co-estradiol -...

STUDIES ON PHENOLIC STEROIDS IN HUMANSUBJECTS.II. THE METABOLICFATE ANDHEPATO-BILIARY-

ENTERIC CIRCULATION OF C14"ESTRONEANDCO-ESTRADIOL IN WOMEN1

By AVERYA. SANDBERGANDW. ROYSLAUNWHITE, JR.(From the Departments of Medicine and Biochemistry, Roswell Park Memorial Institute,

Buffalo, N. Y.)

(Submitted March 13, 1957; accepted April 25, 1957)

It has been known for a number of years thatonly a small percentage of the metabolites of ad-ministered estrone or estradiol can be definitelyidentified in the urine of human subjects (1-6).At the most, 10 to 20 per cent of the injected estro-gen were recovered in the urine either by chemicalor biological assays. The presence of estrogens inbile of animals and in the feces of womenhad beenknown since 1928 (7, 8), but it was not until theefforts of Cantarow, Pearlman, Paschkis, Rakoff,Hansen and Walking that biliary excretion in thedog was shown to constitute a major route ofexcretion of estrogens (9-13). Subsequently,these investigators demonstrated substantial estro-genic activity in the bile of pregnant women (14).Interestingly, it was shown that the estrogens inthe bile of animals existed in the free form, in con-trast to the presence of conjugated steroids in thebile of pregnant women (6, 13). Based on theirextensive experiments, these authors advancedthe theory that estrogens participate in an entero-hepatic circulation.

Significant biliary excretion of estrone labelledwith radioactive bromine and estradiol labelledwith Il31 was demonstrated when these isotopesbecame available (15, 16). Unfortunately, theinclusion of the halogens renders the estrogensbiologically inactive and makes the results of thestudies difficult to compare with those obtainedusing endogenous steroids. Furthermore, pre-cise quantification of biliary and urinary estrogenexcretion was not possible due to lack of knowl-edge of all of the estrogen metabolites and im-perfections of the methods used for their deter-mination.

The availability of C'4-labelled estrone and es-tradiol has made it possible to study the metabolic

1 This study was suported in part by a grant (A-1240)from the National Institute of Arthritis and MetabolicDiseases, National Institutes of Health, U. S. PublicHealth Service.

fate of these steroids more accurately. Such stud-ies have been performed by Beer and Gallagher(17, 18) in human subjects. They demonstratedthat about 65 per cent of the radioactivity of C'4-estrone or C"4-estradiol were excreted in the urinewhen these steroids were injected either intra-muscularly or intravenously. The authors inter-preted their experiments to mean that biliary andintestinal elimination played a minor role in themetabolism of estrogens in human subjects. Itshould be pointed out that while Beer and Gal-lagher (17, 18) determined the radioactivity inthe feces of some of their subjects, no studies onbiliary excretion were performed. No reportshave appeared on the clearance of C14-estrogensfrom the blood, although two reports have indi-cated rapid clearance of injected large amounts ofestrogens (5, 19).

Even though the circulating estrogens have notbeen definitely identified in human subjects, someevidence has been presented to show that consider-able estrogenic activity may be associated withthe erythrocytes of blood (20-22) and may exceedthe levels present in plasma (20, 21).

In this paper we wish to present data on thedistribution of radioactivity in the bile, feces andurine following the intravenous injection of C"-estrone or C"4-estradiol in women. The rate ofclearance from the blood of the free steroids andtheir conjugated metabolites will be presented.In addition, the distribution of radioactivity inerythrocytes will be reported. The isolation andidentification of the various metabolites are thesubjects of separate communications (23).

METHODSAND MATERIALS

One to two /sc. of 16-C1'-estrone (B1) or 16-CM-estra-diol-17,B (E2) 2 (both 1Elc. per 370 pg.) were injected

2C4.-estrone and C"-estradiol were purchased fromCharles E. Frosst & Co., Montreal, Canada. When thepreparations were chromatographed in our laboratory,

1266

STUDIES OF PHENOLIC STEROIDS IN HUMANS

TABLE I

Clinical data pertaining to experiment subjects

Relation toSubject Age Wt. Steroid menstrual cycle Diagnosis

Kg.M. Su. 27 63.0 El 2 days pre-period NormalV. S. 21 53.0 El Middle NormalP. G. 32 53.0 El Middle NormalP. Go. 20 50.0 El 7 days post-period NormalR. C. 23 47.7 El Middle NormalA. D. 24 60.0 Es 4 days post-period NormalM. S. 66 44.5 E1+ Post-menopause Sarcoma of leg (pre-op.)

100 mg. El I.V.M. P. 44 50.9 El 1 day post-period Carcinoma of breast (post-op.)E. K. 52 43.3 E1+ Post-menopause Carcinoma of tongue with metastases

0.5 Gm. I.M.C. M. 56 103.9 El Post-menopause Carcinoma of breast, chronic pyelonephritisD. F. 29 60.9 Et 7 days pre-period Carcinoma of breast (pre-op.)J. B. 32 64.0 El Period began 1 day Carcinoma of breast (pre-op.)

after injectionG. D. 58 69.0 Es Post-menopause Leiomyosarcoma, diabetes mellitusM. B. 39 46.8 Es 7 days post-period Carcinoma of breast (post-op.M. K. 51 49.1 E,+ 7 days post-period Carcinoma of breast (post-op.

100mg. E, I.V.B. A. 30 99.4 El Pregnant Para IV, pregnancy in 8th monthS. R. 25 65.9 E, Pregnant Pam III, pregnancy in 9th monthF. M. 39 68.2 El 1 day pre-period T-tube drainage, cholecystectomy for chronic chole-

cystitisR. D. 60 78.4 El Post-menopause T-tube drainage, cholecystectomy for chronic chole-

cystitisP. C. 66 78.2 El Post-menopause T-tube drainage, cholecystectomy for chronic chole-

cystitisE. H. 49 60.0 E, Post-menopause T-tube drainage, cholecystectomy for hydrops of gall-

bladder with small cancer, diabetes mellitus, bilateraloophorectomy and hysterectomy 7 years ago

C. P. 72 46.6 E2 Post-menopause T-tube drainage, cholecystectomy for hydrops of gall-bladder, hysterectomy 6 months ago, diabetes mellitus

intravenously into 22 women: six normal females, twopregnant women in the last trimester, nine patients withcancer, and five women with bile-fistulas (T-tube drain-age). The clinical data related to these subjects arepresented in Table I. All the subjects were in goodnutritional status and none had evident renal or liverdysfunction or more than a mild anemia (hemoglobinconcentrations not lower than 11 Gm. per cent), exceptwhere indicated. The C'-estrone or C'4-estradiol wasdissolved in 1 to 2 ml. of absolute ethanol and then di-luted with 30 to 50 ml. of saline, which was injected in-travenously over a period of 1 to 2 minutes. In two ofthe subjects (M.S. and M.K.), intravenous infusions of100 mg. of the non-radioactive estrogens, dissolved inpropylene glycol and human serum albumin (24) andthen diluted with physiological saline, were given overa period of one hour. At the end of the infusion, theradioactive steroid was injected intravenously during aperiod of one minute. One patient (E.K.) was injectedintramuscularly, with 500 mg. of an aqueous suspensionof crystalline estrone over a period of five days (20 mg.five times daily). On the fourth day, 5 pc. of estronewere mixed with the injection mixture given at noon.

over 99 per cent of the radioactivity, determined in anActigraph (Nuclear Chicago), were located in the areascorresponding to those of standard compounds.

