Acrolein, a representative carcinogenic aldehyde, that could be ubiquitously generated in biological systems under oxidative stress shows facile reactivity with a nucleophile such as a protein. In this study, to gain a better understanding of the molecular basis of acrolein modification of protein, we characterized the acrolein modification of a model peptide (the oxidized B chain of insulin) by electrospray ionization-liquid chromatography/mass spectrometry method and established a novel acrolein-lysine condensation reaction. In addition, we found that this condensation adduct represented the major antigenic adduct generated in acrolein-modified protein. To identify the modification site and structures of adducts generated in the acrolein-modified insulin B chain, both the acrolein-pretreated and untreated peptides were digested with V8 protease and the resulting peptides were subjected to electrospray ionization-liquid chromatography/mass spectrometry. This technique identified nine peptides, which contained the acrolein adducts at Lys-29 and the N terminus, and revealed that the reaction of the insulin B chain with acrolein gave multiple adducts, including an unknown adduct containing two molecules of acrolein per lysine. To identify this adduct, we incubated N ␣ -acetyllysine with acrolein and isolated a product having the same molecular mass as the unknown acrolein-lysine adduct. On the basis of the chemical and spectroscopic evidence, the adduct was determined to be a novel pyridinium-type lysine adduct, N ⑀ -(3-methylpyridinium)lysine (MP-lysine). The formation of MP-lysine was confirmed by amino acid analysis of proteins treated with acrolein. More notably, this condensation adduct appeared to be an intrinsic epitope of a monoclonal antibody 5F6 that had been raised against acrolein-modified protein.Several lines of evidence indicate that the oxidative modification of protein and the subsequent accumulation of the modified proteins have been found in cells during aging and oxidative stress and in various pathological states including premature diseases, muscular dystrophy, rheumatoid arthritis, and atherosclerosis (1-4). The important agents that give rise to the modification of a protein may be represented by reactive aldehydic intermediates such as ketoaldehydes, 2-alkenals and 4-hydroxy-2-alkenals (3, 5, 6). These reactive aldehydes are considered important mediators of cell damage because of their ability to covalently modify biomolecules, which can disrupt important cellular functions and can cause mutations (5). Furthermore, the adduction of aldehydes to apolipoprotein B in low density lipoproteins has been strongly implicated in the mechanism by which low density lipoproteins is converted to an atherogenic form that is taken up by macrophages, leading to the formation of foam cells (7,8).Acrolein, an unpleasant and troublesome byproduct of overheated organic matter, occurs as a ubiquitous pollutant in the environment, e.g. the incomplete combustion of plastic materials, cigarette smoking, and over...

Acrolein is a representative carcinogenic aldehyde found ubiquitously in the environment and formed endogenously through oxidation reactions, such as lipid peroxidation and myeloperoxidase-catalyzed amino acid oxidation. It shows facile reactivity toward DNA to form an exocyclic DNA adduct. To verify the formation of acrolein-derived DNA adduct under oxidative stress in vivo, we raised a novel monoclonal antibody (mAb21) against the acrolein-modified DNA and found that the antibody most significantly recognized an acroleinmodified 2 -deoxyadenosine. On the basis of chemical and spectroscopic evidence, the major antigenic product of mAb21 was the 1,N 6 -propano-2 -deoxyadenosine adduct. The exposure of rat liver epithelial RL34 cells to acrolein resulted in a significant accumulation of the acrolein-2 -deoxyadenosine adduct in the nuclei. Formation of this adduct under oxidative stress in vivo was immunohistochemically examined in rats exposed to ferric nitrilotriacetate, a carcinogenic iron chelate that specifically induces oxidative stress in the kidneys of rodents. It was observed that the acrolein-2 -deoxyadenosine adduct was formed in the nuclei of the proximal tubular cells, the target cells of this carcinogenesis model. The same cells were stained with a monoclonal antibody 5F6 that recognizes an acrolein-lysine adduct, by which cytosolic accumulation of acrolein-modified proteins appeared. Similar results were also obtained from myeloperoxidase knockout mice exposed to the iron complex, suggesting that the myeloperoxidase-catalyzed oxidation system might not be essential for the generation of acrolein in this experimental animal carcinogenesis model. The data obtained in this study suggest that the formation of a carcinogenic aldehyde through lipid peroxidation may be causally involved in the pathophysiological effects associated with oxidative stress.Lipid peroxidation leads to the formation of a broad array of different products with diverse and powerful biological activities. Among them are a variety of different aldehydes (1). The primary products of lipid peroxidation, lipid hydroperoxides (2), can undergo carbon-carbon bond cleavage via alkoxyl radicals in the presence of transition metals, giving rise to the formation of short chain, unesterified aldehydes (2, 3) or a second class of aldehydes still esterified to the parent lipid. These reactive aldehydic intermediates readily form covalent adducts with cellular macromolecules, including DNA, leading to disruption of important cellular functions and mutations. The important agents that give rise to the modification of DNA may be represented by ␣,␤-unsaturated aldehydic intermediates, such as 2-alkenals and 4-hydroxy-2-alkenals (4, 5). 2-Alkenals represent a group of highly reactive aldehydes containing two electrophilic reaction centers. A partially positive carbon 1 or 3 in such molecules can attack nucleophiles, such as protein and DNA, which leads to the formation of cyclic adducts or cross-links. Among all of the ␣,␤-unsaturated aldehydes, acro...

