Oxidative Stress In Sickle Cell Disease Research Articles

Ferroptosis as an emerging target in sickle cell disease

Sickle cell disease (SCD) is an inherited hemoglobin disorder marked by red blood cell sickling, resulting in severe anemia, painful episodes, extensive organ damage, and shortened life expectancy. In SCD, increased iron levels can trigger ferroptosis, a specific type of cell death characterized by reactive oxygen species (ROS) and lipid peroxide accumulation, leading to damage and organ impairments. The intricate interplay between iron, ferroptosis, inflammation, and oxidative stress in SCD underscores the necessity of thoroughly understanding these processes for the development of innovative therapeutic strategies. This review highlights the importance of balancing the complex interactions among various factors and exploitation of the knowledge in developing novel therapeutics for this devastating disease.

Oxidative stress in sickle cell anemia can be a prognostic marker for disease severity

CONTEXT: Sickle cell anemia (SCA) or sickle cell disease (SCD) is an inherited blood disorder characterized primarily by chronic anemia and periodic episodes of pain. There are reports that increase in oxidative stress may play a significant role in the pathophysiology of SCA. AIM: The present study aims to investigate enzymatic and nonenzymatic antioxidant status in SCA patients to understand the incidence of increased oxidative stress in the populace of tribal Palghar region of Western Maharashtra. SUBJECTS AND METHODS: Patients with SCA (n = 250) and age- and sex-matched healthy persons (n = 250) as controls from the primary health center of Palghar were included in this study. Informed written consent was obtained from all the participants. RESULTS: Activities of enzymatic antioxidants such as glutathione peroxidase, glutathione-S-trasferase, catalase, and level of nonenzymatic antioxidants such as glutathione, Vitamin E and C decreased significantly in SCA participants when compared to controls. The level of lipid peroxides and activity of superoxide dismutase increased significantly above normal in SCA participants. SCA is characterized by the increased generation of reactive-oxygen species resulting in oxidative damage of various cell types, including erythrocytes and this chronically elevated oxidative stress in SCD might play a significant role in the increased autoxidation of Sickle hemoglobin (HbS), increased intravascular hemolysis, ischemia-reperfusion injury, and chronic inflammatory complications. CONCLUSION: The present study indicates that oxidative stress can be considered one of the prognostic markers to evaluate the clinical severity of the SCA participants.

The Role of RBC Oxidative Stress in Sickle Cell Disease: From the Molecular Basis to Pathologic Implications.

Sickle cell disease (SCD) is an inherited monogenic disorder and the most common severe hemoglobinopathy in the world. SCD is characterized by a point mutation in the β-globin gene, which results in hemoglobin (Hb) S production, leading to a variety of mechanistic and phenotypic changes within the sickle red blood cell (RBC). In SCD, the sickle RBCs are the root cause of the disease and they are a primary source of oxidative stress since sickle RBC redox state is compromised due to an imbalance between prooxidants and antioxidants. This imbalance in redox state is a result of a continuous production of reactive oxygen species (ROS) within the sickle RBC caused by the constant endogenous Hb autoxidation and NADPH oxidase activation, as well as by a deficiency in the antioxidant defense system. Accumulation of non-neutralized ROS within the sickle RBCs affects RBC membrane structure and function, leading to membrane integrity deficiency, low deformability, phosphatidylserine exposure, and release of micro-vesicles. These oxidative stress-associated RBC phenotypic modifications consequently evoke a myriad of physiological changes involved in multi-system manifestations. Thus, RBC oxidative stress in SCD can ultimately instigate major processes involved in organ damage. The critical role of the sickle RBC ROS production and its regulation in SCD pathophysiology are discussed here.

The Red Blood Cell-Inflammation Vicious Circle in Sickle Cell Disease.

