Tert-butyl Nitrone Research Articles

Direct measurement of oxygen free radicals during in utero hypoxia in the fetal guinea pig brain

The present study tested the hypothesis that maternal hypoxia induces oxygen free radical generation in the fetal guinea pig brain utilizing techniques of electron spin resonance spectroscopy and alpha-phenyl- tert-butyl nitrone (PBN) spin trapping. Pregnant guinea pigs of 60 days gestation were divided into normoxic and hypoxic groups and exposed to 21% or 7% oxygen for 60 min. Free radical generation was documented by measuring the signal of PBN spin adducts. Fluorescent compounds were determined as an index of lipid peroxidation and the activity of Na +,K +-ATPase was determined as an index of brain cell membrane function. Hypoxic fetal cerebral cortical tissue showed a significant increase in spin adducts (normoxic: 33.8±9.3 units/g tissue vs. hypoxic: 57.9±9.2 units/g tissue, p<0.01) and fluorescent compounds (normoxic: 0.639±0.054 μg quinine sulfate/g brain vs. 0.810±0.102 μg quinine sulfate/g brain, p<0.01) and a decrease in Na +,K +-ATPase activity (normoxic: 43.04±2.50 μmol Pi/mg protein/h vs. hypoxic: 33.80±3.51 μmol Pi/mg protein/h, p<0.001). These results demonstrate an increased free radical generation during hypoxia in the fetal guinea pig brain. The spectral characteristics of the radicals were consistent with those of alkoxyl radicals. The increased level of fluorescent compounds and decreased activity of Na +,K +-ATPase indicated hypoxia induced brain cell membrane lipid peroxidation and dysfunction, respectively. These results directly demonstrate an increased oxygen free radical generation during hypoxia and suggest that hypoxia-induced increase in lipid peroxidation and decrease in membrane function, as indicated by a decrease in Na +,K +-ATPase activity, are consequences of increased free radicals. The nature of predominantly present alkoxyl radical indicates ongoing lipid peroxidation during hypoxia. The direct demonstration of oxygen free radical generation during hypoxia is the critical missing link in the mechanism of hypoxia-induced brain cell membrane dysfunction and damage.

Protective effects of free radical inhibitors in intracerebral hemorrhage in rat

Iron compounds formed in the degradation of a hematoma can accelerate the formation of free radicals in adjacent ischemic or hypoperfused tissue. The purpose of this study was to examine the efficacy of compounds that quench free radicals in improving the outcome in rats with experimental intracerebral hemorrhage. Intracerebral hemorrhage was induced in rats by injection of bacterial collagenase and heparin into the caudate nucleus. Rats were treated with α-tocopherol plus ascorbic acid starting before hemorrhage, or with dimethylthiourea or α-phenyl- N- tert-butyl nitrone starting 2 h after hemorrhage, with treatment continued for 10 days after induction of hemorrhage. Outcome was assessed by behavioral analyses, magnetic resonance imaging, and histopathology. A trend towards behavioral improvement was found for rats treated with α-tocopherol/ascorbic acid, while behavior was significantly improved following intracerebral hemorrhage in rats treated with dimethylthiourea or α-phenyl- N- tert-butyl nitrone. These results suggest that free radicals may play a role in the development of brain injury following intracerebral hemorrhage, and that compounds that interrupt the free radical cascade may improve outcome. However, treatment did not significantly affect edema, resolution of the hematoma, or neuronal injury in tissue adjacent to the hemorrhage.

