Bismuth Compounds React With a Nitrone: Lewis Pair Formation and Quantification of Radical Trapping

Tetracycline-HCl-loaded poly( dl-lactide-co-glycolide) microspheres prepared by a spray drying technique: influence of γ-irradiation on radical formation and polymer degradation

Electron spin resonance (ESR) spectroscopy has been used to investigate hydroxyl radical generation in rats with chronic dietary iron loading. A secondary radical spin-trapping technique was used where hydroxyl radical forms methyl radical upon reaction with DMSO. The methyl radical was then detected by ESR spectroscopy as its adduct with the spin trap alpha-phenyl-N-t-butylnitrone (PBN). This adduct was detected in the bile of rats 10 wk after being fed an iron-loading diet and 40 min after the i.p. injection of the spin trap PBN dissolved in DMSO. Bile samples were collected into a solution of the ferrous stabilizing chelator 2,2'-dipyridyl in order to prevent the generation of radical adducts ex vivo during bile collection. Identification of the ESR spectrum of the major radical adduct as that of PBN/.CH3 provides evidence for the generation of the hydroxyl radical during iron supplementation. Desferal completely inhibited in vivo hydroxyl radical generation stimulated by high dietary iron intake. No radical adducts were detected in rats which were fed the control diet for the same period of time. This is the first evidence of hydroxyl radical generation in chronic iron-loaded rats.

Free radical generation in uncooked carrot ( Daucus carota) root tissue after cell disruption – A model for chemical reactions during mastication

The primary free radical species produced in the sonolysis of aqueous argon-saturated solutions (i.e., H, OH, and O2–) and in N2-containing aqueous solutions (H, OH, O2–, NO) can be identified by electron paramagnetic resonance (EPR) combined with spin trapping. The EPR experiments using cadmium(2+) ions as hydrated electron scavengers show that no detectable level of hydrated electrons is formed in the sonolysis of argon-saturated water at neutral pH. The temperature dependence of the semi-classical treatment of the kinetic isotope effect for H and D formation by O–H and O–D bond scission in 1:1 H2O–D2O mixtures was used to estimate the effective temperatures (2000–4000 K using phenyl t-butyl nitrone spin traps) in collapsing cavitation bubbles. At low concentrations of nonvolatile solutes (e.g., acetate ions, amino acids, and sugars) only radicals formed by H-abstraction were observed. At high concentrations, pyrolysis radicals such as methyl radicals from acetate, amino acids, and sugars could also be spin trapped. For volatile organic solutes, the effect of the vapor pressure of the solute on the effective value of gamma (the ratio of Cp/Cv) in the cavitation bubble is the major determinant of radical yields.

Metabolism of acetaldehyde to methyl and acetyl radicals: in vitro and in vivo electron paramagnetic resonance spin-trapping studies

ESR spin trapping investigation of radical formation from the reaction between hematin and tert-butyl hydroperoxide

Patients undergoing percutaneous transluminal coronary angioplasty (PTCA) were investigated for the production of free radicals and cholesterol hydroperoxides during reperfusion. Fifteen patients were studied. Ischaemia during balloon inflation was assessed by serial coronary sinus lactate analysis (mean maximal increase in anterior descending artery dilation was 130%), and by the demonstration of reperfusion hyperaemia (mean increase of coronary sinus oxygen saturation 74%). Free radicals were detected by electron spin resonance (ESR) spin trapping using the spin trap PBN (N-t-Butyl-alpha-phenylnitrone). Radical adducts were detected in up to 50% of samples taken during reperfusion after anterior descending lesion angioplasty. No radicals were detected in control samples or during the ischaemic phase. Radical detection was positively correlated with the change in coronary sinus lactate (p less than 0.025). Coronary sinus cholesterol hydroperoxide analysis did not show a significant increase over control during reperfusion, due in part to unexpectedly high pre angioplasty levels. This study provides clear evidence for the production of a burst of free radicals and evidence for lipid peroxidation in the minutes following myocardial reperfusion during angioplasty. A relationship between the severity of the ischaemic insult and the detection of radical adducts has also been found.

This study evaluates the use of a pyrroline (DEPMPO) and an imidazole (MCPIO) spin trap for the detection of hydroxyl and biomolecule (a peptide and a phospholipid) free radical adducts by Electrospray Ionization Mass Spectrometry (ESI-MS). The hydroxyl and biomolecule free radical adducts were detected using a QTOF and a linear ion trap (LIT) mass spectrometers. In the presence of hydroxyl radical, the mass spectrum obtained for each of the spin traps, DEPMPO and MCPIO, showed the presence of ions that could be attributed to hydroxyl and peroxyl radicals. Further characterisation by tandem mass spectrometry (ESI-MS/MS) revealed also the presence of hydroxy-hydroxyl adducts. Based on the results here described, we show that DEPMPO is a better spin trap for free radicals trapping and detection by mass spectrometry mainly because adducts show increased signal intensity. The ESI-MS spectra obtained for DEPMPO and MCPIO in the presence of biomolecule radicals (peptide and phospholipid) show molecular ions of DEPMPO and MCPIO adducts, which were characterised by tandem mass spectrometry. Both carbon centered radicals and oxygen centered radicals were efficiently trapped by the two spin traps and analysis of QTOF-MS/MS mass spectra allowed the location of the radical position in either the peptide or in the phospholipid fatty acyl chain. However, the tandem mass spectra of MCPIO adducts were more informative than DEPMPO adducts. The LIT-MS/MS spectra only shows typical peptide and phospholipid fragmentation, which difficult the structural characterisation of the spin adduct. In this study, the DEPMPO and MCPIO adducts were identified either in the nitrone or in the hydroxylamine form, which are ESR silent forms. The results described here show that both spin traps coupled with detection by mass spectrometry are valuable tools for trapping radicals of biomolecules. Furthermore, the acquired data provide valuable information on the presence of adducts (hydroxyl and biomolecule) that are Electron Spin Resonance (ESR) silent. This is especially important considering the complexity of the radical species in biological environment and the presence of reducing compounds that convert the spin adducts to silent ESR forms.

