ESTROGEN METABOLISM 1N HUMAN SUBJECTS

A Thesis by Morry Givner

Subrnitted to the Faculty of Graduate Studies and

Research in partial fulfillment of the Requirements for the degree of Master of Science.

McGill University August 1956. Montreal, Quebec ACKNOWLEDGEMENTS

Grateful acknowledgement is expressed to my research director, Dr. W.S. Bauld for encouragement and supervision during this course of study.

The author wishes to express his gratitude to

Dr. 1. G. Milne of the McGill University Clinic of The Montreal

General Hospital for his co-operation in the selection of subjects and his many valuable suggestions.

To Dr. E. H. Bensley, 1 wish to express my appreciation for his valuable suggestions.

Finally, thanks are offered to Mrs. A. Alfh.eim for her excellent technical assistance, which was neoessary for this work. TABLE OF CONTENTS

ESTROGEN EXCRETION IN PATIENTS WITH PREVIOUS MYOCARDIAL INFARCTION

I. Introduction:

A. Reasons for the Investigation P.l

B. Survey of Previous Investigations on Estrogen Metabolism P.4

C. The relationship of Estrogens to Coronary Artery Disease P.lO

II. Methode:

A. Experimental Procedure for the Study of Patients with Previous Myocardial Infarction P.13

B. Method for Estrogen Analysis P.13

III. Resulta P.16

IV. Discussion P.24

v. Summary P.26 Table of Contents {Contfd)

ESTROGEN METABOLISM IN VARIOUS GYNECOLOGICAL AND ENDOCRINOLOGICAL DISORDERS

I. Experimental P.27

ll. Discussion P.27

Ill. Summary P.28

A METHOD FOR THE DETERMINATION OF ESTRONE IN HUMAN URINE

1. Introduction P.29

ll. Methods P.29

Ill. Resulta P.32

IV. Discussion P.33

V. Summary P.33

GENERAL SUMMARY P.34

REFERENCES

APPENDIX LIST OF FIGURES

Figure 1. Total Blood Cholesterol of Normal and Myocardial Infarction Subjects

Figure 2. Estrogen Excretion in Normal and Myocardial Infarction Subjects

Figure 3. Long Term Study on a Subject with Myocardial Infarction

Figure 4. Estrogen Excretion in a Subject with Infarction and Normal Estriol : Estrone Ratio 1. INTRODUCTION

A. Rea sons for the Investigation.

This phase of the investigation was concerned with a study of

the urinary excretion of estriol, estrone and estradiol -17Jl following the intramuscular injection of estradiol -17J3 in men with myocardial

infarction. The resulta were compared with those of a control group

of male subjects with no clinical evidence of this disease. The study was undertaken for the reasons set forth below.

{1) Rarity of myocardial infarction in females before the menopause.

Myocardial infarction is rare in women before the menopause but postmenopausal women show a high incidence of this disease. The

evidence supporting this statement is of two types:

(a) Clinical evidence - Oliver and Boyd(49) have studied the

sex and age distribution of 1000 consecutive cases of coronary artery disease. From Table 1, we see that the clinical manifestations of coronary artery disease are nineteen times more common in men under the age of 35 and fifteen times more common under the age of

40. In the menopausal and postmenopausal years, the ratio drops to

7:1 to 5:1 to 2:1.

TABLE 1

Sex and Age Distribution of 1000 Consecutive Cases of Coronary Disease; Oliver and Boyd(49) . Age Number of Cases (Year) Men Women Men : Women

Under 35 19 1 19:1 35-39 44 3 15:1 40-49 188 26 7:1 50-59 256 53 5:1 60-69 195 86 2:1 Over 70 68 61 1:1 - 2 -

(b) Pathological evidence - It is recognized, however, that there is considerable limitation in the clinical and electrocardiographie diagnosis of myocardial infarction. However, the striking sex differences in myocardial infarction ls supported by the pathological findings of

Schlesinger and ZoU (59), the resulta of the ir injections and dissections are presented in Table n. TABLE IT

Incidence of Coronary Arter_y Occlusion in Men and Women at Varying Ages; Schlesinger and zou(o9) Age Total Number Hearts with Occlusion {Year) Sex of Hearts Number Per cent

20-39 M 44 1 2.3 F 19 1 5.3

40-59 M 85 20 23.5 F 56 2 3.6

60-79 M 115 43 37.4 F 65 21 32.3

80 on M 9 4 44.4 F 7 2 28.6

Here the resulte are striking. Between the ages of 40 and 59 there are six to seven times more coronary occlusions in males than in females. However, between 60 and 79 the incidence is approximately the same.

(2) Lack of evidence relating sex and atherosclerosis. A detailed study by Sjovall and wihman(60) of 1380 individuals at post- mortem revealed that there are no significant differences between the sexes in so far as general:ized atherosclerosis is concerned. The resulte of their study are indicated in Table III. Even when the study is restricted to the coronary vessels {see Table IV), the difference between males and females in the various age groups is not marked. - 3 -

TABLE ID

Extent of Total Atherosclerosis in Males and Females at Various Ages. Values are expressed as indices derived by combining sepa.rate obser­ vations on the aorta and its main branches; Sjovall and Wihman(60) Age Stockholm Lund (Year) Men Women Mëiï Women 1-20 0.3 0.5 0 0.2 21-30 1.3 1.2 0.6 0.5 31-40 3.5 2.3 2.7 1.9 41-50 8.0 5.6 3.5 5.0 51-60 10.5 10.0 8.4 9.3 61-70 15.7 13.3 13.0 15.0 71-80 16.6 18.5 19.0 23.0 81-90 21.7 20.0 20.8 25.5

TABLE IV

Extent of Coronary Atherosclerosis in Males and Females at Various Ages. Values are indices derived by combining observations in severa! locations in ail individuals of the groups; Ackerman, Dry, and Edwards(l) Age (Year)

Men 1.56 1.74 2.46 2.21 2.32 2.29

Women 1.13 1.30 1.48 1.69 1.99 2.03

Thus, in the ages from 30-39 the degree of coronary atherosclerosis in

men is only 1.4 times that in women. However, from Table 1 we saw that the clinical manifestations of coronary artery disease are 15 times more com.mon in men under the age of 40.

(3) Estrogen as a possible mechanism of protection. The increase in the incidence of myocardial infarction after a natural meno- pause, and prematurely following an artificial menopause suggests develop- ment of this disease may be accelerated by changes in the hormonal equilibrium(49). Clinicat coronary artery occlusion is commonly associated with abnormal circulating lipids and lipoproteins(46)' (51). Fig.l shows the plasma total cholesterol in normal subjects and those with coronary artery disease. FJG.I MEAN PL A$ MA TOTAL CHOLESTEROL VALUES CCORONARY ARTERY DISEASE AND CONTROL GROUPs).

~. ~ 275 (!) ~ -z ..J 0 ~ w t- U) 1&1 ..J CORONARY ARTERY 0 % OISEASE GROUP (6') u c 2 U) c ..J Q. _, c t- 0 t- 150 30 40 5 60 70 -39 -49 -&9 -69 -79 AGE GROUPS - 4 - The administration of estrogen effects changes in the circulating lipids(50).

The evidence of these changes, which will be covered in detail in a subsequent section, emphasizes the importance of hormonal factors in the pathogenesis of the disease.

(4) Availability of a method. The avàilability of a method(7) for the investigation of estrogen metabolism enabled us to determine the response to exogenous estrogen in men with myocardial infarction. Women were not used in this investigation because of cyclic variations in urinary estrogens.

(5) Edinburgh. Study. In a preliminary study on estrogen metabolism in men with myocardial infarction, Bauld(6) found an elevated estriol : estrone ratio. These findings prompted this investigation.

B. Survey of Previous Investigations on Estrogen Metabolism.

This investigation has been confined to the changes in the urinary excretion of estriol, estrone and estradiol -17J3 following the- injection of estradiol -17f3. Consequently this survey will not include a review of the etudies of the formation of estrogens from acetate, testosterone- -

4..C14, or the anabolic interrelationships with other steroid hormones so ably shown by Heard et al. (26-28).

Moreover, the survey will be restricted to estrogen metabolism in the human because of the difference in estrogens in different species of animais. For example, estriol has only been isolated from hum an sources, estradiot -17p is a product of rabbit and equine metabolism and naphtholic estrogens are peculiar to equine species{54). Moreover, the importance of the gastrointestinal tract as a site of estrogen interconversions varies widely with the species of the experimental animal. Heard et al. (26) using Estrone -16-c14 in mice f01m.d the gastrointestinal tract a major pathway of excretion (20. 55% of the total dose administered). V/hen this compolm.d was administered to a pregnant mare, only traces of radio- - 5 - activity were fmmd in the feces(26). Gallagher and Beer(lO) found only

0. 05-4.0 per cent of the ·injected radioactivity in human feces after the administration of Estradiol -17,4-16-c14.

(1) Resulte of isolation etudies. Table V shows the ·classical isolation etudies of estrogens from human sources.

TABLE V

Isolation of Estrogens from Humans

Estrogen Source Reference

Estriol Urine - pregnant Marrian(39) 1930 Meconium Doisy et al. (34) 1956

Estrone Urine - pregnant Doisy(24) 1929 Urine - pregnant Butenandt(16) 1929 Urine - pregnant Laqueur et al. (35) 1930

Estradiol -17J3 Urine - pregnant Huffman et al. {32) 1940 Placenta Huffman et al. (33) 1940 q • Estranediol A Urine - nonpregnant Marker et al. (38) 1938 Estranediol B Urine - nonpregnant Marker et âl. (38) 1938

16 - epiestriol Urine - pregnant Marrian & Bauld(41) 1954

16-p-Hydro?,CYestrone*. Urine - pregnant Watson & Marrian{65h955

Somewhat less conclusive evidence of the existence of estrogens was advanced by Diczfalusy(19) who separa~ed estrone, estradiol -17J3 and estriol from human semen by the use of solvant- partition, cot.m.tercurrent distribution and fluorimetric analysis.

(2) Survey of In Vivo Studies. Heard and Hoffman(29) were the first to demonstrate conclusively the conversion of estradiol -17p to estrone in a human subject. They injected 250 mgm. of estradiol -17p intramuscularly in man over a period of eight days and collected urine during this period and the succeeding ten daye. Estrone (16.2 mgm.) and estradiol -17p

(9. 8 mgm.) were isolated from the pooled urine, thus accounting for only * Subsequent work has shown that 16-ûxoestradiol -17J3 is an artefact, and they were able to isolate 16 p-Hydroxyestrone{42). - 6 - 10.3 per cent of the hormone injected. They did not demonstrate the presence of estriol. This, however, is of no significance since the uriœ was extracted with benzene, and the benzene extract washed with carbonate; these two steps would eliminate any estriol in the extract{43).

The ketonic phenols were separated with Girard'!> Reagent P, and on sublimation gave estrone, which was identified by analysis and mixed melting point of the benzoate. From the non-ketonic phenols, they recovered estradiol by Adsorption Chromatography and sublimation; this was identified by mixed melting point determination.

Isotopes have also been used in the investigation of estrogen metabolism in human subjects. Pearlman et al. (52) studied estrogen metabolism in human pregnancy by the administration of 6, 7-d2- Estrone acetate (99-202 mgm.). The estrogen was dissolved in sesame oil, and injected intramuscularly in equally divided doses. Urine was collected just prior to the administration of estrogen and subsequently for 5 to 12 daye. No marked difference in the Pa.ttern of estrone metabolism was observed in pregnant women when compared to the metabolism of estrone in men. The pere entage of administered estrone recovered in urine during pregnancy was 1. 8-5.4 (as estrone); 3. 3-3. 6 (as estriol); 0. 3-0. 7 (as estradiol); and in men 2.6 (as estrone); 2.7-3.7 (as estriol); 0.5

(as estradiol). The se findings are contrary to expectations based on the concepts of Smith and Smith(61), since a much higher recovery of the respective estrogens during pregnancy would be expected in view of the high production of progesterone. In this experiment, estriol was isolated and identified by mixed melting point determination. However, neither estrone nor estradiol could be isolated because of their low content. Estrone and estradiol were determined by an isotopie dilution technique and analysis (deuterium). - 7 - Schiller and Pincus(58) injected 21.6 mgm. of estracliol -17p into a single male subject and collected three 48-hour post-injection urine specimens. They recovered 0. 21 mgm. estradiot -17:)3, 0. 58 mgm. estrone and 1.17 mgm. estriol. They used solvent-Partition to fractionate the urine extracts. The phenols were taken up in ben.zene and extracted with 0. 3 M Na2co3 in order to obtain the estriol fraction(53), (58). The benzene phase was fractionated into ketone and non-ketones by the use of Girard's Reagent T. .Estrone and estriol were identified by mixed melting point determinations. An attempt to obta.in crystalline estradiol -17p was unsuccessful. The various fractions were assayed with spayed rats

(1 rat unit = 0.125 microgram estracliol -17]3, 1. 0 microgram estrone and 1. 0 microgram estriol).

Stimmel et al. (62) found that oral administration of estradiol

(2·. 0 mgm.i gave higher urinary excretion levels in the first 24 hours than when estrogen was administered intramuscularly. Moreover, it was demonstrated that estriol is not converted to estrone or estradiol, estrone and estracliol -17.J3 are partially interconvertible, and the injection of either leads to excretion of estriol. In their investigation they used adsorption chromatography and colorimetrie estimation.

Beer and Gallagher(ll) studied the fate of large doses of estradiol -17:p-16-c14 (140-350 mgm.) administered intramuscularly to human subjects. The dose was clivided into eight consecutive injections, which were made at 3-hour intervals. Urine collections were made for eight consecutive 24-hour periode; the start of the dose was the first period. The urinary radioactivity corresponded to 65% of the administered dose, and the major portion of this was eliminated in the first four hours after the introduction of the estrogen. Fecal excretion was only 11% of the dose. Estrone was the chief metabolite measured after a massive dose administered intramuscularly or orally. Estrone ranged from 6-7% of the dose or 10% of the radioactivity in the urine during the first four . - 8 - days. Estradiol accounted for 2-3% of the dose or about 4% of the total urinary radioactivity during the first four days. E striol was intermediate between the estrone and estradiol values, from 2-4% of the dose admin­ istered, or about 5% of the total radioactivity in the urine. As time after the estrogen administration progressed, the amount of estrone and estradiol in the urine diminished rapidly, and by the fourth day minor amounts of these metabolites were found in the urine. Estriol was excreted at a constant rate when expressed in terme of urinary radioactivity. For one subject, estriol on the fourth day corresponded to 17% of the urinary radioactivity, compared to 3. 1% for estrone.

