Vitamin D. Basic Research and its Clinical Application: Proceedings of the Fourth Workshop on Vitamin D, Berlin, West Germany, February 1979 [Reprint 2020 ed.] 9783112330029, 9783112330012

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Vitamin D Basic Research and its Clinical Application

Vitamin D Basic Research and its Clinical Application Proceedings of the Fourth Workshop on Vitamin D, Berlin, West Germany, February 1979 Editors A.W. Norman • K. Schaefer • D. v. Herrath H.-G. Grigoleit • J.W. Coburn • H.F. DeLuca E.B. Mawer T.Suda

W DE G Walter de Gruyter • Berlin • New York 1979

Editors A.W. Norman, Ph. D., Department of Biochemistry, University of California. Riverside, Ca 9502, USA K. Schaefer, Priv.-Doz., St. Joseph-Krankenhaus I, Berlin (West), Germany J.W. Coburn, M.D., Nephrology Section, Veterans Administration Wadsworth Hospital Center, Los Angeles, CA 90073, USA H.F. DeLuca, Ph. D., Department of Biochemistry, University of Wisconsin-Madison, Madison, WI53706, USA D. Fräser, M.D., The Hospital for Sick Children, 555 University Avenue, Toronto 2, Canada H.G. Grigoleit, Dr., Medizinische Abteilung Hoechst AG Werk Albert, Wiesbaden, Germany D. von Herrath, Dr., St. Joseph-Krankenhaus I, Berlin (West), Germany E.B. Mawer, Department of Medicine, University of Manchester, England T. Suda, Department of Biochemistry, Showa University Dental School 1-5-8, Hetanodai, Shinagawa-ku, Tokyo 142, Japan CIP-Kuntitelaufnahme

der Deutschen Bibliothek

Vitamin D / ed. A.W. Norman . . . - Berlin, New York: de Gruyter. Früher u.d.T.: Vitamin D and problems related to uremic bone disease. NE: Norman, Anthony W. [Hrsg.] Basic research and its clinical application: proceedings of the 4. Workshop on Vitamin D, Berlin, West Germany, February 1979. 1979. ISBN 3-11-007712-4 NE: Workshop on Vitamin D

Library of Congress Cataloging in Publication Data Workshop on Vitamin D, 4th, Berlin, West Germany, 1979. Vitamin D: basic research and its clinical application. Bibliography: p. Includes index. 1. Vitamin D-Congresses. 2. Vitamin D deficiency-Congresses. I. Norman, Anthony W., 1938 - II. Title. QP772. V53W67 1979 612'.399 79-16793 ISBN 3-11-0077124

© Copyright 1979 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: Dieter Mikolai, Berlin. Printed in Germany.

V

Foreword The Fourth Workshop on Vitamin D was held in Berlin (West), Germany from February 18 through 22,1979. In attendance were 402 official delegates from 26 countries. These include representation from Argentina (1), Australia (4), Austria (6), Belgium (9), Canada (11), Denmark (15), England (42), Finland (5), France (30), Germany (59), Hungary (2), Israel (10), Italy (11), Japan (28), Mexico (1), Netherlands (20), New Zealand (1), Norway (5), Poland (3), South Africa (2), Spain (1), Sweden (14), Switzerland (17), Thailand (1), Turkey (1), and the USA (103); additionally, the proceedings include one paper from the USSR, and a few from the GDR. The growth of interest in the domain of vitamin D and its endocrine system and its associated attendance at vitamin D workshops is literally astounding. The First Workshop on Vitamin D was held as recently as October of 1973 and, as indicated in the table below, interest in the subject of vitamin D has been growing literally exponentially. Workshop Number

Date

Number of Number of Delegates Countries represented

Number of Presentations Formal per Delegates Presentations Talks Posters

I

October 1973

56

3

5

-

0.09

II

October 1974

221

22

84

—

0.39

III

January 1977

332

20

45

124

0.51

IV

February 1979

402

26

80

225

0.76

The highlights of the Fourth Workshop on Vitamin D were many and varied, which is reflective of the diverse interests of the chemists, biochemists, physiologists and clinicians interested in the multiplicity of aspects of the vitamin D endocrine system. Several significant advances were reported in terms of our basic understanding of the vitamin D endocrine system. The structures of three new metabolites of vitamin D were reported. This brings to eight the number of chemically characterized metabolites of vitamin D that are known to occur naturally.ln addition the chemical synthesis and the biological properties of a host of analogs of both 1,25(OH)2D3 and 24,25(OH)2D3were reported by many laboratories. Evidence was presented that the production of 1,25(OH)2D3was affected not only by the peptide hormones, parathyroid hormone, prolactin, and growth hormone but as well by glucocorticoids, estro-

VI gens and androgens. As a consequence of our increasing appreciation of the diversity and multiplicity of vitamin D steroid actions, a major segment of the Fourth Workshop on Vitamin D was devoted to the clinical application of our newer understandings of these relationships. In this regard the clinical efficacy of 1,25-dihydroxyvitamin D in treating certain aspects of renal osteodystrophy now seems to be firmly established. In addition, there is an increasing interest in the possible application of 24,25(OH) 2 D 3 to a variety of disease states. Because of the significantly increased attendance at the Fourth Workshop on Vitamin D and in an effort to retain the "workshop-like demeanor", a number of new features were incorporated into the conduct of this meeting. These included: a special evening Round Table Discussion chaired by Prof. J.W. Coburn concerned with "Unanswered Questions in Renal Osteodystrophy"; (b) a Round Table Discussion chaired by Professors W.H. Okamuraand H.F. DeLuca concerning development of an appropriate nomenclature to apply to the increasing array of vitamin D metabolites and analogs; (c) a provocative evening session on the subject of intracellular receptors chaired by Drs. E.B. Mawer and M.R. Hausslerforvitamin D metabolites; and (d) a session composed of approximately 12 five-minute presentations concerning new vitamin D/Ca/P metabolic diseases and problems. Together these four sessions provided delegates an opportunity to focus on special sub-problems in the vitamin D field. Certainly, however, the highlight of the Fourth Workshop on Vitamin D was the presentation of no less than 225 posters. These were divided into seven separate sessions, each of one and one-half hour duration. They provided convenient coffee-break intervals in the morning and afternoon sessions (where the 80 verbal presentations were given) during each day of the Workshop. The posters were given in the well-lighted foyer of the very beautiful Kongresshalle and it was here that the nitty-gritty of many vitamin D problems was discussed as old friendships were renewed and new friendships were established. Certainly, the Local Organizing Committee is to be congratulated on providing such a perfect setting for the conduct of the Fourth Workshop on Vitamin D. An additional highlight of the Workshop was the attendance of Professor E. Havinga who Is the father of much of the modern chemistry of vitamim D. Professor Havinga played an important role in defining many aspects of the photo-chemistry that is so critical for opening the B-ring and generating vitamin D seco-steroids. A special tribute to him in this volume precedes the section of papers on chemical topics. The Organizing Committee would like to acknowledge the financial support of Der Senator fur Wirtschaft, Berlin, Der Bundesministerfur Jugend, Familie and Gesundheit, Bonn-Bad Godesberg, The National Institute of Arthritis, Metabolism & Digestive Diseases, Hoffmann-La Roche Inc. (Nutley, New Jersey), F. Hoffmann-La Roche & Company (Basle, Switzerland), Firma Gambro GMBH & Company, Hoechst AG, Frankfurt/M, Albert-Roussel Pharma GmbH, Teijin Limited, Leo Pharmaceutical Products, The Upjohn Company, The Proctor & Gamble Company, Firma Diekmann, and Ross Laboratories. Without this generous multi-governmental and multi-corpo-

VII rate financial support it would have been impossible to have a Vitamin D Workshop which included such comprehensive worldwide attendance. A special tribute is also due to the tireless efforts of members of the Local Organizing Committee. These include Dr. Alfred Pauls, Dr. G e m o t Asmus, Dr. Sabine Busse von Cölbe who skillfully and patiently resolved the problems and crises which inevitably arise during the conduct of any meeting as complex as the Workshop. Also thanks are due to Miss Mary Forster who assisted in the compilation of this book. Last but certainly not least, heartfelt thanks are also due to the unstinting work of the Conference secretary in Riverside, Mrs. Wendy Reid, and Frau Dorothee Weisselberg, the Conference secretary in Berlin. Without their devotion and long hours of attention to detail, it would have been impossible for the many facets of the Workshop to proceed as smoothly and as efficiently as they did. Anthony W. Norman, Riverside Klaus Schaefer, Berlin Dietrich von Herrath, Berlin Hans-Günther Grigoleit, Frankfurt Jack W. Coburn. Los Angeles Hector F. DeLuca, Madison E. Barbara Mawer, Manchester Tatsuo Suda, Tokyo June, 1979

IX

Contents

Chemistry, Structure, Function and Biological Assay

1

Synthesis of Some Side Chain Analogues of Vitamin D3 Metabolites N. Ikekawa, M. Morisaki, N. Koizumi, Y. Hirano

13

Structural Transformations on Vitamin D E. Zbiral and W. Reischl

21

Distribution and Activity of a Vitamin D3 Compound in Trisetum Flavescens W.A. Rambeck, A. Wetzel, O. Kreutzberg and H. Zucker

33

Synthesis and Biological Activity of 24R-Hydroxy-25-fluorocholecalciferol and 1a, 24R-Dihydroxy-25-fluorocholecalciferol J.J. Partridge, S.-J. Shiuey, A. Boris, J.P. Mallon and M.R. Uskokovic 37 The Synthesis and Biological Evaluation of Fluorinated Analoges as Probes of Vitamin Da Metabolism J.L. Napoli, W.S. Mellon, M.A. Fivizzani, H.K. Schnoes and H.F. DeLuca

45

19-Substituted-10,19-Dihydrovitamins via Hydrozirconation of Vitamin D3 A.W. Messing, F.P. Ross, M. Miura, A.W. Norman and W.H. Okamura

51

The Synthesis of 1a, 3a-Dihydroxy-25Methylcholesta-5,7-Diene-24-OIC Acid, and of la, 3a-Dihydroxy-25 Methylcholesta-5,7-Diene J.M. Midgley, R.M. Upton, R. Watt and W.B. Whalley

55

Interconversion of Vitamin D and Trans-Vitamin D by Triplet-Sensitized Isomerization H.J.C. Jacobs, J.W.J. Gielen and E. Havinga

61

2

The Use of UV, CD, H-NMR Spectroscopy and Mass Spectrometry for the Investigation of Conformational Mobility of Vitamin D and Previtamin D and their Interconversion E. Berman, N. Friedman, Y. Mazur, M. Sheves and Zeev V.l. Zaretskii 25 (R), 26 and 25 (S), 26-(OH)2D3: Biological Activity in Intact and Nephrectomized Rats. Comparative Effects of 1, 25, 24 (R), 25-(OH)2D3 and I, 24 (R), 25-(OH)3Da M. Thomasset, J. Redel, P. Marche, P. Cuisinier-Gleizes

65

73

Inhibitors of Vitamin D Metabolism and Action B.L Onisko, H.K. Schnoes and H.F. DeLuca

77

C(1)-Hydroxylation of Vitamin D3 and Related Compounds H.E. Paaren, H.K. Schnoes and H.F. DeLuca

81

Vitamin D3 Sulfate in Lactating normally fed Rats and Suckling Pups After Maternal Administration of 3H Vitamin D3 or35S Vitamin D3 Sulfate N. Le Boulch, M. Laromiguiere, C. Marnay-Gulat, L. Miravet and Y. Raoul

85

X

Synthesis of Vitamin D Analogues and their Covalent Binding to Affinity Chromatographic Media F.P. Ross, W.R. Wecksler, W.H. Okamura and A.W. Norman

89

Osteoporosis

97

Calcium Absorption and Plasma 1,25 (OH)2D Levels in Post-Menopausal Osteoporosis B.E.C. Nordin, M. Peacock, R.G. Crilly and D.H. Marshall

99

Osteoporosis and Age-Related Osteopenia: Evaluation of Possible Role of Vitamin D Endocrine System in Pathogenesis of Intestinal Calcium Absorption B.L. Riggs, J.C. Gallagher and H.F. DeLuca

107

Two Years Experience of Oral Treatment with 1a-Hydroxyvitamin D and Calcium in Patients with Senile or Postmenopausal Osteoporosis T.S. Lindholm

115

The Treatment of Osteoporosis with 24,25 Dihydroxycholecalciferol A Pilot Study J. Reeve, R. Hesp, P. Hulme. J.A. Kanis, L. Klenerman, P. Meunier, R.G.G. Russell, M.Tellez-Yudilevich, D. Williams and R. Wootton

119

Osteomalacic Factors in the Osteoporosis and the Treatment of Osteoporotic Patients with Vitamin D2 and 1aOHD3 K. Mizuno and J. Tanaka

123

Transcalciferan/D-Binding Protein(s)

127

Biologic Significance of Genetic Variation in Human Gc (Vitamin D-Binding Protein) S.P. Daiger

129

The Transport of Vitamin D R. Bouillon and H. Van Baelen

137

Structure and Properties of Human Plasma Vitamin D Transport Protein (Group-Specific Component) J. Svasti, A. Kurosky, A. Bennett, R. Surarit and B.H. Bowman

149

Serum Vitamin D Binding and Gc Polymorphism J. Constans, M. Viau, J.P.Moatti and J.L Clavere

153

Sunlight, Skin and Seasonal Effects

157

Identification of in Vivo Generated Previtamin D3, Vitamin D3 and 25-Hydroxyvitamin D3 in the Vitamin D-Deficient Rats Irradiated by Ultraviolet Light T. Kobayashi, TOkano, K. Mizuno, N. Matsuyama, N. Nobuhara, K. Takada and T Takao 159

XI Response of Plasma 250HD to Standardized Ultra Violet Radiation M. Davie and D.E.M. Lawson

163

Effects of Sunlight Exposure on Blood Concentration of Vitamin D Derivatives in Healthy Japanese Adults R. Morita, S. Dokoh, M. Fukunaga, I. Yamamoto and K. Torizuka

165

Contribution of Skin Vitamin D3 Synthesis in Patients Receiving Total Parenteral Nutrition G. Jones, B. Byrnes, D. Duthie and K.N. Jeejeebhoy

169

The Photo-Biochemistry of Vitamin D3 in Vivo in the Skin M.F. Holick, S.A. Holick, S.M. McNeill, N. Richtand, M.B. Clark and J.T. Potts jr.

173

Assays for Vitamin D Metabolites

177

Measurement of Plasma 1,25 (OH)2 Vitamin D by Protein Binding Assay and Radioimmunoassay M. Peacock, G.A. Taylor and W.B. Brown

179

Use of Isotope Dilution Mass Fragmentography in Vitamin D Research I. Björkhem, I. Holmbeg, T. Kristiansen, A. Larsson and J.I. Pedersen

183

Assay of 1,25-Dihydroxyvitamin D and Other Active Vitamin D Metabolites in Serum: Application to Animals and Humans M.R. Haussler, M.K. Drezner, J.W. Pike, J.S. Chandler and L.A. Hagan

189

24,25-Dihydoxyvitamin D in Human Serum C.M.Taylor

197

Radioimmunoassay of Vitamin D Metabolites P.C. Schaefer and R.S. Goldsmith

205

Assays for Vitamin D and its Metabolites R.L. Horst, R.M. Shepard, N.A. Jorgensen and H.F. DeLuca

213

Radioimmunoassay for Circulating 1,25- and 25,26-Dihydroxycholecaliferols in Man J.LH. O'Riordan, T.L Clemens, G.N. Hendy, L J . Fraher, L.M. Sandler and S.E. Papapoulos

221

Hemisuccinates of Vitamin D3 and of its Metabolites K. Lichtwald, E. Mayer, H. Schmidt-Gayk, J. Varga and S. Walch

229

Studies on Antisera against Vitamin D Metabolites H. Schmidt-Gayk, E. Mayer, R. Schjier, K. Lichtwald, R. Bouillon and T.L. Clemens

233

The Measurement of Various Vitamin D Derivatives in Plasma Using High-Pressure Liquid Chromatography S. Dokoh, R. Morita, M. Fukunaga, I. Yamamoto, A. Miyaji and K. Torizuka

239

XII Assay of 1,25- and 24,25 Dihydoxycholecalciferol in Human Serum some Technical Considerations R.S. Mason, D. Lissne, C. Reek, S. Posen

243

Radiocompetitive Protein Binding Assays for 25-Hydroxy Vitamin D, 24,25-Dihydroxyvitamin D and 1,25-Dihydroxy-vitamin D in Human Serum S. Guillemant, R. Kremer, J. Eurin, J. Guillemant

247

Steroid Receptors: What do We not Know? R.J.B. King

251

Vitamin D - Parathyroid Hormone

259

Cholecalcifediol suppression of PTH Secretion from Monolayer Culture of Human Parathyroid Carcinoma Cells M. Fukase, M. Tsutsumi, J. Iwasaki and T. Fujita

261

The Effect of Human Growth Hormone Replacement on Parathyroid Function and Vitamin D Metabolism J.M. Gertner, R.L. Horst, M. Genel and H. Rasmussen

265

Serum Calcium Concentration: Possible Regulator of Renal Parathyroid Hormone Action H.v. Lilienfeld-Toal, K.G. Mackes, E. Keck

267

Effects of Vitamin D3 Metabolites Injection into Thyro-parathyroidectomized Pregnant Rats on Fetal Weight and Liver Glycogen Stores in Mothers and Fetuses J.M. Garel, M. Gilbert and P. Besnard

271

Effects of Vitamin Deficiency on Calcium-Binding Proteins and on Plasma Calcitonin and Parathyroid Hormone Levels in Growing Pigs A. Pointiiiart, M. Thomasset and J.M. Garel

275

I,25 Dihydroxycholecalciferol effect on in Vitro Immunoreactive Human Parathyroid Hormone Secretion from Hyperplastic Glands E. Mallet, J.P. Basuyau, M.C. Tonon, H. Vaudry

279

Quantitative Changes of Rat Thyroid C Cell Population in Hyper- and Hypovitaminosis D M. Petko

285

The Interactions Between Vitamin D Metabolites, Parathyroid Hormone and Calcitonin A.D. Care, D.W. Pickard, S.E. Papapoulos, J.L.H. O'Riordan, J.M. Garel and J. Redel

289

In Vivo Suppression of Parathyroid Hormone Secretion by 24,25 Dihydroxy Cholecalciferol in Hyperparathyroid Dogs J.M. Canterbury, G. Gavellas, J.J. Bourgoignie and E. Reiss

297

XIII

Renal Actions

305

Acute and Chronic Effects of Vitamin D Metabolites on the Renal Handling of Phosphate J.-P. Bonjour, J. Caverzasio, H. Fleisch and U. Trechsel

307

Interaction of Vitamin D Metabolites with Parathyroid Hormone and Vasopressin J.B. Puschett

315

Phosphaturie Effect of Parathyroid Hormone in Phosphate Depleted Man J.C. Pena, J. Guerrero, S. Quiröz, J. Herrera

325

Vitamin D in Maternal, Fetal and Neonatal Conditions

329

Long Term Effects of Low 25-Hydroxyvitamin D (25-OHD) Serum Concentrations in Premature infants: A Preliminary Report LS. Hillmann, D.V. Huebener and J.G. Haddad

331

Vitamin D, Calcium and Phosphorus Requirements and Bone Mineralization in Preterm Infants J.J. Steichen, T.L. Gratton, L Abrams-Becker and R.C. Tsang

335

The Transplacental Movement of Metabolites of Vitamin D in the Sheep R. Ross, A.D. Care, C.M. Taylor, B. Pelc and B.A. Sommerville

341

The Effects of Solanum Glaucophyllum Ingestion by Pregnant Cows on Plasma Calcium Levels in Dams, Fetuses and Neonates and on the Mineral Composition of Colostrum J.P. Barlet, R. Roux and M.-J. Davicco

345

The Vitamin D3 Requirement for Premature Infants H. Wolf, V. Gräff and G. Offermann

349

Calcium Uptake and Binding by Membrane Fractions of Human Placenta: ATP Dependent Calcium Accumulation J .A. Whitsett, M. Costello, D. Lo, J. Bohn and R.C. Tsang

353

Skeletal Actions

357

Influence of 1,25(OH)2D3 on Bone Surface Lining Cells and on cartilage mineralisation in vitro. Ultrastructural studies B. Krempien and F. Klimpel

359

The Role of Vitamin D Metabolites in Calcification of Chicken Epiphysis A. Ornoy, E. Sekeles, R. Cohen and S. Edelstein

363

XIV

Bone Mineral Solubility and its Alteration by 1,25-Dihydroxyvitamin D3 R. Brommage and W.F. Neuman

369

Accelerated Bone Formation in End Stage Renal Disease S.L. Teitelbaum, M.A. Bergfled, J. Freitag and K.A. Hruska

373

24,25-Dihydroxyvitamin D: The Preferred Metabolite for Bone S. Edelstein and A. Ornoy

381

Cellular Location and Regulation of the 24,25-Dihydroxyvitamin D3 Formation in Cultured Cells from Bone and Cartilage M. Carabedian, M. Lieberherr, M.T. Corvol, H. Guiollozo, C.L. Thil and S. Balsan

391

Comparison of the Histological Effect and Metabolism of 25-(OH)D and 1,25-(OH)2D in Rat Bone J .A. Gallagher and D.E.M. Lawson

399

Action of Solanum Malacoxylon on Bone Histology of Vitamin D Deficient Rats M.C. de Vernejoul, M.L. Queille, R.W. Nordin, C.A. Mautalen and L. Miravet

403

Dual Action of Parathyroid Hormone on the Mineralization of Rat Incisor Dentin S. Matsumoto, T. Tsudzki and M. Yamaguchi

411

Influence of Diet on the Response of Bone Cells to 1,25-OH)2D3 in Thyroparathyroidectomized Rats S.E. Weisbrode, C.C. Capen and A.W. Norman

415

Nuclear Binding of 24,25-(OH)2D3 and its Effect on DNA Polymerase Activities in Cultured Chondrocytes M.T. Corvol, M.F. Dumontier, A. Ulmann, M. Garabedian andG. Witmer

419

Effects and Interaction of 1,25 (OH)2Ds and 24,25 (OH)2D3 on Bone H.H. Malluche, H. Henry, W.A. Meyer, D. Sherman, S.G. Massry and A.W. Norman

425

Effects on Mineral Homeostasis of 1,25 (OH)2D3 and 24,25 (OH)2D3, Alone and in Combination, in Rats B.S. Levine, D.B.N. Lee, N. Brautbar, M.W. Walling, C.R. Kleeman and J.W. Coburn

429

Effect of Dihydroxylated Vitamin D Metabolites on Experimental Rickets D. Kraft, G. Offermann, R. Steldinger

431

Evidence for the Presence of 25-OH-D3 Binding Proteins in Embryonic Cartilage F. Vittur, M. Zanetti, N. Stagni, R. Camerotto and B.de Bernard

435

XV

Glucocorticoid Dependence of Cytosol 1,25-Dihydroxycholecalciferol Receptors in Fetal Rat Calvaria S.C. Manolagas, D.C. Anderson and G.A. Lumb

439

Vitamin D Metabolism

443

Recent Developments in the Metabolism of Vitamin D H.F. DeLuca and H.K. Schnoes

445

The Role of Systemic PH in the Bioactivation of Vitamin D LV. Avioli

459

The Site of Synthesis of 1,25(OH)2 Vitamin D3 in the Kidney A Microenzymatic Assay on Isolated Tubules M.G. Brunette, M. Chan, C. Ferriere, K.D. Roberts

463

Response of Chick Kidney Cell Cultures to 1,25-Dihydroxyvitamin Da H.E.Henry

467

Growth Hormone and Prolactin Action on Vitamin D Metabolism in the Rat E.M. Spencer, O. Tobiassen, P. Ling and E. Braunstein

475

Effective Liver 25-Hydroxylation of Vitamin D under Chronic Ethanol Administration in the Rat M. Gascon, B. Hollis and S. Mah

479

Calcium Transport by Rat Colon: Response to 1a,25(OH)2D3 and Dietary Calcium Restriction M.J. Favus, S.C. Kathpalia and F.L. Coe

483

Direct Vitamin D3-Metabolite Regulation of Kidney 25-Hydroxyvitamin D3 Metabolism J.L. Omdahl, L.A. Hunsaker and A.P. Evan

493

The Regulation of 25-Hydroxylation by Vitamin D Intake in Man: A Theoretical Model A.M. Parfitt, M. Mathews, D.S. Rao, M. Kleerekoper

497

Minimal Deprivation of Vitamin D Enables Parathyroid Hormone to Stimulate 25-OH Vitamin D - 1a-Hydroxylase B.E. Booth, H.C. Tsai and R. Curtis Morris jr.

501

Vitamin D Metabolism in Japanese Quail: Effects of Lead Exposure and Dietary Calcium S.N. Baksi and A.D. Kenny

503

Evidence for a Calcium-Dependent, PTH-lndependent Regulation of Plasma 1,25-Dihydroxyvitamin D in Rats U. Trechsel, J.A. Eismann, J.-P. Bonjour and H. Fleisch

511

Computer Modeling of Vitamin Metabolism J.L. Omdahl, R.C. Allen and R.P. Eaton

515

Prolactin, Growth Hormone and Vitamin D Metabolism I. Maclntyre, D.J. Brown and E. Spanos

523

XVI A Study of the Physiologic Significance of 24-Hydroxylation of 25-Hydroxyvitamin D Y. Tanaka, H.F. DeLuca and N. Ikekawa

531

Dynamic Aspects of Vitamin D Metabolism: Formation of Hydroxylated Metabolites from A Physiological Dose of Cholecalciferol in Vitamin D Depleted Man S.W. Stanbury

537

The Regulation of Plasma 1,25-(OH)2-D Concentrations in Healthy Adults R.W. Gray, J. Lemann jr. and N.D. Adams

545

The Role of the Liver in the Control of Vitamin D Metabolism E.B. Mawer

553

Prostaglandins and 25-Hydroxyvitamin D-1a-Hydroxylase J.D. Wark, R.G. Larkins, J.A. Eiman and T.J. Martin

563

Regulation of Plasma Levels of 1,25 Dihydroxy Vitamin D in Growing Dogs with Thyroparathroidectomy and Varying Amounts of Parathyroid Hormone L.A. Nagode and C.L. Steinmeyer

567

Both 24R,25-Dihydroxyvitamin D3 and 1a,25-Dihydroxyvitamin D3 are Indispensable for Normal Calcium and Phosphorus Homeostasis A.W. Norman and H.L. Henry

571

New Renal Metabolites of 24,25-Dihydroxyvitamin Ds in the Chick T. Suda, Y. Takasaki and N. Horiuchi

579

The 1a- and 24-Hydroxylases of 25-Hydroxycholecalciferol: one or two Enzymes? J.G. Ghazarian

587

Regulation of 25-Hydroxycholecalciferol-1 and -24-Hydroxylases in Cultured Kidney Cells U. Trechsel, J.P. Bonjour and H. Fleisch

597

Possible Extra-Renal 25-OHD3-1a-Hydroxylase Activity in the Pregnant Rat G.E. Lester, T.K. Gray and R.S. Lorenc

605

The Absorption and Metabolism of Vitamin D3 from Parenteral Injection Sites M. Davies and E.B. Mawer

609

1,25-(OH)2 Vitamin Da & 24,25-(OH)2 Vitamin Da Synthesis by the Isolated Perfused Rat Kidney A.M. Rosenthal, G. Jones, D. Fraser and S.W. Kooh

613

In Vitro Regulation of 25-Hydroxyvitamin D3 Metabolism by Avian Kidney Cells in Serum-Free Medium R. Turner, G.A. Howard, J.I. Rader, B.L. Bottemiller and D.J. Baylink

617

XVII

Comparative Studies on Mitochondrial and Microsomal NADPHCytochrome c Reductases of Chick Tissues J .A. Kulkoski, J.L. Weber, J.E. Martinez and J.G. Ghazarian

621

Effect of Pregnancy, Acromegaly, Primary Hyperparathyroidism and Prolactinoma on 1,25-Dihydroxyvitamin D in Man D.J. Brown, E. Spanos, P. Raptis and I. Maclntyre

625

Relationship of Dietary Phosphorus Restriction to Calcium Absorption and Vitamin D Metabolism A. Bar and S. Hurwitz

629

Intestinal Actions and Calcium Binding Protein

637

Meaning of Non-Parallel 1,25-Dihydroxycholecalciferol Mediated Response Relationship in Intestine and Bone to Dose and Time D.T. Zolock, R.L. Morrissey and D.D. Bikle

639

Cytoplasmic Localization of the Calcium Binding Protein in Rat Duodenal Absorptive Cells P. Marche, C. Le Guern and P. Cassier

643

Visualization of Vitamin D-Dependent Calcium Binding Protein in Chick Intestine by Immuno-Scanning Electron Microscopy S. Moriuchi, S. Yoshizawa, N. Hosoya, S. Noda, K. Kubota

647

Influence of High Doses of Vitamin D3 on Broilers and Adult Hens P. Garcia Partida, C. Panizo, F.P. Montana and I.D. Prieto

651

Purification of the Intestinal Receptor for 1,25-Dihydroxyvitamin D J.W. Pike and M.R. Haussler

659

Recent Progress on the Application of Affinity and Covalent Chromatography to the Purification of the 1,25-Dihydroxyvitamin Da Receptor from Chick Intestinal Mucosa W. R. Wecksler, F.P. Ross, W.H. Okamura and A.W. Norman

663

1,25-Dihydroxycholecalciferol and the Prevention of Parturient Hypocalcemia and Paresis ("Milk Fever") in Pregnant Dairy Cows C.C. Capen, G.F. Hoffsis and A.W. Norman

667

A Vitamin D-lnduced, Calcium-Binding Protein from Rat Intestinal Brush Border A. Miller III, T.-H. Ueng and F. Bronner

675

Relationship of Intestinal Calcium Absorption to Intestinal and Plasma Calcium Binding Protein Measured by a Radioimmunoassay A. Bar and S. Hurwitz

679

The Effect of 1,25-Dihydroxycholecalciferol and Hydrocortisone on the Development of the Embryonic Chick Duodenum N. Hosoya, S. Moriuchi, S. Yoshizawa, S. Noda and K. Kubota

683

XVIII Theories on the Mechanism of Action of 1,25(OH)3D3 on Active Intestinal Calcium and Inorganic Phosphate Absorption: are the Calcium and Phosphate Transport Processes Coupled, Uncoupled or Both? M.W. Walling and D.B.N. Lee

687

Fundamental Actions of 1,25 Dihydroxycholecalciferol on Intestinal Ion Transport do not Involve Gene Activation O. Fontaine, T. Matsumoto, M. Simoniescu, D.B.P. Goodman and H. Rasmussen

693

The Effect of Cholecalciferol on the Phosphorylation of Intestinal Membrane Proteins R.H. Wasserman and M.E. Brindak

703

Some Biochemical Responses of Chick Intestine to 1,25Dihydroxyvitamin D D.E.M. Lawson, H. Harding, S. Lane, P. Wilson

711

Induction of Metallothionein(s) in Organ-Cultured Duodenum: Relationship to 1a,25-(OH)2D3-lnduced C a B P Synthesis R.A. Corradino, C.S. Fullmer, E. Frelier and S. Maxwell

731

Vitamin D and Calcium Absorption: Computer Simulation S. Hurwitz, H. Talpaz and A. Bar

735

Renal Osteodystrophy

743

Clinical Assessments of Vitamin D3 - Therapy in Patients on Longterm Haemodialysis W. Seeger, P. Müller, K. Buchali, I. Grossmann, C. Hansen

745

Adverse Effects of Vitamin D Metabolites on Osteitis Fibrosa in Patients on Chronic Hemodialysis: Critical Role of Induced Hyperphosphatemia J.L. Sébert, J.F. de Frémont, J. Guéris, B. Coevoet, P. Marie, A. Smadja, D. Kuntz, A. Ryckewaert, P. Meunier, A. Fournier

751

Assessment of Long-Term Effects of 1a(OH)Da on PTH Secretion, Intestinal Calcium Absorption and Bone in Chronic Renal Failure S. Madsen, K. Olgaard and J. Ladefoged

755

Hypergonadotropic Hypogonadism in Renal Failure - A Defect Responsive to 1,25(OH)2D3 W. Kreusser, U. Spiegelberg, E. Ritz

763

Comparative Effects of 1,25-Dihydroxycholecalciferol and Cholecalciferol in non-Dialyzed Chronic Renal Failure M.S. Christensen, F. Meisen, C. Christiansen, B. Hartnack O. Sj0 and P. Rodbro

767

Medium term Effects of 1,25-Dihydroxy-vitamin D in Patients with Renal Osteopathy E. Leicht, G. Biro, H. Baumhöfener, Chr. Blumy

771

XIX The Early Detection and Treatment of Renal Osteodystrophy P.E. Cordy

775

The Combined Use of Human Calcitonin and 1,25-Dihydroxycholecalciferol in the Treatment of Renal Osteodystrophy K. Farrington, Z. Varghese, J.F. Moorhead

781

Intestinal Calcium Transport in Vitro as Influenced by Azotemia, Dietary Calcium, Parathyroidectomy and 1,25-Dihydroxycholecalciferol (1,25-DHCC) Treatment. - Role of Calcium - ATPase H. Schiffl, N. Oswald and U. Binswanger

787

Recurrence of Hyperparathyroidism from Autografted Parathyroid Fragments in Uremic Patients in Spite of Administration of 25(OH)Da and 1a(OH)Da T. Drüeke, J. Zingraff, J. Guéris, N. Chkoff, C. Dubost

791

25 Hydroxycholecalciferol Plasma Levels in Anephric Patients After Oral Loading of Vitamin D D. Brancaccio, G. Coen, C. Galmozzi, S. Casati, G. Lucentini

795

The Histological Study on Experimental Renal Osteodystrophy in Rats R. Niki, H. Ohkawa, S. Suzuki, Y. Takagaki, M. Fukushima, M. Ono, T. Shimizu, Y. Nishii and T. Suda

799

Long Term Effects of Small Doses of 1,25 Dihydroxycholecalciferol in Dialysis Patients B. Hochman, V. Gura, G. Boner, A. Weiss, A.J. Olah, J. Rosenfeld

803

Clinical and Biological Efficiency of 1,25 Dihydrocholecalciferol in Chronic Dialysis Patients with Renal Osteodystrophy R.L. Schmitt, M .A. Dambacher, J. Gunçaga, A.G. Olah, A. Omichi, H.A. Jahn

807

Histomorphometric Evaluation of the effect of 1,25-DHCC in Type II and III of Renal Bone Disorders G. Delling, H. Lühmann, M. Bulla, Ch. Fuchs, H.V. Henning J.L.J. Jansen, R. Ziegler and W. Schulz

811

Histomorphometry of Bone Changes in Renal Osteodystrophy After Treatment with 1,25-Dihydroxycholecalciferol A.J. Olah, H. Jahn, F.W. Reutter, R. König, A. Blumberg and A. Colombi

817

The use of 5,6-trans-25-OH-Cholecalciferol in the Treatment of Patients with Fully Developed Renal Osteopathy Requiring Dialysis: Therapeutical Application and Treatment Results W. Cremer, N. Graben, H.-G. Grigoleit, P. Merguet

821

XX Clinical Experience in Long Term Treatment with 1,25-DHCC in Different Histological Forms of Renal Osteopathy W. Schulz, P. Spiegel, G. Delling

827

Calcium-Phosphorus Metabolism and Renal Function During 1a-OH-D3 Vitamin Treatment of Patients with Chronic Renal Disease H.E. Nielsen, M.S. Christensen, F.K. Romer, H.E. Hansen and F. Meisen

831

Newcastle Bone Disease M.K. Ward, I.S. Parkinson, T.G. Feest, H.A. Ellis, D.N.S. Kerr

835

The Measurement of 1,25-(OH)2-D by Competitive Protein Binding Assay in the Plasma of Anephric Patients: The Effects of Dihydrotachysterol Therapy R.W. Gray, N.D. Adams and J. Lemann jr

839

25-Hydroxycholecalciferol and 1,25 Dihydroxycholecalciferol in the Treatment of Renal Osteodystrophy G. Coen, M. Taccone Gallucci, E. Bonucci, G. Bianchini

843

Bone Histomorphometry in Children with Early Chronic Renal Failure Treated with 25 (OH)D3 R. Baron, M. Norman, A. Mazur, A. Gruskin and H. Rasmussen

847

Hypercalcemia During Treatment of Uremic Patients with I,25(OH)2D3: Analysis of 43 Cases S.N. Winkler, A.S. Brickman, E.G.C. Wong, D.J. Sherrard, R.B. Miller, C.M. Bennet and J.W. Coburn

851

Renal Bone Disorder in Children: Therapy with Vitamin Ü3 or 1,25Dihydroxycholecalciferol M. Bulla, G. Delling, G. Offermann, R. Ziegler.G. Benz, H. Luehmann, A. Sanchez de Reutter

853

Treatment of Predialysis Renal Bone Disease with 1a-(OH) Vitamin D3 and I.25-(OH)2 Vitamin D3. Initial Results of Balance Studies and the Effect on Intestinal Calcium Absorption, Serum PTH and Calcitonin Levels J.R. Juttmann and J.C. Birkenhager

861

The Effect of 1-a-OHD3 on Bone Changes in Non-Dialysed Patients with Chronic Renal Insufficiency F. Meisen, H.E. Nielsen and F.K. Romer

865

25 Hydroxyvitamin D in Renal Osteodystrophy: Long Term Results of A Six-Center Trial R.R. Recker

869

Further Therapeutical Experience with the Vitamin D Analog 5,6-trans-25-OHCC in Renal Osteopathy H.-G. Grigoleit, D. Kraft, K. Schaefer, G. Offermann, D. von Herrath, G. Delling

877

XXI

A New Laboratory Model for Renal Osteodystrophy and Vitamin D Y. Nishii, M. Fukushima, T. Shimizu. M. Nanjo, R. Niki, H. Ohkawa, Y. Hinohara, M. Ogawa, Y. Tohira, H. Nakano, K. Okano and T. Suda

885

The Influence of Uremia, Aluminiumhydroxide and Various Vitamin D Compounds on the Course of Nutritional Rickets in Rats D. von Herrath, K. Schaefer and B. Krempien

893

Effect of Vitamin D Analogs in Rats with Experimental Renal Osteodystrophy K. Ueno, H. Kawashima, Y. Izawa, N. Ohnuma, T. Makita, S. Kurozumi, Y. Hashimoto, S. Ishimoto, Y. Kawaguchi, M. Yamamoto, Y. Kimura, Y. Ogura, Y. Ueda, K. Kushida and T. Inoue

901

Evaluation of Histological Procedures in the Investigation of Bone Disease Related to Vitamin D E. Ritz, B. Krempien, H.H. Malluche

907

Pathophysiology of Vitamin D Related Diseases

913

Effects of in Vitro and in Vivo Sodium Restriction on Corticosteroidinduced Intestinal Calcium Malabsorption J.S. Adams and B.P. Lukert

915

Trisetum Calcinosis: Current Status of Clinical Research G. Dirksen

921

X-Linked Hypophosphatemia is a Disorder of Phosphate Transport in the Renal Brush Border Membrane in the Hypophosphatemic Mouse. Why is Serum 1,25-(OH)2D Low in the Human Homologue? H.S. Tenenhouse, C.R. Scriver and H.F. DeLuca

925

The Effect of Vitamin D3 and Metabolites on Magnesium Metabolism B.S. Levine, N. Brautbar, D.B.N. Lee, M.W. Walling, K. Kurokawa, C.R. Kleeman and J.W. Coburn

933

Subnormal Serum 1,25-Dihydroxyvitamin Da Levels in Children with Glomerular Disease Receiving Corticosteroids R.W. Chesney, R.B. Mazess, A.J. Hamstra, S. O'Regan and H.F. DeLuca

935

Regulation of Serum Phosphorus by 1,25(OH)2D3 D.B.N. Lee, B.S. Levine, M.W. Walling, N. Brautbar, R.K. Lessman, V. Silis and J.W. Coburn

939

Unexplained hypercalcemia:? Sarcoidosis sine Sarcoidosis: with Elevated Serum Level of 1,25 DHCC B. Frame and A.M. Parfitt

941

Vitamin D Metabolites in Hypercalcitoninemic States R. Ziegler, F. Raue, M. Grossmann and H. Schmidt-Gayk

945

XXII Target Cell Resistance in Pseudohypoparathyroidism: Single or Multiple? A.M. Parfitt

949

Vitamin D Metabolism In Patients with Nephrolithiasis J. Lemann Jr., R.W. Gray and N.D. Adams

957

Evidence for Abnormal Metabolism of Vitamin D in Sarcoidosis N.H.Bell

963

Effects of Experimental Diabetes in the Rat on Calcium Transport and Vitamin D Metabolism H.P. Schedl

967

Absorption of Vitamin D and 25-OH Vitamin D in patients with Intestinal Malabsorption E . L Krawitt, E.B. Mawer and M. Davies

975

Effect of Diabetes in Rats on the Metabolism of Cholecalciferol and 25-Hydroxycholecalciferol S. Sulimovici and M.A. Roginsky

979

Do Alkaline Phosphatases Have a Physiological Function? S. Posen

983

Clinical Application of the 1,25-(OH)2D Assay B. Lund. O.H. Sorensen and B. Lund

991

Elevated Plasma 1,25-(OH)2D Following Massive Dosing of Vitamin D3 in Dairy Cattle R.L Horst and E.T. Littledike

999

Vitamin D Metabolism in the Diabetic Rat R.L. Horst, S.L. Spoede and E.T. Littledike

1003

The Effect of Chronic Excess or Deficiency of Growth Hormone on Plasma 1,25-Dihydroxyvitamin D Levels in man R. Kumar, T.J. Merimee, P. Silva and F.H. Epstein

1005

25-Hydroxylation of Vitamin D in the Elderly R.K. Skinner

1011

Intestinal Absorption of Radiophosphate After Physiological Doses of 1,25-Dihydroxyvitamin D in Normals and Pathological Conditions A. Caniggia and A. Vattimo

1015

Calcium Metabolism Following Proximal Gastric Vagotomy in Man D. Scholz, P.O. Schwüle, W. Engelhardt, C. Morcinietz

1019

25-Hydroxyvitamin D Metabolism in Nontropical Sprue S.B. Arnaud, A.D. Newcomer, K.P. Offord and V.L.W. G o

1023

Circulating Vitamin D Metabolites in A Hypoparathyroid Patient Resistent to Vitamin D E. Keck and H. v. Lilienfeld-Toal

1027

XXIII

The Ability of 1a, 25-Dihydroxycholecalciferol to Alter the Fatty Acid Composition of Phosphoglycerides in Rat Intestinal Mucosa and Smooth Muscle W. Alastair, M. Hay, A.G. Hassam, M.A. Crawford, P. Stevens, E.B. Mawer and Ch. Case

1031

Vitamin D Dependent Clinical Disorders

1035

Distinct Subgroups of Patients with Primary Hyperparathyroidism: 1,25(OH)2D3 Levels and Response to Phosphorous Therapy A.E. Broadus, R.L. Horst, R. Lang and H. Rasmussen

1037

The Role of 1-Alpha-Hydroxycholecalciferol in the Treatment of Pseudohypoparathroidism Type 1 J. Kovarik, M. Weissei, G. Neuwirth, P. Kreppler and R. Willvonseder

1041

The Effect of Oral 1 alpha Hydroxy Vitamin Ds and 1,25(OH)2 Da on the Intestinal Absorption of Calcium and Phosphate in Primary Biliary Cirrhosis Z. Varghese, O. Epstein, K. Farrington, S.P. Newman, J.F. Moorhead and S. Sherlock

1045

High Serum 1,25-(OH)2Ü in Acromegaly. Effect of Bromocriptine Treatment Bj. Lund, P.C. Eskildsen, Bi. Lund, A.W. Norman, P.A. Svendsen and O.H. Sorensen

1051

Hypoparathyroidism: Long-Term Treatment with Dihydrotachysterol, 25-Hydroxyvitamin D3 and 1,25-Dihydroxyvitamin D3 B. Argemi, J.P. Vagneur, J.L. Codaccioni and R. Simonin

1057

Treatment of Parathyroprival Tetany with 5,6-trans-25-OH-Cholecalciferol (Findings from 2 Case Reports) W. Cremer, H.-G. Grigoleit and H. Schmidt-Gayk

1061

Cardiomyopathy in Vitamin D Intoxication, Protective Effect of Diphosphonate and Nifedipine Against Myocardial Calcium Overloading H. Schäfer and K. Riesner

1069

Early Effects on Bone of 1,25-Dihydroxy-Vitamin D3 in Normal Subjects and and Paget's Disease Patients C. Gennari, R. Nuti, F. Lore' and M. Galli 1073 Controversies Concerning the Action of 1,25(OH)2D3 and 24,25 (OH)2Ü3 in Man J.A. Kanis, T. Cundy, D. Douglas, A. Andrade, G. Heynen, R. Smith and R.G.G. Russe 1

1077

Preliminary Report of Biological Actions of 24,25(OH)2D3 in Normal Man and Patients with Advanced Renal Failure A.S. Brickman, F.L. Llach and J.W. Coburn

1085

XXIV Oestrogens and Calcium Metabolism in Vitamin D Treated Hypoparathyroidism D. Verbeelen

1091

Bone Disease After Jejuno-Ileal Bypass for Obesity; Response to Oral 1 a-Hydroxyvitamin D3 Therapy J.E. Compston, L.W.L. Horton, M.F. Laker, A.B. Ayers, J.S. Woodhead, T.L. Clemens, L. Fraher, T.R.E. Pilkington

