Enzymatic Hydrolysis of Urinary Sodium Pregnasediol Glucuronidate to Free Pregnanediol*

The available evidence suggests that most of the steriod hormone endproducts which are eliminated from the body by way of the urine are conjugated with an inorganic or organic acid. This conjugation presumably facilitates their transportation and excretion by rendering them soluble in water or urine. Examples of such conjugated steroids which have been isolated from urine as sulfates are androsterone (l), dehydroisoandrosterone (2), and estrone (3), and as glucuronidates, estriol (4) and pregnanediol (5). Convenient methods for measuring the rate of urinary excretion of these conjugated substances depend to a major extent upon hydrolysis of the conjugated steroids to unconjugated steroids. Most of the latter are essentially insoluble in water and can be extracted from aqueous solutions with suitable organic solvents. In a previous paper (6) evidence was presented which showed that the commonly employed hydrochloric acid hydrolysis procedure tends to destroy dehydroisoandrosterone added to water as sodium dehydroisoandrosterone sulfate. On the other hand, it was found that the sodium dehydroisoandrosterone sulfate could be hydrolyzed without losses of dehydroisoandrosterone by barium chloride solution at pH 5. Evidence is available which shows that hydrochloric acid hydrolysis of sodium pregnanediol glucuronidate also is accompanied by an approximately 30 per cent loss of pregnanediol (7). However, trial experiments have revealed that barium chloride does not hydrolyze sodium pregnanediol glucuronidate. The possibility that, sodium pregnanediol glucuronidate could be hydrolyzed satisfactorily with the aid of glucuronidase was suggested by the observations of Fishman (8). He found that an enzyme obtained from beef spleen tended to hydrolyze borne01 glucuronidate and certain other glucuronidates. Because beef spleen enzyme prepared in this laboratory according to Fishman’s directions failed to hydrolyze sodium pregnanediol glucuronidate to pregnanediol satisfactorily, the use of an enzyme prepared from rat liver according to the directions of Astwoodl was investigated.