Except for slight, intermittent nausea and mild swellingand pain in the breasts, none of these three subjectsshowed any untoward effects from the large doses ofestrogens.

All the patients with T-tube drainage of bile hadnormal liver function tests pre-operatively. The steroids

* x TrTlm X75 A OA Os TrorTAL DOSE#4SrTOMOL x wuTOL amicrme

OLESTRONE C'4-ESTRADIOLSUBJECTS SUBJECTS

i.. 120

I so

~40

FIG. 1. THE PERCENTAGEOF RADIoAcTIVTY ExcRrm)IN THE URINE, STOOL AND fILE FOLLowING 16-C0-Es-TRONEOR 16-C4-ESTRADiOL ADMINISTATION

No stool collections were secured from subjects B.A.,E.K., R.C., A.D. and S.R. Subject E.K. received theCo-estrone intramuscularly.

41267

AVERY A. SANDBERGAND W. ROY SLAUNWHITE, JR.

C' ESTRONESUBJECTS

0 COUVLBERATyAW PAT mYa)$6

EXTACTDCOTS

LWO'ASATDCOWS

UU

COV4TSLAoRATde BY COVT EXTnMRACOVAr̂ wCOWSLRATEOBY,&.B- &MOVONSENAd$U

n

l F VI0

4 12144W 4 I234 U 44 ##4Itt 4 1*MIU 0444 44 S1 4 4?*4 Su3 4 431440 mUt4 -443334 4Fa44mS Vs Pe fRC us W EX OA

FIG. 2. EXCRETIONOF RADIoAcTIVrry IN THE URINE FOLLOWINGTHE INJECTION OF C14-ESTRONEIn this, and in the following figures, the numbers on the abscissa refer to the hour, following the injection of the C14-

steroids, when the collection was terminated.

were injected after a patient's condition had become sta-bilized without fever or other complications, after oralintake of fluids and food had been established, and neversooner than three days post-operatively. Complete biliary

45-

collections were assured by a special suction apparatusand by the appearance of acholic stools during and afterthe period of study.

The methods for collection and processing of urine,

C'L ESTRADIOL SUBJECTS

O UNEXTRACEDCOUS* UNCONWATED

3 COUwTSLIBERATED BYACIDrAWEATYrDAPLysI* COMUTLIERAEXDB CONvUO EXTCTrONATPI COaWts LIERATED BY -SWCURONSEN trOYSI$

H

AD CM OF Jo GD Me "K SR

FIG. 3. EXCRETIONOFRADIoAcTIVIY IN THEURINE FOLLOWINGTHEINJECTION OFC4ESTRADIOL

0U

30-

Z 25-aI-F

5

bt1 ff1

I]j

z 35-EzZas

aw4-

hiSao

I.

at *

1268


bile and blood, the determination of radioactivity, andthe various hydrolytic and extraction procedures havebeen described in a previous publication (25). One ad-ditional procedure was added in the latter part of thestudy. The determination of total radioactivity in thebile and urine samples was performed in a dual-channelliquid phosphor scintillation counter (TMC Model LP-24). One ml. of urine or 0.2 ml. of bile was added to10 ml. of a special mixture. The latter consisted of 1,500ml. of dioxane, 250 ml. of anisole, 250 ml. of ethyleneglycol dimethyl ether, 30 Gm. of 2,5-diphenyloxazole and1 Gm. 1,4-di-(2,4-diphenyloxazyl) benzene. The sampleswere then centrifuged and let stand in the dark, refrige-rated chamber before being counted. An internal stand-ard (1 ml. of a dilute solution of C1-acetic acid in thespecial mixture) was then added to each sample to de-termine the extent of "quenching." All samples werecounted for a sufficient length of time to register at least1,000 counts.

The plasma was separated from erythrocytes by centri-fugation and the red cells washed twice with large vol-umes (50 to 150 ml.) of isotonic saline. Total radio-activity was extracted from erythrocytes by addition of ab-solute ethanol to the cells, centrifugation after standingovernight in the cold, and repetition of the extractionprocess. The alcohol was evaporated to dryness and theradioactivity determined in the sample.

The plasma protein residues, following the extraction

40-

W 35z

z- 30

CM

25x

V)0 20a

0

.2

BILE-FISTULA SUBJECTS

UNEXTRACTEDCOUNTS

* UNCOAv/UGATEDCOUNTS

COUNrSLISERATED0 BYACDANOHEATHYDROLYSS

COUNTSLIBERATEDBYm -LUCURONIDASEHYDROLYSIS

COUTSLIBERATEDBY CONrINUOUSEMXRACTIONAr oHi

FM RD PC

C-ESTATE Ce-ESTRONE CZ-ESTRONEEH CP

C'e-ESTRADIOL -EESTfADIOL

35.

ia-Jz

a-

ww

x

nhi

0a

zU.0

[ COUNrSLIBERArED BYP-GLUCURONlDASEHYDROLYSIS

* COUNTSLIBERATED BYCONTINUOUSEXrRACTIONArPHIO COVNTSLIBERATED BYACID ANDHEATHYDROLYSIS

*. UNCONIUGATEDCOUNrS

o UNEXTRACTEDCOUNTS

FM RD PC

C-ESTRONE C-ESTRONE Cd-ESTRONE

l i.

FIG. 4. EXCRETION OF RADIOACTIVITY IN THE URINEOF SUBJECTS WITH BILE-FISTULAS FOLLOWINGTHE IN-JECTION OF C14-ESTROGENS

EH CP

C14-ESTRADIOL C'-ESTRADIOL

FIG. 5. EXCRETION OF RADIOACTIVITY IN THE BILE OFFmv SUBJECTS

Note that most of the extractable radioactive metabo-lites were released following hydrolysis at pH 1 or byhot acid.

of the plasma with chloroform and then with absoluteethanol, were combined and digested in 0.1 N NaOHover a steam-bath. The solution was then neutralizedand extracted with ether.

