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. ? ? ?