Receptor- and Age-Selective Effects of Dopamine Oxidation on Receptor–G Protein Interactions in the Striatum

The striatum contains a high concentration of oxidizable dopamine (DA), and the aged organism shows a decreased ability to respond to oxidative stress (OS), making this area extremely vulnerable to free radical insult. To determine the receptor specificity of this putative increase in OS sensitivity, striatal slices from 6- and 24-month-old animals were incubated (30 min, 37°C) in a modified Krebs medium containing 0 to 500 μM DA with or without a preincubation (15 min) in a nitrone trapping agent, 1 or 5 mM α-phenyl-n- tert-butyl nitrone (PBN), and changes in low K m GTPase activity (an index of receptor–G protein coupling/uncoupling) assessed in muscarinic, 5-HT 1A D 1, and D 2 receptors stimulated with carbachol, 8 OH-DPAT-HBr, SKF 38393, or quinelorane, respectively. DA exposure induced selective decreases in the stimulated activity in all of these receptor systems, and an overall increase in conjugated dienes (56%) of the young. In the case of carbachol and 8 OH-DPAT-HBr, the DA-induced deficits in GTPase stimulation were seen primarily in the young (61 and 32%, respectively), while DA-induced deficits in quinelorane (D 2) stimulation were seen in both age groups. In the case of SKF 38393-stimulation (D 1) the DA-induced deficits were higher in the striatal tissue from the old. The DA-induced decreases in carbachol stimulated GTPase activity in the tissue from the young could be prevented by pretreatment with PBN or the DA uptake inhibitor, nomifensin. No effect of nomifensin was seen in the old, because their DA uptake mechanisms were already compromised. These results suggest that although age-related declines in DA uptake may provide some protection against the OS effects in muscarinic or 5-HT 1A receptors, other factors may increase the vulnerability of DA neurons to OS, even with reductions in DA uptake.

Active Oxygen-Mediated Chromosomal 1–2 M bp Giant DNA Fragmentation Into Internucleosomal DNA Fragmentation in Apoptosis of Glioma Cells Induced by Glutamate

C6 glioma cells treated with 10 mM glutamate reduced intracellular GSH to one-seventh of the initial level, and induced cytolysis accompanied by apoptosis. The treated cells produced extracellular H 2O 2. The cytolysis of the C6 cells induced by glutamate was prevented by antioxidants such as N-acetylcysteine (NAC), ascorbic acid (ASC), catalase, and NaN 3, iron chelators such as deferoxamine and 1,10-phenanthroline, and oxygen radical scavengers such as 5,5′-dimethyl-1-pyrroline-N-oxide (DMPO) and α-phenyl- tert-butyl nitrone (PBN). The effect of these antioxidants, iron chelators, and oxygen radical scavengers on the cytolysis of C6 cells was dependent on the dose and the intracellular GSH level. Furthermore, 1–2 Mbp chromosomal DNA (giant DNA) fragments were observed during cytolysis. The giant DNA fragments were further cleaved into smaller DNA fragments of 200–800 kbp, and then to fragments of less than 300 kbp in size including chromosomal ladder DNA fragments. Such serial chromosomal DNA degradations induced by glutamate were also inhibited by addition of these antioxidants, iron chelators, and oxygen radical scavengers. These findings suggest that glutamate induces GSH depletion, and consequently, apoptosis through endogenously produced active oxygen species in C6 glioma cells and that the apoptosis is accompanied by 1–2 Mbp giant DNA fragmentation prior to the internucleosomal DNA fragmentation.

Neuroprotective effects of pterin-6-aldehyde in gerbil global brain ischemia: comparison with those of α-phenyl- N- tert-butyl nitrone

The superoxide (O 2 ⋅−) scavenging activity and the neuroprotective effects of pterin-6-aldehyde (P6A), a xanthine oxidase inhibitor, were examined and compared with those of α-phenyl- N- tert-butyl nitrone (PBN), a spin trapping agent. The scavenging activity of P6A was more potent than that of PBN by 150-fold in neutrophil/phorbol myristate acetate O 2 ⋅− generating system. P6A attenuated the neuronal damage with a much smaller dose and a greater efficiency than PBN in global brain ischemia in gerbils. These findings suggest that P6A is a more potent neuroprotective agent than PBN and has possible therapeutic effects against various diseases in which O 2 ⋅− is involved.

