Oxidation of cyanide to the cyanyl radical by peroxidase/H2O2 systems as determined by spin trapping

The cyanyl radical was formed during the oxidation of potassium or sodium cyanide by horseradish peroxidase, lactoperoxidase, chloroperoxidase, NADH peroxidase, or methemoglobin in the presence of hydrogen peroxide. The spin adducts of the cyanyl radical with 5,5-dimethyl-1-pyrroline-N-oxide and N-tert-butyl-alpha-phenylnitrone were quite stable at neutral pH. The identity of these spin adducts could be demonstrated using 13C-labeled cyanide and by comparison with the spin adducts of the formamide radical, a hydrolysis product of the cyanyl radical adduct. The enzymatic conversion of cyanide to cyanyl radical by peroxidases should be considered in addition to its well-known role as a metal ligand. Furthermore, since cyanide is used routinely as an inhibitor of peroxidases, some consideration should be given to the biochemical consequences of this formation of the cyanyl radical by the catalytic activity of these enzymes.     

Spin-trapping of superoxide ion by a water-soluble, nitroso-aromatic spin-trap

Spin-trapping of superoxide ion, O2-, which is produced from two different sources (OH(-)-DMSO and xanthine-xanthine oxidase systems), was investigated by use of a water-soluble, notroso-aromatic spin trap, sodium 3,5-dibromo-4-nitrosobenzene-sulfonate (DBNBS). It was found that O2- from all sources was easily trapped by DBNBS to yield the stable O2- adduct showing the ESR spectrum consisting of a triplet of a triplet [aN (1) = 12.63 G and aH (2) = 0.71 G]. Hydroperoxy radical (HO2.), which can be generated from the oxidation of hydrogen peroxide with Ce4+ ion, was not trapped by DBNBS. These results indicate that the trapped radical is O2-, but not HO2..     

Mass spectral analysis of protein-based radicals using DBNBS. Nonradical adduct formation versus spin trapping

Protein-based radicals generated in the reaction of ferricytochrome c (cyt c) with H(2)O(2) were investigated by electrospray mass spectrometry (ESI-MS) using 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS). Up to four DBNBS-cyt c adducts were observed in the mass spectra. However, by varying the reaction conditions (0-5 molar equivalents of H(2)O(2) and substituting cyt c with its cyanide adduct which is resistant to peroxidation), noncovalent DBNBS adduct formation was inferred. Nonetheless, optical difference spectra revealed the presence of a small fraction of covalently trapped DBNBS. To probe the nature of the noncovalent DBNBS adducts, the less basic proteins, metmyoglobin (Mb) and alpha-lactalbumin, were substituted for cyt c in the cyt c/H(2)O(2)/DBNBS reaction. A maximum of two DBNBS adducts were observed in the mass spectra of the products of the Mb/H(2)O(2)/DBNBS reactions, whereas no adducts were detected following alpha-lactalbumin/H(2)O(2)/DBNBS incubation, which is consistent with adduct formation via spin trapping only. Titration with DBNBS at pH 2.0 yielded noncovalent DBNBS-cyt c adducts and induced folding of acid-denatured cyt c, as monitored by ESI-MS and optical spectroscopy, respectively. Thus, the noncovalent DBNBS-cyt c mass adducts observed are assigned to ion pair formation occurring between the negatively charged sulfonate group on DBNBS and positively charged surface residues on cyt c. The results reveal the pitfalls inherent in using mass spectral data with negatively charged spin traps such as DBNBS to identify sites of radical formation on basic proteins such as cyt c.     

Sonolysis, radiolysis, and hydrogen peroxide photolysis of pyrimidine derivatives in aqueous solutions: a spin-trapping study

The sonolysis of aqueous solutions of various dihydropyrimidines and substituted pyrimidines was investigated by ESR and spin trapping with the nonvolatile, water soluble spin trap, 3,5-dibromonitrosobenzene sulfonate (DBNBS) and its deuterated analog to examine the possibility of detecting new radicals specifically generated in the high temperature zones produced by collapsing cavitation bubbles. Similar ESR spectra were obtained from sonolysis of argon-saturated aqueous solutions, from uv photolysis of aqueous solutions containing H2O2, and from gamma radiolysis of nitrous oxide saturated solutions, although sonolysis of aqueous solutions leads to the formation of pyrimidine radicals by H atom as well as OH radical addition to the 5,6 double bond of pyrimidines. No evidence for specific new radicals formed in the high temperature regions induced by cavitation could be found. For the reactions of dihydropyrimidines with hydroxyl radicals additional spin adducts could be detected and identified with the spin trap DBNBS compared to 2-methyl-2-nitrosopropane which was used in previous studies; however, for alkylpyrimidines fewer spin adducts were observed. The use of the deuterated analog of DBNBS is helpful for unambiguous radical structure assignment.     

