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.     

Spin adducts of superoxide,alkoxyl, and lipid-derived radicals with EMPO and its derivatives

The compound 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO) is a hydrophilic cyclic nitrone spin trap, which, in contrast to DMPO, forms a relatively stable superoxide adduct (t(1/2)=8.6 min) with an EPR spectrum similar to the respective DMPO adduct. In order to find the optimal degree of lipophilicity of this novel type of spin trap with respect to the detection of radicals formed during lipid peroxidation, the ethoxy group of EMPO was replaced by alkoxy substituents of increasing chain length, leading to the methoxy- (MeMPO), 1-propoxy- (PrMPO), 1-butoxy- (BuMPO), and 1-octyloxy- (OcMPO) derivatives of EMPO. The stability of their superoxide adducts was found to be strongly dependent on the size of the alkoxycarbonyl group. Increasing chain length of the alkoxyl substituent decreased the stability of alkoxyl radical adducts of MeMPO, EMPO, and PrMPO, but increased the stability of OcMPO adducts. The stability of alkoxyl radical adducts of BuMPO, on the other hand, were practically independent of the size of the alkoxyl group. Detection of lipid alkoxyl radicals formed by peroxidizing linoleic acid in a stationary system was therefore only possible with the most lipophilic spin trap, OcMPO. However, with the more hydrophilic spin traps MeMPO, EMPO, PrMPO, and BuMPO optimal EPR signal intensity could be obtained when a slow-flow system was used. Thus, within this series EMPO is the best spin trap for the detection of superoxide; OcMPO, on the other hand, is most suitable for the detection of lipid alkoxyl radicals.     

Evaluation of spin trapping agents and trapping conditions for detection of cell-generated reactive oxygen species

Electron paramagnetic resonance with spin trapping is a useful technique to detect reactive oxygen species, such as superoxide radical anion (O2*-), a key species in many biological processes. We evaluated the abilities of four spin traps in trapping cell-generated O2*-: 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), 2-diethoxyphosphoryl-2-phenethyl-3,4-dihydro-2H-pyrrole N-oxide (DEPPEPO), 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Optimal experimental conditions for obtaining maximal signal intensity of O2*- adduct in a cellular system were first studied. The maximal intensities of BMPO, DEPMPO, and DMPO adducts were similar while DEPPEPO did not trap cell-generated O2*- induced by 1,6-benzo[a]pyrene quinone in a human mammary epithelial cell line (MCF-10A). BMPO and DEPMPO adducts were more stable, considering the stability of their maximal signal, than DMPO adduct in the tested cellular systems. In addition, we observed that O2*- spin adducts were reduced to their corresponding hydroxyl adducts in the cellular system. The selection of optimal spin trap in trapping cell-generated O2*- is discussed.     

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.     

Activation of the superoxide-generating NADPH oxidase of intestinal lymphocytes produces highly reactive free radicals from sulfite.

The objective of this study was to investigate the ability of immune cells of the small intestine to produce highly reactive free radicals from the food additive sulfites. These free radicals were characterized with a spin-trapping technique using the spin traps 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In the presence of glucose, purified lymphocytes from intestinal Peyer's patches (PP) and mesenteric lymph nodes (MLN) were stimulated with phorbol 12-myristate 13-acetate (PMA) to produce superoxide and hydroxyl DEPMPO radical adducts. The formation of these adducts was inhibited by superoxide dismutase or diphenyleneiodonium chloride, indicating that these cells produced superoxide radical during reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. With the treatment of sodium sulfite, PMA-stimulated PP lymphocytes produced a DEPMPO-sulfite radical adduct and an unknown radical adduct. When DEPMPO was replaced with DMPO, DMPO-sulfite and hydroxyl radical adducts were detected. The latter adduct resulted from DMPO oxidation by sulfate radical, which was capable of oxidizing formate or ethanol. Oxygen consumption rates were further increased after the addition of sulfite to PMA-stimulated lymphocytes, suggesting the presence of sulfiteperoxyl radical. Taken together, oxidants generated by stimulated lymphocytes oxidized sulfite to sulfite radical, which subsequently formed sulfiteperoxyl and sulfate radicals. The latter two radicals are highly reactive, contributing to increased oxidative stress, which may lead to sulfite toxicity, altered functions in intestinal lymphocytes, or both.     

