VII. ESTIMATION 463 



no mention was made of the possible function of the high levels of ferrous 

 sulfate and cysteine other than that they were essential to keep the medium 

 in the "necessary reduced state," it appears quite likely that these com- 

 pounds directly affect vitamin B12. 



The assay method as described by Peeler ci al.*'^ is about one-tenth as 

 sensitive as the L. Icichmannii 313 (7830) method.*^ The authors claim that 

 the method gives quite consistent and reproducible results. Fourteen assays 

 on a vitamin B12 concentrate gave values ranging from 7.69 to 8.54 7 per 

 milliliter, whereas ten assay values on a U.S.P. liver extract ranged from 

 8.18 to 9.44 7 per milliliter. This method has not been applied to a large 

 number of samples or widely used by other workers. 



TABLE XII 

 Sensitivity of Various Assay Methods for Vitamin B12 



Amount of vitamin Interfering substances, maximum 



Bij required per mil- permissible amount per milliliters of 

 Method liliter of sample, m7 diluted sample extract, y 



E. coli pad method^^ 

 E. coli tube method"' ** 

 L. leichmannii (4797)" 



L. leichmannii 313 (7830)" 

 Euglena gracilis*^- *^ 

 L. lactis Dorner (8000)2" 



" Desoxyribosides of thymine, adenine, hypoxanthine, and cytosine." 



Skeggs*^ has recently reported several improvements in the \'itamin B12 

 assay medium used with L. leichmannii (4797). The medium finally de- 

 veloped is shown in Table XI. It was found that the enzymatic digest of 

 casein could be omitted from the medium. Guanylic acid as well as uridylic 

 acid and ribonucleic acid markedly improve the vitamin B12 response curve. 

 Several reducing agents, among which were thioglycollic acid, cysteine, 

 ascorbic acid, ethylenediaminetetraacetic acid, and thiomalic acid, were 

 equally effective in preventing the destruction'*^ of \'itamin B12. Thiomalic 



" H. R. Skeggs, H. M. Nepple, K. A. Valentik, J. W. Huff, and L. D. Wright, /. 



J. Biol. Chem. 184, 211 (1950). 

 " This may be partially an activation as well as a prevention of destruction, as wiL 



be discussed in Section X. 

 " B. D. Davis and E. S. Mingioli, J. Bacieriol. 60, 17 (1950). 

 ** P. R. Burkholder, Science 114, 459 (1951). 

 « S. H. Hutncr, L. Provasoli, E. L. R. Stokstad, C. E. Hoffmann, M. Belt, A. L. 



Franklin, and T. II. Jukes, Proc. Sac. Expil. Biol. Med. 70, 118 (1949). 

 ^6 W. J. Robbins A. Hervey, and M. E. Stebbins, Bull. Torrey Botan. Club 77, 423 



(1950). 



