Recommended GLYCATION, Advanced glycation end-products, ROS (13)

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Mailard reaction and cross liking of collagen    Diabetes 1994 May; 43(5): 676-683.

Glycation, Glycoxidation, and Cross-Linking of Collagen by Glucose: Kinetics, Mechanisms, and Inhibition of Late Stages of the Maillard Reaction

Min-Xin Fu  Kevin J Wells-Knecht


The Maillard or browning reaction between sugar and protein contributes to the increased chemical modification and cross-linking of long-lived 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 reaction of glucose with rat tail tendon collagen in phosphate buffer at physiological pH and temperature. The chemical modifications of collagen that were measured included fructoselysine, the glycoxidation products Nε-(carboxymethyl)lysine and pentosidine and fluorescence. Collagen cross-linking was evaluated by analysis of cyanogen bromide peptides using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by changes in collagen solubilization on treatment with pepsin or sodium dodecylsulfate. Although glycation was unaffected, formation of glycoxidation products and cross-linking of collagen were inhibited by antioxidative conditions. The kinetics of formation of glycoxidation products proceeded with a short lag phase and were independent of the amount of Amadori adduct on the protein, suggesting that autoxidative degradation of glucose was a major contributor to glycoxidation and cross-linking reactions. Chelators, sulfhydryl compounds, antioxidants, and aminoguanidine also inhibited formation of glycoxidation products, generation of fluorescence, and cross-linking of collagen without significant effect on the extent of glycation of the protein. We conclude that autoxidation of glucose or Amadori compounds on protein plays a major role in the formation of glycoxidation products and cross-liking of collagen by glucose in vitro and that chelators, sulfhydryl compounds, antioxidants, and aminoguanidine act as uncouplers of glycation from subsequent glycoxidation and cross-linking reactions.   Journal of clinical investigations   June 1 , 1993

Accumulation of Maillard reaction products in skin collagen in diabetes and aging.

DG Dyer, JA Dunn, et al

To investigate the contribution of glycation and oxidation reactions to the modification of insoluble collagen in aging and diabetes, Maillard reaction products were measured in skin collagen from 39 type 1 diabetic patients and 52 nondiabetic control subjects. Compounds studied included fructoselysine (FL), the initial glycation product, and the glycoxidation products, N epsilon-(carboxymethyl) lysine (CML) and pentosidine, formed during later Maillard reactions. Collagen-linked fluorescence was also studied. In nondiabetic subjects, glycation of collagen (FL content) increased only 33% between 20 and 85 yr of age. In contrast, CML, pentosidine and fluorescence increased five-fold, correlating strongly with age. In diabetic patients, collagen FL was increased threefold compared with nondiabetic subjects, correlating strongly with glycated hemoglobin but not with age. Collagen CML, pentosidine and fluorescence were increased up to twofold in diabetic compared with control patients: this could be explained by the increase in glycation alone, without invoking increased oxidative stress. There were strong correlations among CML, pentosidine and fluorescence in both groups, providing evidence for age-dependent chemical modification of collagen via the Maillard reaction, and acceleration of this process in diabetes. These results support the description of diabetes as a disease characterized by accelerated chemical aging of long-lived tissue proteins.



Glycation (nonenzymatic glycosylation) is a nonenzymatic, posttranslational modification ofprotein, resulting from chemical reactions between glucose and primary amino groups (1).


The major product in tissue proteins is fructoselysine (FL),'formed by glycation of E-amino groups on lysine residues (Fig.  1). The extent of glycation of tissue proteins depends on the ambient glucose concentration and is relatively constant with age (2-4). However, products formed from FL in subsequent Maillard or browning reactions accumulate gradually with advancing age in long-lived tissue proteins (2-4). These products include N'-(carboxymethyl)lysine (CML) and N'-(carboxymethyl) hydroxylysine (CMhL), which are formed by oxidative cleavage of FL and glycated hydroxylysine respectively (3, 4), pentosidine, a fluorescent crosslink formed between arginine and lysine residues (5-7) (Fig. 1), as well as unidentified compounds with characteristic Maillard-type, visible wavelength fluorescence (maxima at Ex = 328 nm, Em = 378 nm) (4). CML, CMhL, and pentosidine have been termed "glycoxidation" products (4, 8) because both glycation and free radical oxidation reactions are required for their formation from reducing sugars. The exact origin of these compounds, i.e., from glucose vs. ribose (5), ascorbate (6, 7, 9), or other sugars (8), is uncertain, although present evidence suggests that they are derived exclusively from reactions between proteins and carbohydrates (8).


According to the "glycation hypothesis" of aging and the pathogenesis of diabetic complications, accumulation of endstage products of the Maillard reaction (also known as advanced glycosylation end-products [AGE products]) alters the structural properties of tissue proteins and reduces their susceptibility to catabolism ( 10, 11 ). These changes contribute to the aging of tissues, and when accelerated by hyperglycemia, to the gradual development of diabetic complications. Despite the attractiveness of this hypothesis, the only Maillard products known to accumulate with age, i.e., CML, CMhL, and pentosidine, result not from glycation alone: oxidation reactions are also necessary for their formation (2-4, 7, 1 1 ). The development of increased fluorescence of proteins in diabetes and aging is also enhanced by oxidation reactions, and carbohydrate or lipid-dependent processes may also be involved (8). These considerations raise questions about the relative importance of the glycation and oxidation components of glycoxidation in the accumulation of Maillard products in tissue proteins.  To our knowledge, no previous studies have made direct comparisons between the concentrations and relative rates of accumulation of the glycoxidation products CML and pentosidine in collagen from control and diabetic populations. There is a similar lack of information on the relative contributions of increased glycation vs. increased oxidation in the accumulation of these products. We have attempted to address these issues in the present study, and show that concentrations of CML and pentosidine, and Maillard-type fluorescence, increase in concert in human skin collagen with age, and that the rate of their increase is accelerated, again in a concerted fashion, in diabetes. Our data also suggest that the accelerated accumulation of glycoxidation products in diabetes can be explained by hyperglycemia and increased glycation alone, without invoking an increase in oxidative stress. 


Our findings support the description of diabetes as a disease characterized by accelerated chemical modification and aging of collagen via the Maillard reaction. The relationships between concentrations of these biomarkers and the presence and severity of complications in diabetes are considered in the following   article ( 12).

Dr. Relman another former editor in chief of the NEJM said this in 2002
“The medical profession is being bought by the pharmaceutical industry, not only in terms of the practice of medicine, but also in terms of teaching and research. The academic institutions of this country are allowing themselves to be the paid agents of the pharmaceutical industry. I think it’s disgraceful”