Immunochemical assay of hemoglobin with N∈-(Carboxymethyl)lysine at lysine 66 of the β chain

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 47; no. 7; pp. 1249 - 1255
• Main Authors: IWAMOTO, Hisahiko, MOTOMIYA, Yoshihiro, MIURA, Keisuke, MORISAWA, Masayo, YOSHIMURA, Yoshimichi, MARUYAMA, Ikuro
• Format: Journal Article
• Language: English
• Published: Washington, DC American Association for Clinical Chemistry 2001
• Subjects: Acetaldehyde - analogs & derivatives; Adult; Aged; Antibodies, Monoclonal; Biological and medical sciences; Hematology; Hemoglobins - analysis; Hemoglobins - chemistry; Humans; Immunoassay - methods; Investigative techniques, diagnostic techniques (general aspects); Lysine - analogs & derivatives; Lysine - chemistry; Lysine - immunology; Medical sciences; Middle Aged; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Peptide Fragments - chemical synthesis; Peptide Fragments - chemistry; Peptide Fragments - immunology; Renal Dialysis; Sensitivity and Specificity; Human; Biochemical analysis; Hemodialysis; Beta-Peptide chain; Glycation; Immunological method; Automatic processing; Extrarenal dialysis; Lysine; Measurement technique; Hemoglobin; Oxidation; Methylation; ELISA assay; Comparative study
• ISSN: 0009-9147

N(epsilon)-(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 beta chain. We aimed to develop an assay for the Hb with a CML (CML-Hb) site specific to Lys-66 on the Hb beta chain and to determine whether the lysine residue at that site is carboxymethylated. Ala-His-Gly-Lys-Lys(CM)-Val-Leu-Gly-Ala-Phe-Ser-Cys, the peptide derived from the beta 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. 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). The latex method for the CML-Hb site specific to Lys-66 on the beta chain can measure large numbers of samples on an automatic analyzer.