Comparison of superoxide detection abilities of newly developed spin traps in the living cells

This study compared the superoxide detection abilities of four spin traps, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), 5-(diphenylphosphinoyl)-5-methyl-1pyrroline N-oxide (DPPMPO) and 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO) in living cells. Electron spin resonance (ESR) signals of the superoxide adducts were observed when spin traps were added to a suspension of human oral polymorphonuclear leukocytes (OPMNs) stimulated by phorbol 12-myristate 13-acetate. The ESR signal of the CYPMPO-superoxide adduct (CYPMPO-OOH) increased for 24 min after the initiation of the reaction, whereas the signals from DMPO-OOH and DPPMPO-OOH peaked at 6 and 10 min, respectively. The maximum concentrations of DMPO-OOH, DPPMPO-OOH and CYPMPO-OOH in OPMNs were 1.9, 6.0 and 10.7 µM, respectively. Furthermore, CYPMPO could more efficiently trap superoxide in blood PMNs compared with DEPMPO. From these results, it was concluded that CYPMPO performs better than DMPO, DPPMPO and DEPMPO for superoxide measurements in living cell systems because it has lower cytotoxicity and its superoxide adduct has a longer lifetime.     

Synthesis and biochemical applications of a solid cyclic nitrone spin trap: a relatively superior trap for detecting superoxide anions and glutathiyl radicals

A novel cyclic nitrone spin trap, 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO) as a pure white solid has been synthesized for the first time. BMPO offers several advantages over the existing spin traps in the detection and characterization of thiyl radicals, hydroxyl radicals, and superoxide anions in biological systems. The corresponding BMPO adducts exhibit distinct and characteristic electron spin resonance (ESR) spectral patterns. Unlike the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-derived superoxide adduct, the BMPO superoxide adduct does not non-enzymatically decompose to the BMPO hydroxyl adduct. This feature is clearly perceived as a definite advantage of BMPO in its biological applications. In addition, the ESR spectrum of the BMPO glutathionyl adduct (BMPO/*SG) does not fully overlap with the spectrum of its hydroxyl adduct. This spectral feature is again distinctly different from that of DMPO because the ESR spectral lines of DMPO glutathionyl and hydroxyl radical adducts largely overlap. Finally, the ESR spectra of BMPO-derived adducts exhibit a much higher signal-to-noise ratio in biological systems. These favorable chemical and spectroscopic features make BMPO ideal for the detection of superoxide anions, hydroxyl and thiyl radicals in biochemical oxidation and reduction.     

Synthesis and characterization of EMPO-derived 5,5-disubstituted 1-pyrroline N-oxides as spin traps forming exceptionally stable superoxide spin adducts

EMPO [5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide] is a highly hydrophilic cyclic nitrone spin trap, whose superoxide adduct is considerably more stable (t 1/2 = 8.6 min) than DMPO (5,5-dimethyl-1-pyrroline N-oxide, t 1/2=45 s). EPR spectra of spin adducts of EMPO and its derivatives are very similar to those of the respective DMPO spin adducts, in contrast to the rather complex spectra obtained using DEPMPO [5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide]. Several EMPO derivatives, with both the ethoxycarbonyl group and the methyl group at position 5 of the pyrroline ring being replaced by other substituents, were synthesized and characterized by 1H and 13C NMR spectroscopy. Thus, a series of derivatives was obtained that exhibit large differences in the stability of their superoxide adducts, ranging from less than one to more than 25 min. The stability of the superoxide adducts was mainly determined by the steric environment of the nitroxyl group: in compounds with less bulky 5-alkoxycarbonyl substituents the nitroxyl group is sterically less shielded, which resulted in a lower stability of the superoxide adducts. The spin density distribution, as obtained from DFT computations, was found to be nearly identical for all compounds, so that in contrast to the steric influences the spin density did not seem to be a crucial factor for the stability of the superoxide adducts.     

