**Abstract**

There is a continuous generation of reactive oxygen species (ROS) in erythrocytes due to their microenvironment. Reactive oxygen species (ROS) and reactive nitrogen species are well known as both harmful and beneficial species. They help in activating the antioxidant enzymes. However, overproduction of ROS can cause fatal damage to cell structures, including lipids and membranes, proteins and cause oxidative stress. Erythrocytes have effective antioxidant defenses to maintain their structure and functions. They protect these cells from damage and maintain their activities. Studies have reported that antioxidant interventions in various situations have proved beneficial to erythrocytes. Therefore, they can be employed as *in vitro* models for antioxidant and free radical interactions and also are ideal cell models for translational studies.

**Keywords:** erythrocytes, oxidative stress, free radicals, antioxidants, reactive oxygen species

### **1. Introduction**

The erythrocyte [red blood cell (RBC)] is an ideal cell to study free-radical-mediated alterations. Approximately 25 trillion erythrocytes course through the human circulatory system. The main function of erythrocytes is the transport of oxygen (O2) and the mediation of carbon dioxide (CO2) production [1]. Reactive oxygen species (ROS) are continuously produced within the erythrocytes due to high O2 tension in arterial blood and heme iron content [2].

The mature erythrocyte contains a variety of enzymes, proteins, carbohydrates, lipids, anions, and cations, to balance the cell's metabolism and functions. An important consequence of erythrocyte imbalance is a reduced ability to deal with oxidative stress, which can lead to degenerative changes in hemoglobin, membrane, and enzymes [3].

Erythrocytes are exposed to circulating inflammatory mediators and related oxidative stress, which cause severe alterations in cellular membrane and functions in a variety of pathological conditions. These alterations have been defined as "erythropathy" [4] and can be observed in conditions of cardiovascular injury [5, 6]. The loss of lipid asymmetry, and thus the exposure of phosphatidyl serine (PS) on the outer monolayer, contributes to the premature destruction of thalassaemic and sickle red cells [7, 8]. Sickle cell disease is distinguished by a change in erythrocyte shape from biconcave discs to elongated and sickle-shaped erythrocytes, consequently leading to loss of function and anemia [9].

Chronic obstructive pulmonary disease (COPD) causes changes in erythrocyte shape, redistribution of microfilaments such as actin and spectrin, and elevations in membrane rigidity [10]. Alterations were also observed in terms of erythrocyte morphology (leptocytes and elliptocytes), elevated membrane F2-isoprostanes and 4-hydroxynonenal (4-HNE) protein adducts, and oxidative damage to actin proteins in Rett syndrome (RTT) and autism spectrum disorder (ASD) [11, 12].

The changes in erythrocyte morphology and stiffness have also been reported in pathologies (type 2 diabetes, obesity, hypertension, and hypercholesterolemia) characterized by consistent oxidative damage followed by reshaping of the lipid distribution and architecture [13]. Erythrocytes participate in physiological and pathological processes associated with oxidative stress, such as aging, Down syndrome, neurodegenerative diseases such as Alzheimer's disease, erectile dysfunction, and cardiovascular disease [14].

The potential clinical application of these erythrocyte alterations as new biomarkers could be useful tools for monitoring a variety of oxidative-stress-related diseases.

## **2. Reactive oxygen species (ROS) in erythrocytes**

Various physiological and pathological conditions, for example, aging, inflammation, and cell death develop through ROS generation. Several factors can lead to the generation of oxidizing radicals such as superoxide anion (O2 •−), hydrogen peroxide (H2O2), and hydroxyl radical (HO• ) in erythrocytes [15].

