Protein Radical Formation Research Articles

Unravelling mechanisms of protein and lipid oxidation in mayonnaise at multiple length scales

In mayonnaise, lipid and protein oxidation are closely related and the interplay between them is critical for understanding the chemical shelf-life stability of mayonnaise. This is in particular the case for comprehending the role of low-density lipoprotein (LDL) particles acting as a main emulsifier. Here, we monitored oxidation and the concomitant aggregation of LDLs by bright-field light microscopy and cryogenic transmission electron microscopy. We further probed the formation of protein radicals and protein oxidation by imaging the accumulation of a water-soluble fluorescent spin trap and protein autofluorescence. The effect of variation of pH and addition of EDTA on the accumulation of the spin trap validated that protein radicals were induced by lipid radicals. Our data suggests two main pathways of oxidative protein radical formation in LDL particles: (1) at the droplet interface, induced by lipid free radicals formed in oil droplets, and (2) in the continuous phase induced by an independent LDL-specific mechanism.

Initiation and Prevention of Biological Damage by Radiation-Generated Protein Radicals.

Ionizing radiations cause chemical damage to proteins. In aerobic aqueous solutions, the damage is commonly mediated by the hydroxyl free radicals generated from water, resulting in formation of protein radicals. Protein damage is especially significant in biological systems, because proteins are the most abundant targets of the radiation-generated radicals, the hydroxyl radical-protein reaction is fast, and the damage usually results in loss of their biological function. Under physiological conditions, proteins are initially oxidized to carbon-centered radicals, which can propagate the damage to other molecules. The most effective endogenous antioxidants, ascorbate, GSH, and urate, are unable to prevent all of the damage under the common condition of oxidative stress. In a promising development, recent work demonstrates the potential of polyphenols, their metabolites, and other aromatic compounds to repair protein radicals by the fast formation of less damaging radical adducts, thus potentially preventing the formation of a cascade of new reactive species.

Free Radical-Mediated Protein Radical Formation in Differentiating Monocytes.

Free radical-mediated activation of inflammatory macrophages remains ambiguous with its limitation to study within biological systems. U-937 and HL-60 cell lines serve as a well-defined model system known to differentiate into either macrophages or dendritic cells in response to various chemical stimuli linked with reactive oxygen species (ROS) production. Our present work utilizes phorbol 12-myristate-13-acetate (PMA) as a stimulant, and factors such as concentration and incubation time were considered to achieve optimized differentiation conditions. ROS formation likely hydroxyl radical (HO●) was confirmed by electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM). In particular, U-937 cells were utilized further to identify proteins undergoing oxidation by ROS using anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibodies. Additionally, the expression pattern of NADPH Oxidase 4 (NOX4) in relation to induction with PMA was monitored to correlate the pattern of ROS generated. Utilizing macrophages as a model system, findings from the present study provide a valuable source for expanding the knowledge of differentiation and protein expression dynamics.

On the Track of Long-Range Electron Transfer in B-Type Dye-Decolorizing Peroxidases: Identification of a Tyrosyl Radical by Computational Prediction and Electron Paramagnetic Resonance Spectroscopy.

The catalytic activity of dye-decolorizing peroxidases (DyPs) toward bulky substrates, including anthraquinone dyes, phenolic lignin model compounds, or 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), is in strong contrast to their sterically restrictive active site. In two of the three known subfamilies (A- and C/D-type DyPs), catalytic protein radicals at surface-exposed sites, which are connected to the heme cofactor by electron transfer path(s), have been identified. So far in B-type DyPs, there has been no evidence for protein radical formation after activation by hydrogen peroxide. Interestingly, B-type Klebsiella pneumoniae dye-decolorizing peroxidase (KpDyP) displays a persistent organic radical in the resting state composed of two species that can be distinguished by W-band electron spin echo electron paramagnetic resonance (EPR) spectroscopy. Here, on the basis of a comprehensive mutational and EPR study of computationally predicted tyrosine and tryptophan variants of KpDyP, we demonstrate the formation of tyrosyl radicals (Y247 and Y92) and a radical-stabilizing Y-W dyad between Y247 and W18 in KpDyP, which are unique to enterobacterial B-type DyPs. Y247 is connected to Y92 by a hydrogen bonding network, is solvent accessible in simulations, and is involved in ABTS oxidation. This suggests the existence of long-range electron path(s) in B-type DyPs. The mechanistic and physiological relevance of the reaction mechanism of B-type DyPs is discussed.

