Characterization of the radical product formed from the reaction of nitric oxide with the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate.

Previously, 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) has been used in combination with electron paramagnetic resonance (EPR) spectrometry to trap nitric oxide (NO(*)). The reaction between DBNBS and NO(*) yields a radical product which gives rise to an EPR signal consisting of three lines with an A(N) = 0.96 mT, but the structure of this product is unknown. A two-stage high-performance liquid chromatography fractionation was performed to isolate the radical product from the other components in the DBNBS/NO(*) reaction mixture. The fractions containing the radical product were identified by the presence of the three-line EPR signal, and then these fractions were analyzed by negative ion fast atom bombardment-mass spectrometry (FAB-MS). Collectively, the FAB-MS data suggested that the radical product is the monosodium electrostatic complex with the dianion, bis(2,6-dibromo-4-sulfophenyl) nitroxyl. Analysis of the Gaussian and Lorentzian linewidths of the EPR signal suggested that bis(2,6-dibromo-4-sulfophenyl) nitroxyl molecules may group together to form micelles. Further studies also indicated that significant amounts of nitrogen and nitrate were produced during the reaction between DBNBS and NO(*). A reaction scheme consistent with these results is presented.     

Nitrite determination in human plasma and synovial fluid using reactions of nitric oxide with 3, 5-dibromo-4-nitrosobenzenesulphonate (DBNBS)

DBNBS (3,5-dibromo-4-nitrosobenzenesulphonate) reacts with nitric oxide (NO) produced from nitrite ions in acid solution to give a radical with a characteristic electron spin resonance spectrum, attributable to a 'DBNBS-NO' product, and comprising a triplet with alphaN=0.96 mT. This is identical with the spectrum obtained when NO, introduced from the gas phase, reacts with DBNBS. Under certain conditions, an additional signal is observed, attributable to oxidation of DBNBS to the radical cation, DBNBS*+ (a triplet with alphaN=1.32 mT). Conditions are described for the determination of nitrite, which avoid this DBNBS oxidation. The height of the low-field signal from the DBNBS-NO product is directly proportional to the nitrite concentration up to about 0.08 mM nitrite. The method has been applied to the measurement of nitrite concentrations in whole blood, plasma and synovial fluid taken from rheumatoid arthritis patients. In order to avoid the oxidation of DBNBS when analysing biological samples of this type, it is necessary to treat the specimen by ultrafiltration as soon as possible after collection and before addition of DBNBS.     

Structure of a substrate radical intermediate in the reaction of lysine 2,3-aminomutase.

Electron paramagnetic resonance (EPR) spectroscopy has been used to characterize an organic radical that appears in the steady state of the reaction catalyzed by lysine 2,3-aminomutase from Clostridium SB4. Results of a previous electron paramagnetic resonance (EPR) study [Ballinger, M. D., Reed, G. H., & Frey, P. A. (1992) Biochemistry 31, 949-953] demonstrated the presence of EPR signals from an organic radical in reaction mixtures of the enzyme. The materialization of these signals depended upon the presence of the enzyme, all of its cofactors, and the substrate, lysine. Changes in the EPR spectrum in response to deuteration in the substrate implicated the carbon skeleton of lysine as host for the radical center. This radical has been further characterized by EPR measurements on samples with isotopically substituted forms of lysine and by analysis of the hyperfine splittings in resolution-enhanced spectra by computer simulations. Changes in the hyperfine splitting patterns in EPR spectra from samples with [2-2H]lysine and [2-13C]-lysine show that the paramagnetic species is a pi-radical with the unpaired spin localized primarily in a p orbital on C2 of beta-lysine. In the EPR spectrum of this radical, the alpha-proton, the beta-nitrogen, and the beta-proton are responsible for the hyperfine structure. Analysis of spectra for reactions initiated with L-lysine, [3,3,4,4,5,5,6,6-2H8]lysine, [2-2H]lysine, perdeuteriolysine, [alpha-15N]lysine, and [alpha-15N,2-2H]lysine permit a self-consistent assignment of hyperfine splittings.(ABSTRACT TRUNCATED AT 250 WORDS)     

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).     

