title: C19-Steroid metabolism by canine prostate, epididymis and perianal glands. Application of the twin-ion technique of gas chromatography/mass spectrometry to establish 7 alpha-hydroxylation.
abstract: The present study applied the twin-ion technique of gas chromatography/mass spectrometry to establish 7 alpha-hydroxylation of 5 alpha-androstane-3 beta, 17 beta-diol by canine prostate, epididymis and perianal glands. 5 alpha-[4-14C, 7 beta-2H0.52]Androstane-3 beta, 17 beta-diol (0.5 microM) was incubated for 60 min at 37 degrees 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 5 alpha-androstane-3 beta, 7 alpha, 17 beta-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 5 alpha-androstane-3 beta, 7 alpah, 17 beta-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 5 alpha-[4-14C, 7 beta-2H0.46]dihydrotestosterone (7 microM) and minced canine perianal glands and NADPH (0.2 mM) gave in 5% yield a transformation product with an RF-value of 5 alpha-androstane-3 beta, 7 alpha, 17 beta-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 7 alpha from the gas chromatography retention time of the free 7 beta-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 5 alpha-dihydro[4-14C]-testosterone with minced canine perianal glands and shoulder skin showed that, whereas both tissues contain a high level of 3 beta-hydroxysteroid oxidoreductase, only the perianal glands were able to transform radioactive testosterone to the 5 alpha-reduced derivatives and thence to the 7 alpha-hydroxylated product.
mesh_terms: D000728:Androgens / Q000378:metabolism*; D015113:Androstane-3,17-diol / Q000378:metabolism; D000818:Animals; D002250:Carbon Radioisotopes; D003903:Deuterium; D013196:Dihydrotestosterone / Q000378:metabolism; D004285:Dogs; D004822:Epididymis / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006900:Hydroxylation; D008297:Male; D009928:Organ Specificity; D010481:Perianal Glands / Q000378:metabolism*; D011467:Prostate / Q000378:metabolism*; D012867:Skin / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism
chemical_list: D000728:Androgens; D002250:Carbon Radioisotopes; D013196:Dihydrotestosterone; D015113:Androstane-3,17-diol; D013739:Testosterone; D003903:Deuterium