Oxygen radical production during ischemia-reperfusion in the isolated perfused rat liver as monitored by luminol enhanced chemiluminescence

We have applied the Luminol enhanced chemiluminescence technique to the isolated perfused rat liver during ischemia and reperfusion to monitor the production of oxygen radicals in tissue. Livers under perfusion with Luminol-containing buffer were subjected to 30 minutes of global ischemia followed by 60 minutes of reperfusion. Their chemiluminescence was continuously monitored to obtain the time course of oxygen radical production. Transient bursts of oxygen radical production were observed in the livers as indicated by chemiluminescence changes on reperfusion. Superoxide dismutase treatment abolished while catalase treatment enhanced the reperfusion-induced chemiluminescence transient.     

Effects of tocopheryl quinone on the heart: model experiments with xanthine oxidase, heart mitochondria, and isolated perfused rat hearts

It is generally accepted that the protection effect of biological tissues by vitamin E is due to its radical scavenging potency in membranes, thereby being transformed to a vitamin E radical. A deficiency of appropriate reductants, which recycle vitamin E radicals back to its antioxidative active form, causes an irreversible degradation of vitamin E leading to tocopheryl quinone (TQ). TQ-like compounds were shown to result from both vitamin E and corresponding hydrophilic analogues of this antioxidant in vitro. In vivo elevated concentrations of tocopheryl quinones were detected after oxidative stress and TQ supplementation as well. Quinones in general are known to be efficient one-electron donors and acceptors. Therefore the question arises whether TQ-like compounds can undergo redox-cycling in conjunction with redox-active enzymes in the heart, thereby producing harmful oxygen radicals, or whether these compounds exhibit antioxidant properties. In order to elucidate this question we focused our interest on the interaction of TQ and a corresponding short-chain homologue (TQ(0)) with xanthine oxidase and heart mitochondria. Furthermore, we tested the influence of TQ on the recovery of isolated perfused rat hearts after ischemia/reperfusion. Our experiments revealed that hydrophilic TQ(0) was univalently reduced by xanthine oxidase (XOD) yielding semiquinone radicals in the absence of oxygen. However, under aerobic conditions TQ(0) enhanced the O(2)(*)(-) radical output of XOD. In the mitochondrial respiratory chain TQ was shown to interact with high potential cytochrome b in the bc(1) complex specifically. In contrast to the system XOD/TQ(0), lipophilic TQ in submitochondrial particles decreased the O(2)(*)(-) radical release during regular respiration possibly due to its interaction with b-cytochromes in the mitochondrial respiratory chain. In isolated rat hearts perfused with liposomes containing lipophilic TQ, it was efficiently accumulated in the heart tissue. When hearts were subjected to conditions of ischemia/reperfusion, infusion of TQ prior to ischemia significantly improved the recovery of hemodynamic parameters. Our results demonstrate that TQ derivatives may induce pro-oxidative and antioxidative effects depending on the distribution of TQ derivatives in the heart tissue and the interacting redox system.     

Uncoupling of mitochondrial oxidative phosphorylation alters lipid peroxidation-derived free radical production but not recovery of postischemic rat hearts and post-hypoxic endothelial cells

