The metabolic clearance and blood production rates of estriol in normal, non-pregnant women.

The Metabolic Clearance and Blood Production Rates of Estriol in Normal, Non-Pregnant Women C. FLOOD, J. HOWARD PRATT, AND C. LONGCOPE The Worcester Foundation for Experimental Biology, Shrewsbury, Mass. 01545, and the Department of Medicine, Boston University School of Medicine, Boston, Mass. 02118 ABSTRACT. The metabolic clearance rate (MCR) and blood production rate (PB) of estriol have been measured in normal, non-pregnant women 21 to 65 years old. 6,7-3H-Estriol was administered as a pulse injection to 4 women between days 5-7 of their menstrual cycle. The disappearance of radioactivity as unconjugated estriol can be described as a function which is the sum of two exponentials. The initial component represents spread into and transfer from a space with a volume of 20.6 ± 5.4 (SE) 1. The mean value for the rate constant of total removal (reversible and irreversible) was 290.2 ± 78.5 units/day of which 0.34 ± 0.06 was irreversible. The mean MCRR was 990 ± 70 1/day/m2. 4-14C-Estriol was infused at a constant rate for 3% hours to 13 women between days 5-7 of their cycle. The mean MCR was 2,100 ± 100 I/day or 1,240 ± 40 1/day/m2. Thirteen women received a constant infusion of 4-14C-estriol between days 20-22 of their cycle. The mean MCR was 2,100 ±115 I/day or 1,280 ± 65 1/day/m2. The mean values for the two phases of the cycle were not significantly different

(P>0.1). The mean value for the MCR in 4 post-menopausal women studied in similar fashion was 1,890 ± 95 I/day or 1,060 ± 35 1/day/m2. The mean concentrations of estriol were 7.0 ± 0.7 and 10.9 ± 0.8 in the follicular and luteal phases of young women, respectively. The mean PB for women in the follicular phase was 14.0 ± 1.6 /itg/day and in the luteal phase was 22.7 ± 1.9 /xg/day. These values were significantly different (P < 0.01). When the PB's for the 11 women studied in both phases of the cycle were compared the luteal phase values were significantly higher 0.02 > P > 0.01) using the paired t test. The PB in the 4 post-menopausal women ranged from 5 to 22 /xg/day. While there was no difference between the MCR of estriol measured in the two phases of the cycle, the PB of estriol was significantly greater in the luteal phase. Estriol probably contributes little to the overall estrogenic activity in normal, non-pregnant, premenopausal women but could make a more significant contribution in some post-menopausal women. (J Clin Endocrinol Metab 42: 1, 1976)

I

T HAS been shown that estriol binds to the nuclear receptors of the rat (1,2,3) and human (4) uterus and can inhibit the stimulatory action of estradiol on uterine weight (5). Estriol administration has been noted to protect animals against carcinogeninduced mammary cancer (6). Despite these, and other evidences of activity, the pathways of estriol metabolism have been largely characterized only by the types of conjugates found and the relative speed of their urinary excretion (7-12). The biologic activity of estriol, however, is probably exerted by the nonconjugated free compound, about which less is known. In view of the possible protective role estriol might play with

regards to breast cancer (6,13,14), we wished to delineate better the metabolic clearance and production rates of unconjugated estriol in normal women of various ages. Materials and Methods All subjects were healthy women 21 to 60 years old, who were on no medication, and who had given informed consent for the studies. All infusions were performed with the subjects supine and, in general began at 8 AM. However, in several instances, infusions began at 4 PM, but we have previously shown that for those steroids studied so far the metabolic clearance rates do not vary with the time of the day (15) provided that the subjects are supine. All solvents, except as noted, were re-distilled prior to use, and di-ethyl ether was passed through an alumina column prior to use. Acetic anhydride and pyridine were prepared as previously described (16). Silica gel HF254 (Brinkman Instruments, Westbury, L.I.) was used for thinlayer chromatography. Estriol was obtained from

Received April 29, 1975. Supported by Contract No. CB-33902 from the National Cancer Institute. A portion of this work was presented at the 56th Meeting of the Endocrine Society, Atlanta, Ga., 1974. Reprints: Dr. C. Longcope, Worcester Foundation for Experimental Biology, Shrewsbury, Mass. 01545. 1