Spin trapping artifacts in DMSO

Spin-trapping experiments in alkaline aqueous dimethyl sulfoxide (DMSO) solution using sodium 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS) yielded a strong signal of the sulfur trioxide anion radical adduct. This radical adduct is identical to that obtained by the oxidation of sulfite with horseradish peroxidase/hydrogen peroxide and subsequent spin trapping with DBNBS. This radical adduct is very stable, and satellite peaks of the natural abundance 13C and 33S could be obtained. Apparently, under alkaline conditions DMSO decomposes in air to form the sulfur trioxide anion radical. A comparison with a recent publication shows that this DMSO-derived radical adduct has been misassigned as a uniquely stable spin adduct of superoxide (Ozawa and Hanaki (1986) Biochem. Biophys. Res. Commun. 136, 657-664).     

Receptor-mediated generation of an EDRF-like intermediate in a neuronal cell line detected by spin trapping techniques

We have studied receptor-mediated generation of an activator of soluble guanylate cyclase in cultured mouse neuroblastoma cells (clone N1E-115) by ESR/spin trapping spectroscopy. A spin adduct was detected during the activation of muscarinic receptors by carbamylcholine in the presence of the spin trap 3,5-dibromo 4-nitrosobenzene sulphonate (DBNBS). The spin adduct does not correspond to that originating from the free radical nitric oxide or hydroxylamine. The same adduct was generated in cytosol preparations from N1E-115 cells incubated with L-arginine, NADPH, in the presence of calcium. The use of isotopically labelled guanidino-N15-L-arginine supported the generation of a DBNBS spin trapped adduct originating from the guanidino moiety of L-arginine. Superoxide dismutase (SOD) stabilized the precursor of the spin adduct as well as the activator of soluble guanylate cyclase derived from L-arginine. Our results provide direct evidence for the receptor-mediated formation of a diffusible precursor of NO. derived from L-arginine.     

Evaluation of dibromonitrosobenzene sulfonate as a spin trap in biological systems

In the present study dibromonitrosobenzene sulfonate (DBNBS) was examined for its suitability for spin trapping for ESR detection of superoxide radicals in biological systems. This nitroso spin trap recently has been reported to yield very persistent spin adducts with O2. as well as with various carbon-centered radicals. In the present work the possible toxicity of DBNBS, the partitioning of its spin adducts into cells, and the stability of the adducts and the parent compound inside cells were studied. No significant toxicity was found. In cellular systems, however, DBNBS did not produce detectable adducts with O2.; it also did not detectably trap superoxide generated in the xanthine/xanthine oxidase system. Both DBNBS and a DBNBS adduct performed extracellularly and then added to cell suspensions were rapidly metabolized by cells. Intracellular spin adducts were not detected under any condition. Evidently, in spite of its promising features, DBNBS will not be useful for spin trapping radicals in cellular systems or for detecting superoxide radicals in any biological system.     

Spin trapping of the superoxide anion: complications in the use of the water-soluble nitroso-aromatic reagent DBNBS

Sodium 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS) is reported to be a useful spin trap for the measurement of superoxide anions in aqueous solution. However, the signal observed arises from interaction of the spin trap with some species other than the superoxide radical or hydrogen peroxide, a product of its dismutation, as the addition of both superoxide dismutase and catalase to a superoxide generating system failed to attenuate the signal. Therefore caution must be employed in the interpretation of results obtained using this spin trap.     

Spin-trapping of sulfite radical anion, SO3-., by a water-soluble, nitroso-aromatic spin-trap

Sulfite radical anion, SO3-., which is generated either by non-enzymatic reaction of hydrogen peroxide (H2O2-) with sulfite (SO3(2-)) or by the oxidation of bisulfite (HSO3) with Ce4+ ion, can be trapped with a water-soluble, nitroso-aromatic spin-trap, sodium 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS, 1), yielding an ESR spectrum with coupling constants [aN (1) = 12.9 G, aH (2) = 0.8 G] and a g-value of 2.0063. The SO3- radical adduct (spin adduct) was observed even in the presence of the very low concentration of H2O2 (1.21 X 10(-2) mumol).     

