Erythrocyte ascorbate recycling: antioxidant effects in blood

Ascorbic acid is an important antioxidant in human plasma, but requires efficient recycling from its oxidized forms to avoid irreversible loss. Human erythrocytes prevented oxidation of ascorbate in autologous plasma, an effect that required recycling of ascorbate within the cells. Erythrocytes had a high capacity to take up dehydroascorbate, the two-electron oxidized product of ascorbate, and to reduce it to ascorbate. Uptake and conversion of dehydroascorbate to ascorbate was saturable, was half-maximal at 400 microM dehydroascorbate, and achieved a maximal intracellular ascorbate concentration of 1.5 mM. In the presence of 100 microM dehydroascorbate, erythrocytes had the capacity to regenerate a 35 microM ascorbate concentration in blood every 3 min. Ascorbate recycling from DHA required intracellular GSH. Depletion of erythrocyte GSH by more than 50% with diamide did not acutely affect the cellular ascorbate content, but did impair the subsequent ability of GSH-depleted cells to recycle dehydroascorbate to ascorbate. Whereas erythrocyte ascorbate recycling was coupled to GSH, an overwhelming extracellular oxidant stress depleted both ascorbate and alpha-tocopherol before the GSH content of cells fell appreciably. Recycled ascorbate was released from cells into plasma, but at a rate less than one tenth that of dehydroascorbate uptake and conversion to ascorbate. Nonetheless, ascorbate released from cells protected endogenous alpha-tocopherol in human LDL from oxidation by a water soluble free radical initiator. These results suggests that recycling of ascorbate in erythrocytes helps to maintain the antioxidant reserve of whole blood.     

Relations between tocopherol depletion and coenzyme Q during lipid peroxidation in rat liver mitochondria

In order to evaluate different mitochondrial antioxidant systems, the depletion of alpha-tocopherol and the levels of the reduced and oxidized forms of CoQ were measured in rat liver mitochondria during Fe++/ascorbate and NADPH/ADP/Fe++ induced lipid peroxidation. During the induction phase of malondialdehyde formation, alpha-tocopherol declined moderately to about 80% of initial contents, whereas the total CoQ pool remained nearly unchanged, but reduced CoQ9 continuously declined. At the start of massive malondialdehyde formation, CoQ9 reaches its fully oxidized state. At the same time alpha-tocopherol starts to decline steeply, but never becomes fully exhausted in both experimental systems. Evidently the oxidation of the CoQ9 pool constitutes a prerequisite for the onset of massive lipid peroxidation in mitochondria and for the subsequent depletion of alpha-tocopherol. Trapping of the GSH by addition of dinitrochlorbenzene (a substrate of the GSH transferase), results in a moderate acceleration of lipid peroxidation, but alpha-tocopherol and ubiquinol levels remained unchanged when compared with the controls. Addition of succinate to GSH depleted mitochondria effectively suppressed MDA formation as well as alpha-tocopherol and ubiquinol depletion. The data support the assumption that the protective effect of respiratory substrates against lipid peroxidation in the absence of mitochondrial GSH is mediated by the regeneration of the lipid soluble antioxidants CoQ and alpha-tocopherol.     

Examples of pitfalls in statistical analysis--6. Evaluation of data consisting of the elapsed time between two events

