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Absorption and metabolism of estrogens from the stomach and duodenum of pigs
DOMESTIC ANIMAL ENDOCRINOLOGY
Vol. 11(2):197-208,1994
ABSORPTION AND METABOLISM OF ESTROGENS FROM THE STOMACH AND DUODENUM OF PIGS 1 W.L. R u o ~ and P.J. Dziuk s,4 University of Illinois Department of Animal Sciences Urbana, Illinois 61801 Received April 29, 1993
ABSTRACT To determine the absorption and metabolism of 17~-estradiol (E2) by the stomach and liver of the pig, crystalline E 2 was placed in the stomach of prepubertal gilts. Blood samples were subsequently obtained from the hepatic portal and jugular veins and plasma was assayed for E2, estrone (El) , 1713estradiol-glucuronide (E2G), estrone-glucuronide (EtG) and estrone-sulfate (E~S). Concentrations of E2, El, E2G and EtS rose in the hepatic portal vein within five rain and remained elevated for several hr. Concentration of E 2 represented only 6% of the total estrogen detected in the hepatic portal vein during the sampling period, indicating that most of the E2 was converted or conjugated prior to entering the hepatic portal vein. The metabolism of E 2 presumably occurred in the stomach mucosa because food had been withheld for 26 hr before infusion of E2. Concentrations of E2G , EtG and E~S, but not E 2 and Et, rose in the jugular vein and remained elevated for several hr. The lack of a rise in E 2 and E 1 in the jugular vein indicates that the E 2 and Et from the hepatic portal vein were completely converted and/or removed by the liver. Most o f F.2 was converted to Et and then to EtG. The infusion of bile containing normal estrogens from pregnant gilts into the duodenum of prepubertal gilts resulted in a peak of E~G and E2G in the hepatic portal and jugular veins within a few minutes. This was followed in about 180 rain by a second sustained rise. The first peak was essentially abolished by extracting E~ and E 2 from the bile before infusion. The second peak failed to occur in gilts given antibiotics orally to reduce gut bacteria before infusion of bile. INTRODUCTION During enterohepatic circulation of estrogens unconjugated estrogens are absorbed from the gut (1, 2, 3) and metabolized by the gut wall (4, 5, 6, 7, 8) prior to entering venous drainage to the liver. The liver then passes them into the bile or peripheral circulation (1, 2, 9, 10). This process may prolong the availability of endogenous estrogens in the peripheral circulation (11, 12) and influence the effect of estrogens on reproduction. The routes and rates of metabolism can be influenced by one or more factors. Bile conjugates of 1713 estradiol (E2) and estrone (E]) are deconjugated in the gut by bacteria (1, 2, 13, 14, 15, 16). The deconjugated E 2 and E I are then reabsorbed from the gut (7, 17, 18). Oral administration of antibiotics that reduce populations of gut bacteria has been found to decrease absorption of biliary estrogens in rats (1, 19, 20). Antibiotics may influence concentrations of estrogens and thereby affect reproduction. These background studies leave unanswered the questions of the proportion of the estrogens that are metabolized by the gut wall and the proportions of each metabolite. This information is needed before more detailed studies of enterohepatic circulation of estrogens can be done. The following experiments were conducted to determine the paths of E 2 administered to the stomach and duodenum and to determine the effect of modifying the gut and bile on the fate of biliary estrogens. MATERIALS AND METHODS E x p e r i m e n t 1. The objective of this study was to establish the timing and the extent of passage of E 2 and its metabolites from the stomach to the blood. Six prepubertal crossbred Copyright © 1994 Butterworth-Heinemann
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gilts, five months of age and weighing 80 kg, were the experimental animals. Jugular (21) and hepatic portal (22) veins were catheterized for repeated sampling of blood. Gilts were kept from food for 24 hr before surgery. Gilts were anesthetized for placement of catheters with pentobarbital sodium followed by maintenance on halothane in a closed circuit system (23). Immediately after exteriorizing the hepatic portal vein catheter, blood was obtained from both the jugular and hepatic portal veins to establish basal concentrations of endogenous estrogens in the gilts. At this time the nylon netting attached to the hepatic portal vein catheter was secured by a single suture to tissue immediately adjacent to the point of insertion of the catheter in the vein. Ten mg of E 2 was dissolved in 1 ml of cocoa butter. Ten min after exteriorizing the hepatic portal vein catheter, the E 2 in cocoa butter was warmed and injected directly into the lumen of the stomach through a 20 gauge needle. This was designated as time zero (0). The incision was then closed and the animal was taken off the anesthetic and moved to a clean, separate pen for subsequent blood samples. Although the animal was not under anesthesia after time 0, it had not recovered completely during the sampling period. During this period animals were allowed water but not food. In preliminary similar experiments on several pigs in which E 2 was administered in a capsule by a boiling gun to conscious animals, the results were the same as regards apparent absorption and metabolism as determined by time-course curves of concentration. Blood was obtained from each of the jugular and hepatic portal veins at +5, 15, 30, 45, 65, 80, 95, 115, 150, 185, 210 and 300 min and kept at +4 ° C until plasma could be separated. Preliminary studies indicated that concentrations of estrogen in the hepatic portal vein would return to near basal levels after a period of 300 min Blood was centrifuged and plasma was stored at -20 ° C. Hormone assays. Concentrations of E2, E 1, estrone sulfate (EIS), 17-13-estradiol glucuronide (E2G) and estrone glucuronide (E1G) were measured to determine the extent of metabolism of E 2. Samples of 200 to 1200 ~tl of plasma were extracted with ethyl ether (10:1) prior to assaying for E 2. Concentrations of E 2 were determined by a single-antibody, charcoal dextran RIA as described and validated previously (24). The antiserum used cross reacts less than 1% with E: and estriol (E3) (25). This antiserum cross reacted less than 1% with 1713-estradiol-17-glucuronide (E2-17G). 