280 PHYSIOLOGICAL CHEMISTRY 



10 to 15 minutes. Rinse the connecting tube; dilute the contents of the receiver 

 to 20 c.c., add 2.5 c.c. of Nessler solution, dilute to the 25 c.c. mark, and make the 

 color comparison in the usual manner. 



Interpretation. Normally from 12-15 ragm. of urea nitrogen are 

 found in 100 c.c. of blood. In early nephritis values of from 12-30 are 

 observed and in severe nephritis, values from 30 up to the 300 seen in 

 some cases of uremia. 



In normal blood 50 per cent of the non-protein nitrogen is in the 

 form of urea. In uremia the percentage may increase to 75 or over. 



4. Determination of Preformed Creatinine. Principle. A portion 

 of the blood filtrate is treated with alkaline picrate solution and the 

 color developed compared with that of a standard in a colorimeter. 



Procedure. Transfer 25 (or 50) c.c. of a saturated solution of purified picric 

 acid 1 to a small, clean flask, add 5 (or 10) c.c. of 10 per cent sodium hydroxide, 

 and mix. Transfer 10 c.c. of blood filtrate to a small flask or to a test tube, 

 transfer 5 c.c. of the standard creatinine solution described below to another 

 flask, and dilute the standard to 20 c.c. Then add 5 c.c. of the freshly prepared 

 alkaline picrate solution to the blood filtrate, and 10 c.c. to the diluted creatinine 

 solution. Let stand for 8 to 10 minutes and make the color comparison in the 

 usual manner, never omitting first to ascertain that the two fields of the colori- 

 meter are equal when both cups contain the standard creatinine picrate solution. 

 The color comparison should be completed within 15 minutes from the time the 

 alkaline picrate was added ; it is, therefore, never advisable to work with more 

 than three to five blood filtrates at a time. tS 



When the amount of blood nitrate available for the creatinine "determination is 

 too small to permit repetition, it is of course advantageous or necessary to start 

 with more than one standard. If a high creatinine should be encountered unex- 

 pectedly without several standards ready, the determination can be saved by 

 diluting the unknown with an appropriate amount of the alkaline picrate solution 

 using for such dilution a picrate solution first diluted with two volumes of water 

 so as to preserve equality between the standard and the unknown in relation to 

 the concentration of picric acid and sodium hydroxide. 



One standard creatinine solution, suitable both for creatin and for creatinine 

 determinations in blood, can be made as follows: 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. 

 Five c.c. of this solution contain 0.03 mg. 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 i to 2 mg. per 100 c.c. In the case of unusual bloods repre- 

 senting retention of creatinine 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 



1 Picric acid may be purified as indicated in the last section of this book. 



