ANALYSIS OF TISSUES 599 



water in a Kjeldahl flask. The distillation and oxidation is best effected by 

 rapidly raising to the boiling-point, heating gently for 30 minutes with little 

 or no distillate passing over and then more rapidly for 15 minutes. 80 to 

 125 c.c. of distillate should be obtained in this time. 2 gm. of sodium 

 peroxide are added to the distillate and the distillation repeated until 80 c.c. 

 have passed over in about 10-15 minutes. The second distillate is collected 

 under 10 c.c. of water in a 100 c.c. measuring flask. It is diluted to 100 c.c. 

 and mixed; 25 or 50 c.c. are removed into another flask containing 25 c.c. 

 water, 15 c.c. of Scott- Wilson reagent are added and the mixture diluted to 

 100 c.c. 



At the same time -5 mgm. acetone are treated in the same way (see under 

 acetone, p. 596). 



i mgm. acetone = 1*78 mgm. hydroxybutyric acid. 



Notes. The use of sodium peroxide obviates the use of lead acetate to 

 remove glucose, glycuronic acid, etc. 



Urines containing no sugar need not be distilled twice. The distillate is 

 collected in 10 c.c. of water in a 100 c.c. measuring flask and diluted to 100 

 c.c. ; 1-2 gm. of sodium peroxide are added to it and mixed with it a few 

 times. This treatment removes the disturbing substances obtained by the 

 distillation of the urine; 25 or 50 c.c. are taken for the estimation. 



The estimation of hydroxybutyric acid in 1-5 c.c. of blood is effected by 

 Marriott in a similar way to the general method, but using a nephelometer 

 to estimate the acetone formed (see under acetone, p. 595). 



The solution after removal of the proteins could be treated equally well 

 by the method of Folin and Denis and it is a little simpler to carry out. 



(3) Extraction with ether and estimation by rotation. 



/?-hydroxy butyric acid is most frequently estimated by this method ; the 

 results are affected by the presence of other substances in the extract which 

 are also optically active, but their amounts are usually so small that they are 

 neglected. 



The urine (100-300 c.c.) may be evaporated and mixed with sand or 

 plaster to form a dry mass, which is extracted in a Soxhlet apparatus with 

 ether, according to the description of Bergell l or Black. 2 



It is preferable to extract the urine which has been acidified with sulphuric 

 aci4 with ether in a special extraction apparatus. 



(a) The urine (300-600 c.c.) is saturated with ammonium sulphate (80- 

 90 gm. per 100 c.c.), strongly acidified with sulphuric acid and ex- 

 tracted for 24, 48, or 72 hours in the extractor with ether according to the 

 rate of the current of ether. The ethereal solution is filtered into a basin and 

 the ether allowed to evaporate spontaneously or rapidly distilled off. The 

 residue is cooled, dissolved in 5-8 c.c. of water and filtered from hippuric acid 

 and oily products into a small measuring flask of 10 or 20 c.c. capacity. The 

 volume is made up and the rotation determined after clearing the solution 

 with a small quantity of "kieselguhr " or charcoal. 



(b) Hurtley 3 treats 100 c.c. urine with 40 per cent, ferric chloride solution 

 which is run in from a burette until ferric phosphate is no longer precipitated, 

 but an amount insufficient for the aceto-acetic acid reaction to appear; 

 1000 c.c. are then treated with the proportional amount. 500 c.c. of the 

 filtrate and the amount corresponding to half the number of c.c. of ferric 

 chloride added are acidified with 13 c.c. of sulphuric acid (i vol. acid + i 



1 Z. Physiol. Chem., 1901, 33, 310. 2 J. Biol. Chem., 1908-9, 5, 208. 



3 1 am much indebted to Dr. Hurtley for telling me his procedure and for much other 

 information respecting " acetone" bodies. 



