Antioxidative activity of hydroxylamines. ESR spectra of radicals derived from hydroxylamines.

The antioxidative activity of hydroxylamines was evaluated for the oxidation of tetralin at 61 degrees C and linoleic acid micelles in an aqueous dispersion at 37 degrees C, induced by an azo initiator. The antioxidative efficacy of the hydroxylamines for the oxidation of tetralin was smaller than that of alpha-tocopherol. However, the hydroxylamines showed more potent antioxidative activity than that of the alpha-tocopherol against the oxidation of linoleic acid micelles. On the basis of the results of an ESR study and the oxidation product obtained, it is suggested that active position in hydroxylamines depend not only on hydroxyl hydrogen-atom, but also on the allylic hydrogen atom.     

Oxidative stress,growth factor production and budding in potato tubers during cold storage

In order to verify the role played by oxidation in the budding of potato tubers (Solanum tuberosum L. cv. Kennebec), the physiological events occurring below bud at 4°C have been studied for a period of 6 months. The low temperature storage induced an increase in the degree of unsaturation and a decrease in the ratio of saturated/unsaturated fatty acids of membrane polar lipids with a subsequent increase of lipid hydroperoxides (LOOH). Cold stress increased both enzymatic antioxidative activities (superoxide dismutase, SOD, E.C.1.15.1.1; catalase, CAT, E.C. 1.11.1.6), and α-tocopherol levels thus protecting membrane's polyunsaturated lipids. Between 0 and 15 days of storage SOD/CAT ratio, α-tocopherol, LOOH levels and the degree of lipid unsaturation showed strong variations. After 30 to 120/150 days the antioxidative system seemed to reach a homeostasis different from that of time 0, accompanied by a constant increase of indole-3-acetic acid (IAA) after 60 days. The antioxidative system, after 150 days, lost its efficiency while LOOH levels were maintained higher than time 0 and IAA concentration was sufficient to allow sprouting.     

Microencapsulation of Linoleic Acid with Low- and High-molecular-weight Components of Soluble Soybean Polysaccharide and Its Oxidation Process

Soluble soybean polysaccharide (SSPS) was fractionated into its low- (LMW) and high-molecular-weight (HMW) components to test their antioxidative and emulsifying properties. Linoleic acid was emulsified with an aqueous solution of SSPS, HMW, a mixture of LMW or HMW with maltodextrin, or maltodextrin alone. The emulsions prepared with SSPS, HWM and the mixture of HMW with maltodextrin were stable. These emulsions were spay-dried to produce microcapsules. The encapsulated linoleic acid was oxidized at 37°C and at various levels of relative humidity. Linoleic acid encapsulated with the mixture of LMW with maltodextrin or HMW was stable to oxidation, and this stability increased as the weight fraction of LMW in the mixture was increased. The LMW components also had high DPPH-radical scavenging activity. These results indicate that LMW played an important role in suppressing or retarding the oxidation of linoleic acid encapsulated with SSPS. The oxidative stability of linoleic acid encapsulated with a mixture of the LMW and HMW components was high at low and high relative humidity, but not at intermediate levels of relative humidity.     

Oxidation of hydroxylamines to nitroxide spin labels in living cells

In the presence of oxygen, cells can oxidize hydroxylamines, which are the products of the reduction of nitroxides in cells, back to nitroxides. Lipid-soluble hydroxylamines are oxidized much more rapidly than water-soluble ones, and most of this oxidation is inactivated by heat or trichloroacetic acid, indicating that the principal mechanism is enzyme-linked. The rates of oxidation of some lipophilic hydroxylamines are comparable to the rates of reduction of the corresponding nitroxides. Hydroxylamines formed by reduction of aqueous soluble nitroxides are not oxidized by cells, except for slight oxidation of some pyrrolidine derivatives. The latter is due to autoxidation. The kinetics of oxidation of reduced lipid-soluble nitroxides are all first-order with respect to hydroxylamines, regardless of the position of the nitroxide group along the carbon backbone, indicating that the oxidation occurs within the membrane. The oxidation of hydroxylamines in cells in inhibited by cyanide but not by antimycin A or SKF-525A. We also describe an effective method to oxidize hydroxylamines and follow this reaction; the method is based on the use of perdeuterated [15N]Tempone.     