1095

Clinical Uses of 1,25-Dihydroxycholecalciferol R.G.G. Russell, D.L Douglas, T. Cundy and J.A. Kanis

1099

Bone Disease - Clinical Aspects

1109

Is there an Osteomalacia Component in Fractures of the Femoral Neck? C.H. Rapin, A. Jung, C. Barras, B. Courvoisier, H. Vasey and J.P. Junod

1109

Idiopathic Hypoparathyroidism: Refractory to Treatment with Vitamin D and its Metabolites D.S. Rao, B. Frame, M. Mathews, D.L Thomson, V. Matkovic and A.M. Parfitt

1113

Hypercalcemia and Hypocalciuria Associated with Calcifying Pancreatitis: Report of a Case A. Ulmann, M. Garabedian. G. Cournot, M. Rieu, R. Doumith, B. Lacour, A. Bourdeau, S. Balsan

1117

The Pathogenesis of Osteomalacia V. Papapoulou and P..D. Byers

1121

Calcium Deficiency Rickets Associated with Elevated 1,25Dihydroxyvitamin D Concentrations in a Rural Black Population J.M. Pettifor, F.P.. Ross, G. Moodley, H.F. DeLuca, R. Travers, and F.H. Glorieux

1125

Bone Changes of Primary Oxalosis in the Uremic Stage A. Poggi, C. Ciccarelli, D. Brancaccio, E. Bonucci and Q. Maggiore

1129

Immigrant Osteomalacia: Occurrence in Turkish Guest Workers in Germany G. Offermann and C. Manhold

1133

Treatment of Osteitis Deformans (Paget's Disease) with Synthetic Salm Calcitonin U.J. Schmidt, K. Trezenschik, K. Abendroth, H. Ansorg, H. Graner, I. Kalbe, K. Lindenhayn and G. Schramm

1137

Vitamin D-Dependent Rickets: A Case of Target Organ Resistance to 1a,25-Dihydroxyvitamin D and Impaired Vitamin D-25-Hydroxylase J.E. Zerweku and C.Y.C. Pak

1139

XXV

Serum 1,25-Dihydroxyvitamin D Concentrations in two Different Types of Pseudo-Deficiency Rickets S. Balsan, M. Garabedian, M. Lieberherr, J. Gueris and A. Ulmann

1143

Congenital Alopecia, Idiopathic or Pseudoidiopathic Hypoparathyroidism and End Organ Resistance to 1,25-(OH)2D3 - an Hereditary Congenital Syndrome U.A. Liberman, R. Samuel, A. Halabe, S. Edelstein, J. Weissmann and R. Kauli

1151

Treatment of Privational Late Rickets and Osteomalacia with the Vitamin D T.C.B. Stamp, W. Perra, S. MacArthur and M.V. Jenkins

1153

The Response of Bone to Oral Phosphate Salts , Ergocalciferol and/or 1a,25-Dihydroxycholecalciferol in Familial Hypophosphatemia F.H. Glorieux, J.P. Bordiert, P. Marie, R. Travers, E.E. Delvin and J.M. Pettifor

1163

Treatment of Mesenchymal Tumor Associated Osteomalcia with 1,25 (OH)2D3: Report of a Case D.F. Nortman, J.W. Coburn, N. Brautbar, D.J. Sherrard, M.R. Haussler, F.R. Singer, A.S. Brickman and R.T. Barton

1167

The Role of 1,25-Dihydroxyvitamin D in the Pathogenesis and Treatment of X-Linked Hypophosphatemic Rickets M.K. Drezner, K.W. Lyles, M.R. Haussler and J.M. Harrelson

1169

Bone Disease Associated with Total Parenteral Nutrition, a New Syndrome C.M. Targoff, J.W. Coburn, M.E. Ament, W.J. Byrne, D.G. Miller, D.J. Sherrard, A.S. Brickman and R. Bluestone

1171

Osteomalacia: Treated with 1a Hydroxy or 1,25 Dihydroxy Vitamin D M. Peacock, P.J. Heyburn, J.E. Aaron, G.A. Taylor, W.B. Brown and R. Speed

1177

Aetiology of Osteomalacia in Chronic Renal Failure and Nutritional Vitamin D Deficiency J.B. Eastwood, D. Memmos and H.E. de Wardener

1185

Bone Disease in Epileptics J.F. Dymling, O. Johnell, L. Lindgren, B.E. Nilsson, A. Wallöe and P.E. Wiklund

1193

Unanswered Questions in Renal Osteodystrophy

1199

Renal Osteodystrophy: Unanswered Questions and Problems J.W. Coburn

1201

XXVI

Pathogenesis of Secondary Hyperparathyroidism in Early Renal Failure: A Multifactorial System including Phosphate Retention, Skeletal Resistance to PTH, and Altered Vitamin D Metabolism S.G. Massry 1203 The Pathogenesis of Secondary Hyperparathyroidism in Early Renal Failure E. Slatopolsky, R. Gray, N.D. Adams, J. Lewis, K. Hruska, K. Martin, S. Klahr, H. DeLuca and J.Lemann 1209 The Renal Osteodystrophies: Clinical Features in Patients Classified by Histologic Features and Response to Treatment with I,25-Dihydroxyvitamin D3 J.W. Coburn, A.S. Brickman, D.J. Sherrard, F.R. Singer, E.G.C. Wong and A.W. Norman

1217

Long-Term Prophylaxis with High Doses of Vitamin D in Patients with Chronic Renal Insufficiency under Treatment with Low Protein Diet P.T. Fröhling, F. Kokot, J. Pietrek, K. Vetter, J. Kuska, I. Kaschube and W.D. Hohmann

1223

Problems of Vitamin D Nomenclature

1229

Vitamin D Nomenclature: A Chemist's Viewpoint W.H. Okamura

1231

Recommendations on Vitamin D Nomenclature H.F. DeLuca and H.K. Schnoes

1233

Observations on a Nomenclature System for the Vitamin D Series of Compounds D.E.M. Lawson

1243

Addendum

1245

Clinical Application of 1,24 (OH)2Ü3 H. Orimo and M. Shiraki

1247

Synthesis of Biologically Active Metabolites and Analoges of Vitamin D Modified in the Side Chain from Ergosterol N.A. Bogoslovsky, G.E. Litvinova, G.I. Samokhvalov

1257

Author Index

1261

Subject Index

1273

Chemistry, Structure, Function and Biological Assay

3

PROFESSOR E. HAVINGA: CONTRIBUTIONS TO VITAMIN D CHEMISTRY

The publication of the Proceedings of the 4th V i t a m i n D Workshop will virtually coincide w i t h Professor Havinga's retirement from the Chair of Organic Chemistry at the University of Leiden. In the context of this W o r k shop it is felt appropriate to present a short survey of Havinga's m a i n contributions to V i t a m i n D chemistry, although his retirement will certainly not put an end to his enthusiastic interest in the exciting adventures of this substance in chemistry as well as in biochemistry and medicine. Born in Amersfoort, the Netherlands, in 1909 Havinga received his academic training at the University of Utrecht. His Ph.D. thesis was concerned w i t h "Monolayers; Structure and Chemical Reactions"

(1939) (promo-

tor: Professor F. Kogl). After having served on the Faculty of Veterinary Sciences in Utrecht (Laboratory of Medicinal Chemistry) he became Professor of Organic Chemistry at the University of L e i d e n in 1946. Apart from V i t a m i n D chemistry H a v i n g a has b e e n active in a number of other areas of research. Perhaps best k n o w n are his contributions to stereochemistry (spontaneous formation of chiral substances, conformational lysis of non-aromatic ring compounds) and photochemistry

substitution, photoreactivity of conjugated dienes and trienes).

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

ana-

(aromatic p h o t o -

k He entered the V i t a m i n D field in the early 1950's w i t h an investigation aimed at exploring the pathways along w h i c h the v i t a m i n exerts its biological activity. It soon became clear, however, that apart from analytical problems the deficiencies in the knowledge of the chemistry of V i t a m i n D w e r e so m a n y at that time as to virtually exclude progress in the study of its biochemical fate. A t t e n t i o n therefore focussed on the sequence of events leading from the provitamin to the vitamin. This study soon expanded into a very broad and general investigation of the photochemical and thermal reactions of V i t a m i n D and its isomers, carried out in close and cordial cooperation w i t h chemists at Philips-Duphar B.V. In c h r o n o logical order the major achievements m a y be listed as follows: 1955-1964: Sequence of reversible photochemical and thermal

isomeri-

zations; central position of p r e v i t a m i n D 1955: Structure of previtamin D, relation to tachysterol;

formation

and structure of trans-Vitamin D 1957: Synthesis of

dihydrotachysterol

1960: F o r m a t i o n and structure of

cis-isotachysterol

1961: Hint to importance of orbital symmetry in thermal and p h o t o chemical reactions in the V i t a m i n D field 1964: Transition state geometry in thermal interconversion of p r e v i t a m i n D and V i t a m i n D; role of ring D in equilibrium 1970: Photosensitized interconversion of tachysterol and p r e v i t a m i n D 1972: Overirradiation products of V i t a m i n D: suprasterols 1977: Overirradiation products of p r e v i t a m i n D/tachysterol:

toxisterols;

role of ground state equilibrium in photoreactions of V i t a m i n D and its isomers 1979: Photosensitized interconversion of V i t a m i n D and trans-Vitamin D Havinga has authored or coauthored about 40 papers on V i t a m i n D and related subjects. Review papers are listed in references

(1) to (4).

References (1) Havinga, E., and Schlatmann, J.L.M.A. (1961) T e t r a h e d r o n ^ , 146-152 (2) Sanders, G.M., Pot, J., and Havinga, E. (1969) Fortschr.Chem.0rg. Naturst. 2_7, 131-157 (3) Havinga, E. (1973) Experientia 29_, 1181-1193 (4) Jacobs, H.J.C., and Havinga, E. (1979) Adv.Photochem. _1_1_, in press H.J.C. Jacobs and J. Lugtenburg University of Leiden, The Netherlands

5 THE VINYLALLENE APPROACH TO la-HYDROXYVITAMIN D ANALOGS*

William H. Okamura, Patrick Condran, Jr., Milton L. Hammond, Antonio Mourino and Alan W. Messing Department of Chemistry, University of California, Riverside, California 92521, USA Classical steroid methods have been used by this laboratory for synthesizing two analogs which differ only at the 3-position from la,25(OH)2-D3, namely 3-d-la,25-(OH)2-D3 and 3-d-3a-Me-la,25-(OH)2-D3 (1,2). Several related analogs without the 25-OH group have also been synthesized by various laboratories and these include 3-d-la-OH-D3, 3-d-3a-Me-la-OHD3 and la-OH-3-epi-D3 (2,3) (Figs 1-2). The classical approach is a linFIGURE 1. Vitamin D3 [D3, Rx=R2=H] and its principal metabolites: 25-hydroxyvitamin D3 [25-OH-D3, Ri=OH, R2=H] and la,25-dihydroxyvitamin D3 [la,25-(OH)2D 3 , R 1 =R 2 =OH].

FIGURE 2. Analogs of la,25-(OH)2-D3: laHydroxyvitamin D3 [la-OH-D3, R^=OH; R2=R3=H], 3-deoxy-la,25-dihydroxyvitamin D3 [3-d-la, 25-(OH)2-D3, R]_=R2=H; R3=0H] , 3-deoxy-lahydroxyvitamin D 3 [3-d-la-OH-D3, R1=R2=R3=H], 3-deoxy-3a-methyl-la-hydroxyvitamin D3 [3-d-3a-Me-la-OH-D3, R I = R 3 = H ; R 2 =CH 3 ], 3deoxy-3a-methyl-la,25-dihydroxyvitamin D3 [3-d-3a-Me-la,25-(OH)2-D3, Ri=H; R 2 =CH 3 ; R3= OH], la-hydroxy-3-epivitamin D3 [la-OH-3epi-D3, R1=R3=H; R2=OH]. ear synthesis, however, and two late synthetic operations (introduction of the A 5 ' 7 -diene usually by a bromination-dehydrobromination scheme; the photochemically induced electrocyclic ring opening of the A 5 » 7 -diene to the previtamin form) are usually inefficient. The greater efficiency and generality of a convergent synthesis, as opposed to a linear one, is well known to synthetic organic chemists (4). By a convergent vitamin D synthetic scheme, we mean one where large fragments (A ring and the C/D/sidechain)are produced separately and then joined to produce the target skeleton at the last possible stage. Our goal was to develop a general scheme for preparing la-hydroxylated analogs of la,25-(OH)2-D3 (with and without the 25-OH group) modified at the 3-position.

*This paper is dedicated to Professor E. Havinga, University of Leiden, the Netherlands, on the occasion of his retirement, 1979.

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

6 The conversion of cis-alkyl vinylallenes to (3Z)-1,3,5-hexatrienes (Fig 3) via a thermally induced suprafacial [1,5]sigmatropic hydrogen shift is a known reaction (5-7). The 3Z-l,3,5-hexatriene stereostructural unit can be found imbedded in a number of natural products including vitamin D, 11-cis-retinal and various carotenoids and pheromones.

»-A

V

FIGURE 3. [1,5]-Sigmatropic Rearrangement of Vinyallenes.

[l,5] - ilgmatrepfc »hltt (•ufrofocloO

The studies of Dauben (8) and Havinga (6,9) have led to the identification of 6 photoproducts of vitamin D. Among these are a pair of minor but isolable vinylallene stereoisomers I and II (Fig 4)(6,9). When either allene in the form of their silyl ether was examined by gas chromatography at 225°, three major peaks appeared. Two of these had the same retention times as the silylether of vitamin D when it was subjected to gas chromatography under identical conditions. It is known that vitamin D affords two substances, isopyrocalciferol and pyrocalciferol, upon heating above 150°C. The possibility that vitamin D is an intermediate in the pyrolysis Vinyl

Alienes

(Havinga)

Isopyrocalciferol

4-

f

.u

Vitomin D s-trans)

*

Pyrocalciferol

ll+-trienes, which differ in configuration only at Cio, thermally equilibrate with one another. Thus, as exemplified for the 1R,10R and the 1R,10S pair, a simple mechanistic pathway involving FIGURE 12. The putative 5Z,7Zvitamin can be envisioned to undergo two consecutive [1,7]sigmatropic shifts through the intermediacy of the 1-hydroxyform of the known cis-isotachysterol3 to afford the observed ¿5,7,1't-trienes.

.5,7,14 A -triene only [1,7]-sigmatropic shifts can be invoked which rationalizes this result (see Fig 13). This equilibrium process is observed for the 1S,10R and 1S,10S pair as well and the results nicely support the mechanistic hypotheses described in Figures 11 and 12. The original synthesis of 3-d-la-OH-D3 from cholesterol required 11 steps (0.2% overall yield). The present convergent synthesis using the cuprate conjugate addition procedure (see also Figs 6-8) involves 6-7 steps giving a 0.7% overall yield from readily available vitamin D3. As an added bonus, the analog 3-d-lf5-OH-D3 is obtained in 3% overall yield in 6 steps from vitamin D3. By incorporating the modified procedure given in Figure 9 the yields have been raised to 3% (3-d-la-OH-D3) and 7% (3-d-lfJOH-D3). The vinylallene scheme which will likely improve on further ex-

11 5 7 11+ FIGURE 13. Pairs 1 or — — — of A —' ' -trienes ^ 3 only 1' 2 or

were delayed after dosing with the fluoro-analog relative to indicated

that la-0H-25-F-D 3 was metabolized prior to manifesting

ical activity. action step.

biolog-

24R-Hydroxylation was the reasonable choice for the activEvidence for this was provided by the evaluation of

hydroxy-25-fluorovitamin D 3

Fluorination and deacetylation of 24R,25-(OH)2D 3 24R-0H-25-F-D 3 which was pure by HPLC analysis M. Uskokovic first prepared

resolution mass spectra

3,24-diacetate

personal

The ultraviolet absorbance

(X

max

265 nm),

(6 1.34, 1.33, 2d, J = 22 Hz each), and

( M + 418.3264) confirmed

the structural

There was a 530 fold difference between l o i , 25-(0H) ^ in the competitive-binding assay, which placed the activity range of 24R-0H-D 3 and 25-0H-D 3

and

(6).

24R-OH-25-F-D 3

This again

( 0 H ) 2 D 3 is less active in the assay than either of the two

high-

assignment.

this fluoro-analog

that the fluorine substituent was behaving as a proton since

monohydroxy compounds.

gave

(8) (J. Partridge and

this compound by another route,

communication to H. F. DeLuca). ' nuclear magnetic resonance

24R-

(24R-0H-25-F-D 3 ).

in

indicated 24R,25-

side-chain

The 24-hydroxyl group was also confirmed as R

since a 24S hydroxy group would have substantially lowered

activity.

24R-OH-25-F-D 3 was quantitatively and qualitatively similar in all respects to 25-OH-D, in mediating calcium homeostasis in vitamin D-deficient

rats.

k7 The two compounds w e r e equipotent in ability to stimulate BCM, ICT, and rachitic cartilage calcification. 24R-OH-25-F-D 3 w a s la-hydroxylated.

This suggested that, like 25-OH-Dj, Evidence for the

la-hydroxylation

of 24R-OH-25-F-D^ was obtained from two different experiments.

The t i m e -

courses of BCM in response to doses of either 25-OH-D 3 or 24R-0H-25-F-D 3 were nearly identical.

In other words, there was a n 18 to 24 hour lag

between dosing and observation of maximum serum calcium concentrations. Morover, bilateral nephrectomy abolished the ability of low doses of 25-OH-D 3 and 24R-0H-25-F-D 3 to stimulate BCM and ICT.

It appeared in-

escapable that the active form of 24R-OH-25-F-D 3 , w a s actually dihydroxy-25-fluorovitamin D 3

(la,24R-(OH)2-25-F-D3>.

Since

la,24R-

24R-OH-25-F-

D 3 was as potent as 25-OH-D 3 it follows that l a , 2 4 R - ( O H ) 2 ~ 2 5 - F - D 3

would

be as potent as la,25-(OH)2^3-

These experiments indicated that both la-OH-25-F-D 3 and 24R-OH-25-F-D.J were converted to the common metabolite l a , 2 4 R - ( 0 H ) 2 - 2 5 - F - D 3 before they acted.

To examine this question, and to exclude the possibility that

the fluoro-analogs were defluorinated in vivo and hydroxylated to la,25(OH)2 D 3»

the following experiment was done (9).

Vitamin D-deficient

rats (three to four per group) were dosed w i t h ethanol, l a - O H - D 3 > 0H-25-F-D 3 , or la-OH-25-F-D

.

24R-

Five hours later, their plasma was 3

collected and tracer l a , 2 5 - ( O H ) 2 [ 2 3 , 2 4 - H ] D 3 was added.

The plasma

samples were extracted w i t h methylene chloride and the soluble material from each group w a s chromtographed separately on Sephadex LH-20 columns (0.9 x 7 cm) and a broad fraction centered around the elution profile was collected.

la,25-(OH)2D3

The samples were then individually

chromatographed through a semi-preparative HPLC column (0.7 x 25 cm) that had b e e n standardized, immediatly before the plasma samples were chromatographed, w i t h l a , 2 4 R - ( O H ) 2 D 3 and la,25-(OH)¿V^.

Fractions

(1.5 ml each)

were collected across the la, 24R- (OH)2 D 3 and the la, 25-(OH) 2I>3 elution profiles.

The 17 fractions from each group were divided and m e a s u r e d

for radioactivity and also for

la, 25-(OH) 2 D 3 ~ l i k e binding

lents by the radioreceptor-binding assay. are expressed as pg/fraction/ml plasma (• of added tracer (0

0).

equiva-

The results shown in the figure •) and dpm/fraction/10,000

In the standarization run, the profile of

dpm

5

10

Fraction

15

5

number

10

(1.5 ml each)

la,24R-(OH)2D3 had peaked in fraction 6, and that of la,25-(OH)2D3 had peaked in fractions 12 and 13.

The animals dosed with ethanol (A) showed

a residual amount of la, 25-(OH) 2 D 3

co_m

ig r a t ing with tracer la, 25-(OH)

The animals dosed with la-OH-D^ (B) showed a greatly increased amount of l a , 2 5 - ( O H ) a n d no material in the l a , 2 4 R - ( O H ) a r e a .

On the other

hand, the animals dosed with either 24R-OH-25-F-D.J (C) or la-0H-25-F-D3 (D) did not have la,25-(0H)2^^ levels greater than the ethanol dosed rats, but did have high levels of metabolites which co-migrated with la,24R(OH)2D3.

Introduction of a 25-fluoro substituent into la,24R-(OH)fl^

would not affect its chromatography.

Additionally, la,24R-(OH)2D3, but

not la,24S-(OH)2D3, binds as well as la,25-(0H)2D3 to the intestinal binding protein used in the assay (unpublished results).

The compounds

in C and D, which co-migrated with la,24R-(OH)2D3 was, therefore, most likely la,24R-(OH)2-25-F-D3.

.

49 These studies support the conclusions that the 25-hydroxy group is not an absolute requirement for vitamin D^ action.

When 25-hydroxylation is

prevented, a 24R-hydroxy group can apparently initiate la-hydroxylation and confer potent calcium-metabolism mediating activity on vitamin D compounds.

They also demonstrate, as was done previously, that la-OH-D^ is

converted to la,25-(OH)2D3 in vivo; and further show that la-OH-D-j is not converted to la, ZAR-^H^DJ in vivo.

Finally, the existence of a vitamin

D^ analog, la,24R-(OH)y-lS-F-D^, as nearly potent as the hormone la,25(OH^DJ has been demonstrated REFERENCES 1.

Kumar, R., Harnden, D., and DeLuca, H. F. (1976) Biochemistry 15, 2420-2423.

2.

Ikekawa, N., Morisaki, M., Koizumi, N., Sawamura, M., Tanaka, Y., and DeLuca, H. F. (1975) Biochem. Biophys. Res. Commun. 62, 485-491.

3.

Tanaka, Y., DeLuca, H. F., Akaiwa, A., Morisaki, M., and Ikekawa, N. (1976) Arch. Biochem. Biophys. 177, 615-621.

4.

Kawashima, H., Hoshima, K., Hashimota, Y., Takeshita, T., Ishimoto, S., Noguchi, T., Ikekawa, N., Morisaki, M. and Orimo, H. (1977) FEBS Lett. 76, 177-181.

5.

Napoli, J. L., Fivizzani, M. A., Hamstra, A. H., Schnoes, H. K. DeLuca, H. F., and Stern, P. H.

(1978) Steroids 32, 453-466.

6.

Eisman, J. A. and DeLuca, H. F. (1977) Steroids 30, 245-257.

7.

Napoli, J. L., Fivizzani, M. A., Schnoes, H. K., and DeLuca, H. F. (1978) Biochemisty 17. 2387-2392.

8.

Napoli, J. L., Mellon, W. S., Fivizzani, M. A., Schnoes, H. K., and DeLuca, H. F.

9.

(1979) J. Biol. Chem. (in press).

Napoli, J. L., Mellon, W. S., Fivizzani, M. A., Schnoes, H. K., and DeLuca, H. F. (1978) Fed. Proc. (abstract).

51 19-SUBSTITUTED-lO,19-DIHYDROVITAMINS VIA HYDROZIRCONATION OF VITAMIN D3. Alan W. Messing, Frederick P. Ross, Michiko Miura, Anthony W. Norman and William H. Okamura Department of Chemistry, University of California, Riverside, California 92521 USA Earlier studies from this laboratory have established the stereochemistry of each of the four stereoisomeric 10,19-dihydrovitamins. These are the (5Z)-(10S) (5Z)-(10R) (5E)-(10S) £ and the (5E)-(10R) 2Zr(H)C1, abbreviated Zr-H] was found to be operationally simpler and faster than the analogous hydroboration procedures used earlier (1). Typically the steroid was mixed with two or three equivalents of Schartz's reagent, dry benzene was added under an argon atmosphere and the mixture stirred at 40°C for one hour (5) (Fig 2). The reaction is highly regioselective but, like hydroboration, is not stereoselective and produces uniformly good yields of 10R and 10S dihydrovitamins on protonation. A very interesting result is that 5Z->-5E

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

52

isomerization occurs during the hydrozirconation-protonation reaction. Significantly, the four stereoisomeric dihydrovitamins 3, 4, 5 and 6 are recovered unchanged upon being subjected to conditions more severe than the conditions used for their preparation. Moreover, there is no evidence of any £ in reactions of and vice versa (5) . Schwartz found that the alkyl zirconium adducts formed by hydrozirconation could be converted to a wide variety of derivatives by treatment with an appropriate electrophilic reagent. We have prepared the 19-chloro, 19-iodo and 19-carboxaldehydes shown in Figure 3. In these reactions 5zj5E isomerization does occur and all four possible dihydro-

vitamin derivatives are detected. The major A 5 stereoisomers, however, are those which retain the original A 5 stereochemistry of the starting triene (1 or 2). Bromination of the alkylzirconate with N-bromosuccinimide has been attempted and appears to lead to a 19-bromo derivative which decomposes on work up. Preliminary bioassays on four of the 19-halodihydrovitamins have been completed and are reported in Table 1. The 19-fluoro dihydrovitamins are currently inaccessible via the hydrozirconation route. The analogs in the fluoro series are exception-

53 AGONIST

5Z-IOS

ANTAGONIST

+

5Z-10R

5Z-IOS

Table 1

+

5Z-IOR H

ally interesting by analogy to the wide variety of biologically active fluorosteroids which have been reported (6). In an entirely different approach, treatment of the readily available 19-hydroxyvitamin D3-3benzoate (both 5Z-10S and 5Z-10R) , with diethylaminosulfur trifluoride (7) (Fig 4), led not to the 19-fluorovitamin, but to the 19-nor-A-homo analogs ^ ^ and (tentative structural assignment based on NMR) . These analogs, after hydrolysis to remove the benzoyl group, are currently undergoing bioassay.

Figure 4. Stereochemistry relative to the starting 10R and 10S dihydrovitamins is as yet unknown. !§ä. Ri»F f?2,H 16g R,.H R2-F We are proceeding with investigations into further reactions of ^ and 8 as well as interesting synthetic possibilities afforded by 13 and Acknowledgments. We are grateful to Professor Michael F. Rettig for helpful discussions and to the National Institutes of Health (USPHS Grants AM-16595 and AM-09012) for generous support of this work. F.P.R. acknowledges the South African Council for Scientific and Industrial Research for a Scholarship. The crystalline vitamin D3 was a generous gift from Dr. M. Rappoldt of Philips-Duphar (Weesp, the Netherlands).

54 (1)

Okamura, W. H., Hammond, M. L., Rego, A., Norman, A. W., Wing, R. M., (1977) J. Org. Chem., 42, 2284-2291. See also Mourino, A. and Okamura, W. H., (1978) ibid., 43, 1653-1656.

(2)

Norman, A. W., Johnson, R. L., Osborn, T. W. , Procsal, D. A., Carey, S. C., Hammond, M. L., Mitra, M. N., Pirio, M. R., Rego, A., Wing, R. M., Okamura, W. H., (1976) Clin. Endocrinology, 5_, Suppl-, 121s-143s.

(3)

Hammond, M. L., Mourino, A., Blair, P., Wecksler, W., Johnson, R. L., Norman, A. W., Okamura, W. H., (1977) Proc. Third Workshop on Vitamin D, Asilomar, Pacific Grove, California, USA.

(4)

Schwartz, J-, Labinger, J. A., (1976) Angew. Chem. Int. Ed. Engl., 15, 333-340 and references cited. The Zr-H used in this study was commercial material (Alfa-Ventron Corp.). Schwartz's reagent was first studied by Wailes and co-workers: Kautzner, B., Wailes, P. C., Weigold H., (1969) Chem. Commun., 1105. Wailes, P. C., Weigold, H., Bell, A. P., (1972) J. Organometal. Chem., 43, C32.

(5)

Messing, A. W., Ross, F. P., Norman, A. W., Okamura, W. H., (1978) Tet. Lett., 39, 3635-3636.

(6)

Schlosser, M., (1978) Tetrahedron, 3±, 3-17.

(7)

Middleton, W. J., (1975) J. Org. Chem., 40, 574-578.

55 THE SYNTHESIS OF la, 3(3-BIHTDROXY-CHOLESTA-5,7-DIEN-24-OIC ACID, AND OF la,3a-DIHYDHOXY-25-METHYlCHOLESTA-5,7-DIENE.

J.M. Midgley, E.M. Upton, E. Watt and W.B. Whalley School of Pharmacy, University of London, London, England. la,25-Dihydroxycholecalciferol is now recognised (l) as one of the major, biologically active metabolites of Vitamin D y

Among the many

investigations concerning the metabolic degradation of the side-chain one (2) appeared to be of particular potential significance, namely that within 4 hours of administration of la, 25-dihydroxy-[26,27-^C]Vitamin D^ (i) to vitamin D-deficient rats, more than 2^% of labelled was expired.

This result may mean (a) that the side-chain

oxidation resulting in the liberation of "^COg is a significant metabolic step in the formation of a biologically active derivative of the vitamin, or (b) that the degradation represents a normal mode of degradation as part of an excretion and control mechanism.

(I)

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

(II)

56 It seemed to us that the immediate product(s) from elimination of Cgg and Cg^ would fee the Cg^ (II) or Cg^-oic acid(s).

We have thus

synthesised la,3P-dihydroxycholesta-5,7-dien-24-oic acid (ill), with a view to its conversion into the corresponding derivative of vitamin D.

CHO

(III)

(IV)

CO^M*.

(v)

(VI)

(P

eoJJ

(VII)

57

KO

Scheme 1

58 Thus, the C-22 aldehyde (IV) obtained (3) reproducibly in 60% yield by the ozonolysis of 5,6-dihydroergosteryl acetate was condensed with the ylid from carbomethoxy-methylenetriphenylphosphonium bromide to yield (up to 85%) the ester ( v ) .

Hydrogénation of (V) gave the saturated

side-chain of the C-24-oic acid (VI).

The sequence of reactions

previously developed (4) for the preparation of la-hydroxycholecalciferol (Scheme l) from the 7-dehydro-steroids gave the desired ester which was saponified to the acid (VIl). In our second area of work in this field we have modified the sidechain of the final cholecalciferol nucleus with the object of inhibiting hydroxylation at C-25, and thus providing a potential antimetabolite which might be useful in therapy.

Previous publications (5) from other

groups have already recorded the synthesis of 25-fluoro-, 25-aza- and 24,25-dehydro-vitamin D,.

59 Our means of blocking 25-hydroxylation is the introduction of a methyl group at C-25, involving the initial synthesis of la-hydroxy-25-methylcholesta-5»7-diene (VIII). The starting point for this synthesis was the C-22 aldehyde (IV) used for the first synthetic objective. Condensation of this aldehyde with 3,J-dimethylbutyltriphenylphosphonium bromide gave the 22,23-unsaturated derivative (IX). This Wittig reaction required careful control of the experimental conditions, especially the rigorous purification of the phosphonium sait by crystallisation from benzene/acetone.

Hydrogénation of the unsaturated

side-chain in (IX), (which did not always proceed smoothly) gave the acetate of 25-methylcholes-7-ene-3|3-ol which was converted, by the established reaction sequence (Scheme l) into the requisite la, 3(3dihydroxy-25-methylcholesta-5,7-diene. A group of other obvious, potential antimetabolites, such as the 25-aza derivative are in the process of synthesis.

60 eeferences 1.

Georghiou, P.E. (1977) Chem.Soc.Hev. 6, 85-102.

2.

Harnden, D, Kumar, R, Holick, M.F., and DeLuca, H.F. (1976)

Science, 12^, 493-494. 3.

Maclean, D, Strachau, V.S., and Spring, P.S. (1933) Chem. and Ind.,

21, 1259-1260. 4.

Hnke, A., Hands, D., Midgley, J.M., Whalley, V.B. and Ahmad, R.

(1977) J.Chem.Soc. Perkin I, 820-822. 5.

Onisko, B.L., Schnoes, H.K., and DeLuca, H.P. (1977) Tetrahedron

Letters, 1107-1108; Yang, S.S., Dorn, C.P., and Jones, H. (1977) Tetrahedron Letters,

2315-2316.

61 INTERCONVERSION OF VITAMIN D AND TRANS-VITAMIN D BY TRIPLET-SENSITIZED ISOMERIZATION

H.J.C. Jacobs, J.W.J. Gielen and E. Havinga Gorlaeus Laboratories, University of Leiden Department of Organic Chemistry, P.O. Box 9502, 2300 RA Leiden The Netherlands The photoreactions of Vitamin D and related dienes and trienes (1), x induced by direct irradiation in the region of the i t - i t absorption band, are generally believed to start from the lowest excited singlet state, intersystem crossing to the triplet state being improbable in view of the large energy separation (^60 Kcal/mole or ^250 kJ/mole) of these states [2]. Experimentally it has been shown [3] that the well-known photocyclization reactions of previtamin D^, occurring upon direct irradiation, to yield 7-dehydrocholesterol and lumisterolg, cannot be brought about by excitation of suitable triplet sensitizers.

In contrast cis/trans-isomerization

of previtamin D to tachysterol and vice versa does occur both upon direct and upon sensitized irradiation, be it with considerably different quantum yields (3).

Two more studies of the sensitized interconversion of previ-

tamin D and tachysterol have recently been reported (4,5). Similar studies of triplet reactions of other dienes and trienes in the Vitamin D field seem not to have been undertaken.

The well-known dye-

sensitized oxidative dimerizations of 7-dehydrocholesterol tor ergosterol) and pyrocalciferol probably do not proceed via the triplet excited state of the dienes but via an entirely different mechanism (1). In this contribution we report the results of an investigation into the triplet-sensitized photoreactions of Vitamin D and its 5E-isomer, trans-Vitamin D.

Apart from its intrinsic photochemical interest this stu-

dy was also initiated because of the potential preparative value of triplet-sensitized interconversion of Vitamin D and trans-Vitamin D. -3

Benzene solutions of Vitamin D 3 or trans-Vitamin D 3 0 2 . 5 x 1 0

M),

purged with nitrogen and containing a five-fold amount of sensitizer, were

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

62

light, using Philips TL 8W/33 lamps. range from ^30 to ^80 Kcal/mole.

The energies of the sensitizers used

The temperature was Kept at 5° C.

The

irradiation was s t o p p e d after reaching the photostationary state. The mixtures were analysed by TLC and GC C25 m capillary columns coated with SE30), the latter method permitting quantitative detection of the components without derivatization [6).

In blank experiments, omitting the sensitizer,

no conversion of either Vitamin D or trans-Vitamin D could be observed. TLC showed the presence of only Vitamin • and trans-Vitamin D in the irradiation mixtures, provided the interference of oxygen was rigorously excluded.

A given sensitizer (except perylene) was found to yield iden-

tical photostationary states starting from either Vitamin D or trans-Vitamin D.

Quantitative results are summarized in Table I and displayed

graphically in Figure 1.

High-energy sensitizers (E.^ 53 kcal/mole) appear

to give stationary states composed of 60% Vitamin • and 40% trans-Vitamin D.

On lowering the energy the amount of Vitamin D gradually increases: at

E^. ^42 kcal/mole selective one-way isomerization is achieved.

At still

lower triplet energy isomerization again occurs in both directions, the photo-stationary state composition showing 55 to 65% of trans-Vitamin D.

Direct irradiation of Vitamin • produces only trace amounts, if any, of trans-Vitamin D (7,8).

The facile formation of this compound from trip-

let excited Vitamin D sugges t s that S^ - T^ crossing following direct irradiation is negligible, in accordance with expectation.

Furthermore, the

absence of suprasterols from the mixtures obtained by sensitized irradiation indicates that the rapid formation of these compounds by direct irradiation of Vitamin D (7) occurs through the singlet excited state.

Note-

worthy is the exclusive isomerization of the central double bond (5-6) of the triene system: no indication of configurational change of the terminal

63 Table 1.

Effect of sensitizer energy on the photostationary state composition of Vitamin D/trans-Vitamin D

Sensitizer

E

T (Kcal/mole)

Acetone Acetophenone Benzophenone Fluorenone Benzanthrone Acridine Phenazine Anthracene Sensitox Perylene Tetracene

% Vitamin D

78 74 68 53 47 45 44 42 39 35 29

%TransVitamin •

62 38 58 42 58 42 62 38 87 13 95 5 95 5 98 2 36 64 very slow conversion 46 54

Rose Bengal bound to a polymeric support (Hydron Laboratories) CIS /

TRANS

50-

40

30

20-

Figure 1. Variation of stationary state composition in photosensitized isomerization of cis- and transVitamin • with various sensitizers

10

20

40

60 Et

80 (KCAL / MOLE)

bond (7-83 has been observed. From the data the triplet energies of trans- and cis-Vitamin • may be estimated to be M 2

and ^50 kcal/mole, respectively.

The occurrence of

isomerization at energi e s below 40 Kcal/mole contrasts with the behaviour of previtamin D and tachysterol (3,5), and is reminiscent of the case of

64 cis- and trans-stilbene (9) • From a preparative point of view the results are promising. By suitable choice of sensitizer the position of the photostationary state can be arbitrarily fixed between M 0 0 / 0 and ^35/65 for cis- and trans-isomer, respectively.

The selective one-way isomerization of trans-Vitamin D at

intermediate triplet energies provides a highly efficient final step in the classical total synthesis of Vitamin • (10), and has potential applicability in the preparation of hydroxylated derivatives and the like. Conversely, the facile geometric isomerization of Vitamin D with Sensitox is recommended as a convenient way of preparing trans-Vitamin D (6,11). When oxygen is insufficiently excluded an additional reaction appears to take place irrespective of the type of sensitizer used.

The product

proved to be a pair of epimeric epidioxides I (MS, H-NMR, C-NMR), the yietl of which can be enhanced considerably by passing a stream of oxygen throigh the solution (6).

Clearly, the epidioxides result from reaction with pho-

tochemically generated singlet oxygen.

Observation of the photoinduced

generation of the epidioxides with time and experiments with chemically generated singlet oxygen suggest that the oxygen adducts are formed from trans-Vitamin D and not from Vitamin • (6,12).

References (1) For a recent survey, see: Jacobs, H.J.C., and Havinga, E. (1979) Adv.Photochem. in press (2) Minnaard, l\l. G . , and Havinga, E. (19 73) Reel.Trav.Chim.Pays-Bas 32, 1179 -1188 (3) Snoeren, A.E.C. , Daha, M.R., Lugtenburg, J., and Havinga, E. (1970) Reel .Trav.Chim.Pays-Bas 89_, 261-264 (4) Eyley, S.C., and Williams, O.H. (1975) J.C.S. Chem.Comm. 858 (5) Denny, M., and L i u , R.S.H. (1978) Nouveau J. de Chimie 2, 637-641 (6) Gielen, J.W.J. (1979 ) Thesis Leiden, to be published (7) Bakker, S.A. , Lugtenburg, J., and Havinga, E. (1972) Reel.Trav.Chim. Pays-Bas 91_, 1459-1464 (8) Ctermet-Bouvier, R. (19 73) Bull. Soc.Chim.France 3023-3026 (9) Saltiel, J. et al. (19 73) Organic Photochemistry 3, 1-113 (10) Inhoffen, H.H., Quinkert, G., Hess, H.J. and Hirschfeld, H. (1957) Chem.Ber. 90, 2544-2553 (11) Cf. Barrett, A. G . M , Barton, D.H.R., Johnson, G., and Nagubandi, S. (1978) Synthesis 741 (12) After completion of our experiments a paper appeared describing the formation and structure elucidation of analogous epidioxides in the Vitamin D 7 series: Yamada, S., Nakayama, K., and Takayama, H. (1978) Tetrahedron Lett. 489 5- 489 8

65 THE USE OF UV, CD, 2 H-NMR SPECTROSCOPY AND MASS SPECTROMETRY FOR THE INVESTIGATION OF CONFORMATIONAL MOBILITY OF VITAMIN D AND PREVITAMIN D AND THEIR INTERCONVERSION E. Berman, N. Friedman, Y. Mazur, M. Sheves, and Zeev V.I. Zaretskii Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot, Israel A remarkable feature of vitamin D and previtamin D is the conformational mobility which distinguishes them from their biogenetic precursor, 7-dehydrocholesterol and other steroids (1,2). The thermal interconversion of previtamin D to vitamin D necessitates the presence of steroidal-like conformations which however, due to steric interaction are energetically unfavourable (Fig. I). This conformational bias suggests a low torsional energy barrier to rotation around the 6,7- and 5,6-single bonds in vitamin D and previtamin D respectively.

4Ì

6

In the following presentation we shall submit experimental evidence for this flexibility, using ultraviolet (UV) and circular dichroic (CD) spectroscopy indicating that both compounds have preferred non-planar conformations in which their triene systems are twisted in specific directions. In addition, using mass spectrometry and 2 H-NMR , we shall show that the transition state for the previtamin D, vitamin D thermal isomerisation has a similar preferred conformation. We have measured the UV spectra of vitamin D at variable temperatures and compared them with the spectra of its 6-methyl analog (3), and observed a small red shift at low temperature for the former and a larger one for the latter. (Fig. II). A different UV temperature dependence is observed in previtamin D which show a larger red shift at low temperature than its 6-methyl analog (Fig. III).

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

66 Temperature dependence of UV spectra

X (nm) max v '

rb 3r

+25°

-180°

264

268

AX(nm) +4

Fig. II

X (nm) max

240

261

+21

+25° -180° X (nm) max '

HO

259

268

AX(nm) +9

Fig. Ill H,c

X (nm) max v 1

250

251

+1

HO'

This temperature dependence of vitamin D, and its 6-methyl derivative may be compared to that of stilbene and its ortho-methyl derivatives respectively (4). The changes in the UV spectra in stilbene system were interpreted by assuming a steric crowding in the planar conformation which is relieved by twisting one of their single bonds. The twist in the hexamethylstilbene was calculated to be ca 50 and in stilbene ca. 5-10 . A red shift was observed in stilbene derivatives at low temperature which was interpreted by the decrease of their twist angles (4). Similar explanation may be put forward for the UV change's in vitamin D and its 6-methyl derivative.

67 The UV-temperature dependence of previtamin D and its 6-methyl analog may be compared to that of 11-cis-retinal and its 14-methyl derivative respectively (5). The 11-cis-retinal is twisted around its 12,13-single bond by ca. 40° and the red shift observed at low temperatures indicates its tendency to become planar. The analogous twist in the 14-methyl-llcis-retinal is larger (ca. 90°). However the steric effect of the 14-methyl group hinders its from becoming planar freezing the twisted conformation. In analogy it may be assumed that previtamin D has also a non-planar conformation around.his 5,6-single bond. Introduction of methyl at C-6 increases the twist angle but also prevents the molecule from changing this conformation. Additional evidence for the flexibility of trans-diene systems in vitamin D, previtamin D and their 6-methyl derivatives may be obtained from their CD spectra which show temperature dependence (Figs. IV and V).

possessing instead of the conjugated triene system, a conjugated diene system. These compounds show temperature independent and identical UV spectra, however their CD spectra (whicjj at room temperature show a low Cotton effect) change dramatically at -100 , giving a bisignate effect, whose amplitude steadily increases with temperature decrease (Fig. VI). We have also measured the CD spectra of analogous trans-dienes lacking a methyl group in ring A, and have found that their low intensity Cotton effect is unchanged down to -180° (Fig. VII).

68

69 Temperature effect in CD R

R

HO-" CD

'OH O)

CD (-)

CD (-) No temperature effect in CD R

Fig.VII

R

In order to interpret these results we postulate that all the transdienes investigated oscillate at room temperature about their 6,7 single bond, but only those having a methyl group in ring A assume at low temperature a conformation, in which the double bonds are twisted in one specific direction. This chiral dienesystem is assumed to be responsible for the strong bisignate Cotton effect.Thus we may distinguish between two groups of flexible chiral trans-dienes: one whose energy in the ground state has a maximum at 0 torsional angle and has two equivalent minima at similar positive and negative twist angles; and the other whose maximum energy is also at 0 but whose minimum on one side of the twist is deeper than on the other. We assume that the temperature dependent CD effect in vitamin D is also derived from the trans-diene system, while the contribution of the exocyclic 10,19 double bond is negligible. Thus in analogy with the transdienes, we suggest that the 5,7-double bonds of vitamin D 3 and 5(10),7double bonds of previtamin D oscillate at room temperature around the single bond, while at low temperature they are preferentially twisted. The origin of the CD bisignate effect is not yet clear. It may be related to the exciton type coupling of two differently polarized tt-tt* transitions, one to the strongly allowed •'By state and the other either to the 1Ag+ state (allowed in the cisoid conformation of the double bonds) or to the forbidden X A - state (the two photon transition) (6). We assume that also in the vitamin D, previtamin D thermal equilibration (Fig. I) the H-migration occurs from a preferential diastereomeric conformation of their cis-triene system. For the investigation of this isomerization we have synthesized a specifically labeled vitamin D_.

