274 PHYSIOLOGICAL CHEMISTRY 



25 c.c. long-necked, round-bottomed Kjeldahl flask, the neck of which has a 

 crook near the top. Add a small piece of acid potassium sulphate, several glass 

 beads, and drive off the alcohol-ether by heating on a hot plate. When the flask 

 is nearly dry add i c.c. of concentrated sulphuric acid and not more than 300 or 

 400 mg. of potassium sulphate. Then place the flask over the direct flame of a 

 micro-burner (resting the bottom of the flask upon an asbestos board which has a 

 circular perforation a little smaller than the circle of i c.c. of sulphuric acid in the 

 digestion flask) and heat until the mixture is colorless. Transfer the digestion 

 mixture to a 100 c.c. flask, neutralize the sulphuric acid with about 3 c.c. of a 30 

 per cent solution of sodium hydroxide, fill the flask to over three-quarters full 

 with ammonia-free water, and add 5 c.c. of Winkler's modification of Nessler's 

 solution (see page 603). Dilute to mark with water and compare the color in the 

 Duboscq colorimeter (see Fig. 153, p. 486) against a standard solution of ammo- 

 nium sulphate. 



2. Urea. The Urease Method. Van Slyke and Cullen's 1 Modification of 

 Marshall's Method. 2 



Principle. See Urease Method, Chapter XXVI. 



Procedure. Run 3 c.c. of fresh blood (carefully measured with an accurate 

 pipette) into a 100 c.c. test-tube containing i c.c. of a 3 per cent solution of potas- 

 sium citrate (to prevent clotting). Add 0.5 c.c. of the urease solution 3 and 2 or 3 

 drops of caprylic alcohol (to prevent foaming). After ten minutes add 15 c.c. 

 of a saturated solution of potassium carbonate, and drive off the ammonia by 

 aspiration into another tube containing 15 c.c. of hundredth-normal hydrochloric 

 or sulphuric acid. Titrate the excess of acid with hundredth-normal sodium 

 hydroxide or potassium hydroxide, 4 using methyl red or alizarin as indicator. 



Calculations. Each cubic centimeter of acid neutralized indicates o.oi gram 

 of urea per 100 c.c. of blood, or 0.0467 gram of urea nitrogen per 100 c. c. of blood. 

 In case the blood should be one of the rare samples containing over 0.15 per cent 

 of urea, all the acid will be neutralized, and it will be necessary to repeat the 

 determinations, using in the determination only i c.c. of blood. Fresh blood 

 contains so little ammonia that it may be disregarded. For further discussion of 

 the urease method see Chapter XXVI. 



3. Uric Acid. Colorimetric Microchemical Method. Principle. 

 Folin and Denis 5 were the first to apply the technic of their method 

 for the determination of small quantities of uric acid to the quan- 

 titative estimation of uric acid in blood. (For a discussion of the 

 principle of the color reaction see the determination of uric acid in urine, 

 page 510.) The procedure necessary for the determination in blood is 

 somewhat different from that used for the determination in urine because 

 of the presence of proteins in the blood. These must be removed first, 

 and then the uric acid may be determined in the protein-free extract, 

 according to the procedure used for the determination in urine. The 



1 Van Slyke and Cullen: /. Am. Med. Ass'n, 62, 1558, 1914. 



2 Marshall: Jour. Biol. Chem., 15, 487, 1913. 



3 The enzyme solution is prepared as described under "Reagents and Solutions," p. 594- 



4 Rose and Coleman (Biochem. Bull., 3, 411, 1914) suggest the colorimetric determina- 

 tion of the ammonia (see page 492). 



5 Folin and Denis: /. Biol. Chem., 13, 469, 1913. 



