**5. Pathological significance of RBC as redox modulators**

The redox-modulating activity of RBC could affect cells and their function throughout the body. We have studied their effect on platelets. Hemolytic anemia, such as thalassemia, is often associated with high incidence of thromboembolic complications (e.g., venous thrombosis and stroke) due to platelet hyperactivation and plasma hypercoagulation [37]. Platelet functioning depends on their redox state. They have an inherent ability to produce ROS by various pathways—as a by-product of the mitochondrial respiratory chain [38] and by the NADH/NADPH oxidase [for a review see [39]] produced mainly in the pentose cycle [40]. ROS, along with NO, adenosine, and prostacyclins, may play a profound role in the regulation of platelet activities [41]. Many studies demonstrated that their functioning during clot formation involves ROS; for example, platelet activators, such as thrombin, increase ROS generation [42–44].

Oxidative stress in platelets may give rise to two pathological outcomes: (I) toxicity, resulting in thrombocytopenia and bleeding and (II) hyperactivation resulting in excess clot formation leading to thromboembolic complications. Exemplifying the latter is hydrogen peroxide that stimulates their oxidative stress [45], and affects their various functions: activation by: (I) arachidonic acid and collagen [46]; (II) thrombin and ADP [47–49]; (III) tyrosine phosphorylation of the platelet αIIbβ3, an independent platelet activation pathway, thereby enhancing their aggregation [50], as well as (IV) through scavenging of the platelet- or endothelium-derived NO—thereby decreasing its aggregationinhibiting effect [51]. Superoxide can also contribute to late clot growth by increasing the bioavailability of ADP and subsequently recruiting additional platelets [49].

Since platelets do not carry known specific inherent redox pathology, it is reasonable to attribute their oxidative stress, at least in part, to continuous exposure to oxidative insults from extra platelet sources, such as their environment, i.e., the blood plasma, and neighboring cells - blood cells and the vascular endothelium. We have shown that incubation of normal platelets with plasma from thalassemia patients, rather than with normal plasma, resulted in their oxidative stress and activation [52]. Potential plasma oxidants are iron-containing compounds such as non-transferrin-bound iron, ferritin, heme, or Hb, all of which are increased in thalassemia patients [47, 53]. Incubation of platelets with iron (ferric ammonium citrate), heme (hemin or heme arginate), or Hb stimulated their oxidative stress. Moreover, addition of the iron-chelator deferoxamine to thalassemic plasma reduced its effect on the platelets' ROS [52].

Interestingly, thalassemic RBC also increased normal platelet oxidative status. In contrast, normal RBC, unless treated with oxidants, decreased it [54]. These results suggest that thalassemic RBC, having a higher than normal ROS level, mediate oxidative stress in platelets directly, probably by contact or close proximity [25]. These results are compatible with studies showing that platelets could be activated by ROS generated by neighboring cells such as RBC, neutrophils [55, 56], fibroblasts, and vascular endothelial and smooth muscle cells [39].

RBC might also affect platelets indirectly by a variety of mechanisms: (I) Release of iron-containing oxidants into the plasma [46, 57, 58], as mentioned above. (II) Release of ROS, e.g., superoxide anions, causing oxidation of low-density lipoprotein [59], which, in turn, might activate platelets [60]. (III) Exposure or shedding of phosphatidylserine moieties, which act as a procoagulant that amplifies the generation of thrombin and thus initiates platelet activation [61]. Thalassemic RBC have been shown to carry and shed higher than normal levels of external phosphatidylserine [35].

#### *Erythrocyte*

Other important function of the RBC-AOC is to scavenge and detoxify NO, an important vasodilating agent. released from the vascular endothelial cells [32] and by inflamed tissues [62].

The role of RBC as redox modulators can be compromised under pathological conditions: when their number decrease (anemia) and when their AOC is defective, both of which may co-exist in many diseases. Anemia may elevate oxidative stress by reducing the oxygen availability (hypoxia) to tissue cells and by reducing the AOC of the RBC.

Various therapeutic modalities may be used to correct anemia:


Both transfusions and EPO have been used pre- and post-major operative procedures that are associated with severe blood loss.

All these therapeutic procedures, on one hand, increase the RBC mass and thereby, supposedly, its AOC. On the other hand, iron supplementation and transfusions might increase the iron load leading to oxidative stress in cells, including RBC, thus compromising their AOC. For example, multi-transfused thalassemic patients, with less severe anemia but higher iron overload, have lower levels of oxidative stress (ROS and lipid hydroperoxides) than un-transfused patients, with more severe anemia but lower iron overload [63]. In cardiovascular diseases, although there is ample clinical evidence for the worsening effect of anemia, RBC transfusions or EPO administration were not always effective [64–66]. As for EPO, it has been demonstrated to have an antioxidative effect on various cells, including RBC [67], and thereby might increase their AOC. The net effect of anemia and iron overload on

#### **Figure 2.**

*The relationship among anemia, its treatment and RBC antioxidative capacity on oxidative stress. Upward red arrows indicate an increase; the downward blue arrows indicate a decrease.*

**57**

at different times.

*Red Blood Cells as Redox Modulators in Hemolytic Anemia*

against its potential reducing effect of the RBC-AOC.

**6.1 Measurement of the redox status in RBC**

cellular fluorescence is proportional to ROS generation [72].

oxidative stress warrants a careful study in transfused and non-transfused patients and favors continuous monitoring of the status of iron and oxidative stress during these treatments. This complex relationship is graphically summarized in **Figure 2**. Some therapeutic protocols are used to reduce the RBC mass (hematocrit). Bloodletting (phlebotomy) is used in cases of polycythemia (erythrocytosis), either primary (polycythemia vera), familiar, or secondary [68], as well as hereditary hemochromatosis—an inheritable disease characterized by iron overload [69]. The benefit of this treatment with respect to decreasing the iron load should be weighed

The oxidative state of RBC depends on intra-RBC factors such as enzymopahtology (e.g., G6PD deficiency), Hb instability (thalassemia and sickle cell disease), membrane pathology (hereditary spherocytosis), glucose metabolism [diabetes [27]], or extra-RBC factors such as in inflammation. Their oxidative state, in turn, may affect their AOC. It was suggested that RBC could be used as bioindicators of prognostic value in clinical practice [19]. They may provide a real-time monitoring of their own conditions as well as those in other parts of the body. This is potentially relevant to RBC-linked and unlinked pathologic conditions associated with oxidative stress.

Measurement of redox parameters in cells and in body fluids, such as the blood plasma, can be accomplished by various methods [3]. These measurements, however, are not a common practice in the clinic mainly because the methodologies are inadequate for the routine clinical laboratory. We have measured redox parameters [54, 70], including the labile iron pool [71], in RBC by flow cytometry, a common methodology in the clinical setting. Various fluorescent probes have been used. For example, ROS were measured by 2′7′-dichlorodihydrofluorescindiacetate, [72]. Following free diffusion into cells, this nonfluorescent compound is esterified and gets trapped intracellularly as 2′7′-dichlorodihydrofluorescein. ROS, mainly peroxides, oxidize it to the fluorescent derivative 2′7′-dichlorodihydrofluorescein that its

Several points should be considered using this method: (I) Since ROS are short-lived, analyses should be performed on fresh samples. (II) The probes used are not specific to a particular ROS—a limitation that does not limit the assessment of general oxidative stress. (III) The intracellular probe content depends on the experimental settings: the concentration of probe added to the composition of the medium and the incubation conditions, such as the temperature. However, it also depends on the cellular uptake of the probe and its esterification, which depends on the different properties of cells (e.g., activated vs. inactivated, pathological vs. normal). To overcome these caveats, we have modified the protocol: Cells were pulsed with the probe, washed, and then re-incubated in probe-free medium. The kinetics of ROS generation was determined by measuring the cellular fluorescence

The method was validated by determining the effect on RBC fluorescence of the ROS-generating agent peroxide, the catalase inhibitor sodium azide, and the ROS scavenger N-acetyl cysteine. When normal RBC were compared with RBC from β-thalassemia patients, both the basal fluorescence and its kinetics were higher in

the latter, confirming that thalassemic RBC were under oxidative stress.

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

**6. RBC as redox bioindicators**

*Red Blood Cells as Redox Modulators in Hemolytic Anemia DOI: http://dx.doi.org/10.5772/intechopen.84498*

oxidative stress warrants a careful study in transfused and non-transfused patients and favors continuous monitoring of the status of iron and oxidative stress during these treatments. This complex relationship is graphically summarized in **Figure 2**.

Some therapeutic protocols are used to reduce the RBC mass (hematocrit). Bloodletting (phlebotomy) is used in cases of polycythemia (erythrocytosis), either primary (polycythemia vera), familiar, or secondary [68], as well as hereditary hemochromatosis—an inheritable disease characterized by iron overload [69]. The benefit of this treatment with respect to decreasing the iron load should be weighed against its potential reducing effect of the RBC-AOC.

### **6. RBC as redox bioindicators**

*Erythrocyte*

by inflamed tissues [62].

AOC of the RBC.

