49 2 



PHYSIOLOGICAL CHEMISTRY 



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air frorrA \ 

 wash bottle V- 



(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. 158), add i drop of caprylic alcohol (to prevent frothing), and i c.c. 

 of enzyme solution. 1 Close "A" with stopper shown in figure, and let the tube 

 stand 15 minutes for the enzyme to act. Measure into Tube U 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, 2 as indicator. Connect "A" and "B" as shown in the figure. At the 



end of 15 minutes aspirate for about one-half 

 minute to remove any ammonia present in the 

 free condition in "A." After this aspiration, 

 open "A" and introduce 5 c.c. of saturated 

 potassium carbonate. Close "A" at once and 

 aspirate until all the ammonia has been re- 

 moved from "A" and carried over into the acid 



in "B." The time needed for the aspiration 

 varies for different pumps from 5 to 30 minutes, 

 and should be determined by trial for the par- 

 ticular apparatus used. At the end of the time 

 needed for the aeration, 3 the pump is discon- 

 nected (care being taken to avoid back suc- 

 tion) and the excess acid in "B" is titrated by 

 means of fiftieth-normal alkali. 



Calculations. The number of cubic centi- 

 meters of fiftieth-normal acid neutralized is 

 multiplied by the factor 0.056 to give the 

 number of grams of urea-plus ammonia-nitro- 

 gen in 100 c.c. of the urine. The ammonia 

 alone may be determined at the same tune as the ammonia plus urea, using 

 the same technic except that 5 c.c. of the undiluted urine, no urease, and the 

 factor 0.0056 are used for the determination of ammonia alone. The am- 

 monia tubes are run in the same series as those for the urea determination, 

 using the same air current for all. 



(b) Colorimetric Modification. Rose and Coleman 4 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 con- 

 tent, as in the Folin-Farmer method for total nitrogen. If this procedure is fol- 

 lowed, the amount of urea and ammonia nitrogen in the solution acted upon by the 



1 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 monopotassmm-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. 



2 Folin states that methyl red is preferable to alizarin for ammonia titration s. 



3 See Fiske (Jour. Biol. Chem. 23, 455, 1915) and Van Slyke and Cullen (Jour. Biol. 

 Chem., 24, 117, 1916) for discussion of details of method. 

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



FIG. 158. VAN SLYKE AND 

 CULLEN APPARATUS. 



