Analytical Sciences

Spectrophotometric Method for the Assay of Steroid 5α-Reductase Activity of Rat Liver and Prostate Microsomes

Atsushi IWAI,*1 Teruki YOSHIMURA,*2 Keiji WADA,*2 Satoshi WATABE,*3 Yuki SAKAMOTO,*4 Etsuro ITO,*4 and Toshiaki MIURA*1

*1 Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita, Sapporo 060-0812, Japan
*2 Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
*3 BL Co., Ltd., Numazu, Shizuoka 410-0042, Japan
*4 Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Sanuki 769-2193, Japan

A simple spectrophotometric method for the assay of steroid 5α-reductase (5α-SR) was developed in which 5α-dihydrotestosterone (5α-DHT) and 5α-androstane-3α,17β-diol (5α-diol), metabolites formed in the NADPH-dependent reduction of testosterone with enzyme sources of 5α-SR, were measured by enzymatic cycling using 3α-hydroxysteroid dehydrogenase in the presence of excess thionicotinamide-adenine dinucleotide (thio-NAD) and NADH. It was found that 5α-SR activity was proportional to the accumulated thio-NADH having an absorption maximum at 400 nm. Because of the high cycling rate (> 600 cycle per min) and no interference from testosterone, enzymatic cycling can determine the sum of 5α-DHT and 5α-diol at the picomole level without separation from excess testosterone. The present method was readily applicable to the assay of 5α-SR activity of rat liver and prostate microsomes as well as to the assay of inhibitory activity of finasteride, a synthetic inhibitor of 5α-SR.

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