Immunochemical Assay of Hemoglobin with Nε-(Carboxymethyl)lysine at Lysine 66 of the β Chain

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Abstract
Background: N ε-(Carboxymethyl)lysine (CML), a well-characterized and major advanced glycation end product structure, is produced via a Maillard reaction by nonenzymatic glycation and/or oxidation. Although few of the carboxymethylation sites of lysine residues on proteins have been identified, it is known that the possible lysine glycation site in hemoglobin (Hb) is Lys-66 on the β chain. We aimed to develop an assay for the Hb with a CML (CML-Hb) site specific to Lys-66 on the Hb β chain and to determine whether the lysine residue at that site is carboxymethylated. Methods: Ala-His-Gly-Lys-Lys(CM)-Val-Leu-Gly-Ala-Phe-Ser-Cys, the peptide derived from the β chain of human Hb, was synthesized as an immunogen, and a monoclonal antibody against the peptide was prepared. A latex immunoassay method was established using the antibody on an automatic analyzer. In this study, 20 samples from healthy subjects and 80 samples from nondiabetic patients undergoing hemodialysis (HD) were analyzed. Results: The latex immunoassay method using the antibody correlated significantly with the ELISA method using the antibody (r = 0.95; P <0.001). Between healthy subjects (n = 20) and nondiabetic HD patients (n = 80), a significant difference was seen in circulating CML-Hb (525 ± 76 vs 778 ± 137 pmol CML/mg of Hb; P <0.0001). Conclusion: The latex method for the CML-Hb site specific to Lys-66 on the β chain can measure large numbers of samples on an automatic analyzer.