Assessment of free radical-induced damage in brain proteins after ischemia and reperfusion.
Brain damage initiated during global ischemia has been shown to be exacerbated by iron-dependent lipid peroxidation during early reperfusion. We hypothesized that other cellular components might be involved in similar free radical reactions. In this study we examined three brain protein fractions and ribosomal RNA for evidence of free radical damage during post-ischemic reperfusion. Global brain ischemia was induced by 20-min cardiac arrest. Dogs were divided into four groups: (1) non-ischemic controls; (2) 20-min cardiac arrest without reperfusion; (3) 20-min cardiac arrest and 2 h reperfusion; (4) 20-min cardiac arrest and 8 h reperfusion. Soluble proteins and proteins from ribosomes and synaptosomes were assayed by a dinitrophenylhydrazine method for carbonyl groups, which are characteristic products of protein peroxidation. The ribosomal RNA was also examined by electrophoresis. When proteins from each fraction were peroxidized in vitro by Fenton reagents, carbonyl content increased as [Fe2+] was increased from 0 to 100 microM. However, following reperfusion there was no significant accumulation of carbonyl content in either the soluble (ANOVA P = 0.92) or ribosome (P = 0.10) protein fractions. There was a significant decrease in the carbonyl content of the synaptosome protein fraction after 8 h of reperfusion (P = 0.03). Similarly, although ribosomal RNA fragmentation was observed in ethidium stained agarose gels following in vitro reaction with Fenton reagents, there was no evidence of ribosomal RNA fragmentation or cross-linking following reperfusion. These results suggest that reperfusion free radical reactions do not involve these cellular proteins or ribosomal RNA.
Krause
G S
GS
Department of Emergency Medicine, Wayne State University School of Medicine, Detroit, MI.
DeGracia
D J
DJ
Skjaerlund
J M
JM
O'Neil
B J
BJ
D000818:Animals; D001921:Brain / Q000378:metabolism*; D004285:Dogs; D005609:Free Radicals; D002546:Ischemic Attack, Transient / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism*; D012151:Resuscitation; D012269:Ribosomal Proteins / Q000378:metabolism*
D005609:Free Radicals; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins