ascorbic-acid and linoleic-acid-hydroperoxide

We aimed to investigate the effects of increased intake of α-linolenic acid (ALA), EPA, or DHA incorporated into a food matrix on the fatty acid composition of erythrocytes and on biomarkers of oxidant/antioxidant status. To this end, a controlled dietary study was conducted in 74 healthy men and women. The participants were randomly assigned to 1 of 3 interventions in which margarines fortified with either 10 weight percent ALA, EPA, or DHA ethyl esters replaced their normal spread for 6 wk. The total intakes of ALA, EPA, and DHA were 4.4, 2.2, and 2.3 g/d, respectively. Consuming EPA increased the erythrocyte proportion of EPA (394%) and the omega-3 index (sum of EPA and DHA, 38%). Consumption of DHA increased erythrocyte DHA (91%), the omega-3 index (98%), and EPA (137%). The omega-3 index increased to a significantly greater extent in the DHA group than in the EPA group. ALA did not increase erythrocyte EPA or the omega-3 index. We found no change in plasma uric acid or antioxidant capacity in any of the groups. Plasma malondialdehyde (MDA) increased with the EPA and DHA interventions. All 3 interventions decreased erythrocyte linoleic acid hydroperoxides but did not affect their MDA concentrations. In conclusion, the intake of both isolated EPA and DHA incorporated into margarine resulted in an enhanced incorporation of EPA and DHA into erythrocytes. Our findings indicate that DHA is quantitatively superior to EPA in view of the EPA+DHA tissue incorporation and also that 4 g/d ALA is not sufficient to increase the omega-3 index over a 6-wk period.

Topics: Adult; alpha-Linolenic Acid; Antioxidants; Ascorbic Acid; Docosahexaenoic Acids; Eicosapentaenoic Acid; Erythrocytes; Fatty Acids; Fatty Acids, Omega-3; Female; Food, Fortified; Humans; Linoleic Acids; Lipid Peroxides; Male; Malondialdehyde; Margarine; Middle Aged; Reference Values; Tocopherols; Uric Acid; Young Adult

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

Phytic acid (PA) has been recognized as a potent antioxidant and inhibitor of iron-catalyzed hydroxyl radical formation under in vitro and in vivo conditions. Therefore, the aim of the present study was to investigate, with the use of HPLC/MS/MS, whether PA is capable of inhibiting linoleic acid autoxidation and Fe(II)/ascorbate-induced peroxidation, as well as Fe(II)/ascorbate-induced lipid peroxidation in human colonic epithelial cells. PA at 100 μM and 500 μM effectively inhibited the decay of linoleic acid, both in the absence and presence of Fe(II)/ascorbate. The observed inhibitory effect of PA on Fe(II)/ascorbate-induced lipid peroxidation was lower (10-20%) compared to that of autoxidation. PA did not change linoleic acid hydroperoxides concentration levels after 24 hours of Fe(II)/ascorbate-induced peroxidation. In the absence of Fe(II)/ascorbate, PA at 100 μM and 500 μM significantly suppressed decomposition of linoleic acid hydroperoxides. Moreover, PA at the tested nontoxic concentrations (100 μM and 500 μM) significantly decreased 4-hydroxyalkenal levels in Caco-2 cells which structurally and functionally resemble the small intestinal epithelium. It is concluded that PA inhibits linoleic acid oxidation and reduces the formation of 4-hydroxyalkenals. Acting as an antioxidant it may help to prevent intestinal diseases induced by oxygen radicals and lipid peroxidation products.

