Oxidative Stress

Corresponding Author: Jean-Charles Preiser, MD, PhD, Department of Intensive Care, Erasme University Hospital, 808 route de Lennik, Brussels, B1070; Belgium; e-mail: Jean-Charles.Preiser@erasme.ulb.ac.be. JPEN J Parenter Enteral Nutr. 2012;36:147-154

Tutorial The issues of oxidative stress and its modulation by antioxidants currently have received more attention than ever before. In the field of nutrition, the content of antioxidants in the components of regular diets is carefully scrutinized by many consumers and healthcare providers. Many antioxidant supplements are available for purchase over the counter in most countries worldwide. These supplements, embedded into pills, syrup, powder, or creams, are usually extracted from vegetals and are claimed to contain high amounts of vitamins with antioxidant properties (mainly vitamins A, C, and E) and trace elements such as selenium, copper, or zinc. In general, these products are not checked as carefully as drugs and pharmaceuticals and can be delivered without medical prescription. The marketing policy of these supplements is based on the claims that antioxidants are able to prevent or treat oxidative stressrelated events or diseases. Similarly, the wave of enthusiasm for the use of antioxidants is further supported by the beneficial effects of several drugs and medications, thought to be related to their recently discovered antioxidant properties. However, in contradiction to the belief that oxidative stress is detrimental and should be treated or prevented, several recent studies, meta-analyses, and systematic 1-4 reviews were unable to confirm the expected benefit of supplemental antioxidants; some of these studies even found deleterious effects related in individuals randomized to receive supplemental antioxidants. These unexpected effects were 5 named the antioxidant paradox by Halliwell in 2000. . Likewise, discrepant findings were also reported from 6 experiments using diverse model organisms, where oxidative stress or antioxidants can increase or decrease life span. These apparently contradictory findings can only be reconciled by the interpretation of increased oxidative stress as an adaptive mechanism in some instances and as a causal mechanism in pathological situations. In humans, markers of oxidative stress are found increased in most if not in all acute and chronic diseases. Even physiological conditions, such as exercise or aging, are associated with increased oxidative stress. Because of this duality in the roles of oxidative stress, its modulation by antioxidants is a more complex issue than previously thought. This tutorial intends to summarize the current understanding of oxidative stress in human medicine to provide some help for clinicians involved in medical nutrition. The following issues will be discussed in this tutorial: 1. Definition of oxidative stress 2. Historical aspects 3. Activation of oxygen and production of reactive oxygen species (ROS) 4. Modulating and stopping the oxidative chain 5. Physiological roles of oxidative stress 6. Pathogenic roles of oxidative stress 7. Biomarkers Abstract Oxidative stress is defined by an imbalance between increased levels of reactive oxygen species (ROS) and a low activity of antioxidant mechanisms. An increased oxidative stress can induce damage to the cellular structure and potentially destroy tissues. However, ROS are needed for adequate cell function, including the production of energy by the mitochondria. Increased oxidative stress has been incriminated in physiological conditions, such as aging and exercise, and in several pathological conditions, including cancer, neurodegenerative diseases, cardiovascular diseases, diabetes, inflammatory diseases, and intoxications. However, prevention by antioxidants has been mostly inefficient. Therefore, a rigorous scientific evaluation in well-defined conditions is mandatory to define the appropriate place for manipulations of the oxidative pathways in human medicine.

