Manganese complexes of curcumin and its derivatives: evaluation for the radical scavenging ability and neuroprotective activity

In this study, three manganese complexes of curcumin (Cp) and related compounds, diacetylcurcumin (AcylCp) and ethylenediamine derivative (CpED), were synthesized and evaluated in vitro for antilipid peroxidation and superoxide dismutase activity. The manganese complexes exhibited a great capacity to protect brain lipids against peroxidation with IC₅₀ of 6.3–26.3 μM. All manganese complexes showed much greater SOD activity than their corresponding antioxidant ligands as well as trolox with IC₅₀ values of 8.9–29.9 μM. AcylCp and curcumin manganese complexes (AcylCpCpx and CpCpx) also gave the highest inhibitory activity to H₂O₂-induced cell damage (oxidative stress) at 0.1 μg/ml (< 0.2 μM) in NG108-15 cells, which were more potent than curcumin and related compounds. The neuropharmacological tests in mice supported the idea that the SOD mimicking complexes were able to penetrate to the brain as well as their role in the modulation of brain neurotransmitters under the aberrant conditions. The complexes significantly improved the learning and memory impairment induced by transient ischemic/reperfusion. AcylCpCpx, CpCpx, and CpEDCpx showed significant protection at 6.25, 25, and 50 mg/kg (i.p.), respectively, whereas manganese acetate and curcumin had no effect at doses of 50 mg/kg. In addition, treatment of AcylCpCpx and curcumin significantly attenuated MPTP-induced striatal dopamine depletion in mice, which was in accordance with the increase in the density of dopaminergic neurons when compared with MPTP-treated mice. These results support the important role of manganese in importing SOD activity and consequently, the enhancement of radical scavenging activity. AcylCpCpx and CpCpx seem to be the most promising neuroprotective agents for vascular dementia.     

Superoxide dismutase-like activities of copper(II) complexes tested in serum

The superoxide dismutase-like activities of a series of coordination complexes of copper were evaluated and compared to the activities of bovine erythrocyte superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) in serum using the nitroblue tetrazolium chloride (NBT)-reduction assay and electron paramagnetic resonance (EPR) spectroscopy. A 40% inhibition was observed for the initial rate of the NBT reduction by superoxide dismutase in serum, but more than 40% inhibition was achieved with CuSO4, Cu(II)-dimethylglyoxime, Cu(II)-3,8-dimethyl-4,7-diazadeca-3,7-dienediamide, Cu2[N,N'-(2-(O-hydroxy-benzhydrylidene)amino)ethyl]2-1,2-ethane dia mine), Cu(II)-(diisopropylsalicylate)2, Cu(II)-(p-bromo-benzoate)2, Cu(II)-(nicotinate)2 and Cu(II)-(1,2-diamino-2-methylpropane)2. The electron paramagnetic resonance technique of spin trapping was used to detect the formation of superoxide (O2-.) and other free radicals in the xanthine-xanthine oxidase system under a variety of conditions. Addition of the spin trapping agent 5,5-dimethylpyrroline 1-oxide (DMPO) to the xanthine-xanthine oxidase system in fetal bovine serum produced the O2-.-spin adduct of DMPO (herein referred to as superoxide spin adduct, DMPO-OOH) as the well known short-lived nitroxyl whose characteristic EPR spectrum was recorded before its rapid decay to undetectable levels. The hydroxyl radical (HO.) adduct of the spin trap DMPO (herein referred to as DMPO-OH) was detected to a very small extent. When CuSO4, or the test complexes of copper, were added to the xanthine-xanthine oxidase system in serum containing the spin trap, the yield of DMPO-OOH was negligible. In addition to their superoxide dismutase-like activity, CuSO4 and the copper complexes also behaved as Fenton-type catalysts as seen by the accumulation of varying amounts of the hydroxyl spin adduct DMPO-OH. Both the Fenton-type catalysis and the superoxide dismutase-like action of these compounds were lost when a chelator such as EDTA was included in the xanthine-xanthine oxidase incubation mixture. Addition of superoxide dismutase instead of the copper compounds to this enzyme system abolished the formation of superoxide adduct DMPO-OOH, and no hydroxyl adduct DMPO-OH was detected. This effect of superoxide dismutase remained unaltered by EDTA.     