beta-Carotene and other carotenoids are widely regarded as biological antioxidants. However, recent clinical trials indicate that beta-carotene supplements are not effective in disease prevention and raise questions about the biological significance of carotenoid antioxidant actions. To further explore this issue, we have reevaluated the antioxidant actions of beta-carotene in liposomal and biological membrane systems. In dilinoleoylphosphatidylcholine liposomes in which 0.35 mol % beta-carotene was incorporated into the bilayer during liposome preparation, the carotenoid inhibited lipid peroxidation initiated by 10 mm azobis[amidinopropane HCl] (AAPH). In carotenoid-free liposome suspensions to which the same amount of beta-carotene was added, no antioxidant effect was observed. Supplementation of rat liver microsomes with beta-carotene in vitro yielded microsomes containing 1.7 nmol beta-carotene mg-1 and 0.16 nmol alpha-tocopherol mg-1 microsomal protein. In beta-carotene supplemented microsomes incubated with 10 mm AAPH under an air atmosphere, lipid peroxidation did not occur until alpha-tocopherol was depleted by approximately 60%. beta-Carotene exerted no apparent antioxidant effect and was not significantly depleted in the incubations. Similar results were obtained when the incubation was done at 3.8 torr O2. In liver microsomes from Mongolian gerbils fed beta-carotene-supplemented diets, beta-carotene levels were 16-37% of alpha-tocopherol levels. The kinetics of AAPH-induced lipid peroxidation were no different in beta-carotene-supplemented microsomes than in microsomes from unsupplemented animals, although the kinetics of beta-carotene and alpha-tocopherol depletion were similar. The results indicate that beta-carotene is ineffective as an antioxidant when added to preformed lipid bilayer membranes and that alpha-tocopherol is a much more effective membrane antioxidant than beta-carotene, regardless of the method of carotenoid-membrane incorporation. These results support a reevaluation of the proposed antioxidant role for beta-carotene in biological membranes.     

Ascorbate recycling in human neutrophils: induction by bacteria

Ascorbate (vitamin C) recycling occurs when extracellular ascorbate is oxidized, transported as dehydroascorbic acid, and reduced intracellularly to ascorbate. We investigated microorganism induction of ascorbate recycling in human neutrophils and in microorganisms themselves. Ascorbate recycling was determined by measuring intracellular ascorbate accumulation. Ascorbate recycling in neutrophils was induced by both Gram-positive and Gram-negative pathogenic bacteria, and the fungal pathogen Candida albicans. Induction of recycling resulted in as high as a 30-fold increase in intracellular ascorbate compared with neutrophils not exposed to microorganisms. Recycling occurred at physiologic concentrations of extracellular ascorbate within 20 min, occurred over a 100-fold range of effector/target ratios, and depended on oxidation of extracellular ascorbate to dehydroascorbic acid. Ascorbate recycling did not occur in bacteria nor in C. albicans. Ascorbate did not enter microorganisms, and dehydroascorbic acid entry was less than could be accounted for by diffusion. Because microorganism lysates reduced dehydroascorbic acid to ascorbate, ascorbate recycling was absent because of negligible entry of the substrate dehydroascorbic acid. Because ascorbate recycling occurs in human neutrophils but not in microorganisms, it may represent a eukaryotic defense mechanism against oxidants with possible clinical implications.     

Receptor distribution and the mechanism of enhanced erythrocyte agglutination by soybean agglutinin

We have examined the role of receptor clustering in intact erythrocyte membranes exhibiting enhanced lectin-mediated cell agglutination by analyzing freeze-fracture and freeze-etch images of human erythrocytes labeled with ferritin-conjugated soybean agglutinin. We find that trypsinization and fixation of intact erythrocytes, in either order, causes no alteration of the random distribution of ferritin-conjugated soybean agglutinin on the surfaces of these cells as compared to their distribution on the surfaces of fixed erythrocytes and untreated erythrocyte ghosts. Furthermore, clustering of the intramembranous particles in the membrane of intact erythrocytes was not found with any of the cells described above. We conclude that clustering of the soybean agglutinin receptors is not a major factor involved in the enhanced agglutination of intact trypsinized erythrocytes. Caution is necessary in transferring information obtained with erythrocyte ghosts, where clustering can be induced, to intact erythrocytes.     

