title: Spectrophotometric method for the assay of steroid 5α-reductase activity of rat liver and prostate microsomes.
abstract: 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.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Iwai
authors.forename: Teruki
authors.initials: T
authors.lastname: Yoshimura
authors.forename: Keiji
authors.initials: K
authors.lastname: Wada
authors.forename: Satoshi
authors.initials: S
authors.lastname: Watabe
authors.forename: Yuki
authors.initials: Y
authors.lastname: Sakamoto
authors.forename: Etsuro
authors.initials: E
authors.lastname: Ito
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Miura
mesh_terms: D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000378:metabolism*; D015113:Androstane-3,17-diol / Q000378:metabolism; D000818:Animals; D013196:Dihydrotestosterone / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D011467:Prostate / Q000166:cytology*; D051381:Rats; D013053:Spectrophotometry / Q000379:methods*
chemical_list: D013196:Dihydrotestosterone; D009243:NAD; D015113:Androstane-3,17-diol; C003451:thionicotinamide adenine dinucleotide; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase