ascorbic-acid and dihydrolipoic-acid

Cytochrome b561 (Cyt-b561) proteins constitute a family of trans-membrane proteins that are present in a wide variety of organisms. Two of their characteristic properties are the reducibility by ascorbate (ASC) and the presence of two distinct b-type hemes localized on two opposite sides of the membrane. Here we show that the tonoplast-localized and the putative tumor suppressor Cyt-b561 proteins can be reduced by other reductants than ASC and dithionite. A detailed spectral analysis of the ASC-dependent and dihydrolipoic acid (DHLA)-dependent reduction of these two Cyt-b561 proteins is also presented. Our results are discussed in relation to the known antioxidant capability of DHLA as well as its role in the regeneration of other antioxidant compounds of cells. These results allow us to speculate on new biological functions for the trans-membrane Cyt-b561 proteins.

Topics: Amino Acid Sequence; Animals; Antioxidants; Arabidopsis; Ascorbic Acid; Cattle; Cytochrome b Group; Mice; Molecular Sequence Data; Oxidation-Reduction; Thioctic Acid

Oxidation of polyunsaturated fatty acids (PUFAs) releases alpha,beta-unsaturated aldehydes that modify deoxyguanosine (dG) to form cyclic 1,N(2)-propanodeoxyguanosine adducts. One of the major adducts detected in vivo is acrolein (Acr)-derived 1,N(2)-propanodeoxyguanosine (Acr-dG). We used a chemical model system to examine the effects of 4 antioxidants known to inhibit fatty acid oxidation on the formation of Acr-dG and 8-oxodeoxyguaonsine (8-oxodG) from the PUFA docosahexaenoic acid (DHA) under oxidative conditions. We found that epigallocatechin gallate (EGCG) and dihydrolipoic acid (DHLA) inhibit both Acr-dG and 8-oxodG formation. In contrast, ascorbic acid and alpha-tocopherol actually increase Acr-dG at high concentrations and do not show a concentration-dependant inhibition of 8-oxodG. We also studied their effects on blocking Acr-dG formation directly from Acr. EGCG and DHLA can both effectively block Acr-dG formation, but ascorbic acid and alpha-tocopherol show weak or little effect. These results highlight the complexity of antioxidant mechanisms and also reveal that EGCG and DHLA are effective at suppressing lipid peroxidation-induced Acr-dG and 8-oxodG formation as well as blocking the reaction of dG with Acr.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Acrolein; alpha-Tocopherol; Antioxidants; Ascorbic Acid; Catechin; Deoxyguanosine; DNA Damage; Docosahexaenoic Acids; Lipid Peroxidation; Thioctic Acid

Myeloperoxidase (MPO)-catalyzed one-electron oxidation of endogenous phenolic constituents (e.g., antioxidants, hydroxylated metabolites) and exogenous compounds (e.g., drugs, environmental chemicals) generates free radical intermediates: phenoxyl radicals. Reduction of these intermediates by endogenous reductants, i.e. recycling, may enhance their antioxidant potential and/or prevent their potential cytotoxic and genotoxic effects. The goal of this work was to determine whether generation and recycling of MPO-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC), by physiologically relevant intracellular reductants such as ascorbate/lipoate could be demonstrated in intact MPO-rich human leukemia HL-60 cells. A model system was developed to show that MPO/H(2)O(2)-catalyzed PMC phenoxyl radicals (PMC*) could be recycled by ascorbate or ascorbate/dihydrolipoic acid (DHLA) to regenerate the parent compound. Absorbance measurements demonstrated that ascorbate prevents net oxidation of PMC by recycling the phenoxyl radical back to the parent compound. The presence of DHLA in the reaction mixture containing ascorbate extended the recycling reaction through regeneration of ascorbate. DHLA alone was unable to prevent PMC oxidation. These conclusions were confirmed by direct detection of PMC* and ascorbate radicals formed during the time course of the reactions by EPR spectroscopy. Based on results in the model system, PMC* and ascorbate radicals were identified by EPR spectroscopy in ascorbate-loaded HL-60 cells after addition of H(2)O(2) and the inhibitor of catalase, 3-aminotriazole (3-AT). The time course of PMC* and ascorbate radicals was found to follow the same reaction sequence as during their recycling in the model system. Recycling of PMC by ascorbate was also confirmed by HPLC assays in HL-60 cells. Pre-loading of HL-60 cells with lipoic acid regenerated ascorbate and thus increased the efficiency of ascorbate in recycling PMC*. Lipoic acid had no effect on PMC oxidation in the absence of ascorbate. Thus PMC phenoxyl radical does not directly oxidize thiols but can be recycled by dihydrolipoate in the presence of ascorbate. The role of phenoxyl radical recycling in maintaining antioxidant defense and protecting against cytotoxic and genotoxic phenolics is discussed.

