ASSAY METHODS 47 



Thiamine 



For physiological research involving thiamine and its functioning, 

 many different organisms and procedures may profitably be used which 

 fall outside the scope of our present discussion. We shall be concerned 

 primarily with methods which are designed to determine thiamine con- 

 tent, and which neglect all other questions involving availability to any 

 particular organism. 



Thiochrome Method. Since the original publication by Jansen 2 which 

 forms the basis of this highly important method, dozens of articles deal- 

 ing with applications, modifications and refinements have been published. 

 Among the more important of these are those of Hennessy and Cerecedo 3 

 and Conner and Straub, 4 who respectively introduced the use of adequate 

 base exchange procedures for eliminating interfering substances and 

 enzyme digestion using commercially available enzyme preparations to 

 free thiamine from its combination in cocarboxylase. 



The experimental essentials of the method as it is now applied include 

 seven steps: 



(1) Quantitative extraction from the material under examination of 

 all of the free thiamine and cocarboxylase present. This is accomplished 

 by the use of acidulated water. 



(2) Enzymatic digestion of the cocarboxylase present by the use of 

 clarase or some other suitable commercial preparation rich in phosphatase. 



(3) Selective quantitative adsorption of the free thiamine by "Decalso" 

 or other suitable agent. 



(4) Quantitative recovery by elution, yielding a thiamine solution 

 from which many interfering substances have been discarded. 



(5) Oxidation of the thiamine to thiochrome by the use of alkaline 

 potassium ferricyanide solution. 



(6) Extraction of thiochrome by isobutyl alcohol. 



(7) Measurement of the fluorescence produced by irradiating the 

 thiochrome solution with ultraviolet light (fluorophotometer) and evalua- 

 tion in terms of thiochrome. 



The key reaction on which the method is based is the quantitative 

 production of the fluorescent pigment from thiamine by oxidation. 



In the application of this method to specific cases, some of the steps 

 may become superfluous. For example, in the assay of wheat and wheat 

 products, Step 2 can be omitted because cocarboxylase is absent, and the 

 results are the same whether or not this step is performed. If one is deal- 

 ing with a vitamin concentrate containing free thiamine and not too much 

 interfering material, steps 1-4 may be dispensed with. If one wishes to 

 determine free thiamine in the presence of cocarboxylase, Step 2 is 



