VII. ESTIMATION 449 



H 



/ 

 C CI 



/ \ / 

 N C CH. N C— CH, 



I. II II II 



CH3C C— NH2HCI HC C— C2H4OH 



\ / \/ 



N S 



Thiamine 



H H2 



/ /- 



C C 



/ \ / \ 

 N C N C— CH3 



I II I II 



CH3C C C C— C.H4OH 



\ / \ / \/ 



N N S 



Thiochrome 



blighted potatoes (infected with Phytophtera) contain a substance with a 

 bkie fluorescence which is soluble in isobutanol. Also urine and other bio- 

 logical fluids contain interfering substances. In such cases a proceeding 

 adsorption of thiamine to Decalso, fuller's earth, or other suitable adsorb- 

 ents, is necessary. 



More elaborate descriptions of this method are available.^' "^ By the use 

 of a suitable fluorometer 0.01 y of thiamine or less maj^ be determined by 

 the thiochrome method. In biological fluids and extracts a large part of the 

 thiamine is present in the form of pyrophosphate. By oxidizing this it is 

 tran.sformed to the pja-ophosphate of thiochrome. This is insoluble in iso- 

 butanol. Therefore the pyrophosphate must first be hydrolyzed. This hy- 

 drolysis can be performed by takadiastase or by a phosphatase, e.g., from 

 yeast. 



Burch et alJ^ elaborated a micromethod to determine thiamine and thia- 

 mine pho.sphates in very small quantities of blood and blood cells. 



Prebluda and ^klcCoUum*- ^) devised a method using the color production 

 of a diazotized aromatic amine (e.g., p-aminoacetophenone) with thiamine. 

 The method has been further de^'eloped by Melnick and Field. ^^^ This 



* Association of Vitamin Chemists, Methods of Vitamin Assay, p. 69. Interscience 



Publishers, New York, 1947. 

 ^ P. Gjorgy, Vitamin Methods, Vol. 1. Academic Press, New York, 1950. 

 '" 11. B. Burch, O. A. Bessey, R. H. Love, and O. H. Lowry, J. Biol. Chein. 198, 477 



(1952). 



8 H. J. Prebluda and E. V. McCollum, Science 84, 488 (1936). 



9 H. J. Prebluda and E. V. McCollum, ./. Biol. Chem. 127, 495 (1939). 

 '"D. Melnick and H. Field, /. Biol. Chem. 127, 505, 515, 531 (1939). 



