IX. KFFKCTS OF DEFICI I;N( V 201 



clavod with water before being adiled to (he assay medium, only l'2% ol 

 the activity was recovered. However, if the cells were autoclaved with 

 reducing agents such as 0.5% sodium glycoUate, 5% ascorbic acid, or in a 

 small amoimt of assay medium, complete recovery was obtained. In each 

 case the residual PGA remaining in the liquid phase after autoclaving was 

 resistant to further autoclaving which showed that a labile form existed in 

 the bacterial cells. This suggests that reducing substances exert a protective 

 influence during extraction from the cell and emphasizes the difficulties 

 that may be encountered in the assay of PGA in microbial cells. This 

 lability of PGA under certain conditions is reminiscent of the acid-labile 

 PGA found in horse liver by Pfiffner et al.^*^ This behavior might also be 

 explained partially by the known properties of the citrovorum factor which 



TABLE X 



Comparative Growth Response of Le. citrovorum, S. faecalis, and L. casei to 

 Leucovorin and Related Compounds 



Amount of compound required per ml. culture medium for 

 half-maximum growth in 24 hr., m7 



Acid-treated Pteroylglutamic 



Organism Leucovorin leucovorin" acid 



Le.citrovornm 0.15 7.5 30,000 



S.faecalis 0.37 0.38 0.18 



L. casei 0.17 0.09 



Prepared by allowing a leucovorin solution (4 mg./ml.) to stand at pH 2 for 24 hours at 25 . 



during its conversion to PGA in slightly acid conditions and in the presence 

 of oxygen goes through certain intermediates, which in turn are converted 

 to PGA in varying yields, depending on the reducing potential and pH of 

 the solution. 



2. Microbiological Activity of Citrovorum Factor 



A summary of the microbiological activity of leucovorin, o-formjd- 

 5 , , 7 , 8-tetrahydro PGA (synthetic citrovorum factor), and related com- 

 pounds for several lactic acid bacteria is shown in Table X. It has been 

 found from a number of microbiological assays that 0.15 m7 of the an- 

 hydrous free acid of leucovorin is required for half-maximum growth of 

 Le. citrovorum ^^-^ however, when the vitamin is exposed to mildly acidic con- 

 ditions as is indicated in the table, only 2 % of the activity remains for Le. 

 citrovorum. About 200,000 times as much PGA as leucovorin is required 

 by Le. citrovorum for a comparable grow'th response ; apparently this organ- 

 ism has lost the ability to convert PGA to CF. It is apparent from the 

 table that leucovorin has PGA activity for N. faecalis and L. casei, although 



»^^ J. J. Pfiffner, S. B. Binkley, E. S. Bloom, and B. L. O'Dell, J. Atti. Chem. Sac. 69, 

 1476 (1947). 



