analogues, with the proteins from some nine mammals binding 25-0K-D2 slightly less efficiently than 25-OH-D^.

In

these last nine mammals the efficiency of 25-OH-D2 binding compared with that of 25-0H-D

was considerably more efficient than that recorded

Secosteroid concentration (ng/ml) 3 Fig. 1. Competitive displacement of H-^-OH-D^ from Green Tree Python and Common Eurasian Mole plasma with increasing amounts of non-labelled 25-OH-D3 (•) and 25-0H-D2 (o). for the reptiles and birds (15).

Results for the Common Eurasian Mole

(TaJ^^europaea) are shown in Fig. 1. If plasma protein binding of vitamin D metabolites does correlate with anti-rachitic efficiency, then the results of our survey suggest that vitamin D^ is less efficient than vitamin D^ in fish, reptiles, birds and the monotreme. There are some anomalies in our findings.

New World primates are unable

to utilise vitamin D^ as efficiently as vitamin D^ (12), yet their plasma transport proteins bind 25-0H-D2 and 25-OH-D^ with equal efficiency (13). Efficient transport of 25-OH-D2 may not be a prerequisite, therefore, for efficient utilisation of vitamin D^ by mammals.

It may only provide

mammals with the potential to utilise vitamin D^, a potential which the New World monkeys have failed to exploit. The change which enabled mammalian transport proteins to bind vitamin D^ metabolites more efficiently may have arisen about 150 million years ago when egg laying was abandoned and mammals were nocturnal (15). An alternative explanation for the evolution of the mammalian transport proteins may be that these proteins have evolved to bind more efficiently.

Support for this suggestion is provided by the obser-

vation of Holick et al (l6) that the plasma clearance rates of 2U,25(OH and 25-OH-D

in the chick are similar, and in the rat the plasma

485 clearance rate of these two analogues is again similar but far slower than that observed in the chick (l6). The two analogues 25-0H-D2 and 2h ,25( OH^D^ both have substituents on position 2k of the vitamin D side chain.

In the case of 25-OH-Dg it is a

methyl group and for 2b,25( OH^D^ an hydroxyl group.

It is possible there-

fore that the evolution of the vitamin D plasma transport proteins observed in the mammals facilitated the efficient binding of vitamin D metabolites with substituent groups on position 2^ of the side chain. To investigate this possibility plasma proteins from 35 vertebrates were examined for their binding properties towards 25-OH-D3, 2UR,25(0H)2D3 and OH)oD_ using the competitive protein binding assay referred to previously (lM.

The results of the binding studies are shown in Table 1

and examples of the differing binding characteristics are shown in Fig. 2 for the Goldfish (Carassius auratus), Alligator (Alligator mississippiensis), Malaysian Fish Owl (Ketupa ketupa) and the Lion (Panthera leo).

Alligator -

-

Ill

1

0.2

0.5

* — •

1—//—\—n—1 1.0 2.0 5.0 Lion

bO Ö -d G •H •H

0.2

0.5

1.0

2.0 5.0

0

1.0

Secosteroid concentration (ng/ml) Fig. 2.

Competitive displacement of t26,27- Hü 25-0H-D3 from Goldfish,

Alligator, Malaysian Fish Owl and Lion plasma proteins by non-labelled 25-0H-D3 (•), 2UR,25(0H)2D3 (o) and

, 2 5 ( O H ^ (x).

486 CO CU T)

w o ir\ OJ

K ~i

CVJ on O i w o I LT\ CM ^ 0

-P O (h ft -P

u o

Ti 01 H H •H m tí bO cd o
9.0 9.9 8.5 8.9 8.5 8.2 9.1 7.8 9.8 9.0 8.0

13 14 21 24 46 61 69 72 74 94 123 140

9.1 + 0.8

The levels of 25-OHD^ and calcium were measured in samples from lactating females. Samples were extracted with chloroform-methanol which, after chromatography, were used in the 25-OHD^ competitive binding radioassay. (Recovery of 25-OHD^ was 70-90% as determined by internal standard.) Milk and plasma were generously donated by: Linda Smith of the Riverside La Leche League; Dr. W. H. Weinfurtner, Riverside, CA; and Dr. J. Saunders, Riverside County Veterinarian. Fore milk is the first 5-10 ml of milk at each lactation. Hind milk is the last of the milk at each lactation. Subject was taking 300 mg of calcium Included in the vitamin D supplement. Subject took last vitamin D supplement 48-72 hours prior. A daily vitamin supplement that contained vitamin D„. Significantly different from hind milk (p 0.05) and plasma (p 0.01). Significantly different from plasma (p 0.001). "25-OHD,-like compound" listed as 25-OHD., equivalents.

chloroform-methanol

and analyzed

an LH-20

column

Sephadex

in the assay after chromatography on

(1x22 cm).

The system employed was

50%

chloroform:50% hexane (6). Two results suggest that the compound specific First,

competitive-binding

human

milk

chromatographed

(pooled

asays

from

(1x80 cm column).

is

several

not

that competes in these identical

subjects) was

to

25-OHD^.

extracted

The 25-OHD 3 peak was bioassayed

the in vivo chick intestinal calcium transport assay.

and in

The milk extract

had no biological activity compared to a vitamin D 3 standard. Second, a pregnant rat was fed a normal diet until the pups were born, and then switched to a vitamin D-free diet.

Fourteen days after birth,

while the pups were still nursing, the lactating rat received 3 I.U. of 3

H-vitamin D 3 (65,000 dpm/IU) daily.

milked. (

14

This milk was

extracted

After 5 days the rat was

and

standard

vitamin D metabolites

C-labeled) from chicks were added as markers before chromatography.

525 No radioactive

25-OHD2 peak was observed; only

a small peak

that

migrated with the vitamin D^ standard. Other

are:

(a)

25-OHD2,

(b) vitamin D sulfate, or (c) a new vitamin D analog.

This

compound

could be 25-OHD2, since the women were taking daily vitamin

D2

possibilities

supplements.

low

activity

for

the

'^S-OHD^-like

25-Hydroxyergocalciferol

in chicks.

However,

has

compound"

been

25-OHD2 would

shown

to

be active

exhibit in

rats;

but milk has been shown, classically, to have low biological activity. If the compound was vitamin D sulfate, which is soluble in organic solvents, it would have to co-migrate on chromatography with 25-OHD and not be biologically active in chicks. vitamin D

sulfate have been reported

Since relatively low levels of in milk,

the

impressively

high

amounts from the competitive-binding assay could be explained if vitamin D

sulfate

was

a better

competitor

(100-

to

200-fold)

than

25-OHD 3 .

One might assume that milk would contain a very active compound to induce intestinal calcium transport.

Milk is usually the only

of nutrition that neonates receive initially.

Therefore, this "25-OHD 3 -

like compound" may be a new form of vitamin D.

The neonate may have an

enzyme that converts this compound to an active form. for

antirachitic

activity

have not detected

source

Classical tests

this compound.

The

rat

line test would not reveal activity because the rats employed have been weaned and, thus, might no longer have an active enzyme. would

not

exposed

be

active

to milk

and

in

the

chick bioassay

thus, would

not have

because

The compound

chicks are never

the enzyme.

Further

work

is in progress to evaluate these possibilities. T.

Deuel, P.

Lipids III, p. 649~l

Interscience Pub., Inc., N.Y. (1957) .

2.

Weckel, K. G.

3.

Sahashi, Y., T. Suzuki, M. Higaki and T. Asano.

J. Dairy Sc. 24, 445 (1941). J. Vitam. 1J5, 78

(1969). 4.

Sahashi, Y., T. Suzuki, M. Higaka and T. Asano.

J. Vitam. L3, 33

(1967). 5.

le Boulch, N., C. Gulat-Marnay and Y. Raoul.

Internat. J. Vit. Nutr.

Res. 44, (1974). 6.

Osborn, T. W., Ph.D. Dissertation, University of CA, Riverside (1976).

SpaaIflo binding of 25-hydroxyoholaoalolfarol in human mammary oanoar

M.S. Hoginaky and A. Malbar Nassau County Madioalflantar,East Maadow, Naw York 11J54 and Stata Univaraity of Naw York at Stony Brook INTRODUCTION Spaoifio raoaptora for 25-hydroxyoholaoaloifarol (25-OHDj) ara widasprsad in animal tiaauaa (1,2), Similar funotional protalna hava not baan daaoribad in human oanosr, It ia tha purpoaa of thia raport to dooumant tha sxistsnos in human braaat oanoar of a apaoiflo high affinity oytoplaamio raoaptor that binda 25-OHD^, MATERIAL AND METHODS Braaat oanoar tiaaua or oontrol aim pit«, inoluding normal braaat, banign braaat tumora, and oanoar of origin othar than tha braaat, wara obtainad at biopsy or oanoar axoision. All spsoimans vara anap frosan in liquid nitrogan until assay, Ths tissua was thawsd and homogsnissd in a ona to four voluma of 10 mM TES buffsr, (N-tria (hydroxymathyl) msthyl-2-aminoathana sulfonio aoid) oontaining thioglyoarol 12 mM and auorosa 250 mM and adjustsd to pH 8,0, Following oantrifugation at 100,000 x g for 60minutss, ths auparnatant oontaining tha oytoaol waa daoantad and kapt at C until ussdi For ths binding assay, an aliquot of ths oytosol fraotion squivalsnt to 0il5 mg protain waa inoubatad at 4° C for 60 minutas with inorsasing amounts of ^H-25-OHDj (sp. aot. 6,9 0i/mM)( Dsxtran ooatad oharooal was ussd to ssparats bound from frss atarol, Binding Indax (B.I.), whioh is ths psrosntags of ths total ^H-25-0HDj bound by ths oytosol dus to ths spsoifio 25-OHDj raoaptor, was dstsrminsd by ths addition of a largs amount (100 fold) of unlabalad ligand, Soatohard plot to datarmina binding affinity (Ka) and maximum binding (Bmax) was oarrisd out (3), Displaosmsnt of ^H sstradiol (E2) binding by 25-0KD^ «nd visa vsrsa was dstsrminsd by ths addition of 100 fold amounts of unlabslsd 25-OHD^ or E 2 , rsspsotivsly. Estrogsn rsosptor aotlvity was sstimatsd aftsr a modifisd prooadura of

528 Menendez-Botet et al

. All mammary tissues were characterized as estro-

gen positive (E+) or estrogen negative (E-), using standard criteria. RESULTS AND DISCUSSION Every tissue examined, estrogen positive and estrogen negative mammary cancers, and control tissues consisting of two normal breast, two breast fibroadenomas and a primary lung and a primary colon cancer, had significant specific receptor activity for 25-OHD^.

There was no detectable

differences between estrogen positive mammary cancers and control tissues in the Ka, Bmax and B.I. values.

See Table I.

TABLE I AFFINITY CONSTANT (Ka), MAXIMUM BINDING (Bmax) & BINDING INDEX (B.I.) IN ESTROGEN POSITIVE & NEGATIVE MAMMARY CANCER & CONTROL TISSUES (MEAN - S.E.M.)

Ka (moles/L)

ESTROGEN

ESTROGEN

CONTROL

POSITIVE (n=12)

NEGATIVE (n=l6)

TISSUES (n=6)

4.99xl0~ 9 - 1.92

4.28xl0~ 9 - 1.87

6.60xl0~ 9 -1.62

1.86 - 0.49

2.58 - 0.57

3.4 - .52

6?M

75.%% - 4.6

7^.7% -

Bmax (pmoles per protein) Binding Index (B.I.)

- 5.7

7.0

3 In no breast cancer tissue did unlabeled E„ displace ^H-25-0HD_ from its receptor, nor did unlabeled 25-OHD^ displace ^H E 2 from its specific receptor in the cytosol. SUMMARY In summary, this report confirms the ubiquitous nature of high affinity receptors for 25-OHD^ in animal tissues, and extends this observation to human breast cancer and probably other human cancers as well.

529 REFERENCES 1.

Haddad, J.G. and Birge, S.J.s

Widespread, specific binding of 25-

hydroxycholecalciferol in rat tissues. 250. 299-303

2.

The J. of Biological Chemistry

(1975).

Lawson, D.E.M., Charman, H., Wilson, P.W. and Edelstein, S.:

Some

characteristics of new tissue-binding proteins for metabolites of Vitamin D other than 1,25-dihydroxyvitamin D. sica Acta VJZ, 403-415

3.

Scatchard, G. : ions.

4.

Biochimica et Biophy-

(1976).

The attractions of proteins for small molecules and

Annals N.Y. Acad. Science =¡1, 660-672 (l95l).

Menendez-Botet, C.J., Nisselbaum, J.S., Fleisher, M., Rosen, P.P., Fracchia, A., Robbins, G., Urban, J.A. and Schwartz, M.K.:

Correla-

tion between estrogen receptor protein and carcinoembryonic antigen in normal and carcinomatous human breast tissue. 1366-1371 (1976).

Clin. Chem. 22.

Bone Organ Culture Bioassay for Determination of 1,25-(0H)2D

Paula H. Stern, Thomas E. P h i l l i p s and Samuel V. Lucas Dept. of Pharmacology, Northwestern U., Chicago, I I . 60611, USA Alan J. Hamstra and Hector F. DeLuca Dept. of Biochemistry, U. of Wisconsin, Madison, Wi. 53706, USA Norman H. Bell Dept. of Medicine, Indiana Univ., Indianapolis, In. 64202, USA Our laboratory has recently been carrying out structure-activity studies on the effects of vitamin D analogs on bone resorption in v i t r o (1-3).

Figure 1.

Effect of vitamin D3 and metabolites on release of 45ca from fetal rat bones in v i t r o . Values are the ratio of release of 45ca from paired metabolite-treated and control bones. Bones were cultured for 48 hours. Details and methods have been described previously (1-3).

Figure 1 i l l u s t r a t e s some of the findings based on the parameter of release of previously incorporated ulnae.

from 19-day fetal rat radii and

In this system, vitamin D3 i s inactive at concentrations up to

532 10-4 M.

25,26-(0H)2D3 is a weak stimulator.

OH-D3 and 24(R),25-(0H)2D3 are between 10"

8

Activity thresholds for 25and 10" 7 M.

1,24,25-(0H) 3 D3

is approximately two orders of magnitude more potent than 25-OH-D3. 1,25-(0H)2D3 has approximately ten times the potency of 1,24,25-(0H)3D3, with an activity threshold of about 1 C H 1

M for 48 hour cultures.

1,25-

(0H)2D2 is slightly, but not significantly, less active than 1,25-(0H)2D3 (3).

Several other laboratories have also examined the effects of some of

these metabolites and their findings with respect to relative potencies are quite similar to ours (4-8).

The sensitivity of the bone cultures to 1,25-(0H)2D2 and 1,25-(0H)2D3 made the use of the system as a bioassay for these metabolites an attractive possibility.

Chromatin (9,10) and cytosol (11,12) radioreceptor binding

assays for 1,25-(OH)2D indicate that normal human plasma concentrations are in the 20-50 pg/ml

0.5-1.2 x 10~10 M) range.

Since bone cultures

are highly responsive at these and lower concentrations, and since each culture requires only 0.5 ml of medium, it seems likely that the bone culture system, in addition to offering the advantage of a biological end organ response for validating the radioreceptor binding methods, might be more sensitive than existing methods for assay of 1,25-(0H)2D. We initially attempted to assay for bone resorbing activity in plasma directly.

There was one obvious pitfall in doing this, the fact that the

25-0H-D, although approximately 1/1000 as active in vitro, was present in plasma at approximately 1000 times the concentration of the 1 -hydroxy!ated metabolite (13-15).

However, we had previously shown that addition of

vitamin D-deficient rat plasma at a concentration of 10% to the culture medium diminished the activity of added 25-OH-D without affecting the activity of added 1,25-(0H)2D3 (1).

Presumably this was due to the pre-

sence of the 25-0H-D binding protein (16).

Thus, it seemed possible that

enough of the 25-OH-D would be bound to prevent it from interfering in the assay.

The actual results (Table 1) revealed an unexpected difficulty.

Neither vitamin D-deficient nor normal rat plasma, when present as 50% of the culture medium had any effect on bone resorption.

It appeared that

whole plasma contained a factor or factors which prevented the effect of 1,25-(0H)2D on the bones.

5

Table 1.

Bone resorbing activities of media prepared with plasma from normal or vitamin D-deficient rats cpm/ml medium

Plasma

+ Plasma (A)

- Plasma (B)

A/B

Normal (+D)

463 ± 95

431 ± 61

1.04 ± .11

D-deficient

472 + 80

415 ± 62

1.11 ± .10

Paired radii or ulnae were cultured for 48 hours. Bones in the A column were cultured in media supplemented with 50% plasma. Values are the means ± standard errors of responses from 5 bone pairs. Medium calcium = 1.3mM, PO4 = ImM for all treatments. We have tested various purification steps to determine the minimum purifi cation required to remove the inhibitory activity.

Neither conventional

(17) chloroform-methanol nor dichloromethane extraction procedures were adequate.

Subsequent chromatography of the extracts on one or even two

LH-20 columns was still insufficient to remove the inhibitory factor.

U1

timately, we found that the same purification procedure which was being used for preparation of plasma extracts for cytosol radioreceptor assay Plasma (5 ml) 4 3x dichloromethane (30, 15, 15 ml) 4evaporate 4 Sephadex LH-20 (0.7 x 9 cm column) Skellysolve B:CHCl3:Me0H (9:1:1) 4 HPLC-Silica (yPorosil) Skellysolve B + 10% isopropanol 4 In vitro bone resorption assay Figure 2.

Plasma purification scheme for 1,25-(0H)2D assay (11,12)

5 3^ (11,12) was satisfactory for preparation of plasma extracts for bone organ culture bioassay.

This involved (Figure 2) extraction with dichlorometh-

ane, Sephadex LH-20 chromatography and finally high pressure liquid chromatography (HPLC) on silica (yPorosil), eluting with 10% isopropanol in hexane.

This final chromatographic step has been shown to separate 1,25-

( O H ^ D from other known metabolites (18).

A fraction is collected which

contains both 1,25-(0H)2D2 and 1,25-(0H)2^3•

Recovery through the purifi-

cation procedure was monitored by addition of [26,27-3h]-1 ,25-(0H)2D3 and averaged 55 ± 4%.

Extracts of vitamin D-deficient rat plasma prepared by

this method were inactive and did not inhibit the effects of added 1,25(0H) 2 D 3 . The sample processing procedure involved the three collaborating laboratories as follows:

Normal human plasma was collected in Indianapolis.

This was packaged in dry ice and sent to Madison, where the chromatographic procedures were carried out.

After purification, the extracts

were evaporated, dissolved in 95% ethanol, sealed under N2, packed in dry ice and shipped to our laboratory.

They were stored at -20° C until use.

We have recently begun carrying out the extraction of 1,25-(0H)2D plasma in our laboratory in Chicago.

from

Our method (Figure 3) is somewhat

Plasma (5 ml) 3x Benzene (5, 5, 5 ml)

+ 2x 0.1 M PO4 buffer, pH 10.5 (5, 5 ml) I save organic phase, evaporate I HPLC-Silica (uPorosil) Hexane + 13% isopropanol •JIn vitro bone resorption assay Figure 3.

Plasma purification scheme for 1,25-(0H)2D assay (Phillips, Lucas and Stern)

535 different from that described above.

Plasma is initially extracted with

benzene and then back extracted with 0.1 M phosphate buffer, pH 10.5. Preliminary results indicate that this removes the inhibitory activity, although it still leaves us with the problem of separation of the metabolites.

This is accomplished by HPLC.

We have chosen to use a slightly

more polar solvent system, 13% isopropanol in hexane, in order to collect the 1,25-(0H) 2 D peak more quickly.

1,25-(0H) 2 D 2 and 1,25-(0H) 2 D 3 are not

resolved and are collected in a single fraction.

1,25-(0H) 2 D3 is ade-

quately separated from the other vitamin D3 metabolites (Figure 4).

A

typical chromatogram of 254 nm UV absorbance in a normal human extract is shown in Figure 5.

The 12-16 min fraction is taken for assay.

Although

our experience with the system is still limited, preliminary results show insignificant levels of 1,25-(0H) 2 D in vitamin D-deficient rat plasma, recovery of 1,25-(0H) 2 D3 added to plasma and carried through the purification procedure and lack of inhibitory activity of the plasma extracts (Table 2).

536

Column^ s i l i c a Porosil") S o l v e n t : 1 3 % isopropanol : 8 7 % h e x a n e . f l o w r a t e : I ml/min Sensitivity: 0 2 AUFS

—.min Figure 5.

UV absorbance at 254 nm from plasma extract purified by the procedures outlined in Figure 3.

Table 2.

Analysis of plasma extracts prepared by HPLC

a) Vitamin D deficient r a t plasma

undetectable

b) 20 pg/ml 1,25-(0H)2D3 added to plasma

17.95 pg/ml

40 pg/ml 1,25-(0H)2D3 added to plasma

38.41 pg/ml

c) Detected amount of a c t i v i t y of 1,25-(0H)2D3 16.64 pg/ml (4 x 10" 1 1 M) added to culture medium containing plasma extract

15.5, 15.6, 18.47 pg/ml

Each of the assay experiments included a 4 or 5 point standard curve for 1,25-(0H) 2 D3.

Plasma samples were run at two d i l u t i o n s , the high concen-

tration being equivalent to approximately 1 ml of plasma/ml culture

537

Table 3.

Normal human plasma levels by bioassay

Dichloromethane, LH-20, HPLC 25.1+

2.2 pg/ml

(N = 15)

Extracts from the same plasma sample, extracted and chromatographed and assayed in separate experiments plasma #1

20.4, 22.3 pg/ml

plasma #2

30.3, 29.8 pg/ml

plasma #3

15.2, 27.9 pg/ml

Benzene, PO4 buffer, HPLC 23.6 + 1.8 pg/ml

(N = 10)

Extracts from the three plasma samples, each of which was divided and the two aliquots extracted and chromatographed separately; the two aliquots were subsequently assayed in the same culture experiment. plasma #1

26.1 , 23.1 pg/ml

plasma #2

28.0, 29.7 pg/ml

plasma #3

22.0, 22.6 pg/ml

Extracts from the two plasma samples, each of which was divided and the aliquots extracted and chromatographed separately; each aliquot was assayed in a different culture experiment. plasma #1

16.0, 19.6 pg/ml

plasma #2

10/12/76 - 19.2 pg/ml 10/19/76 - 27.7 pg/ml 11/08/76 - 22.3 pg/ml 12/14/76 - 24.0 pg/ml 12/21/76 - 18.8 pg/ml

538 medium and the lower concentration a 1:1 d i l u t i o n of t h i s .

Each sample

and standard was assayed in 4-5 separate bone cultures within a given experiment.

The overall c o e f f i c i e n t of v a r i a t i o n ( x / s . e . ) for these r e p l i -

cates was 10.6 ± 0.8%. Our f i n d i n g s on normal human plasma 1,25-(0H)2D concentrations are shown in Table 3.

With the dichloromethane, LH-20, HPLC procedure we f i n d the

mean 1,25-(OH)2D concentration i n normal human plasma to be 25.1 ± 2.2 pg/ml.

We have examined r e p r o d u c i b i l i t y between experiments by comparing

1,25-(0H)2D

l e v e l s in extracts from three blood samples, each of which

was divided and chromatographed and assayed in two separate experiments. In two cases the r e p l i c a t e s were c l o s e , in one other they were quite d i s similar.

With the benzene, phosphate b u f f e r , HPLC procedure the mean was

23.6 ± 1.8 pg/ml.

Three samples which were extracted separately but as-

sayed in the same c u l t u r e experiment gave reasonably reproducible d u p l i cates.

When divided extracts were assayed in d i f f e r e n t experiments the

deviations were g r e a t e r .

Samples from the same pool of blood bank plasma

ranged from 18.0 to 27.7 pg/ml in f i v e d i f f e r e n t a s s a y s , g i v i n g a mean and standard error of 22.4 ± 1.6 pg/ml and a mean difference of 11.8 ± 3.7%. The v a r i a b i l i t y did not appear to be due to l o s s of 1,25-(OH)2D during storage. Our values for 1,25-(OH)2D in normal human plasma are somewhat lower than those reported f o r the binding a s s a y s .

H a u s s l e r ' s laboratory (9,10)

ports a mean of approximately 35 pg/ml and Eisman, et a l . (11,12) 29 ± 2 pg/ml.

Since our N number i s s t i l l

re-

report

small and many of the same i n -

d i v i d u a l s were studied i n the two sets of data I have shown, these d i f f e r ences could be a r e f l e c t i o n of i n d i v i d u a l or population v a r i a t i o n .

On the

other hand they could r e f l e c t an actual d i s p a r i t y between what i s detected by the two a s s a y s .

We are c u r r e n t l y in the process of d i r e c t l y comparing

samples assayed by the two methods. In summary, we have shown that fetal rat bone organ c u l t u r e s can be used as a bioassay f o r 1,25-(0H)2D. to develop s k i l l

Disadvantages of t h i s system are the need

in the t i s s u e c u l t u r e technique and p o s s i b l y greater

v a r i a b i l i t y than with the binding s t u d i e s .

The advantages of our system

539 are that it has potentially greater sensitivity and is based on a biological end organ response.

Since the number of samples which can be assayed

at one time is small, the bioassay is unlikely to be useful for routine work.

However, we feel it is of potential importance for validation of

results from other methods and also as a sensitive assay system when 1,25(0H)2D levels are low or the amount of plasma is limited.

Supported

by USPHS research

Center Grant M01RR750 4

grants AM11262,

and Research

AM14881S

Career Development

Clinical Award

Research

(to P.H.S. )

K04-AM70210. 1. Stern, P.H., Trummel, C.L., Schnoes, H.K. and DeLuca, H.F. (1975) Endocrinol. 97, 1552-1558 2. Stern, P.H., DeLuca, H.F. and Ikekawa, N. (1975) Biochem. Biophys. Res. Comm. 67, 965-971 3. Stern, P.H., Mavreas, T., Trummel, C.L., Schnoes, H.K. and DeLuca, H. F. (1976) Molec. Pharm. 12, 879-886 4. Raisz, L.G., Trummel, C.L., Holick, M.F. and DeLuca, H.F. (1972) Science 175, 768-769 5. Reynolds, J.J., Holick, M.F. and DeLuca, H.F. (1973) Calcif. Tiss. Res. 12, 295-301 6. Reynolds, J.J., Holick, M.F. and DeLuca, H.F. (1974) Calcif. Tiss. Res. 15, 333-339 7. Atkins, D. and Peacock, M. (1974) J_. Endoc. 61, Ixxix 8. Mahgoub, A. (1975) Fed. Proc. 34, 893 9. Brumbaugh, P.F., Haussler, D.H., Bursac, K.M. and Haussler, M.R. (1974) Biochemistry 13, 4091-4097

10. Hughes, M.R., Baylink, D.J., Jones, P.G. and Haussler, M.R. (1976) J. Clin. Invest. 58, 61-70 11. Eisman, J.A., Hamstra, A.J., Kream, B.E. and DeLuca, H.F. (1976) Science 193, 1021-1023 12. Eisman, J.A., Hamstra, A.J., Kream, B.E. and DeLuca, H.F. (1976) Arch. Biochem. Biophys. 176, 235-243 13. Belsey, R.E., DeLuca, H.F. and Potts, J.T., Jr. (1971) J. Clin. Endoc. Metab. 33, 554-557 14. Haddad, J.G. and Chu, K.J. (1971) J. Clin. Endoc. Metab. 33, 992-995

5^0 15. Belsey, R . E . , DeLuca, H.F. arid P o t t s , J . T . , J r . (1974) J_. C l i n . Endoc. Metab. 38, 1046-1051 16. Belsey, R., C l a r k , M.B., Bernât, M., Glowacki, J . , H o l i c k , M.F., DeLuca, H.F. and P o t t s , J . T . , J r . (1974) Amer. J . Med. 57, 50-56 17. B l i g h , E.C. and Dyer, W.J. (1959) Can. J . Biochem. P h y s i o l . 37, 911917 18. Jones, G. and DeLuca, H.F. (1975)

L i p i d Res. 16, 448-453

The M e a s u r e m e n t o f 2k,25_dihydroxycholeca1ciferol

Carol

M.

i n Human S e r u m

Taylor

University

The r e n a l

Department o f M e d i c i n e ,

Manchester,

metabolism of 2 5 " h y d r o x y c h o l e c a l c i f e r o l

i n a c o m p l e x manner by c a l c i u m p h o s p h o r u s ing e i t h e r

1,25-dihydroxycholecalciferol

cholecalciferol under c o n d i t i o n s recently

2CC)

(2k,25(OH) o f normal

become p o s s i b l e

s e r u m by a c o m p e t i t i v e

.

1

(1 ,25 ( O H ) 2 C C )

The m a j o r m e t a b o l i t e

to measure

protein

(25(0H)CC)

and p a r a t h y r o i d

or hypercalcaemia

t o m e a s u r e s e r u m 2k,25(OH)2CC ical

U.K.

binding i n normal

assay.

controlled

hormone

or

of 25(0H)CC

2 4 , 2 5 ( 0 H ) 2 CC and

this 2

metabolite

and

found

it

has

i n human

T h i s method h a s

subjects

produc-

2^,25~dihydroxy-

i

is

levels of

is

been

i n a number o f

used clin-

disorders.

Standard

curves

for

the a s s a y were c o n s t r u c t e d

(OH)CC f r o m t h e b i n d i n g increasing

amounts o f

s e r u m was p r e p a r e d Sephadex

Other

protein

(-5^-5- d i 1 u t e d

u n l a b e l led 2 ^ R , 2 5 ( O H ) 2 C C

for

assay

by d i s p l a c i n g rachitic (Fig.

1).

rat

25- [26,27-3H serum)

with

2k, 2 5 ( O H ) 2 C C

by c h r o m a t o g r a p h y o f a l i p i d e x t r a c t

on

LH 2 0 .

hydroxylated

metabolites

system but w i t h c a r e f u l

of

cholecalciferol

sample e x t r a c t i o n

interfere

and c h r o m a t o g r a p h y

in it

the

assay

is

40

F i g . 1. A typical standard c u r v e f o r the 2 4 , 2 5 ( 0 H ) 2 C C assay. D

o\°

from

0 •05

•5 ng 24R, 25 (OH) a CC

5

5^2 possible

to prepare

metaboli t e s . Using

the method o u t l i n e d

normal "

24,25(0H)2CC

subjects

was

3-39 ng/ml) w i t h

(range

1.1 -

found

t h e mean 2 4 , 2 5 ( O H ) 2 C C

t o be

1 . 6 3 ± 0 . 1 9 ng/ml

corresponding

levels

25(0H)2CC

in p a t i e n t s

be h i g h e r

than

with

is

with

primary

chronic

measure serum

levels

for

being

a s e r u m 25 (OH) CC l e v e l

patients with

interfering

of

renal

14.49 ±

is

ng/ml

positive levels.

correlation

3

Serum

low o r

failure.

renal

between

levels

level,

of

(range

3-7

in

to

ng/ml)

ng/ml).4

"27.8

the serum

therefore of

metabolite,

24,

t h e mean

(range 0.20 - 6.26

undetectable

I t was

0.44

have been found

serum 25(0H)CC

5

nineteen

17.86 ± 2.41

of

1 - 4 9 ng/ml

in

range

hyperparathyroidism their

the o t h e r

level

(mean ± S . E . ,

2 . 3 3 - O . 3 8 ng/ml

1,25(0H)2CC

of

level

and s e r u m 2 5 ( O H ) C C

appropriate

metabolite

25(0H)CC

T h e r e was a s t r o n g

in n i n e t e e n p a t i e n t s

The r e n a l

from o t h e r

above

35-7 ng/ml).

serum 24,25(OH)2CC

value

from serum f r e e

2

of

interest

24,25(OH)2CC,

in

to such

pa t i en t s .

In

four

untreated

from 0 . 4 6 one o f

levels

capable of

Assays

failure

1.26 ng/ml, w i t h

these patients

25(0H)2CC are

to

renal

of sera

rose

was

from s i x

Day

25(0H)CC

treated with

(Table

converting

patients

1).

Thus

25(0H)CC

anephric

to

serum 24,25(0H)2CC

levels

of

levels

range

3-7 to 8.7 ng/ml.

When

1 . 2 5 mg v i t a m i n patients

with

D p e r day s e r u m

chronic

renal

24,

failure

24,25(OH)2CC.

patients

showed no d e t e c t a b l e

25 (OH)CC ng/ml

24,25(0H)2CC

0

6

1 .26

5

20

1.77

13

115

2.52

25

184

4.71

41

195

3.84

24,25(0H)2CC.

ng/ml

T a b l e 1. S e r u m 2 5 ( 0 H ) C C and 2 4 , 2 5 ( 0 H ) 2 C C l e v e l s i n a p a t i e n t w i t h c h r o n i c renal f a i l u r e t r e a t e d with 1 . 2 5 mg v i t a m i n D p e r day f r o m day 0 .

5^3 These p a t i e n t s four

days

were each g i v e n

later.

serum 25(0H)CC 24,25(0H)2CC lation of however, in

this

Again

levels

t h e r e was

had

in a n e p h r i c

25(0H)CC. suggests

state,

100 y g 2 5 ( 0 H ) C C and t h e i r

risen

considerably

patients

Measurement

that

no d e t e c t a b l e

confirms

to

renal

of 24,25(0H)2CC

the 2 ^ - h y d r o x y 1 a s e

in c o n t r a s t

2b , 2 5 ( O H ) 2 C C

(Fig. 2).

the

sera

system

reassayed although

The a b s e n c e

site

in

renal

is

not

of

of

2't-hydroxy-

failure

totally

patients,

suppressed

1-hydroxy 1 at ion.

80 E

60 0» c O U 40 X o Ù) N 20 \

Fig. 2. S e r u m 2 5 ( 0 H ) C C and 2 ^ , 2 5 ( O H ) 2 C C l e v e l s in 6 anephric patients. 100 y g 2 5 ( 0 H ) C C was g i v e n on day 0 .

A

0 E

\

0 c» 2

Day 0

Day 4

T h i s w o r k was s u p p o r t e d by a programme g r a n t Council

to P r o f e s s o r

Dr.R.G.G.Russe11

S.W.Stanbury.

for

sera

from the Medical

I should

like

from the a n e p h r i c

to thank

Research

Dr.J.A.Kanis

and

patients.

References 1.

DeLuca,H.F. endocrine

2.

70, 3.

system.

Taylor,C.M., assay

for

Recent advances

J.Lab.C1in.Med.

Hughes,S.E.

12^3-1249,

Taylor.C.M.,

Taylor,C.M.

Eisman,J.A., cise

7"26,

6 de S i l v a , P .

Jan.

de S i l v a , P .

the v i t a m i n D

1976.

Competitive

& Hughes,S.E.

Biochem.

Tissue,

York,

Proceedings

England,

S e r u m 2b,25_dihydroxycho1eca1 Clin.

Hamstra,A.J.,

and c o n v e n i e n t

in human p l a s m a .

of

protein

binding

Biophys.

Res.Comm.

1976.

hypoparathyroidism. 5.

understanding

2^,25-dihydroxycholecalciferol.

S y m p o s i u m on C a l c i f i e d k.

in our

Sci.

Kream,B.E.

method f o r

Arch.

Molec.

Biochem.

1976.

ciferol Med.

of

In

In

levels the

Biophys.,

of

the in

European

press. primary

press.

& DeLuca,H.F.

determination

the X I I

A sensitive, -

pre-

1,25 dihydroxyvitamin i

1 76, 235"2 »3,

1976.

D

PLASMA 25 (OH) VITAMIN D LEVELS IN NORMAL SUBJECTS THROUGHOUT THE YEAR AND IN PATIENTS WITH RELATED DISEASES C. Velentzas, D.G. Oreopoulos, L. Brandes, D.R. Wilson and W.C. Sturtridge Departments of Medicine, The Toronto Western and Toronto General Hospital, and Department of Medicine, University of Toronto. This work was supported by the Calcium Task Group, University of Toronto and Mr. W. Blackburn (Toronto Kidney Fund). The introduction of Haddad's competitive protein-binding radioassay for the detection of 25-hydroxycholocalciferol (25 (OH) Vitamin D) in plasma (1) has made an important contribution to our understanding of the role of abnormalities of Vitamin D metabolism in various diseases. Because of the important role of Vitamin D in calcium homeostasis, we decided to study plasma 25 (OH) Vitamin D levels in 137 normal controls and 152 patients with a variety of conditions in which Vitamin D metabolism may be abnormal. The patients had diseases of bone - osteoporosis, Paget's disease; kidneys - recurrent calcium stones and renal failure; liver - primary biliary cirrhosis, alcoholism; and epileptics on anticonvulsants, and thyroid - hyperthyroidism. MATERIAL AND METHODS Normal controls consist of 137 persons whose blood was obtained once throughout the year. The study group included 140 patients: 24 with osteoporosis, 30 with recurrent calcium - containing kidney stones, five with Paget's disease of bone, 31 with chronic renal failure - eight were on chronic hemodialysis, 14 on chronic peritoneal dialysis and nine on conservative management, 32 epileptics on anticonvulsants, 15 alcoholics, none of whom had evidence of cirrhosis on liver biopsy, three with primary biliary cirrhosis and 12 patients with hyperthyroidism. Plasma concentration of 25 (OH) Vitamin D was measured by the method of Haddad (1). RESULTS Our normals showed a wide variation over the 12-month period; the lowest mean (+SD) value was during the months of March - April (17+6.7 ng/ml) and the highest was during the months of July - August (28.9+9.4). The overall mean (+SD) was 23.6+9.8 ng/ml. Because of this variation of the normal values throughout the year, whenever we had obtained samples of the disease

546 groups throughout the year we compared the overall msan of each group with the overall mean of normals.

In three groups (hemodialysis, peritoneal

dialysis and hyperthyroidism) that we tested over a period of one or two months, the group mean was compared with the mean normal of the corresponding month (s). Patients with osteoporosis and Paget's disease had a mean 25 (OH) Vitamin D value of 18.9+6.2 and 15.7+9.7 ng/ml respectively. The difference between these values and normal controls was only borderline (p.05).

580 Table 3.

PREVENTION OF PARTURIENT PARESIS (PP) IN DAIRY COWS WITH THE INTRAMUSCULAR ADMINISTRATION OF 25-OH-D3*H20 Holsteins Classified by Phosphorus Consumption

Dose mg 0 2 4 8

Total Animals >50 gm Phosphorus/Day .05).

groups treated with three doses.

The response of non-paresis history

cows to treatment with 25-OH-D3'H20 is in contrast to observations made in similar vitamin D treated animals (V). in combination.

Table 2 illustrates the data

As would be anticipated, treatment with all three doses

of 25-OH-D^ resulted in a significant reduction in the incidence of PP (P 0.05

+ 69 + 112 + 11 + 1.1 + 18

**Wilcoxen Rank Sum Test

After three doses of l,25(OH)2D3 the mean Ca absorption in the six subjects was 323 yH/40 cm compared to the pretreatment level of 142 yM/40 cm, the difference representing a 125% rise in Ca absorption above the pretreatment level. This striking rise in Ca absorption occurred within 4852 hours after the administration of the first dose of 1,25(0H)2D3. The absorption rates of P, Mg, Na and water also increased but these changes were not statistically significant when compared to the pre-treatment levels (Table I). Despite the high Ca content of the diet and the increased Ca absorption the serum Ca levels in these subjects did not rise significantly above their pre-treatment levels. When l,25(OH)2D3 was administered orally instead of by the iv route, the jejunal response in normal subjects ingesting a high Ca diet was similar to that observed after iv administration (Table II). Ca absorption increased in both subjects while P absorption increased in one but decreased in the other (Table II). Table II.

Jejunal Ca and P Absorption in Two Subjects (B.T. & M.M.) Given l,25(OH)2D3 Orally (4 meg daily X 3 dose, 3rd dose given 12 hr prior to the second perfusion). Ca P

before after

B.T. 141 370

M.M. 96 643

- before after

375 301

424

228

625 When the results from the studies with iv and oral administration of l,25(OH)2D3 were combined, it was evident that Ca absorption increased in all of the eight subjects tested while P absorption increased in five of the eight and decreased or was unchanged in the other three subjects. In summary, the intestinal effect of l,25(OH)2Ü3 in normal subjects was measured by triple lumen intestinal perfusion. Jejunal Ca absorption increased 125% above the pre-treatment level within 48 hours after the first dose in subjects ingesting a high Ca diet. No effect on Ca absorption was detected within the first six hours after an iv injection of l,25(OH)2D3 in normal subjects ingesting their customary diets. Jejunal P absorption increased in 5 of the 8 subjects given 1,25(0H)2D3 by the iv or oral route but the m e a n changes in P absorption after the administration of l,25(OH)2Ö3 were not statistically significant. Supported in part by NIH research grant 1-R01-AM17835 and by the National Foundation Clinical Research grant 6-43j also P.H.S. Research Grant RR-46. REFERENCES 1.

DeLuca, H.F., Schnoes, H.K.: Metabolism and Mechanism of Action of Vitamin D. Annual Review of Biochemistry 45_, 631-666 (1976).

2.

Norman, A.W., Henry, H.: 1,25-Dihydroxycholecalciferol - A Hormonally Active Form of Vitamin D. Recent Progress in Hormone Research 3Q, 431-470 (1974).

3.

Brickman, A.S., Coburn, J.W., Friedman, G.R., Okamura, W.H., Massry, S.G., and Norman, A.W.: Comparison of Effects of la-Hydroxy-Vitamin D3 and 1,25-Dihydroxy-Vitamin D3 in Man. J.C.I. 57, 1540-1547 (1976).

4.

Cooper, H., Levitan, R., Fordtran, J.S. and Ingelfinger, F.J.: A method for studying absorption of water and solute from the human small intestine. Gastroenterology 50, 1-8 (1966).

VITAMIN D AND

OSTEOPOROSIS

Robert P. Heaney, M.D. Creighton University, Omaha, Nebraska 68178, U S A

INTRODUCTION Before discussing the relationship, if any, of vitamin D to the osteoporosis problem, it is well to review briefly what is known concerning the nature of osteoporosis, its pathogenesis, and the evidence for involvement of vitamin D anywhere in the process. Osteoporosis, as usually defined, consists of a quantitative reduction in the amount of bony material per unit volume of bone as an organ. This reduction has as its sole consequence of importance, increased bone fragility.

According to this definition, there are no qualitative

abnormalities in the bony tissue which is present.

From a clinical standpoint, osteoporosis consists of a syndrome in which fractures develop, particularly crush fractures of the spine, in association with X-ray evidence of decreased bone mineral density.

Bone biopsy in such patients usually reveals changes consistent with the

customary definition, but occasionally, and particularly in northern Europe, qualitative abnormalities have been found, principally an excess of unmineralized osteoid. There is no agreement as to how common this finding may be — and it is apparently unusual in the United States — or whether it represents an integral part of the syndrome, a malacic component superimposed on an otherwise typical osteoporosis, or some other as yet undefined syndrome. For purposes of this presentation, this is an important distinction, but for the moment I shall set aside, into a special subset, those osteoporotics with wide osteoid seams and concentrate on the vast majority of osteoporotics whose bone seems to fit the usual definition.

DEVELOPMENT OF

OSTEOPOROSIS

In 1965, I proposed a model for the development of osteoporosis in which the elements of calcium homeostasis served as a kind of "final common pathway" by which diverse etiologies might lead to the same result, i.e., net loss of bone mass (1). This model was based in part on the observation that parathyroid hormone (PTH) is the principal determinant of the quantity of bone remodeling, and that, deprived of PTH, animals are protected against osteoporosis; and in part on the fact that PTH exerts its calcium-conserving effects by acting on three otherwise

628 independent end organs.

Differential alteration of the sensitivity or responsiveness of these

end organs to endogenous levels of PTH would — the model predicts — lead to altered skeletal balance. There have been several tests of the predictions of this model in the past 12 years, and to my knowledge all have verified its essential elements (2—5).

I mention the model in this context because it intersects our concern with vitamin D at two significant points, i.e., in the roles vitamin D plays in mediating PTH effects on bone mineral resorption and on intestinal calcium absorption. As we now know, vitamin D is essential for the full resorptive effect of PTH in bone, and mediates, through synthesis of 1,25(OH)2D3 the effect of PTH on calcium absorption. Hence vitamin D and its metabolites find themselves, at least coincidentally, involved in the osteoporotic process.

A primary deficiency of D would be expected to produce some degree of refractoriness of bone resorptive response to PTH, together with some impairment of dietary calcium absorption efficiency; whereas a primary excess of D would be expected to produce hyperabsorption of calcium and some degree of enhanced bone resorption. Is either type of change found in human osteoporosis? And if so, what might be the nature of the association?

VITAMIN D ABNORMALITIES IN OSTEOPOROSIS Very few studies have been directed at vitamin D and its metabolites in osteoporosis. Absorption of the vitamin itself has been determined to be normal (6) but overall, most studies have focused on vitamin effects, rather than on the vitamin itself. Thus some degree of impairment of calcium absorption efficiency has been commonly observed (7—10).

We have recently

reported that this impairment seems to be due specifically to estrogen withdrawal at menopause (4) and that, at any given intake, estrogen-deprived women absorb less efficiently than do those with normal estrogen levels.

Additional D given to osteoporotic women has been

shown to increase absorption (11), and hence some impairment of conversion of D to its active metabolites has been postulated.

Very recently Gallagher and co-workers have de-

scribed reduced circulating levels of 1,25(OH)2D3 in postmenopausal osteoporotics (5).

Do the reduced intestinal absorption efficiency and the lower levels of 1,25(OH)2D3 suggest an effective deficiency of vitamin D as of possible causal or contributory significance? If we recall the generally accepted hallmarks of D deficiency, i.e., wide osteoid seams, impaired osteoclast function, decreased calcium absorption from the intestine, elevated parathyroid hormone levels, hypophosphatemia, decreased urine calcium, and (although not so commonly spoken about) zero calcium balance, we see that this pattern does not well fit the usual picture

629 in osteoporosis. Most significantly, PTH is probably low in post-menopausal osteoporosis (3) — at least it is not high; plasma phosphorus is slightly higher than normal rather than low; urine calcium is not decreased, at least in most patients; and bone resorption is generally felt to be either normal or high, a finding of considerable significance in view of the low-to-normal levels of PTH which appear to characterize the disorder.

What this constellation of findings suggest is not a primary deficiency of vitamin D, but a depression of 1,25(OH)2D3 synthesis as a consequence of excess calcium release from bone. The model, in fact, predicts exactly these findings, i.e., as a result of enhanced osteoclast sensitivity, existing levels of PTH evoke excess calcium release from bone, which leads to reduced PTH secretion, which in turn leads to decreased 1,25(OH)2Dg synthesis and correspondingly reduced intestinal calcium absorption efficiency.

This is in contrast with the

resorptive interference which is a feature of true D deficiency, and which constitutes a protection against calcium wastage, not a mechanism which leads to bone loss.

The lack of

calcification of growth cartilage and of osteoid in frank D deficiency is badly misinterpreted if it is read as an indication of calcium wastage. The real significance of hypomineralization of newly deposited bone is apparent only when taken together with the fully normal mineralization of previously formed bone. In the last analysis vitamin D is necessary for the mobilization of mineral reserves.

Hence I am led to conclude that such abnormalities of vitamin D metabolism as may exist in osteoporosis are not of pathogenetic significance, but occur as a consequence of other changes more directly involved in the disease process. Nevertheless, having said this, I must quickly advert to the almost certain heterogeneity of the disorder. If osteoporosis can develop because of selective enhancement of osteoclastic responsiveness to PTH, could it not also develop because of selective impairment of intestinal response to 1,25(OH)2Dg? predicts that it could.

Indeed the model

One would expect however reflexly elevated levels of 1,25(OH)2Dg

and of PTH, and it is not certain that such changes exist. But for a subset of osteoporotics, this could still be the primary abnormality.

Further, of what significance are the cases which seem to show widened osteoid seams, and those which seem to respond to moderate doses of vitamin D? I cautiously suggest that these represent superimposition of osteomalacia on pre-existing osteoporosis.

Finally, I should

mention a remote connection between osteoporosis and vitamin D noted, to my knowledge, only by Saville (12).

He reported a statistically significant excess among his osteoporotic

women of births occurring in the spring and early summer months. Their gestational periods

630 had thus been during the fall and winter months, when it is known that maternal vitamin D levels are at their lowest. Further, all his patients had been born before vitamin D had been "invented".

He suggested that this perinatal experience of relative D deficiency might, by

unknown mechanisms, have had some influence on later susceptibility to development of osteoporosis.

To my knowledge, this observation has never been repeated, so its validity

cannot be confirmed.

VITAMIN D A N D THE T R E A T M E N T OF OSTEOPOROSIS Most of the reports of the use of vitamin D in the treatment of osteoporosis of which I am aware have employed D as an adjunct, either with calcium alone or with calcium plus fluoride. The rationale, in both instances, has been to insure absorption of calcium, and accordingly the doses of D chosen have usually been high — up to 50,000 units daily.

When used with calcium alone the results have generally been a suppression of bone turnover — both resorption and formation (13), but with no increase in skeletal mass (14). There was little or no difference in the degree of bone turnover suppression whether D was given in doses of 400 units/day or 50,000 units 3x/wk. Since in other series the same suppression of turnover has been produced by calcium alone (15), it is doubtful that vitamin D plays any role at all in this regimen. Calcium absorption, as we have shown elsewhere (16), can be forced simply by increasing intake, and it is probable that the intestinal effect of D in man is of significance primarily in permitting adaptation to reduced calcium intake.

The suppression of bone turnover, coupled with the failure to see bone mass changes, means simply that this form of treatment stabilizes the bone mass — but does not increase it. Hence it is not likely to be of much use as therapy.

However, the combination of calcium, fluoride, and vitamin D produces quite different results. Here bone turnover has not been suppressed; instead osteoblastic surfaces have been increased, and active resorption surfaces decreased (17). These changes are of exactly the character one would want. There have been some reports of increased bone mass (18), but whether this is a regular and consistent finding remains to be seen. The most commonly employed combination regimen includes 50,000 units of vitamin D 3x/wk, but it is simply not known whether the D adds anything significant to this combination.

I suspect that it does not.

As with

calcium therapy alone, the added D does not appear to be necessary to insure calcium absorption so long as the calcium is given in large amounts. Furthermore, the danger of vitamin D intoxication, especially in old people who may misunderstand or misuse the vitamin (19),

631 is real, and it would seem that some effort should be made to see if normal histology can be maintained in these patients without the addition of D to the regimen. VITAMIN D IN PREVENTION OF OSTEOPOROSIS Relatively few studies dealing with calcium or calcium plus D in prevention of osteoporosis have been reported. Our own experience is that calcium can suppress bone turnover in preosteoporotic females, and reduce the usual age-related bone loss of the post-menopausal period (15). It is not known whether supplemental vitamin D would enhance this effect, but it seems doubtful that it would add much. SUMMARY The slightly reduced intestinal absorption of calcium commonly associated with osteoporosis appears to be a consequence of the physiological adjustments which predispose to osteoporosis, rather than a primary defect in D intake or metabolism of causal significance for the disorder. Nevertheless, osteoporosis is very probably a heterogeneous disorder, and the possibility cannot be excluded that a subset of osteoporotics exists in which a defect of D metabolism may be of causal significance. Vitamin D has been employed as a part of regimens containing either calcium alone or calcium plus fluoride in the treatment and/or prevention of osteoporosis.

It is suggested that the

added vitamin D contributes little or nothing to the effects produced by the other agents, and that, in the doses commonly employed, its addition carries a significant risk of toxicity.

632 REFERENCES

1.

Heaney, R.P.: A unified concept of osteoporosis. Am. J. Med. 39, 877-880 (1965).

2.

Riggs, B.L., Jowsey, J., Goldsmith, R.S., Kelley, P.J., Hoffman, D.C., Arnaud, C.D.: Short- and long-term effects of estrogen and synthetic anabolic hormone in postmenopausal osteroporosis. J. Clin. Invest. 51_, 1659-1663 (1972).

3.

Riggs, B.L., Arnaud, C.D., Jowsey, J., Goldsmith, R.S., Kelly, P.J.: Parathyroid function in primary osteoporosis. J. Clin. Invest. 53,181-184 (1973).

4.

Heaney, R.P., Recker, R.R.: Estrogen effects on bone remodeling at menopause. Clin. Res. 23, 535A (1975).

5.

Gallagher, J.C., Riggs, B.L., Eisman, J., Arnaud, S.B., DeLuca, H.F.: Impaired production of 1,25 Dihydroxy vitamin D in post-menopausal osteoporosis.

Clin.

Res. 24, 580A (1976). 6.

Bordier, P.J., Miravet, L., Hioco, D.: Young adult osteoporosis. Clinics Endocrinol. Metab. 2, 277-292 (1973).

7.

Nordin, B.E.C.: Clinical significance and pathogenesis of osteoporosis. Br. Med. J. 571-576 (1971).

8.

Spencer, H., Menczel, J., Lewin, I., Samachson, J.: Absorption of calcium in osteoporosis. Am. J. Med. 37, 223-234 (1964).

9.

Saville, P.D.: The syndrome of spinal osteoporosis.

Clinics Endocrinol. IVletab. 2,

177-185 (1973). 10.

Gallagher, J.C., Aaron, J., Horsman, A., Marshall, D.C., Wilkinson, R., Nordin, B.E.C.: The crush fracture syndrome in post-menopausal women. Clinics Endocrinol. Metab. 2, 293-315 (1973).

11.

Reeve, J., Hesp, R., Veall, N.: Effects of therapy on rates of absorption of calcium from the gut in disorders of calcium homeostasis.

Br. Med. J. 3, 310-313 (1974).

12.

Saville, P.D.: personal communication.

13.

Riggs, B.L., Jowsey, J., Kelly, P.J., Hoffman, D.L., Arnaud, C.D.: Effects of oral therapy with calcium and vitamin D in primary osteoporosis.

J. Clin. Endocrinol.

Metab. 42, 1139-1144 (1976). 14.

Buring, K., Hulth, A.G., Nilsson, B.E., Westlin, N.E., Wiklund, P.E.: Treatment of osteoporosis with vitamin D. Acta Med. Scand. 195,471-472 (1974).

15.

Recker, R.R., Saville, P.D., Heaney, R.P.: Sex hormones or calcium supplements diminish post-menopausal bone loss. Clin. Res. 24, 583A (1976).

16.

Heaney, R.P., Saville, P.D., Recker, R.R.: Calcium absorption as a function of calcium intake. J. Lab. Clin. Med. 85, 881-890 (1975).

633 17.

Jowsey, J., Riggs, B.L., Kelly, P.J., Hoffman, D.L.: Effect of combined therapy with sodium fluoride, vitamin D and calcium in osteoporosis.

Am. J. Med. 53, 43-49

(1972). 18.