70 The rate of deuterium migration from C9 to C19 was monitored by mass spectrometry based on our finding that the characteristic and abundant ion a, which includes ring A, C6 and C7 contained most of its original label (7). We have heated the deuterated vitamin for time periods between 2 and 14 hrs and have determined the deuterium content in both the molecular ion and in the fragment a.

a

a

Hydrogen migration: non selective 50% D in £ stereoselective 66% D in £ Fig. VIII Deuterium label retained in molecular ion

in ion a

2 hrs at 80°

99%

91%

14 hrs at 80°

98%

62%

It may be observed (Fig VIII) that while the molecular ion retained most of the deuterium, the ion ji gradually losses part of the deuterium indicating its migration from C19. Complete distribution of the label between C19 and C9 (corresponding to the loss of 50% deuterium in the ion a) was not reached after 14 hrs heating. The comperatively slow deuterium migration (considering that the equilibrium between vitamin D and previtamin D is reached after 2 hrs at this temperature) indicates a very pronounced kinetic isotope effect. To establish whether the absence of complete distribution of deuterium to the four position at C19 and C9 is due to a kinetic preference in the H-migration, we have measured the 2 H-NMR spectrum of the deuterium labeled vitamin. We have observed two signals whose chemical shifts at 4,7 and 4,9 ppm were identical to those of the protons at C19 in the X H-NMR spectrum of the unlabeled vitamin. After heating for 14 hrs at 80 and separating the vitamin from the reaction mixture, two additional signals appeared at 1.68 and 2.70, the former identified as the deuteron at C9a, and the latter as the deuteron at C9g (Fig. IX). The integration ration of the two signals was found to be ca. 2:1, indicating a preference for euterium migration to the 9a position. This preference was also observed in the 2 H-NMR spectrum after two hours heating which revealed, in addition to the signals due to

71

2 H at C19, also a weak signal at 1.68 ppm of 2 H at C9a, while the signal due to H at C9B was undetectable. We have thus shown that the transition state for the vitamin D,, previtamin D^ isomerisation has a preferred conformation, in which the cis-1,3,5-triene system of the two compounds is twisted in a right-handed sense, ring A lying below the plane of the C/D rings.

The conformational mobilities discussed here may play a role in the biological processes involving the conversion of 7-dehydrocholesterol to vitamin D,. Thus the binding of previtamin D and vitamin D to the receptor protein may cause a conformational changes, the compounds assuming instead of their energetically stable s-transoid a less stable s-cisoid conformation. References 1.

Sanders, G.M., Pot, J. and Havinga, E. (1969) Fortschr. Chem. Org. Naturst. 27_, 131-157; Havinga, E. (1973) Experientia 29, 1181-1193.

2.

La Mar, G.N. and Budd, D.L. (1974) J. Am. Chem. Soc. 96, 7317-7324; Wing, R.M., Okamura, W.H., Pirio, M.R., Sine, S.M. and Norman, A.W. (1974) Science 186, 939-940; Wing, R.M., Okamura, W.H., Rego, A., Pirio, M.R. and Norman, A.W. (1975) J. Am. Chem. Soc. 97_, 4980-4985; Okamura, W.H., Hammond, M.L., Rego, A., Norman, A.W. and Wing, R.M. (1977) J. Org. Chem. £2, 2284-2291; Berman, E., Luz, Z., Mazur, Y. and Sheves, M. (1977) J. Org. Chem. 42, 3325-3330; Berman, E., Friedman, N., Mazur, Y. and Sheves, M. (1978) J. Amer. Chem. Soc. 100, 56265634.

3.

Sheves, N. and Mazur, Y. (1977) J. Chem. Soc. Chem. Commun. 21-22.

72 4.

Jaffe, H.H. and Orchin, M. (1960) J. Chem. Soc. Bromberg, A. and Muszkat, K.A. (1972) Tetrahedron 28_, 1265-1274.

5.

Honig, B., Warshel, A. and Karplus, M. (1975) Accounts of Chem. Res. 8^, 92-100; Chan, W.K., Nakanishi, K., Ebrey, T.G. and Honig, B (1974) J. Amer. Chem. Soc. 96, 3642-3644.

6.

Mason, S.F. and Seal, R.H. (1974) Tetrahedron 3£, 1671-1682; Bouman, T.D. and Hauser, A.E. (1978) Chem. Phys. Letters, 53, 160164; Schulten, K. and Karplus, M. (1972) Chem. Phys. Lett. 14, 305-309. —

7.

Blunt, J.W., DeLuca, H.F. and Schnoes, H.K. (1968) Biochem. ]_, 3317-3322; Okamura, W.H., Hammond, M.L., Jacobs, H.J.C. and van Thuil, I. (1976) Tetrahedron Letters 4807-4810.

73 25(R),26 AND 25(S),26-(0H) 2 D 3 : BIOLOGICAL ACTIVITY IN INTACT AND NEPHRECTOMIZED RATS. COMPARATIVE'EFFECTS OF 1,25, 24(R),25-(OH) 2 D3 and 1,24(R),25(OH) 3 D 3 .

M. Thomasset, J. Redel , P. Marche, P, Cuisi'ni'er-Gleizes. INSERM U120, 44 Chemin de Ronde, 78110 Le Vésinet, France *INSERM U5 (ERA 337 CNRS), Hôpital Cochin, 27 rue du Fg St Jacques, 75674 Paris, France.

Intestinal calcium-binding protein (CaBP) synthesis can be considered as a molecular expression of the hormonal action of l,25-(OH)2D3 on the enterocyte (3). Other dihydroxy-metabolites of vitamin D3 are synthesized in vivo. In the kidney, 25-OH-D3 can be hydroxylated on carbon 24 or 26 to yield 24(R),25-(OH)2D3 or 25(R),26-(OH) 2 D 3 respectively (2). The role of 25,26(0H) 2 D3 is not yet established. In order to elicit its biological activity and the importance of the stereochemical configuration at C-25 position the present study reports the effect of 25(R),26 and 25(S),26-(0H) 2 D 3 to promote intestinal CaBP production as well as bone calcium mobilization in intact and nephrectomized rats and compare it to that of 1,25 and 24(R),25^(0H) 2 D 3 and 1 ,24(R) ,25-(OH) 3 D 3 . To this end, dos-e^response curves have been established for each metabolite. They were based upon the increment in intestinal CaBP and in serum calcium iti vitamin D-rdeficient rats fed a lowcalcium diet. Male weanling rats of the Sprague strain were fed a vitamin D-deficient normal diet (0.50% Ca, 0.36% P) during 4 weeks and then kept on a lowcalcium vitamin D-deficient diet (0.03% Ca, 0.36% P) for 1 week. In dose response experiments all rats were killed 48 hours after the injection of different doses of each vitamin D 3 metabolite. Immediately after surgery, nephrectomized and sham-operated rats received an adequate steroid dose and were sacrificed 30 hours later. All the vitamin D3 metabolites were dissolved in ethanol and administered intravenously. Control animals received an appropriate volume of ethanol. At the sacrifice time the proximal 10 cm of intestine was removed and the mucosal tissue was homogenized in Tris buffer, centrifuged at 100,000 g and the supernatant S100 was stored. In*testinal CaBP was quantitated either in SI00 directly by radioimmunoassay (5) or after Sephadex G-75 chromatography by its + calcium-bindiTig capacity (4) and expressed as ug/wg of protein or nmol Ca bound/tag protein respectively. Bone calcium mobilization is reflected by the increase in serum calcium level (ACas) after the injection of steroid in vitamin D-deficient rats fed a low-calcium diet. Statistical significance was analyzed using Student's t-test. The relative potency of vitamin D3 metabolites resulting from dose-response data was expressed as the coefficient of concentration p. We have recently reported (10) detailed comparison of the biological activity of 25(R),26 and 25(S),26-(0H) 2 D 3 in promoting intestinal CaBP synthesis and bone calcium mobilization. This previous study emphasized that 25R epimer is hardly more effective than 25S epimer on intestine as well as on bone. Both stereisomers exhibited a late onset and a long duration (72h) of

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

74 the calcemi'c r e s p o n s e . S u c h o b s e r v a t i o n s h a v e b e e n also r e p o r t e d w i t h 24 0 0 , 2 5 and 2 4 ( S ) , 2 5 - ( O H ) 2 D 3 (9). F i g u r e a shows a linear r e l a t i o n s h i p b e t w e e n the increment in s e r u m calcium and the logarithm of the a d m i n i s t e r e d d o s e . 25 (R) ,26-KOH) 2 D 3 w a s slightly m o r e active than 2 4 ( R ) , 2 5 - ( O H ) 2 D 3 and about 10-15 times less e f f e c t i v e than l , 2 5 - ( O H ) 2 D 3 or 1 ,24 (R) ,25-1, (1975) Arch. Biochem. Biophys. J_7Q, 620^626. 9 - Thomasset, M. , Redel, J., Marche, P. and Cuivsinier^Glerzes, P, 0 9 7 8 ) J. Ster. Biochem. 9, 159-162. 10 - Thomasset, M. , Redel, J,, Marche, P,, Laborde, K. and Cuisinier-^ Gleizes, P. (1978) Steroids 32, n" 5,

77 INHIBITORS OF VITAMIN D METABOLISM AND ACTION

B. L. Onisko, H. K. Schnoes, and H. F. DeLuca Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 U.S.A. The discovery of the obligatory nature of the two-step metabolic activation of vitamin D made possible the theoretical existence of vitamin D antimetabolites (1). reality.

Our recent work has changed this possibility into

We have prepared four side-chain modified vitamin D analogs—

25-azavitamin Dj (25-N-Dj), 25-fluorovitamin Dj (25-F-D3), 24-dehydrovitamin D^ (A-24-D^) and 25-dehydrovitamin

D^ (¿-25-0^).

These analogs

feature a blocked C-25 position and were specifically designed to be competitive inhibitors of the vitamin D^-25-hydroxylase (2).

We reasoned

that failure of these compounds to be 25-hydroxylated should prevent their subsequent 1-hydroxylation and should thus render them devoid of vitamin D-like biological activity.

We further reasoned that analogs

that are 1) effective 25-hydroxylase inhibitors and 2) biologically inactive themselves should be antagonists of vitamin D action.

Such

agents may prove useful towards reducing the hypercalcemia of a number of diseases of calcium metabolism.

25-N-D3

25-F-D3 A-24-D 3 A-25-D3

25-N-DJ,

25-F-DJ,

A-24-D3

and

hydroxylation of vitamin Dj.

A-25-D3

are indeed

inhibitors

of

the

25-

Male weanling rats were raised on a vitamin-

D-deficient diet and injected with one of the analogs 2 hours before an intrajugular dose of [3- 3 H]D 3 (50 ng).

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

Four hours later serum 25-OH-

78 3 t H]Dj content was measured.

25-N-D 3 and 25-F-D^ decreased serum 25-OH-Dj

to approximately 50% of its normal value when given at 50 or 0.5 ug doses, respectively.

A-24-D^ and A - 2 5 - D ^

appeared even more potent than 25-F-D.j.

The analogs were next tested for antivitamin D activity.

Male weanling

rats were raised on a low calcium, vitamin D-deficient diet.

They were

administered one of the analogs (50 yg/rat) 2 hours before a dose of vitamin D (50 ng).

Twenty hours later, bone calcium mobilization and

intestinal calcium transport were measured.

25-N-D^ significantly

depressed both responses compared to animals receiving vitamin D alone, and is thus a true vitamin D antagonist. A - 2 4 - D ^ nor A - 2 5 - D ^

Surprisingly, neither 25-F-D3>

showed any vitamin D^ antagonist activity.

The

possibility that these three analogs possessed vitamin D agonist activity was then directly examined.

Rats raised as described above were given

graded doses of either 25-F-Dg, A - 2 4 - D 3 , injection.

or A - 2 5 - D j by intrajugular

Bone and intestinal responses were measured 20 hours later.

All 3 compounds showed positive responses in both systems, but only when given at high doses.

The biological activity of 25-F-D 3 is about 100-

300 fold less active than vitamin D^, while the 2 dehydro compounds are about 300-1000 fold less active than vitamin D^. shown by 25-F-D 3 , A - 2 4 - D 3

and A - 2 5 - D 3 ,

The agonist activity

albeit quite low, is evidently

sufficient to rationalize the lack of antivitamin D activity found for these three inhibitors of 25-hydroxylation.

But why are 25-F-D 3>

A-24-D 3 and A-25-D 3 biolgoically active?

25-F-[3-

^H]D 3 was prepared and its metabolism was examined in vitamin D-deficient rats.

The animals, either intact or bilaterally nephrectomized, were 3

given 15 pg of 25-F-[3- H]D 3 and serum was obtained 10 hours later. Chromatography of chloroform extracts of serum on Sephadex LH-20 [chloroform/Skellysolve B (65:35)] showed the presence of small amounts of two metabolites named F-II and F-III.

Peak F-III, when examined by HPLC

(uPorasil, 2-propanol/hexane (1:19)], was resolved into two components, named F-IIIa and F-IIIb.

Peak F-IIIb was identified as la-OH-25-F-D 3 by

its co-chromatography on HPLC with synthetic material, and by its absence in the serum of anephric animals.

Although the yield of la-OH-25-F-D 3

is low, the amount present in serum (7 ng/ml serum at 10 hours after

79 dose) appears adequate to account for the biological response observed due to the high iji vivo potency of la-OH-25-F-D 3

(3) .

A-24-D^ and A - 2 5 - D 3 are biologically active due to conversion, in vivo, to l , 2 5 - ( O H ) 2 D 3 .

Twenty hours after dosing A-24-D.j or A - 2 5 - D 3

yg/rat) to v i t a m i n D-deficient animals, serum l , 2 5 - ( O H ) 2

D

3

(50

c o n t e n t

measured by the method of Eisman et^ al. (4) was found to be 100 pg/ml and 180 pg/ml respectively.

Ethanol-dosed controls had undetectable

pg/ml serum) levels of 1,25-(OH)2°3> whereas rats given v i t a m i n D^ ng/animal) had values of 300 pg/ml.

(OH

. NH.-BSA CH2OH

CHpH

¿PS"*

NH-BSA

Gel-F-NHCH2NH2

Olwralttehyte» Gel- -NHCH2CHCH20H NaBH4 ¿H io;|

Gel 4 - N H C H . H C H . H -Protein V ^ t e ' " Gel-

BHJCN

I

Figure 3 Coupling of Carbohydrates and Proteins to soluble and insoluble matrixes using sodium cyanoborohydride.

In the presence of an aldehyde and any primary or secondary amine and at pH 6-8, reductive coupling occurs with the formation of a more substituted amine (Fig.4).

94 , R-CHO + NH-R1 c

pH

6-8

Na CNBH3

P-

. RCH~ - NH - R1 c

Figure 4 Reductive amination of aldehydes

with sodium cyarioborohydride.

The mixture of aldehydes was bound to either AH Sepharose 4B or bovine serum albumin - AH Sepharose 4B. The reaction was performed in methanol and the efficacy of the process was checked in two ways. F i r s t l y the flurescamine test of Udenfriend et al (16) was negative following treatment as described. Secondly, extensive washing of the gel with methanol f a i l e d to y i e l d any steroid as tested by both TLC and NMR spectroscopy. Methanol washing was shown to remove the steroid from preparations which had not been treated with the reducing agent. The efficacy of the resin as an a f f i n i t y column for the chick i n t e s t i n a l receptor for 1,25-dihydro*yvitamin D was tested using the hydroxyapatite assay of Wecksler and Norman (17). The unsubstituted gel allowed the receptor protein to f e l l through. No receptor was detectable at the same elution volume for the steroid-bound resin, nor could the receptor be removed subsequently from the column. Further experiments are in progress to overcome this problem. Despite the lack of immediate success of this system as an a f f i n i t y column we feel that these results are important on two grounds. F i r s t l y , we have described a method of functionalizing the vitamin D molecule which does not involve regions known to be important for binding to the receptor (18). Secondly and more generally, we have shown that a simple carbonyl function which i s either present or can be introduced into many molecules of biological .interest, can be used for coupling to a f f i n i t y media by reductive a m i n a t i o n . I would l i k e to thank Dr. W.R. Wecksler and Professor W.H. Okamura for their assistance and encouragement in this work and p a r t i c u l a r l y Professor A.W. Norman who made i t possible for me to work in his laboratories at Riverside. I also wish to thank the South African Council for S c i e n t i f i c and Industrial Research for financial support. REFERENCES: 1.

Norman, A.W. (1974) Vitamins and Hormones. 32, 326-384.

2.

De Luca, H.F. and

3.

S i c a , V., Parikh, I . , Nola, E . , Puca, G.A. and Cuatrecasas, P. (1978) 0. B i o l . Chem. 248, 6543-6558.

Schnoes.K. (1976) Ann. Rev. Biochem. 45, 631-666.

95 4.

Kuhn, R.W., Schräder, W.T., Smith, R.G. and O'Malley, B.W. (1975) J. Biol. Chem. 250, 4220-4228.

5.

Govinden, M.V. and Sekeris, C.E. (1976) Steroids 28, 499-507.

6.

Rosner, W. and Bradlow, H.L. (1971) J. Clin. Endocrinol. Metab. 33, 193-198.

7.

Mi ekel son, K.E. and Petra, P.H. (1975) Biochemistry 14-, 957-963.

8.

Sharpe, R., Hillyard, C.J., Szelke, M. and Maclntyre, I. (1977) FEBS Letters 75, 265-271.

9.

McCain, T.A., Haussler, M.R., Okrent, D. and Hughes, M.R. (1975) FEBS Letters 86, 65-70.

10.

Thompson, S.T., Cass, K.H. and Stellwagen, E. (1975) Proc. Nat. Acad. Sei. USA 72, 669-672.

11.

Schwartz, J. and Labinger, J.A. (1976) Angew. Chem. Int. Ed. Engl. J_5, 333-340.

12.

Messing, A.W., Ross, F.P., Norman, A.W. and Okamura, W.H. (1978) Tet. Letters, 3635-3636.

13.

Lane, C.F. (1975) Synthesis. 135-146.

14.

Gray, G.A. (1978) Methods Enzymol. 50, 155-160.

15.

Fiddler, M. and Gray, G.A. (1978) Anal. Biochem. 86, 716-724.

16.

Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W. and Weigele, M. (1972) Science J78, 871-872.

17.

Wecksler, W.R. and Norman, A.W. (1979) Anal. Biochem. In Press.

18.

Wecksler, W.R., Okamura, W.H. and Norman, A.W. (1978) J. Steroid Biochem. 9, 929-937.

Osteoporosis

99 CALCIUM ABSORPTION AND PLASMA 1,25(0H)„D LEVELS IN POST-MENOPAUSAL i OSTEOPOROSIS B.E.C. Nordin, M. Peacock, R.G. Crilly and D.H. Marshall M.R.C. Mineral Metabolism Unit, The General Infirmary, Leeds LSI 3EX, England. Malabsorption of calcium has been a recognised feature of post-menopausal osteoporosis for a long time (1,2), and the idea that it might be due to a deficiency of 1,25(0H)2D was first suggested by us several years ago (3,4). In a small collaborative study with M. Haussler, we were unable to confirm this idea but a slightly not significantly reduced mean plasma 1,25D level in post-menopausal osteoporosis was subsequently reported by Gallagher et al (5). We now report our own results in this field. Clinical material and methods Observations are reported out vertebral compression osteoporotic and 9 of the feeding; and on 16 of the

on 62 post-menopausal women (26 with and 36 withfractures) in the basal state; on 9 of the non-osteoporotic after low and high calcium non-osteoporotics after oestrogen administration.

The plasma and urine samples were all collected after an overnight fast. The low calcium diet (250 mg of calcium) was given for one week and the high calcium diet (800 mg Ca in the diet and 1,200 mg as Sandocal tablets between meals and at night) for a further week. Oestrogen therapy was generally given as ethinyl oestradiol 15 ug daily for 3 weeks out of 4 and the plasma and urine samples obtained after not less than 3 months of treatment. Plasma calcium, phosphate and creatinine and urine calcium, hydroxyproline and creatinine were measured by standard Auto-Analyzer techniques (6). Plasma ionized calcium was measured with a flow-through electrode or (in some cases) calculated from total calcium and albumen (7). Calcium absorption was measured by our standard radiocalcium technique (8), the fractional absorption being calculated from the one-hour plasma activity when repeated tests were being performed on the same subject. Fasting urine calcium and hydroxyproline were expressed as molar concentrations relative to creatinine (Ca/Cr and OHPr/Cr). Plasma 1,25D was measured by radioimmunoassay (9). The data on the 62 subjects are not all complete. There are 60 ionized calcium and plasma 1,25D measurements and only 45 plasma 25D measurements in the basal state. Results The significant correlations derived from the basal data are shown in Table 1. The strongest correlations are the fall in calcium absorption and the rise in plasma creatinine with age, but calcium absorption is not correlated with plasma creatinine and the fall in calcium absorption with age cannot therefore be attributed to declining renal function. There is a strong inverse correlation between calcium absorption and plasma phosphate (Fig. 1) which also cannot be attributed to renal impairment since plasma phosphate does not correlate with plasma creatinine. There is a rather strong positive correlation between plasma phosphate and ionized calcium

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

100 TABLE 1. Correlation maxtrix on basal data on 62 post-aenopausal wanen. Age

Ca absorpt.

1,25

Ca"

P

Cr.

OHPr

Ca absorpt. -.40*** PI.1,25 PI,Ca + +

PI .P

(but not with proline rises positively to tions between

-.36***

PI.Cr

+.38***

Ur.OHPr

+.27*

-.25*

.34**

-.28* -.26*

+.28*

total calcium). Weak correlations suggest that urine hydroxywith age and is inversely related to calcium absorption and plasma P, and there are weak but significant inverse correlathe plasma 1,25D level and plasma P and plasma creatinine.

Some of these relationships are shown in the figures. It is particularly striking that calcium absorption falls with age (Fig. 2) but plasma 1,25D does not (Fig. 3). In fact the mean 1,25D value in our normal postmenopausal women (43 pg/ml) is virtually the same as our pre-menopausal mean (44 pg/ml) (9). It is clear that there are some rather high and some rather low values among the older subjects. This variation is probably due to renal function. Thus if the cases over 75 years are considered, the plasma 1,25D level is an inverse function of the plasma creatinine (Fig. 4). However, the 25D levels in these old subjects tend to be low. Although the correlation matrix did not reveal a significant decline in plasma 25D with age, inspection of the data showed a clear decline with age but two discrepant points (Fig. 5). One of these was in a patient who had previously been given high dose vitamin D for a long period; exclusion of this point produced a significant r value of -0.37 (p

U

•ri

•d

/-V • h ¡3 o ^ tí o

"d (D

•ri

£>

-p h o o cd ra SH CH Ctí

CD ctí ri o SH eh

jH

i—t O a a

H

(D

tí

•ri

•

ra

H O a a

.tíPh

ctí O

CLt

H (d

ctí a

a

cd •H Pi

(d H CU

-—'

ra

(tí

ra

o

(d

a ra

¡tí

i—t (U

H O ÍH Pi K

«

^ !» (d tá

\

O H ^ O •d s !»3 O . t í !h O » tí ON •H -3-P cd •H -P 4) Cd H Ol cd s 0) CH

03 h cd (M i) -P « "í a • r> i vû -p Iß H CU «

§

PH

IO c\J A! 1 , with 25% converting to

D3 at 12 h and 50% converting to D3 by 28 h.

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

Equilibrium was reached

174 after 4 d, w i t h 83% of the previtamin converting to D ^ (Fig. IB).

In c o n -

trast, an analysis of an aliquot of the sample stored at - 2 0 ° C showed that only 2% of the previtamin converted to

Fig. 1: A column (30 aliquot of B Thermal at 37°C — •

even after 7 d of incubation.

High-pressure liquid chromatographic profile o n a y - P o r a s i l cm X 4 m m , developed w i t h 0.25% isopropanol in n-hexane) of an [ 3 a - % ] p r e v i t a m i n D3 that w a s stored at 37°C for 24 h. conversion of previtamin D3 to vitamin D3 as a function of time — , and at - 2 0 C --A--.

To investigate w h a t role, if any, the v i t a m i n D-binding p r o t e i n played in controlling the mobilization of D^ from the skin, w e determined the b i n d ing affinity of this protein for 7-dehydrocholesterol, its three photoisomers and vitamin D ^ using the method of Belsey ^ t al. (6).

Fig. 2 illus-

trates that the DBP has essentially no affinity for p r e D , compared to D~.

DISCUSSION

These observations i n vitro suggest that in vivo the photolytic production of preDj in the skin serves as a unique mechanism responsible for the s y n thesis, storage and thermally-controlled release of D^.

Once preD^ is

made during sun exposure it slowly converts to D ^ and reaches a n equilibrium that is dependent upon the temperature of the skin.

Thus D^ w o u l d be

continually made in the skin from its previtamin, e v e n in the absence of further sunlight for days to w e e k s after the initial sunlight exposure. In warm-blooded animals, the rate of this intramolecular reaction would remain relatively constant since the body temperature is closely

regulated

while in cold-blooded species the rate of this reaction would be h i g h l y

175

nq/TUBE

Fig. 2: Displacement of [26,27-^H]25-hydroxyvitamin D3 from rat v i t a m i n D binding protein b y vitamin D3 and previtamin D 3 . variable.

Since D^ has at least a 1000-fold higher affinity for the D B P

than does preD^

after D ^ is made, it would b e selectively bound to this

transport p r o t e i n to be efficiently carried from the skin into the circulation, leaving preD^ in the skin to continue its thermal conversion to Dj.

However, since lumisterol^ is not bound to the DBP and tachysterol^

is only w e a k l y bound to this transport protein, in the event that small quantities of these biologically-inactive photoisomers w e r e made during excessive sun exposure, they would, like preD^, remain in the skin.

Hence

lumisterolj and tachysterol^, w h i c h are thermally stable photoisomers, would be discarded during the natural turnover of the epidermis.

Although these concepts concerning the control mechanisms involved in the in vivo production of vitamin D ^ are a result of our findings in vitro, the fact that previtamin D^ is present in the skin at least 48 h after exposure to ultraviolet light, while little, if any, is present in the blood at either 24 or 48 h, lends strong support for this theory.

Fur-

thermore, the thermal conversion of preD^ to D^ in vivo appears to occur more rapidly and more efficiently at the same temperature w h e n compared to the in vitro model.

176 Thus it is interesting to reexamine the function of the plasma vitamin D binding protein,

To date it has been believed that the major role of the

DBP is to transport vitamin D through the circulation to the liver and kidney for specific hydroxylatlons and to carry the hydroxylated products to the target tissues to elicit a biological response.

Our recent find-

ings suggest, however, that the DBP also plays an important role in controlling the synthesis of D^ in the skin.

The selective uptake by the

binding protein of D^ from skin would shift the thermal equilibrium to favor the formation of D^ from the previtamin.

In addition, by selec-

tively transporting only D^ out of the skin and not preD^, metabolic degradation of the previtamin in internal organs would be avoided, guaranteeing its storage in the skin prior to its conversion to D^-

This mech-

anism might be of critical importance to cold-blooded vertebrates who live in temperate climates dependent upon the ambient temperature to control this thermal equilibrium, since the rate of formation of D^ from preD^ is dramatically reduced at lower temperatures (from 83% at 37°C to 2% at -20°C after 7 d).

REFERENCES 1.

Rauschkolb, E.W., Davis, H.W., Fenimore, D.C., Black, H.S. and Fabre, L.F. (1969) J. Invest. Dermatol. .53, 289-293.

2.

Okano, T., Yasumura, M., Mizuno, K. and Kobayashi, T. (1977) J. Nutr. Sei. Vitaminol. (Tokyo) 23/2), 165-168.

3.

Esvelt, R.P., Schnoes, H.K. and DeLuca, H.F. (1978) Arch. Biochem. Biophys. 188, 282-286.

4.

Holick, M.F., Frommer, J.E., McNeill, S.C., Richtand, N.M., Henley, J.W. and Potts, J.T., Jr. (1977) Biochem. Biophys. Res. Commun. 76, 107-111.

5.

Havinga, E., Koevoet, A.L. and Verloop, A. (1955) Ree. Trav. Chim. Pays-Bas Belg. J±, 1230-1234.

6.

Belsey, R., Clark, M.B., Bernat, M., Glowacki, J., Holick, M.F., DeLuca, H.F. and Potts, J.T., Jr. (1974) Am. J. Med. 57^, 50-56.

Assays for Vitamin D Metabolites

179 MEASUREMENT OF PLASMA l,25(OH) 2 VITAMIN D BY PROTEIN BINDING ASSAY AND RADIOIMMUNOASSAY M. Peacock, G. A. Taylor and W. B. Brown M.R.C. Mineral Metabolism Unit, The General Infirmary, Leeds LSI 3EX, UK. INTRODUCTION Protein binding assays have been reported which measure plasma 1,25(0H)2D using specific 1,25(0H)2D nuclear (1) or cytosol (2) receptors from D deficient chick intestine.

Antibodies to the 1,25(0H)2D3 linked to

protein have been produced and can replace the cytosol receptor in the assay (3,4). Two antibodies to 1,25(0H)2D3 have been raised and the cross

reactivities

of the D metabolites to the antibodies and to the cytosol binding protein studied.

Plasma 1,25(0H)2D in patients and controls assayed by antibody

have been compared to those assayed by cytosol binding protein. METHODS Preparation p f Cytospl proteinj-

Cytosol protein was prepared from

intestinal mucosa of 4 week old D deficient chicks as previously described (1,2) and stored at -18°C until used i n the assay. Antibody_ production:-

Antisera to la,25(0H) 2 D 3 25 hemisuccinate

(Dr M R Uskokovic) linked to bovine serum albumin (3) were raised in rabbits.

Two of the six rabbits iimiunised produced useful antisera after

four 0.1 mg injections of antigen in complete Freunds given as multiple subcutaneous injections. Label:-

1,25(0H) 2 ( 23,24(n)- 3 H } D3 (Amersham) was used as label.

Standards:-

Crystalline 1,25(0H)2D3 (Roche) 24R,25(0H)2D3 (Roche),

250HD3 (Roussell) and 1a0HD3 (Leo) were diluted in alcohol to give a suitable range of concentrations for establishing standard curves.

The

25,26(0H)2D3 was a g i f t of Dr Redel, Paris. Assay condjtionsj-

The incubation mixture for the cytosol binding

assay consisted of 50 yl of standard or unknown in ethanol, 10yl of labelled 1,25(0H)2D3 in ethanol and 500yl of cytosol binding protein in Tris-HCl buffer (pH 7.4).

The cytosol binding protein was diluted

(usually between 1/1 and 1/4) to bind 40% of the labelled 1,25(0H) ? D V

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

180 The assay was incubated at 25°C for 1 hour.

The incubation mixture for

the antibody assay was similar but 500^1 of diluted antisera (usually between 1/500 - 1/1000) in phosphate buffer (pH 6.0) was substituted for cytosol binding protein. Separation:-

The assay was incubated for 24 hours at 4°C.

Both assays were separated with 0.05% Dextran/0.5%

charcoal in phosphate buffer (pH 7.4) and the bound label counted. P u r i f i c a t i o n o f plasma_D_metabolites.:-

Plasma spiked with 1000 cpm of

labelled 1,25(0H) 2 D 3 was f i r s t extracted with acetone/dichloroethane (3:1) followed by ether (1).

The ether extract was further purified on a 1 x 30

cm column of 5 g of LH-20 eluted with 65% chloroform/35% hexane mixture (1) and the 1,25(0H) 2 D fraction further purified on a 4.6 x 25 cm ZorbaxS i l column (Dupont) using high pressure.

The recovery of labelled

1,25(0H) 2 D 3 was used to estimate the loss of 1,25(0H) 2 D 3 (usually 20-40%) which occurred during extraction and purification. Patients:-

Patients with various disorders of calcium metabolism were

bled after an overnight fast and radiocalcium absorption measured (5). Control subjects aged 18-55 were members of the Unit. RESULTS S p e c i f i c i t y : The cross reactivity of the D metabolites with cytosol binding protein (BP), antibody D g and antibody D 4 are shown in Table 1.

The

data are expressed as the metabolite concentration which gives 50%

dis-

placement of the label under assay conditions.

The concentrations are

compared to what i s considered their normal concentration i n human plasma. Table 1

S p e c i f i t i e s of Antibodies and Binding Protein (B.P.) Plasma (pg/ml)

B.P. 50« d i s p l .

Ab. D8 50% d i s p l .

Ab. D4 50% disp

6.0 x 10 1

9.0 x 10

1,25(0H) 2 D 3

4.4 x 101

9.0 x 10 1

25-OHD3

1.0 x 10 4

2.0

X

105

6.0

10 3

1.9 x 10

24,25(0H) 2 D 3

1.0 x 10 3

1.5

X

106

5.5 x 10 3

2.2 x 10

25,26(0H) 2 D 3

1.0 x 10 3

4.0

X

106

6.0 x 10 2

1.5 x 10

4

2.1 x 10 4

1.5 x 10

1o-0HD3

-

1.7 x 10

X

Antibody D g shows the highest absolute a f f i n i t y for 1,25 and 25,26(0H) 2 D 3 and can measure both i n plasma. The cytosol binding protein showed the greatest relative a f f i n i t y for 1,25(0H) 2 D 3 and i s similar to that seen with bone in tissue culture (6).

Even so separation of 1,25(0H) 2 D from

i t s metabolites i s required before i t can be assayed.

l8i

Assay behaviour:

Table 2

Table 2 shows

Inter assay variation

the inter assay

Detection limit (pg)

variation

50S displacement (1,25(0H)2D3 pg)

the

Assay Characteristics B.P. 41%

Ab. D8

18«

8.3 (SE 2.0 n 5)

4.8 (SE 0.9 n 5)

59.4 (SE 8.2 n 5)

32.2 (SE 1.7 n 5)

the detection limit and the amount of l,25(0H)2Dg causing 50% displacement in 5 consecutive assays with cytosol binding protein and antibody Dg. The assay based on antibody Dg was more reproducible and more sensitive than the cytosol assay in our hands.

Samples assayed by both assays gave a

correlation coefficient of 0.83. Plasma_concentrations of_l¿25(0H}2D:

Fig.l shows the 1,25(0H)2D

concentrations achieved after an oral dose of 2 yg l ^ i O H ^ D g and after 2 yg 1 aOHDg.

The peak blood concentration occurs 6 hours after the dose

FIG 1

FIG 2 RELATIONSHIP BETWEEN RAMOCALCIUM ABSORPTION AM) PLASMA USIOH^O

j40-PLASMA 1,2S(0H),0 RESPONSE JO ORAL %2S40H)^ AND IB-OHC^

J 40 I 2p9l25,° O PENAL FAILURE plasma 1,25(0H)2D and phosphate *4 •OSTEOMALACIC A1°HYPEDPAftATHYH040 y = -26.9 x + 73 (r = -.36,p-26.1a 4-66.8

¿25

normal subjects was 25.7-66.5 pg/ml and there was no di fference between males and females.

182 CONCLUSIONS Two antibodies with different specificities for the D metabolites have been raised in rabbits. Under assay conditions antibody Dg could measure as little as 4 pg of 1,25(0H)2D and in our hands was more reproducible than the assay based on cytosol binding protein. Antibody Dg could also measure normal plasma concentrations of 25,26(0H) 2 D 3 . Using the antibody assay the normal range was 25.7 - 66.5 pg/ml; plasma l,25(0H)2Dg was positively related to radiocalcium absorption and negatively related to plasma phosphate. REFERENCES 1.

Brumbaugh, P.F., Haussler, D.H., Bursac, K.M., and Haussler, M.R. (1974). Biochemistry J3, 4091-4097.

2.

Eisman, J.A., Hamstra, A.J., Kream, B.E., and DeLuca, H.F. (1976). Science J93, 1020-1023.

3.

Fairney, A., Turner, C., Baggiolini, E.G., and Uskokovic, M.R.(1977). In: Vitamin D Biochemical, Chemical and Clinical Aspects Related to Calcium Metabolism. Ed. Norman A.W., Schaefer, K., Coburn, J.W., DeLuca, H.F., Fräser, D., Grigoleit, H.G. and Herrath, D.V. Walter de Gruyter, Berlin.

4.

Clemens, T.L., Hendy, G.N., Graham, R.F., Baggiolini, E.G. Uskokovic, M.R., and 0'Riordan, J.L.H. (1978). Clin. Sei. Mol. Med. 54, 329-333.

5.

Calcium, Phosphate and Magnesium Metabolism (1976). Ed. Nordin, B.E.C. Churchill Livingstone, Edinburgh.

6.

Peacock, M., Taylor, G.A., and Norman, A.W. (1977).

In: Vitamin D

Biochemical, Chemical and Clinical Aspects Related to Calcium Metabolism.

Ed. Norman, A.W., Schaefer, K., Coburn, J.W.,

DeLuca, H.F., Fräser, D., Grigoleit, H.G. and Herrath, D.V. Walter de Gruyter, Berlin.

183 USE OF ISOTOPE DILUTION MASS FRAGMENTOGRAPHY IN VITAMIN D RESEARCH. I. Bjorkhem, I. Holmberg, T. Kristiansen, A. Larsson and J.I. Pedersen. Department of Clinical Chemistry, Huddinge Hospital, Huddinge, Sweden, and Institute for Nutrition Research, School of Medicine, University of Oslo, Oslo, Norway. Assay of vitamin D and its metabolites in biological fluids offers several problems for the investigators, and much of the controversy which still exists may be due to methodological difficulties. The compounds are unstable and occur in a low concentration range. There are several similar compounds with similar properties which may interfere in an assay. When there is a demand for high specificity and accuracy, isotope dilution mass fragmentoqraphy seems to be a method of choice (for reviews, see ref. 1-3). The principle is that an isotope labeled internal standard is added to the biological fluid prior to extraction and chromatography. The ratio between unlabeled and labeled molecules in the purified material is then determined with use of a gas chromatograph - mass spectrometer equipped with a multiple ion detector. In this latter analysis, the multiple ion detector is focused on one ion specific for the unlabeled compound and one ion specific for the labeled internal standard. The specificity of the assay is based on the fact that in order to influence the results, a contaminating compound must both have the same retention time in gas-liquid chromatography and contain the same specific ion in its mass spectrum as the specific compound which is under study. In general there is only a very little risk for such a combination. During the last few years, we have developed specific mass fragmentographic methods for assay of vitamin Dg, 25-hydroxy vitamin D3 and 1,25-dihydroxy vitamin D3 (4-9). In the present paper, these methods as well as some of their applications, will be shortly reviewed. ASSAY OF 25-HYDROXY VITAMIN D 3 BY MASS FRAGMENTOGRAPHY

(4-6).

Methodology. A deuterated internal standard was synthesized according to the following sequence of reactions: 3^-acetoxy-27-norcholest-5-en-25-one-*3^-acetoxy-27-horcholesta-5,7-dien-25-one - » [ 2 6 - 2 H 3 ] cholesta-5,7-diene-3p,25-diol — • 25-hydroxy-f26- 2 H3j vitamin D 3 (4). The deuterated internal standard was purified by thin-layer chromatography (using ethyl acetate/chloroform, 1:3, v/v) as solvent. A typical preparation contains 3 % unlabeled molecules, 0 % mono-deuterated molecules, 5 % di-deuterated molecules and 92 % tri-deuterated molecules. In the standard procedure for mass fragmentography, 100 - 150 ng 25-hydroxy[26- z H3j vitamin D3, dissolved in 50 «1 of ethanol, is added to 1 or 2 ml serum or to an incubation mixture containing subcellular fractions from rat liver (cf. below). After equilibration for 1 hour at room temperature in nitrogen atmosphere, and addition of ammonium carbonate (10), the steroids are extracted with ethyl acetate. After evaporation of the solvent, the

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Researdi and its Clinical Application

18k 3-t-butyl-dimethylsilyl derivative of 25-hydroxy vitamin D3 is formed with use of t^butyldimethylchlorosilane reagent. This compound is then purified by thin-layed chromatography, using toluene/ethyl acetate 3:1 (v/v) as solvent. The application of the sample as well as the development of the chromatoplate must be performed in nitrogen atmosphere. The chromatographic zone containing the 3-t-butyl-dimethylsilyl derivative is detected with the use of l-nitroso-2-naphtol and Sudan-black B as internal standards. These compounds have Rf values of 0.68 and 0.40 respectively under the conditions used. The 3-^t-butyldimethyl si lyl derivative of 25-hydroxy vitamin D3 has a Rf-value of 0.59 and is located between the l-nitroso-2-naphtol spot and the Sudan-black B-spot. This compound is immediately eluted with ethyl acetate and converted into the 3-t-butyl-dimethylsilyl-25-trimethylsilyl derivative. This compound can then be analyzed by combined gas chromatography - mass spectrometry. In our studies we have used either an LKB 2091 or an LKB 9000 instrument equipped with an MID-unit and an 1.5 % SE 30 column. One channel of the MID-unit is focused on the ion at m/e 586 (corresponding to the molecular ion of derivative of unlabeled 25-hydroxy vitamin Do) and one channel is focused on the ion at m/e 589 (corresponding to tne molecular ion of derivative of 26- ^ - l a b e l e d 25-hydroxy vitamin D 3 ). The amount of unlabeled 25-hydroxy vitamin D3 can be calculated from the ratio between the peak height of the two tracings with use of a standard curve. The relative standard deviation of the technique, as calculated from duplicate determinations, is about 3 %. The accuracy of the technique has been ascertained by different recovery experiments. The above technique represents the best modification of those tried (4-6). A modification which gives less interference in the GC-MS step involves reversed phase HPLC on Spherisorb 0DS, followed by rechromatography on Spherisorb silica (8). This latter modification is, however, more time-consuming. Analysis of serum. The mass fragmentographic technique gives values for the concentration of 25-hydroxy vitamin D3 similar to those reported with less specific techniques. The mean value obtained from 12 healthy men and women was 2 1 + 2 ng/ml (S.E.M.) in August and 11 + 1 ng/ml in April. Studies on converison of vitamin D3 into 25-hydroxy vitamin D3 in vitro (5). The above assay is suitable for in vitro determination of the rate of 25hydroxylation of vitamin D3 in subcellular fractions of rat liver. In contrast to previous work the technique allows use of substrate saturation, which gives a much higher degree of conversion. 25-Hydroxylase activity was mainly found in the mitochondrial fraction of a rat liver homogenate. The microsomal fraction was considerably less active. At least part of the mitochondrial 25-hydroxylase activity is located at the outer mitochondrial membranes, since mitochondria imperable for NADH and NADPH could utilize exogenous NADPH to some degree. Part of the enzymatic activity must be located intrafaitochondrially since isocitrate also stimulated the reaction. The degree of conversion obtained with isocitrate and citrate was higher than that obtained with other citric acid cycle intermediates, indicating that the NADP-dependent intramitochondrial isocitrate dehydrogenase is of importance. The mitochondrial NADPH-dependent 25-hydroxylase activity was linear with mitochondrial protein and with incubation time. The enzyme

185 was saturated with about 100 pg of vitamin D3 and with 5 fmol NADPH under the c o n d i t i o n s employed (incubation volume 10 ml). The pH optimum was between 7 and 8. The a c t i v i t y must be catalyzed by a mixed f u n c t i o n o x i d a s e , since ' 8 0 was incorporated i n t o the product when the incubation reaction was performed in ' ^ - a t m o s p h e r e . The vitamin D, s t a t u s had no s i g n i f i c a n t e f f e c t on the 25-hydroxylase a c t i v i t y whereas pnenobarbital treatment i n creased the a c t i v i t y about two-fold. The h y d r o x y l a t i o n was markedly i n h i bited by carbon monoxide. The f i n d i n g that the enzyme a c t i v i t y was i n h i b i t e d by carbon monoxide and stimulated by treatment with phenobarbital, suggests that cytochrome P-450 may beinvolved. Recently, cytochrome P-450 was i s o l a t e d from ihat l i ver mitochondria (11,12). I t was shown that t h i s cytochrome P-450 i s able to c a t a l y z e 2 6 - h y d r o x y l a t i o n of c h o l e s t e r o l and other C 2 7 - s t e r o i d s when combined with f e r r e d o x i n , f e r r e d o x i n reductase and NADPH ( 1 0 - 1 3 ) . We found that such cytochrome P-450 was a c t i v e a l s o towards vitamin D3. The product was i d e n t i f i e d as 25-hydroxy vitamin D3 by d i f f e r e n t means, i n c l u d i n g gas chromatography - mass spectrometry. The rate of c o n v e r s i o n , when assayed as above, varied between 0.01 and 0.02 nmol per nmol cytochrome P-450 per min. ( 8 ) . In accordance with r e s u l t s obtained from other l a b o r a t o r i e s ( 1 5 ) , the microsomal 25-hydroxylase a c t i v i t y was not l i n e a r with the amount of microsomes. T h i s enzyme a c t i v i t y seems to be of high a f f i n i t y - low capacity type whereas the mitochondrial a c t i v i t y seems to be of low a f f i n i t y - high cap a c i t y type ( c f . r e f . 16). In view of the low concentration of vitamin D3 present in v i v o under p h y s i o l o g i c a l c o n d i t i o n s , i t i s p o s s i b l e that the microsomal hydroxylase normally i s more important than the mitochondrial hydroxylase. A proper e v a l u a t i o n of the r e l a t i v e importance of the two s y s tems can f i r s t be made, however, when a l s o the microsomal a c t i v i t y can be assayed under s t r i c t enzymological c o n d i t i o n s . ASSAY OF 1,25-HYDROXY VITAMIN D3 BY MASS FRAGMENTOGRAPHY ( 7 ) . Methodology ? 26- Hn-labeled 1,25-dihydroxy vitamin D 3 was produced b i o s y n t h e t i c a l l y by incubation of 26-^H3-labeled 25-hydroxy vitamin D3 with kidney homogenate from r a c h i t i c c h i c k s . A f i x e d amount of deuterium labeled standard ( u s u a l l y about 20 ng) i s added to a f i x e d amount of serum ( u s u a l l y 20 ml). The s t e r o i d s are extracted with chloroform and p u r i f i e d by chromatography on Sephadex LH-20 and on HPLC, u s i n g f i r s t S p h e r i s o r b S i l i c a and then S p h e r i sorb 0DS ( 7 ) . The p u r i f i e d material i s converted i n t o t r i m e t h y l s i l y l ether and analyzed by gas chromatography - mass spectrometry u s i n g an 1.5 % SE30 column. The m u l t i p l e ion detector i s focused on the ion at m/e 452 ( c o r responding to M-2x90 from d e r i v a t i v e of unlabeled 1,25-dihydroxy vitamin D3) and the ion at m/e 455 (corresponding to M-2x90 from d e r i v a t i v e of 262 H3-labeled 1,25-dihydroxy vitamin D3). E s s e n t i a l l y the same r e s u l t s are obtained when using the more intense ions at m/e 131 and m/e 134 ( c o r r e sponding to cleavage between C-24 and C - 2 5 ) . I n t e r f e r e n c e s from other compounds i n the m a t e r i a l , however, sometimes i v a l i d a t e s the assay when the l a t t e r ions are u t i l i z e d . Under the c o n d i t i o n s employed, the detect i o n l i m i t i s about 5 pg/ml serum. The r e l a t i v e standard d e v i a t i o n of the

186 method, as calculated from duplicate a n a l y s i s of serum samples, i s about 6 %. The accuracy of the method has been ascertained by recovery e x p e r i ments. A n a l y s i s of serum. The serum level of 1,25-dihydroxy vitamin D3 in 15 healthy Norwegian blood donors of both sexes, varying in age between 20 and 62 years (mean 37 years) was 5 5 + 1 0 pg/ml (mean + SD duplicate determinations). According to our knowledge, t h i s represents the f i r s t determination of the serum level of 1,25-dihydroxy vitamin D3 with a method not based on s p e c i f i c protein binding. The values are s l i g h t l y higher than those reported from most previous s t u d i e s (17-19). ASSAY OF VITAMIN D3 BY MASS FRAGMENTOGRAPHY (9). Methodology. In t h i s a n a l y s i s a great excess of vitamin Dp i s used as internal standard. In the determination of the concentration vitamin D3 in serum, about 600 ng vitamin D 2 i s added to 2 ml of serum, followed by freeze-drying and ext r a c t i o n with chloroform/methanol. The vitamin D and previtamin D present in the extract are then h e a t - e q u i l i b r a t e d with each other in stoopered g l a s s tubes f o r 3.5 h at 75°C. The solvent i s evaporated and the residue subjected to preparative t h i n - l a y e r chromatography in No atmosphere, using chloroform/ethyl acetate (17:3, v/v) as solvent. After development of the t h i n - l a y e r chromatoplates, the appropriate chromatographic zone cont a i n i n g both vitamin D2 and D 3 i s scraped o f f and eluted with ethyl acetate. The t r i m e t h y l s i l y l ether i s prepared and analyzed by GC-MS. The multiple ion detector i s focused on the ion at m/e 325 ( c h a r a c t e r i s t i c fragment f o r d e r i v a t i v e of vitamin D3) and on the ion at m/e 337 (charact e r i s t i c fragment f o r d e r i v a t i v e of vitamin Do). The r a t i o between the two t r a c i n g s can be used f o r c a l c u l a t i o n of trie amount of vitamin D 3 , using an appropriate standard curve. The accuracy of the assay has been ascertained by d i f f e r e n t recovery experiments. The r e l a t i v e standard dev i a t i o n , as calculated from duplicate determinations of serum samples containing vitamin D3 in the concentration range 4-6 ng/ i s about 11 %. A n a l y s i s of serum. The level of vitamin D3 in human serum was determined with the above method in samples obtained from 19 healthy men and women. The mean value was 1.6 ng/ml with a range from 0 to 6.3 ng/ml. These values are considerably lower than the values reported previously with l e s s s p e c i f i c techniques (20, 21). Considerably higher values (12-52 ng/ml) were found, however, in four p s o r i a t i c patients subjected to UV-treatment f o r 3-5 weeks.

