540 PHYSIOLOGICAL CHEMISTRY 



gather as acetone and determined by the iodine titration method. 

 The /3-hydroxybutyric acid remains in the residue from distillation 

 and is oxidized by means of potassium bichromate. The product 

 of the oxidation is acetone which is distilled off and determined as 

 such. 



Procedure. Determination of Acetone and Acetoacetic Acid. Measure 

 from 25-100 c.c. 1 or more of urine (usually 50 c.c.) with a pipette into a 500 c.c. 

 volumetric flask containing 200-300 c.c. of water. Add basic lead acetate 

 solution (U. S. P.) in volume equal to that of the urine used 2 and mix well. Add 

 concentrated ammonium hydroxide in amount equal to about one -half that of 

 the lead acetate solution. Dilute the contents of the flask to the mark with 

 water, shake, and after standing a few minutes filter the liquid, preferably 

 through a folded filter. Measure 200 c.c. of the filtrate into a round bottom 

 flask (800 c.c. or liter Kjeldahl flasks are convenient) dilute with water to about 

 600 c.c. and add 15 c.c. of concentrated sulphuric acid and a little talc or a boiling 

 stone. Distil until about 200 c.c. of distillate have been collected. The tube 

 of the condenser should dip beneath the surface of the water in the receiving 

 flask so that no loss of acetone will occur. The distilling flask must also be 

 fitted with a dropping tube or dropping funnel so water may be run in from time 

 to time and the volume of liquid in the flask kept from becoming less than 

 400-500 c.c. A good condenser should be used, but it is not necessary to cool 

 the distillate in ice. 



The distillate thus obtained is transferred to a second Kjeldahl flask and 

 10 c.c. of 10 per cent NaOH added. It is then redistilled for about 20 minutes. 3 

 This second distillate is then titrated with standard iodine and thiosulphate 

 solution as in the method for acetone and acetoacetic acid previously given 

 (see Messinger-Huppert method, page 535), and the calculation made in the 

 same way. The result gives the combined acetone and acetoacetic acid content 

 of the urine expressed as acetone. 



Determination of the /3-Hydroxybutyric Acid. The flask containing the 

 residue from the first distillation above is used in the determination of the 

 /3-hydroxybutyric acid. A new receiver is arranged as before with the tip from the 

 condenser dipping beneath the surface of the water. The distillation is then 

 continued and water added whenever necessary to keep the volume between 

 400 and 600 c.c. A dilute solution (i per cent) of potassium bichromate is added 

 during the distillation. At first 20 c.c. of this i per cent solution is added slowly 

 through the dropping tube and then 10 c.c. portions every 15-20 minutes until 



PI x The amount used depends upon the expected yield of /3-oxybutyric acid. In the case 

 of urines which give a strong ferric chloride reaction for diacetic acid, or when 5-10 grams 

 or more of /3-oxybutyric acid is expected, it is unnecessary to use more than 25-50 c.c. of 

 urine. However, in case only a trace of /3-oxybutyric acid is expected, the volume should be 

 much larger as indicated. Under all conditions, the amount specified is sufficient for dupli- 

 cate determinations. It is desirable to use such a volume of urine as contains the proper 

 amount of /3-oxybutyric acid to yield 25-50 mg. of acetone. 



2 If the urine contains but little or no sugar only half the amount or less of lead acetate 

 should be used. 



3 In many instances when a high degree of accuracy is not required this redistillation 

 may be omitted and the first distillate titrated directly. The results so obtained are 

 slightly higher than those after redistillation from alkali. The object of the redistillation 

 is to get rid of fatty acids of which formic acid is one of the most troublesome. 



