OXYGEN TRANSPORT CYS CELL FREE HEMOGLOBIN

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会导致血管收缩和组织氧合减少。我们认为，无细胞血红蛋白产生的血管收缩是正常的自我调节机制的结果，当血红蛋白在血浆空间中游离时，这种机制参与了对弥漫性氧运输增加的反应。我们发现，导致这种扩散现象的无细胞血红蛋白的关键特性（配体亲和力、粘度和胶体渗透压）显著影响无细胞血红蛋白向组织输送氧气的能力。不同类型的血红蛋白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。这些发现使我们对长期以来的假设提出了质疑，即无细胞血红蛋白必须模仿红细胞的特性才能有效地运输氧气。我们将评估修饰血红蛋白溶液的关键物理和生化特性，以确定决定氧运输生理差异的相关特性。重点将放在配体结合（O2和NO）、溶液性质（粘度和肿瘤压力）和分子大小上。该提案包含4个具体目标：1)根据被认为是O2向组织转移的关键决定因素的配体结合特性，选择和表征模型无细胞血红蛋白进行研究。这些包括O2结合和NO在血红素和亚硝基硫醇上的反应性。2)根据被认为是O2向组织转移的关键决定因素的溶液特性，选择和表征模型无细胞血红蛋白进行研究。这些因素包括粘度、胶体致瘤压力、分子量和摩尔体积，以及改性类型（分子内与表面）的影响。3)在人工毛细管系统中使用这些模型细胞无氧载体，确定载体的物理化学性质对02扩散的改变。该系统不依赖于生物调控机制。4)测量模型携带者等容交换输注及出血后大鼠体内血流动力学心输出量、酸碱平衡、乳酸生成和血容量反应。所选载体对平均动脉血压和心输出量的影响将提供血管活性反应的适应症，以血液稀释这些无细胞氧载体在完整的动物。将监测血容量，以提供对等容交换输血反应中发生的液体转移的指示。动脉乳酸和酸碱状态提供了氧气供需平衡的额外指标。综上所述，这些目标不仅将导致合理设计供临床使用的氧载体，而且对血管活性和组织氧合控制的机制也有重要的见解。

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.