Stereospecific side chain hydroxylations in the biosynthesis of chenodeoxycholic acid.

This paper describes stereospecific side chain hydroxylations in the biosynthesis of chenodeoxycholic acid by liver mitochondria and microsomes. The hydroxylation of 5/3-cholestane-3a,7a-diol was studied in subcellular fractions of rat, guinea pig, and rabbit livers. Incubation of SD-cholestane-3a,7n-diol resulted in hydroxylations at C-12, C-24, C-25, and C-26. The biosynthetic bile alcohols were analyzed by thin layer and gas-liquid chromatography and by mass spectrometry, and optimal assay conditions for the various hydroxylations were tentatively established. With the mitochondrial fraction, 26-hydroxylation predominated and accounted for as high as 60 to 87% of the total hydroxylations. This reaction was stereospecific in that, of the two diastereomers of 5P-cholestane-3a,7c,26-triol, the rate of formation of the 25R isomer was 4 to 8 times higher than that of the 25s compound in all three species. In contrast, in the microsomal fraction, rates of side chain hydroxylations varied widely among the three species but occurred mainly at C-25 forming 5flcholestane-3a,7a,25-triol. There was considerable 12ahydroxylase activity in rat and rabbit, preparations. The microsomes also produced smaller amounts of both diastereomers of 5fl-cholestane-3a,7a,26-triol in ratios of about 1:2 (25R:25S) in rat and guinea pig and about 2:l in rabbit. These findings suggest that in the biosynthesis of chenodeoxycholic acid, the initial step in the side chain oxidation is either the formation of the 25R isomer of SP-cholestane-3cu,7a,26-triol, catalyzed by the mitochondria, or the synthesis of 5/3-cholestane-3a,7a,25-trio1 mediated by the microsomes. On the basis of these results it is not possible to draw firm conclusions concerning the relative importance of the microsomal 25-hydroxylase or the mitochondrial26-hydroxylase in chenodeoxycholic acid synthesis.