MICROCHEMICAL METHODS FOR BLOOD ANALYSIS 303 



lows: Transfer to a liter flask 6 c.c. of the standard creatinine 

 solution used for urine analysis (which contains 6 mg. of 

 creatinine) ; add 10 c.c. of normal hydrochloric acid, dilute to 

 the mark with water, and mix. Transfer to a bottle and add 

 four or five drops of toluene or xylene. 5 c.c. of this solution 

 contain 0.03 nig. of creatinine, and this amount plus 15 c.c. of 

 water represents the standard needed for the vast majority of 

 human bloods, for it covers the range of 1 to 2 mg. per 100 c.c. 

 In the case of unusual bloods representing retention of creati- 

 nine, take 10 c.c. of the standard plus 10 c.c. of water, which 

 covers the range of 2 to 4 mg. of creatinine per 100 c.c. of 

 blood; or 15 c.c. of the standard plus 5 c.c. of water by which 

 4 to 6 mg. can be estimated. By taking the full 20 c.c. volume 

 from the standard solution at least 8 mg. can be estimated ; but 

 when working with such blood it is well to consider whether it 

 may not be more advantageous to substitute 5 c.c. of blood 

 filtrate plus 5 c.c. of water for the usual 10 c.c. of blood filtrate. 



Calculation. The reading of the standard in mm. (usually 

 20) multiplied by 1.5, 3, 4.5, or 6 (according to how much of 

 the standard solution was taken), and divided by the reading 

 of the unknown, in mm., gives the amount of creatinine, in mg. 

 per 100 c.c. of blood. In connection with this calculation it is to 

 be noted that the standard is made up to twice the volume 

 of the unknown, so that each 5 c.c. of the standard creatinine 

 solution, while containing 0.03 mg., corresponds to 0.015 mg. in 

 the blood filtrate. 



Determination of Creatine Plus Creatinine. Transfer 5 c.c. 

 of blood filtrate to a test tube graduated at 25 c.c. These test 

 tubes are also used for urea and for sugar determinations. Add 

 1 c.c. of normal hydrochloric acid. Cover the mouth of the 

 test tube with tin foil and heat in the autoclave to 130 C. for 

 20 minutes or, as for the urea hydrolysis, to 155 C. for 10 

 minutes. Cool. Add 5 c.c. of the alkaline picrate solution and 

 let stand for 8 to 10 minutes, then dilute to 25 c.c. The stand- 

 ard solution required is 10 c.c. of creatinine solution in a 50 c.c. 