1713-estradiol-3-glucuronide (E2-3G) and E1G and less than 3% of these conjugated estrogens were extracted into ethyl ether. Samples of 400 to 1200 ~tl of plasma were extracted with ethyl ether (10:1) prior to assaying for E~. Concentrations of E l were determined by a single-antibody, charcoal dextran RIA as described and validated previously (26). The antiserum used cross reacts less than 1% with E 2 and E 3 (26). This antiserum cross reacted less than 5% with E1G and E~S and less than 3% of these conjugated estrogens were extracted into ethyl ether. Concentrations of E2G were determined by the procedure of Pimentel (27) with modifications. Samples of 50 to 400 ~xl of plasma were added to assay tubes and the volume was increased to 400 ~1 by the addition of phosphate buffered saline-0.1% gelatin, pH 7.5 (PBS-G). To hydrolyze the glucuronide group from E 2, 0.5 ml of acetate buffer (0.01 M, pH 4.8) containing 10 ~tl of 13-glucuronidase from helix pomata was added to plasma and then incubated for 1 hr at 37 ° C. The resulting E 2 was extracted from solution with ethyl ether (10:1), then assayed as described for E 2. The percent recovery of 1000 cpm of 3H-E2-17G tracer from plasma after hydrolysis of the glucuronide and extraction of the free estrogen with ether was 75.1±6.0%, which was not different from the percent recovery of 3H-E2 from plasma. The interassay CV for 32 assays was 18%. The intraassay CV was 9% (n=100). A pool of sera was used to validate the assay for E2G. A known amount of unlabeled E2-17G was diluted to a concentration of 20,000 pg/ ml in PBS-G. When 208, 415, 623 or 829 pg of unlabeled E2-17G were added to 250 ml of plasma, recovery of the unlabeled hormone after correction for procedural losses was 87.8±3.9%. The test for parallelism for effect of volume of plasma assayed resulted in less than 10% variation from theoretical values over the range of volumes used in these experiments. To test whether the assay measured E2-3G accurately, a known amount of unlabeled
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Ee-3G was diluted to a concentration of 20,000 pg/ml in PBS-G, and the concentration of E2-3G was determined as described previously for E2-17G. After correction for procedural losses, the recovery of unlabeled hormone was 89%. The procedure for hydrolysis of the glucuronide from E1G extraction of the free E1 from plasma and reconstitution of the extracted estrogen was the same as for E2G. On the day after extraction, concentrations of E~ in reconstituted solution were determined by RIA as described by Home et al. (26). The interassay CV for 31 assays was 14%. The intraassay CV was 8.1% (n=100). A pool of sera was used to validate the assay for E~G. When 1054, 2134 or 4243 pg of unlabeled E~G were added to 250 ml of plasma, recovery of unlabeled hormone after correction for procedural losses was 84.7+6.2%. The test for parallelism resulted in less than 10% variation for the range of volumes used in the experiment. Concentrations of E~S were determined by the method of Frank et al. (28) with important modifications. Antiserum was generated in sheep using the antigenic protein complex of estrone-3-methylphosphonothioate coupled to methylated bovine serum albumin. In previous validations the antiserum was found to cross react at less than 0.1% with dihydroepiandrosterone sulfate and at non-detectable levels for other sulfates (28). We have determined that this antiserum has undetectable cross reactivity with E2-17G and E2-3G. In contrast, the cross reactivity of the antiserum with E1G ranged from 18% at 64 pg of EIG to 4% at 400 pg of EIG. This cross-reactivity would significantly affect the accuracy of determination of E~S when concentrations of E~G were the same or larger than concentrations of E~S. Preliminary measurements of concentrations of E~G in samples of plasma from this experiment showed the concentration of EjG was 16 fold greater than the concentration of E1S. The E1S values obtained by this method must be corrected for cross reactivity with EIG to be valid. By mathematically correlating the standard curves for the percentage of 3H-E~S bound to antiserum in the presence of E~S or E~S bound to antiserum in the presence of E~S or E~G, Equation 1 was determined: XI = e°'666891nx226585; with X~ =ng of E~G in the sample of X2 = n g of E1S incorrectly determined due to E~G. The theoretical value of EIS in the sample was calculated as the amount of E~S determined by the method of Frank et al. (28) minus X 2. The accuracy of this theoretical correction factor for E~S was tested empirically on 14 samples of plasma obtained sequentially from the jugular vein of a gilt given E2 (Figure 1). In test 1, the concentrations of EIS were determined by the method of Frank et al. (28).
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Figure 1. Concentrations of EIS in 14 samples of plasma from the jugular vein of one prepubertal gilt given 10 mg of E 2. Concentration of E1S in each sample was determined by three different procedures: i. Concentration of E]S determined by direct antibody RIA as described by Frank et al. (28). I1. Concentrations of E1S as determined by the method of Frank et al. (28), followed by theoretical correction for erossreactivity of the antiserum with E1G. IlL Concentration of E]S as determined by the method of Frank et al. (28), after extraction of E]G from plasma.
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In test II, the concentration of E1G in each sample was determined as described above and the theoretical concentration of E1S was determined in each sample of Equation 1. In test III, E1G was removed from each sample by hydrolysis of the glucuronide group followed by extraction of E1 with ether. Following this, the concentration of E~S was determined by the method of Frank et al. (28). When determined by test I, the mean concentration of EtS for all 14 samples was 3.8 fold higher than that of test II. When determined by test III, the mean concentration of the samples was approximately equal to that of test II. Thus, the theoretical removal of the effects of E~G, by calculating cross reaction, produced results similar to those in which E~G was removed prior to assaying for E~S. In the present experiment, 20 to 65 ~tl of plasma were added to assay tubes and the volume was brought to 300 ~tl by the addition of PBS-G. Concentrations of E~S were determined by the addition of antiserum and 3H-E~S (10,000 cpm) directly to plasma, followed by correction for the interference of E1G. The interassay CV for 25 assays was 14%. The intraassay CV was 9% (n=100). Analysis. Pre-treatment samples were taken before placement of E 2 in the stomach. The time course curve for each estrogen was the concentration of estrogen plotted against the interval to sampling. The area under the curve (AUC) for concentrations of estrogen from 0 to 300 min was calculated using the trapezoidal method. Change in AUC was the AUC minus the mean pre-treatment concentration of estrogen multiplied by 300 min: [AAUC = (AUC) - pre-treatment value x 300 min]. The concentration of total estrogen was defined as the sum of the pmol/ml concentrations of each of the five types of estrogen measured. The percentage of each estrogen was determined by dividing the AAUC for each estrogen by the AAUC for total estrogen. To analyze the effects of treatment over time, a repeated measures analysis of variance compared the mean concentration of estrogen at each posttreatment time