Antioxidant defenses in caterpillars: role of the ascorbate-recycling system in the midgut lumen

This study demonstrates that an ascorbate-recycling system in the midgut lumen can act as an effective antioxidant defense in caterpillars that feed on prooxidant-rich foods. In tannin-sensitive larvae of the forest tent caterpillar, Malacosoma disstria (Lasiocampidae), ingested tannic acid is oxidized in the midgut lumen, generating significant quantities of peroxides, including hydrogen peroxide, which readily diffuses across cell membranes and is a powerful cytotoxin. By contrast, in the tannin-tolerant larvae of the white-marked tussock moth, Orgyia leucostigma (Lymantriidae), tannic acid oxidation and the generation of peroxides are suppressed. The superior defense of O. leucostigma against oxidative stress imposed by the oxidation of ingested polyphenols can be explained by the presence of higher concentrations of ascorbate and glutathione in the midgut lumen. In O. leucostigma at least 50% of the ingested ascorbate present in the anterior midgut is still present in the posterior midgut, whereas in M. disstria, only 10% of the ascorbate is present in the posterior half of the midgut. We propose that the maintenance of higher levels of ascorbate in the midgut lumen of O. leucostigma than in M. disstria is explained by the secretion of glutathione into the midgut lumen by O. leucostigma, thereby forming a complete ascorbate-recycling system. The concentration of glutathione in the midgut lumen of O. leucostigma is 3.5-fold higher than in M. disstria and more than double the concentration in the diet. Our results emphasize the importance of a defensive strategy in herbivorous insects based on the maintenance of conditions in the gut lumen that reduce or eliminate the potential prooxidant behavior of ingested phenols.     

Fenton-type reactions and iron concentrations in the midgut fluids of tree-feeding caterpillars

Peroxides are formed in the midgut fluids of caterpillars when ingested tannins and other phenolic compounds oxidize. If these peroxides broke down in the presence of redox-active metal ions, they would form damaging free radicals (Fenton-type reactions). Elemental iron is present in relatively large amounts in leaves and artificial diets, but little is known about its concentration and redox state in midgut fluids, or the extent of Fenton-type reactions in these conditions. This study compared the levels of hydroxyl radicals and iron in the midgut fluids of two species of caterpillars: Orgyia leucostigma, in which phenol oxidation is limited, and Malacosoma disstria, in which phenol oxidation is more extensive. We tested two hypotheses: (1) higher levels of hydroxyl radicals are formed in M. disstria (consistent with the higher concentrations of hydrogen peroxide in this species), and (2) lower concentrations of iron are present in O. leucostigma (providing greater protection of its midgut fluids from oxidative damage). Hydroxyl radical levels increased greatly in M. disstria, but not in O. leucostigma, when they consumed a tannin-containing diet, supporting the first hypothesis. Protein oxidation was also significantly increased in the midgut fluids of M. disstria that ingested tannic acid, consistent with hydroxyl radical damage. Contrary to the second hypothesis, similar concentrations of iron (70 microM) remained in solution or suspension in both species of caterpillars on an artificial diet. Over 90% of this iron appeared to be in the reduced (catalytically active) state in both species. We conclude that tree-feeding caterpillars protect their midgut fluids from oxidative damage caused by Fenton-type reactions by limiting the formation of peroxides, rather than by limiting the availability of reduced iron.     

Hydrolyzable tannins as “quantitative defenses”: Limited impact against Lymantria dispar caterpillars on hybrid poplar