Nitric oxide decreases the stability of DMPO spin adducts.

The effect nitric oxide (NO*) on the stability of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) adducts has been investigated using EPR spectroscopy. We report that the DMPO/HO* adduct, generated by porcine pulmonary artery endothelial cells in the presence of H2O2 and DMPO, or by a Fenton system (Fe(II)+H2O2) is degraded in the presence of the NO*-donor, 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO) or by bolus addition of an aqueous solution of NO*. A similar effect of DEANO was observed on other DMPO adducts, such as DMPO/*CH3 and DMPO/*CH(CH3)OH, generated in cell-free systems. Measurements of the loss of DMPO/HO* in the presence of DEANO in aerated and oxygen-free buffers showed that in both of these settings the process obeys first-order kinetics and proceeds with similar efficacy. This indicates that direct interaction of the nitroxide with NO*, rather than with NO2* (formed from NO* and O2 in aerated media), is responsible for destruction of the spin adduct. These results suggest that the presence of NO* may substantially affect the quantitative determination of DMPO adducts. We also show that NO2* radicals, generated by a myeloperoxidase/H2O2/nitrite system, also degrade DMPO/HO*. Because DMPO is frequently used to study generation of superoxide and hydroxyl radicals in biological systems, these observations indicate that extra caution is required when studying generation of these species in the presence of NO* or NO2* radicals.     

Spin traps inhibit formation of hydrogen peroxide via the dismutation of superoxide: implications for spin trapping the hydroxyl free radical.

To enhance the sensitivity of EPR spin trapping for radicals of limited reactivity, high concentrations (10-100 mM) of spin traps are routinely used. We noted that in contrast to results with other hydroxyl radical detection systems, superoxide dismutase (SOD) often increased the amount of hydroxyl radical-derived spin adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) produced by the reaction of hypoxanthine, xanthine oxidase and iron. One possible explanation for these results is that high DMPO concentrations (approximately 100 mM) inhibit dismutation of superoxide (O2.-) to hydrogen peroxide (H2O2). Therefore, we examined the effect of DMPO on O2.- dismutation to H2O2. Lumazine +/- 100 mM DMPO was placed in a Clark oxygen electrode following which xanthine oxidase was added. The amount of H2O2 formed in this reaction was determined by introducing catalase and measuring the amount of generated via O2.- dismutation as compared to direct divalent O2 reduction. In the presence of 100 mM DMPO, H2O2 generation decreased 43%. DMPO did not scavenge H2O2 nor alter the rate of O2.- production. The effect of DMPO was concentration-dependent with inhibition of H2O2 production observed at [DMPO] greater than 10 mM. Inhibition of H2O2 production by DMPO was not observed if SOD was present or if the rate of O2.- formation increased. The spin trap 2-methyl-2-nitroso-propane (MNP, 10 mM) also inhibited H2O2 formation (81%). However, alpha-phenyl-N-tert-butylnitrone (PBN, 10 mM), 3,3,5,5 tetramethyl-1-pyrroline N-oxide (M4PO, 100 mM), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN, 100 mM) had no effect. These data suggest that in experimental systems in which the rate of O2.- generation is low, formation of H2O2 and thus other H2O2-derived species (e.g., OH) may be inhibited by commonly used concentrations of some spin traps. Thus, under some experimental conditions spin traps may potentially prevent production of the very free radical species they are being used to detect.     

Superoxide dismutase enhanced the formation of hydroxyl radicals in a reaction mixture containing xanthone under UVA irradiation

To clarify the effect of superoxide dismutase (SOD) on the formation of hydroxyl radical in a standard reaction mixture containing 15 microM of xanthone, 0.1 M of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and 45 mM of phosphate buffer (pH 7.4) under UVA irradiation, electron paramagnetic resonance (EPR) measurements were performed. SOD enhanced the formation of hydroxyl radicals. The formation of hydroxyl radicals was inhibited on the addition of catalase. The rate of hydroxyl radical formation also slowed down under a reduced oxygen concentration, whereas it was stimulated by disodium ethylenediaminetetraacetate (EDTA) and diethyleneaminepentaacetic acid (DETAPAC). Above findings suggest that O(2), H(2)O(2), and iron ions participate in the reaction. SOD possibly enhances the formation of the hydroxyl radical in reaction mixtures of photosensitizers that can produce O(2)(-.).     

