- Email: [email protected]
Circulation of estrogens introduced into the rectum or duodenum in pigs
DOMESTIC ANIMAL ENDOCRINOLOGY
Vol. 11(4):383-391,1994
CIRCULATION OF ESTROGENS INTRODUCED INTO THE RECTUM OR DUODENUM IN PIGS 1 W.L. Ruo~ and P.J. Dziuk 3,4 Department of Animal Sciences University of Illinois Urbana, IL 61801 Received February 1, 1994
ABSTRACT To determine the absorption and metabolism of 17[~-estradiol (E2) by the rectum of the pig, 10 mg of crystalline E 2 was placed in the rectum of prepubertal gilts in Experiment 1. Blood samples were subsequently obtained from hepatic portal and jugular veins and plasma was assayed for E2, estrone (El), 17[5-estradiol-glucuronide (E2G), estrone-glucuronide (E1G) and estrone-sulfate (ElS). Concentration of E2, El, E2G, EIG, and EIS rose in the hepatic portal vein within 30 min and remained elevated for several hr. Concentrations of E 2 in the hepatic portal vein represented 3% of the total estrogen detected in the hepatic portal vein during the 5 hr sampling period, indicating that most of the E 2 was metabolized prior to entering the hepatic portal vein after absorption by the rectal mucosa. Concentrations of E2, El, E2G , E~G, and E~S rose in the jugular vein and remained elevated for several hr. The rise in E 2 and Ex in the jugular vein may have come from E 2 and E1 in venous circulation from the rectum that entered the inferior vena cava bypassing the hepatic portal vein and liver. The net result of absorption of E2 from the rectum of gilts was a large rise in unconjugated and conjugated E 2 and E I in the peripheral circulation. In Experiment 2 prepubertal gilts fitted with jugular, hepatic portal, duodenal, and gall bladder catheters were infused into the duodenum with bile from pregnant gilts. Concentrations of E2, El, E2G, and E~G were determined in gallbladder bile of gilts before infusion and at 470 rain. Concentrations of E2G and E,G were determined in hepatic portal and jugular plasma before and after infusion of bile. A cholagogue was given at 480 rain and E2G and E~G were measured in plasma from 490 min to 960 rain. Concentrations of E 2 and E1 in gallbladder bile rose at 470 min and fell to basal concentrations at 970 rain. In gilts given the cholagogue, E2G and E~G in both the jugular and hepatic portal veins rose significantly over those in gilts not given the cholagogue. Bile estrogens circulate via the enterohepatic route and factors that influence secretion of estrogens in bile can influence concentrations of circulating estrogens. INTRODUCTION Enterohepatic circulation of estrogens follows a route from bile in the gallbladder, release of bile into the small intestine, deconjugation of estrogens in the gut lumen, absorption of the biliary estrogens into venous drainage to the liver and then either again into the gallbladder or into the peripheral circulation prolonging the availability of endogenous estrogens (1, 2). In a study by Adlercruetz et al. (3) inhibition of enterohepatic circulation of estrogens in pregnant women resulted in a decrease in the concentrations of estradiol glucuronide (E2G) and estrone glucuronide (EIG), but not estradiol (E2) and estrone (El), implying that enterohepatic circulation does not affect the concentrations of E 2 and E1 in the peripheral circulation. However, apparent inhibition of enterohepatic circulation in women by increased dietary fiber (4) or decreased fat intake (5) resulted in decreased concentrations of E 2 and E~ in plasma indicating reduced availability of E 2 and E r Estradiol administered orally is relatively ineffective biologically because E 2 is inactivated by metabolism during first pass through the gut wall and liver. In pigs administered E 2 in the upper gastrointestinal tract, free estrogens rose in the hepatic portal vein, but concentrations of E 2 and El did not increase in the jugular vein (6, 7). In those studCopyright © 1994 Buttorworth-Hoinomann
383
0739-7240/93/$7.50
RUOFF AND DZIUK
384
ies, most of the E 2 w a s metabolized prior to entering the hepatic portal vein and the liver completely metabolized and/or removed all E 2 and E 1 from the hepatic portal vein. In mammals, some venous drainage from the lower gut bypasses the liver and goes directly to the peripheral circulation via the pudendal and iliac venous systems. Adlercreutz et al. (8) measured high concentrations of free E 2 and E1 in feces of pregnant women, indicating an availability of biologically active estrogen in contents of the lower gut. E 2 and E 1 absorbed from the rectum might bypass the liver and reach the peripheral circulation unmetabolized. The extent of enterohepatic circulation of estrogens might also influence the concentration of peripheral estrogens. One objective of the present study was to investigate in the prepubertal gilt, the fate of E 2 absorbed from the rectum, by determining the extent of metabolism of E 2 during absorption, and the extent to which estrogens absorbed into venous drainage from the rectum enter the peripheral circulation. Exogenous E 2 was placed in the rectum, followed by measurement of the concentrations of conjugated and unconjugated E 2 and E~ in the hepatic portal and jugular veins. A second objective was to determine the fate of biliary estrogens infused into the duodenum followed by stimulation of bile release from the gall bladder. M A T E R I A L S AND M E T H O D S
Experiment 1. Animals and Sampling. Eight prepubertal crossbred gilts, 5 months of age and weighing 80 Kg, were the experimental animals. Jugular and hepatic portal veins were catheterized for repeated sampling of blood as described previously (7). Immediately after exteriorizing the hepatic portal vein catheter, gilts were removed from the anesthesia machine, and blood was obtained from both the jugular and hepatic portal veins to establish basal concentrations of endogenous estrogen in the gilts. Ten mg of E 2 (Sigma, St. Louis, MO) was dissolved in 1 ml of cocoa butter. One hour after exteriorizing the hepatic portal vein catheter, the E 2 in cocoa butter was placed in a gelatin capsule, 25 mm in length and 10 mm in diameter, and the gelatin capsule was inserted 20 cm into the rectum of the gilt. This was designated as time 0. Blood was obtained from both the jugular and hepatic portal veins at 5, 15, 30, 45, 65, 80, 95, 115, 150, 185, 210, and 300 min and was kept at 4 ° C until plasma was separated. Blood was centrifuged and plasma was stored at - 20 ° C. E x p e r i m e n t 2. Eight gilts in two groups of 4 each, similar to those in Experiment 1, had indwelling catheters placed in the jugular and hepatic portal veins and the duodenum and gall bladder. These gilts were given one week to recover and establish normal eating patterns. No antibiotics were given to avoid disturbing the gut microflora. The procedure for the experiment is shown in Figure 1. Briefly, on the day of the experiment, the gilts were fed the normal diet at -60 min. At -20 min samples of bile were taken from 4 gilts in Group II to establish basal concentrations of estrogens in bile of prepubertal gilts. At -10 min 3 blood samples were taken from the 4 gilts in Group I and the 4 in Group II, to establish basal concentrations of estrogens. Bile had been collected from gallbladders of 10 gilts killed at days 27 to 35 of gestation and from 24 gilts killed at days 105 to 116 of gestation. Bile was pooled and stored at -20 ° C (7). At 0 time, 80 ml of the pooled bile was infused into the duodenum of gilts in both groups. The concentrations of E l, E 2, EIG and E2G from time 0 to 500 min have been determined and reported previously (7). Blood samples were taken at +10 min. At 470 min a second bile sample was taken from Group II and at 480 min gilts in Group II were given an infusion of 2.5 ug of a cholagogue (Kinevac, Squibb Pharmaceutical) over a period of 1 min. In preliminary studies with the gall bladder exposed at laparotomy, this dose of cholagogue was seen to cause marked contraction of the gall bladder. Blood samples were then taken at 490, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 780, 840, 900, and 960 min. At 970 min a third sample of bile was obtained. Blood samples
ABSORPTION AND CIRCULATION OF 171~-ESTRADIOL IN PIGS
Group I Omin
,
tit
Blood
t
80 ml
lOmin
,
//
t
490 rain
,;
::
Blood
:
/,
•
17 Blood S w n l ~
385
iW mln a,
,:
:
Group lI ~Sam~
Or,I.
