Role of Superoxide Anions in the Redox Changes Affecting the Physiologically Occurring Cu(I)-Glutathione Complex

The physiologically occurring copper-glutathione complex, [Cu(I)-[GSH]2], has the ability to react continually with oxygen, generating superoxide anions (O2∙−). We addressed here the effects that superoxide removal has on the redox state of Cu(I) and GSH present in such complex and assessed the formation of Cu(II)-GSSG as a final oxidation product. In addition, we investigated the potential of a source of O2∙− external to the Cu(I)-[GSH]2 complex to prevent its oxidation. Removal of O2∙− from a Cu(I)-[GSH]2-containing solution, whether spontaneous or Tempol-induced, led to time-dependent losses in GSH that were greater than those affecting the metal. The losses in GSH were not accompanied by increments in GSSG but were largely accounted for by the cumulative formation of Cu(II)-GSSG molecules. Notably, the redox changes in Cu(I) and GSH were totally prevented when Cu(I)-[GSH]2 was coincubated with hypoxanthine/xanthine oxidase. Data suggest that the generation of O2∙− by Cu(I)-[GSH]2 implies the obliged formation of an intermediate whose subsequent oxidation into Cu(II)-GSSG or back reduction into Cu(I)-[GSH]2 is favoured by either the removal or the addition of O2∙−, respectively

Inhibition of mitochondrial complex i by various non-steroidal anti-inflammatory drugs and its protection by quercetin via a coenzyme Q-like action

Mitochondrial dysfunction plays a major role in the development of oxidative stress and cytotoxicity induced by non-steroidal anti-inflammatory drugs (NSAIDs). A major objective of the present study was to investigate whether in vitro the NSAIDs, aspirin, indomethacin, diclofenac, piroxicam and ibuprofen, which feature different chemical structures, are able to inhibit mitochondrial complex I. All NSAIDs were effective inhibitors when added both, directly to mitochondria isolated from rat duodenum epithelium (50 μM) or to Caco-2 cells (250 μM). In the former system, complex I inhibition was concentration-dependent and susceptible to competition and reversion by the addition of coenzyme Q (32.5-520 μM). Based on reports suggesting a potential gastro-protective activity of quercetin, the ability of this flavonoid to protect isolated mitochondria against NSAIDs-induced complex I inhibition was evaluated. Low micromolar concentrations of quercetin (1-20 μM) protected against such inhibiti

The Cu(I)–glutathione complex: factors affecting its formation and capacity to generate reactive oxygen species

Cu2? ions and reduced glutathione (GSH) swiftly interact to form the physiologically occurring Cu(I)–[GSH]2 complex. Prompted by the recently reported ability of this complex to generate superoxide radicals from molecular oxygen, the present study addressed how the concentration of Cu2? and GSH, the pH, and the temperature affect the formation of the Cu(I)–[GSH]2 complex and its capacity to generate superoxide radicals and hydrogen peroxide. Increasing concentrations of Cu2? and GSH, added at a fixed molar ratio of 1:3, led to a proportionally greater production of superoxide anions, hydrogen peroxide, and oxidized glutathione (GSSG). GSSG formation was found to closely reflect the formation of Cu(I)– [GSH]2. Biologically relevant changes in pH (e.g., from 6.8 to 7.7) and temperature (from 22 to 37 C) did not affect the formation of the Cu(I)–[GSH]2, as assessed by GSSG production. However, production of superoxide radicals increased as the pH values were incremented. An opposite effect was observed regarding hydrogen peroxide production. The ability of a freshly prepared Cu(I)–[GSH]2 complex (assayed within a minute from its formation) to generate superoxide radicals was incremented by as the temperature was increased. Such ability, however, correlated inversely with the temperature when, before assaying for superoxide, the earlier referred preparation was incubated during 30 min in the presence of oxygen. Under the latter condition, hydrogen peroxide linearly accumulated in time, suggesting that an increased autodismutation underlies the apparent time-dependent ‘‘aging’’ of the capacity of the complex to generate superoxide.This work was supported by FONDECYT #3080025 (Postdoctoral grant) and by FONDECYT #1070613