Effects of phospholipids on the antioxidant activity of α-tocopherol in the singlet oxygen oxidation of canola oil

This study evaluated the effects of phosphatidylcholine (PC) or phosphatidylethanolamine (PE) on the antioxidative activity of α-tocopherol during oxidation of canola oil by singlet oxygen at 10°C for seven hours. Singlet oxygen was produced by chlorophyll b (4 ppm) under 1,700 lux. The oxidation of oil was evaluated by headspace oxygen consumption by gas chromatography and peroxide values (POVs). Concentrations of PC, PE, chlorophyll, and α-tocopherol were determined by HPLC. PC and PE protected chlorophyll from degradation, but they accelerated the degradation of α-tocopherol under singlet oxygen. Contents of PC and PE did not change for seven hours under singlet oxygen. α-Tocopherol significantly lowered POV and headspace oxygen consumption of canola oil under singlet oxygen, and its antioxidant activity was increased by the co-presence of PC and PE. PC and PE increased chemical quenching of singlet oxygen by tocopherol in decreasing the oil oxidation.     

Measurement of Binding of Ascorbic Acid to Myrosinase by Rate of Dialysis

The antioxidative activities of l-amino acids on the methylene blue sensitized photooxidation of linoleic acid (LA) were studied in a 40% aqueous ethanol solution at pH 7.0 and 33°C. His and CysH inhibited the photosensitized oxidation of LA, and the antioxidative activity of His was larger than that of CysH. The ratio of the chemical reaction rate constant for His and singlet oxygen (¹O₂) to that for LA and ¹O₂ was similar to the ratio of the ¹O₂ quenching rate constant for His to that for LA. This fact shows that His inhibits the photosensitized oxidation of LA by quenching ¹0₂ chemically.

On the other hand, CysH reacted with linoleic acid hydroperoxides (LAHPO) or with and caused the decomposition of LAHPO or the formation of Cys. This fact suggests that CysH inhibits the photosensitized oxidation of LA by decomposing LAHPO or by quenching ¹O₂ chemically.     

The influence of antioxidants in the thiyl radical induced lipid peroxidation and geometrical isomerization in micelles of linoleic acid

Abstract

The biomimetic model of micelles of linoleic acid containing 2-mercaptoethanol and the antioxidant was examined under gamma irradiation up to 400?Gy in aerobic or deoxygenated conditions where thiyl radicals are the main reactive species. Lipid peroxidation was retarded by ascorbic acid and α-tocopherol, whereas this process was strongly inhibited by resveratrol as effectively as the ascorbic acid/α-tocopherol mixture. Furthermore, antioxidants have a much stronger inhibitory effect on the peroxidation in the presence of 2-mercaptoethanol, and at the same time show protective properties of the double bond, decreasing the cis–trans isomerization. Under anaerobic conditions, cis–trans isomerization occurred and antioxidants efficiency increased along the series: resveratrol < α-tocopherol?<?ascorbic acid. This result is explained taking into account the double bond localization in the hydrophobic core of the micelle and the need of co-localization of the antioxidant in order to get an anti-isomerizing activity and protection of the natural lipid geometry.     

Quantitative Relationship between Alphα-tocopherol and Polyunsaturated Fatty Acids and Its Connection to Development of Oxidative Rancidity in Chicken Skeletal Muscle

Quantitative relationships were investigated between α-tocopherol and either polyunsaturated fatty acids (PUFA) or PUFA > 18:2 (PUFA with three or more double bonds) in chicken dark meat (thigh muscle) and light meat (M. pectoralis profundus). Their effects on the development of oxidative rancidity in precooked meats held at 5°C for 3 days were also investigated. Chicken dark meat had higher concentrations of α-tocopherol (μmol) per gram of PUFA or PUFA > 18 :2 than did chicken light meat. 2-Thiobarbituric acid (TBA) values for the cooked ground meats held at 5°C for 3 days tended to increase at both higher and lower concentrations of α-tocopherol than the concentration of about 1.5 μmol of α-tocopherol per gram of PUFA regardless of the type of chicken skeletal muscle.     