187 REFERENCES 1.

Gordon, A.E., and Frigerio, A. (1972) J. Chromatogr. 73, 401-417.

2.

Falkner, F.C., Sweetman, B.J., and Watson, J.T. (1976) Biomedical applications of selected ion monitoring j n Applied spectroscopy reviews (E.G. Brame, ed.) Vol. 10, Marcel Dekker Inc., New York, 51-116.

3.

Björkhem, I. (1979) Selected Ion Monitoring in Clinical Chemistry. Critical Reviews in Clinical Chemistry, CRC-Press - in press.

4.

Björkhem, I., and Holmberg, I. (1976) Clin. Chim. Acta 68, 215-221.

5.

Björkhem, I., and Holmberg, I. (1978) J. Biol. Chem. 253, 842-849.

6.

Björkhem, I., and Holmberg, I. (1979) Methods of Enzymology - in press.

7.

Björkhem, I., Holmberg, I., Kristiansen, T., and Pedersen, J.I. (1979) Clin. Chem. - in press.

8.

Pedersen, J. I., Holmberg, I., and Björkhem, I. (1979) FEBS Lett. 98, 394-398.

9.

Björkhem, I., and Larsson, A. (1978) Clin. Chim. Acta 88, 559-567.

10.

Stillwell, W.G., Hung, A., Stafford, M., and Horning, M.G. (1973) Anal. Lett. 6, 407-419.

11.

Pedersen, J.I., Oftebro, H., and Vänngärd, T. (.977) Biochem. Biophys. Res. Commun. 76, 666-673.

12.

Sato, R., Atsuta, Y., Imai, Y., Taniguchi, S., and Okuda, K. (1977) Proc. Natl. Acad. Sei. USA 74, 5477-5481.

13.

Pedersen, J.I. (1978) FEBS Lett. 85, 35-39.

14.

Pedersen, J.I., Björkhem, I., and Gustafsson, J. (1979) J. Biol. Chem. - in press.

15.

Delvin, E.E., Arabian, A., and Glorieux, F.H. (1978) Biochem. J. 172, 417-422.

16.

Fukushima, M., Nishil, Y., Suzuki, M., and Suda, T. (1978) Biochem. J. 170, 495-502.

17.

Brumbaugh, P.F., Haussler, D.H., Bursac, K.M., and Haussler, M.R. (1974) Biochemistry U , 4091-4097.

18.

Hughes, M.R., Baylink, D.J., Jones, P.G., and Haussler, M.R. (1976) J. Clin. Invest. 58, 61-70.

19.

Eisman, J.A., Hamstra, A.J., Kream, B.E., and DeLuca, H.F. (1976) Arch. Biochem. Biophys. V76, 235-243.

20.

Belsey, R., DeLuca, H.F., and Potts, J.T. (1971) J. Clin. Endocrinol. Metab. 33, 554-557.

21.

Lambert, P.W., Syverson, B.J., Arnaud, C.D., and Speisberg, T.C. (1977) J. Steroid. Biochem. 8, 929-937.

189 ASSAY OF 1,25-DIHYDROXYVITAMIN D AND OTHER ACTIVE VITAMIN D METABOLITES IN SERUM: APPLICATION TO ANIMALS AND HUMANS

Mark R. Haussler, Marc K. Drezner*, J. Wesley Pike, John S. Chandler, and Laura A. Hagan. Department of Biochemistry, University of Arizona, Tucson, AZ and *Department of Medicine, Duke University, Durham, NC, U.S.A. It is known that vitamin D is metabolized first to 25-hydroxyvitamin D (25-(OH)D) and then subsequently to several dihydroxylated derivatives. Of the dihydroxylated forms, 1,25-dihydroxyvitamin D (1,25-(OH)2D) and 24,25-dihydroxyvitamin D (24,25-(OH)2D) appear to be the most significant, with the former being the calcemic hormone produced in the kidney during hypocalcemic or hypophosphatemic states (1) and the latter being elicited under normal mineral conditions (2). l,25-(OH)2D is the mediator of intestinal calcium and phosphate transport and of bone mineral resorption; the role of 24,25-(OH)2D is less well defined. The biosynthesis in kidney and total circulating level of 1,25-(OH)2D are regulated according to the mineral needs in higher organisms. Thus, low calcium (via PTH), low phosphate, and a number of hormones such as estrogens (3), prolactin (4), and growth hormone (5) all act, ¿n vivo, to stimulate the production and/or blood level of l,25-(OH)2D. Presumably, these latter endocrine factors modulate 1,25-(OH)2D during situations of calcium need, like growth, pregnancy, and lactation. Investigations of the action of l,25-(OH)2D in target tissues have led to the notion that this sterol functions analogously to the classic steroid hormones. Thus, it is thought that 1,25-(OH)2D binds to the genome and influences DNA transcription, with new gene products (proteins) then being functional in mineral translocation (1). Indeed, in chick intestine, a cytoplasmic receptor protein has been detected which translocates the 1,25-(0H)2D hormone into the cell nucleus (6). And that the nucleus is the ultimate site of action of 1,25-(OH)2D has been verified by autoradiography (7,8). Deriving from the above mentioned research, a number of clinical breakthroughs have occurred in the area of bone and mineral disorders. Syndromes normally attributed to antagonism, resistance, or hypersensitivity to vitamin D have been further defined in terms of alterations in vitamin D metabolism and end-organ responsiveness. One key to these advances has been the development of assays for measuring circulating concentrations of vitamin D metabolites. In the present chapter, we describe the refinement of assay techniques for 25-(OH)D, 24,25-(OH)2D, and 1,25(0H)2D. These refinements include the use of high performance liquid chromatography (HPLC) (9) and very high specific activity 1,25-(OH) 2 [ 3 H]D 3 (110 Ci/mmol) in conjunction with the intestinal receptor binding system for l,25-(OH)2D. The resulting sensitive and practical assays are then applied to experimental animals and to humans with various diseases of mineral metabolism. Emphasis is placed upon measurements of l,25-(OH)2D, and several new insights into the physiology, pathology, and pharmacology of this hormone are reported.

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

190 ASSAY METHODOLOGY The procedure for isolation and assay of the three vitamin D metabolites of interest is outlined below; 2-5 ml of serum or plasma are processed. A.

Tracer addition:

1000 CPM each of 25-(OH)[ 3 H]D 3 , 24,25-(0H) 2 t 3 H]D 3 and 1,25-(0H) 2 [ 3 H]D 3 ; Spec. Act = 80-110 Ci/mmol.

B.

Extraction:

Shake with 5 vol acetone, centrifuge, N 2 dry supernatant to 1-2 ml; Extract with 15 ml diethyl ether.

C.

Chromatography: 1.

Silicic Acid Pre-column: 3 ml column vol; elute metabolites with acetone after washing with 75% hexane in ether.

2.

Sephadex LH-20 Resolution: 6 ml column vol; elute with 65% CHCI3 in hexane. Metabolites elute as follows: 25-(0H)D (5-12 ml); 24,25-(OH)2D (12-20 ml); l,25-(OH)2D (20-40 ml).

3.

HPLC Purification (4.6 mm x 25 cm Zorbax-sil column; flow = 1 ml/min; final yields 60-80%). a) b) c)

D.

25-(0H)D: 5% isopropanol in hexane 24,25-(OH)2D: 10% isopropanol in hexane 1,25-(0H)2D: 15% isopropanol in hexane

Assays: 1.

25-(0H)D:

A 2 5^ quantitation (2 ml plasma).

2.

24,25-(OH)2 D:

3.

l,25-(OH)2D: Radioreceptor assay (2 ml plasma in triplicate) Frozen cytosol-chromatin (50 pg protein, 12.5 ug DNA/tube); 20 pg 1,25-(0H) 2 [ 3 H]D 3 plus sample incubated 1 hr at 25° and filtered as described in detail elsewhere (4).

quantitation (5 ml plasma)

In the procedure above, some contaminating lipids are removed via a silicic acid pre-column and the metabolites are then separated with a Sephadex LH-20 column. Figure 1 illustrates typical HPLC traces obtained when each of the three metabolites extracted from normal adult human serum are run in their unique solvent system of isopropanol in hexane. 25-(OH)D3 (Fig. 1, top panel) from 2 ml of serum migrates as an unambiguous peak at 13.5 min; identification is made by comigration with 25-(OH)[ 3 H]D 3 internal standard. No sterol migrating in the position of 25-(OH)D2 was detected in this individual. From other runs with known amounts of 25(0H)D3 and taking into account yield of tritium, the level of 25-(OH)D3 in this subject is calculated as 33 ng/ml. The normal human range for 25(0H)D established by this technique is 15—40 ng/ml and corresponds to data from binding assays (10) and from other HPLC assays (11). Similar quantitation of 24,25-(OH) 2 D 3 is also possible using a 10% isopropanol in hexane elution system (Fig. 1, center panel). However, concentrations of this metabolite are about 1/10 those of 25-(0H)D3, necessitating the use of 5-10 ml of serum to obtain a reasonable peak of absorbance at 254 nm. The normal range of 24,25-(OH) 2 D in humans is approximately 1-4 ng/ml which is consistent with earlier binding (12) and HPLC (13) assays of this metabolite.

191

Fig. 1. Direct HPLC assay of 25-(0H)D3 and 24,25-(OH)2^3; radioreceptor assay (RRA) of HPLC purified l,25-(OH)2l>3.

Fig. 2. Cytosol-chromatin competitive binding assay for l,25-(OH)2D.

l,25-(OH)2D3 circulates in far too low a concentration to be detected via uv absorbance (Fig. 1, bottom panel) and must be quantitated via radioreceptor assay (RRA) (14). Figure 2 illustrates the derivation and sensitivity of the cytosol-chromatin (14) radioreceptor assay when l,25-(OH)2_ [%]D 3 of specific activity 110 Ci/imnol is utilized. Saturation of the receptor occurs when 20 pg of labeled hormone is included in the incubation, yielding a composite dissociation constant (K

VITAMIN DRESISTANT RICKETS (N»2I)

VITAMIN O-OEPENOENT RICKETS! 22yr old 9 lyr R i with l,25-(0H) 2 0 3 elicit* normocolcemio and heals rachitic lotion*

TUMOR-INDUCED OSTEOMALACIA (N-7|

Fig. 5. Serum l,25-(OH)2D in osteomalacic syndromes.

9AM

IIAM

3PM

5PM

7PM

9PM

Fig. 6 Plasma 1,25-(OH)2D in vitamin D-refractory rickets treated with 1,25-(0H)2D3.

Another osteomalacic syndrome we have studied is tumor-induced osteomalacia (TIOM). Figure 5 shows that serum 1,25-(0H)2D is deficient in TIOM and Drezner and Feinglos (24) have shown that the hypophosphatemia and osteomalacia which attend this condition are ameliorated either by resection of the tumor or by treatment with l^S-iOH^D^ (also see Nortman et: al, this volume). Thus, certain tumors probably secrete a factor which inhibits l,25-(OH)2D production and/or blocks renal phosphate reabsorption. Therefore, aberrations in serum 1,25-(OH) D and deranged mineral and bone metabolism result from inherited (VDRR, VDDR) or acquired (CRF, IH, TIOM) renal defects, often those associated with abnormal phosphate reabsorption (IH, VDRR, TIOM). Parathyroid disease is another key factor in abnormal circulating 1,25-(OH)2D. We have been less successful in finding altered serum 1,25-(OH)^D in certain other instances where the hormone might be expected to be changed in humans. For instance, although l,25-(OH).D is markedly lowered in streptozocin diabetic rats (25 and Fig. 7), we nave observed normal serum 1,25-(OH)^T) in a mixed group of juvenile and adult onset diabetics (Fig. 7). The osteopenia of human diabetes is therefore not related to defective 1,25-(OH)^D formation, but could somehow still be related to other aspects of vitamin D metabolism (i.e., 24,25-(OH)„D). Also, although both pregnant and lactating rats exhibit elevated 1,25-tOH)_D(17), we have found increased 1,25-(0H)2D in human pregnancy but not lactation

(Fig. 8), at least at 6 weeks post partum.

195 •(106

Rot*

60

•

:

4 0

î

=

zo o

CM

E

0

40

•

» a

•

n Control

S 3

60

•

0

îocfn " Diabetic Diabetic Insulin

„

Humans

v o « O

v *

•

*

V •

•

#

v Ä

•

« o

• • • • • • •

PRL Tumor Post I m p l a n t Acromegaly Tumoral Calcinoci*

• «î

•

•

CE

:

•

20

I z

_

!

t

g * 9 ® Î î») a.

NORMAL DIABETIC N«2I N-IO

I 1

• g

L O s

g

i l

i

£

is

Fig. 7. Serum 1,25-(OH)^ in diabetes.

Fig. 8. levels.

Human l,25-(OH)2D

DeLuca and collaborators (2) have discovered a subtle but significant reduction of l,25-(OH)2D in postmenopausal osteoporosis, but as can be seen in Figure 8, we have not confirmed this finding. Our 25 subjects with crush fracture osteoporosis (obtained from Dr. Nordin) do not have abnormal 1,25-(OH)2D on the average. Perhaps this difference can be explained by more careful age-matching of controls by DeLuca (2) or by a patient subpopulation? Finally, in several other disorders (Figure 8), we have observed elevated l,25-(OH)2D. These include tumoral calcinosis, acromegaly and galactorrhea associated with prolactin (PRL) secreting tumor. These latter data are consistent with a positive effect of growth hormone and PRL on l,25-(OH)9D synthesis in humans. Of particular interest is the fact that one PRL tumor patient was treated with a radiation implant inserted intranasally to destroy the tumor and serum l,25-(OH)2D fell from 46 to 37 pg/ml (samples courtesy of Dr. Maclntyre). In conclusion, vitamin D metabolism and especially serum l,25-(OH)2D levels are altered in a number of disorders of mineral metabolism as summarized below: Tumoral Calcinosis t Primary Hyperparathyroidism t Hypervitaminose 0 4

I V I T A M I N D|

-

|25-(0H)D|

Idiopathic Hypercalciuria k

l,25-(0H)2D

Nutritional Osteomalacia •

Anticonvulsant Therapy •

Chronic Renal Failure f

Malabsorption •

Hepatobiliary Disorders +

Pseudohypoparathyroidism +

Hypoparathyroidism Vitamin D-dependent Rickets f Postmenopausal Osteoporosis t ( ? ) Tumor I n d u c e d Osteomalacia +

t

196 We have not as yet applied our 24,25^(0H}_2D assay (discussed above) to these diseases and other unsolved metabolic bone maladies, but we plan to do so in the future. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

Haussler MR and McCain TA (1977) N Engl J Med 297, 974-983; 1041-1050. DeLuca HF (1978) Arch Int Med 138, 836-847. Pike JW, Spanos E, Colston KW, Maclntyre I and Haussler MR (1978) Am J Physiol 235, E338-E441. Spanos E, Pike JW, Haussler MR, Colston KW, Evans IMA, Goldner AM, McCain TA, Maclntyre I (1976) Life Sei 19, 1751-1756. Spanos E, Barrett D, Maclntyre I, Pike JW, Safilian EF and Haussler MR (1978) Nature 273, 246-247. Brumbaugh PF and Haussler MR (1975) J Biol Chem 250, 1588-1594. Zile M, Bunge EC, Barsness L, Yamada S, Schnoes HK and DeLuca HF (1978) Arch Biochem Biophys 186, 15-24. Jones PG and Haussler MR (1979) Endocrinol, in press. Jones G and DeLuca HF (1975) J Lipid Res 16, 448-453. Haddad JG and Chyu KJ (1971) J Clin Endocrinol Metab 32, 992-995. Schaefer PC and Goldsmith R (1978) J Lab Clin Med 91, 104-108. Taylor CM, Hughes SE and de Silva P (1976) Biochem Biophys Res Commun 70.» 1243-1249. Lambert PW, Syverson BJ, Arnaud CD and Speisberg TC (1977) J Steroid Biochem 8, 929-937. Brumbaugh PF, Haussler DH, Bursac KM and Haussler MR (1974) Biochemistry 13, 4091-4097. Hughes MR, Brumbaugh PF, Haussler MR, Wergedal JE and Baylink DJ (1975) Science 190, 578-580. Rader JI, Baylink DJ, Hughes MR, Safilian EF and Haussler MR (1979) Am J Physiol, in press. Pike JW, Parker JB, Haussler MR, Boass A and Toverud SU (1979) Science, in press. Harmeyer J, Grabe Cv and Martens H (1977) in Vitamin D: Biochemical, Chemical and Clinical Aspects Related to Calcium Metabolism (AW Norman et^ al, eds.), de Gruyter, Berlin pp. 785-788. Fraser D, Kooh SW, Kind HP, Holick MF, Tanaka Y and DeLuca HF (1973) N Engl J Med 289, 817-822. Haussler MR, Hughes MR, Pike JW and McCain TA (1977) in Vitamin D: Biochemical, Chemical and Clinical Aspects Related to Calcium Metabolism (AW Norman et^ al, eds.) de Gruyter, Berlin, pp. 473-482. Haussler MR and Brickman AS (1979) in Disorders of Mineral Metabolism (F Bronner and JW Coburn, eds.) Academic Press, New York, in press. Bone HG, Zerwekh JE, Haussler MR and Pak CYC (1979) J Clin Endocrinol Metab, in press. Scriver CR, Reade TM, DeLuca HF, Hamstra AJ (1978) N Engl J Med 299, 976-979. Drezner MK and Feinglos MN (1977) J Clin Invest 60, 1046-1053. Schneider L, Schedl HP, McCain TA and Haussler MR (1977) Science 196, 1452-1454.

197 24,25-DIHYDROXYVTTAHIir D IN HUMAN SERUM Carol M.Taylor Department of Medicine, University of Manchester, U.K. Assay methods for the dihydroxylated metabolite vitamin D^, 24,25-dihydroxycholecalciferol (24,25-(0H)„D,), have been described by several groups of workers (1-7). Six of these are similar (1—6) in that they are all competitive protein binding assays, using either rachitic rat serum or kidney cytosol as the binding protein and tritiated 25-hydroxycholecalciferol (25-(OH)Dj)as the radioactive component of the assay. The difference between the methods lies in the preparation of the from serum prior to assay. The seventh method (7) is direct measurement of 24,25-(OH) 2 LJ in a lipid extract of serum by high pressure liquid chromatography (HPLC).

As table 1 shows two questions arise from the results from different groups of workers:a)

What is the normal circulating level of 24,25-(0H)2DJ in man?

b)

Is

24,25-(OH)2DJ

present in the sera of anephric patients?

Four of the groups report serum levels of 24,25-(0H)2Dj of the order of lOfc of the prevailing serum 25-(OH)Dj concentration.

The other three groups,

however, report substantially higher levels of 24,25-(0H)2DJ in serum. The reasons for these differences are not clear, but may be a result of the differing methods of serum extraction and chromatography before the assay step. The second question which arises is whether or not extrarenal 24-hydroxylation of 25-(OH)Dj takes place in man.

It was first thought that 24,25-

(Oil)2Dj was made exclusively in the kidney, but on the basis of the above assay results (2,3), studies of metabolism of radioactively labelled vitamin D^ in anephric man (8) and the production of 24,25-(0H)Dj from 25-(OH)Dj in an in vitro cartilage preparation (9),this has been questioned.

In order to investigate in our laboratory the possibility that 24,25-(OE)rPj

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

198 Table 1 Laboratory

Normal

Anephric

25-(OH)D.3

24,25-(0H)„D, n

Taylor, 35 Manchester,U.K.

18.8 t 9.3

1.73 - 0.93 12

Haddad, 42 S t . L o u i s , Misa.

24.3 - 9.6

3-7 - 1.3

9

20

26.9 - 8 . 4

2.1 Î 0.25

6

Hollis, Guelph,Ontario

4

21.8 i 5.7

5.0 Î 1.2

Kremer, Paris, Prance

10

11.9 - 6.6

6.9 i 3.3

Weisman St.Petersberg, Fla.

20

35.2 i 9.2

3.3 - 1.3

28.6

2.1

n

DeLuca, Madison,Wis.

Arnaud, 20 Rochester, Min.

2.1

0.5

Levels of 25-(0H)D, and 24,25-(0H)2D? in the serum of normal and anephric man as reported by various groups of workers. Mean - S.D. ng/ml serum may be made extrarenally, 6 anephric patients were dosed with 400 jig 25-(OIl)Dj and serum 25-(0H)Dj and 24,25-(OH) 2 DJ levels measured before and after dosage.

Even when serum 25-(OH)Dj concentration was much above

normal, little 24,25-(0H)2DJ was detected in the sera of these patients, confirming the kidney as the major site of 24-hydroxylation of 25-(0H)Dj (l).

These results were supported by a lack of production of ^H/^^C

labelled 24,25-(0H)2D^ from a double isotopically labelled dose of vitamin Dj given to four additional anephric patients (l). Measurement of vitamin D metabolite levels in hypoparathyroid pntient3 has shown that they have normal serum levels of both 25-(OH)Dj and 24,25-(0H)gDjj

when, however, such patients are undergoing therapy with

the hormone 1,25-dihydroxycholecalciferol (l,25-(OH)2Dj) their serum 24,25-(0H) 2 DJ levels became raised (Fig. l).

This, coupled with raised

serum levels of 24,25-(0H)„D, and l,25-(0H)pD, in primary hyperparathyroid

199 HYPOPARATHYROID PATIENTS

10 20 30 SERUM 2S|OH)Dj ng/ml

Fig.

1.

Serum

levels

of

25-(OH)Dj

and

24,25-(OH)2D^

in

hypoparathyroid

in

a

patients. 1.25-(OH)jD, Till/day 80o JS — 60

5Ï

Ts

40208-

n O f

«•
LEI 20 chromatography. 1,25-(0H) 2 D

Lower panel shows the elution profile of unlabelled

and 1, 2k, 25-(CH) D .

anol (85/15, v/v).

or 2 5 , 2 b - ( O H ) ^ , after

Column wa:. eluted with hexane/isoprop-

225 The mean (-SEM) serum concentration of 1,25-(GH)_D, in 2b healthy +

adults (mean age 32 years) was

'

- 2.5 pg^l- with a range of 22-59 pg/ml.

The coefficient of variation within assays wai: 9% and betv/een assays Further valdiation of the assay was obtained by the finding of undetectable ( < 5 pg/ml ) concentrations of 1,25-(0H) D

in anephric patients, unless

they were treated with 1a-hydroxycholecalciferol which is hydroxylated in the liver to 1,25-(QH) 2 D . The application of the radioimmunoassay to the measurment of circulating 1,25-(OH^¿Pj,

man

a

l r e a £ iy been extremely valuable.

For

example, in five patients with Vitamin D deficiency and histologically proven osteomalacia serum 1,25-(0H)^D^ concentrations were found to be below the lower limit of the normal range and were from < 5 to 21.5 Pg/ml. All these patients had low serum 25-OHD concentrations which ranged between < 0 . 8 and 2.b ng/ml.

It was also possible to follow the changes of

circulating 1,25-(OH)_D_ 2 3 concentrations in response to treatment with synthetic 1,25-(0H) o D^ in tv.'o of these patients. It was shown that increases in the serum concentrations of 1,25-(0H)^D^ between 50-90 pg/ml were associated with dramatic symptomatic improvement and healing of the bone disease on a dose of .S^E twice a day. To investigate further the kinetics of exogenously administered 1,25 -(GH)^

its circulating concentration was monitored after an oral dose

of 2ng.

In normal subjects the absorption of orally administered 1,25 (OH)

D^ was shown to be rapid and the concentration rose to approximately ito pg/ml within three hours.

By 2b hours the concentration was almost back

to basal levels. No changes in serum calcium were observed during this period.

In contrast, in a preliminary study in a patient with severe

chronic renal failure, the disappearance of exogenous 1,25-(OH)^D

from

the circulation war; delayed, indicating the possible importance of further metabolism of this compound. Using the radioimmunoassay, it has been recently shown (3) that the development of hypercalcaemia in sarcoidosis is associated with abnormally high concentrations of 1,25-(0H)^D 7 .

A serum concentration of approximat-

ely ikO pg/ml was observed in a patient with sarcoidosis when h- became spontaneously hypercalcaemic and was reproduced in the same patient by administration of a very low dose ( 3300 units daily) of Vitamin D_,.

These

changes occured as a result of small rises in the concentration of 25-OHD^

226 entirely within the normal range.

In another patient with

hypercalcaemic sarcoid who was studied while normocalcaemic, similar changes were demonstrated after administration of 3t000 units of Vitamin D^ daily for 10 days (Fig. 6.).

The inappropriately high concentrations

of 1,25-(0H^Dj account, therefore, for the apparent hypersensitivity to Vitamin D in sarcoidosis.

Whether this is due to overproduction of the

metabolite, or to a failure of its further metabolism, remains to be established. P r e d n i s o n e 5mg/d

W f f f r Vit. D 3 3000 U/d

2.20

L

160 Serum

l,25-(OHI2D3 IpgI mil

0L

i

0

1

1

30

60

90

Days

Fig. 6.

The changes of serum calcium and 1,25-(OH)^D^ in a patient with

sarcoidosis, after administration of Vitamin D^ 3iOOO units daily for 10 days.

Over this period serum 25-OHD^ concentrations were always within the

normal range and rose from9-25ng/riL during Vitamin D^ treatment. with prednisone 5 mg/daily was kept constant throughout. show normal ranges.

Treatment

Stippled areas

227 Very little is known of the role of S S ^ b - C O H j ^ D ^ i n man.

Assays

for this metabolite may therefore help to elucidate its physiological significance.

It has recently been possible to measure the circulating

concentrations of 25,2b- ( O H ) ^

in normal subjects by radioimmunoassay.

Labelled 25,2b- (OH)pD^, was produced biologically and war; used to monitor the recovery of this metabolite through extraction and purification on LH 20 and HPLC.

The reference preparation used in these assays vas a

racaemic mixture of the two stereoisomers of this compound. calibration curve is shown in Fig. h.

A

In normal subjects a mean serum

concentration of 276 pg/ml (range 133-377 pg/ml) was found. Clearly, therefore, the antibodies produced have provided a stable versatile tool for the study of the importance of hydroxylated derivatives of Vitamin D in man and of the regulation of their production. Moreover, there is good oportunity for still further improvement. For example, by immunizing more animals and using different immunogens, antisera of varying structural specificity could be produced.

Fraction-

ization of the antisera is also feasible and might be advantageous. ACKNOWLEDGEMENTS The financial support of the Wellcome Trust is gratefully acknowledged. REFERENCES 1.

Clemens, T.L., Hendy, G.N., Graham, R.F., Basgiolini, E.G., Uskokovic, M.R., and O'Riordan J.L.K., (1978) Clin. Sci. Mol. Med. 5ft, 329-332.

2.

Clemens, T.L., Hendy, G.N., Papapoulos, S.E., Fraher, L.J., Care, A.D., and O'Eiordan J.L.H., (1979) Clin. Endocrinol. In Press.

3.

Tapapoulos, S."., Clemens, T.L., Fraher, L.J., I.ewin, I.G., Sandler, L.M., and C'Riordan, J.L.H., (1979) Lancet, In Press.

229 HEMISUCCINATES OF VITAMIN D 3 AND OF I T S

METABOLITES

K. Lichtwald, E. Mayer 1 , H. Schmidt-Gayk , J. 1Varga 2 and S. Walch1 University of Heidelberg, Department of Pharmacology, Im Neuenheimer Feld 366, D-6900 Heidelberg, F.R.G. 1 Klinisch-Chemisches Labor, Medizinische Universitäts-Klinik, Bergheimer Str. 58, D-6900 Heidelberg, F.R.G. ^Medizinisch-Chemisches Institut der Medizinischen Universität Szeged, Hungary There is still a gap of high quality antisera against vitamin D and its metabolites and also a shortage of them. For raising antibodies against haptens by immunization, it is necessary to make them immunogenic. This is usually done by coupling the low molecular substance covalently to a carrier protein. Prior to coupling, the haptens often have to be derivatised. For vitamin D and its metabolites, hemisuccinates were synthesized and bound to proteins. Gemeiner (1) used the D^-hemisuccinate (D^-HS) and found surprisingly low crossreaction of D^ with the antiserum gained. Fairney et al. (2) reported success in raising antisera against the antigens of D^ HS and 1,25-dihydroxycholecalciferol-25-hemisuccinate

(1,25-

(HO)2"D3"25-HS) . With the loC, 25- (HO)2-D3~25-HS antigen also Clemens et al. (3) communicated successful immunization. They obtained an antiserum of high affinity to the hapten used. One of the most important points in preparing steroid antisera is the need to purify and characterize the steroid derivative before conjugation. Impurities, especially such as isomers, e.g. eis - trans or linked at different positions, may result in less specific antisera. This danger is extremely high for the sensitive compounds around vitamin D. Abbreviations 1o6, 25- (HO) 2 -D 3 : (1oi, 3ß, 5Z , 7E) -9 ,10-Secocholesta-5, 7 ,10(19)triene-1,3,25-triol 25-HO-D3 : (3ß,5Z,7E)-9,10-Secocholesta-5,7,10(19)triene-3,25-diol D3 : (3ß,5Z,7E)-9,1O-Secocholesta-5,7,10(19)triene-3-ol

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

230 The esterification of D 3 , 25-HO-D3 and 1«t,25-(HO)2_D3 with succinic anhydride is the conventional procedure of Schotten-Baumann.

OC-CHJCHJCOOH

R1 = R1 = „1

H; R 2 = H H; R 2 = OH OH; R 2 = OH

2 5-HO-D1eC, 25- (HO) 2 -D 3

The reactions were run at room temperature under nitrogen and in darkness. The reaction time varied from six days to several weeks. The isolation of the hemisuccinates resulted from column or TLC chromatography. D-.-HS: UV. (EtOH; X= 265 nm) £= 18150. NMR. (CDC13) between 7.0 and 0.0 ppm identical with the starting material, except the signal area around 2.0 ppm, where both methylene groups of the succinate part have their resonance. 1R. (KBr) 3090 (C=C-H) 3040 (C=CH2), 2950-2850 (COOH broad), 1740-1710 (COOH and MS. M + - 484. COOR), 1635 (C=C)cm~1. 25-HO-D3-HS(4): NMR. (CDCl-j) the same as U V. (EtOH; X= 265 nm) £= 18220. above. IR. (KBr) 3095 (C=C-H), 3045 (C=CH2), 3400 (HO broad), 2960-2860 (COOH broad), 1740-1710 (COOH and COOR), 1650 and 1600 (C=C)cm~1. MS. M + ' 500. 1oc,25- (HO) 2-D3-HS: Caused by shortage of material UV, NMR and IR characterization was not carried out. MS. M +- -18 (498) .

= OS «O)

Ul 00

100

50

150

u

Sn

200

a

J^J-

250

300

w

°>

i

350

400

450

3o 8 o ui (0

25-OH-D3

Ul

fo 10

a>

100

50

Ul 20 (ng/ml) 1,252D fraction eluted from the Sephadex LH 20 column was rechromatographed on HPLC as described (1).

Results indicate that the 24,25-(OH) 2 D formation is mainly found in the earlier released cell populations, denoted as CT type cells by Luben et al. (8). The lower ability to convert 25-(OH)D3, found in the later isolated populations, is not secondary to a longer exposure to collagenase + trypsin before cell culture.

II IN VITRO REGULATION OF THE CHONDROCYTE 25-(OH)D3 - 24-HYDROXYLASE ACTIVITY.

The experimental protocol followed and the techniques used for this study are schematically represented on figure 2.

394 CULTURED CHONDROCYTES AT THE END OF THE LOGARITHMIC PHASE OF THE GROWTH CURVE (6.10 6 cells/flask)

INCUBATED 2 4 HOURS IN 10 ml MEM - without FETAL C A l f SERUM - with C A L C I U M ACETATE,

Ca

I ETHANOL (0.1%) at ho - with ¡ l , 2 5 - ( O H ) 2 D 3 (10"8M> at ho ( bPTH 1-84 ( 1 0 " % at h o , h 1 0 ,

h23

TRANSFERRED TO GLASS V I A L INCUBATED 1 HOUR IN 4 ml MEM 37 C - with 26.27- 3 H 25-IOHID3 (10-?M, 2 x 1 0 5 dpm)

CELL PELLET EXTRACTED WITH

METHANOL-CHLOROFORM

CHLOROFORM PHASE CHROMATOGRAPHED ON 0 . 5 x 14 cm S E P H A D E X LH 20 COLUMN 55* CHLOROFORM - n H E X A N

24 25-(OH)2D REGION CHROMATOGRAPHED ON HPLC SYSTEM (WATERS, JUBONDAPAK C 1 8 COLUMN) 50-100% METHANOL-WATER

Figure 2 Preincubations w i t h different calcium concentrations did not greatly influence the 25-(OH)D2 drocytes

-

24-hydroxylase activity measured in cultured chon-

(figure 3). Moreover, this activity could be easily detected even

after preincubation w i t h low calcium concentrations has b e e n found w i t h renal tubules

(0.3 mM) unlike what

(7).

The inhibitory effect of parathyroid hormone described in cultured kidney cells

(5) was not found in cartilage cells under the present

conditions. Finally,

l^S-COH^D^

experimental

greatly enhanced the 24-hydroxylation of

25-(OH)D2 in cartilage as in kidney (5,6,7). Yet this effect was found most evident w h e n the presence of 1 ^ S - i O H ^ D ^ w i t h low calcium concentrations.

in the m e d i u m was

associated

395 24.25-(0H)2D3 ( IO"15 mol/60min/106cells ) • Ethanol * PTH (10"8M)

20-

a1.25-(OH)2D3(10"bM)

16-

12-

0.3

1

1.5

3 ( m M Ca+*

0.3

1

1.5

3 ( m M Ca+*

Figure 3 Figure 3 : 25-(OH)D3 - 24-hydroxylase a c t i v i t y in c u l t u r e d chondrocytes incubated for 24 h o u r s in M E M m e d i u m c o n t a i n i n g no a l b u m i n and no fetal calf serum, d i f f e r e n t c a l c i u m c o n c e n t r a t i o n s a n d either 1,25-(OH)2^3> its ethanol solvent or 1-84 bovine p a r a t h y r o i d h o r m o n e (a generous gift from Dr. J.D. P a r s o n s , N . I . H . R . , L o n d o n , England). F o r e a c h c a l c i u m c o n c e n t r a tion, are r e p r e s e n t e d the m e a n and extreme values of two d i f f e r e n t experiments. See experimental procedures o n figure 2.

COMMENTS. Study o n the 2 5 - ( O H ) D 3 c o n v e r s i o n to 2 4 , 2 5 - ( O H ) 2 D 3

in c a r t i l a g e and c a l -

v a r i u m seems especially interesting as the p r o d u c e d h y d r o x y l a t e d

deriva-

tive has b e e n shown to be m o r e active than its p r e c u r s o r 2 5 - ( 0 H ) D 3 these tissues

in

(9, 10). Present results indicate that, in c a l v a r i u m , the

ability to synthetize 2 4 , 2 5 - ( O H ) m a y

be specific of some cell

types

and give preliminary informations on these cells, w h i c h m i g h t prove to be useful for further

identification.

C o m p a r i s o n of the k i d n e y and cartilage 2 5 - ( O H ) D 3 - 2 4 - h y d r o x y l a s e

activi-

ties in r e l a t i o n to the in vitro p r e s e n c e of regulatory factors brings

396 out some differences w h i c h may be of importance in so far as they are not merely secondary to differences in the experimental procedures. B o t h activities are stimulated in the presence of 1 ^ S - C O H ^ D ^ , yet their sensitivity to calcium in the extracellular m e d i u m appears to be different. It is to be remembered that kidney and cartilage cells are very different w i t h respect to their calcium physiological environment and to their calcium sensitivity. One cannot therefore rule out the possibility that b o t h hydroxylases are sensitive to similar intracellular

factors.

The in vitro data demonstrating a stimulation of the 25-(0H)D2 - 24-hydroxylase by low calcium concentration in cartilage b u t not in kidney m a y be used to explain the difference observed b e t w e e n these two tissues,when isolated from chicks fed a v i t a m i n D deficient diet. One could thus propose that in these animals the renal 25-(0H)Dj - 24-hydroxylase is turned off because of secondary hyperparathyroidism and hypocalcemia w h i l e the cartilage enzyme is stimulated b y low calcium concentrations

(and possibly

by high parathyroid hormone levels). Finally, further works are necessary to ascertain the influence of the in vitro observed regulation for the 24,25-(0H)2D3 formation in cartilage on the circulating levels of this metabolite. It is in fact possible that part of the 24,25~(OH)2D found in the serum may derive from cartilage and/or other extrarenal sites of formation, in some clinical Such situation may be the one published elsewhere

situations.

(11) in w h i c h elevated

serum 24,25-(OH)2D concentrations have b e e n found in a child with pseudodeficiency rickets at the beginning of a la-iOlOD^ therapy even w h e n serum calcium, phosphorus and parathyroid hormone levels had entered the limits of the normal ranges.

REFERENCES. 1. Garabedian, M . , Bailly du Bois, M . , Corvol, M . T . , Pezant, E. and Balsan, S. (1978) Endocrinology. \02_, 1262-1268. 2. Garabedian, M. , Lieberherr, M., NGuyen, T.M., Corvol, M.T., Bailly du Bois, M. and Balsan, S. (1978). Clin. Orthop. Rel. Res. 135, 242-248. 3. Kumar, R., Schnoes, H.K. and DeLuca, H.F. (1978). J. Biol. Chem. 253, 3804-3809. 4. Knutson, J.C. and DeLuca, H.F. (1974). Biochemistry. K3, 1543-1548. 5. Juan, D. and DeLuca, H.F. (1977). Endocrinology. _[£]_, 1184-1193.

397 6. Henry, H. (1977). Biochem. Biophys. Res. Commun. 74, 768-774. 7. Omdahl, J.L. and Hunsaker, L.A. (1978). Biochem. Biophys. Res. Commun. 8J_, 1073-1079. 8. Lüben, R.A., Wong, G.L. and Cohn, V.D. (1976). Endocrinology. 99, 526-534. 9. Corvol, M.T., Dumontier, M.F., Garabedian, M. and Rappaport, R. (1978). Endocrinology. H)2, 1269-1274. 10. Lieberherr, M,, Garabedian, M., Guillozo, H., Bailly du Bois, M. and Balsan, S. Calcif. Tiss. Res. Int. (in press). 1 1 . NGuyen, T.M., Guillozo, H., Garabedian, M., Mallet, E. and Balsan, S. Pédiat. Res. (in press).

399 COMPARISON O F THE HISTOLOGICAL EFFECT AND METABOLISM 2 5 - ( O H ) D .AND l , 2 5 - ( O H ) 2 D IN R A T B O N E .

OF

J.A. Gallagher and D.E.M. Lawson Dunn Nutrition Unit, Cambridge, F o l l o w i n g t h e s t u d i e s of B o r d i e r e t al 1 9 7 8 / i 1 ) a n d E d e l s t e i n et a l . 1 9 7 8 / ( 2 ) o n t h e i n e f f e c t i v e n e s s o f 1 , 2 5 - ( 0 H ) 2 D in h e a l i n g all t h e b o n e lesions of v i t a m i n D deficient human subjects and rachitic chicks respectively, w e h a v e c a r r i e d o u t s t u d i e s to i n v e s t i g a t e p o s s i b l e e x p l a n a t i o n s for these findings. Methods G r o u p s of 6 r a c h i t i c r a t s w e r e e i t h e r m a i n t a i n e d o n a l o w P diet or t r a n s f e r r e d to a diet w i t h a higher P level of 0 . 6 5 % a n d g i v e n d o s e s o f l O O n g o r 2 0 0 n g of e i t h e r 2 5 - ( 0 H ) D or 1 , 2 5 - ( 0 H ) 2 D . Growth rate and plasma chemistry w a s followed and the histological changes in b o n e recorded by the s t a i n i n g p r o c e d u r e of T r i p p and M a c k a y , 1972 (3). In other s t u d i e s (1,2-3H) vitamin D w a s a d m i n i s t e r e d to rats and the p a t t e r n of m e t a b o l i t e s m e a s u r e d after 15h and 45h. The m e t a b o l i s m of a daily dose of lOOng of L , 2 5 - ( O H ) 2 D in r a c h i t i c rats w a s followed for 9 days and the c h a n g e s occurring w i t h i n the 24h period b e t w e e n doses recorded for the 2nd and 9th day. P l a s m a v o l u m e of b o n e s w a s m e a s u r e d with 125I-albumin. Results T a b l e 1 s h o w s that l , 2 5 - ( O H ) 2 D i n c r e a s e s the % b o n e ash as reported previously by others but 25-(OH)D w a s found to b e even m o r e effective. Interestingly raising dietary P level also raised the % bone ash without further treatment. Again the osteoid v o l u m e and o s t e o i d surface w e r e d e c r e a s e d by both steroids although a much greater effect was observed if P i n t a k e w a s a l s o r a i s e d . The most successful treatm e n t o f t h e v i t a m i n D - d e f i c i e n c y i n t h e s e r a t s w a s l O O n g of 25-(OH)D with a raisedPi intake. I n c r e a s i n g the dietary Pi level alone r e s u l t e d in a d e c r e a s e in o s t e o i d volume and osteoid s u r f a c e b u t the epiphyseal plate had an abnormally thin appearance and a defective b l o o d supply. Interestingly an i n c r e a s e d d i e t a r y P i i n c o m b i n a t i o n w i t h l , 2 5 ( O H > 2 D treatment w a s less effective in reducing o s t e o i d volume and osteoid surface than increased Pi alone but resulted in a more normal epiphyseal plate. T h e ineffectiveness of 1,25-(0H)2D in h e a l i n g rickets fully w a s not due to its r a p i d m e t a b o l i s m s i n c e in o t h e r e x p e r i m e n t s it w a s s h o w n t h a t l O O n g of t h e s t e r o i d m a i n t a i n e d a p l a s m a l , 2 5 - ( O H ) 2 D l e v e l at l e a s t as h i g h as r e p o r t e d

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Researdi and its Clinical Application

koo n o r m a l v a l u e s (4) t h r o u g h o u t t h e 2 4 h p e r i o d b e t w e e n d o s e s . A two fold i n c r e a s e in d o s e of l , 2 5 - ( O H ) 2 D w a s m o r e e f f e c t i v e t h a n the l o w e r d o s e b u t h e a l i n g w a s s t i l l far from complete. Table 2 p g / g of B o n e * 1,25-(OH)2D 45hrs

25-(OH)D Vit D *

C o r r e c t e d for p l a s m a

pg/mgDNA

It.97

3.71

10.81+

8.11

5.85

3.56

radioactivity

T h e n a t u r e of t h e m e t a b o l i t e s i n b o n e d u r i n g t h e p e r i o d of t r e a t m e n t s h o w e d that 15h a f t e r a d o s e of v i t a m i n D no m o r e l , 2 5 - ( O H ) 2 D w a s p r e s e n t in b o n e t h a n c o u l d b e a c c o u n t e d for by p l a s m a c o n t r i b u t i o n . A f t e r 4 5 h the a m o u n t of 1 , 2 5 - ( 0 H > 2 D p r e s e n t in b o n e w a s s i g n i f i c a n t l y less t h a n that p r e s e n t in i n t e s t i n e e v e n w h e n r e l a t e d to D N A l e v e l s in t h e s e t i s s u e s (Table 2). T h e m a j o r m e t a b o l i t e in b o n e at all t i m e i n t e r v a l s was 25-OHD. C o n s i d e r i n g that l , 2 5 - ( O H ) 2 D is r e p o r t e d l y e f f e c t i v e in b o n e m o b i l i s a t i o n t h e s e low levels of 1 , 2 5 - ( 0 H ) m u s t b e a d e q u a t e for this f u n c t i o n at l e a s t . 24,25-(OH)2D w a s not d e t e c t e d i n b o n e d u r i n g the i n i t i a l p h a s e of h e a l i n g after vitamin D treatment. Reference 1.

