title: Detection, synthesis and characterization of metabolites of steroid hormones conjugated with cysteine.
abstract: The occurrence of several polyunsaturated testosterone related compounds (including 4,6-androstadien-3,17-dione and 4,6-androstadien-17β-ol-3-one) in urine after alkaline treatment of the sample has been recently reported. Although several experiments seem to indicate that they are testosterone metabolites, their origin is still unknown. In this study, it is demonstrated that these metabolites are produced from the degradation of cysteine conjugates. Several testosterone metabolites conjugated with cysteine have been synthesized and characterized by NMR techniques. Their detection in human urine has been performed by LC-MS/MS. The acquisition of several transitions in the SRM mode and the comparison between ion ratios and retention times allowed for the unequivocal confirmation of the presence of cysteine conjugates in urine. The analysis of urine samples collected after testosterone administration confirmed that synthesized cysteine conjugates are testosterone metabolites. The fact that these conjugates result in polyunsaturated compounds in urine after alkaline treatment was demonstrated by fraction collection and alkaline treatment of each fraction. Besides, the presence of these metabolites was also confirmed in human plasma. The formation of these metabolites implies an unreported metabolic biotransformation: 6,7-dehydrogenation as phase I metabolism followed by conjugation with glutathione and subsequent transformation to cysteine conjugates. Finally, the existence of similar metabolites for cortisol and progesterone was also confirmed by LC-MS/MS indicating that the presented metabolic pathway is not exclusively active in androgens, but common to progestagens and glucocorticoids.
authors.affiliation: Bioanalysis Research Group, IMIM, Hospital del Mar, Doctor Aiguader 88, 08003 Barcelona, Spain.
authors.forename: Andreu
authors.initials: A
authors.lastname: Fabregat
authors.forename: Aristotelis
authors.initials: A
authors.lastname: Kotronoulas
authors.forename: Josep
authors.initials: J
authors.lastname: Marcos
authors.forename: Jesús
authors.initials: J
authors.lastname: Joglar
authors.forename: Ignacio
authors.initials: I
authors.lastname: Alfonso
authors.forename: Jordi
authors.initials: J
authors.lastname: Segura
authors.forename: Rosa
authors.initials: R
authors.lastname: Ventura
authors.forename: Oscar J
authors.initials: OJ
authors.lastname: Pozo
mesh_terms: D000730:Androstadienes / Q000097:blood / Q000737:chemistry / Q000652:urine*; D001711:Biotransformation / Q000502:physiology; D002853:Chromatography, Liquid; D003545:Cysteine / Q000097:blood / Q000737:chemistry / Q000652:urine*; D005978:Glutathione / Q000097:blood / Q000737:chemistry / Q000652:urine*; D006801:Humans; D006854:Hydrocortisone / Q000097:blood / Q000737:chemistry / Q000652:urine*; D006878:Hydroxides / Q000737:chemistry; D050217:Metabolic Detoxication, Phase II / Q000502:physiology; D008958:Models, Molecular; D017680:Potassium Compounds / Q000737:chemistry; D011374:Progesterone / Q000097:blood / Q000737:chemistry / Q000652:urine*; D053719:Tandem Mass Spectrometry; D013739:Testosterone / Q000097:blood / Q000737:chemistry / Q000652:urine*
chemical_list: D000730:Androstadienes; D006878:Hydroxides; D017680:Potassium Compounds; D013739:Testosterone; D011374:Progesterone; C038796:4,6-androstadiene-3,17-dione; D005978:Glutathione; D003545:Cysteine; D006854:Hydrocortisone; C029943:potassium hydroxide