518 PHYSIOLOGICAL CHEMISTRY 



Calculation. The standard reading divided by the reading of the 

 unknown gives the number of milligrams of urea N in 0.5 c.c. of un- 

 diluted urine. 



Interpretation. See page 516. 



(e) Marshall's Urease Method. 1 Principle. This is a simple clin- 

 ical method for the determination of urea in urine. It differs from the 

 preceding method in that instead of aspirating off the ammonia formed 

 from the urea by the action of the urease, it is titrated directly in the 

 urine mixture, thus simplifying the procedure. The method is nearly 

 as accurate as the preceding, for normal urine the error being only 

 about 2 per cent which is very satisfactory for a rapid clinical procedure. 

 For diabetic urines the aeration procedure should be used as such urines 

 contain substances which render the titration inaccurate. 



Procedure. Two 5 c.c. portions of the urine are measured into flasks of 

 200-300 c.c. capacity and diluted with distilled water to about 100-125 c.c. 

 One c.c. of a 10 per cent solution of urease 2 prepared as described on page 520 is 

 added to one flask, a few drops of toluene to each and the solution allowed to 

 remain, well stoppered, at room temperature over night (or five hours). The 

 fluid in each flask is titrated to a distinct pink color with N/io hydrochloric acid 

 using methyl orange as an indicator. A few cubic centimeters of the enzyme 

 solution used should also be titrated to determine the amount of N/io hydro- 

 chloric acid required to neutralize i c.c. 



Calculation. The amount of hydrochloric acid required for the contents of 

 the flask containing the urine and enzyme solution, less the amount used for 

 5 c.c. of urine alone and that previously determined for i c.c. of enzyme solution, 

 corresponds to the urea originally present in the sample of urine. Since i c.c. 

 of N/io HC1 is equivalent to 3 mg. of urea, the number of cubic centimeters 

 required, multiplied by 0.6 gives the value of urea expressed in grams per liter 

 of urine. 



Interpretation. Seepage 516. 



(f) Stehle's Gasometric Method for Urea. 3 Shake 25 c.c. of diluted urine 

 (diluted i : 10) with 4 grams of permutit for 4 minutes. Filter. Introduce i c.c. 

 of the nitrate into the Van Slyke CO 2 apparatus, (see p. 312), rinsing with i c.c. of 



When the red iodine solution has begun to become visibly pale, though still red, cool in 

 running water and continue the shaking until the reddish color of the iodine has been re- 

 placed by the greenish color of the double iodide. The whole operation usually does not 

 take more than 15 minutes. Now separate the solution from the surplus mercury by 

 decantation and washing with liberal quantities of distilled water. Dilute the solution 

 and washings to a volume of 2 liters. If the solution is not clear allow it to stand for i or 2 

 days before diluting with alkali to make the finished Nessler solution. 



Preparation of final Nessler Solution. From completely saturated caustic soda solu- 

 tion containing about 55 gm. of NaOH per 100 c.c. decant the clear supernantant liquid 

 and dilute to a concentration of 10 per cent. (It is well to determine by titration that the 

 error in this fo per cent solution is not over 5 per cent). Introduce into a large bottle 

 3500 c.c. of 10 per cent sodium hydroxide solution, and add 750 c.c. of the stock double 

 iodide solution and 750 c.c. of distilled water, giving 5 liters of Nessler's solution. 



1 Marshall: Jour. Biol, Chem., 14, 283, 1913. 



* In this particular method urease free from phosphate should be used as the presence 

 of these salts interferes with the production of a satisfactory end-point. 



3 Private communication from the author. 



