Hormones in normal and abnormal human tissues: Volume 1 9783111447001, 9783110080315

Table of contents :
PREFACE
CONTENTS
CONTRIBUTORS
THE BIOSYNTHESIS AND OCCURRENCE OF 16-ANDROSTENES IN MAN
EFFECTS OF ESTROGENS AND ANTIESTROGENS ON HUMAN BREAST CANCER CELLS IN TISSUE CULTURE
FACTORS CONTROLLING THE ACTION OF TESTOSTERONE ON NORMAL HUMAN SKIN AND IN CONDITIONS SUCH AS ACNE VULGARIS AND HIRSUTISM
IN VITRO PRODUCTION OF THYROID STIMULATORS BY HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS
ANTIESTROGENS AND HUMAN BREAST CANCER: EFFECTS AND MECHANISMS OF ACTION
HUMAN PROLACTIN AND NORMAL AND ABNORMAL BREAST TISSUE
PHYSICOCHEMICAL ANALYSIS OF GLUCO- AND MINERALO- CORTICOID RECEPTORS FROM HUMAN LIVER AND KIDNEY
hCG AND hCG-LIKE SUBSTANCES IN NORMAL AND ABNORMAL HUMAN TISSUE
ECTOPIC PRODUCTION OF GROWTH HORMONE IN HUMAN TISSUES
STEROID HORMONE PRODUCTION IN NORMAL AND ABNORMAL HUMAN ADRENOCORTICAL TISSUE
TRANSPORT OF THYROID HORMONES AND THEIR ENTRY INTO CELLS
SYNTHESIS, RELEASE, AND BIOLOGICAL ACTIONS OF HUMAN PROLACTIN
FATE OF CORTICOSTEROIDS AFTER PERCUTANEOUS ADMINISTRATION
SYNTHESIS AND SECRETION OF ANDROGENS IN HUMAN TESTES WITH SPECIAL REFERENCE TO TESTICULAR DISEASE
OESTRADIOL AND PROGESTERONE IN NORMAL AND ABNORMAL HUMAN UTERINE TISSUE
SYNTHESIS OF PLACENTAL LACTOGEN BY HUMAN PLACENTAE
CALCITONIN PRODUCTION AND CALCITONIN RECEPTORS IN HUMAN CANCERS
INDEPENDENT CONTROL OF LUTEINIZING HORMONE SECRETION BY TESTOSTERONE AND ESTRADIOL IN MALES
INHIBITION BY THYROID HORMONE BINDING PROTEINS AND RELAXATION OF INHIBITION BY THE HORMONES
ENTRY OF INSULIN INTO TARGET CELLS
STEROIDS IN NORMAL AND DISEASED HUMAN PROSTATIC TISSUE
OESTRONE SULPHATE - A MAJOR CIRCULATING OESTROGEN
HUMAN GROWTH HORMONE: ASPECTS OF MEASUREMENT
CATECHOLAMINE SECRETING TISSUES. NORMAL AND PATHOLOGICAL STATES
PROLACTIN DETERMINATIONS IN HEALTH AND DISEASE
References
Subject Index

Citation preview

Hormones in Normal and Abnormal Human Tissues

Hormones in Normal and Abnormal Human Tissues Volume 1 Editors K. Fotherby S.B.Pal

w

c_ DE

Walter de Gruyter • Berlin • New York 1981

Editors: K.Fotherby, Ph.D.,F.R.I.C. Department of Steroid Biochemistry Royal Postgraduate Medical School University of London Ducane Road London W 1 2 0 HS U.K. S. B. Pal, D. Phil., Dr. rer. biol. hum., M . I. Biol. Universität Ulm Department für Innere Medizin Steinhövelstrasse 9 D 7900 Ulm/Donau F. R. Germany

CIP-Kurztitelaufnahme der Deutschen Bibliothek

H o r m o n e s in normal a n d a b n o r m a l h u m a n tissues ed. by K. Fotherby; S. B. Pal. - Berlin, N e w York: de Gruyter. NE: Fotherby, Kenneth (Hrsg.) Vol. 1.-1981. ISBN 3-11-0080031-1

Library of Congress Cataloging in Publication Data

Hormones in normal and abnormal human tissues. Bibliography: v. 1, p. Includes index. 1. Hormones. 2. Hormones, Ectopic. I. Fotherby, K„ 1927- II. Pal,S.B„ 1928[DNLM: 1. Hormones. 2. Disease. W K 1 0 2 H8127] QP571.H663 616.4 80-27070 ISBN 3-11-0080031-1 (v.l)

© Copyright 1981 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. N o 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: Lüderitz & Bauer, Buchgewerbe GmbH, Berlin.Printed in Germany

PREFACE

With the development during the past decade of immunoassay techniques particularly radioimmunoassay, it has been possible to measure with accuracy the circulating levels of most nonpolypeptide hormones although some problems still exist in regard to the measurement of polypeptide and protein hormones. Some of these problems are outlined in these monographs. The techniques used so far have enabled the accumulation of a large amount of information on the levels of these hormones in normal subjects, on changes in various pathological and therapeutic conditions and on the factors controlling the secretion of these hormones. For the non-polypeptide hormones the pathways for their biosynthesis have been elucidated by a number of different techniques. Biosynthetic schemes can be drawn up for a number of species, although there are still many gaps and points needing further substantiation, even for the biosynthesis in human tissues. For most of the polypeptide hormones the biosynthetic pathways and the factors controlling these are still incomplete. Most of these different aspects are touched upon in the various chapters in these volumes. However, we have tried to place the emphasis on the concentration of the various hormones in tissues; those where they are produced and those where they might localise and produce an effect and how these levels are modified under varying circumstances. Little information is available on these points and we hope that these volumes will provide a summary of existing knowledge which will be useful to research workers in various branches of endocrinology. In particular, we have asked contributors to deal in their articles mainly with available information about the human and only to include results from other species where they are relevant to the human situation. We think that these two volumes are the only monographs devoted mainly to this topic.

vi We are grateful to the distinguished contributors for agreeing to undertake the task that was set and we are also grateful to Walter de Gruyter for their willingness to take on the responsibility of publishing these volumes and to Dr. R. Weber who will ensure their speedy publication.

K. Fotherby

S. B. Pal

CONTENTS The Biosynthesis and Occurrence of 16-Androstenes in Man D. B. Gower

1

Effects of Estrogens and Antiestrogens on Human Breast Cancer Cells in Tissue Culture J. C. Allegra, M. E. Lippman

29

Factors Controlling the Action of Testosterone on Normal Human Skin and in Conditions such as Acne Vulgaris and Hirsutism J. B. Hay

43

In Vitro Production of Thyroid Stimulators by Human Peripheral Blood Mononuclear Cells B. Rapoport

61

Antiestrogens and Human Breast Cancer: Effects and Mechanisms of Action Kathryn B. Horwitz

81

Human Prolactin and Normal and Abnormal Breast Tissue H. Nagasawa

115

Physicochemical Analysis of Gluco- and Mineralo- Corticoid Receptors from Human Liver and Kidney J. Paillard, E. Baviera, M. K. Agarwal

145

hCG and hCG-like Substances in Normal and Abnormal Human Tissue B. B. Saxena, P. Rathnam

167

Ectopic Production of Growth Hormone in Human Tissues A. Kaganowicz, A. Blaustein

207

Steroid Hormone Production in Normal and Abnormal Human Adrenocortical Tissue B. J. Whitehouse, G. P. Vinson

215

Transport of Thyroid Hormones and their Entry into Cells B. Ramsden, R. Hoffenberg

257

viii Synthesis, Release, and Biological Actions of Human Prolactin C. Ferrari

281

Fate of Corticosteroids after Percutaneous Administration G. Celasco, F. Galletti, R. Gardi

327

Synthesis and Secretion of Androgens in Human Testes with Special Reference to Testicular Disease H. Oshima, K. Isurugi, B. Tamaoki

351

Oestradiol and Progesterone in Normal and Abnormal Human Uterine Tissue K. Pollow

373

Synthesis of Placental Lactogen by Human Placentae T. Chard

409

Calcitonin Production and Calcitonin Receptors in Human Cancers T. J. Martin, Jane M. Moseley, D. M. Findlay, V. P. Michelangeli

429

Independent Control of Luteinizing Hormone Secretion by Testosterone and Estradiol in Males R. J. Santen

459

Inhibition by Thyroid Hormone Binding Proteins and Relaxation of Inhibition by the Hormones J. S. Orr

491

Entry of Insulin into Target Cells I. D. Goldfine, A. L. Jones, G. Hradek, B. M. Kriz, K. Y. Wong

50 3

Steroids in Normal and Diseased Human Prostatic Tissue R. Vihko, N. Bolton, G. L. Hammond, R. Lahtonen

52 3

Oestrone Sulphate - A Major Circulating Oestrogen K. F. Sttfa, 0. L. Myking

541

Human Growth Hormone: Aspects of Measurement M. A. Vodian, W. P. VanderLaan

573

ix Catecholamine Secreting Tissues. Normal and Pathological States R. E. Coupland

589

Prolactin Determinations in Health and Disease P. A. Kelly

635

CONTRIBUTORS Numbers in parentheses indicate the page on which the authors' articles begin. M. K. Agarwal, Centre National de la Recherche Scientifique, Paris, France (145). J. C. Allegra, Clinical Investigations Branch and Medicine Branch, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205, U.S.A. (29). E. Baviera, Laboratoire Central d'Anatomie Pathologique, Hopital Saint Joseph, Paris, France (145). A. Blaustein, Department of Pathology, Booth Memorial Medical Center, Flushing, New York 11355, U.S.A. (207). N. Bolton, Department of Clinical Chemistry, University of Oulu, SF-90220 Oulu 22, Finland (523). G. Celasco, Vister Research Laboratories, 22064 Casatenovo, Como, Italy (327). T. Chard, Joint Academic Unit of Obstetrics, Gynaecology and Reproductive Physiology, St. Bartholomew's Hospital Medical College and the London Hospital Medical College, London, U.K. (409) . R. E. Coupland, Department of Human Morphology, The University of Nottingham, Clifton Boulevard, Nottingham NG7 2UH, U.K. (589) . A. Ercoli, Via Circo 12, 20123 Milano, Italy (327). C. Ferrari, Second Department of Medicine, Fatebenefrateili Hospital, 23 Corso di Porta Nuova, 20121 Milano, Italy (281). D. M. Findlay, University of Melbourne, Department of Medicine, Repatriation General Hospital, Heidelberg, Victoria, Australia (429) .

xii F. Galletti, Vister Research Laboratories, 22064 Casatenovo, Como, Italy (327) . R. Gardi, Vister Research Laboratories, 22064 Casatenovo, Como, Italy (327). I. D. Goldfine, Cell Biology Laboratory, Mount Zion Hospital and Medical Center, San Francisco, CA 94120, U.S.A. (503). D. B. Gower, Department of Biochemistry, Guy's Hospital Medical School, London SE1 9RT, U.K. (1). G. L. Hammond, Department of Clinical Chemistry, University of Oulu, SF-90220 Oulu 22, Finland (523). J. B. Hay, C.S.I.R.O., Division of Animal Production, Ian Clunies Ross Animal Research Laboratory, Prospect, P.O. Box 239, Blacktown, N.S.W., 2148, Australia (43). R. Hoffenberg, Department of Medicine, University of Birmingham, Queen Elizabeth Hospital, Birmingham, B15 2TH, U.K. (257). Kathryn B. Horwitz, Department of Medicine, University of Colorado Health Science Center, Denver, Colorado 80262, U.S.A. (81) .

G. Hradek, Department of Medicine, University of California, San Francisco, CA 94143, U.S.A. (503) . K. Isurugi, Department of Urology, National Center Hospital, Tokyo, Japan (351). A. L. Jones, Department of Medicine, University of California, San Francisco, CA 94143, U.S.A. (503). A. Kaganowicz, Department of Pathology, Booth Memorial Medical Center, Flushing, New York 11355, U.S.A. (207). P. A. Kelly, Groupe du Conseil de Recherches Médicales en Endocrinologie Moléculaire, Le Centre Hospitalier de l'Université Laval, 2705, Boulevard Laurier, Québec, G1V 4G2, Canada (635). B. M. Kriz, Cell Biology Section, Veterans Administration Hospital, San Francisco, CA 94121, U.S.A. (503).

xiii R. Lahtonen, Department of Clinical Chemistry, University of Oulu, SF-90220 Oulu 22, Finland (523). M. E. Lippman, Clinical Investigations Branch and Medicine Branch, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, 20205, U.S.A. (29) . T. J. Martin, University of Melbourne, Department of Medicine, Repatriation General Hospital, Heidelberg, Victoria, Australia (429) . V. P. Michelangeli, University of Melbourne, Department of Medicine, Repatriation General Hospital, Heidelberg, Victoria, Australia (429). Jane M. Moseley, University of Melbourne, Department of Medicine, Repatriation General Hospital, Heidelberg, Victoria, Australia (429) . 0. L. Myking, Hormone Laboratory, University of Bergen School of Medicine, Bergen, Norway

(541).

H. Nagasawa, Pharmacology Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104, Japan (115). J. S. Orr, Department of Medical Physics, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London, W12 OHS, U.K. (491) . H. Oshima, Department of Urology, Tokyo Medical and Dental University School of Medicine, Tokyo 113, Japan (351). J. Paillard, UER Broussais Hotel Dieu, Paris, France (145). K. Pollow, Department of Experimental Endocrinology, Johannes Gutenberg University Mainz, Langenbeckstrasse 1, 6500 Mainz, Germany

(373) .

B. Ramsden, Department of Medicine, University of Birmingham, Queen Elizabeth Hospital, Birmingham, B15 2TH, U.K. (257) .

xiv B. Rapoport, The Department of Medicine, University of California School of Medicine, San Francisco, California 94143, and, The Veterans' Administration Hospital, San Francisco, California 94121 , U.S.A. (61) . P. Rathnam, Departments of Medicine and Obstetrics and Gynecology, Cornell University Medical College, 1300 York Avenue, New York, N.Y. 10021, U.S.A. (167). R. J. Santen, Department of Medicine, Division of Endocrinology, The Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania, U.S.A. (459). B. B. Saxena, Departments of Medicine and Obstetrics and Gynecology, Cornell University Medical College, 1300 York Avenue, New York, N.Y. 10021, U.S.A. (167). K. F. Sttfa, Hormone Laboratory, University of Bergen School of Medicine, Bergen, Norway (541). B. Tamaoki, Division of Pharmaceutical Sciences, National Institute of Radiological Sciences, Chiba 280, Japan (351). W. P. VanderLaan, Lutcher Brown Center for Diabetes and Endocrinology, Scripps Clinic and Research Foundation, La Jolla, California 92037, U.S.A. (573) . R. Vihko, Department of Clinical Chemistry, University of Oulu, SF-90220 Oulu 22, Finland (523). G. P. Vinson, Department of Biochemistry, St. Bartholomew's Hospital Medical College, London EC1M 6BQ, U.K. (215). M. A. Vodian, Lutcher Brown Center for Diabetes and Endocrinology, Scripps Clinic and Research Foundation, La Jolla, California 92037, U.S.A. (573). B. J. Whitehouse, Department of Physiology, Queen Elizabeth College, Campden Hill Road, London W8 7AH, U.K. (215). K. Y. Wong, Cell Biology Section, Veterans Administration Hospital, San Francisco, CA 94121, U.S.A. (503).

THE BIOSYNTHESIS AND OCCURRENCE OF 16-ANDROSTENES IN MAN

D. B. Gower Department of Biochemistry, Guy's Hospital Medical School, London SE1 9RT., U. K.

Introduction Some of the steroids of the group known as 16-androstenes were originally isolated from boar testes by Prelog and Ruzicka (1) in 1944. At that time these workers were searching for androgens in the pig but discovered that the 16-androstenes were present in much larger quantities. Attention was drawn (2) to the smell of these steroids, which was said to be musk-like, for the alcohols, and urine-like for the ketonic derivatives (see below). Even before this time, several workers (3, 4) had commented on the unpleasant smell and flavour of cooked meat taken from entire or partially-castrated boars. The 'boar-taint' problem subsequently led to intensive research into the biosynthesis of the odorous 16-androstenes in boar testis and these studies have been reviewed (5 - 8). We now know that, because of their smell, some members of this group of steroids act as pheromones in pigs, being produced in boar testes, excreted on the breath via the salivary glands (9, 10) and eliciting the mating stance in oestrous sows (11, 12). This review discusses the results of research published during the past few years showing that the 16-androstenes also occur in human beings.

Structure and Separation of 16-Androstenes Figure 1 shows the formulae of the 16-androstenes. The struc-

Hormones in Normal and Abnormal Human Tissues © Walter de Gruyter • Berlin • New York 1981

2 tures of some odorous macrocycles are also given because of their superficial structural relationship to the 16-androstenes (2) .

As their systematic name implies, the 16-androstenes are characterized by unsaturation at C-16. This lack of substituent at C-17 appears to abolish androgenic activity. Neither androstadienone (androst-4,6-dien-3-one) nor 5a-androstenone (5a-androst-16-en-3-one) has androgenic activity in rats and mice (13) despite the fact that their structures in Ring A and at C-3 are identical with the corresponding androgens testosterone and 5a-dihydrotestosterone. Another interesting feature of the 16-androstenes is their very non-polar character. They can readily be extracted with non-polar solvents but they are also readily adsorbed to glass. This latter property has proved to be a problem in work with these steroids. Because of their non-polar character, solvent systems used in their chromatographic separation must also be relatively non-polar, otherwise the 16-androstenes migrate to the solvent front as a single unresolved spot. Most members of the group (Figure 1) can be separated using systems such as benzene-ether (9:1, v /v) or toluene-ethyl acetate (9:1, V /v) (see reference (5) for review). Andien-3 and an-p (see Figure 1), which only differ by one double bond at C-5,6,

*Abbreviations for steroids names: aetiocholenol (ae-a) , 5(5androst-16-en-3a-ol; oestratetraenol, 1 , 3 , 5 ( 10) ,16-oestratetraen-3-ol; androsterone, 3a-hydroxy-5a-androstan-17-one; aetiocholanolone, 3a-hydroxy-5 3-androstan-17-one; dehydroepiandrosterone (DHA) , 3f3-hydroxy-5-androsten-17-one; oestriol, 1,3,5, ( 10)-oestratriene-3,16a,173-triol; epi-oestriol, 1,3,5(10)oestratriene,3,163,173-triol; 4-androstenedione, 4-androstene3,17-dione; epitestosterone, 17a-hydroxy-4-androsten-3-one. See also Figure 1.

3 are not easily resolvable even by two-dimensional thin layer chromatography acid is used

(14), unless silver nitrate impregnated

silicic

(5, 15). In this case, the diene and mono-ene

can

be separated. This can also be done on columns of silver nitrate-silicic acid (16) and the method has been utilised to 14 14 separate C-labelled andien-3 synthesized from C-pregnenolone by preparations of adrenocortical carcinoma

civetone

(17).

muscone

H 5a - a n d r o s t - 1 6 - e n - 3 a - o l

5a -androst-16-on-30 -ol

H

androsta-4,16-dien-3-one

5a -androst-16-e»-3-one

OH

androsta-5.16-dien-3tf -ol

testosterone

O exaltolide

Figure 1.

Structural formulae of 16-androstenes and odorcxjs macrocylic compounds.

4 Alumina column chromatography (18) has been utilised extensively in separating 16-androstenes from extracts obtained from urine, plasma and tissues (5). In the original method of Brooksbank and Haslewood (18) a single eluant consisting of benzene-light petroleum

b.p. 80-100°C (1:1, V /v) was used

throughout but this has been modified slightly to allow elution of 5a-androstenone first, followed by androstadienone plus an-a and finally andien-p and an-3 (see reference (5) for TM details). More recently, Lipidex (hydroxyalkoxypropylSephadex) has been utilised with n-pentane-cyclohexane (99.5: 0.5, /v) as solvent system (19). The third feature of interest is the relative volatility of the 16-androstenes, and anecdotal evidence has suggested that considerable quantities of these steroids may be lost by evaporating solutions in organic solvents under nitrogen at elevated temperatures. Recently, the losses encountered during various evaporation procedures have been quantified (20). Losses are small providing evaporation is performed with a rotary evaporator using water-pump suction and a temperature of not more than about 40°C. Although the volatility of the 16-androstenes creates problems, they can be eluted as free steroids from a gas-chromatographic column with ease at relatively low temperatures (190-210°C) and, if the trimethylsilyl ethers are used, the operating temperature may be as low as 170°C, providing a non-selective stationary phase is employed (5). The chloromethyldimethyl silyl ethers are useful if an-p and andien-3 are to be resolved on the selective phase XE-60 (21) .

Occurrence of 16-Androstenes in Healthy Humans a) In urine. Brooksbank and Haslewood (18) showed that an-a, conjugated as the glucuronide, was excreted in the urine of normal men (mean value 1.5 mg/day) and women (mean value 0.5 mg/day). In these early experiments, a colorimetric method

5 (resorcylaldehyde/conc.sulphuric acid) was used after the separation of the 'an-a' fraction by alumina chromatography. The results indicated further that excretion of an-a glucuronide was low in pre-pubertal children, increased at puberty and declined somewhat in elderly men and post-menopausal women. Further studies (22) gave support to these observations. Later work (23, 24) showed that, although an-a is the major 16-androstene in human urine, two others, namely, ae-a and andien-3 were excreted as glucuronides in very small quantities. For normal men the values (in kig/g creatinine) were found to be 5.9 and 43.4 respectively and for normal women 4.8 and 20.7 respectively. These small quantities could only be measured by gas-liquid chromatography, and this method, as anticipated, resulted in slightly lower values for an-a relative to those obtained by colorimetric analysis (18). For men aged between 20 and 40 years, the mean value was 1.05 mg/ day and for women aged between 2 0 and 35 years, the mean value was 0.36 mg/day (24). Two more members of the 16-androstene group, an-3 and 5a-androstenone (Figure 1), have been detected in human urine in very small quantities after the administra14 tion of C-androstadienone to one man and one woman (25), while the phenolic 16-unsaturated steroid, oestratetraenol, is excreted as the glucuronide in urine of women in late pregnancy (concentration approximately 100 ug/1 (26) ). b) In plasma. The estimation of the concentrations of 16-androstenes in human peripheral blood plasma was hampered until recently when radioimmunoassays became available, although a method based on gas-liquid chromatography (27) gave values for the concentrations of unconjugated androstadienone (Figure 1) in plasma pools from young men and women of 98 and 36 ng/100 ml respectively. When tritiated 5a-androstenone became available, radioimmunoassays were developed. An antiserum raised against 5aandrostenone-3-(0-carboxymethyl)-oxime to bovine serum albumin (BSA) in rabbits, was used to measure 5a-androstenone levels

6

in plasma, fat and axillary sweat samples from men (28, 29). The mean value for plasma was 3.26 (range 2.13-4.38) ng/ml. An-a levels of human plasma have also been measured (30) using a similar antiserum (31) which cross-reacted to the extent of 42% with an-a. Mean values were 3.08 (range 0.3 14.9) and 0.66 (range 0 - 2.4) ng/100 ml for plasma samples from 31 men and 16 women, respectively. Despite the obvious sex-difference, no correlation was found between the plasma levels of an-a and testosterone (32). Recent attempts to measure the 5a-androstenone content of peripheral plasma of men at 30 min. intervals throughout the day were invalidated because the antiserum reacted with other 16-androstenes and unidentifiable substances in the crude plasma extract (33). c) In sweat. Gower and Llewelyn (cited in ref. 5) showed the presence of 5a-androstenone in the axillary sweat of two men and, more recently, radioimmunoassays (29) have shown that 14.59 +_ 7.39 ng/h/arm-pit (mean +_ S.E.M.) were excreted in the axillary sweat of 13 men. In keeping with the non-polar nature of this steroid, evidence was obtained (29) for the storage of considerable quantities in fatty tissues of the men (mean, 103.33 + 8.78 ng/g) but not in three women (10, 20 and 30 ng/g fat); 5a-androstenone was not detected in the peripheral plasma of these women. In addition to 5a-androstenone, an-a is also found in the axillary sweat of men (34) , and Bicknell (32) found an-a in surface sweat collected from the backs of six men (4.2 _+ 2 1.0 ng/100 cm /12 h; mean + S.D.). These findings are particularly significant in view of the smell of the 16-androstenes, or of a blend of these and other compounds, and this may be consistent with a role in human communication (see subsequent Section) . d) In testis tissue. Few data are so far available on the 16androstenes present in human testis. Ruokonen (35) has analy-

7 sed human cadaver testes and shown that 16-androstenes occur but in similar concentrations to those of the androgens and other steroids. In the free steroid fraction, the relative abundance was an-|3 >andien-3 >an-a, while in the sulphate fraction, andien-p was most abundant followed by an-p and an-a. In both fractions, an-a was only present to the extent of 0.5-1ng/1OOg testis tissue. Results from the author's laboratory (36) showed that 16-androstenes are biosynthesized in human testis from pregnenolone but in lower yields than those found with boar testis (5) (see subsequent Section). It seems significant, therefore, that in boar testis, where the 16-androstenes are quantitatively the most important C^g steroids (37-39), they play such an important role in the reproductive physiology of the pig (11, 12).

Biosynthesis and Metabolism of 16-Androstenes. The suggestion (40) that an-a might be a unique urinary metabolite of testosterone, which was dehydrated to form the 16, 17 double bond, was not confirmed by subsequent work. In humans, little or no labelled urinary an-a was produced after administration of isotopically-labelled 16a-hydroxyprogesterone (41), testosterone and DHA acetate (42), testosterone and DHA (43), testosterone (44) or 17a-testosterone (45). However, 14 an-a was shown to be formed from both [4- C]-cholesterol and [7a- H]-pregnenolone after injection into a woman who had a virilizing adrenal carcinoma (46). More recently, (47) [ H]androstenedione and [1 4C]-progesterone were injected intrave14 3 nously into one man and [ C]-progesterone and [ H]-pregnenolone into another. Urine was collected for four or five days and analysed for 16-androstenes. The percentage conversion (0.026-0.16) to an-a was very low in all cases and indicated that urinary an-a was not formed from circulating C 0 1 or C. n z\ iy precursors but was produced in a compartment in which these precursors do not come into rapid equilibrium from the general

8

circulation.

The c u m u l a t i v e s p e c i f i c r a d i o a c t i v i t y o f

andien-(3 d u r i n g t h e f i v e - d a y p e r i o d

(324 d . p . m . / u g )

urinary

was h i g h

enough t o i n d i c a t e t h a t t h i s m e t a b o l i t e was d e r i v e d from circulating

pregnenolone.

Such f i n d i n g s a r e i n k e e p i n g w i t h r e s u l t s o b t a i n e d boar t e s t i s

i n which p r e g n e n o l o n e i s t h e p r e c u r s o r o f

r a t h e r t h a n 1 7 a - h y d r o x y p r e g n e n o l o n e o r C-19 s t e r o i d s In b o a r t e s t i s , androstadienone,

in

andien-3, (16,

p r o g e s t e r o n e can a l s o s e r v e a s a p r e c u r s o r which i s f u r t h e r m e t a b o l i s e d t o

none and t h e n t o a m i x t u r e o f a n - a and an-f3 (48)

( F i g u r e 2) .

HO**'

Figure 2.

of

5a-androste-

HO

5«-Androst-16-en-3-onc

48).

An-/}

Pathways of biosynthesis of 16-androstenes in boar t e s t i s (based on data from references 16 and 48).

9 These alcohols and andien-3 can all be converted to sulphates in boar testis preparations (49, 50). Similar results for the free 16-androstenes have been obtained with human testis preparations (36) although, as indicated above, the yields of 16-androstenes obtained were less than in boar testis. The subcellular location and some properties of the enzyme 'andien-3 synthetase', which catalyses the formation of andien-3 from pregnenolone in boar testis, have been studied recently (51-53).

Metabolism of 16-Androstenes 3 7a- H-Androstadienone, administered intravenously to humans, disappeared from the peripheral plasma at rates which were consistent with a two-pool distribution (27) (Figure 3). The calculated metabolic clearance rates (1/day) for androstadienone were 991 and 1363 for two men and 1246 for one woman. Corresponding plasma production rates (ng/24h) were 975 and 1341 for the men and 4 56 for the woman. When the urinary production rates of tritiated an-a were calculated, they were found to be much higher than the plasma production values, indicating that much of the urinary an-a was derived from sources other than circulating androstadienone. There was no doubt that the conversion of the latter to an-a was rapid, as judged by the3 excretion of labelled an-a soon after administration of [ H]-androstadienone. 14 Further studies on the metabolism of [4- C]-androstadienone in one healthy man and one healthy woman (25) revealed that the major metabolite was an-a, which accounted for 67% and 46%, respectively, of the total 1 urinary radioactivity in 4 the first 24h. Small quantities of

C were found in ae-a,

andien-3, an-3 and 5a-androstenone. In contrast, considerable amounts of radioactivity were associated with four isomeric androstanetriols : 5a-androstane , 3a, 1 6a, 1 7(3-triol, 5a-androstane-3a,163,17a-triol, 53-androstane-3a,16a,173-triol and 53~ androstane-3a,163,17a-triol.

10

Figure 3.

14 Disappearance of C androsta-4,16-dien-3-one from plasma after a single intravenous injection into a healthy woman. A, intercept on ordinate of initial part of curve extrapolated to zero time; B, intercept on ordinate of later linear part of disappearance curve extrapolated to zero time; 1 / A + B = v x = i n n e r P°°l volime of distribution; = V = volume of an undefined ccnpartment; a, slope of corrected initial curve (first exponential); ¡5, slope of second exponential. (From ref. 27, with permission of authors and Journal of Endocrinology, Ltd.)

It is almost certain that these polar metabolites are formed in the liver. Evidence had been obtained earlier (54) to show that human foetal microsomes contain an oxidase system which catalyses the conversion of androstadienone into an intermediate 16,17-epoxide and further to a 16,17-glycol. Similar results were obtained (55) using microsomal preparations from rats; androstadienone, andien-p, an-a and an-0 were converted by cytochrome P-450 -dependent reactions (56) into isomers of 5a-androstane-3 , 1 63,1 7a-triol and 3 ,1 7|3-dihydroxy5a-androstan-16-one. When 16a,17a-epoxysteroids were used as

11 substrates, 163,1Va-dihydroxylated intermediates were formed. A similar sequence of reactions was shown to occur for oestratetraenol, the weakly-oestrogenic 16-unsaturated steroid (5, 57). This is formed by aromatization of androstadienone in human placental microsomal preparations (58). When oestratetraenol was administered intravenously to human subjects, 16-epi-oestriol was found in the urine (59), the intermediate metabolites probably being 16,17-epoxides, in view of the reactions shown to occur in rat liver preparations (60) (Figure 4). Recently, Kingsbury and Brooksbank (61) have studied the metabolism of [^H]-an-a and [^H] -5a-androstenone in four human 3 subjects. [ H]-An-a was cleared from the plasma of two subjects at rates which were consistent with a two-pool distribution.

Figure 4.

Biosynthesis and metabolism of oestratetraenol. A, androstadienone; B, oestratetraenol; C, 16a,17a-«paxy-oestratrien-3-ol; D, 168,176-epoxyoestratrien-3-ol; E, epi-oestrlol; F, oestrlol.

12

High metabolic clearance rates were calculated (3,790 and 3,120 1/day, respectively, for a man and a woman). The corresponding blood production rates for an-a were (respectively) 3 875 and 1780 ng/24h. The disappearance of [ H]-5a-androstenone from the peripheral circulation of one man and one woman was especially rapid and may indicate that, in view of its extreme non-polarity, the steroid may have been taken up by adipose tissue. Such a situation is well-known in pigs 3(9), and an equally rapid clearance rate was noted when [5a- H]5a-androstenone was administered intravenously to a boar (Y.A. Saat and D.B. Gower, unpublished observations). As 14 confirmation of the earlier study using [ C]-androstadienone 3 3 (25), both [ H]-an-a and [ H]-5a-androstenone were converted into small amounts of an-|3, excreted as a glucuronide, and larger amounts of androstanetriols (61). On the basis of results obtained in man (18, 24, 25, 27, 47, 61), it seems likely that pregnenolone is converted to andien-3 and that this gives rise to androstadienone, presumably through the action of 5-ene -33-hydroxysteroid dehydrogenase and 4,5-isomerase. It is of interest here that this reaction is reversible to some extent since androstadienone gives rise to small quantities of andien-3 (25). Once androstadienone has been formed, it can be reduced to 5a-androstenone by the action of 4-ene-5a-reductase. However, to explain the occurrence and formation of small quantities of ae-a (24, 25), one must invoke the action of the 4-ene-5(3reductase. This 3a, 5|3-isomer would subsequently give rise to androstanetriols of the 5fS-series (25). The predominant 16unsaturated steroid, an-a, must presumably be formed from 5a-androstenone by 3a-hydroxysteroid dehydrogenase action. Further metabolism of an-a then results in the 5a-androstanetriols. In summary, the sequence of reactions may be as follows:

13 pregnenolone 5,16-androstadien-3B-ol

—urine (glucuronide)

I!

4,16-androstadien-3-one

5a-androst-16-en-3-one

5a-androst-16-en-3a-ol

5g-androst-16-en-3a-ol

androstanetriols (5g-series)

\

urine (glucuronide)

5a-androst-16-en-3S~ol

androstanetriols (5a-series)

urine (glucuronide)

urine (glucuronide)

Occurrence and Biosynthesis of 16-Androstenes in Abnormal Situations a) Adrenal disorders. Early work showed that urinary an-a excretion was elevated above normal in women with adrenal tumours (62), adrenal hyperplasia (63) and a luteoma of the ovary (64). In a case of virilizing adenoma studied by Burstein and Dorfman (46), the urinary an-a was as high as 20 mg/24h, compared with the normal range for adult women of 0.23-0.42 mg/24h (24). More recent studies using gas-liquid chromatography

(24) have measured urinary ae-a and andien-p

as well as testosterone and individual 17-oxosteroids. In one woman (65) and two girls (17, 66) with virilizing adrenocortical carcinomas, excretion of 16-androstenes and androgens was grossly elevated; in the woman (65) the plasma an-a sulphate level was approximately 28 ng/ml, a value some ten times higher than the maximum valjie found in normal women (30) . After surgical removal of the diseased adrenals, the urinary excretion of these steroids returned to normal limits and plasma an-a sulphate was undetectable (24). In patients with

14

severe virilization due to adrenal carcinoma, this seems to be an almost invariable pattern - with high correlation between testosterone and an-a excretion, indicative of an over-production of all steroids from pregnenolone. In patients with simple hirsutism, however, no clear correlation emerged (24), suggesting that an-a was not simply derived from testosterone. This would be in keeping with the failure of other workers to show the biosynthesis of an-a from testosterone and other C-19 steroids (42-45). The results cited above (17, 65, 66) clearly indicate that 16-androstenes can be synthesized in the adrenal cortex and the increase in an-a excretion found after ACTH administration (22, 67) would therefore be anticipated. Indeed, biosynthetic studies with the diseased adrenals, removed at surgery from the woman and from one of the children mentioned above (17, 65), showed that pregnenolone and progesterone served as precursors for andien-3 and androstadienone but that no 16-androstenes were formed from testosterone or from DHA. b) Ovarian disorders. The early study of the patient with a luteoma of the ovary (64) has been referred to already. In their report, the authors made no attempts to estimate analytical losses and it is therefore difficult to obtain an accurate assessment of the amount of an-a excreted by this patient. It seems likely, however, that the amount was elevated above the normal female range, or at least at the high end of the range. When normal human ovarian follicles were incubated with labelled pregnenolone (68), not only were the expected androgens and progestogens formed, but also some non-polar steroid metabolites. Two of these were subsequently identified tentatively as andien-p and androstadienone. Incubation of human polycystic ovarian tissue with labelled pregnenolone (69) gave similar results, and a more detailed study (70) showed that andien-p was formed in yields of up to approximately 4% by the end of four hours.

15

P o r c i n e o v a r i a n t i s s u e was r e p o r t e d cytoplasmic

'dehydratase'

androstadienone

system c o n v e r t i n g

in y i e l d s

In c o n t r a s t ,

small y i e l d s stenone

laboratory of

to

repeat

h o w e v e r , were and o f

unsuc-

rise

5a-andro16-

i n sows i s unknown, b o a r a d r e n a l t i s s u e

can

'pool'

t h i s group o f 10% o f

steroids

(37)

the t e s t e s .

but o n l y t o t h e

extent

The s m a l l amounts o f

16-

a n d r o s t e n e s f o r m e d i n sow o v a r i e s

is entirely

in keeping

the

(31)

an-a

low l e v e l s o f

sow p e r i p h e r a l c)

Testicular

5a-androstenone

and o f

f e m i n i z a t i o n syndrome.

Bicknell

an-a and o t h e r

steroids

feminization

me. In two p a t i e n t s ,

ae-a,

l e v e l s of

aetiocholanolone,

an-a,

( f o r adult

females)

of

f o u n d . The f a c t t h a t u r i n a r y a n - a , a f t e r removal of

the t e s t e s ,

the

syndro-

andro-

in urine

pseudohermaphrodite,

a e - a and a n d i e n - 3

indicates that these

administration of

human c h o r i o n i c

t h i s e f f e c t was shown e a r l i e r

decreased

67).

In a d d i t i o n ,

output of

an-a,

gonadotrophin

was

ACTH a d m i n i s t r a t i o n

evidence f o r

Biosynthetic

an a d r e n a l

studies

(22,

(HCG)

increased the 67),

proved that

(22,

urinary

(36),

as

compounds.

(36) w i t h t h e t e s t i c u l a r

tissue

f e m i n i z a t i o n and f r o m

labelled

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

(36);

providing

source f o r these

taken from the p a t i e n t s with t e s t i c u l a r t h e pseudohermaphrodite

thus

the after

i n men

a n d i e n - 3 and a e - a i n t h e p a t i e n t s

w e l l as i n normal men and women indirect

the patients

t o occur normally

were

compounds

s u p p o r t e d by t h e f i n d i n g t h a t t h e u r i n a r y e x c r e t i o n o f i n c r e a s e d i n one o f

were

16-androstenes

may have been f o r m e d i n t h e s e o r g a n s . T h i s p o s s i b i l i t y 16-androstenes

(36) in

(TF)

andien-3,

DHA and t e s t o s t e r o n e

I n a t h i r d p a t i e n t and i n a male

normal amounts

three

in

and Gower

s e v e r a l p a t i e n t s with the t e s t i c u l a r

elevated.

(30)

with

plasma.

have measured t h e e x c r e t i o n o f

sterone,

to

adrenal c o n t r i b u t i o n t o the

synthesize

approximately

(72)

androstadienone

The p o s s i b l e

androstene of

testosterone

p r e g n e n o l o n e was shown t o g i v e

(0.13-0.28%)

(, 483-484 (1 976). 30. Bicknell, D.C., Gower, D.B.: The development and application of a radioimmunoassay for 5a-androst-16-en-3a-ol in plasma. J. Steroid Biochem. ]_, 451-455 (1976) . 31. Andresen, 0.: Development of a radioimmunoassay for 5aandrost-16-en-3-one in pig peripheral plasma. Acta Endocr. (Kbh) 76, 377-387 (1 974) . 32. Bicknell, D.C.: The development and some applications of a radioimmunoassay for 16-androstenes. M.Phil. Thesis (University of London) (1976). 33. Bird, S., Gower, D.B.: Problems encountered in the radioimmunoassay of 5a-androst-16-en-3-one in human plasma. Abstract No. 18, Third Congress, European Chemoreception Organisation, Pavia, Italy. September 11th - 13th, (1978). 34. Brooksbank, B.W.L., Brown, R., Gustafsson, J.8.: The detection of 5a-androst-16-en-3a-ol in human male axillary sweat. Experientia 30, 864-865 (1974). 35. Ruokonen, A.: Free and sulphate-conjugated 16-unsaturated C 1 Q steroids in human testis tissue. Biochim. Biophys. Acta 316, 251-255 (1973).

23

36. Gower, D.B., Bicknell, D.C.: Steroid excretion and biosynthesis, with special reference to 16-unsaturated C.g steroids in cases of testicular feminization and in a male pseudohermaphrodite. Acta Endocr. (Kbh) 567-581 (1 972) . 37. Ahmad, N., Gower, D.B.: The biosynthesis of some androst16-enes from and C.g steroids in boar testicular and adrenal tissue. Biocheili. J. 1 08, 233-241 (1 968). 38. Booth, W.D.: Changes with age in the occurrence of C.g steroids in the testis and submaxillary gland of the boar. J. Reprod. Fert. 42, 459-472 (1975). 39. Hurden, E.L., Gower, D.B., Harrison, F.A.: Biosynthesis of 16-androstenes and androgens in boar testis in vivo. J. Endocr. 8J_, 161P-162P (1 979). 40. Dorfman, R.I.: A system for evaluating the functional status of the adrenal cortex. Metabolism 1_0, 902-91 6 (1 961 ). 41. Calvin, H.I., Lieberman, S.: Studies on the metabolism of 16a-hydroxy progesterone in humans: conversion to urinary 17-isopregnanolone. Biochemistry 1_, 639-645 (1 962). 42. Bulbrook, R.D., Thomas, B.S., Brooksbank, B.W.L.: The relationship between urinary androst-16-en-3a-ol and urinary 1 1-deoxy-17-oxosteroid excretion. J. Endocr. 2j6, 149153 (1963). 43. Wilson, H., Lipsett, M.B., Korenman, S.G.: Evidence that 16-androsten-3a-ol is not a peripheral metabolite of testosterone in man. J. clin. Endocr. Metab. 2_3, 491-492 (1963). 44. Ahmad, N., Morse, W.I.: Metabolites of tritiated testosterone in healthy men. Canad. J. Biochem. 4^, 25-31 (1965). 45. Wilson, H., Lipsett, M.B.: Metabolism of epitestosterone in man. J. clin. Endocr. Metab. 26, 902-914 (1966). 46. Burstein, S., Dorfman, R.I.: Biosynthesis of C.g steroids from 4-1^c-cholesterol and 7-3H-pregnenolone in vivo: Consideration of new pathways. Acta Endocr. (Kbh) 40^, 188202 (1 962) . 47. Brooksbank, B.W.L., Wilson, D.A.: Studies on the in vivo biosynthesis of C.q-Al6-steroids in healthy men. Steroidologia 1_, "113-128 (1 970). 48. Brophy, P.J., Gower, D.B.: 16-Unsaturated C^g 3-oxosteroids as metabolic intermediates in boar testis. Biochem. J. 128, 945-952 (1 972) . 49. Saat, Y.A., Gower, D.B., Harrison, F.A., Heap, R.B.: Studies on the biosynthesis in vivo and excretion of 16unsaturated C i q steroids in the boar. Biochem. J. 129, 657-663 (1 972 J.

24

50. Gasparini, F.J., Hochberg, R.B., Lieberman, S.: Biosynthesis of steroid sulphates by the boar testes. Biochemistry 1_5, 3969-3975 (1 976) . 51. Cooke, G.M., Gower, D.B.: The submicrosomal distribution in rat and boar testis of some enzymes involved in androgen and 16-androstene biosynthesis. Biochim. Biophys. Acta £98, 265-271 (1977). 52. Cooke, G.M., Gower, D.B.: Studies on the involvement of androst-16-enes and other steroids in steroid biosynthesis in boar testis. Biochem. Soc. Trans. 6, 1159-1162 (1978). 53. Cooke, G.M., Gower, D.B.: Attempted solubilization of boar testis microsomal 'Androsta-5,16-dien-3ß-ol Synthetase'. Biochem. Soc. Trans, (in press). 54. Rane, A., Gustafsson, J.8.: Formation of a 16,17-transglycolic metabolite from a 16-dehydro-androgen in human fetal liver microsomes. Clin. Pharm. Therap. 833-839 (1973). 55. Gustafsson, J.8.: The formation of 16,17-dihydroxylated C^g steroids from 16-dehydro C^g steroids in liver microsomes from male and female rats. Biochim. Biophys. Acta 296, 179-188 (1 973) . 56. von Bahr, C., Brandt, K., Gustafsson, J.8.: On the participation of cytochrome P-450 in the formation of 16,17-dihydroxylated C.q steroids from 16-dehydro-C.q steroids. 1 FEBS Lett. 25, 65-68 (1972). 57. Katzman, P.A.: Bioassay of estrogens by intravaginal injection in immature rats. Endocrinology 1 31-138 (1 965) 58. Knuppen, R., Breuer, H.: Biogenese von östratetraenol beim menschen. Acta Endoer. (Kbh) _42 , 129-1 34 (1963). 59. Knuppen, R., Breuer, H., Pangels, G.: Stoffwechsel von 2hydroxy-östradiol-(17ß) und 2-methoxy-östradiol-(17ß) in Geweben des Mensche n und det Ratte . Z . Physio 1 • Chein« 32 4 r 108-117 (1962). 60. Breuer, H., Knuppen, R.: The formation and hydrolysis of 16a,17a-epoxy-oestratriene-3-ol by rat liver tissue. Biochim. Biophys. Acta £9, 620-621 (1961). 61. Kingsbury, A.E., Brooksbank, B.W.L.: The metabolism in man of [3h]-5a-16-androsten-3a-ol and of [3H]-5a-16-androsten3-one. Horm. Res. 9, 254-270 (1978). 62. Mason, H.L., Schneider, J.J.: Isolation of A^®-androsten3(a)-ol from the urine of women with adrenal cortical tumors. J. Biol. Chem. 184, 593-598 (1950). 63. Miller, A.M., Rosenkrantz, H., Dorfman, R.I.: Unsaturated compounds in human urine. Endocrinology 5j3, 238-239 (1953). 64. Engel, L.L., Dorfman, R.I., Abarbanel, A.R.: Neutral steroids in the urine of a patient with luteoma of the ovary. J. Clin. Endocr. Metab. 1_3, 903-910 (1 953).

25 65. Gower, D.B., Stern, M.I.: Steroid excretion and biosynthesis with special reference to androst-16-enes, in a woman with a virilizing adrenocortical carcinoma. Acta Endocr. (Kbh) 60, 265-275 (1969). 66. Gregory, T., Gardner, L.I., Gower, D.B., Bicknell, D.C., Barlow, M.J.: Studies of 16-androstenes in an infant with virilizing adrenal carcinoma. Am. J. Dis. Child. 133, 294297 (1 979). 67. Brooksbank, B.W.L.: Urinary excretion of androst-16-en-3aol levels in normal subjects, and effects of treatment with trophic hormones. J. Endocr. 24, 435-444 (1962). 68. Collins, W.P., Forleo, R., Lefebvre, Y., Sommerville, I.F.: The transformation of isotopically labelled steroid substrates by the testicular tissue of patients with the feminine type of male pseudohermaphroditism. In:'Androgens in Normal and Pathological Conditions', Eds. Vermeulen, A., Exley, D., Excerpta Medica Foundation, Amsterdam, pp. 120129 (1 966) . 69. Inguilla, W., Forleo, R., Bruni, V.: Problems connected with in vitro biosynthesis in Stein-Leventhal ovaries. In: 'Androgens in Normal and Pathological Conditions', Eds. Vermeulen, A., Exley, D., Excerpta Medica Foundation, Amsterdam, pp. 114-119 (1966). 70. Sommerville, I.F., Collins, W.P.: Studies on the biosynthesis of ovarian steroids. Guy's Hospital Reports 118, 329-345 (1969). 71. Armstrong, A.A., Kadis, B.: Steroid dehydrations in porcine subcellular fractions. Steroids J_5, 737-749 (1970). 72. Gower, D.B., Bicknell, D.C.: Studies on the metabolism of C 9 1 and C 1 Q steroids in porcine ovarian preparations. Acta Endocr. (Kbh) Suppl. H 9 , 264 (1975). 73. Brooksbank, B.W.L., Pryse-Phillips, W. : Urinary A^-androst -3a-ol, 17-oxosteroids and mental illness. B.M.J. 16021 606 (1 964). 74. Brooksbank, B.W.L., MacSweeney, D.A., Johnson, A.L., Cunningham, A.E., Wilson, D.A., Coppen, A.: Androgen excretion and physique in schizophrenia. Brit. J. Psychiatry 1 17, 41 3-420 (1 970) . 75. Comfort, A.: The likelihood of human pheromones.In: 'Pheromones', Ed. Birch, M.C., North-Holland Publishing Co., Amsterdam and London, pp. 386-396 (1974). 76. Wiener, H.: External Chemical Messengers. 1. Emission and reception in man. N.Y. State J. Med. 66, 3153-3170 (1966). 77. Birch, M.C.: 'Pheromones'. North-Holland Publishing Co., Amsterdam and London (1974). 78. Lombard!, J.R., Vandenbergh, J.G.: Pheromonally-induced sexual maturation in females; regulation by the social environment of the male. Science, 196, 545-547 (1977).

26

79. Kalogerakis, M.G.: The role of olfaction in sexual development. Psychosom. Med. 25, 420-432 (1963) . 80. McClintock, M.K.: Menstrual synchrony and suppression. Nature, London, 229, 244-245 (1971). 81. Russell, M.J.: Human olfactory communication. Nature, London, 2j>0, 520-522 (1976). 82. Guillot, M.: Physiologie des sensations - Anosmies partielles et odeurs fondamentales. Compt. Rend. Acad. Sci. 226, 1307-1309 (1948). 83. Guillot, M.: Sur les mechanisms psycho-physiologiques de 1 'olfaction. J. Psychol. Norm. Path. 5jS, 1-20 (1958). 84. Kloek, J.: The smell of some steroid sex-hormones and their metabolites. Reflections and experiments concerning the significance of smell for the mutual relation of the sexes. Psychiat. Neurol. Neurochem. 64, 309-344 (1961). 85. Le Magnen, J.: Les phenomenes olfacto-sexuels chez l'homme. Arch. Sci. Physiol. 6, 125-160 (1952). 86. Vierling, J.S., Rock, J.: Variations in olfactory sensitivity to Exaltolide during the menstrual cycle. J. Appl. Physiol. 22, 311-315 (1967). 87. Whissell-Buechy, D., Amoore, J.E.: Odour blindness to musk: simple recessive inheritance. Nature, London, 242 , 271-273 (1973). # 88. Amoore, J.E., Popplewell, J.R., Whissell-Buechy, D.: Sensitivity of women to musk odor: no menstrual variation. J. Chem. Ecol. 291-297 (1975). 89. Griffiths, N.M., Patterson, R.L.S.: Human olfactory responses to 5a-androst-16-en-3-one - principal component of boar taint. J. Sci. Fd. Agric. 2J_, 4-6 (1 970). 90. 'Odour similarity between structurally unrelated odorants'. In: 'Taste and Smell in Vertebrates', Eds. Wolstenholme, G.E.W., Knight, J., J. and A. Churchill, London, pp. 313323 (1 970). 91. Amoore, J.E., Pelosi, P., Forrester, L.J.: Specific anosmias to 5a-androst-16-en-3-one and10-pentadecalactone: The urinous and musky primary odours. Chem. Senses and Flavour 2, 401-425 (1977). 92. Cowley, J.J., Johnson, A.L., Brooksbank, B.W.L.: The effect of two odorous compounds on performance in an assessment of people test. Psychoneuroendocrinology 2, 159-172 (1977). 93. Kirk-Smith, M., Booth, M.A., Carrell, D., Davies, P.: Human social attitudes affected by androstenol. Res. Commun. in Psychol. Psychiatr. and Behav. 3, 379-384 (1978).

27 94. Clark, T.: Whose Pheromone are You? World Medicine, July 26th, pp. 21-23 (1978). 95. Whitten, W.K.: Modifications of the oestrous cycle of the mouse by external stimuli associated with the male. J. Endocr. V3, 399-404 (1956). 96. Eibl-Eibesfeldt, I.: 'Ethology, The Biology of Behaviour1. Holt, Rinehart and Winston, New York, p. 497 (1975).

EFFECTS OF ESTROGENS AND ANTIESTROGENS ON HUMAN BREAST CANCER CELLS IN TISSUE CULTURE

J. C. Allegra, M. E. Lippman Clinical Investigations Branch and Medicine Branch, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, 20205, U.S.A.

