Clinical experiments in relation to the excretion of the estrogens

CJ,INICAII

EXPERIJYIENTS IN RELA.TION OF THE ESTROGENS”

TO THE

I. URISAKY

&TRO(;ENS BEFORE AND AFTER THE IN.JIWTIOPI’ INTO A PATIENT WITH SURGICAL MENOPBUSE

EXCRETION OB &TROSI<

GEORGE VAN S. SMITH, M.D., 0. WATKIXS SMITH, PH.D., AND SARA SCHILLER, B.S., BROORLINE, MASS. (From the Fenri?~g Ilrsenrch T,~borntory, Frw Rnspital for IC.O?WI~)

T

HIS and the following studies represent some of our efforts during the past two years to learn more about, the metabolism of the sex steroids both in normal women, in those with functional disorders, and in those with pregnancy toxemia. METHODS

The technique utilized for hydrochloric acid hydrolysis of urine, for extraction and separation of the estrogens, and for zinc-hydrochloric acid hydrolysis were the same as previously published.l~ ‘t Special precautions must be taken against loss or destruction of estradiol during semicarbazide treatment of the estrone-estradiol fraction. The whole procedure is carried out in a single receptacle. The evaporation after semicarbazone formation is performed in vacua and alcohol is added immediately, since estradiol is rapidly destroyed in concentrated acid solution. Since the difference in estrogenic potency of this fraction before and after the semicarbazide procedure is the estrone value, any 10s~ of estradiol would give a falsely high figure for estrone. Assay by difference is open to objection, especially when bio-assay must be used, but, we have found this method more dependable, when small amounts of estrone are present, than Girard’s separation and assay of the separated fractions. Assays were performed upon mature-spayed rats of known reactivity to a standard dose of crystalline estradiol (0.1 y in 0.1 C.C.of olive oil in one injection) and maintained in a primed condition by the induction of estrus (by test or a priming injection of estradiol) once in every seven to ten days. Each animal used for testing an unknown was in estrus five days previously. The material for a given assay was injected in three subcutaneous doses (0.1 to 0.2 C.C.of olive oil per injection) four hours apart and vaginal smears were t,aken forty-eight hours from the first injection. One rat unit (5 I. IT. by our assay) was considered present in that dosage of the extract which produced positive smears in 4 of 6 rats or in 50 per cent of 8 or more rats, provided no more than 25 per cent of 8 or more rats showed positive smears at the next lower dosage. The difference between dosage levels introduced a range of values over which the end point might lie, amounting to 10 to 25 per *This and the three papers on this subject to be published in succeeding issues presented in brief before the New York Obstetrical Society on Jan. 13, 1942. The Mrs. William Lowell Putnam Investigation of the Toxemias of Pregnancy aided by grants from the Committee on Research in Proble~ms of Rex of the National Research Council. :Thc xferences will accompany the fourth paper of this series.

wwe

455

456

AMERICAN

.IOIlRNAI,

Ok

OBSTETRICS

ASI)

t;\‘KE(‘OI,Ot;Y

rent of the end point figure recorded. ITsing this technique with our colony and with our criteria for int,crpreting smears, we have obtainrtl practically uniform standardization values for the three cr>.stall inch estrogens in 12 assays of each that have been performed during the last three years. With 0.1 y of crystalline estrone as the int.ernaiional lmit. WC find 1 I.IT. in 0.1 7 of crystnllinc cst.riol and in 0.01 'J of (*I*ystallinc~ estradiol. In calculating the number of micrograms of cstrogenic substarlce in urines, these standardization values are used, and it is assumed that all of the potency of each fraction is accountable to the specified estrogen. Although our present knowledge and the accuracy of the met,hods do 1101 entirely justify this procedure, a much closer approximation of the amount of est,rogen excreted results than if activity units alone a.re recorded. A considerable amount of invcsligation has convinced us that t,he methods of ext,raction and assay employed in these studies arc :IS good as any available and sufficiently quantitative to yield results of physiologic significance. The accuracy of all urine collections was checked hy det,ermination of creatinine.3 Specimens were kept cold during t,he period of collectiun and extracted within twelve hours of the last, voiding. ICiPI,AIi,ZTIOP