EPR spin-trapping of a myeloperoxidase protein radical

Incubation of myeloperoxidase (MPO) with H(2)O(2) in the presence of the spin trap DBNBS (3,5-dibromo-4-nitrosobenzenesulfonic acid) results in the EPR-detectable formation of a partially immobilized protein radical. The radical was only formed in the presence of both MPO and H(2)O(2), indicating that catalytic turnover of the protein is required. The changes in the EPR spectrum of the adduct upon treatment with pronase confirm that the spin trap is bound to a protein residue. These results establish that MPO, like lactoperoxidase [Lardinois, O. M., Medzihradszky, K. F., and Ortiz de Montellano, P. R. (1999) J. Biol. Chem. 274, 35441-35448], reacts with H(2)O(2) to give a protein radical intermediate. The protein radical may have a catalytic role, may be related to covalent binding of the prosthetic heme group to the protein, or may reflect a process that leads to inactivation of the enzyme.     

Probing the free radicals formed in the metmyoglobin-hydrogen peroxide reaction

The reaction between metmyoglobin and hydrogen peroxide results in the two-electron reduction of H2O2 by the protein, with concomitant formation of a ferryl-oxo heme and a protein-centered free radical. Sperm whale metmyoglobin, which contains three tyrosine residues (Tyr-103, Tyr-146, and Tyr-151) and two tryptophan residues (Trp-7 and Trp-14), forms a tryptophanyl radical at residue 14 that reacts with O2 to form a peroxyl radical and also forms distinct tyrosyl radicals at Tyr-103 and Tyr-151. Horse metmyoglobin, which lacks Tyr-151 of the sperm whale protein, forms an oxygen-reactive tryptophanyl radical and also a phenoxyl radical at Tyr-103. Human metmyoglobin, in addition to the tyrosine and tryptophan radicals formed on horse metmyoglobin, also forms a Cys-110-centered thiyl radical that can also form a peroxyl radical. The tryptophanyl radicals react both with molecular oxygen and with the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS). The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) traps the Tyr-103 radicals and the Cys-110 thiyl radical of human myoglobin, and 2-methyl-2-nitrosopropane (MNP) traps all of the tyrosyl radicals. When excess H2O2 is used, DBNBS traps only a tyrosyl radical on horse myoglobin, but the detection of peroxyl radicals and the loss of tryptophan fluorescence support tryptophan oxidation under those conditions. Kinetic analysis of the formation of the various free radicals suggests that tryptophanyl radical and tyrosyl radical formation are independent events, and that formation of the Cys-110 thiyl radical on human myoglobin occurs via oxidation of the thiol group by the Tyr-103 phenoxyl radical. Peptide mapping studies of the radical adducts and direct EPR studies at low temperature and room temperature support the conclusions of the EPR spin trapping studies.     

Reaction of the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate with human biofluids

Using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), an oxidant was previously detected in the plasma of patients with renal failure and the synovial tissue of rheumatoid arthritis patients. This oxidant has been shown to react with DBNBS to give a 3-line electron paramagnetic resonance (EPR) spectrum, previously assigned to the DBNBS radical cation (DBNBS*(+). However, confusion has arisen as to whether this paramagnetic species is indeed DBNBS*(+) or, rather, the DBNBS sulfite radical adduct (DBNBS-SO(3)*(-)). In the present study, DBNBS*(+) (a(N)=1.32 mT) was distinguished from DBNBS-SO(3)*(-) (a(N)=1.32 mT with an additional splitting of a(H)=0.06 mT) by (a) using different EPR parameters, (b) determining the effect of addition of sulfite on the EPR spectrum resulting from the incubation of DBNBS with either human biofluids or the horseradish peroxidase (HRP)-hydrogen peroxide (H(2)O(2)) system, and (c) replacing DBNBS with its analogues (DBNBS-d(2,) DBNBS-15N and DBNBS-d(2)-15N) in the two systems.     

N-2-(2-ethoxycarbonyl-propyl) alpha-phenylnitrone: an efficacious lipophilic spin trap for superoxide detection.

The spin trap N-2-(2-ethoxycarbonyl-propyl) alpha-phenylnitrone, EPPN 1, synthesized by methods previously described, has been purified by recrystallization. A measure of its octanol-phosphate buffer partition coefficient (P(oct) = 29.8) indicated that EPPN was quite lipophilic, yet it could be easily solubilized in water up to 60 mmol L(-1). Although this nitrone was unsuitable for detecting hydroxyl radical, it efficiently trapped several carbon-centered radicals as well as superoxide in aqueous media, without yielding any artifactual signals. Kinetic studies of the superoxide adduct decay gave rate constants k(D) of 2 x 10(-3) and 2.1 x 10(-3) s(-1) at pH 5.6 and pH 7, respectively. EPPN can be considered as an easily prepared and highly pure spin trap, allowing efficient detection of superoxide in aqueous environments.     

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)     

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.     