Hansson, T., Roos, B.: Effect of combined therapy with sodium fluoride, calcium, and vitamin D on the lumbar spine in osteoporosis. Am. J. Roentgenol. 126, 1294 (1976).

19.

Libow, L.S., Ross, F., Goldberg, K.: "One tablet per d a y " for osteoporosis: Hypercalcemia. Ann. Int. Med. 81, 120-121 (1974).

LONG-TERM TREATMENT AND C A L C I U M ^

OF O S T E O P O R O T I C

P A T I E N T S WITH

loC-OH-D

T.S. L i n d h o l m , J.A. S e v a s t i k o g l o u , U. Lindgren D e p a r t m e n t of O r t h o p a e d i c S u r g e r y , Huddinge U n i v e r s i t y K a r o l i n s k a I n s t i t u t e , S t o c k h o l m , Sweden.

Hosp it a1

The development of o s t e o p o r o s i s may follow quite different c o u r s e s , most of which are still not clearly u n d e r s t o o d . The p a t h o g e n e s i s of bone loss in the elderly is partly explained by a decline of the i n t e s t i n a l calcium a b s o r p t i o n (l).The absorptive m e c h a n i s m of calcium in the intestine responds to l a - O H - D ^ (h ) . This i n v e s t i g a t i o n was u n d e r t a k e n to study the effect of combined l o n g - t e r m t r e a t m e n t with l a - O H - D ^ and calcium in o s t e o p o rotic p a t i e n t s . CLINICAL M A T E R I A L AND

METHODS

S i x t e e n o s t e o p o r o t i c p a t i e n t s , lU women and 2 m e n , with mean age of 6l years (r.2 U — 7 8 y e a r s ) and with o s t e o p o r o s i s of different g e n e s i s , as g e r o n t o i d : 7 c a s e s , p o s t m e n o p a u s a l : 5 cases, c o r t i c o s t e r o i d : 2 cases and idiopathic: 2 cases were studied. All p a t i e n t s complained of severe lumbar pain and they had m u l t i p l e lumbar c o m p r e s s i o n fractures before a d m i t t a n c e . Before and during the p e r i o d of t r e a t m e n t different p a r a m e t e r s of calcium m e t a b o l i s m and the degree of o s t e o p o r o s i s were examined as follows: radiographic m o r p h o m e t r y , p h o t o n a b s o r p t i o m e t r y , b l o o d and urine c h e m i s t r y , i n t e s t i n a l a b s o r p t i o n of calcium after p e r o r a l a d m i n i s t r a t i o n of ^ 5ca and h i s t o m e t r y of crista b i o p s i e s . T r e a t m e n t . The p a t i e n t s were treated with the synthetic lah y d r o x y c h o l e c a l c i f e r o l ( l a - O H - D ^ ) in oral doses v a r y i n g from 0.5-2 ug per day and 1 - 2 g calcium. The treatment was interrupted or adjusted to lower doses in case of h y p e r c a l c e m i a . The p a t i e n t s have been treated until now for p e r i o d s of 6 - 1 5 months and are still under t r e a t m e n t . RESULTS B e f o r e t r e a t m e n t : D i m i n i s h e d bone mass was found in all patients (Fig.l). The values for serum c a l c i u m , p h o s p h a t e , a l k a line p h o s p h a t a s e and h - p a r a t h y r o i d h o r m o n e were w i t h i n the n o r m a l range levels. The daily e x c r e a t i o n of urinary c a l c i u m , p h o s p h o r o u s and h y d r o x y p r o l i n e was normal. The calcium absorpS u p p o r t e d by grants from the Swedish M e d i c a l C o u n c i l and the K a r o l i n s k a I n s t i t u t e .

Research

636

637 tion rate was lowered osteoporosis .

in all 6 tested

patients with

senile

H i s t o m e t r y v e r i f i e d decreased bone mass in all p a t i e n t s . one there were also signs of o s t e o m a l a c i a . During t r e a t m e n t : The d e n s i t o m e t r i c m e a s u r m e n t s creased values in all patients during treatment (Fig. 2). In some cases decreased bone m i n e r a l recorded after 2 - 3 months of t r e a t m e n t .

In

showed in(p 515-518 (1973). Meema, S. , Bunker, M. L., Meema, H. E.: Preventive effect of estrogen on postmenopausal bone loss: a follow-up study. Arch. Intern. Med. J_35, 1436-1440 (1975). Lafferty, F. W., Spencer, G. E., Pearson, 0. H.: Effects of androgens, estrogens and high calcium intakes on bone formation and resorption in osteoporosis. Am. J. Med. 36, 514-528 (1964). Harris, W. H., Heaney, R. P.: Skeletal renewal and metabolic bone disease. N. Engl. J. Med. _280, 193-202 (1969). Riggs, B. L., Jowsey, J., Kelly, P. J., Jones, J. D., Maher, F. T.: Effect of sex hormones on bone in primary osteoporosis. J. Clin. Invest. 48, 1065-1072 (1969). Riggs, B. L., Jowsey, J., Goldsmith, R. S., Kelly, P. J., Hoffman, D. L., Arnaud, C. D.: Short- and long-term effects of estrogen and synthetic anabolic hormone in postmenopausal osteoporosis. J. Clin. Invest. 51, 1659-1663 (1972).

647 28. 29. 30.

31. 32.

33. 34. 35.

36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.

Orimo, H., Fujita, T., Yoshikawa, M.: Increased sensitivity of bone to parathyroid hormone in ovariectomized rats. Endocrinology 90, 760-763 (1972). Heaney, R. P.: A unified concept of osteoporosis (editorial). Am. J. Med. 3_2> 877-880 (1965). Riggs, B. L., Ryan, R. J., Wahner, H. W., Jiang, N.-S., Mattox, V. R.: Serum concentrations of estrogen, testosterone and gonadotropins in osteoporotic and nonosteoporotic postmenopausal women. J. Clin. Endocrinol. Metab. 36» 1097-1099 (1973). DeLuca, H. F.: Recent advances in our understanding of the vitamin D endocrine system. J. Lab. Clin. Med. 87_, 7-26 (1976). Avioli, L. V., McDonald, J. E., Lee, S. W.: The influence of age on the intestinal absorption of ^'Ca in women and its relation to ^ C a absorption in postmenopausal osteoporosis. J. Clin. Invest. 44, 1960-1967 (1965). Bullamore, J. R., Gallagher, J. C., Wilkinson, R., Nordin, B. E. C.: Effect of age on calcium absorption. Lancet 2, 535-537 (1970). Alevizaki, C. C., Ikkos, D. G., Singhelakis, P.: Progressive decrease of true intestinal calcium absorption with age in normal man. J. Nucl. Med. _14, 760-762 (1973). Szymendera, J., Heaney, R. P., Saville, P. D.: Intestinal calcium absorption: concurrent use of oral and intravenous tracers and calculation by the inverse convolution method. J. Lab. Clin. Med. _79, 570-578 (1972). Caniggia, A., Gennari, C., Bianchi, V., Guideri, R.: Intestinal absorption of ^ C a in senile osteoporosis. Acta Med. Scand. 173, 613-617 (1963). Kinney, V. R., Tauxe, W. N., Dearing, W. H.: Isotopic tracer studies of intestinal calcium absorption. J. Lab. Clin. Med. 66, 187-203 (196b). Lund, B., S/5rensen, 0. H., Christensen, A. B.: 25-Hydroxycholecalciferol and fractures of the proximal femur. Lancet 2, 300-302 (1975). Gallagher, J. C., Riggs, B. L., Eisman, J., Arnaud, S. B., DeLuca, H. F.: Impaired production of 1,25-dihydroxyvitamin D in postmenopausal osteoporosis (abstract). Clin. Res. 24, 580A (1976). Dent, C. E., Smith, R.: Nutritional osteomalacia. Q. J. Med. ¿8, 195-209 (1969). Brickman, A. S., Coburn, J. W., Massry, S. G., Norman, A. W.: 1,25 Dihydroxy-vitamin D^ in normal man and patients with renal failure. Ann. Intern. Med. 8£, 161-168 (1974). Norman, A. W., Henry, H.: The role of the kidney and vitamin D metabolism in health and disease. Clin. Orthop. 98» 258-287 (1974). Alb right, F., Reifenstein, E. C., Jr.: The Parathyroid Glands and Metabolic Bone Disease: Selected Studies. Baltimore, Williams &. Wilkins Company (1948). Kelly, P. J., Jowsey, J., Riggs, B. L., Elveback, L. R.: Relationship between serum phosphate concentration and bone resorption in osteoporosis. J. Lab. Clin. Med. _69, 110-115 (1976). Kenny, A. D.: Vitamin D metabolism: physiological regulation in egg-laying Japanese quail. Am. J. Physiol. 230, 1609-1615 (1976). Tanaka, Y., Castillo, L., DeLuca, H. F.: Control of renal vitamin D hydroxylases in birds by sex hormones. Proc. Natl. Acad. Sci. U.S.A. 73, 2701-2705 (1976).

648 47.

48.

49.

50. 51.

52. 53.

54.

55.

M a l m , 0. J . : A d a p t a t i o n to a l t e r a t i o n s in c a l c i u m intake. _In T h e T r a n s f e r of C a l c i u m and S t r o n t i u m A c r o s s B i o l o g i c a l M e m b r a n e s . E d i t e d by R. H. W a s s e r m a n . N e w Y o r k , A c a d e m i c P r e s s (1963), pp. 143173. R i g g s , B. L., K e l l y , P. J . , K i n n e y , V. R . , Scholz, D. A . , B i a n c o , A. J., Jr.: C a l c i u m d e f i c i e n c y and o s t e o p o r o s i s : o b s e r v a t i o n s in one h u n d r e d and s i x t y - s i x p a t i e n t s and c r i t i c a l r e v i e w of the l i t e r a t u r e . J . B o n e J o i n t Surg. [Am.] 49, 9 1 5 - 9 2 4 (1967). H e a n e y , R. P., R e c k e r , R. R . , Saville, P. D.: C a l c i u m b a l a n c e and c a l c i u m r e q u i r e m e n t s in m i d d l e - a g e d w o m e n ( a b s t r a c t ) . C l i n . Res. _22, 6 4 9 A (1974). S p e n c e r , H., M e n c z e l , J . , L e w i n , I., S a m a c h s o n , J . : A b s o r p t i o n of c a l c i u m in o s t e o p o r o s i s . Am. J. Med. 3_7, 2 2 3 - 2 3 4 (1964). W h e d o n , G. D.: E f f e c t s of h i g h c a l c i u m i n t a k e s o n b o n e s , b l o o d and soft tissue: r e l a t i o n s h i p of c a l c i u m intake to b a l a n c e in o s t e o porosis. Fed. P r o c . _18, 1 1 1 2 - 1 1 1 8 (1959). D e L u c a , H . F.: Personal communication. A l b r i g h t , F., Smith, P. H., R i c h a r d s o n , A . M . : Postmenopausal osteoporosis: its c l i n i c a l f e a t u r e s . J.A.M.A. 116, 2 4 6 5 - 2 4 7 4 (1941). F r a n t z , A . G., R a b k i n , M . T . : E f f e c t s of e s t r o g e n and sex d i f f e r e n c e on s e c r e t i o n of h u m a n g r o w t h h o r m o n e . J. C l i n . E n d o c r i n o l . M e t a b . 2_5:1470-1480 (1965). L u n d , B., H j o r t h , L., K j a e r , I., R e i m a n n , I., F r i i s , T., A n d e r s e n , R . B., S ^ r e n s e n , 0. H . : T r e a t m e n t of o s t e o p o r o s i s of a g e i n g w i t h la-hydroxycholecalciferol. L a n c e t _2, 1168-1171 (1975).

25 HYDROXYVITAMIN D IN RENAL OSTEODYSTROPHY: RESULTS O F A SIX-CENTER TRIAL Preliminary Report

Robert R. Recker Creighton University School of Medicine, Omaha, Nebraska

68108

USA

Patricia Schoenfeld University of California, Artificial Kidney Center, San Francisco, CA Joseph Letteri Nassau County Medical Center, East Meadow, NY Eduardo

Slatopolsky

Washington University School of Medicine, St. Louis, MO Kevin Martin Washington University School of Medicine, St. Louis, MO Lawrence Kleinman Nassau County Medical Center, East Meadow, NY David Hartenbower Veterans Hospital, Wadsworth Center, Los Angeles, CA Arnold Brickman Veterans Hospital, UCLA School of Medicine, Los Angeles, CA Ralph Goldsmith Veterans

Hospital, San Antonio, TX

Harold Frost Southern Colorado Clinic, Pueblo, CO Webster Jee University of Utah, Salt Lake City, UT Steven Teitelbaum Jewish Hospital of St. Louis, St. Louis, MO Don Kimmel University of Utah, Salt Lake City, UT Claude Arnaud Mayo Clinic, Rochester, MN

650 Sara Arnaud Mayo Clinic, Rochester, MN Robert Heaney Creighton University School of Medicine, Omaha, NE

Introduction:

Disturbances in calcium, phosphorus and parathyroid hormone

relationships are almost universal in chronic hemodialysis patients. Resultant symptomatic bone disease is much less common although some authors have found histologic evidence of bone disease in virtually all patients examined by bone biopsy (1).

When the disturbance in skeletal homeostasis

becomes severe enough to cause symptoms, the bone disease becomes a major disability because of skeletal pain, with or without fractures (2).

Recent advances in Vitamin D metabolism led to the availability of 25 hydroxyvitamin D (3), the liver product in the chain of Vitamin D synthesis. Very recent evidence has linked this hormone precursor to the pathogenesis of renal osteodystrophy (4,5).

A multicenter study was designed to test

its efficacy in healing renal osteodystrophy.

This report summarizes the

preliminary findings during the first 17 weeks of treatment.

Methods: Protocol Design: Table I).

Six dialysis centers participated in the study (see

A total of sixty three chronic hemodialysis patients were se-

lected; forty four of these underwent complete clinical and biochemical evaluation; and thirty five of the latter underwent pretreatment and post treatment iliac crest biopsies.

Entry criteria

included the presence

of bone pain and tenderness, elevated alkaline phosphatase and abnormal bone radiographs.

Numerous exclusion criteria

were established in order

to eliminate patients with additional diagnoses and treatments which might affect bone, calcium or vitamin D metabolism.

651 Table I

CENTERS SELECTED FOR CLINICAL TESTING OF 250HD Location

Dialysis Center

Principal

Investigator

1.

San Francisco, CA

U. of California at S.F. General Hospital

P. Schoenfeld, M.D.

2.

East Meadow, NY

Nassau County Hospital

J. Letteri, M.D.

3.

St. Louis, MO

Barnes Hospital

E. Slatopolsky, M.D

4.

Omaha, NE

Creighton University

R. Recker, M.D.

5.

San Antonio, TX

Veterans Hospital Bexar County Hospital Brook Army Hospital

R. Goldsmith, M.D.

6.

Los Angeles, CA

Veterans Hospital

A. Brickman, M.D

Each patient was observed for twenty nine weeks.

The first twelve served

as a control period during which placebo was given and the following 17 weeks served as the period of treatment.

A loading dose of 600 micrograms

of 250HD was given 3 times orally during the first week of therapy immediately following dialysis.

Thereafter a maintenance dose of 200 micrograms

thrice weekly was given immediately following dialysis.

The dose was a d -

justed upwards or downwards so as to avoid severe hypercalcemia or a Ca X P product of over 70.

Vigorous attempts at controlling serum phosphorus

were continued throughout by the use of aluminum containing gels.

Bath

calcium was kept at approximately 6.5 mg% and diet calcium was maintained above at least 500 mgs per day.

Clinical and biochemical

examinations

were performed at 4 week intervals during the control and treatment periods. A total of 4 control and 5 treatment evaluations were obtained for a grand total of 9 evaluations.

Clinical Evaluation Methods:

Symptom evaluation was performed by question-

ing each patient whether he had pain on weight bearing or at rest at 37 different skeletal sites.

Bone tenderness was evaluated at 12 different

sites by gentle fingertip palpation and firm flat pressure.

A clinical

652 score was given to each patient at each evaluation.

Biochemical Evaluation Methods:

Each of the 9 biochemical

evaluations

consisted in serum determinations of total calcium, phosphorus, magnesium, creatinine, 250HD and alkaline phosphatase.

iPTH,

These were all

performed on specimens sent to the Mayo Clinic reference laboratory. The iPTH assays were performed by Dr. Claude Arnaud terminal antibody and the 250HD

assays were performed by Dr. Sara Arnaud

using a modification of Haddad's method

Bone Biopsy Analysis:

(6) using the carboxy

(7).

Details of the method used in this study are de-

scribed in a companion paper (8).

All biopsies were read by both Drs.

Steven Teitelbaum in St. Louis and Donald Kimmel in Salt Lake without knowledge of patient identification or treatment

(pre or post treatment).

The

results of the readings from each center on each patient were averaged. The histomorphometric parameters presented in this report include the percent of bone matrix volume that is unmineralized osteoid, the percent of the linear trabecular surface that is covered with unmineralized osteoid, the absolute linear trabecular surface that is covered with unmineralized osteoid, and the percent of the linear trabecular surface that is covered with fibrous tissue.

Results:

There was wide variation in the severity of disease with clinical

scores ranging from zero to 42.

Certain of the biochemical

parameters

correlated with the severity of symptoms, i.e., baseline serum alkaline phosphatase was generally higher in those with more severe disease and serum 250HD lower.

Baseline serum alkaline phosphatase ranged between

22 and 246 I.U./L, and serum 250HD between 6.7 and 171 mg/ml. Baseline serum calcium averaged 9.27 + .86 (S.D.) mg% and most values were within the normal range.

Serum phosphorus levels showed wider variation with

a mean of 4.53 + 1.05 (S.D.) mg%.

Serum parathyroid hormone (iPTH) levels

were extremely elevated and extremely variable. The levels ranged from approximately 5-fold to 400-fold above the upper limit of normal of 40 microliter equivalents per ml.

There was wide variation in the amount

653 of unmineralized osteoid present in the pretreatment biopsy but overall, there was a mean increase to about 3-fold above normal. Fibrous tissue was present in 26 of the biopsies prior to treatment in some cases to an extensive degree. Treatment effects are summarized in Table II.

For this analysis, the

week 29 value was expressed as a fractional increase or decrease compared to the average baseline value of each parameter.

There was a significant

decrease in pain and tenderness and an improvement in disability. Furthermore, those patients with more severe symptoms tended to have a greater degree of improvement.

Serum calcium and phosphorus both rose in response to treatment.

