The Horseradish Peroxidase Catalysed Oxidation of Deoxyribose Sugars

The ability of horseradish peroxidase (E.C. 1.11.1.7. Donor: H₂O₂ oxidoreductase) to catalytically oxidize 2-deoxyribose sugars to a free radical species was investigated. The ESR spin-trapping technique was used to denionstrate that free radical species were formed. Results with the spin trap 3.5-dibronio-4-nitrosoben-zene sulphonic acid showed that horseradish peroxidase can catalyse the oxidation of 2-deoxyribose to produce an ESR spectrum characteristic of a nitroxide radical spectrum. This spectrum was shown to be a composite of spin adducts resulting from two carbon-centered species, one spin adduct being characterized by the hyperfine coupling constants a^N = 13.6Ganda^H_β = 11.0G, and the other by a^N = 13.4G and a^H = 5.8 G. When 2-deoxyribose-5-phosphate was used as the substrate, the spectrum produced was found to be primarily one species characterized by the hyperfine coupling constants a^N = 13.4G and a^H= 5.2. All the radical species produced were carbon-centered spin adducts with a β hydrogen, suggesting that oxidation occurred at the C(2) or C(5) moiety of the sugar. Interestingly, it was found that under the same experimental conditions, horseradish peroxidase apparently did not catalyze the oxidation of either 3-deoxyribose or D-ribose to a free radical since no spin adducts were found in these cases.

It can be readily seen that 2-deoxyribose and 2-deoxyribose-5-phosphate can be oxidized by HRP/H₂O₂ to form a free radical species that can be detected with the ESR spin-trapping technique. There are two probable sites for the formation of a CH type radical on the 2-deoxyribose sugar, these being the C(2) and the C(5) carbons. The fact that there is a species produced from 2-deoxy-ribose, but not 2-deoxy-ribose-5-phosphate, suggests that there is an involvement of the C(5) carbon in the species with the 1 1.0G β hydrogen. In the spectra formed from 2-deoxy-ribose, there is a big difference in the hyperfine splitting of the β hydrogens, suggesting that the radicals are formed at different carbon centers, while the addition of a phosphate group to the C(5) carbon seems to inhibit radical formation at one site. In related work, the chemiluminescence of monosaccharides in the presence of horseradish peroxidase was proposed to be the consequence of carbon-centered free radical formation (10).     

Identification of a radical formed in the reaction mixtures of oxidized phosphatidylcholine with ferrous ions using HPLC-ESR and HPLC-ESR-MS

Identification of free radicals was performed for the reaction mixtures of autoxidized 1,2-dilinoleoylphosphatidylcholine (DLPC) with ferrous ions (or DLPC hydroperoxide with ferrous ions) and of DLPC with soybean lipoxygenase using electron spin resonance (ESR), high performance liquid chromatography (HPLC)-ESR and HPLC-ESR-mass spectrometry (MS) combined use of spin trapping technique. ESR measurements of the reaction mixtures showed prominent signals with hyperfine coupling constants (a^N=1.58 mT and a^Hβ=0.26 mT). Outstanding peaks with almost same retention times (autoxidized DLPC, 36.9 min; DLPC hydroperoxide, 35.0 min; DLPC with soybean lipoxygenase, 37.1 min) were observed on the elution profile of the HPLC-ESR analyses of the reaction mixtures. HPLC-ESR-MS analyses of the reaction mixtures gave two ions at m/z 266 and 179, suggesting that 4-POBN/pentyl radical adduct forms in these reaction mixtures.     

Free radical formation in the oxidation of malondialdehyde and acetylacetone by peroxidase enzymes.

Malondialdehyde, a product of lipid peroxidation, and acetylacetone undergo one-electron oxidation by peroxidase enzymes to form free radical metabolites, which were detected with ESR using the spin-trapping technique. The structures of the radical adducts were assigned using isotope substitution. With both malondialdehyde and acetylacetone and the enzymes myeloperoxidase and chloroperoxidase, carbon-centered radicals were detected. With horseradish peroxidase, a carbon-centered radical was initially trapped and then disappeared with the concomitant appearance of an iminoxyl radical.     

