Peroxynitrite Induces Formation of N -(Carboxymethyl)Lysine by the Cleavage of Amadori Product and Generation of Glucosone and Glyoxal From Glucose Novel Pathways for Protein Modification by Peroxynitrite

Accumulation of advanced glycation end products (AGEs) on tissue proteins increases with pathogenesis of diabetic complications and atherosclerosis. Here we examined the effect of peroxynitrite (ONOO ) on the formation of N -(carboxymethyl)lysine (CML), a major AGE-structure. When glycated human serum albumin (HSA; Amadori-modified protein) was incubated with ONOO , CML formation was detected by both enzymelinked immunosorbent assay and high-performance liquid chromatography (HPLC) and increased with increasing ONOO concentrations. CML was also formed when glucose, preincubated with ONOO , was incubated with HSA but was completely inhibited by aminoguanidine, a trapping reagent for -oxoaldehydes. For identifying the aldehydes that contributed to ONOO -induced CML formation, glucose was incubated with ONOO in the presence of 2,3-diaminonaphthalene. This experiment led to identification of glucosone and glyoxal by HPLC. Our results provide the first evidence that ONOO can induce protein modification by oxidative cleavage of the Amadori product and also by generation of reactive -oxoaldehydes from glucose. Diabetes 51:2833–2839, 2002 Long-term incubation of proteins with glucose leads, through formation of Schiff bases and Amadori products, to the generation of advanced glycation end products (AGEs), which are characterized by fluorescence, brown color, and intraor intermolecular cross-linking of protein. Although several AGE structures have been reported, our group (1) and Reddy et al. (2) independently demonstrated that N(carboxymethyl)lysine (CML) is a major antigenic AGE structure. CML concentration, adjusted for age and duration of diabetes, is also increased in patients who have diabetes with complications, including nephropathy (3–5), retinopathy (6), and atherosclerosis (7–9). CML is also recognized by receptor for AGE (RAGE), and CML-RAGE interaction activates cell signaling pathways such as NFB and enhances the expression of vascular cell adhesion molecule-1 in human umbilical vein endothelial cells (10). The AGE inhibitors aminoguanidine and pyridoxamine also inhibit CML formation and retard the development of early renal disease in the streptozotocin-diabetic rat (11). Considered together, these studies strongly suggest an association between CML and the development of diabetic complications. Nitric oxide (NO), or endothelium-derived relaxing factor, exhibits several physiological functions, such as inhibition of neutrophil adhesion (12), enhancement of platelet aggregation (13), and regulation of vascular relaxation (14). Furthermore, NO is extremely reactive with superoxide anion radical (O2 ), generating peroxynitrite (ONOO ) (15), which functions as an oxidant to proteins, vitamins, and DNA (16). One hypothesis argues that the balance between NO and O2 generation is a critical determinant in the cause of many human diseases, including atherosclerosis, neurodegenerative diseases, ischemiareperfusion injury, and cancer (17). NO and O2 , both of which can be generated by aortic endothelial cells during hyperglycemia, can disturb the functions of those cells and are known to induce vascular disorders (18). Furthermore, O2 generated from glycated LDL reacts with NO and decreases the NO-induced modulation of cellular cyclic GMP levels (19). These reports suggest that increased From the Department of Biochemistry, Kumamoto University School of Medicine, Kumamoto, Japan; the School of Food and Nutritional Sciences and Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka, Japan; and the Department of Agricultural Chemistry, Meiji University, Kawasaki, Japan. Address correspondence and reprint requests to Dr. Seikoh Horiuchi, Department of Biochemistry, Kumamoto University School of Medicine, Honjo, 2-2-1, Kumamoto 860-0811, Japan. E-mail: [email protected]. Received for publication 15 January 2002 and accepted in revised form 3 June 2002. R.N. and Y.U. contributed equally to this work. AGE, advanced glycation end product; CML, N-(carboxymethyl)lysine; DAN, 2,3-diaminonaphthalene; ELISA, enzyme-linked immunosorbent assay; ESI-MS, electrospray ionization–mass spectrometry; HPLC, high-performance liquid chromatography; HRP, horseradish peroxidase; HSA, human serum albumin; iNOS, inducible nitric oxide synthase; KLH, keyhole limpet hemocyanin; NO, nitric oxide; O2 , superoxide anion radical; ONOO , peroxynitrite; RAGE, receptor for AGE; TGF1, transforming growth factor1.