Abstract
The hydroxylation of lithocholic acid (3 alpha-hydroxy-5 beta-cholanoic acid) by adult male Sprague-Dawley rat liver microsomes supplemented with NADPH was studied. Metabolites were separated by a combination of thin-layer chromatography and high pressure liquid chromatography, both with and without prior methylation and acetylation of the samples. The resulting products were characterized by thin-layer, gas-liquid, and high pressure liquid chromatography by comparison with authentic bile acid standards; final structure determination was by proton nuclear magnetic resonance spectroscopy and by mass spectrometry. The following reaction products were found: 3 alpha, 6 beta-dihydroxy-5 beta-cholanoic acid (80% of total metabolites) and 3 alpha, 6 alpha-dihydroxy-5 beta-cholanoic, 3 alpha, 7 alpha-dihydroxy-5 beta-cholanoic, 3 alpha, 6 beta,7 beta-trihydroxy-5 beta-cholanoic, and 3 alpha-hydroxy-6-oxo-5 beta-cholanoic acids (less than or equal to 5% each). In addition, one unidentified trihydroxylic bile acid and several minor compounds were present. It is concluded that four different hydroxylation reactions of lithocholic acid, namely the predominant 6 beta as well as the minor 6 alpha, 7 alpha, and 7 beta hydroxylations, are catalyzed by rat hepatic microsomes; 7 beta-hydroxylation may occur only with dihydroxylated bile acids but not with lithocholate itself. The presence of the 6-oxo bile acid can be explained either by direct oxidation of a hydroxyl group by cytochrome P-450, or by the action of microsomal dehydrogenase(s) which could also catalyze the epimerization of hydroxyl groups via their oxidation. The results form the basis of a proposed scheme of the oxidative metabolism of lithocholic acid in rat liver microsomes.
Highlights
The hydroxylation of lithocholic acid (3a-hydroxy5&cholanoic acid) by adult male Sprague-Dawley rat liver microsomes supplemented with NADPH was studied
The in vitro incubation of lithocholic acid with rat liver microsomes in the presence of NADPH leads to the formation of a large number of metabolites
Since our main interest was in the hydroxylation reaction, we focused our attention on compounds more polar than the starting material, operationally defined by an Rf lower than that of lithocholic acid in thin-layer chromatography
Summary
The hydroxylation of lithocholic acid (3a-hydroxy5&cholanoic acid) by adult male Sprague-Dawley rat liver microsomes supplemented with NADPH was studied. The resulting products were characterized by thin-layer, gas-liquid, and high pressure liquid chromatography by comparison with authentic bile acid standards; final structure determination was by proton nuclear magnetic resonance spectroscopyand by mass spectrometry. IIt i s concluded that four different hydroxylation reactions of lithocholicacid, namely the predominant 6p as well as the minor 6a,7a,and 78 hydroxylations, are catalyzed by rat hepatic microsomes; 7fl-hydroxylation may occur only with dihydroxylated bile acids but not with lithocholate itself. The results form the basis of a proposed scheme of the oxidative metabolism of lithocholic acid in rat liver micmomes.-Zimniak, P., E. Elucidation of the pathways of oxidative metabolism in rat liver microsomes. J L i ) n d h . 1989. 3 0 907-918
Published Version
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