Free radical metabolites of L-cysteine oxidation

The oxidation of L-cysteine by horseradish peroxidase in the presence of oxygen forms a thiyl free radical as demonstrated with the spin-trapping ESR technique. Reactions of this thiyl free radical result in oxygen consumption, which is inhibited by the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide. Cysteine sulfinic acid, a cysteine metabolite, is a poorer substrate for horseradish peroxidase than cysteine and is oxidized to form both sulfur-centered and carbon-centered free radicals.     

Esr and Spin-Trapping Study of Free Radicals in γ-Irradiated Solid Lysozyme

Free radicals produced by γ-irradiation of solid lysozyme were investigated by a technique combining ESR, spin-trapping and enzymatic digestion. MNP and DMPO were used as spin-trapping reagents. The solid lysozyme was first γ-irradiated and then dissolved in an aqueous solution containing the spin-trapping reagent to stabilize free radicals. The spin adducts of lysozyme were digested to oligopeptides to get ESR spectra having a well-resolved hyperfine structure. The ESR spectra obtained showed that carbon-centered radicals, CH-, at the side chains of amino acids, and thiyl radicals, -CH₂-_-S^-, at disulfide bridges were produced in γ-irradiated solid lysozyme.     

Spin Trapping Using 2,2-Dimethyl-2H-Imidazole-1-Oxides

The ability of novel cyclic nitrones, 4-substituted 2,2-dimethyl-2H-imidazole-1-oxides (IMO's) to trap a variety of short-lived free radicals has been investigated using ESR spectroscopy. IMO's scavenge oxygen-, carbon- and sulfur-derived free radicals to give persistent nitroxides. Compared to the spin trap 5,5-dimethyl-pyrroline-1-oxide, a higher lifetime of hydroxyl radical adducts and a higher selectivity related to the trapping of carbon-centered radicals was found. A reaction between IMO's and superoxide was not observed. ESR parameters of 4-carboxyl-2,2-dimethyl-2H-imidazole-1-oxide (CIMO) spin adducts are highly sensitive to the structure of the trapped radical, e.g., different spectra were detected with radicals derived from Na₂SO₃ and NaHSO₃. From the data obtained, a successful application of these new spin traps in biological systems can be expected.     

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.     

Characterization of the high resolution ESR spectra of the methoxyl radical adducts of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO)

Spin-trapping investigators are largely limited by the instability of the radical adducts. Spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) forms very stable alkoxyl radical adducts. However, the presence of two chiral centers in the DEPMPO alkoxyl radical adduct results in two diastereomers with distinctive ESR spectra, which complicates the interpretation of the ESR spectra. We have analyzed the high resolution ESR spectra of the DEPMPO/_•OCH₃ radical adduct. DEPMPO/_•OCH₃ has been synthesized by the nucleophilic addition of alcohols to DEPMPO. The electron spin resonance (ESR) spectrum of DEPMPO/_•OCH₃ in oxygen-free methanol solution reveals superhyperfine structure with hyperfine coupling constants as small as 0.3 G. In order to simplify the analysis of the electron spin resonance (ESR) spectrum, we synthesized the DEPMPO/_•OCD₃ radical adduct. Computer simulation of the DEPMPO/_•OCD₃ ESR spectrum revealed two diastereomers. Hyperfine coupling constants of γ-protons and ₁₇O from the -OCH₃ group were also determined. ESR spectra of DEPMPO/_•OCH₃ in phosphate buffer have also been characterized. The presence of specific hyperfine couplings from the -OCH₃ group can be used for the unambiguous identification of the DEPMPO/_•OCH₃ radical adducts. We suggest that the analysis of high resolution ESR spectra can be used for the unambiguous characterization of DEPMPO radical adducts.     

