Joy G. Mohanty

Red blood cell oxidative stress impairs oxygen delivery and induces red blood cell aging

Red Blood Cells (RBCs) need to deform and squeeze through narrow capillaries. Decreased deformability of RBCs is, therefore, one of the factors that can contribute to the elimination of aged or damaged RBCs from the circulation. This process can also cause impaired oxygen delivery, which contributes to the pathology of a number of diseases. Studies from our laboratory have shown that oxidative stress plays a significant role in damaging the RBC membrane and impairing its deformability. RBCs are continuously exposed to both endogenous and exogenous sources of reactive oxygen species (ROS) like superoxide and hydrogen peroxide (H2O2). The bulk of the ROS are neutralized by the RBC antioxidant system consisting of both non-enzymatic and enzymatic antioxidants including catalase, glutathione peroxidase and peroxiredoxin-2. However, the autoxidation of hemoglobin (Hb) bound to the membrane is relatively inaccessible to the predominantly cytosolic RBC antioxidant system. This inaccessibility becomes more pronounced under hypoxic conditions when Hb is partially oxygenated, resulting in an increased rate of autoxidation and increased affinity for the RBC membrane. We have shown that a fraction of peroxyredoxin-2 present on the RBC membrane may play a major role in neutralizing these ROS. H2O2 that is not neutralized by the RBC antioxidant system can react with the heme producing fluorescent heme degradation products (HDPs). We have used the level of these HDP as a measure of RBC oxidative Stress. Increased levels of HDP are detected during cellular aging and various diseases. The negative correlation (p < 0.0001) between the level of HDP and RBC deformability establishes a contribution of RBC oxidative stress to impaired deformability and cellular stiffness. While decreased deformability contributes to the removal of RBCs from the circulation, oxidative stress also contributes to the uptake of RBCs by macrophages, which plays a major role in the removal of RBCs from circulation. The contribution of oxidative stress to the removal of RBCs by macrophages involves caspase-3 activation, which requires oxidative stress. RBC oxidative stress, therefore, plays a significant role in inducing RBC aging.

Association of the Red Cell Distribution Width with Red Blood Cell Deformability

The red cell distribution width (RDW) is a component of the automated complete blood count (CBC) that quantifies heterogeneity in the size of circulating erythrocytes. Higher RDW values reflect greater variation in red blood cell (RBC) volumes and are associated with increased risk for cardiovascular disease (CVD) events. The mechanisms underlying this association are unclear, but RBC deformability might play a role. CBCs were assessed in 293 adults who were clinically examined. RBC deformability (expressed as the elongation index) was measured using a microfluidic slit-flow ektacytometer. Multivariate regression analysis identified a clear threshold effect whereby RDW values above 14.0% were significantly associated with decreased RBC deformability (β = -0.24; p = 0.003). This association was stronger after excluding anemic participants (β = -0.40; p = 0.008). Greater variation in RBC volumes (increased RDW) is associated with decreased RBC deformability, which can impair blood flow through the microcirculation. The resultant hypoxia may help to explain the previously reported increased risk for CVD events associated with elevated RDW.

Nitrite enhances RBC hypoxic ATP synthesis and the release of ATP into the vasculature: a new mechanism for nitrite-induced vasodilation

A role for nitric oxide (NO) produced during the reduction of nitrite by deoxygenated red blood cells (RBCs) in regulating vascular dilation has been proposed. It has not, however, been satisfactorily explained how this NO is released from the RBC without first reacting with the large pools of oxyhemoglobin and deoxyhemoglobin in the cell. In this study, we have delineated a mechanism for nitrite-induced RBC vasodilation that does not require that NO be released from the cell. Instead, we show that nitrite enhances the ATP release from RBCs, which is known to produce vasodilation by several different methods including the interaction with purinergic receptors on the endothelium that stimulate the synthesis of NO by endothelial NO synthase. This mechanism was established in vivo by measuring the decrease in blood pressure when injecting nitrite-reacted RBCs into rats. The observed decrease in blood pressure was not observed if endothelial NO synthase was inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME) or when any released ATP was degraded by apyrase. The nitrite-enhanced ATP release was shown to involve an increased binding of nitrite-modified hemoglobin to the RBC membrane that displaces glycolytic enzymes from the membrane, resulting in the formation of a pool of ATP that is released from the RBC. These results thus provide a new mechanism to explain nitrite-induced vasodilation.

Redox Reactions of Hemoglobin

Redox reactions of hemoglobin have gained importance because of the general interest of the role of oxidative stress in diseases and the possible role of red blood cells in oxidative stress. Although electron paramagnetic resonance (EPR) is extremely valuable in studying hemoglobin redox reactions it has not been adequately used. We have focused in this review on the important contributions of EPR to our understanding of hemoglobin redox reactions. We have limited our discussion to the redox reactions thought to occur under physiological conditions. This includes autoxidation as well as the reactions of hydrogen peroxide generated by superoxide dismutation. We have also discussed redox reactions associated with nitric oxide produced in the circulation. We have pinpointed the value of using EPR to detect and study the paramagnetic species and free radicals formed during these reactions. We have shown how EPR not only identifies the paramagnetic species formed but can also be used to provide insights into the mechanism involved in the redox reactions.

Role of the membrane in the formation of heme degradation products in red blood cells

AIMS Red blood cells (RBCs) have an extensive antioxidant system designed to eliminate the formation of reactive oxygen species (ROS). Nevertheless, RBC oxidant stress has been demonstrated by the formation of a fluorescent heme degradation product (excitation (ex) 321 nm, emission (em) 465 nm) both in vitro and in vivo. We investigated the possibility that the observed heme degradation results from ROS generated on the membrane surface that are relatively inaccessible to the cellular antioxidants. MAIN METHODS Membrane and cytosol were separated by centrifugation and the fluorescence intensity and emission maximum were measured. The effect on the maximum emission of adding oxidized and reduced hemoglobin to the fluorescent product formed when hemin is degraded by hydrogen peroxide (H(2)O(2)) was studied. KEY FINDINGS 90% of the fluorescent heme degradation products in hemolysates are found on the membrane. Furthermore, these products are not transferred from the cytosol to the membrane and must, therefore, be formed on the membrane. We also showed that the elevated level of heme degradation in HbCC cells that is attributed to increased oxidative stress was found on the membrane. SIGNIFICANCE These results suggest that, although ROS generated in the cytosol are neutralized by antioxidant enzymes, H(2)O(2) generated by the membrane bound hemoglobin is not accessible to the cytosolic antioxidants and reacts to generate fluorescent heme degradation products. The formation of H(2)O(2) on the membrane surface can explain the release of ROS from the RBC to other tissues and ROS damage to the membrane that can alter red cell function and lead to the removal of RBCs from circulation by macrophages.

The pathophysiology of extracellular hemoglobin associated with enhanced oxidative reactions

Hemoglobin (Hb) continuously undergoes autoxidation producing superoxide which dismutates into hydrogen peroxide (H2O2) and is a potential source for subsequent oxidative reactions. Autoxidation is most pronounced under hypoxic conditions in the microcirculation and for unstable dimers formed at reduced Hb concentrations. In the red blood cell (RBC), oxidative reactions are inhibited by an extensive antioxidant system. For extracellular Hb, whether from hemolysis of RBCs and/or the infusion of Hb-based blood substitutes, the oxidative reactions are not completely neutralized by the available antioxidant system. Un-neutralized H2O2 oxidizes ferrous and ferric Hbs to Fe(IV)-ferrylHb and OxyferrylHb, respectively. FerrylHb further reacts with H2O2 producing heme degradation products and free iron. OxyferrylHb, in addition to Fe(IV) contains a free radical that can undergo additional oxidative reactions. Fe(III)Hb produced during Hb autoxidation also readily releases heme, an additional source for oxidative stress. These oxidation products are a potential source for oxidative reactions in the plasma, but to a greater extent when the lower molecular weight Hb dimers are taken up into cells and tissues. Heme and oxyferryl have been shown to have a proinflammatory effect further increasing their potential for oxidative stress. These oxidative reactions contribute to a number of pathological situations including atherosclerosis, kidney malfunction, sickle cell disease, and malaria. The toxic effects of extracellular Hb are of particular concern with hemolytic anemia where there is an increase in hemolysis. Hemolysis is further exacerbated in various diseases and their treatments. Blood transfusions are required whenever there is an appreciable decrease in RBCs due to hemolysis or blood loss. It is, therefore, essential that the transfused blood, whether stored RBCs or the blood obtained by an Autologous Blood Recovery System from the patient, do not further increase extracellular Hb.

Do Red Blood Cell-β-Amyloid Interactions Alter Oxygen Delivery in Alzheimer’s Disease?

Oxygen delivery requires that Red Blood Cells (RBCs) must be deformable to pass through the microcirculation. Alzheimers disease (AD) is a progressive neurodegenerative disorder characterized by abnormal extracellular deposition of beta-amyloid peptide (Abeta) and neuronal loss. We have analyzed RBC morphology in blood from subjects with AD and found that > 15% of the RBCs are elongated as compared to 5.9% in normal controls (p < 0.0001). To determine whether these morphology changes can be associated with the greater exposure of RBCs to AP in AD subjects, we investigated the in vitro effect of Abeta fibrils on blood. Morphological analysis of RBCs treated with Abeta1-40 or Abeta1-42 fibrils show 8.6% or 11.1% elongated cells, respectively. In contrast, only 2.9% or 1.3% of RBCs are elongated when blood is treated with buffer or mock fibrils generated from Abeta42-1. Elongated RBCs are expected to be less deformable. This prediction is consistent with our earlier studies showing impaired deformability of RBCs treated with Abeta fibrils. An additional factor previously reported by us, expected to impair the flow of RBCs through the microcirculation is their adherence to endothelial cells (ECs) when Abeta1-40 fibrils are bound to either RBCs or ECs. This factor would be more pronounced in AD subjects with elevated levels of Abeta on the vasculature. These results suggest that Abeta interactions with RBCs in AD subjects can result in impaired oxygen transport and delivery, which will have important implications for AD.

A fluorimetric semi-microplate format assay of protein carbonyls in blood plasma.

Oxidative stress, originating from reactive oxygen species (ROS), has been implicated in aging and various human diseases. The ROS generated can oxidize proteins producing protein carbonyl derivatives. The level of protein carbonyls in blood plasma has been used as a measure of overall oxidative stress in the body. Classically, protein carbonyls have been quantitated spectrophotometrically by directly reacting them with 2,4-dinitrophenylhydrazine (DNPH). However, the applicability of this method to biological samples is limited by its low inherent sensitivity. This limitation has been overcome by the development of sensitive enzyme-linked immunosorbent assay (ELISA) methods to measure protein carbonyls. As part of the Healthy Aging in Neighborhoods of Diversity across the Lifespan (HANDL) study, oxidative stress in humans was quantified by measuring blood plasma protein carbonyls using the two commercially available ELISA kits and the spectrophotometric DNPH assay. Surprisingly, two ELISA methods gave very different values for protein carbonyls, both of which were different from the value of the spectrophotometric method. We have developed a fluorescent semi-microplate format assay of protein carbonyls involving direct reaction of protein carbonyls with fluorescein thiosemicarbazide that correlates (R=0.992) with the direct spectrophotometric method. It has a coefficient of variation of 4.99% and is at least 100 times more sensitive than the spectrophotometric method.

Role of peroxiredoxin-2 in protecting RBCs from hydrogen peroxide-induced oxidative stress

Abstract The role of peroxiredoxin-2 (PRDX2) in preventing hydrogen peroxide-induced oxidative stress in the red blood cell was investigated by comparing blood from PRDX2 knockout mice with superoxide dismutase-1 (SOD1) knockout and control mice. Loss of PRDX2 increased basal levels of methemoglobin and heme degradation (a marker for oxidative stress), and reduced red blood cell deformability. In vitro incubation under normoxic conditions, both with and without inhibition of catalase, resulted in a lag phase during which negligible heme degradation occurred followed by a more rapid rate of heme degradation in the absence of PRDX2. The appreciable basal increase in heme degradation for PRDX2 knockout mice, together with the lag during in vitro incubation, implies that PRDX2 neutralizes hydrogen peroxide generated in vivo under the transient hypoxic conditions experienced as the cells pass through the microcirculation.

New insights provided by a comparison of impaired deformability with erythrocyte oxidative stress for sickle cell disease

Sickle cell disease (SCD) is associated with increase in oxidative stress and irreversible membrane changes that originates from the instability and polymerization of deoxygenated hemoglobin S (HbS). The relationship between erythrocyte membrane changes as assessed by a decrease in deformability and oxidative stress as assessed by an increase in heme degradation was investigated. The erythrocyte deformability and heme degradation for 27 subjects with SCD and 7 with sickle trait were compared with normal healthy adults. Changes in both deformability and heme degradation increased in the order of control to trait to non-crisis SCD to crisis SCD resulting in a very significantly negative correlation between deformability and heme degradation. However, a quantitative analysis of the changes in deformability and heme degradation for these different groups of subjects indicated that sickle trait had a much smaller effect on deformability than on heme degradation, while crisis affects deformability to a greater extent than heme degradation. These findings provide insights into the relative contributions of erythrocyte oxidative stress and membrane damage during the progression of SCD providing a better understanding of the pathophysiology of SCD.