THE USE OF STABLE ISOTOPES AND GAS-CHROMATOGRAPHY MASS-SPECTROMETRY IN THE IDENTIFICATION OF STEROID METABOLITES IN THE EQUINE

Stable isotope gas chromatography/mass spectrometry has been used successfully in the elucidation of structures of urinary steroid metabolites in the horse and in the identification of metabolites isolated from in vivo perfusion and in vitro incubation studies using equine tissue preparation. Deuterium-labeled steroids, testosterone, dehydroepiandrosterone, and 5-androstene-3-beta, 17-beta-diol have been synthesized by base-catalyzed isotope exchange methods and the products characterized by gas chromatography/mass spectrometry. [16,16-(H-2)-2]Dehydroepiandrosterone (plus radiolabeled dehydroepiandrosterone) was perfused into a testicular artery of a pony stallion and was shown to be metabolized into (H-2)-2-labeled testosterone, 4-androstenedione, isomers of 5-androstene-3,17-diol, 19-hydroxytestosterone, and 19-hydroxy-4-androstenedione. In further studies, equine testicular minces have been incubated with (H-2)-2-labeled and radiolabeled dehydroepiandrosterone and 5-androstene-3-beta, 17-beta-diol. The metabolites, whose identify was confirmed by stable isotope gas chromatography/mass spectrometry, proved the interconversion of the two substrates, as well as formation of testosterone and 4-androstenedione. The aromatization of dehydroepiandrosterone was also confirmed, together with the formation of an isomer of 5(10)-estrene-3,17-diol from both substrates showing 19-demethylation without concomitant aromatization. In studies of the feto-placental unit, the allantochorion was shown to aromatize ]H-5[-2-testosterone to ]H-2[-4 estradiol, the loss of one H-2 from the substrate being consistent with aromatization of the A ring. The formation of 6-hydroxyestradiol was also confirmed in this study. The same technique has been valuable in determining the structure of two metabolites of nandrolone isolated from horse urine. After deuteration and derivatization, gas chromatography/mass spectrometry analysis provided evidence for the presence of isomers of 3,17-dihydroxyestran-16- and 17-ones and the rearrangement of steroidal alpha-hydroxy-ketones under basic conditions.