-phenyl-tert-butyl-nitrone inhibits free radical release in brain concussion

Traumatic brain injury (TBI) is one of the important causes of mortality and morbidity. The pathogenesis of the underlying brain dysfunction is poorly understood. Recent data have suggested that oxygen free radicals play a key role in the primary and secondary processes of acute TBI. We report direct electron spin resonance (ESR) evidence of hydroxyl (·OH) radical generation in closed-head injury of rats. Moderate brain concussion was produced by controlled and reproducible mechanical, fixed, closed-head injury. A cortical cup was placed over one cerebral hemisphere within 20 min of the concussion, perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent pyridyl-N-oxide-tert-butyl nitrone (POBN, 100 mM), and superfusate samples collected at 10 min intervals for a duration up to 130 min post brain trauma. In addition, POBN was administered systematically (50 mg/kg body wt.) 10 min pretrauma and 20 min posttrauma to improve our ability to detect free radicals. ESR analysis of the superfusate samples revealed six line spectra (_N = 15.4 and ^β_H = 2.5 G) characteristic of POBN-OH radical adducts, the intensity of which peaked 40 min posttrauma. The signal was undetectable after 120 min. Administration of -phenyl-tert-butyl-nitrone (PBN), a spin adduct forming agent systemically (100 mg/kg body wt. IP 10 min prior to concussion) alone or along with topical PBN (100 mM PBN in aCSF),6significantly (P< 0.001) attenuated the ESR signal, suggesting its possible role in the treatment of TBI.     

Alpha-Phenyl-Tert-Butyl-Nitrone (PBN) Attenuates Hydroxyl Radical Production During Ischemia-Reperfusion Injury of Rat Brain: An EPR Study

α-phenyl-tert-butyl-nitrone (PBN) a spin adduct forming agent is believed to have a protective action in ischemia-reperfusion injury of brain by forming adducts of oxygen free radicals including ±OH radical. Electron paramagnetic resonance (EPR) has been used to both detect and monitor the time course of oxygen free radical formation in the in vivo rat cerebral cortex. Cortical cups were placed over both cerebral hemispheres of methoxyflurane anesthetized rats prepared for four vessel occlusion-evoked cerebral ischemia. Prior to the onset of sample collection, both cups were perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent α-(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN 100 mM) for 20 min. In addition 50 mg/kg BW of POBN was administered intraperitoneally (IP) 20 min prior to ischemia in order to improve our ability to detect free radical adducts. Cup fluid was subsequently replaced every 15 min during ischemia and every 10 min during reperfusion with fresh POBN containing CSF and the collected cortical superfusates were analyzed for radical adducts by EPR spectroscopy. After a basal 10 min collection, cerebral ischemia was induced for 15 or 30 min (confirmed by EEG flattening) followed by a 90 min reperfusion. -OH radical adducts (characterized by six line EPR spectra) were detected during ischemia and 90 min reperfusion. No adduct was detected in the basal sample or after 90 min of reperfusion. Similar results were obtained when diethylenetriaminepenta-acetic acid (100 μM; DETAPAC) a chelating agent was included in the artificial CSF. Systemic administration of PBN (100 mg/kg BW) produced a significant attenuation of radical adduct during reperfusion. A combination of systemic and topical PBN (100 mM) was required to suppress -OH radical adduct formation during ischemia as well as reperfusion. PBN free radical adducts were detected in EPR spectra of the lipid extracts of PBN treated rat brains subjected to ischemia/reperfusion. Thus this study suggests that PBN's protective action in cerebral ischemia/reperfusion injury is related to its ability to prevent a cascade of free radical generation by forming spin adducts.     