Mild

hypercalcemia resulted (average at the end of 17 weeks treatment was 10.5 mg%) while mean serum phosphorus,though increased over baseline, remained normal.

However, great variation in serum phosphorus continued during

treatment.

Serum iPTH levels overall did not show a significant change

and remained extremely elevated and variable.

Part of the reason for the

failure of iPTH to fall in spite of hypercalcemia was persistent hyperphosphatemia in some patients.

Serum alkaline phosphatase fell significantly. Table II

Fractional Increase or Decrease at the end of 17 Weeks Treatment with 250HD t

P

Pain

-.307

2.93

ug/wk for 56 wks. Group II: 7 pts requiring hemodialysis received oral 25-OHD 3 times weekly with a mean intake of 60-150/lg/wk for 55 wks. Group III: 16 pts requiring hemodialysis for 36 to 48 wks did not receive 25-OHD. During the study period all pts received aluminum hydroxide gels to control serum P concentration. Dietary Ca and P averaged 700 mg/day and 800 mg/day respectively for Group I and 350 mg/day and 700 mg/day respectively for Group II. Group III received a mean dietary intake of 800 mg/day for both Ca and P in addition to 2.6 to 5.2 gm/day of CaC03. Groups II and III received hemodialysis with a dialysate bath Ca of approximately 6.5 mg percent throughout the study. Techniques: Total body calcium was measured by neutron activation analysis (TBNAA) at the Brookhaven National Laboratory (1). With this procedure the TBCa is measured with a precision (or reproducibilty) of + 1 percent. Thus changes in TBCa of 3 percent or greater can be measured at the 99 percent confidence level (3 S.D.). The BMC and the width of the distal radius were measured by the CameronSorenson densitometric technique with the Norland-Cameron densitometer (2). The precision of the technique was 2.5 percent (S.D.). TBCa and BMC were measured initially in all pts. These measurements were repeated in all pts once during the treatment period and then again at the end of the study.

676 RESULTS In the 2 non dialysis pts (Group I), the BMC remained essentially unchanged throughout the study. After 30 wks TBCa decreased in one and increased in the other. After 55 wks of therapy with 25-0HD TBCa increased in both pts. In the 7 dialysis pts who received 25-OHD (Group II), a biphasic response was noted. After 17 wks TBCa decreased in 2 and remained unchanged in 5, while the BMC remained unchanged in 6 and increased in one pt. At 55 wks, an increase in TBCa was noted in all pts while the BMC increased in 2, decreased in one and remained unchanged in 4 pts. The relationship between percent change in TBCa and BMC in individual pts was poor (Fig. 1). •36 031 ABMC

BMC

/

15-

/

10

5-J

/ /

/

r

/

•

-15 -10 -5_

'

o5

/

/

0 o 10

A TBCa 15

-5

/

s •

l

r

•5 »-5 --10 r "15

1 1— 10 15

A T B C a « % CHANGE IN TOTAL BODY CALCIUM A B M C = % CHANGE IN BONE MINERAL CONTENT

Ho

Fig. 1. Group II, dialysis pts receiving 25-OHD. • 17 wks therapy. 0 - 5 5 wks therapy.

Fig. 2. Group III, dialysis pts not receiving 25-OHD.

In 16 pts receiving hemodialysis, but who did not receive 25-OHD (Group III), no change or a slight decrease in TBCa and BMC was observed after 36 to 48 wks of hemodialysis (3) (Fig. 2). The relationship between percent change in TBCa and BMC in individual pts was poor in Group III. DISCUSSION This study clearly demonstrates that 55 wks of therapy with 25-OHD significantly increased TBCa in all pts (Groups I and II) as measured by TBNAA. These results are especially remarkable when compared to the

677 small group of pts not receiving 25-OHD in whom no significant change in TBCa was generally observed (Group III). The increase in TBCa associated with 25-OHD may be in part due to an increase in intestinal absorption of exogenous Ca and, a decrease in endogenous fecal Ca loss (4). The absence of a significant change in BMC was an unexpected finding. If 25-OHD Increases bone calcification the BMC of the distal radius should increase concomitantly with the observed change in TBCa. The lack of an observed change in BMC may be related to several factors. The BMC measured in the distal radius may not adequately reflect the rate of calcification in other sites of the skeleton. The measurement of BMC by the NorlandCameron densitometer may be insensitive for the detection of significant changes in mineralization of the distal radius. The TBCa measurement includes Ca in soft tissues as well as skeletal Ca; thus, the increase in TBCa may reflect an Increase in soft tissue Ca, and not an increase in the skeletal Ca. It is also possible that there is a delay between an increase in TBCa stores and a concomitant increase in the osseous Ca. In summary, the experimental drug 25-OHD resulted in an increase in TBCa, but the distribution of the retained Ca in the body is uncertain. Concomitant with the adminlfttration of 25-OHD was an elevation of the serum Ca and P which required frequent lowering of the dosage of 25-OHD. Due to the uncertain distribution of the retained Ca, and the associated hypercalcemia observed with this experimental drug it is manditory that uremic pts treated with 25-OHD are monitored closely. BIBLIOGRAPHY 1.

Cohn, S.H., Dombrowski, C.S., and Fairchild, R.G.: In vivo neutron activation analysis of calcium in man. Int. J. Appl. Rad. Isotopes, 22:127 (1970).

2.

Cameron, J. R., Mazess, R.B. and Sorenson, J.A.: Precision and accuracy of bone mineral determination by direct photon absorptionmetry. Invest. Rad., 3, 141-150 (1968).

3.

Cohn, S.H., Ellis, K.J., Caslenova, R.C., Asad, S.N., and Letter!, J. M.: Correlation of radial bone mineral content with total body calcium in chronic renal failure. J. Lab. and Clin. Med. 86: 910-919 (1975).

4.

Letter!, J.M., Kleinman, L.M., Ellis, K.J., Asad, S.N., Caselnova, R.C. and Cohn, S.H.: The effects of 25 hydroxycholecalciferol on calcium metabolism in chronic renal failure. Proceedings of the Second International Conference on Phosphate Metabolism, edited by S. Massry and E. Ritz, Pittman and Company (in press) 1977.

THE EFFECT OF HIGH DOSES 5,6-TRANS-2 5-HYDROXYCHOLECALCIFEROL ON CALCIUM METABOLISM IN RELATIVE VITAMIN D RESISTANCY (HYPOPARATHYROIDISM AND CHRONIC RENAL FAILURE). D.Kraft and G.Offermann Medizinische Klinik, Klinikum Steglitz, Free University of Berlin, Germany Impaired renal production of 1,25-dihydroxycholecalciferol causes a relative vitamin D resistancy in patients with chronic renal failure (CRF) as well as in patients with hypoparathyroidism (hypoPTH). 5,6-trans-25-hydroxycholecalciferol (5,6-trans-250HCC), which has a similar steric configuration as 1,25-dihydroxycholecalciferol, is known to stimulate intestinal calcium transport and to improve hypocalcemia in CRF, while, until now, it has not been used in patients with hypoPTH. In CRF, however, the question is still open, what dosages of 5,6-trans-250HCC are needed to treat renal osteodystrophy in man, because longterm application up to 6,000 units per day could not achieve complete normalization of intestinal calcium absorption and of serum calcium levels. Furthermore, there was disagreement, whether 5,6-trans-250HCC would favorably influence secondary hyperparathyroidism in CRF or, in contrary, even stimulate parathyroid hormone secretion. 7 patients with CRF, all of whom had been treated before with 6,000 U/day of 5,6-trans-250HCC for at least 6 months without sufficient effect on intestinal calcium absorption and serum calcium level, and 8 patients with postsurgical hypoPTH, 4 of whom being pretreated with 6,000 U/d of 5,6trans-250HCC were included in this study. 18,000 U/d of 5,6-trans-250HCC were orally applied to all patients over a period of 2 weeks (A), and then continued at a reduced dosage of 6,000 U/d for further 6 weeks (B). No side effects, besides hypercalcemia in one patients with CRF, were noted. Serum calcium concentration was measured by atomabsorption spectrophotometry. The intestinal calcium absorption was estimated 90 minutes after oral application of a single dose of 10 ,uCi 47-calcium with a specific activity of 200 ,uCi/mg calcium in the fasting patient as percentage of the applied dose per liter serum. Immunoreactive parathyroid hormone (iPTH) was measured by radioimmunoassay as described by ARNAUD et al., using Prof. Arnaud's CH 12 M as antiserum.

680 5fiTrBns-25-OH CC 6000—|l8000|

6000LW-

mmol/l 3.5 "1 5.6-Tr»n9-25 OHCC

Ca~i.S.

|WX>0|—6000 Uid—|

3.5 m mol/l 3,0 2,5-

Ca"i.S.

2,0

1.51,0

% / l 6.0 5,047Ca

Abs.

4.03,0 2.0 1.0

6

8 weeks

• no pretreatment, besides oral calcium • pretreatment 6000 U 5,6-Trans-25 OH CC/day HYPOPARATHYROIDISM 0

2

4

6

8 weeks

CHRONIC RENAL FAILURE

Fig. 1 Fig. 2 Fig. 1 shows the results in patients with CRF: at A there was a significant mean serum calcium increase from 1.95 (+.22) to 2.46 (+.50) mmol/1 (pef»al hydtoxyiafcich (3)2-Patient l's pseudohypoparathyroidism type II(normal blood and urinary phosphorus .reduced blood calcium,normal urinary calcium,elevated iPTH) was shown to be reversed by calcium infusion.Thus ionized calcium is apparently necessary for the PTH to act on the renal tubule. 3-Patient 2's non-homogeneous bone condensation appeared when the iPTH was increased and the 25 OH-D was reduced.Although the radiocompetitive assay might also measure another metabolite,the l,25(OH)2~D is probably almost non-existant in the circulating blood since there is an histological osteomalacia.There is no evidence of hypersecretion of calcitonine to explain this bone condensation. REFERENCES 1-FRASER D.,K00H S.W..KIND H.P..H0LICK M.F.,TANAKA Y.,DeLUCA H.F.-Patho genesis of hereditary vitamin D-dependant rickets.an inborn error of vitamin D metabolism involving defective conversion of 25 hydroxy-vitamin D to 1 ,25 dihydroxy-vitamin D...New Engl.J.Med.,289,817 (1973) 2-GL0RIEUX F.H.,SCRIVER C.R.,H0LICK M.F.,DeLUCA H.F.-X-linked hypophosphatemic rickets : inadequate therapeutic responses to 1,25 dihydroxycholecalciferol...Lancet 2,2g7 (1973) 3-K00H S.H.,FRASER D.,DeLUCA H.F.,H0LICK M.F.,BELSEY R.E.,CLARK M.B., MURRAY T.M.-Treatment of hypoparathyroidism and pseudohypoparathyroidism with metabolite of vitamin D : evidence for impaired conversion of 25 hydroxy-vitamin D to 1,25 dihydroxyvitamin D...New Engl.J.Med293,840(1975)

753

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Proc. Nat. Acad. Sei. USA. 71, 2996-3ooo (1974). 3. Wills, M.R., Zisman, E., Vfortsman, J., Evens, R.G., Pak, C.Y.C., Bartter, F.C.: The measurement of intestinal calcium absorption by external radioisotope counting: application to study of nephrolithiasis. Clin. Sei. 39, 95-lo6 (197o). 4. Kimberg, D.V. Baerg, R.D. Gershon, E., Graudusius, R.T.: Effect of cortisone treatment on the active transport of calcium by the small intestine. J. clin. Invest. 5o, 13o9-1321 (1971). 5. Favus, M.J. Rimberg, D.V., Millar, G.N., Gershon, E.: Effects of corti-

791

6. 7. 8.

9.

sone administration on the metabolism and localization of 25-hydroxycholecalciferol in the rat. J. clin. Invest. 52, 1328-1335 (1973). Lukert, B.P., Stanbury, S.W., Mawer, E.B.: Vitamin D and intestinal transport of calcium: effects of prednisolone. Endocrinology 93, 718722 (1973). Favus, M.J., Walling, M.W., Rimberg, D.V.: Effects of 1,25-dihydroxycholecalciferol on intestinal calcium transport in cortisone-treated rats. J. clin. Invest. 52, 168o-1685 (1973). S0rensen, O.H., Lund, B., Friis, T., Hjorth, L., Reimann, I., Kjasr, I., Andersen, B.: The effect of la-hydroxycholecalciferol in senile osteoporosis and in bone loss following prednisone treatment. Israel med. J. (in press). Hahn, B., Hahn, T.: Reduction of steroid osteopenia by treatment with 25 OH vitamin D and calcium. Arthritis Rheum. 19, 8oo (1976).

BIOLOGICAL EFFECTS OF 24,25-DIHYDROXYCHOLECALCIFEROL IN MAN

J.A. Kanis, G. Heynen, R.G.G. Russell, R. Smith, R.J. Walton, G.T. Warner. Metabolic Unit, Nuffield Orthopaedic Centre, Oxford, U.K. 24,25-dihydroxycholecalciferol

(24,25-DHCC) i s a major renal metabolite of

vitamin D under conditions of vitamin D sufficiency.

In man the concentra-

tion of 24,25-DHCC in plasma i s in the region of 1-4 ng/ml\ which i s considerably higher than that of lo, 85-95

E l l i s , K . J . , Cohn, S.H.: and m u s c l e m a s s in man.

(1976).

The c o r r e l a t i o n b e t w e e n skeletal c a l c i u m m a s s J . Appi. Physiol. ^ 8 , 455-460,

POSTGASTRECTOMIZED Fig. 1.

total-body

(1975).

PATIENTS

L a b o r a t o r y d a t a of p o s t g a s t r e c t o m y

patients.

TUMOR INDUCED la,-25 DIHYDROXYCHOLECALCIFEROL (la,25-(OH)2D3) DEFICIENCY: A CAUSE OF ONCOGENIC OSTEOMALACIA

M. K. Drezner and M. N. Feinglos Veterans Administration Hospital and Duke University Medical Center Durham, North Carolina 27710, USA

Remission of severe unexplained osteomalacia after resection of an osseous or soft tissue tumor has been reported in thirteen patients. Tumors of bone were present in 46% of the reported cases and a variety of mesenchymal tumors in the remainder. In the present study we investigated the mechanism by which a giant cell tumor of bone caused biopsy-proven osteomalacia in a 42 year old woman. The osteomalacia syndrome was characterized by: 1) hypophosphatemia (1.48 + 0.70 mg/dl; normal 2.5-4.5); 2) decreased renal tubular maximum for the reabsorption of phosphate (TmP/GFR = 0 . 8 + 0 . 0 3 mg/dl; normal 2.55-4.5); 3) hyperaminoaciduria; and 4) decreased gastrointestinal absorption of calcium and phosphorus. Malabsorption, hypophosphatasia and acidosis were eliminated as possible causes of the osteomalacia by demonstration of normal fecal fat excretion (2.5 gm/day; normal 7 patients) the mean plasma 25-OH vitamin D level was 7ng/ml; range 4-11ng/ml. In Groups 2 and 3 many of the 25-OII vitamin D levels were low, some at a level found in nutritional rickets. Using the Wilcoxon Sum of Ranks test, Group 1 compared with Group 2, p < 0 . 0 1 ; Group 1 compared with Group 3, p^.0.01. Mean plasma calcium, Group 1, 9.2mg/ml; Group 2, 9-5fne/rr,l; Group 3> 7.4mg/100ml. These findings show that: 1) Osteomalacia only occurred among those patients with lower plasma levels of 25-OH vitamin D

the

2) In those patients with renal failure whose plasma level of 25-OH vitamin D is normal, histological bone mineralisation can be normal, even though there is presumably little or no circulating 1,25-(OH)_ vitamin D„.

This work has now been published.

Lancet

1976,2,

1209-1211

HISTOMORPHOMETRIC ANALYSIS OF TRABECULAR BONE IN RENAL DIALYSIS PATIENTS TREATED WITH 25-HYDROXYVITAMIN D 3 :

PRELIMINARY

REPORT H.M. Frost, M.D.,* W.S.S. Jee, Ph.D,** D. Kimmel, Ph.D.,*** S. Teitelbaum, M.D.**** *Southern Colorado Clinic, Pueblo, CO, Bone Group Sigma **Univ. of Utah School of Medicine, Salt Lake City, UT, Bone Group Sigma ***Univ. of Utah School of Medicine, Salt Lake City, UT ****Assoc. Prof. Pathology, Jewish Hospital, St. Louis, MQ INTRODUCTION Increasing availability of renal dialysis facilities in the last 15 years has escalated interest in the skeletal problems associated with long-term maintenance dialysis.

Many of the most severe and common non-skeletal

problems encountered in the early development of these programs proved manageable by controlling various factors in the diet and in the dialysate fluid.

However, a number of dialysis patients continue to manifest some

clinical, laboratory and x-ray evidence of disordered bone physiology and virtually all have skeletal abnormalities as assessed by histomorphometric analysis.

Oral treatment with vitamin D 2 and D 3 preparations has appar-

ently benefited relatively few dialyzed patients.1"3 Preliminary clinical studies4"8 with 25-OHD3 in the treatment of renal osteodystrophy provided sufficiently encouraging results to indicate the desireability of carrying out a more definitive and larger scale study which included rigorous evaluation of the changes induced in bone. The major participants in the design and execution of this study are listed in Table I. The study was conducted as a single blind evaluation of 25hydroxycholecalciferol monohydrate in the treatment of hemodialysis patients in the dialysis centers indicated in Table I.

Patients selected

for the study were required to present with x-ray evidence of renal osteodystrophy.

In general, the patients selected were the most severely ill

in each center with respect to osseous abnormalities.

However, in some

centers most patients were essentially asymptomatic while in others bone disease was so advanced as to render them seriously incapacitated.

Thus

the patient population studied represented a wide spectrum of severity.

886 A total of 57 patients entered the study after giving informed consent. Each patient served as his own control.

A base-line period of 12 weeks

during which placebo was given was followed by treatment periods, the first of which was 17 weeks in duration.

During week 13 the treatment

period was initiated with three 600-microgram oral doses of 25-0HD3-H20 administered on alternate days.

Thereafter the dose was reduced to 200

micrograms administered orally 3 times per week. At the investigators' discretion the dose was adjusted in accordance with changes in serum calcium and phophorus values.

Evaluation biochemical measurements of serum

iPTH, 25-OHD, Ca, P, Mg, and creatinine were performed centrally at the Mayo Clinic, diet and dialysate composition were carefully controlled and phosphate binders, when required, were administered to minimize hyperphosphatemia.

