Effect of Electron-donating Group on NO Photolysis of {RuNO}6 Ruthenium Nitrosyl Complexes with N2 O2 Lgands Bearing π-Extended Rings.

In this study, we introduced the electron-donating group (-OH) to the aromatic rings of Ru(salophen)(NO)Cl (0) (salophenH2 =N,N'-(1,2-phenylene)bis(salicylideneimine)) to investigate the influence of the substitution on NO photolysis and NO-releasing dynamics. Three derivative complexes, Ru((o-OH)2 -salophen)(NO)Cl (1), Ru((m-OH)2 -salophen)(NO)Cl (2), and Ru((p-OH)2 -salophen)(NO)Cl (3) were developed and their NO photolysis was monitored by using UV/Vis, EPR, NMR, and IR spectroscopies under white room light. Spectroscopic results indicated that the complexes were diamagnetic Ru(II)-NO+ species which were converted to low-spin Ru(III) species (d5 , S=1/2) and released NO radicals by photons. The conversion was also confirmed by determining the single-crystal structure of the photoproduct of 1. The photochemical quantum yields (ΦNO s) of the photolysis were determined to be 0>1, 2, 3 at both the visible and UV excitations. Femtosecond (fs) time-resolved mid-IR spectroscopy was employed for studying NO-releasing dynamics. The geminate rebinding (GR) rates of the photoreleased NO to the photolyzed complexes were estimated to be 0≃1, 2, 3. DFT and TDDFT computations found that the introduction of the hydroxyl groups elevated the ligand π-bonding orbitals (π (salophen)), resulting in decrease of the HOMO-LUMO gaps in 1-3. The theoretical calculations suggested that the Ru-NNO bond dissociations of the complexes were mostly initiated by the ligand-to-ligand charge transfer (LLCT) of π(salophen)→π*(Ru-NO) with both the visible and UV excitations and the decreasing ΦNO s could be explained by the changes of the electronic structures in which the photoactivable bands of 1-3 have relatively less contribution of transitions related with Ru-NO bond than those of 0.

Use of Electron Paramagnetic Resonance (EPR) to Evaluate Redox Status in a Preclinical Model of Acute Lung Injury.

PURPOSE: Patients with hyper- vs. hypo-inflammatory subphenotypes of acute respiratory distress syndrome (ARDS) exhibit different clinical outcomes. Inflammation increases the production of reactive oxygen species (ROS) and increased ROS contributes to the severity of illness. Our long-term goal is to develop electron paramagnetic resonance (EPR) imaging of lungs in vivo to precisely measure superoxide production in ARDS in real time. As a first step, this requires the development of in vivo EPR methods for quantifying superoxide generation in the lung during injury, and testing if such superoxide measurements can differentiate between susceptible and protected mouse strains. PROCEDURES: In WT mice, mice lacking total body extracellular superoxide dismutase (EC-SOD) (KO), or mice overexpressing lung EC-SOD (Tg), lung injury was induced with intraperitoneal (IP) lipopolysaccharide (LPS) (10 mg/kg). At 24 h after LPS treatment, mice were injected with the cyclic hydroxylamines 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) or 4-acetoxymethoxycarbonyl-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid (DCP-AM-H) probes to detect, respectively, cellular and mitochondrial ROS - specifically superoxide. Several probe delivery strategies were tested. Lung tissue was collected up to one hour after probe administration and assayed by EPR. RESULTS: As measured by X-band EPR, cellular and mitochondrial superoxide increased in the lungs of LPS-treated mice compared to control. Lung cellular superoxide was increased in EC-SOD KO mice and decreased in EC-SOD Tg mice compared to WT. We also validated an intratracheal (IT) delivery method, which enhanced the lung signal for both spin probes compared to IP administration. CONCLUSIONS: We have developed protocols for delivering EPR spin probes in vivo, allowing detection of cellular and mitochondrial superoxide in lung injury by EPR. Superoxide measurements by EPR could differentiate mice with and without lung injury, as well as mouse strains with different disease susceptibilities. We expect these protocols to capture real-time superoxide production and enable evaluation of lung EPR imaging as a potential clinical tool for subphenotyping ARDS patients based on redox status.

