title: Identification of a new Sertoli cell metabolite of testosterone: 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol, by HPLC and GC-MS.
abstract: Testosterone metabolism by isolated rat Sertoli cells cultured in vitro was investigated using HPLC and GC-MS techniques. Monolayer cultures of Sertoli cells (greater than 90% pure and free of Leydig cells) were incubated for 3-day periods with a stable labeled [2,2,4,6,6-d5]testosterone prepared and used in a 1:1 proportion with unlabeled testosterone as the substrate (5 X 10(-7)M). After incubation, the metabolites were extracted from the media and reacted with oestradiol-antibodies. The antibody-bound components were separated on reverse phase HPLC and the fraction corresponding to oestradiol was analyzed by GC-MS in the form of TMS-ether. One of the metabolites whose mass spectrum contained d0 + d5 species was detected and interpreted to be a triol with a mol. wt of 308. Mass spectra data indicated that this testosterone metabolite is one of the sixteen possible isomers of 3,16,17-trihydroxy androstane. This substance was identified based on the Vm value (27.81) closely resembling that of 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol TMS-ether (Vm reported = 27.78) [1] and when compared directly with synthesized compounds [2-3]. Recently we have demonstrated that similar Sertoli cell preparations contain two 16 alpha-hydroxylases by their ability to convert oestradiol to oestriol [4] and 5 alpha-androstane-3 alpha,17 beta-diol to 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol [3], where the former conversion is not affected by FSH, the latter is significantly stimulated by the presence of FSH. Presence of this new product represents the first example of testosterone conversion to 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol and confirms our previous observation that 16 alpha-hydroxylation of 5 alpha-reduced androgens can occur in the rat testis.
mesh_terms: D015113:Androstane-3,17-diol / Q000031:analogs & derivatives / Q000378:metabolism*; D000732:Androstanols / Q000378:metabolism*; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D000072467:Cytochrome P450 Family 2; D008401:Gas Chromatography-Mass Spectrometry; D008297:Male; D013058:Mass Spectrometry; D051381:Rats; D011919:Rats, Inbred Strains; D012708:Sertoli Cells / Q000378:metabolism*; D039181:Steroid 16-alpha-Hydroxylase; D013739:Testosterone / Q000378:metabolism*
chemical_list: D000732:Androstanols; D015113:Androstane-3,17-diol; C052627:androstane-3,16,17-triol; D013739:Testosterone; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase