ENE PURRE: 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, .

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 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 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 34 VIII Hydrolysis and extraction of 34 IX Assay of , -17 f.:' and 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

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 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-•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 -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 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 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.

These include besides those already mentioned the C-2 substituted estrogens,

2-hydroxy• and 2-methoxyestrone, 2 hydroxy and 2-methoxy-estrodiol and

2•methoxy estriol (21-25), the c-6 substituted estrogens (26, 27), the

C-11 substituted estrogens (29), the C·l8 substituted estrogens (28-31) and finally the epiestriols (16 & 32-34). Not all the above mentioned compounds have yet been shown with certainty to occur in human urine, but have been found as products of incubation with either , adrenals or some other tissues.

The initial isolations of the estrogenic hormones were from human pregnancy urine almost exclusively but the majority of them have now been detected also in non-pregnant and post-menopausal females and males (35-38).

B. ~mTABOLISM OF THE ESTROGENS

The metabolism of estrogens as known today presents us with a rather complex scheme of interactions. When discussing this, one must bear in mind the reactions of (1) intermediary metabolism, (2) biogenesis, (3) conjugation, and (4) degradation.

Since the present author's work deals mainly with metabolic conversions in health and disease, and particularly in the human male, emphasis will be placed on this part while the other three will be dealt with more briefly.

(1) BIOGENESIS

It is now a well accepted fact that all steroid producing endocrine organs can form the various steroid hormones. The , and during pregnancy the placenta, are still considered to be the major sites of estrogen synthesis, but the testis also secretes this hormone although to a small extent and the s.

adrenal cortex has its part • estrone bas been isolated from beef adrenal (39). As estrogens are structurally related to cholesterol, it was only natural to assume that they can arise from cholesterol. Cholesterol was known to be synthesized from acetate and this bas also been shown to be the primary building block of the estrogens by Heard and his associates (40) in vivo using the pregnant mare; in vitro, this bas been confirmed by

Rabinowitz and Dowben (41) using ovaries and testes. Ryan and Smith (42) have recently repeated this work using human ovaries obtained after in vivo stimulation with ovine FSH and have shown that the conversion of 14 acetate •l • C to both estrone and estradiol-17~ is approximately 0.02%.

Heard (40) in his work in 1954 was unable to show the conversion of cholesterol to estrogens but this bas been proved by Ryan and Smith (43) to occur in ovarian tissue in about 0.1% yield. Solomon et. al. (44) suggested that a pathway exists from progesterone to estrogens since progesterone can be converted to ~4 antrostene -3, 17-dione by ovarian and testicular tissue and the latter is aromatized to estrogens in the same tissues. Again Ryan and Smith (45) studied this problem in human ovaries and showed the yield from progesterone to be about 10%. When ovarian tissue, which bad been stimulated in vivo with gonadotropic hormones, was incubated with A4 androstene

·3, 17-dione, the conversion was 15% (46). In placenta! preparations, the same conversion has been shown to occur to the extent of 50-100% (47).

Already in 1936, Fieser (48) bad suggested that the male hormone, testosterone, can be converted to the female hormones, estrogens. This suggestion of Fieser's was first proved by Heard et. al. (49) in the pregnant mare using radioactive testosterone. They showed the yield of testosterone to estrone to be about 1% and concluded that this must be a straight conversion rather than a breakdown to acetate and reconstruction to estrogens.

Baggett et. al. (50) and Wotiz et. al. (51) have repeated this work in vitro using avaries. The former (50) incubated slices of a normal ovary from a 14 thirty•four year old woman with testosterone -3 • C and isolated radio• active estrodiol-17S ; the latter (51) incubated slices of ovarian tissue showing cortical stroma! hyperplasia removed twenty years after the 14 with testosterone -4- C and found radioactive estrone, estradiol-17~ ' and estriol. Later, Baggett et. al. (52) also showed this conversion in homogenates of horse testes and slices of human feminizing adrenocortical carcenoma and human placenta.

The aromatization mechanism of the neutra! steroids has also been investigated by severa! other workers, among them Ryan (53), Heard (49),

Meyer (54, 55) Hayano (56) and West (57). West (57) injected testosterone 14 • 4 • c 1nto· a woman wh o h a d been b ot h ovarectom i ze d an d a d rena 1ectom1ze · d and showed the conversion of this steroid into estrone and estradiol-17~ in non-endocrine tissue. The biogenesis of the estrogens is schematically summarized in FIGURE II.

(2) METABOLISM

The early work on the isolation and determination of estrogens was carried out on pregnancy urines, and in many cases especially on urines of the pregnant mare which yielded an abundant supply of crystalline estrogens. The early work on the metabolism and inactivation of estrogens posed a more difficult problem, however, as almost the only way of measurement at this time was by bio-assay so that the resulting metabolites could hardly be characterized. Thus in the years from 1930-1955, much time was spent on methodology.

The bases of the lttrich color reaction were laid during these years.

In 1929, Wieland, Straub and Dorfmuller (58) observed that estrogens gave an orange color with green fluorescence in the Lieberman-Burchardt reaction.

Marrian (59) noticed that the same reaction was given merely by warming with e e FIGURE Il BIOGENESIS OF ESTROGENS FROM ACETATE BY OVARIAN TISSUE (REFERENCESIN PARENTHESIS)

o.on. 1 CH3COOH (42) ACETATE HO CHOLESTEROL r%{43) ~H3=0 .. lOt. (45) ""'

HO~ HO 17 Cl HYDROXYPROGESTERONE PROGESTERONE ! ~ 2 15t. {46) d"~ Ho~~~ HO 4 /l ANDROSTENE 3, 17 DIONE ESTRONE ESTRADIOL 17/J 1

TESTOSTERONE

1 PERCENT ON ARROWS INDICATES CONVERSION OF THAl COMPOUND TO ESTROGENS 2 THIS CONVERSION HAS BEEN SHOWN TO OCCUR IN PLACENTAL PREPARATIONS TO -..! THE EXTENT OF 50 -100% (47) .

rrr:tnΠII a.

concentrated sulfuric acid. Kober (60) modified the reaction, which has

borne his name ever since 1 by diluting the orange color with water; he also showed that the addition of a phenol to the sulfuric acid did not eliminate

the green fluorescence but intensified the red color - thus the intensity

of the red color bore a direct but not linear relationship to the amount

of estrogen.

The original Kober reaction has been modified by many workers, among

them Brown (61, 62), Bauld (63), Allen (64) and lastly Ittrich (65, 66).

Ittrich's notable contribution was that he extracted the pink Kober-color

complex with a solution of 2% (w/v) p-nitrophenol and 1% (v/v) ethanol

in tetrachloroethane. This reaction lends itself equally well to both

spectophotometric and fluorimetric analysis depending upon the amount of

estrogens available for measurement.

Back in 1934, Cohen and Marrian (67) had shown the necessity of

separating the neutra! steroids from the phenolic fraction before carrying

out the color test; neutra! steroids, such as cholesterol and pregnandiol,

inhibited the full production of red color and accelerated the rate of

fading. The Girard reaction, as developed by Girard and Sandulesco (68)

was applied to estrogens for the separation of ketonic estrone from non­

ketonic estradiol-17~ and estriol. Engel's (69, 70) most noteworthy

contribution at this time was the application of countercurrent distribution

for the separation of the estrogens. From Dr. Marrian's laboratories in

Edinburgh, two assay methods were developed independently by Brown (71, 72)

and Bauld (73, 74). Brown's method has been used for part of the author's

study and will be described under''tMethods".

The discovery of redioisotopes and the success of the organic chemist

in introducing labelled atoms into the steroid molecule has permitted a much more detailed understanding of the origin and fate of these compounds in 9.

vivo. 131 r was used by Albert et. al. (75) to label estradiol-17fl

and Pearlman et. al. (76) used deuterium in studies of estradiol-17~ 14 metabolism in human pregnancy. C is very suitable for the following

of the metabolic reactions of the estrogens, since this isotope is 3 incorporated into the nucleus of the steroid molecule. H, the radio-

active isotope of hydrogen, has also found wide use in metabolic studies,

especially in in vivo studies, as one can administer higher doses of this

than of 14c since the former is a weaker emitter of /3 rays.

Chromatography is another method which has proven invaluable in the

separation of closely related compounds. The names of Bush (77) and

Zaffaroni (78) must certainly be mentioned in this context for their

valuable work on paper chromatography. Other types of chromatography

such as column partition and adsorption chromatography and thin layer

chromatography have all aided in gaining the information available today.

Tbus, with newer and better methods and radioisotopes, many new

estrogens were discovered and their metabolic interconversions established.

The early knowledge on the metabolism of estrogens has been very ably

reviewed by Pincus and Pearlman (79), Doisy et. al. (80) and Heard (81).

According to Pincus and Pearlman (79), the ovaries and uterus were not

essential for estrogen metabolism as the liver and other non-endocrine

organs could effect the transformation of estrogens; certainly, the liver was the most important site of estrogen inactivation. Their scheme

for estrogen metabolism was as follows:

c< -ESTRADIOL --~ ESTRONE ESTRIOL (ESTRADIOL-178) ! .8 -ESTRADIOL

Heard and Hoffman (82) back in 1941 gave 250 mg of OC -estradiol to

a man during an eight-day period and isolated a total of 26 mg. or 10%

as estrone, estradiol-178 and estriol; they therefore concluded that most 10.

of the activity of the estrogens which are administered is lost within

a short time.

This scheme of estrogen metabolism stil1 holds today but it would

hardly look so simple.

Interconversion of estrone and estradiol-17~ has now been shown both

in vivo and in vitro; it is catalyzed by an enzyme (83), estradiol-17~

dehydrogenase which requires NAD or NADP as coenzymes and has steric

specificity for the 178 group.

Brown (84) injected estrone and estradiol-17~ into humans and

measured the metabolites from urine. When he injected estrone, 10% of

the urinary estrogens (i.e., estrone, estradiol-17~ and estriol) was

excreted as estradiol-17~ and 44% as estrone; when he injected estradiol-17B ,

15% of the urinary estrogens was excreted as estradiol-17E and 41% as

estrone; after the injection of both estrone and estradiol-17fi , 46%

and 44% respectively of the urinary estrogens were excreted as estriol.

His experiments showed that administered estrone and estradiol-178 are

metabolized in the human in an almost identical manner and that equilibrium

between estrone and estradiol-17B must be reached very quickly. Fishman

et. al. (85) made important observations on these rates of conversion. 14 3 They administered estrone -16 - C and estradiol-17B -6, 7 - H to subjects

in varying mass and isotope ratios and compared the isotope ratios of

estrone and estradiol•l7B in successive urine samples. They concluded

that the oxidation of estradiol-178 to estrone is more rapid than the

reduction of estrone to estradiol-17B and also that the highly oxygenated metabolites -estriol and 16-epiestriol - as well as 2•methoxyestrone arise

from estrone.

The conversion of estrone and estradiol-17~ to estriol was first

demonstrated by Pincus and colleagues (86, 87). Brown (84) showed that 11.

when estrone and estradiol•l7B were injected, 45% of the urinary estrogens

(estrone, estradiol•l7~ and estriol) was estriol. Conclusive proof of this

reaction was given by Fishman et. al. (88). These authors injected estradiol-

17/3 labelled with tritium in the ex -orientation of C·l7 to two women.

Most of the tritium appeared promptly in the body water while the estriol

isolated from the urines of both subjects contained only a trivial amount

of radioactivity, thus showing that estrone is the real precursor of estriol.

The formation of estriol from estradiol-17B bas been detected in vitro in

human liver tissue by Engel et. al. (89) and Breuer et. al. (90).

For almost twenty-five years, estriol was the only C·l6 substituted

phenolic steroid known. In 1954, Marrian and Bauld (16 & 91) isolated

16-epiestriol from human pregnancy urine. This opened up a whole new world

in the intermediary metabolism of the estrogens. Three years later, Marrian

et. al. (92), after the isolation of 16~hydroxyestrone (17) and 16~­

hydroxyestrone (18, 19) advanced the hypothesis that both of these compounds

• may be formed metabolically from estrone by 160l- and 16/3 -hydroxylation and

that they are intermediates in the "hydration" of estrone to estriol and

16-epiestriol respectively. The fact that the principal component of

his KC-5 was 16

easily be explained on biogenetic grounds. Since the concentration of

estriol in late pregnancy is about sixty times that of 16-epiestriol, it

might be assumed that the concentration of the immediate precursor of

estriol would be much greater than that of the immediate precursor of

16-epiestriol. Support for this hypothesis came from experiments of

Brown and Marrian (93). They found increased estriol levels in the

urines of two individuals who had received injections of 16 ~-hydroxyestrone.

Final proof came from in vitro studies with human liver slices. Breuer et.

al. (94) incubated 16 ~-hydroxyestrone with human liver slices and isolated 12.

estriol as the main metabolite while the incubation of 16 B· hydroxyestrone

yie1ded mainly 16-epiestriol. In addition (33) 16 ~-hydroxyestrone gave

ri se to 17-epiestrio1 and 16R- hydroxyestrone to 16, 17-epiestrio1.

Nocke et. al. (95) a1so demonstrated the biogenesis of these four epimeric

estriols from 16~-hydroxyestrone and 16,8-hydroxyestrone. They injected

16 ~~hydroxyestrone into a man and iso1ated estrio1 and 17-epiestrio1 in a

ratio of lOO: 1.8. When 16,8- hydroxyestrone was injected, they isolated

16•epiestrio1 and 16, 17~epiestriol in the ratio of 100:6.3.

The evidence for the existence of 16-keto•estradiol•l7B in human urine

has aroused some controversy. As ear1y as 1953, Migeon (96) obtained

evidence by countercurrent distribution and fluorimetry for the existence

of this compound. Wben Marrian, Watson and Panattoni (92) subsequently

iso1ated their new Kober chromogen -KC-5· from enzyme hydro1ysed urine,

they thought at first that this was 16-keto-estradiol-17/3 • The crystalline

material obtained bad the same C and H analysis and me1ting point as 16·

keto-estradiol·17B and did not depress the melting point of authentic

16-keto-estradiol-17~ on admixture. However, the optica1 rotation differed

markedly from that of 16-keto•estradio1-17B and in contrast to the latter,

it developed its maximum color in the Kober reaction without heating in

the first stage, while the spectrum of its solution in concentrated sulfuric

acid differed markedly from that of a similarly prepared solution of

16 .. keto-estradiol-17B • Moreover, the diacetate of KC•5 and 16-keto­

estradiol•17B melted at different temperatures and showed depression of melting point on admixture. Clearly, therefore, KC·5 and 16-keto-estradio1-17B

were not identical. Marrian et. al. (92) suggested that their new compound

was main1y 16~-hydroxyestrone and that the 16-keto-estradio1-17B found in

their ear1ier study (97) arose by isomerization from 16 ~-hydroxyestrone

during exposure to a1ka1i. It was found that synthetic 16 ~-hydroxyestrone 13.

formed 16-ketoestradio1•17B on heating to 216 0 c. Meanwhile, Levitz, 14 Spitzer and Twomb1y (98) administered estradiol-17 -16 - c to two subjects and sought and found 16-ketoestradio1-17P in the urine by isotope dilution technique. 16·Ketoestradio1-17/Jwas found to contain

1.6% of the radioactivity excreted in the first 24 hour urine sample.

The low yield of their product suggested that further studies were needed to establish whether 16-ketoestradio1•17~ was excreted in the human 14 urine. Levitz et.al. (99) then injected estriol-16 - c to human subjects and found radioactive 16·ketoestradiol•l7B and 16-epiestriol. The same reaction in vitro was shown by King (lOO). He incubated estriol with rat homogenates in the presence of NAD and NADP and found that 16-keto• estradiol-17B and 16-epiestrio1 were produced in 20% and 1% yie1d respective1y; he then incubated 16-epiestriol in the same system and found 16-keto• estradiol-17fl in 5% yie1d and estriol in 1%. Thus 16~ketoestradiol-178 seems to arise from 16-epiestrio1 and estrio1 through the mediation of

16-hydroxysteroid dehydrogenase and this compound interconverts

16-epiestriol and e strio1. 16 ... Ketoestradiol•l }( has not yet been iso1ated from urine; it has, however, been shown to be reduced to l~epiestriol and 16, 17-epiestriol by human liver tissue (101).

Finally, last but not 1east in importance among the presently known

C-16 substituted estrogens cornes 16-ketoestrone. As ear1y as 1938,

Marrian (102} had suggested in the Harvey lectures that 16-ketoestrone could arise by oxidation from estrio1. During the following years, severa! authors (103, 104) have reported the presence of 16·ketoestrone in human urine under certain circumstances but none had isolated nor identified it with certainty until the work of Breuer (105). He injected 16-ketoestradiol

·178 into a normal subject and claims to have obtained crystalline 16•keto• estrone. This therefore confirms Marrian's (102) viewpoint since 16·keto• 14.

estradiol-17B arises from estriol. In vitro, Breuer (32) had a1ready shown that 16-keto•estrone is possibly a key substance in the metabolism of C-16 substituted estrogens; he bad incubated 16-keto•estrone with human liver slices and found 16 ~-hydroxyestrone, 16~-hydroxyestrone,

16-keto•estradio1-17~ , estrio1, 16-epiestriol, 17-epiestriol and 16, 17· epiestriol.

2-Hydroxylation and 2-methy1ation also play an important part in the intermediary metabolism of the estrogens. The formation of 2-methoxyestrogens from estrogens requires first hydroxy1ation of the phenolic steroid which can then be methylated; this reaction is mediated by the enzyme o-methyl transferase which has been shown (106) to be the same enzyme as used in the methylation of catecholamines. Engel et. al. (107) have shawn that the conversion to 2-methoxyestrone after the administration of estradiol-17S 14 ·16 - C to be of the order of 8%. lt is not known yet whether these compounds have any physiological significance or are simply inactivation products.

No 6-keto nor 6-hydroxy phenolic steroids have yet been found with certainty in human urine a1though it bas been suggested that the Kober chromogen in human pregnancy urine referred to by Loke et. al. as KC-6B

(31) may be a 6-hydroxyestrone (108). Similarly, KC•6A (31) was thought to be 18-hydroxyestrone as on treatment with sodium hydroxide it yielded formaldehyde and 18-norestrone.

A1though the c~ll substituted neutral steroids are a physiologically important group, the C·ll substituted phenolic steroids certainly seem to play a minor role if any; they have not yet been isolated from human urine.

