|Year : 2016 | Volume
| Issue : 2 | Page : 100-105
Impact of Vitamins C and E supplement on anti-oxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) and lipid peroxidation product (malondialdehyde levels) in sickle subjects
Okot-Asi Thomas Nku-Ekpang1, Ofem Effiong Ofem1, Victor Otu Oka1, Smith I Jaja2
1 Department of Physiology, College of Medical Sciences, University of Calabar, Calabar, Nigeria
2 Department of Physiology, College of Medical Sciences, University of Lagos, Lagos, Nigeria
|Date of Web Publication||5-Jul-2016|
Ofem Effiong Ofem
Department of Physiology, College of Medical Sciences, University of Calabar, Calabar
Aim: This study sought to investigate the effect of combined supplements of Vitamins C and E glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) malondialdehyde (MDA) levels in sickle cell and non-sickle cell patients. Materials and Methods: A total of 27 subjects (16 controls and 11 test subjects) were used for this study. The sickle cell subjects (test) were obtained among the sickle cell patients who visited the out-patient clinic of the Lagos University Teaching Hospital (LUTH) for check-up, their ages ranged from 18 to 27 years. Venous blood samples of the subjects were carefully withdrawn using disposable syringes and needles at the sickle cell anemia (SCA) clinic, LUTH, Nigeria. Vitamins C and E supplementation commenced on the 1 st day of blood collection and lasted for 6 weeks. All the subjects received Vitamin E (1000 IU) in soft gelatinous capsule once daily and 200 mg of Vitamin C orally per day for 42 days. Blood was collected from each subject under aseptic procedure and MDA was determined to assess lipid peroxidation enzyme activity while GPx, SOD, and CAT were estimated for antioxidant enzymes' activity. Results: Results revealed significantly (P < 0.01) lower basal levels of SOD, GPx, and MDA in sickle (SCA) compared with non-sickle anemia (NSCA). After 42 days of Vitamins C and E supplementation, SOD, GPx, and MDA increased significantly in both groups, but the increase was more in SCA compared with NSCA subjects (P < 0.01). Conclusion: Hence, it was found that Vitamins C and E supplementation did not reduce lipid peroxidation enzymes (MDA) activity in SCA subjects despite the increase in scavenging enzymes' (SOD and GPx) activities in SCA.
Keywords: Anti-oxidant enzymes, lipid peroxidation product, sickle cell anemia
|How to cite this article:|
Nku-Ekpang OAT, Ofem OE, Oka VO, Jaja SI. Impact of Vitamins C and E supplement on anti-oxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) and lipid peroxidation product (malondialdehyde levels) in sickle subjects. Trop J Med Res 2016;19:100-5
|How to cite this URL:|
Nku-Ekpang OAT, Ofem OE, Oka VO, Jaja SI. Impact of Vitamins C and E supplement on anti-oxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) and lipid peroxidation product (malondialdehyde levels) in sickle subjects. Trop J Med Res [serial online] 2016 [cited 2020 Jul 9];19:100-5. Available from: http://www.tjmrjournal.org/text.asp?2016/19/2/100/185427
| Introduction|| |
Sickle cell anemia (SCA) is a hereditary blood disorder that causes chronic destruction of red blood cells with higher potential for oxidative damage due to chronic redox imbalance in red cells.  SCA was the first disease to be characterized at the molecular level.  Oxidative stress has been reported in the etiology of sickle anemia. ,,
Normally, oxidation reactions can produce free radicals, which in turn can start chain reactions that damage cells of the body, antioxidants are molecules capable of inhibiting the oxidation of other molecule. Antioxidants terminate these chain reactions by removing free radicals intermediates and inhibit other oxidation reactions.  Low levels of antioxidants or inhibition of the antioxidant enzymes cause oxidative stress and may damage or kill cells.  Antioxidants include Vitamin C and Vitamin E as well as enzymes within the body such as catalase (CAT), superoxide dismutase (SOD), and various peroxidases. 
