Biosynthesis of chenodeoxycholic acid: side-chain hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol by subcellular fractions of guinea pig liver.

Abstract

Side-chain hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha diol was studied in subcellular fractions of guinea pig liver. The purity of the microsomal and the mitochrondrial fractions was determined with marker enzymes, and relatively little cross contamination between the particulate fractions was detected. Methods for the analysis of the incubation mixture by thin-layer chromatography and gas-chromatography-mass spectrometry were developed. Optimal assay conditons were established for the major hydroxylation reactions, namely the mitochondrial 26-hydroxylation and the microsomal 25-hydroxylation, It was found that the most active side-chain hydroxylation in the guinea pig was the microsomal 25-hydroxylation. The mitochondrial omega-hydroxylation was stereospecific, in that the rate of formation of (25R)-5 beta-cholestane-3 alpha, 7 alpha, 26-triol was 8 times greater than that of the 25S isomer. The microsomal "25" hydroxylation was not stereospecific under the conditions employed. It is concluded that the mitochondrial "26" hydroxylation (leading to the formation of (25R)-5 beta-cholestane-3 alpha, 7 alpha, 26-triol) plays an important role in the biosynthesis of chenodeoxycholic acid. The participation of microsomal 25-hydroxylation in the formation of chenodeoxycholic acids requires further investigation.