B o r d i e r , P . , R a s m u s s e n , H., M a r i e , P . , M i r a v e t , L., G u e r i s , J. a n d R y c k w a e r t , A. ( 1978 )J. Clin. Endoc. Met. U6.28U-291».

2.

E d e l s t e i n , S., G o o d w i n , D.,' M e s s e r , Y . a n d G h a z a r i a n , (1978) P r o c 3rd I n t e r - W o r k s h o p o n c a l c i f i e d t i s s u e s (abstr), Israel.

3.

T r i p p , E . J . , and M a c k a y , E . H . 47 1 2 9 - 1 3 6

4.

W e s p i k e , J., T o v e r u d , S., B o a s s , A., M c C a i n , T., and H a u s s l e r , M . R . (1977) P r o c 3rd I n t e r . W o r k s h o p o n V i t . D USA.

J.G.

(1972) S t a i n T e c h n o l .

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ft

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*i0 3 ACTIOS OF SOLANUM MALACOXYLON OM BONE HISTOLOGY OF VITAMIN D DEFICIENT HATS

M.C. de Vernejoul, M.L. Queille, B.W. Nordin, C.A. Mautalen and L. Miravet Unite de Recherche, INSERM N group IV received from the time of weaning to the end of the experiment, 175 ng of vitamin D3 per oa twice a week, to provide an average supply of 2 IU of cholecalciferol per day (positive D fed controls). Twenty four hours after the last dose of either compound, blood was drawn by cardiac puncture, the animals were killed, and the femur and caudal vertebrae were removed. Serum calcium concentration was measured by atomic absorption spectrophotometry. The ninth and tenth caudal vertebrae were embedded in methyl methacrylate and a serie of thin, undecalcified sections (5u) was obtained for quantitative histology, as described elsewhere (2). RESULTS At the time of sacrifice, serum calcium levels were as follows* D deficient controls ( A v t l S E M ) 3.5-0.2* S. Malacoxylon* 8.5±0.2J 25-HO-D3* 9 - 3 + 0 . 2 and D fed controls 1 0 . 0 + 0 . 1 mg/100 ml in all cases. The levels were significantly higher in all treated groups, as compared with the D deficient controls. However, the administration of SM did not restore serum calcium to the level observed in the D fed group (p o +->

CM sE 0) E -O o E 3 c -— z

ce

-a •t— o cu 4-> V) O

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koG The present study does not exclude completely the possibility that the failure of SM to induce a significant stimulation of bone resorption might have been due to the level of dosage given. To clarify this point a suitable assay of the l,25(HO)2D3 potency of the SM should be performed and then,the biological effectB of both compounds should be compared under similar conditions. However, the differences in the biological action between SM and l,25(HO)2D3 found in other studies (10, ll) and infered by the data reported here, suggest that the presence of other metabolites of vitamin I) and/or the sugar molecules attached to its active core, might modify the action of the naturally occuring 1,25(H0)2D3. REFERENCES 1 - WaBserman, R.H., Henion, J.D., Haussler, M.R. and Mc Cain, T.A. (1976) Science 121, 853-855 2 - Bordier, P. and Tun Chot, S. 3 - 219-226 Puche, R. C. and Locatto, M.E.

(1972) J. Clin. End. 1, 197-215 (1974) Calcif. Tiss. Res. 16,

4 - Moorhead, J.F., Humphreys, D.J., Varghese, Z.t Basudde, C.D.K., Jenkins, M.V. and Wills, M.R. (1975) in "Vitamin D and problems related to uremic bone disease" Walter de Qruyter ed., Berlin 725-730 5 - Mautalen, C.A.

(1972) Endocrinology 22.» 563-567

6 - Uribe, A., Holick, M.F., Jorgensen, N.S. and De Luca, H.F. (1974) Biochem. Biophys. Res. Commun. ¿8, 257-262 7 - Ladizesky, M., Fainstein Say, P. and Mautalen, C.A. Medicina (Buenos Aires) 38, 341-346

(1978)

8 - Puche, R.C., Locatto, M.E., Ferretti, J.L., Fernandez, M.C., Orsatti, H.B. and Valenti, J.L. (1976) Calcif. Tiss. Res. 20. 105-119 9 - Queille, M.L., Miravet, L., Bordier, P. and Redel, J. Biomedicine 28, 237-242

(1978)

10- Miravet, L.-, Carre, M., Fisher-Ferraro, C. and Mautalen, C.A. (1977) J.Clin. End. 1230-1234 11- Kraft. £., Herrath, D. Von, Schaefer, K., Wagner, A. and Ott, E. (1977) in "Vitamin D» chemical and clinical aspects related to calcium metabolism", Walter de Gruyter ed., Berlin, 441

407 THE EFFECT OF V I T A M I N D ON THE C O M P O N E N T S OF THE G R O U N D SUBSTANCE

CARTILAGE

1. Foldes , M. K e r n + , and J.A. P a l f r e v + + Department of Anatomy, M e d i c a l University,H-4012 Debrecen,Hungary . ++Department of A n a t o m y , St.Thomas's Hosp., L o n d o n . U.K. Few and contradictory data are available in the literature on the effect of v i t a m i n D on the ground substance of dense connective tissues /l, 2, 3, 6, 7, 7, 9/. In our earlier w o r k /5/ using polarization m i c r o s c o p e for dem o n s t r a t i o n of the ground substance components, g l y c o s a m i n o glycans /GAGs/ and collagen significant differences were found b e t w e e n the structure of these components w h e n investigated in the physiologically mineralizing and non-mineralizing cartilages . In the present experiments further information w a s obtained on the changes of the ground substance components induced by vitam i n D. M a t e r i a l and m e t h o d s Hypervitaminosis was produced in young Wistar rats /100 g/ by administering differents doses of vitamin D /40, 200, 1 000, and 10 0 0 0 IU pro die/ for 12 days through a stomach tube. Hypo^kaminosis w a s induced by keeping the animals on the Steenbock-Black diet for 8 w e e k s . The following tissues were subjected to histological investigations: growing /epiphyseal/ and superficial /articular/ cartilages of vertebrae and tibiae, costal and nasal cartilages and annulus fibrosus of intervertebral discs. Light microscopical histochemistry for demonstration of G A G s and glycoproteins /GPs/ by alciane-blue-PAS staining. Polarization m i c r o s c o p i c a l histochemistry /topo-optical reactions/. 1. Staining with 0,1% toluidine~blue /Chroma/ at p H 3,5 for 10 min. /Romhanyi, 1963/. In some cases testicular hyaluronidase digestion, or 0,4 - 0,7 M MgCl2 w a s added for the demolishtration and identification of different G A G s c o m p o n e n t s /chondroitin sulfates and keratan sulfate/. 2. Phenol reaction /Ebner 1894/ w a s used for collagen d e m o n s t ration. E l e c t r o n m i c r o s c o p i c a l investigations were carried out w i t h standard techniques. E l e c t r o n microscopical results. Significant u l t r a s t r u c t u r a l differences exist b e t w e e n annulus fibrosus, articular, costal cartilages and epiphyseal cartilage under normal c o n d i t i o n s . These differences w h i c h were found in the structure of intracellular organells and in the structure and composition of the extracellular matrix, w e r e partly reduces by v i t a m i n D treatment. In hypervitaminosis the clear area appears around the cell in the articular cartilage /Fig. lb./. In all tissues investigated the interfibrillar matrix distends at the expense of collagen fibers. Like in the cells of the e p i p h y s e a l c a r t i lage of untreated aniftials, the number of Golgi vesicles is increased in the cells of other cartilage tissues as w e l l /Figs, la, b, 2. /.

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

4o8

Fig.l. Middle zone of articular cartilage, a. control, b. 10 OOO IU vitamin D treatment. Fig. 2. Middle part of control epiphyseal cartilage. Scales indicate 1 ^im. Results after toluidine-blue reactions. In hypervitaminosis /10 000 IU vitamin D/ the orientation of GAGs is increased in costal and nasal cartilages /Figs. 3c, 4c,/ as compared with controls /Figs. 3b, 4b./. The toluidine blue reaction after hyaluronidase digestion or with the critical electrolyte concentration technique suggests the predominance of keratan

409 sulfate.Similar effect was evoked by lower doses of vitamin D, but the differences were not as conspicuous, they were not visible in all cartilages investigated. In hypovitaminosis D an increased birefringence induced in the costal cartilage /Fig. 3a/ indicates a high orientation of GAGs which were represented mainly by chondroitin sulfates in this case.

Figs. 3a,b,c, Costal cartilage. P= perichondrium. S=superficial, D=deep zones. Figs 4b,c. Nasal cartilage. Topo-optical reaction after toluidine blue staining. Fig. 3b, Normal costal cartilage. The perichondium is strongly birefringent. The underlying superficial zone of the cartilage shows a weak, and in deeper zone a fair birefringence. Fig. 4b. Normal nasal cartilage. The birefringency shows a network-like distribution. Scales indicate 100 um in Figs. 3a, b, c. and 50 fim in Figs. 1 4b, c. Results after phenol reaction. In hypervitaminosis the optical anisotropy of the cell collagen decreases in the annulus fibrosus /Fig.5c/, and in the superficial and growing cartilages of vertebrae /Fig. 6c/, indicating a decrease of collagen orientation in these structures as compared with controls /Figs. 5b, 6b/. In hypovitaminosis the optical activity decreases in the superficial cartilage of vertebrae /Fig. 6a/, but the orientation of the collagen is increased in the annulus fibrosus Fig. 5a/. From the results it may be concluded that hypervitaminosis D induces a series of alterations in the submicroscopic structure and composition of different cartilages. While the spatial orientation of the GAGs /chiefly keratan sulfate/ is increased, the orientation of the collagen is decreased. Matrix areas containing glycoproteins are extended. In hypovitaminosis D the orientation of GAGs is also enhanced, but mainly the

¿110

Figs.5a,b,c. Annulus fibrosus, Figs 6a,b,c. Superficial /S/ and growing /G/ cartilages of a vertebra, A= annulus fibrosus. Tòpo optical reactions with phenol. Scales indicate 50 pm. chondroitin sulfates. Vitamin D is appeared to be involved in the regulation of the matrix formation; and consequently it can modify the submicroscopic structure of the matrix macromolecules, hereby influencing the mineralization. The increase of Golgi vesicles in hypervitaminosis D is suggestive of an increased biosynthesis of GAGs. References 1./ Canas,F.M., Brand,J.S., Neuman,W.F., Terepka,A.A. /1969 / Amer.J.Physiol. 216, 1092-1096. 2.1 Cipera,J.D., Migikovszky,B.B., Bélanger, L.F. /1960/ Canad.J.Biochem. 38, 807-811. 3.1 Dikshit,P.K. /19597""Nature /Lond/ 183, 334-335. 4./ Ebner V.v. /1894/ Akad.Wiss.Wien, math.nat. K1.103,162-188. 5.1 Földes,I., Kern,M., Matesz,K. /1975/ Biologia 23, 45-54. /In Hungarian/. 6./ Hjertquist, S.O., Bergquist,E., Sevastikoglou;J.A./1970/ Acta orthop.Scand. suppl. 136, 55-72. 7./ Mechanic,S.L., Toverud,S.U., Ramp, W.K., Sonnerman,W.A. /1975/ Biochim.Biophys. Acta 393, 419-425. 8.1 Paterson,C.R., Fourman,P. /1968/ Biochem.J.109, 101-106. 9.1 Rasmussen, H., Feinblatt,J. /1971/ Calcif. Tiss. Res.JL 265-279. 10./ Romhânyi,G. /1963/ Acta histochem. /Jena/ 15, 201-233.

k \ i DUAL ACTION OF PARATHYROID HORMONE ON THE MINERALIZATION OF RAT INCISOR DENTIN.

S. Matsumoto, T. Tsudzuki, and

M. Yamaguchi

Department of Pharmacology, School of Dentistry, Aichi-Gakuin University, Nagoya, 464 JAPAN.

In rats, the incisor dentin requires significant amounts of Ca (and POi») for its mineralization because of its continuous growth.

It is n o w well

known that the serum Ca levels are mainly controlled by 1,25 dihydroxycholecalciferol

(l,25(OH)2D3) and parathyroid hormone

(PTH).

However,

there are few studies on the hormonal regulation of dentin growth at present.

Therefore, an investigation of the roles of these hormones on

the mineralization of incisor dentin has b e e n carried out in parthyroidectomized (PTXed) rats maintained on a v i t a m i n D deficient diet.

When the parathyroidectomy

(PTX) was performed the rats w h i c h had been

maintained on a v i t a m i n D deficient diet and whose serum Ca levels had slipped down from a normal level ( = indication of v i t a m i n D deficiency of the animals), died w i t h i n 24 hrs.

So, w e operated first on rats (

male Wistar strain rats weighing about 150 g) w h i c h had been fed a complete diet and then changed the diet to a v i t a m i n D deficient one ( 0.1 % Ca & 0.42 % P) after the operation to diminish the internal v i t a m i n D content in the animal bodies. About 4 to 5 weeks after PTX, the animals w e r e administered various drugs for 5 days.

Although the vitamin D content of the animals w e r e

not determined, they could be regarded as v i t a m i n D deficient animals as discussed below (cf. 4_) .

During and after the drug administration(s) ,

the serum Ca levels w e r e monitored by taking a small blood sample from the tail veins of the rats.

Five days after the last administration of

drug(s), the rats were killed. The left lower incisor dentin was decalcified by 0.2 N HC1, sectionized, and stained by hematoxylin.

The right lower incisor dentin from the

same animal w a s embedded into a polyester resin, sectionized, and ground

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

412 for contact micro-radiography.

There was a close relationship between

the hematoxylin stainability and the radio-opacity (= indication of metal deposition) in sections of both sides.

Time marking by lead acetate

injections (20 (3 mg/kg i.v. , LD5o (i.p.) = 150 mg/kg) were performed at designated times on the mineralizing region of the dentin (1).

Administrât ion of 25 hydroxycholecalciferol (25(OH)D3) (3.13 n mole/day, for 5 days), as well as that of PTH (20 usp u/day, for 5 days) neither elvated the serum Ca levels nor restored the dentin mineralization which had been inhibited by the lower level of serum Ca (5 mg % or below) after

e n a m e l side

pulp side

hematoxylin s t a i nabi 1 ity

s e r u m Ca ( m g

experi mental schedule

PbiJJj

j | j | j¡ P b ( - 5 , - 2 , 1 )

H

PTX - 1.11 I ¡I -28

day

Pb( 7 )

f 1

* • • • U .. drug administration 1 . J.I L. I J. I .1.I 1. I 1I L..1.. L_L 1 . T 0 2 4 6 8

Fig. 1. Correlation between serum Ca levels and dentin mineralization (1). 1,25 (OH) 2D3 (301 p mole/day) ( • ) or 25(OH)D3 (3.13 n mole/day) + PTH (20 usp u/day)(O) was administered from day 0 to day 4 on PTXed rats maintained on the vitamin D deficient diet. Experimental schedule (lower), changes in serum Ca levels (middle), and hematoxylin stainability of decalcified dentin along with lead apposition (upper) are shown together. The day of lead acetate injections are indicated by the numbers in parentheses . After 1,25(OH)2D3 or 25(OH)D 3 +PTH was administered for 5 days, the serum Ca levels elevated significantly upto 9 mg % and an obvious restoration of dentin remineralization was observed in the treated animal(s) (Fig. 1).

Lead acetate injection on day 4 and day 7 were apposed in the

mineralized region in the dentin(s), indicating the close correlation of dentin remineralization and serum Ca elevation.

These results suggest

that PTH stimulated the activation of 25(OH)D3, probably in the kidneys, and produced 1,25(0H)2D3 which induced the higher levels of serum Ca, which promoted the dentin remineralization.

The direct action of 1,25-

(OH)2D3 on the dentin (re)mineralization is obscure since a durable administration of Ca-gluconate (0.3 mg/kg/12 hr s.c., for 5 days) increased the serum Ca levels and also stimulated the dentin remineralization in rats prepared in the same way as for this study. enamel

side

pulp

side

hematoxyli n Pb(5)

sta i n a b i l i t y

serum Calmg

experimental schedule

%)

T T T T T d r u g I a d m i n i s tl r a t i o n

PTX

i_i_J L_i_

day

-35

Fig. 2. Correlation between serum PTH (60 usp u/day)( • ) or Db-cAMP from day 0 to day 4 on PTXed rats diet. The lead acetate injection further explanation, see Fig. 1.

8

Ca levels and dentin mineralization (2). (100 mg/kg/day)(O) was administered maintained on the vitamin D deficient was performed once on day 5. For

On the other hand, the administration of massive doses of PTH or of dibutylyl cyclic AMP (Db-cAMP) enhanced the dentin mineralization whereas no significant increase of serum Ca levels occurred in the course of the administration (Fig. 2).

This clearly shows the direct action of PTH on

the dentin mineralization which probably couples with the increase of cyclic AMP content in odontobrast cells as has been determined by Suzuki et al (1). As illustrated in Fig. 2, enhanced mineralization in this case occurred in a different region of the dentin compared with the case of l,25(OH)2D3 or 25(OH)D3+PTH administration (Fig. 1).

Lead acetate injected on day

5 apposed in the mineralized region far apart from pulp and wide unmineralized matrix was left behind.

It is unlikely that such a wide unmineral-

ized matrix was formed in only 5 days after drug administration and suggests that PTH (or Db-cAMP) enhanced the mineralization of dentin matrix which had already been formed.

In other words, PTH stimulated the

Ca (or amorphous Ca-POi, compound (cf. _3)) transporting activity of the odontobrast cells to the mineralizing front of the dentin.

4l4 Fig. 2 also indicates that animals prepared in this study could be regarded as vitamin D deficient ones.

If they had any available amounts

of vitamin D3 or 25(OH)D3 in their bodies, the administration of massive doses of PTH or of Db-cAMP would activate them and an elevation of the serum Ca levels would occur.

These effects were not observed.

Another

possible explanation of these results is a depression or loss of activity of the kidneys in response to PTH or Db-cAMP during the long (5 weeks) absense of PTH by PTX.

References (1)

Matsumoto, S., Tsudzuki, T., Yoshida, T., Arai, M., and Yamaguchi, M.

(1977) Folica Pharmacol. Japon. (2)

Okada, M. and Mimura, T.

73, 192. (1938)

Jap. J. Med. Sei. IV Pharmacol.

11,

166.

O)

Ozawa, H. (1972) Anat. Japon.

(4)

Shimura, F. and Tamura, M.

05)

Suzuki, A., Ishikawa, I., and Koyanagi, H.

Biol.

19,

380.

47,

(1978)

76. Vitamins (Japan) _52,

173-181.

(1977) Jap. J. Oral.

415 INLFUENCE OF DIET ON THE RESPONSE OF BONE CELLS TO l,25-(OH)-D, IN THYROPARATHYROIDECTOMIZED RATS. S. E. Weisbrode,* C.C. Capen* and A.W. Norman.** Department of Veterinary Pathobiology,* The Ohio State University, Columbus, OH 43210, USA, and Department of Biochemistry,** University of California, Riverside, CA 92502, USA. The cellular mechanisms by which l,25-(OH)_D, act on bone are unclear and complicated by secondary changes in parathyroid hormone and calcitonin secretion. The objectives of this study were to determine the influence of diet on the response of bone cells to l,25-(OH) 2 D, independent of parathyroid hormone and calcitonin and to correlate these findings to changes in serum and urinary concentrations of calcium and phosphorus. Adult male rats were surgically thyroparathyroidectomized (TXPTX) and supplemented with thyroxine. They were fed a low calcium (0.05%) normal phosphorus (0.3%) diet or a high calcium (1.1%) high phosphorus (0.8%) diet and given either 1 (65 p. moles) or 5 (325 p. moles) units l ^ - t O H ^ D ^ or placebo intraperitoneal^ daily for 7 days. Urine electrolytes were determined on samples collected during the 24-hour period prior to euthanasia. Serum samples were collected terminally. Electron microscopic evaluations were performed on undecalcified preparations of the right tibial metaphysis from each rat. Light microscopic evaluation was performed on EDTA-decalcified preparations of the left tibia from each rat. TABLE 1: EFFECT OF 1. 25-(0H),0, ON SERUM AND URINARY CALCIUM AND PHOSPHORUS CONCENTRATIONS (MG/DL ± SE)IN THYROPARATHYROIDECT-

TABLE 2:

EFFECT OF 1, 26-IOH),D 3 ON MORPHOMETRY PARAMETERS

IN TIBIAL METAPHYSES OF THYROPARATHYROIDECTOMIZED RATS FED

OMIZED RATS FED A HIGH CALCIUM DIET. N - 8/GROUP.

A HIGH CALCIUM DIET: N-8/OHOUP 1. 26-IOH),D, 11 W I T f l M Y l

1, 25—(OH)jDj IS WITS/PAY)

6.42 ± 0.4«

10.99 ±0.27 (PC.001)

12.33 ±0.35 (P

SERUM PHOSPHORUS

11.M 10.46

9.01 ±0.24 (P3 was not restored but showed no further fall over the second 24h (Fig.4).

" HOURS IN CULTURE

«

Other glucocorticoids were tested in this system at 10-7M for 24h and they also significantly preserved binding of 1,25 (011)203 (Table). Their relative efficacies in preserving l,25(OH)2D3 binding were closely related to their relative potencies as glucocorticoids and their affinities for the glucocorticoid receptor. Steroid No steroid Triamcinolone Acetonide Dexamethasone Cortisol Corticosterone Progesterone Estradiol Testosterone

Concentration in medium (moles/L)

_7 IO 7 "

11 11 11

-6 10 b If

Percentage of control l,25(OH)2D3 binding preserved after 24h in culture at 37°C 10.1 98.0 84.0 52.2 37.0 27.0 10.3 10.0

These findings strongly suggest that glucocorticoids at physiological or therapeutic concentrations are important for maintaining the content of cytosol receptors for l,25(OH)2D3 in bone, and favour an inhibition of degradation of existing l,25(OH)2D3 receptors, rather than the induction of synthesis of new receptors. High levels of glucocorticoids cause rapid and severe bone loss in Cushing's Syndrome and in patients on steroid therapy; intestinal calcium absorption(8) and bone formation are reduced and bone resorption increased(9). Numerous studies in amimals and man have so far not fully elucidated the mechanisms of these effects. However, treatment with large doses of cortisone or prednisolone does not reduce the capacity of the vitamin D-deficient rat to produce 1,25(OH)2D3(10). Similarly, cortisone does not influence the localisation of l,25(OH)2D3 in the nuclei of the intestinal target cell(11), nor the capacity of these cells to synthesise the specific vitamin D-dependent, calcium

kkz binding protein(12). Our data indicate that glucocorticoids may influence the stability of cytosol l,25(OH>2D3 receptors in bone. From these experiments we cannot tell if this effect is specific to one population of bone cells or one that affects both osteoblasts and osteoclasts. There is evidence that both types of cell respond to l,25(OH)2D3 at a concentration of 10-9M(13). An augmentation of l,25(OH)2D3 receptors in osteoclasts might enhance bone resorption, since this compound is an exceptionally potent bone-resorbing agent(14,15). The deleterious combination of enhanced resorption, inhibition of growth(16) and augmentation of PTH effects(17) on bone combined with reduced intestinal absorption and hypocalcaemia, could account for the rapid and severe osteoporosis of glucocorticoid excess. Our studies indicate the need for a thorough examination of the effects of glucocorticoids on l,25(OH)2D3 and other receptors in cytosol and nuclei in isolated bone cell populations and in intestine. We thank the North West Regional Health Authority and the Medical Research Council for their support, Mrs C M Case for technical assistance and Dr C M Taylor and Professor S W Stanbury for help and advice. REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Manolagas, S.C. & Anderson, D.C. (1978) J.Endocrinol. 7£, 379-380. Manolagas, S.C., Taylor, C.M. & Anderson, D.C. (1979) J.Endocrinol. 80, 33-39. Feldman, D., Dziak, R., Koehler, R. & Stern, P. (1975) Endocrinology, 96, 29-36. Kream, B.E., Jose, M., Yamada, S. & DeLuca, H.F. (1977) Science, 197, 1086-1088. Norman, A.W. & Wecksler, W.R. (1978) In Receptors in hormone action. Vol.11, Acad.Press. p533-571. Manolagas, S.C., Anderson, D.C. & Lumb, G.A. (1979) Nature, Vol.277, p314-315. Chen, T.L. & Feldman, D. (1978) Endocrinology, 102, 236-244. Harrison, H.E. & Harrison, H.C. (1960) Am.J.Physiol. 199, 265-271. Jowsey, J., Kelly, P.J., Riggs, B.L., Bianco, A.J. & Scholz, D.A. (1965) J.Bone Joint Surg. 47B, 785-806. Lukert, B.D., Stanbury, S.W. & Mawer, E.B. (1973) Endocrinology, 93, 718-722. Favus, M.J., Rimberg, D.V., Miller, G.N. & Gershon, E. (1973) J.Clin. Invest. 52_, 1328-1335. Rimberg, D.V., Baerg, R.D., Gershon, E. & Graudusius, R.T. (1971) J.Clin.Invest. 50, 1309-1321. Wong, G.L. , Lüben, R.A. & Cohn, D.V. (1977) Science. 197, 633-665. Reynolds, J.J. (1974) Biochem.Soc.Spec.Publ. 3_, 91-102. DeLuca, H.F. & Schnoes, H.K. (1976) Ann.Rev.Biochem. 45, 631-666. Chen, T.L., Aronow, L. & Feldman, D. (1977) Endocrinology. 100, 619628. Chen, T.L. & Feldman, D. (1978) Endocrinology. 102, 589-596.

Vitamin D Metabolism

kk5 RECENT DEVELOPMENTS IN THE METABOLISM OF VITAMIN D H. F. DeLuca and H. K. Schnoes Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, U.S.A. The discovery that the kidney (1,2) is the sole site of 1,25-dihydroxyvitamin D^ (1,25-(OH)2^3) production made possible the demonstration that nephrectomized vitamin D-deficient animals do not respond to physiologic doses of 25-hydroxyvitamin D (25-OH-D) whereas they respond normally to I,25-(OH^D^.

These responses are the intestinal calcium (3,4) and phos-

phate (5) transport systems and the mobilization of calcium from bone (6). These experiments demonstrated that 1,25-(OH)

°r a further metabolite

must be the metabolically active form of the vitamin in the three systems. To examine the question of whether 1,25-(OH)20^ must be further metabolized before it functions in these systems, radioactive l,25-(OH)„3

[26,27- H]D^ was administered to vitamin D-deficient animals and at the time the target organs responded, the tissues were removed, extracted and the lipid extracts chromatographed to reveal whether any additional metabolites could be observed (7-9).

In two different laboratories it

was concluded that no further metabolites of 1 , 2 5 - ( O H ) c o u l d be detected at the time the target organs responded, Suggesting that 1,25-(OH) itself was acting to stimulate the target organs.

However, poor recover-

ies of tritium from the terminal side chain label were experienced and furthermore, the presence of water soluble radioactive compounds from 3

the l,25-(OH)2-[2- H]D 3 could be detected in intestinal extracts (7).

To

examine the question further, we chemically synthesized 25-OH-D^ labeled with U C in the 26 and 27 positions (10) . Upon administration of the 2514 14 OH-[26,27- C]D^ to vitamin D-deficient animals, some 7% of the C appeared in expired CO , demonstrating an oxidative cleavage of the vitamin D 14 side chain (11). Nephrectomy prior to injection of the 25-OH-[ C]D_ 14 resulted in abolition of the appearance of the C in CO2, suggesting that the 25-OH-D^ must undergo 1-hydroxylation before it could be oxidatively cleaved to yield carbon dioxide from the terminal carbons of the 14 side chain (11,12). l,25-(OH) -[26,27- C]D was enzymatically prepared from 25-0H-[26,27-

C]D,.

When this compound was injected, between 25

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

446 a n d 30% of t h e

appeared

unaffected by nephrectomy 1,25-(OH)2^3

w a s

i n e x p i r e d CO 2 a n d t h i s e x p i r a t i o n of

(12,13).

T h e s e r e s u l t s c o n f i r m the b e l i e f

w a s

that

u n d e r g o i n g o x i d a t i v e c l e a v a g e of t h e s i d e c h a i n to y i e l d

v i t a m i n D metabolite w i t h a shortened side chain plus carbon 1,24,25-trihydroxyvitamin D 3 intermediate

CO^

(11).

(1,24,25-(OH)3D3)

dioxide;

did not appear

to b e

an

S i n c e e n t e r o c o l e c t o m y b l o c k e d t h e m e t a b o l i s m of

(0H) 2~ [ 2 6 , 2 7 - ^ C ] D 3

to C ^ ,

s i t e of t h i s r e a c t i o n

a

1,25-

the i n t e s t i n e a n d / o r l i v e r a p p e a r e d to b e

the

To examine this m e t a b o l i c pathway further, w e 3 c h e m i c a l l y s y n t h e s i z e d 2 5 - O H - [ 3 a - H ] D . (14). T h i s w a s c o n v e r t e d to 1 , 2 5 3 14 3 ( O H ) 2 ~ [ 3 a - H J D ^ e n z y m a t i c a l l y a n d a m i x t u r e of the [ 2 6 , 2 7 C] a n d [3a- H] 1,25-(OH)was

(13).

administered

to b o t h v i t a m i n D - d e f i c i e n t a n i m a l s

a n i m a l s g i v e n a p r i m i n g d o s e of u n l a b e l e d 1,25-(011)203.

and

A t 3 or 6 h o u r s

a f t e r i n t r a v e n o u s i n j e c t i o n the a n i m a l s w e r e k i l l e d ,

the tissues

removed, homogenized,

methanol-chloroform-

lyophilized and extracted with

w a t e r , p H 8, to y i e l d a n a q u e o u s - m e t h a n o l p h a s e , p H 8, a n d a phase.

T h e c h l o r o f o r m e x t r a c t of l i v e r c o n t a i n e d o v e r

were

chloroform

50% of t h e

tissue

r a d i o a c t i v i t y w h e r e a s 3 9 % w a s f o u n d i n the m e t h a n o l - w a t e r p h a s e ;

the

r e m a i n d e r w a s b o u n d to t i s s u e c o m p o n e n t s .

chloro-

C h r o m a t o g r a p h y of the

f o r m p h a s e o n S e p h a d e x L H - 2 0 r e v e a l e d t h a t the m a j o r f r a c t i o n of r a d i o a c t i v i t y w a s i n the f o r m of 1 , 2 5 - ( 0 H ) 2 D 3

although small amounts

b o t h p o l a r a n d n o n - p o l a r m e t a b o l i t e s c o u l d be f o u n d

(7,8).

The

m e t a b o l i t e c o u l d w e l l be 1 , 2 4 , 2 5 - ( O H ) ^ D ^ w h e r e a s t h e n o n p o l a r although possibly an ester, was not further identified. ents had the original ^H/^^C ratio

indicating

this

metabolite,

All three

t h a t the s i d e c h a i n

product was not found in the chloroform extracts.

intesthe

p H 8, f r a c t i o n a n d the r e m a i n d e r e i t h e r u n e x t r a c t e d

i n the c h l o r o f o r m f r a c t i o n .

C h r o m a t o g r a p h y of the c h l o r o f o r m

a g a i n like liver revealed no ^ H / ^ C of the c h r o m a t o g r a p h e d

enriched m e t a b o l i t e s w i t h 80% or m o r e These

the failure

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

However,

suggesting

to

1,25-

the r a d i o a c t i v e . 3 ,14

c o m p o n e n t s f o u n d i n the a q u e o u s f r a c t i o n w e r e e n r i c h e d w i t h as 3-fold overall,

or

fraction

r a d i o a c t i v i t y a p p e a r i n g a s 1, 2 5 - ( O H ) ^O-^ •

r e s u l t s c o n f i r m the p r e v i o u s i n v e s t i g a t i o n i l l u s t r a t i n g

(OH) D

componcleavage

In the case of

tine, a p p r o x i m a t e l y 4 5 % of t h e t i s s u e r a d i o a c t i v i t y w a s f o u n d in aqueous-methanol,

of

polar

H/

C as much

t h a t the s i d e c h a i n c l e a v e d r e a c t i o n

product

kk7 was found in that fraction.

The aqueous soluble fraction from both

tissues was then passed through the bicarbonate form of a diethylaminoethyl (DEAE) Sephadex column.

Some 90% of the aqueous soluble radio-

activity was bound to the DEAE Sephadex column and could be eluted with 0.3 molar ammonium bicarbonate solution in 90% methanol/10% water.

The

charged aqueous methanol soluble metabolite fraction, therefore, contained a marked enrichment of ^H/^C.

The fraction (charged) which eluted from

DEAE Sephadex column chromatography was then easily methylated with diazomethane. Sephadex.

The methylated product was no longer retained on DEAE

It was chromatographed on straight-phase silica columns using

high-pressure liquid chromatography (HPLC).

This revealed the existence

of two metabolites, a major component and a minor component, both of 14 3 which were enriched with H over C. The major component therefore, is a major metabolite of l ^ S - i O H ^ D ^ istration of l,25-(OH)2D^

to

that appears very rapidly after admin-

vitamin D-deficient animals.

It became

evident, therefore, that its chemical characterization should be of considerable interest to our understanding of the function and regulation of l,25-(OH) 2 D 3 . Although the intestinal tissue and its contents appeared to be the richer source of this metabolite, -the extracts obtained from liver were much less heavily contaminated with extraneous material lending itself to isolation of the metabolite for characterization (14).

However, before

isolation was attempted, co-chromatography on HPLC confirmed that the liver and intestinal charged metabolite in the aqueous phase were identical. 3 Fifty-seven rats were given 1 yg of 1,25-(OH)2~[3a- H]D3-

Six hours

later the animals were killed, their livers removed, homogenized with water and lyophilized.

Lyophilized material was extracted twice with

two liters of 1:1 chloroformrmethanol.

The filtrate was evaporated and

the residue was partitioned in 100 ml of water, 200 ml each of methanol and chloroform at pH 8.

The chloroform phase was removed and back extrac-

ted with an additional quantity of water:methanol, pH 8.

The aqueous

phase was washed once with chloroform and the aqueous phase combined, evaporated to dryness and the residue dissolved in 95% methanol.

The

448 sample was then placed on a DEAE Sephadex column in the bicarbonate form. The column was washed with 200 ml methanol and then subjected to a step gradient of 0.1 M ammonium bicarbonate in 90% methanol and 0.3 M ammonium bicarbonate in 80% methanol. ammonium bicarbonate.

The charged metabolite eluted with 0.1 M

It was collected, the solvent evaporated, and the

residue chromatographed on a Sephadex LH-20 column equilibrated and eluted with methanol.

At this stage the metabolite fraction was subjected

to diazomethane methylation in ether at room temperature.

Over 95% of

the metabolite was rendered neutral (i.e. not retained on DEAE Sephadex), indicating its conversion to the methylated product.

The compound in its

methylated form was then subjected to Sephadex LH-20 chromatography using chloroform:hexane 60:40 and the resultant product purified further on a Zorbax-ODS column eluted with 30% water in methanol by HPLC.

Final

purification was performed on a Zorbax-SIL straight-phase HPLC column 3 using 7% 2-propanol in hexane.

A single component (both by

H and UV

absorbance at 254 nm) was eluted which was homogeneous chromatographically. The metabolite exhibited a typical cis-triene chromophore absorption band characteristic of vitamin D showing a Amax at 264 and a Anun . at 228 with a ratio of A / A . of 1.6. The mass spectrum of the methylated product max min revealed a molecular ion at m/e 388.

Confirmation of the cis-triene

chromophore was provided by mass fragments at 152 (ring A+C-6 & C-7) and 134 (152-H„0), a characteristic of fragmentation of la,3g-dihydroxylated vitamin D compounds.

A mass fragment at m/e 287 (M -side chain) with

ions resulting from the loss of water from this mass fragments at m/e 269 and 251 reveal that the 1,3-diol from 1 , 2 5 - ( O H ) r e m a i n e d during metabolism.

unchanged

Therefore, all modifications from 1, 25-(OH)2D.j during

metabolism to the metabolite occurred on the side chain.

The presence of

a side chain methyl ester function is confirmed by the mass fragment at 357 (M-OCH^) and by the loss of C00CH 3 yielding a fragment of m/e 329 and by the elimination of 74 mass units (C^COOCH^ + H) via a McLafferty type of rearrangment to give m/e 314.

This rearrangement fragmentation speci-

fies a C-23 position for the carbomethoxy function.

These data, there-

fore, clearly establish the structure of the isolated methyl ester product shown in Fig. 1 together with its naturally occurring carboxyl form of the metabolite.

The metabolite is, therefore,

24-nor-5,7,10(19)cholatrien-23-oic acid (14).

la,36"dihydroxy-9,10-secoA trivial name of

OH

^ O M e

CH 2 N 2

HO*'

OH Calcitroic

OH

acid

la-hydroxytetranor-vitamin D - 2 3 - c a r b o x y l i c

acid

la , 3 £ - d i hydroxy - 9 , 1 0 - seco-24 nor - 5 , 7 , 1 0 (l9)-cholatrien-23-oic acid

Fig. 1

calcitroic acid has been assigned to this major metabolite of 1,25(OH)•

Biological activity of this metabolite is not yet fully known.

Therefore, its possible role as a functional metabolite remains to be determined. During the course of our investigation of methods of assaying for 24,25(OH^D^J

the rat plasma transport protein for vitamin D and its metabo-

lites was utilized as a means of detecting this metabolite by competitive 3 protein binding assay using 25-OH-[ H]D^ as the saturating labeled material (15).

It might be expected that the plasma transport protein would

lack particular selectivity for binding of the vitamin D metabolites and

^50 therein lies its utility as a detecting agent for many vitamin D metabolites.

However, great care must be exercised in its use as a detecting

system for vitamin D and its metabolites demanding that the metabolites be highly purified and separated from all others before assay.

Original

methods for determination of 24,25-(OH)2 D 2 had utilized Sephadex LH-20 chromatography to prepurify the samples and to separate 24,25-(OH)^D^ from 25-OH-D 3 and l,25-(OH) 2 D 3 before assay (15, 16).

We subjected the

24,25-(OH)2Dj portion of the Sephadex eluant from one of these methods to HPLC and as 3shown in Fig. 2, four components were found which compete with 25-OH-[ H]D 3 for the plasma transport protein.

One of these peaks,

Peak I, proved to be a nonvitamin D-dependent substance currently being characterized in our laboratory.

The second peak appeared to be residual

25-0H-D 3> the third peak the desired 24,25-(OH) 2 D 3 fraction, and the fourth peak 25,26-(OU)^D^. the 24,25-(OH)^D

When plasma extracts from chicks were examined,

fraction from HPLC appeared to be contaminated with an

50 Peak I - Anephric Plasma -Normal Plasma o

II

CM

(Peak II) 25-0H-D 3

m CJ

(Peak III) 24,25-(0H)'22D.3

(Peak I Y ) 25,26-(0H^D 3

— i 0

5

Vi

10

i

\j

'

•

i

15 2 0 25 3 0 35 4 0 4 5 5 0 55 6 0 Elution

Fig. 2.

•

HPLC of 24,25-(OH) ? D

Volume

(mis)

fraction from Sephadex LH-20 columns.

451 additional metabolite.

This metabolite could be separated from 24,25-

( O H ^ D ^ on reverse-phase HPLC using a Zorbax octadecyl silane column w i t h 25% water in methanol as solvent.

(ODS)

This metabolite, which was

tabbed as Peak X, w a s next examined in the plasma of animals given large amounts of v i t a m i n D.

A s vitamin D dosages increased, the amount of Peak

X is markedly elevated above the amount of 24,25-(OH) 2 D 3 >

Thus Peak X

can become a major circulating metabolite of vitamin D under conditions of hypervitaminosis D.

Because of its presence in large amounts, we under-

took the isolation and identification of this metabolite. the metabolite in pure form from two sources (17).

We isolated

We obtained 16 liters

of chick plasma from the slaughter of broiler chickens for market and from this isolated 8 yg of pure metabolite X.

In addition, we gave

chicks large doses of vitamin D of the order of 10^ units, obtained 1100 m l of plasma from such chicks and isolated 54 pg of the metabolite in pure form.

Both the Peak X from physiologic doses of vitamin D and Peak

X from the h i g h doses proved to be identical both by chromatographic criteria and by physical methods.

The isolation of the physiological

levels is described here.

The 16 liters of plasma was heated to 70° for 1 hour and the pellet collected by centrifugation.

The pellet contained the metabolite.

The

pellet was extracted w i t h methanol-chloroform by the Bligh and Dyer procedure yielding a chloroform extract.

The extract was subjected to

Sephadex LH-20 column chromatography using a 3 x 30 cm column and a solvent system of 9:1:1 hexane:chloroform:methanol.

The entire 24,25-

( O H ^ D J region as revealed by the protein binding assay was collected. It was subjected to another Sephadex LH-20 column using a 70:30 chloroform: hexane solvent system.

The peak fraction was still unresolved from

24,25-(OH) 2 d 3 and was further purified by a n additional Sephadex LH-20 column, again using hexane:chloroform:methanol 9:1:1.

The

24,25-(OH)

binding region w h i c h includes Peak X was subjected to HPLC using a P a r t i sil ODS column and a solvent system of 25% water in methanol. fraction migrating prior to 2 4 , 2 5 - ( 0 H ) w a s

The peak

collected and further puri-

fied o n HPLC using straight phase Zorbax-SIL and a solvent system of 8% isopropanol in hexane.

At this stage 8 pg of metabolite was chromato-

graphically homogenous and subjected to chemical

identification.

The metabolite showed the vitamin D cis-triene ultraviolet absorption spectrum with the

at 264 nm and a minimum at 229 nm.

Mass spectrom-

etry of the metabolite revealed a molecular ion of m/e 428, and fragments at m/e 410, m/e 395, m/e 271, m/e 253, m/e 136, and m/e 118.

High resolu-

tion mass spectrometry gave a molecular weight of 428.2901, which requires the composition C^-jR^^p^.

High resolution mass spectrometry, therefore,

revealed this to be a vitamin D^ compound and not a dihydroxylated derivative of vitamin

The mass fragment at 271 which represents loss of

side chain indicates that the alterations by metabolism occurred entirely on the side chain and that the remainder of the vitamin D molecule has remained intact.

This was confirmed by the mass fragments at 136 and at

118 which reveal a characteristic vitamin D fragmentation spectrum giving ring A plus carbons 6 and 7 while the 118 results from loss of this fragment.

from

The metabolite when subj ec t ed to silylation showed, frag-

ment m/e 572, 482, 467, 208, and 118.

This indicates a ditrlmethyl silyl

ether derivative with one of the silyl groups on ring A and another on the side chain.

This left two oxygen atoms unaccounted for.

The metabolite

was next subjected to nuclear magnetic resonance spectroscopy using a 270 MHz instrument.

The spectrum is typical of known vitamin D compounds,

except for the resonances at S 4.46, 6 1.09, and 6 1.56.

The 6 4.46

resonance (1H) indicates the presence of either an ether or ester function. The doublet at 6 1.09 for the C-21 methyl proton shows that the C-20 carbon is not further functionalized resulting in a doublet for the C-21 protons.

The 6 1.56 resonance obtained at 4°C shows a singlet for the

27-methyl group and only 1 methyl group.

'Ehus, the 26 carbon has no

protons and must be present as a carbonyl functionality.

Also the C-25

carbon must be functionalized since the C-27 resonance is a singlet.

The

NMR data, therefore, show the compound to be 25-hydroxylated, containing a 26-carbonyl function and that it must contain an ether or ester functionality.

From the NMR and mass spectrum as well as the ultraviolet absorp-

tion spectra one can surmize that the compound is a 25-hydroxylated26-lactone derivative of vitamin D^•

The size of the lactone ring,

however, could not be determined by NMR.

The compound was then subjected

to Fourier-Transform infrared spectroscopy to determine the presence of a lactone ring and, if so, the size.

