Biotransformation of Lithocholic Acid by Rat Hepatic Microsomes: Metabolite Analysis by Liquid Chromatography/Mass Spectrometry

Abstract

ABSTRACT: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-5␤-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 6␤-position. This study provides an insight into possible routes of detoxification of lithocholic acid.

Identification of Human Hepatic Cytochrome P450 Enzymes Involved in the Biotransformation of Cholic and Chenodeoxycholic Acid

“…A fixed amount (0.4 g) of murideoxycholic acid, which was the internal standard, was then added to each sample. Sample extraction, evaporation, and reconstitution in preparation for analysis by LC/MS were carried out as described previously (Deo and Bandiera, 2008). Reaction mixtures that were devoid of substrate, NADPH, or microsomes, as well as reaction mixtures that contained defined concentrations of authentic bile acid standards, were routinely included in each assay.…”

“…Formation of chenodeoxycholic acid and cholic acid metabolites was analyzed by LC/MS using a procedure described previously for lithocholic acid metabolites (Deo and Bandiera, 2008), with the following modifications. Chenodeoxycholic acid and cholic acid metabolites were detected using a Waters Acquity Ultra Performance Liquid Chromatograph System (UPLC, Waters, Milford, MA) consisting of a Binary Solvent Manager and Sample Manager and connected to a Waters Quattro Premier XE triple quadrupole mass spectrometer (Waters).…”

3-Ketocholanoic Acid Is the Major in Vitro Human Hepatic Microsomal Metabolite of Lithocholic Acid

“…Internal standard (cholic-2,2,4,4-d 4 acid, 0.4 g) was then added to each sample. Sample extraction, evaporation, and reconstitution in preparation for analysis by LC/MS were carried out as described previously (Deo and Bandiera, 2008a). Reaction mixtures that were devoid of substrate, NADPH, or microsomes, as well as reaction mixtures that contained defined concentrations of authentic bile acid standards, were routinely included in each assay.…”

“…Subsequently, a different metabolite, 3-oxo-5␤-cholan-24-oic (3-ketocholanoic) acid, was identified as the major product of lithocholic acid following incubation with human recombinant CYP3A4 (Bodin et al, 2005). Using rat liver microsomes, we showed that lithocholic acid was extensively metabolized by multiple P450 enzymes to six metabolites (Deo and Bandiera, 2008a). The predominant pathway involved 6␤-hydroxylation and produced murideoxycholic acid as the major metabolite.…”

“…The predominant pathway involved 6␤-hydroxylation and produced murideoxycholic acid as the major metabolite. Formation of hyodeoxycholic acid was a minor pathway (Deo and Bandiera, 2008a). Results from our recent investigation of 3␣,7␣,12␣-trihydroxy-5␤-cholan-24-oic (cholic) acid and chenodeoxycholic acid biotransformation by human liver microsomes (Deo and Bandiera, 2008b) suggest that neither 6␣-hydroxylation nor 6␤-hydroxylation would be the preferred P450-catalyzed oxidation pathway for lithocholic acid in human hepatic microsomes.…”