456 NIACIN 



tinic acid and nicotinamide, as well as related metabolites, can be separated 

 on paper strips with appropriate solvent systems.-^^^- 



3. Separation of Nicotinic Acid and Nicotinamide 



This can be effected by extraction of a water solution with ether, chloro- 

 form, or benzene. The amide dissolves in the organic solvent while the acid 

 remains in the watery phase. Nitranilic acid may also be used for this pur- 

 pose, since it forms an alcohol-soluble compound wdth the acid but an al- 

 cohol-insoluble compound with the amide.^^ Certain amines such as 

 piperidine, n-butylamine, morpholine, or diethylamine will react with a 

 suspension of nicotinic acid, but not with nicotinamide, in a non-aqueous 

 solvent such as benzene to give a soluble amine salt. The unreacted nico- 

 tinamide can be filtered and recovered almost 100 % pure.^^ 



The acid and the amide may be separated using paper strip chromatog- 

 raphy as mentioned previously. This technique also separates free nico- 

 tinamide from that which is bound in coenzymes.^^- ^^ Free nicotinamide 

 may also be separated from coenzyme-bound nicotinamide by acetone ex- 

 traction which dissolves free nicotinamide but not the coenzymes.^^' ^^- ^* 

 This separation must be carried out promptly when extracting mammahan 

 tissues, since many of them contain very active nucleotidases which break 

 down the coenzymes unless inhibited by acetone or some other agent (see 

 p. 507). 



4. Isolation of N^-Methylnicotinamide 



This substance was isolated in pure form from urine by Huff and 

 Perlzweig,^^ using a rather simple procedure. The urine was acidified with 

 acetic acid, clarified with charcoal, partially evaporated, the residue ex- 

 tracted with 95 % ethanol, this extract again evaporated, and the residue 

 extracted with 80 % ethanol. After evaporation of this extract, the residue 

 was taken up in water and adjusted to pH 4 with acetic acid and sodium 



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« E. Leifer, L. J. Roth, D. S. Hogness, and M. H. Corson, /. Biol. Chem. 190, 



595 (1951). 

 ao C. F. Huebner, Nature 167, 119 (1951). 

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



" E. G. Wollish, M. Schmall, and E. G. Shafer, Anal. Chem. 23, 768 (1951). 

 " E. Miiller, Hoppe-Seyler's Z. physiol. Chem. 268, 245 (1941). 

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»" H. von Euler and G. Giinther, Hoppe-Setjler's Z. phxjsiol. Chem. 243, 1 (1936). 

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IV (1937). 

 " J. W. Huff and W. A. Perlzweig, J. Biol. Chem. 150, 395 (1943). 



