156 THE BIOCHEMISTRY OF B VITAMINS 



thiamine to inhibit certain phosphatases present in the brewers' yeast 

 which cause the hydrolysis and inactivation of the intact coenzyme. 114 

 Thus thiamine can appear to be active in the system merely because it is 

 sparing the destruction of its active derivative. The monophosphoric ester 

 and the pyrimidine moiety of the vitamin behave similarly in "activating" 

 the carboxylase system from brewers' yeast. 



These interfering activities of the vitamin in the manometric assay for 

 the coenzyme have been eliminated by using bakers' yeast instead of 

 brewers', 115 by adding sodium iodoacetate to inhibit the phosphorylation 

 of thiamine, 116 or by assaying all preparations in the presence of an excess 

 of thiamine. 117 Concise directions for carrying out the manometric deter- 

 mination of the coenzyme have been published in a standard reference 

 text. 118 An analyst using this procedure on crude materials should be 

 aware of the many complicating effects which the extraneous matter can 

 produce. 



No organisms are known in which the thiamine requirements can be 

 met only by its pyrophosphoric ester. Certain atypical strains of gonococ- 

 cus respond much better to "a cocarboxylase-like substance" than to 

 thiamine, but their requirement for the intact coenzyme is not absolute. 119 



Microbiological and chemical methods can be adapted to coenzyme 

 analysis of tissues and biological products if it is known that all the 

 "bound" thiamine is the pyrophosphate and if an assay method is used 

 in which only the free vitamin is active (yeast growth and thiochrome 

 methods) . If such is the case, the difference in thiamine content of extracts 

 before and after treatment with phosphatases will represent the amount 

 of coenzyme. Due to the differences in solubility of the thiochromes result- 

 ing from the oxidation of thiamine and its phosphate esters, this chemical 

 method can be adapted for the determination of both the vitamin and 

 its phosphorylated derivatives. 120 



The presence of the coenzyme has been directly demonstrated in a 

 number of different plant and animal tissues and in microorganisms. From 

 the results obtained, it would appear that intracellularly most of the 

 thiamine present is in the form of its coenzyme, whereas in plasma and 

 other extracellular fluids (including urine and cerebrospinal fluid) the 

 vitamin occurs predominantly in the free state. 121 Practically all the 

 microorganisms so far tested which cannot synthesize their thiamine 

 requirements can utilize the free vitamin at least as effectively as the 

 phosphorylated derivatives, whereas many organisms cannot use the co- 

 enzyme as a nutrient in place of the free vitamin. It is apparent, then, 

 that the biosynthesis of the coenzyme must take place within the cells 

 of most organisms and tissues. 



Biosynthesis. The presence of enzymes capable of phosphorylating 

 thiamine has been directly demonstrated in a number of different types 