The hormone-dependent nature of some human breast cancers has been appreciated since the pioneering work of Beatson more than 80 years ago (1). Since then, a variety of ablative and additive therapies have been used to induce clinically important remissions in patients with advanced breast cancer (2). Tumour regressions have also been produced in several animal model systems, especially the DMBA induced mammary cancer in rats (3). Nonetheless, data accumulated in clinical and animal studies have failed to define the mechanisms whereby treatment with estrogens and/or antiestrogens leads to tumour regression in some patients. In vivo studies of hormonal effects are difficult to interpret because hormonal pertubations change the levels and/or activity of other trophic factors. Also, indirect hormonal effects on supporting stroma are not readily separable from direct effects on the malignant cells, and indeed, the interaction of the malignant cell and its adjacent stroma may alter the response to physiologic and pharmacologic agents. An alternative approach is to study cultures of human breast-cancer cells, in which specific growth conditions and hormonal milieu can be controlled. This review describes our own studies. It is hoped that an understanding of hormone action in such cultured cells will clarify the mechanisms of hormone action in patients with breast cancer. The two major human breast cancer cell lines used in our studies were the MCF cell line (provided by Dr. M. Rich of the

Hormones in Normal and Abnormal Human Tissues © Walter de Gruyter • Berlin • New York 1981

30 Michigan Cancer Foundation) and the ZR-75-1 cell line (established and propagated by Dr. N. Young and Ms Linda Engel of the National Cancer Institute) (4, 5). Both lines are epithelial and possess microvilli, secretory granules, Golgi apparatus, rough endoplasmic reticulum and desmosomes. They each possess a human karyotype, which resembles that of the original tumour. The cells have receptors for estrogen, progesterone, androgen, glucocorticoid, insulin, thyroid hormone, retinoids and, in the case of MCF-7 cells, epidermal growth factor. The two cell lines are free of any mycoplasma contamination, are cloned, and have been passaged multiple times for more than 3 years in our laboratory . Figure 1 shows the effect of estrogen and anti-estrogens on the MCF-7 human breast cancer cell line. When estradiol was —8 added to the medium at a concentration of 10 M there was a twofold increase in [ H] thymidine incorporation into DNA. Tamoxifen (ICI-46474), a triphenylethylene derivative which blocks estradiol action, inhibited DNA incorporation. When estradiol, at a lower concentration than Tamoxifen, was added to the cells

E s t r a d i o l 1 0i " ' l« VI

T a m o x i f e n 10 +

M

Estradiol 10"8 M

0 FIG. 1. The effects of estrogen human breast cancer in vitro.

and Tamoxifen on thymidine incorporation in

31

together with Tamoxifen at 10 ^M, the inhibitory effects of Tamoxifen were completely obliterated. All incubations were done in 3% serum from which virtually all endogenous steroid was removed by stirring the serum at 55°C with dextran-coated charcoal. Removal of steroid was monitored by adding a trace 3 of [ H]-estradiol at the beginning of the procedure and following the decrease to less than 1% of radioactivity — in8 the supernatant. If untreated serum was used, addition of 10

M estra-

diol did not stimulate the cells. This lack of effect was presumably due to the cells being maximally stimulated by endogenous estradiol in the serum. This may explain many previous failures to demonstrate hormonal responses in in vitro systems (6-9). Tamoxifen strongly inhibited DNA incorporation even when untreated serum was used, but the effect was completely blocked —8 by the addition of 10

M estradiol.

The proliferative effect of estradiol on MCF-7 cells in serum-free medium was demonstrated by the addition of as little as 10— 11M estradiol, and was maximal at 10 — 9M to 10 — 8M estra3 diol (10-11). Incorporation of [ H]-thymidine into acid precipitable material was usually stimulated 1-3 fold after incubation for 24 or 48 h with optimal concentrations of estradiol. Leucine incorporation measured under identical conditions and cell growth measured by daily cell counts over a 1 week period, indicated a lesser, but significant, stimulatory effect of —8

10 M estradiol (10). Concomitant with the increased rate of thymidine incorporation, a comparable increase in the activity of the salvage pathway enzyme, thymidine kinase, was observed (12). Whether or not stimulation of this enzyme activity by estradiol is a critical step in the subsequent growth response of the cells remains to be determined. The progesterone receptor has been shown to be regulated by estradiol in estrogen target tissues such as uterus (13). Horwitz and McGuire found similar results in dimethylbenz(a) anthracene-induced rat mammary tumours (14) and demonstrated that estradiol increases progesterone binding in MCF-7 cells (15).

32 Thus, MCF-7 human breast cancer cells in culture possess estrogen receptors and respond to physiologic concentrations of estrogen with increased macromolecular synthesis, increased thymidine kinase activity and progesterone binding activity as well as increased growth rates. In these earlier experiments, Tamoxifen inhibition of MCF-7 cells below control levels was difficult to interpret. If Tamoxifen acted solely by competing with available estradiol for receptor sites one would not expect inhibition of cell growth below control levels. This suggests that Tamoxifen has some other action in addition to blocking effects mediated through the estrogen receptor. However, —6

10

M Tamoxifen had no effect on thymidine incorporation in the

estrogen receptor deficient MDA-MB-231 cells, and thus did not appear to be non-specifically toxic. If Tamoxifen (10—6M) was given alone for up to 48 h, its effects were completely rever—8

sed by addition of 10

M estradiol. Continuous administration

beyond 48 h, however, was apparently lethal to most MCF-7 cells. One possible explanation for these unusual effects of Tamoxifen has recently been proposed by Zava and McGuire (16, 17). They suggested that the unoccupied nuclear estrogen receptor was able to stimulate estrogenic responses in the absence of estrogen. Tamoxifen may be able to bind to and inactivate this nuclear estrogen receptor which would account for the observed inhibition of thymidine incorporation below control levels. Furthermore, since Tamoxifen appears to be lethal if present for more than 48 h, the activity or gene products resulting from the unoccupied receptor may be critical to cell survival. Obviously, there are many interesting implications of such a hypothesis, yet further speculations should await proof that the unoccupied nuclear estrogen receptors are indeed active. An alternate hypothesis is that Tamoxifen binds to receptor, translocates to the nucleus leading to some alteration in cell function and ultimately to cell death. It is important to note that these experiments were performed with cells maintained and passaged in medium supplemented with charcoal-treated calf serum (CCS) but the stimulation

33 experiments were performed without serum. Although the use of CCS ensures a low concentration of estrogen, prolonged cell growth is not supported in the absence of serum and both controls and estrogen stimulated cells eventually die despite obvious differences between the two. The requirement of virtually all established cell lines for serum has hampered the investigation of many aspects of hormone action. Serum contains growth factors, both known and undefined and a number of different hormones. These hormones and/or growth factors may cloud interpretation of results. Medium containing serum also leads to difficulties in evaluation of hormone receptors of the cells. Two recent papers (17, 18) reported that, in MCF-7 cells grown in CCS, 75% of the total estrogen receptors existed in the unoccupied nuclear form. Cells grown in the presence of estradiol contained exclusively occupied nuclear receptors. Also, since the progesterone receptor of MCF-7 cells is regulated by estradiol, cells grown in serum containing estrogens have higher concentrations of progesterone receptor than cells grown in CCS which contains very small amounts of estrogen (19). It is obvious that a more ideal system for the study of hormone action would include human breast cancer cells growing in long term tissue culture in a totally defined medium without serum supplementation. The availability of a human breast cancer cell line which can be propagated in hormone supplemented medium without serum should aid in the study of the mechanisms by which hormones effect cell proliferation. It is possible to grow cell lines in medium without serum supplementation provided the culture medium is supplemented with hormones and other factors (20, 21). For example, Hayashi et al (22) have shown that the GH^ rat pituitary cell line will grow in Ham's nutrient mixture F-12 supplemented with triiodothyronine, thyrotropin releasing hormone, transferrin, parathyroid hormone, insulin, fibroblast growth factor and somatomedin C. This cell line and several other cell lines are able, even after long-term culture, to be adapted to serum-free growth (23, 24). We have not been able to grow the MCF-7 cell line

34

under serum free conditions in our laboratory. However, we have shown (5) that the ZR-75-1 human breast cancer cell line can be grown in hormone-supplemented medium without serum (25). The factors required for optimal growth, equivalent to that seen in serum supplemented medium, are estradiol, insulin, transferrin, dexamethasone, and triiodothyronine. Thus far most cell lines growing in a hormone-supplemented medium have required insulin and transferrin although their effect on growth at identical concentrations varies among the cell lines. Also, each cell line appears to require a hormone which localizes in the nucleus such as triiodothyronine or a steroid (21, 22). Figure 2 illustrates that ZR-75-1 human breast cancer cells in serum-free hormone-supplemented medium (IMEM-HS) grow rapidly at a rate equivalent to that of cells in medium supplemented with optimal concentrations of serum. This was achieved by ad—7 —8 ding estradiol (10 M), porcine and beef insulin (5 x 10 M), —8 —8 triiodothyronine (10 M), dexamethasdne (10 M) and transferrin (1 |ig/ml) to the medium (26). Nucleosides, non-essential amino acids and fibroblast growth factor were also added when the

0

1

2

3

4

5

6

7

8

9

10

FIG. 2. Growth of the ZR-75-1 cell line in hormone supplemented medium without serum. Cells were plated at a density of 50,000 cells/dish in minimal essential medium supplemented with 5% charcoal treated calf serum. On day 1, the medium was changed to IMEM-HS (Ol and IMEM + 5% fetal calf serum (•).

Arrows

indicate days on which the cells were refed with fresh medium. Standard deviations of triplicate cell counts shown are generally less than 10%.

35

DAY FIG. 3. Requirements for free serum growth of ZR-75-1 human breast cancer cells. Cells were plated at a density of 50,000 cells/dsh in minimal essential medium supplemented with 5% charcoal treated calf serum. On day 1, the medium was changed to IMEM-HS — 170 estradiol

«

, IMEM-HS minus insulin

IMEM-HS minus transferrin

•

, IMEM

• •

• — ,

, IMEM-HS minus T j

IMEM-HS minus O

,

Arrows indicate days on which the cells

were refed with fresh medium. Standard deviations of triplicate cell counts are generally less than 10%.

cells were subcultured to improve plating efficiency. The effects of each of the hormones and transferrin on growth is shown in Figure 3. The cells in IMEM-HS grew rapidly over the 14 day period. Control cells in IMEM alone remained viable for 4-7 days as judged by attachment to the plastic tissue culture dishes, and then detached and died as do cells in IMEM-HS which lacks transferrin. Cells in IMEM-HS minus either estradiol, insulin, or triiodothyronine grew increasingly more slowly for 7 days and then became static although viability was maintained for _ g 14 days. Dexamethasone (10 M), nucleosides, non-essential amino acids, fibroblast growth factor (0.025 mg/ml), dihydro— 8

testosterone (10

M), human placental lactogen (1 mg/ml), oxy-

tocin (1 mp/ml), or vasopressin (1 m^/ml) had no further effect on growth. The establishment of this cell line growing optimally without the unknown effects of serum factors will enable investigators to better study the mechanisms of hormone interaction with breast cancer.

36 THE EFFECT OF ESTRAOIOL AND TAMOXIFEN ON GROWTH OF ZR-75-1 H U M A N BREAST CANCER CELLS GROWING IN IMEM-HS M I N U S 17/! ESTRADIOL 140

120

1 0 - E¡ '

100

?? /

/

s

,

10 " E 2 E,

/

/

» 10 ' F; IMEM-HS Minus E ,

' M E M - H S Minus E ,

40

VS.

20 0 -ii t

P l ^ x

-10 t

-4 t

- 2

t

- 1

r DAYS

RG. 4. The aftea of 17/J nmdol and tamoiitai on Ihe ZB-75-1 huían brast ancv n k Cris « n ptated ¡n MBA suHJhmatel wüh 5 * CCS. Ths medun wss udmiged daüy for 3 day& On dav -í, the nwdum was ctanged to IMEM-HS mnus E ; d ñ medun «ws euftanged driy f v 8 dayi On dsy 0. Nríous u m m u m i m d BMndd and 10 -* M (amonta) m n addad to the cafe. Airare iafcata days on wtádi Ihe ofe wwa retad with hedí medum. Standard denations of tripfcate a * counts an isualy las than 10%.

Figure 4 illustrates the effects of various concentrations of estradiol added to cells growing in IMEM-HS. The cells were plated in MEM plus 5% CCS and this medium was exchanged daily for 3 days. The medium was then changed to IMEM-HS minus estradiol and this medium was exchanged daily for 8 days. This long pretreatment of the cells with charcoal treated calf serum and estradiol free medium is required to completely remove estradiol from these cells. Cells treated with [ H] estradiol and cultured in medium which was exchanged daily for fresh serum-free medium lacking estradiol required 10-14 days for depletion of retained radioactivity (27). In experiments on another cell line, MCF-7, the retained radioactivity was virtually all estradiol, which was specifically bound and largely localized in the nuclear fraction (28). In addition, either charcoal treated serum, bovine serum albumin, antiestrogen, or unlabelled estradiol addition significantly accelerated loss of specifically retained estradiol. On day 0 various concentrations of estradiol —7

(10

— 1 9

- 10

M) or 10

— fi

M Tamoxifen were added. Initially, the

cells in IMEM-HS without estradiol grew slowly and eventually

37

ceased net growth. It is important to note that while there is no change in the number of cells deprived of estradiol, this is not due simply to a cessation of growth for the following reasons: First, estradiol free cells continue to incorporate thymidine though at a lower rate than hormone treated cells; second, if the cells are prelabelled with thymidine there is a loss of radioactivity into the medium in estrogen deprived cells but not in estrogen treated cells (estrogen treated cells, 1% loss; estrogen deprived cells, 60% loss); third, there is an obvious decrease in cell adhesiveness in estrogen deprived cells and detached cells are easily seen in the medium. Thus, it is likely that the ZR-75-1 cells are capable of low growth in estrogen free medium, an effect masked by continued cell loss from the dish and replenishment. Addition of 10-7 - 10-11M estradiol restored growth and adhesiveness of these cells with -9 -10 M being the optimal concentration. No effect was 10 - 10 — 6 — 12 seen with 10 E^. Addition of 10 M Tamoxifen led to cell death. Figure 5 shows a second growth experiment in which the interaction of estrogen and anti-estrogen was examined in more

Rc(E) Incubation Time

Fig. 2. Kinetics of ER distribution after estradiol or antiestrogen treatment. Cells were treated as in Fig. 1 with estradiol (•, 10 nM), tamoxifen (o, 0.1 uM), or nafoxidine (x, 1 txM) ; ER measured by protamine sulfate precipitation. (From 93).

98

Fig. 3. Effect of varying hormone doses on total ER processed. Cells were treated 4 days with the doses shown. Then cells from 4 flasks were pooled, homogenized and cytoplasmic and nuclear ER were measured by the single saturation dose protamine exchange assay. Total cell ER is the sum of unfilled cytoplasmic (Rc, 4°C) and total nuclear sites (filled plus unfilled, 30°C). (Adapted from 93). There appears to be a limit to the amount of receptor loss, however, so that at higher doses processing stops and RnE levels stabilize. With increasing doses of tamoxifen, some processing of total ER occurs. However, the number of sites lost represent only 30% of total and never approach the extent seen with estradiol even at the highest tamoxifen doses. No processing at all is seen with nafoxidine at any dose (93). C. Effect of antiestrogen dose on PgR induction With antiestrogens, as with estradiol, processing parallels PgR induction. Fig. 4 shows that tamoxifen is a potent inducer of PgR. While low doses have only minimal effects, at intermediate doses PgR induction equals or exceeds that obtained with estradiol. When doses are raised further, PgR levels are suppressed even below control levels. This high (1 |j,M) tamoxifen

99

Fig. 4. Estrogenic effects of antiestrogens: effects on PgR induction. Cells were treated 5 days with nafoxidine (•) or tamoxifen (A) at the doses shown and progesterone receptors were measured by dextran-coated-charcoal assay. dose is markedly antiestrogenic; at this dose but not at lower ones cell growth is inhibited and leads to cell death. Nafoxidine, in contrast with tamoxifen, has little or no effect on PgR at any dose studied. The slight increase at high doses may represent an effect on another receptor (45). These results show that in breast cancer cells of human origin, the ER system mediates antiestrogen action. Antiestrogens bind and translocate unfilled cytoplasmic ER. In these respects estrogen antagonists resemble estradiol. However, the subsequent nuclear processing reactions of estrogen and antiestrogen-bound receptors are dissimilar. After estradiol, nuclear hormone-receptor complexes fall rapidly to less than one third of control values. This pathway of receptor processing is either impaired (tamoxifen) or fails entirely (nafoxidine) for the antiestrogen-receptor complex. Our results suggest that

100

processing is an active step in ER function at least in the special case of PgR induction and does not simply serve to return receptor to the cytoplasm. This step appears to be defective when antiestrogens bind the receptor. With estradiol and tamoxifen, processing of receptor occurs despite the continuous presence of the hormone. This may differ from the rat uterus where Clark et al (96) have shown that if estradiol is administered to the rat so as to maintain elevated blood levels of the hormone, nuclear receptors rise to very high levels. Thus, significant differences are found in the early nuclear reactions of the estrogen receptor-hormone complex of human tumour cells compared to the rat uterus, the usual model of estrogen action. Other tissue differences in mechanisms of ER action have also been reported (97) , suggesting perhaps that studies of estrogen action in uteri cannot always be extrapolated to other tissues. Estrogenic and antiestrogenic responses in the rat uterus are characterized as early ( deoxycorticosterone

>

18-hydroxydeoxycorticosterone,

in keeping with relative physiological potencies, they give the false impression that all corticoid molecules are bound to the one and the same component of the receptor. In fact,

separation

of rat kidney MR on DE-52 resins has clearly established they saturate MI^, MR^ and MR^ , respectively

that

(1-3, 9). Recently

we have shown that progestagen antagonism of mineralocorticoid action proceeds via progestin association to MR^ at a time when sites for agonist attachment on MR are concurrently In view of these considerations, it was somewhat

available.

surprising

165

that mineralocorticoid agonist and antagonist action in the human kidney may proceed exclusively via MR^ (Figs. 3, 4). Furthermore, human renal MR. did not bind R-5020 which labels rat kidney MR^ with great avidity (10). Thus, extrapolations from animal to human are hazardous. Furthermore, saturation characteristics would give a false impression of similarity between the rat and the human kidney MR. The species specific differences were not observed however, with liver GR (Figs. 7, 81 7, 8) . What may be the physiological relevance of such studies ? Absence of aldosterone binding during renal cortical atrophy associated with kidney stones or hydronephrosis would suggest that the absence of receptors is compatible with diminution of physiological function. Neoplasia may diminish receptor function slightly or totally (Fig. 2) but this could not be diagnosed by morphological criteria (Fig. 1). With breast cancer, steroid dependent tumours have been shown to benefit from antioestrogen therapy (11). Further studies may reveal that other types of diseases in other tissues may similarly be exploitable by antagonist specific chemotherapy. Indeed, the older physiological observation of progestagen antagonism of aldosterone action has only recently found a defined basis at the receptor level (10, 12). The differences between GR in normal liver and liver metastases (Fig. 8) leaves hope for possible exploitation of GR sites as an adjunct of therapy.

Acknowledgements This work was aided by grants from the CNRS (AI 03 1917) and UER Broussais-Hôtel Dieu. For surgical specimens thanks are due to: Service of Surgery (Pr. Alexandre), Hôpital Broussais; Service of Urology (Pr. Steg), Hôpital Cochin; Service of Urology, (Dr. Brisset), Hôpital St. Joseph; and Service of Nuclear Medicine (Pr. Baillet), Hôpital Broussais. For technical assistance and illustrations, thanks are due to Ms. M. Philippe.

166

References 1. Agarwal, M.K.: Ed. Multiple Molecular Forms of Steroid Hormone Receptors, Elsevier/North Holland,(1 977) . 2. Agarwal, M.K.: Ed. Antihormones, Elsevier/North Holland, (1 979) . 3. Agarwal, M.K.: Ed. Proteases and Hormones, Elsevier/North Holland, (1979). 4. Kornel, L.: On the Effects and the Mechanism of Action of Corticosteroids in Normal and Neoplastic Target Tissues: Findings and Hypotheses. Acta Endocrinologica, Suppl. 178, 7-45 (1973). 5. Fuller, P.J., Funder, J.W.: Mineralocorticoid and Glucocorticoid Receptors in Human Kidney, Kidney International 10, 154-157 (1976). 6. Matulich, D.T., Spindler, B.J., Schambelan, M., Baxter, J.D.: Mineralocorticoid Receptors in Human Kidney. J. Clin. Endocrinol. Metab. £3, 1170-1174 (1976). 7. Agarwal, M.K.: Human Liver Corticosterone Receptors. Biomedicine Express 25^, 73-74 (1 976). 8. Agarwal, M.K.: Human Liver Glucocorticoid Receptors are Similar to those in Rat Liver. Die Naturwissenschaften 63, 50 (1976). 9. Agarwal, M.K.: Physical Characterisation of Cytoplasmic Gluco- and Mineralo- Steroid Receptors, FEBS Letters 85, 1-8 (1978). 10. Agarwal, M.K., Paillard, J.: Paradoxical Nature of Mineralocorticoid Receptor Antagonism by Progestins. Biochem. Biophys. Res. Comm. 89, 77-84 (1979). 11. Leclercq, G., Heuson, J.C., Mettheiem, W.H.: Oestrogen Receptors in Human Breast Cancer. Br. J. Cancer 3£, 177 (1974). 12. Landau, R. ? Progesterone vs Aldosterone. In: "Antihormones", Ed. Agarwal, M.K., Elsevier/North Holland, pp. 153-166 (1979).

hCG AND hCG-LIKE SUBSTANCES IN NORMAL AND ABNORMAL HUMAN TISSUE

B. B. Saxena and P. Rathnam Departments of Medicine and Obstetrics and Gynecology, Cornell University Medical College, 1300 York Avenue, New York, N.Y. 10021, U.S.A.

Introduction Human chorionic gonadotrophin (hCG) is a glycoprotein hormone synthesized and secreted by the syncytiotrophoblast cells of the placenta and is detected in the urine and serum of pregnant women. The main function of hCG early in pregnancy, is to act as a stimulus to the corpus luteum to continue to secrete steroid hormones. The role of hCG in stimulating fetal testicular androgens has also been postulated (1-4). The development of highly specific and sensitive radioimmunoassays (RIA) of hCG using antiserum raised against the hormone-specific 3-subunit (5), and the radioreceptor assay (RRA) of hCG by the use of hCG-specific receptors (6), has provided a reliable detection of as little as 1 mlU hCG. By the use of the radioassays, hCG has not only been detected earlier during pregnancy and trophoblastic disease, but also in patients with ectopic tumours as well as in embryonic cells such as blastocyst and sperm and in the blood and tissue of normal non-pregnant subjects. hCG as well as large immunologic forms of hCG, hCG-a and hCG-3 have also been identified in human chorionic tissues as well as in cancer cell lines cultivated in vitro (7-9). These large forms may represent precursors, which may then be converted to smaller molecular weight species.

Hormones in Normal and Abnormal Human Tissues © Walter de Gruyter • Berlin • New York 1981

168

Chemistry hCG has been isolated from a commercial preparation from first trimester-pregnancy urine and contains a biological activity of 11,000-13,500 IU/mg (10-12). All preparations of hCG appear to be heterogeneous in structure at the NH~—terminal region of the molecule and in carbohydrate content. The carbohydrate moiety appears to be essential for the biological activity of hCG, as removal of sialic acid drastically reduces the metabolic halflife from hours to minutes, because of the binding of asialoglycoproteins to receptors on liver cell membranes, which recognize the exposed underlying galactose residues (13). The removal of the galactose residues restores some of the biological halflife since the liver receptors mainly bind the exposed galactose residues. Removal of sialic acid, however, only slightly diminishes the potency of hCG when assayed by in vitro or bio-assays, RIA or RRA (14, 15). Stepwise removal of sugar does not affect the hCG binding to receptors as much as it reduces the ability of the bound hormone to activate adenyl cyclase. The removal of carbohydrate thus reduces the ability of hCG to maximally stimulate cAMP accumulation as well as steroidogenesis (15). hCG consists of a hormone-nonspecific a-subunit and a hormone-specific p-subunit. The primary amino acid sequence of both the subunits of hCG has been established and the carbohydrate structures have been proposed. The a-subunit of hCG is almost identical to the a-subunits of hFSH, hLH and hTSH and can recombine with the 3-subunits of hFSH, hLH and hTSH to regenerate the respective hormonal activity. The [3-subunits of these hormones on the other hand, show differences. The primary amino acid sequence of hCG-ft has 80% homology with that of hLH-3. hCG-3, however, contains additional 30 amino acids at the C-terminal as shown in Diagram 1. Both LH and hCG bind to the same receptor and have similar biological activity. The antisera produced against hCG or hLH do not discriminate one hormone from the other. This high degree of similarity between hCG and LH accounts for the difficulties

169 10

hCG-3: Ser-Lys-Glu-Pro-Leu-Arg-Pro-Arg-Cys-Arg-Pro-IlehLH-3: Ser-Arg-Glu-Pro-Leu-Arg-Pro-Trp-Cys-His-Pro-Ile20

hCG-3: Asn(CHO)-Ala-Thr-Leu-Ala-Val-Glu-Lys-Glu-Gly-Cys-ProhLH-fJ : Asn(CHO)-Ala-Ile-Leu-Ala-Val-Glu-Lys-Glu-Gly-Cys-ProhCG-3: Val-Cys-Ile-Thr-Val-Asn(CHO)-Thr-Thr-Ile-Cys-Ala-GlyhLH-3: Val-Cys-Ile-Thr-Val-Asn(CHO)-Thr-Thr-Ile-Cys-Ala-Gly40 hCG-3: Tyr-Cys-Pro-Thr-Met-Thr-Arg-Val-Leu-Gln-Gly-ValhCG-3: Tyr-Cys-Pro-Thr-Met-Arg-Met-Leu-Leu-Glx-Ala-Val50 60 hCG-3: Leu-Pro-Ala-Leu-Pro-Gln-Val-Val-Cys-Asn-Tyr-ArghLH-3: Leu-Pro-Pro-Val-Pro-Gln-Pro-Val-Cys-Thr-Tyr-Arg70 hCG-3: Asp-Val-Arg-Phe-Glu-Ser-Ile-Arg-Leu-Pro-Gly-CyshLH-p: Asx-Val-Arg-Phe-Glx-Ser-Ile-Arg-Leu-Pro-Gly-Cys80

hCG-p: Pro-Arg-Gly-Val-Asn-Pro-Val-Val-Ser-Tyr-Ala-ValhLH-p: Pro-Arg-Gly-Val-Asp-Pro-Val-Val-Ser-Phe-Pro-Val90 hCG-3: Ala-Leu-Ser-Cys-Gln-Cys-Ala-Leu-Cys-Arg-Arg-SerhLH-p: Ala-Leu-Ser-Cys-Arg-Cys-Gly-Pro-Cys-Arg-Arg-Ser100

hCG-3: Thr-Thr-Asp-Cys-Gly-Gly-Pro-Lys-Asp-His-Pro-LeuhLH-3: Thr-Ser-Asp-Cys-Gly-Gly-Pro-Lys-Asx-His-Pro-Leu110

120

hCG-3: Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-SerhLH-p: Thr-Cys-Asx-Glx-Asx-Ser-Lys-Gly 130 CHO hCG-p: Ser(CHO)-Lys-Ala-Pro-Pro-Pro-Ser(CHO)-Leu-Pro-Ser-Pro-SérhLH-p: 140 hCG-p: Arg-Leu-Pro-Gly-Pro-Ser(CHO)-Asp-Thr-Pro-Ile-Leu-ProhLH-p: hCG-3: G

Diagram 1.

Amino Acid Sequences of hCG-3 and hLH-p

in the specific measurement of one hormone in the presence of the other. Efforts have, therefore, been made to raise specific antisera against the unique C-terminal fragments of hCG-p as well as against the chemically modified intact hCG-3 to permit the selected measurement of hCG in the presence of LH by RIA. An antisera produced against the chemically synthesized

1 70 C-terminal peptide only binds hCG or hCG-like immunoreactive material. Antibodies that are specific for determinants in the last 15 residues of hCG-3 appear to cross react with native hCG-3, although they do not neutralize the biological activity. The biological determinants do not involve the C-terminal region (10). The choriocarcinoma-hCG and standard-hCG are antigenically similar (16) and have similar amino acid composition (17). A N-terminal analysis of the hydatidiform mole-hCG show Ser and Ala similar to that reported for the standard hCG (18). The hydatidiform mole-hCG is also identical to the standard hCG in immunodiffusion and Immunoelectrophoresis studies. The hydatidiform mole-hCG contains 24,400 IU/mg in bioassay and 4090 IU/ mg in radioimmunoassay. In SDS-polyacrylamide electrophoresis, the molecular weight for the hydatidiform mole-hCG is estimated to be 51,000, for its a-subunit 20,000 and for its 3-subunit 31,000. The carbohydrate constituents of the hydatidiform mole-hCG are similar to the standard hCG, however, with slightly lower amounts of mannose and hexosamines (Table 1) (18, 19). hCG and its subunits derived from placenta, choriocarcinoma, ectopic tumours and cell cultures exhibit heterogeneity.

Function and Biosynthesis hCG, like LH, stimulates the interstitial cells of the preformed follicles converting it into a corpus luteum in the female rat, and the interstitial cells of the testis to secrete androgens in the male rat (22). In the human, hCG stimulates the luteinized cells to produce progestins in early pregnancy that are necessary to support the endometrium (22). Recent studies on the presence of hCG-like material in rabbit blastocysts (23) have suggested a role of this substance in the maintenance of corpus luteum function prior to implantation. Recent results (24) show that hCG has a direct effect on the median eminence in inhibiting the synthesis and release of

171

Table 1. Composition of Choriogonadotrophins. hCG-hydatidiform

hCG (20)

mole (18)

hCG-choriocarcinoma (21)

Based on 4 histidine residues/molecule Amino Acid Lysine Histidine Arginine Aspartic acid

10.0

4.0 10.6

9.3 4.0

10.1

14.2 16.1 15.6 17.9 16.8

14.3

4.0

Glycine

16.9 16.8 16.8 19.3 25.6 13.3

26.3 11.6

17.1 27.4 9.7

Alanine

12.2

11.6

10.9

Half-cystine

11.. 1 17.1

18

17.7

15

16.2

3.5 6.3

3 5 13 5 5

3.6 5.5

Threonine Serine Glutamic acid Proline

Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

15.6 6.3 7.6

Carbohydrate Fucose Galactose

0.4 6.7

Mannose 2.1 N-Acetylgalactosamine 0.5 N-Acetylglucosamine

5.4

Sialic acid Total carbohydrate

6.1 21.2

0.6 5.3 5.3 2.2 8.9 9.0 31 .3

15.6 15.8 17.9

13.7 5.7 5.3

172

FSH and LH. Thus hCG may play a role in the inhibition of ovulation during pregnancy. hCG appears to be the primary stimulus to the fetal Leydig cells which results in testosterone secretion with a maximum during weeks 11-17 of pregnancy and in the differentiation of the male genital tract (25) . The human fetal testis of 16-20 weeks can bind hCG and respond with maximal testosterone production to physiologic levels of hCG in vitro (26). A weak TSH-like activity (27) and a weak FSH-like activity (28) have also been described as intrinsic to the native hCG molecule. A proposed sequence of secretion of hCG in malignant trophoblast cells consists of its production in the rough endoplasmic reticulum (or perinuclear membrane), movement through the cisternae of the endoplasmic reticulum and release from the cell surface (29). Studies in cancer cell lines have indicated that the anti-hCG-3 reactive material is localized predominantly on the plasma membrane of malignant cells. The biosynthesis of hCG is currently being studied in several laboratories. Even though a large excess of a-subunits are found in serum and placenta during pregnancy, information on the details of biosynthesis and assembly of the subunits are at present limited. Using cell-free synthetic techniques, Birken and Canfield (10) report that the hCG-a subunit is synthesized as a precursor with a 24 amino acid addition at the Nl^-terminal of the precursor molecules. The precursor hCG-a synthesized in the ascites system, is cleaved to yield its native hCG-a form in the presence of a membrane extract containing the specific cleavage enzyme. The plant lectin Con-A which binds to specific carbohydrate groups on the cell membrane (30) stimulates hCG and hCG-a secretion by cultured human choriocarcinoma cells (31). The stimulation is prevented by a-methyl-D-mannopyranoside. The gradual decline of the hCG level with time in a monolayer culture of trophoblast cells is retarded with theophylline and cAMP. Exposure to methotrexate (MTX) does not increase hCG secretion in normal trophoblast cells in contrast to a five-fold

173

stimulation of MTX in the JAR line of choriocarcinoma cells (32). There is evidence for the de novo biosynthesis of hCG by the malignant cell.

Measurement a) Bioassays. Aschheim and Zondek in 1927 first introduced qualitative biological detection of hCG which was later modified as the A-Z mouse test, rat hypermia test, Friedman test, and the frog and toad tests (33) . The quantitative-biological determination of hCG has been performed by the use of the ovarian hypermia assay, by graded ovarian increments in weights of ovaries, uterus, seminal vesicles and ventral prostates, in mice or rats as well as by depletion of ascorbic acid content of ovaries of pseudopregnant rats. These bioassays of hCG are, however, relatively insensitive and cumbersome and measure both LH and hCG-like activity simultaneously (34, 35). b) Immunoassays. By 1960, sufficient purification of the hCG molecule had been achieved so that a potent anti-hCG serum could be raised in rabbits. The use of hCG coupled to red cells and latex particles provided the basis of the hemeand latex-agglutination inhibition slide or tube tests (36, 37). The simplicity and reduced cost of heme- and latexagglutination tests has significantly improved the clinical availability of hCG detection as an index of pregnancy. The sensitivity of the immunological tests is not significantly greater than that of the bioassays and thus a large number of early normal pregnancies, prior to six weeks after the last menstrual period, threatened and missed abortions, 50% of ectopic pregnancies, and trophoblastic tumours, with levels of hCG below 500 mlU/ml cannot be detected (35).

174

c) Radioimmunoassay. The introduction of radioimmunoassay techniques by Yalow and Berson (38) has resulted in the development of the RIA for hCG with a sensitivity of 6 IU/litre. The RIA, however, requires 48 to 72 h of incubation to achieve the higher sensitivity. The cross reaction between hCG and LH, especially when low levels of hCG are present, is overcome by the use of an antiserum raised against the hormone-specific, but biologically inactive, hCG-p subunit (5) . The hCG-fJ antiserum recognizes intact hCG and its f5subunit but not hLH. The affinity of such antisera also varies for hCG and hCG-p from batch to batch. Fragments of hCG-3 immunologically compatible amino acid sequences also cross-react with the antibody. The hCG-3 subunit and its fragments are essentially devoid of biological activity, hence, interference by these immunoreactive materials necessitates biological, physiological and clinical validation of the radioimmunoassay values. Different antisera detect varying degrees of non-specific interference and thereby give different values for serum hCG. (Tables 2 and 3). Hence, each antiserum requires a different cut-off point to detect clinically meaningful levels of hCG, especially in early pregnancy or in conditions where the levels of hCG are low. The nonuniformity in the sensitivity and specificity of the antibody in the estimation of varying levels of hCG in the same samples require extreme caution in the comparison of the interlaboratory data, especially when low levels of the hormone are present (35). The immunization of animals with hCG and its 3-subunits, followed by careful selection of specific antibodies has made it possible to measure hCG in the presence of LH (39). A receptorimmunoassay (40) is described for the evaluation of the specificity, affinity and the ability of anti-hCG-p sera. d) Radioreceptorassay (RRA). The presence of specific binding sites for hCG in the plasma membranes of cow corpora lutea (41) has been used in the development of a rapid, sensitive

175

Table 2. Variability in Serum hCG Levels Measured by Different Antisera to hCG-3 hCG (Ng/Ml) Subjects

Antisera I Mean S.E.

Antisera II Mean S.E.

Antisera III Mean S.E.

RRA Mean S.E.

Females Gonadal Tumours Occult Pregnancy Males Testicular Tumours

4.0

0.3

3.6

0.5

5.5

0.7

4.0

0.8

2.4

1.0

4.8

2.0

12.3

5.7

7.9

7.2

3.3 0.9

0.5 0.02

6.6

2.6

12.4

1.7

0.7

0.2

2.0

0.2

21.5 3.0

3.5 0.7

1.1

0.1

3.9

0.8

4.8

0.6

7.9

5.2

Table 3. Variability in Serum hCG Levels Measured by Different Antisera to hCG-p No. of Samples with Detectable hCG No. RIA with RIA with KKA RIA with Subjects studied Antisera I Antisera II Antisera III Normal Females Females with Ovarian Tumours Malignant Benign Suspected Occult Pregnancies Normal Males Males with Testicular Tumours

29

20

22

5 4

0 2

3 4

3 4

3 4

19

19

18

14

15

28

5

5

17

14

7

176

and biospecific RRA pregnancy test for the detection of hCG. The quantitative RRA of hCG can detect hCG as early as one week after the documented conception (42, 43). Even though the radioreceptor-assay is highly sensitive and measures essentially bioreactive material, the receptor also detects LH. Fortunately, it takes 2-5 times more LH than hCG to achi125 eve the same degree of inhibition of binding of I-hCG to the receptor. The levels of LH during pregnancy or trophoblastic disease are also suppressed due to excessive secretion of gonadal steroids. Nonetheless, interference with unexpected rises in LH above basal levels cannot be precluded. The interference from LH is minimized by the addition of normal serum with basal levels of LH to the control and the standards. If interference from LH is suspected, repeat determinations and consideration of the clinical symptoms provide a high degree of correct diagnoses. For example, if the repeat test is negative, the previous high level was likely due to LH which, because of its short half-life, disappears from the circulation within one day. If the levels increase when the repeat test is performed, pregnancy may be the most probable cause, provided choriocarcinoma or molar disease is ruled out. Attempts are currently being made to avoid the use of radioisotopes in the performance of RIA and RRA methods by using enzymeimmunoassays (44-46). An early and specific measurement of hCG in blood and urine by current RIA and RRA procedures has played an important role in the detection and management of normal and abnormal pregnancies, trophoblastic disease as well as in the detection of ectopic secretion of hCG-like substances by normal and neoplastic tissues.

hCG in Normal Tissues a) Placenta. Normally hCG is produced during pregnancy and circulates in the blood of both the mother and the fetus. The

177

blood and urine levels of hCG during a normal pregnancy are shown in Figs. 1 and 2 (35, 47). Radioimmuno- and radioreceptor assays have permitted the detection of hCG in the blood and urine as early as 7-10 days after documented conception (35). Concentrations of hCG then increase rapidly, reaching levels of 100 to 200 IU/ml between the 8th and 12th week. During mid-pregnancy, hCG levels decline to a mean of approximately 10 IU/ml and remain relatively constant until the occurrence of a slight rise during the third trimester (Fig. 3) (48, 49). The levels of hCG-3 rise similarly to those of hCG, to reach a maximum between the eighth and the twelfth week and subsequently diminish. The levels of hCG-a, however, are low at the beginning of pregnancy and increase at the end of gestation. Thus in early pregnancy the ratio of hCG-a to hCG or hCG-p is 2 (50). The ratio of biological to immunological activities of hCG varies throughout gestation (51). This may be due to the measurement of subunits of hCG and fragments in the RIA, which are immunoreactive but are inactive in bio- or receptor assays. Both pregnant serum and placental tissue contain an excess of a-subunit relative to 3-subunit (51, 52). The absolute amount of hCG and hCG-a in placental extracts decreases after the first trimester of pregnancy but the relative quantity of hCG-a exceeds that of immunoreactive hCG by more than 10-fold during the last two trimesters. In addition to native hCG-a, a large molecular weight species of hCG-a has been detected during the second trimester (51). A "large" immunoreactive species of hCG containing low receptor activity, is present in the extracts of human chorionic tissues cultured in vitro (53) . Gel filtration profiles suggest that the large form is predominant in chorionic tissue cultured for a short period and the authentic form of hCG is predominant in the culture media. The large form cannot be distinguished immunologically from purified hCG and yields an hCG-like component on trypsinization. These results suggest that hCG is also synthesized as a prohormone

178

in placenta. Free a-subunit is also produced in primary cultures of the placenta (54).

1,200

mftJ hCG p « r ml SERUM

Fig. 1. Detection of ovulation and pregnancy by RRA of LH and hCG (35). 10-0

5*0 -4-0 c 3*0 3 O

« 5*0 K (C 3*0

2*0

3 hLO

E 1*0

0*8

0-8 I

X

0*6

LH SURGE

0*6

•^RIA

f

3

3) >

0*4

0.4 » O

0*2

0*2

0

-10 0 +10 20 30 40 50 Day Of Cycle

Fig. 2. LH-hCG levels, following the midcycle LH surge in pregnancy, measured in 24 h samples by RRA, and, hCG levels measured by RXA (47).

179

Fig. 3. Serum hCG levels during pregnancy determined by a solid phase RIA and by RRA (48). b) Fetus. Maximum levels of 6 to 550 ng hCG/ml are found in fetal serum at 11-14 weeks and they are not influenced by the sex of the fetus (25, 55). In fetuses of 12-20 weeks, the meconium, ovary, kidney, thymus, and testes are found to contain respectively, 356 + 104, 46.9 + 4.3, 20.3 + 2.8, 11.5 _+ 1.2 and 8.2 _+ 1.7 pg hCG/mg wt. of tissue, whereas the adrenal, lung, liver, spleen or muscle contain approximately 1.4 - 3.4 pg hCG/mg wt. of tissue (56). hCG is not detectable in fetal pituitary gland (25). However, the immunoreactive a-subunit is first identifiable during the 8th week of gestation in the fetal pituitary tissue (57) and is present throughout gestation (58). c) Amniotic fluid. Levels of hCG up to 7400 ng/ml are measurable in amniotic fluid prior to 12 weeks (25). d) Normal subjects. Small quantities of hCG have been detected in normal, nonpregnant females and males. Extracts of human testes obtained at autopsy (59) , urinary concentrates and pituitary extracts, as well as plasma obtained from non-

180

pregnant subjects (60, 61) contain a substance which is immunologically similar to hCG, elutes from columns of Sephadex G-100 and Ultrogel ACA-54 with Kd similar to hCG, has isoelectric point similar to that of hCG in isoelectric focusing and exhibits in vitro biological activity similar to hLH and hCG. A gonadotrophin preparation from urine of postmenopausal women was also reported to contain hCG-like biological activity in the radioreceptor assay. The source of this hCG is assumed to be the pituitary. A large concentration of immunoreactive a-subunit co-chromatographing with TSH-, LH- or FSH-a, has also been detected in extracts of normal pituitary tissue (58, 62, 63). e) Nonendocrine Sources. An immunoreactive hCG-like substance has been identified in extracts of liver and colon (64). The colon and liver CG do not bind to Con-A-Sepharose columns indicating little carbohydrate content, which may explain the lack of in vivo bioactivity of the material. f) Blastocyst. An hCG-like substance has recently been detected in pre-implanted mouse, rat and rabbit blastocysts (6567). The estimate of hCG-like material in rabbit is 87 ng/ml blastocyst fluid by the radioreceptor assay (68) and 20 ng/ ml by radioimmunoassay (69) on day 6 following fertilization. However, in the absence of the secretory and clearance rates, the exact quantity of hCG-like substance produced before implantation in the rabbit is difficult to estimate at the present time. The exact chemical nature, time of appearance and role of the blastocyst gonadotrophin may vary phylogenetically and caution should be exercised in the interpretation of results between species. The absence of hCG-like material in unfertilized ova (70) strongly suggests that the hCG-like substance is produced by the morulae themselves and is not a component of the uterine fluid that coats the early embryo. The secondary rise of hCG-like material in the serum of pregnant rabbits on days 3 and 5

181

following the ovulatory LH surge is consistent with the presence of luteotropic substances prior to or at the time of implantation. If an hCG-like substance is present in the rabbit blastocyst, its detection in blood suggests an active transport of material through the uterine wall before implantation and prior to the establishment of vascular connections. It is interesting that exogenous hCG introduced into rabbit uteri appears in the peripheral circulation within 30 minutes. Current observations in our laboratory also indicate the presence of a PRL-LH-like material in the preimplanted rabbit blastocyst fluid. It is conceivable that the gonadotrophin-like material in the pre-implanted rabbit blastocyst may act as a barrier to immunologic rejection. The possibility of a direct or indirect role of pre-implantation gonadotrophin in the maintenance of the corpus luteum of gestation, however, remains to be established (Fig. 4). Sperm. An hCG-like material has been detected in sperm of various species including human. The p-subunit of chorionic gonadotrophin was demonstrated using a fluorescein-labelled double-antibody technique, in the spermatozoa of seven volunteers. The hCG-like substance was present in 5-7% of spermatozoa in all specimens analyzed and could provide the precursor for the production of hCG-like material by the conceptus prior to implantation (71, 72). hCG, possibly from the germ cells (Fig. 5), was also demonstrated in human

non-neoplastic testicular homogenates by radioimmuno-

assay . hCG-like material in women using intrauterine devices (IUDs). Recently, 200 serum samples have been obtained randomly during the luteal phase from women of known fertility who were using IUDs. hCG-like material was found in almost 20% of them by radioimmunoassay using antibody against hCG-3 with a sensitivity of 6IUhCG/l, and a radioreceptor assay using membranes from the bovine corpus luteum with a sensi-

182

Fig. 4. A portion of rabbit blastocyst stained with FITCconjugated y-globulin isolated from antibodies to 3~hCG. A: Zona Pellucida, B: Layer of trophoblastic cells (67). (Reproduced with the permission of the publisher, The American Fertility Society).

%

Fig. 5. Photomicrograph of human spermatozoa using the immunofluorescence double antibody technique. Note the positive fluorescence reaction in different segments of the spermatozoa (x400) (72). (Reproduced with the permission of the publisher, The American Fertility Society).

183

tivity of 6 IU/1, (73, 74) thus suggesting the presence of a blastocyst. However, these women do not develop clinical signs of pregnancy. A heme-agglutination inhibition test with a sensitivity of 30 IU/1 was applied to urine samples collected during the second half of the cycle and 32 of the 73 samples contained hCG (75). Both urine and blood samples were examined for hCG by radioimmunoassay and radioreceptor assay; there were 14 positives in 92 women of whom 2 demonstrated clinical signs of pregnancy and hCG could also be demonstrated in serum when urinary levels exceeded 100 IU/ml. These results were not confirmed by another group of workers (76), hCG was not found in either blood or urine of women with an IUD using radioimmunoassay with an antibody to hCG-p crossreacting with LH in the range of 15 to 50 mlU. However, in the absence of specific assays of LH and hCG, these observations do not allow any final conclusions as to the presence or absence of hCG in these samples. In another recent collaborative study (77), a more specific radioimmunoassay of hCG, and confirmatory bio- and radioreceptor assay, detected transitory hCG-like activity in the urine of women with IUDs. The inability to detect hCG in serum in this study is intriguing since "today's urine is yesterday's blood" and may have been due to the relative insensitivity of the immunoassay used in blood as compared to RRA used on urine (RRA:RIA ratio, 13:1). The current disagreement about the presence of hCG in women with IUDs appears to be partly due to the use of non-specific assays at the limit of their sensitivity. The IUD is widely used today but its mode of action is not understood. It could interfere with several aspects of conception, since it induces an inflammatory reaction in the uterus. Some recent evidence has indicated that it suppresses the growth of an implanted embryo because peaks of hCG are found in the postovulatory phase in the plasma of women wearing IUDs• The questions whether IUD interferes with the implantation of the blastocyst, acts as an abortificant soon after implantation, or interferes with

184

the sperm migration and fertilization and thus provides a contraceptive effect, remain to be answered.

hCG in Normal Tissues In general, excluding pregnancy, serum hCG levels over 1 ng/ml, serum hCG-3 levels over 1.5 ng/ml and serum hCG-a levels over 3.5 ng/ml are considered pathological (50). Ectopic secretion of hCG is found in 42% of patients with the adenocarcinoma of the ovary and in 51% of patients with testicular tumours (78). In patients with non-gonadal tumours, the highest incidence of hCG is found in association with pancreatic carcinoma (33%), gastric adenocarcinoma (22%) and lung (9%). The presence of hCG with a-fetoprotein has been demonstrated in testicular, ovarian and extra-gonadal malignant germ cell tumours (79). a) Abnormal pregnancies. Altered patterns of hCG levels are present in patients with ectopic pregnancies, threatened and spontaneous abortions, and in occult pregnancies (35). Serum hCG levels greater than twice those normally found at 4 to 8 weeks gestation indicate twin pregnancy (80) provided the existence of trophoblastic disease is ruled out. b) Trophoblastic tumours. There is evidence for the secretion of hCG and either one or both subunits of hCG by a variety of ectopic tumours. In one study of trophoblastic disease, all hCG-positive serum samples showed a-subunit by RIA, but no isolated a-subunit elevation was encountered (81), but in another study, a tumour secreting exclusively hCG-a has also been reported (10). Women with gestational trophoblastic tumours have widely different ratios of hCG and its subunits in serum and tumour extracts (82). No a or 3-subunits are detected in patients responding to chemotherapy (54). However, Franchimont et al (50) have consistently found free a and 3-subunits in trophoblastic tumours responsive

185

or unresponsive to chemotherapy. The profile of hCG and its subunits found in patients who failed to respond to chemotherapy is indistinguishable from that found in patients with nongestational trophoblastic tumours as well as patients with tumours ectopically secreting hCG and its subunits (54). Hydatidiform mole, a disorder of the human placenta, is characterized by hyperplasia of the trophoblast, hydropic degeneration of the villous stroma and disappearance of villous blood vessels resulting in early death of the embryo; the most striking biochemical change is the excessive production of hCG by the syncytiotrophoblast (18, 83). The level of hCG-f} is also considerably increased in hydatidiform mole and is proportionally greater than that of native hCG. At 10-18 weeks, the ratio of hCG-p to hCG is 3.7 in a patient with hydatidiform mole as compared to 1.8 in a normal patient. Since the increase in the concentration of hCG-a is relatively slight, the ratios of hCG-a to hCG and hCG-a to hCG-p are much lower in molar pregnancy (50). Sera from patients with a hydatidiform mole show normal hCG and two peaks of hCG-a, one normal and a second coinciding with hCG on gel filtration. Similarly, 2 peaks of hCG-3 are detected, eluting just before and just after hCG. The larger forms of the a-subunits are found in greater quantities than the monomeric forms. Free a-subunit was also detected in two of four patients with hydatidiform mole (81). In some patients with choriocarcinoma who eventually develop cerebral metastases, increased levels of hCG-a are detected during periods when the concentrations of hCG and hCG-3 are decreased or are not detectable (78) • Rutanen (81) found free a-subunits in 3 out of 6 patients with choriocarcinoma. These results suggest that the monitoring of trophoblastic tumours can be improved by assaying both subunits of hCG. c) Nontrophoblastic tumours. Eleven of 130 patients with testicular seminoma have increased serum hCG levels (84) . The

186

hCG molecules are localized in the synctytiotrophoblastic component of the testicular choriocarcinoma and in the syncytiotrophoblastic giant cell that is occasionally found in association with embryonal carcinoma, teratoma and seminoma (85). Schultz et al (86) studied 67 patients with malignant germ cell neoplasia of the testis and found hCG elevation in 38% of 34 patients with nonseminoma. In 60 patients with seminoma, 4 exhibited elevated serum hCG levels (87). After surgical correction and chemotherapy, hCG levels returned to normal. d) Nonendocrine tumours. Gonadotrophin production is not limited to trophoblastic

neoplasms but also occurs in a number

of nonendocrine tumours (54), such as hepatoblastoma (88, 89) and bronchogenic carcinoma (90-92) (Table 4). The gonadotrophin produced by these tumours is similar to hCG or LH (93, 94). Gynecomastia has been associated with ectopic gonadotrophin secretion in patients with carcinoma of the lung (92), adrenal (95) and liver (96). hCG, hCG-3 and hCG-a were detected in blood, urine and tissue from the malignant neoplasm of a patient with bladder carcinoma (97). Tumours of the gastrointestinal tract have been among those associated with the highest circulating levels of ectopically secreted hCG. Vaitukaitis (54) reports an adenocarcinoma of the stomach in a postmenopausal woman producing quantities of hCG comparable to those observed in the first trimester of pregnancy. Hattori et al (98) reported that 10% of plasma samples from patients with malignant tumours contained hCG. The amine precursor uptake and decarboxylation tumours (APUD) produce less amounts of hCG than do non-APUD tumours. In studies of pineal tumours, the concentration of the subunits of hCG may be increased, but is not sufficiently abnormal to provide a useful index of tumour activity (99). e) Cell culture lines. Pattillo and Gey (100) explanted trophoblastic tissue from a choriocarcinoma which has been serially

187 Table 4.

Site

hCG and

hCG-B

in P l a s m a ,

Di s o r d e r s Tested

cell

Ca

A d e n o c a rc i n o m a

Extracts

for

Tested

for

hCG*

hCG

0

12

4

2

3

1

Large

Cell

Ca

1

0

6

4

Cell

Ca

6

0

14

3

1

0

7

0

3

Squamous

Stomach

Adenocarci noma

34

3

8

4

Adenoacanthoma

1

1

1

1

1

1

1

1

1

1

Cell

Ca

C a r c i noi d 73

Carcinoma Liver

Pancreas

1

82

14

6

2

Adenoca

Rectum Colon

and

42

1

1

1

1

0

1

102

Retroperi toneum

Teratoma

1

1

Lymph

Lymphosarcoma

1

0

Mediastinum

Carcinoid

1

0

Uterus

Oat

Ca

3

2

Adrena1

Cortical

Adenoma

2

0

Testi s

Pheochromocytoma Carcinoma

1

0

Bladder

98

50**

443

4**

"

102

Tissue

ng/ml

ng/g

wet

Urine

Reference

ng/ml

tissue

280 .0

5414.0

489.0

hCG-a

41 .0

5188.0

5 6 9 .0

hCG-B

72 .0

10910.0

501 .0

hCG

Normal

98

»

B1 o o d

Carcinoma

98

0

1

Controls

"

13** 1

Normal

98 102

Carcinoma

Cell

98

1

Duodenum

Node

"

102

0

112

Carcinoma

"

14**

9

Adenocarcinoma

»

2

102

Insu1inoma Carcinoma

"

16**

6

Hepatoma Hepatocel1ular carcinoma

98

"

Oesophagus

Squamous

Malignancy

hCG-B**

1 3

Ca

with

Reference

Positive

Small

Cell

Patients

Ti s s u e Samples

14

Carcinoid

of

Positive or

Squamous

and Tissue

Blood Samples

Lung

Urine

97

"

Males hCG

1 .25 .

hCG-a

1 .0 not

hCG-B •Plasma "Samples

samples with

with

hCG/LH

elevated

hCG-p

ratio

not

1 .2

97

4.3

detectable exceeding

detectable

0.25

M

ii

not

detectable

"

188

transplanted in the hamster cheek pouch by Hertz (101). This cytotrophoblastic pure culture called BeWo is the first human hormone synthesizing cell culture system to be established in continuous cultivation (103). The clones called JEG-1, JEG-2B, JEG-3, JEG-7 and JEG-8 have continued to produce several hormones including hCG. In cultures of BeWo human malignant trophoblast cell, hCG and hCG-f3 of normal size and hCG-a of apparently larger size are present (8). There is approximately twice as much hCG as the subunits in the culture fluid. The cell homogenate consists mainly of hCG-|3. The major hCG-3 and hCG-a present are of apparently smaller molecular weights than standard hCG-3 and hCG-a. In the BeWo line of malignant trophoblast, only 6% of the cells produce hCG-|3 and a in the unstimulated conditions (29) ; however, 70% and 18% stimulation of hCG production is achieved with dibutyryl cAMP and theophylline. A cervical carcinoma (Caski) producing only the 3-hCG and a choriocarcinoma producing only a-hCG were recently established (104). The a-subunit production was induced in Hela (105) and choriocarcinoma cells (106) with sodium butyrate. Bordelon et al (107) first reported continued hCG production in hybrids. The JEG-3 cell line with high levels of hCG production was fused with human VA-2, mouse 3T34ED and mouse LMTK-(C11D) cells. Some clones from each fusion series showed low levels of hCG production. The cultured human choriocarcinoma cell line, JEG-3 secreted substantial quantities of both biologically active hCG and an immunoreactive a-subunit (JEG-a) (9). The JEG-hCG is apparently similar in size to that of purified hCG (108). The apparent molecular weight of the JEG-a is greater than hCG-a. Both JEG-a and hCG-a exhibit heterogeneity on electrofocusing. JEG-a contains a major component with an isoelectric point of pH 4.8, which is a minor component in hCG-a. A minor JEG-a component with an isoelectric point of pH 7.0

189

chromatographs similar to the standard. The possibilty of JEG-a being a precursor of hCG-a has been suggested. A human lung cancer cell line (ChaGO) produces hCG and hCG-a. The control doubling time averaged 4 days. Weintraub et al (109) isolated an a-subunit secreted by a gastric carcinoid tumour (AL-a) and an a-subunit from tissue culture of bronchogenic carcinoma cell lines (ChaGO-a). The AL-a and dTTreduced ChaGO-a exhibit lower molecular weights of 15,000 and 13,000, respectively in SDS gel electrophoresis as compared to 22,000 obtained with standard hCG-a. However, on gel chromatography, the apparent molecular weights of AL-a (27,000) and ChaGO-a (30,000) were slightly higher than that of standard hCG-a (23,000). No differences were found between them on ion-exchange chromatography. The amino acid composition of AL-a showed less phenylalanine and more valine compared to hCG-a. Glucosamine was identified in AL-a. AL-a recombined with hCG-3 to regenerate only 2% of the expected activity whereas ChaGO-a did not produce any detectable activity on incubation with hCG-p. The secretion of hCB and the p-subunit by a cell line from an epidermoid carcinoma of the human cervix and the presence of hCG or hCG-p in the serum of the patient from whom the tumour was derived has recently been reported (104). HeLa and HEp-2 cells released hCG and subunits in tissue culture and hCG-3 was localized in the plasma membrane of tumorigenic cells (105, 110, 111). A high intensity immunoreaction of hCG-p is characteristic of most tumorigenic cells. Acevedo et al (112) demonstrated the presence of a membrane-associated hCG-like immunoreactive protein in 15 strains of bacteria isolated from tissues of patients bearing malignant neoplasms.

