The kinetics of the reaction of superoxide radical with Fe(III) complexes of EDTA, DETAPAC and HEDTA

AbstractTo gain an understanding of the mechanism by which the hydroxyl free radical can arise in superoxide generating systems and learn how different chelaters of iron can inhibit this reaction, a pulse radiolysis kinetic study of the reaction of O−2 with Fe(III)EDTA, Fe(III)HEDTA and Fe(III)DETAPAC (or DTPA) was undertaken. Superoxide reacts readily with Fe(III)EDTA and Fe(III)HEDTA with a pH-dependent second-order rate constant having values of 1.9 × 106 M−1.s−1 and 7.6 × 105 M−1.s−1 at pH 7, respectively. However, the rate constant for the reaction of O−2 with Fe(III)DETAPAC was found to be much slower, the upper limit for the rate constant being 104 M−1.s−1. These results in conjunction with spin-trapping experiments with Fe(II)EDTA, Fe(II)HEDTA, Fe(II)DETAPAC and H2O2 suggests that DETAPAC inhibits the formation of OH by slowing the reduction of Fe(III) to Fe(II) and not by inhibiting the Fenton reaction

Surface charge, fluidity, and calcium uptake by rat intestinal brush-border vesicles

AbstractBiological membrane outer surfaces are negatively charged and interact with positively charged calcium ion during calcium uptake. Positively charged polycations such as polyarginine bind to membranes with high affinity, displacing bound calcium from the membrane. We tested the effect of polyarginine on uptake of calcium by brush-border membrane vecicles and examined the responses in terms of membrane fluidity by electron paramagnetic resonance (EPR). Polyarginine inhibited the saturable component of calcium uptake by a mechanism combining inhibition characteristics of strontium (competitive) and magnesium (non-competitive). Unlike the inhibition of non-saturable calcium uptake by strontium and magnesium, polyarginine increased kD, the rate constant for non-saturable calcium uptake, by a concentration dependent mechanism. These effects of polyarginine on calcium uptake were associated with decreased membrane fluidity at the uptake temperature. These findings are consistent with a role for surface negative charge in determining both saturable and non-saturable calcium uptake. Increased membrane fluidity is associated with decreased saturable and increased non-saturable calcium uptake. Although increased fluidity might be involved in the increased kD for non-saturable uptake, the concentration-specific stimulating effect of polyarginine suggests a gating mechanism

Direct Measurement of Supra-Physiological Levels of Ascorbate in Plasma using a Nanophotometer

High dose intravenously administered vitamin C (ascorbate) is currently being tested in clinical trials as an adjuvant to current standard of care therapies in a variety of cancers. Intravenous infusion is used with a goal to achieve supraphysiological ascorbate concentrations in blood of at least 20 mM, 300 to 500 times normal healthy concentrations (0.04-0.08 mM). These trials need quick and easy access to information on the levels of ascorbate achieved in the blood to make clinical decisions. Previous methods that quantify ascorbate levels in blood require extensive preparation, time, and materials that may not always be present in clinical settings. We developed a new approach to meet this need using direct UV spectroscopy with a nanophotometer. The only preparation required is centrifugation of whole blood to separate the red blood cells from plasma. No more than 3 microliters of plasma are needed; the approach can determine the concentration of ascorbate in the range of 3 – 35 mM; the method is fast and efficient. This approach has already been deployed to gather this information in a clinical trial with lung cancer patients

Minimization of free radical damage by metal catalysis of multivitamin/multimineral supplements

Multivitamin/multimineral complexes are the most common dietary supplements. Unlike minerals in foods that are incorporated in bioorganic structures, minerals in dietary supplements are typically in an inorganic form. These minerals can catalyze the generation of free radicals, thereby oxidizing antioxidants during digestion. Here we examine the ability of a matrix consisting of an amino acid and non-digestible oligosaccharide (AAOS) to blunt metal-catalyzed oxidations. Monitoring of ascorbate radical generated by copper shows that ascorbate is oxidized more slowly with the AAOS matrix than with copper sulfate. Measurement of the rate of oxidation of ascorbic acid and Trolox® by catalytic metals confirmed the ability of AAOS to slow these oxidations. Similar results were observed with iron-catalyzed formation of hydroxyl radicals. When compared to traditional forms of minerals used in supplements, we conclude that the oxidative loss of antioxidants in solution at physiological pH is much slower when AAOS is present

