Bioformation of boldenone and related precursors/metabolites in equine feces and urine, with relevance to doping control

Boldenone (1-dehydrotestosterone) is an exogenous anabolic-androgenic steroid (AAS) but is also known to be endogenous in the entire male horse and potentially formed by microbes in voided urine, the gastrointestinal tract, or feed resulting in its detection in urine samples. In this study, equine fecal and urine samples were incubated in the presence of selected stable isotope labeled AAS precursors to investigate whether microbial activity could result in 1-dehydrogenation, in particular the formation of boldenone. Fecal matter was initially selected for investigation because of its high microbial activity, which could help to identify potential 1-dehydrogenated biomarkers that might also be present in low quantities in urine. Fecal incubations displayed Delta 1-dehydrogenase activity, as evidenced by the use of isotope labeled precursors to show the formation of boldenone and boldione from testosterone and androstenedione, as well as the formation of Delta 1-progesterone and boldione from progesterone. Unlabeled forms were also produced in unspiked fecal samples with Delta 1-progesterone being identified for the first time. Subsequent incubation of urine samples with the labeled AAS precursors demonstrated that Delta 1-dehydrogenase activity can also occur in this matrix. In all urine samples where labeled boldenone or boldione were detected, labeled Delta 1-progesterone was also detected. Delta 1-progesterone was not detected any non-incubated urine samples or following an administration of boldenone undecylenate to one mare/filly. Delta 1-progesterone appears to be a candidate for further investigation as a suitable biomarker to help evaluate whether boldenone present in a urine sample may have arisen due to microbial activity rather than by its exogenous administration.