47O 48O lo.min , I ~n .rain
. . . . . . smp~ ~ ~
)0 eknm I.v.
,m0
m
~n
rain.
17Bo~l Smgl~
Se~e
Figure 1. Experimental procedure for gilts in Experiment 2.
were not taken from 10 to 480 min because the pattern of concentration of estrogens for gilts receiving 80 ml of bile infused into the duodenum had been established previously (7). Samples were taken 10 rain after injection of bile into the duodenum to confirm the expected increase in estrogens in the blood at this time. The present experiment was designed to determine the extent that estrogens from infused bile could be recirculated and reappear in bile for a second pass. Hormone Assays. Concentrations of E2, E 1, E]S, E2G, and E:G in plasma and bile were measured by radioimmunoassay as described previously (7). Definitions and Statistical Analysis. The time-course curve for each estrogen was the concentration of estrogen plotted against the interval to sampling. The change in area under the curve (AAUC) for concentration of estrogen from 0 to 300 rain in Experiment 1 was calculated using the trapezoidal method. In Experiment 2 the AAUC was calculated from 490 to 960 min. Change in AUC was the AUC minus the mean pre-treatment concentration of estrogen multiplied by the period in rain: {AAUC = (AUC) - (pre-treatment value x 300 or 470 rain)}. The concentration of total estrogen was defined as the sum of the pmol/ml concentration of each of the five types of estrogen measured. The percentage of each estrogen was determined by dividing the AAUC for each estrogen into 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 post-treatment 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. Because there were missing values, the data were analyzed by analysis of variance to estimate LSMEANS (SAS, PROC GLM) (9). RESULTS Experiment 1. Catheters in the jugular vein remained patent for the duration of the experiment in all eight gilts. Catheters in the hepatic portal vein were patent at the beginning of the experiment in seven of the gilts. One gilt became ill during the experiment and blood samples were obtained for only 95 rain after treatment. Data from the eight gilts were used for calculation of results. Concentrations in pmol/ml (meaneSE) of each type of estrogen in the jugular and hepatic portal veins of untreated gilts are shown in Table 1. After 10 mg of E 2 w a s put in the rectum, mean concentrations of E2, El, E]S, E2G , and E:G in the hepatic portal vein increased within 30 min, reaching peak values at sara-
386
RUOFF AND DZIUK
TABLE 1. CONCENTRATIONOF ESTROGENS IN THE JUGULAR VEIN AND HEPATIC PORTAL VEINS OF UNTREATED PREPUBERTAL GILTS.
VEIN Estrogen
Jugular (pmol/ml) a
E2 E1 E1S E2G E1G
.09 .43 .04 .27 .94
± ± + ± ±
Total
1.78 + .08
H e p a t i c Portal ( p m o l / m l )
.01 .02 .02 .03 .05
.12 .38 .04 .37 .87
+ .02 ± .02 ± .02 __ .04 ± .07
1.82 ± .12
'Mean :t: SE
... 3[ o E2
0
b
50
A E1
100
150
200
250
300
Time (minutes) Figure 2. C o n c e n t r a t i o n of E2 ( m e a n + SE, n = 7) and E 1 (mean ± SE, n = 7) in the hepatic portal vein in g i l t s g i v e n 10 m g o f E 2 in the r e c t u m at t i m e 0. L e t t e r s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e b e t w e e n m e a n concentration at post-treatment and that of pre- treatment, w i t h i n estrogen: a = P < .05, b = P < .01, c = P < .001.
~'~ElS
b
A E2G
a E1G
15.
5
0
50
100
150
2O0
250
30O
T i m e (minutes) Figure 3. C o n c e n t r a t i o n of E]S ( m e a n ± SE, n = 5), E2G (mean + SE, n = 6) and E1G (mean ± SE, n = 6) in the hepatic portal vein in g i l t s g i v e n 10 m g of E 2 in the rectum at t i m e 0. Letters indicate s i g n i f i c a n t difference b e t w e e n m e a n c o n c e n t r a t i o n at post-treatment and that of pre-treatment, w i t h i n estrogen: a = P < .05, b = P < .01, c = P < .001.
ABSORPTION AND CIRCULATION OF 1713-ESTRADIOL IN PIGS
387
piing times ranging from 65 to 115 m i n (Figures 2 and 3). Peak concentrations of estrogens ranged from 2 to 51X those of pre- treatment and AAUC for total estrogens in the hepatic portal vein was 10563 pmol. The mean proportion of each type of estrogen in the hepatic portal vein after E 2 was placed in the rectum was: E 2 - 3%; E] - 1%; EIS - 2%; E2G - 32%; E]G - 62%. Thus, 97% of the E 2 absorbed from the rectum was metabolized before reaching the hepatic portal vein, 96% of the estrogen was conjugated to glucuronide or sulfate and 65% of the estrogen was E~, either unconjugated or conjugated. Before treatment, the mean concentration of each type of estrogen in the j u g u l a r vein was similar to that in the hepatic portal vein (Table 1). After treatment, the mean concentrations of E 2, E 1, E~S, E2G, and E~G in the j u g u l a r vein increased within 30 min to peak at times ranging from 95 to 150 min (Figures 4 and 5). Peak concentrations of estrogens ranged from 1.5 to 55X those of pre-treatment.
o.8
oF..2
A E1
0.6
O. 0.4
~,1 0 . 2 . 0.1 o
0
50
100
150
200
250
300
Time (minutes) Figure 4. Concentration of E2 (mean ± SE, n = 8) and E] (mean ± SE, n = 8) in the jugular vein in gilts given 10 mg of E2 in the rectum at time 0. Letters indicate significant difference between mean concentration at post-treatment and that of pre-treatment, within estrogen: a = P < .05, b = P < .01.