Identification of a radical formed in the reaction mixture of rat brain homogenate with a ferrous ion/ascorbic acid system using HPLC-EPR and HPLC-EPR-MS

Identification of a free radical is performed for the reaction mixture of rat brain homogenate with a ferrous ion/ascorbic acid system using EPR, high performance liquid chromatography-electron paramagnetic resonance spectrometry (HPLC-EPR) and high performance liquid chromatography-electron paramagnetic resonance-mass spectrometry (HPLC-EPR-MS). EPR measurements of the reaction mixtures showed prominent signals with hyperfine coupling constants (alpha(N) = 1.58 mT and alpha(H)beta = 0.26 mT). No EPR spectrum was detectable without rat brain homogenate, suggesting that the radical is derived from rat brain homogenate. An HPLC-EPR analysis of the reaction mixture showed a peak with retention time of 33.7 min. An HPLC-EPR-MS analysis of the peak gave two ions at m/z 224 and 137, suggesting that alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN)/ethyl radical adduct forms in the reaction mixture.     

Formation of long-lived hydroxyl free radical adducts of proline and hydroxyproline in a Fenton reaction

Proline and hydroxyproline when exposed to the hydroxyl free radical generating system of ADP-Fe(II)-H2O2 yielded long-lived free radicals. An analysis of the electron paramagnetic resonance spectra of the long-lived hydroxyl free radical adducts of proline and hydroxyproline is consistent with a free electron on a nitroxyl group interacting with the nitrogen atom as well as with three separate protons. In the case of proline, nitroxide formation was observed under the influence of tert-butyl-hydroperoxide, giving a similar EPR spectrum (Lin, J.S., Tom, T.C. and Olcott, H.S. (1974) J. Agr. Food Chem. 22, 526-528); however, the hydroxyl free radical adduct of hydroxyproline has not been described yet. In the case of the proline nitroxide radical, two of the three protons involved interact with the free electron equivalently. The coupling constants for the hydroxyl free radical adduct of proline are AN = 1.58 mT, AH1 beta = AH2 beta = 2.13 mT, AH3 beta = 1.77 mT and for hydroxyproline are AN = 1.54 mT, AH1 beta = 2.56 mT, AH2 beta = 2.03 and AH3 beta = 1.51. The data are consistent with the amine nitrogen of proline and hydroxyproline being oxidized to a nitroxyl group and the free electron of the nitroxyl interacting with the beta-protons of these amino acid hydroxyl free radical adducts.     

Identification of a radical formed in the reaction mixture of rat brain homogenate with a ferrous ion/ascorbic acid system using HPLC–EPR and HPLC–EPR–MS

Identification of a free radical is performed for the reaction mixture of rat brain homogenate with a ferrous ion/ascorbic acid system using EPR, high performance liquid chromatography–electron paramagnetic resonance spectrometry (HPLC–EPR) and high performance liquid chromatography–electron paramagnetic resonance–mass spectrometry (HPLC–EPR–MS). EPR measurements of the reaction mixtures showed prominent signals with hyperfine coupling constants (α^N = 1.58 mT and α^Hβ = 0.26 mT). No EPR spectrum was detectable without rat brain homogenate, suggesting that the radical is derived from rat brain homogenate. An HPLC–EPR analysis of the reaction mixture showed a peak with retention time of 33.7 min. An HPLC–EPR–MS analysis of the peak gave two ions at m/z 224 and 137, suggesting that α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN)/ethyl radical adduct forms in the reaction mixture.     

The free radical of pyruvate formate-lyase. Characterization by EPR spectroscopy and involvement in catalysis as studied with the substrate-analogue hypophosphite

The first-derivative EPR spectrum of the active form of Escherichia coli pyruvate formate-lyase shows an asymmetric doublet with partially resolved hyperfine splittings (g = 2.0037). Isotope substitution studies demonstrated couplings of a carbon-centered unpaired electron to a solvent-exchangeable proton (a = 1.5 mT) and to further hydrogen nuclei (a = 0.36 and 0.57 mT). By selective incorporation of unlabelled tyrosine into 2H-labelled enzyme protein, a tyrosyl radical structure has been ruled out. Circumstantial evidence indicates that the organic free radical, which also displays an ultraviolet absorption signal at 365 nm, is located on a standard amino acid residue of the polypeptide chain. EPR signal quantification found a stoichiometry of 1 spin per active site. The formate analogue hypophosphite has been characterized as a specific kcat inhibitor of pyruvate formate-lyase which destroys the enzyme radical. Protein-linked 1-hydroxyethylphosphonate was previously described as the dead-end product after reaction of the analogue with the intermediary acetyl-enzyme form of the catalytic cycle [W. Plaga et al. (1988) Eur. J. Biochem. 178, 445-450]. EPR spectroscopy of this system has now identified the corresponding alpha-phosphoryl radical as a reaction intermediate [g = 2.0032; a(P) = 2.72 mT, a(3H) = 1.96 mT]; it showed a half-life of about 20 min at 0 degrees C. This finding proves that the enzyme radical is a hydrogen-atom-transferring coenzymic element.     

