598 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



In Tissues. 



Marriott 1 has adapted the method for the estimation of hydroxybutyric 

 acid in tissues : 



100 c.c. blood (or 50 to 100 gin. of minced tissue) are diluted with 400 

 c.c. of water and put through a dropping funnel into a 2-3 litre flask which is 

 connected with a condenser and receiving flask containing 500 c.c. of water, 

 the end of the condenser dipping into the water. The large flask containing 

 500 c.c. of water, 3-5 c.c. of glacial acetic acid and some powdered talc is 

 heated to boiling. The blood is added at such a rate that boiling does not 

 cease. The contents are distilled until 300 c.c. distillate have passed over ; 

 a small amount of foam which may pass over is of no consequence as the 

 distillate is redistilled. The distillate contains acetone (pre-formed and from 

 aceto-acetic acid). It is redistilled after adding a little dilute sulphuric acid 

 which retains ammonia. It is again distilled after adding 20 c.c. of 3 per 

 cent, hydrogen peroxide and a slight excess of alkali. This serves to destroy 

 and hold back aldehydes, hydrogen sulphide and volatile acids. The distillate 

 is titrated with iodine and thiosulphate (see p. 593). 



The contents of the large flask whilst still hot are treated with about 15 c.c. 

 of 20 per cent, sodium carbonate solution. As soon as sufficient has been 

 added, the dark colour changes to brown and a precipitate settles, leaving a clear 

 straw-coloured liquid of amphoteric reaction. The contents of the flask are 

 boiled for 1-2 minutes, allowed to cool, transferred to a 1000 c.c. measuring 

 cylinder and diluted to 1000 c.c. The contents are mixed and filtered on 

 a Buchner funnel. 700 c.c. of the filtrate are put into a 1000 c.c. graduated 

 flask, 30 c.c. basic lead acetate solution (U.S. P.) and 15 c.c. of strong ammonia 

 are added and the volume made up to 1000 c.c. The contents are mixed, 

 allowed to stand for a time and filtered through a dry folded paper. 900 c.c. 

 of the filtrate are taken, boiled to remove the greater part of the ammonia 

 and concentrated to 500 c.c. After cooling, excess of dilute sulphuric acid is 

 added to remove the lead and then 30 c.c. of 50 per cent, sulphuric acid. 

 The solution is put in a 1000 c.c. flask furnished with a dropping funnel and 

 connected to a condenser. The contents are distilled, running in bichromate 

 so that the liquid always retains a yellow colour and a volume of about 

 400-500 c.c. ; '5 gm. bichromate is usually sufficient. Slow distillation is 

 continued for 2 hours and 600-800 c.c. of distillate are collected, the end of 

 the condenser being kept under water. The distillate is redistilled with 20 c.c. 

 of peroxide and 5 c.c. of 10 percent, sodium hydroxide. The distillate is 

 titrated with iodine and thiosulphate (p. 597). 



Micro- A nalysis. 



The oxidation method of Shaffer has been found by Folin and Denis to 

 give correct results if the amount of hydroxybutyric acid to be oxidised is about 

 2-5 mgm. 



Folin and Denis estimate hydroxybutyric acid in a similar way to acetone 

 and aceto-acetic acid (see p. 593) : 



The urine is diluted 10-50 times so that the volume to be taken con- 

 tains about 2 mgm. The volume of diluted urine is put into a 500 c.c. round 

 bottom flask with 200 c.c. of water and 5 c.c. of 10 per cent, sulphuric acid 

 and boiled for 10 minutes to remove acetone and aceto-acetic acid. 25 

 c.c. of a solution containing 2 per cent, of potassium bichromate and 35 per 

 cent, of sulphuric acid are added. The solution is distilled for 40-60 minutes, 

 using an efficient condenser, the end of which is under 75-100 c.c. oi 



1 T. Biol. Chetn.. TOI?. 16. 20*. 



