Objective: Research in the pathophysiology of ischemia/reperfusion or redox signaling is hindered by lack of simple methodology to measure short-lived oxygen radicals. In the presence of hydroxyl radical ((*)OH), d-phenylalanine (d-Phe) yields para-, meta- and ortho-tyrosine. We have previously demonstrated that d-Phe can accurately detect (*)OH formation in chemical, enzymatic and cellular systems by simple HPLC methodology [Anal Biochem 290:138;2001]. In the present study, we tested whether d-Phe hydroxylation can be used to detect (*)OH formation in intact organs.
Methods: Rat hearts were perfused with buffer containing 5 mM d-Phe and subjected to 30 min of total global ischemia at 37 degrees C followed by 45 min of reperfusion. Quantitative analysis of the three hydroxytyrosine isomers was achieved by HPLC-based electrochemical detection of cardiac venous effluent, with the analytical cells operating in the oxidative mode. The detection limit of this assay was <10 fmol.
Results: Under baseline conditions, hydroxytyrosine release from the heart was very low ( congruent with0.8 nmol/min/g). However, a prominent tyrosine burst occurred immediately upon post-ischemic reflow. In cardiac effluent collected 40 s into reperfusion, the hydroxytyrosine concentration was more than 40 times greater than at baseline; hydroxytyrosine concentration then progressively declined, to return to pre-ischemic values by 5 min of reperfusion. In parallel experiments, formation of hydroxytyrosines was markedly reduced in hearts reperfused in the presence of the (*)OH scavenger mannitol. Inclusion of 5 mm d-Phe in the perfusion medium altered neither basal cardiac function nor coronary vascular tone, but it enhanced recovery of myocardial function during post-ischemic reperfusion, consistent with direct reaction with (*)OH.
Conclusion: Our results demonstrate that d-Phe is a sensitive method for detection of (*)OH generation in the heart. Since d-Phe is not a substrate for endogenous enzymes, it can be exploited as a reliable method to measure (*)OH formation under a variety of pathophysiological conditions.