Time courses of LPS-induced cytokine and transcription factor expression in rats and the inhibitory effect by phenyl n- tert-butyl nitrone

To kill invading bacteria, viruses, and fungi, phagocytes secrete hydrogen peroxide (H2O2) and the heme enzyme myeloperoxidase. We have explored the possibility that myeloperoxidase might use H2O2 to convert L-tyrosine to tyrosyl radical. Activated human neutrophils and monocytes used the system to oxidize free L-tyrosine to o,o′-dityrosine, a stable product of tyrosyl radical. Protein-bound tyrosyl residues exposed to myeloperoxidase, H2O2, and l-tyrosine were also oxidized to o,o′-dityrosine. The cross-linking reaction required free L-tyrosine, suggesting that myeloperoxidase converts the amino acid to a diffusible radical catalyst that promotes protein oxidation. We used electron paramagnetic resonance to provide direct evidence that the oxidizing intermediate is free tyrosyl radical. Myeloperoxidase-generated tyrosyl radical also initiates lipid peroxidation, suggesting that activated phagocytes might also be able to oxidize lipids in host tissues. Moreover, myeloperoxidase is present and active in human atherosclerotic tissue, and levels of protein-bound dityrosine are elevated in such lesions. Our recent studies indicate that activated neutrophils use oxidants generated by the phagocyte NADPH oxidase to produce protein-bound dityrosine during acute inflammation. Collectively, these findings suggest that generation of tyrosyl radical by myeloperoxidase allows activated phagocytes to damage both proteins and lipids. Elevated levels of o,o′-dityrosine have been detected in inflammatory lung disease, neurodegenerative disorders, and aging. Thus, oxidation of tyrosine to tyrosyl radical might play a role in the pathogenesis of many diseases.

Inhibition by phenyl N- tert-butyl nitrone of early phase hepato-carcinogenesis in rats fed a choline-deficient diet

Inhibition by phenyl N- tert-butyl nitrone of early phase hepato-carcinogenesis in rats fed a choline-deficient diet

Induction of free radicals in hepatocytes, mitochondria and microsomes of rats by ochratoxin A and its analogs

Oxidative damage may be one of the manifestations of cellular damage in the toxicity of ochratoxin A (OA). OA; its three natural analogs, OB, OC and Oα; and three synthetic analogs, the ethyl amide of OA (OE-OA), O-methylated OA (OM-OA), and the lactone-opened OA (OP-OA) were used to study free radical generation in hepatocytes, mitochondria and microsomes from rats. Electron paramagnetic resonance spectroscopy (EPR) using α-(4-pyridyl-1-oxide)- N- tert-butyl nitrone (4-POBN) as a spin trapping agent showed an enhanced free radical generation due to the addition of NADPH to the microsomes. An EPR signal was not observed in the mitochondria and hepatocyte samples when they were treated with a variety of agents. Addition of OM-OA together with NADPH and Fe 3+ to the microsomes resulted in a strong EPR signal compared with the other analogs, whereas the signal could be quenched by the addition of catalase. OM-OA does not have a dissociable phenolate group and does not chelate Fe 3+. The spin adduct hyperfine splitting constants indicated the presence of α-hydroxyethyl radicals resulting from generated hydroxyl radicals, which were trapped by 4-POBN. The results also suggested that the production of hydroxyl radicals by OA does not require a dissociable phenolate group or the prior formation of an OA-Fe complex.

