Studies on Phenolic in Human Subjects. VII. Metabolic Fate of and Its Glucuronide

Avery A. Sandberg, W. Roy Slaunwhite Jr.

J Clin Invest. 1965;44(4):694-702. https://doi.org/10.1172/JCI105181.

Research Article

Find the latest version: https://jci.me/105181/pdf Journal of Clinical Investigation Vol. 44, No. 4, 1965

Studies on Phenolic Steroids in Human Subjects. VII. Metabolic Fate of Estriol and Its Glucuronide * AVERY A. SANDBERG t AND W. RoY SLAUNWHITE, JR. (From the Roswell Park Memorial Institute, Buffalo, N. Y.)

Estriol has been considered a metabolic product of capable of oxidizing the hydroxyl of the more active , (1, 2) and group at position 16, a finding not reported in the indirectly . Recently, an alternative path- past. way has been proposed (3) based on the observa- The attention of investigators has recently been tions that 16a-hydroxydehydroepiandrosterone is directed toward conjugates, not only be- present in high concentrations in cord blood and cause they are excreted in that form, but owing to that it is aromatized by placental enzymes. During the demonstration that steroid sulfates may, in the has been thought the some instances, serve as biosynthetic intermediates source of the mother's urinary estriol, but recent (12-14), that sulfate is evidence indicates that the fetus (4-6) and, in par- secreted by the adrenal cortex (15), that estrone ticular, the fetal (7) may play an important circulates in the blood as a sulfate (16), and that role in the conversion of the estrone to estriol steroid sulfates appear to be biologically active (8). Although estriol has been considered an (17). end product of , its fate and In previous studies we have reported on the metabolism have received considerable attention clearance of C'4-labeled estrone and estradiol from because it does have some estrogenic effects, al- the plasma of human subjects and on the signifi- though they are much smaller in some respects and cance of the enterohepatic circulation as a meta- larger in others than those of its precursors, es- bolic pathway for these steroids (18). The tradiol and estrone (9). present study extends these observations to C14- Several interesting studies have appeared on estriol and the glucuronide of this estrogen. In the fate of estriol administered to human subjects. this paper are presented data on the distribution Schiller and Pincus (10) administered estriol tri- of radioactivity in the bile, feces, and fol- acetate and recovered 57% of the estriol in the lowing the intravenous injection of C14-estriol or urine, as shown by bioassay of the material in the C14-estriol glucuronide. The rate of clearance urine. The metabolism of estriol was reinvesti- from the blood of the free and conjugated steroids gated by Levitz, Spitzer, and Twombly (11), us- and their metabolites is compared to observations ing C14-labeled steroid. They injected about 5 uc obtained with C14-labeled estrone and estradiol. of estriol-16-C14 in 7 ml of propylene glycol intra- In addition, the isolation and identification of the muscularly into two adult women. The recovery metabolites of the injected steroids are described of the radioactivity in the urine in 3 days was 58 and the results compared with those present in and 90%, respectively. A most interesting ob- the literature. servation was the appearance of "small but sig- nificant amounts of radioactive 16-ketoestradiol Methods and 16-" in the urine of the subjects. One to 5 ,uc of 16-C'4-estriol or 16-C"-estriol glucuro- This finding pointed to the presence in the body nide (SA, 22 ,uc per mg)1 was injected intravenously. The glucuronide was synthesized in our laboratory by bio- * Submitted for publication November 6, 1964; accepted synthetic means described previously (19). All the ster- December 31, 1964. oids injected were shown to be pure by chromatographic This study has been supported in part by grants A-1240 and countercurrent distribution techniques. The noncon- and CA-03559 from the National Institutes of Health. t Send requests for reprints to: Dr. Avery A. Sand- 1The C"-estriol was synthesized by Dr. Mortimer berg, Roswell Park Memorial Institute, Buffalo, N. Y. Levitz from whom it was obtained through the courtesy 14203. of the American Cancer Society. 694 STUDIES ON PHENOLIC STEROIDS 695

TABLE I of radioactivity in the urine and stools of women injected with C14-estriol and the radioactivity extractable following various hydrolytic procedures*

