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Hepatic microsomal bile acid biotransformation : identification of metabolites and cytochrome p450 enzymes involved

University of British Columbia

Bile acids are end-products of cholesterol metabolism and essential for absorption of dietary lipids in the body. Impaired bile flow leads to hepatic bile acid accumulation and liver damage. Hepatic microsomal oxidation offers a potential mechanism for efficient elimination of bile acids. The present study investigated the cytochrome P450 (P450)-mediated hepatic microsomal biotransformation profiles of lithocholic acid, cholic acid and chenodeoxycholic acid using a liquid chromatography-mass spectrometry (LCIMS) based assay. Incubation of lithocholic acid with rat hepatic microsomes resulted in the formation of a major 6β-hydroxylated metabolite, murideoxycholic acid, followed by isolithocholic acid and 3-ketocholanoic acid. Ursodeoxycholic acid, hyodeoxycholic acid and 6-ketolithocholic acid were identified as minor metabolites. Studies using P450-specific antibodies, chemical inducers, and rat recombinant enzymes showed that formation of murideoxycholic acid and 3-ketocholanoic acid were mediated by CYP3A2 and CYP2C 11. Similar metabolite profiles were obtained by incubation of lithocholic acid with mouse hepatic microsomes generating murideoxycholic acid as the major metabolite. Studies using P450 inducers and chemical inhibitors suggested the involvement of murine CYP3A in murideoxycholic acid and 3-ketocholanoic acid formation, and CYP1A, CYP2B and CYP3A enzymes in ursodeoxycholic acid, hyodeoxycholic acid and 6-ketolithocholic acid formation by mouse liver microsomes. Biotransformation of lithocholic acid by human hepatic microsomes generated 3-ketocholanoic acid as the major metabolite, and hyodeoxycholic acid, ursodeoxycholic acid, 6-ketolithocholic acid and murideoxycholic acid, as minor metabolites. Studies with chemical inhibitors and human recombinant enzymes demonstrated that CYP3A4 catalyzed the formation of all five metabolites. The biotransformation of cholic acid and chenodeoxycholic acid by human hepatic microsomes revealed the formation of a single cholic acid metabolite, 3-dehydrocholic acid. Chenodeoxycholic acid biotransformation generated 7α-hydroxy-3 -oxo-5β-cholan-24-oic acid as the major metabolite followed by у-muricholic acid, 7-ketolithocholic acid and cholic acid, respectively. CYP3A4 was found to be the major enzyme involved in the biotransformation of cholic acid and chenodeoxycholic acid in human liver microsomes. A comparison of metabolite profiles demonstrated the dominant role of human CYP3A4 in the oxidation of bile acids at the C-3 position. In contrast, 6β-hydroxylation catalyzed by multiple P450 (CYP1A, CYP2B, CYP2C and CYP3A) enzymes was the preferred biotransformation pathway in rodent liver microsomes.

Thesis/Dissertation

application/pdf

2009-11-18

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/15222

Pharmaceutical Sciences

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/