No abstract

THE vitamin B, , concentrations in the serum of normal subjects, of patients with niegaloblastic anaemia due to vitamin B, , deficiency and of patients with various other diseases have been previously reported by us Ross, 1952, 1954). Assays of vitamin B,, were made with Euglena gracilis var. bacillaris as test organism.The total vitamin B, , concentration of the serum of 126 healthy normal subjects aged 15 to 70 years ranged from IOO to 900 wg./ml. with a mean of 362 wg./ml. Almost all the vitamin was in the combined form. The mean serum B,, concentration in each of 190 patients with pernicious anaemia in relapse was much lower than those of the normal subjects, being less than 100 !.wg./ml.; in the majority of patients it was less than 50 wg./ml. In the patients with other blood disorders the serum B,, concentrations were not significantly different from those of normal subjects. This group included some patients with acute and chronic leukaemia (Mollin and Ross, 1952).More detailed studies of the serum B,, concentrations of patients with different types of leukaemia have shown that while the concentrations in chronic lymphocytic leukaemia and in acute undifferentiated leukaemia were normal, those in chronic myelocytic leukaemia were in all cases very much higher. The mean serum B,, concentrations of patients yith certain other conditions have also been found to be significantly higher than normal. The results of our more recent observations are given in this paper, a summary of which was coniniunicated to the Fifth International Congress of Haematology, which was held in Paris in September 1954. The data now presented have been obtained from 56 patients with leukaemias of various types, 32 with polycythaemia rubra Vera, eight with myelosclcrosis, two with leukaemoid reactions, six with myelomatosis, seven with aplastic anaemia, and one with chronic agranulocytosis. Except where stated in the text, the patients were UIItreated at the time when the assays were made. METHODS HaenintologicalThe methods used were those described by Dacie (1950). Needle biopsies of the bonemarrow were made on all patients, a diagnosis of myelosclerosis being supported by histological examination of bone-marrow removed by surgical biopsy. A diagnosis of polycythaemia rubra Vera was based on the demonstration of hyperplasia of the erythropoietic, granulopoietic and platelet-forming tissues of the marrow associated with an increased peripheral red-cell count, leucocyte count and/or platelet count. Differential counts on bonemarrow films were based on counts of at least 500 cells.

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.-The vitamin 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.