FreeRadicalBiology& Medicine. Vol. 15, pp. 667-670, 1993 0891-5849/93 $6.00 + .00 Printed in the USA. All rights reserved. Copyright © 1993 Pergamon Press Ltd. Brief Communication INHIBITION OF POSTCARDIAC ARREST BRAIN PROTEIN OXIDATION BY ACETYL-L-CARNITINE YUANBIN LIU,* ROBERT E. ROSENTHAL, *t PAMELA STARKE-REED, • and GARY FISKUM *t *Department of Biochemistry and Molecular Biology and *Emergency Medicine, The George Washington University School of Medicine, Washington, DC, USA; and *National Institutes of Health, Bethesda, MD, USA (Received 24 August 1992; Revised 10 February 1993; Accepted 21 April 1993) Abstract--Free radical mediated, site-specific protein oxidation has been implicated in the pathophysiology ofischemia/reper- fusion brain injury. The purpose of this study was to determine whether this form of molecular damage could be detected in a clinically relevant model employing 10-min cardiac arrest in dogs followed by restoration of spontaneous circulation for up to 24 h. The effects of postischemic acetyl-L-carnitine administration on protein oxidation were also tested due to its previously reported improvement of brain energy metabolism and neurological outcome in this model. Following the experimental period, soluble proteins were extracted from a sample of frontal cortex and reacted with dinitrophenylhydrazine for spectrophotometric measurement of protein carbonyl groups. The most important results of this study were that brain protein carbonyl groups were significantly elevated following 2 and 24 h of reperfusion compared to nonischemic controls, and that postischemic IV adminis- tration of acetyl-L-carnitine eliminated the increase in carbonyl groups observed at the 24-h period. These results indicate that brain protein oxidation does occur in a clinically relevant model of complete global cerebral ischemia and reperfusion, and that oxidation is inhibited under treatment conditions that improve neurological outcome. Keywords--Protein oxidation, Carbonyl groups, Cerebral ischemia, Acetyl-L-carnitine, Free radicals INTRODUCTION It is well known that ischemia/reperfusion brain in- jury has been linked to the generation of free radicals.I Most studies have demonstrated a role for free radical damage to lipids; z however, the role of oxidative pro- tein and enzyme damage in reperfusion injury has not been clearly defined. It has previously been deter- mined from in vitro experiments that many key meta- bolic enzymes are oxidatively inactivated by metal cat- alyzed oxidation systems, including biological sys- tems) It appears that oxidative enzyme inactivation is a site-specific process involving the binding of Fe 2÷ to generate an activated oxygen species which oxi- dizes amino acids, e.g., proline and histidine, at or near the metal binding site. 4 Site-specific, free radical-mediated modification of proteins and inactivation of enzymes is accompanied by the generation of protein carbonyl groups that can Address correspondence to: Gary Fiskum, Department of Bio- chemistry and Molecular Biology, George Washington University School of Medicine, Washington, DC 20037, USA. react with dinitrophenylhydrazine to form stable pro- tein-hydrazone derivatives. Oliver et al. 5 utilized a spectrophotometric assay of these derivatives to dem- onstrate a twofold increase in the level of protein car- bonyl groups in the brains of gerbils that underwent 15 min of complete ischemia followed by 2 h ofreper- fusion. A more recent study by Krause et al. 6 reported no increase in protein oxidation following ischemia/ reperfusion in dogs when a high affinity iron chelator was added to brain homogenates to minimize protein oxidation that might occur in vivo. The purpose of the present study was to determine what tissue pro- cessing conditions are necessary and sufficient for minimizing in vitro protein oxidation and to charac- terize the degree of brain protein oxidation present following 10 min of canine cardiac arrest and up to 24 h of restoration of spontaneous circulation. Another aim was to determine whether postischemic adminis- tration of acetyl-L-carnitine, which is a drug that has been shown to improve neurological outcome in this model (7), would lower potentially elevated levels of carbonyl groups present in the soluble protein frac- tion of canine cerebral cortex. 667