Sickle cell disease (SCD) is a genetic disease caused by a single mutation in the β-globin gene, leading to the production of an abnormal hemoglobin called hemoglobin S (HbS), which polymerizes under deoxygenation, and induces the sickling of red blood cells (RBCs). Sickled RBCs are very fragile and rigid, and patients consequently become anemic and develop frequent and recurrent vaso-occlusive crises. However, it is now evident that SCD is not only a RBC rheological disease. Accumulating evidence shows that SCD is also characterized by the presence of chronic inflammation and oxidative stress, participating in the development of chronic vasculopathy and several chronic complications. The accumulation of hemoglobin and heme in the plasma, as a consequence of enhanced intravascular hemolysis, decreases nitric oxide bioavailability and enhances the production of reactive oxygen species (ROS). Heme and hemoglobin also represent erythrocytic danger-associated molecular pattern molecules (eDAMPs), which may activate endothelial inflammation through TLR-4 signaling and promote the development of complications, such as acute chest syndrome. It is also suspected that heme may activate the innate immune complement system and stimulate neutrophils to release neutrophil extracellular traps. A large amount of microparticles (MPs) from various cellular origins (platelets, RBCs, white blood cells, endothelial cells) is also released into the plasma of SCD patients and participate in the inflammation and oxidative stress in SCD. In turn, this pro-inflammatory and oxidative stress environment further alters the RBC properties. Increased pro-inflammatory cytokine concentrations promote the activation of RBC NADPH oxidase and, thus, raise the production of intra-erythrocyte ROS. Such enhanced oxidative stress causes deleterious damage to the RBC membrane and further alters the deformability of the cells, modifying their aggregation properties. These RBC rheological alterations have been shown to be associated to specific SCD complications, such as leg ulcers, priapism, and glomerulopathy. Moreover, RBCs positive for the Duffy antigen receptor for chemokines may be very sensitive to various inflammatory molecules that promote RBC dehydration and increase RBC adhesiveness to the vascular wall. In summary, SCD is characterized by a vicious circle between abnormal RBC rheology and inflammation, which modulates the clinical severity of patients.

Decreased erythrocyte binding of Siglec-9 increases neutrophil activation in sickle cell disease

Oxidative stress and inflammation promote vaso-occlusion in sickle cell disease (SCD). CD33-related Sialic acid-binding immunoglobulin-type lectins (CD33rSiglecs) are cell surface proteins that recognize sialic acids inhibit innate immune cell functions. We have shown that Siglec-9 on human neutrophils interact with erythrocyte sialic acids (prominently glycophorin-A (GYPA) to suppress neutrophil reactive oxygen species (ROS). We hypothesized that altered sickle erythrocyte membrane sialic acid leads to decreased Siglec-9 binding capability, and thus a decreased neutrophil oxidative burst. SS erythrocytes express significantly more sialic acid than AA erythrocytes (p = 0.02). SS erythrocytes displayed significantly less Siglec-9-Fc binding 39% ± 11 (mean ± SEM) compared to AA erythrocytes 78% ± 5 (p = 0.009). Treatment of AA erythrocytes with sialidase to remove sialic acid decreased binding to 3% ± 7.9 (p ≤ 0.001). When freshly isolated neutrophils were incubated with AA erythrocytes, neutrophils achieved 16% ± 6 of the oxidative burst exhibited by a stimulated neutrophil without erythrocytes. In contrast, neutrophils incubated with SS erythrocytes achieved 47% ± 6 of the oxidative burst (AA versus SS, p = 0.03). Stimulated neutrophils incubated with AA erythrocytes showed minimal NET formation while with SS erythrocytes NETs increased. SS erythrocytes are deficient in binding to neutrophil Siglec-9 which may contribute to the increased oxidative stress in SCD.

Decreased Activity and Stability of Pyruvate Kinase in Hereditary Hemolytic Anemia: A Potential Target for Therapy By AG-348 (Mitapivat), an Allosteric Activator of Red Blood Cell Pyruvate Kinase

