Free Radicals and Sod Activity of Jaw Cyst. Direct Measurement and Spin Trapping Studies by Esr

Free radicals produced in the fluid of jaw cysts were directly measured at room temperature using ESR. With these samples, SOD activity of the cyst fluid was measured by the ESR spin trapping method with DMPO as a trapping agent. Freeze-dried samples of cyst fluid showed a broad ESR signal at g = 2.005. Relative signal intensity of samples from jaw cysts with inflammation was higher than jaw cysts without inflammation. SOD activity of cyst fluid with high viscosity showed higher values than that of cyst fluid with low viscosity. We suggest that free radicals produced in jaw cyst damage tissues while higher SOD activity of cyst fluid play a role in a self-defense mechanism against free radicals.     

Carnivorous pitcher plant uses free radicals in the digestion of prey

Abstract

A study of the involvement of free oxygen radicals in trapping and digestion of insects by carnivorous plants was the main goal of the present investigation. We showed that the generation of oxygen free radicals by pitcher fluid of Nepenthes is the first step of the digestion process, as seen by EPR spin trapping assay and gel-electrophoresis. The EPR spectrum of N. gracilis fluid in the presence of DMPO spin trap showed the superposition of the hydroxyl radical spin adduct signal and of the ascorbyl radical signal. Catalase addition decreased the generation of hydroxyl radicals showing that hydroxyl radicals are generated from hydrogen peroxide, which can be derived from superoxide radicals. Gel-electrophoresis data showed that myosin, an abundant protein component of insects, can be rapidly broken down by free radicals and protease inhibitors do not inhibit this process. Addition of myoglobin to the pitcher plant fluid decreased the concentration of detectable radicals. Based on these observations, we conclude that oxygen free radicals produced by the pitcher plant aid in the digestion of the insect prey.     

Free Radicals in Rabbit Spinal Cord Ischemia: Electron Spin Resonance Spectroscopy and Correlation with SOD Activity

1. In nonanesthetized rabbits temporal occlusion of the abdominal aorta was used to induce oxidative stress in the lower part of the body including distal segments of the spinal cord.2. Spinal cord samples were taken from the animals exposed to 25-min aortic occlusion (AO ) or to occlusion followed by 1- or 2-hr reperfusion (AO/R1 or AO/R2, respectively) or from sham-operated animals (C). The presence of free radicals (FR) in the spinal cord samples frozen in liquid N₂ was assessed by ESR spectroscopy without spin trapping. Moreover, superoxide dismutase (SOD) activity and conjugated diene (CD) levels were measured in the samples.3. In the AO group FR were detected in the spinal cord regions close to the occlusion (lower thoracic and distal segments) along with a decrease in SOD activity. The calculated g value (g = 2.0291) indicated that the paramagnetic signal recorded might be attributed to superoxide radicals. FR were absent in the AO/R1 group. Concurrently, the SOD activity revealed a significant tendency to return to the control level. FR appeared again in the AO/R2 group, mostly in the upper and middle lumbar regions, along with a decrease in SOD activity. No sample from the C group revealed FR. A significant increase in CD levels was observed in the thoracolumbar region only in the AO/R2 group. The temporary absence of FR in the AO/R1 group suggests activation of defense antioxidant mechanisms (e.g., specific enzymatic systems such as SOD), which might have been exhausted later.4. Changes in SOD activity similar to those observed in the thoracolumbar region, though less noticeable, occurred in the obviously noncompromised tissue (upper cervical region). This points to a kind of generalized reponse of the animal to aortic occlusion.5. Direct ESR spectroscopy revealed the presence of FR as well as their time course in the spinal cord during the early phase of ischemia/reperfusion injury and the inverse relationship between FR and SOD activity.     

ESR spin trapping studies of free radicals generated from nitrofuran derivative analogues of nifurtimox by electrochemical and Trypanosoma cruzi reduction

Electron spin resonance (ESR) spectra of radicals obtained from two analogues of the antiprotozoal drug nifurtimox by electrolytic and Trypanosoma cruzi reduction were analyzed. The electrochemistry of these compounds was studied using cyclic voltammetry. STO 3-21G ab initio and INDO molecular orbital calculations were performed to obtain the optimized geometries and spin distribution, respectively. The antioxidant effect of glutathione on the nitroheterocycle radical was evaluated. DMPO spin trapping was used to investigate the possible formation of free radicals in the trypanosome microsomal system. Nitro1 and Nitro2 nitrofuran analogues showed better antiparasitic activity than nifurtimox. Nitro2 produced oxygen redox cycling in T. cruzi epimastigotes. The ESR signal intensities were consistent with the trapping of either the hydroxyl radical or the Nitro2 analogue radicals. These results are in agreement with the biological observation that Nitro2 showed anti-Chagas activity by an oxidative stress mechanism.     

Carnivorous pitcher plant uses free radicals in the digestion of prey

A study of the involvement of free oxygen radicals in trapping and digestion of insects by carnivorous plants was the main goal of the present investigation. We showed that the generation of oxygen free radicals by pitcher fluid of Nepenthes is the first step of the digestion process, as seen by EPR spin trapping assay and gel-electrophoresis. The EPR spectrum of N. gracilis fluid in the presence of DMPO spin trap showed the superposition of the hydroxyl radical spin adduct signal and of the ascorbyl radical signal. Catalase addition decreased the generation of hydroxyl radicals showing that hydroxyl radicals are generated from hydrogen peroxide, which can be derived from superoxide radicals. Gel-electrophoresis data showed that myosin, an abundant protein component of insects, can be rapidly broken down by free radicals and protease inhibitors do not inhibit this process. Addition of myoglobin to the pitcher plant fluid decreased the concentration of detectable radicals. Based on these observations, we conclude that oxygen free radicals produced by the pitcher plant aid in the digestion of the insect prey.     

Generation and recycling of radicals from phenolic antioxidants

Hindered phenols are widely used food preservatives. Their pharmacological properties are usually attributed to high antioxidant activity due to efficient scavenging of free radicals. Butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) also cause tissue damage. Their toxic effects could be due to the production of phenoxyl radicals. If phenoxyl radicals can be recycled by reductants or electron transport, their potentially harmful side reactions would be minimized. A simple and convenient method to follow phenoxyl radical reactions in liposomes and rat liver microsomes based on an enzymatic (lipoxygenase + linolenic acid) oxidation system was used to generate phenoxyl radicals from BHT and its homologues with substitutents in m- and p-positions. Different BHT-homologues display characteristic ESR signals of their radical species. In a few instances the absence of phenoxyl radical ESR signals was found to be due to inhibition of lipoxygenase by BHT-homologues. In liposome or microsome suspensions addition of ascorbyl palmitate resulted in disappearance of the ESR signal of phenoxyl radicals with concomittant appearance of the ascorbyl radical signal. After exhaustion of ascorbate, the phenoxyl radical signal reappears. Comparison of the rates of ascorbyl radical decay in the presence or absence of BHT-homologues showed that temporary elimination of the phenoxyl radical ESR signal was due to their reduction by ascorbate. Similarly, NADPH or NADH caused temporary elimination of ESR signals as a result of reduction of phenoxyl radicals in microsomes. Since ascorbate and NADPH might generate superoxide in the incubation system used, SOD was tested. SOD shortened the period, during which the phenoxyl radicals ESR signal could not be observed. Both ascorbyl palmitate and NADPH exerted sparing effects on the loss of BHT-homologues during oxidation. These effects were partly diminished by SOD. These data indicate that reduction of phenoxyl radicals was partly superoxide-dependent. It is concluded that redox recycling of phenoxyl radicals can occur by intracellular reductants like ascorbate and microsomal electron transport.     

Endothelial Cells as A Source of Oxygen-Free Radicals An Esr Study

Endothelial cells were subjected to anoxia/reoxygenation in order to simulate some of the free radical mechanisms occurring in ischaemialreperfusion. With ESR and spin trapping using the spin traps 5.5-dimethyl-l-pyrroline-l-oxide (DMPO) and 3,3,5,5-dimethyl-l-pyrroline-l-oxide (M₄PO), the results show that upon reoxygenation of endothelial cells, following a period of anoxia, these cells generate superoxide (0₂). Cytotoxicity of the spin traps was measured by standard trypan blue exclusion methods. Cell injury or death was measured at various times during reoxygenation by lactate dehydrogenase (LDH) release. Experiments using oxypurinol, SOD, CAT and a combination of SOD and CAT show that while oxypurinol partially prevents spin adduct formation. the combination of SOD and CAT is more effective in doing so. These results suggest that the majority of the oxygen radicals produced by endothelial cells are done so exogenously. The results also suggest that endothelial cells are not only a source of oxygen radicals but also a target.     

