Electron Paramagnetic Resonance and Spin Trapping Study of Radicals Formed During Reaction of Aromatic Amines with isoAmyl Nitrite Under Aprotic Conditions

Diazotization of primary aromatic amines with isoamyl nitrite in benzene at room temperature was studied employing EPR and spin trapping techniques. Nitrosodurene (ND). 2-methyl-2-nitrosopropane (MNP). and 5,5-dimethyl-pyrroline N-oxide (DMPO) were used as spin trapping agents. Aryl radicals were detected employing ND and MNP. Using DMPO as a spin trap most of the amines produced EPR spectra ascribed to adducts with aniline-type radicals (N-centred radicals). The assignments were verified using ¹⁵JN-labeled anilines. Similar spectra of DMPO adducts were recorded from amines treated with benzoyl peroxide or benzophenone plus UV. Possible mechanisms of formation of these adducts (radical trapping versus nucleophilic addition to DMPO followed by oxidation) during treatment of the amines with isoamyl nitrite are discussed.     

Immuno-spin trapping of hemoglobin and myoglobin radicals derived from nitrite-mediated oxidation

The reaction of nitrite with hemoglobin has become of increasing interest due to the realization that plasma nitrite may act as an NO congener that is activated by interaction with red blood cells. Using a combination of spectrophotometry, immuno-spin trapping, and EPR, we have examined the formation of radicals during the oxidation of oxyhemoglobin (oxyHb) and oxymyoglobin (oxyMb) by inorganic nitrite. The proposed intermediacy of ferryl species during this oxidation was confirmed by spectrophotometry using multiple linear regression analysis of kinetic data. Using EPR/spin trapping, a protein radical was observed in the case of oxyMb, but not oxyHb, and was inhibited by catalase. When DMPO spin trapping was combined with Western blot analysis using an anti-DMPO-nitrone antibody, globin/DMPO adducts of both oxyHb and oxyMb were detected, and their formation was inhibited by catalase. Catalase effects confirm the intermediacy of hydrogen peroxide as a heme oxidant in this system. Spectrophotometric kinetic studies revealed that the presence of DMPO elongated the lag phase and decreased the maximal rate of oxidation of both oxyHb and oxyMb, which suggests that the globin radical plays an active role in the mechanism of autocatalysis. Interestingly, the oxidation of oxyHb or oxyMb by nitrite, but not by hydrogen peroxide, produced a diffusible radical that was able to generate spin adducts on a bystander protein. This indicates that the oxidation of oxyhemeproteins by nitrite may cause more widespread oxidative damage than the corresponding oxidation by hydrogen peroxide. The immuno-spin trapping technique represents an important new development for the study of the range and extent of protein oxidation by free radicals and oxidants.     

Evidence for the formation of strand-break precursors in hydroxy-attacked thymidine 5'-monophosphate by the spin trapping method

A method combining spin trapping, ESR, and HPLC was employed to obtain evidence for the formation of sugar radicals in OH-attacked TMP with special emphasis on the detection of strand-break precursors of DNA. OH radicals were produced by irradiating an N2O-saturated aqueous solution with X-rays. When an N2O-saturated aqueous solution containing TMP and a spin trapping reagent, MNP, was irradiated with X-rays, it was estimated on the basis of theoretical calculations using rate constants that 94% of the TMP radicals were induced by OH radicals. Since several spin adducts between TMP radicals and MNP, as well as the byproducts of the spin trapping reagent itself, were produced, reverse-phase HPLC was used to separate them. The presence of six spin adducts was confirmed by ESR examination. Further examination of these spin adducts by UV absorbance spectrophotometry showed the presence of a chromophore at 260 nm in three adducts. Since a gradual increase in the release of unaltered base from these adducts was observed when they were allowed to stand for 0-22 h at room temperature, they could be regarded as the spin adducts of sugar radicals and MNP. ESR spectra from the spin adducts were consistent with hydrogen abstraction radicals at the C1', C4', and C5' positions of the sugar moiety. These radicals appeared to be precursors of AP sites and strand breaks. In addition to these spin adducts, ESR spectra that were consistent with the spin adducts of base radicals (the C5 and C6 radicals) and MNP were observed.     