OF

TERMS

T, symbolizes the total urinary estrogens excreted as such, t,hat is, the total estrogenic potency of the urine after hydrochloric acid hydrolysis. It is the sum of the activities of the estradiol, estrone, and estriol fractions and is expressed in activity units (international units or estronc equivalents). T,,, also expressed in activity units, symbolizes the total estrogenic potency of the urine after prolonged hydrochloric acid hydrolysis in the presence of powdered zinc. This procedure has been shown2 60 convert any estrone in the urine into some compound approximately five times as active as estrone, to have no effect’ upon the potency of estradiol or estriol, and to render est,rogenically active certain urinary constituents which are excreted in an inactive form and which are not rendered estrogenie by simple hydrochloric arid hydrolysis. “Unaccounted for” T,, activity, therefore, is the difference between the total T,,, value and the sum of the potencies of the cstradiol, estriol, and five times t,hc estrone fractions. EXPERIMENT

The subject, Miss M.. aged 50 years, and in excellent health, had undergone supravaginal hysterectomy with bilateral salpingo-oophorcctomy nearly eight years previously. She had taken estrogemc substances intermittently until one month before the inception of this study, t.he results of which are presented in Table I. Three days after the injection of estrone a series of severe hot flushes supervened. The seventy-two-hour control specimen contained 90 I.U. of estrogcnic activity, which was located in the estradiol fraction. Small amounts of estrogenic activity in menopausa.1 urine have been reported before and assumed to come from the adrenal glands. During the three days following the in jcction of 5 mg. of estrone, 3,350 I.U. were recovered, 64 per cent as estradiol, 19 per cent as estriol! and

I

141.5

Y

(2.8%)

3,350

I.U.

(6.7%)

47.0

Y

ESTRYD

ESTROGENS

CASTRATE.

1

/

I

(152)

(64%)

(15%) 700

0.9

5,000

I

minus

(212)

(50%) 100 (9%)

0

ESTYNE

HC1

~RIKARY AFTER

1 (100%) (

1

SURGICAL

I-RINARY

0.9

TOTAL;;)

M.

1,100

1

MISS

I.U.

Y

I.U.

Y

I.U.

I.

141.5

1

=

HYDROLYSIS

0

4,860

(64;)

I

j

URINARY

AFTER

I.U.

(20.3%)

10,150

5,800

TOTAL,($

AND

y or 48,600

BF,FOR&

(27%)

(35%) 300

EST;

ESTROGENS

lost

1

estrone

c

1,500$

(26%)

“RO”,g”

ESTROGENS

INJECTION

*This value, obtained by difference in potency before and after semicarbazide treatment of the estrone-estradiol Derformine a seoaration with Girard’s reaeent. tZinc-hydrochloric acid hydrolysis of the ketonic fraction after Girard’s separation (474 I.U. estrone) yielded in potency.z Therefore, of the total 4,350 I.U. after zinc-hydrochloric acid hydrolysis of the urine itself, 2,375 I.U. 1.775 I.U. from estradiol and estriol, neither of which is affected by zinc-hydrochloric acid hydrolysis. :Similarly, this value is the sum of 700 I.U. estradiol. 300 1.17. estriol, and 5x100 I.U. estrone.

2/11-13/41 Total 72” recovery after injection 2/10-13/41

48” 24.72’ after inj.

2/10-11/41

2/10/41 First. 24” after inj.

control 2/3-6/41

72”

TABLE Zi’-HC1

fraction,

4,500 from

2 375 I U weke der’irgd

I

4,300 (74%)

/ COU;;f;FOR

AFTER

OF ESTRONE

5.3

confirmed

‘,a

by a fivefold increase from estrone and

was

/

1

HYDROLYSIS

458

AMERICAN

.JOTRNAl,

01”

OBSTJWRICS

.\SD

GYNECOLOt;I

17 per cent as e&one. In t,crms of activity, 6.7 per cent of the inject,ecI material found its way int,o the urine. This figure agrees with reportetl tuinary recovery of injected estronc in dogs.‘:, S Tn terms of -weight. however, only 2.8 per rent of ,thc injected cstrone was recovered, 1,; per cent as estradiol, 40 per cent as estrone, and 45 per cent as est,riol. after zinc-hydrochloric acid hydrolysis, 10,150 I.U., 20.3 per cent of the injected material in terms of potcncv, WI’C rccovercd. Fit?;\--six per cent (5,650 KU.) of this total T,,, activity came from the estrogens present before zinc-hydrochloric acid hydrolysis, as explained in the footnotes of the table, leaving 4,500 T.U., or 44 per cent, unac~counted for. DISCT-SSIOX ,, ,

