Vit. B12 occupies an important place in the nutrition of microorganisms, lower animals, and man, but as yet neither minimal nor optimal daily requirements of this vitamin have been established for humans. Accordingly, studies of the distribution of vit. B12 in tissues and circulation of healthy and diseased persons seem justified. Previous studies have employed assay methods that raise the question as to whether total vit. B12 was measured, or they have dealt with a limited number of patients. The present investigation was therefore directed toward (a) evaluation of the Lactobacillus leichmannii assay method when applied to serum and (b) accumulation of sufficient data in "normal" individuals of various ages to justify statistical treatment of the results obtained. All persons reported in this study were free of overt laboratory or physical evidence of disease, and all were amhulatory. Individuals with cardiac, hepatic, renal, or pulmonary disease were eliminated. Estimations of serum vit. B12 were performed on more than 1000 persons, but, after eliminating all subjects with some deviation from "normal" health, 5 28 individuals constitute the series reported here. Microbiological methods for the determination of vit. B12 which require a turbidimetric measurement of the response to vit. B12, such as is essential in the Euglena gracilis method( 1,2), require clear and colorless samples for assay. Methods for the determination of vit. B12 where a titrimetric response is employed, such as the L. Zeichmannii method ( 3 ) , permit the direct assay of turbid and colored solutions. Preliminary to the present studies, a number of serum samples were assayed by the L. Zeichmannii assay (a) after clarification by centrifugation following heat denaturation of the serum proteins near their isoelectric point and (b) after simple dilution without preliminary removal of proteins. In the assay of "clarified" samples the denatured proteins are dis-carded and do not come in contact with the assay organism, whereas in the assay of "diluted" samples coagulation of the serum proteins takes place directly in the culture tubes. These preliminary studies demonstrated that the results obtained on the "clarified" samples were considerably lower than those obtained with the "diluted" samples. Vit. BIZ added to serum prior to clarification could not be recovered quantitatively, as noted by previous investigators, whereas vit. Bl2 added to serum prior to dilution could be quantitatively accounted for. Apparently even heat denatured proteins are capable of combining with, or adsorbing, the vitamin, since the values obtained from clarified samples of heat coagulated sera were low with respect to original vit. B12 as well as to added vit. Biz. The results suggest the desirability of distinguishing clearly between (a) the usually small amount of "free" vit. B12 present in serum, (b) the major portion of vit. B12 bound dynamically to blood proteins, chiefly to alpha globulin(4), that can be rendered free for microbiological assay by protein denatur...