Ozone exposure generates free radicals in the blood samples in vitro. Detection by the ESR spin-trapping technique

Generation of free radicals in the reaction of ozone with blood samples and related salt solutions was investigated in vitro by using ESR spin-trapping technique with DMPO. In the reactions of low levels of ozone, a carbon-centered radical was spin-trapped with DMPO, giving rise to the 6-line ESR signal in both whole blood and blood plasma. In the blood plasma, DMPO-spin adduct of hydroxyl radical (DMPO-OH) was detected together with the spin adduct of carbon-centered radical. The present spin-trapping study demonstrates that, when exposed to ozone, 0.9% NaCl solution in the presence of DMPO gives rise to the formation of DMPO-OH. The addition effects of ethanol, which is a ^·OH scavenger, into the NaCl solution reveal that DMPO-OH is produced by the reaction of DMPO with both ^·OH and unidentified oxidants originated from the reaction of Cl^- and ozone. Based on these observations, we consider that ^·OH is generated similarly in the blood plasma exposed to ozone. The ESR study of DMPO-spin adducts in the ozone-exposed aqueous solution of NaOCl indicates that Cl^- reacts with ozone to give ClO^-. Presumably, further oxidation of ClO^- by ozone leads to the formation of ^·OH and the unidentified oxidants.     

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.     

A spin label study of horseradish peroxidase.

The topography of the active sites of native horseradish peroxidase and manganic horseradish peroxidase has been studied with the aid of a spin-labeled analog of benzhydroxamic acid (N-(1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxy)-p-aminobenzhydroxamic acid). The optical spectra of complexes between the spin-labeled analog of benzhydroxamic acid and Fe3+ or Mn3+ horseradish peroxidase resembled the spectra of the corresponding enzyme complexes with benzhydroxamic acid. Electron spin resonance (ESR) measurement indicated that at pH 7 the nitroxide moiety of the spin-labeled analog of benzhydroxamic acid became strongly immobilized when this label bound to either ferric or manganic horseradish peroxidase. The titration of horseradish peroxidase with the spin-labeled analog of benzhydroxamic acid revealed a single binding site with association constant Ka approximately 4.7 . 10(5) M-1. Since the interaction of ligands (e.g. F-, CN-) and H2O2 with horseradish peroxidase was found to displace the spin label, it was concluded that the spin label did not indeed bind to the active site of horseradish peroxidase. At alkaline pH values, the high spin iron of native horseradish peroxidase is converted to the low spin form and the binding of the spin-labeled analog of benzhydroxamic acid to horseradish peroxidase is completely inhibited. From the changes in the concentration of both bound and free spin label with pH, the pK value of the acid-alkali transition of horseradish peroxidase was found to be 10.5. The 2Tm value of the bound spin label varied inversely with temperature, reaching a value of 68.25 G at 0 degree C and 46.5 G at 52 degrees C. The dipolar interaction between the iron atom and the free radical accounted for a 12% decrease in the ESR signal intensity of the spin label bound to horseradish peroxidase. From this finding, the minimum distance between the iron atom and nitroxide group and hence a lower limit to the depth of the heme pocket of horseradish peroxidase was estimated to be 22 A.     

Fatty acid radical formation in rats administered oxidized fatty acids: in vivo spin trapping investigation.

We report in vivo evidence for fatty acid-derived free radical metabolite formation in bile of rats dosed with spin traps and oxidized polyunsaturated fatty acids (PUFA). When rats were dosed with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and oxidized PUFA, the DMPO thiyl radical adduct was formed due to a reaction between oxidized PUFA and/or its metabolites with biliary glutathione. In vitro experiments were performed to determine the conditions necessary for the elimination of radical adduct formation by ex vivo reactions. Fatty acid-derived radical adducts of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) were detected in vivo in bile samples collected into a mixture of iodoacetamide, desferrioxamine, and glutathione peroxidase. Upon the administration of oxidized 13C-algal fatty acids and 4-POBN, the EPR spectrum of the radical adducts present in the bile exhibited hyperfine couplings due to 13C. Our data demonstrate that the carbon-centered radical adducts observed in in vivo experiments are unequivocally derived from oxidized PUFA. This in vivo evidence for PUFA-derived free radical formation supports the proposal that processes involving free radicals may be the molecular basis for the previously described cytotoxicity of dietary oxidized PUFA.     

