Monitoring Red Cell Metabolism using a Lab on a Chip

描述（由申请人提供）：在本提案中，我们描述了分析系统的开发，该系统将能够阐明红细胞（RBC）在 II 型糖尿病引起的并发症中的作用。具体来说，我们建议使用芯片实验室方法来检查红细胞通过其抗氧化防御系统维持固有变形性的能力。最近，已经证明红细胞变形能力是变形诱导的三磷酸腺苷（ATP）释放的决定因素。此外，这种 ATP 是一种已知的刺激物，可促进微循环阻力血管内皮细胞中一氧化氮的产生。当释放时，这种内皮衍生的一氧化氮最终导致阻力血管周围的平滑肌细胞松弛，并随后扩张循环血管。这种扩张可以增加红细胞流量，将氧气输送到所需的组织并维持适当的血压。有文献报道，II型糖尿病患者的红细胞比健康患者的红细胞变形程度更小。因此，II 型糖尿病患者红细胞变形能力的降低可能与红细胞来源的 ATP 减少（这反过来可能导致内皮细胞中 NO 产生减少）以及随后高血压或循环问题的增加（这两者都是大多数 II 型糖尿病患者所遭受的并发症）有关。在这里，我们建议 1) 在微流体装置上开发一种电流检测方案，以确定兔子红细胞中 GSH:GSSG 的比率，2) 在微流体装置上开发一种电流测定法，以确定兔子红细胞中 NADPH 的水平，3) 开发一种基于芯片的方法，用于测定红细胞变形性，4) 采用 1-3 中开发的测量方案来确定这些分子的水平，同时同时定量测定家兔红细胞和II型糖尿病患者红细胞释放的ATP量。重要的是，该设备不仅仅用于监测终点代谢物；相反，它将首次能够实时监测关键的代谢途径（特别是磷酸戊糖途径），同时测量身体结果（ATP 释放）。这些测定将有助于确定红细胞在糖尿病并发症中的作用，从而改善糖尿病患者的健康。

DESCRIPTION (provided by applicant): In this proposal, we describe the development of an analytical system that will enable the elucidation of the role of red blood cells (RBCs) in complications resulting from type II diabetes. Specifically, using a lab on a chip approach, we propose to examine the ability of erythrocytes to maintain inherent deformability via their antioxidant defense system. Recently, it has been demonstrated that erythrocyte deformability is a determinant of deformation-induced release of adenosine triphosphate (ATP). Moreover, this ATP is a known stimulant of nitric oxide production in endothelial cells that line resistance vessels in the microcirculation. When released, this endothelium-derived NO results in the eventual relaxation of smooth muscle cells surrounding the resistance vessels and subsequent dilation of the circulatory vessel. This dilation allows for an increase in erythrocyte flow delivering oxygen to required tissues and maintaining proper blood pressure. It has been reported in the literature that the erythrocytes of patients with type II diabetes have erythrocytes that are less deformable than erythrocytes of healthy patients. Thus, it is possible that the decreased deformability of erythrocytes from type II diabetics may be related to a decrease in erythrocyte-derived ATP (which in turn may result in diminished NO production in endothelial cells) and a subsequent increase in hypertension or circulation problems (both of which are complications suffered by most type II diabetics). Here, we propose 1)To develop an amperometric detection scheme on a microfluidic device to determine the ratio of GSH:GSSG in the RBCs of rabbits, 2) To develop an amperometric assay on a microfluidic device to determine the levels of NADPH in the RBCs of rabbits, 3) To develop a chip-based method for the determination of RBC deformability, 4) To employ the measurement schemes developed in 1-3 to determine the levels of those molecules while at the same time quantitatively measuring the amount of ATP released from rabbit RBCs and the RBCs from patients with Type II diabetes mellitus. Importantly, this device will not be employed solely for monitoring endpoint metabolites; rather, it will enable, for the first time, the ability to monitor a crucial metabolic pathway (specifically, the pentose phosphate pathway) in real time while simultaneously measuring the physical outcome (ATP release). Such determinations will help identify the role of RBCs in diabetic complications, thus improving the health of patients with diabetes.