**2. Oxidative stress and its involvement in pathology**

The cellular redox status represents the balance between generation of free radicals, such as the reactive oxygen species (ROS) and reactive nitrogen species (RNS), and the ability to detoxify them or to repair their resultant damage by antioxidants, such as the reduced glutathione (GSH), the major intracellular scavenger of ROS. ROS are generated in cells mainly during energy production: In the mitochondria, about 2% of the total oxygen (O2) consumption results in the free radical superoxide anion (O2 <sup>−</sup>) [1]. While not particularly reactive, superoxide can act as a reductant toward divalent metal ions, mainly iron and copper, and can react with itself by spontaneous or enzymatic (e.g., by the reducing enzyme superoxide dismutase, SOD) dismutation to form hydrogen peroxide (H2O2). The latter is a mild oxidant, but in the presence of divalent metals, it can generate the reactive hydroxyl (˙OH) radical.

In mature RBC, which are devoid of mitochondria, the hemoglobin (Hb) is the major source of ROS generation [2]. The heme iron, which is in the Fe(II) ferrous state in the oxygenated Hb, is oxidized to the Fe(III) ferric state in metHb—a reaction that normally occurs at a rate of about 3% of the Hb per day. This process results in the production of superoxide that in turn generates hydrogen peroxide and oxygen as products of dismutation by SOD [3]. The metHb is then restored back to oxyHb by the NADH-cytochrome b5 reductase [4].

An additional pathway of oxygen to superoxide reduction is by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Phagocytic cells, such as polymorphonuclear (PMN) neutrophils and macrophages, have an NADPH oxidase complex that generates ROS as part of the innate immune response to infection. Non-phagocytic cells contain NADPH oxidases that generate ROS, at lower levels than phagocytes, for signaling responses [5]. ROS can also arise as the indirect byproduct of enzymatic activities, such as that of monooxygenases (e.g., cytochrome P450) [6].

RNS originate from the gaseous molecule nitric oxide (NO). The latter is synthesized by constitutive or inducible nitric oxide synthase enzymes by oxidation of L-arginine to L-citrulline. NO can react rapidly with the superoxide anion to form the oxidant peroxynitrite (ONOO<sup>−</sup>), nitrogen dioxide (NO2), nitroxyl (HNO), and nitrosonium cation (NO+ ) (for review see [7]).

The cellular prooxidants are tightly controlled by restricting the magnitude and the location of their generation and by elaborating antioxidant mechanisms that scavenge their excess and correct their toxic consequences (for review see [1]). In addition to these intracellular antioxidant mechanisms, extracellular mechanisms function as well. For example, the blood serum contains many molecules with AOC such as bilirubin, albumin, ascorbic acid, as well as diet-derived antioxidants such as polyphenols.

Under certain conditions, excess oxidants my override the AOC and generate a state of oxidative stress. This may occur due to external factors (e.g., certain food components, air pollution, sun exposure, environmental radiation, as well as radioand chemotherapeutic regimes) or internal factors such as various pathological circumstances (e.g., inflammation, iron overload, Hb instability). Excess ROS react quickly with bio-molecules such as the DNA, proteins, and lipids, interfering with

**51**

*Red Blood Cells as Redox Modulators in Hemolytic Anemia*

cellular functions. As such, even if it is not the primary etiology, oxidative stress is believed to mediate the symptoms of many diseases, such as cancer, atherosclerosis, diabetes, cardiovascular, thromboembolic and neurodegenerative disorders, as well

We have studied oxidative stress in hemolytic anemias [8]. The anemia in these hereditary or acquired diseases is the result of augmented destruction (hemolysis) of mature RBC and their immature progenitors/precursors that is not balanced by compensatory overproduction. Among these diseases are: (I) The hemoglobinopathies—caused by mutations in the globin genes, leading to insufficient production (thalassemia) or production of aborted (sickle cell disease) globin chains [9]. (II) RBC membrane/cytoskeletal disorders such as hereditary spherocytosis, elliptocytosis, and stomatocytosis—caused by mutations in genes leading to abnormal RBC shape and propensity for hemolysis [10]. (III) Inherited enzymatic defects in RBC such as glucose-6-phosphate dehydrogenase (G6PD) deficiency and pyruvate kinase deficiency. G6PD is a key enzyme of the pentose pathway (hexose monophosphate shunt) which supplies NADPH—a reducing agent that is important for the regulation of the redox state, especially in RBC [11]. Patients with G6PD deficiency exhibit hemolytic anemia in response to infection and certain medications or foods. (IV) Paroxysmal nocturnal hemoglobinuria—a clonal disease caused by an acquired somatic mutation in the phosphatidylinositol glycan complementation class A gene. This gene encodes the enzyme responsible for the first step in the production of the glycosylphosphatidylinositol anchor, by which various proteins are linked to the plasma membrane. In this disease, the mutation occurs in a hematopoietic stem cell and is expressed in its progeny, affecting various membrane proteins including the complement (C′) inhibitors: CD55 (decay-accelerating factor) which inhibits the C3 component of the C′, and CD59 (membrane inhibitor of reactive lysis) which inhibits terminal C′ components (C5b-9) from forming the hemolytic membrane pore [12]. This leads to hemolysis and platelet activation, leading to anemia and to venous thrombosis, respectively [13]. (V) Congenital dyserythropoietic anemias—a heterogeneous group of diseases characterized by anemia due to abnormalities of erythroid precursor cells and reduced erythropoiesis [14]. (VI) Autoimmune hemolytic anemia such as ABO mismatch transfusion reaction and severe idiopathic autoimmune hemolytic anemia—caused by autoantibodies against antigens expressed on the surface of RBC. Once formed, these antibodies bind to the surface of RBC marking them for destruction through C′-mediated lysis (intravascular hemolysis) and/or Fc-mediated phagocytosis (extravascular hemolysis). Autoimmune hemolytic anemia can occur alone, but is often seen in association with other autoimmune diseases, cancer, drug treatment, transfusion, and pregnancy. (VII) Myelodysplastic syndromes (MDS)—diverse conditions that involve ineffective production (dysplasia) of hematopoietic cells. The patients often develop severe anemia and require frequent blood transfusions. In most cases, the disease worsens, and the patient develops cytopenias due to progressive bone marrow failure. In about one third of patients, the disease transforms

into acute myelogenous leukemia, usually within months to a few years.

Although oxidative stress is not the primary etiology of these diseases, except for G6PD deficiency, it mediates their symptoms, including anemia, recurrent infections, and thromboembolic complications [8]. The main causes of oxidative stress in these diseases are: (I) Degradation of abnormal Hbs in the mature RBC and their precursors (in the hemoglobinopathies), leading to the production of hemichromes

*DOI: http://dx.doi.org/10.5772/intechopen.84498*

as physiological aging [1].

**3. Hemolytic anemia**

cellular functions. As such, even if it is not the primary etiology, oxidative stress is believed to mediate the symptoms of many diseases, such as cancer, atherosclerosis, diabetes, cardiovascular, thromboembolic and neurodegenerative disorders, as well as physiological aging [1].