Extracellular Protein Radical Formation Increases After Damage to Articular Cartilage

Extracellular Protein Radical Formation Increases After Damage to Articular Cartilage

Immuno-spin Trapping of Copper Nanoparticles for Beta-amyloid Protein Radical Formation: Implication of Protein Radical Formation in Alzheimer's Disease

Immuno-spin Trapping of Copper Nanoparticles for Beta-amyloid Protein Radical Formation: Implication of Protein Radical Formation in Alzheimer's Disease

Characterization of Protein Radicals in Arabidopsis.

Oxidative modification of proteins in photosystem II (PSII) exposed to high light has been studied for a few decades, but the characterization of protein radicals formed by protein oxidation is largely unknown. Protein oxidation is induced by the direct reaction of proteins with reactive oxygen species known to form highly reactive protein radicals comprising carbon-centered (alkyl) and oxygen-centered (peroxyl and alkoxyl) radicals. In this study, protein radicals were monitored in Arabidopsis exposed to high light by immuno-spin trapping technique based on the detection of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) nitrone adducts using the anti-DMPO antibody. Protein radicals were imaged in Arabidopsis leaves and chloroplasts by confocal laser scanning microscopy using fluorescein conjugated with the anti-DMPO antibody. Characterization of protein radicals by standard blotting techniques using PSII protein specific antibodies shows that protein radicals are formed on D1, D2, CP43, CP47, and Lhcb3 proteins. Protein oxidation reflected by the appearance/disappearance of the protein bands reveals that formation of protein radicals was associated with protein fragmentation (cleavage of the D1 peptide bonds) and aggregation (cross-linking with another PSII subunits). Characterization of protein radical formation is important for better understating of the mechanism of oxidative modification of PSII proteins under high light.

Antioxidant efficiency and mechanisms of green tea, rosemary or maté extracts in porcine Longissimus dorsi subjected to iron-induced oxidative stress

Plant extracts from rosemary (RE), green tea (GTE), and maté (ME) were compared for the protection against iron-induced oxidation in porcine homogenates at total phenolic concentrations from 25 to 250 ppm. Lipid oxidation as indicated by TBARS was in all cases sufficiently suppressed, especially for RE. Hydrophobic RE retarded overall oxidation in the homogenates with an inverted dose-dependent response. Optimum delay of oxygen consumption was found at the lowest concentration applied, similar to protection against thiols and formation of protein radicals as measured by ESR, whereas the high concentration increased oxygen consumption and caused additionally thiol loss possibly due to thiol-quinone interactions, generating protein-phenol complexes. Hydrophilic ME or GTE increased the initial oxygen consumption rate as an indication of prooxidant activities at elevated concentrations. However, they were found to protect myoglobin and protein at those high concentrations with GTE being more efficient, possibly due to better chelation effect.

LanCL1 attenuates ischemia-induced oxidative stress by Sirt3-mediated preservation of mitochondrial function

Lanthionine synthetase C-like protein 1 (LanCL1) is homologous to prokaryotic lanthionine cyclases, and has been shown to have novel functions in neuronal redox homeostasis. A recent study showed that LanCL1 expression was developmental and activity-dependent regulated, and LanCL1 transgene protected neurons against oxidative stress. In the present study, the potential protective effects of LanCL1 against ischemia was investigated in an in vitro model mimicked by oxygen and glucose deprivation (OGD) in neuronal HT22 cells. We found that OGD exposure induced a temporal increase and persistent decreases in the expression of LanCL1 at both mRNA and protein levels. Overexpression of LanCL1 by lentivirus (LV-LanCL1) transfection preserved cell viability, reduced lactate dehydrogenase (LDH) release and attenuated apoptosis after OGD. These protective effects were accompanied by decreased protein radical formation, lipid peroxidation and mitochondrial dysfunction. In addition, LanCL1 significantly stimulated mitochondrial enzyme activities and SOD2 deacetylation in a Sirt3-dependent manner. The results of western blot analysis showed that LanCL1-induced activation of Sirt3 was dependent on Akt-PGC-1α pathway. Knockdown of PGC-1α expression using small interfering RNA (siRNA) or blocking Akt activation using specific antagonist partially prevented the protective effects of LanCL1 in HT22 cells. Taken together, our results show that LanCL1 protects against OGD through activating the Akt-PGC-1α-Sirt3 pathway, and may have potential therapeutic value for ischemic stroke.

Biocompatibility of new materials based on nano-structured nitinol with titanium and tantalum composite surface layers: experimental analysis in vitro and in vivo.