Cellular metabolism of proxyl nitroxides and hydroxylamines

Previous data from model systems indicated that the proxyl nitroxides should be especially resistant to bioreduction and therefore could be an effective solution to this often problematic characteristic of nitroxides. Therefore, we investigated the rate of reduction by cells and by the usual model system, ascorbate, of four proxyl nitroxides and three reference nitroxides. We found that, while the rate of reduction by ascorbate of the proxyl nitroxides was slower than the rate of a prototypic pyrrolidine nitroxide (PCA), the reverse was true for reduction by cells. We also studied the rate of oxidation of the corresponding hydroxylamines. The rate of oxidation by cells of the proxyl hydroxylamines was relatively fast, especially for the most lipophilic derivative. These results indicate that: (i) proxyl nitroxides may not be unusually resistant to bioreduction by functional biological systems; (ii) accurate knowledge of relative rates of metabolism of nitroxides and hydroxylamines in cells and tissues will require direct studies in these systems because the rates may not closely parallel those observed in model (chemical) systems; and (iii) proxyl nitroxides show potential value as agents to measure oxygen concentrations by the rates of oxidation of their corresponding hydroxylamines.     

Effects of α-tocopherol analogs on lysosome membranes and fatty acid monolayers

The surface pressures of α-tocopherol analogs, fatty acids, and their mixtures were measured in their spread monolayers at an air—water interface. The surface pressure—area isotherms for the mixed monolayers of α-tocopherol and either stearic acid, oleic acid or linoleic acid deviated positively from those calculated on the basis of the additivity rule, and the magnitude depended on the length of the phytyl side chain in α-tocopherol and on the degree of unsaturation of the fatty acid chains. Lysosome membranes of mouse liver were stabilized by addition of α-tocopherol. A decrease in the length of the phytyl side chain in α-tocopherol reduced its ability to stabilize lysosome membranes. A good correlation was obtained between the extent of stabilizing activity of α-tocopherol analogs on lysosome membranes and the degree of positive deviation of the surface pressure for their mixtures with fatty acids.     

Fused heterocyclic antioxidants: antioxidative activities of hydrocoumarins in a homogeneous solution.

We compared the antioxidative activities of seven hydrocoumarins with those of alpha-tocopherol for the oxidation of tetralin and linoleic acid in a homogeneous solution. Hydrocoumarins exhibited a higher induction period than that of alpha-Toc in both systems. However, the rate of oxygen absorption during the induction period for alpha-Toc was slower than that of the hydrocoumarins in both systems. In addition, 6,7-dihydroxy-4,4-dimethylhydrocoumarin showed less cytotoxicity toward human fibroblasts than did 2,6-di-t-butyl-4-methylphenol.     

α-Tocopherol Application Modulates the Response of Wheat (Triticum aestivum L.) Seedlings to Elevated Temperatures by Mitigation of Stress Injury and Enhancement of Antioxidants

Wheat seedlings (4 days old) were subjected to varying temperatures of 25, 30, and 35 °C for 7 days in a growth chamber under hydroponic conditions in the absence or presence of α-tocopherol (5 μM). The growth of shoots and roots was inhibited severely at 35 °C. The endogenous α-tocopherol increased in the shoots at 30 °C over the controls but decreased significantly at 35 °C over the previous temperature. The exogenous application of α-tocopherol elevated the endogenous levels in the heat-stressed plants, which were consequently able to maintain significantly greater growth associated with reduction in damage to membranes, cellular oxidizing ability, chlorophyll content, and photochemical efficiency in shoots. The relative leaf water content and stomatal conductance were not affected significantly with the application of tocopherol. The oxidative stress induced by high temperature (35 °C) in terms of malondialdehyde and hydrogen peroxide contents was significantly lower in the presence of α-tocopherol. The enzymatic antioxidants such as superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase showed considerable reduction in their activities at 35 °C compared to those at 30 °C, with greater effects on APX and GR. The nonenzymatic antioxidants like ascorbate, glutathione, and proline increased at 30 °C but decreased appreciably at 35 °C, suggesting impairment in their synthesis at stressful temperatures. α-Tocopherol-treated plants, especially those growing at 35 °C, had improved levels of enzymatic and nonenzymatic antioxidants. These observations provided evidence about the involvement of α-tocopherol in governing heat sensitivity in wheat and suggested manipulation of its endogenous levels to induce heat tolerance in this crop.     

