456 PHYSIOLOGICAL CHEMISTRY 



with ether by stirring and decantation. Any /?-hydroxybutyric acid present 

 will be extracted by the ether. Evaporate the ether extract spontaneously or 

 on a water-bath, dissolve the residue in water, and neutralize it with barium 

 carbonate. To 5 to 10 c.c. of this neutral fluid hi a test-tube add 2 to 3 drops 

 of ordinary commercial acid hydrogen peroxide. Mix by shaking and add a 

 few drops of Black's reagent. 1 Permit the tube to stand and note the gradual 

 development of a rose color which increases to its maximum intensity and then 

 gradually fades. 2 



In carrying out the test care should be taken to see that the solution 

 is cold and approximately neutral and that a large excess of hydrogen 

 peroxide and Black's reagent are not added. In case but little 

 /3-hydroxybutyrlc acid is present the color will fail to appear or will 

 be but transitory if the oxidizing agents are added in too great excess. 

 It is preferable to add a few drops of the reagent and at intervals of 

 a few minutes repeat the process until the color undergoes no further 

 increase in intensity. One part of /3-hydroxybutyric acid in 10,000 

 parts of the solution may be detected by this test. 



2. Polariscopic Examination. Subject some of the urine (free from protein) 

 to the ordinary fermentation test (see page 437). This will remove glucose 

 and fructose, which would interfere with the polariscopic test. Now examine the 

 fermented fluid in the polariscope and if it is levorotatory the presence of /?-hy- 

 droxybutyric acid is indicated. This test is not absolutely reliable, however, since 

 conjugate glycuronates are also levorotatory after fermentation. 



CONJUGATE GLYCURONATES 



Glycuronic acid does not occur free in the urine, but is found, for 

 the most part, in combination with phenol. Much smaller quantities 

 are excreted in combination with indoxyl and skatoxyl. The total 

 content of conjugate glycuronates seldom exceeds 0.004 P er cen t under 

 normal conditions. The output may be very greatly increased as 

 the result of the administration of antipyrin, borneol, camphor, 

 chloral hydrate, menthol, morphine, naphthol, turpentine, etc. The 

 glycuronates as a group are levorotatory whereas glycuronic acid is 

 dextrorotatory. Most of the glycuronates reduce alkaline metallic 

 oxides and so introduce an error in the examination of urine for sugar. 

 Conjugate glycuronates often occur associated with glucose in glyco- 

 suria, diabetes mellitus, and in some other disorders. As a class the 

 glycuronates are non-fermentable. 



EXPERIMENTS 



i. Naphthoresorcinol Reaction (Tollens). Introduce 5 c.c. of urine in a 

 test-tube and add 0.5-1 c.c. of a i per cent solution of naphthoresorcinol in 



1 Made by dissolving 5 grams of ferric chloride and 0.4 gram of ferrous chloride in 100 

 c.c. of water. 



1 This disappearance of color is due to the further oxidation of the acetoacetic acid. 



