VITAMIN AND GROWTH FACTOR RESEARCH 



CONHCHCOOH 



N N^ II I CH^ 



OH H ' COOH 



(VII) Tetrahydrofolic acid 



labile di- and tetrahydrofolic acids (formula VII)) during 

 the isolation procedure, remains to be established. Pfiffner 

 and co-workers (45) in their paper describing isolation of folic 

 acid (vitamin Be) demonstrated clearly the existence of a more 

 labile compound which promoted growth of Streptococcus faecalis 

 and which closely resembled in properties those later established 

 for folinic acid. However, the clear-cut differentiation of this 

 latter substance required a specific assay method. This was 

 discovered by Sauberlich and Baumann (56) who found that 

 Leuconostoc citrovorum (more recently identified as Pediococcus 

 cerevisiae) required extremely high levels of folic acid for growth, 

 and then grew only slowly, but grew rapidly in the presence of 

 small amounts of liver extracts. A second assay procedure was 

 independently discovered by Shive and co-workers (4), who found 

 that inhibition of 5". faecalis by a folic acid analogue was prevented 

 much more effectively by liver extracts than could be accounted 

 for by their folic acid content. Just as pyridoxamine and 

 pyridoxal were earlier produced by empirical procedures from 

 pyridoxine, and their structure clarified without their isolation 

 from natural materials, so it proved possible to form folinic acid 

 from folic acid by empirical procedures, isolate the active reaction 

 product, and establish its structure (11,47). In this instance, 

 however, two difliicultly separable diastereoisomeric modifications 

 of the growth factor were produced, and final proof of the identity 

 of the naturally occurring and synthetic growth factor required 

 separation of the diastereoisomers and comparison with the 

 product isolated from natural materials. 



Incomplete evidence indicates that in addition to these two 

 substances with folic acid activity, di- and tetrahydrofolic acid 



95 



