530 PHYSIOLOGICAL CHEMISTRY 



ture, introduce it into a 500 c.c. volumetric flask and determine its creatinine con- 

 tent according to Folin's Colorimetric Method (see page 526). 

 For calculation and interpretation see the foregoing method. 



3. Method of Folin. 1 Water-bath Procedure. Heat 10 c.c. of urine with 5 c.c. 

 of normal hydrochloric acid on the boiling water-bath or at 9OC. for three hours. 

 The creatine is transformed into creatinine. Some darkening takes place but this 

 does not interfere because of the subsequent dilution. The mixture is made up to 

 50 c.c., 25 c.c. of this is taken, neutralized, and creatinine plus creatine determined 

 just as in the case of creatinine alone. The creatine is obtained by difference. 

 This procedure may be used for diabetic urines which is not the case with the auto- 

 clave procedure nor with the Benedict modification. It is perhaps not quite so 

 accurate as the autoclave procedure. 



4. Microchemical Modification of Folin. 2 By greatly diluting the urine the 

 time required for the conversion of creatine to creatinine is decreased, and picric 

 acid can be substituted for mineral acid. 



Procedure. Enough urine to give 0.7-1.5 mg. of creatinine is measured into 

 a weighed Erlenmeyer Jena flask (capacity 200 c.c.); 20 c.c. of saturated picric acid 

 solution, about 130 c.c. of water, and a few very small pebbles to promote even 

 boiling are added and the mixture is gently boiled, preferably over a micro-burner 

 for about one hour. At the end of this time the heat is increased and the solution 

 is boiled down to rather less than 20 c.c. The flask is transferred to the scales 

 and enough water is added to make the total solution equal to 20-25 grams. The 

 solution is cooled in running water, 1.5 c.c. of 10 per cent sodium hydroxide are 

 added, and the total creatinine is determined as in the preformed creatinine deter- 

 mination using i mg. of creatinine as a standard. The method has been found to 

 give good results in the presence of glucose and other sugars. 



Morris 3 has suggested that in the case of diabetic urines the total creatinine 

 be determined after precipitation of the creatine and creatinine with picric acid. 

 The method is not recommended as a regular procedure. 



V 



Uric Acid 



i. Microchemical Colorimetric Method. Method of Folin and 

 Wu* Principle. The principle of the method depends upon the fact, 

 first noted by Folin and Macallum 5 and further investigated by Folin 

 and Denis, 6 and Benedict and Hitchcock, 7 that uric acid gives, with 

 phosphotungstic acid and alkali, a deep blue color the depth of which is 

 proportional to the amount of uric acid present. Since certain other 

 substances present in urine produce a similar blue color with the phos- 

 photungstic acid, it is necessary to separate the uric acid from them. 

 This is accomplished by precipitation as the silver salt. The silver 

 urate is subsequently dissolved and treated with the uric acid reagent. 



1 Folin: Zeitschr. f. physiol. Chem., 41, 222, 1904. 



2 Folin: Jour. Bid. Chem., 17, 469, 1914. 



3 Morris: Jour. Biol. Chem., 21, 201, 1915. 



4 Folin and Wu: Jour. Biol. Chem., 38, 459, 1919. 



6 Folin and Macallum: Jour. Biol. Chem., 13, 363, 1912. 



6 Folin and Denis: Jour. Biol. Chem., 14, 95, 1913; ibid., 13, 469, 1913. 



7 Benedict and Hitchcock: /. Biol. Chem., 20, 619, 1915; Benedict: ibid, t 20, 629, 1915. 