Evidence for a Novel Pentyl Radical Adduct of the Cyclic Nitrone Spin Trap MDL 101,002

3,4-Dihydro-3,3-dimethyl-isoquinoline-2-oxide (MDL 101,002) is a conformationally constrained cyclic analog of the known spin trap α-phenyl N- tert-butyl nitrone (PBN). Because of PBN's ability to scavenge free radicals, MDL 101,002 is currently being evaluated in stroke models as a means to ameliorate the oxidative insult associated with reperfusion injury. To augment our understanding of the radical scavenging mechanism of this potential drug, MDL 101,002 was incubated with soybean lipoxygenase in the presence of linoleic acid to study the interaction between MDL 101,002 and free radicals formed during lipid peroxidation. Analysis of the reaction mixture was performed by high performance liquid chromatography using normal phase conditions with detection by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Similar to the work by Iwahashi et al. [ Arch. Biochem. Biophys., 1991, 285, 172], who studied the spin trap α-(4-pyridyl-1-oxide)-N- tert-butyl nitrone (4-POBN), an adduct that suggested the trapping of pentyl radicals by MDL 101,002 was observed. However, the apparent molecular ion for this adduct (246 Da) was 1 Da lower than would be predicted if a pentyl radical had simply added to MDL 101,002. In addition, the adduct exhibited significant absorbance at 304 nm, consistent with the unsaturated nitrone structure of MDL 101,002. To account for these observations, it is postulated that, after the initial capture of a pentyl radical, subsequent abstraction of a hydrogen atom by a neighboring radical occurs to regenerate a nitrone (1-pentyl analog of MDL 101,002). We present evidence for this adduct and offer a mechanism for its formation. These findings indicate that mass spectroscopic analysis of stable nitrone radical adducts may be useful in the identification of radical-dependent damage in vivo and possibly in clinical development of MDL 101,002 as an antioxidant pharmaceutical. Copyright © 1997 Elsevier Science Inc.

Necrosis of the Substantia Nigra, Pars Reticulate, in Flurothyl-Induced Status Epilepticus is Ameliorated by the Spin Trap α Phenyl-N- tert-butyl Nitrone

The objective of the present study was to explore whether a diffusible free radical scavenger can ameliorate the pan-neurotic lesions of the substantia nigra, pars reticulate (SNPR), which are incurred in rats subjected to status epilepticus of more than 30 min duration. Vehicle-injected animals had flurothyl seizures induced for 45 min. The seizures were then terminated and the animals were recovered for 7 d to allow histopathological evaluation of the SNPR lesions. Drug-treated animals, which were otherwise treated identically, were given either 100–800 mg/kg of dimethylthiourea (DMTU), a diffusible hydroxyl ion scavenger, or the diffusible spin trap α-phenyl N- tert-butyl nitrone (PBN) in a dose of 100 mg/kg IP. All animals given DMTU died 2 to 8 h after status epilepticus, but PBN was tolerated well by the animals. The amount of flurothyl required to sustain the electrographic seizures was identical in the vehicle- and drug-injected groups, demonstrating that PBN did not suppress seizure activity. Vehicle-injected animals had large bilateral infarcts localized to the SNPR. Of the six animals treated with PBN, one had a small, unilateral lesions, and in all other animals the SNPR had a normal histological appearance. The results strongly suggest that the pan-necrotic lesions of the SNPR incurred during ongoing seizure activity represent a free radical-mediated lesion. Copyright © 1997 Elsevier Science Inc.

Optimal Time and Dosage of Phenyl N- Tert-Butyl Nitrone (PBN) for the Inhibition of Nitric Oxide Synthase Induction in Mice

We have previously reported that phenyl N- tert-butyl nitrone (PBN) inhibits the induction of inducible nitric oxide synthase (iNOS) and, thus, prevents the overproduction of nitric oxide (NO), resulting in the reduction of endotoxin-mediated death in mice. In this study, to examine the effect of PBN in detail, we investigated the dose- and administration-timing dependence of PBN on endotoxin-induced NO generation in mice. NO generation was monitored in the mouse liver after administration of lipopolysaccharide (LPS) by the in vivo NO-spin trapping method using the iron complex of N-methyl- d-glucamine dithiocarbamate (MGD) as a spin trap, followed by ex vivo EPR measurement of the liver tissue. PBN was effective in reducing liver NO generation monitored 6 h after endotoxin injection when it was administered shortly before or after LPS injection. The maximum inhibition of liver NO was obtained when PBN was administered 30 min before LPS injection. ID 50 for the inhibition was estimated to be approximately 200 mg/kg when the LPS dose of 50 mg/kg was used. Expression of mRNA for iNOS in the liver as estimated by reverse transcription polymerase chain reaction was decreased when PBN was given 30 min before LPS injection, indicating that the reduction of expression of iNOS protein by PBN, which has been shown previously, is at least in part caused by a decrease in mRNA expression. Copyright © 1996 Elsevier Science Inc.

