Kinetic study of the reaction between vitamin E radical and alkyl hydroperoxides in solution

Kinetic study of the reaction between vitamin E radical and alkyl hydroperoxides has been performed, as a model for the reactions of lipid hydroperoxides with vitamin E radical in biological systems. The rates of reaction of hydroperoxides (n-butyl hydroperoxide 1, sec-butyl hydroperoxide 2, and tert-butyl hydroperoxide 3) with vitamin E radical (5,7-diisopropyl-tocopheroxyl 4) in benzene solution have been determined spectrophotometrically. The second-order rate constants, k-1, obtained are 1.34 x 10(-1) M-1s-1 for 1, 2.42 x 10(-1) M-1s-1 for 2, and 3.65 x 10(-1) M-1s-1 for 3 at 25.0 degrees C. The result indicates that the rate constants increase as the total electron donating capacity of the alkyl substituents at alpha-carbon atom of hydroperoxides increases. The above rates, k-1, are about seven order of magnitude lower than those, k1, for the reaction of vitamin E with peroxyl radical.     

Stopped-flow investigation of the reaction of vitamin C with tocopheroxyl radical in aqueous triton X-100 micellar solutions. The structure-activity relationship of the regeneration reaction of tocopherol by vitamin C

A kinetic study of the reaction between vitamin C (L-ascorbic acid, AsH2) and a tocopheroxyl radical (7-tert-butyl-5-isopropyltocopheroxyl) in Triton X-100 micellar solution has been performed using stopped-flow spectrophotometry. The second-order rate constants (k2) obtained showed notable pH dependence with a broad maximum around pH 8. For instance, the k2 values obtained were 26 M-1 S-1 at pH 3, 322 M-1 S-1 at pH 7, and 273 M-1 S-1 at pH 10. A good correlation between the rate constants and the mole fraction of ascorbate monoanion (AsH-) was observed, showing that ascorbate (AsH-) can regenerate the tocopherol from tocopheroxyl in biological systems. Furthermore, the results indicate that reduced ascorbic acid (AsH2) does not have the ability to regenerate the tocopherol in aqueous solution. On the other hand, it was found that AsH2 can reduce the tocopheroxyl to tocopherol in benzene/ethanol (2:1) mixtures, although the rate of reaction is only approximately 15% of that observed in micellar solution at pH 7.     

Kinetic study of the reaction of vitamin C with vitamin E radicals (tocopheroxyls) in solution

New stable vitamin E radicals (7-tert-butyl-5-isopropyltocopheroxyl (4), 5,7-diisopropyltocopheroxyl (5), 7-tert-butyl-5-methyltocopheroxyl (6), and 5,7-diethyltocopheroxyl (7] with two bulky alkyl substituents at ortho positions (C-5 and C-7) have been prepared, and the reaction rates of vitamin C (ascorbic acid (1) and 6-O-stearyl ascorbic acid (2] with these tocopheroxyl radicals in benzene/ethanol/water (2:1:0.1, v/v) solution have been determined spectrophotometrically, using a stopped-flow technique. The second-order rate constants, k2, obtained vary in the order of 10(3), and decrease dramatically in the order 7 greater than 6 greater than 5 greater than 4, as the size of two ortho-alkyl groups in tocopheroxyl increases. The result suggests that the effect of steric hindrance on the reaction rate is considerable. These reaction rates were compared with those of vitamin C with alpha-tocopheroxyl reported by Packer et al. (Nature 278 (1979) 737-738) and Scarpa et al. (Biochim. Biophys. Acta 801 (1984) 215-219).     

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.     

Stopped-flow kinetic study of vitamin E regeneration reaction with biological hydroquinones (reduced forms of ubiquinone, vitamin K, and tocopherolquinone) in solution.

A kinetic study of the regeneration reaction of vitamin E (tocopherol) with eight biological hydroquinones (HQs) (ubiquinol-10 (Q10H2 1); ubiquinol-0 (Q0H2 2); vitamin K1 HQ (VK1H2 3); vitamin K3 HQ (VK3H2 4); alpha-, beta-, and gamma-tocopherol-HQs (alpha-, beta-, and gamma-TQH2 5-7); and 2,3,5-trimethyl-1,4-HQ (TMQH2 8)) in solution was performed. The second-order rate constants (k4) for the reaction of HQs 1-8 with alpha-tocopheroxyl and 5,7-diisopropyltocopheroxyl radicals in ethanol, benzene, and isopropyl alcohol/water (5:1, v/v) solutions were measured with a stopped-flow spectrophotometer. The order of magnitude of k4 values obtained for HQs is VK1H2 > VK3H2 > alpha-TQH2 > beta-TQH2 approximately gamma-TQH2 approximately TMQH2 > Q10H2 > Q0H2, being independent of the kinds of tocopheroxyl radicals and the polarity of the solvents. The log of the k4 values obtained for HQs was found to correlate with their peak oxidation potentials. Comparing the k2 value (2.68 x 10(6) M-1 s-1 obtained for the reaction of alpha-tocopheroxyl with vitamin C (sodium ascorbate) with those (k4 = 2.54 x 10(5) and 8.15 x 10(5) M-1 s-1) obtained for the reaction of alpha-tocopheroxyl with Q10H2 and alpha-TQH2 in isopropyl alcohol/water mixtures, the former is approximately 11 and 3 times as reactive as the latter, respectively. On the other hand, the k2 value obtained for sodium ascorbate is smaller than the k4 values obtained for VK1H2 and VK3H2. These results suggest that mixtures of vitamin E and these HQs (as well as those of vitamins E and C) may function synergistically as antioxidants in various tissues and mitochondria.     