Topics: Antioxidants; Ascorbic Acid; Cell Survival; Chromans; Chromatography, High Pressure Liquid; Electron Spin Resonance Spectroscopy; Free Radicals; HL-60 Cells; Humans; Hydrogen Peroxide; Oxidation-Reduction; Peroxidase; Phenols; Spectrophotometry; Substrate Cycling; Thioctic Acid

The heme enzyme myeloperoxidase (MPO) has recently been implicated in hydrogen peroxide H(2)O(2)-induced apoptosis of HL-60 human leukemia cells. The purpose of this study was to investigate the molecular mechanism(s) of MPO-mediated apoptosis, in particular caspase-3 activation, and to determine the effects of the antioxidants ascorbate and (dihydro)lipoic acid. Incubation of HL-60 cells (1 x 10(6) cells/ml media) with H(2)O(2) (0-200 microM) resulted in dose-dependent stimulation of caspase-3 activity, DNA fragmentation, and morphological changes associated with apoptosis. Caspase-3 activity, DNA fragmentation and apoptosis were maximal at approximately 50 microM H(2)O(2). Pre-incubation of the cells with the MPO-specific inhibitor 4-aminobenzoic acid hydrazide (ABAH) and the heme enzyme inhibitor 3-aminotriazole (100 microM each) resulted in complete and partial inhibition, respectively, of intracellular MPO, caspase-3 activity, and apoptosis following addition of 50 microM H(2)O(2). Enhancement of cellular antioxidant status by pre-incubation of the cells with dehydro-ascorbic acid and lipoic acid, which are reduced intracellularly to ascorbate and dihydrolipoic acid, respectively, afforded protection against caspase-3 activation and apoptosis following addition of H(2)O(2). Addition of high concentrations of H(2)O(2) (200 microM) to cells pre-incubated with lipoic acid, however, resulted in cytotoxicity. Overall, our data indicate that MPO-derived oxidants, rather than H(2)O(2) itself, are involved in caspase-3 activation and apoptosis in HL-60 cells, and the antioxidants ascorbate and (dihydro)lipoic acid inhibit caspase-3 activation and apoptosis in these cells, likely via scavenging the MPO-derived oxidants.

Topics: Antioxidants; Apoptosis; Ascorbic Acid; Caspase 3; Caspases; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; HL-60 Cells; Humans; Hydrogen Peroxide; Oxygen; Peroxidase; Thioctic Acid

In the redox antioxidant network, dihydrolipoate can synergistically enhance the ascorbate-dependent recycling of vitamin E. Since the major endogenous thiol antioxidant in biological systems is glutathione (GSH) it was of interest to compare the effects of dihydrolipoate with GSH on ascorbate-dependent recycling of the water-soluble homologue of vitamin E, Trolox, by electron spin resonance (ESR). Trolox phenoxyl radicals were generated by a horseradish peroxidase (HRP)-hydrogen peroxide (H2O2) oxidation system. In the presence of dihydrolipoate, Trolox radicals were suppressed until both dihydrolipoate and endogenous levels of ascorbate in skin homogenates were consumed. Similar experiments made in the presence of GSH revealed that Trolox radicals reappeared immediately after ascorbate was depleted and that GSH was not able to drive the ascorbate-dependent Trolox recycling reaction. However, at higher concentrations GSH was able to increase ascorbate-mediated Trolox regeneration from the Trolox radical. ESR and spectrophotometric measurements demonstrated the ability of dihydrolipoate or GSH to react with dehydroascorbate, the two-electron oxidation product of ascorbate in this system. Dihydrolipoate regenerated greater amounts of ascorbate at a much faster rate than equivalent concentrations of GSH. Thus the marked difference between the rate and efficiency of ascorbate generation by dihydrolipoate as compared with GSH appears to account for the different kinetics by which these thiol antioxidants influence ascorbate-dependent Trolox recycling.

