OxyFile #321

Hydrogen peroxide changes in ischemic and reperfused heart. 
Cytochemistry and biochemical and X-ray microanalysis.

Author:  Slezak J; Tribulova N; Pristacova J; Uhrik B; Thomas T; 
         Khaper N; Kaul N; Singal PK

Source:  Am J Pathol 1995 Sep; 147(3):772-81


Active oxygen species including hydrogen peroxide (H2O2) 
play a major role in ischemia-reperfusion injury. In the 
present study, changes in myocardial H2O2 content as well 
as its subcellular distribution were examined in rat hearts 
subjected to ischemia-reperfusion. Isolated perfused rat 
hearts were made globally ischemic for 20 or 30 minutes 
and were reperfused for different durations. H2O2 content 
in these hearts was studied biochemically and changes were 
correlated with the recovery of function. These hearts 
were also analyzed for subcellular distribution of H2O2. 
Optimal conditions of tissue processing as well as incubation 
medium were established for reacting cerium chloride with 
H2O2 to form cerium perhydroxide, an insoluble electron-dense 
product. The chemical composition of these deposits was 
confirmed by x-ray micro-analysis. Global ischemia caused 
complete contractile failure in minutes and after 30 minutes 
of ischemia, these was a > 250% increase in the myocardial 
H2O2 content. Depressed contractile function recovery in 
the early phase of reperfusion was accompanied by approximately 
a 600% increase in the myocardial H2O2 content. Brief pre-fixation 
with low concentrations of glutaraldehyde, inhibition of 
alkaline phosphatase, glutathione peroxidase, and catalase, 
post-fixation but no post-osmication, and no counterstaining 
yielded the best cytochemical definition of H2O2. In normal 
hearts, extremely small amounts of cerium hydroperoxide 
precipitates were located on the endothelial cells. X-ray 
microanalysis confirmed the presence of cerium in the reaction 
product. Ischemia resulted in a stronger reaction, particularly 
on the sarcolemma as well as abluminal side of the endothelial 
cells; and upon reperfusion, cerium precipitate reaction 
at these sites was more intense. In the reperfused hearts, 
the reaction product also appeared within mitochondria 
between the cristae as well as on the myofibrils, but Z-lines 
were devoid of any precipitate. The data support a significant 
increase in myocardial H2O2 during both the phase of ischemia 
and the first few minutes of reperfusion. A stronger reaction 
on the sarcolemma and abluminal side of endothelial cells 
may also indicate enhanced H2O2 accumulation as well as 
vulnerability of these sites to oxidative stress injury.