Detection of peroxyl and alkoxyl radicals produced by reaction of hydroperoxides with heme-proteins by electron spin resonance spectroscopy

ESR spin trapping using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has been used to directly detect alkoxyl radicals (with hyperfine coupling constants aN 1.488, aH 1.600 mT and aN 1.488, aH 1.504 mT for the tBuO. and PhC(CH3)2O. adducts, respectively) and peroxyl radicals (aN 1.448, aH 1.088, aH 0.130 mT and aN 1.456, aH 1.064, aH 0.128 mT for the tBuOO. and PhC(CH3)2OO. adducts, respectively) produced from t-butyl or cumene hydroperoxides by a variety of heme-containing substances (purified cytochrome P-450, metmyoglobin, oxyhemoglobin, methemoglobin, cytochrome c, catalase, horseradish peroxidase) and the model compound hematin. The observed species exhibit a complicated dependence on reagent concentrations and time, with maximum concentrations of the peroxyl radical adducts being observed immediately after mixing of the hydroperoxide with low concentrations of the heme-compound. Experiments with inhibitors (CN-, N3-, CO, metyrapone and imidazole) suggest that the major mechanism of peroxyl radical production involves high-valence-state iron complexes in a reaction analogous to the classical peroxidase pathway. The production of alkoxyl radicals is shown to arise mainly from the breakdown of peroxyl radical spin adducts, with direct production from the hydroperoxide being a relatively minor process.     

DMPO-Alkyl radical spin trapping: an in situ radiolysis steady-state ESR study

Short-lived free radicals formed in the reaction of 11 substrates and radiolytically produced hydroxyl radicals were trapped successfully with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) in dilute aqueous solution. The in situ radiolysis steady-state ESR spectra of the spin adducts were analyzed to determine accurate ESR parameters for these spin adducts in a uniform environment. Parent alkyl radicals include methyl, ethyl, 1-propyl and 2-propyl (1-methylethyl). Hydroxyalkyl parent radicals were hydroxymethyl, hydroxyethyl, 2-hydroxy-2-propyl (1-methyl-1-hydroxyethyl), 1-hydroxypropyl and 2-hydroxy-2-methylpropyl. Carboxyl radical (carbon dioxide anion, formate radical) and sulfite anion radical were the sigma radicals studied. The DMPO spin adduct of 1-propyl was identified for the first time. For most spin adducts, g factors were also determined for the first time. In DMPO spin adducts of hydroxyalkyl radicals, nitrogen and C(2)-proton hyperfine coupling constants are smaller than those of alkyl radical adducts; the hydroxyalkyl spin adducts possess larger g values than their unsubstituted counterparts. These changes are ascribed to the spread of pi conjugation to include the hydroxyl group. Strong evidence of spin addend-aminoxyl group interaction can be seen in the asymmetrical line shapes in the hydroxyethyl and the hydroxypropyl spin adducts.     

Chemical reactivity of the carbon-centered free radicals and ferrous iron in coals: Role of bioavailable Fe2+ in coal workers' pneumoconiosis

