**3. Oxidative stress in erythrocytes**

Erythrocytes are well endowed to combat oxidative stress due to their continuous contact with oxygen, as their inherent carrier function. When the concentration of ROS in cells or tissues exceeds the antioxidant protection, oxidative stress occurs [21]. RBC properties have been shown to change as a result of oxidative damage. Oxidative damage can also alter membrane permeability resulting in hemolysis [29, 30]. Oxidative cross-linking of spectrin can cause increased membrane rigidity and decreased erythrocyte deformability. Erythrocytes can be recognized by the immune system as a result of oxidative damage [31].

#### **3.1 Extracellular hemoglobin as a source of oxidative stress**

Extracellular Hb, which results from erythrocyte hemolysis or the infusion of cell-free Hb-based blood substitutes, can be a major source of oxidative stress. This potential source of oxidative stress is minimized under normal conditions by haptoglobin and hemopexin, which bind Hb and free heme, respectively. They inhibit the oxidative reactions of Hb and heme, allowing them to be removed from circulation. Elevated levels of free Hb and heme, which cannot be neutralized by haptoglobin and hemopexin, cause a variety of adverse clinical effects [32].

Autoxidation of Hb produces superoxide as well as methemoglobin. Hb is known to react with hydrogen peroxide to form ferrylhemoglobin, a strong oxidant [33]. Hb binds to RBC membrane proteins, especially under hypoxic conditions. The ROS produced by bound Hb may be inaccessible to cellular antioxidants, allowing the production of heme degradation products close to the membrane [34–36]. Extensive lipid peroxidation results in changes in fluidity such as a drop in membrane potential and an increase in permeability to different ions, which eventually leads to hemolysis. Thus, perturbations in erythrocyte function and structure can result in an increased flow of prooxidant generation that can lead to oxidative stress.

Oxidative stress and ROS accumulation in RBCs during aging may induce hemolysis. As a result, the plasma proteins haptoglobin and hemopexin can render free Hb and heme relatively inactive and deliver them safely to macrophages for phagocytosis [37, 38]. Oxidized Hb on the other hand exhibits impaired plasma clearance, due to its low affinity for haptoglobin protein.

Erythrocyte membranes exposed to oxidative stress undergo cellular component modifications such as oxidative denaturation of Hb, peroxidation of lipids, and high-molecular-weight cross-linked membrane proteins. The erythrocyte membrane is rich in sulfhydryl (∙SH) groups, which help to maintain cellular oxidative balance [39]. Changes in the membrane elasticity may occur due to oxidative damage to the membrane ∙SH groups [40]. Oxidative damage to erythrocytes can occur and manifest itself in a variety of ways, including potassium release, increase in malondialdehyde (MDA), phosphatidylserine externalization [41], decrements in glutathione, superoxide dismutase, glutathione peroxidase, glutathione S-transferase, and glutathione reductase as well as total antioxidant activity of

#### *Reactive Oxygen Species and Antioxidant Interactions in Erythrocytes DOI: http://dx.doi.org/10.5772/intechopen.107544*

plasma [42–44]. Oxidant stress is a key component to both normal RBC aging and pathological dysfunction [32].

Oxidative damage to a specific protein, particularly at the active site, can result in the progressive loss of a specific biochemical function [45]. Peroxidation causes globin cross-linking to proteins such as spectrin and band 3. These processes further lead to decreased phospholipid symmetry, formation of cross-linked spectrin and Hb, aggregation of band 3 protein, and increase in advanced glycation end products leading to deformability and morphologic and surface changes in the erythrocyte [15, 46, 47]. During erythrocyte aging, an irreversible oxidative complex is formed between the Hb globin chain and spectrin [20]. Iron release may be accompanied by the generation of senescent antigens (SCA) and oxidative alteration of membrane proteins [15, 48, 49]. Another event associated with red cell homeostasis disruption is an increased inflammatory state in the bloodstream, since erythrocytes are constantly exposed to inflammatory molecules transported in the vascular system, their membranes may be particularly vulnerable to their interaction. A large number of inflammatory mediators, including tumor necrosis factor-α, interleukins, interferons, and C-reactive protein (CRP), have been proposed as potential inflammatory response markers [50].

