Evidence suggests an imbalance between antioxidant and oxidant-generating systems resulting in oxidative stress in uremic patients. As plasma proteins are critical targets for oxidants, we developed a novel spectrophotometric assay which allows to detect advanced oxidation protein products (AOPP) in uremic plasma. By size-exclusion chromatography AOPP are retrieved in two distinct peaks at 600 and below 80 kDa in uremic plasma, while no such peaks are found in control plasma. Further biochemical characterization revealed that AOPP are carried by oxidized plasma proteins, especially albumin and do not have oxidant properties. AOPP increased in a dose-dependent manner following in vitro exposure of plasma or purified human serum albumin (HSA) to hypochlorous acid. Advanced glycation end products of human serum albumin (AGE-HSA) also increased AOPP levels. In vivo, plasma level of AOPP was the highest in patients on hemodialysis, followed by those on peritoneal dialysis and by undialyzed patients with advanced chronic renal failure. AOPP levels correlated with plasma concentrations of dityrosine and AGE-pentosidine, as indices of oxidant-mediated protein damage, but not with thiobarbituric reactive substances as lipid peroxidation markers. A close correlation was also found between AOPP and neopterin levels, suggesting that AOPP could be part in the monocyte-mediated inflammatory disorders associated with uremia. In conclusion, we propose the measurement of AOPP as a reliable marker to estimate the degree of oxidant-mediated protein damage in uremic patients and to predict the potential efficacy of therapeutic strategies aimed at reducing such an oxidative stress.

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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...