Interaction of tert -butyl Hydroperoxide with Hypochlorous Acid. A Spin Trapping and Chemiluminescence Study

The formation of radical species during the reaction of tert-butyl hydroperoxide and hypochlorous acid has been investigated by spin trapping and chemiluminescence. A superposition of two signals appeared incubating tert-butyl hydroperoxide with hypochlorous acid in the presence of the spin trap &#102 -(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). The first signal (a_N = 1.537mT, a^&#103_H = 0.148mT) was an oxidation product of POBN caused by the action of hypochlorous acid. The second spin adduct (a_N = 1.484mT, a^&#103_H = 0.233mT) was derived from a radical species that was formed in the result of reaction of tert-butyl hydroperoxide with hypochlorous acid. Similarly, a superposition of two signals was also obtained using the spin trap N-tert-butyl- &#102 -phenylnitrone (PBN). tert-Butyl hydroperoxide was also treated with Fe²⁺ or Ce⁴⁺ in the presence of POBN. Using Fe²⁺ a spin adduct with a _N= 1.633mT and a^&#103_H = 0.276mT was observed. The major spin adduct formed with Ce⁴⁺ was characterised by α_N = 1.480mT and a^&#103_H = 0.233mT. The reaction of tert-butyl hydroperoxide with hypochlorous acid was accompanied by a light emission, that time profile and intensity were identical to those emission using Ce⁴⁺. The addition of Fe²⁺ to tert-butyl hydroperoxide yielded a much smaller chemiluminescence. Thus, tert-butyl hydroperoxide yielded in its reaction with hypochlorous acid or Ce⁴⁺ the same spin adduct and the same luminescence profile. Because Ce⁴⁺ is known to oxidise organic hydroperoxides to peroxyl radical species, it can be concluded that a similar reaction takes place in the case of hypochlorous acid.     

Spin Trapping Study in the Lungs and Liver of F344 Rats after Exposure to Ozone

Fischer 344 rats were injected with the spin traps C-phenyl N-tert-butyl nitrone (PBN, 150 mg/kg bw, ip) or 4-pyridine-N-oxide N-tert -butyl nitrone (POBN, 775 mg/kg bw, ip), and exposed to clean air or 2 ppm ozone for two hours. The presence of spin adducts was determined by electron paramagnetic resonance (EPR) spectroscopy of chloroform extracts of lung and liver homogenates. No significant levels of adducts were detected in the lungs of air control animals. Benzoyl N-tert-butyl aminoxyl, attributed to direct reaction of ozone with PBN, and tert-butyl hydroaminoxyl, the scission product of the hydroxyl adduct of PBN, were detected in the lungs of ozone exposed rats. EPR signals for carbon-centred alkoxyl and alkyl adducts were also detected with PBN in the lungs and liver of animals exposed to ozone. With POBN, only carbon-centred alkyl radicals were detected. Senescent, 24 months old rats were found to retain about twice more ¹⁴C-PBN in blood, heart and lungs by comparison to juvenile, 2 months old animals. Accordingly, the EPR signals were generally stronger in the lungs of the senescent rats by comparison to juvenile rats. Together, the observations were consistent with the previously proposed notion that a significant flux of hydrogen peroxide produced from the reaction of ozone with lipids of the extracellular lining, or from activated macrophages in the lungs could be a source of biologically relevant amounts of hydroxyl radical.     

Alpha-Phenyl-Tert-Butyl-Nitrone (PBN) Attenuates Hydroxyl Radical Production During Ischemia-Reperfusion Injury of Rat Brain: An EPR Study

-phenyl-tert-butyl-nitrone (PBN) a spin adduct forming agent is believed to have a protective action in ischemia-reperfusion injury of brain by forming adducts of oxygen free radicals including ±OH radical. Electron paramagnetic resonance (EPR) has been used to both detect and monitor the time course of oxygen free radical formation in the in vivo rat cerebral cortex. Cortical cups were placed over both cerebral hemispheres of methoxyflurane anesthetized rats prepared for four vessel occlusion-evoked cerebral ischemia. Prior to the onset of sample collection, both cups were perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent -(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN 100 mM) for 20 min. In addition 50 mg/kg BW of POBN was administered intraperitoneally (IP) 20 min prior to ischemia in order to improve our ability to detect free radical adducts. Cup fluid was subsequently replaced every 15 min during ischemia and every 10 min during reperfusion with fresh POBN containing CSF and the collected cortical superfusates were analyzed for radical adducts by EPR spectroscopy. After a basal 10 min collection, cerebral ischemia was induced for 15 or 30 min (confirmed by EEG flattening) followed by a 90 min reperfusion. -OH radical adducts (characterized by six line EPR spectra) were detected during ischemia and 90 min reperfusion. No adduct was detected in the basal sample or after 90 min of reperfusion. Similar results were obtained when diethylenetriaminepenta-acetic acid (100 μM; DETAPAC) a chelating agent was included in the artificial CSF. Systemic administration of PBN (100 mg/kg BW) produced a significant attenuation of radical adduct during reperfusion. A combination of systemic and topical PBN (100 mM) was required to suppress -OH radical adduct formation during ischemia as well as reperfusion. PBN free radical adducts were detected in EPR spectra of the lipid extracts of PBN treated rat brains subjected to ischemia/reperfusion. Thus this study suggests that PBN's protective action in cerebral ischemia/reperfusion injury is related to its ability to prevent a cascade of free radical generation by forming spin adducts.     

