5IO PHYSIOLOGICAL CHEMISTRY 



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 1 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. 



Uric Acid 



i. Microchemical Colorimetric Method. Benedict and Hitchcock 

 Modification of the Folin-Macallum-Denis Procedure. The principle 

 of the method depends upon the fact, first noted by Folin and Macallum 2 

 and further investigated by Folin and Denis, 3 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. 



Benedict and Hitchcock 4 have examined the method of Folin and 

 Denis and have suggested a number of important modifications. 



Procedure. Measure such an amount of urine as will contain from 0.7 

 to 1.3 mg. of uric acid (2 to 4 c.c. is usually the correct amount) into a centrifuge 

 tube, dilute with water to about 5 c.c., and add 15 to 20 drops of an ammoniacal 

 silver magnesium solution. 5 Mix the contents of the tube with a small stirring 

 rod and centrifuge the tube for one or two minutes. Pour off the supernatant 

 liquid, as completely as possible, by inverting the tube, allowing it to drain a 

 moment, and then touching the inside of the lip of the tube with a towel or piece 

 of filter paper. Add to the residue in the tube two drops of a 5 per cent solution 

 of potassium cyanide to dissolve the silver urate, stir the mixture thoroughly 

 with a thin rod, for half a minute, add a few drops (0.5 to i.o c.c.) of water, and 

 stir again. 6 Two c.c. of the uric acid reagent 7 are added and the mixture stirred 



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



2 Folin and Macallum: /. Biol. Chem., 13, 363, 1912. 



3 Folin and Denis: /. Biol. Chem., 14, 95, 1913; ibid., 13, 469, 1913. 



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

 6 This solution has the following composition: 



3 per cent silver lactate solution ................................. 70 c.c. 



Magnesia mixture ............................................. 30 c.c. 



Concentrated ammonium hydroxide solution ...................... 100 c.c. 



6 At this point perfectly clear solutions are obtained with pure uric acid solutions in phos- 

 phate mixture or in pyridin. With urines some magnesium ammonium phosphate is precipi- 



tated with the uric acid, which does not dissolve in the cyanide. After adding the two 

 subsequent reagents, however, a perfectly clear solution is obtained. 



7 Preparation of the Uric Acid Reagent. Place 100 grams of sodium tungstate, 80 c.c. 

 of 85 per cent phosphoric acid, and 750 c.c. of distilled water in a liter flask. Boil the mix- 

 ture with a reflux condenser for two hours, cool and dilute to i liter, filtering if necessary. 



