URINE 



515 



carefully regulated heating procedures, and is applicable to diabetic 

 urines. 



The procedure for the determination in urine consists in treating 

 the urine sample with urease, aerating the ammonia formed into fiftieth- 

 normal acid, and titrating the excess of acid with fiftieth-normal 

 alkali. (For colorimetric procedure see page 517.) 



Preparation of Solid Urease. 1 Digest one part of soy bean meal with five parts 

 of water at room temperature, with occasional stirring, for an hour, and clear the solu- 

 tion by filtration through paper pulp or centri- 

 fugation. Pour this extract slowly, with stirring, 

 into at least 10 volumes of acetone. The ace- 

 tone dehydrates the enzyme preparation. Filter, 

 dry in vacuum, and powder. The activity of 

 the preparation is retained indefinitely. Thus 

 prepared it is not perfectly soluble in water, but 

 this fact interferes in.no way with its use. 



Standardization of the Enzyme Preparation. 

 Make up accurately a 3 per cent solution of 

 pure urea. Treat this solution exactly as the 

 urine is treated in the following method, using 

 % c.c. of the solution. The ammonia formed 

 should neutralize 25 c.c. of N/5o acid. If it does 

 so the preparation is of sufficient strength to use 

 as indicated. If not, more of the preparation 

 must be used for a determination. 



The ground soy bean may also be used 

 directly in this determination. It should pass 

 through a 2o-mesh sieve. Rose and Coleman for 

 their micro-procedure (see below) use 0.2-0.4 gram of bean flour acting in a 

 water-bath at 50-60 for five minutes. In their macro-method, using 5 c.c. of 

 urine they dilute with 30 c.c. of water warmed to 50-60 and then add 5 grams 

 of the soy bean flour and let stand for 30 minutes. They then add 5 c.c. of 

 saturated sodium carbonate solution and aerate as usual. 



(a) Procedure of Van Slyke and Cullen. Dilute 5 c.c. of urine to 50 c.c. with 

 ammonia-free water. Measure 5 c.c. of the diluted urine into Tube "A" (see 

 Fig. 1 66), add i drop of caprylic alcohol (to prevent frothing), and i c.c. 

 of enzyme solution. 2 Close "A" with stopper shown in figure, and let the tube 

 stand 15 minutes for the enzyme to act. Measure into Tube "B" 25 c.c. of N/SO 

 HC1 or H 2 SO 4 . Add i drop of caprylic alcohol and i drop of a i per cent alizarin 

 solution, 3 as indicator. Connect "A" and "B" as shown in the figure. At the 



x Van Slyke and Cullen: Jour. Biol. Chem., 19, 211, 1914. Satisfactory preparations 

 of Urease in powder or tablet form may be obtained from the Arlington Chemical Company, 

 Yonkers, N. Y., and from^Hynson, Westcott and Dunning, Baltimore. 



2 The enzyme solution is prepared by dissolving 2 grams of the enzyme preparation, 0.6 

 gram of dipotassium-hydrogen phosphate, and 0.4 gram of monopotassium-dihydrogen 

 phosphate in 10 c.c. of water. Solution is aided by stirring with a glass rod. The slightly 

 opalescent solution should be covered with toluol and may be kept for two weeks without 

 losing activity. 



3 Folin states that methyl red is preferable to alizarin for ammonia titrations. 



FIG. 166. VAN SLYKE AND 

 CULLEN APPARATUS. 



