Detection of nitrosyl-iron complexes by proton-electron-double-resonance imaging.

The nitrogen monoxide radical (NO*) forms paramagnetic mono- and dinitrosyl-iron complexes in biologic tissues. To establish a noninvasive technique for in vivo NO* imaging, we evaluated the suitability of these complexes as magnetic resonance (MR) contrast agents, making use of the ability of the unpaired electrons of the complexes to enter into dynamic nuclear polarization with water protons and hence produce enhancement on images generated by the technique of proton-electron-double-resonance imaging (PEDRI). Phantom solutions of synthetic nitrosyl-iron complexes (NICs) altered the signal intensity of PEDRI images. The dinitrosyl-iron complex (DNIC) with serum albumin induced a significantly larger signal alteration than the mononitrosyl-iron complex (MNIC) with dithiocarbamate. Exposure of rat liver to sodium nitroprusside (SNP) by ex vivo and in situ perfusion induced a composite X-band electron spin resonance (ESR) spectrum of the isolated liver characteristic of a MNIC and DNIC. On storage of the tissue, the MNIC signal disappeared and the DNIC signal intensity increased. Correspondingly, in cross-sectional PEDRI images taken at room temperature, the SNP-exposed livers initially exhibited a weak signal that strongly increased with time. In conclusion, NICs can be detected using PEDRI and could be exploited for in vivo NO* imaging.     

Formation of protein tyrosine ortho-semiquinone radical and nitrotyrosine from cytochrome c-derived tyrosyl radical

Oxidative alteration of mitochondrial cytochrome c (cyt c) has been linked to disease pathophysiology and is one of the causative factors for pro-apoptotic events. Hydrogen peroxide induces a short-lived cyt c-derived tyrosyl radical as detected by the electron spin resonance (ESR) spin-trapping technique. This investigation was undertaken to characterize the fate and consequences of the cyt c-derived tyrosyl radical. The direct ESR spectrum from the reaction of cyt c with H(2)O(2) revealed a single-line signal with a line width of approximately 10 G. The detected ESR signal could be prevented by pretreatment of cyt c with iodination, implying that the tyrosine residue of cyt c was involved. The ESR signal can be enhanced and stabilized by a divalent metal ion such as Zn(2+), indicating the formation of the protein tyrosine ortho-semiquinone radical (ToQ.). The production of cyt c-derived ToQ. is inhibited by the spin trap, 2-methyl-2-nitrosopropane (MNP), suggesting the participation of tyrosyl radical in the formation of the ortho-semiquinone radical. The endothelium relaxant factor nitric oxide is well known to mediate mitochondrial respiration and apoptosis. The consumption of NO by cyt c was enhanced by addition of H(2)O(2) as verified by inhibition electrochemical detection using an NO electrode. The rate of NO consumption in the system containing cyt c/NO/H(2)O(2) was decreased by the spin traps 5,5-dimethyl pyrroline N-oxide and MNP, suggesting NO trapping of the cyt c-derived tyrosyl radical. The above result was further confirmed by NO quenching of the ESR signal of the MNP adduct of cyt c tyrosyl radical. Immunoblotting analysis of cyt c after exposure to NO in the presence of H(2)O(2) revealed the formation of 3-nitrotyrosine. The addition of superoxide dismutase did not change the cyt c nitration, indicating that it is peroxynitrite-independent. The results of this study may provide useful information in understanding the interconnection among cyt c, H(2)O(2), NO, and apoptosis.     

Nitric oxide generation in aqueous solutions of cigarette smoke and approaches to its origin.

By using the ESR spin trapping technique with the N-methyl-D-glucamine dithiocarbamate (MGD)2-Fe(II) complex, the generation of nitric oxide (NO), a gaseous free radical, was observed in NO spin trapping solution bubbled with the filtered main-stream of cigarette smoke. The ESR signal with a three-line spectrum characteristic of an NO radical, which was not observed immediately after bubbling of smoke, started rapidly increasing with time up to around 25 min after the last addition of ferrous ions Fe(II), and then slowly approached a peak value dependent on the burned cigarette mass and on the smoking speed. The production of NO was, however, much affected by air oxidation and enhanced by the addition of ascorbic acid. A certain concentration of sodium nitrite (NaNO2) solution, in which nitrite NO2- is assumed as the main origin of the NO, mimicked closely the time course of NO generation resulting from the smoke of one cigarette. The cigarette smoke that was passed through alkaline pyrogallol solution as a deoxidizer; however, it exhibited an unchanged intensity of NO signal throughout the measurement. These results strongly suggest that NO would be gradually reproduced from NO2- in the reductive aqueous solution containing excess Fe(II) through NO2, which is initially formed and is concomitantly oxidized from NO in cigarette smoke.     

