Analysis of androgen-5 alpha-reductase by an enzyme kinetic method

Analysis of androgen-5 alpha-reductase (A-5 alpha-R) is commonly done by measuring distinct testosterone (T) metabolites after tissue incubation. In the present study, A-5 alpha-R analysis was performed according to the principles of Michaelis-Menten, i.e. by evaluation of KM and Vmax data of the enzyme. 48 tissue samples (foreskin) of healthy boys in the age group 6 months to 8 years were examined. Incubation was carried out using increasing concentrations of 3H-T (8-208 nM) as substrate, followed by extraction and thin layer chromatography (TLC) of the reaction products. Zone detection and quantitation of radioactivity was done by a computing TLC scanner. The specific radioactivity of the metabolites was evaluated by high resolution radio gas chromatography. KM values ranged from 81.8 to 118.1 nM and Vmax from 8.9 to 30.1 pmol/mg . h. Coefficient of variation was smaller for KM (0.1) than for Vmax (0.38). It is recommended to do A-5 alpha-R analysis by evaluation of the enzyme kinetics as this is a reproducible method giving accurate biochemical data of the qualitative and quantitative characteristics of the enzyme.