Throughout this paper the word "unconjugated," or"free," will refer to steroids which are extractable withether or chloroform before any of the specimens werehydrolyzed; the "glucuronidate fraction" to steroidswhich become extractable with ether or chloroform fol-lowing hydrolysis with fi-glucuronidase; and the "sulfatefraction" to steroids extracted continuously with etherfor 48 hours at pH 1.

RESULTS

The excretion of injected radioactivity in urine,feces and bile is shown in Figure 1. The amountexcreted in the urine during the 96 to 120 hoursfollowing intravenous injection of C14-estrone orC14-estradiol ranged (exclusive of the bile-fistulapatients) from 41 to 109 per cent (average 80 percent) of the injected dose. When the C14-estrone

-1

1269

-4J-


UCUTECOUN 3 cONs LIErAED Bor ACID NDm ADRYSs0 L"vxnucr~v coam

0 COUNTSLIBER 5YA-GLUCUNNDASEYD&ROYSISo usWRACE COUTSs * CSaLIBEsuRATEDBYoCNTNpEXTRACTIONAr p/fl

CZ-ESTRONESUBJECTS

g

Ct-ESTRADIOLSUBJECTS

I

F

IIS

toLKO

BILE-FISTULASUBJECTS

F.uK

iIRCS.

FIG. 6. RADIOACTIVITY ExcRETED IN THE STOOLS OF SUBJECTS INJECTEDWITH C*-ESTROGENS

Note that most of the extractableunconjugated form.

was injected intramuscularly in a mixture withlarge amounts of estrone in an aqueous suspension,only 31 per cent of the radioactivity was excretedin the urine of Subject E.K. in 144 hours. Thetotal radioactivity excreted in the urine of nor-

mal subjects did not differ significantly from thatof patients with cancer. Of interest was thelow excretion of radioactivity in the pregnantwomen, B.A. and S.R. The rate of excretion ofradioactivity in the urine following the adminis-tration of C14-estrogens is shown in Figures 2 and3. A comparison with the rates following theadministration of other C14-steroids (25-31) indi-cates much slower excretion of radioactivity in theurine following C14-estrogen administration thanthose following other C14-steroids. This was par-ticularly marked following C'l-estradiol adminis-tration when radioactivity was excreted in theurine as late as 4 to 5 days after intravenousinjection.

The urinary excretion of total radioactivity dur-ing certain time periods following injection isshown in Figures 2, 3, and 4, as well as theamounts of steroid metabolites extractable follow-ing various hydrolytic procedures. It is apparentthat a preponderant part of the injected G14-estroneor Cl"-estradiol is excreted in conjugated form,primarily as the glucuronidate. Except for a few

radioactive metabolites occur in the

urine samples where, for some reason as yet un-

known, large amounts of free metabolites were

present, unconjugated steroids were present invery small amounts (less than 4 per cent). Sul-fate-conjugated metabolites of C14-estrone or C14-estradiol constituted, in general, an unimpressivepart of extractable metabolites in the urine. Aninsignificant part of the radioactivity was extractedfrom the urine after strong acid hydrolysis. Onthe average, about 72 per cent of the counts in theurine were extractable following the various hy-drolytic procedures, the highest percentage (79per cent) being extracted from urines of patientsreceiving estradiol.

Patients with bile-fistulas excreted 35 to 52 per

cent (average 47 per cent) of the injected radio-activity in the urine (Figure 4). The amount ofradioactivity excreted during the initial 12 hourswas similar to that seen in non-fistula patients.Following that period, however, the urinary excre-

tion of radioactivity was much less than in theother subjects of the study. It should be pointedout that the bile-fistula patients constituted a

mixed group-: three injected with C14-estrone andtwo with C"4-estradiol. The amount of radio-active metabolites extractable from the urines ofthe bile-fistula patients was similar to that of thetotal group, i.e., the largest amount being pres-

~20-

Isf-

ci

91 I

1270


ent as the glucuronidate and the smallest as free.The amount of radioactivity excreted in the

bile is shown in Figure 5. The excretion rangedfrom 31 to 68 per cent (average 52 per cent) andoccurred primarily during the first 12 hours. Itis interesting to note that most of the extractablemetabolites were liberated either with continuousextraction at pH 1 or by hot acid. The amountsof free and glucuronidate metabolites were small.This contrasts with the pattern present in theurine. In addition, a larger percentage of thecounts in the bile (59 per cent) was non-extract-able after hydrolysis as compared to the urine (36per cent).

The radioactivity excreted in the stools (Fig-ure 6) of non-fistula patients varied from 1 to 18per cent (average 7 per cent). In the patientswith T-tube drainage, the fecal radioactivity ex-creted ranged from 0 to 2.2 per cent (average 0.9per cent). Most of the extractable radioactivityin the stool was present in the free form.

The clearance of free radioactive steroids from

I-

o Z

z

Luw

80.0lojeo

4.0-

3.0-

LO-

0.6-0.5-

0.4-

03.

02-

0%

0

TOTAL GROUPo---O C-ESTRADIOL SUBJECTS

-- CJ4ESTRONESUBECTSX---X BLE-FtSTULA SUBJECTS

6o 120

TIME IN MINUTES

0 15 30

FIG. 7. THE CLEARANCEOF "FREE STERom" RADio-ACTIVITY FROM THE PLASMA FOLLoWING THE INTRAVE-NOUSINJECTION OF CU4_ESTRONEOR CM-ESTRADIOL

Note initial rapid rate and subsequent slower rate ofclearance.

TABLE II

Radioactivity levels in the free steroid fraction of plasmafollowing injection of 16-C4-estrone

or 16-C4L-estradiol *

%of injected dose present in total plasma volume

Subject 15 min. 30 min. 60 mi 120 mln. 240 min.