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.     

The influence of vitamin E on membrane lipids of mouse fibroblasts in culture

A tissue culture system, in which the composition of the medium, with respect to vitamin E, linoleic acid, and cholesterol, can be manipulated at will, was used to study the effect of vitamin E on the fatty acid profiles of fibroblast membrane phospholipids. The effect was studied of α-tocopherol, and of butylated hydroxytoluene, on the uptake of isotopically labeled linoleic acid and cholesterol, and of the effect of these antioxidants on the metabolic interconversion of linoleic acid with other unsaturated fatty acids. Butylated hydroxytoluene was without effect on any of the parameters measured. α-Tocopherol caused a large enhancement in the content and radioactivity of the arachidonyl residues of phosphatidyl choline, phosphatidyl serine, and phosphatidyl ethanolamine, generally at the expense of linoleic acid in the same phospholipids. There was no effect of α-tocopherol on the unsaturated fatty acids of the neutral lipids, suggesting that there was no general effect on the chain elongation and desaturation of linoleic acid. The results are thought to demonstrate a specific effect of α-tocopherol upon the architecture of membrane phospholipids by controlling the profiles of their unsaturated fatty acid components. The uptake of radioactive cholesterol, and the content of cholesterol and cholesterylesters in cultured cells was also significantly increased by the presence of α-tocopherol in the medium. Possible reasons for these phenomena are discussed in the light of present knowledge of the biological function of vitamin E.     

Antioxidative effect of α-tocopherol incorporation into lecithin liposomes on ascorbic acid-Fe2+-induced lipid peroxidation

The antioxidative effect of α-tocopherol incorporated into lecithin liposomes was studied. Lipid peroxidation of liposome membranes, assayed as malondialdehyde production, was catalyzed by ascorbic acid and Fe²⁺. The peroxidation reaction, which did not involve the formation of singlet oxygen, superoxide, hydrogen peroxide, or a hydroxyl radical, was inhibited by α-tocopherol and a model compound of α-tocopherol, 2,2,5,7,8-pentamethyl-6-hydroxy-chroman (TMC), but not by phytol, α-tocopherylquinone, or α-tocopheryl acetate. One mole of α-tocopherol completely prevented peroxidation of about 100 moles of polyunsaturated fatty acid. Decrease in membrane fluidity by lipid peroxidation, estimated as increase of fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) embedded in the membrane, was also inhibited by α-tocopherol and TMC, reflecting their antioxidant functions. Cholesterol did not act as an antioxidant, even when incorporated in large amount into the liposome membranes, but it increased the antioxidative efficiency of α-tocopherol. When a mixture of liposomes with and without α-tocopherol was incubated with Fe²⁺ and ascorbic acid, α-tocopherol did not protect the liposomes not containing α-tocopherol from peroxidation. However, preincubation of the mixture, or addition of Triton X-100 allowed the α-tocopherol to prevent peroxidation of the liposomes not containing α-tocopherol. In contrast, in similar experiments, liposomes containing TMC prevented peroxidation of those without TMC without preincubation. Tocopherol in an amount so small as to exhibit only a slight antioxidative effect was oxidized when incorporated in egg lecithin liposomes, but it mostly remained unoxidized when incorporated in dipalmitoyllecithin liposomes, indicating that oxygen activated by ascorbic acid-Fe²⁺ does not oxidize α-tocopherol directly. Thus, decomposition of α-tocopherol may be caused by its interaction with peroxy and/or alkoxyl radicals generated in the process of lipid peroxidation catalyzed by Fe²⁺ and ascorbic acid.     

