ESTIMATION 



biologically active nicotinic acid derivatives. On the other hand, in 

 studying the metabolism of nicotinic acid (see page 252), it is essential 

 to estimate the urinary trigonelline derived from dietary nicotinic 

 acid, whilst at the same time making due allv^wance for the trigonelline 

 originally present as such in the diet — a problem to which no simple 

 solution exists ! 



Fortunately, comparatively few foods contain sufficiently large 

 amounts of trigonelline to interfere seriously with the estimation of 

 the other metabolites ; such foods should be avoided by subjects about 

 to take part in metabolic experiments. 



Another nicotinic acid derivative present in urine is nicotinuric 

 acid (page 252). This does not exist in foodstuffs, but is formed from 

 nicotinic acid in vivo by conjugation with glycine ; its estimation in 

 urine is essential in studying the metabolism of nicotinic acid. 



Another pyridine compound formed in vivo from nicotinamide is 

 N^-methylnicotinamide, the amide corresponding to trigonelline 

 (page 254). It forms a strongly fluorescent compound (" Fg ") on 

 treatment with alkali, so that its separate estimation, where necessary, 

 presents no great difficulty. Conversion to N^-methylnicotinamide 

 can actually be used for the estimation of nicotinamide. This is 

 brought about by leaving a dilute methanolic solution of nicotin- 

 amide overnight with excess methyl iodide and evaporating. On 

 treatment with alkali and isobutanol a fluorescence develops, the 

 intensity of which is proportional to the nicotinamide concentration. ^^ 

 Alternatively, nicotinamide is treated with cyanogen bromide and 

 alkali, and the fluorescent product extracted with isobutanol. The 

 second procedure gives a more intense fluorescence than the first (see 

 also page 226). 



The latter method was modified by Huff et al.,^^ who used acetone 

 in a single phase at an alkaline _/)H instead of extracting into isobutanol. 

 E. Kodicek used methyl ethyl ketone instead of acetone, but subse- 

 quently found that the use of a solvent was unnecessary. ^^ 



N^-Methylnicotinamide is not the end-product of nicotinamide 

 metabolism, an oxidation product of the former, N^-methyl-6-pyridone- 

 3-carboxylamide, being excreted at the same time (see page 255). ^^^ 



Preparation of Extracts 



A variety of methods have been employed for the preparation of 

 extracts suitable for the estimation of nicotinic acid by means of the 

 Konig reaction. Many workers followed the principle employed by 

 E. Bandier 10 and by Vilter et al.^ and used hydrolysis to convert 

 nicotinamide and other nicotinic acid derivatives into free nicotinic 

 acid, whilst V. H. Cheldelin and R. R. Williams ^^ found that many 



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