Formation of Δ1 and Δ6 testosterone metabolites by human hepatocytes

• Δ1-T, Δ1-AED, Δ6-T and Δ6-AED appeared after hepatocytes incubation with T.
• Time course production of selected metabolites was determined.
• Results were compared with those observed for the major T metabolites.
• 1,2- and 4,6-dehydrogenation has been confirmed as phase I metabolic pathway for T.

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 Δ1 and Δ6 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 Δ1 and Δ6 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.

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