Comparative investigation of superoxide trapping by cyclic nitrone spin traps: the use of singular value decomposition and multiple linear regression analysis

The kinetics of the reaction between superoxide and the spin trapping agents 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), and 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) were re-examined in the superoxide-generating xanthine/xanthine oxidase system, by competition with spontaneous dismutation. The approach used singular value decomposition (SVD), multiple linear regression, and spectral simulation. The experiments were carried out using a two-syringe mixing arrangement with fast scan acquisition of 100 consecutive EPR spectra. Using SVD analysis, the extraction of both temporal and spectral information could be obtained from in a single run. The superoxide spin adduct was the exclusive EPR active species in the case of DEPMPO and BMPO, and the major component when DMPO was used. In the latter case a very low concentration of hydroxyl adduct was also observed, which did not change during the decay of the DMPO-superoxide adduct. This indicates that the hydroxyl radical adduct is not formed from the spontaneous decay of the superoxide radical adduct, as has been previously suggested [correction]. It was established that in short-term studies (up to 100 s) DMPO was the superior spin trapping agent, but for reaction times longer than 100 s the other two spin traps were more advantageous. The second order rate constants for the spin trapping reaction were found to be DMPO (2.4 M(-1)s(-1)), DEPMPO (0.53 M(-1)s(-1)), and BMPO (0.24 M(-1)s(-1)) determined through competition with spontaneous dismutation of superoxide, at pH 7.4 and 20 degrees C.     

Reassignment of organic peroxyl radical adducts.

The study of the important role of peroxyl radicals in biological systems is limited by their difficult detection with direct electron spin resonance (ESR). Many ESR spectra were assigned to 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/peroxyl radical adducts based only on the close similarity of their ESR spectra to that of DMPO/superoxide radical adduct in conjunction with their insensitivity to superoxide dismutase, which distinguishes the radical adduct from DMPO/superoxide radical adduct. Later, the spin-trapping literature reported that DMPO/peroxyl radical adducts have virtually the same hyperfine coupling constants as synthesized alkoxyl radical adducts, raising the issue of the correct assignment of peroxyl radical adducts. However, using 17O-isotope labelling, the methylperoxyl and methoxyl radical adducts should be distinguishable. We have reinvestigated the spin trapping of the methylperoxyl radical. The methylperoxyl radical was generated in aerobic solution with 17O-molecular oxygen either in a Fenton system with dimethylsulfoxide or in a chloroperoxidase system with tert-butyl hydroperoxide. Two different spin traps, DMPO and 2,2,4-trimethyl-2H-imidazole-1-oxide (TMIO), were used to trap methylperoxyl radical. 17O-labelled methanol was used to synthesize methoxyl radical adducts by nucleophylic addition. It was shown that the 17O hyperfine coupling constants of radical adducts formed in methylperoxyl radical-generating systems are identical to that of the methoxyl radical adduct. Therefore, methylperoxyl radical-producing systems form detectable methoxyl radical adduct, but not detectable methylperoxyl radical adducts at room temperature. One of the possible mechanisms is the decomposition of peroxyl radical adduct with the formation of secondary alkoxyl radical adduct. These results allow us to reinterpret previously published data reporting detection of peroxyl radical adducts. We suggest that detection of 17O-alkoxyl radical adduct from 17O-labelled molecular oxygen can be used as indirect evidence for peroxyl radical generation.     

Improved method for EPR detection of DEPMPO-superoxide radicals by liquid nitrogen freezing

5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) is frequently used as a spin trap for the measurement of superoxide by EPR spectrometry. However, its half life is fairly short in room temperature. We here show that superoxide radicals trapped by DEPMPO can be successfully recorded at -196 degrees C. Moreover, we show that the signal intensity remains unaltered for up to 7 days, when the samples are stored in liquid nitrogen. Our new approach for measurement of superoxide should greatly simplify the studies of this important radical in biological systems.     

Studies on the photolytic breakdown of hydroperoxides and peroxidized fatty acids by using electron spin resonance spectroscopy. Spin trapping of alkoxyl and peroxyl radicals in organic solvents.