The contribution of mitochondrial free radical production towards the initiation of lipid peroxidation (LPO) and functional injury in the post-ischemic heart is unclear. Using the isolated rat heart model, the effects of the uncoupler of mitochondrial oxidative phosphorylation dinitrophenol (DNP, 50 M final) on post-ischemic lipid peroxidation-derived free radical production and functional recovery were assessed. Hearts were subjected to 30 min total global ischemia followed by 15 min of reperfusion in the presence of DNP. As expected, DNP enhanced oxygen consumption before (11.3 ± 0.9 mol/min, p < 0.001) and during reperfusion (at 10 min: 7.9 ± 0.7 umol/min), compared to the heart with control treatment (8.2 ± 0.5 and 6.7 ± 0.3, respectively). This effect was only associated with a higher incidence of ventricular tachycardia during reperfusion (80 vs. 50% for control treatment, p < 0.05). Electron spin resonance spectroscopy (ESR) and spin trapping with u.-phenyl-tert-butylnitrone (PBN, 3 mM final) were used to monitor free radical generation during reperfusion. The vascular concentration of PBN-radical adducts (untreated: 6.4 ±1.0 nM, at 10 min) decreased in the presence of DNP (1.7 ± 0.4 nM, p < 0.01). The radical concentration inversely correlated with myocardial oxygen consumption. Total liberation of free radical adducts during the initial 10 min of reperfusion was reduced by DNP (0.59 ± 0.09 nmol, p < 0.01) compared to the respective control treatment (1.26 ± 0.16 nmol). Similar effects, prevention of PBN adduct formation and unchanged viability in the presence of DNP, were obtained with endothelial cells during post-hypoxic reoxygenation. Since inhibition of mitochondrial phosphorylation can inhibit the formation of LPO-derived free radicals after an ischemic/hypoxic interval, mitochondria may represent an important source of free radicals capable of initiating lipid peroxidative injury during reperfusion/reoxygenation. (Mol Cell Biochem 160/161: 167–177, 1996)     

Effect of ischemia/reperfusion sequence on cytosolic iron status and its release in the coronary effluent in isolated rat hearts

The hypothesis that oxygen-derived free radicals play an important role in myocardial ischemic and reperfusion injury has received a lot of support. In the presence of catalytic amounts of transition metals such as iron, superoxide anions, and hydrogen peroxide can be transformed into a highly reactive hydroxyl radical °OH (Haber-Weiss reaction). In view of this, we have undertaken this study to investigate whether iron is involved in the reperfusion syndrome and therefore could aggravate free radicals injury. Coronary effluent iron concentrations and cardiac cytosolic iron levels were evaluated in rat hearts subjected to an ischemia/reperfusion sequences. In the case of total ischemia, iron concentration in coronary effluents peaked immediately in the first sample collected upon reperfusion. However, in the case of partial ischemia, iron concentration in coronary effluents peaked rather exclusively during ischemia period. Cardiac cytosolic iron level augmented significantly after 30 min of total ischemia and non significantly in the other ischemia protocols compared to perfused control hearts. It also appears that the iron released is not protein-bound, and could therefore have a marked catalytic activity. The results of the present study suggest that in the oxygen paradox, iron plays an important role in inducing alterations during reoxygenation.     

Oxygen radicals in cardiac surgery

Most cardiac surgical procedures require the use of prolonged induced myocardial ischemia. Experimental models of global myocardial ischemia which mimic cardiac surgical techniques have been developed to investigate the possibility of oxygen free radical development during prolonged myocardial ischemia or upon reperfusion. In such experiments, various free radical scavenging agents, including superoxide dismutase, catalase, and mannitol, have been shown to improve the tolerance of the heart to protracted global ischemia. Use of these agents has improved cardiac functional recovery and has attenuated the biochemical and structural changes which occur due to prolonged ischemia and reflow. In a recently developed porcine experimental model, the effects of preexisting regional myocardial ischemia with superimposed global ischemia and reperfusion have been studied, with free radical scavenging agents administered in an attempt to reduce myocardial infarction and improve regional functional recovery. In most such studies completed to date, free radical scavenging agents have resulted in better myocardial preservation, suggesting, at least indirectly, that there may be an oxygen free radical-mediated component of the ischemia-reperfusion injury seen in such models. Techniques for directly measuring myocardial oxygen free radical levels may allow for early clarification of the development of such toxic species in the clinical cardiac surgical setting.     