Effect of salicylic and abscisic acid administered through detached tillers on antioxidant system in developing wheat grains under heat stress

The mechanism imparting thermotolerance by salicylic acid (SA) and abscisic acid (ABA) is still unresolved using either spraying technique or in vitro conditions. Alternative way of studying these effects under near in vivo conditions is through the use of liquid culturing technique. Effects of SA and ABA (100 μM) on antioxidative enzymes, antioxidants and lipid peroxidation were studied in detached tillers of three wheat (Triticum aestivum L.) cultivars PBW 343, C 306 (heat tolerant) and WH 542 (heat susceptible) cultured in a liquid medium. Ears were subjected to heat shock treatment (45°C for 2 h) and then maintained at 25°C for 5 days. Heat shock treatment resulted in increased peroxidase (POD) activity, while superoxide dismutase (SOD) and catalase (CAT) activities were reduced compared to control. The decrease in CAT activity was more significant in susceptible cultivar WH 542. Concomitantly, content of α-tocopherol and lipid peroxides increased in heat-treated wheat ears, whereas contents of total ascorbate level were reduced. Following treatment with SA and ABA, activities of all three antioxidative enzymes increased in correspondence with an increase in ascorbate and α-tocopherol content. Apparently, lipid peroxide content was reduced by SA in heat tolerant cultivars (PBW 343 and C 306) whereas in susceptible cultivar it was decreased by ABA. The up-regulation of the antioxidant system by SA and ABA possibly contributes to better tolerance against heat shock-induced oxidative damage in wheat grains.     

Antioxidant activity of 3-methyl-1-phenyl-2-pyrazolin-5-one

Summary

The antioxidant activity of an anti-ischemic agent, 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), was examined. The pKa value of MCI-186 is 7.0 and the rate of oxidation of MCI-186 initiated with an azo compound increased with increasing pH, suggesting that the anionic form of MCI-186 is much more reactive than the non-ionic form. The major products were 3-methyl-1-phenyl-2-pyrazolin-4,5-dione (4,5-dione) and 2-oxo-3-(phenylhydrazono)-butanoic acid (OPB). Hydrolysis of 4,5-dione gave OPB. The minor intermediate product was 4-hydroxy-4-(3-methyl-1-phenyl-1H-pyrazolin-5-on-4-yl)-3-methyl-1-phenyl-1H-pyrazolin-5-one (BPOH). The nucleophilic attack of the anionic form of MCI-186 to 4,5-dione is likely to give BPOH. MCI-186 (50 μM) inhibited the aerobic oxidation at 37°C of 5.2 mM unilamellar soybean phosphatidylcholine (PC) liposomal membranes, initiated with a water-soluble initiator, as efficientlyas did ascorbate (100 μM). MCI-186 (50 μM) also inhibited the oxidation of the same PC liposomal membranes, this time initiated with a lipid-soluble initiator, almost as efficiently as did α-tocopherol (2 μM). Furthermore, the combination of MCI-186 with ascorbate or α-tocopherol showed almost complete inhibition of PC oxidation induced by both initiators. These data suggest that MCI-186 may work as a good antioxidant in cellular systems as well as in cell-free systems.     

Effects of ascorbic acid,alpha-tocopherol,and glutathione on microspore embryogenesis in Brassica napus L.