Failure to detect hydroxyl radical (.OH)-derived spin adducts of 5,5-dimethyl-1-pyrroline N-oxide in electron spin resonance (ESR) spin trapping experiments has been offered as evidence for the lack of the endogenous capacity of stimulated human phagocytes (neutrophils, monocytes, and monocyte-derived macrophages (MDM] to generate .OH. Recent reports that 5,5-dimethyl-1-pyrroline N-oxide spin adducts are unstable in the presence of superoxide-generating systems such as stimulated neutrophils has raised concerns regarding the sensitivity of spin trapping techniques for assessment of phagocyte free radical formation. Consequently, we have employed a new approach that uses the spin trap N-t-butyl-alpha-phenyl-nitrone (PBN) and dimethyl sulfoxide. In the presence of dimethyl sulfoxide and PBN, the formation of .OH via three different mechanisms in air-saturated aqueous solutions all yielded a single nitroxide species whose ESR peak amplitude remained stable in the presence of superoxide (.O2-). This nitroxide, which we have assigned as PBN/.OCH3, appears to be an oxygen-centered radical derived from the spin trapping of the reaction product of O2 and methyl radical. When neutrophils, monocytes, or MDM were stimulated with phorbol 12-myristate 13-acetate or opsonized zymosan in the presence of exogenous iron, catalase-inhibitable PBN/.OCH3 was the sole nitroxide detected. In the absence of exogenous iron, no nitroxide was observed, providing evidence for the lack of the endogenous capacity of neutrophils, monocytes, and MDM to generate .OH.

9] Spin trapping of hydroxyl radicals in biological systems

The Spin-Trap N-tert-α-Phenylbutylnitrone Prolongs the Life Span of the Senescence-Accelerated Mouse

Reactive nitrogen species (RNS) such as nitrogen dioxide ((•)NO(2)), peroxynitrite (ONOO(-)), and nitrosoperoxycarbonate (ONOOCO(2)(-)) are among the most damaging species present in biological systems due to their ability to cause modification of key biomolecular systems through oxidation, nitrosylation, and nitration. Nitrone spin traps are known to react with free radicals and nonradicals via electrophilic and nucleophilic addition reactions and have been employed as reagents to detect radicals using electron paramagnetic resonance (EPR) spectroscopy and as pharmacological agents against oxidative stress-mediated injury. This study examines the reactivity of cyclic nitrones such as 5,5-dimethylpyrroline N-oxide (DMPO) with (•)NO(2), ONOO(-), ONOOCO(2)(-), SNAP, and SIN-1 using EPR. The thermochemistries of nitrone reactivity with RNS and isotropic hfsc's of the addition products were also calculated at the PCM(water)/B3LYP/6-31+G**//B3LYP/6-31G* level of theory with and without explicit water molecules to rationalize the nature of the observed EPR spectra. Spin trapping of other RNS such as azide ((•)N(3)), nitrogen trioxide ((•)NO(3)), amino ((•)NH(2)) radicals and nitroxyl (HNO) were also theoretically and experimentally investigated by EPR spin trapping and mass spectrometry. This study also shows that other spin traps such as 5-carbamoyl-5-methyl-pyrroline N-oxide, 5-ethoxycarbonyl-5-methyl-pyrroline N-oxide, and 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide can react with radical and nonradical RNS, thus making spin traps suitable probes as well as antioxidants against RNS-mediated oxidative damage.

Detection of the formyl radical by EPR spin-trapping and mass spectrometry.

Using a competitive spin trapping method, relative spin trapping rates were quantified for various short-lived radicals (methyl, ethyl, and phenyl radicals). High static pressure was applied to the competitive spin-trapping system by employing high-pressure electron spin resonance (ESR) equipment. Under high pressure (490 bar), spin trapping rate constants for alkyl and phenyl radicals increased by 10 to 40%, and the increase was dependent on the structure of nitrone spin traps. A maximum increase was obtained when tert-butyl(4-pyridinylmethylene)amine N-oxide (4-POBN) was used as a spin trap. Activation volumes (DeltaDeltaV(double dagger)) for the two spin trapping reactions were calculated to be -17-(-9) cm(3) mol(-1) for the 4-POBN system.

In Vivo ESR Spin Trapping Evidence for Hydroxyl Radical-Mediated Toxicity of Paraquat and Copper in Rats