Is oxidative stress primarily involved in reperfusion injury of the ischemic heart?

Abstract

Reperfusion injury of ischemic organs is suggested to result from metabolic derangements initiating an imbalanced formation of free oxygen radicals. Most investigators in this field have used the spin-trap 5,5′-dimethyl-N-pyrroline-N-oxide (DMPO) to stabilize these short-lived radicals and make them visible by means of the electron spin resonance (ESR) technique. ESR signals obtained from intravascular DMPO were reported to indicate the formation of free OH . radicals and, in some cases, also carbon-centered radicals. We were unable to confirm these findings. Carbon-centered radicals were not obtained irrespectively of conditions studied, while oxygen-centered DMPO-adducts could only be detected in minor amounts. Instead, we observed an ascorbyl-related ESR signal. The addition of ethylenediaminetetraacetic acid (EDTA), which was used by many investigators in this field, was found to greatly influence ESR-spectra of the reperfusion fluid. The ascorbyl radical concentration was clearly reduced and the DMPO-OH . adduct became more prominent. The addition of iron further stimulated this change eliciting a Fenton-type reaction responsible for DMPO-OH .-related ESR spectra in the perfusate after ischemia. Accordingly, we observed the release of iron and ascorbic acid into the perfusate as a consequence of ischemia. We could demonstrate that iron in the presence of ascorbate and EDTA causes both types of radicals detected in the perfusate. DMPO-OH . generation in the presence of EDTA was found to result from free OH . radicals that were not generated in the absence of EDTA. While intravascular oxygen activation seems unlikely to be primarily involved in reperfusion injury, we were able to demonstrate extravascular radical formation using appropriate spin-trapping methods. Although nothing can be said about the identity of the extravascular radical source, it is concluded that these radicals are responsible for the liberation of tissue iron and its diffusion together with ascorbate across the normally impermeable vascular wall.