ascorbic-acid and dityrosine

The effect of ascorbate treatment on apheresis-induced oxidative stress in uremic and dyslipidemic patients was evaluated.. We developed a chemiluminescence-emission spectrum and high-performance liquid chromatography analysis to assess the effect of ascorbate supplement on plasma reactive oxygen species (ROS) scavenging activity and oxidized lipid/protein production in hyperlipidemic and uremic patients undergoing apheresis. Apheresis was efficient in reduction of atherogenic lipoproteins, complement, fibrinogen, soluble intercellular adhesion molecule-1, and oxidative parameters including phosphatidylcholine hydroperoxide (PCOOH), malonaldehyde, methylguanidine, and diotyrosine. Apheresis itself, however, activated leukocytes to increase ROS activity and reduced the plasma ROS scavenging activity. Ascorbate administration selectively diminished apheresis-enhanced H2O2 and inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein-1. Chronically dyslipidemic and uremic patients undergoing biweekly apheresis plus ascorbate treatment had lower levels of C-reactive protein and PCOOH than did those without ascorbate treatment during a 6-month follow-up study period.. We demonstrate that apheresis with ascorbate treatment provides a therapeutic potential in reducing atherosclerotic risk via inhibition of H2O2-induced oxidative stress in patients with uremia or dyslipidemia.

Topics: Adult; Antioxidants; Ascorbic Acid; Biomarkers; C-Reactive Protein; Combined Modality Therapy; Drug Therapy, Combination; Female; Humans; Hyperlipidemias; Hypolipidemic Agents; Lipid Peroxidation; Luminescent Measurements; Male; Malondialdehyde; Methylguanidine; Middle Aged; Neutrophils; Oxidative Stress; Plasmapheresis; Reactive Oxygen Species; Respiratory Burst; Treatment Outcome; Tyrosine; Uremia; Vitamin E

Pyroglutamate-modified amyloid-β (pE-Aβ) is a highly neurotoxic amyloid-β (Aβ) isoform and is enriched in the brains of individuals with Alzheimer disease compared with healthy aged controls. Pyroglutamate formation increases the rate of Aβ oligomerization and alters the interactions of Aβ with Cu(2+) and lipids; however, a link between these properties and the toxicity of pE-Aβ peptides has not been established. We report here that Aβ3pE-42 has an enhanced capacity to cause lipid peroxidation in primary cortical mouse neurons compared with the full-length isoform (Aβ(1-42)). In contrast, Aβ(1-42) caused a significant elevation in cytosolic reactive oxygen species, whereas Aβ3pE-42 did not. We also report that Aβ3pE-42 preferentially associates with neuronal membranes and triggers Ca(2+) influx that can be partially blocked by the N-methyl-d-aspartate receptor antagonist MK-801. Aβ3pE-42 further caused a loss of plasma membrane integrity and remained bound to neurons at significantly higher levels than Aβ(1-42) over extended incubations. Pyroglutamate formation was additionally found to increase the relative efficiency of Aβ-dityrosine oligomer formation mediated by copper-redox cycling.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Ascorbic Acid; Calcium Signaling; Cell Membrane Permeability; Cells, Cultured; Copper; Humans; Lipid Peroxidation; Mice, Inbred C57BL; Neurons; Peptide Fragments; Protein Aggregates; Pyrrolidonecarboxylic Acid; Reactive Oxygen Species; Tyrosine

There is a great interest in the role of free radicals and oxidative stress in Alzheimer's disease and for the role of transition metals in the generation of oligomers of Aβ peptides. In the literature, there are a multitude of varying methods that can be used to create soluble oligomers of Aβ, however, the processes that create these oligomers are often stochastic by nature and thus reproducibility is an issue. Here we report a simple and reproducible method for the production of radically derived dityrosine cross-linked oligomers of Aβ, through reaction with copper and ascorbic acid.

Topics: Amyloid beta-Peptides; Ascorbic Acid; Copper; Cross-Linking Reagents; Hydroxyl Radical; Immunoblotting; Kinetics; Oxidation-Reduction; Peptide Fragments; Protein Multimerization; Protein Structure, Secondary; Spectrometry, Fluorescence; Tyrosine

Using albumin as model, we conducted series of in vitro glycation experiments to examine role of zinc in glycation using glucose at 4-100 mg/ml, incubations at 37°C or 60°C, duration of 2 or 4 weeks and in presence of zinc or ascorbic acid (AA) or folic acid (FA). Modifications of bovine serum albumin (BSA) were examined by using fluorescence of advanced glycation end products (AGEs) and dityrosine, UV, and Fourier transformed infrared spectroscopy. Adding zinc (0 to 768.5 μmol/l) resulted in significant inhibition of albumin glycation by glucose with a linear fit, y = -0·0895x + 230·99(R² = 0·7676, p = 0·013). The glycation by fructose was greater than that of glucose with stronger inhibitory effect by zinc in fructose-glycation (t= -5.8, p=0.002). Addition of zinc significantly decreased fluorescence as seen in Zn + FA or Zn + AA sets as compared to sets of FA alone (p=0.00056) or AA alone (p=0.037). The fluorescence for dityrosine and AGE had a correlation of 0.897 (p<0.01). The data from fluorescence, UV, and FTIR spectra collectively suggested inhibitory effect of zinc in BSA glycation alone or in presence of FA and AA, showing new dimension for the protective action of zinc in hyperglycemic conditions.

Topics: Ascorbic Acid; Folic Acid; Glycation End Products, Advanced; Glycosylation; Spectroscopy, Fourier Transform Infrared; Tyrosine; Zinc

We recently developed two biomarker sets for oxidative damage: one for determination of lipid peroxidation (LPO) degradation products; acetaldehyde, propanal, butanal, pentanal, hexanal, heptanal, octanal, nonanal, malondialdehyde and acetone, by a gas chromatography-electron capture detection method, and the other for protein oxidation products such as o,o'-dityrosine, by an isotope dilution high performance liquid chromatography-tandem mass spectrometry method. In the present study, we explored the possibility to utilize these biomarkers for determining the oxidative damage in liver mammalian cells in vitro. Two different treatments were chosen for inducing oxidative stress in Chinese Hamster ovary cells: menadione and copper plus hydrogen peroxide (Cu2+/H2O2). Cells were incubated with the model compounds in the presence or absence of vitamin E and C, and cytotoxicity was evaluated by a nuclear-dye method. Results were compared to two fluorescent probes, H2DCF-DA and C11 -BODIPY581/591, which have been used for determining the formation of free radicals in the cells. From ten LPO degradation products, eight were increased significantly following incubation with menadione in cell lysate or incubation media. Menadione-induced oxidative stress was also confirmed by oxidation of fluorescent probes. However, no increased formation of protein oxidation products was observed. Vitamin E and C did not diminish the formation of LPO degradation products that were increased by menadione. Although Cu2+/H2O2 did not induce oxidation of fluorescent probes, it induced formation of six out of ten LPO degradation products. Vitamin E and C did not diminish the formation of LPO degradation products; vitamin C even substantially increased the formation of acetaldehyde and propanal, which is in line with its reported prooxidant action under certain conditions. Vitamin C also caused two-fold increase in Cu2+/H2O2-induced o,o'-dityrosine formation when applied simultaneously. In conclusion, our present results show that the LPO biomarker set can be used for evaluation of oxidant capacity and the toxic potential of various chemicals in an in vitro cell model. These biomarkers might even be more sensitive than measuring protein oxidation products or oxidation of fluorescent probes.

