|Year : 2017 | Volume
| Issue : 3 | Page : 110-115
Low serum glutathione-S-transferase activity and vitamin e levels do not correlate with disease severity in steady state adults with sickle cell anemia
Patrick O Manafa1, Chide E Okocha2, John C Aneke2, Ujunwa Obiano1, Nancy C Ibeh1, George O Chukwuma1, Vera I Manafa3
1 Department of Medical Laboratory Science, College of Health Sciences, Nnamdi Azikiwe University, Nnewi Campus, Nnewi, Anambra State, Nigeria
2 Department of Haematology, Nnamdi Azikiwe University Teaching Hospital, Nnewi, Anambra State, Nigeria
3 Department of Chemical Pathology, Nnamdi Azikiwe University Teaching Hospital, Nnewi, Anambra State, Nigeria
|Date of Web Publication||18-Sep-2017|
John C Aneke
Department of Haematology, Nnamdi Azikiwe University Teaching Hospital, Nnewi, Anambra State
Source of Support: None, Conflict of Interest: None
Introduction: Adequate levels of antioxidants are essential in subjects with sickle cell anaemia (SCA) to counter the effects reactive oxygen species generated during normal red cell metabolism. Objective: This study evaluated serum glutathione transferase activity and vitamin E levels in comparison with disease severity in steady state adult SCA subjects.
Subjects And Methods: Five milliliters of venous blood was collected from each of 30 homozygous haemoglobin SS, 30 heterozygous haemoglobin AS and 30 haemoglobin AA subjects for glutathione transferase activity, vitamin E levels, haemoglobin phenotype and full blood count determination. The objective score of disease severity was calculated using white cell count, haemoglobin concentration and number of life time disease related complications and correlated with serum glutathione transferase activity and vitamin E levels, using the Spearman's correlation; P < 0.05 was taken as significant.
Results: The median serum Glutathione transferase activity and vitamin E levels in all study participants were 22 U/L (Q1 –Q3; 15:00 U/L – 28:50 U/L) and 32:00 ug/ml (Q1 – Q3; 17.00 ug/ml – 54.00 ug/ml, respectively). Glutathione transferase enzyme activity was significantly lower in HbSS compared with HbAA (P = 0.001) and HbSS compared with HbAS study subjects (P = 0.01). Enzyme activity and vitamin E levels did not show significant correlations with disease severity in subjects with HbSS (r = 0.07; P = 0.94 and r = -0.04; P = 0.12, respectively.
Conclusion: Serum glutathione transferase activity and vitamin E levels may not be predictors of disease severity in SCA patients.
Keywords: Disease severity, oxidant stress, sickle cell anemi
|How to cite this article:|
Manafa PO, Okocha CE, Aneke JC, Obiano U, Ibeh NC, Chukwuma GO, Manafa VI. Low serum glutathione-S-transferase activity and vitamin e levels do not correlate with disease severity in steady state adults with sickle cell anemia. J Appl Hematol 2017;8:110-5
|How to cite this URL:|
Manafa PO, Okocha CE, Aneke JC, Obiano U, Ibeh NC, Chukwuma GO, Manafa VI. Low serum glutathione-S-transferase activity and vitamin e levels do not correlate with disease severity in steady state adults with sickle cell anemia. J Appl Hematol [serial online] 2017 [cited 2018 Jul 22];8:110-5. Available from: http://www.jahjournal.org/text.asp?2017/8/3/110/214995
| Introduction|| |
Ssickle cell anemia (SCA) is an inherited disease caused by an amino acid substitution (glutamic acid is substituted by valine) in the position 6 of the beta globin chain, this leads to the formation of a defective form of hemoglobin, known as hemoglobin S (HbS). In the presence of reduced oxygen tension, the deoxygenated sickle cell hemoglobin (HbS) molecules undergo polymerization leading to red cell distortion and assumption a sickle shape.
Normal red cells have long been known to be subjected to oxidative stress as a result of the generation of reactive oxygen species (ROS) during normal metabolism, this becomes particularly marked in individuals with SCA due to the auto-oxidation of the hemoglobin molecule. Consequently, SCA has been designated a condition that is associated with chronic inflammation, characterized by the continuous inflammatory response and oxidative stress., ROS mediate damage to red blood cell membrane components and contribute significantly to erythrocyte rigidity and fragility in SCA. It has been earlier hypothesized that erythrocyte ROS generation, hemolysis, vaso-occlusion and the inflammatory response to tissue damage may form critical positive feedback loops that drive organ dysfunction in SCA. These findings emphasize the unique pathogenetic mechanism of SCA and may herald new therapeutic interventions that could counter inflammation, red cell rigidity and fragility and ultimately organ dysfunction in patients with SCA.
