Spin Trapping Of Radicals Research Articles

Use of Rapid-Scan EPR to Improve Detection Sensitivity for Spin-Trapped Radicals

The short lifetime of superoxide and the low rates of formation expected in vivo make detection by standard continuous wave (CW) electron paramagnetic resonance (EPR) challenging. The new rapid-scan EPR method offers improved sensitivity for these types of samples. In rapid-scan EPR, the magnetic field is scanned through resonance in a time that is short relative to electron spin relaxation times, and data are processed to obtain the absorption spectrum. To validate the application of rapid-scan EPR to spin trapping, superoxide was generated by the reaction of xanthine oxidase and hypoxanthine with rates of 0.1–6.0 μM/min and trapped with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO). Spin trapping with BMPO to form the BMPO-OOH adduct converts the very short-lived superoxide radical into a more stable spin adduct. There is good agreement between the hyperfine splitting parameters obtained for BMPO-OOH by CW and rapid-scan EPR. For the same signal acquisition time, the signal/noise ratio is >40 times higher for rapid-scan than for CW EPR. Rapid-scan EPR can detect superoxide produced by Enterococcus faecalis at rates that are too low for detection by CW EPR.

Comparative Study of the Physicochemical Properties of Ortho-Substituted Aromatic Nitroso Compounds

C-Nitroso compounds represent a class of chemical species with radical spin trapping capacity. They are widely employed to scavenge nitric oxide (NO) in biological and chemical systems and prospectively could be applied to reduce and control NO formation from important chemical processes. In this study, we examined the physicochemical properties of four ortho-substituted aromatic nitroso compounds, 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS), 4-nitrosobenzenesulfonate (NBS), 3,5-dimethyl-4-nitrosobenzenesulfonate (DMNBS), and 3,5-dichloro-4-nitrosobenzenesulfonate (DCNBS). These compounds, like most C-nitroso spin traps, exist in a monomer–dimer equilibrium, and only the monomeric form is active as a free radical spin scavenger. The influence of the aromatic ring substituent(s) on the dissociation of the dimer to give the monomer was investigated using UV–vis spectrophotometry. We describe the thermodynamic and kinetic parameters associated with the monomer–dimer equilibria, which allow us to provide a mechanistic understanding of NO trapping by C-nitroso compounds.

キノコ熱水抽出物のESRスピントラップ法によるラジカル捕捉活性評価とORAC法

キノコ熱水抽出物のESRスピントラップ法によるラジカル捕捉活性評価とORAC法

Oxidative stress and nitric oxide in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting 5–10% of premenopausal women. In addition to its well-established reproductive effects, PCOS is a metabolic disorder characterised by insulin resistance, hyperandrogenism and dyslipidaemia but the effects of these disturbances on oxidative stress and nitric oxide metabolism are not known. We thus sought to compare measures of oxidative stress and nitric oxide metabolites in patients with PCOS and healthy volunteers (HV). Preliminary data from banked frozen plasma samples obtained from a phenotypically detailed cohort of PCOS patients (age 29.4 ± 6.6 years, BMI 33.2 ± 7.4 kg/m 2 ) and age/BMI-matched HV showed similar nitrite (PCOS [ n = 25] 153 ± 16.57 nmol/l, HV [ n = 17] 187.8 ± 26.59nmol/l, p = NS) but lower nitrate levels in subjects with PCOS (PCOS [ n = 33] 7.3 ± 1.92 μmol/l, HV [ n = 27] 24.54 ± 2.59μmol/l, p < 0.001). The PCOS and control group displayed similar lipid profiles (TC: 4.79 ± 0.1 and 4.5 ± 0.06, HDL: 1.35 ± 0.1 and 1.38 ± 0.05 and LDL: 2.93 ± 0.15 and 2.76 ± 0.15, respectively). Plasma nitrite correlated inversely with LDL concentrations in subjects with PCOS ( r = −0.531, p < 0.05) but not in HVs. There were no significant relationships between plasma nitrate and lipid levels. Inspired by these results, data will be presented at this meeting from a fresh group of subjects in which these parameters are compared in the context of an increased free radical formation (detected as direct spin-trapping of lipid-derived radicals in patient blood) and ORAC status that result from dysfunctional lipid metabolism within PCOS. Abbreviatons: PCOS, polycystic ovary syndrome; HV, healthy volunteers; NS, non-significant; TC, total cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; ORAC, oxygen radical absorbance capacity.

