Spectrophotometric method for the assay of steroid 5α-reductase activity of rat liver and prostate microsomes

Anal Sci. 2013;29(4):455-9. doi: 10.2116/analsci.29.455.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3-Oxo-5-alpha-Steroid 4-Dehydrogenase / metabolism*
  • Androstane-3,17-diol / metabolism
  • Animals
  • Dihydrotestosterone / metabolism
  • Enzyme Assays / methods*
  • Male
  • Microsomes, Liver / enzymology*
  • NAD / analogs & derivatives
  • NAD / metabolism
  • Prostate / cytology*
  • Rats
  • Spectrophotometry / methods*

Substances

  • Dihydrotestosterone
  • NAD
  • Androstane-3,17-diol
  • thionicotinamide adenine dinucleotide
  • 3-Oxo-5-alpha-Steroid 4-Dehydrogenase