Marina V. Ivanova

How do calcium ions induce free radical oxidation of hydroxy-1,4-naphthoquinone? Ca2+ stabilizes the naphthosemiquinone anion-radical of echinochrome A ☆

A surprising effect is the direct action of Ca(2+) on redox reactions of ortho-quinoid compounds. The effect of Ca(2+) on oxidation of the sea urchin pigment 6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone (echinochrome A) has been studied by electron paramagnetic resonance (EPR) spectroscopy, by UV/VIS absorbance spectroscopy, and by measurement of oxygen consumption. Echinochrome A per se reacted with dioxygen only in an alkaline solution; 2,3-semiquinone anion-radical of echinochrome A and superoxide anion-radical were the intermediates of the oxidation. Addition of calcium ions sharply increased the rate of echinochrome A autooxidation at alkaline pH and provoked oxidation at neutral pH. To explain this phenomenon we have focused on changes of the acid-base properties of echinochrome A in the presence of calcium and on stabilization of 2,3-semiquinone anion-radical of echinochrome A by Ca(2+). Dissociation constants (pK(a1), pK(a2), and pK(a3)) of echinochrome A determined by potentiometric titration were 5.20, 6.78, and >10 in calcium-free solution and 5.00, 6.10, and 7.15 in the presence of Ca(2+). We have found that Ca(2+) forms an insoluble adduct with the 2,3-semiquinone anion-radical. Thus, the effect of redox-inert calcium on the free radical reactions could be explained (i) by additional deprotonation of echinochrome A and (ii) by formation of a Ca(2+)-naphtho-2,3-semiquinone complex (calcium semiquinonate). Additionally, we have shown that the dried red spines from Strongylocentrotus intermedius possess paramagnetic properties; the EPR signal of the natural spines was similar to that of calcium semiquinonate obtained in our artificial chemical system.

IRON CHELATORS AND FREE RADICAL SCAVENGERS IN NATURALLY OCCURRING POLYHYDROXYLATED 1,4-NAPHTHOQUINONES

The cardioprotective effect of polyhydroxylated 1,4-naphthoquinones on the experimental model of myocardial ischemia-reperfusion has been demonstrated previously. In this study, using different models, such as bulk organic phase, liposomes and sarcoplasmic reticulum (SR) vesicles, we have shown the ability of naturally occurring polyhydroxynaphthoquinones, echinochrome (Ech), spinochromes C, D and E (SpC, SpD and SpE) to inhibit free-radical oxidation induced by heme iron (hemin) or by free iron ions (in ferrous/ascorbate system). The polyhydroxy-1,4-naphthoquinones (PHNQs) were more effective in inhibiting the phosphatidyl choline liposome peroxidation induced by ferrous/ascorbate than that induced by hemin. The iron chelating ability of PHNQs was determined spectrophotometrically. Prevention of the ferrous/ascorbate-induced leakage of calcium by Ech was demonstrated in isolated SR vesicles from rabbit skeletal muscle. The PHNQs displayed high scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. We concluded that iron chelation predominates in the overall antioxidant potential of the polyhydroxynaphthoquinones.

Calcium-dioxolene complexes: Rate constants of pyrocatechol oxidation in the presence of Ca2+

The effect of calcium ions on the rate of pyrocatechol autoxidation at pH 9.0 has been studied by mathematical modeling. The effect of Ca2+ on the oxygen absorption rate has been studied, and a kinetic model has been suggested, which takes different stages of interaction of pyrocatechol and its radical form with oxygen into account. It has been shown that the prooxidant action of Ca2+ is related to an abrupt increase (approximately by three orders of magnitude) in the rate constant of comproportionation (reaction of chain branching and formation of o-semiquinonates) and a marked decrease (by two orders of magnitude, from 1.4 · 107 to 0.6 · 105 M−1 s−1) in the rate constant of disproportionation of o-semiquinones. The system can be used as a model for studying the prooxidant action of calcium ions.

Paramagnetic calcium melanins

Treatment of catechol, pyrogallol, DOPA, dopamine, norepinephrine, and natural polyhydroxy-1,4-naphthoquinone echinochrome with solution of potassium superoxide (KO2) in the presence of CaCl2 leads to the formation of water-insoluble dark pigments with stable paramagnetic properties (“calcium melanins”). In control experiments in the same procedure without Ca2+, the pigments were not formed. EPR spectra of the calcium melanins had little difference from each other and from known melanins in shape, line width, and the g factor about 2.004. Addition of EDTA water solution to dried paramagnetic pigments leads to their fast dissolving and disappearing of EPR signal. Formation of similar polymers is also observed during autoxidation of o-diphenols in Ca2+-containing alkaline buffer solution, however, this process takes a few days instead of few seconds in the presence of KO2. Thus, calcium (and other divalent cation M2+) can be considered as a key structural element in formation of M2+-catecholate paramagnetic polymer. We assume the existence of two types of paramagnetic centers in melanin-like polymer: M2+-stabilized o-semiquinone radical or bi-radical complex containing o-semiquinone and superoxide anion radicals, stabilized by M2+.

