Medium-throughput ESR detection of superoxide production in undetached adherent cells using cyclic nitrone spin traps.

Spin trapping with cyclic nitrones coupled to electron spin resonance (ESR) is recognized as a specific method of detection of oxygen free radicals in biological systems, especially in culture cells. In this case, the detection is usually performed on cell suspensions, which is however unsuitable when adhesion influences free radical production. Here, we performed ESR detection of superoxide with four spin traps (5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DEPMPO; 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DIPPMPO; (4R*, 5R*)-5-(diisopropyloxyphosphoryl)-5-methyl-4-[({[2-(triphenylphosphonio)ethyl]carbamoyl}oxy)methyl]pyrroline N-oxide bromide, Mito-DIPPMPO; and 6-monodeoxy-6-mono-4-[(5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide)-ethylenecarbamoyl-(2,3-di-O-methyl) hexakis (2,3,6-tri-O-methyl)]-ß-cyclodextrin, CD-DIPPMPO) directly on RAW 264.7 macrophages cultured on microscope coverslip glasses after phorbol 12-myristate 13-acetate (PMA) stimulation. Distinct ESR spectra were obtained with each spin trap using this method. CD-DIPPMPO, a recently published phosphorylated cyclic nitrone bearing a permethylated ß-cyclodextrin moiety, was confirmed as the most specific spin trap of the superoxide radical, with exclusive detection of the superoxide adduct. ESR detection performed on cells attached to coverslips represents significant advances over other methods in terms of simplicity, speed, and measurement under near-physiological conditions. It thus opens the way for numerous applications, such as medium-throughput screening of antioxidants and reactive oxygen species (ROS)-modulating agents.

High binding yet accelerated guest rotation within a cucurbit[7]uril complex. Toward paramagnetic gyroscopes and rolling nanomachines.

The (15-oxo-3,7,11-triazadispiro[5.1.5.3]hexadec-7-yl)oxidanyl, a bis-spiropiperidinium nitroxide derived from TEMPONE, can be included in cucurbit[7]uril to form a strong (K(a)∼ 2 × 10(5) M(-1)) CB[7]@bPTO complex. EPR and MS spectra, DFT calculations, and unparalleled increased resistance (a factor of ∼10(3)) toward ascorbic acid reduction show evidence of deep inclusion of bPTO inside CB[7]. The unusual shape of the CB[7]@bPTO EPR spectrum can be explained by an anisotropic Brownian rotational diffusion, the global tumbling of the complex being slower than rotation of bPTO around its "long molecular axis" inside CB[7]. The CB[7] (stator) with the encapsulated bPTO (rotator) behaves as a supramolecular paramagnetic rotor with increased rotational speed of the rotator that has great potential for advanced nanoscale machines requiring wheels such as cucurbiturils with virtually no friction between the wheel and the axle for optimum wheel rotation (i.e. nanopulleys and nanocars).

2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide: evaluation of the spin trapping properties.

The 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-l-oxide (EMPO), an easily prepared pyrroline-N-oxide has been tested as a free radical scavenger. Spin adducts of superoxide, hydroxyl radical, and other free radicals were characterized in phosphate buffer at pH 7.0 and 5.6. At pH 7 in phosphate buffer, the EMPO/O(2)(-*) spin adduct was estimated to be about five times more persistent than its DMPO analogue. Furthermore, its decay does not produce the EMPO/HO&z.rad; adduct.

beta-phosphorylated nitroxides in the pyrrolidine series: reduction by ascorbate.

Reduction-resistant nitroxides are particularly interesting for biomedical applications. beta-Phosphorylated pyrrolidinyl nitroxides, a new series of stable pyrrolidinoxyl radicals prepared in our laboratory, were tested toward ascorbate reduction in phosphate buffer at pH 7.4. The kinetics of decay were monitored by ESR and compared to those of two reference nitroxides, PCA and Proxyl. The introduction of a beta-phosphoryl group on a pyrrolidinoxyl structure resulted in a moderate increase of the reduction rate constant. However, inside the phosphorylated series, slight structural modifications can induce significant changes in the rate constants.

