Estrogen Metabolism in the Human Estrogen Metabolism in the Human
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ENE PURRE: ESTROGEN METABOLISM IN THE HUMAN ESTROGEN METABOLISM IN THE HUMAN A THESIS BY ENE PURRE SUBMITTED TO THE FACULTY OF GRADUATE STUDIES AND RESEARCH IN PARTIAL FULFILMENT FOR THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE McGILL UNIVERSITY MONTREAL, CANADA. DEPARTMENT OF EXPERIMENTAL MEDICINE AUGUST 1965 TABLE OF CONTENTS Page No. I INTRODUCTION A. Early history and isolation of the e strogens 2 B. Metabolism of the estrogens 4 1. Biogenesis 4 2. Metabolism 6 3. Degradation 14 4. Conjugation 15 c. Altered states of estrogen metabolism 18 Purpose of the Investigation II MATERIALS AND METHODS 25 Materials I Radioactive compounds 25 II Non-radioactive compounds 25 Ill Enzymes 26 IV Reagents 26 Methods I Purification of radioactive steroids 30 II Preparation of non-radioactive standards 31 III Spectophotometric and f1uorimetric readings 31 IV Counting methods 32 V Enzyme assays 33 VI Injections 33 VII Collection of urine 34 VIII Hydrolysis and extraction of urines 34 IX Assay of estrone, estradiol-17 f.:' and estriol 36 X Attempts at separa ting ring 011( keto1s 3 7 XI Assay of ring D..: ketols in the urine 39 XII Recrysta1lization studies 40 ---------------···---- TABLE OF CONTENTS (cont.) Page No._ Ill RESULTS Section I - Studies with control subjects and those with myocardial infarction 41 Section II- A. Recovery of 16~-hydroxyestrone and 16-ketoestradiol-17~ from toluene/ propylene glycol system 50 B. Pre 1 imi nary study on urinary ring De>< ketols 51 c. Urinary ring D<K ke to 1 s in nonnal males 53 D. Studies of the excretion and metabolism of eight estrogen metabolites from urine 59 IV DISCUSSION 71 SUMMARY 87 BIBLIOGRAPHY 89 • LIST OF FIGURES Figure No. Title Page No. I Structural formulae and systematic names of the natural estrogens 1 II Biogenesis of e strogens from acetate by ovarian tissue 1 III Intermediary metabolism of the estrogens 17 • LIST OF TABLES Table No. Title Page No. I Endogenous estrogen excretion in normal males 42 II Endogenous and exogenous estrogen excretion in normal males 44 III Endogenous and exogenous estrogen excretion in subjects with myocardial infarction 45 IV Clinical history of subjects with myocardial infarction 46 v Ratios of endogenous and exogenous estrio1/ estrone and estriol/estrone+estradio1•178 47 VI Average percent conversion of estradiol-178 .3H to estrone and estrio1 and percent estradiol-17 tS .3H excreted unchanged in normal subjects and those with myocardial infarction 48 VII Recovery of 16-ketoestradio1•17/3 from toluene/propylene glycol chromatographie system 50 VIII Recovery of 16-ke toe stradiol-1713 and 160(-hydroxyestrone from to1uene/propylene glycol chromatographie system 51 IX Results of preliminary study of ring D« keto1s from pregnancy urine 52 x Results of the isolation of urinary ring D 0( ketols in normal males 54 XI Endogenous and exogenous levels of ring D01, ketols in normal male urine 55 XII Urinary ring Dol. ketol and estrone levels in normal male urine 57 XIII Percent conversion of injected estradio1-17~ .3H to the three ring D 0( ketols 58 XIV Resu1ts of the isolation of eight urinary estrogen metabolites in subject B injected • with estradio1-17 f3 .. 3a and 16- ketoestradiol- 17/3 .. 14c 60 -------------···········--··· LIST OF TABLES (cont.) Table No. Title Page No. xv Recrystallization data of the eight urinary estrogen metabolites of subject B 61 XVI Results of the isolation of eight urinary estrogen metabolites in subject M injected with estradiol-17~ .3H and 16-epiestriol•l4c 64 XVII Recrystallization data of the eight urinary estrogen metabolites of subject M 65 3 XVIII Percent conversion of estradiol-17~ • H, 16·ketoestradiol-17~ .14c and 16-epiestriol .14c to various estrogen metabolites 66 XIX Resulta of the isolation of eight urinary estrogen metabolites in subject G injected with estradiol-17B .3H and testosterone-14c 68 xx Recrystallization data of the eight urinary metabolites of subject G 69 3 XXI Percent conversion of estradiol-178 - H and testosterone•l4c to various estrogen metabolites 70 ACKNOWLEDGEMENTS First and foremost, 1 wish to express my sincerest thanks to my research director, Dr. R. Hobkirk, for his invaluable advice and encouragement, for his laboratory assistance during the course of these investigations and his helpful criticism in the preparation of this thesis, as well as the provision of laboratory space in the University Medical Clinic of the Montreal General Hospital. Thanks are due to the Medical Research Council of Canada for financial aid through a grant made to Dr. R. Hobkirk. I am also grateful to Dr. o. J. Lucis for his keen interest and advice and to Mr. R. Raud, a fellow graduate student. The author wishes to thank the medical and nursing staff of the Montreal General Hospital, especially Ors. D. L. Thompson and 1. w. D. Henderson, for the injection of patients and the collection of urine samples. The excellent technical assistance of Miss M. Nilsen is gratefully acknowledged. Special thanks are extended to Mrs. E. McCaffrey, who typed this manuscript, and to my husband for his encouragement, patience and endurance during the course of this work. • INTRODUCTION • 1. FIGURE 1 STRUCTURAL FORMULAE AND SYSTEMATIC NAMES OF THE NATURAL ESTROGENS -?'1 HO~ ES TRONE ESTRADIOL 17{1 3 HYDROXY 1, 3, 5 (10) 3, 17{1 DIHYDROXY 1, 3, 5 (10) ESTRATRIEN 17 ONE ESTRATRIENE ES TRIOL 16 EPIESTRIOL 3, 16«, 17{1 TRIHYDROXY 3, 16{1, 17/J TRIHYDROXY 1,3,5 (10) ESTRATRIENE 1,3,5 (10) ESTRATRIENE 0 0 ••• OH OH ri HO HO~ 16a HYDROXYESTRONE 16{1 HYDROXYESTRONE 1, 3, 5 (10) 3, 16« DIHYDROXY 3, 16/l DIHYDROXY ESTRATRIEN 17 ONE ESTRATRIEN 17 ONE HO HO 16 KETOESTRADIOL 17/J 16 KETOESTRONE 3, 17/J DIHYDROXY 1,3,5(10) 3 HYDROXY 1, 3,5 (10) ESTRATRIEN 16 ONE ESTRATRIENE 16, 17 DIONE HO~ 2 METHOXY ESTRONE 2 METHOXY 3 HYDROXY 1,3,5 (10) ESTRATRIEN 17 ONE 2. CAAPmRI INTRODUCTION A. EARLY HISTORY AND ISOLATION OF THE ESTROGENS The history of the estrogenic hormones has its start at the turn of the century. In the year 1900, Knauer (1) showed that auto-transplanted ovaries in spayed aniruals could renew the estrous cycle. From this he concluded that the female gonads played an important part in the regulation of estrus and the production of ova and were thus organs of internai secretion. Thirteen years later, Fellner (2) showed that extracts of human placenta were potent in producing estrus in spayed animais; therefore the placenta was also invo1ved in the metabolism of estrus•producing materia1. It remained, however, for Allen and Doisy (3) to prove that only one substance was responsible for this phenomenon and that the active principle was a chemical substance. With the discovery in 1927 by Aschheim and Zondek (4) that large amounts of estrogenic activity are present in the urines of pregnant women, there was a shift to studies on urine rather than tissues such as ovaries and placenta. Thus this vast work of more than a quarter of a century's duration culminated in 1929-30 in the isolation of two crystalline compounds. Estrone, isolated independent1y by Doisy (5) in the United States, Butenandt (6) in Germany and Laquer (7) in Holland was not only the first estrogenic hormone but also the first steroid hormone to be isolated in a pure form. Almost at the same time, Marrian (8-11) in Eng1and in a series of experiments, isolated another estrus-producing substance (with a molecu1ar 3. formula H )from urines of pregnant women and named it c18 24 o3 "trihydroxyoestrin"; this compound is now known as estriol. It was weakly acidic in nature due to a phenolic hydroxyl group as was Butenandt's compound but a comparison of melting point and analytical data clearly showed that the two compounds were not the same. Doisy and Thayer (12) in the following year confirmed the identity of estrone and estriol; they called the compounds theelin and theelol from the Greek "THEELUS" meaning female. They showed that theelin was two times as active in adult spayed rats but theelol was six to seven times as active in immature female rats. The review of this early work must not omit mention of Cook and Girard (13) who in their brilliant work provided complete proof of the structure of the natural estrogens. Five years lapsed again; then a third estrogenic compound was isolated by Doisy (14) and his group from liquor folliculi of sow ovaries. This was the most potent of all the estrogens by the vaginal smear technique of Allen and Doisy (3) and was named dihydroxytheelin (now known as estradiol-17,8 ). Shortly afterwards, Huffman et. al. (15) found estradiol•l7~ in human pregnancy urine. Estrone, estradiol-17P and estriol thus became known as the three classical estrogens since no new compounds of the estrogen family were isolated for the next twenty years. Starting in 1954, Marrian and his colleagues have, however, changed this situation considerably. In conjunction with the late Dr. Bauld (16) he 1so1ated a new Kober-chromogen from human pregnancy urine in the yield of 0.1 mg/1 which was called 16-epiestriol; then, with Loke, Watson and Panatonni (17) he isolated 16~·hydroxy estrone also from human pregnancy urine and together with Layne (18, 19) in 1958 16.P·hydroxy estrone and 16·ketoestradiol-17.P from the same source. At the same time, Brown, Fishman and Gallagher (20) 4. reported on the isolation of 16P-hydroxy estrone from the urine of a 14 woman who had received an injection of estradiol-17fl -16 - c. By about 1960, most of the estrogens known today had been isolated.