NICOTINIC ACID (NIACIN) 



through the body. The simplest procedure is one in which all these 

 substances are converted into free nicotinic acid, but errors will then 

 be introduced if substantial amounts of the trigonelline present have 

 been derived from the diet. 



Early attempts to estimate nicotinic acid in urine by this method 

 are those of E. Bandier ^^ and L. J. Harris and W. D. Raymond/^ 

 already referred to. J. C. Roggen ^^ heated the sample of urine with 

 nitric acid to hydrolyse nicotinamide and nicotinuric acid to 

 nicotinic acid and then added saturated potassium permanganate 

 solution, which does not attack nicotinic acid, in order to oxidise 

 interfering substances. The resulting solution was adsorbed on 

 frankonite and the adsorbate was eluted with barium hydroxide 

 solution. The colour was developed in the usual way with cyanogen 

 bromide and aniline. 



More satisfactory methods of estimating nicotinic acid in urine, 

 capable of giving information of greater value in metabolic studies 

 than a mere knowledge of the total free and combined nicotinic acid, 

 are those in which nicotinic acid and each of its metabolites are separ- 

 ately estimated. Melnick and his colleagues ^^> ^'' ^^ worked out a 

 method of estimating total nicotinic acid, nicotinuric acid and tri- 

 gonelline based on the fact that nicotinamide is hydrolysed when 

 the urine is heated With 4N acid for half an hour, whereas nico- 

 tinuric acid is more stable and requires heating for five hours with 

 4N acid whilst trigonelline can only be hydrolysed to nicotinic 

 acid by treatment with alkali. They used this method to study 

 the fate of nicotinic acid in the organism (see page 253) . F. W. Lamb ^^ 

 noted that the maximum extinction coefficient of the colour, and the 

 time required for its development, varied for nicotinic acid, nicotin- 

 amide, pyridine and ^-picoline, and was characteristic for each sub- 

 stance. D. Melnick and B. L. Oser ^^ devised a method of differenti- 

 ating between nicotinamide and nicotinic acid, based on the observation 

 that, when cyanogen bromide and aniline were added in quick succes- 

 sion, the two substances gave colours of different intensity. By 

 evaluating the colour before and after acid hydrolysis, it was possible 

 to obtain an approximate idea of the relative amounts of the two 

 substances present in the mixture. 



Perlzweig et al.'^^ devised a method of estimating total nicotinic 

 acid and trigonelline separately in urine. Total nicotinic acid (ex- 

 cluding trigonelline) was estimated by boiling the urine with hydro- 

 chloric acid to hydrolyse all the nicotinic acid derivatives, with the 

 exception of trigonelline, to nicotinic acid. This was then adsorbed 

 on Lloyd's reagent, eluted with alkali and the colour developed as in 

 Bandier and Hald's method. Trigonelline was estimated by treating 

 with urea and alkali, which convert it into a substance giving a colour 



224 



