Potential Modulation of Vascular Function by Nitric Oxide and Reactive Oxygen Species Released From Erythrocytes.

The primary role for erythrocytes is oxygen transport that requires the reversible binding of oxygen to hemoglobin. There are, however, secondary reactions whereby the erythrocyte can generate reactive oxygen species (ROS) and nitric oxide (NO). ROS such as superoxide anion and hydrogen peroxide are generated by the autoxidation of hemoglobin. NO can be generated when nitrite reacts with hemoglobin forming an HbNO+ intermediate. Both of these reactions are dramatically enhanced under hypoxic conditions. Within the erythrocyte, interactions of NO with hemoglobin and enzymatic reactions that neutralize ROS are expected to prevent the release of any generated NO or ROS. We have, however, demonstrated that partially oxygenated hemoglobin has a distinct conformation that enhances hemoglobin-membrane interactions involving Band 3 protein. Autoxidation of the membrane bound partially oxygenated hemoglobin facilitates the release of ROS from the erythrocyte. NO release is made possible when HbNO+, the hemoglobin nitrite-reduced intermediate, which is not neutralized by hemoglobin, is bound to the membrane and releases NO. Some of the released ROS has been shown to be transferred to the vasculature suggesting that some of the released NO may also be transferred to the vasculature. NO is known to have a major effect on the vasculature regulating vascular dilatation. Erythrocyte generated NO may be important when NO production by the vasculature is impaired. Furthermore, the erythrocyte NO released, may play an important role in regulating vascular function under hypoxic conditions when endothelial eNOS is less active. ROS can react with NO and, can thereby modulate the vascular effects of NO. We have also demonstrated an inflammatory response due to erythrocyte ROS. This reflects the ability of ROS to react with various cellular components affecting cellular function.

Near-Wall Migration Dynamics of Erythrocytes in Vivo: Effects of Cell Deformability and Arteriolar Bifurcation.

Red blood cell (RBC) deformability has a significant impact on microcirculation by affecting cell dynamics. Despite previous studies that have demonstrated the margination of rigid cells and particles in vitro, little information is available on the in vivo margination of deformability-impaired RBCs under physiological flow and hematocrit conditions. Thus, in this study, we examined how the deformability-dependent, RBC migration alters the cell distribution under physiological conditions, particularly in arteriolar network flows. The hardened RBCs (hRBCs) were found to preferentially flow near the vessel walls of small arterioles (diameter = 47.1-93.3 µm). The majority of the hRBCs (63%) were marginated within the range of 0.7R-0.9R (R: radial position normalized by vessel radius), indicating that the hRBCs preferentially accumulated near the vessel walls. The laterally marginated hRBCs maintained their lateral positions near the walls while traversing downstream with attenuated radial dispersion. In addition, the immediate displacement of RBCs while traversing a bifurcation also contributes to the near-wall accumulation of hRBCs. The notable difference in the inward migration between the marginated nRBCs and hRBCs after bifurcations further supports the potential role of bifurcations in the accumulation of hRBCs near the walls.

Red blood cell membrane-facilitated release of nitrite-derived nitric oxide bioactivity.

The reduction of nitrite by deoxyhemoglobin to nitric oxide (NO) has been proposed as a mechanism for the transfer of NO bioactivity from the red blood cell (RBC) to the vasculature. This transfer can increase vascular dilatation. The major challenge to this hypothesis is the very efficient scavenging of NO by hemoglobin, which prevents the release of NO from RBCs. Previous studies indicate that the reaction of nitrite with deoxyhemoglobin produces two metastable intermediates involving nitrite bound to deoxyhemoglobin and a hybrid intermediate [Hb(II)NO(+) ↔ Hb(III)NO] where the nitrite is reduced, but unavailable to react with hemoglobin. We have now shown how unique properties of these intermediates provide a pathway for the release of NO bioactivity from RBCs. The high membrane affinity of these intermediates (>100-fold greater than that of deoxyhemoglobin) places these intermediates on the membrane. Furthermore, membrane-induced conformational changes of the nitrite-reacted intermediates facilitate the release of NO from the hybrid intermediate and nitrite from the nitrite-bound intermediate. Increased membrane affinity, coupled with facilitated dissociation of NO and nitrite from the membrane-bound intermediates, provides the first realistic mechanism for the potential release of NO and nitrite from the RBC and their potential transfer to the vasculature.