Theoretical and experimental studies of the spin trapping of inorganic radicals by 5,5-dimethyl-1-pyrroline N-oxide (DMPO). 3. Sulfur dioxide, sulfite, and sulfate radical anions.

Radical forms of sulfur dioxide (SO(2)), sulfite (SO(3)(2-)), sulfate (SO(4)(2-)), and their conjugate acids are known to be generated in vivo through various chemical and biochemical pathways. Oxides of sulfur are environmentally pervasive compounds and are associated with a number of health problems. There is growing evidence that their toxicity may be mediated by their radical forms. Electron paramagnetic resonance (EPR) spin trapping using the commonly used spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), has been employed in the detection of SO(3)(•-) and SO(4)(•-). The thermochemistries of SO(2)(•-), SO(3)(•-), SO(4)(•-), and their respective conjugate acids addition to DMPO were predicted using density functional theory (DFT) at the PCM/B3LYP/6-31+G**//B3LYP/6-31G* level. No spin adduct was observed for SO(2)(•-) by EPR, but an S-centered adduct was observed for SO(3)(•-)and an O-centered adduct for SO(4)(•-). Determination of adducts as S- or O-centered was made via comparison based on qualitative trends of experimental hfcc's with theoretical values. The thermodynamics of the nonradical addition of SO(3)(2-) and HSO(3)(-) to DMPO followed by conversion to the corresponding radical adduct via the Forrester-Hepburn mechanism was also calculated. Adduct acidities and decomposition pathways were investigated as well, including an EPR experiment using H(2)(17)O to determine the site of hydrolysis of O-centered adducts. The mode of radical addition to DMPO is predicted to be governed by several factors, including spin population density, and geometries stabilized by hydrogen bonds. The thermodynamic data supports evidence for the radical addition pathway over the nucleophilic addition mechanism.

Characteristics of the spin-trapping reaction of a free radical derived from AAPH: further development of the ORAC-ESR assay

The characteristics of the spin-trapping reaction in the oxygen radical absorbance capacity (ORAC)-electron spin resonance (ESR) assay were examined, focusing on the kind of spin traps. 2,2-Azobis(2-amidinopropane) dihydrochloride (AAPH) was used as a free radical initiator. The spin adducts of the AAPH-derived free radical were assigned as those of the alkoxyl radical, RO· (R=H(2)N(HN)C-C(CH(3))(2)). Among the spin traps tested, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5,5-dimethyl-4-phenyl-1-pyrroline N-oxide (4PDMPO), 5-(2,2-dimethyl-1,3-propoxycyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO), and 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) were applicable to the ORAC-ESR assay. Optimal formation of spin-trapped radical adduct was observed with 1 mM AAPH, 10 mM spin trap, and 5 s UV irradiation. The calibration curve (the Stern-Volmer's plot) for each spin trap showed good linearity, and their slopes, k (SB)/k (ST), were estimated to be 87.7±2.3, 267±15, 228±9, and 213±16 for DMPO, 4PDMPO, CYPMPO, and DEPMPO, respectively. Though the k (SB)/k (ST) values for selected biosubstances varied with various spin traps, their ratios to Trolox (the relative ORAC values) were almost the same for all spin traps tested. The ORAC-ESR assay also had a very good reproducibility. The ORAC-ESR assay was conducted under stoichiometric experimental conditions. The present results demonstrate the superiority of the ORAC-ESR assay.

α-Phenyl-N-tert-butylnitrone (PBN) Prevents Light-induced Degeneration of the Retina by Inhibiting RPE65 Protein Isomerohydrolase Activity

α-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5-12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC(50) = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with K(i) = 53 μm. PBN had no effect on the activity of other visual cycle enzymes, lecithin retinol acyltransferase and retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-α-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.

