URINE 505 



Procedure. Measure into a 500 c.c. Erlenmeyer flask 200 c.c. of a 24-hour 

 urine which has been diluted to 2000 c.c. (or its equivalent). Add an equal volume 

 of 10 per cent phosphotungstic acid (Merck) 1 in 2 per cent HCL Let stand at least 

 three hours, better over night. Pour off 250 c.c. of the clear fluid, add i c.c. of a 

 0.5 per cent solution of phenolphthalein and then barium hydroxide in substance 

 until the whole fluid turns decidedly pink. The barium hydroxide should be added 

 a very little at a time. Let stand one hour. Filter off two 100 c.c. samples (=50 

 c.c. urine). Neutralize these samples to litmus (using good quality litmus paper) 

 with N/5 HC1. Add at once 10-20 c.c. of neutral formalin 2 and titrate cautiously, 

 to a deep red color, i.e., until the drop produces no additional color with N/io 

 NaOH. Deduct from the result thus obtained the amount of N/io NaOH neces- 

 sary to produce the same depth of color in an equal quantity of water, freed from 

 carbon dioxide by boiling and cooling, and to which an equal volume of neutral 

 formalin has been added. 



Calculation. One c.c. of N/io NaOH is equivalent to 1.4 mg. of amino-acid 

 nitrogen. Multiply the number of cubic centimeters of N/io NaOH used (after 

 deducting for control as indicated above) by 1.4 and by 2 (as the equivalent of 50 

 c.c. of urine was used) to obtain the number of milligrams of amina-acid nitrogen 

 in 100 c.c. of the urine. 



Interpretation. See page 504. 



3. Method of Frey-Gigon. 3 Principle. The ammonia is removed from the 

 urine by aspiration after treatment with barium hydroxide and the formol titration 

 performed in the usual manner. 



Procedure. Treat 50 c.c. of urine in an aerometer cylinder such as used in the 

 Folin ammonia method, with 20 c.c. of saturated barium hydroxide solution and 

 15 c.c. of alcohol. Aspirate for two or three hours, using a slow current of air. 

 The ammonia is carried off. (It may be collected in standard acid solution and 

 determined as in the Folin method (see page 499) if desired.) Transfer the urine 

 mixture quantitatively to a 250 c.c. flask and make to mark with distilled water. 

 Shake well, allow to settle, filter. Take 100 c.c. of the nitrate and just neutralize 

 with N/5 hydrochloric acid, using rosolic acid as an indicator. Then to another 

 100 c.c. portion of the filtrate add an amount of the standard acid equal to that 

 added to the first portion. Next add 10 c.c. of neutral formalin solution, a few 

 drops of phenolphthalein and titrate in the usual manner to a red-violet color. 

 Calculate as in the preceding method. 



The amino-acid nitrogen may also be approximately determined by carrying 

 out the titration for ammonia + and amino-acid nitrogen as given under Ammonia, 

 page 502, making a separate determination of ammonia, and subtracting the latter 

 result from the former. 



4. Van Slyke's Method for Total Amino-Acid Nitrogen. 4 Take 25 c.c. of 

 urine 5 and mix with i c.c. of concentrated sulphuric acid and heat in an auto- 

 clave at 1 80 (oil bath temperature) for one and one-halt hours. Transfer to a 

 50 c.c. flask and add 2 grams powdered calcium hydroxide. Shake thoroughly, 

 make up to 50 c.c. and filter through a dry folded filter. Transfer 20 c.c. of the 



1 Kahlbaum's preparation is a very different substance. 



2 To 50 c.c. commercial formalin solution (30^-40 per cent) add i c.c. of phenolphthalein 

 solution and then N/5 NaOH to a very faint pink color. The solution should be freshly 

 prepared. 



3 Frey and Gigon: Biochem. Zeit., 22, 309, 1909. 



4 Van Slyke: Jour. Biol. Chem., 16, 125, 1913. 



5 See (Van Slyke: Proc. Soc. Exp. Biol. and Med., 13, 63, 1915) for treatment of urines 

 containing glucose or albumin. 



