Myocardial tissue preparation for ESR spectroscopy: some methods may cause artifactual generation of signals.

It has been suggested that some techniques of tissue preparation for esr spectroscopy may artifactually generate radicals. We have investigated this, together with the possibility that the susceptibility of the tissue to preparation artifacts may be altered by ischaemia and reperfusion. Three different methods of tissue processing have been assessed: (i) freeze-clamping (-196 degrees C), using grooved, aluminium tongs which produce frozen cylinders of tissue (3 mm diameter) which fit directly into esr tubes; (ii) grinding of freeze-clamped tissue with a porcelain pestle and mortar; (iii) lyophilization of ground, freeze-clamped, tissue. Isolated rat hearts (n = 7 or n = 5/group) were subjected to aerobic perfusion (10 min, 37 degrees C), total, global ischaemia (15 min) and reperfusion (30 sec). Hearts were freeze-clamped at the end of each period. Tissue was prepared by each of the three methods and esr spectra recorded at -100 degrees C. In spectra from tissue which had been freeze-clamped only, broad high- and low-spin iron III signals (g = 1.9, g = 2.2-2.9 and g = 4.6) were seen together with a narrow, well-defined signal (g = 2.005), possibly from a semiquinone radical.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the effects of antioxidants and inhibitors of radical generation on free radical production in the reperfused rat heart using electron spin resonance spectroscopy.

Reperfusion of the heart after a period of ischaemia can precipitate ventricular arrhythmias and lead to an exacerbation of tissue injury. Direct evidence to suggest the involvement of free radicals has been obtained using electron spin resonance (esr) spectroscopy and the spin trap N-tert. butyl-alpha-phenyl nitrone (PBN). In the present study, we have used esr spectroscopy and PBN to examine the individual effects of superoxide dismutase (SOD), catalase, allopurinol or desferal on radical production in the isolated, reperfused rat heart. A burst of radical production was observed in the control group during the first 5 minutes of reperfusion; the peak occurred during the first minute, when signal intensity had increased by almost 300%, but returned to the baseline by 15 minutes of reperfusion. The esr signals were consistent with the trapping of either alkoxyl or carbon-centered radicals (aN = 13.6 and aH = 1.56 G). In the desferal-treated group, a burst of radical production was observed during the first five minutes of reperfusion; this was maximal during the second minute, when signal intensity had increased by almost 200%, but had returned to the baseline value by 30 minutes of reperfusion. In the SOD-treated group, a burst of radical production was observed during the first 10 minutes of reperfusion; signal intensity was maximal during the tenth minute of reperfusion, when signal intensity had increased by almost 200%, but had returned to the baseline value by 30 minutes of reperfusion. In the allopurinol- and catalase-treated groups, no significant burst of radical production could be detected. These data further support the concept that cytotoxic, oxygen-derived species are formed upon reperfusion and that hydrogen peroxide and/or hydroxyl radicals, are likely to be involved.

Manipulation of myocardial alpha-tocopherol levels fails to affect reperfusion arrhythmias or functional recovery following ischemic challenge in the rat heart.

Antioxidants that act as free radical scavengers have the potential to inhibit lipid peroxidation. It has previously been proposed that a relationship exists between free radicals, lipid peroxidation and reperfusion-induced arrhythmias. We have therefore examined the ability of the lipid-soluble antioxidant, alpha-tocopherol, to decrease the incidence of reperfusion-induced arrhythmias. We have shown that the myocardial alpha-tocopherol content can be significantly increased from its control value of 65.3 +/- 5.6 nmoles/g wet wt of heart to 115.0 +/- 15.6 nmoles/g wet wt of heart (p less than 0.01) by chronic intraperitoneal pretreatment and that it can be decreased, to 21.1 +/- 3.7 nmoles/g wet wt of heart (p less than 0.01), by chronic dietary manipulation. Rat hearts isolated from either of these groups, or from placebo-treated control animals, were subjected to 5 or 10 min coronary artery occlusion and were subsequently reperfused; there were no significant differences between the incidence and duration of VF and VT or the incidence and number of VPBs in these three groups. The effect of alpha-tocopherol manipulation on metabolic and functional recovery of working hearts subjected to 20 min global ischemia was subsequently examined and no significant changes were observed. Cardiac output recovered to 82 +/- 4, 81 +/- 6 and 76 +/- 5% of its preischemic value in the control, enriched and depleted groups, respectively. In conclusion, myocardial alpha-tocopherol content appears to bear no relation to the severity of reperfusion-induced arrhythmias or to the resistance of the heart to ischemic injury.

Electron spin resonance and pulse radiolysis studies on the spin trapping of sulphur-centered radicals.

Thiyl radicals are shown to be readily trapped with the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (TMPO) giving characteristic spin adducts with hyperfine coupling constants aN 1.52-1.58, aH 1.52-1.80 mT, and g values in the range 2.0065-2.0067 for the DMPO adducts and aN 1.50-1.56, aH 1.70-1.92 mT, g 20049-2.0051 for the TMPO adducts. Kinetic data obtained from pulse radiolysis studies show that, in general, thiyl radicals react rapidly with these spin traps with rate constants of the order of 10(7)-10(8) dm3 mol-1 s-1. The tetramethylated spin trap TMPO though giving slightly less intense electron spin resonance (ESR) spectra, produces longer lived adducts, and is suggested to be of greater utility due to the more characteristic nature of the coupling constants of the observed adducts; reaction of certain thiyl radicals with DMPO produces adducts which are superficially similar to the hydroxyl radical adduct to the same trap.