In the present study, to investigate the contribution of n-3 PUFAs in the oxidative modification of protein in vivo, we characterize the covalent binding of 4-hydroxy-2-hexenal (HHE), a potent cytotoxic aldehyde originating from the peroxidation of n-3 PUFAs, to protein and describe the production of this aldehyde in oxidatively modified LDL and in human atherosclerotic lesions. Upon incubation with BSA, HHE was rapidly incorporated into the protein and generated the protein-linked carbonyl derivative, a potential marker of oxidatively modified proteins under oxidative stress. To detect the protein-bound HHE in vivo, we raised monoclonal antibody HHE53 (MAb HHE53) directed to the HHE-modified protein and identified the Michael addition-type HHE-histidine adduct as the major epitope. This antibody reacted with copper-oxidized LDL, suggesting that HHE was produced during the oxidative modification of LDL. In addition, we demonstrated that the materials immunoreactive to MAb HHE53 indeed constituted the atherosclerotic lesions, in which intense positivity was associated primarily with macrophage-derived foam cells. The results of this study suggest that the reaction between oxidized n-3 PUFAs and protein might represent a process common to the formation of degenerative proteins during aging and its related diseases. -Yamada, S., T. Funada, N. Shibata, M. Kobayashi, Y. Kawai, E. Tatsuda, A. Furuhata, and K. Uchida. Protein-bound 4-hydroxy-2-hexenal as a marker of oxidized n-3 polyunsaturated fatty acids. J. Lipid Res. 2004. 45: 626-634.

Acrolein, a representative carcinogenic aldehyde that could be ubiquitously generated in biological systems under oxidative stress, shows facile reactivity with the ⑀-amino group of lysine to form N ⑀ -(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) as the major product (Uchida, K., Kanematsu, M., Morimitsu, Y., Osawa, T., Noguchi, N., and Niki, E. (1998) J. Biol. Chem. 273, 16058 -16066). In the present study, we determined the electrophilic potential of FDP-lysine and established a novel mechanism of protein thiolation in which the FDP-lysine generated in the acrolein-modified protein reacts with sulfhydryl groups to form thioether adducts. When a sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase, was incubated with acrolein-modified bovine serum albumin in sodium phosphate buffer (pH 7.2) at 37°C, a significant loss of sulfhydryl groups, which was accompanied by the loss of enzyme activity and the formation of high molecular mass protein species (>200 kDa), was observed. The FDP-lysine adduct generated in the acrolein-modified protein was suggested to represent a thiol-reactive electrophile based on the following observations. (i) N ␣ -acetyl-FDP-lysine, prepared from the reaction of N ␣ -acetyl lysine with acrolein, was covalently bound to glyceraldehyde-3-phosphate dehydrogenase. (ii) The FDP-lysine derivative reacted with glutathione to form a GSH conjugate. (iii) The acroleinmodified bovine serum albumin significantly reacted with GSH to form a glutathiolated protein. Furthermore, the observation that the glutathiolated acroleinmodified protein showed decreased immunoreactivity with an anti-FDP-lysine monoclonal antibody suggested that the FDP-lysine residues in the acrolein-modified protein served as the binding site of GSH. These data suggest that thiolation of the protein-bound acrolein may be involved in redox alteration under oxidative stress, whereby oxidative stress generates the increased production of acrolein and its protein adducts that further potentiate oxidative stress via the depletion of GSH in the cells.Several lines of evidence indicate that the oxidative modification of protein and the subsequent accumulation of the modified proteins have been found in cells during aging and oxidative stress and in various pathological states including premature diseases, muscular dystrophy, rheumatoid arthritis, and atherosclerosis (1-4). The important agents that give rise to the modification of a protein may be represented by reactive aldehydic intermediates such as 2-alkenals and 4-hydroxy-2-alkenals (3, 5, 6). These reactive aldehydes are considered important mediators of cell damage because of their ability to covalently modify biomolecules, which can disrupt important cellular functions and cause mutations (5). 2-Alkenals represent a group of highly reactive aldehydes containing two electrophilic reaction centers. A partially positive carbon 1 or 3 in such molecules can attack a nucleophile such as a protein. Among all the ␣,␤-unsaturated aldehydes, acrolein is by far the strongest ...

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