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FLOOD, PRATT AND LONGCOPE

JCE & M • 1976 Vol 42 • No 1

taken up in 2 ml CC14:CHC13 (5:1, vol/vol) and extracted 2 x with 1.0 ml vol of lN-NaOH (19). The NaOH extracts containing the estrogens were pooled, adjusted to pH 7.0 with 1.0 ml 3N acetic acid and extracted 2 x with 5 ml vol of diethyl ether. The ether extracts were pooled, washed 2 x with 1 ml vol H2O, and the ether removed under nitrogen. The dried residue was spotted and developed in TLC system no. 1, CHCl3:acetone (90:10, vol/vol). Following development, the estriol area was located under UV and eluted with acetone. The acetone was removed under nitrogen and the dried residue spotted and developed in TLC system no. 2, ethyl acetate:n-hexane:ethanol (80:15:5 vol/vol/vol). Following development, the estriol area was Pulse injection studies located and eluted. The acetone was removed, These studies were carried out as described and the dried residue spotted and developed in (17,18) and all subjects were between days 5-7 of TLC system no. 3, CHCl3:EtOH (85:15). The 3 their cycle. Subjects received 15 fxCi of 6.7- Hestriol area was located and eluted. The acetone estriol in 10 ml of an 8% ethanol in isotonic saline was removed and the dried residue chromatosolution in one arm vein. Heparinized blood graphed on LH-20 Sephadex as described above. samples were then drawn from the opposite arm The estriol fraction was collected, the tube dried at increasing time intervals up to 300 min. The under nitrogen, and the residue counted. samples were centrifuged and the plasma stored In order to show radio-chemical purity of the at —15 C until analyzed as described below. estriol, random samples were treated as follows: following chromatography on LH-20 Sephadex, Constant infusion studies the estriol fraction was split into 2 parts, one part With the subjects supine, a priming dose of 2.5 was counted as usual, the other part was acetyfjuCi of 4-14C-estriol in 8 ml of an 8% ethanol in lated overnight with 0.1 ml pyridine and 0.1 ml isotonic saline solution was given in one arm vein. acetic anhydride. The following day the acetylaThen, into the same vein 4.0-6.0 fid of 4-14C- tion was stopped with 0.05 ml ethanol, and the estriol in 14 ml of the solution was infused at a tube was dried under nitrogen. The residue was constant rate for 210 min. Heparinized blood spotted and developed in TLC system no. 4, CHCl3:acetone (98:2 vol/vol). Following desamples, 30 ml, were obtained after 150,180, and velopment, the estriol triacetate area was located 210 min of the infusion. The samples were cenunder UV, eluted, and counted. The 3H/14C ratios trifuged and the plasma was stored at - 1 5 C until of the estriol and estriol triacetate were not analyzed as described below. significantly different indicating radiochemical purity had been achieved following Sephadex Analysis of plasma samples chromatography. a) To the samples obtained following pulse b) To the samples obtained during the constant injections, 200 dpm 4-14C-estriol, 100 fjug estriol, infusions, 200 /xg of nonradioactive estriol were and 4 drops of 50% NH4OH were added, the added. (The reason for not using radioactive samples being agitated to insure adequate mix- estriol to correct for losses during the procedure 3 ing. The samples were poured into 250 ml was that in many infusions H-precursors to esin the separatory funnels, to which 5 ml H2O and 30 ml triol were also infused so that the estriol 3 14 diethyl ether were added, and were then shaken final samples already contained both H and C). The samples were then processed as described vigorously. The contents were extracted twice with 100 ml volumes of cold dichloromethane above with the following modification: following (CH2C12). The CH2Cl2:ether extracts were pooled chromatography on LH-20 Sephadex, the estriol and washed once with 10 ml saturated NaHCO3 fraction was dried under nitrogen. The residue and once with 10 ml H2O, and the CH2Cl2:ether was then taken up in ethanol and the optical removed under vacuum. The dried residue was density measured at 265, 280, and 295 nm's using

Steraloids, Inc., Pawling, N.Y. and was crystallized from methanol prior to use. 4-14C-Estriol (SA 50 mCi/mmole) and 6,7-3Hestriol (SA 50 Ci/mmole) were obtained from New England Nuclear Corp. They were purified prior to use by column chromatography on Sephadex, LH-20 (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.). The estriol was applied successively with 50 /x\ methanol, 0.1 ml benzene and 0.1 ml benzene:methanol (70:30, vol/vol). The column was then developed using benzene:methanol (85:15 vol/vol); the first 2.5 ml run through was discarded, and the next 3.0 ml, containing the estriol, was saved.