The effect of intermittent pneumatic compression of legs on the levels of nitric oxide related species in blood and on arterial function in the arm.

BACKGROUND: Intermittent pneumatic compression (IPC) of legs exerts beneficial local vascular effects, possibly through local release of nitric oxide (NO). However, studies demonstrating systemic transport of nitrogen oxide species and release of NO prompt the question of whether IPC could also exert nonlocal effects. We tested whether IPC (1) affects systemic levels of nitrite, S-nitrosothiols and red blood cell (RBC) NO, and (2) exerts vasoactive effects in the brachial artery (BA), although this hypothesis-generating pilot study did not investigate cause and effect relationship between (1) and (2). METHODS: In 10 healthy subjects, ages 24-39 years, we measured plasma nitrite, plasma S-nitrosothiols and RBC-NO from venous blood samples drawn before and after IPC treatment. We also measured BA responses to 5 min of upper arm occlusion at rest and during 1 h of leg IPC. RESULTS: There was a significant decrease in plasma nitrite (112±26 nM to 90±15 nM, p=0.0008) and RBC-NO (129±72 nM to 102±41 nM, p=0.02). Plasma S-nitrosothiols were unchanged (5.79±4.81 nM to 6.27±5.79 nM, p=0.3). BA occlusion-mediated constriction (OMC) was significantly attenuated with IPC treatment (-43±13% to -33±12%, p=0.003). High-flow mediated BA dilation was unchanged (13.3±9.4% to 11.5±7.2%, p=0.2). CONCLUSION: Plasma nitrite, RBC-NO, and BA OMC decreased with leg IPC. We hypothesize that this decrease in circulatory pool of plasma nitrite and RBC-NO may result from the transfer of their NO-bioactivity from blood to the hypoxic arm tissue, to be stored and released under hypoxic stress and oppose OMC. Future studies should investigate whether IPC-induced decreases in brachial OMC are caused by the changes in systemic NO activity, and whether leg IPC could benefit distant arterial function in systemic cardiovascular disease.

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.

Nitroprusside inhibits calcium-induced impairment of red blood cell deformability.

BACKGROUND: Red blood cell (RBC) deformation is critical for microvascular perfusion and oxygen delivery to tissues. Abnormalities in RBC deformability have been observed in aging, sickle cell disease, diabetes, and preeclampsia. Although nitric oxide (NO) prevents decreases in RBC deformability, the underlying mechanism is unknown. STUDY DESIGN AND METHODS: As an experimental model, we used ionophore A23187-mediated calcium influx in RBCs to reduce their deformability and investigated the role of NO donor sodium nitroprusside (SNP) and KCa3.1 (Gardos) channel blockers on RBC deformability (measured as elongation index [EI] by microfluidic ektacytometry). RBC intracellular Ca(2+) and extracellular K(+) were measured by inductively coupled plasma mass spectrometry and potassium ion selective electrode, respectively. RESULTS: SNP treatment of RBCs blocked the Ca(2+) (approx. 10 µmol/L)-induced decrease in RBC deformability (EI 0.34 ± 0.02 vs. 0.09 ± 0.01, control vs. Ca(2+) loaded, p < 0.001; and EI 0.37 ± 0.02 vs. 0.30 ± 0.01, SNP vs. SNP plus Ca(2+) loaded) as well as Ca(2+) influx and K(+) efflux. The SNP effect was similar to that observed after pharmacologic blockade of the KCa3.1 channel (with charybdotoxin or extracellular medium containing isotonic K(+) concentration). In RBCs from KCa3.1(-/-) mice, 10 µmol/L Ca(2+) loading did not decrease cellular deformability. A preliminary attempt to address the molecular mechanism of SNP protection suggests the involvement of cell surface thiols. CONCLUSION: Our results suggest that nitroprusside treatment of RBCs may protect them from intracellular calcium increase-mediated stiffness, which may occur during microvascular perfusion in diseased states, as well as during RBC storage.