Definition of Oxidative Stress A popular definition of oxidative stress can be found in Wikipedia: an imbalance between the production and manifestation of reactive oxygen species (ROS) and a biological systems ability to readily detoxify the reactive intermediates 7 8 or to repair the resulting damage. In a textbook on oxidative stress published in 1985 by Sies, a very similar definition can be found: the imbalance between oxidants and antioxidants potentially leading to damage. In any case, 3 distinct issues are suggested in these definitions: (1) a production of reactive or activated forms of oxygen (ROS) (2) with potential toxicity and (3) the presence of detoxifying systems. Oxygen is not used by the cell if not activated as ROS, implying that life is impossible in most organisms without ROS, which are needed for several important cell functions. The physiological roles of ROS are balanced with their deleterious effects in case of excessive release of ROS overwhelming the regulatory antioxidant mechanisms. Indeed, when produced in excess and not tightly kept under control by the antioxidant detoxifying systems, ROS can potentially damage all components of the cell. Historical Aspects Oxygen has not always been present in the Earths atmosphere: several lines of evidence indicate that oxygen, produced by cyanobacteria and plants, appeared in the Earths atmosphere about 2.8 billion years ago and took up residence around 2.45 billion years ago. Because of its toxicity, oxygen eliminated the living organisms unable to develop antioxidant systems. A Darwinian-type evolutionary process selected the organisms most adapted to their environmentthat is, only the organisms able to use the activated form of oxygen (ROS) and neutralize the oxygen-related toxicity were able to survive. Eukaryotic cells engulfed bacteria able to use oxygen to produce energy with the formation of ROS as a component of this process. Once incorporated into cells, these symbiotic bacteria evolved as mitochondria, able to generate a large 9 amount of energy from oxygen via the generation of ROS. Very importantly, mitochondria also developed regulatory systems able to protect themselves against oxidative injuries 10 related to these ROS. . Oxygen itself was discovered and described by the Swedish chemist Scheele in 1772; 2 years later, Priestley reported the production of oxygen by vegetals; in 1778, Lavoisier discovered the presence of oxygen in the ambient air. A role for oxygen in combustion was described. In 1927, toxic effects of oxygen were reported in several 11 animal species when present in the atmosphere at high concentrations (over 70%). In the mid-1950s, similarities between radiation-induced toxicity and oxygen-related damages were reported. Gerschman et als free radical theory of oxygen toxicity, published in 1954, speculated that the toxicity of oxygen was due to partially 12 reduced forms of oxygen, later identified as ROS. Currently, the dual roles of oxygen and ROS are highlighted in different experimental and clinical settings (discussed in the sections on physiological and pathogenic roles of oxidative stress). In parallel, the various aspects of the activation of oxygen as ROS were progressively unravelled. Activation of Oxygen and Production of ROS ROS are the active form of oxygen (Table 1), needed for several cellular functions at rest and in response to different 13-16 stimuli. . Indeed, the diatomic oxygen molecule (O2) does not react spontaneously with other molecules, as it contains 2 unpaired electrons (biradical). To allow the reaction of oxygen with organic molecules, there are 2 possibilities: (1) the organic molecule is transformed into a monoradical (a molecule containing 1 unpaired electron) by the removal of 1 electron (oxidation), and/or (2) oxygen is converted into a monoradical by the addition of 1 electron (reduction). As these processes require a considerable energy supply, catalytic systems are needed to drop the energy barrier by multiple subsequent low-energy steps. In vivo, these systems are metallic complexes or enzymes, the oxygenases and oxidases, which respectively transform organic molecules or oxygen into monoradicals. The oxygenases (like cyclooxygenases) catalyze the extraction of H (a hydrogen atom with its electron) from a molecule. The oxidases (like NADPHoxidase) transfer one electron from a donor molecule to oxygen, thereby generating superoxide (O2 ) (Figure 1).

