The C242T CYBA polymorphism as a major determinant of NADPH oxidase activity in patients with cardiovascular disease.

Single nucleotide polymorphisms (SNP) in the CYBA gene encoding p22(phox) have been associated with respiratory burst and cardiovascular phenotypes. We previously reported a reduced phagocytic respiratory burst activity in healthy adults with the C242T SNP, but found no correlation between CYBA SNPs and coronary artery disease (CAD) phenotype. Using lymphoblastoid cells, we hypothesized that CYBA SNPs affect enzyme activity in patients with cardiovascular disease (CVD), but would not be associated with angiographic severity of CAD due to confounding by risk factors. We established lymphoblastoid cell lines from patients with CVD and genotyped the study cohort for CYBA SNPs and phenotyped each subject's coronary angiogram for CAD severity. As quantified by electron spin resonance, superoxide production in picomoles per 10(6) resting lymphoblastoid cells per minute for the CC, CT, and TT genotypes of the C242T SNP were 16.2+/-1.4, n=70, 11.9+/-0.7, n=87, and 11.9+/-1.5, n=28, respectively (P=0.002). The -930(A/G) and A640G SNPs did not affect superoxide production (P > 0.2). Expression of p22(phox) was not affected as determined by real-time RT-PCR and Western blot analysis. The C242T CYBA SNP is associated with altered NADPH oxidase activity in lymphoblastoid cells of patients with CVD. By reducing the influence of confounding environmental factors, lymphoblastoid cell lines could serve as a tool to assess direct genotype/phenotype interactions of candidate genes known to affect atherosclerosis.

Overexpression of cytochrome P450 CYP2J2 protects against hypoxia-reoxygenation injury in cultured bovine aortic endothelial cells.

CYP2J2 is abundant in human heart and its arachidonic acid metabolites, the epoxyeicosatrienoic acids (EETs), have potent vasodilatory, antiinflammatory and cardioprotective properties. This study was designed to examine the role of CYP2J2 in hypoxia-reoxygenation-induced injury in cultured bovine aortic endothelial cells (BAECs). Early passage BAECs were exposed to 24-h hypoxia followed by 4-h reoxygenation (HR). HR resulted in cell injury, as indicated by significant increases in lactate dehydrogenase (LDH) release and trypan blue stained cells (p < 0.01) and was associated with a decrease in CYP2J2 protein expression. Transfection of BAECs with the CYP2J2 cDNA resulted in increased CYP2J2 expression and arachidonic acid epoxygenase activity, compared with cells transfected with an irrelevant green fluorescent protein (GFP) cDNA. HR induced significant injury in GFP-transfected BAECs, as indicated by increases in LDH release and trypan blue-stained cells (p < 0.01); however, the HR-induced injury was markedly attenuated in CYP2J2-transfected cells (p < 0.01). HR increased cellular 8-iso-prostaglandin F(2alpha) (p < 0.05), and decreased eNOS expression, L-arginine uptake and conversion, and nitrite production (p < 0.01) in GFP-transfected BAECs. CYP2J2 transfection attenuated the HR-induced increase in 8-iso-prostaglandin F(2alpha) (p < 0.05) and decreased the amount of extracellular superoxide detected by cytochrome c reduction under normoxic conditions (p < 0.05) but did not significantly affect HR-induced decreases in eNOS expression, L-arginine uptake and conversion, and nitrite production. Treatment of BAECs with synthetic EETs and/or epoxide hydrolase inhibitors also showed protective effects against HR injury (p < 0.05). These observations suggest: (1) HR results in endothelial injury and decreased CYP2J2 expression; (2) transfection with the CYP2J2 cDNA protects against HR injury; and (3) the cytoprotective effects of CYP2J2 may be mediated, at least in part, by antioxidant effects.

Spin trapping of polyunsaturated fatty acid-derived peroxyl radicals: reassignment to alkoxyl radical adducts.