The Fourier-Transform infrared spec-

trum shows an intense absorbance at 1787 cm

This indicates a five-

453 membered lactone ring and the structure of the compound must be 36,25dihydroxy-9,10-seco-5,7,10(19)cholestatrieno-26,23-lactone ly 25-OH-D2~26,23-lactone. lactone.

or alternative-

A trivial name for the compound is calcidiol

The structure of the compound was further confirmed by the

reaction of its ditrimethylsilyl ether derivative w i t h methyl lithium to produce a methyl lactol.

The ditrimethylsilyl ether derivative of the

lactol gave a mass spectrum w i t h peaks at m/e 588, 570, 528, 498, 208, 118.

The molecular ion indicates the addition of a methyl group w i t h

conversion of the C-26 carbonyl to an alcohol.

This lactol was further

silylated to form the trimethylsilyl ether derivative showing m/e 660, 645, 570, 555, 528, 208, and 118.

The molecular ion at m / e 660 indicates

the formation of a third silyl ether on the C-26 hydroxyl group.

The ion

at m / e 528 shows loss of AcOTMS resulting from the fragmentation through the lactol ring system characteristic of this compound.

The structure of

the calcidiol lactone or the 25-OH-D2-26,23-lactone is shown in Fig. 3. Its biological activity is under study.

Calcidiol 25 h y d r o x y

lactone

vitamin

D3-26,23-lactone

3/3,25 d i h y d r o x y - 9 , I O - s e c o - 5 , 7 , I O ( l 9 ) cholestatrieno-26,23 lactone

Fig. 3

k5h There has been considerable interest in the metabolic role played by 24,25-(OH)•

Little is known concerning its further metabolism except

its conversion under special circumstances to 1,24,25-(OH)^D^ (18). Claims for its extraordinary biological activity as a calcifying agent in bone (19) and as a repressant of parathyroid hormone secretion and size (20) have further kindled interest in the metabolite.

Regardless of

its possible biological activity, its role as a significant metabolite of vitamin D is obvious.

We have, therefore, begun our investigation of its

metabolism in animals.

Previous work has revealed its conversion to

1,24,25-(OH)3D3 when given to vitamin D-deficient animals (18).

This

abnormal situation obtains because the 1-hydroxylase is maximally activated and the absence of other vitamin D compounds predisposes the conversion of 24,25-(OH)2D3 to 1,24,25-(0H)3D3; a situation unlikely to occur under normal circumstances.

A more appropriate question is:

what is the metab-

olism of 2 4 , 2 5 - ( O H ) i n approximately normal animals, a condition under which it is metabolized ^ v l v o ? W e have therefore prepared 25-OH3 [3a- H]D 3 as a substrate and utilizing the 24-hydroxylase found in animals given high calcium diets plus vitamin D we have produced 24,25(OH)2-[3a-3H]D3.

When 24,25-(OH) 2 ~ [3ct-3H]D3 is administered intravenously

to normal animals, a substantial amount of the compound is rapidly converted to water solubility.

Furthermore, the 24,25-(OH)2D3 rapidly

disappears from the lipid extracts, making its appearance in the aqueous phase.

The aqueous soluble radioactivity when subjected to DEAE Sephadex

chromatography shows it to be 70-80% bound, illustrating that the major metabolite is charged.

We, therefore, proceeded to the isolation and

identification of the charged metabolite derived from 24,25-(OH)2D3-

To

generate substantial amounts of the compound, we have incubated rat kidney homogenates in the presence of NADPH, molecular oxygen and 24,253 (OH)2~[3a- H]D 3 which readily produces substantial amounts of the watersoluble metabolite.

Incubations were carried out with the tissues obtain-

ed from normal rats given vitamin D.

The reactions were terminated with

methanol:chloroform and the extractions were carried out at pH 8, revealing a chloroform phase and an aqueous-methanol phase.

The aqueous phase

was subjected to DEAE Sephadex column chromatography demonstrating the compound to be charged.

The isolation procedure was then carried out as

described for the side chain cleaved product of 1 , 2 5 - ( O H ) , namely,

^55 preliminary purification by reverse-phase HPLC through Zorbax ODS and water:methanol solvent systems. purified further metabolite.

The product was then methylated and

on straight-phase HPLC.

Final HPLC yielded a pure

This metabolite gave the expected ultraviolet absorption

spectrum of the vitamin D-cis-triene chromophore, namely ^ m a x at 264 nm and a ^ ^

at 229 nm.

The methylated metabolite was then subjected to

mass spectrometry, giving characteristic ions as follows:

molecular ion

m/e 386, loss of water giving m/e 368; m/e 327 representing the loss of a carbomethoxy function; m/e 353 arising from elimination of water plus methyl; fragments at 271 (loss of side chain) and 253 (271~H20).

The 271

fragment demonstrates that the vitamin D nucleus has remained intact. Furthermore, a fragment at 136 with a base peak at 118 demonstrates clearly that the cis-triene structure is still intact and that the A ring Tlas only one hydroxyl function.

These results strongly suggested that

the structure must be the methylated C-24 acid derived from cleavage of the diol of 24,25-(0H)2D,J.

We, therefore, chemically synthesized the C-

24 acid by periodate cleavage followed by Ag 2 0 oxidation of synthetic 2 4 , 2 5 - ( O H ) a v a i l a b l e by the kindness of Milan Uskokovic of the HoffmannLaRoche Company.

The methyl ester of this compound gave a mass spectrum,

and ultraviolet spectrum identical to the isolated methylated metabolite. The synthetic methylated sample also exactly co-chromatographed on HPLC with the isolated, methylated metabolite.

These results proved that the

product is 9,10-secochola-5,7,10(19)-trien-24-oic acid (Fig. 4).

This

compound has now been assigned the trivial name of chola-calcioic acid or 25,26,27-trisnor-vitamin D2~24-carboxylic acid.

The biological activity

of this 24-carboxylic acid is unknown at the present time although previous tests of la-hydroxylated 24-carboxylic acid showed a lack of activity in the intestinal and bone systems.

The 24-carboxylic acid is

likely to be a major metabolite of 24,25-(OH)2D3, although other metabolites of 24,25-(OH)2Dj have been isolated and their structures are undergoing determination.

456 O II

C \

HO

OH

x

cholacalcioic

acid

25, 26,27-trinorvitamin D-carboxylic

acid

3 / 3 - h y d r o x y - 9, 1 0 - s e c o c h o l a - 5 , 7 , 1 0 ( 1 9 ) - t r i e n - 2 4 - o i c

acid

Fig. A

This report, therefore, describes and identifies unequivocally three new major metabolites of v i t a m i n D.

The first is a major metabolite of the

known and p r o v e n hormonal form of v i t a m i n D, namely 1 , 2 5 - ( O H ) .

The

second is a major blood component found in large amounts in v i t a m i n D intoxication and w h i c h m a y accumulate under other pathological or physiologic circumstances.

The third is a m e t a b o l i t e of the major alternate

metabolite to l,25-(OH) 2 I> 3 , namely 24, 2 5 - ( O H ) 2 D 3 .

Their biological

activity profiles are n o t yet completed and thus their overall p h y s i o l o g ical and m e t a b o l i c significance is unknown.

It is likely, however,

that

at least two of these will be of major importance in our final u n d e r standing of the metabolism and function of v i t a m i n D.

k57 REFERENCES 1.

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(1970)

Nature 228, 764-766.

2.

Gray, R., Boyle, I. T. and DeLuca, H. F.

(1971)

Science 172, 1232-

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Boyle, I. T., Miravet, L., Gray, R. W., Holick, M. F. and DeLuca, H. F.

4.

(1972)

Endocrinology 90, 605-608.

Wong, R. G., Norman, A. W., Reddy, C. R. and Coburn, J. W.

(1972)

J. Clin. Invest. 51, 1287-1291. 5.

Chen, T. C., Castillo, L., Korycka-Dahl, M. and DeLuca, H. F.

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Arch. Biochem. Biophys.

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Science 193, 493-494. 11.

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(1976)

Biochemistry 15,

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Kumar, R. and DeLuca, H. F.

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Biochemistry, submitted for publication. 15.

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Taylor, C. M., Hughes, S. E. and deSilva, P.

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Holick, M. F., Kleiner-Bossaller, A., Schnoes, H. K., Kasten, P. M., Boyle, I. T. and DeLuca, H. F.

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6696. 19.

Rasmussen, H. and Bordier, P. Related Research

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459 THE ROLE OF SYSTEMIC PH IN THE BIOACTIVATION OF VITAMIN D

L. V. Avioli Division of Bone and Mineral Metabolism, The Jewish Hospital of St. Louis and Washington University School of Medicine, St. Louis, Mo 63110, USA Within the last decade a considerable amount of knowledge has accumulated with regard to the bioactivation of vitamin D, a substance recognized as essential for normal skeletal growth and the modeling and remodeling processes which condition bone maturation and turnover. The majority of experiments designed to evaluate the metabolism of vitamin D have been performed in animal models (primarily chicks and rats) which have been either calcium-or phosphate-depleted and subjected to diets deficient in vitamin D. The data derived from experiments in these hypophosphatemic, hypocalcemic and D-depleted animal models have been subsequently extrapolated to humans in order to explain a variety of clinical disorders characterized by either a resistance to vitamin D (i.e. chronic renal disease, familial hypophosphatemic rickets, or renal tubular acidosis), a sensitivity to the hypercalcemic or hypercalciuric action of vitamin D (i.e. sarcoidosis, and idiopathic hypercalciuric states), and syndromes wherein a definite antagonism exists between vitamin D and certain therapeutic agents (i.e. cortisone and anticonvulsant therapy). Although the majority of these extrapolations have proven both efficacious and satisfactory with regard to detailing the underlying pathogenesis in vitamin D metabolism in these varied clinical disorders, it may be appropriate to re-evaluate the animal experimental data which perpeturally conditions our clinical analyses of vitamin D metabolism in man. Despite well-documented reports of hypophosphatemic-stimulation of 1,25(0H)2D formation and calcium absorption in animal models (1-3) others, having reported either less calcium absorption (4) or no change in either intestinal calcium binding protein or 1,25(0H)2D production (5) in animals on low-calcium and low-phosphorus diets, have concluded that blood phosphate concentration does not appear to regulate the bioactivation of vitamin D (5,6). Despite well-appreciated controversies with regard to experimental design and animal models, the objective investigator must by necessity be often confused by these seemingly paradoxical conclusions obtained from jm vivo rat or chick studies and jjn vitro experiments with either chick homogenates, renal tubules or renal mitochondria. In 1977, Booth et al^ reported that a metabolic acidosis developed in hypocalcemic vitamin D-deficient chicks (7). These investigators also noted that within 24 hr of vitamin D administration, both acidosis and hypocalcemia were attentuated; during the subsequent 72 hr, the acidosis was further normalized although a mild hypocalcemia persisted (7). They suggested that vitamin D deficiency per se may have been a prerequisite for the development of acidosis and concluded that one should consider the attendent acidosis when interpreting results of studies of vitamin D metabolism performed in vitamin D-deficient chicks. Stimulated by these initial observations, we proceeded to analyze the

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

k(>0 effect of alterations in dietary vitamin D, calcium and phosphate on the blood pH of the chick and rat. In these experiments, 1-day old White Leghorn cockerels and 3-week old female Holtzman rats were placed on either vitamin D-depleted or vitamin D - s u f f i c i e n t , 0.2% phosphate diets. Animals were also subjected to either high (1.3%)or low (chick-0.4%, and rats-0.1%) calcium diets. Both chicks and rats were followed for predetermined intervals and arterial blood obtained for pH, pC02, HCOo", calcium and phosphate determinations. In the vitamin D-depleted chicks on low calcium intakes, the arterial pH decreased from 7.33 + 0.06 to 7.29 + 0.08 by the third week. During this interval, serum calcium values also decreased from 9.25 + 0.56 to 5.90 + 0.54 mg/dl. The pH changes noted in the v i t a min D-depleted chicks on low (0.4%) calcium intakes were also observed in the vitamin D-depleted chicks on high calcium (1.3%) diets. Of note was the additional observation that vitamin D feeding prevented the acidosis in the chicks on diets containing 0.4% calcium whereas i t failed to do so in those chicks receiving 1.3% calcium diets. In contrast to the results obtained in chicks, vitamin D-depleted rats on 1.3% calcium and 0.2% phosphorus diets became acidotic within 4 weeks with arterial pH values decreasing from 7.48 + 0.66 to 7.25 + 0.05; however rats on 0.1% calcium and 0.2% phosphate diets were able to maintain normal systemic pH values despite vitamin D depletion intervals which were identical to those on 1.3% calcium and 0.2% phosphate diets. As anticipated, the serum levels of calcium and phosphate in the vitamin D depleted rats was conditioned by the dietary intake of those elements; calcium and phosphate values of rats fed the high calcium diet were 9.02 + 0 . 8 1 and 2.11 + 1.21 mg/dl, respectively; calcium and phosphate values of animals on the low calcium diet were 4.53 + 0.31 and 7.67 + 1.07 mg/dl, respectively. Arterial pH remained within normal limits in vitamin D-repleted rats on the 0.2% phosphate diet subjected to either high or low calcium diets despite s i g n i f i c a n t l y higher values for serum phosphate in the rats on low (0.1%) calcium diets. These experiments revealed that perturbations in calcium and phosphate intake affects acid-base balance and systemic pH in the growing chick independent of the vitamin D status of the animal; in contrast, similar perturbations are relatively ineffective in altering blood pH in the vitamin D-repleted rat (8). With these observations in hand, how does one interpret the results of experiments designed to evaluate the functional metabolism of vitamin D in chicks on low calcium (0.2%) and low phosphorus (0.3%) diets which lead some authors to conclude that the intestine and kidney, but not bone are the main target organs for vitamin D in the maintenance of calcium homeos t a s i s (9)? Vitamin D-deficient chicks on low calcium diets become hypocalcemia (9); the latter should by definition also stimulate parathyroid gland a c t i v i t y resulting in increased circulating levels of parathyroid hormone. Given the premise that vitamin D-depleted phosphate-deficient chicks are acidotic (7,8) until proven otherwise, and recognizing the combined effects of phosphate-depletion and acidosis per se on bone resorption (10), as well as the reported blunted renal (1~T7 and exaggerated skeletal (12) response to parathyroid hormone in metabolic a c i d o s i s , how can one legitimately relate the observed changes in bone metabolism and serum calcium in D-depleted acidotic animal models to vitamin D action alone?

461 In addition to the effects of phosphate depletion on skeletal metabolism and mineral mobilization (10), and the direct effects of a c i d o s i s on bone metabolism (13), the effects of systemic pH changes on vitamin D metabolism must also be considered. In 1977, Sauveur e^t al_ reported studies in vitamin D - d e f i c i e n t chicks subjected to a diet containing 3% ammonium chloride, T% calcium, and 0.7% phosphorus (14). When kidney homogenates prepared from the acidotic chick, ( with blood pH 7.31 + 0.03 vs control r a c h i t i c chicks with pH values of 7.39 + 0.02),were incubated with @H)-250Hr)3, (3H)-1,25(0H)2D3 was reduced by 40%. Moreover, during in vivo experiments designed to study the metabolic fate of (3H) vitamin D3, (^H)-L,25(0H)2Ü3 was also reduced in blood, i n t e s t i n e and bone of the acidotic chicks (14). Since the acidotic chicks reared on a 0.7% phosphorus diet were also hyperphosphatemia (9.5 + 0 . 5 mg/dl), these investigators concluded that the observed reduction in 1,25(0H)2D3 production in the acidotic chicks stemmed from the attendant a c i d o s i s (14). Although s i m i l a r observations were made by Lee e_t al_ in the acidotic rat in the same year (15), these l a t t e r authors concluded that in t h e i r acidotic animal model, impaired enzymatic hydroxylation of 25OHD3 occurred independent of c i r c u l a t i n g calcium and phosphate concentrations. The r e s u l t s of the aforementioned in vivo studies of Lee et £]_ only documented decreased c i r c u l a t i n g and i n t e s t i n a l (3H)-1,25(0H)2D3 accumulation following an intravenous bolus of ( 3 H ) 250HDo in acidotic animals. As such the data did not necessarily implicate defective ( 3 H)-250HD3 — ( 3 H ) - 1 , 2 5 ( 0 H ) 2 D 3 conversion alone; decreased t i s s u e a f f i n i t y and/or increased degradation of pre-formed ( 3 H)-1,25(0H^D3 had to be entertained. In an attempt to resolve these queries, 1-day old White Leghorn cockerels were placed on d i e t s depleted of vitamin D which contained 0.8% calcium and 0.8% phosphorus. Metabolic a c i d o s i s was induced in one subgroup by the addition of 1.8% NH4CI to the drinking water, and respiratory a c i d o s i s in another by CO2 inhalation. Some chicks from each subgroup (and appropriate pair-fed controls) were injected with (3H)-250HD3 and i n t e s t i n a l ( 3 H)-1,25(0H)2D isolated. When compared to control animals with systemic pH values of 7.42 + 0.08, pH values of chicks with metabolic or r e s p i r a tory a c i d o s i s were 7.30 + 0.08 and 7.32 + 0.06, respectively. Serum c a l cium of chicks with metabolic (6.8 + 1.2 mg/dl) and r e s p i r a t o r y (6.1 + 0.4 mg/dl) a c i d o s i s were s i g n i f i c a n t l y higher than those of the control a n i mals (4-6 + 0.5 mg/dl), although blood phosphate values were s i m i l a r . The r e s u l t s of the in vivo (3H)-250HD3 studies in the a c i d o t i c chicks confirmed our observations made e a r l i e r in r a t s , i . e . the c i r c u l a t i n g l e v e l s and i n t e s t i n a l concentration of (3H)-1,25(0H)2D3 was decreased when compared to non-acidotic pair-fed control animals (15). Since, in v i t r o estimates of renal 1-hydroxylation rats can be performed in the chick (and not the rat) we proceeded to duplicate the above experimental design in chicks with both respiratory and metabolic a c i d o s i s , with emphasis on i s o l a t i n g renal mitochondria and determining the effects of a systemic a c i d o s i s (either metabolic or r e s p i r a t o r y ) on 1-hydroxylase a c t i v i t y . When the chicks were s a c r i f i c e d , blood pH, calcium and phosphorus were comparable to those obtained from the acidotic chicks used in aforementioned in vivo studies. Despite the s i g n i f i c a n t l y elevated serum calcium of the a c i d o t i c chicks,

462 mitochondrial 1-hydroxylase activity (in pmoles/mg protein/min x 100) was increased in either metabolic (8.9 + 0.4, p < 0.005) or respiratory (9.5 + 0.2, p < 0.001) acidotic animals when compared to control values of 5.6 j^O.l. There was no difference in 1-hydroxylase activity between respiratory or metabolic-acidotic chicks despite significant increments in both pC02 and HCO3" in those animals with respiratory acidosis. Thus, despite in vivo evidence of decreased circulating (3H)-1,25(0H)2D3 in both the acidotic chick and rat, in vitro data from acidotic chick revealed a paradoxical stimulation of 1-hydroxylase activity which was independent of circulating calcium, phosphate, bicarbonate and pC02 levels. One can only conclude from these experiments that the acidotic state results in either accelerated degradation of 1,25(0H)2D3 and/or a decreased target organ affinity. The relevance of the combined data accumulated in acidotic animals given radioactive 250HDj preparation to the vitamin D resistance observed in non-uremic acidotic states (16), and the healing of rachitic or osteomalacic lesions in vitamin D-resistant patients with renal tubular acidosis by alkali administration (17) is presently still conjectural. One must recognize however, that vitamin D-and phosphate-depleted animals so commonly used to evaluate the bioactivation of vitamin D may actually represent acidotic models. Consequently the data derived from experiments with vitamin D-depleted-acidotic rats or chicks with regard to intestinal calcium absorption, renal handling of calcium and phosphate, and bone resorption should not only be interpreted with caution but also acknowledged only when presented with full recognition of the acid-base status of the animals. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Hughes, M.R. and Haussler, M.R. (1975) Science 190, 578-580. Haussler, M., Hughes, M., Bay!ink, D. (1977) in Phosphate Metabolism (Massry, S. & Ritz, E., eds.) pp. 233-250, Plenum Press, New York. Ribowich, M.L. & DeLuca, H.F. (1975) Arch. Biochem. Biophys. 170, 529-535. Hurwitz, S., Stacey, R.E. (1969) Am. J. Physio. 216., 254-262. Edelstein, S., Noff, D. (1978) Biochem. J. 170, 227-233. Swaminathan, R., Sornnerville, B.A. (1978) Clin. Sci. 54, 197-200. Booth, B.E., Tsai, H.C. (1977) Metabolism 26, 1099-1105. Lee, S.W., Cooke, N.E., Birge, S.J. (1978) Clin. Res. 26, 530A. Edelstein, S., Harell, A. (1975) Biochim. Biophys. Acta 385, 438-442. Emmett, M., Goldfarb, S. (1977) J. Clin. Invest. 5£, 291-298. Beck, N., Kim, H.P., Kim, K.S. (1975) Am. J. Physiol. 228, 1483-1488. Beck, N. and Webster, S.K. (1976) Am. J. Physiol. 230, 127-131. Nguyen, V.V. and Jowsey, J. (1970) J. Bone Joint Surg. 52A, 1041-1049. Sauveru, B., Garabedian, M., Fellot, C., Mongin, P. and Balsan, S. (1977) Calcif. Tiss. Res. 23, 121-124. Lee, S.W., Russell, J. andAvioli, L.V. (1977) Science 195, 994-996. Albright, F., Consolazio, W.V., Coombs, H.W. and Sulkowitch, J.J. (1940) 66, 7-11. Foss, G.L., Perry, C.B., Wood. (1972) Lancet 2, 1204-1205.

463 THE SITE OF SYNTHESIS OF 1,25(OH) ? VITAMIN D, IN THE KIDNEY. A MICROENZYj MATIC ASSAY ON ISOLATED TUBULES. M.G. Brunette, M. Chan, C. Ferriere, K.D. Roberts Univ. of Montreal, Dept. of Pediatrics, Maisonneuve Hospital Montreal HIT 2M4, P.Q. Canada In 1970 Fraser and Kodicek reported that the kidney is the exclusive site of synthesis of 1,25 hydroxycholecalciferol (1,25(0H)2D3) and that the la hydroxylation of 25 OH D3 occurs in mitochondria. These findings were confirmed by several laboratories (5, 6, 7, 8). However, 8 years later, the identity of the cells within the kidney, responsible for this synthesis, is still unknown. Attempts have been made in this laboratory to measure la hydroxylase activity, in the various segments of the chicken nephron, using a single nephron micro-assay of this enzyme. Results demonstrated that the proximal tubule is the main and probably exclusive site of synthesis of the 1,25(0H) 2 D 3 . METHOD White leghorn cockerels were raised in darkness, and fed a calciferol deficient diet containing 1.74% Ca and 1.24% inorganic phosphorus, for 42 to 64 days. Controls were maintained in daylight, on a normal diet. After a systemic injection of heparin, the animals were bled, and renal arteries perfused in situ with a cold solution of collagenase. Left kidney was removed and minute fragments involving superficial and deep parts of the lobes were further digested in oxygenated collagenase solution for lh at room temperature. Chicken kidney consists of three lobes, each being divided in numerous lobuli. These lobuli themselves consist of superficial subunits (sublobuli) containing nephrons devoid of loops of Henle, and a deep portion where nephrons have well defined loops of Henle. A superficial nephron consists of a glomerulus, a proximal convoluted tubule (PCT), followed by a hairpin shaped segment (thick loop cortex), a hairpin shaped very thin segment (thin loop cortex) and finally a distal convoluted tubule (DCT) which connects to the collecting tubule (Coll). The deep portion of the lobuli is less readily digested by collagenase and could not be properly dissected. Therefore, no deep nephrons were assayed. Only small bundles of terminal collecting tubules in the medulla were isolated satisfactorily (Collecting papilla). After dissection, fragments of individual tubules were grouped according to their morphological characteristics, until a total length of 4-16 mm was obtained. Tubules were photographed for length measurement, and incubated in 0.6 to 1.0 ul of incubation medium for 60 min at 35° C. The final composition of the incubation medium was malate 10 mM, Tris HC1 100 mM, MgCl2 4 mM, EDTA 0.25 mM, glucose 6 phosphate 5 mM, NA-DP 0.33 mM, glucose 6 phosphate dehydrogenase 600 unit/L, bovin serum albumin 1%. 25, hydroxy 26.27 methyl 3h cholecalciferol (3h 25 OH D3 - Roche), 95-110 Ci mmol"! was added to the preparation. The 25 OH D3 concentration in the

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

k6k incubation medium varied from 135 to 560 nmol After incubation, the metabolites of vitamin D were separated by chromatography on paper impregnated with s i l i c a g e l (Whatman SG 81) using the technique of Bikle and Rasmussen (1). Papers were cut in 2cm pieces and dispersed into s c i n t i l l a tion v i a l s . The amount of metabolites was expressed in fmol, based on the original specific a c t i v i t y of the 3h 25 OH Dg. RESULTS The levels of la hydroxylase a c t i v i t y within the nephron segments are given in Fig. 1. The only s i g n i f i c a n t production of 1,25(0H)2D3 occurred in the PCT and the thick loops of the cortex, in rachitic chickens: 12.3 ± 1.3 fmol and 12.4 ± 2.4 fmol/ug tubular protein. No 1,25(0H)2D3 was produced in the remaining nephron segment tested. This absence of enzyme a c t i v i t y in the segments other than the proximal one may be assumed, unless other active segments are too short to produce detectable amounts of enzyme a c t i v i t y with this technique.

Fig. 1. 1,25(0H) 2 D 3 synthesised by various segments of the nephron of normal and rachitic chickens. Columns i n dicate the mean values and bars the standard errors. A s i g n i f i c a n t amount of the vitamin D3 metabolite was found only in PCT and thick loops of the rachitic chickens. The results are expressed in fmol of 1,25(0H) 2 D 3 per ug of tubular protein, a, PCT; b, thick loop cortex; c, thin loop cortex; d, DCT; e, collecting papilla. Similar experiments have been performed in vitamin D depleted mice and rats. Despite several attempts to overcome the inhibitor of the la hydroxylase already described in mammal kidney by Botham (2, 3), results remained poor and non reproducible. In r a t s , 1/3 of PCT and 1/4 of glomerul i preparations exhibited some la hydroxylase a c t i v i t y , whereas the other

k(>5 segments remained always s i l e n t . IN CONCLUSION These studies demonstrate that in vitamin D deficient chickens, la hydroxylase a c t i v i t y i s found exclusively in the proximal tubule of the kidney. The localization of the la hydroxylase a c t i v i t y within the proximal tubule of the rachitic chicken, where most of inorganic phosphorus (Pi) reabsorption probably occurs, and where PTH greatly influences this transport through c y c l i c AMP production, suggests that 1,25(0H)„D 3 may participate in the regulation of tubular Pi transport. BIBLIOGRAPHY 1.

Bikle, D. D., & Rasmussen, H. (1974) Biochem & Biophys Acta 362, 425-438.

2.

Botham, K. M., Tanaka, Y., De Luca, H. F. (1974) Biochem 13, 49614966.

3.

Botham, K. M., Ghazarian, J. G. I . , Kream, B. E., De Luca, H. F. (1976) Biochem 15, 2130-2135.

4.

Fraser, D.R., Kodicek, E. (1970) Nature 228, 764-766.

5.

Gray, R., Boyle, I . , De Luca, H.F. (1971) Science 172, 1232-1234.

6.

Gray, R.W., Omdahl, J . L . , Ghazarian, J.G., and De Luca, H.F. (1972) J. B i o l . Chem. 247, 7528-7532.

7.

Norman, A.W., Midgett, R.J., Myrtle, J . F . , and Nowicki, H.G. (1971) Biochem and Biophys Res Commun 42, 1082-1087.

8.

Shain, S.A. (1972) J. Biol Chem 247, 4393-4408.

k67 RESPONSE OF CHICK KIDNEY CELL CULTURES TO 1 ,25-DIHYDROXYVITAMIN Dj Helen L. Henry Department of Biochemistry, University of C a l i f o r n i a , California 92521 U S A

Riverside,

I t is now well recognized that the a b i l i t y of the kidney to produce l,25(OH) 2 D i or 24,25(0H) 2 D 3 from 25-OH-D3 is modulated by l,25(OH),D 3 itself. This is clear from several studies in intact animals ( 1 - 3 ) . Work in this laboratory with primary cultures of chick kidney c e l l s has e s t a b l i s h e d that l , 2 5 ( O H ) , D 3 can e x e r t i t s e f f e c t s d i r e c t l y on the renal c e l l jjn v i t r o ( 4 - 6 ; . That i s , kidney c e l l s c u l t u r e d in the presence o f 1,25(OH)-D, exhibit a markedly decreased 25-hydroxyvitamin D^-l-hydroxylase (1-hydroxylase) a c t i v i t y and increased 25-hydroxyvitamin D 3 ~24-hydroxylase ( 2 4 - h y d r o x y l a s e ) a c t i v i t y . The l e v e l s of these enzymes and their response to the presence of l^SCOH^D^ in tissue culture and in kidney homogenates following treatment of the intact animal, are depicted in Figure 1. Here we see that whether l,25(OH)_D„ is given to chicks or added to kidney cultures, there is a 5-10 f o l a reduction measurable 1-hydroxylase a c t i v i t y . In the intact chick and in c e l l cultures, the 24-hydroxylase is undetectable in the absence of l,25(OH)„D3. Note a l s o the q u a n t i t a t i v e l y s i m i l a r v a l u e s f o r both enzymes (pmol/min/mg protein) obtained in kidney homogenates and in cultured c e l l s . This f i g u r e demonstrates that primary c u l t u r e s of chick kidney c e l l s are, in terms of the metabolism 25-OH-Dj and response to 1,25(OH) 2 D.j , a v a l i d experimental model f o r f u r t h e r b i o c h e m i c a l investigation.

p m o l / m i n / m g protein -Hydroxylase

24-Hydroxylase

1-2

I I I +I 0.1-0.6 -D Chick

0.1-0.3

O

1-1.5 ¿H,25(0H) 2 D 3

I

0.1-0.4

0.3-0.6

Figure 1. Comparison of the rate of metabolism of H-25-OH-Dj by the two renal hydroxylases before and a f t e r treatment of chick (upper) or chick kidney c e l l cultures (lower) with 1 ^ S i O H ^ D j .

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

468 The methods for the preparation and culture of chick kidney cells have been previously published (4,6). Briefly, the kidney is removed following perfusion ^ n situ, dissected into small fragments and digested for 12 minutes with collagenase and hyaluronidase and for an additional minute with 0.25% trypsin. A suspension of singly cells, obtained by a series of cenrifugations, is diluted to 5 x 10 cells/ml of Minimal Essential Medium (Earles) and 10% fetal bovine serum. Twenty-four hours after plating, medium is changed, and the cells allowed to grow for another 36 hours. At that time serum is removed from the medium and 1^-20 hours later assays for the metabolism of H-25-hydroxyvitamin D^ These are iniitatecL following a change ( H-25-OH-D-) are carried out. of serum-free medium, by the addition of 5 x 10 M H-25-OH-D 3 (100 mCi/ mmol). Following a 30 minute incubation, lipids are extracted from cells and medium. Separation of tritiated metabolites is accomplished by high pressure liquid chromatography. —8 As described above and depicted in Figure 1 the addition of 5 x 10 M 1,25(0H)„I1- in serum-free medium to cultures for 20 hours prior to the assay of^ H-25-OH-D, metabolism results in a marked decrease in the production of H-l,25foH) 2 D 3 and an increase in that of H-24,25(OH> 2 D 3 from undetectable to quite substantial levels. The effect of varying the concentration of l,25(OH) 2 D 3 is shown by the solid circles in Figure 2. The 1-hydroxylas^ was decreased and the 24-hydroxylase increased by as little as 10 M l,25(OH)-D,. Interestingly, as shown by the open circles in Figure 2, 24,25(oH)„D 3 added to cultures prior to assay, has the same effect as l,25(OH72l>3 but requires 3-5 times the concentration. Experiments are currently underway to determine whether this effect of 24,25(0H).D, is of physiological significance.

E o

[l,25(OH)2D3]

M IO"7M [24,25(0H)2D3] O

Figure 2. Response of chick kidney cell cultures to the presence of l,25(OH) 2 D 3 (closed circles) and 24,25(OH) 2 D 3 (open circles). Steroids were added in serum-|ree medium 20 hours prior to asay. Solid lines show the production of H - 1 , 2 5 ( O H ) - D , and dashed lines indicate that of 3 H-24,25(OH) 2 D 3 .

469

Time After Removol of 1,25(01^03 (Hours) Figure Effect of removal of 1,25(011)203 from medium on the metabolism of H-25-OH-D2 by chick kidney cell cultures. For all time points fetal calf serum was removed 24 hours prior to assay and l ^ S i O H ^ D ^ was present for twelve hours. The mean +_ SEM of triplicate cultures is shown.

Jo determine whether the effect of 1,25(011)203 on the metabolism of H-25-OH-D3 is a reversible one, cultures were exposed to the steroid for twelve hours at which time the medium containing the steroid was replaced after washing the culture^ twice. The cultures were assayed H-25-OH-D3 at several time points. for their ability to metabolize Production of H-24,25(011)203 continued to rise for four hours after removal of l,25(OH)„D3 ^ r o m medium at which time it began to decrease as the synthesis of .^1-1,25 (OlO-Dj increased. In a separate experiment (data not shown) H-l, 25(011)203 was added to cultures and four hours later medium was removed and cells washed. The amount of steroid remaining in the cells was thirty percent of that originally added, suggesting that the four-hour lag in the reversibility of the effects of l,25(OH)2D3 seen in Figure 3 may be due to steroid remaining in the cell which is subsequently metabolized to an inactive form and/or released. Figure 4 shows that when either the inhibitor of protien synthesis, cycloheximide, or that of RNA synthesis, actinomycin D, is added to cultures at the same time (6 hours prior to assay) as l,25(OH)2D,, the effect of the steroid on 25-OH-D3, metabolism is altered. The presence of either inhibitor resulted in an approximate 50% loss in 1-hydroxylase activity whether 1,25(011)203 was present or not. However, the effect of l,25(OH),D, on the remaining activity was the same regardless of whether an inhibitor was present. In contrast, the presence of either cycloheximide or actinomycin D virtually completely abolished the

470

3 Figure 4. E f f e c t of l , 2 5 ( O H ) 2 D , on the metabolism of H - 2 5 - 0 H - D , by chick kidney c e l l c u l t u r e s in the presence of cyclohe^amide ( l e f t ; or actinomycin D ( r i g h t ) . The i n h i b i t o r s were added (10 M) at the same time as l , 2 5 ( O H ) „ D , , 6 hours p r i o r to a s s a y .

r i s e in 2 4 - h y d r o x y l a s e a c t i v i t y induced by l , 2 5 ( O H ) 2 D 2 in the absence of i n h i b i t o r . T h e s e r e s u l t s d e m o n s t r a t e t h a t the i n c r e a s e i n 2 4 h y d r o x y l a s e a c t i v i t y , but not the d e c r e a s e in 1 - h y d r o x y l a s e a c t i v i t y ,

Tim« In Presence of l,25(OH)2D3 (Hours) F i g u r e 5. Time course o f the e f f e c t of l ^ S i O H ^ D j on kidney c e l l c u l t u r e metabolism of 3 H-25-OH-D 3 . F e t a l c a l f serum_^as removed from a l l c u l t u r e s 20 h o u r s p r i o r to a s s a y and 5 x 10 M l,25(OH)2D, was added at the i n d i c a t e d times p r i o r to a s s a y . The p r o d u c t i o n or H - l , 2 5 ( O H ) 2 D , i s shown b y * — • o f the two m e t a b o l i t e s b y A

; 24,25(OH> 2 D 3 .

by t>

o

;

and

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471 The time course of the effect of l,25(OH)^D^ on the metabolism of H2 5 - O H - D 3 by chick kidney cell cultures is shown in Figure The results are expressed as percent of the maximal production of H-1,25( O H ^ D , (87 pmol/30 min/culture obtained in the absence of added 1,25(OH) 2 D 3 ) and H-24,25(0^-0.3 (26 pmol/30 min/culture obtained 20 hours after the addition of lj'zSvOH^D- to the culture medium). Previous experiments had established that 20 hours is sufficient time to^achieve the maximal change in 25-OH-D. metabolism. The production of H-1,25(0H).D 3 began to fall within "30 minutes of the addition of l,25(OH) 2 D 3 to the culture medium and was reduced to 50% of its original level in three hours. The production of H-24,25(OH).D, was not detectable until after four hours in the presence of 1,25TOH) 2 D 3 and thereafter rose nearly linearly. The rapidity of the effect of l,25(OH) 2 D 3 suggested the possibility that the steroid was acting, during the early part of the time course, as a product exerting feedback inhibition on the enzyme which produces it. In many cases of product inhibition, the inhibition observed is of the competitive type with respect to the substrate. If this is the case, then higher substrate ( H-25-OH-D.) concentrations during the assay period should diminish or^abolish tne inhibitory effect of the product. Therefore four different H-Z5-0H-D. concentrations were used following a 2 hour incubation with 10 or lu~ M l,25(OH) 2 D 3 . The results are shown in Figure 6. The light dashed line shows the expected relationship between subustrate concentration and inhibition by product if the product inhibition is of the competitive type. Clearly in the case of o cc

1

1

1

1

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24.8

C 3 H-25-0H-D 3 ] HT® M Figure 6. Inhibition of the metabolism of H-25-OH-D 3 by l,25(OH) 2 D 3 in cultured kidney cells. Cultures were exposed to 1,25(OH) 2 d 3 f o r 2 hours. Medium was then removed, th^ cells washed, and fresh medium containing varying concentrations of H-25-OH-D, was added. Results are expressed as the percent of control (no l,25(OH) 2 D 3 added 2 hours prior to assay) and the dashed line indicates the pattern expected if competitive product inhibition is occurring.

472 the effect of 1,25(0H)^D2 on the 1-hydroxylase, this is not the situation; in fact, inhibition was more pronounced at the higher substrate concentrations. It must be kept in mind, of course, that this system involves an intramitochondrial enzyme within a whole cell rather than an isolated enzyme protein . Therefore, it is possible that the effect of l,25(OH)2D^ is being exerted elsewhere in the cell than directly on the 1-hydroxylase itself. An experiment was carried out to determine whether prior treatment with l,25(OH)2D, for two ^hours had an effect on the ability of cells to subsequently obtain H-25-OH-D, from the medium. N e i t h e r 10 nor 10 M l , 2 5 ( O H ) 2 D 3 had such an e f f e c t . Of the H-25-OH-Dj added to cultures, 65% was associated with cells within 5 minutes regardless of whether l ^ S C O H ^ D ^ was present or not. Whether the intracellular localization of the substrate was affected remains to be examined but the results in Figure 6 strongly suggest that the early marked effect of l ^ S i O H ^ D j on the 1-hydroxylase in kidney cell culture involves a more complex mechanism than competitive product inhibition. In v i e w of the r e s p o n s i v e n e s s of the c^iick k i d n e y c e l l c u l t u r e s to l ^ S C O H ^ D ^ in terms of the metabolism of H-25-OH-D2, it was of interest to explore other possible effects of the steroid on these cells. For many decades it has been known that primary tissue and cell cultures r e q u i r e the p r e s e n c e of c e r t a i n u n d e f i n e d factors for s u r v i v a l and growth. Recently the appropriate factors for several cell types have b e e n d e f i n e d (7). In a p r e l i m i n a r y a t t e m p t to d e t e r m i n e w h e t h e r l ^ S C O H ^ D ^ affects kidney cell growth the experiment shown in Figure 7 Kidney Cells 24

hours

in

culture

10% FBS

24

-FBS

•FBS

+ I,25(0H) 2 D 3

hours

l-Hydroxylase: pmol/30 min/IO5 cells 2.0±0.l #

cells/culture (xl05)

24

13.4+3.6

2.8+0.2 6.6+1.0

l.5±0.l I3.4H.2

hours 11

#

cells/culture (xlO 5 )

28.4±4

0.9±0.3

6.4+0.9

Figure 7. Effect of l,25(OH)„D2 on the survival and growth of chick kidney cells in culture. Cells were prepared and cultured as usual in 10% fetal calf serum. Twenty-four hours later this medium was replaced with serum free medium in two sets of cultures, to one of which 10 - ^ M l ^ S C O H ^ D ^ was added. Control cultures received medium containing serum. Cells were counted 24 at^d 48 hours after this change and the ability of the cells to produce H - l , 2 5 ( O H ^ D ^ was determined 24 hours after serum removal.

^73 was carried out. In the absence of serum, cells died rapidly and by 48 hours after the removal of serum from medium these cultures had only 2% of the number of cells as did the control cultures. The addition of l,25(0H)2Dg to the medium maintained growth for 24 hours and even after 48 hours these cultures had 25% the number of cells in control cultures. The effect of 1,25(OH)2D.J on H-25-OH-D 3 metabolism was as expected during the first 24-hour period, with a decrease in the measurable 1-hydroxylase activity per cell. These results indcate that, even though significant cell death occured during the second 24 hours in the absence of serum and presence of 1, 25 (OH)„D.,, the steroid markedly reduced death and actually maintained growtn during the first 24 hour period. The effect of l ^ S C O H ^ D j on parathyroid hormone-stimulated cyclic AMP production was also determined as shown in Figure 8. Pre-incubation of cultures with 10 M l ^ i i O H ^ D ^ for one hour significantly reduced subsequent cyclic AMP production in response to PTH.

[I,25(0H)2D3] M

Figure 8. cyclic AMP exposed to Then bovine AMP content

Effect of l,25(0H)2Do on parathyroid hormone stimulated production by cultured chick kidney cells. Cultures were the indicated concentration of 1 , 2 5 ( 0 ^ 2 ^ for one hour. PTH was added and following a 20 minute incubation the cyclic of the medium and cells was determined by radioimmunoassay.

The results obtained thus far with primary cultures of kidney cells can be summarized as follows: (1) l,25(OH)2D, inhibits its own production by a mechanism which does not require RNA or protein synthesis but which also may not be simple product inhibition of the 1-hydroxylase.

k7k (2) L,25(OH) 2 D 3 induces the production of 24,25(0H) 2 D 3 by a

mechanism which requires new RNA and protein synthesis. (3) 1,25(0H)„D, affects the viability of culture and may moaulate cyclic AMP production. Thus, the possibility is emerging that metabolism of 25-OH-D3 by the kidney other functions of the cell which are survive and grow in culture and to

chick

kidney

cells

in

in addition to its effects on the cell, 1,25(OH)2D.J may well have reflected in the cells ability to respond to parathyroid hormone.

References 1.

Henry, H., Midgett, R. J., and Norman, A. W. (1974) J. Biol. Chem. 249, 7684-7692.

2.

Galante, L. H., Colston, K. W., Evans, I. M. A., Byfield, P. G. H., Matthews, E. W., and Maclntyre, I. (1973) Nature 244, 438-440.

3.

Horiuchi, N., Suda, T., Sasaki, S., Izawa, I., Sano, Y., and Ogata, E. (1974) FEBS Lett. 43, 353-356.

4.

Henry,.H. L. (1977) Biochem. Biophys. Res. Commun. 74, 768-775.

5.

Henry, H. L. (1977) in Vitamin D: Biochemical, Chemical and Clinical Aspects Related to Calcium Metabolism (Ed. A. W. Norman, ^t ; Walter de Gruyter, 1977) p. 125.

6.

Henry, H. L. (1979) J. Biol. Chem. (in press).

7.

Rizzino, A., and G. Sato (1978) Proc. Natl. Acad. Sci. 75., 1844-1848.

G R O W T H H O R M O N E A N D P R O L A C T I N A C T I O N ON V I T A M I N D M E T A B O L I S M IN T H E R A T

E.M. Spencer, 0. Tobiassen, P. Ling, and E. Braunstein General Clinical Research Center, Metabolic Unit, and Cancer Research Institute, University of California, San Francisco, CA 94143, USA INTRODUCTION Growth hormone (GH) was first shown to increase intestinal absorption of calcium in humans by Beck et al (1). This observation was subsequently confirmed in several laboratories. Finkelstein and Schachter demon^ strated that hypophysectomy decreased duodenal absorption of calcium as measured by the everted gut sac method; GH administration to hypophysectomized (Hypox) rats could correct this defect (2). The first study identifying the mechanism of GH action was done by Spencer and Tobiassen who suggested that GH stimulated the renal 25-OH-Dj - lahydroxylase activity (3). They presented evidence that hypophysectomy sharply decreased the in vivo conversion of 3H-25-OH-D3 to 3H-1,25-(OH)2D3 in rats and that GH treatment of Hypox rats significantly stimulated this conversion. These authors pointed out that since the recovery of ,25-(OH)2D3 was used as a measure of lahydroxylase activity, an increase in the peripheral metabolism of l,25-(OH)2D3 could cause a decreased recovery of 3H-1,25-(OH)2D3 after 3H-25-OH-D3 injections. The low plasma l,25-(OH)2D3 levels subsequently reported in Hypox rats did not establish a mechanism, for the low l,25-(OH)2D3 levels could also be caused by either increased catabolism of l,25-(OH)2D3 or decreased lahydroxylase activity. Therefore, we have measured the metabolic clearance of ,25-(OH)2D3 in Hypox animals in order to distinguish between these two possible mechanisms of GH action: i.e., either on the renal lahydroxylase activity or on the peripheral metabolism of 1,25(OH)2D3 . Additional studies were done to: 1) compare the intestinal mucosal localization of iv ,25-(OH)2D3 in Hypox and intact rats and 2) test whether the other hormones lost by virtue of hypophysectomy had an effect on renal lahydroxylase activity. Concerning the former, a mucosal defect had been suggested by the much lower recovery of 3H-1,25-(0H)2D3 from intestinal mucosa than serum in the studies of Spencer and Tobiassen (3). Concerning the latter, Finkelstein and Schachter found that ovine prolactin (oPRL) administration to Hypox rats had a weak stimulatory action on calcium transport in the everted gut sac (2). In view of the reported effects of PRL on renal lahydroxylase activity in birds (5, and Bikle and Spencer, unpublished) it was decided to look more carefully at its action in rats. These investigations have shown that the metabolic clearance of 3H-1,25(OH)2D3 in Hypox rats is normal. This indicates that GH stimulates the activity of the renal lahydroxylase. PRL, cortisone, thyroxin, and testosterone had no activity on lahydroxylase activity. The reported effects of PRL on calcium absorption in rats are probably due to direct action on the intestinal mucosa (2).