Topics: Animals; Antioxidants; Ascorbic Acid; Cell Extracts; Chromans; Electron Spin Resonance Spectroscopy; Free Radicals; Glutathione; Hydrogen Peroxide; Kinetics; Mice; Peroxidase; Phenols; Skin; Thioctic Acid; Vitamin E

Coenzyme Q (Q10) and alpha-tocopherol cooperatively delay the onset of diene conjugation in isolated human low density lipoprotein if supplied in water-soluble preparations to blood serum. Both copper ions and morpholino sydnonimine (in the presence of glucose; SIN-1-glucose) -driven diene conjugation is measurable as soon as both reduced Q10 and tocopherol are oxidized, where tocopherol oxidation starts after 80-90% consumption of reduced Q10. LDL-bound Q10 in turn can be rapidly reduced by dihydrolipoic acid (thioctic acid). This reaction is at least 10 times faster than reduction by ascorbic acid.

Topics: Antioxidants; Ascorbic Acid; Coenzymes; Copper; Humans; Ions; Lipoproteins, LDL; Male; Middle Aged; Models, Biological; Molsidomine; Nitric Oxide Donors; Oxygen; Protein Binding; Thioctic Acid; Time Factors; Ubiquinone; Vitamin E

One-electron oxidation of the antiatherogenic and antiatherosclerotic drug probucol has been studied in relation to its activity as an antioxidant. Oxidation by triplet excited states of duroquinone and benzophenone, and by the inorganic radicals Br2.- and N3., lead to the formation of a transient absorption at 500 nm. This was identified by time-resolved resonance Raman spectroscopy as the phenoxyl radical from probucol, formed by hydrogen atom or electron plus proton loss from one of the phenolic groups of probucol. The reactivity of probucol with triplet duroquinone and triplet benzophenone, and as a quencher of singlet oxygen, was compared with the reactivities of other antioxidants (alpha-tocopherol, palmitoyl ascorbic acid, dihydrolipoic acid and N-acetyl cysteine). In quenching of the triplet states the reactivity of probucol was comparable with that of alpha-tocopherol, whereas as a quencher of singlet oxygen probucol (k < 10(6) M-1 s-1) was less effective than alpha-tocopherol (k = 2.0 x 10(8) M-1 s-1) by more than two orders of magnitude. This difference in reactivity may allow the contribution of singlet oxygen towards oxidative stress to be quantified separately.

Topics: Acetylcysteine; Antimutagenic Agents; Antioxidants; Ascorbic Acid; Benzophenones; Benzoquinones; Free Radicals; Oxidation-Reduction; Photolysis; Probucol; Reactive Oxygen Species; Spectrophotometry; Spectrum Analysis, Raman; Thioctic Acid; Vitamin E

Due to its strained five-membered ring, alpha-lipoic acid (LA) has an absorption band around 330 nm, which is used to quantify its concentration. In order to obtain information for the homolytic rupture of the S-S bond and the formation of dihydrolipoic acid (DHLA), the photochemical reaction of lipoic acid was examined in the presence or absence of ascorbic acid upon exposure to UVA light. The absorption band of alpha-lipoic acid at around 330 nm disappeared by photoirradiation, which corresponds to the rupture of S-S bond of the 1,2-dithiolane ring in lipoic acid. HPLC-Electrochemical Detection (ECD) analysis of lipoic acid showed significant formation of dihydrolipoic acid and other thiols. The formation of thiols from the photoreaction of lipoic acid was also confirmed by the Ellman method. The formation of thiols from lipoic acid was completely time-dependent and the formation of the thiols increased upto 55%. Similar results were obtained in the photochemical reactions of short-chain analogues, bisnor- and tetranor-lipoic acid. On the other hand, beta-lipoic acid was quite stable, no photodecomposition of beta-lipoic acid was observed in the UV region. The formation of thiols including dihydrolipoic acid from lipoic acid can be explained by considering the rupture of S-S bond, which results in the formation of the dithiyl radicals of alpha-lipoic acid. It is proposed that intra- and intermolecular hydrogen abstraction of the dithyl radical produces thiols including dihydrolipoic acid as final products.

