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Clinica Chimica Acta 390 (2008) 1 11 www.elsevier.com/locate/clinchim

Invited critical review

Free radical metabolism in human erythrocytes

M.Y. Burak imen
Mersin University, Medical Faculty, Department of Biochemistry, 33079 Mersin/Turkey Received 1 October 2007; received in revised form 13 December 2007; accepted 21 December 2007 Available online 18 January 2008

Abstract As the red cell emerges from the bone marrow, it loses its nucleus, ribosomes, and mitochondria and therefore all capacity for protein synthesis. However, because of the high O2 tension in arterial blood and heme Fe content, reactive oxygen species (ROS) are continuously produced within red cells. Erythrocytes transport large amount of oxygen over their lifespan resulting in oxidative stress. Various factors can lead to the generation of oxidizing radicals such as O, H2O2, HO in erythrocytes. Evidence indicates that many physiological and pathological conditions such as 2 aging, inflammation, eryptosis develop through ROS action. As such, red cells have potent antioxidant protection consisting of enzymatic and nonenzymatic pathways that modify highly ROS into substantially less reactive intermediates. The object of this review is to shed light on the role of ROS both at physiological and pathological levels and the structural requirements of antioxidants for appreciable radical-scavenging activity. Obviously, much is still to be discovered before we clearly understand mechanisms of free radical systems in erythrocytes. Ongoing trends in the field are recognition of undetermined oxidant/antioxidant interactions and elucidation of important signaling networks in radical metabolism. 2008 Elsevier B.V. All rights reserved.
Keywords: Erythrocyte; Reactive oxygen species; Antioxidant

Contents Organization of the human red cells . . . . . . . . . Free radicals in metabolism. . . . . . . . . . . . . . Oxidative stress in human erythrocytes . . . . . . . . Formation of ROS from molecular oxygen. . . . . . Iron redox status and oxidative stress in erythrocytes Oxidative aging in mature erythrocytes. . . . . . . . Cellular antioxidant defense systems against ROS . . 7.1. Enzymatic antioxidants . . . . . . . . . . . . 7.2. Non-enzymatic antioxidants . . . . . . . . . . 7.3. Exogen antioxidants . . . . . . . . . . . . . . 8. Antioxidant properties of hemoglobin . . . . . . . . 9. Role of ROS in necrotic/apoptotic erythrocyte death . 10. Conclusion . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . 1. 2. 3. 4. 5. 6. 7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 2 2 3 4 5 5 5 7 8 8 8 9 9

Tel.: +90 324 337 43 00 / 1527; fax: +90 324 337 43 05. E-mail address: mybcimen@mersin.edu.tr. 0009-8981/$ - see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.12.025

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and singlet oxygen as well as radicals superoxide anion (O2 ), hydroxyl radical (HO ), and nitric oxide (NO). ROS are continuously formed in small amounts by normal metabolic pro cesses. The addition of one electron to O2 produces the O2 wheres addition of two electrons results in formation of H2O2. H2O2 can react with O2 and ferric or cupric ions to produce the highly reactive HO [6,7]. High ROS concentration, formed under pathological conditions, can overwhelm cellular defenses leading to cellular damage. Interestingly, ROS can act as both oxidizing and reducing agents. Although the initial free radical produces only local effects, secondary radicals and degradation products can have biological effects at distant sites [7]. Oxidants such as O2 , H2O2, HO , and lipid peroxides play important roles in biological processes such as phagocytosis, aging, inflammation, tissue repair and intracellular messenger pathways [710]. Oxidative stress is a disturbance in the prooxidantantioxidant balance in favor of the former. This imbalance can lead to damage at the macromolecular level including DNA strand breakage, damage to membrane ion transport systems, enzymes and other proteins and lipid peroxidation [7,11]. Noncovalent bonds that maintain the three-dimensional structure of proteins are generally weak and susceptible to ROS action. It can be appreciated that even subtle changes in macromolecular structure or at the level of single amino acid residues may cause drastic changes in protein function [7]. Polyunsaturated fatty acids have become an area of interest in the biochemistry of oxidative reactions. Oxidizing radicals also target cell membranes rich in polyunsaturated fatty acids [6]. Specific enzymatic oxidation of polyunsaturated fatty acids leads to the formation of extremely potent and biologically important compounds such as prostaglandins and leukotrienes. In contrast, nonspecific oxidation of polyunsaturated fatty acids can lead to lipid peroxidation via a radical mediated pathway [7].

