Biosynthesis of dihydrotestosterone-1,2-T

Dihydrotestosterone-1,2-T was obtained by biosynthesis using as substrate Testosterone-1,2-T. Labelled testosterone was synthesized by selective hydrogenation of 1Δ Testosterone acetate. In this study enzymatic activity of 5α reductase was analyzed. Enzymatic activity was studied using human skin and prostate tissue homogenates incubated 1 h at 37 deg. C with labelled tritium testosterone. As cofactor we used NADPH. At the end of reaction, testosterone metabolites were extracted with a mixture of cyclohexane/ethyl acetate. Product separation was carried out using Celite chromatographic columns. As mobile phase we used a mixture of isooctane/toluene. Labelled testosterone used as substrate in enzymatic reactions was purified by column chromatography using a previous system. Enzymatic activities of the collected fractions as a result of chromatographic elution were determined using a Packard liquid scintillation counter. For metabolites identification were used testosterone, dihydrotestosterone, androstenedione 3α and 3β and androstenediol C-14 labelled as internal standards. From radiochromatographic profiles the base peak of testosterone used as substrate were identified, as well as the peaks of dihydrotestosterone, androstenedione and also the peaks of 3α and 3β androstenediol with very low intensity. This study confirms the possibility of dihydrotestosterone and androstenedione biosynthesis. Androstenediol can be also synthesised with low yields. (authors)