For a long time, superoxide generation by an NADPH oxidase was considered as an oddity only found in professional phagocytes. Over the last years, six homologs of the cytochrome subunit of the phag...

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The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology

Publisher Summary This chapter discusses the role of free radicals and catalytic metal ions in human disease. The importance of transition metal ions in mediating oxidant damage naturally leads to the question as to what forms of such ions might be available to catalyze radical reactions in vivo . The chapter discusses the metabolism of transition metals, such as iron and copper. It also discusses the chelation therapy that is an approach to site-specific antioxidant protection. The detection and measurement of lipid peroxidation is the evidence most frequently cited to support the involvement of free radical reactions in toxicology and in human disease. A wide range of techniques is available to measure the rate of this process, but none is applicable to all circumstances. The two most popular are the measurement of diene conjugation and the thiobarbituric acid (TBA) test, but they are both subject to pitfalls, especially when applied to human samples. The chapter also discusses the essential principles of the peroxidation process. When discussing lipid peroxidation, it is essential to use clear terminology for the sequence of events involved; an imprecise use of terms such as initiation has caused considerable confusion in the literature. In a completely peroxide-free lipid system, first chain initiation of a peroxidation sequence in a membrane or polyunsaturated fatty acid refers to the attack of any species that has sufficient reactivity to abstract a hydrogen atom from a methylene group.

Role of free radicals and catalytic metal ions in human disease: an overview.

Lethal reperfusion injury is a paradoxical type of myocardial injury caused by the restoration of coronary blood flow after an ischemic episode. This review focuses on the mechanisms of the injury, on attempts to protect the heart against it, and on promising new approaches to cardioprotection during percutaneous coronary intervention.

Myocardial Reperfusion Injury

Byproducts of normal mitochondrial metabolism and homeostasis include the buildup of potentially damaging levels of reactive oxygen species (ROS), Ca2+, etc., which must be normalized. Evidence suggests that brief mitochondrial permeability transition pore (mPTP) openings play an important physiological role maintaining healthy mitochondria homeostasis. Adaptive and maladaptive responses to redox stress may involve mitochondrial channels such as mPTP and inner membrane anion channel (IMAC). Their activation causes intra- and intermitochondrial redox-environment changes leading to ROS release. This regenerative cycle of mitochondrial ROS formation and release was named ROS-induced ROS release (RIRR). Brief, reversible mPTP opening-associated ROS release apparently constitutes an adaptive housekeeping function by the timely release from mitochondria of accumulated potentially toxic levels of ROS (and Ca2+). At higher ROS levels, longer mPTP openings may release a ROS burst leading to destruction of mitochondria, and if propagated from mitochondrion to mitochondrion, of the cell itself. The destructive function of RIRR may serve a physiological role by removal of unwanted cells or damaged mitochondria, or cause the pathological elimination of vital and essential mitochondria and cells. The adaptive release of sufficient ROS into the vicinity of mitochondria may also activate local pools of redox-sensitive enzymes involved in protective signaling pathways that limit ischemic damage to mitochondria and cells in that area. Maladaptive mPTP- or IMAC-related RIRR may also be playing a role in aging. Because the mechanism of mitochondrial RIRR highlights the central role of mitochondria-formed ROS, we discuss all of the known ROS-producing sites (shown in vitro) and their relevance to the mitochondrial ROS production in vivo.

Mitochondrial Reactive Oxygen Species (ROS) and ROS-Induced ROS Release

This review focuses on the role of oxidative processes in atherosclerosis and its resultant cardiovascular events. There is now a consensus that atherosclerosis represents a state of heightened oxidative stress characterized by lipid and protein oxidation in the vascular wall. The oxidative modification hypothesis of atherosclerosis predicts that low-density lipoprotein (LDL) oxidation is an early event in atherosclerosis and that oxidized LDL contributes to atherogenesis. In support of this hypothesis, oxidized LDL can support foam cell formation in vitro, the lipid in human lesions is substantially oxidized, there is evidence for the presence of oxidized LDL in vivo, oxidized LDL has a number of potentially proatherogenic activities, and several structurally unrelated antioxidants inhibit atherosclerosis in animals. An emerging consensus also underscores the importance in vascular disease of oxidative events in addition to LDL oxidation. These include the production of reactive oxygen and nitrogen species by vascular cells, as well as oxidative modifications contributing to important clinical manifestations of coronary artery disease such as endothelial dysfunction and plaque disruption. Despite these abundant data however, fundamental problems remain with implicating oxidative modification as a (requisite) pathophysiologically important cause for atherosclerosis. These include the poor performance of antioxidant strategies in limiting either atherosclerosis or cardiovascular events from atherosclerosis, and observations in animals that suggest dissociation between atherosclerosis and lipoprotein oxidation. Indeed, it remains to be established that oxidative events are a cause rather than an injurious response to atherogenesis. In this context, inflammation needs to be considered as a primary process of atherosclerosis, and oxidative stress as a secondary event. To address this issue, we have proposed an "oxidative response to inflammation" model as a means of reconciling the response-to-injury and oxidative modification hypotheses of atherosclerosis.