Bicarbonate enhances the hydroxylation, nitration, and peroxidation reactions catalyzed by copper, zinc superoxide dismutase. Intermediacy of carbonate anion radical

The effect of bicarbonate anion (HCO(3)(-)) on the peroxidase activity of copper, zinc superoxide dismutase (SOD1) was investigated using three structurally different probes: 5, 5'-dimethyl-1-pyrroline N-oxide (DMPO), tyrosine, and 2, 2'-azino-bis-[3-ethylbenzothiazoline]-6-sulfonic acid (ABTS). Results indicate that HCO(3)(-) enhanced SOD/H(2)O(2)-dependent (i) hydroxylation of DMPO to DMPO-OH as measured by electron spin resonance, (ii) oxidation and nitration of tyrosine to dityrosine, nitrotyrosine, and nitrodityrosine as measured by high pressure liquid chromatography, and (iii) oxidation of ABTS to the ABTS cation radical as measured by UV-visible spectroscopy. Using oxygen-17-labeled water, it was determined that the oxygen atom present in the DMPO-OH adduct originated from H(2)O and not from H(2)O(2). This result proves that neither free hydroxyl radical nor enzyme-bound hydroxyl radical was involved in the hydroxylation of DMPO. We postulate that HCO(3)(-) enhances SOD1 peroxidase activity via formation of a putative carbonate radical anion. This new and different perspective on HCO(3)(-)-mediated oxidative reactions of SOD1 may help us understand the free radical mechanism of SOD1 and related mutants linked to amyotrophic lateral sclerosis.     

5,5-dimethyl-1-pyrroline-N-oxide alone enhances the spontaneous superoxide generation by primaquine.

Primaquine (PQ), a well-known antimalarial drug, has been reported to generate superoxide (O2-) in the presence of reducing agents such as NADPH. In the present study, chemiluminescence was detected by adding only PQ to aqueous 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo-[1,2-alpha] pyrazin-3-one (MCLA), which is a specific chemiluminescent probe for O2-, and was quenched by superoxide dismutase (SOD), indicating that PQ alone can generate O2- in aerobic conditions. Furthermore, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) enhanced the O2- generation by PQ. Superoxide spin adduct, DMPO-OOH, was also detected by ESR both in aqueous solutions and in dimethyl sulfoxide with DMPO. The level of O2- generation showed a linear correlation with the DMPO concentration, and SOD competitively inhibited the DMPO-OOH formation. The results suggested that in aerobic conditions PQ is autoxidized to 5-hydroxy-PQ, which generates O2-, and DMPO accelerates the autoxidation process by trapping O2-. DMPO or M4PO alone enhances the spontaneous O2- generation by PQ, therefore cautious evaluation is necessary in all studies using the ESR/spin trapping technique to elucidate the mechanism of PQ-related radical generation.     

Lipophilic ionophore complexes as superoxide dismutase mimetics

A wide range of metal ion complexes exhibit superoxide dismutase like activities as detected by inhibition of nitroblue tetrazolium reduction. Mn(II) and Cu(II) complexes of EDTA, EHPG, and EGTA exhibit SOD like activities commensurate with many of the purpose-built SOD mimics. Here, we report analogous lipophilic chelators that localise metal ions (Cu(II), Mn(II), and Fe(III)) in the lipid membranes and lipoproteins to protect them from superoxide mediated oxidative damage. Spectroscopic titrations and Jobs method confirm that both 1:1 and 2:1 metal ion monensin complexes form. The cupric complexes are the most active exhibiting IC(50) values of 0.09 and 0.18 microM for 2Cu(II)-monensin and Cu(II)-monensin, respectively, for superoxide destruction. In addition, the IC(50) value for Mn(II)-monensin is 0.31 microM. In conclusion, Mn(II) and Cu(II) complexes of the ionophore monensin exhibit considerable superoxide scavenging activities and represent a novel class of catalytic antioxidants for the protection of lipid structures.     

