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

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-product s of oxidation of polyunsaturated fatty acids, and are frequently meas ured as indicators of lipid peroxidation and oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues and cross-links pr oteins in vitro; HNE also reacts with lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4, reduction t o stabilize these adducts to conditions used for acid hydrolysis of pr otein, and have prepared reduced forms of lysine-MDA [3-(N-epsilon-lys ino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [ 1,3-di(N-epsilon-lysino)propane (LML)] and lysine-HNE [3-(N-epsilon-ly sino)-4-hydroxynonan-1-ol (LHNE)]. Gas chromatography/MS assays have b een developed for quantification of the reduced compounds in protein. RNase incubated with MDA or HNE was used as a model for quantification of the adducts by gas chromatography/MS There was excellent agreement between measurement of MDA bound to RNase as LM and LML, and as thiob arbituric acid-MDA adducts measured by HPLC; these adducts accounted f or 70-80% of total lysine loss during the reaction with MDA. LM and LM L (0.002-0.12 mmol/mol of lysine) were also found in freshly isolated low-density lipoprotein (LDL) from healthy subjects. LHNE was measured in RNase treated with HNE, but was not detectable in native LDL. LM, LML and LHNE increased in concert with the formation-of conjugated die nes during the copper-catalysed oxidation of LDL, but accounted for mo dification of < 1% of lysine residues in oxidized LDL. These results a re the first report of direct chemical measurement of MDA and HNE addu cts to lysine residues in LDL. LM, LML and LHNE should be useful as bi omarkers of lipid peroxidative modification of protein and of oxidativ e stress in vitro and in vivo.