Metabolism of lithocholic acid in the rat: formation of lithocholic acid 3-0-glucuronide in vivo

Milligram amounts of [3P-3H]lithocholic (3a-hydroxy5P-cholanoic) acid were administered by intravenous infusion to rats prepared with a biliary fistula. Analysis of sequential bile samples by thin-layer chromatography (TLC) demonstrated that lithocholic acid glucuronide was present in bile throughout the course of the experiments and that its secretion rate paralleled that of total isotope secretion. Initial confirmation of the identity of this metabolite was obtained by the recovery of labeled lithocholic acid after P-glucuronidase hydrolysis of bile samples. For detailed analysis of biliary metabolites of [ 3H]lithocholic acid, pooled bile samples from infused rats were subjected to reversed-phase chromatography and four major labeled peaks were isolated. After complete deconjugation, the two major compounds in the combined first two peaks were identified as murideoxycholic (3a,6@-dihydroxy-5P-cholanoic) and P-muricholic (3a,6~,7@-trihydroxy-5~-cholanoic) acids and the third peak was identified as taurolithocholic acid. The major component of the fourth peak, after isolation, derivatization (to the methyl ester acetate), and purification by high pressure liquid chromatography (HPLC), was positively identified by proton nuclear magnetic resonance as lithocholic acid 3a-O-(P-D-glucuronide). These studies have shown, for the first time, that lithocholic acid glucuronide is a product of in vivo hepatic metabolism of lithocholic acid in the rat. -Little, J. M., P. Zimniak, K. E. Shattuck, R. Lester, and A. Radominska. Metabolism of lithocholic acid in the rat: formation of lithocholic acid 3-0glucuronide in vivo. J. Lipid Res. 1990. 31: 615-622. Supplementary key words bile acid cholestasis glucuronide Lithocholic acid is a hydrophobic, toxic compound that has been shown to cause cholestasis in experimental animals (1-4). One result of cholestasis is a dramatic increase in the concentration of bile acids, including lithocholic acid, in serum and liver. The deleterious effects of this accumulation of lithocholic acid (and other bile acids) can be attenuated by two detoxification pathways available in the liver: oxidative reactions, mainly hydroxylations, and conjugation with sulfuric or glucuronic acids. The relative importance of these two pathways appears to be strongly species-specific. In the human, the 3a-hydroxyl group of lithocholic acid is efficiently sulfated (5-9), but not glucuronidated (10). Alternatively, lithocholic acid can undergo hydroxylation in position 6or (11-13); the newly formed 6a-hydroxylated bile acids are rapidly conjugated with glucuronic acid in position 6 (10, 14, 15) and are efficiently excreted in urine (16). The conspicuous lack of the formation of lithocholic acid glucuronide is understandable in light of the somewhat unexpected finding that, at least in the rat, the compound is not less, but more toxic (cholestatic) than lithocholic acid itself (17). The metabolism of lithocholic acid in the rat presents a more complex picture. In contrast to the human, formation of the 3-sulfate is a minor reaction (18-20). Lithocholic acid is known to be efficiently hydroxylated by the rat, both in vivo (19, 21) and in vitro by liver microsomes (22-24). Multiple hydroxylation reactions of various specificities occur, with 6P-hydroxylation predominating (24). The 60-hydroxylation of lithocholic acid yields murideoxycholic acid (3q6P-diOH) which by further hydroxylation can be converted to the muricholates (amuricholic acid, 3a,GP,7a-triOH, and P-muricholic acid, Sa,6/3,7/3-triOH), normal constituents of rat bile which comprise approximately 20% of the bile acid pool (25). Therefore, 6P-hydroxylation in the rat not only detoxifies lithocholic acid but is also a salvage pathway that returns the additionally hydroxylated products to the bile acid The scheme of lithocholic acid metabolism in rat liver described above does not incorporate glucuronidation reactions. Indeed, the formation of bile acid glucuronides after administration of lithocholic acid to the rat has not been reported previously. However, studies from our and other laboratories have established that rat liver microsomes have a high capacity for the formation of lithocholic acid 3-0-glucuronide (26, 27), as well as glucuronides of pool. Abbreviations: TLC, thin-layer chromatography; HPLC, high pressure liquid chromatography; NMR, nuclear magnetic resonance. 'To whom correspondence should be addressed at: Division of Gastroenterology, University of Arkansas for Medical Science, 4301 West Markham, Slot 567, Little Rock, AR 72205. Journal of Lipid Research Volume 31, 1990 615 by gest, on S etem er 9, 2017 w w w .j.org D ow nladed fom the 6-hydroxylated bile acids that are the products of microsomal hydroxylation of lithocholic acid (24, 28). Other bile acids that are substrates of UDP-glucuronosyltransferases in vitro, when administered intravenously to rats, have been shown to be secreted in bile as glucuronide conjugates (29, 30). Faced with this apparent contradiction, we considered the possibility that the UDP -glucuronosyltransferase activities seen by us in hepatic microsomes may be latent or otherwise downregulated in vivo. Alternatively, it was conceivable that bile acid glucuronides have not been found previously because methods of isolation and analysis were, in many cases, not devised for the identification of glucuronides. To examine these possibilities, we used methodology developed specifically for the purpose of analysis of bile acid glucuronides to evaluate the role of the glucuronidation reaction in the hepatic response to a lithocholic acid