Topics: Aldehydes; Ascorbic Acid; Caco-2 Cells; Humans; Ions; Iron; Iron Chelating Agents; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Oxidation-Reduction; Phytic Acid

It is possible that oxidative stress causes several retinal diseases. However, the natural biogenic role of antioxidants in the retina is not clear.. This study investigates the change in concentration of vitamin E (VE), ascorbate and glutathione (GSH) in the retina following vitreous injection of 600 mug 18:2 linoleic acid hydroperoxide (LHP) in male New Zealand rabbits.. LHP was injected above the retinal surface. The animals were sacrificed and the eyes enucleated before LHP injection, 1, 3, 6, 12, 24 h and 4 and 7 days after LHP injection. Retinas were removed, VE and ascorbate measured by HPLC, and GSH determined by a fluorometric method.. The concentration of VE in the retina decreased from pretreatment levels of 154.6 +/- 29.7 nmol/g wet weight (n = 7) and was lowest at 6 h (61.1 +/- 18.1 nmol/g wet weight, n = 4, p < 0.05), then increased gradually, returning slowly to pre-LHP levels by 7 days. The concentration of ascorbate in control retinas decreased at 6 h from pretreatment levels of 7.33 +/- 0.93 micromol/g wet weight (n = 7) to 2.74 +/- 0.16 micromol/g wet weight (n = 4, p < 0.05) and returned to pretreatment levels rapidly by 24 h after injection. The concentration of GSH in retinas decreased from baseline levels of 109.53 +/- 8.19 microg/g wet weight (n = 9), was lowest at 12 h (72.40 +/- 11.17 microg/g wet weight, n = 5, p < 0.05) and returned to pretreatment levels by 7 days.. The results suggest that intravitreous LHP injection is a contributor to oxidative stress in the rabbit retina by causing a reduction in antioxidant capacity.

Topics: Animals; Ascorbic Acid; Biomarkers; Chromatography, High Pressure Liquid; Glutathione; Injections; Linoleic Acids; Lipid Peroxides; Male; Oxidative Stress; Rabbits; Retina; Spectrometry, Fluorescence; Vitamin E

There is strong evidence that the oxidation of plasma lipoproteins plays an important role in atherogenesis. The exact mechanisms by which lipoprotein oxidation occurs in the presence of other plasma constituents, however, remains unclear. To investigate the role of different antioxidants for this process, we studied the oxidation of human plasma supplemented in vitro with physiological amounts of major plasma antioxidants alpha-tocopherol, ubiquinol-10 ascorbate, urate, bilirubin and albumin. The plasma was diluted 2-fold and oxidized by 3.75 mM Cu(II). The concentrations of the antioxidants, fatty acids, linoleic acid hydroperoxides and oxycholesterols in oxidizing plasma were measured. The oxidation was characterized by three consecutive phases similar to the known lag, propagation, and decomposition phases of low density lipoprotein oxidation. The rate of the initiation of oxidation as calculated from antioxidant consumption rates was raised by supplementation with alpha-tocopherol or ascorbate. The oxidation rate in the lag phase was lowered by supplementation with any of the antioxidants, whereas in the propagation phase the oxidation rate was slightly higher in supplemented than in unsupplemented plasma. The kinetic chain length in the lag phase was less than one in supplemented plasma and about one in unsupplemented plasma. The chain length in the propagation phase was between three and six for all plasma samples. A higher rate of urate consumption and a reduced rate of alpha-tocopherol consumption were found in plasma supplemented with ascorbate in comparison with unsupplemented plasma. These data suggest that: (i) the reduction of Cu(II) by alpha-tocopherol and ascorbate is a major initiating event in Cu(II)-catalyzed oxidation of human plasma; (ii) the following lag phase is caused by radical-scavenging effects of all antioxidants with alpha-tocopherol as a major lipophilic and urate as a major hydrophilic scavenger; (iii) interactions between antioxidants, such as regeneration of ascorbate by urate and of alpha-tocopherol by ascorbate, take place during the lag phase; (iv) in the absence of added antioxidants the oxidation in the lag phase can occur via a chain reaction; and (v) in the propagation phase the oxidation is not inhibited by antioxidants and occurs autocatalytically.