Formation of radicals during heating lysine and glucose in solution with an intermediate water activity

Abstract

Heating glucose with lysine under alkaline conditions (pH 7.0–10.0) was found to take place with consumption of oxygen together with formation of brown-colored compounds. Highly reactive intermediary radicals were detected when lysine and glucose were heated at intermediate water activity at pH 7.0 and 8.0. The detection was based on initial trapping of highly reactive radicals by ethanol followed by spin trapping of 1-hydroxyethylradicals with α-(4-pyridyl N-oxide)-N-tert-butylnitrone (POBN) and Electron Spin Resonance (ESR) spectroscopy. The generation of reactive intermediary radicals from the Maillard reactions was favored by enhancing alkaline conditions (pH 8.0) and stimulated by presence of the transition metal ion Fe²⁺. The stability of the nitrone spin traps, N-tert-butyl-α-phenylnitrone and POBN was examined in buffered aqueous solutions within the pH range 1–12, and found to be less temperature dependent at acidic pH compared to alkaline conditions. A low rate (k_obs) of hydrolysis of POBN was found at the used experimental conditions of 70°C and pH 7.0 and 8.0, which made this spin trap method suitable for the detection of radicals in the Maillard reaction system.     

Metabolism of Ethanol to 1-Hydroxyethyl Radicals in Rat Liver Microsomes: Comparative Studies with Three Spin Trapping Agents

Metabolism of ethanol to 1-hydroxyethyl radicals by rat liver microsomes was studied with three nitrone spin trapping agents (POBN, PBN, and DMPO) under essentially comparable conditions. The data indicate that POBN was the superior spin trapping agent for 1-hydroxyethyl radicals, and that DMPO was least efficient. Addition of deferoxamine completely prevented detection of 1-hydroxyethyl radicals with PBN or DMPO, but caused only 50% decrease in EPR signals when POBN was the spin trap. However, superoxide dismutase only decreased 1-hydroxyethyl radical formation when POBN was the spin trap. Other experiments demonstrated that POBN was the most effective of these nitrones for reduction of Fe(III) in aqueous solutions. Furthermore, 1-hydroxyethyl radical adducts were formed when POBN was added to mixtures of ethanol, phosphate buffer, POBN and FeCl₃, but this effect did not occur with either PBN or DMPO. Thus, these data indicate that undesirable effects of POBN on iron chemistry may influence results of spin trapping experiments, and complicate interpretation of the resulting data.     

Lack of Protection of Pbn in Isolated Heart During Ischemia and Reperfusion: Implications for Radical Scavenging Mechanism