METABOLISM AND PRODUCTION OF ESTRIOL a Beckman DB Spectrophotometer (Beckman Instruments, Palo Alto, Calif.). The optical densities of the samples were compared with those obtained from known concentrations of estriol after applying the Allen correction (20), and the mass of estriol in the sample was determined, and the losses incurred in the procedure were calculated. The ethanol was then removed under nitrogen, the samples were assayed for radioactivity (21) and the final counts determined after correction for procedural losses. For a number of the later infusions, 2 or occasionally all 3 samples were pooled and analyzed collectively, as part of a continuing study on the precursors of estriol. Random samples were divided after Sephadex ehromatography, and one aliquot was measured and counted while the other aliquot was acetylated and handled as described above. Following isolation of the estriol triacetate the OD was measured by UV and the mass determined from the OD of known standards. The specific activity of estriol triacetate was not significantly different from that of the estriol, indicating radiochemical purity after the Sephadex ehromatography. Radioimmunoassay of estriol was carried out on blood samples obtained prior to the infusion as described in a previous report (22). Analysis of data a) Pulse Injection. The CPM/1 of estriol were analyzed by the approach described by Rizkallah (23), and used by us in describing the metabolism of estrone sulfate (17), estrone, and estradiol (18). b) Constant Infusion. Using the data from the pulse injection and the approach of Shipley and Clark (24) we calculated that a priming dose equal to twice the hourly infusion rate would result in an isotopic steady state within 2V2 hr. For each

infusion in which 3 samples were analyzed separately the concentrations in plasma of radioactivity as estriol were normalized as per cent of the mean concentration. An estimate of variance was then obtained for the groups of data by a one-way analysis of variance done on the normalized data in order to test the significance and determine the confidence limits for the mean slope (21). The metabolic clearance rate (MCR) was calculated as described (25). The blood production rate (PB) was calculated as PB = MCR x i where i is the concentration of endogenous estriol. Student's t test and paired t test were used for statistical analysis of the data. Results All values will be given as mean ± SE unless stated otherwise. Pulse injection data (Table 1) For the 4 subjects studied the disappearance of radioactivity as free estriol could be described as a function which is the sum of two exponentials. The mean value for the initial volume of distribution was 20.6 ± 5.4 liters. Based on a two exponential function, the mean MCR for estriol was 1,600 ± 100 I/day or 990 ± 70 1/day/m2. Constant infusion data (Tables 2, 3, and 4) The mean value of the slope determined as noted above was 3.2% per 100 minutes with 95% confidence limits of -7.4 to +13.8%.

TABLE 1. Pulse injection data

A* Subject 1 2 3 4

Mean ±SE

B

Fraction of dose

at

MCR R

/3

units/day

R

ktt

V

MCR / kv**

I/day

1/day/m2

0.035 0.043 0.025 0.148

0.0061 0.0078 0.0062 0.0099

166.8 350.1 262.5 537.6

12.70 19.23 11.45 24.61

144.2 298.8 212.0 505.7

24.3 19.7 32.0 6.3

0.41 0.31 0.21 0.45

1,450 1,900 1,580 1,470

930 1,140 1,050 830

0.0627 0.028

0.0075 0.0008

392.2 78.9

17.00 3.06

290.2 78.5

20.6 5.4

0.34 0.06

1,600 100

990 70

* A and B are the intercepts of each exponential extrapolated to '0' time. f a and /3 are the respective slopes of each exponential. tf k = rate constant of total removal (22). ** MCRR/kv = fraction of total removal that is irreversible.


JCE & M • 1976 Vol 42 • No 1

F L O O D , PRATT AND L O N G C O P E TABLE 2. Data from infusions of l4C-estriol in the follicular phase Minutes from start of infusion Subject # 11 12 13 14 15 16 17 18 19 20

21 22 23

rate cpm/day x 10-6 14.8 14.7 15.6 14.1 27.6 21.3 11.2 15.5 19.5 19.2 22.3 17.2 21.8