A technology for obtaining materials from nanostructured nitinol with titanium- or tantalum-enriched surface layers was developed. Surface layers enriched with titanium or tantalum were shown to provide a decrease in the formation of reactive oxygen species and long-lived protein radicals in comparison to untreated nitinol. It was determined that human peripheral vessel myofibroblasts and human bone marrow mesenchymal stromal cells grown on nitinol bases coated with titanium or tantalum-enriched surface layers exhibit a nearly two times higher mitotic index. Response to implantation of pure nitinol, as well as nano-structure nitinol with titanium or tantalum-enriched surface layers, was expressed though formation of a mature uniform fibrous capsule peripherally to the fragment. The thickness of this capsule in the group of animals subjected to implantation of pure nitinol was 1.5 and 3.0-fold greater than that of the capsule in the groups implanted with nitinol fragments with titanium- or tantalum-enriched layers. No signs of calcinosis in the tissues surrounding implants with coatings were observed. The nature and structure of the formed capsules testify bioinertia of the implanted samples. It was shown that the morphology and composition of the surface of metal samples does not alter following biological tests. The obtained results indicate that nano-structure nitinol with titanium or tantalum enriched surface layers is a biocompatible material potentially suitable for medical applications.

Peroxiredoxin-3 attenuates traumatic neuronal injury through preservation of mitochondrial function

Peroxiredoxins (PRDXs) are a highly conserved family of thiol peroxidases that scavenge peroxides in cells. PRDX3 is one member of PRDXs localized in the mitochondria, and has been shown to be involved in antioxidant defense and redox signaling. In this study, we investigated the role of PRDX3 in neuronal trauma using a traumatic neuronal injury (TNI) model in primary cultured cortical neurons. We found that TNI significantly decreased the expression of PRDX3 at both mRNA and protein levels. Overexpression of PRDX3 by lentivirus (LV-PRDX3) transfection attenuated lactate dehydrogenase (LDH) release and neuronal apoptosis after TNI. The results of immunostaining showed that LV-PRDX3 transfection markedly reduced TNI-induced intracellular ROS production, protein radical formation and lipid peroxidation. In addition, overexpression of PRDX3 preserved mitochondrial membrane potential (MMP) levels and ATP generation, and inhibited mitochondrial cytochrome c release in TNI-injured neurons. The results of polymerase chain reaction (PCR) showed that PRDX3 overexpression also increased mitochondrial DNA (mtDNA) content and upregulated the expression of mitochondrial biogenesis-related factors. Taken together, our data demonstrate that PRDX3 protects against TNI insult by preserving mitochondrial function and mitochondrial biogenesis, and may have potential therapeutic value for traumatic brain injury (TBI).

Dose-Dependent Effects of Green Tea or Maté Extracts on Lipid and Protein Oxidation in Brine-Injected Retail-Packed Pork Chops.

Background: Phenolic plant extracts are added as antioxidants in meat to prevent lipid oxidation, but depending on the concentration applied, may affect proteins either through covalent interactions or by serving as a prooxidant. Methods: Brine-injected pork chops prepared with green tea extract (25–160 ppm gallic acid equivalents (GAE)), or maté extract (25–160 ppm GAE) and stored (5 °C, 7 days) in high-oxygen atmosphere packaging (MAP: 80% O2 and 20% CO2) were analyzed for color changes, lipid oxidation by thiobarbituric acid reactive substances (TBARS), and protein oxidation evaluated by thiol loss and protein radical formation by electron spin resonance (ESR) spectroscopy, and compared to a control without antioxidant. Results: Extract of maté and green tea showed significant and comparable antioxidative effects against formation of TBARS in brine-injected pork chops for all concentrations applied compared to the control. Protein radical formation decreased significantly by addition of 25 ppm maté extract, but increased significantly by addition of 80–160 ppm green tea extract, when monitored as formation of protein radicals. Meanwhile, protein thiol groups disappeared when applying the extracts by reactions assigned to addition reactions of oxidized phenols from the extracts to protein thiols. Conclusion: Maté is accordingly a good source of antioxidants for protection of both lipids and proteins in brine-injected pork chops chill-stored in high-oxygen atmosphere, though the dose must be carefully selected.

