GLYCATION, GLYCOXIDATION, AND CROSS-LINKING OF COLLAGEN BY GLUCOSE - KINETICS, MECHANISMS, AND INHIBITION OF LATE STAGES OF THE MAILLARD REACTION

The Maillard or browning reaction between sugar and protein contribute s to the increased chemical modification and cross-linking of long-liv ed tissue proteins in diabetes. To evaluate the role of glycation and oxidation in these reactions, we have studied the effects of oxidative and antioxidative conditions and various types of inhibitors on the r eaction of glucose with rat tail tendon collagen in phosphate buffer a t physiological pH and temperature. The chemical modifications of coll agen that were measured included fructoselysine, the glycoxidation pro ducts N-epsilon-(carboxymethyl)lysine and pentosidine, and fluorescenc e. Collagen cross-linking was evaluated by analysis of cyanogen bromid e peptides using sodium dodecyl sulfate-polyacrylamide gel electrophor esis and by changes in collagen solubilization on treatment with pepsi n or sodium dodecylsulfate. Although glycation was unaffected, formati on of glycoxidation products and cross-linking of collagen were inhibi ted by antioxidative conditions. The kinetics of formation of glycoxid ation products proceeded with a short lag phase and were independent o f the amount of Amadori adduct on the protein, suggesting that autoxid ative degradation of glucose was a major contributor to glycoxidation and crosslinking reactions. Chelators, sulfhydryl compounds, antioxida nts, and aminoguanidine also inhibited formation of glycoxidation prod ucts, generation of fluorescence, and cross-linking of collagen withou t significant effect on the extent of glycation of the protein. We con clude that autoxidation of glucose or Amadori compounds on protein pla ys a major role in the formation of glycoxidation products and cross-l inking of collagen by glucose in vitro and that chelators, sulfhydryl compounds, antioxidants, and aminoguanidine act as uncouplers of glyca tion from subsequent glycoxidation and cross-linking reactions.