Histidinyl radical formation in the self-peroxidation reaction of bovine copper-zinc superoxide dismutase.

In the absence of suitable oxidizable substrates, the peroxidase reaction of copper-zinc superoxide dismutase (SOD) oxidizes SOD itself, ultimately resulting in its inactivation. A SOD-centered free radical adduct of 2-methyl-2-nitrosopropane (MNP) was detected upon incubation of SOD with the spin trap and a hydroperoxide (either H(2)O(2) or peracetic acid). Proteolysis by Pronase converted the anisotropic electron paramagnetic resonance (EPR) spectrum of MNP/(center dot)SOD to a nearly isotropic spectrum with resolved hyperfine couplings to several atoms with non-zero nuclear spin. Authentic histidinyl radical (from histidine + HO(center dot)) formed a MNP adduct with a very similar EPR spectrum to that of the Pronase-treated MNP/(center dot)SOD, suggesting that the latter was centered on a histidine residue. An additional hyperfine coupling was detected when histidine specifically (13)C-labeled at C-2 of the imidazole ring was used, providing evidence for trapping at that atom. All of the experimental spectra were convincingly simulated assuming hyperfine couplings to 2 nearly equivalent nitrogen atoms and 2 different protons, also consistent with trapping at C-2 of the imidazole ring. Free histidinyl radical consumed oxygen, implying peroxyl radical formation. MNP-inhibitable oxygen consumption was also observed when cuprous SOD but not cupric SOD was added to a H(2)O(2) solution. Formation of 2-oxohistidine, the stable product of the SOD-hydroperoxide reaction, required oxygen and was inhibited by MNP. These results support formation of a transient SOD-peroxyl radical.     

The interaction of reduced glutathione with active oxygen species generated by xanthine-oxidase-catalyzed metabolism of xanthine

The interaction of reduced glutathione (GSH) with active oxygen species generated during xanthine-oxidase-catalyzed metabolism of xanthine was investigated. The only GSH-derived product detected in this system was oxidized glutathione (GSSG). Catalase inhibited the oxidation of GSH to GSSG by more than 80%, whereas superoxide dismutase exerted a smaller but significant inhibition of GSSG formation. Hydroxyl radical (OH) scavengers or desferrioxamine (1 mM) had no effect on GSSG formation. Using EPR spectroscopy and the spin trap 5,5-dimethylpyrroline-N-oxide (DMPO), the production of superoxide was observed by the detection of a DMPO-OOH radical adduct. This spectrum was altered by the inclusion of GSH (5 - 20 mM) in the reaction mixture, indicating the generation of a different radical species consistent with DMPO-glutathionyl radical adduct generation.     

Direct evidence for inhibition of free radical formation from Cu(I) and hydrogen peroxide by glutathione and other potential ligands using the EPR spin-trapping technique.

Copper-induced oxidative damage is generally attributed to the formation of the highly reactive hydroxyl radical by a mechanism analogous to the Haber-Weiss cycle for Fe(II) and H2O2. In the present work, the reaction between the Cu(I) ion and H2O2 is studied using the EPR spin-trapping technique. The hydroxyl radical adduct was observed when Cu(I), dissolved in acetonitrile under N2, was added to pH 7.4 phosphate buffer containing 100 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Formation of the hydroxyl radical was dependent on the presence of O2 and subsequent formation of H2O2. The kscav/kDMPO ratios obtained were below those expected for a mechanism involving free hydroxyl radical and reflect the interference of nucleophilic addition of H2O to DMPO to form the DMPO/.OH adduct in the presence of nonchelated copper ion. Addition of ethanol or dimethyl sulfoxide to the reaction suggests that a high-valent metal intermediate, possibly Cu(III), was also formed. Spin trapping of hydroxyl radical was almost completely inhibited upon addition of Cu(I) to a solution of either nitrilotriacetate or histidine, even though the copper was fully oxidized to Cu(II) and H2O2 was formed. Bathocuproinedisulfonate, thiourea, and reduced glutathione all stabilized the Cu(I) ion toward oxidation by O2. Upon addition of H2O2, the Cu(I) in all three complexes was oxidized to varying degrees; however, only the thiourea complex was fully oxidized within 2 min of reaction and produced detectable hydroxyl radicals. No radicals were detected from the bathocuproinedisulfonate or glutathione complexes. Overall, these results suggest that the deleterious effects of copper ions in vivo are diminished by biochemical chelators, especially glutathione, which probably has a major role in moderating the toxicological effects of copper.     

