THE COLORIMETRIC ASSAY OF TOTAL, a-, AND p-17- KETOSTEROIDS IN EXTRACTS OF HUMAN URINE*

The present paper reports a method for determining the respective concentrations of total, o(-, and /3-17-ketosteroids in urine which is based upon the previously reported method of assaying mixtures of crystalline androsterone (a-steroid) and dehydroisoandrosterone (p-steroid) (1) and a simple means of eliminating the interfering effect of the non-ketonic substances from the calorimetric determination. The commonly employed procedure for “clearing” urine extracts with decolorizing charcoal (2) may result in the loss of significant amounts of hormones (3). On the other hand, urine contains non-ketonic substances which have a color with an absorption spectrum in the same region as that developed by 17ketosteroids with m-dinitrobenzene and potassium hydroxide in alcoholic solution. Therefore, either these non-ketonic substances must be eliminated or the light absorption due to them must be subtracted in the calorimetric assay. The method reported here adopts the latter procedure. In presenting the evidence for the validity of this correction, a procedure is also described for eliminating the non-ketonic substances. The application of the digitonin precipitation procedure to the detailed quantitative analysis of 17-ketosteroids in the neutral fraction of urine extracts is based upon the fact that steroids which have a hydroxyl group in the /3 position (4) on carbon atom 3 are selectively precipitated with this reagent. The latest evidence to be found in the literature (5, 6) suggests that only two P-17ketosteroids exist in significant amounts in the neutral fraction