The high levels of tannins in many tree leaves are believed to cause decreased insect performance, but few controlled studies have been done. This study tested the hypothesis that higher foliar tannin levels produce higher concentrations of semiquinone radicals (from tannin oxidation) in caterpillar midguts, and that elevated levels of radicals are associated with increased oxidative stress in midgut tissues and decreased larval performance. The tannin-free leaves of hybrid poplar (Populus tremula × P. alba) were treated with hydrolyzable tannins, producing concentrations of 0%, 7.5% or 15% dry weight, and fed to Lymantria dispar caterpillars. As expected, larvae that ingested control leaves contained no measurable semiquinone radicals in the midgut, those that ingested 7.5% hydrolyzable tannin contained low levels of semiquinone radicals, and those that ingested 15% tannin contained greatly increased levels of semiquinone radicals. Ingested hydrolyzable tannins were also partially hydrolyzed in the midgut. However, increased levels of semiquinone radicals in the midgut were not associated with oxidative stress in midgut tissues. Instead, it appears that tannin consumption was associated with increased metabolic costs, as measured by the decreased efficiency of conversion of digested matter to body mass (ECD). Decreased ECD, in turn, decreased the overall efficiency of conversion of ingested matter to body mass (ECI). Contrary to our hypothesis, L. dispar larvae were able to maintain similar growth rates across all tannin treatment levels, in part, because of compensatory feeding. We conclude that hydrolyzable tannins act as “quantitative defenses” in the sense that high levels appear to be necessary to increase levels of semiquinone radicals in the midguts of caterpillars. However, these putative resistance factors are not sufficient to decrease the performance of tannin-tolerant caterpillars such as L. dispar.     

Oxygen levels in the gut lumens of herbivorous insects

Oxygen levels were measured in the foregut and midgut lumens of ten species of caterpillars and three species of grasshoppers. In most species, the foregut was nearly anoxic, with oxygen levels ranging from 0 to 2.5 mm Hg. However, two caterpillar species with large foreguts (Malacosoma disstria and Lymantria dispar) had elevated oxygen levels (27.9 and 32.1 mm Hg) in this region when they were fed artificial diet. In all of the species surveyed, the anterior and posterior midgut were nearly anoxic, with oxygen levels ranging from 0 to 7.3 mm Hg. Oxygen levels in the midgut lumen of Helicoverpa zea did not differ when caterpillars were fed artificial diet or tomato foliage, suggesting that the insect is capable of reducing the level of ingested oxygen in its gut. An examination of the radial microgradient of oxygen in the gut lumen demonstrated that the midgut epithelium is not a sink for ingested oxygen. However, the midgut contents of larvae fed artificial diet were capable of depleting oxygen. This capacity was reduced by boiling, suggesting that the nearly anoxic state of the midgut lumen in some insects is maintained by endogenous chemical processes. We conclude that low oxygen levels in the gut lumens of most herbivorous insects may greatly reduce the rates of oxidation of ingested plant compounds by oxygen-dependent reactions.     

Antioxidant defense of the midgut epithelium by the peritrophic envelope in caterpillars

The peritrophic envelope (PE) is an extracellular matrix that is secreted by the midgut epithelium in most arthropods. In addition to protecting the midgut epithelium from abrasive food particles and microbial pathogens, in vitro experiments have suggested that the PE functions as a radical-scavenging antioxidant in caterpillars. This study tested the hypothesis that the PE is a "sacrificial antioxidant" in vivo in caterpillars. As a sacrificial antioxidant, the PE would (1) bind catalytic metal ions, (2) become oxidized itself, and (3) protect the midgut epithelium from oxidative damage. Each of these functions was supported by our results: the PE in Malacosoma disstria adsorbed increased amounts of iron as the concentration of iron was increased in its diet. Iron adsorption by the PE helped protect the midgut epithelium of M. disstria from oxidative damage over a wide range of ingested iron concentrations. Secondly, while the midgut epithelium was protected, protein oxidation in the PE increased 108% when tannic acid was oxidized in the endoperitrophic space. Finally, when the formation of the PE was inhibited by Calcofluor, protein carbonyls in the midgut epithelia of M. disstria and Orgyia leucostigma increased by two- to threefold. We conclude that the PE functions as an effective iron-binding and radical-scavenging antioxidant that protects the midgut epithelia of caterpillars.     

