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ORIGINAL ARTICLE
Year : 2017  |  Volume : 10  |  Issue : 5  |  Page : 406-411  

Serum superoxide dismutase activity: A predictor of disease severity in nigerian sickle cell anemia patients in steady state


1 Department of Haematology, Nnamdi Azikiwe University Teaching Hospital, Nnewi, Nigeria
2 Department of Medical Laboratory Science, College of Health Sciences, Nnamdi Azikiwe University, Nnewi, Anambra, Nigeria
3 Department of Chemical Pathology, Nnamdi Azikiwe University Teaching Hospital, Nnewi, Nigeria

Date of Submission28-Apr-2017
Date of Acceptance06-Jul-2017
Date of Web Publication14-Nov-2017

Correspondence Address:
C John Aneke
Department of Haematology, Nnamdi Azikiwe University Teaching Hospital, Nnewi, Anambra
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/MJDRDYPU.MJDRDYPU_90_17

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  Abstract 


Background: Sickle cell anemia (SCA) is associated with intense oxidative stress; optimal antioxidant levels are essential to prevent oxidant tissue damage. Objective: The objective of this study was to evaluate superoxide dismutase (SOD) activity and Vitamin C levels in individuals with SCA, heterozygous sickle cell (heterozygous hemoglobin AS [HbAS]), and normal (hemoglobin AA [HbAA]) hemoglobin phenotypes in comparison with objective scores of disease severity (in those with SCA). Subjects and Methods: A total of ninety participants were recruited, including thirty SCA (in steady state), thirty HbAS, and thirty HbAA. From each participant, 5 ml of venous blood was collected; 3 ml was dispensed into plain tubes and serum was extracted for the estimation of SOD activity and Vitamin C level. Serum SOD activity was measured using a semi-automated spectrophotometric procedure, while serum Vitamin C level was estimated by the enzyme-linked immunosorbent assay technique. The remaining 2 ml was used for hemoglobin electrophoresis and full blood count estimation. Objective score of disease severity was calculated for SCA individuals using a scoring system. Results: The mean serum activity of SOD was significantly lower in SCA compared with HbAS and HbAA participants (9.45 ± 3.39 U/ml vs. 12.87 ± 2.17 U/ml and 13.24 ± 2.10 U/ml, P < 0.001, respectively). No significant differences were observed between the mean serum Vitamin C levels of SCA, HbAS, and HbAA participants (1922.59 ± 771.56 ng/ml vs. 1631.10 ± 526.57 ng/ml and 2029.17 ± 902.99 ng/ml P > 0.05, respectively). Serum SOD activity was significantly correlated with objective score of disease severity in SCA participants, while Vitamin C level was not (r = −0.529, P = 0.02 and r = −0.349, P = 0.14, respectively). Conclusion: Serum SOD activity is a predictor of disease severity in Nigerian individuals with SCA.

Keywords: Antioxidants, disease severity, sickle cell anemia


How to cite this article:
Okocha E C, Manafa O P, Aneke C J, Onwuzuruike E C, Ibeh C N, Chukwuma O G. 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

How to cite this URL:
Okocha E C, Manafa O P, Aneke C J, Onwuzuruike E C, Ibeh C N, Chukwuma O G. Serum superoxide dismutase activity: A predictor of disease severity in nigerian sickle cell anemia patients in steady state. Med J DY Patil Univ [serial online] 2017 [cited 2017 Nov 23];10:406-11. Available from: http://www.mjdrdypu.org/text.asp?2017/10/5/406/218203




  Introduction Top


Sickle cell anemia (SCA) is an inherited hemoglobinopathy which occurs due to a single point mutation in the β-globin gene that results in the formation of the sickle hemoglobin (HbS).[1] The disorder has a wide distribution in parts of Africa, Middle East, and Mediterranean countries and carries significant morbidity, particularly in the African population.[2],[3]

Phenotypic expression of individuals with SCA is complex and widely varied; commonly characterized by intermittent vaso-occlusive events, hemolytic crisis, increased susceptibility to infections, chronic inflammatory state as well as widespread microvascular damage.[4] Even though the molecular basis for HbS formation has been well characterized, the sickle mutation alone cannot sufficiently explain the heterogeneous phenotypes observed in individuals, including episodic painful crisis, acute chest syndrome, neurological complications, chronic leg ulceration, and a number of other disease-related complications. Following the description of sickle-shaped erythrocytes by Herrick in 1910, the understanding of the pathophysiology of the disease has profoundly increased. New body of evidence has suggested that oxidative stress is a significant pathway to sickle-related organ dysfunction and morbidity; this could offer some insight into the heterogeneous phenotypic expression of the disease.[5]

