278 PHYSIOLOGICAL CHEMISTRY 



test tube; dilute to about 60 c.c. and add 30 c.c. of Nessler's solution. The 

 unknown and the standard should be Nesslerized simultaneously. 



Calculation. If the standard is set at 20 mm. for the color comparison, 

 20 divided by the reading and multiplied by 0.3 gives the non-protein nitrogen in 

 i c.c. of blood, because 0.5 e.c, (the amount of blood represented in 5 c.c. of the 

 blood filtrate) Nesslerized at a^olume of 50 c.e. is equivalent to i c.c. Nesslerized 

 at a volume of 100 c/c. 



The, non-protein nitrogen per 100 c.c. of blood is, therefore, 20 divided by the 

 reading and multiplied by 30 (0.3 times 100). 



If the standard containing 0.5 mg. N is used the calculation becomes 20, 

 divided by R, times 50. 



Alternate Procedures. Instead of Nesslerizing, it is possible^ lo^distill or 

 aspirate off the ammonia into standard acid, and titrate using apparatus of the' 

 types used in the micro -determinations of total nitrogen in urine (see Chapter 

 XXVII). Stehle has suggested 1 a gasometric method along the line of his 

 method for urea in urine (see Chapter XXVII). 



Interpretation. Normal blood contains 25-30 mgms. of non-pro- 

 tein nitrogen per 100 c.c. In early interstitial nephritis values of 

 30-50 may be obtained, and in severe nephritis much higher values, up 

 to the 400 mgms. occasionally found in uremia. 



The non-protein nitrogen of the blood includes nitrogen present in 

 urea, uric acid, creatinine, ammonia, and other substances. The 

 nitrogen in undetermined forms is called "rest N" and makes up about 

 46 per cent of the normal non-protein nitrogen. In uremia this 

 percentage may fall to 20. 



3. Determination of Urea. Principle. The urea is decomposed to 

 ammonium carbonate by means of the enzyme urease, in the presence 

 of phosphate, which maintains suitable reaction in the mixture. The 

 ammonia is distilled off and determined colorimetrically after Nessler- 

 ization. Alternate aeration and autoclave procedures- are given. 



Procedure. /Transfer 5 c.c. of the tungstic acid blood filtrate to a Pyrex 

 ignition tube (200 X 25 mm.) This test tube must be rinsed with nitric acid 

 and then with water if it has contained Nessler Solution. Add 2 drops of buffer 

 mixture 2 and then introduce i c.c. of urease solution. 3 Immerse the test tube 

 in warm water, 40 to 55C., and leave it there for 5 minutes, or let stand at room 

 temperature for 15 minutes. 



The ammonia formed from the urea is most conveniently obtained by distil- 

 lation, without a condenser, and using a test tube graduated at 25 c.c. and con- 



l Jour. Biol. Chem., 45, 223, 1920. According to the author, the pyrogallate treatment 

 may be dispensed with if copper sulphate is omitted. 



2 Made by dissolving 69 gm. of monosodium phosphate and 179 gm. of crystallized 

 disodium phosphate in 800 c.c. of warm distilled water and diluting to one liter. 



3 Urease solution. Wash about 3 gm. of permutit in a flask, once with 2 per cent acetic 

 acid, then twice with water; add 5 gm. of fine jack bean meal and 100 c.c. of 15 per cent 

 alcohol. Shake gently but continuously for 10-15 minutes, pour on large filter, and cover 

 with a watch glass. The solution may be kept about a week at room temperature or 

 4-6 weeks in an ice box. 