Generation and recycling of radicals from phenolic antioxidants

Hindered phenols are widely used food preservatives. Their pharmacological properties are usually attributed to high antioxidant activity due to efficient scavenging of free radicals. Butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) also cause tissue damage. Their toxic effects could be due to the production of phenoxyl radicals. If phenoxyl radicals can be recycled by reductants or electron transport, their potentially harmful side reactions would be minimized. A simple and convenient method to follow phenoxyl radical reactions in liposomes and rat liver microsomes based on an enzymatic (lipoxygenase + linolenic acid) oxidation system was used to generate phenoxyl radicals from BHT and its homologues with substitutents in m- and p-positions. Different BHT-homologues display characteristic ESR signals of their radical species. In a few instances the absence of phenoxyl radical ESR signals was found to be due to inhibition of lipoxygenase by BHT-homologues. In liposome or microsome suspensions addition of ascorbyl palmitate resulted in disappearance of the ESR signal of phenoxyl radicals with concomittant appearance of the ascorbyl radical signal. After exhaustion of ascorbate, the phenoxyl radical signal reappears. Comparison of the rates of ascorbyl radical decay in the presence or absence of BHT-homologues showed that temporary elimination of the phenoxyl radical ESR signal was due to their reduction by ascorbate. Similarly, NADPH or NADH caused temporary elimination of ESR signals as a result of reduction of phenoxyl radicals in microsomes. Since ascorbate and NADPH might generate superoxide in the incubation system used, SOD was tested. SOD shortened the period, during which the phenoxyl radicals ESR signal could not be observed. Both ascorbyl palmitate and NADPH exerted sparing effects on the loss of BHT-homologues during oxidation. These effects were partly diminished by SOD. These data indicate that reduction of phenoxyl radicals was partly superoxide-dependent. It is concluded that redox recycling of phenoxyl radicals can occur by intracellular reductants like ascorbate and microsomal electron transport.     

Redox cycles of caffeic acid, alpha-tocopherol, and ascorbate: implications for protection of low-density lipoproteins against oxidation

This study addresses the dynamic interactions among alpha-tocopherol, caffeic acid, and ascorbate in terms of a sequence of redox cycles aimed at accomplishing optimal synergistic antioxidant protection. Several experimental models were designed to examine these interactions: UV irradiation of alpha-tocopherol-containing sodium dodecyl sulfate micelles, one-electron oxidations catalyzed by the hypervalent state of myoglobin, ferrylmyoglobin, and autoxidation at appropriate pHs. These models were assessed by ultraviolet (UV) and electron paramagnetic resonance (EPR), entailing direct- and continuous-flow experiments, spectroscopy and by separation and identification of products by HPLC. The alpha-tocopheroxyl radical EPR signal generated by UV irradiation of alpha-tocopherol-containing micelles was suppressed by caffeic acid and ascorbate; in the former case, no other EPR signal was observed at pH 7.4, whereas in the latter case, the alpha-tocopheroxyl radical EPR signal was replaced by a doublet EPR spectrum corresponding to the ascorbyl radical (A*-). The potential interactions between caffeic acid and ascorbate were further analyzed by assessing, on the one hand, the ability of ascorbate to reduce the caffeic acid o-semiquinone (generated by oxidation of caffeic acid by ferrylmyoglobin) and, on the other hand, the ability of caffeic acid to reduce ascorbyl radical (generated by autoxidation or oxidation of ascorbate by ferrylmyoglobin). The data presented indicate that the reductive decay of ascorbyl radical (A*-) and caffeic acid o-semiquinone (Caf-O*) can be accomplished by caffeic acid (Caf-OH) and ascorbate (AH-), respectively, thus pointing to the reversibility of the reaction Caf-O* + AH- <--> Caf-OH + A*-. Continuous-flow EPR measurements of mixtures containing ferrylmyoglobin, alpha-tocopherol-containing micelles, caffeic acid, and ascorbate revealed that ascorbate is the ultimate electron donor in the sequence encompassing transfer of the radical character from the micellar phase to the phase. In independent experiments, the effects of caffeic acid and ascorbate on the oxidation of two low-density lipoprotein (LDL) populations, control and alpha-tocopherol-enriched, were studied and results indicated that alpha-tocopherol, caffeic acid, and ascorbate acted synergistically to afford optimal protection of LDL against oxidation. These results are analyzed for each individual antioxidant in terms of three domains: its localization and that of the antioxidant-derived radical, its reduction potential, and the predominant decay pathways for the antioxidant-derived radical, that exert kinetic control on the process.     

