534 NIACIN 



VII. Estimation 



Biological, chemical, fliiorometric, photometric, and microbiological tech- 

 niques have been employed for the estimation of nicotinic acid and related 

 substances. 



A. BIOLOGICAL ASSAY 



J. M. HUNDLEY 



Animal assays offer little opportunity for the exact determination of 

 nicotinic acid unless the substances to be tested are completely free from 

 protein and amino acids, especially tryptophan. Biological tests might offer 

 possibilities in assessing the over-all pellagra-protective potency of foods if 

 a suitable way to quantitatively express and compare the results could be 

 devised. Young nicotinic acid-deficient animals do show a growth response 

 proportional, within a narrow range, to the amount of nicotinic acid admin- 

 istered. This has been demonstrated with pure, or relatively pure, solutions 

 of nicotinic acid or nicotinamide in puppies,^ weanling rats,^"^ and chicks.^ 

 Some idea of the nicotinic acid content of test solutions may be gained by 

 attempting to cure blacktongue in adult dogs, but precise quantitative re- 

 sults are almost impossible. Test substances may also be evaluated by 

 their ability to prevent blacktongue in dogs on suitable diets, but this tech- 

 nique also has many variables. 



B. CHEMICAL METHODS 



J. M. HUNDLEY 



L Nicotinic Acid 



The chemical determination of nicotinic acid is, in theory, quite simple. 

 Utilizing the Konig reaction,^ nicotinic acid is treated with cyanogen bro- 

 mide and then with an aromatic amine to yield a colored compound which 

 can be quantitated photometrically. The underlying reaction proceeds in 

 two steps: first, the intermediate production of a pyridinium derivative 

 by reaction of the nicotinic acid with cyanogen bromide, and, second, the 

 production of a colored glutaconic dialdehyde derivative by reaction with 

 the aromatic amine. ^' ^ 



1 H. A. Waisman, O. Mickelsen, J. M. McKibbin, and C. A. Elvehjem, J. Nutrition 

 19, 483 (1940). 



2 W. A. Krehl, P. S. Sarma, L. J. Teply, and C. A. Elvehjem, J. Nutrition 31, 85 

 (1946). 



3 J. M. Hundley, /. Biol. Chem. 181, 1 (1949). 



* L. J. Harris and E. Kodicek, Brit. J. Nutrition 4, xiii (1950). 



6 M. E. Coates, S. K. Kon, and E. E. Shepheard, Brit. J. Nutrition 4, 203 (1950). 



6 W. Konig, /. praki. Chem. 69, 105 (1904); 70, 19 (1904). 



' T. Zincke, Ann. 330, 361 (1904). 



