PRACTICAL EXERCISES 519 



mixed, 2 drops of caprylic alcohol are added to prevent foaming during 

 the subsequent aeration (by Folin's method, p. 521), and the stopper 

 bearing the aerating tubes is put into place. Let stand twenty minutes 

 at room temperature of 15, or fifteen minutes at 20 C. or above, for 

 complete decomposition of urea. This time must not be shortened 

 unless more enzyme is used, but no harm is done if it is longer. Mean- 

 while measure into tube B 25 c.c. |^ hydrochloric or sulphuric acid, 

 i drop of i per cent, sodium alizarine sulphonate indicator, and 

 i drop caprylic alcohol, and connect the 

 tubes as shown in Fig. 194. After the 

 twenty minutes for decomposition of urea 

 has elapsed, the air current is passed for 

 half a minute to sweep into B, a small 

 amount of ammonia which has escaped 



into the air space of A during the decom- 

 position. Then A is opened, and 4 to 



5 grammes potassium carbonate added to 



liberate the ammonia from the ammonium 



carbonate formed. Now let the air cur- 

 rent pass gently through the tubes for 



the first minute and then rapidly until 



all the ammonia has been brought into 



the acid in B. The time necessary for 



this will depend upon the speed of the 



air current (varying from five minutes to 



half an hour, according to the efficiency 



of the pump or vacuum by which the air 



is moved) and should be determined once 



for all. The excess- acid in B is now 



titrated with ^ sodium hydroxide. The 



Fig. 194. Apparatus for deter- 

 mining Urea Content by means 

 of Urease. (After Van Slyke.) 



weight of nitrogen contained in the urea of 100 c.c. of urine is 

 (25 - x) x 0-056, where x is the number of c.c. of the sodium hydroxide 

 solution necessary to neutralize the acid. Included in the urea 

 nitrogen is the nitrogen of any ammonia originally present in the 

 urine. This can be determined separately, if desired, by putting 

 5 c.c. of urine into A without urease, adding the carbonate at once, 

 and then aerating through the acid in B. The acid neutralized in 

 this case is multiplied by 0-0056 to give the ammonia nitrogen in 100 c.c. 

 of urine, and this being deducted from the previous result, the net urea 

 nitrogen is obtained. 



The method as above described is adapted for urine containing not 

 more than 3 per cent, urea, which is about the maximum found in human 

 urine. Cat's or dog's urine should first be diluted to reduce the urea 

 below 3 per cent. 



Clinical Urease Method by Direct Titration of Urine. Two 5 c.c. 

 portions of urine are put into flasks a and b of 200 to 300 c.c. capacity, 

 and diluted with distilled water to about 100 to 125 c.c. One c.c. of 

 10 per cent, urease is added to flask a, and a few drops of toluene to 

 each. The flasks are allowed to remain, well stoppered, at room tempera- 

 ture over night, then titrated to a distinct pink colour with ^ hydro- 

 chloric acid, using methyl orange as indicator. From the amount of 

 hydrochloric acid needed for a is deducted the amount needed for b; 

 and also the amount previously determined to be necessary to neutra- 

 lize the alkalinity of the enzyme solution. The remaining number of 

 c.c. multiplied by 0-6 gives the urea in gvammes per litre of urine, since 