Vitamin C (ascorbic acid) helps to maintain healthy collagen in the skin, repair damaged tissues, boost immune system, and promote healthy teeth and bones. This vitamin is one of the most powerful and well-known antioxidants.  It is a free radical fighter which helps ward off wrinkles and many illnesses linked to oxidation. Vitamin C can be regenerated to an antioxidant form of Vitamin E. 
Vitamins C and E supplements have been observed to increase markers of hemolysis in SCA patients following a randomized, double-blind, placebo-controlled trial.  Vitamin E (alpha tocopherol) has been claimed to be the most important fat-soluble antioxidant that protects the cell membrane forms oxidation by reacting with lipid radicals produced in the lipid peroxidation chain reaction. ,
Lipid peroxidation is a well-established mechanism of cellular injury in both plants and animals; lipid peroxidation is used as an indicator of oxidative stress in cells and tissues. The measurement of malondialdehyde (MDA) and 4-hydroxyalkenals has been used as an indicator of lipid peroxidation.  It is important, therefore, to investigate the possible effects of Vitamins C and E supplementation on the concentrations of some antioxidant enzymes (SOD, CAT, and glutathione peroxidase [GPx]) and lipid peroxidation product (MDA) in sickle patients.
The aim of this study was to compare the effect of supplements with Vitamins C and E on antioxidant enzymes (SOD, CAT, and GPx) and lipid peroxidation enzyme (MDA) levels in sickle cell and non-sickle cell subjects.
| Materials and Methods|| |
A total of 27 subjects (16 controls and 11 test subjects) were used for this study. The healthy control subjects (nonsickle cell anemic [NSCA] control) were medical students of the College of Medicine of the University of Lagos who never had symptoms of SCA. Their ages ranged between 19 and 30 years. Eleven of them were males whereas five were females. The SCA subjects (test) were obtained among sickle cell patients who visited the sickle cell clinic of the Lagos University Teaching Hospital (LUTH) for their routine checks and to obtain their medication, hydroxycarbamide/hydroxyurea (HU). Their ages ranged from 18 to 27 years. Seven of them were females and four were males.
Ethical approval was obtained from the College Ethical Committee, and all the subjects were handled according to the standard procedure.
Venous blood samples of the subjects were withdrawn using disposable syringes and needles under aseptic technique. The collection of blood was done at the SCA clinic, LUTH, for sickle cell subjects and in the research laboratory of the Physiology Department, LSU, Nigeria, for the control subjects. From each subject, 4 mL of blood was collected from the antecubital vein of the right arm by a medical doctor into plain sample bottles and sera were extracted for the assay of SOD, CAT, GPx, and MDA.
Administration of experimental drugs
Vitamin C (100 mg) tablets (manufactured by Emzor Pharmaceuticals, Lagos, Nigeria) and Vitamin E (1000 IU) capsules (manufactured by International Health Services; Baltimore, Maryland) were purchased from BEZ Pharmacy, Etta Agbor Road, Calabar, Nigeria.
Vitamins C and E supplementation commenced on the 1 st day of blood collection and lasted for 6 weeks. The subjects were given a daily dose of 1000 IU (i.e., 833.7 mg) of Vitamin E in soft gelatinous capsule form and also received daily doses of 200 mg tablets of Vitamin C orally per day, the treatment regimens lasted for 42 days (6 weeks).
Determination of lipid peroxidation
MDA, a secondary product of lipid peroxidation, was measured spectrophotometrically as an evidence of lipid peroxidation by the method of Reilly and Aust. 
Determination of antioxidant enzymes
GPx activity was measured by a method described by Ates et al.  The optical densities were read at 412 nm using Optima Spectrophotometer, SP 3000 Plus.
GPx (μmol/mL) was derived from the equation:
Where, ∑ (molecular extinction coefficient) = 13,600
V (total reactive volume) = 4.5 mL
v (volume of sample) = 0.1 mL.