(3) DEGRADATION

Since the first studies on the intermediary metabo1ism of estrogens, it has been noticed that there is a low recovery of phenolic steroids from 15.

the urine after the administration of estrone or estradiol•l7B (109, llO); also the phenolic steroids disappear very rapidly after incubation with animal tissues. From this it was concluded that phenolic steroids are converted to non•steroidal compounds. Jellinck (111) from his work on estrone degradation products reached the conclusion that the liver probably forms non-steroidal metabolites. No radioactivity was present in the carbon dioxide collected during the incubation of liver slices, showing that ring D must remain intact at least. Also these products remain in the acqueous layer after hydrolysis with acid or enzyme and subsequent extraction with ether. Breuer (112) has suggested that the following types of substances might conceivably be among those present in the acqueous layer:

1. Unhydrolysed conjugates of unknown nature,

2. Phenolic steroids with their structure intact but very

soluble in water on account of a larger number of

hydroxyl groups,

3. Degradation products of marrianolic or doisynolic acid

in which the D ring is oxidized to carboxyl groups (these

have not yet been detected in natural materials) and

lastly,

4. Degradation products in which the steroid structure has

been extensively modified and highly oxygenated.

(4) CONJUGATION

Excretion of the biologically active phenolic steroids is made possible by conjugation with slucuronic or sulfuric acid. Steroids combined with these acids are more soluble in water and can be eliminated through the kidney. Certainly in the organs where they are elaborated, estrogens seem to occur mainly in the free form but in other parts of the organism they are present as conjugates. 16.

The first estrogen isolated in the conjugated form was estriol

monoalucosiduronate from pregnancy urine by Cohen and Marrian (113 ).

It was shown that the phenolic group of the conjugate is free and that the glucuronic acid is attached at position C-16 or C·l7 (114). It is known now that all the other estrogens are also excreted in this form.

For a long time, the potassium salt of estrone hydrogen sulfate, isolated by Schachter and Marrian (115) in 1938 from the urine of the pregnant mare, was the only sulfuric acid ester of estrogens known to occur in nature. Recently, estrone hydrogen sulfate (116) and estriol-3- hydrogen sulfate (117) have been isolated from human pregnancy urine.

Conjugation was considered a means of detoxification until it was shown that conjugated steroids were also present in plasms. Thus, Engel and his co1leagues (118) identified estrone hydrogen sulfate in extracts of human plasma and showed it to be an important form of estrogen transport. In pregnancy plasma, the was present in approximately four times the molar concentration of unconjugated estrone; this is therefore the predominant estrogen metabolite in blood. Estrogen hydrogen sulfates are probably formed in the same manner as other sulfate esters. The sulfate ester linkage is attached to the phenolic hydroxyl, i.e., at position three, as it requires a phenol sulfatese to split it and phenol sulfatases do not hydrolyse alcholic sulfates (119).

The occurrence of a double conjugate has a1so been reported (120); this compound is estriol containing one molecule of glucuronic acid at position 16 or 17 and one of sulfuric acid at position 3.

Estrogens are also known to be bound to plasma proteins (121) but this is not true conjugation but a 11bindingn of sorne kind. e e FIGURE Ill INTERMEDIARY METABOLISM OF THE ESTROGENS (ACCORDING TO BREUER)

HO'~ ESTRADIOL- 11/J A

~ffiOH :o:J ·' 17 EPIESTRIOL 0/ 16 EPIESTRIOL H ~ ' ••.OH '

HO HO' 16œ HYDROXYESTRONE 16/J HYDROXYESTRONE / ~ .... OH /,~~··· .-.....-..""o

HO' 16 KETOESTRONE /... .. OH ?H ~ ,~o

...... HO HO ...., 16 KETOESTRADIOL 17/J 16 KETOESTRADIOL 17a. .

FIGU\E III 18.

C. ALTERED STATES OF ESTROGEN .METABOLISM

Although levels of estrogen excretion may vary considerably from individual to individual, the pattern of the excretion of estrone, estradiol-17B and estriol in the normal human is relatively constant.

Thus, premenopausal females tend to have higher levels of estriol than estrone (ratio of almost 3:1) while the male ratio is closer to unity; estradiol-178 is always the lowest of the three. These patterns of excretion as well as total excretion have been shown to vary in different dise a se states.

(1) Since the liver is the site of the intermediary metabolism and inactivation of the estrogens, naturally it was assumed that patients with chronic liver disease would show alterations in estrogen metabolism.

Patients with chronic liver disease frequently also show alterations in their secondary sexual characteristics and sexual function such as loss of libido, diminished sexual potency, testicular atrophy and gynecomastia in the male and menstrual disturbances in the female.

Thus, on the assumption that reduced inactivation by the liver might lead to increased excretion of biologically active estrogens, assays were carried out on total and unconjugated estrogens in the urines of patients with liver disease. Glass, Edmondson and Soll in 1940 (122), using bioassay, reported increase in estrogen excretion, especially free estrogens, thereby showing a failure of the diseased liver to conjugate or to "inactivate". The same au thors, in a la ter experiment (123}, administered estrone and estradiol-17a to patients with liver disease and recovered 83 - 86% of the biological activity of the administered dose in the urine whereas 10% would have been expected for normal individuals. Later workers, using colorimetrie measurements, have found raised values in only about 20% of the cases and no value has exceeded 19.

twice the normal maximum (124, 125). The major metabolite in Cameron 1 s study (124) was often estriol which is the least biologically active of the three thereby completely disagreeing with bioassay results. Lyngbye and Mogenson (125) in five cases found normal overall recoveries but in two the recovery of estradiol-178 was increased at the expense of estriol.

Brown et. al. (126) have recently confirmed and explained many of these findings. They obtained several very high estrogen levels by a chemical method such as bad previously only been shown by bioassay. The proportion of estriol to the other endogenous estrogens excreted was higher than normal in many of their patients although in two instances the increase was only in the estrone fraction. They obtained no high recoveries of estrogens as bad been reported by Glass et. al. (123); on the contrary, sorne of their recoveries were rather lower than normal. They concluded that the "increased output of urinary estrogens was due to increased secretion rates of the primary estrogenic hormone rather than to impaired estrogen metabolism by the liver. The abnorma1ities of estrogen output were most marked and common in terminal liver failure but they also occurred in mild cirrhosis."

(2) The growth of sorne mammary tumors is assumed to be under the influence of estrogens; therefore a study of estrogen production and metabolism in patients with breast cancer should yield valuable information concerning this disease. However, investigations carried out so far on the endogenous excretion of estrogens have not shown much difference between postmenopausal women with and without mammary carcinome (127). Cancer patients as a group were excreting slightly more estriol than the control group which in turn reflected in higher levels of total estrogens.

Similarly, not much difference in the total amounts of estrogens recovered following the injection of estradiol-178 was observed. The proportion of estriol to total estrogens recovered after the injection of estradiol•l7~ 20.

was higher in the breast cancer group (72%) as compared to the contro1s (58%).

In vitro expertments have also been carried out with cancerous breast tissue, but here a1so the results have been varied and far from conclusive. Breuer and Nocke (128) have found that ma1ignant mammary tissue may have a higher steroid turnover rate as measured by oxygen uptake (Q0 ). 2

(3) Adrena1 cortical tumors are sometimes capable of elaborating excessive amounts of estrogens both in males and females. Diczfalusy and

Luft (129) studied one such case and found the presence of large amounts of free estriol as well as conjugated estriol. In 250 ml of urine they found

34.5~g free estriol, 857~g conjugated estriol, 68.5pg estrone and 18.4pg estradiol-17B , thus raising the estriol/estrone + estradiol-17B ratio to about 10 which, as reported earlier, is far from the male pattern.

The authors attributed the high estriol level to increased production of progesterone, but as metastases of the liver was also shown in this patient at autopsy, this could a1so have some bearing on the results obtained.

(4) The remarkably beneficia! effect of estrogen therapy in patients with carcinoma of the prostrate bas brought interest to the study of estrogen metabolism by such patients. May and Stimmel (130) carried out investigations on such patients and found what bas been shown in the other types of cancer. Patients with cancer of the prostrate seem to have a tendency to convert therapeutic doses of estrone to estriol more readily than non•cancerous patients but there was no consistent difference in the total estrogen excretion.

(5) The leve! of thyroid hormone changes the metabolic transformation of steroid hormones by affecting the enzymes involved in these transformations.

The effect of thyroid hormone on estrogens was studied by Fishman et. al. (131).

They studied schizophrenies and normals, and patients with myxedema and 14 untreated spontaneous hyperthyroidism. Estradiol•l7P •16 - C was injected 21.

into all patients before treatment and urinary estrogens were measured.

Normal patients were made hyperthyroid and myxedema patients were made euthyroid with 3, 5, 31.tri•iodothyronine while the hyperthyroids were treated with 131 I and thereby made euthyroid. Two major changes produced by elevated thyroid hormone were noted; first, a decreased oxidation of

C·l6 in ring D decreasing estrio1 and second1y an increased oxidation of

C·2 in aromatic ring A leading to a large increase in 2•methoxy-estrone and 2·0H estrogens; estrone remained relatively constant. The schizo- phrenic patients responded and behaved in a similar fashion to the other groups showing that glandular production and peripheral metabolism are not significantly altered in this mental disorder.

(6) It has been noted for some time that there is a higher incidence of myocardial infarction in men of all ages and in women after the menopause as compared to women in the child bearing age. Oliver and Boyd (132) studied 1000 consecutive patients with clinica1 and electrocardiographie evidence of myocardial infarction or ischemia and found that coronary artery disease is eight times more frequent in men than in women under the age of fifty and that this sex difference decreases markedly in the older age group. The same kind of evidence has also been found by other workers (133, 134). These data show that the lack of estrogens might play an important role in the onset of this disease and thereby suggest a ttprotective 11 action of the functioning ovary.

The concentration of plasma lipids, especially cholesterol, has been known to be altered in coronary artery disease. Patients with myocardial infarct and arteriosclerosis usually have an elevated plasma cholesterol leve! and the plaque•s which form on arterial walls consist mainly of cholesterol. Studies have also been carried out on the effect of estrogens on plasma lipids (135, 136). It has been shown that plasma lipid concentration 22.

as well as urinary estrogen excretion both vary during the normal menstrual cycle {137). Synthetic estrogens, such as ethinyl estradiol, are used to correct abnormal levels of plasma lipids and lipoproteins in male subjects with myocardial infarction (138).

The foregoing evidence, thus, bas prompted severa! others as well as the present author to investigate in a more detailed manner, this relationship of estrogens and myocardial infarction. Bauld et. al. {139) injected small doses of estradiol•l7B (350-500 p~ intramuscularly into males with previous myocardial infarcts and analysed the urine for estrone, estradiol•l78 and estriol. There was no significant difference in two indices of estrogen metabolism between the two groups. The increase in estriol + estrone + estradiol•l7~ excretion in the myocardial infarction group was similar to that in the control group. Also, the relative proportions of estriol, estrone, and estradiol•l7B excreted on the day before the injection, i.e., the endogenous urinary levels, were not different in the two groups. The proportional increase in urinary estriol to the increase in urinary estrone and estradiol•l7B was however significantly greater in patients with previous myocardial infarction than in controls. Therefore they stated, there is a quantitative difference in estrogen metabolism in subjects with and without previous myocardial infarction. The period from the episode of myocardial infarction to the beginning of the experiment ranged from one week to two years, showing that the time since the infarct does not influence the response.

Similarly, the mean age of the two groups was the same, eliminating the factor of age.

In another study, the same authors (140) injected ACTH, chorionic and to control and myocardial infarction groups and again followed the urinary levels of estrone, estradiol-178 and estriol. Although an increase in the estrogen level was noted following 23.

the injections, they could not demonstrate a difference in response between

the control group and infarction group. They concluded that the "estrogen

metabolic defect in myocardial infarction can be demonstrated only when

estradiol-178 is adrninistered."

An interesting contribution to this question cornes from Bersohn and

Oelofse working in South Africa (141, 142). They had observed that the

male South African Bantu, who frequent1y shows signs of estrogenic changes

such as gynecomastia, was re1ative1y immune to coronary artery disease.

Hence, they cornpared the estrogen excretion of healthy European men,

European men with myocardial infarction and Bantu men and found differences

between these groups. The hea1thy European group consisted of two age

groups • one with mean age of 31, the other 55, while the infarction group

had a mean age of 57 and the Bantus of 31. In the hea1thy Europeans, the

older age group had a higher estrogen excretion than the younger group, especia11y

in the estrio1 fraction. Mean values for estrone, estradiol-178 and estriol

were 4.3 pg, 1.1 pg, 2.68 pg respectively in the younger group and 6.3 pg,

2.1 pg and 6.0 pg respectively for the older group. The subjects with previous

myocardial infarction, compared to the ir own age group excreted le ss e strone

and estradiol-178 (3.7 pg and 0.7 pg respectively) than the control group but

an equivalent amount of estriol (6.3 pg). This raised their ratio of estriol

to estrone and estradiol-17/3 higher than the normal. On the other hand, the

total excretion of estrogens as wel1 as the individual fractions were higher

in the Bantu than in the healthy European of comparable age; estrone mean value was 5.5 ~g, estradiol-178 2.5 pg and estriol 3.5 pg. The increase was most noticeable in the estradiol-178 fraction, which is the most active of

the estrogens. Cornparing the Bantus to the myocardial infarct group, the picture is different. Here there is an increase in estrone and estradio1•178

fractions also, but the mean estrio1 value is only about one half of the 24.

infarction group's. One conclusion that bas been drawn from this is that the Bantu is less efficient in metabolizing estradiol-17B than the

European.

Thus, differences in estrogen metabolism have been found in patients with myocardial infarction by different investigators. The findings of

Bersohn and Oelofse (141, 142) were made for endogenous estrogen levels.

Bauld et. al. (139) were unable to show a difference in endogenous metabolism between infarct group and control group; only when estradiol-17~ was injected, could they demonstrate this difference. Also, a higher than normal estriol to estrone and estradiol-17~ ratio is not confined only to this disease but bas also been shown in liver disease (124-126) and various types of cancer (127,129, 130). Could it be that this "high estriol", be it of endogenous or exogenous origin, is only a reflection of the general health of the body? PURPOSE OF THE INVESTIGATION

Since estrogen metabolism bas been shown to be altered in patients with myocardial infarction, the object of the present investigation was to study, by a well•established technique, this alteration, if any, in more detail.

ln addition, a study of the intermediary metabolism of eight estrogen metabolites, including the ring DO\ ketols was carried out with the aim of gaining more information regarding their source, relative interconversion and the percent conversion from estradiol-17/3. EXPERIMENTAL

MATERIALS AND METHODS 25.

CHAPTER Il

MATERIALS AND METHOOS

MATERIAI.S

I. RADIOACTIVE COMPOUNDS

1. Two batches of estradiol-17~ -6, 7 • 3H were used:

(a) Batch 1 • this was obtained from the New England Nuclear 3 Corporation, Boston, as estrone-6, 7 - H with a specifie

activity of 150 ~c/pg and was reduced to estradiol-17~ 3 -6, 7 • H.

(b) Batch 2 - obtained from Merck, Sharp and Dohme {Montreal), 3 as estradiol-17~ -6, 7 - H with a specifie activity of

40 pc/J~.g. 14 2. 16-keto-estradiol-17~ -16 • c was a gift from Dr. M. Levitz (N.Y.U.) and had a specifie activity of 44 pc/mg. 14 3. 16-Epiestriol-16 - C was prepared from 16-keto-estradiol-17~ 14 -16 • C by reduction with sodium borohydride. 14 4. Testosterone -4 - c with specifie activity of 140 pc/mg was

bought from Merck, Sharp and Dohme, Montreal.

Standard solutions of these steroids were prepared in absolute alcohol and used for the injection of patients.

Saline used for the dilution of injection doses was obtained from

Abbott l.aboratories, Montreal, in sterilized bottles.

Il. NON•RADIOACTIVE STEROIDS

Pure, crystalline (1) estrone and (2) estradiol-17~ were donations by the late Dr. Bauld, (3) estriol was gifted by Parke, Davis and Co., Ann

Arbour, Michigan. Both (4) 2•methoxyestrone and (5) 16-epiestriol were 26.

purchased from Organon lnc. while (6) 16 ~-hydroxyestrone was obtained from Dr. A. E. Kellie, Courtauld Institute of Biochemistry, Middlesex

Hospital, London, England and (7} 16-keto-estradiol•l7B was again donated by Dr. M. Levitz (N.Y.U.). The (8) 16S-hydroxyestrone diacetate was purchased from Southeastern Biochemicals, Morristown, Minnesota.

(9) Estrone-3•methy1ether, (10) estradiol-17~ ·3-methylether and (11) estriol-3-methylether were prepared from free crystalline compounds by the method of Brown (72).

Solutions of these and all other materials in A. R. grade methanol yielded standard solutions for such purposes as alumina standardization, use in the Ittirch color reaction, or as standards for paper chromatography.

III. ENZYMES

Three different kinds of enzymes were used in the hydro1ysis of urine:

1. Limpet enzyme containing 1,000,000 U ~ •glucuronidase / gm

plus phenolsulfatase was prepared in Dr. Brown's laboratories

in Edinburgh. 2. B ·Glucuronidase enzyme from the Sigma Chemical Company, St. Louis, Missouri, containing 1,000,000 U B -glucuronidase

/2.7 gm and phenolsulfatase was also a preparation from limpets.

3. Helicase enzyme from Industrie Biologique Francaise, S.A., ~ Gennevilliers (Seine), France, prepared from Helix Pomatia,

contained 1,000,000 F.U. E -g1ucuronidase / gm and 15,000,100 Roy. u. sulfatase / gm.

Df. REAGENTS

All reagents used were distilled in an all•glass apparatus before use and stored in brown glass botties. 27.

A. The following reagents were obtained from Anachemia Chemicals

Limited:

1) Diethyl ether, reagent grade, was freed from peroxides by shaking with

approximately 0.3M Feso in 0.4 N H S04 (lOOml/1), and distilled within 4 2 six hours of ,use as described by Bauld (140).

2) Hexane, c. P. grade. 3) Methanol, aldehyde and ketone free.

4) 1bluene, A. c. S .. reagent grade.

5) Propylene glycol - required no further purification.

