X. EFFECTS OF DEFICIENCY 483 



L. k'icfinuuDiii 4797,^ L. Icichmannii 313, and L. acidophilus 213,^ vitamin 

 Bi2b ^vas found to be less active than vitamin B12. These results were ob- 

 tained on a medium without added reducing agents. Hoffman et al}^ had 

 demonstrated considerably earlier that vitamin B12 and vitamin Bi2b con- 

 tained in liver extract were protected from destruction by thioglycollic 

 acid. The apparent destruction took place when pure or crude samples 

 were autoclaved in the medium without a reducing agent. The protective 

 property of thioglycollic a(dd is most apparent in combination with basal 

 medium'" and is evidently of greater importance for vitamin Bi2b than for 

 vitamin B12. In spite of earlier reports^- '^ that vitamin B12 and vitamin 

 Bi2b were of approximately equal activity when added aseptically to micro- 

 biological assay medium, it appears well established at present that vitamin 

 Bi2b is considerably more potent under these conditions.^- '^■''* In fact, for 

 at least one organism, L. leichmannii 327 (A.T.C.C. 7831), vitamin B^b 

 is 20 % to 80 % more active than vitamin B12 under all conditions of assay 

 and addition.'^ Loy and Kline^* reported for L. leichmannii 313 that asepti- 

 cally added vitamin Bi2b was about 30% more active than vitamin B12, 

 whereas the two forms were of equal activity when autoclaved in the me- 

 dium containing ascorbic acid. A subsequent study by Broquist et alP 

 revealed certain points regarding the nature of the action of reducing agents 

 such as thiomalic acid. Both vitamins B12 and B^b had very low activity 

 for L. leichmannii 4797 when autoclaved with medium without thiomalic 

 acid. Thiomalic acid was added to samples of vitamin B12 and vitamin Bi2b 

 in a series of tubes which had been autoclaved once without thiomalic acid 

 and the tubes were re-autoclaved. Vitamin B12 regained full microbiological 

 activity, and vitamin B^b regained a large part of its activity. This demon- 

 strated that thiomalic acid was not protecting vitamin B12 but, when 

 present in the medium during autoclaving, actually caused the formation 

 of a microbiologically more potent form. By paper chromatography and 

 microbiological activity this form was shown to be different from vitamin 

 Bi2b. Vitamin Bi2b autoclaved in the medium with thiomalic acid was 60 % 

 to 70% as active as the product derived from vitamin B12 under these 

 conditions. When both forms were added aseptically to previously heated 

 medium containing thiomalic acid, vitamin Bi2b was about five times more 



8 E. A. Kaczka, R. G. Denkewalter, A. Holland, and K. Folkers, J . Am. Chem. Soc. 



73, 335 (1951). 

 »» C. E. Hoffmann, E. L. R. Stokstad, B. L. Hutchings, A. C. Dornbush, and T. H. 



Jukes, J. Biol. Chem. 181, 635 (1951). 

 '• D. Hendlin and M. H. Soars, J. Biol. Chem. 188, 603 (1951). 

 12 H. P. Broquist, E. L. R. Stokstad, and T. H. Jukes, Proc. Soc. Exptl. Biol. Med. 



76, 806 (1951). 

 '3 H. E. Scheid and B. S. Schweigert, J. Biol. Chem. 193, 299 (1951). 

 " H. W. Loy, Jr., and O. L. Kline, J. Assoc. Offic. Agr. Chemists 34, 225 (1951). 



