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

Abstract

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 N epsilon-(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.

Carboxymethyllysine in Dermal Tissues of Diabetic and Nondiabetic Patients with Chronic Renal Failure: Relevance to Glycoxidation Damage

“…CML formation from Amadori products is currently thought to be the major pathway in vivo [30]. Since CML formation is inhibited by antioxidants and antioxidative conditions, the oxidative mechanism has been suggested to be involved in this process [10, 12]. Since oxidative stress is increased in patients with CRF [20, 21, 22], it is likely that collagen oxidation plays an important role in CML formation in patients with CRF.…”

“…Previous studies using model systems have shown that the AGEs pentosidine and carboxymethyllysine (CML), which are known to accumulate in tissue proteins, require autoxidative conditions (molecular oxygen and metal ion catalysis) for their formation from glucose [10]. Furthermore, the formation of these compounds is inhibited under anaerobic and aerobic conditions by various chelators, reducing agents, and oxygen radical scavengers [11, 12]. Since both glycation and oxidation are required for their formation, CML and pentosidine are also known as ‘glycoxidation’ products [10], a subclass of AGEs.…”

Calcification of the Medial Layer of the Internal Thoracic Artery in Diabetic Patients: Relevance of Glycoxidation

“…A close relationship between glycation and oxidation has been proposed, and the resulting hypothesis suggests that the term ‘glycoxidation’ is used [17]. CML and pentosidine are two glycoxidation products.…”

“…Glycoxidative modification of collagen (gCol) was performed according to the method of Fu et al [17]with minor modifications. Briefly, type I collagen from porcine skin (3 mg/ml) was obtained from Iwaki Inc. (Tokyo, Japan) and was dissolved with 200 m M D -glucose in 0.2 M sodium phosphate buffer (pH 7.4).…”

Molecular Mechanisms of Diabetic Angiopathy – Clues for Innovative Therapeutic Interventions

“…1a). Oxidation that accompanies glycation in vivo supports the formation of permanent irreversible chemical modifications [3, 7]. Examples are the glycooxidation products (3-4)Nε-(carboxymethyl)-lysine [7] or pentosidine [8].…”