Formation Of Ascorbate Free Radical Research Articles

Vitamin C-Induced Loss of Redox-Dependent Viability in Lung Microvascular Endothelial Cells

Recent clinical trials have shown that vitamin C, at pharmacological concentrations (milligram to approximately gram), upon infusion into circulation, modulates vasodilation and vascular tone in humans. This also results in the elevated concentrations of vitamin C in circulation in the millimolar range. Here, it was hypothesized that vitamin C at pharmacological concentrations (millimolar) would induce oxidative stress and cause loss of redox-dependent cell viability in vascular endothelial cells (ECs). To test the hypothesis, bovine lung microvascular ECs (BLMVECs) in monolayer cultures were exposed to vitamin C (0-10 mM) for different time periods (0-2 h). Electron paramagnetic resonance spectroscopy revealed the intracellular formation of ascorbate free radical in a dose- and time-dependent fashion. Vitamin C also induced formation of intracellular reactive oxygen species in a dose-dependent fashion. It was observed that vitamin C induced morphological alterations and loss of cell viability in a dose- and time-dependent fashion, as measured by light microscopy and Alamar Blue redox cell viability assay, respectively. Vitamin C analogues failed to induce such changes. Vitamin C depleted cellular GSH levels in a dose-dependent fashion, suggesting that vitamin C altered thiol-redox status in BLMVECs. Antioxidants, intracellular iron chelator, and catalase protected cells against vitamin C-induced loss of redox-dependent cell viability, confirming the role of hydrogen peroxide and iron during redox cycling of vitamin C. These results, for the first time in detail, established that vitamin C at pharmacological doses induced oxidative stress and loss of redox-dependent cell viability in microvascular ECs.

Cupric–amyloid β peptide complex stimulates oxidation of ascorbate and generation of hydroxyl radical

A growing body of evidence supports an important role for oxidative stress in the pathogenesis of Alzheimer's disease. Recently, a number of papers have shown a synergistic neurotoxicity of amyloid β peptide and cupric ions. We hypothesized that complexes of cupric ions with neurotoxic amyloid β peptides (Aβ) can stimulate copper-mediated free radical formation. We found that neurotoxic Aβ (1-42), Aβ (1-40), and Aβ (25-35) stimulated copper-mediated oxidation of ascorbate, whereas nontoxic Aβ (40-1) did not. Formation of ascorbate free radical was significantly increased by Aβ (1-42) in the presence of ceruloplasmin. Once cupric ion is reduced to cuprous ion, it can be oxidized by oxygen to generate superoxide radical or it can react with hydrogen peroxide to form hydroxyl radical. Hydrogen peroxide greatly increased the oxidation of cyclic hydroxylamines and ascorbate by cupric–amyloid β peptide complexes, implying redox cycling of copper ions. Using the spin-trapping technique, we have shown that toxic amyloid β peptides led to a 4-fold increase in copper-mediated hydroxyl radical formation. We conclude that toxic Aβ peptides do indeed stimulate copper-mediated oxidation of ascorbate and generation of hydroxyl radicals. Therefore, cupric–amyloid β peptide-stimulated free radical generation may be involved in the pathogenesis of Alzheimer's disease.

Protective effects of vitamins C and E on the number of micronuclei in lymphocytes in smokers and their role in ascorbate free radical formation in plasma

Cigarette smoke is widely believed to increase free radical concentrations causing subsequent oxidative processes that lead to DNA damage and hence, to several diseases including lung cancer and atherosclerosis. Vitamin C is a reducing agent that can terminate free-radical-driven oxidation by being converted to a resonance-stabilized free radical. To investigate whether short-term supplementation with the antioxidants vitamin C and E decreases free-radical-driven oxidation and thus decreases DNA damage in smokers, we determined the frequency of micronuclei in lymphocytes in 24 subjects and monitored the electron paramagnetic resonance signal of ascorbate free radical formation in plasma. Further parameters comprised sister-chromatid exchanges and thiobarbituric acid-reactive substances. Twelve smokers and twelve non-smokers took 1000 mg ascorbic acid daily for 7 days and then 1000 mg ascorbic acid and 335.5 mg RRR-α-tocopherol daily for the next 7 days. Baseline concentrations of both vitamins C and E were lower and baseline numbers of micronuclei were higher (p < 0.0001) in smokers than in non-smokers. After 7 days of vitamins C and E, DNA damage as monitored by the number of micronulei was decreased in both, smokers and non-smokers, but it was more decreased in smokers as indicated by fewer micronuclei in peripheral lymphocytes (p < 0.05). Concomitantly, the plasma concentrations of vitamin C (p < 0.001) as well as the ascorbate free radical (p < 0.05) were increased. The corresponding values in non-smokers, however, did not change. Our findings show that increased ascorbate free radical formation in plasma after short-term supplementation with vitamins C and E can decrease the number of micronuclei in blood lymphocytes and thus DNA damage in smokers.

