Biotransformation of 1-dehydrotestosterone in the equine male castrate: Identification of the neutral unconjugated and glucuronic acid conjugated metabolites in horse urine

Abstract

The in vivo biotransformation of (1,2(n)-³H)1-dehydrotestosterone was studied in three equine male castrates and a number of neutral metabolites were identified in the urinary unconjugated and glucuronic acid conjugate fractions by gas chromatography/mass spectrometry. The metabolites were extracted from aliquots of the 0–24 h urine samples by Amberlite XAD-2 and separated into combined unconjugated plus glucuronic acid conjugated and sulphoconjugated fractions by Sephadex LH-20 column chromatography. After enzymatic hydrolysis of the glucuronides, the crude neutral unconjugated steroids plus the aglycones were partially purified by Kieselgel H chromatography and identified as their methyloxime trimethylsilyl derivatives. In the unconjugated fraction, the major metabolites were isomers of androsta-1,4-diene-6,16,17-triol-3-one. In the aglycone fraction a small amount of the parent steroid was present but the major metabolite was the 17α isomer androsta-1,4-dien-17α-ol-3-one. Other metabolites containing the 1,4-dien-3-one group were isomers of androsta-1,4-diene-16,17-diol-3-one and androsta-1,4-diene-6,16-diol-3-one. Reduction of the 4-ene functionality leading to the formation of 5-androst-1-en-16-ol-3,17-dione, 5-androst-1-ene-16,17-diol-3-one and of the 1-ene functionality leading to the formation of 5-androst-1-en-16-ol-3,17-dione, 5-androst-1-ene-16,17-diol-3-one and of the 1-ene functionality leading to the formation of testosterone and its further reduction leading to the formation of C₁₉O₂ and C₁₉O₃ androstane metabolites was observed. Some interesting features on the electron impact fragmentations of the methyloxime trimethlysilyl derivatives of steroids containing a 1,4-dien-3-one group were also observed.