It has been shown that SCA is associated with marked increase in serum activity of several oxidases such as nicotinamide adenine dinucleotide phosphate oxidase and endothelial xanthine oxidase.[6] The exaggerated oxidant state leads to auto-oxidation of hemoglobin, heme iron release, increased levels of asymmetric dimethyl arginine, uncoupling of nitric oxide (NO) synthase activity, and decreased NO levels.[7] The antioxidant defense systems in SCA have been observed to be suboptimal, thus ineffective in neutralizing the excess pro-oxidant species produced.[8] Consequently, a state of chronic oxidative stress is established; this is critical in the development of endothelial dysfunction, peroxidation of membrane phospholipids as well as multiple end-organ damage.[9]

To counter oxidative stress, erythrocytes have evolved a number of self-sustaining activities of antioxidant defense enzymes, such as superoxide dismutase (SOD) and low molecular weight antioxidants, which includes Vitamin C.[10]

SOD is a key enzyme in the dismutation of superoxide radicals (which result from cellular oxidative metabolism) into hydrogen peroxide. It is an important antioxidant defense system, particularly in all cells that are involved in aerobic cellular metabolism.

Vitamin C is a potent water-soluble micromolecule whose antioxidant function in humans is largely dependent on its ability to function as an electron donor. The important role of Vitamin C as an antioxidant (especially in ameliorating the adverse effects of reactive oxygen and nitrogen radicals) has been well established in a number of disease entities.[11]

The aim of this study was to evaluate serum SOD activity and Vitamin C levels in steady state SCA patients in comparison with objective scores of disease severity. This is the first bold attempt to compare antioxidant status with objective score of disease severity in the Nigerian SCA patients; the results have important potential prognostic and therapeutic implications. The authors are hopeful that this work will bridge identified knowledge gaps in this field, particularly as regards Nigerian patients with SCA.


  Subjects and Methods Top


This is a case–control study designed to assess SOD activity and Vitamin C levels in steady-state SCA patients at the hematology outpatient clinic of our hospital. Study participants comprised thirty SCA, thirty heterozygous hemoglobin AS [HbAS], and thirty hemoglobin AA [HbAA] individuals. Steady-state clinical condition for SCA was described in this study following the criteria previously enunciated by Akinola et al., which depict the absence of any form of crises or any symptom of ill health for at least 3 weeks and no history of blood transfusion in the preceding 3 months, prior to recruitment.[12] Information used for severity scoring in homozygous hemoglobin SS (HbSS) study participants (and for study exclusion) was obtained by means of questionnaires as well as by full blood count estimation.

Collection of samples/laboratory work-up

Each participant had 5 ml of venous blood collected by venipuncture, following standard protocols; 3 ml was dispensed into plain container, centrifuged at 5000 rpm for 5 min, and serum was extracted for the estimation of SOD activity and Vitamin C levels. The remaining 2 ml was delivered into ethylenediaminetetraacetic acid specimen bottle for hemoglobin electrophoresis and full blood count estimation (for HbSS individuals only).

SOD activity was estimated using the method described by Akinduko et al.[13] This is a semi-automated spectrophotometric procedure which is based on the ability of SOD generated by the xanthine oxidase reaction to inhibit the auto-oxidation of adrenaline at pH of 10.2. The reaction leads to the formation of an adenochrome (which gives off a yellowish coloration) which is measured spectrophotometrically at a wavelength of 480 nm.

Serum Vitamin C level determination was performed using the method described by Pancewicz et al.[14] This is basically an enzyme-linked immunosorbent assay which employs the competitive enzyme inhibition immunoassay technique. A monoclonal antibody specific to Vitamin C was used to precoat microplate wells. The competitive inhibition reaction was launched between biotin-labeled Vitamin C and biotin-unlabeled Vitamin C (which can be either a standard or the actual sample) with the precoated antibody. After incubation, the unbound conjugate was washed off, followed by the addition of avidin conjugated with horseradish peroxidase to each microplate well and further incubated. The intensity of color developed was inversely proportional to the concentration of Vitamin C in the sample.

The hemoglobin phenotype of each participant was confirmed by doing cellulose acetate paper hemoglobin electrophoresis, as previously described,[15] while full blood count was determined (for HbSS individuals only) using the Sysmex automated hematology analyzer (KX21N ® model, Sysmex Corporation Kobe, Japan).

Exclusion criteria

Study participants and controls who had known conditions which could affect SOD activity and Vitamin C levels such as myocardial infarction, history of smoking, and chronic Vitamin C therapy were excluded from the study.

