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Dicarbonyl compounds, such as 3-deoxyglucosone (3DG) and glyoxal (GO) can be produced in vivo by several metabolic pathways or by Maillard reaction and they react rapidly with proteins to form advanced glycation end products (AGEs). The generation of these compounds in the early stage of the reaction was investigated. Lysozyme was incubated with fructose for 14 days at 50 °C, and the benzo[g]quinoxaline derivatives of dicarbonyls were analyzed by HPLC. It revealed that 3DG was predominant in this reaction system, whereas lower amounts of other dicarbonyls [3-deoxyxylosone (3DX), glyoxal, glucosone (GLCO), tetrosone (TSO), and methylglyoxal (MG)] were also generated. These results suggest the important role of 3DG in the formation of AGEs. Lysyl-pyrropyridine (LPP) is a fluorescent product of the reaction between 3DG and lysine residues. LPP is supposed to be involved in the protein cross-linking, but has been identified only in the in vitro model reaction system. A sensitive assay was developed to detect LPP formed in vivo. Lysozyme incubated with 3DG was digested completely with proteases. Liberated LPP was separated by HPLC and detected by its fluorescence (Ex. 370 nm, Em. 448 nm). This assay allowed us to detect LPP in the tail collagen from aged rats. This study shows that 3DG-mediated lysine–lysine cross-links occur in vivo and provides a method for assessing protein–lysine modification by 3DG in aging and diabetes. |
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