SOD was assayed following the technique of Kakkar et al.  The optical density/absorbance was read at 480 nm using Optima Spectrophotometer, SP 3000 Plus, Japan. Initial reading was taken at 0 min, 1 min, 2 min, and 3 min.
where, ∑ (molecular extinction coefficient) = 4020
V (total reactive volume) = 3.5 mL
V (volume of sample) = 0.02 mL.
CAT was assayed colorimetrically at a 620 nm absorbance reading and expressed as μmoles of hydrogen peroxide (H 2 O 2 ) consumed per minute per milligram protein as described by Sinha.  The readings were taken spectrophotometrically at 620 nm at 0 min, 1 min, 2 min, and 3 min.
CAT (μmol/mL) =
[change in absorbance per minute/ε] × [V/v] × 10 3
Where ε (molar extinction coefficient) = 40
V (total reactive volume) = 1.50 mL
V (volume of sample) = 0.10 mL.
Values were presented as mean ± standard error of mean. Before and after comparison was done using paired Student's t-test, analysis involving more than two groups was done using one-way ANOVA, then followed with a least significant difference post hoc test. The software used for the analysis was SPSS version 15.0, Microsoft Inc., Chicago, IL, USA) and also Excel for windows 8, P < 0.05 was considered statistically significant.
| Results|| |
Baseline values of SOD (μmol/mL) show a significant (P < 0.001) reduction in SCA compared with NSCA. After Vitamins C and E supplementation, SOD levels increased significantly (P < 0.001) above the baseline both in SCA and NSCA, the increase was higher in SCA compared with NCSA [Table 1] and [Figure 1].
|Figure 1: Comparison of superoxide dismutase concentration before and after Vitamins C + E supplements in sickle cell anemic and non-sickle cell anemic subjects. Values are expressed as mean ± standard error of mean. ***P < 0.001 versus before|
Click here to view
|Table 1: Summary of antioxidant enzymes levels and lipid peroxidation before and after vitamin supplement in non-sickle cell and sickle cell anaemic subjects |
Click here to view
The basal levels of CAT (μmol/mL) were significantly lower in SCA compared with NSCA. The levels of CAT significantly (P < 0.01) reduced following Vitamins C and E supplementation in both SCA and NSCA compared with the basal levels. CAT levels in SCA and NSCA were not significant after Vitamins C and E supplementation, although the reduction was more in NSCA [Table 1] and [Figure 2].
|Figure 2: Comparison of catalase concentration before and after Vitamins C + E supplements in sickle cell anemic and nonsickle cell anemic subjects. Values are expressed as mean ± standard error of mean. **P < 0.01 versus before|
Click here to view
As shown in [Table 1], before Vitamins C and E supplementation, GPx levels (μmol/mL) in SCA was significantly (P < 0.01) reduced compared with NSCA, but after vitamin supplementation, GPx levels increased significantly (P < 0.001) in both SCA and NSCA compared to their basal levels, but the increase was significantly (P < 0.01) higher in SCA compared with the NSCA [Figure 3].
|Figure 3: Comparison of glutathione peroxidase concentration before and after Vitamins C + E supplements in sickle cell anemic and nonsickle cell anemic subjects. Values are expressed as mean ± standard error of mean. ***P < 0.001 versus before|
Click here to view
Basal levels of MDA (μmol/mL) in SCA was significantly (P < 0.05) higher compared with NSCA subjects before vitamin supplementation; after vitamins C and E supplementation, MDA levels increased significantly (P < 0.001) in both groups, but the increase was relatively higher in NSCA compared with SCA subjects [Table 1] and [Figure 4].
|Figure 4: Comparison of malondialdehyde concentration before and after Vitamins C + E supplements in sickle cell anemic and nonsickle cell anemic subjects. Values are expressed as mean ± standard error of mean. ***P < 0.001 versus before|
Click here to view
| Discussion|| |
This study attempted to investigate the role of Vitamins C and E supplement on antioxidant enzymes levels and lipid peroxidation in SCA and NSCA subjects. Our findings indicated that the different free radical scavenging enzymes' (SOD, CAT, and GPx) levels and MDA concentrations were significantly lower in the SCA patients relative to the normal subjects. We also observed that Vitamins C and E supplement raised the levels of SOD and GPx levels (while CAT levels were decreased) in both subjects, but the increases in SOD and GPx were remarkable in the sickle cell subjects.
