ascorbic-acid and 4-5-epoxy-2-decenal

Polyunsaturated fatty acids are highly susceptible to oxidation induced by reactive oxygen species and enzymes, leading to the formation of lipid hydroperoxides. The linoleic acid (LA)-derived hydroperoxide, 13-hydroperoxyoctadecadienoic acid (HPODE) undergoes homolytic decomposition to reactive aldehydes, 4-oxo-2(E)-nonenal (ONE), 4-hydroxy-2(E)-nonenal, trans-4,5-epoxy-2(E)-decenal (EDE), and 4-hydroperoxy-2(E)-nonenal (HPNE), which can covalently modify peptides and proteins. ONE and HNE have been shown to react with angiotensin (Ang) II (DRVYIHPF) and modify the N-terminus, Arg(2), and His(6). ONE-derived pyruvamide-Ang II (Ang P) alters the biological activities of Ang II considerably. The present study revealed that EDE and HPNE preferentially modified the N-terminus and His(6) of Ang II. In addition to the N-substituted pyrrole of [N-C4H2]-Ang II and Michael addition products of [His(6)(EDE)]-Ang II, hydrated forms were detected as major products, suggesting considerable involvement of the vicinal dihydrodiol (formed by epoxide hydration) in EDE-derived protein modification in vivo. Substantial amounts of [N-(EDE-H2O)]-Ang II isomers were also formed and their synthetic pathway might involve the tautomerization of a carbinolamine intermediate, followed by intramolecular cyclization and dehydration. The main HPNE-derived products were [His(6)(HPNE)]-Ang II and [N-(HPNE-H2O)]-Ang II. However, ONE, HNE, and malondialdehyde-derived modifications were dominant, because HPNE is a precursor of these aldehydes. A mixture of 13-HPODE and [(13)C18]-13-HPODE (1:1) was then used to determine the major modifications derived from LA peroxidation. The characteristic doublet (1:1) observed in the mass spectrum and the mass difference of the [M+H](+) doublet aided the identification of Ang P (N-terminal α-ketoamide), [N-ONE]-Ang II (4-ketoamide), [Arg(2)(ONE-H2O)]-Ang II, [His(6)(HNE)]-Ang II (Michael addition product), [N-C4H2]-Ang II (EDE-derived N-substituted pyrrole), [His(6)(HPNE)]-Ang II, [N-(9,12-dioxo-10(E)-dodecenoic acid)]-Ang II, and [His(6)(9-hydroxy-12-oxo-10(E)-decenoic acid)]-Ang II as the predominant LA-derived modifications. These modifications could represent the majority of lipid-derived modifications to peptides and proteins in biological systems.

Topics: Aldehydes; Angiotensin II; Ascorbic Acid; Aspartame; Carbon Isotopes; Epoxy Compounds; Isomerism; Linoleic Acids; Lipid Peroxides; Malondialdehyde; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

The role of amino phospholipids in the removal of 4-hydroxy-2-alkenals and 4,5-epoxy-2-alkenals was studied to determine the fate of highly toxic oxygenated aldehydes produced in foods as a consequence of lipid oxidation. The results obtained showed that phosphatidylethanolamine rapidly removed both exogenously added aldehydes as well as the endogenously produced aldehydes when its fatty acid chains were oxidized in the presence of an oxidative stress inducer. This removal, which was always produced in the range of pH (6-9) and temperature (25-60 degrees C) studied, produced the corresponding carbonyl-amine reaction products between the aldehydes and the amino group of the amino phospholipid. These results suggest that, in the presence of amino phospholipids, the oxidation of polyunsaturated fatty acid chains is not likely to produce free oxygenated aldehydes in enough concentration to pose a significant risk for human health. On the other hand, these compounds contributed to the formation of specific carbonyl-amine reaction products whose toxicity is mostly unknown at present.

Topics: Aldehydes; Ascorbic Acid; Epoxy Compounds; Food Analysis; Gas Chromatography-Mass Spectrometry; Hydrogen-Ion Concentration; Iron; Lipids; Oxidation-Reduction; Phosphatidylethanolamines; Phospholipids; Pyrroles; Temperature

Epidemiological data suggest that dietary antioxidants play a protective role against cancer. This has led to the proposal that dietary supplementation with antioxidants such as vitamin C (vit C) may be useful in disease prevention. However, vit C has proved to be ineffective in cancer chemoprevention studies. In addition, concerns have been raised over potentially deleterious transition metal ion-mediated pro-oxidant effects. We have now determined that vit C induces lipid hydroperoxide decomposition to the DNA-reactive bifunctional electrophiles 4-oxo-2-nonenal, 4,5-epoxy-2(E)-decenal, and 4-hydroxy-2-nonenal. The compound 4,5-Epoxy-2(E)-decenal is a precursor of etheno-2'-deoxyadenosine, a highly mutagenic lesion found in human DNA. Vitamin C-mediated formation of genotoxins from lipid hydroperoxides in the absence of transition metal ions could help explain its lack of efficacy as a cancer chemoprevention agent.

Topics: Aldehydes; Antioxidants; Ascorbic Acid; Buffers; Copper; Cyclooxygenase 1; Cyclooxygenase 2; DNA Adducts; DNA Damage; Epoxy Compounds; Ferrous Compounds; Humans; Isoenzymes; Linoleic Acids; Lipid Peroxides; Membrane Proteins; Metals; Mutagens; Oxidants; Oxidation-Reduction; Prostaglandin-Endoperoxide Synthases

The Ehrlich reaction was optimized to determine pyrrolized proteins produced as a consequence of lipid peroxidation and oxidative stress. The procedure consisted of the treatment of the modified protein with p-(dimethylamino)benzaldehyde at a controlled acidity and temperature, and the determination of adducts produced against the blank obtained in the absence of the reagent. The extinction coefficient of Ehrlich adducts was calculated by using epsilon-N-pyrrolylnorleucine (Pnl) as standard and was 35,000 M-1 cm-1. The response was linear and reproducible within the range 0. 16-20 microM Pnl. The assay was applied to determination of pyrrole content in bovine serum albumin, bovine alpha-globulins, bovine gamma-globulins, and mixtures of them, incubated overnight with 1 mM of 4,5(E)-epoxy-2(E)-heptenal, obtaining results similar to those from determination of Pnl by capillary electrophoresis after basic hydrolysis of the protein. The method was also applied to pyrrole determination in bovine plasma proteins either incubated with epoxyalkenals, hydroxyalkenals, lipid hydroperoxides, and secondary products of lipid peroxidation, or oxidized with Fe3+/ascorbate. All these treatments produced pyrrolization of plasma proteins and all Ehrlich adducts gave very similar absorbance spectra with the exception of that produced in the treatment with hydroxyalkenals. The above results suggest that protein pyrrolization is a normal consequence of the lipid peroxidation process and of oxidative stress, and that Ehrlich adducts may be valid to determine this pyrrolization.

Topics: Aldehydes; Amino Acids; Animals; Ascorbic Acid; Benzaldehydes; Blood Proteins; Cattle; Epoxy Compounds; Ferric Compounds; Globulins; Kinetics; Lipid Peroxidation; Lipid Peroxides; Molecular Structure; Oxidation-Reduction; Oxidative Stress; Proteins; Pyrroles; Serum Albumin; Spectrophotometry