We evaluated the ability of α-phenyl-tert-butyl nitrone (PBN) to trap free radicals and to protect the rat myocardium during ishcemia and reperfusion. Isolated bicarbonate buffer-perfused hearts (n = 8) were subjected to 20 min global ishcemia (37°C) followed by reperfusion with 0.4 to 4.0 mM PBN. Coronary effluent containing the PBN adduct was extracted in toluene. Electron spin resonance analysis of the toluene extract revealed a PBN-hydroxyl adduct. To verify this assignment, a Fenton system was used to generate an authentic PBN-hydroxyl adduct (n = 8), which yielded the same ESR spectra as the reperfusion-derived adduct. The structure of the adduct formed in the Fenton system was confirmed by gas chromatography-mass spectrometry. The ESR parameters of the PBN-hydroxyl adduct were exquisitely sensitive to solvent polarity during extraction of the adduct. Extraction of an authentic PBN-hydroxyl adduct into chloroform, chloroform:methanol, and toluene closely matched the ESR parameters obtained during reperfusion of ischemic myocardium in other animal models. To determine whether PBN could confer any protective effect during ischemia or reperfusion, hearts (n = 8/group) were subjected to 35 min global ischmia at 37°C with the St. Thomas' II cardioplegic solution followed by 30 min reperfusion. Percent recovery (mean ± SEM) of developed pressure, rate pressure product, and leakage of lactate dehydrogenase during reperfusion in control hearts were 58 ± 3%, 48 ± 4% and 3.2 ± 0.5 IU/15 min/g wet wt. PBN at a concentration of 0.4 mM or 4.0 mM when present either during ischemia alone or reperfusion alone did not exert any effect upon recovery of developed pressure, rate pressure product or post-ischemic enzyme leakage. We conclude that PBN fails to improve contractile recovery and reduce enzyme leakage during reperfusion of myocardium subjected to global ischemia.     

α-2,6-Difluorophenyl-N-Tert-Butylnitrone: A Spin Trap for Distinguishing Different Types of Alkyl Radicals Based on Long-Range Fluorine Hyperfine Splitting

α-2.6-Difluorophenyl-N-tert-butylnitrone (F₂PBN) was synthesized and evaluated. A number of alkyl adducts of F₂PBN were studied by ESR/ENDOR. An additional hyperfine splitting (a triplet of doublets of doublets) is reported. The existence of two (one large, one small) F-hfsc's from the ortho-fluorine atoms in the phenyl ring of most alkyl adducts was confirmed by ENDOR spectroscopy.     

Mass Spectroscopy and Chromatography of the Trichloromethyl Radical Adduct of Phenyl Ter T-Butyl Nitrone

Positive structural identification of the PBN-trichloromethyl spin adduct in vim was accomplished with the use of high pressure liquid chromatography and/or gas chromatography coupled with mass spectrome-try. Both thin layer and liquid chromatography were used to separate a complex mixture of compounds from rat liver extracts treated with CCI, in vitro and in vivo. Deuterated PBN's (PBN-d, text-butyl deuteration, or PBN-d₁₄; both phenyl and tert-butyl deuteration) were also used to aid in the mass spectral analysis of spin adducts from liver extracts of CCI, exposed rat livers, since the rerr-butyl group fragment ion, C₄D₉+ (m/z = 66) is always present for PBN and PBN spin adducts. In addition, the masses of the ion peaks increase by the amount of deuteration, i.e. an increase of 9 for PBN-d, or PBN-d₁₄ in comparison to normally synthesized PBN.     

Comparison of the radical trapping ability of PBN,S-PBN and NXY-059

The nitrones α-phenyl-N-tert-butyl nitrone (PBN), sodium 2-sulfophenyl-N-tert-butyl nitrone (S-PBN) and disodium 2,4-disulfophenyl-N-tert-butyl nitrone (NXY-059) are neuroprotective in a variety of rodent models. The objective of the current studies was to compare the ability of PBN, S-PBN, and NXY-059 to form radical adducts and to prevent salicylate oxidation in an aqueous system. For the electron spin resonance (ESR) studies, hydroxyl radicals were generated with ultraviolet (UV) light and hydrogen peroxide. Secondary radicals were then produced by the addition of methanol, ethanol, isopropanol, dimethylsulfoxide, tetrahydrofuran or 1,4-dioxane. In addition, competition spin trapping studies were performed using PBN-α-¹³C and either S-PBN or NXY-059. In the salicylate studies, PBN, S-PBN and NXY-059 were compared to a variety of other antioxidants and reference compounds (cysteine, glutathione, ascorbate, uric acid, Tempo, Trolox, and Tirilizad) for their ability to prevent 2,3- and 2,5-dihydroxybenzoic acid formation induced by hydroxyl radical generating systems. All 3 nitrones trapped carbon- and oxygen-centered radicals to produce ESR-detectable radical adducts. Each nitrone also prevented salicylate oxidation, with PBN being the most effective. The ability of these 3 nitrones to prevent salicylate oxidation resembled that of most of the other compounds tested.     

