ascorbic-acid and 2-2--azobis(2-amidinopropane)

Chain breaking antioxidants scavenge the chain carrying oxygen radicals and suppress the peroxidation of liposomal and biological membranes in aqueous dispersions. Vitamin E scavenges peroxyl radicals rapidly and its lateral diffusion is suggested to be fast, but its antioxidant efficiency in the liposomal and bio-membranes appears to be considerably smaller than in homogeneous solution. Water soluble chain breaking antioxidants, such as uric acid, cysteine, glutathione, and vitamin C, scavenge radicals in the aqueous region and suppress the peroxidation. However, they cannot scavenge the peroxyl radicals within the lipid region of the membranes. Nevertheless, vitamin C can interact with vitamin E radical, probably at membrane-water interface, and regenerate vitamin E.

Topics: Amidines; Antioxidants; Ascorbic Acid; Free Radicals; Lipid Peroxides; Vitamin E

To evaluate the in vivo radical scavenger activity of vitamin E, vitamin C, and beta carotene on erythrocyte membranes.. A prospective, open trial without placebo.. Department of Clinical Pharmacy.. Ten healthy volunteers being supplemented with beta carotene, vitamin E, and vitamin C.. Erythrocytes were incubated in water bath with 2,2' azobis (2 amidinopropane) hydrochloride (AAPH). AAPH decomposes spontaneously at 37 degrees C to generate free radicals inducing membrane cellular damage and hemolysis. The absorbance was measured at 405 nm at 0, 30, and 60 min, and then every 20 minutes for four hours. The time for 50 percent of maximal hemolysis (T50%), which expresses the radical scavenger activity of erythrocytes, was determined.. The physiologic T50% value determined in 52 healthy volunteers is 117 +/- 12 min. Patients receiving these supplements have a higher value of T50% (143.2 +/- 11.6 min at 30 d and 145.7 +/- 10.5 min at 60 d) than the physiologic value (p < 0.001).. These data suggest that vitamin C, vitamin E, and beta carotene stimulate the radical scavenger activity of erythrocyte membranes after 30 days.

Topics: Amidines; Ascorbic Acid; beta Carotene; Carotenoids; Erythrocyte Membrane; Free Radical Scavengers; Humans; Male; Prospective Studies; Vitamin E

Emerging viruses are a public health threat best managed with broad spectrum antivirals. Common viral structures, like capsids or virion envelopes, have been proposed as targets for broadly active antiviral drugs. For example, a number of lipoperoxidators have been proposed to preferentially affect viral infectivity by targeting metabolically inactive enveloped virions while sparing metabolically active cells. However, this presumed preferential virion sensitivity to lipoperoxidation remains untested. To test whether virions are indeed more sensitive to lipoperoxidation than are cells, we analyzed the effects of two classic generic lipoperoxidators: lipophilic 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) and hydrophilic 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) on Vero and human foreskin fibroblasts (HFF) cell viability, HSV-1 plaquing efficiency, and virion and cell lipoperoxidation. Cells or virions were incubated with the lipoperoxidators at 37°C for 2 h or incubated in atmospheric O

Topics: Antiviral Agents; Ascorbic Acid; Humans; Lipid Peroxidation; Serum Albumin, Bovine; Virion; Water

Topics: Amidines; Animals; Ascorbic Acid; Benzhydryl Compounds; Biphenyl Compounds; Butylated Hydroxyanisole; Butylated Hydroxytoluene; Chromans; Erythrocytes; Free Radical Scavengers; Hydrazines; Hydrogen Peroxide; Imidazolines; Nitric Oxide; Picrates; Rats; Structure-Activity Relationship

Oxygen radical absorbance capacity (ORAC) assay in 96-well multi-detection plate readers is a rapid method to determine total antioxidant capacity (TAC) in biological samples. A disadvantage of this method is that the antioxidant inhibition reaction does not start in all of the 96 wells at the same time due to technical limitations when dispensing the free radical-generating azo initiator 2,2'-azobis (2-methyl-propanimidamide) dihydrochloride (AAPH). The time delay between wells yields a systematic error that causes statistically significant differences in TAC determination of antioxidant solutions depending on their plate position. We propose two alternative solutions to avoid this AAPH-dependent error in ORAC assays.

Topics: Amidines; Antioxidants; Ascorbic Acid; Biological Assay; Chromans; Fluorescence; Gallic Acid; Oxidants; Oxygen Radical Absorbance Capacity; Reactive Oxygen Species

The stable ascorbic acid (AA) derivative, 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), exhibits vitamin C activity after enzymatic hydrolysis to AA. The biological activity of AA-2G per se has not been studied in detail, although AA-2G has been noted as a stable source for AA supply. The protective effect of AA-2G against the oxidative cell death of human dermal fibroblasts induced by incubating with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) for 24 h was investigated in this study. AA-2G showed a significant protective effect against the oxidative stress in a concentration-dependent manner. AA-2G did not exert a protective effect during the initial 12 h of incubation, but had a significant protective effect in the later part of the incubation period. Experiments using a α-glucosidase inhibitor and comparative experiments using a stereoisomer of AA-2G confirmed that AA-2G had a protective effect against AAPH-induced cytotoxicity without being converted to AA. Our results provide an insight into the efficacy of AA-2G as a biologically interesting antioxidant and suggest the practical use of AA-2G even before being converted into AA as a beneficial antioxidant.

Topics: Amidines; Ascorbic Acid; Cytotoxins; Drug Stability; Fibroblasts; Free Radical Scavengers; Free Radicals; Humans; Oxidative Stress; Skin; Time Factors

Resveratrol (RES) is a well-known antioxidant, yet in combination with other antioxidant vitamins, it was found to be more effective than any of these antioxidants alone. Present work aims to compare the antioxidant actions of resveratrol with and without vitamin C following delivery as liposomes tested using chemical and cellular antioxidative test systems.. Liposomes were prepared by the thin film hydration method and characterised for percent drug entrapment (PDE), Z-average mean size (nm), polydispersity index (PDI) and zeta potential. Antioxidative capacity was determined by studying the inhibition of AAPH induced luminol enhanced chemiluminescence and inhibition of ROS production in isolated blood leukocytes. Intracellular oxygen-derived radicals were measured using flow cytometry with buffy coats (BC) and human umbilical vein endothelial cells using H2DCF-DA dye.. Particle size varied from 134.2 ± 0.265 nm to 103.3 ± 1.687 nm; PDI ≤ 0.3; zeta potential values were greater than -30 mV and PDE ≥ 80%. Radical scavenging effect was enhanced with liposomal systems; oxidative burst reaction in BC was inhibited by liposomal formulations, with the effect slightly enhanced in presence of vitamin C. Reduction in reactive oxygen species (ROS) production during spontaneous oxidative burst of BC and incubation of HUVECs with H2O2 further intensified the antioxidative effects of pure RES and liposomal formulations.. The present work clearly shows that the antioxidative effects of resveratrol loaded into liposomes are more pronounced when compared to pure resveratrol. Liposomal resveratrol is even active within the intracellular compartments as RES could effectively quench the intracellular accumulation of ROS.

Topics: Amidines; Antioxidants; Area Under Curve; Ascorbic Acid; Cell Survival; Coloring Agents; Drug Carriers; Free Radical Scavengers; Humans; Indicators and Reagents; Leukocytes; Liposomes; Luminescence; Luminol; Oxidative Stress; Particle Size; Reactive Oxygen Species; Resveratrol; Stilbenes; Tetrazolium Salts; Thiazoles; Vitamins

High levels of oxidative stress were reported in obesity-linked type 2 diabetes and were associated with elevated formation of advanced glycation end products (AGEs). Many studies have focused on the effect of antioxidants on vascular and circulating cells such as macrophages. However, despite the major role of adipocytes in the etiology of diabetes, little is known about the effect of natural antioxidants on adipocyte response to oxidative stress. The present study reports the differential protective effects of plant nutrients toward adipose cells subjected to oxidative stress. Caffeic acid, quercetin, L: -ascorbic acid, and alpha-tocopherol were tested on SW872 liposarcoma cells subjected to a free radical generator or to AGEs. Proliferation, viability, free radical formation, and superoxide dismutase expression were assessed in treated cells. Caffeic acid and quercetin appeared as the most potent antioxidant nutrients. Our findings clearly show a novel antioxidant role for caffeic acid and quercetin at the adipose tissue level. These new data confirm the beneficial role of phytotherapy as an interesting alternative mean for the development of novel therapeutical and nutritional strategy to prevent metabolic disorders inherent to obesity-linked diabetes.

Topics: Adipocytes; Amidines; Animals; Antioxidants; Ascorbic Acid; Caffeic Acids; Cattle; Cell Line; Cell Proliferation; Cell Survival; Diabetes Mellitus, Type 2; Free Radicals; Glycation End Products, Advanced; Humans; Obesity; Oxidants; Oxidative Stress; Phytotherapy; Quercetin; Serum Albumin, Bovine; Superoxide Dismutase; Tocopherols; Up-Regulation

The effects of oxidative stress (OS) on the rat erythrocytes (RBCs) that were fractionated on the percoll/BSA gradient into young and old cells were studied to find out if the altered Na+/H+ and Cl⁻/ HCO3⁻ antiporters and in turn the intracellular pH (pHi) could act as one of the promoters of cell death. Old cells were more spherical with lesser surface area, more fragile osmotically and had lesser protein sulphydryl content than the young cells. OS was induced in RBCs by 2,20-azobis (2-amidinopropane) dihydrochloride (AAPH). AAPH increased the superoxide dismutase (SOD) activity and MDA level and, the changes between the young and old. Interestingly, vitamin C was effective in reducing MDA in the old. Further, in the old a rapid Na+-dependent acidification in the presence of AAPH and a marginal acidosis in the presence of vitamin C were evident. Old RBCs exhibited higher acidosis and vitamin C was less effective in lowering the stress-induced acidosis compared to the young. Our studies suggest that increased acidosis followed by low intracellular pH could be one of the determinant factors for the disappearance of old RBCs from circulation, and perhaps of the young too under OS.

Topics: Amidines; Analysis of Variance; Animals; Antioxidants; Ascorbic Acid; Cellular Senescence; Chloride-Bicarbonate Antiporters; Erythrocytes; Hydrogen-Ion Concentration; Lipid Peroxidation; Male; Malondialdehyde; Osmotic Fragility; Oxidants; Oxidative Stress; Rats; Sodium-Hydrogen Exchangers; Superoxide Dismutase

Hepatocytes expressing liver fatty acid binding protein (L-FABP) are known to be more resistant to oxidative stress than those devoid of this protein. The mechanism for the observed antioxidant activity is not known. We examined the antioxidant mechanism of a recombinant rat L-FABP in the presence of a hydrophilic (AAPH) or lipophilic (AMVN) free radical generator. Recombinant L-FABP amino acid sequence and its amino acid oxidative products following oxidation were identified by MALDI quadrupole time-of-flight MS after being digested by endoproteinase Glu-C. L-FABP was observed to have better antioxidative activity when free radicals were generated by the hydrophilic generator than by the lipophilic generator. Oxidative modification of L-FABP included up to five methionine oxidative peptide products with a total of approximately 80 Da mass shift compared with native L-FABP. Protection against lipid peroxidation of L-FABP after binding with palmitate or alpha-bromo-palmitate by the AAPH or AMVN free radical generators indicated that ligand binding can partially block antioxidant activity. We conclude that the mechanism of L-FABP's antioxidant activity is through inactivation of the free radicals by L-FABP's methionine and cysteine amino acids. Moreover, exposure of the L-FABP binding site further promotes its antioxidant activity. In this manner, L-FABP serves as a hepatocellular antioxidant.

Topics: alpha-Tocopherol; Amidines; Animals; Antioxidants; Ascorbic Acid; Azo Compounds; Escherichia coli; Fatty Acid-Binding Proteins; Fluoresceins; Fluorescence; Free Radicals; Glutathione Transferase; Lipid Peroxidation; Nitriles; Rats; Recombinant Proteins

In mammals, aging is linked to a decline in the activity of citrate synthase (CS; E.C. 2.3.3.1), the first enzyme of the citric acid cycle. We used 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), a water-soluble generator of peroxyl and alkoxyl radicals, to investigate the susceptibility of CS to oxidative damage. Treatment of isolated mitochondria with AAPH for 8-24 h led to CS inactivation; however, the activity of aconitase, a mitochondrial enzyme routinely used as an oxidative stress marker, was unaffected. In addition to enzyme inactivation, AAPH treatment of purified CS resulted in dityrosine formation, increased protein surface hydrophobicity, and loss of tryptophan fluorescence. Propyl gallate, 1,8-naphthalenediol, 2,3-naphthalenediol, ascorbic acid, glutathione, and oxaloacetate protected CS from AAPH-mediated inactivation, with IC(50) values of 9, 14, 34, 37, 150, and 160 muM, respectively. Surprisingly, the antioxidant epigallocatechin gallate offered no protection against AAPH, but instead caused CS inactivation. Our results suggest that the current practice of using the enzymatic activity of CS as an index of mitochondrial abundance and the use of aconitase activity as an oxidative stress marker may be inappropriate, especially in oxidative stress-related studies, during which alkyl peroxyl and alkoxyl radicals can be generated.

Topics: Aconitate Hydratase; Amidines; Antioxidants; Ascorbic Acid; Catechin; Citrate (si)-Synthase; Enzyme Activation; Glutathione; Inhibitory Concentration 50; Mitochondria; Naphthols; Oxaloacetic Acid; Oxidants; Oxidation-Reduction; Oxidative Stress; Peroxides; Propyl Gallate

The antioxidant activity of a provitamin C agent, 2-O-beta-D-glucopyranosyl-L-ascorbic acid (AA-2betaG), was compared to that of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) and ascorbic acid (AA) using four in vitro methods, 1,1-diphenyl-picrylhydrazyl (DPPH) radical-scavenging assay, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS(*+))-scavenging assay, oxygen radical absorbance capacity (ORAC) assay, and 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte hemolysis inhibition assay. AA-2betaG slowly and continuously scavenged DPPH radicals and ABTS(*+) in roughly the same reaction profiles as AA-2G, whereas AA quenched these radicals immediately. In the ORAC assay and the hemolysis inhibition assay, AA-2betaG showed similar overall activities to AA-2G and to AA, although the reactivity of AA-2betaG against the peroxyl radical generated in both assays was lower than that of AA-2G and AA. These data indicate that AA-2betaG had roughly the same radical-scavenging properties as AA-2G, and a comprehensive in vitro antioxidant activity of AA-2betaG appeared to be comparable not only to that of AA-2G but also to that of AA.

Topics: Amidines; Animals; Antioxidants; Ascorbic Acid; Benzothiazoles; Erythrocytes; Hemolysis; Hydrogen-Ion Concentration; Molecular Structure; Sheep; Sulfonic Acids

We have previously demonstrated that acute arginine administration induces oxidative stress and compromises energy metabolism in rat hippocampus. In the present study, we initially investigated the effect of intracerebroventricular infusion of arginine (0.1, 0.5 and 1.5 mM solution) on Na(+),K(+)-ATPase activity and on some parameters of oxidative stress, namely thiobarbituric acid-reactive substances (TBA-RS) and total radical-trapping antioxidant parameter (TRAP) in the hippocampus of rats. Results showed that 1.5 mM arginine solution significantly increases TBA-RS and reduces Na(+),K(+)-ATPase activity and TRAP in the rat hippocampus. We also evaluated the influence of the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), and antioxidants, namely alpha-tocopherol plus ascorbic acid, on the effects elicited by arginine on Na(+),K(+)-ATPase activity, TBA-RS and TRAP. Results showed that treatment with alpha-tocopherol plus ascorbic acid per se did not alter these parameters but prevented these effects. Furthermore, intracerebroventricular infusion of L-NAME prevented the inhibition caused by arginine on Na(+),K(+)-ATPase activity, as well as the increased of TBA-RS. Our findings indicate that intracerebroventricular infusion of arginine induces oxidative stress in rat hippocampus and that the inhibition of Na(+),K(+)-ATPase activity caused by this amino acid was probably mediated by NO and/or its derivatives ONOO(-) and/or other free radicals. Finally, we suggest that the administration of antioxidants should be considered as an adjuvant therapy to specific diets in hyperargininemia.

Topics: alpha-Tocopherol; Amidines; Analysis of Variance; Animals; Antioxidants; Arginine; Ascorbic Acid; Brain Injuries; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Hippocampus; Injections, Intraventricular; Male; NG-Nitroarginine Methyl Ester; Oxidative Stress; Rats; Rats, Wistar; Sodium-Potassium-Exchanging ATPase; Thiobarbituric Acid Reactive Substances

A new method has been developed for the determination of total vitamin C in foods. The method requires less time than the traditional methodologies and uses a radical oxidation of L-ascorbic acid (AA) to obtain dehydro-L-ascorbic acid (DHAA) by means of a peroxyl radical generated in situ by thermal decomposition of an azo-compound, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The dehydro-L-ascorbic acid is condensed with benzene-1,2-diamine (o-phenylenediamine, OPDA) to form its highly fluorescent quinoxaline derivative, 3-(1,2-dihydroxyethyl)furo[3,4-b]quinoxaline-1-one (DFQ), which is then separated on a C(18) column eluted with a mobile phase of 80 mM phosphate buffer and methanol at pH=7.8 and detected fluorometrically at lambda(ex)=355 nm and lambda(em)=425 nm. The reaction conditions for the complete conversion of AA to DFQ were 56 degrees C, 36 min and a mumol AAPH/AA ratio of 60. The sample, extracted with an aqueous metaphosphoric acid solution, was analyzed after being filtered through a 0.45 microm membrane. The method has shown good repeatability, sensitivity and accuracy compared to the results obtained with the reference method. The response of the detection system was linear within a range of 0.5-8.0 microg/mL with a correlation coefficient of 0.9997. The limit of detection was 0.27 microg/mL and the limit of quantification was 0.83 microg/mL. The AA contents of some selected foods were analyzed.

Topics: Amidines; Ascorbic Acid; Chromatography, High Pressure Liquid; Dehydroascorbic Acid; Fluorometry; Food Analysis; Oxidation-Reduction

Vitamin C is a popular antioxidant; however, its water solubility limits its function in the lipid environment. As a result, the antioxidative properties of its lipophilic derivatives have aroused research attention, especially L-ascorbyl-6-laurate (VC-12). We have investigated the effect of a high concentration of VC-12 on 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH)-induced haemolysis of human erythrocytes. The findings indicated that VC-12 was capable of protecting erythrocytes against AAPH-induced haemolysis when its concentration was below 60 microM. With an increase in the concentration of VC-12 and a decrease in the concentration of AAPH, VC-12 promoted haemolysis remarkably, the mechanism of which has been proposed as VC-12-mediated peroxidation. When the concentration of VC-12 was increased to above 150 microM, VC-12 promoted haemolysis by its function as a surfactant, to unbalance the osmotic pressure within and outside erythrocytes. High concentrations of VC-12 may have generated radicals via autooxidation, resulting in eventual haemolysis. Therefore, the double-faced effect of VC-12 on haemolysis of human erythrocytes was due to its concentration. This information may be useful for the safe use of lipophilic vitamin C.

Topics: Amidines; Antioxidants; Ascorbic Acid; Dose-Response Relationship, Drug; Erythrocytes; Free Radicals; Hemolysis; Humans; Lipid Peroxidation; Osmotic Pressure; Oxidants; Oxidation-Reduction

Inhibitory effects of 2-O-substituted ascorbic acid derivatives, ascorbic acid 2-glucoside (AA-2G), ascorbic acid 2-phosphate (AA-2P), and ascorbic acid 2-sulfate (AA-2S), on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of sheep erythrocytes were studied and were compared with those of ascorbic acid (AA) and other antioxidants. The order of the inhibition efficiency was AA-2S> or =Trolox=uric acid> or =AA-2P> or =AA-2G=AA>glutathione. Although the reactivity of the AA derivatives against AAPH-derived peroxyl radical (ROO(*)) was much lower than that of AA, the derivatives exerted equal or more potent protective effects on AAPH-induced hemolysis and membrane protein oxidation. In addition, the AA derivatives were found to react per se with ROO(*), not via AA as an intermediate. These findings suggest that secondary reactions between the AA derivative radical and ROO(*) play a part in hemolysis inhibition. Delayed addition of the AA derivatives after AAPH-induced oxidation of erythrocytes had already proceeded showed weaker inhibition of hemolysis compared to that of AA. These results suggest that the AA derivatives per se act as biologically effective antioxidants under moderate oxidative stress and that AA-2G and AA-2P may be able to act under severe oxidative stress after enzymatic conversion to AA in vivo.

Topics: Amidines; Animals; Antioxidants; Ascorbic Acid; Erythrocyte Membrane; Free Radicals; Hemolysis; Sheep; Time Factors

Aqueous extract of Andrographis paniculata was examined for antioxidant activity using rat liver subcellular organelles as model systems. The study deals with two important biological oxidative agents, ascorbate-Fe(+2) and AAPH generating hydroxyl and peroxyl radical, respectively. Oxidative damage was examined against the inhibition of membrane peroxidation, protein oxidation and restoration in decreased SOD and catalase activity. The antimutagenic activity of Ap was examined following inhibition in AAPH induced strand breaks in plasmid pBR322 DNA. Extract was a potent scavenger of DPPH, ABTS radicals, exemplified by ESR signals, O2-*, *OH and H2O2, displayed excellent reducing power, FRAP potentials to reduce Fe (III) --> Fe (II) and had considerable amount of phenolics/ flavonoids contents, an effective antioxidant index. The observed antioxidant effect might be primarily due to its high scavenging ability for ROS. Effect was confirmed ex vivo following inhibition in peroxidation, restoration in SOD enzyme, SOD band intensity and protein degradation in Ap fed liver homogenate. Based on these results, it was concluded that the aqueous extract of Andrographis paniculata might emerge as a potent antiradical agent against various pathophysiological oxidants.

Topics: Amidines; Andrographis; Animals; Ascorbic Acid; DNA Damage; Female; Free Radical Scavengers; Free Radicals; In Vitro Techniques; Liver; Mice; Microsomes, Liver; Oxidants; Oxidative Stress; Plant Extracts; Rats; Rats, Wistar; Subcellular Fractions

Free-radical-induced peroxidation in-vivo is regarded as the aetiology of some diseases and free-radical-scavenging drugs, also called antioxidants (AH), have been widely used to overcome oxidative stress. An in-vitro experimental method, 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH)-induced haemolysis of human erythrocytes can be applied to assess the free-radical-scavenging activity of a drug. The major objectives of this work were focused on three aspects. Firstly, introduction of the chemical kinetic deduction of free-radical-initiating reaction to AAPH-induced haemolysis of human erythrocytes, by which the number of free radicals trapped by an antioxidant, n, can be obtained after finding the quantitative relationship between the inhibition period (t(inh)) and the concentration of the antioxidant, t(inh) = (n/Ri) [AH]. Ri, the free-radical-initiating rate, was initially confirmed by using alpha-tocopherol (VE) whose n was taken as 2. Secondly, the free-radical-scavenging activity of diclofenac acid (DaH) and its sodium salt (DaNaH) was assessed. It has been found that DaH and DaNaH protect human erythrocytes against AAPH-induced haemolysis dose-dependently. In particular, the n values of DaH and DaNaH (4.96 and 3.60) were much higher than some traditional antioxidants, such as 6-hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a water-soluble structural analogue of VE, n = 0.30) and L-ascorbic acid (VC, n = 0.25), and L-ascorbyl-6-laurate (VC-12, a lipophilic structural analogue of VC, n = 1.11). Moreover, the free-radical-scavenging activity of lipophilic antioxidants is higher than the corresponding water-soluble species. Thirdly, the free-radical-scavenging activity of mixed antioxidants, VE + DaH, VC-12 + DaH, Trolox + DaNaH and VC + DaNaH, was revealed. The n value of VC, VC-12, VE and Trolox increase in the case of mixed usage with DaH and DaNaH, implying that diclofenac acid can repair the radical of these antioxidants. Thus, a mutual antioxidant effect between diclofenac acid and these antioxidants prolongs the lifespan of VC, VC-12, VE and Trolox, respectively.

Topics: Amidines; Ascorbic Acid; Chromans; Diclofenac; Dose-Response Relationship, Drug; Drug Combinations; Erythrocytes; Free Radical Scavengers; Free Radicals; Hemolysis; Humans; In Vitro Techniques; Solubility

Reactive oxygen species (ROS) are continuously formed in biological systems. Any increase in radical production or decrease in the defense against ROS induces oxidative stress. This imbalance between ROS formation and ROS detoxification is believed to be involved in a variety of pathogenic processes, including ischemia-reperfusion injury. Various markers indicating oxidative stress has been used in experimental and clinical studies. One of them is ascorbate free radical (AFR), electron spin resonance intensity of which correlates with the severity of radical formation. We investigated the impact of alkyl peroxyl radicals produced by 2,2-Azobis (2-amidinopropane) dihydrochloride decomposition on the magnitude of the AFR signal. Our data confirmed the principal applicability of AFR as a nontoxic marker of radical generation.

Topics: Amidines; Ascorbic Acid; Electron Spin Resonance Spectroscopy; Free Radicals; Oxidants; Oxidative Stress; Oxygen Consumption; Reactive Oxygen Species

Ascorbic acid is present as a primary antioxidant in plasma and within cells, protecting both cytosolic and membrane components of cells from oxidative damage. The effects of intracellular ascorbic acid on F(2)-isoprostanes (biomarkers of oxidative stress) and monocyte chemoattractant protein-1 (marker of inflammatory responses) production in monocytic THP-1 cells were investigated under conditions of 2,2'-Azobis(2-methylpropionamidine)dihydrochloride (AAPH) induced oxidative stress. Cells cultured under normal conditions have extremely low ascorbate levels and the intracellular ascorbate can be augmented significantly by adding ascorbate to the culture medium. While AAPH treatment reduced cell viability, increased F(2)-isoprostanes and MCP-1 production, the presence of intracellular ascorbic acid maintained high cell viability and attenuated both F(2)-isoprostanes and MCP-1 production. Measurement of intracellular ascorbic acid and its oxidised products showed that intracellular ASC was oxidised to a significantly greater extent during AAPH treatment and may be utilised to protect the cells under conditions of oxidative stress. This study demonstrates the importance of intracellular ascorbate, which may be lacking under normal cell culture conditions, under conditions of increased oxidative stress.

Topics: Amidines; Ascorbic Acid; Cell Culture Techniques; Cell Line; Cell Membrane; Cell Survival; Chemokine CCL2; Cytosol; F2-Isoprostanes; Humans; Inflammation; Monocytes; Oxidative Stress; Time Factors

Assessment of the antioxidant activity of vitamins and other compounds is of interest in the understanding of their in vivo effects. In this study, we have investigated the activity of several lipid and water-soluble vitamins in human whole blood. Measurements were carried out using a biological test that enables the evaluation of both red blood cells and plasma resistance against free radical activity induced by 2,2'-azobis (2-amidinopropane)hydrochloride (AAPH). Antioxidant activity of vitamins has been determined by using the biological test versus chemical methods (chemiluminescence, DMPD radical). We have observed strong anti-oxidant potentials for vitamins B6 and B9 with biological tests, but not with chemical methods. At 10 microM, the vitamin B9 efficiency in inhibiting radical-induced red blood cell hemolysis was almost three times higher than vitamin C efficiency and two times higher than alpha-tocopherol efficiency. Antioxidant activity was not observed for vitamins B1 or B2, nor for retinol. The weak activity of beta-carotene still remains to be investigated particularly in relation to oxygen pressure. Our study demonstrated that the biological test is more useful than the chemical methods employed in this instance, for the evaluation of antioxidant capacity of lipophilic and putatively biologically active compounds.

Topics: alpha-Tocopherol; Amidines; Antioxidants; Ascorbic Acid; beta Carotene; Biological Assay; Caffeic Acids; Dose-Response Relationship, Drug; Fluorescent Dyes; Hemolysis; Humans; Luminescent Measurements; Oxidants; Phenylenediamines; Reference Values; Solubility; Vitamin A; Vitamin B Complex; Vitamins

The aims of the study are to analyze the interaction between 5-aminosalicylic acid (5-ASA) and peroxyl radicals and to evaluate the effect of some endogenous compounds such as ascorbic acid and amino acids on the oxidation of 5-ASA induced by 2,2'-azo-bis(2-amidinopropane) dihydrochloride.. The consumption and/or the recovery of 5-ASA (7.6 microM) exposed to a peroxyl radical source [2,2'-azo-bis(2-amidinopropane)] was followed by techniques such as spectrofluorescence, high-performance liquid chromatography, and differential pulse voltammetry.. 5-Aminosalicylic acid was found to readily react with peroxyl radicals at micromolar concentrations and to protect c-Phycocyanin in a very similar fashion to that shown by Trolox. Exposure of 5-ASA to peroxyl radicals led to its oxidation into the corresponding quinone-imine. Disappearance of 5-ASA was prevented by tryptophan, cysteine, glutathione, and ascorbic acid. Furthermore, some of these compounds induced the partial (cysteine and glutathione) or total (ascorbic acid) recovery of 5-ASA when added after its almost total consumption.. 5-Aminosalicylic acid is a very efficient peroxyl radical scavenger. The 5-ASA oxidation by peroxyl radicals was prevented by ascorbic acid, cysteine, and glutathione. In addition, 5-ASA can be regenerated by these endogenous compounds, which would be a valuable mechanism to preserve 5-ASA in tissues undergoing oxidative stress conditions.

Topics: Amidines; Anti-Inflammatory Agents, Non-Steroidal; Ascorbic Acid; Cysteine; Electrochemistry; Mesalamine; Oxidants; Oxidation-Reduction; Peroxides; Phycocyanin; Tryptophan

Insulysin (IDE) and neprilysin (NEP) were found to be inactivated by oxidation with hydrogen peroxide, an iron-ascorbate oxidation system, and by treatment with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). In each case reaction led to the introduction of protein carbonyl groups as judged by reaction with 2,4-dintrophenylhydrazine. IDE was inactivated by reaction with 4-hydroxy-2-nonenal (HNE) with the concomitant formation of protein adducts. NEP was not inactivated to a significant extent by HNE, but some HNE-adduct formation did occur. Prior reaction with hydrogen peroxide or AAPH led to enhanced formation of HNE adducts. Treatment of IDE with AAHP or hydrogen peroxide increased its susceptibility to proteolysis, while treatment of NEP with iron/ascorbate or hydrogen peroxide increased its susceptibility to proteolysis. Since IDE and NEP play a prominent role in the clearance of amyloid beta peptides, their oxidative inactivation and enhanced proteolysis can contribute to the onset and/or progression of Alzheimer's disease.

Topics: Aldehydes; Alzheimer Disease; Amidines; Amyloid beta-Peptides; Ascorbic Acid; Chlorides; Chymotrypsin; Ferric Compounds; Hydrogen Peroxide; Insulysin; Neprilysin; Oxidation-Reduction; Trypsin

Antioxidant activity of L-ascorbic acid (AsA) against oxidative modification of apolipoprotein (apo) E in human very-low-density lipoprotein (VLDL) was investigated. The VLDL oxidation induced by peroxyl radicals and peroxynitrite led to lipid peroxidation and oxidative modification of apoE. The binding activity of apoE to heparin was decreased by the oxidative modification. AsA (200, 500, and 1,000 microm) inhibited both the lipid peroxidation and the oxidative modification of apoE. These results suggest that AsA prevents the biological function of apoE in VLDL from the oxidation by free radicals.

Topics: Amidines; Antioxidants; Apolipoproteins E; Ascorbic Acid; Heparin; Humans; Lipid Peroxidation; Lipoproteins, VLDL; Molsidomine; Oxidants; Peroxides; Peroxynitrous Acid

Resveratrol (3,5,4'-trans-trihydroxystilbene) is a natural phytoalexin present in grapes and red wine, which possesses a variety of biological activities including antioxidative activity. To find more efficient antioxidants by structural modification, resveratrol analogues, that is, 3,4-dihydroxy-trans-stilbene (3,4-DHS), 4,4'-dihydroxy-trans-stilbene (4,4'-DHS), 4-hydroxy-trans-stilbene (4-HS) and 3,5-dihydroxy-trans-stilbene (3,5-DHS), were synthesized and their antioxidant activity studied for the free radical-induced peroxidation of rat liver microsomes in vitro. The peroxidation was initiated by either a water-soluble azo compound 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH) or Fe(2+)/ascorbate, and monitored by oxygen uptake and formation of thiobarbituric acid reactive substances (TBARS). It was found that all of these trans-stilbene derivatives are effective antioxidants against both AAPH- and iron-induced peroxidation of rat liver microsomes with an activity sequence of 3,4-DHS>4,4'-DHS>resveratrol>4-HS>3,5-DHS. The remarkably higher antioxidant activity of 3,4-DHS is discussed.

Topics: Amidines; Animals; Antioxidants; Ascorbic Acid; Drug Design; Free Radicals; Kinetics; Lipid Peroxidation; Microsomes, Liver; Molecular Structure; Oxygen Consumption; Rats; Resveratrol; Stilbenes; Structure-Activity Relationship; Thiobarbituric Acid Reactive Substances

Melatonin is a well-known hydroxyl radical (*OH) scavenger that protects DNA and lipids from free radical attack. In this paper, we studied the ability of melatonin to prevent oxidative damage to bovine serum albumin (BSA) induced by two different paradigms: the metal-catalyzed oxidation (MCO) induced by Cu(2+)/H(2)O(2) and the alkoxyl and alkylperoxyl radicals formed by the azo initiator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH, 40 mM). The protective effects of melatonin were compared with 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), glutathione (GSH), ascorbate, 3,4',5-trihydroxy-trans-stilbene (resveratrol, 0.1 microM-4 mM) and mannitol (50 microM-100 mM). Melatonin efficiently prevented protein modification induced by both models, as assayed by polyacrylamide gel electrophoresis and carbonyl content. Both trolox and ascorbate had an obvious pro-oxidant effect in the Cu(2+)/H(2)O(2) model, whereas both prevented BSA damage induced by AAPH. In the MCO model, the efficacy of GSH in terms of protein protection was higher than melatonin at relatively high concentrations (250 microM-4 mM); however, at lower concentrations (50-250 microM), the efficacy of melatonin was superior to GSH. D-Mannitol (50 microM-100 mM) and resveratrol did not protect BSA from the site-specific damage induced by Cu(2+)/H(2)O(2). On the other hand, the relative protective efficiency in the AAPH model was melatonin approximately trolox>GSH>ascorbate.

Topics: Amidines; Animals; Antioxidants; Ascorbic Acid; Cattle; Chromans; Copper; Electrophoresis, Polyacrylamide Gel; Hydrogen Peroxide; Melatonin; Metals; Oxidants; Peroxides; Serum Albumin, Bovine; Time Factors

4-(4-Hydroxyphenyl)-5-(4-hydroxyphenylmethyl)-2-hydroxyfurane-2-one 1 was prepared by an acidic dimerisation of 4-hydroxyphenylpyruvic acid and some of its antioxidant and spectroscopic properties have been measured and compared to that of ascorbic acid. 1 is as good an antioxidant as ascorbic acid in the DPPH (2,2-diphenyl-1-picryl hydrazyl radical) test and the inhibition of hydroxyl radical and a powerful inhibitor of the Cu(2+) or AAPH (2,2'-azobis-(2-amidinopropane) dihydrochloride) induced oxidation of human LDL. 1 gives a stable radical characterised by its ESR spectrum similarly to ascorbic acid but in lower concentration and with a different reactivity towards nitroxides. Theoretical calculations allow us to propose the structure for the radical formed from 1, to explain its lower stability than ascorbyl radical and to evaluate the lipophilicity of 1.

Topics: Amidines; Antioxidants; Ascorbic Acid; Biphenyl Compounds; Chemical Phenomena; Chemistry, Physical; Cholesterol, LDL; Copper; Electromagnetic Fields; Electron Spin Resonance Spectroscopy; Furans; Humans; Hydroxyl Radical; Indicators and Reagents; Lipids; Models, Molecular; Molecular Conformation; Picrates; Spectrophotometry, Ultraviolet

The antioxidant activity of hydroxytyrosol, hydroxytyrosol acetate, oleuropein, 3,4-dihydroxyphenylelenolic acid (3,4-DHPEA-EA) and 3,4-dihydroxyphenylelenolic acid dialdehyde (3,4-DHPEA-EDA) towards oxidation initiated by 2,2'-azobis(2-amidinopropane) hydrochloride in a soybean phospholipid liposome system was studied. The antioxidant activity of these olive oil phenols was similar and the duration of the lag phase was almost twice that of alpha-tocopherol. Trolox, a water-soluble analogue of alpha-tocopherol, showed the worst antioxidant activity. However, oxidation before the end of the lag phase was inhibited less effectively by the olive oil phenols than by alpha-tocopherol and Trolox. Synergistic effects (11-20% increase in lag phase) were observed in the antioxidant activity of combinations of alpha-tocopherol with olive oil phenols both with and without ascorbic acid. Fluorescence anisotropy of probes and fluorescence quenching studies showed that the olive oil phenols did not penetrate into the membrane, but their effectiveness as antioxidants showed they were associated with the surface of the phospholipid bilayer.

Topics: alpha-Tocopherol; Amidines; Antioxidants; Ascorbic Acid; Benzothiazoles; Chromans; Fluorescence Polarization; Glycine max; Kinetics; Liposomes; Membrane Fluidity; Olive Oil; Oxidation-Reduction; Phenols; Plant Oils; Sulfonic Acids

Thermal decomposition by the azo initiator 2,2' azobis(2-amidinopropane) dihydrochloride (AAPH) has been widely used as a water-soluble source of free radical initiators capable of inducing lipid peroxidation and protein damage. Here, in a lipid-free system, AAPH alone (40 mM) rapidly induced protein modification and inactivation of the enzyme catalase (EC 1.11.1.6). Using SDS-PAGE, it was shown that protein band intensity is dramatically reduced after 4 h of incubation with AAPH, leading to protein aggregation. Several antioxidants including melatonin, glutathione (GSH) and trolox prevented catalase modification when used at a 250 microM concentration whereas ascorbate was only effective at 1 mM concentration. All the antioxidants tested reduced carbonyl formation although melatonin was the most effective in this regard. Enzyme inactivation caused by AAPH was also significantly reduced by the antioxidants and again melatonin was more efficient than the other antioxidants used in this study. Results shown here demonstrate that alkyl peroxyl radicals inactivate catalase and reduce the effectiveness of cells to defend against free radical damage; the damage to catalase can be prevented by antioxidants, especially melatonin.

Topics: Amidines; Antioxidants; Ascorbic Acid; Catalase; Chromans; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Glutathione; Melatonin; Oxidants; Oxidation-Reduction; Peroxides; Time Factors

The antioxidant and prooxidant activities of flavonoids belonging to several classes were studied to establish their structure-activity relationships against different oxidants. Special attention was paid to the flavonoids quercetin (flavone), taxifolin (flavanone) and catechin (flavanol), which possess different basic structures but the same hydroxylation pattern (3,5,7,3'4'-OH). It was found that these three flavonoids exhibited comparable antioxidant activities against different oxidants leading to the conclusion that the presence of ortho-catechol group (3',4'-OH) in the B-ring is determinant for a high antioxidant capacity. The flavone kaempferol (3,5,7,4'-OH), however, in spite of bearing no catechol group, also presents a high antioxidant activity against some oxidants. This fact can be attributed to the presence of both 2,3-double bond and the 3-hydroxyl group, meaning that the basic structure of flavonoids becomes important when the antioxidant activity of B-ring is small.

Topics: Amidines; Antioxidants; Ascorbic Acid; Biphenyl Compounds; Catechin; Edetic Acid; Flavonoids; Flavonols; Free Radicals; Iron; Kaempferols; Liposomes; Metmyoglobin; Picrates; Quercetin; Structure-Activity Relationship

Antioxidative activities of the extracts from Sargassum siliquastrum were determined using the inhibition of red blood cell (RBC) hemolysis induced by 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) radicals, suppression of lipid peroxidation using rat brain homogenate, and scavenging activity of superoxide radicals. The dichloromethane fraction isolated from the methanol crude extract by differential solvent extractions exhibited the strongest antioxidant activity in both RBC hemolysis and lipid peroxidation assays. This fraction was further fractionated into four subfractions F1-F4 by silica gel column chromatography. F1 was found to be most effective in protecting RBC against AAPH radicals and in inhibiting lipid peroxidation. On the basis of thin-layer chromatography and UV and IR spectra analyses, all subfractions contained phenolic compounds. However, there was no correlation between the above antioxidant potency and total phenolic compounds estimated by using the Folin-Ciocalteau method.

Topics: Amidines; Animals; Antioxidants; Ascorbic Acid; Brain Chemistry; Chemical Fractionation; Chromatography, Thin Layer; Ferrous Compounds; Free Radicals; Hemolysis; Lipid Peroxidation; Male; Methylene Chloride; Phaeophyceae; Phenols; Rats; Rats, Sprague-Dawley; Seaweed; Spectrophotometry, Ultraviolet

Four spectrophotometric methods of determination of antioxidant capacity: a method based on the scavenging of the l,1-diphenyl-2-picrylhydrazyl (DPPH) free radical, the "ferric-reducing ability of plasma" (FRAP), a method based on reduction of the 2.2'-azinobis (3-ethylbenzthiazolinesulfonate) free radical (ABTS.+) and a kinetic method based on the oxidation of dihydro-2,7-dichlorofluorescein by 2,2'-azobis(2-amidopropane) (ABAP) were compared with respect to standard antioxidants (ascorbate, glutathione, Trolox and urate) and human blood plasma. Various reactivities of standard antioxidants in different tests were found. glutathione showing a low reactivity in the FRAP assay. Kinetic measurements show that the reduction of indicators, especially by blood plasma, may not be complete at recommended times of the assays and the time of measurement is an important parameter when comparing the results.

Topics: Adult; Amidines; Antioxidants; Ascorbic Acid; Benzothiazoles; Biphenyl Compounds; Chemistry, Clinical; Female; Glutathione; Humans; Kinetics; Male; Middle Aged; Oxidative Stress; Picrates; Plasma; Spectrophotometry; Sulfonic Acids; Uric Acid

This study was carried out to investigate sequel of oxidative insult to human erythrocytes induced by a water-soluble radical initiator, 2,2'-azobis-(amidinopropane) dihydrochloride (AAPH) and the effect of a commercially available mixed antioxidant (Blackmores, BioAce Excel), containing alpha-tocopherol, ascorbic acid, beta-carotene and some herbal extracts (containing grape seed catechins and milk thistle derived silybin), on lipid peroxidation, degradation of membrane proteins and haemolysis. We performed this study in order firstly to clarify aspects of the mechanism of AAPH induced free radical damage in human erythrocytes and secondly to establish in vitro conditions by which the efficacy of mixed antioxidant preparations may fairly and objectively be compared. In the process of oxidation initiated by peroxyl radical, a rapid loss of reduced glutathione occurred in the first 60 min. Formation of thiobarbitric acid-reactive substances indicative of lipid peroxidation increased subsequently and almost reached maximal levels at 180 min before significant apparent degradation of membrane proteins was detected. At this point, a significant haemolysis occurred. This sequence of events is consistent with the idea that haemolysis is a consequence of lipid peroxidation and the degradation of membrane proteins. The mixed commercial antioxidant, which suppressed lipid peroxidation and protected membrane proteins against degradation induced by peroxyl radicals, also effectively delayed AAPH induced haemolysis. The system we describe provides a sound objective basis for the in vitro comparison of the potential efficacy of the hundreds of antioxidant nutritional supplements currently available in the market place.

Topics: Amidines; Antioxidants; Ascorbic Acid; beta Carotene; Catechin; Drug Combinations; Erythrocytes; Fruit; Glutathione; Hemolysis; Humans; In Vitro Techniques; Kinetics; Lipid Peroxidation; Membrane Proteins; Methemoglobin; Oxidants; Plant Extracts; Seeds; Silymarin; Thiobarbituric Acid Reactive Substances; Vitamin E

Changes in ascorbate content in primary cultured rat hepatocytes exposed to oxidative stress derived from water soluble radical initiator 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) were examined. Cells were exposed to 0.05 and 5 mg/ml of AAPH as 'mild' and severe' oxidative stresses, respectively. Lipid peroxidation in hepatocytes was induced by 'severe' oxidative stress, but not by 'mild' oxidative stress. Ascorbate decreased at 6 hr after administration of both mild' and severe' oxidative stresses, and recovered to the control level after a further 6 hr. In cells treated with 'severe oxidative stress, however, total ascorbate (reduced form plus oxidized form) had increased 24 hr after administration. These results indicated that consumption alone did not account for the increase of ascorbate in hepatocytes under oxidative stress.

Topics: Amidines; Animals; Ascorbic Acid; Hepatocytes; Male; Oxidants; Oxidative Stress; Rats; Rats, Wistar; Thiobarbituric Acid Reactive Substances

The relationship between the antioxidant effect of acidic phospholipids, phosphatidic acid (PA), phosphatidylglycerol (PG) and phosphatidylserine (PS), on iron-induced lipid peroxidation of phospholipid bilayers and their abilities to bind iron ion was examined in egg yolk phosphatidylcholine large unilamellar vesicles (EYPC LUV). The effect of each acidic phospholipid added to the vesicles at 10 mol% was assessed by measuring phosphatidylcholine hydroperoxides (PC-OOH) and thiobarbituric acid-reactive substances. The addition of dipalmitoyl PS (DPPS) showed a significant inhibitory effect, although the other two acidic phospholipids, dipalmitoyl PA (DPPA) and dipalmitoyl PG (DPPG), did not exert the inhibition. Neither dipalmitoyl PC (DPPC) nor dipalmitoyl phophatidylethanolamine (DPPE) showed any remarkable inhibition on this system. None of the tested phospholipids affected the lipid peroxidation rate remarkably when the vesicles were exposed to a water-soluble radical generator. The iron-binding ability of each phospholipid was estimated on the basis of the amounts of iron recovered in the chloroform/methanol phase after separation of the vesicle solution to water/methanol and chloroform/methanol phases. EYPC LUV containing DPPS, DPPA, and DPPG had higher amounts of bound iron than those containing DPPC and DPPE, indicating that these three acidic phospholipids possess an iron-binding ability at a similar level. Nevertheless, only DPPS suppressed iron-dependent decomposition of PC-OOH significantly. Therefore, it is likely that these three acidic phospholipids possess a significant iron-binding ability, although this ability per se does not warrant them antioxidative activities. The ability to suppress the iron-dependent decomposition of PC-OOH may explain the unique antioxidant activity of PS.

Topics: Amidines; Antioxidants; Ascorbic Acid; Iron; Lipid Bilayers; Lipid Peroxidation; Liposomes; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Thiobarbituric Acid Reactive Substances

We evaluated the interaction between ascorbic acid (AA) and (+)-catechin (CTCH) in potassium phosphate solution, pH 7.4 (PPS) and in human plasma. In both systems, the oxidation was started by adding 2,2'-azobis-(2-amidinopropane) clorhidrate (AAPH). The concentrations of AA and CTCH were determined by HPLC using electrochemical detection. In PPS, CTCH was oxidized by AAPH (50 mM), in either the absence or presence of different initial concentrations of AA (25-200 microM). In the presence of AA, CTCH depletion was delayed, an effect that was dependent upon the initial concentration of AA. When 100 microM AA was added after the oxidation had begun, CTCH depletion was arrested for 30 min. The kinetics of AA oxidation by AAPH was also characterized in PPS. AA (100 microM) was completely consumed after 60 min of reaction at 37 degrees C, in both the absence and presence of 100 mM CTCH. When human plasma was incubated with 50 mM AAPH in the absence of added CTCH, AA was completely consumed after 45-60 min. CTCH did not prevent AA depletion in human plasma at the concentrations tested (10, 50 100 microM). The results point out that AA is able to protect other aqueous soluble antioxidants, e.g.: CTCH.

Topics: Amidines; Antioxidants; Ascorbic Acid; Catechin; Chromatography, High Pressure Liquid; Drug Interactions; Humans; Kinetics; Oxidants; Phosphates; Plasma; Potassium Compounds; Time Factors

The reactions of nitric oxide ((.)NO) and alpha-tocopherol (alpha-TH) during membrane lipid oxidation were examined and compared with the pair alpha-TH/ascorbate. Nitric oxide serves as a more potent inhibitor of lipid peroxidation propagation reactions than alpha-TH and protects alpha-TH from oxidation. Mass spectrometry, oxygen and (.)NO consumption, conjugated diene analyses, and alpha-TH fluorescence determinations all demonstrated that (.)NO preferentially reacts with lipid radical species, with alpha-TH consumption not occurring until (.)NO concentrations fell below a critical level. In addition, alpha-TH and (.)NO cooperatively inhibit lipid peroxidation, exhibiting greater antioxidant capacity than the pair alpha-TH/ascorbate. Pulse radiolysis analysis showed no direct reaction between (.)NO and alpha-tocopheroxyl radical (alpha-T(.)), inferring that peroxyl radical termination reactions are the principal lipid-protective mechanism mediated by (.)NO. These observations support the concept that (.)NO is a potent chain breaking antioxidant toward peroxidizing lipids, due to facile radical-radical termination reactions with lipid radical species, thus preventing alpha-TH loss. The reduction of alpha-T(.) by ascorbate was a comparatively less efficient mechanism for preserving alpha-TH than (.)NO-mediated termination of peroxyl radicals, due to slower reaction kinetics and limited transfer of reducing equivalents from the aqueous phase. Thus, the high lipid/water partition coefficient of (.)NO, its capacity to diffuse and concentrate in lipophilic milieu, and a potent reactivity toward lipid radical species reveal how (.)NO can play a critical role in regulating membrane and lipoprotein lipid oxidation reactions.

Topics: Amidines; Antioxidants; Ascorbic Acid; Free Radicals; Linoleic Acid; Lipid Peroxidation; Nitric Oxide; Oxidation-Reduction; Vitamin E

The effect of beta-carotene on protein oxidation was examined under different oxygen (O(2)) tensions and with other antioxidants: alpha-tocopherol, ascorbic acid, and mixtures of antioxidants. Human serum albumin (HSA) was incubated with 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) to induce protein oxidation (carbonyl formation), under 15, 150, and 760 torr of O(2) tension. Antioxidant activity was related to O(2) tension, antioxidant concentrations and interaction between mixtures of antioxidants: (1) Under 15 torr of O(2), incubating HSA with AAPH, 1. 6 microM beta-carotene, 80 microM alpha-tocopherol, 160 microM ascorbic acid, and mixtures (0.1 microM beta-carotene, 5.0 microM alpha-tocopherol and 10 microM ascorbic acid) resulted in 24, 29, 39, and 44% reduction of carbonyl formation, respectively. (2) Under 150 torr of O(2) tension, the antioxidant effect of beta-carotene was decreased by 4% but increasing O(2) tension did not diminish the antioxidant effects of alpha-tocopherol, ascorbic acid, or antioxidant mixtures. (3). Under 760 torr of O(2) tension, adding 1. 6 microM beta-carotene resulted in 26% more carbonyl formation. (4) Under 760 torr of O(2) tension, the antioxidant effect of ascorbic acid was decreased 32% compared to what was observed at 150 torr of O(2) tension. Changes in O(2) tension had no effect on the antioxidant effect of alpha-tocopherol. The mixture of antioxidants inhibited carbonyl formation by 37% and was 7% less effective than that of 15 and 150 torr of O(2) tension. High concentration of beta-carotene produces more protein oxidation in the presence of high O(2) tension by a prooxidant mechanism. Mixtures of beta-carotene, alpha-tocopherol, and ascorbic acid provided better protective effects on protein oxidation than any single compound.

Topics: Amidines; Antioxidants; Ascorbic Acid; beta Carotene; Enzyme-Linked Immunosorbent Assay; Humans; Oxidants; Oxidation-Reduction; Oxygen; Phenylhydrazines; Serum Albumin; Vitamin E

Low-molecular weight antioxidants (LMWAs) play a major role in protecting biological systems against reactive oxygen-derived species and reflect the antioxidant capacity of the system. Cyclic voltammetry (CV), shown to be convenient methodology, has been validated for quantitation of the LMWA capacity of blood plasma, tissue homogenates, and plant extracts. Analysis of the CV tracing yields the values of (i) the biological oxidation potential, E and E(1/2), which relate to the nature of the specific molecule(s); (ii) the intensity (Ia) of the anodic current; and (iii) the area of the anodic wave (S). Both Ia and S relate to the concentration of the molecule(s). LMWA components of human plasma and animal tissues were identified and further validated by reconstruction of the CV tracing and by high-performance liquid chromatography-electrochemical detection. To reflect the oxidative stress status, the use of an additional parameter, R, has been proposed. R represents the level (%) of oxidized ascorbate (compared with total ascorbate) and is measured by high-performance liquid chromatography-electrochemical detection. All these parameters were monitored in healthy human subjects as well as in chronic (diabetes mellitus) and acute care patients (subjected to total body irradiation before bone marrow transplantation). The electroanalytical methodologies presented here could be widely employed for rapid evaluation of the status of subjects (in health and disease) for monitoring of their response to treatment and/or nutritional supplementation as well as for screening of specific populations.

Topics: Amidines; Antioxidants; Ascorbic Acid; Chromans; Copper; Electrochemistry; Humans; Microelectrodes; Oxidative Stress; Peroxides; Plants, Edible

The relative activities of the antioxidants Trolox, ascorbic acid, uric acid, quercetin, and rutin, and the activities of total antioxidants in serum samples were determined using a fluorometric assay based on the dye 6-carboxyfluoroscein (6C-Fl) as a fluorescent indicator; 2,2'-azobis-2-amidinopropane hydrochloride (AAPH) as a peroxyl radical generator; 6-hydroxy-2,5,7, 8-tetramethyl-1-chroman-2-carboxylic acid (Trolox) as a calibrator; and phosphate buffer (pH 7.0) as a solvent. Incubation of 6C-Fl in 0. 075 M phosphate buffer, in the presence of AAPH at 37 degrees C, resulted in loss of its fluorescence signal at 520 nm with excitation at 495 nm. The antioxidants Trolox, ascorbic acid, and uric acid provided protection of the fluorescence of 6C-Fl, and the relative antioxidant activities, determined by the net protection area under curve technique, were found to be 1:0.4:1, respectively. Trolox and ascorbic acid were used to validate this assay. A linear correlation of the net protection value with the concentration of serum, Trolox, ascorbic acid, and uric acid was demonstrated. Quercetin and rutin were shown to have strong antioxidant activities, nearly 10 times those of vitamin C. This assay is simple, reliable, and suitable for automation to handle many samples and requires few microliters of serum samples.

Topics: Amidines; Animals; Antioxidants; Area Under Curve; Ascorbic Acid; Chromans; Dogs; Fluoresceins; Free Radicals; Humans; Oxygen; Quercetin; Rutin; Spectrometry, Fluorescence; Time Factors; Uric Acid

A novel post-addition method, based on the trapping of ABTS-radicals, is applied for studying the total antioxidant capacity of seminal plasma. A remarkable profile is observed, in which seminal plasma quenches radicals in a continuous, relatively slow fashion. Five putative antioxidants present in seminal plasma were studied using the same assay. Some of the compounds such as ascorbic acid, alpha-tocopherol and uric acid exert immediate, fast radical trapping, whereas hypotaurine and tyrosine give rise to the same slow radical trapping curve as seminal plasma. Due to this slow, continuous radical trapping, quantification of the total antioxidant capacity (expressed as trolox equivalent antioxidant capacity, TEAC) strongly depends on the chosen time point after onset of radical trapping. When determined during the slow antioxidant trapping phase, tyrosine has a powerful antioxidant capacity, which in combination with its relatively high plasma concentration makes it an important contributor to the total antioxidant capacity of seminal plasma.

Topics: Amidines; Antioxidants; Ascorbic Acid; Benzothiazoles; Drug Synergism; Free Radicals; Humans; Indicators and Reagents; Male; Oxidants; Oxidation-Reduction; Semen; Spectrophotometry; Sulfonic Acids; Taurine; Tyrosine; Uric Acid; Vitamin E

The objective of this study was to investigate the ability of endothelial cells (EC) to incorporate anthocyanins and to examine their potential benefits against various oxidative stressors. Endothelial dysfunction has been proposed to play an important role in the initiation and development of vascular disease, with studies having shown that administration of antioxidants improves endothelial function. Elderberry extract contains 4 anthocyanins, which where incorporated into the plasma membrane and cytosol of EC following 4 h incubation at 1 mg.ml(-1). However, incorporation within the cytosol was considerably less than that in the membrane. Uptake within both regions appeared to be structure dependent, with monoglycoside concentrations higher than that of the diglucosides in both compartments. The enrichment of EC with elderberry anthocyanins conferred significant protective effects in EC against the following oxidative stressors: hydrogen peroxide (H(2)O(2)); 2, 2'-azobis(2-amidinopropane) dihydrochloride (AAPH); and FeSO(4)/ascorbic acid (AA). These results show for the first time that vascular EC can incorporate anthocyanins into the membrane and cytosol, conferring significant protective effects against oxidative insult. These findings may have important implications on preserving EC function and preventing the initiation of EC changes associated with vascular diseases.

Topics: Amidines; Animals; Anthocyanins; Aorta; Ascorbic Acid; Cattle; Cell Division; Cells, Cultured; Endothelium, Vascular; Ferric Compounds; Fruit; Humans; Kinetics; Mitochondria; Oxidants; Oxidative Stress; Plant Extracts

The antioxidant activity of catechin monomers and procyanidin (dimers to hexamers) fractions purified from cocoa was studied in two in vitro systems: liposomes and human LDL. Liposome oxidation (evaluated as formation of 2-thiobarbituric acid reactive substances) was initiated with 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN), iron/ascorbate, or UV-C; LDL oxidation (evaluated as formation of conjugated dienes) was initiated with Cu(2+) or AAPH. Catechin monomers and procyanidin fractions inhibited both liposome and LDL oxidation. Monomers, dimers, and trimers fractions were the most effective antioxidants when liposome oxidation was initiated in the aqueous phase. When oxidation was initiated in the lipid domains, higher molecular weight procyanidins were the most effective. All fractions significantly inhibited Cu-mediated LDL oxidation; no significant effect of procyanidin molecular weight was observed. The hexamer fraction was the least effective with respect to preventing AAPH initiated LDL oxidation. Results reported herein give further evidence on the influence of the oligomer chain length on the antioxidant protection by procyanidins.

Topics: Amidines; Antioxidants; Ascorbic Acid; Azo Compounds; Biflavonoids; Cacao; Catechin; Copper; Dimerization; Egg Yolk; Humans; Inhibitory Concentration 50; Iron; Lipoproteins, LDL; Liposomes; Molecular Weight; Nitriles; Oxidants; Oxidation-Reduction; Proanthocyanidins; Protein Binding; Thiobarbituric Acid Reactive Substances; Ultraviolet Rays

We have extended the application of our previously reported total oxidant scavenging capacity (TOSC) assay (Winston et al., Free Radical Biol. Med. 24, 480-493, 1998) to permit facile quantification of the absorbance capacity of antioxidants toward three potent oxidants, i.e., hydroxyl radicals, peroxyl radicals, and peroxynitrite. Respectively, these oxidants were generated by the iron plus ascorbate-driven Fenton reaction, thermal homolysis of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (ABAP), and 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). Each of these oxidants reacts with alpha-keto-gamma-methiolbutyric acid (KMBA), which is oxidized and yields ethylene. The antioxidant capacity of the compounds tested is quantified from their ability to inhibit ethylene formation relative to a control reaction. Assay conditions were established in which control reactions give comparable yields of ethylene with each of the oxidants studied. Thus, the relative efficiency of various antioxidants could be compared under conditions of quantitatively similar KMBA oxidizing capability by the three oxidants. Reduced glutathione was an efficient scavenger of peroxyl radicals, but scavenged peroxynitrite and hydroxyl radicals relatively poorly. Uric acid, Trolox, and ascorbic acid were comparable scavengers of peroxynitrite and peroxyl radicals. Uric acid and Trolox were approximately an order of magnitude less efficient as scavengers of hydroxyl radicals. The classical hydroxyl radical scavenging agents mannitol, dimethyl sulfoxide, and benzoic acid had much higher TOSC values with hydroxyl than with peroxyl radicals or peroxynitrite. The very different chemical reactivity toward KMBA by the SIN-1- and iron-ascorbate-generated oxidants indicates that hydroxyl radical is not a major oxidant produced by the SIN-1 system. The data show that the TOSC assay is useful and robust in distinguishing the reactivity of various oxidants and the relative capacities of antioxidants to scavenge these oxidants.

Topics: Amidines; Antioxidants; Ascorbic Acid; Chromatography, Gas; Free Radical Scavengers; Free Radicals; Hydroxyl Radical; Methionine; Molsidomine; Nitrates; Nitric Oxide Donors; Oxidants; Oxidation-Reduction; Peroxides

We evaluated the antioxidant effect of (+)-catechin (CTCH), in the presence of physiological antioxidant levels of ascorbic acid (AA), alpha-tocopherol (AT) and beta-carotene (BC), in human plasma oxidised with AAPH. Following a five-hour incubation, the formation of lipid oxidation products (TBARS) was almost doubled, and the concentrations of lipid soluble antioxidants were 10 to 30% from the initial levels. In these conditions, AA was consumed within the first hour of incubation. The addition of CTCH prevented AT and BC depletion and TBARS formation, but had no effect on AA consumption. When the kinetics of oxidation were analysed CTCH oxidation preceded lipid soluble antioxidant depletion, but no consumption of CTCH was associated to AA oxidation. Considering that CTCH could contribute to the antioxidant activity of red wine, we first characterised both the antioxidant capacity and CTCH content of several wines. The wines with highest content of CTCH and antioxidant activity were also the most effective in preventing AAPH-mediated oxidation of plasma vitamin E. Results support the idea that CTCH could have a role as a physiological antioxidant in human plasma, and that CTCH of wine could contribute to the antioxidant status of human plasma.

Topics: Amidines; Antioxidants; Ascorbic Acid; beta Carotene; Catechin; Humans; In Vitro Techniques; Kinetics; Oxidants; Oxidation-Reduction; Thiobarbituric Acid Reactive Substances; Vitamin E; Wine

A chemical model based on the thermal decomposition of AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride is used for the production of peroxyl radicals. Peroxyl radicals induces the decarboxylation of [7-13C]benzoic acid and the production of 13CO2, which is measured by gas chromatography-isotope ratio mass spectrometry (GC-IRMS). The decarboxylation depends on temperature, AAPH, and benzoic acid concentrations. The decarboxylation also depends on the presence of oxygen. Electron spin resonance studies are performed to confirm the presence of peroxyl radicals under oxygen and of carbon-centered radicals in the absence of oxygen. Decarboxylation rates are measured in the presence of various antioxidants: ascorbate, dimethylsulfoxide, mannitol, and uric acid. It turns out that the decarboxylation is inhibited by each of these antioxidants. The ratio of decarboxylation rates, with and without the antioxidant, varies linearly with the antioxidant concentration. HPLC and GC-MS analyses of reaction products between benzoic acid and AAPH-derived radicals do not detect the presence of radical substitution products on the aromatic ring or the products derived from benzoic acid. There is no doubt that GC-IRMS is a powerful technique to investigate the effects of peroxyl radicals on benzoic acid. In addition, it is possible to follow the degradation of 13C-labeled chemical targets exposed to peroxyl radicals through the production of 13CO2.

Topics: Amidines; Antioxidants; Ascorbic Acid; Benzoates; Benzoic Acid; Carbon Dioxide; Chromatography, High Pressure Liquid; Decarboxylation; Dimethyl Sulfoxide; Electron Spin Resonance Spectroscopy; Gas Chromatography-Mass Spectrometry; Hot Temperature; Mannitol; Methanol; Oxidation-Reduction; Peroxides; Uric Acid

Ascorbic acid can recycle alpha-tocopherol from the tocopheroxyl free radical in lipid bilayers and in micelles, but such recycling has not been demonstrated to occur across cell membranes. In this work the ability of intracellular ascorbate to protect and to recycle alpha-tocopherol in intact human erythrocytes and erythrocyte ghosts was investigated. In erythrocytes that were 80% depleted of intracellular ascorbate by treatment with the nitroxide Tempol, both 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and ferricyanide oxidized alpha-tocopherol to a greater extent than in cells not depleted of ascorbate. In contrast, in erythrocytes in which the intracellular ascorbate concentration had been increased by loading with dehydroascorbate, loss of alpha-tocopherol was less with both oxidants than in control cells. Protection against AAPH-induced oxidation of alpha-tocopherol was not prevented by extracellular ascorbate oxidase, indicating that the protection was due to intracellular and not to extracellular ascorbate. Incubation of erythrocytes with lecithin liposomes also generated an oxidant stress, which caused lipid peroxidation in the liposomes and depleted erythrocyte alpha-tocopherol, leading to hemolysis. Ascorbate loading of the erythrocytes delayed liposome oxidation and decreased loss of alpha-tocopherol from both cells and from alpha-tocopherol-loaded liposomes. When erythrocyte ghosts were resealed to contain ascorbate and challenged with free radicals generated by AAPH outside the ghosts, intravesicular ascorbate was totally depleted over 1 h of incubation, whereas alpha-tocopherol decreased only after ascorbate was substantially oxidized. These results suggest that ascorbate within the erythrocyte protects alpha-tocopherol in the cell membrane by a direct recycling mechanism.

Topics: Amidines; Antioxidants; Ascorbic Acid; Cyclic N-Oxides; Dehydroascorbic Acid; Erythrocyte Membrane; Erythrocytes; Ferricyanides; Glutathione; Humans; Kinetics; Liposomes; Oxidants; Oxidation-Reduction; Phosphatidylcholines; Spin Labels; Vitamin E

Antioxidative activity of carp blood plasma was estimated by measuring hydroperoxides formed by liposome peroxidation during the exposure of liposomes to AAPH. Ascorbic acid of high concentration, uric acid of low content, and tocopherol formed special protective system against lipid poroxidation in fish plasma. The decrease of uric acid, ascorbic acid, and tocopherol showed synergism of ascorbic acid and tocopherol, uric acid, and tocopherol. Carp blood plasma with a low concentration of protein (about 2%) and SH groups (88 microM) had a great effect on the antioxidative activity, as the effects of ascorbic acid, uric acid, and tocopherol were dramatically extended. Dialysed carp protein also displayed a very strong antioxidative activity on lipid peroxidation of a multilayer liposome system.

Topics: Adult; Amidines; Animals; Antioxidants; Ascorbic Acid; Blood Proteins; Carps; Chromatography, High Pressure Liquid; Female; Humans; Lipid Peroxidation; Liposomes; Male; Peroxides; Phosphatidylcholines; Plasma; Uric Acid; Vitamin E

Induced erythrocyte membrane peroxidation (EMP) is considered as an accurate model of reperfusion injuries and as such was used to investigate protective effects of various drugs. EMP was induced by an azo initiator and monitored by oxygen uptake. Both hydrophilic (ascorbic acid) and lipophilic (alpha-tocopherol, probucol, nicanartine) chain-breaking antioxidants as well as ferriheme-bound drugs (deferoxamine, chloroquine) inhibited EMP. When antioxidants and ferriheme-bound drugs were combined, synergistic effects were observed. It is proposed that ferriheme compounds which catalyse peroxide induced lipid peroxidation were blocked by deferoxamine and/or chloroquine. So these drugs inhibited at least partly the membrane peroxidation process and added their effects to the ones of chain-breaking antioxidants.

Topics: Adult; Amidines; Antioxidants; Ascorbic Acid; Chloroquine; Deferoxamine; Drug Synergism; Erythrocyte Membrane; Fatty Acids; Female; Hemin; Humans; Linoleic Acid; Lipid Peroxidation; Male; Membrane Lipids; Oxidation-Reduction; Oxygen Consumption; Oxyhemoglobins; Probucol; Pyridines; Reperfusion Injury; Vitamin E

We found that ticlopidine, at therapeutically relevant concentrations (2.5-10 microM), but not aspirin nor salicylate, significantly counteracted copper-driven human LDL oxidation. Ticlopidine, at 5 and 10 microM, was also antioxidant on peroxyl radical-induced LDL oxidation; yet it was ineffectual on thiol and ascorbate oxidation mediated by peroxyl radicals themselves, suggesting that drug antioxidant capacity is somehow related to the lipoprotein nature of the oxidizable substrate, but not to radical scavenging. The drug could not indeed react with the stable free radical 1,1-diphenyl-2-pycrylhydrazyl, nor had apparent metal complexing-inactivating activity. Thus, ticlopidine has antioxidant effects on LDL oxidation, which, together with its anti-platelet activity, could confer peculiar antiatherogenic properties to the drug in vivo.

Topics: Amidines; Antioxidants; Ascorbic Acid; Aspirin; Free Radical Scavengers; Free Radicals; Humans; Kinetics; Lipoproteins, LDL; Oxidants; Oxidation-Reduction; Peroxides; Ticlopidine

The peroxidation of human low density lipoprotein (LDL) was initiated by water-soluble initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH) and copper and inhibited by vitamin C (VC) and its lipophillic derivatives ascorbyl-6-caprylate (VC-8), 6-laurate (VC-12) and 6-palmitate (VC-16), respectively. The peroxidation was monitored by oxygen uptake, by decay of alpha-tocopherol and by formation of lipid oxidation products. Kinetic analysis of the antioxidation process demonstrates that the VCs can work synergistically with endogenous antioxidants (vitamin E, ubiquinol-10, beta-carotene etc.) in LDL to suppress the peroxidation and that the synergistic effect of the lipophilic VCs is appreciably higher than that of their lipophobic parent molecule. It is also shown that VC and especially the lipophilic VCs, are much more effective in inhibition of copper-induced than AAPH-induced LDL peroxidation.

Topics: Amidines; Antioxidants; Ascorbic Acid; Copper; Free Radicals; Humans; Lipid Metabolism; Lipid Peroxidation; Lipoproteins, LDL; Oxidants; Oxygen Consumption; Solubility; Spectrophotometry, Ultraviolet

A simple spectrophotometric method of determination of peroxyl radical-trapping capacity (PRTC) of body fluids and food products is proposed. In this method, decomposition of 2,2'-azobis(2-amidopropane) hydrochloride (ABAP) is the source of peroxyl and alkoxyl radicals which oxidize 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to a green cation radical. Antioxidant present in a sample inhibit the reaction; the induction time of the reaction is proposed as a parameter enabling determination of antioxidant content. Standard assay conditions are: 20 mM ABAP and 150 microM ABTS in 0.1 M phosphate buffer, pH 7.0, at 37 degrees C; absorbance is monitored at 414 nm. A 10-min assay allows for determination of the induction time of appropriately diluted sample. As examples of application of this method, PRTC values of several types of beverages are reported.

Topics: Alcoholic Beverages; Amidines; Amino Acids; Antioxidants; Ascorbic Acid; Benzothiazoles; Chromans; Epinephrine; Free Radicals; Indicators and Reagents; Oxidation-Reduction; Peroxides; Plasma; Spectrophotometry; Sulfonic Acids; Time Factors

The heme precursor 5-aminolevulinic acid (ALA), acting as a prooxidant, has been proposed to underlie the clinical manifestations of various porphyric disorders. Accordingly, ALA-generated oxyradicals where shown to cause oxidative lesions in biomolecules and isolated cell organelles and to release iron from ferritin. In rats, administered ALA triggered oxidative stress in liver, brain and red muscles. We now study the correlation between the plasma antioxidant capacity and tissue oxidative damage, after acute (one and two doses) and prolonged (eight doses) ALA treatment of rats (one dose of ALA = 40 mg/kg body weight). The in situ spontaneous chemiluminescence intensity increased 5-fold in brain, 50% in liver and 4-fold in soleus muscle upon two dose-treatment, indicating tissue response to oxidative injury by ALA. Chemiluminescence reached the highest intensity after one or two doses of ALA and decreased after eight doses in all tissues. The plasma trapping capacity, evaluated by the luminol/2-amidinopropane system, gave a parallel response: maximum values after two doses and decreased values after prolonged treatment. After eight doses, the ALA concentration was found to be 3-fold above the normal value in plasma, 48% higher in liver and 38% higher in total brain. These data indicate that the plasma antioxidant system responds to ALA treatment and is correlated with tissue chemiluminescence. In vitro studies showed that ALA does not interfere with the antioxidant plasma capacity, neither promoting oxidation of plasma elements nor binding to plasma proteins.

Topics: Amidines; Aminolevulinic Acid; Animals; Antioxidants; Ascorbic Acid; Blood Proteins; Brain; Chromans; Glutathione; Liver; Luminescent Measurements; Luminol; Male; Muscle, Skeletal; Oxidation-Reduction; Oxidative Stress; Plasma; Rats; Rats, Wistar; Sulfhydryl Compounds; Uric Acid

The antioxidant activities of compounds endogenous to bovine rod outer segments (ROS) were investigated by measuring the loss of polyunsaturated fatty acids (PUFA's) from membranes exposed to the water-soluble oxidant 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Osmotically intact ROS, ROS membranes, and unilamellar liposomes prepared from ROS phospholipids (PL) were compared. Intact ROS were most resistant to oxidative loss of PUFA's, followed by ROS membranes and then PL liposomes. The development of a model membrane system allowed the investigation of putative antioxidants singly and in combination. These lipid-soluble compounds were incorporated into PL liposomes, and it was found that normal physiological concentrations of alpha-tocopherol (vitamin E) and free fatty acids (16:0, 18:0, 18:1, 22:6) significantly decreased oxidative loss of PUFA's. When all the major free fatty acids were added to PL liposomes at the same concentrations found when ROS phospholipase A is stimulated, the oxidative loss of PUFA's was reduced by 31%. The antioxidant effect of free fatty acids suggests that endogenous phospholipase A's may act to protect membranes by releasing esterified fatty acids in proportions and concentrations that afford protection to membrane lipids.

Topics: Amidines; Animals; Antioxidants; Ascorbic Acid; Cattle; Esterification; Fatty Acids, Monounsaturated; Fatty Acids, Nonesterified; Fatty Acids, Unsaturated; Liposomes; Membrane Lipids; Oxidation-Reduction; Phospholipids; Rod Cell Outer Segment; Vitamin E

2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butyl-benzofuran (BO-653) is a novel antioxidant synthesized by theoretical designing based on the previous experimental findings and consideration. The antioxidant activities of BO-653 against the oxidative modification of low-density lipoprotein (LDL) induced by free radicals were studied. BO-653 was consumed faster than endogenous alpha-tocopherol and inhibited the formation of lipid hydroperoxides, which was observed during the consumption of alpha-tocopherol. Doxyl stearic acids incorporated into LDL as spin probes competed with the antioxidants in scavenging radicals. It was found that the efficacy of radical scavenging by alpha-tocopherol became smaller as the radical went deeper into the interior of LDL particle, whereas that by BO-653 did not change. Ascorbic acid in the aqueous phase spared alpha-tocopherol efficiently during oxidation. On the other hand, the sparing effect of ascorbic acid for BO-653 was not remarkable, unlike that for alpha-tocopherol, which implied different locations of radicals derived from BO-653 and alpha-tocopherol within the LDL particle. It was concluded that BO-653 protected LDL from oxidative modification efficiently by scavenging peroxyl radicals and by reducing alpha-tocopheroxyl radicals and that this novel antioxidant might act as a potent inhibitor of development of atherosclerosis.

Topics: Amidines; Animals; Antioxidants; Apolipoproteins B; Arteriosclerosis; Ascorbic Acid; Azo Compounds; Benzofurans; Cyclic N-Oxides; Drug Design; Free Radical Scavengers; Humans; Kinetics; Lipid Peroxidation; Lipid Peroxides; Lipoproteins, LDL; Nitriles; Oxidants; Oxidation-Reduction; Peroxides; Rabbits; Vitamin E

Oxidative modification of low-density lipoprotein (LDL) is thought to play a key role in the formation of foam cells and in initiation and progression of atherosclerotic plaque. The hypolipidemic 3-thia fatty acids contain a sulfur atom and might therefore possess reducing (antioxidant) properties. Consequently, the effects of 3-thia fatty acids on the susceptibility of LDL particles to undergo oxidative modification in vitro were studied. Tetradecylthioacetic acid (TTA), incorporated into the LDL particle and increased the lag time of copper ion induced LDL oxidation in a dose-dependent manner, 80 mumol/L TTA reduced the generation of lipid peroxides during copper ion induced LDL oxidation (for 2 hours) by 100%, 2,2'-azobis-(2,4-dimethylvaleronitrile) induced LDL oxidation by 64%, and 2,2'-azobis-(2-amidinopropane hydrochloride) induced LDL oxidation (for 6 hours) by 21%. The electrophoretic mobility of the oxidized LDL was reduced by TTA in both copper ion and azo-compounds initiated oxidation. This fatty acid analogue was effectively able to reduce in a dose dependent manner the formation of 8-hydroxydeoxyguanosine from 2-deoxyguanosine with ascorbic acid as the radical producer. TTA bound copper(II) ions and did not reduce copper(II) to copper(I). It failed to scavenge the 1,1-diphenyl-2-picrylhydrazyl radicals. The results suggest that the modification of LDL in the lipid and protein moieties can be significantly reduced by TTA. This acid may exert its antioxidant effect partially through metal ion binding and through free radical scavenging.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Amidines; Antioxidants; Ascorbic Acid; Azo Compounds; Copper; Deoxyguanosine; DNA Damage; Dose-Response Relationship, Drug; Free Radical Scavengers; Free Radicals; Humans; Kinetics; Lipid Peroxidation; Lipoproteins, LDL; Nitriles; Oxidants; Oxidation-Reduction; Oxidative Stress; Sulfides

The purpose of this study was to determine the role of reducing agents in maintaining the integrity of vitamin E-deficient red cells. Three groups of one-month-old male Wistar rats were fed a basal vitamin E-deficient diet supplemented with either 0, 10 or 100 mg d, 1-alpha-tocopheryl acetate per kg diet for up to 12 weeks. Washed red blood cells (5%) were resuspended in saline-phosphate buffer, pH 7.4, and were incubated at 37 degrees C with or without containing 12.5 mM 2, 2'-azobis (2amino- propane) dihydrochloride (AAPH), 2.8 mM glucose, 1 mM ascorbic acid, 10 mM hydrogen peroxide (H2O2), 250 microM dimethylsulfoxide (DMSO) or 2.8 mM deoxyribose (DR) for up to 20 hours. Addition of either glucose, AAPH, ascorbic acid or H2O2 markedly accelerated the rates of hemolysis and lipid peroxidation in the red cells of vitamin E-deficient rats. On the contrary, both glucose and ascorbic acid were protective against oxidative damage to the red cells of vitamin E-supplemented rats in a dose-dependent manner. Also, vitamin E-supplemented red cells were more resistant to AAPH and H2O2 than the deficient cells. DMSO or. DR had no significant effects on the rates of hemolysis or lipid peroxidation. Glucose, but not others, maintained or slowed down the loss of glutathione (GSH) during incubation. The results obtained suggest a dual role of ascorbic acid and GSH in the function of vitamin E in maintaining red cell integrity: these reducing agents may exert antioxidant function by participating in vitamin E regeneration when certain levels of vitamin E is maintained, but promote oxidative damage by enhancing free radical generation when vitamin E is low or depleted.

Topics: alpha-Tocopherol; Amidines; Analysis of Variance; Animals; Antioxidants; Ascorbic Acid; Deoxyribose; Dimethyl Sulfoxide; Erythrocytes; Glucose; Hydrogen Peroxide; In Vitro Techniques; Kinetics; Male; Rats; Rats, Wistar; Tocopherols; Vitamin E; Vitamin E Deficiency

We investigated the inhibition mechanism of lipid peroxidation by estrogens. Estradiol and 2-hydroxyestradiol showed strong inhibitory activities toward NADPH and ADP-Fe(3+)-dependent lipid peroxidations in the microsomes from rat livers only when the steroids were added to the reaction system before the start of the peroxidation reaction. These steroids also strongly inhibited oxygen uptake only when added before the start of the reaction. These results suggest that estradiol and 2-hydroxyestradiol inhibit the initial stage of microsomal lipid peroxidation. Lipid peroxidation of erythrocyte membranes induced by the systems of xanthine oxidase-hypoxanthine and ascorbate was strongly inhibited by 2-hydroxyestradiol, but not by estradiol. Lipid peroxidation of erythrocyte membranes induced by 2.2'-azobis- (amidinopropane) dihydrochloride was not markedly inhibited by estradiol and 2-hydroxyestradiol, suggesting that the steroids have low reactivity with lipid peroxyl radicals. However, lipid peroxidation induced by t-butyl hydroperoxide-Fe3+ was strongly inhibited only by 2-hydroxyestradiol. It seems that 2-hydroxyestradiol may interact with alkoxyl rather than with peroxyl radicals during lipid peroxidation.

Topics: Adenosine Diphosphate; Amidines; Animals; Ascorbic Acid; Butylated Hydroxytoluene; Dose-Response Relationship, Drug; Erythrocyte Membrane; Estradiol; Free Radical Scavengers; Hypoxanthine; Iron; Lipid Peroxidation; Microsomes, Liver; NADP; Rats; Rats, Wistar; Thiobarbituric Acid Reactive Substances; Xanthine Oxidase

Alpha-Tocopherol is a classical lipophilic antioxidant well known as a scavenger of free radicals in a hydrophobic milieu. However, it can develop both anti- and prooxidant activity in isolated low density lipoprotein (LDL). It is unknown how these activities are balanced in vivo in human plasma. We studied oxidation of plasma and LDL isolated from healthy donors or from a patient with familial isolated vitamin E deficiency and supplemented with alpha-tocopherol in vivo or in vitro. We found that alpha-tocopherol supplementation decreased plasma and LDL oxidizability under strong oxidative conditions when oxidation was initiated by high amounts of Cu2+ or 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH). The effect was independent of the presence of ascorbate in the samples. Under conditions of mild oxidation by low amounts of Cu2+ or AAPH, alpha-tocopherol supplementation decreased plasma oxidizability only in the presence of physiological amounts of ascorbate. A prooxidant effect of alpha-tocopherol was found under mild oxidative conditions in highly diluted (150-fold) plasma and in isolated LDL. These results indicate that the level of oxidative stress and concentration of co-antioxidants, such as ascorbate, capable of regenerating alpha-tocopherol in the oxidizing lipoprotein particle, appear to represent major factors determining alpha-tocopherol activity towards oxidation both in human plasma and LDL. In vivo, in the presence of high concentrations of co-antioxidants and under mild oxidative conditions, alpha-tocopherol should normally behave as an antioxidant. This antioxidant activity is also expected to prevail under strong oxidative conditions independently of the presence of co-antioxidants but it may evolve into prooxidant, when the co-antioxidants are exhausted under conditions of mild oxidation. It remains to be shown whether such a transformation is physiologically relevant and can occur in vivo.

Topics: Adult; Amidines; Antioxidants; Ascorbic Acid; Copper; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Humans; Lipid Peroxidation; Lipoproteins, LDL; Ubiquinone; Vitamin E; Vitamin E Deficiency

The oxidation of low density lipoprotein (LDL) was carried out aiming specifically at elucidating the anti-oxidant action of alpha-tocopherol. Lipophilic and hydrophilic azo compounds and copper induced the oxidation of LDL similarly to give cholesterol ester and phosphatidylcholine hydroperoxides as major products. The antioxidant potency of alpha-tocopherol in LDL was much poorer than in homogeneous solution. Doxyl stearic acids were used as spin probe and incorporated in LDL. The rate of reduction of doxyl nitroxide in LDL by ascorbate decreased with increasing distance from the LDL surface. From the competition between the spin probe and alpha-tocopherol in scavenging radical, it was found that the efficacy of radical scavenging by alpha-tocopherol became smaller as the radical went deeper into the interior of LDL. On the other hand, 2,2,5,7,8-pentamethyl-6-chromal spared the spin label regardless of the position of nitroxide. The antioxidant activity of chromanols against LDL oxidation increased with decreasing length of isoprenoid side chain at the 2-position. All these results were interpreted by location and low mobility of alpha-tocopherol in LDL. The tocopherol mediated propagation was observed notably at low rate of radical flux, but this was suppressed by reductant such as ascorbic acid and ubiquinol.

Topics: Amidines; Antioxidants; Ascorbic Acid; Azo Compounds; Copper; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Humans; Lipoproteins, LDL; Nitriles; Oxidation-Reduction; Solutions; Stearic Acids; Ubiquinone; Vitamin E

Oxidative damage to biological membranes is an important cause of tissue injury in inflammatory bowel disease. 5-Aminosalicylic Acid (5ASA) has therapeutic efficacy in Ulcerative colitis, which may be based on its antioxidant properties. We used Parinaric acid as a fluorescent marker of oxidation in an intestinal microvillous brush border membrane preparation. Various concentrations of the antioxidants 5ASA, ascorbate, and tocopherol were added, and oxidation was initiated from within the membrane by 2,2' azobis (2.4-dimethylvaleronitrile) (AMVN) and from solution by 2,2' azobis (2-amidinopropane) hydrochloride (AAPH). Tocopherol was able to inhibit oxidation from either source. Ascorbate was only able to inhibit oxidation initiated from solution. 5ASA was able to inhibit oxidation initiated from either site, and was more effective than tocopherol against AAPH, but similarly effective against AMVN. We postulate that water soluble 5ASA preferentially associates with membrane surface, allowing chain-breaking antioxidant activity when peroxidation is initiated within the membrane. Likewise, it is effective against aqueous oxidants because its position allows it to interact with AAPH before lipid peroxidation can be initiated as well as breaking the lipid peroxidation chain once it is initiated. This dual capacity may be important for therapeutic effect of 5ASA and may suggest other candidate antioxidants for clinical trials.

Topics: Amidines; Aminosalicylic Acids; Animals; Antioxidants; Ascorbic Acid; Azo Compounds; Fatty Acids, Unsaturated; Fluorescent Dyes; Guinea Pigs; Intestines; Male; Mesalamine; Microvilli; Nitriles; Oxidation-Reduction; Spectrometry, Fluorescence; Vitamin E

The kinetics of oxidation of endogenous antioxidants such as vitamins C and E and thiols as well as membrane cholesterol in isolated rat brain mitochondria were studied. Oxidation was induced by incubating the mitochondria at 37 degrees C with the free radical generators 2,2' azobis (2'-amidinopropane) dihydrochloride (ABAPH) and 2,2' azobis (2,4-dimethyl) valeronitrile (ABDVN) which undergo thermal decomposition to yield free radicals. An approximate order for the in vitro ease of oxidation was: ascorbate >> alpha-tocopherol > sulfhydryls >> cholesterol. However, small amounts of ascorbate were present in the mitochondria when alpha-tocopherol and sulfhydryl compounds were getting oxidized. This observation is different from those with more homogeneous biological substrates like blood plasma or serum. The order of oxidation of the various compounds is a function of not only the redox potentials but also the (a) concentrations of the oxidized and reduced species, (b) compartmentation of the compounds and (c) enzymatic and nonenzymatic systems for the repair or regeneration of the individual antioxidants. Even though ascorbate levels are quite low within mitochondria this nutrient may play a major role as a first line of defense against oxidative stress. The lipid-soluble ABDVN was much more potent in oxidizing membrane alpha-tocopherol and thiols than the water-soluble ABAPH. With both free radical generators the rate of oxidation of the antioxidants consisted of two phases. The initial phase, that is more rapid, may represent a pool of antioxidant that is involved in immediate antioxidant protection of the organelle with the slower compartment being responsible for replenishing the faster pool whenever needed.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amidines; Animals; Ascorbic Acid; Azo Compounds; Brain; Cholesterol; Intracellular Membranes; Male; Mitochondria; Nitriles; Oxidation-Reduction; Rats; Rats, Inbred F344; Sulfhydryl Compounds; Vitamin E

The objective of this study was to determine whether subjecting human plasma to oxidant stress reduces the activity of lecithin/cholesterol acyltransferase (LCAT, EC 2.3.1.43). Plasma was incubated for 4 h with 2.25-45 mM of 2,2'-azobis(2-amidinopropane)HCl (AAPH), a source of peroxyl radicals. A time- and concentration-dependent reduction of LCAT activity occurred, relative to control samples incubated in the absence of AAPH. Reduction of LCAT activity was disproportionate to elevation of thiobarbituric acid-reactive substances (TBARS) in the plasma. Added ascorbate was able to significantly prevent reduction of LCAT activity, but this effect was unrelated to blockage of TBARS formation by the antioxidant. The results suggest that LCAT activity can be down-modulated by oxidant stress, but not necessarily by lipid peroxidation.

Topics: Amidines; Antioxidants; Ascorbic Acid; Enzyme Inhibitors; Free Radicals; Humans; Kinetics; Lipid Peroxidation; Oxidative Stress; Peroxides; Phosphatidylcholine-Sterol O-Acyltransferase; Thiobarbituric Acid Reactive Substances

Hemolysis of human erythrocytes induced by free radicals initiated from water-soluble, 2,2'-azobis(amidinopropane) dihydrochloride (AAPH) has been investigated. The formation of the radical detected as DMPO (5,5-dimethyl-1-pyrroline N-oxide) adduct depended on temperature and AAPH concentration in a similar manner as hemolysis. The curve for the formation of DMPO--radical adduct, however, did not correspond directly to the hemolysis curve. The product of thiobarbituric acid-reactive materials, which reflect the extent of lipid peroxidation, could not be related directly to the hemolysis curve, too. During the hemolysis, the fluidity of the erythrocyte membrane did not change in appearance. To study whether band 3 proteins participate in the hemolysis or not, eosin-5-maleimide (EMI)-labeled ghosts were incubated in the presence of AAPH. High molecular weight band 3 was formed, and the induced circular dichrosim spectrum of the bound EMI was changed, indicating a conformational change of band 3. It was observed that ascorbic acid suppressed the hemolysis and the oxidation of band 3 dose dependently to produce an induction period. This result shows that specifically blocking band 3 oxidation inhibits the hemolysis, despite lipid peroxidation. Further, it was observed that the EMI-labeled erythrocytes revealed distinct clusters by incubation with AAPH. This means a redistribution of band 3 proteins to form hemolytic holes in the membrane. However, the time course of the conformational change of band 3 during the redistribution was not also correspondent to the hemolysis curve. These results indicate that either lipid peroxidation or redistribution of oxidized band 3 is not attributed only by itself to the hemolysis. Thus, the hemolysis was interpreted by a simple competitive reaction model between lipid peroxidation and redistribution of oxidized band 3. This model explained well the hemolysis curves.

Topics: Amidines; Anion Exchange Protein 1, Erythrocyte; Ascorbic Acid; Circular Dichroism; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Erythrocytes; Free Radicals; Hemolysis; Humans; In Vitro Techniques; Kinetics; Lipid Peroxidation; Membrane Lipids; Oxidation-Reduction; Protein Conformation; Solubility; Spin Labels; Thermodynamics; Water

We studied in vitro the ability of nitecapone, 3-[(3,4-dihydroxy-5-nitrophenyl)methylene]-2,4-pentanedione, a novel water-soluble compound with antioxidative properties, to inhibit the LDL oxidation promoted by copper ions, the aqueous free radical generator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH), and mouse peritoneal macrophages. In these three oxidation systems, the extent of LDL oxidation was determined by measuring the formation of conjugated dienes, the formation of thiobarbituric acid-reactive substances, the change in the electrophoretic mobility of LDL, and the uptake of LDL by macrophages. When LDL oxidation was promoted by copper ions, the reaction was found to be inhibited by nitecapone added in a three- to five-molar excess of the concentration of copper ions. The mechanism by which nitecapone exerted its antioxidative effect in copper-mediated LDL oxidation depended on binding and redox inactivation of the copper ions. Moreover, nitecapone released LDL-bound copper ions and so rendered the LDL particles more resistant to oxidation. In contrast to a water-soluble alpha-tocopherol analogue that was rapidly consumed during the oxidative process, nitecapone retained its inhibitory effect for at least 2 days. Using immobilized metal ion affinity chromatography, we showed that nitecapone binds both copper and iron ions, whereas its affinity for zinc ions is low. Nitecapone also inhibited LDL oxidation in the free radical-mediated oxidation system (AAPH). In this system, nitecapone showed synergistic antioxidative action with ascorbic acid. Finally, nitecapone inhibited macrophage-mediated LDL oxidation. Accordingly, nitecapone appears to have a unique antioxidative profile in that it both selectively chelates pro-oxidative transition metals and scavenges free radicals.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amidines; Animals; Antioxidants; Ascorbic Acid; Catechols; Copper; Female; Free Radicals; Humans; Iron; Lipid Peroxidation; Lipoproteins, LDL; Macrophages, Peritoneal; Mice; Oxidation-Reduction; Pentanones; Thiobarbituric Acid Reactive Substances

The Total Radical-Trapping Antioxidant Parameter (TRAP) of 10 freshly prepared human plasmas was measured by a new fluorometric assay. In this method, the rate of peroxidation induced by 2,2'-diazobis (2-amidinopropane) dihydrochloride (ABAP) was monitored through the loss of fluorescence of the protein R-Phycoerythrin (R-PE). The lag-phase induced by plasma was compared to that induced by 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid (Trolox, a water-soluble analogue of vitamin E). Proteins (but not their sulphydryl groups) interfere with the analysis, partially protecting R-PE when all plasma antioxidants are exhausted. A Trolox-induced lag-phase must therefore be measured on each plasma sample. We found that ascorbate (2.5-5.3%), alpha-tocopherol (2.9-8.5%), urate (19.6-61.0), and thiol groups (17.3-42.3%) jointly explain up to 70% of TRAP. Thus, either other compounds present in plasma are likely to exert antioxidant action, or a marked synergistic action between antioxidants should be postulated to exist. This latter hypothesis is supported by the finding that the simultaneous inactivation of ascorbate and thiol groups produces a loss in antioxidant capacity of plasma greater (26%) than the sum of the decreases produced by the separate inactivation of each of the two compounds. The proposed method appears simple, reliable, and allows the rapid handling of a reasonable number of freshly prepared plasma samples. Given the rapid loss of TRAP upon storage, the latter characteristic is crucial in studies on humans, involving a large number of subjects.

Topics: Adult; Amidines; Antioxidants; Ascorbic Acid; Chromans; Free Radicals; Humans; Peroxides; Phycoerythrin; Spectrometry, Fluorescence; Sulfhydryl Compounds; Uric Acid; Vitamin E

Free radical-induced oxidation of vitamins C, E, sulfhydryl compounds, and cholesterol in brain synaptosomes from Fisher 344 rats was studied. The synaptosomes were incubated at 37 degrees C with 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH), which undergoes thermal decomposition to yield free radicals. After incubation, the synaptosomes were sedimented, saponified, and extracted with hexane to isolate tocopherol and cholesterol. Ascorbate and tocopherol were assayed by liquid chromatography, cholesterol by gas chromatography, and total sulfhydryls by spectrophotometry. Under the in vitro conditions used in this study, the approximate order for the ease of oxidation of the various compounds was: ascorbate >>tocopherol > sulfhydryl compounds >>> cholesterol. However, tocopherol and sulfhydryl oxidation occurred even before all of the ascorbate had been consumed. Therefore, the fate of a specific antioxidant at a particular cellular location cannot be predicted with complete accuracy using the in vitro order for ease of oxidation shown here. Ascorbate may play a major role in protecting brain against oxidative damage because: (i) ascorbate concentration is high in brain, (ii) it can regenerate vitamin E from its radical oxidation product, and (iii) it is one of the first antioxidants to be consumed during oxidative reactions.

