BLOOD ANALYSIS 273 



Determination of Total Non-protein Nitrogen. Transfer 5 c.c. of the alcoholic 

 filtrate to a large Jena test-tube of the same kind as is used in urine analysis 

 (see page 485). Add i drop of concentrated sulphuric acid, i drop of kerosene, 

 and a small pebble or glass bead to prevent bumping. Immerse the test-tube 

 in a beaker of boiling water for five or ten minutes to drive off the methyl alcohol. 

 When the alcohol is removed add i c.c. of concentrated sulphuric acid, i gram of 

 potassium sulphate and i drop of copper sulphate solution. Boil, cool, dilute and 

 aerate the solution as described in the determination of total nitrogen hi urine 

 (see page 485), except that the ammonia is collected in a large test-tube instead 

 of the 100 c.c. flask. Nesslerize the solution, using 7 to 8 c.c. of diluted Nessler 

 reagent (dilution 1:5), dilute to 25 or 50 c.c. according to the amount of color, 

 and compare with a standard solution containing i mg. of ammonia nitrogen, 

 Nesslerized and diluted to 100 c.c. and the colorimeter prism set at 20 mm. 



Calculations. If 5 c.c. of blood are diluted to 50 c.c. and 10 c.c. of the alcoholic 

 extract (equivalent to i c.c. of blood) are used for the determination, the amount 

 of non-protein nitrogen (as milligrams per 100 c.c. of the blood) can be obtained 



20 

 by use of the formula p X D, in which R stands for the reading of the unknown 



and D represents the volume to which its ammonia has been diluted. If the 

 equivalent of 0.4 c.c. of blood has been taken for the determination the formula 



So 



*n X D is used, and if the equivalent of 0.5 c.c. of blood has been taken the 



jc 



formula becomes >, X D. 



K. 



(b) Colorimetric Method for Finger Blood. Principle. Taylor and 

 Hulton 1 have suggested a modification for the determination of non- 

 protein nitrogen using from 4 to 8 drops of blood. It is based upon the 

 observation of Gulick 2 that the presence of small amounts of potassium 

 sulphate does not appreciably interfere with the Nesslerization of solu- 

 tions containing ammonium salts. The Nesslerization is accordingly 

 carried out directly upon the oxidized material without removal of the 

 ammonia by aspiration or distillation. The authors use a mixture of 

 ether and alcohol for the precipitation of proteins. The authors claim 

 only approximate accuracy for the method and the small amount of 

 lipoid nitrogen included with the other non-protein nitrogen may be 

 disregarded. 



Procedure. Place 10 c.c. of a mixture of absolute alcohol and ether (3:1) 

 in a small Erlenmeyer flask. Stopper and weigh the flask with its contents. 

 Remove the stopper and allow from 4 to 8 drops of blood (depending upon the 

 amount of non-protein nitrogen) to drop from the finger which has been cleaned 

 and pricked with a sharp lance. The blood should drop freely when the hand is 

 held down. Insert the stopper and weigh the flask and contents. The increase 

 in weight is of course the weight of blood. Within a half -hour filter the mixture 

 into the digestion flask and wash the filter with 5 c.c. of alcohol-ether. The 

 filtrate is clear and practically free from protein, although it probably contains 

 traces of nitrogen-containing lipoids. The Kjeldahl digestion is carried out in a 



1 A. E. Taylor and F. Hulton: /. Biol. Chem., 22, 63, 1915. 



2 Gulick: /. Biol. Chem., 18, 541, 1914. 



18 



