Electron paramagnetic resonance (EPR) spectroscopy was used to investigate whether (i) the free radicals produced in the "stunned" myocardium (myocardium with postischemic contractile dysfunction) are derived from O2, (ii) inhibition of radical reactions improves function, and (iii) i.v. spin traps are effective. Open-chest dogs undergoing a 15-min coronary occlusion received an i.v. infusion of the spin trap, alpha-phenyl N-tert-butylnitrone (PBN) (50 mg/kg). In group I (n = 6), EPR signals characteristic of radical adducts of PBN appeared in the coronary venous blood during ischemia and increased dramatically after reperfusion. In group II (n = 6), which received PBN and i.v. superoxide dismutase (SOD; 16,000 units/kg) plus catalase (12,000 units/kg), myocardial production of PBN adducts was undetectable during ischemia (delta = -100%, P less than 0.01 vs. group I) and markedly inhibited after reperfusion (delta = -86%, P less than 0.001). This effect was seen at all levels of ischemic zone flow but was relatively greater in the low-flow range. In group III (n = 8), the same dosages of SOD and catalase without PBN markedly enhanced contractile recovery (measured as systolic wall thickening) after reperfusion [P less than 0.01 at 3 hr vs. controls (group IV, n = 7)]. Systemic plasma activity of SOD and catalase averaged 127 +/- 24 and 123 +/- 82 units/ml, respectively, 2 min after reperfusion. PBN produced no apparent adverse effects and actually improved postischemic contractile recovery in group I (P less than 0.05 at 3 hr vs. controls). This study shows that (i) SOD and catalase are highly effective in blocking free radical reactions in vivo, (ii) the radicals generated in the "stunned" myocardium are derived from univalent reduction of O2, and (iii) inhibition of radical reactions improves functional recovery. The results provide direct, in vivo evidence to support the hypothesis that reactive oxygen metabolites play a causal role in the myocardial "stunning" seen after brief ischemia.
The toxic gas phosgene reacts with the spin trap C-phenyl N-tert-butyl nitrone (PBN) to produce spontaneously N-tert-butyl-N-chloroacylaminoxyl as the main radical product identified by EPR and mass spectrometry. The reaction mechanisms for the formation of N-tert-butyl-N-chloroacylaminoxyl and other radicals are proposed. Reactions of chlorine gas and bromine with PBN have also been investigated. First α-chloro- or α-bromo-PBN is formed followed by chlorination or bromination of the aryl ring of these compounds, respectively.
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-d14 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.
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Abstract The EPR spectra of a variety of different acyl radical adducts of PBN ( C ‐phenyl N‐tert ‐butyl nitrone) and MNP (2‐methyl‐2‐nitrosopropane) were measured and their hyperfine splitting constants determined. Included are benzoyl, acetyl and other alkylacyl, alkoxyacyl, aminoacyl and alkylaminoacyl adducts. All examples gave relatively large β‐hydrogen hyperfine splittings ranging from 2.71 to 5.93 G in the PBN spin adducts. Long‐range hyperfine splitting was found to both nitrogen and amino‐hydrogen atoms in the aminoacyl adducts, indicating specific preferred conformations of the spin adducts. The MNP adducts have the well known 7‐8 G nitrogen hyperfine splitting with little else. The existence of hydrogen atom abstraction from benzaldehyde by nitroxides was established, indicating that nitroxides cannot be considered as inert reagents when sent to probe systems containing aldehydes. For example, the EPR signal due to a nitronyl nitroxide was rapidly quenched in the presence of benzaldehyde in benzene. Also, a surprising increase in intensity of benzoyl PBN adduct was noted after photolysis when the light was turned off. Double acyl adduct formation is proposed.
The spin-trapping method is introduced and discussed. Some chemistry of nitroxides and nitrones is reviewed. Pattern recognition of ESR spectra of nitroxides is outlined. Factors controlling the magnitude of hyperfine splitting constants are mentioned. Methods of assigning spin adducts are listed. Review articles in the literature are referenced. Results in the electrochemical reduction of halocarbons are presented and some parallels with superoxide chemistry shown. Various speculative reactions are given. The in vitro and in vivo experiments where halocarbon radicals have been detected by spin trapping are reviewed and some new results reported. A comparison for different animals is added.
