DETERMINATION OF UREA IN URINE 41 



Procedure. Two 5 c.c. portions of the urine are measured 

 into flasks of 200 to 300 c.c. capacity and diluted with distilled 

 water to about 100 to 125 c.c. One c.c. of a 10 per cent solution 

 of urease is added to one flask, a few drops of toluene to each 

 and the solution allowed to remain, well stoppered, at room 

 temperature over night (or five hours). The fluid in each flask 

 is titrated to a distinct pink color with N/10 hydrochloric acid 

 using methyl orange as an indicator. A few cubic centimeters 

 of the enzyme solution used should also be titrated to determine 

 the amount of N/10 hydrochloric acid required to neutralize 1 c.c. 



Calculation. The amount of hydrochloric acid required for 

 the contents of the flask containing the urine and enzyme solution, 

 less the amount used for 5 c.c. of urine alone and that previously 

 determined for 1 c.c. of enzyme solution, corresponds to the urea 

 originally present in the sample of urine. Since 1 c.c. of N/10 

 HC1 is equivalent to 3 mg. of urea, the number of cubic centi- 

 meters required, multiplied by 0.6 gives the value of urea expressed 

 in grams per liter of urine. 



DETERMINATION OF UREA IN URINE BY DIRECT 

 NESSLERIZATION * 



Principle. By using a urease preparation sufficiently free 

 from nitrogenous materials the urea nitrogen can be Nesslerized 

 without .treatment by charcoal for purposes of purification. 



Procedure. Wash 3 gms. of permutit in a flask, once with 

 2 per cent acetic acid, and twice with water; add 5 gms. of fine 

 jack bean meal and 100 c.c. of 30 per cent alcohol. Shake gently 

 but continuously for ten to fifteen minutes, and filter. The 

 filtrate contains practically the whole of the urease and extremely 

 little of other materials. Add 1 c.c. of this urease solution to 

 1 c.c. of diluted urine (dilution usually 1:10) in a test-tube, and 

 digest in a beaker of warm water (40 to 55 C.) for five minutes, 

 or at room temperature for fifteen minutes. It is preferable, 

 but not necessary, to add a drop of a suitable phosphate solution 

 to the mixed contents in the test-tube at the beginning of the 

 digestion. The buffer mixture is particularly desirable if the 

 digestion is to be made at room temperature. At the end of the 



1 Folin and Youngburg: Jour. Biol. Chem., 1919, 38, 111. 