Characterization of radicals in polysorbate 80 using electron paramagnetic resonance (EPR) spectroscopy and spin trapping.

Polysorbates are an important class of nonionic surfactants that are widely used to stabilize biopharmaceuticals. The degradation of polysorbate 20 and 80 and the related particle formation in biologics are heavily discussed in the pharmaceutical community. Although a lot of experimental effort was spent in the detailed study of potential degradation pathways, the underlying mechanisms are only sparsely understood. Besides enzymatic hydrolysis, another proposed mechanism is associated with radical-induced (auto)oxidation of polysorbates. To characterize the types and the origin of the involved radicals and their propagation in bulk material as well as in diluted polysorbate 80 solutions, we applied electron paramagnetic resonance (EPR) spectroscopy using a spin trapping approach. The prerequisite for a meaningful experiment using spin traps is an understanding of the trapping rate, which is an interplay of (i) the presence of the spin trap at the scene of action, (ii) the specific reactivity of the selected spin trap with a certain radical as well as (iii) the stability of the formed spin adducts (a slow decay rate). We discuss whether and to which extent these criteria are fulfilled regarding the identification of different radical classes that might be involved in polysorbate oxidative degradation processes. The ratio of different radicals for different scenarios was determined for various polysorbate 80 quality grades in bulk material and in aqueous solution, showing differences in the ratio of present radicals. Possible correlations between the radical content and product parameters such as the quality grade, the manufacturing date, the manufacturer, the initial peroxide content according to the certificate of analysis of polysorbate 80 are discussed.

A new photoactivable NO-releasing {Ru-NO}6 ruthenium nitrosyl complex with a tetradentate ligand containing aniline and pyridine moieties.

A new type of photoactivable NO-releasing ruthenium nitrosyl complex, [Ru(EPBP)Cl(NO)], with a tetradentate ligand, N,N'-(ethane-1,2-diyldi-o-phenylene)-bis(pyridine-2-carboxamide) (= H2 EPBP) was synthesized. Single crystal X-ray crystallography revealed that the complex has a distorted octahedral coordination geometry and NO is positioned at cis to Cl- ion. NO-photolysis was observed under a white room light. The photodissociation of Ru-NO bond was identified by various techniques including X-ray crystallography, IR, UV/Vis absorption, electron paramagnetic resonance (EPR), and NMR spectroscopies. Quantum yields for the NO-photolysis of the complex in CH3 OH, CHCl3 , DMSO, CH3 CN, and CH3 NO2 were measured to be 0.19-0.36 with 400 (±5) nm excitation. Density functional theory (DFT) and time-dependent density functional theory (TDDFT) calculations were performed to understand the details of the photodissociation of the complex. The calculations suggest that the NO photolysis is most likely initiated by the electronic transition from the aniline moiety π MOs (π (aniline)) of the EPBP2- chelating ligand to the π-antibonding MO of Ru-NO (π*(Ru-NO)). Experimental and theoretical investigations indicate that the EPBP2- ligand provides an effective platform forming ruthenium nitrosyl complexes useful for NO-photoreleasing.

Liquid-Phase Cyclohexene Oxidation with O2 over Spray-Flame-Synthesized La1-x Srx CoO3 Perovskite Nanoparticles.