Sulfite-induced protein radical formation in LPS aerosol-challenged mice: Implications for sulfite sensitivity in human lung disease

Exposure to (bi)sulfite (HSO3–) and sulfite (SO32–) has been shown to induce a wide range of adverse reactions in sensitive individuals. Studies have shown that peroxidase-catalyzed oxidation of (bi)sulfite leads to formation of several reactive free radicals, such as sulfur trioxide anion (.SO3–), peroxymonosulfate (–O3SOO.), and especially the sulfate (SO4. –) anion radicals. One such peroxidase in neutrophils is myeloperoxidase (MPO), which has been shown to form protein radicals. Although formation of (bi)sulfite-derived protein radicals is documented in isolated neutrophils, its involvement and role in in vivo inflammatory processes, has not been demonstrated. Therefore, we aimed to investigate (bi)sulfite-derived protein radical formation and its mechanism in LPS aerosol-challenged mice, a model of non-atopic asthma. Using immuno-spin trapping to detect protein radical formation, we show that, in the presence of (bi)sulfite, neutrophils present in bronchoalveolar lavage and in the lung parenchyma exhibit, MPO-catalyzed oxidation of MPO to a protein radical. The absence of radical formation in LPS-challenged MPO- or NADPH oxidase-knockout mice indicates that sulfite-derived radical formation is dependent on both MPO and NADPH oxidase activity. In addition to its oxidation by the MPO-catalyzed pathway, (bi)sulfite is efficiently detoxified to sulfate by the sulfite oxidase (SOX) pathway, which forms sulfate in a two-electron oxidation reaction. Since SOX activity in rodents is much higher than in humans, to better model sulfite toxicity in humans, we induced SOX deficiency in mice by feeding them a low molybdenum diet with tungstate. We found that mice treated with the SOX deficiency diet prior to exposure to (bi)sulfite had much higher protein radical formation than mice with normal SOX activity. Altogether, these results demonstrate the role of MPO and NADPH oxidase in (bi)sulfite-derived protein radical formation and show the involvement of protein radicals in a mouse model of human lung disease.

87 - Induction of Beta amyloid Radical fFrmation through Immuno-spin trapping of Copper nano particles: Implication of Protein Radicals in Alzheimer's Disease

Alzheimer’s disease (AD) is a debilitating progressive neurodegenerative disease that usually starts slowly and worsens over time. Though AD etiology is believed to be multifactorial, there is considerable evidence showing the role of copper exposure in beta-amyloid (βA) aggregation and AD pathogenesis. Owing to the fact that βA aggregation takes place in the synaptic cleft where copper is found in high concentrations, copper exposure especially in the form of nano particles can be more detrimental. Though copper-induced oligomeric forms of βA accumulate and significantly damage the brain cells, inducing cognitive dysfunctions, the mechanism that initiates and promotes βA oligomerization remains unknown. We hypothesized protein radical formation as an initiating mechanism for βA oligomerization. Therefore, we used the highly sensitive immuno-spin trapping technique to investigate protein radical formation as a possible mechanism of βA oligomerization. Here, we have investigated copper nano particles induced βA radical formation and results clearly showed that both variants of nano particles (25 and 70 nm sizes) significantly produced βA radical in presence of hydrogen peroxide. To further validate copper nano particle-induced protein radical formation in biological context, we exposed IMR-32 hippocampal neuroblastoma cells expressing βA to both variants of copper nano particles and analyzed protein radical formation using confocal microscopy. Confocal microscopy results clearly showed protein radical formation in IMR-32 cells exposed to either variant of copper nano-particles in presence of hydrogen peroxide. Taken together, these results clearly suggest that both variants of copper nano particles induce βA radical formation which can lead to βA oligomerization, and therefore contributes to AD pathogenesis.

205 - Switch of Mitochondrial Superoxide Dismutase (SOD2) into a Prooxidant Peroxidase in Manganese-deficient Cells and Mice

Reactive oxygen species (ROS), in particular the superoxide radical anion O2•‒, are constantly produced during respiration and are increased in many human diseases. In mitochondria, the dismutation of O2•‒ is accelerated by the mitochondrial superoxide dismutase 2 or MnSOD2, an enzyme that has been traditionally associated with antioxidant protection. However, increases in SOD2 expression promote oxidative stress, indicating that there may be a prooxidant role for SOD2 in mitochondria. We show that SOD2, which is normally bound to manganese, can incorporate iron and generate an alternative isoform with peroxidase activity, the FeSOD2. In this regard, a switch in metal cofactor changes SOD2 from an antioxidant to a prooxidant enzyme that utilizes H2O2 to promote oxidative stress. Moreover, we found that the formation of FeSOD2, which happens in cellulo and in vivo, causes changes in overall cellular metabolism and gene expression profiles. Cells in culture with FeSOD2 show a shift in metabolism from oxidative phosphorylation to glycolysis due to mitochondrial dysfunction. Mice showed accumulation of FeSOD2 in liver when fed a manganese-deficient diet or an iron-enriched diet. Their liver mitochondria showed high levels of protein radicals, including those of Complex I. More importantly, levels of protein radical formation correlated with FeSOD2. Hence, we provide evidence in support of the identification of the first mitochondrial metal peroxidase which is produced by the incorporation of iron into SOD2. We also show that the formation of FeSOD2 converts an antioxidant defense (MnSOD2) into a prooxidant enzyme that leads to cellular changes seen in multiple human diseases.