Effects of oxygen radicals on substrate oxidation by cardiac myocytes

Freshly isolated adult rat heart cells were used to study the effects of oxygen-free radicals on the myocardial oxidation of different substrates. The calcium-tolerant quiescent cells were incubated with xanthine plus xanthine oxidase as the source of free radicals. The oxidation of exogenous glucose, lactate and octanoate was severely inhibited (approx. 70%) by products of xanthine oxidase activity. Superoxide dismutase plus catalase effectively prevented the inhibition of oxidation. Cellular high energy phosphate levels were decreased in the presence of the oxygen free radical generating system although cell viability determined by Trypan blue exclusion and light microscopic assessment of normal morphology was not affected. These data suggest that oxygen free radicals decrease myocardial substrate oxidation which may contribute to the functional and ultrastructural changes in the myocardium under conditions such as reoxygenation after hypoxia and reperfusion after ischemia.     

Identification and quantification of free radicals during myocardial ischemia and reperfusion using electron paramagnetic resonance spectroscopy

There is general agreement that free radicals are involved in reperfusion injury. Electron paramagnetic resonance (EPR) spectroscopy can be considered as the more suitable technique to directly measure and characterize free radical generation during myocardial ischemia and reperfusion. There are essentially two approaches used in the detection of unstable reactive species: freezing technique and spin traps. The detection of secondary free radicals or ascorbyl free radicals during reperfusion might provide an index of oxidative stress. Spin trapping can also characterize nitric oxide. EPR spectroscopy can provide important data regarding redox state and free radical metabolism but ideally, the spin traps must not interfere with cell or organism function.     

Detection of ischemia-reperfusion cardiac injury by cardiac muscle chemiluminescence

Various methods have been used in the past to assess the implication of oxygen free radicals (OFR) in ischemia-reperfusion-induced cardiac injury. Luminol-enhanced tert-butyl-initiated chemiluminescence in cardiac tissue reflects oxidative stress and is a very sensitive method. It was used to elucidate the role of OFR in cardiac injury due to ischemia and reperfusion. Studies were conducted on perfused isolated rabbit hearts in three groups (n = 8 in each): I, control; II, submitted to global ischemia for 30 min; III, submitted to ischemia for 30 min followed by reperfusion for 60 min. The heart tissue was then assayed for chemiluminescence (CL); content of malondialdehyde (MDA), an indicator of OFR-induced cardiac injury; and activity of tissue levels of antioxidants [superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px)].The control values for left and right ventricular CL and malondialdehyde were 81.1 ± 15.4 (S.E.) and 182.4 ± 50.3 (S.E.), mv-min-mg protein^–1; and 0.024 ± 0.006 (S.E.) and 0.324 ± 0.005 (S.E.) nmoles-mg protein^–1 respectively. Ischemia produced an increase in the cardiac CL (3.3 to 4.4 fold) and MDA content (2 to 2.6 fold). Reperfusion following ischemia also produced similar changes in CL and MDA content. The control values for activity of left ventricular SOD, catalase, and GSH-Px were 45.77 ± 1.73 (S.E.) U-mg protein^–1 5.35 ± 0.51 (S.E.) K-10^–3-sec^–1-mg protein^–1, and 77.50 ± 7.70 (S.E.) nmoles NADPH-min^–1-mg protein^–1 respectively. Activities of SOD and catalase decreased during ischemia but were similar to control values in ischemic-reperfused hearts. The GSH-Px activity of left ventricle was unaffected by ischemia, and ischemia-reperfusion. GSH-Px activity of the right ventricle increased with ischemia, and ischemic-reperfusion.These results indicate that cardiac tissue chemiluminescence would be a useful and sensitive tool for the detection of oxygen free radical-induced cardiac injury.     

Application of high-dose propofol during ischemia improves postischemic function of rat hearts: effects on tissue antioxidant capacity

Previous studies have shown that reactive oxygen species mediated lipid peroxidation in patients undergoing cardiac surgery occurs primarily during cardiopulmonary bypass. We examined whether application of a high concentration of propofol during ischemia could effectively enhance postischemic myocardial functional recovery in the setting of global ischemia and reperfusion in an isolated heart preparation. Hearts were subjected to 40 min of global ischemia followed by 90 min of reperfusion. During ischemia, propofol (12 microg/mL in saline) was perfused through the aorta at 60 microL/min. We found that application of high-concentration propofol during ischemia combined with low-concentration propofol (1.2 microg/mL) administered before ischemia and during reperfusion significantly improved postischemic myocardial functional recovery without depressing cardiac mechanics before ischemia, as is seen when high-concentration propofol was applied prior to ischemia and during reperfusion. The functional enhancement is associated with increased heart tissue antioxidant capacity and reduced lipid peroxidation. We conclude that high-concentration propofol application during ischemia could be a potential therapeutic and anesthetic strategy for patients with preexisting myocardial dysfunction.     

