CO,~O VIT. B12 SERUM BINDING CAPACITY 169 adult mast cells of the mesentery of rats after moderate intraperitoneal doses of the histamine liberator, compound 48/80. After larger doses, which cause disintegration of mesenteric mast cells, mitoses are found in mast cells of the subcutaneous tissue. 2) Replacement of cells after disintegration appears to be by heteroplastic differentiation of lymphocytes or undifferentiated mesenchymal cells or both.Recent studies ( 1-4) of plasma concentration and transport mechanisms of vit. B12 have included microbiological measurements of its serum in vitro binding capacity. Results have varied widely. The present investigations are concerned with estimation of Co60 vit. B12 serum binding capacity.Methods. Sera were obtained from 40 normal persons. The CoSo vit. Bl2 had a specific activity of 0.786 mc/mg. Its physiological activity was proved when 3 pg given intravenously to a patient with pernicious anemia in relapse induced a moderate reticulocyte response and clinical improvement. To 1 ml of serum was added an aliquot of CoB0 vit. B12 ranging from 1 to 500 pg. This was done either by adding the chosen amount of vitamin directly, or by making an initial concentrated solution in serum, then rediluting with more serum at once. These methods gave equal results. Specimens were prepared in duplicate, one to serve as a standard. Each was pipetted into a Visking bag. Samples were incubated at room temperature for 2 hours, then subjected to cons tan t agitation *C060 Vit. B , , was obtained from Merck & Co, Rahway, N. J. and exhaustive dialysis in cold, running tap water for 48 hours. Standards were placed in 4 ml vials at once; samples were placed in similar vials at conclusion of dialysis. Each vial was filled with concentrated sulfuric acid, dissolving the bag and allowing complete mixing within the vial. Counting was done in a well-type scintillation counter. "Bound" vit. B12 was calculated as activity retained in the bag after dialysis. Parallel studies were made with CoGoC12 and sera from 14 normal subjects. The specific activity and cobalt content of the CoS0Cl2 solution were equal to that of the radioactive vit. BI2. Continuing dialysis for 72 or 96 hours did not decrease residual activity further than the 48-hour period used. Incubation of the serum-B12 mixture for more than 2 hours prior to dialysis did not alter amount of vit. B12 bound. Dialysis for 48 hours in a reservoir of normal saline, 6% dextran in normal saline, or human plasma 50x the volume of the samples and changed at 24 hours gave the same results as exhaustive dialysis in tap water. CoB0 B12 in saline dialyzed for 48 hours in tap water showed residual activity within the Visking bag of less than 2% at any concentraby guest on July 28, 2015 ebm.sagepub.com Downloaded from

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CSF CoGO BI2 BINDING CAPACITY 60 7 animal. In our experiments doses several times the lethal dose elicited no responses which suggested release of significant amounts of histamine, Summary. 1) In anesthetized rabbits, lethal doses of compound 48/80 produced marked depression of respiration with ensuing blood pressure changes that could be altered by administering artificial respiration. Blood pressure and electrocardiographic records in animals artificially respired soon after receiving doses several times the lethal dose revealed no evidence of adverse effects on the vascular system or on cardiac rate and rhythm. 2 ) Under similar conditions, however, a lethal dose of histamine produced a prompt and profound fall in blood pressure, marked changes in cardiac rate and rhythm and death despite artificial respiration. The effect appears to be primarily on the cardiovascular system. Respiratory effects are prob-aldy secontlary to this main action. 3 ) The lethal action of 48/80 in rabbits is apparently not mediated by release of endogenous histamine since large doses do not elicit histaminelike responses.