Radioactivity in hydrolytic fractions Total Weak Total hydro- radio- acid lyzable Radio- activity 6-Glucu- (pH 1) Strong radio- activity Subject Age in urine Free ronidase "sulfates" acid activity in stools 1 67 81.1 1.0 49.4 20.6 3.0 74.0 0.8 2 69 88.8 2.6 43.0 5.5 10.1 61.2 2.8 3 71 82.0 3.4 56.8 9.1 4.5 73.8 1.3 4 56 84.8 2.8 54.3 9.2 8.8 74.1 0.0 5 43 88.0 0.8 37.9 10.1 2.2 51.0 4.4 * Results are shown as percentages of injected dose excreted in 72 hours. jugated C"-estriol was dissolved in 1 to 2 ml of absolute period of 72 hours following the injection of C14- alcohol and then diluted with 30 to 50 ml of saline, which estriol, was approximately 23%o (Table II). Since was injected intravenously over a period of 1 to 2 min- only of the radioactivity could be accounted utes. The C"-estriol glucuronide was easily dissolved in 2% 30 ml of saline and injected similarly. The free C'4- for in the stools of the nonfistula subjects, the estriol was injected into ten female subjects, five with findings would indicate that the preponderant part biliary fistulas (T-tube drainage). One pregnant woman of the biliary radioactivity following C14-estriol was injected in the last trimester. The estriol glucuronide administration is reabsorbed in the gastrointestinal was administered to six women, three of whom had biliary tract and excreted in the urine. The fact that the fistulas. All the subjects were in good nutritional state, and none had evident renal or liver disease. biliary fistula subjects excreted significantly lower All the patients with T-tube drainage were injected amounts of radioactivity in the urine (70% vs. no sooner than the third postoperative day and had no 85%o) than did the nonfistula women would sup- evidence of impaired liver function. In each case the pa- port this statement. The percentage of radioac- tient's condition had been stabilized without fever or tivity excreted in the bile other complications after oral intake of fluids and food following the injection had been established. Complete biliary collections were of C14-estriol was much less than that obtained assured by a special apparatus and by the appearance of following the injection of C14-estrone or C'4-es- acholic stools. tradiol (50%o).. The conditions for the collection of bile and urine and Of the 85% of injected radioactivity excreted the procedures used for the determination of radioac- in the urine of the nonfistula subjects, nearly tivity, separation of the plasma from the erythrocytes, 70% and hydrolytic methodologies employed have been de- could be extracted following a combination of scribed in previous publications (19, 20). ,f-glucuronidase incubation (48%o) and weak acid (11%) and strong acid hydrolyses (6%o) (Table Results I). A little over 2%o of the radioactivity could be extracted from the urine before any hydrolytic Studies with C14-estriol. Nearly 85%o of the procedure was performed. The results of hy- radioactivity injected was excreted within 72 drolysis and extraction of the urine samples from hours in the urine of subjects given C14-estriol the biliary fistula patients were similar to those (Table I), with about 30%o being excreted within 4 hours and 50%o within 12 hours. This pattern TABLE II contrasts with that observed to Excretion of radioactivity in the urine, bile, and stools of subsequent the women with biliary fistulas injected with Cl4-estriol* injection of C14-estrone on C14-estradiol; when these two estrogens were injected, the peak ex- Subject Age Urine Bile Stools Total cretion of urinary radioactivity often occurred 6 44 70.0 22.2 0.0 92.2 24 to 72 hours following the injection. In the 7 36 71.3 17.3 0.2 88.8 8 54 82.9 19.6 2.2 104.7 case of C14-estradiol significant excretion of radio- 9 68 74.2 16.2 0.0 90.4 activity in the urine was present 72 to 120 hours 10 67 50.4 38.5 0.0 88.9 after the administration of the steroid The (18). Results are shown as percentages of injected dose average excretion of radioactivity in the bile, in a excreted in 72 hours. 