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Estrone : Production Rate and in Man

Henry J. Ruder, … , Lynn Loriaux, M. B. Lipsett

J Clin Invest. 1972;51(4):1020-1033. https://doi.org/10.1172/JCI106862.

Research Article

Since sulfate (E1S) is present at high concentration in plasma, we have examined the parameters of the plasma estrone, , E1S system. The metabolic rate of E1S was 157 liter/day (range 70-292) in men and women. Estimated plasma production rates of E1S were (μgrams per day): men, 77; women, early , 95; women, early , 182. The conversion of plasma estrone and estradiol to E1S was measured and from these data and E E S E E S the metabolic clearance rates of the , the transfer factors were ρ 1 1 = 0.54 and ρ 2 1 = 0.65. Using average production rates, all plasma E1S could be shown to be derived from plasma estrone and estradiol.

E SE The conversion of plasma E1S to plasma estrone and estradiol was studied. The calculated transfer factors were: ρ 1 1 E SE = 0.21, ρ 1 2 = 0.014. Essentially, similar data were obtained when E1S was given by mouth to two subjects.

We conclude: (a) E1S is a major circulating plasma and has a long plasma half-life; (b) the large contributions of estrone and estradiol to plasma E1S are more than sufficient to account for all the circulating plasma E1S.

Find the latest version: https://jci.me/106862/pdf : Production Rate and Metabolism in Man

HENRY J. RuDER, LYNN LORLAUX, and M. B. LIPSETr From the Reproduction Research Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20014

A B S T R A C T Since estrone sulfate (E1S) is present life in blood (1), and Purdy, Engel, and Oncley (2) at high concentration in plasma, we have examined the found that it was the major plasma of estra- parameters of the plasma estrone, estradiol, E1S system. diol and that it was present in high concentration in The metabolic clearance rate of EiS was 157 liter/day . During previous studies of estrogen metabo- (range 70-292) in men and women. Estimated plasma lism we had postulated (3) that there was another production rates of E1S were (/Agrams per day): men, plasma estrogen compartment in equilibrium with 77; women, early follicular phase, 95; women, early plasma estrone (E1) and estradiol (E2). Since it seemed luteal phase, 182. The conversion of plasma estrone probable that this compartment was estrone sulfate, we and estradiol to EiS was measured and from these data initiated studies of the origin, rates of metabolism, and the metabolic clearance rates of the estrogens, the blood levels, and conversions of estrone sulfate. These transfer factors were p51'18 = 0.54 and p'2'1' = 0.65. studies have confirmed the finding that E1S is an im- Using average production rates, all plasma E1S could portant plasma metabolite of estradiol and have defined be shown to be derived from plasma estrone and the production rate of E1S and its interconversions estradiol. with estrone and estradiol. The conversion of plasma E1S to plasma estrone and estradiol was studied. The calculated transfer factors METHODS were: p'1`81 = 0.21, pBis2 = 0.014. Essentially, similar Materials. Estrone-6,-7-'H, estradiol-17fi-6,7-8H, estrone- data were obtained when E1S was given by mouth to 3-NH4-sulfate-6,-7-8S (40 Ci/mmole), and estrone-4-1'C and two subjects. estradiol-17fi-4-"'C (45 mCi/mmole) were purchased from New England Nuclear Corp., Boston, Mass. Portions of 8H- We conclude: (a) EiS is a major circulating plasma and '4C-labeled were combined and radiochemical estrogen and has a long plasma half-life; (b) the large purity demonstrated before use by constancy of 8H : "C contributions of estrone and estradiol to plasma E1S ratio through a series of derivatives as shown in Fig. 1. are more than sufficient to account for all the circulat- Estrone-3-NH4-sulfate-4-14C (45 mCi/mmole) was prepared ing plasma E1S. from estrone-4-"C (45 mCi/mmole) by a modification of the method of Mumma, Hoiberg, and Weber (4). Estrone- 4-4C was dried in a 1 ml centrifuge tube and redissolved in 20 ,ul of dimethyl formamide (DMF) and chilled to 0°C in INTRODUCTION an ice-water bath. To the estrone in DMF, 10 ,ul of chilled 14% concentrated sulfuric acid in DMF (v/v) was added Estrone-3-sulfate (E1S)1 is the major component of the and mixed quickly with a vortex mixer. This was followed conjugated equine estrogens and of other estrogenic by addition of 25 ,l of 3 M dicyclohexylcarbodiimide (DCC) preparations and has been used for over 30 years for in DMF prepared by adding 0.150 g DCC to 0.0240 ml DMF. treatment of the postmenopausal woman. Although After mixing, the solution turned a thick white, and was then incubated at 0'C for an additional 15 min. 1 ml of estrone sulfate has been identified in human , little 1 M NH40H in methanol was added, the solution mixed, is known of its blood levels and metabolism. Twombly and centrifuged. The supernatant was chromatographed by and Levitz showed that estrone sulfate had a long half- thin-layer chromatography (TLC) (5) to separate mono- sulfate from free steroids. Received for publication 24 September 1971 and in revised Radiochemical purity of estrone-3-sulfate-4-1'C was dem- form 22 November 1971. onstrated by mixing it with portions of estrone-3-NH4- 1 Abbreviations used in this paper: DCC, dicyclohexylcar- sulfate-6,7-'H and demonstrating constancy of 3H : 14C ratios bodiimide; DHS, dehydroeipandrosterone sulfate; DMF, di- through TLC, isolation, solvolysis of E1S, and recrystalliza- methyl formamide; E1, estone; E2, estradiol; E1S, estrone-3- tion of estrone to constant specific activity. Before any study sulfate; MCR, metabolic clearance rate; RBC, red blood involving the use of estrone sulfate-"H, the was dis- cells; TLC, thin-layer chromatography. solved in sterile saline and preextracted with ether to re- 1020 The Journal of Clinical Investigation Volume 51 1972 14C ESTROGENS ADDED