Transilial bone biopsies were taken at the end of the control

period (week 12) and after 17 weeks of therapy (week 29). All the bone biopsies were performed by Dr. Teitelbaum using the trephine technique of Bordier.9

Forty-two patients completed the study and 35 patients provided

sequential evaluable biopsies.

Their mean duration on dialysis was 2.5

years and their ages ranged from 26-64.

One objective of this communica-

tion is to summarize some aspects of the histomorphometric findings after the first 17 weeks of treatment.

This study yielded many other signifi-

cant observations and measurements.

An additional preliminary report on

this study was provided by Recker et al10 in this volume. Table I Major Participants in 6-Center Study Dialysis Centers: A.S. Brickman, D.L. Hartenbower, J.W. Coburn, UCLA School of Medicine, V..A. Wadsworth Hospital Center, Los Angeles, CA E. Slatopolsky, K. Martin, Washington Univ. School of Medicine, Barnes Hospital, St. Louis, MO R.R. Recker, R. P. Heaney, Creighton Univ. School of Medicine, Omaha, NE J.M. Letteri, L. Kleinman, State Univ. of New York at Stony Brook, Nassau County Medical Center, East Meadow, L.I., NY R.S. Goldsmith, Univ. of Texas Health Science Center, Audie Murphy V.A. Hospital, San Antonio, TX P. Schoenfeld, Univ. of California Renal Center, San Francisco General Hospital, San Francisco, CA

887 Table I (cont.) Histomorphometry: H.M. Frost, Southern Colorado Clinic, Pueblo, CO W.S.S. Jee, D. Kimmel, U. of Utah School of Medicine, Salt Lake City, UT S.L. Teitelbaum, The Jewish Hospital of St. Louis, St. Louis, MO Clinical Biochemistry: C.D. Arnaud, S. Arnaud, Mayo Clinic, Rochester, MN Water and Skin Analyses: R.R. Recker, Creighton Univ. School of Medicine, Omaha, NE

MATERIALS AND METHODS Tetracycline labelling - In order to properly assess the status of the bone after the stabilization (control) period but prior to 25-OHD3 therapy, and again after 17 weeks of therapy, the tetracycline double labelling technique11 was employed.

Three doses of a tetracycline antibiotic (to-

talling 450 to 750 mg per day) were administered for 3 days. After an interval of 14 days, the same tetracycline dosage was repeated for 3 days, and 4 to 10 days later the transilial biopsy was performed under local anesthesia. Preparation of bone biopsies.- The cylindrical biopsy cores were placed immediately in modified Millonig's fixative. After 24 hours the cores were dehydrated, embedded in methyl methacrylate, and sectioned using a Jung microtome.12»13

Sections to be stained were approximately 5 microns

in thickness; those to be used for histodynamic measurements of tetracycline labels were cut to 10 microns thickness. cortices and the intervening trabeculae. sections were quantitated from each core.

Each section included both

Two sets of widely separated Each histology laboratory mea-

sured adjacent sections from each site. All measurements for static parameters resulted from counts of the entire trabecular surface of each section. Measurement of entire sections was not necessary for dynamic parameters.

The preliminary results in this report are based on data from

one set of sections only.

The data for the complete report will be forth-

coming when the measurements for the other sections are available. Measurements of the biopsy sections.- Trained personnel from the laboratories of Drs. Jee and Teitelbaum were tested to determine uniformity of histologic recognition and measurement.

Sections were apportioned for

888 measurement to each laboratory, identified only by code numbers.

Some

sections were remeasured (again blind) to determine reproducibility of measurement.

The code was not broken until after all measurements had

been completed by both groups. 11 14

followed those of Frost, »

The techniques of measurement in general

Meunier15 and Merz and Schenk.16

The con-

ceptual basis for the analysis and mathematical reduction of the data were devised specifically for analysis of trabecular bone tissue turnover parameters.14

These included correction of marker-separation measurements,

the section plane orientation problem,17 and the use of a scaling constant.14

Perimeters and areas were measured with Merz grids, and basic

stereological principles19 were used to obtain measurements made on thin sections.11'19»22»23

The groups doing measurements used identical optics,

magnifications, grids and procedures, Histomorphometric Parameters - These pertain solely to the lamellar trabecular bone in the biopsy cores. a) Osteoid fractional volume (v„).- The decimal part of trabecular bone plus bone marrow space constituting unmineralized osteoid as measured by a point count grid technique11 and expressed as a decimal fraction. b) Bone volume (Vv).- The total bone mass of trabecular bone in a cubic millimeter of whole trabecular space.

This was measured using the point

count method and is expressed as decimal fraction. c) The appositional rate (M).- The mean thickness of new bone lying between the two markers, divided by the interval between them expressed in years and corrected for the section plane orientation problem.

This para-

meter was measured with a micrometer eyepiece and expressed in millimeters per year.11>14 d) Fractional labelled surface (S).- The decimal fraction of the whole trabecular perimeter which accepted double tetracycline labels as measured using the parallel-line grid intersect technique.11 e) The bone formation rate (sVf).- This parameter was computed by multiplying the values of c and d and expressed in surface referent as cubic millimeters of new bone produced per typical square millimeter of trabecular surface per year.11 f) The volume to surface ratio (V/S).- The volume (mm3) of bone lying beneath a representative area (mm2) of trabecular bone surface as measured using grid techniques.11»14

This parameter can provide an assessment of

889 the bone balance in order to calculate the resorption rate according to previously described strategems.11>18 c g) Active resorption surface ( r).- The decimal fraction of the whole trabecular surface directly beneath and in contact with osteoclasts. This parameter, measured with a parallel-line grid intersect technique,11'14»16 constitutes the only fraction of the trabecular surface on which resorption occurred or could have been occurring at the time of biopsy. h) Resorption rate on active resorption surface (sVr).- This parameter is computed14 from values of e, f and g and expressed as mm3/mm2/year.

It re-

presents cubic millimeters of bone resorbed per square millimeter of active resorption surface per year. This is a derived parameter based upon three others, each with a wide range of variation. Precision and accuracy in the study.- Inter-observer reproducibility was determined by having the technicians involved measure the same 5 parameters in each of 5 coded sections. The parameters included V/S, V D , S, M and S^. The mean of all measurements made of each parameter for each section by the 3 technicians generally varied by no more than 5%. Variation within a bone sample provided most of the "noise" in the data, as anticipated. RESULTS Table II shows the mean of the paired differences (A) for each patient for the various parameters. A paired T-test was used to determine statistically significant differences and the associated significance levels are also given (P). To help in interpreting these differences the pre-treatment and post-treatment group means are also listed for each parameter.

Parameter

Table II Changes In Histomorphometric Parameters Pre Rx Post Rx A

P

Vo

.0394

.0161

-.023

.004*

V V M

.2344

. 1665

-.068

.0001*

.325mm/yr

.335mm/yr

S

.087

.009

.647

.068

-.019

.033*

%

.030mm /mm /yr

.025mm"Vmm^/yr

-.006

.09

V/S

.049

.044

-.005

.026*

sr

.028

.021

-.007

.012*

s

vr

3

2

2

3.28mm3/mm /yr

o o 3.47mm /mm /yr

.184

.866

890 '^Statistically significant change

Initially 90% of the patients had supernormal amounts of osteoid.

In the

post-treatment biopsies, V Q , the osteoid fractional volume declined a significant 59%.

Pre-treatment bone volume, V , with a mean value of 0.2344 '

v'

was high compared to age and sex-matched normal values. 24 > 25 > 26 weeks of treatment the mean V

v

After 17

declined toward normal to a value of 0.1665.

The mean appositional rate, M, was normal both before and after treatment with no significant change.

The fraction of trabecular perimeter lying

over double tetracycline markers (S) averaged .087 before treatment, .068 after and the change was significant. s

rate ( V f ) varied widely.

The surface-based bone formation

The mean value pre-treatment was .030 mm 3 of

bone made/mm2 trabecular surface/year and .025 mm3/mm2/year post-treatment. This difference was not statistically significant.

v/S, the volume to

surface ratio, decreased significantly from .049 to .044 mm.3

The decimal

fraction of whole trabecular surface lying beneath and in contact with osteoclasts, Howship's lacunae, (Sr) was .028 before and .021 after treatment.

The bone resorbed per unit of active resorption surface ( s V r ) rose

from 3.28 mm 3 of bone resorbed/mm2 of active resorption surface/year pretreatment to 3.47 mm3/mm2/year after treatment.

Because of the wide varia-

tion in individual values used to calculate this parameter, the change, using a paired T-test, was not significant. DISCUSSION The primary objectives of this portion of the study were to: 1) Characterize the histomorphometric features of hemodialysis renal osteodystrophy in the U.S.A.,

2) Evaluate the usefulness of 25-hydroxycholecalciferol in

the treatment of this disorder, and

3) Devise quantitative histomorpho-

metric methods of general utility in evaluating changes in trabecular bone. This preliminary report, based on only a fraction of the data collected, is an interim evaluation useful for determining whether or not the patients benefited sufficiently to justify continuation of the treatment. Pre-treatment Findings.- The parameters reported here along with those considered by Recker et al10 show that at the end of the 12-week pretreatment control period these patients demonstrated the following osseous abnormalities.

As others have reported,6,7,20-22

in nearly all of our patients.

we

f ounc j osteoid excesses

This abnormality was independent of geo-

891 graphical location and occurred even in patients without clinical evidence Bone volume,v , was generally high. 2 5 » 2 6

of underlying bone disease.

This

was somewhat unexpected and in contrast with the popular view that hemodialysis patients' bones are being "dialyzed away."

However, this finding

2t

is consistent with a report by Malluche et al ° that bone volume tends to increase with declining GFR.

Quite predictably, fibrous tissue was pre-

sent in three-quarters of the pre-treatment biopsies. 10

Values for v/s,

S, and S r for comparably aged normals are not known with precision, but it is believed that pre-treatment values for these parameters were higher than normal. 1 1 ' 1 2

Normal appositional rate (M) values for human trabecular bone

have been reported 23 to be in the range of 0.25-0.32mm/year. On this basis, the pre-treatment M for these patients appear to be in the normal range. In other words, when the osteoblasts in these patients made bone, they did so at normal rates.

Other information to be presented elsewhere, reveals,

however, that the osteoblasts spent more than 2/3 of their existence inactive. Post treatment Changes.- The surfeit of osteoid declined significantly with 25-OHD3 treatment.

Since this occurred without any measurable change

in appositional rate (M), this beneficial effect upon osteoid mineralization may not depend solely upon changing the amounts of new osteoid deposited by osteoblasts. edly and significantly. fibrous tissue.

10

The increased bone volume (Vv) was reduced markSimilarly there was a significant reduction in

The significant decline in volume to surface ratio (v/S)

signified a net loss of trabecular bone.

This suggests the slight increase

observed in resorption per unit active resorption surface ( s V r ) may be meaningful even though the latter change was not statistically significant. The most logical explanation of how bone vol. (Vv) decreased while linear extent of resorption (Sr) also decreased, would be that the Howship's lacunae were eroded deeper following the 17 week treatment.

Perhaps when

the analysis of the entire data has been completed, better numerical verification of this logic will be provided.

It is also consistent with the

preliminary assessment of the sigma parameter 11 in these biopsies which indicated that the 17-week post-treatment biopsy occurred during the resorptive phase of a transient period rather than during therapeutic steady state (sigma approximated 3.4 years for the group). 11

perties of the bone remodeling unit (i.e., BMU) '

23

The known pro-

plus the findings of

892 this study suggest that the osteoid surfeit should continue to decrease with further treatment and the net loss of trabecular bone should prove non-progressive.

Whole body neutron activation analysis27 of the body

calcium after 12 months of treatment in some of these patients strongly supports the later inference. Summary and Conclusions 1. Histomorphometric analysis of trabecular bone from transilial biopsies of 35 hemodialysis patients in the USA revealed that their osseous abnormality was characterized by an excess of osteoid, increased bone volume, trabecular fibrosis, normal appositional rates, and elevated bone formation rate and surface to volume ratio.

Sigma, the time required for an

osteon to complete resorption and formation, was abnormally prolonged. 2. After 17 weeks of treatment with 25-OHD3 (cumulative dose approximately 10 mg) the osteoid was reduced significantly, bone volume declined, fibrous tissue was dramatically reduced, the appositional rate remained normal, the volume to surface ratio decreased significantly (trabeculae were ca. 101 thinner) and there was no detectable change in sigma. We conclude that significant improvement occurred in the osseous abnormalities observed in these patients.

There is potential for further improvement

and continuation of treatment is justified.

The findings suggest that

after 17 weeks of treatment the biopsy occurred during a transient period and that analysis of a second post-treatment biopsy after 85-90 weeks of treatment will be important to answer questions which the study has presented. 3. An effort has been made to define the static and dynamic nature of human trabecular bone and to devise reliable methods for evaluating changes induced by an experimental treatment. Acknowledgements We wish to express our sincere thanks to Dr. Jack W. Hinman and Mr. Ron P. McCandlis of the Upjohn Company, who planned and coordinated the efforts of all collaborators and literally made the thing work; and to Mr. Steve Francom of the Upjohn Biostatistics Unit, who reduced the primary data into interpretable and statistically evaluated results.

893 References 1. Lumb, G.A., Mawer, E.B., Stanbury, S.W.: The apparent vitamin D resistance of chronic renal failure. Am. J. Med. J50, 421-441 (1971). 2. Avioli, L.V., Birge, S.J., Slatopolsky, E.: The nature of the vitamin D resistance of patients with chronic renal disease. Arch. Intern. Med. 124, 451-454 (1969). 3. Editorial: Vitamin D3 metabolism in renal osteodystrophy. J.A.M.A. 210, 1094 (1969). 4. DeLuca, H.F., Avioli, L.V.: Treatment of renal osteodystrophy with 25-hydroxycholecalciferol, Arch. Intern. Med. 126, 896-899 (1970). 5. Slatopolsky, E., Hruska, K., Rutherford, W.E.: Current concepts of parathyroid hormone and vitamin D metabolism: Perturbations in chronic renal disease. Kidney Int. 1_ (Suppl. 2), 590-596 (1975). 6. Bordier, P., Marie, J.P., Arnaud, C.D.: Evolution of renal osteodystrophy: Correlation of bone histomorphometry and serum mineral and immunoreactive parathyroid hormone values before and after treatment with calcium carbonate or 25-hydroxycholecalciferol. Kidney Int. (Suppl. 2), 5102-5112 (1975). 7. Teitelbaum, S.L., Bone, M.J., Stein, P.M., Gilden, J.J., Bates, M., Boisseau, V.C., Avioli, L.V.: Calcifediol in chronic renal insufficiency: skeletal response. J.A.M.A. 2J35, 164-167 (1976). 8. Witmer, G., Margolis, A., Fontaine, 0., Fritsch, J., Lenois, G., Broyer, M., Balsan, S.: Effects of 25-hydroxycholecalciferol in bone lesions of children with terminal renal failure. Kidney Int. 1(3, 395-408 (1976). 9. Bordier, P.J., Tun Chot, S.: Quantitative histology of metabolic bone disease. Clinics Endocrinol, and Metab. j^, 197-215 (1972). 10. Recker, R.R., Schoenfeld, P., Letteri, J., Slatopolsky, E., Martin, K., Kleinman, L., Hartenbower, D., Brickman, A., Goldsmith, R., Frost, H., Jee, W., Teitelbaum, S., Kimmel, D., Arnaud, C., Arnaud, S., Heaney, R.: 25-Hydroxyvitamin D in renal osteodystrophy: Results of a six-center study. Preliminary report. Presented at the Third Workshop on Vitamin D. Asilomar, January, 1977. 11. Frost, H.M.: Tetracycline based analysis of bone dynamics. Calc. Tiss. Res. 3, 211-237 (1969). 12. Arnold, J.S., Jee, W.S.S.: Embedding and sectioning undecalcified bone and its application to radioautography. Stain Technology 29_, 225-239 (1954). 13. Schenk, R.K.: Zur histologischen Verarbeitung von unentkalkten knochen. Acta Anatomica 60, 3-19 (1965).

894 14. Frost, H.M.: Bone histomorphometry: Analysis of trabecular bone dynamics. In Proc. Second Workshop on Bone Morphometry, ed. P. Meunier, Univ. Claude Bernard, Lyon, France, 1977. in press. 15. Meunier, P., Vignon, A., Vauzelle, J.L.: Methodes histogiques quantitatives en pathologic osseuse. Rev. Lyon Med. _18, 133-142 (1969). 16. Merz, W.A., Schenk, R.K.: A quantitative histological study on bone formation in human cancellous bone. Acta Anatomica lb, 1-11 (1970). 17. Frost, H.M.: Bone histomorphometry: Theoretical correction of appositional rate measurements in trabecular bone. In Proc. Second Workshop on Bone Morphometry, ed. P. Meunier, Univ. Claude Bernard, Lyon, France, 1977. in press. 18. Frost, H.M., Villanueva, A.P., Jaworski, Z.F.G., Meunier, P., Shimizu, A.G.: Evaluation of cellular-level haversian bone resorption in human hyperparathyroid states. Henry Ford Hosp. Med. J. 1J_, 259-266 (1969). 19. Schenk, R.: Basic Stereological Principles. In Proc. First Workshop on Bone Morphometry, ed. Z.F.G. Jaworski, Univ. Ottawa Press, pp. 1-395, 1976. 20. Garner, A., Ball, J.: Quantitative observations on mineralized and unmineralized bone in chronic renal azotaemia and intestinal malabsorption syndrome. J. Pathol. 91, 545-553 (1966). 21. Hill, 0., Jaworski, Z.F.G., Shimizu, A.G., Frost, H.M.: Tissue-level bone formation rates in chronic renal failure measured by means of tetracycline bone labelling. Canad. J. Physiol, and Pharm. 48, 824-828 (1970). 22. Meunier, P., Edouard, C., Bressot, C., Valat, J.W., Courpron, P., Zech, P.: Histomorphometrie osseuse dans l'insuffisance renale aiguë et chronique. J. Urol, and Nephrol. 12, 931-940 (1975). 23. Bressot, C., Courpron, P., Edouard, C., Meunier, P.: Histomorphometrie des Osteophathies Endrocriniennes, Univ. Claude Bernard, Lyon, France pp. 1-260, 1976. 24. Schenk, R.: Trabecular bone volume in iliac crest biopsies. In Proc. First Workshop on Bone Morphometry, ed. Z.F.G. Jaworski, Univ. Ottawa Press, pp. 97-99, 1976. 25. Meunier, P., Courpron, P.: Iliac trabecular bone volume in 236 controls - representativeness of iliac samples. In Proc. First Workshop on Bone Morphometry, ed. Z.F.G. Jaworski, Univ. Ottawa Press, pp. 100^105, 1976. 26. Malluche, H.H., Ritz, E., Lange, H.P., Kut-chera, J., Hodgson, M., Seiffert, U., Schoeppe, W. : Bone histology in incipient and advanced renal failure, Kidney Int. 9, 355-362 (1976).