6) Benzene, purified as described by Bauld (140).

7) Ethanol • 95%, was allowed to stand over 2, 4-dinitrophenylhydrazine

for 24 hours and distilled twice.

8) Ethyl acetate, distilled and stored over Na so • 2 4 9) Hydrogen peroxide - 30%, A. c. s. reagent grade.

B. Obtained from Fisher Scientific Company were: 1) Sodium sulfate, c. P. reagent grade.

2) Sodium hydroxide, A. R. electrolytic pellets.

3) Sodium bicarbonate, c. P. reagent grade. 4) Glacial acetic acid, A. R. grade. 5) Sulfuric acid, c. P. grade. 6) Hydrochloric acid, C. P. reagent grade.

7) Alumina, Brockman Activity 1, 80-200 mesh.

8) Girard's reagent "Tu, A. R. grade, stored over calcium chloride in

an evacuated dessicator.

9) Borie acid, c. P. grade. 10) Pyridine, c. P. grade.

11) Acetic anhydride. 28.

c. 1) Dimethyl sulfate was obtained from Eastman Organic Chemicals.

2) Sodium borohydride from L. Light and Company.

3) Silica gel H, according to Stahl, for thin layer chromatography

from E. Merck, A.G., Darmstadt, Germany.

4) Celite 545 and asbestos from Johns-Manville Company, Ltd., both

purified and prepared as described by Bauld (143).

5} Whatman paper No. 42 for paper chromatography was purchased from

Fisher Scientific Company.

D. Reagents for detecting standards on chromatograms:

1) Turnbull 1 s blue reagent for paper chromatography consisting of equal

volumes of 1% potassium ferricyanide, A. c. S. reagent grade,

Anachemia and 1% ferric chloride, analytical reagent, Mallinckrodt.

2) Pauli's stain for thin layer chromatography consisting of:

(a) 0.9 gm sulfanilic ac id 9 ml HCl 90 ml H 2o (b) 1% NaN0 in H 0 2 2 (c) 10% Na in H 0 2co3 2 • the above are mixed in proportions of 1:1:2 respective1y and were

all obtained from Fisher Scientific Company, Montreal.

E. Scintillation Fluid

Toluene Scintillation Fluid for the counting of radioactive samples was prepared by dissolving 3 gm of 2, 5 - diphenyloxazole and 100 mg of

p•Bis (2 • C5-phenyloxazolyl) l·benzene, both purchased from Pilot Chemicals

Inc., Watertown, Massachusets asC. P. reagents, in 1 litre of reagent grade

sulfur free to~ene from British Drug Houses, Montreal; this was stored away

from light in dark glass botties. 29.

F. Ittrich Reagents

Ittrich reagents for the accurate determination of weight of

estrogen present (66): a) Hydroquinone • from British Drug Houses, Ltd., Montreal, asc. P.

reagent, was further powdered for use. b) p~Nitrophenol, from Fisher Scientific Company, Montreal, as c. P.

grade; this was further crystallized two times from double•distilled

benzene and stored in a dark brown bottle. c) Tetrachloroethane (for spectophotometric determination). d) Tetrabromoethane (for fluorimetric determination).

- both from Anachemia Chemicals Ltd., as analytic reagent grade did

not require further purification.

G. Chromatographie Solvents a) Brown's (72) alumina columns for separating estrogen methylethers­

benzene and hexane from same source as previously described. b) Givner et. al. (144) celite partition columns using benzene, hexane

and methanol also from source previously described. c) Toluene-propylene glycol system for paper chromatographie separation

of ring D~ketols- solvents again from previously described source. d) Lisboa • Diczfalusy System A (145) for thin layer chromatography,

consisting of: ethyl acetate, 45: cyclohexane, 45: ethanol, 10:

all solvents were obtained from Anachemia Chemicals Ltd. as c. P.

grade. Cyclohexane was used as received but ethyl acetate and

ethanol were distilled in ali-glass apparatus, ethanol as previously

described. 30.

METHODS

I. PURIFICATION OF RADIOACTIVE STEROIDS

3 1. (a) Batch 1 was bought as estrone -6, 7 - H with a specifie 3 activity 150 pc/pg. This was converted to estradiol-17B -6, 7 - H by sodium borohydride reduction (146) and purified on the celite partition column 3 of Givner et. al. (144). The purity of the estradio1-17a -6, 7 • H so obtained was proven as follows: 3 100,000 Cpm of estradiol-178 6, 7 - H was mixed with 26.8 mg of non-radioactive, crysta1line estradiol•l78 giving a ca1culated specifie activity of 4730 cpm/mg. Crystal1ization was then done from methanol and the specifie activities of the crystals and mother liquor determined.

Specifie activities were 4700 cpm/mg and 4640 cpm/mg respectively. The close agreement of the two specifie activities and the calculated specifie activity was taken as proof of the purity of the compound. 3 (b) Batch 2 was bought as estradio1-17B -6, 7 - H with a specifie activity of 40 pc/pg. This was a1so chromatographed on the celite partition column of Givner et. al. (144) and its purity proven in the same way as 3 for Batch 1. 555.000 cpm of estradiol-17~ -6, 7 - H was mixed with

31.0 mg of non-radioactive estradiol-178 , giving a calculated specifie activity of 17,900 cpm/mg. The specifie activities of the crystals and mother liquor were in excellent agreement, being 17,900 cpm/mg for both.

14 2. 16-Epiestrio1-16 • c was prepared by sodium borohydride reduction 14 of 16-keto-estradiol-17~ ·16 - c by the method of Diczfa1usy and 14 6 Munstermann (146). 16•Keto-estradio1•17B -16 - c, about 1.0 x 10 cpm, was dissolved in 10 ml of 50% aqueous ethanol with 10 mg/ml of sodium borohydride added and left in the dark overnight. The next morning. the reaction was stopped by the addition of glacial acetic acid, drop by drop, 31.

until the effervescence disappeared and the pH was below 7. The solution

was then diluted with water and extracted with ether four times; the

combined ether extracts were washed with sodium bicarbonate and water~

dried over sodium sulfate and evaporated to dryness. The 16-epiestriol

-16 • 14C so produced was purified on the non-ketonic celite column of 14 6 Givner et. al. (144). The pure 16-epiestriol-16 - c, 1.0 x 10 cpm,

was then transferred to an autoclaved vial by means of a sterile pipette 3 6 and estradiol-17B -6, 7 - H, 6.6 x 10 cpm, was added.

II PREPARATION OF NON-RADIOACTIVE STANDARDS

All non-radioactive standards were obtained in pure, crystalline form.

Since 16/3- hydroxyestrone was bought as its diacetate, this had to be

hydrolysed for use as a chromatographie standard. The sample was dissolved

in 20 ml of methanol and 5 ml of 5N H so was added and hydrolysis was 2 4 carried out for five days in the dark. Then, 140 ml of ethyl acetate

were added and the sample washed with NaHCo and water. Ethyl acetate was 3 dried over Naso and evaporated off. The sample was then transferred to 4 a volumetrie flask with methanol and stored at 4°C.

III SPECTOPHOTOMETRIC AND FLUORIMETRIC READINGS

The quantity of estrogen present in a sample was determined by the method of Ittrich (6o). The optical densities or fluorescence were measured

against similarly treated reagent blanks; solutions (in duplicate)

containing known amounts of pure standards were treated in the same way

and the corrected values were used to determine the amount of estrogen

in the experimental fraction by simple proportion. Fluorescence was

measured in a G.K. Turner Associates Model 110 Fluorometer and optical

densities were determined in a Unie~ Spectophotometer, Model SP600.

Optical density readings (O.D.) were corrected for non-specifie pigments 32.

by the use of the Allen (64) correction equation •

O.D. Corrected • O.D. 538 mp • ~ (O.D. 506 mp + O.D. 570 rn~)

IV COUNTING METHODS

All counting of radioactive samples was done by liquid scintillation spectrometry but two different instruments were used.

1. Packard Triwcarb Liquid Scintillation Spectrometer Model 314 AX

was used in the studies of normal controls and myocardial infarct

patients where only the three classical e strogens were determined.

The machine was set by discriminator controls from 10-100 and 10·100 3 at a high voltage of 1160 (tap reading 9.5) for H and 1100 volts 14 (tap reading 5.5) for c in the case if single isotopes. The

efficiency for the two isotopes (in toluene) in this instrument was 3 14 16% for H and 60% for c when counted separately.

2. Nuclear Chicago Liquid Scintillation Spectrometer 725 series was

used in all double labelled experiments and in studies of the ring

D 1)1. ketols. The discriminator controls were set as follows:

Data attenuator 1.6 x 3.2, L (level one) was fixed at 0.5, 1 L was at 9.9, L at 9.5 and L at 9.0; the gate M.V. was at 2 3 4 3 1490 volts and data at 1320 volts. These settings count H at an 14 efficiency of 32% and C at about 80%. 3 14 Calculations of H and c from double labelled samples were done

according to the formulae of Okita et. al. (147) 3H N 14c .. Nl • ..1_ ... N2 .. aN b 1

where Nl .. total counts in channel I

N2 = total counts in channel II 33.

3 counts per minute H in channel 11 a = counts per minute 3n in channel 1 counts per minute 14c in channel II b = counts per minute 14c in channel I

Both a and b values were calculated by counting samples with single isotope and these values were redetermined with each experimental sample.

The radioactivity for all samples was determined to within 5 percent probable error and corrected for background radiation. Quenching was corrected where necessary by adding standards of known radioactivity to the experimental sample and recounting.

The assay of radioactivity was carried out by transferring a known aliquot of the sample in methanol to a 20 ml liquid scintillation spectrometer vial and evaporating this under a stream of air, care being taken to exclude any water. The sample, if the weight present was negligible, was then dissolved in 5 ml of toluene scintillation fluid. In the expertments where crystallization studies were performed, weighed portions of the crystalline material and evaporated mother liquors were dissolved in counting vials in 0.1 - 0.3 ml of methanol with heating if necessary and 10 • 15 ml of toluene scintillation fluid were then added.

V ENZYME ASSAYS

Both commercially bought enzymes were assayed for activity by the method of Talalay, Fishman and Huggins (148). Limpet enzyme was found to contain the same activity as stated by the manufacturer while the activity of the helicase enzyme was considerably less. The activity of different batches ranged from 300,000 F.U.- 600,000 F.u. per gram of ~glucuronidase instead of 1,000,000 F.U. as stated. Therefore each batch of this enzyme was assayed and used according to results obtained.

VI INJECTIONS

' 3 0 Estradiol-17B -6 -7 • H was stored in benzene at 4 c. This was taken 34.

to dryness and redissolved in a known amount of absolute alchol • 99% • obtained from the pharmacy of the Montreal General Hospital. From 6 6 6.0 x 10 - 8.0 x 10 cpm (equivalent to 1 ml usually) were taken out for each injection and placed in vials. All glassware that came into contact with the injection material was sterilized by means of steam autoclaving.

In the double labelled experiments, the two steroids were mixed in the 3 14 vials and 1% of the mixture was removed for the determination of HJ c prior to injection. The vials were prepared on the day prior to the injection and kept covered at 4 0 c. On the day of the injection, the vials were warmed to room temperature and the contents diluted with a few milliliters of sterile saline which was drawn into a syringe; the vial was rinsed out two times with saline, bringing the volume up to 10 ml.

After the patient bad voided, the injection was given intravenously into the arm.

The vials and syringes were washed with saline and extracted with ethyl acetate after the injection and the radioactivity remaining determined to correct for !osses of the original material.

VII COLLECTION OF URINE

Urines were collected on the wards of the Montreal General Hospital starting at the time of the injection in separate 24 hour specimens for four consecutive days. They were stored in plastic botties at 40 C until the completion of the collection and then immediately processed; where delays of more than four days from the start of the collection were necessary, the samples were stored in a deep freeze until required.

VIII HYDROLYSIS AND EXTRACTION OF URINES

Suitable aliquots from each of four 24 hour specimens were combined to give a "pool of urine" for the measurement of estrogens; usually one 35.

quarter of each 24 hour specimen was used, making the working "pooln equal to one day's urine.

The urines were adjusted (with glacial acetic acid) to the pH required by the enzyme used. Three different enzymes were used as described under

Materials.

1. The urines of all non-injected control patients were hydrolysed with the limpet enzyme prepared in Dr. Brown•s laboratories, Edinburgh.

The activity of this enzyme was 1,000,000 p/gm of /3 glucuronidase plus sulfatase and 600 u were used per milliliter of urine. The pH optimum was 4.5 and incubation was carried out for 96 hours.

2. The urines of all patients with myocardial infarcts were hydrolysed with limpet enzyme from the Sigma Chemical Company containing

370,000 u/gm of ~glucuronidase plus phenol•sulfatase, 600 u being again used per milliliter of urine. Incubation was carried out for 96 hours at pH 4.5.

3. All other urine samples were hydrolysed with helicase enzyme from Industrie Biologique Fran~aise. As different batches of this enzyme were found to have different activity, it was used according to the activity found, 500 F.u. being used per milliliter of urine. The optimum activity of this enzyme is at pH 5.2 and incubation was carried on for 24 hours.

Five ml of suitable acetate buffer per 100 ml of urine were added and incubation was performed in an incubator at 37°C for the time required.

When the hydrolysis was complete, the urines were cooled at 40 C prior to extraction. Extraction was done with diethyl.ether:' (purified as described under Materials). Each sample was extracted three times • once with a volume of ether equivalent to one half the volume of urine and two times with a volume of ether equivalent to one quarter the volume of urine. The portions of ether were combined in a separatory funnel and washed with alkali and water. 36.

From here on, the urines of the control subjects and myocardial

infarcts where only estrone, estradiol-17/3 and estriol were measured, were processed by different techniques from the urines where the ring D

~ ketols were also determined.

IX ASSAY OF ESTRONE, ESTRADIOL-1713 AND ESTRIOL

The urines from control subjects and myocardial infarction patients were assayed by the method of Brown et. al. (72). The combined ether extract was shaken with concentrated carbonate solution of pH 10.5 which was discarded; then with 8% NaOH which after shaking was partly neutralized with 8% NaHco and then discarded; finally the ether was washed with 8% 3 NaHC0 and water. The ether was then dried over Na so4 and evaporated 3 2 to dryness over a water bath. Estrone and estradiol-l7S were separated from estriol by means of a benzene/water partition, saponified in NaOH and

boiled for 30 min. under reflux. The pH was then lowered to 9.0 - 9.5 with concentrated HCl and NaHC0 and the estrogens suitably extracted. 3 Methylation was done with dimethylsulfate overnight at room temperature.

The contaminants were then oxidized with hydrogen peroxide and the methylated estrogens again extracted. Estrone and estradiol-17~ methyl ethers and estriol methyl ethers were then separated and further purified on alumina columns, the three compounds being collected in round bottom flasks. Each of the three fractions was then analysed for radioactivity present as previously described and for weight present by the Ittrich reaction (66). As the weight of estrogen present in the urine of the male is rather small, measurements were done by fluorimetry thereby allowing for at least duplicate measurements. The amount of estrogen methyl ether present was then converted to the corresponding amount of free estrogen by multiplying by the ratio of the molecular weights - 0.95.

The alumina used was standardized as described by Brown et. al. (72) 3 7.

and stored in completely airtight containers. The amount of water required for deactivation of our alumina was 10% (i.e., 10 ml per 100 gm alumina). Recovery experimenta were carried out and found to be very satisfactory - 96% for estrone, 97% for estradiol-178 and 96% for estriol.

Recovery experiments were also done on 30 minute methylation at 370 C versus overnight methylation at room temperature and both were found to give the same recoveries.

X ATTEMPTS AT SEPARATING RING D ~ KETOLS

Before a method for the determination of the ring D ~ keto1s could be chosen, a technique whereby 16~-hydroxyestrone, 16 8-hydroxyestrone and

16-ketoestradiol-178 could be separated from each other had to be established. A paper chromatographie chloroform-formamide system had been used by Marrian et. al. (17·19) when they first isolated these compounds.

1. Previous attempts at using the ch1oroform-formamide system in this laboratory had not given satisfactory results; however, it was decided to try again with this system. Again, however, the author failed to achieve separation of the three compounds by means of this system.

2. Toluene-propylene glycol was then chosen as another potential system as it is a good system for polar compounds and bad been used for the purification of estrogens ear11er. Estradio1•178 has an Rf of 0.2 in this system, but since the ring D ~ ketols are more polar than estradio1•178 , a running ttme of 16 hours was chosen. The paper was impregnated with propylene glycol:methanol • 1:1 and blotted between fi1ter papers; standards were applied with methanol on the starting line and the paper was placed in a chromatography jar which had previous1y been saturated with toluene for at least 24 hours and allowed to run for 16 hours.

The chromatograph was then dried in air for a few hours and the standards visua1ized by spraying with Turnbull 1 s Blue. In this time, the standards 38.

had barely moved from the origin. Even a running time of 60 hours was found not to be sufficient (16-ketoestradiol-178 moved 7.5 cm from the origin and 16 CM.-hydroxyestrone 4.5 cm).

3. Ethylene dichloride would increase the Rf's of these compounds but certainly just an ethylene dichloride/propylene glycol system would not separate them. A mixture of toluene: ethylene dichloride/propylene glycol (75:25/100) was then tried but without success. Even changing the proportions to 90:10/100 respectively did not meet with too great a success.

The running time was certainly decreased as expected but so also was the resolving power.

The toluene/propylene glycol system was then tried over again as previously described, but the chromatograph was allowed to run tor 7 days.

In this long running time, the three compounds did separate from each other very satisfactorily. The distances from the origin for 25~ of 16-keto­ estradiol-178 , 25~ of 16~-hydroxyestrone and 40~ of 16 8-hydroxyestrone were 26.5 cm. 20.5 cm and 9.0 cm respectively and no streaking or diffusion of the compounds was observed.

To obtain the percent recovery from this system, a known amount of radioactive plus non-radioactive 16-ketoestradiol-178 and non-radioactive

16 ~-hydroxyestrone were chromatographed in the usual manner. After the standards had been sprayed with Turnbull's Blue, areas corresponding to the compounds were eluted by cutting the paper into small pieces and soaking them in methanol overnight. The eluates were then filtered through sintered glass filters, evaporated and redissolved in ethyl acetate. The ethyl acetate was in turn washed with small amounts (5 - 10 ml) of water to remove the propylene glycol which would interfere in the Ittrich reaction, dried over

Na 2so4 and evaporated to dryness. The weight recovered was then determined by the lttrich reaction (66) and counts recovered by counting a small aliquot of the eluate. 39.