Ether Extraction

Ether (E1, E2) Plasma + 4 Vol MEOH /Hr -150C

Centrifuge

LH 20 Column Dry Discard ppt. Add 10 ml H20 Sat. with (NH4)2SO4 t Extract with Ethyl Acetate 3Vol x3 t Dry, TLC System III Solvolysist I LH 20 E2 El IAc IAc E2Ac* El Ac* |Ac I Reduc. E2(AC)2o* E2Ac*F |Saponification |AC E2* E2(AC)2 *

Fraction counted for 3H/14C FIGURE 1 Flow chart for measurement of tritium in plasma estrone, estradiol, and ES during infusion of 3H estrogens. move any contaminating free estrogen. The ether phase was maining in the basal state until the end of the study. The discarded, excess ether removed by an air stream, and por- infusion techniques were performed as described previously tions taken in triplicate for determination of E1S-8H. An (7). appropriate volume of sterile saline was then drawn up in All short infusions were begun with a 10 ,uCi bolus a glass syringe for or addition to an infusion bottle. given i.v. over 1 min, 30 min later an infusion of approxi- Chromatoquality estrone and estradiol (Calbiochem, Los mately 10 ,uCi/hr was begun and maintained at a constant Angeles, Calif.) were used without further recrystallization. rate until the end of the study. The duration of the short Solvents were spectroquality and used without further puri- infusions was usually 165 min from the time of the bolus fication. TLC was performed on Merck precoated silica gel and varied from 135 min in earlier studies to 210 min in GF-254 20 X 20 cm glass plates (Merck Chemical Division, later studies, 3040 ml of blood was drawn at various times Merck & Co., Inc., Rahway, N. J.). The systems used for after the bolus was given; the heparinized plasma was sepa- separations of various steroid and the Rr values are pre- rated by centrifugation and stored at - 16°C until extracted. viously described (3, 5, 6). Column chromatography was To measure the MCR of E1S, 20-40 ,uci of ES-3H was performed on small Sephadex LH-20 columns (Pharmacia injected over 1 min at 9:00 a.m. via an antecubital vein. Fine Chemicals Inc., Piscataway, N. J.) which separate El 10-ml blood samples were taken at i, 1, 2, 4, 6, and 8 hr and E2 as described previously (5). and 20-35-ml blood samples were taken at 11, 14, 19, and Subjects. Normal subjects, (Nos. 2-18), ages 18-30 yr 24 hr. Blood was heparinized, centrifuged immediately, and were hospitalized at the National Institutes of Health and the plasma frozen at - 15°C until extracted. Subjects were received no . Each woman had normal cyclic kept at bed rest with bathroom privileges and fed light meals menses. Subject 16 was a 28 year old euthyroid subject with as desired for the duration of the study except for subjects a benign nodule. Subject 1 was a normal man, age 1 and 2, who were allowed normal daily activities. 48, in good health. Calculationts. The symbols and calculations for the sev- Clearance rates. Determinations of metabolic clearance eral parameters of the system are essentially those used by rate (MCR) were started at 7:30 a.m. with subjects re- Horton and Tait (8). Since all measurements were made in

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1022 H. J. Ruder, L. Loriaux, and M. B. Lipsett plasma, the compartment is not indicated and the superscript tubes at 2000 g for 20 min. The yellow supernatant was indicates the steroids. Production rate equals MCR X plasma decanted and either stored as described above or processed. concentration. The conversion ratio, CE1ElS, is the ratio of The supernatant was dried under vacuum until only 2-5 ml radioactivity of product ElS, to precursor, El, when equi- of cloudy yellow water remained. This was quantitatively librium has been reached. The transfer factor, p'1ME1 = transferred to a 40 ml glass-stoppered tube (final vol. = 10 CEi~iS X MCRzsE/MCRE1, is the fraction of plasma E1 pro- ml H20), alkalinized with 5 drops of concentrated NH4OH, duction rate converted to plasma E1S. saturated with dry (NH)2SO4, and extracted three times Isolation of labeled estrogens. All plasma samples were with 2 vol of ethyl acetate. The ethyl acetate quantitatively processed within 3 wk of collection and were treated identi- removed the E1S. Recovery to this point is 60-70%. The cally, as shown in Fig. 1. To monitor recovery appropriate ethyl acetate was combined and reduced in volume, and the amounts (100-1000 cpm) of XC estrogens were added to residue was spotted onto TLC plates and the monosulfate each plasma sample. All samples from a single study were fraction isolated (5). Dehydroeipandrosterone sulfate processed together. After alkalinizing with 4 drops of (DHS) was used as a marker on both sides of the plate NH4OH, the plasma was extracted three times with 2 vol and the DHS identified by streaking with Allen's reagent. of ether. The ether extracts containing the neutral steroids After elution from the TLC plate, the samples were dried were under air and stored until further processing in 5 ml and then resuspended in 0.2 ml of absolute ethanol. Sol- of absolute ethanol at - 15'C. Previous tests had shown volysis was performed by addition of 5 ml of 10%o glacial that only 1-2% of estrone sulfate is removed by the ether, acetic acid in ethyl acetate and incubation in a tightly probably in the small amount of water soluble in the ether. capped tube for 16 hr at 50°C. The solvents were removed Estrone sulfate proved stable in the cold so that any con- under Ns at 40°C. No washing was necessary, and previous taminating E1S was separated from free estrogens by the tests demonstrated that the estrone was not altered by this subsequent column chromatography. The ether-extracted procedure. From this point on, derivative formation and plasma containing the conjugated steroids, including E1S, portioning for determination of 'H :1'4C ratios is as pre- was warmed to 40'C and the residual ether removed under viously described for estrone (6). Recovery of ES-Y4C to an air stream. Recovery of estrone sulfate was found to be the isolation of estrone averaged 50%. In sample studies this inversely related to the quantity of ether remaining at this procedure was shown to separate completely E1 and E1S in point. To the plasma was then added 4 vol of absolute plasma since addition of a 100-fold excess of E1-8H did not methanol to precipitate . Each sample was mixed influence the values obtained for tritiated E1S; and con- by shaking for 30 sec and stored at - 15'C for at least versely, addition of 100-fold excess of E1S-2H did not in- 16 hr to precipitate lipids. Centrifugation and decanting of fluence the values obtained for tritiated E1. the supernatant yielded a yellow methanol solution which Processing E1 and E,. Sample tubes containing neutral contained about 80% of the starting E1S. After addition of steroids in ethanol were dried under N2 at 40°C and estrone 4 more drops of NH4OH, samples could be stored in- and estradiol separated by column chromatography on Sepha- definitely in the cold until further processing. dex LH-20 (5). To the appropriate fraction was added Processing ES. After 16 hr at -15°C, the methanol- 200 ,ug estrone or estradiol, and derivative formation car- precipitated plasma was centrifuged in 100-ml centrifuge ried out as shown on Fig. 1 and as previously described