Extracellular superoxide dismutase (SOD3) regulates oxidative stress at the vitreoretinal interface

Oxidative stress is a pathogenic feature in vitreoretinal disease. However, the ability of the inner retina to manage metabolic waste and oxidative stress is unknown. Proteomic analysis of antioxidants in the human vitreous, the extracellular matrix opposing the inner retina, identified superoxide dismutase-3 (SOD3) that localized to a unique matrix structure in the vitreous base and cortex. To determine the role of SOD3, Sod3-/- mice underwent histological and clinical phenotyping. Although the eyes were structurally normal, at the vitreoretinal interface Sod3-/- mice demonstrated higher levels of 3-nitrotyrosine, a key marker of oxidative stress. Pattern electroretinography also showed physiological signaling abnormalities within the inner retina. Vitreous biopsies and epiretinal membranes collected from patients with diabetic vitreoretinopathy (DVR) and a mouse model of DVR showed significantly higher levels of nitrates and/or 3-nitrotyrosine oxidative stress biomarkers suggestive of SOD3 dysfunction. This study analyzes the molecular pathways that regulate oxidative stress in human vitreous substructures. The absence or dysregulation of the SOD3 antioxidant at the vitreous base and cortex results in increased oxidative stress and tissue damage to the inner retina, which may underlie DVR pathogenesis and other vitreoretinal diseases

Free radicals produced by the oxidation of gallic acid: An electron paramagnetic resonance study

<p>Abstract</p> <p>Background</p> <p>Gallic acid (3,4,5-trihydroxybenzoic acid) is found in a wide variety of plants; it is extensively used in tanning, ink dyes, as well as in the manufacturing of paper. The gallate moiety is a key component of many functional phytochemicals. In this work electron paramagnetic spectroscopy (EPR) was used to detect the free radicals generated by the air-oxidation of gallic acid.</p> <p>Results</p> <p>We found that gallic acid produces two different radicals as a function of pH. In the pH range between 7-10, the spectrum of the gallate free radical is a doublet of triplets (a^H= 1.00 G, a^H= 0.23 G, a^H= 0.28 G). This is consistent with three hydrogens providing hyperfine splitting. However, in a more alkaline environment, pH >10, the hyperfine splitting pattern transforms into a 1:2:1 pattern (a^H(2) = 1.07 G). Using D₂O as a solvent, we demonstrate that the third hydrogen (<it>i.e</it>. a^H= 0.28 G) at lower pH is a slowly exchanging hydron, participating in hydrogen bonding with two oxygens in <it>ortho </it>position on the gallate ring. The p<it>K</it>_aof this proton has been determined to be 10.</p> <p>Conclusions</p> <p>This simple and novel approach permitted the understanding of the prototropic equilibrium of the semiquinone radicals generated by gallic acid, a ubiquitous compound, allowing new insights into its oxidation and subsequent reactions.</p

The rate of cellular hydrogen peroxide removal shows dependency on GSH: Mathematical insight into in vivo H2O2 and GPx concentrations

Although its concentration is generally not known, glutathione peroxidase-1 (GPx-1) is a key enzyme in the removal of hydrogen peroxide (H2O2) in biological systems. Extrapolating from kinetic results obtained in vitro using dilute, homogenous buffered solutions, it is generally accepted that the rate of elimination of H2O2 in vivo by GPx is independent of glutathione concentration (GSH). To examine this doctrine, a mathematical analysis of a kinetic model for the removal of H2O2 by GPx was undertaken to determine how the reaction species (H2O2, GSH, and GPx-1) influence the rate of removal of H2O2. Using both the traditional kinetic rate law approximation (classical model) and the generalized kinetic expression, the results show that the rate of removal of H2O2 increases with initial GPxr, as expected, but is a function of both GPxr and GSH when the initial GPxr is less than H2O2. This simulation is supported by the biological observations of Li et al.. Using genetically altered human glioma cells in in vitro cell culture and in an in vivo tumour model, they inferred that the rate of removal of H2O2 was a direct function of GPx activity × GSH (effective GPx activity). The predicted cellular average GPxr and H2O2 for their study are approximately GPxr ≤ 1 μm and H2O2 ≈ 5 μm based on available rate constants and an estimation of GSH. It was also found that results from the accepted kinetic rate law approximation significantly deviated from those obtained from the more generalized model in many cases that may be of physiological importance