In general, erythrocytes respond to oxidative stress by activating tyrosine kinases [51, 52], resulting in tyrosine phosphorylation at the cytoplasmic domain of band 3 protein, which mediates interactions with ankyrin, leading to membrane destabilization. This also seen in β-thalassemia disease with the loss of stability between cytoskeleton and membrane complexes, such as band 3 protein [53]. Oxidative damage to band 3 has been linked to RBC aging including the exposure of senescent specific neo-antigens that bind autologous IgG triggering RBC removal [54].

Erythrocytes also contain NADH oxidases, which can generate endogenous ROS [55]. However, some forms of NADH oxidase were also detected in normal RBCs. L-isoaspartyl groups were discovered on erythrocyte membrane proteins in response to aging or pathological oxidative stress, such as glucose 6-phosphate dehydrogenase deficiency or Down syndrome [56–59]. Thus, L-isoaspartyl group accumulation in RBC proteins correlates with RBC dysfunction and pathology.

The effects of oxidative stress observed in various pathological conditions in erythrocytes are depicted in **Table 1**.



*Reactive Oxygen Species and Antioxidant Interactions in Erythrocytes DOI: http://dx.doi.org/10.5772/intechopen.107544*


#### **Table 1.**

*Effects of oxidative stress in erythrocytes during diseases.*

## **4. Antioxidant defense in erythrocytes**

The antioxidant defense system of erythrocytes prevents oxidative cell damage. This implies that the erythrocyte antioxidant defenses operate in a balanced manner. As a result, an appropriate redox state, balanced antioxidant action is required for ROS homeostasis.

Antioxidants are molecules that prevent or delay cellular damage by inhibiting or quenching ROS reactions [94]. Antioxidants can be synthesized in the body or obtained from the environment, such as through diet. Erythrocytes are well equipped to fight against oxidative stress, i.e., mechanisms to scavenge and detoxify ROS, prevent their production, and sequester transition metals [95]. Erythrocytes contain both enzymatic and non-enzymatic antioxidants to combat oxidative stress.

#### **4.1 Enzymatic antioxidants**

Enzymatic antioxidants include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), and peroxiredoxin-2 (PRX-2). Their coordinated actions protect the erythrocytes from free-radical-mediated damage. Since there is no *de novo* synthesis of antioxidant enzymes in mature erythrocytes, their defense capacity is limited. Free radicals affect the capacities of antioxidative enzymes as well as the overall antioxidative system [96]. Under normal conditions, erythrocytes contain sufficient levels of scavenger enzymes such as Cu,Zn-SOD, CAT, and selenium-dependent GPX to protect from free radical injury.

Superoxide dismutase, a ubiquitous metal-containing enzyme, involves in the detoxification of O2 •− into O2 and H2O2.

$$\text{O}\_2\text{"}^- + \text{O}\_2\text{"}^- + 2\text{H}^+ \xrightarrow{\text{SOD}} \text{H}\_2\text{O}\_2 + \text{O}\_2\tag{3}$$

SOD family comprises CuSOD, ZnSOD, MnSOD, and extracellular SOD, which protect from particularly O2 •−. Cu,Zn-SOD catalyzes the dismutation of O2 •− to H2O2, which is later converted to water by CAT or GPX [21, 27, 97]. The activity of these enzymes in erythrocytes is highest than that of other tissues in the body [98].

Erythrocytes are well protected against ROS due to the abundance of Cu,Zn-SOD, which scavenges free radicals and thus prevents metHb formation [43]. Cu, Zn-SOD synthesis is induced by O2 •− formation through the activation of regulatory genes [15]. SOD scavenges O2 •− and inhibits the formation of peroxynitrite, thereby preventing injury and regulating the bioavailability of NO [27]. Erythrocytes contain an abundant quantity of Cu,Zn-SOD, which maintains intra-erythrocyte O2 •− levels at concentrations as low as 10−13 mol/L [96].

Catalases catalyze the direct decomposition of H2O2 to water and O2 [21].

$$\text{2H}\_2\text{O}\_2 \xrightarrow{cAT} \text{2H}\_2\text{O} + \text{O}\_2\tag{4}$$

