C19-Steroid Metabolism by Canine Prostate, Epididymis and Perianal Glands. Application of the Twin-Ion Technique of Gas Chromatography/Mass Spectrometry to Establish 7alpha-Hydroxylation

Abstract

The present study applied the twin-ion technique of gas chromatography/mass spectrometry to establish 7α-hydroxylation of 5α-androstane-3β,17β-diol by canine prostate, epididymis and perianal glands. 5α-[4-¹⁴C,7β-²H_0.52]Androstane-3β,17β-diol (0.5 μM) was incubated for 60 min at 37°C with minced canine prostate and epididymis in 50 ml 0.067 M phosphate buffer (pH 7.4) containing NADPH (0.2 mM). The principal radioactive metabolite fraction was isopolar in thin-layer chromatography with 5α-androstane-3β,7α,17β-triol and contained 24% (prostate incubation) and 23% (epididymis incubation) of the radioactivity added. Following gas chromatography of the trimethylsilyl ether derivative of these metabolites, the peak with the retention time of the derivative of 5α-androstane-3β,7α,17β-triol yielded a mass spectrum consistent with that of the authentic standard triol and gave the characteristic twin-ion, though with some loss of deuterium. Incubation of 5α-[4-¹⁴C,7β-²H_0.46]dihydrotestosterone (7 μM) with minced canine perianal glands and NADPH (0.2 mM) gave in 5% yield a transformation product with an R_F-value of 5α-androstane-3β,7α,174bT-triol. One half of the chromatographic fraction was subjected to gas chromatography/mass spectrometry as the free steroid, the other as the CrO₃-oxidation product. The site of hydroxylation was identified as 7α from the gas chromatography retention time of the free 7β-deuterated (twin-ion) triol and mass-spectrometry loss of deuterium in the 3,7,17-trione produced by mild CrO₃ oxidation. Results of a comparative study of the metabolism of [4-¹⁴C]testosterone and 5α-dihydro[4-¹⁴C]-testosterone with minced canine perianal glands and shoulder skin showed that, whereas both tissues contain a high level of 3β-hydroxysteroid oxidoreductase, only the perianal glands were able to transform radioactive testosterone to the 5α-reduced derivatives and thence to the 7α-hydroxylated product.