OXYGEN TRANSPORT CYS CELL FREE HEMOGLOBIN

用于临床使用的基于血红蛋白的氧载体仍然难以捉摸，主要是因为无细胞血红蛋白诱导血管收缩和减少组织氧合。我们建议，无细胞血红蛋白产生的血管收缩是正常的自动调节机制的结果，这些机制是在响应增加扩散O2运输血红蛋白是免费的血浆空间。我们已经发现，负责这种扩散现象的无细胞血红蛋白的关键属性（配体亲和力，粘度和胶体渗透压）显着影响无细胞血红蛋白向组织输送O2的能力。血红蛋白的不同类型的化学修饰引起一系列生理效应，从血管活性和酸碱状态到动物血容量的变化。这些发现使我们质疑长期存在的假设，即无细胞血红蛋白要有效地运输O2，它们必须模仿RBC的特性。我们将评估改性血红蛋白溶液的关键物理和生化特性，以确定确定O2运输生理差异的相关特性。具体的重点将是配体结合（O2和NO），溶液性质（粘度和oncurrentpressure），和分子大小。该提案包含四个具体目标。1)根据被认为是O2转移到组织的关键决定因素的配体结合特性，选择和表征用于研究的模型无细胞血红蛋白。这些包括O2结合和在血红素和亚硝基硫醇的NO反应性。2)选择和表征模型无细胞血红蛋白的基础上的解决方案的性质被认为是氧气转移到组织的关键决定因素的研究。这些因素包括粘度、胶体吸附压力、分子量和摩尔体积，以及改性类型（分子内与表面）的影响。3)在人工毛细血管系统中使用这些模型无细胞氧载体，以确定由载体的物理化学性质引起的对O2扩散的改变。该系统独立于生物调节机制。4)测定大鼠体内血液动力学心输出量、酸碱平衡、乳酸产生和对模型载体等容交换输血及随后出血的血容量反应。所选载体对平均动脉血压和心输出量的影响将提供完整动物中对这些无细胞氧载体血液稀释的血管活性反应的指示。将监测血容量，以提供等容交换输血后发生的液体转移的指示。动脉乳酸和酸碱状态提供了O2供需平衡的额外指标。总之，这些目标将不仅导致合理设计的氧载体用于临床使用，而且重要的洞察血管活性和组织氧合的控制机制。

A hemoglobin-based oxygen carrier for clinical use remains elusive, primarily because cell-free hemoglobin induces vasoconstriction and reduced tissue oxygenation. We propose that vasoconstriction produced by cell-free hemoglobin is the result of normal autoregulatory mechanisms which are engaged in response to increased diffusive O2 transport when hemoglobin is free in the plasma space. We have discovered that the critical properties of cell-free hemoglobin responsible for this diffusive phenomenon, (ligand affinity, viscosity and colloid osmotic pressure) markedly influence the ability of cell-free hemoglobin to deliver O2 to tissue. Different types of chemical modification of hemoglobin elicit a range of physiological effects from vasoactivity and acid-base status to changes in blood volume in animals. These findings have led us to question the long-standing assumption that for cell-free hemoglobins to effectively transport O2, they must mimic the properties of RBCs. We will evaluate key physical and biochemical characteristics of modified hemoglobin solutions to identify pertinent properties that determine physiologic differences in O2 transport. Specific focus will be on ligand binding (O2 and NO), solution properties (viscosity and oncotic pressure), and molecular size. The proposal contains 4 Specific Aims. 1) To select and characterize model cell-free hemoglobins for study on the basis of ligand binding properties believed to be critical determinants of O2 transfer to tissue. These include O2 binding and NO reactivity at the hemes and as nitrosothiol. 2) To select and characterize model cell-free hemoglobins for study on the basis of solution properties believed to be critical determinants of O2 transfer to tissue. These include viscosity, colloid oncotic pressure, molecular weight and molar volume, and the influence of the type of modification (intramolecular versus surface). 3) To use these model cell-free oxygen carriers in an artificial capillary system to determine the alteration to 02 diffusion resulting from the physicochemical properties of the carriers. This system is independent of biological regulatory mechanisms. 4) To measure in vivo hemodynamic cardiac output, acid-base balance, lactate production, and blood volume responses to isovolemic exchange transfusion with the model carriers and subsequent hemorrhage in rats. The effect of the selected carriers on mean arterial blood pressure and cardiac output will provide indications of vasoactive response to hemodilution with these cell-free oxygen carriers in intact animals. Blood volume will be monitored to provide an indication of the fluid shifts that occur in response to isovolemic exchange transfusion. Arterial lactic acid and acid-base status provides additional indicators of the balance between O2 supply and demand. Taken together, these aims will not only lead to rationally-designed oxygen carriers for clinical use, but also important insight into mechanisms of vasoactivity and control of tissue oxygenation.