895 27. Kleinman, L., Letteri, J.M., Ellis, K.N., Caselnova, R., Akhtar, M., Cohn, S.M.: Effects of 25-hydroxycholecalciferol (25-OHD) on calcium metabolism in uremia. Abstracts, Third Workshop on Vitamin D. Asilomar, CA p. 151, 1977.

Vitamin D Metabolites and Bone Mineralization In Man

Philippe Bordier, Antoine Ryckwaert, Pierre Marie, L i v i a Miravet, Anthony Norman, and Howard Rasmussen Unite de Recherche Andre Lechwitz, Hopital Lariboisiere, Paris, France, Department of Biochemistry, University of California, Riverside; and Department of Internal Medicine, Yale University, New Haven, Connecticut In the short time since i t s discovery (1-4), la-25-hydroxyvitamin D^ 1,25(0H)2D3, has become recognized as the major, i f not sole, physiol o g i c a l l y active metabolite of vitamin D involved in the regulation of intestinal calcium transport (1,2,5-7).

Furthermore the demonstration

of i t s very marked potency as a stimulator of bone resorption both in vivo (5-7,9) and in vitro (8) has led to the conclusion that i t i s probably the most important vitamin D metabolite in the control of bone mineral metabolism as well (5-7,9,10).

Strong support for this conclusion

comes from the observations of Tanaka and DeLuca showing that this metabolite i s an effective a n t i r a c h i t i c agent in the D-deficient rat (11). However, there are other data obtained from studies in this same species that suggest a difference in the effects of 25(0H)D3 and 1,25(0H)2D3 upon bone metabolism (10).

The question most relevant to the present study i s

that of whether 1,25(0H)2D3 and 25(0H)D3 are equally effective in stimulating the rate of bone mineralization in vitamin D-deficient adult man. Patient Selection The diagnosis of osteomalacia was made on c l i n i c a l , radiographics, and biochemical c r i t e r i a .

All of the patients complained of muscle weakness

particularly marked in the proximal part of the lower extremity that was associated with a waddling gait.

Bone pain was present in 75% of patients,

but s i g n i f i c a n t skeletal deformity was rare. of hypocalcemia or tetany. when examined by x-ray.

None had signs or symptoms

Over 85% had one or more pseudofractures

The characteristic biochemical findings were

898 a slightly low serum calcium (circa 8.5), a low serum phosphate, a moderately elevated serum alkaline phosphatase, a low or undetectable serum 25(0H)D3 concentration, a high normal or slightly high serum immunoreactive parathyroid hormone (IPTH) except four patients with values over 2.5 times normal, a normal or moderately low rate of urinary phosphate excretion, a slightly elevated rate of urinary calcium excretion, a slightly elevated rate of excretion of urinary hydroxyproline-containing peptides, and a normal creatinine clearance. Bone Histology The clinical diagnosis was confirmed in all cases by quantitative histological evaluation of undecalcified, stained sections of bone obtained from a transileal bone biopsy performed under local anesthesia (14). Classically, the histological criterion employed to establish the diagnosis of vitamin D deficiency osteomalacaia is an excess of osteoid, i.e. an increase in osteoid volume.

However, this criterion has proven inadequate.

An increase in osteoid volume is seen in patients with Paget's disease, primary hyperparathyroidism, thyrotoxicosis, and in normal elderly individuals.

In all of these instances even though the osteoid volume is

increased there is no evidence of vitamin D deficiency, and in all the extent of the mineralization front (see below) measured either by staining with Toluidine blue or by labeling with tetracycline is normal.

Converse-

ly, there are patients with mild or subclinical vitamin D deficiency with low serum 25(0H)Dg and inorganic phosphate concentrations in which one does not observe an increase in osteoid volume, but does invariably observe a decrease in the extent of the mineralization front. An increase in the extent of the osteoid surface has also been employed as an histological criterion for osteomalacia.

Whenever osteoid volume is

increased this surface is increased, but there are conditions in which the extent of this surface is increased without an increase in osteoid volume.

This is particularly true in the bones of patients treated with

corticosteroids, and some patients with thyrotoxicosis and primary hyperparathyroidism.

However, in these instances, the extent of the minerali-

zation front is normal.

899 The most specific histological criterion of vitamin D deficiency osteomalacia is a decrease in the extent of the mineralization front (formerly called by us the calcification front (10) measured either as an absolute value or as the percentage of the osteoid surface. A cruicial aspect of making such measurements is that of carrying them out at a precise time after tetracycline administration because the extent of front labeled is a function of the time after methylchlortetracycline administration in both normal individuals and those with vitamin D deficiency osteomalacia.

The rate of mineralization is slower in the D-

deficient patient, the maximal difference between the extent of the mineralization front measured in control and D-deficient subjects is seen at 24 hours after tetracycline administration. In the present study, the method was standardized by administering methylchlortetracycline (15 mg/Kg body wt) to the patients 24 hours before the bone biopsy was obtained. In concluding this discussion of the mineralization front and its usefulness as a criterion of vitamin D deficiency, it is necessary to note that there are some other conditions which may also lead to a decrease in the extent of the mineralization front.

These include hypophosphatasia,

renal tubular acidosis, administration of the diphosphonate, EHDP or fluoride administration, and vitamin D resistant rickets or osteomalacia. It is not year clear, particularly in the latter five types of subjects, whether this is secondary to some disorder of vitamin D metabolism, or whether there are local factors at the bone mineralization site which determine the change in mineralization front. Method of Procedure After diagnosis and preliminary evaluation patients were placed on a standard hospital diet containing approximately 600 mg calcium and 1000 mg of phosphate. (10).

A transileal bone biopsy was obtained under local anesthesia

The patients were then given either 25(0H)D 3 (50-150 ug/d),

l,25(0H)2r)3 or la(0H)D3 (0.5-2.5 ug/d), oral phosphate (1500-2100 mgP/day), or vitamin D3 (50-200 ug/d).

The response to treatment was assessed by

900 clinical and radiological examination, and by biochemical analysis.

The

following parameters were measured: a) serum calcium, phosphate, IPTH, 25(0H)D3

and alkaline phosphatase concentrations; and b) the rates of

excretion of calcium and phosphate.

At appropriate intervals of weeks

or months a repeat bone biopsy was obtained.

All biopsies were processed

and stained in a standard fashion (10) and the various parameters of bone mineralization determined by two independent observers.

The biopsies

were coded so that the observers had no knowledge of whether the biopsy represented a pretreatment one, or if from a treated patient, the form of therapy.

In some patients, successive courses of 1,25(0H) 2 D 3 followed

by 25(0H)D3 were given and a third biopsy obtained after this second period of metabolite administration.

All procedures were carried out

with the informed consent of the patients. Results:

Mineralization Parameters

Prior to treatment all the patients had a significant reduction in the extent of mineralization front, and all but two had an increase in osteoid volume.

These patients were divided randomly into groups who received

either oral phosphate (seven patients), vitamin D3 (ten patients), 25(0H)D3 (17 patients), or either 1,25(0H) 2 D3 or lct(0H)D3 (12 patients).

The re-

sults with 1,25(0H)2D3 and la(0H)D3 were indistinguishable and are considered as one group throughout this presentation. The results of the different forms of treatment on the extent of the mineralization front as shown in Figure 1.

Both vitamin D3 and 25(0H)D3

caused a significant increase (P< 0.01),phosphate therapy had no effect, and 1,25(0H) 2 D3 and la(0H)D3 had a barely significant effect on the mineralization front. The data in Figure 1 are a composite of all the patients treated for varying periods of time, hence it might be argued that the results may be skewed in some fashion.

The data showed that duration of treatment did

not explain the difference between the effects of 1,25(0H)2D3 and those of 25(0H)D3.

Patients treated for 21 or more days with either vitamin

D3 or 25(0H)D 3 had the same response as those treated for longer periods of time.

Also, there was no difference in response in patients given

901

«> 8 0 r-

o

o

•

70

\ •

Ò N 1 1 25 0

o « 60 M

O

»

Y

50

Y

f

•

o oc

40 30

%

• Aa

»< 20 Kb T j ^ A

N

-I < a: 10 UJ

•

-

•

A

b*

AAA A ^ A A A

•

AA

cf • •

CONTROL ORAL P 0 4 VALUES EACH GROUP

VITD3

25(0H)D3

1-25 (0H)2D3

Figure 1. The extent of the tetracycline-labeled mineralization front in adult humans with vitamin D deficiency osteomalacia before (control values) and after treatment with either oral phosphate ( • ) , vitamin D3 ( • ) , 25-hydroxy vitamin D3 ( • ) or either l3 (unpublished data) indicating that the 1,25(0H) 2 D 3 was exerting an effect on bone; third, giving doses of la(OH)Dg as large as 2.5 ug/day for several months did not cause any progressive increase in the degree of mineralization; fourth, these doses of 1,25(0H) 2 D 3 and lct(OH)D3 have been shown to be effective in hypoparathyroidism and pseudohypoparathyroidism; fifth, in relation to their relative concentrations in normal serum, the amounts of the two types of metabolites given in the present study are a reasonable estimate of that needed for 'replacement' therapy;sixth, the minimal effective doses of 25(0H)D3 (37.5-50 ug/d) caused

a

rise in the serum 25(0H)D 3 concentration to approximately normal values indicating that massive over-treatment with this metabolite was not the explanation; seventh, Brickman et al (16) have reported that 1,25(0H) 2 D 3 or la(0H)D 3 in doses employed in this study will not produce healing of the osteomalacia seen in patients with renal disease or chronic hemodialysis even though there was a marked reduction in the degree of hyperparathyroid bone disease (i.e. osteitis fibrosa) and studies by one of us (P.B.) using 2 ug of la(OH)D 3 or 1,25(0H) 2 D 3 every two days in patients with this disease confirm these observations; eight, the observations in the present study showing that 25(0H)D 3 was effective when given to patients who had little response to several months of 1,25(0H) 2 D 3 or la(0H)D 3 treatment indicates that a bias in the selection of the patient population can not explain our results; and ninth, in patients with hypoparathyroidism and pseudohypoparathyroidism even though the serum

907 1,25(0H)D3 concentrations are below normal, the extent of the mineralization front is normal in the bones of these individuals (unpublished data). On the basis of the present evidence we believe it is reasonable to conclude that 25(0H)D 3 or a further metabolite of this compound, other than 1,25(0H) 2 D 3 , plays a role in the mineralization of bone in man.

Be-

cause of the increasing evidence that phosphate plays a key role in bone formation (10) and the previous data showing that oral phosphate or a combination of calcium and phosphate can cure certain types of osteomalacia in man (17), the proposed effect of 25(0H)D 3 or one of its metabolites upon bone mineralization may not be direct, but may act indirectly by influencing the renal retention of calcium and phosphate and thus elevating the product of these ions in the extracellular fluids. The present data tend to support this conclusion.

The administration of

25(0H)D 3 led to a decrease in the excretion of both calcium and phosphate, an immediate (within days) increase in serum phosphate concentration and a later rise in serum calcium concentration.

These data indicate that

25(0H)D 3 probably has a direct renal effect of increasing the tubular reabsorption of calcium and phosphate in man, as suggested by the studies of Puschett et al (18 ).

It does not seem possible to explain the rise in

serum phosphate concentration and the fall in urinary phosphate excretion solely on the basis of a fall in IPTH because, the fall in IPTH after 1,25(0H)D3 treatment was as great as that seen after 25(0H)D 3 treatment but the changes in serum phosphate concentration and urinary phosphate excretion after 1,25(0H)2D3 administration were considerably less.

Also, if

PTH falls and serum calcium concentration does not change or rises as seen after 25(0H)D 3 therapy then one would expect an increase in the urinary excretion of calcium rather than the observed decrease. In spite of the impressive evidence that 25(0H)D 3 increases the renal retention of phosphate and calcium, it does not seem likely that these effects can entirely account for the striking effects of 25(0H)D 3 on bone mineralization seen in the present study.

Both in the present

study and in previous works (10) it has been shown that phosphate administration to hyponhosohatemic patients with osteomalacia does not

908 increase the extent of the mineralization front even though long continued administration of phosphate will definitely decrease osteoid volume.

However, if one examines bone biopsy material from such phosphate-

treated individuals there is a striking difference in the pattern of mineralization (10) from that seen in vitamin D-treated patients. patients treated either with vitamin Dg

In

or 25(0H)Dg the most impressive

feature of the remineralization of the osteoid is its regularity. mineralization front first appears on the osteoid lamella

The

closest to the

old mineralized bone, and then moves progressively toward the external bone surface.

In contrast, the mineralization of the osteoid seen after

phosphate therapy appears in random patches often close to the external bone surface, extents irregularly, and commonly leaves areas of unmineralized osteoid amongst those fairly well or completely mineralized ones. It is quite possible that the mobilization of mineral from old bone by 1,25(0H)2D 3 , the renal retention of calcium and phosphate stimulated by 25(0H)D 3 , and the direct effect of 25(OH)D 3 (or one of its metabolites) upon the osteoid osteocytes involved in bone mineralization are all integrated to produce the observed changes in rate of bone mineralization after treatment of patients with osteomalacia with either vitamin D 3 or 25(0H)D 3 . In addition to their obvious interest in terms of our understanding the physiology of vitamin D and its metabolites in man, the present results are of practical importance as well.

If our conclusions are correct,

then it is quite likely that treatment of patients with the osteomalacia of chronic renal disease with either 1,25(0H) 2 D 3 or 25(0H)D 3 alone may not lead to the desired improvement

in bone mineralization, but this

may be achieved if 1,25(0H)2D 3 and the proper metabolite specific for bone mineralization are given simultaneously.

In this regard, the recent

report of Pierides et al (19) concerning long term treatment of having hemodialysis osteodystrophy with la(OH)D 3 is of interest.

patients They

found that those patients exhibiting predominantly osteitis fibrosa responded favorably to la(OH)D 3 , but that those exhibiting predominantly osteomalacia did not respond well to la(0H)D 3 in terms of healing their bone disease even though they showed a response in terms of an increase

909 in intestinal calcium absorption, and a rise in serum calcium concentration.

Pierides et al attribute the difference in responsiveness to

a lower serum phosphate concentration in the unresponsive group.

It

is also possible that the difference could be due to a difference in availability of a second vitamin D metabolite.

However, it is of

interest that in two of their 'unresponsive' patients cessation of aluminium hydroxide therapy, at a time when they were still receiving la(OH)Dj, led to a rise in serum alkaline phosphatase activity. The possibility that a separate metabolite of vitamin Do, other than 1,25(0H)2D2 regulates the process of bone mineralization and possibly bone formation has implications beyond the area of renal osteodystrophy. The major metabolic bone disease is clearly osteoporosis.

The decrease

in bone mass seen in patients with any form of this disorder results from a long sustained imbalance between bone formation and bone resorption.

It

is possible that an inhibition of the synthesis or action of the proposed bone metabolite is a common pathogenic factor in the etiology of all forms of osteoporosis.

If so, a specific agent which could restore bone re-

modeling balance by enhancing the rate of bone formation would be of great therapeutic value.

910

References 1.

Lawson, D.E.M., Wilson, P.W., and Kodicek, E.: New Vitamin D Metabol i t e Localized in Intestinal Cell Nuclei. Nature 222, 171-172 (1969).

2.

Haussler, M.R., Boyce, D.W., L i t t l e d i k e , E.T. and Rasmussen H.: A Rapidly Acting Metabolite of Vitamin D. Proc. Natl. Acad. Sei. USA 68, 177-181 (1971).

3.

Lawson, D.E.M., Fräser, D.R., Kodicek, E., Morris, H.R. and Williams, D.H.:

Identification of 1,25-dihydroxycholecalciferol, A New

Kidney Hormone Controling Calcium Metabolism. Nature 230, 228-230 (1971). 4.

Holick, M.F., Schmoes, H.K, and DeLuca, H.F.:

Identification of

1,25-dihydroxycholecalciferol - A form of Vitamin D Metabolically Active in the Intestine.

Proc. Natl. Acad. Sei. USA 68, 803-804

(1971). 5.

DeLuca, H.F.: Vitamin D - 1973. Am. J. Med. 57, 1-12 (1974).

6.

Pechet, M.M. and Hesse, R.H.: Metabolic and Clinical Effects of Pure Crystalline la-hydroxyvitamin Dg and la,25-dihydroxyvitamin Dg. Am. J. Med. 57, 13-20 (1974).

7.

Norman, A.W.: 1,25-Dihydroxyvitamin D^: A Kidney-Produced SteroidHormone Essential to Calcium Homeostasis. Am. J. Med. 57^, 21-27 (1974).

8.

Raisz.L.G., Trummel, C.L., Holick, M.F. and DeLuca, H.F.: 1-25-Dihydroxycholecalciferol: A Potent Stimulator of Bone Resorption in Tissue Culture.

9.

Science 175., 768-769 (1972).

Haussler, M.R., Zerwekh, J . E . , Hesse, R.H., Rizzardo, E. and Pechet, M.M.:

Biological Activity of la-hydroxycholecalciferol - A Synthetic

Analog of the Hormonal Form of Vitamin Dg. Proc. Natl. Acad. Sei. USA 70, 2248-2254 (1973). 10. Rasmussen, H.and Bordier, P.: The Physiological and Cellular Basis of Metabolic Bone Disease.

Williams and Wilkens Company, Baltimore

(1974). 11. Tanaka, Y. and DeLuca, H.F.: Role of 1,25-dihydroxyvitamin Dg in Maintaining Serum Phosphorus and Curing Rickets. Sei. USA 71, 1040-1044 (1974).

Proc. Natl. Acad.

911

12.

Rasmussen, H. and DeLuca, H . F . : Calcium Homeostasis. Ergebnisse Des Physiologie.

13.

B i o l . Chem. Exp. Pharmakol. 53^, 108-173 (1963).

Steenbock, H. and H e r t i n g , D.C.: Vitamin D and Growth. J .

Nutr.

57, 449-468 (1966). 14.