1. Organization of the human red cells The anuclear mature human erythrocyte is the most abundant and one of the most specialized cells in the body. Approximately 25 trillion red blood cells course through the human circulatory system. The main function of erythrocytes is transport of oxygen (O2) and mediation of carbon dioxide (CO2) production. [1] As the red blood cell emerges from the bone marrow, it loses its nucleus, ribosomes, and mitochondria and therefore all capacity for cell division, protein synthesis, and mitochondrial-based oxidative reactions [1,2]. The erythrocyte membrane consists of a lipid bilayer composed of 50% protein, 40% lipid, and 10% carbohydrate. More than 95% of cytoplasmic protein is hemoglobin (Hgb) [3]. Hgb is one of the most widespread and specialized hemecontaining proteins that exist in nature. These unique proteins permit the reversible binding to O2 to heme while maintaining iron in the +2 oxidation state. This protein also facilitates exchange of CO2 produced in tissues with the lungs. Heme iron must be maintained in the reduced ferrous form in order to bind O2 reversibly [3,4]. Membrane-associated cytoskeletal proteins include spectrin, ankyrin, band 3 (anion exchanger protein), glycophorin C, and protein band 4.1 have important roles in control of cell shape, attachment to other cells and substrates, and in organization of specialized membrane domains [5]. All lipids in the mature erythrocyte are found in the membrane bilayer and consist of phospholipid and cholesterol in 1.2:1 molar ratio. Approximately one-half of the fatty acids in the membrane are unsaturated [3]. Interestingly, outer surface lipids exchange freely with the plasma lipid compartment [2]. In addition, the structure of the lipid bilayer is critical to the cytoskeletal network organization within the red blood cell [5]. Glucose, the only fuel utilized by mature red cells, is primarily metabolized via anaerobic glycolysis. Following facilitated diffusion, glucose is immediately converted to glucose-6 phosphate. Approximately 8090% percent is then converted to lactate via the glycolytic pathway. The remaining 10% undergoes oxidation via the pentose phosphate shunt. Glucose metabolism effectively maintains glutathione in the reduced form thereby protecting hgb sulfhydryl groups and red cell membranes from oxidation. A significant portion of the adenosine triphosphate (ATP) generated by glycolysis is spent in operating the sodium potassium pump necessary to preserve the cytoplasmic ionic milieu thus preventing colloidal osmotic lysis. In addition, some metabolic energy is expended on maintenance and repair of the red cell membrane [2]. Aged erythrocytes are ultimately removed from circulation by phagocytic cells. Each day, less than 1% of these cells are destroyed and replaced by virtually identical numbers of new cells [1]. 2. Free radicals in metabolism Free radicals are chemical species possessing an unpaired electron [6]. Reactive Oxygen Species (ROS) are formed by the one or two electron reduction of O2. ROS are oxygen-centered molecules that include non-radicals hydrogen peroxide (H2O2)

3. Oxidative stress in human erythrocytes ROS are of increasing interest as agents of pathologic states including atherosclerosis, hypertension, Parkinson's disease, nephropathy, inflammatory arthritis and diabetes [1215]. In most cells, mitochondria are major source of ROS [16]. Despite their lack of mitochondria, ROS are continuously produced in the red cells due to the high O2 tension in arterial blood and their abundant heme iron content [17]. Various factors lead to generation of oxidizing radicals such as O2 , H2O2, HO in erythrocytes [18]. The source of ROS in erythrocytes is the oxygen carrier protein Hgb that undergoes autoxidation to produce O2 . Since the intraerythrocytic concentration of oxygenated Hgb is 5 mM, even a small rate of autoxidation can produce substantial levels of ROS. Occasional reduction of O2 to O2 is accompanied by oxidation of Hgb to metHgb, a rustbrown-colored protein that does not bind or transport O2. Although oxidative stress may damage the red cell itself, the mass effect of large quantities of ROS leaving the red cell have a tremendous potential to damage other components of the circulation [16]. Thus, it is of special interest to determine the extent of this oxidant challenge and ROS balance in the erythrocyte.

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As a consequence of their physiologic role, erythrocytes are exposed to continuous oxidant stress. Although the normal red cell reducing capacity is greater than 250 times its oxidizing potential several erythrocyte abnormalities have been identified that circumvent or overwhelm the erythrocyte oxidant defense system. Complex aerobic organisms have assured an adequate and continuous flow of oxygen to their tissues, while simultaneously protecting themselves from the inherent toxicity of oxygen. This occurs by two mechanisms: oxygen-carrying proteins, ie, Hgb, and oxidant defense systems [19]. Polyunsaturated fatty acids within the membrane, an oxygenrich environment, and iron-rich Hgb make reds cells susceptible to peroxidative damage [20]. ROS initiate lipid peroxidation reactions that lead to loss of membrane integrity and cell death [17]. Malondialdehyde (MDA), a highly reactive bifunctional molecule, is an end product of membrane lipid peroxidation. MDA has been shown to cross-link erythrocyte phospholipids and proteins. This process results in impairment of the membrane-related functions that ultimately leads to diminished survival. MDA accumulation can affect the anion transport and function of the band 3 associated enzymes, glyceraldehyde-3phosphate dehydrogenase and phosphofructokinase [18]. Several reports have documented that in vitro exposure to oxidants increases erythrocyte membrane instability by damaging protein band 4.1 and forming a defective spectrin-band 4.1-actin tertiary complex. Membrane-bound proteinases, the secondary antioxidant defense mechanism, protect erythrocytes by preferentially degrading oxidatively damaged proteins [18]. Although many membrane components are possible targets for oxidants, calcium ATPase may be of crucial importance for the survival of red cells. Ca-ATPase contains one or more reactive sulfhydryl groups that are susceptible to oxidation with resultant loss of enzyme activity. Because this enzyme is instrumental to maintaining the very steep gradient between extracellular and intracellular calcium, loss of activity is associated with decreased red cell deformability and premature destruction [21]. 4. Formation of ROS from molecular oxygen The addition of one electron to O2 produces the superoxide anion radical (O2 ) (Fig. 1) [7]. O2 is implicated as a potential source of oxidative damage and as a mediator of oxidative hemolysis in numerous studies [19]. At least two sources of O2 generation within red cell have been identified. First, oxyHgb autoxidizes at a relatively slow rate to yield metHgb and O2 +3 [22]. Second, the oxidation state of hemicrom iron (Fe ) indicates that an electron has been lost during its formation and, therefore, that O2 has probably been generated or has been derived from exogenous sources, ie, drugs [23]. This reactive species is capable of attacking the red cell membrane directly and causing alterations in lipid and protein structure [20]. O O 2H H2 O2 O2 2 2 As seen from the above equation, dismutation of O2 will readily generate excessive amounts of H2O2 (Fig. 1) [23]. H2O2 can cross cell membranes almost as readily as water while the

Fig. 1. Main free radical metabolism pathways in human erythrocytes.

