VII. ESTIMATION 451 



^^'(^st('nl)I•ink''' workcHl out a niiciomctliod which pcnnils the (Ictcrmiiui- 

 tioii of al)()iit ().(){)()().") 7 ot" cocarhowlasc and about ().()()().') 7 of thiamine 

 separately in a mixture ot both comijounds. 



Westenbrink c( a/.'-' used alkaU-washed yeast as an adsorbant and at the 

 same time as the apoenzyme for the cocarboxylase preparation from blood. 

 In tliis way, using the "cartesian diver" technique, it was possible to deter- 

 mine the cocarboxylase content of 0.01 ml. of blood (Parv6-''). 



E. MICROBIOLOGICAL 



In 1935 tiieSwiss investigator Schopfer found tiiat the growthof the mould 

 Phycomyccs blakesleeanus required the presence of thiamine.'-' He used this 

 fact to develop "a method for the estimation of thiamine, by measuring its 

 influence on the growi,h of Phycomyces blakesleeanus P 



This method is very sensitive and especially suitable for series analysis of 

 foodstuffs, etc. A drawback is the fact that it takes 8 to 10 days before 

 growth is complete. The split products of thiamine, the pyrimidine plus the 

 thiazole part, are active also. (For further details, see ref. 7, p. 485.) 



In his search for the components of Wildiers' "bios," Williams and 

 Roehm-^ found that thiamine greatly stimulates the growth of yeast {Sac- 

 charomyces cerevisiae). So thiamine may be estimated by its influence on 

 the growth of yeast. Here again the pyrimidine and the thiazole parts are 

 both active on yeast growth ,^^ 



In 1944 Sarett and Cheldelin introduced the use of Lactobacillus jermenti. 

 This microorganism is extremely sensitive to traces of thiamine.^^- ^® 



In this way it is possible to determine quantities of 5 to 50 m7 of thiamine. 

 Cocarboxylase is about 30 % more active than an equimolecular quantity 

 of thiamine. The pyrimidine and thiazole moieties are inactive if the incuba- 

 tion is not prolonged beyond the usual time of 16 to 18 hours at 37°. (For 

 details and for the use of other lactic acid bacteria, see ref. 7, p. 372.) 



Often chemical or microbiological methods are used for thiamine estima- 

 tion in the urine of men or animals to establish the state of thiamine nutri- 

 tion. Mickelsen et al.,^'' in their extensive research on the thiamine excre- 



J8 H. G. K. Westenbrink, Enzymologia 8, 97 (1940). 



'^ H. G. K. Westentjrink, E. P. S. Parv4, A. C. van der Linden, and W. A. van den 



Broek, Z. Vitaminforsch. 13, 218 (1943). 

 -" E. P. S. Parv^, Thesis, Sctieltema en Holkema, Amsterdam, 1945. 

 =' W. H. Schopfer, Z. Vitaminforsch. 4, 67, 187 (1935). 

 " W. H. Schopfer, Ergeb. Biol. 16, 1 (1939). 



" R. J. Williams and R. R. Roehm, J. Biol. Chem. 87, 581 (1950). 

 " R. J. Williams, J. R. Mc.Mahan, and R. E. Eakin, Univ. Texas Publ. 4157, 31 (1941). 

 " H. P. Sarett and V. H. Cheldelin, ./. Biul. Chem. 155, 153 (1944). 

 2« H. P. Sarett and V. H. Cheldelin, ./. Nutrition 30, 25 (1945). 

 " O. Mickelsen, W. O. Caster, and A. Keys, ./. Biol. Chem. 168, 415 (1947). 