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.

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.

Enhanced Aß(1-40) production in endothelial cells stimulated with fibrillar Aß(1-42).

Amyloid accumulation in the brain of Alzheimer's patients results from altered processing of the 39- to 43-amino acid amyloid ß protein (Aß). The mechanisms for the elevated amyloid (Aß(1-42)) are considered to be over-expression of the amyloid precursor protein (APP), enhanced cleavage of APP to Aß, and decreased clearance of Aß from the central nervous system (CNS). We report herein studies of Aß stimulated effects on endothelial cells. We observe an interesting and as yet unprecedented feedback effect involving Aß(1-42) fibril-induced synthesis of APP by Western blot analysis in the endothelial cell line Hep-1. We further observe an increase in the expression of Aß(1-40) by flow cytometry and fluorescence microscopy. This phenomenon is reproducible for cultures grown both in the presence and absence of serum. In the former case, flow cytometry reveals that Aß(1-40) accumulation is less pronounced than under serum-free conditions. Immunofluorescence staining further corroborates these observations. Cellular responses to fibrillar Aß(1-42) treatment involving eNOS upregulation and increased autophagy are also reported.

Routes for formation of S-nitrosothiols in blood.

S-nitrosothiols (RSNO) are involved in post-translational modifications of many proteins analogous to protein phosphorylation. In addition, RSNO have many physiological roles similar to nitric oxide ((•)NO), which are presumably involving the release of (•)NO from the RSNO. However, the much longer life span in biological systems for RSNO than (•)NO suggests a dominant role for RSNO in mediating (•)NO bioactivity. RSNO are detected in plasma in low nanomolar levels in healthy human subjects. These RSNO are believed to be redirecting the (•)NO to the vasculature. However, the mechanism for the formation of RSNO in vivo has not been established. We have reviewed the reactions of (•)NO with oxygen, metalloproteins, and free radicals that can lead to the formation of RSNO and have evaluated the potential for each mechanism to provide a source for RSNO in vivo.

Erythrocytes induce proinflammatory endothelial activation in hypoxia.

Although exposure to ambient hypoxia is known to cause proinflammatory vascular responses, the mechanisms initiating these responses are not understood. We tested the hypothesis that in systemic hypoxia, erythrocyte-derived H(2)O(2) induces proinflammatory gene transcription in vascular endothelium. We exposed mice or isolated, perfused murine lungs to 4 hours of hypoxia (8% O(2)). Leukocyte counts increased in the bronchoalveolar lavage. The expression of leukocyte adhesion receptors, reactive oxygen species, and protein tyrosine phosphorylation increased in freshly recovered lung endothelial cells (FLECs). These effects were inhibited by extracellular catalase and by the removal of erythrocytes, indicating that the responses were attributable to erythrocyte-derived H(2)O(2). Concomitant nuclear translocation of the p65 subunit of NF-κB and hypoxia-inducible factor-1α stabilization in FLECs occurred only in the presence of erythrocytes. Hemoglobin binding to the erythrocyte membrane protein, band 3, induced the release of H(2)O(2) from erythrocytes and the p65 translocation in FLECs. These data indicate for the first time, to our knowledge, that erythrocytes are responsible for endothelial transcriptional responses in hypoxia.

Considering the vascular hypothesis of Alzheimer's disease: effect of copper associated amyloid on red blood cells.

The vascular hypothesis of Alzheimer's disease (AD) considers cerebral hypoperfusion as a primary trigger for neuronal dysfunction. We have previously reported that red blood cells (RBCs) bind amyloid, which are the characteristic deposits found in AD brains, and interact with amyloid on the vasculature [1-3]. Oxidative stress triggered by these RBC/amyloid interactions could impair oxygen delivery. Recent literature has implicated copper bound amyloid-ß peptide (CuAß) and the associated production of reactive oxygen species (ROS) as one of the primary factors contributing to AD pathology. In this work, we have investigated CuAß generated RBC oxidative stress. Aß(1-40) peptide with a stoichiometric amount of copper bound was produced and compared to the metal-free form of the peptide. Different aggregation states of the peptides were isolated and incubated with RBCs for 15 h. Interestingly, CuAß stimulated a pronounced increase in red cell oxidative stress as indicated by increased hemoglobin (Hb) oxidation, increased formation of fluorescent heme degradation products, and a decrease in RBC deformability. These findings demonstrate a potential role for CuAß in promoting vascular oxidative stress leading to impaired cerebral oxygen delivery, which may contribute to neurodegeneration associated with AD.