C&L.estroneM.Su. 3.4 2.4 1.2 0.5 0.2V. S. 2.5 1.5 0.7 0.4 0.2P. G. 2.5 2.4 0.9 0.4 0.2M.S. 5.0 4.3 2.2 1.2 0.2M. P. 1.8 0.9 0.3 0.1 0.0B. A. 3.0 0.9 0.4 0.3F. M. 2.1 1.2 0.5 0.2 0.1R. D. 2.6 0.8 0.7 0.4 0.0P. C. 3.2 2.1 0.9 0.5 0.4

C1'estradiolC.M. 5.7 3.1 0.8 0.4 0.0D. F. 7.8 3.1 0.9 0.6 0.1J.B. 8.2 4.3 1.5 0.6 0.1G. D. 6.9 3.5 1.6 0.6 0.4M. B. 4.9 2.0 1.2 0.4 0.1M. K. 6.3 3.8 2.0 0.7 0.2E. H. 9.4 4.5 2.6 0.8 0.6C. P. 4.5 2.2 1.3 0.8 0.3

* In this, and the following tables, the number of countspresent in samples representing values less than 1 were ofsuch magnitude that their counting did not involve astandard error from the mean greater than 5 per cent; insamples representing values less than 0.5 the counting didnot involve a standard error from the mean greater than10 per cent.

the plasma of the injected subjects is shown inFigure 7 and Table II. For the sake of compari-son and uniformity, it was assumed that each sub-ject's blood volume constituted 9 per cent of hisbody weight, with 5 per cent as the plasma volumeand 4 per cent as the total red blood cell mass.The results are expressed as the percentage of theinjected radioactivity present in the total plasmavolume or red cell mass. From Figure 7 it can beseen that the free steroids were cleared from theplasma at two different rates, with half-lives ofapproximately 20 and 70 minutes. Even thoughthe radioactivity in the free steroid fraction tendedon the average to be lower (but not statisticallydifferent) following C14-estrone administrationthan that following C14-estradiol injection, theclearance rates were nearly identical. The pres-ence of a bile-fistula had no effect upon the levelof radioactive free steroid. Negligible radioac-tivity was present in the free steroid fractioneight hours following injection and none could bedemonstrated 24 hours later. In Figure 8 andTable III are shown steroid glucuronidate plasma

1271


&O.

4.0-

a0-

2.0-

2 je

bZonA:

* -- *TOTAL GROUP

o--.- 0C- ESTRADIOL SUBJECTS

_--* C- ESTRONESUBJECTSX--X ILE-FISTULA SUSECTS

0io Go 240

TIME IN MINUTESFIG. 8. RADIOACTIVE GLUCURONIDATESTEROID LEVEAS

IN THE PLASMA FOLLOWING THE ADMINISTRATION OF

RADIOACTIVE ESTRONEOR ESTRADIOL INTRAVENOUSLY

levels. Within 30 minutes following injection ofthe C14-estrogens, the radioactivity in the plasma"glucuronidate fraction" exceeded that present inthe unconjugated fraction. Activity declinedslowly thereafter. In contrast to the radioactivityin the free steroid fraction, a significant number

TABLE III

Radioactivity levels in steroid glucuronidate fraction ofplasma following injection of 16-C1Lestrone

or 16-C14-estradiol


Subject 15 min. 30 min. 60 min. 120 min. 240 min.

C14-estrone

M.Su. 0.8 1.3 2.4 1.8 1.3V. S. 1.1 1.5 2.6 2.1 2.3P. G. 1.4 1.9 2.9 1.6 1.1M. S. 4.2 5.5 4.5 4.0 1.5M. P. 1.9 2.5 1.6 0.8 0.4B.A. 3.6 4.3 2.5 1.8F. M. 1.4 2.1 1.9 0.8 1.2R. D. 2.2 3.1 4.1 2.5 0.8P. C. 1.8 2.2 2.4 2.0 1.6

C14-estradiol

C. M. 0.8 0.8 0.9 1.7 2.3D. F. 2.6 2.7 3.4 5.7 2.2J. B. 3.8 4.1 4.5 6.0 3.4G. D. 4.8 6.6 3.4 3.3 4.8M. P. 4.3 7.8 9.2 3.7 1.3M. K. 7.2 5.1 4.9 3.4 2.4E. H. 2.8 2.1 2.0 0.6 0.9C. P. 2.0 1.9 1.9 2.0 1.0

TABLE IV

Radioactivity levels in steroid sulfate fraction of plasmafollowing injection of 16^Cl&estrone

or 16-C04-estradiol


Subject 15 min. 30 min. 60 min. 120 min. 240 min.

C'4-estroneM. Su. 0 0.2 0.4 0.2 0.2V. S. 0.4 (combined specimens)*P. G. 0.8 (combined specimens)M. S. 1.4 3.6 2.7 1.6 0.7M. P. 0.3 (combined specimens)B. A. 1.1 (combined specimens)F. M. 0.7 1.0 2.2 0.5 1.1R. D. 2.0 (combined specimens)P. C. 0.1 0.2 0.3 0.3 0.2

Cl4-estradiolC. M. 1.2 0.5 1.3 0.5 0.2D. F. 1.1 1.1 1.1 1.6 0.5J. B. 0.4 0.4 1.1 1.4 1.2G. D. 0.1 0.2 1.1 1.1 1.0M. P. 1.2 1.4 1.3 1.1 0.5M. K. 0.2 0.8 0.4 0.3 0.1E. H. 1.0 1.3 2.4 1.0 0.8C. P. 0.8 1.0 0.8 0.6 1.2

* The values shown for the "combined specimens" referto the radioactivity present in the pooled plasma samplesobtained from the five blood samples drawn from eachpatient.

of counts was present in the glucuronidate fractionas late as one to two days after the injection of theC14-estrogen, particularly Cl4-estradiol. For ex-ample, Subject G.D. had 3.5 and 1.4 per cent ofthe injected radioactivity in the total plasma vol-ume 24 and 48 hours, respectively, following theinjection. Patient J.B. had 1 and 0.5 per cent 24and 48 hours, respectively, following the injectionof C-4-estradiol. It is interesting to note, again,that the radioactivity in the glucuronidate frac-tion following C"4-estrone administration waslower than that following C14-estradiol injection.On the other hand, the levels in the plasma of thebiliary fistula patients were the lowest of thegroup.

The counts present in the plasma "sulfate frac-tion" are shown in Table IV. The levels were onthe average one-third to one-tenth as high asthose in the glucuronidate fraction. In some sub-jects (F.M., M.S., C.M. and E.H.), however, theradioactivity in the sulfate fraction approachedthat present in the glucuronidate fraction of thesepatients.

The radioactivity present in the plasma frac-tions following hydrolysis by strong acid and heat

1272


was negligible. The radioactivity associated withthe red blood cells is shown in Figure 9 and TableV. It should be pointed out that the radioactivityextracted from the erythrocytes represents thetotal number of counts without subsequent hy-drolytic procedures. From the data it can be seen

that substantial radioactivity may be associatedwith the red blood cells, although the amount was

much lower than that present in the plasma.The radioactivity present in the protein residues

is shown in Table V. Since the calculations are

based upon the counts present in the combinedprotein residues, the results are only a rough ap-

proximation of the radioactivity associated withthe plasma proteins. In general, the radioactivityassociated with the proteins tended to be ratherlow in comparison with the radioactivity extractedfrom the plasma.

