Electron Spin Resonance Spectroscopy Signal Research Articles

Free-Radical Generation from Bulk Nanobubbles in Aqueous Electrolyte Solutions: ESR Spin-Trap Observation of Microbubble-Treated Water.

Microbubbles are very fine bubbles that shrink and collapse underwater within several minutes, leading to the generation of free radicals. Electron spin resonance spectroscopy (ESR) confirmed the generation of hydroxyl radicals under strongly acidic conditions. The drastic environmental change caused by the collapse of the microbubbles may trigger radical generation via the dispersion of the elevated chemical potential that had accumulated around the gas-water interface. The present study also confirmed the generation of ESR signals from the microbubble-treated waters even after several months had elapsed following the dispersion of the microbubbles. Bulk nanobubbles were expected to be the source of the spin-adducts of hydroxyl radicals. Such microbubble stabilization and conversion might be caused by the formation of solid microbubble shells generated by iron ions in the condensed ionic cloud around the microbubble. Therefore, the addition of a strong acid might cause drastic changes in the environment and destroy the stabilized condition. This would restart the collapsing process, leading to hydroxyl radical generation.

Quantitative detection method of semiquinone free radicals on particulate matters using electron spin resonance spectroscopy

Semiquinone free radicals (SFRs) exist significantly in ambient particulate matters and threaten human health due to their genotoxicity and stability compared to other radicals. Efficient quantitative detection methods for SFRs on particles are in great need. In this study, we proposed a quantitative detection method for semiquinone free radicals on particles using electron spin resonance spectroscopy (ESR). We established the relationship between free electron number (which is equal to the number of SFRs as one mole SFRs contain one mol free electron) and corresponding ESR signal intensity, thus SFRs can be quantified by obtaining the ESR signal intensity. The calibration curve was obtained using 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution and has good linearity with r2 of 0.9998. The proposed quantitative detection method reaches repeatability precisions of 2.69% ˜ 4.80% in three concentration levels and recovery ratios of 75.42% ˜ 86.99% for four kinds of SFRs. We successfully used the method to determinate semiquinone free radicals on particles in two northern China cities, Beijing and Jinzhong. The proposed quantitative detection method provides basis for further monitoring of semiquinone free radicals that are existed on ambient particles as well as epidemiologic researches on human exposure risk.

Monosodium glutamate for accidental, retrospective, and medical dosimetry using electron spin resonance.

The risk of a radiation episode has increased in the last years due to several reasons. In case of a nuclear incident, as with the use of an improvised nuclear device, determination of the radiation doses received by the victims is of utmost importance to define the appropriate medical treatment or to monitor the late effects of radiation. Dose assessment in case of accidents can be performed using commonplace materials found in the accident area. In this paper, the dosimetric properties of monosodium glutamate are investigated by electron spin resonance spectroscopy (ESR), for retrospective and accidental dosimetry. The spectroscopic parameters were optimized to achieve higher signal intensity and better signal-to-noise ratio. As a result, the lowest detectable dose was 0.1Gy, and monosodium glutamate showed a linear dose-response curve for doses ranging from 0.1Gy to 10kGy. The dosimetric signal was monitored from minutes right after irradiation, until 1year. No changes in the signal intensity were observed over this period, meaning that doses could be assessed immediately after radiation exposure and can still be reconstructed long after the accident. This property also implies that late effects due to victim's radiation exposure could be better monitored and understood. ESR signal intensity for samples irradiated with a photon energy below 100keV was decreased by only 27% and no dose-rate dependence was noticed. Therefore, the ability to measure doses as low as 0.1Gy, the high stability of the dosimetric signal, as well as independence on dose rate, tissue equivalence, low-cost, and wide commercial availability make monosodium glutamate a very good dosimetric material not only for retrospective and accidental but also for medical dosimetry.

Electron spin resonance spectroscopy for immunoassay using iron oxide nanoparticles as probe.

With the help of iron oxide nanoparticles, electron spin resonance spectroscopy (ESR) was applied to immunoassay. Iron oxide nanoparticles were used as the ESR probe in order to achieve an amplification of the signal resulting from the large amount of Fe3+ ion enclosed in each nanoparticle. Rabbit IgG was used as antigen to test this method. Polyclonal antibody of rabbit IgG was used as antibody to detect the antigen. Iron oxide nanoparticle with a diameter of either 10 or 30nm was labeled to the antibody, and Fe3+ in the nanoparticle was probed for ESR signal. The sepharose beads were used as solid phase to which rabbit IgG was conjugated. The nanoparticle-labeled antibody was first added in the sample containing antigen, and the antigen-conjugated sepharose beads were then added into the sample. The nanoparticle-labeled antibody bound to the antigen on sepharose beads was separated from the sample by centrifugation and measured. We found that the detection ranges of the antigen obtained with nanoparticles of different sizes were different because the amount of antibody on nanoparticles of 10nm was about one order of magnitude higher than that on nanoparticles of 30nm. When 10nm nanoparticle was used as probe, the upper limit of detection was 40.00μgmL-1, and the analytical sensitivity was 1.81μgmL-1. When 30nm nanoparticle was used, the upper limit of detection was 3.00μgmL-1, and the sensitivity was 0.014 and 0.13μgmL-1 depending on the ratio of nanoparticle to antibody. Graphical abstract Schematic diagram of procedure and ESR spectra.

Reactive radical-driven bacterial inactivation by hydrogen-peroxide-enhanced plasma-activated-water

The combined effects of plasma activated water (PAW) and hydrogen peroxide (H2O2), PAW/HP, in sterilization were investigated in this study. To assess the synergistic effects of PAW/HP, S. aureus was selected as the test microorganism to determine the inactivation efficacy. Also, the DNA/RNA and proteins released by the bacterial suspensions under different conditions were examined to confirm membrane integrity. Additionally, the intracellular pH (pHi) of S. aureus was measured in our study. Electron spin resonance spectroscopy (ESR) was employed to identify the presence of radicals. Finally, the oxidation reduction potential (ORP), conductivity and pH were measured. Our results revealed that the inactivation efficacy of PAW/HP is much greater than that of PAW, while increased H2O2 concentration result in higher inactivation potential. More importantly, as compared with PAW, the much stronger intensity ESR signals and higher ORP in PAW/HP suggests that the inactivation mechanism of the synergistic effects of PAW/HP: more reactive oxygen species (ROS) and reactive nitrogen species (RNS), especially OH and NO radicals, are generated in PAW combined with H2O2 resulting in more deaths of the bacteria.

Reversible photochromic effect in the TiO2—polymer hybrid system

The photochromism of poly methyl methacrylate–TiO2 composites have been investigated in this research. Amorphous titanium oxide was synthesized by sol–gel using titanium isopropoxide. The titanium solution was mixed with a dissolved polymer. The composite shows photo-response under UV radiation by changing, generally, to a dark brown tonality. This effect was reversible in different time ranges according to the intensity and exposure time. The mechanism for this photochromic change was proposed as a reduction of TiOOH domains to Ti3+ and not directly from amorphous TiO2, which provide electron-hole pairs after absorbing UV. The backscattered electrons image shows the clusters or domains of titanium compounds as white dots, ranging from 0.1 to 1 μm. The electron spin resonance spectroscopy signals indicated a titanium valence change from Ti(IV) to Ti(III). The X-ray photoelectron spectroscopy peaks positions for Ti 2p1/2 and Ti 2p3/2 were assigned as Ti4+. The values were obtained for Ti 2p3/2 and the identified species were Ti4+ and Ti3+, that correspond to TiO2, Ti2O3, and Ti(OOH). Few and small crystalline domains were identified by Transmission Electron Microscope as orthorhombic TiO2.’

ESR investigation of radicals formed in γ-irradiated vinylidene fluoride based copolymer: P(VDF-co-HFP)

Samples of copolymer based on vinylidene fluoride and hexafluoropropene P(VDF-co-HFP) were exposed to γ-radiation performed under an inert atmosphere and the total amount of radicals was quantified by electron spin resonance spectroscopy (ESR). An in-depth study of recorded ESR spectra allowed the identification of several types of radical species formed during radiolysis. Starting from an ESR simulation model established for irradiated PVDF, seven radical species have been identified in the case of P(VDF-co-HFP): five of them are related to the VDF units while the two others are derived from the HFP unit. The model used to simulate the complex superimposed ESR signals is presented. The proportions of each species are discussed and correlated to the amount of HFP units contained in the copolymer, and to the stability of each species depending on their local environment. Furthermore, the evolution of radical density with radiation dose and the decay resulting from annealing at a given temperature are presented. Corresponding spectral evolution shows the progressive predominance of most stable species.