Detection of superoxide anion using an isotopically labeled nitrone spin trap: potential biological applications.

We describe the synthesis and biological applications of a novel nitrogen-15-labeled nitrone spin trap, 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide ([(15)N]EMPO) for detecting superoxide anion. Superoxide anion generated in xanthine/xanthine oxidase (100 nM min(-1)) and NADPH/calcium-calmodulin/nitric oxide synthase systems was readily detected using EMPO, a nitrone analog of 5,5'-dimethyl-1-pyrroline N-oxide (DMPO). Unlike DMPO-superoxide adduct (DMPO-OOH), the superoxide adduct of EMPO (EMPO-OOH) does not spontaneously decay to the corresponding hydroxyl adduct, making spectral interpretation less confounding. Although the superoxide adduct of 5-(diethoxyphosphoryl)-5-methyl-pyrroline N-oxide is more persistent than EMPO-OOH, the electron spin resonance spectra of [(14)N]EMPO-OOH and [(15)N]EMPO-OOH are less complex and easier to interpret. Potential uses of [(15)N]EMPO in elucidating the mechanism of superoxide formation from nitric oxide synthases, and in ischemia/reperfusion injury are discussed.

5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide: a new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygen-centered radicals.

5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO, 2), a new spin trap, has been synthesized via a two-step synthetic route, and its ability to spin trap oxy radicals in biological milieu has been addressed. The in vitro spin trapping of hydroxyl and superoxide radicals was investigated in a phosphate buffer 0.1 M, and the hyperfine coupling constants of the spin adducts were determined. The rates of spin trapping of hydroxyl and superoxide radicals with 2 were found to be close to those reported for 5,5-dimethyl-1-pyrroline N-oxide (DMPO). However, the DEPMPO-superoxide spin adduct was shown to be significantly more persistent (15 times at pH 7) than the DMPO--superoxide spin adduct. Using 2 as a spin trap, the production of superoxide has been clearly characterized during the reperfusion of ischemic isolated rat hearts.

Calcium-dependent free radical generation in cultured retinal neurons injured by kainate.

Cultured rat retinal neurons exposed to kainate produced free radicals, as demonstrated by electron spin resonance (ESR) spin trapping using the nitrone 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and the generation of DMPO hydroxyl adduct (DMPO-OH). This DMPO-OH production was abolished by EGTA, nitro-arginine and oxypurinol, suggesting that it was dependent on Ca2+ influx and subsequent activation of nitric oxide synthase and xanthine oxidase. Moreover, kainate induced a receptor-mediated Ca2+ influx and neuronal injury assessed by lactate dehydrogenase release. Neuroprotection afforded by nitro-arginine and oxypurinol shows that calcium-dependent free radical production plays a major role in kainate retinal toxicity.

Effect of redox-active drugs on superoxide generation from nitric oxide synthases: biological and toxicological implications.

In this article, we address the mechanism of superoxide formation from constitutive nitric oxide synthases (NOS). Merits and drawbacks of the various superoxide detection assays are reviewed. One of the most viable techniques for measuring superoxide from NOS is electron spin resonance (ESR) spin-trapping using a novel phosphorylated spin trap. Implications of superoxide and peroxynitrite formation from NOS enzymes in cardiovascular and cerebrovascular disorders are discussed.

Force field calculations on five membered ring aminoxyl radicals.

Two force fields (MM2 and Genmol) have been applied to the modeling of five membered ring aminoxyl radicals. For the six molecules which were investigated the geometry of the conformation with the lowest strain energy was in very good agreement with the X-ray geometry. However owing to the high flexibility of five membered rings other conformations were shown to have a strain energy close to the energy minimum.

Characterization of sulfur-centered radical intermediates formed during the oxidation of thiols and sulfite by peroxynitrite. ESR-spin trapping and oxygen uptake studies.