DISCUSSION

It is apparent from the data presented that thepreponderant part of the radioactivity of C14_estrone or C14-estradiol is excreted in the urine.Our findings are similar to those of Beer andGallagher (17, 18), who recovered about 65 per

cent of the radioactivity in the urine following in-travenous or intramuscular injection of radioac-tive estrone or estradiol. These findings contrastmarkedly with the results obtained in the past us-

ing large and unphysiological amounts of non-

radioactive estrogens. Thus, following injectionof 25 mg. or more of estrone or estradiol, usuallyless than 20 per cent was recovered in the urineas the three known estrogens (estrone, estradiol,or estriol) (1-6, 32). Without knowledge of allthe metabolites of estrone or estradiol, it was im-possible to determine how much of the injected

i,_27- DF. 29 YRS.

st _J 6- o of- oGLUCURONWESU S

- - LPHATES

j D 5- I \RE--x D BLOODCELLS0

4-.

0_2

012 4 24HOURS

FIG. 9. COMPARISONOF THE TOTALRADIOACTIVITY LEV-ELS PRESENTIN THE ERYTHROCYTESTO THOSE PRESENTIN THE PLASMA FREE, GLUCURONIDATEAND SULPHATEFRACTIONS OF SUBJECT D.F. FOLLOWINGTHE INJECTIONOF C14-ESTRADIOL

steroid appeared in the urine in some other form.The introduction of /8-glucuronidase hydrolysishas resulted in the liberation of more extractablefree phenolic metabolites following estrogen ad-ministration than was possible in the past (17).Labelling of the estrogens with carbon-14 makesit possible to measure recoveries more preciselythan with chemical or bioassay techniques.

In our studies about 45 per cent of the radio-activity were extractable from the urine followingB8-glucuronidase hydrolysis. Somewhat highervalues (about 60 per cent) were obtained by Beerand Gallagher (17, 18). It is possible that in our

TABLE V

Total radioactivity levels associated with the erythrocytes and plasma proteins following injection of16-C04-estrone or 16-Cl&estradiol

%associated%of dose present in total red cell volume with proteins

(combinedSubject 15 min. 30 min. 1 hr. 2 hr. 4 hr. 8 hr. 12 hr. 24 hr. 48 hr. samples)

M. S. 2.3 0.6 0.3 0.2 0.4 0.3 0.2 0.4M. K. 2.7 2.4 1.8 0.8 0.7 0.1 0.9M. B. 0.5 0.3 0.2 0.1 0.1 0.1 0.0 2.1M. P. 0.5 0.7 0.8 0.8 0.7 0.5 0.5B. A. 0.2 0.2 0.1 0.1 0.4G. D. 1.1 0.9 0.4 0.9 0.2 0.6 0.3 0.4 0.5J. B. 0.9 1.4 0.6 1.6 2.3 3.3 0.4 0.6 1.0D. F. 2.8 0.7 0.7 3.1 1.9 2.4

1273


study the re-incubation of urine with /8-glucuroni-dase could have resulted in further liberation ofmetabolites. In some of the urine specimens it wasindeed demonstrated that an additional 10 to 20per cent of the radioactivity become extractablefollowing such re-incubation. In addition, it ispossible that the incubation of the urine with,8-glucuronidase for longer periods of time, suchas employed by Beer and Gallagher (120 hours),than used in our experiments (48 hours) may re-sult in a greater percentage of extractable counts.Beer and Gallagher, in addition, demonstratedthat following the injection of G14-estrone or C14-estradiol 8 to 10 per cent of the radioactivity in theurine were present as estrone and 11 to 31 percent were present as estriol. Even though therecent identification of new metabolites (23, 33-35) of estrone and estradiol has enlarged ourknowledge of the metabolism of these steroids, asignificant part of the metabolites of the injectedestrogens remains unknown, especially those re-maining in the urine following the various hydro-lytic and extraction procedures.

Intravenous injection of large amounts of es-trone or estradiol with the radioactive steroids didnot affect the rate of their excretion. On theother hand, the intramuscular administration of asuspension of crystalline estrone with C14-estroneresulted in only 31 per cent excretion of the radio-activity in the urine over a period of many days.Beer and Gallagher (18) found 65 and 72 percent of the radioactivity in the urine following theintramuscular injection of C14-estradiol with largeamounts of carrier steroid, and 65 per cent fol-lowing oral ingestion. The discrepancy betweenthe results in our Subject E.K., and those of Beerand Gallagher may be due to the fact that theseauthors administered the steroid in solution inbenzyl alcohol and sesame oil, whereas SubjectE.K. was injected with an aqueous suspension ofestrone resulting in slower absorption of thesteroid.

Radioactivity is excreted much more slowly fol-lowing C14-estrone or C"4-estradiol administrationthan that following other C14-steroids. Similarobservations were made by Beer and Gallagher(17, 18). Thus, following C14-labelled cortisol(27-29), testosterone (25, 26), 1 1,8-hydroxy-androstenedione (31, 36) or corticosterone (30)administration, over one-third of the radioactivity

is excreted in the urine in the first four hours andover 60 to 70 per cent in the first 12 hours. Onthe other hand, following C14-estrone or C"-estradiol administration, only 12 per cent are ex-creted in the first four hours and about 33 per centafter 12 hours. Following the administration ofthe former steroids, radioactivity excretion in theurine is usually essentially complete in 48 hours,whereas following C14-estrone or C14-estradiol sig-nificant radioactivity was being excreted as lateas 96 to 120 hours after the administration of thesteroids intravenously.

Even though, in general, the importance of thebiliary excretion of estrogens and their entero-hepatic circulation had been accepted in the pastto be true for animals, e.g., dogs, cows (6, 37),such has not been the case for human subjects.Our data demonstrate, however, that not only wasmore of the radioactivity of C4-estrone or C"-estradiol excreted in the bile than that seen fol-lowing the injection of any other known steroidin human subjects (25, 28-30, 38), but in addi-tion more of the radioactivity appeared in the bilethan in the urine of three of the bile-fistula sub-jects. This establishes the importance of biliaryexcretion in the metabolism of estrogens in women.The importance of the biliary route of excretion ofestrogens is in accord with the earlier findings indogs and in pregnant women (9-14). Similarresults were obtained by Albert, Heard, Leblond,and Saffran in animals using I'll-labelled estradiol(16). In addition, Twombly and Schoenewaldt(15) demonstrated considerable excretion of ra-dioactivity in the bile of human subjects followingthe injection of estrone labelled with radioactivebromine. Since the introduction of a halogeninto the steroid molecule may affect its metabo-lism in an unknown fashion, it is difficult to com-pare these results with those obtained using C14-labelled naturally occuring estrogens.