1,000,000

-- A t1/2 9 * A'~~~~~A2_ 100,000 _

cpm/liter t,,2 =5.;

HOUR FIGURE 2 Representative data from a low and high MCRES. Infusion started at time 0. The points are the data points; the lines were determined by a computer program.

Estrone Sulfate: Production Rate and Metabolism in Man 1023 600,000 r described (5) and normal values for our laboratory are shown in Table III. These values include all patients used MCRR 207 liters/day in the infusion studies and agree well with those obtained . by others (9-11).

MCR= 194 liters/day Plasma concentration of estradiol monosulfate. To de- MC94ltrsdy termine if estradiol monosulfate was an important metab- olite of estradiol or if it was present in plasma in sig- nificant amounts, the following studies were performed. cpm/liter 100,000 : (a) We attempted to isolate E2S-8H from plasma ob- tained from two subjects during E2-'H infusion. The en- tire monosulfate fraction was solvolyzed and the E1 and E2 fractions isolated. Radiochemical purity was demonstrated ,S, H only for E1-3H, but tritium present in the E28H fraction t ENS- IV was approximately 10% that present as Ei-8H. Hence con- version of E2 to E2S could occur maximally at only 1is the 0 1 2 4 6 8 10 12 14 16 rate of E2 conversion to ES. (b) 15 random plasma sam- HOUR ples were examined for E2 monosulfate. No recovery trace was available for Ee-8H-3-sulfate or E2-8H-17-sulfate, but FIGURE 3 MCRi1s measuired after injection of E1S-'H at recovery was assumed to be similar to that for E1S deter- time 0 and at 8 hr follo)wed then by a continuous infu- mined simultaneously. After solvolysis of the monosulfate sion of E1S-8H (subject 6 fraction from TLC, both E1 and E2 were isolated on Sepha- dex LH-20. The E2 fractions were assayed and found to be indistinguishable from blank values pg). Assuming (3, 6). After the first fou ir studiese were completed, it was recovery (25-50 evident that in no case dlitdI: eCratioschangeedigiwas the same for E2 monosulfate as for E1S, this would lid in mean that E2S was present at less than 100-150 pg/ml cantly after the first derivative waswCahsisolatedhandgehat in plasma whereas E1S was measured at concentrations of all cases the 'H :14C raltio of the inrstfirst derivativeade thati was 1000 pg/mI. 250- theLt%,i sameOCA11%, ascad thatL11ML ofUL theLLIK LI111 UVL SV4 Li V 11wV1tC LL1s U- after, only two derivatives were counted; the second was the same as the first in the remainder of the studies. RESULTS In no instance was fewer than 15 cpm above background of ES metabolic clearance rates. The data for the dis- 4C present in the second derivative, and except in the case appearance of E1S-3H from plasma are shown in Table of the Es levels during E1S infusions where conversion to E2 was quite low, the 8H :14C ratio was greater than 1.0 I. When the data for each subject were analyzed, it and less than 8.0 in all samples. was found that in all cases, disappearance curves could Estrone sulfate binding to red blood cells (RBC). Por- be fitted by a single straight line, consistent with a tions of packed RBC taken during E1S-3H infusions were one-compartment system described by a single Y inter- examined for binding of E1S. We found little (< 5%) of cept (volume of distribution) and a single the E1S-8H present in whole blood to be associated with slope (frac- RBC. tional pool turnover rate). Two representative studies Counting methods. Radioactivity was measured as pre- are shown in Fig. 2. The lines of best fit were calcu- viously described (3, 6). All samples were counted to either lated by computer program, and the points shown are a minimum of 3000 counts in the carbon channel or for 100 the data points. Metabolic clearance rates were calcu- min. Samples were spot checked for differences in quenching and none were found. lated, assuming a one-compartment model, by the for- Plasma estrogen concentrations. Methods for measure- mula MCR= VY, where V = the volume of distribu- ment of plasma concentration of E,, E2, and ES have been tion and ' = fractional pool turnover rate per unit

TABLE I I Plasma Concentrations and Production Rates (PR) of Estrone, Estradiol, and Estrone-3-Sulfate EiS MCR EiS EiS PR El MCR* Ei Ei PR E2 MCR* E2 E2 PR liter/day pg/mi pg/day liter/day pg/mi pg/day liter/day pg/mi pg/day Men 167 460 77 2380 47 112 1700 34 58 (1177)t (892) Women Follicular phase 146 654 95 1750 62 109 1055 110 116 (1070) (642) Women Luteal phase 146 1,246 182 1750 86 151 1055 193 204 * Average MCR for this laboratory. 1 Numbers in parentheses, average MCR for this laboratory, expressed as liter/day per m2. 1024 H. J. Ruder, L. Loriaux, and M. B. Lipsett ew 0 * 60 0 066o0

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Estrone Sulfate: Production Rate and Metabolism in Man 1025 TABLE IV Estrone-6,7-

