Biotransformation of lithocholic acid by rat hepatic microsomes: metabolite analysis by liquid chromatography/mass spectrometry.
Lithocholic acid is a lipid-soluble hepatotoxic bile acid that accumulates in the liver during cholestasis. A potential detoxification pathway for lithocholic acid involves hydroxylation by hepatic cytochrome P450 (P450) enzymes. The purpose of the present study was to identify the hepatic microsomal metabolites of lithocholic acid by liquid chromatography/mass spectrometry and to determine the P450 enzymes involved. Incubation of lithocholic acid with rat hepatic microsomes and NADPH produced murideoxycholic acid (MDCA), isolithocholic acid (ILCA), and 3-keto-5beta-cholanic acid (3KCA) as major metabolites and 6-ketolithocholic acid and ursodeoxycholic acid as minor metabolites. Experiments with hepatic microsomes prepared from rats pretreated with P450 inducers and with inhibitory antibodies indicated that CYP2C and CYP3A enzymes contribute to microsomal MDCA formation. Results obtained with a panel of recombinant P450 enzymes and CYP2D6 antiserum showed that CYP2D1 can also catalyze MDCA formation. Similar experimental evidence revealed that formation of 3KCA was mediated primarily by CYP3A enzymes. ILCA formation appeared to be catalyzed by a distinct pathway mediated largely by microsomal non-P450 enzymes. Based on the results obtained using lithocholic acid and 3KCA as substrates, a mechanism for the formation of ILCA involving a geminal diol intermediate is outlined. In conclusion, lithocholic acid was extensively metabolized by multiple P450 enzymes with the predominant biotransformation pathway being hydroxylation at the 6beta-position. This study provides an insight into possible routes of detoxification of lithocholic acid.
Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver, BC, Canada V6T 1Z3.
Anand K
AK
Deo
Stelvio M
SM
Bandiera
D000818:Animals; D001711:Biotransformation; D002853:Chromatography, Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005260:Female; D008095:Lithocholic Acid / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000378:metabolism*; D051381:Rats; D020318:Rats, Long-Evans; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
D065607:Cytochrome P-450 Enzyme Inhibitors; D011994:Recombinant Proteins; D008095:Lithocholic Acid; D003577:Cytochrome P-450 Enzyme System