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Abbreviations
- eDAMP
- erythrocytic danger‐associated molecular pattern molecules
- GPx
- glutathione peroxidase
- HbAA
- hemoglobin A for normal subjects (controls)
- HbAC
- hemoglobin C trait
- HbAS
- hemoglobin S trait
- HbSC
- hemoglobin SC for sickle cell disease
- HbSS
- hemoglobin SS for sickle cell disease
- MDA
- malondialdehyde
- ROS
- reactive oxygen species
- SCD
- sickle cell disease
- SOD
- superoxide dismutase
- TAS
- total antioxidant status
Hemoglobin (Hb) and iron released from erythrocytes in biological systems are associated with uninterrupted prooxidant generation attributed to superoxide anion via autoxidation of Hb and Fenton's reaction . Iron is a key element that sometimes prompts Hb oxidation. Increased free iron in Hb variants is linked with increase in reactive free radicals that may in turn set off oxidative stress. However, there are contradictory reports about the iron level in sickle cell patients compared to the normal Hb phenotype. Both increased iron and reduced levels that have been conflictingly reported in sickle cell disease (SCD). Besides, studies show that sickle red cells generate larger amounts of O2•−, H2O2, and •OH compared to the normal Hb phenotype. Meanwhile, antioxidant molecules, both enzymatic and nonenzymatic, systemically function to form a defensive system in preventing erythrocyte membrane attack. Despite the function of antioxidant systems, reports show rising and continuous prooxidant generation in SCDs. These oxidative agents perhaps prompt consumption of antioxidant reserve very rapidly and consequently result in antioxidant insufficiency, thus building up oxidative stress and possible complications. Recent suggestions have exclusively linked abnormal hemoglobins S and C to many complications with multiple prooxidant processes.
However, in spite of the associated complications in a majority of these common Hb traits, very few studies focused attention on prooxidant and antioxidant system interactions in sickle cell and HbC carriers. This is in opposite to the ample available data on SCD, although without resolute agreement at present. More so, there are still conflicting reports on the rate of activities of various antioxidant enzymes such as SOD, CAT, and GPx in SCDs. We therefore aimed to assess the interplay between erythrocyte lipid peroxidation and antioxidant defense system in all common Hb variants—both SCDs and the carriers in relation to possible links to iron mediation.
Participants were recruited at Obafemi Awolowo University Teaching Hospital, Ile‐Ife and Ladoke Akintola University of Technology Teaching Hospital Osogbo, the only two tertiary hospitals in Osun State, Nigeria. The study protocol was carried out in accordance with Helsinki Declaration as revised in 2000. The study was also approved by the Ethics Committees of the two study centers. The participants were informed of the purpose and procedures of the study, and thus voluntarily gave written consents.
The study population comprised a total number of one‐hundred ninety‐three (n = 193) overall participants with confirmed different Hb genotypes using Hb electrophoresis. The participants included were consecutive patients with sickle cell anemia (SCA, n = 32) and HbSC disease (n = 28) regularly followed up in a steady state. Other participants included were subjects in the same geographical locations with abnormal Hb traits (HbAS, n = 50; HbAC, n = 33). The control subjects were apparently healthy subjects with normal Hb phenotype (HbAA, n = 50), all confirmed with the same method before enrollment, respectively.
Patients were excluded from the study if they were transfused within the preceding 3 months or have had any sickle cell crisis within 2 weeks before the time of recruitment. Patients on contraceptive pills, vitamin C, iron‐containing supplements, or other hematinics were also excluded. Other exclusion criteria involved smoking, pregnancy, and history of surgical or other medical conditions such as renal diseases, leukemia, or other malignancies.
The sample size was obtained using:[Image Omitted. See PDF]where
N = Minimum sample size.
d = Desired level of significance taken as 0.05.
Z α = Confidence interval (1.96) from statistical table.
P = Prevalence rate (1.1%) of the HbSC obtained from the literature.
Therefore,[Image Omitted. See PDF]
Minimum sample size for each group = 17.
Ten milliliters (10 mL) of blood was withdrawn aseptically from antecubital vein from each subject. Four milliliters each was put into sterile lithium heparin and EDTA bottles while the remaining 2 mL was put in plain bottles, respectively. The blood samples were centrifuged at 3000 rpm for 10 minutes. The supernatant was extracted and stored in plain tubes at −20°C until the time of analyses.
The method of analyzing Hb and hematocrit levels was based on the instruction manual of the SYSMEX Automated Hematology Analyzer (KX‐21).
Determination of the iron level was done following the method of Tietz. Four test tubes, labeled as blank, standard, test and control, containing fresh unhemolyzed serum were prepared. An acidic buffer containing hydroxylamine was added. This caused the oxidized form of iron (Fe3+) to be converted into its reduced form (Fe2+). A chromogenic agent was then added which produced a color‐complexed solution (Fe2+‐complex). The end product was measured using a spectrophotometer at 560 nm.
Total antioxidant status (mmol/L) determination followed a modified method of Koracevic et al. In this method, a standard solution of the Fe‐EDTA complex was reacted with hydrogen peroxide using Fenton's reaction. The reaction resulted in the formation of hydroxyl radicals (•OH), which degraded sodium benzoate and released thiobarbituric acid reactive substances (TBARS) from sodium benzoate. Antioxidants from the blood specimen were expected to cause suppression of the production of TBARS and inhibition of color development. This reaction was measured using deionized water at 532 nm using a spectrophotometer.
Malondialdehyde (MDA) estimation (μmol/L) followed a modified method of Gutteridge et al. MDA is a resultant product of lipid peroxidation in plasma. Estimation was based on the reaction with thiobarbituric acid (TBA). The presence of MDA formed a complex pinkish chromogenic solution known as MDA‐TBA adducts, which was measured by its absorbance at 532 nm using a spectrophotometer.
Assay of glutathione peroxidase (GPx) activity (mU/mL) followed the method of Rotruck et al. GPx normally converts reduced glutathione (GSH) into the oxidized form using hydrogen peroxide during reaction. In this reaction, the amount of GSH utilized before and after the enzyme activity was estimated in the assay mixture. GSH reacted with the dithiobisnitrobenzoic acid (DTNB) reagent to give a yellow color compound, which was then measured at 412 nm using a spectrophotometer. The enzyme activity was then expressed as mU/mL of GPx consumed.
Estimation of the superoxide dismutase (SOD) level (ng/mL) followed the method of Misra and Fridovich and was based on the ability of SOD to inhibit the autooxidation of epinephrine at pH 10.2. The superoxide (O2−) radical evolved the triggered oxidation of epinephrine yielding adenochrome per increased O2− molecule with increasing pH. The increase was then monitored every 30 seconds to 150 seconds at 480 nm. One unit of SOD activity was given as the amount of SOD necessary to cause 50% inhibition of the oxidation of adrenalin (ng/mL).
The study data were statistically analyzed using the Statistical Package for Social Science version program (SPSS program version 10.0 ‐ SPSS Inc., Chicago, Illinois). The data were expressed as mean (±SD). Between‐group comparisons were assessed for nominal variables using the χ2 test, while analysis of variance (ANOVA) and post hoc for multiple comparisons were used to compare the quantitative variables for all the groups. Statistical significance was assessed at P < 0.05. All calculated P values were two‐tailed.
Table shows demographic, hematological, and biochemical parameters of erythrocyte lipid peroxidation and antioxidant defense systems in different groups of Hb variants. There were no significant differences (P > 0.05) in the gender ratio and mean ages, both across and in‐between the groups.
Demographic, hematological, and biochemical parameters of erythrocyte lipid peroxidation and antioxidant defense systems in different groups of hemoglobin variants| Parameters | HbSS (n = 32) | HbSC (n = 28) | HbAC (n = 33) | HbAS (n = 50) | HbAA (n = 50) | P value |
| Demographic status | ||||||
| Gender ratio (M/F) | 12/20 | 12/16 | 15/18 | 23/27 | 24/26 | 0.8063 |
| Age (years) | 23.75 ± 4.25 | 24.10 ± 2.10 | 23.40 ± 3.60 | 24.10 ± 4.90 | 24.80 ± 3.80 | 0.5260 |
| Hematological parameters | ||||||
| Hct (%) | 23.78 ± 4.85a,b,c,d | 30.33 ± 3.52a,b,c | 39.45 ± 3.24 | 39.06 ± 3.11a | 40.47 ± 3.50 | <0.001* |
| Hb conc. (g/dL) | 7.86 ± 1.74a,b,c,d | 10.56 ± 1.69a,b,c | 12.82 ± 1.48 | 12.67 ± 1.35a | 13.42 ± 1.73 | <0.001* |
| Iron (μg/dl) | 74.02 ± 13.38a,b,c,d | 104.02 ± 13.48a,b,c | 155.29 ± 18.89b | 145.75 ± 18.55a | 155.33 ± 19.03 | <0.001* |
| Biochemical parameters | ||||||
| MDA (μmol/L) | 3.06 ± 0.82a,b,c | 2.63 ± 0.44a,b,c | 1.70 ± 0.49a,b | 1.24 ± 0.33 | 1.05 ± 0.30 | <0.001* |
| SOD (ng/mL) | 3.42 ± 0.49a,b,c,d | 2.89 ± 0.44a,b | 2.89 ± 0.19a,b | 2.33 ± 0.46a | 1.60 ± 0.53 | <0.001* |
| TAS (mmol/L) | 0.59 ± 0.18a,b,c,d | 1.13 ± 0.24a | 0.96 ± 0.23a,b | 1.38 ± 0.41 | 1.68 ± 0.41 | <0.001* |
| GPx (mu/mL) | 8.08 ± 0.36a,b,c | 8.37 ± 0.77a,c | 9.28 ± 0.61a,b | 8.78 ± 1.30a | 10.34 ± 1.16 | <0.001* |
The hematocrit level and Hb concentrations showed significant reductions (P < 0.001) in SCDs (HbSS and HbSC) and Hb S trait (HbAS) against the controls (HbAA). Similarly, the serum iron level showed significant reductions (P < 0.001) in SCDs (HbSS and HbSC) and Hbs S and C traits (HbAS and HbAC) against the controls (Table and Figure ).
Figure shows considerable reductions in hematocrit, hemoglobin, and iron levels in all Hb variants (HbSS, HbSC, HbAC, and HbAS) against normal controls. However, the reductions were less notable in HbAC.
Conversely, there were significant increases (P < 0.001) both across and in‐between the groups comparing the mean plasma values of malondialdehyde (MDA) in HbSS, HbSC, and HbAC groups against the controls, respectively. Nevertheless, comparison of mean plasma values of MDA in the HbAS group against the controls showed no statistical significance (P > 0.05), even though an increase in the mean value was observed (Figure ).
Similarly, there were significant increases (P < 0.001) comparing the mean plasma values of SOD in all Hb variants against the controls, both across and in‐between the groups. However, comparison of the mean plasma values of SOD in the HbSC group against the HbC trait showed no significant difference (P > 0.05).
On the contrary, there were significant reductions (P < 0.001) across the groups comparing the mean plasma values of total antioxidant status (TAS) in abnormal HbSS, HbSC, HbAC, and HbAS groups against the control group, respectively. In the same vein, in‐between group comparisons involving the mean plasma values of TAS in the HbSS group against the other groups (HbSC, HbAC, HbAS, and HbAA) independently showed significant reductions (P < 0.001). In addition, in‐between comparisons involving the mean plasma values of TAS in the HbAC group against each group of HbAS and the controls equally showed significant reductions (P < 0.001), respectively.
Furthermore, there were significant reductions (P < 0.001) in‐between and across the groups comparing the mean plasma values of glutathione peroxidase (GPx) in the individual group of Hb variants (HbSS, HbSC, HbAC, and HbAS) against the controls. However, comparison of the HbSC group against the HbS trait showed no significant difference (P > 0.05).
The graphical interpretation of biochemical parameters in different hemoglobin phenotypes are shown in Figure .
Figure shows the elevated plasma levels of MDA and SOD in all hemoglobin variants (HbSS, HbSC, HbAC, and HbAS) when compared against normal controls. In contrast, there were reductions in the plasma levels of TAS and GPx in different Hb variants relative to normal controls.
In this present study, we observed reductions in serum iron concentrations in common Hb variants against normal controls. This was in agreement with an earlier study. Report shows that intravascular hemolysis coupled with urinary losses of iron causes iron deficiency in SCD, and thus no evidence of iron overload other than iron deficiency is expected. A contrary study exists: report shows increased iron status in SCD but reduced in HbS trait. Although it appears that varying reports of iron status is as a result of different screening method or recruitment criteria involving transfusion experience by the participants and the interpretation of what seems to be normal. However, the present study suggests that SCDs, without prior transfusion, are likely to be iron deficient in tropical countries other than iron overload Meanwhile, our study is in agreement with previous reports in tropical areas including Nigeria and India, where the cases of iron deficiency have been repeatedly reported among Hb variants.
Similarly, we observed reductions in red blood cells (RBCs) indicated by low hematocrit levels as well as in the Hb concentrations, among the SCD patients and Hb S traits (HbAS) (Figure ). Report indicates that the reduced number of RBCs and Hb levels may trigger lower partial pressure of oxygen, while partially oxygenated Hb prompts autooxidation of Hb, thus converting to methemoglobin with resultant production of reactive superoxide ions. In this process, the evolved superoxide ion is fast converted to hydrogen peroxide (H2O2) accumulating free radicals, which are detrimental to the erythrocyte membrane and induction of lipid radicals, aldehydes, and other reactive products.
The current study also showed elevation of malondialdehyde (MDA) in blood levels of all common Hb variants when evaluated against the controls. The degree of increase in the MDA blood level may be consequently attributed to vulnerability of lipid peroxidation, which is in agreement with earlier studies. MDA is a major aldehyde and the end product of lipid peroxidation of erythrocytes and reveals the damage level to their membrane lipids. Similarly, our study showed that no considerable difference existed in the plasma values between HbSC and HbSS groups regarding the build‐up of plasma MDA levels. However, there was a remarkable increase in the HbSS group (Figure ). Meanwhile, accumulation of MDA in the blood system is another key factor that may disrupt organization of the phospholipid bilayer within the erythrocyte membrane. The increased MDA level in this study was evidence of increased lipid peroxidation and might be exacerbated upon depletion of some iron‐containing antioxidant enzymes, as earlier mentioned. More so, subjects with sickle cell (SCT) and Hb C traits are identified as carriers, and they are usually considered to be benign and asymptomatic conditions owing to the level of MDA in their blood system. Previous report suggests that these carrier groups should be reclassified as disease states with a number of associated clinical conditions, including exercise‐related deaths, pregnancy‐related complications, splenic infarction, hematuria, acute chest syndrome, thromboembolic disease, and many renal associated problems such as renal medullary carcinoma and renal papillary necrosis. Indeed, SCT is linked with various complications at rest and during exercise, particularly under hypoxic conditions as a cofactor for morbidity and mortality. This is attributed to increased autooxidation and worsened by reduced antioxidant capacity.
There are many studies on the activities and functions of SOD with conflicting reports. The present study, however, assessed the relative functional status of SOD in abnormal Hb variants. The comparative assessment from this study showed that SOD has increased activities from normal Hb to abnormal phenotypes involving both SCDs and Hb traits, which were in line with a previous study. Conversely, report shows the reduced activity of SOD in sickle cell anemia as opposed to Hb S trait and normal subjects. The current study showed that there was no substantial difference in SOD levels between HbAC and HbSC subjects (Figure ). This may be due to the related genetic expression pattern in Hb C. Meanwhile, the enhanced activity or increased plasma level of SOD does not minimize the intensity of oxidative damage, rather it increases the concentration of H2O2. This may also inhibit the activity of erythrocytic catalase enzymes, thereby leading to denaturation of Hb and the formation of Heinz bodies. Consequently, these processes add to sickling and/or hemolytic effects, as earlier reported by Scott et al.
Moreover, considerable reductions in total antioxidant status (TAS) in SCDs and HbC traits, as observed in this present study, were in agreement with previous findings. The low level of TAS is associated with manifestations of SCD, such as increased susceptibility to infections, acute chest syndrome, and chronic hemolytic anemia. Conversely, we noted the substantial reduction in the TAS level in subjects with the HbC trait against the HbS trait, contrary to the previous study. However, the current study signified that HbC carriers were more vulnerable to lipid peroxidation, possibly due to the low TAS level which, therefore, could be suggestive of the increase and overburden of reactive oxygen species (ROS) more than in HbAS carriers. In addition, assessment between HbSC and HbAC, and between HbSC and HbAS groups revealed that there was no remarkable difference in the mean values of TAS. However, there was a slight reduction in the HbAC trait compared with the HbAS trait and HbSC disease. What contributed to the reduced TAS in the HbC trait relative to HbSC disease is currently unknown.
The study shows that a number of factors may be culprit of free radical induced oxidative damage in Hb variants, with and without iron effects. Excluding iron effects, vaso‐occlusion induced ischemia‐reperfusion injury is a notable inducer of xanthine oxidase, NADPH oxidase, and nitric oxide synthase enzymes, which may trigger many oxidative events and ultimately damage erythrocytes, especially in SCDs. Besides, irregularities in the architecture of erythrocyte membrane proteins of Hb variants and chronic inflammation (in SCDs) are other plausible factors. In addition, reduced antioxidant capacity has been linked with the reduced serum iron level that may in turn affect the production of iron‐containing proteins, particularly antioxidant enzymes such as catalase (CAT) and peroxidase levels. Moreover, there is possibility of increased O2.– species, while the resultant generation of reactive metabolites is indispensable. Consequently, this may have a causal relationship with TAS depletion in the HbC trait, hitherto unidentified.
Again, the defined basis for hemolysis in some HbC carriers without sickle Hb is presently unclear. However, chronic hemolytic anemia has been reported in SCDs, clinical indications such as microcytosis have been noted in the HbC trait and have been further associated with crystallization and RBC rigidity, compared with normal RBCs. The HbC trait forms rod‐like crystals under hypoxic conditions. Moreover, owing to their rigid shape, small blood vessels may be blocked, thus resulting in ischemia and endothelial cell damage. Consequently, ischemia‐reperfusion injury may worsen oxidative stress by the generation of more ROS.
Furthermore, our findings revealed that glutathione peroxidase, a symbolic antioxidant enzyme, was considerably reduced in HbSS, HbSC, and HbAC groups against the normal group. This observation was consistent with some previous studies. Meanwhile, there was no substantial difference between HbAC and HbAS traits, although there was a slight increase in HbAC.
It is evident that both SCDs and Hb traits may be predisposed to enhanced oxidative stress against the normal phenotype, even so at varying degrees. In other words, our findings revealed that abnormal HbSS, HbSC, HbAC, and HbAS subjects were more at risk of lipid peroxidation attributable to overburden of reactive oxygen metabolites than in normal HbAA subjects. Moreover, the deficient antioxidant system in these subjects may probably serve the reason for ineffective counteracting the augmented oxidative stress rather than effect of iron‐mediated oxidation. At this instance, a further study may be required to suggest whether the oral intake of antioxidant supplements could be of help in ameliorating the antioxidant shortfalls other than needless iron‐chelating drugs to reduce the associated clinical complications.
The authors appreciate the staff members of the Obafemi Awolowo University Teaching Hospital, Ile‐Ife, and Ladoke Akintola University of Technology Teaching Hospital Osogbo, Osun State, Nigeria, who gave their immense support during participant recruitments and laboratory analyses. The authors equally acknowledge the roles of Dr. Bamigbade Waheed (of the Department of English, Obafemi Awolowo University, Ile‐Ife, Nigeria) and Mr. Adebiyi Rasheed (of the Department of Mass Communication, Fountain University, Osogbo, Nigeria) on language editing.
All authors declare no conflicts of interest.
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; Oduola, Taofeeq 3 ; Oparinde, Dolapo P 1 ; Ayelagbe, Olubunmi G 1 ; Ojokuku, Hammed O 4 1 Department of Chemical Pathology, Ladoke Akintola University of Technology, Osogbo, Osun State, Nigeria
2 Department of Chemical Pathology, College of Medicine, University of Ibadan, Ibadan, Oyo State, Nigeria; Department of Oral Pathology, King Saud University Medical City, DUH, Riyadh, Saudi Arabia
3 Department of Chemical Pathology, Faculty of Medical Laboratory Sciences, Usmanu Danfodiyo University, Sokoto, Nigeria
4 Department of Medical Laboratory, Reddington Multispecialist Hospital, Victoria Island, Lagos, Nigeria