The reduction in the levels of CAT, PGx, and SOD in sickle subjects observed in this study agrees with earlier reports by Manfredini and his colleagues,  but contradicts reports by other investigators, ,, who reported that sickle cell patients have significantly higher GPx and SOD activities than healthy subjects.
From our study, low levels of SOD and GPx in sickle subjects suggest that there is less scavenging activities on the continuously generated reactive oxygen species (ROS), which is a steady state cellular events in respiring cells,  and less presence of these antioxidants is grossly amplified in response to a variety of pathophysiology conditions such as inflammation, immunological disorders, hypoxia, hyperoxia, metabolism of drugs or alcohol, and exposure to ultraviolet radiation in sickle cell patients.  Antioxidant enzyme defense system in SCA subjects is low and oxidative stress can develop because of the imbalance between enhanced generation of ROS and low cellular content of antioxidants. , Reduction in the levels of GPx observed in the sickle cell subjects will hinder their normal functions of disposing organic peroxides rather than removal of H 2 O 2 and may not protect the membrane and hemoglobin from peroxidative damage. 
The increase in SOD and GPx levels in these subjects following Vitamins C and E supplementation could boost redox reactions, thereby removing ROS, which is a steady-state cellular event in respiring cells, thereby terminating their harmful activity.  The increase in these antioxidant enzymes will also reduce the damage of cellular macromolecules (DNA, protein, and lipids). , The combined supplement shows enhanced levels of SOD and GPx, which scavenge the free radicals, reduce H 2 O 2 concentration in blood via SOD reaction with superoxide ion to form H 2 O 2 , which is then removed by GPx or CAT. 
On the other hand, this study will add to the existing knowledge that low levels of CAT observed in both study groups after Vitamins C and E supplement are rather compensated by elevated levels of SOD and GPx.
The basal levels of malondialdehyde (MDA), which are products of lipid peroxidation, were observed to be significantly lower in SCA than in NSCA subjects, but after Vitamins C and E supplement, MDA levels in SCA rose more than levels in NSCA. The reduced basal levels of MDA in sickle cell patients observed in this study contradicts earlier reports,  in which higher basal levels of MDA in SCA patients were documented. The reason these researchers proffered for the possible increase in MDA in sickle cell patients was auto-oxidation of unstable hemoglobin, which produces superoxide radicals in addition to the presence of high erythrocyte iron. 
The reduction in MDA levels of SCA in our study could probably be due to the drug HU given to SCA subjects in the clinic. HU has been implicated in alleviating complications of SCA such as vaso-occlusive painful crisis.  The high levels of MDA in both SCA and NSCA subjects following Vitamins C and E supplement observed in this study are contrary to the previous findings that Vitamin C supplement reduced peroxidative damage (i.e., reduced MDA levels) in SCA subjects and non-sickle cell subjects ,, and that Vitamin E supplement reduced lipid peroxidation. ,,
Previous studies have also shown that in sickle cell patients, there is depletion of Vitamins C and E, , which could worsen the state of sickling in sickle cell patients as a result of more susceptibility to oxidative stress. ,, Hence, there is a need for antioxidant vitamin supplementation in sickle cell patients.
