DISTURBANCES OF CARBOHYDRATE METABOLISM 297 



But with the discovery of (3-hydroxybutyric acid and of the conjugated 

 glycuronic acids it became clear that more direct evidence was required. 



In some cases, it is true, the difference between the results obtained by 

 reduction or fermentation methods and by observing the optical rotation 

 were too great to be accounted for by any likely amount of (3-hydroxy- 

 butyric acid or of conjugate glycuronic acids. But the preparation of 

 the urine for polariscopic examination may have removed some of the 

 glucose. In most cases, the urine must be clarified. Basic lead acetate 

 was formerly extensively used for this purpose and no particular attention 

 seems to have been paid as to the amount so used. It has since been 

 shown that basic lead acetate will, under certain conditions, precipitate 

 sugars. These conditions are generally realized in urine. The normal 

 acetate is less effective in this regard and, in small quantities, may be used 

 in acid urine without danger of loss of sugar. 



Still another source of error is the faulty character of the reduction 

 methods employed. Most of these are by no means so accurate as was 

 formerly supposed to be the case. Funk (a) showed that the difference be- 

 tween the values obtained by Bang's method and by the use of the 

 polariscope disappeared when Bertrand's method was substituted for the 

 former. These results were confirmed by Borchardt(a) (&). 



It is essential that other methods be employed to establish the presence 

 of fructose. The levorotation, or the difference between reduction and 

 polariscopic methods, must disappear after fermentation. Seliwanoff's 

 reaction and the formation of the methylphenylosazone, both under care- 

 fully controlled conditions, should be employed as confirmatory tests. 



The former of these depends upon the production of a red color upon 

 heating with hydrochloric acid and resorcin. The test is probably best 

 carried out as follows: The urine is mixed with one-half its volume of 

 concentrated hydrochloric acid, heated to boiling for half a minute and 

 divided into two portions, one of which serves as a control. To the other 

 are added a few crystals of resorcin and it is again heated to boiling for a 

 few seconds. The presence of fructose is indicated by the appearance of a 

 red color. The mixture should be cooled, made alkaline with solid 

 sodium carbonate and extracted with ethyl acetate, which will take on a 

 yellow color. Prolonged boiling or the addition of a greater quantity of 

 acid should be avoided or glucose will also react. For this reason the test 

 should be controlled by another, using normal urine containing added 

 glucose in amount equivalent to the reducing action of the urine under 

 examination. 



Methylphenylhydrazin was recommended by Neuberg(e)(d) (1902, 

 1904) as reacting specifically with ketoses, such as fructose, and not with 

 aldoses, such as glucose, to form the osazone. However, Ofner showed 

 that, under certain conditions, glucose will also react with methylphenyl- 

 hydrazin. The osazones formed from the two sugars are, of course, 