with the mean concentration of estrogen at pre-treatment, within blood vessel and type of estrogen. This method was preferred because of the variation between pigs. Each pig serves as its own control because it would be difficult to sample two groups of pigs at exactly the same time or to make valid companions between samples taken at slightly different times. Because there were missing values, the data were analyzed by LSMEANS (SAS, PROC GLM) (29). Experiment2. This study was conducted to establish the fate in prepubertal gilts of estrogens in bile of pregnant gilts for subsequent experiments on enterohepatic recycling of estrogens. Bile was obtained from the gallbladders of 10 pregnant gilts killed at days 27 to 35 of gestation and from 24 gilts killed at days 105 to 116 of gestation. Bile was removed immediately and stored at -20 ° C. Samples of bile near 30 d and 114 d of gestation and a pooled sample were assayed by RIA for concentration of E 2, E~, E~S, E2G and E~G as described in Experiment 1. Pooled bile was then divided into 30 aliquots of 80 ml each. Seven prepubertal gilts were fitted with catheters in the jugular and hepatic portal veins for obtaining blood as in Experiment 1 and also had a duodenal catheter for infusion of bile. No antibiotics were given to avoid interference of effect on gut bacteria. Gilts were given about 7 d from surgery to experimentation. The normal diet was given 60 min before infusion of bile at time 0. This simulates the usual order of a meal followed by secretion of bile. Blood was obtained at -10 rain to determine basal concentrations of estrogens. At time 0, 80 ml of pooled bile was infused into the duodenum over a period of one min Blood samples of about 10 ml each were obtained from the jugular and hepatic portal catheters at +10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 300, 360, 420 and 480 min. Plasma was stored and assayed as described in Experiment 1 except that E~S was not analyzed separately as it gave similar patterns to E~G. Experiment 3. This experiment tested the effect of oral antibiotics intended to reduce the population of gut bacteria on the metabolism of bile estrogens from pregnant gilts. Six prepuberal gilts were prepared as in Experiment 2 with the exception that at days -3, -2, -1 gilts
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were infused into the duodenum three times daily with 3 gm of neomycin sulfate and I gm of lincomycin dissolved in 8 ml of water to reduce gut bacteria. Infusion of bile, sampling and assays were as in Experiment 2. Experiment 4. This study determined the effect of reducing the free estrogens in bile from pregnant gilts on the pattern of estrogen metabolites after infusion of the bile into prepubertal gilts. Six gilts were prepared as in Experiment 2. The 480 ml of bile to be infused was divided into 480 samples of 1 ml each and extracted with 6 ml of ether to remove free estrogens. The extracted bile was then pooled into six equal samples for infusion. Other procedures were as described in Experiment 2. RESULTS The concentrations of each type of estrogen in the jugular and hepatic portal veins of eleven prepubertal gilts before treatment are shown in Table 1. The concentrations of estrogens in the hepatic portal vein of five gilts given E 2 in the stomach in Experiment 1 are shown in Figures 2 and 3. Concentration of all estrogens rose within a few min after administration of E 2. Concentration of E 2 reached a peak within a few min after administration of E 2 whereas E 1 peaked at about 30 min and the conjugates: EIS, EIG and E2G peaked near 120 min. Ninety-four percent of the E in the hepatic portal vein was some metabolite of E 2. Eighty-eight percent of E was conjugated to glucuronide or sulfonate, and 79% of E was E 1 either free or conjugated. Concentrations of estrogens in the jugular vein are shown in Figure 4. Concentrations of E~ and E 2 did not differ between blood samples taken before and after administration of E2, data are not shown. Concentration of conjugates rose within a few min after administration of E 2, reaching a peak in 60 to 100 min. All of the E in the jugular was conjugated and 91% was conjugated E. The comparison of the concentrations of each estrogen in the hepatic portal and jugular veins over the entire 300 min sampling period is shown in Figure 5. Concentrations of estrogens in the bile of pregnant gilts are shown in Table 2. The amount of E contained in one dose of bile infused into the duodenum is shown in Table 3. Concentrations of estrogens in the hepatic portal vein of gilts infused with pooled bile are shown in Figures 6 and 7. The interval from infusion to a significant rise in E 2 and E 1 was about 180 min. There was a significant peak of E2G and EIG within a few min of infusion with a second more sustained peak at about 180 min. There was no rise in concentration of E~ and E 2 in the jugular vein and the rise of E2G and E~G was very similar to that in the hepatic portal vein, data not shown. Ninety-seven percent of E in the hepatic portal was conjugated to glucuronide. The results of infusion of bile into gilts pre-treated with oral antibiotics are shown in Figure 8. There was no difference in AUC of E~G and E2G between those animals treated with antibiotics and those not treated during the period from 0 to 80 min. There was, however, significantly less (p<.01) AUC from 80 to 480 min in animals given antibiotics. The first peaks of E1G and E2G mimicked those of untreated gilts, but the second peak was absent.
TABLE 1. CONCENTRATION OF ESTROGENS IN THE JUGULAR AND HEPATIC PORTAL VEINS OF UNTREATED PRE-PUBERTAL GILTSa
Estrogen E2 E1 EIS E2G EIG 'mean ± SE
Jugular (pmol/ml) .09 .,- .01 .36 ± .02 .02 ± .01 .32 ± .05 .87 ± .07
Portal (pmol/ml) .08 + .01 .41 ± .04 .02 ± .02 .37 ± .06 .96 ± .07
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TABLE 2. MEAN CONCENTRATIONOF ESTROGENS IN BILE FROM PREGNANTPIGS Stage of Gestation Estrogen
Day 30 a (pmol/ml)
E2 E1 E1S E2G E1G Total
0.14 7.47 45.62 16.47 749.98 819.68
Day l l 4 b (pmol/ml)
Pooled c (pmol/ml)
41.25 3311.87 741.39 788.82 87129.95 92013.28
23.16 862.96 598.21 18674.98 20159.31
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TABLE 3. ESTROGENSIN BILE POOLED FROMPREGNANTPIGS BEFOREAND AFTER EXTRACTIONWITHETHER (EE) Estrogen
Pooled Bile (80 ml) (pmol)
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1840 69840 47840 1491840
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When the bile was extracted with ether to reduce E t and E 2 before infusion the first peaks of EIG and E2G were absent while the second peak occurred as with untreated bile (Figure 9). The AUC was significantly less (p<.05, E2G; p<.01, E1G) for the period from 0 to 80 min with no difference from 80 to 480 min. In both Experiments 3 and 4 the peaks of E1G and E2G in the jugular vein were at the same intervals and significance as in the hepatic portal vein.