Electron Spin Resonance Analyses of Grinding- and Radiation-Induced Signals in Raw and Refined Sugars

Irradiated food containing crystalline sugar may be identified using the electron spin resonance spectroscopy (ESR) in accordance with the European standard EN 13708 2001. The method is based upon the detection of sugar radicals induced by irradiation giving a typical dose-dependent spectrum. Grinding, a common industrial process, can also produce ESR signals. In this study, the grinding-induced ESR signals were investigated for their dependence on sugar purity, grinding time, particle size, and grinding method. The results were compared with radiation-induced ESR signals. The mechanical grinding, depending mainly on grinding time, provided clear signals comparable to radiation-induced signals. The specific ESR signal corresponding to sugar radical appeared at 1 min of mechanical grinding which became clearer with the increase in grinding time and sample purity. All sugar samples upon 6 min of mechanical grinding showed comparable results to ≥10 Gy-irradiated samples. Similar results with negligible differences were found using samples of different origins. The results elaborated the detection limits of sugar containing irradiated food based on ESR analysis.

Synthesis and Spectroscopic Characterization of Undoped Nanocrytalline ZnO Particles Prepared by Co-Precipitation

A series of undoped nanocrystalline ZnO particles were successfully synthesized at various dry temperatures (100?C - 600?C) using coprecipitation method. The samples were characterized using a variety of experimental methods such as x-ray diffraction (XRD), energy dispersive x-ray spectroscopy (EDX), thermal analysis TG-DTA, UV-vis spectroscopy, infrared absorption spectroscopy (FTIR) and electron spin resonance spectroscopy (ESR). According to XRD analysis, all of our ZnO samples posses the hexagonal wurzite structure with average crystallite size increased ranging from 19 - 23 nm as dry temperature increased. Optical absorption spectra show that the band gap shifted to the lower energy with increasing crystallite size. ESR measurements showed the resonance of electron centers with the g values of about 1.96. With increasing dry temperature we observed the decrease of the g values and the increase of the intensities of the ESR signal. In addition an increase in dry temperature results in a pronounce decrease of OH local vibrational modes. The results from ESR measurements are well supported by the results obtained from Infrared absorption spectroscopy and thermal analysis measurements.

The reduction of Ag+ in metallic silver on pseudomelanin films allows for antibacterial activity but does not imply unpaired electrons

Dopamine–melanin films produced through the oxidation of dopamine in the presence of oxygen as an oxidant allow to reduce silver ions onto silver particles as already described in the paper by Lee et al. (H. Lee, S.M. Dellatore, W.M. Miller, P.B. Messersmith, Science 318 (2007) 426.). This reduction process has to occur through the oxidation of moieties present in the melanin film. This investigation shows that the free radicals present in the pseudomelanin film, quantified by means of electron spin resonance spectroscopy (ESR) for the first time, are not used in the transformation of Ag+ cations to deposit silver. The ESR signal is hardly affected by the deposition of silver particles. On the other hand, X-ray photoelectron spectroscopy shows a small increase in the density of quinone groups and a small decrease of catechol groups on the surface of the film during the deposition of silver. This suggests that the deposited pseudomelanin films contain a significant fraction of catechol groups able to trigger reduction processes of metallic cations. These silver nanoparticles remain adherent to the melanin films and allow for a quantitative killing of Escherichia coli over a broad range of bacterial dilutions. However, the presence of the bacteria induces a release of the nanoparticles. The pseudomelanin films cannot be reused again for a silver ion reduction step. Nevertheless, the easy preparation of the pseudomelanin–silver composite and its effective one shot bacterial killing activity renders the strategy presented in this paper attractive. Some fundamental questions about redox process allowed by the pseudomelanin films will also be asked.

Identification of γ-ray irradiated medicinal herbs using pulsed photostimulated luminescence, thermoluminescence, and electron spin resonance spectroscopy