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

476 MATERIALS AND METHODS Nineteen day weanling male Wistar rats were fed a partially D-deficient diet containing 0.3% Ca and 0.47% P. Partial D-deficiency was achieved by feeding an ICN custom diet containing no vitamin D and supplementing this diet with 1.0 U of vitamin D3 in 50 ul of Wesson oil by gavage daily beginning on day 26. A partially D-deficient diet was used to allow the conversion of 3H-25-OH-D3 to 3H-1,25-(OH)2D3 to be measured. Growth was 80% of normal and serum Ca and P were normal in control rats. Hypophysectomies and sham procedures were done on day 28. For the metabolic clearance and intestinal mucosal localization of l,25-(OH)2D3, 0.6 uCi of 3H-1,25-(OH)2D3 ( a gift from Dr. James Hamilton, Hoffman-LaRoche, Nutley, NJ) was given iv on day 44. For plasma disappearance curves, aliquots of blood were taken from each of 4 rats at 3.75, 7.5, 15, 30, 60, 120, 240, and 720 minutes. After separation of the heparinzed plasma, the red cells were reinfused to maintain a constant blood volume. Plasma was extracted with methanol:chloroform (2:1,v:v) and the 3H-1 ,25-(OH>2D3 recovered was counted after Sephadex LH-20 chromatography with chloroform:hexane (65:35). For intestinal mucosal localization of ,25-(OH)2D3 studies, the mucosa was extracted at 1, 4, and 12 hours with methanol:chloroform and chromatographed on Sepadex LH-20. Intestinal mucosal and serum 3H-1,25-(OH)2D3 were found to be 90% intact after 12 hours. The conversion of 3h-25-OH-D3 to 3h-1,25-(OH)2D3 was measured by giving 1.0 uCi of 3H-25-(OH)2D3 (specific activity 92 Ci/mmol, a gift from Hoffmann-LaRoche, Nutley, NJ) by intrajugular injection to rats under light ether anesthesia at 1700 hours on day 43. After 16 hours, the time recovery of 3h-1,25-(OH)2D3 from serum and mucosa had been determined to be maximal, rats were etherized and sacrificed by bleeding from the inferior vena cava. The proximal 50 cm of small intestine was removed from each rat and rinsed with ice cold 0.9% saline. The mucosa was scraped free using a glass microscope slide, weighed, and transferred to ice cold distilled water. Homogenates were extracted with methanol: chloroform (2:1, v:v). 3H-25-OH-D3 metabolites were resolved on Sephadex LH-20 (Pharmacia Fine Chemicals, Piscatoway, NJ) columns equilibrated with 65% chloroform - 35% hexanes (Mallinkrodt Chemical Works, St. Louis, MO). Fractions of 4 ml were evaporated to dryness and counted in a toluene based scintillation fluid containing 4 g of 2,5-dephenyloxazole (PPO) and 0.4 g l,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene (dimethyl POPOP) per liter. Radioactivity was determined on a Packard Tri-Carb (Model 2003) liquid scintillation spectrometer. Peak identifications were verified by chromatography of standard 3H-1,25-(OH)2D3, periodate treatment, and chromatography on SG-81 paper (Whatman, W.R. Balston Ltd., England). The administration of the hormones to be tested was begun on day 38. The daily sc doses were: bGH 250 ug, oPRL (courtesy of Dr. C.H. Li) 250 ug, thyroxin 1 ug, testosterone 100 ug, and cortisone acetate 20 ug. Hormones were given at 1400 hours. Groups of 3-5 rats were studied. + 0 . 4 7 % Ca and 0 . 3 % P.

^77 RESULTS The metabolic clearance of iv ,25-(OH)2D3 was the same in Hypox and intact rats. The half disappearance times were both 4.0 minutes. Twentyfive percent of the radioactivity remained in plasma at 1 hour and 8% at 12 hours. No defect in intestinal mucosal localization of iv

, 25-(OH)2D3 was observed in Hypox rats. Localization was maximal at 4 hours in Hypox rats and 3 times that of intact rats when expressed as radioactivity/gm. These values were not corrected for differences in the pool sizes of l,25-(OH) (OH) 2 D 3 . 2D3. At 12 hours Hypox rats still localized 50% more 3H-1,25-

The effect of various hormones on the conversion o

f 3H-25-OH-D3 to 3Hl,25-(OH)2D3 in Hypox animals was compared. bGH was the most effective stimulator of this conversion. With newly synthesized l,25-(OH)2D3 expressed as per cent of eluted radioactivity, conversion was: 36.7 ± 4.1 with controls; 10.0 ± 4 with Hypox; 33.6 ± 2.1 with bGH; 32 ± 4 with bGH and cortisone; 41 ± 2 with bGH and oPRL; 21 with bGH, oPRL, cortisone, thyroxin and testosterone; and 10 ± 2.1 with oPRL. Hypophysectomy and hormone treatments had no effect on the serum Ca. Hyposec tomy decreased the serum P from 8.86 ± 0.34 to 6.86 ± 0.24 mg/dl (n = 12) and GH treatment of Hypox rats raised the serum P to 7.89 ± 0.32. DISCUSSION

The metabolic clearance of

,25-(OH)^D3 was not increased in Hypox rats. The intestinal mucosal localization of , 25-(OH)2D3 was only slightly greater than controls after an approximate correction for the decreased l,25-(OH)2D3 pool based on data in the literature for D-deficient rats (4). These studies established that the conversion of 3H-25-

OH-D3 to 3 H-1

, 25— (0H)2D3 could be used as a measure of renal la hydroxylase activity. Since no defect in intestinal mucosal localization of l,25-(OH)2D3 was found, the recovery of ,25-(OH)2D3 from intestinal mucosa was used to estimate relative changes in lahydroxylase activity. Our results indicate that after hypophysectomy only GH returned the decreased lahydroxylase activity to normal. The addition to GH of other hormones lost subsequent to hypophysectomy, including PRL, thyroxine, cortisone, and testosterone, had no additional effect on the lahydroxylase activity. PRL alone had no effect on lahydroxylase activity. We propose that GH regulates intestinal Ca absorption by an action on the renal lahydroxylase increasing the amount of l,25-(OH)2D3 formed. GH does not appear, however, to be involved in short term regulation of lahydroxylase activity, but may be responsible for the increased intestinal Ca absorption and l,25-(OH)2D3 plasma levels of growing versus adult animals. The mechanism of action of GH in stimulating lahydroxylase activity is unknown. In view of unpublished studies that avian GH does not stimulate lahydroxylase activity of chick renal tubules (Bikle, Spencer, and Burke) it is possible that this action is mediated by soma-

tomedin (a family of low molecular weight polypeptides thought to mediate the growth-promoting action of GH). In in vitro studies with chick renal tubules, Bikle, Spencer, and Burke (unpublished observations) have found that PRL has a direct action on lahydroxylase activity. Contrary to these experiments we have found that PRL has no effect on the renal lahydroxylase activity in rats. This difference could be explained by the unique Ca requirements of birds for egg laying. The reported effects PRL on intestinal Ca absorption in rats are undoubtedly direct effects on the intestine and represent a persistence of the fluid and ion transport function of PRL evident in lower species. SUMMARY The metabolic clearance of

,25-(OH)2D3 is not increased in Hypox rats nor is there a defect in the intestinal mucosal localization of 3H-1,25(OIO2D3 in spite of the severe mucosal atrophy attendant to hypophysectomy. Thus the recovery of 3H-l,25-(OH)2D3 from intestinal mucosa after 3H-25-OH-D3 injection allows an indirect relative measurement of renal lahydroxylase activity to be made. Hypophysectomy was found to decrease lahydroxylase markedly. Only GH of the pituitary hormones, or pituitary dependent hormones, could restore this activity to normal. PRL was inactive in Hypox rats. REFERENCES 1.

Beck, J.C., McGarry, E.E., Dyrenfurth, D. and Venning, E.H. (1957) Science 125, 884-887

2.

Finkelstein, J.D. and Schachter, D. (1962) 876.

3.

Spencer, E.M. and Tobiassen, 0. (1977) In Vitamin D Biochemical, Chemical and Clinical Aspects Related to Calcium Metabolism, Walter de Gruyter, Berlin, p. 197-199.

4.

Spanos, E., Barrett, D., Maclntyre, I., Pike, J.W., Safilian, E.F. and Haussler, M.R. (1978) Nature 273, 246-247.

5.

Spanos, E., Colston, K.W., Evans, I.M.S., Galante, L.S., Macauley, S.J. and Maclntyre, I. (1976) Molecular and Cellular Endocrinology 5, 163-167.

Am. J. Phy.

203, 873-

^79 EFFECTIVE

LIl/ER 25-HVDROXVLATION

OF VITAMIN

ADMINISTRATION McwLdULz

IN THE

V UNDER CHRONIC

ETHANOL

RAT*

Gcu>c.(m-BN O) M &

tu rH

•

o

MH O

« ra tU n 4-1

tM

m NO

a

ra 4-1 H tí O, O)

•

O

Vi tu •H

ra ra o ra•Otí e ra ra

e o •H 4J

•

m CM CO < r t>j ON NO iH CN

ra 1-1 S ra ra rH

ra rH

< CL

z

a: 3 UJ

H I in O a

I

4

8

12

IS

36

48

TIME (HOURS)

Fig. 2

Computer fit ( 0 — 0 ) of experimental data (A) for appearance of 1,25(OH)2(3H)D3 in the urine. Use of the generator function is described in the text.

520 then used to model the appearance of radioactivity in the urine. Using the generated disappearance data, we were able to fit the urine radioactivity data points to a very close approximation (Figure 2). The estimated appearance of 1,25-(OH)2D in the urine was 0.019 nmol per day (Figure 3) which represents approximately 13% of the daily 1,25-(OH)2D loss from the plasma and exchangeable pools. Given the appropriate data, it would also be possible to model the 1,25-(OH)2D appearance in feces, the major excretory pathway for the metabolite (4).

HUMAN COMPARTMENTAL ANALYSIS 1,25-DIHYDROXYVITAMIN D

Fig. 3

Four compartment model for disappearance of plasma 1 , 2 5 (0H)2(^H)DJ and its distribution into exchangeable tissue pools and irreversible outs, such as the urine.

DISCUSSION - The 3-compartment model which was used to simulate the plasma disappearance of vitamin (3H)D 3 , 25-OH(3H)D3 and l , 2 5 - ( O H ) 2 (3h)D3 appear to meet both analytic and physiological constraints. Computer fitting of the experimental data through use of the KABIS computer program was quite good. In addition, the model addresses the minimum number of exchange and irreversible pathways which are required to describe the vitamins' physiological processes. A model similar to ours, but lacking an irreversible out from the plasma compartment, has been proposed (4) for the plasma disappearance of 1,25-(0H)2(^H)D3 during the 4 h following injection. However, when considering the complete timeframe, a direct out ( A 0,1) from the plasma compartment is required in order to account for the irreversible metabolism of 1,25-(OH)2(^H)D3.

521 Each analytic process for the modeling of vitamin D or metabolite plasma disappearance was performed independently. Yet, vitamin D and 25-OHD fluxes through the irreversible metabolism-pathway (A 0,1) were large enough to meet the subsequent metabolites turnover demands. In other words, the generated kinetic parameters are compatible with the endogenous metabolic sequence of vitamin D to 25-OHD to 1,25-(OH)2D. The physiological relevance of the flux calculations are demonstratable for both vitamin D and 1,25-(OH)2D. Urine and fecal losses (X 0,3) in the vitamin D model (normal levels) were -v. 14 yg/day which is quite close to the 10 yg RDA in the United States. Likewise, the 0.5 yg of l,25-(OH)2D3 which turnovers each day is reasonable since exogenous supplements of 0.5 to 1 yg/day can meet the physiological needs of anephric patients. From this study it is now evident that the normal kinetics for plasma disappearance of vitamin D, 25-OHD and 1,25-(OH)2D can be determined. It seems appropriate, therefore, to consider using the compartment model and the computer simulation capabilities to predict the kinetics of vitamin D metabolism in metabolic disease states. We are currently investigating such computer applications using animal-models which have defined abnormalities in vitamin D metabolism. ACKNOWLEDGMENTS

- Biomedical Research Support Grant # RR05583-13.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

Meyer, M., S. Wechsler, S. Shibolet, M. Jedwah, A. Harell and S. Edelstein (1978) J. Mol. Med. 3, 29-37. Mawer, E.B., G.A. Lumb, K. Schaeler, and S.W. Stanberg (1971) Clin. Sci. 40, 39-53. Bee, P.H., F. Bayard and J.P. Louvet (1972) Rev. Europ. Etudes Clin. Et Biol. 17, 793-796. Gray, R.W., A.E. Caldas, D.R. Wilz, J. Lemann, Jr., G.A. Smith and H. F. DeLuca (1978) J. Clin. Endocrinol. Metab. 46, 756-765. Jones, G. (1978) Clin. Chem. 24, 287-298. Berman, M.,E. Shahn, and M.F. Weiss (1962) Biophys. J. 2, 275-287. Gray, R.W., H.P. Weber, J.H. Dominguez and J. Lemann, Jr. (1974) J. Clin. Endocrinol. Metab. 39, 1045-1056.

523 PROLACTIN, GROWTH HORMONE AND VITAMIN D METABOLISM

I . Maclntyre, D.J. Brown and E. Spanos Endocrine U n i t , Royal Postgraduate Medical School, Du Cane Road, London W12 OHS, England. INTRODUCTION In t h i s account we f i r s t summarize what i s known of the action of p r o l a c t i n and growth hormone on vitamin D metabolism, and l a t e r describe our recent work which explains how p r o l a c t i n a c t s . We also discuss the regulation of vitamin D in general, including new data on parathyroid hormone, so that p r o l a c t i n and growth hormone are seen i n context. Our findings resolve many apparent discrepancies and suggest a rational hypothesis f o r the regulation of vitamin D metabolism i n h e a l t h . METHODS Our techniques f o r measuring 1- and 24-hydroxylase a c t i v i t i e s i n kidney, c i r c u l a t i n g l e v e l s of 1,25(0H)2D3, and the required separation techniques have been described elsewhere ( 1 , 2 , 3 , 4 ) . A primary chick kidney c e l l culture technique was used e x t e n s i v e l y (5) and has proved results. extremely useful i n explaining in vivo RESULTS AND DISCUSSION (a)

Known Regulators

Any idea that the conversion of 25 OH D3 to i t s most active metabolite, 1,25(0H)2D3, i s under only one trophic control i s completely untenable. It i s now recognized that there i s a group of substances, each of which influences the production of the hormonal form of vitamin D from the kidney. These substances are: 1,25(0H)2D3 i t s e l f ( 6 , 5 ) ; the calcium and phosphorus content of the d i e t (7,8); parathyroid hormone (9,10); oestrogen (11,12,13); and the p i t u i t a r y hormones, growth hormone (14,15) and p r o l a c t i n (to be discussed below). And the length of t h i s l i s t should not be s u r p r i s i n g . The regulation of calcium, in which vitamin D plays such an important p a r t , i s so important f o r many v i t a l functions that we must expect multiple c o n t r o l s . Thus, although parathyroid hormone helps to regulate vitamin D metabolism, i t s e f f e c t may be overruled by other agents i n appropriate p h y s i o l o g i c a l circumstances. The main function of 1,25(0H)2D3 appears to be the enhancement of calcium and phosphorus supplies under p h y s i o l o g i c a l calcium s t r e s s . This occurs during growth, pregnancy and l a c t a t i o n , and plasma l e v e l s of 1,25(0H)2D3 are elevated i n each of these s i t u a t i o n s (3,16,13). It may be best to consider 1,25(0H)2D3 as a hormone whose most v i t a l role i s to

© 1979 Walter de Gruyter Sc Co., Berlin New York Vitamin D , Basic Research and its Clinical Application

52*4 provide the calcium and phosphorus supplies of the foetus and growing c h i l d . The destructive osteoclast-enhancing effect of 1,25(0H)2D3 on bone may be normally prevented by c a l c i t o n i n , but able to provide needed calcium and phosphorus for the foetal tissues should the mother's intake be grossly deficient. The role of parathyroid hormone in these physiological changes of vitamin D metabolism may be e s s e n t i a l , permissive or n e g l i g i b l e depending on each case. For example, the effect of growth hormone in increasing plasma 1,25(0H)2D3 levels i s not due to a change in parathyroid hormone secretion (Figure 1). 200-,

. 2000

T

160 -

1600

, 120 -

1200

•

m Sham

Figure

I (redraun

from

C.HPX

HGH1

HGH2

oPRL

IS)

E f f e c t of growth hormone on plasma lex>els of ls25(OH)2D3 and PTH. Shamj sham operated; C3HPX} control hypophysectomisedj HGHlj 100 y.g himan growth hormcme/d.; HGH2, 500 pc? human growth hormcne/d.; oPRL, IOO \ig ovine prolactin/d. The hormones were given to hypophyseatomized animals only. S i m i l a r l y , the great enhancement of plasma 1,25(0H)2D3 levels in pregnancy (3) i s not due to parathyroid hormone. But i t i s possible that parathyroid hormone levels may increase immediately before delivery and exert some effect. Further, although prolactin plays an important role in l a c t a t i o n , independent of parathyroid hormone, i t seems l i k e l y that parathyroid hormone i s also actively involved, p a r t i c u l a r l y in the later stages. Some years ago we reported experiments which provoked a great deal of argument (17). These showed that when rats on a low calcium intake were placed on a high calcium diet there was a change i n vitamin D metabolism.

525

Unexpectedly, this change was accelerated i f parathyroid hormone was also given. The e f f e c t was a further decrease in 1,25(0H)2D3 and an increase in the l e s s active metabolite, 24,25(0H)2D3 (Figure 2 ) . CONTROL

+PTH

f 40.5

n* 44.4

"

V o l u m e of eluate

(mil

Figure 2 (redrawn from 17) Chromatography an LH20 Sephadex of serum extracts from D-de fiaient 25-H.C.C. The PTH group received 200 I.U. 8 hours after tritiated parathyroid extract/d.

rats

How are we to account for the apparent contradiction between these r e s u l t s and others which appear to show the reverse (9,10)? Recent experiments on the e f f e c t s of calcium and parathyroid hormone in c e l l culture provide the answer: elevation of calcium in the culture medium depresses the 1-hydroxylase enzyme markedly while enhancing 24-hydroxylase (Figure 3 ) ; but parathyroid hormone has the opposite e f f e c t (Figure 4 ) . 30-,

0

O.H

0.8

1.2

2.1 Ca (innioI ' 11

Figure 3 3.6 (legend overleaf).

526 Figure 3 The effect of medium ca.lei.um concentration on the ratio of hydroxylase enzymes in 36 hr. chick kidney cell cultures. Two experiments are shown.

Similar conclusions about calcium have been drawn from in vivo experiments ( 7 ) , and our new results confirm that the effect of dietary calcium i s due in part to a direct action of the circulating level of calcium on the renal c e l l . I t has been d i f f i c u l t to demonstrate an effect of parathyroid hormone in kidney cell cultures. We have been unable to show any convincing effect of parathyroid hormone in the isolated renal tubule. And parathyroid hormone has no clear acute effect in chick kidney cell cultures. However, when cultures are exposed to parathyroid hormone for 24 hours the effect i s marked (Figure 4). 10

5 -

1

2

50

—I

500

PTH (ng/ml)

Figure 4

OPTH chronic

«PTH acute

The effect of bovine parathyroid hormone for 24 hr. and I hr. on the hydroxylase enzymes ratio in chick kidney cell culture.

The hormone dramatically depresses the activity of 24-hydroxylase and although the s l i g h t enhancement of 1-hydroxylase i s s t i l l unconvincing, this may reflect experimental conditions rather than absence of any action on this enzyme. Clearly, further study i s necessary, but there i s no doubt that parathyroid hormone has a direct effect on the kidney to suppress 24-hydroxylase, and probably also to enhance 1-hydroxylase. This conclusion i s in agreement with recent work from DeLuca (18). But parathyroid hormone has another effect in vivo which i s not dependent on the presence of vitamin D: to enhance calcium resorption by the renal tubule. Therefore, when parathyroid hormone i s given to rats on a high calcium diet (17) i t w i l l have two actions: stimulation of 1-hydroxylase and depression of 24-hydroxylase; and a further

527 e l e v a t i o n of serum calcium due to diminished renal calcium e x c r e t i o n . This l a t t e r e f f e c t may predominate and thus, i f the d i e t a r y calcium i s s u f f i c i e n t l y high, reverse the d i r e c t action of parathyroid hormone to increase 1,25(0H)2D3. The conclusion drawn from our e a r l y experiments (17) that parathyroid hormone could not be the s o l e r e g u l a t o r of vitamin D metabolism i s c e r t a i n l y consistent with most recent work. (b)

Prolactin

I n j e c t i o n of ovine p r o l a c t i n in chicks markedly stimulates the renal 1-hydroxylase enzyme within one hour ( 1 9 ) . Repeated i n j e c t i o n maintains this e f f e c t , and the action is accompanied by a marked elevation of 1,25(0H)2D3 l e v e l s in plasma ( 2 0 ) . Further, administration of ovine p r o l a c t i n in rats is associated with a moderate but d e f i n i t e increase in c i r c u l a t i n g l e v e l s ( 2 1 ) . But the c l e a r e s t evidence of the physiological importance of p r o l a c t i n in mammals i s the e f f e c t of bromocryptine on plasma l e v e l s of 1,25(0H)2D3 in e a r l y l a c t a t i o n (14) (Figure 5 ) . LACTATING N0N-1ACTATING 120-,

Figure 5 (redraun from 14) The effect of bromocryptine (CB-154) and bromocryptine in lactating and control rats.

plus

prolactin

Our measurements of plasma PTH l e v e l s in this experiment suggested that the e f f e c t of p r o l a c t i n could not be due to changes in the secretion of parathyroid hormone. We therefore studied the e f f e c t of p r o l a c t i n in our kidney culture system. The r e s u l t s are d e f i n i t e (Figure 6 ) .

5 28

W

I 0

I Figure

1

10

100

0 Prolactin (ng/ml) I 24-OHase la-OHase

6

The effect of ovine prolactin for in chick kidney cell culture.

one hour an the hydroxylase

enzymes

P r o l a c t i n causes a marked and rapid enhancement of 1 - h y d r o x y l a s e activity. The e f f e c t s are seen w i t h i n one hour as in the a c t i o n of p r o l a c t i n -in vivo. This i s an important f i n d i n g and i t i s tempting t o e x t r a p o l a t e t o mammals. But caution i s n e c e s s a r y : p r o l a c t i n i s known to have d i f f e r e n t a c t i o n s depending on the s p e c i e s s t u d i e d . And i t i s p o s s i b l e t h a t e f f e c t s seen in c u l t u r e may not occur in vivo. N e v e r t h e l e s s , there i s now s t r o n g evidence t h a t p r o l a c t i n e l e v a t e s 1,25(0H)2D3 l e v e l s in the chick by a d i r e c t a c t i o n on the kidney t o enhance 1 - h y d r o x y l a s e , and t h a t t h i s e f f e c t i s p h y s i o l o g i c a l l y important. The evidence in mammals i s l e s s c o n c l u s i v e , but we may hypothesize t h a t p r o l a c t i n plays a r o l e in l a c t a t i o n in mammals, and t h a t t h i s i s a l s o e x e r t e d by a d i r e c t a c t i o n on the kidney c e l l . The marked e l e v a t i o n s o f c i r c u l a t i n g 1,25(0H)2D3 l e v e l s in pregnancy probably depend on s e v e r a l f a c t o r s , but we b e l i e v e t h a t p r o l a c t i n , and perhaps a l s o human p l a c e n t a l l a c t o g e n , are l i k e l y t o be of major importance. ACKNOWLEDGEMENTS This work was supported by the Medical Research C o u n c i l , the Wellcome Trust and the N u f f i e l d Foundation.

529 REFERENCES 1.

C o l s t o n , K.W., Evans, I . M . A . , Galante, L . , Maclntyre, Moss, D.W. (1973) Biochem. J . J 3 4 , 817-820.

2.

Brumbaugh, P . F . , H a u s s l e r , D . H . , Bursac, K.M. and H a u s s l e r , M.R. (1974) Biochemistry 13., 4091-4097.

3.

Brown, D . J . , Spanos, E . , i n t h i s volume.

4.

Matthews, E.W., B y f i e l d , P . G . H . , C o l s t o n , K.W., Evans, I . M . A . , Galante, L . S . and Maclntyre, I . (1974) FEBS L e t t e r s 48, 122-125.

5.

Spanos, E . , B a r r e t t , D . I . , Biochem. J . 174, 231-236.

6.

L a r k i n s , R . G . , MacAuley, S . J . Nature 252, 412-414.

7.

L a r k i n s , R . G . , MacAuley, S . J . , C o l s t o n , K.W., Evans, I . M . A . , Galante, L . S . and Maclntyre, I . (1973) Lancet ii_, 289-291.

8.

H a u s s l e r , M . R . , B a y l i n k , D . J . , Hughes, M . R . , Brumbaugh, P . F . , Wergedal, J . E . , Shen, F . H . , N i e l s e n , R . L . , Counts, S . J . , Bursac, K.M. and McCain, T . A . (1976) C l i n . Endoc. 5, S u p p l . , 151s-165s.

9.

F r ä s e r , D.R. and Kodicek, E. 163-166.

R a p t i s , P. and Maclntyre,

I.,

I.

(1979)

Chong, K . T . and Maclntyre,

and Maclntyre,

I.

I.

(1978)

(1974)

(1973) Naturö new B i o l .

241,

10.

Garabedian, M., H o l i k , M . F . , DeLuca, H . F . and Boyle, (1972) Proc. N a t l . Acad. S e i . U.S. 6£, 1673-1676.

I.T.

11.

Kenny, A.D.

12.

Maclntyre I . (1977) i n Vitamin D: B i o c h e m i c a l , Chemical and C l i n i c a l Aspects Related to Calcium Metabolism (Norman, A.W., S c h a e f e r , K . , Coburn, J . W . , DeLuca, H . F . , F r ä s e r , D . , G r i g o l e i t , H.G. and Herrath D . V . , eds) pp. 155-164, Walter de G r u y t e r , B e r l i n .

13.

P i k e , J . W . , Spanos, E . , C o l s t o n , K.W., Maclntyre, I . and H a u s s l e r , M.R. (1978) Am. J . P h y s i o l . 235(3), E338-E343.

(1976) Am. J . P h y s i o l . 230,

1609-1615.

530 14.

Maclntyre, I . , Colston, K.W., Robinson, C.J. and Spanos, E. (1977) i n Molecular Endocrinology: Proceedings of Endocrinology '77 (Maclntyre, I . and Szelke, M., eds) pp. 73-85, Elsevier/North Holland Biomedical Press, Amsterdam.

15.

Spanos, E . , B a r r e t t , D., Maclntyre, I . , Pike, J.W., S a f i l i a n , E. and Haussler, M.R. (1978) Nature 273, 246-247.

16.

Pike, J.W., Toverud, S . , Boass, A . , McCain, T. and Haussler, M.R. (1977) in Vitamin D: Biochemical, Chemical and C l i n i c a l Aspects Related to Calcium Metabolism (Norman, A.W., Schaefer, K., Coburn, J.W., DeLuca, H.F., Fräser, D., G r i g o l e i t , H.G. and Herrath, D.V., eds) pp. 186-189, Walter de Gruyter, B e r l i n .

17.

Galante, L . , MacAuley, S . , Colston, K. and Maclntyre, I . (1972) Lancet i_, 985-988.

18.

Juan, D. and DeLuca, H.F. (1977) Endocrinol. J01_, 1184-1193.

19.

Spanos, E . , Colston, K.W., Evans, I . M . A . , Galante, L . S . , MacAuley, S . J . and Maclntyre, I . (1976) Mol. and Cell. Endoc. 5, 163-167.

20.

Spanos, E . , Pike, J.W., Haussler, M.R., Colston, K.W., Evans, I . M . A . , Goldner, A.M., McCain, T.A. and Maclntyre, I . (1976) Life Sciences J_9, 1751-1756.

21.

Spanos, E . , Maclntyre, I . and Haussler, M.R. unpublished.

per se on the p l a s m a l e v e l of 1,25 (OH) 2 D.

531 A STUDY OF THE PHYSIOLOGIC SIGNIFICANCE OF 24-HYDROXYLATION OF 25-HYDROXYVITAMIN D Y. Tanaka, H. F. DeLuca and N. Ikekawa Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 USA The production of 24,25-dihydroxyvitamin D 3 (24,25-(OH)2D3) is controlled by 1,25-dihydroxyvitamin D^ ( 1 , 2 5 - ( O H ) ( 1 ) , serum calcium (2), serum phosphorus (3) and parathyroid hormone (4) and is increased under the same conditions in which l,25-(OH)2D3 production is decreased (5).

It is the

major dihydroxy metabolite of the vitamin in normal man (6) and animals (2).

The compound has significant biological activity in vitamin D-

deficient rats in the stimulation of intestinal calcium transport (7), bone calcium mobilization (7) and curing rickets (8).

It is inactive at

physiologic doses in nephrectomized rats suggesting 1-hydroxylation is essential to its activity (7).

Thus, measurement of biological activity

of 2 4 , 2 5 - ( O H ) i n intact, vitamin D-deficient animals cannot provide information on the specific biological activity of 24,25-(OH)^D^• Recently 24,25-(OH)2D-j

^ e e n suggested to have specific actions in

the mineralization of bone (9), suppression of parathyroid hormone (10), and for embryo development (11).

To study the biological importance of

24-hydroxylation in the function of vitamin D, 25-hydroxyvitamin D^ (25-OH-D3) blocked at the 24 position with fluorine atoms (24,24-difluoro25-OH-D^) has been synthesized (12).

Biological responses of this fluoro

analog were compared to those of 25-OH-D^.

A single dose of 650 pmole of 24,24-difluoro-25-OH-D3 was used to test its intestinal calcium transport activity in rats that had been fed a low calcium, vitamin D-deficient diet.

The difluoro analog stimulated the

calcium transport at least as much as does 25-OH-Dj at any time point tested as shown in Fig. 1.

When given daily for one week, both compounds

stimulated calcium transport equally as well at a level of 32.5 pmoles/day while neither compound was effective at the 6.5 pmole level.

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

532 I O T— O

v»

o

0> (O

o o m 25-OH-D3

o i/i o

1

o

£

control

^

o o

I»

J

0

I

I

I

10

I

I

I

L

hr after

20

J

L

30

dose

Fig. 1 Response of Intestinal Calcium Transport to 24,24-F 2 ~25-OH-D 3 or 25-OH-D 3 . The ability of the difluoro analog to mobilize calcium from bone in comparison to that of 25-OH-D^ is shown in Table 1.

TABLE 1.

Increase in serum calcium concentration in response to a single dose of 24,24-difluoro-25-OH-D^ or 25-OH-D3-

Compound given Control

Serum calcium (mg/100 ml) 8 hours 29 hours 3.7 + 0.2*

3.9 + 0.1

24,24-difluoro-25-OH-D3

4.9 + 0.2

b

5.2 + 0.2 b

25-OH-D,

4.7 + 0.3 b

5.3 + 0.2 b

b from a

b from a

P

•—' *

E 3 •H « O m T-l 4-1 co c O M

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• i—1

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+

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CM + CN • rH rH

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a> a> ¿s > o Ü rH U ai rH tO tí cO ÍH •ri u ra•4-1 C A! a. 3 h ai 3 ai • O O ti 4-1 X. rH ¡H ai Ih a. E oo rarH rH raO P. ra o X. - O 4J C O to 0. o ÍH C 4J M ti » c tí 4-1 o •rl O » rH o ai E u 3 •a C O ai ai o •ri -a CJ > e rH 4-1 rH o O ratí ran) O tí cO-o ra•H to ai ai •rl rH 1—1 E o •O ra ci 4-1 ai tí co ai rH -O J3 •H .c a. tí ctí 4-1 4J 4-1 C O 3 ai O ti ai UH h a. • ai 4-1 O ai E ra4-1 tí 3 o M UH •ri tí ra Cl ai tO UH ai o 1-1 4-1 m 3 raO 4-1 co ai ti to h J 3 CN 3 4-1 •rl 4-1 0 tí > 00 i H h J3 ai ai tí ai O E UH ti ai a •rl rH tí 3 ti O 3 •o tí ai MC O> C OUH m co ai •H •tí 1 co oo >. ai •o 3 c >> i-l E C O ai Ih tí 4-1 lu ai ai M CA ai > S O » ai H UH

535 TABLE 3:

Increase of Serum Inorganic Phosphorus and

Antirachitic Activity in Response to a Single Dose of 24,24-difluoro-25-0H-D3 or 25-Hydroxyvitamin D 3 Compound Given

Dosage (pmol)

Serum Inorganic Line Test Score phosphorus (mg/100 ml) (unit)

Control

0

1.6+0.2

24,24-difluoro-25-OH-D^

25-0H-D3

130

3.0 + 0.2 b

325

33.5 + 0.4 b

130

3.3 + 0.1b

2..6 + 0.6 b

325

3.6 + 0.4 b

3..5 + 0.6

4..4 ± I-*® >5

*

Standard deviation of the mean. Rats fed a low phosphorus, vitamin D-deficient diet for 3 weeks were given a single dose of either compound dissolved in 0.05 ml 95% ethanol intrajugularly. Rats in the control group received ethanol only. They were killed by decapitation, blood was collected and their radii and ulnae were removed to determine antirachitic activity. Blood was immediately centrifuged to yield serum. Serum inorganic phosphorus was determined by the colorimetric method. Each group has 5-6 rats.

tainly equal to and in some cases somewhat more active than 25-OH-D^ in intestinal calcium transport, mobilization of calcium from bone and the prevention and healing of rickets argues strongly against either possibility.

Our results, therefore, do not provide support for the claims

that 24-hydroxylation plays an important role in the known functions of vitamin D.

Especially, it is important to note that vitamin D-deficient

bone and epiphyseal plate cartilage calcifies equally well in response to 24,24-difluoro-25-OH-D3 and 25-OH-D.j.

If 24,25-(OH) 2 D plays a role in

matrix synthesis (16) or preparation of the matrix of cartilage or bone, the resulting defect must not affect its calcifiability.

Furthermore, it

appears unlikely that 24-hydroxylation of 25-OH-Dj plays a role in bone mineralization in the rat as has been claimed in the chicken (17), or man (9).

536 This report is not addressed to the possibility that 2 4 , 2 5 - ( O H ) m a y play some specialized role such as suppression of parathyroid secretion and embryonic development.

This plus the mineralization responses in

man and chick are currently under study in our laboratory. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Tanaka, Y., and DeLuca, H. F. (1974) Science 183, 1198-1200. Boyle, I. T., Gray, R. W. , and DeLuca, H. F. (1971) Proc. Nat. Acad. Sei. 68, 2131-2134. Tanaka, Y-, and DeLuca, H. F. (1973) Arch. Biochem. Biophys. 154, 566-574. Garabedian, M., Holick, M. F., DeLuca, H. F., and Boyle, I. T. (1972) Proc. Nat. Acad. Sei. 69, 1673-1676. Tanaka, Y., Lorenc, R. S., and DeLuca, H. F. (1975) Arch. Biochem. Biophys. 171, 521-526. Taylor, C. M. , Hughes, S. E., and deSilva, P. (1976) Biochem. Biophys. Res. Coimnun. 70, 1243-1249. Boyle, I. T., Omdahl, J. L., Gray, R. W., and DeLuca, H. F. (1973), J. Biol. Chem. 248, 4174-4180. Tanaka, Y., DeLuca, H. F., Ikekawa, N., Morisaki, M., and Koizumi, N. (1975) Arch. Biochem. Biophys. 170, 620-626. Rasmussen, H., and Bordier, P. (1978). Metabol. Bone Dis. & Related Res. 1, 7-13. Bates, R. F. L., Care, A. D., Peacock, M., Mawer, E. B., and Taylor, C. M., (1974) J. Endocrin. 64, 6-9. Henry, H. L., and Norman, A. W. (1978) Science 201, 835-837. Kobayashi, Y., Taguchi, T., Ikekawa, N., and Morisaki, M. (1979) Tetrahedr. Lett, (in press). Peters, R. A. (1957) Adv. Enzmol. 18^ 113-159. Heidelberger, C., Griesbach, L., Montag, B. J., Morren, D., and Cruz, D. (1958) Cancer Res. 18, 305-317. Napoli, J. L., Mellon, W- S., Fivizzani, M. A., Schnoes, H. K. and DeLuca, H. F. (1979) Proc. 4th Workshop on Vitamin D, in press. Garabedian, M., DuBois, M. B., Corvol, M. T., Pezant, E., and Balsan, S. (1978) Endocrinology 102, 1262-1268, Goodwin, D., Noff, D., and Edlstein, S. (1978) Nature 276, 516-522.

537 DYNAMIC ASPECTS OF V I T A M I N D METABOLISM: FORMATION OF HYDROXYLATED METABOLITES FROM A PHYSIOLOGICAL DOSE OF CHOLECALCIFEROL IN V I T A M I N D DEPLETED MAN. S.W.Stanbury Department of

Medicine,

Royal

Infirmary,

Manchester

Ml 3 9WL,

England.

We h a v e r e c e n t l y a n a l y s e d a mass o f d a t a , f r o m d i f f e r e n t p e r s o n a l s t u d i e s i n man, i n an a t t e m p t t o e x t r a c t a p i c t u r e o f t h e e n d o c r i n e f u n c t i o n o f t h e s k i n and i t s i n t e g r a t i o n w i t h t h e 2 5 - h y d r o x y 1 a t I o n o f c h o l e c a l c i f e r o l in t h e l i v e r ( l ) . The o b s e r v a t i o n s i n v o l v e d m e a s u r e m e n t s o f s e r u m 2 5 " ( 0 H ) D a f t e r e x p o s u r e t o sun and s u b s e q u e n t s e c l u s i o n f r o m s u n s h i n e ; c o m p a r a b l e m e a s u r e m e n t s d u r i n g and a f t e r p e r i o d s o f a d m i n i s t r a t i o n o f s m a l l ( 2 2 . 5 y g ) o r l a r g e ( 2 - 5 - 3 - 7 5 rng) d a i l y o r a l doses o f c a l c i f e r o l ; and m e a s u r e m e n t s o f t h e mass c o n c e n t r a t i o n s o f c h o l e c a l c i f e r o l and 2 5 ~ ( 0 H ) D 3 i n s e r u m a f t e r i . v . i n j e c t i o n o f d i f f e r e n t masses o f l a b e l l e d c h o l e c a l c i f e r o l ( 5 vg 2 mg) i n t o v i t a m i n D d e f i c i e n t s u b j e c t s ( 2 ) . The scheme f o r m u l a t e d f r o m a n a l y s i s o f t h e s e d a t a is summarised in F i g . 1. S t a r t i n g w i t h the premiss t h a t the s k i n is the o n l y s t r i c t l y p h y s i o l o g i c a l source of the v i t a m i n in the a d u l t , i t p o s t u l a t e s t h a t c h o l e c a 1 c i f e r o l slowly released from the s k i n i s 2 5 - h y d r o x y 1 a t e d q u a n t i t a t i v e l y i n t h e l i v e r , so t h a t i t s p h y s i o l o g i c a l c o n c e n t r a t i o n in plasma is m a i n t a i n e d low. The c o n s e q u e n t i a l l y p r o d u c e d 2 5 " ( 0 H ) 2 D 3 b u i l d s up i t s c o n c e n t r a t i o n i n p l a s m a ( a n d t i s s u e s ) t o t h e maximum l e v e l recorded an a p p a r e n t l y f i n i t e l i m i t o f 5 0 - 80 \ig/H, a f t e r prolonged s o l a r exposure ( 3 ) . I t is f u r t h e r p o s t u l a t e d t h a t t h i s c o n c e n t r a t i o n o f 2 5 - ( 0 H ) D 3 , by c o m p e t i n g f o r t h e c a r r i e r transporting c h o l e c a l c i f e r o l from epidermis to b l o o d , a r r e s t s the continued cutaneous release of c h o l e c a 1 c i f e r o l . This would imply a p h y s i o l o g i c a l c o n t r o l o f 2 5 - h y d r o x y 1 a t i o n by i n d i r e c t r e g u l a t i o n o f t h e a v a i l a b i l i t y o f s u b s t r a t e . The d a i l y d e l i v e r y o f v i t a m i n D 3 f r o m s k i n t o b l o o d f o l l o w i n g U V - i r r a d i a t i o n o f t h e w h o l e body i s e s t i m a t e d t o be ^ 5 0 0 i . u . ( 1 ) . I t is a l s o

ORAL V I T A M I N D

7 • dehydrocholesterol

o

precholecalolerol

&

cholecalciferol

-A

SPECULATION!

SPECULATION! Circulating 25- Hydroxycholecalciferol

m m

Fig,

1

A c o n c e p t o f t h e e n d o c r i n e f u n c t i o n o f t h e s k i n and i t s integration w i t h c o n t r o l o f the 25-hydroxy1 a t i o n of c h o l e c a l c i f e r o l . (Continuous arrows — ^ represent c h o l e c a l c i f e r o l , broken arrows > 2 5 " ( 0 H ) D 3 and t h e i r t h i c k n e s s c r u d e l y i n d i c a t e s relative concentrations).