Oxidation of DOPAC by nitric oxide: effect of superoxide dismutase

This study aimed to characterize the redox interaction between 3,4-dihydroxyphenylacetic acid (DOPAC) and nitric oxide (.NO), and to assess the reductive and oxidative decay pathways of the DOPAC semiquinone originating from this interaction. The reaction between DOPAC and.NO led to the formation of the DOPAC semiquinone radical, detected by electron paramagnetic resonance (EPR) and stabilized by Mg(2+), and the nitrosyl anion detected as nitrosylmyoglobin. The EPR signal corresponding to the DOPAC semiquinone was modulated as follows: (i) it was suppressed by glutathione and ascorbic acid with the formation of new EPR spectra corresponding to the glutathionyl and ascorbyl radical, respectively; (ii) it was enhanced by Cu,Zn-superoxide dismutase; the enzyme also accelerated the decay of the semiquinone species to DOPAC quinone. These results are interpreted as a one-electron oxidation of DOPAC by.NO; the reductive decay of the semiquinone back to DOPAC was facilitated by reducing agents, such as glutathione and ascorbate, whereas the oxidative decay to DOPAC quinone was facilitated by superoxide dismutase. The latter effect is understood in terms of a reversible conversion of nitrosyl anion to.NO by the enzyme. The biological relevance of these reactions is also discussed in terms of the reactivity of peroxynitrite towards DOPAC as a model with implications for aerobic conditions.     

Spin trapping endogenous radicals in MC-1010 cells: Evidence for hydroxyl radical and carbon-centered ascorbyl radical adducts

Incubation of MC-1010 cells with the spin-trapping agent 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) followed by brief treatment with the solid oxidant lead dioxide (PbO₂) yielded, after filtration, a cell-free solution that contained two nitroxyl adducts. The first was the hydroxyl radical adduct, 5,5-dimethyl-2-hydroxypyrrolidine-1-oxyl (DMPO-OH), which formed immediately upon PbO₂ oxidation. The second had a 6-line EPR spectrum typical of a carbon-centered radical (A_N=15.9 G; A_H=22.4 G) and formed more slowly. No radical signals were detected in the absence of either cells or PbO₂ treatment. The 6-line spectrum could be duplicated in model systems that contained ascorbate, DMPO and DMPO-OH, where the latter was formed from hydroxyl radicals generated by sonolysis or the cleavage of hydrogen peroxide with Fe²⁺ (Fenton reaction). In addition, enrichment of MC-1010 cells with ascorbate prior to spin trapping yielded the 6-line EPR spectrum as the principal adduct following PbO₂ oxidation and filtration. These results suggest that ascorbate reacted with DMPO-OH to form a carbon-centered ascorbyl radical that was subsequently trapped by DMPO. The requirement for mild oxidation to detect the hydroxyl radical adduct suggests that DMPO-OH formed in the cells was reduced to an EPR-silent form (i.e., the hydroxylamine derivative). Alternatively, the hydroxylamine derivative was the species initially formed. The evidence for endogenous hydroxyl radical formation in unstimulated leukocytes may be relevant to the leukemic nature of the MC-1010 cell line. The spin trapping of the ascorbyl radical is the first report of formation of the carbon-centered ascorbyl radical by means other than pulse radiolysis. Unless it is spin trapped, the carbon-centered ascorbyl radical immediately rearranges to the more stable oxygen-centered species that is passive to spin trapping and characterized by the well-known EPR doublet of A_H4=1.8 G.Abbreviation EPR Electron Paramagnetic Resonance     

Pyridoxal isonicotinoyl hydrazone inhibits iron-induced ascorbate oxidation and ascorbyl radical formation

Previous work from our laboratory demonstrated that pyridoxal isonicotinoyl hydrazone (PIH) has in vitro antioxidant activity against iron plus ascorbate-induced 2-deoxyribose degradation due to its ability to chelate iron; the resulting Fe(III)-PIH(2) complex is supposedly unable to catalyze oxyradical formation. A putative step in the antioxidant action of PIH is the inhibition of Fe(III)-mediated ascorbate oxidation, which yields the Fenton reagent Fe(II) [Biochim. Biophys. Acta 1523 (2000) 154]. In this work, we demonstrate that PIH inhibits Fe(III)-EDTA-mediated ascorbate oxidation (measured at 265 nm) and the formation of ascorbyl radical (in electron paramagnetic resonance (EPR) studies). The efficiency of PIH against ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation was dose dependent and directly proportional to the period of preincubation of PIH with Fe(III)-EDTA. The efficiency of PIH in inhibiting ascorbate oxidation and ascorbyl radical formation was also inversely proportional to the Fe(III)-EDTA concentration in the media. When EDTA was replaced by the weaker iron ligand nitrilotriacetic acid (NTA), PIH was much more effective in preventing ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation. Moreover, the replacement of EDTA with citrate, a physiological chelator with a low affinity for iron, also resulted in PIH having a higher efficiency in inhibiting iron-mediated ascorbate oxidation and 2-deoxyribose degradation. These results demonstrate that PIH removes iron from EDTA (or from either NTA or citrate), forming an iron-PIH complex that cannot induce ascorbate oxidation effectively, thus inhibiting iron-mediated oxyradical formation. These results are of pharmacological relevance because PIH has been considered for experimental chelating therapy in iron-overload diseases.     

The oxidation of 2′,7′-dichlorofluorescin to reactive oxygen species: A self-fulfilling prophesy?

The oxidation of 2',7'-dichlorofluorescin (DCFH) and its diacetate form (DCFHDA) by the HRP/peroxynitrite system was investigated. Both DCFH and DCFHDA were oxidized to fluorescent products. A major anomaly, however, was the observation that fluorescence continued to build up long after peroxynitrite total decomposition and the initial HRP compound I reduction, suggesting the production of oxidants by the system. Indeed, preformed HRP compound I was instantly reduced by DCFH and DCFHDA to compound II with the obligate formation of DCF(-) semiquinone and DCFHDA-derived radicals. Catalase strongly inhibited fluorescence and EPR signals, suggesting the intermediate formation of H2O2. Taken together the data indicate that peroxynitrite rapidly oxidizes HRP to HRP compound I, which is reduced by DCFH and its diacetate form with the concomitant formation of DCF(-) semiquinone and DCFHDA-derived radicals. These are oxidized by O2, producing O2(-) (as demonstrated by EPR and oxygen consumption experiments), which dismutates to produce H2O2, which serves to fuel further DCFH/DCFHDA oxidation via HRP catalysis. Also DCFHDA was shown to be considerably more resistant to oxidation than its hydrolyzed product DCFH, presumably because of the absence of the easily oxidizable phenol moieties. DCFHDA/DCFH have been used to study free radical production in a variety of systems. Our findings demonstrate that this assay is subject to a serious artifact in that it produces what it is purported to measure; therefore, its use in biological systems should be approached with caution.     