Spin trapping of hydroxyl radicals on Cu/HY zeolites suspended in aqueous solution

Hydroxyl radical intermediates are trapped in calcined Cu/HY zeolites in the presence of oxygen and water. This suggests that hydrogen peroxide is formed in situ from oxygen. Brønsted acids enhance the formation of the radicals.

CeeTox™ Analysis of CNB-001 a Novel Curcumin-Based Neurotrophic/Neuroprotective Lead Compound to Treat Stroke: Comparison with NXY-059 and Radicut

In the present study, we used a comprehensive cellular toxicity (CeeTox) analysis panel to determine the toxicity profile for CNB-001 [4-((1E)-2-(5-(4-hydroxy-3-methoxystyryl-)-1-phenyl-1H-pyrazoyl-3-yl)vinyl)-2-methoxy-phenol)], which is a hybrid molecule created by combining cyclohexyl bisphenol A, a molecule with neurotrophic activity and curcumin, a spice with neuro-protective activity. CNB-001 is a lead development compound since we have recently shown that CNB-001 has significant preclinical efficacy both in vitro and in vivo. In this study, we compared the CeeTox profile of CNB-001 with two neuroprotective molecules that have been clinically tested for efficacy: the hydrophilic free radical spin trap agent NXY-059 and the hydrophobic free radical scavenger edaravone (Radicut). CeeTox analyses using a rat hepatoma cell line (H4IIE) resulted in estimated C(Tox) value (i.e., sustained concentration expected to produce toxicity in a rat 14-day repeat dose study) of 42 μM for CNB-001 compared with >300 μM for both NXY-059 and Radicut. The CeeTox panel suggests that CNB-001 produces some adverse effects on cellular adenosine triphosphate content, membrane toxicity, glutathione content, and cell mass (or number), but only with high concentrations of the drug. After a 24-h exposure, the drug concentration that produced a half-maximal response (TC(50)) on the measures noted above ranges from 55 to 193 μM. Moreover, all CNB-001-induced changes in the markers were coincident with loss of cell number, prior to acute cell death as measured by membrane integrity, suggesting a cytostatic effect of CNB-001. NXY-059 and Radicut did not have acute toxic effects on H4IIE cells. We also found that CNB-001 resulted in an inhibition of ethoxyresorufin-o-deethylase activity, indicating that the drug may affect cytochrome P4501A activity and that CNB-001 was metabolically unstable using a rat microsome assay system. For CNB-001, an estimated in vitro efficacy/toxicity ratio is 183-643-fold, suggesting that there is a significant therapeutic safety window for CNB-001 and that it should be further developed as a novel neuroprotective agent to treat stroke.

The role of phenolic compounds during formation of turbidity in an aromatic bitter

An aromatic bitter produced from alcoholic extracts of botanicals were studied over 2½ years. The concentrations of gallic acid and the flavonoids catechin, epicatechin, eriodictyol, and taxifolin decreased during the first year of storage in the presence of oxygen, whereas the turbidity stayed below the limit for visible detection. After one year the turbidity increased linearly with time and the total concentration of phenolic compounds decreased. In contrast, the concentrations of eugenol and phenolic acids with two or one hydroxyl groups remained constant or increased, and these compounds are probably not involved in formation of turbidity. The oxidative properties of the phenolic compounds were evaluated in an assay based on iron-catalysed Fenton reactions and spin trapping of radicals. The fresh bitter and bitter stored for one year increased the amount of radicals detected in the assay, demonstrating an overall prooxidative effect of the phenolic compounds. It is concluded that easily oxidised phenolics in the bitter are converted into soluble intermediates that slowly react with proteins and carbohydrates forming haze and deposits.

