The relationship between glucose concentration and rate of lactate production by human erythrocytes in an open perfusion system |
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Authors: | Philip W Kuchel Bogdan E Chapman VAlbert Lovric Julia E Raftos IMalcolm Stewart David R Thorburn |
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Institution: | 1. Department of Biochemistry, University of Sydney, Sydney, New South Wales, 2006, Australia;2. Red Cross Blood Transfusion Service, 153 Clarence Street, Sydney, New South Wales, 2000, Australia |
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Abstract: | A thermodynamically open system, based on an assembly of capillaries with semi-permeable walls was constructed in order to study glycolysis in human erythrocytes in high haematocrit suspensions. A phenomenological expression for the rate of lactate production as a function of glucose concentration was obtained. The rate was measured under steady-state conditions with low substrate concentrations (approx. 50 μmol/l). In a corresponding closed system, this concentration of glucose would be exhausted within a few minutes. A mathematical model of the whole system consisted of five differential equations, and involved parameters relating to flow rates, volumes of reaction chambers, the rates of lactate efflux from erythrocytes and the expression for the rate of lactate production by red cells. The binding of 14C]pyruvate to haemoglobin and the rate of efflux of 14C]lactate from red cells were measured to yield additional information for the model. The concentrations of ATP and 2,3-bisphosphoglycerate were measured during the perfusion experiments, and a detailed analysis of a model of red cell hexokinase was carried out; the former two compounds inhibit hexokinase and alter the apparent Km and Vmax for glucose in vivo. These steady-state parameters were similar to the glucose concentration at the half-maximal rate of lactate production and the maximal rate, respectively. These findings are consistent with the known high control-strength for hexokinase in glycolysis in human red cells. The practical and theoretical validation of this perfusion system indicates that it will be valuable for NMR-based studies of red cell metabolism using a flow-cell in the spectrometer. |
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Keywords: | Glycolysis Lactate formation Computer model Hexokinase Glucose (Human erythrocyte) Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid Mes 4-morpholineethanesulphonic acid Tes 2-{[2-hydroxy-1 1-bis(hydroxymethyl)ethyl]amino}ethanesulphonic acid |
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