Stopped-flow kinetic study of the regeneration reaction of tocopheroxyl radical by reduced ubiquinone-10 in solution

Kinetic study of the reaction between tocopheroxyl (vitamin E radical) and reduced ubiquinone, n = 10) has been performed. The rates of reaction of ubiquinol with α-tocopheroxyl 1 and seven kinds of alkyl substituted tocopheroxyl radicals 2–8 in solution have been determined spectrophotometrically, using a stopped-flow technique. The result shows that the rate constants decrease as the total electron-donating capacity of the alkyl substituents on the aromatic ring of tocopheroxyls increases. For the tocopheroxyls with two alkyl substituents at ortho positions (C-5 and C-7), the second-order rate constants, k₁, obtained vary i n the order of 10², and decrease predominantly, as the size of two ortho-alkyl groups (methyl, ethyl, isopropyl and tert-buty) in tocopheroxyl increases. On the other hand, the reaction between tocopheroxyl and ubiquinone-10 (oxidized ubiquinone) has not been observed. The result indicates that ubiquinol-10 regenerates tocopherol by donating a hydrogen atom of the 1-OH and/or 4-OH group to the tocopheroxyl radical. For instance, the k₁ values obtained for α-tocopheroxyl are 3.74 · 10⁵ M^?1 · s^?1 and 2.15 · ⁵ M^?1 · s^?1 in benzene and ethanol solution at 25°C, respectively. The above reaction rates, k₁, obtained were compared with those of vitamin C with α-tocopheroxyl reported by Packer et al. (k₂ = 1.55 · 10⁶ M^?1 · s^?1) and Scarpa et al. (K₂ = 2 · 10⁵ 10⁵ M^?1 · s^?1), which is well known as a usual regeneration reaction of tocopheroxyl in biomembrane systems. The result suggests that ubiquinol-10 also regenerates the tocopheroxyl to tocopherol and prevents lipid peroxidation in various tissues and mitochondria.     

Electron spin resonance study of the role of vitamin E and vitamin C in the inhibition of fatty acid oxidation in a model membrane

The neutral α-tocopheroxyl radicals, generated in monolayers on silica gel containing α-tocopherol and partly autoxidised methyl linoleate at 90°C, were detected and identified by ESR spectroscopy. Addition of ascorbic acid to the monolayer resulted in the complete quenching of the α-tocopheroxyl radical spectrum. This lends support to the view that ascorbate transfers hydrogen to α-tocopheroxyl radicals thus regenerating α-tocopherol.     

On-line EPR study of free radicals induced by peroxidase/H2O2 in human low-density lipoprotein

The aim of this study was to use direct electron paramagnetic resonance (EPR) spectroscopy at 37 °C and spin trapping techniques to study radical species formed during horseradish peroxidase/H₂O₂-initiated low-density lipoprotein (LDL) oxidation. Using direct EPR, we obtained evidence for the formation not only of the α-tocopheroxyl radical but also of a protein radical(s), assigned to a tyrosyl radical(s) of apolipoprotein B-100 (apo B-100). Spin trapping with 2-methyl-2-nitrosopropane revealed (i) the formation of a mobile adduct with β-hydrogen coupling assigned to a lipid radical and (ii) a partially immobilised adduct detected in LDL as well as in apo B-100, assigned after proteolytic digestion to the trapping of a radical centred on a tertiary carbon atom of an aromatic residue, probably tyrosine. Our results support the hypothesis that radicals are initiators of the oxidative process, and show that their formation is an early event in peroxidase-mediated oxidation. We also tested the effects of resveratrol (RSV), a polyphenolic antioxidant present in red wine. Our data indicate that 1–10 μM RSV is able to accelerate α-tocopherol consumption, conjugated dienes formation and the decay kinetics of LDL-centred radicals. Since phenols are substrates for peroxidases, this result may be ascribed to a RSV-mediated catalysis of peroxidase activity.     

Antioxidant effect of FeAOX-6 on free radical species produced during iron catalyzed breakdown of tert-butyl hydroperoxide

There is a body of evidences demonstrating, in biological systems, a cooperative interaction between tocopherols and carotenoids. FeAOX-6 is a novel antioxidant that combines the chroman head of α-tocopherol and a fragment of the isoprenyl chain of lycopene. We have tested its antioxidant effect on different radical species generated in a chemical system, where peroxyl, alkoxyl and methyl radicals are generated by the ferrous ion-mediated decomposition of tert-butyl hydroperoxide. We found that FeAOX-6 has the same effectiveness of α-tocopherol in quenching peroxyl radical with no contribution by lycopene. The antioxidant activity of FeAOX-6 on alkoxyl and methyl radicals is comparable to that of the equimolar mixture of the parent compounds. Lycopene is able to quench alkoxyl radical, while it has no effect on peroxyl radical, showing a different antioxidant activity compared to other carotenoids, such as β-carotene and lutein.     