Metabolism of Ethanol to 1-Hydroxyethyl Radicals in Rat Liver Microsomes: Comparative Studies with Three Spin Trapping Agents

Metabolism of ethanol to 1-hydroxyethyl radicals by rat liver microsomes was studied with three nitrone spin trapping agents (POBN, PBN, and DMPO) under essentially comparable conditions. The data indicate that POBN was the superior spin trapping agent for 1-hydroxyethyl radicals, and that DMPO was least efficient. Addition of deferoxamine completely prevented detection of 1-hydroxyethyl radicals with PBN or DMPO, but caused only 50% decrease in EPR signals when POBN was the spin trap. However, superoxide dismutase only decreased 1-hydroxyethyl radical formation when POBN was the spin trap. Other experiments demonstrated that POBN was the most effective of these nitrones for reduction of Fe(III) in aqueous solutions. Furthermore, 1-hydroxyethyl radical adducts were formed when POBN was added to mixtures of ethanol, phosphate buffer, POBN and FeCl₃, but this effect did not occur with either PBN or DMPO. Thus, these data indicate that undesirable effects of POBN on iron chemistry may influence results of spin trapping experiments, and complicate interpretation of the resulting data.     

Metabolism of Ethanol to 1-Hydroxyethyl Radicals in Rat Liver Microsomes: Comparative Studies with Three Spin Trapping Agents

Metabolism of ethanol to 1-hydroxyethyl radicals by rat liver microsomes was studied with three nitrone spin trapping agents (POBN, PBN, and DMPO) under essentially comparable conditions. The data indicate that POBN was the superior spin trapping agent for 1-hydroxyethyl radicals, and that DMPO was least efficient. Addition of deferoxamine completely prevented detection of 1-hydroxyethyl radicals with PBN or DMPO, but caused only 50% decrease in EPR signals when POBN was the spin trap. However, superoxide dismutase only decreased 1-hydroxyethyl radical formation when POBN was the spin trap. Other experiments demonstrated that POBN was the most effective of these nitrones for reduction of Fe(III) in aqueous solutions. Furthermore, 1-hydroxyethyl radical adducts were formed when POBN was added to mixtures of ethanol, phosphate buffer, POBN and FeCl₃, but this effect did not occur with either PBN or DMPO. Thus, these data indicate that undesirable effects of POBN on iron chemistry may influence results of spin trapping experiments, and complicate interpretation of the resulting data.     

Spin trapping of polyunsaturated fatty acid-derived peroxyl radicals: reassignment to alkoxyl radical adducts

Polyunsaturated fatty acid (PUFA) peroxyl radicals play a crucial role in lipid oxidation. ESR spectroscopy with the spin-trapping technique is one of the most direct methods for radical detection. There are many reports of the detection of PUFA peroxyl radical adducts; however, it has recently been reported that attempted spin trapping of organic peroxyl radicals at room temperature formed only alkoxyl radical adducts in detectable amounts. Therefore, we have reinvestigated spin trapping of the linoleic, arachidonic, and linolenic acid-derived PUFA peroxyl radicals. The slow-flow technique allowed us to obtain well-resolved ESR spectra of PUFA-derived radical adducts in a mixture of soybean lipoxygenase, PUFA, and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). However, interpretation of the ESR spectra was complicated by the overlapping of the PUFA-derived alkoxyl radical adduct spectra. In order to understand these spectra, PUFA-derived alkoxyl radical adducts were modeled by various alkoxyl radical adducts. For the first time, we synthesized a wide range of DMPO adducts with primary and secondary alkoxyl radicals. It was found that many ESR spectra previously assigned as DMPO/peroxyl radical adducts based on their close similarity to the ESR spectrum of the DMPO/superoxide radical adduct, in conjunction with their insensitivity to superoxide dismutase, are indeed alkoxyl radical adducts. We have reassigned the PUFA alkylperoxyl radical adducts to their corresponding alkoxyl radical adducts. Using hyperfine coupling constants of model DMPO/alkoxyl radical adducts, the computer simulation of DMPO/PUFA alkoxyl radical adducts was performed. It was found that the trapped, oxygen-centered PUFA-derived radical is a secondary, chiral alkoxyl radical. The presence of a chiral carbon atom leads to the formation of two diastereomers of the DMPO/PUFA alkoxyl radical adduct. Therefore, attempted spin trapping of the PUFA peroxyl radical by DMPO at room temperature leads to the formation of the PUFA alkoxyl radical adduct.     

A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions

Superoxide anions (O₂^.−) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O₂^.−. Mixtures of xanthine, xanthine oxidase, DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O₂^.−, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O₂^.−, generated by concentrations of xanthine as low as 0.05 μM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 μM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.     

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.     

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.     

The interaction of 5,5-dimethyl-1-pyrroline-N-oxide with human myeloperoxidase and its potential impact on spin trapping of neutrophil-derived free radicals

Activation of human neutrophils leads to secretion of myeloperoxidase (MPO) with resulting generation of several oxidant species including OCl-. Spin trapping techniques employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) are being applied increasingly to the investigation of free radical production by in vitro and in vivo experimental systems which contain neutrophils. Because such knowledge is critical to the interpretation of these data, we examined the impact of MPO and MPO-derived oxidants on DMPO spin adduct formation and stability. Addition of increasing concentrations of OCl- to DMPO yielded a number of EPR-detectable products including DMPO-OH. However, the concentration of OCl- required was in excess of that expected under physiologic conditions. Addition of purified human MPO and H2O2 to DMPO yielded EPR spectra consisting of small DMPO-OH peaks. The addition of MPO and H2O2 to preformed DMPO-OH and DMPO-CH3 resulted in rapid destruction of these spin adducts. Thus MPO/H2O2 appeared to both generate and destroy DMPO spin adducts. Neutrophils stimulated with phorbol myristate acetate or opsonized zymosan generated large DMPO-OOH and DMPO-OH peaks as well as small DMPO-CH3 peaks. Addition of the MPO inhibitor azide to the reaction mixture had no effecting on resulting DMPO-OH or DMPO-CH3 peak amplitudes but increased that of DMPO-OOH. These data suggest that MPO-derived oxidants likely have little impact on the nature of EPR spectra resulting from DMPO spin trapping of free radical species following neutrophil stimulation. Because MPO oxidants did appear to react with DMPO the ability of DMPO to protect a biologic target from in vitro MPO injury was examined. DMPO (greater than 10 mM) significantly decreased MPO/H2O2/Cl- -mediated erythrocyte hemolysis as assessed by 51Cr release. The experimental and/or pharmacologic implications of this observation require further study.     

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

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

Lymphocytes can produce respiratory burst and oxygen radicals as polymorphonuclear leukocytes.

The respiratory burst and production of oxygen radicals by lymphocytes stimulated with phorbol myristate acetate (PMA) was studied and compared with that of polymorphonuclear leukocytes (PMN) by electron paramagnetic resonance (EPR) and spin trapping technique. Superoxide anion and hydroxyl radicals spin adducts of DMPO were detected in the stimulated PMN system, but only hydroxyl radical spin adducts of DMPO were detected in the stimulated lymphocyte system. It was proved by superoxide dismutase (SOD) and catalase that the hydroxyl radicals produced in the stimulated lymphocyte system came from superoxide anions, just like the hydroxyl radicals produced in the stimulated PMN.     

Lymphocytes can produce respiratory burst and oxygen radicals as polymorphonuclear leukocytes

The respiratory burst and production of oxygen radicals by lymphocytes stimulated with phorbol myristate acetate (PMA) was studied and compared with that of polymorphonuclear leukocytes (PMN) by electron paramagnetic resonance (EPR) and spin trapping technique. Superoxide anion and hydroxyl radicals spin adducts of DMPO were detected in the stimulated PMN system, but only hydroxyl radical spin adducts of DMPO were detected in the stimulated lymphocyte system. It was proved by Superoxide dismutase (SOD) and catalase that the hydroxyl radicals produced in the stimulated lymphocyte system came from Superoxide anions, just like the hydroxyl radicals produced in the stimulated PMN.     

