URINE 491 



at once or at least within an hour in a colorimeter, using a depth of standard of 

 2030 mm. 



Urea 



i. Urease Methods. Principle. These methods depend upon the 

 principle that the enzyme urease is able, at ordinary temperatures, to 

 transform urea, quickly and completely, into ammonium carbonate. 

 Takeuchi 1 in 1909 discovered the presence of this enzyme in the soja 

 or soy bean. The application of this enzyme to the determination 

 of urea in urine, blood, etc., was first proposed by Marshall, 2 whose 

 methods have been modified by Van Slyke and Cullen. 3 These latter 

 investigators prepared a permanent preparation of the enzyme, in a 

 water-soluble form, the use of which makes more convenient the rapid 

 and accurate determination of urea in urine, blood and other biological 

 fluids. 



The urease method is probably the most satisfactory of all methods 

 for the determination of urea. Other nitrogenous constituents such 

 as allantoin are not decomposed by urease. The method involves no 

 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 492.) 



Preparation of Solid Urease. 4 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 centrifugation. Pour this extract slowly, 

 with stirring, into at least 10 volumes of acetone. The acetone 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/SO 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 



1 Takeuchi : Journ. Coll. Agr. Tokyo, 1909, Part i. 



2 Marshall, E. K., Jr.: /. Biol Chem., 14, 283, 1913; 15, 495, 1913; 15, 487, 1913; 17, 

 351, 1914. 



3 Van Slyke, D. D., and Cullen, G. E.: /. Am. Med. Ass'n, 62, 1558, 1914. See, also, 

 /. BioL Chem., 19, 141, 1914. 



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

 of Urease may be obtained from the Arlington Chemical Company, Yonkers, N. Y., and 

 from Hynson, Westcott and Co., Baltimore. 



