The Vitamin B ₁₂ Concentrations of Serum and Urine of Normals and of Patients with Megaloblastic Anaemias and Other Diseases

Until recently deficiency of vitamin B12 in man could only be recognized by finding the characteristic clinical and haematological signs of the deficiency and by demonstrating that these signs were cured or relieved by treatment with the vitamin.Changes in the vitamin B12 content of the tissues could only be inferred from the responses of patients to treatment.The recent introduction of highly sensitive methods of microbiological assay has made it possible to measure the actual vitamin B12 content of body.fluids.Euglena gracilis var.bacillaris is the most sensitive organism for microbiological assay (Hutner, Provasoli, Stokstad, Hoffmann, Belt, Franklin, and Jukes, 1949; Ross, in the press), and the purpose of this paper is to report the results of assays with this organism and to demonstrate the value of the assay method in the study of megaloblastic anaemias.The serum-and urinary vitamin B12 concentrations of patients with pernicious anaemia have been found to be much lower than those of both normal subjects and of patients suffering from other diseases.The concentrations found in patients suffering from megaloblastic anaemias other than pernicious anaemia varied.In some the concentrations were as low as those found in pernicious anaemia, but in others the vitamin B12 concentrations were normal.Materials and Methods Serum.-Blood was obtained by venepuncture, using a dry syringe, and was allowed to clot in a sterile glass container at 370 C. The serum was removed after one hour at this temperature and kept frozen until assayed.Assays were usually made within three days of collection.Urine.-Twenty-four hourly collections were made, using toluene as preservative.In most instances each sample was stored at 4°C. immediately after being passed.Assays were usually made within three days of collection.X Method of Assay.-Thevitamin B12 concentration in the serum and urine was measured by a modification of the assay method introduced by Hutner et al. (1949), in which the green alga Euglena gracilis was used as the test organism.Details of the technique will be published in the next issue of this journal.As little as 10 ppg. per ml. of vitamin B,2 are detectable in serum and urine with this method.