ADVANCED CHEMICAL PHYSIOLOGY 317 



0-03 mg. creatinine and this amount plus 15 c.c. water represents the 

 standard required for most human bloods, approximately 1-2 mg. per 

 100 c.c. If blood is known to be rich in creatinine a smaller volume of 

 blood filtrate may be used. 



Calculation. The reading of the standard in mm. (usually 20) 

 multiplied by 1 -5 and divided by the reading of the unknown in mm. 

 gives the amount of creatinine in mg. in 100 c.c. blood. As standard is 

 made up to twice the volume of the unknown 5 c.c. while actually 

 containing 0-03 mg. creatinine corresponds to 0-015 mg. in the blood 

 nitrate. 



Folin recommends that in all cases where the colorimeter is used a 

 preliminary comparison be always made between two amounts of the 

 standard solution employed in order to ascertain that the two fields 

 of the colorimeter are equal. 



Determination of Total Creatinine (Creatine and Preformed Creatin- 

 ine). Place 5 c.c. of the blood filtrate in a test tube graduated at 

 25 c.c. Add 1 c.c. normal hydrochloric acid. Cover mouth of test tube 

 with tinfoil and heat in autoclave at 130 C. for twenty minutes (155 C. 

 for ten minutes suffices). Cool, then add 5 c.c. of the alkaline picrate 

 solution as above, let stand eight to ten minutes and then dilute to 

 25 c.c. The standard solution is 10 c.c. of the creatinine solution in a 

 50 c.c. flask, add 2 c.c. normal HC1 and 10 c.c. alkaline picrate. Allow 

 to stand for ten minutes, then dilute to 50 c.c. 



Calculation. Reading of standard in mm. (usually 20) divided by 

 the reading of the unknown and multiplied by 6 gives total creatinine in 

 mg. in 100 c.c. blood. Average value is 56 mg. per 100 c.c. 



Determination of Uric Acid. 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. Stir with a very fine glass rod. Centrifuge ; 

 add a drop of the silver lactate solution to the supernatant fluid, which 

 should remain clear. Decant off as completely as possible the clear 

 fluid and then add to each tube 1 c.c. of a 10 per cent, solution of sodium 

 chloride in 0-1 normal HC1 ; stir thoroughly. Then add 5-6 c.c. water, 

 again stirring, and the centrifuge till clear. (Addition of salt solution 

 frees uric acid from precipitate. ) Transfer the two supernatant fluids to 

 a 25 c.c. volumetric flask by decantation, add 1 c.c. 10 per cent, sodium 

 sulphite solution, 0-5 c.c. 5 per cent, sodium cyanide solution and 3 c.c. 

 20 per cent, sodium carbonate solution. 



Prepare now two standard solutions of uric acid containing different 

 amounts of uric acid. Add 1 c.c. standard uric acid solution 1 to one 

 50 c.c. volumetric flask and 2 c.c. to another similar flask. To the 

 first flask add in addition 1 c.c. of 10 per cent, sodium sulphite solution 

 and then to each flask 4 c.c. of the acid solution of sodium chloride, 

 1 c.c. of the sodium cyanide solution, 6 c.c. of the sodium carbonate 

 solution and finally dilute with water to about 45 c.c. Solutions 

 contain 0-1 or 0-2 mg. uric acid. 



Now add to the standard solutions and to the unknown 0-5 c.c. of the 

 uric acid phosphotungstic reagent (v. p. 283) to the unknown and 1 c.c. 



1 Standard Uric Acid Solution. Dissolve 1 gm. uric acid in 150 c.c. water by 

 help of 0-5 gm. lithium carbonate in a 500 c.c. flask. Dilute to 500 c.c. and 

 mix. Of this solution take 50 c.c. in a litre flask, add 500 c.c. 20 per cent, 

 sodium sulphite solution, dilute to mark and mix. Store in tightly stoppered 

 bottles. 



