Joe M. McCord

Oxygen-Derived Free Radicals in Postischemic Tissue Injury

It is now clear that oxygen-derived free radicals play an important part in several models of experimentally induced reperfusion injury. Although there are certainly multiple components to clinical ischemic and reperfusion injury, it appears likely that free-radical production may make a major contribution at certain stages in the progression of the injury. The primary source of superoxide in reperfused reoxygenated tissues appears to be the enzyme xanthine oxidase, released during ischemia by a calcium-triggered proteolytic attack on xanthine dehydrogenase. Reperfused tissues are protected in a variety of laboratory models by scavengers of superoxide radicals or hydroxyl radicals or by allopurinol or other inhibitors of xanthine oxidase. Dysfunction induced by free radicals may thus be a major component of ischemic diseases of the heart, bowel, liver, kidney, and brain.

Free Radicals and Inflammation: Protection of Synovial Fluid by Superoxide Dismutase

Enzymatically genierated superoxide radical. by reactitng with hydrogen peroxide to prduce the hydroxyl radical, depolymerized puirified hyaluronic acid and bovine synovial flulid. Since phagocytizing polymorphonuclear leukocytes produce superoxide radicals, this reac-tion is sutggested and shown to be quantitatively feasible as the ini vivo mechanism of synovial fluid degradation in anl inflamed joint. Superoxide dismutase, and catalase protect synaovial fluid against such degradation in vitro.

Superoxide-dependent production of hydroxyl radical catalyzed by iron-EDTA complex.

Their proposal was supported by the following observations: (i) ethylene production could be inhibited by superoxide dismutase, (ii) by catalase, or (iii) by scavengers of hydroxyl radical such as ethanol and benzoate; and (iv) a lag in ethylene production was found to be due to the time required for hydrogen peroxide to accumulate to a reactive concentration. Reaction 1 was the simplest explanation accommodating all the observed facts, and the reaction had been proposed many years earlier by Haber and Weiss during their studies of the catalytic decomposition of hydrogen peroxide by iron salts [4] . In the last seven years the reaction has become known in the biological literature as the Haber-Weiss reaction, and has been invoked by many investigators to explain diverse biological phenomena which are characterized by the same inhibitory criteria described by Beauchamp and Fridovich and outlined above. On the other hand, attempts to demonstrate directly the simple biomolecular reaction shown as reaction 1 have met with uniform failure [5-71, leading to the conclusion that, although reaction 1 may indicate the stoichiometry of the overall process, it cannot be construed as the reaction mechanism. Under chemically well-defined conditions, it has in fact been shown that reaction 1 does not proceed at any appreciable rate. Koppenol, however, has shown the reaction to be quite feasible thermodynamically [8], even if the oxygen molecule which appears as a product is in the electronically excited ‘AgOz singlet state, as Kellogg and Fridovich have suggested [9] .

[41] Preparation and assay of superioxide dismutases

Publisher Summary This chapter discusses the preparation and assay of superoxide dismutase (SOD). SODs are found in all oxygen-utilizing organisms and constitute a defense against oxygen toxicity. SODs were first isolated from erythrocytes as a copper protein of unknown function. Thus, some SODs contain copper and zinc, others contain manganese, and still others contain iron. Assay techniques for each of these enzymes are similar, but distinct isolation procedures are used in their purification. Most mammalian tissues contain both a cuprozinc and a mangano superoxide dismutase. SODs are unique among enzymes in that their substrate is an unstable free radical. This complicates the measurement of their catalytic activity. Convenient assays of SODs have necessarily been of the indirect type. Such assays consist of two components: a superoxide generator and a superoxide detector. The control reaction rate can be completely inhibited by large amounts of SOD if a xanthine oxidase of high quality is being used. Partially degraded xanthine oxidase can, to a small extent, reduce cytochrome c by a nonsuperoxide mediated mechanism.

Xanthine oxidase as a source of free radical damage in myocardial ischemia.

Experiments were performed to determine if xanthine oxidase is a source of free radicals during myocardial ischemia. Open chest dogs were subjected to 1 h of total occlusion of the left anterior descending coronary artery followed by 4 h of reperfusion. Directly after coronary artery occlusion, Ce141 microspheres were injected into the left atrium to mark the ischemic bed. At the end of reperfusion, the hearts were removed and sectioned. Autoradiography determined the ischemic myocardium at risk, and the necrotic zone was determined by triphenyl-tetrazolium staining. Animals were divided into three groups: control, allopurinol (24-h oral pretreatment 400 mg, then 50 mg/kg IV bolus on occlusion); and superoxide dismutase starting with occlusion (15 000 U/kg). The size of the infarct as a percentage of the tissue at risk was: 23.1 +/- 4.1 for the control; 8.7 +/- 1.2 for the allopurinol group; and 5.4 +/- 1.2 for the superoxide dismutase group. The infarcts in the allopurinol and superoxide dismutase groups were significantly smaller than those in the control groups. In a second series of experiments we determined the xanthine oxidase/xanthine dehydrogenase content of dog myocardium. The left anterior descending branch was ligated for 30 min and then biopsies were removed from both the normal and the ischemic regions. Total enzyme content did not differ between the two regions averaging 0.259 U/g protein for the ischemic tissue and 0.225 U/g protein for the normal region. Only 9.8% of the enzyme was in the oxidase form in the normal region while 32.8% was in the oxidase form in the ischemic zone.(ABSTRACT TRUNCATED AT 250 WORDS)

