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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 7  |  Issue : 1  |  Page : 99-103

Oxidative stress in sickle cell anemia can be a prognostic marker for disease severity: A case − control study in the western region population of Maharashtra


1 Department of School of Biotechnology and Bioinformatics, D Y Patil Deemed to be University, Navi Mumbai, India
2 Department of Biochemistry, Grant Government Medical College and Sir JJ Group of Hospitals, Mumbai, Maharashtra, India

Date of Submission02-Jun-2021
Date of Acceptance26-Oct-2021
Date of Web Publication27-Jun-2022

Correspondence Address:
Dr. Deepa Garg
Department of School of Biotechnology and Bioinformatics, D Y Patil Deemed to be University, Sector 15, CBD Belapur, Navi Mumbai - 400 614, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bjhs.bjhs_55_21

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  Abstract 


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.

Keywords: Antioxidant, oxidative stress, reactive oxygen species, sickle cell anemia


How to cite this article:
Satam NN, Patil VW, Marar T, Garg D. Oxidative stress in sickle cell anemia can be a prognostic marker for disease severity: A case − control study in the western region population of Maharashtra. BLDE Univ J Health Sci 2022;7:99-103

How to cite this URL:
Satam NN, Patil VW, Marar T, Garg D. Oxidative stress in sickle cell anemia can be a prognostic marker for disease severity: A case − control study in the western region population of Maharashtra. BLDE Univ J Health Sci [serial online] 2022 [cited 2022 Oct 3];7:99-103. Available from: https://www.bldeujournalhs.in/text.asp?2022/7/1/99/348274



Sickle cell disease (SCD) is a genetic disorder the molecular basis being substitution of valine in the position of glutamic acid at the 6th position of the hemoglobin beta chain.[1] It shows dominant inheritance from parents, whereas sickle cell trait or heterozygous (HbAS) result from inheritance of sickle gene from one parents and normal gene from other parent.[2] It is a disease affecting approximately 5% of the world's population.

The predominant pathology of sickle cell anemia (SCA) is sickling of red blood cells (RBCs) due to the aberrant polymerization of mutant deoxygenated hemoglobin. Sickle-shaped RBC decreases the elasticity of RBC. During decreased oxygenation or higher oxygen demand sickling becomes rapid, further causing chronic hemolytic anemia, vasoocclusive crisis, and intravascular hemolysis.[3]

For efficient oxygen transport, RBCs need to maintain its flexible shapes, thus establishing the need to reduce polymerization or gelling, mostly during deoxygenation. This eventually distorts the RBCs leading to the formation of irreversible sickled cells. In SCD, impaired hemoglobin stability makes RBC vulnerable to severe oxidative stress which overcomes the antioxidant defense.[4] Hence, SCD is emerging toward a new concept of oxidative stress in the pathogenesis, which may contribute toward irreversible sickle cell-mediated membrane change, leading to increase in the prevalence of hemolytic episodes.[5]

SCA leads to decrease in hemoglobin level and reticulocyte proliferation which may lead to nexus among oxidative stress and hematological response in SCD. There are many studies on SCA with respect to oxidative stress and antioxidant mechanisms.[6] However, participants from the Western zone of Maharashtra specifically Palghar region have got scant attention; therefore, the pathophysiological conditions of this population are still unknown. Hence, this study aims to examine if oxidative stress can be a marker for understanding the pathophysiology of these participants.


  Subjects and Methods Top


In the present study, 250 SCA participants, who were attending the Primary Health Center at Palghar from August 2015 to August 2017 were included. Out of the 250, 49 participants were with SCD (homozygous recessive) and 201 were sickle cell trait or heterozygous in steady state, as diagnosed by the high-performance liquid chromatography (HPLC). It included 124 males and 126 females aged between 5 and 55 years. Patients with vaso-occlusive crisis and those on treatment with hydroxyurea or any vitamin supplementation were excluded.