5°[0 ¢1s
"Az2¢
o£;o
c
20
b
~w 0
b c ¢¢
O'
0
50
.
(;
100 Time
150
200
250
300
(minutea|
Figure 5. Concentration of EzS (mean ± SE, n = 7), E2G (mean ± SE, n = 8) and E]G (mean ± SE, n = 8) in the jugular vein in gilts given 10 mg of E2 in the rectum at time 0. Letters indicate significant difference between concentration at post-treatment and that of pre-treatment, within estrogen: a = P < .05, b = P < .01, c = P < .001.
388
RUOFF AND DZIUK
Ilqpatic-Porlal ~'soo I OBlle ==Bile,thencholagogue ~<4(~
I-
~300
_E2oo
~
100
Ill "100
E2G
EIG
Type of Estrogen Figure 6. Comparison of A U C (mean ± SE) from 490 min to 960 min for E2G and EIG in the hepatic portal vein after infusion of 80 ml of pooled bile in the d u o d e n u m at time 0 between gilts treated with a cholagogue at 480 min and untreated gilts. Letters indicate significant differences: a = P < .05, b = P < .01.
Jugular E -~ 300
E
nBile ==Bile,then cholagogue
2OO
o
f,
E2G
EIG
Type of Estrogen Figure 7. Comparison of A U C (mean + SE) from 490 min to 960 min for E2G and EIG in the jugular vein after infusion of 80 ml of pooled bile in the duodenum at time 0 between gilts treated with a cholagogue at 480 rain and untreated gilts. Letters indicate significant differences: a = P < .05, b = P < .01.
The mean proportion of each type of estrogen in the jugular vein after E 2 w a s placed in the rectum was: E 2 - .4%; E I - .6%; E1S - 4%; E2G - 20%; E1G - 75%. Thus 99% of the estrogen found in the jugular vein was conjugated to glucuronide or sulfate and 79.6% of the estrogen was El, either unconjugated or conjugated. Experiment 2. The concentrations of E2G and E~G in both the hepatic portal and jugular veins rose significantly in groups I and II within 10 min of infusion of bile into the duodenum. From 560 to 960 rain, the concentrations of those estrogens were not different from the basal concentrations as observed previously (7) in Group I. The AAUC of E2G and EIG in plasma from the hepatic portal vein of gilts of Group II that received the cholagogue at 480 min were significantly higher than in Group I from 490 to 960 min
ABSORPTION AND CIRCULATION OF 1713-ESTRADIOLIN PIGS
389
(Figure 6). The AAUC of E2G in jugular veins of gilts for Group II from 490 to 960 min was significantly greater than that for Group I (Figure 7). Although the AAUC of E1G in the jugular in Group II was 2.5X that in Group I, the difference was not significant. The bile from the gall bladder of Group II before time 0 had mean concentrations of estrogens (pg/ml) of: E 2 - .17; E l - 7.48; E2G - 11.6; E1G - 107.6. At 470 rain concentrations of estrogens in bile from Group II were (pg/ml): E 2 - .74; E 1 - 35.8; E2G - 70.1; E~G 1175.1. Each of these concentrations were significantly higher than at time 0. At 970 min the concentrations of estrogens in bile were not significantly different from those at time 0. -
DISCUSSION Placement of E 2 in the rectum of prepubertal gilts resulted in a rise in the concentration of total estrogens in the hepatic portal vein within minutes. Moore et al. (6) reported that 14-C-estrogens were present in the hepatic portal vein one min after placing 14-CE 2 in the colon of gilts. In the present study, the AAUC for concentrations of total estrogens in the hepatic portal vein was 10563 pmol, indicating that there was extensive absorption of E 2 from the rectum during the 300 min sampling period. Concentrations of E 2 in the hepatic portal vein represented a very small proportion (3%) of the total estrogen detected in the hepatic portal vein during the sampling period. Apparently, 97% of the E 2 absorbed from the rectum was metabolized to E 1, E2G, E1G, and EIS before entering the hepatic portal vein. Moore et al. (6) reported that nearly all of 14-C-E 2 absorbed from the colon of pigs was converted to conjugated E 2 and E 1 prior to entering the hepatic portal vein. We have reported that 94% of E 2 absorbed from the stomach of gilts was metabolized to E l, E2G, EIG, and E~S prior to entering the hepatic portal vein (7). A soluble estrogen glucuronyltransferase which conjugates E 2 and E 1 to glucuronide has been isolated from the mucosa of the small intestine of pigs (10). Incubation of E 2 and E 1 with intestinal microsomes yielded E2G and EIG. Enzymes that metabolize E 2 between the lumen of the gut to the hepatic portal vein are likely in the gut mucosa (11). In the current study, these enzymes converted 65% of E 2 to E 1 and conjugated 96% to glucuronide or sulfate. As noted after E 2 was placed in the stomach (7) and after E 2 was placed in the rectum, about 4% of estrogen in the hepatic portal vein was in an unconjugated form; E 2 or E r Placement of 14-C-E 2 in the colon of immature pigs has also led to a small rise in unconjugated estrogens in the hepatic portal vein (6). Apparently under these conditions the enzymes in the posterior gut do not metabolize all of the E 2 allowing passage of some unconjugated estrogen into the posterior vena cava. After E 2 w a s placed in the rectum, concentrations of E 2 and E 1 in the jugular vein rose and remained elevated for several hr. This is in contrast to the results of placing E 2 in the stomach of pigs in other studies (7, 12), where concentrations of unconjugated estrogen in the jugular vein did not rise. Bottoms et al. (13) and Moore et al. (6) also reported no rise in E 2 and E~ in the jugular vein in pigs administered E 2 in the upper gastrointestinal tract. In mammals, the hepatic portal vein carries all blood and components from the stomach to the liver where metabolism occurs prior to entering the peripheral circulation. In mammals, veins from the rectum go either to the hepatic portal vein or alternatively via the pudendal and iliac veins to the inferior vena cava. This latter route would permit unconjugated estrogen to appear in the peripheral circulation by bypassing the first pass effects of the liver. This may explain why concentrations of unconjugated estrogen rose in the jugular vein when E 2 was placed in the rectum, but not when placed in the stomach (6, 7, 12, 13). Endogenous estrogens enter the gut in the bile (14), are deconjugated or converted by gut bacteria (8, 15) and are present as free estrogens in the rectum (8).