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Time (minutes) Figure 9. Mean concentration of E~G and E2G (n = 4) in the hepatic portal vein in pre-pubertal gilts infused with 65 ml of bile extracted with ether, in the duodenum at time 0. Letters indicate significant difference between mean concentrationat post-treatment and that of pre-treatment: a = P<.05, b = P<.01, c = P<.001. SE: Standard error of the mean. DISCUSSION Although metabolites of E 2 constitute a very significant proportion of total E they have been largely ignored in many studies. They provide a reservoir of E and may contribute to effects of E on reproduction. The very short period between injection of E 2 in the stomach and a sharp rise in E metabolites in the hepatic portal vein would indicate that the stomach contents or wall were very active in metabolizing E. The extent of conversion and subsequent conjugation of E 2 and E 1 indicates very efficient metabolism. In a similar study (8) no unconjugated E was found in the hepatic portal vein following infusion of radiolabeled E 2. In that study the experimental pigs were much younger with perhaps a more active enzyme system, the E 2 w a s dissolved in alcohol and the dose of E 2 w a s much lower than in the present study. Each of these factors may have contributed to detection of unconjugated E in the present study. After infusion of E2 in the stomach, F r e e E 2 and E1 were detected in the hepatic portal vein but not in the jugular although the concentration of conjugates rose markedly in the jugular vein. The potential effects and fate of these conjugates are not well known. If they were stored and deconjugated or converted to an active form they could influence reproduction. The concentration of E and conjugates in the bile of pregnant gilts at day 30 and increasing 100 fold to day 114 may be the first such report. Although the conjugates make up the bulk of E there is free E as detected by RIA and also by the response following infusion of bile extracted to remove free E. Infusion of bile into the duodenum provides a relatively normal route of delivery of normally occurring forms of E. The timing of the infusion shortly after the ingestion of a meal also mimics the normal sequence of bile secretion after a meal. The first rise in conjugates is quite likely from free Et and E 2 in the bile. This conclusion is supported by the observa-
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tion of Experiment 1 of the almost immediate rise of conjugates in the hepatic portal vein when E 2 was placed in the stomach. This together with an essential absence of a first peak after infusion of bile that had been extracted with ether to reduce the E l and E 2 substantiates the conclusion. The peaks of conjugates occurring at about 180 min after infusion of normal bile into untreated gilts might appear to be the result of direct absorption of conjugates. The results of other studies would indicate that conjugates are poorly absorbed and require deconjugation before absorption and conjugation (2, 30, 31, 32). It therefore seems more reasonable that the delay from infusion to the peak would indicate that deconjugation by gut bacteria took place before absorption (33). The extent and consistency with which E2 was converted to E l as evidenced by the relatively greater proportion of E 1 conjugates is somewhat at variance with previous reports (33). The predominance of E l conjugates over E 2 conjugates was consistent whether E 2 was instilled in the stomach or bile was infused into the duodenum. Because 96% of estrogens in bile were conjugates and 96% of estrogens in the second peak in the hepatic portal were conjugates would indicate that the bile conjugates were the main source of hepatic portal conjugates. The marked reduction in the second peaks of conjugates in the hepatic portal of gilts pretreated with oral antibiotics would indicate that conjugates are not absorbed directly and that reduction of gut bacteria reduces deconjugation, absorption and enterohepatic recycling. The finding of estrogens in both jugular and hepatic portal veins after infusion of normal bile is one of the first direct demonstrations of the possibility of enterohepatic circulation of natural estrogens. The observation that the concentrations of circulating estrogens can be influenced by antibiotics supports the clinical observations of failure of oral contraceptives following exposure to various xenobiotics. Conjugated estrogens may be deconjugated in the periphery (30, 34, 35), but the extent and significance has not yet been determined. We conclude that most estrogens administered orally or entering the gut from the bile are conjugated by the gut wall, pass to the liver where they can be put into the bile or passed into the peripheral circulation. These estrogens in the periphery have the potential to influence reproduction. ACKNOWLEDGEMENTS/FOOTNOTES ~Supported in part by Biomedical Research Grant, University of Illinois and Illinois Pork Producers Association. 2present address: 1 Holiday Drive, Village Terrace Apts. #A-109, Cortland, NY 13045. 3312 Animal Sciences Lab, University of Illinois, 1207 W. Gregory Dr., Urbana, IL 61801. 4Correspondence and reprints.
REFERENCES 1. Brewster D, Jones RS, and Symons AM. Effects of neomycin on the biliary excretion and enterohepatic circulation of mestranol and 17[3-oestradiol. Biochem Pharm 26:943-946, 1977. 2. Back DJ, Chapman GR, May SA, Rowe PH. Absorption of oestrone sulphate from the gastrointestinal tract of the rat. J Steroid Biochem 14:347-356, 1981. 3. Sire SM, Back DJ. Intestinal absorption of oestrone, oestrone glucuronide and oestrone sulphate in the rat in situ. I. Importance of hydrolytic enzymes on conjugate absorption. J Steroid Biochem 22:781-788, 1985. 4. Meli A, Cargill DI, Giannina T, Steinetz BG. Studies on the transport of estrogens by the rat small intestine in vivo. Proc Soc Exp Biol Med 129:937-944, 1968. 5. Smith FR, Tapley DF, Ross JE. Glucuronide formation in the transport of estradiol by rat intestine in vitro. Biochem Biophys Acta 69:68-73, 1963. 6. Diczfalusy E, Franksson C, Martinsen B. Oestrogen conjugation by the human intestinal tract. Acta Endocrinol 38:59-72, 1961. 7. Diezfalusy E, Franksson C, Lisboa BP, Martinsen B. Formation of oestrone glucuronide by the human intestinal tract. Acta Endoerinol 40:537-51, 1962. 8. Moore AB, Bottoms GD, Coppoc GL, Pohland RC, Roesel OF. Metabolism of estrogens in the gastrointestinal tract of swine. I. Instilled estradiol. J Anim Sci 55:124-134, 1982.