Dried herbal samples consisting of root, rhizome, cortex, fruit, peel, flower, spike, ramulus, folium, and whole plant of 20 different medicinal herbs were investigated using pulsed photostimulated luminescence (PPSL), thermoluminescence (TL), and electron spin resonance spectroscopy (ESR) to identify gamma-ray irradiation treatment. Samples were irradiated at 0-50 kGy using a 60Co irradiator. PPSL measurement was applied as a rapid screening method. Control samples of 19 different herbs had photon counts less than the lower threshold value (700 counts 60 s(-1)). The photon counts of non-irradiated clematidis radix and irradiated evodia and gardenia fruits were between the lower and upper threshold values (700-5,000 counts 60 s(-1)). TL ratios, i.e., integrated areas of the first glow (TL1)/the second glow (TL2), were found to be less than 0.1 in all non-irradiated samples and higher than 0.1 in irradiated ones providing definite proof of radiation treatment. ESR spectroscopy was applied as an alternative rapid method. In most of the irradiated samples, mainly radiation-induced cellulosic, sugar, and relatively complicated carbohydrate radical ESR signals were detected. No radiation-specific ESR signal, except one intense singlet, was observed for irradiated scrophularia and scutellaria root and artemisiae argyi folium.

The Suitability of FISH Chromosome Painting and ESR-spectroscopy of Tooth Enamel Assays for Retrospective Dose Reconstruction

A comparative analysis of two groups of highly irradiated victims was carried out in order to evaluate the suitability of two assays for retrospective dose assessment: late translocations and electron spin resonance (ESR) dosimetry. The first group comprised 24 subjects who exhibited acute radiation syndrome (ARS) due to overexposure as a result of nuclear submarine accidents during the period 1961-1985. Their grades of ARS and individual doses were ascertained by Navy physicians who carried out primary examinations and treatment of the exposed seamen. Cytogenetic analyses were made 16-40 y after their accidents. During medical treatment seven tooth samples were collected for ESR analysis from this group. The second group consisted of ten highly irradiated men from the Chernobyl accident. Comparison was made between estimates of their average whole-body penetrating radiation doses derived from several biological parameters. In three cases ESR measurements on tooth enamel from this group were also made. Retrospective dosimetry using FISH translocations was attempted 10-13 y later. Yields of late translocations were in good agreement with initially estimated doses and with doses obtained by ESR spectroscopy analysis of tooth enamel long after exposure. It was concluded that both persisting stable translocations and ESR spectroscopy signals are suitable with similar efficiencies for retrospective biodosimetry after acute whole-body exposure.

The fate of structure-bound Mn2+during the decomposition of dolomite and in the resulting conversion products: An EPR study

Dolomite from the Jhamarkotra phosphate mine (India) contains 680 ppm Mn 2+ as indicated by electron-spin resonance spectroscopy (EPR). Dipolar broadening of the EPR signal prevents a quantitative assignment of Mn 2+ to the Ca and Mg sites. Upon heating to 700 °C, over 99% of the dolomite is decomposed and all the Mn 2+ is released from the dolomite structure. Approximately 95% of the original Mn 2+ is oxidized and forms Mn-oxide. The remaining Mn 2+ preferentially migrates into CaO. This behavior is interpreted by the Goldschmidt rule stating that smaller ions are taken up at the sites of larger ions. During subsequent hydration of CaO into Ca(OH) 2 , the Mn 2+ remains stable. The protection of Mn 2+ against oxidation is explained by the topotaxical alteration of the host minerals. Carbonation of the Ca(OH) 2 leads to Mn 2+ oxidation caused by the dissolution of the hydroxide prior to the formation of CaCO 3 . Divalent Mn monitored by EPR can be used to unravel the behavior of trace elements on the molecular level during mineral alteration

Electron and hole transfer from DNA base radicals to oxidized products of guanine in DNA.

An investigation of electron and hole transfer to oxidized guanine bases in DNA is reported. Guanine in DNA was preferentially oxidized by Br(2)(*-) at 298 K to 8-oxo-7,8-dihydro-guanine (8-oxo-G) and higher oxidation products. HPLC-EC analysis of irradiated DNA shows that the formation of 8-oxo-G could not be increased above the ratio of one 8-oxo-G to 127 +/- 6 bp regardless of dose. 8-oxo-G can be produced only at low levels because it is further oxidized to other species. These oxidation products of guanine have been extensively investigated and identified by others. Our electron spin resonance studies suggest that at 77 K 8-oxo-G is a trap for radiation-produced holes, but certain further oxidation products of 8-oxo-G (G(ox)) are found to be efficient electron traps. Gamma irradiation of oxidized DNA samples in frozen (D(2)O) aqueous ices and glassy 7 M LiBr solutions resulted in radicals formed by electron attachment to the G(ox) sites that were monitored by electron spin resonance spectroscopy (ESR) at 77 K. These ESR spectra suggest that those oxidation products of 8-oxo-G containing alpha-diketo groups account for the electron traps (G(ox)) in oxidized DNA with oxaluric acid a likely major trap. Electron transfer from DNA anion radicals to G(ox) was followed by monitoring of their ESR signals with time at 77 K. Using typical values for the tunneling constant beta estimates of the relative amount of G(ox) to base pairs were obtained. Radicals formed by UV photolysis of oxidized DNA in 8 M NaClO(4) glassy aqueous solutions were also investigated. The 8-oxo-G cation accounts for less than 10% of all the radicals observed after either gamma irradiation of oxidized DNA in frozen (D(2)O) aqueous solution or UV photolysis of oxidized DNA in 8 M NaClO(4) glassy aqueous solutions. We estimate hole transfer distances of about 7 +/- 1 bp at 1 min from G(*+) to 8-oxo-G.