Plasma tritium levels Sex and Estrone- Subject age 'H-infusion 'H-steroid 1.75 hr 2.0 hr 2.25 hr

cpm/day X 10-8 cpm X 10/l1iter [111 M 21 1.048 El 41.7 44.6 47.2 ElS 86.5 99.4 110 12 F 19 0.965 El 45.4 43.5 43.5 E1S 89.3 101 117 13 F 20 0.992 El 43.8 45.2 49.0 E1S 60.6 67.0 75.6 8 F 18 1.08 El (59.2) E1S 222 Mean * Value for MCRElB performed in the same patient (Table II) used for calculation of p; otherwise average values for our laboratory used (Table III). time. The average MCRisS in five men was 167 liter/ rates to equivalent E1 production rates, multiply EiS day or 87.4 liter/day per m2. The average MCR of five values by 0.71. women was 146 liter/day or 94.1 liter/day per mi. The Conversion ratios. CEiSEl and CE1S2 were measured large differences among normal subjects are related in five subjects (Table III). Shown in Table III are both to differences in fractional turnover rate, 'y (range the plasma levels of tritium in E1,E2, and ElS; con- 0.0679-0.219 pools/hr) and volume of distribution, V version ratios have been calculated at each time point. (range 20-93 liters). The range of volumes of distribu- Though plasma levels of E1S-3H, Ei-'H, and E2-'H are tion was roughly correlated with body surface area. increasing, the conversion ratios are constant and ap- The validity of calculation of MCRiS based on the pear to represent equilibrium values. Each series of assumption of a one-compartment system was tested by conversion ratios was examined for systematic trends comparing in the same subject the MCR obtained in or deviations from the mean. Only in subject 11 was this way with that obtained by the constant infusion there a suggestion that CE1S`l was increasing during the technique. In this study, we injected a bolus of E1S into period of observation. In subject 6, plasma levels of a normal subject. No. 6, and established a MCREsS from El-3H and E1S-'H reached equilibrium as did the con- the plasma disappearance curve over 8 hr. Subsequently, version ratios and the conversion ratio of 0.025, was a calculated bolus of E1S-'H was given followed by a the same as the average of the other subjects. The continuous infusion of ES-3H for a further 8 hr achiev- conversion ratio (CEi1"i) averaged 0.017 in five studies. ing a constant plasma level of E1S-'H (Fig. 3). From Transfer factors (p) were calculated using these con- the data obtained after the injection of E1S-'H (Table version ratios and values for MCREs and MCREnss either I), a MCR of 207 liter/day was calculated. The MCR measured in the same subject (Table IV) or using calculated from the constant infusion data (Table III) average values from our laboratory (Table III) (3). was 194 liter/day. These two MCR are not different. Transfer factor (pEiSEL) averaged 0.21 (range 0.13- The production rate of E1S was calculated from the 0.30) (Table III). MCREs and the measured plasma concentrations of E1S The conversion ratio (C'1"s2) averaged 0.0023 in (Table II). Average values for MCR and plasma EiS four studies, about 1/10 that for the conversion of E1S concentrations in both men and women were used. In to estrone. Transfer factors were then calculated using men, ES production averaged 77 Ag/day, not greatly these conversion ratios and values for MCRE, and different from E1 and E2 production rates. In women, MCREs either measured in the same subject (Table V) ES production averaged 95 Ag/day during the early or using average values from our laboratory (Table follicular phase; and 182 ,Ag/day during the luteal II) (3). The transfer factor ( pusu2) averaged 0.014 phase. Follicular and luteal phase ES production rates (range 0.010-0.019) (Table III). are compared with follicular and luteal E, and E2 pro- Since ES is effective by mouth in contrast to estrone duction rates (Table II). To convert EiS production and estradiol, the metabolism of EiS taken orally to

1026 H. J. Ruder, L. Loriaux, and M. B. Lipsett 3H Infusions

Plasma tritium levels Equilibrium CE1E1S 2.75 hr 3.0 hr 3.5 hr 3H levels EHS-3H/Ei-H MCRE1 MCREI pElEIB* cpm X 10-3/liter cpm/liter X 10-3 liter/day liter/day/n;2 49.2 45.7 10.3 2290 1350 0.67 123 471 45.0 44.3 8.68 2180 1400 0.58 142 385

47.4 46.3 5234 2140 1310 0.38 83.2 247 66.7 67.6 67.2 12.4 1610 1020 0.54 288 335 833

9.18 0.54

plasma ES, estrone, and estradiol was studied. When librium values for ES during a short E1 infusion. 30 ACi of E1S-3H was given by mouth. there was a There was agreement between this value and that ob- prompt rise in plasma levels of tritiated E1S, peaking tained by the extrapolation shown above. around 1 hr as shown in Fig. 4. Plasma levels of tri- The average of four determinations of CE1E1S in tiated E1 and E2 were 1.0% and 0.1%, respectively, plasma was 9.2 (range 5.34-12.4) (Table IV). Trans- that of E1S-3H, although E2-3H was detectable only at fer factors were calculated using the conversion ratios, 80 min. These values are in close agreement with the measured values for MCRE,, and average male and conversion ratios found during i.v. infusions of tritiated MCRE1s from Table I (an MCREs was measured ElS shown in Table III. Conversion of E1 to E.S. The data from the estrone- 1,000,000 F 3H infusions are shown in Table IV. Plasma level of the infused estrone became constant in all cases. How- ever, conversion of estrone to ES did not reach equi- librium in any infusion, as shown by increasing plasma levels of E1S-3H. Thus the conversion ratio, could not 100,000 be calculated directly since equilibrium conditions were EIS not met. Estimates of equilibrium values for estrone-3- sulfate and hence conversion ratios, C 1EIS, were calcu- lated using the formula, cpm/liter 10,000

(El - 3H))((1 - e-y- where E=-(H is th -eTX) (see Appendix) where Ei-3H is the equilibrium value for E1-3H during E1-3H infusions and EiStx-'H is the plasma level of 1,000j 7- tritium in ES at time T.; e is the natural logarithm, T. is the time after beginning the infusion; and y is the fractional pool turnover of ES per unit time. In subject 8, y was measured directly and average values E2A 1w- l for y from Table II were used for subjects 11-13. In 20 40 80 one subject, No. 18, in whom MCRE,, MCREs, and MINUTES plasma levels of all estrogens had been measured, the SAAM program of Berman and Weiss (12) was used FIGURE 4 Plasma levels of tritium in ES; estrone (E,), and estradiol (E2) after of E1S-3H at to perform a computer simulation and calculate equi- time 0 (subjects 1 and 2).