Polyunsaturated fatty acid (PUFA) peroxyl radicals play a crucial role in lipid oxidation. ESR spectroscopy with the spin-trapping technique is one of the most direct methods for radical detection. There are many reports of the detection of PUFA peroxyl radical adducts; however, it has recently been reported that attempted spin trapping of organic peroxyl radicals at room temperature formed only alkoxyl radical adducts in detectable amounts. Therefore, we have reinvestigated spin trapping of the linoleic, arachidonic, and linolenic acid-derived PUFA peroxyl radicals. The slow-flow technique allowed us to obtain well-resolved ESR spectra of PUFA-derived radical adducts in a mixture of soybean lipoxygenase, PUFA, and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). However, interpretation of the ESR spectra was complicated by the overlapping of the PUFA-derived alkoxyl radical adduct spectra. In order to understand these spectra, PUFA-derived alkoxyl radical adducts were modeled by various alkoxyl radical adducts. For the first time, we synthesized a wide range of DMPO adducts with primary and secondary alkoxyl radicals. It was found that many ESR spectra previously assigned as DMPO/peroxyl radical adducts based on their close similarity to the ESR spectrum of the DMPO/superoxide radical adduct, in conjunction with their insensitivity to superoxide dismutase, are indeed alkoxyl radical adducts. We have reassigned the PUFA alkylperoxyl radical adducts to their corresponding alkoxyl radical adducts. Using hyperfine coupling constants of model DMPO/alkoxyl radical adducts, the computer simulation of DMPO/PUFA alkoxyl radical adducts was performed. It was found that the trapped, oxygen-centered PUFA-derived radical is a secondary, chiral alkoxyl radical. The presence of a chiral carbon atom leads to the formation of two diastereomers of the DMPO/PUFA alkoxyl radical adduct. Therefore, attempted spin trapping of the PUFA peroxyl radical by DMPO at room temperature leads to the formation of the PUFA alkoxyl radical adduct.

A new approach for extracellular spin trapping of nitroglycerin-induced superoxide radicals both in vitro and in vivo.

Anti-ischemic therapy with nitrates is complicated by the induction of tolerance that potentially results from an unwanted coproduction of superoxide radicals. Therefore, we analyzed the localization of in vitro and in vivo, glyceryl trinitrate (GTN)-induced formation of superoxide radicals and the effect of the antioxidant vitamin C and of superoxide dismutase (SOD). Sterically hindered hydroxylamines 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidin (PP-H) can be used for in vitro and in vivo quantification of superoxide radical formation. The penetration/incorporation of CP-H or PP-H and of their corresponding nitroxyl radicals was examined by fractionation of the blood and blood cells during a 1-h incubation. For monitoring in vivo, GTN-induced (130 microg/kg) O2*- formation CP-H or PP-H were continuously infused (actual concentration, 800 microM) for 90 to 120 min into rabbits. Formation of superoxide was determined by SOD- or vitamin C-inhibited contents of nitroxide radicals in the blood from A. carotis. The incubation of whole blood with CP-H, PP-H, or corresponding nitroxyl radicals clearly shows that during a 1-h incubation, as much as 8.3% of CP-H but only 0.9% of PP-H is incorporated in cytoplasm. Acute GTN treatment of whole blood and in vivo bolus infusion significantly increased superoxide radical formation as much as 4-fold. Pretreatment with 20 mg/kg vitamin C or 15,000 U/kg superoxide dismutase prevented GTN-induced nitroxide formation. The decrease of trapped radicals after treatment with extracellularly added superoxide dismutase or vitamin C leads to the conclusion that GTN increases the amount of extracellular superoxide radicals both in vitro and in vivo.

Reassignment of organic peroxyl radical adducts.

The study of the important role of peroxyl radicals in biological systems is limited by their difficult detection with direct electron spin resonance (ESR). Many ESR spectra were assigned to 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/peroxyl radical adducts based only on the close similarity of their ESR spectra to that of DMPO/superoxide radical adduct in conjunction with their insensitivity to superoxide dismutase, which distinguishes the radical adduct from DMPO/superoxide radical adduct. Later, the spin-trapping literature reported that DMPO/peroxyl radical adducts have virtually the same hyperfine coupling constants as synthesized alkoxyl radical adducts, raising the issue of the correct assignment of peroxyl radical adducts. However, using 17O-isotope labelling, the methylperoxyl and methoxyl radical adducts should be distinguishable. We have reinvestigated the spin trapping of the methylperoxyl radical. The methylperoxyl radical was generated in aerobic solution with 17O-molecular oxygen either in a Fenton system with dimethylsulfoxide or in a chloroperoxidase system with tert-butyl hydroperoxide. Two different spin traps, DMPO and 2,2,4-trimethyl-2H-imidazole-1-oxide (TMIO), were used to trap methylperoxyl radical. 17O-labelled methanol was used to synthesize methoxyl radical adducts by nucleophylic addition. It was shown that the 17O hyperfine coupling constants of radical adducts formed in methylperoxyl radical-generating systems are identical to that of the methoxyl radical adduct. Therefore, methylperoxyl radical-producing systems form detectable methoxyl radical adduct, but not detectable methylperoxyl radical adducts at room temperature. One of the possible mechanisms is the decomposition of peroxyl radical adduct with the formation of secondary alkoxyl radical adduct. These results allow us to reinterpret previously published data reporting detection of peroxyl radical adducts. We suggest that detection of 17O-alkoxyl radical adduct from 17O-labelled molecular oxygen can be used as indirect evidence for peroxyl radical generation.