Kinetic study on ESR signal decay of nitroxyl radicals,potent redox probes for in vivo ESR spectroscopy,caused by reactive oxygen species

The effect of the chemical structure of nitroxyl spin probes on the rate at which ESR signals are lost in the presence of reactive oxygen species (ROS) was examined. When the spin probes were reacted with either hydroxyl radical (.OH) or superoxide anion radical (O(2)(.-)) in the presence of cysteine or NADH, the probes lost ESR signal depending on both their ring structure and substituents. Pyrrolidine nitroxyl probes were relatively resistant to the signal decay caused by O(2)(.-) with cysteine/NADH. Signal decay rates for these reactions correlated with reported redox potentials of the nitroxyl/oxoammonium couple of spin probes, suggesting that the signal decay mechanism in both cases involves the oxidation of a nitroxyl group. The apparent rate constants of the reactions between the spin probe and .OH and between the spin probe and O(2)(.-) in the presence of cysteine were estimated using mannitol and superoxide dismutase (SOD), respectively, as competitive standards. The rate constants for spin probes and .OH were in the order of 10(9) M(-1) s(-1), much higher than those for the probes and O(2)(.-) in the presence of cysteine (10(3)-10(4) M(-1) s(-1)). These basic data are useful for the measurement of .OH and O(2)(.-) in living animals by in vivo ESR spectroscopy.     

NMR spin trapping: detection of free radical reactions with a new fluorinated DMPO analog

Electron spin resonance (ESR) and nuclear magnetic resonance (NMR) spin trapping were used for detection of free radical reactions utilizing a new fluorinated analog of DMPO, 4-hydroxy-5,5-dimethyl-2-trifluoromethylpyrroline-1-oxide (FDMPO). The parent FDMPO spin trap exhibits a single 19F-NMR resonance at -66.0 ppm. The signal to noise ratio improved 10.4-fold compared to 31P-NMR sensitivity of the phosphorus-containing spin trap, DEPMPO. The spin adducts of FDMPO with .OH, .CH3, and .CH2OH were characterized. Competitive spin trapping of FDMPO with DMPO showed that both have similar rates of addition of .OH and C-centered radicals. The corresponding paramagnetic spin adducts of FDMPO were extremely stable to degradation. In the presence of ascorbate, reaction products from C-centered radicals resulted in the appearance of two additional 19F-NMR signals at -78.6 and -80 ppm for FDMPO/ .CH(3) and at -74.6 and -76.75 ppm for FDMPO/ .CH(2)OH. In each case, these peaks were assigned to the two stereoisomers of their respective, reduced hydroxylamines. The identification of the hydroxylamines for FDMPO/ .CH3 was confirmed by EPR and 19F-NMR spectra of independently synthesized samples. In summary, spin adducts of FDMPO were highly stable for ESR. For NMR spin trapping, FDMPO showed improved signal to noise and similar spin trapping efficiency compared to DEPMPO.     

Hydroxyl Radical Generation in the Nadh/Microsomal Reduction of vanadate

ESR spin trapping measurements demonstrate generation of hydroxyl (.OH) radical from reduction of vanadate by rat liver microsomes/NADH without exogenous H₂O₂. Catalase decreases the .OH signal while increasing a vanadium(4+) signal. Addition of superoxide dismutase (SOD) or measurements under an argon atmosphere show decreased .OH radical production. The results suggest that during the one-electron vanadate reduction process by microsomes/NADH, molecular oxygen is reduced to H₂O₂, which then reacts with vanadium (4+) to generate .OH radical via a Fenton-like mechanism.     

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.     

Hydroxyl Radical Generation Caused by the Reaction of Singlet Oxygen with a Spin Trap,DMPO, Increases Significantly in the Presence of Biological Reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (¹O²) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ¹O² with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (?OH) adduct of DMPO (DMPO-OH) resulting from ¹O²-dependent generation of ?OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its ?OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of ?OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of ?OH from ¹O², and that spin trap-mediated ?OH generation hardly occurs with DEPMPO.     