Striking differences in the prevalence of coal workers' pneumoconiosis (CWP) exist between different coal mine regions. The major factors responsible for the observed regional differences in CWP have not yet been identified. In the present study, chemical reactivity of the carbon-centered free radicals in coals and lung tissues, as well as ferrous iron in the coals, were studied by ESR techniques. The ESR spectra clearly demonstrated the presence of at least two types of carbon-centered free radical species, which might respectively attribute to the macromolecular phase and the molecular phase of coal. Grinding produced free radicals in coals. Exposure of freshly ground coal to air for 28 h induced a slight increase of free radicals for most of the coals, and a slight decrease after 4 months' exposure. The lung tissue samples of coal workers deceased of CWP showed similar ESR spectra as coal samples, and these radicals were highly stable in the lung. After incubation of coals with glutathione, hydrogen peroxide, sodium formate or oxygen, the coal sample from the Gardanne mine which has never induced CWP, and thus is the least hazardous coal, showed the most significant change in the carbon-centered free radical concentration. No significant changes were observed among other coals reported to induce CWP. On the other hand, we found that the coals released different amounts of Fe²⁺ in an acidic medium. Interestingly, the prevalence of CWP correlates positively with the released Fe²⁺ content in these coals and with the amount of oxygen radicals produced by the interaction of Fe²⁺ with O₂ in the acidified coal filtrates. Our studies indicate that the carbon-centered free radicals may not be biologically relevant to coal dust-induced pneumoconiosis, whereas the acid soluble Fe²⁺, which may be dissolved in the phagolysosomes of macrophages, can then lead to Fe²⁺-induced oxidative stress and eventual CWP development.     

EPR spin trapping detection of carbon-centered carotenoid and beta-ionone radicals

Free radical intermediates were detected by the electron paramagnetic resonance spin trapping technique upon protonation/deprotonation reactions of carotenoid and beta-ionone radical ions. The hyperfine coupling constants of their spin adducts obtained by spectral simulation indicate that carbon-centered radicals were trapped. The formation of these species was shown to be a result of chemical oxidation of neutral compounds by Fe(3+) or I(2) followed by deprotonation of the corresponding radical cations or addition of nucleophilic agents to them. Bulk electrolysis reduction of beta-ionone and carotenoids also leads to the formation of free radicals via protonation of the radical anions. Two different spin adducts were detected in the reaction of carotenoid polyenes with piperidine in the presence of 2-methyl-2-nitroso-propane (MNP). One is attributable to piperidine radicals (C(5)H(10)N*) trapped by MNP and the other was identified as trapped neutral carotenoid (beta-ionone) radical produced via protonation of the radical anion. Formation of these radical anions was confirmed by ultraviolet-visible spectroscopy. It was found that the ability of carotenoid radical anions/cations to produce neutral radicals via protonation/deprotonation is more pronounced for unsymmetrical carotenoids with terminal electron-withdrawing groups. This effect was confirmed by the radical cation deprotonation energy (H(D)) estimated by semiempirical calculations. The results indicate that the ability of carotenoid radical cations to deprotonate decreases in the sequence: beta-ionone > unsymmetrical carotenoids > symmetrical carotenoids. The minimum H(D) values were obtained for proton abstraction from the C(4) atom and the C(5)-methyl group of the cyclohexene ring. It was assumed that deprotonation reaction occurs preferentially at these positions.     

Nitrobenzyl radical metabolites from microsomal reduction of nitrobenzyl chlorides

The o-, m-, and p-nitrobenzyl chlorides are reduced aerobically and anaerobically by NADPH and rat hepatic microsomes. Under aerobic conditions, these nitro anion radicals reduce oxygen to superoxide as demonstrated by oxygen consumption and spin trapping of superoxide with 5,5-dimethyl-1-pyrroline N-oxide. At low oxygen concentration, the p- and o-nitro anion radicals undergo intramolecular electron transfer and decompose to carbon-centered nitrobenzyl radicals, which can be spin-trapped with t-nitrosobutane. The p-nitrobenzyl (o-nitrobenzyl) radical adduct was characterized by a nitrogen hyperfine splitting of 16.5 G (17.1 G) and two equivalent beta-hydrogen hyperfine splittings of 10.6 G (14.4 G). The spin trap 5,5-dimethyl-1-pyrroline N-oxide also yields adducts characteristic of carbon-centered free radicals. This unimolecular decomposition is much faster than the disproportionation decay, which is characteristic of most nitro anion radicals, and the primary o- and p-nitrobenzyl chloride anion radicals never achieve detectable concentrations. The nitrobenzyl radical trapping is not inhibited by metyrapone or CO. In contrast, the m-nitrobenzyl anion radical does achieve a detectable steady-state concentration, which is increased 20% by either metyrapone or a CO atmosphere.     