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

538 implied that high plasma l e v e l s o f c a l c i f e r o l would a r i s e only a f t e r u n p h y s i o l o g i c a l d o s a g e by t h e u n p h y s i o l o g i c a l o r a l r o u t e . Such dosage is known t o i n c r e a s e s e r u m 2 5 ~ ( 0 H ) D a b o v e t h e p h y s i o l o g i c a l l i m i t , a l t h o u g h i n a n o n - l i n e a r manner ( 4 ) ; a n d we s u g g e s t t h a t t h i s n o n - l i n e a r i t y i s c a u s e d by t h e h i g h p l a s m a c a l c i f e r o l c o m p e t i n g f o r a c a r r i e r r e s p o n s i b l e f o r t r a n s f e r r i n g 25"(0H)D from l i v e r to blood (1; F i g . 1). A l t h o u g h t h i s s c h e m e ( F i g . 1) f i t s o u r own a n d much o t h e r p u b l i s h e d d a t a , i t is o f c o u r s e s p e c u l a t i v e , but the f e a s i b i l i t y o f i t s primary tenet has been d e m o n s t r a t e d . A depot o f l a b e l l e d c h o l e c a l c i f e r o l i n human s u b c u t aneous f a t (which i s a n a l o g o u s to the e x c e s s mass o f e p i d e r m a l cholecalc i f e r o l p r o d u c e d by UV e x p o s u r e ( 5 ) c a n p r o d u c e a s l o w i n c r e a s e o f s e r u m 2 5 " ( 0 H ) D 3 by 12 y g / £ w i t h o u t i n c r e a s i n g s e r u m c h o l e c a l c i f e r o l by more t h a n 1.5 y g / £ ( 6 ) . An o u t c o m e o f t h i s a n a l y s i s h a s b e e n t o e m p h a s i s e t o u s t h e e x t e n t t o w h i c h s t u d i e s o f v i t a m i n D m e t a b o l i s m in any s p e c i e s - and a l s o d e d u c t i o n s c o n c e r n i n g t h e e f f e c t s o f i t s i n d i v i d u a l m e t a b o l i t e s - h a v e d e p e n d e d on the use o f a r b i t r a r y and o f t e n u n p h y s i o l o g i c a 1 d o s e s a d m i n i s t e r e d a s a b o l u s by u n p h y s i o l o g i c a l r o u t e s . C o n s e q u e n t l y , we h a v e e x a m i n e d ( 7 ) t h e f o r m a t i o n o f the 3 d i h y d r o x y l a t e d m e t a b o l i t e s o f c h o l e c a l c i f e r o l i n man i n 2 8 s u b j e c t s , 19 o f whom w e r e v a r i a b l y d e p l e t e d o f v i t a m i n D ( s e r u m 2 5 " ( 0 H ) D ^ 10 \ig/l) . Doubly l a b e l l e d chol e c a 1 c i f e r o l as p u l s e t r a c e r (8) h a s the advantage of p r o v i d i n g a r e l a t i v e l y s t a b l e serum c o n c e n t r a t i o n of p h y s i o l o g i c a l l y produced l a b e l l e d endogenous s u b s t r a t e f o r p r o d u c t i o n o f d i h y d r o xylated metabolites (Fig. k). The s m a l l m a s s - d o s e u s e d (25 U 9 ! 1000 i . u . ) is s u f f i c i e n t to repair, the p r e v a i l i n g v i t a m i n D d e f i c i e n c y ; and i n t h e v i t a m i n D d e p l e t e d s u b j e c t s , i t i n c r e a s e d t h e mean s e r u m 2 5 - ( 0 H ) D a by 1 . 7 5 y g / £ i n 24 h ( 7 ) . I t was thus p o s s i b l e t o m o n i t o r the t e m p o r a l prod u c t i o n and r e l a t i o n s h i p s o f i n d i v i d u a l m e t a b o l i t e s d u r i n g 6 - 1 0 days a f t e r c o r r e c t i n g v i t a m i n D d e f i c i e n c y by a p h y s i o l o g i c a l d o s e o f t h e vitamin; and t o c a l c u l a t e t h e i r m o l a r c o n c e n t r a t i o n s in serum. (Table). The s e q u e n t i a l

changes

of

mean s e r u m c o n c e n t r a t i o n s

(± SEM)

of

radioactive

CONTROLS

T I M E A F T E R I N J E C T I O N C H O L E C A L C I F E R O L ( 8 . 9 m g / d l ) ; in the o t h e r 8 , w i t h p r o d u c t i o n d e l a y e d 6 d a y s o r l o n g e r , 75% were i n i t i a l l y h y p o c a l c a e m i c ( F i g . 6 ) . In o t h e r s e v e r e c a s e s o f h y p o c a l c a e m i c o s t e o m a l a c i a , g i v e n a d a i l y dose o f v i t a m i n D (450 i . u , ) adequate to r e s t o r e r a p i d l y normal a s s a y e d l e v e l s o f 2 5 " ( 0 H ) D and 1 , 2 5 - ( 0 H ) 2 D , t h e r e was no a s s a y a b l e 2 4 , 2 5 " ( 0 H ) 2 D in s e r u m a f t e r 16 days o f t r e a t m e n t ( C . M , T a y l o r and S . U . S t a n b u r y , to be p u b l i s h e d ) . Convers e l y , o f 16 ' v i t a m i n D d e p l e t e d 1 c a s e s o f h y p e r c a l c a e m i c p r i m a r y h y p e r p a r a t h y r o i d i s m ( c f . F i g , 4 ) , 12 (75%) p r o d u c e d r a d i o a c t i v e 2 4 , 2 5 - ( 0 H ) 2 D 3 w i t h i n 24 h o f r e c e i v i n g the l a b e l l e d p u l s e o f c h o l e c a 1 c i f e r o l ; in the I

0.28

;

0.24

s

0.20

°

0.16

I

o

F i g . 5 The r e l a t i o n s h i p between serum r a d i o a c t i v e 2 4 , 2 5 " ( O H ) 2 D 3 on day 6 and 1 , 2 5 - ( 0 H ) 2 D 3 on day 2 . (D-depleted subjects)

o

O 0.12 •

>

O

o

0.08

o o

< O

o

0.02

QD

O

O

0.04

0.06

0.08

0.10

0.12

S E R U M R A D I O A C T I V E 1.25-(OHl 2 D 3 |day 2] I % of d o » / litre I

5^2 [^PRIMARY HYPERPARATHYROIDISM

m " CONTROLS I"! HYPOCALCAEMIC NORMOCALCAEMIC

F i g . 6 The time o f f i r s t a p p e a r a n c e o f r a d i o a c t i v e 2 4 , 2 5 - ( O H ) 2 D 3 in the c i r c u l a t i o n of vitamin D depleted subjects

1 I

other 4, with osteomalacia complicating t h e i r primary d i s e a s e , production was d e l a y e d u n t i l days 3 o r 6 ( F i g . 6 ) . T h i s s u g g e s t s to us t h a t the c r i t i c a l f a c t o r d e t e r m i n i n g 2*1,25-(OH) 2D3 p r o d u c t i o n i s the ' i n t r a r e n a l c o n c e n t r a t i o n o f C a 2 + I ; the l a t t e r w i l l be i n f l u e n c e d by the C a 2 + i n c i r cumambient f l u i d (ECF) and by the ' v i t a m i n D s t a t u s 1 . On c o r r e c t i n g v i t a m i n D d e f i c i e n c y , renal u p t a k e o f C a 2 + under the i n f l u e n c e o f newly s y n t h e s i s e d 1 , 2 5 " ( O H ) 2 D 3 w i l l i n c r e a s e i n t r a r e n a l C a 2 + to a c r i t i c a l l e v e l t h a t i n h i b i t s l a - h y d r o x y 1 a t i o n and a c t i v a t e s 2 4 - h y d r o x y 1 a t i o n , Or the c u m u l a t i v e u p t a k e o f C a 2 + i n d u c e s a l l o s t e r i c c h a n g e s in the l a - h y d r o x y 1 a s e r e s u l t i n g s i m u l t a n e o u s l y i n g a i n o f 2 4 - h y d r o x y l a s e and l o s s o f l a - h y d r o x y 1ase a c t i v i t y . Calciferol

TABLE.

and 2 5 ~ ( 0 H ) D .

The c a l c u l a t e d

initial

mass c o n c e n t r a t i o n

of

CALCULATED MOLAR CONCENTRATIONS OF CALCIFEROL AND ITS METABOLITES IN SERUM BEFORE THE INJECTION OF LABELLED CHOLECALCIFEROL Group 1 mean ± SD range

Calciferol

(nmol/A)

2 5 " (OH) D"

(nmol/i.)

I,25-(OH)2D

(pmol/i)

2.55(2.0)

Group 2 mean ± SD range

0.27-6.53

367(455)

42-1203

15-00(5.3)

5"25

68.5(32.3)

98.9(39.8)

44-188

not

30-125

calculable

2 5 , 2 6 - (OH) 2D (nmol/S,) 0 . 3 2 ( 0 . 1 3 5 )

0.14-0.55

3.59(4.97)

0.82-16

2 4 , 2 5 " (OH) 2 D

0.017-0.165

4.81(5.69)

0.72-14.0

(nmol/Jl) 0 . 1 2 ( 0 . 0 6 )

* Serum 2 5 - ( 0 H ) D was measured d i r e c t l y . indi rectly ( 1 , 7 ) .

All

o t h e r v a l u e s were

derived

5^3 c a l c i f e r o l in serum v a r i e d from 6 . 5 to 6 3 - 5 pg/& in v i t a m i n D d e p l e t e d s u b j e c t s ; and from 16 t o 462 pg/l in the v i t a m i n D r e p l e t e , the v e r y h i g h values being in s u b j e c t s r e c e i v i n g d a i l y m i l l i g r a m doses o f c a l c i f e r o l o r a l l y (Table). There was a c u r v i l i n e a r r e l a t i o n s h i p between the i n i t i a l ( t 0 ) c a l c u l a t e d serum c a l c i f e r o l and measured serum 2 5 ~ ( 0 H ) D , w h i c h c o u l d be r e n d e r e d r e c t i l i n e a r by l o g / l o g t r a n s f o r m a t i o n . The f i t t e d r e g r e s s i o n ( l o g / l o g ) p r e d i c t e d v a l u e s o f serum c a l c i f e r o l f o r a p a r t i c u l a r serum 2 5 " (0H)D w h i c h a g r e e d c l o s e l y w i t h p u b l i s h e d p a i r e d d i r e c t measurements made by HPLC ( 1 2 , 1 3 ) . In e x a m i n i n g the ' k i n e t i c s ' o f p r o d u c t i o n o f 2 5 " ( 0 H ) D 3 in the 2k h a f t e r g i v i n g the p u l s e o f c h o l e c a l c i f e r o l , the i n i t i a l ( t o ) c a l c u l a t e d serum c a l c i f e r o l and the serum t o t a l ( i . e . s t a b l e + r a d i o a c t i v e ) 2 5 " ( O H ) D a t 2k h have been r e g a r d e d as h a v i n g a s i m p l e s u b s t r a t e / p r o d u c t

Fig.

7

R e l a t i o n s h i p between the r e c i p r o c a l c o n c e n t r a t i o n s o f t o t a l 2 5 " ( 0 H ) D a t 2k h (measured and c a l c i f e r o l a t time z e r o ( c a l c u l a t e d ) ( O vitamin D depleted; £ D-replete)

relationship. A r e c i p r o c a l p l o t of t h e i r molar c o n c e n t r a t i o n s y i e l d e d s e p a r a t e l i n e a r r e g r e s s i o n s f o r the v i t a m i n D d e p l e t e d and D - r e p l e t e s u b j e c t s ( F i g . 7) w i t h ' a p p a r e n t K m v a l u e s ' d i f f e r i n g by two o r d e r s o f magnitude. Q u a l i t a t i v e l y , these f i n d i n g s are i d e n t i c a l w i t h those o b t a i n e d by Suda et a 1 (k) w i t h the p e r f u s e d r a t l i v e r and i n t e r p r e t e d by them t o imply two d i f f e r e n t t y p e s o f 2 5 " h y d r o x y 1 a t i on (? 2 enzymes) dependi n g on the p r e v a i l i n g s u b s t r a t e c o n c e n t r a t i o n . I t i s s u g g e s t e d (1) t h a t the scheme i n F i g . 1 p r o v i d e s an e q u a l l y f e a s i b l e e x p l a n a t i o n . Thus the c o r r e c t i o n o f an e x i s t i n g v i t a m i n D d e f i c i e n c y i n man a p p e a r s to r e s u l t i n the r a p i d p r o d u c t i o n o f 2 5 ~ ( 0 H ) D and 2 5 , 2 6 - (OH) 2D3 in amounts d e t e r m i n e d by the a v a i l a b i l i t y o f t h e i r r e s p e c t i v e p r e c u r s o r s ; and in t h e c o n t r o l l e d p r o d u c t i o n o f a f i n i t e q u a n t i t y o f 1 , 2 5 - ( 0 H ) 2 D 3 , as d e t e r m i n e d by t h e p r e v a i l i n g l e v e l o f p a r a t h y r o i d f u n c t i o n and s e l f - i n h i b i t i o n o f i t s own f o r m a t i o n . W i t h i n 1 0 - 1 5 days o f c o r r e c t i n g the d e f i c i e n c y i n an o s t e o m a l a c i c p a t i e n t , n e t c a l c i u m a b s o r p t i o n may be > 80% o f d i e t a r y i n t a k e (14). I f some m e t a b o l i t e o t h e r than 1 , 2 5 - ( 0 H ) 2 D i s r e q u i r e d to f a c i l i t a t e d i r e c t l y the d e p o s i t i o n o f t h i s a b s o r b e d c a l c i u m i n t o b o n e , 2 5 - ( 0 H ) D and

544 2 5 > 2 6 - ( O H ) z D become r a p i d l y a v a i l a b l e t o meet t h i s h y p o t h e t i c a l requirement. G e n e r a l l y , 2 4 , 2 5 " ( O H ) 2 D i s not a v a i l a b l e in t h i s time and c o u l d t h e r e f o r e not f u n c t i o n to promote m i n e r a l i s a t i o n in these c i r c u m s t a n c e s . The a p p a r e n t s e l f - i n h i b i t i o n o f 1 , 2 5 ~ ( 0 H ) 2 D 3 p r o d u c t i o n w i t h i n d a y s o f c o r r e c t i n g v i t a m i n D d e f i c i e n c y p o s e s t h e q u e s t i o n o f how h i g h r a t e s o f c a l c i u m a b s o r p t i o n a r e s u s t a i n e d d u r i n g t h e m o n t h s r e q u i r e d t o h e a l human osteomalacia. I t is s u g g e s t e d t h a t , once m i n e r a l i s a t i o n o f the o s t e o m a l a c i c s k e l e t o n h a s been i n i t i a t e d , c o n t i n u e d d e p o s i t i o n o f c a l c i u m o v e r a v a s t s u r f a c e o f b o n e may e x e r t a d r a g e f f e c t t e n d i n g t o d e p l e t e s o f t t i s s u e s o f c a l c i u m and r e c r e a t e i n t r a r e n a l c o n d i t i o n s f a v o u r i n g l a - h y d r o x y l a t i o n - c o n c e i v a b l y p h a s i c a l l y so t h a t the s y n t h e s i s o f 1 , 2 5 " ( 0 H ) D i s r e p e a t e d l y t u r n e d o n and o f f . This

r e s e a r c h was

s u p p o r t e d by

the B r i t i s h

Medical

Research

Council.

REFERENCES 1. 2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12.

13. 14.

S t a n b u r y , S , W . , M a w e r . E . B . , T a y l o r , C . M . , de S i l v a . P . ( 1 9 7 9 ) M i n e r a l E l e c t . Metab. In the p r e s s . Mawer.E.B. (1979) In C l i n i c a l and N u t r i t i o n a l A s p e c t s o f V i t a m i n D (Norman,A.U. ed.) M a r c e l D e k k e r I n c . , New Y o r k . Haddad , J . G . , C h y u . K . J . (1971) J . C I i n . E n d o c r . 33, 992-995. S u d a , T . , H o r i uch i , N . , F u k u s h i m a , M . , N i s h i i . Y . I J s i l ) In V i t a m i n D : B i o c h e m i c a l , Chemical and C l i n i c a l A s p e c t s R e l a t e d t o C a l c i u m M e t a b o l i s m ( N o r m a n , A . W. e t a l , e d s . ) 2 0 1 - 2 1 0 . de G r u y t e r , B e r l i n (1977). B e a d l e , F . C . (1977) Ibid. 549-551. D a v i e s , M . , Mawer,E.B. (1979) T h i s volume p . . . . S t a n b u r y , S . W . , Mawer.E.B. (1979) Submitted for p u b l i c a t i o n . M a w e r . E . B . , B a c k h o u s e , J . , H i l l . L . F . , Lumb ,G. A . , de S i l v a . P . , T a y l o r , C . M . , S t a n b u r y , S , W . (1975) CIin.Sci.molec.Med. 48, 349-365. B i l e z e k i an , J . P . , C a n f i e 1 d , R . E , , J a c o b s , T , P . , P o l a y . J . S . , D 1 A d a m o , A . P . , E i sman , J . A . , D e L u c a ,H . F. ( 1 9 7 8 ) New E n g l a n d J . Med. 299., 4 3 7 " 4 4 l . Mawer.E.B., Backhouse,J., Davies,M,, H i l l . L . F . , Taylor,C.M. (1976) L a n c e t i_. 1 2 0 3 " 1 2 0 6 . C o l s t o n ~K.W,, E v a n s , I . M . A . , S p e l s b e r g , T , C . , M a c l n t y r e . l . (1977). B i o c h e m . J . 1 6 4 , 83-89. Lambert,P.W., S y v e r s o n , B . F . , T o f t , D . 0 . , Spe1sberg,T.C., A r n a u d , S . B . , Arnaud,C.D, (1978) In E n d o c r i n o l o g y o f C a l c i u m M e t a b o l i s m ( C o p p , D . H . , T a l m a g e , R , V . e d s . ) p. 3 9 8 , E x c e r p t a M e d i c a , Amsterdam. Jones,G. (1978) C l i n . Chem. 2 4 , 2 8 7 - 2 9 8 . S t a n b u r y , S . W . (1979) In C l i n i c a l and N u t r i t i o n a l A s p e c t s o f V i t a m i n D M e t a b o l i s m ( N o r m a n , A . W . e d . ) M a r c e l D e k k e r I n c . , New Y o r k .

545 THE REGULATION OF PLASMA l , 2 5 - ( O H ) 2 ~ D CONCENTRATIONS IN HEALTHY ADULTS

R i c h a r d W. Gray, Jacob Lemann, J r . and Nancy D. Adams Departments o f B i o c h e m i s t r y and M e d i c i n e and The C l i n i c a l M e d i c a l C o l l e g e o f W i s c o n s i n , Milwaukee, WI 53226, USA

Research

Center,

Plasma 1 , 2 5 - ( 0 H ) 2 - v i t a m i n D c o n c e n t r a t i o n s i n h e a l t h y a d u l t s have now been measured i n s e v e r a l l a b o r a t o r i e s and a v e r a g e about 80 pmol/L o r about 33 pg/ml ( 1 - 4 ) . The plasma c o n c e n t r a t i o n o f t h e hormone i s d e t e r m i n e d by t h e b a l a n c e between i t s r a t e o f s y n t h e s i s by t h e r e n a l 1 - a - h y d r o x y l a s e and i t s rate of degradation. To o b t a i n more i n f o r m a t i o n c o n c e r n i n g t h e plasma dynamics o f 1 , 2 5 - ( O H ^ - D , we s t u d i e d t h e d i s a p p e a r a n c e o f r a d i o a c t i v i t y f r o m t h e plasma i n s e v e n h e a l t h y v o l u n t e e r s i n j e c t e d i n t r a v e n o u s l y w i t h doses o f 3 H - 1 , 2 5 - ( 0 H ) 2 - D j r a n g i n g f r o m 1/10 t o 10 t i m e s t h e q u a n t i t y endog e n o u s l y p r e s e n t i n t h e c i r c u l a t i n g plasma p o o l ( 5 ) . 100,

50

Percent 3

dose H

remaining in

20

%%

rr

plasma

*hi•

pool

I :

30 60 90 CO 150 180 210 240 6 minutes

12 TIME

24

30

36

hours

F i g u r e 1. Percentage of administered H r e m a i n i n g i n t h e plasma p o o l ( e s t i m a t e d as 5% o f body w e i g h t ) as a f u n c t i o n o f t i m e a f t e r i n j e c t i o n o f H-l,25-(0H)2_Do. Note change i n time s c a l e a t 240 m i n u t e s . Reproduced f r o m r e f e r e n c e 5 by p e r m i s s i o n o f J . B . L i p p i n c o t t Company. The d a t a i n F i g u r e 1 show t h a t r e g a r d l e s s o f t h e d o s e o f 1 , 2 5 - ( O H ^ - D ^ a d m i n i s t e r e d , t h e h a l f - t i m e f o r d i s a p p e a r a n c e o f t h e r a d i o a c t i v i t y was 8 minutes o r l e s s i n a l l s u b j e c t s . Only 10 - 16% o f t h e i n j e c t e d r a d i o -

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

5^6 activity remained in the plasma pool 4 hours after dosing. This would suggest that the plasma level of l,25-(OH>2-D is probably determined primarily by its rate of synthesis and that removal of the hormone from the plasma pool is not rate-limiting, at least over a 10-fold change in the circulating plasma pool size. In the two subjects who received the smallest dose of 1,25-(OH^-DJ, a compartmental analysis of the kinetic data for the first 240 minutes was performed. During this period, most of the hormone was cleared from the plasma and essentially all of the plasma radioactivity was still present as 1,25-(OH^-Dj (5). A multiexponential fit of the plasma tracer data during this period required a function composed of at least three exponential terms. From compartmental analysis theory, a model having at least three compartments is required to adequately characterize the kinetics of 1,25-(011)2-1)3 disappearance from the plasma. The model which we have proposed is shown in Figure 2 (5).

Figure 2. Model for compartmental analysis of injected 3H-1,25-(OIO2-D3 disappearance from plasma in man. Reproduced from reference 5 with permission of J.B. Lippincott Company. Analysis of the rate constants from this model provided estimates of the endogenous rate of l,25-(OH)„-D synthesis in the two subjects receiving the smallest doses of 1,25-(uH^-D^. These were 2.4 and 0.82 nmol/day or 1.0 and 0.34 pg/day. These estimates are in reasonable agreement with the daily doses of 1,25-(OH^-D^ that are required for a biological effect in patients with chronic renal failure who have little or no endogenous 1,25(0H)2-D synthesis. In addition, it was also possible to estimate relative compartment sizes in these individuals. Thus, about 20 to 25 percent of the total body 1,25-(0H)2~D is circulating and it would appear that body stores of 1,25-(0H)2"D turnover 1.4 to 2.3 times each day (5). Finally, it is also possible to predict from the model how rapidly one would expect to see changes in the circulating plasma level of 1,25-(0H)„-D in response

5^7 to a change in the rate of synthesis of the hormone. For example, a 50% reduction in the rate of l,25-(OH)2~D synthesis would result in a 50% decrease in the plasma level in about 24 hours, while a 90% reduction in the rate of synthesis would lower the plasma concentration to undetectable levels within the same time. These predicted changes in plasma l,25-(OH)2" D levels are in good agreement with the dissipation of the biological effects of the hormone after its administration is discontinued in normal subjects (6). Since changes in the plasma level of l,25-(OH)2~D probably reflect changes in the rate of synthesis of the hormone, measurement of plasma 1,25-(OH) D levels is an important tool which can be used to study the control of l,25-(OH)2-D synthesis. It is well known from experiments in animals that calcium and phosphorus are important regulators of l,25-(OH>2-D synthesis. The elevation in plasma 1,25-(OH) -D levels in rats in response to calcium deprivation has been shown to be dependent upon PTH since a very blunted response to calcium deprivation was observed in thryoparathyroidectomized animals (7). On the other hand, the response to phosphorus deprivation was shown to be independent of PTH (7). The importance of calcium in regulating plasma l,25-(OH)2~D concentrations in man can be seen by examination of plasma concentrations of the hormone in relation to urinary calcium excretion/creatinine where a significant inverse correlation is observed.

43 HEALTHY ADULTS EATING NORMAL DIETS 20Ch P L A S M A

I, 2 5 - ( 0 H ) 2 - D ,

»128-142

pmol/liter

U C o V mmol/mmol creatinine

r = 0.56 p 0 . 6 mmol/kg/day) w e r e a c h i e v e d by s u p p l e m e n t i n g a normal d i e t w i t h c a l c i u m c a r b o n a t e ( 0 . 4 4 t o 1 . 5 mmol CaC0 3 /kg/day). Phosphorus has a l s o been shown t o be an i m p o r t a n t d e t e r m i n a n t o f plasma 1 , 2 5 - ( O H ) - D l e v e l s i n man. E v i d e n c e f o r an i n v e r s e r e l a t i o n s h i p b e t w e e n plasma I , z 5 - ( 0 H ) 2 _ D l e v e l s and serum PO^ was f i r s t o b t a i n e d when c i r c u l a t i n g l e v e l s o f t h e hormone were measured i n p a t i e n t s w i t h n e p h r o l i t h i a s i s , many o f whom a r e hypophosphatemic and have h i g h plasma l e v e l s of 1 , 2 5 (OH^-D. Thus, when d a t a from normal s u b j e c t s and c a l c i u m s t o n e f o r m e r s are considered t o g e t h e r , the f o l l o w i n g r e l a t i o n s h i p i s observed: Plasma 1,25 pmol/L = 256 - 125 x serum PO^ mmol/L, r = - 0 . 4 2 ( 4 ) . A similar re-

549 lationship was observed by Shen et al (9). Dietary PO^ deprivation in healthy volunteers results in a fall in plasma PO, concentrations in women and a correlated rise in plasma l,25-(OH)2-D levels (4). Th is occurred despite a fall in circulating PTH levels in the women. For reasons which are not yet entirely clear, no changes in serum PO or plasma l,25-(OH)2_D were observed in the men even after 16 days of dietary PO^ deprivation. However, in both men and women, the change in plasma l,25-(OH)2_D levels were significantly and inversely correlated to the change in serum PO^: Aplasma 1,25 pmol/L = 1 - 72 x ¿serum PO, mmol/L, r = -0.59 (4). The mechanism for the regulation of 1,25-(0H)2-D synthesis by phosphate in man remains to be determined. Although calcium and phosphate are probably important regulators of 1,25(0H)2~D synthesis in man, it can be seen from the data in Figure 4 that there is a wide variation in plasma l,25-(OH)2-D levels in the normal population at normal dietary calcium intakes. This can be seen more clearly in Figure 5 which shows the distribution of plasma l,25-(OH)2_D levels in 97 healthy individuals all eating normal diets. Also shown are measurements in 24 anephric patients all with undetectable plasma l,25-(OH)2~D levels again emphasizing the absolute requirement for renal tissue for the synthesis of this hormone in man.

PLASMA

lt25-(0H)

2

20

pg/ml

60

40

Anephrics

9 7 HEALTHY ADULTS MEAN ± SD = 8 9 ± 2 5

30

Number of 2 Subjects

-D

pmol/liter

0

v

10«

i

1 I

0

25 P L A S M A

50

100

75

l . 2 5 - ( 0 H )

2

- D

125

150

pmol/liter

Figure 5. Distribution of plasma l,25-(OH)2-D levels in 97 healthy adults eating normal diets. The undetectable levels in 24 anephric patients are also shown.

550 The stability of plasma l,25-(0H) 2 -D concentrations

150-iwith time in 7 healthy adults

•60

A

PLASMA

ioo-

o

•40

l,25-{0H)z-D pmol / liter

~~

0

pg/ml

*

•

50-

1975

1976

1977

PLASMA , , l,25-(0H) 2 -D

1978

•20

0

YEAR F i g u r e 6. Plasma l , 2 5 - ( O H ) 2 _ D c o n c e n t r a t i o n s as a f u n c t i o n o f time i n 7 healthy adults. Although plasma l e v e l s o f the hormone v a r y w i d e l y i n the normal p o p u l a t i o n , The the l e v e l i n any g i v e n i n d i v i d u a l tends t o remain r e l a t i v e l y c o n s t a n t . data i n F i g u r e 6 show measurements o f plasma 1,25-(OH) -D c o n c e n t r a t i o n s on 2 t o 9 s e p a r a t e occasions i n each o f 7 h e a l t h y i n d i v i d u a l s o v e r p e r i o d s ranging from 13 t o 40 months. I t i s apparent t h a t i n d i v i d u a l plasma 1 , 2 5 (0H) 2 -D l e v e l s tend t o be s t a b l e . These data are i n c o n t r a s t t o the plasma l e v e l s o f 25-OH-D which can v a r y w i t h s u n l i g h t exposure and d i e t a r y v i t a m i n D intake. Thus, d e s p i t e the complex homeostatic mechanisms t h a t appear t o i n f l u e n c e r e n a l l,25-(OH)2~D p r o d u c t i o n , the plasma l e v e l o f t h i s hormone i s s t r i c t l y defended. Acknowledgements Supported by USPHS RR-00058, AM-15089, AM-22014, and HL-05949 and The Medical C o l l e g e o f Wisconsin Kidney Research Fund. We a p p r e c i a t e the a s s i s t a n c e o f the s t a f f o f the Medical C o l l e g e o f Wisconsin C l i n i c a l Research Center. References 1.

Brumbaugh, P . F . , H a u s s l e r , H.D., Bursac, K.M., and H a u s s l e r , M.R. (1974) Biochem 13:4091-4097.

2.

Eisman, J . A . , Hamstra, A . F . , Science 193:1021-1023.

3.

Lambert, P.W., Syverson, B . F . , Arnaud, C . D . , and S p e l b e r y , (1977) J. S t e r o i d Biochem. 929-932.

Kream, B . E . , and DeLuca, H.F.

(1976)

T.C.

551 4.

Gray, R.W., Wilz, D.R., Caldas, A.E., and Lemann, J., Jr. J. Clin. Endrocrinol. 45:299-306.

(1977)

5.

Gray, R.W., Caldas, A.E., Wilz, D.R., Lemann J., Jr., Smith, G.A., and DeLuca, H.F. (1978) J. Clin. Endocrinol. Metab. 46:756-765.

6.

Brickman, A.S., Cobum, J.W., Friedman, G.R., Okamura, W.H., Massry, S.G., and Norman, A.W. (1976) J. Clin. Invest. 57:1540-1547.

7.

Hughes, M.R., Brumbaugh, P.F., Haussler, M.R., Wergedal, J.E., and Baylink, D.T. (1975) Science 190:578-570.

7.

Bilezikian, J.P., Canfield, R.E., Jacobs, T.P., Polay, J.S., D'Amato, A.P., Eisman, J.A., and DeLuca, H.F. (1978) NEJM 299:437-441.

9.

Shen, F.H., Baylink. D.J., Nielsen, R.L., Sherrard, D.J., Ivey, J.L., and Haussler, M.R. (1977):90:955-960.

553 THE ROLE OF THE LIVER IN THE CONTROL OP VITAMIN II METABOLISM E. Barbara Mawer Department of Medicine, University of Manchester, England. The relatively high incidence of bone disease associated with disorders of the liver and gastro-intestinal tract has led to speculation that the hepatic phase of vitamin D metabolism might be impaired.

The hepatic

phase has usually been equated with the 25-hydroxylation whilst other functions of the liver concerned with vitamin D metabolism have received less attention. HYDROXYLATION OF VITAMIN D IN LIVER DISEASE We have been able t.o show in primary biliary cirrhosis (PBC) that although 25-hydroxylation of vitamin Dj (D^) was normal, intestinal absorption was decreased and urinary excretion of polar metabolite significantly increased, thus making vitamin D-deficiency more likely (l). However, the correction of such deficiency by vitamin D treatment does not necessarily cure osteomalacia in PBC patients (2). To explain this lack of response Long and co-authors have suggested that either the active renal metabolite 1,25-dihydroxy vitamin D^ (l,25(0H)2Dj)is not formed normally in PBC or that there is a selective impairment in the metabolism of vitamin DgCDg), the usual form of therapy (2). We have investigated both these suggestions and have confirmed the recent finding of Long et al (3) that labelled l,25(OH)2D^ is formed in PBC. To study the metabolism of D^ in PBC we gave patients simultaneous equimolar doses of

- Dj and ^H - Dg by i.v. injection and studied the time

course of appearance of metabolites in serum, urine and faeces. Vie made 3 H-labelled marker metabolites of D 2 in rats and gave each peak produced on chromatography of serum extracts a tentative identity based on relative mobility on LH20 and HPLC in comparison to the respective D^ metabolites. This identification agrees well with that in a recent paper by Hay and Jones ( 4 ) .

A chromatograph of serum extract from a patient with severe

PBC is shown in Fig. 1.

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

55>i

8ERUM EXTRACT FROM PATIENT GIVEN* H-fl, AND "C-D,

Fig. 1. Serum extract from patient given ^H-Dg and "^C-Dj

Dp and D^ are metabolised to a very similar extent.

Faecal excretion was

low or undetectable in the 4 patients studied but urinary excretion was high, confirming our earlier study (l); in 2 patients almost twice as much radioactivity from Dg was excreted than from D^, (Table l), always in the form of polar metabolites. Table 1.

Excretion of radioactivity in urine (days 0 - 3 ) after i.v. injection of D^, and D? in PBC patients. fa dose

»3 patient

control

1

31.5

16.7

2

12.6

6.7

3

6.9

8.1

4

9.3

10.0

2.7

1.2

Thus it seems unlikely that either of the two postulated hydroxylation defects exist in PBC although in severe cases the urinary loss , particularly of D 2 metabolites, may be considerable.

555 THE EXCRETION ROUS OF THE LIVER It has been further suggested that both in PBC patients and. those with gastro-intestinal disease, vitamin D-deficiency might be excerbated byinterruption of the enterohepatic circulation of vitamin D metabolites in addition to the primary malabsorption of the vitamin (5)

(6).

This suggestions have arisen from a study in man in which evidence was presented in support of an extensive entero-hepatic circulation of injected 25-hydroxy vitamin D^ (25(0H)Dj) ( 7 ) .

Radioactivity entering the

duodenum was measured and assumed to be in the form of 25(OH)Dj although no characterisation was carried out.

The validity of this assumption and

the linked conclusion that 25(OH)Dj undergoes entero-hepatic circulation can be challenged on several grounds.

The implication that large amounts

of 25(OH)Dj per se normally enter the bile, is not borne out by published data from other sources. Very little of the radioactivity secreted in bile from labelled D^ is lipid-soluble.

Avioli and co-workers found only

10$ of biliary radioactivity could be extracted by CHCl^ (8); a similar study in our own laboratory revealed only minor amounts of D^ and 25(0H)Dj in bile from a total biliary fistula in man (9), and this was only present during the first 24 h after injection of D^ (Fig. 2).

Fig. 2

Chromatogram of extract of human bile collected between 8 and 20 h after i.v. injection of

NUMBER OF 20ml FRACTIONS

At all times polar metabolites were the main form of radioactivity. This lack of endogenously formed 25(OH)Lj in bile is significant because the concentration in serum was high - only 0.2fo of the injected dose was secreted into bile in 3 days during which time the serum content remained

556 constant at about

of the dose.

Much of the biliary radioactivity in man

was in the form of glucuronide (8), an observation confirmed in the rat by Bell and Kodicek (10).

Little unchanged sterol enters the bile even after

injection of 25(OH)D,5 Gray et al showed that only 5 h after injection

y

^

of

H-25(OH)D, most of the biliary radioactivity was in the form of polar

metabolites

(ll).

Gray also found much less radioactivity in bile than

Arnaud (12$> of the dose in 24 h. compared with 35 In an attempt to elucidate further the role of biliary secretion in vitamin D metabolism, we compared the excretion of ^ C - Dj and ^H-25(OH)Dj

in

3 types of experiments- a) human subjects with ileostomies, b) isolated perfused pig livers, c) rats with ligated or cannulated bile ducts. RESULTS a) Human subjects.

Equimolar doses of

and the excretory products collected.

"^C - Dj and ^H-25(OH)Dj were given 2 subjects with established ileo-

stomies and known malabsorption of fat showed a similar excretion pattern to that seen in the faeces of a control subject.

In all 3 subjects how-

ever, excretion of ^H from 25(OH)Dj was twice that of "^C from D^, Table 2. Table 2.

Excretion of radioactivity in faeces or ileostomy fluid {jo dose per day) from

Da

y Ileostomy

1

patient 1

3.55

patient 2 Control

14

C - J

2

from \

- 25(0H)D,

3

1

2

3

O.49

O.90

7.15

2.38

0.97

2.31

1.75

O.72

3-30

5-23

1.73

2.91

0.88

0.83

5.48

I.46

1.19

Ileostomy

Less than 1$ of the administered 25(OH)Dj appeared in ileostomy fluid in unchanged form but a further Tfo was excreted as more-polar and 2$ as lesspolar metabolites.

These results suggest that loss of 25(OH)Dj as such

through interruption of the entero-hepatic circulation are minimal. only

Since

of the ^ C D, was excreted as polar metabolites it seems likely

557 that the greater formation of polar products from ^H-25(OH)Dj may represent the hepatic reaction to a bolus of injected. 25(OH)Dj entering the liver from the circulation.

Although 25(OH)d^

is synthesised in liver

it is rapidly released into circulation and very little remains in the organ ( 1 2 ) ( 1 3 ) .

It is possible that administered 25(OH)Dj entering by an

unphysiological route does not immediately equilibriate with the serum pool (perhaps on account of the nature of its binding) and on entering the liver is recognised as a different pool to which the response is the formation of conjugates for excretion. b) Pig liver perfusion.

Labelled D^ or 25(OH)Dj was added to the blood

used to perfuse isolated pig livers (14). were analysed for metabolites.

The perfusate, bile and liver

In both cases polar metabolites formed the

main component of biliary radioactivity and began to appear as early as half an hour after starting the perfusion (Fig. 3). PIG LIVER PERFUSED WITH 2WOHID,

Fig. 3

LH20

M(

.

u v t

„ ,ERFUSED

M T

H VITAMIN D,

Chromatography of bile from pig liver perfusions

These experiments provide little support for the concept that large amounts of 25(OH)Dj are secreted into bile. c) Rats with modified biliary function.

Rats maintained on diets either

deficient or adequate in vitamin D were given high (400 i.v-.) or low (20 i.u.) i.v. doses of both

- Dj and \

- 25(OH)Dy

were cannulated so that bile could be collected:

The bile ducts

to avoid interference

with the entero-hepatic circulation of bile salts, replacement bile from donor animals not given labelled D^ was introduced onto the intestine. Biliary radioactivity was greatest between 4 and 6 h after injection and

558 results of collection at this time are shown in Table 3-

Table 3»

Excretion of radioactivity in rat bile 4 - 6 h after i.v. injection. jo ^ C dose excreted —3

2

5(OH)D3

jo ^H

metabs.

polar

dose excreted metabs.

+ D rats high dose

0.48

0.10

1.75

0.78

0.37

2.25

low dose

0.14

0.09

0.08

0.10

0.14

O.54

- D rats high dose

0.14

0.05

0.01

0.24

0.16

0.11

low dose

0.06

0.05

0

0.07

0.20

0.11

As in the human experiment above, excretion of ^H always exceeded that of In D -deficient animals there is little difference in the excretion pattern between high and low doses and little excretion of polar metabolites.

D - replete rats on the other hand formed mainly polar metabolites

from high doses of D^ and 25(OH)Dj but from the low doses only of 25(OH)Dj.

These results indicate that the extent and nature of biliary

secretion is affected both by the size of the administered dose and by the vitamin D-status of the animals.

In D - replete rats there appears to be

an enzyme system in operation that conjugates 25(OH)Dj and to a lesser extent, D^ itself, whereas in the I) -deficient animals this pathway did not seem open even to the high dose of 25(OH)Dj. The appearance in serum of the polar metabolites formed from 25(OH)Dj was followed in intact rats compared with those with either cannulated or ligated bile ducts.

The serum content of these metabolites at 6 h in

intact rats was 2.5/» of the injected ^H dose, compared with 0.7/j in rats with biliary cannulation; the bile collected between 4 and 6 h however contained 2.3cJc dose (Table3),suggesting that in the intact rats the polar metabolites were being partially reabsorbed from the small intestine.

Rats

with ligated bile ducts had only 0.5io dose in serum as polar metabolites

559 but a further 1.1 fo appeared in urine, compared with O.157Ó in urine of intact rats.

In this case interruption of the entero-hepatic circulation

seems to have promoted urinary excretion in a manner similar to that seen in the PBC patients. Other metabolites, e.g. 1,25(0H)2DJ, when given as an i.v.bolus appear to enter a comparable hepatic pathway, Pig. 4; unchanged 1,25(oh)2D^ enters the intestine across the intestinal wall, but the polar metabolites are presumably mainly of liver origin since their concentration decreases on biliary ligation.

to

30

M

20 ml FRACTIONS

Pig. 4«

LH20

Chromatography of intestinal contents of rats with intact or ligated bile ducts, after intravenous l ^ s C O H ^ D ^

CONCLUSIONS Any enterohepatic circulation of vitamin D^ metabolites is likely to involve the more-polar metabolites rather than 25(OH)Dy

This type of

circulation has been described for metabolites of vitamin A (15). Dj or 25(OH)Dj probably enter bile in those forms only at the time when the liver content is high, shortly after i.v. injection, a circumstance which may have no physiological parallel.

The polar metabolites are

thought to have little biological activity (16) so their re-circulation would have no function in conserving the physiologically active pool of vitamin D.

Our long-term studies have shown that the concentration of

polar metabolites in human serum whilst always low, increases slowly with time, becoming eventually the only detectable form of radioactivity from labelled D, (9)5

this is not a pattern that would be expected for biolo-

56o gically useful metabolites.

The results of the rat experiments show that

hiliary excretion is neglible in D -deficient animals and suggest that the function of biliary secretion may be to dispose of, rather than to conserve the vitamin.

The formation of polar metabolites from large doses indicates

the existence of a mechanism for the rapid excretion of excess vitamin D^ or 25(0H)D^ from liver.

Results from variety of sources thus provide

little evidence to support the concept of an extensive enterohepatic circulation of

and it seems unnecessary to invoke the breakdown

of such mechanism as a contributory factor to vitamin D -deficiency in human gastro-intestinal disease. This work was supported by a Programme Grant from the Medical Research Council to Professor S.W. Stanbury. I am grateful for the clinical co-operation of Dr. T. Warnes, Dr. H. Klass and Dr. H. Davies. REFERENCES 1.

Krawitt, 3.L., Grundnan, II. J. and Kawer, E.IJ. (1977) Lancet II, 1246 - 1249-

2.

Long, R.G., Varghese, Z., Keinhard, 3.A., Skinner, R.K., Wills, K.R. and Sherlock, S. (1978)

Brit. Hed. J. 1, 75 - 77-

3.

Long, R.G., Skinner, R.K., Wills, K.R. and Sherlock, S. (1978)

4.

Hay, A.W.H. and Jones, G. (1979)

5.

Compston, J.2., Horton, L.W.L., Ayers, A.B., Tighe, J.R. and

Clin. Chim. Acta

311 - 317-

Creamer, B. (1978)

Clin. Chem. (in press).

Lancet I, 9 - 12.

6.

Lancet I (1978)

1138.

7.

Arnaud, S.3., Goldsmith, R.S., Lambert, P.W. and Go, V.L.W. (1975) Proc. Soc. Exp. Biol. Iled.

G.

J. Clin. Invest.

46, 983 - 992.

Kawer, E.B., Backhouse, J., Holman, C.A., Lumb, G.A. and Stanbury, S.W. (1972)

10.

570 - 572.

Avioli, L.V., Lee, S.W., McDonald, J.E., Lund, J. and LeLuca, H.E. (1967)

9.

1^9,

Cli. Sci. Al,

413 - 431.

Bell, P.A. and Kodicek, E. (19^9)

Biochem. J. 115.

663 -

669.

56i 11.

Gray, R.V., Weber, 5!.P., Domínguez, J.H. and. Lemann, J. (1974) J. Clin. Endocrinol. lie tab.

_39,

lo45 - 1056.

12.

Ponchon, C-. and DeLuca, H.F. (1969)

13.

Mawer, E.B. and Reeve, A. (1977)

14.

Hamilton, C.A. (1977)

15.

Zachman, R.D. and Olson, J.A. ( 1 9 6 4 )

16.

Lumb, G.A. and Mawer, E.B.

J. Clin. Invest. 48, 1275 - 1279-

Calc. Tis. Res. 22S, 24 - 28.

Ph.D. Thesis, Univ. of Manchester. Nature (Lond) 201, 1222 - 1224.

unpublished observations.

563 PROSTAGLANDINS AND 25-HYDROXYVITAMIN D-1 crHYDROXYLASE. Wark, J.D., Larkins, R.G., Eisman, J.A. and Martin, T.J. Department of Medicine, Repatriation General Hospital, West Heidelberg, 3081, Victoria, Australia. The regulation of 1-hydroxylation is complex and remains poorly understood although a number of agents have been implicated. However the mechanism (s) by which these agents may exert regulatory effects is obscure partly because of the difficulty in demonstrating direct in vitro effects on 1-hydroxylation. Nevertheless cyclic AMP (c'AMP) is Furthermore renal a potent stimulus to 1-hydroxylation in vitro (1). tissue is rich in c'AMP and a range of prostaglandins (PGs) and these are known to interact in a variety of ways in other situations. Therefore these studies were initiated to explore a possible role of PGs, perhaps acting as local tissue mediators, in the regulation of 25-OHD-lhydroxylation. The levels of PGs in the experimental system were manipulated by adding exogenous precursor or exogenous PG and inhibitors of PG metabolism were used to alter endogenous PG levels. The effects of these manipulations on 1,25-(OH)formation were then examined. Fig. 1. prostaglandin metabolism

membrane phospholipid | arachidonic acid

. thromboxane A PGDH

PL

cyclic endoperoxides 1 I I 1 I | PGE. | ¡_f I

I

15-ketO PGE. 2

PGF

, prostacyclin PKR

2a

Precursor arachidonic acid (AA) is generated from membrane phospholipid by phospholipase (PL) a step that may be rate-limiting in the pathway (Fig. 1.). This represents a site where c'AMP could alter PG production since it is known to modulate PL activity in some tissues. PL can be inhibited by bromophenacylbromide (BPB), reducing the availability of endogenous AA for PG synthesis (2). The cyclic endoperoxides which give rise to primary PGs and the prostanoids are formed by the action of cyclooxygenase (CO). Aspirin-like drugs, of which indomethacin (IN) is a prominent example, are thought to act mainly by inhibition of this enzyme, thus blocking PG synthesis. Conversely, endogenous PGE concentrations may be elevated by a recently described anti-inflammatory agent, MK447 (3). This agent, which acts as a free radical scavenger, elicits an increased synthesis of PGEs and has also been found to decrease levels of the cyclic endoperoxide PGG2 in an in vitro enzyme system. The enzymes 15-hydroxyprostaglandin dehydrogenase (PGDH) and 9-ketoreductase (PKR) each may be important in PGE catabolism. Frusemide (F) and ethacrynic acid (EA) have been reported to inhibit both of these enzymes in mammalian renal tissue (4). IN was shown to inhibit the same enzymes although this action required a higher concentration than that expected for CO inhibition.

© 1979 Walter de Gruyter & Co., Berlin New York Vitamin D, Basic Research and its Clinical Application

564 METHODS. Kidneys were obtained from 3-to 6-week old vitamin D deficient White Leghorn-Australorp cross cockerels, and isolated renal tubules prepared as described previously (1). After equilibration tubules were preincubated with test substances for 30 minutes before a further 30 minute incubation with substrate ^H-25-OHD2. The reaction was terminated with methanol-chloroform. Vitamin D metabolites were extracted into chloroform and separated by HPLC (Waters yPorasil, 0.4 x 30 cm, solvent 12.5% isopropanol in n-hexane). H-1,25-(OH)2D3 production was determined from the percentage radioactivity which eluted as 1,25-(OH)2D3. RESULTS AND DISCUSSION. There was no detectable degradation of l,25-(OH)2D3 during 1 hour's incubation with tubules. Therefore the metabolite recovered was regarded as a reliable index of the amount formed. Cyclic AMP induced a marked stimulation of 1,25-(OH)2D3 production to approximately 150% of basal at a concentration of 2 x 10~4M or 10 -3 M. This response was used as a positive control. No alteration of basal l,25-(OH)2D3 production was observed in the presence of the following agents: AA (1.6 x 10 -4 M) , PGE-L (o.l yg/ml, 10 yg/ml), PGE2 (10 yg/ml), PGF2a ( 1 0 yg/ml). While the failure of these exogenous agents to alter basal 1,25-(OH) production provided some evidence against a regulatory role of PGs on 1-hydroxylation, it remained possible that an effect might be missed using these exogenous PGs or precursor because (i) these agents might not gain access to an intracellular site of action, (ii) they themselves or active metabolites subsequently formed might be labile or unstable under experimental conditions, or (iii) the regulatory PG(s) might be other than those tested. BPB

c'AMP

MK447

IN

-4 2x10 M

-3 10 M

-6 5x10 M

-5 1.4x10 M

0. lUg/ml

lOUg/ml

152.6 ±2.4

165.9 ±3.8

94.5 ±0.7

119.6 ±3.9

108.1 ±5.2

100.7 ±6.7

P