As a further proof of the identity of the eluates, the 16 ~-hydroxy­ estrone and 16-ketoestradiol-178 eluted were reduced with sodium boro­ hydride (146) and the reduction products identified on thin layer system A

(145) as estriol and 16-epiestriolrespectively. A1so, 16 ~ hydroxyestrone gave a pink colour ünmediately upon the addition of sulfuric acid and heating in the Ittrich reaction which is characteristic of this compound.

XI ASSAY OF RING D OC KETOLS IN THE URINE

Having thus obtained a system for the separation of the three compounds -

16 ~-hydroxyestrone, 16 B-hydroxyestrone and 16-ketoestradiol-17S , the fol1owing method was then used for their assay from urine samples.

1. The urine was hydrolysed and extracted as described before (Section

VIII - 3, P• 34-36).

2. The ether extract was washed with NaHC0 only, as any stronger 3 alkali such as NaOH would destroy the ring D~ ketols.

3. A modified Girard reaction as described by Givner et. al. (144) was used for the separation of the non-ketonic from the ketonic fraction.

4. The non-ketonic fraction was then saponified as described by

Brown (72) to remove urinary pigments.

5. For the separation of the ring D ~ ketols from the ketonic fraction, two systems were tried:

(a) Lisboa-Diczfalusy system A (145) for thin layer chromatography.

(b) Celite partition columns for the ketonic fraction as described

by Givner et. al. (144).

The recoveries from the ce1ite columns were better and were therefore adopted as part of the method. On these columns, 2 methoxyestrone and estrone are also separated.

6. The non-ketonic fraction was separated into estradiol-17fl ,

16-epiestriol and estriol on the non•ketonic celite column of Givner et. al. 40,.

1. Finally, the ring 01:1{ keto1s were separated on toluene/proplyene glycol system as described.

It must be noted here that as the chromatographie jars became better saturated and as the temperature and humidity in the room increased, the time required to separate the three compounds decreased considerably. The final running time when most of the experiments were performed was between

3 .. 3~ days. Als~ all chromatography was done on Whatman No. 42 paper.

The method described above was used when only ring Do<. ketols were assayed and also when the other five metabolites -2 methoxyestrone, estrone, estradiol-178 , 16-epiestriol and estriol were determined.

XII RECRYSTALLIZATION STUDIES

In the double labelled experiments, an additional step was employed for the achievement of radiochemical purity. In these experimenta, a total of eight metabolites .. 2 methoxyestrone, estrone, estradio1•17/3,

16-ketoe stradiol-17 J9 , 16 C(.-hydroxye strone, 16,$-hydroxyestrone, 16-epie striol and estriol • were iso1ated. Pure, crystalline, unlabe1led carrier of known weight was added to each metabolite (of known radioactivity) and the samples recrystallized from methanol unti1 the specifie activities of the crystals became constant and equal to that of the mother liquor. In some cases, further purification or proof of purity was achieved by derivative formation such as acety1ation fo1lowed by recrystal1ization. Samp1es which were suitable were reduced by sodium borohydride (146) and the reduction products recrysta11ized.

From the specifie activities thus obtained, percent conversions from estradiol-178 and from either 16-ketoestradio1•17B, 16-epiestrio1 or testosterone were calculated and metabo1ic pathways established. RESULTS 41.

Cl:f.APTER III

RESULTS

Two different methods were used as described in the last chapter, one for the determination of estrone, estradiol-17 /3 and estriol alone, and the other where either all eight metabolites or only ring D~ ketols were studied. The results will therefore also be described in two parts, according to the method used.

SECTION I

All the studies of control subjects and those with myocardial infarction were performed using the method of Brown et. al. (72) as described in Section VIII of nMethods". The final measurements of estrone, estradiol-17~ and estriol methyl ethers were done both by spectophotometry and fluorimetry for the non-injected control group as shown in Table I.

The results obtained by the two procedures were in good agreement with each other and in later experiments, therefore, only fluorimetric determinations were carried out.

The first series of experimenta consisted of a measurement of the three endogenous "classicn estrogens in a group of six young, healthy males. This served as a check on the author's use of the Brown method as well as providing a good control level of estrogen excretion in the male. As can be seen from the results in Table I, the average value of estrone was 3.3 pg/

24 hours, estradiol-17~ 1.4 pg/24 hours and estriol 4.7 pg/24 hours, with a total of 9.6 pg/24 hours. Table I also shows Brown 's (149) results (which compare very favourably with those of the present investigation) and those of Givner et. al. (144) assayed by the method of Bauld (74). Also shown are Bersohn and Oelofse•s (141) results assayed again by the Brown method. e e

TABLE I

ENDOGENOUSESTROGEN EXCRETION IN NORMALMALES (PG/24 HOURS)

Subject Age Es trone Estradiol•17/3 Es triol Total -- (Years) F* S** F* S** F* S** F* S** 1 34 3.8 3.4 1.3 1.5 2.7 3.0 7.8 7.9 2 30 2.5 2.7 0.9 1.0 3.1 3.0 6.5 6.7 3 31 7.2 7.5 3.3 2.2 10.0 10.2 20.5 19.9 4 24 2.0 1.8 0.8 o.8 4.7 4.5 7.5 7.1 5 30 2.1 2.1 1.0 1.0 5.0 4.8 8.1 7.9 6 24 2.2 2.3 1.1 1.3 2.7 2.6 6.0 6.2

Average 29 3.3 3.3 1.4 1.3 4. 7 5.0 9.6 9.3 (Range) (2.0- 7.l) (0.8-3.3) (2. 7-10.0) (6.0-20.5) Brown (149) 20-50 5.4 1.5 3.5 10.4 (3.0-8. 2) (o.o .. 6.3) (0.8-11.0) (6.0·17 .8) Givner et. 3.9 2.9 10.0 16.8 al. (144) (3.2-4.8) (l.0-4.8) (6.0-20.0) (10.3-28.4)

Bersohn & 20-48 4.3 1.1 2.6 8.o Oelefse (141, (1. 7-9.8) (o.o ...3.1) (0.6·9.9) (3.8-15.4) 142) 45-65 6.3 2.1 6.0 14.4 (2.8-12.5) (0.5-3.8) (1.3-12. 7) (5.6-21.1)

F1uorimetric Readings * .f:­ ** Spectophotometric Readings •N 43.

When comparing Givner's results to the present investigation, it is at once evident that the values by Bauld 1 s technique, particularly for estriol, are much higher, giving an average total estrogen excretion of 16.8 pg/24 hours which is almost twice that found by the author. Bersohn and Oelofse

(141) found different levels of estrogen excretion in two different age groups of normal males. Total average estrogens in the younger age group

(20 • 48 years) were 8.0 pg/24 hours. This is almost the same as the author's for a similar age group. Total estrogens for the older age group

(45 - 65 years) were 14.4 pg/24 hours, which is much higher, suggesting that estrogen excretion in the male rises with increasing age.

A second control group was also studied. This consisted of six hospitalized male patients who were free of heart as weil as liver disease and whose mean age was about the same as that of the myocardial infarct 3 group. These subjects received a tracer dose of estradiol-17~ -6, 7 - H 6 (about 6.0 x 10 cpm) and their urines were handled in an identical manner to that of the infarct group.

Table II shows the results of this study.

The total average estrogen excretion of this group (8.8 pg/24 hours) is of the same order as that of the younger, non-injected group as are the excretions of estrone, estradiol•l7~ and estriol individually. Thus, this investigation did not reveal a rise in the level of urinary estrogens with increasing age as Bersohn and Oelofse (141) had shown.

Table III shows the results of the patients with myocardial infarcts 3 who received a tracer dose of estradio1-17S ~ H similar to that given to the subjects of Table II. Their clinical history is shown in Table IV.

The levels of SGOT, LDH and serum cholesterol given are those at the time of admission and are all elevated, which is typical of myocardial infarction.

Electrocardiograms (ECG's) were done on all patients and provided further t:

•

hrs

e

7,800

26

413,000 524,100 464,800

440,000

496,800

324,400

cpm724

Total

hrs

7.3

9.9 5.5 5.1

8.8 cpm

13.1

11.5

pg/24

6

(5.1-13.1)

10

*

x

hrs

6.0

,ooo

MALES

99,600

•

143,200

283,400 197

187,900

203,500

264,300

cpm/24

H

3

NORMAL

Estriol

7•

hrs

IN

6,

7.2

2.5 2.5

5.1 2.4

6.5

4.4

pg/24

(2.4-7.2)

hrs

EXCRETION

!3

II

79,300

79,800

45,800

61,200

85,600

83,800

143,300

estradiol-17~

cpm/24

ESTROGEN

of

TABLE

hrs

Estradio1-17

7-1.4)

1.4

1.1

1.2

1.0

0.9

0.9

0.7

eg/24

(O.

injection

EXOGENOUS

AND

hrs

700

J

71,000

120,000

177,300 148

136,200

197,600

•122,400

intravenous

cmn/24

trone

ENDOGENOUS

an

Es

h!"S

7

1.

7-5.0)

2.3

5.0

3.8

3.8

3.5 3.3

pg/24

{1.

received

49 55

24 61

57

67

Age

38

subject

(Years)

Each

e

*

1

2

5

4

6

3

Subject

{Range)

Average e e

TABLE III

ENDOGENOUSAND EXOGENOUS ESTROGEN EXCRETION IN SUBJECTS WITH MYOCARDIALINFARCTION

Subject Age Es trone Estradio1-17/3 Estrio1 Total (Years) pg/24 hrs cPîîî/24 hrs iîs/24 hrs cpm/24 hrs pâ/24 hrs cpm/24 hrs jlgj24 hrs ~24 hrs 1 38 1.1 97,500 0.6 101,700 5.0 306,600 6.7 505,800 2 37 3.0 177,300 1.5 150,300 6.0 283,600 10.5 611,200 3 63 4.8 122,800 0.7 84,320 6.8 203,000 12.3 410,100 4 82 4.8 204,300 1.8 128,700 3.4 153,600 10.0 486,600 5 60 2.8 18 7' 700 0.7 74,200 3.6 131,300 7.1 393,200 6 52 1.0 55,800 0.5 3 7,100 2.4 150,400 3.9 243,300 7 48 2.0 175,400 0.5 81,300 2.3 274,500 4.8 531,200 8 34 3.0 153,300 1.2 95,800 2.8 232,500 7.0 480,600 9 34 4.7 128,600 1. 7 76,000 3.3 128,400 9.7 328,000 10 54 2.8 100,600 1.0 108,700 3.0 84,200 6.3 293,500 11 47 3.6 109,200 0.9 62,200 4.0 249,400 9.5 520,800 12 46 3.5 182,560 1.3 112,540 6.6 302,500 11.4 597,600

Average 50 3.1 141' 100 1.0 92,600 4.1 200,000 8.2 433,700 (Range) (34-82) (l.0-4.8) (0.5-1.8) (2.3-6.8) (3.9-12.3)

e; • e e

TABLE IV

CLINICAL HISTORY OF SUBJECTS WITH MYOCARDIALINFARCTION

Subject Age Days After SGOT (u) CHOL. (mgm %) l.DH (u) (Years) Infarct lnjected (10 - 40) (150 - 200) (200 - 500)

1 38 23 86 274 440 2 37 11 105 290 850 3 63 5 240 308 2780 4 82 4 160 285 770 5 60 2 106 312 740 6 52 1 108 266 1270 7 48 5 360 320 4450 8 34 3 172 278 2950 9 34 19 188 219 1430 10 54 25 llO 262 980 11 47 3 181 265 1260 12 46 15 175 282 1130

Note: Leve1s of SGOT, CHOL, LDH are those at time of admission. Normal values are given in parenthesis at top of each column.

•~ 4 7.

proof of diagnosis. Also shown on this table is the number of days after the infarct that the patient received the intravenous injection of estradiol-17 8-3 H; as can be seen, this ranged from 1 to as many as 25 days. The total endogenous estrogens for this group were found to be

8.2 pg/24 hours which is slightly but not significantly less than for either control group. The exogenous metabolism, as shown by the excretion of radioactivity, is also in close agreement and again, excretions of estrone, estradiol-178 and estriol individually are almost identical.

TABLE V

RATIOS OF ENDOGENOUS AND EXOGENOUS ESTRIOL/ESTRONE AND ESTRIOL/ESTRONE+ESTRADIOL-178

Es triol (EJ) Estriol (EJ) Es trone (El) Es trone (EJ) + Estradiol-17Ë(E2) Endogenous Exogenous Endogenous Exogenous

Control Group 1 1.4 1.0 Control Group 2 1.3 1.4 1.0 0.91 Myocardial Infarcts 1.3 1.4 1.0 0.90

In Table V are shown the ratios of estriol/estrone and of estriol/ estrone + estradiol-17 /3. A glanee at these results reveals that here also no difference in estrogen metabolism between the two groups of subjects was observed. The ratios, both for endogenous and exogenous metabolites, of E /E were about 1.4 while the ratios of E /E + E were 3 1 3 1 2 unity or less for all groups.

If ratios of this type are calculated from the data of Givner et. al.

(144) as shown in Table 1, again a difference between the latter's work and the present results is quite evident. Givner et. al. (144) found an endogenous E /E ratio of 2.6 (vs. 1.4) and an E /E + E ratio of 1.5 3 1 3 1 2 (vs. 1.0) in a control group. Bauld et. al. (139) in their study of patients with myocardial infarction found different ratios between the 48.

two groups in exogenous metabolism on1y. 'rheir ratios of endogenous

E /E for control and myocardial infarct groups were 3.2 and 4.0 3 1 respectively, while & /& + E for both groups were 1.7 and 2.8 respectively, 3 1 2 which they did not consider significant1y different. The exogenous E /E 3 1 ratio for the two groups was 1.5 and 4.3 respectively and E /E + E ratio 3 1 2 was 1.0 and 2.6 respectively. These latter ratios show indeed a significant difference between normal subjects and those with myocardial infarction.

TABLE VI

3 AVERAGE. PERCENT CONVERSION OF ESTRADIOL-17 /3 .. H TO ESTRONE AND ESTRIOL AND PERCENT ESTRADIOL-178 - 3H EXCRETED UNCHANGED lN NORMAL SUBJECTS AND THOSE WITH MYOCARDIAL INFARCTION

Subject % Conversion from E -17/] to % Unchanged 2 El + E3 E2

Control Group * 9.0 13.1 5.3 (12 subjects) Myocardial Infarcts * 9.4 13.3 6.1 (12 subjects)

Myocardial Infarcts ** Subject B 4.3 5.2 2.2 Subject G 6.8 3.2 2.6 Subject M lO.O 9.6 3.0

Average 7.0 6.0 2.6 * Resu1ts by Brown' s me thod. ** These three sets of results are from the experimente described under Section II D of "Resultsu where eight metabolites of the estrogens, including ring D~ ketols, were determined. The urines were not analysed by Brown's method but by a technique described in Section VIII of "Methods". + Explanation of abbreviations used in the present and following tables. 2 ME1 • 2-methoxyestrone E2 • estradiol•l78 E1 "" estrone 16epiE3= 16 epiestrio1 E3 = estriol 16KE2 "" 16 ketoestradiol-17B 160\0HEl • 16~ hydroxyestrone l6/30HE1 = 16/3 hydroxye strone -'

49.

The percent conversion of estradiol•l7B• 3 H to either estrone or estriol and the percent of radioactive estradio1•178 which was excreted unchanged, i.e., strict1y metabolism of exogenous material, is shown in

Table VI. The handling of such exogenous1y administered estradiol-17~ is again almost identica1 for both groups although individual differences exist as the results of the last three subjects show. The conversion of estradiol•l7a to estriol seems to be higher than the conversion of estradiol-178 to estrone (as determined by the method of Brown) but this is true of both groups. ln the last three subjects, however, the conversion of estradiol-178 to estrio1 is less than or equal to its conversion to estrone. This difference may be due to the fact that in these experimenta much more rigorous purification of metabolites was carried out by crystallization to constant specifie activity with carrier. The figures on this table point out rather clearly that the metabolism of a tracer dose of estradiol-178 and the conversion of this compound to estrone and estriol is identical between normal male subjects and those with previous myocardial infarctions. so.

SECTION II

STUDIES ON RING D ~ KETOLS

A. RECOVERY OF 16~-HYDROKYESTRONE AND 16-KETOESTRADIOL-178 FROM

TOLUENE: PROPYLENE GLYCOL CHROMATOGRAPHie SYSTEM

When the toluene: propy1ene glycol system was found to give satisfactory separation of the three ring D ~ ketols, recovery of these compounds from the chromatographie system was determined.

Recovery Experiment 1. 14 40 pg of pure 16-ketoestradiol•17B were mixed with 9000 cpm of C label1ed 16·ketoestradio1-17B , app1ied to a paper with methanol and the chromatograph was a1lowed to run for four days. The compound was eluted as described in Methods and the recovery determined.

TABLE VII

RECOVERY OF 16·KETOESTRADIOL-178 FROM TOLUENE/PROPYLENE GLYCOL CHROMATOGRAPHie SYSTEM

App1ied to Recovered from Chromatogram Chromatogram % Recovery

c~ 9000 7148 79.6

pg 40 30 75.0

Table VII shows the actual recovery of radioactivity and weight as well as the percent recovery. 79.6% and 75.0% of the radioactivity and weight respectively was recovered.

Recovery Experiment 2. Shown in Table VIII. 51.

TABLE VIII

RECOVERY OF 16-lŒTOESTRADIOL-17 /3 AND l~HYDROXYESTRONE FROM TOLUENE/PROPYLENE GLYCOL CHROMATOGRAPHie SYSTEM

16 o<.-Hxdroxxestrone 16-Ketoestradiol-17 S CPM ...ElL CPM .1!L Applied to Chromatogram .. 40 7000 56 Eluted from Chromatogram .. 5050 - Recovered after E t AC/H20 partition 25 4900 36 % Recovery 62 70 64

The recovery in this second ezperiment was lower, ranging from

62 • 70% for radioactivity and weight for both compounds. However, this is not unusual with a Zaffaroni type system.

