Abstract
3α-Hydroxy-5β-cholan-24-oic (lithocholic) 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-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-5β-cholan-24-oic (3-ketocholanoic) acid, 3α,6α-dihydroxy-5β-cholan-24-oic (hyodeoxycholic) acid, 3α,7β-dihydroxy-5β-cholan-24-oic (ursodeoxycholic) acid, 3α,6β-dihydroxy-5β-cholan-24-oic (murideoxycholic) acid, and 3α-hydroxy-6-oxo-5β-cholan-24-oic (6-ketolithocholic) acid. 3-Ketocholanoic acid was the major metabolite, exhibiting apparent Km and Vmax values of 22 μM and 336 pmol/min/mg protein, respectively. Incubation of lithocholic acid with a 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.
Footnotes
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This work was supported by the Canadian Institutes of Health Research [MOP-81174]. Partial support was provided by a training grant from Merck Research Laboratories (Merck & Co., Whitehouse Station, NJ) (to A.K.D.).
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.109.027763.
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ABBREVIATIONS: VDR, vitamin D receptor; PXR, pregnane X receptor; lithocholic, 3α-hydroxy-5β-cholan-24-oic; P450, cytochrome P450; hyodeoxycholic, 3α,6α-dihydroxy-5β-cholan-24-oic; chenodeoxycholic, 3α,7α-dihydroxy-5β-cholan-24-oic; murideoxycholic, 3α,6β-dihydroxy-5β-cholan-24-oic; 3-ketocholanoic, 3-oxo-5β-cholan-24-oic; cholic, 3α,7α,12α-trihydroxy-5β-cholan-24-oic; LC/MS, liquid chromatography/mass spectrometry; SKF 525A, 2′-diethylaminoethyl 2,2-diphenylpentanoate hydrochloride; α-muricholic, 3α,6β,7α-trihydroxy-5β-cholan-24-oic; β-muricholic, 3α,6β,7β-trihydroxy-5β-cholan-24-oic; γ-muricholic, 3α,6α,7α-trihydroxy-5β-cholan-24-oic; 3-dehydrocholic, 7α,12α-dihydroxy-3-oxo-5β-cholan-24-oic; ursodeoxycholic, 3α,7β-dihydroxy-5β-cholan-24-oic; deoxycholic, 3α,12α-dihydroxy-5β-cholan-24-oic; 6-ketolithocholic, 3α-hydroxy-6-oxo-5β-cholan-24-oic; 7-ketolithocholic, 3α-hydroxy-7-oxo-5β-cholan-24-oic; isolithocholic, 3β-hydroxy-5β-cholan-24-oic.
- Accepted May 27, 2009.
- Received March 31, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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