A QUANTITATIVE EVALUATION OF REDOX-ACTIVE COMPOUNDS IN HUMAN BLOOD LIPIDS

Endogenous low molecular weight redox active compounds (RACs) comprise antioxidants, pro-oxidants, transition metal cations and metal chelators. Traditional electrochemical methods of measuring RACs are limited to aqueous solutions, thus providing information of only hydrophilic RAC pools. In a large number of diseases associated with oxidative stress and/or with metal toxicity, redox states of hydrophilic as well as hydrophobic compartments are modified, and therefore development of methods for their detection is both necessary and important. The pools of lipid soluble RACs in reduced and oxidized forms in n-hexane extracts obtained from blood plasma, erythrocytes and whole blood of healthy donors were determined by spectrophotometric detection of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, which stoichiometrically interacts with hydrogen donors in non polar solutions. Measurements of RACs in extracts before and after treatment with NaBH4 provided information about the levels of both reduced and oxidized RACs. Vitamin E was also determined using a fluorescence method. The results have shown that vitamin E is the major RAC in blood plasma lipids but not in blood cell lipids, where other phenols and quinones appear to predominate.

Superoxide Formation in Cardiac Mitochondria and Effect of Phenolic Antioxidants

Since mitochondria are the main cellular source of reactive oxygen species, it is important to study the effect of dietary phenolic compounds on the level of ROS in these organelles. Using the EPR spectroscopy and TIRON probe, the ability of the investigated phenols (quercetin, rutin, caffeic acid, curcumin, and resveratrol) to scavenge superoxide anion radicals generated by isolated heart mitochondria of Wistar rats under variable oxygen partial pressure was studied. It was shown that during a 10 min incubation, caffeic acid in concentrations of 10–500 μM most effectively scavenged superoxide radicals formed in the complex III of the mitochondrial respiratory chain. A comparable antioxidant effect of rutin under these experimental conditions was observed at higher concentrations of 1–10 mM. The antioxidant activity of quercetin in the concentration range of 10–500 μM during the first minutes of incubation was higher than that of caffeic acid. Of the phenolic compounds studied, curcumin had the least effect on the superoxide radicals.

Dinitrosyl iron complexes: Formation and antiradical action in heart mitochondria

Mitochondria are widely known as a major source of reactive oxygen and nitrogen species for the cardiovascular system. Numerous studies established that superoxide anion radical production by heart mitochondria is only slightly suppressed under conditions of deep hypoxia, but is completely blocked under anoxia. It was found also that dinitrosyl iron complexes (DNIC) compare favourably with other physiologically active derivatives of nitric oxide (NO). DNIC with glutathione effectively scavenge superoxide radicals generated by mitochondria at different partial pressures of oxygen. Under conditions of simulated hypoxia, the synthesis of thiol-containing DNIC takes place in mitochondria and is concomitant with a significant decrease in the concentration of NO metabolites at the reoxygenation step. Free NO required for DNIC synthesis is generated in the reaction of S-nitrosothiols with superoxide or during single-electron oxidation of the nitroxyl radical (HNO) by coenzyme Q. Plausible mechanisms of antiradical effects of DNIC and their protective role in oxidative stress induced by hypoxia/reoxygenation of myocardial tissues are considered.

The generation of superoxide radicals by complex III in heart mitochondria and the antioxidant effect of dinitrosyl iron complexes at different partial pressures of oxygen

EPR spin trapping and EPR oximetry were used to study the superoxide radical generation in heart mitochondria from Wistar rats at various oxygen concentrations. Lithium phthalocyanine and TEMPONE-15N-D16 were chosen to determine the oxygen content in a gas-permeable capillary containing mitochondria. TIRON was used as a spin trap. Several oxygen concentrations in the incubation mixture were tested; heart mitochondria were found to generate superoxide in complex III at various partial pressures of oxygen, including deep hypoxia (<5% O2). Dinitrosyl iron complexes with glutathione (the drug Oxacom) exerted an antioxidant effect regardless of the partial pressure of oxygen; the magnitude and kinetic characteristics of the effect depended on the drug concentration.