Two hundred and fifty healthy, age-matched individuals with no relevant history of illness and normal Hb pattern confirmed with HPLC were considered as control group, of which 114 were male and 136 were female.

The demographics data and clinical examination reports were recorded from all participants, and informed consents was obtained before enrollment in the study. The ethical committee of Grant Government Medical College and J. J. Hospitals approved this study protocol.

Materials and Methods

Sample collection

Approximately 5 ml of venous blood sample was collected from all participants of SCD and control groups. Half was collected in ethylenediaminetetraacetic acid vial and divided into two parts, of which one part was used to perform RBC morphology and the rest for preparation of hemolysate for hemoglobin HPLC, and remaining blood samples were used to separate serum for the estimation of enzymatic and nonenzymatic parameters. The level of lipid peroxides was assayed by the method of Yagi.[7] Superoxide dismutase (SOD) activity was assayed by the method of Kakkar et al.[8] The activity of catalase (CAT) was assayed by the method of Sinha.[9] The activity of glutathione peroxidase (GPx) was determined the method of Rotruck et al. with slight modifications.[10] The activity of glutathione S transferase (GST) was assayed by the method of Habig et al.[11] The reduced glutathione (GSH) level was determined by the method of Beutler et al.[12] The level of Vitamin C was estimated by the method of Omaye et al.[13] Vitamin E content was estimated by the method of Palan et al.[14] Automated hematology analyzer was used to study the morphology of RBC's.[15]

Statistical analysis

Comparative analysis for the significant difference in the means of various parameters between case and respective control groups was performed by the Student's t-test and analysis of variance. Pearson correlation analysis was carried out to detect any association between individual oxidative stress and hematological parameters. Statistical analysis of the data was carried out by statistical software.


  Results Top


Percent hemoglobin (Hb %) [Table 1] was found statistically significantly (P < 0.001) decreased in SCD participants compared to control but heterozygotes (AS) showed a lower reduction in Hb levels. All participants were grouped into anemia classes based on hemoglobin level according to the WHO guidelines.[16] Levels of lipid peroxide and activity of SOD showed an increase in the study participants, but CAT and GST activity showed a decrease in SCA participants when compared to controls. Level of nonenzymic antioxidants such as GSH, Vitamin E, and Vitamin C also reduced significantly in SCA participants when compared to control. The reduction in GSH and Vitamin E was extremely statistically significant (P < 0.001). All other enzymatic and nonenzymatic antioxidants showed a decrease in SCA participants when compared to control [Table 1].
Table 1: Enzymatic and nonenzymatic antioxidant parameters and hemoglobin in homozygous and heterozygous sickle cell disease participants compared to the respective control levels

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In heterozygous SCA participants, oxidative stress parameters showed significant (P < 0.001) alterations, but their condition was better as compared to SCD participants. SOD activity was significantly increased as compared to control and CAT activity shows significant decrease as compared to control [Table 1]. Other enzymatic and nonenzymatic parameters the values were reduced than control. Even in hematological parameters, there were lower values than control. The correlation of serum level of malondialdhyde (MDA) with antioxidant levels (enzymatic and nonenzymatic antioxidant and SOD) performed in SCD patients [Table 2]; only Vitamin C, Vitamin E, and super oxide dismutase show significant results.
Table 2: Correlation of serum MDA with enzymatic and nonenzymatic antioxidant and superoxide dismutase levels in sickle cell disease (homozygous) participants