EPR spectroscopic detection of free radical outflow from an isolated muscle bed in exercising humans.

There is no direct evidence to support the contention that contracting skeletal muscle and/or associated vasculature generates free radicals in exercising humans. The unique combination of isolated quadriceps exercise and the measurement of femoral arterial and venous free radical concentrations with the use of electron paramagnetic resonance (EPR) spectroscopy enabled this assumption to be tested in seven healthy men. Application of ex vivo spin trapping using alpha-phenyl-tert-butylnitrone (PBN) resulted in the detection of oxygen- or carbon-centered free radicals (a(N) = 1.38 +/- 0.01 mT and a(beta)(H) = 0.17 +/-0.01 mT, where a(N) and a(beta)(H) are the nitrogen and beta-hydrogen coupling constants, respectively) with consistently higher EPR signal intensities of the PBN spin adduct observed in the venous compared with the arterial circulation (P < 0.05). Incremental exercise further increased the venoarterial intensity difference [85 +/- 58 arbitrary units (AU) at 24 +/- 6% maximal work rate (WR(max)) vs. 387 +/- 214 AU at 69 +/- 7% WR(max); P < 0.05]. When combined with measured changes in femoral venous blood flow (Q), this resulted in a net adduct outflow of 130 +/- 118 and 1,146 +/- 582 AU/min (P < 0.05), which was positively associated with leg oxygen uptake (r(2) = 0.47, P < 0.05) and Q (r(2) = 0.47, P < 0.05). These results provide the first evidence for oxygen- or carbon-centered free radical outflow from an active muscle bed in humans.     

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)     

Direct probing of copper active site and free radical formed during bicarbonate-dependent peroxidase activity of bovine and human copper, zinc-superoxide dismutases. Low-temperature electron paramagnetic resonance and electron nuclear double resonance studies

Using X-band electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopy at liquid helium temperatures, the Cu(II) coordination geometry at the active site of bovine and human copper,zinc-superoxide dismutases (bSOD1 and hSOD1) treated with H(2)O(2) and bicarbonate (HCO(3)(-)) was examined. The time course EPR of wild type human SOD1 (WT hSOD1), W32F hSOD1 mutant (tryptophan 32 substituted with phenylalanine), and bSOD1 treated with H(2)O(2) and HCO(3)(-) shows an initial reduction of active site Cu(II) to Cu(I) followed by its oxidation back to Cu(II) in the presence of H(2)O(2). However, HCO(3)(-) induced a Trp-32-derived radical from WT hSOD1 but not from bSOD1. The mutation of Trp-32 by phenylalanine totally eliminated the Trp-32 radical signal generated from W32F hSOD1 treated with HCO(3)(-) and H(2)O(2). Further characterization of the free radical was performed by UV irradiation of WT hSOD1 and bSOD1 that generated tryptophanyl and tyrosyl radicals. Both proton ((1)H) and nitrogen ((14)N) ENDOR studies of bSOD1 and hSOD1 in the presence of H(2)O(2) revealed a change in the geometry of His-46 (or His-44) and His-48 (or His-46) coordinated to Cu(II) at the active site of WT hSOD1 and bSOD1, respectively. However, in the presence of HCO(3)(-) and H(2)O(2), both (1)H and (14)N ENDOR spectra were almost identical to those derived from native bSOD1. We conclude that HCO(3)(-)-derived oxidant does not alter significantly the Cu(II) active site geometry and histidine coordination to Cu(II) in SOD1 as does H(2)O(2) alone; however, the oxidant derived from HCO(3)(-) (i.e. carbonate anion radical) reacts with surface-associated Trp-32 in hSOD1 to form the corresponding radical.     