B. PRELIMINARY STUDY ON URINARY EXCRETION OF RING D ~ KETOLS

The ring D ct. ketols, being precursors of the triols, are, like the latter, a1so elevated in pregnancy; therefore to facilitate measurement, a pregnancy urine was chosen for this preliminary study. The subject was in her second trimester of a normal pregnancy. The urine was analysed as described in Section X of "Methods" and the estrogens determined at various stages by the Ittrich reaction followed by spectophotometric readings. After elution of the ketols from the paper chromatogram, an a1iquot (equal to about 10%) was removed for the determination of weight present and the remaining portions of 16f)(.·hydroxyestrone, 16-ketoestradiol-17 B and 16 /3·hydroxyestrone were reduced with sodium borohydride. The se reduction products were then subjected to thin layer chromatography on Diczfalusy

System A and areas corresponding to 16-epiestriol and estriol eluted. The results of this study are recorded in Table IX. 52.

TABLE IX

RESULTS OF PRELIMINARY STUDY OF RING D~ KETOLS FROM PREGNANCY URINE

MGM. Estrogen/24 hours

1. Total ketonic fraction 5. 72

2. Ring D~ ketols (from column) 3.88

3. Toluene:propylene glycol 16 o<.-OHestrone 1. 76 16-ketoestradiol-178 0.92 16 /;3-0Hestrone 0.10 Total 2. 78

4. ETAC/H20 partition to remove prop. glycol 16· o<.-oHe strone 1.56 16-ketoestradiol-178 0.83 16 8-0Hestrone 0.09 Total 2.48

Na BH reduction 5. 4 From 16~-hydroxyestrone a) 16-epiestriol a rea 0.15 (presumab1y 17-epiestriol) b) estrio1 area 1.36

From 16-ketoestradio1•17/3 a) 16-epiestriol a rea o.ao b) estriol area ...

From 16 8-hydroxyestrone a) 16·epiestriol a rea 0.07 b) estriol a rea

Total 2.23

Losses in the ethyl acetate/water partition are about 10%, but as propylene glycol interferes in the lttrich reaction, it is very important to remove it by means of this partition. ln later experimenta, the losses at this stage were determined by counting an aliquot before and after partition of labelled fractions and were found to be almost negligible.

16 o<·Hydroxyestrone, 16-ketoestradiol-17 B and 16 B-hydroxyestrone were found to be 1.56 mgm, 0.83 mgm and 0.09 mgm respectively, making

160(;-hydroxyestrone the major metabolite of the ring Do( keto1s, at least 53.

in pregnancy. This is not surprising at all, however, as 16~-hydroxy•

estrone is the precursor to estriol which certainly is the major metabolite

of all the estrogens in the pregnant woman. The reduction of the 16~hydroxy•

estrone area yielded mainly estriol and 16~-hydroxyestrone and 16-keto•

estradiol-17B areas mainly 16-epiestriol. This then is additional proof

of the identity of the compounds eluted from the paper chromatograph as

well as the observation that 16~-hydroxyestrone gave its characteristic

pink colour in the lttrich reaction immediately upon heating.

C. UlUNARY RING D tl( IŒTOLS IN NORMAL MALES

As the preliminary study bad shown that the method for the measurement

of the ring D ~ ketols was a feasible one, a determination of the excretion

of these compounds in six normal males was undertaken. Each one of these 6 3 subjects received a tracer dose of 6.0 x 10 cpm/.ug of estradiol-17 /3 6, 7- H,

thereby allowing a study of exogenous metabolism as well as measurement of

endogenous levels. Aliquots for counting were taken of 1} the ether extract

of the hydrolysed urine, 2) the non•ketonic and ketonic fractions after the

Girard reaction, 3) estrone and total ring D ~ ketols from the celite column,

and 4) 16 o<-hydroxyestrone, 16 13-hydroxyestrone and 16~ketoestradiol-171J after

their elution from paper and also after partition between ethyl acetate/water

and these results are shown in Table X. In the case of the total ether extracts and the non-ketonic fractions, no attempts to correct the effect of quenching were made.

Table Xl contains data on the endogenous levels of these compounds measured in pg/24 hours, as well as information on the exogenous metabolism

as shown by cpm excreted per 24 hours, and also estrone levels of the same

subjects. lt must be stated that the results on this table are uncorrected

for any experimental !osses. ln recovery experiments discussed earlier,

these compounds were recovered from the toluene/propylene glycol chromato- e e

TABlE X

R.ESULTSOF THE ISOLATION OF URINARYRING D ri.. KETOLS IN NORMALMAIES

CPML24 Hours Subject l Subject 2 Subject 3 Subject 4 Subject 5 Subject 6 Ether Extract 1,100,000 1,034,000 400 ,ooo 350,000 610,000 480,000 t Non Ketonic 400,000 420,000 150,000 140,000 96,500 83,000 Ketonic 620,000 540,000 250,000 200,000 426,000 350,000 [: Estrone - 350,000 122,500 41,200 133,420 187,900 Ring D oe.Ketols 440,000 255,000 74,560 41,000 145,000 136,000

16- Ketoestradiol -17/3 70,000 37 ,ooo 11,000 4, 700 E1uted - 60,000 60,000 5, 700 10,600 from t 16 0(-l!ydroxye strone - • Paper 16 8-Hydroxyestrone 42.000 u.ooo 1,500 1,500 Total 172,000 108 ,ooo 28,200 16,800

After E16- Ketoestradio1 -17/3 60,000 34,000 10,700 4,600 15,400 31 ,ooo ETAC/H 0 16 o<-Hydroxyestrone 52,000 56,000 5,200 10,000 22,700 20,000 Partition 16 8-Hydroxyestrone 42.000 11 1000 1,400 1t300 1,100 ~00 Total 154,000 101 ,ooo 27,300 15,900 49,200 62,900

% of Ether Extract 14.0 10.0 4.2 4.6 8.0 13.0

VI •.t::- e e

TABLE XI

ENDOGENOUSAND EXOGENOUS LEVELS OF RING D IX l

16l

3 0.82 10' 700 0.35 5,200 0.23 1,400 1.40 27,300 3.40 122,500

4 0.47 4,600 0.51 10,000 0.18 1,300 1.16 15,900 1.68 41,200

5 0.85 15,400 0.30 22,700 0.14 1,100 1.29 49,200 3.50 133,420

6 o. 77 31,000 1.30 20,000 0.15 1,900 2.22 62,900 s.oo 87,900

Average 1.00 27,600 o. 73 28,900 0.30 9,800 2.03 66,200 3.16 107 ,ooo

Vl •Vl 56.

graphie system to the extent of about 70% only; it would not be too incorrect to assume, therefore, that their recovery from urine would not be more than 50% or thereabouts. 16-Ketoestradiol-178 was found to equal on an average 1.0 pg/24 hours, 16 ~-hydroxyestrone 0.73 pg/24 hours and 16 ~-hydroxyestrone 0.30 pg/24 hours, giving an average total of

2.03 pg/ 24 hours. Individual differences did occur though; one subject excreted a total of 3.54 pg/24 hours, while the lowest excretion level was 1.16 pg/24 hours. These resulta are much lower than those found by other investigators in the past, although the methods used have also differed. Hobkirk (155) measuring these compounds as their sodium borohydride reduction products, found average levels of 5.6 pg/24 hours, i.e., more than twice the amount found in the present investigation.

It is doubtful that the age of the subjects (males) should make any difference regarding the amounts excreted; the mean age of the present group was 57 years while that of Hobkirk's was 32 years. It is more likely that the difference in results found is due to the method used although whether the lower results are due to a more extensive purification on the chromatographie system or simply due to large losses on this system is presently unknown. Hobkirk (155) also found that the total ring D~ ketols were about equal to the level of estrone in the urine. This again is not quite so according to this study, estrone being higher than the total ring

D ~ ketols. However, it must not be forgotten that greater losses occurred in the ring D ~ ketone fraction than in the estrone fraction due to further purification of the former on paper. Therefore, to really compare these compounds, the amounts of ring DKketols found should be corrected for the

losses known to occur on the paper system. Taking this into account, with a mean recovery from paper of 70%, the levels of the ring D~ ketols approach those of estrone as shown in Table XII. 57.

TABU: XII

URINARY RING DOC. IŒTOL AND ESTRONE U:VELS IN NORMAL .MALE URINE

Subject Ring D Ketols * Estrone ** pg/24 hrs pg/24 hrs

1 5.00 2 3.50 2.24 3 2.00 3.40 4 1. 70 1.68 5 1.80 3.50 6 3.10 5.00

Average 2.90 3.16

* These values are corrected for lasses known to occur on the paper chromatogram. ** Estrone values are from celite column and are uncorrected.

Looking at the relative values of these compounds excreted in the

urine, there seems to be an equal amount of 16-ketoestradiol•l7 13 and

16tkhydroxyestrone plus 16/3-hydroxyestrone; 16-ketoestradiol.. l7/3 thus

·seems to be the main metabolite in the urine of the three compounds and

16 B·hydroxye strone the minor one. An abso1ute determination of 16/3-hydroxy• estrone is very difficult though as this compound rearranges very readily

to 16-ketoestradiol-17/3 in its free form.

The exogenous metabolism of these compounds as reflected by the cpm/24 hours excreted in each fraction is shown in Table Xl a1so. Almost equal

amounts of cpm/24 hours were excreted in both the 16-ketoestradiol-178

and 160(.-hydroxyestrone fractions; 16/3·hydroxyestrone fraction contained

the least amount of radioactivity as would be expected from endogenous

levels. lt would perhaps be more meaningful to look at these exogenous

results as the percent conversion from the injected estradiol-17/3. 3a

rather than just absolute counts. These percent conversions are given 58.

in Table XIII.

TABLE XIII 3 PERCENT CONVERSION OF INJECTED ESTRADIOL-17 /3 - H TO THE THREE RING D 0( IŒTOLS

3 % Conversion of Estradiol•l7n- H to Subject 16-Ketoestradiol•l7E 16 Q(-Hydroxye strone 16 B-Hxdroxye strone Total

1 1.00 0.90 o. 70 2.60 2 0.58 0.93 0.18 1.69 3 0.18 0.09 0.02 0.29 4 0.08 0.17 0.02 0.27 5 0.26 0.38 0.02 0.66 6 0.51 0.33 o.o3 0.87

Average 0.46 0.48 0.16 1.10

Resu1ts from the last Table indicate that the precent conversion of 3 estradiol-178- H to both 16-ketoestradiol•l7/3 and 16o<.-hydroxyestrone is about equal and to 168-hydroxyestrone rouch lower. The percent conversions are 0.46, 0.48 and 0.16 respectively with a total average conversion of

1.10%. Levitz, Spitzer and Twornbly (98) have also studied the conversion of estradiol-17~ to 16-ketoestradiol-l7E and have shown a figure of 1.6% for this reaction. It is difficult to compare their work to the present one, however, as their resu1ts were expressed in terrns of the percentage of cprn excreted in the first 24 hour urine following the injection of estradiol•l7 B.

Hobkirk (155) also found a higher conversion of adrninistered estrone • 14c to total ring D~ ketols in males; his conversion figure was between 6 •7%.

However, there is no doubt in the author's mind that the cornpounds she was measuring were really 16-ketoestradiol-178, 16ot_-hydroxyestrone and 168-hydroxy• estrone. Perhaps a thorough recovery study of the method used wou1d exp1ain better the differences between the past studies and the present one. 59.

D. STUDIES OF THE EXCRETION AND METABOLISM OF EIGHT ESTROGEN METABOLITES FROM URINE

In addition to the determination of ring D~ketols in normal males described in the previous section, a detailed study of eight metabolites of the estrogens was carried out, also in males. These were all hospital patients, and although no attempt was made at chosing certain types of

patients, all happened to have some type of coronary disease • either arteriosclerosis or myocardial infarction. All patients received intra­ 3 venous tracer doses of estradiol-17n- H, in addition, patient No. 1 14 (subject B) received 16·ketoestradiol•l78 - c, patient No. 2 (subject M) 14 14 16-epiestriol - c and patient No. 3 (subject G) testosterone - c, also 3 as intravenous doses given in the same injection as the estradiol-17n- H.

By analysing the urines of these subjects for estrogen metabolites, the author hoped to gain a better understanding of the intermediary metabolism of the estrogens, especially of the ring D~ ketols, and also to determine

the conversion of neutra! steroids such as testosterone, if any, to estrogens. These three experimenta will be described separately for the

sake of convenience and clarity as each one involves a great deal of data.

The results of subject B are recorded in Tables XIV and xv. Aliquots for the determination of the distribution of radioactivity were taken from the ether extract, from the non•ketonic and ketonic fractions after the

Girard reaction, from the six fractions separated from the celite columns and from the three ring D~ keto1s eluted from the paper chromatograph.

The cpm given are for the total four day urine, even though in this actual experiment on1y 1/16 of the mixed four day urine was ana1ysed. The 3H/14c ratio of the injected estradiol-17~-3 H and 16-ketoestradiol•178-14c was

9.2. The isotope ratios of 16-epiestriol, estriol, 16~-hydroxyestrone,

16B·hydroxyestrone and 16-ketoestradiol•l78 before recrystallization were 5.2, 3.1, 36.0, s.o and 1.1 respectively. The 2 methoxyestrone, 60.

TABlE XIV

RESULTS OF THE ISOLATION OF EIGHT URINARY ESTROGEN 3 METABOLITES IN SUBJECT B INJEC'fED WITH ESTRADIOL·l78- H AND 16·IŒTOESTRADIOL-17B .. 14c

3 14 CPM H CPM c 3HL14c

Injection Material 9,576,000 1,042,000 9.2

Total Ether Extract 3,993,000 375,000

Ke tonie Fraction 1,832,000 135,000

Non•Ketonic Fraction 1,956,000 313 ,ooo

Ketonic Fraction 2 ME 82,840 1 El 1,213,000 .. Ring DQlketols 341,000 86,000

Non .. Ketonic Fraction E2 368,000 16 EPIE 233,900 45,000 5.2 3 E3 635,000 204,000 3.1 Ring Dol Ketols 16ot. OHE 146,000 4,000 36.0 1 16 !3 OHE 30,000 6,000 5.0 1 16 IΠ70,000 2 80,000 1.1

The cpm given are for total four day urine; in the actua1 experiment only 1/16 of four days was used. TABLE XV

RECRYSTALLIZATION DATA OF THE EIGHT URINARY ESTROGEN METABOLITES OF SUBJECT B

Urine CPM for MG of CALC S.A. of 3H s.A .. of 14c Fraction Cryst Carrier s.A. xs -ML xs ML 3H/4c 2 ME 55,900 20.0 2800 (1) 1960 ... 1 * (2) 1870 (3) 1850+ • (4) 1730+ 1900 - El 76,000 25.0 3040 (l) 1000 1950 .. (2) 950 1000 • (3) 960 970

E2 23,000 10.0 2300 (1) 1380 1560 .. (2) 1360 1400

1460 (l) 755 1000 290 270 2.6 16 EPIE3 14,620 10 .. 0 (2) 730 810 310 270 2.4 (3) 720 750 270 270 2.6

E3 33,450 15.0 2230 (1) 1970 2000 870 1000 2.3 (2) 1960 1960 800 840 2.5

0( 80,000 15.5 4500 (1) 3300 100 3.3 16 OHE 1 * • (2) 2400 30 - 8.0 (3) 29oo+ -.. 64 .. 4.5 (4) 3000+ • - • 13 ,ooo 1500 (1} 850 x 280 16/3 OHE 1 * 11.4 3.0 (2} 790 x 280 2.9 diacetate (3) 780 x - 270 2.9 (4) 680 x 230 - 2.9 KE 40,000 1860 (1) 1430+ 1380 16 2 * 17.0 1.0 (2) 1360+ .. 1380 - 0.96

* Crystal1ized by Dr. Hobkirk. Results given are for glucuronide fraction only. + Crystallized as acetates but calcu1ations done for free compound. x Re-crystallized as the diacetate all the way and ca1cu1ations also given for the diacetate.

XS = crystals ML = mother liquor S.A. = cpm/mgm 62.

14 estrone and estradiol-178 fractions did not contain any c. Crystalline carrier, as shown in Table XV, was then added to each of the eight fractions and a series of recrystallizations to constant specifie activity performed.

Such rigorous purification obtained by crystallizing to constant specifie

activity of both crystals and mother liquors is still the best method of

purification known presently. All compounds were crystallized from methanol, except the acetylated ones, which were crystallized from methanol: water mixtures. The recrystal1ization data of the 2 methoxyestrone,

160{-hydroxyestrone, 16/1-hydroxyestrone and 16-ketoestradiol-178 in this experiment is from Dr. Hobkirk and the use of these resulta is gratefully

acknowledged here both for the present experiment and also for the two

following ones. The reason for this is that in the present experiment the

author only used 1/16 of the total four day urine and did not obtain

sufficient counts in the above fractions for repeated recrystallizations although attempts were made; in the following experiments contaminations must have occurred in sorne fractions to explain the results obtained.

After repeated recrystallizations, the 2 methoxyestrone, estrone, 14 estradiol-178 and 16~~hydroxyestrone contained no c, showing that no conversion of 16-ketoestradiol-178 to these compounds occurred. The 3 14 H/ C ratios of estriol and 16-epiestriol are almost equal being 2.5 and

2.6 respective1y. 16-Ketoestradio1-17S therefore is converted to both of these compounds, although not to the same extent as the contribution of 3 H from estradiol-178 is much greater to estriol than to 16-epiestriol.

The 168-hydroxyestrone fraction also contained 14C which is perhaps the most

surprising finding of the whole experiment. 16- Ketoestradio1•17 /3 therefore

is apparently converted to 168·hydroxyestrone through some pathway, the most

1ikely of which is through 16-ketoestrone. The percent conversions to a11

these compounds from estradiol•17/3 and from 16-ketoestradiol-17/3 are shown 63.

together with data from the following experiment for easier comparison

of results in Table XVIII.

Tables XVI and XVII contain the data from subject M who was given 3 14 estradio1-178 and 16-epiestriol; the ratio of H/ c of the injection material here was also 9.2. Again, aliquots of the ether extract, the non•

ketonic and ketonic fractions and of all eight metabolites were counted

and the results shown on Table XVI are calculated for the total four day

urine. 2 Methoxyestrone, estrone, estradiol-17 f3 and 16oC-hydroxyestrone

contained again no 14c, showing the lack of conversion of 16-epiestriol 14 to these compounds. All other fractions contained c. The conversion of

16-epiestriol to estriol and 16-ketoestradio1·17~ was certainly to be expected, but the conversion to 168-hydroxyestrone was again a somewhat

surprising finding; however, this agrees well with the results of the

previous experiment, suggesting maybe a rather selective reduction at

the fi position.