Metabolism of Ethanol to 1-Hydroxyethyl Radicals in Rat Liver Microsomes: Comparative Studies with Three Spin Trapping Agents

Metabolism of ethanol to 1-hydroxyethyl radicals by rat liver microsomes was studied with three nitrone spin trapping agents (POBN, PBN, and DMPO) under essentially comparable conditions. The data indicate that POBN was the superior spin trapping agent for 1-hydroxyethyl radicals, and that DMPO was least efficient. Addition of deferoxamine completely prevented detection of 1-hydroxyethyl radicals with PBN or DMPO, but caused only 50% decrease in EPR signals when POBN was the spin trap. However, superoxide dismutase only decreased 1-hydroxyethyl radical formation when POBN was the spin trap. Other experiments demonstrated that POBN was the most effective of these nitrones for reduction of Fe(III) in aqueous solutions. Furthermore, 1-hydroxyethyl radical adducts were formed when POBN was added to mixtures of ethanol, phosphate buffer, POBN and FeCl₃, but this effect did not occur with either PBN or DMPO. Thus, these data indicate that undesirable effects of POBN on iron chemistry may influence results of spin trapping experiments, and complicate interpretation of the resulting data.     

Atypical effect of some spin trapping agents: reversible inhibition of acetylcholinesterase

N-tert-butyl-alpha-phenylnitrone (PBN), a widely used nitrone-based free radical trap was recently shown to prevent acetylcholinesterase (AChE) inhibitors induced muscle fasciculations and brain seizures while being ineffective against glutamergic or cholinergic receptor agonist induced seizures. In the present study we compared the effects on AChE activity of four free radical spin traps PBN, alpha-(4-pyridil-1)-N-tert-butyl nitrone (POBN), N-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The kinetics of AChE inhibition were studied in vitro using a spectrophotometric kinetic assay with AChE from rat brain, diaphragm, electric eel and mouse brain. Spin trapping compounds S-PBN and DEPMPO, in concentrations up to 3 mM did not inhibit hydrolysis of ACh, while PBN and POBN inhibited hydrolysis of ACh in a reversible and concentration-dependent manner. Double reciprocal plots of the reaction velocity against varying ACh concentrations at each inhibitor concentration were linear and generally indicated mixed type inhibition. PBN was the most potent inhibitor of mouse AChE with Ki and Ki' of 0.58 and 2.99 mM, respectively, and the weakest inhibitor of electric eel AChE. In contrast, POBN showed the highest affinity for electric eel enzyme, with Ki and Ki' values of 1.065 and 3.15 mM, respectively. These findings suggest that the effect of PBN and POBN on AChE activity does not depend on trapping of damaging reactive oxygen and that in addition to their antioxidant action other pharmacological effects of these compounds should be considered when neuroprotective actions of PBN or POBN are investigated.     

Combined antibiotic and free radical trap treatment is effective at combating Staphylococcus-aureus-induced septic arthritis

Although early antibiotic treatment of patients with septic arthritis eradicates bacteria, joint destruction commonly results from the unregulated host inflammatory responses to infection. The spin trap compound phenyl-N-tert-butyl nitrone (PBN) has been shown to have both anti-inflammatory and antioxidant effects. The aim of this study was to assess the effect of combined systemic administration of PBN and cloxacillin on the development of Staphylococcus aureus arthritis.     

