450 THIAMINE 



azocoloric method is less sensitive and less specific than the thiochrome 

 method, but it is somewhat simpler and quicker and may be useful in 

 special instances, e.g., for clinical determination in urine. ^^ 



C. BIOLOGICAL 



Obviously the earliest methods that were used for thiamine estimation 

 were biological ones. In the beginning when polished rice, an easily obtain- 

 able thiamine-free food, was taken as a basal diet, hens and cocks were used 

 as experimental animals. Soon smaller birds, especially pigeons, were taken, 

 and Jansen worked with very small birds, nonnetjes (Munia maja). When 

 mammals (rats) were the experimental animals, polished rice could no 

 longer be used as a basal diet because of its deficiencies in other nutrients. 

 Therefore a complex basal diet was essential. The experiments may be pro- 

 phylactic or curative. Both are apt to give not very accurate results, and 

 both require large amounts of thiamine-rich extracts. These are serious 

 handicaps, and therefore when the chemical and microbiological methods 

 were brought about, the biological methods were not applied very much 

 any longer. The advantages of the biological methods are that they do not 

 require cumbersome extractions and that they give specific results. For de- 

 tails see, e.g., Coward^^ and Gyorgy.^^ 



D. FERMENTATION 



Schultz et al}'^ found that yeast fermentation is enhanced by the pres- 

 ence of free thiamine. They utilized this fact for a quantitative method for 

 the estimation of thiamine. L^sed with a Warburg apparatus, the method 

 is suitable for a microestimation.^^ 



Nearly all the thiamine in blood and in animal tissues is present in the 

 form of thiamine pyrophosphate (cocarboxylase). This cocarboxylase can 

 be measured by its activity as a coenzyme. Ochoa and Peters^^ were the 

 first to use this method for the quantitative determination of thiamine and 

 cocarboxylase in boiled tissue extracts. Goodhart and Sinclair^^ applied 

 this method to the determination of cocarboxylase in blood. All the blood 

 cocarboxylase was found within the blood cells, particularly in the nucleated 

 cells. 



" M. Hochberg and D. Melnick, /. Biol. Chem. 156, 53 (1944). 



1^ K. H. Coward, The Biological Standardisation of Vitamins. William Wood and 



Co., Baltimore, 1947. 

 " P. Gyorgy, Vitamins Methods, Vol. II, pp. 45, 179, 448. Academic Press, New York, 



1951." 

 14 A. S. Schultz, L. Atkin, and C. N. Frey, Ind. Enq. Chem. Anal. Ed. 14, 35 (1942). 

 16 L. Atkin, A. S. Schultz, and C. N. Frey, ./. Bioi. Chem. 129, 471 (1939). 

 IBS. Ochoa and R. A. Peters, Biochem. J. 32, 1501 (1938). 

 1^ R. S. Goodhart and H. M. Sinclair, Biochem. J. 33, 1099 (1939). 



