Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein

Abstract

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-products of oxidation of polyunsaturated fatty acids, and are frequently measured as indicators of lipid peroxidation and oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues and cross-links proteins in vitro; HNE also reacts with lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4 reduction to stabilize these adducts to conditions used for acid hydrolysis of protein, and have prepared reduced forms of 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)] and lysine-HNE [3-(N epsilon-lysino)-4-hydroxynonan-l-ol (LHNE)]. Gas chromatography/MS assays have been 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 thiobarbituric acid-MDA adducts measured by HPLC; these adducts accounted for 70-80% of total lysine loss during the reaction with MDA. LM and LML (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 dienes during the copper-catalysed oxidation of LDL, but accounted for modification of < 1% of lysine residues in oxidized LDL. These results are the first report of direct chemical measurement of MDA and HNE adducts to lysine residues in LDL. LM, LML and LHNE should be useful as biomarkers of lipid peroxidative modification of protein and of oxidative stress in vitro and in vivo.

Effect of antioxidants and ACE inhibition on chemical modification of proteins and progression of nephropathy in the streptozotocin diabetic rat

“…Only pyridoxamine exhibited a treatment effect, limiting the amount of this adduct to 20% of that in all other diabetic groups; this is consistent with its unique ability to normalise plasma lipids. The 4-hydroxynonenal adduct to lysine [29] was not detectable in the collagen of any group. For all adducts, there was a trend for lower AGE/ALE in the vitamin E group, but the differences did not reach statistical significance.…”

“…Fructose-lysine, a measure of glycation of collagen, was measured in unreduced samples. Fructose-lysine, CML, CEL and MDA-lysine were converted into their trifluoroacetyl-methyl ester derivatives and quantified by isotope dilution, selected ion monitoring gas chromatography-mass spectrometry as described previously [28,29]. Pentosidine was measured in collagen samples reduced with 100 mmol/l NaBH 4 , hydrolysed in acid as above, and analysed by reversed-phase HPLC [30].…”

Protein and lipid oxidative damage and complex I content are lower in the brain of budgerigar and canaries than in mice. Relation to aging rate

“…Protein samples were immediately reduced by overnight incubation with 500 mM NaBH 4 in 0.2 M borate buffer at pH 9.2, and containing one drop of hexanol as an anti-foam reagent. Protein was reprecipitated by adding 1 ml of 20% (v/v) (Knecht et al 1991;Ahmed et al 1997;Requena et al 1997); and [ 2 H 5 ]5-hydroxy-2-aminovaleric acid (for GSA quantification), and [ 2 H 4 ]6-hydroxy-2-aminocaproic acid (for AASA quantification), prepared as previously described , were then added. The samples were hydrolysed at 155 -C for 30 min in 1 ml of 6 M HCl, then dried in vacuo.…”

The AGE Inhibitor Pyridoxamine Inhibits Development of Retinopathy in Experimental Diabetes

“…Data were sent to South Carolina for unmasking. An ϳ2-cm 2 section of abdominal skin was also removed for isolation of insoluble collagen and measurement of collagen glycation and AGE/ALEs by gas chromatography/mass spectrometry as described previously (35). Retinal vascular digests.…”