Estrone Sulfate: Production Rate and Metabolism in Man 10:27 TABLE V Estradiol-6, 7-

Plasma tritium levels and Estradiol- Sex hr Subject age 'H infusion 'H-steroid 1.75 hr 2.0 hr 2.25 hr 2.75 cpm/day X 10'- cpm X 1O-'/liter 7 F 18 1.32 E2 111 EiS 206 9 F 22 0.919 E2 EiS 15 F 22 1.27 E2 140 129 136 148 E1S 106 111 122 154 16 M 26 1.14 E2 69.3 73.2 72.1 E1S 26.7 38.9 17 M 26 1.06 E2 58.1 58.9 62.6 EiS 135 167 211 18 M 23 1.26 E2 60.1 60.9 64.9 EiS 78.4 79.2 100 Mean * Value for CMREs1 performed in the same patient (Table II) used for calculation of p; otherwise average values for our laboratory used (Table III). and used for calculations in subject 8). The transfer subject (13, 14). E1S has been the major urinary estro- factor (pElEis) averaged 0.54 (Table IV). genic metabolite. As Purdy et al. (2) showed, it is Conversion of Es to ES. The data obtained from also the major plasma metabolite of estradiol. We had estradiol-6,7-3H infusions are shown in Table V. Plasma postulated that there was a large plasma compartment levels of the infused steroid, estradiol, were at equi- that slowly exchanged with plasma estrone and estra- librium in all cases, but as with E1 the conversion of E2 diol (3), and it seemed likely that this was E1S. This to ES did not reach equilibrium. The extrapolation to and the lack of information concerning origin, clear- equilibrium values was performed as for E1. Again, the ance rates, and metabolism of ES prompted the studies SAAM program of Berman and Weiss (12) was used reported here. to confirm the validity of the estimates of equilibrium The MCREss was obtained by fitting the data obtained values calculated by the above formula. This was done after rapid injection of E1S-2H to a straight line thereby in subject 7 in whom we had sufficient data about MCR assuming a one-compartment model. Frequent samples and plasma estrogen concentrations to allow the com- were not obtained during the first hr so that a rapid been puter to derive equilibrium solutions. There was good component of the disappearance curve would have between the two methods. missed. In two studies, the 30 min sample suggested agreement little average of six determinations of C2Ei1s is 7.1; an early faster component but this could exert The MCR since the total range 2.19-13.6) (Table V). Transfer factors were effect on the calculation of the these conversion ratios, actual mea- rate of removal of E1S from plasma is so slow. The calculated using the MCR ob- values for MCRH2, and average male and female study in subject 6 directly compared sured into a compartment values for MCRjss from Table I (MCRxis was mea- tained assuming distribution single in and that obtained during constant infusion when plasma sured and used in the calculation of transfer factor Since 7 and 9). The transfer factor (PE2E1') averaged concentrations of E1S-2H were at equilibrium. subjects method are independent 0.65 (Table V). results obtained by the latter of the compartmental distribution of the isotope, the DISCUSSION agreement between these data is validation of our mea- In the study of the clearance Estrone sulfate was identified in urine over 30 years ago surement of MCRss. single role in the economy of DHS, a one-compartment model also was adequate but little is known of its estrogen the steroid The urinary estrogens are generally to explain the distribution of (15). of the body. Although of 150 is in agreement with excreted as the glucuronide conjugates, in an occasional estimated MCRuss liter/day 1028 H. J. Ruder, L. Loriaux, and M. B. Lipsett 3H Infusions

Plasma tritium levels Equilibrium CE2E1S 3.0 hr 3.5 hr 5.0 hr 7.0 hr 2H levels ElS-'H/E2-'H MCRE2 MCRE2 pE2E1S* cpm X 10-3/1iter cpm/liter X 10-3 liter/day liter/day/rn 111 108 110 11.0 1200 739 0.59 293 318 1210 58 68 81 74.5 5.8 1230 832 0.81 108 200 275 432 138 425 3.08 920 520 0.56

1.5 2.19 1594 886 0.22

815859.9 13.6 1770 983 1.20 62.0 6.68 2032 1129 0.52 414 17.06 3 0.65

data reported recently by Longcope (16) and confirms same binding sites. However, the association constants the early work of Twombly and Levitz (1) who showed were influenced by the nature of the steroid. The asso- that ES is cleared from the blood slowly. ciation constant of DHS with albumin has been re- The plasma MCREs of 150 liter/day is considerably ported as 0.6-2 X 10' liter/mole (20, 22), whereas the higher than the plasma MCR of DHS (7 liter/day) E1S binding constant was estimated at 2 X 104 liter/ (15), and of the blood MCR of DHS (16-30 liter/day) mole (24). Although these binding affinities were not (17), sulfate (25 liter/day) (17), 17- obtained in the same system, the association of lower acetoxypregnenolone sulfate (37 liter/day) (18). A affinity constants with higher volumes of distribution recent report gives values for MCR is apparent. It therefore seems probable that the clear- of 75-240 liter/day which are in the same range as ance rates of the steroid vary inversely with our estimates of MCRElS (19). However, the fractional the tightness of binding to albumin which in turn de- pool turnover rate of E1S of 3.3 pools/day is about the termines the volume of distribution. same as the fractional pool turnover rates of other Using the average MCRE1s and mean ELS plasma steroid sulfates (15, 17-19). Thus, the higher MCREXS concentrations, we found that plasma production rates must be associated with a larger volume of distribution of E1S were similar to those of estrone and estradiol of E1S, and in fact its volume of distribution of about in men and women. Although we have not looked for 50 liters is higher than those of the other steroids (15, possible variations of E1S concentrations throughout 17, 18) which have lower MCR. Only sul- the day, it can be shown that will not fate they vary greatly had a higher MCR than E1S but it had a relatively because of the low fractional pool turnover rate small (ay). volume of distribution (7 liters) and a higher Thus, the E1S production rates need not fractional he corrected turnover rate almost equal to that of some for a mean plasma EiS concentration, and should closely unconjugated steroids (18). approximate the 24-hr production rates of E1S. The volume of distribution of the Since sulfokinases are present in peripheral depends on tissues the binding of the sulfates to plasma pro- as well as in the and , we could teins, in particular to albumin. Binding to albumin in not know to what extent E1S was either secreted' or man has been demonstrated by Puche and Nes (20), Sandberg and Slaunwhite (21), and Plager (22). 2 The term "" is used to indicate entry of the Puche and Nes, Plager, and Wang and Bulbrook (23) steroid into the plasma from the glands. The term "produc- tion" signifies the rate of entry of the steroid from all noted that all steroid sulfates tested competed for the sources.

