Chapter IMA 



COMBINED FORMS— EXTRACTION 



It is an experimental fact that in spite of their relatively high water 

 solubility in the free state, the B vitamins often resist aqueous extraction 

 from plant and animal materials. 



In the case of at least one of the B vitamins (biotin), its distribution 

 in various tissues was originally determined quantitatively on the assump- 

 tion that it would be extracted by water, since it exhibited a hydrophilic 

 character in' the free state. Subsequent investigations showed that a small 

 and uneven fraction of the vitamin in the various tissues had been ex- 

 tracted. 1 Since the extraction of each of the B vitamins presents special 

 problems, we must consider them separately. 



Thiamine 



Historically, yeast and rice polish have been the most important mate- 

 rials used in attempts to concentrate and purify this vitamin. These two 

 sources offer considerable contrast, because yeast requires autolysis before 

 it becomes a favorable source while rice polish can be effectively extracted 

 directly with acidulated water. 2 



Since the work of Auhagen 3 and Lohmann and Schuster, 4 it has been 

 recognized that a considerable part of the thiamine in yeast and in animal 

 tissues is combined in the form of diphosphothiamine (cocarboxylase) , 

 and this may undergo cleavage to produce free thiamine. Actually the 

 diphosphothiamine is itself in combined form in the hoioenzyme, car- 

 boxylase. Carboxylase has been isolated in pure form and contains 0.46 

 per cent diphosphothiamine and 0.13 per cent magnesium. 5 In the presence 

 of high salt concentrations the cocarboxylase is firmly bound, but in dilute 

 salt solutions or in alkaline or acid ammonium sulfate solution it is almost 

 completely dissociated. 6 



Various commercial enzyme preparations (taka-diastase, malt diastase, 

 clarase, polidase, high phosphatase mylase, etc.) catalyze the hydrolysis 

 of diphosphothiamine, usually at pH 4.5 7 - 8 and hence can be used to free 

 thiamine more or less completely from its natural combination in various 

 tissues. 



Westenbrink 9 postulated the existence of more than one type of com- 

 bination in undissociated carboxylases, and Sarett and Cheldelin, 10 by 