Background: Reactive oxygen species (ROS) play an important role in the complex and multifactorial pathophysiology of hereditary hemolytic anemia like sickle cell disease (SCD), β-thalassemia and hereditary xerocytosis (HX). Increased intracellular levels of oxidative stress disrupt normal cell functioning and may contribute to premature red blood cell (RBC) clearance from the circulation. Pyruvate kinase (PK) is a key regulatory enzyme of glycolysis, the cell's main source of energy. Because PK is very sensitive to redox balance we hypothesized that increased levels of oxidative stress in SCD, β-thalassemia and HX impairs proper enzyme function, thereby compromizing RBC energy metabolism. This may contribute to disease pathophysiology. Aims: To investigate if secondary deficiency of PK is common in SCD, thalassemia, and HX, and to investigate if PK in these disorders is able to respond to treatment with the allosteric PK activator AG-348 (mitapivat). Methods: Enzymatic activities of red cell PK and hexokinase (HK) were measured together with PK-thermostability in order to assess relative PK activity and enzyme stability. Purified RBCs were incubated with AG-348 (3.33μM) for 24 hours after which PK activity and ATP response was measured. RBCs of SCD patients were also analyzed with the oxygenscan, a newly developed method that characterizes individual sickling behavior by oxygen gradient ektacytometry (Rab et al, Am J Hematol, 2019). Individual tendency to sickle is reflected by Point-of-Sickling (PoS) that indicates the specific pO2 at which RBCs start to sickle during deoxygenation under shear stress. Results: Thirty-eight patients and 21 healthy controls (HC) were included. The patient cohort consisted of patients homozygous for HbS (HbSS, n=26), patients compound heterozygous for HbS and HbC (HbSC, n=4), β-thalassemia major (regularly transfused, n=3), and hereditary xerocytosis (n=5). Patients showed reticulocytosis and, in line with this, a concomitant increase in HK activity. In contrast however, relative PK activity was decreased significantly compared to HK in HbSS, β-thalassemia and HX patients, but not in HbSC patients (Figure 1A). PK thermostability was significantly decreased compared to healthy controls in HbSS patients and patients with HX (Figure 1B). In HbSC and β-thalassemia patients, PK-thermostability was comparable to HC. PK thermostability strongly correlated with absolute reticulocyte count (ARC), indicating that patients displaying the highest degree of PK instability had the highest reticulocyte count (Figure 1C). This suggests that in general, a higher degree of PK instability is associated with more severe anemia due to a high hemolytic rate. In SCD patients, PK-thermostability inversely correlated with PoS, indicating that decreased PK stability is associated with sickling at higher pO2 (r=-0.646, p<0.001, Figure 1F). When purified RBCs were incubated with 3.33μM of the allosteric PK-activator AG-348, an increase in PK activity was seen in all patients and HCs, with a mean increase of 122% in HbSS (range 111-139%, n=6), 137% in β-thalassemia (n=1), 163% in HX (range 152-174%, n=2) and 143% in HC (range 113-173%, n=9, Figure 1E). Accordingly, ATP-levels increased in all patients and HCs, with a mean increase of 133% in HbSS (range 125-141%, n=5), 144% patient with β-thalassemia (n=1), 121% in HX (range 112-129, n=3), and 132% in HCs (range 101-149%, n=9, Figure 1E). Conclusion: PK enzyme activity and stability is compromised in patients with various forms of hereditary hemolytic anemia. This implies that PK stability and, hence, compromised red cell metabolism could contribute to the complex pathophysiology of these diseases. In SCD patients, reduced PK-thermostability is associated with higher PoS, which we previously have shown to be associated with more severe disease (Rab et al, Am J Hematol, 2019, ASH 2019 abstract ID128870). This is confirmed by the correlation of decreased PK-thermostability with increased reticulocyte count as presented in this study. Current studies are in progress to further substantiate the underlying mechanism(s) involved, and to investigate whether AG-348 may ameliorate clinical features such as hemolysis, sickling tendency and iron overload. Disclosures Rab: RR Mechatronics: Research Funding. Bos:RR Mechatronics: Research Funding. Kosinski:Agios Pharmaceuticals, Inc: Employment, Other: Stakeholder. Kung:Agios Pharmaceuticals, Inc: Employment, Other: Stakeholder. van Beers:Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding. van Wijk:Agios Pharmaceuticals: Consultancy, Research Funding; RR Mechatronics: Research Funding.