Topics: Animals; Ascorbic Acid; Biomarkers; Boron Compounds; Cell Survival; CHO Cells; Cricetinae; Fluoresceins; Fluorescent Dyes; Lipid Peroxidation; Malondialdehyde; Oxidation-Reduction; Proteins; Tyrosine; Vitamin E

To establish its significance during commercial breadmaking, dityrosine formation was quantified in flours and doughs of six commercial wheat types at various stages of the Chorleywood Bread Process. Dityrosine was formed mainly during mixing and baking, at the levels of nmol/g dry weight. Good breadmaking flours tended to exhibit a higher dityrosine content in the final bread than low quality ones, but no relationship was found for dityrosine as a proportion of flour protein content, indicating that the latter was still a dominant factor in the analysis. There was no correlation between gluten yield of the six wheat types and their typical dityrosine concentrations, suggesting that dityrosine cross-links were not a determinant factor for gluten formation. Ascorbic acid was found to inhibit dityrosine formation during mixing and proving, and it has no significant effect on dityrosine in the final bread. Hydrogen peroxide promoted dityrosine formation, which suggests that a radical mechanism involving endogenous peroxidases might be responsible for dityrosine formation during breadmaking.

Topics: Ascorbic Acid; Bread; Cross-Linking Reagents; Flour; Food Handling; Glutens; Hydrogen Peroxide; Proteins; Tyrosine

A method for the quantitation of dityrosine in wheat flour and dough by high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) using an isotope dilution assay with the internal standard 3,3'-(13)C(2)-dityrosine in the single-reaction monitoring mode was developed. The method consisted of the release of protein-bound dityrosine by hydrolysis in 4 mol/L hydrochloric acid/8.9 mol/L propionic acid for 24 h at 110 degrees C after addition of the internal standard, cleanup by C(18) solid-phase extraction, and HPLC-MS/MS. The limit of detection of dityrosine was 80 ng/g of sample (0.22 nmol/g), and the limit of quantitation was 270 ng/g of sample (0.75 nmol/g). The method was sensitive enough to analyze wheat flour and dough and to study the effect of flour improvers on the dityrosine content. Furthermore, the effect of the mixing time was studied. The dityrosine concentration in the flour was 0.66 nmol/g. After we mixed a dough to peak consistency, the dityrosine concentration doubled and remained constant on further mixing. Overdoses of hydrogen peroxide and hexose oxidase (HOX, E.C. 1.1.3.5) resulted in a strongly increased dityrosine content, whereas no increase of the dityrosine concentration was found after the addition of ascorbic acid and potassium bromate. Calculation of the percentage of dimeric tyrosine showed that less than 0.1% of the tyrosine residues of wheat protein were cross-linked. Therefore, dityrosine residues seem to play only a very minor role in the structure of wheat gluten.

Topics: Alcohol Oxidoreductases; Antioxidants; Ascorbic Acid; Bread; Bromates; Chromatography, High Pressure Liquid; Flour; Hydrogen Peroxide; Mass Spectrometry; Oxidants; Triticum; Tyrosine

Recent reports by Galeazzi and co-workers demonstrated the susceptibility of Abeta(1-42) to undergo dityrosine formation via peroxidase-catalyzed tyrosine cross-linking. We have formed dityrosine cross-links in Abeta(1-40) using these enzymatic conditions as well as a copper-H(2)O(2) method. The efficiency of dityrosine cross-link formation is strongly influenced by the aggregation state of Abeta; more dityrosine is formed when copper-H(2)O(2) or horseradish peroxidase-catalyzed oxidation is applied to fibrillar Abeta vs soluble Abeta. Once formed, dityrosine cross-links are susceptible to further oxidative processes and it appears that cross-links formed in soluble Abeta react through these pathways more readily than those formed in fibrillar Abeta. Because preorganization of fibrils affects the efficiency of dityrosine formation, we examined the effect of dityrosine formation upon local peptide conformation by assessing the solution structure of a small dityrosine dimer derived from Abeta(8-14). Two-dimensional (1)H NMR studies of the short dityrosine dimer offer no evidence of structure. Thus, the fibrillar structure of Abeta enhances formation of dityrosine cross-links, but dityrosine cross-links do not seem to enhance local secondary structure.