In another experiment, Beer and Gallagher(là) determined the fate of small doses of estrone-16-c14 and estradiol -17J3-16-c14 (0. 25 mgm.).

Estriol was the major product in the urine of the first day after a tracer dose of either estrone or estradiol. Estriol made up 22% of the urinary radioactivity whereas estrone was 8. 5%. There was no difference in the excretion of radioactivity after the intravenous administration of estrone or estradiol -11p. The total urinary recovery of radioactivity corresponded to about 65% of the hormone given. This is the same figure they obtained with massive doses of estrogen. Beer and Gallagh.er found that the chief difference between the small and large doses of estradiol -17p is the estriol to estrone ratio. With the small doses, estriol was excreted in greater amounts than estrone. In.tramuscular or oral administration of large doses of estradiol -17:p resulted in estrone being excreted in excess of estriol.

In their experimenta, Beer and Gallagh.er used solvent-partition to fractionate the estrogens. The estrone fractions were characterized by infra-red spectrometry and recrystallized to constant specifie activity from ethanol. The non-ketonic phenolic fraction was acetylated with pyridine and acetic anhydride and chromatographed on silica gel. The eluates con­ taining estriol triacetate, as judged by infra-red spectra, were hydrolyzed - 9 - and recrystallized to constant specifie activity. The amount of radio­ active metabolite was calculated br a formula. Glass et al. (25) stu.died the urinary estrogen excretion in patients with Laennec rs cirrhosis of the liver. The se patients appear to have impaired capacity to metabolize exogenous estradiol or estrone, since Glass et al. recovered 86% of the estradiol administered and 83-85% of the estrone. One subject excreted mainly "Free" estrogen hence liver failure was considered complete.

In their experiment, endogenous estrogen was determined on 24-hour urine specimens before the injection of the estrogens. They injected 600,000 I.U. of the estradiol proprionate and 150,000 I. U. estrone in two daily doses. The urine was extracted with carbon tetrachloride, and assayed on spayed rats. The free estrogens were recovered by the omission of the usual acid hydrolysis prior to the extraction with carbon tetrachloride.

May et al. (45) studied estrogen excretion of patients with carcinoma of the prostate. These patients showed a tendency (75% of the eight cases) to convert exogenous estrone into estriol, whereas only 20% of the patients without cancer behaved in this way. Jn their experiment the patient was given 2. 0 mgm. estrone in a single dose.

Diczfalusy and Luft(20) studied estrogen excretion in a male patient with adrenal cortical tumor. The hydrolysis of the urine revealed

68.5 micrograms of estrone, 18.4 micrograms of estradiol and 85.7 micrograms of estriol. They found 34.5 micrograms of free estriol in

250 ml. urine. Counter-current distribution and fluorimetric analysis were used for their investigation.

(3) Survey of Jn Vitro Studies. Ryan and Engel{57) showed that tissues of the digestive, reproductive and endocrine system of man can convert estradiol -17J3 to estrone. Incubation of estradiol with sUces of terminal ileum resulted in an 11% yield of estrone. Placenta! tissue has - 10 -

the greatest activity of ali the tissues they studied, 11 and 38% yields of estrone being realized with four-month and term placental tissue

respectively. However, the reduction of estrone to estradiol is minimal by this tissue. The testis in marked contrast to the other tissues

studied, converts estrone to estradiol to a greater extent than the

reverse reaction. Adrenal cortex and kidney sUces can effect the

interconversion of estrone to estradiol. Liver and human pregnancy

breast can oxidize estradiol to estrone. Under the experimental con-

ditions employed (0. 01 - 0. 02 mgm. of hormone/mgm. tissue/ml.

buffer) no conversion of estrone or estradi9l to estriol was observed.

Human feces can reduce estrone to estradiol -1rrp(44).

C The Relationship of Estrogens to Coronary Artery Disease.

(1) Plasma Lipids and Coronary Artery Disease. There is strong ' evidence that coronary artery disease is associated with abnormalities of

the circulating llpids and lipoproteins. Controlled investigations of subjects with coronary artery disease have indicated that there is elevation of the

plasma total cholesterol(46). These workers also showed a rise in the

values of plasma total-cholesterol phospholipid ratio and of the concen­ tration of the cholesterol attached to the p lipoprotem(51). This is summarized in Table VI.

TABLE VI Distribution of Cholesterol between the Alpha and Beta Lipoprotein Fractions expresred as the Mean o< :p Ratio, in Coronary Artery Disease; Oliver and Boyd(5 ) • Mean Mean Plasma Total Mean C/P ~~~~----~;;~~~~~~--~~~~~~~~~~~~~----~Ra=tioSubjects Number of Cases c;~... :)3 Ratio Cholesterol (mg.%) Normal Men 50 28:72 193 0.82

Men with Coronary Artery Disease 50 9:91 270 1.04

Note that 72 per cent of the cholesterol is attached to the p lipoprotein fraction in the control group, whereas the corresponding figure is 91 per cent in the group of comparable age with coronary artery disease. - 11 - Sim.Uar findings have been reported by other groups{4)' (36)' (63).

(2) Plasma Lipide and Estrogen. The apparent protection of premenopausal women from clinical corona.ry disease emphasizes the importance of hormonal factors in the pathogenesis of the disease. There is a striking fall in plasma ester cholesterol, a less marked fall in the plasma phospholipide, and therefore, a decrease in the plasma total- cholesterol:phospholipid·:·ratio at the time of ovulation, and immediately prior to menstruationC47). Moreover, there is lees cholesterol attached to the p lipoprotein fraction,. and thus a higher d-:p ratio at ovulation tban at any other time during the cycle(49). Brown(12), in a study on the ur:lnary excretion of estrogens during the menstrual cycle, found two well-defined peaks at ovulation (14th day) and one at the luteal phase

(21st day). The estrogen levels then gradually decrease before the onset of menstruation. Therefore, the decrease in the plasma total-cholesterol: phospholipid ratio prior to menstruation cannot be attributed to a sudden ohange in the circulating estrogen.

(3) Estrogen and Coronary Disease. In men with myocardial infarction, the administration of ethinyl estradiol is accompan.ied by changes in the distribution of lipide in the plasma(48)' (50). A daily dose of 200 micrograms of estradiol caused depression of the plasma cholesterol, the total-cholesterol:phospholipid ratio, and the concentration of cholesterol on the p lipoprotein fraction. The se changes are shown in Table VII. TABLE vn

Changes in the Circulating Lipide and Lipoproteine of Men wtth Coronary Az:tery Disease; Oliver and Boyd{49). Plasma T'otâl Plasma Serum Cholesterol mg.% C/P Ratio o<:]3 Ratio 1 Ethinyl E stradiol (20 men) a) Mean Control Value 251 1. 01 9:91 b) 6 Months Later 223 0.74 18:82 1 Inert Tablets (20 men) a) Mean Control Value 236 0.95 10:90 b) 6 Months Later 230 0.93 9:91 - 12 -

These affects have also been shown by other worlœrs(5)' (3~.

The efficiency of estradiol administration in decreasing the recurrence of coronary artery occlusion must await assessment in terms of morbidity and mortality rates. Such etudies are now in progress(50). - 13 -

II METHODS

A. Experimental Procedure for the Study of Myocardial Jnfarction.

The experimental procedure is described below.

{1) Collection of 24-hour urine specimen (Control Day 1).

(2) Injection of 350-450 micrograms of estradiol -17J3

to a control or myocardial infarction subject.

(3) Collection of 24-hour urine specimens for 4 days (Day

2, 3, 4, 5).

(4) Analysis of 5 speciniens for estriol, estrone and

estradiol was carried out by the chromatographie method

of BauldF) .

(5) Calculation of estriol:estrone ratio:

1) (6) Calculation of the per cent . recovecy:

E striol, Estrone and E stradiol -17:,3 excreted on days 2, 3, 4, 5 - 4(Estriol, Estrone and Estradiol -17~ excreted on Day 1) Estradiol -17p injected

B Method for Estrogen Analysis. The method(7) used in this phase of the investigation was critically discussed at an International "Symposium on Estrogen Methodology" convened in London in June 1956 tmder the auspices of the British Empire Cancer Campaign{9). The following conclusions were reached:

(a) Specificity - The method does not measure 16-epiestriol or

16- bydroxyestrone. The specificity thus depends upon the procedures for the purification and separation of estriol, estrone and estradiol (which remove more than 95% of the background color) and upon the specificity of the Kober r eaction for estrogens. That these factors are adequate is suggested by increase in these fractions in subjects given estradiol, by fluctuations in the se fractions during the menstrual cycle, by decrease in - 14 - the se fractions on castration and adrenalectomy of female patients, and by recovery in the respective fractions of pure estrogens added to urine.

A high degree of specificity is also borne out by the Unearity of the background color of the Kober reaction over the range of 480-550 mJl. in which The Allen Color Correction is applied(7). Proof of the ab solute specificity must await countercurrent distribution analyses of the final fractions in the method in the manner carried out by Diczfalusy(21) for

Brownts method(13). It is planned to carry out tb.ese etudies in co­ operation with Dr. Diozfalusy in September, 1956 and at the same time to correlate the bioassay and colorimetrie estimation resulta in co"""Ûperation with Dr. John Lorraine of Edinburgh. Since, however, these etudies have already been completed on Dr. Brown ts meth.od(21) and Binee a comptrison of the methode of Bauld and Brown has already been published, indirect but conclusive evidence of the specüicity of the method used in this investigation is available. Study of the comparison(40) shows that in the

11 analyses carried out simultaneously by the two methods, the average düferences between the re8Ults were -0. 3 microgram per day for estriol,

-0. 2 micro gram for estrone and j.o. 5 microgram for estradiol. (The negative sign means lower value in the Brown method). The average düference between the two methode of analyses does not exceed its own variation. Thus there is no evidence that the two methods differ.

{b) Accuracy - Since the conjugated forma in which estrogens are excreted in human. urine are not lmown, accuracy was defined as

"the average percentage recovery of pure estrogens added to urine after a cid hydrolysis". This was shown to be(7) 81% for estriol, 90% for estrone and 84% for estradiol. Pure estrogens added to urine undergo some losa. Brown(9) demonstrated that this losa varied with dilution of the urine and that with the urine dilution employed in this investigation

(24-hour volume diluted to 2. 5 litera) this losa was 0-10% for estriol - 15 - and estrone and 5-15% for estradiol. Since in this method we are determining organic compounds at a dilution of lesa than one in a

1umdred million, there is a possibility of laboratory error completely invalidating the resulta. The precautions necessary for accurate determinations have been clearly defined(7) and these have been care­ fully followed in the present investigation. In addition, in the majority of cases control and myocardial infarction cases have been determined simultaneously.

(c) Precision and Sensitivity - These factors depend upon the instrumental error of the spectrophotometer. Dr. Bauld(6) feels that corrected optical densities below 0. 005 cannot be distinguished in replicate analyses with any degree of certainty. In the method this corresponds to 1 microgram of estrone per day and 1. 5 microgram of estradiol and estriol. This value is slightly above that obtained by statistical analyses of sixty-six consecutive determinations and it is felt that these values represent a conservative estimate of the precision of the method. Sensitivity was set by the conference at double the values for precision. This means that the sensitivity of the method is set at 2 micrograms of estrone and 3 micrograms of estradiol and estriol. - 16 - .l ill RESULTS The resulta from the controlled investigation of estrogen meta.bolism in hwnan subjects with myocardial infarction are shown in detail in the

Appendix (pp.1-52). Fig. 2 illustrates our findings in patterns of estrogen excretion

in a subject with previous myocardial infarction and in a control subject (see Appendix: p. 32 and p. 3). The excretion pattern of a 52 year old

male who suffered a myocardial infarction one month prior to study is

seen in Fig. 2a. On Day 1, the control day, the amoun.ts of the three estrogens are a reflection of the endogenous production in this patient.

There is an increased amoun.t of estriol excreted when compared to estrone and estradiol. This finding has been generally present in the infarction

group studied, and is seen in Table vm where the mean estriol:estrone

ratio of 5:3 on Day 1 of the infarction group is higher than the value 2:5 of the control group. This difference on Day 1 is not statistically

significant, and this is due to the low estrone excretion of one patient (S. T.). However, the photometrie determination is inaccurate at such

low levels of estrogen excretion. This inaccuracy may be a factor

in the lack of significance in the difference of the mean values. Follow­

ing injection of 412 micrograms of estradiol there is a small rise in

estrone and estradiol which reaches a peak on Day 3. The estriol :

estrone ratio in this subject is 7. 6, that is, following administration

there is 7. 6 times as much estriol excreted as estrone.

Fig. 2b illustrates the findings in a 68 year old male subject without evidence of previous myocardial infarction {see p. 3 of

Appendix for details) . The final diagnosis was made on the basie of

physical and laboratory procedures supported by electrocardiographie

investigations. In comparison to the subject with myocardial infarction there is a lesser amount of estriol excreted on the control day. FIG.2 B tf 52 Y R S. POST~ R 1 0 R d 68 YRS. CONTROL INFARCTION 40 r

f'GM. OF ESTROGE N

ESTRIOL :0·!5 ESTI'ONE 20 . ES TRIOL = 7 6 ESTRONE ESTRIOL -

ESTI'IOL

E STRONE ESTRADIOL EITIUDIOL UTRONE

4 DAYS DAYS ESTR ADIOL 429 JAGM. ESTRADIOL - 17 -

TABLE VIII

Estriol:Estrone Ratio* on Day 1

Control Group Myocardial Infarction Group

Subject Estriol:Estrone Subject E striol :E strone 74- B.R. 2.4 W.I. 1.3

E.G. 1.8 S. T. 3.8

F. I. 3.3 S.A. 0.8 F.O. 1.6 P.A. 3.0

M.G. 4.2 M.A.R. 4.6

H.A. 2.0 M.A.C. 0.7

S.H. 2.2 L.A. 4.3

W.E. 0.9 H.A. 2.8

W.A. 1.3 D.E. 6.4

Y.A. 3.1 B.E. 3.6

B.O. 1.5 R.E. 5.3

P.O. 2.6 M.G. L. 1.8

C.H. 5.2 F.R. 5.4

W.B. 1.2 T.H. 2.8 W.R. 1.5 c.o. 0.3 R.E.I. 0;7 G.R. 12.0

R.B. 5.0 W. Y. 3.8

S.R. 2.5 S. M. 3.6

J.M. 0.9 T.H.O. 2.5

A. P. 6.5 M.A. 5.2 H.Q. 4.7 S.T. 38.5.

Mean 2 . 5 Mean 5.3

* E striol excreted on Day 1 Estrone excreted on Day 1 - 18 - Following estradiol administration, the major metabolite is estrone which

reaches a peak excretion on Day 2. The estriol:estrone ratio in this

subject is O. 5, that is following estradiol administration there is only 1/2

as much estriol as estrone being excreted.