Using a novel phosphorylated spin trap, 5-diethoxy-phosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), an analog of the commonly used trap 5,5'-dimethyl-1-pyrroline N-oxide (DMPO), we have investigated the reactions of sulfur-centered radicals produced from the oxidation of thiols and sulfite by peroxynitrite. The predominant species trapped in all cases are the corresponding sulfur-centered radicals, i.e. glutathionyl radical (GS) from glutathione (GSH), N-acetyl-DL-penicillamine thiyl radical (S-NAP) from N-acetyl-DL-penicillamine (NAP) and sulfate anion radical (SO3-) from sulfite. These radicals consume molecular oxygen forming either peroxyl or superoxide anion radicals. GS, S-NAP, and (SO3-)-derived radicals react with ammonium formate to form the carbon dioxide anion radical (CO2-). Further support of spin adduct assignments and radical reactions are obtained from photolysis of S-nitrosoglutathione and S-nitroso-N-acetyl-DL-penicillamine. We conclude that the direct reaction of peroxynitrite with thiols and sulfate forms thiyl and sulfate anion radicals, respectively, by a hydroxyl radical-independent mechanism. Pathological implications of thiyl radical formation and subsequent oxyradical-mediated chain reactions are discussed. Oxygen activation by thiyl radicals formed during peroxynitrite-mediated oxidation of glutathione may limit the effectiveness of GSH against peroxynitrite-mediated toxicity in cellular systems.

Quantitative measurement of superoxide generation using the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide.

Measurement and quantitation of superoxide by electron paramagnetic resonance (EPR) using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) have been limited by the short half-life of the superoxide adduct DMPO-OOH (approximately 50 s at pH 7). Recently a beta-phosphorylated nitrone, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), was developed and reported to form a more stable superoxide adduct with a half-life of approximately 15 min. We evaluated the use of DEPMPO for quantitative measurement of superoxide in chemical and biochemical systems. To estimate the efficiency of trapping, EPR oximetry was used to measure oxygen consumption and the intensity of the DEPMPO-OOH signal to measure superoxide generation and adduct decay. With the superoxide-generating systems, riboflavin/light and xanthine/xanthine oxidase, DEPMPO trapped approximately 65% of the superoxide produced. The efficiency of superoxide trapping by DEPMPO was compared to the commonly used cytochrome c reduction method. When superoxide production was > 20 microM, cytochrome c detected approximately 100% of the superoxide produced, while DEPMPO trapped 60 to 70%. However, EPR detection with DEPMPO was 40-fold more sensitive than cytochrome c. Thus, DEPMPO is an efficient spin trap which enables specific and sensitive detection and quantitation of superoxide generation.

Quantitative measurement of superoxide generation and oxygen consumption from leukocytes using electron paramagnetic resonance spectroscopy.

In view of the important role of superoxide in cellular injury, there has been a great need for methods suitable for quantitation of superoxide production from cells. Previous methods have had limited sensitivity or specificity as well as problems with side reactions in cellular systems. Recently, we have shown that the new spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide has ideal properties for quantitative superoxide measurement in chemical/biochemical systems; however, its suitability and potential for measurements in cellular systems has not been determined. Therefore, we evaluated the use of DEPMPO for quantitative measurement of superoxide formed by polymorphonuclear leukocytes. After activation of these cells with the phorbol ester (PMA, 200 ng/ml) or opsonized zymosan (1 mg/ml) at 24 degrees C a strong signal of the superoxide adduct, DEPMPO-OOH, was observed. This technique was highly sensitive and enabled measurement of superoxide generation from as few as 2 x 10(3) cells. The kinetics of adduct formation and decay were measured which enabled quantitation of superoxide formation. Spin label electron paramagnetic resonance (EPR) oximetry was used to measure the oxygen consumption from these cells. With PMA activation rapid onset of superoxide generation occurred with a rate of 0.78 nmol/min/10(6) cells while with zymosan a slower gradual onset of activation was seen to a peak rate of 0.061 nmol/min/10(6) cells. With both stimulators the ratios of superoxide production to oxygen consumption were similar with values of approximately 50% obtained. Thus, EPR spin trapping with DEPMPO together with EPR oximetry methods can be used to provide sensitive and specific quantitation of cellular superoxide generation and oxygen consumption. These methods provide a promising new approach for the measurement of oxygen reduction and superoxide generation in cellular systems.