From superoxide, hydrogen peroxide (H2O2) is formed by dismutation (spontaneous or enzymatic). Hydrogen peroxide is relatively stable and can therefore easily diffuse far from its production site. Possible subsequent steps include (1) detoxification into water by the enzymes glutathione peroxidases or catalase and/or by the mitochondrial respiratory chain complexes, (2) formation of hydroxyl OH) in the presence of iron (eg, ferrous in heme groups), or (3) conversion into hypochlorous acid (HOCl) by peroxidases. Hypochlorous acid is a powerful oxidant, with a lifetime sufficient to diffuse in and out of the cell and to oxidize almost all types of biomolecules. Close connections also exist between the metabolism of ROS and the metabolism of reactive nitrogen species, mainly nitric oxide (NO; Figure 1). NO is also a free radical, able to freely diffuse, and its activity is limited by its short lifetime. Importantly, the most toxic derivate of NO is peroxynitrite ONOO, which results from the combination of NO with superoxide. Peroxynitrite is a powerful oxidant for all types of molecules and is therefore highly cytotoxic. Peroxynitrite is unstable and can be converted into new active species, such as hydroxyl radical (OH) and nitryl radical (NO2), esponsible for hydroxylation and nitration. NO can also react with O2 to generate nitrite (NO2) in appropriate conditions. In Vivo Production of ROS In vivo, ROS are produced by different pathways and released from several cell types, with important differences in the 15-17 amount produced upon stimulation. The phagocytes (monocytes/macrophages and polymorphonuclear neutro18 phils) are the most important producers of ROS in acute conditions, as a component of the immune response to designed to neutralize invading particles and micro-organisms. A continuous production of low amounts of ROS is present in cells equipped with active mitochondria, and ROS are continuously produced in much lower amounts in the respiratory 9,15,16 chain to generate energy. Enzymatic Pathways Enzymes of the NADPH oxidase (NOX) family. In resting conditions, these enzymes are not active but are quickly activated by the assembly of the protein subunits after binding by soluble and receptor binding mediators to readily convert O2 into O2 (monoelectronic reduction). NOX2 is an essential enzyme of phagocytes, but NOX enzymes are present in many cell types, such as lymphocytes, fibroblasts, endothelial cells, myocytes, and chondrocytes, where moderate amounts of ROS are produced and serve as a regulator of cell responses.
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 Myeloperoxidase (MPO), an enzyme present in the azurophilic granules of neutrophils and at lower levels in monocytes and eosinophils, uses H2O2 and Cl to form HOCl. When the enzyme MPO is released from neutrophils, its concentration in blood is a marker of neutrophil activation. Mitochondrial enzymes. In mitochondria, O2 is reduced in H2O by the mitochondrial respiratory chain complexes, via an intermediate step of O2 production by an electron leak at different levels of the electron transport chain (the complex I, the ubiquinone/cytochrome c intersection, and the cytochrome oxidase). The mitochondrion is equipped with a rapidly inducible SOD (Mn-SOD) but also with an NOS, suggesting that an intramitochondrial production of ONOO is possible. Xanthine-oxidase, a cytosolic, nonheme enzyme, which converts xanthine or hypoxanthine into uric acid, derives from xanthine dehydrogenase by proteolytic activation during hypoxia. It uses O2 as an electron acceptor, forming O2 and H2O2 . Its activity is probably regulated by NO NO synthases (NOS) produce NO from O2 and L-arginine, in the presence of NADPH, calcium, and/or biopterin as cofactors. 19. In particular conditions related to substrate and cofactor concentrations, NOS produce O2 together with NO , increasing the risk of in situ generation of ONOO . Endothelial cells and neuronal cells contain a constitutive enzyme (eNOS and nNOS respectively), considered a housekeeping enzyme, producing physiological levels of NO needed for vascular tone and for the regulation or neurotransmission. During inflammation, an inducible isoform of the NOS enzyme (iNOS) is activated in many cells (macrophages, neutrophils, endothelial cells, smooth muscle cells, hepatocytes) and releases much higher amounts of NO. Mitochondria themselves also contain an iNOS enzyme. Numerous radical enzymes are present in many cell types and tissues, such as the mixed-function oxidases of the endoplasmic reticulum, the cytosolic enzymes (lipoxygenases, prostaglandin H synthases, or cyclooxygenases), the peroxysomic enzymes (glycolate oxidase, Damino acid oxidase, urate oxidase, fatty acyl Co-A oxidase), and even DNA methylating enzymes and enzymes involved in the synthesis of hormones and neurotransmitters. In normal conditions, production of ROS by these enzymes is low. Some metallic ions (iron, copper), released in case of cell lysis, amplify oxidative stress, as these serve as cofactors of the conversion of hydrogen peroxide into hydroxyl. Modulating and Stopping the Oxidative Chain Once an organic molecule has been oxidized and becomes a free radical (R), ROS may further react with R to highly reactive peroxyl radicals (ROO). These ROS could then initiate a chain of reactions toward certain chemical entities or cellular components, which can result in cell death and tissue injury. A classical example of this chain reaction is lipid peroxidation of cell membrane lipids leading to cellular injury. Only reducing molecules (noncellular components with the capacity to accept the unpaired electron from the free radical) can interrupt the process by the transfer of 1 electron. Among potential reducing systems, the antioxidant mechanisms were specifically developed to balance and neutralize the oxidative effects of ROS. In vivo, the mechanisms of inactivation of ROS include successive steps: the acceleration of the dismutation of O2 anion into H2O2 under the influence of SOD and the conversion of H2O2 into water under the