EPR kinetic studies of superoxide radicals generated during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium, a bioactivated intermediate of parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

1-Methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), a metabolic product of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been shown to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected as a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).O2- spin adduct by spin trapping in combination with EPR techniques. The rate of formation of spin adduct was dependent not only on the concentrations of MPDP+ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP+, and of superoxide dismutase to effectively compete with this reaction for the available O2-, has been used as a convenient competition reaction to quantitatively determine various kinetic parameters. Thus, using this technique the rate constant for scavenging of superoxide radical by superoxide dismutase was found to be 7.56 x 10(9) M-1 s-1. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP+ was found to be 4.48 x 10(-10) M s-1. The rate constant (K1) for MPDP+ making superoxide radical was found to be 3.97 x 10(-6) s-1. The secondary order rate constant (KDMPO) for DMPO-trapping superoxide radicals was found to be 10.2 M-1 s-1. The lifetime of superoxide radical at pH 10.0 was calculated to be 1.25 s. These values are in close agreement to the published values obtained using different experimental techniques. These results indicate that superoxide radicals are produced during spontaneous oxidation of MPDP+ and that EPR spin trapping can be used to determine the rate constants and lifetime of free radicals generated in aqueous solutions. It appears likely that the nigrostriatal toxicity of MPTP/MPDP+ leading to Parkinson's disease may largely be due to the reactivity of these radicals.     

Thiol-induced hydroxyl radical formation and scavenger effect of thiocarbamides on hydroxyl radicals

The effects of thiols and thiocarbamides on hydroxyl radical (.OH) formation by the hypoxanthine(HYP)-xanthine oxidase(XOD)-Fe3+ .EDTA system were investigated in the range of 0.5-5 mM by colorimetrically measuring salicylate hydroxylation. Thiocarbamides powerfully inhibited the hydroxylation while thiols showed a paradoxical effect, enhancing it at low concentrations, but inhibiting it at high ones. Thiols in the presence of Fe3+ .EDTA generated superoxide anions (O2-.) and .OH during the oxidation, but thiocarbamides did not. A study of the effect of ergothioneine, a thiocarbamide present in mammals, on the .OH spin adduct of 5,5-dimethyl-1-pyrroline-N-oxide(DMPO) by EPR spectrometry showed that it effectively decreased the .OH spin adduct without causing the appearance of other signals. Reaction mechanisms are proposed for the O2-. evolution and .OH formation by the thiols themselves in the presence of Fe3+ .EDTA and .OH with thiols and thiocarbamides.     

Insulin-like growth factor I receptor is expressed at normal levels in Nijmegen breakage syndrome cells

Curcumin (diferuloylmethane) is a major component of food flavoring turmeric (Curcuma longa), and has been reported to be anticarcinogenic and anti-inflammatory. Although curcumin was shown to have antioxidant properties, its exact antioxidant nature has not been fully investigated. In this report we have investigated the possible antioxidant properties of curcumin using EPR spectroscopic techniques. Curcumin was found to inhibit the (1)O(2)-dependent 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) formation in a dose-dependent manner. (1)O(2) was produced in a photosensitizing system using rose bengal as sensitizer, and was detected as TEMP-(1)O(2) adducts by electron paramagnetic resonance (EPR) spectroscopic techniques using TEMP as a spin-trap. Curcumin at 2.75 microM caused 50% inhibition of TEMP-(1)O(2) adduct formation. However, curcumin only marginally inhibited (24% maximum at 80 microM) reduction of ferricytochrome c in a xanthine-xanthine oxidase system demonstrating that it is not an effective superoxide radical scavenger. Additionally, there was minor inhibition of DMPO-OH adduct formation by curcumin (solubilized in ethanol) when an ethanol control was included in the EPR spin-trapping study, suggesting that curcumin may not be an effective hydroxyl radical scavenger. Together these data demonstrate that curcumin is able only to effectively quench singlet oxygen at very low concentration in aqueous systems.     