charged O2 can cross membranes only via transmembrane anion channels. H2O2, is not especially toxic to the cell macromolecules, but it can pass through membranes and this feature is potentially important because the extracellular environment possesses few antioxidant defense mechanisms [7]. O2 generating agents may indirectly produce metHgb by generation of H2O2, in the course of normal cellular events [19,24]. OxyHgb undergoes a slow autooxidation, producing O2 , which yields H2O2. Therefore, Hgb is constantly exposed to an intracellular flux of H2O2 as well as to an extracellular flux, due to the high permeability of this metabolite. Exposure of oxyHgb to H2O2 leads to oxidative modifications that have been proposed as selective signals for proteolysis in erythrocytes [25]. As H2O2 concentration is increased, a dose-dependent increase in metHgb, lipid peroxidation, and spectrin-Hgb complexes are seen. Peroxidation, which results in globin cross-linking to any one or all of these interrelated proteins, such as spectrin and band 3, may lead to a decreased deformability, as well as morphologic and surface changes in the erythrocyte. Synder et al. [26], demonstrated that H2O2 induces a covalent complex of spectrin and Hgb as well as a myriad of cellular changes that include alterations in cell shape, membrane deformability, phospholipid organization, and cell surface characteristics.

O H2 O2 O2 OH OH HaberWeiss Reaction 2 Fe H2 O2 Fe OH OH Fenton Reaction

H2O2 can react with O2 and ferric or cupric ions to produce OH , the most active ROS (Fig. 1). This arises from a much higher reduction potential in comparison to other ROS. As a result of its reactivity, the OH does not travel far due to its half-life of a few nanoseconds [7,27]. The mechanism of red cell OH generation is, however, not as straightforward [23]. Due to its charge, O2 is concentrated in the intracellular compartment. As such, OH is produced predominantly from H2O2 by HaberWeiss reactions whereas the Fenton reaction is more important extracellularly [7]. Substantial evidence supports the view that NO is a key component of the respiratory cycle, a third gas transported together with O2 and CO2 by red cells. NO, which is an important mediator of endothelial vasorelaxation, immunity and

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inflammation, and the inhibition of platelet adhesion, regulation of cell growth and differentiation of vascular cells is also known as endothelium-derived relaxing factor [2830]. It has been reported that erythrocytes possess endothelium-type NOS [31] and erythrocyte NO production is diminished in patients with several diseases, possibly due to decreased NOS activity [32]. Recent studies have demonstrated the importance of Hgb in control of vascular tone mediated by NO [3]. In the circulation, erythrocytes are the major scavenger of NO, because they contain high Hgb concentration [28]. NO is sequestered via reactions with the heme prosthetic groups of Hgb and with cysteine residues in the -chain in erythrocytes. HgbFe+ 2O2 converts NO to nitrate, whereas HgbFe+ 2 binds to NO to form HgbFe+ 2NO. The consumption of NO has generally been considered to be unregulated as Hgb efficiently consumes NO at high rates. Han et al. [28], demonstrated that NO consumption by erythrocytes under hypoxic conditions can be regulated by HgbFe++NO formation. Loss of oxygen in the peripheral tissues results in transition of Hgb to the T state and release of NO [3]. It is known that partially nitrosylated Hgb (Hgb[Fe++]NO) enters the lung in the T form. Stamler et al. [33] reported that S-nitrosylation is facilitated by the O2-induced conformational change in Hgb. SNOoxyHgb (SNOHgb[Fe++]O2) enters the systemic circulation in the R form. NO released from Hgb may be transferred directly to the endothelium and exported from erythrocytes. Thus, the O2 gradient in arterioles serves to enhance O2 delivery. It promotes an allosteric transition in Hgb which releases (S)NO to improve blood flow. It has been suggested that diffusion barriers exist between the site of NO production and erythrocyte-encapsulated Hgb. The cytoskeleton and associated NO-inert proteins may act as members of a submembrane resistance to the entry of NO [34]. Liu et al. [35], showed that even high levels of NO generation (100 nmol/s) would lead to an intravascular NO concentration that would be far too low to exert functional effects. Reaction of NO with erythrocyte Hgb greatly limits intravascular NO concentration. Thus, it is unlikely that NO is directly exported or generated from the red blood cell as an intravascular signaling molecule. If red cells did export NO bioactivity, this would most likely occur via a chemical species that could release or form NO, rather than NO itself [35]. Huang et al. [36], demonstrated that chemical modifications to the red cell result in the modulation of NO bioavailability by altering the NO consumption rate. The potential mechanism by which NO uptake was increased may lie with the membrane skeleton, because HgbFe++NO was more abundant in the membrane versus cytosol [28]. Under physiological conditions, a reaction of vascular-derived NO with Hgb is supposed to be the most important pathway for limiting NO bioactivity. Intravascular hemolysis releases Hgb from the erythrocyte into the plasma compartment. This plasma Hgb is not confined by the diffusional barriers that limit the reaction of intraerythrocytic Hgb with NO, resulting in rapid rates of NO consumption. The rapid dioxygenation of NO by Hgb results with the formation of nitrate and metHgb and thereby prevents the diffusion of NO from plasma to smooth muscle. Consequently, smooth muscle guanylyl cyclase is not activated and vascular relaxation and vasodilation are inhibited [37].

Red cells shield NO bioactivity from elimination reactions generating nitrate [38]. NO is produced in increased amounts in inflammatory conditions and may cause tissue injury by reacting with O2 to yield peroxynitrite [30]. Peroxynitrite formed in the intravascular space does not only oxidize plasma components and decompose to secondary radicals that promote tyrosine nitration but also reacts with intracellular components [29]. In particular, nitration of protein tyrosine residues that gives 3-nitrotyrosine thus provoking gain or loss of protein function. Intravascular peroxynitrite can diffuse into the erythrocyte and undergo a fast reaction with oxyHgb, which mainly results in its isomerization to nitrate [29]. In human erythrocytes approximately 3% of total Hgb is cycled to the metHgb every day, mainly through slow reaction of Hgb with O2. However, due to metHgb reduction mechanisms the steady-state level of metHgb is approximately 1%. In vivo, metHgb is predominately reduced by the NADHcytochrome b 5-metHgb reductase system, and minor pathway such as the NADPH-dependent metHgb reductase [39]. Glucose-6-phosphate-dehydrogenase (G6PD) deficiency is the most common inherited enzyme abnormality in humans and results in increased sensitivity to H2O2 generating agents. The enhanced oxidant sensitivity of G6PD deficient cells is not due to intracellular reduced glutathione (GSH); rather, it is most likely due to the absence of NADPH and the functional impairment of both GSH/glutathione peroxidase (GSHPx) and catalase (CAT) mediated catabolism of H2O2 [24,40]. In the deficiency of GSHPx, erythrocytes can have very low GSH concentrations. Because of its role as a cofactor for normal enzyme activity, functional loss of GSHPx activity has been believed to be responsible for the enhanced sensitivity of G6PD deficient erythrocytes to H2O2 generating redox active drugs [24]. As a result, NADPH appears to be essential for the catalytic activity of both major H2O2 catabolizing pathways. Kirkman et al. [41], reported that human CAT actually contains four tightly bound molecules of NADPH necessary for enzymatic activity. Several recent studies have implicated an important role for NADPH in not only sustaining GSH but also in maintaining the catalytic activity of CAT. [24,42] Gaetani et al. [40], also determined the NADPH concentration in G6PD deficient erythrocytes and demonstrated that decreased NADPH levels were correlated to loss of CAT activity. CAT-bound NADPH is not essential for mammalian catalase function but offsets inactivation of CAT by its substrate H2O2 [43]. Entrapment of G6PD in deficient cells restores apparent metHgb reductase activity. These data suggest that NADPH concentration may be important in preventing metHgb generation. Loss of NADPH and GSH are thought to account for the enhanced rates of metHgb generation and lipid peroxidation [24]. 5. Iron redox status and oxidative stress in erythrocytes Iron is the most abundant, important, and essential transition metal in biochemical reactions. A heterogeneous group of proteins contain iron in a variety of molecular forms [1]. Iron not only binds oxygen reversibly but also participates in a number of vital oxidationreduction reactions. To bind oxygen, heme iron must be maintained in the reduced state. If these

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mechanisms fail, Hgb becomes non-functional [3]. As a result of this process, iron is released from Hgb (or its derivatives) and the release is accompanied by metHgb formation. If erythrocytes are depleted of GSH, the release of iron is accompanied by lipid peroxidation and hemolysis [44]. Iron levels in the cell must be delicately balanced, as iron loading leads to free radical damage. Copper and iron cations present in some toxic material can promote ROS formation by Fenton reaction which occurs when excess iron reacts with H2O2 to generate OH [45,46]. To achieve appropriate levels of cellular iron and to avoid ironloading, transport, storage and regulatory proteins have evolved [47]. Iron released from its storage macromolecules represents the source of iron-catalyzed oxidative stress, such as lipid, protein and DNA oxidation. It is also believed that such processes occur not only in pathological but also in physiological conditions such as those regulating the signal transduction pathways [44]. Because of the abundance of O2 in aqueous media, ferrous ion autoxidation may be an important route for initiation of free radical oxidation. This reaction would result in the formation of FeO complexes, named as ferryl or perferryl ions. Due to their high electron affinity, these ions would have reactivities approaching those effects of OH [48]. Ferryl species are strong oxidants for several biomolecules including vitamin E, vitamin C, cholesterol, catecholamines, lipoproteins, and membrane lipids [25]. Release of iron in a reactive form may be relevant to the generation of senescent antigen (SCA). In fact, Signorini et al. [49], demonstrated the relationship between iron release, oxidation of membrane proteins and binding of autologous IgG, ie, formation of SCA, in an in vitro model of rapid erythrocyte aging. It has been reported that the aerobic incubation of erythrocytes in buffer markedly accelerate erythrocyte aging as measured by vesciculation. Iron release was also accompanied by oxidative alteration of membrane proteins. Polyacrylamide gel electrophoresis (PAGE) demonstrated the appearance of new bands in the range of 4566 kDa. These bands, not observed following anaerobic incubation, were considered an index of erythrocyte aging and were thought to originate from the oxidative degradation of protein band 3. Moreover, similar results have been obtained by using an iron chelator as a protective agent [44]. Use of red cells as carriers of bioactive substances has been explored as a new field of research. From a therapeutic perspective, erythrocytes may act as a drug reservoir, providing sustained release into the body. Erythrocyte encapsulation in vitro of Cu+ 2 complexes causes slight oxidative stress, compared to the unloaded and native cells. Carrier erythrocytes for the administration of desferrioxamine and other iron chelators may be useful for improving iron chelation efficiency [50]. 6. Oxidative aging in mature erythrocytes Mature erythrocytes have a finite lifespan. Although the exact molecular mechanisms that determine removal of cells from the circulation remain unknown, red cells provide a unique model for the study of cellular aging. Studies of red cell turnover in young hosts have led to several hypotheses about the mechanisms

