92 PTEROYLGLUTAMIC ACID 



preparation from spinach was described in a series of papers by Mitchell 

 and coworkers at Texas.^'^ These workers employed repeated adsorption 

 and elution from activated charcoal, precipitation with heavy metals, and 

 chromatographic adsorption on alumina. Since folic acid from spinach has 

 never been crystallized, it is not possible to establish its identity with 

 PGA. However, comparison of samples of folic acid concentrates of known 

 potencies with pure PGA^- ^ indicates that the most active preparation ob- 

 tained by the Texas workers had essentially the same biological potency as 

 pure PGA. 



The isolation of PGA from liver has been reported from two laboratories. 

 Pfiffner et al.^ obtained this compound by starting with liver in which the 

 conjugates had been converted into free PGA by autolysis.^" The isolation 

 process involved extraction with boiling water, adsorption and elution 

 using first the ion exchange resin Amberlite 4R and later activated charcoal, 

 extraction of an aqueous solution of the free acid with butanol at pH 3 to 4, 

 formation of a barium salt and a zinc salt, and finally crystallization of the 

 free acid from water. 



Stokstad^^ reported the isolation of the methyl ester of PGA and later of 

 the free acid.^^ The starting material was an 80% alcohol precipitate of an 

 aqueous extract of liver. The method involved adsorption and elution, first 

 with Norit and then with Super Filtrol, precipitation of the barium salt 

 with methanol, esterification in acid methanol, extraction of the methyl 

 ester from aqueous solution with butanol, chromatographic adsorption of 

 the methyl ester on Super Filtrol, and fractional precipitation of the ester 

 from water and methanol. The free acid was obtained by saponification of 

 the ester and crystallization from hot aqueous dilute acetic acid solution. 



Pteroylglutamic acid has also been isolated from yeast extracts by Pfiffner 

 et al}^ after treatment with hog kidney enzyme which hydrolyzed the con- 

 jugated vitamin to free PGA. 



3 H. K. Mitchell, E. E. Snell, and R. J. Williams, J. Am. Chem. Soc. 66, 267 (1944). 

 * E. Frieden, H. K. Mitchell, and R. J. Williams, J. Am. Chem. Soc. 66, 269 (1944). 

 6 H. K. Mitchell and R. J. Williams, J. Am. Chem. Soc. 66, 271 (1944). 



6 H. K. Mitchell, /. Am. Chem. Soc. 66, 274 (1944). 



7 B. C. Johnson, ./. Biol. Chem. 163, 255 (1946). 



8 V. Mims and M. Laskowski, J. Biol. Chem. 160, 493 (1945). 



9 J. J. Pfiffner, S. B. Binkley, E. S. Bloom, R. A. Brown, O. D. Bird, A. D. Em- 

 mett, A. G. Hogan, and B. L. O'Dell, Science 97, 404 (1943). 



10 J. J. Pfiffner, S. B. Binkley, E. S. Bloom, and B. L. O'Dell, /. Ajn. Chem. Soc. 69, 



1476 (1947). 

 " E. L. R. Stokstad, J. Biol. Chem. 149, 573 (1943). 

 12 E. L. R. Stokstad, B. L. Hutchings, and Y. SubbaRow, /. Am. Chem. Soc. 70, 3 



(1948). 



