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.
ASJC Scopus subject areas
- Analytical Chemistry