190

Application a) Abnormal pregnancies. Altered serum levels of hCG and its subunits have been useful markers in the detection of ectopic pregnancies, spontaneous, threatened and induced abortions (35, 113, 114) . b) Tumour marker. hCG has served as a cancer marker in the diagnosis of trophoblastic neoplasms (50, 54, 115, 116). Use of highly sensitive RIA and RRA of serum or urinary concentrates further increases the success rate of early diagnosis and detection of the recurrence of trophoblastic disease. The hCG or hCG-3 is frequently used in the diagnosis as well as a guide for the success of therapy for hCGsecreting neoplasms. A good correlation was found between RRA (47) and RIA of hCG in patients with trophoblastic disease (117). With the RRA, patients with hydatidiform mole can be monitored for up to 10 weeks after evacuation and have the hCG titer available on the day the specimen is obtained. To rule out active disease, specific radioimmunoassays for hCG and its free a and 3-subunits can be performed when hCG levels fall below 10 mIU/ml (35). hCG and afetoprotein (AFP) have been used as biological markers for detecting, staging and monitoring the management of nonseminomatous testicular tumours (85, 102, 118). The followup of patients with testicular cancer is almost as important as treatment itself, since early detection of metastases may result in exact timing of surgical and/or chemotherapeutic approaches with improved results as well as in the regulation of the dosage of chemotherapeutic agents which in excessive use may be toxic and detrimental. Measuring both AFP and hCG, pre- as well as post-operatively(79) is better than measuring either one alone. A normal level does not exclude active disease being present. These markers are of limited usefulness in undiagnosed testicular masses. The discordance sometimes found between AFP and hCG can be explained on the

191

findings that the cells producing hCG and AFP are different (119, 120). The hCG-3 RIA has increased the sensitivity of this test so significantly that up to 60% of patients with non-seminomatous testis tumours were found to have elevated levels of hCG (118) and the RIA of hCG in the urine of patients with non-seminomatous testicular tumours was found to be of significant prognostic value. The a-subunit of hCG has a short half-life (20 min.) compared to the prolonged half-lives of serum AFP and hCG (5 days and 24 h, respectively) and has been used to localize a recurrent metastatic tumour (120). In addition to trophoblastic and testicular tumours, elevated levels of hCG have been used as a guide in nonendocrine tumours such as gastric, pancreatic and breast carcinoma. The concept of specific tumour markers has been extended to the subunits. A few tumours have been found to ectopically secrete only one of the subunits of hCG. Isolated production of hCG-p was found in a patient with pancreatic carcinoma (121, 122). The serum concentration of immunoreactive hCG-a in a patient with an adenocarcinoma of the stomach exceeded 20,000 ng/ml (123). hCG-p is the predominant form present in a patient with a lung tumour (54). Serum hCG levels are elevated in 48.5% of patients with breast carcinoma (124). c) Induction of ovulation. hCG has been used as a substitute for LH in the induction of ovulation (125) . In one series of 200 pregnancies, the overall multiple pregnancy rate (mainly twins) was 18%, the abortion rate was 17% and the ectopic pregnancy rate was 3% (126). More recently, this technique has been applied to the superovulation of ovulating women with occluded Fallopian tubes, with the aim of collecting multiple mature oocytes for in vitro fertilization (127) .

1 92 Biological Significance Clinically the serial measurement of hCG, pre- and postoperatively has a high potential for changing the survival of patients with cancer, since more accurate staging and early detection of metastases are possible and may lead to earlier and more aggressive treatment. The serial measurement of hCG also aids in the diagnosis and treatment of abnormal pregnancies. Early, specific and rapid detection of pregnancy is helpful in protecting the embryo from radiation and surgery during early gestation, drastically reducing doubt and anxiety of rape victims, facilitating the management of infertility, improving the accuracy of artificial insemination and evaluating contraceptive agents that do not prevent ovulation. The detection of hCG by RIA and RRA has provided an effective research tool for the determination of pregnancy wastage during early implantation and for the evaluation of the specificity of antibodies obtained from women immunized against hCG-3 for contraception (35) . hCG associated with the microvillous border of the syncytial trophoblast is in direct contact with maternal blood and may contribute to the immunoregulatory processes operating during pregnancy (128) . By determination of the membraneassociated domains from the amino acid sequence of hCG, it has been proposed that the protective effect of hCG on transplanted trophoblast and skin cells may represent antigenic modulations (129). It has also been suggested that since the trophoblast produces organ-specific antigens, hCG may be involved in the modulation of their expression. hCG in the neoplastic cell membrane might, therefore, perform a similar role altering the potentially immunogenic antigens expressed by neoplastic cells. Recently, using a solid phase RIA, antibodies to hCG and hLH have been detected in normal human sera (130) . In contrast with antibodies to hCG resulting from immunization procedures, natural antibodies to hCG/LH do not interfere with conception. Low affinity antibodies to the hormone have been

1 93 found to protect it from rapid destruction. The possibility of antibodies provoked by microorganisms which share some antigenic sites with hCG has been suggested. Ectopic pregnancies in many different sites in the body, are normally successful for considerable periods, even in presensitized hosts; their demise, when it eventually occurs is not immunologically mediated (128). Choriocarcinoma of gestational origin, a malignant derivative of the trophoblast is also completely refractory to transplantation immunity directed against paternal antigens in affected women. hCG-like material has been reported in bacteria (131), in plant materials (132), in extracts of the stomach and hepatopancreas of the lady crab (133) as well as in placental extracts of the rat, mouse, hamster and rabbit (134, 135). Fifteen strains of bacteria isolated from tissues of patients bearing malignant neoplasms exhibited the presence of a membrane-associated, immunoreactive protein similar to hCG (136). The hCG-like antigen was not present in every 'cancer associated bacteria'. However, besides trypsin-like proteases, high salt and protein content can also interfere in in vitro RIA and RRA systems for hCG to give rise to false positive results (133, 137). Hence, supportive in vivo or in vitro assays in conjunction with RIA and/or RRA are necessary to confirm the presence of hCG-like material. For many years, reproductive endocrinologists assumed that the only non-neoplastic, extra-pituitary sources of hCG-like material were the trophoblastic tissues of placentae of primates and equines. The production of hCG-like material has, however, been demonstrated in neoplasms as well as in normal human testes, liver and colon. These findings suggest that the genome for production of CG is present in normal adult cells. The trophoblastic hormone is a common chemical denominator of every cancer cell. The high concentration observed in the most tumorigenic cells may be one of the reasons for the failure of the immune mechanisms to reject tumour growth (110, 138). These observations clearly suggest that the striking biochemical and functional similarities between the cancer cell and the syncy-

1 94 tiotrophoblast is more than casual. The presence of hCG-like material in human spermatozoa and in the rabbit blastocyst has raised interesting biological speculations in the maintenance of the corpus luteum of gestation prior to implantation and protection of the embryo from immuno-rejection by the mother. The detection of hCG-like material may also serve as surface markers in the study of the early stages of embryonal development in the rabbit. The presence of an hCG-like material in human tissues may have serious implications for current endeavours to control fertility based on immuno-interference with trophoblastic hCG by active immunization with specific hCG-fS subunit. Research in this area will provide new information on one physiological role of hCG in human reproduction and may well open new approaches to fertility control in humans. In a recent study by Yoshimoto et al (139), hCG-like material devoid of carbohydrate moiety was demonstrated in normal human tissues. However, serum and tissue from cancer patients as well as placental tissue and serum from pregnant subjects show significantly higher levels of hCG-like material. It is suggested that the trophoblastic cell is not unique in its ability to synthesize hCG but has developed the ability to glycosylate hCG. These observations have an important bearing on the use of this hormone as a tumour marker. Serial determinations of hCG in patients with tumours showing increasing quantities above the levels in the normal tissue should provide more reliable evidence of tumorogenesis. The ubiquitous presence of hCG appears to be of evolutionary significance and the precise role of hCG at the molecular level in human reproduction as well as in the genesis, maintenance, diagnosis and treatment of neoplasms needs to be further explored.

195

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203 110. Acevedo, H.F., Slifkin, M., Pouchet, G.R., Rakshan, M.: Human chorionic gonadotropin in cancer cells. I. Identification in in vitro and in vivo cancer cell systems. In: "Detection and prevention of cancer", Ed. Nieburg, H.R., Marcel Dekker, Inc., New York, pp. 937-963 (1978). 111. Slifkin, M. , Acevedo, H.F., Pardo, M., Pouchet, G.R., Rakshan, M.: Human chorionic gonadotropin in cancer cells. II. Ultrastructural localization. In: "Detection and Prevention of Cancer", Ed. Nieburg, H.R., Marcel Dekker, Inc., New York, pp. 965-979 (1978). 112. Acevedo, H.F., Slifkin, M., Pouchet, G.R., Pardo, M.: Immunohistochemical localization of a choriogonadotropinlike protein in bacteria isolated from cancer patients. Cancer 1217-1229 (1 978). 113. Mandelin, M., Rutanen, E.M., Heikinheimo, M., Jalanko, H., Seppala, M.: Pregnancy-specific ß-1-glycoprotein and chorionic gonadotropin levels after first trimester abortions. Obstet. Gynecol. 52, 314-317 (1978). 114. Lahteenmaki, P.: The disappearance of hCG and return of pituitary function after abortion. Clinical Endocrinology 9, 101-112 (1978). 115. Bagshawe, K.: Ed. Medical Oncology, Medical Aspects of Malignant Disease, Blackwell Scientific Publications, Oxford (1975). 116. Weintraub, B.D., Rosen, S.W.: Competitive radioassays and specific tumor markers. Metabolism 22, 1119-1127 (1973). 117. Kletzky, O.A., Scott, J.Z., Morrow, C.P., Mishell, D.R.: A comparative study between RRA and RIA for hCG in patients with trophoblastic disease. Obstet. Gynecol. 52^, 328-331 (1 978) . 118. Wajsman, Z., Murphy, G.P.: The current management of advanced testicular cancers. Urological Survey 28, 127-133 (1 978) . 119. Moore, M.R., Garret, P.R.Jr., Walton, K.N., Walmann, T.A., Mclntire, R.K., Vogel, C.L.: Evaluation of human chorionic gonadotropin and a fetoprotein in benign and malignant testicular disorders. Surgery Gynec. Obstet. 147, 167-174 (1 978) . 120. Javadpour, N., Mclntire, K.R., Waldmann, T.A., Scardino, P.T., Bergman, S., Anderson, T.: The role of RIA of serum AFP and hCG in intensive chemotherapy and surgery of metastatic testicular tumors. J. Urol. 119, 759-762 (1978). 121. Weintraub, B.D., Kadasky, Y.M., Rosen, S.W.: Ectopic production of human chorionic gonadotropin (hCG) and its free ß-subunit. Clin. Res. 20, 444 (1972). 122. Weintraub, B.D., Rosen, S.W.: Ectopic production of the isolated ß-subunit of human chorionic gonadotropin. J. clin. Invest. 52, 3135-3142 (1973).

204 123. Rosen, S.W., Weintraub, B.D.: Ectopic production of the isolated a-subunit of the glycoprotein hormones. A quantitative marker in certain cases of cancer. New Engl. J. Med. 290, 1441-1447 (1974). 124. Tormey, D.C., Waalkes, T., Simon, R.M.: Biological Markers in Breast Carcinoma. Cancer 39, 2391-2396 (1977). 125. Gemzell, C.A., Diczfalusy, E., Tillinger, K.G.: Clinical effects of human pituitary FSH. J. Clin. Endocrin. 18, 1333-1348 (1958). 126. Brown, J.B.: Pituitary control of ovarian function concepts derived from gonadotropin therapy. Aust. N.Z. J. Obstet. Gynecol. JJ5, 47-54 (1978). 127. Talbot, J.M., Dooley, M., Leeton, J., Lopata, A., McMaster, R., Wood, C., Brown, J.B., Evans, J.H.: Gonadotropin stimulation for oocyte recovery and in vitro fertilization. Aust. N.Z. J. Obstet. Gynecol. J_6 , 1 1 1-1 18 (1 976). 128. Beer, A.E., Billingham, R.E.: Immunoregulatory aspects of pregnancy. Fedn. Proc. 37.' 2374-2378 (1 978). 129. Whyte, A.: hCG and trophoblast antigenicity. Lancet 2, 1003 (1978) . 130. Wass, M., McCann, K., Bagshawe, K.D.: Isolation of antibodies to hCG/LH from human sera. Nature 274, 368-370 (1 978) . 131. Cohen, H., Strampp, A.: Bacterial synthesis similar to human chorionic gonadotropin. Proc. Soc. exp. Biol. Med. 152, 408-410 (1 976) . 132. Geschwind, I.I.: The chemistry and immunology of gonadotropins. In: "Gonadotropins", Ed. Cole, H.H., W.H. Freeman and Company, San Francisco, pp. 1-39 (1964). 133. Maruo, T., Segal, S.J., Koide, S.S.: Studies on the apparent human chorionic gonadotropin-like factor in the Crab Ovalipes ocellatus. Endocrinology 104, 932-939 (1979). 134. Wide, L., Hobson, B.: Presence of chorionic gonadotropin and free a- and 8 subunits in placental extracts of the rat, mouse and hamster. Acta Endocrinol. 85^, Supplement 212, p. 31 (1 977) . 135. Asch, R.H., Fernandez, E., Siler-Khodr, T., Pauerstein, C.J.: Presence of chorionic gonadotropin in the rabbit placenta. Abst. 256, Soc. of Gynec. Investigation (1979). 136. Acevedo, H.F., Slifkin, M. , Pouchet, G.R., Pardo, M.: Immunohistochemical localization of a choriogonadotropinlike protein in bacteria isolated from cancer patients. Cancer £2, 1217-1229 (1978). 137. Reichert, N.D., Ryan, R.J.: Specific gonadotropin binding to Pseudomonas maltophilia. Proc. natn. Acad. Sei. U.S.A. 74, 878-882 (1977).

205 138. Acevedo, H.F., Slifkin, M., Pouchet, G.R., Rakhshan, M.: Choriogonadotropin in cancer cells. III. Further studies in in vitro and in vivo cell systems. Endocrine Society Meeting, San Francisco, USA, Abstract No. 420, p. 267 (1 976) . 139. Yoshimoto, Y., Wolfsen, A.R., Hirose, F., Odell, W.D.: Human chorionic gonadotropin-like material: Presence in normal human tissues. Am. J. Obstet. Gynec. 134, 729-733 (1979).

ECTOPIC PRODUCTION OF GROWTH HORMONE IN HUMAN TISSUES

A. Kaganowicz and A. Blaustein Department of Pathology, Booth Memorial Medical Center, Flushing, New York 11355, U.S.A.

Introduction Polypeptide hormones are complex structures that are normally produced in the anterior lobe of the pituitary gland, thyroid, parathyroid glands and islets of Langerhan's. Recently, a number of syndromes of ectopic polypeptide hormone production from non-endocrine tumours have been described. This paper reviews some of the sites of ectopic growth hormone production and suggests some of the possible mechanisms.

Lung Cancer Steiner et al (1) reported a case of adenocarcinoma of the lung, pulmonary osteoarthropathy and a high basal level of human growth hormone (hGH) which returned to normal on removal of the tumour. The tumour, however, was not analyzed for hGH. In a patient with adenocarcinoma of the lung and painful osteoarthropathy who had normal serum hGH levels, the tumour contained 169 mug hGH/g wet wt. tissue compared with a level of 6.3 m|ig/g in normal tissue adjacent to the tumour (2) . The tumour cells exhibited fluorescence when treated with hGH antibody conjugated with fluorescence isothiocyanate, whereas normal lung tissue did not. Sparagana et al (3) assayed normal lung and lung tumour from 28 patients with lung cancer for hGH; only one tumour had

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a grossly elevated hGH concentration. This tumour was an adenocarcinoma and the patient also had pulmonary osteoarthropathy. Using radioimmunoassay (4) significant amounts of immunoreactive hGH (IRhGH) were demonstrated in 7 of 18 lung cancers studied. Three of the tumours were adenocarcinoma, two were later called large-cell carcinoma and two small-cell carcinoma. Six of the seven tumours were poorly differentiated. Three patients had pulmonary osteoarthropathy. The authors could not relate the presence of hGH to the pulmonary osteoarthropathy since all patients with this complication had IRhGH in their tumours. If lack of differentiation leads to de-repression of genomes, the association of elevated levels of hGH in poorly differentiated tumours is interesting. No evidence was presented as to whether the tumours synthesized the hormonal polypeptide or whether they took it up from the circulation. Greenberg et al (5) found hGH in the tissue culture of one lung tumour that had been in culture for one month. Assuming that the culture media did not contain any GH, initially one must conclude that the lung tumour synthesized it.

Stomach Cancer Eight gastric neoplasms were assayed for IRhGH (4); in 5, high levels of GH were found, ranging from 216-1775 ng/g of tissue with levels of 6-62 ng/g in tissue surrounding the tumour. The degree of

differentiation of the adenocarcinoma was variable,

but tended to be poor. One squamous cell carcinoma of the stomach

and one lymphosarcoma did not contain high levels of

IRhGH.

Breast Cancer Some cell cultures of breast tumours were found to grow at an accelerated rate after addition of hGH to the culture media (6).

209 This led Kaganowicz, Farkouh, Frantz and Blaustein (7) to assay benign and malignant breast tumours for IRhGH. One adenofibroma in a woman contained 340 ng IRhGH/g tissue and 276 ng hGH/g by radioreceptor assay (RRA) using rabbit and rat liver membranes. Immunoperoxidase techniques using antihuman hGH in a dilution of 1:100 revealed dense cytoplasmic deposits of hGH in the ductular component of the adenofibroma (Fig. 1). The dense deposits were above and below nuclei, but not within them (Fig. 2). The enormously high levels of hGH suggest synthesis rather than

absorption by the tumour. A second case was that of a

scirrhous carcinoma of the breast with metastases to the ovaries The breast carcinoma contained 370 ug hGH/g (radioreceptor assay using

rat liver) and the ovarian metastases contained 51 ug

IRhGH/g. In both cases, immunoreactive hGH in effluent fractions

Fig. 1. Adenofibroma in a 23-year-old woman, immunostained for growth hormone, revealed dense cytoplasmic deposits of hGH in the ductular component of the adenof ibroma.

SHHi ¿7

'mm %

S.J!' 57 H Fig. 2. Section from adenofibroma immunostained for hGH. The dark reaction product is found above and below nuclei, but not within them.

Fig. 3. Immunoperoxidase staining of the metastatic scirrhous carcinoma of the breast to the ovaries showing isolated cells in the cortex that contain IRhGH in the cytoplasm.

.2.1 1

of tissue extracts chromatographed similarly on Sephadex G-100, 131 to I-labelled hGH tracer. Immunoperoxidase studies of ovarian tissue in case 2 showed isolated cells in the cortex that contained hGH in the cytoplasm (Fig. 3).

Ovary Normally the ovary does not contain more than blood circulating levels of hGH and is not an organ geared to the production of peptide hormones. Kaganowicz et al (7) found that of 118 ovaries removed surgically, 8 contained significantly high levels of IRhGH (from 50 to 51,000 ng/g). Similar findings

were confir-

med by radioreceptor assay using rabbit and rat liver membranes. The elevated levels were not specifically found in tumours, but were found in normal ovaries, corpus luteum cysts, endometriosis, hyperthecosis, mucinous cyst, serous cystadenoma and Leydig cell tumour. The authors concluded that these studies provided evidence that hGH or a molecule closely resembling it can be found in some human ovaries. The comparatively high concentrations found in some of the specimens suggest formation by the gland or tumour itself rather than absorption from the blood stream, though the latter mechanism could not be excluded. The hGH molecule, as most polypeptides, are large molecules and in the past it was felt they could not cross into the cell by absorption. Recent experiments (8) with tagged insulin have shown that polypeptides can be absorbed by cells. The findings of hGH in the cells of the ovarian cortex, by immunoperoxidase techniques, confirm the RIA and RRA findings, but does not help to distinguish synthesis from absorption.

Discussion The presence of hGH in significant levels, in tissue sites other than the anterior lobe of the pituitary gland, raises

212

two questions: 1) Is it being synthesized in the ectopic sites or being selectively absorbed ? 2) What role, if any, does it have within normal or cancerous tissue ? The answer to the first question may be that both mechanisms are operative. If the ectopic tissue is synthesizing hGH, it is assumed that derepression has occurred. Cancers of the lung and stomach containing significant amounts of hGH were predominantly poorly differentiated. It is possible that poorly differentiated tumours could evidence de-repression more often than well differentiated tumours. In the ovary, however, the evidence would appear to contradict this theory, since well differentiated tissue appear to contain significantly high levels of hGH. The answer to the second question is equally elusive. Samples of venous blood draining organs or tumours rich in hGH do not reflect the high levels found within the tissue and there is no evidence for an effect of hGH peripherally. It is possible that de-repression precedes tumour formation or that hGH elaborated within a tumour accelerates its growth. Evidence for this comes from tissue culture of selected breast cancers, in which growth was accelerated

by the addition of hGH to the

culture media.

References 1. Steiner, H., Dahlback, 0., Waldenstrom, J.: Ectopic growthhormone production and osteoarthropathy in carcinoma of the 783-785 (1968). bronchus. Lancet 2. Cameron, D.P., Burger, H.G., De Kretzer, D.M., Catt, K.J., Best, J.B.: On the presence of immunoreactive growth hormone in a bronchogenic carcinoma. Australian Ann. Med. 18, 143-146 (1969). 3. Sparagana, M., Phillips, G., Hoffman, C., Kucera, L.: Ectopic growth hormone syndrome associated with lung cancer. Metabolism 20, 730-736 (1971). 4. Beck, C., Burger, H.G.: Evidence for the presence of immunoreactive growth hormone in cancers of the lung and stomach. Cancer 30, 75-79 (1 972) .

213

5. Greenberg, P.B., Beck, C., Martin, T.J., Burger, H.G.: Synthesis and release of human growth hormone from lung carcinoma in cell culture. Lancet 1_, 350-352 (1 972). 6. DeSouza, J., Morgan, L., Lewis, U.J., Raggatt, P.R., Salih, H., Hobbs, J.R.: Growth hormone dependence among human breast cancers. Lancet 2, 182-184 (1974). 7. Kaganowicz, A., Farkouh, N.H., Frantz, A.G., Blaustein, A.U.: Ectopic Human Growth hormone in ovaries and breast cancer. J. clin. Endocrinol. Metab. 48, 5-8 (1979). 8. Kolata, G.B.: Polypeptide Hormones: What are they doing in cells ? Science 201, 895-897 (1978).

STEROID HORMONE PRODUCTION IN NORMAL AND ABNORMAL HUMAN ADRENOCORTICAL TISSUE

B. J. Whitehouse and G. P. Vinson Department of Physiology, Queen Elizabeth College, Campden Hill Road, London W8 7AH, and Department of Biochemistry, St. Bartholomew's Hospital Medical College, London EC1M 6BQ, U. K.

While the biochemistry of the adrenal cortex in animals has been studied by in vitro experimentation with preparations of excised tissue, the adult human adrenal cortex for obvious reasons is less well investigated, and concepts of its cellular and intracellular function frequently derive from indirect parameters such as the levels of hormones in circulating plasma. Nevertheless, there is some information regarding the biochemistry of "normal" tissue, often from patients with breast cancer, and of tissue from two disease states: Cushing1s syndrome and Conn's syndrome. The purpose of this chapter is to review the literature and to examine the question to what extent the biochemistry of the tissues in vitro correlates with or explains the observed features of the diseases.

Normal Human Adrenal Tissue The morphology of the normal human adrenal cortex has been ably described (1, 2). It is likely that the classically described three types of cells of the adrenal cortex are arranged into zones as in other species, namely the zona glomerulosa, zona fasciculata and zona reticularis, although somewhat different in appearance from other species. In particular the zona

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216

glomerulosa is sparse and arranged into islets. Ultrastructurally the cells of the glomerulosa contain abundant tubular smooth endoplasmic reticular and are distinguished by their elongated mitochondria with parallel cristae, while the mitochondria of the fasciculata are spherical and filled with vesicles or short tubules. They also contain considerable SER (3). Symington (1) distinguishes a second type of fasciculata cell at the interface with the reticularis (type II fasciculata) in which the mitochondria show great variation in size and the cells contain increased lysosomes and microvilli. Symington refers to the fasciculata cells as "clear" cells, and reticularis cells as "compact" cells on the basis of their staining characteristics in conventional light microscopic preparations. In the compact cells of the reticularis, which are of intermediate size between glomerulosa and fasciculata cells, there is densely packed SER and varying sizes of mitochondria with both short and long tubular cristae.

Nature of Hormones Secreted by the Adult Human Adrenal Cortex A wide range of steroids have been obtained from normal adult human adrenal tissue preparations (see Table 1), or are inferred to come from the adrenal cortex from data on circulating levels of steroids. They include Cortisol, the major glucocorticoid of the human adrenal, and compounds involved in its synthesis (see below), and certain metabolites of Cortisol, notably cortisone, and some arising through further hydroxylation, such as 6(3-hydroxycortisol. Similarly, a range of steroids associated with aldosterone formation and metabolism have been isolated. While these compounds are concerned with the production of hormones associated with the most widely studied human adrenocortical functions they by no means constitute its total product.lt is perhaps ironic that one of the major pro-

217 Table 1. Steroids Extracted from Normal Human Adrenal Tissue or Adrenal Incubations Precursor

Products Glucocorticoids, precursors and metabolites

Reference

Endogenous

Cortisol, cortisone, corticosterone, 11-deoxycortisol

5-11

Pregnenolone

Cortisol, corticosterone, deoxycorticosterone, progesterone, 17a-hydroxypregnenolone, 17ahydroxyprogesterone, 16a-hydroxypregnenolone, pregnenolone sulphate, 17a-hydroxypregnenolone sulphate, 5a-pregnane-3,20-dione, 3a-11ß, 17a, 21-tetrahydroxy-5a-pregnane-20-one.

12-18

Progesterone

Cortisol, 17a-hydroxyprogesterone, 11-deoxycortisol, cortisone, corticosterone, deoxycorticosterone, 16a-hydroxyproge8terone, 5a-pregnane3,20-dione.

12,13,15, 16,18-20

17a-hydroxypregnenolone

Cortisol, 17a-hydroxyprogesterone, 11-deoxycortisol

21 ,22

Cortisol

Cortisone, 20a-dihydrocortisol, 20a-dihydrocortisone, 2a-hydroxycortisol, 6ß-hydroxycortisol

23

Mineralcorticoids precursors and metabolites Endogenous

Aldosterone

8,24

Corticosterone

Aldosterone 18-hydroxycorticosterone

25-27

Progesterone

Aldosterone, 18-hydroxycorticosterone

26,27

18-hydroxycorticosterone Deoxycorticosterone

Aldosterone

28

18-hydroxydeoxycorticosterone

29,30

18-hydroxydeoxycorticosterone

16a-,18-dihydroxy-11-deoxycorticosterone Aldosterone

54

218

ducts, dehydroepiandrosterone (DHA) and its sulphate, second only to Cortisol in amounts produced (see Kime et al (4) ), should be so little studied or understood. Indeed, other C^g steroids including androstenedione and C^g compounds such as oestrone appear on the whole to have received greater study, although in comparison produced only in trace amounts. The production of C^g and C^g steroids, which has recently been reviewed (4), is not covered in the present chapter.

Sites of Hormone Production in the Human Adrenal Cortex: Functional Zonation The relationship of zonation and function in the adrenal cortex has also been reviewed (31). Of the variety of theories proposed on histological evidence to account for the zonation in mammals, it now appears likely that the "zonal theory" in which cell type has its own distinctive functional characteristics is most plausible, although the possibility of transformation of one cell type into another, coupled with centripetal migration from the outermost layers to the inner also exists. In man it is likely that the glomerulosa is the exclusive site of aldosterone production, and a major site for production of C2^, 17-deoxysteroids including deoxycorticosterone, corticosterone, and 18-hydroxycorticosterone, while products arising through 17a-hydroxylation including Cortisol and its precursors, and C^g and C^g steroids, are confined to the inner zones. There is little evidence in man for functional differences between the fasciculata and reticularis(31-34) . The different zones show differences in response to stimulation. Thus the glomerulosa in experimental animals responds to a variety of stimuli, including angiotensin II and potassium as well as to ACTH (31, 35, 36), while the fasciculata responds almost exclusively to ACTH, and has a greater secretory reserve. The response of the reticularis to ACTH stimulation in man and experimental animals is relatively slight compared to the

219

fasciculata (33, 34, 37). There is a suggestion that DHA-sulphate production in the reticularis is preferentially stimulated by ACTH (33). There is no functional evidence for the view of Symington (1) that the reticularis is the source of all corticosteroids except aldosterone, androgens and oestrogens produced by the adrenal cortex, and that the fasciculata does not produce steroids but acts only as a store of steroid precursors such as cholesterol or cholesterol esters. These conjectures are based on histology and histochemistry alone.

Biosynthesis of Corticosteroids While the cortex can synthesise steroids from acetate (10) it is widely thought that the immediate precursor for steroidogenesis is cholesterol, which may be contained in mitochondria or as esters in lipid droplets after being synthesised de novo or taken up from the blood (10, 38-42). It is likely that in man as in other species, hydroxylation at C-20 and C-22 precedes side chain cleavage of cholesterol, although the possibility of further hydroxylation at C-17 also exists (43). In normal human adrenal tissue from a patient with hypertension, Burstein and workers (44) showed the transformation of cholesterol to 22R -22-hydroxycholesterol and 22R -20a,22-dihydroxycholesterol, and thence to pregnenolone, but 20a-hydroxycholesterol was not formed in sufficient yields for identification. Thus there are alternative possibilities for the order of these hydroxylations. Similar findings were obtained with guinea pig and bovine preparations (44). There is no information for human tissue on the mechanism postulated for bovine adrenals, in which the yields of hydroxylated products from cholesterol are small, suggesting that the conversion of cholesterol occurs on an organised steroid-enzyme complex which prevents the release of the intermediates (45-47). That such transient intermediates may occur generally has also been suggested (48).

220 CHjOH

17a-llylkuj(yimpMnolom C* SMuMi

Ffc 1

Pathway ior cortiiol Production from pregnenolone in normal human adrenal tisaje (ef ref. 1 0 ) ; — " major p a t h w a y , — m i n o r pathway (18. 721.

From pregnenolone (see Fig. 1) the major pathway involves hydroxylation at C-17 (13, 16, 18, 22, 49, 50) giving 17-hydro5 xypregnenolone which is then subjected to the A , 3(3-hydroxysteroid dehydrogenase/isomerase system, yielding 17a-hydroxyprogesterone before sequential hydroxylation at C-21 and C-11fS to give Cortisol. For the 17-deoxysteroids, pregnenolone is converted to progesterone, then hydroxylated at C-21 and C-11fJ, yielding corticosterone. Similar findings were reported in perfusion studies in vivo (51). Aldosterone was believed to arise by dehydrogenation of 18-hydroxycorticosterone (18-OH-B), which is itself formed from corticosterone; these conversions have been demonstrated in human tissue (see Table 1). However, this scheme has recently been subjected to scrutiny. The essential role of corticosterone in the biosynthetic pathway has been reexamined, and in the rat 18-hydroxy-deoxycorticosterone (18-OH-DOC) appears to be an effective precursor of aldosterone under some conditions (52, 53). The conversion of 18-OH-DOC to aldosterone in small

221

yield has also been demonstrated in normal human tissue (54) (Fig. 2a). In addition, the role of 18-OH-B as an obligatory

Fig. 2a

Pathway« for aldottarona biotyntha» (rem praywoo tone in human adrenal ttaue band on proven conversion« (10. 52-55). («'. Fig. 3).

M - Enz HO CH,

CHj

R

OH

V

R

\ M - Enz STEP 2

M - Enz STEP 1 CORTICOSTERONE

OH

0

I

»

CHj

R

18-HYDROXY CORTICOSTERONE

CH

R

ALDOSTERONE

Fig. 2b Postulated alternative route for aldosterone and 18-hydroxycorticosterone biosynthesis. From Ulick (56).

222

intermediate in aldosterone biosynthesis has also been questioned, firstly because the yields of aldosterone from 18-OH-B have almost always been found to be smaller than would be expected from an immediate precursor, and secondly because there is no real evidence for a dehydrogenase enzyme system, which should be reversible, being involved in aldosterone biosynthesis (58). The problem has not been fully resolved. However, it has been suggested that aldosterone formation from corticosterone involves a double hydroxylation at C-18 followed by spontaneous dehydration, with 18-OH-B being formed independently from a common intermediate, which is postulated to be a steroidenzyme complex (Fig 2b) (55, 56).

Abnormalities of Human Adrenocortical Tissue Diseases arising from or associated with adrenocortical malfunction include conditions of an excess of hormone output (Cushing's syndrome, Conn's syndrome), deficiency (Addison's disease), or an inappropriate profile of steroid secretion arising from individual enzyme defects. Of these, detailed studies correlating hormone output and the biochemistry of adrenocortical tissue have only been carried out in tissue removed because of excess production, and the remainder of this chapter will be devoted to these studies. In passing it should be pointed out that because adrenal tissue is not normally removed from patients with adrenocortical insufficiency or enzyme defects, there remains a very great deal which is conjectural or simply unknown about these conditions. This is particularly true, perhaps rather surprisingly, with regard to the literature on hyperplasia arising from enzyme deficiencies: although these conditions are universally referred to as enzyme deficiencies, there is in fact no direct evidence on this point and the story has been almost entirely built on indirect evidence from plasma or urinary studies (see ref. 57). In what ways the enzymes are deficient is simply not known.

223

Cushing's Syndrome The hypersecretion of glucocorticoids usually, although not invariably, without diurnal variation (58-63) is generally recognised to result from one of three causes (64): (i) the adrenal may secrete Cortisol autonomously, without stimulation by ACTH. Such excess secretion by an adrenal neoplasm usually suppresses pituitary ACTH secretion; (ii) the excessive secretion of Cortisol arises because the otherwise normal glands are stimulated by overproduction of ACTH from an ectopic source. The tumour secreting ectopic ACTH is not generally susceptible to suppression by glucocorticoids; (iii) the adrenals are overstimulated by excess ACTH secretion from a pituitary tumour: this may be suppressed by large doses of glucocorticoid. Differential diagnosis is thus based on the dexamethasone suppression test (65) with measurement of circulating ACTH and steroids (60, 64, 65). Biosynthesis of steroids in tissue from Cushing's syndrome patients Conversion of radioactive precursors by abnormal tissue from patients with Cushing's syndrome has been studied, and while there is considerable data from individual experiments available, the direct comparison of normal and abnormal tissue in a single laboratory, using identical techniques, has only rarely been recorded because of the basic difficulties of obtaining tissue. The review which follows must be interpreted in this light, and some differences between the findings of different groups can be attributed to procedural differences and others to different foci of attention. Some reported cases are clearly dealing with very rare conditions of enzymic insufficiency. It is difficult therefore to draw general conclusions about the nature of changes in the adrenal tissue from the normal condition using these methods. Indeed it is likely on the evidence from conversion of radioactive precursors that such differences

224 are quite subtle, and the statistical conditions are such that from present evidence they are unlikely to be revealed. This evidence for tumours and for hyperplastic tissue will now be discussed. Tumour s The basic pathways for the formation of Cortisol from pregnenolone in tumours, of what in most cases appear to be zona fasciculata ("clear cell") tissue, appear to be the same as in normal tissue. Thus Weliky and Engel (66) and Cohn (67) showed that 17a-hydroxypregnenolone was efficiently converted to Cortisol and to other steroids, and Griffiths et al (68) showed that pregnenolone and also pregnenolone sulphate could be utilised. Cameron et al (69) indeed obtained a set of product yield-time curves from pregnenolone and progesterone with adenomatous tissue very similar to those of Whitehouse and Vinson (16) with normal tissue, leading to the same conclusions on the nature of the major pathways (Fig. 1). Cameron et al (70) further raised the possibility that 17a-hydroxyprogesterone is itself not an obligatory intermediate, following other work (71) with hyperplastic glands in which it was shown that 17a,21-dihydroxypregnenolone could be converted to Cortisol. This conclusion, however, is at variance with that of Whitehouse and Vinson (72) because in normal (foetal) adrenals the pathway did appear to be via 17a-hydroxyprogesterone (Fig. 1). In addition to these findings on the major pathways, further C-21 products found in tumour tissue include the 17-deoxysteroids, leading to corticosterone through the pathway involving progesterone (69, 73), various sulphates, including those of Cortisol and corticosterone and 16a-hydroxyprogesterone (69, 75, 76). The finding that these transformations are essentially the same as occur in normal tissue was emphasised by Oshima et al (77), who found no significant difference in the conversion of pregnenolone and progesterone by a fasciculata ("clear cell") adenoma compared to adjacent normal tissue.

225

Less work has been done on the conversion of cholesterol, presumably because of the difficulties of substrate penetration into the cell. Nishida et al (78) found very poor yields of pregnenolone (0.75%) and progesterone (0.17%) from cholesterol in a virilising tumour slice preparation. It is still not completely excluded however that some differences in steroidogenesis remain to be discovered in tumours. In work on circulating levels of hormones, McKenna et al (79) showed that 17a-hydroxypregnenolone was in the normal range in patients with excess Cortisol caused by hyperplasia, ectopic ACTH secretion, or adrenal adenoma, but it was significantly elevated in patients with carcinoma. Pregnenolone levels were similar in all patients. It is not clear therefore whether this is directly due to biosynthetic changes, or is consequent on changes in clearance or other circulatory effects. Using an acetone powder preparation, Burstein et al (44) found similar conversion of cholesterol to 22R-22-hydroxycholesterol and (22R)-20a-22-dihydroxycholesterol in both an androgen secreting carcinoma and in normal tissue, but in normal adrenals the yield of pregnenolone was more than five-fold higher than in the carcinoma. This apparent reduction in steroidogenesis in a tumour is still the only recorded difference in steroid biosynthesis between normal and abnormal inner zone tissue. Steroid conversions in hyperplastic adrenal tissue from Cushing's patients As in the case of Cushing's syndrome resulting from adrenal tumours, there is little reason to suppose that steroidogenesis is significantly different in qualitative terms in hyperplastic tissue, resulting from excess ACTH secretion, either from the pituitary or from an ectopic site, and normal human adrenal tissue. The major pathways for Cortisol production from pregnenolone and progesterone appear to be identical with those depicted in Fig. 1 for normal tissue, with the probability that, while progesterone is an intermediate in 17-deoxysteroid formation, for the production of 17a-hydroxysteroids, 17a-hydroxy-

226

pregnenolone, not progesterone, is the first intermediate formed after pregnenolone (18, 22, 49). Further hydroxylation at C-21 may occur before A 5 , 33~hydroxysteroid dehydrogenase iso4 merase action yields the A -3-ketone,11-deoxycortisol (71). Other possible products are 113-hydroxyandrostenedione and other C1f, steroids, 16a-hydroxyprogesterone (18, 80, 81) and iy 5a-pregnane-3,20-dione (15). There may be some interconversion among the sulphates, and Calvin and Lieberman (82, 83) demonstrated the transformation of pregnenolone sulphate to 17ahydroxypregnenolone sulphate. As in the case of the tumour work, it is often difficult to decide whether apparent differences between the results reported by different groups arise from methodological variation, or from differences between tissues. Wherever direct comparisons have, been made, however, between normal and hyperplastic tissue, or between hyperplastic and adenomatous tissue, little difference has been detected (15, 18, 81). As discussed earlier however, it may still be that such differences which exist are too subtle to be revealed without a series of experiments with rigorous controls. Control of steroidogenesis in adrenocortical tumours from patients with Cushing's syndrome Despite the fact that the biosynthesis of corticosteroids from added precursors such as pregnenolone and progesterone proceeds in tumours or hyperplastic tissue from patients with Cushing's syndrome at least as well as in normal tissue, there exists a wide range of biochemical features which are different in normal and abnormal tissue. Mostly these relate to the mechanisms involved in the control of steroid secretion at the cellular level. One problem that arises however, is that the abnormal conditions are highly heterogeneous, and the range of deficiencies recorded thus far by no means describe the entire field. One of the earliest observations in this field is that abnormal tissue generally shows a refractory response to stimula-

227 tion, for example by ACTH (84), consistent with the concept of "autonomy", and there is no distinction between carcinoma and adenoma in this respect. Similar findings have been recorded in studies with experimental animals and it is worth briefly reviewing current information on control of steroidogenesis in certain animal tumours before looking more closely at the situation in the human species. In a transplantable adrenocortical carcinoma in OsborneMendel rats, designated tumour 494, biosynthesis of corticosterone takes place along the conventional pathway, but the tissue is not responsive to stimulation by ACTH or cyclic nucleotides (85-87). Indeed, ACTH appears to inhibit the conversion of progesterone to deoxycorticosterone in tumour cells, an effect not seen in normal cells (88) . Despite this the tumours do contain an active adenyl cyclase (86) which is stimulated by ACTH (and also by epinephrine and TSH (89), effects not seen in normal tissue). The block to stimulation by ACTH therefore lies between the generation of cyclic AMP and increased steroidogenesis. According to Sawhney and Sharma (90), it is possible by preloading normal cells with tritiated deoxycorticosterone to demonstrate increased conversion to corticosterone with cyclic AMP stimulation, which is however without effect on this transformation in tumour tissue. Studies with 3-adrenergic agonists and antagonists show that they compete for binding to tumour cell membrane receptors. These ectopic 3-adrenergic receptors, not found in normal tissue, presumably confer on the neoplastic tissue the catecholamine sensitivity of its adenylate cyclase. The block in the steroidogenic response to ACTH and other stimulants seems to be the presence in the tumour cells of a defective cyclic AMP-binding protein kinase. When studied in a partially purified form by Sharma et al (91), it was found that the enzyme could bind to histone but not phosphorylate it and could undergo phosphorylation, and was therefore rightly classified as a kinase, but was independent of cyclic AMP. In some ways these carcinoma cells appear to offer a model of human adrenocortical tumour function, closer perhaps than

228

certain other animal tumour cell lines. In others, for example, in the mouse tumour cells Y1, stimulation of steroidogenesis by ACTH and cyclic nucleotides appears to be at least as efficient as in normal tissue but the identity of the major steroid product, 20a-dihydroprogesterone and 11 ¡3-hydroxy-20a-dihydroprogesterone is changed (92-98) . The truth is however that human tumours are quite heterogeneous, and only some are reflected in function by rat tumour 494 cells. The autonomy of human adrenal tumours, and the partial autonomy of nodular hyperplasia seen in vivo is largely also reflected in the functions of cells or tissues incubated in vitro (compare refs. 84, 99 with 100-101), although in the study by Voigt et al (102) a tumour causing Cushing's syndrome which was apparently not suppressible by dexamethasone or stimulable by ACTH in vivo, produced only small amounts of Cortisol when incubated as a cell suspension in vitro under control conditions and responded to ACTH stimulation. This is an unusual finding however. Normal adrenal tissue incubated or maintained in culture is very responsive to ACTH. In culture, ACTH modifies the characteristic pattern of foetal adrenal steroidogenesis, which yields largely A , 3f5-hydroxysteroids, towards the adult pattern (103) reflecting the processes seen in similar cultures of rat foetal cells (104-105). In cultured adult cells, although the presence of ACTH is not necessary for continued steroidogenesis , it can still stimulate steroid output by 10-15 times (106-107). In diced adrenal tissue from breast cancer patients, steroidogenesis was maximally stimulated by 1 mU ACTH/ml (appro-9 -1 ximately 3 x 10 moles 1 ), with Cortisol output some 4-5 times higher than controls (108). ACTH also stimulated cyclic AMP, but only at the higher doses did significantly increased cyclic AMP formation precede steroid output. Cyclic AMP output was further increased at higher concentrations of ACTH (10— 100 mU/ml) although Cortisol was not: findings which reflect those in rat adrenal cell suspension incubations (109).

229

Stimulation of normal adrenal tissue by prostaglandins has also been studied. Prostaglandin receptors exist in human adrenal cell membranes (110) and prostaglandins E^ and E 2 (PGE^, PGE2) both stimulate adenylate cyclase, but their effects at maximal stimulation are not additive, whereas the effects of PGE and ACTH are additive. The binding of PGE is inhibited only by further prostaglandin, and not by ACTH. Further distinctions between ACTH and prostaglandin actions are that the ACTH effect is not inhibited by indomethacin whereas stimulation by PG is, and the PG effect is not affected by reduced calcium levels, whereas the ACTH effect is (110). In diced tissue, the two prostaglandins PGE^ and PGE2 gave dose related responses of both cyclic AMP and Cortisol, whereas PGF 2a and PGF^reduced Cortisol output at all dose levels (111). In the view of these authors, prostaglandins may have variable effects on the response to ACTH, depending on the temporal sequence of application: initially they potentiate the response to ACTH, and later they inhibit it. PGE1 exerts its effect significantly more rapidly than ACTH under these conditions (2 minutes for a significant response with PGE^ compared with 8 minutes for ACTH). The depressive effect of PGF 2a on Cortisol is not seen on cyclic AMP, which may be stimulated, thus apparently revealing a dissociation between these products. Complex responses to PGA^ and PGA2 were reported (112) which at a low concentration (1 ng per ml) depressed both cyclic AMP and Cortisol, but at higher concentrations (10-100 ug per ml) stimulated the two products. PGB^ and PGB2 appear to be largely similar in their actions to PGE. There were variable effects on aldosterone. The somewhat curious effects of PGF_ have been ex2a plained on the basis that higher levels may interact with specific PGE receptors (113). In cell suspensions, which as in other species appear to give enhanced sensitivity to ACTH and other stimulants, Kolanowski and Crabbe(11) found that half maximal stimulation of Cortisol production from normal cortical cells taken from a patient with phaeochromocytoma, occurred at 20 pg/ml, with

230 maximal rates of output over a 2 h incubation of 188 ng for Cortisol, 106 ng for corticosterone, 11 ng for 11-deoxycortisol and 32 ng for cortisone. Dibutyryl cyclic AMP stimulated steroidogenesis to a similar extent, but cyclic AMP stimulation by ACTH showed that as in the rat, further increases in cyclic AMP output could be induced by ACTH at higher concentrations than produce maximal stimulation of steroid. Thus the half maximal stimulation of cyclic AMP output was obtained at an ACTH concentration of 437 pg/ml. These values and the amounts (though not the types) of steroid produced are similar to those obtained in rat preparations. In contrast with the rat, there were concomitant increases in cyclic AMP and Cortisol at lower levels of stimulation, despite the non-parallelism of the curves (cf. ref. 109) . The literature suggests that while abnormal tissues show great variability in their biochemistry, most are less responsive to ACTH than normal tissue. In conventional incubations of cell suspension Cowan et al (101) found that hyperplastic tissue was less responsive than normal tissue at lower concentrations of ACTH (up to 6 JJ.U per ml), but responded at higher levels (up to 150 uU per ml). In focal hyperplastic tissue, in which basal steroid outputs were high and cytochrome P450 content was high (114), Honn et al (115) found that ACTH stimulated neither Cortisol nor cyclic AMP. In cell suspensions of adenomas Kolanowski et al (11) attributed the low sensitivity to ACTH to decreased cyclic AMP generation, although the pathway for Cortisol production was unaffected. Detailed investigations into these responses, and their underlying biochemical origins have been conducted by Saez and co-workers. Investigating a series of 10 carcinomas and 3 adenomas (100), they found the high 17-hydroxysteroid output from these patients in vivo was not increased by ACTH or decreased by dexamethasone, and that this was reflected by the activities of the glands in vitro. Generally too, the cyclic AMP output was low compared with normal glands, but both normal and abnor-

231

mal adenylate cyclase activity was increased by stimulation -5 -1 with NaF: ACTH was maximally effective at 10 moles 1 in normal tissue but was without effect in 7 tumours. The additive stimulatory effect of PGE and SCTH on adenyl cyclase seen in normal tissue was only apparent in those tumours which responded to both agents separately. For various reasons they considered it unlikely that the lack of response in other tumours was due to increased phosphodiesterase activity, abnormal adenylate cyclase distribution, or increased degradation of ACTH. In at least some of the tumours there was evidence that although ACTH receptor binding was. as specific as in normal tissue, the binding affinity was reduced. In the view of these workers, the 1-10 moeity of ACTH contributes to receptor binding as well as being the active site for stimulation of adenylate cyclase (116) and in some of the tumours there was evidence that binding was impaired in this, as well as in the 11-24, region. In one tumour a deficiency in PGE^ binding was observed, while in others some resemblances to the rat tumour 4 94 were seen in that ACTH stimulated cyclic AMP output as in normals, but that the steroidogenic response was nevertheless impaired. Clearly these studies confirm the impression of considerable variability in the nature of tumour deficiencies in response to stimulation. In further studies on four tumours for protein kinase deficiencies as seen in rat tumour 494, Riou et al (117) showed that two which exhibited ACTH binding receptor abnormalities as detailed above contained normal protein kinase activities. In another tumour the response to both ACTH and dibutyryl cyclic AMP was higher than normal both in steroidogenesis and in protein kinase activity, although activation constants for both cyclic AMP and cyclic GMP were similar to normal. In the fourth tumour on the other hand, both basal and cyclic AMP stimulated protein kinase activities were lower than normal, as well as the activation constant for cyclic AMP. Of the three protein kinases normally resolved by fractionation of human tissue on DEAE cellulose chromatography, two are normally cyclic AMP dependent and one of these was absent from the tumour, while

232 the other did not respond to cyclic AMP. Thus non-responsive tumours may fall into one of two classes, one in which the receptor is abnormal, and the other in which, as the rat tumour 494, protein kinases are aberrant. One problem with interpreting the range of data recorded overall however, is that as well as the existence of heterogeneity in the nature of tumours, there is also considerable heterogeneity in methodology employed and it is sometimes difficult to ascribe differences in findings to either of these variables with assurance. We have already drawn attention to the responsiveness of adenoma cells to ACTH in the work of Voigt (192). Considerable work has also been performed by O'Hare and colleagues on cultured tumour cells

(107). In contrast to other

authors they find that in their system

(in a series of 20 benign

and 5 malignant tumours), the following generalities could be noted: 1. All benign tumours were responsive to ACTH in culture, and secreted a qualitatively and quantitatively normal range of steroids, whereas 4 out of the 5 malignant tumours showed abnormal or non-existent response to ACTH was a 125 g tumour of debatable

(the exception

malignancy).

2. In contrast to the adenomas, malignant tumour cells

secreted

a steroid profile deficient both in the total amounts of steroid formed per cell, and in the relative amounts of 11 |3-hydroxysteroids. Such distinctions between adenomas and carcinomas have not previously been recorded, and it would be extremely

interesting

to know whether this results just from a fortuitous supply of adenomas of the ACTH responsive type, or whether they reflect the choice of culture as the in vitro method. There is comparatively little data on the

responsiveness

of hyperplastic tissue from Cushing's patients; however it may well be that such tissue shows many features distinct from the tumours. In cell suspensions of hyperplastic tissue Kolanowski and Crabbe

(11) found that there was enhanced response of ste-

233

roids to dibutyryl cyclic AMP and to ACTH compared to normals by a factor of 2-3. The ACTH stimulated increase in Cortisol, calculated per mole of cyclic AMP formed was also higher: maximally at 10 pg ACTH/ml by a factor of about 3. It seemed possible that there was an effect of prolonged ACTH stimulation at a stage beyond the membrane step of cyclic AMP generation. One effect of this is that the curves for response of steroid and cyclic AMP to ACTH are more closely parallel in hyperplastic cells than in normals. In a recent study in which adenomas and hyperplastic tissue were compared Nishikawa et al (180) described ACTH unresponsive and responsive adenomas. Basal steroid production and cholesterol ester content were high and cyclic AMP was low in the former, whereas in the ACTH responsive tumour, cyclic AMP levels were relatively high and adenylate cyclase activity was low with phosphodiesterase activity high. The hyperplastic tissue resembled the ACTH responsive tumour in these respects.

Conn's Syndrome Conn (118, 119) first recognised that the syndrome which bears his name could be ascribed to the overproduction of a potent mineralocorticoid. The removal of an adrenocortical adenoma rapidly relieved the symptoms (120). By 1964 it was possible to analyse the results of 145 case reports of treatment of aldosterone producing adenomas (121). Aldosterone producing carcinomas of the adrenal cortex also occur, but are much more rare. Conn's syndrome, or primary aldosteronism, must be distinguished from secondary aldosteronism where the normal adrenal is responding to excess stimulation, usually due to increased activity of the renin-angiotensin system. In general this occurs where there is reduced extracellular fluid or plasma volume, where blood is retained on the venous side of the circulation or when the circulation of the kidney is impaired for whatever reasons (see ref. 124). Renin-secreting tumours of

234 the kidney may also occur but are very rare (125). In contrast, in primary aldosteronism, suppressed renin-angiotensin activity combined with high plasma aldosterone are diagnostic features. Primary aldosteronism was subsequently shown to be caused not only by a discrete adenoma as described by Conn, but also by bilateral hyperplasia of the zona glomerulosa without the presence of a tumour (126, 127). The symptoms of the two diseases are the same: hyperaldosteronism, hypokalaemia, suppressed plasma renin activity with normal 17-hydroxysteroid secretion. Thus, distinguishing between the two conditions is difficult but necessary, particularly as even total adrenalectomy can fail to cure the hypertension in the case of hyperplasia (128, 129). The biochemical signs tend to be more abnormal with an adenoma than with hyperplasia, but the two can only be distinguished on a statistical basis with computer-assisted analysis (130). Ganguly (131)reported that the normal postural rise in aldosterone is absent in patients with an adenoma but not in those with hyperplasia and suggested that this could be used as a diagnostic criterion. Adrenal venography together with sampling of adrenal vein blood is also possible, but difficult (132). Scanning the adrenals following the administration of 131 I-labelled cholesterol is proving a useful method of visualising the adrenals and of identifying areas of high biosynthetic activity (133, 134). Very recently it has been suggested that plasma 18-hydroxycorticosterone

(18-OH-B) levels can serve

as an indicator of an adenoma, since these were found to be six times higher in patients with an adenoma than in patients with hyperplasia after overnight recumbency (135). A further sub-group of patients has been recognised where the hyperaldosteronism is correctable by dexamethasone therapy (136-138), but these are easily distinguishable from patients with either the adenoma or hyperplasia, where dexamethasone has no sustained effect (139). The aetiology of the lesion in adrenal hyperplasia is unknown, but since there is usually bilateral involvement of the adrenals the existence of a trophic humoral factor has been

235 suspected (128). Prolactin seemed a plausible candidate in view of its ion regulatory effects in lower vertebrates, but current evidence is not in favour of its having this role (140). If this factor were to be isolated then the condition should be reclassified as a secondary aldosteronism: the term 'pseudoprimary aldosteronism1 has also been suggested (128). In the absence of any knowledge of the nature or origin of this factor (141), the terminology idiopathic hyperaldosteronism is equally appropriate. Steroid biosynthesis in aldosterone producing tumours Aldosterone In general it has been found that adenomatous tissue removed from patients with Conn's syndrome shows a high capacity to synthesise aldosterone, judged by a variety of criteria. Thus, the aldosterone content of tumours was found to be at least five times higher than that of adjacent normal tissue (142, 143), and the basal aldosterone production rate by dispersed cells from an adenoma was reported to be at least twenty times higher than 'normal' zona glomerulosa cells (24). In addition, good yields of aldosterone were formed by whole tissue and subcellular preparations of adenoma from a whole range of added radioactive precursors: these included pregnenolone (142), progesterone, DOC and corticosterone (144-149), 18-hydroxycorticosterone (28) and also 18-hydroxy DOC (54, 150). Where comparisons have been made in the same experiments with adjacent normal tissue, the yields of aldosterone obtained from the adenoma appear to be 8-10 times higher. However, only individual experiments have been performed and no strict statistical criteria can be applied (see Fig. 3). Nevertheless, it seems reasonable to draw the conclusion that in Conn's syndrome, in contrast with Cushing's syndrome (see p. 225, 226), increased steroid synthesis is due to both an increased biosynthetic capacity as well as increased tissue mass. This may explain why tumour size is not necessarily related to the extent of aldosterone hyperproduction (34).

236

Precursor:-

Pregnenolone

18-OHCorticotterone

Corticosterone

18-OH-DOC

Progesterone

Progesterone Pregnenolone

5

%

Yield

4 3

2 1 0

r—

\ Produet:-

Aldosterone

Cortisol

Fig. 3 Yields of aldosterone and Cortisol produced by a human adrenal adenoma adjacent normal tissue I I from radioactive precursors. From Fattah (150). 18-Hydroxycorticosterone

/

V

and

(18-OH-B)

The production of this steroid by human adrenal tissue has not been extensively studied, probably due to the inherent difficulties in handling this unstable steroid (ref. 151) and the fact that authentic material has only recently become commercially available. In an early study Sandor and Lanthier (27) demonstrated that 18-OH-B could be formed from both corticosterone and progesterone, and found the yields to be several times higher than those of aldosterone. In vivo 18-OH-B levels appear to increase roughly in step with aldosterone in Conn 1 s syndrome (56, 151-153) although the ratio of 18-OH-B to aldosterone can vary from 0.8 to 3 (135) and the same seems to be true in vitro. Equivalently high yields of aldosterone and 18-OH-B were obtained in adenoma incubations in several of the studies mentioned above (54, 142-144, 146). However, unlike aldosterone, 18-OH-B is synthesised in both the zona glomerulosa and a significant -proportion may be synthesised in the inner zones of the adrenal cortex. In some studies it was found

237 that 18-OH-B was synthesised in tumour and adjacent tissue at similar rates, and since the adjacent zona glomerulosa is probably suppressed, it has been suggested that much of the 18-0HB produced by the adjacent 'normal' tissue originated from inner zone cells (54 , 148) . Other steroids It seems probable that adenomatous tissue can produce compounds other than those of the aldosterone biosynthetic pathway, although some doubt must be expressed since it is uncertain how complete the separation of adenoma from the adjacent so-called normal tissue can be. In contrast with normal tissue 17-deoxysteroids are usually the most prominent products (142, 145, 154), but Cortisol production in some (145, 150) but not all (142) has been described. Histologically the cells of the tumour show a mixture of zona glomerulosa and zona reticularis characteristics (154, 156). Biosynthetic pathways for aldosterone Few authors have attempted to delineate the biosynthetic pathway for aldosterone in human adrenal tissue. On the basis of the similarity of the isotope ratios after incubation with ^H14 DOC and C-corticosterone Zogbi and co-workers (147) concluded that the conventional route through corticosterone was being followed. The same conclusions are implicit in most of the studies of the conversion of radioactive precursors mentioned above. However, Grekin et al (54) found a substantial yield of aldosterone from 18-0H-D0C in adenomatous tissue, some five times higher than that found in adjacent 'normal' tissue. It was therefore suggested that 18-0H-D0C might represent a significant precursor of aldosterone in an adenoma. However, in another study the yields of aldosterone obtained from corticosterone were ten fold higher than from 18-OH-DOC (150). If judgements on the relative importance of precursors can be made on the results of individual incubations this would suggest that

238 the pathway via 18-OH-DOC is of minor importance in human tissue. Resolution of this point must await more extensive studies (10). Control of aldosterone production In contrast to the cell of the zona fasciculata, the zona glomerulosa cell is under the control of more than one agent. As part of a physiological control system aldosterone secretion increases in response to sodium lack or potassium excess, and also when decreases in effective extracellular fluid volume occur. A number of agents act directly on the adrenal to increase aldosterone secretion, of which the most important are angiotensin II, potassium ions and ACTH (156). However, it is still a matter of argument as to what extent changes in aldosterone secretion seen in the whole animal can be accounted for by changes in these factors, and the existence of other as yet unidentified stimulators of aldosterone biosynthesis is frequently postulated (e.g. ref. 141). What role these might play in adrenal hyperplasia is an interesting point. Studies with rat adrenal tissue show that the stimulation of aldosterone biosynthesis occurs at a minimum of two stages in the pathway: between cholesterol and pregnenolone ('early pathway1) and between corticosterone and aldosterone ("late pathway') (35, 157). In vitro studies of human adrenal function are relatively rare and studies of the control of aldosterone production even more so. Thus much of the data discussed will, of necessity, have been obtained in vivo. However, it does appear that the acute responses of normal human adrenal tissue to stimulation by angiotensin II, ACTH and potassium are similar to those previously described for rat adrenal cells (24) . Cells from an adenoma showed a considerably greater basal production of corticosterone and aldosterone and greater conversion of added corticosterone to aldosterone than 'normal cells' suggesting that part of the increased biosynthetic activity lay in the 'late pathway'. It was significant that the adenomatous cells also showed increased production of aldosterone, in res-

239 ponse to angiotensin II, ACTH and potassium with dose response relationships that were reasonably similar to those obtained with normal cells, although the overall production was higher. This limited data is consistent with that obtained in vivo, where it appears that the secretion of aldosterone in patients with Conn's syndrome is not autonomous. However, significant differences in the responses of adenomatous and hyperplastic glands to stimulation have also been described. True autonomy only seems to occur with an aldosterone producing carcinoma(158). Generally speaking, aldosterone production by an adenoma does not respond to changes in activity of the renin-angiotensin system. Thus, basal aldosterone production is high and not suppressed by saline infusion, by a high salt intake or by treatment with exogenous mineralocorticoid, manoeuvres which should decrease renin activity (149, 159, 160). Sodium depletion stimulates aldosterone production only to a minor extent or not at all; however, the response to a high potassium intake is almost normal (149, 160). It is thus possible that some of the reported variations in the responses to sodium depletion are due to differences in potassium intake. Adenomatous tissue appears to be generally unresponsive to both endogenous and exogenous angiotensin II; thus aldosterone does not rise after spironolactone treatment, or when the patient stands (131). Contradictory results have been obtained in studies with direct stimulation of aldosterone production by exogenous angiotensin II partly due to excessive pressor responses to the peptide before a dose adequate to stimulate aldosterone synthesis could be administered (159-161). However, Spark and co-workers (162) found that aldosterone excretion increased markedly with a 6 hour infusion of a sub-pressor dose of angiotensin II and also that acute stimulation increased aldosterone and 18-OH-B secretion into the adrenal vein in patients whose plasma renin was elevated following spironolactone therapy. Recently, a diffe1

rential response to [des-aspartyl ] angiotensin II, chosen because of its lower pressor activity, has been described, two of seven patients with an adenoma responded with a greater

240 increase in aldosterone concentration than normotensive controls (163). The authors suggest that either there are two distinct types of adenoma, one responsive to angiotensin, the other not, or that alterations in tumour responsiveness occur throughout the course of the disease. In the absence of a longitudinal study it is impossible to choose between these alternatives. However, a decrease in angiotensin sensitivity as the disease progresses would be consistent with the decreased responsiveness of normal human adrenal tissue to angiotensin II when endogenous angiotensin is suppressed by a high sodium diet (164). This decrease is explained by a decrease in number of angiotensin receptors in the zona glomerulosa, where the peptide exerts a trophic influence on its own receptors (165, 166). Exogenous ACTH can stimulate aldosterone production by an adenoma, but only in the short term. Sensitivity to ACTH appears to be greater in patients with an adenoma than in normal subjects on a sodium replete diet (167). Continuous administration of ACTH leads to a fall in aldosterone production, sometimes to lower than control levels at the end of stimulation (149, 161, 168, 169). Conversely, acute dexamethasone suppression decreases aldosterone concentrations (170) but only on the first day of its administration (139). This change in response was not due to changes in plasma potassium or plasma renin activity and was postulated to be due to an intrinsic alteration in aldosterone biosynthesis within the adenoma in the continued absence of ACTH. An analogous transient stimulation of aldosterone biosynthesis by ACTH has been reported for normal human adrenal tissue (171). It is interesting that in the rat chronic ACTH treatment directly inhibits aldosterone biosynthesis, possibly by conversion of the zona glomerulosa cell to a functional zona fasciculata cell (172-174). In spite of this endogenous ACTH appears to exert a definite regulatory influence on aldosterone secretion by the adenoma, since a circadian rhythm for aldosterone which parallels that of Cortisol has been described in several studies (158, 170, 175-177). In addition, the influence of ACTH appears to over-ride any postural stimuli

241

since the morning fall in aldosterone concentration is still seen in standing patients. In adrenal hyperplasia on the other hand, not only are aldosterone levels on average lower than with an adenoma, they generally show no diurnal rhythm nor any correlation with plasma Cortisol levels (131, 158, 170). Aldosterone is however responsive to ACTH when injected or when secretion is outside the normal physiological range, as in stress (160, 169). Plasma aldosterone levels rise on standing, and during spironolactone treatment and in general it appears that angiotensin II levels are more readily increased and have themselves more influence on aldosterone in subjects with hyperplasia than in those with an adenoma (131, 158, 178). Schambelan and co-workers (158) suggested that the large increases in aldosterone produced by • small increases in renin in response to upright posture suggested a greater sensitivity than normal to angiotensin II in hyperplasia. This has now been shown to be the case (159). The differential sensitivity of the adenomatous and hyperplastic tissue to angiotensin need not be a fundamental characteristic of the tissue and may be related to differences in the number of angiotensin receptors produced as a result of the trophic influence of angiotensin on its own receptors (165, 166). Thus it might be expected that the hyperplastic condition with typically lower aldosterone levels and somewhat higher angiotensin activity would show a greater sensitivity to angiotensin. However, the increased sensitivity to angiotensin compared with normal subjects would not be predicted on this basis, and suggests alterations in the nature of the receptor and/or its interaction with the biosynthetic process. In summary, some of the discrepancies in this account may be explained on the basis of the heterogeneity of the conditions which are responsible for hyperproduction of aldosterone. Nevertheless, it is obvious that much more fundamental work needs to be carried out, and it is hoped that further interaction between scientists and physicians will enable a systematic approach to the investigations to take place.

242 References 1.

Symington, T.: Adult adrenal cortex. In: "Functional Pathology of the Human Adrenal Gland", Ed. Symington, T., Livingstone, Edinburgh, pp. 3-181 (1969).

2.

Idelman, S.: The structure of the mammalian adrenal cortex. In: "General Comparative and Clinical Endocrinology of the Adrenal Cortex", Eds. Chester Jones, I., Henderson, I.W., Academic Press, London, Vol. II, pp. 2-199 (1978).

3.

Long, J.A., Jones, A.L.: Observations on the fine structure of the adrenal cortex of man. Lab. Invest. V7, 355370 (1967).

4.

Kime, D., Vinson, G.P., Major, P.W., Kilpatrick, R.: Adrenal-gonad relationships. In: "General Comparative and Clinical Endocrinology of the Adrenal Cortex", Eds. Chester Jones, I., Henderson, I.W., Academic Press, London, Vol.Ill (in press).

5.

Siebenmann, R.E.: Zur lokalisation der aldosteronbildung in der menschlichen nebennierenrinde. Schweiz, med. Wschr. 89, 837-841 (1959). Davignon, J., Tremblay, G., Genest, J.: Failure to isolate Cortisol from a pheochromocytoma. J. clin. Endocrinol. Metab. 20, 1515-1520 (1960).

6. 7.

Neher, R.: Aldosterone and other adrenocortical hormones in human adrenals and adrenal tumours. In: "Aldosterone", Eds. Müller, A.F., O'Connor, C.M., Churchill, London, pp. 1 1-28 (1 958) .

8.

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252 137. Sutherland, D.J.A., Ruse, J.L., Laidlaw, J.C.: Hypertension, increased aldosterone secretion and low plasma renin activity relieved by dexamethasone. Canad. med. Ass. J. 95, 1109-1119 (1966). 138. Miura, K., Yoshing, K., Goto, K., Katsushima, I., Maebashi, M., Demura, H., lino, M., Demura, R., Torikai, T.: A case of glucocorticoid responsive hyperaldosteronism. J. clin. Endocrinol. Metab. 28, 1807-1815 (1966). 139. Ganguly, A., Chavarri, M. , Luetscher, J.A., Dowdy, A.J.: Transient fall and subsequent return of high aldosterone secretion by adrenal adenoma during continued dexamethasone administration. J. clin. Endocrinol. Metab. 44^, 775779 (1977). 140. Holland, O.B., Gomez-Sanchez, E., Kem, D.E., Weinberger, M.H., Kramer, N.J., Higgins, J.R.: Evidence against prolactin stimulation of aldosterone in normal human subjects and patients with primary aldosteronism. J. clin. Endocrinol. Metab. 45, 1064-1076 (1977). 141. Coghlan, J.P., Blair-West, J.R., Denton, D.A., Fei, D.T., Fernley, R.T., Hardy, K.J., McDougall, J.G., Puy, R., Robinson, P.M., Scoggins, B.A., Wright, R.D.: Control of aldosterone secretion. J. Endocr. 81_, 55P-67P (1 979). 142. Dufau, M.L., Villee, D.B., Kliman, B.: Pregnenolone metabolism in an aldosterone secreting tumour of the adrenal and its adjacent adrenal tissue. J. clin. Endocrinol. Metab. 28, 983-991 (1968). 143. Dahl, V., Scattini, C.M., Lantos, C.P.: Comparative biosynthetic studies in a case of primary aldosteronism. J. Steroid Biochem. 7, 715-717 (1976). 144. Raman, P.B., Sharma, D.C., Dorfman, R.I., Gabrilove, J.L.: Biosynthesis of C-18-oxygenated steroids by an aldosteronesecreting human adrenal tumour. Metabolism of [4-14c]progesterone, [1,2-3H]-11-deoxycorticosterone and [4-14c]pregnenolone. Biochemistry 1376-1385 (1965). 145. Fazekas, A.G., Kokai, W., Webb, J.L., Symington, T.: Biosynthesis of aldosterone and other corticosteroids by aldosterone secreting adrenocortical tumours in vitro. Biochim. biophys. Acta 3, 53-67 (1968). 146. Legrand, J.C., Aupetit, B., Zogbi, F., Malmejal, A., Desgrez, P.: Synthèse in vitro de 18-hydroxy-corticostérone par trois adénomes de la zone glomerulêe surrênalienne (syndrome de Conn). I. Rendements de conversion. Bull. Soc. Chim. biol. 51_, 307-320 (1 969). 147. Zogbi, F., Legrand, J.C., Desgrez, P.: Synthèse in vitro de 18-hydroxycorticostêrone et d 1 aldosterone par trois adénomes de la zona glomerulêe surrénalienne (syndrome de Conn). II. Voies métaboliques. Bull. Soc. Chim. biol. 51, 321-326 (1969).

253 148. Marusic, E.T., Mulrow, P.J.: 18-hydroxycorticosterone biosynthesis in an aldosterone secreting tumour and in the surrounding non-tumorous adrenal gland. Proc. Soc. exp. Biol. (N.Y.) V3±, 778-780 (1969). 149. Cain, J.P., Tuck, M.L., Williams, G.H., Dluhy, R.L., Rosenoff, S.H.: The regulation of aldosterone secretion in primary aldosteronism. Am. J. Med. 5^, 627-637 (1972). 150. Fattah, D.I.: Control of aldosterone synthesis in the rat adrenal cortex. PhD Thesis, University of London (1977). 151. Fräser, R., Lantos, C.P.: 18-hydroxycorticosterone: a review. J. Steroid Biochem. 9, 273-286 (1978) . 152. Ulick, S., Nicolis, G.L., Vetter, K.K.: Relationships of 18-hydroxycorticosterone to aldosterone. In: "Aldosterone. A symposium". Eds. Baulieu, E.E., Röbel, P., Blackwell, Oxford, pp. 3-17 (1964). 153. Biglieri, E.G., Slaton, P.E., Schambelan, M., Kronfield, S.J.: Hypermineralocorticoidism. Am. J. Med. 45, 170-175 (1 968) . 154. Brode, E., Grant, J.K., Symington, T. : A biochemical investigation of adrenal tissues from patients with Conn's syndrome. Acta endocr. 4J_, 41 1-431 (1962). 155. Neville, A.M., Symington, T.: Pathology of primary aldosteronism. Cancer J_9, 1855-1868 (1966). 156. Williams, G.H., Dluhy, R.G.: Aldosterone biosynthesis. Interrelationships of regulatory factors. Am. J. Med. 53, 595-605 (1 972) . 157. Müller, J.: Regulation of aldosterone biosynthesis. Springer-Verlag, Berlin (1971). . 158. Schambelan, M., Brust, N.L., Chang, B.C.F., Slater, K.L., Biglieri, E.G.: Circadian rhythm and effect of posture on plasma aldosterone concentration in primary aldosteronism. J. clin. Endocrinol. Metab. _£3, 115-131 (1976). 159. Horton, R.: Stimulation and suppression of aldosterone in plasma of normal man and in primary aldosteronism. J. clin. Invest. 48, 1230-1236 (1969). 160. Slaton, P.E., Schambelan, M., Biglieri, E.G.: Stimulation and suppression of aldosterone secretion in patients with an aldosterone producing adenoma. J. clin. Endocrinol. Metab. 29, 239-250 (1969). 161. Wenting, G.J., Man In't Veld, A.J., Derkx, F.H., Brummelen, P.V., Schalekamp, M.A.D.H.: ACTH-dependent aldosterone excess due to adrenal cortical adenoma: a varient of primary aldosteronism. J. clin. Endocrinol. Metab. £6, 326335 (1978). 162. Spark, R.F., Dale, S.L., Kahn, P.C., Melby, J.C.: Activation of aldosterone secretion in primary aldosteronism. J. clin. Invest. 48, 96-104 (1969).

254 163. Carey, M., Ayers, C.R., Vaughan, E.D., Peach, M.J., Herf, S.M.: Activity of [des-aspartyl1] angiotensin II in primary aldosteronism. J. clin. Invest. 63^, 718-726 (1 979). 164. Oelkers, W., Schoneshofer, M., Schultze, G., Brown, J.J., Lever, A.F., Robertson, J.I.S.: Effect of prolonged lowdose angiotensin II infusion on the sensitivity of adrenal cortex in man. Circulat. Res. 36-37, (suppl. II) 14 9-156 (1975). 165. Hauger, R.L., Aguilera, G., Catt, K.J.: Angiotensin II regulates its receptor sites in the adrenal glomerulosa zone. Nature (Lond.) 271, 176-177 (1978). 166. Catt, K.J., Harwood, J.P., Aguilera, G., Dufau, M.L.: Hormonal regulation of peptide receptors and target cell responses. Nature (Lond.) 280, 109-116 (1979). 167. Kem, D.C., Weinberger, M.H., Higgins, J.R., Kramer, N.J., Gomez-Sanchez, C., Holland, O.B.: Plasma aldosterone response to ACTH in primary aldosteronism and in patients with low renin hypertension. J. clin. Endocrinol. Metab. £6 , 552-560 (1978) . 168. Biglieri, E.G., Schambelan, M., Slaton, P.E.: Effect of adrenocorticotrophin on desoxycorticosterone, corticosterone and aldosterone excretion. J. clin. Endocrinol. Metab. 29, 1090-1101 (1969). 169. Newton, M.A., Laragh, J.A.: Effect of corticotrophin on aldosterone excretion and plasma renin in normal subjects, in essential hypertension and primary aldosteronism. J. clin. Endocrinol. Metab. 28, 1006-1013 (1968). 170. Kem, D.C., Weinberger, M.H., Gomez-Sanchez, C., Higgins, J.R., Kramer, N.J.: The role of ACTH in the episodic release of aldosterone in patients with idiopathic adrenal hyperplasia, hypertension and hyperaldosteronism. J. Lab. clin. Med. 88, 261-270 (1976). 171. Tucci, J.R., Espiner, E.A., Jagger, G.L., Lauler, D.P.: ACTH stimulation of aldosterone secretion in normal subjects and in patients with chronic adrenocortical insufficiency. J. clin. Endocrinol. Metab. 27, 568-575 (1967). 172. Manueldis, L., Mulrow, P.J.: ACTH effects on aldosterone production and mitochondrial ultrastructure in adrenal gland cultures. Endocrinology 9_3/ 1 104-1 1 08 (1 973). 173. Hornsby, P.J., O'Hare, M.J., Neville, A.M.: Functional and morphological observations on rat adrenal zona glomerulosa cells in monolayer culture. Endocrinology 9_5, 12401251 (1974). 174. Miiller, J.: Suppression of aldosterone biosynthesis by treatment of rats with adrenocorticotrophin: comparison with glucocorticoid effects. Endocrinology 103, 20612068 (1978).

255 175. Katz, F.H., Romfh, P., Smith, J.A.: Diurnal variation of plasma aldosterone, Cortisol and renin activity in supine man. J. clin. Endocrinol. Metab. £0, 125-134 (1975). 176. Kem, D.C., Weinberger, M.H., Gomez-Sanchez, C., Kramer, N.J., Levman, R., Furuyama, S., Nugent, C.A.: Circadian rhythm of plasma aldosterone concentration in patients with primary aldosteronism. J. clin. Invest. 52, 22722277 (1973). 177. Vetter, H.M., Berger, M., Armbruster, H., Siegenhalter, W., Werning, C., Vetter, W.: Episodic secretion of aldosterone in primary aldosteronism. J. clin. Endocrinol. Metab. 3^ 41-48 (1974). 178. Biglieri, E.G., Schambelan, M., Brust, N., Chang, B., Hogan, M.: Plasma aldosterone concentration: further characterisation of aldosterone-producing adenomas. Circulât. Res. 34-35, (suppl. 1 I) 183-192 (1974). 179. Wisgerhof, M., Carpenter, P.C., Brown, R.D.: Increased adrenal sensitivity to angiotensin II in idiopathic hyperaldosteronism. J. clin. Endocrinol. Metab. 938944 (1978). 180. Nishikawa, M., Mikami, K., Tamura, Y., Yamamoto, M., Kumagai, A.: Comparative study of cyclic AMP-generating system and lipid metabolism in vitro in ACTH responsive and unresponsive adrenal tumours. Endocr. jap. 26_, 9-17 (1979).

TRANSPORT OF THYROID HORMONES AND THEIR ENTRY INTO CELLS

B. Ramsden and R. Hoffenberg Department of Medicine, University of Birmingham, Queen Elizabeth Hospital, Birmingham, B15 2TH, U.K.

Introduction Three thyroxine binding proteins have been recognised in human serum: thyroxine binding globulin (TBG), thyroxine binding prealbumin (TBPA) and albumin (alb). Because TBG binds the major part of thyroid hormone in plasma, most attention has been paid to it in this chapter, relatively little to TBPA and still less to albumin. Although many of the biochemical properties of TBG are well-known, the literature contains many contradictions. The biochemistry of TBPA is well understood but in contrast to the simple transport role of TBG, the physiological role of TBPA is multifaceted and the significance of its different functions awaits elucidation.

Thyroxine-Binding Globulin Methods of isolation The first report of a successful attempt to isolate 'pure' TBG from human serum was that of Tata (65). There has since been a succession of similar claims, using methods that encompass the usual range of protein fractionation techniques such as salt precipitation, gel filtration, ion-exchange chromatography and gel electrophoresis (60, 26, 43). The singularly difficult nature of the task has even led to the development of some novel variants of these methods e.g. reverse polyacrylamide gel elec-

Hormones in Normal and Abnormal Human Tissues © Walter de Gruyter • Berlin • New York 1981

258 trophoresis (62). No one fractionation technique, however, possesses sufficient resolving power to achieve isolation in a single step, and therefore all these attempts have used combinations of techniques. More recent regimes have been based on an affinity chromatography step which utilises the hormone binding properties of the protein: T4 or occasionally T3 is coupled to a solid matrix to form the affinity adsorbent with which the solution containing TBG is allowed to mix. Proteins not capable of interacting with the coupled hormone are washed off by mild elution buffers and more firmly bound protein is desorbed under more vigorous conditions. This latter step involves the inhibition of hormone protein binding in the affinity chromatography column by altering pH or introducing either hydrogen bond breaking agents or a competitive binding species such as excess T4 with a T3 affinity matrix. Usually the desorbed material requires further purification indicating that the interaction of affinity matrix and protein is not specific (51, 39, 36, 24, 34, 47, 10). Molecular weight The multiplicity of attempts to isolate pure TBG is reflected in the range of molecular weights reported for the protein. These vary between 36,500 (62) and 65,000 (34). In attempting to shed light on what at first appears to be a confusing situation there is a natural inclination to take the mean of the reported values. This should be resisted for a number of reasons. First of all methods to determine molecular weight possess intrinsic error and it is often difficult to assess the accuracy of the individual values reported. This is compounded by the fact that the entity frequently measured is molecular radius and molecular weight is calculated by assuming a degree of spherical character in both sample and standards. Secondly, the chemical nature of TBG should be taken into account. TBG appears to be a delicate protein exemplified by irreversible structural changes resulting from brief exposure to a pH of

259 less than 5 (22) . Often lower molecular weights are associated with more vigorous extraction regimes. The lowest molecular weight was found (62) using polyacrylamide gel electrophoresis where a change in pH between 4.5 and 8.9 occurred. Even where no drastic conditions are employed in the isolation, chemical modification of the structure would seem likely to occur. Evidence for this contention can be gained by inspection of the reported analyses of TBG summarised in Table 1. Of particular interest are the differences in sialic acid content. TBG is known to be a glycoprotein with several sialic acid moieties. Loss of these leads to rapid clearance from plasma in normal individuals, yet some so-called pure native TBG preparations appear to have few if any sialic acid moieties per molecule. These values should be regarded with caution, as should those unsupported by analytical data for this essential component. Even taking these considerations into account universal agreement is not obtained although the range is considerably reduced (54,000-61,000). Composition and structure of TBG The difficulty of isolating pure intact TBG from serum is reflected in studies of composition and structure. Even simple amino acid analyses, some of which are summarised in Table 1 (a), vary widely, although there tend to be more areas of agreement in recent reports. Nevertheless, a marked discrepancy still exists concerning the percentage of the molecule made up of amino acid or carbohydrate residues. The carbohydrate content is said to vary from 13 to 24%. The points which do gain wide or universal agreement are: i)

TBG is a glycoprotein possessing several sialic acid residues as the terminal moieties of the carbohydrate chains in the molecule,

ii)

it is sufficiently acidic to possess an a^-a^ mobility under standard electrophoretic conditions,

260

Table 1. Amino Acid and Carbohydrate Content of TBG Amino acid moities/mol TBG Reference Alanine

39

47

22

34

62

60

35

30

28

29

32

Arginine

16

17

6

7

4

27 22

Aspartic Acid

42

45

36

38

26

25

1/2 Cystine

11 63

8 61

5 42

3

29 12

19

Histidine

32 14

11

13

Isoleucine Leucine

19 47

17 37

18

20 41

28

8 43

Lysine

22

29

18

18

12

Methionine

11 22

8

28 12

15

6

Glutamic Acid Glycine

Phenylalanine

46 20

30

5 34

23

25

7 12

11

17

22

23

12

3 14

32

20 49

15 29

8 31

16 24

27 19

26

28

25

26

20

18

4

4

4 12

Proline Serine

29

Threonine Tryptophan

38

-

Tyrosine

12

15

9

10

3 4

Valine

27

25

27

24

21

-

-

7

iii) the molecule possesses only one hormone binding site, and iv)

the protein section is made up of a single polypeptide chain (22) By circular dichroism techniques approximately half the

polypeptide chain has an ordered array, equally divided between a-helix and 3-structures (24). Using a similar preparation, initial studies to determine the primary sequence of the polypeptide chain were described (11). The carboxyl terminal residue

261

Table 1. Amino Acid and Carbohydrate Content of TBG b) Carbohydrate residues /mol TBG Reference Galactose Glucose

39

47

22

62

6

7

13

6

6

2

7 1

6

5

7

22

11

10

4

2

Mannose Fucose (N-Acetyl)

12

Glucosamine

12

Galactosamine

4

Sialic Acid

6

70, 71 9 12

19

Xylose Total % of Molecule

60

17 9

9

1 13

23

14.6

was found to be leucine (22). With a different preparation of TBG, the carbohydrate section of the molecule was found to be composed of four branched chains, two of which were thought to be identical (70, 71). The primary sequence of one glycopeptide chain (A chain) is shown in Fig. 1. The oligosaccharide section of the two identical chains (B-chain) possesses a similar structure to the abovementioned, differing only in the mode of linkage between the GlcNAc and Man residues indicated by the broken vertical lines. The fourth oligosaccharide unit is similar to B chains but appears also to possess an additional NeuAc-Gal-GlcNAc branch from the Man* moiety (Fig. 1). Korcek and Tabachnick (39) reported that their preparation of TBG had a number of a long-chain fatty acid residues non-

262

NeuNAc^-^Gal- P1 ,4 GlcNAc--11^-Ma \ct1 ,3 R1 4 R1 4 R \ Man ' GlcNAc ' GlcNAc-^-Asn 'a1 ,6 a2,6 ,2„* NeuNAcGal- 31 ,4 -GlcNAc-11 — ! —Man i i i Fig. 1. Structure of A chain of thyroxine binding globulin. NeuNAc = N-Acetylneuramic acid - Gal = Galactose Man = Mannose - GlcNAc = N Acetylglucosamine Asn = Asparagine covalently associated with the structure. Although it seems clear that TBG possesses only one polypeptide chain, there have been a number of reports demonstrating microheterogeneity by isoelectric focusing of pure TBG (44, 34). The reasons for this microheterogeneity are still purely speculative but may represent either different phenotypes or fragments arising from the initial stages of TBG catabolism. The suggestion by Gershengorn et al. (24) that they may arise as a methodological artefact of either the isolation procedure or the process of electrofocusing seems relatively unlikely, as microheterogeneity has been shown in TBG unextracted from serum, using a combination of electrofocusing and immunoprecipitation. This microheterogeneity was qualitatively unaffected by the conditions of electrofocusing i.e. focusing from acidic, alkaline or neutral regions of the gel (5). Since the isoelectric point is below the point of pH sensitivity, artefactual generation cannot be wholly excluded. The actual isoelectric points reported by authors vary as do the number of bands. These differences may represent the technological difficulties associated with measuring pH in thin layers. The different number of subspecies reported may also represent the

263 selective influence of isolation regimes on populations present in whole serum. Finally in this brief review of TBG structure, reference should be made to a species of TBG present in some pathological sera, known as 'slow TBG' in view of its markedly slower electrophoretic mobility. Although 'slow TBG' possesses similar hormonal binding properties to those of 'native' TBG, it has a lower molecular weight, 5-10% smaller than its parent molecule (39). Also it possesses few if any sialic acid moieties. 'Slow TBG' would appear to be a fragment formed during catabolism of TBG, not usually observed due to its rapid clearance in normal subjects. Assay of TBG Early methods of assessing serum concentrations of TBG were estimates of T4 or T3 binding capacity rather than concentration. Indeed these are still popular for routine clinical use, in the form of modified versions of the T3 uptake test which originally used red blood cells (31). Developments in immunological techniques have now led to a wide variety of assay methods including a range of radioimmunoassays (42, 34, 32), single radial immuno diffusion (37) , quantitative Immunoelectrophoresis (4) and, most recently, nephelometric assay (54). Competitive ligand binding assays have also been described (45). Characteristics of the various methodological approaches are well-recognised, and vary between extremes of highly automated, large throughput, capital intensive radioimmunoassays to ones suitable for much more modest budgets but requiring greater manual involvement e.g. single radial diffusion. The choice of technique is now primarily dependent on local circumstances surrounding its use, rather than on research expertise to prepare the basic components of the assay. Normal range of serum TBG concentration Because of the difficulty associated with isolation of TBG, a

264 number of assays have arisen around any one source of pure TBG reference material. Thus families of assays may be classified by their source of standard. Those based on the preparation of Pensky and Marshall (51), which was used in the first radioimmunoassay described for the protein (42), tend to have high values for the normal range (approx 30 mg/1) whereas subsequent assays quote values 40-70% lower. The values are summarised in Table 2 and refer usually to young/middle-aged adults. Despite this difference in standards, assays often show good ranking agreement of samples with varying TBG concentrations (2). Most but not all groups report a greater concentration in normal females not taking oral contraceptives than in normal males. At birth TBG concentration is higher than in adult life (35), and most authors report a steady decrease with advancing age until stable adult levels are attained. Stubbe et al. (64) however found levels maintained until adolescence whereupon an abrupt decrease occurred with the onset of puberty. Although this has not been confirmed by other workers (18), the number of subjects studied in all cases is relatively small and the issue requires further clarification. Old-age is also said to be associated with increased TBG concentration compared to young/middle aged adults, often with a lower T4 concentration than would be anticipated from results from younger subjects (33). Again the numbers studied are small and it is difficult to differentiate between the intrinsic effect of the ageing process and socio-economic and medical differences between elderly and younger populations. Factors regulating metabolism of TBG Although synthesis of TBG by liver was suggested by earlier studies, the first unambiguous demonstration was by Glinoer et al. (28), using isolated monkey hepatocytes. Despite the short life-span of these cells in culture, over a six hour period an increase in amount and concentration of TBG in the culture medium was demonstrated. That this represented de novo synthesis

265

Table 2. Serum TBG Concentration in Normal Healthy Euthyroid Adults (Values are mean + SD, * denotes range) Concentration (mg/1) Method* Standard

Reference

both sexes

males

females

30 .1+0.6

27.7+0.6

RIA

A

42

34+0.7

CLB

A

12

28.5+0.6

RIA

X

10

17.8+2.1

RIA

B

23

14.8+4.6

13.7+3.7

16.6+5.6

IEP

B

8

11.6+0.6

11 . 2 + 0.5

12.5+8

CLB

A

1

31.6+5.4

RIA

X

32

9.7+1.4

RIA

X

34

20.1+4.4

RID

X

14

19.7+3.9

18.3+0.45

CLB

A

45

34 + 6

32 + 6

RID

X

37

36 + 7

9.8-17.8*

A - Standard used in the assay was based on the TBG preparation of Pensky and Marshall (51). B - Standard used in the assay was based on the TBG preparation of Gershengorn et al. (23). X - Standard used in the assay was based on the authors' own TBG preparation. * RIA - radioimmunoassay, CLB - competitive ligand binding, IEP - Immunoelectrophoresis, RID - radial immunodiffusion.

266

rather than leakage of preformed material from damaged cells 14 was shown by the incorporation of C-leucine into the protein. In the human a similar site of synthesis is assumed. The plasma half-life of TBG is approximately 5 days in the normal healthy adult human (56), whilst in the monkey it is about 2.7 days (29). A variety of pathological conditions, hormones, drugs and genetic factors give rise to altered serum concentrations of TBG (Table 3). Oestrogen whether endogenous as in pregnancy or exogenous results in higher than normal concentrations. These have been shown to be almost wholly due to oestrogen action on hepatic synthesis (27, 29). Asparaginase is reported to bring about an abrupt cessation of TBG synthesis in humans (20). Numerous reports have claimed that TBG concentrations are altered in thyroid disease, although there is not universal agreement as to the nature of the changes. In our experience TBG concentration is elevated in hypothyroidism and normal or low normal in hyperthyroidism. Replacement therapy in myxoedema restores TBG to normal, whilst antithyroid treatment for hyperthyroidism results in an increase in TBG concentration although this still remains within the normal range (6). The plasma halflife of TBG is longer in hypothyroid subjects (10). Gershengorn et al. (21) using isolated monkey hepatocarcinoma cells have shown increasing sub-physiological doses of T4 enhance TBG synthesis whilst supraphysiological doses inhibit it. It would appear therefore that thyroid dysfunction affects both the synthesis and catabolism of TBG. Most instances of congenitally altered TBG levels have been shown to be inherited by an X-linked co-dominant mode. In preliminary investigation of three families with elevated TBG concentrations, no male to male transmission was observed and affected heterozygotic females had levels ranging between hemizygotic males and normal subjects (7). This finding was confirmed in a further seven families with elevated TBG and six with congenital TBG deficiency (unpublished results W.A. Burr). That these changes are due to altered synthesis rather

267 Table 3. Instances of Altered Serum TBG Concentration or Capacity Increased Concentration

Reduced Concentration

Congenital

Congenital

Pregnancy

Thyrotoxicosis

Hypothyroidism

Acute Stress

Viral hepatitis

Protein-losing enteropathies

Acute Intermittent Porphyria

Renal dialysis and nephrotic

Hepatocellular Carcinoma

syndrome

Oestrogen therapy

Starvation and malnutrition

5-Fluorouracil therapy

Androgen therapy

Methadone therapy

Asparaginase therapy

Phenothiazines, perphenazine

Corticosteroids, anabolic

Clofibrate

steroids Reduced Capacity Phenytoin therapy Salicylate therapy Theophylline therapy Barbiturate therapy Heparin therapy

than catabolism may be inferred from the finding of Refetoff 125 131 et al. (56) that the catabolism of I or I-TBG was not different in subjects with inherited TBG deficiency or excess when compared to normal subjects. The incidence of congenitally absent or low TBG is about 1 in 10,000 live births. These figures stem from the results of screening programmes for neonatal hypothyroidism using a T4 assay as the primary screen. In such programmes congenital TBG deficiency is a major non-methodological cause of false positive detection of hypothyroidism. Hitherto detection of altered TBG concentration was largely for-

268

tuitous as neither absence nor elevation of TBG concentration is inherently associated with thyroid dysfunction.

Thyroxine Binding Prealbumin (TBPA) Whereas there is considerable doubt about the physico-chemical properties of TBG, little uncertainty remains concerning TBPA whose primary amino acid sequence (38) and three dimensional structure (3) has been largely determined. TBPA is composed of four identical polypeptide chains held together primarily by non-covalent bonds. It contains no carbohydrate or lipid residues. The symmetrical tetrameric structure has a cavity running through it formed by amino acid residues in 3~pleated sheets, giving two identical hormone binding sites. These display the phenomenon of negative co-operativity, in that as one site is occupied the affinity of the second is reduced (17). The binding of T3 to TBPA is much weaker than that of T4 (13). In normal serum less than 10% of total T3 is bound to TBPA. The absence of an amino group in the hormone increases its relative affinity for TBPA compared to TBG so that the naturally occurring acetic acid analogue of T4

(3,5,3151-tetraiodothyroacetic

acid, tetrac T4A) and the synthetic propionic acid analogue are bound in serum exclusively by TBPA. Tetrameric TBPA also has found binding sites for another protein, retinol binding protein (67), although in serum these two combine in complexes with molecular ratios of one to one. TBPA or the TBPA-RBP complex may also be involved in transport of Zn + + ions in plasma (59). In contrast to the relatively simple physiology of TBG, that of TBPA is more complex and little understood. It binds several different entities, its role in T4 and T3 transport being more limited than that of TBG in quantitative terms. As with TBG, it is synthesised by the liver and has a very short plasma half-life, approximately 1.5 days. In most states of acute shock and chronic illness serum concentrations are

269 reduced, largely due to diminished hepatic synthesis (48, 64). When TBPA levels are very low this is associated with a very poor prognosis. One conspicuous exception is renal insufficiency, where serum TBPA levels may be markedly elevated (66) . Glucocorticoid administration increases serum TBPA concentration as a result of increased synthesis (50). We are unaware of any reported

case of congenital TBPA deficiency or excess.

Interaction of Ligands Binding Proteins As mentioned above there are three major binding proteins for iodocompounds in serum and they display different types of binding. TBG has only one binding site per molecule (40), whereas TBPA has two interactive sites and albumin has one high affinity site and up to six lower affinity sites (61). There is no conclusive evidence as to the precise number of secondary sites on albumin, nor whether they interact amongst themselves or with the primary high affinity site. The approximate distribution of the various iodocompounds in serum is shown in Table 4. It can be seen that loss of the amino group from the amino acid side-chain favours binding to TBPA and abolishes binding to TBG and the presence of three iodine atoms or less reduces binding to TBPA to a greater extent than to TBG, although binding to both is

diminished.

Because only T4 and T3 are of known clinical importance and they are present in plasma in much higher concentrations than any of the others, only the binding of these two has been dealt with on a theoretical basis to any extent (57, 15, 69). In vitro, where equilibrium conditions prevail, the distribution of T4 and T3 among the various types of binding sites and free hormone concentrations may be computed from a knowledge of total hormone and binding protein concentrations (52). This takes into account effects such as negative co-operativity between binding sites, the presence of non-interactive sites on the same protein and the competition.between the two hormones. It is also capable

270 Table 4. Relative Distribution of Iodocompounds Among Serum Proteins as shown by Immunoprecipitation* or Immunoelectrophoresis+. (Results from Burr, Scott and Ramsden (unpublished) and from Woeber and Ingbar (1 968) + ) . TBG

TBPA

Alb

T4

70*

20* +

10* +

T3

75-80*

reverse T3

47*

Split T2

52*

T4A

631-638 (1974). 43. Winters, S.J., Janick, J.J., Loriaux, D.L., Sherins, R.J.: Studies on the Role of Sex Steroids in the Feedback Control of Gonadotropin Concentrations in Men. II. Use of the Estrogen Antagonist, Clomiphene Citrate. J. Clin. Endocrinol. Metab. 48, 222-227 (1979).

INHIBITION BY THYROID HORMONE BINDING PROTEINS AND RELAXATION OF INHIBITION BY THE HORMONES

J. S. Orr Department of Medical Physics, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London, W12 OHS, U. K.

Introduction Hormones, by definition, stimulate and activate. One important recognised role of the thyroid hormones results in a stimulation of protein synthesis and a general enhancement of metabolism. The physiological insight gained from the conventional view of this role has proved invaluable in the diagnosis and treatment of pathological conditions affecting the control of the production of thyroid hormones. In striking contrast to the successes of thyroid endocrinology is the very disappointing progress that has been made towards an understanding of the role of the thyroid hormones in the metabolic response to trauma, including surgical trauma. Current dogma has been empirically very useful where the thyroid gland and its control are malfunctioning in an otherwise healthy body. However, it has been apparent for many years that observation is inconsistent with dogma where the thyroid gland and its controls function correctly in a sick or abnormal body (1-11). In the latter case, it is not at all clear whether the changes seen in thyroid hormones are causal, permissive, or simply responsive with respect to alterations in metabolism. The changes are certainly closely linked with changes in protein metabolism and the purpose of this article is to examine the problems from the other side of the fence which marks the territorial limits of thyroid endocrinology. The first part of the discussion is in very general terms.

Hormones in Normal and Abnormal Human Tissues © Walter de Gruyter • Berlin • New York 1981

492 Turnover and Stability A considerable portion of body weight is composed of regulated amounts of proteins, many of which are turning over rapidly (12). For example, rat liver replaces 70% of its protein every 4-5 days from dietary sources. This turnover is largely intracellular, as most cells have a much longer life than their protein constituents. The high rates of both synthesis and degradation must be closely controlled to maintain the correct stable steady state levels. Some indications of the nature of the control of the system can be drawn from a consideration of its stability as material pours through the synthetic processes to form structural or functioning proteins which last only a few days, hours, or even minutes before being degraded and lost or recycled. Both the synthetic and degradative processes must be able to respond rapidly to changes in supply and demand. These responses to outside influences must also be closely co-ordinated and linked internally to provide the stability of function which is required for the maintenance of the internal environment.

Speed of Compensation It is ironic that many studies of the homeostatic state can be made only by disturbing it. When the steady flow of synthesis into a pool of functioning protein is disturbed, the level or concentration starts to change. To compensate for the disturbance and restore normal levels, the homeostatic mechanism must either counteract the disturbance of synthesis or modify the rate of degradation. The rate at which levels change after an alteration in synthesis rate reflects the normal fractional rate of random degradation. Some interesting indications of mechanism can be found from a consideration of the requirements imposed by a need for stability in a system tending to react at the degradation rate.

