Study on mechanism of ischemia-reperfusion injury in ischemic pancreas, and development of its preventive modality and viability assessment method.

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The mechanism of ischemia-reperfusion injury and impaired microcirculation in ischemic pancreas was was investigated with respect to the biochemical and structural analysis of red cell membrane and cytoskeletal protein, and red cell rouleau formation, deformability and filerability. And followings were clarified.1.The deformability and membrane stability of red blood cells (RBCs) taken from healthy control (group A), nondiabetic dialysis patients (group B), nonuremic diabetic patients (group C) and diabetic dialysis patients (group D) were investigated using Ektacytometry. No difference in deformability index (DI) of whole blood and RBC was observed between group B and A, while DI of whole blood and RBC in group C and D was depressed as treatment with phenyhydrazine was depressed. The DI of RCG containing oxidized hemoglobin was depressed and its membrane stability was augmented. These findings suggested that the interaction among cytoskeletal red cell proteins would be altered. The fr … More agmentation of RBC was examined with RCGs exposed to shear stress (750dyn/cm^2) and T50, when the DI became 0.5 (50%), was 15.6+/-23sec in group A, 20.25+/-2.63sec in group B, 17.9+/-4.02sec in group C and 16.25+/-1.44sec in group D.2.The filterability of diluted RBCs solution through nickel mesh (pore size=4μm) was studied under the continuous negative pressure at -50mmH_2O. As in group B, C and D, the flow rate-pressure curve was shifted to the right as compared with group A, the filterability of RBCs was depressed in group B, C and D.3.Along with spectrin and actin, cytoskeletal red cell protein 4.1 organizes hexagonal lattice complex (mesh-like network) in the horizontal plane of membrane cytoskeletal structure of RBC, The biochemical and structural study of skeletal red cell protein 4.1 proved the binding site of calmodulin/Ca^<++> to protein 4.1(30kDa domain), at which protein 4.1 was hydrophobically bound to fatty acid in the lipid layer of the surface membrane.4..No difference in the band pattern of cytoskeletal red cell proteins by SDS-PAGE was noticed among 4 groups.5.Anisocytosis was seen by microscopic observation of RBC smear stained with Giemsa in groups B and D, although it was confined to little difference.6.As the percentage of annexin V-positive red cells by Flow cytometry was significantly increased in group D, it was suggested that the appearance of phosphatidyl-serine, normally confined to the inner lipid layer, at the outer lipid layer of red cell surface membrane was increased and that the composition of phospholipids in the lipid layer and the asymmetry of the lipid bilayer of red cell surface membrane were deranged.7.Although RBCs repel each other by the negative charge elicited by sciatic acids at the side chain of glycoprotein (glycophorin etc) on red cell surface membrane, the decrease in siaic acids and the defect of glycophorin caused the decreased negative charge and the aggregation of RBCs. The membrane electronic charge was studied by examining the absorbability of RBCs in each group using anion and cation exchange resin. Among 4 groups, no difference in absorbability of RBCs to anion/cation exchange resin was observed. The treatment with endo-β-galactosidase almost completely removed glycoprotein from red cell surface membrane and RBCs aggregated each other. Additionally the treatment with rectin, bound to glycoprotein on red cell surface membrane specifically, depressed the deformability by the observation using laser diffraction method. These findings implied that rouleau formation of RBCs would be mediated by the reduction or the defect of glycoprotein on red cell surface membrane.In conclusion, the deformability and the filerability of RBCs were depressed in diabetic dialysis patients. The asymmetry of lipid b*layers of red cell surface membrane was deranged by the increase in phosphatidylserine in the outer lipid layer in diabetic dialysis patients. The removal of glycoprotein from red cell surface membrane caused the decreased negative charge of red cell surface membrane and RBCs easily aggregated each other. The exposure to the oxidative stress depressed the deformability of RBCs and altered the interaction of cytoskeletal red cell proteins. Less