High-performance liquid chromatography assay for 3 beta-hydroxy-delta 5-C27-steroid oxidoreductase activity in rat liver microsomes.

A quantitative HPLC method has been developed for measuring 3 beta-hydroxy-delta 5-C27-steroid oxidoreductase activity in rat liver microsomes. Apparent Km values of 23 and 27 microM were estimated for 5-cholestene-3 beta,7 alpha-diol and NAD+, respectively. A temperature optimum of 45 degrees C was estimated using standard assay conditions. It was observed that feeding cholesterol (2%) or cholestyramine (5%) to rats increased (twofold) specific activity. This method should prove useful in studies of the regulation of bile acid synthesis and complement the HPLC assay technique recently developed for measuring cholesterol 7 alpha-hydroxylase activity.