493 If the compensatory mechanism comes into action at a slower rate than the change of the protein being controlled, there will be a tendency for a much greater deviation from the optimal level to occur than would be the case with a rapid compensatory mechanism. Further, with slow compensation there will tend to be a build up of oscillations, since after the level has been started to return towards normality, it will be given time to overshoot, and to overshoot by a progressively greater amount, before the compensatory process catches up each time and reverses the direction of change. Therefore stability of the system will normally require that the compensatory or control mechanisms react more rapidly than the system they control. The fast compensation is able to reduce oscillations by catching up and turning the level back at a smaller deviation from normal each swing. When the compensation required is a change in the rate of degradation this may be brought about by a change in the concentration of a specific degradation system. Such a degradative enzyme, according to the above argument, would require to have a shorter half life than the protein whose level it controlled. Similarly the level of the degradative enzyme may be controlled by its own degradative enzyme. Taking such a model mechanism to its extreme, it would be possible to propose that the degradation of each protein requires another specific degradative protein. However, the continual replacement of a wide variety of proteins would then require that for each there exists a degradative protein of shorter half life, and that for each such degradative protein there exists another of even shorter half life, etc., Big fleas have little fleas Upon their backs to bite 'em; Little fleas have lesser fleas And so ad infinitum. The unreasonableness of the consequences of such a proposal make it reasonable to accept that changes in the operation

494

of a series of control steps do not depend on changes in the concentrations of a complete sequence of separate species of molecules, although a small number of such steps may exist. These arguments apply equally to degradative and to synthetic processes, which must contain a damped and stable mechanism for maintaining homeostasis.

Conformational Change One way for a control system to avoid these dilemmas would be for some of the controlling molecular species to alter their activity as a result of conformational change in the molecular structure of a single species of molecule. Another would be a rapid change in the assembly arrangement of a complex of several molecules. Such changes in form could be rapid and reversible, providing very sensitive control of activity without excessive consumption of material. Changes in form could produce a large effect on the chemical reaction being controlled for a small change in the concentration of the substance or substances causing the form change.

Inhibitors and

Negative Feedback

An essential feature of any homeostatic system is that a change in one direction should produce a tendency to change in the opposite direction. In the preceding discussion of stability, it was assumed that an increase in the amount of the protein being controlled brought about a tendency for the amount to decrease. This property by which a change in any direction tends to cause a counteracting change in the opposite direction, commonly termed negative feedback, is essential for stability. When the activity of a degradative process is too low and insufficient, allowing an increase in the amount of the substance being controlled, the control on the degradative process must

495

allow or cause an increase in its activity. If synthetic activity is too great it must be reduced. Thus negative feedback is required at many of the stages of control. Negative feedback can be directly provided either by inhibitors or activators. Overproduction can be countered by either an increase of inhibitor or a decrease of activator. However, a decrease of activator requires an inhibition of synthesis of activator or an inhibition of whatever controls the degradation of activator. Thus, negative feedback requires inhibitors, which may act either directly or indirectly.

Local and Systemic Control Systems consisting of functional proteins must not only have local internal control systems particular to their function, whose properties have been discussed above, but must be subject to complementary controls responsive to the requirements of the whole organism. The systems rely on common pools of raw materials and are subjected to common changes in physiological conditions. The local controls can be expected to be organ specific, tissue specific, cell specific, or even more localised. They must, however, have certain common features, so that the systemic controls can be economical and efficient as well as effective. Where the action of a systemic control is to change the stable equilibrium quantities of proteins, the magnitude of the effects on the synthetic and on the degradative systems must be different. Otherwise, the equilibrium quantity would remain constant and only turnover rate would increase. The effects of growth hormone must be of this nature. Although the magnitude of the effects must be different, they must nevertheless remain closely co-ordinated. This would be simpler to achieve if the systemic control acted by modifying the local control, rather than independently.

496

Nearly all the systemic controls studied in the field of endocrinology are exercised by small and simple molecules. These molecules are categorised generally as hormones, that is, as activators. Their function must therefore be associated with other molecular species functioning as inhibitors. Since the systemic control is exercised by activators, it is not unlikely that the locally effective control is exercised by inhibitors.

Thyroid Hormones It is possible to regard the role of the thyroid hormones as a systemic mechanism which affects a wide range of local control systems, adjusting and tuning the effectiveness of these local tissue specific controls to suit overall physiological requirements. Since the thyroid hormones are by definition activators, the primary and tissue specific control on which they act must contain an inhibitor as an essential part of the system. A simple and economical arrangement would be one in which essential negative feedback for a system is provided by an inhibitor, the effectiveness of which is reduced when bound to a thyroid hormone molecule. The hormone, by this means, would act to produce stimulation and activation. The simplicity of thyroid hormone molecules would allow them to bind in a similar way to a wide range of specific inhibitor protein molecules. Each thyroid hormone benzenoid ring acts as a rigid mounting for the heavy iodine atoms which must have particular spacings and relative positions to be functional. A degree of freedom is allowed by the ability of the two rings to change the angular relationship of their mean planes by rotation of the linking bonds. The iodine atoms, when located accurately in receptor sites of the inhibitor molecules, could cause changes in inhibitor structure.

497 Properties of Thyroxine Binding Pre-Albumin (TBPA) In the light of the general discussion above, it is of interest to review some of the recent suggestions regarding the properties of human TBPA. This molecule also binds T 3 > The X-ray crystallographic studies of the structure of the whole molecule and the properties of its thyroxine binding sites have agreed very well with chemical studies of binding reactions (13-18). The picture to emerge is one of a tetramer of four identical sheet-like sub-units linked as a double sandwich to form a structure with a hollow channel through the centre. Two external features are of particular interest: 1) The presence of four specific sites which bind retinol binding protein (RPA). TBPA has a molecular weight of about 55000. and that of RPA is about 21000. The significance of this association of two binding proteins, and of the association of the hormone and the vitamin, is not known, but the presence of retinol affects the binding of the two proteins and binding appears to produce conformational changes. 2) The presence of two arms which can lie close together, and whose adjacent faces appear to have surfaces which would fit snugly around a length of helical molecule. This concave helical structure arises naturally from the right hand twist of the sheets and the molecular symmetry. Although this feature suggests a number of ways in which TBPA could act as an inhibitor of protein synthesis, there is no clear evidence that it enters the cells of any particular tissue, nor that it has a special affinity for a particular cellular constituent. A large number of receptor sites of differing specificity for thyroid hormones have been found in the cytoplasm and nuclei of many types of cells. It has not been suggested that any of these could be identified with TBPA, but there may be a close relationship. The sites within the TBPA molecule which are receptive to both T. and T.. lie within the central channel. There are two 4 3

498 sites, apparently identical, but normally only one is occupied. This may be due to negative co-operativity indicating conformational change. The hormone molecule enters well inside the protein lattice, with the benzenoid rings innermost. As the planes of the two rings are at right angles (19), the hormone molecule must turn when partly in. The iodine atoms fit into parts of the protein structure which are connected to the projecting arms. The 3' iodine of T^ can fit into either of the 3" and 5' T^ iodine sites. It appears possible that the effect of the presence of certain iodine atoms may be the cause of flexure of the structure which results in a considerable alteration in the separation of the arms.

Picture of Action A pictorial analogy to this possible action of the hormone can be obtained by visualising the TBPA molecule or its near relations as a sort of padlock. The hoop of the closed padlock prevents transcription from whatever synthetic template is locked in. When the hormone molecule key is inserted with a half turn into the keyhole in the centre of the TBPA molecule, the iodine atoms act as the projections on the key bit. By pressing on the tumblers in an appropriate direction and extent, the lock is released and the hoop can open to allow the controlled synthesis to proceed. Thus the hormone molecules would produce their effect as activators by relaxing or preventing the inhibiting action of a proportion of the inhibitor molecules that control the synthetic production of the system.

Some Implications The value in medicine of any view of physiological processes must be measured by its ability to co-ordinate observations and predict or guide diagnostic or therapeutic procedures.

499 The view presented in this article has some implications which can be briefly explored here. One implication is that the synthesis within a system will tend to be inversely proportional to the concentration of free protein inhibitor. The more inhibitor the less will be the output. The production will tend, on the other hand, to be directly proportional to the concentration of inhibitor molecules which have been inactivated by the binding of a hormone molecule. The more inactivation of inhibitor by hormone the more will be the output. Thus the production will be approximately proportional to the ratio of inactivated hormone bound inhibitor to active free inhibitor. That is to say, when all other factors are in a standard condition; * 4- • "4Activity

co

T

_ _ B

P

H

where TBPH is hormone bound to specific proteins and UTBP is the residual binding capacity of these proteins. Using the concept of a primary protein inhibitor relaxed by thyroid hormone, the possible effects of variations in other factors due to illness, trauma, or other abnormalities, can be explored. Acute changes in

dietary intake, liver disease, ab-

normal muscle conditions, stress and other metabolic disturbances, are fields for study. Some of the discrepancies between observation and theory referred to in the introduction may be resolved. Another implication is that the receptor site in the inhibitor could be occupied by a naturally produced molecule which did not inactivate the inhibitor but which prevented an active hormone molecule from entering. Structural response to the binding of other constituents of a complex could also prevent inactivation. Thus the simple relationship between inhibitor and hormone could be modulated if required by local or temporary needs. The existence of such modulation opens the possibility of utilising its mechanisms for diagnostic or therapeutic purposes.

500 References 1.

Robbins, J., Rail, J.E.: Hormone transport in circulation: Interaction of thyroid hormones and proteins in biological fluids. Recent Prog. Horm. Res. J_3, 161-208 (1957).

2.

Hanbury, E.M.: Thyroid function after trauma in man. Metabolism 8, 904-912 (1959).

3.

Inada, M., Sterling, K.: Thyroxine turnover and transport in active acromegaly. J. clin. Endocr. Metab. 21_, 1019— 1027 (1967).

4.

Bellabarba, D., Inada, M., Varsano-Aharon, N., Sterling, K.: Thyroxine transport and turnover in non-thyroidal illness. J. clin. Endocr. Metab. 28, 1023-1030 (1968).

5.

Hollander, C.S., Bernstein, G., Oppenheimer, J.H.: Abnormalities of thyroxine binding in analbuminaemia. J. clin. Endocr. Metab. 28, 1064-1066 (1968).

6.

Irvine, C.H.G.: Effect of exercise on thyroxine degradation in athletes and nonathletes. J. clin. Endocr. Metab. 28, 942-948 (1968).

7.

Oppenheimer, J.H.: Role of plasma proteins in the binding, distribution and metabolism of the thyroid hormones. New Engl. J. Med. 278, 1153-1162 (1968).

8.

Harland, W.A., Orr, J.S., Richards, J.R.: Increased thyroxine secretion following surgical operation. Scott, med. J. V7, 92-97 (1 972) .

9.

Kirby, R., Clark, F., Johnston, I.D.A.: The effect of surgical operation of moderate severity on thyroid function. Clinical Endocrinology 2, 89-99 (1973).

10. Harland, W.A., Orr, J.S.: The effect of Clofibrate on thyroxine metabolism. In: "Thyroid Hormone Metabolism", Eds. Harland, W.A., Orr, J.S., Academic Press, London, pp. 6587 (1975). 11. Prescott, R.W.G., Yeo, P.P.B., Watson, M.J., Johnston, I. D.A., Ratcliffe, J.G., Evered, D.C.: Total and free thyroid hormone concentrations after elective surgery. J. clin. Path. 32, 321-324 (1979). 12. Schimke, R.T.: On the properties and mechanisms of protein turnover. In: "Intracellular Protein Turnover", Eds.Schimke, R.T., Katunuma, N., Academic Press, London, pp.173-186 (1975) 13. Blake, C.C.F., Swan, I.D.A., Rerat, C., Berthou, J.,Laurent, A., Rerat, B.: An X-ray study of the subunit structure of prealbumin. J. Mol. Biol. 61^, 217-224 (1971 ). 14. Blake, C.C.F., Swan, I.D.A., Geisow, M.J., Oatley, S.J., Rerat, C., Rerat, B.: Crystal structure of human plasma prealbumin and its interaction with thyroxine. In: "Thyroid Hormone Metabolism",Eds. Harland, W.A., Orr, J.S., Academic Press, London, pp. 23-34 (1975).

501

15. Blake, C.C.F., Oatley, S.J.: Protein-DNA and protein-horraone interactions in prealbumin: a model of the thyroid hormone nuclear receptor ? Nature 268, 115-120 (1977). 16. Blake, C.C.F.: X-ray studies of protein-DNA-hormone interactions. Physics Med. Biol. 23, 373-384 (1978). 17. Robbins, J.: Structure and function of thyroxine-transport proteins. In: "Thyroid Hormone Metabolism", Eds. Harland, W.A., Orr, J.S., Academic Press, London, pp. 1-22 (1975). 18. Robbins, J., Cheng, S-Y., Gershengorn, M.C., Glinoer, D., Cahnmann, H.J., Edelnoch, H.: Thyroxine transport proteins of plasma. Molecular properties and biosynthesis. Recent Prog. Horm. Res. 34, 477-517 (1978). 19. Cody, V.: Thyroid hormones: crystal structure, molecular conformation, binding, and structure-function relationships. Recent Prog. Horm. Res. 3£, 437-469 (1978).

ENTRY OF INSULIN INTO TARGET CELLS

I.D. Goldfine Cell Biology Laboratory, Mount Zion Hospital and Medical Center, San Francisco, CA 94120, U.S.A. A.L. Jones, G. Hradek Department of Medicine, University of California, San Francisco, CA 94143, U.S.A. B.M. Kriz, K.Y. Wong Cell Biology Section, Veterans Administration Hospital, San Francisco, CA 94121, U.S.A.

Introduction Insulin regulates the metabolism of most cells in man. The actions of insulin range from rapid effects on membrane transport (seconds), through intermediate effects on protein synthesis and enzyme activation (minutes), to delayed effects on RNA and DNA synthesis (hours) (Table 1). Although insulin has been studied for over five decades, the mechanism through which insulin regulates these diverse effects on or in target tissues is still unknown. In contrast, glucagon, a hormone discovered later than insulin, is known to act via the intracellular second messenger, cyclic AMP. No specific intracellular second messenger system, however, has been established for insulin (6). Insulin binds to receptors on the plasma membrane and it is likely that this binding leads in turn to changes in cell surface transport. The exact nature of the insulin receptor is unknown. It appears, however, to have a subunit structure, one subunit being a glycoprotein (7-9). Recent studies also indicate that insulin enters the interior of target cells and then interacts with several

Hormones in Normal and Abnormal Human Tissues © Walter de Gruyter • Berlin • New York 1981

504 intracellular structures. These findings raise the possibility, therefore, that insulin or a fragment of the hormone may act as an intracellular second messenger to regulate events such as protein and RNA synthesis. IM-9 human lymphocytes in permanent culture have been an important tool for studying insulin receptors (10, 11). These cells have large numbers of insulin receptors with binding characteristics identical to those of receptors on other cells (10, 11). In addition, IM-9 lymphocytes are easily grown in Table 1: Actions of Insulin at Various Subcellular Levels(1-5)

Rapid Cell membrane Stimulation of transport Change of m e m b r a n e potential Intermediate Cy tosol Activation and inhibition of enzymes Endoplasmic reticulum A c t i v a t i o n and inhibition of enzymes Ribosome Increased protein synthesis Mitochondria A c t i v a t i o n of enzymes Lysosome Inhibition of protein

degradation

Delayed Nucleus M o d u l a t i o n of DNA and R N A

synthesis

sizable quantities. Further, the insulin binding characteristics of these lymphocytes remain stable even after many passages. The cells have recently been employed in our laboratory to study both the internalization of insulin and the subsequent binding of insulin to intracellular structures (12-13).

505 Direct binding of insulin to lymphocyte nuclei. Studies from our laboratory

(14-17) and the laboratories of Horvat et al.

(18) and Goidl

(19) demonstrated that highly purified

from liver have specific binding sites for insulin. insulin binding sites were also detected in purified prepared from IM-9 human-cultured lymphocytes

nuclei

Specific nuclei

(Fig. 1). Neither

electron micrographs of the nuclei nor studies of plasma membrane marker enzymes revealed contamination of the nuclei with other cellular components

(12).

.08-

13 .06

z

.04-

0

1

1 10°

1

1 102

1

1 104

1

1 106

INSULIN CONCENTRATION (ng/ml)

Figure Nuclei with 1 nuclei

. . 125 1. Specific binding of I-insulin to isolated lymphocyte nuclei. were first isolated from IM-9 lymphocytes (12) and then incubated ng I-insulin/ml for two hours at 24° as described for liver (14).

506 Binding of insulin to intact cells and subsequent translocation of insulin to the nucleus. When IM-9 human-cultured lymphocytes were incubated at 37° in complete Eagle's culture medium with 10% fetal calf serum, the uptake of insulin by the intact cells was very rapid. One-half maximal uptake occurred within 30 sec. and maximal uptake occurred within five min. (Fig. 2). If the cell concentration was kept below 10^/ml, degradation of insulin in the supernatant even after 2 h of incubation was minimal

MINUTES

Figure 2. Whole cell uptake of I^insulin and its subsequent translocation to the nucleus. IM-9 lymphocytes (10 /ml) were incubated with 0.1 nm insulin and whole cell uptake measured (top). Cells were then subfractionated and nuclear binding measured (bottom). Taken from reference 12.

507 (less than 10%). In addition, the radioactivity associated with cells after extraction twice with 0.1% Triton X 100, 5M urea, and 1M acetic acid and filtration over Sephadex G-50 as described for liver cells (20), represented intact insulin (Fig. 3). When nuclei were isolated from cells washed free of extracellular insulin and lysed in MgCl_, the insulin radioactivity was

Void •

Insulin •

Soil •

o c • w k«

a

20 ML ELUTED

Top: Gel filtration profile of X-insulin not reacted with lymphocytes. Middle: Gel filtration profile of 125x-i n sulin extracted ( 2 0 ) from lymphocytes after 30 minutes of incubation. Bottom: Gel filtration profile of I-insulin extracted ( 2 0 ) from lymphocyte nuclei after 30 minutes of incubation.

508

associated with these nuclei (12). Extraction of the radioactivity revealed that it represented intact hormone (Fig. 3). The time course of this translocation of insulin to the nucleus lagged behind total cellular uptake; nuclear binding was onehalf maximal within 5 min. and maximal within 30 min. Both whole 125 cell uptake and subsequent nuclear binding of I-insulin were inhibited by increasing concentrations of unlabelled insulin (Fig. 4); the concentration of insulin that produced one-half maximal inhibition of both functions was approximately 10-20 ng/ ml.

Figure 4. Effect of unlabeled insulin on specific whole cell uptake and subsequent nuclear binding of 0.1 nM 125 I-insulin. Taken from reference 12.

While insulin entered IM-9 lymphocytes and then translocated to the nucleus, this uptake process was also reversible. When lymphocytes were preincubated with insulin and then washed free of extracellular hormone, the insulin rapidly left the cells (Fig. 5). The rate of efflux, however, differed as a

509

Minutes

125 Figure 5. Efflux of I-insulin from IM-9 lymphocytes. Cells were incubated with 0.1 nM 125 I-insulin for the indicated times and indicated temperatures, washed, and resuspended in insulin-free buffer. Efflux was followed at 24°.

function of both the preincubation time and temperature. If cells were preincubated with insulin for either 1 min. at 37°C or 60 min. at 4°C, there was a rapid efflux. In contrast, if the cells were preincubated with insulin for 60 min. at 37°C, efflux was decreased. This effect of a 60 min. preincubation at 37°C most likely reflected the translocation of insulin from the cell surface to intracellular organelles which, in turn, then delayed the subsequent efflux of the hormone.

510

Autoradiographic studies. In order to locate more precisely the intracellular binding sites for insulin, quantitative electron microscopic (EM) autoradiography was utilized. In these studies, 125 IM-9 cultured lymphocytes were incubated with 1nM I-insulin, washed to remove the extracellular insulin, fixed in glutaraldehyde, postfixed in osmium, and prepared for EM autoradiography (13). When autoradiographs were examined after a 30 sec. incubation period, the majority of the silver grains representing insulin appeared over the plasma membrane (Fig. 6). In contrast, after 30 min. incubation, a large number of grains were seen over several intracellular organelles including the nucleus (Fig. 6). In order to determine the 125 resolution of the autoradiographic method, a line source of I-insulin was established. For 125 these studies a thin (0.01 p.m) layer of I-insulin was embedded in plastic resin, sectioned, layered with emulsion, and

F i g u r e 6. EM a u t o r a d i o g r a p h s of IM-9 l y m p h o c y t e s incubated with 1 nM I - i n s u l i n for 30 seconds and 30 m i n u t e s . T a k e n from reference 13.

511

developed as described by Salpeter et al. (21) (Fig. 7). The location of grains around the source was noted and a grain den1 25 sity histogram for I was constructed (Fig. 8). As expected, the number of grains fell off rapidly as the distance from the source increased.The half-distance from the source was approximately 0.085 nm. Less than 2% of the radioactivity was found at a distance of 1 nm or greater from the source. This type of analysis indicates that if a cell studied by EM autoradiography has a source of 125 I-radioactivity localized only at the plasma

Line

Source

125 Figure 7. EM autoradiographs of a 0.01 pm line source of I-insulin. The line source was prepared as described by Salpeter et al (21).

512

membrane, fewer than 2% of the total cellular grains will be found in the cell interior 1 urn from the cell surface. Profiles of grain distribution relative to the plasma membrane were prepared from the autoradiographs of human-cultured lymphocytes (Fig. 8). After 30 sec. incubation, the histogram resembled

Source

1.0

>1.0

1.0

>1.0

Distance from Source (jum)

Distance from PM (>im)

1.0 >1.0 • 0.1 PM Distance from PM (,/um)

Distance from PM (/im)

60-1

1.0

>1.0

F i g u r e 8. G r a i n density h i s t o g r a m s of the I - i n s u l i n line source (A, 289 grains c o u n t e d ) and l y m p h o c y t e s incubated 30 seconds (B, 235 grains), 5 minutes (C, 385 grains), and 30 m i n u t e s (D, 273 g r a i n s ) w i t h ^ ^ j - i n s u l i n . Taken from reference 13.

the line source indicating that most of the grains were on the plasma membrane (Fig. 8). However, approximately 14% of the grains were located in the cell interior at a distance of 1 from the plasma membrane, which suggested that insulin can

513

very rapidly enter these cells. The amount of intracellular insulin increased with time and after 30 min. incubation, nearly 40% of the grains were inside the cells at a distance of 1 urn or greater from the plasma membrane (Fig. 8). The cellular distribution of the grains was then determined (Table II). After 30 sec. incubation, the majority of grains were found over the plasma membrane and the rest were distributed among various cellular organelles and cytoplasm. After 5 and 30 min. incubation, however, there was a progressive decrease in grains over the plasma membrane and an increase in grains over all cellular compartments. Treatment of the lymphocytes with trypsin (10) to remove the plasma membrane insulin receptor completely blocked the up1 25 take and internalization of I-insulin. This finding provided additional evidence that the cell surface receptor plays a role in the internalization of insulin. It was reported that when IM-9 lymphocytes are incubated with insulin, the number of insulin receptors on the plasma membrane rapidly decreases (22). It is possible, therefore, that the entry of insulin into these cells also requires the entry of the insulin receptor. Table II.

G r a i n distribution analysis of (

125

I) insulin in lymphocytes.

Cellular

location (percentage of total g r a i n s )

PM

CY

30 seconds

70.6

19.8

30 minutes

39.1

26.4

Time ER

GO

MI

NM

NU

3.5

0.3

1.7

0.8

3.1

11.4

0.5

3.6

3.3

15.6

Autoradiographs w e r e analyzed with the electron microscope. The location of grains was noted and appropriate organelles credited. W h e n grains were located over two or more organelles, partial credit was given to each organelle. Extracellular grains w e r e assigned to the plasma m e m b r a n e . Abbreviations: PM, plasma m e m b r a n e ; CY, cytoplasm; GO, Golgi; MI, mitochondria; NM, nuclear membrane; reference 13.

ER, endoplasmic reticulum; and NU, nucleus. Taken from

514

To determine whether the grains in the cell interior after 30 min.incubation were associated with intracellular organelles in proportion to the relative volumes (volume density) of these organelles, we conducted a stereologic analysis (23) (Fig. 9). After 30 min. incubation, the percentage of grains associated with both the nuclear membrane and endoplasmic reticulum was greater than the volume densities of these structures. On the other hand, the percentage of grains associated with the Golgi, cytoplasm and nucleus, was less than their respective volume densities. These analyses indicate that the interaction of insulin with intracellular organelles is not a random process. Our studies employing IM-9 cultured lymphocytes incubated with radiolabelled insulin indicate that insulin rapidly enters the interior of these cells and then interacts with intracellular organelles. Carpentier and coworkers have confirmed our observations that insulin is internalized by these cells (25) . 5.0n

E

>o

3.0-

o 6 1.0

0-

NM

ER

Ml

Intracellular

CY

NU

GO

Organelle

Figure 9. Ratio of the percentage of intracellular grains to intracellular organelle volume density, from autoradiographs of IM-9 lymphocytes incubated 30 minutes with 1 nM insulin. Grain concentration occurs in organelles that have a ratio greater than 1:0. Data adapted from reference 13.

515 In contrast to our findings, however, Carpentier et al. have calculated that insulin penetrates into IM-9 lymphocytes no further than 0.9 urn from the plasma membrane and is not associated with intracellular organelles. In our studies lymphocytes were incubated in 380 milliosmolar growth medium which contained serum. Carpentier et al. however, incubated cells in a 435 milliosmolar Hepes-buffered

serum-free medium devoid of

calcium, potassium, and phosphate. The sizes of

IM-9 lympho-

cytes incubated in these two solutions are markedly

different

(Table III). The average diameter of the cells incubated

in

culture medium containing serum was 17.4 |im whereas the diameter of the cells incubated in Hepes buffer was 14.5 |j,m (Fig.10). Thus, the differences in both content and osmolality of the incubation solutions employed in the two studies may have resulted in the reported differences in intracellular of

distribution

insulin.

Table III.

Characteristics of IM-9 lymphocytes incubated different

solutions

Complete Medium

Osmolality (milliosmolar) Cell Size J* (|im) Internalization of insulin

in

380

17.4 - 2.4

complete

Hepes

Buffer

435

14.5 - 2.0

partial

Cell size was measured on 100 fixed cells from each incuba|ion f solution, as previously described (13). Values are the mean - S. D.

516

Figure 10. M i c r o g r a p h s of IM-9 l y m p h o c y t e s incubated in either c o m p l e t e Eagle's m e d i u m w i t h 10% fetal calf serum (13) or the Hepes b u f f e r of Carpentier et al (25).

Freeze fracture studies. It has been reported by Carpentier et al. that incubation of adipocytes with radiolabelled insulin causes an increase in the number of particles in the plasma membrane as measured by freeze fracture studies (26). Since the uptake of insulin into cells appears to be receptor-mediated, and the entry of insulin into cells may deplete the cell surface of insulin receptors, we investigated whether incubation of IM-9 lymphocytes with insulin altered the distribution of particles in the plasma membrane. Lymphocytes were incubated with unlabelled insulin at varying concentrations for 2 h, the cells were then freeze fractured and intramembranous particles were quantitated (Table IV). No change was observed in the number of particles on either the P or E face of the plasma membrane.

517

Table IV:

Lack of an effect of insulin on numbers of intramembranous particles

in IM-9

lymphocytes.

Particles/vjm

2

plasma m e m b r a n e

P face

E face

Control

1218 - 65 (15)

488 - 27 (21)

Insulin 167 pM

1086 - 40 (21)

4 4 4 - 36

Insulin 167 nM

1137 - 61 (15)

389 - 38 (9)

(15)

Cells were treated for two hours with insulin, washed in 154 mM NaCl at 4°, fixed in glutaraldehyde-paraformaldehyde (13), glycerinated, freezefractured without etching, and shadowed with platinum and carbon, according to a modification of the method described by Moor (27). A Balzers BAF301 freeze-fracture apparatus was employed. Replicas were viewed and photographed with a Philips 300 electron microscope, and intramembranous particles in a 20 cm^ area of each micrograph were counted. The number of particles per ym^ plasma membrane was then calculated. Mean values are given - SEM. Number of cells evaluated is given in parentheses. No statistically significant changes in particle number were detected.

Studies with rat liver in vivo. In order to determine whether insulin can enter target cells in vivo, the uptake of into the liver of fasted rats was studied

insulin

(24, 28). Animals

were anesthetized, an abdominal incision was made, and radio125 labelled

I-insulin

(0.75 ng) was injected directly into the

portal vein and followed at various times by

perfusion-fixation

with glutaraldehyde. Electron microscopic autoradiographs of liver were prepared similar to those of lymphocytes. Insulin was associated with the hepatocyte plasma membrane 1 min. after 1 25 injection but at 10 min. I-insulin had entered the interior of the hepatocytes and was associated with the nuclear membrane, endoplasmic reticulum, and Golgi

(Fig. 11). These studies

indi-

cate, therefore, that insulin can also enter the interior of other cell types in vivo.

518

1

m i n

10

m i n

I"

'iGo N I *

^ S ^ y | p

m ^ làgKr I tdêÊKH ^ M ^ •W A p j ip y M E ^. 1 1 tliMlAllfr:-. .y •'•S-.' fl

i ^ K l m *

|

f sJ

s

R: • V '"fT'r i

V

'vu.-

SIVî

m*

nm

R E R

N u

f

F i g u r e 11. E M a u t o r a d i o g r a p h s of rat liver one and 10 m i n u t e s after the injection of 0.75 yg of 1 2 5 i - i n s u i i n into the p o r t a l v e i n followed by p e r f u s i o n - f i x a t i o n (24).

Conclusion The biological significance of the uptake of insulin into target cells is unknown. We and others have described specific binding sites for insulin on intracellular structures including the nucleus, nuclear membranes, smooth and rough endoplasmic reticulum, and Golgi apparatus.

519 S i n c e i n s u l i n h a s a v a r i e t y of e f f e c t s o n s u b c e l l u l a r

or-

g a n e l l e s a n d s i n c e the h o r m o n e e n t e r s c e l l s a n d b i n d s to

these

organelles, we postulate that this intracellular

may

participate

insulin

in the r e g u l a t i o n of i n s u l i n - d e p e n d e n t

lar f u n c t i o n s

(Fig.

intracellu-

12).

Figure 12. Possible m e c h a n i s m of a c t i o n of insulin on target cells. Insulin binds to its r e c e p t o r on the cell surface leading in turn to changes in a m i n o a c i d a n d glucose transport. I n s u l i n a n d its receptors then enter the cell together. Insulin dissociates from its receptor and then binds to intracellular organelles such as the endoplasmic reticulum and nucleus, w h e r e it possibly regulates subsequent intracellular events such as p r o t e i n and RNA synthesis. The insulin receptor is then either recycled or degraded.

520 Acknowledgements This research was supported by NIH grants # AM 26667 (I.D.G.), # AM 25878 (A.L.J.) and # P50AM 18520 (A.L.J.), the Elise Stern Haas Research Fund, Harold Brunn Institute, Mount Zion Hospital and Medical Center (I.D.G.) and the Dr. John A. Kerner Foundation, Mount Zion Hospital and Medical Center (B.M.K.). We wish to thank Dr. S. Grayson for conducting the technical aspects of the freeze fracture studies.

References 1.

Krahl, M.E.: Endocrine Function of the Pancreas. In: Annual Review of Physiology", Eds. Comroe, J.H., Sonnenscheen, R.R., Zerler, K.L., Annual Reviews, Inc., Palo Alto, Vol. 36, pp. 331-360 (1 974) .

2.

Pilkis, S.J., Park, C.R.: Mechanism of Action of Insulin. In: "Annual Review of Pharmacology", Eds. Elliot, H.W., Okur, R., George, R., Annual Reviews, Inc., Palo Alto, Vol. U , pp. 365-388 (1 974).

3.

Fain, J.: Biochemistry of hormones. In: "Biochemistry Series One, MTP International Review of Science", Ed. Pickenberg, H.V., University Park Press, Baltimore, pp. 1-23 (1974).

4.

Czech, M.P.: Molecular basis of insulin action. Ann. Rev. Biochem. 46, 359-384 (1977).

5.

Goldfine, I.D.: Minireview: Insulin receptors and the site of action of insulin. Life Sciences 23, 2639-2648 (1978) .

6.

Goldfine, I.D.: Does insulin need a second messenger? Diabetes 26, 148-155 (1977). Ginsberg, B.H., Kahn, C.R., Roth, J.: The insulin receptor of the turkey erythrocyte. Characterization of the membranebound receptor. Biochim. Biophys. Acta 443, 227-242 (1976).

7. 8.

Maturo, J.M., III, Hollenberg, M.D.: Insulin receptor: Interaction with nonreceptor glycoprotein from liver cell membranes. Proc. Nat. Acad. Sei. U.S.A. 75, 3070-3074 (1978).

9.

Krupp, M.N., Livingston, J.N.: Insulin binding to solubilized material from fat cell membranes: Evidence for two binding species. Proc. Nat. Acad. Sei. U.S.A. 75, 2593-2597 (1978).

10. Gavin, J.R., III, Görden, P., Roth, J., Archer, J.A., Buell, D.W.: Characteristics of the human lymphocyte insulin receptor. J. Biol. Chem. 248, 2202-2207 (1973).

521

11. Gavin, J.R., III.: Polypeptide Hormone Receptors on Lymphoid Cells.In: "Immunopharmacology", Eds. Hadden, J.W., Coffey, R.G., Spreafico, F., Plenum Medical Book Company, New York, pp. 357-387 (1977). 12. Goldfine, I.D., Smith, G., Wong, K.Y., Jones, A.L.: Cellular uptake and nuclear binding of insulin in human cultured lymphocytes: evidence for potential intracellular sites of insulin action. Proc. Nat. Acad. Sci. U.S.A. 7£, 1368-1372 (1 977) . 13. Goldfine, I.D., Jones, A., Hradek, G., Wong, K.Y., Mooney, J.: Entry' of insulin into human cultured lymphocytes: Electron microscopic autoradiographic analysis. Science 202, 760-763 (1978). 14. Goldfine, I.D., Smith, G.: Binding of insulin to isolated nuclei. Proc. Nat. Acad. Sci. U.S.A. 73, 1427-1431 (1976). 15. Vigneri, R., Goldfine, I.D., Wong, K.Y., Smith, G.J., Pezzino, V.: The nuclear envelope. The major site of insulin binding in rat liver nuclei. J. Biol. Chem. 253, 20982103 (1978). 16. Goldfine, I.D., Vigneri, R., Cohen, D., Pliam, N.B., Kahn, C.R.: Intracellular binding sites for insulin are immunologically distinct from those on the plasma membrane. Nature 269, 698-700 (1977). 17. Vigneri, R., Pliam, N.B., Cohen, D.C., Pezzino, V., Wong, K.Y., Goldfine, I.D.: In vivo regulation of cell surface and intracellular binding sites by insulin. J. Biol. Chem. 253, 8192-8197 (1978). 18. Horvat, A.: Insulin binding sites on rat liver nuclear membranes: Biochemical and immunofluorescent studies. J. Cell Physiol. 97, 37-48 (1978). 19. Goidl, J.A.: Insulin binding to isolated liver nuclei from obese and lean mice. Biochemistry _1_8, 3674-3679 (1 979). 125 20. Terris, S., Steiner, D.: Retention and degradation of Iinsulin by perfused livers from diabetic rats. J. Clin. Invest. 52, 885-896 (1976). 21. Salpeter, M., Fertuck, H., Salpeter, E.: Resolution in electron microscope autoradiography III. Iodine - 125, the effect of heavy metal staining and a reassessment of critical parameters. J. Cell. Biol. 12_, 161-173 (1977). 22. Gavin, J.R.,III, Roth, J., Neville, D.M.,Jr., DeMeyts, P., Buell, D.N.: Insulin-dependent regulation of insulin receptor concentrations: a direct demonstration in cell culture. Proc. Nat. Acad. Sci. U.S.A. 7J_, 84-88 (1974). 23. Weibel, E.R., Bolender, R.P.: Stereological Techniques for Electron Microscopic Morphometry. In: "Principles and Techniques of Electron Microscopy", Ed. Hayat, M.A., Van Nostrand/Reinhold, New York, Vol. 3, pp. 237-296 (1973).

522 24. Renston, R.H., Maloney, D.G., Jones, A.L., Goldfine, I.D., Mooney, J.S.: Bile secretory apparatus: Evidence for a vesicular transport mechanism for proteins. J. Cell Biol. 79, 379a (1978). 25. Carpentier, J., Gordon, P., Amherdt, M., Van Obberghen, E., Kahn, C., Orci, L.: 125i-i n sulin binding to cultured human lymphocytes. J. Clin. Invest. 6J[, 1057-1070 (1978). 26. Carpentier, J.L., Perrelet, A., Orci, L.: Effects of insulin, glucagon, and epinephrine on the plasma membrane of the white adipose cell: a freeze-fracture study. J. Lipid Res. V7, 335-342 (1976). 27. Moor, H.: Platiim-Kohle-Abdruck-Technik angewandt auf den feinbau der milchröhen. J. Ultrastructure Research 2, 393422 (1959). 28. Goldfine, I.D., Wong, K.Y., Korc, M., Hradek, G., Jones, A.L.: Entry of insulin into target cells: EM autoradiographic studies in vitro and in vivo. J. Cell Biol. 79., 194a (1978).

STEROIDS IN NORMAL AND DISEASED HUMAN PROSTATIC TISSUE

R. Vihko, N. Bolton, G.L. Hammond*, R. Lahtonen Department of Clinical Chemistry, University of Oulu, SF-90220 Oulu 22, Finland

Introduction Prostatic development, growth and function are androgen mediated (1). Benign and malignant tumours of the prostate are seen almost exclusively in man and dog, and are also androgen-dependent (2), but the mechanisms leading to unregulated growth of the prostate are not well understood. Clarification of the exact nature of the hormone dependency of the normal and diseased prostate has attracted extensive research, but progress has not been very substantial. There is no doubt that by far the most important source of androgen for the prostate is the testis (3). However, it is also clear that extensive prostatic metabolism of circulating steroids takes place before active compounds, important for growth and function of this organ, are formed (see Fig. 1). At present, the concensus of opinion is that 5a-dihydrotestosterone

(DHT) is the most potent of

these metabolites. It is assumed that it exerts its effect by way of receptor-mediated mechanisms (4).

*Present address: Reproductive Endocrinology Center, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco, San Francisco, California 94143, U.S.A.

Hormones in Normal and Abnormal Human Tissues © Walter de Gruyter • Berlin New York 1981

524

TESTOSTERONE 4

5O-ANDR0STANE-3B,176-DIOL «

5O-ANDR0STANE-3B,7a,178-TRIOL

I I

> ANDROSTENEDIONE

» 5a~DIHYDROTESTOSTERONE « IRO'

» 5CI-ANDROSTANEDIONE

5a-ANDROSTANE-3a,173-DIOL
ANDROSTERONE

Fig. 1. Major pathways of androgen metabolism in the human prostate. The unregulated growth in association with benign prostatic hypertrophy (BPH) or carcinoma of the prostate may be due to some abnormality in the production of androgenic hormones, their access to or metabolism in the prostate gland, or in some of the steps in the receptor-mediated actions. Several reports have appeared in which the circulating concentrations of steroid hormones have been measured and the only consistent finding in well-controlled series has been a small but significant elevation in serum concentrations of DHT in BPH patients (5-7). In addition, several recent studies have been unable to identify any consistent quantitative differences in the receptor content of normal or diseased prostate tissues (8). Indeed, the only consistent differences observed between normal, BPH and carcinomatous prostate tissues have been in their profiles of endogenous steroids. It is therefore our purpose to summarize the data available on the levels of endogenous steroids

525 in normal, BPH and carcinomatous human prostate, in an effort to try to understand their role in the aetiology of these diseases.

Normal Prostate Information on the steroid content of the normal human prostate is limited to studies in which steroids have been measured in prostates removed from cadavers shortly after death (9-14). In these reports, normal autopsy prostate tissues have been used as a reference in investigations of the steroid content of prostate tissues removed surgically from patients with BPH or prostatic cancer. It has been assumed that the concentrations of various steroids are not affected by changes in prostate tissue after death, and this is supported by evidence which indicates that the steroid composition of BPH tissue removed at surgery does not change upon storage for at least 12 hours at room temperature before refrigeration (9, 15). However, it is unknown whether normal prostate tissue behaves similarly to BPH tissue under these conditions. Therefore, studies of normal prostate tissues obtained during surgery, e.g. for bladder cancer, are needed to confirm this assumption. The immature prostate Recently, the concentrations of testosterone, androstenedione, DHT, 5a-androstanedione, 5a-androstane-3a,173-diol, androsterone, progesterone and 17-hydroxyprogesterone have been measured in prostates taken from subjects aged between 6 days and 75 years at the time of death (11). In Fig. 2, the mean concentrations of these steroids in the human prostate are shown with respect to various phases of development and maturity. As a result of neonatal exposure to high concentrations of steroids, the prostates of newborn infants contain high concentrations of DHT and other androgen metabolites, in addition to very high

526

M l ru4Aon

DHT

5aA

3a Diol

I70HP

Fig. 2. Age-related changes in the concentrations of steroids in normal prostatic tissue of males from birth to old age. Age groups for each steroid, from left to right: newborn (6-10 days), children (0.25-10 years), pubertal (13-14 years), adults (20-49 years), aged adults (50-75 years). T, testosterone; 4A, androstenedione; DHT, 5a-dihydrotestosterone; 5aA, 5a-androstanedione; 3aDiol, 5a-androstane-3a,17p-diol; A, androsterone; P, progesterone; 170HP, 17-hydroxyprogesterone. Data from ref. 11. concentrations of progesterone and 17-hydroxyprogesterone. This finding seems to indicate that the latter progestins do not exert an effective competitive inhibition of the 5a-reductase enzyme system in the infant prostate, as it reportedly does in BPH tissues in vitro (16-18). It is also of interest to note that the prostate during the first 3 months of life is characterised by a period of proliferative growth and secretory activity (19). In addition to the in utero exposure to high levels of steroids, this is most probably a consequence of the shortlived phase of testicular testosterone secretion and target cell metabolism during the first months of life, which results in adult serum concentrations of both testosterone and DHT (20).

527 After this neonatal period, the serum concentrations of adrenal and testicular androgens are very low until the approach of puberty (21, 22). During this phase of infancy, the prostate is not functionally active, but undergoes a limited degree of nonproliferative growth (23). The levels of testosterone and androstenedione in the prostate are low in pre-pubertal infants, but DHT and 5a-androstane-3a,173-diol are present in appreciable concentrations. In contrast to the presence of these 17(3-hydroxysteroids, the concentrations of 5a-androstanedione and androsterone are low in pre-pubertal prostatic tissue. During puberty in boys, the adrenal and testicular production of androgens increases over a period of several years (21, 22). The steroid profiles in pubertal prostatic tissue are similar to those observed in pre-pubertal prostates, with the exception that the tissue concentrations of androsterone appear to increase. The prostatic concentrations of androgens and their metabolites increase after puberty, the most marked increases being associated with 17-oxosteroids, and in particular 5a-androstanedione. It is perhaps pertinent to note that the prostate undergoes a period of rapid growth during puberty (19). The results therefore suggest that 5a-androstanedione and/or androsterone may play a role in the function of the mature prostate. The adult prostate The relative amounts of 3-oxo-4-ene-steroids (testosterone and androstenedione) and their 3-oxo-5a-reduced (DHT and 5a-androstanedione) and 3a-hydroxy-5a-reduced

(5a-androstane-3a,17|3-

diol and androsterone) metabolites are closely correlated in individual autopsy samples from normal mature prostates (Fig. 3). The relationships observed demonstrate the particular characteristics of normal prostate, in which the concentrations of 3a-hydroxy-5a-reduced androgens are greater than those of DHT and 5a-androstanedione (11). Recently, others (12, 13) have confirmed the observation that the concentration of 5aandrostane-3a-173-diol is higher than that of DHT in normal prostate. The observations confirm the original finding that

528

Fig. 3. Comparison of the combined concentrations (in pairs) of testosterone (T), androstenedione (4A), 5a-dihydrotestosterone (DHT), 5a-androstanedione (5aA), 5a-androstane-3a,173-diol (3aDiol) and androsterone (A) in normal prostatic tissue (closed squares) and tissue from patients with benign prostatic hypertrophy (open squares) . Regression lines are shown with correlation coefficients (r). Modified from ref. 11, with permission from the Journal of Endocrinology.

529 normal prostate concentrations of 5a-androstane-3a(33),173diols are higher than those of DHT (10). These observations have led to the concept that the 3a(33)-hydroxy-5a-reduced metabolites represent an important step in the further metabolism and clearance of active androgens from the prostate. Recently it has been suggested that 7a-hydroxysteroids may be the principal 33-hydroxy-5a-reduced metabolites in the prostate (24), but as yet no reports of the tissue concentrations of these compounds have appeared. In addition to testosterone and androstenedione, dehydroepiandrosterone is quantitatively an important androgen in blood and it has been reported that the BPH prostate contains relatively high concentrations (6.6 ng/g) of this adrenal androgen (9). We have also observed dehydroepiandrosterone concentrations of 2-13 ng/g tissue wet weight in samples taken from normal prostates, using radioimmunoassay

(unpublished

data). The significance of the presence of high concentrations of dehydroepiandrosterone in the human prostate is unknown. It has, however, been demonstrated that dehydroepiandrosterone may act as a precursor of more active androgens in vitro (25). One of the major active metabolites of dehydroepiandrosterone in vitro is 5a-androstane-3 3,173-diol (25), and it has been suggested that this metabolite may have a functional role in the human prostate (26). Direct measurements of the relative concentrations of 5a-androstane-3a,173-diol and 5a-androstane33,173-diol by gas chromatography-mass spectrometry have indicated that the 3a-epimer is probably the quantitatively most important form in the human BPH prostate, although significant concentrations of 5a-androstane-33,173_diol were also found (27). Moreover, recent measurements of the concentrations of 5a-androstane-3a,173-diol by radioimmunoassay

(11-13), are at

least 50% lower than those reported earlier for the measurement of the combined concentrations of both epimers in normal and BPH prostate tissues by radioimmunoassay

(10).

The steroid content of normal prostates taken at autopsy from men 20-75 years of age did not show any age-related change

530 Table 1 - Summary of the concentrations of steroids measured in the normal and diseased human prostate. Method

Tissue

Ref.

type Double isotope derivative

N

9

BPH

Mean concentration (ng/g tissue wet weight) T

4A

CHT

0.90

0.90

1.3

0.90

0.40

6.0

5aA

3oDiol

3BDiol

A

CHEA

Prog

17CHP

6.6

Gas-liquid chronatographymass spectroscopy

BPH

27

1.70

12.7

1.06

41

3.4

10.6

3.0

3.9

13.5

3.1

Ccnipetitive protein

BPH

binding using SHBG

Ca

Radioimmunoassay

N

10

BPH Radioijinunoassay

Radioijinunoassay

N

Radioimmunoassay

Radioijinunoassay

2.30

0.13

1.22

1.31

4.32

4.15

0.39

0.42

0.27

0.16

5.33

1.70

1.40

0.80

0.56

0.38

Ca

1.75

0.50

4.20

0.20

35.03

1.20

0.80

0.60

Cae

0.20

0.10

1.20

0.20

2.40

0.75

0.60

0.40

1.30

2.50

1.00

4.00

0.70

0.32

12

0.20

1.60

1.7

BPH

0.30

4.50

0.6

Ca

1.20

3.90

1.6

N

N

13

14x

0.57

0.60

BPH

0.65

2.10

Ca

2.12

1.21

BPH

15

Ca^ Radioijinunoassay

10.20

5.60 0.25

BPH Radiolnrnunoassay

2.10

BPH

N

11

0.53

BPH Ca

42XX

0.15

0.64

5.80

0.07

0.74

0.88

0.76

1.62

2.46

2.14

2.27

1.21

2.70 3.50

Abbreviations: N, noimal prostate; BPH, benign prostatic hypertrophy; Ca, carcinone; T, testosterone; 4A, androstenedione; DOT, 5a-dihydrotestosterone, 5uA, Su-androstanedione; 3aDiol, 5a-androstane-3a, 176-diol; 36Diol, 5a-androstane-3B,176-diol; A, androsterone; CHEA, dehyroepiandrosterone; Prog, progesterone; 170HP, 17-hydroxyprogesterone. e

Oestrogen treated; C+€castrated and oestrogen treated; Xestinated from the published figures, expressed as ng/g dry weight;

""estimated frcin the published figures, expressed as pmol/g dry weight.

531

(11) nor was. any difference observed in the profile of androgens in tissues removed from the periurethral and outer regions of five normal prostates. Our own results corroborate these findings. Although the prostate is thought of primarily as an androgen dependent target organ, progestin (28) and oestrogen (28, 29) receptors have been found in the human prostate. The concentrations of progesterone and 17-hydroxyprogesterone are very low in normal and diseased adult prostate tissues (11), and there is at present no information on the prostatic concentrations of oestrogens. Moreover, there is little evidence that these steroids exert a significant physiological impact on the human prostate.

Benign Prostatic Hypertrophy Many laboratories have recently confirmed the original observation (9) that the concentration of DHT is elevated in hypertrophic prostates compared to normal prostates taken at autopsy (10-14). The accumulation of this androgen in hypertrophic prostate is thought to be of direct aetiological significance, because of its potential for stimulating growth in vitro (30). Recently, it has been observed that the accumulation of DHT occurs in concert with a depletion in the tissue concentrations of 5a-androstane-3a(33),1V3-diols

(10-13) and androsterone (11,

12). The accumulation of DHT in hypertrophic tissue may be due to an increase in the activity of 5a-reductase (31) but others have considered that a reduction in the activity of 3a-hydroxysteroid dehydrogenase is of more significance (10, 32). It is also possible that 5a-androstane-3a,173~diol is more extensively metabolised to DHT in hyperplastic than in normal tissues (26). This could explain the failure to observe any difference in 5areductase and 3a-hydroxysteroid dehydrogenase activity between normal and hypertrophic tissue (33).

532 Unlike DHT, there is no significant accumulation of 5aandrostanedione (11) suggesting a specific bias towards DHT accumulation in hypertrophic tissue. Thus, if the latter is simply a result of a decrease in the 3a-hydroxysteroid dehydrogenase system, the results also indicate that the deficiency must be rather specific towards DHT, or that most of the 5aandrostanedione is metabolised to DHT in hypertrophic tissue. A close relationship between the combined concentrations of testosterone and androstenedione and their respective 5areduced and 3a-hydroxy-5a-reduced metabolites is seen in both normal and hypertrophic tissue (Fig. 3). However, it is evident that despite similar levels of testosterone and androstenedione in normal and hypertrophic tissue, the concentration of their metabolites differs markedly. The combined concentrations of these androgens are very similar in both normal and hypertrophic tissue. This supports the suggestion that the accumulation of DHT in hypertrophic tissue is a result of a decrease in its conversion to 3a-reduced metabolites and not an increase in its formation or retention. Separated cells In the hypertrophic prostate steroid metabolism is markedly different in the two main cellular compartments, the stromal, essentially fibromuscular cells, and the actively secreting epithelial cells. In cell culture experiments, epithelial cells from hypertrophic tissue could not be maintained in a viable condition without the presence of stromal tissue (34). Moreover, studies of the development of the foetal prostate in animal models have demonstrated the close association between epithelial and stromal elements as well as the importance of an androgen environment (35, 36). Recently, a number of reports have appeared in which biochemical parameters have been studied in separated stromal and epithelial cells of hypertrophic tissue. It seems that 5a-reductase activity in the stroma is increased compared to the epi-

533 thelium (37, 38, 39). However, DHT is more evenly distributed between these two cell compartments and it has been suggested that the prostatic stroma is involved in the supply of androgens to the epithelium (37). In contrast, sulphatase activity for dehydroepiandrosterone sulphate was found to be localized mainly in the epithelium (37), while the reductive and oxidative activities of 3a(33)-hydroxysteroid dehydrogenase was found to be more evenly divided between stroma and epithelium (39).

Sex hormone binding globulin (SHBG) in separated cells

of hypertrophic tissue (40) was predominantly a component of the interstitial fluid within the fibromuscular stroma and might function as a steroid reservoir for the prostate or may influence the exit of steroids from cells. Recently we have separated the stromal and epithelial elements. Although the epithelial cells were histologically pure the stromal cells were always contaminated with some epithelial cells. The concentrations of six androgens (testosterone, DHT, androstenedione, 5a-androstanedione, androsterone and 5a-androstane-3a,173-diol) were measured in these separated cell preparations by radioimmunoassays. Testosterone and 5aandrostanedione were found to be present in about equal concentrations in both cell types, when expressed either on a wet weight or a DNA content basis. All the other androgens were found to be more concentrated in the stroma, this being particularly marked in the case of androstenedione.