Determination of anti-oxidant activity by electron spin resonance spectroscopy

A method to assess anti-oxidant activity quantitatively is presented. In this method, free radicals (OH·) were produced from the Fenton reaction. Electron spin resonance spectroscopy (ESR) was then used to determine the effectiveness of five anti-oxidants to scavenge the free radicals. Anti-oxidant activity was assessed as the percentage reduction of the ESR signal intensity relative to that of a control after 10 min. The order of the potency of the antioxidants at 4.8 mM was: caffeic acid > o-coumarin > 6,7-dihydroxy-4-methylcoumarin > catechin > scopoletin. In addition, pro-oxidant activity (a higher ESR signal intensity than that of the control) was observed for catechin and scopoletin at low concentrations (below 3.6 mM). © 2000 Society of Chemical Industry

Gamma radiation effects on an amine antioxidant added in an ethylene-propylene copolymer

The aim of this work was to compare the gamma radiation induced effects on samples of an ethylene-propylene copolymer antioxidant free with samples loaded with an antioxidant characterised by the presence of an -NH functional group. The employed techniques were Electron Spin Resonance spectroscopy (ESR) and High Performance Liquid Chromatography (HPLC). Stable radicals RNO° due to the interaction of free radicals produced in the irradiated polymer with the antioxidant have been observed by ESR at room temperature. The time evolution of the ESR signals following the irradiation was examined at different doses. The amount of antioxidant not involved in the oxidation reactions has been determined using HPLC.

Effect of Reoxygenation with 21% or 100% Oxygen on Free Radical Formation Following Hypoxia in the Cerebral Cortex of Newborn Piglets. † 166

The present study tests the hypothesis that reoxygenation after cerebral tissue hypoxia with 100% oxygen increases generation of oxygen free radicals compared to reoxygenation with room air. Studies were performed in anesthetized, ventilated newborn piglets subjected to 1 hr of hypoxia(FiO2 0.05-0.07, PaO2 < 20 mmHg). Following hypoxia, animals were randomized to Group 1 (reoxygenation with 21% O2, n=8) or Group 2(reoxygenation with 100% O2, n=8). Near infrared spectroscopy (NIRS) was used with wavelengths of 716 nm and 850 nm to monitor tissue oxygenation using a phase modulation spectrometer with an interprobe distance of 22 mm. A phase shift in the NIRS signal was observed at 50±10 sec following initiation of reoxygenation. Cortical biopsies were obtained via cranial windows for free radical measurements using electron spin resonance spectroscopy (ESR) immediately following appearance of the phase shift. ESR spectra of electron spin adducts were obtained in duplicate using a Varian-E-109 spectrometer. Signal heights were measured from the resultant graph and divided by tissue weight. Reference ESR signals were measured from tissues incubated at room temperature for 10 min to confirm the absence of free radicals. Tissue was homogenized in 100 mMα-phenyl-tert-butyl-nitrone (PBN) and extracted with 2:1 chloroform:methanol. Intensity of the ESR signal from the PBN extract was expressed as signal height (mm)/g tissue after subtracting the reference value determined in control animals. Free radical signal height was 130±30 mm/g brain in Group 1 and 230±90 mm/g brain in Group 2 (p <0.05). Reoxygenation with 100% O2 was associated with significantly more free radical generation than reoxygenation with 21% O2. We speculate that mitochondria which were anaerobic during the hypoxic phase produce a surge of free radicals during reoxygenation and that the amount of free radicals produced increases with increased oxygen concentration. (NIH#HD-20337 and AAP/AHA/Neonatal Resuscitation Program)

