Isolated perfused rat hearts release secondary free radicals during ischemia reperfusion injury: cardiovascular effects of the spin trap alpha-phenyl N-tert-butylnitrone.

Free radicals produced during myocardial post-ischemic reperfusion are aggravating factors for functional disturbances and cellular injury. The aim of our work was to investigate the significance of the secondary free radical release during non ischemic perfusion and post-ischemic reperfusion and to evaluate the cardiovascular effects of the spin trap used. For that purpose, isolated perfused rat hearts underwent 0, 20, 30 or 60 min of a total ischemia, followed by 30 min of reperfusion. The spin trap: alpha-phenyl N-tert-butylnitrone (PBN) was used (3 mM). Functional parameters were recorded and samples of coronary effluents were collected and analyzed using Electron Paramagnetic Resonance (EPR) to identify and quantify the amount of spin adducts produced. During non ischemic perfusion, almost undetectable levels of free radical release were observed. Conversely, a large and long-lasting (30 min) release of spin adducts was detected from the onset of reperfusion. The free radical species were identified as alkyl and alkoxyl radicals with amounts reaching 40 times the pre-ischemic values. On the other hand, PBN showed a cardioprotective effect, allowing a significant reduction of rhythm disturbances and a better post-ischemic recovery for the hearts which were submitted to 20 min of ischemia. When the duration of ischemia increased, the protective effects of PBN disappeared and toxic effects became more important. Our results have therefore confirmed the antioxidant and protective properties of a spin trap agent such as PBN. Moreover, we demonstrated that the persistent post-ischemic dysfunction was associated with a sustained production and release of free radical species.

The 5/2,3/2 Spin Admixture in the Chloroiron(III) Derivative of the Sterically Crowded 2,3,7,8,12,13,17,18-Octaethyl-5,10,15,20-tetraphenylporphyrin.

Despite similar ring deformations in solution and in the solid state, the chloroiron(III) derivative of 2,3,7,8,12,13,17,18-octaethyl-5,10,15,20-tetraphenylporphyrin ([FeCl(oetpp)], shown schematically) prepared in this study exhibits only a very weak quantum-mechanical admixture of spin S=3/2 (only 4-10 %) with spin S=5/2. In contrast, for the variety of [FeCl(oetpp)] studied earlier by other researchers a 40 % contribution of the S=3/2 state was found.

Electron spin resonance detection of nitric oxide generation in major organs from LPS-treated rats.

The increased production of nitric oxide (NO) has been implicated as the basis for myocardial dysfunction and the lack of response to vasoconstrictors during endotoxin shock induced by lipopolysaccharide (LPS). Our objective was to evaluate and compare NO production in major organs of rats treated with LPS, 1 or 14 mg/kg. A NO spin-trapping technique using electron spin resonance (ESR) spectroscopy has been used to study NO production in the liver, the kidney, the aorta, and the heart. The method was based on the trapping of NO by a metal-chelator complex consisting of N-methyl-D-glucamine dithiocarbamate (MGD) and reduced iron (Fe2+) to form a stable [(MGD)2-Fe2+-NO] complex, giving rise to a characteristic triplet ESR spectrum with g = 2.04 and aN = 12.65 G: Iron was quantified in the different organs to study the [(MGD)2-Fe2+] complex distribution. Six hours after intravenous injection of 1 or 14 mg/kg of LPS, we observed large increases in the [(MGD)2-Fe2+-NO] adduct signal in the liver, the kidney, and in the aorta, strongly suggesting an increased production of NO in these organs. The [(MGD)2-Fe2+-NO] adduct was also detected in the heart, 6 h after injection of LPS. Moreover, we observed dose-dependent increases in [(MGD)2-Fe2+-NO] adduct in the heart, whereas no changes were observed in the other organs. Concurrently, the [(MGD)2-Fe2+-NO] adduct was not detected in the blood from rats treated with LPS, although circulating nitrosylhemoglobin, nitrite, and nitrate levels increased. The spin-trapping technique allowed us to monitor organ-specific formation of NO after LPS administration and for the first time demonstrated direct NO production in aorta and heart of LPS-treated animals.

Evolution of free radical formation during low-flow ischemia and reperfusion in isolated rat heart.

Free radicals have been implicated in several aspects of cellular injury, both during ischemia and reperfusion of the myocardium. In this study, formation of free radicals in the isolated rat heart was measured a) directly using electron paramagnetic resonance (EPR) spectroscopy and b) indirectly using the generation of thiobarbituric acid reactants as an index of lipid peroxidation. EPR spectra of frozen heart powder recorded at 100 degrees K show several lines and consist of different components separated by temperature studies: signal C disappears after warming the sample 1 minute at 190 degrees K and is suggestive of a triplet signal g = 2.001, aN = 25 Gauss; signal B g parallel = 2.034, g perpendicular = 2.007, disappears after 1 min at 240 degrees K, and is similar to those previously reported for oxygen alkylperoxyl free radical; the remaining signal, signal A with g = 2.004 is identical to that of a carbon-centered ubiquinone free radical. The total free radical concentration in isolated rat heart perfused at a constant flow rate of 12 ml/min was increased by 44% compared with control (p less than 0.05) after 10 minutes of normothermic global ischemia with a 10% residual flow, and by only 31% compared with control after 20 seconds of reflow with oxygenated perfusate (p less than 0.05). Compared with the reperfused group, trimetazidine 10(-5) M administered 15 minutes before the ischemic period decreased the free radical concentration (-20%). However, this free radical generation in heart was not associated with a concomitant increase of lipid peroxides.