Comparison of glyceryl trinitrate-induced with pentaerythrityl tetranitrate-induced in vivo formation of superoxide radicals: effect of vitamin C.

Glyceryl trinitrate (GTN) and pentaerythrityl tetranitrate (PETN) are among the most known organic nitrates that are used in cardiovascular therapy as vasodilators. However, anti-ischemic therapy with organic nitrates is complicated by the induction of nitrate tolerance. When nitrates are metabolized to release nitric oxide (NO), there is considerable coproduction of superoxide radicals in vessels leading to inactivation of NO. However, nitrate-induced increase of superoxide radical formation in vivo has not been reported. In this work, the authors studied the in vivo formation of superoxide radicals induced by treatment with PETN or GTN and determined the antioxidant effect of vitamin C. The formation of superoxide radicals was determined by the oxidation of 1-hydroxy-3-carboxy-pyrrolidine (CP-H) to paramagnetic 3-carboxy-proxyl (CP) using electron spin resonance spectroscopy. CP-H (9 mg/kg intravenous bolus and 0.225 mg/kg per minute continuous intravenous GTN or PETN 130 microg/kg) were infused into anesthetized rabbits. Every 5 min, blood samples were obtained from Arteria carotis to measure the CP formation. Both PETN and GTN showed similar vasodilator effects. Formation of CP in blood after infusions of GTN and PETN were 2.0+/-0.4 microM and 0.98+/-0.23 microM, respectively. Pretreatment with 30 mg/kg vitamin C led to a significant decrease in CP formation: 0.27+/-0.14 microM (vitamin C plus GTN) and 0.34+/-0.15 microM (vitamin C plus PETN). Pretreatment of animals with superoxide dismutase (15,000 units/kg) significantly inhibited nitrate-induced nitroxide formation. Therefore, in vivo infusion of GTN or PETN in rabbits increased the formation of superoxide radicals in the vasculature. PETN provoked a minimal stimulation of superoxide radical formation without simultaneous development of nitrate tolerance. The data suggest that the formation of superoxide radicals induced by organic nitrate correlates with the development of nitrate tolerance. The effect of vitamin C on CP formation leads to the conclusion that vitamin C can be used as an effective antioxidant for protection against nitrate-induced superoxide radical formation in vivo.

Amyloid beta peptides do not form peptide-derived free radicals spontaneously, but can enhance metal-catalyzed oxidation of hydroxylamines to nitroxides.

Amyloid beta (Abeta) peptides play an important role in the pathogenesis of Alzheimer's disease. Free radical generation by Abeta peptides was suggested to be a key mechanism of their neurotoxicity. Reports that neurotoxic free radicals derived from Abeta-(1-40) and Abeta-(25-35) peptides react with the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) to form a PBN/.Abeta peptide radical adduct with a specific triplet ESR signal assert that the peptide itself was the source of free radicals. We now report that three Abeta peptides, Abeta-(1-40), Abeta-(25-35), and Abeta-(40-1), do not yield radical adducts with PBN from the Oklahoma Medical Research Foundation (OMRF). In contrast to OMRF PBN, incubation of Sigma PBN in phosphate buffer without Abeta peptides produced a three-line ESR spectrum. It was shown that this nitroxide is di-tert-butylnitroxide and is formed in the Sigma PBN solution as a result of transition metal-catalyzed auto-oxidation of the respective hydroxylamine present as an impurity in the Sigma PBN. Under some conditions, incubation of PBN from Sigma with Abeta-(1-40) or Abeta-(25-35) can stimulate the formation of di-tert-butylnitroxide. It was shown that Abeta peptides enhanced oxidation of cyclic hydroxylamine 1-hydroxy-4-oxo-2,2,6, 6-tetramethylpiperidine (TEMPONE-H), which was strongly inhibited by the treatment of phosphate buffer with Chelex-100. It was shown that ferric and cupric ions are effective oxidants of TEMPONE-H. The data obtained allow us to conclude that under some conditions toxic Abeta peptides Abeta-(1-40) and Abeta-(25-35) enhance metal-catalyzed oxidation of hydroxylamine derivatives, but do not spontaneously form peptide-derived free radicals.