696 AVERY A. SANDBERG AND W. ROY SLAUNWHITE, JR. 10.0 higher fecal radioactivity subsequent to the in- 5.0 jection of either C14-estrone or C14-estradiol, -J which averaged 7%o. The difference may be re- 0 lated to the greater amounts of radioactivity ex- creted via the bile into the gastrointestinal tract z LWL 1.0 _2 in the case of the latter two steroids (18). W 0.5 (/,-iD The free (unconjugated) radioactivity follow- 00 0 > ing C14-estriol administration was cleared from 20o 04< the plasma at an initial rapid rate (t4 ca. 20 min- F- n O. I utes) and at a subsequent slower rate (tj ca. 60 IL 0.05 minutes) (Figure 1). These two rates were very z similar to those observed following the injection -, of C14-labeled estrone or estradiol. The lower 0 the in- 0e 0.Q1 levels of radioactivity observed following 5 30 60 o20 240 jection of C14-estriol may indicate a larger volume TIME IN MINUTES of distribution for this steroid or more ready egress of the steroid from the plasma due to lesser protein FIG. 1. CLEARANCE FROM THE PLASMA OF THE RADIO- ACTIVITY FOUND IN THE FREE (UNCONJUGATED) FRACTION binding of this steroid as compared to that of FOLLOWING THE INJECTION OF C14-LABELED ESTRONE (E1), estrone and estradiol or both. Within 15 min- ESTRADIOL (E2), OR ESTRIOL (E3). Note similarity in the utes after the injection of C"4-estriol, the radio- rates of clearance, but lower concentration of radioac- activity in the plasma "glucuronide fraction" tivity following estriol injection. In each case only the (Figure 2) exceeded that of the "free fraction" total radioactivity in the free fractions is represented. In the case of estriol this fraction probably consists pre- (Figure 1) at least fourfold. The activity in the dominantly of estriol itself, but in the case of estrone "glucuronide fraction" declined fairly rapidly and estradiol, some metabolites of these steroids (estriol within the 4-hour period of study. Comparisons and others) may constitute a significant moiety of the free fraction, and the results should be interpreted with 10* these points in mind. Similarily, this statement applies 9. to the glucuronide and sulfate fractions of estrone and -J 8' I- 77 estradiol as shown in Figures 2 and 3. 0 H 6 ZW 5- just described. These findings do not differ ma- D en 4- -GLUC. terially from those observed following the injec- 0 0 0 > tion of C14-labeled estrone or estradiol. In the 3- bile only about 50%o of the radioactivity present ALLJ could be extracted following the various hydrolytic procedures, most of the radioactivity being pres- -'a- 2. ent in the fraction hydrolyzed with strong acid. z LL. A 25-year-old woman in the third trimester of 0 pregnancy was injected with 3 Auc of C14-estriol and the urine collected for 96 hours. Over 67%o of the injected radioactivity was excreted in the Is 30 60 120 240 urine within 24 hours and over 83%o during the TIME IN MINUTES period of urinary collection. The results of the FIG. 2. DISAPPEARANCE OF RADIOACTIVITY FROM THE various hydrolytic procedures were similar to PLASMA GLUCURONIDE FRACTION AS A FUNCTION OF TIMEX those described for the group of patients in the The results shown were obtained following the injec- tion of C14-labeled estrone (E1), estradiol (E2), estriol previous paragraph. (Es), and estriol glucuronide (E3-gluc.). Note that the The average excretion of radioactivity in the glucuronides of the last two were cleared at almost stools was approximately 2%o after the adminis- identical rates, whereas those of E1 and E2 showed an tration of C14-estriol. This contrasts with the initial different rate. STUDIES ON PHENOLIC STEROIDS 697

10 triol glucuronide synthesized in our laboratory was injected intravenously into six women, three -J 5 E3 -GLUC. of whom had biliary fistulas. Over 50% of the 0 radioactivity appeared in the urine within 4 hours 2- radio- Zw.] .__EI + E2p - "SULFATE" following the injection. The remaining ZWI- - --- in the urine in 48 hours. No -o- activity appeared radioactivity (Table III) could be detected in the 00 0.5 E3- "SULFATE" 0o> bile of the three subjects with biliary T-tube drain- age. Thus, it would appear that the glucuronide of estriol is not excreted in the bile. In the sub- Z M- Wj jects without fistulas, from 60 to about 70%o of _- 0.05- the injected radioactivity could be extracted with ether following hydrolysis of the urine with 0 ,8-glucuronidase; 80% was extractable in the urine of the subjects with biliary fistulas. The amount 15 30 60 120 240 of radioactivity in the urinary free fraction was FIG. 3. THE CLEARANCE FROM PLASMA OF THE FREE, negligible and indicates that hydrolysis of the GLUCURONIDE, AND SULFATE FRACTIONS OF INJECTED C14- glucuronic acid moiety from the estriol had not ESTRIOL. For comparison, the clearance of the sulfates of occurred to any significant extent in the body. E1 and E2 is shown. The clearance of the injected C14-estriol glucu- ronide from the plasma was almost identical to of the radioactivity levels in the plasma following that shown by the estriol glucuronide fraction the injection of C14-estrone, estradiol, and estriol present following the injection of C14-estriol (Fig- are shown in Figures 1 to 3. The possible reasons ure 2). For all practical purposes, no radioac- for the differences in the curves shown in those tivity could be found in the free fraction from figures will be discussed later in the paper. plasma following the administration of the estriol In Figure 3 are shown the radioactivity levels glucuronide, a finding consonant with the lack of in the various fractions of the plasma, i.e., free radioactivity in the free fraction of the urine. (unconjugated), "sulfate" (radioactivity extract- Neither in the urine nor in the plasma was sig- able by solvolysis with ether for 48 hours at pH nificant radioactivity found following hydrolysis 1), and the glucuronide moiety, following the in- at pH 1 for 48 hours or after exposure to hot jection of C14-estriol. It is interesting to note strong acid. Interestingly, when an alcoholic ex- that the radioactivity in the glucuronide fraction tract of the plasma, which contained almost all of exceeded that in the sulfate fraction by a factor the radioactivity in the plasma, was subjected to of over 4 at 15 minutes following the injection ,8-glucuronidase hydrolysis, over 90%o of the ra- and by nearly sevenfold at the end of 4 hours. dioactivity became ether extractable. Thus, it As in the urine, so in the plasma, estriol glucu- would appear that the injected C14-estriol glu- ronide is the predominant conjugate of the es- A comparison of the radioac- trogen injected. TABLE III tivity in the sulfate plasma fraction following es- Excretion of radioactivity in the urine and bile of women trone and estradiol injection with that after es- injected with C14-estriol glucuronide* triol administration reveals that the former rises Bile for a period of 1 hour, similar to the curve for Subject Age Urine the plasma glucuronides of estrone and estradiol 11 52 92.0 64 88.0 a level higher than that 12 metabolites, and maintains 13 62 86.3 of the sulfate of estriol. In addition, the ratio of 14 59 78.6 0.0 glucuronide to sulfate radioactivity following es- 15 60 80.0 0.0 52 91.4 0.0 trone and estradiol is significantly lower than 16 that observed after estriol injection. *Results are shown as percentages of injected dose Studies with C14-estriol glucuronide. C14-es- excreted in 48 hours. 698 AVERY A. SANDBERG AND W. ROY SLAUNWHITE, JR. curonide exists as such in the plasma and is ex- tive identification and better yields of the metabo- creted as such in the urine and that no splitting lites to be studied. In our experience the use of of the conjugate occurs in the body to any sig- countercurrent distribution has given us more nificant extent. reliable results and better recoveries of the mate- The steroid present in the urine following C14- rials studied than afforded by either paper or estriol glucuronide injection was shown to be adsorption chromatography. unchanged estriol glucuronide by countercurrent Extraction of a major portion of the urine distribution studies, with systems similar to those containing 1.8 X 106 cpm with ether removed a described later in the paper. In addition, the negligible amount of radioactivity. Glucuronides aglycone was shown to be estriol following in- were hydrolyzed by incubation with 300 U per cubation with /8-glucuronidase. The estriol was ml of fl-glucuronidase (Ketodase) at pH 5.0 for identified by countercurrent distribution and by 5 days at 370 C. Continuous extraction with paper chromatography. In each case the steroid ether at pH 5.0 for 48 hours yielded 918,000 cpm. was shown to behave identically with that of An additional 20% of the radioactivity was ex- standard estriol. No significant radioactivity was tracted from the urine following mild and strong found in any other fraction, indicative that the acid hydrolysis, but only the pooled free and glu- C'4-estriol had not been metabolized in the body curonide extracts were used for the identifica- to any significant degree, if at all. tion of the metabolites. Association with erythrocytes. The amount of To the ether extract containing 918,000 cpm radioactivity associated with the erythrocytes fol- was added 10 mg each of estriol, 16-epiestriol, lowing the injection of C14-estriol did not differ and 16-ketoestradiol. The mixture was subjected materially from that observed for C'4-labeled to countercurrent distribution (n = 100) in the estrone and estradiol. Thus, 15 minutes following system H20: CH30H/CC14: CHC13, 30: 