Severe oxidative stress in sickle cell disease patients with uncomplicated Plasmodium falciparum malaria in Kampala, Uganda

BackgroundOxidative stress plays a vital role in the pathogenesis of both Sickle Cell Disease (SCD) and Plasmodium falciparum malaria. However, there are limited studies on the effect of P. falciparum malaria infection on oxidative stress in SCD patients.MethodsA cross-sectional study was undertaken to compare levels of biomarkers of oxidative stress in isolates from SCD patients with uncomplicated P.falciparum malaria. The biomarkers namely: malondialdehyde (MDA), reduced glutathione (GSH), catalase (CAT) and glutathione peroxidase (GPx) were determined in plasma samples from SCD malaria positive, malaria positive, SCD malaria negative and healthy control participants. The genetic diversity of P.falciparum was determined by nested polymerase chain reaction of merozoite surface protein-2 (MSP-2) gene.ResultsOut of 207 participants, 54 (26%) were SCD malaria positive, 51 (24%) malaria positive, 51 (24%) SCD controls and 51 (24%) healthy control individuals. The mean concentration of MDA was significantly higher in SCD malaria positive than SCD controls (P < 0.0001). In contrast, the mean concentration of GSH (P < 0.0001) and GPx (P < 0.0001) were significantly lower in SCD malaria than SCD controls. Although not significantly different, the mean concentration of MDA was higher (P = 0.0478), but the geometric mean parasite density (P = 0.2430) and multiplicity of infection (P = 0.3478) were lower in SCD malaria samples than in malaria samples. The most prevalent MSP2 allelic family was IC3D7 in SCD malaria (72%) and Malaria (76%) samples. The biomarkers of oxidative stress were not significantly different between IC3D7 and FC27 allelic families of MSP2.ConclusionWe identified severe oxidative stress in Sickle cell disease patients with uncomplicated P.falciparum malaria.

A Pilot Study of High-Dose N-Acetylcysteine Infusion in Patients with Sickle Cell Disease

▪We have completed a clinical trial evaluating the safety of N-acetylcysteine (NAC) infusion in adult patients with sickle cell disease (SCD) at disease baseline (NCT01800526). This trial was inspired by the hypothesis that von Willebrand factor (VWF) has an important role in the pathophysiology of vaso-occlusion and hemolysis in SCD and that NAC can reduce signs and symptoms of the disease by reducing the activity of VWF. A secondary hypothesis was that NAC could also act by reversing or preventing some of the oxidative changes associated with SCD.VWF is stored in the Weibel-Palade bodies of endothelial cells and α-granules of platelets and released from endothelial cells upon activation and mediates the attachment of platelets to the vessel wall, and secondarily, of erythrocytes and leukocytes. We previously showed that VWF levels and adhesive activity in SCD plasma were elevated and that total active VWF correlated with the extent of hemolysis (determined by plasma LDH levels), suggesting that VWF participates in the pathophysiology of SCD (Chen et al, Blood. 2011; 117:3680‐3). In another study, we showed that NAC was able to reduce VWF multimer size and platelet-binding activity ex vivo in human plasma and in vivo in mice (Chen et al, JCI, 2011, 121:522).In this clinical trial, we enrolled 5 subjects at disease baseline, 4 with SCD and 1 with sickle trait. All subjects received two high-dose NAC infusions: initially 150 mg/kg and 4 weeks later 300 mg/kg. NAC infusion was administered in subjects 1 and 2 as a bolus infusion of half of the dose in the 1sthour and the remaining half in 7 hr. Subjects 3-5 received NAC as constant infusion for 8 hr. Blood was collected immediately before infusion, during infusion (at 1 and 4 hr), immediately after infusion (8 hr), and 24 and 72 hr after infusion. We examined blood for red blood cell (RBC) fragments, dense cells, platelet activation status, platelet-monocyte complexes and the concentrations of reduced and oxidized glutathione. We also examined plasma for VWF concentration and multimer distribution, ADAMTS13 antigen and activity, and evaluated plasma redox status by measuring the concentrations of NAC and cysteine in their reduced, oxidized, and mixed disulfide forms, as well as the concentration of protein-bound cysteine. The small molecule thiols were measured by mass spectrometry.Results:1) All subjects tolerated NAC infusion well, except that subjects 1 and 2 experienced pruritus during the bolus infusion at the 300 mg/kg dose. We therefore changed the protocol to deliver the drug over 8 hr by continuous infusion for subjects 3 through 5. 2) During NAC infusion, the percentage of dense cells and concentration of RBC fragments decreased rapidly (average decrease: low dose 44%; high dose 31%) and the change persisted up to 72 hr. 3) Platelet activation also decreased, as determined by reduced P-selectin expression (low dose, 7.7 ± 0.7% to 5.0 ± 1.9%; high dose, 17.8 ± 0.07% to 7.3% ± 0.4), PAC-1 binding (evaluates activated αΙΙbβ3), and formation of platelet-monocyte complexes (monocytes with attached platelets: low dose, 52 ± 16% to 27 ± 12%; high dose, 77 ± 12% to 50 ± 17%). 4) The highest molecular weight VWF multimers transiently disappeared during the NAC infusion and recovered by 24 hr. There were no appreciable changes in VWF antigen or ADAMTS13 antigen or activity with NAC infusion. 5) With NAC infusion, the concentrations of Cys and NAC increased in plasma, both total and reduced forms, with a concomitant decrease in protein-bound Cys in the one patient studied (free thiol Cys increased 23 fold with the high dose infusion at 8 hr; protein-bound Cys decreased 87%). Similarly, the concentrations of reduced and oxidized glutathione increased in whole blood.Summary: NAC infusion in SCD patients at disease baseline appears safe and is well tolerated. NAC infusion decreases RBC fragments, dense cells, and the size of large VWF multimers, and inhibits platelet activation and formation of platelet-monocyte complexes. In addition, NAC infusion increases the total and free-thiol concentrations of cysteine and glutathione in blood, relieving oxidative stress in SCD. Our data suggest that NAC may be of benefit for SCD patients during vaso-occlusive crisis through a variety of mechanisms, which include improvements in red cell parameters, decreased platelet adhesion, reduced adhesive activity of VWF, and protection against oxidative damage. DisclosuresNo relevant conflicts of interest to declare.

