URINE 439 



acetone and acetoacetic acid may be present in the urine. Aceto- 

 aceturia is most frequently observed in children, especially accompany- 

 ing fevers and digestive disorders ; it is perhaps less frequently observed 

 in adults, but when present, particularly in fevers and diabetes it is 

 frequently followed by fatal coma. 



Acetoacetic acid is a colorless liquid which is miscible with water, 

 alcohol and ether, in all proportions. It differs from acetone in giving 

 a violet-red or Bordeaux-red color with a dilute solution of ferric 

 chloride. 



EXPERIMENTS 1 



1. Le Nobel Reaction. 2 Make 10 c.c. of urine acid with acetic acid, add a 

 few drops of a dilute aqueous solution of sodium nitroprusside and stratify con- 

 centrated ammonium hydroxide upon the mixture. In the presence of aceto- 

 acetic acid a violet ring forms at once. 



Acetone also responds to this test, but the test is more delicate for aceto- 

 acetic acid and the response is more prompt. 



2. Ferric Chloride Test (Gerhardt). To 5 c.c. of urine in a test-tube add 

 ferric chloride solution, drop by drop, until no more precipitate forms. In the 

 presence of acetoacetic acid a Bordeaux-red color is produced ; this color may be 

 somewhat masked by the precipitate of ferric phosphate, in which case the fluid 

 should be filtered. 



A positive result from the above manipulation simply indicates the possible 

 presence of acetoacetic acid. Before making a final decision regarding the pres- 

 ence of this body make the two following control experiments : 



(a) Place 5 c.c. of urine in a test-tube, small beaker, or Erlenmeyer flask 

 and boil it vigorously for 3-5 minutes. Cool the vessel and, with the boiled 

 urine, make the test as given above. As has been already stated, acetoacetic 

 acid yields acetone upon decomposition and acetone does not give a Bordeaux- 

 red color with ferric chloride. By boiling as indicated above, therefore, any 

 acetoacetic acid present would be decomposed into acetone and carbon dioxide 

 and the test upon the resulting fluid would be negative. If positive, the color 

 is due to the presence of bodies other than acetoacetic acid. 



(b) Place 5 c.c. of urine in a test-tube, acidify with H 2 SO 4 , to free aceto- 

 acetic acid from its salts, and carefully extract the mixture with ether by shaking. 

 If acetoacetic acid is present it will be extracted by the ether. Now remove 

 the ethereal solution, evaporate it to dryness, dissolve the residue in 1-2 c.c. 

 of water and add 3-5 drops of 3 per cent ferric chloride. Acetoacetic acid is 

 indicated by the production of the characteristic Bordeaux-red color. 



This color disappears spontaneously in 24-48 hours. Such sub- 

 stances as antipyrin, kairin, phenacetin, salicylic acid, salicylates, 

 sodium acetate, thiocyanates, and thallin yield a similar red color 



1 To prepare a diacetic acid solution which may be added to urine, if urines containing 

 this acid are not available, proceed as follows: Treat 13 grams of ethyl acetoacetate with 

 500 c.c. of N/5 sodium hydroxide. Allow to stand for 48 hours to hydrolyze the ester. 

 In preparing urine for tests add i part of this solution to 10 parts of urine. 



2 Harding and Ruttan: Biochem. Jour., 6, 445, 1912; also Biochem. Bull., 2, 223, 1913. 