La1-x Srx CoO3 (x=0, 0.1, 0.2, 0.3, 0.4) nanoparticles were prepared by spray-flame synthesis and applied in the liquid-phase oxidation of cyclohexene with molecular O2 as oxidant under mild conditions. The catalysts were systematically characterized by state-of-the-art techniques. With increasing Sr content, the concentration of surface oxygen vacancy defects increases, which is beneficial for cyclohexene oxidation, but the surface concentration of less active Co2+ was also increased. However, Co2+ cations have a superior activity towards peroxide decomposition, which also plays an important role in cyclohexene oxidation. A Sr doping of 20 at. % was found to be the optimum in terms of activity and product selectivity. The catalyst also showed excellent reusability over three catalytic runs; this can be attributed to its highly stable particle size and morphology. Kinetic investigations revealed first-order reaction kinetics for temperatures between 60 and 100 °C and an apparent activation energy of 68 kJ mol-1 for cyclohexene oxidation. Moreover, the reaction was not affected by the applied O2 pressure in the range from 10 to 20 bar. In situ attenuated total reflection infrared spectroscopy was used to monitor the conversion of cyclohexene and the formation of reaction products including the key intermediate cyclohex-2-ene-1-hydroperoxide; spin trap electron paramagnetic resonance spectroscopy provided strong evidence for a radical reaction pathway by identifying the cyclohexenyl alkoxyl radical.

EPR and NMR study of molecular components mobility and organization in goat milk under ultrasound treatment.

The effect of ultrasound treatment on molecular mobility and organization of the main components in raw goat milk was studied by EPR and NMR spectroscopies. NMR relaxation studies showed an increase in the spin-lattice T1 and spin-spin T2 relaxation times in goat milk products (cream, anhydrous fat) and change in the diffusion of proton-containing molecules during ultrasound treatment. The diffusion became more uniform and could be rather accurately approximated by one effective diffusion coefficient Deff, which indicates homogenization of goat milk components, dispersion of globular and supermicellar formations under sonication. EPR studies have shown that molecular mobility and organization of hydrophobic regions in goat milk are similar to those observed in micellar formations of surfactants with a hydrocarbon chain length C12-C16. Ultrasound treatment did not affect submicellar and protein globule organization. Free radicals arising under ultrasound impact of milk reacted quickly with components of goat milk (triglycerides, proteins, fatty acids) and were not observed by spin trapping method.

Study of Translocation of Stabilized NO Forms through Rat Skin using Electron Paramagnetic Resonance Method.

We studied the effect of dinitrosyl-iron complexes with N-acetyl-L-cysteine as a thiol-containing ligand (DNIC-Acc) after transdermal administration to rats. Electron paramagnetic resonance spectroscopy with a lipophilic NO spin trap (a complex of iron and diethyldithiocarbamate ions) showed that DNIC-Acc administration significantly increased the total level of NO in the lung and liver tissues of the animal, which was accompanied by a slight decrease in the mean BP (<10%).

Redox Interactions of Vitamin C and Iron: Inhibition of the Pro-Oxidant Activity by Deferiprone.

Ascorbic acid (AscH2) is one of the most important vitamins found in the human diet, with many biological functions including antioxidant, chelating, and coenzyme activities. Ascorbic acid is also widely used in a medical practice especially for increasing the iron absorption and as an adjuvant therapeutic in the iron chelation therapy, but its mode of action and implications in the iron metabolism and toxicity are not yet clear. In this study, we used UV-Vis spectrophotometry, NMR spectroscopy, and EPR spin trapping spectroscopy to investigate the antioxidant/pro-oxidant effects of ascorbic acid in reactions involving iron and the iron chelator deferiprone (L1). The experiments were carried out in a weak acidic (pH from 3 to 5) and neutral (pH 7.4) medium. Ascorbic acid exhibits predominantly pro-oxidant activity by reducing Fe3+ to Fe2+, followed by the formation of dehydroascorbic acid. As a result, ascorbic acid accelerates the redox cycle Fe3+ ↔ Fe2+ in the Fenton reaction, which leads to a significant increase in the yield of toxic hydroxyl radicals. The analysis of the experimental data suggests that despite a much lower stability constant of the iron-ascorbate complex compared to the FeL13 complex, ascorbic acid at high concentrations is able to substitute L1 in the FeL13 chelate complex resulting in the formation of mixed L12AscFe complex. This mixed chelate complex is redox stable at neutral pH = 7.4, but decomposes at pH = 4-5 during several minutes at sub-millimolar concentrations of ascorbic acid. The proposed mechanisms play a significant role in understanding the mechanism of action, pharmacological, therapeutic, and toxic effects of the interaction of ascorbic acid, iron, and L1.

Quantitation of spin probe-detectable oxidants in cells using electron paramagnetic resonance spectroscopy: To probe or to trap?

Electron Paramagnetic Resonance (EPR) spectroscopy coupled with spin traps/probes enables quantitative determination of reactive nitrogen and oxygen species (RNOS). Even with numerous studies using spin probes, the methodology has not been rigorously investigated. The autoxidation of spin probes has been commonly overlooked. Using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH), the present study has tested the effects of metal chelators, temperature, and oxygen content on the autoxidation of spin probes, where an optimized condition is refined for cell studies. The apparent rate of CMH autoxidation under this condition is 7.01 ± 1.60 nM/min, indicating low sensitivity and great variation of the CMH method and that CMH autoxidation rate should be subtracted from the generation rate of CMH-detectable oxidants (simplified as oxidants below) in samples. Oxidants in RAW264.7 cells are detected at an initial rate of 4.0 ± 0.7 pmol/min/106 cells, which is not considered as the rate of basal oxidants generation because the same method has failed to detect oxidant generation from the stimulation of phorbol-12-mysirate-13-acetate (PMA, 0.1 nmol/106 cells) in cells (2.5 ± 0.9 for PMA vs. 2.1 ± 1.5 pmol/min/106 cells for dimethyl sulfoxide (DMSO)-treated cells). In contrast, the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), which exhibits minimal autoxidation, reveals differences between PMA and DMSO treatment (0.26 ± 0.09 vs. -0.06 ± 0.12 pmol/min/106 cells), which challenges previous claims that spin probes are more sensitive than spin traps. We have also found that low temperature EPR measurements of frozen samples of CMH autoxidation provide lower signal intensity and greater variation compared to RT measurements of fresh samples. The current study establishes an example for method development of RNOS detection, where experimental details are rigorously considered and tested, and raises questions on the applications of spin probes and spin traps.

The role of chelating agents and amino acids in preventing free radical formation in bleaching systems.

The control of bleaching reaction is important in hair bleaching and laundry detergents to ensure quality of the final product. A better understanding of the reaction mechanisms is needed to minimize product failures. 31P NMR-spectroscopy-based spin trap technique was employed to detect and quantify the free radical species that were generated in different bleaching solutions. These solutions contained the key actives in an alkaline hair colorant/bleaching product, an ammonium salt and hydrogen peroxide at pH = 10. Generally, the main radical species detected in hair oxidative coloring or bleaching processes, were hydroperoxyl/superoxide radicals HO2·/O2.-, amino radicals ·NH2 and hydroxyl radicals ·OH. Their amounts showed a variation based on the chemical composition of the bleaching systems and the metal ion content. The generation of free radicals from reactions between transition metal ions, such as copper, and hydrogen peroxide at pH = 10 was evaluated. In the absence of chelating agents, the copper ions generated a significant level of hydroxyl radicals in a Fenton-like reaction with hydrogen peroxide at pH = 10. Besides that, an increase in copper ion content led to an increase of amino radical ·NH2, whereas the concentration of superoxide radical O2·- decreased which was not yet well reported in the previous literature. The effect of chelating agents like ethylenediaminetetraacetic acid (EDTA), tetrasodium-iminodisuccinate (IDS), a mixture of basic amino acids and dicarboxylic acid on free radical formation was investigated in the presence of binary Cu2+-Ca2+ bleaching systems. As expected, in the binary Cu2+-Ca2+ ion system EDTA did not suppress hydroxyl radical formation effectively, but the mixture containing sodium succinate, lysine and arginine reduced hydroxyl radical formation, whereas IDS (nearly) completely inhibited hydroxyl radical formation. The results indicated that each bleaching solution has its characteristic performance and damage profile. Whereas the reactivity can be controlled by the usage of chelating agents.