Glutathione (GSH) transferases are a group of enzymes (known as antioxidants) which are involved in the conjugation of the reduced form of GSH to xenobiotic substrates for detoxification of a variety of potentially toxic (and carcinogenic) electrophilic compounds., These enzymes have been shown to display significant peroxidative activity; thus they protect cells (particularly red cells) from oxidative damage.
Vitamin E is a fat soluble vitamin and an antioxidant which acts as a peroxyl radical scavenger. It reacts with free radicals in tissues and prevents their propagation and consequent peroxidation (auto-oxidation) of cellular lipids through the formation of tocopheryl radical (which are subsequently reduced to a harmless substance through the process of hydrogenation). Vitamin E has been reported to improve vascular endothelial integrity through inhibition of lipid peroxidation, protein kinase activation, and enhancement of other nitric oxide dependent mechanisms. In SCA patients, in particular, it has equally been linked with increased femoral blood flow and decrease in forearm vascular resistance.
Earlier studies had emphasized the importance of adequate serum levels of antioxidant levels in conditions associated with significant inflammation such as SCA.,, Gizi et al. had reported significant impairment of the GSH system and consequent alteration of redox status and increased predisposition to tissue damage in a population of SCA patients in Athens Greece. This finding was attributed to the continuous generation of ROS which occur in these patients. Fasola and Tukur independently corroborated these earlier findings in SCA subjects in Ibadan, South-West and Maiduguri, North-East Nigeria, respectively., The Ibadan study reported a 50% lower total antioxidant status (TAS) in SCA patients compared with the controls. Interestingly, the study equally observed that markers of disease (such as higher frequency of bone pain crises) were present in SCA patients with lower TAS. The Maiduguri study reported lower serum levels of Vitamin E in steady state SCA patients, compared with controls.
There is a paucity of literature on antioxidant status and its relationship with disease severity in Nigerian SCA patients. This study was therefore aimed at evaluating the anti-oxidant status (GSH-S-transferase Activity and serum Vitamin E Levels) in adult Nigerians with homozygous (HbSS) and heterozygous (HbAS) hemoglobin phenotypes with a view to correlating these with disease severity in SCA subjects.
| Subjects and Methods|| |
This was a case-controlled study carried out from July 2015 to September 2015, at the Hematology Department of the Nnamdi Azikiwe University Teaching Hospital, Nnewi, Anambra State, Nigeria. Ethical approval for this study was obtained from our institutional review board (the Nnamdi Azikiwe University Teaching Hospital Research and Ethics Committee), and all participants gave written informed consent.
Study subjects included 30 confirmed homozygous hemoglobin SS (HbSS) patients in the steady state, 30 heterozygous hemoglobin AS (HbAS) subjects and 30 hemoglobin AA subjects, who served as controls. The selection of steady state patients was based on the criteria earlier described by Akinola et al., which stipulates that subjects be clinically stable in the past 3 weeks and had not received any blood transfusion in the preceding 3 months. Exclusion criteria included subjects on Vitamin E supplementation and those with other comorbid conditions such as any form of cancer, which could affect the activity of GSH transferase.
Each participant had 5 ml of venous blood collected through venipuncture following standard protocols, this included applying a soft tubing tourniquet to the upper arm after which the subject was asked to make a fist, a sufficiently large and straight vein was then selected. The area was disinfected with 70% alcohol, whereas a 20 gauge needle (attached to 5 ml plastic syringe) was carefully introduced into the vein (with the bevel of the needle directed upwards) and 5 ml of blood gently aspirated. Of this, 3 ml was dispensed into a plain container, allowed to clot at room temperature, centrifuged at 5000 rpm for 5 min and serum extracted for the estimation of GSH transferase activity and Vitamin E levels. Serum was stored at −20°C for batch analysis. The remaining 2 ml was dispensed into ethylene diamine tetra acetic acid containers for hemoglobin phenotype and full blood count (FBC) determination.
The measurement of GSH transferase activity was done following the protocol earlier described by Anosike et al., using GSH transferase assay kits manufactured by Sigma Aldrich and BDH, England ®. The protocol involved preparation of the GSH-S-tranferase stock reagent (by mixing 50 ml of phosphate buffer (pH 6.5) with 20 ml of 1-chloro-2,4-dinitrobenzene (CDNB) and incubating at 20°C for 10 min. To 0.8 ml of the stock solution, 0.1 ml of sample and GSH substrate, respectively, was added and thoroughly mixed. The reaction mix produced the compound dinitrophenyl thioether, which was measured spectrophotometrically at a wavelength of 340 nm. A similar protocol was applied to the blank tube, except that 0.1 ml of a buffer solution was used to replace the sample. The principle of the assay is based on the GSH transferase catalyzed reaction between GSH substrate and CDNB.