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9. Schwenk M, Lopez del Pino V, Bolt HM. The kinetics of hepatocellular transport and metabolism of estrogens (comparison between estrone sulfate, estrone and ethinylestradiol). J Steroid Biochem 10:3741, 1979. 10. Back DJ, Breckenridge AM, Crawford FE, Giles M, Orme MLE, Rowe PH. The pharmacokinetics of oestrone sulfate in the rat. J Steroid Biochem 14:1045-1047, 1981. 11. Sandberg AA, Slaunwhite WR. Studies on phenolic steroids in human subjects. I1. The metabolic fate and hepato-biliary-enteric circulation of 14-C-estrone and 14-C-estradiol in women. J Clin Endocrinol 18:12661278, 1957. 12. Sandberg AA, Kirdani RY, Back N, Weyman P, Slaunwhite WR. Biliary excretion and enterohepatic circulation of estrone and estriol in rodents. Am J Physiol 213:1138-1142, 1967. 13. Adlercreutz H, Martin F, Pulkkinen M, Dencker H, Rimer U, Sjoberg N-O, Tikkanen MJ. Intestinal metabolism of estrogens. J Clin Endocrinol Metab 43:497-505, 1976. 14. Adlercreutz H, Martin F, Jarvenpaa P, Fotsis T. Steroid absorption and enterohepatic recycling. Contraception 20:201-223, 1979. 15. Adlercreutz H, Martin F. Biliary excretion and intestinal metabolism of progesterone and estrogens in man. J Steroid Biochem 13:231-244, 1980. 16. van Eldere J, Parmetier G, Robben J, Eyssen H. Influence of an estrone-desulfating intestinal flora on enterohepatic circulation of estrone-sulfate in rats. J Steroid Biochem 26:235-239, 1987. 17. Anderson ABM, Sklovsky E, Sayers L, Steele PA, Turnbull AC. Comparison of serum oestrogen concentrations in post-menopausal women taking oestrone sulphate and oestradiol. Br Med J 1:140-144, 1978. 18. Sim SM, Back DJ. Intestinal absorption of oestrone, oestrone glucuronide and oestrone sulphate in the rat in situ. II. Studies with the doluiso technique. J Steroid Biochem 24:1085-1089, 1986. 19. Back DJ, Breckonridge AM, Challiner M, Crawford FE, Orme MLE, Rowe PH, Smith E. The effect of antibiotics on the enterohepatic circulation of ethinylestradiol and norethisterone in the rat. J Steroid Biochem 9:527-531, 1978. 20. Back DJ, Breckenridge AM, Cross KJ, Orme MLE, Thomas E. An antibiotic interaction with ethinyloestradiol in the rat and rabbit. J Steroid Biochem 16:407---413, 1982. 21. Pond W J, Houpt KA. The Biology of the Pig. New York: Cornell University Press. p. 204, 1978. 22. Yen JT, Killefer J. A method for chronically qualifying net absorption of nutrients and gut metabolites into hepatic portal vein in conscious swine. J Anim Sci 64:923--934, 1987. 23. Dziuk PJ, Phillips TN, Graber JW. Halothane closed-circuit anesthesia in the pig. Am J Vet Res 25:17731775, 1964. 24. Dial GD, Dial OK, BeVier GW, Glenn SD, Dziuk PJ. Estrous behavior and circadian discharge of luteinizing hormone in the prepubertal gilt in response to exogenous estrogen. Biol Reprod 29:1047-1056, 1983. 25. Bahr JM, Gardner R, Schenck P, Shahabi N. Follicular steroidogenesis: effect of reproductive condition. Biol Reprod 22:817-826, 1980. 26. Home C, Chew BP, Wiseman BS, Dziuk PJ. Relationship between the level of estrone sulfate in the plasma and the number of fetuses during pregnancy in the gilt. Biol Reprod 29:56--62, 1983. 27. Pimentel CA, Progesterone and estrogen production by the placenta of late pregnant and periparturient cows following lutectomy. Ph.D. Thesis, University of Illinois, 1982. 28. Frank GR, Noble RC, Esch MW, Green CA, Bahr JM. Direct estimation of estrone sulfate in serum and urine of pregnant swine as indicators of litter size at birth. Anita Reprod Sci 15:121-129, 1987. 29. SAS Institute Inc. SAS User's Guide; Statistics, Version 5 Edition. Cary NC:SAS Institute Inc., 956 pp, 1985. 30. Back DJ, Breckenridge AM, Crawford FE, Maclver M, Orme MLE, Rowe PL. lnterindividual variation and drug interactions with hormonal steroid contraceptives. Drugs 21:46-61, 1981. 31. Sim SM, Huljghebaert S, Back DJ, Egssen HJ. Gastrointestinal absorption of estrone sulfate in germ-free and conventional rats. J Steroid Biochem 18:499-503, 1983. 32. Adlercreutz H, Martin F, Pulkkinen M, Dencker H, Rimer U, Sjolieng NO, Tikkaren MJ. Intestinal metabolism of estrogens. J Clin Endocrinol Metab 43:497-505, 1976. 33. Pohland RC, Coppoc GL, Bottoms GD, Moore AB. Metabolism of estrogens in the gastrointestinal tract of swine. 1II. Estradiol-171~-D-glucuronide instilled into sections of intestine. J Anim Sci 55:145-152, 1982. 34. Collins DC, Balikian HM, Preedy JRK. Evidence for the simultaneous uptake and release of certain estrogens by the splanchnic area in the dog. Endocrinology 99:420--428, 1976. 35. Gurpide E, Stolee A, Tseng L. Quantitative studies of tissue uptake and disposition of hormones. Karolinska Symposia on Research Methods in Reproductive Endocrinology. 3rd Symposium: In vitro methods in reproductive cell biology, p247, 1971.
Vol. 11(2):197-208,1994
ABSORPTION AND METABOLISM OF ESTROGENS FROM THE STOMACH AND DUODENUM OF PIGS 1 W.L. R u o ~ and P.J. Dziuk s,4 University of Illinois Department of Animal Sciences Urbana, Illinois 61801 Received April 29, 1993
ABSTRACT To determine the absorption and metabolism of 17~-estradiol (E2) by the stomach and liver of the pig, crystalline E 2 was placed in the stomach of prepubertal gilts. Blood samples were subsequently obtained from the hepatic portal and jugular veins and plasma was assayed for E2, estrone (El) , 1713estradiol-glucuronide (E2G), estrone-glucuronide (EtG) and estrone-sulfate (E~S). Concentrations of E2, El, E2G and EtS rose in the hepatic portal vein within five rain and remained elevated for several hr. Concentration of E 2 represented only 6% of the total estrogen detected in the hepatic portal vein during the sampling period, indicating that most of the E2 was converted or conjugated prior to entering the hepatic portal vein. The metabolism of E 2 presumably occurred in the stomach mucosa because food had been withheld for 26 hr before infusion of E2. Concentrations of E2G , EtG and E~S, but not E 2 and Et, rose in the jugular vein and remained elevated for several hr. The lack of a rise in E 2 and E 1 in the jugular vein indicates that the E 2 and Et from the hepatic portal vein were completely converted and/or removed by the liver. Most o f F.2 was converted to Et and then to EtG. The infusion of bile containing normal estrogens from pregnant gilts into the duodenum of prepubertal gilts resulted in a peak of E~G and E2G in the hepatic portal and jugular veins within a few minutes. This was followed in about 180 rain by a second sustained rise. The first peak was essentially abolished by extracting E~ and E 2 from the bile before infusion. The second peak failed to occur in gilts given antibiotics orally to reduce gut bacteria before infusion of bile. INTRODUCTION During enterohepatic circulation of estrogens unconjugated estrogens are absorbed from the gut (1, 2, 3) and metabolized by the gut wall (4, 5, 6, 7, 8) prior to entering venous drainage to the liver. The liver then passes them into the bile or peripheral circulation (1, 2, 9, 10). This process may prolong the availability of endogenous estrogens in the peripheral circulation (11, 12) and influence the effect of estrogens on reproduction. The routes and rates of metabolism can be influenced by one or more factors. Bile conjugates of 1713 estradiol (E2) and estrone (E]) are deconjugated in the gut by bacteria (1, 2, 13, 14, 15, 16). The deconjugated E 2 and E I are then reabsorbed from the gut (7, 17, 18). Oral administration of antibiotics that reduce populations of gut bacteria has been found to decrease absorption of biliary estrogens in rats (1, 19, 20). Antibiotics may influence concentrations of estrogens and thereby affect reproduction. These background studies leave unanswered the questions of the proportion of the estrogens that are metabolized by the gut wall and the proportions of each metabolite. This information is needed before more detailed studies of enterohepatic circulation of estrogens can be done. The following experiments were conducted to determine the paths of E 2 administered to the stomach and duodenum and to determine the effect of modifying the gut and bile on the fate of biliary estrogens. MATERIALS AND METHODS E x p e r i m e n t 1. The objective of this study was to establish the timing and the extent of passage of E 2 and its metabolites from the stomach to the blood. Six prepubertal crossbred Copyright © 1994 Butterworth-Heinemann
