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.     

-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.     

Isolated perfused rat hearts release secondary free radicals during ischemia reperfusion injury: Cardiovascular effects of the spin trap α-phenyl N-tert-butylnitrone

Free radicals produced during myocardial post-ischemic reperfusion are aggravating factors for functional disturbances and cellular injury. The aim of our work was to investigate the significance of the secondary free radical release during non ischemic perfusion and post-ischemic reperfusion and to evaluate the cardiovascular effects of the spin trap used. For that purpose, isolated perfused rat hearts underwent 0, 20, 30 or 60 min of a total ischemia, followed by 30 min of reperfusion. The spin trap: α-phenyl N-tert-butylnitrone (PBN) was used (3 mM). Functional parameters were recorded and samples of coronary effluents were collected and analyzed using Electron Paramagnetic Resonance (EPR) to identify and quantify the amount of spin adducts produced. During non ischemic perfusion, almost undetectable levels of free radical release were observed. Conversely, a large and long-lasting (30 min) release of spin adducts was detected from the onset of reperfusion. The free radical species were identified as alkyl and alkoxyl radicals with amounts reaching 40 times the pre-ischemic values. On the other hand, PBN showed a cardioprotective effect, allowing a significant reduction of rhythm disturbances and a better post-ischemic recovery for the hearts which were submitted to 20 min of ischemia. When the duration of ischemia increased, the protective effects of PBN disappeared and toxic effects became more important. Our results have therefore confirmed the antioxidant and protective properties of a spin trap agent such as PBN. Moreover, we demonstrated that the persistent post-ischemic dysfunction was associated with a sustained production and release of free radical species.     

α-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 (α_N = 15.4 and α^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 α-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.     

Use of Spin Traps in Intact Animals Undergoing Myocardial Ischemia/Reperfusion: A New Approach to Assessing the Role of Oxygen Radicals in Myocardial “Stunning”

Numerous studies have indirectly, suggested that oxygen-derived free radicals play an important path-ogenetic role in the prolonged depression of contractile function observed in myocardium reperfused after reversible ischemia (myocardial “stunning”). In order to provide direct evidence for the oxy-radical hypothesis of stunning, we administered the spin trap, α-phenyl N-tert-butyl nitrone (PBN), to open-chest dogs undergoing a 15-min coronary artery occlusion followed by reperfusion. Plasma of local coronary venous blood was analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR signals characteristic of radical adducts of PBN appeared during ischemia and increased dramatically in the first few minutes after reperfusion. After this initial burst, the production of adducts abated but did not cease, persisting up to 3 h after reflow. The production of PBN adducts after reperfusion was inversely related to collateral flow during ischemia. PBN itself enhanced recovery of contractile function. indicating that the radicals trapped may play a pathogenetic role in myocardial stunning. Superoxide dismutase plus catalase attenuated PBN adduct production and, at the same time, improved recovery of contractile function. Antioxidant therapy given 1 min before reperfusion suppressed PBN adduct production and improved contractile recovery; however, the same therapy given 1 min after reperfusion did not suppress early radical production and did not attenuate contractile dysfunction. After i.v. administration, the elimination half-life of PBN was estimated to be approximately 4–5 h. The results demonstrate that 1) free radicals are produced in the stunned myocardium in intact animals; 2) inhibition of free radical production results in improved contractile recovery; and 3) the free radicals important in causing dysfunction are produced in the first few minutes of reperfusion. Taken together, these studies provide cogent evidence supporting the oxy-radical hypothesis of stunning in open-chest dogs. It is now critical to determine whether these results can be reproduced in conscious animal preparations.     

Isolated perfused rat hearts release secondary free radicals during ischemia reperfusion injury: cardiovascular effects of the spin trap alpha-phenyl N-tert-butylnitrone.

Free radicals produced during myocardial post-ischemic reperfusion are aggravating factors for functional disturbances and cellular injury. The aim of our work was to investigate the significance of the secondary free radical release during non ischemic perfusion and post-ischemic reperfusion and to evaluate the cardiovascular effects of the spin trap used. For that purpose, isolated perfused rat hearts underwent 0, 20, 30 or 60 min of a total ischemia, followed by 30 min of reperfusion. The spin trap: alpha-phenyl N-tert-butylnitrone (PBN) was used (3 mM). Functional parameters were recorded and samples of coronary effluents were collected and analyzed using Electron Paramagnetic Resonance (EPR) to identify and quantify the amount of spin adducts produced. During non ischemic perfusion, almost undetectable levels of free radical release were observed. Conversely, a large and long-lasting (30 min) release of spin adducts was detected from the onset of reperfusion. The free radical species were identified as alkyl and alkoxyl radicals with amounts reaching 40 times the pre-ischemic values. On the other hand, PBN showed a cardioprotective effect, allowing a significant reduction of rhythm disturbances and a better post-ischemic recovery for the hearts which were submitted to 20 min of ischemia. When the duration of ischemia increased, the protective effects of PBN disappeared and toxic effects became more important. Our results have therefore confirmed the antioxidant and protective properties of a spin trap agent such as PBN. Moreover, we demonstrated that the persistent post-ischemic dysfunction was associated with a sustained production and release of free radical species.     

高氧预适应对大鼠心肌缺血─再灌注时自由基的影响

为了解决高氧预适应（ＨｙｐｅｒｏｘｉｃｐｒｅｃｏｎｄｉｔｉｏｎｉｎｇＨＯＰ）对大鼠心肌缺血再灌注时自由基的影响,本实验将实验组大鼠放入高压氧舱内,每日吸８０－８５％氧气（１ａｔｍ）６ｈ,连续７ｄ。利用Ｌａｎｇｅｎｄｏｒｆ装置做成心肌缺血再灌注模型,采用电子自旋共振技术测定自由基含量。实验动物随机分为二组,第一组为对照组：缺血１０ｍｉｎ,再灌注６０ｍｉｎ。第二组为ＨＯＰ组：缺血１０ｍｉｎ,再灌注６０ｍｉｎ。实验观察冠脉回流液中自由基ＰＢＮ加合物含量。结果表明：在再灌注过程中,１、５、１０ｍｉｎ３个时间点,ＨＯＰ组ＰＢＮ加合物含量较对照组明显减少。提示：ＨＯＰ能减少缺血再灌注时自由基的产生。     

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.     

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.     

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.     

Detection of lipid radicals by electron paramagnetic resonance spin trapping using intact cells enriched with polyunsaturated fatty acid.

Electron paramagnetic resonance (EPR) spin trapping was used to detect lipid-derived free radicals generated by iron-induced oxidative stress in intact cells. Using the spin trap alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), carbon-centered radical adducts were detected. These lipid-derived free radicals were formed during incubation of ferrous iron with U937 cells that were enriched with docosahexaenoic acid (22:6n-3). The EPR spectra exhibited apparent hyperfine splittings characteristic of a POBN/alkyl radical, aN = 15.63 +/- 0.06 G and aH = 2.66 +/- 0.03 G, generated as a result of beta-scission of alkoxyl radicals. Spin adduct formation depended on the FeSO4 content of the incubation medium and the number of 22:6-enriched cells present; when the cells were enriched with oleic acid (18:1n-9), spin adducts were not detected. This is the first direct demonstration, using EPR, of a lipid-derived radical formed in intact cells in response to oxidant stress.     

Protective effect of a new anti-oxidant on the rat brain exposed to ischemia-reperfusion injury: inhibition of free radical formation and lipid peroxidation.

A new oligomeric derivative was synthesized from prostaglandin B2 and ascorbic acid, and its effect on rat brain ischemia-reperfusion injury was studied. Brain ischemia was produced in the rat by the combination of bilateral common carotid artery occlusion and hemorrhagic hypotension (30 mmHg, 20 min). The cerebral cortex was homogenized in the presence of the spin trap agent, N-tert-butyl-alpha-phenyl-nitrone (PBN). Spin-adducts were detected using an electron spin resonance spectrometer (EPR). Lipid peroxidation was estimated from the amounts of both thiobarbituric acid reactive substances (TBAR) and conjugated diene. In control experiments, reperfusion induced a burst of free radical formation which peaked at 5 min reperfusion time (238 +/- 41%). Lipid peroxidation increased significantly after 20 min of reperfusion (TBAR, 161 +/- 50%; conjugated diene, 160 +/- 29%). When the oligomeric derivative was administered (9 mg/kg i.p. 30 min before ischemic insult), it significantly reduced both spin adduct formation (103 +/- 13%) and lipid peroxidation (TBAR, 109 +/- 14%; conjugated diene, 97 +/- 33%).     

