508 PHYSIOLOGICAL CHEMISTRY 



urine gives directly the number of milligrams of creatinine per cubic centimeter of 

 urine. 



Folin's Microchemical Modification. 1 Principle. The principle is the same 

 as that of the original colorimetric method (see page 506). This procedure is to be 

 recommended particularly where only small amounts of material are available. 



Procedure. One c.c. of the standard creatinine (see above) solution (i mg. per 

 c.c.) is measured into a 100 c.c. volumetric flask and i c.c. of urine into another; 

 20 c.c. of saturated picric acid solution (measured with a cylinder) are added to 

 each and then 1.5 c.c. of a 10 per cent solution of sodium hydroxide. At the end 

 of ten minutes the flasks are filled up to the mark with tap water and the color 

 of the unknown is determined. The reading of the standard divided by the 

 reading of the unknown gives directly the number of milligrams of creatinine in 

 the amount of urine taken for analysis. 



3. Shaffer's Modification for the Determination of Creatinine in Very Dilute 

 Solutions. 2 The regular Folin procedure is not accurate when applied to urines 

 containing less than 20 mg. of creatinine per 100 c.c. By a slight modification it 

 becomes applicable to creatinine solutions containing as little as i mg. or less per 

 100 c.c. 



Procedure. To the solution under examination add an equal volume of satu- 

 rated picric acid solution and one- tenth this volume of 10 per cent sodium hydroxide 

 solution. After standing 6-10 minutes the liquid is diluted to a definite volume 

 depending upon the intensity of the color developed. With very dilute solutions 

 one may add solid picric acid equivalent to half saturation (0.6 per cent) and when 

 dissolved, one-twentieth the volume of sodium hydroxide. Provided the creatinine 

 solution itself has not sufficient color to interfere, the results by this method appear 

 to be as accurate as the original procedure. The colorimetric readings and calcu- 

 lations are made in the same way as in the preceding methods. 



Creatine 



Folin-Benedict Method. 3 Principle. Creatinine on boiling with 

 acid is transformed into creatinine. By determining the content of 

 creatinine before and after the acid treatment we are able to calculate 

 the amount of creatinine originally present in the urine. The Folin 

 colorimetric method (page 506) is used for determining the creatinine 

 in both cases. The method is not applicable to diabetic urines. 



Procedure. Introduce into a small flask or beaker 10 c.c. of the urine to 

 be examined. (If 10 c.c. contains more than 12 or less than 7 mg. of total 

 creatinine use a correspondingly smaller or larger volume of urine.) Add from 

 10-20 c.c. of normal HC1, and a pinch or two of powdered or granulated lead. 

 Boil the mixure over a free flame as slowly or as rapidly as may be desired, until 

 very nearly down to dryness, when the heating should be continued to dryness 

 either on the water-bath or very easily by simply holding the vessel in the hand 

 and heating carefully for a moment or two. Let the residue stand on the water- 

 bath for a few minutes until most of the excess of hydrochloric acid gas has been 



1 Folin: Jour. Biol. Chem., 17, 469, 1914. 



2 Shaffer: Jour. Biol. Chem., 18, 525, 1914. 



3 Benedict: Jour. Biol. Chem., 18, 191, 1914. 