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 (ß = -0.24; p = 0.003). This association was stronger after excluding anemic participants (ß = -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.

SOD2 deficiency in hematopoietic cells in mice results in reduced red blood cell deformability and increased heme degradation.

Among the three types of super oxide dismutases (SODs) known, SOD2 deficiency is lethal in neonatal mice owing to cardiomyopathy caused by severe oxidative damage. SOD2 is found in red blood cell (RBC) precursors, but not in mature RBCs. To investigate the potential damage to mature RBCs resulting from SOD2 deficiency in precursor cells, we studied RBCs from mice in which fetal liver stem cells deficient in SOD2 were capable of efficiently rescuing lethally irradiated host animals. These transplanted animals lack SOD2 only in hematopoietically generated cells and live longer than SOD2 knockouts. In these mice, approximately 2.8% of their total RBCs in circulation are iron-laden reticulocytes, with numerous siderocytic granules and increased protein oxidation similar to that seen in sideroblastic anemia. We have studied the RBC deformability and oxidative stress in these animals and the control group by measuring them with a microfluidic ektacytometer and assaying fluorescent heme degradation products with a fluorimeter, respectively. In addition, the rate of hemoglobin oxidation in RBCs from these mice and the control group were measured spectrophotometrically. The results show that RBCs from these SOD2-deficient mice have reduced deformability, increased heme degradation products, and an increased rate of hemoglobin oxidation compared with control animals, indicative of increased RBC oxidative stress.

Hemoglobin redox reactions and red blood cell aging.

SIGNIFICANCE: The physiological mechanism(s) for recognition and removal of red blood cells (RBCs) from circulation after 120 days of its lifespan is not fully understood. Many of the processes thought to be associated with the removal of RBCs involve oxidative stress. We have focused on hemoglobin (Hb) redox reactions, which is the major source of RBC oxidative stress. RECENT ADVANCES: The importance of Hb redox reactions have been shown to originate in large parts from the continuous slow autoxidation of Hb producing superoxide and its dramatic increase under hypoxic conditions. In addition, oxidative stress has been shown to be associated with redox reactions that originate from Hb reactions with nitrite and nitric oxide (NO) and the resultant formation of highly toxic peroxynitrite when NO reacts with superoxide released during Hb autoxidation. CRITICAL ISSUES: The interaction of Hb, particularly under hypoxic conditions with band 3 of the RBC membrane is critical for the generating the RBC membrane changes that trigger the removal of cells from circulation. These changes include exposure of antigenic sites, increased calcium leakage into the RBC, and the resultant leakage of potassium out of the RBC causing cell shrinkage and impaired deformability. FUTURE DIRECTIONS: The need to understand the oxidative damage to specific membrane proteins that result from redox reactions occurring when Hb is bound to the membrane. Proteomic studies that can pinpoint the specific proteins damaged under different conditions will help elucidate the cellular aging processes that result in cells being removed from circulation.

Internal Spin Trapping of Thiyl Radical during the Complexation and Reduction of Cobalamin with Glutathione and Dithiothrietol.

