Analysis of Androgen-5α-Reductase by an Enzyme Kinetic Method

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Abstract
Analysis of androgen-5.alpha.-reductase (A-5.alpha.-R) is commonly done by measuring distinct testosterone (T) metabolites after tissue incubation. In this 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 age 6 mo.-8 yr 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 81.8-118.1 nM and Vmax from 8.9-30.1 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 the enzyme kinetics as this is a reproducible method giving accurate biochemical data of the qualitative and quantitative characteristics of the enzyme.