390
RUOFF AND DZIUK
The absorption of fecal estrogens from the rectum may affect the concentrations of E 2 and E 1 in the peripheral circulation. This route may help explain the phenomenon of postpartum estrus in sows (16). The high concentration of estrogens in plasma of sows just before parturition could contribute to high concentrations of bile estrogens. These estrogens in turn could pass down the intestines during the 48-hr ingesta passage period either as conjugates that could be deconjugated in the rectum by the gut bacteria or deconjugated in the upper gut. The free estrogens could then be absorbed directly from the rectum as free estrogens, bypass the liver and influence behavior. The extent of deconjugation could be influenced by antibiotics in the diet (3, 7). Alternatively the conjugated estrogens could be deconjugated by lungs or brain and reenter the systemic circulation as free estrogens (17,18). In Experiment 2 the immediate rise in E2G and E1G in both the hepatic portal and jugular veins after infusion of bile into the duodenum was as reported previously (7). The detection of estrogens in the bile at concentrations more than 10X those before infusion demonstrates the extent of recirculation of bile estrogens. After induction of bile secretion with a cholagogue at 470 min, the concentrations of estrogens were again significantly higher in the jugular and hepatic portal veins than before infusion and in gilts at the same interval from infusion but not given a cholagogue. The rise in concentration of estrogens persisted for 210 min. This indicates that the original bile estrogens could recirculate at least twice. The series of steps in recirculation (19, 20) and the number of possible points in the series provide the potential for influencing the extent of recycling by many different means. Dietary components and nature of the ingesta, xenobiotics and antibiotics each have the potential for influencing the rate and route of estrogen metabolism and excretion. Because of estrogen's ubiquitous effects on reproduction and gonadotropin secretion, it is important to consider these aspects when evaluating effects of treatments that seem unrelated to 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. #A109, Cortland, NY 13045. 3312 Animal Sciences Lab, 1207 W. Gregory Dr., Urbana, 1L 61801. 4Correspondence and reprints
REFERENCES 1. Sandberg AA, Slaunwhite WR. Studies on phenolic steroids in human subjects. II. The metabolic fate and hepato-biliary-enteric circulation of 14-C-estrone and 14-C-estradiol in women. J Clin Endocrinol 18:1266-1278, 1957. 2. Sandberg AA, Kirdani RY, Back N, Weyman P, Slaunwhite WR. Biliary excretion and enterohepatic circulation of estrone and estriol in rodents. Am J Physiology 213:1138-1142, 1967. 3. Adlercreutz H, Martin F, Lehtinen T, Tikkanen MJ, Pulkkinen MO. Effect of ampicillin administration on plasma conjugated and unconjugated estrogen and progesterone levels in pregnancy. Am J Obstet Gynecol 128:266-271, 1977. 4. Goldin BR, Adlercreutz H, Dwyer JT, Swenson L, Warram JH, Gorbach SL. Effect of diet on excretion of estrogens in pre- and postmenopausal women. Cancer Res 41:3771-3772, 1981. 5. Rose DP, Boyar AP, Cohen C, Strong LE. Effect of a low-fat diet on hormone levels in women with cystic breast disease. I. Serum steroids and gonadotropins. J Nat Cancer Inst 78:623- 626, 1987. 6. Moore AB, Bottoms GD, Coppic GL, Pohland RC, Roesel OF. Metabolism of estrogens in the gastrointestinal tract of swine. I. Instilled estradiol. J Anita Sci 55:124--134, 1982. 7. Ruoff WL, Dziuk PJ. Absorption and metabolism of estrogens from the stomach and duodenum of pigs. Domest Anita Endocrinol 11:197-208, 1994. 8. Adlercreutz H, Martin F, Pulkkinen M, Deneker H, Rimer U, Sjoberg N-O, Tikkanen MJ. Intestinal metabolism of estrogens. J Clin Endocrinol Metab 43:497-505, 1976. 9. SAS Institute Inc. SAS User's Guide; Statistics, Version 5 Edition. Cary, NC:SAS Institute, Inc., 956 pp. 1985.
ABSORPTION AND CIRCULATION OF 17~-ESTRADIOL IN PIGS
391
10. Rao GS, Breuer H. Partial purification and kinetic properties of a soluble estrogen glucuronyltransferase from pig intestine. J Biol Chem 20:5521-5527, 1969. 11. Back DJ, Breckenridge AM, Crawford FE, Maclver M, Orme MLE, Rowe PH. Interindividual variation and drug interactions with hormonal steroid contraceptives. Drugs 21:46-61, 1981. 12. Dziuk PJ, Elsaesser F, Eilendorff F. Models and results of studies on levels of sex steroids in the hepatic portal and jugular veins of the conscious pig. The Endocrines and the Liver. Academic Press, New York. p. 413-414, 1982. 13. Bottoms GD, Coppoc GL, Monk E, Moore AB, Roesel OF, Regnier FE. Metabolic fate of orally administered estradiol in swine. J Anim Sci 46:674--685, 1977. 14. Adlercreutz, H, Ervast HS, Tenhunen A, Tikkanen. Gas chromatographic and mass spectrometric studies on oestrogens in bile: Part I. Pregnant women. Acta Endocrinol 73:.543- 554, 1973. 15. Lombardi P, Goldin B, Boutin E, Gorbach SL. Metabolism of androgens and estrogens by human fecal microorganisms. J Steroid Biochem 9:795-802, 1978. 16. Holness DH, Hunter RHF. Post-partum oestrus in the sow in relation to the concentration of plasma oestrogens. J Reprod Fertil 45:15-20, 1975. 17. Collins DC, Balikian HPM, Preedy JRK. Hydrolysis of estrogen conjugates by the lungs of the dog in vivo. Endocrinology 96:1543-1551, 1975. 18. Collins DC, Balikian HM, Preedy .IRK. Evidence for the simultaneous uptake and release of certain estrogens by the splanchnic area in the dog. Endocrinology 99:420-428, 1976. 19. Rao GS, Rao ML, Haueter G, Breuer H. Steroidglucuronyltransferases, V. Formation and hydrolysis of oestrogen glucuronides by the liver, kidney and intestine of the pig. Hoppe- Seyler's Physiol Chem 355:881-890, 1974. 20. Schumann W, Hillesheim HG, Gina G. Model systems for pharmokinetics of steroid drugs subject to enterohepatic circulation. Exp Clin Endocrinol 87:118-124, 1986.