Characterization of oxidative stress in Leishmaniasis-infected or LPS-stimulated macrophages using electrochemical impedance spectroscopy

The physiological changes caused by external stimuli can be employed as parameters to study pathogen infection in cells and the effect of drugs. Among analytical methods, impedance is potentially useful to give insight into cellular behavior by studying morphological changes, alterations in the physiological state, production of charged or redox species without interfering with in vitro cellular metabolism and labeling. The present work describes the use of electrochemical impedance spectroscopy to simply monitor by modeling impedance plots (Nyquist diagram) in appropriate equivalent circuit, the changes affecting murine macrophage cell line (RAW 264.7) in response to parasite infection by Leishmania amazonensis or to lipopolysaccharide (LPS) treatment. These results demonstrate the ability of electrochemical impedance spectroscopy to discriminate between two opposite cell responses associated to two different stimuli, one caused by the internalization of a parasite, and the other by activation by a bacterium component. Indeed, the study has allowed the characterization, from an electrical point of view, of the extra-cellular NO radical produced endogenously and in great quantities by the inducible form of NO-synthase in the case of LPS-stimulated macrophages. This production was not observed in the case of Leishmania-infected macrophages for which to survive and multiply, the parasite itself possesses mechanisms which may interfere with NO production. In this latest case, only the intracellular production of ROS was observed. To confirm these interpretations confocal microscopy analysis using the ROS (reactive oxygen species) fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate and electron paramagnetic resonance experiments using Fe(DETC) 2 as NO radical spin trap were carried out.

Essential Role of the Redox-Sensitive Kinase p66shcin Determining Energetic and Oxidative Status and Cell Fate in Neuronal Preconditioning

Ischemic preconditioning is a phenomenon in which low-level stressful stimuli upregulate endogenous defensive programs, resulting in subsequent resistance to otherwise lethal injuries. We previously observed that signal transduction systems typically associated with neurodegeneration such as caspase activation are requisite events for the expression of tolerance and induction of HSP70. In this work, we sought to determine the extent and duration of oxidative and energetic dysfunction as well as the role of effector kinases on metabolic function in preconditioned cells. Using an in vitro neuronal culture model, we observed a robust increase in Raf and p66(Shc) activation within 1 h of preconditioning. Total ATP content decreased by 25% 3 h after preconditioning but returned to baseline by 24 h. Use of a free radical spin trap or p66(shc) inhibitor increased ATP content whereas a Raf inhibitor had no effect. Phosphorylated p66(shc) rapidly relocalized to the mitochondria and in the absence of activated p66(shc), autophagic processing increased. The constitutively expressed chaperone HSC70 relocalized to autophagosomes. Preconditioned cells experience significant total oxidative stress measured by F(2)-isoprostanes and neuronal stress evaluated by F(4)-neuroprostane measurement. Neuroprostane levels were enhanced in the presence of Shc inhibitors. Finally, we found that inhibiting either p66(shc) or Raf blocked neuroprotection afforded by preconditioning as well as upregulation of HSP70, suggesting both kinases are critical for preconditioning but function in fundamentally different ways. This is the first work to demonstrate the essential role of p66(shc) in mediating requisite mitochondrial and energetic compensation after preconditioning and suggests a mechanism by which protein and organelle damage mediated by ROS can increase HSP70.

The Assessment of Artifacts Originating from the Non-Radical Forrester-Hepburn Mechanism as a Side Reaction in Sulfite Radical Spin Trapping by DMPO and its 15N Analog

The Assessment of Artifacts Originating from the Non-Radical Forrester-Hepburn Mechanism as a Side Reaction in Sulfite Radical Spin Trapping by DMPO and its 15N Analog

Probing the Antimalarial Mechanism of Artemisinin and OZ277 (Arterolane) with Nonperoxidic Isosteres and Nitroxyl Radicals

Peroxidic antimalarials such as the semisynthetic artemisinins are critically important in the treatment of drug-resistant malaria. Nevertheless, their peroxide bond-dependent mode of action is still not well understood. Using combination experiments with cultured Plasmodium falciparum cells, we investigated the interactions of the nitroxide radical spin trap, 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), and four of its analogs with artemisinin and the ozonide drug development candidate OZ277. The antagonism observed for combinations of artemisinin or OZ277 with the TEMPO analogs supports the hypothesis that the formation of carbon-centered radicals is critical for the activity of these two antimalarial peroxides. The TEMPO analogs showed a trend toward greater antagonism with artemisinin than they did with OZ277, an observation that can be explained by the greater tendency of artemisinin-derived carbon-centered radicals to undergo internal self-quenching reactions, resulting in a lower proportion of radicals available for subsequent chemical reactions such as the alkylation of heme and parasite proteins. In a further mechanistic experiment, we tested both artemisinin and OZ277 in combination with their nonperoxidic analogs. The latter had no effect on the antimalarial activities of the former. These data indicate that the antimalarial properties of peroxides do not derive from reversible interactions with parasite targets.

