Glycolaldehyde (GA) is formed from serine by action of myeloperoxidase and reacts with proteins to form several products. Prominent among them is N ⑀ -(carboxymethyl)lysine (CML), which is also known as one of the advanced glycation end products. Because CML is formed from a wide range of precursors, we have attempted to identify unique structures characteristic of the reaction of GA with protein. To this end, monoclonal (GA5 and 1A12) and polyclonal (non-CML-GA) antibodies specific for GA-modified proteins were prepared. These antibodies specifically reacted with GA-modified and with hypochlorous acid-modified BSA, but not with BSA modified by other aldehydes, indicating that the epitope of these antibodies could be a specific marker for myeloperoxidase-induced protein modification. By HPLC purification from GA-modified N ␣ -(carbobenzyloxy)-L-lysine, GA5-reactive compound was isolated, and its chemical structure was characterized as 3-hydroxy-4-hydroxymethyl-1-(5-amino-5-carboxypentyl) pyridinium cation. This compound named as GA-pyridine was recognized both by 1A12 and non-CML-GA, indicating that GA-pyridine is an important antigenic structure in GA-modified proteins. Immunohistochemical studies with GA5 demonstrated the accumulation of GA-pyridine in the cytoplasm of foam cells and extracellularly in the central region of atheroma in human atherosclerotic lesions. These results suggest that myeloperoxidase-mediated protein modification via GA may contribute to atherogenesis.Modification of proteins with reactive aldehydes is thought to play a role in the pathogenesis of several diseases, including diabetes and atherosclerosis. Products of nonenzymatic glycation, Maillard reactions, such as 3-deoxyglucosone (1, 2), glyoxal (3), and methylglyoxal (4) are formed in tissue proteins in vivo and are considered to be precursors of advanced glycation end products (AGE). 1In a parallel pathway involving both enzymatic and nonenzymatic reactions during inflammation, leukocytes are activated to secrete myeloperoxidase, which mediates the formation of hypochlorous acid (HOCl) from hydrogen peroxide and chloride (5). Aldehydes such as GA, which is formed by reaction of HOCl with serine, then react to form chemical modifications in protein. Myeloperoxidase has been detected immunohistochemically in lipid-rich advanced atherosclerotic lesions, and the active myeloperoxidase was purified from atherosclerotic arteries (6). A subsequent study by Hazell et al. (7) demonstrated that human atherosclerotic lesions were positively stained by a monoclonal antibody against HOCl-modified proteins, suggesting the presence of an epitope(s) specific for HOCl-modified proteins. HOCl generated by the myeloperoxidase system was shown to react with L-threonine to generate acrolein via 2-hydroxypropanol, whereas the similar reaction with L-serine led to formation of glycolaldehyde (GA) (8). An immunohistochemical study demonstrated the accumulation of an acrolein-protein adduct(s) in macrophage-derived foam cells as well in thickened neointi...

SUMMARY:Accumulation of advanced glycation end products (AGE) of the Maillard reaction increases by aging and in age-enhanced diseases such as atherosclerosis and diabetic complications. Immunohistochemical analysis has been used to demonstrate AGE in vivo. In immunochemistry, the heat-induced epitope retrieval technique is extensively used with formalin-fixed, paraffin-embedded tissue sections. Here we examined whether AGE could be formed artificially through the heating process. Normal rat skin and liver samples were divided into two groups, one rapidly frozen, the other formalin-fixed, paraffin-embedded and submitted to heat-induced epitope retrieval treatment. In heat-treated sections, the cytoplasm of rat epidermal cells and hepatocytes were strongly stained by monoclonal antibody against N ⑀ -(carboxymethyl)lysine (CML), while the staining was negligible in either frozen sections or in paraffin-embedded but heat-untreated sections. To clarify the mechanism, we conducted heat treatment to glycated human serum albumin (HSA), a model Amadori protein, and generation of CML was determined by immunochemical and HPLC analysis. CML was generated from glycated HSA by heat treatment (above 80°C) and increased in a time-dependent manner. In contrast, generation of CML from glycated HSA was significantly inhibited in the presence of NaBH 4 , a reducing agent, diethylenetriamine pentaacetic acid, a chelator of transition metal ion, or aminoguanidine, a trapping reagent for ␣-oxoaldehydes. Furthermore, heat-induced CML formation in rat liver samples determined by HPLC was markedly reduced by pretreatment with NaBH 4 . Reactive intermediates such as glucosone, 3-deoxyglucosone, methylglyoxal, and glyoxal were formed upon heat treatment of glycated HSA at 100°C, indicating that these aldehydes generated from Amadori products by oxidative cleavage can contribute to further CML formation. CML generated by heating, directly from Amadori products or via these aldehydes, might serve as an artifact upon immunohistochemistry. (Lab Invest 2002, 82:795-807). L ong-term incubation of proteins with glucose leads, through early-stage products such as Schiff base and Amadori products, to the formation of advanced glycation end products (AGE), which are characterized by fluorescence, brown color, and intra-or intermolecular cross-linking (Maillard, 1912). Several AGE structures have been identified including pyrraline (Hayase et al, 1989), pentosidine (Sell and Monnier, 1989), N ⑀ -(carboxymethyl)lysine (CML) (Ahmed et al, 1986), crosslines (Nakamura et al, 1992), imidazolone (Konishi et al, 1994;Lo et al, 1994), methylglyoxal lysine dimer (Frye et al, 1998), N ⑀ -(carboxyethyl)lysine (Ahmed et al, 1997), vesperlysine A, B, and C (Nakamura et al, 1997), glyoxal lysine dimer (Frye et al, 1998), argpyrimidine (Oya et al, 1999), and carboxymethylarginine (Iijima et al, 2000). Among these products, several in vitro experiments demonstrated that CML (Ikeda et al, 1996;Reddy et al, 1995) is a major antigenic AGE structure in vivo. CML is gen...

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