Detection of a Stable Free Radical in the B2 Subunit of the Manganese Ribonucleotide Reductase (MN-RRase) of Corynebacterium ammoniagenes

Ribonucleotide reductases catalyze the irreversible reductive formation of 2′-deoxyribonucleotides required for DNA replication and cell proliferation, and a radical mechanism was assumed to be involved in this reaction. In order to search for a radical in the aerobic manganese ribonucleotide reductase (Mn-RRase) by electron paramagnetic resonance (EPR) the native metal-containing 100 kDa B2 subunit was deliberately prepared from the wild type strain Coynebacterium ammoniagenes ATCC 6872. Enrichment by 2′5′-ADP Sepharose 4B affinity chromatography, fast protein liquid chromatography (FPLC) with Superose 12 and concentration by vacuum evaporation allowed for the first time the detection of a stable free radical by EPR spectroscopy at 77 K. The EPR spectrum exhibits an easily saturable doublet of 1.8 mT splitting and a line width of 1.3 mT at g = 2.0040. The EPR signal intensity showed a clear correlation with the enzymatic activity upon long-time storage at ambient temperature (294 K) and inactivation by the specific RRase inhibitor hy-droxyurea (HU). This leads to the assumption of a protein-linked radical, with functional significance, in the metal-containing 100 kDa 82 subunit of the Mn-RRase of Corynebacteriurn ammoniagenes.     

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.     

Identification of radicals formed in the reaction mixtures of rat liver microsomes with ADP, Fe3+ and NADPH using HPLC EPR and HPLC EPR MS

The reaction of rat liver microsomes with Fe(3+), ADP and NADPH was examined using EPR, HPLC-EPR and HPLC-EPR-MS combined use of spin trapping technique. A prominent EPR spectrum (alpha(N) = 1.58 mT and alpha(H)beta = 0.26 mT) was observed in the complete reaction mixture. The EPR spectrum was hardly observed for the complete reaction mixture without rat liver microsomes. The radicals appear to be derived from microsomal components. The EPR spectrum was also hardly observed in the absence of Fe(3+). Addition of some iron chelators such as EDTA, citrate and ADP resulted in the dramatic change in the EPR intensity. Iron ions seem to be essential for this reaction. For the complete reaction mixture with boiled microsomes, a weak EPR spectrum was observed, suggesting that enzymes participate in the reaction. Five peaks were separated on the HPLC-EPR elution profile of the complete reaction mixture of rat liver microsomes with ADP, Fe(3+) and NADPH. The retention times of the peaks 1 to 5 were 19.4, 22.5, 27.3, 29.8 and 31.4 min, respectively. To identify the radical adducts, HPLC-EPR-MS analyses were performed for the three prominent peaks. The HPLC-EPR-MS analyses showed that a new radical adduct, 4-POBN/1-hydroxypentyl radical, in addition to 4-POBN/ethyl radical adducts, forms in a reaction mixture of rat liver microsomes with ADP, Fe(3+) and NADPH.     

Oxidation of tetrahydrobiopterin by peroxynitrite or oxoferryl species occurs by a radical pathway.

The molecular mechanisms of tetrahydrobiopterin (BH4) oxidation by peroxynitrite (ONOO-) was studied using ultra-weak chemiluminescence, electron paramagnetic resonance (EPR) and UV-visible diode-array spectrophotometry, and compared to BH4 oxidation by oxoferryl species produced by the myoglobin/hydrogen peroxide (Mb/H2O2) system. The oxidation of BH4 by ONOO- produced a weak chemiluminescence, which was altered by addition of 50 mM of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN). EPR spin trapping demonstrated that the reaction occurred at least in part by a radical pathway. A mixture of two spectra composed by an intense six-line spectrum and a fleeting weak nine-line one was observed when using ONOO-. Mb/H2O2 produced a short-living light emission that was suppressed by the addition of BH4. Simultaneous addition of POBN, BH4 and Mb/H2O2 produced the same six-line EPR spectrum, with a signal intensity depending on BH4 concentration. Spectrophotometric studies confirmed the rapid disappearance of the characteristic peak of ONOO- (302 nm) as well as substantial modifications of the initial BH4 spectrum with both oxidant systems. These data demonstrated that BH4 oxidation, either by ONOO- or by Mb/H2O2, occurred with the production of activated species and by radical pathways.     

Direct modification of low density lipoprotein by the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid.