Topics: Antioxidants; Ascorbic Acid; Disulfides; Free Radicals; Models, Chemical; Molecular Structure; Photochemistry; Photolysis; Reactive Oxygen Species; Serum Albumin; Spectrophotometry, Ultraviolet; Sulfhydryl Compounds; Thioctic Acid; Ultraviolet Rays

Vitamin E (alpha-tocopherol) is the major lipid-soluble antioxidant of retinal membranes whose deficiency causes retinal degeneration. Its antioxidant function is realized via scavenging peroxyl radicals as a result of which phenoxyl radicals of alpha-tocopherol are formed. Our hypothesis is that alpha-tocopherol phenoxyl radicals can be reduced by endogenous reductants in the retina, providing for alpha-tocopherol recycling. The results of this study demonstrate for the first time that: (i) endogenous ascorbate (vitamin C) in retinal homogenates and in rod outer segments is able to protect endogenous alpha-tocopherol against oxidation induced by UV-irradiation by reducing the phenoxyl radical of alpha-tocopherol, (ii) in the absence of ascorbate, neither endogenous nor exogenously added glutathione (GSH) is efficient in protecting alpha-tocopherol against oxidation; (iii) GSH does not substantially enhance the protective effect of ascorbate against alpha-tocopherol oxidation; (iv) exogenous dihydrolipoic acid (DHLA), although inefficient in direct reduction of the alpha-tocopherol phenoxyl radical, is able to enhance the protective effect of ascorbate by regenerating it from dehydroascorbate. Thus, regeneration of alpha-tocopherol from its phenoxyl radical can enhance its antioxidant effectiveness in the retina. The recycling of alpha-tocopherol opens new avenues for pharmacological approaches to enhance antioxidants of the retina.

Topics: Animals; Ascorbic Acid; Chromatography, High Pressure Liquid; Dehydroascorbic Acid; Electron Spin Resonance Spectroscopy; Free Radicals; Glutathione; Male; Oxidation-Reduction; Rats; Rats, Sprague-Dawley; Retina; Thioctic Acid; Ultraviolet Rays; Vitamin E

A detailed evaluation of the antioxidant and pro-oxidant properties of lipoic acid (LA) and dihydrolipoic acid (DHLA) was performed. Both compounds are powerful scavengers of hypochlorous acid, able to protect alpha 1-antiproteinase against inactivation by HOCl. LA was a powerful scavenger of hydroxyl radicals (OH.) and could inhibit both iron-dependent OH. generation and peroxidation of ox-brain phospholipid liposomes in the presence of FeCl3-ascorbate, presumably by binding iron ions and rendering them redox-inactive. By contrast, DHLA accelerated iron-dependent OH. generation and lipid peroxidation, probably by reducing Fe3+ to Fe2+. LA inhibited this pro-oxidant action of DHLA. However, DHLA did not accelerate DNA degradation by a ferric bleomycin complex and slightly inhibited peroxidation of arachidonic acid by the myoglobin-H2O2 system. Under certain circumstances, DHLA accelerated the loss of activity of alpha-antiproteinase exposed to ionizing radiation under a N2O/O2 atmosphere and also the loss of creatine kinase activity in human plasma exposed to gas-phase cigarette smoke. Neither LA nor DHLA reacted with superoxide radical (O.2-) or H2O2 at significant rates, but both were good scavengers of trichloromethylperoxyl radical (CCl3O2.). We conclude that LA and DHLA have powerful antioxidant properties. However, DHLA can also exert pro-oxidant properties, both by its iron ion-reducing ability and probably by its ability to generate reactive sulphur-containing radicals that can damage certain proteins, such as alpha 1-antiproteinase and creatine kinase.