If urine had been colIect,ed for another day following injection :r Iittie more estrogenic activity would probably have been recovered, hut not enough t,o alter significantly the presented figures. From reports now appearing435 and our own as yet incomplete dat,a, it is certain thal some, Icss than 10 per cent, of the administered hormone was excreted in the bile and possibly through the intest,inal murosa. We have repeatedly presented evidenee”-l’ that, estrone to estriol (*oilversion is a gauge of progestin activity, but. this subject following injection of estrone excreted a, goodly amount of activity in the estriol fraction in the absence of any known endogenous progestin. However, WV have also demonstrated that administered testosterone and adrenal (ortical extract act like propest,eronc in inhibiting destruction anal en hancing conversion of &radio1 to estronc to estriol.‘~ I’) Furt~hermor(~, WChave found that pregnancdiol glucrtronidate, a physiologically iilcrt excretion product of progesterone, augments the effect of progesteronc~ upon estrogen metabolism, through protecting progesterone against dostruction.lo It begins to appear that retardation of steroid degradation may be the primary factor in the caonversion mechanism. Thus, in the present experiment, the quantity ot’ e&one administered may it,sclC have clogged the estrogen destruct,ivc process enough to permit estronc? to estriol conversion, or it, may have had t,hc same final effect. indirectly through protecting adrenal steroids from destruction. That the high Icvel of estrogen was either direcilp or indirectly prevent.ing degradat,ion is indicated by the difference in partit,ion of urinary met,abolites during the first twentyfour hours injection, lvhen total estrogens were high, and the last forty-eight, hours, when the excretion per Iwent,:-four-hour volume was one-fourth as great. Jn the second specimen, evidence for a marked increase in the rate of estrogen destruction is found in the drop in cstronc and rise in T,,, to T,, ratio. The rate of estriol excretion decreased only 50 per cent during t’he last fort,y-eight hours, as compared with a 90 per (a(>ntdrop in e&one, an observation which is in keeping with the known stability of cstriol as compared with estrone to oxidative destruction. ifler

SMITH

ET

AI,.

:

EXCRETION

OF

ESTROGENS

459

The thesis that the partition of urinary estrogens provides an index of progestin activity, therefore, must be modified, especially when ovarian and placental secretions are absent or arc not the predominating factors in steroid metabolism. In the nonpregnant female with active ovaries, however, and in pregnancy the thesis still holds, as demonstrated by the urinary findings after administration of progesterone (see secoud paper of this series), during the luteal phase of ovulatory cycles 6, 11, 2nd third p:~per rd thiq series and the parallel curves of estriol and pregi&iediol in pregnancy.8-10 Contrary to our previous assumptions,’ based on the work of Pincus and Zahl, j2, I3 the ut,erus is not necessary for estrone-to-estriol conversion and the ovaries are not needed for the reversible estrone-to-estradiol reaction. Acid hydrolysis of urine with the addition of powdered zinc has in our st,udies consistently given higher estrogenic activity than acid hydrolysis alone. We have shown that this procedure does not change the activity of estradiol or estriol but results in about a fivefold increase in the potency of estrone, ascribable t,o conversion of this ketone into estradiol, probably a mixture of the LYand ,8 forms.2 The method, however, yields more estrogenic activity than can be accounted for even by maximum conversion of estrone, and this we have designated “unaccounted for. ” Although more complete hydrolysis with the longer boiling period of the zinc-hydrochloric acid procedure may contribute to this fraction, we have considerable evidence in favor of the assumption that resynthesis of estrogenic substance from inactive estrogen metabolites is the source of a large part of it, especially in urines with high T,, to T, ratios.2 Certainly in the present experiment the 4,500 I.U. of ‘ ‘ unaccounted for ” activity must have come from the injected estrone and at t,hat represents only about 9 per cent of the lost activity. CONCLUSIOss

From this experiment we would conclude that in the castrate female: Mrone is converted in the body both to estradiol and estriol; this conversion mechanism is favored by ret,ardation of steroid degradation; the internal genitalia are not necessary for these processes; less than 3 pei cent by weight of injected estrone reaches the urine in recognized forms; hydrolysis with the addition of zinc recovers more of the injected estrogen, a finding consistent with our hypothesis t,hat a good part of the additional activity from zinc-hydrochloric acid hydrolysis represents rehydrogenation of inactive products of estrogen oxidation.