We have previously reported that the spin trap alpha-phenyl-tert-butyl nitrone (PBN) inhibited the oxidative modification of low density lipoprotein (LDL) (Kalyanaraman, B., Antholine, W.E. and Parthasarathy, S. (1990) Biochim. Biophys. Acta 1035, 286-292). In the present study, we report that 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS), a water-soluble spin trap, also inhibited the oxidation of LDL as measured by the formation of thiobarbituric acid reactive substances (TBARS). However, when compared with LDL incubated without DBNBS, the DBNBS-incubated LDL showed increased negative charge on agarose gel electrophoresis and was avidly degraded by mouse peritoneal macrophages. Despite the suggestion of biological modification, there was no decrease in lysine-amino groups in DBNBS-incubated LDL. Furthermore, reductively methylated LDL in which more than 85% of the amino group of lysines was blocked, was also modified by DBNBS. A sulfonic acid analog of PBN failed to modify LDL in a similar manner, suggesting that the presence of sulfonic acid alone does not ensure modification. When LDL was incubated with DBNBS, radical adducts associated with both lipid and protein were detected by electron paramagnetic resonance (EPR) technique. It is suggested that DBNBS may bind to the apoprotein B100 and lipids of LDL by a lysine-independent mechanism resulting in increased recognition and degradation by macrophages. The present work offers a novel approach for rapid modification of LDL.     

Antioxidant properties of tea investigated by EPR spectroscopy

The antioxidant properties of green, black and mixed (fruit) tea samples of different origin were investigated by means of EPR spectroscopy. A six line EPR spectrum of solid tea samples indicates the presence of Mn(II) ions and it is superimposed with a sharp singlet line attributed to semiquinone radical species (Delta H(pp)=1 mT; g=2.0022). Antioxidant properties of aqueous tea extracts in H(2)O(2)/NaOH/dimethylsulfoxide system generating reactive radicals (*OH, O(2)*-), *CH(3)) were followed by spin trapping technique. In addition, antioxidant capacity of these samples was assessed using stable radicals 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPOL). Typically, the highest antioxidant potential to terminate superoxide radicals was found in green teas, followed by black and fruity teas. The pro-oxidant activity of green teas evidenced by spin traps was promoted in samples with higher Mn(II) and ascorbic acid concentrations. Various sources of free radicals used in the antioxidant tests due to their specific action show different termination rates in the presence of the individual tea samples.     

New reactive oxidizing species causes formation of carbon-centered radical adducts in organic extracts of blood following liver transplantation

-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN) radical adducts from Folch (chloroform:methanol) extraction of blood of transplanted livers exhibited a large 6-line electron paramagnetic resonance (EPR) spectrum. Slow EPR sample preparation involving freezing and thawing prior to extraction over 15 min yielded a spectrum assigned as a lipid-derived free radical species, whereas rapid (< min) extraction without a freeze-thaw cycle yielded a mixture of radicals, one with coupling constants similar to the -hydroxymethyl-4-POBN adduct (4-POBN/^.CH₂OH). Extraction with purified chloroform, however, yielded a much weaker, probably lipid-derived signal. Use of ¹³C-methanol in the Folch extracting solution yielded a 12-line EPR spectrum, indicating that a new, highly reactive oxidant species from blood following liver transplantation can convert organic solvents used in tissue extractions to free radicals. This hypothesis was supported by simulation of EPR spectra of free radicals extracted rapidly with Folch, which indicated that the spectrum contained two carbon-centered species, one with hyperfine coupling constants similar to the -methylhydroxyl-4-POBN adduct, the other probably lipid-derived. Because the former originates from methanol in the Folch, extraction of samples with alcohol-free organic solvent is most likely superior when the potential for formation of stable oxidant species exists, such as after liver transplantation.     

In vitro electron paramagnetic resonance characterization of free radicals: relevance to exercise-induced lipid peroxidation and implications of ascorbate prophylaxis

This study tested the hypothesis that exercise-induced oxidative stress is caused by free radical-mediated damage to polyunsaturated fatty acids (PUFA) which can be prevented following ascorbate prophylaxis. Hyperfine coupling constants (HCC) of alpha-phenyl-tert-butylnitrone (PBN)-adducts were measured via room temperature electron paramagnetic resonance (EPR) spectroscopy in the venous blood of 12 subjects at rest and following maximal exercise during a randomized double-blind placebo-controlled trial and compared to those observed following room-air incubation (2 h at 37 degrees C) of L-alpha-phosphatidycholine, linoleic acid, alpha-linolenic acid and arachidonic acid. All adducts exhibited similar HCC [a(N) 13.6 Gauss (G) and a beta(H) 1.8 G] with the exception of L-alpha-phosphatidycholine [a(N1)=13.4 G, a beta(H1)=1.6 G (37%) and a(N2)=14.9 G, a beta(H2)=0.3 G (63%)] consistent with the trapping of lipid-derived alkoxyl and oleate radicals, respectively. Ascorbate pre-treatment ablated radical formation in both systems. These findings identify circulating PUFA as a potential source of secondary radicals that are capable of initiating oxidative stress in the exercising human.