Oxidation of alpha-tocopherol during the peroxidation of dilinoleoylphosphatidylcholine in liposomes

Liposomal suspensions of dilinoleoylphosphatidylcholine (DLPC) containing alpha-tocopherol (0.1 mol%, based on DLPC were oxidized at 37 degrees C. The oxidation was initiated by a lipid-soluble or water-soluble free radical initiator, or by the addition of CuSO4 and fructose. In all the oxidation systems, alpha-tocopherol suppressed the formation of DLPC hydroperoxides until all the alpha-tocopherol had been depleted. The oxidation products of alpha-tocopherol were 8a-alkyldioxy-alpha-tocopherones, 5,6-epoxy-alpha-tocopherylquinone, 2,3-epoxy-alpha-tocopherylquinone, and alpha-tocopherylquinone. The 8a-alkyldioxy-alpha-tocopherones were decomposed in the liposomes primarily by being hydrolyzed to produce alpha-tocopherylquinone. The results indicate that alpha-tocopherol can trap peroxyl radical to form 8a-alkyldioxy-alpha-tocopherones which are hydrolyzed to alpha-tocopherylquinone in phospholipid bilayers. In another oxidation pathway, alpha-tocopherol may be oxidized by peroxyl radicals to form isomeric epoxy-alpha-tocopherylquinones.     

Definition of conditions for the detection of genotoxic chemicals in the adult rat-liver epithelial cell/hypoxanthine-guanine phosphoribosyl transferase (ARL/HGRPT) mutagenesis assay

1. Fresh human erythrocytes were treated with lytic and non-lytic combinations of phospholipases A2, C and sphingomyelinase. The 31P-NMR spectra of ghosts derived from such erythrocytes show that, in all cases, the residual phospholipids and lysophospholipids remain organized in a bilayer configuration. 2. A bilayer configuration of the (lyso)phospholipids was also observed after treatment of erythrocyte ghosts with various phospholipases even in the case that 98% of the phospholipid was converted into lysophospholipid (72%) and ceramides (26%). 3. A slightly decreased order of the phosphate group of phospholipid molecules, seen as reduced effective chemical shift anisotropy in the 31P-NMR spectra, was found following the formation of diacyglycerols and ceramides in the membrane of intact erythrocytes. Treatment of ghosts always resulted in an extensive decrease in the order of the phosphate groups. 4. The results allow the following conclusions to made: a. Hydrolysis of phospholipids in intact red cells and ghosts does not result in the formation of non-bilayer configuration of residual phospholipids and lysophospholipids. b. Haemolysis, which is obtained by subsequent treatment of intact cells with sphingomyelinase and phospholipase A2, or with phospholipase C, cannot be ascribed to the formation of non-bilayer configuration of phosphate-containing lipids. c. Preservation of bilayer structure, even after hydrolysis of all phospholipid, shows that other membrane constitutents, e.g. cholesterol and/or membrane proteins play an important role in stabilizing the structure of the erythrocyte membrane. d. A major prerequisite for the application of phospholipases in lipid localization studies, the preservation of a bilayer configuration during phospholipid hydrolysis, is met for the erythrocyte membrane.     

High D-glucose does not affect binding of alpha-tocopherol to human erythrocytes

The role of alpha-tocopherol uptake system in human erythrocyte in the uptake of plasma alpha-tocopherol has been suggested. However no information is available on alpha-tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of alpha-tocopherol to human erythrocytes, the binding characteristics of alpha-tocopherol to these cells were established first. Binding of [3H]alpha-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of alpha-tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 microM and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the alpha-tocopherol binding activity. Competition for the binding of alpha-tocopherol to human erythrocytes was effective with other homologues of alpha-tocopherol (beta-tocopherol, gamma-tocopherol and delta-tocopherol) and their potency was almost equal to alpha-tocopherol itself. The order of preference was alpha-tocopherol > beta-tocopherol > or = gamma-tocopherol > or = delta-tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect alpha-tocopherol uptake activity. Our data demonstrate the presence of an alpha-tocopherol uptake system in human erythrocytes and that the alpha-tocopherol uptake activity is not modulated by the presence of D-glucose.     