Free radical generation by ultrasound in aqueous solutions of nucleic acid bases and nucleosides: an ESR and spin-trapping study

Direct evidence for the detection of intermediate radicals of nucleic acid constituents induced by ultrasound in argon-saturated aqueous solution is presented. The method of spin trapping with 3,5-dibromo-4-nitrosobenzene sulphonate, which is a water-soluble, non-volatile, aromatic nitroso spin trap, combined with ESR, was used for the detection of sonochemically induced radicals. Spin adducts were also generated by OH radicals produced by UV photolysis of aqueous solution containing H2O2. ESR spectra observed from these photolysis experiments were identical to those after sonolysis. The ESR spectra of the spin adducts suggest that the major spin-trapped radical of thymine and thymidine was the 5-yl radical, and that of cytosine, cytidine, uracil, and uridine was the 6-yl radical. To compare the radicals induced by sonolysis and photolysis, the decay of the ESR spectra of the thymine and thymidine spin adducts was investigated. The decay curves of thymine and thymidine after sonolysis indicated biphasic decay. However, after photolysis the spin adducts from both compounds showed very little decay. These results suggest that the observed spin adducts in the sonolysis of pyrimidine bases and nucleosides were formed by OH radical and H atom addition to the 5,6 double-bond.     

Hydroxyl radical generation caused by the reaction of singlet oxygen with a spin trap, DMPO, increases significantly in the presence of biological reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (₁O₂) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ₁O₂ with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (•OH) adduct of DMPO (DMPO-OH) resulting from ₁O₂-dependent generation of •OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its •OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of •OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of •OH from ₁O₂, and that spin trap-mediated •OH generation hardly occurs with DEPMPO.     

Mitochondrial aconitase is a source of hydroxyl radical. An electron spin resonance investigation

Mitochondrial aconitase (m-aconitase) contains a [4Fe-4S](2+) cluster in its active site that catalyzes the stereospecific dehydration-rehydration of citrate to isocitrate in the Krebs cycle. It has been proposed that the [4Fe-4S](2+) aconitase is oxidized by superoxide, generating the inactive [3Fe-4S](1+) aconitase. In this reaction, the likely products are iron(II) and hydrogen peroxide. Consequently, the inactivation of m-aconitase by superoxide may increase the formation of hydroxyl radical ((*)OH) through the Fenton reaction in mitochondria. In this work, evidence for the generation of (*)OH from the reaction of m-aconitase with superoxide is provided using ESR spin trapping experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide and alpha-phenyl-N-tert-butylnitrone. Formation of free ( small middle dot)OH was verified with the (*)OH scavenger Me(2)SO, which forms methyl radical upon reacting with (*)OH. The addition of Me(2)SO to incubation mixtures containing m-aconitase and xanthine/xanthine oxidase yielded methyl radical, which was detected by ESR spin trapping. Methyl radical formation was further confirmed using [(13)C]Me(2)SO. Parallel low temperature ESR experiments demonstrated that the generation of the [3Fe-4S](1+) cluster increased with increasing additions of superoxide to m-aconitase. This reaction was reversible, as >90% of the initial aconitase activity was recovered upon treatment with glutathione and iron(II). This mechanism presents a scenario in which (*)OH may be continuously generated in the mitochondria.     

Vanadyl-induced Fenton-like reaction in RNA. An ESR and spin trapping study.

Vanadyl (VO2+) complexed to RNA reacts with hydrogen peroxide in a Fenton-like manner producing hydroxyl radicals (.OH). The hydroxyl radicals can be spin trapped with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) forming the DMPO-OH spin adduct. In addition, in the presence of ethanol the formation of the hydroxyethyl radical adduct of DMPO (DMPO-ETOH) confirms the production of hydroxyl radicals by the RNA/VO2+ complex. When the reaction between the RNA/VO2+ complex and H2O2 is carried out in the presence of the spin trap 2-methyl-2-nitrosopropane (MNP), radicals produced in the reaction of .OH with RNA are trapped. Base hydrolysis of the MNP-RNA adducts (pH 12) followed by a reduction in the pH to pH 7 after hydrolysis is complete, yields an MNP adduct with a well-resolved ESR spectrum identical to the ESR spectrum obtained from analogous experiments with poly U. The ESR spectrum consists of a triplet of sextets (aN = 1.48 mT, a beta N = 0.25 mT and a beta H = 0.14 mT), indicating that the unpaired nitroxide electron interacts with the nuclei of a beta-nitrogen and beta-hydrogen. The results suggest that the .OH generated in the RNA/VO2+ reaction with H2O2 add to the C(5) carbon of uracil forming a C(6) carbon centered radical. This radical is subsequently spin trapped by MNP.     