Direct ESR detection and spin trapping of radicals generated by reaction of oxygen radicals with sulfonated poly(ether ether ketone) (SPEEK) membranes

The stability of membranes under the strong oxidizing conditions in fuel cells is one of the major challenges in the development of fuel cells based on proton exchange membranes (PEMs). This study is centered on the determination of the susceptibility to degradation of SPEEK membranes exposed to OH radicals, using both direct ESR and spin trapping with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). In order to achieve a complete picture on SPEEK degradation, two types of experiments were performed: 1. UV irradiation at 77 K of SPEEK membranes swollen by aqueous solutions of H 2O 2; 2. UV irradiation of SPEEK membranes swollen by aqueous solutions of H 2O 2 in the presence of DMPO as a spin trap. UV irradiation without oxygen of SPEEK at 77 K in acid or basic form in the presence of H 2O 2/H 2O produced phenoxyl radicals as the predominant radicals detected by direct ESR or spin trapping methods. At pH 4, the oxygen radicals produced phenyl radicals as the predominant species detected by spin trapping methods. The hydroperoxyl radical, as DMPO/OOH adduct, was detected only when the DMPO/OH adduct was absent. The appearance of phenyl and phenoxyl radicals provides the evidence that OH radicals react with the aromatic ring of SPEEK or leading to the scission of its ether bridge.

Visualizing Chemical Reactions and Crossover Processes in a Fuel Cell Inserted in the ESR Resonator: Detection by Spin Trapping of Oxygen Radicals, Nafion-Derived Fragments, and Hydrogen and Deuterium Atoms

We present experiments in an in situ fuel cell (FC) inserted in the resonator of the ESR spectrometer that offered the ability to observe separately processes at anode and cathode sides and to monitor the formation of HO and HOO radicals, H and D atoms, and radical fragments derived from the Nafion membrane. The presence of the radicals was determined by spin-trapping electron spin resonance (ESR) with 5,5-dimethylpyrroline N-oxide (DMPO) as a spin trap. The in situ FC was operated at 300 K with a membrane-electrode assembly (MEA) based on Nafion 117 and Pt as catalyst, at closed and open circuit voltage conditions, CCV and OCV, respectively. Experiments with H(2) or D(2) at the anode and O(2) at the cathode were performed. The DMPO/OH adduct was detected only at the cathode for CCV operation, suggesting generation of hydroxyl radicals from H(2)O(2) formed electrochemically via the two-electron reduction of oxygen. The DMPO/OOH adduct, detected in this study for the first time in a FC, appeared at the cathode and anode for OCV operation, and at the cathode after CCV FC operation of >or=2 h. These results were interpreted in terms of electrochemical generation of HOO at the cathode (HO + H(2)O(2) --> H(2)O + HOO) and its chemical generation at the anode from hydrogen atoms and crossover oxygen (H + O(2) --> HOO). DMPO/H and DMPO/D adducts were detected at the anode and cathode sides, for CCV and OCV operation; H and D are aggressive radicals capable of abstracting fluorine from the tertiary carbon in the polymer membrane chain and of leading to chain fragmentation. Carbon-centered radical (CCR) adducts were detected at the cathode after CCV FC operation; weak CCR signals were also detected at the anode. CCRs can originate only from the Nafion membranes, and their presence indicates membrane fragmentation. Taken together, this study has demonstrated that FC operation involves processes such as gas crossover, reactions at the catalyst surface, and possible attack of the membrane by reactive H or D that do not occur in ex situ experiments in the laboratory, thus implying different mechanistic pathways in the two types of experiments.

Light-fluorous TEMPO: reagent, spin trap and stable free radical

The synthesis of two light-fluorous TEMPO derivatives is reported, along with their fluorous-organic solvent partition coefficients and their ESR spectra. Applications of the fluorous-TEMPO reagents in oxidation reactions and as radical traps are discussed.

