54 THE BIOCHEMISTRY OF B VITAMINS 



whole wheat which contained very little riboflavin but a relatively good 

 supply of other B vitamins required by rats. Hence, when a riboflavin- 

 containing material was supplied, its effect was largely due to the ribo- 

 flavin. The method, of course, was particularly useful in connection with 

 assaying relatively rich sources. Rats were first depleted for about two 

 weeks and then fed supplements containing control and unknown 

 amounts of "vitamin G," and the comparisons evaluated on the basis of 

 the growth rates over a period of several weeks. Diets of substantially 

 different character have been used more recently for the assay of ribo- 

 flavin using rat growth, but we shall not go into detail here. 57, 58> 59 



Extensive use has not been made of chicks in the assay of riboflavin, 

 though methods were early proposed. 60 - 61 An improved method was 

 devised by Jukes 62 in which the growth response at lower levels was 

 found to be approximately proportional to the amount of riboflavin fed. 

 The basal medium contained in addition to yellow corn, wheat middlings, 

 casein, and supplements, 7 per cent of a "rice bran filtrate," which served 

 to supply the chicks with the "filtrate factor" (pantothenic acid), and 

 other unknowns. This was designed to make the test more specific and 

 was apparently successful. 



Nicotinic Acid, Nicotinamide 



Two types of methods have been used almost exclusively for the assay 

 of nicotinic acid or its amide: colorimetric methods and microbiological 

 methods. Biological methods are relatively unimportant. 



Colorimetric Methods. The colorimetric methods which have been 

 applied widely to nicotinic acid assays all involve the interaction be- 

 tween it, cyanogen bromide and an aromatic amine. The chemistry of 

 the reactions is not well known, but rupture of the pyridine ring is thought 

 to be involved. 63 Among the aromatic amines used for this purpose 

 are p-aminoacetophenone, 64 metol 65 (p-methylaminophenol), aniline, 66 

 /3-naphthylarnine, 67 and p-phenylenediamine. 68 



One of the more extensively used amines has been p-aminoaceto- 

 phenone. This reagent has been investigated by Kodicek, who agrees 

 with the originators that it is 3 to 5 times as sensitive as aniline or 

 metol; he has applied it with success to a variety of plant and animal 

 products. 69 It has been applied principally to cereal products by Bina 

 and co-workers. 70 



Metol has been used by a number of workers, including Perlzweig and 

 co-workers, 71 Dann and Handler, 72 and Steel and collaborators. 73 The 

 color produced is said to be more stable than that obtained with p-amino- 

 acetophenone, 73 and the reaction is reported to be more specific for 

 nicotinic acid. 72 Aniline has been used by Melnick and Field 74, 75 and 