A new nitroxyl-probe with high retention in the brain and its application for brain imaging

In order to estimate free radical reactions and image them in the brain of living animals, a nitroxyl spin-probe, carboxy-PROXYL acetoxymethyl ester (CxP-AM) was newly synthesized. CxP-AM was designed to be hydrolyzed by esterase, but not by lipase, so that it would pass through the blood-brain barrier and be retained in the cytosolic phase of parenchymal cells in the brain after intravenous injection. The pharmacokinetics of CxP-AM was compared with those of carboxy-PROXYL (CxP) and its methyl ester (CxP-M). Carboxyl esterase almost completely hydrolyzed CxP-AM within 3 min. After intravenous injection, the brain retained 1.8 times more CxP-AM than CxP-M, and retained it for more than 30 min. Electron spin resonance computed tomographic (ESR-CT) imaging of CxP-AM in the heads of mice produced marked contrast in the encephalon region, while CxP was distributed only in the extracranial region and CxP-M was distributed in both regions, confirming the pharmacokinetics of CxP-AM. The decay rate of CxP-AM determined with time-resolved ESR-CT imaging was different in the two brain regions, suggesting regional differences in the total reducing capability. CxP-AM should become a powerful probe for the investigation and diagnosis of free radical reactions and their imaging in the brain.     

Direct evidence for in vivo hydroxyl radical generation in blood of mice after acute chromium (VI) intake

Although it is assumed from in vitro experiments that the hydroxyl radical (*OH) may be responsible for chromium(VI) toxicity/carcinogenicity, no electron spin resonance (ESR) evidence for the generation of *OH in vivo has been reported. In this study, we have employed an ESR spin-trapping technique with 5,5-dimethylpyrroline-N-oxide (DMPO), a selective *OH trap, to detect *OH in blood. The ESR spectrum of spin adduct observed in the blood of mice given 4.8 mmol Cr(VI)/kg body weight exhibited the 1:2:2:1 intensity pattern of a quartet with a hyperfine coupling constant A(N) = A(H) = 14.81 G and g-value = 2.0067. The concentration of the spin adduct detected in the blood was 7.37 microM. The adduct production was inhibited by the addition of specific *OH scavengers such as sodium benzoate and methional to the blood. The results indicate that the spin adduct is nitroxide produced by the reaction of *OH with DMPO. This is the first report of ESR evidence for the in vivo generation of *OH in mammals by Cr(VI).     

Free-radical reactions induced by OH-radical attack on cytosine-related compounds: a study by a method combining ESR, spin trapping and HPLC.

Free-radical reactions induced by OH-radical attack on cytosine-related compounds were investigated by a method combining ESR, spin trapping with 2-methyl-2-nitrosopropane and high-performance liquid chromatography (HPLC). Cytidine, 2'-deoxycytidine, cytidine 3'-monophosphate, cytidine 5'-monophosphate, 2'-deoxycytidine 5'-monophosphate and their derivatives, of which 5,6-protons at the base moiety were replaced by deuterons, and polycytidylic acid (poly(C] were employed as samples. OH radicals were generated by X-irradiating an N2O-saturated aqueous solution. Five spin adducts were separated by HPLC. Examination of them by ESR spectroscopy and UV photospectrometry showed that spin adducts assigned to C5 and C6 radicals due to OH addition to the 5,6 double-bond, a deaminated form of the spin adduct derived from a C5 radical due to the cyclization reaction between C5' of the sugar and C6 of the base, and a spin adduct assigned to the C4' radical due to H abstraction by OH radicals were produced. From these results the sites of OH-radical attack and the subsequent radical reactions in cytosine-related compounds were clarified.     

Deferoxamine inhibition of Cr(V)-mediated radical generation and deoxyguanine hydroxylation: ESR and HPLC evidence.