Superoxide radical scavenging by phenolic bronchodilators under aprotic and aqueous conditions.

Asthmatic airway disease is accompanied by the appearance of inflammatory cells which produce reactive oxygen species (ROS). Therefore, the radical scavenging properties of the bronchodilators reproterol, fenoterol, salbutamol and terbutaline toward superoxide anion radicals and hydroperoxyl radicals were investigated in a model system by electron paramagnetic resonance spectroscopy (EPR) and photometric approaches. The substances under study showed activity in superoxide radical scavenging under aprotic and protic conditions as well. The efficiency of the reaction decreased in the order: fenoterol > salbutamol > reproterol > terbutaline > oxyfedrine when DMSO was used as an aprotic solvent. In an aqueous system, the rate constants decreased in the order: fenoterol > reproterol > salbutamol. It is suggested that the antioxidant effect of these beta2-agonists is an additional advantage in treatment of asthmatic lung disease, reducing the negative consequences of airway inflammation.

Formation of reactive oxygen species by pentaerithrityltetranitrate and glyceryl trinitrate in vitro and development of nitrate tolerance.

Anti-ischemic therapy with organic nitrates is complicated by tolerance. Induction of tolerance is incompletely understood and likely multifactorial. Recently, increased production of reactive oxygen species (ROS) has been investigated, but it has not been clear if this is a direct consequence of the organic nitrate on the vessel or an in vivo adaptation to the drugs. To examine the possibility that nitrates could directly stimulate vascular ROS production, we compared the development of nitrate tolerance with the formation of ROS induced by pentaerithrityltetranitrate (PETN) or nitroglycerin (GTN) in vitro in porcine smooth muscle cells, endothelial cells, washed ex vivo platelets and whole blood. By examining cGMP formation, it was found that 24-hr treatment with GTN but not PETN induced significant nitrate tolerance, which was prevented by parallel treatment with Vit C. Incubation of vascular cells acutely with 0.5 mM GTN doubled the rate of ROS generation, whereas PETN had no such effect. The rate of ROS (peroxynitrite and O2) formation detected by specific spin traps in tolerant smooth muscle cells, treated for 24 hr with 0.01 mM GTN, was substantially higher (30.5 nM/min) than in control cells acutely treated with 0.5 mM GTN (25 nM/min). In contrast to PETN, GTN induces nitrate tolerance and also increases the formation of ROS both in vascular cells and in whole blood. ROS formation is minimally stimulated by PETN comparable to data obtained in Vit C-suppressed GTN tolerance. ROS formation induced by organic nitrates seems to be a key factor in the development of nitrate tolerance.

Oxygen radical generation and enzymatic properties of mitochondria in hypoxia/reoxygenation.

The time-dependence of oxygen radical formation and development of enzymatic dysfunction after hypoxia/reoxygenation was investigated in isolated rat liver mitochondria. Generation of oxygen radicals was studied by electron paramagnetic resonance (EPR) spectroscopy using the spin trap DMPO (5,5-dimethyl-l-pyrroline-N-oxide). The spin adduct DMPO-OH was found to be formed from the primarily generated adduct of DMPO with the superoxide anion radical (DMPO-OOH). Hypoxic storage followed by reoxygenation at room temperature resulted in an increased decay rate of the DMPO-OH spin adduct while its steady state concentration remained unchanged. This finding strongly suggests an increased rate of DMPO-OH formation which originally derived from enhanced superoxide anion radical production due to hypoxia/reoxygenation. The enhanced superoxide radical formation seems to be due to dysfunction of respiratory chain enzymes, resulting in increased levels of reductive components. In agreement with that, we found the decrease of respiration control and ATP synthesis activity at a similar time scale as that for DMPO-OH adduct formation. The increase of superoxide radical formation and of the reductive capacity of mitochondria was accompanied by a decrease in membrane order at the polar interface. Oxidative phosphorylation was completely abolished after 30 min of hypoxic storage, whereas ATP synthesis decreased significantly after 15 min of hypoxia.

