NMR STUDIES OF SICKLE ERYTHROCYTE METABOLISM

镰状细胞性贫血（SSA）是一种慢性、使人衰弱的疾病， 疼痛、血管闭塞性危象和进行性器官衰竭。 知识 在完整的红细胞中调节镰状化过程的因子将 提供了对体内镰状化的病理生理学的更好理解， 并将提供用于测试潜在抗镰状病药物的方法。 核 磁共振（NMR）光谱是一种强大的设备， 非破坏性地从完整细胞中获取生化信息。 P-31 NMR光谱将用于将细胞内pH和2，3-DPG-Hb 结合细胞体积，血红蛋白浓度和血红蛋白-氧亲和力， 完整、正常和镰状细胞。 这些关系将首先 在一个模型系统中探索，其中细胞体积被改变 调节细胞的渗透压和溶质组成 悬浮液 该模型将与红细胞体积 已经增加了潜在的抗镰状细胞药物，使细胞 膜渗透Na+（亚碲酸盐，cetiedil），或减少其他 增加K+渗透性的药物（缬氨霉素）。 的红细胞 SSA患者将通过不连续的 阿拉伯半乳聚糖梯度密度离心分离成亚群 不断增加的密度。 使用P-31 NMR，细胞内pH， 2，3-DPG浓度和2，3-DPG - HbS结合的程度 将估计亚群，并与预测值进行比较 基于细胞体积、Hb浓度和Hb -氧的模型系统 亲和力 这些研究将验证以下假设： SSA具有较低的pH值，这是由于进行性失水和HbS增加 浓度，以及用于降低HbS的抗氧化剂， 浓度可以通过提高 这些亚群中的平均细胞年龄将是 根据丙酮酸激酶活性估计，C-13 NMR将用于 研究糖酵解代谢率。 将决定是否 糖酵解活性是镰状细胞平均年龄较年轻的反映， 细胞，或者这种活动是否被镰状化过程改变。 通过 在SSA患者上进行这些实验， 在危机和健康状况良好的情况下，将确定是否改变 镰状细胞亚群的生理特性可以与 血管闭塞状态的发展。

Sickle cell anemia (SSA) is a chronic, debilitating illness that leads to painful, vaso-occlusive crises and progressive organ failure. Knowledge of the factors which regulate the sickling process in the intact red cell will provide a greater understanding of the pathophysiology of sickling in vivo, and will provide methods for testing potential antisickling drugs. Nuclear magnetic resonance (NMR) spectroscopy is a powerful device for nondestructively obtaining biochemical information from intact cells. P-31 NMR spectroscopy will be used to relate intracellular pH, and 2,3-DPG -Hb binding to cell volume, Hb concentration and Hb - oxygen affinity in intact, normal and sickle cells. These relationships will first be explored in a model system in which the cell volumes are altered by adjustment of the osmolarity and solute composition of the cell suspension. This model will be compared to one in which red cell volumes have been increased by potential antisickling drugs that render cell membranes permeable to Na+ (tellurite, cetiedil), or decreased by other drugs that increase the permeability to K+ (valinomycin). Red cells from SSA patients will then be separated by means of discontinuous arabinogalactan gradient density centrifugation into subpopulations of succeedingly increasing density. Using P-31 NMR, the intracellular pH, 2,3-DPG concentration and the degree of 2,3-DPG - HbS binding for each subpopulation will be estimated, and compared to the values predicted by the model systems, based on cell volume, Hb concentration and Hb - oxygen affinity. These studies will test the hypotheses that the dense cells in SSA have lower pH as a result of progressive water loss and increasing HbS concentration, and that antisickling agents that act to decrease HbS concentration may provide additional antisickling action by raising intracellular pH. The average cell ages in these subpopulations will be estimated from pyruvate kinase activities, and C-13 NMR will be used to study rates of glycolytic metabolism. It will be determined whether glycolytic activity is a reflection of the younger average age of sickle cells, or whether this activity is altered by the sickling process. By performing these experiments on SSA patients both during vaso-occlusive crisis and during good health, it will be determined whether alterations in the physiologic properties of the sickle cell subpopulations can be related to the development of the vaso-occlusive state.