In Vivo Electron Paramagnetic Resonance: Radical Concepts for Translation to the Clinical Setting.

Electron paramagnetic resonance (EPR)-based spectroscopic and imaging techniques allow for the study of free radicals-molecules with one or more unpaired electrons. Biological EPR applications include detection of endogenous biologically relevant free radicals as well as use of specially designed exogenous radicals to probe local microenvironments. This Forum focuses on recent advances in the field of in vivo EPR applications discussed at the International Conference on Electron Paramagnetic Resonance Spectroscopy and Imaging of Biological Systems (EPR-2017). Although direct EPR detection of endogenous free radicals such as reactive oxygen species (ROS) in vivo remains unlikely in most cases, alternative approaches based on applications of advanced spin traps and probes for detection of paramagnetic products of ROS reactions often allow for specific assessment of free radical production in living subjects. In recent decades, significant progress has been achieved in the development and in vivo application of specially designed paramagnetic probes as "molecular spies" to assess and map physiologically relevant functional information such as tissue oxygenation, redox status, pH, and concentrations of interstitial inorganic phosphate and intracellular glutathione. Recent progress in clinical EPR instrumentation and development of biocompatible paramagnetic probes for in vivo multifunctional tissue profiling will eventually make translation of the EPR techniques into clinical settings possible. Antioxid. Redox Signal. 28, 1341-1344.

Spin Trapping: A Review for the Study of Obesity Related Oxidative Stress and Na+/K+-ATPase.

Reactive oxygen species (ROS) have gained attention with mounting evidence of their importance in cell signaling and various disease states. ROS is produced continuously as a natural by-product of normal oxygen metabolism. However, high levels ROS causes oxidative stress and damage to biomolecules. This results in loss of protein function, DNA cleavage, lipid peroxidation, or ultimately cell injury or death. Obesity has become a worldwide epidemic; studies show fat accumulation is associated with increased ROS and oxidative stress. Evidence exists supporting oxidative stress as a factor driving forward insulin resistance (IR), potentially resulting in diabetes. Na+/K+-ATPase signaling is also a potential source of ROS promoting oxidative stress. The best way to observe radical species in biological systems is electron paramagnetic resonance spectroscopy with spin trapping. EPR spin trapping is an important technique to study the mechanisms driving disease states attributed to ROS.

Imaging free radicals in organelles, cells, tissue, and in vivo with immuno-spin trapping.

The accurate and sensitive detection of biological free radicals in a reliable manner is required to define the mechanistic roles of such species in biochemistry, medicine and toxicology. Most of the techniques currently available are either not appropriate to detect free radicals in cells and tissues due to sensitivity limitations (electron spin resonance, ESR) or subject to artifacts that make the validity of the results questionable (fluorescent probe-based analysis). The development of the immuno-spin trapping technique overcomes all these difficulties. This technique is based on the reaction of amino acid- and DNA base-derived radicals with the spin trap 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) to form protein- and DNA-DMPO nitroxide radical adducts, respectively. These adducts have limited stability and decay to produce the very stable macromolecule-DMPO-nitrone product. This stable product can be detected by mass spectrometry, NMR or immunochemistry by the use of anti-DMPO nitrone antibodies. The formation of macromolecule-DMPO-nitrone adducts is based on the selective reaction of free radical addition to the spin trap and is thus not subject to artifacts frequently encountered with other methods for free radical detection. The selectivity of spin trapping for free radicals in biological systems has been proven by ESR. Immuno-spin trapping is proving to be a potent, sensitive (a million times higher sensitivity than ESR), and easy (not quantum mechanical) method to detect low levels of macromolecule-derived radicals produced in vitro and in vivo. Anti-DMPO antibodies have been used to determine the distribution of free radicals in cells and tissues and even in living animals. In summary, the invention of the immuno-spin trapping technique has had a major impact on the ability to accurately and sensitively detect biological free radicals and, subsequently, on our understanding of the role of free radicals in biochemistry, medicine and toxicology.