Photoaccumulation of two ascorbyl free radicals per photosystem I at 200 K

Illumination of photosystem I (PSI) from the cyanobacterium Synechocystis sp. PCC 6803 at 200 K in the presence of ascorbate leads to the formation of two ascorbyl radicals per PSI, which are formed by P700(+) reduction by ascorbate. During photoaccumulation, one half of the ascorbyl radicals is formed with a halftime of 1 min and the other half with a halftime of 7 min. Pulsed electron paramagnetic resonance (EPR) experiments with protonated/deuterated PSI show that a PSI proton/deuteron is strongly coupled to the ascorbyl radical. Our data indicate that reactive ascorbate molecules bind to PSI at two specific locations, which might be symmetrically located with respect to the pseudo-C(2) axis of symmetry of the heterodimeric core of PSI. Reduction of P700(+) by ascorbate leads to multiple turnover of PSI photochemistry, resulting in partial photoaccumulation of the doubly reduced species (F(A)(-), F(B)(-)). A modified form of F(B)(-)-in accordance with Chamorovsky and Cammack [Biochim. Biophys. Acta 679 (1982) 146-155], but not of F(A)(-), is observed by EPR after illumination at 200 K, which indicates that reduction of F(B) at 200 K is followed by some relaxation process, in line with this cluster being the most exposed to the solvent.     

Effect of the microsomal system on interconversions between hydroquinone, benzoquinone, oxygen activation, and lipid peroxidation

Our previous results indicated that cytochrome P450 destruction by benzene metabolites was caused mainly by benzoquinone (Soucek et al., Biochem. Pharmacol. 47 (1994) 2233-2242). The aim of this study was to investigate the interconversions between hydroquinone, semiquinone, and benzoquinone with regard to both spontaneous and enzymatic processes in order to test the above hypothesis. We have also studied the participation of hydroquinone and benzoquinone in OH radicals formation and lipid peroxidation as well as the role of ascorbate and transition metals. In buffered aqueous solution, hydroquinone was slowly oxidized to benzoquinone via a semiquinone radical. This conversion was slowed down by the addition of NADPH and completely stopped by microsomes in the presence of NADPH. Benzoquinone was reduced to semiquinone radical at a significantly higher rate and this conversion was stimulated by NADPH and more effectively by microsomes plus NADPH while semiquinone radical was quenched there. In microsomes with NADPH. both hydroquinone and benzoquinone stimulated the formation of OH radicals but inhibited peroxidation of lipids. Ascorbate at 0.5-5 mM concentration also produced significant generation of OH radicals in microsomes. Neither hydroquinone nor benzoquinone did change this ascorbate effect. On the contrary, 0.1-1.0 mM ascorbate stimulated peroxidation of lipids in microsomes whereas presence of hydroquinone or benzoquinone completely inhibited this deleterious effect of ascorbate. Iron-Fe2+ apparently played an important role in lipid peroxidation as shown by EDTA inhibition, but it did not influence OH radical production. In contrast, Fe3+ did not influence lipid peroxidation, but stimulated OH radical production. Thus, our results indicate that iron influenced the above processes depending on its oxidation state, but it did not influence hydroquinone/benzoquinone redox processes including the formation of semiquinone. It can be concluded that interconversions between hydroquinone and benzoquinone are influenced by NADPH and more effectively by the complete microsomal system. Ascorbate, well-known antioxidant produces OH radicals and peroxidation of lipids. On the other hand, both hydroquinone and benzoquinone appear to be very efficient inhibitors of lipid peroxidation.     

Redox regulation of copper-metallothionein

Copper (Cu) is an essential element whose localization within cells must be carefully controlled to avoid Cu-dependent redox cycling. Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotective effects during metal exposure and oxidative stress. The specific role of MTs, however, in modulating Cu-dependent redox cycling remains unresolved. Our studies utilized a chemically defined model system to study MT modulation of Cu-dependent redox cycling under reducing (Cu/ascorbate) and mild oxidizing (Cu/ascorbate + H2O2) conditions. In the presence of Cu and ascorbate, MT blocked Cu-dependent lipid oxidation and ascorbyl radical formation with a stoichiometry corresponding to Cu/MT ratios 相似文献   

The effects of short-term selenium stress on Polish and Finnish wheat seedlings-EPR, enzymatic and fluorescence studies

