Detoxification of lithocholic acid. Elucidation of the pathways of oxidative metabolism in rat liver microsomes.
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.
Zimniak
P
P
Department of Medicine, University of Rochester School of Medicine, NY 14642.
Holsztynska
E J
EJ
Lester
R
R
Waxman
D J
DJ
Radominska
A
A
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D008401:Gas Chromatography-Mass Spectrometry; D006900:Hydroxylation; D008095:Lithocholic Acid / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D006899:Mixed Function Oxygenases; D009252:NADPH Dehydrogenase; D010084:Oxidation-Reduction; D051381:Rats; D013997:Time Factors
D008095:Lithocholic Acid; D006899:Mixed Function Oxygenases; D009252:NADPH Dehydrogenase