One consequence of high blood sugar in diabetes is increased non-enzymatic glycation of proteins throughout the body. Glycation is recognized as the first step in a complex series of Maillard reactions, which lead to the browning, crosslinking, denaturation and insolubilization of proteins by reducing sugars. These reactions contribute to structural and functional changes in proteins which occur naturally with age. The acceleration of Maillard reactions during hyperglycemia in diabetes and accumulation of advanced glycation end products in proteins, particularly in long-lived lens crystallins and basement membrane and connective tissue collagens, is thought to contribute to the development of long-term complications of diabetes, such as blindness, renal failure and vascular disease. Our recent work indicates that both glycation and oxidation reactions are involved in the irreversible modification and crosslinking of proteins by glucose, yielding products which have termed glycoxidation products. The focus of the proposed research is on the relationship between glycoxidation, crosslinking and complications in diabetes. Major objectives include: (1) determination of the structure and mechanism of formation of the predominant Maillard reaction products in sugar-amino acid and sugar-protein model systems; (2) characterization of routes of oxidative degradation of sugars under physiological conditions and identification of major products formed on reaction of sugar degradation products with protein; (3) application of novel techniques for isolation and identification of Maillard reaction products and assaying their concentrations in tissue proteins, including the use of immunoaffinity and immunoassay techniques, liquid chromatography - and capillary electrophoresis - mass spectrometry (4) characterization of products formed during inhibition of the Maillard by aminoguanidine, in order to identify and compare the reactive intermediates trapped by aminoguanidine in vitro and in vivo; (5) assessment of the effects of changes in the status of oxidative stress in control and diabetic rats on the rates of formation of glycoxidation products in tissue proteins and the development of diabetic nephropathy; and (6) studies on the glycation of lipids in plasma lipoproteins and cell membranes. With a better understanding of mechanisms and products of the Maillard reaction, and the role of oxidative stress in the chemical modification of proteins by sugars, it will be possible to assess the role of this reaction in the development of pathophysiology in diabetes and then to develop better therapeutic approaches for the treatment of diabetes.
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