The Biology and Pathology of Oxygen Radicals

Superoxide radicals (O2-) are commonplace products of the biological reduction of oxygen. Their intrinsic reactivity and ability to generate other more reactive entities constitute a threat to cellular integrity. Superoxide dismutases, enzymes that catalytically scavenge these radicals, have evolved to meet this threat. These metalloenzymes are essential for respiring organisms to survive. Several compounds, such as the antibiotic streptonigrin and the herbicide paraquat, augment the production rate of O2- inside cells. This accounts for the oxygen-enhancement of their lethality. Some bacteria respond to this artificially increased rate of O2- production by synthesizing additional superoxide dismutase. Ionizing radiation generates O2- in its passage through oxygenated aqueous media, and superoxide dismutase added to the suspending medium, decreases the oxygen-enhancement of the lethality of such irradiation of the bacterium Escherichia coli. Production of O2- by activated neutrophils is clinically significant, since it is an important component of the bactericidal actions of these cells and the inflammatory process. Superoxide dismutases exert an anti-inflammatory action that may be useful in managing inflammations.

Free radicals and myocardial ischemia: Overview and outlook☆

Much evidence suggests that free radicals and active oxygen species derived from molecular oxygen (superoxide, hydrogen peroxide, and hydroxyl radical) contribute to the tissue injury which accompanies myocardial ischemia and reperfusion. Three possible sources have been identified for the production of active oxygen species: the enzyme xanthine oxidase; the activated polymorphonuclear leukocyte; the disrupted mitochondrial electron transport system. These sources may be mutually interactive. Once triggered, they may lead to the loss of antioxidant enzymes and to the release of iron, both of which are exacerbatory events.

Xanthine oxidase inhibitors attenuate ischemia-induced vascular permeability changes in the cat intestine

Previous reports indicate that allopurinol, a xanthine oxidase inhibitor, largely prevents the injury produced by reperfusion of ischemic tissues. In order to further assess the role of xanthine oxidase in ischemia-reperfusion injury, we examined the influence of another inhibitor of the enzyme (pterin aldehyde) on the increased vascular permeability produced by intestinal ischemia. Vascular permeability estimates in autoperfused segments of cat ileum were derived from the relationship between lymph-to-plasma protein concentration ratio and lymph flow. One hour of intestinal ischemia increased vascular permeability to 0.43 +/- 0.02 from a control (nonischemic) value of 0.08 +/- 0.005. In ischemic ileal segments pretreated with purified pterin aldehyde, vascular permeability increased to only 0.15 +/- 0.02. Pretreatment with commercially prepared folic acid, which is contaminated with pterin aldehyde, also attenuated the ischemia-induced increase in vascular permeability (0.16 +/- 0.04). These findings support the hypothesis that xanthine oxidase is a major source of oxygen-free radicals produced during reperfusion of the ischemic small bowel.

MITOCHONDRIAL GENERATION OF OXYGEN RADICALS DURING REOXYGENATION OF ISCHEMIC TISSUES

Ischemia and reperfusion causes severe mitochondrial damage, including swelling and deposits of hydroxyapatite crystals in the mitochondrial matrix. These crystals are indicative of a massive influx of Ca2+ into the mitochondrial matrix occurring during reoxygenation. We have observed that mitochondria isolated from rat hearts after 90 minutes of anoxia followed by reoxygenation, show a specific inhibition in the electron transport chain between NADH dehydrogenase and ubiquinone in addition to becoming uncoupled (unable to generate ATP). This inhibition is associated with an increased H2O2 formation at the NADH dehydrogenase level in the presence of NADH dependent substrates. Control rat mitochondria exposed for 15 minutes to high Ca2+ (200 nmol/mg protein) also become uncoupled and electron transport inhibited between NADH dehydrogenase and ubiquinone, a lesion similar to that observed in post-ischemic mitochondria. This Ca(2+)-dependent effect is time dependent and may be partially prevented by albumin, suggesting that it may be due to phospholipase A2 activation, releasing fatty acids, leading to both inhibition of electron transport and uncoupling. Addition of arachidonic or linoleic acids to control rat heart mitochondria, inhibits electron transport between Complex I and III. These results are consistent with the following hypothesis: during ischemia, the intracellular energy content drops severely, affecting the cytoplasic concentration of ions such as Na+ and Ca2+. Upon reoxygenation, the mitochondrion is the only organelle capable of eliminating the excess cytoplasmic Ca2+ through an electrogenic process requiring oxygen (the low ATP concentration makes other ATP-dependent Ca2+ transport systems non-operational).(ABSTRACT TRUNCATED AT 250 WORDS)

Cardioprotection by Cu, Zn-superoxide dismutase is lost at high doses in the reoxygenated heart

Limited dose-response curves for superoxide dismutase (SOD) were assessed in isolated and in vivo hearts. SOD at 2.3, 7, 20, or 50 mg/L suppressed CK release in Langendorff rat hearts by 61%, 63%, 72%, and 30%, respectively. SOD at 0.5, 1, 5, and 50 mg/L suppressed LDH release in Langendorff rabbit hearts by 32%, 48%, 54%, and -12%, respectively. In rabbit hearts subjected to coronary artery ligation and reperfusion in vivo, SOD at 2, 5, or 15 mg/kg reduced infarct size by 10%, 30% or 19%, respectively, while 50 mg/kg increased infarct size by 28%. In conclusion, while SOD was protective at low doses in all models, protection was lost at higher doses in the isolated rat and rabbit hearts, and exacerbation of damage was seen in the in vivo rabbit hearts.