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

Abstract

Analysis of 5&-reductase(5&R) is commonly done by measurement of distinct testosterone(T) metabolites, following tissue incubation. Interlaboratory comparison is difficult, as different individual methods are used. In order to overcome this disadvantage, 5&R analysis was performed by evaluation of enzyme kinetics, according to Michaelis-Menten (Km- and Vmax-values), and by comparing the results with a conventional method. 17 tissue specimens (foreskin) of healthy boys (1 to 8 yrs) were examined with two methods (A and B). A: Conventional assay, using 8 pmol 3H-T as substrate. B: Evaluation of enzyme kinetics with increasing concentrations of 3H-T (8 to 208 pmol). Separation of metabolites by thin layer chromatography (TLC). Zone detection by computing radio TLC scanner. Control of purity and specific radioactivity by high resolution radio gas chromatography. Results showed age dependent values of 5&R (x̄+SEM): Vmax=18.49+2.57 pmol/mg.h; Km= 102.82+11.41 nM, with a maximum at age 5 yrs: Vmax=36.33+4.32 pmol/mg.h; Km=171.77+23.53 nM. Conclusion: It is recommended to evaluate 5&R-analysis by the enzyme kinetic method. The results of Vmax correspond to those obtained with the single point assay (method A, r=0.98). The advantage of method B is a reproduceable method giving biochemically absolute data for quality and quantitiy of the enzyme.