536 NIACIN 



Ciusa^^ described a procedure which depends on the Konig reaction be- 

 fore and after the unknown solution is boiled with chlorobenzene. Since 

 chlorobenzene reacts with nicotinamide, but not with nicotinic acid, the 

 two forms of the vitamin can be determined by difference. Scudi-^ described 

 a fluorometric procedure for the determination of nicotinamide which in- 

 volves the conversion of nicotinamide to a fluorescent N^-methyl derivative. 

 Chaudhuri and Kodicek-* have developed a somewhat related method which 

 measures only nicotinamide in biological materials. They used cyanogen 

 bromide and treatment with strong alkali to yield a fluorescent derivative 

 which could be quantitated in a photoelectric fluorometer. Kato and 

 Shimizu^^ had difficulty with this method, because of interference from 

 kynurenine, 3-hydroxykynurenine, and other substances. These interfer- 

 ing substances could be removed by chromatographing on a carboxylic- 

 type cation exchange resin and a strong-base-type anion exchange resin. 



Ellinger and Abdel Kader-^ have developed chemical methods with which 

 they could differentially determine nicotinic acid, nicotinamide, nicotinuric 

 acid, N^-methylnicotinamide, and trigonelline. The basis of the procedure 

 was hydrolysis of sample aliquots in various strengths of acid, alkali, and 

 alkali plus urea, with subsequent application of the Konig reaction. (N^- 

 methylnicotinamide was assayed fluorometrically.) 



3. N^-Methylnicotinamide 



Several methods have been devised to measure this substance in urine.-^'^^ 

 Some of these measured trigonelline as well as N^-methylnicotinamide. The 

 method in most general use at present is that devised by Huff et alP This 

 method is sensitive, accurate, and rapid and has given excellent results 

 in many hands. It depends on the reaction of N'-methylnicotinamide and 

 acetone in the presence of alkali to yield a fluorescent derivative which can 

 be quantitated against an internal standard of synthetic N^-methylnico- 



19 C. F. Heubner, Nature 167, 119 (1951). 



20 E. Kodicek and K. K. Reddi, Nature 168, 475 (1951). 



21 E. G. Wollish, M. Schmall, and E. G. Schafer, Anal. Chem. 23, 768 (1951). 



22 W. Ciusa, Ann. chim. appl. 39, 93 (1949). 



23 J. V. Scudi, Science 103, 567 (1946). 



24 P. K. Chaudhuri and E. Kodicek, Biochem. J. 44, 343 (1949). 

 26 M. Kato and H. Shimizu, Science 114, 12 (1951). 



26 P. Ellinger and M. M. Abdel Kader, Biochem. J. 44, 77, 627 (1949). 

 2T J. W. Huff and W. A. Perlzweig, /. Biol. Chem. 150, 395 (1943). 



28 M. Hochberg, D. Melnick, and B. L. Oser, /. Biol. Chem. 158, 265 (1945). 



29 V. A. Najjar, Bull. Johns Hopkins Hosp. 74, 392 (1944). 



3» R. A. Coulson, P. Ellinger, and M. Holden, Biochem. J. 38, 150 (1944). 



31 H. P. Sarett, /. Biol. Chem. 150, 159 (1943). 



32 J. W. Huff, W. A. Perlzweig, and M. W. Tilden, Federation Proc. 4, 92 (1945); 

 /. Biol. Chem. 167, 157 (1947). 