Oxidative Stress in Developmental Origins of Disease: Teratogenesis, Neurodevelopmental Deficits, and Cancer

In the developing embryo and fetus, endogenous or xenobiotic-enhanced formation of reactive oxygen species (ROS) like hydroxyl radicals may adversely alter development by oxidatively damaging cellular lipids, proteins and DNA, and/or by altering signal transduction. The postnatal consequences may include an array of birth defects (teratogenesis), postnatal functional deficits, and diseases. In animal models, the adverse developmental consequences of in utero exposure to agents like thalidomide, methamphetamine, phenytoin, benzo[a]pyrene, and ionizing radiation can be modulated by altering pathways that control the embryonic ROS balance, including enzymes that bioactivate endogenous substrates and xenobiotics to free radical intermediates, antioxidative enzymes that detoxify ROS, and enzymes that repair oxidative DNA damage. ROS-mediated signaling via Ras, nuclear factor kappa B and related transducers also may contribute to altered development. Embryopathies can be reduced by free radical spin trapping agents and antioxidants, and enhanced by glutathione depletion. Further modulatory approaches to evaluate such mechanisms in vivo and/or in embryo culture have included the use of knockout mice, transgenic knock-ins and mutant deficient mice with altered enzyme activities, as well as antisense oligonucleotides, protein therapy with antioxidative enzymes, dietary depletion of essential cofactors and chemical enzyme inhibitors. In a few cases, measures anticipated to be protective have conversely enhanced the risk of adverse developmental outcomes, indicating the complexity of development and need for caution in testing therapeutic strategies in humans. A better understanding of the developmental effects of ROS may provide insights for risk assessment and the reduction of adverse postnatal consequences.

Influence of changing pulse repetition frequency on chemical and biological effects induced by low-intensity ultrasound in vitro

This study was undertaken to examine ultrasound (US) mechanisms and their impact on chemical and biological effects in vitro as a function of changing pulse repetition frequency (PRF) from 0.5 to 100 Hz using a 1 MHz-generator at low-intensities and 50% duty factor (DF). The presence of inertial cavitation was detected by electron paramagnetic resonance (EPR) spin-trapping of hydroxyl radicals resulting from sonolysis of water. Non-cavitational effects were evaluated by studying the extent of sucrose hydrolysis measured by UV spectrophotometry. Biological effects were assessed by measuring the extent of cell killing and apoptosis induction in U937 cells using Trypan blue dye exclusion test and flow cytometry, respectively. The results indicate significant PRF dependence with respect to hydroxyl radical formation, cell killing and apoptosis induction. The lowest free radical formation and cell killing and the highest cell viability were found at 5 Hz (100 ms pulse duration). On the other hand, no correlation was found between sucrose hydrolysis and PRF. To our knowledge, this is the first report to be devoted to study the impact of low PRFs at low-intensities on US-induced chemical and biological effects and the mechanisms involved. This study has introduced the role of “US streaming” (convection); a forgotten factor in optimization studies, and explored its importance in comparison to standing waves.

Electron Paramagnetic Resonance Spin Trapping of Glutathiyl Radicals by PBN in the Presence of Cyclodextrins and by PBN Attached to β-Cyclodextrin

The reaction of the glutathiyl radical (GS*) with a widely used spin trap N- tert-butyl-alpha-phenylnitrone (PBN) has been studied in the presence of various methylated beta-cyclodextrins and with PBN covalently bound to dimethylated beta-cyclodextrin (PBN-DIMEB) and permethylated beta-cyclodextrin (PBN-TRIMEB). Scavenging rate constants for GS* by PBN were obtained in the presence of randomly methylated cyclodextrin (RAMEB) and PBN-TRIMEB and found to be close to the rate constant previously measured for PBN. RAMEB and 2,6-di- O-Me-beta-cyclodextrin (DIMEB) were found to be the most efficient in the increasing PBN/GS* lifetime, by a factor of 5.5 for RAMEB and 6.8 for DIMEB compared with the lifetime of PBN/GS*. It is concluded that the presence or "attachment of" beta-cyclodextrins does not influence the scavenging rate constant of GS* but it does lead to stabilization of the spin adducts formed.