Investigation of the existence and biological role of L-arginine/nitric oxide pathway in human platelets by spin-trapping/EPR studies.

The aim of the present study was to apply spin trapping/EPR spectroscopy to investigate the existence and biological role of the L-arginine/nitric oxide pathway in human platelet aggregation. Three different spin traps were used: two nitroso, 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS) and 2-methyl-2-nitrosopropane (MNP), and a nitrone, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The effect of spin-trap concentration on the collagen-induced human platelet aggregation was compared to the anti-aggregatory effect caused by L-arginine. The results show that the nitroso spin traps (DBNBS and MNP) are more effective than L-arginine in preventing platelet aggregation. DMPO has virtually no effect on the collagen-induced aggregation except at a high concentration (300 mM). Furthermore, activation of platelets with a low concentration of collagen (17 micrograms/ml) and in the presence of DBNBS or MNP yields several EPR-detectable spin adducts. Some of the observed spin adducts do not correspond to those originating from the interaction of a free radical, nitric oxide (NO.) gas, with the spin traps [Arroyo, C.M. & Kohno, M. (1991) Free Radical Res. Commun. 14, 145-155]. Only one adduct of DBNBS, with a relative intensity of 0.1, observed in the washed-platelet experiment and in the presence of superoxide dismutase, is similar to the EPR spectrum obtained following a reaction of pure NO. gas with DBNBS. This suggests that the EPR spectrum of the DBNBS adduct consisting of a triplet may originate from the production of NO. by these cells. Additional DBNBS and MNP spin adducts were generated during platelet activation in the presence of Ca2+ and of a cytosol-depleted L-arginine preparation from washed platelets to which L-arginine was subsequently added. The formation of these DBNBS and MNP spin adducts were inhibited by N omega-methyl-L-arginine (MeArg, 100 microM), suggesting that these originated from a product of NO synthase. Furthermore, the formation of DBNBS and MNP spin adducts in platelet suspensions was enhanced by the presence of superoxide dismutase; however, their formation was prevented by the endothelial-derived relaxing factor (EDRF) inhibitors methylene blue and hemoglobin. The results from the MeArg and EDRF inhibitor experiments support the existence of the L-arginine/NO pathway in platelets. In addition, the prevention of spin-adduct formation by EDRF inhibitors, suggests that the mechanisms of EDRF formation and the L-arginine/NO pathway in endothelial cells and platelets are similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solvent Effects in the Spin Trapping Of Lipid Oxyl Radicals

Studies documenting spin trapping of lipid radicals in defined model systems have shown some surprising solvent effects with the spin trap DMPO. In aqueous reactions comparing the reduction of H₂O₂ and methyl linoleate hydroperoxide (MLOOH) by Fe^z+, hydroxyl (HO·) and lipid alkoxyl (LO·) radicals produce identical four-line spectra with line intensities 1:2:2:1. Both types of radicals react with commonly-used HO· scavengers, e.g. with ethanol to produce ·C(CH₃)HOH and with dirnethylsulfoxide (DMSO)togive ·CH₃. However, DMSO radicals (either ·CH₃or ·OOCH₃) react further with lipids, and when radicals are trapped in these MLOOH systems, multiple adducts are evident. When acetonitrile is added to the aqueous reaction systems in increasing concentrations, ·CH₂CN radicals resulting from HO· attack on acetonitrile are evident, even with trace quantities of that solvent. In contrast, little, if any, reaction of LO· with acetonitrile occurs, even in 100% acetonitrile. A single four-line signal persists in the lipid systems as long as any water is present, although the relative intensity of the two center lines decreases as solvent-induced changes gradually dissociate the nitrogen and β-hydrogen splitting constants. Extraction of the aqueous-phase adducts into ethyl acetate shows clearly that the identical four-line spectra in the H₂0₂ and MLOOH systems arise from different radical species in this study, but the lack of stability of the adducts to phase transfer may limit the use of this technique for routine adduct identification in more complex systems. These results indicate that the four-line 1:2:2:1. a_N = a_H = 14.9G spectrum from DMPO cannot automatically be assigned to the HO· adduct in reaction systems where lipid is present, even when the expected spin adducts from ethanol or DMSO appear confirmatory for HO-. Conclusive distinction between HO· and LO· ultimately will require use of ¹³C-labelled DMPO or HPLC-MS separation and specific identification of adducts when DMPO is used as the spin trap.     