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gilts, five months of age and weighing 80 kg, were the experimental animals. Jugular (21) and hepatic portal (22) veins were catheterized for repeated sampling of blood. Gilts were kept from food for 24 hr before surgery. Gilts were anesthetized for placement of catheters with pentobarbital sodium followed by maintenance on halothane in a closed circuit system (23). Immediately after exteriorizing the hepatic portal vein catheter, blood was obtained from both the jugular and hepatic portal veins to establish basal concentrations of endogenous estrogens in the gilts. At this time the nylon netting attached to the hepatic portal vein catheter was secured by a single suture to tissue immediately adjacent to the point of insertion of the catheter in the vein. Ten mg of E 2 was dissolved in 1 ml of cocoa butter. Ten min after exteriorizing the hepatic portal vein catheter, the E 2 in cocoa butter was warmed and injected directly into the lumen of the stomach through a 20 gauge needle. This was designated as time zero (0). The incision was then closed and the animal was taken off the anesthetic and moved to a clean, separate pen for subsequent blood samples. Although the animal was not under anesthesia after time 0, it had not recovered completely during the sampling period. During this period animals were allowed water but not food. In preliminary similar experiments on several pigs in which E 2 was administered in a capsule by a boiling gun to conscious animals, the results were the same as regards apparent absorption and metabolism as determined by time-course curves of concentration. Blood was obtained from each of the jugular and hepatic portal veins at +5, 15, 30, 45, 65, 80, 95, 115, 150, 185, 210 and 300 min and kept at +4 ° C until plasma could be separated. Preliminary studies indicated that concentrations of estrogen in the hepatic portal vein would return to near basal levels after a period of 300 min Blood was centrifuged and plasma was stored at -20 ° C. Hormone assays. Concentrations of E2, E 1, estrone sulfate (EIS), 17-13-estradiol glucuronide (E2G) and estrone glucuronide (E1G) were measured to determine the extent of metabolism of E 2. Samples of 200 to 1200 ~tl of plasma were extracted with ethyl ether (10:1) prior to assaying for E 2. Concentrations of E 2 were determined by a single-antibody, charcoal dextran RIA as described and validated previously (24). The antiserum used cross reacts less than 1% with E: and estriol (E3) (25). This antiserum cross reacted less than 1% with 1713-estradiol-17-glucuronide (E2-17G). 1713-estradiol-3-glucuronide (E2-3G) and E1G and less than 3% of these conjugated estrogens were extracted into ethyl ether. Samples of 400 to 1200 ~tl of plasma were extracted with ethyl ether (10:1) prior to assaying for E~. Concentrations of E l were determined by a single-antibody, charcoal dextran RIA as described and validated previously (26). The antiserum used cross reacts less than 1% with E 2 and E 3 (26). This antiserum cross reacted less than 5% with E1G and E~S and less than 3% of these conjugated estrogens were extracted into ethyl ether. Concentrations of E2G were determined by the procedure of Pimentel (27) with modifications. Samples of 50 to 400 ~xl of plasma were added to assay tubes and the volume was increased to 400 ~1 by the addition of phosphate buffered saline-0.1% gelatin, pH 7.5 (PBS-G). To hydrolyze the glucuronide group from E 2, 0.5 ml of acetate buffer (0.01 M, pH 4.8) containing 10 ~tl of 13-glucuronidase from helix pomata was added to plasma and then incubated for 1 hr at 37 ° C. The resulting E 2 was extracted from solution with ethyl ether (10:1), then assayed as described for E 2. The percent recovery of 1000 cpm of 3H-E2-17G tracer from plasma after hydrolysis of the glucuronide and extraction of the free estrogen with ether was 75.1±6.0%, which was not different from the percent recovery of 3H-E2 from plasma. The interassay CV for 32 assays was 18%. The intraassay CV was 9% (n=100). A pool of sera was used to validate the assay for E2G. A known amount of unlabeled E2-17G was diluted to a concentration of 20,000 pg/ ml in PBS-G. When 208, 415, 623 or 829 pg of unlabeled E2-17G were added to 250 ml of plasma, recovery of the unlabeled hormone after correction for procedural losses was 87.8±3.9%. The test for parallelism for effect of volume of plasma assayed resulted in less than 10% variation from theoretical values over the range of volumes used in these experiments. To test whether the assay measured E2-3G accurately, a known amount of unlabeled
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Ee-3G was diluted to a concentration of 20,000 pg/ml in PBS-G, and the concentration of E2-3G was determined as described previously for E2-17G. After correction for procedural losses, the recovery of unlabeled hormone was 89%. The procedure for hydrolysis of the glucuronide from E1G extraction of the free E1 from plasma and reconstitution of the extracted estrogen was the same as for E2G. On the day after extraction, concentrations of E~ in reconstituted solution were determined by RIA as described by Home et al. (26). The interassay CV for 31 assays was 14%. The intraassay CV was 8.1% (n=100). A pool of sera was used to validate the assay for E~G. When 1054, 2134 or 4243 pg of unlabeled E~G were added to 250 ml of plasma, recovery of unlabeled hormone after correction for procedural losses was 84.7+6.2%. The test for parallelism resulted in less than 10% variation for the range of volumes used in the experiment. Concentrations of E~S were determined by the method of Frank et al. (28) with important modifications. Antiserum was generated in sheep using the antigenic protein complex of estrone-3-methylphosphonothioate coupled to methylated bovine serum albumin. In previous validations the antiserum was found to cross react at less than 0.1% with dihydroepiandrosterone sulfate and at non-detectable levels for other sulfates (28). We have determined that this antiserum has undetectable cross reactivity with E2-17G and E2-3G. In contrast, the cross reactivity of the antiserum with E1G ranged from 18% at 64 pg of EIG to 4% at 400 pg of EIG. This cross-reactivity would significantly affect the accuracy of determination of E~S when concentrations of E~G were the same or larger than concentrations of E~S. Preliminary measurements of concentrations of E~G in samples of plasma from this experiment showed the concentration of EjG was 16 fold greater than the concentration of E1S. The E1S values obtained by this method must be corrected for cross reactivity with EIG to be valid. By mathematically correlating the standard curves for the percentage of 3H-E~S bound to antiserum in the presence of E~S or E~S bound to antiserum in the presence of E~S or E~G, Equation 1 was determined: XI = e°'666891nx226585; with X~ =ng of E~G in the sample of X2 = n g of E1S incorrectly determined due to E~G. The theoretical value of EIS in the sample was calculated as the amount of E~S determined by the method of Frank et al. (28) minus X 2. The accuracy of this theoretical correction factor for E~S was tested empirically on 14 samples of plasma obtained sequentially from the jugular vein of a gilt given E2 (Figure 1). In test 1, the concentrations of EIS were determined by the method of Frank et al. (28).