Vol. 11(4):383-391,1994
CIRCULATION OF ESTROGENS INTRODUCED INTO THE RECTUM OR DUODENUM IN PIGS 1 W.L. Ruo~ and P.J. Dziuk 3,4 Department of Animal Sciences University of Illinois Urbana, IL 61801 Received February 1, 1994
ABSTRACT To determine the absorption and metabolism of 17[~-estradiol (E2) by the rectum of the pig, 10 mg of crystalline E 2 was placed in the rectum of prepubertal gilts in Experiment 1. Blood samples were subsequently obtained from hepatic portal and jugular veins and plasma was assayed for E2, estrone (El), 17[5-estradiol-glucuronide (E2G), estrone-glucuronide (E1G) and estrone-sulfate (ElS). Concentration of E2, El, E2G, EIG, and EIS rose in the hepatic portal vein within 30 min and remained elevated for several hr. Concentrations of E 2 in the hepatic portal vein represented 3% of the total estrogen detected in the hepatic portal vein during the 5 hr sampling period, indicating that most of the E 2 was metabolized prior to entering the hepatic portal vein after absorption by the rectal mucosa. Concentrations of E2, El, E2G , E~G, and E~S rose in the jugular vein and remained elevated for several hr. The rise in E 2 and Ex in the jugular vein may have come from E 2 and E1 in venous circulation from the rectum that entered the inferior vena cava bypassing the hepatic portal vein and liver. The net result of absorption of E2 from the rectum of gilts was a large rise in unconjugated and conjugated E 2 and E I in the peripheral circulation. In Experiment 2 prepubertal gilts fitted with jugular, hepatic portal, duodenal, and gall bladder catheters were infused into the duodenum with bile from pregnant gilts. Concentrations of E2, El, E2G, and E~G were determined in gallbladder bile of gilts before infusion and at 470 rain. Concentrations of E2G and E,G were determined in hepatic portal and jugular plasma before and after infusion of bile. A cholagogue was given at 480 rain and E2G and E~G were measured in plasma from 490 min to 960 rain. Concentrations of E 2 and E1 in gallbladder bile rose at 470 min and fell to basal concentrations at 970 rain. In gilts given the cholagogue, E2G and E~G in both the jugular and hepatic portal veins rose significantly over those in gilts not given the cholagogue. Bile estrogens circulate via the enterohepatic route and factors that influence secretion of estrogens in bile can influence concentrations of circulating estrogens. INTRODUCTION Enterohepatic circulation of estrogens follows a route from bile in the gallbladder, release of bile into the small intestine, deconjugation of estrogens in the gut lumen, absorption of the biliary estrogens into venous drainage to the liver and then either again into the gallbladder or into the peripheral circulation prolonging the availability of endogenous estrogens (1, 2). In a study by Adlercruetz et al. (3) inhibition of enterohepatic circulation of estrogens in pregnant women resulted in a decrease in the concentrations of estradiol glucuronide (E2G) and estrone glucuronide (EIG), but not estradiol (E2) and estrone (El), implying that enterohepatic circulation does not affect the concentrations of E 2 and E1 in the peripheral circulation. However, apparent inhibition of enterohepatic circulation in women by increased dietary fiber (4) or decreased fat intake (5) resulted in decreased concentrations of E 2 and E~ in plasma indicating reduced availability of E 2 and E r Estradiol administered orally is relatively ineffective biologically because E 2 is inactivated by metabolism during first pass through the gut wall and liver. In pigs administered E 2 in the upper gastrointestinal tract, free estrogens rose in the hepatic portal vein, but concentrations of E 2 and El did not increase in the jugular vein (6, 7). In those studCopyright © 1994 Buttorworth-Hoinomann
383
0739-7240/93/$7.50
RUOFF AND DZIUK
384
ies, most of the E 2 w a s metabolized prior to entering the hepatic portal vein and the liver completely metabolized and/or removed all E 2 and E 1 from the hepatic portal vein. In mammals, some venous drainage from the lower gut bypasses the liver and goes directly to the peripheral circulation via the pudendal and iliac venous systems. Adlercreutz et al. (8) measured high concentrations of free E 2 and E1 in feces of pregnant women, indicating an availability of biologically active estrogen in contents of the lower gut. E 2 and E 1 absorbed from the rectum might bypass the liver and reach the peripheral circulation unmetabolized. The extent of enterohepatic circulation of estrogens might also influence the concentration of peripheral estrogens. One objective of the present study was to investigate in the prepubertal gilt, the fate of E 2 absorbed from the rectum, by determining the extent of metabolism of E 2 during absorption, and the extent to which estrogens absorbed into venous drainage from the rectum enter the peripheral circulation. Exogenous E 2 was placed in the rectum, followed by measurement of the concentrations of conjugated and unconjugated E 2 and E~ in the hepatic portal and jugular veins. A second objective was to determine the fate of biliary estrogens infused into the duodenum followed by stimulation of bile release from the gall bladder. M A T E R I A L S AND M E T H O D S
Experiment 1. Animals and Sampling. Eight prepubertal crossbred gilts, 5 months of age and weighing 80 Kg, were the experimental animals. Jugular and hepatic portal veins were catheterized for repeated sampling of blood as described previously (7). Immediately after exteriorizing the hepatic portal vein catheter, gilts were removed from the anesthesia machine, and blood was obtained from both the jugular and hepatic portal veins to establish basal concentrations of endogenous estrogen in the gilts. Ten mg of E 2 (Sigma, St. Louis, MO) was dissolved in 1 ml of cocoa butter. One hour after exteriorizing the hepatic portal vein catheter, the E 2 in cocoa butter was placed in a gelatin capsule, 25 mm in length and 10 mm in diameter, and the gelatin capsule was inserted 20 cm into the rectum of the gilt. This was designated as time 0. Blood was obtained from both the jugular and hepatic portal veins at 5, 15, 30, 45, 65, 80, 95, 115, 150, 185, 210, and 300 min and was kept at 4 ° C until plasma was separated. Blood was centrifuged and plasma was stored at - 20 ° C. E x p e r i m e n t 2. Eight gilts in two groups of 4 each, similar to those in Experiment 1, had indwelling catheters placed in the jugular and hepatic portal veins and the duodenum and gall bladder. These gilts were given one week to recover and establish normal eating patterns. No antibiotics were given to avoid disturbing the gut microflora. The procedure for the experiment is shown in Figure 1. Briefly, on the day of the experiment, the gilts were fed the normal diet at -60 min. At -20 min samples of bile were taken from 4 gilts in Group II to establish basal concentrations of estrogens in bile of prepubertal gilts. At -10 min 3 blood samples were taken from the 4 gilts in Group I and the 4 in Group II, to establish basal concentrations of estrogens. Bile had been collected from gallbladders of 10 gilts killed at days 27 to 35 of gestation and from 24 gilts killed at days 105 to 116 of gestation. Bile was pooled and stored at -20 ° C (7). At 0 time, 80 ml of the pooled bile was infused into the duodenum of gilts in both groups. The concentrations of E l, E 2, EIG and E2G from time 0 to 500 min have been determined and reported previously (7). Blood samples were taken at +10 min. At 470 min a second bile sample was taken from Group II and at 480 min gilts in Group II were given an infusion of 2.5 ug of a cholagogue (Kinevac, Squibb Pharmaceutical) over a period of 1 min. In preliminary studies with the gall bladder exposed at laparotomy, this dose of cholagogue was seen to cause marked contraction of the gall bladder. Blood samples were then taken at 490, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 780, 840, 900, and 960 min. At 970 min a third sample of bile was obtained. Blood samples
ABSORPTION AND CIRCULATION OF 171~-ESTRADIOL IN PIGS
Group I Omin
,
tit
Blood
t
80 ml
lOmin
,
//
t
490 rain
,;
::
Blood
:
/,
•
17 Blood S w n l ~
385
iW mln a,
,:
:
Group lI ~Sam~
Or,I.