Roseoflavin Formation by Streptomyces davawensis

We previously found that one of the pharmacological effects of N-tert-butyl-α-phenylnitrone (PBN) is the release of nitric oxide (NO) under oxidative conditions. However, to confirm this hypothesis in vivo, NO released from PBN must be distinguished from NO produced in biological systems, and therefore we undertook the synthesis of PBN using labeled ¹⁵N to identify its corresponding ¹⁵NO in vivo. The properties were examined with an ESR spectrometer. To synthesize ¹⁵N-PBN, the starting material, ammonium-¹⁵N chloride, was converted to 2-amino-¹⁵N-2-methylpropane, oxidized to 2-methyl-2-nitropropane-¹⁵N, and finally reacted with benzaldehyde to give ¹⁵N-PBN. The final product was purified by repeated sublimation. With ferrous sulfate-methyl glucamine dithiocarbamate complex, Fe (MGD)₂, as a trapping agent to measure the NO levels of ¹⁵N-PBN or ¹⁴N-PBN in vitro, the peak intensity of ¹⁵NO[Fe(MGD)₂] was over 50% stronger than that of ¹⁴NO[Fe(MGD)₂], and that ¹⁵NO and ¹⁴NO had the corresponding two-and three line hyperfine structures due to their nuclear spin quantum numbers. Subsequently, the ESR spectrum of ¹⁵NO derived from ¹⁵N-PBN was significantly different than that of lipopolysaccharide (LPS)-induced NO, which was derived from biological cells, and therefore we have demonstrated the possibility to distinguish ¹⁵NO from PBN and ¹⁴NO generated from cells. These results suggested that ¹⁵N-PBN is a useful molecule, not only as a spin-trapping agent, but also as an NO donor to explore the pharmacological mechanisms of PBN in vivo.     

PBN spin trapping of free radicals in the reperfusion-injured heart. Limitations for pharmacological investigations

Post-ischemic reperfusion causes cardiac dysfunction and radical-induced lipid peroxidation (LPO) detectable by ESR spin trapping. This study deals with the applicability of the spin trap technique to pharmacological investigations during myocardial reperfusion injury. The use of the spin trap phenylbutylnitrone (PBN, 3 mM) in isolated rat hearts demonstrated the release of alkoxyl radicals (a_N = 1.39 mT, a_H = 0.19 mT) formed particularly within the first 15 min of reperfusion following 30 min of ischemia. The decline of radicals, after 10 min of reperfusion, was accompanied by recovery of function in 80% of the hearts. The radical concentration in the coronary effluent (maximum after 7.5 min) was reduced by the infusion of 1 mM mercaptopropionylglycine (MPG, 2.7 ± 0.5 U/ml, p < 0.001) or 5 M vitamin E (11.7 ± 0.8 U/ml, p < 0.001), compared to the (PBN-containing) control (29.7 ± 4.3 U/ml). Moreover, functional recovery (left ventricular developed pressure, LVDP 91.6 ± 20% of pre-ischemic level, p < 0.05) was improved by the hydrophilic radical scavenger MPG, compared to the (PBN-containing) control (LVDP 50.5 ± 15.7% of baseline). PBN alone led to higher functional recovery (p < 0.05) and reduced VF (duration of ventricular fibrillation; 7.10 ± 0.36 min/30 min, p < 0.05), compared to the untreated (PBN-free) control (LVDP 26.6 ± 11.8%; VF 19.42 ± 3.64 min/30 min). The Ca antagonist verapamil (0.1 M), MPG, and the lipophilic vitamin E showed cardioprotection in the absence of PBN: post-ischemic recovery of LVDP was 25.4 ± 6.8% (p < 0.05), 39.6 ± 12.7% (p < 0.05) and 52.4 ± 2.6% (p < 0.01), respectively, compared to the corresponding untreated control (13.3 ± 6.6%). Whereas verapamil and vitamin E were able to protect the heart when present alone, they offered no additive effect in the presence of PBN. Therefore, PBN can be used to estimate the radical scavenger properties of an agent in the heart. However, because of the protective properties of PBN itself, the results of simultaneous investigations of the effects of other compounds, such as Ca antagonists or lipophilic radical scavengers, on heart function may be limited.     

The diffusion-solubility of oxygen in lipid bilayers

The product, D_oα, of the oxygen diffusion coefficient, D_o, and the oxygen solubility, α, is determined in phosphatidylcholine bilayers at temperatures above the lipid phase transitions from ESR spin-exchange measurements. The resulting values of D_oα are in good agreement with those obtained from fluorescence-quenching experiments. The use of fatty acid spin labels makes it possible to measure D_oα as a function of the coordinate perpendicular to the bilayer surface. The results indicate that D_oα is a strong function of this coordinate; it is greatest in the bilayer center and least near the bilayer head groups.     