Stopped-flow investigation of antioxidant activity of estrogens in solution

A kinetic study of the reaction between estrogens (female hormone) and substituted phenoxyl radical has been performed, as a model for the reactions of estrogens with lipid peroxyl radical in biological systems. The rates of reaction of estrogens (estrone 1, estradiol 2, 2-methoxyestrone 3, 3-methoxyestrone 4, and 2-hydroxyestrone 5) with substituted phenoxyl radical in benzene have been determined spectrophotometrically, using stopped-flow technique. The second-order rate constants, k2, obtained are 84 M-1.s-1 for 1, 138 M-1.s-1 for 2, 520 M-1.s-1 for 3, less than 10(-4) M-1.s-1 for 4, and 2.6 X 10(5) M-1.s-1 for 5 at 25.0 degrees C. 2-Hydroxyestrone 5 was found to be 2.9-times more active than alpha-tocopherol, which has the highest antioxidant activity among natural tocopherols. The order of magnitude of k2 value (1 less than 2 less than 3 less than alpha-Toc less than 5) is in agreement with that of in vitro tests of their antioxidant activities, as measured by the inhibition of lipid peroxidation. Further, similar measurements have been performed for the reaction between the above estrogens 1-5 and tocopheroxyl 6 in benzene solution. It was found that the estrogens having an OH group at the aromatic ring have an ability to regenerate the tocopheroxyl 6 to tocopherol. Especially, the 2-hydroxyestrone 5 showed about three orders of magnitude higher reactivity than ascorbic acid.     

Stopped-flow ESR study on the reactivity of vitamin E, vitamin C and its lipophilic derivatives towards Fremy's salt in micellar systems

The reaction between Fremy's salt and alpha-tocopherol (VE), ascorbic acid (VC) and its lipophilic derivatives ascorbyl-6-caprylate (VC-8), 6-laurate (VC-12) and 6-palmitate (VC-16) were studied by stopped-flow ESR spectroscopy in cetyl trimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) micelles, as a model reaction of these antioxidants with alkyl peroxy radicals in biological systems. The second order rate constants for the reaction of Fremy's salt with VE in CTAB and SDS micelles were found to be 7.9 x 10(3) and 2.2 M-1 s-1, respectively, with as high as a 3600-fold variation. Rate constants for VC, VC-8, VC-12 and VC-16 are 4.3, 35, 53 and 56 x 10(3) M-1 s-1 and 3.3, 2.7, 1.2 and 0.86 x 10(3) M-1 s-1 in CTAB and SDS micelles, respectively. The results demonstrate remarkable effects of the charge type of the micelles and the side-chain of the antioxidants on the antioxidation reactivity in the micelles. It reveals that the inter-micellar diffusion may be the rate-limiting step for antioxidation carried out in micelles.     

Inhibition of oxidation of methyl linoleate in solution by vitamin E and vitamin C

The oxidation of methyl linoleate in solution initiated with azo compounds has been studied in the absence and presence of vitamin E and vitamin C. Both vitamin E and vitamin C acted as a chain-breaking antioxidant and they suppressed the oxidation and produced an induction period. The inhibition rate constant for the scavenging of peroxy radical was calculated at 37 degrees C as kinh = 5.1 X 10(5) M-1 s-1 and 7.5 X 10(4) M-1 s-1 for vitamin E and vitamin C, respectively. It was suggested that each vitamin E could trap two peroxy radicals, whereas vitamin C could trap only one peroxy radical under the reaction conditions employed in this study. When both vitamin E and vitamin C were present, the oxidation was suppressed quite efficiently and the apparent inhibition rate constant was obtained as kinh = 4.0 X 10(5) M-1 s-1. Furthermore, vitamin E remained almost unchanged and only vitamin C was consumed at the initial stage and vitamin E was consumed after vitamin C was exhausted. It was concluded that vitamin E trapped the peroxy radical and the resulting alpha-chromanoxy radical reacted with vitamin C to regenerate vitamin E.     

Kinetics of the reaction by which natural vitamin E is regenerated by vitamin C

The rate constant and activation energy of the regeneration reaction of natural vitamin E by vitamin C were determined with a double-mixing stopped-flow spectrophotometer. The formation of vitamin C radical was observed in the absorption spectrum. The kinetic effect of methyl substitution on the aromatic ring of vitamin E radical indicates that partial charge-transfer plays a role in the reaction. Since a substantial deuterium kinetic isotope effect was not found, the tunneling effect may not play an important role under the present experimental conditions.     

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.     

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.     