Concentration-Dependant Antioxidant Activity of Pentoxifylline in Nitrite-induced Hemoglobin Oxidation Model

Free radical formation in heme proteins is recognized as a factor in mediating the toxicity of many chemicals. The present study was designed to evaluate the dose-response relationship of the free radical scavenging properties of pentoxifylline in nitrite-induced Hb oxidation. Different concentrations of pentoxifylline were added at different time intervals of Hb oxidation in erythrocytes lysate, and formation of methemoglobin (MetHb) was monitored spectrophotometrically. The results showed that in this model, pentoxifylline successfully attenuates Hb oxidation after challenge with sodium nitrite; this protective effect was found to be not related to the catalytic stage of Hb oxidation, though such effect was reported to be more prominent when the compound was at the same time of induction of Hb oxidation with nitrite. In conclusion, pentoxifylline can effectively, in concentration-dependent pattern, attenuate sodium nitrite-induced Hb oxidation in vitro.
 Key words: pentoxifylline, radical scavenging, hemoglobin oxidation

Role of cytochrome c in α-synuclein radical formation: implications of α-synuclein in neuronal death in Maneb- and paraquat-induced model of Parkinson's disease.

BackgroundThe pathological features of Parkinson’s disease (PD) include an abnormal accumulation of α-synuclein in the surviving dopaminergic neurons. Though PD is multifactorial, several epidemiological reports show an increased incidence of PD with co-exposure to pesticides such as Maneb and paraquat (MP). In pesticide-related PD, mitochondrial dysfunction and α-synuclein oligomers have been strongly implicated, but the link between the two has not yet been understood. Similarly, the biological effects of α-synuclein or its radical chemistry in PD is largely unknown. Mitochondrial dysfunction during PD pathogenesis leads to release of cytochrome c in the cytosol. Once in the cytosol, cytochrome c has one of two fates: It either binds to apaf1 and initiates apoptosis or can act as a peroxidase. We hypothesized that as a peroxidase, cytochrome c leaked out from mitochondria can form radicals on α-synuclein and initiate its oligomerization.MethodSamples from controls, and MP co-exposed wild-type and α-synuclein knockout mice were studied using immuno-spin trapping, confocal microscopy, immunohistochemistry, and microarray experiments.ResultsExperiments with MP co-exposed mice showed cytochrome c release in cytosol and its co-localization with α-synuclein. Subsequently, we used immuno-spin trapping method to detect the formation of α-synuclein radical in samples from an in vitro reaction mixture consisting of cytochrome c, α-synuclein, and hydrogen peroxide. These experiments indicated that cytochrome c plays a role in α-synuclein radical formation and oligomerization. Experiments with MP co-exposed α-synuclein knockout mice, in which cytochrome c-α synuclein co-localization and interaction cannot occur, mice showed diminished protein radical formation and neuronal death, compared to wild-type MP co-exposed mice. Microarray data from MP co-exposed wild-type and α-synuclein knockout mice further showed that the absence of α-synuclein per se or its co-localization with cytochrome c confers protection from MP co-exposure, as several important pathways were unaffected in α-synuclein knockout mice.ConclusionsAltogether, these results show that peroxidase activity of cytochrome c contributes to α-synuclein radical formation and oligomerization, and that α-synuclein, through its co-localization with cytochrome c or on its own, affects several biological pathways which contribute to increased neuronal death in an MP-induced model of PD.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0135-y) contains supplementary material, which is available to authorized users.

Differential heme release from various hemoglobin redox states and the upregulation of cellular heme oxygenase-1.