Serum Vitamin E levels were estimated using Vitamin E enzyme linked immunosorbent assay kits, manufactured by Elabscience Biotech, China ®, following the methodology described by Meydani et al. This assay employed the competitive enzyme inhibition immunoassay technique, where a monoclonal antibody specific to tocopherol was precoated onto a microplate. A standard or sample of 50 uL concentration was added to each well while the blank well was added with reference standard and sample diluents. A volume of 50 uL of biotinylated detection antibody working solution was added to each well and incubated for 45 min at 37°C. Each well was then aspirated and washed repeatedly for 3 times. Horse radish peroxidase conjugate (100 uL) was added to each well and further incubated for 30 min at 37°C. The aspiration/washing process was repeated 5 times, thereafter 90 uL concentration of substrate reagent was added to each well and incubated for 15 min at 37°C. A 50 uL concentration of stop solution was subsequently added to each well, following which a yellow color developed, the optical density value of each well was determined immediately using a microplate reader set to 450 nm wavelength. The kits had installed sensitivity and specificity of 100%, respectively, whereas the reference standard had a linear curve.
FBC and hemoglobin phenotype of each participant were done using automated hematology analyzer (Mythic 22, Switzerland ®) and cellulose acetate paper electrophoresis (Helena biosciences, UK ®), respectively.
Calculation of disease severity score
Calculation of disease severity score for SCA subjects was based on the method earlier described by Okocha et al. Indices such as total white blood cell count, hemoglobin concentration and number of complications present were assigned scores; subjects with scores of <3 were considered to be mild disease, those with scores of 3 ≥ 5 were considered to have moderate disease, whereas those with scores of >5 were categorized as severe disease.
All data analysis was performed using SPSS Version 20 computer software (SPSS Inc., Chicago, IL, USA) because the data set did not pass normality test, results of GSH transferase activity and serum Vitamin E levels were presented as medians (and interquartile range). Comparison of medians of parameters was carried out using the Mann–Whitney U-test, whereas the relationship between substrate activity/levels and objective scores of disease severity was assessed using the Spearman's correlation; P < 0.05 was considered to be statistically significant.
| Results|| |
The median serum GSH transferase activity in all the study participants was 22 U/L (Q1–Q3; 15:00 U/L – 28:50 U/L). Enzyme activity was significantly lower in HbSS than HbAA (P= 0.01) and HbSS compared HbAS (P = 0.01) but not significantly different in HbAS versus HbAA (P = 0.44).
The median serum Vitamin E levels in all study subjects was 32:00 ug/ml (Q1–Q3; 17.00 ug/ml– 54.00 ug/ml. Serum Vitamin E levels were not significantly different in HbSS versus HbAA (P = 0.48), HbSS versus HbAS (P = 0.50) and HbAS versus HbAA (P = 0.77).
Serum GSH transferase activity and Vitamin E levels did not show significant correlations with disease severity in subjects with HbSS (r = 0.07; P = 0.94 and r = −0.04; P = 0.12), respectively [Figure 1] and [Figure 2].
|Figure 1: Comparison of glutathione transferase activity with disease severity in homozygous sickle cell anemia patients|
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|Figure 2: Comparison of serum Vitamin E levels with disease severity in homozygous sickle cell anemia patients|
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| Discussion|| |
The study evaluated serum GSH transferase activity and Vitamin E levels in subjects with different hemoglobin phenotypes and compared enzyme activity and vitamin levels with an objective score of disease severity in study subjects with SCA.
The median serum activity of GSH transferase was significantly lower in SCA subjects compared with HbAA and HbAS subjects (P = 0.001 and 0.01, respectively). Our finding is in agreement with the earlier report of Reid et al., which recorded a significantly decreased GSH concentration in SCA patients compared with the controls; the lower enzyme activity was thought to be due to increased consumption, occasioned by high predisposition to oxidant stress in SCA. In addition, it has been reported that some endogenous compounds such as bilirubin could inhibit the activity of GSH transferase. Even though our study subjects were selected while in steady state, the chronic hemolysis which occurs in SCA subjects, even in steady clinical conditions, might, in fact, lead to varying degrees of plasma hyperbilirubinemia, this could result in low enzyme activity. Similarly, the activity of GSH transferase is reportedly determined by the serum levels of GSH (a cofactor for the enzyme activity). The significance of this observation vis-à-vis our research finding may not be entirely known currently because the level of GSH in our population of SCA patients has not yet been studied. Our findings appear to be in conflict with some earlier reports; whereas, Ezeriuaku et al. observed a significantly increased activity in SCA, Chiekezie and Silva independently reported no significant difference in serum activity between HbSS and controls.,, A careful analyses of the above previous studies revealed marked differences in study design compared with the present study; the study by Ezeriuaka et al. consisted of a significant pediatric population (as young as 2 years of age), whereas the study subjects in that of Chikezie and Silva were on medications at the point of recruitment (fansidar/quinine and hydroxyurea, respectively). This may have contributed in the observed discrepancies with the findings of the present study because our subjects were all adults and none was on similar medications as in the previous studies.