Topics: Amyloid beta-Peptides; Antibodies, Monoclonal; Ascorbic Acid; Blotting, Western; Copper; Cross-Linking Reagents; Dimerization; Humans; Hydrogen Peroxide; Magnetic Resonance Spectroscopy; Models, Molecular; Peptide Fragments; Protein Structure, Secondary; Structure-Activity Relationship; Tyrosine

The antioxidative effects of urate on peroxidase-induced protein oxidation and light-induced riboflavin degradation and lipid oxidation in whole milk were studied. In addition, experiments using ascorbate were conducted to directly compare the antioxidative activity of urate and ascorbate. The presence of urate and/or ascorbate (10-30 mg/L) lowered peroxidase-induced formation of dityrosine by 44-96% in unpasteurized whole milk. No synergistic effect of urate and ascorbate on peroxidase-induced dityrosine formation was registered, but merely an additive effect. Light exposure of pasteurized whole milk showed that ascorbate was oxidized at the expense of urate, which indicated ascorbate-mediated recycling of the urate radical. Moreover, both urate and ascorbate (30 mg/L) retarded light-induced lipid oxidation in pasteurized whole milk as measured by formation of lipid hydroperoxides with urate being the most effective (28% reduction in lipid hydroperoxides) compared with ascorbate (14%). Finally, addition of urate or ascorbate (300 mg/L) to pasteurized whole milk showed a slight protective effect against light-induced degradation of riboflavin with urate being the most effective.

Topics: Animals; Ascorbic Acid; Cattle; Food Preservation; Hydrogen Peroxide; Light; Milk; Milk Proteins; Peroxidases; Riboflavin; Tyrosine; Uric Acid

In the NO2-exposure of tyrosine in 70% dioxane/phosphate buffer (pH 7.4), beta-carotene enhanced the degradation of tyrosine and/or 3-nitrotyrosine produced, whereas alpha-tocopherol and ascorbyl palmitate inhibited the transformation of tyrosine into 3-nitrotyrosine. Generation of certain active species in the interaction of beta-carotene with NO2 was suggested. Ascorbyl palmitate effectively and alpha-tocopherol slightly inhibited the transformation of tyrosine in the NO2-exposure in the presence of beta-carotene. In the reaction of tyrosine with ONOO-/ONOOH, beta-carotene enhanced the degradation of 3-nitrotyrosine produced suggesting generation of certain active species, whereas alpha-tocopherol and ascorbyl palmitate completely suppressed the transformation of tyrosine into 3-nitrotyrosine.

Topics: Ascorbic Acid; beta Carotene; Dioxanes; Dose-Response Relationship, Drug; Free Radical Scavengers; Nitrogen Dioxide; Nitrous Acid; Peroxynitrous Acid; Time Factors; Tyrosine; Vitamin E

In this paper we demonstrate that NADPH-initiated oxidative damage of microsomal proteins occurs in the absence of free metal ions and that this protein oxidation is mediated by cytochrome P450 (cyt P450). Oxidized proteins are rapidly degraded by proteases. Ascorbate (AH2) specifically inhibits free metal ion-independent cyt P450-mediated protein oxidation and thereby prevents subsequent proteolytic degradation. Other scavengers of reactive oxygen species including superoxide dismutase, catalase, and glutathione are ineffective. This is in variance with free metal ion-catalyzed protein oxidation, which is accelerated by AH2 and inhibited by catalase. Oxidative damage of proteins has been assessed by the production of carbonyl groups, bityrosine formation, and tryptophan loss. The mechanism of protein oxidation has been studied using a reconstituted system comprised of purified NADPH-cyt P450 reductase, cyt P450, and isolated microsomal proteins as well as model polypeptides, e.g. poly-L-proline and poly-L-lysine. Cyt P450 Fe3+ is reduced by NADPH-cyt P450 reductase to cyt P450 Fe2+, which consumes oxygen in a stoichiometric proportion to produce cyt P450 Fe2+ O2, the resonance form of which is a perferryl moiety, cyt P450 Fe3+.O2-.. It is proposed that cyt P450 Fe3+.O2-. abstracts hydrogen from amino acid side chains leading to the production of carbonyl derivatives. Tentatively, AH2 prevents protein oxidation by interacting with cyt P450 Fe3+.O2-..