A l b r i g h t , F. and R e i f e n s t e i n , E . C . , J r . : The P a r a t h y r o i d Glands and Metabolic Bone Disease.

W i l l i a m s and W i l k i n s Company, B a l t i m o r e ,

1948. 15.

B o r d i e r , P . , Pechet, M.M., Hesse, R. and Rasmussen, H.: Response of A d u l t P a t i e n t s w i t h Osteomalacia to Treatment w i t h C r y s t a l l i n e hydroxyvitamin D3.

16.

la-

New Engl. J . Med. 291, 866-869 (1974).

Brickman, A . S . , Coburn, J.W., Norman, A.W., and Massry, S . G . : Short-Term E f f e c t s o f 1 , 2 5 - d i h y d r o x y c h o l e c a l c i f e r o l on Disordered Calcium Metabolism of Renal F a i l u r e . Am. J . Med. 57, 28-33 (1974).

17.

Popovtzer, M.M., Matthay, R., A l f r e y , A . C . , B l o c k , M., Beck, M i l e s , J . and Reeve, E . B . :

P.,

Vitamin D d e f i c i e n c y osteomalacia -

Healing of the Bone Disease i n the Absence o f Vitamin D w i t h Intravenous Calcium and Phosphorus I n f u s i o n . In C l i n i c a l of Metabolic Bone Disease.

Aspects

E d i t o r s : Frame, B, P a r f i t t , A.M. and

Ducan, H. Excerpta Medica, Amsterdam, 1973 pp. 382-387. 18.

Puschett, J . B . , Moranz, J . and Kurnick, W.S.¡Evidence f o r a D i r e c t Action of C h o l e c a l c i f e r o l and 2 5 - h y d r o x y c h o l e c a l c i f e r o l on the Renal Transport of Phosphate, Sodium, and Calcium. J . C l i n .

Invest. 51_,

373-385 (1972). 19.

P i e r i d e s , A.M., Simpson, W., Ward, M.K., E l l i s , H . A . , Dewarr, J . H . , and Kerr, D.N.S.: ciferol

V a r i a b l e Response to Long Term l a - h y d r o x y c h o l e c a l -

i n Hemodialysis Osteodystrophy. Lancet i : 1092-1095 (1976).

The Effect of la-0H-D3 Treatment on the Structure and Function of Chick Intestine Brush Border Membrane

Howard Rasmussen, Edward E. Max, and David B.P. Goodman Department of Cell Biology and Internal Medicine, Yale University Medical School, New Haven, Connecticut

06510

Since the pioneering work of Nicolaysen and coworkers (1), i t has been repeatedly demonstrated that a major physiological effect of vitamin D is to increase the active transport of calcium in the duodenum and upper jejuncem of

nearly all mammalian and avian species.

In spite of these

demonstrations and numerous studies at the c e l l u l a r and subcellular level, the biochemical and c e l l u l a r basis of this action of vitamin D is not known. One major hypothesis has been that vitamin D increases the rate of synthesis of a specific calcium-binding protein (2).

This change does

occur after vitamin D administration, but i t has not been possible to relate i t directly to the change in transcel1ular transport of calcium ion. The problem i s that even though the physiological studies lead to the conclusion that a major c e l l u l a r s i t e of vitamin D action i s at the level of brush border or microvillar membrane of the intestinal mucosal c e l l s , i t has not been possible to show any s i g n i f i c a n t amount of calcium binding protein associated with this subcellular fraction, or any change in the content of calcium-binding protein in this fraction after vitamin D administration.

On the other hand vitamin D-induced changes in this cell

fraction have been reported and include: a) an increase in a c t i v i t y (3,4) and apparent molecular weight (5) of alkaline phosphatase; b) an increase in the a c t i v i t y of a calcium-activated ATPase (6); c) an increase in the content of a calcium-binding complex (7); and d) an alteration in the l i p i d composition of t h i s fraction (8). A major shortcoming of all of these studies, including our own, i s that even though most were carried out in the D-deficient chick or rat, the

9\k technique for preparation of the brush border fraction of the intestinal mucosal c e l l s was that o r i g i n a l l y developed by Forstner et al (9).

Docu-

mentation of i t s adequacy for the i s o l a t i o n of purified brush border fractions from D-deficient chicks or rats was never made.

As a prelude

to the present study, we undertook such an analysis and found that when the method of Forstner et al (9) was applied to intestinal mucosa from the D-deficient chicks the brush border fraction was inadequate in terms of y i e l d and purity as assessed both by electron microscopy and by the use of enzyme markers for various subcellular fraction. Preparation Of Isolated Brush Borders From The Mucosa Of D-deficient Chicks This result necessitated the development of a more adequate method for the orenaration of the brush border fraction from the intestinal mucosa of the D-deficient chick.

The method f i n a l l y developed i s summarized in

Figure 1. PURIFICATION SCHEME —PURIFIED BRUSH BORDERS FROM CHICK DUODENUM 2 4 - H r - S t o r v e d Rachitic Chicks decapitate, exsanguinate; remove d u o d e n u m and flush with 0 . 9 % N a C I ; s c r a p e

2g

mucosal scrapings

homogenize ( T e f l o n - g l a s s ) in 2 0 0 ml E H Buffer

Supernatant (discord)

Pellet - " p u r i f i e d brush b o r d e r s "

915 Figure 1. Purification scheme for isolation of chick duodenal brush border. Details of procedure are described in the text. In order to obtain reproducible results it was found necessary to starve the animals for at least 24 hours.

A highly purified brush border could

then be prepared from the mucosal scrapping from these animals by 1) initial homogenization in a teflon-glass homogenizer using 2mM HEPES2.5 mM NaEGTA, pH 7.4 (EH buffer) and 1 gm of mucosa for each 100 ml of buffer; 2) this homogenate was subjected to low speed centrifugation (400 g) for 20 minutes; 3) the pellet was resuspended in a 10 ml of MEH buffer (5 m M Mg CI2 - 2.5 mM Ma EGTA, pH 7.4 - 2 mM HEPES) and layered over a discontinuous gradient of sucrose - 10 ml of 50% sucrose - MEH 10 ml of 63% sucrose - MEH, and centrifuged for 90 minutes at 80,000 x g in a swinging bucket rotor; 4) the material at the lower interface (between the 50% and 63% sucrose layers) was collected and resuspended in 34 ml of MEH buffer and collected by centrifugation at 80,000 g for 30 minutes. Both by electron microscopic examination and by analysis of marker enzymes (Figure 2), this fraction (I) was highly purified and represented approximately 50% of the original sucrase activity.

B R U S H BORDER BRUSH

MEMBRANE

BORDER

10 calcifero1

930

Fig. 2 - Bond lengths, bond angles, and torsional angles for 25-0H-D 3 .

Fig. 3 - An [010] projection of the monoclinic unit cell of crystalline C27Hi,i,02-H20 showing the arrangement of the two 25-0H-D 3 and two water molecules per cell under P2-] symmetry. Infinite chains of hydrogen-bonded moleculues are formed into a sheet (or layer) which forms a double layer with an adjacent sheet through hydrogen bonding with water molecules. Hence, all hydrogen atoms of both hydroxyl groups per 25-0H-D 3 molecule and of the one independent water molecule participate in hydrogen bonding in the infinite double layer (parallel to the b£ plane) such that there is no interaction between double layers other than van der Waals contacts.

931

11 C(4)' C(!9)'

CO)'I,

\ 0(3') C(2)' 4 - The D3a and D36 conformers of vitamin D 3 .

5 - Hydrogen bonding scheme showing the a and B conformers of vitamin Dj connected in the crystalline state by their single hydroxyl groups to form an infinite spiral-like hydrogen-bonded oxygen chain. Each helical chain is constrained about a 2] axis in the b direction, with the two conformers occuDying alternating positions. 0 ( 3 ^ and 0 ( 3 1 1 ) are related to 0(3) and 0(3'), respectively, by the screw axis, while 0(3'11) is related to 0(3'•) by a whole lattice translation along the b axis. The unusually large thermal elliposoids for the isopropyl carbon atoms at the end of the side chain of the a conformer relative to those for the other nonhydrogen atoms are attributed at least in part to these atoms possessing more than one crystal orientation.

932 favorable C(6)=C(5)-C(10)=C(19) cis-diene arrangment gives rise to a o

H(7-l)••-H(19-l)

contact distance of 2.50 A which is sufficiently long to imply no significant repulsive interaction.

The large degree of distortion of the exocyclic C(6)=C(5)-C(10)=C(19)

cis-diene fragment from planarity is evidenced by its torsional angle of 56.7° (vs^ 0° for a planar system).

The fact that similar angles of twisting of the corresponding

cis-diene fragment are found in D3a (53.6°), D3B (55.2°), D2a (49°), and D2B (46°) and that this torsional angle is essentially invariant to the two different chair-shaped conformations adopted by ring A (vide infra) indicates that the effect of the nonplanarity of the A ring on the twisting of the above exocyclic cis-diene system is practically the same in both conformations of ring A. Conformation of Ring A.

The geometrical nature of ring A is of particular interest

in that its two chair conformations (readily differentiated from each other by the torsional angles) correspond to the two rapidly equilibrating solution conformations which were deduced 9 ' 9 from recent analyses by 'H nmr spectroscopy of vitamin D 2 of

vitamin D 3 , 1,25-(0H) 2 -D 3 , and other metabolites.'

8

and

Figure 1 shows that the solid-

state conformation of ring A in 25-0H-D 3 corresponds to the a chair form, also found in D3a and in D2a, with the exocyclic CH 2 group situated below the mean cyclohexanelike ring plane and with the 3-OH group occupying the equatorial position. formation was initally discovered benzoate substituent

2

This con-

for the A ring in INC with the bulky 4-iodo-5-nitro-

at C(3) in an equatorial

position.

The other conformation of

ring A (6 chair form) with the CH 2 group situated above the mean ring plane and with the 3-OH group in the axial position was first observed 3 in the crystalline state in ECF (which has a dioxolane ring substituted at C(3)) and later in D38 and in D2B.

The

existence of two adjacent exocyclic double bonds at the C(5) and C(10) atoms leads to a pronounced deformation of ring A from an idealized cyclohexane conformation.

The

relative flatness of ring A in 25-0H-D 3 is apparent from its mean torsional angle of 51.8°, which is significantly less than that of 55.9° found in a gaseous cyclohexane molecule.

Analogous ring flattening from a regular cyclohexane geometry was also

observed to occur in other vitamin D molecules with corresponding mean torsional angles of 53.8° in D3a, 50.1° in D3B, 52° in D2a, and 51° in D2BConformation of the C,D-Ring System.

To a first approximation the irregular chair

conformation of ring C and the trans-fusion of the cyclopentane ring D to ring C at C(13) and C(14) (Figure 1) are similar in vitamin D molecules, and the ring torsional angles are expectedly in reasonable agreement with those in representative steroids. 7 The flexible conformation of ring D is indicated below from a geometrical

description 1 0

of this five-membered ring system in terms of a maximum torsional anole and a max conformational parameter A (denoted as the phase angle of pseudorotation). 25-OH-D3

Ideal:

D3a

D3B

45.7°

49.9°

45.5°

+ 6.7°

+ 0.6°

+19.1°

D2a 45°

D2B 49'

+27c A=0°, regular half chair (C 2 -2 symmetry); A=+36°, regular C(13)-12°

envelope (C s -m symmetry); A=-36°, regular C(14)-envelope (C -m symmetry).

933 Whereas the narrow range of

values in these vitamin D molecules indicates a

similar puckering of ring D in all of these molecules, the wide variation in their pseudorotation parameters A indicates the flexibility of ring D to assume a variety of conformations in the solid state ranging from a near standard C(13)-envelope form in D2B,to a regular half-chair form in D3a, to a near half-chair form in 25-0H-D 3 , to an intermediate conformation between a half chair and a C(14)-envelope in D2a. Conformation of the Side Chain.

The prime structural characteristic of the side

chain of 25-0H-D 3 is the close conformity of its carbon skeleton to a planar zigzag o

conformation, which results in a long distance of 7.48 A between C(17) and C(27). Figure 4 shows that the nonhydroxylated side chain in D3a also possesses a nearly planar zigzag conformation (except for the crystal-disordered isopropyl fragment at the end of the side chain), whereas the corresponding nonhydroxylated side chain in D3B is not in such a regular form.

The net consequence is not only much smaller

C(17)•••C(27) distances of 6.74 A in D3a and 6.15 A in D3B relative to that of 7.48 A in 25-0H-D 3 but also that these C(17)-•*C(27) distances are considerably smaller than o

o

the corresponding C(17)-•-C(26) distances of 7.24 A in D3a and 6.74 A in D3B, respectively.

These differences in side chain conformations for the D3a and D3B molecules

show that nonbonded repulsive interactions can cause a marked change in geometry of the side chian.

Nevertheless, these variable distances still reflect the extended

nature of the side chains in the crystalline state for vitamin D 3 molecules. solid-state conformation of the side chain of each of the vitamin D 2 and D2B, is likewise not in a planar zigzag form.

The

conformers, D2a

However, its particular arrangement,

which is partly influenced by the steric requirements of its double bond between C(22) and C(23) and

a methyl group substituent at C(24), also produces an extendedo conformao

tion as indicated by the C ( 1 7 ) — C ( 2 7 ) separation of 6.42 A in D2a and 6.49 A in D2fi. Relationship Between Structure and Activity of Vitamin D Metabolites and Analogs, (a) Requirements of the Side Chain. The fully extended side-chain feature of 25-0H-D 3 correlates with the markedly reduced physiological activity of the 24-nor-25-0H-D 3 1 1 and the lack of activity of 22-27-hexanor-20-0H-D 3 .

12

It may be that analogs of

25-OH-D 3 with shorter side chains are not transported as effectively on the 25-0H-D 3 binding protein to the kidney for lo-hydroxylation.

Apparently the 25-hydroxyl group

must also be at the spatial position elicited by a fully extended side chain for the conversion into the more polar 1-hydroxylated metabolite.

Any difference in the

physiological activity between a vitamin D 2 metabolite and a corresponding vitamin D 3 metabolite may also be correlated to a fully extended C(17)-C(25) side chain in a D 3 o

molecule being approximately 0.2 A longer than that in a D 2 molecule. o

The C(17)•••C(25)

intramolecular distance of 6.41 A observed in the fully extended side chain of 25-0H-D 3 o

o

is longer by 0.44 A than the corresponding distance of identical value 5.97 A in both crystal 1 i ne D2c and D28 due to their side chains not being fully extended.

The esti-

mated C(17)- • -C(25) distance at full extension of the side chain in 24-nor-25-0H-D 3 (corresponding to the C(17)••-C(24)o distance in 25-0H-D 3 as the side chain contains one less carbon atom) is only 5.0 A, which represents an apparent shortening by ca_.

93k o 1.4 A compared to that in 25-0H-D 3 . (b) Requirements of the Ring Systems.

The fact that the C,D-ring system and the

seco-B ring fragment which bridges rings A and C of 25-0H-D 3 , D3a, D3B. D2a, and D28 display analogous s o l i d - s t a t e conformations (other than the f l e x i b l e nature of ring D) leads to the conclusion that t h i s part of the molecule i s conformationally s i m i l a r in a l l b i o l o g i c a l l y active vitamin D molecules.

Okamura, et^ al_. 13 recently proposed

that the la-hydroxyl group and concomitantly the conformation of ring A play a key role in producing a l l of the biological functions attributed to vitamin D 3 .

They also

suggested that the minimal requirements for vitamin-D b i o l o g i c a l a c t i v i t i e s do not necessitate the presence of either the 36-hydroxyl group or the C(19)H 2 group because active analogs such as 3 - d - l a - O H - D s , 1 1 " 1 5 DHT 3 , 1 6 5 , 6 - t r a n s - D 3 , "

and isotachysterol

3

( i - T 3 ) 1 7 do not possess a hydroxyl substituent at the 36 p o s i t i o n , while the l a t t e r three compounds do not possess a methylene group at the carbon atom which geometrically corresponds to the C(10) position of ring A.

I t should be noted that the three l a t t e r

compounds are at least two orders of magnitude less active than the l a , 2 5 - ( 0 H ) 2 - D 3 , and furthermore, while the 3-d-lc.-OH-D 3 i s found to be active in the intestine, 1 1 4 i s r e l a t i v e l y inactive on calcium mobilization or mineralization of bone. 1 8

it

The

25-0H-5,6-trans-D 3 was also found to be unable to stimulate bone calcium m o b i l i z a t i o n . 1 1 These considerations lead us to suggest that the 3-hydroxyl group may be important for a l l functions, and further research i s obviously needed to c l a r i f y t h i s point. The very large reduction but not complete elimination of b i o l o g i c a l a c t i v i t y in the 5 , 6 - t r a n s - D 3 , DHT3, and i s o - T 3 which do not possess a methylene group at the pseudo-19 p o s i t i o n indicates that either t h i s methylene group i s important or the substituent group (methyl in DHT3 and i s d - T 3 , and methylene in 5 , 6 - t r a n s - D 3 ) which occupies the pseudo-4 position s t e r i c a l l y hinders an interaction with the receptor.

To differentiate

between these two p o s s i b i l i t i e s , biological assays with compounds such as the epimeric pair of 4a- and 46-methyl-la,25-dihydroxyvitamin D 3 ( I I I ) which have both a methylene group at the 10 position and a methyl substituent at the 4 p o s i t i o n , and the 19-nor3-deoxy-lc.,25-dihydroxyvitamin D3 (IV) which has neither group at the 4 and 10 p o s i t i o n s , should be carried out.

I f the two former compounds are active then the

C(10)=C(19)H 2 fragment i s necessary, while the 4-methyl group d e f i n i t e l y constitutes a' s t e r i c hindrance i f the biological a c t i v i t i e s of these two compounds are much reduced.

S i m i l a r l y , i f the 19-nor-3-dexoy-la,25-dihydroxyvitamin D 3

i s not active,

then the C(19)H 2 substituent of vitamin D molecules i s important for physiological responses, while i f the 19 -nor-3-deoxy-la,25-dihydroxvvitamin D 3 (which i s also the 19-nor-5,6-trans-25-0H-D 3 ), i . e . , the 5,6-trans_-

v

25-0H-D 3 with the methylene group at the pseudo-4

f^L

position elminated) i s more active than the 5,6trans-25-0H-D 3 , then a methylene or methyl s u b s t i tuent at the pseudo-4 position of 5,6-trans-25-0H-D 3 and 25-0H-DHT3 i s not desirable.

H c

s *VjX^ H