Impaired Ca2+-induced tyrosine phosphorylation and defective lipid scrambling in erythrocytes from a patient with Scott syndrome: a study using an inhibitor for scramblase that mimics the defect in Scott syndrome

Scott syndrome is an hereditary bleeding disorder characterized by a deficiency in platelet procoagulant activity. Unlike normal blood cells, Scott platelets, as well as erythrocytes and lymphocytes, are strongly impaired in their ability to scramble their membrane phospholipids when challenged with Ca2+. In normal cells this collapse of membrane asymmetry leads to surface exposure of phosphatidylserine. Here we report that Scott erythrocytes show an apparent defect in tyrosine phosphorylation on treatment with Ca2+-ionophore. Diminished tyrosine phosphorylation was also apparent in activated Scott platelets, but much less pronounced than observed in red blood cells. On the other hand, tyrosine phosphorylation profiles observed in Scott red blood cell ghosts after sealing in the presence of adenosine triphosphate (ATP) were indistinguishable from those obtained from normal ghosts. Several observations argue in favor of a mechanism in which tyrosine phosphorylation in red blood cells is facilitated by, rather than required for scrambling of membrane lipids. Staurosporin blocks tyrosine phosphorylation in normal red blood cells, but does not inhibit the lipid scrambling process. White ghosts from normal erythrocytes, resealed in the absence of ATP, exhibit Ca2+-induced lipid scrambling without tyrosine phosphorylation. A selective inhibitor of Ca2+-induced lipid scrambling also showed an apparent inhibition of tyrosine phosphorylation in ionophore-treated normal red blood cells, similar to that observed in Scott erythrocytes. While this inhibitor also suppressed Ca2+-induced lipid scrambling in ghosts that were sealed in the presence of ATP, it did not inhibit tyrosine kinase activity. We conclude that the apparent deficiency in tyrosine phosphorylation in Scott cells is an epiphenomenon, possibly associated with a defect in phospholipid scrambling, but not causal to this defect.     

Lipid peroxidation and antioxidant status in the liver, erythrocytes, and serum of rats after methanol intoxication

Lipid peroxidation products measured as a malondialdehyde and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), and concentrations of ascorbic acid, alpha-tocopherol, and glutathione (GSH) were measured in the liver, erythrocytes, and serum of rats 6, 14, and 24 h and 2, 5, and 7 d after treatment with 3 g methanol/kg. GSH-Px and GSSG-R activities, GSH level, and ascorbate concentration in the liver, erythrocytes, and blood serum were significantly decreased. In addition, SOD and alpha-tocopherol in erythrocytes were diminished, while malondialdehyde (MDA) in liver, erythrocytes, and serum were elevated. Further, erythrocyte counts, hemoglobin levels, hematocrit, and mean corpuscular volume (MCV) were reduced. These results indicate that methanol intoxication in rats leads to an increase in the lipid peroxidation and impairment in the antioxidant mechanisms in liver, erythrocytes, and blood serum.     

Reduction of the ascorbyl free radical to ascorbate by thioredoxin reductase

Recycling of ascorbic acid from its oxidized forms is required to maintain intracellular stores of the vitamin in most cells. Since the ubiquitous selenoenzyme thioredoxin reductase can recycle dehydroascorbic acid to ascorbate, we investigated the possibility that the enzyme can also reduce the one-electron-oxidized ascorbyl free radical to ascorbate. Purified rat liver thioredoxin reductase catalyzed the disappearance of NADPH in the presence of low micromolar concentrations of the ascorbyl free radical that were generated from ascorbate by ascorbate oxidase, and this effect was markedly stimulated by selenocystine. Dehydroascorbic acid is generated by dismutation of the ascorbyl free radical, and thioredoxin reductase can reduce dehydroascorbic acid to ascorbate. However, control studies showed that the amounts of dehydroascorbic acid generated under the assay conditions used were too low to account for the observed loss of NADPH. Electron paramagnetic resonance spectroscopy directly confirmed that the reductase decreased steady-state ascorbyl free radical concentrations, as expected if thioredoxin reductase reduces the ascorbyl free radical. Dialyzed cytosol from rat liver homogenates also catalyzed NADPH-dependent reduction of the ascorbyl free radical. Specificity for thioredoxin reductase was indicated by loss of activity in dialyzed cytosol prepared from livers of selenium-deficient rats, by inhibition with aurothioglucose at concentrations selective for thioredoxin reductase, and by stimulation with selenocystine. Microsomal fractions prepared from rat liver showed substantial NADH-dependent ascorbyl free radical reduction that was not sensitive to selenium depletion. These results suggest that thioredoxin reductase can function as a cytosolic ascorbyl free radical reductase that may complement cellular ascorbate recycling by membrane-bound NADH-dependent reductases.     