N-Acetylcysteine in Patients with Sickle Cell Disease: A Randomized Controlled Trial

Patients with sickle cell disease (SCD) suffer from frequent and severe episodes of pain that are associated with hospitalizations, impaired quality of life and increased mortality. Current treatment options are scarce. Oxidative stress appears to play a pivotal role in the pathophysiology of SCD. Pilot studies have demonstrated that administration of the antioxidant N-acetylcysteine (NAC) effectively reduces markers of oxidative stress in SCD and may decrease the hospitalization rate for painful crises. NAC is a safe, inexpensive and well-tolerated drug that has been used for years for various indications. The primary aim of this study was to evaluate the effect of NAC on the frequency of daily pain in patients with SCD.We conducted a randomized (1:1), double-blind, placebo-controlled, parallel-group trial at 11 sites across The Netherlands, Belgium and the United Kingdom. Patients were eligible for participation if they were ≥12 years of age, had either HbSS, HbSC, HbSβ°- or HbSβ+-thalassemia with a history of at least 1 painful crisis per year over the 3 years prior to enrolment. At randomization patients were assigned to receive either oral NAC 600 mg twice daily or placebo for a total duration of 6 months. Patients had monthly checkups during the study. The primary endpoint was the rate of SCD related pain days per patient year, assessed daily with the use of pain diaries. Secondary endpoints included the rate of days with painful crises, admission days, hospitalizations and days with home analgesic use, the severity of pain, the time to first painful crisis and hospitalization, the number of adverse events and the effect on quality of life and various blood markers. The primary intention-to-treat analysis of this study was limited to patients with a minimal completed diary observation time of 110 days. Sensitivity analyses were done in both all randomized patients as well as a subset with ≥80 days of observation time. Lastly, an additional per protocol analysis was performed on patients with adequate adherence (≥80% of tablets used) and ≥110 days of completed diary observation time.A total of 96 patients were randomized of which 67 patients met the minimum observation time of 110 days (27 in the NAC and 40 in the placebo arm). Inclusions were stopped before reaching the estimated sample size of 116 patients due to imposed time restrictions by the main funder of this study. Groups were well balanced for baseline characteristics. The proportion of patients adherent to the assigned study medication regimen was low in both treatment groups (53% in the NAC and 50% in the placebo arm). The rate of SCD related pain days per patient year was 61.4 in the placebo group and 61.6 in the NAC group (rate ratio 0.98; 95% confidence interval [CI] 0.54-1.78; P=.98). Moreover, there were no significant differences in the secondary endpoints between groups. These findings were consistent in the sensitivity analyses. In the total study population, more patients reported gastro-intestinal events in the NAC group (42%) as compared to the placebo group (15%, P<0.01). In addition, we performed a per protocol analysis in the patients with adequate adherence (n=27), demonstrating a significantly lower rate of painful crisis days in the NAC group as compared to the placebo group, respectively 20.5 versus 40.1 events per patient year (rate ratio 0.50; CI 0.27-0.94; P=.03). Event rates of pain days (rate ratio 0.85; CI 0.33-2.20), admission days (rate ratio 0.33; CI 0.08-1.34) and hospitalizations per patient year (rate ratio 0.65; CI 0.19-2.15) appeared to be lower in the NAC group, but did not reach statistical significance. Moreover, NAC seemed to decrease the risk of a first hospitalization for painful crisis (hazard ratio 0.56; CI 0.23-1.39).In conclusion, treatment with oral NAC did not provide any clinical benefit over placebo in this phase 3 study, possibly due to low compliance rates. Therefore, a potential effect of NAC in SCD cannot fully be excluded based on these results. In an adherent subset of patients we did observe a reduction of days with painful crises and a trend to reduction in other pain related endpoints in the NAC treatment arm. To corroborate these findings additional blood sample analysis of pathophysiological markers is currently being performed. This may further elucidate on the dose-effect relation of NAC in SCD. DisclosuresFijnvandraat:Bayer: Research Funding; CSL Behring: Research Funding.