Detection of superoxide radicals and peroxynitrite by 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine: quantification of extracellular superoxide radicals formation.

The reactions of the new sterically hindered hydroxylamine 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PP-H) with superoxide radical and peroxynitrite have been studied. These reactions produce the nitroxide 4-phosphonooxy-2,2,6, 6-tetramethyl-piperidinyloxy. The rate constant for reaction of superoxide with PP-H is determined as (8.4+/-0.6).10(2) M-1s-1. It was found that PP-H provides almost the same spin trapping efficacy as 1-hydroxy-3-carboxy-pyrrolidine (CP-H). The background oxidation of PP-H in blood is much less than for CP-H. The extremely slow PP-H penetration into the cells makes possible the study of extracellular formation of superoxide radical. The acute treatment of blood with nitroglycerin is shown to induce an extracellular superoxide radical formation. PP-H is more sensitive for detection of reactive oxygen species as compared with CP-H. PP-H is an effective scavenger of superoxide radical and of peroxynitrite, and can be used to quantify the extracellular formation of these reactive oxygen species.

Formation of Reactive Oxygen Species in Various Vascular Cells During Glyceryltrinitrate Metabolism.

BACKGROUND: Anti-ischemic therapy with organic nitrates as nitric oxide (NO) donors is complicated by the induction of tolerance. When nitrates are metabolized to release NO, there is a considerable coproduction of reactive oxygen species (superoxide radical and peroxynitrite) in vessels leading to inactivation of NO, to diminished cyclic quanosine monophosphate production in smooth muscle cells (SMC), to impaired vasomotor responses to the endothelium-derived relaxation factor (EDRF), and to formation of nitrotyrosine as a marker of glyceryltrinitrate (GTN)-induced formation of peroxynitrite. The aim of the study was to analyze in vitro the formation of superoxide radicals and of peroxynitrite in GTN-treated endothelial and smooth muscle cells and in washed ex vivo platelets using electron spin resonance and spin-trapping techniques. METHODS AND RESULTS: Using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap, it was shown that in platelets, smooth muscle, and endothelial cells incubated acutely for 15 minutes with 0.5 mM GTN, the rate of generation of reactive oxygen species (ROS) was twice as high as under control conditions. Using the new spin-trap 2H-imidazole-1-oxide (TMIO), a GTN-induced peroxynitrite formation was detected in SMC and in platelets incubated with 0.5 mM GTN for 15 minutes. Spin-trap 1-hydroxy-3-carboxy-pyrrolidine (CP-H) was used to estimate the rate of ROS formation in platelets incubated for 15 minutes with 0.5 mM GTN; the rate amounted to 14.6 +/- 1.1 nM/min/mg protein compared with 4.0 +/- 0.4 nM/min/mg protein in controls. The rate of ROS formation in SMCs was substantially increased (240 +/- 16%) after initiation of GTN tolerance by treatment of the cells in culture with 100 µM GTN for 24 hours. CONCLUSIONS: GTN increases the formation of superoxide radicals in endothelial cells, SMCs, and platelets. Peroxynitrite is formed during GTN metabolism in vascular cells and may contribute to the development of tolerance. A decrease in the nitrate-induced inhibition of platelet aggregation during GTN tolerance is associated with oxidative actions of ROS formed in platelets during GTN metabolism.

Spin trapping study of antioxidant properties of neopterin and 7,8-dihydroneopterin.

Previously, it has been shown that pteridine derivatives are capable of modulating the action of free radicals and both prooxidant and antioxidant properties have been described. However, the mechanism of manifestation of these properties is still unclear. We studied the radical scavenging properties of 7,8-dihydroneopterin and neopterin using the spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). It was found that dihydroneopterin acts generally as a radical scavenger. In the presence of dihydroneopterin the ESR signal was reduced by 30 to 90% compared to the control signal. The rate constants for the reactions of 7,8-dihydroneopterin with superoxide (10(3) M(-1) s(-1)) and peroxyl radicals (10(7) M(-1) s(-1)) were determined. Neopterin in contrast showed no reduction of the ESR signal except with superoxide radicals produced by xanthine oxidase. However, this effect was shown to be due to an inhibition of enzyme rather than to radical scavenging. Our results provide a basis for understanding previous observations of radical scavenger activity of 7,8-dihydroneopterin.