A liposome-encapsulated spin trap for the detection of nitric oxide.

Electron paramagnetic resonance (EPR) is one of the few methods that allows for the unambiguous detection of nitric oxide (NO). However, the dithiocarbamate-iron spin traps employed with this method inhibit the activity of nitric oxide synthase and catalyze NO production from nitrite. These disadvantages limit EPR's application to biological NO detection. We present a liposome-encapsulated spin-trap (LEST) method for the capture and in situ detection of NO by EPR. The method shows a linear response for [NO]≥4µM and can detect [NO]≥40nM in a 500µL sample (≥20 pmol). The kinetics of NO production can be followed in real time over minutes to hours. LEST does not inhibit the activity of inducible nitric oxide synthase or nitrate reductase and shows minimal abiotic NO production in the presence of nitrite and NADH. Nitrate reductase-like activity is detected in cell lysates of the coccolithophore Emiliania huxleyi and is elevated in virus-infected culture. This method shows particular promise for NO detection in cell lysates and crude preparations of NO-producing tissues.

Detection of Nitric Oxide by Electron Paramagnetic Resonance Spectroscopy: Spin-Trapping with Iron-Dithiocarbamates.

Electron paramagnetic resonance (EPR) spectroscopy is the ideal methodology to identify radicals (detection and characterization of molecular structure) and to study their kinetics, in both simple and complex biological systems. The very low concentration and short life-time of NO and of many other radicals do not favor its direct detection and spin-traps are needed to produce a new and persistent radical that can be subsequently detected by EPR spectroscopy.In this chapter, we present the basic concepts of EPR spectroscopy and of some spin-trapping methodologies to study NO. The "strengths and weaknesses" of iron-dithiocarbamates utilization, the NO traps of choice for the authors, are thoroughly discussed and a detailed description of the method to quantify the NO formation by molybdoenzymes is provided.

Enhanced NADPH oxidases and reactive oxygen species in the mechanism of methanol-initiated protein oxidation and embryopathies in vivo and in embryo culture.

Methanol (MeOH) teratogenicity in rodents may be mediated in part by reactive oxygen species (ROS), the source of which is unknown. To determine if MeOH enhances embryonic ROS-producing NADPH oxidases (NOXs), p22phox mRNA and protein and oxidatively damaged protein were measured in gestational day 12 MeOH-exposed CD-1 mouse embryos with or without pretreatment with the free radical spin trap phenylbutylnitrone (PBN) or the NOX inhibitor diphenyleneiodonium chloride (DPI). MeOH exposure upregulated p22phox mRNA and protein expression, and enhanced protein oxidation, within 3-6 h. Compared to embryos exposed to MeOH alone, PBN and DPI pretreatment decreased MeOH-enhanced p22phox mRNA expression, DPI but not PBN blocked p22phox protein expression, and both blocked protein oxidation. To assess developmental relevance, mouse embryos were exposed in culture for 24 h to MeOH or vehicle with or without pretreatment with PBN, DPI, or the prostaglandin H synthase (PHS) inhibitor eicosatetraynoic acid (ETYA), and evaluated for abnormalities. ETYA did not prevent MeOH embryopathies, despite blocking phenytoin embryopathies (ROS-initiating positive control), precluding bioactivation of MeOH or its metabolites by PHS. Concentration-dependent MeOH embryopathies were blocked by both DPI and PBN pretreatment, suggesting that enhanced embryonic NOX-catalyzed ROS formation and oxidative stress may contribute to the mechanism of MeOH embryopathies.

Tea extracts protect normal lymphocytes but not leukemia cells from UV radiation-induced ROS production: An EPR spin trap study.

