A Method for the Quantitative Estimation of Indoxyl Compounds in Urine

In 1913 Jolles (1) introduced alcoholic thymol as a reagent for the detection of indican in urine, and later, based upon its use, developed a calorimetric method (2) for the estimation of indican in both urine and blood. His method for urine consisted in adding to 10 cc. of urine filtrate, after clarification with lead acetate, 1 cc. of 5 per cent alcoholic thymol, and 10 cc. of concentrated hydrochloric acid containing ferric chloride. This mixture was allowed to stand for 15 minutes, after which it was thoroughly shaken with 4 cc. of chloroform. The chloroform extracted a purple pigment (indoxyl-thymol compound) whose color intensity was alleged to be proportional to the amount of indican present. Jolles claimed to detect by this method 0.0032 mg. of indican in 10 cc. of urine. For quantitative measurements by the usual comparison calorimetry, pure indican was necessary for the production of pigment intensities of known indican value. Repetition and manipulation of this procedure with both urine and indican solution disclosed that Jolles’ method fails to be a satisfactory quantitative method, that the use of many times the amount of acid advised produces pigments of greater color intensity per unit of indican, and that the addition of trichloroacetic acid to the acid reagent induces a red value in the purple hue which increases in direct proportion to the increase of indican in t,he reaction mixture. It was possible to adjust the amount of trichloroacetic acid used to form readily within the reaction mixture ethyl trichloroacetate which served as an organic solvent for the indoxyl-thymol condensate produced. The procedure indicated below makes use of the findings just mentioned and is a quantitative method for indoxyl at, least 23 times as sensitive as Jolles’ reaction. For