Spin trapping of superoxide radicals and peroxynitrite by 1-hydroxy-3-carboxy-pyrrolidine and 1-hydroxy-2,2,6, 6-tetramethyl-4-oxo-piperidine and the stability of corresponding nitroxyl radicals towards biological reductants.

The reactions of new spin trap 1-hydroxy-3-carboxy-pyrrolidine (CP-H) with superoxide radicals and peroxynitrite were studied. The rate constants were determined as 3.2 x 10(3) and 4.5 x 10(9) M-1s-1, respectively. It was found that 2mM of spin trap CP-H or 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine (TEMPONE-H) provide almost the same spin trapping efficacy. In contrast to TEMPONE-H the reaction of CP-H with peroxynitrite was inhibited by 20 mM DMSO. This simplifies the quantification of peroxynitrite formation. During the reaction of CP-H and TEMPONE-H with superoxide radicals or peroxynitrite the stable nitroxide radicals 3-carboxy-proxyl (CP) and 2,2,6,6-tetramethyl-4-oxo-piperidinoxyl (TEMPONE) are formed. It was found that the rate of reduction of CP by glutathione or by smooth muscle cells was two-fold slower and the reduction of CP by ascorbate was 66-fold slower than corresponding rates of reduction of TEMPONE. Therefore quantification of the formation of superoxide radicals and of peroxynitrite by CP-H is much less hindered by a variety of biological reductants than in case of TEMPONE-H. Thus, CP-H is more suitable for spin trapping of superoxide radicals and peroxynitrite in biological systems than the TEMPONE-H.

Synthesis and spin trapping applications of 2,2-dimethyl-d6-4-methyl-2H-imidazole-1-oxide-1-15N.

A new spin trap, 2,2-dimethyl-d6-4-methyl-2H-imidazole-1-oxide-1-15N (lTMIO), was synthesized and characterized. Hyperfine splitting (HFS) constants of spin adduct ESR spectra of this compound with oxygen-centered, carbon-centered, thiyl and sulfite-derived radicals were determined and compared with the data of the unsubstituted compound. The increase in ESR spectral intensity and the accompanying decrease of the spectral linewidth result in resolution of the HFS due to interaction with alpha-protons of alkyl radicals trapped by lTMIO. Trapping of the formate radical in deoxygenated aqueous solution revealed a very low spectral linewidth (delta Bpp = 0.028 mT) of the corresponding adduct. A strong dependence of the ESR spectra on pH was observed when the autoxidation product of sulfite, SO3-, was trapped. The pKa was found to be 5.8 +/- 0.3. In comparison to other nitrones, application of this spin trap provides more detailed information on the structure of the species trapped, especially for carbon-centered radicals.

Quantification of peroxynitrite, superoxide, and peroxyl radicals by a new spin trap hydroxylamine 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine.

The reactions of hydroxylamine 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine hydrochloride (TEMPONE-H) with peroxynitrite, superoxide and peroxyl radicals were studied. It was shown that under these reactions TEMPONE-H is oxidized into a stable nitroxide 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidi-noxyl (TEMPONE). The reactivity of TEMPONE-H towards reactive oxygen species was compared with the spin traps DMPO and TMIO as well as with DMSO and SOD. The rate constants of reactions of TEMPONE-H with peroxynitrite and superoxide radicals were 6 x 10(9) M(-1)s(-1) and 1.2x10(4) M(-1)s(-1), respectively. Using TEMPONE-H the sensitivity in the detection of peroxynitrite or superoxide radical was about 10-fold higher than using the spin traps DMPO or TMIO. Thus, TEMPONE-H may be used as a spin trap in chemical and biological systems to quantify peroxynitrite and superoxide radical formation.

Quantification of superoxide radicals and peroxynitrite in vascular cells using oxidation of sterically hindered hydroxylamines and electron spin resonance.