Severity scoring protocol

This was done for HbSS individuals only and involved combining the anemia, complication, white cell and transfusion scores to generate an objective score of disease severity as follows:

Anemia score:

  • Hb ≥ 10 g/dl → 0
  • Hb ≥ 8 g/day < 10 g/dl → 1
  • Hb ≥ 6 < 8 g/dl → 2
  • Hb ≥ 4 < 6 g/dl → 3
  • Hb < 4 g/dl → 4.


Complications' score:

Each complication was scored 1 except

  • Nephropathy → 2
  • Stroke → 2.


White cell count score:

  • Count < 9 × 103 → 0
  • Count ≥ 9 < 11 × 103 → 1
  • Count ≥ 11 < 15 × 103 → 2
  • Count ≥ 15 × 103 → 3.


Transfusion score:



Transfusion rate was approximated to the nearest whole number.

Disease severity was scored as;

  • Mild (<3)
  • Moderate (>3 ≤ 5)
  • Severe (>5).


Data analysis was done using the Statistical Package for the Social Sciences (SPSS) version 20 computer software (SPSS Inc., Chicago, IL, USA) and results were presented as means ± standard deviation. Means of serum SOD activity and Vitamin C levels were compared using the Student's t-test, while comparison with objective scores of disease severity was by the Pearson's linear regression for bivariate correlation. Values were deemed statistically significant at P < 0.05.

Ethical approval for this research was obtained from the Nnamdi Azikiwe University Teaching Hospital Ethics and Research Committee and each participant gave informed consent prior to recruitment.


  Results Top


The means of serum SOD activity in HbSS, HbAS, and HbAA individuals were 9.45 ± 3.39 U/ml, 13.24 ± 2.10 U/ml, and 12.87 ± 2.17 U/ml, respectively [Table 1]. There were statistically significant differences in serum enzyme activity in HBSS and HbAS and between HbSS and HbAA individuals [P< 0.001, respectively, [Table 1].
Table 1: Comparison of serum superoxide dismutase activity among different hemoglobin phenotypes

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The means of serum Vitamin C levels in HbSS, HbAS, and HbAA individuals were 1922.59 ± 771.56 ng/ml, 2029.17 ± 902.99 ng/ml, and 1631.10 ± 526.57 ng/ml, respectively, these were nonsignificantly different (P > 0.05) [Table 2].
Table 2: Comparison of serum Vitamin C among different hemoglobin phenotypes

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The means of packed cell volume (PCV), white cell, and platelet counts in HbSS individuals were 24.41 ± 5.77 × L/L, 10.57 ± 3.48 × 109/L, and 441.83 ± 226.13 × 109/L, respectively.

Serum SOD activity was lowest in SCA individuals with severe diseases and showed significant correlation with objective score of disease severity [r = −0.529, P = 0.02, [Figure 1] and [Figure 2]. Serum Vitamin C level was not significantly correlated with objective score of disease severity in SCA individuals (r = −0.349, P = 0.14).
Figure 1: Scatter plot of serum superoxide dismutase activity and objective score of disease severity in homozygous sickle cell (sickle cell anemia) group

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Figure 2: Correlation of mean serum superoxide dismutase levels with disease severity in homozygous sickle cell (sickle cell anemia) group

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There were no significant differences in the serum SOD activity and Vitamin C levels in male and female SCA individuals.


  Discussion Top


Reactive oxygen species (ROS) have been reported to mediate inflammatory processes and are thus involved in oxidative reactions such as lipid peroxidation and protein oxidation (particularly hemoglobin).[16] Vascular endothelial cell damage, with the activation of adhesion molecules leading to inflammation, release of C-reactive protein (and other inflammatory mediators), and enhancement of vasculopathy and subsequent ischemia have also been shown to occur following ROS activation.[17] C-reactive protein is produced as part of the nonspecific acute-phase response to inflammation and tissue necrosis and has been shown to be significantly higher in SCA patients compared with controls.[18] The above lines of evidence thus suggest that SCA is associated with significant chronic inflammation.

In the recent years, the role of ROS in the pathophysiology of SCA has become well established and this has given more insight into the diverse manifestations of the disease.[19] The development of a good number of sickle-related complications has since been linked with the sustained generation of oxidative stress and widespread impairment of antioxidant protection.[20] Indeed the relationship between markers of oxidative stress and more debilitating complications of SCA such as acute chest syndrome and pulmonary arterial hypertension had been emphasized in an earlier study.[21]

In this study, the mean serum activity of the antioxidant enzyme, SOD, was significantly lower in SCA patients compared with those of the HbAS and HbAA groups [P< 0.001, respectively, [Table 1]. Traditionally, progression of disease has been associated with conditions that predispose to high production of free radical and ROS as well as those that predispose to the depletion of antioxidants.[22] Therefore, the observed lower antioxidant level could potentially compromise the ability of SCA patients to scavenge free radicals and ROS, which might potentially increase the risk of disease progression.