influence of catalase and glutathione peroxidase. Importantly, trace elements (copper/manganese/zinc, iron, and selenium) are respectively required for the activity of SOD, catalase, and glutathione peroxidase. The bioavailability of these trace elements is often the ratelimiting factor of the antioxidant enzymes. The major nonenzymatic defense mechanisms include endogenous molecules (glutathione, urate, ubiquinones/ubiquinol, albumin, and bilirubin) and vitamins (ascorbic acid, -tocopherol, -carotene). Functionally, the antioxidant systems can be classified as stoichiometric (scavengers of ROS) and catalytic (detoxifying enzymatic systems). The localization of these antioxidants (into the cytoplasm or extracellularly) and the lipophilic properties are other features used to characterize their site and type of action. Physiological Roles of Oxidative Stress The production and release of ROS are involved in several physiological pathways, and the intracellular concentrations of ROS are tightly controlled by the antioxidant defense mechanisms. Interestingly, the efficiency of the antioxidant mechanisms is not stable over time, implying a natural increase of oxidative stress at the extreme stages of life. 15,16 ROS are involved numerous physiological functions extensively reviewed elsewhere.

Figure 2 shows that the intracellular concentration of ROS (y-axis, zones 2 and 3) is transiently elevated in response to one stimulus such as cytokine, growth factor, or hormone. This pattern is very typical in physiological situations, where the release of ROS is rapidly controlled by the antioxidant regulatory mechanisms. When sustained or not counterbalanced (zones 46), the increased oxidative stress has probably overwhelmed the antioxidant capabilities, and ROS can induce damage (see the next section on pathogenic roles). The release of ROS is involved in major pathways of cell signalling, allowing the transduction of extracellular stimuli into changes in cell physiology by the modulation of the transcription of some genes or by posttranscriptional modulation. So far, redox-responsive signaling pathways have been involved in important functions such as the production of NO , regulation of vascular tone and neurotransmission, cell adhesion, the immune response, and the sensing of hypoxia and 15,16 apoptosis. However, ROS are often viewed as mostly toxic molecules because a reduction of oxidative stress is associated with prolongation of the life span in various species. For instance, the free radical theory of aging states that 20 any organisms age because cells accumulate damage related to oxidative damage induced by ROS over time. An imbalance between the antioxidant defense mechanisms and the amount of ROS produced by mitochondria was 21 suggested later to further support this theory. The success of this model resulted in huge enthusiasm for dietary antioxidant supplements and for topical treatments containing antioxidants supposed to hide the signs of aging. More 6 recently, the effects of the manipulation of oxidative stress on the life span of various organisms have differed widely, suggesting that the inhibition of oxidative stress with antioxidants could be deleterious in some species and/or some