Role of Oxidative Modifications in Atherosclerosis

The purpose of this study was to use a direct method, that of electron spin resonance (ESR) spectroscopy, to demonstrate that reperfusion after a period of ischemia results in a sudden increase in the production of free radicals in the myocardium. The isolated buffer-perfused rat heart was used with N-tert-butyl-alpha-phenylnitrone (PBN) as a spin-trapping agent. Samples of coronary effluent were taken and extracted into toluene for detection of radical adducts by ESR spectroscopy. After 15 minutes of total, global ischemia, aerobic reperfusion resulted in a sudden burst of radical formation that peaked at 4 minutes. When hearts were reperfused with anoxic buffer, no dramatic increase in radical production was observed. Subsequent reintroduction of oxygen, however, resulted in an immediate burst of radical production of a similar magnitude to that seen in the wholly aerobic reperfusion experiments. The ESR signals obtained (aN = 13.60 G, aH = 1.56 G) are consistent with the spin-trapping by PBN of either a carbon-centered species or an alkoxyl radical, both of which could be formed by secondary reactions of initially-formed oxygen radicals with membrane lipid components.

Direct detection of free radicals in the reperfused rat heart using electron spin resonance spectroscopy.

Reperfusion-induced arrhythmias: Mechanisms and prevention

To determine residual flow to ischaemic tissue, which is the primary determinant of the rate of development and ultimate size of the myocardial infarct resulting from coronary artery occlusion, the coronary collateral circulation was quantified during acute myocardial ischaemia in eight species in vivo using the radiolabelled microsphere technique. In each case, a prominent branch of the left coronary artery was ligated, and within 5 min microspheres (141Ce labelled, 15 micron diameter) were injected intra-atrially. Hearts were then excised, frozen, and sliced perpendicular to the septum. Using autoradiograms as a guide, tissue samples were obtained from non-ischaemic and ischaemic tissue and the radioactivity of the ischaemic samples measured and expressed as a percentage of the activity in the non-ischaemic myocardium. In the guinea pig heart, despite ligation of a major artery, no zone of significant underperfusion was detected. In the hearts from other species, coronary collateral flow (as a percentage (mean(SEM)) of non-ischaemic flow) was: dog 15.9(1.8) (n = 6); cat 11.8(1.1) (n = 16); rat 6.1(0.7) (n = 6); ferret 2.4(0.6) (n = 6); baboon 2.1(0.3) (n = 6); rabbit 2.0(0.5) (n = 9); pig 0.6(0.2) (n = 6). The dog and cat hearts both possessed transmural gradients of collateral flow with greatest delivery in the epicardium. The patterns of flow distribution in the guinea pig heart were further examined in a Langendorff perfused preparation. Blue dye was injected into the coronary circulation and its distribution over 5 s recorded on cine film. After ligation of the left anterior descending or circumflex arteries, or both, the perfusion field of these arteries was seen to fill retrogradely within seconds. It is concluded that a wide spectrum of collateral flow exists between various mammalian species, a fact that should be taken into account in the study of the pathophysiology and control of regional ischaemia and myocardial infarction.

Species variation in the coronary collateral circulation during regional myocardial ischaemia: a critical determinant of the rate of evolution and extent of myocardial infarction

David J. Hearse

Papers

Direct detection of free radicals in the reperfused rat heart using electron spin resonance spectroscopy.

Reperfusion-induced arrhythmias: Mechanisms and prevention

Species variation in the coronary collateral circulation during regional myocardial ischaemia: a critical determinant of the rate of evolution and extent of myocardial infarction

The oxygen paradox and the calcium paradox: Two facets of the same problem?

Reperfusion of the ischemic myocardium.