A study of the percent conversions to all metabolites of the injected materials as shown in Table XVIII reveals some interesting observations.

16-Epiestriol, estriol and 16-ketoestradiol•17~ are all readily inter•

converted. The metalolism of l6-ketoestradiol•l78 must occur rapidly

as only 3.9% of it was excreted in unchanged form; it is mainly converted

to estriol to the extent of 19% although some of it (4.6%) is converted to

16-epiestriol. Of the injected 16-epiestrio1 in this experiment, 20% was excreted in an unchanged form suggesting that the metabolism of this

compound is not as rapid as that of 16-ketoestradiol-17 !3. 16-Epiestriol was converted to estrio1 to the extent of 10% but to 16-ketoestradiol-178

only to 1.8%. Both 16-ketoestradio1-178 and 16-epiestriol seem to contribute equal amounts to 168-hydroxyestrone • 0.5% and 0.6% respectively. 64

TABLE XVI

RESULTS OF THE ISOLATION OF EIGHT URINARY ESTROGEN 3 METABOLI1'ES IN SUBJECT M INJECTED WITH ESTRADlOL-17/3 • H AND 16-EPIESTRIOL 14c

3 14 CPM H CPM c 38;14c

Injection Materia1 9,407,000 1,026,000 9.2

Total Ether Extract 4,150,000 396,000

Ketonic Fraction 1,980,000 122,560

Non•Ketonic Fraction 1,864,000 473,440

Ketonic Fraction 2 ME 133,800 1 El 1,536,000 Ring DIX ketols 644,000 ..

Non-I

Ring D 01. Ketols 16 c( OHE 90,200 1 16 BOHEl 26,400 15,000 1. 7 16 KE 2 77 2 ,ooo 161,200 1. 7 65.

TABLE XVII

RECRYSTALLIZATION DATA OF THE EIGHT URINARY ESTROGEN METABOLITES OF SUBJECT M

3 14 S.A. of H s.A. of c Urine CPM for MG of Cale Fraction Cryst Carrier S.A. xs ML xs ML 3H/14c

2 MEl 16,700 15.0 1117 (1) 600 910 • (2) 485 690 (3) 460 460

El 192,000 25.0 7700 (1) 5750 6550 (2) 5100 6150 - (3) 5250 5050 (4) 5000 5200 -

E2 54,200 25.0 2200 (1) 1520 (2) 1680 1640 (3) 1550 1540

16 EPIE 30,200 25.0 1200 (1) 1200 1130 1700 2030 3 (2) 860 910 1480 1760 (3) 770 800 1440 1260 o.s

E3 538,000 10.4 51000 (1) 51500 4850 10.6 (2) 51000 - 4400 11.6 (3) 52000+ 5000 10.4 (4) soooo+ 4900 10.4

161X OHE 11,280 1100 (1) 510 1 10.0 690 (2) 500 560 ..

168 OHE 3,300 15.0 220 (1) 73 x 147 40 1 40 (2) 76 x 100 47 42 (3) 86 x 86 50 52 l. 7

(1) 16 KE 2 * 13,000 11.7 1100 1060 .. 580 1.8 (2) 970 560 1. 7 (3) 860 500 1. 7

N.B. See Table XV for exp1anation of symbols 66.

TABLE XVIII

3 PERCENT CONVERSION OF ESTRADIOL-17 fl - H, 16 KETO· ESTRADIOL·l7B-14c AND 16 EPIESTRIOL-14c TO VARIOUS METABOLITES

Subject B Subject M % Ez % 16 KE2 % E2 % 16 EPIE3 Metabolite Converted Converted Converted Converted

2 MEl 0.6 0.5

El 4.3 • 10.0

E2 2.2 .. 3.0 16 EPIE 1.2 3 4.6 1.5 20.0 E3 5.2 19.2 13.2 10.4

16 o< OHE 0.9 1 0.4 .. 16BOHE 1 0.15 0.5 0.1 0.6 16 KE2 0.45 3.9 0.6 1.8

Total 16.0 28.1 29.3 32.8 67.

The isolation of 16-ketoestrone in experiments of this kind would

indeed be an interesting thing, since this compound is claimed to hold a

key position in the metabo1ism of ali these c-16 substituted estrogens and

could help in explaining the results obtained in 168-hydroxyestrone fractions of both experiments.

The conversion of estradiol-17n to all these metabolites is also

contained in Table XVIII. According to the present studies, estradiol-178 conversion to 2 methoxyestrone is lower than has been reported earlier.

Enge1 (107) has shown this to be about 8% while the author found only about

0.5% conversion. The conversions to estrone and estriol respective1y were

4.3% and 5.2% in subject B and 10.0% and 9.6% in subject M. Both converted estradiol"l78 to 16-epiestriol to almost the same extent, namely 1.2% and

1.5% respectively. Estradiol•l7B conversion to the ring D c{ keto1s was

rather low again, but these results agree very well with those described

in the previous section. 3 The third of these three subjects was injected with estradiol-178- H 14 and testosterone •4 - c. The experiment was performed in an identical manner to the previous two and the data is recorded in Tables XIK and xx.

All the fractions except estriol, when eluted from the column, contained 14 c, but as the recrystallization data shows rather clearly, these counts all disappeared after only one crystallization. Therefore this experiment was quite unsuccessful in showing any conversion of this male hormone to any of the estrogens. Finally, the conversions of estradiol•l7fi to all eight metabolites is shown in Table XXI.

All these conversions were of the same order as found in the two

previous experiments. The conversion to 2 methoxyestrone was higher here, but certainly such individual differences do exist. The conversions to 68.

the ring D~ ketols were sti11 as low as in all the other experiments.

Very sma11 conversions of testosterone to ewtrogens may have been

obscured by the counting techniques used, as all samp1es regardless of 14 whether they contained any c, were counted at settings for a doubly

labelled sample. Tiny amounts of an isotope may easily go undetected

by such a counting method.

TABLE XIX

RESULTS OF THE ISOLATION OF EIGHT URINARY ESTROGEN 3 METABOLITES IN SUBJECT G INJECTED WITH ESTRADIOL-17 B • H AND TESTOSTERONE-14c

3 14 CPM H CPM c

Injection Material 10,470,000 3,922,000

Total Ether Extract 3,411,200 1,105,600

Ketonic Fraction 1,904,000 1,618,000

Non-Ketonic Fraction 970,000 18,000 (Phenolic)

Ketonic Fraction 2 MEl 165,900 1,433,680 0.12

El 1,892,000 104,560 17 .o Ring Do( ketols 495,600 21,840 22.0

Non-Ketonic Fraction E2 431,000 12,000 36.0 16 EPIE 135,000 32,800 3 4.1 E3 906,000 ..

Ring Do< Ketols 16 0( OHE 88,000 1 16 !3 OHE 32,000 1 16 IΠ64,000 2 - 69.

TABLE XX

RECRYSTALLIZATION DATA OF THE EIGHT URINARY ESTROGEN METABOLITES OF SUBJECT G

3 14 S.A. of H S.A. of C Urine CPM for MG of Cale Fraction Cryst Carrier S.A. xs ML xs ML 3H;14c

2 ME 20,740 15.0 1380 (1) 1490 lOO 1 • 14.9 (2) 1400 1400 (3) 1500 1490 - El 153,800 20.0 7690 (1) 4850 7850 600 (2) 4380 5230 • - (3) 4460 4850 - - (4) 4300 4410 - - E2 160,000 26.8 6000 (1) 3800 * (2) 3300 - (3 > 3 7oo+ - (4) 3600+ -.. -

16 EPIE3* 46,000 15.2 3000 (1) 2200 (2) 2000 .. - (3) 2ooo+ .. - - (4) 2000+ - E3 3 7,500 25.0 1500 (1) 1380 1230 • (2) 1600 1700 - - 16 0( OHE 11,600 10.0 1160 (1) 1 730 738 (2) 727 630 16/3 OHE 1 4,100 15.0 164 (1) 132x 171 (2) 112x 113 (3) llO x 111 .. 16 KE 7, 700 2 15.0 515 (1) 360 350 - (2) 330 330 -

N. B. See Table XV for explanation of symbols 70.

TABLE XXI

3 PERCENT CONVERSION OF ESTRADIOL-17 /3 - H AND TESTO­ STERONE·l4c TO VARIOUS ESTROGEN METABOLITES

Subject G Metabolite % E2 Converted % Test. Converted

2 ME 1.8 1 El 6.8 ..

E2 2.4 .. 16 EPΠ0.9 3 - E3 3.2 - 16 o( OREl 0.58 16 /3 OREl 0.13 ..

16 IΠ0.39 2

Total 16.1 .. DISCUSSION 71.

CHAPTER IV

DISCUSSION OF THE RESULTS

It has been noted for a long time that myocardial infarction is rare

in women in the child•bearing age; however, after the menopause, the

incidence of this disease in females is comparable to that in men of all

ages. This observation has led to the suggestion that the lack of estrogens might play an important role in the onset of this disease as the level of

this hormone is quite comparable between men and post•menopausal women but much higher in the pre•menopausal female. Further support for this

observation comes from the work of Bersohn and Oelofse (141, 142) in South

Africa. These authors noted that the male South African Bantu is relatively

immune to coronary artery disease; also, he frequently shows signs of such

estrogenic changes as gynecomastia.

This question of the relationship between estrogens and myocardial

infarction has thus raised much interest and also controversy during the

past years. A difference between the metabolism of estrogens in normal males and those with myocardial infarction has been pointed out by several

investigators (139, 141, 142).

Bauld et. al. (139) administered from 350 • 500 pg of estradiol-178

to both normal males and those with myocardial infarction and studied the

levels of the estrogens excreted in the urine both before and following

such a dose. In certain indices of estrogen metabolism, no significant

difference between the control and myocardial infarction group was found.

The relative amounts (i.e., endogenous levels) of estrone, estradiol-178

and estriol appearing in the urine were almost equal in both groups. Also,

the mean percentages of administered estrogens appearing in the urine as 72.

estriol plus estrone plus estradiol-178 during the four days after the

injection did not differ. Urinary estriol1 estrone as well as estradiol-17B all increased after the injection of the latter hormone. A significant

quantitative difference between the two groups was, however, found. The

proportional increase in urinary estriol to the increase in urinary estrone

and estradiol•l7fi was much greater in patients with previous myocardial

infarction than in the control group.

The question of age as a determining factor was ruled out by them, as

both groups were of about equal age. Similarly, the time after the infarction

did not influence the response; the period from the episode of myocardial

infarction to the beginning of the experiment ranged from one week to two

years.

Differences in behaviour were shown by individual subjects, howeverJ

two of their control cases, after estradiol•l7S administration, excreted estrogens in the proportions of the myocardial infarct group. This makes

the present author wonder whether these two subjects, in the years to come, ever suffered from myocardial infarction; it would have been interesting

if their history could have been followed. In all probability, the change

in estradiol-178 metabolism in this disease is not an inborn error but most

likely manifes~itself at some stage in life, if it does at all; whether it occurs before the infarction or is observed as a result of the infarction is an unanswered question.

Three of the subjects with myocardial infarction metabolised estradiol•l78 in the normal manner. Therefore, myocardial infarction can still occur even

though the conversion of administered estradiol•l7S to estriol is a normal one.

In a later study (140) the same authors attempted to demonstrate a

similar difference in estrogen metabolism between subjects with and without 73.

previous myocardial infarction after the administration of ACTH, chorionic

gonadotropin and testosterone propionate. From this study they concluded

though "that the estrogen metabolic 11defect11 in myocardial infarction can

be demonstrated only when estradiol-17 8 is administered •••• •"

Summarising then, Bauld et. al. (139) could demonstrate a difference

in estrogen metabolism between healthy males and myocardial infarction

patients only in the conversion of exogenously administered estradiol-17a

to estriol.

The work of Bersohn and Oelofse (141, 142) deals exclusively with endogenous metabolism of estrogens. These authors determined the urinary

estrogen levels in two age groups of healthy Europeans, a group with previous myocardial infarction and a group of Bantus. ln the healthy European groups,

the total estrogen output was much higher in the older age group than in the

younger, the increase being found in all three estrogens but being most marked in the estriol fraction. These findings, then, suggest an increase

in estrogen excretion with advancing age, an observation that has not been

made by other workers. Comparing the men with myocardial infarction to the

healthy group of comparable age, the former excreted less total estrogens, estrone and estradiol-17~ than the latter, but the same amount of estriol.

They even excreted less estrone and estradiol•l7B than the younger age

group, but about 2.5 times as much estriol. This then bears out very

favourably the theory that low estrogens, especially low estradiol-17 13 which

is the most active of the naturally occurring estrogens, can be related to myocardial infarction. Also, according to the evidence of Bauld et. al.

(139), there is a trend towards a higher proportion of estriol than estrone

and estradiol-17/3. The total estrogen excretion in the Bantu was higher

than in the healthy European, again of comparable age. Ali three estrogens were elevated but the most noteworthy rise occurred in the estradiol•l7B 74.

fraction. The explanation that the authors offered was that the Bantu is less efficient in metabolizing estradiol•l7n than is the European. The total estrogen excretion of the Bantu was almost equal to the myocardial infarction group; this then certainly cannot explain why the Bantus are relatively immune to heart attacks. The individual metabolites, though, showed variability. Estriol in the Bantu was much lower than the same metabolite in myocardial infarcts, while the greatest increase was in the estradiol-178 fraction. Again, the present autbor wonders what the estrogen excretion of an older group of Bantus, comparable to the older

European group, would have been. If they follow the pattern of healthy

Europeans as shown in Bersohn and Oelofse's (141, 142) study, i.e., an increase in total estrogens with advancing age, then the difference between the myocardial infarct group and the Bantus could have been significant and one might then apply the theory of the protective action of the estrogens in myocardial infarction.

With all this information available then, the present study was undertaken.

It was hoped to establish a more definite answer to this problem of a difference in estrogen metabolism between the two groups.

Three groups of subjects were studied; two control groups of different age and one group with previous myocardial infarction. The first control group consisted of young healthy males and in these only endogenous estrogen levels were determined. The second control group was of the same age as the myocardial infarction group and here the urines were analysed for both endogenous excretion and exogenous excretion of estrogen metabolites.

It is well to bear in mind that the groups studied in the present investigation were quite small, consisting of a total of 12 control subjects and an equal number of patients with myocardial infarction. If an investigation of a much larger group (a few hundred subjects) bad been carried out, it may 75.

well be that significantly different findings would have resulted.

First of all, it must be stated, that there was no apparent difference between the urinary levels of estrogens of the younger and the older groups. Total estrogens were equal as well as estrone, estradiol•l78 and estriol values individually.

Both the older control group and the myocardial infarction group 3 received an intravenous tracer dose of estradiol-178 • H and their urines were handled in an identical manner. As far as is known presently, such tracer doses of radioactive steroids are assumed to be metabolized in an identical manner to the endogenously secreted hormone. The results of this

study can be expressed simply as follows no difference in estrogen metabolism between the two groups was found, either in endogenous levels or in any index of exogenous metabolism. The urinary levels of estrone, estradiol•l7~ and estriol were more or less the same for all groups.

This is in contrast to the findings of Bersohn and Oelofse (141, 142) who used the same method as in the present study. The handling of exogenously administered materia1 was also similar in both groups. In

the study of Bauld et. al. (139), the ratios of endogenous estriol to estrone and endogenous estriol to estrone and estradiol-17n were similar in both groups as they were in this investigation. However, the relative increase in estriol to that in estrone and estradiol•l78 after the injection of estradiol•l7~ was higher in the myocardial infarction group in the study of Bauld et. al. (139). In the present work, the radioactivity excreted in the estriol fraction as compared to the radioactivity excreted in the estrone and estradiol-17B fraction after the administration of isotopically labelled estradiol•l7~ was similar in both groups, although individual subjects showed varied behaviour. Considering yet another interpretation of results, the percent of radioactive estradiol•l78 converted to estrone 76.

and estriol and the percent estradiol-178 excreted unchanged was no different nor was there any trend towards a higher conversion to estriol than to estrone in the subjects with myocardial infarction.

lt is difficult to expla~n the results of Bersohn and Oelofse (141,

142) in comparison to the present work, since the method used was almost the same and the ages of all the groups virtually the same also.

More variables exist between the investigation of Bauld et. al. (139) and the present one, some of which could explain the discrepancies in results. First of all, many investigators (151, 152) have observed that the method of Bauld (74) or modifications of it overestimate the levels of estradiol-17~ and particularly of estriol in the male. This is also evident from the present study; total estrogens were found to be between

8.0 - 9.0 pg/24 hours with estriol around the 4.5 pg level, while Bauld and Givner (74, 144) have reported average levels around 16.0 • 17.0 pg/

24 hours with an average estriol of 10.0 pg/24 hours, and even much higher in individual subjects.

The route of administration as well as the amount of estrogen

~dministered was also quite different. Bauld et. al. (139) gave doses of 350 • 500 ~g of estradiol-17~ dissolved in peanut oil intramuscularly, while the author injected true tracer doses of radioactive estradiol-178 of negligible weight intravenously. Doses as high as the former are certainly far from physiological amounts especially in the male and when the system is so overloaded with a hormone, different routes of metabolism may conceivably come into function due to the saturation of an enzyme or limitation of a cofactor. Tracer doses on the other band, as pointed out earlier, are con­ sidered to be handled in a manner indentical to the endogenous hormone as they by no means contribute any excess weight or presumably physiological activity. The author is aware that controversy does exist as to whether n.

radioactive material is metabolized in an identical manner to endogenous

material, but until conclusive proof is obtained, this assumption will be maintained in the present work.

The relative proportions of estrone, estradiol•l7 /3 and estrtol excreted

in the urine in response to administered estradiol-178 depend upon the amount

given according to Beer and Gallagher (llO, 153). This conclusion was

reached by the above authors with small (250 pg) and large U40 • 350 mgm) 14 doses of estradiol-17 B -16 - C administered to female subjects.. With small

doses (which compare to those given by Bauld et. al. (139) ), estriol was

found to be the main urinary excretion product. This finding could perhaps

partly explain the results of Bauld et. al. (139) even though the small doses

in Beer and Gallagher 1 s (110, 153) studies were given intravenously. With

the larger doses, the principal urinary excretion product was found to be

estrone. May and Stimmel (130), as well as Brown (154) have confirmed this

finding.

