Copyright © 2006, European Society of Cardiology
Hydroxylation of D-phenylalanine as a novel approach to detect hydroxyl radicals: Application to cardiac pathophysiology
aDepartment of Clinical and Experimental Medicine, University of Perugia School of Medicine, Perugia, Italy
bAzienda Ospedaliera S. Maria, Terni, Italy
cThe Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine and Center for Biomedical EPR Spectroscopy and Imaging, The Ohio State University, Columbus, OH, USA
* Corresponding author. Cardiology, Ospedale Silvestrini, S. Andrea delle Fratte, 06156 Perugia, Italy. Fax: +39 755271244. Email address: giuseppe.ambrosio{at}ospedale.perugia.it
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 °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 (
0.8 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.
KEYWORDS Ischemia; Reperfusion; Oxygen radicals; Redox signaling