150

180

210

Mean

cpm/1 iter plasma* 8,080 —

8,820 7,740 14,500 7,180 5,830 8,310 7,070 — —

5,910 —

7,400 Pool 7,570 — — — — — 8,350 9,170 Pool 6,650 Pool

8,140 —

9,070 6,710 14,920 8,290 5,410 6,540 6,050 9,320 —

7,270 —

7,870 7,570 8,490 7,070 14,710 7,730 5,620 7,420 7,160 9,240 11,960 6,610 12,500

Mean ± SE

MCR

I/day

1/day/m2

pg/ml

it

Pet /i.g/day

1,880 1,940 1,840 1,990 1,880 2,750 1,990 2,090 2,720 2,080 1,860 2,600 1,740

1,170 1,270 1,010 1,260 1,120 1,110 1,270 1,220 1,640 1,330 1,240 1,290 1,080

8

15 15 11 18 15

2,100

1,240

7.0

14.0

100

40

0.7

1.6

8 6

9 8 3 6 3 8 11

22 23

7

9

3 11

8 19

8 12

6

* 14C (cpm/1) corrected for losses with non-radioactive indicator. f i = plasma concentration. | PB = blood production rate.

Since the confidence limits include zero, the mean slope was not significant, and we concluded that the isotopic steady state was reached during the 3Vi h infusion. The mean value for the MCR of estriol for the 13 women in the follicular phase was 2,100 ± 100 I/day or 1,240 ± 401/day/m2. The

mean value for the MCR of estriol for the 13 women in the luteal phase was 2,100 ±115 I/day or 1,280 ± 65 1/day/m2. The mean values for the follicular phase were not significantly (P > 0.1) different from those of the luteal phase. Eleven of the 13 women were studied in both phases of the cycle, and

TABLE 3. Data from infusions of 14C-estriol in the luteal phase Minutes from start of infusion

no. 11 12 13 14 15 16 17 18 19 20 21

24 25

Infusion rate cpm/day x 10~6 28.5 20.5 22.1 25.4 20.3 20.9 30.0 17.6 14.3 11.5 14.8 20.7 20.6

150

180

210

Mean

cpm/liter plasma* 14,220 — 14,570 —

9,440 —

16,030 8,830 — —

6,150 6,740 —

13,730 14,510 10,940 11,630 9,610 7,810 14,750 9,170 Pool 5,200 6,770 7,160 Pool

14,010 14,570 13,620 12,020 9,930 7,420 17,010 9,310 — 5,370 6,740 6,510 —

13,990 14,240 13,040 11,820 9,660 7,610 15,930 9,100 6,880 5,280 6,550 6,800 10,660

Mean ± SE

MCR

it

I/day

1/day/m2

2,040 1,440 1,690 2,150 2,100 2,750 1,880 1,930 2,080 2,180 2,260 3,040 1,930

1,270 1,430 1,260 1,180 1,200 1,130 1,280 1,400 1,510 1,830 1,280

10 10

2,110 115

1,280 65

10.9 0.8

940 930

pg/ml 10 11

8 11 11 7 16 16 6 14 12

Pel /ig/day 20 16 13 24 23 19 30 31 12 30 28 30 19

22.7 1.9

* 14C (cpm/1) corrected for losses with non-radioactive indicator, t i = plasma concentration. | PB = blood production rate.


METABOLISM AND PRODUCTION OF ESTRIOL TABLE 4. Data from infusions of MC-estriol in post-menopausal women Minutes from j;tart of infusion Subject no.

Age

Infusion rate cpm/day x 10~6

31 32 33 34

60 58 58 53

17.5 28.9 12.4 16.1

150

14,080 7,580

180

Mean

210

cpm/liter plasma* 8,690 8,570

8,130 16,280 7,570 7,830

8,410 15,180 7,580 8,200

Mean ± SE

MCR I/day

1/day/m2

it pg/ml

fig/day

2,080 1,900 1,630 1,960

1,110 1,110 960 1,080

3 7 3 11

7 13 5 22

1,890 95

1,060 35

* I4C (cpm/1) corrected for losses with non-radioactive indicator. f i = plasma concentration. \ PB = blood production rate.

when these values were compared by the paired t test, there was also no significant (P>0.1) difference between the follicular and luteal phase values. The mean values for the 4 postmenopausal women were 1,890 ± 95 I/day and 1,060 ± 35 1/day/m2. The latter value was significantly (0.05 > P > 0.02) less than the mean MCR in the follicular phase values. Plasma concentrations (Tables 2, 3, and 4) The mean concentration of estriol in the plasma was significantly (P < 0.01) higher in the luteal, 10.9 ± 0.8 pg/ml, than in the follicular phase, 7.0 ± 0.7 pg/ml. In the 11 subjects in whom measurements were made in both follicular and luteal phases of the cycle the values were compared using the paired t test. The luteal phase values were significantly (P < 0.01) higher than the follicular phase values. In the small group of postmenopausal women the values ranged from 3 to 11 pg/ml. Blood production rates (Tables 2, 3, and 4) The mean blood production rate of estriol in the follicular phase was 14.0 ± 1.6 /u,g/day which was significantly (P < 0.01) less than the mean value of 22.7 ±1.9 /xg/day in the luteal phase. In the 11 subjects in whom measurements were made in both phases of the cycle, the values were significantly (0.02 > P > 0.01) higher in the luteal compared with the follicular phase using the paired t test.