Accumulation and metabolism of low density lipoprotein-derived cholesteryl linoleate hydroperoxide and hydroxide by macrophages

Cholesteryl linoleate hydroperoxide (CLOOH) and hydroxide (CLOH) are present in human atheroma. The intracellular metabolism of low density lipoprotein (LDL)-derived CLOOH and CLOH remain undefined because extensive free radical-mediated LDL oxidation, which modifies LDL apolipoprotein B sufficiently to allow endocytosis by the scavenger receptor (ScR), also degrades CLOOH and CLOH. This problem was approached by first acetylating LDL lysine residues (AcLDL) to achieve protein modification, then exposing AcLDL to the aqueous radical donor 2,2'-azobis(2-amidinopropane) HCl (AAPH), to generate mildly oxidized AcLDL (OxAcLDL). Murine peritoneal macrophages incubated with OxAcLDL accumulated large quantities of CE and small, non-toxic quantities of CLOOH and CLOH in a time- and concentration-dependent manner, and accumulation was inhibited by fucoidin. Inhibition of acyl CoA: cholesterol acyltransferase during loading did not inhibit the accumulation of either CLOOH or CLOH, whereas NH4Cl decreased intracellular clearance of accumulated CLOOH from 68.3 +/- 1.7% to 35.3 +/- 1.0% over 12 h, suggesting lysosomal or pre-lysosomal accumulation. Intracellular clearance of unoxidized lipoprotein-derived CE decreased from 84.0 +/- 5.9% to 43.1 +/- 2.3% over 12 h when cells were loaded with AcLDL or OxAcLDL, respectively. Aggregation of mildly oxidized LDL, even without acetylation, also promoted cellular accumulation of CLOOH and CLOH. We conclude that intracellular accumulation of cholesteryl linoleate hydroperoxide and cholesteryl linoleate hydroxide can follow charge modification or aggregation of mildly oxidized LDL, and that LDL-derived oxidation products may inhibit hydrolysis of LDL-derived CE in foam cell macrophages.     

Peroxidase-catalyzed effects of indole-3-acetic acid and analogues on lipid membranes, DNA, and mammalian cells in vitro

This study aimed to explore the mechanisms and molecular parameters which control the cytotoxicity of derivatives of indole-3-acetic acid (IAA) when oxidatively activated by horseradish peroxidase (HRP). Lipid peroxidation was measured in liposomes, damage to supercoiled plasmid DNA assessed by gel electrophoresis, free radical intermediates detected by EPR following spin trapping, binding of IAA-derived products demonstrated by 3H labelling, stable products measured by HPLC, and cytotoxicity in hamster fibroblasts measured by clonogenic survival. IAA, and nine analogues more easily oxidized by HRP, caused lipid peroxidation in liposomes, but not detectably in membranes of hamster fibroblasts, and were cytotoxic after HRP activation to varying degrees. Cytotoxicity was not correlated with activation rate. The hydrophilic vitamin E analogue, Trolox, inhibited cytotoxicity, whereas loading fibroblasts with vitamin E was ineffective, consistent with an oxidative mechanism in which radical precursors to damage are intercepted by Trolox in the aqueous phase. However, two known oxidation products were nontoxic (the 3-carbinol and 3-aldehyde, both probably produced from 3-CH2OO* peroxyl radicals via the 3-CH*2 [skatolyl] radical following decarboxylation of the radical cation). The skatolyl radical from IAA was shown by EPR with spin trapping to react with DNA; electrophoresis showed binding to occur. Treatment of hamster fibroblasts with 5-3H-IAA/HRP resulted in intracellular bound 3H. Together with earlier results, the new data point to unknown electrophilic oxidation products, reactive towards intracellular targets, being involved in cytotoxicity of the IAA/HRP combination, rather than direct attack of free radicals, excited states, or membrane lipid peroxidation.     

