Gas chromatography-mass spectrometric determination of activity of human placental aromatase using 16 alpha-hydroxyandrostenedione as a substrate

Aromatization of 16 alpha -chydroxyandrostenedione (16 alpha -N AD) with ar omatase in human placental microsomes was studied by gas chromatography-mas s spectrometry (GC-MS) using [2,4,6,6,9 alpha ,16 beta ,17 alpha (2)-H-7]es triol as an internal standard. 16 alpha -OH AD was incubated with the micro somes in the presence of NADPH in air. The metabolite was extracted with et hyl acetate and treated with NaBH4. The reduced product, estriol, was isola ted by Sep-Pak C-18 cartridge and then analyzed as the tris(trimethylsilyl) ether by a GC-RIS (EI mode). The production of estriol was dependent upon p rotein concentration and incubation time. Apparent K-m and V-max values of the microsomal aromatase for 16 alpha -OH AD were 568 nl and 25.5 pmol/min/ mg protein, respectively: In this assay, aromatase activity, estriol format ion, could be determined at a level as low as 1 pmol/min/mg protein. Aromat ase inhibitors, 4-hydroxy- and 6-oxo-androstenediones, prevented the estrio l formation in a competitive manner,vith 25 and 30 nM of apparent K-i value s, respectively.