Electron spin resonance study on the formation of ascorbate free radical from ascorbate: The effect of dehydroascorbic acid and ferricyanide

Ascorbate free radical is considered to be a substrate for a plasma membrane redox system in eukaryotic cells. Moreover, it might be involved in stimulation of cell proliferation. Ascorbate free radical can be generated by autoxidation of the ascorbate dianion, by transition metal-dependent oxidation of ascorbate, or by an equilibrium reaction of ascorbate with dehydroascorbic acid. In this study, we investigated the formation of ascorbate free radical, at physiological pH, in mixtures of ascorbate and dehydroascorbic acid by electron spin resonance spectroscopy. It was found that at ascorbate concentrations lower than 2.5 mM, ascorbate-free radical formation was not dependent on the presence of dehydroascorbic acid. Removal of metal ions by treatment with Chelex 100 showed that autoxidation under these conditions was less than 20%. Therefore, it is concluded that at low ascorbate concentrations generation of ascorbate free radical mainly proceeds through metal-ion-dependent reactions. When ascorbate was present at concentrations higher than 2.5 mM, the presence of dehydroascorbic acid increased the ascorbate free-radical signal intensity. This indicates that under these conditions ascorbate free radical is formed by a disproportionation reaction between ascorbate and dehydroascorbic acid, having aK equil of 6 × 10−17 M. Finally, it was found that the presence of excess ferricyanide completely abolished ascorbate free-radical signals, and that the reaction between ascorbate and ferricyanide yields dehydroascorbic acid. We conclude that, for studies under physiological conditions, ascorbate free-radical concentrations cannot be calculated from the disproportionation reaction, but should be determined experimentally.

Interactions between vitamin E homologues and ascorbate free radicals in murine skin homogenates irradiated with ultraviolet light.

The mechanism of oxidation of ascorbic acid in mouse skin homogenates by UV light was investigated by measuring ascorbate free radical formation using electron spin resonance signal formation. Addition of vitamin E (alpha-tocopherol or alpha-tocotrienol) had no effect, whereas short-chain homologues (2,5,7,8-tetramethyl-6-hydroxychroman-2-carboxylic acid [Trolox] and 2,2,5,7,8-pentamethyl-6-hydroxychromane [PMC]) accelerated ascorbate oxidation. The similar hydrophilicity of ascorbate, Trolox and PMC increased their interaction, thus rapidly depleting ascorbate. When dihydrolipoic acid was added simultaneously with the vitamin E homologues, the accelerated ascorbate oxidation was prevented. This was due to the regeneration of ascorbate and PMC from their free radicals by a recycling mechanism between ascorbate, vitamin E homologues and dihydrolipoic acid. Potentiation of antioxidant recycling may be protective against UV irradiation-induced damage. The rate of ascorbate oxidation in the presence of vitamin E homologues was enhanced by a photosensitizer (riboflavin) but was not influenced by reactive oxygen radical quenchers, superoxide dismutase or 5,5-dimethyl-1-pyrroline-N-oxide. These experimental results suggest that the UV irradiation-induced ascorbate oxidation in murine skin homogenates is caused by photoactivated reactions rather than reactive oxygen radical reactions.

Vitamin E Slows the Rate of Free Radical-Mediated Lipid Peroxidation in Cells

Much of what is known about the antioxidant mechanism of vitamin E has been learned from studies of lipid dispersions, solutions, or subcellular organelles. We have investigated the effect of vitamin E supplementation on intact live eucaryotic cells. L1210 murine leukemia cells were exposed to an oxidative stress induced by 20 μMFe2+and 100 μMascorbic acid introduced immediately before oxidative measurements were begun, and the kinetics of the generation of lipid-derived free radicals, as measured by EPR spin trapping (a product) and O2consumption (a reactant) were measured. Cells grown for 24 h with supplemental (5–100 μM) vitamin E in their media had a slower rate of lipid radical generation compared to cells grown without vitamin E supplementation; this inhibition in the rate of oxidation was generally dependent upon the amount of vitamin E supplementation. In complementary studies measuring O2consumption, 5–100 μMvitamin E slowed the rate of oxidation (10-fold with 100 μMsupplemental vitamin E) consistent with the EPR studies. The membrane active drug edelfosine accentuated the vitamin E effects; vitamin E introduced a discernible lag phase (time delay) in both lipid radical generation and O2consumption that was not seen in the absence of edelfosine. Vitamin E supplementation of cells also altered the kinetics of ascorbate free radical formation. We conclude that vitamin E inhibits lipid peroxidation in cells by slowing the rate of lipid peroxidation; but with iron/ascorbate as the initiating system, vitamin E does not delay the onset of peroxidation. Of special interest is that these free radical peroxidation events parallel cell membrane damage as detected using trypan blue exclusion. These observations are consistent with the free radical events preceding and causing the observed membrane damage.

Iron-induced ascorbate oxidation in plasma as monitored by ascorbate free radical formation. No spin-trapping evidence for the hydroxyl radical in iron-overloaded plasma.