Biochemical analyses of antioxidant content were compared with measurements of fluorescence and electron paramagnetic resonance (EPR) to examine the alteration of radicals in wheat seedlings exposed to 2 days of selenium stress. Two genotypes of Polish and one of Finnish wheat, differing in their tolerance to long-term stress treatment, were cultured under hydroponic conditions to achieve the phase of 3-leave seedlings. Afterwards, selenium (sodium selenate, 100 μM concentration) was added to the media. After Se-treatment, all varieties showed an increase in carbohydrates (soluble and starch), ascorbate and glutathione content in comparison to non-stressed plants. These changes were more visible in Finnish wheat. On the basis of lipid peroxidation measurements, Finnish wheat was recognized as the genotype more sensitive to short-term Se-stress than the Polish varieties. The antioxidant enzyme activities (superoxide dismutase, ascorbate peroxidase and glutathione reductase) increased in Polish genotypes, whereas they decreased in Finnish wheat plants cultured on Se media. The action of reactive oxygen species in short-term action of Se stress was confirmed by the reduction of PSII and PSI system activities (measured by fluorescence parameters and EPR, respectively). EPR studies showed changes in redox status (especially connected with Mn(II)/Mn(III), and semiquinone/quinone ratios) in wheat cell after Se treatment. The involvement of the carbohydrate molecules as electron traps in production of long-lived radicals is postulated.     

Generation of Probucol Radicals and Their Reduction by Ascorbate and Dihydrolipoic Acid in Human Low Density Lipoproteins

Probucol, 4.4'-[(1-methylethylidene)bis(thio)]bis-[2,6-bis(1.1-dimethyl)phenol], is a lipid regulating drug whose therapeutic potential depends on its antioxidant properties. Probucol and x-tocopherol were quantitatively compared in their ability to scavenge peroxyl radicals generatcd by the thermal decomposition of the lipid-soluble azo-initiator 2,2'-azo-bis(2,4-dimethyl-valeronitrile), AMVN, in dioleoylphos-phatidylcholine (DOPC) liposomes. Probucol showed 15-times lower peroxyl radical scavenging efficiency than x-tocopherol as measured by the effects on AMVN-induced luminol-dependent chemiluminescence. We suggest that probucol cannot protect x-tocopherol against its loss in the course of oxidation, although probucol is known to prevent lipid peroxidation in membranes and lipoproteins. In human low density lipoproteins (LDL) ESR signals of the probucol phenoxyl radical were detected upon incubation with lipoxygenase + linolenic acid or AMVN. Ascorbate was shown to reduce probucol radicals. Dihydro-lipoic acid alone was not able to reduce the probucol radical but in the presence of both ascorbate and dihydrolipoic acid a synergistic effect of a stepwise reduction was observed. This resulted from ascorbate-dependent reduction of probucol radicals and dihydrolipoic acid-dependent reduction of ascorbyl radicals. The oxidized form of dihydrolipoic acid, thioctic acid, did not affect probucol radicals either in the presence or in the absence of ascorbate.     

Generation of Probucol Radicals and Their Reduction by Ascorbate and Dihydrolipoic Acid in Human Low Density Lipoproteins

Probucol, 4.4′-[(1-methylethylidene)bis(thio)]bis-[2,6-bis(1.1-dimethyl)phenol], is a lipid regulating drug whose therapeutic potential depends on its antioxidant properties. Probucol and x-tocopherol were quantitatively compared in their ability to scavenge peroxyl radicals generatcd by the thermal decomposition of the lipid-soluble azo-initiator 2,2′-azo-bis(2,4-dimethyl-valeronitrile), AMVN, in dioleoylphos-phatidylcholine (DOPC) liposomes. Probucol showed 15-times lower peroxyl radical scavenging efficiency than x-tocopherol as measured by the effects on AMVN-induced luminol-dependent chemiluminescence. We suggest that probucol cannot protect x-tocopherol against its loss in the course of oxidation, although probucol is known to prevent lipid peroxidation in membranes and lipoproteins. In human low density lipoproteins (LDL) ESR signals of the probucol phenoxyl radical were detected upon incubation with lipoxygenase + linolenic acid or AMVN. Ascorbate was shown to reduce probucol radicals. Dihydro-lipoic acid alone was not able to reduce the probucol radical but in the presence of both ascorbate and dihydrolipoic acid a synergistic effect of a stepwise reduction was observed. This resulted from ascorbate-dependent reduction of probucol radicals and dihydrolipoic acid-dependent reduction of ascorbyl radicals. The oxidized form of dihydrolipoic acid, thioctic acid, did not affect probucol radicals either in the presence or in the absence of ascorbate.     

