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 Table of Contents  
Year : 2020  |  Volume : 11  |  Issue : 2  |  Page : 59-63

Superoxide dismutase activity in sickle cell anemia patients during crisis and in steady State

1 Department of Haematology and Blood Transfusion, Lagos State University College of Medicine, Lagos, Nigeria
2 Department of Haematology and Blood Transfusion, Faculty of Clinical Sciences, College of Medicine, University of Lagos, Lagos, Nigeria
3 Department of Haematology and Blood Transfusion, Faculty of Medicine, Ahmadu Bello University, Zaria, Kaduna State, Nigeria
4 Department of Community Health and Primary Care, Faculty of Clinical Sciences, College of Medicine, University of Lagos, Lagos, Nigeria
5 Department of Haematology and Blood Transfusion, Federal Medical Center, Abeokuta, Ogun State, Nigeria

Date of Submission19-Dec-2019
Date of Decision23-Feb-2020
Date of Acceptance28-Feb-2020
Date of Web Publication28-Jul-2020

Correspondence Address:
Dr. Ebele Uche
Department of Haematology and Blood Transfusion, Lagos State University College of Medicine, Lagos
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/joah.joah_87_19

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INTRODUCTION: Sickle cell anemia is associated with intense oxidative stress, and optimal antioxidant levels are essential to prevent continuous oxidant tissue damage. The role of oxidant damage to red blood cells in sickle cell anemia (SCA) has been of interest in recent years, and some available results indicate that sickled red blood cells produce almost twice as much superoxide, hydrogen peroxide, and hydroxyl free radicals compared with normal red cells. To counter the effects of oxidants, red blood cells have evolved a number of self-sustaining activities of antioxidant defense enzymes including but not limited to superoxide dismutase (SOD). This study aimed to measure serum SOD activity in patients SCA patients (as a measure of antioxidant defense) during the crisis and in steady state and to compare this with the activity in HbAA normal controls.
SUBJECTS AND METHODS: Eighty-eight participants were recruited into this case control study and comprised 34 HbAA controls, 12 HbSS patients in crisis, and 42 HbSS patients in steady state. Five milliliter of venous blood was collected from each participant and 3 ml was dispensed into plain tubes and centrifuged. The serum was used for the estimation of SOD activity. The remaining 2 ml was used for hemoglobin electrophoresis.
RESULTS: SOD levels were highest in the HbAA control group (1.928 ± 0.051 U/mL) followed by the HbSS patients in steady state (1.844 ± 0.118 U/mL) and lowest in the HbSS patients in crisis (1.755 ± 0168 U/mL). This difference was statistically significant with P = 0.001.
CONCLUSION: SOD levels are reduced during vaso-occlusive crisis.

Keywords: Crisis, sickle cell anemia, steady state, superoxide dismutase

How to cite this article:
Uche E, Olowoselu F, Augustine B, Suleiman A, Ismail A, Oluwole E, Akinbami A, Onwah L. Superoxide dismutase activity in sickle cell anemia patients during crisis and in steady State. J Appl Hematol 2020;11:59-63

How to cite this URL:
Uche E, Olowoselu F, Augustine B, Suleiman A, Ismail A, Oluwole E, Akinbami A, Onwah L. Superoxide dismutase activity in sickle cell anemia patients during crisis and in steady State. J Appl Hematol [serial online] 2020 [cited 2023 Feb 4];11:59-63. Available from: https://www.jahjournal.org/text.asp?2020/11/2/59/290968

  Introduction Top

Sickle cell disease (SCD) is a group of inherited disorders in which the sickle mutation is inherited with a mutation at the other beta globin allele, leading to a reduction or absence of normal beta-globin production. These include sickle cell anemia (HbSS; homozygous sickle mutation), HbSC disease, and sickle beta-thalassemia. The point mutation which occurs in the beta-globin gene leads to the production of sickle hemoglobin (HbS) which is less soluble than normal fetal or adult hemoglobin.

The clinical presentation of SCD is varied and the major features are related to hemolysis leading to hemolytic anemia and vaso-occlusion which results in painful episodes (acute or chronic) and tissue ischemia/infarction.