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In test II, the concentration of E1G in each sample was determined as described above and the theoretical concentration of E1S was determined in each sample of Equation 1. In test III, E1G was removed from each sample by hydrolysis of the glucuronide group followed by extraction of E1 with ether. Following this, the concentration of E~S was determined by the method of Frank et al. (28). When determined by test I, the mean concentration of EtS for all 14 samples was 3.8 fold higher than that of test II. When determined by test III, the mean concentration of the samples was approximately equal to that of test II. Thus, the theoretical removal of the effects of E~G, by calculating cross reaction, produced results similar to those in which E~G was removed prior to assaying for E~S. In the present experiment, 20 to 65 ~tl of plasma were added to assay tubes and the volume was brought to 300 ~tl by the addition of PBS-G. Concentrations of E~S were determined by the addition of antiserum and 3H-E~S (10,000 cpm) directly to plasma, followed by correction for the interference of E1G. The interassay CV for 25 assays was 14%. The intraassay CV was 9% (n=100). Analysis. Pre-treatment samples were taken before placement of E 2 in the stomach. The time course curve for each estrogen was the concentration of estrogen plotted against the interval to sampling. The area under the curve (AUC) for concentrations of estrogen from 0 to 300 min was calculated using the trapezoidal method. Change in AUC was the AUC minus the mean pre-treatment concentration of estrogen multiplied by 300 min: [AAUC = (AUC) - pre-treatment value x 300 min]. The concentration of total estrogen was defined as the sum of the pmol/ml concentrations of each of the five types of estrogen measured. The percentage of each estrogen was determined by dividing the AAUC for each estrogen by the AAUC for total estrogen. To analyze the effects of treatment over time, a repeated measures analysis of variance compared the mean concentration of estrogen at each posttreatment time with the mean concentration of estrogen at pre-treatment, within blood vessel and type of estrogen. This method was preferred because of the variation between pigs. Each pig serves as its own control because it would be difficult to sample two groups of pigs at exactly the same time or to make valid companions between samples taken at slightly different times. Because there were missing values, the data were analyzed by LSMEANS (SAS, PROC GLM) (29). Experiment2. This study was conducted to establish the fate in prepubertal gilts of estrogens in bile of pregnant gilts for subsequent experiments on enterohepatic recycling of estrogens. Bile was obtained from the gallbladders of 10 pregnant gilts killed at days 27 to 35 of gestation and from 24 gilts killed at days 105 to 116 of gestation. Bile was removed immediately and stored at -20 ° C. Samples of bile near 30 d and 114 d of gestation and a pooled sample were assayed by RIA for concentration of E 2, E~, E~S, E2G and E~G as described in Experiment 1. Pooled bile was then divided into 30 aliquots of 80 ml each. Seven prepubertal gilts were fitted with catheters in the jugular and hepatic portal veins for obtaining blood as in Experiment 1 and also had a duodenal catheter for infusion of bile. No antibiotics were given to avoid interference of effect on gut bacteria. Gilts were given about 7 d from surgery to experimentation. The normal diet was given 60 min before infusion of bile at time 0. This simulates the usual order of a meal followed by secretion of bile. Blood was obtained at -10 rain to determine basal concentrations of estrogens. At time 0, 80 ml of pooled bile was infused into the duodenum over a period of one min Blood samples of about 10 ml each were obtained from the jugular and hepatic portal catheters at +10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 300, 360, 420 and 480 min. Plasma was stored and assayed as described in Experiment 1 except that E~S was not analyzed separately as it gave similar patterns to E~G. Experiment 3. This experiment tested the effect of oral antibiotics intended to reduce the population of gut bacteria on the metabolism of bile estrogens from pregnant gilts. Six prepuberal gilts were prepared as in Experiment 2 with the exception that at days -3, -2, -1 gilts
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were infused into the duodenum three times daily with 3 gm of neomycin sulfate and I gm of lincomycin dissolved in 8 ml of water to reduce gut bacteria. Infusion of bile, sampling and assays were as in Experiment 2. Experiment 4. This study determined the effect of reducing the free estrogens in bile from pregnant gilts on the pattern of estrogen metabolites after infusion of the bile into prepubertal gilts. Six gilts were prepared as in Experiment 2. The 480 ml of bile to be infused was divided into 480 samples of 1 ml each and extracted with 6 ml of ether to remove free estrogens. The extracted bile was then pooled into six equal samples for infusion. Other procedures were as described in Experiment 2. RESULTS The concentrations of each type of estrogen in the jugular and hepatic portal veins of eleven prepubertal gilts before treatment are shown in Table 1. The concentrations of estrogens in the hepatic portal vein of five gilts given E 2 in the stomach in Experiment 1 are shown in Figures 2 and 3. Concentration of all estrogens rose within a few min after administration of E 2. Concentration of E 2 reached a peak within a few min after administration of E 2 whereas E 1 peaked at about 30 min and the conjugates: EIS, EIG and E2G peaked near 120 min. Ninety-four percent of the E in the hepatic portal vein was some metabolite of E 2. Eighty-eight percent of E was conjugated to glucuronide or sulfonate, and 79% of E was E 1 either free or conjugated. Concentrations of estrogens in the jugular vein are shown in Figure 4. Concentrations of E~ and E 2 did not differ between blood samples taken before and after administration of E2, data are not shown. Concentration of conjugates rose within a few min after administration of E 2, reaching a peak in 60 to 100 min. All of the E in the jugular was conjugated and 91% was conjugated E. The comparison of the concentrations of each estrogen in the hepatic portal and jugular veins over the entire 300 min sampling period is shown in Figure 5. Concentrations of estrogens in the bile of pregnant gilts are shown in Table 2. The amount of E contained in one dose of bile infused into the duodenum is shown in Table 3. Concentrations of estrogens in the hepatic portal vein of gilts infused with pooled bile are shown in Figures 6 and 7. The interval from infusion to a significant rise in E 2 and E 1 was about 180 min. There was a significant peak of E2G and EIG within a few min of infusion with a second more sustained peak at about 180 min. There was no rise in concentration of E~ and E 2 in the jugular vein and the rise of E2G and E~G was very similar to that in the hepatic portal vein, data not shown. Ninety-seven percent of E in the hepatic portal was conjugated to glucuronide. The results of infusion of bile into gilts pre-treated with oral antibiotics are shown in Figure 8. There was no difference in AUC of E~G and E2G between those animals treated with antibiotics and those not treated during the period from 0 to 80 min. There was, however, significantly less (p<.01) AUC from 80 to 480 min in animals given antibiotics. The first peaks of E1G and E2G mimicked those of untreated gilts, but the second peak was absent.
