Determination of aromatization of 19-oxygenated 16 alpha-hydroxyandrostenedione with human placental microsomes by high-performance liquid chromatography coupled with coulometric detection.

A sensitive assay of aromatization of 16 alpha-hydroxylated androgens, 16 alpha-hydroxyandrostenedione (16 alpha-OHA), 16 alpha,19-dihydroxyandrostenedione [16 alpha,19-(OH)2A], and 16 alpha-hydroxy-19-oxo androstenedione (16 alpha-OH-19-oxo A), was developed using reversed phase high-performance liquid chromatography with a coulometric detector. The estrogens, estriol and 16 alpha-hydroxyestrone, were simultaneously detected in quantities as low as 300 pg of the estrogens formed in an assay by an internal standard method. Apparent Km and Vmax of the microsomal aromatase for 16 alpha-OHA, 16 alpha,19-(OH)2A or 16 alpha-OH-19-oxo A were 1.06, 4.00 or 571 microM and 0.014, 0.087 or 1.67 pmol/min/micrograms protein, respectively. The results show that the 19-oxo steroid has extremely low affinity for aromatase relative to the other substrates.