Topics: alpha 1-Antitrypsin; Animals; Antioxidants; Arachidonic Acid; Ascorbic Acid; Bleomycin; Brain; Cattle; Deoxyribose; DNA Damage; Free Radical Scavengers; Hydrogen Peroxide; Hypochlorous Acid; Lipid Peroxidation; Liposomes; Myoglobin; Superoxides; Thioctic Acid

The azo initiator of peroxyl radicals 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) induces oxidative hemolysis in human erythrocytes and subsequent hemoglobin oxidation. Using the degree of hemolysis versus time as an indication of the oxidative damage it was found that i) both reduced and oxidized alpha-lipoic acid protected against oxidative damage; ii) simultaneous treatment of erythrocytes with ascorbate and dihydrolipoate or alpha-lipoate has a synergistic tendency to protect cells against hemolysis; iii) glutathione in combination with dihydrolipoic acid or alpha-lipoic acid has an additive effect on hemolysis protection. The spin trapping reagent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) formed an adduct with the peroxyl/alkoxyl radicals produced by thermal decomposition of AAPH in the presence of oxygen. The formation of this adduct was prevented by reduced or oxidized lipoic acid, reduced glutathione or ascorbate. It is concluded that AAPH-peroxyl radicals progressively damage the cells and the released hemoglobin is subsequently oxidized to methemoglobin which might further enhance the oxidative damage. The protective effect of antioxidants is exerted outside the cells by directly scavenging AAPH-alkoxyl radicals.

Topics: Amidines; Ascorbic Acid; Cyclic N-Oxides; Drug Synergism; Electron Spin Resonance Spectroscopy; Erythrocytes; Free Radicals; Glutathione; Hemolysis; Humans; Methemoglobin; Oxidation-Reduction; Stereoisomerism; Structure-Activity Relationship; Thioctic Acid

Phenoxyl radicals are intermediates in the oxidation of phenolic compounds to quinoid derivatives (quinones, quinone methides), which are known to act as ultimate mutagenic, carcinogenic, and cytotoxic agents by directly interacting with macromolecular targets or by generating toxic reactive oxygen species. One-electron reduction of phenoxyl radicals may reverse oxidative activation of phenolic compounds to quinoids, thus preventing their cytotoxic effects. In the present work, we studied interactions of ascorbate, thiols (glutathione, dihydrolipoic acid, and metallothioneins), and combinations thereof with the phenoxyl radical generated by tyrosinase-catalyzed oxidation of VP-16 [etoposide, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucop yra noside)], a hindered phenol widely used as an antitumor drug. We found by liquid chromatography-ionspray mass spectrometry and electron spin resonance (ESR) that tyrosinase caused oxidation of VP-16 to its o-quinone and aromatized derivative via intermediate formation of the phenoxyl radical. Both ascorbate and thiols (GSH, dihydrolipoic acid, and metallothioneins) were able to directly reduce the VP-16 phenoxyl radical and prevent its oxidation. The characteristic ESR signal of the VP-16 phenoxyl radical was quenched by the reductants. The semidehydroascorbyl radical ESR signal was detected in the presence of ascorbate; thiols did not produce signals in the ESR spectra. In combinations, ascorbate plus GSH and ascorbate plus metallothionein acted independently and additively in reducing the VP-16 phenoxyl radical. Ascorbate was more reactive: the VP-16-dependent oxidation of GSH or metallothionein commenced only after complete oxidation of ascorbate. The semidehydroascorbyl radical ESR signal preceded the quenching of the VP-16 phenoxyl radical by GSH and metallothionein. In the presence of ascorbate plus dihydrolipoic acid, ascorbate was also more reactive toward the VP-16 phenoxyl radical than dihydrolipoic acid, but the ascorbate concentration was maintained at the expense of its regeneration from dehydroascorbate by dihydrolipoic acid. In ESR spectra, the semidehydroascorbyl radical ESR signal was continuously detected and then was abruptly substituted by the VP-16 phenoxyl radical signal. When VP-16 and tyrosinase were incubated in the presence of retina or hepatocyte homogenates, a two-phase lag period was observed by ESR for the appearance of the VP-16 radical signal: an ascorbate-dependent par

Topics: Animals; Ascorbic Acid; Electron Spin Resonance Spectroscopy; Etoposide; Free Radicals; Liver; Male; Monophenol Monooxygenase; Oxidation-Reduction; Phenols; Rats; Rats, Sprague-Dawley; Retina; Subcellular Fractions; Sulfhydryl Compounds; Thioctic Acid