To determine the consistency of patterns of estrogen metabolism,

patient D. E. was chosen for a long-term investigation {see Appendix:

pp. 40-41). This subject was a 66 year old male who bad suffered a

myocardial infarction one month prior to the initial study. The first

determinations were made while the patient was on anticoagulants and

at bed rest in hospital. He showed the pattern of increased estriol

excretion after estradiol administration with a urinary estriol:estrone

ratio of 4. 2. This is illustrated in Fig. 3a. The second determination

was made six weeks after the initial one or ten weeks following infarction.

He aga:ln shows similar curves with a urinary estriol:estrone ratio of 3.8

(Fig. 3b). The patient was then placed on a 30 gm. fat diet and Premarin

{2. 5 mgm. daily) which was continued for a period of two months. The

Premarin was discontinued five days prior to the last study. The last

determinations were made slightly more than three months following the

infarction. Again he shows similar curves with a urinary estriol:estrone

ratio of 4.4 (Fig. 3c).

These findings would indicate that the pattern of estrogen metabolism

remains constant in the individual over the time studied. Similarly, a fixed pattern of estrogen excretion is known to occur after pregn.ancy{15).

They would also indicate that the pattern seen in patients with myocardial

infarction is not influenced by bed rest, anticoagulants, nor did two months

of estrogen therapy alter the pattern in this patient. These three experi­

menta also show that over a period of time his endogenous excretion as

shown in each of the control days remains relatively constant, showing high endogenous levels of estriol with small amomts of estrone and estradiol. ,.... ,r fi yea. pQ1Ip;;B10R INfARCTION, ANTICOAfULANTS ANBULANT AFTl R PlU N AlUN 12/11/SS 1/21/58 1/18/58 so

,)1 GM. OF 40 I:ITit OGlN

20 l STR IOL

ES TRIOL "' . ES TRIOL ES!RIOL . 4 2 3 8 : 4 4 ESTRONE ESTRONE '" ' ES TRONE 10

ESTRONE ~-,--~--~--~~~-r--,---~~~-,---r--~~~-,---r--,--=~EITRADIOL 2 3 4 5 Lt 2 3 4 5 't 0 AY8. UT ltA Dl OL aa4 )IGM. 3t4 JIIN. - 19 -

Similar etudies have been completed in 42 male subjects.

Twenty-two ~tients have bad clinical and electrocardiographie evidence of myocardial infarction. The time of study following infarction ranged from two weeks to two years with the majority being studied one month after infarction. Twenty other subjects in whom there was no evidence of infarction were used as controle. This group consisted of six laboratory workers and 14 hospital patients with varying clinical diagnoses.

Significant renal and hepatic disease was excluded in ali subjects.

The over-all resulte are summarized in Tables IX and X. The ages of the control subjects varied from 23 to 81 years With a mean of

51 years. This is slightly lower than the infarction group where the range was 36 to 77 years with a mean of 59 years. The percentage recovery is the sum of the three estrogens excreted in the urine expressed as a percentage of the amount of administered estradiol

(e.g. see Appendix: p.2). The mean of 17.5% in the control group is not significantly different from the 20. 8% recovery in the infarction group.

The percentage recoveries are in line with previous estrogen metabolism experimente in humans(29)' (58). The urinary estriol:estrone ratio in the control group varied from 0. 3-5. 3 with the mean for the group of 1. 4.

In 18 of the 20 control subjects the estriol:estrone ratio was 0.3-2.1.

In the other two subjects the ratios were 4.1 and 5.3. One was a patient witb. recurrent hemoptyses of unknown origin who bad numerous previous admissions for alcoholism. The other was a patient with advanced pulmonary emphysema and a benign gastric ulcer.

In the infarction group the estriol:estrone ratio ranged from

0. 9-16.0 with a mean of 4. 7. The mean therefore in the infarction group is 3. 4 times greatw than in the control group. The P value for these resulte is less than O. 01 and therefore the difference between the findings in the two groups is significant. Thus in 18 of the infarction group and in 18 of the control group there was no overlap in the urinary - 20 -

TABLE IX

Estriol:Estrone Ratio* and Per Cent Recove!l: in Control GrouE Subject E striol/E strone % Recovecy Age Re marks

B.R. 5.3 21 62 Gastric meer

E.G. 0.5 11 68 Bronchitis

F.I. 1.7 26 53 Coronary Insufficiency

F.O. 1.3 18 63 Rheumatoid Arthritis

M.G. 0.4 14 23 Normal

H.A. 2.1 16.8 64 Gastric Ulcer

S.H. 1.9 30.0 61 P. T. C • Deficiency

W.E. 1.4 15.0 81 Bronchogenic Carcinoma

W.A. 0.8 16.0 69 Peripheral Arteriosclerosis

Y.A. 1.0 17.0 51 Mitral stenosis

B.O. 0.5 15.0 52 Coronacy Insufficiency

P.O. 1.0 9.0 55 Cirrhosis

C.H. 4.1 23.0 44 Alcoholism

W.B. 0.6 18.0 35 Normal

W.R. 0.9 18.0 39 Normal

R.E.I. 0.3 17.0 48 Normal

R.B. 0.9 8.0 25 Coarction of Aorta

S.R. 0.5 21.0 28 Normal

J.M. 1.6 24.0 35 Normal

A. P. 1.5 13.0 54 Coronary Insufficiency

Mean 1.4 17.5 50.5

* Calculated by equation on p. 13. - 21 -

TABLE X

Estriol:Estrone Ratio* and Per Cent Recovery in Myocardial Infarction Group

Subject E striol/Estrone % Recovery Age W.I. 1.5 28 50

S.T. 3.3 29 51

S.A. 0,9 25 67

P.A. 3.2 27 52

M.A.R. 7.6 24 52

M.A.C. 0.9 29 60

L.A. 9.2 24 45

H.A. 3.4 10 63

D.E. 3.8 17 66

B.E. 16.0 25 77

R.E. 5.3 29 64

M.C.L. 0,9 16 65 F.R. 3.6 24 63

T.H. 3.4 16 75 c.o. 4.9 44 58

G.R. 6.3 12 60

W. Y. 2.4 13 52

S. M. 3.1 15 59

T.H.O. 3.0 17 36

M.A. 4.1 12 55 H.O. 6.0 14 64

S.T. 10.5 8 69

Mean 4.7 20.8 58.7 - 22 - estriol:estrone ratios. In four cases in the infarction group the estriol: estrone ratios are between O. 9 and 1. 5 and hence in the range of the control group. However, in two of them, despite their low estriol;estrone ratios, they did present abnormal patterns of estrogen metabolism. Fig.4 illustrates the estrogen excretion of one such patient (see Appendix: p. 24).

The patient was a 50 year old male studied one month following an acute anterior myocardial infarction. In response to estradiol administration a large amount of estriol is excreted which reaches a peak of 56 gamma on the 4th day, but unlike the infarction group he also excretes large amounts of estrone on the second and third day. Thus despite the high estriol excretion his urinary estriol:estrone ratio (1. 5) is low because of the high estrone levels.

However, the percentage of estrogen recovered as estriol in the infarction group is higher than the value of the control group. These resulta are summarized in Table XI. In the infarction group the per c-ent recovery ranged from 5.8-32.1% with a mean of 14.5%. In the control group the range of values was from 2. 7-16.9% with a mean of 7. 5%. The P value for the se observations is less than 0. 01 and therefore the difference between the findings in the two groups is significant. FIG•4 tf 0 0 Y R 8. ANTE R 10ft 1N FA R C Tl 0 N..

ESTRIOL - 1· 0 ESTRONE -

.)-( G M. 0 F ESTROGEN

ESTRIOL 20

r L ESTRONE 10 1 1 ..._____ E8TRADIOL

2 4 DAY 8 ESTRADIOL. - 23 -

TABLE Xl

~ of Estrogen Recovered as Estriol

Control Gro:!;!E Myocardial Infarction Grou,e

Subject Jg Subject ~ B.R. 15.1 W.I. 14.8

E.G. 2.7 S. T. 19.1

F .1. 13.3 S.A. 10.4

F.O. 7.6 P.A. 17.8

M.G. 2.7 M.A.R. 18.3

H.A. 7.8 M.A.C. 11.6

S.H. 15.6 L.A. 20.2

W.E. 7.0 H.A. 5.8

W.A. 6.0 D.E. 11.4 Y.A. 7.0 B.E. 22.6

B.O. 4.4 R.E. 19.7

P.O. 4.7 M.C. L. 6.3

C.H. 16.9 F.R. 17.7

W.B. 6.4 T.H. 11.4

W.R. 9.0 c.o. 32.1

R.E.I. 3.6 G.R. 10.6

R.B. 3.3 W.Y- 10.7 S.R. 6.6 S. M. 10.2

J.M. 3.6 T.H.D. 11.2

A. P. 7.4 M.A. 9.0

H.O. 11.0

S. T. 16.8

Mean 7.5 14.5 - 24 -

IV. DISCUSSION

In response to estradiol administration, patients with previous myocardial :lnfarction bad significantly higher estriol:estrone

ratios than the control subjects. This disturbance :ln estrogen

metabolism does not appear to be related to the age of the patient, anticoagulant therapy or the duration of time following :lnfarction.

The relationship of these f:lndings to myocardial :lnfarction is at present

unknown.

The high estriol excretion of the infarction group cannot be

attributed to renal damage overlooked at exammatf.on, since the recovery

of estrogen is the same in both groups.

In liver disease, there is high estradiol excretion(25), whereas

in myocardial infarction a high estriol level is found. It bas been shown

that liver can "inactivate" and "activate" estrogen{56). It is possible

that in liver disease exogenous and endogenous estrogens are activated

which resulta in the appearance of large amounts of estradiol in the ur:lne,

whereas in patients with myocardial infarction the "inactivat:lng"

mechanism is predom:lnant resulting in elevated estriol excretion. In the human, estriol is esterified as a glucuronide .and linked to a beta-globul.in

found in the plasma beta-lipoprotein fraction{55). It is possible that the

liver functions :ln the formation of this complex which may be necessary

for the transport of endogenous or exogenous estrogen. It bas recently

been shown that the liver is an organ of great importance in the regu­ lation of the circulating amount of poly-estradiol phospbate(22). A dis­

turbance :ln liver function may be reflected by abnormalities in the

circulating lipide, lipoproteine and estrogen metabolism, giv:lng the

pattern found :ln subjects with previous myocardial infarction.

It is known that adrenocorticotrophin administration accompanied by adrenal cortical hyperactivity bas a powerful :lnhibitory influence on the responsiveness of the castrate uterus to adm:lnistered estradiol(64). - 25 -

In addition, it is lmown (23) that 50, 000 I. U. human chorionic gonadotrophin administered to a castrated male causes a small increase in estrone and estradiol excretion but a large rise in estriol. It is possible that patients with previous myocardial infarction suffer from a chronic stress condition resulting in rapid conversion of estradiol to estrone to estriol by adrenals stimulated by ·an ACTH level which is perma.nently elevated. This would explain the stable pattern of estrogen metabolism shown by patients With myocardial infa.rction (e.g., subject D.E.). Moreover, a high estriol concentration may decrease the biological effect(31) of estradiol and estrone which would explain the inhibitory influence of ACTH on the action of estradiol on the castrate uterus.

In the course of an investigation of steroids excreted by a boy with adrenocortical carcinoma A5 - androstenetriol-3 {f3), 16, 17 was isolated(30). This triol of the androstene series carries the hydroxyl groups in the same position as estriol. The structural similarity of these two compounds raises the question whether these two compounds are formed by an analogous reaction in the adrenals. If it is assumed a single pathway exista which converts estradiol to estrone to estriol, it follows that the administration of estradiol would result in a rise of the three estrogens in the urine. An elevation of the se estrogens is found in this investigation. The hypothetioal existence of an adrenal pathway where only estriol is formed, would preclude by its very nature a rise of estriol excretion after estradiol administration. The rise in estriol found is evidence that such a route is not the only means of estriol formation. Therefore, it is possible that both pathways are found in the adrenal gland, and that stress would stimulate an hypo­ thetical mechanism, which converts an adrenal corticoid directly to estriol. It is well lmown that stress can cause an increased production of adrenal steroids. - 26 -

V. SUMMARY

The resulta of this controlled investigation of estrogen

metabolism in human subjects with previous myocardial infarction have been presented. The patients showed a significantly higher estriol:estrone ratio after the administration of estradiol. Moreover, the endogenous estriol:estrone ratio is elevated in the infarction group. The estriol recovered from the urine of the infarction group is significantly higher. The characteristic pattern of estrogen excretion does not vary. These findings are discussed. - 27 -

Estrogen Metabolism in Various Gynecological and Endocrinological Disorders: This phase of the investigation was concerned with a study of the urinary excretion of estrogen by patients with different gyneco- logical and endocrinological disorders.

I. Experimental. The estrogen levels in urine were deter- mined by the same method used for the study of estrogen metabolism in men with myocardial infarction. The resulta are shown in Table XII.

TABLE XII Excretion of E strone, E stradiol -1 7:p and E striol in Different Gynecological and Endocrinolog!cal Disorders. microa!ams per dal Subject E strone Ëstradiol Estriol Re marks

B 8.3-8.6 3.2-3.5 30. 8-31. 2 pgm. Cushing's Syndrome (Male) H 218.2 103.2 11.5 mgm. Adrenalectomy, 5-6 months pregnant. M 1. 5-2.0 1.0 3.0 -5.5 pgm. Early Menopause (13 years old). w 14.0 6.0 14.0 Jlgm. Adenomatous Endometrium . L 2.8 3.0 10.0 J,lgm. Absence of Uterus and Vagina. s 1.5 0.5 7; 0 )lgm. Ovariectomy. s 1.0 1.0 4.3 J,lgm. Adrenalectomy.

IT. Discussion: Table XIT shows that Subject B with Cushing's

Syndrome excreted much more estriol than either estrone or estradiol.

This result is significant when it is remembered that Diczfalusy (see p. 9) found a high estriol excretion in a patient with adrenal cortical tumor.

Subject H bad Cushingts Syndrome for nine years, and bad a bilateral adrenalectomy performed. Following her operation she bad no menstrual period, and was 5... 6 months pregnant when her estrogen excretion was determined. This woman excreted large quantities of estriol (11.5 mgm.) when compared to estrone (1.5-2.0 pgm.) or estradiol (1. 0 pgm.). It is lmown that the patients with late toxemia of pregnancy excrete normal amounts of estrone and estradiol but show low estriol excretion{18). The disturbance in estrogen metabolism by - 28 - this subject is the low estrone excretionC15).