The Cancerous Prostate Far less information is available on the concentrations of steroids in the cancerous prostate, primarily due to the limited number of untreated samples available. There is an accumulation of DHT in the untreated cancerous prostate, which is as great as (41), or slightly less than (11, 13-15, 42), in hypertrophic tissue. In addition, an accumulation of testosterone was reported in untreated cancerous tissue,•compared to its concentration

534 in hypertrophic (11, 13, 14, 42) and normal (11, 13, 14) prostate tissue. This accumulation of testosterone in the untreated cancerous prostate may be related to a decrease in the activity of the 5a-reductase (13). However, this does not explain the accumulation of DHT in the untreated cancerous prostate. Moreover, it has also been reported that the concentration of 5aandrostane-3a,173-diol is much higher in cancerous than in hypertrophic (11, 13) or normal (11) prostate. The concentrations of 5a-androstanedione and androsterone were found to be extremely low in tissue removed from a cancerous prostate (11). Thus, the available data indicate that the mechanism behind the accumulation of DHT in the cancerous prostate is probably very different to that in BPH. The influence of oestrogen treatment on the concentration of androgens in the cancerous prostate has not been studied in detail. In cancerous tissue removed from men who had been treated with oestrogens, the testosterone and DHT concentrations were similar to those in normal prostatic tissues (11), while their concentrations in cancerous prostate taken from castrated and oestrogen-treated men were similar to those in androgen independent tissues (15). The present data indicate that although a reduction in the concentrations of various androgens occurs in oestrogen treated patients, their relative amounts do not change (11). This does not support the concept that oestrogens reduce the activity of the 5a-reductase system (16, 17), but suggests that oestrogens act primarily by reducing the concentrations of available androgens or by limiting their uptake into the target cells.

Conclusions Age-related changes in the androgen concentrations in the human prostate during maturation are reflected in the growth and function of this organ. In the normal mature prostate there do not appear to be any changes in androgen concentrations with

535 advancing age, nor do there appear to be any significant

diffe-

rences in the concentrations and profiles of steroids in the peri-urethral and outer gland regions of the normal prostate. The combined concentrations of androgens stenedione, DHT, 5a-androstanedione,

(testosterone,

andro-

5a-androstane-3a,173-diol

and androsterone) are similar in both normal and hypertrophic tissues, but their relative concentrations are very

different.

This is primarily reflected in an accumulation of DHT and a depletion of 5a-androstane-3a,173-diol

and androsterone in the

hypertrophic tissue. These differences may be explained by changes in the activities of enzymes responsible for androgen metabolism and may be directly related to the aetiology of the disease. Although very few androgen measurements have been made on the cancerous prostate, it is evident that the androgen

profi-

les in this tissue are very different from both normal and hypertrophic tissues; there is an accumulation of both testosterone and DHT, while the levels of

5a-androstane-3a,173-diol

are as high if not higher than in normal tissue. The mechanism of DHT accumulation in the cancerous prostate seems to be different from that occurring in hypertrophic

tissue.

In future studies, steroid determinations in various cellular and subcellular compartments of normal and diseased human prostates may provide a greater understanding of the mechanism of androgen action in these target tissues. In addition,

future

studies on the relative steroid concentrations in prostatic carcinoma tissue must take into account the degree of differentiation of the tissue. This type of information may

indicate

whether any changes in androgen metabolism or accumulation are related to anaplastic changes in prostatic

tissues.

References 1. Geller, J.: Medical treatment of benign prostatic hypertrophy. In: "The treatment of prostatic hypertrophy and neoplasia", Ed. Castro, J.E., Medical and Technical Publishing Co. Ltd., Lancaster, England, pp. 27-58 (1974).

536 2.

Gloyna, R.E., Siiteri, P.K., Wilson, J.D.: Dihydrotestosterone in prostatic hypertrophy. II. The formation and content of DHT in the hypertrophic canine prostate and the effect of DHT on prostatic growth tin the dog. J. clin. Invest. 49, 1746-1753 (1970).

3.

Mostofi, F.K.: Benign hyperplasia of the prostate gland. In: "Urology", Vol. 2, Eds. Campbell, M.F. and Harrison, J.H., W.B. Saunders Co., Philadelphia, 1065-1129 (1970).

4.

Liao, S., Fang, S., Tymoczko, J.L., Liang, T.: Androgen receptors, antiandrogens, and uptake and retention of androgen in male accessory organs. In: "Male accessory sex organs. Structure and function in mammals", Ed. Brandes, D., Academic Press Inc., New York, 237-265 (1974).

5.

Vermeulen, A., De Sy, W.: Androgens in patients with benign prostatic hyperplasia before and after prostatectomy. J. clin. Endocrinol. Metab. £3, 1250-1254 (1976).

6.

Chisholm, G.D., Ghanadian, R.: Comparison between the changes in serum 5a-dihydrotestosterone and testosterone in normal men and patients with benign prostatic hypertrophy. V International Congress of Endocrinology, Hamburg, July 18-24, Abstract 455 (1976).

7.

Hammond, G.L., Kontturi, M., Vihko, P., Vihko, R.: Serum steroids in normal males and patients with prostatic diseases. Clinical Endocrinology 9, 113-121 (1978).

8.

Shain, S.A., Boesel, R.W.: Human prostate steroid hormone recffeptor quantitation. Current methodology and possible utility as a clinical discriminant in carcinoma. Invest. Urol. 1_6 , 169-174 (1 978) .

9.

Siiteri, P.K., Wilson, J.D.: Dihydrotestosterone in prostatic hypertrophy. 1. The formation and content of dihydrotestosterone in the hypertrophic prostate of man. J. clin. Invest. 49, 1737-1745 (1970).

10. Geller, J., Albert, J., Lopez, D., Geller, S., Niwayama, G.: Comparison of androgen metabolites in benign prostatic hypertrophy and normal prostate. J. clin. Endocrinol. Metab. 43, 686-688 (1976). 11. Hammond, G.L.: Endogenous steroid levels in the human prostate from birth to old age: A comparison of normal and diseased tissues. J. Endocr. 78, 7-19 (1978). 12. Meikle, A.W., Stringham, J.D., Olsen, D.C.: Subnormal tissue 3a-androstanediol and androsterone in prostatic hyperplasia. J. clin. Endocrinol. Metab. 47, 909-913 (1978). 13. Krieg, M., Bartsch, W., Janssen, W. , Voigt, K.D.: A comparative study of binding, metabolism and endogenous levels of androgens in normal, hyperplastic and carcinomatous human prostate. J. Steroid. Biochem. JM_, 615-624 (1979).

537 14. Habib, F.K., Mason, M.K., Smith, P.H.,Stitch, S.R.: Cancer of the prostate: early diagnosis by zinc and hormone analysis ? Br. J. Cancer 39, 700-704 (1979). 15. Albert, J., Geller, J., Geller, S., Lopez, D.: Prostate concentrations of endogenous androgens by radioimmunoassay. J. Steroid Biochem. 7, 301-307 (1976). 16. Jenkins, J.S., McCaffery, V.M.: Effect of oestradiol and progesterone on the metabolism of testosterone by human prostatic tissue. J. Endocr. 6J3, 51 7-526 (1 974). 17. Tan, S.Y., Antonipillai, I., Pearson Murphy, B.E.: Inhibition of testosterone metabolism in the human prostate. J. clin. Endocrinol. Metab. 29^, 936-941 (1974). 18. Morfin, R.F., Bercovici, J.P., Charles, J.F., Floch, H.H.: Testosterone and progesterone metabolism and their interaction in the human hyperplastic prostate. J. Steroid Biochem. 6, 1347-1352 (1975). 19. Zondek, T., Zondek, L.H.: The fetal and neonatal prostate. In: "Normal and abnormal growth of the prostate", Ed. Goland, M., Charles C. Thomas, Springfield, Illinois, pp. 5-28 (1975) . 20. Hammond, G.L., Koivisto, M., Kouvalainen, K., Vihko, R.: Serum steroids and pituitary hormones in infants with particular reference to testicular activity. J. clin. Endocrinol. Metab. 49, 40-45 (1979). 21. Pakarinen, A., Hammond, G.L., Vihko, R.: Serum pregnenolone, 17-hydroxyprogesterone, androstenedione, testosterone, 5adihydrotestosterone and androsterone during puberty in boys. Clinical Endocrinology 1_1_, 465-474 (1979). 22. Sizonenko, P.C.: Endocrinology in pre-adolescents and adolescents. I. Hormonal changes during normal puberty. Am. J. Dis. Child. 1_32, 704-712 (1 978). 23. Moore, R.A.: The histology of the newborn and pre-pubertal prostate gland. The Anatomical Record 66_, 1-7 (1 936). 24. Morfin, R.F., Di Stefano, S., Charles, J.-F., Floch, H.H.: Precursors for 6 3- and 7a-hydroxylations of 5a-androstane33,173-diol by human normal and hyperplastic prostates. Biochimie 59, 637-644 (1977). 25. Harper, M.E., Pike, A., Peeling, W.B., Griffiths, K.: Steroids of adrenal origin metabolized by human prostatic tissue both in vivo and in vitro. J. Endocr. 60^, 117-125 (1 974) . 26. Morfin, R.F., Di Stefano, S., Bercovici, J.-P., Floch, H.H.: Comparison of testosterone, 5a-dihydrotestosterone and 5aandrostane-33, 173-diol metabolisms in human normal and hyperplastic prostates. J. Steroid Biochem. 9_, 245-252 (1978).

538 27. Millington, D.S., Buoy, M.E., Brooks, G., Harper, M.E., Griffiths, K.: Thin-layer chromatography and high resolution selected ion monitoring for the analysis of C-|g steroids in human hyperplastic prostate tissue. Biomedical Mass Spectrometry 2, 219-224 (1975). 28. Ekman, P., Snochowski, M., Dahlberg, E., Bression, D., Hogberg, B., Gustaffson, J.-A.: Steroid receptor content in cytosol from normal and hyperplastic human prostates. J. clin. Endocrinol. Metab. 49, 205-215 (1979). 29. Bashirelahi, N., O'Toole, J.H., Young, J.D.: A specific 170-estradiol receptor in human benign hypertrophic prostate. Biochemical Medicine 1_5, 254-261 (1976). 30. Laznitzki, J., Whitaker, R.H., Withycombe, J.F.R.: The effect of steroid hormones on the growth pattern and RNA synthesis in human benign prostatic hyperplasia in organ culture. Br. J. Cancer 32, 168-178 (1975). 31. Bruchovsky, N., Lieskovsky, G.: Increased ratio of 5areductase: 3a(0)-hydroxysteroid dehydrogenase activities in the hyperplastic human prostate. J. Endocr. 8£, 289-301 (1 979) . 32. Krieg, M., Bartsch, W., Herzer, S., Becker, H., Voigt, K.D.: Quantification of androgen binding, androgen tissue levels, and sex hormone-binding globulin in prostate, muscle and plasma of patients with benign prostatic hypertrophy. Acta endocr. 86, 200-215 (1977). 33. Morfin, R.F., Charles, J.-F., Floch, H.H.: C 1 9 0 2 -steroid transformations in the human normal, hyperplastic and cancerous prostate. J. Steroid Biochem. Y\_, 599-607 (1 979). 34. Franks, L.M., Riddle, P.N., Carbonell, A.W., Gey, G.O.: A comparative study of the ultrastructure and lack of growth capacity of adult human prostate epithelium mechanically separated from its stroma. J. Path. 100, 113-119 (1970). 35. Cunha, G.R., Lung, B.: The importance of stroma in morphogenesis and functional activity of urogenital epithelium. In Vitro 5, 50-71 (1979). 36. Lasnitzki, I., Mizuno, T.: Role of the mesenchyme in the induction of the rat prostate gland by androgens in organ culture. J. Endocr. 82, 171-178 (1979). 37. Cowan, R.A., Cowan, S.K., Grant, J.K., Elder, H.Y.: Biochemical investigations of separated epithelium and stroma from benign hyperplastic prostatic tissue. J. Endocr. 74, 111-120 (1977). 38. Cowan, R.A., Cook, B., Cowan, S.K., Grant, J.K., Sirett, D.A.N., Wallace, A.M.: Testosterone 5a-reductase and the accumulation of dihydrotestosterone in benign prostatic hyperplasia. J. Steroid Biochem. V\_, 609-61 3 (1979).

539 39. Bruchovsky, N., Rennie, P.: Cellular factors contributing to the high concentration of dihydro-testosterone in hyperplastic and carcinomatous human prostates. Abstract, Symposium 4, First International Congress on Hormones and Cancer Rome (1979). 40. Cowan, R.A., Cowan, S.K., Giles, C.A., Grant, J.K.: Prostatic distribution of sex hormone-binding globulin and cortisol-binding globulin in benign hyperplasia. J. Endocr. 21, 121-131 (1976) . 41. Farnsworth, W.E., Brown, J.R.: Metabolism of testosterone by the human prostate. J. Am. med. Ass.183, 436-439 (1963). 42. Habib, F.K., Lee, I.R., Stitch, S.R., Smith, P.H.: Androgen levels in the plasma and prostatic tissues of patients with benign hypertrophy and carcinoma of the prostate. J. Endocr. 71, 99-107 (1976).

OESTRONE SULPHATE - A MAJOR CIRCULATING OESTROGEN

K. F. St0a and 0. L. Myking Hormone Laboratory, University of Bergen School of Medicine, Bergen, Norway

Introduction The fact that steroid hormones, including oestrogens, are excreted in the urine mainly as glucuronides, was established several years ago. Furthermore, it was a general concept that the purpose of the conjugation process was to render superfluous compounds physiologically less active and more water soluble in order to promote their excretion. Only small amounts of oestrone sulphate are excreted in the urine. The enzyme oestrone sulphatase has the important function of contributing to the transformation of oestrone sulphate to oestrogen glucuronides, which are excreted more rapidly by the kidneys. Due to this enzyme, the oestrogen sulphates are metabolized considerably easier than by a more generalized detoxication process. Comparatively high concentrations of steroid hormones are present in blood as conjugates, either glucuronides or sulphates. To what extent these circulating conjugates are primarily involved in metabolic interconversions or are only transported from one organ to another prior to the final excretion, is not completely clear. Oestrone sulphate comprises a major portion of the circulating oestrogens (1). Since several tissues contain the enzymes necessary to transform this conjugate to oestradiol (2, 3), oestrone sulphate may act as a potential oestrogen which can be activated by hydrolysis and hydrogenation, particularly in the liver, but possibly also in other tissues (4).

Hormones in Normal and Abnormal Human Tissues © Walter de Gruyter • Berlin • New York 1981

542 This chapter will deal primarily with the biochemistry of oestrone sulphate as it pertains to man. Observations made in animal experiments will be included only when data exist which may be relevant to the understanding of conditions existing in man.

Analytical Methods Analysis of oestrogen conjugates generally requires elaborate preparation and multiple chromatographies. A thorough review of methods for the isolation of steroid conjugates has been published by Siiteri (5). a) Extraction. The most widely employed principle of preliminary fractionation of steroid conjugates from crude biological samples has been organic solvent extraction. Of the many solvents employed for this purpose, n-butanol (6, 7), ethanol, with (8) and without (9) admixture of ethyl ether, ethyl acetate (10) and tetrahydrofuran (11, 12) are the most important. Other methods for obtaining steroid conjugates from plasma or tissue are also available. As examples of procedures for extraction of oestrogen conjugates should be mentioned Sephadex gel filtration (13, 14) and the use of the ion exchange resin Amberlite XAD-2 (15). In this laboratory the procedures usually employed have been ethanol extraction or absorption to Amberlite XAD-2. b) Isolation of oestrone sulphate. A number of procedures are available for the isolation and purification of individual oestrogen conjugates. Possible choices are countercurrent distribution (CCD), partition or adsorption chromatography, paper or thin layer chromatography (TLC), electrophoresis and high performance liquid chromatography (HPLC). The following review aims at evaluating each of these principles with respect to the detection and determination of oestrone sulphate.

543 Schneider and Lewbart (16) were the first to report on the extensive application of CCD to the isolation of steroid conjugates, and rigorous statistical methods for the analysis of such data, obtained with oestrone sulphate, have been presented by Sheps et al (17). A correspondingly rigorous characterization of radioactive oestrone sulphate in plasma 14 extracts from women after the administration of [ C]-oestradiol has been obtained by Purdy et al (18) largely on the basis of critical CCD analysis. Separation of individual components is achieved in a CCD system provided their K values (K = distribution coefficient) are sufficiently different (19). Thus the selection of a solvent system which will achieve the desired separation can be made on the basis of simple preliminary experiments. This property of CCD affords a distinct advantage over many purification methods, such as adsorption chromatography, in which

systems are

based on empirical findings. On the other hand, CCD has its practical limits caused by factors such as extract overloading, operation time and expenses of equipment and solvents. Several types of column chromatography have been applied to the purification of steroid conjugates. For adsorption chromatography a variety of adsorbents such as alumina, Florisil and silica gel have been used. The resolution of adsorption columns is generally poor, so that separation of closely related compounds is seldom achieved. Incomplete recoveries may also arise from interactions of conjugates with the adsorbents, the properties of which can be variable. Nevertheless, adsorption columns have been very useful for the separation of classes of conjugates. One of the first applications of adsorption chromatography to steroid conjugate isolation is that of Grant and Beall (20), who some 30 years ago obtained pure oestrone sulphate from pregnant mare's urine with the aid of columns containing alumina and Celite. These authors utilized solvent systems consisting of benzene and increasing amounts of methanol for the conjugate elution.

544 Column liquid-liquid partition chromatography (GLLC) (21), is a valuable tool for isolating steroid conjugates (5). Although the separation of individual steroid sulphates by this method is more difficult than the separation of the corresponding glucuronides, a number of solvent systems have been devised which separate many important steroid sulphates (22), including oestrogen sulphates (23). In 1965 Hahnel reported the use of diethylaminoethyl (DEAE)Sephadex for purification and separation of steroid conjugates (24). This anion-exchanger has subsequently been used for the same purpose by Hobkirk (25) and in the laboratory of the present authors (26). Gel filtration through columns of Sephadex has also been applied for the separation of oestrogen sulphates (13,27). In studies reported in 1969, Hobkirk and his colleagues (25) improved Hahnel's original technique of DEAE-Sephadex chromatography of oestrogen conjugates using a linear NaCl gradient. However, dextran or cellulose based ion exchangers have the disadvantage of being soft easily collapsable gels in which the bed volume changes continuously with changes in pH, ionic strength and pressure. In this situation, there is no fixed relationship between elution volume and retention of the oestrogen conjugate. This problem was partly solved by development of an isocratic mode of separation (28), which allows calculation of the elution volume of a conjugate from the molarity of the eluent. The introduction of rigid permanently bonded supports and the speed advantage provided by high pressure liquid chromatography

(HPLC) has

lately made possible a separation of complex mixtures of oestrogen conjugates using a high pressure system (Fig. 1). This system permits not only a separation but also a quantitative, sensitive and specific estimation of steroid conjugates. It is probably more convenient and faster than conventional methods, the chromatography of these conjugates

545

15

EIG

E217G

O12 O

EI S

CL û

w

I

0

5

10 15 2 0

I

I—I

25 30

35

TIME (MIN )

Fig. 1. Separation of radioactive metabolites in plasma after i.v. administration of [3H]-oestrone into human subject. Technique used: High pressure liquid chromatography. Column: n Partisil SAX (4 mm x 2 5 cm). Eluent: 0.01 M KH2PO4, pH 4.2. Flow rate: 0.6 ml/min. Inlet pressure: 1000 psi. Effluent collected directly into scintillation vials on a fraction collector. Musey, I.I., Collins, D.C., Preedy, J.R.K.: Steroids _31_# 583592 (1978). E 2 17G: oestradiol-17-glucuronide, E-|G: oestrone-3-glucuronide, E-|S: oestrone sulphate. being complete in about 30 min, whereas DEAE Sephadex chromatography takes over 18 h (28). The use of paper chromatography and thin layer chromatography in studies of steroid conjugates has been reported in several publications. In 1955 Cavina (29) described three solvent systems for paper chromatography (butanol: H_0,

546 ethyl acetate:butanol:0.2 N acetic acid, 90:10:100, and isoamyl alcohol:NH4OH, 55:27:18) as applied to various synthetic conjugates. Lewbart and Schneider (30) also described a number of systems containing butyl acetate for use in paper chromatography with both glucuronides and sulphates. Improved systems were devised by Bush (21), Lewbart and Schneider (30) and Baulieu (31). Alkaline systems are preferred when dealing with steroid sulphates because of the ease with which they are hydrolyzed in organic solvents containing acid (32). The use of thin layer chromatography (TLC) systems has been suggested by Oertel et al (33), Wusteman et al (34) and Sarfaty and Lipsett (35). The last mentioned authors used silica gel H (Merck) and the solvent system ethyl acetate: ethanol:15 N-NH.OH, 5:5:1. Two-dimensional TLC has been applied by Crepy et al (36) and Fishman et al (37). A number of workers have used high voltage electrophoresis to achieve the separation of conjugated oestrogens (23, 38). A complete scheme for the separation and identification of urinary oestrogen sulphates after i.v. injection of labelled oestrone sulphate, including both chromatography and electrophoresis, was presented in 1969 by Jirku and Levitz (23) . Part of the scheme, pertinent to oestradiol-3-sulphate and oestrone sulphate, is shown in Fig. 2. c) Direct estimation of oestrone sulphate. Up to now, methods for quantitative determination of oestrone sulphate have involved removal of the sulphate group and estimation of the free oestrogen. The introduction of high pressure liquid chromatography has made possible a sensitive and specific direct measurement of oestrone sulphate in extracts from biological material. Another possibility for assay of the intact conjugate is the raising of an antiserum to oestrone sulphate. Generation of such antisera has been reported (39, 40).

547 EXTRACT IN 95 % ETHANOL

I

ALUMINA CHROMATOGRAPHY ^ ELUATE 90 - 80 % ETHANOL I CHROMATOGRAPHY

PAPER

n - BUTANOL : ETHYLACETATE : NH .OH : H . O

\

'

Z O N E A ( MOBILITY : 2.4 cm / h ) ( " LEAST POLAR SULPHATES " ) 1

=

I

RID

2" 4r RID

Fig. 2. Isolation and identification of oestrone and oestradiol sulphates. Alumina chromatography was carried out with gradient elution technique. The mixing chamber contained 95 % ethanol and the upper chamber contained water (pH 7.0-7-4, adjusted with NH 4 0H). Paper chromatography system: n-Butanol: Ethyl acetate: NH4OH: HoO (1:9:1:9). Electrophoresis carried out in pyridine:acetic acid:H 2 0 (125:5:2500), pH 6.4. Jirku, H., Levitz, M.: J. Clin. Endocr. 29, 615 (1969). EtS = Oestrone sulphate. E 2 S = Oestradiol-3-sulphate. RID = Reverse isotope dilution.

Oestrone Sulphate in Plasma Until recently, the state of our knowledge of the concentration of oestrone sulphate in human plasma was incomplete, probablydue to methodological difficulties. However, during the last decade assays of this oestrogen conjugate have been performed in plasma throughout the menstrual cycle by at least four groups of workers (41-44). In each of these studies the levels of oestrone sulphate exhibited a marked cyclic pattern with a pre-ovulatory peak and a secondary increase during the luteal

548 2000

pg/ml

1500

IM i'11

1000

500

0

'

8 6 4 2

1

0 2 4

-LH*

6

HH 8

4

2

0

2

4

- M»

Fig. 3. Pattern of the levels of oestrone sulphate in 8 normally menstruating women. Geometric means are indicated by filled circles, 95 % confidence limits by vertical bars. LH = days around the LH peak. M = days around the onset of menstruation. Oestrone sulphate was solvolysed prior to chromatographic purification (celite column) of oestrone and radioimmunoassay. Values expressed in terms of the steroid moiety of the conjugate. From Nunez et al. (45).

phase (Fig. 3). The mean levels of the steroid moiety of oestrone sulphate were found to be 10-15 times as high as those of unconjugated oestrone and 2-3 times as high as oestradiol sulphate, unconjugated oestrone and unconjugated oestradiol together. A quantitative relationship between oestrone sulphate and other plasma oestrogen fractions similar to that of menstruating women has recently been found also in the human male. In 53 normal men of different age (20-87 years) the levels of the steroid moiety of plasma oestrone sulphate has been found to vary from 117 pg/ml to 566 pg/ml (Fig. 4). This concentration range exceeded any of the other plasma oestrogen components by 5-20 times (45). The range of levels is in good agreement with that obtained recently by Loriaux et al (12) in their study of 30 men, but clearly lower than the results reported by Hawkins

549

pg/iml 500

U 00

t

300 200 100

20

1

I

40 60 age (years)

L_

80

Fig. 4. Individual concentrations of plasma oestrone sulphate in 53 normal human males, spearman rank correlation coefficient: -0.279 (2P-C0.05). Steroid conjugates were absorbed to Amberlite XAD-2 and eluted with methanol. After evaporation of the solvent and hydrolysis with arylsulphatase (Mylase p), oestrone was determined with radioimmunoassay.

and Oakey (42) from repeated measurements in 6 men. Remarkably, the concentration of oestrone sulphate in human male plasma was found to decrease significantly with increasing age, while, on the other hand, unconjugated plasma oestrone increases with age (45). This may support the suggestion of Skoldefors et al (46) that a drop in low-polar oestrogen excretion at about 6 0 years reflects a decrease in plasma oestrone sulphate.

Formation Since the work of De Meio and Lewycka in 1955 (47) it has been known that steroid sulphates can be formed in vitro by crude

550

preparations from liver. However, it is only recently that the details of this conjugation process have been clarified. This transfer of the sulphuryl group is catalyzed by steroid sulphotransferases, the reactions being irreversible. The authorized nomenclature recognizes two such steroid sulphotransf erases : 33-hydroxysteroid sulphotransferase (EC 2.8.2.2) and

3'-phosphoadenylyl-sulphate:oestrone

sulphotransferase

(EC 2.8.2.4). There are several such enzymes, which are often known as the steroid sulphokinases. The first separation of enzymes belonging to this group was achieved by Nose and Lipmann (48) who showed that oestrone sulphate and dehydroepiandrosterone sulphate were formed by different enzymes. Banerjee and Roy (49) separated the two responsible enzymes, oestrone sulphotransferase and androstenolone sulphotransferase, from guinea pig liver and produced evidence for the occurrence of two more related enzymes, testosterone sulphotransferase and deoxycorticosterone sulphotransferase. The oestrone sulphotransferase from ox adrenal has recently been obtained devoid of other sulphotransferase activity (50). With regard to the tissue distribution of the steroid sulphotransferases, it should be stated that these enzymes, including the oestrone sulphotransferase, occur in significant amounts in the liver, the adrenal gland and the jejunal mucosa (51). Smaller amounts undoubtedly occur in other tissues, and more sensitive methods have shown their existence in the human ovary (52) and testis (53) where they probably are of physiological importance. Recently, studies by Buirchell and Hahnel (54) revealed that oestrogen sulphurylation was also a property of human endometrium, the sulphotransferase activities being greatly stimulated during the secretory phase (55). An increased dehydrogenation of oestradiol with a concurrent increase in sulphurylation was found to be accompanied by a decreased uptake of the nuclear oestrogen receptor. As it is considered that oestrogen sulphates do not bind to the cytoplasmic oestrogen receptor (56) and, in fact, are preferentially excreted from uterine

551

tissue, it can be suggested that the increase of the sulphotransferase activity within the endometrium facilitates the conversion of a proliferative to a secretory tissue under the control of progesterone. This premise is fortified by the fact that the uterine oestrogen metabolizing enzymes are induced by progesterone (57-60), their activity being maintained by this hormone throughout the implantation process in porcine uterus (61) .

During their studies of oestrogen metabolism, Lipsett and his colleagues (62) postulated that there exists another plasma oestrogen compartment in equilibrium with plasma oestrone and plasma oestradiol. It was considered probable that this compartment might be identical with oestrone sulphate. Later studies (1) confirmed that oestrone sulphate was an important plasma metabolite of oestradiol and defined the production rate of oestrone sulphate in men and women (Table 1). The average plasma production rates of oestrone sulphate, calculated from plasma concentrations and metabolic clearance rates, were 77 g,g/ day in men, 95 ug/day in early follicular phase and 182 ng/day in early luteal phase, in women. All plasma oestrone sulphate could be shown to be derived from plasma oestrone and plasma oestradiol. Hence it is unlikely that oestrone sulphate is secreted in amounts sufficient to make it a significant precursor of plasma oestrone, as suggested by Baird et al (63).

Binding to Serum Albumin In the early work of Engel and his colleagues (18) it was shown that oestrone sulphate bound to plasma albumin with a large number of binding sites. More recently, the existence of two independent sets of binding sites for oestrone sulphate was suggested by Rosenthal et al (64) , who obtained data which indicated that different steroid sulphates can displace each other from albumin. Since binding curves for oestrone sulphate with dilute blood bank plasma and human serum albumin were not

552 Table 1. Average plasma concentrations and production rates (PR) of oestrône, oestradiol-17p, and oestrone sulphate in four normal men and five normal women 3 '. b > pg/ml

V

E^ S PR ^g/day

E 1 pg/ml

E 1 PR pg/day

E 2 pg/ml

E 2 PR (ig/day

Hen

460

77

47

112

34

58

Women Follicular phase

654

95

62

109

110

116

1246

182

86

151

193

204

Women Luteal phase

From Ruder, H.J., Loriaux, L., Lipsett, M.B.: J. Clin, invest. 51, 1020-1033 (1972). E. = Oestrone. E. S = Oestrone sulphate. E. = 0estradiol-17fi.

appreciably different, it was concluded by the same authors that all of the binding of oestrone sulphate was due to albumin. It was further suggested that there was one strong binding site and several weaker ones. At physiological levels of oestrone sulphate, the strong binding

site alone will account for about

85% of all oestrone sulphate binding. Unless the steroid conjugate is displaced by competitors, it can be expected that about 85% of oestrone sulphate in blood will be bound to the strong binding site of albumin at physiological oestrogen levels. The per cent bound at 37°C of a tracer amount of oestrone sulphate in whole plasma with a human serum albumin concentration of 4.6% would be expected to be 98.5%, a value which has been confirmed by binding experiments (64) . 0estradiol-3-sulphate, oestradiol-17-sulphate, oestradiol3,17-disulphate, oestriol-3-sulphate, androsterone sulphate, and dehydroepiandrosterone sulphate have all been shown to compete for oestrone sulphate binding. Under physiological conditions, however, their concentrations are too low to have a measurable effect on oestrone sulphate binding in plasma. Under normal conditions, the metabolic clearance rate of

553 oestrone sulphate is less than 10% of that of unconjugated oestrone. Similar ratios between the metabolic clearance rates of the sulphate conjugated and unconjugated steroid have been noted for dehydroepiandrosterone

(65) and for testosterone (66).

The low metabolic clearance of these steroid sulphates are obviously due to their strong binding to serum albumin. Unconjugated oestrone and oestradiol are mainly bound to specific steroid-binding 3-globulin, sex hormone binding globulin (SHBG). The affinity of this protein for oestradiol is about 50-60% of that for testosterone, and oestrone is only weakly bound (67). The influence of the albumin-binding of oestrone sulphate on its enterohepatic circulation and on its transport and uptake will be discussed further in subsequent sections of this chapter.

Enterohepatic Circulation The importance of enterohepatic circulation to the metabolism of oestrone was first indicated by Cantarow et al (68). The quantitative significance of this phenomenon in the human was later demonstrated by Sandberg and Slaunwhite (69, 70) and by Levitz and coworkers (71, 72). In an exhaustive study by Jirku and Levitz (23) of the metabolism of injected, radioactively labelled oestrone sulphate in a 4 0-year-old woman whose bile was being drained via a T-tube, it was found that oestriol-3sulphate, with lesser amounts of oestrone sulphate, predominated in the urine, whereas sulphates of oestrone, ring D a-ketols, 15a-hydroxyoestrone and 15a-hydroxyoestradiol were found in comparable amounts in the bile. Glucuronides were more concentrated in the urine than in the bile. On the other hand, double conjugates with sulphuric acid and glucuronic acid were higher in bile than in urine. Significant amounts of the sulpho-N-acetylglucosaminides of 15a-hydroxyoestrone and 15a-hydroxyoestradiol were found in the bile.

554 Uptake and Metabolism in Tissues Studies both in vitro (73) and in vivo (74) have revealed that the metabolism of oestrone differs from that of its sulphate ester. The reason for this is not yet known, and there are still controversies about the physiological role of oestrone sulphate (75). Whereas some authors, for example Longcope (76), emphasize that oestrone sulphate is not a precursor for free oestrogens, others (1) regard oestrone sulphate as a transport form of oestrogen in human plasma. It has even been claimed (77) that sulphation of oestrone is a prerequisite of the stimulatory effects of the hormone in target tissues. New light has been shed on these problems by Breuer and his coworkers in perfusion experiments with isolated rat livers (75). In these studies, which were carried out partly by cyclic perfusion and partly by once through perfusion of equimolecular amounts of 14 3 [4- C] oestrone and of [6,7- H] oestrone sulphate, it was shown that the metabolites of oestrone and of oestrone sulphate were differently distributed between the3 three compartments, 14 liver, medium and bile. A ratio of 1.7( H/ C) for total radioactivity in the perfusion medium suggested a slower uptake of oestrone sulphate than of oestrone. Further analysis of the sulphate fraction of the perfusion medium after cyclic 3perfusion revealed that this fraction consisted mainly of [ H] oest14 rone sulphate. Only small amounts of newly formed [ C] oestrone sulphate had been delivered to the medium. A slow uptake of oestrone sulphate was also demonstrated in the once-through perfusions, which showed that the hepatic clearance rate of oestrone was three to four times that of oestrone sulphate. It was suggested that the reason for this phenomenon was that more oestrone sulphate than unconjugated oestrone was bound to albumin. When dextran was used as macromolecular component in oncethrough perfusions, oestrone sulphate was almost quantitatively extracted by the liver from the medium, indicating that the liver cell membrane was permeable for oestrone sulphate. It is evident that the metabolism of oestrone and oestrone sulphate

555 depends on the interaction between albumin and these compounds. Whereas albumin serves to keep the plasma level of oestrone sulphate high, it apparently counteracts the tissue uptake of this steroid conjugate. On the basis of transport studies in isolated rat liver cells, Schwenk and Lopez del Pino (78) postulated that oestrone sulphate uptake was mediated by an energy-dependent anion carrier. Their conclusion was based on observations of initial rates of uptake, which was found to be saturable, with a K^ of approximately 1 |j.M. It was inhibited by SH reagents (mersalyl) taurocholate and oestrone sulphate competed for uptake. The activation energy was found to be 67 kcal/mol. There was a pH optimum in the physiological range. Inhibition of mitochondrial respiration lowered uptake. It could be demonstrated that oestrone sulphate was accumulated inside the cells, where it was partly cleaved to the free steroid. It may be suggested that an anion-transport protein, like that described for the human erythrocyte membrane (79), takes part in the uptake process.

Metabolic Reactivation In a previous section of this chapter, it was mentioned that oestrogen sulphates do not bind to the cytoplasmic receptor, and it was shown (80) that unconjugated oestrone was only weakly bound to this receptor. Therefore, in order to become biologically active, sulphate-conjugated oestrone must undergo an enzymatic activation, i.e. hydrogenation at C-17 and hydrolysis. It was repeatedly demonstrated that mammalian extrahepatic tissues possess the enzyme activities necessary for bringing about these reactions (81-84). The enzyme responsible for the biotransformation of oestrone to oestradiol-173 belongs to the 173-hydroxysteroid dehydrogenases. Inano and Tamaoki (85) have purified a 173-hydroxysteroid dehydrogenase solubilized from the microsomal fraction of porcine testis. Studies of the specificity of this enzyme

556 showed that androstenedione, dehydroepiandrosterone and oestrone were reduced at similar rates when NADPH was employed as cofactor. A similar steroid specificity of the corresponding enzyme in human endometrium has been demonstrated by Tseng and Gurpide (86). Uterine tissue is capable of both reduction of oestrone and oxidation of oestradiol (87-89), in accordance with the reversible character of the 173-hydroxysteroid dehydrogenase activity. However, experiments by Gabb and Stone (90), 3 who incubated rabbit uterine tissue with [ H]-17^-oestradiol and ["^H] oestrone, demonstrated an equilibrium of the reaction which favoured the reduction of oestrone. It was also shown by these authors that proportionately less oestrone than oestradiol was bound to the nuclear oestrogen receptor. These findings provided further evidence for oestradiol being the oestrogen which is active in the uterus. Interestingly, it has been shown both in perfusion experiments with rat liver (75) and in incubation experiments with guinea pig and human kidney slices (91) that intact oestrogen sulphates can be involved in direct oxidoreduction. In the perfusion experiments with isolated rat liver previously referred to (75), it was found that intracellular oestrone sulphate was preferably reduced to oestradiol sulphate, thus indicating that oestrone sulphate was a better substrate for 17 3-hydroxysteroid dehydrogenase than oestrone. With regard to the incubation experiments of Hobkirk and coworkers (91) with kidney tissue, also previously referred to, it is interesting to speculate upon the possibility that oestradiol-3-sulphate may form a source of active hormone, via sulphatase action, for use at some tissue site. It should be noted that oestrogen sulphatase activity has been reported to be present in the kidney by Zuckerman and Hagerman (92) and that some evidence for the presence of oestrogen receptors has been obtained in rat kidney (93). The scheme in Fig. 5 illustrates the central roles played by the two enzymes 17[5-hydroxysteroid dehydrogenase and arylsulphatase C in the intracellular metabolism of oestrone sul-

557

PLASMA

CYTOPLASMA

ARYLSULPHATASE C

Fig. 5. Uptake and intracellular metabolism of oestrone sulphate in target tissue. E-| = Oestrone. E2= 17p-0estradiol. E-|S= Oestrone sulphate. EgSs Oestradiol-3-sulphate. E2Rc= Cytoplasmic oestradiol-receptor complex. E 2 RM= Nuclear oestradiolreceptor complex. 17p-0HDH= 17P~Hydroxysteroid dehydrogenase.

phate. As stated previously, the presence of these enzymes in mammalian tissues has been repeatedly demonstrated. Both enzymes have been detected in uterine tissue, and the activities of both of them are influenced by the hormonal milieu (86, 94). The activities of the endometrial oestradiol dehydrogenase and 20a-dihydroprogesterone dehydrogenase vary during the menstrual cycle (59) reaching a maximum at the midluteal phase. A synchronization of these activities during the cycle and the reported identity of the enzymes involved in the conversion of oestradiol to oestrone and 20a-dihydroxyprogesterone to progesterone in the human placenta (95) suggest the possibility that the 173- and 20a-dehydrogenase activities in human endometrium correspond to a single enzyme. Furthermore, the same 17 3-hydroxysteroid dehydrogenase apparently catalyzes the interconversion between oestradiol and oestrone, testosterone and androstene-

558 dione, and 5-androstene-30,173-diol and dehydroepiandrosterone (59). In a recent study, Tseng and Gurpide (86) showed experimentally that progestins enhanced the activity of dehydrogenases involved in the metabolism of oestrogens, androgens and progestins in human endometrium and provided evidence that these activities resided in a single dehydrogenase. The stimulation of endometrial 1 7(3-oestradiol dehydrogenase activity by progestins has been considered to have a physiological role in the regulation of intracellular levels of oestradiol and oestrogenic effects in the endometrium (57). It has also been related to the therapeutic action of progestins in endometrial cancer (96). Of the arylsulphatase enzymes (A, B and C), arylsulphatase C has been suggested to be identical with oestrone sulphatase (2, 97). This type of enzyme activity is present in various mammalian tissues, e.g. liver (2, 97), placenta (98), kidney (92), brain (99) and pituitary (100). Little information is available regarding quantitative assays of oestrone sulphatase in uterus, which is a prominent target organ for oestrogenic hormones. As part of studies of the organ distribution of oestrone sulphatase in rats, two groups, Zuckerman and Hagerman (92) and Dolly et al (2) made one single assay each of the enzyme in the uterus. Both groups found considerable enzyme activity, using oestrone sulphate as substrate, the mean activities being 42 and 14 nmol (mg protein) ^h \

respectively.

In the authors' laboratory, oestrone sulphatase activity has been studied in uteri from rats in different hormonal states (94). Since it has been demonstrated that oestrone sulphatase is localized in the microsomal fraction (101), the 12500 g supernatant, containing this fraction (97) , has been used as the enzyme medium in these assays. Using this medium, the mean uterine oestrone sulphatase activity of normal rats was found to - 1

be 5.1 nmol (mg protein)

- 1

h

(Table 2). In ovariectomized rats

the corresponding mean enzyme activity was significantly higher, whereas treatment of ovariectomized animals with oestradiol resulted in a significant decrease of oestrone sulphatase activity

559 Table 2. oestrone sulphatase activity in rat uterus as determined in the 12 500 x g supernatants of tissue homogenates a Group of animals

Number of animals

Number Uterus Oestrone sulof weight (mg) phatase, nmol-1 assaysb x ± S.D. Jug frotein)"^

6

6

505 - 115

Ovariectomized rats

12

6

305 -

32

14.5 - 1.0

Hypo phys ec t omized rats

20

10

105 -

18

18.7 - 2.8

Ovariectomized rats treated with oestradiol0

4

4

402 -

78

4.7 i 0.4

Intact rats treated with, progesterone

5

5

427 - 136

8.3 - 0.8

Hypophysectomized rats treated with oestradiol

4

4

307 -

19

6.0 i 0.5

Hypophysectomized rats treated with progesterone

8

4

112 ±

17

17.1 - 4.2

Intact rats

5.1 - 0.7

Assay according to Iwamori et al. (101), slightly modified. [6,7-3h] Oestrone sulphate solution, 5 mM, 0.1 ml (about 120000 CPM), was mixed with 0.4 ml of 0.14 M imidazole-HCl buffer, pH 8.0, and 0.5 ml supernatant (homogenization in 0.32 M sucrose, 100-300 mg tissue per ml). After incubation for 30 min at 37°C, the reaction was stopped by the addition of 2.0 ml of ice-cold acetone. After centrifugation, the supernatant was decanted off and the acetone evaporated at 37°C in a stream of nitrogen. Liberated [3H]oestrone was extracted with ether and its radioactivity counted in a liquid scintillation spectrometer. Protein was determined in the supernatants by the method of Lowry et al. (106). b

Uteri weighing less than about 300 mg were analyzed in arbitrary pairs.

c

Dosage in all experiments: 2 ^g l7p-oestradiol s.c. in 0.2 ml sesamAoil daily for 5 days, control animals were given only sesamtoil.

d

Dosage in all experiments: Progesterone, 1 mg s.c. in sesame, oil daily for 5 days.

Uterine oestrone sulphatase activity of hypophysectomized rats was found to be somewhat higher than that of the ovariectomized animals and treatment of these animals with oestradiol resulted in a significant decrease of the enzyme activity. Treatment of the hypophysectomized animals with progesterone gave no significant change in enzyme activity. On the other hand, intact rats given injections of progesterone showed increased oestrone

560 sulphatase activity as compared with the controls. These results, although not easily interpreted, show clearly the influence of the levels of ovarian hormones on the uterine oestrone sulphatase activity. In analogy with the 17(3~ hydroxysteroid dehydrogenase, the arylsulphatase C activity is apparently also stimulated by progestin. This effect is noticeable in intact, but not in hypophysectomized animals, which can be explained by the fact that progestin action is dependent on the action of oestrogen (103). The low sulphatase activities found in the presence of relatively high levels of endogenous or exogenous oestrogen, may possibly be explained as a result of product inhibition.

Possible Role of Oestrone Sulphate in Neoplasia The formation of steroid sulphates by breast tumours was reported in 1964 by Adams (104) and in 1968 by Dao and Libby (105). In further studies, it was suggested that the sulphotransferase levels in the tumour could be correlated with the patient's response to bilateral adrenalectomy (106, 107). Thus, patients whose breast cancers did not possess measurable steroid sulphotransferase activity rarely responded to bilateral adrenalectomy, whereas breast tumours having the capacity to synthesize more dehydroepiandrosterone sulphate than oestrogen sulphates often regressed after adrenalectomy. It was also shown (108) that about 36% of primary breast cancers had appreciable oestrone sulphatase activity. In addition to steroid sulphotransferases and sulphatases, the presence of 173-hydroxysteroid dehydrogenase has been demonstrated in breast cancer tissue (109). There are, therefore, reasons to assume that a system of enzymes stimulating formation and metabolism of oestrone sulphate, exists in breast tumour tissue. In a study of steroid transformation by human breast cancer it was found (109) that malignant breast tumour tissue

561

contained the enzymes necessary for transforming C^g precursor steroids, e.g. dehydroepiandrosterone sulphate, into oestrogen. Furthermore, it has recently been shown that cultured human breast cancer cells possess aromatase activity and the ability to convert testosterone to oestradiol (110). Epidemiological studies have established that a family history of breast cancer increases an individual's predisposition for the disease (111). The factor responsible for the increased risk is unknown. The evidence of hormonal participation in the etiology of breast cancer (112) suggests, however, that the inherited predisposition for the disease is mediated through a genetically transmitted endocrine factor. In a recent report, Fishman and coworkers (113) present data showing that young women considered at risk for breast cancer because of a family history of the disease exhibit a urinary oestrogen profile which is significantly different from that of carefully matched controls. Steroids in the urine were extracted after glucuronidase hydrolysis and measured by radioimmunoassay. Differences were observed only in the case of oestrone and oestradiol, with the high-risk subjects exhibiting lower values than the controls. According to the authors, a possible explanation of the abnormally low values for urinary oestrone and oestradiol in the high-risk subjects may be that only the oestrogen glucuronides and not the oestrogen sulphates have been measured. A change in the conjugative pattern of oestrone and oestradiol in the high-risk group with decreased glucuronidation and increased sulphation would result in lower urinary oestrone and oestradiol values with the technique applied. The possibility that a change in conjugation might be an endocrine feature distinguishing women at risk for familial breast carcinoma would imply that the risk factor is linked not to the total amount of oestrogen secreted but to its metabolic fate, which is dependent on an enzymatic spectrum under genetic control. The hypothesis of Fishman and his coworkers is interesting in view of a demonstrated tissue uptake preference for the sulphated oestrogen molecule (114) and its

562 biological activity (77). However, the hypothesis has not, as yet, been corroborated by oestrone sulphate assays, either in plasma or in urine. The uptake and intracellular metabolism of oestrone sulphate in endometrial tissue have been discussed earlier in this chapter. Since exposure to oestrogen has been considered to be the causal factor in development of adenocarcinoma, it would not be unexpected that oestrone sulphate might play a role in the etiology of uterine cancer. A close relationship seems to exist between the extraglandular production of oestrone from androstenedione and the occurrence of endometrial neoplasia. Both in premenopausal and postmenopausal women an increased occurrence of endometrial carcinoma is found in women with an increased oestrone production as a consequence of increased availability of its prehormone, androstenedione. There is also a 15-20 fold increase in the extent of extraglandular conversion of androstenedione to oestrone in markedly obese older women compared to slender younger women, and it is known that obesity is another factor predisposing to endometrial hyperplasia (115). Under normal conditions the increase in plasma oestrone or oestradiol will be followed by an increase in oestrone sulphate, and it is not known to which oestrogenic component the increase in neoplasias is related. Treatment with conjugated oestrogens has been shown to be followed by an increased frequency both in endometrial carcinoma (116) and breast cancer (117). Since the principal (more than 50%) steroid component of conjugated oestrogens is oestrone sulphate, both oestrone and oestrone sulphate must be considered as possible carcinogenic factors (118, 119).

Concluding Remarks The formation of plasma oestrogens varies considerably with age and sex. In premenopausal women they are mainly secreted directly from the ovaries, whereas in postmenopausal women

563 and in men they are mainly produced from secreted androgen precursors. The close relationship between oestrone, oestradiol and oestrone sulphate in circulating plasma, the 5-10 fold higher concentration and a much stronger protein binding make oestrone sulphate the major oestrogen storage component. Because various

types of tissue exhibit sulphatase and 173-

hydroxysteroid dehydrogenase activity, oestrone sulphate can be considered as a potentially active oestrogenic component. In premenopausal women, plasma oestrone sulphate fluctuates in the same manner as the unconjugated oestrogens throughout the menstrual cycle. In men, the oestrone/oestrone sulphate ratio will increase with age as a consequence of the decrease in oestrone sulphation which accompanies ageing. Determination of oestradiol and perhaps also oestrone in plasma without a simultaneous measurement of oestrone sulphate can be considered to be incomplete.

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565 22. Calvin, H.I., Roberts, K.D., Weiss, C., Bandi, L., Cos, J.J., Lieberman, S.: Column liquid-liquid partition chromatography of steroidal sulfates. Anal. Biochem. _1_5, 426-436 (1966). 23. Jirku, H., Levitz, M.: Biliary and urinary metabolites of estrone-6,7-3H-sulfate-35s in a woman. J. clin. Endocrinol Metab. 29, 615-637 (1969). 24. Hahnel, R.: Use of Sephadex ion exchanger for the separation of conjugated urinary estrogens. Anal. Biochem. 10, 184-192 (1965). 25. Hobkirk, R., Musey, P., Nilsen, M.: Chromatographic separation of estrone and 17|3-estradiol conjugates on DEAESephadex. Steroids U , 191-206 (1969). 26. St^a, K.F., B^rjesson, B.W.: Metabolism of oestradiol in the guinea pig. Biochim. biophys. Acta 239, 337-344 (1971). 27. Sjovall, J., Vihko, R.: Determination of androsterone and dehydroepiandrosterone sulfate in human serum by gas-liquid chromatography. Steroids 6, 597-604 (1965). 28. Musey, P.I., Collins, D.C., Preedy, J.R.K.: Xsocratic separation of estrogen conjugates on DEAE-Sephadex. Steroids 29, 657-668 (1977). 29. Cavina, G.: Chromatografia su carta di esteri solforici e glucuronosidi di steroidi. Boll. Soc. ital. Biol. sper. 31_, 1668-1670 (1955). 30. Lewbart, M.L., Schneider, J.J.: Paper chromatography of steroid glucuronosides and sulphates. Nature 176, 1175 (1955). 31. Baulieu, E.-E.: Studies of conjugated 17-ketosteroids in a case of adrenal tumor. J. clin. Endocrinol. Metab. 22, 501-510 (1962). 32..Burstein, S., Lieberman, S.: Kinetics and mechanism of solvolysis of steroid hydrogen sulfates. J. Am. chem. Soc. 80, 5235-5239 (1958). 33. Oertel, G.W., Tornero, M.L., Groot, K.: Thin-layer chromatography of steroid conjugates. J. Chromatogr J_4, 509-511 (1 964) . 34. Wusteman, F.S., Dodgson, K.S., Lloyd, A.G., Rose, F.A., Tudball, N.: Thin-layer chromatography in the study of ester sulphates. J. Chromatogr J_6, 334-339 (1 964). 35. Sarfaty, G.A., Lipsett, M.B.: Separation of free and conjugated 11-deoxy-17-oxosteroids by thin-layer chromatography. Anal. Biochem. Jj5, 184-186 (1 966). 36. Crepy, O., Judas, O., Lachese, B.: Detection of conjugated steroids separated by thin-layer chromatography. J. Chromatogr J_6, 340-344 (1964).

566 37. Fishman, W.H., Harris, F., Green, S.: Two dimensional thin layer chromatography of estradiol and estradiol glucosiduronic acids extracted from buffered solution. Steroids 5^, 375-383 (1965). 38. Fujii, K., Mizota, S., Takama, T., Mijamoto, S., Ozaki, T.: High voltage electrophoresis of free and conjugated estrogens. J. Biochem. (Tokyo) _51_, 167-168 (1962). 39. Wright, K., Collins, D.C., Musey, P.I., Preedy, J.R.K.: A specific radioimmunoassay for estrone sulfate in plasma and urine without hydrolysis. The Endocr. Society, 6 0th Annual Meeting, Abstr. No. 365, p. 257 (1978). 40. Sanyaolu, A.A., Eccles, S.S., Oakey, R.E.: An antiserum for oestrone sulphate. J. Endocr. 69, 11P (1976). 41. Brown, J.B., Smyth, B.J.: Oestrone sulphate - the major circulating oestrogen in the normal menstrual cycle ? J. Reprod. Fertil. 24, 142 (1971). 42. Hawkins, R.A., Oakey, R.E.: Estimation of oestrone sulphate, oestradiol-173 and oestrone in peripheral plasma: Concentrations during the menstrual cycle and in man. J. Endocr. 60, 3-17 (1974). 43. Dyrenfurth, I., Jewelewicz, R., Warren, M., Ferin, M., Van de Wiele, R.L.: Temporal relationships of hormonal variables in the menstrual cycle. In: "Biorhythms and Human Reproduction", Eds. Ferin, M., Halberg, F., Richart, R.M., Van de Wiele, R.L., John Wiley and Sons, New York, pp. 171201 (1974). 44. Nunez, M., Aedo, A.-R., Landgren, B.-M., Czecan, S.Z., Diczfalusy, E.: Studies on the pattern of circulating steroids in the normal menstrual cycle. 6. Levels of oestrone sulphate and oestradiol sulphate. Acta Endocrinol. 8£, 621633 (1977). 45. Myking, O.L., Thorsen, T., St^a, K.F.: Conjugated and unconjugated plasma oestrogens - oestrone, oestradiol and oestriol - in normal human males. Submitted for publication. 46. Skoldefors, H., Carlstrom, K., Furuhjelm, M.: Influence of aging upon the urinary hormone excretion in the male. Acta obstet. gynec. scand. 55, 119-123 (1976). 47. De Meio, R.H., Lewycka, C.: In vitro synthesis of dehydroepiandrosterone sulfate. Endocrinology 56^, 489-490 (1955). 48. Nose, Y., Lipman, F.: Separation of steroid sulfokinases. J. biol. Chem. 233, 1348-1351 (1958). 49. Banerjee, R.K., Roy, A.B.: The sulfotransferases of guinea pig liver. Mol. Pharmacol. 2, 56-66 (1966). 50. Adams, J.B., Poulos, A.: Enzymic synthesis of steroid sulphates. 3. Isolation and properties of estrogen sulphotransferase of bovine adrenal glands. Biochim. biophys. Acta 146, 493-508 (1967).