EPR detection of lipid-derived free radicals from PUFA, LDL, and cell oxidations

We have used the spin trap 5,5-dimethyl-pyrroline-1-oxide (DMPO) and EPR to detect lipid-derived radicals (Ld*) during peroxidation of polyunsaturated fatty acids (PUFA), low-density lipoprotein (LDL), and cells (K-562 and MCF-7). All oxygen-centered radical adducts of DMPO from our oxidizable targets have short lifetimes (<20 min). We hypothesized that the short lifetimes of these spin adducts are due in part to their reaction with radicals formed during lipid peroxidation. We proposed that stopping the lipid peroxidation processes by separating oxidation-mediator from oxidation-substrate with an appropriate extraction would stabilize the spin adducts. To test this hypothesis we used ethyl acetate to extract the lipid-derived radical adducts of DMPO (DMPO/Ld*) from an oxidizing docosahexaenioc acid (DHA) solution; Folch extraction was used for LDL and cell experiments. The lifetimes of DMPO spin adducts post-extraction are much longer (>10 h) than the spin adducts detected without extraction. In iron-mediated DHA oxidation we observed three DMPO adducts in the aqueous phase and two in the organic phase. The aqueous phase contains DMPO/HO* aN approximately aH approximately 14.8 G) and two carbon-centered radical adducts (aN1 approximately 15.8 G, aH1 approximately 22.6 G; aN2 approximately 15.2 G, aH2 approximately 18.9 G). The organic phase contains two long-chain lipid radical adducts (aN approximately 13.5 G, aH approximately 10.2 G; and aN approximately 12.8 G; aH approximately 6.85 G, 1.9 G). We conclude that extraction significantly increases the lifetimes of the spin adducts, allowing detection of a variety of lipid-derived radicals by EPR.     

Investigating the free radical trapping ability of NXY-059, S-PBN and PBN

The spin trapping ability of the nitrones 2,4-disulphophenyl-N-tert-butyl nitrone (NXY-059), 2-sulphophenyl-N-tert-butyl nitrone (S-PBN) and α-phenyl-N-tert-butyl nitrone (PBN) for both hydroxyl and methanol radicals was investigated using electron paramagnetic resonance (EPR) spectroscopy. The radicals of interest were generated in situ in the spectrometer under constant flow conditions in the presence of each nitrone. The spin adducts formed were detected by EPR spectroscopy. This approach allowed for quantitative comparison of the EPR spectra of the spin adducts of each nitrone. The results obtained showed that NXY-059 trapped a greater number of hydroxyl and methanol radicals than the other two nitrones, under the conditions studied.     

Identification of free radicals of glycerophosphatidylcholines containing omega-6 fatty acids using spin trapping coupled with tandem mass spectrometry