47O 48O lo.min , I ~n .rain
. . . . . . smp~ ~ ~
)0 eknm I.v.
,m0
m
~n
rain.
17Bo~l Smgl~
Se~e
Figure 1. Experimental procedure for gilts in Experiment 2.
were not taken from 10 to 480 min because the pattern of concentration of estrogens for gilts receiving 80 ml of bile infused into the duodenum had been established previously (7). Samples were taken 10 rain after injection of bile into the duodenum to confirm the expected increase in estrogens in the blood at this time. The present experiment was designed to determine the extent that estrogens from infused bile could be recirculated and reappear in bile for a second pass. Hormone Assays. Concentrations of E2, E 1, E]S, E2G, and E:G in plasma and bile were measured by radioimmunoassay as described previously (7). Definitions and Statistical Analysis. The time-course curve for each estrogen was the concentration of estrogen plotted against the interval to sampling. The change in area under the curve (AAUC) for concentration of estrogen from 0 to 300 rain in Experiment 1 was calculated using the trapezoidal method. In Experiment 2 the AAUC was calculated from 490 to 960 min. Change in AUC was the AUC minus the mean pre-treatment concentration of estrogen multiplied by the period in rain: {AAUC = (AUC) - (pre-treatment value x 300 or 470 rain)}. The concentration of total estrogen was defined as the sum of the pmol/ml concentration of each of the five types of estrogen measured. The percentage of each estrogen was determined by dividing the AAUC for each estrogen into 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 post-treatment 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. Because there were missing values, the data were analyzed by analysis of variance to estimate LSMEANS (SAS, PROC GLM) (9). RESULTS Experiment 1. Catheters in the jugular vein remained patent for the duration of the experiment in all eight gilts. Catheters in the hepatic portal vein were patent at the beginning of the experiment in seven of the gilts. One gilt became ill during the experiment and blood samples were obtained for only 95 rain after treatment. Data from the eight gilts were used for calculation of results. Concentrations in pmol/ml (meaneSE) of each type of estrogen in the jugular and hepatic portal veins of untreated gilts are shown in Table 1. After 10 mg of E 2 w a s put in the rectum, mean concentrations of E2, El, E]S, E2G , and E:G in the hepatic portal vein increased within 30 min, reaching peak values at sara-
386
RUOFF AND DZIUK
TABLE 1. CONCENTRATIONOF ESTROGENS IN THE JUGULAR VEIN AND HEPATIC PORTAL VEINS OF UNTREATED PREPUBERTAL GILTS.
VEIN Estrogen
Jugular (pmol/ml) a
E2 E1 E1S E2G E1G
.09 .43 .04 .27 .94
± ± + ± ±
Total
1.78 + .08
H e p a t i c Portal ( p m o l / m l )
.01 .02 .02 .03 .05
.12 .38 .04 .37 .87
+ .02 ± .02 ± .02 __ .04 ± .07
1.82 ± .12
'Mean :t: SE
... 3[ o E2
0
b
50
A E1
100
150
200
250
300
Time (minutes) Figure 2. C o n c e n t r a t i o n of E2 ( m e a n + SE, n = 7) and E 1 (mean ± SE, n = 7) in the hepatic portal vein in g i l t s g i v e n 10 m g o f E 2 in the r e c t u m at t i m e 0. L e t t e r s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e b e t w e e n m e a n concentration at post-treatment and that of pre- treatment, w i t h i n estrogen: a = P < .05, b = P < .01, c = P < .001.
~'~ElS
b
A E2G
a E1G
15.
5
0
50
100
150
2O0
250
30O
T i m e (minutes) Figure 3. C o n c e n t r a t i o n of E]S ( m e a n ± SE, n = 5), E2G (mean + SE, n = 6) and E1G (mean ± SE, n = 6) in the hepatic portal vein in g i l t s g i v e n 10 m g of E 2 in the rectum at t i m e 0. Letters indicate s i g n i f i c a n t difference b e t w e e n m e a n c o n c e n t r a t i o n at post-treatment and that of pre-treatment, w i t h i n estrogen: a = P < .05, b = P < .01, c = P < .001.
ABSORPTION AND CIRCULATION OF 1713-ESTRADIOL IN PIGS
387
piing times ranging from 65 to 115 m i n (Figures 2 and 3). Peak concentrations of estrogens ranged from 2 to 51X those of pre- treatment and AAUC for total estrogens in the hepatic portal vein was 10563 pmol. The mean proportion of each type of estrogen in the hepatic portal vein after E 2 was placed in the rectum was: E 2 - 3%; E] - 1%; EIS - 2%; E2G - 32%; E]G - 62%. Thus, 97% of the E 2 absorbed from the rectum was metabolized before reaching the hepatic portal vein, 96% of the estrogen was conjugated to glucuronide or sulfate and 65% of the estrogen was E~, either unconjugated or conjugated. Before treatment, the mean concentration of each type of estrogen in the j u g u l a r vein was similar to that in the hepatic portal vein (Table 1). After treatment, the mean concentrations of E 2, E 1, E~S, E2G, and E~G in the j u g u l a r vein increased within 30 min to peak at times ranging from 95 to 150 min (Figures 4 and 5). Peak concentrations of estrogens ranged from 1.5 to 55X those of pre-treatment.
o.8
oF..2
A E1
0.6
O. 0.4
~,1 0 . 2 . 0.1 o
0
50
100
150
200
250
300
Time (minutes) Figure 4. Concentration of E2 (mean ± SE, n = 8) and E] (mean ± SE, n = 8) in the jugular vein in gilts given 10 mg of E2 in the rectum at time 0. Letters indicate significant difference between mean concentration at post-treatment and that of pre-treatment, within estrogen: a = P < .05, b = P < .01.