Estrone Sulfate: Production Rate and Metabolism in Man 1029 produced from peripheral transformation of plasma is also evident from our data for pE1ElsI p 2 1 , and precursors. We therefore calculated the contributions MCREls. Since our study included only a small sample of plasma estrone and estradiol to the E1S production from a population with a large normal range, it is rate. To do this, we used our values for transfer fac- possible that our average values for pE1E1S, p 2B1S could tors, pE1E1S, pE8; pE1SE1 p1SE2 (Tables II-V) as well be considerably higher than the true mean for the as the average transfer factors, pEB2A, pE2A1 (25, 26). normal population. Agreement between the calculations The following three equations give solutions for the would be possible only if all parameters were mea- net amount of steroid entering the plasma from all sured in the same normal subjects. Secondly, average sources (shown by [ ] ) other than from conversion values for estradiol concentrations in men reported by of the two other steroids: us previously (5) and used for our calculations of E2 production rates are somewhat higher than more recent [E1] = PRE1 - pE1ESEi[E1S] - pE2E'[E2] values of 20-25 pg/ml reported by other laboratories [E2] = PRE2 - - pE1SE2[EEs] pE1E2[E1] (9-11). The errors in measurement of low estrogen [EE5] = - - pE2EjS[E2] PREIS pE1E1S[Ei] concentrations by radioligand assays tend to cause Since the production rates (PR) and transfer factors high values which would result in overestimation of are known, the equations can be solved for the [ ] production rates. The consistent finding that estrogen values. These values are not true secretion rates since production rates, calculated from dilution of labeled they would include any steroid produced from other estrogen into urinary , are smaller than precursors. For example, [EL] would include that those calculated from the MCR and plasma concentra- estrone derived from plasma and tes- tion is further evidence that the latter method over- tosterone as well as that moiety that is secreted. estimates the production rate. Next, the known diurnal The derived data are given in Table VI. For each variations in plasma estrone concentration (27, 28) and subgroup, [E1S] was negative. This means that the possibly of estradiol, mean that production rates ob- production rate calculated to result from conversion of tained using morning plasma estrogen concentrations plasma estrone and estradiol is higher than that cal- will systematically overestimate the 24 hr estrogen culated by direct measurement of the MCRisu and production rate of these unconjugated estrogens. plasma E1S concentration. Hence, there is no need to Finally, since plasma estradiol and estrone have plasma postulate glandular secretion of E1S. The lack of cor- turnovers of about 50 pools/day and E1S turnover rate respondence of the data must be explained and serves is 3.3 pools/day, plasma concentrations of E1S will to point out the many sources of discrepancies in all lag behind plasma estrone and estradiol by 6-12 hr. studies such as this. Therefore, it will be difficult to arrive at correct sam- The first and most important reason for lack of cor- pling times to estimate the contributions of estrone respondence in calculations such as these is not really and estradiol to E1S production rates. "error" but relates to the large range of normal aver- Since most of the errors discussed will cause over- age values for metabolic clearance rates and transfer estimation of estrone and estradiol blood production factors that are used. Inspection of published data for rates, the amount of EiS calculated to arise from these metabolic clearance rates of E1 and E2 (3, 25, 26) re- sources will also be overestimated. Nevertheless, all E1S veals not only a large range of values for normals but found in plasma can be accounted for merely by using also for groups of normals studied in the same labora- mean values ± standard deviations for production rates tory (25, 26). This same large range of normal values and p used in the calculations. Hence, it is untenable

