276 PHYSIOLOGICAL CHEMISTRY 



after coagulation in dilute acetic acid. Myers and Fine 1 suggest the 

 use of aluminium cream for this purpose. Benedict's procedure is as 

 follows: 



To 20 c.c. 2 of oxalated blood are added 100 c.c. of boiling N/ioo acetic acid 

 in a casserole, and the mixture is heated to boiling for a moment. Remove the 

 casserole from the flame and add 200 c.c. of boiling distilled water. Pour the 

 mixture upon a folded filter and wash the residue with 50 c.c. of boiling water 

 (heated in the same casserole hi which the original coagulation took place). 

 Transfer the whole filtrate to a casserole and boil down rapidly to a volume of 

 about 25 c.c. Pour this solution into a small flask roughly marked to indicate a 

 volume of 50 c.c. Transfer the contents of the casserole to the flask quantita- 

 tively, with the help of two or three portions of water, heating vigorously to 

 boiling and rubbing the sides of the casserole with a rubber-tipped stirring rod 

 each time. The total volume in the flask should not exceed 50 c.c. after the ad- 

 dition of the washings. Thoroughly cool the turbid solution in the flask under 

 nmning water, and add 2 c.c. 3 of colloidal iron solution (Merck's "Dialyzed Iron," 

 5 per cent solution) while the flask is being gentry rotated. Filter the mix- 

 ture through a small folded filter into a 100 c.c. Jena Florence flask, and wash 

 the residue twice with distilled water. The filtrate obtained here should be as 

 clear and colorless as distilled water. Boil the solution down to a volume of from 

 i to 2 c.c. (care being taken hi the early stages to prevent bumping), then care- 

 fully pour into a small centrifuge tube and wash out the flask with three portions 

 of water (i or 2 c.c. each), heating each to boiling in the flask and shaking thor- 

 oughly prior to transferring it to the centrifuge tube. The volume of liquid in the 

 tube at this point should be from 5 to 10 c.c. Cool the liquid, add 20 drops of the 

 ammoniacal silver magnesium solution and proceed with the determination as 

 described under Urine (Chapter XXVI). If the amount of uric acid present is 

 very small the addition of i drop of cyanide solution, i c.c. of uric acid re- 

 agent, 5 c.c. of 20 per cent sodium carbonate solution, and dilution to 25 c.c. 

 are carried out rather than using the larger quantities given for the determina- 

 tion hi the urine. 



4. Creatine and Creatinine. Methods ofFolin. 4 Preformed Creatinine. Meas- 

 ure 10 c.c. of blood into a 50 c.c. volumetric flask or, better, into a 50 c.c. shaking 

 cylinder which can be closed with a glass stopper. Fill to the 50 c.c. mark with satu- 

 rated picric acid solution and shake a few times. Add about i gram of dry picric 

 acid to the mixture and shake for five minutes. Transfer the mixture to centrifuge 

 tubes, throw down the sediment and precipitate and pour the supernatant liquid 

 through a filter. This- is the most economical process where but little blood is avail- 

 able. If desired, however, double quantities of blood and reagents may be taken and 

 filtration carried out without preliminary centrifugation. This process removes the 

 protein materials and leaves the creatine and creatinine in the filtrate which is a satu- 



1 Myers and Fine: "Blood in Health and Disease," 1915, p. 14. 



2 Smaller amounts of blood may be employed, and the quantity of acetic acid and 

 water correspondingly reduced. Unless the quantity of uric acid present is very large, 

 the results are far more accurate when 20 c.c. of blood are used. 



3 With old samples of blood it may be necessary to add 3 or 4 c.c. of the iron solution 

 and a little 10 per cent sodium chloride solution. When the precipitate separates in large 

 flocculent masses the right amount of iron has been added. Any excess of iron must be 

 avoided, as it would oxidize some of the uric acid later on in the process. 



4 Folin: Jour. Biol. Chem.. 17, 475, 1914. 



