Synthetic and endogenous testosterone conjugates: acid hydrolysis studied by radioimmunoassay

The acid hydrolysis of synthetic testosterone sulphate and glucuronide by sulphuric acid has been studied by measuring the release of uncojugated testosterone by radioimmunoassay. The sulphate was hydrolyzed at lower concentration of acid but no conditions were found where only the sulphate was completely hydrolyzed without any hydrolysis of the glucuronide. Results of similar studies on human urine, however, suggested that human urine contains three conjugates of testosterone and 5 alpha-dihydrotestosterone: the sulphate and glucuronide and also a very acid labile conjugate hitherto unknown. Hydrochloric acid has stronger hydrolyzing effect at the same normality and released more unconjugated testosterone in urine at the same pH than did sulphuric acid. A complete hydrolysis of urinary conjugates in 3 molar sulphuric acid was accomplished without destruction of immunoassayable testosterone and constitutes a suitable method for the determination of the total testosterone content in urine. The method was compared with other hydrolyzing methods by measuring the testosterone release from urinary conjugates after their extraction and separation by by thin-layer chromatography.