The low estrogen levels of subject M appear to have caused the early menopause.

Subject L had congenital absence of uterus and vagina, but at operation the ovaries appeared normal. The low estrogen excretion

found is normal for the first daye of the menstrual cycle(12).

Subject S suffered from carcinoma of the breast with skeletal

metastases. The first urine analysis followed radiological castration

of the ovaries. Table XII shows a high estriol and a low estrone and

estradiol. The estriol excretion was reduced by adrenalectomy. The estrogen values following adrenalectomy are not significant since the

photometrie estimation is inaccurate under 5 micrograms per day.

Brown(l4) found no abnormality of estrogen metabolism in patients with

carcinoma of the breast. m. Summ.ary: Estrogen excretion by subjects with different gynecological and endocrinological disorders was studied. A subject

with Cushing's syndrome had an elevated estriol excretion. A low

estrone level in urine was found in a woman 5-6 months pregnant and who had a previous adrenalectomy. Disturbances in estrogen metabolism were found in a subject with no uterus or vagina but who had normal ovaries. - 29 -

A Method for the Determination of Estrone in Human Urine by Partition-;­ Chromatography and Colorimetrie Estimation

1. INTRODUCTION The purpose of this investigation was to devise a short method for the determination of estrone in human urine, suitable for routine use. There were two reasons which prompted the development of this method. Firstly, the method which we used for the study of myocardial infarction required extensive fractionation to separate out estrone, estriol and estrad.iol from the crude urine extract. Secondly, the bioassay of a mixture of estrogens on castrated adult rats is inaccurate, since Hisaw et al. {31), showed that estriol even in small doses (0.1 ugm.) decreased the effect of estrad.iol and estrone on uterine growth when the hormones were given simultaneously. A method for the accurate deter- mination of estrone is of value in the study of the menstrual cycle, pregnancy and adrenalectomy in human subjects.

II. METHODS (a) Purification of materials. Reagentsare A.R., unless otherwise stated. Solvents are d.istilled in aH-glass apparatus.

Benzene - This reagent (Mallinckrodt, thiophene-free) is distilled from liquid paraffin (50 mL/liter), and redistilled. Ethylene-dichloride - (Anachemia, Technical Grade) The solvent is poured through a column of silica gel {1 in. in d.iameter, 12 in. of length being used for each liter) at a rate of 4-6 ml./min. and d.istilled within 24 hours of use.

Celite-535 - This is heated at 4000 C. for 4 hours, stirred with an excess of 12~ HCl and allowed to stand overnight. The solid is washed with distilled water until they are free of Cl- (AgN03 test), Feu"' (SCN- test), and neutral. The material is then dried at 110° C. - 30 -

for 48 hours, cooled in a vacuum desiccator and stored in containers

with tightly fitting glass stoppera.

(b) Cleaning of glassware. Glassware is rinsed after use

with tap and distilled water. When visibly dirty it is allowed to stand

in chromic acid in sulphuric acid mixture for 12 hours. It is then rinsed with water, soaked in ethanol to destroy traces of chromic acid, washed with a vigorous stream of water for 2-3 minutes and finally

rinsed in distilled water.

{c) Preparation of estrone color reagent. NaN03 (10 mgm.)

and quinone {certified chemical, 20 mgm.) in 1 liter of H2So4 {66%, v/v) are warmed until the solution turns light green and opalescent. Quinol

(20 gm. of British Drug House Laboratory Reagent) is added, dissolved by further warming, and shaking, and the solution {after cooling) is filtered through sintered glass {porosity No.4, fine).

{d) Collection of urine. A complete 24-hour urine specimen is collected without preservative and stored at 4°C . if necessary, and analyses are begun within 48 hours.

{e) Hydrolysis. The 24-hour urine specimen is diluted to 2. 5 litera with distilled water and mixed thoroughly, and two 500 ml. portions are placed in 1 liter round-bottomed flasks. The sampl es are brought to the boiling point under reflux condensers, 75 ml. of concentrated HCl is added and holling continued for 20 minutes.

(f) Separation of the acid fraction. The cooled solutions are extracted with ether (2 x 250 ml.), interfaoial solid re sidues being dis­ carded after vigorous shaldng of the combined extracts. The ether solutions are submitted to the following procedures:

(i) Washed With 0.1~ NaOH containing 20% NaCl (3 ~ 50 ml.), which is then discarded.

(ü) Washed With 20% NaCl (1 x 100 ml.), the aqueous phase is again discarded. - 31 -

(iii) Washed with water (1 x 100 ml. , 1 x 50 ml.), the aqueous phases are discarded. The ether solutions are then distilled to dryness.

These solvents were chosen because of their favorable partition coefficients(3).

{g) Chromatography of the impure estrone fraction. Chromatograms are prepared as previously described by Bauld(8) with the following characteristics:

Mobile phase, benzep.e; stationary phase, O. 8~ NaOH; columns

1 cm. x 12 cm. are packed from a slurry containing O. 8 ml. of stationary phase/gm. of celite; diameter of holes in packing plunger,

1. Omm.; temp. 18 t O. soc., percolation rate of solvent, 10-12 ml. /br. The residues are dissolved in 1 ml. of mobile phase, transferred by pipettes carefully to the tops of the chromatograms and allowed to pass through the column until the top is just dry. The transfer procedure is repeated twice with 1 ml. washings of mobile phase. When the second wash entera the chromatogram, collection of eluate is begun and a reservoir containing 40 ml. of mobile phase is fitted to the top of the column. The eluate 0 to 8 ml. is discarded, the end of the column rinsed with a stream of benzene, and the eluate 10-30 ml. collected, giving the impure-estrone-fraction.

(h) Final purification of the impure-estrone-fraction. This fraction is evaporated in vacuo, boiled under reflux for 30 minutes with

10 ml. of l.O!i" NaOH, acidified with 1 ml. of 12.0~ H2S04 and extracted with benzene (1 x 20 ml.). The extracts are washed with 0. 5M_-NazCOa

{1 x 4 ml.) and water (2 x 4 ml.) and transferred to color-reaction tubes.

Quinol (50 t 5 mg.) is added and the solvent removed by heating in a boiling water-bath at 80-900C. in a stream of filtered air.

(i) Color development on purified extracts. The reagent for estrone (3. 0 ml.) is added to the estrogen and quinol residues contained in test tubes (23 mm. x 150 mm.). These are heated for 20 minutes to - 32 - ensure adequate mixing and solution of the quinol. The solutions are then cooled in a bath of cold water (approximately 15°C.), 50 r 5 mgm. of quinol is added, dilution carried out with 0. 3 ml. of water. The tubes are then shaken 5-10 times and reheated for 15 minutes with two shaldngs during this time to d,issolve the quinol. The cooled solutions are then transferred to 1 cm. cuvettes and their optical densities measured at 480, 512.5 and 545 mp. These measurements were clone with a Beckman Model B spectrophotometer. Optical density readings are corrected by applying the following equation of Allen(2) :estrone corrected readings "' D. - i(D. J. D. ) 512 mp. ( 480 mp. 545 mp.).

0> Precautions taken in analyses. In this method estrogens are being determined at a concentration of approximately 5 pgm. /liter of the original urine. Meticulous care must therefore be taken to avoid contamination of glassware, mouthe of reagent botties and separating funnels, ground-glass joints, stoppera, and air-jets.

m. RESULTS This phase of the investigation was restricted to the recovery of added estrone to different male urine collections. The resulte of

12 recoveries (in duplicate) are shown in Table XTII .

TABLE Xlll % Recovery of Estrone Added to Male Urine Sample ;m No. 5. 0 p~. Estrone Added 2.0 pgm. Estrone Added Recovery % Recovery 1. 85. 0%; 95.0% 80. 0%; 91.4%

2. 83.3%; 82.5% 81.1%; 78.4%

3. 89. 2%; 90.8% 82. 9%; 77.2%

4. 83.5%; 89.~% 79.7%; 73.0%

5. 90.2%; 90.9% 79. 8%i 81. 2%

6. 89. 5%; 90. ~% 8~. 5%; 81. O%

Mean 88.3 80.7 - 33 -

IV. DISCUSSION

The recovery experimenta appear to justify the future use of this method in the study of estrone excretion in various endo­ crinological and gynecological disorders.

V. SUMMARY A method for the determination of estrone in urine has been described. Solvent-partition, partition chromatography and colorimetrie estimation are the essential elements of this method. The recovery of added estrone was sufficient to justify the use of the method in the study of various endocrinological and gynecological disorders. - 34 -

GENERAL SUMMARY

The resulte of this controlled investigation of estrogen metabolism in human subjects with previous myocardial infarction have been presented. These patients showed a significantly higher estriol:estrone ratio in their estrogen excretion after the administration of estradiol. The estriol recovered from the urine of these patients was significantly higher.

The pattern of estrogen excretion was determined in subjects suffering from various endocrinological and gynecological disorders.

A short method for the determination of estrone excretion has been presented.

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53. PEARLMAN, W.H., and PINCUS, G., J. Biol. Chem., 147, 379 (1943).

54. PINCUS, G., and PEARLMA.N, W.H., In Vitamine and Hormones, ed. by R. S. Harris and K. V. Thimann, Academie Press, N.Y., Vol.1, 293 (1943).

55. ROBERTS, S., and SZEGO, C.M., Endocrinology, ~ 183 (1946).

56. ROBERTS, S., and SZEGO, C.M., Endocrinology, 40, 73 {1947).

57. RYAN, K.J., and ENGEL, L.L., Endocrinology, 52, 287 {1953). 58. SCHILLER, J., and PINCUS, G., Arch. Biochem., !, 317 (1943).

59. SCHLESINGER, M.J., and ZOLL, P.M., Arch. Path., ~ 178 (1941).

60. SJOVALL, H., and WIHMAN, G., Acta. Path. et Microbiol. Scandinav., SJ.pp. ~ 1 (1934).

61. SMITH, G.V.S., and S:MITH, O.W., Physiol. Rev., ~' 1 (1948). - 4 -

62. STEALY, C.L., and STIMMEL, B.F., J. Clin. Endocrinol., ~. 67 (1948).

63. SWAHN, B., Scandinav. J. Clin. & Lab. Jnvest., .2_, supp. , !, 1 (1953). 64. SZEGO, C.M., and ROBERTS, S., Am. J. Physiol., 152' 131 {1948}.

65. WATSON, E.J.D., and MARRIAN, G.F., Biochem. J., 61, XXIV (1955). APPENDIX Control Subjectr B.R. Age: 62 Diagnosis: Gastric Ulcer

Control Day (480f 545) Micrograms Es trone: 480 IDJI, 512.5 IDl!• 545 IDJ!. 512.5 - per day *K = 38.8 0.848 0.730 0.524 0.044 1. 7 p.gm. 8.5 0.703 0.618 0.448 0.042 1.6 p.gm.

(480 t..550) Estradiol: 480 IDI!· 515.0 IDJ!. 550 ffil!· 515.0 - 2 K = 43.5 0.319 0.294 0.254 0.007 o. 3 Jlgm. 1.5 0.312 0.274 0.221 0.007 O. 3 p.gm.

(480 t. 545) Estriol: 480 IDl!· 512.5 IDJ!. 545 IDl!· 512.5 - 2 K • 50.0 0.380 0.369 0.252 0.053 .080 4.0 p.gm. 20.0 0.408 0.393 0.271 0.053 .080 4. 0 pgm. Day2 Estrone: 0.519 0.488 0.332 0.062 2.4 15.5 0.622 0.567 0.352 0.080 3.1 Estradiol: 0.199 0.202 0.146 0.029 1.3 6.5 0.218 0.219 0.161 0.029 1.3 Estriol: 0.268 0.271 0.180 0.047 .071 4.0 20.0 0.229 0.239 0.155 0.047 .071 4.0

*The factor '~" is determined by carrying out a Kober colour reaction on 5 micrograms of standard solution (50 ugm. of estrone/ml. of solution). - 2 - Micrograms Day 3 ~gay Es trone: 0.452 0.420 0.248 0.070 2 7 13.5 K = 38.8 Estradiol: 0.239 0.227 0.179 0.018 0 . 8 4.5 K = 43.5 0. 218 0.208 0.155 0.021 0 . 9 Estriol: 0.333 0.432 0.240 0.145 0 . 219 11.7 58.5 K • 53.2 0.299 0.402 0.212 0.146 Day4 Es trone: 0. 595 0.525 0.370 0 042 1.6 8.0 0 . 499 0.453 0.331 0.038 1.5 Estradiot: 0.282 0.252 0.210 0.006 0.3 2.0 0.378 0.351 0.300 0.012 0.5

Estriol: 0.369 0.418 0.252 0.107 0. 161 8.1 44.5 0.570 0.598 0 . 371 0.127 0.191 9.6 Day 5 Es trone: 0.453 0.393 0.262 0.035 1.4 7.0 K = 40.0 Estradiol: 0.270 0.237 0.174 0 . 015 0.6 3.5 K = 40.0 0.366 0.320 0.232 0.021 0 .8 Estriol: 0.338 0.319 0.233 0.033 0.050 2.5 11.5 K = 50.0 0.319 0.296 0.218 0.027 0.041 2.1

Es triol = 38.5 .;. 24.5 = 63 = 5.3 Es triol = 63 - 63 - 2 8 Es trone 7 7 5 12 Estrone 7 Estradiol 12 7 57 3 7 0.5 7 2 - 22.5 - .