TABLE VI Origin of Estrone Sulfate PR EiS PR EiS [ El]* pElISC[Ei] [E2] pE2EiS[E2] CEiS] (from Ei and E2) (MCR X i) Men 153t 82.6 74.5 48.5 -54 131 77 Women Follicular phase 137 74 157 102 -81 176 95 Women Luteal phase 175 94.5 279 181 -94 275 182 * [I = the amount of steroid entering the plasma from all sources other than the two other steroids. $ All values are micrograms per day, expressed as E1S. 1030 H. J. Ruder, L. Loriaux, and M. B. Lipsett to suggest (29) that E1S may be secreted in amounts levels. Thus it may be that the conversion of orally ad- sufficient to make it a significant precursor of plasma ministered estrone sulfate to plasma estrone and estradiol estrone. is sufficient to account for its biologic effect. In this re- The transfer factor, pE 11 of 0.21 was derived from gard, it is of interest that Gurpide, Stolee, and Tseng the conversion ratios obtained from the short infusions (31) reported that human endomentrium rapidly metab- of ES during which plasma levels of E1S-3H, E1-3H, olized estrone sulfate to estrone and estradiol. and E2-3H were increasing in four of the five studies. In summary: (a) EiS is a major circulating plasma However, pE1SEi and pE1SE2 appeared to be at equilibrium. estrogen and has a long plasma half-life; (b) the large The pE1SE1 of 0.21 agrees closely to that of 0.15 re- contributions of estrone and estradiol to plasma ES ported recently by Longcope (16). The transfer factor, are more than sufficient to account for all the circulating pESE2 was only 10% of that for estrone (Table IV). plasma EiS; (c) orally administered E1S is metabo- Since pE1E2 is also about 10% (25, 26), it is probable lized to plasma estrone and estradiol to the same extent that E1S reaches the plasma estradiol compartment as i.v. administered ES. via plasma estrone. Although the mass of estrone sul- fate available for exchange with E1 is large, about 25- APPENDIX. 50 Ag (plasma concentration X volume of distribution), only 5-10 /Ag could enter the plasma volume Because of the slow fractional turnover rate of EIS, measure- daily as ment of pElElS and at values of estrone. This is a small pE2EiS equilibrium El, E2, and fraction of estimated estrone EIS was impossible unless infusions were carried out for longer production rates of 40-200 ,Ag/day in men and women. than 24 hr. Since the dissappearance of E1S from plasma can Although pE1SE1 and pE1SE2 were apparently at equilib- be adequately described by a single exponential, this same rium, computer simulations of these studies' using the single exponential describes the appearance of E1S in plasma an infusion of - or E2- 3H. SAAM program of Berman and Weiss during E1 3H When plasma levels (12) have of El - 3H and E2 - 3H are at equilibrium, a constant shown that these conversion ratios cannot be at equi- amount of E1 - 3H or E- 3H is being metabolized to E1S. librium and that the true conversion ratio may be Thus when estrone or estradiol is infused at rates such that greater by about 15%. Short sampling times and vari- their plasma levels reach equilibrium values, ElS enters the at a rate to the or ability in the determinations mask this trend. For the plasma equal pElEIS pE2EiS X the infusion rate of E1 - 3H or E2- 3H. We were therefore able to purposes of understanding these facets of estrone sul- estimate CEIE S, CE2EI' during short infusions of either fate metabolism, the values used in our calculations E -3H or E2- 3H by making the following assumptions: are sufficiently accurate although they are not equilib- (a) The bolus given before the El or E2 infusions adequately rium values. sets the plasma E1 - 3H or E2- 3H at an equilibrium level that is then maintained by the constant E -3H or E2- 3H In contrast to the low biologic of orally ad- infusions. (b) A constant amount of El - 3H or E2 - 3H is ministered estrone and estradiol, EiS is an active oral being metabolized to E1S- 3H from the time 0 when the bolus estrogen. Our data confirm that it is rapidly absorbed is given and hence a constant amount of E1S- 3H is entering from the gut and reaches the peripheral circulation the plasma E1S pool during the entire time of the short El or without E2 infusion. To verify these assumptions, we performed com- significant metabolism by the . Since 0.5 puter simulations of typical El - 3H or E2 - 3H infusions mg of estrone sulfate is an adequate replacement dose utilizing the computer model program of Berman and Weiss for many postmenapausal women, we have calculated (12). We were able to show that the dose of E1 - 3H or that the plasma estrone production rate resulting from E2- 3H given as a bolus to begin the short infusions ade- set the level this dose is 70 /Lg/day (0.5 X 0.7 x 0.2).' quately plasma of E -3H or E2- 3H at near Similarly, equilibrium levels; and that in addition, a constant amount of the resultant estradiol production rate would be 7 gg/ E1S - 3H is entering the plasma EIS pool from the time of day. The minimal estrogen production rates that cause the bolus at time 0. either vaginal cornification or endometrial hyperplasia Thus, the instantaneous change in E1S- 3H is described are unknown but Grodin and McDonald 5 found that by the following, an estrone production rate greater than 40 ,g/day (by urinary isotope dilution) induced endometrial hyper- dEiSt = k1Eit, - yE1St., plasia in women. The estrone production rate of normal postmenapausal women calculated from plasma concentra- where E1St. is the instantaneous level of E1S- 3H at time t,; El is the instantaneous - tions and metabolic clearance rates is about 40 level of E1 3H at time t,; k1 is the Ag/day fractional turnover of El - 3H to E1S- 3H; and y is the total (30) and there is little biologic estrogenic effect at these rate of removal of ElS- 3H as given in Table I. Integrating and solving for ElSt. yields, "Ruder, H. J., L. Loriaux, M. B. Lipsett, and M. Ber- man. Manuscript in preparation. E =Sk=Elt (x1 --yTx), 'Dose of E1S is 0.5 mg/day; the transfer constant, pES1 is 0.2 and only 70% of E1S is estrone by weight. where e is natural 6Grodin and McDonald. Personal communication. logarithm; T. is time after beginning the studies. By definition, however CEIEIS = ElS- 3H/El - 3H