5°[0 ¢1s
"Az2¢
o£;o
c
20
b
~w 0
b c ¢¢
O'
0
50
.
(;
100 Time
150
200
250
300
(minutea|
Figure 5. Concentration of EzS (mean ± SE, n = 7), E2G (mean ± SE, n = 8) and E]G (mean ± SE, n = 8) in the jugular vein in gilts given 10 mg of E2 in the rectum at time 0. Letters indicate significant difference between concentration at post-treatment and that of pre-treatment, within estrogen: a = P < .05, b = P < .01, c = P < .001.
388
RUOFF AND DZIUK
Ilqpatic-Porlal ~'soo I OBlle ==Bile,thencholagogue ~<4(~
I-
~300
_E2oo
~
100
Ill "100
E2G
EIG
Type of Estrogen Figure 6. Comparison of A U C (mean ± SE) from 490 min to 960 min for E2G and EIG in the hepatic portal vein after infusion of 80 ml of pooled bile in the d u o d e n u m at time 0 between gilts treated with a cholagogue at 480 min and untreated gilts. Letters indicate significant differences: a = P < .05, b = P < .01.
Jugular E -~ 300
E
nBile ==Bile,then cholagogue
2OO
o
f,
E2G
EIG
Type of Estrogen Figure 7. Comparison of A U C (mean + SE) from 490 min to 960 min for E2G and EIG in the jugular vein after infusion of 80 ml of pooled bile in the duodenum at time 0 between gilts treated with a cholagogue at 480 rain and untreated gilts. Letters indicate significant differences: a = P < .05, b = P < .01.
The mean proportion of each type of estrogen in the jugular vein after E 2 w a s placed in the rectum was: E 2 - .4%; E I - .6%; E1S - 4%; E2G - 20%; E1G - 75%. Thus 99% of the estrogen found in the jugular vein was conjugated to glucuronide or sulfate and 79.6% of the estrogen was El, either unconjugated or conjugated. Experiment 2. The concentrations of E2G and E~G in both the hepatic portal and jugular veins rose significantly in groups I and II within 10 min of infusion of bile into the duodenum. From 560 to 960 rain, the concentrations of those estrogens were not different from the basal concentrations as observed previously (7) in Group I. The AAUC of E2G and EIG in plasma from the hepatic portal vein of gilts of Group II that received the cholagogue at 480 min were significantly higher than in Group I from 490 to 960 min
ABSORPTION AND CIRCULATION OF 1713-ESTRADIOLIN PIGS
389
(Figure 6). The AAUC of E2G in jugular veins of gilts for Group II from 490 to 960 min was significantly greater than that for Group I (Figure 7). Although the AAUC of E1G in the jugular in Group II was 2.5X that in Group I, the difference was not significant. The bile from the gall bladder of Group II before time 0 had mean concentrations of estrogens (pg/ml) of: E 2 - .17; E l - 7.48; E2G - 11.6; E1G - 107.6. At 470 rain concentrations of estrogens in bile from Group II were (pg/ml): E 2 - .74; E 1 - 35.8; E2G - 70.1; E~G 1175.1. Each of these concentrations were significantly higher than at time 0. At 970 min the concentrations of estrogens in bile were not significantly different from those at time 0. -
DISCUSSION Placement of E 2 in the rectum of prepubertal gilts resulted in a rise in the concentration of total estrogens in the hepatic portal vein within minutes. Moore et al. (6) reported that 14-C-estrogens were present in the hepatic portal vein one min after placing 14-CE 2 in the colon of gilts. In the present study, the AAUC for concentrations of total estrogens in the hepatic portal vein was 10563 pmol, indicating that there was extensive absorption of E 2 from the rectum during the 300 min sampling period. Concentrations of E 2 in the hepatic portal vein represented a very small proportion (3%) of the total estrogen detected in the hepatic portal vein during the sampling period. Apparently, 97% of the E 2 absorbed from the rectum was metabolized to E 1, E2G, E1G, and EIS before entering the hepatic portal vein. Moore et al. (6) reported that nearly all of 14-C-E 2 absorbed from the colon of pigs was converted to conjugated E 2 and E 1 prior to entering the hepatic portal vein. We have reported that 94% of E 2 absorbed from the stomach of gilts was metabolized to E l, E2G, EIG, and E~S prior to entering the hepatic portal vein (7). A soluble estrogen glucuronyltransferase which conjugates E 2 and E 1 to glucuronide has been isolated from the mucosa of the small intestine of pigs (10). Incubation of E 2 and E 1 with intestinal microsomes yielded E2G and EIG. Enzymes that metabolize E 2 between the lumen of the gut to the hepatic portal vein are likely in the gut mucosa (11). In the current study, these enzymes converted 65% of E 2 to E 1 and conjugated 96% to glucuronide or sulfate. As noted after E 2 was placed in the stomach (7) and after E 2 was placed in the rectum, about 4% of estrogen in the hepatic portal vein was in an unconjugated form; E 2 or E r Placement of 14-C-E 2 in the colon of immature pigs has also led to a small rise in unconjugated estrogens in the hepatic portal vein (6). Apparently under these conditions the enzymes in the posterior gut do not metabolize all of the E 2 allowing passage of some unconjugated estrogen into the posterior vena cava. After E 2 w a s placed in the rectum, concentrations of E 2 and E 1 in the jugular vein rose and remained elevated for several hr. This is in contrast to the results of placing E 2 in the stomach of pigs in other studies (7, 12), where concentrations of unconjugated estrogen in the jugular vein did not rise. Bottoms et al. (13) and Moore et al. (6) also reported no rise in E 2 and E~ in the jugular vein in pigs administered E 2 in the upper gastrointestinal tract. In mammals, the hepatic portal vein carries all blood and components from the stomach to the liver where metabolism occurs prior to entering the peripheral circulation. In mammals, veins from the rectum go either to the hepatic portal vein or alternatively via the pudendal and iliac veins to the inferior vena cava. This latter route would permit unconjugated estrogen to appear in the peripheral circulation by bypassing the first pass effects of the liver. This may explain why concentrations of unconjugated estrogen rose in the jugular vein when E 2 was placed in the rectum, but not when placed in the stomach (6, 7, 12, 13). Endogenous estrogens enter the gut in the bile (14), are deconjugated or converted by gut bacteria (8, 15) and are present as free estrogens in the rectum (8).