On deoxygenation, HbS molecules polymerize into intracellular fibers, leading to the formation of a high molecular weight gel and causing the sickle cell deformity. This, in turn, stimulates an incompletely understood pathophysiological cascade including hemolysis (extravascular and intravascular), adhesive interactions among sickle cells, endothelial cells, other blood cells, and plasma with resultant microvascular occlusion, leading to localized pain and in many cases, death of the affected individual.

There are several features of sickle cell anemia (SCA) which are not accounted for by polymerized based explanations alone. These include the diversity in the clinical presentation among patients with identical hemoglobin genotypes, the different frequencies of chronic complications, as well as the variable rate of hemolysis among patients. Even though this variance in phenotypic expression may partly be due to the effects of modifier genes such as those that control the expression of HbF, there are other less-documented polymerization-independent mechanisms that may be contributory.

Available reports show that the sickled red blood cells produce twice as much superoxide, hydrogen peroxide, and hydroxyl radicals, compared to normal healthy controls.[1]

This increased oxidant state causes auto-oxidation of hemoglobin, heme iron release, increased levels of asymmetric dimethylarginine, uncoupling of nitric oxide (NO) synthase activity, and decreased NO levels.[2] The anti-oxidant defense systems in SCA have been observed to be suboptimal, thus ineffective in neutralizing the excess pro-oxidant species produced.[3] This leads to chronic oxidative stress and the subsequent development of endothelial dysfunction, peroxidation of membrane phospholipids as well as multiple end-organ damage.[4]

There are contradictory reports on the activity of various antioxidants in patients with SCA with some researchers reporting increased activity whilst others report reduced activity.

This study therefore aimed to assess the activity of serum superoxide dismutase (SOD) in SCA patients in steady state and crisis and to compare these values with HbAA individuals.

  Subjects and Methods Top

Study population

This comprised SCA patients (study group) attending the adult sickle cell clinic of Lagos State University Teaching Hospital (LASUTH) and HbAA volunteer participants attending general outpatients and blood donor clinics (control group). The study group was further sub-divided into SCA patients in steady state and in crises.

Steady state was defined as the period free of crisis extending from at least 3 weeks since the last clinical event and 3 months or more since the last blood transfusion, to at least 1 week before the start of a new clinical event.[5]

Study design

This was a case–control study involving consenting adult SCA patients attending the Haematology clinic, LASUTH and consenting patients of the general outpatient department and blood donor clinics with HbAA Phenotype.

Study period

This study was done between November 2019 and December 2019.


  1. Adults who are HbSS phenotype attending LASUTH hematology clinic
  2. General outpatient and blood donor clients of LASUTH who have HbAA phenotype served as controls.

Inclusion criteria

Adult HbSS phenotype patients

  1. Alkaline hemoglobin electrophoresis showing the HbSS phenotype
  2. Age ≥18 years.

General outpatient and donor clinic clients

  1. Alkaline hemoglobin electrophoresis showing the HbAA phenotype
  2. Age ≥18 years.

Exclusion criteria

Adult HbSS phenotype patients

  1. Nonconsenting HbSS patients
  2. Other Hb phenotypes (e.g., HbSC, standard deviation [SD], etc.,)
  3. Participants with a documented history of concomitant illness, for example, systemic lupus erythematosus (SLE), rheumatic heart disease, diabetes mellitus, and hypertension
  4. Participants on hydroxyurea.

General outpatient and donor clinic clients

  1. Nonconsenting participants
  2. Other Hb phenotypes e.g., AS, SC, AC
  3. Participants with a documented history of concomitant illness, for example, SLE, rheumatic heart disease, diabetes mellitus, and hypertension.

Ethical considerations and clearance

Ethical approval was obtained from the Health Research Ethics committee of LASUTH (No LREC/06/10/1285).

Sample size determination

The sample size for SCD patients was determined by the formula designed for the determination of a single proportion noncomparative study in a given population.

Sample size was determined using the Daniel formula.[6]

n = Z2 pq/d2


n = Sample size

Z = Z statistic for a level of confidence of 95%, which is conventional, Z value is 1.96

p = Expected prevalence

q = 1 − p

d = precision

Z = 1.96

p = Prevalence of sickle cell anemia in our population = 3%[7]

P = 0.03

q = 1 − p

d = 0.05

n = (1.96) 2 × 0.03× (1 − 0.03)/(0.05) 2

n = 3.8416 × 0.03 × 0.97/0.0025

n = 0.07529536/0.0025

n = 44.7

Because of attrition, a total of 54 sickle cell anemia patients (In steady state and during crisis) and 34 HbAA controls were used.