Blood Antioxidant Defenses and Sickle Cell Disease: Analyzing Cytochrome b Reductase 1 Protein Levels and Structure in Erythrocyte Membranes

Introduction: The pathology of sickle cell disease (SCD) includes ischemia / reperfusion events in the vasculature, compromised blood antioxidant defenses and elevated risk of cardiovascular complications. Vitamin C (ascorbate) is a major blood antioxidant; ascorbate inside red blood cells (RBC) furnishes an electron (e-) to recycle plasma ascorbate to its active, reduced state (Fig. 1). A key step, transmembrane e- transfer, involves cytochrome b reductase 1 (CYBRD1). Thus, a deficiency or defect in red cell CYBRD1 could plausibly increase oxidative stress and interconnected inflammatory processes, contributing to cardiovascular complications in SCD patients. Testing for CYBRD1 deficiency requires a quantitative assay for the protein in RBC, so we developed a suitable assay and used it to screen a small number of SCD patients and healthy individuals.Materials and methods: Blood samples were collected from steady state adult SCD patients, healthy African American adults, and steady state pediatric SCD patients before and 2-5 months on hydroxyurea (HU) therapy. Protocols were approved by institutional review.The CYBRD1 assay workflow is shown in Fig. 2. RBC membrane proteins were separated on two SDS-PAGE gels, along with a mixed standard (1.0 µg bovine serum albumin (BSA) + 1.9 ng of His6-tagged recombinant human CYBRD1 (rCYBRD1; PMID 21501687)). One gel was Coomassie-stained for quantitation of total membrane protein minus membrane-bound hemoglobin (Hb) (standard: BSA). Proteins in the other gel were transferred to a nitrocellulose filter, probed with CYBRD1 antibody (GeneTex #104793) and visualized with secondary antibody and chromogenic substrate for quantitation of CYBRD1 (standard: rCYBRD1), which was normalized to membrane protein. Gels, blots and calibrated density step tablets were scanned (transmittance mode) with an Epson V550; digitized images were analyzed using ImageJ. Two independent samples and paired t tests were used to compare between- and within-group mean differences in RBC CYBRD1 levels, respectively.Results: The Coomassie-stained gels exhibited the usual profile of RBC membrane proteins (Fig. 2). In the immunoblots, the rCYBRD1 standard showed the expected narrow monomer band at ~30 kDa and faint dimer band at ~60 kDa (Fig. 2). However, RBC membrane samples revealed several CYBRD1 monomer bands, indicating the presence of previously unsuspected isoforms of CYBRD1. Further, the CYBRD1 isoform pattern in RBC from healthy controls was quite different from those seen in SCD patients. The functional impact of these differences remains to be investigated.Assay of progressively diluted control RBC membranes produced a linear response for CYBRD1 from 0.7-7.2 ng (r2 = 0.975) and for total protein from 1 to 10 µg (r2 = 0.993). The sub-nanogram detection of CYBRD1 protein makes the assay usable with as little as 0.25 ml of blood. The coefficient of variation was ~15%, quite reasonable precision for quantitative immunoblotting.CYBRD1 levels in RBC from healthy African American adults averaged 0.68 ± 0.13 ng/µg membrane protein (N1 = 10; Fig. 3). CYBRD1 levels in RBC of adult SCD patients averaged 0.35 ± 0.14 ng/µg membrane protein (N2=7). This difference in means was highly statistically significant by two-tailed t-test (p < 0.0002), supporting our hypothesis that a deficiency in RBC CYBRD1 contributes to elevated oxidative stress in SCD patients.RBC CYBRD1 levels in pediatric SCD patients not on HU therapy averaged 0.27 ± 0.14 ng/µg protein (N3=12; Fig. 3). Pediatric patients on HU therapy averaged 0.41 ± 0.18 ng CYBRD1/µg membrane protein (N4=7). For the five pediatric patients examined both before and during HU therapy there was a marginally significant increased mean CYBRD1 level on HU (p < 0.099 by paired, two-tailed t test).Conclusions: Our newly developed assay allows quantitation of the RBC CYBRD1 protein level and analysis of the CYBRD1 isoform pattern using a small blood sample. The pilot screening results suggest that adult SCD patients have lower levels of CYBRD1 in their RBC, and a different pattern of CYBRD1 isoforms. Analysis of the CYBRD1 protein may be useful in assessing the state of individual SCD patients' blood antioxidant defenses and the antioxidant effects of HU. Thus, it will be worthwhile to examine the RBC CYBRD1 protein and activity in larger numbers of patients and controls, and to explore the origins of any differences between the groups. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Triterpenoid inducers of Nrf2 signaling as potential therapeutic agents in sickle cell disease: a review.

