Assessment of oxidative stress in patients with sickle cell disease: The glutathione system and the oxidant–antioxidant status

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Abstract

Continuous reactive oxygen species (ROS) production in individuals with sickle cell disease (SCD) may alter their overall redox status and cause tissue damage. The aim of this study was to evaluate oxidative stress in patients with SCD using two new assays, FORT (free oxygen radical test) and FORD (free oxygen radical defense) along with assessment of glutathione system including superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GPx) activities, vitamins A, C and E, malondialdehyde (MDA), non-transferrin bound iron (NTBI) and nitric oxide (NO) concentrations.

A total of 40 patients with SCD and 25 apparently healthy volunteers (control group) were enrolled in the study. Components of glutathione system, vitamins A, C, and E, and malondialdehyde were determined with reverse-phase HPLC, non-transferrin bound iron (NTBI) was assessed with atomic absorption spectroscopy using graphite furnace, superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GPx) activities were determined spectrophotometrically in red cell lysates, nitric oxide (NO) was detected colorimetrically, while FORT and FORD using colorimetric assays, as two point-of-care tests. The findings revealed significant impairment of the glutathione system indicated by reduced GSHtotal (p < 0.00001), GSHreduced (p < 0.00001) and GSSG (p > 0.056) values of SCD patients compared to the control group. ROS expressed as FORT were significantly increased (p < 0.00001), while antioxidant defense expressed as FORD was significantly reduced (p < 0.02) in SCD group compared to the control group. Age and genotype of the patients as well as therapy of their disease appeared to play no role in their oxidative status.

Introduction

The polymerization of sickle hemoglobin is a remarkably dynamic event. When SS red cells are slowly or partially deoxygenated, a single nucleus of aggregated molecules of deoxygenated hemoglobin S is formed. This nucleation is followed by the growth and alignment of fibers, transforming the cell into a classic sickle shape. The distortion of the shape of the cell by projections of aligned hemoglobin S fibers has a critical role in perturbing the structure and function of the membrane in SS red cells, mediated in part by oxidant stress. Anything that retards the transit of SS red cells in the microcirculation can have a critical effect on the pathogenesis of vaso-occlusion in sickle cell disease. Accordingly, there has been considerable interest in studies of the interaction between SS red cells and vascular endothelium. Measurements performed under both static and dynamic conditions have demonstrated that SS red cells have a sticky surface and attach more readily than normal cells to monolayers of cultured endothelial cells. The degree of adherence is strongly correlated with the severity of the disease in patients with SS disease or other sickle genotypes [1].

The production of reactive oxygen species is a steady-state event in respiring cells. Red cell hemoglobin is a quantitatively significant source of superoxide generation in biological systems. There is an electron transfer in the bonding interaction between the heme and oxygen in oxygenated hemoglobin. When hemoglobin deoxygenates, the heme iron normally remains in the Fe(II) ferrous state. In this exchange, alterations wherein hemoglobin auto-oxidizes, result in the formation of methemoglobin and superoxide. There is a normal “physiologic” rate of red cell methemoglobin formation that provides a continual source of superoxide production that in turn generates hydrogen peroxide and oxygen as byproducts of dismutation. Compared with normal RBC, HbS RBC spontaneously generate significantly increased amounts of O2, H2O2, and radical dotOH. In hemoglobin preparations obtained from red cells of sickle cell patients (HbS), autoxidation of oxygenated hemoglobin is 1.7 times faster than oxygenated HbA and sickle red cells have been reported to generate ~ 2-fold greater extent of superoxide, hydrogen peroxide, hydroxyl radical and lipid oxidation products compared with HbA-containing red cells. Oxidative damage to hemoglobin has been shown to cause irreversible hemoglobin denaturation, precipitation and propensity for further autoxidation [2], [3], [4], [5], [6], [7].

An improved understanding of the abnormal oxidative processes that occur in sickle cell disease has led to new insights into the mechanism of action of some currently accepted therapies and suggest new therapies for this disease. Hydroxyurea, which is widely used in sickle cell disease, may have beneficial effects on vascular functions [2], [8], [9], [10].

