De novo biosynthesis of 6 beta-hydroperoxyandrostenedione in human placental microsomes

Fresh human placental microsomes were incubated at 37 degrees C with a homogeneous mixture of tritium labeled 4-androstene-3,17-dione and unlabeled 6 beta-hydroperoxy-androstenedione in the presence, or absence, of various inhibitors. In all cases, radioactive 6 beta-hydroperoxyandrostenedione could be trapped in amounts significantly different from carefully conducted controls. The radioactive purity of the hydroperoxide formed was established by repetitive HPLC purifications whereby a constant ratio of radioactive counts versus peak area was obtained. The amount of tritiated 6-bèta-hydroperoxyandrostenedione produced was dependent both on the incubation time and on the microsomal protein concentration used. It was also found that in the complete absence of air, the unlabeled hydroperoxide could act as oxygen donor, supporting the formation of radioactive, 6-oxygenated metabolites. These findings are fully consistent with the concept that 6-bèta-hydroperoxyandrostenedione acts as an endogenous precursor in the formation of the corresponding 6 beta -hydroxy and 6-oxo androstene metabolites in the human placenta; a fact not previously recognized.