The large excretion of radioactivity in the bilefollowing C4-estrogen administration is at vari-ance with results obtained by others when excre-tion of non-labelled estrogens was studied in hu-man subjects (39, 40). This is not surprisingsince even with labelled C14-estrone and C4-estradiol only about 40 per cent of the radioac-tivity could be released and extracted from thebile following various hydrolytic procedures. Inaddition, the estimation of the biliary excretion of

1274


metabolites following non-radioactive administra-tion depended on biological assay or determinationof a few known estrogen metabolites. Neitherprocedure can estimate all the metabolites of estro-gens in the bile. Thus, although Rubin, Dorfmanand Miller (39) found at least a 30-fold increasein the urinary and an 8- to 10-fold increase inbiliary excretions of the three naturally occuringestrogens following the administration of 200 mg.of estradioldipropionate, the total recovery wasvery small. Somewhat similar findings were ob-served by Stimmel (40).

In contrast to the findings in the bile of animals,where the estrogens occur in unconjugated form,almost all of the estrogen metabolites present inhuman bile were in conjugated form. In addition,the percentage of glucuronidate conjugated me-tabolites was small when compared to similar me-tabolites in the urine. Most of the extractablemetabolites in the bile were released by hydrolysisat pH 1 (with continuous ether extraction) andby hot acid. It should be pointed out that most ofthe radioactivity in the bile was not extractablefollowing all hydrolytic and extraction procedures.

Since, on the average, only about 7 per centof the radioactivity appeared in the stool of thenon-fistula patients, it is apparent that a consider-able reabsorption of estrogen metabolites excretedin the bile must occur in the gastrointestinal tract.In addition, patients with bile-fistulas excretednegligible amounts of radioactivity in the stools.The small amounts that were present may pos-sibly be due to escape of small amounts of bileor to the excretion of steroid metabolites in thegastrointestinal juices. It was interesting to notethat most of the extractable radioactivity in thestools was in the free form. Since the steroidsoccur in the bile in conjugated form, it is possiblethat the free steroids present in the stool are dueto the fi-glucuronidase and phenolsulfatase ac-tivities of the feces. Such activities have beendemonstrated by several observers (28, 41).

Radioactive estrone or estradiol was clearedfrom the plasma rapidly and at somewhat fasterrates than the free steroid clearance following theadministration of C"4-cortisol or Cl(-testosterone(25, 29). In addition, there was no significantdifference in the clearance of the free steroidsamong the various subjects studied. The clear-ance of the free steroids from the plasma occurred

FIG. 10. SCHEMATIC REPRESENTATIONOF THE VARI-OUS POSSIBLE PATHWAYSOF ESTROGENMETABOLISM INHUMANSUBJECTS

The thickness of the arrows indicates roughly thequantity of steroid metabolites involved in the metabolicpathway.

at two different rates, an initial fast rate with ahalf-life of approximately 20 minutes, which isprobably due to mixing of the free steroid withina body pool, and a slower rate, with a half-life of approximately 70 minutes, which prob-ably represents the true metabolic clearance ofthe free steroid metabolites. The conjugates ofthe radioactive estrogens reached their peak ofradioactivity within 30 minutes and declinedslowly thereafter. Following the initial 15 to 30minutes, the levels of the glucuronidate markedlyexceeded those of the free steroids. In addition,no free steroid radioactivity was demonstrable 24hours following the injection of the radioactiveestrogens. On the other hand, substantial radio-activity in the conjugated fractions was demon-strable as late as 48 to 72 hours following the in-jection. Of importance was the demonstrationthat the conjugated steroids in the plasma of pa-tients with bile-fistulas tended to be the lowest ofthe group.

Based on the data of the present experimentsand those published in the papers cited, a clearerpicture of the metabolic fate and the importance ofthe hepato-biliary-enteric circulation in the metab-olism of administered radioactive estrogens in hu-man subjects can now be advanced. The meta-bolic pathways of estrogen metabolism are sche-matically represented in Figure 10. Following in-jection of radioactive estrone or estradiol a cer-tain amount (usually one-third) is excreted inthe urine conjugated with glucuronic acid. Thisexcretion occurred within the first 24 hours fol-lowing administration and was evident not only inthe non-fistula patients but also in the patientswith T-tube drainage. About one-third to over

1275

AVERY A. SANDBERGAND W. ROY SLAUNWHITE, fR.

one-half of the radioactivity of the administeredestrogens appeared in the bile, mostly as conju-gates which are still unknown. We would liketo advance the hypothesis that the conjugates aresplit in the gastrointestinal tract and then reab-sorbed, as indicated by arrow 4 in Figure 10.Following reabsorption, some of the metabolitesare conjugated again in the liver with glucuronicacid and excreted in the urine, but the preponde-rant part is reconjugated and re-excreted in thebile to repeat the cycle just described. It is pos-sible that a small amount of metabolites reabsorbedfrom the gastrointestinal tract escapes conjuga-tion by the liver and enters the circulation to beexcreted free in the urine. This is indicated byarrows 5 and 1 in Figure 10. The fact that glu-curonidate-conjugated estrogens are not excretedappreciably in the bile, in contrast to the excre-tion in the urine, lends further support to the hy-pothesis that the metabolites which appear in thebile must be hydrolyzed in the gastrointestinaltract and reabsorbed as unconjugated steroids,which upon re-entry into the liver are conjugatedwith glucuronic acid, and are excreted in the urine.An alternate hypothesis would have the steroidmetabolites absorbed from the gastrointestinaltract as conjugates, which in the liver under dy-namic equilibrium are partially converted to adifferent type of conjugate.

This continuous recirculation of the metabolitesof estrone and estradiol through the liver to thebile and the gastrointestinal tract and the excre-tion of a certain small percentage of this reab-sorbed material in the urine accounts for the slowand delayed excretion in the urine of the radio-activity following the administration of C"4-estroneor C14-estradiol. In contrast, steroids which areexcreted rapidly in the urine are minimally ex-creted in the bile. This has been demonstratedto be true for such steroids as cortisol, cortisone,1 l-,8-hydroxyandrostenedione and testosterone.The re-entry of metabolites through the gastro-intestinal tract into the general circulation prob-ably accounts for the persistence of high levels ofglucuronidated metabolites in the plasma of thesubjects studied. In addition, the low levels ofplasma glucuronidated steroids in the T-tubedrainage patients are probably due to failure ofreabsorption of the steroids present in the bile(Figure 8).