Metal-catalysed radical oxidation of diacyl-glycerophosphatidylcholines (GPC) with ω-6 acyl polyunsaturated fatty acids (PAPC, palmitoyl-arachidonoyl-glycerophosphatidylcholine and PLPC, palmitoyl-lineloyl-glycerophosphatidylcholine) was studied. Free radical oxidation products were trapped by spin trapping with 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) and identified by electrospray mass spectrometry (ES-MS). The spin adducts of oxidised GPC containing one and two oxygen atoms and one and two DMPO molecules were observed as doubly charged ions. Structural characterisation by tandem mass spectrometry (MS/MS) of these ions revealed product ions corresponding to loss of the acyl chains (sn-1-palmitoyl and sn-2-oxidised spin adduct of lineloyl or arachidonoyl), loss of the spin trap (DMPO) and product ions attributed to oxidised sn-2 fatty acid spin adduct (lineloyl and arachidonoyl). Product ions formed by homolytic cleavages near the spin trap and also from 1,4 hydrogen elimination cleavages involving the hydroxy group in the sn-2 fatty acid spin adduct allowed to infer the nature of the radical. Altogether, the presence of GPC hydroxy-alkyl/DMPO and hydroxy-alkoxyl/DMPO spin adducts was proposed.     

Solvent Effects in the Spin Trapping Of Lipid Oxyl Radicals

Studies documenting spin trapping of lipid radicals in defined model systems have shown some surprising solvent effects with the spin trap DMPO. In aqueous reactions comparing the reduction of H₂O₂ and methyl linoleate hydroperoxide (MLOOH) by Fe^z+, hydroxyl (HO·) and lipid alkoxyl (LO·) radicals produce identical four-line spectra with line intensities 1:2:2:1. Both types of radicals react with commonly-used HO· scavengers, e.g. with ethanol to produce ·C(CH₃)HOH and with dirnethylsulfoxide (DMSO)togive ·CH₃. However, DMSO radicals (either ·CH₃or ·OOCH₃) react further with lipids, and when radicals are trapped in these MLOOH systems, multiple adducts are evident. When acetonitrile is added to the aqueous reaction systems in increasing concentrations, ·CH₂CN radicals resulting from HO· attack on acetonitrile are evident, even with trace quantities of that solvent. In contrast, little, if any, reaction of LO· with acetonitrile occurs, even in 100% acetonitrile. A single four-line signal persists in the lipid systems as long as any water is present, although the relative intensity of the two center lines decreases as solvent-induced changes gradually dissociate the nitrogen and β-hydrogen splitting constants. Extraction of the aqueous-phase adducts into ethyl acetate shows clearly that the identical four-line spectra in the H₂0₂ and MLOOH systems arise from different radical species in this study, but the lack of stability of the adducts to phase transfer may limit the use of this technique for routine adduct identification in more complex systems. These results indicate that the four-line 1:2:2:1. a_N = a_H = 14.9G spectrum from DMPO cannot automatically be assigned to the HO· adduct in reaction systems where lipid is present, even when the expected spin adducts from ethanol or DMSO appear confirmatory for HO-. Conclusive distinction between HO· and LO· ultimately will require use of ¹³C-labelled DMPO or HPLC-MS separation and specific identification of adducts when DMPO is used as the spin trap.     

Oxygen Radical Formation in Well-Washed Rat Liver Microsomes: Spin Trapping Studies

The generation of hydroxyl radicals by rat liver microsomes was monitored by spin trapping with 5, 5-dimethylpyrroline N-oxide (DMPO). The results confirm and extend previous data which demonstrated that hydroxyl radicals are produced by microsomes in the presence of NADPH and O₂, and without the exogenous addition of iron. No EPR signals could be detected unless catalase activity which was associated with the microsomes could be substantially diminished. Addition of azide was the most effective means of eliminating catalase activity, but azide also reacted rapidly with hydroxyl radicals, forming azidyl radicals which were in turn trapped by DMPO. Extensive washing and preincubation of microsomes with 3-amino-1, 2,4-triazole in the presence of H₂O₂ were evaluated as alternative methods of decreasing the catalase contamination of microsomes. Although neither method completely eliminated microsomal catalase activity, addition of azide was no longer necessary for hydroxyl radical detection with DMPO. When highly washed microsomal preparations were tested, weak signals of the superoxide radical adduct of DMPO could also be detected. These data indicate that the sensitivity of spin trapping in microsomal systems can be improved substantially when care is taken to eliminate cytosolic contaminants such as catalase.