circumstances. A meta-analysis of the effects on some antioxidants (-carotene, vitamins A and E) in healthy humans 1 (more than 180,000 participants) showed an increased risk of mortality in the treated group (relative risk, 1.02). These 2-4 data are consistent with some findings in some subgroups of patients at specific risk. In any case, a prolongation of human life span by the intake of antioxidant supplements is not supported. However, a 5 healthy lifestyle, including the consumption of antioxidant-rich food, is clearly beneficial. Similarly, the effects of antioxidants on the signs of aging or senescence do not necessarily imply a longer life. Another common physiological condition associated with increased oxidative stress is physical exercise. Indeed, increased mitochondrial activity in muscles will result in enhanced production and leakage of ROS. In parallel, ischemia-reperfusion and activation of neutrophils during exercise further enhance the production of ROS by the xanthine oxidase and NOX enzymes, respectively. Intense exercise or exercise in untrained individuals is more likely to be associated with increased oxidative stress, as compared with regular and moderately intense aerobic physical activity. Not surprisingly, a beneficial role of antioxidant supplementation before exercise has not been confirmed, even though antioxidant supplementation 22 decreases the measurable levels of biomarkers of oxidative stress. In fact, long-term regular physical activity might indeed improve some antioxidant defense mechanisms and thereby could 23,24 also limit the oxidative damage to mitochondria (mitohormesis). A similar mechanism was postulated to explain the increase in life span of several species submitted to calorie restriction. The effects of calorie restriction (not associated with malnutrition) in humans could indeed prolong life, as suggested by the high number of centenarians in Okinawa, Japan, where caloric restriction is unavoidable, whereas deficiencies in noncaloric nutrients are efficiently prevented. 25-28 Recent evidence demonstrates that calorie restriction reduces oxidative stress. This issue is presently under investigation in humans. Pathogenic Roles of Oxidative Stress The involvement of excessive oxidative stress has been suggested for several pathological conditions, as increased levels of several biomarkers of oxidative damage were found in many chronic diseases, as well as associated with risk factors for the development of these diseases. The balance between ROS and antioxidants is a critical factor. Specifically, a number of antioxidants (eg, vitamins C and E) themselves have demonstrated a potent pro-oxidant effect in the absence of a sufficient amount of free radicals for them to neutralize. Therefore, the physiological excess of antioxidant concentration at a given time, either by supplementation or by other factors, may also complicate clinical outcomes. Increased oxidative stress is found in cases of chronic high alcohol intake, obesity, smoking, and chronic exposure to air pollutants. The finding of higher levels of oxidative stress during diabetes or stress hyperglycemia, chronic obstructive pulmonary disease, chronic and acute inflammation, cancer, and ischemia-reperfusion further support the hypothesis of a pathogenetic role of oxidative stress. Likewise, epidemiological findings showed a link between depletion of the stores of antioxidants with an increased prevalence of various disorders, including cancer, heart disease, accelerated aging, and neurodegenerative diseases. Conversely, increased consumption of fruits and vegetables exerts synergistic effects on antioxidant activities and reduces the risk of chronic disease, at least for cancer and heart disease. However, primary and secondary prevention with dietary supplementations of antioxidant vitamins and trace elements in the general population is 1,2 mostly inefficient. Contrasted findings were also reported from large-scale studies of supplementation, stressing the complexities of the 29 roles and the regulation of the components of oxidative stress in vivo. Obviously, the type of antioxidant used and the dose, timing, and duration of administration are important issues that could partially explain the failures of some reventive strategies. Therefore, the involvement of oxidative stress can only be considered established, probable, or plausible, depending on the robustness of the supporting evidence. Established Role At the very early stage of life, especially in premature babies, high concentrations of oxygen are clearly detrimental for the 30 retina (retrolental fibroplasia described by Ashton et al in 1954) and for the lungs (diffuse alveolar damage 31,32 bronchopulmonary dysplasia). Therefore, the avoidance of high concentrations of oxygen during resuscitation of neonates is a standard of care. Similarly, some acute and chronic intoxications are associated with oxidative damage, as evidenced by the success of anti-

oxidant strategies to reverse the deleterious effects of the poison. For instance, the toxic effects of some metals (iron, chromium, cadmium, arsenic), chemicals (paraquat, menadione, doxorubicin), drugs (acetaminophen, troglitazone), and 33 34 airborne particles and fibers are clearly related to oxidative damage (see Ma and Roberts et al for complete updated reviews). Probable Role In contrast to the oxygen-related toxicity in premature and neonates and the toxicity of chemical compounds, several conditions are associated with increased oxidative stress, and a causal link is suspected but not established. The tumorigenic effects of ROS are established and support the hypothesis of a pathogenic role of oxidative stress in cancer. Indeed, ROS can increase cell proliferation, migration, and survival and can also induce damage to the DNA. Conversely, some oncogenes can also modulate the intracellular redox status and its regulation. Importantly, some antioxidant 35 treatments have increased the risk of developing cancers. Neurodegenerative diseases such as Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis are all associated with markers of increased oxidative stress. In experimental models, there is evidence that the inhibition of oxidative stress inhibits the progression of these diseases. Possible Role Cardiovascular diseases and atherosclerosis could be partly induced by oxidative stress, as the pathways of ROS production are upregulated and oxidative damage to phospholipids and proteins has been consistently reported. However, large-scale 36 trials of antioxidants failed to demonstrate any protective effect in at-risk individuals. Although oxidative stress contributes to the development of type 2 diabetes and related complications, high glucose also increases the production 37,38 of ROS and oxidative stress. To date, there is little evidence that inhibiting ROS production prevents diabetes. Chronic inflammatory diseases (asthma, rheumatoid arthritis, chronic obstructive pulmonary disease, inflammatory bowel diseases, psoriasis, lupus) all share evidence of increased oxidative stress at the site of inflammation. Several clinical trials of the inhibition of ROS production or the enhancement of antioxidants failed to confirm the expected effect of inhibiting the progression of these chronic 39 diseases. Acute inflammatory conditions such as critical illnesses are also associated with increased oxidative stress. 40,41 Some protective effects could be afforded by the preventive use of antioxidants in critically ill patients, particularly , 42,43 after trauma although the most adequate composition, dose, and timing for antioxidant cocktails are still undefined. In some conditions, early administration of antioxidants may represent a logical approach to prevent tissue damage and dysfunction. Indeed, after trauma, burn injury, or during sepsis, a shortage of cofactors for the endogenous antioxidant mechanisms has been reported. In contrast, a prolonged administration of antioxidants carries the risk of interference with the physiological roles of ROS.

