594 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



The urine, 20 c.c., or more if it contain little acetone, is mixed with 

 150 c.c. water and 2 c.c. of 50 per cent, acetic acid and distilled for 25 

 minutes ; during this time about 60 c.c. of liquid should pass over ; the 

 vapours are cooled in an efficient condenser, through which a rapid stream 

 of water must be passed, and collected in a 150 c.c. water. The end of the 

 condenser should reach below the surface of the water as described above. 



The titration is carried out with this distillate. 30 c.c. of 33 per cent, 

 sodium hydroxide solution and excess of 'iN iodine solution from a burette 

 are added and the liquids mixed by gentle shaking. The presence of excess 

 of iodine can be ascertained by the appearance of a brown coloration at the 

 point of contact on adding a drop of hydrochloric acid. lodoform separates 

 out. After 5 minutes the contents are acidified with 25 per cent, hydrochloric 

 acid and titrated with 'iN sodium thiosulphate solution using starch as 

 indicator. 



Alcohol, if present in the urine, would pass over with the acetone, but it 

 is converted into iodoform very slowly in the cold. Its presence scarcely 

 affects the estimation. 



Acetaldehyde, which may arise by decomposition of substances in the 

 urine, seriously affects the estimation. It is removed by redistilling the dis- 

 tillate with sodium hydroxide solution and 20 c.c. of hydrogen peroxide solu- 

 tion. It is not present in the distillate except under special circumstances. 



In Tissues. 



The estimation of acetone and aceto-acetic acid in tissues is carried out 

 in a similar way: 150 c.c. of blood or 150 gm. of minced tissue are mixed 

 with 4-5 volumes of water, acidified and distilled. 



To avoid bumping during the distillation it is advisable to remove the 

 protein. The blood or tissue is mixed with an equal volume of water and 

 twice the volume of 2 per cent, hydrochloric acid and 5 per cent, mercuric 

 chloride solution and well stirred. The solution is filtered when the pre- 

 cipitate has settled, or after about 1 2 hours, and the estimation carried out 

 with 500 c.c. of the filtrate. 



Marriott also gives a method in conjunction with his method for estimating 

 hydroxybutyric acid in tissues (see p. 598). 



Small Amounts. 



Small amounts of acetone are best estimated by the method of Scott- 

 Wilson. 1 The acetone is distilled from the solution and precipitated as a 

 keto-mercury cyanide compound 2 : 



C 3 H 6 + 2Hg(CN) 2 + 3 HgO = C 3 OHg 5 (CN) 4 + 3H 2 O. 



The mercury contained in this compound is estimated by titration with 

 thiocyanate after oxidation of the organic matter. 



The liquid (100 c.c. normal urine) is placed in a flask of about 500 c.c. 

 capacity together with i c.c. of concentrated sulphuric acid and 25 gm. of 

 anhydrous sodium sulphate. The flask is provided with a 2 -holed rubber 

 cork ; through one opening a bent tube reaching to the bottom of the flask is 

 inserted ; at its outer end it is fitted with a piece of rubber tube closed by a 

 piece of glass rod ; through the other opening passes a bent tube connecting 

 this flask to another of similar capacity and reaching to the bottom of the 

 latter. This flask contains 10 c.c. of 40 per cent, sodium hydroxide solution 

 and is connected by tubing to a condenser. By means of an adapter the 

 distillate from the condenser is conducted below the surface of the reagent 3 

 contained in a conical flask. The liquids in the flask are heated to boiling, 

 that containing the alkali being made to reach this temperature sooner than 

 the other so as to prevent condensation of vapours in it. 



1 J. Physiol., IQII, 42, 444; cf. Marriott, J. Biol. Chem., 1913, 16, 281. 



<J The precipitation is effected by the following reagent which is prepared by dissolving 

 10 gm. of mercuric cyanide in 600 c.c. of water and adding 180 gm. of sodium hydroxide in 

 600 c.c. of water. To the co d solution are added slowly with constant stirring 400 c.c. of 

 a 7268 per cent, solution of silver nitrate (2~g gm. in 400 c.c. of water). The solution ia 

 set aside for about 3 days and the supernatant liquid poured off from the sm.aH sediment. 



a 30 c,c. are required for each merm, of acetone. 



