368 RIBOFLAVIN 



the mononucleotide, which fluoresce equally, is utilized for the estimation 

 of FAD. Benzyl alcohol extractions'^- ^^ is used to separate riboflavin from 

 the nucleotides. From 70 to 90 % of the riboflavin in tissues was found to 

 be present as FAD, and essentially all the remainder was the mononucleo- 

 tide. Fujita and Matsuura^^ used benzyl alcohol separation to distinguish 

 between riboflavin and esterified riboflavin and examined a large number 

 of plant and animal tissues. 



Crammer^^ described the separation of the three forms by paper chroma- 

 tography using "collidine" or butanol-acetic acid as the developing solvent. 

 He detected only FAD in heart, brain, kidney, and liver, whereas only free 

 riboflavin was found in the spleen. These results are not consistent with 

 those of Bessey et at. The latter authors report the distribution of riboflavin 

 compounds in one sample of spleen to be similar to that found in other tis- 

 sues mentioned above. Yagi^ has extended the qualitatively chromato- 

 graphic method of Crammer to a quantitative estimation of the three con- 

 stituents. After separation, the position on the paper was determined under 

 ultraviolet light, and the spots extracted and analyzed by the lumiflavin 

 procedure. It would appear that his results are comparable to those re- 

 ported by Bessey et at. 



Since the only known functions of riboflavin involve the nucleotides 

 rather than free riboflavin, these methods should be of great use and inter- 

 est. Critical evaluation of the various procedures must, of course, await 

 further comparative work. 



2. COLORIMETRIC AND POLARIGRAPHIC METHODS 



Since riboflavin is a colored compound, it can obviously be determined 

 by colorimetry.^' *" A few authors apparently use this procedure,^' but it 

 is about 100 times- less sensitive than the fluorometric method and there- 

 fore not applicable to most biological materials. Its usefulness in the assay 

 of pharmaceuticals should apparently depend upon the equipment of the 

 laboratory and the specificity of the separation from other yellow materials 

 as compared to separation from fluorescent materials. Recently^^ a colori- 

 metric method based on the production of a red color in the presence of 

 silver nitrate or Deniges reagent has been described. Sensitivity is much 

 below that of fluorometric methods. 



" A. Emmorie, Nature 141, 416 (1938). 



38 A. Emmerie, Rec. trav. chim. 58, 290 (1939). 



39 J. C. Crammer, Nature 161, 349 (1948). 



^^ H. R. Rosenberg, Chemistry and Physiology of the Vilamins, Interscience Pub- 

 lishers, New York, 1945. 

 41 F. Weygand and K. Wacker, Klin. Wochschr. 24/25, 438 (1947). 

 « M. Z. Barakat and N. Bodran, J. Pharm. and Pharmacol. 3, 501 (1951). 