The postulate was advanced by Beer and Gal-lagher (17, 18) that the slow urinary excretion ofradioactivity following C"4-estrogen administra-tion, as compared to the excretion following otherC"4-steroid injections, e.g., testosterone, is relatedto the binding of the estrogens and their metabo-lites to plasma and liver proteins (42, 43) withresultant retardation of their renal excretion. Inaddition, these authors felt that the "relativelyminor excretion of estrogen metabolites in thefeces is suggestive that biliary and intestinal elimi-nation played but minor roles in the metabolism ofthe estrogen by human subjects." These authorsdid not present data, however, on biliary excretionof radioactivity following administration of C4_estrogens. The results of our experiments do notsupport either of their concepts. Wehave previ-ously been able to demonstrate that those steroidswhich are most avidly bound to human plasmaproteins have a substantial biliary elimination,whereas those least bound were excreted insig-nificantly in the bile (44). As in the presentstudy, it was shown that the exclusion of the bilefrom the gastrointestinal tract results in rapidexcretion of radioactivity in the urine. Thus,nearly all of the radioactivity following the in-jection of C"4-estrone or C14-estradiol appeared inthe urine and bile within 24 hours. Of the radio-activity excreted in the urine almost 90 per centappeared in the urine in 24 hours and over 60per cent in the first 12 hours, in contrast to 60 percent and 35 per cent, respectively, in the non-fistula group. It should be pointed out that theresults of studies indicate avid binding of the es-trogenic conjugates to human plasma proteins(44), but the exact role that this binding plays inthe rate of urinary excretion of estrogenic meta-bolites awaits more conclusive and extensivestudies.

The appearance of about 7 per cent of radio-activity in the feces, an amount equivalent to thatfound by Beer and Gallagher in their studies (17,18), indicates that reabsorption from the gastro-intestinal tract of the biliary metabolites may notbe complete under normal circumstances, sinceless than 1 per cent of radioactivity appeared inthe feces of patients with bile-fistulas. Since neg-ligible radioactivity is present in the unconjugatedfraction in the bile, one would predict that theclearance of the free steroids from patients with

1276


T-tube drainage would resemble that seen in pa-tients without fistulas. That such is the case isdemonstrated in Figure 7. The clearance of thefree steroids from the plasma of patients withbile-fistulas was almost identical to that of thetotal group.

The association of radioactivity with the redblood cells would appear to indicate that a sig-nificant percentage of endogenous or injected es-trone or estradiol and their metabolites are as-sociated with the erythrocytes (20-22, 45). Thebulk of the radioactivity was present in the plasma,however. Our findings are at variance with previ-ously published reports in which more estrogenicactivity was found in the red blood cells than inthe plasma (20, 21). The nature of the metabo-lites associated with the red blood cells and theeffects of these cells on the metabolism of estrogensis a subject which deserves further study.

SUMMARY

C14-estrone and C14-estradiol have been injectedintravenously into 22 women. A little over 80per cent of the administered radioactivity were ex-creted in the urine during the 96 to 120 hoursfollowing the injection of these steroids. On theaverage, 72 per cent of the counts in the urine wereextractable folowing various hydrolytic proce-dures, with the preponderant part being present inthe glucuronidate form. About 50 per cent of theradioactivity administered appeared in the bile,collected through T-tube drainage, mostly as con-jugates released by hydrolysis at pH 1 or by thehot acid. Approximately 7 per cent of the radio-activity appeared in the stools of non-fistula pa-tients, indicating gastrointestinal reabsorption ofmetabolites excreted in the bile. The radioactivesteroids were cleared from the plasma rapidlywith at least two separate rates having half-livesof approximately 20 and 70 minutes, respectively.The conjugated metabolites of C"4-estrone andC14-estradiol reached their peak in the plasmawithin 15 to 30 minutes following the injection ofthe steroids and retained high levels for as longas three days. The levels of the conjugated ster-oids in the plasma of patients with T-tube drain-age of bile were the lowest of the group. Thesignificance of the hepato-biliary-enteric circula-tion of estrogens in relation to the slow urinaryexcretion of the radioactivity is discussed.

ACKNOWLEDGMENTS

Wewish to thank Dr. John Paine, Professor of Sur-gery, University of Buffalo Medical School, for his in-terest in the study and for his cooperation in makingavailable to us patients with T-tube drainage. Wewishto thank Mrs. Lucille Zablotny, Miss Maria Foldes andMr. Lawrence Beecher for technical assistance.

REFERENCES

1. Smith, G. V., and Smith, 0. W., Observations con-cerning the metabolism of estrogens in women.Am. J. Obst. & Gynec., 1938, 36, 769.

2. Pearlman, W. H., and Pincus, G., The metabolism ofestrone in men. J. Biol. Chem., 1943, 147, 379

3. Pincus, G., and Pearlman, W. H., Metabolism of es-trone in men and non-pregnant women. Endo-crinology, 1942, 31, 507.

4. Heard, R. D. H., and Hoffman, M. M., Steroids IV.The fate in man of injected a-estradiol. J. Biol.Chem., 1941, 141, 329.

5. Zondek, B., and Black, R., Estrone clearance test ininfectious hepatitis. J. Clin. Endocrinol., 1947, 7,519.

6. Paschkis, K. E., and Rakoff, A. E., Some aspects ofthe physiology of estrogenic hormones. Rec. Prog.Hormone Research, 1950, 5, 115.

7. Gsell-Busse, M. A., Thelykinine in der Galle. Klin.Wchnschr., 1928, 7, 1606.

8. Dohrn, M., and Faure, W., Uber die Ausscheidungdes weiblichen Sexual hormones. Klin. Wchnschr.,1928, 7, 943.

9. Cantarow, A., Rakoff, A. E., Paschkis, K. E., Hansen,L. P., and Walking, A. A., Excretion of estrogenin bile. Endocrinology, 1942, 31, 515.

10. Cantarow, A., Rakoff, A. E., Paschkis, K. E., Hansen,L. P., and Walking, A. A., Excretion of exogenousand endogenous estrogen in bile of dogs and hu-mans. Proc. Soc. Exper. Biol. & Med., 1943, 52,256.

11. Pearlman, W. H., Paschkis, K. E., Rakoff, A. E.,Cantarow, A., Walking, A. A., and Hansen, L. P.,A note on the biliary excretion of endogenousestrogens. Endocrinology, 1945, 36, 284.