Specimen collection

From an intravenous access, under aseptic conditions using a vacutainer needle, 5 ml of venous blood was collected and 3 ml dispensed into a lithium heparin vacutainer bottle, centrifuged at 5000 rpm for 5 min and serum extracted which was used for estimation of SOD activity. SOD activity was determined using the ELISA kit manufactured by ElabScience USA.

The remaining 2 ml of blood was dispensed into a vacutainer ethylenediaminetetraacetic acid bottle and thoroughly mixed with the anticoagulant. This was used for alkaline Haemoglobin electrophoretic analysis in all participants.

Statistical analysis

Data was analyzed by IBM SPSS (Statistical Package for Social Sciences, Inc.) statistics for windows version 20.0 Armonk, New York, USA. The continuous variables are presented as means ± SD. The Pearson Chi-squared tested for association between discrete variables. Independent t-test and analysis of variance (ANOVA) were used between the three groups. P value was considered to be statistically significant when <0.05.

  Results Top

Sociodemographic characteristics of study participants

A total of 84 subjects were recruited into the study. This was made up of 34 HbAA controls (24 males and 10 females) with a mean age of 26.76 ± 6.22 years and 54 HbSS patients (34 males and 20 females). Of the 54 HbSS patients, 42 were in steady state (27 males and 15 females) with a mean age of 22.95 ± 7.96 years whilst 12 (7 males and 5 females) with a mean age of 25.25 ± 4.20 years were seen during a vaso-occlusive crisis.

[Table 1] and [Table 2] show the distribution and the sociodemographic characteristics of the study participants, respectively.
Table 1: Distribution of the study participants

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Table 2: Sociodemographic characteristics of the study participants

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The overall mean age of the study participants was 24.74 ± 7.07 years.

Superoxide dismutase activity levels

The levels of SOD were highest in the HbAA control group and lowest in the HbSS patients in crisis [Table 3]. Using ANOVA to compare the ratios, this difference was statistically significant with P = 0.001.
Table 3: Superoxide dismutase activity among the study participants

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Correlation between age/sex and superoxide dismutase activity levels

Using one-way ANOVA, there was no significant correlation between participants' age (F = 0.898; P = 0.644 and sex (F = 0.778; P = 0.788) with SOD activity levels [Table 4].
Table 4: Correlation between age/sex and superoxide dismutase levels in the participants

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  Discussion Top

Following the discovery and description of sickle-shaped red blood cells in 1910 by Herrick, there has been an improved understanding of the pathophysiology of SCA, and recent studies suggest that oxidative stress plays an important role in sickle cell-related organ dysfunction, morbidity, and mortality.[8]

The link between oxidative stress and disease complications are well known and has triggered the need for its study in sickle cell anemia.

There is an increase in serum activity of several oxidases[9] and this increased oxidant state leads to auto-oxidation of hemoglobin, the release of heme iron with a subsequent reduction in NO levels.[10]

The antioxidant systems in SCA have been observed to be supoptimal and so ineffective in neutralizing the excess pro-oxidant species produced;[11] this results in a state of chronic oxidative stress, leading to endothelial dysfunction and peroxidation of membrane phospholipids, with consequent multiple end-organ damage.[12]

As a counter mechanism to oxidative stress, red blood cells have evolved a number of self-sustaining activities of antioxidant defense enzymes such as SOD.[13]

SOD is a key enzyme in the dismutation of superoxide radicals into hydrogen peroxide. It is an important antioxidant defense system, especially in cells involved in aerobic metabolism.

The progressive microvascular damage in organs seen in SCA is caused by chronic activation and damage of endothelial cells by sickled red blood cells, as well as inflammatory mediators.[14],[15],[16] There is an increase in oxidative stress and this might play a significant role in the pathophysiology of SCA-related microvascular dysfunction, vaso-occlusion, and organ damage.[1],[3],[5],[17],[18]

Excessive superoxide anion (O2−) as well as other reactive oxygen species (ROS) play an important role in vascular dysfunction. SOD is the body's major antioxidant defense system against these ROS.

This is done by catalyzing the conversion of O2 − H2O2, which may participate in cell signaling. Furthermore, SOD is important in the inhibition of oxidative inactivation of NO, thus preventing peroxynitrite formation and endothelial and mitochondrial dysfunction. This occurs particularly in cells that are involved in aerobic cellular metabolism.

