High-performance liquid chromatographic determination of aromatization of 16 alpha-hydroxylated androgens with human placental microsomes.

A sensitive nonradiometric assay of aromatization of 16 alpha-hydroxylated androgens, 16 alpha-hydroxy-4-androstene-3,17-dione (16 alpha-OHA), and 16 alpha-hydroxytestosterone (16 alpha-OHT), has been developed using reverse-phase high-performance liquid chromatography with voltametric detector. The estrogens produced by human placental microsomes, estriol (E3) and 16 alpha-hydroxyestrone (16 alpha-OHE1), were simultaneously detected in quantities as low as 1-2 ng using 3-methoxy-1,3,5(10)-estratriene-2, 16 alpha,17 beta-triol as an internal standard. E3 was the only estrogen detected from the incubate of 16 alpha-OHT with the microsomes and NADPH, while 16 alpha-OHA gave 16 alpha-OHE1 and E3 under the same conditions. Apparent Km and Vmax of the microsomal aromatase for 16 alpha-OHA and 16 alpha-OHT were 2.56 microM and 71.4 pmol/min/mg and 13.33 microM and 15.4 pmol/min/mg, respectively.