Electron spin resonance and electron nuclear double resonance studies of cation radicals derived from tocopherol model compounds

Electron spin resonance (ESR) and electron nuclear double resonance (ENDOR) measurements were performed for the cation radicals obtained from the model compounds of α-, β-, γ- and δ-tocopherol (vitamin E) by oxidizing the tocopherol precursors in an AlCl₃-CH₂Cl₂ solution. The proton hyperfine coupling constants g-values were precisely determined. The ENDOR spectra of the cation radicals of α-, β-, γ- and δ-tocopherol models in CH₂Cl₂ at ?100°C clearly show 10, 6, 6 and 12 different proton hyperfine couplings, respectively. By varying the temperature, the ESR spectra of the α- and δ-tocopherol model cations exhibit line-width alternation phenomena characteristic of the hindered rotation of the OH group. However, neither the β- nor the γ-tocopherol model cation radical ESR spectra show any sign of an alternative line-width effect. These results are interpreted by assuming that the β- and γ-tocopherol model cations are stabilized in the trans and cis conformations, respectively. On tocopherol model cations are stabilized in the trans and cis conformations, respectively. On the other hand, both the α- and δ-tocopherol model cations exist as cis and trans isomers.     

Effect of CPTA and cycocel on the biosynthesis of carotenoids by Phycomyces blakesleeanus mutants

CPTA and cycocel cause accumulation of lycopene and γ-carotene, simultaneously inhibiting the formation of β-carotene and β-zeacarotene in Phycomyces blakesleeanus mutant strain C115. Phytoene synthesis is enhanced. CPTA is more effective than cycocel. Kinetic studies show that with increasing concentrations of CPTA, lycopene and γ-carotene increase with the concomitant decrease in β-carotene, the total of these three carotenes being almost equal to β-carotene present in the control. When CPTA-treated mycelium is washed free of the chemical and resuspended in phosphate buffer solution containing 2·5% glucose (pH 5·6), β-carotene is formed at the expense of both γ-carotene and lycopene. β-Zeacarotene, which is not present in the mycelium, reappears upon resuspension. These results indicate that CPTA is inhibiting the enzymes causing cyclization both at neurosporene and lycopene levels. Studies on the effect of CPTA on the high lycopene mutant strain C9 reveal that with increasing concentrations of the compound, lycopene increases slightly and both β-carotene and γ-carotene decrease. Phytoene synthesis is stimulated up to a certain level of CPTA and then becomes steady. In the albino mutant strain C5, there is a slight increase in phytoene formation on the addition of CPTA to the medium. No other carotenoid is formed, suggesting that CPTA cannot remove the block caused by genetic mutation and exerts its influence in an already existing biosynthetic pathway.     

Electron nuclear double resonance studies of radicals produced by the PbO2 oxidation of α-tocopherol and its model compound in solution

The electron nuclear double resonance (ENDOR) spectra of chromanoxyl radicals obtained by the PbO₂ oxidation of α-tocopherol and its model compound were observed in t-butylbenzene, and the proton hyperfine coupling constants were correctly determined. Each of the two β- and γ-methylene protons in the chromanoxyl ring shows an equivalent hyperfine splitting, suggesting that the heterocyclic ring attached to the aromatic ring are coplanar with the plane of the aromatic system. A comparison of the hyperfine couplings in α-tocopheroxyl radical and its model shows that the introduction of a long-isoprenoid-chain in the α-tocopherol in place of a methyl group in the model compound has very little effect on the unpaired spin distribution or molecular structure of the chromanoxyl skeleton. The results of McLachlan molecular orbital (MO) calculations were found to be in satisfactory agreement with the ‘experimental’ spin densities evaluated from the hyperfine coupling constants.     