It is very important that optimal antioxidant status is maintained, particularly in SCA, so as to prevent significant oxidant tissue damage. Amer et al. had previously shown that sickled red cells generate two-fold greater quantities of superoxide, hydrogen peroxide, and hydroxyl radicals than normal cells. ROS cause toxic cellular injury in the form of oxidation of haemoglobin, and cellular membrane as well as deoxyribonucleic acid damage. Oxidized haemoglobin forms a number of intermediate compounds such as methemoglobin (MetHb), reversible hemichromes, and irreversible hemichromes (iHCRs). These intermediates (particularly MetHb and iHCRs) bind to spectrin, leading to the formation of cross-linked hemoglobin-spectrin complexes and alteration of the cytoskeletal organization of the red cell lipid bi-layer. This leads to altered membrane potential and fluidity, with eventual cell lysis. Interestingly, these changes have been reported to occur even in subjects in steady state. In addition, oxidation of hemoglobin causes methaemoglobinemia with resultant reduced oxygen delivery and tissue hypoxemia; reduced tissue oxygen tension is a recognized trigger for red cell sickling and other related complications in subjects with SCA.,
The GSH transferase enzyme systems play significant roles in protecting against oxidant stress and other potentially toxic and carcinogenic electrophilic compounds by conjugating these substances to GSH., These enzyme systems include GSH peroxidases, GSH S-transferases and the GSH-S-conjugate efflux pumps, which function in an integrated fashion to allow cellular adaption to oxidative stress. Other line of body antioxidant defense is thought to be provided by the superoxide dismutase and catalase systems. These defenses may however not function optimally in certain conditions, such as in selenium deficiency (selenium is a cofactor for GSH peroxidase, therefore serum activity may become impaired) and in a number of infective processes., Manafa et al. had earlier shown that serum selenium concentration was significantly lower in SCA patients in Nnewi (our study site) compared with controls. Despite these findings, our study did not show any significant correlation between serum GSH tranferase activity and disease severity score, in SCA subjects [Figure 1]. This could imply that the other mediators of antioxidant defense in our patients are probably working in optimal capacity to ensure that subjects with low GSH transferase activity did not present with more adverse disease outcome. The evaluation of the serum activity of superoxide dismutase, catalase, and GSH peroxidase in our patient population will be an interesting subject of future research.
This study observed no significant difference in serum levels of Vitamin E in HbSS compared with HbAA and HbAS subjects (P = 0.48 and 0.50, respectively). This contrasts with a number of earlier studies which reported decreased levels of Vitamin E in sickle cell disease (SCD) patients., This is thought to be due the depletion of the vitamin in proportion to the severity of oxidative stress and nutritional status in SCD., It has also been reported that transfusional iron overload, which occurs in SCD, may increase the potential for oxidative damage and reduced Vitamin E antioxidant capacity. Hydroxyurea therapy has been shown to boost antioxidant capacity by mitigating the tendency for red cell sickling, thereby ameliorating oxidative stress., None of the subjects in this study was however on treatment with hydroxyurea at the point of recruitment.
| Conclusion|| |
The low GSH tranferase activity observed in our population of SCA subjects does not appear to impact negatively on disease severity. This could be as a result of the activity of other antioxidants in our patient population or that our data set was not powered enough to detect this impact on disease severity. In addition, the absence of significant difference in serum Vitamin E level of HbSS compared with HbAA subjects in this study could imply reduced Vitamin E degradation (by oxidant stress) in the former group, most probably due to the protective effect of other antioxidant defense systems in the body.
Strength of the study
This is the first (published) study that evaluated antioxidant status in comparison with objective scores of disease severity in Nigerian subjects with SCA. Even though the result is a negative one, we believe it should open up new vista of further research and intellectual brainstorming on this topic.
Limitation of the study
This study is limited by the small number of SCA subjects as well as the noninclusion of assay for serum bilirubin.
Future lines of study
- To replicate this study using larger number of SCA subjects, in steady state and in crises
- To evaluate serum activity of first-line of antioxidant defense (superoxide dismutase, catalase and GSH peroxidase systems) in steady state SCA subjects.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]