Biomarkers
Measurement of Biomarkers of Oxidative Stress The numerous effects of ROS on several biological molecules can be quantified by markers of oxidative stress in vivo (Table 2). Many indirect techniques are available, but none of these can be considered the gold standard for the evaluation of oxidative stress. Direct measurement of ROS. The ideal method would be to measure directly the ROS, but these are labile compounds, and their direct detection and quantification are difficult. Free radicals are particularly short-living species; they can be measured only by electron paramagnetic resonance (EPR), most often coupled to spin trapping to increase their lifetime and the sensibility of the detection method. EPR has been successfully applied in animal models but is very difficult to use in vivo in humans. Stimulated production of ROS. The global production of oxidant species produced by stimulated phagocytes can be measured by chemiluminescence on freshly isolated cells, stimulated ex vivo. Unfortunately, this method does not allow an identification of ROS that have been produced. Moreover, to increase the sensitivity, chemiluminescence enhancers are used, which are highly sensible to incidental (non-ROS-dependent) oxidation. The same criticism can be
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addressed to the fluorescence techniques developed for ROS identification on isolated cells (fluorescein, rhodamine). Assessment of oxidative damage. Many indirect methods have been developed to measure stable by-products of ROS activity on biomolecules: isoprostanes, hydroxyl-nonenal, nitrated proteins, chlorinated lipids, lipid peroxides, conjugated dienes, oxidized glutathione, malondialdehyde (MDA, detected as thiobarbituric acid reactants), and other breakdown products of cell membranes. However, these techniques have limitations. These are not specific of an oxidative stress and are at risk of artifacts. For example, isoprostanes can be produced by platelets independently from oxidant stress, and the chemical reaction of MDA detection is influenced by the iron present in the sample. Moreover, the intensity of the oxidative stress measured indirectly is also dependent on the type of ROS released. For instance, nitrated proteins will be much more influenced by the presence of peroxynitrite than by any other ROS.

Antioxidant status. Additional approaches use the assessment of the antioxidant status and the oxidant capacity. The antioxidant status evaluates the consumption of antioxidants or the changes in activity or expression of the antioxidant enzymes. It claims to evaluate the capacity of healthy humans to fight a potential oxidative stress, regardless of the type of antioxidant involved. The total antioxidant capacity measures the capacity of a biological sample (plasma, tissue extract) to inhibit the transformation of a selected substrate by an in vitro generated free radical. However, the total antioxidant capacity measurement also raises questions: the radical used in the technique does not represent the ROS that are produced in vivo, and the capacity of a biological sample to inhibit it can be unrelated to the in vivo situation. But the most important problem is the signification of this kind of technique when applied to plasma samples, in which the concentration of albumin, an excellent barrier against ROS, is high. And finally, as there are no reference values for antioxidant status in humans, how should we interpret the changes? Circulating antioxidant levels. The easily measurable plasma or serum levels of some antioxidant molecules have been used as biomarkers. However, the relation between these circulating levels and the magnitude of intracellular oxidative stress is not established. In addition, the level of a single molecule is unlikely to reflect the complex cascade of mechanisms implying numerous players. Practical issues. In addition to the limitations of each of these methods, the origin of samples and the timing are unsolved issues. As ROS react rapidly, they react in situ, at the site of production. For instance, if oxidative stress is suspected in the lungs, biomarkers should thus be measured ideally in bronchoalveolar lavage but not in blood. If ROS are produced in membranes, lipid derivatives should be searched.

Conclusion
Oxidative stress is a physiological pathway closely regulated by antioxidant mechanisms. Dysregulation of oxidative stress is associated with many frequent pathological or physiological conditions. In some circumstances, overenthusiastic use of supplemental or dietary antioxidants to prevent or treat these diseases has been detrimental, in relation with an inhibition of the physiological roles played by ROS. A rigorous scientific evaluation in well-defined conditions is mandatory to define the appropriate place for manipulations of the oxidative pathways in human medicine.45 Glossary Free radical: chemicals with a molecular or ionic structure that includes an unpaired electron that commonly confers high

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