Progressive Effect of α-Phenyl-N- Tert-Butyl Nitrone (PBN) on Rat Embryo Development in Vitro

In the present study we demonstrated the effects of the spin-trapping agent α-phenyl-N- tert-butylnitrone (PBN) on the in vitro development of rat embryos at the early stage. In rat embryos, PBN increased the speed of the first cleavage and had no toxicity during pregnancy after embryo culture. These results showed that reactive oxygen species (ROIs) that were formed by activating molecular oxygens through redox reactions regulated the speed of development for early-stage embryos. Thus, PBN caused a decrease in the level of ROIs and toxicity and an in increase in the level of the development of rat embryos. On the other hand, PBN could not decrease the 2-cell block in vitro nor increase the blastulation rate, in contrast to the fact that a scavenger of superoxide anions, SOD, is effective in doing so for mouse embryos. From these results it was concluded that free radicals play an important role in the in vitro development of rat embryos at the early stage, but play no role in the decrease of the 2-cell block or their blastulation rate. It should be noted that PBN had no toxicity for embryonic development at the 2-cell stage.

Hydroxyl Radical is the Major Causative Factor in Stress-Induced Gastric Ulceration

The role of the metal-catalyzed production of hydroxyl radicals (OH ) on gastric ulceration caused by restraint-cold stress in rat was studied. Stress causes a 50% increase in the thiobarbituric acid reactive species (TBARS) as a measure of the lipid peroxidation, nearly 70% increase in protein oxidation as measured by its carbonyl content and about 40% decrease in the glutathione content of the fundic stomach, suggesting oxidative damage by stress. Stress also causes a time-dependent increase in the mitochondrial superoxide dismutase activity and a decrease in the peroxidase activity, both of which correlate well with the increase in the severity of ulceration as measured by the ulcer index. Specific OH scavengers such as benzoate or dimethylsulfoxide (DMSO) and the free radical trap such as α-phenyl N- tert-butyl nitrone (PBN) significantly inhibit gastric ulceration suggesting the role of OH in this oxidative damage. Desferrioxamine (DFO), a nontoxic transition metal ion chelator, protects the mucosa against stress-ulceration dose dependently. Increased level of TBARS and the inactivation of gastric peroxidase are also prevented by DFO or by antioxidants such as glutathione or vitamin E, suggesting the critical role of metal ion and OH in the oxidative damage. A metal-catalyzed OH generating system constituted by Cu 2+, H 2O 2 and ascorbate (reducing equivalent of O 2 −) causes inactivation of the purified gastric peroxidase in vitro, which can be effectively prevented by DFO. The stress-induced activation of the superoxide dismutase is completely blocked by pretreatment with α-amanitin indicating an increased synthesis of the enzyme by increased transcription of its m-RNA. Quantitative measurement indicates that stress causes a fivefold increase in the generation of OH , which correlates well with the increase in ulcer index with the progress of stress. The results indicate that the stress-induced gastric ulceration is a consequence of the oxidative damage of the gastric mucosa. This is caused by the OH generated through the metal-catalyzed Haber–Weiss reaction between O 2 − and H 2O 2, the latter being formed by the stimulation of the superoxide dismutase and inactivation of the gastric peroxidase. © 1997 Elsevier Science Inc.

Trichloroethylene metabolism in vitro: an EPR/SPIN trapping study.