The above findings concur with previous reports by Schacter and Ren who observed that SOD enzyme activity in individuals with HbAA hemoglobin phenotype was significantly higher compared with that of SCA patients.[23],[24] A number of factors are thought to contribute to this observation; ranging from some trace element deficiency to increased consumption of antioxidants in SCA patients. Manafa et al. had earlier reported a significantly lower concentration of serum zinc in SCA patients compared with HbAA controls.[25] This could lead to decrease in SOD activity since zinc is an important metallic co-factor of the enzyme, which is necessary for optimal activity. An earlier study has equally shown that sickle erythrocytes produce twice as much superoxide, hydrogen peroxide, and hydroxyl radical than normal (HbAA) control red cells.[26] It is, therefore, likely that the difference in enzyme activity in our study participants may also be due to increased demand for and rapid degradation of SOD from excess levels of ROS. In contrast to our finding, Das et al. demonstrated an increase in SOD in SCA patients and concluded that this may be an adaptive defense mechanism to counter increased oxidative stress.[27]

The role of Vitamin C as an antioxidant is well known. Chou et al. proposed that, while ascorbic acid may act synergistically with Vitamin E, in isolation, its antioxidant effect may be due to the quenching of singlet oxygen in the aqueous medium.[28] The accelerated oxygen radical production in SCA could have serious adverse effects on cell membrane proteins and lipids, resulting in thiol oxidation and lipid peroxidation. An important role of Vitamin C in preventing lipid peroxidation and potentiating the cardioprotective ability of high-density lipoprotein fraction has equally been demonstrated.[29] Some studies have shown lower serum Vitamin C levels in patients with SCA, in comparison with controls; this is thought to arise mainly from excessive consumption of the vitamin due to increased oxidative stress.[30] In contrast, Adelekan et al. reported no significant difference in the levels of Vitamin C between SCA patient group and HbAA control group,[31] this latter observation is in keeping with result of this study; no significant difference was observed between the mean serum Vitamin C levels of SCA patients compared with HbAS and HbAA individuals [P = 1.00 and 0.40, respectively, [Table 2]. Even though the reasons for these discrepant observations were not entirely clear from our data set, we suspect that this may be related to variations in dietary intake of the vitamin among the different SCA populations.

An inverse significant correlation was observed between serum SOD activity and objective score of disease severity in SCA patients (r = −0.529, P = 0.02), this is in keeping with the earlier report of Schacter et al. which showed that SCA patients with more severe manifestations had lower levels of SOD activity than those with milder symptoms.[23] The pathway of the above observation appears to be related to the increased levels and activity of ROS (which occur following depletion of SOD), leading to acceleration of complications such as hemolysis, hypercoagulability, endothelial damage, glomerulopathy, and ultimately worse disease severity.[32] It is, therefore, important that measurement of serum SOD activity should be incorporated into the routine work-up for disease stratification, particularly for SCA patients in Nigeria. Serum Vitamin C level was not significantly correlated with disease severity in SCA patients (r = −0.349, P = 0.14), this is contrary to earlier reports. Ohnishi et al. had showed that the addition of (ascorbic acid) supplements inhibited the formation of dense red blood cells and lipid peroxidation in patients with SCA.[33] Correspondingly, Amer et al. reported that supplementation of Vitamin C decreased ROS production by almost 4 folds, while increasing the concentration of glutathione mediated antioxidant defense by up to 2 folds in patients with SCA.[8]

The moderate anemia observed in HbSS individuals in this study (mean PCV of 24.41 ± 5.77 × 109/L) is in keeping with previous observations and is due to the background chronic red cell destruction (hemolysis) which occurs in these individuals, even during steady clinical conditions.[34] Correspondingly, elevated platelet count (mildly in this study) is thought to be due to the negative feedback effect of chronic anemia on erythropoietin (which is structurally related to thrombopoetin) production as well as the absence of splenic sequestration of platelets due to atrophy in SCA.[35],[36]


  Conclusion Top


The lower serum SOD activity observed in patients with SCA in this study as well as the significant correlation with objective score of disease severity is in keeping with earlier reports, related to progressive enzyme degradation and consumption as a result of high oxidant stress. It may therefore be important to include the measurement of serum SOD activity in the routine work-up and disease stratification of patients with SCA.

Limitation of the study

It will be desirable to replicate this study with larger number of SCA patients in steady state.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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