ANALYSIS OF TISSUES 593 



F. ACETO-ACETIC ACID. 



Since acetone is usually present in urine and tissues together with aceto- 

 acetic acid, the estimation gives the amount of these two compounds together. 

 Acetone may be estimated separately by the method of Folin l or of Embden 

 and Engel.' 2 The separation and estimation of acetone is usually not required 

 as it arises by the decomposition of aceto-acetic acid. 



In Urine. 



The procedure usually adopted in the estimation is that of Messinger as 

 applied by Huppert (Messinger-Huppert). Since urine on distillation with 

 acid may yield phenol, ammonia, nitrous acid and formic acid, substances 

 which also react with iodine, the distillation is carried out with acetic acid 

 to prevent the hydrolysis of phenol sulphuric acid ; the distillate is redistilled 

 after shaking it with calcium carbonate to remove nitrous and formic acids 

 and with sulphuric acid to retain the ammonia. 



20 c.c. of diabetic urine (human) ^ 



50-100 c.c. of febrile ,, I are the quantities usually 



,, of urine passed during starvation j required. 



500 c.c. of normal urine J 



The urine is placed in a flask of about 600 or yog c.c. capacity. 2 c.c. 

 of 50 per cent, acetic acid for every 100 c.c. of urine are added and distilled 

 water, if necessary. The flask is connected with a condenser which is most 

 conveniently arranged in a vertical position so that the end may be made to 

 dip beneath the surface of some water contained in another flask of 600 c.c. 

 used as receiver. The contents of the flask are distilled until they become 

 almost dry and |- have passed over. The distillate is shaken with 10 gm. of 

 calcium carbonate ; 2 c.c. of dilute sulphuric acid (2N) are added and it is 

 again distilled. To prevent frothing about 20 c.c. of liquid paraffin may be 

 added. 



The distillate is collected below the surface of some water made faintly 

 alkaline with sodium hydroxide and contained in a 1000 c.c. bottle which 

 can be closed with a ground glass stopper ; 4 are again distilled over. 



The acetone in the distillate is converted into iodoform by adding 30 c.c. 

 of 15 per cent, sodium hydroxide and excess of *iN iodine solution, shaking 

 for J of a minute and allowing to stand for 15 minutes in the closed bottle. 

 The presence of excess of iodine may be ascertained by adding a drop of 

 hydrochloric acid ; a brown coloration due to separation of iodine will 

 appear. If insufficient has been added, a further quantity must be run in and 

 allowed to react for 5 minutes. 



The excess of iodine is determined by acidifying the solution with hydro- 

 chloric acid and titrating with 'iN sodium thiosulphate solution until the 

 solution is only slightly yellow. Starch solution is added and the titration 

 continued until the blue colour disappears and a clear or milky white solution 

 results on standing for i minute. 



102 gm. aceto-acetic acid ) 

 58 |m. acetone } = 762 gm. I. 



i c.c. -iN iodine solution = '0127 gm. iodine. 



= '0017 gm. aceto-acetic acid. 

 = -000967 gm. acetone. 



A blank experiment should be made with the reagents on account of the 

 presence of sodium nitrite in some samples of caustic soda. 



A rather more complicated manipulation is used by Shaffer and Marriott 

 who subsequently estimate hydroxybutyric acid (see p. 597). 



Embden has found that the same result is obtained by a single short 

 distillation from acid solution. By thus avoiding the high concentration of 

 the urine, glucose, if present, is not decomposed with the formation of pro,- 

 ducts which absorb iodine. The procedure is the following : 



3 J. Biol. Chem,, 1907, 3, 177. 



*fleiu. chem. Physiol, Path,, 1908, n, 324, 