The route of steroid administration may well be important in determining

the amount of estrogen catabolism. It will obviously take rouch longer time

for an intramuscular dose to reach the blood stream and be transported to the

sites of metabolism.

There is no doubt that Bauld et. al. (139) found a difference in estrogen metabolism between normal males and those with myocardial infarction. The question arising concerns the significance of such a difference which could

only be demonstrated by injecting large, unphysiological amounts of estradiol-17 8 • Certainly, where estradiol•l7fi was not administered,

there was no difference observed. The authors themselves attribute this to

the fact that such differences may have been obscured by the lack of

sensitivity of the method for values less than three micrograms per day.

This, of course, may also be true to a certain extent, of the present work even though the limits of sensitivity of the Brown technique followed 78.

by the lttrich reaction and fluorimetric readings are much lower. The

smallest amount of estrogens which could be measured with reasonable

accuracy by the present method is about 0.5 pg total estrogens pér day.

The amounts of estrogens found in normal male urine are much higher than

these limits of sensitivity.

Sufficient radioactivity was administered to each subject, however,

to allow for an accurate determination of the isotope content of estrone,

estradiol-17 !3 and estriol fractions from the urines.

Givner et. al. (140) also suggested that an examination of the

precursors of the triols • 16({-hydroxyestrone and 16/3·hydroxyestrone •

could reveal more clearly this "defect" in estrogen.metaboUsm. These metabolites have been studied by the author (these results are described

in Section II c, D of "Results"). The conversion to these compounds from

administered radioactive estradiol•l7B seems to be very low • indeed lower

than bad been anticipated from previous data. It is too early to draw any

definite conclusions regarding the metabolism of 16~-hydroxyestrone and

168-hydroxyestrone in normal males and those with previous myocardial

infarction from this investigation, but there seems to be no detectable

difference present.

The author, by presenting this work, does not by any means wish to

deny the existance of a relationship between estrogen metabolism and the

incidence of myocardial infarction. This study is presented to show that with a more sensitive technique perhaps and the use of tracer isotopes, such a relationship could not be illustrated. If a difference does exist, in the

author's opinion at least, it is not of such a simple nature, involving maybe more than the three classic estrogens, their relative tates of conversion as well as the enzyme systems concerned in the interconversions of the estrogens.

Since their discovery by Marrian and his colleagues in the 1950's, the 79.

ring D~ keto1s have aroused much interest and speculation. Their existence in human pregnancy urine has been known for some time and they have been shown to be quantitatively important metabolites in such urines (150}.

In vitro studies have contributed much information regarding the metabo1ism of these three compounds. The name of Breuer (94) must not go unmentioned in this respect. This author incubated 16~-hydroxyestrone with human

1iver s1ices and iso1ated estriol as the main metabolite; when he incubated

16~hydroxyestrone in the same system the main metabolite isolated was

16-epiestrio1. In addition, 16~·0Hestrone gave rise to 17-epiestrio1 and

168-hydroxyestrone to 16, 17-epiestrio1. King (lOO) incubated estrio1 with rat kidney homogenates in the presence of NAD and NADP and found that 16-keto• estradio1-17B and 16-epiestriol were produced in 20% and 1% yie1d respective1y; the incubation of 16-epiestriol in the same system gave rise to 5% 16-keto• estradio1•178 and 1% estriol. From these in vitro studies it was then readily concluded that 16~·hydroxyestrone and 168-hydroxyestrone were precursors of the epimeric estriols, at 1east in in vitro systems; also estriol, 16-epiestrio1 and 16-ketoestradiol-178 are readi1y interconverted compounds and that 16-ketoestradiol•17B holds a central position among these three. The possible position of 16-ketoestrone as a key substance among all the CM16 substituted estrogens was also shown by Breuer (32) in an in vitro experiment. After the incubation of this latter compound with human liver slices, he and his associates, iso1ated a11 the C-16 substituted estrogens -

16~-hydroxyestrone, 16B·hydroxyestrone, 16-ketoestradiol•l7P , estriol,

16-epiestriol, 17-epiestriol and 16, 17-epiestriol. This experiment suggested that the ring D~ketols may be formed from estrone or estradiol-178 via such an intermediate as 16~ketoestrone.

The endogenous ring D~ ketols have previously been measured in the non­ pregnant human by Hobkirk (155) and as metabolites of radioactive estrogens 80.

by Levitz, Spitzer, and Twomb1y (98), by Brown, Fishman and Ga11agher (20) and Nocke et. al. (95), also in non-pregnant fema1es and males. Hobkirk

(155) measured these compounds in terms of their reduction products, i.e., as estriol from 161X.·hydroxyestrone and 16-epiestrio1 from 16/3-hydroxyestrone and 16-ketoestradio1-17~. Brown, Fishman and Gal1agher (20) reported the isolation of radioactive 16/3-hydroxyestrone from the urine of a woman who 14 had received an injection of estradio1-178 -16 - c. The studies of Levitz,

Spitzer and Twombly (98) dealt to a greater extent with 16-ketoestradiol-17 f3.

Even before the isolation of this compound from human pregnancy urine by

Layne and Marrian (18, 19) the former authors reported on the detection of

16-ketoestradio1•17B in human urine after the injection of estradiol-1713·16 _14c.

In another experiment Levitz and his co•workers (99) injected estriol-16

.. 14c into humans and found radioactive 16-ketoestradiol•l7B and 16-epiestriol.

Nocke et. al. (95), after the injection of 16~-hydroxyestrone into a man, were able to isolate estriol as weil as l~epiestriol in the ratio of 100:1.8 from the urine; after the injection of 16lrhydroxyestrone, 16-epiestrio1 and 16, 17-epiestriol were obtained in the ratio of 100:6.3

These findings then are in complete agreement with those of King (100) and Breuer (94) for in vitro studies. Since 16-ketoestradiol-17,8 itself is reduced to estriol and 16-epiestriol in vitro by human liver tissue (94) as we11 as in vivo by man (155 and present study), the interconversion of estriol and 16-epiestriol can be exp1ained by oxido reduction via 16-keto­ estradio1 .. 17 13 •

The results of the present investigation are in agreement in some respects with the information obtained previously regarding the ring DrA ketols.

In the present preliminary investigation on pregnancy urine, 16X-hydroxy- 81.

estrone emerged as the main metabolite of the ring D~ ketols. As this compound is assumed to be the precursor of estriol this finding was to be expected, since estriol is the most abondant of the estrogen metabolites in human pregnancy. The compound second in quantitative significance in this series was found to be 16-ketoestradiol-17 8 , it being equal to about one half of 16ol.·hydroxyestrone in this subject. This leaves 16 8-hydroxye strone as the minor of the three metabolites. lt is difficult to say how true this value of 0.09 mgm for 16/3-hydroxyestrone is, as this compound is extremely unstable, rearranging rather readily to 16-ketoestradiol-178 even upon standing.

Proof of the identity of these compounds was achieved by showing that the reduction product of 16~-hydroxyestrone was mainly estriol and the reduction products of 16 /3-hydroxyestrone and 16- ke toe stradiol .. I7/3 mainly

16- epie striol.

The determination of the endogenous leve1s of these three estrogen metabolites in male urine revealed them as rather lower than had been shown previously. Hobkirk (155) found total ring D~ ketols/24 hours to be equal to about 5.6 pg while in the present study this figure was 2.03 pg. As already mentioned in the results, the age of the subjects is unlikely to have made such a difference in the results obtained. The most reasonable explanation for this discrepancy is that either a more extensive purification of these compounds was achieved in the author 1 s study by means of the toluene/propylene glycol chromatographie system or that large !osses of these compounds occurred on this system. The recovery of pure standards from this chromatograph was about 70%, but even when applying this correction factor the results still do not appear equal. According to the present results, the total ring D~ ketols appear to be lower than estrone in male urine. 82.

An explanation for this may be that possibly the ring D~ ketols are not hydrolysed as easily by enzyme preparations as is estrone. The enzyme used in the present study was a lyophilized preparation from the snail Helix

Pomatia, which contained both~ glucuronidase as well as sulfatase activity.

This question of differtial hydrolysis of the compounds involved by any enzyme is difficult to prove as one 1acks a standard procedure of comparison; acid hydrolysis cannot be used in the studies with ring D~ketols, as these compounds are unstable to hot acid. Therefore, the proportion of ring D~ ketols hydrolysed to that of estrone by the enzyme may be responsible for the results found.

Undoubtedly also, much greater !osses of the ring D~ketols occurred in the present study than in that of Hobkirk (155) or the estrone fraction.

The main metabolite of the three in male urine in the present study was

16-ketoestradiol-178 which was found to be equal to the sum of the other two; however, individua1 differences did occur. The conversion of estradiol•l7fito these compounds was also lower than had been shown previously.

(155). Levitz et. al. (98) studied the conversion of estradiol•l7n to

16·ketoestradiol•l78 and showed this to be equal to 1.6%; their figure was ca1culated as the percent of radioactivity excreted in the first 24 hour urine sarnple as 16-ketoestradiol-17$ following the injection of 14 estradiol-17~ -16 c. The conversion in the present study was found to be

0.46%, but this is expressed as the percent of 16-ketoestradiol•l7S isolated after hydrolysis from a pooled four-day urine sample following the injection 3 of estradio1•17~ •6, 7• H. Thus, the percent conversions from these two experimenta are hardly comparable. The conversion to 16~~hydroxyestrone was of the same order (0.48%) and to 16~-hydroxyestrone only 0.16%. However, as these percent conversions were determined in the same urine samples as the endogenous levels; the same arguments for low values can be used as discussed previously. 83.

A more detailed study of the intermediary metabolism of the estrogens was carried out in two subjects fo11owing the administration of estradiol-17~ 3 H 14 14 and either 16-ketoestradio1-17S - c or 16-epiestriol- c. 16-Ketoestradiol-17B

in this experiment, was converted, in addition to estriol and 16-epiestrio1

to 16 /3-hydroxyestrone but not to 16 oC.-hydroxyestrone. Breuer et. al. (32) have shown the in vitro conversion of 16-ketoestrone to ail the C-16 substituted estrogens. In vivo (105), he c1aims to have shown the conversion of 16-keto• estradiol-17~ to 16-ketoestrone fo11owing the injection of the former compound. If 16-ketoestradiol-17~ is at 1east partially oxidized to

16-ketoestrone, ~hich in turn has been shown to be converted to al1 the C·16

substituted estrogens, it is difficu1t to exp1ain this lack of conversion to

16 0\·hydroxyestrone in our experiment. A study of our data would almost

indicate that 16(1(.-hydroxyestrone arises solely by the 16 o<-hydroxylation of estrone while 16/3-hydroxyestrone arises by a pathway via the triols and

16-ketoestrone, and perhaps partially or not at all by the 16 /J-hydroxy1ation of estrone. It is too ear1y to draw any definite conclusion from just one experiment of this kind although the triols have previously been suggested

as the precursors to 16~hydroxyestrone and 16~hydroxyestrone by Breuer et. al. (32), and the findings from the subject who was injected with 16-epiestriol-

14c do not seem contradictory either. In this case, 16-epiestriol (which is

readily interconverted with 16-ketoestradiol-17/3) was also converted to

16/3-hydroxye strone as well as 16-ketoestradio1-17/3 and e striol but again not

to 16 ci.-hydroxye strone. In his experiments, Hobkirk (155) also found unlike ly

the production of 16 o<.-bydroxyestrone from the triols.

The conversion of 16- ketoestradiol .. l7 B is greater to estriol than to

16-epiestriol in vivat a finding which bas been reported before (155) and

the conversion of 16-epiestriol to estriol is about the same or greater

than that of 16-ketoestradiol-17 B to estriol. 84.

The conversion of estradiol-17/3 to 2 methoxyestrone, as found in these experiments, was lower than that reported earlier by other investigators.

Engel et. al. (107) has shown that the radioactivity in the 2 methoxy fraction 14 was about equal to 8% of the administered estradiol-178 -16 - c. Hobkirk and Nilsen (157) detected no rqdioactive 2 methoxyestrone in one subject and 6% in another following the administration of 2 methoxyestrone itself.

According to the work of Fishman et. al. (131), the level of thyroid hormone can produce changes in regard to the 2 methoxylation of estrogens. Elevated thyroid hormone levels tend to decrease the levels of the oxidation of C-16 in ring D decreasing estriol while at the same time increasing the oxidation of C·2 in aromatic ring A leading to a large increase in 2 methoxyestrone. lt is well to bear in mind this observation when studying the conversions of various compounds to 2 methoxyestrone. The subjects in this investigation showed normal thyroid function.

The conversion of estradiol-178 to both estrone and estriol appears to be almost equal, although individual subjects showed different percent conversions ranging from 4.0% to 10%. The conversion of estradiol-178 to ring D ~ ketols found in the se two subjects was very much of the same order as found in the previous group discussed earlier and will not be dealt with to a great extent. ln two of the subjects, a predominant conversion to

16~-hydroxyestrone was found but in the third subject the 16-ketoestradiol-178 metabolite was highest.

Lastly, the conversion of the male hormone, testosterone, to estrogens was investigated. This conversion has been shown to occur with certainty in ovarian and testicular tissue following the in vitro incubation of these 14 tissues with testosterone"4 • c. Baggett et. al. (50) incubated slices of a normal ovary from a 34 year old woman with radioactive testosterone and isolated radioactive estradiol-17 B. lvotiz et. al. (51) showed the same 85.

reaction with ovarian slices removed twenty years after the menopause; in addition to estradiol-178, he also was able to find estrone and estriol.

Later, the former author also showed this conversion in homogenates of horse testes and slices of human feminizing adrenocortical carcinoma and human placenta. The conversion of testosterone to estrogens in vivo has been shown in the pregnant mare by Heard et. al. (49). In vivo conversion has also been shown by West and associates (57); they injected testosterone into a woman who had been both ovarectomized and adrenalectomized and showed the conversion of this steroid to estrone and estradiol-178. Such a conversion in their study must then have occurred in non-endocrine tissue.

Givner et. al. (140), in their studies on estrogen excretion in normal male subjects and those with myocardial infarctions, injected 200 mgm of testosterone propionate to their subjects. Following such large doses of this male hormone, an increased level of urinary estrone, estradiolwl7$ and estriol was found.

Recently, Morse and co•workers (158) have also studied the inter• conversion of these hormones and have shown the transformation of testosterone to estrogens to be about 0.3% in the human.

The results of the present investigation, however, contradict all the foregoing in vivo evidence as no conversion from testosterone was obtained.

Certainly, the interconversion of these compounds can occur in vitro and the present study does not by any means try to exclude this reaction. In vitro and in vivo work is not always comparable though. It seems that perhaps Morse et. al. (158) did not apply a rigorous enough purification such as was done by the author by repeated crystallizations to constant specifie activity of all the estrogen metabolites. However, tiny conversions 86.

of the male hormone to the female ones may still occur and may be beyond the limits of detection of the method in use presently. 87.

SUMMARY

1. Estrogen metabolism in subjects with and without previous myocardial infarction bas been investigated.

6 3 2. Tracer doses of about 6.0 x 10 cpm of estradio1-17B 6, 7 • H were injected intravenously to the subjects and the urinary excretion of estrone, estradio1-178 and estrio1 was determined by the method of Brown et.al.

3. Contrary to previous evidence, no difference in either endogenous or exogenous metabolism of the three classical estrogens between a control group and one with previous myocardial infarction was found in the present study.

4. Urinary 1evels of the ring DO\ ketols, 16 oe,-hydroxyestrone, 16/3-hydroxy- estrone and 16·ketoestradiol•l7~ were a1so determined in a group of males.

5. The average amount of 16-ketoestradio1-17B excreted was 1.00 pg/24 hours, 16~-hydroxyestrone 0.73 pg/24 hours and 168-hydroxyestrone 0.30 pg/24 hours with a total of 2.03 pg/24 hours.

6. The excretion of total ring D~ ketols (2.03 pg/24 hours) was compared to the excretion of estrone (3.16 pg/24 hours) in the same subjects and was found to be lower than that of estrone.

7. Each subject in this group was also injected with about 6.0 x 10 6 cpm 3 of estradiol-178 -6, 7 • H and the percent conversion of this compound to the ring De< ketols was determined.

8. The average percent conversion to 16·ketoestradiol-178 , 16~-hydroxy• estrone and 168rhydroxyestrone were respectively 0.46, 0.48 and 0.16 with an average total conversion of 1.10%. 88.

9. ln addition, the metabolism of eight urinary metabolites of the estrogens including 2 methoxyestrone, estrone, estradiol-178, 16-epiestriol, estriol, 16-ketoestradiol-17N, 16~·hydroxyestrone and 16~·hydroxyestrone was studied. 3 10. Three subjects were injected with estradiol-178 -6, 7 - H and either 14 14 14 16·ketoestradio1·17n -16 - c, 16-epiestrio1-16 - c, or testosterone-4 - c.

11. 16-Ketoestradio1•17B was converted to estriol (19.2%), 16-epiestriol

(4.6%) and 168-hydroxyestrone (0.5%) but not to 16~-hydroxyestrone nor any of the other metabolites.

12. 16-epiestriol was similarly converted to estriol (10.4%), 16-keto• estradiol•17~ (1.8%) and 16S·hydroxyestrone (0.6%).

13. Estradiol-17~ was converted to all the metabolites studied and the percent conversions were on an average 0.5% to 2 methoxyestrone, 7.0% to estrone, 1.5% to 16-epiestrio1, 9.0% to estriol, 0.7% to 16~-hydroxyestrone,

0.1% to 168·hydroxyestrone and 0.5% to 16-ketoestradiol-178.

14. From this it was concluded that 16~·hydroxyestrone and 16/.5flydroxy• estrone may arise by separate pathways, the former from estrone and the latter perhaps on1y via the triols and 16-ketoestrone.

15. The conversion of the male hormone testosterone to any of the estrogen metabolites studied could not be detected. 89.

BIBLIOGRAPHY

(1) KNAUER, E. Arch. F. Gynakol. 60, 322, 1900. (2) FELLNER, O. o. Arch. F. Gynako1. 100, 641, 1913.

