THE FOLIC ACID COMPLEX 



results were obtained by the microbiological assay of milk following 

 digestion with chick pancreas than by assay with chicks.^ 



Chemical Methods 



A chemical method of estimating folic acid was described by 

 Hutchings et al}^ It was based on the observation that pteroyl- 

 glutamic acid and related compounds were cleaved by reduction with 

 zinc and acid giving a pteridine and an aromatic amine. The latter 

 was estimated colorimetrically by reaction with N-naphthylethylene 

 diamine. Interference due to the presence of adenine, adenosine and 

 nucleic acid was eliminated by reducing with titanous chloride instead 

 of with zinc and acid.^^ 



Pteroylglutamic acid can also be estimated fiuorimetrically.^^ Qn 

 oxidation with potassium permanganate it is converted into 2-amino- 

 4-hydroxypteridine-6-carboxylic acid, which fluoresces strongly at 470 

 m/x. when irradiated with light of wave-length 365 m/x. ; the intensity 

 of the fluorescence is proportional to the concentration. When inter- 

 fering pigments are present, the oxidation product is isolated chromato- 

 graphically. 



Pteroylglutamic acid can also be estimated polarographically, 

 except when iron is present. ^^ With i % tetramethylammonium 

 hydroxide solution, pYl 9-0 to 9-5, as the base solution, it has a half- 

 wave potential versus the S.C.E. of 0-98 volt. With cadmium as an 

 internal standard the error of the estimation is only i 2 %. 



References to Section 7 



1. H. K. Mitchell, E. E. Snell and R. J. WilHams, /. Amer. Chem. 



Soc, 1941, 63, 2284 ; H. K. Mitchell and E. E. Snell, Univ. 

 Texas Publ., 1941, No. 4137, 36. 



2. T. D. Luckey, G. M. Briggs and C. A. Elvehjem, /. Biol. Chem., 



1944, 152, 157 ; L. J. Teply and C. A. Elvehjem, ihid., 1945, 

 157, 303. 



3. M. Landy and D. M. Dicken, /. Lah. Clin. Med., 1942, 27, 1086. 



4. V. H. Cheldelin, M. A. Eppright, E. E. Snell and B. M. Guirard, 



Univ. Texas Publ., 1942, No. 4237, 32. 



5. M. Laskowski, V. Mims and P. L. Day, /. Biol. Chem., 1945, 157, 



731- 



6. O. D. Bird, B. Bressler, R. A. Brown, C. J. Campbell and A. D. 



Emmett, ibid., 1945, 159, 631. 



7. O. E. Olson, E. E. C. Eager, R. H. Burris and C. A. Elvehjem, 



Arch. Biochem., 1948, 18, 261. 



8. A. Sreenivasan, A. E. Harper, and C. A. Elvehjem, /. Biol. Chem., 



1949, 177, 117. 



9. A. Z. Hodson, /. Nutrition, 1949, 38, 25. 



482 