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.     

Involvement of free radicals in cerebral vascular reperfusion injury evaluated in a transient focal cerebral ischemia model of rat

Free radicals have been suggested to be largely involved in the genesis of ischemic brain damage, as shown in the protective effects of alpha-phenyl-N-tert-butyl nitrone (PBN), a spin trapping agent, against ischemic cerebral injury. In the present study, the effects of PBN as well as MCI-186, a newly-developed free radical scavenger, and oxypurinol, an inhibitor of xanthine oxidase, were evaluated in a rat transient middle cerebral aretery (MCA) occlusion model to clarify the possible role of free radicals in the reperfusion injury of brain. The volume of cerebral infarction, induced by 2-h occlusion and subsequent 2-h reperfusion of MCA in Fisher-344 rats, was evaluated. The administration of PBN (100 mg/kg) and MCI-186 (100 mg/kg) just before reperfusion of MCA significantly reduced the infarction volume. In contrast, oxypurinol (100 mg/kg) failed to show any preventive effect on the infarction. These results suggest that free radical formation is involved in the cerebral damage induced by ischemia-reperfusion of MCA, and that hydroxyl radical is responsible for the reperfusion injury after transient focal brain ischemia. It is also suggested that xanthine oxidase is not a major source of free radicals.     

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.     

Acute methanol intoxication generates free radicals in rats: an ESR spin trapping investigation

Electron spin resonance (ESR) spectroscopy has been used to investigate free radical generation in rats with acute methanol poisoning. The spin trapping technique was used where a spin trapping agent, alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), reacted with the corresponding alcohol-derived or alcohol-dependent radical to form radical adducts. One radical adduct was detected in both bile and urine samples 2 h after acute methanol poisoning in male Sprague Dawley rats. The hyperfine coupling constants for the radical adduct from [(13)C]-labeled methanol detected in the bile were a(N) = 15.58, a(beta)(H) = 2.81 G, and a(beta)(13C) = 4.53 G, which unambiguously identified this species as POBN/*CH@OH. The same radical adduct was detected in urine. The identification of a methanol-derived radical adduct in samples from bile and urine provided strong direct evidence for the generation of the alcohol-derived radicals during acute intoxication by methanol. Simultaneous administration of the alcohol dehydrogenase inhibitor 4-methylpyrazole and methanol resulted in an increase in the generation of the free radical metabolite detected in the bile. This is the first ESR evidence of methanol-derived free radical generation in an animal model of acute methanol intoxication.     

Inhibitory Effect of a Hydrophilic α-Tocopherol Analogue,MDL 74, 405, on Generation of Free Radicals in Stunned Myocardium in Dogs

A previous study has demonstrated that the hydrophilic (α-tocopherol analogue, MDL 74, 405, attenuates postischemic myocardial dysfunction (“stunning”) in dogs. The present study was undertaken to determine directly whether the salutary effect of this drug on myocardial stunning results from inhibition of the generation of oxygen-derived free radicals. Open-chest dogs undergoing a 15-min coronary artery occlusion and 3 h of reperfusion received an intravenous infusion of either saline (controls, n = 7) or MDL 74, 405 (n = 6) starting 30 min before coronary occlusion and ending 60 min after reflow at a dose of 0.3 mg/kg/h. To measure free radical production, all dogs received an intravenous infusion of the spin trap α-phenyl N-tert-butyl nitrone (PBN) and local coronary venous plasma was analyzed by electron paramagnetic resonance (EPR). In control dogs, the myocardial production of PBN adducts exhibited an initial burst immediately after the onset of reflow and remained elevated until 10 min after reperfusion. Dogs treated with MDL 74, 405 demonstrated a marked decrease in PBN adduct production. This effect of MDL 74, 405 could not be attributed to nonspecific factors such as differences in ischemic zone size, collateral flow, arterial pressure, heart rate, coronary flow or other hemodynamic variables. These results demonstrate that the hydrophilic vitamin E analogue, MDL 74, 405, inhibits free radical generation after myocardial ischemia-reperfusion in vivo. This finding provides direct evidence that the salutary effects of MDL 74, 405 on myocardial stunning are due to attenuation of oxidative stress.     