In the post-menopausal women the blood production rates of estriol ranged from 5 to 22 pg/ml. Discussion Although it has been known for some time that estriol possesses biologic activity of its own (5,26), most studies of its metabolism have been concerned with pathways and routes of metabolism (7-12), or the speed of urinary excretion (7,8,12,27). Sandberg and Slaunwhite (7) found that following the intravenous administration of 14C-estriol the disappearance of free radioactivity in the plasma could be described by a function which was the sum of two exponentials. Barlow and Logan (28), using urinary methods, noted that the production rate of estriol was higher in the luteal than in the follicular phase and raised the possibility of estriol secretion. Our data following the pulse injection of 3 H-estriol are consistent with the findings of Sandberg and Slaunwhite concerning the disappearance of estriol from the plasma, in that we also noted a two-exponential type of curve. This is somewhat different than our findings for the other free estrogens (18) for which we have postulated a slowly turning over sulfate pool. The shape of the disappearance curve for estriol suggests that there is little interconversion between free estriol and a more slowly metabolized compound. This conclusion is also compatible with the data of several groups who noted a rapid


FLOOD, PRATT AND LONGCOPE recovery of radioactivity in the urine following the pulse injection of labeled estriol (7,8,12). Thus not only are the conjugates of estriol cleared relatively rapidly from the body but they probably do not interconvert with the free estriol pool in the blood to any significant degree. The initial volume of distribution of estriol was greater than the plasma volume which suggests that estriol is not bound strongly to a plasma protein in vivo. This confirms the in vitro studies of Murphy (29) and Vermeulen and Verdonck (30). Although the rate constants (23) for total removal, k, were in the same range as those we noted for estrone sulfate (17), the fraction of the total removal that was irreversible, i.e. 0.34, was far higher for estriol than for estrone sulfate, and was in keeping with estriol's higher MCR. The MCR of estriol measured by either pulse injection or constant infusion was in the. same range as we have reported for estrone (16,18,21).and we and others have reported for androstenedione (15,25,31,32). This relatively high MCR was another indication that there was no high-affinity plasma protein binding of estriol. The MCR for estriol measured in plasma was above the estimated splanchnic plasma flow, indicating a considerable degree of extrasplanchnic metabolism. Metabolism of estriol by the kidney has been reported (33), and it is probable that other extra-splanchnic tissues might be involved. The mean MCR, as measured by pulse injection and based on a two-exponential disappearance curve, was significantly less (P < 0.01) than the mean MCR measured by constant infusion when both are expressed as 1/day/m2. It is unlikely that this difference was due to the differences in the mass of estriol administered in the pulse injections, ~90 ng, compared with the constant infusions, ~37/Ag. Hembree et at. (34) noted that up to 50 fig of estrone could be infused without altering the MCR of estrone. The difference in the MCR's could be interpreted as indicating that our model was incorrect or that we had not reached an

JCE & M • 1976 Vol 42 • No 1

isotopic steady state during the constant infusion. There is no evidence for the latter, and the ratio of the primer to the infusion dose which we used was based on the two-exponential model and would have lead to an isotopic steady state within IVi hours. In addition, 1 subject was studied by both pulse injection and constant infusion and the MCR's were not different, 1900 vs 1880 I/day. It is thus probable that the lower mean MCR determined by pulse injection was due to the small sample size and the inclusion of a number of subjects in the low normal range. We were unable to find any difference in the mean MCR's of estriol measured in the follicular and luteal phases. The stability of the MCR through the cycle has also been noted for estrone and estradiol (16,35). Although we found a higher concentration oi: estriol in the luteal phase, the increase over the follicular phase value appeared not to be sufficient to alter the dynamics of estriol, an alteration which has been noted to occur for testosterone (36) and estradiol dynamics when their plasma concentrations increase excessively. We did not study any subjects at mid-cycle so cannot say whether the midcycle peak of estriol which we noted in some women (22) would be sufficient to alter the metabolism. The mean MCR that we found in a small sample of post-menopausal women was significantly smaller than the mean MCR of a larger group of younger women in the follicular phase of their cycle. In an older group of women we reported a marked decrease, 25% in the MCR of estrone and estradiol (37), but the decrease we found for estriol was somewhat less, 13%. This lesser difference in the MCR for estriol between young and old women may be a reflection of sample size, but could represent the decrease due to aging which has been noted for the MCR's of a number of steroids (37,38). We have used the product of the estriol concentration times the MCR to determine the blood production rate, PB, and as has been noted previously (15,39), this could result in a maximal value for PB. We were


