Effect of ethynylestradiol on biliary excretion of bile acids, phosphatidylcolines, and cholesterol in the bile fistula rat1

The effects of ethynylestradiol on endogenous bile acids, their capacity to conjugate and excrete intravenously infused cholic acid, the concentrations of biliary cholesterol and lecithin, and the individual molecular species of phosphatidylcholine have been determined in male and female Sprague-Dawley rats. Endogenous biliary bile acids were analyzed by gas-liquid chromatographymass spectrometry. Eleven bile acids were identified and several minor bile acids, primarily muricholates, could not be completely characterized. After 5 days of treatment with ethynylestradiol (1 mg/kg per day), the percentage of cholic acid decreased and the percentage of GB-hydroxylated bile acids, including several monounsaturated species, increased. Ethynylestradiol caused a decrease in bile acidindependent bile flow. Intravenous infusion of cholic acid at a high concentration caused cholestasis in control animals but, after ethynylestradiol treatment, cholestasis developed during the infusion of a much lower concentration of cholate, indicating a lowered threshhold for bile acid-induced cholestasis. In the treated rats, there was a slight increase in excretion of unconjugated endogenous bile acids, and a striking impairment of conjugation of intravenously administered cholic acid. One of the few sex-related differences observed was an increased concentration of biliary phospholipids in untreated male rats. Both phospholipid and cholesterol concentrations in the bile were higher in the treated animals. The molar percentage of cholesterol was always 1-296, but it was slightly higher in treated animals, especially males. Ethynylestradiol treatment also affected biliary phospholipid by causing a marked increase of phosphatidylcholine species containing palmitic and oleic acid residues and a decrease of species containing stearic and linoleic acid residues. There was no increase in biliary excretion of long chain polyunsaturated species, which might have indicated damage to membranes, in response to ethynylestradiol either alone or with cholic acid infusion. Some of these ethynylestradiol-induced changes in biliary bile acid and lipid excretion are probably peculiar to the rat, but others, such as the increase in molar percentage of cholesterol and cholestasis, may be relevant to disorders in man, especially cholesterol gallstones and idiopathic cholestasis of pregnancy. Supplementary key words gas-liquid chromatography-mass spectrometry . biliary cholesterol * bile acid conjugation * [24-'4C]cholic acid infusion * cholestasis . bile flow Many investigators have studied the hepatic effects of natural and synthetic estrogens, especially ethynylestradiol, a common estrogenic component of contraceptive steroids. In the rat, estrogens increase liver size and water content, RNA, DNA, and protein content. They decrease the bile salt-independent fraction of bile flow and the maximal transport capacity for bile acids, bilirubin, and bromosulfophthalein (BSP) (1 -6). Ethynylestradiol reduces hepatic cytochrome P-450 activity (7) and inhibits microsomal hydroxylation of steroids in vitro (8). It is not known, however, to what extent it affects hydroxylation and conjugation reactions involved in bile acid formation in vivo and whether changes in bile acid metabolism, especially conjugation, are related to the cholestatic effect of ethynylestradiol. In this study, we examined the effect of ethynylestradiol on bile acid metabolism in vivo by analyzing bile acids in bile of both male and female rats given this drug. We also determined the capacity of these rats to conjugate and excrete cholic acid infused intravenously and related the results to the cholestatic effect of ethynylestradiol. Ethynylestradiol, other estrogens, and contraceptive steroid preparations are also known to alter the composition of biliary lipids in several animal species, including man, so that the bile becomes more saturated with cholesterol (911). This is consistent with the clinically important observation that cholesterol gallstones occur more frequently in women during the childbearing age than in men (12, 13) and that contraceptive steroids cause an increase in ' Published in part in Advances in Bile Acid Research, 1975. S. Matern, J. Hackenschmidt, P. Back, and W. Gerok, editors. F. K. Schattauer Verlag, Stuttgart, Germany, and in abstract form in Gastroenterology 67: A-251802, 1974. * Permanent address: University of Colorado Medical Center, 4200 East Ninth Avenue, Denver, CO 80220. Abbreviations: GLC, gas-liquid chromatography; MS, mass spectrometry; EE, ethynylestradiol, TMS, trimethylsilyl. Journal of Lipid Research Volume 18, 1977 623 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 3, 2013 w w w .j.org D ow nladed fom the incidence of gallstones (14, 15). The effect of ethynylestradiol on biliary lipid composition in the rat has not been carefully studied. In the experiments reported here we measured the concentrations of cholesterol and lecithin in the bile of treated rats. The nature of the cellular effects of ethynylestradiol on the liver is unknown. It is possible that this synthetic estrogen damages cell membranes either directly or indirectly, even though such damage is not seen by electron microscopy (8). Injury to membranes might result in the biliary excretion of phosphatidylcholine species with long chain polyunsaturated fatty acids, derived from membranes. The individual molecular species of biliary phosphatidylcholine were, therefore, determined. These studies provide a fairly comprehensive understanding of the effects of a large dose of ethynylestradiol on bile acids and biliary lipids in the rat. MATERIALS AND METHODS