involved in the generation of senescence signals and the removal of aged erythrocytes by splenic macrophages [51]. The erythrocyte aging process is a multifactorial event, and understanding of the interrelationship between various cellular changes is essential to define the complex process of senescent cell recognition. Free radical theory is a widely accepted chemical theory of aging. The origins of this theory date to the mid 20th century when oxygen free radicals, traditionally thought too reactive to biologically exist, were discovered [52]. Free radical theory treats aging as the results of cumulative oxidative damage to biomolecules such as proteins, lipids, glucoconjugates and nucleic acids [53,54]. Other proposed mechanisms include senescence antigens by phagocytes, mechanical fatigue, ATP depletion, and calcium accumulation. Human erythrocyte membranes exposed to oxidative stress in the circulation undergo various modifications of cellular components. These include formation of oxidatively denatured Hgb, peroxidized lipids, high molecular weight cross-linked membrane proteins, desialylation of glycoproteins. These processes lead to decreased phospholipid symmetry, formation of cross-linked spectrin and Hgb, aggregation of band 3 protein, and increased advanced glycation end products [51,55]. Synder et al. [26], reported that an irreversible complex between the globin chain of Hgb and spectrin was formed oxidatively during the erythrocyte aging. Erythrocyte study in aged individuals may provide a better understanding of the factors involved in this process [51]. 7. Cellular antioxidant defense systems against ROS The human erythrocyte, due its role as O2 and CO2 transporter, is under constant exposure to ROS and oxidative stress. Oxidative stress occurs in cells or tissues when ROS concentration exceeds antioxidant protection [7]. Extracellular antioxidant capacity and reduction of extracellular oxidants allows erythrocytes to respond to stress. The mobility of the erythrocyte makes it an ideal antioxidant not only for its own membrane and local environment, but also as an oxidant scavenger throughout the circulation. Although O2 can act as an electron acceptor for transmembrane redox reactions in some cell lines and may be a physiological electron acceptor, red cells do not possess this activity [56]. Red cells from newborns, especially premature infants, have previously been shown to be more sensitive to peroxidative damage in vitro than adults, due in part to deficiencies of antioxidant capacity [20]. Oxidant/antioxidant equilibrium can change in the erythrocyte in several diseases. Erythrocytes are exposed to high oxidant stress may result in accelerated peroxidation reactions and cellular aberration [13]. Protective mechanisms exist to scavenge and detoxify ROS, block production, or sequester transition metals [11]. The antioxidant system consists of enzymatic and nonenzymatic pathways in human red cells. 7.1. Enzymatic antioxidants Enzymes for preventing oxidative denaturation in erythrocytes include superoxide dismutase (SOD), CAT, GSHPx, GSH

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reductase-dependent regeneration of GSH, and NADH metHgb reductase [3,19]. O O 2H Y H2 O2 O2 2 2 Superoxide anion is converted to O2 and H2O2 by SOD a ubiquitous metal-containing enzyme (Fig. 1) [7,57]. SOD is a family of enzymes, comprising CuSOD, ZnSOD, MnSOD and extracellular SOD, whose function is protection from ROS, particularly O2 [30]. Due to their lack of mitochondria, cytoplasmic Cu,ZnSOD plays a much more important role in erythrocytes. Zinc appears to stabilize the enzyme, while the copper atom and histidine amino acid are required for enzymatic activity [7]. Physiologically, erythrocytes are well protected against ROS by abundant Cu,ZnSOD which scavenges free radicals thus preventing metHgb formation [18]. In addition, Cu,ZnSOD synthesis is induced by O2 . Its activity is increased in the presence of O2 with activation of regulatory genes. The efflux of oxygen to tissues is drastically reduced, but probably sufficient to generate ROS as a result of incomplete reduction of O2. Erythrocyte Cu,ZnSOD activity tends to be decreased in critical ischemia because production of O2 may be lower than during moderate ischemia. It seems reasonable that diminished synthesis of Cu,ZnSOD is caused by decreased formation of O2 . Both inflammation and marginal Cu intake are negatively affected by the activity of this enzyme, and as a result, the resistance to oxidative stress is changed. In contrast, it has been suggested that toxic exposure, ie, smoking, causes no impairment in the enzymatic antioxidant defense systems and does not lead to erythrocyte oxidant stress due to their potent antioxidant defense. Similarly in our previous study, no statistically significant difference was noted in erythrocyte antioxidant enzymes in smokers [45]. Cell damage may also be due to the superoxide itself or, indirectly, even more ROS, such as OH, formation of which, via the Fenton reaction, is favored by excess O2 [58]. Several researches have reported decreased erythrocyte SOD activity during therapeutic applications and pathophysiologic conditions [59]. In previous studies [60,61], we observed decreased SOD activity with acetyl salycylic acid and nonsteroidal anti-inflammatory drugs. SOD scavenges O2 and inhibits the formation of peroxynitrite, thereby suppressing injury and regulating the bioavailability of NO [30]. 2 GSH H2 O2 Y 2 H2 O GSSG 2 H2 O2 Y 2 H2 O O2 H2O2 is produced by normal metabolic pathways. Two enzyme systems exist to catalyze H2O2 (Fig. 1) and are present at high activity in human red cells [24]. Low levels of H2O2 (10 9 M) are removed by GSH to form oxidized glutathione (GSSG) and water, a reaction catalyzed by GSHPx [7]. Because the direct reaction between H2O2 and GSH is very slow, GSHPx reduces H2O2 by oxidizing GSH to GSSG. Cytoplasmic, gastrointestinal and lipophilic enzymes are able to reduce hydroperoxides of complex lipids in membranes.
CAT GSHPx SOD