Nitrone spin traps and their pyrrolidine analogs in myocardial reperfusion injury: hemodynamic and ESR implications--evidence for a cardioprotective phosphonate effect for 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide in rat hearts.

Formation of free radicals during reperfusion of the isolated ischemic heart has often been demonstrated by detecting hydroxyl radical spin adducts of the nitrone 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in coronary effluents. However, questions still remain regarding (a) whether the reported cardiovascular effects of nitrone perfusion may affect the formation of spin adducts, and (b) the primary generation of superoxide (O2.-), because of the short persistency of O2.-/DMPO spin adduct. We therefore compared the effects of perfusing 5 mM of two nitrones, DMPO and 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) or the two structurally related pyrrolidines, diethyl (2-methyl-2-pyrrolidinyl) phosphonate (DEPMPH) and pyrrolidine (PyH), on postischemic functional recovery of rat hearts subjected to 10 min of low-flow ischemia, 30 min of global ischemia and 60 min of reperfusion. All compounds were added to the perfusate before ischemia, throughout low-flow ischemia and during the initial 10 min of reflow. In one additional group, hearts received DEPMPO only at reflow. Hemodynamic and in vitro ESR evidence is presented indicating that the phosphonate group of DEPMPO and DEPMPH confers these molecules with an enhanced cardioprotective efficacy, unrelated to radical scavenging, acting in synergy with the intrinsic radical trapping effects of the nitronyl group. Continuous-flow ESR spin trapping using 5.7 mM DEPMPO administered at reflow, but not before ischemia, demonstrated for the first time extended formation of O2.- in the reperfused myocardium.

Spin-trapping of free radicals formed during the oxidation of glutathione by tetramethylammonium peroxynitrite.

Glutathionyl radical (GS.) formed during the oxidation of glutathione by tetramethylammonium peroxynitrite ([NMe4][ONOO]) was spin-trapped with 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). This radical reacted with ammonium formate to form the carbon dioxide anion radical (CO2-.). The superoxide anion formed during oxidation of GSH by peroxynitrite salt was trapped with DMPO and detected as the DMPO-hydroxyl adduct. Addition of SOD mimic completely abolished the spectrum of the hydroxyl adduct but not the spectrum of the DMPO-glutathionyl radical adduct. Addition of seleno-DL-cystine or its reduced form caused a dramatic inhibition in the formation of spin adducts, suggesting that seleno-DL-cysteine is a more effective scavenger of peroxynitrite. The oxygen uptake observed during oxidation of GSH by peroxynitrite salt was inhibited by spin traps. In the presence of catalase, approximately 50% of the oxygen consumed was restored, indicating stoichiometric conversion of O2 to H2O2 during oxidation of GSH by peroxynitrite salt. Results indicate that nitrite and glutathione disulfide are formed as the major products during oxidation of GSH by peroxynitrite.

Beta-phosphorylated five membered ring nitroxides. Synthesis and EPR study.

A series of stable beta-phosphorylated five membered ring nitroxides was prepared by intramolecular aminomercuration of alkenyl alpha-aminophosphonates. The structure of these nitroxides was deduced from their 13C and 31P coupling constants and from force field calculations.

Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors.

The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium/calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium/calmodulin binding, superoxide formation is increased. Heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical inhibit superoxide generation. No inhibition is observed after addition of L-arginine, NG-hydroxy-L-arginine, L-thiocitrulline, and L-NG-monomethyl arginine to activated eNOS. These results demonstrate that superoxide is generated from the oxygenase domain by dissociation of the ferrous-dioxygen complex and that occupation of the L-arginine binding site does not inhibit this process. However, the concomitant addition of L-arginine and tetrahydrobiopterin (BH4) abolishes superoxide generation by eNOS. Under these conditions, L-citrulline production is close to maximal. Our data indicate that BH4 fully couples L-arginine oxidation to NADPH consumption and prevents dissociation of the ferrous-dioxygen complex. Under these conditions, eNOS does not generate superoxide. The presence of flavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. Our data indicate that modulation of BH4 concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS.