STUDIES ON PHENOLIC STEROIDS IN HUMAN SUBJECTS: EVALUATION OF ENZYMATIC HYDROLYSIS OF ESTROGEN CONJUGATES12

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Abstract
A comparison of the ability of three enzyme preparations of bacterial, mammalian and molluscan origin to hydrolyze urinary and bilary conjugates of the metabolites of C14-labelled estrone, estradiol-17β and estriol revealed the last to be slight superior in hydrolyzing urinary conjugates and markedly superior in hydrolyzing biliary conjugates. It is recommended that for the hydrolysis of urinary estrogen conjugates, incubation with 300 units of molluscan β-glucuronidase per milliliter of urine be conducted at pH 5.5 and 37° C for 96 hours. For the hydrolysis of biliary estrogen conjugates, the concentration should be increased to 1,000 units/ml. A combination of enzymatic hydrolysis and continuous extraction at pH 1 will liberate 30–50% more conjugates than will rcfluxing urine for 45 minutes with 5% (v/v) concentrated sulfuric acid. All three preparations contained enzymes capable of modifying non-conjugated estrone, estradiol-17β and estriol. In the case of estradiol-17β, conversions were quite extensive. Thus, the interpretation of metabolic experiments is complicated when one of the preparations is employed for hydrolysis of conjugates. Although the inhibition by Hg++ and CU++ of enzymatic activity toward phenolphthalein glucosiduronate was reversed by ethylenediaminetetracetic acid, this substance had no effect upon the inhibition produced by urine. On the basis of the thermal stability, ultrafiltrability and the insolubility in ethyl acetate of the inhibitor(s), it was concluded that the inhibition is probably due to competition by other glucosiduronates present in urine and bile.