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Drug Metabolism and Disposition Fast Forward
First published on June 1, 2009; DOI: 10.1124/dmd.109.027763

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Received for publication April 1, 2009.
Revised May 19, 2009.
Accepted for publication May 27, 2009.

3-Ketocholanoic Acid Is The Major In Vitro Human Hepatic Microsomal Metabolite of Lithocholic Acid

Anand K Deo 1 Stelvio M Bandiera 2*

1 Universitry of British Columbia 2 University of British Columbia

* Address correspondence to: E-mail: bandiera{at}interchange.ubc.ca

Abstract

Lithocholic (3{alpha}-hydroxy-5{beta}-cholan-24-oic) acid is a relatively minor component of hepatic bile acids in humans but is highly cytotoxic. Hepatic microsomal oxidation offers a potential mechanism for effective detoxification and elimination of bile acids. The aim of the present study was to investigate the biotransformation of lithocholic acid by human hepatic microsomes and to assess the contribution of cytochrome P450 (P450) enzymes in human hepatic microsomes, using human recombinant P450 enzymes and chemical inhibitors. Metabolites were identified and metabolite formation was quantified using a liquid chromatography/mass spectrometry (LC/MS)-based assay. Incubation of lithocholic acid with human liver microsomes resulted in the formation of five metabolites, which are listed in order of their rates of formation, 3-oxo-5{beta}-cholan-24-oic (3-ketocholanoic) acid, 3{alpha},6{alpha}-dihydroxy-5{beta}-cholan-24-oic (hyodeoxycholic) acid, 3{alpha},7{beta}-dihydroxy-5{beta}-cholan-24-oic (ursodeoxycholic) acid, 3{alpha},6{beta}-dihydroxy-5b-cholan-24-oic (murideoxycholic acid) and 3{alpha},hydroxy-6-oxo-5{beta}-cholan-24-oic (6-ketolithocholic) acid. 3-Ketocholanoic acid was the major metabolite exhibiting apparent Km and Vmax values of 22 mM and 336 pmol/min/mg protein, respectively. Incubation of lithocholic acid with a panel of human recombinant P450 enzymes revealed that all five metabolites were formed by recombinant CYP3A4. Chemical inhibition studies with human liver microsomes and recombinant P450 enzymes confirmed that CYP3A4 was the predominant enzyme involved in hepatic microsomal biotransformation of lithocholic acid. In summary, the results indicate that oxidation of the third carbon of the cholestane ring is the preferred position of oxidation by P450 enzymes for lithocholic acid biotransformation in humans and suggest that formation of lithocholic acid metabolites leads to enhanced hepatic detoxification and elimination.


Key words: bile acid metabolism, CYP3A, cytochrome P450, cytochrome P450 function, human CYP enzymes, mass spectrometry




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