Topics: Amidines; Animals; Antioxidants; Ascorbic Acid; Brain; Cholesterol; Free Radicals; Kinetics; Lipid Peroxidation; Male; Malondialdehyde; Oxidation-Reduction; Rats; Rats, Inbred F344; Sulfhydryl Compounds; Synaptosomes; Thiobarbituric Acid Reactive Substances; Vitamin E

We have compared the peroxyl radical scavenger ability to melatonin with that of vitamin E, vitamin C and reduced glutathione (GSH). In the assay system, beta-phycoerythrin (beta-PE) was used as fluorescent indicator protein, 2-2'-azo-bis(2-amidinopropane)dihydrochloride as a peroxyl radical generator and the water soluble vitamin E analogue. Trolox, as reference standard. Results are expressed as oxygen radical absorbing capacity (ORAC(perox)) units, where 1 ORAC unit equals the net protection produced by 1 microM Trolox. A linear correlation of ORAC values with concentration (0.5-4 microM) of all the substances tested has been observed. However, on molar basis, the relative ORAC(perox) of Trolox, vitamin C, GSH and melatonin was 1:1.12:0:68:2.04, respectively. Thus, melatonin, which is a lipid-soluble compound, was twice more active than vitamin E, believed to be the most effective lipophilic antioxidant.

Topics: Amidines; Ascorbic Acid; Free Radical Scavengers; Glutathione; Melatonin; Peroxides; Phycoerythrin; Time Factors; Vitamin E

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

Tamoxifen (TAM) is the antiestrogen most widely used in the chemotherapy and chemoprevention of breast cancer. It has been reported that TAM and its more active metabolite 4-hydroxytamoxifen (OHTAM) induce multiple cellular effects, including antioxidant actions. Here sarcoplasmic reticulum membranes (SR) were used as a simple model of oxidation to clarify the antioxidant action type and mechanisms of these anticancer drugs on lipid peroxidation induced by Fe2+/ascorbate and peroxyl radicals generated by the water-soluble 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) and by the lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). Peroxidation was monitored by different assay systems, namely cis-parinaric acid (PnA) fluorescence quenching, production of thiobarbituric acid-reactive substances, polyunsaturated fatty acids (PUFA) degradation and oxygen consumption. TAM and OHTAM are efficient inhibitors of lipid peroxidation induced by Fe2+/ascorbate and strong intramembraneous scavengers of peroxyl radicals generated either in the water or lipid phases by AAPH and AMVN, respectively. However, these drugs are not typical chain-breaking antioxidant compounds as compared with vitamin E. Additionally, their antioxidant effectiveness enhances the protective capacity of vitamin E against lipid peroxidation induced by AMVN. OHTAM is a more powerful intramembraneous inhibitor of lipid peroxidation as compared with TAM; this effectiveness not correlating with alterations on membrane fluidity may be due to the presence of a hydrogen-donating HO-group in the OHTAM molecule and its preferential location in the outer bilayer regions where it can donate the hydrogen atom to quench free radicals capable of initiating the membrane oxidative degradation. The stronger OHTAM intramembraneous scavenger capacity over TAM also correlates with its higher partition in biomembranes. Therefore, the strong peroxyl radical scavenger activity of OHTAM in the hydrophobic membrane phase may putatively contribute to the mechanisms of cytostatic and chemopreventive action of its promoter TAM on development of breast cancer.

Topics: Amidines; Animals; Ascorbic Acid; Azo Compounds; Ferrous Compounds; Free Radical Scavengers; Hydrogen Peroxide; Intracellular Membranes; Lipid Peroxidation; Nitriles; Oxygen Consumption; Rabbits; Sarcoplasmic Reticulum; Tamoxifen; Time Factors

We have recently shown that the mitochondrial transcription system is extremely sensitive to inhibition by peroxyl radicals generated by either 2,2'-azobis(2-amidinopropane) (AAPH) or 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN), and that this inhibition occurs prior to detectable evidence of lipid peroxidation as measured by thiobarbituric acid (TBA)-reactive substances, 4-hydroxynonenal accumulation, and oxygen consumption. In this report, we further confirm that mitochondrial transcription is sensitive to oxidative stress. We also demonstrate that alpha-tocopherol, ascorbate, and glutathione can partially attenuate these effects, but none of the three, nor the three together, are capable of completely preventing this oxidant-induced repression. This suggests that these physiological antioxidants, while capable of preventing lipid peroxidation chain reactions, are less effective at protecting the mitochondrial transcriptional machinery against this oxidative insult.

Topics: Amidines; Animals; Antioxidants; Ascorbic Acid; Azo Compounds; Glutathione; Male; Mitochondria, Liver; Nitriles; Peroxides; Rats; Rats, Inbred F344; Transcription, Genetic; Vitamin E

Extracellular fluids contain low-molecular-weight antioxidants that are actively involved in the defense against reactive oxygen species. The antioxidant activity of these compounds is largely due to their ability to trap oxygen radicals. Less known is the ability of extracellular antioxidants to scavenge carbon-centered free radicals (C-radicals). These radicals can be involved in the damage under hypoxic/anoxic conditions as well as in ischemia/reperfusion injury. We studied the reactivity of some plasma antioxidants toward a water-soluble C-radical generated by the azocompound 2,2'-azobis(2-amidinopropane) hydrochloride (AAP) under anaerobic conditions. The AAP C-radical in plasma was trapped by the spin trap 3,5-dibromo-4-nitrosobenzene-sulfonic acid (DBNBS) and produced a DBNBS radical. The scavenging properties of urate, cysteine, glutathione, natural amino acids, and serum albumin were assessed by the inhibition of the intensity of DBNBS radical. The antioxidant activity of ascorbate and that of vitamin E was measured directly by the formation of their free radicals. Urate, vitamin E and non-SH amino acids were ineffective and ascorbate was a poor scavenger of AAP C-radical. At variance, cysteine and glutathione (0.1-1.0 mM) were effective scavengers of AAP C-radicals and, importantly, protected plasma ascorbate from oxidation under both aerobic or anaerobic conditions. Our data show that ascorbate in aerobic plasma can reduce vitamin E radical and the oxidized ascorbate may be recycled by a thiol antioxidant cycle. Low-molecular-weight antioxidants accounted only partially for plasma scavenging activity of C-radicals. Plasma strongly reduced the intensity of DBNBS radical and, after dialysis, its activity was reduced by approximately 10%. Serum albumin showed an antioxidant activity comparable to dialyzed plasma. Also the cysteine residue of serum albumin was an efficient scavenger of C-radicals as shown by approximately 20% decrease in the protein scavenging activity after thiol alkylation. These results suggest that elevation in the concentration of total reduced thiols in plasma may improve its antioxidant activity under hypoxic/anoxic conditions. This may be particularly useful since other important antioxidant mechanisms such as urate, ascorbate, and vitamin E appear to be inefficient.

Topics: Aerobiosis; Amidines; Anaerobiosis; Antioxidants; Ascorbic Acid; Benzenesulfonates; Blood Proteins; Carbon; Cysteine; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Free Radicals; Glutathione; Humans; Molecular Weight; Nitroso Compounds; Serum Albumin; Spin Labels; Sulfhydryl Compounds; Uric Acid; Vitamin E

Topics: 1,2-Dipalmitoylphosphatidylcholine; Amidines; Animals; Antioxidants; Ascorbic Acid; Erythrocytes; Free Radicals; Hemolysis; In Vitro Techniques; Mice; Solubility

The pyridoindole derivative stobadine [(-)-cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-1H-pyrido(4,3b)indole] has been described as a drug with antihypoxic and antiarrhythmic cardioprotective properties. Here its reactivity with peroxyl radicals in liposomes using a lipid-soluble azo-initiator of peroxyl radicals, 2,2'-azo-bis(2,4-dimethyl-valeronitrile) (AMVN), was examined. Stobadine exerted scavenging as evidenced by the inhibition of: (i) cis-parinaric acid fluorescence decay (half-maximal effect at 20 microM), or (ii) luminol-sensitized chemiluminescence (half-maximal effect at 33 microM). In rat liver microsomes, stobadine was equally efficient in inhibiting lipid peroxidation induced by lipid-soluble (AMVN) or water-soluble 2,2'-azo-bis(2-aminopropane)-HCl (AAPH), azo-initiators of peroxyl radicals with half-maximal effect at 17 microM. Stobadine partitions in a two-phase system (octanol-water) with the coefficient log P = 0.57 +/- 0.03, explaining its ability to quench peroxyl radicals in both lipid and aqueous phases. Stobadine is not an efficient scavenger of superoxide radicals. The second order rate constant for the reaction of stobadine with superoxide was estimated to be 7.5 x 10(2) M-1 sec-1 as measured by superoxide-induced lucigenin-amplified chemiluminescence. ESR measurements showed that stobadine in liposomes does not reduce the chromanoxyl radical of a vitamin E homologue with a 6-carbon side-chain, 2,5,7,8-tetramethyl-2-(4'-methylpentyl)chroman-6-ol(chromanol++ +-alpha-C6), in agreement with pulse-radiolysis results obtained using Trolox in homogeneous solution (Steenken et al., Chem Res Toxicol 5: 355-360, 1992). Stobadine increased the magnitude of the chromanoxyl and ascorbyl radical ESR signal generated by lipoxygenase+arachidonate. This was interpreted to be due to the interaction of stobadinyl radicals with the chromanol ring and ascorbate, respectively. It is suggested that high reactivity of stobadine radicals requires the presence of reducing antioxidants (vitamin E, vitamin C) to exhibit its antioxidant effects in physiological systems.

Topics: Amidines; Animals; Antioxidants; Ascorbic Acid; Azo Compounds; Carbolines; Electron Spin Resonance Spectroscopy; Fatty Acids, Unsaturated; Female; Free Radical Scavengers; Free Radicals; Luminescent Measurements; Luminol; Microsomes, Liver; Nitriles; Peroxides; Phenols; Rats; Rats, Sprague-Dawley; Superoxides; Vitamin E

The ascorbate free radical (A.-) is a resonance-stabilized tricarbonyl species that is readily formed from the one-electron oxidation of ascorbate, AH-. Because of the low reduction potential of the A.-/AH- couple, Eo' = +282 mV, nearly every oxidizing radical that can arise in a biological system will bring about the one-electron oxidation of AH-, forming A.-. Thus, the steady-state concentration of A.- can be used as a measure of oxidative stress in chemical, biochemical, and biological systems. In this article, we discuss the energetics of ascorbate free radical reactions and demonstrate that the A.- electron paramagnetic resonance (EPR) signal intensity can serve as a marker for the degree of oxidative stress occurring in a system.

Topics: Amidines; Ascorbic Acid; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Free Radicals; In Vitro Techniques; Oxidation-Reduction; Riboflavin; Spin Labels; Thermodynamics; Xanthine Oxidase

We exposed human blood plasma and low-density lipoprotein (LDL) to many different oxidative challenges and followed the temporal consumption of endogenous antioxidants in relation to the initiation of oxidative damage. Under all types of oxidizing conditions, ascorbic acid completely protects lipids in plasma and LDL against detectable peroxidative damage as assessed by a specific and highly sensitive assay for lipid peroxidation. Ascorbic acid proved to be superior to the other water-soluble plasma antioxidants bilirubin, uric acid, and protein thiols as well as to the lipoprotein-associated antioxidants alpha-tocopherol, ubiquinol-10, lycopene, and beta-carotene. Although these antioxidants can lower the rate of detectable lipid peroxidation, they are not able to prevent its initiation. Only ascorbic acid is reactive enough to effectively intercept oxidants in the aqueous phase before they can attack and cause detectable oxidative damage to lipids.

Topics: Amidines; Ascorbic Acid; Free Radicals; Humans; Lipid Metabolism; Lipid Peroxides; Lipids; Lipoproteins, LDL; Neutrophils; Nicotiana; Oxidation-Reduction; Peroxides; Plants, Toxic; Smoke; Tetradecanoylphorbol Acetate

Topics: Amidines; Ascorbic Acid; Copper; Free Radicals; Indicators and Reagents; Peroxides; Phycoerythrin; Rhodophyta; Spectrometry, Fluorescence

Topics: Amidines; Antioxidants; Ascorbic Acid; Bilirubin; Erythrocytes; Free Radicals; Hemoglobins; Hemolysis; Humans; Potassium; Sulfhydryl Compounds; Uric Acid; Vitamin E

Liposomes comprising dimyristoylphosphatidylcholine and benzo[a]pyrene (B[a]P) were incubated at 37 degrees C in the presence of a water-soluble azo initiator. B[a]P 1,6-, 3,6- and 6,12-quinone were formed with the generation of peroxyl radicals by the thermal decomposition of the initiator in an aqueous phase of the suspension. Vitamin E showed little inhibitory effect on B[a]P quinone formation. Uric acid was found to suppress B[a]P quinone formation completely at a concentration lower than that of vitamin C, indicating that uric acid in an aqueous phase traps peroxyl radicals more effectively.

Topics: Amidines; Antioxidants; Ascorbic Acid; Benzo(a)pyrene; Free Radicals; Quinones; Uric Acid; Vitamin E