ESTIMATION 



7. ESTIMATION OF FOLIC ACID 

 Microbiological Methods 



Although chicks were used extensively in studying the relation- 

 ship between different members of the folic acid group of factors, the 

 methods used to assay preparations of these factors and foodstuffs 

 containing them have invariably involved the use of micro-organisms, 

 particularly L. helveticiis and S. faecalis R. These, as already noted, 

 respond to different degrees with the different factors. Pteroic acid 

 and the SLR factor, for instance, stimulate 5. faecalis R but not 

 L. helveticus, pteroylglutamate stimulates both, pteroyltriglutamate 

 (fermentation L. casei factor) stimulates L. helveticus but not 5. 

 faecalis R, and pteroylheptaglutamate (vitamin Be conjugate) stimu- 

 lates neither organism. 



In their original method, Mitchell et al} used S. faecalis R which 

 was also used by Elvehjem and his colleagues, ^ who modified the 

 medium in order to obtain greater acid production and thus increase 

 the titre, 



L. helveticus was used by M. Landy and D. M. Dicken.^ The 

 medium was the same as that used for the assay of five other members 

 of the vitamin B complex, but had the disadvantage of requiring a 

 large nmnber of constituents. 



As might be expected, the chief difficulty in assaying folic acid 

 arises from the differences in the response elicited by the different 

 forms of the factor. The object of assaying foodstuffs, for example, 

 is to determine the total folic acid content, that is, the amount of 

 material that could be utilised by the healthy animal for growth and 

 haemopoiesis and, to this extent, a biological method using the chick 

 or the monkey, would be superior to any microbiological assay method. 

 Such a method does not appear to exist, however, and workers have 

 therefore been forced to use microbiological methods in combination 

 with some process of hydrolysis to convert the microbiologically in- 

 active forms into forms that would produce a response. Cheldelin 

 et al.^ advocated digestion with takadiastase, which they claimed to 

 give maximum folic acid values, whilst Laskowski et al.'" recommended 

 digestion with an extract of chick pancreas for yeast extracts and 

 autoclaving at _^H 4 for twelve hours for liver preparations. 



Bird et al.^ described a method of hydrolysing vitamin Bo con- 

 jugate by means of vitamin Be conjugase prepared from hog kidney 

 or almonds ; the total folic acid was then estimated microbiologically 

 with L. helveticus. 



Although the conjugase from hog kidney is said to give satisfactory 

 results with plant materials,"^ a combination of hog kidney and chick 

 pancreas enzymes gave higher results than either alone. ^ Lower 

 31 481 



