ESTIMATION 



Kynurenine and 3-hy(iroxyanthranilic acid, two intermediates in 

 the conversion of tryptophan into nicotinic acid (page 250), did not 

 stimulate the growth of L. arabinosus, L. mesenteroides, S. faecalis, Pr. 

 vulgaris or Torula cremoris, and therefore do not interfere with the 

 estimation of nicotinic acid by any of these organisms. ^^" 



Estimation of Coenzymes I and II 



Several methods are available for the estimation of coenzymes I 

 and II, di- and triphospho-p5n:idine nucleotides (see page 274). O. 

 Warburg and W. Christian ^^ estimated codehydrogenase II by 

 measuring the amount of carbon dioxide evolved by the action on 

 sodium bicarbonate of glycerophosphoric acid produced enzymatic- 

 ally with hexose monophosphate (Robison ester) as substrate ; in 

 addition to the coenz3mie, the specific apoenzyme and the yellow 

 ferment must also be present. Jandorf et al."^^ adopted the same 

 method in principle, but used the more readily accessible hexose 

 diphosphate as substrate. D. E. Green and J. Brasteaux '^^ used 

 oxidation of lactic acid by animal tissues as a method of estima- 

 tion. 



Haemophilus parainfluenzae, which cannot synthesise codehydrogen- 

 ase I or II from its constituents and cannot grow without one or the 

 other, can be used to estimate " factor V ", the coenzyme-like substance 

 in blood or yeast. ^2, 73 ^^lis is presumably a mixture of the two 

 coenzymes. Vilter et al.'^^ used Bacillus influenzae to estimate the 

 factor V content of blood, whilst K. Myrback '^ and A. E. Axelrod and 

 C. A. Elvehjem ^^ used a yeast-growth method, which estimates 

 coenz3mie I, but not coenzyme II. It is not certain whether these 

 two methods of estimating factor V give comparable results. 



A rapid method for the estimation of pyridine nucleotides in blood 

 was developed by Levitas et alJ"^ In this method, the nucleotide was 

 converted into N^-methylnicotinamide by treatment with alkali, and 

 this was then condensed with acetone to give the fluorescent com- 

 pound referred to above (page 226). Wlien attempts were made to 

 apply this method to tissues, low values were obtained.''^ The losses 

 were due to the action of nucleotidases (page 279) and were avoided 

 by the addition of nicotinamide, which is a specific inhibitor of nucleo- 

 tidase. This modification gave results in excellent agreement with 

 those obtained by the microbiological method using Haemophilus 

 parainfluenzae. When the values thus obtained for pyridine nucleo- 

 tides were compared with the total nicotinic acid content of tissues, 

 as determined microbiologically with the aid of L. arabinosus, it was 

 found that all the nicotinic acid in rat tissues was in the form of 

 pyridine nucleotide. 



229 