Isoenzymes are also present in red blood cells [62]. The catalytic activity of CAT appears to be a special case of peroxidase activity in which the electron donor is a second molecule of H2O2. The mechanism of CAT action is similar to SOD, wherein one molecule of H2O2 is reduced to water and the other oxidized to oxygen (Fig. 1) [7,30]. That CAT and SOD react synergistically to protect each other was observed earlier in hemolysis studies of erythrocytes [63]. Gaetani et al. [42], demonstrated that CAT and GSHPx are equally active in the detoxification of H2O2 in normal erythrocytes. Nonetheless, several researchers found that the GSH-dependent activity of GSHPx has been generally viewed as the primary defense against H2O2 in erythrocytes [24]. The data from the GSHPxdeficient red cells clarify the issue of intraerythrocytic Hgb oxidation and H2O2 generation [16]. In conclusion, GSHPx are important for dealing with the endogenous H2O2 produced by Hgb autoxidation, while CAT plays an increasingly important role as the erythrocyte is exposed to increased H2O2 flux. One might therefore anticipate that elevated SOD activity would be protective against O2 generating agents. However, red cells with 5 to 9-fold increased activity showed no enhanced resistance to O2 generators. Consequently, O2 is more likely to reduce metHgb to regenerate oxyHgb. Indeed, metHgb levels were slightly higher in SOD-loaded erythrocytes, indicating perhaps that O2 mediated reduction of metHgb was inhibited. Consequently, it is difficult to distinguish the specific roles of the various antioxidants in protecting the erythrocyte from oxidant stress [19]. Glutathione-S-transferases play a major role in detoxification of electrophilic xenobiotics such as herbicides, insecticides, chemical carcinogens, and other organic and inorganic environmental pollutants. These enzymes catalyze the conjugation of GSH with exogenous and endogenous toxic compounds or their metabolites, rendering them more water soluble, less toxic, and easier to excrete. In addition, they are responsible for various resistance mechanisms including chemotherapeutic or antibiotic drug resistance [64]. G6PD is essential to protect the red cell from oxidative damage. The absence of this protection can result in severe hemolysis [65]. In erythrocytes, b1% of Hgb is normally present as metHgb. Via metHgb reductase activity, metHgb may be reduced to normal ferroHgb [66]. In vivo, metHg is predominantly reduced via the NADH-cytochrome b5-metHgb reductase system that requires NADH and cytochrome b5 as cofactors. MetHgb may also be reduced through minor pathways such as the NADPH-dependent metHgb reductase and direct reduction by intracellular antioxidants such as ascorbate and GSH [56]. Individuals with decreased enzymatic activity are more susceptible to metHgb formation caused by oxidant drugs and chemicals [66]. In addition to primary antioxidant defense systems that prevent the generation of free radicals or radical chain reactions, secondary systems have been proposed. These include proteases that preferentially degrade oxidatively damaged proteins. A multicatalytic proteolytic complex appears responsible for degradation of oxidized intracellular proteins in erythrocytes [67]. Fujino et al. [68], demonstrated the presence of an 80-kDa serine protease in the oxidized erythrocyte membranes that

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preferentially degrades oxidized proteins specifically protein hydrolase. This cytoplasmic protein, becomes adherent to membranes when cells are oxidized thus promoting membrane protein degradation. The protease is characterized by its inhibition by a serine protease inhibitor [68]. It is endogenously present in oxidized or aged erythrocyte membranes and plays a role in removal of the oxidation-induced membrane protein aggregates and in reducing the oxidation-induced anti-band 3 binding in aging. Oxidized protein hydrolase (OPH) functions as a secondary defense system by removal of oxidized protein aggregates. This process has some beneficial effects on circulating red cells since it may reduce anti-band 3 autoantibody binding and macrophage recognition. Evidence indicates that the amount of membrane protein aggregates and bound antiband 3 autoantibody in senescent erythrocytes are increased in aged erythrocytes [68]. Peroxiredoxins (PRX), a group of ubiquitous thiol-containing enzymes, are erythrocyte proteins that could also contribute as H2O2 and peroxynitrite scavengers in the circulation. PRXs have a reductive capacity for hydroperoxides via a reductant thiol. Studies have shown that PRXs could also act as peroxynitrite reductases catalytically. It has been also reported that PRX-II is present in the cytosol of red cells. The catalytic cycle involves the reduction of oxidized Prx by thioredoxin and reduction capacity of NADPH via NADPH-thioredoxin reductase [29,69]. As can be expected, mitochondrial cyctochrome oxidase is absent in human red cells. 7.2. Non-enzymatic antioxidants Endogenous non-enzymatic antioxidants are defined in two phases: lipophylic (vitamin E, carotenoids, ubiquinon, melatonin, etc.) and water soluble (vitamin C, glutathione, uric acid, ceruloplasmin, transferin, haptoglobulin, etc.). Three antioxidant vitamins, A, C, and E, provide defense against oxidative damage. Vitamin C acts in the aqueous phase whereas vitamin E acts in the lipid phase act as a chain breaking antioxidants. Vitamin C reduces O2 and lipid peroxyl radical, but is also a well-known synergistic agent for vitamin E [17,70]. It exists as the enolate anion at physiologic pH. Dehydroascorbate, formed by a second reduction or dismutation reaction, is recycled by dehydroascorbate reductase, a GSH dependent enzyme. Dehidroascorbyl radical may also dismutate to ascorbate and dehydroascorbate [17]. It has been shown to play a protective role against peroxidation of erythrocyte membrane lipids and tocopherols by t-butylhydroperoxide, preserving lipids by up to 92% and vitamin E by 50% and 65%, respectively [56]. Vitamin E is the most widely distributed antioxidant in nature. When vitamin E donates an electron to a lipid peroxyl radical, it is converted to free radical stabilized by resonance structure [5]. The enolate anion reduces O2 , organic and tocopheroxy radicals, forming a dehidroascorbyl radical. Vitamin C and E work together to inhibit lipid peroxidation reactions in plasma lipoproteins and membranes. Vitamin A, a potent free radical scavenger, is a lipophylic antioxidant [17]. Carotenoids, the precursor of vitamin A, can exert antioxidant effects, as well as

