Abstract
4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, reacts with histidine to form a stable HNE-histidine Michael addition-type adduct possessing three chiral centers in the cyclic hemiacetal structure. In the present study, we characterized configurational isomers of a HNE-N(alpha)-acetylhistidine adduct by NMR spectroscopy and by molecular orbital calculations. In addition, we raised monoclonal antibodies against (R)-HNE-histidine and (S)-HNE-histidine adducts, characterized their specificities, and examined in vivo localizations of each adduct under oxidative stress. To facilitate structural characterization of the configurational isomers of an HNE-histidine adduct, we prepared the (R)-HNE-histidine and (S)-HNE-histidine adducts by incubating N(alpha)-acetylhistidine with each HNE enantiomer, both of which provided two peaks (Ra and Rb from (R)-HNE-histidine and Sa and Sb from (S)-HNE-histidine adducts) in reversed-phase high-performance liquid chromatography. The NMR analysis showed that each peak was a mixture of two diastereomers. In addition, the analysis of the nuclear Overhauser effect enabled the determination of configurations of the eight isomers. The relative amounts of these isomers in the NMR analysis correlated with the relative energies calculated by molecular orbital methods. On the other hand, using (R)-HNE-modified and (S)-HNE-modified keyhole limpet hemocyanins as the antigens, we raised the monoclonal antibodies, mAbR310 and mAbS412, which enantioselectively recognized the (R)-HNE-histidine and (S)-HNE-histidine adducts, respectively. Among the mixtures (Ra, Rb, Sa, and Sb) of diastereomers, mAbR310 showed the highest immunoreactivity to Rb (the mixture of 2R,4S,5R and 2S,4S,5R isomers), whereas mAbS412 preferentially recognized Sa (the mixture of 2R,4S,5S and 2S,4S,5S isomers). The presence of (R)-HNE and (S)-HNE epitopes in vivo was immunohistochemically examined in the kidney of rats exposed to the renal carcinogen, ferric nitrilotriacetate, by which nuclear and cytosolic stainings with mAbR310 and mAbS412, respectively, were detected.
Highlights
4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, reacts with histidine to form a stable HNE-histidine Michael addition-type adduct possessing three chiral centers in the cyclic hemiacetal structure
The adduction of aldehydes to apolipoprotein B in low density lipoproteins has been strongly implicated in the mechanism by which low density lipoprotein is converted to an atherogenic form that is taken up by macrophages, leading to the formation of foam cells [5, 6]
The liquid chromatography-mass spectrometry analysis of these peaks gave a pseudomolecular ion peak at m/z 354 (MϩH)ϩ, which would be expected from the Michael addition-type HNE-histidine adducts, suggesting that they all represent the products derived from the Michael addition of the imidazole nitrogen atom to the C-3 of HNE
Summary
Materials—N␣-Acetyl-L-histidine and bovine serum albumin (BSA) were obtained from Sigma. After incubation with 100 l of the hybridoma supernatants, and with intervening washes with Tris-buffered saline, pH 7.8, containing 0.05% Tween 20 (TBS-Tween), the wells were incubated with alkaline phosphatase-conjugated goat anti-mouse IgG, followed by a substrate solution containing 1 mg/ml p-nitrophenyl phosphate. Immunoblot Analysis—The gel was transblotted onto a nitrocellulose membrane, incubated with Block Ace (40 mg/ml) for blocking, washed, and treated with the primary antibody This procedure was followed by the addition of horseradish peroxidase conjugated to a goat anti-mouse IgG F(abЈ) fragment and ECL reagents (Amersham Biosciences, Buckinghamshire, UK). Ferric nitrate enneahydrate and the nitrilotriacetic acid disodium salt were each dissolved in deionized water to form 80 and 160 mM solutions, respectively They were mixed at the volume ratio of 1:2 (molar ratio, 1:4), and the pH was adjusted with sodium hydrogen carbonate to 7.4. Procedures, using phosphate-buffered saline or the IgG fraction (0.5 g/ml) of normal mouse serum instead of mAbR310 and mAbS412 antibodies, showed no or negligible positive responses
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