LACK OF PROTECTION OF PBN IN ISOLATED HEART DURING ISCHEMIA AND REPERFUSION - IMPLICATIONS FOR RADICAL SCAVENGING MECHANISM

We evaluated the ability of alpha-phenyl-tert-butyl nitrone (PBN) to t rap free radicals and to protect the rat myocardium during ishcemia an d reperfusion. Isolated bicarbonate buffer-perfused hearts (n = 8) wer e subjected to 20 min global ishcemia (37 degrees C) followed by reper fusion with 0.4 to 4.0 mM PBN. Coronary effluent containing the PBN ad duct was extracted in toluene. Electron spin resonance analysis of the toluene extract revealed a PBN-hydroxyl adduct. To verify this assign ment, a Fenton system was used to generate an authentic PBN-hydroxyl a dduct (n = 8), which yielded the same ESR spectra as the reperfusion-d erived adduct. The structure of the adduct formed in the Fenton system was confirmed by gas chromatography-mass spectrometry. The ESR parame ters of the PBN-hydroxyl adduct were exquisitely sensitive to solvent polarity during extraction of the adduct. Extraction of an authentic P BN-hydroxyl adduct into chloroform, chloroform:methanol, and toluene c losely matched the ESR parameters obtained during reperfusion of ische mic myocardium in other animal models. To determine whether PBN could confer any protective effect during ischemia or reperfusion, hearts (n = 8/group) were subjected to 35 min global ischmia at 37 degrees C wi th the St. Thomas' II cardioplegic solution followed by 30 min reperfu sion. Percent recovery (mean +/- SEM) of developed pressure, rate pres sure product, and leakage of lactate dehydrogenase during reperfusion in control hearts were 58 +/- 3% 48 +/- 4% and 3.2 +/- 0.5 IU/15 min/g wet wt. PBN at a concentration of 0.4 mM or 4.0 mM when present eithe r during ischemia alone or reperfusion alone did not exert any effect upon recovery of developed pressure, rate pressure product or post-isc hemic enzyme leakage. We conclude that PBN fails to improve contractil e recovery and reduce enzyme leakage during reperfusion of myocardium subjected to global ischemia.