METABOLISM AND PRODUCTION OF ESTRIOL unable to demonstrate any major secretory spikes for estriol during the day (22), and it would appear that the concentration of estriol is relatively stable over this time period. We (21) and others (40) have shown that the MCR's for steroids are not dependent on the time of day so that it is probable that any overestimation of PB for estriol is not large. We found a significant increase in the PB for estriol in the luteal phase of the cycle. Barlow and Logan (28) and others (41) have shown that estradiol and estrone can be precursors for free estriol, and thus the rise in PB in these precursors would lead to a concomitant rise in the PB of the product, estriol. Although we suggested that estriol is directly secreted in a number of women (22), our blood production rate data neither confirm nor deny that assumption. However, since the MCR's for estradiol and estriol do not appear to change through the cycle, it is probable that the varying ratios in the concentrations of estradiol to estriol which we noted (22) to occur during the cycle, are best explained by variations in the secretion rates of these estrogens. Anderson et at. (2) reported that estriol, when given to rats as a single injection, stimulated early uterotrophic responses but not the true uterine weight increases seen after estradiol injection. They felt this transient effect of estriol was due to the estriolnuclear receptor complex dissociating very rapidly in comparison with the estradiolnuclear receptor complex. Recently, however, Anderson et al. (42) noted that when estriol was given to rats by repetitive injections, uterine weights increased to the same extent as when estradiol was injected repetitively. Tseng and Gurpide (4) reported that estriol will bind to human endometrial nuclei, and it is thus probable that endogenous estriol could exert an estrogenic effect in humans. However, in young women of reproductive age the calculated PB of estradiol and estrone (18) far exceeds our calculated PB for estriol, and it is thus likely that the estrogenic effect of estriol is minimal in this age group. In post-menopausal

women, the calculated PB's of estriol were in the same range as those that we have previously reported for estradiol (37). It is possible,1 therefore, that estriol could exert a more significant estrogenic effect'in some older women. Acknowledgments The authors would like to thank Mrs. K. Rotti and J. Stevens for their excellent assistance in the measurement of estriol concentrations. The antibody used in the estriol immunoassay was a kind gift of Drs. S. Burstein, K. I. H. Williams, and W. Stylos of the Worcester Foundation. Portions of this study were performed in the Clinical Research Center of Boston City Hospital, Boston University Division, which is supported by a j Grant (RR-533) from the General Clinical Research Center's Program of the Division of Research Resources, National Institutes of Health.

References 1. Brecher, P. I., and H. H. Wotiz, Competition between estradiol and estriol for end organ receptor proteins, Steroids 9: 431, 1967. 2. Anderson, J. N., J. H. Clark, and E. J. Peck, Jr., The relationship between nuclear receptor-estrogen binding and uterotrophic responses, Biochem Biophys Res Comm 48: 1460, 1972. 3. Gorski, J., and B. Baker, Estrogen action in the uterus: the requisite for sustained estrogen binding in the nucleus, Gynecol Oncol 2: 249, 1974. 4. Tseng, L., and E. Gurpide, Nuclear concentration of estriol in superfused human endometrium; competition with estradiol, J Steroid Biochem 5: 273, 1974. 5. Hisaw, F. L., J. T. Velardo, and C. M. Goolsby, Interaction of estrogens on uterine growth, / Clin Endocrinol Metab 14: 1134, 1954. 6. Lemon, H. M., D. M. Miller, and J. F. Foley, Competition between steroids for hormonal receptor, National Cancer Institute Monograph 34: 77, 1971. 7. Sandberg, A. A., and W. R. Slaunwhite, Jr., Studies on phenolic steroids in human subjects. VII. Metabolic fate of estriol and its glucuronide,./ Clin Invest 44: 694, 1965. 8. Stoa, K. F., and M. Levitz, Comparison of the conjugated metabolites of intravenously and intraduodenally administered oestriol, Ada Endocrinol (Kbh) 57: 657, 1968. 9. Fishman, J., B. Zumoff, L. Hellman, and T. F. Gallagher, Metabolism of estriol-17a-3H in man, Steroids 11: 337, 1968. 10. Goebelsmann, U., and R. B. Jaffe, Oestriol metabolism in pregnant women, Ada Endocrinol (Kbh) 66: 679, 1971.


