Evidence for precursors of norandrogens as 3-enol sulfates from porcine Leydig cell incubations

Norandrogens are significant anabolic-androgenic steroids used initially for the treatment of debilitating conditions and later as performance-enhancing drugs. Regulatory challenges of their abuse in human, equine, and canine sports stem from the question of endogenous norandrogens. Evidence from mass spectrometry for 19-norandrostenedione (19-norA4) in the sulfate fraction of conjugated steroids in equine yolk-sac fluid suggested a need for reexamination. In vitro biosynthesis of 19-norandrogens was examined in highly purified preparations of porcine Leydig cells from mature pigs by incubation in -medium containing [3H]-androstenedione (A4) or nonradioactive A4. Steroids were recovered from media by solid-phase extraction, separately as unconjugated and conjugated fractions. High performance liquid chromatographic (HPLC) profiles were obtained from liquid-scintillaion counting of radioctivity. Several peaks in the conjugated fractions were investigated further by HPLC. Two peaks widely separated on HPLC as conjugated steroids yielded evidence of norandrostenedione (19-norA4) after solvolysis. Other peaks identified by HPLC retention times matching standards were 19-hydroxy-A4, A4 (substrate), and epiandrosterone. Incubations with non-radioactive A4 provided further evidence from radioimmunoassay of sulfated steroids as precursors of 19-norA4. With known high sulfotransferase activity in porcine Leydig cells, it is proposed that two 3-enol sulfates were formed as stable "precursors" of 19-norA4 by the distinctive action of the third aromatase isoform of the boar testes. Although their identities remain unknown, it was shown that 19-norA4 itself was not present as a 3-enol sulfate. These findings have implications regarding endogenous anabolic agents in normal growth and relevance to illegal use of anabolic androgens in sports and animal production.