Our study documents a significantly lower mean serum SOD levels in HbSS individuals compared with HbAA individuals, and this value was even lower in HbSS patients during the crisis.

Various studies have documented reduced SOD activity among SCA patients when compared to HbAA controls.[19],[20] Various reasons have been postulated for this difference and include zinc deficiency which has been documented in SCA patients.[21] Zinc deficiency may lead to a reduction in SOD activity since this enzyme requires zinc as a cofactor for its optimal activity.

However, it is likely that the difference in enzyme activity when comparing SCA patients with HbAA controls may also be due to increased demand for and rapid degradation of SOD from excess levels of ROS.[22] During vaso-occlusive crisis, large amounts of ROS are generated which lead to oxidative damage. The activities of antioxidants including SOD help to ameliorate the effects of the ROS, and thus, their levels will be lowest during vaso-occlusive crisis. In addition, it has been shown that HbS auto-oxidizes faster than its HbA counterpart; this may lead to the production of more ROS, leading to more lipid peroxidation and resultnant consumption of antioxidant enzymes including SOD.[23],[24]

Using an objective disease severity score, in addition to lower SOD levels seen in HbSS patients, Okocha et al. documented that SOD activity was lowest in HbSS individuals with severe disease, and in addition, they found a significant correlation between SOD activity and disease severity.[22]

In contrast, Das and Nair showed that SOD levels were increased in patients with sickle cell anemia[25] and postulated that this increase may be an adaptive defense mechanism to counter the increased oxidative stress.

Similar to the findings of Titus et al.,[26] our study showed no significant effect of age and sex on SOD activity levels.

  Conclusion Top

Serum SOD activity was lower in HbSS patients when compared with HbAA control and was even lower during the VOC crisis when compared with HbSS patients in a steady state. This confirms the increased oxidant activity in sickle cell anemia which is worsened during periods of crises. The use of antioxidant vitamin supplementation may result in enhanced antioxidant activity of serum as well as a decrease in the oxidant levels. A larger-scale randomized study will be useful in confirming or refuting the findings in our study.


The authors are grateful to Mr. Oloko who coordinated the ELISA analysis.

Financial support and sponsorship

The research was solely self-funded by the authors; resulting in the small sample size used for the study due to financial constraints.

Conflicts of interest

There are no conflicts of interest.

  References Top

Essien EU. Increased susceptibility of erythrocyte membrane lipids to peroxidation in sickle cell disease. Cent Afr J Med 1994;40:217-20.  Back to cited text no. 1
Nath KA, Grande JP, Croatt AJ, Frank E, Caplice NM, Hebbel RP, et al. Transgenic sickle mice are markedly sensitive to renal ischemia-reperfusion injury. Am J Pathol 2005;166:963-72.  Back to cited text no. 2
Morris CR, Suh JH, Hagar W, Larkin S, Bland DA, Steinberg MH, et al. Erythrocyte glutamine depletion, altered redox environment, and pulmonary hypertension in sickle cell disease. Blood 2008;111:402-10.  Back to cited text no. 3
Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci U S A 1987;84:9265-9.  Back to cited text no. 4
Akinola NO, Stevens SM, Franklin IM, Nash GB, Stuart J. Subclinical ischaemic episodes during the steady state of sickle cell anaemia. J Clin Pathol 1992;45:902-6.  Back to cited text no. 5
Naing L, Winn T, Rusli BN. Practical issues in calculating the sample size for prevalence studies. Arch of OrofSci 2006;1:9-14.  Back to cited text no. 6
Nwogoh B, Adewoyin AS, Iheanacho OE, azuaye GN. Prevalence of haemoglobin variants in Benin City Nigeria. Ann Biomed Sci 2012;11:60-4.  Back to cited text no. 7
Rees DC, Gibson JS. Biomarkers in sickle cell disease. Br J Haematol 2012;156:433-45.  Back to cited text no. 8
Wood KC, Hebbel RP, Granger DN. Endothelial cell NADPH oxidase mediates the cerebral microvascular dysfunction in sickle cell transgenic mice. FASEB J 2005;19:989-91.  Back to cited text no. 9
Morris CR, Kato GJ, Poljakovic M, Wang X, Blackwelder WC, Sachdev V, et al. Dysregulated arginine metabolism, hemolysis-associated pulmonary hypertension, and mortality in sickle cell disease. JAMA 2005;294:81-90.  Back to cited text no. 10
Amer J, Ghoti H, Rachmilewitz E, Koren A, Levin C, Fibach E. Red blood cells, platelets and polymorphonuclear neutrophils of patients with sickle cell disease exhibit oxidative stress that can be ameliorated by antioxidants. Br J Haematol 2006;132:108-13.  Back to cited text no. 11
Hebbel RP, Osarogiagbon R, Kaul D. The endothelial biology of sickle cell disease: Inflammation and a chronic vasculopathy. Microcirculation 2004;11:129-51.  Back to cited text no. 12
Perrone S, Tataranno ML, Stazzoni G, Del Vecchio A, Buonocore G. Oxidative injury in neonatal erythrocytes. J Matern Fetal Neonatal Med 2012;25:104-8.  Back to cited text no. 13
van Beers EJ, van Tuijn CF, Mac Gillavry MR, van der Giessen A, Schnog JJ, Biemond BJ, et al. Sickle cell disease-related organ damage occurs irrespective of pain rate: Implications for clinical practice. Haematologica 2008;93:757-60.  Back to cited text no. 14
Hebbel RP. The systems biology-based argument for taking a bold step in chemoprophylaxis of sickle vasculopathy. Am J Hematol 2009;84:543-5.  Back to cited text no. 15
Aslan M, Ryan TM, Adler B, Townes TM, Parks DA, Thompson JA, et al. Oxygen radical inhibition of nitric oxide-dependent vascular function in sickle cell disease. Proc Natl Acad Sci U S A 2001;98:15215-20.  Back to cited text no. 16
Nur E, Brandjes DP, Schnog JJ, Otten HM, Fijnvandraat K, Schalkwijk CG, et al. Plasma levels of advanced glycation end products are associated with haemolysis-related organ complications in sickle cell patients. Br J Haematol 2010;151:62-9.  Back to cited text no. 17
Klings ES, Farber HW. Role of free radicals in the pathogenesis of acute chest syndrome in sickle cell disease. Respir Res 2001;2:280-5.  Back to cited text no. 18
Schacter LP, DelVillano BC, Gordon EM, Klein BL. Red cell superoxide dismutase and sickle cell anemia symptom severity. Am J Hematol 1985;19:137-44.  Back to cited text no. 19
Ren H, Ghebremeskel K, Okpala I, Lee A, Ibegbulam O, Crawford M. Patients with sickle cell disease have reduced blood antioxidant protection. Int J Vitam Nutr Res 2008;78:139-47.  Back to cited text no. 20
Manafa PO, Okocha EC, Nwogbo SC, Chukwuma GO, Ibim AC, Ebugosi RS, et al. The status of some trace elements in sickle cell homozygous and heterozygous subjects attending Nnamdi Azikiwe university teaching hospital (NAUTH), Nigeria. Arch Basic Appl Med 2013;1:95-8.  Back to cited text no. 21
Okocha C, Manafa P, Aneke J, Onwuzurike C, Ibeh N, Chukwuma O. Serum superoxide dismutase activity: A predictor of disease severity in Nigerian sickle cell anemia patients in steady state. Med J DY Patil Univ 2017;10:406-11.  Back to cited text no. 22
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Steinberg MH. Sickle cell anemia, the first molecular disease: Overview of molecular etiology, pathophysiology, and therapeutic approaches. ScientificWorldJournal 2008;8:1295-324.  Back to cited text no. 23
Oyeyemi A, Oyebanji G, Akinlua I. Evaluation of antioxidant enzymes in children with sickle cell anaemia in Ekiti State, Nigeria. J Med Dent Sci Res 2016;3:1-4.  Back to cited text no. 24
Das SK, Nair RC. Superoxide dismutase, glutathione peroxidase, catalase and lipid peroxidation of normal and sickled erythrocytes. Br J Haematol 1980;44:87-92.  Back to cited text no. 25
Titus J, Chari S, Gupta M, Parekh N. Pro-oxidant and anti-oxidant status in patients of sickle cell anaemia. Indian J Clin Biochem 2004;19:168-72.  Back to cited text no. 26


  [Table 1], [Table 2], [Table 3], [Table 4]


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