Trichloroethylene (TCE) was hypothesized to produce free radicals which could be detected using electron paramagnetic resonance spectroscopy with the spin trap, PBN (alpha-phenyl tert-butyl nitrone). The free radicals detected following incubation of precision cut liver slices in media containing 10 mM PBN had hyperfine coupling constants aN = 1.61 mT and aH = 0.325 mT. There was a linear increase in free radicals detected in the bathing media when the headspace TCE concentration was increased from 2500-10000 p.p.m. The levels of conjugated dienes measured in the slices incubated in PBN supplemented media were less than slices exposed to TCE in incubation media without PBN. The PBN trap may act as a scavenger preventing the propagation of free radicals and inhibiting lipid peroxidation. The experiments suggest that free radical formation by TCE leads to a concomitant increase in conjugated dienes in liver slices which may contribute to the pathological changes which occur in liver following TCE exposure.

Antioxidant treatment with phenyl-α- tert-butyl nitrone (PBN) improves the cognitive performance and survival of aging rats

Accumulating evidence has implicated free radical production and resultant oxidative damage as a major contributing factor in brain aging and cognitive decline. In the present study, aging 24-month-old rats were chronically treated with the synthetic spin-trapping antioxidant phenyl-α- tert-butyl nitrone (PBN) for up to 9.5 months. Chronic PBN treatment (1) improved the cognitive performance of aged rats in several tasks, (2) resulted in greater survival during the treatment period, and (3) decreased oxidative damage within brain areas important for cognitive function. These results not only provide a direct linkage between free radicals/oxidative damage and cognitive performance in old age, but also suggest that synthetic brain antioxidants could be developed to treat or prevent agea-associated cognitive impairment and Alzheimer's disease.

The effects of α-phenyl- tert-butyl nitrone (PBN) on copper-induced rat fulminant hepatitis with jaundice

In the present study we demonstrated the protective effects of the spin-trapping agent α-phenyl- tert-butyl nitrone (PBN) against fulminant hepatitis with jaundice in LEC rats. In LEC rats an excess amount of copper is accumulated in the liver and causes hepatitis with severe jaundice. PBN was subcutaneously administered every 2 d at the concentration of 128 mg/kg, beginning with 13-week-old rats and continuing for 17 weeks. PBN prevented the loss of body weight, reduced death rate, and suppressed the increase in GTP and GOT values reflecting hepatic cell destruction. Ocular inspection also confirmed the suppressive effects of PBN on jaundice. In parallel with these phenomena, the amounts of thiobarbituric acid-reactive substances (TBARS) in livers of PBN-administered rats were found to be lower than those of non-PBN-administered rats. Little histological changes were observed in PBN-administered rats in comparison with non-PBN-administered rats. The protective effect of PBN on the formation of oxidative damage in liver DNA was observed but not so remarkable as that on lipid peroxidation. From these results, it was concluded that PBN had the liver-protective effects against fulminant hepatitis with jaundice. This suggested that free radicals play an important role in abnormally accumulated copper-induced liver injury and that PBN potentially has therapeutic value for the treatment of hepatitis.

Aβ(25–35) peptide displays H 2O 2-like reactivity towards aqueous FE 2+, nitroxide spin probes, Ant) synaptosomal membrane proteins