Antioxidant activity of carotenoids: An electron-spin resonance study on β-carotene and lutein interaction with free radicals generated in a chemical system

β-Carotene is thought to be a chain-breaking antioxidant, even though we have no information about the mechanism of its antioxidant activity. Using electron-spin resonance (ESR) spectroscopy coupled to the spin-trapping technique, we have studied the effect of β-carotene and lutein on the radical adducts of the spin-trap PBN (N-t -butyl-α-phenylnitrone) generated by the metal-ion breakdown of different tert -butyl hydroperoxide (t BOOH) concentrations in methylene chloride. The peroxyl radical, along with an oxidation product of PBN (the PBNOx), trapped at room temperature from the breakdown of high concentration of t BOOH (1 M), were quenched by β-carotene or lutein, in competition with the spin-trapping agent. However, carotenoids were not able to quench the alkoxyl and methyl radicals generated in the reaction carried out in the presence of low t BOOH concentration (1 mM). The reaction between carotenoids and the peroxyl radical was also carried out in the absence of the spin trap, at 77 K: Under these different experimental conditions, we did not detect any radical species deriving from carotenoids. In the same system, a further evidence of the peroxyl radical quenching by β-carotene and lutein was obtained. The antioxidant activity of vitamin E was also tested, for comparison with the carotenoids. In the presence of α-tocopherol, peroxyl and alkoxyl radicals were quenched, and the tocopheroxyl radical was detected. Our data provide the first direct evidence that carotenoids quench peroxyl radicals. Under our experimental conditions, we did not detect any carotenoid radical species that could derive from the interaction with the peroxyl radical. The radical-trapping activity of β-carotene and lutein demonstrated in this chemical reaction contributes to our understanding carotenoid antioxidant action in biological systems. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 299–304, 1998     

Formation of cyclodextrin inclusion compounds of stable nitroxide radicals as monitored by electron-spin resonance spectra

Hyperfine coupling constants and rotational correlation times, calculated from electron-spin resonance spectra of cyclodextrins incubated with stable nitroxide radicals, indicate inclusion compound formation of β- and γ-cyclodextrin with certain nitroxide radicals. In contrast, α-cycledextrin exhibits no effect on the spectra of the radicals, probably because its central cavity is too small to form such inclusion compounds. Furthermore, one 1:1 molar ratio complex of β-cyclodextrin and a nitroxide radical (isolated as crystalline precipitate and identified both by combustion analysis and ir measurements) is shown by electron-spin resonance data to be an inclusion compound.     

Reduction of oxidized guanosine by dietary carotenoids: A pulse radiolysis study

Time-resolved pulse radiolysis investigations reported herein show that the carotenoids β-carotene, lycopene, zeaxanthin and astaxanthin (the last two are xanthophylls - oxygen containing carotenoids) are capable of both reducing oxidized guanosine as well as minimizing its formation. The reaction of the carotenoid with the oxidized guanosine produces the radical cation of the carotenoid. This behavior contrasts with the reactions between the amino acids and dietary carotenoids where the carotenoid radical cations oxidized the amino acids (tryptophan, cysteine and tyrosine) at physiological pH.     

Mitochondrial electron transport-linked tocopheroxyl radical reduction

alpha-Tocopherol (vitamin E) is a lipophilic chain-breaking antioxidant which inhibits lipid peroxidation in isolated mitochondrial membranes and protects membranes from oxidative damage. The primary oxidation product of vitamin E is the tocopheroxyl radical. Reduction of the tocopheroxyl radical can occur by reactions with water-soluble anti-oxidants such as ascorbate or glutathione, resulting in the recycling of vitamin E. Physiological concentrations of vitamin E are too low to allow detection of tocopheroxyl radical by ESR. After dietary supplementation with vitamin E, a 10-20-fold increase in the rat liver mitochondrial membrane content of vitamin E was achieved and this allowed for direct detection of the tocopheroxyl radical by ESR, after treatment with an oxidizing system composed of lipoxygenase and arachidonic acid. By using submitochondrial particle membranes, it was shown that NADH, succinate, and reduced cytochrome c-linked oxidation reduce the tocopheroxyl radical, preventing both accumulation of the radical and vitamin E consumption. As the electron transport chain can reduce tocopheroxyl radical it may have an important physiological role in recycling vitamin E.     

Biosynthesis of carotenoids by Phycomyces blakesleeanus mutants in the presence of nitrogenous heterocyclic compounds

Pyridine, imidazole and some of their derivatives stimulate lycopene and γ-carotene synthesis-simultaneously inhibiting β-carotene formation in Phycomyces blakesleeanus Strain C115. Isonicotinoly-hydrazine has a toxic effect on Strains C9 and C115 and 1-methylimidazole on Strain C115 in the concentrations of 1 g/l. Compounds which cause an accumulation of lycopene and γ-carotene usually cause an increase in phytoene synthesis and the disappearance of β-zeacarotene. The effect of succinimide, 4-hydroxypyridine, and isonicotinoylhydrazine on Strain C9 has also been studied. When β-picoline and 2-methylimidazole treated C115 mycelia were washed and resuspended in phosphate buffer at pH 5·6 β-zeacarotene reappeared and β-carotene increased with the simultaneous decrease in lycopene and γ-carotene. The sum of β-carotene, γ-carotene up to 3days of resuspension was almost equal to the total of these at zero time. These results show that the inhibitory action of these compounds is on the enzymes responsible for cyclization of acyclic carotenes. This inhibition varies with the nature of the substituent on the heterocyclic ring and pyridine derivatives having pK_a values of 6 ± 1 show the greatest degree of inhibition.     