Spin trapping using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has been used to detect and distinguish between the carbon-centred, alkoxyl, and peroxyl radicals produced during the photolytic decomposition of hydroperoxides. Photolysis of tert-butyl and cumene hydroperoxides, and peroxidized fatty acids, in toluene, with low levels of u.v. light, is shown to lead to the initial production of alkoxyl radicals by homolysis of the oxygen-oxygen bond. Subsequent reaction of these radicals with excess hydroperoxide leads, by hydrogen abstraction, to the production of peroxyl radicals that can be detected as their corresponding adducts with the spin trap. Subsequent breakdown of these adducts produces alkoxyl radicals and a further species that is believed to be the oxidized spin-trap radical 5,5-dimethyl-1-pyrrolidone-2-oxyl. No evidence was obtained at low hydroperoxide concentrations, with either the cumene or lipid alkoxyl radicals, for the occurrence of beta-scission reactions; the production of low levels of carbon-centred radicals is believed to be due to the alternative reactions of hydrogen abstraction, ring closure, and/or 1,2 hydrogen shifts. Analogous experiments with 3,3,5,5-tetramethyl-1-pyrroline N-oxide (TMPO) led only to the trapping of alkoxyl radicals with no evidence for peroxyl radical adducts, this is presumably due to a decreased rate of radical addition because of increased steric hindrance.     

Detection of peroxyl and alkoxyl radicals produced by reaction of hydroperoxides with rat liver microsomal fractions.

E.s.r. spin trapping using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to detect peroxyl, alkoxyl and carbon-centred radicals produced by reaction of t-butyl hydroperoxide (tBuOOH) with rat liver microsomal fraction. The similarity of the hyperfine coupling constants of the peroxyl and alkoxyl radical adducts to those obtained previously with isolated enzymes suggests that these species are the tBuOO. and tBuO. adducts. The effects of metal-ion chelators, heat denaturation, enzyme inhibitors and reducing equivalents demonstrate that these species arise from reaction of tBuOOH with a haem enzyme such as cytochrome P-450 or cytochrome b5. In the absence of NADPH or NADH the previously undetected peroxyl radical adduct is the major species observed. In the presence of these reducing equivalents the alkoxyl and carbon-centred radical adducts predominate, which is in accord with product studies on similar systems. These results demonstrate that both reductive and oxidative decomposition of tBuOOH can occur in rat liver microsomal fraction with the reductive pathway favoured in the presence of NADH or NADPH.     

Evaluation of DEPMPO as a spin trapping agent in biological systems

Cellular toxicity, pharmacokinetics, and the in vitro and in vivo stability of the SO3*- spin adduct of the spin trap, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-n-oxide (DEPMPO), was investigated, and the results were compared with those of the widely used spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Similar to DMPO, DEPMPO was quickly taken up (<15 min) after intraperitoneal injection, and distributed evenly in the liver, heart, and blood of the mice. In the presence of ascorbate the in vitro stability of the adduct DEPMPO/SO3*- was 7 times better than DMPO/SO3*-. Under in vivo conditions, the spin adduct DEPMPO/SO3*- was 2-4 times more stable than DMPO/ SO3*-, depending on the route of administration of the adducts. Using a low frequency EPR spectrometer, we were able to observe the spin trapped SO3*- radical both with DMPO and DEPMPO directly in the intact mouse. DEPMPO had a detectable spin adduct signal at a concentration as low as 1 mM, as compared to 5 mM for DMPO. We conclude that DEPMPO is potentially a good candidate for trapping radicals in functioning biological systems, and represents an improvement over the commonly used trap DMPO.     

Trapping of free radicals with direct in vivo EPR detection: a comparison of 5,5-dimethyl-1-pyrroline-N-oxide and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide as spin traps for HO* and SO4*-.

To spin trap hydroxyl radical (HO*) with in vivo detection of the resultant radical adducts, the use of two spin traps, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) (10 mmol/kg) has been compared. In mice treatment with 5-aminolevulinic acid and Fe3+ resulted in detection of adducts of hydroxyl radicals (HO*), but only with use of DEPMPO. Similarly, 'HO* adducts' generated via nucleophilic substitution of SO4*- adducts formed in vivo could be observed only when using DEPMPO as the spin trap. The reasons for the differences observed between DEPMPO and DMPO are likely due to different in vivo lifetimes of their hydroxyl radical adducts. These results seem to be the first direct in vivo EPR detection of hydroxyl radical adducts.     