% recovery = ~ • 21% 418 - 3 - Control Subject: E. G. Age: 68 Diagnosis: Bronchitis Micrograms Control Da;ï per day Es trone: 0. 341 0.268 0.187 0.004 0.1 0.5 K = 30 . 1 0.339 0.260 0.179 0.001 Estradiol: 0.209 0.175 0.136 0.002 0.1 1.0 K = 38.4 0.356 0.289 0.212 0.005 0.2 Estriol: 0.309 0.252 0.145 0.02'5 0.038 1.8 9.0 K = 47.1 0.251 0.207 0.113 0.025 Day 2 Estrone: 0.301 0.319 0.133 0.102 3.1 16.0 0.350 0.361 0.156 .0.108 3.3 Estradiol: 0.265 0.252 0.156 0.041 1.6 8.0 0.196 0.195 0.116 0.040 1.5 Estriol: 0.293 0.243 0.141 0.026 0.039 1.7 10.0 0.245 0.210 0.107 0.034 0.051 2.2

Day 3 Estrone: 0.277 0.246 0.144 0.035 1.0 6.0 0.236 0.226 0. 132 0.042 1.3

Estradiol: 0.147 0.124 0.085 0.008 0.4 2.0 0.161 0.136 0.098 0.006 0.3 Estriol: 0.295 0.269 0.138 0.052 0.078 3.4 19.0 0.320 0.289 0.133 0.062 0.093 4.1 - 4 - Micrograms Day4 per day Estrone: 0.226 0.198 0.137 0.016 0.5 3.0 0.324 0.278 0.195 0.018 0.6 Estradiol: 0.186 0.170 0.134 0.010 0.8 2.5 0.200 0.178 0.148 0.004 0.3 Estriol: 0.397 0.326 0.200 0.027 0.041 1.8 9.5 0.298 0.244 0.143 0.028 0.042 1.9

Day 5 Estrone: 0.219 0.185 0.127 0.012 0.4 1.5 0.260 0.211 0.145 0.008 0.2 Estradiol: 0.180 0.171 0.145 0.008 0.7 2.5 0.160 0.140 0.111 0.004 0.3 Estriol: 0.297 0.253 0.174 0.017 0.026 1.2 6.0 0.290 0.241 0.162 0.015 0.023 1.1

Estriol _ 1 /10 1 0.5. = 11.5 - o 5 E strone 15 . 5 7 5. 5 7 2. 5 7 1. 0 24. 5 • Estri.W. - 11.5 - 11.5 = 0.3 Estrone f Estradiol 24.5 7 7} 1 7 1.5 f 1.5 35.5 Recovery = 47 = 11% 429 - 5 - Control Subjeot: F. I. Age: 53 Diagnosis: Coronary Sufficiency Micrograms Control Da;y: per day Estrone: .321 .301 .255 .013 0.7 4.5 K : 57.5 • 3:2! .S94 .322 . 018 1.0 Estradiol: .269 .241 .193 .010 0.6 3.5 K = 57.5 .354 .309 .237 .013 0.7 Estriol: .249 .248 .150 . 048 .072 3.0 15

Da;y2 Estrone: .504 .528 .428 .061 4.4 21.8 K = 72. .461 .491 .400 .060 4.3 Estradiol: .349 .339 .245 .042 2.7 13.5 K = 63.2 Estriol: .270 .279 .201 .043 .065 4.7 24 K • 72 .300 .328 .268 ..044 .066 4.8 Day 3 Estrone: . 553 .551 .460 .044 3.2 15.5 .471 .469 .384 .041 3.0 Estradiol: .351 .312 .239 .017 1.1 6.0 .410 .364 .280 .019 1.2

Estriol: .528 .555 .410 .086 .129 9.3 41 .288 .330 .240 .066 .099 7.1 Estriol

Estradiol:

Estriol:

Estrone: Da;y:5 Estrone Estrone: Da;y:4 % Estriol Estradiol: E

Estriol:

strone

recovery

1

-

E

1.74

•

stradiol

100.3

396

o. o:543 0.469 0.460

0.360

.318

.542 .549

55 .519 .585 •

.220

539

538

=

=

1

·

1

26%

0.480 0.488

0.400 0

0

.452

.469

.340

.486

.246

.

.

357

390

0.305

0.399 0.400

0.304

0.272

.260

.304

• .186

.400 .418

359

- 6 -

0.39

0.011 0.016

0.013 0.008

0.041

.025

.018 •

.051 .043

.017

016

.077 .065

.062 .059

1.2 1.2 1.2

1.1 1.6

0.8

4.5 4.3 0.8 4.7

5.5

0.5

Micrograms

per

25.5 6.0

3.5 7.0

5.0 2

"

2.0

day - 7 - Control Subject: F. 0. Age: 63 Diagnosis: Rheumatoid Arthritis Micrograms Control Da;r per day Estrone: 0.287 0.261 0.181 0.027 1.2 7.0 K = 43.4 0.301 0.283 0.194 0.035 1.6 Estradiol: 0.250 0.220 0.180 0.005 0.2 1.5 K = 47.6 0.252 0.230 0.194 0.007 0.3 Estriol: 0.452 0.409 0.310 0.028 0.042 2.3 11.0 K = 54.4 0.337 0.302 0.218 0,024 0.036 2.0 Day2 Estrone: 0,288 0.318 0.202 0.073 3.3 18.0 0.295 0.332 0.202 0.083 3.8

Estradiol: 0.291 0.289 0.174 0.056 2.7 13.0 0.272 0.279 0.180 0.053 2.5 Estriol: 0.378 0.344 0.218 0.046 0.069 3.6 17.5 K : 52.6 0.310 0.292 0.188 0.043 0.065 3.4 Day3 Estrone: 0.479 0.510 0.340 0.100 4.5 23 0.439 0.478 0.310 0.103 4. 7 Estradiol: 0.379 0.359 0.272 0.033 1.6 8.0 0.379 0.349 0.255 0.032 1.5 Estriol: 0.361 0.377 0.238 0.077 0.116 6.0 30.8 0.$38 0.522 0.342 0.082 0.123 6.3 - 8 - Micrograms Da;y4 per day Estrone: 0.360 0.339 0.235 0.041 1.8 9.5 0.345 0.330 0.230 0.042 1.9 Estradiol: 0.279 0.258 0.207 0.015 0.7 3.5 0.251 0.238 0.196 0.014 0.7

Estriol: 0.388 0.365 0.243 0.049 0.074 4.1 19.8 K = 55.6 0.319 0.472 0.332 0.046 0.069 3.8

Da;y 5 Estrone: 0,695 0.608 0.473 0.024 1.0 4.5 0.568 0.495 0.382 0.020 0.8

Estradiol: 0.282 0.251 0.212 0.004 0.2 0.5 0.242 0.214 0.185 0 0

Estriol: 0.328 0.308 0.210 0.037 0.056 14.0

Estriol 6.5 ~19.8t 8.8 t 3 = ~= 13 Estrone = 11 16 f 2. 5 29.5 . Estriol • 38.1 = 38.1 = 0 8 Estrone f Estradiol 29.5 f 20 49.5 .

% recovery. 87.6 18% 501 = - 9 - Control Subject: M. G. Age: 23 Diagnosis: Normal Micrograms Control Da~ per day Estrone: 0.253 0.227 0.171 0.015 0.6 3.0 K = 42. 0.267 0.227 0.156 0.015 0.6 Estradiol: 0.169 0.150 0.117 0.007 0.3 2.0 K = 37.9 0.249 0.220 0.170 0.010 0.4 Estriol: 0.172 0.177 0.120 0.031 .047 2.3 12.5 K = 49.8 0.220 0.223 0.153 0.036 .054 2.6

Dat2 Es ne: 0.390 0.431 0.291 0.090 3.5 18.0 K = 38.8 0.385 0.426 0.278 0.094 3.6 Estradiol: 0.300 0.313 0.200 0.063 2.6 13.0 K = 40.8 0.282 0.302 0.199 0.061 2.5 Estriol: 0.238 0.242 0.161 0.042 0.063 2.8 14.5 K = 44.2 0.225 0.233 0.151 0.045 0.068 3.0 Day 3 Estrone: 0.375 0.375 0.250 .062 2.4 11.5 Estradiol: 0.221 0.216 0.174 .018 0.7 3.5 0.257 0.248 0.200 .019 0.7 Estriol: 0.251 0.264 0.179 .049 .074 3.3 16.5 0.237 0.252 0.170 .048 .072 3.2 - 10 - Micro gram a Da;r4 per day Estrone: 0.319 0.298 0.211 0.033 1.3 7.0 0.291 0.277 0,201 0.040 1.5

Estrad.iol: 0.212 0.192 0.150 0.011 0.4 2.5 0.220 0.199 0.152 0.013 0.5

Estriol: 0.251 0.258 0.179 0.043 0.065 3.1 17.0 0.268 0.278 0.187 0.050 0. 075 3.6 Da;r5 Estrone: 0.256 0.229 0.167 0.017 0.7 3.5 0.238 0.210 0.154 0.016 0.6

Estrad.iol: 0.159 0.139 0.109 0.005 0.2 1.0 0.141 0.124 0.097 0.005 Estriol: 0.199 0.194 0.137 0.026 0.039 1.9 9.8 0.263 0.250 / 0.181 0.028 0.042 2.0

Estriol 2 f 4 1 4.5 = 10.5 = 0 4 Estrone = 16 7 8.5 f 4 f 0.5 29 . Es triol = 10.5 - 10.5 0.2 Estrone 7 E stradiol 29 7 11 f 1.5 7 0.5 ~ - % recovery • 14 - 11 - Control Subject: H.A. Age: 64 Diagnosis: Chronic Lung Disease Micrograms Control Da;ï Eer da;ï Estrone: 0.279 0.241 0.184 0.009 0.4 2 K = 40.0 0.254 0.218 0.164 0.009

Estradiol: 0.222 0.196 0.157 0.006 0.2 1 K = 39.7 0.231 0.200 0.159 0.005 0.2 Estriol: 0.240 0.207 0.140 0.017 0.026 1.3 6 K • 49.0 0.212 0.187 0.134 0.014 0.021 1.0 Day 2 Estrone: 0.363 0.399 0.220 0.107 4.3 21.0 0.319 0.352 0.184 0.100 4.0 Estradiol: 0.201 0.199 0.114 0.041 1.6 8.5 0.221 0.212 0.117 0.043 1.7 Estriol: 0.341 0.351 0.186 0.087 0.131 6.5 32.5 Day 3 Estrone: 0.267 0.231 0.165 0.015 0.6 3.5 0.300 0.259 0.184 0.017 0.7 Estradiot: 0.115 0.096 0.069 0.004 0 2 1.0 Estriol: 0.277 0.270 0.163 0.050 0.075 3.6 17.5 0.239 0.240 0.144 0.048 0.072 3.4 - 12 - Micrograms Da~4 ~day Estrone: 0.269 0.223 0.163 0.007 0.3 1.5 0.272 0.224 0.163 0.006 0.2

Estradiol: 0.·154 0.122 0.088 0.001 0 0.179 0.141 0.104 0.001 Estriol: 0.208 0.185 0.112 0.025 0.038 2.0 9.5 K = 51.6 0.602 0.518 0.390 0.022 0.033 1.7 Day5 Estrone: 0.231 0.196 0.144 0.008 0.3 1.5 0.278 0.231 0.171 0.006 0.2 Estradiol: 0.181 0.150 0.115 0.002 0.1 1.0 0.161 0.142 0.114 0.004 0.2 Estriol: 0.261 0.230 0.159 0.020 0.030 1.4 6.5 K = 47 ~2 0.238 0.209 0.147 0.016 0.024 1.1

E striol = 26. 5 i. 11. 5 i_ 3. 5 t_ 0. 5 _ '!!_ - 2.1 Estrone 19 f 1.5 20.5 Estriol _ 42 = 1.5 E strone j: E stradtol 2 8

Recovery = 16.8% - 13 - Control Subject: S. H. Age: 61 Diagnosis: P. T. C. Deficiency Micrograms Control Day Eer da;r Estrone: 0.170 0.154 0.114 0.012 0.5 2.5 K .. 37.8 0.174 0.161 0.119 0.014 0.5 Estradiol: 0.110 0.094 0.064 0.007 0.3 2.0 K • 49.0 0.127 0.105 0.072 0.009 0.4 Estriol: 0.125 0.119 0.081 0.016 o. 024 1.2 5.5 K • 51.2 0.128 0.119 0.083 0.013 0.020 1.0 Day2 Estrone: 0.237 0.279 0.175 .073 2.8 x 9 25.2 Estradiol: 0.149 0.167 .0.094 0.045 2.2 20.7 0.129 0.149 0.074 0.049 2.4 x 9

Estriol: 0.123 0.124 0.079 0.023 0.035 1.8 17.6 0.159 0.157 0.101 0.027 0.041 2.1 x 9

Da;r 3 Estrone: 0.209 0.208 0,144 0.031 1.2 8.8 0.263 0.258 0.184 0.034 1.3 x 7

Estradiol: 0.150 0.142 0.101 0.016 0.8 7 6.6 0.162 0.155 0.104 0.022 1.1x Estriol: 0.122 0.142 0.071 0.045 0.068 3.5 24.5 0.116 0.137 0,065 0.046 0. 069 3.5

Estriol Estriol

Estrone Estrone

Estriol Estriol

% %

Estriol: Estriol:

Estradiol: Estradiol:

Estrone Estrone

Estradiol: Estradiol:

Day5 Day5

Estriol: Estriol:

Estrone: Estrone:

Estrone: Estrone: Day4 Day4

recovery recovery

7 7

• •

Estradiol Estradiol

318 318

612 612

.. ..

30 30

• •

0.240 0.240

0.133 0.133

0.095 0.095

0.201 0.201

0.197 0.197

0.312 0.312

0.283 0.283

0.275 0.275

0.200 0.200

0.211 0.211

0.232 0.232

1

. .

93 93

=1.11 =1.11

0.228 0.228

0.287 0.287 0.124 0.124

0.086 0.086

0.251 0.251

0.243 0.243 0.210 0.210

0.183 0.183

0.203 0.203

0.182 0.182

0.179 0.179

0.071 0.071

0.028 0.028

0.205 0.205

0.191 0.191 0.167 0.167

0.146 0.146

0.156 0.156

0.172 0.172

0.136 0.136

0.132 0.132

0.139 0.139

-

14 14

-

0.014 0.014

0.061 0.061

0.022 0.022

0.010 0.010 0.006 0.006

0.010 0.010

0.019 0.019

0.026 0.026 0.013 0.013

0.024 0.024

0.007 0.007

0.033 0.033

0.036 0.036

.092 .092

1.7 1.7

1.8 1.8

4.7 4.7

0.9 0.9

0.5 0.5

0.3 0.3

0.4 0.4

0.2 0.2

0.4 0.4

0.6 0.6

0.4 0.4

x x

x x

x x x x

x . . x

x x

6 6

7. 7.