Spin trapping of free radical species produced during the microsomal metabolism of ethanol

Liver microsomes incubated with a NADPH regenerating system, ethanol and the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) produced an electron spin resonance (ESR) signal which has been assigned to the hydroxyethyl free radical adduct of 4-POBN by using 13C-labelled ethanol. The free radical formation was dependent upon the activity of the microsomal monoxygenase system and increased following chronic feeding of the rats with ethanol. The production of hydroxyethyl free radicals was stimulated by the addition of azide, while catalase and OH. scavengers decreased it. This suggested that hydroxyl radicals (OH.) produced in a Fenton-type reaction from endogenously formed hydrogen peroxide were involved in the free radical activation of ethanol. Consistently, the supplementation of iron, under various forms, also increased the intensity of the ESR signal which, on the contrary, was inhibited by the iron-chelating agent desferrioxamine. Microsomes washed with a solution containing desferrioxamine and incubated in a medium treated with Chelex X-100 in order to remove contaminating iron still produced hydroxyethyl radicals, although at a reduced rate. Under these conditions the free radical formation was apparently independent from the generation of OH. radicals, whereas addition of cytochrome P-450 inhibitors decreased the hydroxyethyl radical formation, suggesting that a cytochrome P-450-mediated process might also be involved in the activation of ethanol. Reduced glutathione (GSH) was found to effectively scavenge the hydroxyethyl radical, preventing its trapping by 4-POBN. The data presented suggest that ethanol-derived radicals could be generated during the microsomal metabolism of alcohol probably through two different pathways. The detection of ethanol free radicals might be relevant in understanding the pathogenesis of the liver lesions which are a consequence of alcohol abuse.     

Changes of the Antioxidant Status and System of Generation of Free Radicals in Hemolymph of Galleria mellonella Larvae at Microsporidiosis

Changes of superoxide dismutase (SOD) and glutathione-S-transferase (GST) activities as well as of the content of SH-containing compounds were revealed in hemolymph of the native and the Vairimorpha ephestiae microsporidian-infected greater wax moth Galleria mellonella larvae. The SOD and GST activities in hemolymph of infected insects decreased at the stage of merogony, whereas during massive sporulation the enzymatic antioxidant activity in host tissues was higher than in control. By the ESR spectroscopy method, using the 1-hydroxy-3-carboxy-pyrrolidinespin-trap, generation of free radicals in hemolymph of infected insects was shown to decrease only at the stage of sporogony. The phenoloxidase activity in lymph was lower at acute microsporidiosis than in native larvae. The hemolymph concentration of thiol-containing proteins in infected insects did not differ from that in control. We suggest that decrease of generation of free radicals in hemolymph of the greater wax moth larvae at the stage of sporogony is due to a suppression of the prophenoloxidase system and an elevation of the antioxidant activity.     

Hydroxyl radical production by H2O2 plus Cu,Zn-superoxide dismutase reflects the activity of free copper released from the oxidatively damaged enzyme.

To elaborate the catalytic activity of Cu2+ of Cu,Zn-superoxide dismutase (SOD) in the generation of hydroxyl radical (.OH) from H2O2, we investigated the mechanism of inactivation of alpha 1-protease inhibitor (alpha 1-PI), mediated by H2O2 and Cu,Zn-SOD. When alpha 1-PI was incubated with 500 units/ml Cu,Zn-SOD and 1.0 mM H2O2, 60% of anti-elastase activity of alpha 1-PI was lost within 90 min. ESR spin trapping using 5,5-dimethyl-1-pyrroline N-oxide showed that free .OH was indeed generated in the reaction of Cu,Zn-SOD/H2O2; this was substantiated by the almost complete eradication of .OH by either ethanol or dimethyl sulfoxide accompanied by the generation of carbon-centered radicals. .OH production and alpha 1-PI inactivation in the H2O2/SOD system became apparent at 30 min or later. Dimethyl sulfoxide and 5,5-dimethyl-1-pyrroline N-oxide protected inactivation of alpha 1-PI significantly in this system, indicating that alpha 1-PI inactivation was mediated by .OH. SOD activity decreased rapidly during the reaction with H2O2 for the initial 30 min. Time-dependent changes in the ESR signal of SOD showed the destruction of ligands for Cu2+ in SOD by H2O2 within this initial period. Thus we conclude that inactivation of alpha 1-PI is mediated in the H2O2/Cu,Zn-SOD system via the generation of .OH by free Cu2+ released from oxidatively damaged SOD.     

维生素E和硒水平对肉鸡不同自由基代谢的影响

选用200羽14日龄健康AA肉鸡,以电子自旋共振(ESR)捕集法和生物化学法对肉仔鸡血液和组织器官的不同自由基进行直接或间接测定,探讨VE和Se对肉鸡不同自由基代谢的作用及其动态变化.结果表明:组织一氧化氮(NO)自由基水平随日粮VE含量升高而降低,二者呈负相关关系,日粮高水平Se有诱导产生NO自由基的倾向;高VE和Se日粮显著提高血清和肝脏中SOD和GSH-Px的活性,但随处理时间的延长,组织SOD活性逐渐降低,而GSH-Px活性逐渐升高,说明日粮VE和/或Se不足均会诱导机体产生O.2-、H2O2自由基,且O2.-自由基会持续大量产生,而H2O2自由基仅在缺乏初期大量产生,而后趋于缓和;低VE和/或低Se均显著提高组织MDA含量,且低Se比低VE更为显著.VE和Se对肉鸡NO、O.2-和H2O2自由基代谢的作用存在协同效应.     

Ischemia and reperfusion of skeletal muscle lead to the appearance of a stable lipid free radical in the circulation

Both ischemia and reperfusion injury and contractile activity are associated with the generation of reactive oxygen species and free radicals by skeletal muscle. In addition, exercise has been reported to lead to the formation of a circulating free radical species that is detectable in the blood by spin trapping before analysis by electron-spin resonance (ESR) techniques. Previous analysis of the ESR signal indicated that the circulating species is either a carbon- or oxygen-centered lipid-derived free radical. The current data indicate that this species is present in the blood of anesthetized rats after 4-h ischemia and 1 h of reperfusion of a single hindlimb. During 4 h of ischemia, the species was also present in microdialysates from the tibialis anterior muscle but was unchanged in magnitude compared with control tissue. During 1 h of reperfusion, the signal intensity increased by a mean of 420% (P < 0.05, n = 4). Hydroxyl radical activity in the interstitial fluid also significantly increased during ischemia and further increased by a mean of 210% (P < 0.05, n = 4) during reperfusion. No changes in interstitial superoxide levels were seen, but interstitial PGE(2) content also increased during reperfusion. A significant positive correlation was found between the magnitude of the ESR signal and both the hydroxyl radical activity and PGE(2) content of microdialysis fluids. These data support the hypothesis that the circulating free radical species is formed in the interstitial fluid by hydroxyl radical interaction with a lipid that may be released from reperfused tissue with a similar pattern to prostanoids.     

Endothelial Cells as A Source of Oxygen-Free Radicals An Esr Study

Endothelial cells were subjected to anoxia/reoxygenation in order to simulate some of the free radical mechanisms occurring in ischaemialreperfusion. With ESR and spin trapping using the spin traps 5.5-dimethyl-l-pyrroline-l-oxide (DMPO) and 3,3,5,5-dimethyl-l-pyrroline-l-oxide (M₄PO), the results show that upon reoxygenation of endothelial cells, following a period of anoxia, these cells generate superoxide (0₂). Cytotoxicity of the spin traps was measured by standard trypan blue exclusion methods. Cell injury or death was measured at various times during reoxygenation by lactate dehydrogenase (LDH) release. Experiments using oxypurinol, SOD, CAT and a combination of SOD and CAT show that while oxypurinol partially prevents spin adduct formation. the combination of SOD and CAT is more effective in doing so. These results suggest that the majority of the oxygen radicals produced by endothelial cells are done so exogenously. The results also suggest that endothelial cells are not only a source of oxygen radicals but also a target.     

An electron spin resonance study on alkylperoxyl radical in thin-sliced renal tissues from ferric nitrilotriacetate-treated rats: the effect of alpha-tocopherol feeding.

Formation of excess free radical causes cellular oxidative stress, which has been shown to be associated with a variety of pathologic conditions. While electron spin resonance (ESR) spectroscopy has been the only method to demonstrate the presence of free radicals, its application to tissue samples has been challenging. We report here the successful ESR detection in thin-sliced fresh tissues or frozen sections in a rat model. Ferric nitrilotriacetate (Fe-NTA) induces oxidative renal tubular damage that ultimately leads to high incidence of renal carcinoma in rodents. Twenty minutes after administration of 5 mg iron/kg Fe-NTA to rats, a thin-slice of the kidney was mounted on a tissue-type cell and analyzed by ESR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). An ESR signal from alkylperoxyl radical adduct was obtained, and the signal was inversely proportional to renal alpha-tocopherol content which was modulated through diet. Furthermore, we undertook ex vivo study using frozen sections. Fe-NTA (1 mM) was added to a rat kidney frozen section for 10 min. After washing the specimen was mounted on a tissue-type cell and analyzed with ESR spin trapping using DMPO. Alkylperoxyl radical signal was dependent on thickness, incubation time and renal tissue levels of alpha-tocopherol, and was reduced by preincubation with catalase or dimethyl sulfoxide but not with alpha-tocopherol outside tissue. This versatile method facilitates identification of free radicals in pathologic conditions, and may be useful for selection of antioxidants.     

The inhibitory effect of extracts of cigarette tar on electron transport of mitochondria and submitochondrial particles.

Acetonitrile extracts of cigarette tar inhibit state 3 and state 4 respiration of intact mitochondria. Exposure of respiring submitochondrial particles to acetonitrile extracts of cigarette tar results in a dose-dependent inhibition of oxygen consumption and reduced nicotinamide adenine dinucleotide (NADH) oxidation. This inhibition was not due to a solvent effect since acetonitrile alone did not alter oxygen consumption or NADH oxidation. Intact mitochondria are less sensitive to extracts of tar than submitochondrial particles. The NADH-ubiquinone (Q) reductase complex is more sensitive to inhibition by tar extract than the succinate-Q reductase and cytochrome complexes. Nicotine or catechol did not inhibit respiration of intact mitochondria. Treatment of submitochondrial particles with cigarette tar results in the formation of hydroxyl radicals, detected by electron spin resonance (ESR) spin trapping. The ESR signal attributable to the hydroxyl radical spin adduct requires the presence of NADH and is completely abolished by catalase and to a lesser extent superoxide dismutase (SOD). Catalase and SOD did not protect the mitochondrial respiratory chain from inhibition by tar extract, indicating that the radicals detected by ESR spin trapping are not responsible for the inhibition of the electron transport. We propose that tar causes at least two effects: (1) Tar components interact with the electron transport chain and inhibit electron flow, and (2) tar components interact with the electron transport chain, ultimately to form hydroxyl radicals.     

Identification of A Free Radical and Oxygen Dependence of Ribonucleotide Reductase in Yeast

Ribonucleotide reductase is a key enzyme for DNA biosynthesis. The enzymes isolated from animal and plant cells possess a stable tyrosyl free radical which is essential for catalysis. Fungal ribonucleotide reductases are little known; the partially characterized enzyme from yeast cells proved exceptionally shortlived, and a free radical could not as yet be demonstrated. We here show that a doublet ESR signal centered at g = 2.0046 can be measured below 60°K in rapidly purified protein samples which is very similar to the ESR spectra of the tyrosine radicals present in other eukaryotic ribonucleotide reductases in structure, microwave saturation, and quenching by hydroxyurea. Because generation of these radicals requires oxygen, anaerobic yeast cultures were also studied. No change in ribonucleotide reductase was observed at 50ppm residual oxygen in the gas phase, but cell proliferation ceased entirely under complete anaerobiosis.     

The involvement of ethanol in the free radical reaction of 6-hydroxydopamine

ESR spin trapping technique was used to detect and analyze free radical formation. When 6-hydroxydomine (6-OHDA) was incubated alone or in the presence of a free radical generating system (H₂O₂ and FeSO₄), hydroxyl free radicals were observed in a concentration-dependent manner. Glutathione was found to be the most effective scavenger of the ESR signal when compared with vitamin E or Mannitol. The addition of ethanol resulted in the formation of the pure hydroxyethyl free radicals. The amount of hydroxyethyl free radicals in the system was dependent upon the concentration of ethanol and the formation of hydroxyethyl free radicals correlated well with the extent of lipid peroxidation and the loss of enzymic activity of the membrane-bound (Na⁺, K⁺)-ATPase. We suggest that in the biological system ethanol may potentiate the neurotoxicity of 6-OHDA with the formation of hydroxyethyl free radicals, which are longer-lived and far more damaging to membranes that the hydroxyl radicals. These data lead us to further hypothesize that the neuronal degeneration caused by 6-OHDA and other compounds that generate free radicals could be potentiated in the presence of ethanol.     