Electron spin resonance (ESR) and high-performance liquid chromatography (HPLC) techniques were utilized to investigate the effect of deferoxamine on free radical generation in the reaction of Cr(V) with H2O2 and organic hydroperoxides. ESR measurements demonstrated that deferoxamine can efficiently reduce the concentration of the Cr(V) intermediate as formed in the reduction of Cr(VI) by NAD(P)H or a flavoenzyme glutathione reductase/NADH. ESR spin trapping studies showed that deferoxamine also inhibits Cr(V)-mediated .OH radical generation from H2O2, as well as Cr(V)-mediated alkyl and alkoxy radical formation from t-butyl hydroperoxide and cumene hydroperoxide. HPLC measurements showed that .OH radicals generated by the Cr(VI)/flavoenzyme/NAD(P)H enzymatic system react with 2'-deoxyguanine to form 8-hydroxy-2'-deoxyguanine (8-OHdG), a DNA damage marker. Deferoxamine effectly inhibited the formation of 8-OHdG also.     

A time-resolved electron spin resonance study of the oxidation of ascorbic acid by hydroxyl radical.

Time-resolved electron spin resonance (ESR) spectroscopy for the study of radicals produced by pulse radiolysis is illustrated by a study of the oxidation of ascorbic acid by OH radical in aqueous solution. In basic solution, the direct oxidation product, the ascorbate mono-anion radical, is formed within less than 2 mus of the radiolysis pulse. In acid solutions (pH 3(-4.5), N(2)O:saturated) three radicals are initially formed, the ascorbate mono-anion radical, an OH adduct seen also in steady-state ESR experiments, and an OH adduct at C2 with the main spin density at C3 of the ring. The first OH adduct decays with an initial half-life of about 100 mus, probably by biomolecular reaction. The second OH adduct, which shows one hyperfine splitting about a(H) = 24.4 +/- 0.3 G and g = 2.0031 +/- 0.0002, decays with a half-life of about 10 mus. On this same time scale the concentration of the ascorbate radical approximately doubles. It is concluded that the adduct at C2, but not the other adduct, loses water rapidly to form the ascorbate radical.     

-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.     

In vivo imaging of free radicals: applications from mouse to man

Free radicals and other paramagnetic species, play an important role in cellular injury and pathophysiology. EPR spectroscopy and imaging has emerged as an important tool for non-invasive in vivo measurement and spatial mapping of free radicals in biological tissues. Extensive applications have been performed in small animals such as mice and recently applications in humans have been performed. Spatial EPR imaging enables 3D mapping of the distribution of a given free radical while spectral-spa-tial EPR imaging enables mapping of the spectral information at each spatial position, and, from the observed line width, the localized tissue oxygenation can be determined. A variety of spatial, and spectral-spatial EPR imaging applications have been performed. These techniques, along with the use of biocompatible paramagnetic probes including particulate suspensions and soluble nitroxide radicals, enable spatial imaging of the redox state and oxygenation in a variety of biomedical applications. With spectral-spatial EPR imaging, oxygenation was mapped within the gastrointestinal (GI) tract of living mice, enabling measurement of the oxygen gradient from the proximal to the distal GI tract. Using spatial EPR imaging, the distribution and metabolism of nitroxide radicals within the major organs of the body of living mice was visualized and anatomically co-registered by proton MRI enabling in vivo mapping of the redox state and radical clearance. EPR imaging techniques have also been applied to non-invasively measure the distribution and metabolism of topically applied nitroxide redox probes in humans, providing information regarding the penetration of the label through the skin and measurement of its redox clearance. Thus, EPR spectroscopy and imaging has provided important information in a variety of applications ranging from small animal models of disease to topical measurement of redox state in humans.     

Nucleotide Modification,A Radical Mechanism of Oxidative Toxicity

Reduced nicotinamide adenine dinucleotide (NADH) reacts rapidly with hypochlorite to form five major products separable by reversed-phase high-pressure liquid chromatography (HPLC). The involvement of a free radical mechanism is indicated by an electron spin resonance (ESR) signal as well as unusual pH changes and the uptake of oxygen. The present work suggests that hypochlorite may contribute to the cytotoxic activity of phagocytic cells through its ability to modify important cellular components by means of radicals generated by its reaction with reduced pyridine nucleotides.