282 PHYSIOLOGICAL CHEMISTRY 



is set free by means of chloride solution and determined colorimetric- 

 ally after addition of phosphotungstic acid which gives a blue solution. 



Procedure.^l-To 10 c.c. of blood filtrate in each of two centrifuge tubes add 

 2 c.c. of a 5 per cent solution of silver lactate in 5 per cent lactic acid, and stir 

 with a very fine glass rod. Centrifuge ; add a drop of silver lactate to the super- 

 natant solution, which should be almost perfectly clear and should not become 

 turbid when the last drop of silver solution is added. Remove the supernatant 

 liquid by decantation as completely as possible. Add to each tube i c.c. of a 

 solution of 10 per cent sodium chloride in o.i normal hydrochloric acid and stir 

 thoroughly with the glass rod. Then add 5 to 6 c.c. of water, stir again, and 

 centrifuge once more. By this chloride treatment the uric acid is set free from 

 the precipitate. Transfer the two supernatant liquids by decantation to a 25 c.c. 

 volumetric flask. Add i c.c. of a 10 per cent solution of sodium sulphite, 0.5 

 c.c. of a 5 per cent solution of sodium cyanide, and 3 c.c. of a 20 per cent solution 

 of sodium carbonate. Prepare simultaneously two standard uric acid solutions 

 as follows : 



Transfer to one 50 c.c. volumetric flask i c.c. and to another 50 c.c. flask 

 2 c.c. of the standard uric acid sulphite solution described above. To the first 

 flask add also i c.c. of 10 per cent sodium sulphite solution. Then add to each 

 flask 4 c.c. of the acidified sodium chloride solution, i c.c. of the sodium cyanide 

 solution, and 6 c.c. of the sodium carbonate solution. Dilute with water to 

 about 45 c.c. When the two standard solutions and the unknown have been 

 prepared as described they are ready for the addition of the uric acid reagent. 

 Add 0.5 c.c. of this reagent to the unknown and i c.c. to each of the standards, 

 and mix. Let stand for 10 minutes, fill to the mark with water, mix, and make 

 the color comparison.) 



Calculation. In connection with the calculation it is to be noted (a) that 

 the blood filtrate taken corresponds to 2 c.c. of blood, (b) that the standard is 

 diluted to twice the volume of the unknown, and (c) that the standard used con- 

 tains o.i or 0.2 mg. of uric acid. The blood filtrate from blood containing 

 2.5 mg. of uric acid will be just equal in color to the weaker standard. Twenty 

 times 2.5 divided by the reading of the unknown gives, therefore, the uric acid 

 content of the blood when the weaker standard is set at 20 mm. 



The uric acid may sink to as low as i mg. of uric acid per 100 c.c. of blood. 

 It seems hardly worth while to prepare a third and weaker standard regularly in 

 order to provide for such low acid values. 



A standard corresponding to the color obtained from 1.25 mg. of uric acid per 

 100 c.c. of blood can be prepared within a couple of minutes as follows : Transfer 



in unopened bottles, because the sulphite prevents the spontaneous oxidation of the uric 

 acid. In used bottles the standard usually remains good for 2-3 months. 



2. A 10 per cent sodium sulphite solution. 



3. A 5 per cent sodium cyanide solution, to be added from a burette. 



4. A 10 per cent solution of sodium chloride in o.i normal hydrochloric acid. 



5. A uric acid reagent prepared according to Folin and Denis. This may be made as 

 follows: Introduce into a flask 



750 c.c. of water, 

 -'* 100 g. of sodium tungstate, 



80 c.c. of phosphoric acid (85 per cent H 3 PO 4 ). 



Partly close the mouth of the flask with a funnel and small watch glass and boil gently for 

 two hours. Dilute to a liter. A still stronger reagent is obtained by heating for 24 hours, 

 instead of 2 hours; but the advantage gained, about 20 per cent, is not needed. 



6. A solution of'5 per cent silver lactate in 5 per cent lactic acid. 



