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Following intraperitoneal injection of a mixture of testosterone-7-3-H-17-sulfate and testosterone-4-14-C into male and female rats with bile fistulas, biliary metabolites were separated and purified by a combination of column chromatography, enzymic hydrolysis or solvolysis of the conjugate fractions and identification of the liberated aglycones. The injected steroids were extensively metabolized and excreted predominantly in the bile. The major portion of the 3H was excreted in the disulfate fraction in both sexes. Solvolysis of the disulfate revealed the sex-specific aglycone pattern: 5alpha-Androstane-3beta,17beta-diol was the major metabolite in the male rat, whereas 5alpha-androstane-3alpha,17beta-diol and polar steroids were found in the female. In marked contrast, testosterone was metabolized in a different way than testosterone sulfate. 14-C radioactivity was distributed in monoglucosiduronate, monosulfate, and diconjugate fractions. Analysis of the aglycones showed that polar steroids were the main metabolites in the male. In the female, testosterone was metabolized to polar steroids, androsterone, and 5alpha-androstane-3alpha,17beta-diol.

The chemical synthesis of 16beta-hydroxyandrosterone was described preparatory to studies of the disulphates of the 16-oxygenated ketonic C19 steroids present in the bile of female rats dosed with [3H]androsterone sulphate. The biliary metabolites were separated by chromatography on Sephadex LH-20 to afford monosulphate and diconjugate fractions. After solvolysis of the diconjugate fraction, the liberated steroids were separated by partition chromatography on Celite 545 and analyzed by gas chromatography-mass spectometry. In addition to 3alpha, 17beta-dihydroxy-5alpha-androstan-16-one isolated previously, 16beta-hydroxyandrosterone was identified as a disulphate.