Exposure of human plasma to gas phase cigarette smoke (CS) produces a depletion of ascorbic acid, peroxidation of lipids (Frei et al. Biochem J 1991; 277: 133-8), and protein modification (as measured by protein carbonyl accumulation and loss of sulfhydryl groups) (Reznick et al. Biochem J 1992; 286: 607-11). CS contains both saturated and unsaturated aldehydes. The contribution of these aldehydes to the damaging effects of CS on human plasma was investigated. Aldehydes present in CS did not cause a depletion of plasma antioxidants such as ascorbic acid or alpha-tocopherol and did not induce plasma lipid peroxidation. Aldehydes decreased plasma protein sulfhydryl concentrations but increased protein carbonyls. The thiols glutathione and dihydrolipoic acid had a significant effect in reducing aldehyde-induced protein modifications.

Topics: Adult; Aldehydes; Antioxidants; Ascorbic Acid; Glutathione; Humans; Lipid Peroxidation; Male; Middle Aged; Smoking; Sulfhydryl Compounds; Thioctic Acid; Vitamin E

Etoposide (VP-16) is an antitumor drug currently in use for the treatment of a number of human cancers. Mechanisms of VP-16 cytotoxicity involve DNA breakage secondary to inhibition of DNA topoisomerase II and/or direct drug-induced DNA strand cleavage. The VP-16 molecule contains a hindered phenolic group which is crucial for its antitumor activity because its oxidation yields reactive metabolites (quinones) capable of irreversible binding to macromolecular targets. VP-16 phenoxyl radical is an essential intermediate in VP-16 oxidative activation and can be either converted to oxidation products or reduced by intracellular reductants to its initial phenolic form. In the present paper we demonstrate that the tyrosinase-induced VP-16 phenoxyl radical could be reduced by ascorbate, glutathione (GSH) and dihydrolipoic acid. These reductants caused a transient disappearance of a characteristic VP-16 phenoxyl radical ESR signal which reappeared after depletion of the reductant. The reductants completely prevented VP-16 oxidation by tyrosinase during the lag-period as measured by high performance liquid chromatography; after the lag-period VP-16 oxidation proceeded with the rate observed in the absence of reductants. In homogenates of human K562 leukemic cells, the tyrosinase-induced VP-16 phenoxyl radical ESR signal could be observed only after a lag-period whose duration was dependent on cell concentration; VP-16 oxidation proceeded in cell homogenates after this lag-period. In homogenates of isolated nuclei, the VP-16 phenoxyl radical and VP-16 oxidation were also detected after a lag-period, which was significantly shorter than that observed for an equivalent amount of cells. In both cell homogenates and in nuclear homogenates, the duration of the lag period could be increased by exogenously added reductants. The duration of the lag-period for the appearance of the VP-16 phenoxyl radical signal in the ESR spectrum can be used as a convenient measure of cellular reductive capacity. Interaction of the VP-16 phenoxyl radical with intracellular reductants may be critical for its metabolic activation and cytotoxic effects.

Topics: Ascorbic Acid; Cell Nucleus; Chromatography, High Pressure Liquid; Deferoxamine; Electron Spin Resonance Spectroscopy; Etoposide; Free Radicals; Glutathione; Humans; Leukemia; Monophenol Monooxygenase; Oxidation-Reduction; Phenols; Thioctic Acid; Tumor Cells, Cultured

