COMBINED FORMS— EXTRACTION 35 



case of some cooked meats. The differences in comparable results from 

 different laboratories are probably due in part to the fact that crude 

 enzyme preparations are not uniform. Willerton and Cromwell 62 found 

 that clarase digestion of yeast and liver preparations caused a several- 

 fold increase in the available pantothenic acid in some cases, and brought 

 the assay values for these materials up to the point where they agreed 

 substantially with chick assay values. In all laboratories phosphatase 

 preparations are effective in releasing pantothenic acid, and except in the 

 case of cooked meats, release by this method is at least near the maximum. 

 Recently Neilands and Strong 63 have made combined use of liver enzyme 

 and alkaline phosphatase to release pantothenic acid from foodstuffs. 

 They emphasize the incomplete release of this vitamin by previously used 

 procedures. 



The form (or forms) in which pantothenic acid is bound in natural mate- 

 rials is largely unknown. On the basis of the fact that pantothenic acid 

 itself is readily hydrolyzed by acid or alkaline hydrolysis but that 

 /^-alanine is not readily released from tissues by this means, Williams 

 postulated that combination, presumably with proteins, takes place 

 through the /^-alanine portion of the molecule. 64 Since esters of panto- 

 thenic acid are readily hydrolyzed (this can be accomplished without 

 cleaving the pantothenic acid) and since pantothenic acid has not been 

 removed from tissues by this means, the amide linkage suggests itself as 

 most probable. The question of whether pantoic acid is readily split from 

 the naturally combined forms of pantothenic acid has apparently never 

 been determined. 65 



The fact that the coenzyme of Lipmann was found to contain about 10 

 per cent pantothenic acid has a tremendous bearing upon the problem of 

 the combined form or forms of pantothenic acid. 66 Since the functioning 

 of pantothenic acid supposedly centers in this coenzyme, it may be pre- 

 sumed that pantothenic acid occurs naturally combined in this form, 

 which constitutes the prosthetic group of one or more enzymes. It is inter- 

 esting that pantothenic acid was freed only very slowly from this co- 

 enzyme by clarase-papain digestion, according to Cheldelin et al. 8 After 

 /^-alanine had been found as a significant hydrolytic product, a combina- 

 tion of a liver enzyme and an alkaline phosphatase which together had 

 previously been found to inactivate the coenzyme was found to release 

 the pantothenic acid quantitatively. 



B 6 Group: Pyridoxal, Pyridoxine, and Pyridoxamine 



Although the chemistry of vitamin B 6 appeared to be cleared up with 

 the isolation and synthesis of pyridoxine, a biologically active vitamin, 

 in 1938 and 1939, it was shown conclusivelv vears later 67 - 68 that "vitamin 