6 6

7 4 4 7 6 6

7.4 7.4

4 4

12.9 12.9

4.5 4.5

Micrograms Micrograms

~~ ~~

3.3 3.3

28.2 28.2

2.1 2.1 2.2 2.2 - 15 - Control Subject: W. E. Age: 81 Diagnosis: Bronchogenio Carcinoma Mic:rograms Control Da;ï Wsday Estrone: 0.402 0.395 0.265 0.061 2.6 . K = 42.4 0.330 0.330 0.215 0.057 2.4 Estradiol: 0.248 0.225 0.181 0.010 0.5 3.0 K = 47.6 0.281 0.250 0.195 0.012 0.6 Estriol: 0.370 0.342 0.258 0.028 0.042 2.2 12.0 K ... 53.1 0.312 0.290 0.205 0.031 0.042 2.5 Day2 Estrone: 0.412 0.481 0.291 0.129 5.5 28.0 0.402 0.477 0.285 0.133 5.6 Estradiol: 0,308 0.298 0.212 0,038 1.8 9.5 0.339 0.322 0.223 0.041 2.0 Estriol: 0.300 0.293 0.188 0.049 0.074 4.0 19.5 0.261 0.270 0.186 0.046 0.069 3.8 Day3 Estrone: 0.379 0.399 0.257 0.081 3.4 16.5 0,397 0.410 0.271 0.076 3.2 Estradiol: 0,388 0.308 0.215 0.031 1.5 7.0 0.260 0.237 0.160 0.027 1.3

Estriol: 0.257 0.262 0.164 0.051 0.077 ,.. 21.5 0.262 0.270 0.169 0.054 0.081 - 16 - Micrograms Da;r 4 12er da;r Es trone: 0.398 0.392 0.261 0.062 2.6 13.5 0.492 0.472 0.319 0.066 2.8 Estradiol: 0.292 0.251 0.179 0.015 0.7 4.0 0.329 0.293 0.220 0.018 0.9

Estriol: 0.358 0.339 0.222 0.049 0.074 3.9 21.5 0. 500 0.460 0.302 0.059 0.089 4.7

Da;r 5 Estrone: 0.499 0.419 0.231 0.054 8.0 0.495 0.420 0.239 0.053 Estradiol: 0.351 0.388 0.190 0.017 0.7 3.0 0.239 0.199 0.139 0.010 0.4 Es triol: 0.545 0.443 0.270 0.035 0.053 2.5 13.5 0.486 0.409 0.252 0.040 0.060 2.8

Estriol = 7.5 t 9.5 t 9.5 t 1.5 -~ =14 Estrone 15.5 1 4 7 1 20.5 .

Estriol = 28 • 28 =0 Estrone 7 Estradiol 20.5 f 6. 5 7 4 7 1 32 · 9

% recovery • 60 = 15% 401 ° - 17 - Control Stibject: W.A. Age: 69 Diagnosis: Peripheral Arteriosclerosis Micrograms Control Da;ï per day Estrone: 0.341 0.312 0.222 0.030 1.2 6.0 K - 39.4 0.358 0.329 0.235 0.032 Estradiol: 0.220 0.195 0.144 0.013 0.5 2.5 K = 42.4 0.191 0.175 0.137 0.011 Estriol: 0.212 0.200 0.140 0.024 0.036 7.5 K • 42.7 0.200 0.189 0.130 0.024 Day 2 Estrone: 0.459 0.514 0.299 0.140 5.6 28.0 0.399 0.475 0.272 0.139

Estradiol: 0.254 0.272 0.172 0.059 2.4 12.0 0.320 0.330 o. 219 0.060 Estriol: 0.250 0.241 0.166 0.033 .050 2.4 12.0 0.305 0.288 0.202 0.033 .050

Day 3 Estrone: 0.245 0.266 0.165 0.061 2.4 11.5 0.246 0.260 0.165 0.054 2.1

Estradiol: 0.196 0.191 0.147 0.019 0.8 4.5 0.218 0.204 0.146 0.022 0.9 Estriol 0.281 0.283 0.177 0.054 .081 3.9 18.8 0.220 0.230 0.140 0.050 .075 3.6 - 18 - Micrograms Da:t:4 per day Estrone: 0.309 0.289 0.212 0.028 1.2 5.8 0.360 0.331 0.250 0.026 1.1 Estradiol: 0.200 0.175 0.127 0.011 0.4 2.5 0.213 0.196 0.152 0.013 0.5 Estriol: 0.233 0.225 0.147 0.035 .053 2.6 11.8 0.208 0.200 0.136 0.028 .042 2.1 Day5 Estrone: 0.279 0.252 0.189 0.018 0.7 3.5 0.253 0.232 0.179 0.016 0.7 Estradiol: 0.188 0.165 0.124 0.011 0.4 2.0 0.185 0.163 0.122 0.009 Estriol: 0.229 0.214 0.146 0.026 0.039 1.9 10.0 0.250 0.233 0.161 0.027 0.041 2.0

Estriol = 4.5 l 11.3 l 4.3 l 2.5 - 22.6 = 0 8 Estrone 22 7 5. 5 27.5 •

Estriol - 22.6 0.6 Estrone 7 Estradiol - 27 . 5 7 9. 5 7 2. 0 = 2i86 = % recovery = 16 - 19 - Control Subject: Y .A. Age: 51 Diagnosis: Mitral Stenosis Micrograms Control Dal ~day Estrone: 0.319 0.291 0.209 0.027 0.9 4.5 K - 32.4 0.369 0.330. 0.239 0.026 0.8 Estradiol: 0.177 0.166 0.138 0.008 0.3 1.5 K = 40.9 0.181 0.162 0.127 0.008 Estriol: 0.401 0.361 0.229 0.046 0.063 2.9 14.0 K = 45~9 0.245 0.231 0.139 o·.039 0.059 2.7 Day 2 Estrone: 0.543 0.594 0.341 0.152 4.9 25.0 0.522 0.574 0.319 0.153 5.0 Estradiol: 0.321 0.331 0.194 0.073 3.0 15.0 0.299 0.309 0.173 0.073 Estriol: 0.223 0.241 0.125 0.067 0.101 4.7 23.0 0.204 0.223 0.113 0.064 0.096 4.5

Day 3 Estrone: 0.351 0.338 0.211 0.057 1.8 9.5 0.381 0.371 0.234 0.063 2.0

Estradiol: 0.166 0.145 0.100 0.012 0.5 2.5 Estriol: 0.263 0.300 0.142 0.097 0.146 6.5 32 0.331 0.349 0.180 0.093 0.140 6.3 - 20 - Micrograms Dai4 ~eblY Estrone: 0.330 0.298 0.205 0.030 1.0 5.0 0.289 0.263 0.181 0.028 0.9 Estradiol: 0.189 0.153 0.107 0,005 0.2 1.5 0.262 0.220 0.161 0,008 0.3 Estriol: 0,239 0.232 0.136 0.044 0.066 3.0 15.5 0.215 0.211 0.117 0.045 0.068 3.1 Dai5 Estrone: 0.290 0.271 0.194 0,029 0.9 4.5 0.319 0.308 0.241 0.028 0.9 Estradiol: 0.272 0.237 0.180 0.011 0.4 2.0 0.241 0.201 0.143 0,009 0.4 Estriol: 0.240 0.222 0.136 0.034 0.051 2.3 11.5 0.221 0,209 0.132 0.032 0,048 2.2

Estriol 9 18 5 = i. i. 1. :r: ~- 1.1 Estrone 20.5 1 5 f 0.5 26

Estriol - 28.5 = 28.5 = o. 7 Estrone 7 Estradiol 26 f 13.5 f 1.0 f 0.5 41 % recovery .. 17 Age:

K

K.

K

Diagnosis:

Estrone: Estriol Estradiol:

Estriol:

Dal1! Estriol Estrone: Estrone

Estradiol: Estrone

Estriol:

Control

% Control

=

•

recovery

49.5

43.1

40.7

52

2-6

Subject:

Da~

f

=

{Pooled

Estradiol

Coronary

5(16.3- 5{17.5

=

.

15

0.469 0.430 0.251 0.249 0.270 0.233 0.353 0.360 0.368

0.450 0.462

B.

Urine)

-

0.

13.5)

-

9)

Jnsufficiency

20

36.5

•

f

0.432 0.410 0.240 0.229 0.331 0.204 0.338 0.342 0.329

0.462 0.479

.

~

36.5

5(5

-

=

2.

0.5

5)

...

0,301

0.172

0.228 0.220

0.318 0.329 0.318 0.184 0.163 0.230 0.148

49

20

=

.

0.4

-

21

-

0.044

0.010

0.048 0.035

0.079 0,080 0.042 0.022 0,023 0.013 0.046

0.072 0.053

0.069

--

1.8

3.6

1.7 3.2 2.7 1.0 3.3

0.95 0.4 s-. 0.6

4

~rda~

Micrograms

13.5

2.5 16.3

9.0

17.5

5.0 - 22 - Control Subject: P. 0. Age: 55 Diagnosis: Cirrhosis Micrograms Control Da;y: per day Estrone: 0.152 0.140 0.104 0.012 0.036 1.5 7.0 K,.. 40.5 0.230 0.204 0.156 0.011 0.033 1.3 Estradiol: 0.218 0.192 0.144 0.011 0,033 1.4 7.0 K • &3.1 0.180 0.164 0.127 0.010 0. 030 1.3

Estriol: 0.164 0.151 0.107 0.015 0.075 3.8 18.5 .. K = 51.1 0 . 120 0.114 0.080 0. 014 0.070 3.6

Dai:S 2-6 (Eooled Urine) Estrone: 0.139 0.144 0.113 0.018 0.054 2.2 11.0 0.101 0.099 0.062 0.017

Estradiol: 0.076 0.069 0.048 0.007 0.021 0.8 4.0 0.091 0. 083 0.061 0. 007 Es triol: 0.168 0.164 0.101 0.029 0.159 0.158 0.096 0.030 0.090 4.5 22.5

Estriol • 5(22. 5 - 18. 5) = 20 = 1 Estrone 5(11 - 7) 20

% recovery - 40 = 9 422 - 23 - Control Subject: C. H. Age: 44 Diagnosis: Alcoholism Micrograms Control Dal:: per day Estrone: 0.373 0.343 0.268 0.022 0.9

K == 40.7 0.399 0.358 0.272 0.022 Estradiol: 0.288 0.242 0.190 0.003 0.1 1.5 K ... 43.1 0.302 0.259 0.188 0.009 0.4 Estriol: 0.452 0.420 0.271 0.058 0.087 4.7 23.5 \.._ K = 54.6 DaiS 2-6 (Pooled Urine) Es trone: 0.422 0.411 0.300 0.040 1.6 8.0 K = 40.7 0.469 0.432 0.319 0.038 1.5 Estradiol: 0.262 0.231 0.167 0.016 0.7 3.5 K = 43.1 0.236 0.209 0.150 0.016 0.7 Estriol: 0.518 0.513 0.301 0.103 0.155 7.7 38 K = 49.5 0.450 0,461 0.272 0.100 0.150 7.4

Estriol ... 5(38 - 23. 5) = 72. 5 = 4 1 Estrone 5(8 - 4. 5) 17.5 ·

Estriol - 72.5 - 2.6 E strone f E stradiol 17.5 7 5(3.5- 1.5)-

% recovery • 100 = 23 429 - 24 - M;Eocardial Infarction Subject: W. I. Age: 50 Micrograms Control Da;E Eer da;E Es trone: 0.318 0.300 0.221 0.030 1.2 9.8 K = 40.0 0.420 0.399 0.302 0.038 1.5 Estradiol: 0.228 0.208 0.175 0.006 0.2 2.1 K = 40.0 0.288 0.260 0.212 0.010 0.4 Estriol: 0.243 0.219 0.150 0.022 0.033 1.8 12.6 K = 53.7 Day2 Es trone: 0~382 0.461 0.300 0.120 4.8 34.3 0.380 0.459 0.288 0.125 5.0 Estradiol: 0.223 0.231 0.162 0.038 1.5 10.5 0.210 0.220 0.153 0.038 1.5 Estriol: 0.209 0.199 0.130 0.029 0.044 2.3 14.7 0.221 0.201 0.136 0.022 0.033 1.8 Day3 Estrone: 0.500 0.544 0.353 0.117 4.7 34.3 0.523 0.583 0.389 0.128 5.1 Estradiol: 0.292 0.271 0.201 0.024 1.0 6.3 0.241 0.220 0.160 0.019 0.8

Estriol: 0.373 0.364 0.239 0.058 0.087 4.4 33.6 0.402 0.394 0.251 0.067 0.101 5.2

% %

Estrone Estrone

Estriol Estriol

Estradiol: Estradiol: Estriol: Estriol:

Estrone Estrone

Estrone: Estrone: Estriol Estriol

Da!5 Da!5

Estriol: Estriol:

Estrone: Estrone:

Estradiol: Estradiol:

Da!4 Da!4

recovery recovery

= =

f f

24.5 24.5

2.1 2.1

Estradiol Estradiol

= =

144. 144.

1 1

f f

520 520

0.398 0.398

0.059 0.059

0.420 0.420 0.218 0.218

0.323 0.323

0.378 0.378

21 21

24.5 24.5

9 9

1 1

• •

= =

43.4 43.4

f f

28 28

53.2 53.2

77 77

2.8 2.8

/10.5 /10.5

0.281 0.281

0.192 0.192 0.379 0.379

0.378 0.378

0.126 0.126

0.359 0.359

7 7

7 7

8.4 8.4

1.4 1.4

7 7

:: ::

42 42

53.2 53.2

77 77

f f

0.280 0.280

0.142 0.142 0.249 0.249

0.252 0.252 0.218 0.218

0.036 0.036

1.4 1.4

.., ..,

1.5 1.5

f f

-

0.7 0.7

25 25

= =

-

0.012 0.012 0.044 0.044

0.044 0.044

0.010 0.010

0.039 0.039

0.078 0.078

'11. '11.

