Vitamin B ₁₂ Assay in Body Fluids using <i>Euglena gracilis</i>

Abstract

The methods of assay of vitamin B12 most generally used depend on the fact that some lacto- bacilli require this vitamin as an essential growth factor and that growth is proportional to the amount available over a certain range of concentration.Some desoxynucleotides (Shive, Sibley, and Rogers, 1951), desoxyribonucleosides and thymidine particularly, and many other substances too, have, however, been found to replace vitamin B12, although in relatively large amounts only.Assays with lactobacilli then are not entirely specific and this fact, combined with rather low sensitivity, makes these methods unsatisfactory for detecting the low concentrations of vitamin B12 found in some body fluids.The results of lacto- bacillus assays of whole blood of a number of animal species (Couch, Olcese, Witten, and Colby, 1950), of milk of a number of animal species (Collins, Harper, Schreiber, and Elvehjem, 1951), of urine of subjects receiving vitamin B12 therapy (Chow, Lang, Davis, Conley, and Ellicott, 1950), and of human urine (Girdwood, 1951) have, how- ever, been reported.Plate assays with a Bact. coli mutant requiring vitamin B12 as a growth factor (Bessell, Harrison, and Lees, 1950) are also lacking in sensitivity, but, because of the simplicity of the method and rapidity of growth, are useful for the assay of large concentrations such as are found in urine after injection of vitamin B12 (Chesterman, Cuthbertson, and Pegler, 1951).Chemical methods of assay are being developed (Boxer and Rickards, 1951; Fantes, Ireland, and Green, 1950), but are still insufficiently sensitive to detect concentrations normally found in body fluids.The use of an alga, Euglena gracilis var.bacillaris, for the assay of vitamin B12, crystalline and in liver extract, was described by Hutner, Provasoli, Stokstad, Hoffmann, Belt, Franklin, and Jukes (1949).Assays with the Euglena by both the tube and plate method of a variety of material