Ascorbyl free radical as a reliable indicator of free-radical-mediated myocardial ischemic and post-ischemic injury. A real-time continuous-flow ESR study

The real-time kinetics of the release of ascorbyl free radicals in the coronary perfusate from isolated rat hearts submitted to an ischemia/reperfusion sequence has been achieved by continuous-flow ESR using high-speed acquisition techniques. Enhanced ESR detection of ascorbyl free radicals was obtained by addition of dimethyl sulfoxide (Me2SO), a strong cation chelator and oxidizing agent. A continuous-flow device allowed a direct monitoring of the ascorbyl free radical and/or ascorbate leakage in coronary perfusate by observation of the ascorbyl radical doublet (aH = 0.188 mT and g = 2.0054). 1. The results showed that ascorbyl free radical release occurred mainly during sequences of low-flow ischemia (90 min) coupled or not with 30 min of zero-flow ischemia followed by reperfusion (60 min). The kinetic profiles of ascorbyl-free-radical detection confirm in quantitative terms the expected correlation between the duration of the ischemic insult and the magnitude of ascorbate extracellular release upon reperfusion. There is indication that ascorbyl free radical depletion could be secondary to oxygen-derived-free-radical-induced cellular damage. 2. The amount of residual ascorbic acid was quantitated on myocardial tissue at the end of reperfusion using Me2SO as extracting solvent. Intense oxidation of ascorbate and chemical stabilization of the resulting free radical species provided by Me2SO allowed ESR measurement of a marked tissue ascorbate depletion related to the duration of ischemia. 3. Perfusion of superoxide dismutase during low-flow ischemia and the first 10 min of reperfusion greatly inhibited both extracellular release and endogenous ascorbate depletion. These results suggest that the ascorbate redox system constitutes a major protective mechanism against free-radical-induced myocardial injury. 4. The proposed direct ESR detection of ascorbyl free radicals in the coronary perfusates or in tissue extracts does not require extensive chemical preparation and conditioning of effluent or tissue samples. It provides an interesting straightforward alternative to the evaluation of detrimental free radical processes affecting the myocardium during ischemia and reperfusion.     

Evaluation of the role of xanthine oxidase in myocardial reperfusion injury

The free radical-generating enzyme xanthine oxidase has been hypothesized to be a central mechanism of the injury which occurs in postischemic tissues; however, its importance remains controversial. Much attention has focused on the role of this enzyme in myocardial reperfusion injury. While xanthine oxidase has been observed in ischemic tissue homogenates, the presence and importance of radical generation by the enzyme in intact tissues are unknown. Therefore, we performed electron paramagnetic resonance, nuclear magnetic resonance and hemodynamic studies to measure the presence and significance of xanthine oxidase-mediated free radical generation in the isolated rat heart. When isolated perfused rat hearts were reperfused after 30 min of global ischemia, myocardial function and coronary flow were significantly improved in the presence of the definitive xanthine oxidase blocker oxypurinol. Free radical concentrations measured by spin-trapping with 5,5'-dimethyl-1-pyrroline-N-oxide were significantly decreased by oxypurinol and the energetic state of the heart was improved as reflected by an increased recovery of phosphocreatine and a higher phosphocreatine/Pi ratio. ATP recovery, however, was not altered, indicating that the improved functional and metabolic state of the heart was not due to ATP salvage. Spectrophotometric assays for the enzyme showed an increase in the amount of xanthine oxidase relative to dehydrogenase following ischemia, and a total available xanthine oxidase pool in the rat heart of approximately 150 milliunits/g of protein. Thus, xanthine oxidase is a significant source of the oxidative injury which occurs upon reperfusion of the ischemic rat heart.     