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.     

Free radical formation from organic hydroperoxides in isolated human polymorphonuclear neutrophils.

We have demonstrated with electron paramagnetic resonance (EPR) that organic hydroperoxides are decomposed to free radicals by both human polymorphonuclear leukocytes (PMNs) and purified myeloperoxidase. When tert-butyl hydroperoxide was incubated with either PMNs or purified myeloperoxidase, peroxyl, alkoxyl, and alkyl radicals were trapped by the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In the case of ethyl hydroperoxide, DMPO radical adducts of peroxyl and alkyl (identified as alpha-hydroxyethyl when trapped by tert-nitrosobutane) radicals were detected. Radical adduct formation was inhibited when azide was added to the incubation mixture. Myeloperoxidase-deficient PMNs produced DMPO radical adduct intensities at only about 20-30% of that of normal PMNs. Our studies suggest that myeloperoxidase in PMNs is primarily responsible for the decomposition of organic hydroperoxides to free radicals. The finding of the free radical formation derived from organic hydroperoxides by PMNs may be related to the cytotoxicity of this class of compounds.     

Desulfonation of a colitis inducer 2,4,6-trinitrobenzene sulfonic acid produces sulfite radical

2,4,6-Trinitrobenzene sulfonic acid (TNBS) has been used in vivo to induce colitis. With the nitroreductase of intestinal cells, TNBS underwent redox cycling to produce TNBS-nitro and superoxide radical anions which are thought to be involved in initial oxidative reactions that lead to colonic injury. In this study, we demonstrated that the TNBS desulfonative reaction with tissue amino acids produces sulfite which is subsequently oxidized to sulfite radical. Sulfite radical was measured using a spin trapping methodology. Sulfite radical adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) were detected in a mixture of TNBS and lysine, xanthine oxidase, red blood cells, colonic mucosal or submucosal muscle tissues. TNBS alone did not produce sulfite radical, indicating that its formation required the presence of amino acids. Because sulfite radical is the precursor of highly reactive sulfiteperoxyl and sulfate radicals, our data imply that these sulfite-derived free radicals may also contribute to oxidative reactions leading to colonic injury in TNBS-induced colitis.     

Generation of free radicals from organic hydroperoxide tumor promoters in isolated mouse keratinocytes. Formation of alkyl and alkoxyl radicals from tert-butyl hydroperoxide and cumene hydroperoxide

The organic hydroperoxides tert-butyl hydroperoxide and cumene hydroperoxide are tumor promoters in the skin of SENCAR mice, and this activity is presumed to be mediated through the activation of the hydroperoxides to free radical species. In this study we have assessed the generation of free radicals from organic hydroperoxides in the target cell (the murine basal keratinocyte) using electron spin resonance. Incubation of primary isolates of keratinocytes from SENCAR mice in the presence of spin traps (5,5-dimethyl-1-pyrroline N-oxide or 2-methyl-2-nitrosopropane) and either tert-butyl hydroperoxide or cumene hydroperoxide resulted in the generation and detection of radical adducts of these spin traps. tert-Butyl alkoxyl and alkyl radical adducts of 5,5-dimethyl-1-pyrroline N-oxide were detected shortly after addition of tert-butyl hydroperoxide, whereas only alkyl radical adducts were observed with cumene hydroperoxide. Spin trapping of the alkyl radicals with 2-methyl-2-nitrosopropane led to the identification of methyl and ethyl radical adducts following both tert-butyl hydroperoxide and cumene hydroperoxide exposures. Prior heating of the cells to 100 degrees C for 30 min prevented radical formation. The radical generating capacity of subcellular fractions of these epidermal cells was examined using 5,5-dimethyl-1-pyrroline N-oxide and cumene hydroperoxide, and this activity was confined to the 105,000 X g supernatant fraction.     

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.