460 VITAMIN Bi2 



Hoffmann et alP found that for routine assays with L. leichmaniih 313 

 it was advisable to replace the enzymatic digest of casein with a butanol- 

 extracted asparagus juice concentrate. This resulted in less growth in the 

 blank tubes containing no added vitamin B12. These investigators also 

 describe a method for correcting for the growth due to desoxy ribosides 

 occurring m liver extracts together with vitamin B]2. Vitamin B12 was de- 

 stroyed by heating with 0.2 iV NaOH at 100° for 30 minutes under which 

 conditions the desoxyribosides of thymine, guanine, and hypoxanthine are 

 not affected. Liver extracts were assayed before and after this treatment, 

 and the difference was ascribed to vitamin B12. Assay of several liver ex- 

 tracts revealed that only about 3 % of the growth-promoting activity of 

 liver extracts was due to desoxyribosides. L. leichmannii (4797) was also 

 tested on this medium devised for L. leichmannii 313 (7830) and was found 

 to require 48 hours to reach maximum growth as compared to 20 hours 

 for L. leichmannii 313 (7830). 



The U.S. P. microbiological assay method for vitamin B12 has continued 

 to be the subject of study. Campbell et al.^^'^ suggested a change in the 

 experimental design of the method. These workers selected a portion of the 

 titrimetric response curve which, when plotted on log-log paper, gave a 

 straight line. Only three dilutions of the standard and three dilutions or 

 less of the samples were used. Using such modifications as the randomiza- 

 tion of tubes, the omission of tomato juice, and the range method of calcu- 

 lation, Campbell et at. were able to obtain limits of error as small as ±5%. 

 Frost et al.^^ compared a rat-growth assay for vitamin B12 with the U.S.P. 

 microbiological method. Studying the same U.S.P. liver extracts which 

 were used in extensive collaborative tests of the U.S.P. microbiological 

 method for vitamin B12 , these investigators found good correlation betw'een 

 the rat-growth method and the U.S.P. microbiological method. They further 

 reported that vitamins B12 and Bi2b gave equal and full response by both 

 methods and that alkali destroyed over 90 % of the vitamin B12 as measured 

 microbiologically or by the rat assay. The investigations of the I'niversity 

 of Reading group on vitamin B]2-like compounds active for microorganisms 

 but not for animals are of great importance with regard to valid micro- 

 biological assays for vitamin B12 . It must be recognized that all the organ- 

 isms now in use for the assay of vitamin B12 respond to great or lesser 

 extent to factors A, B, and C and pseudovitamin B12 . This includes bacteria 

 of the lactic acid group, E. coli and Euglena gracilis. It is therefore of great 

 importance to consider carefully all new organisms offered for the assay 

 of vitamin B12 . Hamilton et al.^^" discussed briefly the possibility of using 



=»" J. A. Campbell, J. M. McLaughlan, G. A. Clark, aud C. W. Dunnett, J. A771. 



Pharm. Assoc. Sci. Ed. 42, 276, 1953. 

 38b D. V. Frost, II. H. Fricke, aud H. C. Spruth, J. Nutrition 49, 107, 1953. 

 38° L. D. Hamilton, S. H. Hutner, and L. Provasoli, Analyst 77, 618, 1952. 