Sickle cell disease (SCD) is an inherited disorder of hemoglobin in which the abnormal hemoglobin S polymerizes when deoxygenated. This polymerization of hemoglobin S not only results in hemolysis and vasoocclusion but also precipitates inflammation, oxidative stress and chronic organ dysfunction. Oxidative stress is increasingly recognized as an important intermediate in these pathophysiological processes and is therefore an important target for therapeutic intervention. The transcription factor nuclear erythroid derived-2 related factor 2 (Nrf2) controls the expression of anti-oxidant enzymes and is emerging as a protein whose function can be exploited with therapeutic intent. This review article is focused on triterpenoids that activate Nrf2, and their potential for reducing oxidative stress in SCD as an approach to prevent organ dysfunction associated with this disease. A brief overview of oxidative stress in the clinical context of SCD is accompanied by a discussion of several pathophysiological mechanisms contributing to oxidative stress. Finally, these mechanisms are then related to current management strategies in SCD that are either utilized currently or under evaluation. The article concludes with a perspective on the potential of the various therapeutic interventions to reduce oxidative stress and morbidity associated with SCD.

Sensitivity to cisplatin-induced mutations and elevated chromosomal aberrations in lymphocytes from sickle cell disease patients

Sickle cell disease (SCD) is an inherited disorder caused by a single nucleotide substitution in the beta-globin gene. The clinical heterogeneity observed in SCD patients has been attributed to environmental and genetic factors. The patients are subjected to increased oxidative stress, particularly during vaso-occlusive crises and acute chest pain. Another possible cause of oxidative stress in SCD is the high concentration of iron in the patients' plasma. The increase in oxidative stress could be a relevant risk factor for mutagenesis and carcinogenesis. Studies on the frequency of basal chromosomal aberrations in cultured lymphocytes from SCD patients have not been reported so far. In order to contribute to the understanding of the role of the different biomarkers and their relationship with the extremely variable clinical manifestation of SCD, we investigated the frequency of chromosome damage in peripheral lymphocytes from sickle cells patients and healthy controls. We found an increased frequency of chromosome damage and percentage of aberrant metaphases in these patients when compared with control subjects, even at basal values (p<0.05). In the cytogenetic sensitivity assay, the results showed that these patients presented a marked decrease in the mitotic index values compared with healthy controls. Cisplatin-induced chromosomal damage in lymphocytes from these patients was significantly higher than the frequency measured in healthy controls. The results obtained in the present study showed that more investigations are needed in order to elucidate the susceptibility to genomic instability of SCD patients.

