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

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

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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 Km and Vmax 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 Ki values, respectively.

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