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11. Klopper, A., G. Masson, D. Campbell, and G. Wilson, Estriol in plasma: a compartmental study, Am] Obstet Gynecol 117: 21, 1973. 12. Stoa, K. F., and P. A. Skulstad, Biliary and urinary metabolites of intravenously and intraduodenally administered 17/3-oestradiol and oestriol, Steroids Lipids Res 3: 299, 1972. 13. Lemon, H. M., H. H. Wotiz, L. Parsons, and P. J. Mozden, Reduced estriol excretion in patients with breast cancer prior to endocrine therapy,JAMA 196: 1128, 1966. 14. Lemon, H. M., Endocrine influences on human mammary cancer formation, Cancer 23: 781, 1969. 15. Flood, C , S. A. Hunter, C. A. Lloyd, and C. Longcope, The effects of posture on the metabolism of androstenedione and estrone in males, J Clin Endocrinol Metab 36: 1180, 1973. 16. Longcope, C., D. S. Layne, and J. F. Tait, Metabolic clearance rates and interconversions of estrone and 17/3-estradiol in normal males and females, J Clin Invest 47: 93, 1968. 17. Longcope, C., The metabolism of estrone sulfate in normal males, J Clin Endocrinol Metab 34: 113, 1972. 18. Longcope, C , and K. I. H. Williams, The metabolism of estrogens in normal women after pulse injections of 3H-estradiol and 3H-estrone, / Clin Endocrinol Metab 38: 602, 1974. 19. Baird, D. T., A method for the measurement of estrone and estradiol-17/3 in peripheral human blood and other biological fluids using 35S pipsyl chloridej Clin Endocrinol Metab 28: 244, 1968. 20. Allen, W. M., A simple method for analyzing complicated absorption curves, of use in the colorimetric determinations of urinary steroids, J Clin Endocrinol Metab 10: 71, 1950. 21. Longcope, C , and J. F. Tait, Validity of metabolic clearance and interconversion rates of estrone and 17/3-estradiol in normal adults,/ Clin Endocrinol Metab 32: 481, 1971. 22. Rotti, K., J. Stevens, D. Watson, and C. Longcope, Estriol concentrations in plasma of normal, nonpregnant women, Steroids 25: 807, 1975. 23. Riskallah, T., E. Gurpide, and R. L. Vande Wiele, Metabolism of HCG in man, J Clin Endocrinol Metab 29: 92, 1969. 24. Shipley, R. A., and R. E. Clark, Tracer Methods for In Vivo Kinetics, Academic Press, N.Y.C., 1972, Chap. 9, p. 145. 25. Baird, D., R. Horton, C. Longcope, and J. F. Tait, Steroid prehormones, Perspect Biol Med 11: 384, 1968. 26. Merrill, R. C , Estriol: a review, Physiol Rev 38: 463, 1958. 27. Brown, C. H., B. D. Saffan, C. M. Howard, and J. R. K. Preedy, The renal clearance of endogenous

