110 pteroylglutamic acid 



2. Chemistry of Citrovorum Factor (Leucovorin, Folinic Acid) 

 a. Biochemical Relations between Citrovorum Factor and PGA 



In 1948 Sauberlich and Baumann'^" noted that the organism Leuconosioc 

 citrovorum failed to grow in a chemically defined medium containing all the 

 known amino acids and vitamins, but the addition of small amounts of 

 natural materials such as liver extract or yeast to the medium promoted 

 luxuriant growth. The substance(s) in these natural materials necessary for 

 growth of Le. citrovorum was termed the citrovorum factor (CF). It was 

 also noted by these workers that the organism responded to thymidine or 

 to massive doses of PGA, but because the response to these substances was 

 delayed and submaximum, it appeared that these substances could not be 

 the citrovorum factor. It soon became apparent, however, that a close 

 relationship existed between PGA and CF. Thus Sauberlich^^ noted that 

 rats on a PGA-deficient diet excreted very little CF in the urine, but the 

 administration of PGA resulted in large increases in the CF content of the 

 urine. Subsequently it was noted by Sauberlich''^ and confirmed by Broquist 

 et al?^ that concentrates of CF can competitively overcome the toxicity of 

 aminopterin (4-aminopteroylglutamic acid), an inhibitory analog of PGA, 

 for Le. citrovorum. These latter workers also showed that CF had approxi- 

 mately the same distribution coefficient as PGA between butanol and water 

 at pH 2. It appeared then that PGA might be a biological precursor of CF 

 and that a close chemical relationship existed between the two substances. 



In a seemingly unrelated series of investigations. Bond et al."^* provided 

 evidence from microbiological studies for the existence of an unrecognized 

 form of PGA in natural materials. These workers studied the toxic effect 

 of "x methyl PGA" for growth of L. casei and observed that liver extract 

 was approximately fifteen times more active than PGA in counteracting 

 the toxicity of "x methyl PGA" than could be accounted for on the basis of 

 its PGA content when assayed in the absence of inhibitor. With the use of 

 this inhibition assay highly refined concentrates of the factor from liver, 

 termed folinic acid by the Texas group, were prepared and found to be 

 highly active for Leuconostoc citrovorum. It thus seems very likely that 

 folinic acid and CF are identical. 



Gordon et al.^^ had previously reported the preparation of 10-formyl- 

 pteroylglutamic acid and observed that it was more active than PGA in 

 counteracting the inhibitory action of "x methyl PGA" for S. faecalis R^ 



^» H. E. Sauberlich and C. A. Baumann, J. Biol. Chem. 176, 165 (1948). 

 71 H. E. Sauberlich, /. Biol. Chem. 181, 467 (1949). 

 " H. E. Sauberlich, Arch. Biochem.. 24, 224 (1949). 



" H. P. Broquist, E L. R. Stokstad, and T. H. Jukes, J. Biol. Chem. 185, 399 (1950). 

 -'* T. J. Bond, T. J. Bardos, M. Sibley, and W. Shive, /. Am. Chem. Soc. 71, 3852 

 (1949). 



I 