New evidence that the Alzheimer beta-amyloid peptide does not spontaneously form free radicals: an ESR study using a series of spin-traps

The direct formation of free radicals from Abeta has been suggested to be a key neurotoxic mechanism in Alzheimer's disease (AD). We have explored the possibility of the spontaneous formation of peptide-derived free radicals during the incubation of Abeta 1-40 by ESR spectroscopy using N-tert-butyl-alpha-phenylnitrone (PBN), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), and 3,5-dibromo-4-nitrosobenzenesulfonic acid sodium salt (DBNBS) as spin traps. Employing PBN, we observed spectra during the incubation of beta-amyloid peptide, at 37 degrees C, which included adducts of 2-methyl-2-nitrosopropane (MNP), despite rigorous purification of the PBN before incubation. The formation of some of these adducts was found to be enhanced by ambient laboratory light. Our experiments have led us to propose a hypothesis that PBN undergoes hydrolysis and decomposition in the presence of oxidants, which explains the origin of all of the PBN and MNP adducts observed (even when the PBN is highly purified). Hydrogen peroxide, formed during incubation, could play a major role as an oxidant in these experiments. Of the other three spin traps, only DMPO gave (very weak) spectra, but these could be assigned to its hydroxyl radical adduct, formed as an artifact by the nucleophilic addition of water to DMPO, catalyzed by trace levels of iron ions. Thus, while spectra are observed during our experiments, none of them can be assigned to adducts of radicals derived from the peptide and, therefore, our data do not support the suggestion that radicals are spontaneously formed from beta-amyloid peptide.     

Detection of Free Radicals by Microdialysis/Spin Trapping Epr Following Focal Cerebral Ischemia-Reperfusion and a Cautionary Note on the Stability of 5,5-Dimethyl-1-Pyrroline N-Oxide (DMPO)

We have examined free radical production in a rat model of focal cerebral ischemia using microdialysis coupled with EPR analysis. A microdialysis probe was inserted 2 mm into the cerebral cortex, supplied by the right middle cerebral artery (MCA), and after a 2-hour washout period with artificial cerebral spinal fluid (ACSF), the perfusate solution was changed to ACSF containing the spin trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). No free radicals were detected by DMPO during the pre-ischemia period. Both common carotid arteries and the right MCA were then ligated for 90 minutes. Microdialysate collected every 15 min during the ischemic period demonstrated predominantly superoxide or peroxyl radical production. After release of the occlusive sutures, hydroxyl radical became apparent initially, then thiyl and carbon centered radicals appeared later in samples collected every 15 min for two hours following cortical reperfusion. Careful studies on the purification and stability of DMPO solution were performed to circumvent artifacts and spurious signals.     

Evidence of in vivo Free Radical Generation by Spin Trapping with α-Phenyl N-Tert-Butyl Nitrone During Ischemia/Reperfusion in Rabbit Kidneys

By using α-phenyl N-tert-butyl nitrone (PBN) as spin trap molecule and the electron paramagnetic resonance (EPR) technique, we obtained the first direct evidence of in vivo intervention of free radicals during an ischemia (50 minutes) reperfusion phenomenon in kidney of an intact rabbit.

An EPR signal (triplet of doublets) characterized by coupling constants a_N = 14.75–15 G and a^H_s = 2.5–3 G was detected in blood samples. The signal was consistent with a nitroxyl-radical adduct resulting from the spin trapping by PBN of either oxygen-or carbon-centered radicals. Control experiments indicated that the EPR signal was not due to a toxic effect of the spin trap molecule.