URINE 517. 



a thin filter paper which must not contain any appreciable amount of ammonia. 

 If there is no permutit " dust " the urine may be decanted without filtering. To 

 5 c.c. of the ammonia free filtrate add 2 c.c. of alcoholic urease solution 1 and 2 

 drops of buffer solution. 2 Allow 15 minutes for decomposition of urea by the 

 urease solution and proceed as in the Van Slyke and Cullen method. 



Calculation. The number of cubic centimeters of fiftieth-normal 

 acid neutralized by ammonia during aspiration multiplied by the 

 factor 0.056 gives the number of grams of urea nitrogen in 100 c.c. of 

 the original urine. In case the original dilution was 10 to 50 this value 

 must be divided by 2. 



Interpretation. See page 516. 



(c) Colorimetric Modification of Van Slyke and Cullen's Method. Rose and 

 Coleman 3 suggest the colorimetric determination of the ammonia which is carried 

 over by the aspiration, rather than titration of the excess of acid. They Nesslerize 

 the solution in "B," and compare the color produced with the color of a Nesslerized 

 solution of known ammonia content, as in the Folin-Farmer method for total 

 nitrogen. If this procedure is followed, the amount of urea and ammonia nitrogen 

 in the solution acted upon by the urease must not exceed 2 mg. This procedure 

 has been found useful where small quantities of urea are to be estimated. 



(d) Direct Nesslerization Method of Folin and Youngburg. 4 



Principle. The ammonia is removed from urine by permutit and 

 direct Nesslerization carried out on the ammonia free filtrate follow- 

 ing the decomposition of the urea by use of the permutit treated urease 

 solution. 



Procedure. Place i c.c. of the ammonia free urine filtrate (prepared as 

 described in the preceding method) in a test tube, add i c.c. of the alcoholic 

 urease solution, and i drop of buffer solution. Digest in a beaker of warm 

 water (4O-55C.) for 5 minutes or at room temperature for 15 minutes, at the 

 end of which tune transfer the contents of the test tube to a 200 c.c. volumetric 

 flask, diluting to a volume of about 150 c.c. Prepare a standard in another 

 200 c.c. flask by adding i mg. of N in the form of ammonium sulphate, i c.c. 

 of urease solution, and enough ammonia free water to make about 150 c.c. 

 Then add 20 c.c. of Nessler solution 5 to each flask, dilute to volume and compare 

 the two solutions in a colorimeter. 



1 To prepare the alcoholic urease solution place 3 grams of permutit in a flask, wash 

 once with 2 per cent acetic acid, then twice with water; and 5 grams of fine jack bean meal 

 and 100 c.c. of 30 per cent alcohol. Shake gently but continuously for 10 to 15 minutes 

 and filter. The filtrate contains practically all of the urease and extremely little of other 

 materials. 



2 Dissolve and make up to 1000 c.c. 142 ,gms. Na 2 HPO 4 and 120 gms. NaH 2 PO 4 or 

 equivalent amounts of the crystalline salts. 



3 Rose and Coleman: Biochem. Bull., 3, 411, 1914. 



4 Folin and Youngburg: Jour. Biol. Chem., 38, in, 1919; and Youngburg: ibid., 45, 

 319, 1921. 



6 The Nessler solution is prepared according to the procedure of Folin and Wu : Jour. 

 Biol. Chem., 38, 89, 1919, in the following manner. 



Mercuric Potassium Iodide Preparation. Transfer 150 gm. of potassium iodide and 

 no gm. iodine to a 500 c.c. Florence flask; add 100 c.c. of water and an excess of metallic 

 mercury, 140 to 150 gm. Shake the flask continuously and vigorously for 7 to 15 minutes 

 or until the dissolved iodine has nearly disappeared. The solution becomes quite hot. 



