212 Messrs. G. Graham and E. P. Poulton. The Alleged 



acetic acid produces a considerable error in the estimation of creatinine, so 

 that the result obtained is too low. 



II. A Method for Removing Aceto-acetic Acid from Urine Preliminary to the 



Estimation of Creatinine. 



In Folin's method for estimating the creatinine + creatine, the creatine is 

 converted into creatinine by heating on a water-bath for three hours with 

 normal hydrochloric acid. This procedure removes all the aceto-acetic acid 

 from the urine by converting it into acetone, which is distilled away. Thus 

 the aceto-acetic acid could not be detected by Rothera's (21)* nitroprusside 

 test after one hour's heating. Consequently the estimation of the creatinine 

 + creatine will not be disturbed by the presence of any aceto-acetic acid and 

 will be accurate, but the result of the creatinine estimation which is carried 

 out in the presence of aceto-acetic acid will be too low. Consequently the 

 result obtained for the creatinine + creatine will be higher than that for the 

 creatinine and will lead to the conclusion that creatine is present in the urine 

 whether this is actually the case or not. 



The aceto-acetic acid must, therefore, be removed from the urine before the 

 determinations are made. Greenwald (12) extracted the urine with ether for 

 two hours and found that the aceto-acetic acid was all removed by that 

 process ; the ether was subsequently removed by aeration for one hour. This 

 process involved some dilution of the urine and in order to get over this 

 difficulty Greenwald added twice the amount of picric acid and soda. This 

 method takes some time to carry out and in our experience it is better if 

 possible to avoid all dilution of the urine, especially when the urine is dilute 

 to begin with, in order to get correct results. 



The method which we employed (10) is very much simpler and we have 

 now tested it carefully and modified it slightly (11). 



1 c.c. of 10-per-cent. phosphoric acid is added to 10 c.c. of urine in a 

 boiling tube 200 mm. long and 30 mm. wide. The mixture is then heated in 

 a water-bath of which the temperature is between 65° and 70° C. and at 

 a pressure of about 210 mm. of mercury produced by means of a filter pump. 

 Bumping is prevented by allowing air to bubble slowly through the liquid by 

 means of a capillary tube dipping into it. The temperature must not rise 

 above 70° C. nor the pressure fall below 210 mm. or else concentration of the 

 urine takes place. If the above directions are followed only a few drops of liquid 

 are distilled over into the receiver, as the result of three-quarters of an hour's 

 distillation. At the end of this time the process is stopped and the solution 



* Rothera's test has been shown by Hurtley(14) to be a test for aceto-acetic acid as 

 well as for acetone. 



