44 PHYSIOLOGICAL CHEMISTRY 



under these conditions, but when due to the presence of any of these 

 substances the color does not disappear spontaneously but may re- 

 main permanent for days. Many of these disturbing substances are 

 soluble in benzene or chloroform and may be removed from the urine 

 by this means before extracting with ether as above. Acetoacetic 

 acid is insoluble in benzene or chloroform. 



3. Sodium Nitrite Ferrous Sulphate Reaction (Hurtley). Place 10 c.c. 

 of urine in a large test-tube, add 21/2 c.c. of concentrated hydrochloric acid 

 and i c.c. of fresh i per cent sodium nitrite. Shake the tube and permit it to 

 stand for two minutes. Add 15 c.c. of concentrated ammonium hydroxide and 

 5 c.c. of 10 per cent ferrous sulphate. Shake the tube and permit it to stand. 

 Note the slow development of a violet or purple color in the presence of aceto- 

 acetic acid. 



This test serves to detect ace toace tic acid when present in a dilu- 

 tion of i to 50,000. The concentration of the acetoacetic acid regulates 

 the speed at which the color develops. If the concentration be very 

 low an interval of five hours may elapse before the color appears. 

 The test is believed to be specific for acetoacetic acid. 



4. Arnold-Lipliawsky Reaction. This reaction is somewhat more delicate than 

 Gerhardt's test (2) and serves to detect acetoacetic acid when present in the pro- 

 portion of i : 25,000. It is also negative toward acetone, /3-hydroxybutyric acid 

 and the interfering drugs mentioned as causing erroneous deductions in the applica- 

 tion of Gerhardt's test. If the urine under examination is highly pigmented it 

 should be partly decolorized by means of animal charcoal before applying the test 

 as indicated below. 



Place 5 c.c. of the urine under examination and an equal volume of the Arnold- 

 Lipliawsky reagent 1 in a test-tube, add a few drops of concentrated ammonia and 

 shake the tube vigorously. Note the production of a brick-red color. Take 1-2 

 c.c. of this colored solution, add 10-20 c.c. of hydrochloric acid (sp. gr. 1.19), 3 c. c. 

 of chloroform, and 2-4 drops of ferric chloride solution and carefully mix the fluids. 

 Acetoacetic acid is indicated by the chloroform assuming a violet or blue color; if 

 acetoacetic acid is absent the color may be yellow or light red. 



H OHH 



I I I 



/3-HYDROXYBUTYRIC ACID, H C C C COOH 



I I I 

 H H H 



This acid occurs in normal urine in traces, e.g., 20-30 mg. per day. 2 

 It is found under certain pathological conditions in larger quantities 



1 This reagent consists of two definite solutions which are ordinarily preserved separately 

 and mixed just before using. The two solutions are prepared as follows: 



(a) One per cent aqueous solution of potassium nitrite. 



(b) One gram of />-amino-acetophenon dissolved in 100 c.c. of distilled water and 

 enough hydrochloric acid (about 2 c.c.) added, drop by drop, to cause the solution, which 

 is at first yellow, to become entirely colorless. An excess of acid must be avoided. 



Before using, a and b are mixed in the ratio 1:2. 



2 Shaffer and Marriott: Jour. Biol. Chem., 16, 265, 1913. 