Over the past decade, there has been substantial interest in oxidative stress and its potential role in the development of disease related pathophysiological complications in many diseases. “Oxidative stress” refers to an imbalance between antioxidant and oxidant-generating systems, a disruption in the cellular pro-oxidant antioxidant balance. As the balance shifts toward pro-oxidants, potential damage in the form of oxidized DNA, proteins and lipids can occur. An increase in oxidative stress can have a profound effect on lipoprotein modification, transcription and cell function and metabolism. Oxidative stress can arise via various mechanisms associated with excessive oxygen radical production [11]. Unbalanced production of reactive oxygen species (ROS) is thought to be related to the pathogenesis of several human diseases, such as ischemic stroke, atherosclerosis, cardiovascular disease, and neurodegenerative diseases. Several studies have shown that many aspects of the abnormalities in sickle cell disease result from the oxidative stress of RBCs, WBCs and endothelial cells [9], [12], [13]. Biological macromolecules can be damaged by oxidative insult, leading to oxidized products that act as biomarkers of oxidative stress status. Oxygen radicals may provoke oxidative stress that can induce lipid peroxidation. Lipid peroxidation can be defined as the oxidative deterioration of lipids containing two or more carbon–carbon double bonds. The prime targets of peroxidation by ROS are the polyunsaturated fatty acids (PUFA) in the membrane lipids. The propensity of PUFAs to undergo lipid oxidation (LPO) is due to the bis-allylic methylene hydrogens, which are more susceptible to hydrogen abstraction by oxidants than fully saturated lipids. Furthermore, the decomposition of these peroxidized lipids yields a variety of products, including lipid hydroperoxides (LHP) and malondialdehyde (MDA). Malondialdehyde, which is an intermediate product of lipid peroxidation, is elevated in thalassemia. Lipid peroxidation and MDA formation are detrimental to cellular integrity and function. The levels of these lipid peroxides indicate the extent of lipid peroxidation in general and serve as markers of cellular damages due to free radicals [13], [14], [15].

A different approach to evaluation of oxidative stress is the analysis of antioxidant concentrations. Antioxidant defense systems work cooperatively to alleviate the oxidative stress caused by enhanced free radical production. To protect against oxidative damage, the body has endogenous defense mechanisms that are supported by dietary antioxidants. The lipid-soluble vitamin E (tocopherols) antioxidants, including α- and γ-tocopherol, are an important front line defense. Other cellular antioxidants include ascorbic acid (vitamin C) and β-carotene (vitamin A). These antioxidants are better known as oxygen radical scavengers [4], [13]. A ROS attack can lead to a major depletion of antioxidants such as vitamin E, vitamin C, reduced glutathione (GSH), and urate [13], [16]. SCD patients have approximately a 40% reduction in plasma carotene level and a 30% reduction in vitamin E levels. Additionally, there is an inverse correlation between vitamin E levels and the percentage of irreversibly sickled RBCs [4], [16]. GSH can be oxidized, mainly to glutathione disulfide (GSSG), or can form glutathionylated proteins (PSSG) [17], [18]. The intracellular thiol reductant that directly scavenges reactive species and serves as a GPx (glutathione peroxidase) cofactor, GSH, contributes to the reduction of hydrogen peroxide, lipid peroxides and other oxidizing species, thus protecting red cells against oxidative damage [2], [19], [20], [21]. Blood measurement of both reduced glutathione (GSH) and glutathione disulfide (GSSG) in the blood has been considered a sensitive index of whole-body GSH status and a useful indicator of oxidative stress status in humans [22]. On the other hand, glutathione interacts with hemoglobin, leading to the formation of glutathionyl hemoglobin (G-Hb). This formation can be induced within erythrocytes, leading to a marked reduction in the propensity for sickling in the erythrocytes of individuals homozygous for HbS [17], [23]. Analytical methods based on spectrophotometry, HPLC, capillary electrophoresis, nuclear magnetic resonance, and mass spectrometry have been reported for the determination of glutathione in biological samples [24]. Enzymatic antioxidant defense systems include superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR). Antioxidant enzymes provide the first line of cellular defense against toxic free radicals. These enzymes react directly with oxygen free radicals to yield non-radical products [15].