TABLE 1. CONCENTRATION OF ESTROGENS IN THE JUGULAR AND HEPATIC PORTAL VEINS OF UNTREATED PRE-PUBERTAL GILTSa
Estrogen E2 E1 EIS E2G EIG 'mean ± SE
Jugular (pmol/ml) .09 .,- .01 .36 ± .02 .02 ± .01 .32 ± .05 .87 ± .07
Portal (pmol/ml) .08 + .01 .41 ± .04 .02 ± .02 .37 ± .06 .96 ± .07
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TABLE 2. MEAN CONCENTRATIONOF ESTROGENS IN BILE FROM PREGNANTPIGS Stage of Gestation Estrogen
Day 30 a (pmol/ml)
E2 E1 E1S E2G E1G Total
0.14 7.47 45.62 16.47 749.98 819.68
Day l l 4 b (pmol/ml)
Pooled c (pmol/ml)
41.25 3311.87 741.39 788.82 87129.95 92013.28
23.16 862.96 598.21 18674.98 20159.31
"n=9 an=11 CBile pooled from 34 pregnant pigs at approximately day 30 or day 114 of gestation = estrogen not measured -
TABLE 3. ESTROGENSIN BILE POOLED FROMPREGNANTPIGS BEFOREAND AFTER EXTRACTIONWITHETHER (EE) Estrogen
Pooled Bile (80 ml) (pmol)
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T i m e (minutes) Figure 6. Mean concentrations of E t and E 2 (n = 6) in the hepatic portal vein in pre-pubertal gilts infused with 80 ml of pooled bile from pregnant pigs in the duodenum at time 0. Letters indicate significant difference between mean concentration at post-treatment and that of pre-treatment: a = P<.05, c = P<.001. SE: Standard error of the mean.
When the bile was extracted with ether to reduce E t and E 2 before infusion the first peaks of EIG and E2G were absent while the second peak occurred as with untreated bile (Figure 9). The AUC was significantly less (p<.05, E2G; p<.01, E1G) for the period from 0 to 80 min with no difference from 80 to 480 min. In both Experiments 3 and 4 the peaks of E1G and E2G in the jugular vein were at the same intervals and significance as in the hepatic portal vein.
ABSOR PT I ON A N D M E T A B O L I S M OF E S T R O G E N S
205
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Time (minutes) Figure 7. Mean concentrations of E1G and E2G (n = 6) in the hepatic portal vein in pre-pubertal gilts infused with 80 ml of pooled bile from pregnant pigs in the duodenum at time 0. Letters indicate significant difference between mean concentration at post-treatment and that of pre-treatment: a = P<.05, c = P<.001. SE: Standard error of the mean.
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Time (minutes) Figure 8. Mean concentrations of EIG and E2G (n = 5) in the hepatic portal vein in pre-pubertal gilts treated with oral antibiotics then infused with 80 ml of pooled bile from pregnant pigs in the duodenum at time 0. Letters indicate significant difference between mean concentration at post-treatment and that of pre-treatment: a = P<.05, b = P<.01, c = P<.001. SE: Standard error of the mean.
206
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Time (minutes) Figure 9. Mean concentration of E~G and E2G (n = 4) in the hepatic portal vein in pre-pubertal gilts infused with 65 ml of bile extracted with ether, in the duodenum at time 0. Letters indicate significant difference between mean concentrationat post-treatment and that of pre-treatment: a = P<.05, b = P<.01, c = P<.001. SE: Standard error of the mean. DISCUSSION Although metabolites of E 2 constitute a very significant proportion of total E they have been largely ignored in many studies. They provide a reservoir of E and may contribute to effects of E on reproduction. The very short period between injection of E 2 in the stomach and a sharp rise in E metabolites in the hepatic portal vein would indicate that the stomach contents or wall were very active in metabolizing E. The extent of conversion and subsequent conjugation of E 2 and E 1 indicates very efficient metabolism. In a similar study (8) no unconjugated E was found in the hepatic portal vein following infusion of radiolabeled E 2. In that study the experimental pigs were much younger with perhaps a more active enzyme system, the E 2 w a s dissolved in alcohol and the dose of E 2 w a s much lower than in the present study. Each of these factors may have contributed to detection of unconjugated E in the present study. After infusion of E2 in the stomach, F r e e E 2 and E1 were detected in the hepatic portal vein but not in the jugular although the concentration of conjugates rose markedly in the jugular vein. The potential effects and fate of these conjugates are not well known. If they were stored and deconjugated or converted to an active form they could influence reproduction. The concentration of E and conjugates in the bile of pregnant gilts at day 30 and increasing 100 fold to day 114 may be the first such report. Although the conjugates make up the bulk of E there is free E as detected by RIA and also by the response following infusion of bile extracted to remove free E. Infusion of bile into the duodenum provides a relatively normal route of delivery of normally occurring forms of E. The timing of the infusion shortly after the ingestion of a meal also mimics the normal sequence of bile secretion after a meal. The first rise in conjugates is quite likely from free Et and E 2 in the bile. This conclusion is supported by the observa-
ABSORPTION AND METABOLISM OF ESTROGENS
207
tion of Experiment 1 of the almost immediate rise of conjugates in the hepatic portal vein when E 2 was placed in the stomach. This together with an essential absence of a first peak after infusion of bile that had been extracted with ether to reduce the E l and E 2 substantiates the conclusion. The peaks of conjugates occurring at about 180 min after infusion of normal bile into untreated gilts might appear to be the result of direct absorption of conjugates. The results of other studies would indicate that conjugates are poorly absorbed and require deconjugation before absorption and conjugation (2, 30, 31, 32). It therefore seems more reasonable that the delay from infusion to the peak would indicate that deconjugation by gut bacteria took place before absorption (33). The extent and consistency with which E2 was converted to E l as evidenced by the relatively greater proportion of E 1 conjugates is somewhat at variance with previous reports (33). The predominance of E l conjugates over E 2 conjugates was consistent whether E 2 was instilled in the stomach or bile was infused into the duodenum. Because 96% of estrogens in bile were conjugates and 96% of estrogens in the second peak in the hepatic portal were conjugates would indicate that the bile conjugates were the main source of hepatic portal conjugates. The marked reduction in the second peaks of conjugates in the hepatic portal of gilts pretreated with oral antibiotics would indicate that conjugates are not absorbed directly and that reduction of gut bacteria reduces deconjugation, absorption and enterohepatic recycling. The finding of estrogens in both jugular and hepatic portal veins after infusion of normal bile is one of the first direct demonstrations of the possibility of enterohepatic circulation of natural estrogens. The observation that the concentrations of circulating estrogens can be influenced by antibiotics supports the clinical observations of failure of oral contraceptives following exposure to various xenobiotics. Conjugated estrogens may be deconjugated in the periphery (30, 34, 35), but the extent and significance has not yet been determined. We conclude that most estrogens administered orally or entering the gut from the bile are conjugated by the gut wall, pass to the liver where they can be put into the bile or passed into the peripheral circulation. These estrogens in the periphery have the potential to influence reproduction. ACKNOWLEDGEMENTS/FOOTNOTES ~Supported in part by Biomedical Research Grant, University of Illinois and Illinois Pork Producers Association. 2present address: 1 Holiday Drive, Village Terrace Apts. #A-109, Cortland, NY 13045. 3312 Animal Sciences Lab, University of Illinois, 1207 W. Gregory Dr., Urbana, IL 61801. 4Correspondence and reprints.
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