12. Pearlman, W. H., Rakoff, A. E., Cantarow, A., andPaschkis, K E., The isolation of estrone from thebile of pregnant cows. J. Biol. Chem., 1947, 170,173.

13. Pearlman, W. H., Rakoff, A. E., Paschkis, K. E.,Cantarow, A., and Walking, A. A., The metabolicfate of estrone in bile-fistula dogs. J. Biol. Chem.,1948, 173, 175.

14. Pearlman, W. H., and Rakoff, A. E., A note on theestrogens in the bile of pregnant women. Endo-crinology, 1949, 44, 199.

15. Twombly, G. H., and Schoenewaldt, E. F., Themetabolism of radioactive dibromoestrone in man.Cancer, 1950, 3, 601.

1277


16. Albert, S., Heard, R. D. H., Leblond, C. P., andSaffran, J., Distribution and metabolism of iodo-a-estradiol labeled with radioactive iodine. J. Biol.Chem., 1949, 177, 247.

17. Beer, C. T., and Gallagher, T. F., Excretion of estro-gen metabolites by humans. I. The fate of smalldoses of estrone and estradiol-17p. J. Biol. Chem.,1955, 214, 335.

18. Beer, C. T., and Gallagher, T. F., Excretion of estro-gen metabolites by humans. II. The fate of largedoses of estradiol-17p8 after intramuscular and oraladministration. J. Biol. Chem., 1955. 214, 351.

19. Rakoff, A. E., Cantarow, A., Paschkis, K. E., Han-sen, L. P., and Walking, A. A., Removal ofexogenous estrogens from the circulation. Endo-crinology, 1944, 34, 370.

20. Albrieux, A. S., The distribution of estrogens in theblood of pregnant and non-pregnant women. J.Clin. Endocrinol., 1941, 1, 893.

21. Albrieux, A. S., Estrogen content of the blood atfour-hour intervals. Proc. Soc. Exper. Biol. &Med., 1941, 47, 380.

22. Bischoff, F., and Katherman, R. E., Distribution ofestradiol between serum and red cells. Am. J.Physiol., 1948, 152, 189.

23. Slaunwhite, W. R., Jr., and Sandberg, A. A., Studieson phenolic steroids in human subjects. I. Theconversion of 16-C4-estrone to C4-16-ketoesterone.Arch. Biochem., 1956, 63, 478.

24. Rothchild, I., A simple method for the preparation ofaqueous solutions of steroid hormones for intra-venous administration. Endocrinology, 1952, 50,583.

25. Sandberg, A. A., and Slaunwhite, W. R., Jr., Metabo-lism of 4-C4-testosterone in human subjects. I.Distribution in bile, blood, feces and urine. J. Clin.Invest., 1956, 35, 1331.

26. Fukushima, D. K., Bradlow, H. L., Dobriner, K., andGallagher, T. F., The fate of testosterone infusedintravenously in man. J. Biol. Chem., 1954, 206,863.

27. Hellman, L., Bradlow, H. L., Adesman, J., Fuku-shima, D. K., Kulp, J. L., and Gallagher, T. F.,The fate of hydrocortisone-4-C' in man. J. Clin.Invest., 1954, 33, 1106.

28. Peterson, R. E., Wyngaarden, J. B., Guerra, S. L.,Brodie, B. B., and Bunim, J. J., The physiologicaldisposition and metabolic fate of hydrocortisone inman. J. Clin. Invest., 1955, 34, 1779.

29. Migeon, C. J., Sandberg, A. A., Decker, H. A., Smith,D. F., Paul, A. C., and Samuels, L. T., Metabolismof 4-C'4-cortisol in man: Body distribution andrates of conjugation. J. Clin. Endocrinol. & Me-tab., 1956, 16, 1137.

30. Migeon, C. J., Sandberg, A. A., Paul, A. C., andSamuels, L. T., Metabolism of 4-C4-corticosteronein man. J. Clin. Endocrinol. & Metab., 1956, 16,1291.

31. Bradlow, H. L., Hellman, L., and Gallagher, T. F.,Metabolism of 11-hydroxy-A'-androstene-3,17-dione-4-C14. Federation Proc., 1956, 15, 223.

32. Pearlman, W. H., Pearlman, M. R. J., and Rakoff,A. E., Estrogen metabolism in human pregnancy; astudy with the aid of deuterium. J. Biol. Chem.,1954, 209, 803.

33. Levitz, M., Spitzer, J. R., and Twombly, G. H., Theconversion of estradiol-17f#-16-C'4 to radioactive16-ketoestradiol-17p in man. J. Biol. Chem., 1956,222, 981.

34. Marrian, G. F., and Bauld, W. S., The isolation of 16-epioestriol from the urine of pregnant women.Biochem. J., 1955, 59, 136.

35. Marrian, G. F., Watson, E. J. D., and Panattoni, M.,The isolation of a ketonic dihydroxy Kober chro-mogen from the urine of pregnant women. Bio-chem. J., 1957, 65, 12.

36. Sandberg, A. A., and Slaunwhite, W. R., Jr., Un-published observations.

37. Longwell, B. B., and McKee, F. S., The excretionof estrogens in the bile and urine after the adminis-tration of estrone. J. Biol. Chem., 1942, 142, 757.

38. Hellman, L., Bradlow, H. L., Frazell, E. L., andGallagher, T. F., Tracer studies of the absorp-tion and fate of steroid hormones in man. J.Clin. Invest., 1956, 35, 1033.

39. Rubin, B. L., Dorfman, R. I., and Miller, M., Andro-gen excretion in human bile. Endocrinology, 1952,51, 463.

40. Stimmel, B. F., Simultaneous biliary and urinaryexogenous estrogen excretion studies in man.Federation Proc., 1951, 10, 254.

41. Stimmel, B. F., Reduction of radioactive estrone toestradiol by human feces. Federation Proc., 1954,13, 305.

42. Szego, C. M., The influence of the liver upon estro-gen-protein binding in vitro. Endocrinology, 1953,52, 669.

43. Riegel, I. L., and Mueller, G. C., Formation of aprotein-bound metabolite of estradiol-16-C4' by ratliver homogenates. J. Biol. Chem., 1954, 210, 249.

44. Sandberg, A. A., and Slaunwhite, W. R., Jr., Biliaryexcretion and protein binding of radioactive ster-oids in human subjects: Their possible relation-ship. J. Clin. Endocrinol. & Metab., 1956, 16, 923.

45. Gray, C. L., and Bischoff, F., Conversion of estroneto estradiol by mammalian red cells. Am. J.Physiol., 1955, 180, 279.

1278

co-estradiol -...

Documents