Catalase and SOD react synergistically to protect each other [99]. CAT and GPX are equally active in the detoxification of H2O2 in normal erythrocytes [100]. At physiological concentrations, GPX acts as a primary defense in H2O2 degradation by reducing H2O2 while also converting GSH to its oxidized form (GSSG). However, under H2O2 overproduction, CAT exhibits increased enzymatic activity, as measured by the Michaelis-Menten constant (Km). The Km for CAT (2.4 × 10−4 M) is significantly greater than the Km for GPX (1 × 10−6 M), which indicates that CAT scavenges H2O2 efficiently at higher concentrations [101–103]. GPX is important in dealing with endogenous H2O2 produced by Hb autoxidation, whereas CAT becomes increasingly important when erythrocytes are exposed to increased H2O2 flux [15]. H2O2 readily crosses erythrocyte membranes and can protect other tissues against extracellular H2O2 by "absorbing" and destroying it. Reduced glutathione (GSH) is used by GPX to detoxify hydrogen peroxide during normal antioxidant defense system function. Furthermore, glutathione reductase is required to convert H2O2 to GSH, which contributes to H2O2 detoxification [104].

Glutathione-S-transferases are important in the detoxification of electrophilic xenobiotics. This enzyme catalyzes the conjugation of GSH with exogenous and endogenous toxic compounds or their metabolites, making them more water-soluble, less toxic, and easier to excrete. In addition, they are responsible for various resistance mechanisms such as chemotherapeutic or antibiotic drug resistance [15].

Glucose-6-phosphate dehydrogenase (G6PD) is an important antioxidant enzyme in erythrocytes, which is the regulatory enzyme of the pentose-phosphate pathway (PPP). As erythrocytes lack mitochondria, the PPP pathway is the only source of NADPH, and it plays an important role in NADPH-dependent antioxidant defense [105, 106]. G6PD is required to protect erythrocytes from oxidative damage. The lack of this protection can lead to severe hemolysis [107].

In addition to primary antioxidant defense systems that prevent the generation of free radicals or radical chain reactions, secondary systems have been proposed. Proteases that preferentially degrade oxidatively damaged proteins are among them. In erythrocytes, a multicatalytic proteolytic complex appears to be responsible for the degradation of oxidized intracellular proteins [47]. The presence of an 80-kDa serine protease in the oxidized erythrocyte membranes preferentially degrades oxidized proteins specifically protein hydrolase. When cells are oxidized, this cytoplasmic protein

#### *Reactive Oxygen Species and Antioxidant Interactions in Erythrocytes DOI: http://dx.doi.org/10.5772/intechopen.107544*

becomes adherent to membranes, promoting membrane protein degradation. The protease is characterized by its inhibition by a serine protease inhibitor [108]. It is endogenously present in oxidized or aged erythrocyte membranes and plays a crucial role in the removal of the oxidation-induced membrane protein aggregates and in reducing the oxidation-induced anti-band 3 binding in aging. Oxidized protein hydrolase (OPH) acts as a secondary defense system by removing oxidized protein aggregates.

Peroxiredoxins (PRX), a class of thiol-containing enzymes, act as H2O2 and peroxynitrite scavengers in circulation. PRXs have a reductive capacity for hydroperoxides via a reductant thiol. Peroxiredoxins have been shown in studies to be catalytic peroxynitrite reductases. It has been also reported that PRX-II is present in the cytosol of erythrocytes. The catalytic cycle involves the reduction of oxidized PRX by thioredoxin and the reduction capacity of NADPH via NADPH-thioredoxin reductase [28, 109].

#### **4.2 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 as a chain-breaking antioxidant. Vitamin C reduces O2 •− and lipid peroxyl radical, but is also a well-known synergistic agent for vitamin E [2]. Uric acid is an endogenous antioxidant with metal-chelating properties and scavenges nitrogen radicals and superoxide in plasma, thereby blocking the generation of peroxynitrite. Uric acid in erythrocytes quenches the free radicals and ROS. Uric acid maintains the smooth membrane surface of RBCs, thus preventing echinocyte formation [110].

Additionally, erythrocytes have a plasma membrane redox system (PMRS) that transfers electrons from intracellular substrates to extracellular electron acceptors, which may be NAD+ or/and vitamin C [111].


Many studies have demonstrated the influence of different antioxidants on erythrocytes during oxidative stress (**Table 2**).


#### **Table 2.**

*The effects of antioxidants on erythrocytes during oxidative stress.*

There are *in vitro* studies on oxidative stress in erythrocytes reporting the protective effects of antioxidants from plant extracts (**Table 3**).


*Reactive Oxygen Species and Antioxidant Interactions in Erythrocytes DOI: http://dx.doi.org/10.5772/intechopen.107544*


**Table 3.**

*The modulations of antioxidants from plant extracts in erythrocytes.*