Stopped-flow kinetic analysis of the bacterial luciferase reaction.

The kinetics of the reaction catalyzed by bacterial luciferase have been measured by stopped-flow spectrophotometry at pH 7 and 25 degrees C. Luciferase catalyzes the formation of visible light, FMN, and a carboxylic acid from FMNH2, O2, and the corresponding aldehyde. The time courses for the formation and decay of the various intermediates have been followed by monitoring the absorbance changes at 380 and 445 nm along with the emission of visible light using n-decanal as the alkyl aldehyde. The synthesis of the 4a-hydroperoxyflavin intermediate (FMNOOH) was monitored at 380 nm after various concentrations of luciferase, O2, and FMNH2 were mixed. The second-order rate constant for the formation of FMNOOH from the luciferase-FMNH2 complex was found to be 2.4 x 10(6) M-1 s-1. In the absence of n-decanal, this complex decays to FMN and H2O2 with a rate constant of 0.10 s-1. The enzyme-FMNH2 complex was found to isomerize prior to reaction with oxygen. The production of visible light reaches a maximum intensity within 1 s and then decays exponentially over the next 10 s. The formation of FMN from the intermediate pseudobase (FMNOH) was monitored at 445 nm. This step of the reaction mechanism was inhibited by high levels of n-decanal which indicated that a dead-end luciferase-FMNOH-decanal could form. The time courses for these optical changes have been incorporated into a comprehensive kinetic model. Estimates for 15 individual rate constants have been obtained for this model by numeric simulations of the various time courses.     

Effect of simultaneous administration of vitamin C, L-cysteine and vitamin E on the melanogenesis

The effect of simultaneous administration of vitamin C (ascorbic acid), L-cystein (Cys) and vitamin E (tocopherol) on the melanogenesis in vivo and in vitro was studied. Forty-eight brownish guinea pigs were divided into 4 groups as follows: VC group, VC+Cys group, VC+Cys+VE group and control group. They were given these vitamins by oral administration every day. UV-B exposure (0.384 J/cm2) on their depleted back skin was done at the day 8, 10, 12, 15 17 and 19. After UV-B irradiation, vitamins were administrated further 3 weeks. The luminosity score was measured using a Color Reader CR-11 (Minolta, Co) and the numbers of DOPA-positive melanocytes of their back skin were counted. B16 melanoma cells were incubated with VC, N-acetyl cystein (NAC) and VE. After 4 days of incubation, cells were harvested. The melanin contents and the tyrosinase activities in cells were measured. The luminosity score in the VC+VE+Cys group was higher than those in the other groups. The numbers of DOPA-positive melanocytes of guinea pigs treated with VC, VE and Cys were significantly decreased compared with those in VC group. In B16 melanoma cells, simultaneous treatment of VC, VE and NAC was the most effective to decrease the melanin contents and to inhibit tyrosinase activity.     

Vitamin E radical reaction with antioxidants in rat liver membranes

The Japanese herbal medicine Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ-960) has been demonstrated to have an antioxidant action by quenching free radicals. The effects of TJ-960 on the tocopheroxy radicals generated by an arachidonic acid and lipoxygenase oxidation system were compared with those of the ascorbate and glutathione in vitamin E-enriched rat liver microsomes and submitochondrial membrane particles (SMP). Using electron spin resonance spectrometry, the disappearance of the tocopheroxy radicals after addition of glutathione and ascorbate was detected in microsomes and SMP, withh ascorbate displaying a more potent action than glutathione. Addition of TJ-960 demonstrated a similar effect on the tocopheroxy radicals in microsomes and SMP. In the presence of TJ-960, ascorbate, and glutathione, the loss of vitamin E in the vitamin E-enriched microsomes of rat liver undergoing oxidation was slowed down. In this paper, we introduced TJ-960 as another replenisher of vitamin E in membrane, increasing the membrane's resistance against oxidative damage.     

Perturbations in cerebral oxygen radical formation and membrane order following vitamin E deficiency

The effects of dietary vitamin E deficiency on mouse cerebral membrane order and oxygen reactive species were studied. Quantitation of vitamin E levels in several brain regions showed greatest deficiencies in striatum and cerebellum, followed by substantia nigra, and cortex. Vitamin E deficiency increased central-core membrane order in cerebral P2 fraction, but was without effect in the superficial hydrophilic membrane domain. Oxygen radical formation was studied using the probe 2',7'-dichlorofluorescein diacetate. Basal generation rates of oxygen reactive species were 2.5-fold higher when compared to control animals. While hepatic levels of vitamin E are much more reduced than brain levels, in deficient mice, the rate of oxygen radical formation in the liver was unaltered. This implies an special susceptibility of the brain to deficiency of this lipophilic antioxidant vitamin. Data demonstrate that endogenous levels of free radical scavengers, such as vitamin E, may play an important role in maintaining basal oxygen radical levels and membrane integrity. The dietary vitamin E depletion paradigm suggests that a relation exists between elevated levels of oxygen radicals and more rigid hydrophobic central-cores in cerebral membranes, effects that may play a role in mechanisms underlying the neuropathologic lesions observed following vitamin E deficiency.