ABSTRACTRed blood cells (RBCs) are known to play an important role in regulating microvascular circulation by releasing the signaling molecule adenosine triphosphate (ATP). Extracellular ATP in the bloodstream reacts with endothelial cells lining the blood vessel via a P2Y purinergic receptor, causing the endothelial cells to create and release nitric oxide, a known vasodilator. Interestingly, RBCs from Type-1 diabetic (T1D) subjects release significantly less ATP than RBCs from non-diabetic subjects. This effect is believed to impair microvascular blood flow and be a potential cause of the prevalent downstream microvascular complications seen in T1D. In T1D, the immune system destroys the pancreatic b-cells, therefore creating a deficiency of the hormones normally secreted by these cells. Since the early 1920’s, T1D patients have been administering the pancreatic b-cell secretion insulin to manage their blood sugar and stay alive. However, insulin treatment alone with diet and exercise does not prevent the microvascular complications seen in the disease. C-peptide is a 31-amino acid peptide that is co-secreted with insulin from the pancreatic b-cells along with Zn2+. Researchers have found that treating T1D RBCs with a combination of C-peptide and Zn2+ in the presence of albumin, a prevalent bloodstream protein, can significantly increase the amount of ATP released from the cells. However, RBCs are unaffected when treated with C-peptide and Zn2+ in the absence of albumin. Researchers have shown that albumin from diabetic patients has different properties than albumin from non-diabetic subjects. Therefore, the hypothesis of the work in this thesis is that diabetic albumin interferes with the interaction of RBCs with C-peptide and Zn2+.The work in this dissertation is focused on the role of albumin and its interactions with C-peptide, Zn2+, and RBCs. 3D-printing technology was used in order to create novel devices to study interactions between albumin and C-peptide and Zn2+ under diabetic conditions. A 3D-printed equilibrium dialysis device was used to measure the binding affinity between human serum albumin (HSA) and Zn2+ (Kd= 562 ± 93 nanomolar). It was found that the affinity of this interaction was decreased by glycation of albumin, but not by immediate addition of glucose. The device was also used to measure that C-peptide does not alter the affinity of Zn2+to albumin. The dialysis device is compatible with plate-reader technology and enables automated and direct measurements of certain analyte ligands. Further, a 3D-printed ultrafiltration device was created in order to measure the binding affinity between HSA and C-peptide (Kd= 2.4 ± 0.3 micromolar). The affinity of this interaction was not altered by Zn2+, glucose, or glycation of albumin. Novel 3D-printing techniques were developed in order to create these devices, such as the Print-Pause-Print method for integrating membranes directly into 3D-printed devices, and a support-free Polyjet printing technique.Lastly, the interaction of RBCs with C-peptide and Zn2+ was measured in the presence of normal and glycated albumin. The ability of RBCs to release ATP under flow conditions was increased significantly in the presence of C-peptide/Zn2+ and normal albumin (p<0.05); whereas the RBCs treated with C-peptide/Zn2+ and glycated albumin did not release statistically more ATP than the control (n=4 blood draws). The ability of C-peptide to bind to RBCs in the presence of normal and glycated albumin was also measured. Approximately 1.8 ± 0.1 picomoles of C-peptide bound to RBCs in the presence of normal albumin, whereas 0.8 ± 0.1 picomoles bound in the presence of glycated albumin (n=3 blood draws, p<0.05). Therefore, the ability of C-peptide to bind and affect RBCs was affected by the presence of glycated albumin, such as seen in diabetes. The results of the studies reported in this thesis should be taken into consideration by those studying C-peptide as a replacement therapy in T1D.
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