QUANTIFICATION OF MALONDIALDEHYDE AND 4-HYDROXYNONENAL ADDUCTS TO LYSINE RESIDUES IN NATIVE AND OXIDIZED HUMAN LOW-DENSITY LIPOPROTEIN

摘要: Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-products of oxidation polyunsaturated fatty acids, frequently measured as indicators lipid peroxidation oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues cross-links proteins vitro; HNE also reacts lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4 reduction to stabilize these conditions used for acid hydrolysis protein, prepared reduced lysine-MDA [3-(N epsilon-lysino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [1,3-di(N epsilon-lysino)propane (LML)] lysine-HNE epsilon-lysino)-4-hydroxynonan-l-ol (LHNE)]. Gas chromatography/MS assays been quantification compounds protein. RNase incubated or was model by gas chromatography/MS. There excellent agreement between measurement bound LM LML, thiobarbituric acid-MDA HPLC; accounted 70-80% total loss during reaction MDA. LML (0.002-0.12 mmol/ mol lysine) were found freshly isolated low-density lipoprotein (LDL) from healthy subjects. LHNE treated HNE, but not detectable native LDL. LM, increased concert formation conjugated dienes copper-catalysed LDL, modification < 1% oxidized These results first report direct chemical should be useful biomarkers peroxidative protein vitro