Evidence for a free radical mechanism of styrene-glutathione conjugate formation catalyzed by prostaglandin H synthase and horseradish peroxidase

We have proposed, using styrene as a model, a new mechanism for the formation of glutathione conjugates that is independent of epoxide formation but dependent on the oxidation of glutathione to a thiyl radical by peroxidases such as prostaglandin H synthase or horseradish peroxidase. The thiyl radical reacts with styrene to yield a carbon-centered radical which subsequently reacts with molecular oxygen to give the styrene-glutathione conjugate. We have used electron spin resonance spin trapping techniques to detect the proposed free radical intermediates. A styrene carbon-centered radical was trapped using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and t-nitrosobutane. The position of the carbon-centered radical was confirmed to be at carbon 7 by the use of specific 2H-labeled styrenes. The addition of the spin trap DMPO inhibited both the utilization of molecular oxygen and the formation of styrene-glutathione conjugates. Under anaerobic conditions additional styrene-glutathione conjugates were formed, one of which was identified by fast atom bombardment mass spectrometry as S-(2-phenyl)ethylglutathione. The glutathione thiyl radical intermediate was observed by spin trapping with DMPO. These results support the proposed free radical-mediated formation of styrene-glutathione conjugates by peroxidase enzymes.     

Lipid radicals: properties and detection by spin trapping

Unsaturated lipids are rapidly oxidized to toxic products such as lipid hydroperoxides, especially when transition metals such as iron or copper are present. In a Fenton-type reaction Fe2+ converts lipid hydroperoxides to the very short-lived lipid alkoxyl radicals. The reaction was started upon the addition of Fe2+ to an aqueous linoleic acid hydroperoxide (LOOH) emulsion and the spin trap in the absence of oxygen. Even when high concentrations of spin traps were added to the incubation mixture, only secondary radical adducts were detected, probably due to the rapid re-arrangement of the primary alkoxyl radicals. With the commercially available nitroso spin trap MNP we observed a slightly immobilized ESR spectrum with only one hydrogen splitting, indicating the trapping of a methinyl fragment of a lipid radical. With DMPO or 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) adducts were detected with carbon-centered lipid radical, with acyl radical, and with the hydroxyl radical. We also synthesized lipophilic derivatives of the spin trap DEPMPO in order to detect lipid radical species generated in the lipid phase. With all spin traps studied a lipid-derived carbon-centered radical was obtained in the anaerobic incubation system Fe2+/LOOH indicating the trapping of a lipid radical, possibly generated as a secondary reaction product of the primary lipid alkoxyl radical formed. Under aerobic conditions an SOD-insensitive oxygen-centered radical adduct was formed with DEPMPO and its lipophilic derivatives. The observed ESR parameters were similar to those of alkoxyl radical adducts, which were independently synthesized in model experiments using Fe3+-catalyzed nucleophilic addition of methanol or t-butanol to the respective spin trap.     

In vivo spin trapping of free radicals generated in brain, spleen, and liver during gamma radiation of mice

Spin trapping techniques combined with electron spin resonance spectroscopy were used to capture and detect free radicals generated in vivo during exposure to ionizing radiation. Tissue extracts of mice given an intraperitoneal or intragastric dose of the spin trap, alpha-phenyl-t-butyl nitrone prior to exposure to gamma radiation (2 to 5 Gy), contained a radical adduct with hyperfine splitting constants characteristic of spin adducts of carbon-centered lipid radicals. Considerably more radicals were trapped in tissues when the trap was given 3 h before radiation as compared to 30 min before exposure. The radicals observed may either be secondary species resulting from an attack on cellular components by products of water radiolysis, or primary radicals resulting from direct interaction of the radiation with biological molecules. The results indicate that the spin trapping agent is able to penetrate well into animal tissues, and to capture radical species under conditions where the latter would be expected to occur.