While the free radical generating enzyme xantUne oxidase (XO) has been proposed as a central mechanism of injury in postischemic tissues, questions remain regarding how the burst of XO mediated radical generation is triggered during ischemia and reperfusion. There is controversy regarding whether radical generation is caused by enzyme formation or that of its substrates xanthine and hypoxanlMne. Therefore, studies were performed in isolated rat hearts to correlate the magnitude and time course of radical generation with the alterations in XO and its substrates. Radical generation was measured by electron paramagnetic resonance spectroscopy using the spin trap DMPO and correlated with assays of tissue XO activity and HPLC measurement of tissue and effluent concentration of XO substrates and products. XO was present in preisctenic hearts, with an activity of 8.4 ±1.2 mU/g protein, and slightly increased during 30 min global ischemia, followed by a further 2 fold increase during subsequent reperfusion. HypoxantHne and xanthine were not present prior to ischemia but accumulated during ischemia with values of 2.2 ±0.3 and 2.4 ±0.2 nmoi/mg protein, respectively, due to ATP degradation. These substrate concentrations rapidly declined over the first S min of reperfusion matching the observed time course of radical generation while the concentration of XO was largely unchanged. Both substrates were also observed in the coronary effluent during the first S min of reflow along with XO product uric acid. These data demonstrated that alterations of XO activity did not trigger postischemic radical generation. In contrast, the increased concentrations of hypoxanthine and xarahine in postischemic myocardium exactly matched the time course of free radical generation. Thus, the burst of XO mediated free radical generation upon reperfusion is triggered and its time course controlled by a large increase in substrate formation due to the degradation of ATP during ischemia.
|Original language||English (US)|
|Journal||Journal of Investigative Medicine|
|State||Published - 1996|
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)