Vitamin C is a vital antioxidant, it is a reducing agent that decreases the production of lipid peroxidation products such as MDA.  However, Vitamin E as a useful antioxidant vitamin may not be effective to prevent cellular damage alone by inhibiting the peroxidation of polyunsaturated fatty acids contained in cellular and sub-cellular membrane phospholipid which may be attributed to insufficient antioxidant enzymes. Hence, the cells of these subjects who received vitamin supplements may still be prone to cellular damage, lipid peroxidation, and the consequent effects of increased cell rigidity, inactivation of membrane-bound receptors and enzymes,  degradation of proteins, and the promotion of DNA-stand breakage,  which culminates in oxidative stress.
| Conclusion|| |
Vitamins C and E combined supplement reverses the reduced levels of free radical scavenging enzymes (SOD and GPx) activity and also leads to enhanced lipid peroxidation activity (MDA level) in SCA subjects, hence lipid peroxidation activity in cells of SCA subjects was not attenuated despite increases in free radical scavenging enzymes activities in SCA following Vitamins C and E supplementation.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Smith WR, Penberthy LT, Bovbjerg VE, McClish DK, Roberts JD, Dahman B, et al.
Daily assessment of pain in adults with sickle cell disease. Ann Intern Med 2008;148:94-101.
Strasser BJ. Linus pauling's "molecular diseases": Between history and memory. Am J Med Genet 2002;115:83-93.
Conran N, Franco-Penteado CF, Costa FF. Newer aspects of the pathophysiology of sickle cell disease vaso-occlusion. Hemoglobin 2009;33:1-16.
Silva DG, Belini Junior E, de Almeida EA, Bonini-Domingos CR. Oxidative stress in sickle cell disease: An overview of erythrocyte redox metabolism and current antioxidant therapeutic strategies. Free Radic Biol Med 2013;65:1101-9.
Hierso R, Waltz X, Mora P, Romana M, Lemonne N, Connes P, et al.
Effects of oxidative stress on red blood cell rheology in sickle cell patients. Br J Haematol 2014;166:601-6.
Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O. Oxidative stress and antioxidant defense. World Allergy Organ J 2012;5:9-19.
Baillie JK, Thompson AA, Irving JB, Bates MG, Sutherland AI, Macnee W, et al.
Oral antioxidant supplementation does not prevent acute mountain sickness: Double blind, randomized placebo-controlled trial. QJM 2009;102:341-8.
Arrigoni O, De Tullio MC. Ascorbic acid: Much more than just an antioxidant. Biochim Biophys Acta 2002;1569:1-9.
Huang HY, Appel LJ, Croft KD, Miller ER 3 rd
, Mori TA, Puddey IB. Effects of Vitamin C and Vitamin E on in vivo
lipid peroxidation: Results of a randomized controlled trial. Am J Clin Nutr 2002;76:549-55.
Arruda MM, Mecabo G, Rodrigues CA, Matsuda SS, Rabelo IB, Figueiredo MS. Antioxidant Vitamins C and E supplementation increases markers of haemolysis in sickle cell anaemia patients: A randomized, double-blind, placebo-controlled trial. Br J Haematol 2013;160:688-700.
Herrera E, Barbas C. Vitamin E: Action, metabolism and perspectives. J Physiol Biochem 2001;57:43-56.
Traber MG, Atkinson J. Vitamin E, antioxidant and nothing more. Free Radic Biol Med 2007 1;43:4-15.
Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malondialdehyde and related aldehydes. Free Radic Biol Med 1991;11:81-128.
Reilly CA, Aust SD. Measurement of lipid peroxidation. In: Current Protocols in Toxicology. UK.; John Wiley and Sons, Inc.; 2001.
Ates B, Ercal BC, Manda K, Abraham L, Ercal N. Determination of glutathione disulfide levels in biological samples using thiol-disulfide exchanging agent, dithiothreitol. Biomed Chromatogr 2009;23:119-23.
Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys 1984;21:130-2.
Sinha AK. Colorimetric assay of catalase. Anal Biochem 1972;47:389-94.
Manfredini V, Lazzaretti LL, Griebeler IH, Santin AP, Brandão VD, Wagner S, et al.
Blood antioxidant parameters in sickle cell anemia patients in steady state. J Natl Med Assoc 2008;100:897-902.
Chiu D, Lubin B. Abnormal Vitamin E and glutathione peroxidase levels in sickle cell anemia: Evidence for increased susceptibility to lipid peroxidation in vivo
. J Lab Clin Med 1979;94:542-8.
