X. EFFECTS OF DEFICIENCY 487 



tmtcd from pit; manure l)y Wymcngti"" was found to be a mixture of 

 ]is(>u(lovitamiii Bi.. and factor B with a small amount of vitamin Bi-^ itself. 

 Factor WR of \\'\'monga is also a crude mixture containing factor A, 

 l)soudovitamin Bi- , and vitamin Bio . The significance of factor WR was 

 its preparation from beef liver. Thus, the designations of vitamin liijm , 

 factor WR, and \itamin Biof should be discarded in favor of the substances 

 l>ropared in pure form and characterized as to their position after electro- 

 phoresis on paper under defined conditions. None of the forms of vitamin 

 Bi? , factor A, B, C, and pseudovitamin B12 possesses significant activity 

 for animals. Those forms most commonly occur in the species of many 

 different animals and in several cases have been demonstrated to exist in 

 urine. For example, the vitamin B12 activity of cow urine is made up entirely 

 of substances other than vitamin B12. Calf feces contain fraction A with 

 some pseudovitamin B12 and a trace of vitamin B12 . 



5. Antimetabolites of Vitamin B12 



Woolley-^ found that l,2-dichloro-4,5-diaminobenzene was inhibitory to 

 organisms which do not show a nutritional requirement for riboflavin or 

 \itamin B12. It failed to retard growth of organisms which require both 

 these vitamins. Woolley-^ presents convincing evidence that dichlorodi- 

 aminobenzene is an antimetabolite of 1 , 2-dimethyl-4 , 5-diaminobenzene 

 contained within the structure of the two vitamins and pictured as a 

 metabolic precursor of them. Dimethyldiaminobenzene was found to have 

 some activity in replacing vitamin B12 for L. leichmannii 313 and E. gracilis. 

 Mice whose nutritional needs may be compared to the lactobacilli which 

 reciuire both riboflavin and vitamin B12 were likewise very resistant to 

 dichlorodiaminobenzene. They tolerated a level of 0.5 % in their ration in 

 addition to daily injections of 6 mg. Table XVITI from the excellent book 

 of WooUey-'' on antimetabolites shows the unique correlation of the toxicity 

 of dichlorodiaminobenzene with nutritional requirements for riboflavin and 

 vitamin B12. Direct evidence that dimethyldiaminobenzene participates in 

 the biosynthesis of vitamin B12 is the observation-*^ that in growing Bacillus 

 megatherium cultures additions of the compound increased synthesis of the 

 vitamin without affecting growth. Further structural analogs of dimethyl- 

 diaminobenzene were synthesized by WooUey and Pringle,-^- •'" many for 



^^° H. G. Wymenga, Vitamin Bu and Other Factors, Thesis, University of Utrecht, 



1951. 

 2" D. W. Woolley, J. Exptl. Med. 93, 13 (1951). 

 2' D. W. Woolley, A Study of Antimetabolites. John Wilej' and Sons, New York, 



1952. 

 " D. W. Woolley, Proc. Soc. Exptl. Biol. Med. 75, 745 (1950). 

 29 D. W. Woolley and A. Prinsle, Federation Proc. 10, 272 (1951). 

 3« D. W. Woolley and A. Pringle, ./. Biol. Chem. 194, 729 (1952). 