The reactions of two hydroxylamines, 1-hydroxy-3-carboxy-pyrrolidine (CP-H) and 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine (TEMPONE-H), with superoxide radicals and peroxynitrite were studied. In these reactions corresponding stable nitroxyl radicals 3-carboxy-proxyl (CP) and 1-hydroxy-2,2,6,6-tetramethyl-4-oxopiperidinoxyl (TEMPONE) are formed and the amount of them can be quantified by electron spin resonance (ESR). It was found that CP-H and TEMPONE-H provide almost the same efficacy in assaying peroxynitrite by ESR in vitro at pH 7.4. The formation of superoxide radicals in suspensions of cells was discriminated from that of peroxynitrite using superoxide dismutase or dimethyl sulfoxide as competitive reagents. The stability of the radicals CP and TEMPONE in the presence of ascorbate or thiols was studied in vitro. The reduction rate of CP by ascorbate was 66-fold slower than the rate of reduction of TEMPONE. Therefore, the quantification of the formation of superoxide radicals and of peroxynitrite is much less affected by ascorbic acid when CP-H, but not TEMPONE-H, is used. Both TEMPONE-H and CP-H were used to determine the formation rates of superoxide radicals and peroxynitrite in suspensions of cultured aortic smooth muscle cells and endothelial cells, in washed ex vivo platelets, and in blood treated with glycerol trinitrate (GTN) as an NO donor. It was shown that both the acute addition of GTN (0.5 mM) to vascular cells and the incubation of smooth muscle or endothelial cells in culture with 0.1 mM GTN for 24 h enhance significantly the formation of reactive oxygen species in cells. The rates of of superoxide radical formation were increased at least in two times and peroxynitrite was detected. Hydroxylamines TEMPONE-H and CP-H can be used as nontoxic compounds in ESR assay capable of quantifying the formation of superoxide radicals and peroxynitrite in suspensions of cells and in the whole blood with high sensitivity.

Determination of rate constants of the reactions of thiols with superoxide radical by electron paramagnetic resonance: critical remarks on spectrophotometric approaches.

Two new EPR approaches were developed for determination of rate constants of reaction glutathione (GSH), N-(2-mercaptopropionyl) glycine (MPG), dihydrolipoic acid (BNL), and tetranor-dihydrolipoic acid (TNL) with superoxide radical. In both cases the competition between thiols and spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) for superoxide radical was used. In the first method the dependence of amplitude of EPR spectrum of DMPO-OOH spin adduct on concentration of thiols in a superoxide-generating system was studied. In the second approach the changes in reduced thiol concentration due to reaction with superoxide radical were measured by nitroxide biradical containing disulfide bond. Observed rate constants were the following: GSH, 1.8 x 10(5) M-1s-1; MPG, 2.2 x 10(5) M-1s-1; TNL, 1.2 x 10(5) M-1s-1; BNL, 2.5 x 10(5) M-1s-1; DHL, 4.8 x 10(5) M-1s-1. The determination of the rate constants of reaction of superoxide radical with thiols by spectrophotometrical cytochrome C assay could result in an underestimation of the values due to the reduction of cytochrome C by thiols. Use of epinephrine for this purpose could lead to an overestimation of experimental rate constants because the adrenochrome formed in the reaction of epinephrine with superoxide radical reacts with thiols.

Spin trapping of O-, C-, and S-centered radicals and peroxynitrite by 2H-imidazole-1-oxides.

Spin trapping with 2H-imidazole-1-oxides (2,2,4-trimethyl-2H-imidazole-1-oxide (TMIO); 4-carboxy-2,2-dimethyl-2H-imidazole-1-oxide, potassium salt (CDMIO); 2,2-dimethyl-4-phenyl-2H-imidazole-1-oxide (DMPIO)) was studied. It was found that these compounds form spin adducts with different short lived free radicals, such as OH, CH3, CH2OH, CH3(CH)OH, C(CH3)3O, HOCH2CH2S, glutathionyl and cysteinyl radicals. 2H-imidazole-1-oxides did not trap superoxide radical. Therefore, the detection of OH and thiol radicals is not complicated by the presence of superoxide radical. It was shown that 2H-imidazole-1-oxides are effective scavengers of peroxynitrite forming the same spin adducts as with OH-radicals. Thus, TMIO, CDMIO and DMPIO can be used for spin trapping studies in chemical and biological systems.