Other important function of the RBC-AOC is to scavenge and detoxify NO, an important vasodilating agent. released from the vascular endothelial cells [32] and

The role of RBC as redox modulators can be compromised under pathological conditions: when their number decrease (anemia) and when their AOC is defective, both of which may co-exist in many diseases. Anemia may elevate oxidative stress by reducing the oxygen availability (hypoxia) to tissue cells and by reducing the

• Administration of erythropoiesis-stimulating agents such as erythropoietin (EPO) in cases of reduced erythropoiesis. This includes patients with chronic kidney disease where there is insufficient EPO production due to renal dysfunction, patients with malignancies during the course of chemotherapy, and patients with myelodysplastic syndrome. In most of these cases, the treatment

• Blood transfusion is used in the event of acute, severe, hemorrhage, or in chronic hemolysis. An example of the latter is β-thalassemia major where patients are transfused with packed RBC every 3 weeks for their entire life.

Both transfusions and EPO have been used pre- and post-major operative

All these therapeutic procedures, on one hand, increase the RBC mass and thereby, supposedly, its AOC. On the other hand, iron supplementation and transfusions might increase the iron load leading to oxidative stress in cells, including RBC, thus compromising their AOC. For example, multi-transfused thalassemic patients, with less severe anemia but higher iron overload, have lower levels of oxidative stress (ROS and lipid hydroperoxides) than un-transfused patients, with more severe anemia but lower iron overload [63]. In cardiovascular diseases, although there is ample clinical evidence for the worsening effect of anemia, RBC transfusions or EPO administration were not always effective [64–66]. As for EPO, it has been demonstrated to have an antioxidative effect on various cells, including RBC [67], and thereby might increase their AOC. The net effect of anemia and iron overload on

*The relationship among anemia, its treatment and RBC antioxidative capacity on oxidative stress. Upward red* 

*arrows indicate an increase; the downward blue arrows indicate a decrease.*

Various therapeutic modalities may be used to correct anemia:

• Iron supplementation in the case of deficiency.

comprises both EPO and iron supplementation.

procedures that are associated with severe blood loss.

**56**

**Figure 2.**

The oxidative state of RBC depends on intra-RBC factors such as enzymopahtology (e.g., G6PD deficiency), Hb instability (thalassemia and sickle cell disease), membrane pathology (hereditary spherocytosis), glucose metabolism [diabetes [27]], or extra-RBC factors such as in inflammation. Their oxidative state, in turn, may affect their AOC. It was suggested that RBC could be used as bioindicators of prognostic value in clinical practice [19]. They may provide a real-time monitoring of their own conditions as well as those in other parts of the body. This is potentially relevant to RBC-linked and unlinked pathologic conditions associated with oxidative stress.

#### **6.1 Measurement of the redox status in RBC**

Measurement of redox parameters in cells and in body fluids, such as the blood plasma, can be accomplished by various methods [3]. These measurements, however, are not a common practice in the clinic mainly because the methodologies are inadequate for the routine clinical laboratory. We have measured redox parameters [54, 70], including the labile iron pool [71], in RBC by flow cytometry, a common methodology in the clinical setting. Various fluorescent probes have been used. For example, ROS were measured by 2′7′-dichlorodihydrofluorescindiacetate, [72]. Following free diffusion into cells, this nonfluorescent compound is esterified and gets trapped intracellularly as 2′7′-dichlorodihydrofluorescein. ROS, mainly peroxides, oxidize it to the fluorescent derivative 2′7′-dichlorodihydrofluorescein that its cellular fluorescence is proportional to ROS generation [72].

Several points should be considered using this method: (I) Since ROS are short-lived, analyses should be performed on fresh samples. (II) The probes used are not specific to a particular ROS—a limitation that does not limit the assessment of general oxidative stress. (III) The intracellular probe content depends on the experimental settings: the concentration of probe added to the composition of the medium and the incubation conditions, such as the temperature. However, it also depends on the cellular uptake of the probe and its esterification, which depends on the different properties of cells (e.g., activated vs. inactivated, pathological vs. normal). To overcome these caveats, we have modified the protocol: Cells were pulsed with the probe, washed, and then re-incubated in probe-free medium. The kinetics of ROS generation was determined by measuring the cellular fluorescence at different times.

The method was validated by determining the effect on RBC fluorescence of the ROS-generating agent peroxide, the catalase inhibitor sodium azide, and the ROS scavenger N-acetyl cysteine. When normal RBC were compared with RBC from β-thalassemia patients, both the basal fluorescence and its kinetics were higher in the latter, confirming that thalassemic RBC were under oxidative stress.