70/50: the injection the average amount associated with 50 (by volume). Analysis of radioactivity and the total erythrocyte mass in the body was a little Kober chromogen showed that the 16-ketoestra- less than 2%, at 1 hour about 0.5%o, and at 4 diol, epiestriol, and estriol had partition coef- hours slightly less than 0.2%o of the injected ficients of 1.0, 1.5, and 4.1, respectively. The dose. On the other hand, following the injection preponderant amount of the radioactivity was as- of C14-estriol glucuronide the radioactivity asso- sociated with the estriol and was not identified ciated with the erythrocytes was about threefold further. There was a minor peak with a parti- that observed following C14-estriol administration tion coefficient of 0.63 which had a shoulder that (15 minutes, 9%o; 1 hour, 3.3%o; 4 hours, 1.7%o trailed into the 16-ketoestradiol region. of the injected dose). Since the three standards were incompletely Identification of metabolites of injected C14- separated, tubes 40 to 72 were pooled (254,000 estriol. Levitz and associates (11) had reported cpm), and the residue, after flash evaporation, the presence of 16-ketoestradiol and 16-epiestriol was treated by the method of Marrian and Bauld in the urine of two subjects injected with C14- (21) for acetonide formation. Countercurrent estriol intramuscularly. The urinary extracts of distribution in the same system showed the three all the subjects studied were pooled and sub- standards to be completely separated with the mitted to countercurrent distributions, as de- 16-epiestriol acetonide having a partition coef- scribed below, but neither 16-ketoestradiol nor 16- ficient of 0.04. The major peak of radioactivity epiestriol was found to be present in detectable was again associated with the estriol standard, amounts. To further substantiate our observa- and the minor peak was unchanged. The three tions a normal female subject, age 32 years, was areas were separately pooled and counted: epi- given 3 ,uc of estriol-16-C14 intravenously. The estriol acetonide, 13,000 cpm; 16-ketoestradiol, urine was collected for 48 hours and submitted 21,000 cpm; estriol, 124,000 cpm. to the various hydrolytic procedures described The 16-epiestriol acetonide was redistributed previously. In this study the work-up employed (n = 50) in the system n-hexane/CH3OH: H20, countercurrent distribution rather than adsorption 100/50: 50. All of the radioactivity remained in chromatography in order to afford a more defini- tubes 0 to 3, leaving the standard (K = 0.9) de- STUDIES ON PHENOLIC STEROIDS 699 void of radioactivity (no counts above back- jection of estradiol, and the steroid and its metab- ground). olites then become involved in an enterohepatic The 16-ketoestradiol was redistributed (n = circulation that may trap a considerable portion 200) in the system H2O: CH3OH/CC14: CHCl3, of the radioactivity for prolonged periods of time. 30: 70/20: 80. The same pattern prevailed, i.e., The different degrees to which the estrogens the radioactivity peaked (K = 0.20) lower than studied are involved in an enterohepatic circula- the standard (K = 0.36), but trailed into the 16- tion are further indicated by the recovery of radio- ketoestradiol area. The radioactivity in this activity in the stools. Thus, following the in- area was now 3,000 cpm and showed no indica- jection of C14-estrone or C14-estradiol an average tion of being identical with 16ketoestradiol. of 7%, and as much as 18go, was recovered in We concluded, therefore, that although estriol the stools, whereas only 2%o was found when C"- is metabolized to a small extent, it was not con- estriol was administered. The repeated recircula- verted in the subjects studied according to the tion in the enterohepatic system of the former scheme proposed by Levitz and associates (11). two steroids and their greater excretion in the bile (40 to 60%) probably account for the sub- in the stools when es- Discussion stantial radioactivity found trone or estradiol is administered. In the case of Recent studies on estriol metabolism have not estriol the radioactivity excreted in the bile is only concerned themselves with the metabolic rapidly and almost quantitatively reabsorbed, as derivation of this steroid, both in pregnant and witnessed by the less than 2% of radioactivity nonpregnant subjects, but also with its metabolic found in the stools and the relatively rapid ex- pathways and fate. To further clarify the lat- cretion in the urine. That a small amount of the ter