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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK071888-05
Application #
7746354
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Castle, Arthur
Project Start
2007-01-15
Project End
2011-06-30
Budget Start
2010-01-01
Budget End
2011-06-30
Support Year
5
Fiscal Year
2010
Total Cost
$228,951
Indirect Cost
Name
Michigan State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
Raththagala, Madushi; Karunarathne, Welivitya; Kryziniak, Matthew et al. (2010) Hydroxyurea stimulates the release of ATP from rabbit erythrocytes through an increase in calcium and nitric oxide production. Eur J Pharmacol 645:32-8
Oblak, Teresa D'Amico; Meyer, Jennifer A; Spence, Dana M (2009) A microfluidic technique for monitoring bloodstream analytes indicative of C-peptide resistance in type 2 diabetes. Analyst 134:188-93
Tolan, Nicole V; Genes, Luiza I; Subasinghe, Wasanthi et al. (2009) Personalized metabolic assessment of erythrocytes using microfluidic delivery to an array of luminescent wells. Anal Chem 81:3102-8
Meyer, Jennifer A; Subasinghe, Wasanthi; Sima, Anders A F et al. (2009) Zinc-activated C-peptide resistance to the type 2 diabetic erythrocyte is associated with hyperglycemia-induced phosphatidylserine externalization and reversed by metformin. Mol Biosyst 5:1157-62
Batz, Nicholas G; Martin, R Scott (2009) Selective detection of endogenous thiols using microchip-based flow analysis and mercury/gold amalgam microelectrodes. Analyst 134:372-9
Meyer, J A; Froelich, J M; Reid, G E et al. (2008) Metal-activated C-peptide facilitates glucose clearance and the release of a nitric oxide stimulus via the GLUT1 transporter. Diabetologia 51:175-82
Subasinghe, Wasanthi; Spence, Dana M (2008) Simultaneous determination of cell aging and ATP release from erythrocytes and its implications in type 2 diabetes. Anal Chim Acta 618:227-33
Ku, Chia-Jui; D'Amico Oblak, Teresa; Spence, Dana M (2008) Interactions between multiple cell types in parallel microfluidic channels: monitoring platelet adhesion to an endothelium in the presence of an anti-adhesion drug. Anal Chem 80:7543-8
Tolan, Nicole V; Genes, Luiza I; Spence, Dana M (2008) Merging Microfluidics with Micro-titre Technology for More Efficient Drug Discovery. JALA Charlottesv Va 13:275-279
Moehlenbrock, Michael J; Martin, R Scott (2007) Development of an on-chip injector for microchip-based flow analyses using laminar flow. Lab Chip 7:1589-96