PURPOSE: An ex vivo method for detection of free radicals and their neutralization by aqueous tea in human normal lymphocytes and MEC-1 leukemia cells under ultraviolet (UV) irradiation was investigated. MATERIALS AND METHODS: This method is based on the electron paramagnetic resonance (EPR) spectroscopy spin-trapping technique. 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO) was used as the spin trap. Normal human lymphocytes and leukemia cells were exposed to UVB radiation (290-315 nm) at 47.7 and 159 mJ/cm(2) and to UVA radiation (315-400 nm) at 53.7 J/cm(2). RESULTS: No significant radical production at 47.7 mJ/cm(2) UVB dose in both cell lines was observed. In normal cells, free radical production was observed at 159 mJ/cm(2) UVB and 53.7 J/cm(2) UVA doses. However, both UV sources did not significantly produce free radicals in leukemia cells. A radical scavenging property of tea extracts (black, green, sage, rosehip) was observed in normal lymphocytes after both UVB and UVA exposure. In leukemia cells, the intensities of EPR signals produced in BMPO with tea extracts were found to be increased substantially after UVA exposure. CONCLUSION: These results showed that UV radiation induced free radical formation in normal human lymphocytes and indicated that tea extracts may be useful as photoprotective agents for them. On the other hand, tea extracts facilitated free radical production in leukemia cells.

Detecting and monitoring NO, SNO and nitrite in vivo.

The detection and quantification of nitric oxide and related reactive nitrogen species in vivo is vital to the understanding of the pathology and/or treatment of numerous conditions. To that end, several detection and quantification methods have been developed to study NO, as well as its redox relatives, nitrite and S-nitrosothiols. While no single technique can offer a complete picture of the nitrogen cycle in a given system in vivo, familiarity with the benefits and limitations of several common tools for NOx determination can assist in the development of new diagnostics and therapeutics.

Visualization of oxidative stress in ex vivo biopsies using electron paramagnetic resonance imaging.

PURPOSE: The purpose of this study was to develop an X-Band electron paramagnetic resonance imaging protocol for visualization of oxidative stress in biopsies. METHODS: The developed electron paramagnetic resonance imaging protocol was based on spin trapping with the cyclic hydroxylamine spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine and X-Band EPR imaging. Computer software was developed for deconvolution and back-projection of the EPR image. A phantom containing radicals of known spatial characteristic was used for evaluation of the developed protocol. As a demonstration of the technique electron paramagnetic resonance imaging of oxidative stress was performed in six sections of atherosclerotic plaques. Histopathological analyses were performed on adjoining sections. RESULTS: The developed computer software for deconvolution and back-projection of the EPR images could accurately reproduce the shape of a phantom of known spatial distribution of radicals. The developed protocol could successfully be used to image oxidative stress in six sections of the three ex vivo atherosclerotic plaques. CONCLUSIONS: We have shown that oxidative stress can be imaged using a combination of spin trapping with the cyclic hydroxylamine spin probe cyclic hydroxylamine spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine and X-Band EPR imaging. A thorough and systematic evaluation on different types of biopsies must be performed in the future to validate the proposed technique.

Redox equilibration after one-electron reduction of cytochrome c oxidase: radical formation and a possible hydrogen relay mechanism.

Kinetic studies using UV/visible and EPR spectroscopy were carried out to follow the distribution of electrons within beef heart cytochrome c oxidase (CcO), both active and cyanide-inhibited, following addition of reduced cytochrome c as electron donor. In the initial one-electron reduced state the electron is shared between three redox centers, heme a, CuA and a third site, probably CuB. Using a rapid freeze system and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) a protein radical was also detected. The EPR spectrum of the DMPO adduct of this radical was consistent with tyrosyl radical capture. This may be a feature of a charge relay mechanism involved in some part of the CcO electron transfer system from bound cytochrome c via CuA and heme a to the a3CuB binuclear center.