Topics: Adult; Antioxidants; Ascorbic Acid; Carotenoids; Cholesterol; Copper; Fatty Acids; Humans; Kinetics; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Lipoproteins; Lipoproteins, HDL; Lipoproteins, LDL; Oxidation-Reduction; Ubiquinone; Uric Acid; Vitamin E

A new amperometric analytical technique for measuring lipid hydroperoxides is described. The technique is based on the measurement of cathodic current due to the reduction of ferricinium ion formed as result of the oxidation of ferrocene by lipid hydroperoxides. The effects of pH and applied potential were investigated to determine the optimum pH and working potential for the determination of linoleic acid and linolenic acid hydroperoxides. The analysis, performed in pH 5.5, 0.1 M phosphate buffer and at -100 mV (vs Ag/AgCl) applied potential, responded linearly to linoleic acid hydroperoxide and linolenic acid hydroperoxide up to 1.5 and 1.2 microM, respectively. The lower detection limits were 20 nM for linoleic acid hydroperoxide and 25 nM for linolenic acid hydroperoxide. Reductants such as ascorbate and urate present in the biological samples, as well as other peroxides, did not interfere in the amperometric analyses of lipid hydroperoxides.

Topics: Ascorbic Acid; Chemical Phenomena; Chemistry, Physical; Electric Conductivity; Electrochemistry; Electrodes; Ferrous Compounds; Hydrogen-Ion Concentration; Linoleic Acids; Linolenic Acids; Lipid Peroxides; Metallocenes; Organometallic Compounds; Oxidation-Reduction; Oxygen; Sensitivity and Specificity; Spectrophotometry; Uric Acid

Rat myocardial membranes exposed to the free radical-generating systems, Fe2+/ascorbate, Cu2+/t-butylhydro-peroxide, linoleic acid hydroperoxide, and soybean lipoxygenase (Type I) undergo lipid peroxidation. This is evidenced by the accumulation of thiobarbituric acid-reactive substances and the loss of both extractable phospholipids and their polyunsaturated acyl groups. Lipid peroxidation is accompanied by alterations of membrane proteins including the general loss of polypeptides and accumulation of high-molecular weight material. The most sensitive protein is a polypeptide with a molecular weight of 28 kDa. At low levels of oxidation, this protein moves incrementally to slightly higher apparent molecular weight. At higher oxidant levels or longer periods of oxidation, the protein disappears completely from the SDS-PAGE gel. The "28K reaction" occurs prior to the massive, oxidant-induced lipid alterations and may thus indicate specific adduct formation between this protein and certain peroxidized membrane phospholipids.

Topics: Animals; Ascorbic Acid; Cations, Divalent; Copper; Ferrous Compounds; Free Radicals; Glycine max; Heart; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Lipoxygenase; Membrane Lipids; Membrane Proteins; Molecular Weight; Myocardium; Peroxides; Rats; tert-Butylhydroperoxide

Simultaneous addition of ascorbic acid and organic hydroperoxides to rat liver microsomes resulted in enhanced lipid peroxidation (approximately threefold) relative to incubation of organic hydroperoxides with microsomes alone. No lipid peroxidation was evident in incubations of ascorbate alone with microsomes. The stimulatory effect of ascorbate on linoleic acid hydroperoxide (LAHP)-dependent peroxidation was evident at all times whereas stimulation of cumene hydroperoxide (CHP)-dependent peroxidation occurred after a lag phase of up to 20 min. EDTA did not inhibit CHP-dependent lipid peroxidation but completely abolished ascorbate enhancement of lipid peroxidation. Likewise, EDTA did not significantly inhibit peroxidation by LAHP but dramatically reduced ascorbate enhancement of lipid peroxidation. The results reveal a synergistic prooxidant effect of ascorbic acid on hydroperoxide-dependent lipid peroxidation. The inhibitory effect of EDTA on enhanced peroxidation suggests a possible role for endogenous metals mobilized by hydroperoxide-dependent oxidations of microsomal components.

Topics: Animals; Ascorbic Acid; Benzene Derivatives; Edetic Acid; Iron; Kinetics; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Male; Microsomes, Liver; Rats; Rats, Inbred F344; Thermodynamics