COMBINED FORMS— EXTRACTION 41 



Lampen and Peterson 119 made a careful study of hydrolytic conditions 

 using as the principal material to be assayed a "dry powdered liver 

 sample." They found liberation by acid hydrolysis to be rapid, but that 

 it never appeared to be complete. Subsequent digestion of the same soluble 

 material with alkali caused an increase in the total p-aminobenzoic acid, 

 indicating that the acid treatment had extracted the compound from the 

 liver but had not rendered it available to the test organism (CI. aceto- 

 butylicum) . Freeing of the available form took place more slowly under 

 alkaline conditions, but proceeded considerably further. The curve for 

 autoclaving (15 lbs.) with 2N NaOH had not reached a plateau even at 

 20 hours. Some destruction was observed as a result of long alkaline treat- 

 ments. The question of whether the p-aminobenzoic acid being formed 

 during drastic alkaline treatment might be an artifact was investigated, 

 with negative indications. Preliminary treatment with acid seemed to 

 cause the subsequent liberation to be more rapid under alkaline con- 

 ditions. 



These four studies cited involved the use of four different assay organ- 

 isms, and this may be partially responsible for the differences observed. 

 It is possible that the "aminobenzoicless" Neurospora used in the study 

 of Thompson et al. 120 responds to a conjugate produced by acid treatment, 

 and that the Clostridium acetobutylicum does not. This would help to 

 explain the difference between the last two studies cited. 



The numerous observations, as well as those relating to folic acid, 

 suggest that p-aminobenzoic acid may be combined in nature in a number 

 of ways and that some of these combinations are extraordinarily stable. 

 Further study is required to clarify the picture. 



A comparison of available p-aminobenzoic acid assay values for beef 

 liver, spinach and egg with corresponding folic acid assay values indicates 

 that there is 3-10 times as much p-aminobenzoic acid present as could be 

 derived from the folic acid present. Lampen and Peterson 119 found a 

 maximum of 8 fxg per gram of p-aminobenzoic acid in liver powder. This 

 would require 25.6 ixg/gvo. of folic acid if it were all in this form. The 

 folic acid content of beef liver (no conjugase used) on a dry basis was 

 2.8 ng/gm (calculated on the basis of 160,000 potency). Even if this 

 value were to be doubled or tripled by conjugase action, there would 

 still be more than half of the total p-aminobenzoic acid present in liver 

 in some conjugated form other than folic acid and its conjugates. 



Bibliography 



1. Kogl, F., and Hasselt, W. van, Z. physiol. Chem., 243, 189-94 (1936). 



2. Williams, R. R., Waterman, R. E., and Keresztesy, J. C, J. Am. Chem. Soc. 



56, 1187-91 (1934). 