567 51. Bostrom, H., Wengle, B.: Studies on ester sulphates. 23. Distribution of phenol and steroid sulphokinase in adult human tissues. Acta Endocrinol 691-704 (1967). 52. Wallace, E., Silberman, N.: Biosynthesis of steroid sulfates by human ovarian tissue. J. biol. Chem. 239, 2809-2812 (1964). 53. Dixon, W.R., Vincent, V., Kase, N.: Biosynthesis of steroid sulfates by normal human testis. Steroids 6>, 757-769 (1965) 54. Buirchell, B.J., Hahnel, R. : Metabolism of estradiol in human endometrium during the menstrual cycle. J. Steroid Biochem. 6, 1489-1494 (1975). 55. Pack, B.A., Tovar, R., Booth, E., Brooks, S.C.: The cyclic relationship of estrogen sulfurylation to the nuclear receptor level in human endometrial curettings. J. clin. Endocrinol Metab. 4j3, 420-424 (1979). 56. Hahnel, R., Twaddle, E., Ratajazak, T.: The specificity of the estrogen receptor of human uterus. J. Steroid Biochem. 4, 21-31 (1973). 57. Tseng, L., Gurpide, E.: Changes in the in vitro metabolism of estradiol by human endometrium during the menstrual cycle. Amer. J. Obstet. Gynecol. 114, 1002-1008 (1972). 58. Tseng, L., Stolee, A., Gurpide, E.: Quantitative studies on the uptake and metabolism of estrogens and progesterone by human endometrium. Endocrinology ^0, 390-404 (1972). 59. Tseng, L., Gurpide, E.: Estradiol and 20a-dihydroprogesterone dehydrogenase activities in human endometrium during the menstrual cycle. Endocrinology 94, 41 9-423 (1 974). 60. Tseng, L., Gurpide, E.: Induction of human endometrial estradiol dehydrogenase by progestins. Endocrinology 97, 825-833 (1975). 61. Pack, B.A., Brooks, C., Dukelow, W.R., Brooks, S.C.: The metabolism and nuclear migration of estrogen in porcine uterus throughout the implantation process. Biol. Reprod. (1979) (in press) . 62. Hembree, W.C., Bardin, C.W., Lipsett, M.B.: A study of estrogen metabolic clearance rates and transfer rates. J. clin. Invest. 48, 1809-1819 (1969). 63. Baird, D., Horton, R., Longcope, C., Tait, J.F.: Steroid prehormones. Perspect. Biol. Med. V\_, 384-421 (1968). 64. Rosenthal, H.E., Pietrzak, E., Slaunwhite, W.R., Sandberg, A.A.: Binding of estrone sulfate in human plasma. J. clin. Endocrinol Metab. 34/ 805-813 (1972). 65. Sandberg, E., Gurpide, E., Lieberman, S.: Quantitative studies on the metabolism of dehydroisoandrosterone sulfate. Biochemistry

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568 66. Wang, D.Y., Bulbrook, R.D., Sneddon, A., Hamilton, T.: The metabolic clearance rates of dehydroepiandrosterone, testosterone and their sulphate esters in man, rat and rabbit. J. Endocr. 38, 307-318 (1967). 67. Bidlingmaier, F., Knorr, D., Oestrogens, Physiological and Clinical Aspects. S. Karger AG., Basel (1978). 68. Cantarow, A., Rakoff, A.E., Paschkis, K.E., Hansen, L.P., Walking, A.A.: Excretion of estrogen in bile. Endocrinology 3_1_, 51 5-51 9 (1 942) . 69. Sandberg, A.A., Slaunwhite, W.R.: Phenolic steroids in human subjects. II. The metabolic fate and hepato-biliaryenteric circulation of carbon-14 and carbon-14 estradiol in women. J. clin. Invest. 36, 1266-1278 (1957). 70. Sandberg, A.A., Slaunwhite, W.R.: Studies on phenolic steroids in human subjects. VII. Metabolic fate of estriol and its glucuronide. J. clin. Invest. 4_4, 694-702 (1 965). 71. Twombley, G.H., Levitz, M.: Metabolism of estrone-sulfate in women. Amer. J. Obstet. Gynecol. 80, 889-898 (1960). 72. Emerman, S., Twombley, G.H., Levitz, M.: Biliary and urinary metabolites of estriol-15-3H-3-sulfate-35s in women. J. clin. Endocrinol Metab. 21_, 539-548 (1 967). 73. Dahm, K., Breuer, H.: Vergleichende Untersuchungen über den Stoffwechsel von Oestron-sulfat und Oestron in Zellfraktionen der Rattenleber. Biochim. biophys. Acta 137, 196-198 (1967). 74. Fishman, J., Hellman, L.: Comparative fate of oestrone and oestrone sulfate in man. J. clin. Endocrinol Metab. 36, 160-164 (1973). 75. Höller, M., Grochtmann, W., Napp, M., Breuer, H.: Studies on the metabolism of oestrone sulphate. Comparative perfusions of oestrone sulphate through isolated rat livers. Biochem. J. 166_r 363-371 (1 977). 76. Longcope, C.: The metabolism of estrone sulfate in normal males. J. clin. Endocrinol Metab. X4, 113-122 (1972). 77. Brooks, S.C., Leithauser, G., De Locker, W.C., De Wever, F.: In vitro stimulation of protein synthesis in uterine microsomal supernatant by estrone sulfate. Endocrinology 84, 901-907 (1969). 78. Schwenk, M., Lopez del Pino, V.: Transport and metabolism of estrone sulfate in isolated rat liver cells. HoppeSeyler's Z. physiol. Chem. 359, 322-323 (1978). 79. Williams, D.G., Jenkins, R.E., Tanner, M.J.A.: Structure of the anion-transport protein of the human erythrocyte membrane. Further studies on the fragments produced by proteolytic digestion. Biochem. J. 181, 477-493 (1979).

569 80. Korenman, S.G.: Comparative binding affinity of estrogens and its relation to estrogenic potency. Steroids _1_3, 16 3 — 177 (1969). 81. Ryan, K.J., Engel, L.L.: The interconversion of estrone and estradiol by human tissue slices. Endocrinology 52, 287-291 (1953). 82. Gray, C.L., Bischoff, F.: Conversion of estrone to estradiol by mammalian red cells. Am. J. Physiol. 180, 279-281 (1955). 83. Fishman, J., Bradlow, H.L., Gallagher, T.F.: Oxidative metabolism of estrogens. J. Am. chem. Soc. 2273 (1959). 84. Breuer, H., Breuer, J., Schmähl, D.: Stoffwechsel von Östradiol-17ß in dem durch 7,12-Dimethyl-benzanthrazen induzierten Mammaecarcinom der Ratte. Z. Krebsforsch. 247-254 (1 965) . 85. Inano, H., Tamaoki, B.: Purification and properties of NADP-dependent 17ß-hydroxysteroid dehydrogenase solubilized from porcine-testicular microsomal fraction. Europ. J. Biochem. 44, 13-23 (1974). 86. Tseng, L., Gurpide, E.: Stimulation of various 17ß- and 2Oa-hydroxysteroid dehydrogenase activities by progestins in human endometrium. Endocrinology 104, 1745-1748 (1979). 87. Jensen, E.V.: Mechanism of estrogen action in relation to carcinogenesis. Can. Cancer Conf. 6^, 143-165 (1 966). 88. Jütting, G.: Die Wirkung von Östrogenen auf die 17-betaHydroxysteroid: NAD-Oxydoreduktase des Myometriums von Kaninchen. Geburtsh. u. Frauenheilk. .26, 636-639 (1966). 89. Macartney, J.C., Thomas, G.H.: NADP-linked 173- and 20asteroid reductase activity in the rabbit uterus. J. Endocr. 43, 247-252 (1 969) . 90. Gabb, R.G., Stone, G.H.: Uptake and metabolism of tritiated oestradiol and oestrone by human endometrial and myometrial tissue in vitro. J. Endocr. 109-123 (1 974). 91. Hobkirk, R., Nilsen, M., Jennings, B.: 17-Oxidoreduction of 17ß-estradiol, estrone and their 3-sulfates by kidney slices from guinea pig and human. Can. J. Biochem. 53, 1333-1336 (1975). 92. Zuckerman, N.G., Hagerman, D.D.: The hydrolysis of estrone sulfate by rat kidney microsomal sulfatase. Arch. Biochem. Biophys. J_35, 410-41 5 (1 969). 93. King, R.J.B., Mainwaring, W.I.P.: Steroid-Cell Interactions. Butterworth, London (1974). 94. Utaaker, E., St$a, K.F.: Oestrone sulphatase activity of the rat uterus in different hormonal states. Submitted for publication.

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Purdy, R.H., Halla, M., Little, B.: 2Oa-Hydroxysteroid dehydrogenase activity, a function of human placental 173hydroxysteroid dehydrogenase. Biochim. biophys. Acta 89, 557-560 (1964).

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Tseng, L., Gusberg, S.B., Gurpide, E.: Estradiol receptor and 173-dehydrogenase in normal and abnormal human endometrium. Ann. N.Y. Acad. Sci. 286, 190-198 (1977).

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Iwamori, M., Moser, H.W., Kishimoto, Y.: Solubilization and partial purification of steroid sulfatase from rat liver: Characterization of estrone sulfatase. Arch. Biochem. Biophys. 174, 199-208 (1976).

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Pulkkinen, M.O.: Arylsulphatase and the hydrolysis of some steroid sulphates in developing organism and placenta. Acta physiol. scand. 52, Suppl.180, 1-92 (1961).

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Kishimoto, Y., Sostek, F.: Activity of sterol-sulphate sulphohydrolase in rat brain: Characterization, localization and change with age. J. Neurochem. J_9, 123-130 (1972).

100. Payne, A.H., Lawrence, C.C., Foster, D.L., Jaffe, R.B.: Intranuclear binding of 173-estradiol and estrone in female ovine pituitaries following incubation with estrone sulphate. J. biol. Chem. 248, 1598-1602 (1973). 101. Dodgson, K.S., Spencer, B., Thomas, J.: The localization of arylsulphatase in the rat liver cell. Biochem. J. 56, 177-181 (1964). 102. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. J_93, 265-275 (1951 ). 103. Rao, B.R., Wiest, W.G., Allen, W.M.: Progesterone receptor in human endometrium. Endocrinology 95, 1275-1281 (1974). 104. Adams, J.B.: Enzymic synthesis of steroid sulfates. II. Presence of steroid sulfokinase in human mammary carcinoma extracts. J. clin. Endocrinol Metab. 24^, 988-996 (1964). 105. Dao, T.L., Libby, P.R.: Conjugation of steroid hormones by normal and neoplastic tissues. J. clin. Endocrinol Metab. 28, 1431-1439 (1968). 106. Dao, T.L., Libby, P.R.: Conjugation of steroid hormones by breast cancer tissue and selection of patients for adrenalectomy. Surgery 66^, 162-166 (1969). 107. Dao, T.L., Libby, P.R.: Steroid sulfate formation in human breast tumors and hormone dependency. In: "Estrogen Target Tissues and Neoplasia", Ed. Dao, T.L., The University of Chicago Press, Chicago, pp. 181-200 (1972). 108. Dao, T.L., Hayes, C., Libby, P.R.: Steroid sulfatase activities in human breast tumors. Proc. Soc. exp. Biol. Med. 146, 381-384 (1 974) .

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109. Dao, T.L., Varela, R., Morreal, C.: Metabolic transformation of steroids by human breast cancer. In: "Estrogen Target Tissues and Neoplasia", Ed. Dao, T.L., The University of Chicago Press, Chicago, pp. 163-179 (1972). 110. Maclndoe, J.H.: Estradiol formation from testosterone by continuously cultured human breast cancer cells. J. clin. Endocrinol Metab. 4_9, 272-277 (1 979). 111. Petrakis, N.L.: Genetic factors in the etiology of breast cancer. Cancer 39, 2709-2715 (1977). 112. Henderson, B.E., Gerkins, V.R., Pike, M.C., Casagrande, J.T.: Endocrine function and breast cancer. In:"Genetics of Human Cancer", Eds. Mulville, J.J., Miller, R.W., Fraumeni, J.F., Raven Press, New York, pp. 291-295 (1977). 113. Fishman, J., Fukushima, D.K., O'Connor, J., Lynch, H.T.: Low urinary estrogen glucuronides in women at risk for familial breast cancer. Science 204, 1089-1091 (1979). 114. Brooks, S.C., Pack, B.A., Horn, L.: The influence of sulfation on estrogen metabolism and activities. In: "Estrogen Target Tissues and Neoplasia", Ed. Dao, T.L., The University of Chicago Press, Chicago, pp. 221-236 (1972) . 115. MacDonald, P.C., Siiteri, P.K.: The relationship between the extraglandular production of oestrone and the occurrence of endometrial neoplasia. Gynecol. Oncol. 2, 2 5 9263 (1974) . 116. Ziel, H.K., Finkl, W.D.: Association of oestrone with the development of endometrial carcinoma. Amer. J. Obstet. Gynecol. J_24 , 735-740 (1976). 117. Hoover, R., Gray, L.A., Cole, P., MacMahon, B.: Menopausal estrogens and breast cancer. New Engl. J. Med. 295, 401-405 (1976). 118. Hayward, J.: Hormones and the aetiology of human breast cancer. Guys Hospital Rep. 121, 51-61 (1972). 119. Ryan, K.J.: Cancer risk and estrogen use in the menopause. New Engl. J. Med. 293, 1199-1200 (1975).

HUMAN GROWTH HORMONE: ASPECTS OF MEASUREMENT

M.A. Vodian and W.P. VanderLaan Lutcher Brown Center for Diabetes and Endocrinology, Scripps Clinic and Research Foundation, La Jolla, California 92037, U. S. A.

Although great advances have been made in the chemistry and physiology of human growth hormone, there persist unresolved problems. These will be reviewed and results from human studies will be considered in parallel with results obtained in other species. Most of the reviews concerning neuroendocrine control of growth hormone secretion (1-4) deal with human growth hormone and rely upon radioimmunoassays (RIA) to measure changes in plasma growth hormone levels. These assays, which are widely accepted as measuring physiologically meaningful changes, indicate that stimuli such as fasting, hypoglycemia, stress, exercise and arginine can produce elevated plasma growth hormone levels. In rodents however, immunoassayable growth hormone does not appear to respond as in man. Garcia and Geschwind (5) studied numerous stimuli in rats, rabbits and mice and found that except for rises with fasting, plasma GH measured by RIA was virtually unresponsive. Later studies have added only nembutal as an effective stimulus to GH secretion. The results in rodents are particularly perplexing in contrast with data obtained by bioassay (tibia test) of pituitary tissue after various stimuli. Studies in rats show that all agents which increase plasma GH levels in humans are effective in depleting pituitary GH. This discrepancy between pituitary and plasma GH changes has led some to question the bioassay as a specific measure of pituitary and plasma GH content (6). An alternate hypothesis, however, and one which will be discussed

Hormones in Normal and Abnormal Human Tissues © Walter de Gruyter • Berlin • New York 1981

574 herein, is that at the time of secretion, pituitary growth hormone is modified such that the circulating form retains biological activity but is no longer immunologically fully active. Several reports demonstrating the existence of a growth hormone-like substance in human plasma were published prior to RIA studies (7). Gemzell et al. showed "tibial activity" in plasma from one acromegalic subject (8). Segaloff et al. (9) later demonstrated values of 0-40ug equivalents bovine GH per ml plasma in normal individuals and 41, 61 and 55ug/ml in three individuals considered to have active acromegaly. Following X-ray therapy, plasma GH concentrations decreased in two of these patients. Moreover, seven other successfully treated acromegalics showed levels below those found in normal individuals, while no hormone could be found in plasmas of six patients with panhypopituitarism. Similar results were found by Gemzell (8);5 acromegalics had growth activity levels of 4-12 jig hGH/ml and undetectable levels after X-ray therapy or hypophysectomy. In contrast with these studies using bioassay methods. Hunter (10) on 81 untreated acromegalic samples showed levels by immunoassay no higher than 0.3 ug/ml. Early reports of bioassayable GH activity also exist for species other than human. Gemzell noted levels in young pig and calf plasmas of 0.37 and 0.53 ng/ml respectively (7), while Cotes and Young found activity in pregnant goat plasmas (11). In the rat, where more extensive studies have been done, ng/ml quantities of growth activity have also been found. Levels of 700 ng/ml, decreasing after 7 days starvation, were reported (12). Contopoulos and Simpson found the equivalent of 500-1000 ng/ml bovine GH in normal rat plasma by the 4-day tibia test (13). Even more impressive in this report were values of 2.5 3.5 ng/ml plasma from pregnant rats using a weight gain bioassay. A daily dose of 3 ml plasma from either 17 or 21 day pregnant rats caused a body weight gain of 10 to 13 g and a tail length increase of 4 - 5 mm; 5 ml/day increased body weight by 1317 g and tail length by 8 mm.

The animals also showed thymus

weight increases up to 100% with no evidence of thyroid stimu-

575 lation, as judged histologically and by

131

I uptake.

Thus, it appears that plasma contains by bioassay GH in ng/ml quantities. Such levels are for the rat at least 50 times those measured by RIA and for man 200 times (14). This discrepancy between the two assays suggests major differences between the pituitary form from which RIAs are developed and the major circulating form. This idea was explored by Ellis and Grindeland (14) who mixed antibodies to rat GH with purified growth hormone, pituitary homogenates, or plasma before injection into hypophysectomized test animals; results showed that tibial responses were blocked in the first two preparations, but plasma growth bioactivity was unaltered. The measurements of plasma growth bioactivity suggest an altered form of GH; if this is so there should be differences between plasma draining the pituitary and the peripheral circulation. In one study (15), bioactive hormone levels were about 4 times greater in jugular vein plasma than in cardiac plasma (1700 ng/ml vs 400 ng/ml, respectively) while immunoactive hormone levels were only 1.6 ng/ml and 5.5 ng/ml respectively. Injection of insulin (2U/kg) into fasted rats caused an 1100 ng/ml increase (in terms of equivalence to GH standards) in jugular plasma bioactive hormone levels while cardiac plasma bioactive levels and all immunoactive levels were not significantly altered. In the second study (16), exposure to 5°C for 1 h caused a small (400 ng/ml) but not significant increase in jugular bioactive levels with no change in immunoactive GH. In animals that had been anesthetized with nembutal, there was a 1900 ng/ml increase in bioactivity and a 10 ng/ml increase in immunoactivity. The changes in immunoactivity were similar to those detailed by Howard and Martin (17). In the second study, somatomedin C levels were also measured. Whereas tibial responses to jugular and trunk plasma supported the notion of a pituitary origin for the growth promoting activity there was no difference in somatomedin C concentrations, making it unlikely that somatomedin C is the growth factor under consideration. Indeed, we know of no reliable

576 evidence that somatomedin can cause growth of the epiphyseal cartilage in the hypophysectomized rat, and a recent study (18) clearly shows that somatomedin A does not stimulate bone growth in the hypophysectomized rat. Similar studies which also suggest a conversion of pituitary GH to a circulating form were done by Vodian and Nicoll (19). In their studies, three groups of rats were either 1) uninjected or 2) injected intravenously with a putative synthetic GRF and killed after 15 or 30 min. Pituitary and plasma GH levels were estimated by bioassay and RIA. Results showed that the bioassayable pituitary growth hormone was depleted by 75% (142 mU) after 15 min. This change was accompanied by an increase of 3.2 mU/ml in plasma bioactive hormone. Such changes in pituitary and plasma hormone levels have been reported previously (20, 21); however, concurrent measurements by both bioand immuno-assays allowed for a more thorough analysis. Quantitative analysis of the relationships between depletion and secretion of bioactive growth hormone by the methods of Garcia and Geschwind (5) showed an excellent balance between the processes if the amount of hormone that was depleted during 15 min. was secreted at a relatively constant rate. Depletion of 142 mU of bioactive growth hormone at a constant rate over 15 min gives an average secretion rate of 9.5 mU/min. With an initial plasma level of 1 mU/ml using the formula of Garcia and Geschwind the anticipated concentration in the plasma after 15 min was 4.1 mU/ml, similar to the observed level of 4.2 mU/ml. Depletion of 4.2 mU of immunoactive growth hormone during 5 min gives an average secretion rate of 2.8 mU/min. With an initial plasma level of 0.03 mU/ml, the concentration after 15 min should be 0.94 mU/ml. which is almost 12 times the amount measured. Thus, from results presented so far, it is clear that plasma contains GH which has a high bioassay/immunoassay ratio— varying from 50-400. This form is of pituitary origin and responds to physiological stimuli. Furthermore, evidence suggests that it is not a somatomedin.

577 The disparity between bioassay and immunoassay measurements in plasma and in particular, jugular plasma of rats, suggests the possibility that the plasma bioactive hormone may be generated in the pituitary by a chemical conversion which results in a relative loss of immunoactivity. This possibility is indirectly supported by the work of Singh et al. (25) who showed that cx^-hGH, has an 8-fold increase in bioactivity but no change in immunoactivity. Further evidence comes from the work of Russell et al. (22) in which rat pituitary tissue and pituitary incubation media were processed by gel electrophoresis and different segments were analyzed by bioassay and RIA. A number of forms of GH with different bio-to immuno-assay ratios were found. Tissue contained a slower migrating form with a ratio of 11.1 - 25.7 while incubation medium contained a faster migrating form with a ratio of 32.7 - 33.8. This supports the possibility that plasma GH may be a converted form of the pituitary hormone with enzymatically attenuated immunoreactivity. Additional evidence for the conversion theory is available from analysis of media in which rat pituitary tissue had been incubated (23). While bio- and immuno-assay measurements of several pituitary samples showed a correlation of 1.2 _+ 0.1, the regression coefficient for secreted GH in incubation media was 2.1 _+ 0.1. The slight increase in the bioassay/RIA ratio of GH spontaneously secreted in vitro does not account for the large discrepancy between bioassay and immunoassay measurements of plasma or serum. Hence, ratios in excess of 50 reported for rat plasma cannot be due to a dramatic change in the property of GH that accompanies secretion. Nevertheless, experiments on the metabolism of GH in vivo and in vitro indicate that secreted GH differs from the intraglandular form in other important respects. Secreted GH was immunologically less stable than purified rat GH when incubated with slices of various rat tissues in vitro. Similarly, the former was cleared from the circulation of hypophysectomized rats more rapidly than the latter (tiof 2.3 and 6.5 respectively). The fact that pretreatment of

578

the rats with trasylol, an inhibitor of trypsin-like enzymes, slowed the rate of clearance of both forms significantly (t^. of 5.1 and 13.2 respectively) indicates that proteolysis is involved, at least in part, in the clearance process. Additional evidence that secreted GH differs from the purified form was obtained from experiments on the effect of trasylol on biological activity. This drug inhibits the tibial epiphyseal response to purified bovine, ovine and rat GH (24) but does not inhibit responses to rat pituitary incubation medium (23). It also does not block responses to rat or human plasma or to purified rat GH which was digested with plasmin. Thus, it appears that purified forms of GH undergo proteolytic modification in vivo before they can be fully biologically active and that trasylol blocks this change. Neither purified GH digested with plasmin nor plasma GH requires this modification since trasylol failed to inhibit their biological activity. Furthermore, since trasylol did not affect the bioactivity of rat GH secreted in vitro, it appears that this form has properties intermediate between those of the major storage form in the pituitary and those found in plasma. The purification of a form of GH similar to that found in plasma was first attempted using cytosol isolated from 0.25M sucrose homogenates of rat anterior pituitary glands (16). Sephadex G-100 gel filtration of the extract produced two bioassayable components with very low immunoactivity. The first peak appeared at the breakthrough volumn while the second and more active peak had a molecular weight of 68,000. Gel filtration of rat pituitary incubation medium showed a very similar distribution of bio- and immuno- activities except that the content of bioassayable hormone was 2-3 times greater in the protein peaks preceding the major storage form. Ellis et al. (16) also examined the pituitary cytosol of bovine glands and found results similar to those in the rat. By Lephacryl L-200 gel filtration, three peaks of bioassayable hormone emerged before the immunoactive form, and comprised about 18% of the total bioactivity in the cytosol. The bioassay-

579 able/RIA ratios ranged from 400 to 1600. Only the breakthrough peak showed somatomedin C activity (1.1 serum units/mg). The second and third peaks (MW-150,000 and 78,000) contained between 20 and 40 ng bovine GH-equivalents per mg. When the second peak, which contained the majority of the activity, was subjected to isoelectric focusing, the bioassayable hormone migrated in the region between pH 5-6 and had bioassayable/RIA ratios in excess of 1500. The immunoactive hormone appeared in the region of pH 8 which is consistent with the previously published pi of bovine growth hormone. Thus, the results clearly show that both the rat and ox pituitary glands contain bioassayable-nonimmunoactive GH. The bovine material more closely resembles human plasma activity with a pi of 5-6 and low immunoactivity. Sephadex G-200 gel filtration of whole plasma (16) showed the bioactive material to elute with albumin, i.e., a molecular weight of 60,000-80,000. Metaphosphate precipitation produced a fraction in which the bioactive material was enriched from 4 ng/mg (whole plasma) to 50 ng/mg; immunoactivity was only 0.24 ng/mg. Further purification by free flow electrophoresis at pH 5.5 produced enrichment to 1.2 p.g/mg. The activity in this fraction could be potentiated 3-fold by the concurrent administration of L-thyroxine to the assay animals; a property also seen with pituitary GH (26). They also evaluated the use of Dowex-50 as an adsorbant for the bioactive hormone. By this technique, activity in human and rat plasma could be enriched to the equivalents 0.8 ng/mg and 0.9 ug/mg respectively. Interestingly, bovine jugular plasma activity was enriched to 2.5 ng/mg, a value that is consistent with pituitary origin. Further studies have been undertaken using a radioimmunoassay directed toward cleaved or two-chain hGH (27). The motivation for the development of such an assay came from the observation that the human pituitary contained cleaved forms, the one lacking residues 135-146 had potentiated growth activity (25). The small amounts of this material in the gland led to subsequent work with bacterial fibrinolysin (28) or subtilisin (29) to produce similar modifications of hGH with the resulting

580

2-chain structure. This 2-chain material was shown to have enhanced metabolic effects in GH deficient individuals (30) and enhanced insulin-secreting properties in isolated pancreatic islets from hypophysectomized rats (31). Antiserum used in the development of a 2-chain RIA was produced 21 days after the injection of the 2-chain material into the Evans Blue dyed popliteal lymph nodes of rabbits (27). The radioiodinated tracer and standard were produced from a subtilisin digest of hGH (hGH-S). After radioiodination by either oxidative or non-oxidative methods, only the high molecular weight, acidic peak proved suitable for RIA. The characteristics of this assay have been detailed more fully (27). Basically, however, the 2-chain (hGH-S) assay was similar in sensitivity to the assay for single chain 191-amino acid hGH. Moreover, none of the single chain forms, including the interchain disulfide dimers, significantly displaced the hGH-S standard. Only certain cleaved molecules and an F^ fragment (residues 1-139) had activity. In studies using the single and two-chain assay systems to measure levels in normal subjects (32), it was found that the basal levels were higher in the hGH-S assay; however, in response to several

stimuli, hGH-S levels were unchanged while

single chain hormone levels were elevated as expected. An interesting response of hGH-S levels was noted in one individual, who suffered a growth arrest during a developing craniopharyngioma. After removal of some but not all of the pituitary gland, the individual resumed growth but showed no hGH responses to multiple stimuli as judged by conventional RIA. Interestingly, by the hGH-S assay, basal levels were significantly elevated and a significant rise occurred in response to arginine infusion (32) suggesting, but not proving, a growth promoting function of hGH-S activity. In additional studies, the molecular weight of plasma hGHS activity was investigated (32). After chromatography of normal plasma on Sephadex G-200, hGH activity, by conventional assay, appeared with the expected molecular weight whereas

581

hGH-S activity emerged at the breakthrough volumn. When a portion of this high molecular weight material was treated with 6M urea and rechromatographed on Sephadex G-100 there was a general enhancement of hGH-S activity throughout the chromatograph and a specific and marked elevation of activity in the low molecular weight range suggesting the unfolding or dissociation of the large molecule. Results from the hGH-S immunoassay suggest the existence of a high molecular weight, acidic and 2-chain form of human GH in plasma. These characteristics are not inconsistent with those presented earlier with regard to the existence and nature of a growth promoting substance in plasma with very little immunoactivity by conventional RIA. There is no evidence, however, to relate hGH-S RIA measurement to biological activity. To this end, studies were undertaken to correlate hGH-S and tibial activities. Table 1 shows the hGH-.S activities of several plasmas and their Dowex-50 fractions after chromatography according to Poffenbarger (33) and Ellis et al. (13). The results show an average enrichment in GH equivalents of hGH-S activity from 0.24 ng/mg in the starting material to 5.6 ng/mg in the adsorbed fraction. These changes, although somewhat smaller than those found by Ellis (13) for bioassayable growth activity, are consistent. Table 1. Plasma

Chromatography of hGH-S activity from plasma Initial hGH-S ng/mg

Dowex Adsorbed

Unadsorbed

ng/mg

ng/mg 0.12

L.L.

0.16

4.2

W.O. S.P.

0.20

12.0

0.27

2. 9

0.084

Pooled

0.31

3.08

0.077

Average

0.235

5.55

0.094

0 .096

582 Table 2 shows the growth hormone activités of Dowex-50 fractions produced from a pool of whole plasma and from a Cohn fraction IV supernatant made by isoelectric precipitation of a crude Cohn IV paste at pH 4.9 - 5.0. The results show, for whole plasma, a 2 5-fold increase in the adsorbed bioactivity and a 10-fold increase in the hGH-S activity. With the Cohn IVpH5 supernatant the increases were 21- and 14-fold, respectively. The significance of these findings is, firstly that the Cohn IV fraction is enriched in hGH-S activity

(unpublished

observation) and in bioactive growth hormone (discussed earlier) and secondly, that both activities adsorbed to Dowex-50. Table 2.

Growth hormone activity of plasma Bioassay

Whole plasma

hGH-S

32 ng/mg

0 .33 ng/mg

Dowex adsorbed Cohn IV-pH5 super

0 .79 ng/mg 0 . 1 Hg/mg

3 .10 ng/mg

Dowex adsorbed

2 . 1 ng/mg

Dowex unadsorbed

0 .05 ng/mg

0 .35 ng/mg 14 .0

ng/mg

0 .063 ng/mg

Fig. 1 shows the Sephadex G-200 chromatograph of a sample of the Dowex adsorbed fraction of whole plasma described in Table 2. The results show the peak hGH-S activity to have a molecular weight of 60,000 to 80,000, a value quite consistent with that of the bioactive GH (13), although different from the studies discussed earlier in which hGH-S activity emerged at the void volumn. When tubes 42-43 were chromatographed on Sephadex G-100 with and without being made 6M in urea, the results were identical to those obtained above: namely all the hGH-S activity was retained and eluted in the low molecular weight region. These results further suggest that although the molecular weight of the hGH-S activity may vary, possible

583

Fig. 1. Sephadex G-200 (2.5 x 90 cm) chromatography of a sample of the Dowex-50 fraction in 0.14M NaCl, 0.01M phosphate buffer pH 6.3 ( , protein concentration; , hGH-S immunoactivity). dissociating or other effects, it yields a smaller and more uniform molecular species. Thus, human and rat plasma contain 50 to 200 times more bioassayable growth activity than suggested by immunoassay. The parallelism between the dose responses for plasma and pituitary GH suggests the plasma activity is derived from pituitary GH. Further evidence comes from the fact that the activity is much higher in jugular than aortic plasma and responds to stimuli such as insulin and nembutal. Thus, it appears that the transformation occurs at or near the pituitary to produce a form with a molecular weight of 60,000-80,000 and a loss of immunoactivity. Numerous accounts have shown immunoactive GH in both plasma and pituitary to be heterogeneous with respect to size. Some larger forms dissociate with urea while others require disruption of interchain disulfide bonds. However, none of these forms can account for the high bioactive but low immunoactivities in plasma.

584

Tube Number

Fig. 2. Sephadex G-200 chromatography of tubes 42-43 from figure 1, before and after treatment with 6M urea. Fractions were analyzed by hGH RIA and hGH-S RIA. The existence of cleaved 2-chain growth hormones in the pituitary has complicated, at least potentially, our understanding of the structures of plasma GH. Studies with human plasma and Cohn IV fractions using the 2-chain immunoassay showed that the tibial line activity adsorbed to the Dowex resin. Moreover, hGH-S immunoactivity also adsorbed to the resin showing approximately the same enrichment of hGH-S and tibial activities. Thus it appears at this time the hGH-S assay may be a better indicator of bioactivity than conventional RIA. In conclusion, the finding of cleaved or 2-chain growth hormones in the pituitary gland stimulated radioimmunoassay studies to identify the circulating form of hGH. However, more studies are needed to define the structure of circulating human growth hormones.

585 Acknowledgements This is Publication #32 from the Lutcher Brown Center for Diabetes and Endocrinology. W.P. VanderLaan is an Olive H. Whittier Fund Investigator.

This work was supported by NIH

Grants CA-14025, HL-20517, AM-09537, American Cancer Society Grant BC-104 and Biomedical Research Support Grant RR-05514.

References 1. Pecile, A., Müller, E.E.: Control of Growth Hormone Secretion.In: "Neuroendocrinology", Eds. Martini, L., Ganong, W.F., Academic Press, New York, pp. 537-564 (1967). 2. Reichlin, L.: Regulation of Somatotrophic Hormone Secretion. In: "The Pituitary Gland", Eds. Harris, G.W., Donovan, B.T., Butterworths, London, pp. 270-298 (1966). 3. Glick, S.M.: The Regulation of Growth Hormone Secretion. In: "Frontiers in Neuroendocrinology", Eds. Ganong, W.F., Martini, L., Oxford, London, pp. 141-182 (1969). 4. Reichlin, S.: Regulation of Somatotrophic Hormone Secretion. In: "Handbook of Physiology", Section 7, Vol. IV, part 2, American Physiological Society, Washington, pp. 405-448 (1 974) . 5. Garcia, J.F., Geschwind, I.I.: Investigation of Growth Hormone Secretion in Selected Mammalian Species. In: "Growth Hormone", Ed. Pecile, A., Müller, E.E., Excerpta Medica Foundation, New York, pp. 217-291 (1968). 6. Rodger, N.W., Beck, J.C., Burgus, R., Guillemin, R.: Variability of response in the bioassay for a hypothalamic somatotrophin releasing factor based on rat pituitary growth hormone content. Endocrinology 84^, 1 373-1383 (1969). 7. Gemzell, C.A., Heijkenskjold, F., Strom, L.: A method for demonstrating growth hormone activity in human plasma. J. clin. Endocrinol. Metab. 1_5, 537-546 (1 955). 8. Gemzell, C.A.: Demonstration of growth hormone in human plasma. J. clin. Endocrinol. Metab. 1_9# 1 049-1054 (1 959). 9. Segaloff, A., Komrad, E.L., Flores, A., Hardesty, M.: The growth hormone content of human plasma. Endocrinology 57, 527-530 (1955). 10. Hunter, W.M., Gillingham, F.J., Harris, P., Kanis, J.A., McGurk, F.M., McLelland, J., Strong, J.A.: Serial assays of plasma growth hormone in treated and untreated acromegaly. J. Endocrinol. 63, 21-34 (1974).

586

11. Cotes, P.M., Young, F.G.: Growth hormone in blood and urine. Biochem. J. 49, lix (1951). 12. Dickerman, E., Negro-Vilar, A., Meites, J.: Effects of starvation on plasma GH activity, pituitary GH and GH-RF levels in the rat. Endocrinology 84, 814-819 (1969). 13. Contopoulos, A.N., Simpson, M.E.: Increased growth promoting substance in the plasma of pregnant rats. Endocrinology 6_1_, 765-773 (1 957) . 14. Ellis, S., Grindeland, R.E.: Dichotomy Between Bio and Immunoassayable Growth Hormone. In: "Advances in Human Growth Hormone Research", Ed. Raiti, S., U.S. Govt. Printing Office, Washington, D.C., pp. 409-433 (1974). 15. Ellis, S., Grindeland, R.E., Reilly, T.J., Yang, S.H.: Studies on the Nature of Plasma Bioassayable Growth Hormone. In: "Growth Hormone and Related Peptides", Proc. 3rd Int. Symp. Growth Hormone, Milan, Eds. Müller, E.E., Pecile, A., Excerpta Medica Foundation, Amsterdam, pp. 75-115 (1976). 16. Ellis, S., Vodian, M.A., Grindeland, R.E.: Studies on the bioassayable growth hormone-like activity of plasma. Recent Prog. Horm. Res. 34' 213-238 (1978). 17. Howard, N., Martin, J.M.: A stimulatory test for growth hormone in the rat. Endocrinology 88, 497-499 (1971). 18. Thorngren, K.G., Hanson, L.I., Fryklund, L., Sievertssen,H.: Human somatomedin A and longitudinal bone growth in the hypophysectomized rat. Molec. Cell. Endocrinol. 6, 217 (1 977) . 19. Vodian, M.A., Nicoll, C.S.: Growth hormone releasing factor and the bioassay-radioimmunoassay paradox revisited. Acta endocr. 86, 71-80 (1977). 20. Müller, E.E., Schally, A.V., Cocchi, 0.: Increase in plasma growth hormone-like activity after administration of porcine GH releasing hormone. Proc. Soc. Exp. Biol. 137, 489-494 (1971) . 21. Sawano, L. , Arimura, A., Bowers, C.Y., Redding, T.W., Schally, A.V.: Pituitary and plasma growth hormone-like activity after administration of a highly purified pig growth hormone-releasing factor. Proc. Soc. Exp. Biol. 127, 1 01 0-1014 (1 968) . 22. Russell, S.M., Vodian, M.A., Hughes, J.P., Nicoll, C.S.: Electrophoretic separation of forms of rat growth hormone with different bioassay and radioimmunoassay activities: Comparison of intraglandular and secreted forms. Life Sciences 23, 236-237 (1978). 23. Vodian, M.A., Nicoll, C.S.: Evidence to suggest that rat growth hormone is modified when secreted by the pituitary gland. J. Endocrinol. 80, 69-81 (1979).

587 24. Grindeland, R.E., Ellis, S.: Effect of trasylol on plasma concentrations, biological activity and immunological half life of growth hormone in rats. The Physiologist J_9, 210 (1976). 25. Singh, R.N.P., Seavey, B.K., Rice, V.P., Lindsey, T.T., Lewis, U.J.: Modified forms of human growth hormone with increased biological activities. Endocrinology 94^, 883-898 (1 974) . 26. Geschwind, I.I., Li, C.H.: The Tibia Test for Growth Hormone. In: "The Hypophyseal Growth Hormone, Nature and Actions" Eds. Smith, R.W., Gaebler, O.H., Long, C.N.H., McGraw Hill, New York, pp. 28-53 (1955). 27. Sigel, M.B., VanderLaan, W.P., VanderLaan, E.F., Lewis, J.J.: Measurement of multiple forms of hGH: Cross reactivities in conventional and 2-chain radioimmunoassays. Endocrinology (in press). 28. Lewis, U.J., Pence, S.J., Singh, R.N.P., VanderLaan, W.P.: Enhancement of the growth promoting activity of human growth hormone. Biochem. biophys. Res. Commun. 6J7, 617-624 (1975). 29. Lewis, U.J., Singh, R.N.P., VanderLaan, W.P., Tutwiler, G.F.: Enhancement of the hyperglycemic activity of human growth hormone by enzymatic modification. Endocrinology 101, 15871597 (1977). 30. Bunner, D.L., Lewis, U.J., VanderLaan, W.P.: Comparative potency of subtilisin cleaved and intact human growth hormone measured in growth hormone deficient human subjects. J. clin. Endocrinol. Metab. 48, 293 (1979). 31. Larson, B.A., Williams, T.L., Lewis, U.J., VanderLaan, W.P.: Insulin secretion from pancreatic islets: Effect of growth hormone and related proteins. Diabetologia 2_5, 129-135 (1978). 32. Lewis, U.J., Singh, R.N.P., Tutwiler, G.F., Sigel, M.B., VanderLaan, E.F., VanderLaan, W.P.: Human growth hormone: A complex of proteins. Recent Prog. Horm. Res.(1979) in press. 33. Poffenbarger, P.L.: The purification and partial characterization of an insulin-like protein from human serum. J. clin. Invest. 56, 1455-1463 (1975).

CATECHOLAMINE SECRETING TISSUES.

NORMAL AND PATHOLOGICAL STATES

R. E. Coupland Department of Human Morphology, The University of Nottingham, Clifton Boulevard, Nottingham NG7 2UH, U.K.

Introduction Catecholamines of importance in human tissues include dopamine, noradrenaline and adrenaline and are synthesized using a common pathway from either tyrosine or dopa (Fig. 1). In lower forms

TYRAMINE

OCTOPAMINE

Fig. 1. Main pathway involved in synthesis of catecholamines with possible alternative routes. 1=tyrosine hydroxylase, 2=dopa decarboxylase, 3=dopamine-3-hydroxylase, 4=phenylethanolamine-n-methyltransferase.

Hormones in Normal and Abnormal Human Tissues © Walter de Gruyter • Berlin • New York 1981

590 including molluscs and arthropods octopamine is also an important catecholamine and recent work suggests that the latter functions as a neurotransmitter along with dopamine, which is the predominant catecholamine, and noradrenaline (1, 2). Although small quantities of octopamine have been reported to occur in the vertebrate peripheral and central nervous systems any function is likely to be associated with nervous activity in a transmitter role or as a chance side product of the catecholamine synthesis rather than as a normal secretion having either paracrine or endocrine activities and is, in consequence, beyond the scope of this article. This account will be restricted to a consideration of the distribution of catecholamine synthesizing and secreting tissues of an endocrine or paracrine nature and of the localization and nature of catecholamine secretion by tumours arising from endocrine paraganglionic and nervous tissues but will exclude an account of normal adrenergic neurons. This account is, therefore, primarily concerned with the distribution, function and pathology of cells that are commonly referred to as chromaffin cells, a term justified by almost a century of usage and the fact that true chromaffin cells synthesize and store catecholamines in sufficient concentrations to result in a brown-yellow chromaffin reaction being produced when they are fixed in an aqueous mixture of aldehydes (formaldehyde or glutaraldehyde) and potassium dichromate; the amines are secreted in endocrine or paracrine fashion after appropriate stimuli. These cells are developed from the neural crest (3, 4, 5), are usually innervated by pre-ganglionic sympathetic nerve fibres (6, 7) though in some forms, such as rabbit, large collections of extra-adrenal cells may be non-innervated (8, 9). In addition to chromaffin cells other elements such as the carotid body, paraganglia, glomus cells and enterohormone-secreting cells that store catecholamines will be considered as will neural tumours that are characterized by their synthesis of relatively large amounts of catecholamines (neuroblastomas etc.).

591

Chromaffin Cells These elements may occur singly but more commonly in groups in close association with the sympathetic nervous system - in the paravertebral chains and in the prevertebral plexuses and their extensions to individual organs - as well as in the well-accepted site, the adrenal medulla. In adult vertebrates chromaffin cells synthesize and store catecholamines mainly in the form of either noradrenaline or adrenaline and the type of amine stored is determined by the presence or absence of the methylating enzyme phenyl-ethanolamine-n-methyltransferase

(PNMT)

which is responsible for the conversion of noradrenaline into adrenaline (10, 11, 12, 13). The amine is synthesized from tyrosine or dopa by enzymes present in the membrane-bound chromaffin granules or in the cytosol (14) and does not traverse the Golgi zone during the synthetic process (15). In consequence all existing chromaffin granules, according to storage capacity will bind recently synthesized catecholamines and hence in terms of amine content, but not content of binding substance, there are no such things as recently synthesized amine storage granules. However, within chromaffin granules catecholamines are bound to the protein chromogranin and ATP (14, 16) both of which are strongly anionic, and the granule protein does traverse the endoplasmic reticulum and the Golgi zone during its synthesis and prior to packaging in the usual way (17, 18) and in consequence so far as protein content is concerned one may recognize recently synthesized and older granules. Within the normal cell catecholamines are stored in membrane-bound granules (6, 18, 19) along with binding protein that is mainly water-soluble chromogranin but includes dopamine3-hydroxyla se (DBH) (14, 16). A substantial amount of insoluble DBH is found in the granule membrane. Substantial quantities of lipid are also found in chromaffin granules and these characteristically have a high cholesterol level relative to phospholipid (21). The content of lysolecithin is relatively great, about 16.8% total lipidphosphorus (16, 21). More recently the

592 carbohydrate content including sugars, glycoproteins and glycosaminoglycans has been analysed and as with proteins and lipids has been shown to exist in various proportions within the granule and its membrane. The granule contents are probably mainly discharged by exocytosis (6, 20, 22) during which the limiting membrane of the granule fuses with the plasma membrane of the cell and in consequence protein (chromogranin and some dopamines-hydroxylase) , ATP and catecholamine are discharged simultaneously into tissue spaces, adjacent to blood capillaries. However, there is good evidence from exocytotic profiles that in chromaffin tissue exocytosis is not restricted to the plasma membrane of the capillary face of the cell but also occurs elsewhere into intercellular spaces and there is good evidence that after drug treatment, e.g. in vivo administration of reserpine, significant amine loss occurs by diffusion across granule and plasma membranes rather than by exocytosis (23, 24). In normally innervated chromaffin tissue amine discharge normally follows preganglionic sympathetic nerve stimulation, the stimulus being conveyed to the cells via a synaptic-type contact between a cholinergic nerve ending and the chromaffin cell (6, 20). The distribution of chromaffin cells in man was first described in detail by Zuckerkandl (25) and Kohn (26, 27). These endocrine elements were observed to develop in intimate association with the sympathetic nervous system and discrete collections of cells were referred to by Kohn as 'paraganglia' due to their close association with prevertebral and paravertebral sympathetic ganglia. The largest collections of chromaffin cells are observed in the abdomen within the prevertebral sympathetic plexuses adjacent to the abdominal aorta. This association prompted the writer to refer to them as para-aortic bodies (28, 29). Chromaffin bodies also develop in the region of the coeliac and superior mesenteric sympathetic plexuses and are often in cellular continuity with the developing adrenal medulla for a limited time. Smaller bodies are noted in association with all

593 parts of the prevertebral sympathetic plexuses, adjacent to the paravertebral chains, splanchnic nerves and pelvic (inferior hypogastric) plexuses, while discrete chromaffin cells or variable sized aggregates are present in all paravertebral sympathetic ganglia and most prevertebral ganglia. The continuity of developing intra-adrenal and extra-adrenal chromaffin tissue is still more evident in some other forms such as the rabbit (6, 30) and may persist into the postnatal period. In man there is a marked and relatively precocious development of extra-adrenal chromaffin tissue during foetal life, while the adrenal medulla is relatively poorly developed even at birth and still contains a mixture of cell types (primitive sympathetic and chromaffin cells). The largest paraganglia in man, para-aortic chromaffin bodies, are found in the vicinity of the origin of the inferior mesenteric artery and are often referred to as the organs of Zuckerkandl (Fig. 2). These may be paired structures or they may be united by an isthmus crossing the aorta. The adrenal medulla only reaches adult proportions later during childhood while the extra-adrenal elements appear to achieve maximum size by 3-5 years of age after which the larger bodies elongate and disintegrate. It is apparent however, that although the major extra-adrenal bodies disintegrate as anatomical entities in later childhood chromaffin cells persist in this situation and in all others in which they are found at the time of birth even in older adults (6 , 29, 31 , 32) . The encapsulated extra-adrenal chromaffin bodies (paraganglia) become more numerous and larger as one moves in a craniocaudal direction and in consequence the smallest are found in the cervical region and the largest in the pelvis. In the pelvis, adjacent to the sacral ganglia encapsulated chromaffin bodies up to 1 mm in diameter are frequently observed and similar sized chromaffin bodies extend along the medial face of the inferior hypogastric plexus on each side as far as the base of the bladder and are particularly numerous lateral to the prostate in the newborn male. Similar but usually smaller (about 0.5 mm diameter) chromaffin bodies are observed along the

594

Fig. 2. Reconstruction of distribution of chromaffin tissue in the infant. CA=coeliac axis, SM=superior mesenteric artery, IM=inferior mesenteric artery. The largest discrete masses are the adrenal medullas and organs of Zuckerkandl. greater thoracic splanchnic nerve in the thorax. The pelvis also contains smaller, often non-encapsulated, groups of chromaffin cells on the visceral branches of the pelvic plexuses adjacent to midline viscera and embedded within the genitourinary organs, especially the prostatic capsule and the

595 f ibromuscular stroma of the gland, anteriorly (Fig-. 3) ''•«•• 11

«

.

• ; • : • - *; ^.fSBiS jflfMPf 'f gMMHIll >

.

pi®

l ^ H ^ H ^ H H H R I i H i H

; -•"*>••,^

' v' „ w

ilk

•• >

*.

.

CSV

v

-'. 'Si P n 'Vi

i ,

4iv- -C-••

:f1

•

>:

.

Fig. 3. Section through pelvis of neonate. Arrows indicate groups of chromaffin cells within and adjacent to pelvic sympathetic nerve fibres and in the capsule of the prostate. In all the above situations, whether the groups of chromaffin cells are encapsulated or not they lie in intimate relation with capillary blood vessels and present an endocrine appearance. Furthermore, the nature and colour of the chromaffin reaction suggests that they store noradrenaline. In the opinion of the writer the cells described above in man are typical noradrenaline-secreting chromaffin cells and are best referred to as such since over the past seventy years the term chromaffin has acquired descriptive, embryological significance (6). However, some workers (33, 34) refer to the non-encapsulated granule containing cells of sympathetic ganglia as small intensely fluorescent cells (SIF cells) (35), since they fluoresce intensely after exposure to formaldehyde

596 vapour, and to the encapsulated groups of cells lying in the retroperitoneal tissues as paraganglia. In order to give these chromaffin or SIF cells functional relevance it must be stressed that in man they store catecholamines, principally noradrenaline, and that they are innervated (36) and hence almost certainly are functionally active. As stressed previously by the writer (6, 37) the extra-adrenal chromaffin tissue in man and many other mammals including guinea-pig, rabbit, cat and dog, persists throughout life although some cells may be lost in consequence of adaptive growth changes during the period between birth and sexual maturity. Since the cells involved are similar to, but not identical with, the undoubtedly endocrine cells of the adrenal medulla, it seems reasonable to assume that the collections of extra-adrenal chromaffin cells (paraganglia) also have an endocrine function. In keeping with this conclusion is the fact that although they usually lie adjacent to sympathetic nerve fibres in the retroperitoneal or retropleural connective tissues they are usually some distance away from sympathetic neurons or likely target organs and hence almost certainly secrete their stored amines into the associated capillary blood vessels. An interesting relationship of human extra-adrenal chromaffin bodies is their occasional close association with lymph nodes (28, 31). Thus lymphocytes and chromaffin cells may share a common connective tissue capsule with the two cell types lying on opposite sides and intermingling in the mid—zone (Fig. 4). Clearly these normal associations should be recognized since, in cases of phaeochromocytoma, the findings of normal chromaffin cells within a common capsule with lymphoid tissue could be misinterpreted as a secondary deposit. Ivanoff (38) considered that lymphocytic invasion of the extra-adrenal chromaffin bodies occurred in man during childhood and was responsible for the disappearance of the large discrete bodies often referred to as the organs of Zuckerkandl. In the writer's opinion this cannot be substantiated (6, 29) and the dispersal and possibly partial loss of extra-adrenal chromaffin cells is a consequence of

597

Fig. 4. Chromaffin body (right) fused with lymph node (left). The arrow indicates a group of chromaffin cells surrounded by lymphocytes. growth changes possibly combined with hormonal influences especially the concentration of glucocorticoids - since the extra-adrenal masses are exposed to a lower concentration of adrenocorticosteroid than the cells of the adrenal medulla (39). A hormonal effect is strongly supported by the observations of Lempinen (40) on the rat, in which, unlike man, rabbit, guineapig and mouse (37) the extra-adrenal chromaffin cells normally degenerate shortly after birth. Lempinen (40) observed that when cortisone or Cortisol was administered to neonatal animals not only was the disappearance of the extra-adrenal chromaffin body prevented but the cells increased in number; furthermore, the amine content changed from almost entirely noradrenaline to predominantly adrenaline (41). The latter finding is entirely in keeping with the biochemical findings (9, 10), in vitro work (11, 42) and in vivo observations (13, 43).

