BLOOD ANALYSIS 277 



rated picric acid solution. The preformed creatinine is then determined colorimet- 

 ricaily. For this purpose a standard solution of creatinine for comparison is 

 necessary. Prepare this from the standard creatinine stock solution as used in the 

 analysis of urine (see chapter on Quantitative Analysis of Urine) by diluting an 

 amount of this solution equivalent to i mg. of creatinine to 500 c.c. with saturated 

 picric acid solution. We have then a standard solution containing 0.2 mg. of 

 creatinine in 100 c.c- f saturated picric acid solution. 



Take 20 c.c. portions each of the filtrate and of the standard solution. To each 

 solution then add exactly i c.c. of 10 per cent NaOH from a burette. (If the blood 

 filtrate becomes turbid on addition of alkali it must be centrifuged or filtered.) 

 Allow to stand for 10 minutes and compare the colors directly in the colorimeter 

 without further dilution. The standard creatinine solution may be set advantage- 

 ously at 20 mm., although this is not necessary. 



Calculation. Since the blood was diluted five times in the precipitation pro- 

 cedure and as the standard for comparison contains 0.2 mg. of creatinine per 100 c.c., 

 it is merely necessary to divide the reading of the standard by the reading of the 

 unknown to obtain without further calculation the number of milligrams of creatin- 

 ine in 100 c.c. of blood. 



Creatine Plus Creatinine. For determining the total creatinine plus creatine 

 in the blood carry out the preliminary precipitation with picric acid just as in the 

 determination of creatinine above. Take 10 c.c. of this filtrate for the determina- 

 tion. Transfer it to a small Erlenmeyer flask or large test-tube. Cover the flask 

 or test-tube with tin foil, transfer to an autoclave and heat to about i2oC. for 

 about 20 minutes. The autoclave should not be opened until the temperature has 

 fallen below iooC. Cool the solution to room temperature, rinse into a 25 c.c. 

 volumetric flask with saturated picric acid solution. Add 1.25 c.c. of 10 per cent 

 NaOH for the development of the color. 



On account of the variations in the creatine content of normal blood two 

 standard creatinine solutions are used. In working on pathological cases a third 

 standard is desirable. These standards contain 0.5, i, and 2 mg. of creatinine 

 respectively per 100 c.c. of saturated picric acid solution. To 20 c.c. of each of 

 these solutions in measuring cylinders add i c.c. of 10 per cent NaOH and allow 

 to stand for 10 minutes. By inspection determine which standard corresponds 

 most nearly in color with the unknown and use this for comparison. The standard 

 is usually set at 10 mm. in the Duboscq colorimeter. 



Calculation. Multiply the reading of the standard by 125 and by 0.5, i, or 2, 

 according to which standard is used, and divide by the reading of the unknown in 

 millimeters. The result gives the number of milligrams of creatine + creatinine 

 in 100 c.c. of the blood examined. 



5. Amino-acid Nitrogen, (a) Method of Van Slyke and Meyer. 1 Principle. 

 The protein of the blood is removed by precipitation with alcohol and the ammo- 

 acid nitrogen determined in the filtrate by the nitrous acid method. 



Procedure. Thirty to 50 c.c. of freshly drawn blood are mixed with 9 or 10 

 volumes of 95 per cent alcohol to precipitate the proteins. The volume of the 

 alcohol-blood mixture must be known, but in case it is not convenient to use a 

 graduated cylinder for the mixture, its volume can be taken as the sum of the 

 volumes of the alcohol and blood without essentially affecting the results. The 

 alcohol and blood are thoroughly mixed, the vessel containing them is closed and 



1 Van Slyke and Meyer: Jour. Biol. Chem., 12, 399, 1912. 