67.9 67.9

= =

1 1 1 1

• •

0.066 0.066

0.117 0.117

1.6 1.6

3.3 3.3

8.0 8.0

1.8 1.8

0.5 0.5

0.4 0.4

11.2 11.2

23 23

2.8 2.8

Micrograms Micrograms

12.6 12.6 3.5 3.5

per per

56 56 day day - 26 - M;ïocardial Infarction Subject: S. T. Age: 51 Micrograms Control day 12er da~ Estrone: 0.262 0.229 0.176 0.010 0.4 2.0 3. K = 42 0.202 0.185 0.137 0.015 0.7 3.5 Estradiol: 0.209 0.179 0.132 0,008 0.3 1.5 K. 40 0.218 0.183 0.132 0.008 0.3 Estriol: 0.219 0.201 0.132 0.025 0.038 1.9 11.5 K =50 0.358 0.317 0.204 0.036 0.054 2.7 Day2 Es trone: 0.42!0 0.450 0.318 0.071 3.0 15.0 0.442 0.448 0.312 0.071 3.0 Estradiol: 0.311 0.307 0.195 0.054 2.2 11.5 0.352 0.349 0.225 0.060 2.4 Estriol: 0.337 0.381 0.217 0.104 0.156 7.8 39.5 0.343 0.389 0.222 0.106 0.159 8.0 Day3 Estrone: 0.376 0.362 0.249 0.049 1.9 10.0 0.327 0.328 0.220 0.054 2.1 Estradiol: 0.300 0.272 0.178 0.033 1.3 7.5 0.399 0.350 0.222 0.039 1.6 Estriol: 0.352 0.400 0.221 0.113 0.170 8.4 41.8 0.428 0.460 0.268 0.112 0.168 8.3 - 27 - Micrograms Dax4 per day Estrone: 0.272 0.243 0.184 0.015 0.6 3.5 0.229 0.209 0.156 0.016 0.7 Estradiol: 0.258 0.210 0.180 0.006 0.2 1.5 0.209 0.176 0.120 0.011 0.4 Estriol: 0.312 0.306 0.201 0.049 0.074 4.2 21.5 0.295 0.292 0.184 0.052 0.078 4.4

Day 5 Estrone: 0.290 0.263 0.202 0.017 0.7 4.0 0.330 0.298 0.221 0.022 0.9

Estradiol: 0.303 0.264 0.197 0.014 0.6 2.5 0.264 0.231 0.175 0.011 0.4 Estriol: 0.258 0.258 0.166 0.046 0.069 3.9 19 0.230 0.238 0.154 0.046

Estriol • 27.5 1 29.8 1 9.5 1 7 =r 73.8 = 3 . 6 Estrone 12 f 7 f 0.5 Jf 1.0 20.5

Estriol = 73.8 = 2 0 Estrone f Estradiol 37.5 . % recovery - 29 - 28 - M;ïocardial Infarction Subject: S.A. Age: 67 Micrograms Control Da;ï per day Estrone: 0.330 0.310 0.213 0.038 1.3 6.5 K = 33.8 0.288 0.279 0.201 0.034 1.2 Estradiol: 0.500 0.432 0.302 0.031 1.2 K = 40.0 Estriol: 0.202 0.176 0.115 0.017 0.026 1.1 5.5 K- 41 0.199 0.176 0.116 0.018 0.027 1.1 Day2 Estrone: 0.522 0.601 0.329 0.175 5.9 31 0.483 0.581 0.292 0.193 6.5 Estradiol: 0.273 0.259 0.133 0.056 2.2 11.8 0.291 0.286 0.154 0.063 2.5 Estriol: 0.199 0.184 0.117 0.026 0.039 1.6 8.5 0.257 0.231 0.144 0.030 0.045 1.8 Day3 Es trone: 0.429 0.502 0.261 0.156 5.3 24.8 0.306 0.381 0.186 0.135 4.6 Estradiol: 0.285 0.260 0.186 0.024 1.0 5.0 Estriol: 0.409 0.391 0.232 0.070 0.105 4.7 23.5 ";" 29 - Micrograms Day4 ~day Estrone: 0.378 0.361 0.232 0.056 1.9 9. 0.319 0.311 0.205 0.049 1.7 Estradiol: 0.519 0.432 0.292 0.026 1.0 5.0 0.530 0.436 0.286 0.026 1.0 Estriol: 0.386 0.350 0.222 0.048 0.072 3.2 16.0 0.396 0.352 0.210 0.049 0.074 3.2 Day5 Estrone: 0.245 0.241 0.168 0.034 1.1 5.5 0.306 0.297 0.217 0.035 1.1

Estradid~: 0.196 0.178 0.136 0.012 0.5 2.5 0.194 0.174 0.133 0.010 0.4 Estriol : 0,302 0.291 0.194 0.0 43 0.065 3.2 15.5 0,222 0.221 0.142 0,039 0.059 2.9

Estriol = 41.5 = o. 9 Estrone 45.3

Estriol = 41.5 - 0.6 Estrone } Estradiot 45.3 7 19.5

% recovery = 106,3 = 25 400 - 3 0 - My8cardial Infarction Subject: P .A. Age: 52 Micrograms Control Da;ï Eer da;ï Estrone: 0.321 0.289 0.223 0.017 0.5 2.5 K. 31.1 0.508 0.437 0.334 0.016 0.5 Estradiol: -0.262 0.240 0.204 0.007 0.3 1.5 K • 38.5 0.303 0.272 0.224 -o.008 0.3

Estriol.: 0.499 0.418 0.290 0.023 0.035 1.7 7.5 K = 57.8 0.372 0.312 0.216 0.018 0.027 1.3 Day2 Es trone: 0.518 0.529 0.369 0.085 2.7 16.2 0.517 0.529 0.362 0.088 2..7

Estradiol: 0.315 0.33!) 0.234. 0.055 2.1 12.3 0.320 0.330 0.238 0.051 2.0 Estriol: 0.227 0.293 0.182 0.088 0.132 6.6 37.2 0.308 0.330 0.195 0.078 0.117 5.8 Day3 Estrone: 0. 480 -<>.429 0.324 0.027 1.0 6.6 0.432 0.398 0.302 0.031 1.2 Estradiol: 0.228 0.203 0.160 0.009 0.4 2.4 0.212 0.193 0.150 0.010 0.4 Estriol: 0.251 0.284 0.151 0.083 0.125 6.2 34.5 0.245 0.269 0.151 0.071 0.107 5.3 - 31 - Micrograms .. Day4 Estrone: 0.486 0.422 0.320 0.019 0.7 ~~0day 0.552 0.480 0.360 0.024 0.9 Estradiol: 0.243 0.216 0.165 0.012 0.5 2.5 0.217 0.184 0.134 0.008 0.4 Estriol: 0.195 0.187 0.105 0.037 0.056 2.8 15.3 0.518 0.454 0.302 0.044 0.066 3.3 ry5 ne: 0.454 0.392 0.290 0.020 0.8 4.8 0.513 0.445 0.339 0.019 0.8 Estradiol: 0.362 0.326 0.264 0.013 0.6 3.6 0.411 0.370 0.300 0.014 0.6 Estriol: 0.310 0.281 0.186 0.033 0.050 2.2 12.6 0.287 0.262 0.176 0.030 0.045 2.0

Estriol - 29.7 5.1 = 69.6 = 3 2 Estrone - 13.7 2.3 21.6 •

Estriol 69.6 • 1 9 Estrone f Estradiol • 21.6 7 10.8 f O. 9 f 1. 0 f 2.1 •

% recovery • ~ _ 27 391 - - 32 - MI:ocardial Infarction Subject: M.A.R. Age: 52 Micrograms Control Dai: Eer dai: Estrone: 0.465 0.409 0.312 0.020 0.8 4.0 3.8 K = 39.4 0.421 0.371 0.287 0.017 0.7 3.5 Estradiol: 0.298 0.268 0.209 0.014 0.6 3.0 K • 42.3 0.359 0 . 312 0.242 0.011 0.5 Estriol: 0.349 0.340 0.222 0.054 0.081 3.5 17.5 K • 42.7 Day2 Estrone: · 0.519 0. 498 0.362 0.057 2.3 0.518 0.490 0.348 0.057

Estradiol: 0.332 0.335 0.222 0.058 2.4 0.364 0.389 0.282 0.081 2.5 Estriol: 0.472 0.520 0.313 0.126 0.189 8.9 43.8 0.469 0.518 0.323 0.122 0.183 8.6 Day3 Estrone: 0.341 0.320 0.238 0.030 1.2 6.0 0.306 0.284 0.202 0.030 Estradiol: 0.261 0.233 0.179 0.013 0.5 3.0 0.269 0.239 0.179 0.015 0.6

Estriol : 0.400 0.451 0.255 0 .123 0.185 9.0 45.0 - 33 - Micrograms Day4 ~day Estrone: 0.432 0.384 0.301 0.017 0.7 3.5 0.459 0. 412 0.331 0.017 Estradiol: 0.319 0.296 0.240 0.016 0.6 3.5 0.330 0.298 0.230 0.018 0.7 Estriol: 0.393 0.413 0.260 0.086 0.129 6.4 32 0.417 0.430 0.273 0.085 0.128 6.4 Day5 Estrone: 0.352 0.304 0. 231 0.0 12 0.5 2.5 0.383 0.332 0.254 0.013 Estradiol: 0.246 0.221 0.180 0.008 0.3 1.5

0.282 0.253 0.20~ 0.008

Estriol: 0.361 0. 367 0.232 0.070 0.105 5.2 24. 5 0.339 0.341 0.221 0.061 0.092 4.6

Estriol = 26.3 é 27.5 é 14.5 t 7.0 ..... 7.6 Estrone 7.7 7 2.2 Estriol _ 75.3 Estrone f Estradiol - 22.4• 3 · 4 % recovery • 97.7 _ 24 412 - - 34 - Myocardial Jnfarction Subject: M. A. C. Age: 60 Micrograms Control Da! per day Estrone: 0.299 0.299 0.201 0.049 2.0 9.5 K = 40.2 0.329 0.330 0.220 0.045 1.8 Estradiol: 0.343 0.302 0.232 0.014 0.6 3.0 K • 40.1 0.231 0.208 0.157 0.014 0.6 Estriol: 0.193 0.178 0.124 0.019 0.029 1.4 7.0 K = 47.4 0.199 0.181 0.125 0.019 0.029 Day2 Estrone: 0.548 0.678 0.398 0.205 8.1 48 0.604 0.780 0.425 0.265 10.8 9.6

Estradio1: 0.251 0.297 0.164 0.089 3.4 16.8 0.282 0.322 0.189 0.086 3.3 Estriol: 0.320 0.315 0.202 0.054 0.081 4.0 19.8 0.280 0.281 0.176 0.053 0.080 3.9 Day3 Es trone: 0.429 0.466 0.287 0.108 4.2 0.433 0.471 0.294 0.107 Estradiol: 0.279 0.259 0.164 0.037 1.4 6.8 0.290 0.272 0.181 0.036 1.3 Estriol: 0.390 0.400 0.259 0.075 0.113 5.6 27.3 0.309 0.327 0.200 0.072 0.108 5.3 - 35 - Micrograms Day4 ~day Es trone: 0.449 0.463 0.308 0.084 3.0 15 Estradiol: 0 . 290 0.252 0.170 0.022 0.9 4.5 0.294 0.261 0.181 0.023 0.9 Estriol: 0.289 0.282 0.175 0.050 0.075 3.4 17.0 0.324 0.308 0.192 0.050 Day5 Estrone: 0. 370 0.362 0.248 0.053 2.1 10.5 0.399 0.392 0.279 0.053 Estradiol: 0.309 0.269 0.187 0.021 0.8 4.0 0.262 0.239 0.178 0.019 0.7 Estriol: 0.341 0.307 0.194 0.039 0.059 2.7 13.5 0.371 0.331 0.212 0.039

Estriol ... 12.8 ~ 20.3 ~ 10 t 6.5 • 0.9 Estrone 38.5 11.5 5.5 f 1.0 % recovery • 29 - 36 - Mlocardial Infarction Subject: L .A. Age: 45 Micrograms Control Dal ;eer dal Estrone: 0.303 0.254 0.195 0.005 0.2 1.8 K = 42,0 0.341 0.292 0.227 0,008 0,3 Estradiol: 0.282 0.237 0.175 0.008 0.3 1.2 K • 37.9 0,300 0.239 0.172 0,003 0.1 Estriol: 0.222 0.204 0.149 0,018 0.027 1.3 7.8 0.181 0.167 0,121 0.016 0.024 1.2 Day2 Es trone: 0.320 0.309 0.208 0.045 1.7 9.0 0.388 0.371 0.261 0,046 1.8 Estradiol: 0".320 0.305 0,209 0.040 1.6 8.5 0,370 0.340 0.225 0.042 1.7 Estriol: 0,331 0.391 0.219 0.116 0.174 7.7 38.5

Day 3 Estrone: 0.360 0,309 0.226 0.016 0.6 3,0

Estradiol: 0,228 0, 189 0.138 0,006 0,3 1.5 0.252 0.189 0,116 0.005 0.2 Estriol: 0.268 0.329 0.208 0.091 0.151 34.0 0.310 0.365 0.217 0.101 0.152 - 37 - Micrograms Day4 ~~ Es trone: 0.370 0.313 0,238 0.009 0.3 1.5 0.250 0.219 0.170 0.009 Estradiol: 0.261 0.219 0.162 0.007 0.3 1.5 0.237 0.197 0.149 0,004 0.2 Estriol: 0.185 .210 .124 0.055 0.083 20.0

Day5 Estrone: 0.335 0.281 0.209 0.009 0.3 2.0 0.360 0.309 0.232 0.013 0.5 Estradiol: 0.195 0.159 0.125 0 0.220 0.182 0.143 0

Estriol: 0.193 0.207 . 0.127 0.047 0.071 17.5 0.239 0.250 0.169 0.046 0.069

Estriol = 30. 7 ~ 26. 2 J. 12. 2 l. 9. 7 = 9.2 E strone 7. 2 1. 2 To. 2 % recovery = 24 - 38 - Mrocardial Infarction Subject: H.A. Age: 63 Micrograms Control Dar day Estrone: 0.329 0.280 0.210 0.010 0.4 n2. K = 37.9 0.308 0.260 0.196 0.008 0.3 Estradiol: 0.226 0.202 0.159 0.009 0.4 2.0 K = 39.4 0.191 0.172 0.137 0.008 0.3 Estriol: 0.230 0.202 0.144 0.015 0.023 1.1 5.5 K • 45.9 0.194 0.174 0.121 0.016 0.024 1.1 Day2 Estrone: 0.362 0.331 0.229 0.035 1.4 7.3 0.313 0.300 0.210 0.038 1.5 Estradiol: 0.262 0.288 0.194 0.060 2.4 11.5 0. 278 0.301 0.211 0.056 2.2 Estriol: 0.262 0.253 0.162 0.041 0.063 2.9 15.3 0.252 0.250 0.155 0.046 0.069 3.2

Day 3 Estrone: 0.439 0.373 0.282 0.012 0.5 3.0 0.412 0.353 0.262 0.016 0.6

Estradiol: 0.218 0.194 0.144 0.013 0.5 2.5 0.222 0.209 0.171 0.012 0.5

Estriol: 0.392 0.360 0.248 0.040 0.060 3.0 14.3 0.349 0.320 0.218 0.036 0.054 2.7 - 39 - Micrograms Day4 ~day Estrone: 0.314 0.271 0.209 0.009 0.4 2.0 0,302 0.264 0,208 0.009 Estradiol: 0.221 0.202 0.164 0,009 0.4 2.5 0.275 0.248 0.192 0.014 0.6 Estriol: 0.282 0.247 0.172 0.020 0.030 1.5 7.5 0.262 0.233 0.163 0.020 Day5 Es trone: 0.362 0.312 - 0.236 0.011 0.4 2.5 0.392 0.338 0.252 0.016 0.6 Estradiol: 0.229 0.209 0.167 0.011 0.5 2.0 0.186 0.166 0.132 0.007 0,3 Estriol: 0.429 0.372 0.269 0,024 0.036 1.8 9.3 0.312 0.278 0.194 0,025 0.038 1.9