The impact of culture conditions and addition of antioxidants to media on microspore embryogenesis in rapeseed (Brassica napus cv. ‘PF704’) was investigated. Different concentrations of ascorbic acid (0, 5, 10, 20, 50, 100, and 200 mg l^?1) and alpha (α)-tocopherol (0, 5, 10, 20, 50, 100, and 200 mg l^?1) were evaluated along with two temperature pretreatments (18 d at 30°C; 2 d at 32.5°C followed by 16 d at 30°C). In addition, combinations of reduced glutathione (0, 10, 50, and 100 mg l^?1) and ascorbic acid (5 and 10 mg l^?1) were tested. Microspore embryogenesis was significantly enhanced using 10 mg l^?1 ascorbic acid (334 embryos per Petri dish) compared with untreated cultures (184 embryos per Petri dish) at 30°C. α-Tocopherol (5 and 10 mg l^?1) enhanced (312 and 314 embryos per Petri dish, respectively) microspore embryogenesis relative to untreated cultures (213 embryos per Petri dish) at 30°C, although there were no significant differences among cultures treated with 5–50 mg l^?1 α-tocopherol. When 50 mg l^?1 α-tocopherol was combined with 5 or 10 mg l^?1 ascorbic acid, embryogenesis was significantly enhanced (308 and 328 embryos per Petri dish, respectively) relative to other ascorbic acid levels. Moreover, 10 mg l^?1 of reduced glutathione and 5 mg l^?l ascorbic acid enhanced microspore embryogenesis (335 embryos per Petri dish) compared to cultures without reduced glutathione (275 embryos per Petri dish). Microspore embryogenesis could be improved by adding ascorbic acid, α-tocopherol, and reduced glutathione when the appropriate combination and temperature pretreatment were selected.     

Antioxidant Effect of Tocopherols on Autoxidation of Milk Fat

Antioxidant activity of d-α-, dl-β-, d-γ- and d-δ-tocopherol was investigated with fatty acid methylester of milk fat from which unsaponifiable matter had been removed. Autoxidation was carried out at 50°C and its degree was indicated by peroxide value, α- or β-Tocopherol was more effective at lower concentrations (0.003 and 0.01%) than at higher concentrations (0.05, 0.1 and 0.5%). The antioxidant activity of γ- and δ-tocopherol was increased with the increase of tocopherol concentration within the range of 0.001 to 0.5%. The order of antioxidant activity of these tocopherols, which was compared in terms of the time to reach 30 meq of peroxide value, varied with the concentration; γ > β > δ > α at 0.001%, α > γ > β > δ at 0.003%, γ > δ > β > α at 0.01%, and δ > γ > β > α at the concentrations more than 0.05%. α-Tocopherol at the concentration of 0.003%, which corresponded to the concentration in original milk fat, was more effective than other tocopherols at the same concentration and α-tocopherol at other concentrations. Synergism due to the combination of β-, γ-, or δ-tocopherol with 0.003% of α-tocopherol was not observed.     

Airborne limonene confers limited thermotolerance to Quercus ilex

The purpose of the study was to test the possible and controversial thermotolerance role of monoterpene production and emission and the related responses of antioxidants. Quercus ilex seedlings were exposed to a ramp of temperatures of 5°C steps from 25 to 50°C growing with and without limonene fumigation (7.5 µl l⁻¹). Net photosynthetic rates, maximal photochemical efficiency of PSII (F_v/F_m), oxidation state of ascorbic acid, and lipid peroxidation estimated by malondialdehyde concentrations of limonene-fumigated (LF) plants did not significantly differ from control (C) plants. No consistent changes in emissions of the other monoterpenes, α-pinene, β-phellandrene, β-pinene or β-myrcene were found. However, slight differences were found in the concentration of antioxidants. The amounts of α-tocopherol did not change or even tended to decrease at high temperatures in LF plants whereas they tended to increase by approximately 60% at 45 and 50°C relative to 25°C in C plants. Ascorbic acid reached its maximum concentration only at 45°C in LF plants whereas it reached its maximum at 35°C in C plants. β-Carotene did not decrease at high temperatures in LF plants whereas it decreased by approximately 15% at 45–50°C in C plants. Brown pigment index (BPI), an optical indicator of tissue oxidative processes, was lower in LF plants than in C plants. The photochemical reflectance index (PRI), an optical indicator of photosynthetic light use efficiency, was higher for LF plants than for C plants at elevated temperatures. Visual leaf damage (browning) tended to be less in LF plants than in C plans although not significantly (26.5 ± 8.5 versus 16.2 ± 4.8%). These results show that limonene does not confer clear and strong thermotolerance but might have some minor role. These results are in agreement with previous indications of weaker thermotolerance effect of monoterpenes than of isoprene.