598 Histogenesis of Catecholamine-Storing Chromaffin Cells Following the elegant work of Nicole Le Douarin and her colleagues using quail-chick grafts there is now no doubt that adrenal medullary chromaffin cells arise from neural crest. Under normal circumstances in the chick and quail adrenal chromaffin cells develop from neural crest of somites (18-24, 44). Since neural crest cells derived from other parts of the neural axis are also capable of differentiating in this way, providing they are transplanted to the appropriate level (S10-24), it is apparent that local factors influence differentiation: from the observations of Le Douarin et al. (44) this is probably notochord or a substance derived therefrom. The common developmental origin of the suprarenal bodies, equivalent of the mammalian adrenal medulla, and sympathetic neurons in the elasmobranch was described by Balfour (45) and since that time numerous workers have confirmed this work with respect to sympathetic neurons and adrenal chromaffin cells in amphibia, reptiles, birds and mammals (for recent reviews see Coupland 6, 28). It is generally agreed that the first identifiable precursor cell is a small cell, not unlike a small lymphocyte in size and appearance in the light microscope - the primitive sympathetic cell - which presents a basophilic nucleus about 6 |!in in diameter and scanty cytoplasm. In 8 mm rabbit and human embryos these cells can be traced from the neural crest and neural tube to the sites of definitive sympathetic chain and ganglia and adrenal gland, as well as to the abdominal preaortic region in which the major extra-adrenal chromaffin bodies will develop (6, 28). In these situations the primitive sympathetic elements differentiate into either neuroblasts and subsequently neurons or phaeochromoblasts and subsequently chromaffin cells (phaeochromocytes). Using the highly sensitive formaldehyde-induced fluorescence (FIF) reaction for catecholamines in mice, Fernholm (46) has shown that the primitive sympathetic elements lying in the position of the sympathetic chain and extending ventrally lateral to the aorta, give a

599 positive FIF. Subsequently, fluorescence is observed in sympathetic neurons or granule-containing cells storing catecholamines. In the chick fluorescence is noted even earlier (stage 20) in cells in the dorsal root ganglion and subsequently in the primary sympathetic trunks (47). At this early stage none of the cells would give a positive chromaffin reaction though in consequence of the positive FIF reaction they could appropriately be referred to as SIF cells. Some of these elements may develop into typical chromaffin cells depending on the nature of the amine and its concentration within the cell. However, all would give a positive FIF reaction. In addition to depending upon the concentration of amine stored in a cell chromaffinity depends in part on the type of amine stored adrenaline and noradrenaline giving a stronger reaction than dopamine-storing cells. This difference in the intensity of the chromaffin reaction with respect to tissues containing dopamine may in part reflect the greater difficulty in retaining dopamine in the tissues during the fixation process (48). The cells first described by Eranko as SIF cells were catecholamine-storing elements in the superior cervical ganglion of the rat that failed to give a positive chromaffin reaction and are now known to be dopamine-storing elements (49, 50, 51). According to their proximity to sympathetic neurons or blood vessels, in some forms SIF cells can be divided into two types (52), Type I, that are interneurons, often with long processes synapsing or adjacent nerve cells and Type II that are associated with blood capillaries, into which their secretory products are discharged. From the work of Santer et al. (53) and Lever et al. (54) it is apparent that the Type II cells are also chromaffin cells. There is currently no evidence to suggest that the chromaffin cells of the adrenal medulla, encapsulated extra-adrenal chromaffin bodies (paraganglia) and those scattered in small groups adjacent to the sympathetic chains, plexuses and branches to viscera, are other than endocrine cells contributing to the noradrenergic output of the sympatho-adrenal system. Furthermore, all evidence to date points to these endo-

600

crine elements storing and secreting noradrenaline in the normal in vivo situation. Since there is now general agreement that chromaffin cells develop from neuroectodermal elements first identifiable as primitive sympathetic cells and there is evidence that the primitive cell contains catecholamine (46) it is of interest to follow the differentiation of these elements up to the stage of the mature chromaffin cell. Soulie (55) described three stages in the histogenesis of the chromaffin cell and Poll (56), confirming these findings, suggested that cells typical of each stage should be designated sympathogonia, phaeochromoblasts and phaeochromocytes. More recent accounts of the changes as observed in the light microscope have been published by Coupland (6, 28, 29, 30), crowder (57), Ito (58) and Boyd (59), while Coupland and Weakley (8, 9) and Hervonen (60) followed the changes by electron microscopy in rabbit and man respectively. From these works it is apparent that three distinct stages exist in the histogenesis of chromaffin cells as judged by nuclear morphology, cytoplasmic characteristics and chromaffin reaction. The cells involved are, as in order of appearance: (a) primitive sympathetic cells, (b) phaeochromoblasts, and (c) chromaffin cells (phaeochromocytes). In a number of species such as man, marmoset and rabbit, adrenal medulla and extra-adrenal chromaffin tissue show continuity during development. Even so, the intra- and extra-adrenal chromaffin tissues show different rates of differentiation. In man extra-adrenal chromaffin bodies differentiate before the cells lying within the adrenal gland and at birth the adrenal medulla is a rudimentary structure that still possesses a few primitive cells as well as typical chromaffin cells. In man there is a marked increase in size of the adrenal medulla between birth and 3 years of age (6) accompanied by the disappearance of primitive cells and an increase in the intensity of the chromaffin reaction in the parenchymal elements. In lower forms the chromaffin reaction of cells in the adrenal medulla is usually only weakly positive at birth even though it may have a

601

substantial size, usually the extra-adrenal tissue gives a more intense reaction. This may in part reflect the mainly noradrenaline content of extra-adrenal tissue. Recent electron microscopic observations on developing chromaffin cells by the writer indicate that although at an early stage primitive sympathetic cells possess no cytoplasmic amine storage granules, occasional granules are observed just prior to the appearance of phaeochromoblasts. The latter cells are characterized by an elongated nucleus, large aggregates of chromatin, more abundant cytoplasm with small highly electron dense membrane-bound granules lying adjacent to the plasma membrane and a negative chromaffin reaction; they also tend to form whorls (6, 8, 28). Thereafter the nuclei become more rounded, granules increase in number and size and become more generally distributed throughout the cytoplasm except in a paranuclear zone adjacent to the Golgi complex in NA cells, and a positive chromaffin reaction develops - at this stage the cells can appropriately be designated chromaffin cells (phaeochromocytes). During the histogenesis of chromaffin cells an interesting difference in the development of the amine-synthesis pathway is noted between adrenal and extra-adrenal cells: adrenaline synthesis first occurring in chromaffin cells situated within the adrenal medulla while extra-adrenal elements always synthesize and store mainly noradrenaline irrespective of age. An apparent correlation between the relative size of the adrenal cortex and the proportion of adrenaline (cf. noradrenaline) stored within the adrenal medulla prompted Shepherd and West (61) to suggest that the cortex played some role in determining the synthesis of adrenaline. However, later Shepherd and West (62) largely abandoned this idea when they identified adrenaline in the suprarenal bodies of dogfish which are separate from cortical (inter-renal) tissue and when they found some adrenaline in the extra-adrenal bodies (organs of Zuckerkandl) of man (6 3). However/ by separating intra— and extra—adrenal chromaffin tissue in young rabbits aged between birth and 14

602

days and assaying the adrenal glands or extra-adrenal bodies of pre- and postnatal specimens of man, Coupland (39) showed that adrenaline synthesis and storage occurs in the foetal adrenal from about mid-term and continues to increase in proportion to total catecholamine content after birth until the proportion characteristic of the species is attained prior to sexual maturity - in man the adult proportions of the two amines are obtained by 3 years of age. Although with the bioassay techniques used at the time of the earlier work the small amount of adrenaline ( vagal > jugulotympanic > laryngeal and aorticopulmonary. As with phaeochromocytoma it appears to reflect the size of the normal population of specific cells in the various situations. Other sites include the lateral wall of the nasal cavity and orbit. Ahmed et al. (155) reporting a case of chemodectoma of the orbit noted its relatively anterior as well as lateral position and suggested the possibility that paraganglionic tissue may normally exist in such a situation as well as adjacent to

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the ciliary ganglion. Once again it is worth emphasizing that in view of the extensive contribution of neural crest tissue and mesectoderm to the head and neck (143), it is possible for such tissue to occur virtually anywhere in association with arteries derived from branchial arteries and the branches of sensory cranial nerves. Virtually all reports on glomus tumours (paragangliomas) of head and neck refer to their non-chromaffinity, even when catecholamines are readily revealed by assay or amine-storing granules are evident by electron microscopy. Extensive experience of fixation of phaeochromocytomas and normal adrenal medulla (human) by the writer has demonstrated repeatedly that the majority of amines are lost from intracellular storage sites during the operation due to handling of the tissues and a variable period of deprivation of blood supply prior to final removal. Even masses containing up to 0.5 - 1 mg/g catecholamine may give a patchy or weak chromaffin reaction in consequence of amine loss from the intracellular storage site. In consequence of the above, chromaffinity is not a good criterion for determining the functional type of these elements and electron or light microscopy combined with chemical assay are essential. The report of Matsuguchi et al. (145) make it apparent that it is worth considering central venous blood catecholamine determinations in the identification of these tumours and larger masses would be likely to result in abnormal levels of metanephrine, VMA and/or catecholamines in the urine.

The APUD Concept and Catecholamine Producing Tumours In 1966 and 1969 Pearse described collections of cells in various parts of the body that contained various esterases and had in common an ability to produce a peptide hormone and to take up amine precursors and decarboxylate them (156, 157). Initially these cells were also thought to develop from neural crest; however, subsequent work, in particular by Le Douarin and colleagues

621

(158) made it apparent that only some members of what are now called the APUD series are neural crest in origin. A recent review of the APUD concept and series was published by Pearse (159), these elements are widely distributed from brain to the peripheral tissues and the majority of endocrine cells that store preformed hormone belong to this series. While all APUD cells, under specific condition, may be able to take up amine precursors, in particular dopa and 5-hydroxytryptophan, and decarboxylate them forming dopamine or 5-hydroxytryptamine in the adult few of these cells show evidence of amine storage. Their most characteristic feature is the presence of cytoplasmic granules that contain a protein or peptide that may be a recognised hormone, as in the case of the gut endocrine cells, or may be without hormonal activity, chromogranins associated with catecholamine storage. However, a further important fact that has emerged is that tumours composed of APUD cells may synthesize and secrete not only the peptide hormone normally associated with the particular cell type but also peptides normally associated with other members of the APUD series. Furthermore, a tumour composed of one type of APUD cell may occur simultaneously with tumours of other members of the series. Examples of multiple hormone production by phaeochromocytomas are provided by the reports of calcitonin and catecholamine production by phaeochromocytomas (160, 161, 162) while a tumour of the adrenal medulla may secrete both ACTH and 3*~MSH (163). Examples of the occurrence of phaeochromocytomas as part of multiple endocrine neoplasia syndromes (adenomatosis) include in particular type 2A (Sipple's syndrome) involving medullary thyroid carcinoma and hyperplasia or adenomatosis of parathyroid glands with phaeochromocytoma, and type 2B, medullary thyroid carcinoma, rarely parathyroid lesions, mucosal neuromas and Marfanoid habits with phaeochromocytoma (for recent discussions see Manger and Gifford, 88 and Lips, 164). Thus in the clinical and pathological context catecholamine secreting tissues must be considered not only in terms of

622

a common developmental origin from neural crest but also in terms of their possession of common functional and morphological features reflecting their ability to synthesize and store peptides within cytoplasmic granules.

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PROLACTIN DETERMINATIONS IN HEALTH AND DISEASE

P. A. Kelly Groupe du Conseil de Recherches Médicales en Endocrinologie Moléculaire, Le Centre Hospitalier de l'Université Laval, 2705, Boulevard Laurier, Québec, G1V 4G2, Canada

Introduction Prolactin, a hormone secreted by the anterior pituitary gland, was first isolated from ovine pituitary glands in 1932 (1, 2). In subsequent years, prolactin was identified and purified in many other species. However, in humans there was serious question concerning its existence as a separate molecule, since human growth hormone was shown to possess, in addition to its growth promoting activity, substantial bioassayable "prolactin activity" (3). There was, however, increasing evidence which supported the existence of a separate prolactin molecule in the human. An in vitro bioassay was developed (4) which measured elevated lactogenic activity in patients with abnormal lactation, while the same patients had growth hormone levels which were normal. Subsequently, it was shown that pregnant monkey pituitaries secreted a protein which was immunologically different from human growth hormone, but which crossreacted with an antiserum to ovine prolactin (5), and that incubations of human pituitaries synthesize and secrete prolactin (6). Human prolactin was isolated and purified independently in the laboratories of Lewis (7) and Friesen (8) and a specific radioimmunoassay was developed (9)

Hormones in Normal and Abnormal Human Tissues © Walter de Gruyter • Berlin • New York 1981

636 Measurement of Prolactin A. Radioimmunoassay The ability of an extract of pituitary glands to stimulate the crop sacs of hypophysectomized doves was the initial assay method for measuring prolactin (PRL) activity (1, 2). Using this assay, Riddle et al (2) successfully isolated and purified prolactin. Probably the most widely known action of prolactin in mammals is its effect on the mammary gland. In addition, prolactin is luteotropic in rodents (10). Although these and other bioassay techniques have been successfully applied to the measurement of prolactin in pituitary extracts, none of the assays was suitable to measure normal circulating levels of prolactin, since the assay sensitivity was between 0.2 and 10 (ig prolactin. The identification of human prolactin was not made until 1971, due in part to the inherent lactogenic activity of the growth hormone molecule. Another

assay technique, namely iji vitro

bioassay using mammary tissue explants from mice or rabbits, was important in the identification of human prolactin as a separate molecule (4). These assays, which measure activity between 2 to 100 ng/ml plasma, were much more sensitive than those previously utilized. However, a major drawback was the amount of work and time required to assay only a few samples. Yalow and Berson (11) revolutionized endocrinology in 1959 with the development of a radioimmunoassay to measure plasma insulin. In the years that followed, specific radioimmunoassays for numerous protein as well as non-protein hormones were developed including radioimmunoassays for prolactins of cow, sheep, goat, pig, mouse, rat and dog. The initial radioimmunoassays for human prolactin were heterologous and used the cross-reaction of prolactin from one species with an antiserum of another. The first assay utilized the cross-reaction of primate prolactins with antiserum to ovine prolactin (5). With the subsequent isolation and purification of human prolactin, specific homologous radioimmunoassays

637 were developed (9, 12). This development of a simple and reliable means of measuring human prolactin enabled a great expansion of knowledge of both the physiological and pathological control of prolactin secretion. 1. Antibody. The Hormone Distribution Program of the National Pituitary Agency of the National Institutes of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, provide both purified human prolactin and anti-hPRL antiserum to qualified investigators. 125 2. Iodination. The incorporation of [ I] as an isotopic marker is routinely carried out by the chloramine-T (13) or lactoperoxidase method (14). Human prolactin is slightly more labile than other anterior pituitary hormones and care must be taken with the iodination. In our laboratory, much lower concentrations of chloramine T are used and the reaction is allowed to go to completion (15). This procedure involves the addition of 20 ul 0.5 M phosphate buffer pH 7.4 and 10 ill [ 1 2 5 I]Na (750 |iCi) to 2-5 ng of prolactin. The reaction is started by the addition of 10 |il chloramine T (800 ng) and is allowed to proceed for 3-4 minutes. The reaction goes to completion, obviating the necessity to add sodium metabisulfate. The incubation volume is diluted by the addition of 1 ml buffer (see below). The radioiodinated prolactin is purified on a column (0.9 x 50 cm) of Sephadex G-75 . 3. Assay conditions. For assay 10 mM phosphate buffer containing 150 mM NaCl, 0.25% BSA and 0.1% sodium azide is used. Each assay tube contains 500 m- 1 (15,000-20,000 cpm) iodinated hormone and 100 m- 1 of standard solutions from 0.1 to 20 ng hPRL/tube or of unknown samples. Antiserum (200 (il) , at an initial dilution of 1:60,000 in phosphate buffer containing 1% normal rabbit serum are added and the tubes incubated for 48 h at 4°C. Second antibody (200 nl), at a concentration sufficient to precipitate the bound prolactin-

638 antibody complex, is added and the tubes incubated overnight at 4°C. Bound and free hormone are separated by low speed centrifugation (2000 x g) for 15 min, after which the supernatants are decanted and the tubes left inverted to dry. At the concentration of labelled hormone and antibody used, 4050% of the hormone is bound in the absence of any unlabelled PRL. Quality control samples are included in each assay. B. Radioreceptor assay A radioreceptor assay (RRA) has been developed to measure prolactin and lactogenic hormones (16, 17). This assay utilizes specific, high affinity receptors for prolactin from rabbit mammary glands. A major advantage of the prolactin radioreceptor assay is that it is not species specific as are most radioimmunoassays, since presumably the biologically active part of the molecule binds to the receptor. This assay can be used to screen active fragments of the prolactin molecule in the search for smaller active peptides. The other advantages of the RRA are that it is simple and quick (samples can be assayed in 6 hours) and the reaction is highly specific to hormones with lactogenic activity, including prolactin, primate growth hormones and placental lactogens (PL). The RRA has in fact been successfully applied to quantitate PL in species which had not been recognized as possessing PL activity (18). Data on PL activity in a number of species have recently been reviewed (19). It should be mentioned that the ability of the receptor to recognize all 3 types of lactogenic hormones necessitates, when the levels are sufficiently high to interfere, measuring the level of the other two hormones by RIA. This is not a problem when measuring human placental lactogen since hPL levels often exceed 2000-3000 ng/ml, whereas hGH levels are usually below 5 ng/ml and PRL levels below 30 ng/ml, and not higher than 200 ng/ml at term.

639 Circulating Forms of Prolactin For many years, hormones circulating in the blood were thought to exist with a single molecular size. A number of polypeptide hormones, including insulin (20), parathyroid hormone (21), gastrin (22), adrenocorticotropic hormone (23) and growth hormone (24), have been reported to be heterogeneous and appear in the circulation in more than one form. Native human prolactin has a molecular weight of approximately 21,000 daltons (7, 8). In normal subjects, two forms of human prolactin could be separated by gel filtration, "little" hPRL, which accounted for 80-90% of the total activity and the "big" form which was less than 20% (25). Aubert et al. (26) reported a third form, "big-big" prolactin which constituted 0.8 to 7.9% of the total prolactin activity. The molecular form of prolactin varies under different physiological conditions and following various stimuli. The greatest amounts of "big" prolactin, up to 31%, are found during pregnancy (25). Kataoka et al. (27) found the major circulating form of human prolactin to be "big", and after stimulation with thyroid releasing hormone (TRH), the predominant form was "little". Others (25) failed to observe any change in the distribution of human prolactin after

TRH injection, breast

stimulation or L-Dopa inhibition. The distribution pattern in serum of patients with pituitary secreting tumours was altered compared to that of normal patients, in that there was a shift to the larger molecular weight forms "big" and "big-big" (25, 26). Freezing and thawing, urea or mercaptoethanol treatment have been reported to convert "big" prolactin to the "little" form. Aubert et al. (26) compared the forms of human PRL by radioimmunoassay and radioreceptor assay and reported that "big" prolactin had 25% less activity in the RRA than when measured by RIA. Guyda (28) failed to confirm this, reporting comparable immunologic and receptor activity for all forms of human prolactin .

640 These results indicate that prolactin is found in the circulation in several forms, albeit the major form is "little" prolactin. During periods of increased secretion such as pregnancy or in patients with tumours, a larger proportion of the bigger forms is found. These larger forms may be storage or precursor forms.

Prolactin Receptors A. Assay of prolactin receptors For the quantification of receptor levels in a tissue, crude plasma membrane fractions are prepared by differential centrifugation, and ovine prolactin is iodinated to a low specific activity (20-60 p,Ci/ng) . Prolactin binding is assayed using a fixed quantity of membrane preparation (200 ug) with a fixed 1 25 quantity of [ I] ovine prolactin (approximately 100,000 counts per minute). The differences between incubates not containing and those containing excess unlabelled prolactin indicate prolactin specifically bound to the binding sites from which the "percent specific binding" can be calculated. In addition to these "single point assays", saturation curves or displacement curves can be carried out on representative membrane preparations and the data transformed into Scatchard plots (29) yielding affinity constants and binding capacities of the membranes. B. Tissue distribution Binding of polypeptide hormones to specific receptors located in the cell membrane is the first event in the action of these hormones in their target tissues. Specific receptors for a large number of polypeptide hormones have already been identified (30) . Specific prolactin binding has been identified in plasma membrane fractions of choroid plexus, liver, kidney, mammary gland, mammary tumour, adrenal, ovary, testis, prostate, seminal

641

vesicle and uterus (17, 31-35). One of the tissues binding the greatest quantity of prolactin was liver. The presence of prolactin binding sites in liver was observed in a number of species (36) . C. Regulation of prolactin receptors The concept that hormone receptors are not static systems, but change according to the physiological state of the animal has interesting implications in terms of control of cellular activity. The binding sites in rat liver have been shown to be specific for lactogenic hormones, i.e. prolactin, primate growth hormones or placental lactogens (31). The level of these binding sites is very low in male rats and quite high in female animals. Furthermore, when these binding sites were studied as a function of the developmental state of the rat, a marked increase of receptor levels was observed after puberty and during pregnancy (37). The concentration of these binding sites fluctuated during the estrous cycle and declined after ovariectomy or hypophysectomy (38). The hormonal regulation of prolactin receptors is complex. Estradiol injection into male or female rats leads to an increase in hepatic prolactin binding sites (38, 39). The fact that prolactin binding can be stimulated by estrogens, fluctuates with the estrous cycle and is reduced by ovariectomy implies a direct physiological involvement of estradiol. The loss of prolactin binding in rat liver after hypophysectomy implied the importance of a pituitary factor in the maintenance of these binding sites (38, 39). A direct effect of prolactin on its own receptor was first implied by the finding that prolactin binding to liver in hypophysectomized rats with a pituitary implant under the kidney capsule began to increase approximately 3 days after the increase in serum prolactin levels (40). Costlow et al. (41) have also shown that direct administration of 2 mg prolactin to hypophysectomized female rats increased prolactin binding in liver.

642 Androgen administration to both males and females results in a reduction of prolactin binding in rat liver (36, 42).Testosterone and dihydrotestosterone (DHT) reduced basal as well as estrogen stimulated prolactin binding without affecting plasma prolactin levels. The afternoon peak of prolactin induced in ovariectomized rats by estrogen administration is inhibited when estrogen is combined with DHT (42). However, the fact that DHT reduces prolactin binding in males without affecting plasma prolactin levels indicates that this androgen may have a direct effect on prolactin receptors. D. Dissociation of endogenously bound prolactin As a result of elevated plasma levels of prolactin or other lactogenic hormones, often exceeding 1000 ng/ml, occurring for example during pregnancy (18) or during a spontaneous afternoon surge (42), a large fraction of the prolactin receptors in a target cell might be occupied with endogenous prolactin. Although the procedures of homogenization and fractionation offer opportunities for prolactin to dissociate from its binding sites, a large portion of the prolactin still remains bound to the receptors at the end of the preparation. It thus became of interest to develop a technique to remove this endogenously bound prolactin. Two possible approaches were tried. The first involved an in vivo desaturation of the prolactin receptor by lowering the circulating level of plasma prolactin for a short period prior to removal of the tissue. This approach was successfully applied to the quantitation of prolactin receptors in the mammary gland of the rabbit during pregnancy (43) , particularly since the rabbit does not produce a placental lactogen (18, 19). In rat mammary glands there was a low level of receptors during pregnancy with a marked increase during lactation. When the source of placental lactogen was removed by surgical removal of the placenta, prolactin binding approached that observed during lactation (44).

643 The level of placental lactogen in rats during the secondhalf of pregnancy is approximately 800 ng/ml (16, 45). Holdaway et al. (46) using rats infused with ovine prolactin demonstrated that wh n levels exceeded 300 ng/ml, receptor concentrations in both mammary tumour and liver began to decline. Therefore, the hypothesis that placental lactogen was occupying a majority of the prolactin receptor sites and lowering available receptor levels is reasonable. The second technique involves an in vitro desaturation of the

hormone from the receptor (15). Several chiotropic agents

were examined including high molar concentrations of magnesium chloride, ammonium thiocyanate and sodium trifluoroacetate as well as urea and acidic or basic pH but few maintained the integrity of the receptor. Magnesium chloride at a concentration of 4 or 5 M is capable of removing 90-95% of the labelled hormone from rabbit mammary gland prolactin receptors and retain their ability to specifically bind prolactin (15). The control of prolactin receptors in all prolactin responsive tissues is not uniform. Rat ventral prostate has abundant prolactin receptors (46). Castration of male rats results in a reduction of prolactin receptors of the prostate (47, 48) but an increase in those of the liver (42). Prolactin receptors in the prostate are enhanced in animals injected with testosterone propionate or DHT (48, 49) and hepatic prolactin binding is reduced (42) . Estrogens do not appear to stimulate mammary gland prolactin receptors (43) whereas prolactin itself has been shown to stimulate and progesterone inhibit, prolactin binding in lactating rabbit mammary glands (50) . E. Down-regulation In contrast with the inhibitory effect of a large number of hormones on the level of their own receptor (51-53), a stimulatory effect of prolactin on its receptor in both rabbit mammary gland and rat liver has been observed (40, 50). Using 4 M MgCl„

644 to dissociate bound prolactin from its receptor, the short-term action of prolactin on its receptor was investigated to determine

if prolactin, in addition to its ability to up-regulate

prolactin receptors, is, like most other hormones studied so far, capable of inducing a down-regulation of its own receptor. This in turn would lead some support to recent views (54) suggesting that down-regulation, and possibly up-regulation as well, are ubiquitous events which might be intimately linked to the very mechanism of hormone action. Injection of lactating rabbits with 3 mg ovine prolactin resulted in maximal saturation within 15 min of free prolactin receptors, after which receptor levels returned to normal at 1 to 6 h. However, total prolactin receptor levels, measured after in vitro desaturation with 4 M M g C ^ , declined progressively up to 6 h after the intravenous injection of prolactin and returned to normal at 24 to 30 h. A similar down-regulation of prolactin receptors was observed in rat liver (55). These studies show that prolactin, as for a number of other hormones, is capable of inducing a transient down-regulation of its own receptor, in addition to the well established long-term stimulatory effect on its receptor.

Prolactin and Human Breast Cancer A. Evidence for role of prolactin Although the role of prolactin in the development and promotion of experimental breast cancer in rodents is well documented, it has been much more difficult to ascertain whether prolactin is involved in mammary carcinoma in humans, even after the identification and isolation of a separate human prolactin molecule. Hypophysectomy had been reported to induce clinical remission in a substantial number of patients with carcinoma of the breast (56, 57), although the role of prolactin in such remissions has been questioned (58).

645 In a retrospective study, prolactin has been implicated indirectly in human breast cancer in a report by the Boston Collaborative Drug Surveillance Program, which reported an association between regular reserpine use, which is a known stimulator of prolactin secretion, and newly diagnosed breast cancer (59). A similar correlation was found by Heinonen et al. (50), although the association was evident only for women below 50 years of age. These findings although not conclusive, at least suggest that a causal relationship between reserpine use and breast cancer be considered. There is little evidence of elevated plasma prolactin levels in the etiology of human breast cancer. Basal levels have been measured in several groups of patients with established mammary carcinoma and these studies conclude plasma prolactin levels are not significantly different from values observed in normal women (61-63). In a study reported by Kwa et al. (64) plasma prolactin levels in breast cancer patients were similar to those of hospital matched controls. Interestingly, however, they observed elevated prolactin levels in a large number of family members with a high risk of developing cancer (more than two first degree relatives with a previous history of breast cancer). In a more recent study, daughters of patients with breast cancer had significantly elevated prolactin levels in the luteal phase compared to control patients (65). Elevated plasma prolactin levels in both pre- and postmenopausal Caucasian breast cancer patients have been observed (66). A British group has recently reported breast cancer patients had greater prolactin concentrations than normal controls at the follicular and periovulatory stages of the menstrual cycle, although in general no major differences were observed in these patients which had undergone mastectomy 3 months previously (67). Another group reported 15 cases of advanced breast cancer in which plasma prolactin levels were consistently higher (although generally within the normal range) than control levels (68). Finally, in patients with benign disease of the

646 breast, prolactin concentrations were abnormally elevated, especially in women over the age of 30 years, indicating a possible involvement of prolactin in this condition (69). Because of possible stimulatory effects of elevated prolactin levels, L-Dopa, which lowers prolactin, has been given to women with metastatic breast cancer. Minton (70) reported 10 of 30 patients with severe bone pain became "pain free" when treated with L-Dopa. Patients with low fasting prolactin levels who did not respond to L-Dopa by a decrease in prolactin also had no relief of bone pain. B. Prolactin receptors If prolactin does stimulate human mammary tumours, the tissue should contain prolactin receptors, as is the case for other prolactin responsive tissues as well as for experimental mammary tumours (30-34). Holdaway and Friesen (71) have reported on the specific binding of prolactin to human breast tumours. Specific binding of greater than 1 % of the added radioactivity (which the author

considered significant) occurred in 8 of

41 tumours (19.5%). For one tumour, enough material was present to perform a Scatchard plot, and an affinity constant (Ka) of 2.5 nM was determined, which is similar to that of other prolactin receptors (31, 37). Morgan et al. (72) have reported that 15/55 (27%) of human breast tumours showed specific prolactin binding of which 64% were prolactin dependent in culture. Prolactin binding sites in human breast tumours were also localized immunohistochemically. Of 80 cases studied, 4 5 were prolactin dependent in culture, whereas 30 showed positive staining for prolactin (73). In another study of 20 tumours, 70% were described as having measurable prolactin binding (74). In our laboratory, we have examined over 500 biopsies of human mammary carcinoma, both primary lesions and metastases. We consider that values for specific binding above 0.5% reflect the presence of receptors. On this basis, slightly under 50%

647 of the tumours are "prolactin responsive" in terms of possessing prolactin receptors and theoretically being able to respond to prolactin.

References 1. Riddle, 0., Bates, R.W., Dykshorn, S.W.: A new hormone of the anterior pituitary. Proc. Soc. exp. Biol. Med. 29, 1211-1212 (1932). 2. Riddle, 0., Bates, R.W., Dykshorn, S.W.: The preparation, identification and assay of prolactin - a hormone of the anterior pituitary. Am. J. Physiol. 105, 191-216 (1933). 3. Forsyth, I.A., Folley, S.J., Chadwick, A.: Lactogenic and pigeon crop-stimulating activities of human pituitary growth hormone preparations. J. Endocrinol. 3_1_f 115-126 (1 965) . 4. Kleinberg, D.L., Frantz, A.G.: Human prolactin: measurement in plasma by in vitro bioassay. J. clin. Invest. _50, 15571568 (1971). 5. Guyda, H., Hwang, P., Friesen, H.G.: Immunologic evidence for monkey and human prolactin. J. clin. Endocrinol. Metab. 32, 120-123 (1971 ) . 6. Friesen, H., Guyda, H., Hardy, J.: Biosynthesis of human growth hormone and prolactin. J. clin. Endocrinol. Metab. 3±, 611-624 (1970). 7. Lewis, U.J., Singh, R.N.P., Sinha, Y.N., Vanderlaan, W.P.: Electrophoretic evidence for human prolactin. J. clin. Endocrinol. Metab. 33, 153-156 (1971). 8. Hwang, P., Guyda, H., Friesen, H.G.: Purification of human prolactin. J. biol. Chem. 247, 1955-1958 (1972). 9. Hwang, P., Guyda, H., Friesen, H.G.: A radioimmunoassay for human prolactin. Proc. Nat. Acad. Sci. USA 68, 1902-1906 (1971) . 10. Bern, H.A., Nicoll, C.S.: The comparative endocrinology of prolactin. Recent Prog. Horm. Res. 24, 681-720 (1968). 11. Yalow, R.S., Berson, S.A.: Assay of plasma insulin in human subjects by immunologic methods. Nature 184, 1648-1649 (1959) 12. Sinha, Y.N., Selby, F.W., Lewis, U.J., Vanderlaan,.W.P.: A homologous radioimmunoassay for human prolactin. J. clin. Endocrinol. Metab. 36, 509-516 (1973).

648 13. Hunter, W.M. , Greenwood, F.C.: Preparation of iodine 131labelled human growth hormone of high specific activity. Nature J_94, 495-496 (1 962). 14. Thorell, J.I., Johansson, B.G.: Enzymatic iodination of polypeptides with 1251 to high specific activity. Biochim. biophys. Acta 25J[, 363-369 (1971). 15. Kelly, P.A., Leblanc, G., Djiane, J.: Estimation of total prolactin binding sites by in vitro desaturation. Endocrinology 104, 1631-1638 (1979). 16. Shiu, R.P.C., Kelly, P.A., Friesen, H.G.: Radioreceptor assay for prolactin and other lactogenic hormones. Science J_8_0 , 968-971 (1973). 17. Kelly, P.A., Posner, B.I., Tsushima, T., Shiu, R.P.C., Friesen, H.G.: Tissue distribution and ontogeny of growth hormone and prolactin receptors. In: "Advances in Human Growth Hormone Research", Ed. Raiti, S., General Printing Office, Washington, pp. 567-584 (1974). 18. Kelly, P.A., Tsushima, T., Shiu, R.P.C., Friesen, H.G.: Lactogenic and growth hormone like activities in pregnancy determined by radioreceptor assays. Endocrinology 9_9, 765774 (1 976) . 19. Kelly, P.A.: Secretion and biological effects of placental lactogens. In: Proceedings of the V International Congress of Endocrinology, Ed. James, V.H.T., Vol. 2, Excerpta Medica, Amsterdam, pp. 298-302 (197~6). 20. Roth, J., Gorden, P., Pastan, I.: "Big insulin": A new component of plasma insulin detected by immunoassay. Proc. Nat. Acad. Sci. USA 61_, 1 38-1 45 (1 968). 21. Berson, S.A., Yalow, R.S.: Immunologic heterogeneity of parathyroid hormone in plasma. J. clin. Endocrinol. Metab. 28, 1037-1047 (1968). 22. Yalow, R.S., Berson, S.A.: Size and charge distinction between endogenous human plasma gastrin in peripheral blood and heptadeca-peptide gastrins. Gastroenterology 58, 609-615 (1970). 23. Yalow, R.S., Berson, S.A.: Size heterogeneity of immunoreactive human ACTH in plasma extracts of pituitary glands and ACTH-producing thymoa. Biochem. biophys. Res. Commun. 44, 439-445 (1971). 24. Bala, R.M., Ferguson, K.A., Beck, J.C.: Plasma biological and immunoreactive human growth hormone-like activity. Endocrinology 87, 506-516 (1970). 25. Suh, H.K., Frantz, A.G.: Size heterogeneity of human prolactin in plasma and pituitary extracts. J. clin. Endocrinol. Metab. 39, 928-935 (1974).

649 26. Aubert, M.L., Garnier, P.E., Kaplan, S.L., Grumbach, M.M.: Heterogeneity of circulating human prolactin (LPRL); decreased radioreceptor activity of "big" LPRL. Endocrinology 96, 80A (1975). 27. Kataoka, K., Imai, Y. , Hollander, C.S.: Altered molecular heterogeneity of circulating prolactin following thyrotropin releasing hormone. Clin. Res. 2J3, 238A (1975). 28. Guyda, H.J.: Heterogeneity of human growth hormone and prolactin secreted In Vitro: Immunoassay and radioreceptor assay correlation. J. clin. Endocrinol. Metab. 4_1_, 953-967 (1975). 29. Scatchard, G.: The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sei. 5_1_, 660-672 (1949) . 30. Roth, J.: Peptide hormone binding to receptors: a review of direct studies in vitro. Metabolism 22, 1059-1073 (1973). 31. Posner, B.I., Kelly, P.A., Shiu, R.P.C., Friesen, H.G.: Studies of insulin, growth hormone and prolactin binding: tissue distribution, species variation and characterization. Endocrinology 96, 521-531 (1974). 32. Frantz, W.L., Maclndoe, J.H., Turkington, R.W.: Prolactin receptors: characteristics of the particulate fraction binding activity. J. Endocrinol. 60, 485-497 (1974). 33. Turkington, R.W.: Prolactin receptors in mammary carcinoma cells. Cancer Res. 34' 758-763 (1974). 34. Costlow, M.E., Buschow, R.A., McGuire, W.L.: Prolactin receptors in an estrogen receptor-deficient mammary carcinoma. Science 184, 85-86 (1974). 35. Walsh, R.J., Posner, B.I., Kopriwa, B.M., Brawer, J.R.: Prolactin binding sites in rat brain. Science 201, 10411043 (1978). 36. Kelly, P.A., Ferland, L., Labrie, F., De Lean, A.: Hormonal control of liver prolactin receptors. In: "Hypothalamus and Endocrine Functions", Eds. Labrie, F., Meites, J., Pelletier, G., Plenum Press, New York, pp. 321-335 (1976). 37. Kelly, P.A., Posner, B.I., Tsushima, T., Friesen, H.G.: Studies of insulin, growth hormone and prolactin binding: ontogenesis, effects of sex and pregnancy. Endocrinology 9j5, 532-539 ( 1974). 38. Kelly, P.A., Posner, B.I., Friesen, H.G.: Effects of hypophysectomy, ovariectomy and cycloheximide on specific binding sites for lactogenic hormones in rat liver. Endocrinology 97, 1408-1415 (1975). 39. Posner, B.I,, Kelly, P.A., Friesen, H.G.: Induction of a lactogenic receptor in rat liver: influence of estrogen and the pituitary. Proc. Nat. Acad. Sei. USA 71_, 24072410 (1974).

650 40. Posner, B.I., Kelly, P.A., Friesen, H.G.: Prolactin receptors in rat liver: possible induction by prolactin. Science 187, 57-59 (1 975) . 41. Costlow, M.E., Bushcow, R.A., McGuire, W.L.: Prolactin stimulation of prolactin receptors in rat liver. Life Sciences 1_7 , 1457-1 466 (1 975) . 42. Kelly, P.A., LeBlanc, G., Ferland, L., Labrie, F., De Lean, A.: Androgen inhibition of basal and estrogen-stimulated prolactin binding in rat liver. Mol. Cell. Endocrinol. 195-204 (1977). 43. Djiane, J., Durant, P., Kelly, P.A.: Evolution of prolactin receptors in rabbit mammary gland during pregnancy and lactation. Endocrinology 100, 1348-1356 (1977). 44. Holcomb, H.H., Costlow, M.E., Buschow, R.A., McGuire, W.L.: Prolactin binding in rat mammary gland during pregnancy and lactation. Biochim. biophys. Acta 428, 104-112 (1976). 45. Kelly, P.A., Shiu, R.P.C., Robertson, M.C., Friesen, H.G.: Characterization of rat chorionic mammotropin. Endocrinology 96 , 1 187-1 195 (1975) . 46. Holdaway, I.M., Deegan, M., Friesen, H.G.: Influence of infused prolactin on hormone binding to tissue slices. Can. J. Physiol. Pharmacol. 5_5, 1 93-195 (1 977). 47. Aragona, C., Friesen, H.G.: Specific prolactin binding sites in the prostate and testis of rats. Endocrinology 97, 677-684 (1 975) . 48. Kledzik, G.S., Marshall, S., Campbell, G.A., Gelato, M. , Meites, J.: Effects of castration, testosterone, estradiol, and prolactin on specific prolactin-binding activity in ventral prostate in male rats. Endocrinology 98^, 373-379 (1976) . 49. Charreau, E.H., Attramadal, A., Torjesen, P.A., Calandra, R., Purvis, K., Hansson, V.: Androgen stimulation of prolactin receptors in rat prostate. Mol. Cell. Endocrinol. 1_, 1-7 (1 977) . 50. Djiane, J., Durand, P.: Prolactin-progesterone antagonism in self-regulation of prolactin receptors in the mammary gland. Nature 26J5, 641-643 ( 1977). 51. Gavin, J.R., Roth, J., Neville, D.M., De Meytes, P., Buell, D.N.: Insulin-dependent regulation of insulin receptor concentrations: a direct demonstration in cell culture. Proc. Nat. Acad. Sei. USA 71_, 84-88 (1974). 52. Lesniak, M.A., Roth, J.: Regulation of receptor concentration by homologous hormone: effect of human growth hormone on its receptor in IM-9 lymphocytes. J. biol. Chem. 251, 3720-3729 (1976). 53. Roff, M.: Self regulation of membrane receptors. Nature 259, 265-266 (1976) .

651 54. P o s n e r , B . I . , R a q u i d a n , D., J o s e f b e r g , Z., B e r g e r o n , J . M . : D i f f e r e n t r e g u l a t i o n of insulin r e c e p t o r s in i n t r a c e l l u l a r (Golgi) and p l a s m a m e m b r a n e s from l i v e r s of o b e s e and lean m i c e . P r o c . N a t . A c a d . Sci. U S A 75> 3302-3306 (1978). 55. D j i a n e , J., C l a u s e r , H., K e l l y , P . A . : Rapid down r e g u l a t i o n of p r o l a c t i n r e c e p t o r s in m a m m a r y gland and l i v e r . B i o c h e m . b i o p h y s . R e s . C o m m u n . (in p r e s s ) . 56. M a n n i , A . , P e a r s o n , O . H . , B o r d k l e y , J., M a r s h a l l , J . S . : T r a n s s p h e n o i d a l H y p o p h y s e c t o m y in b r e a s t c a n c e r : E v i d e n c e for an i n d i v i d u a l role of p i t u i t a r y and g o n o d a l h o r m o n e s in s u p p o r t i n g tumor g r o w t h . C a n c e r £ 4 , 2 3 3 0 - 2 3 3 7 (1979). 57. P e a r s o n , O . H . , Ray, B . S . : H y p o p h y s e c t o m y in the t r e a t m e n t of m e t a s t a t i c m a m m a r y c a n c e r . A m . J. S u r g . 9ji, 5 4 4 - 5 5 2 (1 960) . 58. M c M i l l i n , J.M., Seal, U . S . , T h e o l o g i d e s , A . : P r o l a c t i n d y n a m i c s f o l l o w i n g t r a n s p h e n o i d a l h y p o p h y s e c t o m y for m e t a static c a r c i n o m a of the b r e a s t . C a n c e r 39' 2 2 5 4 - 2 2 5 7 (1977). 59. B o s t o n C o l l a b o r a t i v e Drug and B r e a s t C a n c e r . L a n c e t

Surveillance Program, 2, 669-671 (1974).

Reserpine

60. H e i n o n e n , O . P . , S h a p i r o , S., T u o m i n e n , L., T u r u n e n , M . I . : R e s e r p i n e use in r e l a t i o n to b r e a s t c a n c e r . L a n c e t 2, 6 7 5 677 (1974). 61. W i l s o n , R.G., B u c h a n , R., R o b e r t s , M . M . , F o r r e s t , A . P . M . , Boyns, A.R., Cole, E.N., Griffiths, K.: Plasma prolactin and b r e a s t c a n c e r . C a n c e r J33' 1325-1327 (1974). 62. S h e t h , N . A . , R a n a d i v e , K . J . , Suraiya, J . N . , S h e t h , A . R . : C i r c u l a t i n g l e v e l s of p r o l a c t i n in h u m a n b r e a s t c a n c e r . B r . J. C a n c e r 32^, 160-167 (1 975). 63. B o y n s , A . R . , C o l e , E . N . , G r i f f i t h s , K . , R o b e r t s , M . M . , B u c h a n , R., W i l s o n , R.G., F o r r e s t , A . P . M . : P l a s m a p r o l a c t i n in b r e a s t c a n c e r . E u r o p . J. C a n c e r 9, 99-102 (1973). 64. K w a , H.G., De J o n g - B a k k e r , M . , E n g l e s m a n , E., C l e t o n , F . J . : P l a s m a p r o l a c t i n in h u m a n b r e a s t c a n c e r . L a n c e t _1_, 433-435 (1 974) . 65. Kwa, H . G . , C l e t o n , F., J o n g - B a k k e r , M. , B u l b r o o k , R.D., H a y w a r d , J . L . , W a n g , D . Y . : P l a s m a p r o l a c t i n and its r e l a t i o n s h i p to risk f a c t o r s in h u m a n b r e a s t c a n c e r . Int. J. C a n c e r V7, 4 4 1 - 4 4 7 (1976) 66. H i l l , P., W y n d e r , E . L . , K u m a r , H., H e l m a n , P., Rona, G . , K u n o , K . : P r o l a c t i n l e v e l s in p o p u l a t i o n s at risk for b r e a s t c a n c e r . C a n c e r R e s . 36_, 4 1 0 2 - 4 1 0 6 (1976). 67. C o l e , E.N., E n g l a n d , P . C . , S e l l w o o d , R . A . , G r i f f i t h s , K . : Serum p r o l a c t i n c o n c e n t r a t i o n s t h r o u g h o u t the m e n s t r u a l c y c l e of n o r m a l w o m e n and p a t i e n t s w i t h r e c e n t b r e a s t c a n c e r . E u r o p . J. C a n c e r 1_3, 6 7 7 - 6 8 4 (1977). 68. R o l a n d i , E., B a r r e c a , T., M a s t u r z o , P., P o l l e r i , A . , I n d i v e r i , F., B a r a b i n o , A . : P l a s m a p r o l a c t i n in b r e a s t c a n c e r . L a n c e t 2, 845-846 (1974).

652 69. Cole, E.N., Seelwood, R.A., England, P.G., Griffiths, K.: Serum prolactin concentrations in benign breast disease throughout the menstrual cycle. Europ. J. Cancer 597603 (1977). 70. Minton, J.P.: The response of breast cancer patients with bone pain to L-Dopa. Cancer 3^3 , 358-363 (1974). 71. Holdaway, I.M., Friesen, H.G.: Hormone binding by human mammary carcinoma. Cancer Res. 37_, 1946-1 951 (1977). 72. Morgan, L., Raggatt, P.R., de Souza, I., Salih, H., Hobbs, J.R.: Prolactin receptors in human breast tumors. J. Endocrinol. 73, 17P (1 977) . 73. De Souza, I., Hobbs, J.T., Morgan, L., Salih, H.: Localization of prolactin in human breast tumors. J. Endocrinol. 73, 17P (1977) . 74. Stagner, J.I., Jochimsen, P.R., Sherman, B.M.: Lactogenic hormone binding to human breast cancer: correlation with estrogen receptor. Clin. Res. 25, 302A (1977).

Subject

Index

Abnormal

167

Absorption

327

Acne

43

ACTH receptor

231

Actinomycin D

101

Adenocarcinoma

148

Adrenals

13

Adrenal cortex

215

Adrenal medulla

591

Affinity chromatography

258

Albumin

257

Albumin, human serum

551

Aldosterone

216

Androgens

43,351,527

Androstenedione

46,556

16-Androstenes

1

A n g i o t e n s i n II

218

Antagonist specific chemotherapy

165

Anti-Estrogen

36,82

A n t i s e r u m to o e s t r o n e s u l p h a t e

54 6

Aromatization

475

Arylsulphatase C

556

Assay

26 3

Autoradiography

510

Baldness

44

Benign breast tumours

132

Benign prostatic hypertrophy

524

Binding proteins

491

Bioassay

573

Biosynthesis

351

Blastocyst

180

Breast cancer

29,208

Breast cancer, animal models

81

654 Breast cancer cells in tissue culture

33

Breast cancer, human

81

Breast tissue

115

Calcitonin

429

Calcitonin receptors

429

Cancer, breast

560

Cancer of the colon

146

CB-154

121

Cell culture

186

Cell membrane

272

Cells in culture, breast cancer

94

Chromaffin cell

590

Chromatography

146

Chromatography, high pressure liquid

544

Cleaved forms

579

Conn's syndrome

215

Corticosteroids

327

Cortisol

216

Cushing's syndrome

215

Cyclic AMP

227

Cytosol

272

DEAE-Sephadex

544

Defined environment

40

Degradation

492

Dehydroepiandrosterone

44,553

Dehydrogenase, 20a-dihydroprogesterone

557

Dehydrogenase, 17ß-hydroxysteroid -

560

Dihydrotestosterone

463

5a-Dihydrotestosterone

43,523

Ectopic growth hormone

207

Ectopic hormone

438

Electron microscopic

510

Electrophoresis, high voltage

546

Endometrial carcinoma

383

Endometrial hyperplasia

381

Enterohepatic circulation

553

655 Enzyme deficiencies

362

Estradiol

459

Estrogen

30,81

Estrogen receptor, cytoplasmic

97

Estrogen receptor, nuclear

97

Free hormone

26 9

Freeze fracture

516

Glomus tumour

617

Glucocorticoid receptor

159

Gonadotropin

359

Graves' disease

61

Growth hormone

573

Half-life

266

hCG

167

Hirsutism

43

Human kidney

150

Human liver

145

Human placenta

412

Human prolactin

115

Human prolactin, biological actions

296

Human prolactin, molecular forms

288

Human prolactin, release

287

Human prolactin, secretion throughout the life

292

cycle Human prolactin, synthesis

281

Human uterus

37 3

Hydronephro sis

148

Hypogonadal men

481

Hypogonadism

363

Hypophysectomy

129

Hypothalamic-pituitary-axis

480

Immunoassay

574

Immunoreactive hGH

208

Inhibition

491

Insulin

503

Klinefelter's syndrome

360

656 Leiomyoma

385

Liver

505

Liver metastases

146

Lung cancer

207

Luteinizing hormone

459

Lymphocytes

504

Male infertility

364

Malignant breast cancer

129

Man

9

Measurement

577

Medullary thyroid carcinoma

431

Metabolism

45,264,339

Mineralocorticoid receptor

152

Molecular structure

4 94

Molecular weight

258

Negative feedback

459

Neoplasia, endometrial

562

Nephrolithiasis

148

Neural crest

590

Neuroblastoma

590

Nonendocrine

180

Non-thyroid tumours

438

Normal

167

Normal human endometrium

387

Nuclear receptor processing

95

Nuclei

272

Oestrogens

541

Oestrogen metabolism

386

Oestrogen receptor

378

Oestrone

550

Oestrone sulphate

541

Oral contraceptives

121

Ovaries

15

Ovary

211

Paraganglia

590

Peripheral blood mononuclear cells

63

657 Phaeochromocyte

598

Phaeochromocytoma

5 96

Pheromones

1

Placental lactogen

409

Plasma

4

Pregnancies

173

Progesterone

551

Progesterone metabolism

393

Progesterone receptor

103,376

Prolactin

635

Prolactin concentration in serum, clinical

308

applications Prolactinomas

284

Prolactin receptor (s)

130,293

Prostaglandins

68

Prostate

523

Prostatic carcinoma

535

Protein synthesis

415

Pulsatile LH release

465

Radioimmunoassay

529,6 35

Radioreceptor assay

6 38

Receptor

503

Reserpine

127

Reservoir in the skin

336

Reverse-T3

270

Secreted

577

SIF Cell

595

Skin

43

Skin penetration

335

Somatomedin

5 76

Sperm

181

Steroids

52 3

Steroid conjugates

543

Steroidogenesis

225

Stomach cancer

208

Sulphotransferase

550

658 Sweat

6

Sympathetic nervous system

591

Synthesis

4 92

T3

258

T4

258

T.B.P.A.

497

Testes

1,351,463

Testosterone

4 3,45 9

3,5,3',5'-Tetraiodothyroacetic

acid

(T4A)

268

Thyroid hormones

4 91

Thyroid-stimulating-immunoglobulin

62

Thyroxine

4 97

Thyroxine binding globulin

(TBG)

Thyroxine binding prealbumin

(TBPA)

257 257

Tibia test

573

Trophoblast

172

Tumours

167

Two-chain

579

Urine

1

Uterus

551