Estrone Sulfate: Production Rate and Metabolism in Man 1031 when both are equilibrium values. And in fact, Et. - 3H is at 7. Bardin, C. W., and M. B. Lipsett. 1967. Testosterone equilibrium from time 0 as explained above and will be written and androstenedione blood production rates in normal El- H. Hence, Et. - 3H = El- H. At time T. = oo, E1S women with idiopathic hirsutism or polycystic . is also at equilibrium by defintion and eYTz = 0 and yields: J. Clin. Invest. 46: 891. 8. Horton, R., and J. F. Tait. 1966. Androstenedione pro- duction and interconversion rates measured in peripheral ElS =klEl (1 -O). blood and studies on the possible site of its conversion to testosterone. J. Clin. Invest. 45: 301. Rearranging yields: 9. Baird, D. T. 1968. A method for the measurement of estrone and estradiol-17 in peripheral human blood and E1S ki other biological fluids using '3S pipsyl chloride. J. Clin. El z Endocrinol. Metab. 28: 244. 10. Korenman, S. G., L. E. Perrin, and T. P. McCallum. Since CEiELS = E1S- 3H/El -3H; CElE1s = kl/-y. Thus, 1969. A radio- binding assay system for estradiol measurement in human plasma. J. Clin. Endocrinol. ElSt. -3H = CElElS . (E1 - 3H) (1- ei7T.). Metab. 29: 879. 11. Nagai, N., and C. Longcope. 1971. Estradiol-17 and And rearranging: estrone: studies on their binding to rabbit uterine and their concentration in plasma. Steroids. 17: 631. CElEiS = - 12. Berman, M., and M. F. Weiss. 1967. Users manual for (E1 - 3H) (1 - eyTx) SAAM. U. S. Government Printing Office, Washington, The same formula holds for CE2EIS substituting appropriate D. C. Publication No. 1703. values for E2 and -y. All conversion ratios shown in Tables IV 13. Howard, G. M., H. Robinson, F. H. Schmidt, J. R. Mc- and V were calculated using this formula and, where possible, cord, and J. R. Preedy. 1969. Evidence for a two-pool the actual measured value for oy. Otherwise, average values for system governing the of radioactive urinary oy were used from Table I. Since each infusion had at least three estrogen conjugates during the first eight hours follow- plasma samples at different time points, conversion ratios were ing the administration of estrone-6,7-'H to male sub- calculated at each time point during a single infusion and jects. Probable role of the enterohepatic circulation. J. averaged, yielding the values shown in Tables IV and V. Clin. Endocrinol. Metab. 29: 1618. Validity of these calculations was confirmed by using data 14. Hobkirk, R., M. Nilsen, and P. R. Blahey. 1969. Con- from subjects 7 and 8 in whom we had estimates of all MCR jugation of urinary phenolic steroids in the nonpregnant and plasma concentrations for computer simulation using the human female with particular reference to estrone sul- SAAM program of Berman and Weiss (12). There was close fate. J. Clin. Endocrintol. Metab. 29: 328. agreement between conversion ratios predicted by the com- 15. Sandberg, E., E. Gurpide, and S. Lieberman. 1964. Quan- puter program and the conversion ratios calculated by the titative studies on the metabolism of dehydroisoandro- above formula. sterone sulfate. . 3: 1256. 16. Longcope, C. 1972. The metabolism of estrone sulfate ACKNOWLEDGMENTS in normal males. J. Clin. Endocrinol. Metab. 34: 113. authors 17. Wang, D. Y., R. D. Bulbrook, A. Sneddon, and T. The assistance of Dr. Monas Berman in teaching the of program and of Dr. David Hamilton. 1967. The metabolic clearance rates dehy- the use of the SAAM computer droepiandrosterone, testosterone and their sulfate Rodbard for his aid in the derivation of the mathematical Endocrinol. 38: 307. formulas is gratefully acknowledged. in man, rat and rabbit. J. 18. Wang, D. Y., D. R. Bulbrook, F. Ellis, and M. M. REFERENCES Coombs. 1967. Metabolic clearance rates of pregnenolone, 17-acetoxypregnenolone and their sulphate esters in man 1. Twombly, G. H., and M. Levitz. 1960. Metabolism of and in rabbit. J. Endocrinol. 39: 395. estrone-C"-16 sulfate in women. Amer. J. Obstet. Gyne- 19. Saez, J. M., J. Bertrand, and C. J. Migeon. 1971. Meta- col. 80: 889. bolic clearance rate, urinary and plasma production rates 2. Purdy, R. H., L. L. Engel, and J. L. Oncley. 1961. The of testosterone sulfate in man. Steroids. 17: 435. characterization of estrone sulfate from human plasma. 20. Puche, R. C., and W. R. Nes. 1962. Binding of dehydro- J. Biol. Chem. 236: 1043. sulfate to serum albumin. Endocrinology. 3. Hembree, W. C., C. W. Bardin, and M. B. Lipsett. and 70: 857. 1969. A study of estrogen metabolic clearance rates 21. Sandberg, A. A., and W. R. Slaunwhite. 1957. The bind- transfer rates. J. Clin. Invest. 48: 1809. ing of steroids and steroid conjugates to human plasma 4. Mumma, R. O., C. P. Hoiberg, and W. W. Weber. proteins. Recent Progr. Res. 13: 209. 1969. Preparation of sulfate esters. The synthesis of medicated 22. Plager, J. E. 1965. The binding of sulfate, steroid sulfates by a dicyclohexyl-carbodiimide sulfate, and dehydroisoandrosterone sul- . Steroids. 14: 67. Invest. 44: 1234. M. B. 1971. fate by human plasma . J. Clin. 5. Loriaux, D. L., H. J. Ruder, and Lipsett. R. D. Bulbrook. 1967. Binding of the measurement of estrone sulfate in plasma. Steroids. 23. Wang, D. Y., and The sulfate esters of , testosterone, 18: 463. 17-acetoxypregnenolone and pregnenolone in the plasma 6. Ruder, H. J., P. Corvol, J. A. Mahoudeau, G. T. Ross, Endocrinol. 39: 405. and M. B. Lipsett. 1971. Effects of induced hyperthy- of man, rabbit and rat. J. 24. Sandberg, A. A., H. Rosenthal, S. L. Schneider, and roidism on steroid metabolism in men. J. Clin. Endocri- interactions and their nol. Metab. 33: 382. W. R. Slaunwhite. 1966. Steroid 1032 H. J. Ruder, L. Loriaux, and M. B. Lipsett role in the transport of steroids. In Steroid Dynamics. castrate and postmenopausal women and in men. J. Clin. G. Pincus, T. Nokao, and J. F. Tait, editors. Academic Endocrinol. Metab. 29: 149. Press, Inc., New York. 1-61. 28. Tulchinsky, D., and S. G. Korenman. 1970. A radio- 25. Longcope, C., D. S. Layne, and J. F. Tait. 1968. Meta- ligand assay for plasma estrone; normal values and varia- bolic clearance rates and interconversions of estrone and tion during the . J. Clit. Endocrinol. 17-estradiol in normal males and . J. Clin. Invest. Metab. 31: 76. 47: 93. 29. Baird, D., R. Horton, C. Longcope, and J. F. Tait. 1968. 26. Longcope, C., and J. F. Tait. 1971. Validity of metabolic Steroid prehormones. Perspect. Biol. Med. 11: 384. clearance and interconversion rates of estrone and 17- 30. Longcope, C. 1971. Metabolic clearance and blood pro- estradiol in normal . J. Clin. Endocrinol. Metab. duction rates of estrogens in postmenopausal women. 32: 481. Amer. J. Obstet. Gynecol. 111: 778. 27. Baird, D. T., and A. Guevara. 1969. Concentration of 31. Gurpide, E., A. Stolee, and L. Tseng. 1971. Quantitative unconjugated estrone and estradiol in peripheral plasma studies of uptake and disposition of . in nonpregnant women throughout the menstrual cycle, Acta Endocrinol. Suppl. 153.

Estrone Sulfate: Production Rate and Metabolism in Man 1033