Identification of the myoglobin tyrosyl radical by immuno-spin trapping and its dimerization

5,5-Dimethyl-1-pyrroline N-oxide (DMPO) spin trapping in conjunction with antibodies specific for the DMPO nitrone epitope was used on hydrogen peroxide-treated sperm whale and horse heart myoglobins to determine the site of protein nitrone adduct formation. The present study demonstrates that the sperm whale myoglobin tyrosyl radical, formed by hydrogen peroxide-dependent self-peroxidation, can either react with another tyrosyl radical, resulting in a dityrosine cross-linkage, or react with the spin trap DMPO to form a diamagnetic nitrone adduct. The reaction of sperm whale myoglobin with equimolar hydrogen peroxide resulted in the formation of a myoglobin dimer detectable by electrophoresis/protein staining. Addition of DMPO resulted in the trapping of the globin radical, which was detected by Western blot. The location of this adduct was demonstrated to be at tyrosine-103 by MS/MS and site-specific mutagenicity. Interestingly, formation of the myoglobin dimer, which is known to be formed primarily by cross-linkage of tyrosine-151, was inhibited by the addition of DMPO.     

EPR kinetic studies of superoxide radicals generated during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium, a bioactivated intermediate of parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

1-Methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), a metabolic product of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been shown to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected as a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).O2- spin adduct by spin trapping in combination with EPR techniques. The rate of formation of spin adduct was dependent not only on the concentrations of MPDP+ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP+, and of superoxide dismutase to effectively compete with this reaction for the available O2-, has been used as a convenient competition reaction to quantitatively determine various kinetic parameters. Thus, using this technique the rate constant for scavenging of superoxide radical by superoxide dismutase was found to be 7.56 x 10(9) M-1 s-1. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP+ was found to be 4.48 x 10(-10) M s-1. The rate constant (K1) for MPDP+ making superoxide radical was found to be 3.97 x 10(-6) s-1. The secondary order rate constant (KDMPO) for DMPO-trapping superoxide radicals was found to be 10.2 M-1 s-1. The lifetime of superoxide radical at pH 10.0 was calculated to be 1.25 s. These values are in close agreement to the published values obtained using different experimental techniques. These results indicate that superoxide radicals are produced during spontaneous oxidation of MPDP+ and that EPR spin trapping can be used to determine the rate constants and lifetime of free radicals generated in aqueous solutions. It appears likely that the nigrostriatal toxicity of MPTP/MPDP+ leading to Parkinson's disease may largely be due to the reactivity of these radicals.     

Spin trapping of lipid radicals with DEPMPO-derived spin traps: detection of superoxide, alkyl and alkoxyl radicals in aqueous and lipid phase

The spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) forms a superoxide adduct with a half-life of almost 15 min. DEPMPO is very hydrophilic and its use for the detection of radicals in the lipid phase (lipid-derived radicals and superoxide generated in the lipid phase) is therefore limited due to its very low concentration in the lipid phase. For the detection of lipid-derived radicals, three derivatives of DEPMPO with increasing degree of lipid solubility have been investigated: 5-(di-n-propoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DPPMPO), 5-(di-n-butoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DBPMPO), and 5-(bis-(2-ethylhexyloxy)phosphoryl)-5-methyl-1-pyrroline N-oxide (DEHPMPO). As compared with the spin trap DMPO, the half-lives of the respective superoxide adducts were clearly higher in aqueous solutions of the spin traps, which facilitates qualitative ESR measurements. The stability of the superoxide spin adducts formed with the various lipophilic spin traps in aqueous buffer were similar to those observed with DEPMPO (half-life: 7-11 min.). In model experiments using Fe(3+)-catalyzed nucleophilic addition of methanol or tert-butanol to the respective spin trap the respective alkoxyl radical adducts were formed in aqueous solution as transient species in the presence of high concentrations of the alcohol. Upon dilution with water the alkoxyl group was substituted by water, giving the respective hydroxyl adduct of the spin trap. Care must therefore be taken when Fenton-type reactions are used for the generation of radicals such as the use of Fe(2+) complexes with phosphate or DTPA or inactivation of iron by addition of "Desferal" (Novarti's Pharma GmbH, Vienna, Austria) after a short incubation time. Addition of Fe(2+) under anaerobic conditions to an aqueous suspension of linoleic acid hydroperoxide and the spin trap resulted in the detection of three different species: a carbon-centered radical adduct, an acyl radical adduct, and the hydroxyl adduct. In the presence of oxygen a different species was observed with DEPMPO, DPPMPO, and DBPMPO, which was only slightly suppressed upon the addition of SOD, possibly the respective spin adduct of either the alkylperoxyl radical or, in analogy to DMPO, a secondary alkoxyl radical.     

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.     

Identification of free radicals of glycerophosphatidylcholines containing ω-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.