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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.

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Summary. Isolated, surviving sacs of everted small intestine were used to characterize ammonia transport in the golden hamster. Jejunal and ileal sacs incubated aerobically in ammonia-free test solution liberated the same quantity of ammonia as did sacs that were filled and immediately emptied of their contents, indicating no significant evolution of metabolic ammonia.Under aerobic conditions, ileal sacs transferred a solution of high ammonia content from the mucosal surface to the serosal surface against a concentration gradient. This transport was not glucose dependent and exhibited first-order Michaelis-Menten kinetics. Inhibition of absorption occurred with anaerobiosis, 2,4-dinitrophenol, and sodium cyanide. In jejunal segments ammonia was not transported against an adverse chemical gradient. Ileal ammonia absorption was accompanied by bicarbonate secretion and acidification of the serosal solution. Both bicarbonate movement and pH gradients were abolished by inhibitors of ammonia transport. In the jejunum, the absence of ammonia movement occurred in association with minimal bicarbonate secretion and no appreciable change in serosal pH.Despite the creation of hydrogen ion gradients tending to augment or to retard ammonia absorption by nonionic diffusion, ammonia movement was unaffected, i.e., relative acidification of serosal contents did not augment ammonia absorption, and relative alkalinization of serosal fluid caused no inhibition of ammonia transport. In the absence of bicarbonate ion, ammonia transport did not occur. The significance of these findings is discussed with consideration of both ionic and nonionic mechanisms of ammonia movement.

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Vitamin B12 in doses of 500 to 3000 µg. was given by mouth or by rectum to patients with megaloblastic anemia due to B12 deficiency and to normal subjects. Its absorption was studied by following the hematologic response and/or the changes in serum and urinary B12 concentrations by microbiologic assay with Euglena gracilis var. bacillaris. After oral doses of 1000 to 3000 µg. of B12 there was a definite but variable hematologic response in the thirty-five patients. The serum B12 concentrations, measured in eighteen of these patients, increased in the first twenty-four hours after the dose. The urinary excretion of B12 increased in fifteen out of sixteen patients, but the average amount excreted was only one quarter of that excreted after intramuscular injections producing comparable hematologic responses. The lower urinary excretion after the oral doses was associated with a lower total and uncombined B12 concentration in the serum. In the majority of patients serum B12 concentrations and urinary B12 excretion reached a maximum in the first six hours after the dose. After oral doses of 3000 µg. there was a rise in serum and urinary B12 in three out of four normal subjects. After doses of 500 µg., there was an increase in the serum B12 concentrations of two normal subjects and of one out of two patients with megaloblastic anemia, but there was no change in urinary B12 excretion. After rectal doses of 1000 or 3000 µg. there was evidence of absorption of the vitamin in seven out of eight patients with pernicious anemia.