Hypoxia depletes ascorbate in the cat carotid body

Vitamin C or l-ascorbate is an effective endogenous reducing agent influencing the functions that are involved with the redox mechanism. Detection of oxygen tension changes by the carotid body chemoreceptors is one such function. In this study we investigated the hypothesis that the level of ascorbate, if present, could change in the carotid body as a result of ascorbate's interaction in the chemosensing process, which would be reflected in the production of the ascorbyl radical. We addressed this issue by examining changes of the ascorbyl radical, using the in vitro electron spin resonance spectroscopy (ESR), in the carotid bodies dissected from pentobarbitone anesthetized cats exposed in vivo to three eucapnic, breathing gas mixtures of different O 2 concentration: normoxic, 21% O 2; hypoxic, 7% O 2; and hypoxic-reoxygenated, 7% O 2 followed by 100% O 2. Each group consisted of five cats, yielding five pairs of carotid bodies for the ESR signal recording. We found that the intensity of ESR signals, measured as peak-to-peak amplitude, was diminished by 39 and 43% in the hypoxic and hypoxic-reoxygenated groups, respectively, compared with that in normoxia. The study shows that ascorbate was present in the normoxic carotid body and was depleted by hypoxia. We conclude that ascorbate is part of free radical mechanisms operative in the carotid body in hypoxia.

Effects of sphingosine and sphingosine analogues on the free radical production by stimulated neutrophils: ESR and chemiluminescence studies

Sphingolipids inhibit the activation of the neutrophil (PMN) NADPH oxidase by protein kinase C pathway. By electron spin resonance spectroscopy (ESR) and chemiluminescence (CL), we studied the effects of sphingosine (SPN) and ceramide analogues on phorbol 12-myristate 13-acetate (PMA, 5 × 10-7M) stimulated PMN (6 × 106 cells). By ESR with spin trapping (100 mM DMPO: 5,5-dimethyl-1-pyrroline-Noxide), we showed that SPN (5 to 8 × 10-6M), C2-ceramide (N-acetyl SPN) and C6-ceramide (N-hexanoyl SPN) at the final concentration of 2 × 10-5 and 2 × 10-4M inhibit the production of free radicals by stimulated PMN. The ESR spectrum of stimulated PMN was that of DMPO-superoxide anion spin adduct. Inhibition by 5 × 10-6M SPN was equivalent to that of 30 U/ml SOD. SPN (5 to 8 × 10-6M) has no effect on in vitro systems generating superoxide anion (xanthine 50 mM/xanthine oxidase 110 mU/ml) or hydroxyl radical (Fenton reaction: 88 mM H2O2, 0.01 mM Fe2+ and 0.01 mM EDTA). SPN and N-acetyl SPN also inhibited the CL of PMA stimulated PMN in a dose dependent manner (from 2 × 10-6 to 10-5M), but N-hexanoyl SPN was less active (from 2 × 10-5 to 2 × 10-4M). These effects were compared with those of known PMN inhibitors, superoxide dismutase, catalase and azide. SPN was a better inhibitor compared with these agents. The complete inhibition by SPN of ESR signal and CL of stimulated PMN confirms that this compound or one of its metabolites act at the level of NADPH-oxidase, the key enzyme responsible for production of oxygen-derived free radicals.

Microsurgical model of ischemia reperfusion in rat muscle: evidence of free radical formation by spin trapping.

The purpose of developing the experimental model described in this study was to verify the hypothesis that free radicals are formed during ischemia-reperfusion of skeletal muscle. Spin trapping technique, along with electron spin resonance spectroscopy (ESR), directly indicates the presence of reactive radicals, which are widely considered to be important in tissue injury. The experimental model was a rat pedicled rectus femoris muscle flap. The femoral artery and vein were cannulated to inject the "spin trap" and collect the effluent flow. The spin trap agent was phenyl-t-butyl nitrone (PBN) and Hank's balanced salt solution. Three injections and collections were made: a) before ischemia; b) after ischemia of 15, 30, 60, 120, and 180 minutes, but before blood flow had been restored; and c) after blood flow had been restored. No ESR signal was detected either before the ischemic period or after only 15 minutes of ischemia. PBN radical adducts were detected after 30, 60, 120, and 180 minutes of ischemia. A similar signal was detected when PBN was injected during reperfusion 10 minutes after the ischemic periods. The study demonstrated the presence of free radicals in an in vivo intact skeletal muscle ischemia-reperfusion model.