ASSAY METHODS 55 



Pearson, 76 who indicates that it is four times as sensitive as /J-napthyla- 

 mine. 



There have been at least two obstacles in the way of coming to an 

 agreement upon a chemical method which will be generally recognized 

 as acceptable. One is the fact that the amount of nicotinic acid appear- 

 ing in the extract depends upon the method of extraction, and there has 

 not been common agreement on this point. It is evident that some un- 

 known substance is converted to nicotinic acid by certain extraction 

 procedures. 69, 77 



Another more serious obstacle is the fact that the fundamental reactions 

 involved in producing the dye or dyes are not understood. The reaction 

 is not specific for nicotinic acid, 75 but with different aromatic reagents 

 other pyridine derivatives such as nicotinamide, nicotinuric acid and 

 nicotine as well as pyridine itself react to produce more or less color. 



In spite of these obstacles, it is probable that several aromatic amines 

 can be used successfully for particular types of products with good 

 success. The colorimetric methods in general are not as sensitive, how- 

 ever, as the microbiological method which has now become pretty well 

 standardized and accepted. 



Microbiological Method. The principle of this method is the same as 

 for the riboflavin method, but the microbiological procedure for nico- 

 tinic acid is in an especially favorable position because of the lack of 

 any very satisfactory competing chemical method. 



As with the microbiological methods generally, the substance to be 

 assayed must first be extracted and brought into solution. In the Snell 

 and Wright method, 78 which has received wide acceptance, 79 - so nicotinic 

 acid, nicotinamide, nicotinuric acid, and cozymase all have the same 

 biological activity when tested in equimolecular amounts. 78 This makes 

 it unnecessary to bring about complete hydrolysis of the combined 

 forms. The formation of nicotinic acid by acid and alkali treatment from 

 precursors which may or may not have vitamin activity, 77 presents an 

 uncertainty which has not been overcome. 



The organism used in this assay is Lactobacillus arabinosis 17-5 and 

 the evaluation of the response involves titration, usually at the end of 

 72 hours. When this method is used, the turbidity of the extracts does 

 not interfere. The method is 20 to 100 times as sensitive as the chemical 

 methods, 78 and once the nicotinic acid or its derivative is in solution, 

 there is no obstacle to its satisfactory determination in amounts down 

 to 0.05 /xg or less if drop cultures are used. 81 The organism used for this 

 assay is not as sensitive to fat acids as is L. casei. Krehl and co-workers 

 found that with the possible exception of linoleic acid, they did not 

 interfere. 



