ASSAY METHODS 59 



Lipmann et al. 120 with respect to the release of pantothenic acid from 

 coenzyme A, the subject needs to be reinvestigated. Certainly there is 

 some hazard involved in using crude, non-uniform enzyme preparations, 

 just as there is in the use of chemicals of low purity. 



More recently a rat growth method for the estimation of pantothenic 

 acid has been proposed in which wheat germ, in addition to thiamine, 

 riboflavin, pyridoxine, inositol, nicotinic acid and choline, is included in 

 the basal diet. 121 This material is relatively deficient in pantothenic acid 

 but contains other vitamins essential to rapid growth, and hence is said 

 to serve excellently as a constituent of the basal diet. 



Vitamin B — Pyridoxal, Pyridoxamine, Pyridoxine 



The available methods for determining these forms of the vitamin 

 either individually or together are chemical, microbiological and biologi- 

 cal. Much material which is primarily of historical interest, that ante- 

 dating the discovery of pyridoxal and pyridoxamine, 122 will be omitted 

 from the present discussion. 



Chemical Methods. Since each of the three forms of vitamin B G pos- 

 sesses a phenolic group in the 3 position and the 6 position is unsubsti- 

 tuted, it is not surprising that they can be condensed with various 

 reagents to yield dyes. Among the reagents which have been used in 

 attempts to develop colorimetric methods for vitamin B 6 are diazotized 

 sulfanilic acid, 123 2,6-dichloroquinone chlorimide 124, 125> 126 and diazotized 

 p-amino acetophenone. 127 The earlier attempts were, of course, based 

 upon the assumption that vitamin B G and pyridoxine are identical. 



In natural materials there are numerous phenolic and other reactive 

 substances, however, and the application of such color reactions to natural 

 products is a quite different matter from their application to the pure 

 substances. In developing colorimetric methods it has been necessary to 

 eliminate or rule out other reacting substances, and in some cases this 

 has required an elaborate procedure. 123 The results obtained using such 

 precautions have often agreed substantially with animal assays of similar 

 materials, though actually the number of available animal assay values 

 on materials which are in any sense duplicable is exceedingly small. The 

 question of the reliability of the animal assays will be discussed in a later 

 paragraph. 



One of the interesting facts which has a bearing upon the applicability 

 of this colorimetric procedure is that all three forms of the vitamin give 

 colors when treated with diazotized sulfanilic acid. Pyridoxal gives a 

 bright yellow, pyridoxamine yields orange to pink, and pyridoxine gives 

 an orange color. 128 It is easy to see that a natural mixture might give a 

 a color for which all three components might be partially responsible. 