Ascorbyl free radical as a reliable indicator of free-radical-mediated myocardial ischemic and post-ischemic injury. A real-time continuous-flow ESR study

The real-time kinetics of the release of ascorbyl free radicals in the coronary perfusate from isolated rat hearts submitted to an ischemia/reperfusion sequence has been achieved by continuous-flow ESR using high-speed acquisition techniques. Enhanced ESR detection of ascorbyl free radicals was obtained by addition of dimethyl sulfoxide (Me2SO), a strong cation chelator and oxidizing agent. A continuous-flow device allowed a direct monitoring of the ascorbyl free radical and/or ascorbate leakage in coronary perfusate by observation of the ascorbyl radical doublet (aH = 0.188 mT and g = 2.0054). 1. The results showed that ascorbyl free radical release occurred mainly during sequences of low-flow ischemia (90 min) coupled or not with 30 min of zero-flow ischemia followed by reperfusion (60 min). The kinetic profiles of ascorbyl-free-radical detection confirm in quantitative terms the expected correlation between the duration of the ischemic insult and the magnitude of ascorbate extracellular release upon reperfusion. There is indication that ascorbyl free radical depletion could be secondary to oxygen-derived-free-radical-induced cellular damage. 2. The amount of residual ascorbic acid was quantitated on myocardial tissue at the end of reperfusion using Me2SO as extracting solvent. Intense oxidation of ascorbate and chemical stabilization of the resulting free radical species provided by Me2SO allowed ESR measurement of a marked tissue ascorbate depletion related to the duration of ischemia. 3. Perfusion of superoxide dismutase during low-flow ischemia and the first 10 min of reperfusion greatly inhibited both extracellular release and endogenous ascorbate depletion. These results suggest that the ascorbate redox system constitutes a major protective mechanism against free-radical-induced myocardial injury. 4. The proposed direct ESR detection of ascorbyl free radicals in the coronary perfusates or in tissue extracts does not require extensive chemical preparation and conditioning of effluent or tissue samples. It provides an interesting straightforward alternative to the evaluation of detrimental free radical processes affecting the myocardium during ischemia and reperfusion.     

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.     

An electron spin resonance (ESR) study on the mechanism of ascorbyl radical production by metal-binding proteins

The mechanism of ascorbate oxidation by metal-binding proteins (ceruloplasmin, albumin and transferrin) was investigated in vitro and in isolated plasma by the measurement of the ascorbyl free radicals (AFR) by electron spin resonance (ESR). In plasma of 13 healthy volunteers, a spontaneous and variable pro-duction of AFR was detected, which was increased by a 10 M ascorbate overloading; however, this increase was not correlated to the intensity of the spontaneous AFR signal. The addition of Cu and ceruloplasmin to plasma increased the ESR signal, while the addition of transferrin decreased the signal intensity in a dose-dependent manner. In vitro, we demonstrated that ascorbate was oxidized by human serum albumin and by ceruloplasmin, and that this oxidase-like activity was lost by trypsin or heat treatment of these proteins. These two proteins positively interacted in the oxidation of ascorbate, since addition of crude albumin to a solution of ascorbate and ceruloplasmin increased the intensity of ESR signal in a dose-dependent manner. The treatment of albumin by a metal chelator (DDTC) abolished these positive inter-actions. The respective roles of copper and iron in ascorbate oxidation were studied and showed a dose-dependent effect of these ions on ascorbate oxidation. The role of iron was confirmed by the inhibiting effect of metal-free transferrin on iron-dependent ascorbate oxidation. Concerted actions between iron carrying albumin and copper carrying ceruloplasmin appear responsible for the production of AFR in vitro and in vivo. © Rapid Science 1998