Received for publication May 7, 2008.
Revised June 25, 2008.
Accepted for publication June 25, 2008.

IDENTIFICATION OF HUMAN HEPATIC CYTOCHROME P450 ENZYMES INVOLVED IN THE BIOTRANSFORMATION OF CHOLIC AND CHENODEOXYCHOLIC ACID

Abstract

Cholic and chenodeoxycholic acid are the predominant hepatic and biliary bile acids of most mammalian species including humans. Cholic and chenodeoxycholic acid are synthesized from cholesterol and accumulate in the liver during cholestasis. Biotransformation by hepatic cytochrome P450 (P450) enzymes represents a potentially effective pathway for elimination of these lipid soluble bile acids. We developed a liquid chromatography/mass spectrometry-based assay to identify and quantify the human hepatic microsomal metabolites of cholic acid and chenodeoxycholic acid, and using a panel of recombinant human P450 enzymes, we determined the P450 enzymes involved. Incubation of cholic acid with human hepatic microsomes and NADPH produced a single metabolite, 3-dehydrocholic acid. Of the recombinant CYP enzymes tested, only CYP3A4 catalyzed 3-dehydrocholic acid formation. Similar experiments with chenodeoxycholic acid revealed the formation of 7-hydroxy-3-oxo-5-cholan-24-oic acid and -muricholic acid as major metabolites and 7-ketolithocholic acid and cholic acid as minor metabolites. Among the recombinant human P450 enzymes examined, CYP3A4 exhibited the highest rates of formation for 7-hydroxy-3-oxo-5-cholan-24-oic acid and -muricholic acid from chenodeoxycholic acid. Formation of 7-ketolithocholic acid and cholic acid from chenodeoxycholic acid has not been reported previously and could not be attributed to any of the recombinant P450 enzymes tested. In conclusion, the predominant pathway for the biotransformation of both cholic and chenodeoxycholic acids in human hepatic microsomes was oxidation at the third carbon of the cholestane ring. This study highlights a major role for CYP3A4 and suggests a possible route for the elimination of these two bile acids.

Key words: bile acid metabolism, CYP3A, cytochrome P450, cytochrome P450 catalyzed oxidations, cytochrome P450 function, enzyme kinetics, hepatobiliary disposition, human CYP enzymes, liver microsomes, mass spectrometry