A study was made of the interaction of plasma ascorbate and ascorbate free radical (AFR) with exogenously added iron. The quantitative determination of AFR has the advantage that transient increases in ascorbate oxidation can be directly monitored by e.p.r. spectroscopy. An AFR signal was found in the plasma of all donors and was unaffected by superoxide dismutase, catalase and the strong iron chelator deferoxamine. These findings and the rapid decrease in AFR under a nitrogen atmosphere suggest that plasma AFR is probably a result of air auto-oxidation. Iron loading of plasma did not affect the intensity of the AFR signal until the iron concentration approached or exceeded the plasma latent iron-binding capacity. In iron-overloaded plasma, the intensity of the AFR signal increased to about 10 times the normal level before decreasing rapidly to undetectable levels after 15-20 min. Determination of plasma ascorbate showed that the disappearance of AFR was due to a complete loss of the vitamin. When 50 microM-ascorbate was loaded with iron in iso-osmotic phosphate buffer there was an increase in the AFR signal, independent of the iron concentration, which was stable at least for 15 min. Thus the rate of ascorbate loss in the iso-osmotic phosphate buffer was considerably lower than in iron-overloaded plasma. The addition of different iron chelators produced comparable effects on the intensity of the AFR signal in both iron-overloaded plasma and ascorbate solution. These results suggest that the characteristic behaviour of plasma AFR after iron loading is due to its specific iron-binding capacity and to plasma ferroxidase activity. The ferroxidase activity of plasma is important to promote the transfer of Fe2+ into transferrin without a transient ascorbate oxidation. Spin-trapping studies with 5,5-dimethyl-1-pyrroline N-oxide and N-t-butyl-alpha-phenylnitrone revealed that iron-overloaded plasma was unable to produce spin-trap adducts even in the presence of 50-300 microM-hydrogen peroxide or 100 microM-azide. Evidence of OH. radical formation was obtained only after the addition of EDTA. Therefore, iron-overloaded plasma itself does not produce a Fenton reaction and, if ascorbate does indeed have a free-radical-mediated pro-oxidant role, it is not detectable in plasma by spin-trapping experiments.

Effect of omeprazole on ascorbate free radical formation.

In order to study the effect of omeprazole on the K/[H+] value of ascorbic acid (K = the ascorbic acid-ascorbic free radical equilibrium constant), changes of the concentration of ascorbate free radical in guinea pig sera were examined after intraperitoneal administration of omeprazole. Furthermore, to see the in vitro effect of omeprazole on ascorbate free radical, changes were examined in ascorbate solutions after addition of omeprazole. It was found that the K/[H+] value in the serum increased significantly after administration of omeprazole and also that the drug amplified the electron spin resonance (ESR) signal intensity of ascorbate free radical in vitro. These results suggested that omeprazole acts like oxygen radicals.

Evidence from the acceleration of cytochrome c reduction for the formation of ascorbate free radical by dopamine β-monooxygenase

This is believed to be the physiologically important reaction in mammalian tissues, but the purified enzyme is also an efficient catalyst for similar hydroxylations with a number of alternative substrates. Dopamine can be replaced by several other derivatives of phenylethylamine, with tyramine frequently being used for assays of the enzyme. Ascorbate can be replaced by other two-electron (2 e-) donors, such as catechol, or by the oneelectron (1 e-) donor Fe(CN)z -. The path of electron transfer is believed to involve the enzyme-bound copper (reviewed [ 11). Classification of ascorbate as a 2 e-donor in. enzymic oxidations describes the overall stoichiometry correctly. It leaves open, however, the question of whether: (i) Each ascorbate ion transfers 2 eto the enzyme and is released as dehydroascorbate; or (ii) Each ascorbate ion transfers 1 eand is released as the unstable ascorbate free radical (semidehydroascorbate), followed by nonenzymic dismutation of two radicals to give one ascorbate and one dehydroascorbate. Most discussions of the mechanism of dopamine fl-monooxygenase have assumed alternative (i), but we have discussed alternative (ii) [ 11. Compelling evidence for 1 emechanisms (alternative (ii)) in the cases of ascorbate oxidation by ascorbate oxidase and by peroxidase has been presented [2]. The experimental methods used were EPR spectroscopy [3] and a scavenger method which employed oxidized cytochrome c as the scavenger of radicals [4]. Ascorbate free radical reduces cytochrome c more rapidly than the fully reduced ascorbate, and hence an acceleration of cytochrome c reduction upon the addition of oxidizing enzyme was interpreted as evidence for free radical formation in the enzymic oxidations. Although the question of 1 eor 2 etransfer in the oxidation of ascorbate by dopamine &monooxygenase must be answered for correct interpretation of the kinetics and mechanism of this enzyme, the problem had not yet been addressed experimentally. We now report evidence obtained with the cytochrome c scavenger method that dopamine /3-monooxygenase also employs a 1 emechanism, releasing ascorbate free radical as the product of enzymic oxidation. Our experimental evidence includes measurements with ascorbate oxidase for the purpose of comparing with a standard reaction [2 1, and the results reveal close correspondence between these two enzymes in the cytochrome c reduction assay.