Identification of all classes of spin-trapped carbon-centered radicals in soybean lipoxygenase-dependent lipid peroxidations of omega-6 polyunsaturated fatty acids via LC/ESR,LC/MS,and tandem MS

Using the combined techniques of on-line high performance liquid chromatography/electron spin resonance (LC/ESR) and mass spectrometry (MS), we previously identified spin-trapped adducts of all expected carbon-centered lipid-derived radicals ((*)L(d)) formed in linoleic acid peroxidation. In the present study, spin trapped lipid-derived carbon-centered radicals formed from the reactions of two omega-6 polyunsaturated fatty acids (PUFAs: linoleic and arachidonic acids) with soybean lipoxygenase in the presence of alpha-[4-pyridyl 1-oxide]-N-tert-butyl nitrone (POBN) were identified using a combination of LC/ESR and LC/MS. All expected lipid-derived carbon-centered radicals in lipoxygenase-dependent peroxidations of linoleic acid and arachidonic acid were detected and identified by the combination of LC/ESR and LC/MS with confirmation by tandem mass spectrometry (MS/MS). The five classes of (*)L(d) formed from both omega-6 PUFAs including lipid alkyl radicals (L(*)), epoxyallyic radicals (OL(*)), dihydroxyallyic radicals ((*)L(OH)(2)), and a variety of R(*) and (*)RCOOH from beta-scission of lipid alkoxyl radicals, gave distinct retention times: POBN/(*)L(OH)(2) approximately 4-6 min, POBN/R(*) and POBN/(*)RCOOH approximately 8-22 min, POBN/L(*) and PBON/OL(*) approximately 25-36 min. The major beta-scission products in peroxidations of omega-6 PUFAs were the pentyl radicals. The ratio of beta-scission products, however, varied significantly depending on pH, [PUFA], as well as [O(2)].     

ESR characterization of thallium(III)-mediated nitrones oxidation

We characterized the reactions between Tl (III) and the usual nitrone spin traps (DMPO, POBN, and PBN). The ESR signal obtained with DMPO corresponded to DMPOX which was avoided neither by the enzymes superoxide dismutase or catalase, nor by the hydroxyl radical scavengers’ mannitol, ethanol, or methanol. Only when methanol concentration rose to 90% (v/v), a significant decrease in DMPOX formation was observed while no detectable DMPOX signal was present at 100% methanol solution. POBN rendered an unexpected adduct, while no oxidation products were obtained with PBN. Together, the experimental evidence demonstrates that Tl(III)-supported DMPO oxidation is not mediated by oxygen reactive species. Instead, we propose that Tl(III) interacts with the probe, favoring the addition of water. The intermediate is susceptible to be further oxidized by Tl(III) yielding a keto group. Kinetic studies showed that Tl(III) disappearance rate was higher (1.6 times) than DMPOX formation rate, and equal to Tl(I) formation rate, suggesting a stoichiometry 1.5:1 for Tl(III):DMPOX and 1:1 for Tl(III):Tl(I). The process, an example of the Forrester-Hepburn’s mechanism, was demonstrated for DMPO while the reactions of Tl(III) with POBN and PBN are still under elucidation.     

Effect of high hydrostatic pressure on the formation of radicals in maize starches with different amylose content

Native and high pressure-treated (water suspensions, 650 MPa) waxy maize starch, containing mainly amylopectin, and Hylon VII, rich in amylose, were studied for their ability to generate free radicals upon thermal treatment at 180–230 °C. The electron paramagnetic resonance (EPR) spectroscopy was used to characterize the nature, number and stability of radicals. Various stable and short living (stabilized by N-tert-butyl-α-phenylnitrone (PBN) spin trap) radical species were formed. It was found, that at given conditions the waxy maize starch reveals higher ability to generate radicals, than Hylon VII. The presence of water and high pressure pretreatment of starches, both resulted in the reduction of the amount of thermally generated radicals. The decrease in crystallinity of waxy maize starch and of Hylon VII, occurring upon high pressure treatment, leads to the increase of the relative amount of fast rotating component in the EPR spectrum of both types of starches.