Generation of reactive oxygen species by a sufficient, insufficient and varicose vein wall

Despite numerous theories, the etiology and pathogenesis of primary varicose veins remain unclear. The etiology of chronic venous diseases (CVDs) known as chronic venous insufficiency (CVI) is related to leukocyte trapping. Leukocyte trapping involves trapping of white cells in vessel walls followed by their activation and translocation outside the vessel. Release of reactive oxygen species (ROS) from trapped white cells has been documented. Superoxide dismutase (SOD) directly inhibits the generation of free radicals and compounds that are produced during oxidation by ROS, such as malonyldialdehyde (MDA). The aim of this study was to determine the involvement of free radicals in the etiology of venous changes. The following material was used for the study: fragments of sufficient or insufficient venous system and varices from 31 patients diagnosed with chronic venous disease in the 2nd or 3rd degree, according to clinical state, etiology, anatomy and pathophysiology (CEAP), which were qualified for surgical procedure. The levels of oxidative stress markers strongly correlated with lesions observed by USG in insufficient and varicose veins. In both a higher concentration of MDA was observed, which is a sign of lipid peroxidation. Antioxidative mechanisms, SOD activity and total antioxidative power expressed as FRAP were inversely proportional to MDA concentration. In insufficient and varicose veins both FRAP and SOD activities were significantly lower than in normal veins. The severity of clinical changes was inversely dependent on the efficiency of scavenging of ROS, which additionally proves the participation of free radicals in pathogenesis of CVDs.     

Prediction of viability of porcine neurocysticercosis with proton magnetic resonance spectroscopy: correlation with histopathology

Neurocysticercosis (NCC) is the most frequent parasitic disease of central nervous system. In our earlier study, we had observed creatine [(creatine + phosphocreatine); (tCr)] on ex vivo proton MR spectroscopy (1H MRS) in some of the cysticercus cyst fluid samples obtained from swine's brain parenchyma. In current study, swine brains of freshly slaughtered animals naturally infected with NCC were subjected to ex vivo magnetic resonance (MR) imaging on a 1.5Tesla MR system. Cysticercus cysts (n = 12) were removed from these brains and were labeled depending upon presence or absence of edema around cysts as observed on imaging. Cysticercus cyst fluid (100 microl) was subjected to different 1H MRS experiments and results were compared with histopathological examinations to look for any relationship between tCr and parameters like quantification of musculature, and cellular infiltration in wall of the parasite. Histopathology of cyst wall was categorized into two groups based on cellular characteristics and the amount of musculature. Grade I cysts (n = 5) with no or minimal inflammation and large amount of musculature showed tCr on 1H MRS. However, grade II cysts (n = 7) with profuse inflammation and less amount of musculature in the cyst wall lacked tCr. Higher amount of musculature in grade I cysts was associated with higher concentration of tCr in the cyst fluid (r2 = 0.93, P = 0.007). Creatine appears to be a marker of innocuous and viable NCC.     

ESR measurement of rapid penetration of DMPO and DEPMPO spin traps through lipid bilayer membranes

The passive permeation rates of DMPO and DEPMPO spin traps and their hydroxyl radical adducts through liposomal membranes were measured using ESR spectroscopy. For the spin traps, we measured the time-dependent change in the signal intensity of the OH-adduct, which is formed by a reaction between the penetrated spin trap and hydroxyl radicals produced by the UV-radiolysis of H(2)O(2) inside the liposomes. The hydroxyl radicals produced outside the liposomes were quenched with polyethylene glycol. For the OH-adduct, pre-formed adduct was mixed with liposomes and the time-dependent change of the ESR signal was measured in the presence of a line-broadening reagent outside the liposomes to make the signal outside the liposomes invisible. Both the spin traps and their OH-adducts diffused across the lipid membranes rapidly and reached equilibrium within tens of seconds. These findings suggest that if used for the detection of free radicals inside cells, these spin traps should be well distributed in cells and even in organelles.