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estrogens in late pregnancy, J Clin Invest 43: 295, 1964. Barlow, J. J., and C. M. Logan, Estrogen secretion, biosynthesis and metabolism: Their relationship to the menstrual cycle, Steroids 7: 309, 1966. Murphy, B. E. P., Binding of testosterone and estradiol in plasma, Can] Biochem 46: 299, 1968. Vermeulen, A., and L. Verdonck, Studies on the binding of testosterone to human plasma, Steroids 11: 609, 1968. Longcope, C , T. Kato, and R. Horton, Conversion of blood androgens to estrogens in normal adult men and women, J Clin Invest 48: 2191, 1969. Horton, R., and J. F. Tait, Androstenedione production and interconversion rates measured in peripheral blood and studies on the possible site of its conversion to testosterone, J Clin Invest 45: 301, 1966. Kirdani, R. Y., D. Sampson, G. P. Murphy, and A. A. Sandberg, Studies on phenolic steroids in human subjects. XVI. Role of the kidney in the disposition of estriol, J Clin Endocrinol Metab 34: 546, 1972. Hembree, W. C , C. W. Bardin, and M. B. Lipsett, A study of estrogen metabolic clearance rates and transfer factors,; Clin Invest 48: 1809, 1969. Baird, D. T., and I. S. Fraser, Blood production and ovarian secretion rates of estradiol-17/3 and estrone in women throughout the menstrual cycle, J Clin Endocrinol Metab 38: 1009, 1974. Southren, A. L., G. G. Gordon, and S. Tochimoto, Further study of factors affecting the metabolic clearance rate of testosterone in man, J Clin Endocrinol Metab 28: 1105, 1968. Longcope, C , Metabolic clearance and blood production rates of estrogens in postmenopausal women. Amy Obstet Gynecol 111: 778, 1971. Flood, C , C. Gherondache, G. Pincus, J. F. Tait, S. A. S. Tait, and S. Willoughby, The metabolism and secretion of aldosterone in elderly subjects,; Clin Invest 46: 960, 1967. Baird, D. T., R. Horton, C. Longcope, and J. F. Tait, Steroid dynamics under steady-state conditions. Recent Prog Horm Res 25: 611, 1969. Lipsett, M. B., H. Wilson, M. A. Kirschner, S. G. Korenman, L. W. Fishman, G. A. Sarfaty, and C. W. Bardin, Studies on Leydig cell physiology and pathology: Secretion and metabolism of testosterone, Recent Prog Horm Res 22: 245, 1966. Fishman, J., L. Hellman, B. Zumoff, and J. Cassouto, Pathway and stereochemistry of the formation of estriols in man, Biochemistry 5: 1789, 1966. Anderson, J. N., E. J. Peck, Jr., and J. H. Clark, Estrogen-induced uterine responses and growth: Relationship to receptor estrogen binding by uterine nuclei, Endocrinology 96: 160, 1975.


Metabolic clearance and blood production rates of estradiol in hyperthyroidism.

Metabolic clearance and production rates of prolactin in man.

Effect of adrenocorticotropin on production rates and metabolic clearance rates of testosterone and estradiol.

Blood ethanol clearance rates.

Metabolic clearance and production rates of 3,3',5-triiodothyronine in hyperthyroid, euthyroid, and hypothyroid subjects.

Metabolic clearance and production rates of progesterone in non-pregnant and pregnant baboons (Papio papio).

Serum concentrations, metabolic clearance rates, and production rates of reverse triiodothyronine, triiodothyronine, and thyroxine in starved rabbits.

Metabolic clearance and secretion rates of subunits of human thyrotropin.

Plasma levels of unconjugated estrone, estradiol and estriol and of HCS throughout pregnancy in normal women.

Production and metabolic clearance rates of 1,25-dihydroxyvitamin D3 during maturation in rats: studies using a rapid, primed-infusion technique.

Direct radioimmunoassay of specific urinary estrogen glucosiduronates in normal men and nonpregnant women.

Metabolic clearance rates of oxyntomodulin and glucagon in the rat: contribution of the kidney.

Esophageal manometry in pregnant and nonpregnant women.

Subfoveal choroidal thickness in preeclampsia: comparison with normal pregnant and nonpregnant women.

Aldosterone metabolic clearance is normal in low-renin essential hypertension.

Blood mercury concentrations in pregnant and nonpregnant women in the United States: National Health and Nutrition Examination Survey 1999-2006.

[Levels of HCG, HPL, progesterone, and estriol in the blood of pregnant women following ovulation induced by drugs (proceedings)].

Comparison of gestational diabetes mellitus rates in women with increased and normal white blood cell counts in early pregnancy.

Fracture rates calculated from fracture histories in normal postmenopausal women.

[Composite microassays of plasma progesterone, 17alpha-OH-progesterone, estrone, 17beta-estradiol and estriol in normal adult women. II.Steroid patterns in normal pregnancy, labor and the puerperium (author's transl)].

Blood and milk progesterone in pregnant and nonpregnant buffalo.

Recurrent urinary tract infections in healthy and nonpregnant women.

Plasma immunoreactive relaxin levels in pregnant and nonpregnant women.

Metabolic clearance rate, production rate, and mammary uptake and metabolism of progesterone in cows.