Enzymatic detection of urinary conjugated steroids after gel chromatography

An enzymatic detection method is described for urinary conjugated steroids after chromatographic fractionation with Sephadex G-25. The principle of the method is as follows. Part of a 24-h urine sample, (1-2 ml of urine) is applied directly, to a short column of Sephadex G-25 and eluted with acetate buffer solution. Steroid conjugates in each fraction are hydrolyzed with steroid sulfatase--beta-glucuronidase. After enzymatic hydrolysis, an enzymatic color development reagent for steroids, either 3 alpha-hydroxysteroid dehydrogenase or 3 beta-hydroxysteroid oxidase, are added and the dye formed is measured spectrophotometrically. Excretion patterns of steroid-3 beta-sulfates, and steroid-3 alpha-glucuronides and steroid-3 alpha-sulfates ae shown with some patients' samples. A precision of the assay values for steroid-3 alpha-glucuronide, steroid-3 alpha-sulfate and steroid-3 beta-sulfates in urine samples and assay values for normal subjects are also studied. This simple enzymatic method for detecting the excretion patterns of urinary conjugated steroids may have a diagnostic value for clinical tests.