Amyloid beta peptides (Aβs) are found in abnormally high accumulations in brains of persons with Alzheimer's disease, and are believed to contribute to cognitive decline in this disorder. Synthetic Aβ and its peptide fragment 25–35 [Aβ(25–35)] are toxic to cells in culture; however, the exact mechanism of amyloid peptide toxicity is not known. An emerging hypothesis contends that Aβ toxicity results from peptide-mediated free radical reactions and generation of reactive oxygen species [Hensley, et al., Proc. Natl. Acad. Sci. USA 19 : 3270–3274 (1994); Harris, et al., Exp. Neurol. 131 : 193–202 (1995)]. Recently, we reported that reactivity of Aβ toward the oxidation-sensitive enzyme glutamine synthetase is related to the peptide's reactivity toward the spin trap phenyl- tert-butyl nitrone (PBN) [Hensley, et al., Neuroreport 6 : 489–492 (1995)]. Neuronal damage may be due, in part, to oxidative processes initiated by amyloid-derived free radicals species. This work presents evidence from electron paramagnetic resonance (EPR) spin labeling techniques and spectrophotometric assays that a portion of synthetic Aβ(25–35) demonstrates hydrogen peroxide-like reactivity toward Fe 2+, nitroxide spin probes, and neocortical synaptasomal membrane proteins. These results are discussed with reference to free radical membrane damage and neurotoxicity in Alzheimer's disease.

Biological spin trapping II. Toxicity of nitrone spin traps: dose-ranging in the rat

To obtain the strongest possible free radical spin adduct signal using the electron paramagnetic resonance spectroscopy-spin trapping technique, it is desirable to load an animal with the highest dose of spin trap possible. One hundred and twenty six male Sprague-Dawley rats were used to establish the toxic dose range for PBN (α-phenyl N- tert butyl nitrone) and 18 other similar spin traps. The lethal dose of PBN was found to be approximately 100 mg/100 g BW (0.564 mmol/100 g). The 18 other compounds were then tested, and their toxicities were gauged in terms of molar equivalents to PBN. Of these spin traps, DMPO (5,5-dimethyl-1-pyrroline-N-oxide) was found to be the least toxic (no toxic signs at twice the lethal dose for PBN) while 2,6-difluoro-PBN and M 4PO (3,3,5,5-tetramethyl-1-pyrroline-N-oxide) were the most toxic, both causing death at one eighth the PBN-equivalent lethal dose. Nine of the 18 nitrones appeared non-toxic at the 0.25 PBN-equivalent lethal dose level.

Tandem mass spectrometry study of C-phenyl- N-tert-butyl nitrone spin adducts from in vitro rat liver microsomal metabolism of bromotrichloromethane and carbon tetrachloride

Electron ionization and thermospray were used in conjunction with tandem mass spectrometry methods to identify trichloromethyl/C-phenyl-N-tert-butyl nitrone (PBN) spin adducts produced in rat liver microsomal dispersions that had been treated with reduced nicotinamide adenine dinucleotide phosphate (NADPH)-generating system and BrCCl3 (or CCl4). In the identification of PBN spin adducts, a scan of precursors of m / z 57 was utilized to confirm the presence of PBN spin adducts, because PBN spin adducts produce m / z 57 from tert-butyl as a characteristic fragment. Use of deuterated PBN (PBN-d9 deuterated at tert-butyl; PBN-d 14 deuterated at both phenyl and tert-butyl) improved the recognition of PBN adducts in mixtures by precursor ion scans, because m / z 66 (which corresponds to the deuterated tert-butyl group) is characteristic and, unlike m / z 57, it is not a common fragment for any other compounds. Two new PBN spin adducts that were not detected before by electron paramagnetic resonance or mass spectrometry were identified by these methods for the first time.

Peroxynitrite-mediated oxidation of albumin to the protein-thiyl free radical

Nitric oxide reacts with superoxide to produce peroxynitrite, which may be an important mediator of oxidant-induced cellular injury. Here we report that peroxynitrite is able to oxidize a protein, bovine serum albumin (BSA), to the corresponding protein-thiyl free radical as demonstrated by electron paramagnetic resonance (EPR)-spin-trapping experiments with both α-phenyl- N- tert-butyl nitrone (PBN) and 5,5-dimethyl-1 -pyrroline- N-oxide (DMPO). BSA radical adduct yields increased with pH indicating peroxynitrite anion as its main forming agent. Reaction with peroxynitrite may be another aspect of the antioxidant action of albumin in extracellular fluids.