Preparation of [1, 2-3H, 4-14C] 16α-Hydroxyandrostenedione and Its Use for Radiometric Determination of Human Placental Aromatase Activity

[1, 2-3H, 4-14C] 16α-Hydroxyandrostenedione (4) (3H, 3.20 mCi/mmol; 3H/14C = 222) was synthesized from commercially available [1, 2-3H, 4-14C] dehydroepiandrosterone (1) through bromination at C-16α of the 17-ketone 1 and controlled alkaline hydrolysis of the 16α-bromoketone 3, obtained from the brominated product 2 by 8 N CrO3 oxidation followed by p-toluenesulfonic acid treatment, as key reactions. The tritium distribution of the labeled 16α-ketol 4 was determined by chemical and biochemical methods to be 47% at C-1α, 18% at C-2α, and 35% at the β-side of C-1 and C-2. When the labeled ketol 4 was incubated with human placental microsomes and reduced nicotinamide adenine dinucleotide phosphate, the rate of 3H2O release into the medium was dependent upon protein concentration and incubation time. Aromatase activity obtained by the radiometric assay was comparable to that determined by the high-performance liquid chromatographic method.