VII. ESTIMATION 361 



normally a dominant organism in human intestinal flora, jiroduccd the 

 most riboflavin. Reviews by Knight'-' and Van Lanen and Tanner'^ on 

 growth factors in microbiology have covered other aspects of riboflavin 

 synthesis by microorganisms. 



The synthesis of riboflavin in green plants is of major importance in 

 supplying human riboflavin requirements. The locus of this synthesis is 

 not known, but there is apparently a higher concentration in the leaves 

 than in the remainder of the plant. 



It is apparent that the biosynthesis of riboflavin phosphate and flavin 

 adenine dinucleotide from riboflavin is a property possessed by all living 

 organisms which have need of this vitamin."- '* 



The work of Plant' ^ has thrown some light on the mechanism of ribo- 

 flavin biosynthesis by Ashbya goss2jpii. The addition of C'^-formate to flask 

 cultures of yeast gave rise to riboflavin tagged in the carbon-2 position. 

 The tracer atom from C'''-bicarbonate ended up in the carbon-4 position. 

 C'^HsCOOH, CHsC'^OOH, and totally labeled glucose produced riboflavin 

 containing C'^ in both the side chain and o-xylene portions of the molecule. 



VII. Estimation 



D. M. HEGSTED 



The most commonly used methods for the estimation of riboflavin are 

 undoubtedly the various modifications of the fluorometric and microbio- 

 logical methods. These methods have largely replaced the more laborious, 

 expensive, and generally less exact biological assays using chicks and rats. 

 In fact, the early development of the rapid and fairly satisfactory methods 

 discouraged the development of careful animal assays for riboflavin. Colori- 

 metric and polarigraphic methods have been proposed, but these do not 

 appear to be generally applicable to biological materials. 



A. PHYSICAL AND CHEMICAL METHODS 



D. M. HEGSTED 



1. P'luorometric Methods 



The properties of riboflavin have been discussed in an earlier section of 

 this chapter. Those which have been utilized for the separation and estima- 



'^ B. C. J. G. Knight, Vitamins and Hormones 3, 108 (1945). 



'* J. M. Van Lanen and F. W. Tanner, Jr., Vitamins and Hormones 6, 163 (1948). 

 »' E. B. Kearney and S. Englard, /. Biol. Chcm. 193, 821 (1951). /rvGlC/TT 



'8 J. R. Klein and H. I. Kohn, /. Biol. Chem. 136, 177 (1940). /c^5^-'~^ ^ 



G. W. E. Plaut, Federation Proc. 12, 254 (1953) /^/S^^^ '^5\'^ 