Free radicals can be formed in three ways:


The latter, electron transfer, is a common process in biological systems [16]. Free radicals and ions are formed as illustrated below: Radical formation by electron transfer: A + e− → A•− Radical formation by homolytic fission: X: Y → X• + Y• Ion formation by heterolytic fission: X: Y — > X: <sup>−</sup> + Y+

#### **2.1 Nature of reactive oxygen species**

ROS are defined as oxygen-containing species, which are highly reactive. O2 undergoes one or two-electron reduction to form ROS, which reacts quickly with other compounds, attempting to capture the required electron in order to gain

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

stability. ROS are oxygen-centered molecules that include hydrogen peroxide, singlet oxygen, superoxide anion, hydroxyl radical, and nitric oxide (NO) [16]. ROS are constantly produced in small quantities by normal metabolic processes. The addition of one electron to O2 forms O2 •−, whereas the addition of two electrons results in the production of H2O2.

There are two causes for O2 •− generation in erythrocytes.

Firstly, Oxyhemoglobin (oxyHb) autoxidizes at a relatively slow rate to yield methemoglobin (metHb), and O2 •−, which, further produces H2O2. Hemoglobin (Hb) is constantly exposed to an intracellular and extracellular flux of H2O2. When oxyHb is exposed to H2O2, it undergoes oxidative modifications that have been proposed as selective signals for proteolysis in erythrocytes [17]. Secondly, the oxidation state of trivalent iron (Fe3+) has lost an electron during its formation; consequently, O2 •− has been generated from exogenous sources, such as drugs, etc. [18].

Hydrogen peroxide is hydrophilic; however, recent studies reported that aquaporins are not involved in facilitating H2O2 diffusion across RBC membranes; rather, diffusion occurs through the lipid fraction or an unidentified membrane protein [19]. While charged, O2 •− can only cross membranes via transmembrane anion channels. MetHb, lipid peroxidation, and spectrin-Hb complexes increase with H2O2, which further generates a covalent complex of spectrin and Hb, leading to changes in cell shape, membrane deformability, phospholipid organization, and cell surface characteristics [20].

The Fenton reaction occurs when H2O2 reacts with ferrous iron to produce OH• . H2O2 can react with O2 •− to generate OH• , the most active ROS that cannot travel far due to its short half-life of a few nanoseconds known as Haber-Weiss Reaction [21, 22].

$$Fe^{++} + \text{H}\_2\text{O}\_2 \rightarrow Fe^{++} + OH^- + OH^\bullet \text{(Fenton Reaction)}\tag{1}$$

$$\text{O}\_2\text{ }^\bullet + \text{H}\_2\text{O}\_2 \rightarrow \text{O}\_2 + \text{OH}^- + \text{OH}^\bullet \text{ (Haber-Weiss Reaction)}\tag{2}$$

ROS have the ability to act as both oxidizing and reducing agents. ROS are capable of directly attacking the red cell membrane and causing changes in lipid and protein structure [23]. ROS also alter mechanical properties, increase rigidity, and RBC interactions with other cells and coagulation factors, as well as stimulate microparticle (MP) generation and phosphatidylserine (PS) exposure [24]. Human red cell aging could be attributed to oxidative damage. RBC deformability, membrane permeability, and surface antigenicity abnormalities, on the other hand, have been recognized as defects in cellular properties that contribute to RBC senescence [25].

Nitric oxide, along with O2 and CO2, is the third gas transported by erythrocytes. Erythrocytes are the primary NO scavengers in circulation due to their high Hb concentration. NO is taken up by heme prosthetic groups of Hb-chain cysteine residues. NO is converted to nitrate by oxyhemoglobin (HbFe+2O2), whereas deoxyhemoglobin (HbFe2+) binds to NO to form iron-nitrosylhemoglobin (HbFe2+NO). NO consumption by erythrocytes can be regulated by HbFe2+NO formation under hypoxic conditions [26]. NO reaction with Hb greatly limits intravascular NO concentration. As a result, it is unlikely that NO is directly exported or produced by red blood cells as an intravascular signaling molecule. The rapid deoxygenation of NO by Hb results in the formation of nitrate and metHb, preventing NO diffusion from plasma to smooth muscle [15]. NO is produced in large amounts in inflammatory conditions and reacts

with O2 •− to generate peroxynitrite [27]. Peroxynitrite oxidizes plasma components, releasing secondary radicals that promote tyrosine nitration, leading to gain or loss of protein function [28].
