Kuchel P W, Chapman B E, Lovric V A, Raftos J E, Stewart I M, Thorburn D R
Biochim Biophys Acta. 1984 Oct 12;805(2):191-203. doi: 10.1016/0167-4889(84)90168-x.
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 mumol/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.
构建了一个基于具有半透膜壁的毛细血管组件的热力学开放系统，以研究高血细胞比容悬浮液中人类红细胞的糖酵解。获得了乳酸产生速率作为葡萄糖浓度函数的唯象表达式。该速率是在低底物浓度（约50 μmol/l）的稳态条件下测量的。在相应的封闭系统中，这种葡萄糖浓度会在几分钟内耗尽。整个系统的数学模型由五个微分方程组成，涉及流速、反应室体积、红细胞乳酸流出速率以及红细胞乳酸产生速率的表达式等参数。测量了[14C]丙酮酸与血红蛋白的结合以及[14C]乳酸从红细胞的流出速率，为该模型提供额外信息。在灌注实验期间测量了ATP和2,3 - 二磷酸甘油酸的浓度，并对红细胞己糖激酶模型进行了详细分析；前两种化合物抑制己糖激酶并改变体内葡萄糖的表观Km和Vmax。这些稳态参数分别类似于乳酸产生半最大速率和最大速率时的葡萄糖浓度。这些发现与已知的人类红细胞糖酵解中己糖激酶的高控制强度一致。该灌注系统的实际和理论验证表明，它对于使用光谱仪中的流通池进行基于NMR的红细胞代谢研究将是有价值的。
