402 PHYSIOLOGICAL CHEMISTRY. 



acetone by Folin's method (see p. 400). Subtract the value deter- 

 mined by the second method from that obtained in the first method 

 to secure data regarding the diacetic acid content of the urine, in 

 terms of acetone. 



XVII. /?-Oxybutyric Acid. 



i. Shaffer's Method. Introduce 25-250 c.c. of urine 1 into a 

 500 c.c. volumetric flask and add an excess of basic lead acetate and 

 10 c.c. of concentrated ammonium hydroxide. Dilute the mixture 

 to the 500 c.c. mark, shake the flask thoroughly and filter. Transfer 

 200 c.c. of the filtrate to an 800 c.c. Kjeldahl distilling flask, add 

 300-400 c.c. of water, 15 c.c. of. concentrated sulphuric acid and a 

 little talcum and distil the mixture until 200 to 250 c.c. of distillate 

 has been collected (A). 2 To this distillate (A), which contains 

 acetone (both preformed and that produced from diacetic acid), 

 and volatile fatty acids is added 5 c.c. of 10 per cent potassium 

 hydroxide and the distillate redistilled in order to remove the vola- 

 tile fatty acids. 3 This second distillate (A 2 ) is then titrated with 

 standard iodine and thiosulphate (see p. 399). The urine-sulphuric 

 acid residue from which distillate A was obtained, is again distilled, 

 400-600 c.c. of a 0.1-0.5 per cent potassium bichromate solution 

 being added, by means of the dropping tube, during the process of 

 distillation. 4 In adding the bichromate, care should be taken not 

 to add it faster than the distillate collects except in cases where 

 the boiling fluid assumes a pure green color, thus indicating that 

 the bichromate is being used up more rapidly. After about 500 

 c.c. of distillate (B) has collected 20 c.c. of a 3 per cent solution 

 of hydrogen peroxide and a few cubic centimeters of potassium 



1 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 jS-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 duplicate determina- 

 tions. It is desirable to use *such a volume of urine as contains the proper 

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



2 This distilling flask should be provided with a dropping tube, by means of 

 which water may be introduced in order to prevent the contents of the flask 

 from becoming less than 400 c.c. in volume. Care should be taken to use a 

 good condenser in the distillation, but it is not necessary to cool the distillate 

 with ice. 



3 Formic acid is one of the most troublesome. 



4 Generally the addition of 0.5 gram of potassium bichromate is sufficient. In 

 case the urine contains a high concentration of sugar or when a large volume 

 of urine is used, it may be necessary to use 2-3 grams of the bichromate. 



