Separation of stereoisomers of C27-bile acid CoA esters by liquid chromatography and its application to the study of the stereospecificities of D- and L-bifunctional proteins in bile acid biosynthesis

The separation and quantitation analysis of the stereoisomers of 3α,7α,12α,24-tetrahydroxy- and 3α,7α,24-trihydroxy-5β-cholestan-26-oyl CoAs (VA-CoA and DVA-CoA), known as one of the intermediates for bile acid biosynthesis, were achieved by liquid chromatography. The above stereoisomers of C27-bile acids were effectively separated by a linear gradient solvent system of methanol/acetonitrile/sodium acetate buffer (pH 5) and acetonitrile/sodium acetate buffer (pH 5) using a reversed phase C18 column. Application for the study of the stereochemistry of the products by enzymatic reaction related to the β-oxidation of bile acids in rat liver was also demonstrated. The products (VA-CoA and DVA-CoA) from the corresponding 3α,7α,12α-trihydroxy- and 3α,7α-dihydroxy-5β-cholest-24-en-26-oyl CoAs (24E-THCA-CoA and 24E-DHCA-CoA) from a catalytic reaction with rat liver peroxisomal D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase or enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase were quantitatively determined. The results indicated different stereospecificities of these enzymes which might prove useful for the evaluation of their role and function.