There is great interest in the role of nitric oxide (oxidonitrogen(S) or NO S) in biology because it can be a signaling molecule, a toxin, a pro-oxidant, and a potential antioxidant. Nitric oxide (NOradical dot) is a free radical, an uncharged molecule with an unpaired electron and it plays multiple roles in both intracellular and extracellular signaling mechanisms. Nitric oxide can serve as a chain-terminating antioxidant by reacting with chain-carrying peroxyl radicals. This highly reactive, yet simple molecule can be produced in the body both nonenzymatically and enzymatically by the isoenzyme nitric oxide synthase (NOS) using l-arginine as a substrate. The family of NOS enzyme mediates the five-electron oxidation of l-arginine to l-citrulline producing NO. There are three isoforms of NOS: neuronal (NOS1, nNOS), inducible (NOS2, iNOS) and endothelial (NOS3, eNOS). Because SCD is associated with oxidative stress, increased expression of endothelial cell adhesion molecules and blood cell adhesion, which are characteristic features of an inflammatory response, several groups have evaluated the contribution of inducible NOS in this disorder. The role of endothelial NOS in SCD vasculopathy remains controversial. The most compelling evidence favoring eNOS comes from reports describing impaired endothelium-dependent vasodilation in SCD mice and exaggerated vasoconstriction of aortic rings derived from SCD mice following NOS inhibition. Given the capacity of all NOS isoforms to function as both superoxide and NO producers, neuronal NOS represents a potential, yet unexplored, source of oxidative and nitrosative stress in SCD [9]. Reaction of NOradical dot with oxygen or other free radicals generates reactive nitrogen species (RNS), which cause multiple biological effects. Cells within different tissues display varying responses to NOradical dot, which may relate to the presence of cellular antioxidants such as GSH, GPx and SOD. Thus, the biological outcome of the NO mediated effects is complex and depends on the internal and external environments of the target and generation sites of the cells as well as the concentration of NOradical dot generated [15]. Several studies have suggested that alterations in NO metabolism occur during SCD, particularly during vaso-occlusive crisis or acute chest syndrome. Serum levels of nitrite NO2 and nitrate NO3 are used to estimate the level of NOradical dot formation, since NOradical dot is highly unstable and has a very short half life [4], [9], [14], [15], [25].

A significant biomarker of tissue injury or inflammation in SCD revealing the iron burden is non-transferrin bound iron (NTBI). NTBI measurement estimates the possible availability of free iron and therefore the iron stress during transfusions [13].

In this study we assessed oxidative stress by means of the glutathione system impairment, antioxidant enzymes and lipid peroxidation products in patients with HbS disease syndromes and also using new methodologies of oxidant–antioxidant status.

Section snippets

Patients

A total of 40 patients with SCD and 25 apparently healthy volunteers (control group) were enrolled in the present study. In the SCD group sixteen (40%) individuals were male and twenty four (60%) were female, while eight (32%) were male and seventeen (68%) were female in the control group. The SCD subjects ranged in age from 5 to 63 years (4 children) and the control group from 4 to 45 years (4 children).

The SCD sample comprised 30 βs/βthal and 10 βs/βs subjects. 21 of them (17 βs/βthal and 4 βs/β

Determination of hematological parameters

Hematological parameters and red blood cell indices were measured using a Siemens-ADVIA 120 whole blood auto-analyzer (Siemens Healthcare Diagnostics, Tarrytown, NY, USA). Hemoglobins were characterized and quantitated using weak cation-exchange high-pressure liquid-chromatography (CE-HPLC) with the Bio-Rad Variant Hemoglobin Testing system and the β-Thalassemia Short Program (Bio-Rad Laboratories, Hercules, CA, USA). Ferritin levels were measured in duplicate using a two site chemiluminescence

Results

The hematological parameters determined from SCD patients and healthy volunteers are presented as mean ± SD in Table 1.

NTBI values as well as the lipid peroxidation marker MDA were significantly higher in patients with SCD compared to controls (p < 0.001 and p < 0.001, respectively) (Table 2). Impairment of the glutathione system was indicated by reduced GSHtotal levels in patients with SCD compared to controls (748.9 ± 135.9 vs. 1125 ± 180.0 μmol/L, respectively, p < 0.001). Similarly, GSHreduced levels

Discussion

HbS is autoxidized 1.7 times more than HbA and precipitates, and SCD-RBCs undergo oxidative stress because they produce greater quantities of O2, H2O2 and radical dotOH than normal RBCs. In the present study, we have evaluated the oxidative status of red blood cells by investigating the glutathione system. Reduced glutathione (GSH) is an important scavenger of free radicals and a potent endogenous antioxidant, which helps to protect cells from oxidative injury. Besides its role in the maintenance of the

Acknowledgments

Grant/funding support: Funding was received from Athens University Medical School and Menarini Hellas SA (to I.P. and E.K.). The funding sources played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

References (32)

  • C.K. Lii et al.

    Protein thiol modifications of human red blood cells treated with t-butyl hydroperoxide

    B B A

    (1997)
  • K. Wood et al.

    Sickle cell disease vasculopathy: a state of nitric oxide resistance

    Free Radic. Biol. Med.

    (2008)
  • F. Epstein

    Pathogenesis and treatment of sickle cell disease

    N. Engl. J. Med.

    (1997)
  • M. Aslan et al.

    Reactive species in sickle cell disease

    Ann. N.Y. Acad. Sci.

    (2000)
  • E. Klings et al.

    Role of free radicals in the pathogenesis of acute chest syndrome in sickle cell disease

    Respir. Res.

    (2001)
  • R. Hebbel et al.

    Accelerated autoxidation and heme loss due to instability of sickle hemoglobin

    Proc. Natl Acad. Sci. USA

    (1988)
  • Cited by (0)

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