Michael addition-type 4-hydroxy-2-nonenal adducts in modified low-density lipoproteins: markers for atherosclerosis.
It has been proposed that plasma low-density lipoprotein (LDL) undergoes oxidative modification before it can give rise to foam cells in atherosclerosis. Oxidation of LDL generates a variety of reactive aldehyde products including 4-hydroxy-2-nonenal (HNE), which may covalently attach to the LDL apolipoproteins. We here present direct evidence that HNE derivatization of LDL forms Michael addition-type adducts of HNE with histidine and lysine residues of apolipoprotein B-100 (apoB) and also demonstrate the utility of an antibody specific to the HNE adducts generated in the LDL treated with HNE or oxidatively modified by Cu2+ or cultured endothelial cells. HNE adducts present in the LDL that had been treated with HNE were attested to be Michael addition-type adducts on the basis of the fact that incubation of LDL with 1 mM HNE (2 h, 37 degrees C) resulted primarily in the formation of Michael addition-type HNE-histidine (39.9 mol/mol of LDL) and HNE-lysine (19.3 mol/mol of LDL) adducts. An enzyme-linked immunosorbent assay (ELISA) and an SDS-polyacrylamide gel electrophoresis (SDS-PAGE)/immunoblot analysis of HNE-modified LDL demonstrated that these HNE adducts were detectable with the HNE-specific antibody affinity-purified with the Michael adduct (HNE-histidine) as a ligand. The following lines of evidence indicated the presence of Michael addition-type HNE adducts in the oxidatively modified LDL in vitro: (i) Amino acid analysis of LDL that had been treated with Cu2+ (24 h, 37 degrees C) demonstrated the presence of a Michael addition-type HNE-histidine adduct (7-9 mol/mol of LDL).(ABSTRACT TRUNCATED AT 250 WORDS)
Uchida
K
K
Laboratory of Food and Biodynamics, Nagoya University School of Agriculture, Japan.
Toyokuni
S
S
Nishikawa
K
K
Kawakishi
S
S
Oda
H
H
Hiai
H
H
Stadtman
E R
ER
D000447:Aldehydes / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001011:Aorta; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000378:metabolism; D001161:Arteriosclerosis / Q000378:metabolism*; D001667:Binding, Competitive; D002417:Cattle; D002478:Cells, Cultured; D003300:Copper / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006639:Histidine / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008315:Malondialdehyde / Q000378:metabolism; D010084:Oxidation-Reduction; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism
D000447:Aldehydes; D000906:Antibodies; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D008077:Lipoproteins, LDL; D017392:Thiobarbituric Acid Reactive Substances; D006639:Histidine; D008315:Malondialdehyde; D003300:Copper; C027576:4-hydroxy-2-nonenal; D008239:Lysine