Despite advances in our understanding of the oxidative pathways mediated by free hemoglobin (Hb), the precise contribution of its highly reactive redox forms to tissue and organ toxicities remains ambiguous. Heme, a key degradation byproduct of Hb oxidation, has recently been recognized as a damage‐associated molecular pattern (DAMP) molecule, able to trigger inflammatory responses. Equally damaging is the interaction of the highly redox active forms of Hb with other biological molecules. We determined the kinetics of heme loss from individual Hb redox states—ferrous (Fe2+), ferric (Fe3+), and ferryl (Fe4+)—using two different heme receptor proteins: hemopexin (Hxp), a naturally occurring heme scavenger in plasma, and a double mutant (H64Y/V86F), apomyoglobin (ApoMb), which avidly binds heme released from Hb. We show for the first time that ferric Hb (Fe3+) loses heme at rates substantially higher than that of ferryl Hb (Fe4+). This was also supported by a higher expression of heme oxygenase‐1 (HO‐1) when ferric Hb was added to cultured lung alveolar epithelial cells (E10). The reported cytotoxicity of Hb may therefore be attributed to a combination of accelerated heme loss from the ferric form and protein radical formation associated with ferryl Hb. Targeted therapeutic interventions can therefore be designed to curb specific oxidative pathways of Hb in hemolytic anemias and when Hb is used as an oxygen‐carrying therapeutic.

Effect of Skin Wine Pomace and Sulfite on Protein Oxidation in Beef Patties During High Oxygen Atmosphere Storage

Meat storage in high oxygen atmosphere has been reported to induce protein oxidation reactions decreasing meat quality. The incorporation of antioxidants has been proposed to reduce the extent of these reactions. In this study, the ability of red and white skin wine pomaces as well as sulfites to inhibit protein oxidation were tested in beef patties stored for up to 15 days at 4 °C in a high oxygen atmosphere (70 % O2 and 30 % CO2). SO2 (300 ppm) effectively protected against protein oxidation measured as radical formation by electron spin resonance (ESR) spectroscopy, as thiol loss by the DTNB assay and as myosin heavy chain (MHC) disulfide cross-linking by SDS-PAGE. Pomace from red wine production with a total phenol of 9.9 mg gallic acid equivalent/g protected against protein radical formation and against MHC cross-linking, but not against thiol loss by addition of 2.0 % (w/w) to the beef patties. Pomace from white wine production with a total phenol of 4.0 mg gallic acid equivalent/g only protected against MHC cross-linking. For both types of wine pomace, protein modifications not seen for sulfite addition were observed and were proposed to involve covalent phenol addition to proteins. Red wine pomace may be an alternative to sulfite as a meat additive for protection of beef patties against protein oxidation.

Immuno-spin trapping detection of antioxidant/pro-oxidant properties of zinc or selenium on DNA and protein radical formation via hydrogen peroxide.

Trace elements can participate in the catalysis of group-transfer reactions and can serve as their structural components. However, most of them including zinc and selenium have multifunctional roles in biological environments such as antioxidant and/or pro-oxidant effects, as concentration-dependent manner. Although it has been demonstrated the antioxidant actions of either selenium or zinc compounds, there are several documents pointing out their pro-oxidant/oxidant roles in biological systems. Here we have used ELISA-based immuno-spin trapping, a method for detection of free radical formation, to detect whether or not a zinc compound, Zn3(PO4)2, or a selenium compound, Na2SeO3, has antioxidant and/or pro-oxidant effect on 5,5-Dimethyl-1-Pyrroline-N-Oxide (DMPO)-DNA nitrone adducts induced with Cu(II)-H2O2-oxidizing system in in vitro preparations. Second, we examined whether this technique is capable to demonstrate the different DMPO-protein nitrone adduct productions in isolated protein crude of hearts from normal rats (CON) or rats with metabolic syndrome (MetS). Our data demonstrated that either Zn(2+) (100 µM) or SeO3(-2) (50 nM) has very strong antioxidant action against 200 µM H2O2-induced DMPO-DNA nitrone adduct production, whereas their higher concentrations have apparent pro-oxidant actions. We also used verification by Western blotting analysis whether immuno-spin trapping can be used to assess H2O2-induced DMPO-protein nitrone adducts in heart protein crudes. Our Western blot data further confirmed the ELISA-data from proteins and demonstrated how Zn(2+) or SeO3(-2) are dual-functioning ions such as antioxidant at lower concentrations while pro-oxidant at higher concentrations. Particularly, our present data with SeO3(-2) in DMPO-protein nitrone adducts, being in line with our previous observation on its dual-actions in ischemia/reperfusion-induced damaged heart, have shown that this ion has higher pro-oxidant actions over 50 nM in MetS-group compared to that of CON group.