title: Maillard reactions by alpha-oxoaldehydes: detection of glyoxal-modified proteins.
abstract: 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. Recent studies indicate that several alpha-dicarbonyl compounds, including glyoxal (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 antibodies by chromatography on GXL-modified bovine serum albumin (BSA-GXL) coupled to EAH-Sepharose. This preparation was then processed on a human serum albumin-carboxymethyllysine (HSA-CML)-NHS-Sepharose to remove CML-specific antibodies. We used the resulting purified antibody in a competitive ELISA to probe GXL-AGEs in vitro and in vivo. We found increasingly greater antibody binding with increasing concentrations of GXL-modified BSA, but the antibody failed to react with either free CML or protein-bound CML. Incubation experiments with BSA revealed that glyceraldehyde, ribose and threose could be precursors of GXL-AGEs as well. Experiments in which GXL was incubated with N-alpha-acetyl amino acids showed that the antibody reacts mostly with lysine modifications. The GXL-derived lysine-lysine crosslinking structure, 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 accumulate intracellular GXL-AGEs. These studies underscore the importance of GXL for extracellular AGE formation (except in lens where it is likely to be formed intracellularly) and suggest that changes associated with age and diabetes might be prevented by alteration of GXL-AGE formation.
authors.affiliation: Department of Opthalmology, Center for Vision Research, Case Western Reserve University and University Hospitals of Cleveland, OH 44106, USA.
authors.forename: C
authors.initials: C
authors.lastname: Sady
authors.forename: C L
authors.initials: CL
authors.lastname: Jiang
authors.forename: P
authors.initials: P
authors.lastname: Chellan
authors.forename: Z
authors.initials: Z
authors.lastname: Madhun
authors.forename: Y
authors.initials: Y
authors.lastname: Duve
authors.forename: M A
authors.initials: MA
authors.lastname: Glomb
authors.forename: R H
authors.initials: RH
authors.lastname: Nagaraj
mesh_terms: D000368:Aged; D000375:Aging / Q000378:metabolism; D000906:Antibodies / Q000276:immunology; D002386:Cataract / Q000378:metabolism; D003920:Diabetes Mellitus / Q000097:blood / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D017127:Glycation End Products, Advanced / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D006031:Glycosylation; D006037:Glyoxal / Q000737:chemistry*; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism; D008239:Lysine / Q000031:analogs & derivatives* / Q000276:immunology; D015416:Maillard Reaction; D011506:Proteins / Q000737:chemistry*; D012709:Serum Albumin / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000276:immunology; D000075462:Serum Albumin, Human
chemical_list: D000906:Antibodies; D017127:Glycation End Products, Advanced; D011506:Proteins; D012709:Serum Albumin; C081628:advanced glycation end products-bovine serum albumin; C109748:advanced glycation end products-human serum albumin; D012710:Serum Albumin, Bovine; D006037:Glyoxal; C048496:N(6)-carboxymethyllysine; D008239:Lysine; D000075462:Serum Albumin, Human