Characterization of a membrane-associated, receptor and G-protein responsive phosphoinositide-specific phospholipase C from avian erythrocytes

We describe the reconstitution and purification of a membrane-associated phosphoinositide-specific phospholipase C (PIC) from turkey erythrocyte ghosts. This PIC is responsive to a G-protein coupled to P2y purinergic receptors which are expressed in turkey erythrocytes. Reconstitution is achieved by adding partially purified PIC to [3H]inositol-prelabelled turkey erythrocyte membranes depleted of their endogenous PIC (acceptor membranes). PIC activity is associated with a 52 kDa polypeptide on SDS-polyacrylamide gel electrophoresis. Addition of a 307-fold purified enzyme to the acceptor membranes has no effect on basal PIC activity, but markedly increases the response to GTP gamma S and P2y-purinergic receptor activation.     

Irreversible deformation of the spectrin-actin lattice in irreversibly sickled cells

Irreversibly sickled cells (ISC's) are circulating erythrocytes in patients with sickle cell disease that retain a sickled shape even when oxygenated. Evidence points to a membrane defect that prevents the return of these cells to the normal biconcave shape. The erythrocyte membrane protein spectrin is believed to help control erythrocyte shape and deformability. Recent studies suggest that normally spectrin and an erythrocyte actin form a self-supporting, fibrillar, lattice-like network on the cytoplasmic membrane surface. When normal erythrocyte ghosts are extracted with Triton X-100 all the integral membrane proteins and most of the membrane lipids are removed, leaving a ghost-shaped residue composed principally of spectrin and actin. We concentrated ISC's from patients with sickle cell anemia and compared the morphology and protein composition of ghosts and Triton-extracted ghost residues prepared from these ISC's with similar preparations of reversibly sickable cells and normal cells. (a) Many ISC's formed ISC-shaped ghosts. (b) All ISC-shaped ghosts formed ISC-shaped Triton residues. (c) Spectrin, erythrocyte actin (Band 5), an unidentified Band 3 component, and Band 4.1 were the major protein components of the Triton residues. All membrane-associated sickle hemoglobin was removed by the Triton treatment. (d) No ISC-shaped ghosts or ISC-shaped Triton residues were formed when deoxygenated, sickled RSC's were lysed or Triton-extracted. ISC-shaped ghosts and Triton residues were never formed from normal cells. These observations suggest that a defect of the "spectrin-actin lattice" may be the primary abnormality of the ISC membrane. Since ISC's are rigid cells, the data support the postulate that spectrin is a major determinant of membrane deformability. Finally, they provide direct evidence that spectrin is important in determining erythrocyte shape.     

The antioxidant properties of lycopene

The antioxidant properties of the carotenoid lycopene were compared in three different model oxidative systems. In egg yolk liposomes, in the presence of 2.5 mM FeSO4 and 200 mM ascorbate, lycopene, alpha-tocopherol, and beta-carotene inhibited the accumulation of lipid peroxidation products reacting with 2-thiobarbituric acid (TBARS) in a dose-dependent mode, with the concentration of half-inhibition being 80, 30 and 130 mM, respectively. In the liposomes subjected to illumination with a He-Ne laser (632.8 nm) at a dose of 10.5 J/cm2, in the presence of 32.5 micrograms/ml hematoporphyrin derivatives (Fotogem, NIOPIC, Russia) TBARS accumulated, and this effect was inhibited by lycopene, alpha-tocopherol, and dihydroquercetin with approximately equal efficiencies (the half-inhibition concentrations were 10(-5) mM). In both systems studied, sodium azide at a concentration of 10 mM inhibited the TBARS accumulation by no more than 20%. Apparently, the inhibitory action of not only alpha-tocopherol, but also beta-carotene and lycopene was the result of their antiradical action, rather than quenching of the singlet oxygen in an aqueous medium. The introduction of lycopene, as well as beta-carotene in liposomes subjected to Fe(2+)-induced lipid peroxidation decreased the chemiluminescence (CL) intensity at the stage of CL slow flash, with no essential influence on the lag period. These data suggest that the effect of lycopene on lipid peroxidation was the result of its interaction with free radicals rather than chelating ferrous ions. The antiradical activity of lycopene was also confirmed by the method of luminol photochemiluminescence (PCL). Lycopene increased the PCL lag period (L) and decreased the PCL amplitude (A), which implies its antiradical and SOD-like activity in this system.     

