Immunochemical assay of hemoglobin with N(epsilon)-(carboxymethyl)lysine at lysine 66 of the beta chain.
BACKGROUND
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.


METHODS
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.


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).


CONCLUSIONS
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.
Iwamoto
H
H
R&D for Diagnostics, A&T Corporation, 2023-1, Endo, Fujisawa-City, Kanagawa 252-0816, Japan. iwamoto@alice.aandt.co.jp
Motomiya
Y
Y
Miura
K
K
Morisawa
M
M
Yoshimura
Y
Y
Maruyama
I
I
D000079:Acetaldehyde / Q000031:analogs & derivatives; D000328:Adult; D000368:Aged; D000911:Antibodies, Monoclonal; D006454:Hemoglobins / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000379:methods; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000276:immunology; D008875:Middle Aged; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D006435:Renal Dialysis; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D006454:Hemoglobins; D010446:Peptide Fragments; C049826:2-methoxyacetaldehyde; C048496:N(6)-carboxymethyllysine; D000079:Acetaldehyde; D008239:Lysine