Degradation of DMPO Adducts from Hydroxyl and 1-Hydroxyethyl Radicals by Rat Liver Microsomes

Hydroxyl and 1-hydroxyethyl radical adducts of 5, 5-dimethylpyrroline N-oxide (DMPO) were prepared by photolysis, and mechanisms for loss of their EPR signals in rat liver microsomal suspensions were evaluated. Rates of NADPH-dependent EPR signal loss were more rapid in phosphate buffer than in Tris buffer. Addition of superoxide dismutase (SOD) partially protected the adducts when Tris was used as a buffer, but was relatively ineffective in the presence of phosphate. The ferrous iron chelator bathophenanthrolene partially protected the spin adducts in the presence and absence of phosphate, but complete protection was observed when SOD was also added. The spin adducts were unstable in the presence of Fe⁺² and K₃Fe(CN)₆, but Fe⁺³ alone had little effect on the EPR signals. The data are consistent with two mechanisms for microsomal degradation of DMPO spin adducts under these conditions. Microsomes form superoxide in the presence of oxygen and NADPH, which attacks these DMPO spin adducts directly. The spin adducts are also degraded in the presence of Fe⁺², and phosphate stimulates this iron-dependent destruction of DMPO spin adducts.     

Evaluation of DEPMPO as a spin trapping agent in biological systems

Cellular toxicity, pharmacokinetics, and the in vitro and in vivo stability of the SO3*- spin adduct of the spin trap, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-n-oxide (DEPMPO), was investigated, and the results were compared with those of the widely used spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Similar to DMPO, DEPMPO was quickly taken up (<15 min) after intraperitoneal injection, and distributed evenly in the liver, heart, and blood of the mice. In the presence of ascorbate the in vitro stability of the adduct DEPMPO/SO3*- was 7 times better than DMPO/SO3*-. Under in vivo conditions, the spin adduct DEPMPO/SO3*- was 2-4 times more stable than DMPO/ SO3*-, depending on the route of administration of the adducts. Using a low frequency EPR spectrometer, we were able to observe the spin trapped SO3*- radical both with DMPO and DEPMPO directly in the intact mouse. DEPMPO had a detectable spin adduct signal at a concentration as low as 1 mM, as compared to 5 mM for DMPO. We conclude that DEPMPO is potentially a good candidate for trapping radicals in functioning biological systems, and represents an improvement over the commonly used trap DMPO.     

Spin-Trapping of the Superoxide Radical in Aprotic Solvents

The superoxide adduct of 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) has been detected by EPR spectroscopy in aprotic solvents using KO₂ solubilized in 18-crown-6-ether as a source of superoxide. The EPR hyperfine splitting constants of the DMPO-superoxide adduct were as follows: benzene/toluene (a_N = 12.65 G; a^β_β = 10.4G; a_γ = 1.3G); heptane (a_N = 12.49G; a_β^β = 10.29G; a^γ_H = 1.2g); and acetone (a_N = 12.6G; a_β^β = 10.17 G; a_γ = 1.3 G). The EPR parameters for benzene, toluene and heptane differ significantly from previously reported values. A plot of the hyperfine splitting constants for the DMPO superoxide adduct as a function of solvent polarity (Kosower Z value) indicates that while a_N and a_β^β both decrease by about 1 G on going from water to ethanol, further decreases in polarity do not greatly affect these EPR parameters.     