Oxidation and antioxidation of human low-density lipoprotein and plasma exposed to 3-morpholinosydnonimine and reagent peroxynitrite

As peroxynitrite is implicated as an oxidant for low-density lipoprotein (LDL) in atherogenesis, we investigated this process using reagent peroxynitrite (ONOO-) and 3-morpholinosydnonimine (SIN-1, which produces peroxynitrite via generation of NO. and O2.-). LDL oxidation was assessed by the consumption of ubiquinol-10 (CoQ10H2) and alpha-tocopherol (alpha-TOH), the accumulation of cholesteryl ester hydro(pero)xides, the loss of lysine (Lys) and tryptophan (Trp) residues, and the change in relative electrophoretic mobility. Exposure to ONOO- or SIN-1 resulted in rapid (<1 min) and time-dependent oxidation, respectively, of LDL's lipids and protein. Manipulating the alpha-TOH content by in vivo or in vitro means showed that when ONOO- or SIN-1 was used at oxidant-to-LDL ratios of <100:1 the extent of LDL lipid peroxidation increased with increasing initial alpha-TOH content. In contrast, in vivo enrichment with the co-antioxidant CoQ10H2 decreased LDL lipid peroxidation induced by SIN-1. At oxidant-to-LDL ratios of >200:1, alpha-TOH enrichment decreased LDL lipid peroxidation for both SIN-1 and ONOO-. In contrast to lipid peroxidation, altering the alpha-TOH content of LDL did not affect Trp or Lys loss, independent of the amounts of either oxidant added. Aqueous antioxidants inhibited ONOO--induced lipid and protein oxidation with the order of efficacy: 3-hydroxyanthranilate (3-HAA) > urate > ascorbate. With SIN-1, these antioxidants inhibited Trp consumption, while only the co-antioxidants ascorbate and 3-HAA prevented alpha-TOH consumption and lipid peroxidation. Exposure of human plasma to SIN-1 resulted in the loss of ascorbate followed by loss of CoQ10H2 and bilirubin. Lipid peroxidation was inhibited during this period, though proceeded as a radical-chain process after depletion of these antioxidants and in the presence of alpha-TOH and urate. Bicarbonate at physiological concentrations decreased ONOO--induced lipid and protein oxidation, whereas it enhanced SIN-1-induced lipid peroxidation, Trp consumption, and alpha-tocopheroxyl radical formation in LDL. These results indicate an important role for tocopherol-mediated peroxidation and co-antioxidation in peroxynitrite-induced lipoprotein lipid peroxidation, especially when peroxynitrite is formed time-dependently by SIN-1. The studies also highlight differences between ONOO-- and SIN-1-induced LDL oxidation with regards to the effects of bicarbonate, ascorbate, and urate.     

