526 PHYSIOLOGICAL CHEMISTRY 



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

 in 100 c.c. of the urine. 



Interpretation. See page 525. 



3. Method of Frey-Gigon. The ammonia is removed from the urine by aspir- 

 ation after treatment with barium hydroxide and the formol titration performed in 

 the usual manner (see Frey and Gigon: Biochem. Zeit., 22, 309, 1909). 



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

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

 page 521, making a separate determination of ammonia, and subtracting the latter 

 result from the former. 



4. Van Slyke's Method for Total Amino-Acid Nitrogen. 1 Take 25 c.c. of 

 urine 2 and mix with i c.c. of concentrated sulphuric acid and heat in an auto- 

 clave at 180 (oil bath temperature) for one and one-half 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 

 filtrate to a Jena glass evaporating dish and concentrate to dryness on the water - 

 bath. This requires about half an hour. The residue is moistened with i c.c. 

 of 50 per cent acetic acid to bring the calcium hydroxide and carbonate into 

 solution, and is then washed into a 10 c.c. flask and filled up to the mark. One 

 can use the entire solution for determination of the amino-nitrogen in the large 

 amino-apparatus, or use 2 c.c. portions for the micro-apparatus. (See Van 

 Slyke Apparatus, Figs. 34 and 35, p. 87 in Chapter IV on Proteins.) 



The length of time which the nitrous acid solution should be shaken in order 

 to drive off all the amino-nitrogen depends somewhat on the temperature. 

 When the latter is 15-20 the time should be five to four minutes; for 20-25 it 

 is three minutes, for 25-30, two and a half to two minutes. It is preferable 

 that the solution should be shaken vigorously with a motor and the time kept 

 down to these limits, for the sake not only of rapidity but of accuracy. 



Van Slyke's Method for Free Amino-Acid Nitrogen. To 25 c.c. of urine 2 

 hi a 50 c.c. flask add urease solution and allow to stand for one and one-half 

 times the interval which has been found necessary to effect the maximum de- 

 composition of urea, as observed by titration of the ammonia. The last traces 

 of urea are decomposed. At the end of the digestion period 10 c.c. of a 10 per 

 cent suspension of calcium hydroxide are added, the mixture shaken and made 

 up to 50 c.c. Then filter, evaporate, and complete the determination according 

 to the method outlined under total amino-acid nitrogen, above. 



Creatinine 



Folin's Colorimetric Method. Principle. This method is based 

 upon the characteristic property possessed by creatinine, of yielding a 

 certain definite color-reaction in the presence of picric acid in alkaline 

 solution. The picric acid is reduced to picramic acid. 



Procedure. Place 10 c.c. of urine in a 500 c.c. volumetric flask, add 15 c.c. of 

 a saturated solution of picric acid and 5 c.c. of a 10 per cent solution of sodium hy- 

 droxide, shake thoroughly and allow the mixture to stand for five minutes. Dur- 



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



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

 containing glucose or albumin. 