Identification of free radicals in myocardial ischemia/reperfusion by spin trapping with nitrone DMPO

The spin trapping ESR technique was applied to investigate oxygen-derived radicals in ischemic and post-ischemic rat hearts. Using 5,5'-dimethyl-l-pyrroline-N-oxide, carbon-centered radicals were identified during ischemia and oxy-radical adducts (superoxide anion radical, O.-2 and hydroxyl radicals, .OH) in post-ischemic rat heart. The formation of these spin adducts was inhibited by superoxide dismutase, suggesting that superoxide plays a role in the adducts' formation. The results demonstrate that oxygen derived free radicals are important byproducts of abnormal oxidative metabolism during myocardial ischemic and reperfusion injuries.     

Role of xanthine oxidase inhibitor as free radical scavenger: a novel mechanism of action of allopurinol and oxypurinol in myocardial salvage

Xanthine oxidase (XO) has been hypothesized to be a potential source of oxygen-derived free radicals during reperfusion of ischemic myocardium based on the fact that allopurinol, a XO-inhibitor, can reduce reperfusion injury. In this communication we report that both allopurinol and oxypurinol, the principle metabolite of allopurinol, prevent the reperfusion injury in isolated pig heart. However, we found that neither pig heart nor pig blood contain any XO activity. Our study showed a direct free radical scavenging action of these XO-inhibitors during ischemia and reperfusion, as judged by the reduction of free radical signals when compared using an Electron Paramagnetic Resonance Spectrometer. Using a Luminometer, we also confirmed that both allopurinol and oxypurinol can scavenge ClO2, HOCl, and significantly inhibit free radical signals generated by activated neutrophils. These XO-inhibitors, however, failed to scavenge O2. and OH. radicals. Our results suggest that these XO-inhibitors salvaged the ischemic-reperfused myocardium by scavenging free radicals, and not by inhibiting XO in the pig heart.     

Detection of hydroxyl radical in the mitochondria of ischemic-reperfused myocardium by trapping with salicylate

Although the presence of free radicals has been indicated in ischemic-reperfused heart, the exact nature and source of these free radicals are not known. The present study utilized a chemical trap, salicylic acid, to trap hydroxyl radical which could be detected as hydroxylated benzoic acid using high pressure liquid chromatography. Since the hydroxylated product is extremely stable, heart was subjected to subcellular fractionation after ischemia and reperfusion, and each fraction was separately examined for the presence of hydroxyl radical. The results indicated for the first time the presence of hydroxyl radical in the mitochondrial fraction during early reperfusion, which decreased in intensity as the reperfusion progressed.     

Maintenance of left ventricular function (90%) after twenty-four-hour heart preservation with deferoxamine.

During 24-h in vitro heart preservation and reperfusion, irreversible tissue damage occurs caused by reactive oxygen intermediates, such as superoxide radicals, singlet oxygen, hydrogen peroxide, hydroperoxyl, hydroxyl radicals, as well as the peroxynitrite radical. Reduction of the related oxidative damage of reperfused ischemic tissue by free radical scavengers and metal chelators is of primary importance in maintaining heart function. We assessed whether deferoxamine (DFR) added to a cardioplegia solution decreased free radical formation during 24-h cold (5 degrees C) heart preservation and normothermic reperfusion (37 degrees C) in the Langendorff isolated perfused rat heart. The deferoxamine treated hearts were significantly (p less than .001) better preserved than the control hearts after 24 h of preservation with regard to recovery of left ventricular diastolic pressure, contractility (+dP/dt), relaxation (-dP/dt), creatine kinase release, and lipid peroxidation. DFR preserved cell membrane integrity and maintained 93% of left ventricular contractility. The evidence suggests that DFR reduces lipid peroxidation damage by reducing free radical formation and thereby maintaining normal coronary perfusion flow and myocardial function.     