Prevention of phorbol myristate acetate-induced acute lung injury by alpha-tocopherol liposomes

Phorbol-myristate acetate (PMA) is commonly used to produce experimental edema and other tissue injuries in the lung. Lung injuries induced by the administration of PMA has been shown to be mediated mainly by neutrophils. Neutrophils recruited to the lower respiratory tract may damage lung tissues by releasing reactive oxygen species, neutral proteases, and lysosomal enzymes. The present study was conducted to investigate whether alpha-tocopherol, entrapped in dipalmitoylphosphatidylcholine liposomes and delivered directly to the lung, could counteract some of the PMA-induced lung injuries. Plain liposomes or alpha-tocopherol containing liposomes (8 mg alpha-tocopherol/kg body weight) were intratracheally instilled into the lungs of rats 24 hr prior to PMA exposure (25 micrograms/kg) and treated rats were killed 3 hr later. Lungs of control animals exposed to PMA developed an increase in lung weight and lipid peroxidation as well as a decrease in lung angiotensin converting enzyme (ACE) and alkaline phosphatase (AKP) activities. PMA treatment also caused an increase in myeloperoxidase (MPO) activity in the lung, suggestive of neutrophil infiltration. Pretreatment of PMA-treated rats with plain liposomes had no effect on PMA-induced injuries. In contrast, pretreatment of rats with liposomal alpha-tocopherol, 24 hr prior to PMA administration, resulted in a significant elevation of pulmonary alpha-tocopherol concentration, accompanied by a concomitant reduction in MPO activity and reversal of PMA-induced changes in lung edema, lipid peroxidation, ACE and AKP activities. These results appear to demonstrate that the intratracheal administration of a liposome-associated lipophilic antioxidant, such as alpha-tocopherol, can significantly ameliorate the toxic effects of reactive oxygen species, putatively released from PMA-stimulated pulmonary target cells and infiltrating neutrophils.     

Effect of antioxidant in endothelial cells exposed to oxidized low-density lipoproteins

Antioxidants such as probucol and alpha-tocopherol have been shown to attenuate the oxidation of low-density lipoproteins (LDL) and atherosclerotic lesions in animal models of atherosclerosis. The purpose of this study is to determine the protection effect of antioxidants on endothelial cells when exposed to oxidized and native LDL. In a cell-free system, we found that probucol, alpha-tocopherol, and ascorbic acid inhibited copper-induced LDL oxidation by a dose-dependent fashion (from 1 microM to 10 mM). In porcine aortic endothelial cells, antioxidants alone did not change basal endothelin-1 (ET-1) secretion. When porcine aortic endothelial cells were exposed to LDL and oxidized-LDL, both of them stimulated ET-1 secretion dose-dependently, whereas oxidized-LDL elicited higher ET-1 secretion. However, probucol, alpha-tocopherol, and ascorbic acid did not prevent LDL or oxidized-LDL induced ET-1 secretion. Furthermore, nimodipine inhibited both of native and oxidized LDL induced ET-1 secretion. Since Ca2+ channel blocker reduced the elevation of induced ET-1 secretion, the [Ca2+]i is possibly involved for the regulation of ET-1 secretion. Our results suggest that antioxidants can only prevent the oxidation of LDL rather than oxidized and native LDL-induced ET-1 secretion in vascular endothelial cells. The increase in the [Ca2+]i of endothelial cells through the opening of voltage-dependent Ca2+ channels may be involved in the LDL-induced ET-1 release.     

Calcium induces phospholipid redistribution and microvesicle release in human erythrocyte membranes by independent pathways

The increase in intracellular Ca2+ concentration in erythrocytes and platelets results in simultaneous phospholipid scrambling and microvesicle shedding. Microvesicle formation involves membrane fusion events which were proposed either to be tightly linked to phospholipid transversal redistribution or to occur by a separate mechanism. We report here that in erythrocytes incubated in high K+ medium, or in resealed ghosts, phospholipid scrambling can be fully induced by intracellular Ca2+ without microvesicle formation. Furthermore, in ghosts resealed in the presence of spermine, intracellular Ca2+, at low concentration, was able to induce microvesicles, whereas scrambling was drastically inhibited. Surprisingly, in spermine-containing ghosts prepared from erythrocytes of a patient with a bleeding disorder, due to a lack of Ca2+-induced phospholipid scrambling and vesicle shedding (characterized as a Scott syndrome), Ca2+ also promoted microvesicle release. Data show that phospholipid scrambling and microvesicle production, although closely regulated, proceed by independent pathways.