Gas Chromatography-Mass Spectrometric Determination of Activity of Human Placental Aromatase Using 16α-Hydroxyandrostenedione as a Substrate

Abstract

Aromatization of 16α-hydroxyandrostenedione (16α-OH AD) with aromatase in human placental microsomes was studied by gas chromatography-mass spectrometry (GC-MS) using [2,4,6,6,9α, 16β, 17α-²H₇]estriol as an internal standard. 16α-OH AD was incubated with the microsomes in the presence of NADPH in air. The metabolite was extracted with ethyl acetate and treated with NaBH₄. The reduced product, estriol, was isolated by Sep-Pak C₁₈ cartridge and then analyzed as the tris(trimethylsilyl)ether by a GC-MS (EI mode). The production of estriol was dependent upon protein concentration and incubation time. Apparent K_m and V_max values of the microsomal aromatase for 16α-OH AD were 568 nM and 25.5 pmol/min/mg protein, respectively. In this assay, aromatase activity, estriol formation, could be determined at a level as low as 1 pmol/min/mg protein. Aromatase inhibitors, 4-hydroxy- and 6-oxo-androstenediones, prevented the estriol formation in a competititve manner with 25 and 30 nM of apparent K_i values, respectively.