was the major purpose of this study. Our estriol may be involved in an enterohepatic circu- salient findings are related to the biliary excretion, lation is evidenced by the average recovery of clearance from plasma, and rate of conjugation of 93% of the radioactivity injected in the biliary C14-estriol, and the metabolic fate of C14-estriol fistula subjects and only 87% in the nonfistula glucuronide. Each aspect will be discussed sepa- women. rately. A second factor affecting the rate of urinary The rate of urinary excretion for the various excretion of the various estrogens is related to estrogens is dependent on the amount of the the metabolism of these steroids. The metabolic steroid involved in the enterohepatic circulation. conversion of estrone and estradiol and subse- Of the three estrogens studied here, estriol ap- quent conjugation may consume a substantially pears to be excreted least in the bile. The pre- longer period of time than that required for the ponderant part of the estriol (and its possible direct conjugation of estriol. A combination of metabolites) in the bile is reabsorbed via an en- the two factors just discussed may, in fact, be the terohepatic circulation; about 85% of the injected major factors involved in the delayed urinary estriol is recovered in the urine within 72 hours, excretion of radioactivity following the injection with most of that being excreted within the first of C"4-labeled estrone and estradiol, as compared 24 hours. This contrasts with the urinary excre- to estriol. tion of radioactivity following the administration Examination of Figure 2 reveals a phenomenon of estrone or estradiol, an excretion characterized that may be related to one of the statements made by a peak in the second 24 hours and consum- in the preceding paragraph. The Figure shows ing 4 to 6 days for 90% of the radioactivity to that the level of radioactivity in the "glucuronide appear in the urine. fraction" of estrone or estradiol rose to a peak These differences in the rates of urinary ex- at about 60 minutes and then declined at a rate cretion between estriol on the one hand and es- similar to that following the injection of either trone and estradiol on the other may be due to estriol or estriol glucuronide. It is possible that at least two factors. One factor is related to the rising levels in the glucuronide fraction are the biliary excretion. At least 50% of the radio- due to the time required for conversion of estrone activity is excreted in the bile following the in- and estradiol to their major metabolite, estriol (1, 700 AVERY A. SANDBERG AND W. ROY SLAUNWHITE, JR. 2), a reaction that is slower than conjugation The studies with C14-estriol glucuronide re- (19). vealed several interesting features. Urinary ex- The decline in radioactivity in the "free frac- cretion of the conjugated steroid was prompt, tion" of the plasma following the injection of C`4- and practically none of it was excreted in the bile. estriol was characterized by an initial rapid and This lack of biliary excretion contrasts with the a subsequent slower rate of clearance; both rates significant (20%) excretion of radioactivity in were very similar to those following the injection the bile following C14-estriol injection. Thus, of estrone or estradiol. As a matter of fact, these these two observations and others point to the rates were not greatly dissimilar from those ob- fact that the estriol glucuronide was not split sig- served for some other steroids, including non- nificantly in the body. Almost all of the radio- phenolic ones [deoxycorticosterone (22), proges- activity in the urine could be recovered following terone (23), 17a-hydroxyprogesterone (24)], ,8-glucuronidase hydrolysis, and the steroid agly- and may indicate that removal of steroids from cone was shown to be estriol. Ideally, the study the circulating blood, probably by the liver, is a should have utilized a doubly labeled estriol con- process whose rate limiting step is similar for a jugate, i.e., one label in the steroid and another on number of steroids. The lower concentration of the glucuronide. However, such a doubly labeled radioactivity in the plasma following the injection compound is not available. These observations of C14-estriol may be due to a larger volume of plus the fact that no significant radioactivity could distribution in the body, possibly related to the be obtained in the "free steroid" fraction of the lesser binding of estriol to plasma protein when urine point to the