Oxidative modification of low density lipoproteins (LDL) and their unrestricted scavenger receptor-dependent uptake is believed to account for cholesterol deposition in macrophage-derived foam cells. It has been suggested that vitamin E that is transported by LDL plays a critical role in protecting against LDL oxidation. We hypothesize that the maintenance of sufficiently high vitamin E concentrations in LDL can be achieved by reducing its chromanoxyl radicals, i.e., by vitamin E recycling. In this study we demonstrate that: i) chromanoxyl radicals of endogenous vitamin E and of exogenously added alpha-tocotrienol, alpha-tocopherol or its synthetic homologue with a 6-carbon side-chain, chromanol-alpha-C6, can be directly generated in human LDL by ultraviolet (UV) light, or by interaction with peroxyl radicals produced either by an enzymic oxidation system (lipoxygenase + linolenic acid) or by an azo-initiator, 2,2'-azo-bis(2,4-dimethylvaleronitrile) (AMVN; ii) ascorbate can recycle endogenous vitamin E and exogenously added chromanols by direct reduction of chromanoxyl radicals in LDL; iii) dihydrolipoic acid is not efficient in direct reduction of chromanoxyl radicals but recycles vitamin E by synergistically interacting with ascorbate (reduces dehydroascorbate thus maintaining the steady-state concentration of ascorbate); and iv) beta-carotene is not active in vitamin E recycling but may itself be protected against oxidative destruction by the reductants of chromanoxyl radicals. We suggest that the recycling of vitamin E and other phenolic antioxidants by plasma reductants may be an important mechanism for the enhanced antioxidant protection of LDL.

Topics: Ascorbic Acid; beta Carotene; Carotenoids; Chromans; Drug Synergism; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Humans; Linolenic Acids; Lipid Peroxidation; Lipoproteins, LDL; Lipoxygenase; Thioctic Acid; Ultraviolet Rays; Vitamin E

Vitamin E (alpha-tocopherol) is the major lipid-soluble chain-breaking antioxidant of membranes. Its UV-absorbance spectrum (lambda max 295 nm) extends well into the solar spectrum. We hypothesize that in skin alpha-tocopherol may absorb solar UV light and generate tocopheroxyl radicals. Reduction of tocopheroxyl radicals by other antioxidants (e.g. ascorbate, thiols) will regenerate (recycle) vitamin E at the expense of their own depletion. Hence, vitamin E in skin may act in two conflicting manners upon solar illumination: in addition to its antioxidant function as a peroxyl radical scavenger, it may act as an endogenous photosensitizer, enhancing light-induced oxidative damage. To test this hypothesis, we have illuminated various systems (methanol-buffer dispersions, liposomes and skin homogenates) containing alpha-tocopherol or its homologue with a shorter 6-carbon side chain, chromanol-alpha-C6 with UV light closely matching solar UV light, in the presence or absence of endogenous or exogenous reductants. We found that: (i) alpha-tocopheroxyl (chromanoxyl) radicals are directly generated by solar UV light in model systems (methanol-water dispersions, liposomes) and in skin homogenates; (ii) reducing antioxidants (ascorbate, ascorbate+dihydrolipoic acid) can donate electrons to alpha-tocopheroxyl (chromanoxyl) radicals providing for vitamin E (chromanol-alpha-C6) recycling; (iii) recycling of UV-induced alpha-tocopheroxyl radicals depletes endogenous antioxidant pools (accelerates ascorbate oxidation); (iv) beta-carotene, a non-reducing antioxidant, is not active in alpha-tocopherol recycling, and its UV-dependent depletion is unaffected by vitamin E.

Topics: Animals; Antioxidants; Ascorbic Acid; beta Carotene; Carotenoids; Electron Spin Resonance Spectroscopy; Free Radicals; Liposomes; Methanol; Mice; Mice, Hairless; Models, Biological; Neoplasms, Radiation-Induced; Oxygen; Phosphatidylcholines; Radiation Tolerance; Skin; Skin Neoplasms; Suspensions; Thioctic Acid; Ultraviolet Rays; Vitamin E; Water

