Determination of estradiol 2- and 16 alpha-hydroxylase activities in rat liver microsomes using high-performance liquid chromatography.

We have developed a sensitive and nonradiometric assay of estradiol 2- and 16 alpha-hydroxylase activities using reverse-phase high-performance liquid chromatography with voltametric detector. The 2- and 16 alpha-hydroxylated estrogens produced by the incubation of estradiol with rat liver microsomes were initially separated into the catechol and phenolic fractions using a QAE-Sephadex A-25 borate column. The metabolites were detected in quantities as low as 0.5-1 ng using 3-methoxy-1,3,5(10)-estratriene-2,16 alpha,17 beta-triol or 4-hydroxyestrone 17-oxime as an internal standard. Apparent Km and Vmax of the 2- and 16 alpha-hydroxylases were 41.9 microM and 1.3 nmol/mg protein/min, and 82 microM and 480 pmol/mg protein/min, respectively.