Isolated perfused rat hearts release secondary free radicals during ischemia reperfusion injury: Cardiovascular effects of the spin trap α-phenyl N-tert-butylnitrone

Free radicals produced during myocardial post-ischemic reperfusion are aggravating factors for functional disturbances and cellular injury. The aim of our work was to investigate the significance of the secondary free radical release during non ischemic perfusion and post-ischemic reperfusion and to evaluate the cardiovascular effects of the spin trap used. For that purpose, isolated perfused rat hearts underwent 0, 20, 30 or 60 min of a total ischemia, followed by 30 min of reperfusion. The spin trap: α-phenyl N-tert-butylnitrone (PBN) was used (3 mM). Functional parameters were recorded and samples of coronary effluents were collected and analyzed using Electron Paramagnetic Resonance (EPR) to identify and quantify the amount of spin adducts produced. During non ischemic perfusion, almost undetectable levels of free radical release were observed. Conversely, a large and long-lasting (30 min) release of spin adducts was detected from the onset of reperfusion. The free radical species were identified as alkyl and alkoxyl radicals with amounts reaching 40 times the pre-ischemic values. On the other hand, PBN showed a cardioprotective effect, allowing a significant reduction of rhythm disturbances and a better post-ischemic recovery for the hearts which were submitted to 20 min of ischemia. When the duration of ischemia increased, the protective effects of PBN disappeared and toxic effects became more important. Our results have therefore confirmed the antioxidant and protective properties of a spin trap agent such as PBN. Moreover, we demonstrated that the persistent post-ischemic dysfunction was associated with a sustained production and release of free radical species.     

Isolated perfused rat hearts release secondary free radicals during ischemia reperfusion injury: cardiovascular effects of the spin trap alpha-phenyl N-tert-butylnitrone.

Free radicals produced during myocardial post-ischemic reperfusion are aggravating factors for functional disturbances and cellular injury. The aim of our work was to investigate the significance of the secondary free radical release during non ischemic perfusion and post-ischemic reperfusion and to evaluate the cardiovascular effects of the spin trap used. For that purpose, isolated perfused rat hearts underwent 0, 20, 30 or 60 min of a total ischemia, followed by 30 min of reperfusion. The spin trap: alpha-phenyl N-tert-butylnitrone (PBN) was used (3 mM). Functional parameters were recorded and samples of coronary effluents were collected and analyzed using Electron Paramagnetic Resonance (EPR) to identify and quantify the amount of spin adducts produced. During non ischemic perfusion, almost undetectable levels of free radical release were observed. Conversely, a large and long-lasting (30 min) release of spin adducts was detected from the onset of reperfusion. The free radical species were identified as alkyl and alkoxyl radicals with amounts reaching 40 times the pre-ischemic values. On the other hand, PBN showed a cardioprotective effect, allowing a significant reduction of rhythm disturbances and a better post-ischemic recovery for the hearts which were submitted to 20 min of ischemia. When the duration of ischemia increased, the protective effects of PBN disappeared and toxic effects became more important. Our results have therefore confirmed the antioxidant and protective properties of a spin trap agent such as PBN. Moreover, we demonstrated that the persistent post-ischemic dysfunction was associated with a sustained production and release of free radical species.     

Effects of dimethyl sulfoxide on the globally ischemic heart: Possible general relevance to hypothermic organ preservation