Thioctic (lipoic) acid is used as a therapeutic agent in a variety of diseases in which enhanced free radical peroxidation of membrane phospholipids has been shown to be a characteristic feature. It was suggested that the antioxidant properties of thioctic acid and its reduced form, dihydrolipoic acid, are at least in part responsible for the therapeutic potential. The reported results on the antioxidant efficiency of thioctic and dihydrolipoic acids obtained in oxidation models with complex multicomponent initiation systems are controversial. In the present work we used relatively simple oxidation systems to study the antioxidant effects of dihydrolipoic and thioctic acids based on their interactions with: (1) peroxyl radicals which are essential for the initiation of lipid peroxidation, (2) chromanoxyl radicals of vitamin E, and (3) ascorbyl radicals of vitamin C, the two major lipid- and water-soluble antioxidants, respectively. We demonstrated that: (1) dihydrolipoic acid (but not thioctic acid) was an efficient direct scavenger of peroxyl radicals generated in the aqueous phase by the water-soluble azoinitiator 2,2'-azobis(2-amidinopropane)-dihydrochloride, and in liposomes or in microsomal membranes by the lipid-soluble azoinitiator 2,2'-azobis(2,4-dimethylvaleronitrile); (2) both dihydrolipoic acid and thioctic acid did not interact directly with chromanoxyl radicals of vitamin E (or its synthetic homologues) generated in liposomes or in the membranes by three different ways: UV-irradiation, peroxyl radicals of 2,2'-azobis(2,4-dimethylvaleronitrile), or peroxyl radicals of linolenic acid formed by the lipoxygenase-catalyzed oxidation; and (3) dihydrolipoic acid (but not thioctic acid) reduced ascorbyl radicals (and dehydroascorbate) generated in the course of ascorbate oxidation by chromanoxyl radicals. This interaction resulted in ascorbate-mediated dihydrolipoic acid-dependent reduction of the vitamin E chromanoxyl radicals, i.e. vitamin E recycling. We conclude that dihydrolipoic acid may act as a strong direct chain-breaking antioxidant and may enhance the antioxidant potency of other antioxidants (ascorbate and vitamin E) in both the aqueous and the hydrophobic membraneous phases.

Topics: Animals; Antioxidants; Ascorbic Acid; Azo Compounds; Chromans; Female; Free Radicals; Intracellular Membranes; Lipid Peroxidation; Liposomes; Microsomes, Liver; NAD; NADP; Nitriles; Oxidation-Reduction; Peroxides; Rats; Rats, Sprague-Dawley; Thioctic Acid

Microsomes from rat liver were used to investigate the mechanisms by which thiol compounds protect cellular membranes against damage from oxidants. Glutathione (GSH), dihydrolipoate and dithioerythritol, but not cysteine, ameliorated the loss of thiol groups of microsomal proteins attacked by Fe/ADP/NADPH or Fe/ADP/ascorbate prooxidant systems. The protection by GSH, but not dihydrolipoate or dithioerythritol, appeared to be enzymic since it was lost after microsomes were heated or treated with trypsin. The blocking of microsomal protein thiols with N-ethylmaleimide also diminished the protective effect of GSH. Lipid peroxidation, as assessed by chemiluminescence and vitamin-E loss, was inhibited in parallel with the protection of protein thiols. In microsomes lacking vitamin E, the protection of protein thiols by exogenous thiols was diminished. However, the GSH-dependent protection of vitamin E showed no preference for alpha-tocopherol over other tocopherol homologs. It is suggested that a GSH-dependent enzyme maintains protein thiols in the face of oxidative damage during microsomal peroxidation. A maintenance of protein thiols might not only protect important metabolic functions, but may also afford an antioxidant capacity to membranes, and account for one facet of the GSH-dependent inhibition of lipid peroxidation.

Topics: Adenosine Diphosphate; Animals; Ascorbic Acid; Cysteine; Dithioerythritol; Glutathione; Iron; Lipid Peroxidation; Luminescent Measurements; Microsomes, Liver; NADP; Rats; Sulfhydryl Compounds; Thioctic Acid; Vitamin E

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 alpha-tocopherol were quantitatively compared in their ability to scavenge peroxyl radicals generated by the thermal decomposition of the lipid-soluble azo-initiator 2,2'-azo-bis(2,4-dimethyl-valeronitrile), AMVN, in dioleoylphosphatidylcholine (DOPC) liposomes. Probucol showed 15-times lower peroxyl radical scavenging efficiency than alpha-tocopherol as measured by the effects on AMVN-induced luminol-dependent chemiluminescence. We suggest that probucol cannot protect alpha-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. Dihydrolipoic 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.

Topics: Antioxidants; Ascorbic Acid; Electron Spin Resonance Spectroscopy; Free Radicals; Humans; Hydrogen Peroxide; Lipoproteins, LDL; Luminescent Measurements; Oxidation-Reduction; Phenols; Spectrometry, Fluorescence; Thioctic Acid; Vitamin E