The activation of cobalamin requires the reduction of Cbl(III) to Cbl(II). The reduction by glutathione and dithiothreitol was followed using visible spectroscopy and electron paramagnetic resonance. In addition the oxidation of glutathione was monitored. Glutathione first reacts with oxidized Cbl(III). The binding of a second glutathione required for the reduction to Cbl(II) is presumably located in the dimethyl benzimidazole ribonucleotide ligand cavity. The reduction of Cbl(III) by dithiothreitol, which contains two thiols, is much faster even though no stable Cbl(III) complex is formed. The reduction, by both thiol reagents, results in the formation of thiyl radicals, some of which are released to form oxidized thiol products and some of which remain associated with the reduced cobalamin. In the reduced state the intrinsic lower affinity for the benzimidazole base, coupled with a trans effect from the initial GSH bound to the ß-axial site and a possible lowering of the pH results in an equilibrium between base-on and base-off complexes. The dissociation of the base facilitates a closer approach of the thiyl radical to the Co(II) α-axial site resulting in a complex with ferromagnetic exchange coupling between the metal ion and the thiyl radical. This is a unique example of 'internal spin trapping' of a thiyl radical formed during reduction. The finding that the reduction involves a peripheral site and that thiyl radicals produced during the reduction remain associated with the reduced cobalamin provide important new insights into our understanding of the formation and function of cobalamin enzymes.

Oxidative damage to DNA and single strand break repair capacity: relationship to other measures of oxidative stress in a population cohort.

It is well accepted that oxidative DNA repair capacity, oxidative damage to DNA and oxidative stress play central roles in aging and disease development. However, the correlation between oxidative damage to DNA, markers of oxidant stress and DNA repair capacity is unclear. In addition, there is no universally accepted panel of markers to assess oxidative stress in humans. Our interest is oxidative damage to DNA and its correlation with DNA repair capacity and other markers of oxidative stress. We present preliminary data from a small comet study that attempts to correlate single strand break (SSB) level with single strand break repair capacity (SSB-RC) and markers of oxidant stress and inflammation. In this limited study of four very small age-matched 24-individual groups of male and female whites and African-Americans aged 30-64 years, we found that females have higher single strand break (SSB) levels than males (p=0.013). There was a significant negative correlation between SSB-RC and SSB level (p=0.041). There was a positive correlation between SSBs in African American males with both heme degradation products (p=0.008) and high-sensitivity C-reactive protein (hs-CRP) (p=0.022). We found a significant interaction between hs-CRP and sex in their effect on residual DNA damage (p=0.002). Red blood cell reduced glutathione concentration was positively correlated with the levels of oxidized bases detected by endonuclease III (p=0.047), heme degradation products (p=0.015) and hs-CRP (p=0.020). However, plasma carbonyl levels showed no significant correlation with other markers. The data from the literature and from our very limited study suggest a complex relationship between measures of oxidative stress and frequently used clinical parameters believed to reflect inflammation or oxidative stress.

Racial discrimination is associated with a measure of red blood cell oxidative stress: a potential pathway for racial health disparities.

BACKGROUND: There are racial health disparities in many conditions for which oxidative stress is hypothesized to be a precursor. These include cardiovascular disease, diabetes, and premature aging. Small clinical studies suggest that psychological stress may increase oxidative stress. However, confirmation of this association in epidemiological studies has been limited by homogenous populations and unmeasured potential confounders. PURPOSE: We tested the cross-sectional association between self-reported racial discrimination and red blood cell (RBC) oxidative stress in a biracial, socioeconomically heterogeneous population with well-measured confounders. METHODS: We performed a cross-sectional analysis of a consecutive series of 629 participants enrolled in the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study. Conducted by the National Institute on Aging Intramural Research Program, HANDLS is a prospective epidemiological study of a socioeconomically diverse cohort of 3,721 Whites and African Americans aged 30-64 years. Racial discrimination was based on self-report. RBC oxidative stress was measured by fluorescent heme degradation products. Potential confounders were age, smoking status, obesity, and C-reactive protein. RESULTS: Participants had a mean age of 49 years (SD = 9.27). In multivariable linear regression models, racial discrimination was significantly associated with RBC oxidative stress (Beta = 0.55, P < 0.05) after adjustment for age, smoking, C-reactive protein level, and obesity. When stratified by race, discrimination was not associated with RBC oxidative stress in Whites but was associated significantly for African Americans (Beta = 0.36, P < 0.05). CONCLUSIONS: These findings suggest that there may be identifiable cellular pathways by which racial discrimination amplifies cardiovascular and other age-related disease risks.