Isolated perfused rabbit hearts were made globally ischemic for 2 hr, then reperfused. For 5 min before and after ischemia hearts were perfused with hypothermic (20 or 27 °C), hypoxic, substrate-free cardioplegic solutions, some of which contained 70 mM dimethyl sulfoxide. Postischemic ventricular pressure development, spontaneous heart rate, coronary flow, lactate dehydrogenase release, tissue Ca²⁺ content, and in vitro mitochondrial oxidative phosphorylation were used to evaluate the protective effects of the various solutions. Aside from the expected observations that cold cardioplegia lessens ischemic damage, we found that dimethyl sulfoxide gave no indication that it exacerbated ischemic damage or lessened the protection afforded by cardioplegia. We also found that, compared to values measured in comparable drug-free treated hearts, dimethyl sulfoxide significantly improved mitochondrial State 3 respiratory rates, respiratory control, and oxidative phosphorylation rates, and essentially prevented mitochondrial changes due to ischemia and reperfusion. We propose that dimethyl sulfoxide may act as a “scavenger” of cytotoxic free radicals, many of which are known to be generated by mitochondria during reoxygenation. Since hypoxia, ischemia, and reoxygenation are common accompaniments of most organ preservation protocols, we suggest that low concentrations of dimethyl sulfoxide might serve as a useful adjunct to organ preservation in the nonfrozen state, when cryoprotective concentrations are not needed.     

Oxygen radical injury in the immature isolated rabbit heart.

We speculated that the increased vulnerability of the immature rabbit heart to global ischemia might be due to an increased susceptibility to free radical injury. To evaluate this, we exposed newborn (age 2.4 +/- 0.3 days, n = 20) (mean +/- SEM), juvenile (2 to 3 weeks, mean 16.6 +/- 0.5 days, n = 20), and adult (5 to 7 months old, n = 20) isolated, isovolumic, Krebs perfused rabbit hearts to oxygen radicals or cumene hydroperoxide. Control hearts showed no deterioration in left ventricular developed pressure over 60 min (newborns = 104 +/- 11%, juveniles = 101 +/- 7%, and adults = 113 +/- 12% of baseline, n = 5 for each age group). After only 30 min of oxygen radical exposure, the newborn group developed pressure decreased to 49 +/- 6% of the baseline value, while juveniles and adults were functioning at 70 +/- 10% and 83 +/- 6% of baseline, respectively (n = 10 for each age group) (P less than 0.05, newborn different from adult group). In contrast to the oxygen radical protocol, the hearts exposed to cumene hydroperoxide showed no significant difference between the age groups in deterioration of left ventricular function. There was no significant difference between the age groups in ATP content or thiobarbituric reactive substances following the oxygen radical exposure. We conclude that the newborn rabbit heart is significantly more vulnerable than the adult heart to the toxic effects of oxygen radicals. This may account, in part, for age related differences in response to global ischemia and reperfusion.     

Free radical scavenging is involved in the protective effect of L-propionyl-carnitine against ischemia-reperfusion injury of the heart.

L-Propionyl-carnitine is known to improve the recovery of myocardial function and metabolic parameters reduced in the course of ischemia-reperfusion of the heart. The mechanism of this protective effect of L-propionyl-carnitine is not fully understood. The purpose of this study was to elucidate the effects of L-propionyl-carnitine in Langendorff perfused rat hearts subjected to 40 min of ischemia followed by 20 min of reperfusion. We tested the hypothesis that L-propionyl-carnitine suppresses generation of oxygen radicals and subsequent oxidative modification of myocardial proteins during reperfusion. Our data show that the protective effect of L-propionyl-carnitine in the course of ischemia-reperfusion is highly significant in terms both of mechanical properties of the heart (developed pressure) and of high-energy phosphates (ATP, creatine phosphate). Myocardial creatine phosphokinase (CPK) activity decreased in the course of the reperfusion period. The loss of CPK activity was partially prevented by L-propionyl-carnitine. Two other effects were observed when L-propionyl-carnitine was present in the perfusion solution: (i) the reperfusion-induced sharp increase in oxidative protein modification was completely prevented as detected by the formation of protein carbonyls, and (ii) generation of hydroxyl radicals was significantly inhibited as detected by the formation of the adducts with the spin trap 5,5-dimethyl-1-pyrroline-1-oxide. We conclude that the protective effect of L-propionyl-carnitine against ischemia-reperfusion injury of the heart is at least due in part to its ability to suppress the development of oxidative stress and free radical damage.