(3) ALLEN, E., DOISY, E. A. J. Am. Med. Assoc. 81, 819, 1923. (4) ASCHHEIM, s., ZONDEK, B. Klin. Wschr. 6, 1322, 1927.

(5) DOISY, E. A., VELER, C. D., THAYER, S.A. Am. J. Phys. 90, 329, 1929.

(6) BUTENANDT, A. Naturwissenschaften 17, 879, 1929.

(7) DINGEMANSE, E., de JOHGH, S. E., KOBER, S., LAQUER, E. Dtsch. Med. Wschr. 56, 301, 1930.

(8) MAR.RIAN, G. F. Biochem. J. 23, 1090, 1929.

(9) MAR.RIAN, G. F. Biochem. J. 23, 1233, 1929.

(10) MARRIAN, G. F. Biochem. J. 24, 435, 1930.

(11) MARRIAN, G. F. Biochem. J .. 24,. 1021,. 1930.

(12) DOISY, E. A., THAYER, S. A. J. Biol. Chem. 91, 641, 1931.

(13) COOK, J. W., GIRARD, A. Nature 133' 3 77' 1934. (14) MACCORQUOOALE, D. W., THAYER, S. A., DOISY, E. A. J. Biol. Chem. 115, 435, 1936.

(15) HUFFMAN, M. N., MACCORQUODAL&, D. W., THAYER, S. A., DOISY, E. A., SMITH, O. W., SMITH, S. S. J. Biol. Chem. 134, 591, 1940.

(16) MARRIAN, G. F ., BAULD, W. S. Biochem. J. 59, 136, 1955.

(17) MARRIAN, G. F., LOKE, K. H., WATSON, E. J. D., PANATONNI, M. Biochem. J. 66, 60, 1957. 90.

(18) LAYNE, O.S., MARRIAN, G. F. Nature 182, 50, 1958.

(19) LAYNE, O. S., MARRIAN, G. F. Biochem. J. 70, 244, 1958.

(20) BROWN, B. T., FISHMAN, J., GALLAGHER, T. F. Nature 182, 50, 1958.

(21) KRACHY, S., GALLAGHER, T. F. J. Am. Chem. Soc. 79, 754, 1957.

(22) KRACHY, S., GALLAG~ T. F. J. Biol. Chem. 229, 519, 1957.

(23) FISHMAN, J., GALLAGHER, T. F. Arch. Biochem. Biophys. 77, 511, 1958.

(24) LOKE, K. H., MARRIAN, G. F. Biochem. Biophys. Acta. 27, 213, 1958.

(25) FRANSOEN, V. A. Acta. Endocr. 31, 603, 1959. (26) MUELLER, G. c., RUMNEY, G. J. Am. Chem. Soc. 79, 1004, 1957.

(27) BREUER, H., NOCKE, L., KNUPPEN, R. Hoppe-Seyler 1 s z. Physiol. Chem. 315, 72, 1959. (28) LOKE, K. H., WATSON, E. J. o., MARRIAN, G. F. Biochem. Biophys. Acta. 26, 230, 1957.

(29) KNUPPEN, R., BREUER, H. Biochem. Biophys. Acta. 58, 147, 1959.

(30) LOKE, K. H., MARRIAN, G. F., JOHNSON, t.J'. S., MEYER, W. L., CAMERON, O. D. Biochem. Biophys. Acta. 28, 214, 1958.

(31) LOKE, K. H., MARRIAN, G. F., WATSON, E. J. D. Biochem. J. 71, 43, 1959.

(32) BREUER, H., KNUPPEN, R., PANGELS, G. Acta. Endocr. 30, 247, 1959.

(33) BREUER, H., KNUPPEN, R., NOCKE, L. Biochem. J. 71, 26P, 1959.

(34) BREUER, H., KNUPPEN, R., PANGELS, G. Hoppe-Seyler's z. Physiol. Chem. 321, 57, 1960. (35) BROWN, J. B. Biochem. J. 60, 185, 1955. (36) BAULD, W. s. Biochem. J. 63, 488, 1956. 91.

(37) ITTRICH, G. Hoppe-Seylers z. Physio1. Chem. 312, 1, 1958. (38) DINGEMANSE, E. Nature 141, 927, 1938.

(39) BEALL, D. Nature 144, 76, 1939.

(40) HEARD, R.D.H., JACOBS, R., O'DONNELL, U.J., PERON, G.F., SAI<'FRAN, J .C., SOLOMON, S.S., THOMPSON, L.M., WILLOUGHBY, H., YATER, C.H. Rec. Prog. Horm. Res. 9, 383, 1954.

(41) RABINOWITZ, J. L., DOWBEN, R. M. Biochem. Biophys. Acta 16, 96, 1955.

(42) RYAN, K. J., SMITH, O. W. J. Biol. Chem. 236, 705, 1961.

(43) RYAN, K. J., SMITH, O. W. J. Biol. Chem. 236, 2204, 1961.

(44) SOLOMON, S. S., VAN DE WIELE, R., LIEBERMAN, S. J. Am. Chem. Soc. 78, 5453, 1956.

(45) RYAN, K. J., SMITH, O. W. J. Biol. Chem. 236, 710, 1961. (46) SMITH, O. w., RYAN, K. J. Endocrinology · 69, ·869, 1961.

(47) RYAN, K. J. J. Biol. Chem. 234, 268, 1959.

(48) FIESER, L. F. "The Chemistry of Natural Products Related to Phenanthrene" p. 252. Reinhold, New York 1936.

(49) HEARD, R.D.H., JELLINCK, P. H., O'DONNELL, U. J. Endocrinolody 57, 200, 1955.

(50) BAGGETT, B., ENGEL, L.L., SAVARD, K., DORFMAN, R. 1. J. Biol. Chem. 221, 931, 1956.

(51) WOTIZ, H. H., DAVIS, J. W., LEMON, H. M., GUT, M. J. Biol. Chem. 222, 487, 1956.

(52) BAGŒTT, B., ENGEL, L.L., BAIDERAS, L., LANMAN, G., SAVARD, K., DORFMAN, R. I. Endocr. 64, 600, 1959.

(53) RYAN, K. J. Biochem. Biophys. Acta 27, 658, 1958.

(54) MEYER, A. S. Experimentia 11, 99, 1955. 92.

(55) MEYER, A. S. Biochem. Biophys. Acta 17, 441, 1955.

(56) HAYANO, M., LONGCHAMPT, J., KELLY, W., GUAL, c., DORF~N, R. I. Acta Endocr. Suppl. 51, 699, 1960.

(57) WEST, C. D., DAMAST, B. L., SARRO, S. D., PEARSON, O. H. J. Biol. Chem. 218, 409, 1956.

(58) HIE LAND, H. , STRAUB, W. , DORlï'MULLER, T• z. Physiol. Chem. 186, 97, 1929.

(59) MARRIAN, G. F. Biochem. J. 24, 435, 1930.

(60) KOBER, S. Biochem. z. 239, 209, 1931. (61) BROWN, J. B. in G. E. w. Wolstenholme, Ed. CIBA Foundation Coloquia on Endocrinology, Vol. II, J. and A. Churchill Ltd., London, 1952, P• 132.

(62) BROWN, J. B. J. Endocr. 8, 196, 1952.

(63) BAULD, W. S. Biochem. J. 56, 426, 1954.

(64) ALLEN, W. W. J. Clin. Endocr. 10, 71, 1960.

{65) ITTRICH, G. Hoppe•Sey1ers z. Physio1. Chem. 312, 1, 1958. (66) ITTRICH, G. Acta Endocr. 35, 34, 1960.

(67) COHEN, S. L.. , MARRIAN, G. F. Biochem. J. 28, 1603, 1934.

(68) GIRARD, A., SANOULESCO, G. Helv. Chim. Acta 19, 1095, 1936.

{69) ENGEL, L.L. Rec. Prog. Horm. Res. 5, 335, 1950.

(70) ENGEL, L.L., SLAUNWHITE, W.R. Jr., CARTER, P., NATHANSON, I. T. J. Biol. Chem. 185, 225, 1950.

(71) BROWN, J. B. Biochem. J. 60, 185, 1955.

{72) BROWN, J. B., BULBROOK, R. D., GREENWOOD, F. C. J. Endocr. 16, 41, 1957. 93.

(73) BAULD, W. S. Bi oc hem. J. 59, 294, 1955.

(74) BAULD, W. S. Biochem. J. 63, 488, 1956. (75) ALBERT, s.' HEARD, R.D.H., LEBLOND, C. P., SAFFRAN, J. E. J. Biol. Chem. 177' 24 7' 1949. (76) PEARLMAN, W. H., PEARLMAN, M.R .. J., RAKOFF, A. E. J. Biol. Chem. 209, 803, 1954.

(77) BUSH, I. E. Biochem. J. 50, 370, 1951.

(78) BURTON, R. B., ZAFFARONI, A., KEUTMAN, H. E. J. Biol. Chem. 188, 763, 1951.

(79) PINCUS, G., PEARLMAN, W. H. Vitamins and Hormones 1, 326, 1943.

(80) DOISY, E. A., THAYER, S. A., VAN BRUGGEN, J. T. Fed. Proc. 1, 202, 1942.

(81) HEARD, R.D.H. Rec. Prog. Horm. Res. 4, 25, 1949.

(82) HEARD, R.D.H., HOFFMAN, M.M. J. Biol. Chem. 141, 329, 1941.

(83) LANGER, L. J., ENGEL, L.L. J. Biol. Chem. 233, 583, 1958.

(84) BROWN, J. B. J. Endocr. 16, 202, 1957. (85) FISHMAN, J., BRADLOW, H. L., GALLAGHER, T. F. J. Biol. Chem. 235, 3104, 1960.

(86) PEARLMAN, W. H., PINCUS, G. J. Biol. Chem. 144, 569, 1942. (87) SCHILLER, J., PINCUS, G. Arch. Biochem. 2, 317, 1943.

(88) FISHMAN, J., BRADLOW, H. L., ZUMOFF, B., HELLMAN, L., GALLAGHER, T.F. Acta Endocr. 37, 57, 1961.

(89) ENGEL, L.L., BAGGETT, B., HALLA, M. Biochem. Biophys. Acta 30, 435, 1958.

(90) BREUER, H., KNUPPEN, R., ORTLEPP, R., PANGELS, G., PUCK, A. Biochem. Biophys. Acta 40, 560, 1960. 94.

(91) MARRIAN, G. F., BAULD, W. S. Biochem. J. 58, 35, 1954.

(92) MARRIAN, G. F., WATSON, E.J.D., PANATTONI, M. Biochem. J. 65, 12, 1957.

(93) BROWN, J. B., MARRIAN, G. F. J. Endocr. 15, 307, 1957.

(94) BREUER, H., NOCI

(95) NOCKE, W., BREUER, H., KNUPPEN, R., Acta Endocr. 36, 393, 1961.

(96) MlGEON, C. J. J. Clin. Endocr. & Met. 13, 674, 1953.

(97) WATSON, E.J.D., MARRIAN, G. F. Biochem. J. 61, 241, 1955.

(98) LEVITZ, M., SPITZER, J. R., TWOMBLY, G. H. J. Biol. Chem. 222, 981, 1956.

(99) LEVITZ, M., SPITZER, J. R., TWOMBLY, G. H. J. Biol. Chem. 231, 787, 1958. (lOO) KING, R.J.B. Biochem. J. 76, 7p, 1960.

(101) BREUER, H., NOCKE, W. Biochem. Biophys. Acta 36, 271, 1959.

(102) MARRIAN, G. F. Harvey Lectures, pp. 37-54 Williams & Williams & Co., Baltimore, 1938·39. (103) SLAUNWHITE, W. R., SANDBERG, A. A. Arch. Biochem. Biophys. 63, 478, 1956. (104) MIGEON, C. J. J. Clin. lnvest. 38, 619, 1959. (105) BREUER, H. To be published. Quoted in Vitamines and Hormones 20, 307, 1962.

(106) BREUER, H., VOGEL, W., KNUPPEN, R. Hoppe-Sey1ers z. Physiol. Chem. 327, 217, 1962.

(107) ENGEL, L. L., BAGGETT, B., CARTER, P. Endocr. 61, 113, 1957.

(108) MARRIAN, G. F., SNEDDON, A. Biochem. J. 74, 430, 1960. 95.

(109) VALCOURT, A. J., THAYER, S. A., DOISY, E. A., Jr., ELLIOTT, W. H., DOISY, E. A. Endocr. 57, 692, 1955.

(110) BEER, Ch. T., GALLAGHER, T. F. J. Biol. Chem. 214, 335, 1955.

(111) JELLINCK, P. H. Biochem. J. 71, 665, 1959.

(112) BREUER, H. Vit. and Horm. 20, 320, 1962.

(113) COHEN, S. L., MARRIAN, G. F. Biochem. J. 30, 57, 1936.

(114) CARPENTER, J.G.D., KELLIE, A. E. Biochem. J. 78, 1p, 1961.

(115) SCHACHTER, B., MARRIAN, G. F. J. Biol. Chem. 126, 663, 1938.

(116) MCKENNA, J., MENINI, E., NORYMBERSKI, J. K. Biochem. J. 79, 11p, 1961.

(117) ADLERCREUTZ, H., BELING, C. G. Persona! Comm. 1961.

(118) PURDY, R. H., ENGEL, L. L., ONCLEY, J. L. J. Biol. Chem. 236, 1043, 1961.

(119) COHEN, H., BATES, R. W. Endocr. 45, 86, 1949.

(120) STRAW, R. F., KATZMAN, P. A., DOISY, E. A. Endocr. 57, 87, 1955.

(121) RAKOKOFF, A. E., PASCHKIS, K. E., CANTAROW, A. Amer. J. Obstet, Gynec. 46, 856, 1943.

(122) GLASS, S. J., EDMONDSON, H. A., SOLL, S. N. Endocr. 27, 748, 1940.

(123) GLASS, S. J., EDMONDSON, H. A., SOLL, S. N. J. Clin. Endocr. 4, 54, 1944.

(124) CAMERON, C. B. J. Endocr. 15 ' 199' 19 57. (125) LYNGBYE, J., MOGENSON, E. F. Acta Endocr. 36, 350, 1961.

(126) BROWN, J. B., CREAN, G. P., GINSBURG, J. Gvt 5, 56, 1964. 96.

(127) BROWN, J. B. In ''Endocrine Aspects of Breast Cancer11 (A. R. Currie, Ed.) pp. 197-208, Livingstone, Edinburgh and London, 1958.

(128) BREUER, H., NOCKE, L. Acta Endocr. 31, 69, 1959.

(129) DICZFALUSY, E., LUFT• R. Acta Endocr. 9, 327, 1952.

(130) MAY, J. A., STIMMEL, B. F. J. Uro1. 59, 396, 1948.

(131) FISHMAN, J., HELLMAN, L., ZUMOFF, B., GALLAGHER, T. F. J. Clin. Endocr. Met. 22, 389, 1962.

(132) OLIVER, M. F., BOYD, G. S. Minne·sota Med. 38, 794, 1955. (133) ALDERSBERG, D., SCHAEFER, L. A., STEINBERG, A. G., WANG, C. J. Am. Med. Assoc. 162, 619, 1956.

(134) SCHLESINGER, M. J., SOLL, P. M. Arch. Patho1. 32, 178, 1941.

(135) GOFMAN, J. W., JONES, H. B., STRISOWER, B., TAMPLIN, A. R. Circulation 14, 714, 1956.

(136) LEWIS, L. A., OLMSTED, F., PAGE, I. H., LAt•JRY, E • Y. , MANN, G. v., STARE, F. J., HANIG, M., LAUFFER, M. A., MOORE, F. E. Circulation 14, 720, 1956.

( 137) OLIVER, M. F ., BOYD, G. S. Clin. Sc. 12 , 21 7 , 19 53 •

(138) OLIVER, M. F., BOYD, G. S. Am. Heart J. 47, 348, 1954.

(139) BAULD, W. S., GIVNER, M. L., MILNE, I. G. Can. J. Biochem. Physiol. 35, 1277, 1957.

(140) GIVNER, M. L., BAULD, W. s., HALE, T., VAGI, K., NILSEN, M. J. Clin. Endocr. Met. 20, 665, 1960.

(141) BERSOHN, I., OELOFSE, P.J. s. African Med. J. 31, 1174, 1957.

(142) BERSOHN, I., OELOFSE, P. J. s. African Med. J. 32, 979, 1958. (143) BAULD, w. s., GREENWAY, R. M. Methods of Biochem. Anal. 5, 33 7, 19 57.

(144) GIVNER, M. L., BAULD, W. S., VAGI, K. Biochem. J. 77, 400, 1960. 97.

(145) LISBOA, B. P., DICZFALUSY, E. Acta Endocr. 40, 60, 1962.

(146) DICZFALUSY, E., MUNSTERMANN, A. M. Acta Endocr. 32, 195, 1959.

(147) OKITA, G. T., KABARA, J. J., RICHARDSON, F., LEROY, G. V. Nucleonic 15, 111, 1957.

(148) TALALAY, P., FISHMAN, t-1. H., HUGGINS, C. J. Biol. Chem. 166, 757, 1946.

(149) BROWN, J. B. quoted by J. A. LORAINE in The Clinical Application of Hormone Assay, Loraine, J. A., Livingstone, Edinburgh, p 180, 1958.

(150) HOBKIRK, R., NILSEN, M. J. Clin. Endocr. Met. 22, 134, 1962.

(151) MARRIAN, G. F. Mem. Soc. Endocr. 3, 48, 1955.

(152) HOBKIRK, R., NILSEN, M. Steroids 3:4, 453, 1964.

(153) BEER, C. T., GALLAGHER, T. F. J. Biol. Chem. 214, 351, 1955.

(154) BROWN, J. B. Mem. Soc. Endocr. 3, 1, 1955.

{155) HOBKIRK, R. J. Clin. Endocr. Met. 23 , 2 79 , 1963.

(156) BELING, C. G., BREUER, J. A., BREUER, H. Acta Endocr. 43, 87, 1963.

(157) HOBKIRK, R., NILSEN, M. J. Clin. Endocr. Met. 23' 2 74, 1963. {158) AHMAD, N., MORSE, w. I. Can. J. Biochem. 43, 25, 1965. •