quench singlet O2. There is considerable in vitro evidence for interaction of -carotene with free radicals, for its properties as a chain-breaking antioxidant and in scavenging and quenching singlet oxygen [71]. In all cell types, -glutamylcysteinylglycine is the most important nonenzymatic regulator of intracellular redox homeostasis. Several recent studies have shown that red cells are important as biological carriers of GSH by de novo synthesis and as such appear to provide an important detoxifying system within the circulation [72,73]. GSH synthesis in erythrocytes is limited by the availability of the substrate amino acids, especially cysteine [11,72]. It is synthesized by two ATP-dependent reactions, catalyzed by -glutamylcysteine synthetase and GSH synthetase. Defects of both enzymes are rare, but in severe cases, hemolytic anaemia and neurological abnormalities occur [62]. Glutathione exists either in reduced GSH or oxidized GSSG forms and participates in redox reactions by the reversible oxidation of its active thiol. GSH may covalently bind to proteins through a process called glutathionylation and can act as a coenzyme for various cell defense enzymes [74]. It can thus directly scavenge free radicals or act as a substrate for GSHPx and GST during the detoxification of H2O2, lipid hydroperoxides and electrophilic compounds [11]. In erythrocytes the major antioxidant is GSH which protects important proteins such as spectrin the oxidation of which can lead to increased membrane stiffness [75]. GSH not only supports antioxidant defense, but is also an important sulfhydryl buffer, maintaining SH groups in Hgb and enzymes in the reduced state [17]. Dumaswala et al. [72], observed that in vitro augmentation of the erythrocyte endogenous antioxidant reserve, especially GSH, provides protection against cell damage induced by oxidative stress. A higher demand for GSH causes increased degenerative oxidative modifications of proteins or lipids. On the other hand, a number of compounds synthesized endogenously function as non enzymatic antioxidants. While uric acid can directly scavenge OH and peroxy radicals, melatonin, the chief secretory product of the pineal gland, scavenges O2 , H2O2, HO, peroxynitrite anion and lipid peroxides [5,76,77]. Bilirubin is a sensitizer of singlet O2 production. It behaves as an antioxidant especially the albumin bound fraction. Erythrocyte bilirubin acts as photosensitizer in the presence of phototherapy and causes oxidative damage [78]. Although bilirubin is regarded as toxic when present at high concentrations, it has been postulated as a transitional antioxidant in the first few days of life before other antioxidant defense mechanisms mature [79]. A number of different electron transport processes are present in the erythrocyte membrane. Redox cycling of drugs or other xenobiotics can generate ROS in red cells [7]. Some of these membrane transport systems have an antioxidant role. These processes appear sensitive to their immediate environment and cellular redox state as large variations in oxidoreductase (NADH and ascorbate oxidoreductases) activities have been reported in erythrocyte membranes. NADH, arachidonic acid (AA), flavonoids, ubiquinone, and a-tocopherolquinone act as electron donors to membrane redox systems [56].

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7.3. Exogen antioxidants Several exogen compounds such as inhibitors of NADPHOxidase, allopurinol, and flavonoids have antioxidant properties. It is known that flavanoids are good exogen antioxidants against free radical initiated lipid peroxidation in human red cells and that the antioxidant activity of flavanoids depends significantly on molecular structure and initiation conditions [80]. Many studies have suggested that flavonoids exhibit biological activities, including anti-allergenic, anti-viral, anti-inflammatory, and vasodilation [81]. The capacity of flavonoids to act as antioxidants in vitro has been the subject of several studies that demonstrate their structure-activity relationships. Most ingested flavonoids are extensively degraded to various phenolic acids, some of which still possess a radical-scavenging ability [82,83]. Absorbed flavonoids and their metabolites may display an in vivo antioxidant activity as demonstrated by increased plasma antioxidant status, the sparing effect on vitamin E of erythrocyte membranes and preservation of erythrocyte membrane polyunsaturated fatty acids [84,85]. 8. Antioxidant properties of hemoglobin In various hemolytic anemias associated with red cell defects, similar but more pronounced membrane changes lead to premature cell destruction [51,86]. In -thalassemia the persistence of HgbF is related to the lack or deficiency of chains and therefore to an excess of chains. The observed correlation between free iron and HgbF is in agreement with the hypothesis that an excess of chains represents a prooxidant state. In -Hgb chains loaded erythrocytes membrane bound heme and iron were markedly elevated and the cells were more prone to oxidative stress [87]. Inside-out membranes from -thalassemic and sickle cells, elevated amounts of heme iron and, especially, free iron were observed. This form of iron may thus represent the trigger for the oxidative damage seen in thalassemic cells. It may also represent the mechanism of formation of sickle cell anemia or any other membrane event responsible for the early removal of erythrocytes from the blood stream [44]. Unstable sickle red blood cells are particularly vulnerable to oxidative stress because they generate significantly increased O2 , H2O2, and HO [23,88]. It is known that, HgbS red cell membranes have vitamin E deficiency which results with abnormal peroxidation. In addition, HgbS red cells contain increased amounts of MDA. Abnormal amino acid cross-linking by MDA has been demonstrated in lipid extracts of these cell membranes [23]. A stable blood supply is of increased importance as medical applications evolve. To improve stability more expensive and laborious methods are generally required [89]. In addition, risk of infection makes the development of an alternative blood substitute an important goal. Storage of red cell products are compromised due to decrease erythrocyte antioxidant defense during storage may damage erythrocyte membranes through oxidative modifications of lipids and proteins [18,90]. Red cells are the major scavenger of NO in circulation, because erythrocytes contain high concentrations of Hgb. OxyHgb converts NO to nitrate, whereas HgbFe+ 2 binds to NO to form