Topics: Adenosine Diphosphate; Animals; Ascorbic Acid; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Guinea Pigs; Hydrolysis; Iron; Male; Microsomes, Liver; NADP; Oxidation-Reduction; Proteins; Tryptophan; Tyrosine

Enzymatic removal of the cell wall induces vegetative Chlamydomonas reinhardtii cells to transcribe wall genes and synthesize new hydroxyproline-rich glycoproteins (HRGPs) related to the extensins found in higher plant cell walls. A cDNA expression library made from such induced cells was screened with antibodies to an oligopeptide containing the (SP)x repetitive domains found in Chlamydomonas wall proteins. One of the selected cDNAs encodes an (SP)x-rich polypeptide that also displays a repeated YGG motif. Ascorbate, a peroxidase inhibitor, and tyrosine derivatives were shown to inhibit insolubilization of both the vegetative and zygotic cell walls of Chlamydomonas, suggesting that oxidative cross-linking of tyrosines is occurring. Moreover, insolubilization of both walls was concomitant with a burst in H2O2 production and in extracellular peroxidase activity. Finally, both isodityrosine and dityrosine were found in hydrolysates of the insolubilized vegetative wall layer. We propose that the formation of tyrosine cross-links is essential to Chlamydomonas HRGP insolubilization.

Topics: Amino Acid Sequence; Animals; Ascorbic Acid; Base Sequence; Cell Wall; Chlamydomonas reinhardtii; Cloning, Molecular; Cross-Linking Reagents; Epitopes; Glycoproteins; Hydrogen Peroxide; Molecular Sequence Data; Peroxidase; RNA, Messenger; Solubility; Tyrosine

Dimerization of tyrosine phenoxyl radical yields bityrosine (BT) which can easily be monitored by its characteristic fluorescence at 400 nm. The reactivity of tyrosine phenoxyl radical with O2 was examined by a variety of techniques. BT fluorescence was measured as a function of O2 concentration. Over a range of pH values (4-12) there was no effect of oxygen on BT production ([O2] less than or equal to 0.72 mM). In addition, oxygen uptake by the phenoxyl radical was measured directly with an oxygen electrode. It was determined by this technique that oxygen does not react with the phenoxyl radical with a rate constant greater than 10(3) M-1 s-1. Tyrosine phenoxyl radical "repair" by superoxide and physiological antioxidants was examined by BT fluorescence quenching as well as pulse radiolysis. Implications of these results as to the fate of tyrosine phenoxyl radicals produced in biological systems is discussed.

Topics: Antioxidants; Ascorbic Acid; Chromans; Free Radicals; Oxygen; Spectrometry, Fluorescence; Superoxides; Tyrosine

A method for determining relative tyrosyl radical scavenging activity of antioxidants which requires only a standard fluorometer and commercially available materials is presented. Ultraviolet irradiation of aqueous tyrosine solutions containing superoxide dismutase and catalase produces fluorescent dityrosine residues via dimerization of photogenerated tyrosyl radicals. Added antioxidants suppress the buildup of fluorescence by scavenging the tyrosyl radicals. A correlation exists between the ability of a substance to suppress dityrosine formation and the substance's one-electron oxidation potential. This method demonstrates that ovothiol A scavenges tyrosyl radicals much more efficiently than glutathione or cysteine, resembling instead the known biological radical scavengers uric acid and ascorbic acid and the alpha-tocopherol analog trolox.

Topics: Animals; Antioxidants; Ascorbic Acid; Cattle; Chromans; Free Radicals; Methylhistidines; Spectrometry, Fluorescence; Tyrosine; Uric Acid