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Isolation of Metabolites of 4-¹⁴C-Corticosteroids from Urine of an Ovariectomized Heifer¹

Department of Animal Sciences, Purdue University, Lafayette, Indiana 47907

ABSTRACT

The purpose of this study was to isolate and characterize major ¹⁴C-labeled compounds excreted in urine of an ovariectomized heifer following intravenous injections of 4-¹⁴C-corticosterone (4.96 µCi) and 4-¹⁴C-cortisoI (4.28 µCi). Only 7.4% and 11.7% of the injected radioactivity were recovered in urine during the first 12 hours after injection of corticosterone and Cortisol, respectively, and 53% and 62% of this urinary radioactivity were extracted. Enzymatic hydrolysis and sol-volysis procedures were used in sequence. The urine was extracted with ethyl acetate before and after each hydrolysis. Isolation of distinct zones of radioactivity was accomplished by column (one system) and paper chromatography (three systems). Partial identifications of labeled compounds were made by matching their mobilities to those of authentic standards during paper chromatography. Ultraviolet light, blue tetrazolium, and phosphomolybdic acid were used as indicators of certain reactive groups. Due to small quantities of labeled metabolites only tentative identifications were possible. Isolations corresponding to standards of 6-hydroxycortisol represented 35% of the radioactivity recovered after injection of ¹⁴C-cortisol as compared to 19% tetrahydrocortisol, 6.4% cortols, 4.5% cortolones, and 1.6% tetrahydrocortico-sterone. Similarly, for ^I4C-corticosterone, 34% of the radioactivity recovered and isolated was from an unknown highly polar compound(s) as compared to 17.6% tetra-hydrocorticosterone, 15.7% tetrahydrocortisol, 3.6% 6-hydroxycortisols, 2.4% cortolones and 2.2% cortols.

¹ Paper 4456, Purdue University Agricultural Experiment Station.

² Fellow, National Aeronautical and Space Agency, Grant 2779-82; present address: Ohio Agricultural Eesearch and Development Center, Wooster 44691.