Maillard reactions by alpha-oxoaldehydes: detection of glyoxal-modified proteins

Proteins can be chemically modified by sugars by glycation, or the Maillard reaction. The Maillard reaction produces irreversible adducts on proteins that are collectively known as advanced glycation end products, or AGEs. Re cent studies indicate that several a-dicarbonyl compounds, including glyoxa l (GXL), are precursors of AGEs in vivo. We developed antibodies against a GXL-modified protein (GXL-AGE) and purified a mixture of GXL-AGE-specific a ntibodies by chromatography on GXL-modified bovine serum albumin (BSA-GXL) coupled to EAH-Sepharose. This preparation was then processed on a human se rum albumin-carboxymethyllysine (HSA-CML)-NHS-Sepharose to remove CML-speci fic antibodies. We used the resulting purified antibody in a competitive EL ISA to probe GXL-AGEs in vitro and in vivo. We found increasingly greater a ntibody binding with increasing concentrations of GXL-modified BSA, but the antibody failed to react with either free CML or protein-bound CML. Incuba tion experiments with BSA revealed that glyceraldehyde, ribose and threose could be precursors of GXL-AGEs as well. Experiments in which GXL was incub ated with N-alpha-acetyl amino acids showed that the antibody reacts mostly with lysine modifications. The GXL-derived lysine-lysine crosslinking stru cture, GOLD was found to be one of the antigenic epitopes for the antibody. Analysis of human plasma proteins revealed significantly higher levels of GXL-AGE antigens in type II diabetic subjects compared with normal controls (P < 0.0001). We also found GXL-AGEs in human lens proteins. Bovine aortic endothelial cells cultured for 7 days with 30 mM glucose did not accumulat e intracellular GXL-AGEs. These studies underscore the importance of GXL fo r extracellular AGE formation (except in lens where it is likely to be form ed intracellularly) and suggest that changes associated with age and diabet es might be prevented by alteration of GXL-AGE formation. (C) 2000 Elsevier Science B.V. All rights reserved.