528 PHYSIOLOGICAL CHEMISTRY 



cular atrophy and muscular weakness. It increases with increased 

 tissue catabolism as in fever. 



By the "creatinine coefficient" is meant the number of milligrams 

 of creatinine nitrogen excreted daily per kilo of body weight. This 

 varies under normal conditions from 7-11. 



Use of Pure Creatinine Standards. Instead of using as a standard a potassium 

 dichromate solution as above indicated, a solution of pure creatinine is to be recom- 

 mended. By using this certain arbitrary factors are eliminated and the method 

 becomes of more general applicability. The standard need not be set at a definite 

 mark as is necessary in the case of dichromate and temperature and time have less 

 influence on the accuracy of the'results. A stock solution of pure creatinine (made 

 according to Benedict's directions; see Chapter XXIII in Physiological Constituents 

 of Urine) is made by dissolving i gram of the substance in sufficient N/io HC1 to 

 make a liter. This solution contains i mg. of creatinine per cubic centimeter. In 

 carrying out the determination treat 10 c.c. of the stock solution in the same way and 

 at the same time as the 10 c.c. sample of urine. Compare in the colorimeter. The 

 calculation is simple. The reading of the standard divided by the reading of the 

 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 526). 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. 



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

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