Analytical parameters of the microplate-based ORAC pyrogallol red assay

The analytical parameters of the microplatebased oxygen radicals absorbance capacity (ORAC) method using pyrogallol red (PGR) as probe (ORAC-PGR) are presented. In addition, the antioxidant capacity of commercial beverages, such as wines, fruit juices, and iced teas, is estimated. A good linearity of the area under the curve (AUC) versus Trolox concentration plots was obtained [AUC = (845 ± 110) + (23 ± 2) [Trolox, μM], R = 0.9961, n = 19]. QC experiments showed better precision and accuracy at the highest Trolox concentration (40 μM) with RSD and REC (recuperation) values of 1.7 and 101.0%, respectively. When red wine was used as sample, the method also showed good linearity [AUC = (787 ± 77) + (690 ± 60) [red wine, μL/mL]; R = 0.9926, n = 17], precision and accuracy with RSD values from 1.4 to 8.3%, and REC values that ranged from 89.7 to 103.8%. Additivity assays using solutions containing gallic acid and Trolox (or red wine) showed an additive protection of PGR given by the sample

Redox-active complexes formed during the interaction between glutathione and mercury and/or copper ions

Prompted by the recently reported capacity of the physiologically occurring Cu(I)-[glutathione]2 complex (Cu(I)-[GSH)]2) to reduce oxygen, the effect of various GSH-binding metals (Co2+, Cd2+, Zn2+, Pb2+, Al3+, Hg2+ and Ni2+) on the superoxide-generating capacity of such complex was investigated. Amongst all tested metals, only Hg2+ was able to substantially affect the capacity of Cu(I)-[GSH]2 to generate superoxide. When Hg2+ and Cu(I)-[GSH]2 were mixed equimolarly, the superoxide formation, assessed through the cytochrome c reduction and dihydroethidium oxidation, was increased by over 50%. Such effect was totally inhibitable by SOD. Based on the reportedly higher affinity of Hg2+ for GSH and the observed ability of Hg2+ to lower the concentration of Cu(I)-[GSH]2 (spectroscopically assessed), we suggest that Hg2+ displaces Cu(I) from Cu(I)-[GSH]2, to release Cu(I) ions and form a Hg(II)-[GSH]2 complex. The latter species would account for the Hg2+-induced exacerbation of the superox

Redox-changes associated with the glutathione-dependent ability of the Cu(II)-GSSG complex to generate superoxide

The intracellularly-occurring Cu(I)-glutathione complex (Cu(I)-[GSH] 2) has the ability to reduce molecular oxygen into superoxide. Removal of such radicals leads to the irreversible conversion of Cu(I)-[GSH] 2 into the redox-inactive Cu(II)-GSSG complex. The present study addressed the potential of reduced glutathione, ascorbate and superoxide to reductively regenerate Cu(I)-[GSH] 2 from Cu(II)-GSSG, and investigated the redox changes involved in such process. Results show that: (i) among the three tested reductants, only GSH is able to reduce the Cu(II) bound to GSSG; (ii) during the reduction of Cu(II)-GSSG, a Cu(I)-GSSG intermediate would be formed (supported here by Cu(I) and GSSG recovery data and by NMR studies); (iii) when GSH is present in a molar excess equal or greater than 1:3, the reduction of Cu(II)-GSSG into Cu(I)-[GSH] 2 is quantitative and complete. Under such conditions, the Cu(II)-GSSG complex acquires a superoxide-generating capacity which is identical to that see

Application of a microplate-based orac-pyrogallol red assay for the estimation of antioxidant capacity: First action 2012.03

The method was approved by the Expert Review Panel on Strategic Foods Analytical Methods as First Action. See "Standards News," (2012) Inside Laboratory Management, March/April issue. The AOAC Stakeholder Panel on Strategic Foods Analytical Methods (SPSFAM) invites method users to provide feedback on the First Action methods. Feedback from method users will help verify that the methods are fit for purpose and are critical to gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author

Reactivity of benzohydrazide derivatives towards acetylation reaction. Experimental and theoretical studies

We herein report an experimental and theoretical study on the acetylation reaction of benzohydrazide derivatives towards p-nitrophenyl acetate (NPA). The kinetic data are consistent with a stepwise mechanism with the nucleophilic attack as the rate determining step. From the theoretical analysis it is found that benzohydrazide derivatives establish intramolecular proton rearrangement. The enol form appears as the active species for nucleophilic attack. A reaction mechanism incorporating keto-enol pre-equilibria is proposed. The study is completed with a local reactivity analysis describing the most reactive centers for nucleophilic attack together with a site activation analysis describing inductive substituent effects.This work received financial support from project Clínica Alemana- Universidad del Desarrollo 8011046 and Fondecyt Projects: 1070715 and 11060195