Nutritional Deficiencies in Chronically Transfused Hemoglobinopathy Patients.

Vitamin and mineral deficiencies have been well described in non-transfused patients with sickle cell disease (SCD). Levels of biomarkers of oxidant damage are increased in both SCD and thalassemia major (TM), and levels of antioxidants have been shown to be lower in both diseases. Hypermetabolism, increased hemolysis and denaturation of unpaired globin chains contribute to oxidative stress in SCD and TM. Chronic transfusions, initially thought to be protective against oxidation by marrow suppression, may have the opposite effect due to the well-known propensity of iron to promote free radical generation. Methods: To test this hypothesis, we measured levels of vitamins A, B1, B6, C, D, E as well as selenium, zinc, copper and carnitine and compared them to liver iron concentrations in chronically transfused SCD and TM patients. All patients were required to fast overnight and to hold their iron chelator for 24 hours prior to blood draws. Samples were processed by routine clinical processing (Quest Nichols Institute, San Juan Capistrano, CA). Results were expressed as the percentage of patients outside the reference range for the referral laboratory. Twenty four patients with TM and 43 patients with SCD were studied.Abnormal Values In Transfused PatientsSC (%abn)TM (%abn)A73.752.4B138.537.5B634.234.8Folate28.637.5C56.766.7D2574.450.0D1, 2548.639.1Se67.575.0Zn24.38.3Carnitine (Free)22.241.7Results: The results in the table demonstrate that over half the patients were deficient in A, C, D and selenium. A third of the patients had low levels of B vitamins and folate (despite replacement doses in the latter), as well as carnitine. B12 levels(not shown) were normal. E levels (not shown) were low in only a few patients. Deficiency profiles were remarkably similar between the two diseases: only copper was significantly higher in the SCD patients (34.2% elevated vs 0% in TM). B6 and folate were weakly correlated with hepatic iron concentration (r2 = −0.12, −0.18). B6, folate, D25-OH, and D1,25 OH levels declined with age. These results demonstrate that chronically transfused patients have significant deficiencies in nutrients which are involved in buffering oxidant stress, regardless of their diagnosis. Severity of iron overload was not a strong predictor of these abnormalities; although the precise explanation is not known, chronic anemia may be the unifying factor. The magnitude of B1, B6, C and D deficiencies in some patients was well into the range known to cause serious complications. These combined abnormalities may contribute to the morbidity of chronically transfused patients with SCD and TM.

Protective effect of arginine on oxidative stress in transgenic sickle mouse models

Sickle cell disease (SCD) is characterized by reperfusion injury and chronic oxidative stress. Oxidative stress and hemolysis in SCD result in inactivation of nitric oxide (NO) and depleted arginine levels. We hypothesized that augmenting NO production by arginine supplementation will reduce oxidative stress in SCD. To this end, we measured the effect of arginine (5% in mouse chow) on NO metabolites (NOx), lipid peroxidation (LPO), and selected antioxidants in transgenic sickle mouse models. Untreated transgenic sickle (NY1DD) mice (expressing ∼ 75% β S-globin of all β-globins; mild pathology) and knockout sickle (BERK) mice (expressing exclusively hemoglobin S; severe pathology) showed reduced NOx levels and significant increases in the liver LPO compared with C57BL mice, with BERK mice showing maximal LPO increase in accordance with the disease severity. This was accompanied by reduced activity of antioxidants (glutathione, total superoxide dismutase, catalase, and glutathione peroxidase). However, GSH levels in BERK were higher than in NY1DD mice, indicating a protective response to greater oxidative stress. Importantly, dietary arginine significantly increased NOx levels, reduced LPO, and increased antioxidants in both sickle mouse models. In contrast, nitro-L-arginine methylester, a potent nonselective NOS inhibitor, worsened the oxidative stress in NY1DD mice. Thus, the attenuating effect of arginine on oxidative stress in SCD mice suggests its potential application in the management of this disease.