The generation of ROS starts during ischemia in the mitochondria. Although it might be expected that ischemia, leading to a low intracellular partial pressure of oxygen, would decrease oxyradical production, the paradoxic increase in ROS is secondary to the altered redox balance of the mitochondria. With ischemia, the metabolic reduction of the adenine nucleotide pool leaves the respiratory chain cytochromes in a more fully reduced state, allowing them to directly transfer (i.e., “leak”) electrons to the residual molecular oxygen entrapped within the inner mitochondrial membrane. This electron leakage appears capable of producing large amounts of superoxide radical. While reintroduction of oxygen with reperfusion reenergizes the mitochondria, the low ADP content also allows further electron leakage, thus enabling more reactions with molecular oxygen and yielding the excessive “burst” of ROS.

Within a few minutes of reperfusion, the pHi returns to normal values. However, in this altered milieu of calcium overload, high phosphate concentrations, and depletion of adenine nucleotides, the pH normalization favors the opening of the mitochondrial permeability transition pore, a highconductance channel whose opening leads to an increase of mitochondrial inner membrane permeability to solutes with molecular masses up to 1500 Da. This event plays a major role in the progression toward cell death. The opening of these pores releases stored calcium into the cytosol, and dissipation of the mitochondrial inner transmembrane potential uncouples the electron transport system from ATP hydrolysis. These events further abolish mitochondrial ATP production, leading to energy failure, enhanced production of ROS, and the release of apoptotic factors, thus contributing to cell death by apoptosis (4,5).

The xanthine oxidoreductase (XOR) enzyme system is an important enzymatic complex for the catabolism of purines, catalyzing the oxidation of hypoxanthine to xanthine and the oxidation of xanthine to uric acid. The system consists of two interconvertible forms, XO and xanthine dehydrogenase (XDH) (6). During ischemia, XDH is converted to the oxidase form by a protease activated by the intracellular overload of calcium. At the same time, degradation of ATP leads to an accumulation of hypoxanthine in the ischemic tissue. During reperfusion, with the presence of large quantities of molecular oxygen and high concentrations of hypoxanthine, XO yields a burst of superoxide (Fig. 21.4). During ischemia, the intracellular acidic conditions also allow the XOR enzyme system to catalyze the reduction of nitrates and nitrites to nitrites and NO, respectively, thus contributing to nitrosative stress.

Furthermore, ischemia and reperfusion in any tissue leads to the release of XOR into the circulation. Elevated plasma levels of XOR have been reported in adults subjected to limb ischemia-reperfusion (7) or liver transplantation (8). In critically ill infants and children with shock subsequent to severe sepsis, trauma, or major surgery, high XO activity has been detected in urine. This elevation in the XO activity index appears to correlate with high blood levels of oxidized glutathione (a marker of oxidative stress) and with the clinical symptoms of an excessive proinflammatory response (9). It has been suggested that, once in the circulation, XOR may contribute to the development of MODS. These circulating levels may, in fact, bind to the endothelial cells of distant sites and contribute to the initiation of oxidative damage in organs remote from the original ischemic tissue (6). The hypothesis that generation of superoxide by XO may play an important pathogenetic role in reperfusion injury has been supported by experimental studies in animals demonstrating that the inactivation of XO with allopurinol ameliorates reperfusion-induced tissue damage (10,11). A recent clinical study reported that allopurinol treatment might afford neuroprotective effects in moderately asphyxiated infants (12). Despite this human study and the beneficial effects reported in animal models, there is no other evidence of favorable effects of XOR inhibition in human conditions of reperfusion injury (10,11,13).

The NADPH oxidase family consists of seven Nox proteins: Nox1 to Nox5, Duox1 and Duox2. These proteins differ in their cell type expression, subunit composition, and function. In addition to phagocytic cells and neutrophils, where they mediate bacterial killing by producing a burst of superoxide anion, Nox enzymes are highly expressed in endothelial cells, vascular smooth muscle cells, cardiomyocytes, and renal tubular cells (14). A considerable number of experimental studies on mice genetically deficient of Nox proteins or their complex subunits suggest that this class of enzymes contribute to ischemia and reperfusion injury. Nox2 appears to contribute to lung, and brain reperfusion injury, as well as the remodeling process in the heart after infarction (14). Contribution of Nox enzymes in ischemia and reperfusion injury is suggested by clinical data demonstrating an upregulation of Nox2 and Nox5 in cardiomyocytes and intramyocardial blood vessels of patients who had died after myocardial infarction (15,16). Other clinical studies have reported that platelet-derived microparticles or exosomes, which were obtained from patients with septic shock, contained several Nox subunits and appeared to contribute to vascular cell apoptosis (17). At the present, there are no specific Nox inhibitors available. It has been reported that statins block activation of the Nox2 in addition to their HMG-CoA reductase inhibitory action (18,19); and polyphenols and anthocyanins, apart from their direct scavenging properties, can prevent Nox expression and reduce activity (20,21). However, whether the Nox system may be a therapeutic target in shock and reperfusion injury needs to be investigated in preclinical and clinical studies.

NO is a highly reactive gas that is synthesized from L-arginine by a family of enzymes referred as to NOSs. Three isoforms of NOS have been characterized: neuronal (nNOS, or type 1), inducible (iNOS, or type 2), and endothelial (eNOS, or type 3) (22). The eNOS and nNOS isoforms are constitutively expressed, are calcium/calmodulin dependent, and produce low amounts of NO. The designation of these constitutive isoforms does not strictly reflect their tissue expression because eNOS and nNOS are not only found in endothelial and neuronal cells but also in other cell types, including cardiomyocytes and muscle cells. The iNOS isoform is a calcium/calmodulin-independent enzyme that is responsible for the production of large amounts of NO. Although not detected under normal conditions, the expression of the iNOS isoform is induced in response to microbial products, inflammatory cytokines, and growth factors in almost every cell type during diverse pathologic conditions, including sepsis, hemorrhagic shock, trauma, and ischemia (22).

Under normal conditions, the constitutive forms of NOS release low concentrations of NO, which are critical to normal physiology. For example, the nNOS-derived NO acts as a neurotransmitter and a second messenger. The eNOS-derived NO is the physiologic mediator of normal vascular tone. Once formed by vascular endothelial cells, NO diffuses to adjacent smooth muscle cells, activates soluble guanylate cyclase, produces cyclic guanosine monophosphate (cGMP), and reduces intracellular calcium concentration, thus resulting in vasodilatation. Apart from its direct vasodilatory effect, the endothelium-derived NO also maintains normal tissue perfusion and vascular permeability by inhibiting platelet aggregation, leukocyte adherence, and smooth muscle proliferation in the vascular system. In cardiomyocytes, under physiologic conditions, both constitutive forms of NOS (types 1 and 3) have been described to release NO, which regulates cardiac function through their direct effects on several aspects of cardiomyocyte contractility, from the fine regulation of excitation -contraction coupling (with positive inotropic and lusitropic effects) to modulation of autonomic signaling and mitochondrial respiration (22,23).