Involvement of Hydrogen Peroxide in Collagen Cross-linking by High Glucose in Vitro and in Vivo

Abstract

The Maillard reaction has been implicated in crosslinking and fluorescence formation of collagen exposed to high glucose in vitro. However, several pharmacologic agents, whose action seems unrelated to pathways of nonenzymatic glycation, have been demonstrated to prevent cross-linking in diabetes. To clarify this discrepancy, kinetic changes in glycation, glycoxidation (carboxymethyllysine, CML), and cross-linking (measured as tendon breaking time, TBT) were evaluated in rat tail tendons incubated in 5 and 30 mM glucose in vitro and in tendons implanted in vivo into diabetic rat peritoneal cavity. In vitro, rates were found to be both O 2 -and glucose-dependent. Tendon preglycation and presence of added 2 mM glycosylamine and Amadori compounds (Amadori product of glucose and propylamine) catalyzed these changes in a primarily O 2 -dependent manner. In the presence of Amadori compounds, kinetic changes were dramatically increased and were preventable by addition of catalase to the medium. Tendons implanted into diabetic rat peritoneum became more rapidly glycoxidized and cross-linked when implanted at day 30 from diabetes onset (high tissue glycation) compared to day 3 (low tissue glycation) in spite of similar glycation kinetics, suggesting a mechanistic dissociation between glycation, glycoxidation, and crosslinking in diabetes. Indeed, intraperitoneal injection of catalase and other antioxidants dramatically suppressed cross-linking, fluorescence formation, and, to some extent, glycoxidation, without affecting glycation. This study confirms the role of oxidative stress in protein cross-linking by the Maillard reaction in vitro and provides the first evidence for a role of H 2 O 2 in crosslinking in diabetes. Whereas Amadori products are a potent source of H 2 O 2 formation in vitro, their precise contribution to H 2 O 2 generation and the actual role of Maillard reaction products in collagen cross-linking in diabetes requires further investigation.Considerable efforts have been devoted in recent years toward understanding of the pathogenetic mechanisms leading to collagen insolubilization in diabetes and aging (1-3). The rationale for this research lies in the hope that understanding the biochemical basis of stochastic mechanisms of damage to collagen will help elucidate their role in diabetic complications and aging. One key proposition is that protein cross-links are generated in the advanced Maillard reaction (4, 5). According to the original concept proposed by Hodge (6), the central molecule responsible for the advanced Maillard reaction is the Amadori product which, upon dehydration and rearrangement, forms highly reactive deoxyosones Ϫ potent precursors of protein cross-links. This concept was challenged by Namiki (7) who found that sugar fragmentation can also occur at the stage of the glycosylamine, or Schiff base, i.e. prior to the Amadori rearrangement. Glyoxal is then formed which can serve as a protein cross-link (8 -10). More recently, Wolff et al. (11) obtained evidence from in vitro experimenta...

Silymarin, a flavonoid antioxidant, protects streptozotocin-induced lipid peroxidation and β-Cell damage in rat pancreas

“…Measurement of HbA 1c has proven to be particularly useful in monitoring the effectiveness of therapy in diabetes (Goldstein, 1995). Agents with antioxidant or free radical scavenging property may inhibit oxidative reactions associated with glycation (Elgawish, 1996). Administration of silymarin (25 mg/kg and 50 mg/kg) to diabetic rats significantly reduced the glycosylation of hemoglobin by virtue of its free radical scavenging property and thus may decreases the level of HbA 1c .…”

Histone carbonylation in vivo and in vitro

“…To determine if the carbonylation of histone H1 by ADPribose involved oxidative reaction pathways, several compounds known to interfere with the glycoxidation of proteins by -glucose [26] were examined for their effects on protein carbonylation by ADP-ribose ( Figures 5C and 5D). Different reagents including the transition metal ion chelator DTPA in the absence of oxygen (lane 3), the nucleophilic antioxidant N α -acetyl--cysteine (lane 5), the hydroxyl radical scavenger mannitol (lane 6), and the presence of catalase for the removal of traces of hydrogen peroxide (lane 7) did not interfere with histone carbonylation by ADP-ribose.…”

Isolation, Purification, and Characterization of Amadoriase Isoenzymes (Fructosyl Amine-oxygen Oxidoreductase EC 1.5.3) from Aspergillus sp.

“…Except for the Pseudomonas enzyme described earlier (17), whose isolation was recently described (34), all amadoriase enzymes that (35,36), the question thus arises as to whether amadoriase enzymes occur in human tissues. The sequence homology between amadoriase isoenzymes and mammalian D-amino acid oxidase suggests that deglycating or related enzymes could occur in the human.…”