Comparative investigation of superoxide trapping by cyclic nitrone spin traps: the use of singular value decomposition and multiple linear regression analysis

The kinetics of the reaction between superoxide and the spin trapping agents 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), and 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) were re-examined in the superoxide-generating xanthine/xanthine oxidase system, by competition with spontaneous dismutation. The approach used singular value decomposition (SVD), multiple linear regression, and spectral simulation. The experiments were carried out using a two-syringe mixing arrangement with fast scan acquisition of 100 consecutive EPR spectra. Using SVD analysis, the extraction of both temporal and spectral information could be obtained from in a single run. The superoxide spin adduct was the exclusive EPR active species in the case of DEPMPO and BMPO, and the major component when DMPO was used. In the latter case a very low concentration of hydroxyl adduct was also observed, which did not change during the decay of the DMPO-superoxide adduct. This indicates that the hydroxyl radical adduct is not formed from the spontaneous decay of the superoxide radical adduct, as has been previously suggested [correction]. It was established that in short-term studies (up to 100 s) DMPO was the superior spin trapping agent, but for reaction times longer than 100 s the other two spin traps were more advantageous. The second order rate constants for the spin trapping reaction were found to be DMPO (2.4 M(-1)s(-1)), DEPMPO (0.53 M(-1)s(-1)), and BMPO (0.24 M(-1)s(-1)) determined through competition with spontaneous dismutation of superoxide, at pH 7.4 and 20 degrees C.     

Degradation of DMPO Adducts from Hydroxyl and 1-Hydroxyethyl Radicals by Rat Liver Microsomes

Hydroxyl and 1-hydroxyethyl radical adducts of 5, 5-dimethylpyrroline N-oxide (DMPO) were prepared by photolysis, and mechanisms for loss of their EPR signals in rat liver microsomal suspensions were evaluated. Rates of NADPH-dependent EPR signal loss were more rapid in phosphate buffer than in Tris buffer. Addition of superoxide dismutase (SOD) partially protected the adducts when Tris was used as a buffer, but was relatively ineffective in the presence of phosphate. The ferrous iron chelator bathophenanthrolene partially protected the spin adducts in the presence and absence of phosphate, but complete protection was observed when SOD was also added. The spin adducts were unstable in the presence of Fe⁺² and K₃Fe(CN)₆, but Fe⁺³ alone had little effect on the EPR signals. The data are consistent with two mechanisms for microsomal degradation of DMPO spin adducts under these conditions. Microsomes form superoxide in the presence of oxygen and NADPH, which attacks these DMPO spin adducts directly. The spin adducts are also degraded in the presence of Fe⁺², and phosphate stimulates this iron-dependent destruction of DMPO spin adducts.     

Evaluation of a new copper(II)-curcumin complex as superoxide dismutase mimic and its free radical reactions

A mononuclear (1:1) copper complex of curcumin, a phytochemical from turmeric, was synthesized and examined for its superoxide dismutase (SOD) activity. The complex was characterized by elemental analysis, IR, NMR, UV-VIS, EPR, mass spectroscopic methods and TG-DTA, from which it was found that a copper atom is coordinated through the keto-enol group of curcumin along with one acetate group and one water molecule. Cyclic voltammetric studies of the complex showed a reversible Cu(2+)/Cu(+) couple with a potential of 0.402 V vs NHE. The Cu(II)-curcumin complex is soluble in lipids and DMSO, and insoluble in water. It scavenges superoxide radicals with a rate constant of 1.97 x 10(5) M(-1) s(-1) in DMSO determined by stopped-flow spectrometer. Subsequent to the reaction with superoxide radicals, the complex was found to be regenerated completely, indicating catalytic activity in neutralizing superoxide radicals. Complete regeneration of the complex was observed, even when the stoichiometry of superoxide radicals was 10 times more than that of the complex. This was further confirmed by EPR monitoring of superoxide radicals. The SOD mimicking activity of the complex was determined by xanthine/xanthine oxidase assay, from which it has been found that 5 microg of the complex is equivalent to 1 unit of SOD. The complex inhibits radiation-induced lipid peroxidation and shows radical-scavenging ability. It reacts with DPPH radicals with rate constant 10 times less than that of curcumin. Pulse radiolysis-induced one-electron oxidation of the complex by azide radicals in TX-100 micellar solutions produced strongly absorbing ( approximately 500 nm) phenoxyl radicals, indicating that the phenolic moiety of curcumin remained intact on complexation with copper. The results confirm that the new Cu(II)-curcumin complex possesses SOD activity, free radical neutralizing ability, and antioxidant potential. Quantum chemical calculations with density functional theory have been performed to support the experimental observations.