ANALYSIS OF TISSUES 597 



G. /J-HYDROXYBUTYRIC ACID. 



Three methods have been proposed for the estimation of /?-hydroxy- 

 butyric acid : 



(1) Conversion into crotonic acid by distillation with sulphuric acid. 

 This method originally described by Darmstaedter has not been found entirely 

 satisfactory ; Ryffel described another procedure in I9O5. 1 



(2) Conversion into aceto-acetic acid by oxidation and estimation of the 

 acetone by decomposition of the aceto-acetic acid. 



This method has been worked out by Shaffer. The results are too low 

 by about 10 per cent, as was shown later by Shaffer and Marriott 2 and also 

 by Kennaway. 8 The error appears to be constant so that the method can be 

 employed if 10 per cent, be added to the values obtained. 



In Urine. 



Shaffer and Marriott's procedure is as follows : 



25 to 100 c.c. of urine (50 c.c. average) are placed in a 500 c.c. 

 measuring flask containing 200-300 c.c. of water. A volume of basic lead 

 acetate solution (U.S. P.) equal to the volume of urine is added and 

 the liquids well mixed. A volume of strong ammonia water equal to half of 

 the basic lead acetate solution is poured in and the volume made up to 500 

 c.c. with water. The contents are mixed and filtered after standing for a few 

 minutes through a fluted filter paper. This treatment removes glucose 

 and pigments. 200 c.c. of the filtrate are measured out into an 800 or 

 looo c.c. round bottom flask, diluted to about 600 c.c. and 15 c.c. of con- 

 centrated sulphuric acid are added together with some talc or a piece of por- 

 celain. The solution is distilled and 200 c.c. distillate collected in another 

 800 c.c. flask, the end of the condenser being placed under some water in 

 this receiver. In order that the volume in the flask does not become less 

 than 400-500 c.c., water is occasionally added through a tap funnel fitted in 

 the neck of the flask. 



This distillate contains acetone from aceto-acetic acid. It is redistilled 

 after adding 10 c.c. of 10 per cent, sodium hydroxide solution and the distil- 

 late collected and titrated as described under aceto-acetic acid. 



The residue of urine is again distilled and at the same time oxidised 

 with potassium bichromate, of which '5-1*0 gm. is generally sufficient; if 

 the liquid turns green more will be required. The volume must be kept be- 

 tween 400-500 c.c. It is most convenient to keep a 10 per cent, solution of 

 bichromate and to dilute 10 c.c. to 100 c.c. for each determination. 20 c.c. 

 of the diluted solution are added through the tap funnel and alternately water, 

 and 10 c.c. portions of bichromate are added every 15-20 minutes. If the 

 liquid turns green, the bichromate must be added at shorter intervals. The 

 oxidation and distillation is continued at a moderate rate for 2-3 hours. 



The distillate is collected in a 1000 c.c. round bottom flask as before 

 and redistilled after adding 10 c.c. of 10 per cent, sodium hydroxide and 25 

 c.c. of 3 per cent, hydrogen peroxide, ithe heating being cautious until the 

 hydrogen peroxide is decomposed. Acetaldehyde obtained by the oxidation 

 of lactic acid is removed by this second distillation. This distillate is titrated 

 with iodine and thiosulphate. 



i c.c. 'iN iodine solution = 0-001793 gm. hydroxybutyric acid. 



1 J. Physiol., 32, Proc. LVI. 2 J. Biol. Chem., 1913, 16, 265. 



3 Biochem. J., 1914, 8, 230. 



