|Year : 2020 | Volume
| Issue : 2 | Page : 59-63
Superoxide dismutase activity in sickle cell anemia patients during crisis and in steady State
Ebele Uche1, Festus Olowoselu2, Benjamin Augustine3, Aisha Suleiman3, Ayobami Ismail1, Esther Oluwole4, Akinsegun Akinbami1, Lawretta Onwah5
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 Submission||19-Dec-2019|
|Date of Decision||23-Feb-2020|
|Date of Acceptance||28-Feb-2020|
|Date of Web Publication||28-Jul-2020|
Dr. Ebele Uche
Department of Haematology and Blood Transfusion, Lagos State University College of Medicine, Lagos
Source of Support: None, Conflict of Interest: None
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 2020 Sep 23];11:59-63. Available from: http://www.jahjournal.org/text.asp?2020/11/2/59/290968
| Introduction|| |
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.
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. The anti-oxidant defense systems in SCA have been observed to be suboptimal, thus ineffective in neutralizing the excess pro-oxidant species produced. This leads to chronic oxidative stress and the subsequent development of endothelial dysfunction, peroxidation of membrane phospholipids as well as multiple end-organ damage.
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|| |
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.
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.
This study was done between November 2019 and December 2019.
- Adults who are HbSS phenotype attending LASUTH hematology clinic
- General outpatient and blood donor clients of LASUTH who have HbAA phenotype served as controls.
Adult HbSS phenotype patients
- Alkaline hemoglobin electrophoresis showing the HbSS phenotype
- Age ≥18 years.
General outpatient and donor clinic clients
- Alkaline hemoglobin electrophoresis showing the HbAA phenotype
- Age ≥18 years.
Adult HbSS phenotype patients
- Nonconsenting HbSS patients
- Other Hb phenotypes (e.g., HbSC, standard deviation [SD], etc.,)
- Participants with a documented history of concomitant illness, for example, systemic lupus erythematosus (SLE), rheumatic heart disease, diabetes mellitus, and hypertension
- Participants on hydroxyurea.
General outpatient and donor clinic clients
- Nonconsenting participants
- Other Hb phenotypes e.g., AS, SC, AC
- 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.
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%
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.
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.
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|| |
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.
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.
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|
Click here to view
| Discussion|| |
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.
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 and this increased oxidant state leads to auto-oxidation of hemoglobin, the release of heme iron with a subsequent reduction in NO levels.
The antioxidant systems in SCA have been observed to be supoptimal and so ineffective in neutralizing the excess pro-oxidant species produced; this results in a state of chronic oxidative stress, leading to endothelial dysfunction and peroxidation of membrane phospholipids, with consequent multiple end-organ damage.
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.
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.,, 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.,,,,
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., Various reasons have been postulated for this difference and include zinc deficiency which has been documented in SCA patients. 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. 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.,
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.
In contrast, Das and Nair showed that SOD levels were increased in patients with sickle cell anemia 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., our study showed no significant effect of age and sex on SOD activity levels.
| Conclusion|| |
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.
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