Dailly E, Urien S, Barré J, Reinert P, Tillement JP. Role of bilirubin in the regulation of the total peroxyl radical trapping antioxidant activity of plasma in sickle cell disease. Biochem Biophys Res Commun 1998;248:303-6.
Halliwell B, Gutteridge JM. Cellular responses to oxidative stress, adaptation, damage, repair, senescence and death. 3 rd
ed. New York: Oxford University Press; 2007. p. 187-267.
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.
Cesquini M, Torsoni MA, Stoppa GR, Ogo SH. t-BOOH-induced oxidative damage in sickle red blood cells and the role of flavonoids. Biomed Pharmacother 2003;57:124-9.
Antunes F, Salvador A, Marinho HS, Alves R, Pinto RE. Lipid peroxidation in mitochondrial inner membranes. I. An integrative kinetic model. Free Radic Biol Med 1996;21:917-43.
Stadtman ER, Berlett BS. Reactive oxygen-mediated protein oxidation in aging and disease. Chem Res Toxicol 1997;10:485-94.
Dröge W. Free radicals in the physiological control of cell function. Physiol Rev 2002;82:47-95.
Charache S, Terrin ML, Moore RD, Dover GJ, Barton FB, Eckert SV, et al.
Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia. Investigators of the multicenter study of hydroxyurea in sickle cell anemia. N Engl J Med 1995;332:1317-22.
Walter PB, Fung EB, Killilea DW, Jiang Q, Hudes M, Madden J, et al.
Oxidative stress and inflammation in iron-overloaded patients with beta-thalassaemia or sickle cell disease. Br J Haematol 2006;135:254-63.
Lu ZH, Steinberg MH. Fetal hemoglobin in sickle cell anemia: Relation to regulatory sequences cis to the beta-globin gene. Multicenter study of hydroxyurea. Blood 1996;87:1604-11.
Jialal I, Fuller CJ, Huet BA. The effect of alpha-tocopherol supplementation on LDL oxidation. A dose-response study. Arterioscler Thromb Vasc Biol 1995;15:190-8.
Cadenas E, Davies KJ. Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med 2000;29:222-30.
Gbenebitse S, Jaja SI, Kehinde MO. Effect of changes in plasma Vitamin E level of vascular responses and lipid peroxidation in sickle cell anaemia subjects. Niger Postgrad Med J 2005;12:81-4.
Heitzer T, Ylä Herttuala S, Wild E, Luoma J, Drexler H. Effect of Vitamin E on endothelial vasodilator function in patients with hypercholesterolemia, chronic smoking or both. J Am Coll Cardiol 1999;33:499-505.
Huang C, Huang Y, Li J, Hu W, Aziz R, Tang MS, et al.
Inhibition of benzo(a)pyrene diol-epoxide-induced transactivation of activated protein 1 and nuclear factor kappaB by black raspberry extracts. Cancer Res 2002;62:6857-63.
Clarkson PM, Thompson HS. Antioxidants: What role do they play in physical activity and health? Am J Clin Nutr 2000;72 2 Suppl: 637S-46S.
Debes D, Kalyan G, Neelam G. Antioxidant Vitamin levels in sickle cell disorders. Natl J India 2007;20:11-3.
Behera S, Dixit S, Bulliyya G, Kar SK. Vitamin A status and hematological values in sickle cell disorder cases. Indian J Med Sci 2012;66:169-74.
Stuart MJ, Nagel RL. Sickle-cell disease. Lancet 2004;364:1343-60.
Switzer JA, Hess DC, Nichols FT, Adams RJ. Pathophysiology and treatment of stroke in sickle-cell disease: Present and future. Lancet Neurol 2006;5:501-12.
Ugonabo MC, Onwuamaeze IC, Okafor EN, Ezeoke AC. Plasma cholesterol of sickle cell anaemia patients in Enugu Nigeria. Bio Res 2007;5:241-3.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]