Formation of Δ(1) and Δ(6) testosterone metabolites by human hepatocytes

The existence of urinary testosterone (T) metabolites conjugated with cysteine has been recently reported. The formation of a ring double bond by a phase I metabolic transformation and the subsequent nucleophilic conjugation with glutathione was proposed as a putative metabolic pathway for the occurrence of these metabolites in urine. The main goal of the present study was to confirm the first step of the postulated pathway. For that purpose, human hepatocyte cells systems were incubated with a pure T standard. The cell culture supernatants were analyzed by liquid chromatography coupled to mass spectrometry using a selected reaction monitoring method. Major T metabolites such as androsterone and 4-androstene-3,17-dione, together with the recently reported Δ(1) and Δ(6) metabolites were simultaneously quantified. The formation of 1,4-androstadien-3,17-dione, 4,6-androstadien-3,17-dione, 17β-hydroxy-4,6-androstadien-3-one and 17β-hydroxy-1,4-androstadien-3-one (boldenone) after incubation of T in hepatocyte cell cultures was demonstrated by comparing the retention times and the ion ratios of the metabolites with those obtained by analysis of commercial standards. Thus, the formation of double bonds Δ(1) and Δ(6) by hepatic phase I metabolism of T was confirmed. Analogously to T, this pathway might also be present in other steroids, opening the possibility of targeting additional biomarkers.