40 THE BIOCHEMISTRY OF B VITAMINS 



form has a total of seven glutamic acid residues and has been called 

 "vitamin B c conjugate." 10S 



For the liberation of free folic acid (which is active alike in S. fecaelis, 

 L. casei and chick tests) from tissues, a special enzyme (or enzymes) is 

 necessary 109 - 110 ; drastic acid or alkaline treatment, of course, causes 

 destruction of the vitamin. Even autoclaving certain bacteria in the 

 presence of water destroys most of it. 111 Treatment with commercial 

 phosphatase-containing and proteolytic enzymes or autolysis frees only 

 a fraction of the vitamin present. 



Specific enzymatic treatments yield sufficiently divergent results, when 

 applied to different tissues, to suggest the probability that folic acid 

 exists in different types of combination. 112 The fact that folic acid is 

 particularly associated with green leaves, 113, 114 where it probably func- 

 tions in a special way, suggests that special combined forms exist in 

 leaves. The observation of Bird and co-workers 115 that no enzymatic 

 treatment of plant extracts was found that would cause the microbiolog- 

 ical assay values to equal those obtained by chick assays, is in line with 

 this suggestion. 



p-Aminobenzoic Acid 



Three well defined naturally occurring combined forms of p-amino- 

 benzoic acid are known: folic acid (including conjugates), rhizopterin 116 

 and p-aminobenzoylpolyglutamic acid. 117 In addition, the acetyl deriva- 

 tive may occur in blood and urine. 118 - 119 Other information regarding com- 

 bined forms is based upon indirect evidence. Most of the p-aminobenzoic 

 acid of yeast (about 90 per cent) is in the free state; that is, it is extract- 

 able and utilizable by microorganisms. 120, 121 In different tissues tested, the 

 amounts of "bound" versus total p-aminobenzoic acid varied from 6 per 

 cent in potatoes to 93 per cent in rat kidney. Eight animal tissues averaged 

 about 80 per cent bound, whereas miscellaneous materials mostly of plant 

 origin averaged 44 per cent bound. 



The quantitative extraction of p-aminobenzoic acid from tissues offers 

 difficulties that have not been fully overcome. 



Landy and Dicken 122 autoclaved the material to be assayed with water 

 and obtained maximum yields. No data were given as to what other 

 procedures were used for comparison. Lewis 123 found greater destruction 

 with acid than with alkali, and autoclaved samples with N NaOH for 

 30 minutes to obtain assay values. Thompson et al. 120 obtained yields 

 about 3 times as high when the material (beef liver and kidney) was 

 autoclaved for 1 hour with 6N H 2 S0 4 , than when alkaline hydrolysis 

 (mild compared to that used by Lampen and Peterson below) was used. 

 Under these acid conditions they found about a 15 per cent destruction. 