maintenance of the integrity of compared to that of estrone and estradiol (25). the estriol glucuronide in the body. This finding That estriol is bound less to human serum al- is in striking contrast to the behavior of estrone bumin (and plasma) has been previously demon- sulfate, which was hydrolyzed extensively when strated in our laboratory (25), and it is possible injected into women (26, 27). that this may be responsible for egress of some Our failure to find detectable amounts of radio- of the C14-estriol from the blood, resulting in active 16-ketoestradiol and 16-epiestriol contrasts lower concentrations of radioactivity in the with the results described by Levitz and co- plasma. The disparity cannot be ascribed to as- workers (11). These differences are difficult sociation with the erythrocytes, since the three to explain. It is possible that the mode of ad- estrogens studied were bound to about the same ministration they utilized (11), intramuscularly extent by the red blood cells. in propylene glycol, may have been an important Examination of Figure 2 reveals that estriol factor. The intravenous route of administration was rapidly conjugated in the body and initially that we used may have obviated the influence of (15 minutes) reached levels of radioactivity at the intramuscular milieu on the metabolism of least threefold those seen following estrone and estriol. The hydrolytic procedures utilized by estradiol administration. The rapidity of conju- Levitz and associates (11) and the recoveries of gation can be judged from the fact that the clear- radioactivity resulting from these procedures ance curve of estriol glucuronide from the plasma (approximately 50% and 67% in the two sub- following estriol glucuronide administration was jects studied, respectively) did not differ sub- almost identical to that when estriol was injected. stantially from the circumstances and results of Although the concentration of radioactivity pres- our experiments. On the other hand, it is pos- ent as the glucuronide in the plasma was es- sible that the approaches used for the identification sentially identical following estriol or estriol of the metabolites may have contributed to the glucuronide administration, there was a substan- divergence in the observations. We relied pri- tially higher amount of estriol glucuronide asso- marily on countercurrent distribution and re- ciated with the erythrocytes than following estriol. verse isotopic dilution, whereas Levitz and asso- This finding may be due to greater water solu- ciates (11) employed chromatography for separa- bility of the estriol glucuronide and ready pene- tion of steroid mixtures that did contain other tration into the erythrocytes. radioactive components of nearly identical polar- STUDIES ON PHENOLIC STEROIDS 701 ity. We cannot state whether the other metabo- References lites they mentioned (11) (16-ketoestrone and 1. Fishman, J., H. L. Bradlow, and T. F. Gallagher. 16a-hydroxyestrone, not rigorously identified by Oxidative metabolism of estrogens. J. Amer. them due to the small quantities available) were chem. Soc. 1959, 81, 2273. present in the urine of the patients of our study, 2. Fishman, J., H. L. Bradlow, and T. F. Gallagher. since no effort was made to isolate and identify Oxidative metabolism of estradiol. J. biol. Chem. these compounds. However, the countercurrent 1960, 235, 3104. distribution results indicate that estriol is changed 3. Magendantz, H. G., and K. J. Ryan. Isolation of an estriol precursor, 16a-hydroxydehydroepiandros- structurally only to a small extent. terone, from umbilical sera. J. clin. Endocr. 1964, 24, 1155. Summary 4. Cassmer, 0. production of the isolated hu- man placenta. Acta endocr. (Kbh.) 1959, suppl. The metabolic fate of C14-estriol and C14-estriol 45. glucuronide has been studied following their in- 5. Frandsen, V. A., and G. Stakemann. Urinary ex- travenous injection into, women, including sub- cretion of estriol during normal pregnancy. Dan. jects with biliary fistulas. The radioactivity fol- med. Bull. 1960, 7, 95. lowing C14-estriol injection is excreted more 6. Coyle, M. G., M. Greig, and J. Walker. Blood- rapidly in the urine than previously observed and urinary and cestro- gens in foetal death from severe pre-eclampsia. when C14-estrone or C04-estradiol was adminis- Lancet 1962, 2, 275. tered. The preponderant part of the radioactivity 7. Engel, L. L., B. Baggett, and M. Halla. In vitro of the former steroid is excreted in the urine metabolism of estradiol-17,3 