The hydrolysis of steroid glucuronides with calf spleen glucuronidase.

It has long been recognized that the relatively severe acid hydrolysis necessary to release steroids from their conjugates in urine may result in considerable losses or transformations (3, 4). The isolation of steroid glucuronides (5-7) and of steroid sulfates (8, 9) from human urines has led to the belief that most, if not all, of the urinary steroid conjugates exist as glucuronides or as sulfates. The separate hydrolysis of these two types of conjugates seems to offer the greatest possibility of development of less drastic hydrolytic procedures than those employing strong acids at high temperatures. The development of such specific hydrolytic procedures was felt highly desirable in connection with our studies designed to permit the isolation of a greater number of urinary steroid conjugates than have hitherto been characterized. The concentration of a specific group of steroid conjugates could thus be readily followed. The possibility of an enzymatic hydrolysis of conjugated steroids was first apparent in the observation of Cohen and Marrian in 1935 (10) that prolonged exposure of pregnancy urine to bacterial action resulted in a complete hydrolysis of the conjugated estrogens. Subsequent identification of a considerable portion of this estrogen as estriol glucuronide indicated the involvement of some bacterial glucuronidase in this hydrolysis. Shortly thereafter Patterson (11) used preparations of Escherichicz coli to effect a hydrolysis of the conjugated estrogens in early pregnancy urines, thus obtaining estrogen concentrates which on calorimetric assay gave a considerably reduced amount of interfering background chromogen. In 1939 Fishman (12) reported on the ability of preparations of ox spleen p-glucuronidase to effect hydrolysis of pure sodium estriol monoglucuronidate. Rat liver preparations were subsequently used by Talbot et al. (13) and by Mason and Kepler (14) for the hydrolysis of C19and CZ1steroid glucuronides. In 1949 Buehler et al. (15) reported on the successful