Enzymatic hydrolysis of steroid hormone esters by blood serum.

In recent years the concept of the specificity of enzyme action has been subjected to revision. The proteolytic enzymes (1) have been shown to have esterase activity and the concept of a blood esterase as distinct from a blood lipase is no longer tenable (2). The enzymatic hydrolysis of the steroid hormone esters by blood sera (3) had not been suspected until solubility determinations of the esters in sera accidentally established the phenomenon, possibly because the nearest chemical analogues, the cholesterol esters, are not hydrolyzed to any measurable extent by serum esterases (4). The present report is a quantitative study of the enzymatic hydrolysis of esters of two phenolic estrogens, estradiol and estrone, by mammalian sera and a comparison with analogous phenol and naphthol esters, fats, and cholesterol esters. It was necessary to develop a method based on the Folin-Ciocalteu (5) phenol reaction for the quantitative estimation of enzyme activity. The esters studied were the bensoates of phenol, P-naphthol and estradiol, phenyl stearate, estradiol dipropionate, the acetates of estrone and phenol, sodium estrone sulfate, and estrone palmitate. Analytical Principle-To undiluted sera or sera diluted with 6 per cent bovine albumin solution is added the substrate ester in ethanol, so that the final concentration of ethanol is 10 per cent and the ester is completely dispersed. The enzymatic reaction is terminated by precipitation at 80 per cent ethanol concentration, proteins being removed which would react with the phenol reagent. Liberated phenols are determined in aliquots of the ethanol filtrate, and details of the procedures and proofs for recovery follow. Analytical Determination of Phenol-The extracted residue of the 80 per cent ethanol filtrate of undiluted sera was found to produce a precipitate or heavy opalescence when subjected to the Folin-Ciocalteu reaction. Filtration or centrifugation removed some of the blue color. The use of 0.5 per cent lauryl sodium sulfate (SLS) as the reaction medium was found to