by human fetal liver: within 24 hours, whereas the peak excretion with formation of estriol, 16-epiestriol, estrone and an the latter two steroids occurs 48 to 72 hours sub- estriol glucosiduronic acid. Endocrinology 1962, sequent to injection. In all probability, the in- 70, 907. volvement of estrone and estradiol in an entero- 8. Slaunwhite, W. R., Jr., M. A. Karsay, K. Niswander, hepatic circulation, owing to the fact that over and A. A. Sandberg. of estrone and estradiol by human placenta and fetal liver. half of their radioactivity is excreted in the bile, Program of the 46th meeting of the Endocrine may be the major reason for the differences in Society, 1964, 151. the urinary excretory rates. Only 20%o of the 9. Merrill, R. C. Estriol: a review. Physiol. Rev. C'4-estriol was excreted in the bile. C'4-estriol 1958, 38, 463. was conjugated more rapidly than estrone or 10. Schiller, J., and G. Pincus. The fate of a-estradiol estradiol, and only a very small amount of the and of estriol injected into a human male subject. aglycone was metabolized. By reverse isotopic Arch. Biochem. 1943, 2, 317. dilution, 16-ketoestradiol and 16-epiestriol could 11. Levitz, M., J. R. Spitzer, and G. H. Twombly. In- not be shown to be among the metabolites. C14_ terconversions of 16-oxygenated estrogens. I. The estriol is excreted in the synthesis of estriol-16-C" and its metabolism in glucuronide rapidly 787. urine, and no evidence was obtained that the man. J. biol. Chem. 1958, 231, 12. Calvin, H. I., R. L. Vande Wiele, and S. Lieberman. conjugate had been split to any extent in the Evidence that steroid sulfates serve as biosynthetic body. No biliary excretion of the estriol glucuro- intermediates: in vivo conversion of - nide occurred. sulfate-STM to dehydroisoandrosterone-sulfate-S'. Biochemistry 1963, 2, 648. 13. Calvin, H. I., and S. Lieberman. Evidence that Acknowledgments steroid sulfates serve as biosynthetic intermediates. We wish to thank Dr. Mortimer Levitz and the II. In vitro conversion of pregnenolone-'H sulfate- American Cancer Society for generous supplies of 16-C"- 8'S to 17a-hydroxypregnenolone-'H sulfate-TmS. estriol. Mrs. Maria Karsay, Mrs. Anne Agacz, and Biochemistry 1964, 3, 259. Elek Karsay gave valuable technical assistance. We are 14. Roberts, K. D., L. Bandi, H. I. Calvin, W. D. particularly grateful to various staff members of the Drucker, and S. Lieberman. Evidence that cho- Buffalo General Hospital for allowing us to study their lesterol sulfate is a precursor of steroid . patients with T-tube drainage of bile. J. Amer. chem. Soc. 1964, 86, 958. 702 AVERY A. SANDBERG AND W. ROY SLAUNWHITE, JR.

15. Baulieu, E.-E. Studies of conjugated 17- 21. Marrian, G. F., and W. S. Bauld. The isolation of in a case of adrenal tumor. J. clin. Endocr. 1962, 16-epiOestriol from the urine of pregnant women. 22, 501. Biochem. J. 1955, 59, 136. 16. Purdy, R. H., L. L. Engel, and J. L. Oncley. The 22. Sandberg, A. A., W. R. Slaunwhite, Jr., and E. characterization of from human Chang. Metabolism of C"-deoxycorticosterone in plasma. J. biol. Chem. 1961, 236, 1043. human subjects. In preparation. 17. Baulieu, E. Studies on metabolism. Recent 23. Sandberg, A. A., and W. R. Slaunwhite, Jr. The Progr. Hormone Res. 1965, 21, in press. metabolic fate of C"-progesterone in human sub- 18. Sandberg, A. A., and W. R. Slaunwhite, Jr. Studies jects. J. clin. Endocr. 1958, 18., 253. on phenolic steroids in human subjects. II. The 24. Slaunwhite, W. R., Jr., and A. A. Sandberg. Dis- metabolic fate and hepato-biliary-enteric circulation position of radioactive 17a-hydroxyprogesterone, of C"-estrone and C14-estradiol in women. J. clin. 6a-methyl-17a-acetoxyprogesterone and 6a-methyl- Invest. 1957, 36, 1266. prednisolone in human subjects. J. clin. Endocr. 19. Slaunwhite, W. R., Jr., M. A. Lichtman, and A. A. 1961, 21, 753. Sandberg. Studies on phenolic steroids in human 25. Slaunwhite, W. R., Jr., H. Rosenthal, and A. A. subjects: VI. Biosynthesis of estriol-glucosiduronic Sandberg. Interactions of steroids with human acid-16-"C by human liver. J. clin. Endocr. 1964, plasma proteins. Arch. Biochem. 1963, 100, 486. 24, 638. 26. Twombly, G. H., and M. Levitz. Metabolism of 20. Sandberg, A. A., and W. R. Slaunwhite) Jr. Me- estrone-C4-16 sulfate in women. Amer. J. Obstet. tabolism of 4-C"- in human subjects. Gynec. 1960, 80, 889. I. Distribution in bile, blood, feces and urine. J. 27. Sandberg, A. A., and W. R. Slaunwhite, Jr. Un- din. Invest. 1956, 35, 1311. published results.