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  Discussion Top


Unlike earlier studies that measure antioxidants, the present study has given more focused on details analysis of enzymatic and nonenzymatic parameters. In accordance to known biology of SCD, the present study also shows approximately 50% reduction in the total antioxidant capacity of serum in SCD patients in the steady state compare with healthy Hb A control. Autooxidation is faster in Hb S-containing red cells. In SCD, the formation of superoxide, hydrogen peroxide radical is accelerated since HbS oxidizes at high rate than Hb A by “Haber-Weiss” reaction. H2O2 in turn produces hydroxyl radicals and damages red cell membrane, proteins and lipids by lipid and thiol oxidation.[17] Oxidative degradation of fatty acids of the cell membrane and archidonic acid leads to the production of MDA, F2-isoprostanes, and 4-hydroxynonenal. These can be measured as oxidative stress biomarkers in urine and blood, to indicate the degree of oxidative stress. A significant increased level of MDA and SOD indicates the high rate of free radical formation. Our results are in accordance with those reported by Prakash et al. in 2011[18] Many other studies have also reported increased levels of MDA in SCA.[17],[19],[20] Few studies have also reported increased level of MDA in homozygous as well as in heterozygous.[21] Enzymatic and nonenzymatic antioxidant parameters (GST, GPx, CAT, Vitamin C, and Vitamin E) have shown reduction as compared to control which can be attributed to its consumption or inactivation. This, therefore, places SCD patients at increased risk of oxidative stress and injury, even with sickle cell trait. Increased oxidative stress can be a factor causing progressive sickling process of erythrocyte, in addition to shortening of RBC life span and the formation of vaso-occlusion.[20],[22],[23]

The Vitamin E levels in SS and AS decreased significantly which indicate pronounced oxidative stress in homozygous as well as heterozygous sickle cell patients. A study published by Hoewitt reports significantly increased hydrogen peroxide induced hemolysis following a drop in the level of Vitamin E.[24] Vitamin E antioxidant activity is very effective even at high oxygen concentrations thus it is concentrated in the lipid structure which are exposed to higher partial pressure of oxygen like erythrocyte.[25] Some studies have also suggested that hemolysis can induce hydrogen peroxide increase, which can ultimately lead to a drop in level of Vitamin E.[20] In SCD participants, decreased Vitamin E level might also behave as a cause or an effect of increased hemolysis.

Another important antioxidant Vitamin C also decreased significantly in both SS and AS group when it compared with control. Vitamin C is directly accepting electrons from superoxide hydroxyl anion and from other lipid hydroxyl peroxides as it is a free radical scavenger.[20] Some studies have observed a similar trend of decrease in the levels of Vitamin C in SS patients when compared with AS and control group (P < 0.001).[23]

In SCA, an exhausted status of Vitamin C along with other antioxidant level may be explained as an attempt to annihilate increased free radicals. Being an aqueous phase, antioxidant Vitamin C has excellent protective role in the regeneration of reduced form of other powerful antioxidants such as GSH peroxide and Vitamin E and it also helps to inhibit free radical chain reactions.[23] In SCD due to hemolysis and rapid formation of free radical has decreased the Vitamin C level. The Vitamin C levels decrease and show a negative correlation with serum MDA level, as SCA (SS) patients are more prone to hemolysis and have a greater propensity for the formation of superoxide and hydrogen peroxide. CAT and GPx shows lower activity levels in SCD, as compare to Hb AA.

Activities of antioxidants, like Catalase, help to combat the damaging effects of Reactive oxygen species.[26] This study reports that levels of CAT are significantly lower in patients with SCD as compared to controls. This is in line with other studies[27],[28],[29] that also reported levels of antioxidant enzymes to decrease in sickle cell patients. The reduction in the levels of GPx in sickle participants observed in this study agrees with earlier reports by Manfredini.[30] Reduction in the levels of GPx observed in the sickle cell participants will hinder their normal functions of disposing organic peroxides rather than removal of H2O2 and may not protect the membrane and hemoglobin from peroxidative damage.[31]


  Conclusion Top


The increased in the oxidative stress level, especially lipid peroxides in sickle cell homozygous and heterozygous participants as compared to healthy individual confirms that oxidative stress involved in the pathogenesis of SCD. High level of free radical formation increases the rate of hemolysis, which can lead to further clinical complications. Vitamin supplementation might play a significant role in delaying the progression and complications of SCA disease.

Acknowledgments

The authors are grateful to the School of Biotechnology and Bioinformatics, D.Y. Patil Deemed to be University, Navi Mumbai, for providing the research facility to carry out this work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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