Succinate-ubiquinone reductase linked recycling of alpha-tocopherol in reconstituted systems and mitochondria: requirement for reduced ubiquinone.

Studies have demonstrated that accumulation of mitochondrial tocopheroxyl radical, the primary oxidation product of alpha-tocopherol, accompanies rapid consumption of tocopherol. Enzyme-linked electron flow lowers both the steady-state concentration of the radical and the consumption of tocopherol. Reduction of tocopheroxyl radical by a mitochondrial electron carrier(s) seems a likely mechanism of tocopherol recycling. Succinate-ubiquinone reductase (complex II) was incorporated into liposomes in the presence of tocopherol and ubiquinone-10. After inducing formation of tocopheroxyl radical, it was possible to show that reduced ubiquinone prevents radical accumulation and tocopherol consumption. There was no evidence of direct reduction of tocopheroxyl radical by succinate-reduced complex II. These reactions were also measured using ubiquinone-1 and alpha-C-6-chromanol (2,5,7,8-tetramethyl-2-(4'-methylpentyl)-6-chromanol) which are less hydrophobic analogues of ubiquinone-10 and alpha-tocopherol. Mitochondrial membranes were made deficient in ubiquinone but sufficient in alpha-tocopherol and were reconstituted with added quinone. With these membranes it was shown that mitochondrial enzyme-linked reduction of ubiquinone protects alpha-tocopherol from consumption, and there is a requirement for ubiquinone. This complements the observations made in liposomes and we propose that reduced mitochondrial ubiquinones have a role in alpha-tocopherol protection, presumably through efficient reduction of the tocopheroxyl radical.     

Poduction of free radicals from phenol and tocopherol by bleomycin-iron(II) complex.

Oxygen-bubbling of 1:1 bleomycin(BLM)-Fe(II) complex efficiently generates hydroxy radical which is inhibited by catalase. Hydroxy radical produced from BLM-Fe(II)-O₂ system oxidizes 2,6-di-tert-butyl-p-cresol and α-tocopherol to form the corresponding phenoxy and tocopheroxyl radicals, respectively. These free radicals were identified by the ESR hyperfine structures.     

Interaction of vitamin C and vitamin E during free radical stress in plasma: An ESR study

To study the interaction of the antioxidant vitamins C and E in a biological system, we used electron spin resonance (ESR) spectroscopy to make serial measurement of ascorbate tocopheroxyl free radicals in plasma subjected to continuous free radical-mediated oxidative stress. Upon initiation of a continuous oxidative stress, we observed an immediate increase in the concentration of ascorbate radical, which reached a peak, and then steadily declined. Only after the virtual disappearance of the ascorbate radical did we observe the appearance of the tocopheroxyl radical. These data are consistent with the hypothesis that ascorbate is the terminal small-molecule antioxidant in biological systems. This is the first experimental demonstration that the predicted thermodynamic hierarchy of ascorbate, -tocopherol, and their free radicals holds in a biological system containing endogenous levels of these antioxidant vitamins.     

Alpha-tocopherol amplifies benzoyl peroxide free radical decomposition in a chemical system

Benzoyl peroxide is commonly used in the treatment of acne, even though some adverse effects have been reported, probably mediated by the formation of peroxide-derived free radicals and the depletion of antioxidants. In the present work we have studied, in a chemical system, the effect of alpha-tocopherol on benzoyl peroxide radical decomposition to analyse the presence of an interaction between these two compounds, leading to an enhanced peroxide-cytotoxicity, as we have previously reported. Under our experimental conditions alpha-tocopherol strongly amplified the peroxide free radical decomposition occurring either in the presence or in the absence of UV irradiation, and lead to the formation of an unknown radical species in addition to benzoyloxy, phenyl and tocopheroxyl free radicals. The results of this study show that the enhancement of benzoyl peroxide toxicity in cells exposed simultaneously to this peroxide and alpha-tocopherol, is likely due to the generation of the detected radical species.     

Selenium vitaminology: The connection between selenium,vitamin C,vitamin E,and ergothioneine

Selenium is connected to three small molecule antioxidant compounds, ascorbate, α-tocopherol, and ergothioneine. Ascorbate and α-tocopherol are true vitamins, while ergothioneine is a “vitamin-like” compound. Here we review how selenium is connected to all three. Selenium and vitamin E work together as a team to prevent lipid peroxidation. Vitamin E quenches lipid hydroperoxyl radicals and the resulting lipid hydroperoxide is then converted to the lipid alcohol by selenocysteine-containing glutathione peroxidase. Ascorbate reduces the resulting α-tocopheroxyl radical in this reaction back to α-tocopherol with concomitant production of the ascorbyl radical. The ascorbyl radical can be reduced back to ascorbate by selenocysteine-containing thioredoxin reductase. Ergothioneine and ascorbate are both water soluble, small molecule reductants that can reduce free radicals and redox-active metals. Thioredoxin reductase can reduce oxidized forms of ergothioneine. While the biological significance of this is not yet realized, this discovery underscores the centrality of selenium to all three antioxidants.     

Electron transport between cytochrome c and alpha tocopherol.

Using liposomes we have demonstrated an electron transfer between tocopherol (vitamin E) and cytochrome c. Reduced cytochrome c protects vitamin E from oxidation induced either directly by ultraviolet light or indirectly by soybean lipoxygenase-catalyzed oxidation of arachidonic acid. Oxidized cytochrome c is reduced by tocopherol and tocopherol homologues (chromanols) resulting in accumulation of tocopheroxyl radicals which we detected by ESR. The peak height of the ESR spectrum of tocopheroxyl radicals (which is proportional to the amount of radical present) is proportional to the ratio of reduced to oxidized cytochrome c. In mitochondrial membranes succinate-cytochrome c reduction is inhibited by antimycin A. Addition of exogenous chromanols facilitates a by-pass of the antimycin A blocked electron pathway, and succinate-dependent cytochrome c reductase activity is restored. Cytochrome c may act as a water-soluble complement to the lipid-soluble ubiquinol in regenerating mitochondrial tocopherol from tocopheroxyl radical.     

Sugar radicals in DNA: isolation of neutral radicals in gamma-irradiated DNA by hole and electron scavenging

In this investigation of the radical formation and the reaction of radicals in gamma-irradiated DNA, we report the isolation of putative neutral radicals by the scavenging of holes by Fe(CN)6(4-) and of electrons by Fe(CN)6(3-). Experiments are performed under conditions that emphasize direct and quasi-direct effects (collectively called direct-type effects.) Samples containing Fe(CN)6(4-) show effective scavenging of holes and the ESR spectra obtained arise principally from DNA anion radicals and neutral radicals. On the other hand, for samples containing Fe(CN)6(3-), electron scavenging is highly efficient, and the resulting spectra arise principally from guanine cation radicals and neutral radicals. When both Fe(CN)6(4-) and Fe(CN)6(3-) are present, a near complete scavenging of cation radicals and anion radicals is observed at 77 K, and the ESR spectra that result originate predominantly with neutral radicals which are assigned predominantly to radicals on the sugar phosphate backbone. A notable finding is the presence of spectral components that indicate the formation, through the rupture of the C3'-O bond, of a neutral deoxyribose radical; a concurrent strand break must accompany formation of this radical. This radical was previously reported in argon-ion-irradiated DNA and now, for the first time, is reported in DNA irradiated with low-LET radiation.     

Cation radicals of xanthophylls

Carotenes and xanthophylls are well known to act as electron donors in redox processes. This ability is thought to be associated with the inhibition of oxidative reactions in reaction centers and light-harvesting pigment–protein complexes of photosystem II (PSII). In this work, cation radicals of neoxanthin, violaxanthin, lutein, zeaxanthin, β-cryptoxanthin, β-carotene, and lycopene were generated in solution using ferric chloride as an oxidant and then studied by absorption spectroscopy. The investigation provides a view toward understanding the molecular features that determine the spectral properties of cation radicals of carotenoids. The absorption spectral data reveal a shift to longer wavelength with increasing π-chain length. However, zeaxanthin and β-cryptoxanthin exhibit cation radical spectra blue-shifted compared to that of β-carotene, despite all of these molecules having 11 conjugated carbon–carbon double bonds. CIS molecular orbital theory quantum computations interpret this effect as due to the hydroxyl groups in the terminal rings selectively stabilizing the highest occupied molecular orbitals of preferentially populated s-trans-isomers. The data are expected to be useful in the analysis of spectral results from PSII pigment–protein complexes seeking to understand the role of carotene and xanthophyll cation radicals in regulating excited state energy flow, in protecting PSII reaction centers against photoinhibition, and in dissipating excess light energy absorbed by photosynthetic organisms but not used for photosynthesis.