390
RUOFF AND DZIUK
The absorption of fecal estrogens from the rectum may affect the concentrations of E 2 and E 1 in the peripheral circulation. This route may help explain the phenomenon of postpartum estrus in sows (16). The high concentration of estrogens in plasma of sows just before parturition could contribute to high concentrations of bile estrogens. These estrogens in turn could pass down the intestines during the 48-hr ingesta passage period either as conjugates that could be deconjugated in the rectum by the gut bacteria or deconjugated in the upper gut. The free estrogens could then be absorbed directly from the rectum as free estrogens, bypass the liver and influence behavior. The extent of deconjugation could be influenced by antibiotics in the diet (3, 7). Alternatively the conjugated estrogens could be deconjugated by lungs or brain and reenter the systemic circulation as free estrogens (17,18). In Experiment 2 the immediate rise in E2G and E1G in both the hepatic portal and jugular veins after infusion of bile into the duodenum was as reported previously (7). The detection of estrogens in the bile at concentrations more than 10X those before infusion demonstrates the extent of recirculation of bile estrogens. After induction of bile secretion with a cholagogue at 470 min, the concentrations of estrogens were again significantly higher in the jugular and hepatic portal veins than before infusion and in gilts at the same interval from infusion but not given a cholagogue. The rise in concentration of estrogens persisted for 210 min. This indicates that the original bile estrogens could recirculate at least twice. The series of steps in recirculation (19, 20) and the number of possible points in the series provide the potential for influencing the extent of recycling by many different means. Dietary components and nature of the ingesta, xenobiotics and antibiotics each have the potential for influencing the rate and route of estrogen metabolism and excretion. Because of estrogen's ubiquitous effects on reproduction and gonadotropin secretion, it is important to consider these aspects when evaluating effects of treatments that seem unrelated to 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. #A109, Cortland, NY 13045. 3312 Animal Sciences Lab, 1207 W. Gregory Dr., Urbana, 1L 61801. 4Correspondence and reprints
REFERENCES 1. Sandberg AA, Slaunwhite WR. Studies on phenolic steroids in human subjects. II. The metabolic fate and hepato-biliary-enteric circulation of 14-C-estrone and 14-C-estradiol in women. J Clin Endocrinol 18:1266-1278, 1957. 2. Sandberg AA, Kirdani RY, Back N, Weyman P, Slaunwhite WR. Biliary excretion and enterohepatic circulation of estrone and estriol in rodents. Am J Physiology 213:1138-1142, 1967. 3. Adlercreutz H, Martin F, Lehtinen T, Tikkanen MJ, Pulkkinen MO. Effect of ampicillin administration on plasma conjugated and unconjugated estrogen and progesterone levels in pregnancy. Am J Obstet Gynecol 128:266-271, 1977. 4. Goldin BR, Adlercreutz H, Dwyer JT, Swenson L, Warram JH, Gorbach SL. Effect of diet on excretion of estrogens in pre- and postmenopausal women. Cancer Res 41:3771-3772, 1981. 5. Rose DP, Boyar AP, Cohen C, Strong LE. Effect of a low-fat diet on hormone levels in women with cystic breast disease. I. Serum steroids and gonadotropins. J Nat Cancer Inst 78:623- 626, 1987. 6. Moore AB, Bottoms GD, Coppic GL, Pohland RC, Roesel OF. Metabolism of estrogens in the gastrointestinal tract of swine. I. Instilled estradiol. J Anita Sci 55:124--134, 1982. 7. Ruoff WL, Dziuk PJ. Absorption and metabolism of estrogens from the stomach and duodenum of pigs. Domest Anita Endocrinol 11:197-208, 1994. 8. Adlercreutz H, Martin F, Pulkkinen M, Deneker H, Rimer U, Sjoberg N-O, Tikkanen MJ. Intestinal metabolism of estrogens. J Clin Endocrinol Metab 43:497-505, 1976. 9. SAS Institute Inc. SAS User's Guide; Statistics, Version 5 Edition. Cary, NC:SAS Institute, Inc., 956 pp. 1985.
ABSORPTION AND CIRCULATION OF 17~-ESTRADIOL IN PIGS
391
10. Rao GS, Breuer H. Partial purification and kinetic properties of a soluble estrogen glucuronyltransferase from pig intestine. J Biol Chem 20:5521-5527, 1969. 11. Back DJ, Breckenridge AM, Crawford FE, Maclver M, Orme MLE, Rowe PH. Interindividual variation and drug interactions with hormonal steroid contraceptives. Drugs 21:46-61, 1981. 12. Dziuk PJ, Elsaesser F, Eilendorff F. Models and results of studies on levels of sex steroids in the hepatic portal and jugular veins of the conscious pig. The Endocrines and the Liver. Academic Press, New York. p. 413-414, 1982. 13. Bottoms GD, Coppoc GL, Monk E, Moore AB, Roesel OF, Regnier FE. Metabolic fate of orally administered estradiol in swine. J Anim Sci 46:674--685, 1977. 14. Adlercreutz, H, Ervast HS, Tenhunen A, Tikkanen. Gas chromatographic and mass spectrometric studies on oestrogens in bile: Part I. Pregnant women. Acta Endocrinol 73:.543- 554, 1973. 15. Lombardi P, Goldin B, Boutin E, Gorbach SL. Metabolism of androgens and estrogens by human fecal microorganisms. J Steroid Biochem 9:795-802, 1978. 16. Holness DH, Hunter RHF. Post-partum oestrus in the sow in relation to the concentration of plasma oestrogens. J Reprod Fertil 45:15-20, 1975. 17. Collins DC, Balikian HPM, Preedy JRK. Hydrolysis of estrogen conjugates by the lungs of the dog in vivo. Endocrinology 96:1543-1551, 1975. 18. Collins DC, Balikian HM, Preedy .IRK. Evidence for the simultaneous uptake and release of certain estrogens by the splanchnic area in the dog. Endocrinology 99:420-428, 1976. 19. Rao GS, Rao ML, Haueter G, Breuer H. Steroidglucuronyltransferases, V. Formation and hydrolysis of oestrogen glucuronides by the liver, kidney and intestine of the pig. Hoppe- Seyler's Physiol Chem 355:881-890, 1974. 20. Schumann W, Hillesheim HG, Gina G. Model systems for pharmokinetics of steroid drugs subject to enterohepatic circulation. Exp Clin Endocrinol 87:118-124, 1986.