Estriol = 9.8 ~ 8.8 ~ 2.0 t 2.8 = 3.4 Estrone 5.3 1. 0 O. 5 % recovery = 40.7 40.5 = 10 - 40 - ~ocardialInfarction Subject: D. E. Age: 66 Micrograms Control Da~ per day Estrone: 0.598 o.uo 0.4:09 0.016 0.7 3.5 K = 43.9 Estradiol: 0.401 0.349 0.271 0.013 0.5 2.5 K = 40.7 0.391 0.338 0.262 0.011 0.5 Estriol: 0.460 0.444 0.305 0.061 0.092 4.6 22.5 K = 50.5 0.470 0.462 0.339 0.057 0.086 4.3 Day2 Estrone: 0.335 0.346 0.225 0.066 2.4 12.3 0.300 0.315 0.195 0.067 2.5 Estradiol: 0.311 0.308 0.194 0.055 2.2 11.5 0.279 0.281 0.165 0.059 2.4 Estriol: 0.311 0.360 0.189 0.110 0.165 6.9 35.5 0.350 0.397 0.212 0.116 0.175 7.3

Da~3 Estrone: 0.320 0.289 0.203 0.027 0.9 4.5 0.322 0.289 0.204 0.026 0.9 Estradiol: 0.379 0.322 0.220 0.022 0.8 3.5 0.212 0.271 0.196 0.017 0.6 Estriol: 0.435 0.507 0.260 0.159 0.239 10.2 51.0 - 41 - Micrograms Da;r4 per day Estrone: 0.316 0.279 0.208 0.017 0.7 4.0 0.360 0.319 0.230 0.024 0.9 Estradiol: 0.269 0.231 0.175 0.009 0.3 2.0 0.248 0.216 0.160 0.012 0.5 Estriol: 0.362 0.379 0.225 0.085 0.127 5.4 26.0 0.362 0.374 0.229 0.078 0.117 5.0

Da;r 5 Estrone: 0.350 0. 307 0.222 0.021 0.8 4.0 0.358 0.312 0.230 0.018 0.7 Estradiot: 0.232 0.206 0.148 0.016 0.6 2.5 0.222 0.194 0.143 0.011 0.4 Estriol: 0.291 0.292 0.190 0.051 0.077 3.3 16.5 0.367 0.354 0.238 0.051

Estriol 13 l 28 . 5 ~ 3. 5 - 4. 2 Estrone = 8.8 f. 1.0 .57 .5

% recovery ::: 19 - 42 - M;ïocardial Infarction Subject: B. E. Age: 77 Micrograms Control Da;ï ~-~y Estrone: 0.281 0.248 0.187 0.014 0.4 2.5 K = 40.2 0.200 0.181 0.141 0.010 0.6 Estradiol: 0.204 0.184 0.146 0.009 0.4 1.5 K = 40.1 0.236 0.211 0.173 0.006 0.2 Estriol: 0.162 0.164 0.116 0.025 0.038 1.8 9.0 K • 47.4 0.156 0.156 0.105 0.025 Day2 Es trone: 0.353 0.328 0.235 0.034 1.3 6.5 0.381 0.351 0.252 0.034 Estradiol: 0.232 0.212 0.159 0.016 0.6 3.5 0.227 0.207 0.148 0.019 0.7

Estriol: 0.398 0.409 0.253 0.083 0.125 61 31.0 0.392 0.412 0.262 0.085 0.128 6.3 Day3 Estrone: 0.268 0.242 0.179 0.018 0.7 4.0 0.370 0.331 0.249 0.021 0.8 Estradiol: 0.298 0.261 0.203 0.010 0.4 2.0 0.289 0.238 0.167 0.010 Estriol: 0.427 0.469 o.eris 0.116 0.174 8.5 42.5 0.419 0.462 0.272 0.116 - 43 - Micrograms Dax4 ~day Estrone: - 0.253 0.219 0.169 0.008 0.3 1.5 0.289 0.242 0.181 0.007 0.2

Estradiol: 0.200 0.169 0.131 0.003 0~1 0.5 0.220 0.179 0.138 0. 0 Estriol: 0.300 0,321 0.189 0.076 0.114 5.2 29.0 0.309 0.338 0.184 0.091 0.137 6.3

Day 5 Estrone: 0,302 0.268 0.209 0.012 0.5 2.5 0.379 0.330 0.258 0.011 0.4 Estradiol: 0.209 0,175 0.140 0 0.266 0.231 0 . 192 0.002

Estriol: 0.337 0.349 0.208 0.076 0.114 5.2 23.0 0.308 0,299 0.178 0.056 0.084 3.9

Es triol 22 !..33 f. 20 t. 14 • 1.6 Estrone = 4.0 f1.5

% recovery = 25 -44- M;y:ocardial Infarction Subject: R. E. Age: 49 Micrograms Control Da;y: per day Estrone: 0.603 0.515 0.368 0.029 1.0 5.5 K = 35.7 0.668 0.565 0.402 0.030 1.1 Estradiol: 0.422 0.358 0.262 0.016 0.6 3.0 K • 39.4 0.381 0.321 0.232 0.014 0.6 Estriol: 0.372 0.380 0.222 0.083 0.125 5.8 29.0 K = 46.4 Day2 Estrone: 0.440 0.428 0.262 0,077 2.7 13.5 0.411 0.400 0.241 0.074 2.6 Estradiol: 0.352 0.341 0.212 0.059 2.3 11.0 0.392 0.363 0.228 0,053 2.1 Estriol: 0.422 0.452 0.249 0.111 0.167 7.8 39.0

Day 3 Estrone: 0.624 0.592 0.362 0.099 3.5 17.5 0.603 0.583 0.370 0.096 3.5 Estradiol: 0.439 0.401 0.255 0.054 2.1 11.0 0.471 0.440 0.292 0.058 2.3 Estriol: 0.522 0.643 0.282 0.241 0.362 16.8 84.5 0.578 0.683 0.302 0.243 0.365 16.9 - 45 - Micrograms nax4 per day Es trone: 0.360 0.298 0.218 0.009 0.3 4.5 0.453 0.371 0.273 0.008 0.3 Estradiot: 0.332 0.272 0.198 0.007 0.3 4.5 o: 358 0.287 0.202 0.007 0.3 Estriol: 0.269 0.258 0.156 0.045 0.068 3.2 46.5 0.278 0.252 0.140 0.043 0,065 3.0 nax5 Es trone: 0,600 0.502 0.352 0.026 0.9 4.5 Estradiol: 0.320 0.269 0.181 0.018 0.7 3.5 0.452 0.361 0.233 0.7 Estriol: 0.220 0.232 0.132 0.056 0.084 3.9 20.0 0.278 0.279 0.162 0.059 0.089 4.1

Estriol 10 ~ 55.5 17.5 = l = 4.2 Estrone 8 1?12

% recovery = 121 _ 29 422 - - 46 - Miocardial Jnfarction Subject: M. C • L. Age: 68 Micrograms Control Day :eer dai Estrone: 0.222 0.194 0.139 0.013 0.4 3.0 K = 32.4 0.211 0.186 0.133 0.014 0.5 Estrad.iol: 0.196 0.172 0.144 0.002 0.1 0.6 K = 40.9 0.111 0.115 0.084 0.002 0.1 Estriol: 0.195 0.164 0.106 0.013 0.020 0.9 5.4 K= 0.179 0.153 0,103 0,012 0.018 0.8 Day2 Estrone: 0.390 0.431 0.231 0.120 3.9 23.4 0.312 0.359 0.164 0.121 3.9 Estrad.iol: 0.252 0.231 0.131 0.039 1.6 10.2 0.320 0.292 0.179 0.042 1.7

Estriol: 0.127 0.124 0.075 0 ..023 0.035 1.6 10.2 0.240 0.215 0.141 0.024 0.036 1.7

Day 3 Estrone: 0.319 0.300 0.180 0.050 1.6 9.6 0.312 0.297 0.180 0.051 1.6

Estrad.iol: 0.195 0.155 0.104 0.005 0.2 1.2 0.182 0.144 0,095 0.005 Estriol: 0.231 0.219 0.129 0.039 0.059 2.6 15.6 0.202 0.196 0.111 0.039 - 47 - Micrograms Day4 12er dai Es trone: 0.319 0.281 0.186 0.028 0.9 5.4 0.341 0.300 0.201 0.029 0.9 Estradiol: 0.247 0.187 0.131 0.002 0 0.194 0.150 0.106 Q Estriol: 0.298 0.267 0.170 0.033 0.050 2.3 14.4 0.330 0.299 0.195 0.036 0.054 2.5 Day5 Estrone: 0.219 0.189 0.132 0.013 0.4 2.4 0.194 0.170 0.122 0.012 0.4

Estradiol: 0.213 0.167 0.116 0.002 0.1 0.6 0.262 0.208 0.151 0.001 Estriol: 0.223 0.193 0.125 0.019 0.029 1.3 8.4 0.241 0.211 0.138 0.021 0.032 1.5

Estriol _ 4.8 t. 10.2 ~ 9.0 t_ 3 Estrone 20.4 f 6.6 2.4 = 0.9

% recovery ... 16 - 48 - M~ocardialInfarction Subject: F .R. Age: 63 Micrograms Control Da~ ,eer da;r Estrone: 0.442 0.382 0.295 0.013 0.5 2.5 K = 40.5 0.392 0.340 0.262 0.013 0.5 Estradiol: 0.332 0.300 0.229 0.019 0.8 3.5 K"" 43.1 0.311 0.272 0.208 0,012 0.5 Estriol: 0.409 0.362 0.252 0.031 0.047 2.4 13.5 K = 51.5 0.302 0.288 0.196 0.039 0.059 3.0

Da~s2-6 (Pooled Urine) Estrone: 0.439 0.402 0.302 0.031 1.3 6.5 0.494 0.442 0.330 0.030 1.2 Estradiol: 0.261 0.232 0.160 0.021 0.9 5.0 0.253 0.234 0.164 0.025 1.1 Estriol: 0.453 0.450 0.298 0.074 0.111 5.6 28.0

Estriol = 28. 5 - 13. 5 = 14. 5 • 3. 6 Estrone 6. 5 - 2. 5 4

% reoovery • 24 - 49 - M;y:ocardial Infarction Subject: T .H. Age: 75 Micrograms Control Dal:: per day Estrone : 0.379 0.320 0.220 0.020 2.1 K = 35.7 0.353 0.300 0.208 0.019 Estradiol: 0.240 0.208 0.157 0.009 0.4 0.9 K = 39.4 0.220 0.185 0.137 0.006 0.2 Estriol: 0.137 0.139 0.085 0.028 0.042 2.0 5.7 K • 46.4 0.124 0.129 0.083 0.025 0.038 1.8 Da.y2 Estrone: 0.362 0.371 0.221 0.079 2.8 11.6 0.378 0.388 0.231 0.083 3.0 Estradiol: 0.189 0.189 0.112 0.038 1.5 6.0 0.194 0.192 0.115 0.037 1.5 Estriol: 0.137 0.161 0.081 0.052 0.078 3.6 15.2 0.201 0.224 0.132 0.057 0.086 4.0

Day 3 Estrone: 0.279 0.272 0.169 0.048 1.7 5.4 0.352 0.332 0.209 0.051 1.8 Estradiol: 0.222 0.192 0.131 0.015 0.6 1.8 0.243 0.213 0.154 0.014 0.6

Estriol: 0.208 0.255 0.130 0.086 0.129 6.0 17.7 0.170 0.222 0.106 0.084 0.126 5.8 - 50 - Miorograms Day4 day Es trone: 0.351 0.305 0.203 0.028 1.0 n3. 0.318 0.280 0.187 0.027 1.0 Estradiol: 0.258 0.212 0.154 0.006 0.2 0.9 0.255 0.219 0.165 0.009 0.4 Estriol: 0.237 0.278 0.136 0.091 0.137 6.4 18.3 0.345 0.362 0.213 0.083 0.125 5.8 Day5 Es trone: 0.332 0.280 0.196 0.016 0.6 1.8 0.431 0.371 0.278 0.016 Estradiol: 0.231 0.191 0.122 0.014 0.6 1.8 0.309 0.249 0.164 0.012 0.5 Estriol: 0.101 0.149 0.049 0.074 0.111 5.1 15.0 0.182 0.211 0.102 0.069 0.104 4.8

Estriol • 9.5 ~ 12 t 16.2 l.. 9.3 = 3 4 E strone 9 . 5 3. 3 f 0. 9 •

% recovery = 67. 6 = 16 T12 - 51 - Myocardial Infarction Subject: C. 0. Age: 53 Micrograms Control Da;ï: per day Estrone: .519 .471 • 359 .032 1.2 6.0 K = 37.1 .503 .439 .312 • 031 Estradiol: .305 .247 .176 .006 0.2 1.0 K = 40.4 .342 .281 .210 .005 Estriol: .162 .139 .106 .005 .008 0.4 1.5 K: 47.2 .140 .122 .098 .003 - .005 0.2 Day2 Estrone: . 665 .739 • .459 .177 6.6 32.0 .688 .742 .459 .168 6.2

Estradiol: .481 .469 .269 .094 3.8 19.5 .503 .500 .303 .097 3.9

Estriol: .452 .490 .271 .128 .192 9.1 46.0 .482 .520 .300 .129 .194 9.2 Day3 Estrone: .419 .379 .252 .043 1.6 8.0 Estradiol: .289 .243 .165 .016 0.6 3.5 .390 .329 .231 • 018 0.7

Estriol: .389 .398 .221 .093 .140 6.6 33.0 .449 .449 .262 .093 .140 - 52 - Micrograms Da~4 per day Estrone: .500 .432 .301 0.031 1.2 6.0 .605 .520 .369 0.033 1.2 Estradiol: .428 .359 .253 .013 0.5 3.0 .428 .348 .240 .014 0.6 Estriol: .538 .510 .305 .088 .132 6.2 33.5 .410 .421 .229 .101 .152 7.2

Da~5 Estrone: .480 .435 .325 .032 1.2 6.0 .465 .449 .369 .032 1.2

Estradiol: .245 .229 .200 .006 0.2 1.0 .306 .253 .189 ,005

Estriol: .369 .380 .208 .091 .137 6.5 31.5 .349 .361 .200 • 086 .129 6.1

Estriol =~= 4.9 Estrone 28 % recovery • 189 _ 430- 44