Diabetics slowly fall prey to a variety of serious, even lethal health complications which are apparently unrelated to insulin levels but have been clearly associated with hyperglycemia. We have studied the chemical molecular and cellular mechanisms which underly these diabetic sequelae for the past twenty years, and developed the central hypothesis that diabetic complications are caused in part by the covalent addition of reducing sugars, particularly glucose, to proteins by non-enzymatic reactions. Non- enzymatic glycation begins with the Amadori rearrangement of sugar-protein condensation products, and proceeds through a complex series of chemical rearrangements to generate a wide variety of Advanced Glycosylation Endproducts (AGEs) which are, as a class, permanent, fluorescent, cross- linking adducts that accumulate on cells, soluble proteins and tissue components exposed to glucose either in vitro or in vivo. Despite our prior success in identifying several AGE-related entities, the chemistry and structure of AGE adducts are known at only a rudimentary level. We now propose to extend certain novel approaches we have applied to this structural work, emphasizing the use of specially synthesized starting materials and AGE-trapping reagents to further elucidate the chemistry of AGEs. We believe, and are now demonstrating, that these chemical insights will lead to therapeutic innovations against diabetic complications. We also propose new experimental approaches to better understand the mechanism of action of one such potential therapeutic agent, aminoguanidine, discovered under the support of our last grant. Another long-standing priority has been to develop quantitative assay systems for AGEs on plasma or tissue components. We propose to refine our current assay technologies for AGEs in biological samples, primarily by developing an AGE receptor-based solid-phase competitive assay capitalizing on our recent success with a competitive whole-cell assay based on the same receptor.
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