Biosynthesis of cholic acid in rat liver. 24 Hydroxylation of 3α,7α,12α trihydroxy 5β cholestanoic acid

Conversion of 3α, 7α, 12α trihydroxy 5β [7β ³H] cholestanoic acid into 3α, 7α, 12α, 24 tetrahydroxy 5β cholestanoic acid in rat liver was catalyzed either by the mitochondrial fraction fortified with the 100,000 x g supernatant fluid or the microsomal fraction fortified with 100,000 x g supernatant fluid and ATP. The microsomal system was more active than the mitochondrial system. With the microsomal system the rate of reaction was considerably faster with free 3α, 7α, 12α trihydroxy 5β cholestanoic acid as substrate than with the corresponding coenzyme A ester. Addition of coenzyme A inhibited the activity. Addition of cofactors other than ATP and coenzyme A did not markedly influence the reaction. The 100,000 x g supernatant fluid could be substituted with a protein fraction obtained by ammonium sulfate precipitation and Sephadex chromatography of the 100,000 x g supernatant fluid. The reaction was not catalyzed by a mixed function oxidase since there was no incorporation of ¹⁸O into the product when the reaction was performed in an atmosphere containing ¹⁸O₂. On the other hand, oxygen may be obligatory since there was almost complete inhibition when the reaction was performed in an atmosphere consisting of nitrogen. Carbon monoxide did not inhibit the reaction. One atom of deuterium was incorporated into the product when the reaction was performed in a medium containing deuterated water. It was concluded that microsomal 24 hydroxylation of 3α, 7α, 12α trihydroxy 5β cholestanoic acid involves the combined action of a desaturase and a hydratase. The reaction catalyzed by the hydratase appears to be stereospecific since the 24α epimer of 3α, 7α, 12α trihydroxy 5β cholestanoic acid was the predominant product. In contrast to the microsomal system, the mitochondrial system was not stimulated by the addition of ATP and was not inhibited by coenzyme A. The coenzyme A ester of 3α, 7α, 12α trihydroxy 5β cholestanoic acid was 24 hydroxylated more efficiently than the free acid.