Use of aromatic hydroxylation of phenylalanine to measure production of hydroxyl radicals after myocardial ischemia in vivo: Direct evidence for a pathogenetic role of the hydroxyl radical in myocardial stunning

A pathogenetic role of ·OH in myocardial stunning has been inferred from the protective effects of ·OH scavengers and iron chelators. However, conclusive demonstration of the ·OH radical hypothesis of myocardial stunning requires direct verification of three major, but still unproven, assumptions: (1) ·OH is produced in the stunned myocardium in vivo; (2) antioxidant therapy inhibits ·OH production; and (3) such inhibition results in enhanced recovery of contractility (ie, ·OH is necessary for the development of myocardial stunning). Since phenylalanine (Phe) reacts with ·OH to form the hydroxylated products ortho-, meta-, and para-tyrosines (o-, m-, and/p-tyr), we used aromatic hydroxylation of Phe to detect ·OH formation in the stunned myocardium. Open-chest dogs undergoing a 15-minute coronary occlusion followed by reperfusion received an intravenous infusion of Phe (54.3 mg/kg for 11.5 minutes beginning 90 seconds before reperfusion); these animals were given either no antioxidant therapy (group I, n=15), N-2-mercaptopropionyl glycine (MPG) (group II, n=11), or MPG combined with superoxide dismutase, catalase, and desferrioxamine (group III, n=12). In addition, group IV (nonischemic control group, n=6) received Phe but did not undergo coronary occlusion, whereas group V (ischemic control group, n=16) underwent a 15-minute occlusion but did not receive Phe or antioxidants. The plasma concentrations of tyrosines in the local venous effluent and in the arterial blood were measured with high-performance liquid chromatography. In group I, production of o- and m-tyr, which are specific markers of ·OH formation, began during coronary occlusion but increased dramatically immediately after reperfusion, peaking at 1 minute and continuing up to 10 minutes of reperfusion. In group II, the production of o- and m-tyr was markedly decreased throughout the first 10 minutes of reperfusion. In group III, the production of m-tyr was decreased to levels similar to those in group II, whereas the production of o-tyr was almost completely abolished. There was no appreciable production of o- or m-tyr in group IV. Recovery of contractile function (assessed as systolic wall thickening) was increased in group I vs group V. Recovery of function was further enhanced in group II, with only a slight additional improvement in group III. This study demonstrates that (1) ·OH is produced in the stunned myocardium in vivo after brief regional ischemia, (2) antioxidants, such as MPG or MPG combined with superoxide dismutase, catalase, and desferrioxamine, actually inhibit ·OH activity in vivo, (3) this inhibition results in attenuation of myocardial stunning, (4) there is an inverse relation between the magnitude of the inhibition of ·OH activity soon after reflow and the severity of the subsequent contractile dysfunction, and (5) Phe, which itself acts as an ·OH scavenger, attenuates postischemic dysfunction. These results provide direct evidence that ·OH is an important mediator of myocardial stunning and suggest that aromatic hydroxylation of Phe may be a useful technique to investigate the role of ·OH in intact animals or in humans.