HgbFe+ 2NO. The intercellular junctions of the endothel allow tetrameric Hgb molecules to move to the extravascular space. Tetrameric Hgb molecules, on leaving the circulation, act as an effective NO scavenger. Decreased NO levels result in vasoconstriction and other effects on smooth muscle [89]. On the other hand, oxyHgb autoxidizes at a relatively slow rate to yield metHgb and O2 , which dismutates to H2O2. It is known that prolonged reaction of Hgb with H2O2, also leads to Hgb damage in vitro and in vivo [22]. These data emphasize the importance of oxidant/antioxidant balance in erythrocytes. Erythrocyte GSH synthesis can be manipulated by supplementing the storage medium with GSH precursors. The effect of GSH synthesis on oxidative changes that may diminish erythrocyte oxygen transport as well as free radical scavenging function [72]. Modified Hgbs have thus been developed and tested clinically. These include cross-linked polyHgb, crosslinked tetrameric Hgb, conjugated Hgb, and recombinant Hgb. Most of the modified-Hgb blood substitutes are prepared using ultrapure Hgb. They are effective as oxygen carriers in conditions without prolonged ischemia in routine medication. However, in condition with prolonged ischemia, there is potential for ischemia reperfusion injury [91]. Reperfusion process, as in strokes, myocardial infarction, hemorrhagic shock, can result in the release of ROS and other reactions leading to tissue injury. It is known that SOD and CAT in erythrocytes decreases this effect by removing O2 and H2O2 [89]. As a result of this finding, Hgbs modified by antioxidant enzymes have been investigated. PolyHgb-superoxide dismutase-catalase (polyHgbSODCAT) is formed by cross-linking CAT, SOD, and Hgb [91]. In contrast to polyHgb, polyHgbSODCAT removes significantly more free radicals and peroxides and stabilizes the cross-linked Hgb. This phenomenon results in decreased release of oxidative iron and heme. During reperfusion, polyHgbSOD CAT also significantly reduces the ROS when compared to polyHgb [89,92]. Chang et al. [93], showed that polyHbSOD CAT effectively reduces hepatic damage by diminishing ROS mediated damage after liver ischemia reperfusion injury. Another group have studied the use of polynitroxylated Hgb with antioxidant activity. This chemical modification gives rise to SOD CAT-like activities [94]. Chang et al. [91], were able to coencapsulate SOD, CAT and metHgb reductase with the Hgb. It was also found that these nanocapsules were also permeable to glucose and other small hydrophilic molecules. 9. Role of ROS in necrotic/apoptotic erythrocyte death Cell death may occur via apoptosis or necrosis. Cell death induced by excessive oxidative stress has been assumed to occur by necrosis [95]. ROS mediated cellular damage causes a destruction of membrane integrity and a loss of cellular homeostasis. GSH levels are decreased during apoptosis, which may indicate that an increase in oxidative stress induces cell death. It has been also shown that antioxidants such as SOD and CAT can inhibit apoptosis. However, Shimizu et al. [96], have suggested that the intracellular ROS increase in apoptotic systems may be caused by apoptosis, rather than being involved in its induction, thus, the detected increase in ROS could be a secondary response

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[7] The mechanisms underlying erythrocyte apoptosis are apparently important for the tuning of the erythrocyte life span. Similar mechanisms may be operative in nucleated cells where they may be hidden by the more complex apoptotic machinery. This mechanism may be termed eryptosis. Cellular stress, e. g. osmotic shock, oxidative stress or energy depletion, activate Ca+ 2 sensitive K+ channels in the erythrocyte cell membrane presumably via generation of prostaglandins, which stimulate eryptosis [97]. While a high degree of oxidative stress can cause necrosis, lower levels will trigger apoptosis [95]. Three types of proteins control apoptosis or programmed cell death: 1) bcl-2 (a family of anti-apoptotic proteins); 2) interleukin converting enzymes (ICE), ie, caspases; and 3) tumor suppressor gene (p53). Caspases are present in the cell cytoplasm as inactive forms called pro-caspases, which become activated during apoptosis. It is known that ROS can activate the pro-caspase and lead to apoptosis. p53 is a transcription regulator that plays a role in the control of normal cell proliferation and induces apoptosis [7]. Foxo3, which is a member of transcription factor Foxos, may regulate the lifespan of red cells [65]. Kane et al. [98], have suggested that bcl-2 inhibits apoptosis by reducing ROS generation or its effect while the activation of ICE can be prevented by antioxidants. In the light of these data, it can be accepted that ROS may contributed to the progress of the normal metabolic process by taking a part in the process of programmed cell death. 10. Conclusion Evidence indicates that many physiological and pathological conditions such as aging, inflammation, and cell death develop through reactive oxygen species (ROS) action. Due to their fundamental role in the transport of oxygen, erythrocytes provide a unique opportunity to study oxidative defense systems at the cellular level. Erythrocytes possess potent antioxidant activity consisting of both enzymatic and nonenzymatic pathways that together effectively function to modify highly ROS into substantially less reactive and more tolerable intermediate forms. Although much work is still required, investigation of the structural requirements of antioxidants within erythrocytes provides an important framework for elucidating radical-scavenging activity, understanding oxidant/antioxidant interactions and identifying signaling networks in radical metabolism in general. Despite the advances of the last half century, further study is clearly warranted to decipher the exact molecular mechanisms involving ROS under both physiologic and pathophysiologic conditions. References
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