SJMLS The Antioxidant Status of Patients with Sickle Cell Anaemia in the Niger Delta Region of Nigeria

Transcription

1 Sokoto Journal of Medical Laboratory Science 1; 1(1): - Orginal Article SJMLS-1-1- The Antioxidant Statu of Patient with Sickle Cell Anaemia in the Niger Delta Region of Nigeria Ezeiruaku, F. C. 1, Eze, E. M. and Ukaji, D. C. 3 Department of Medical Laboratory Science, Faculty of Baic Medical Science, College of Health Science, Niger Delta Univerity, Bayela State, Nigeria 1, Department of Medical Laboratory Science, Faculty of Science, River State Univerity of Science and Technology, Nkpolu, Port Harcourt, Nigeria, Department of Medical Laboratory Science, Faculty of Baic Medical Science, Univerity of Nigeria, Enugu Campu, Enugu, Nigeria 3. Correponding Author: feddyezeh@yahoo.com; Abtract Sickle cell Anaemia (SCA) i an inherited blood diorder that i aociated with everal complication, including oxidative tre and generation of free radical. Thi cla of haemoglobinopathy caue a diruption of the normal activitie in different ytem. The role of the antioxidant compound in the treatment of certain pathophyiological dyfunction ha been of interet in the management of the dieae. Thi tudy invetigated the level of the antioxidant vitamin A, C, E and the enzyme uperoxide dimutae, glutathione peroxidae and catalae in the three human haemoglobin electrophoretic pattern (HBAA,HbAS and HbSS) in the Niger Delta region of Nigeria. Thi i with a view to acertain the influence of ickling phenomenon on the activitie of thee antioxidant compound. A total of 13 ubject were included in thi tudy. Subject included 3 HbSS, HbAS and HbAA individual of different gender and age bracket. The antioxidant vitamin A, C, E and the antioxidant enzyme were meaured uing the Enzyme linked immunoorbent Aay (ELISA). The reult howed a ignificantly (p<.) lower level of the antioxidant vitamin A,C, E and the antioxidant enzyme in HbSS ubject compared to thoe who were HbAA and HbAS. There wa no ignificant difference (p>.) in the value of thee antioxidant compound in HbAA and HbAS ubject. There wa no gender and age- related effect on the value obtained for the variou antioxidant parameter. The decreae in the level of the compound etimated howed a poitive correlation in ickle cell patient with repect to the control group. Sickle cell anaemia i aociated with different pathophyiological condition with altered oxidative tre and thi could account for the lower level of the antioxidant vitamin and enzyme, uggeting a therapeutic approach that can be applied in the management of the dieae. Key word: Sickle cell anaemia; Antioxidant; Oxidative tre; Haemoglobinopathy; Catalae; Superoxide dimutae; Glutathione peroxidie; Haemoglobin electrophorei; Vitamin. Introduction Sickle cell dieae i the mot common genetic diorder in peron of African Origin (Huiman, 181) and the diorder comprie a pectrum of yndrome that range from the almot completely benign trait or carrier tate (HbAS) to the mot evere yndrome, the ickle cell anaemia due to homozygou preence of haemoglobin S (HbSS). Sickle cell anaemia, an inheritance of mutant haemoglobin gene from both parent i globally widepread. According to World Health Organiation (WHO) report,, about % of the world population carrie the gene reponible for haemoglobinopathie and each year about 3, infant are born with major haemoglobin diorder. Sickle cell anaemia ha it highet prevalence in tropical Africa where approximately % of the population are carrier of the ickle cell gene (Beutler, ). In Nigeria, the mot populou country in the ub region with about 1 million inhabitant (Nigeria Cenu, ), 4% of the population are carrier of the mutant gene and the prevalence of the ickle cell anaemia i about per 1 birth (Faola et al., ). There ha been a growing body of evidence uggeting that an increae in oxidative tre and Page Inaugural Edition: Volume 1: Number 1 July 1

2 abnormal oxidant/ antioxidant balance are implicated in the pathophyiology of everal dyfunction oberved in ickle cell anaemia (Nur et al., 11; Lamia et al., 1) reulting in variou haematological and biochemical change (Odievre et al., 11) that include production of reactive oxygen pecie (ROS) like uperoxide radical, hydrogen peroxide, hydroxyl radical and nitric oxide (Yohikawa et al., ). The red blood cell reoxygenation phae in ickle cell anaemia i a major ource of free radical production. During thi period, normal RBC can generate a ignificant amount of uperoxide due to an electron tranfer between the heme iron and oxygen (Alan et al., ). The intracellular polymeriation of HbSS during deoxygeneration i the primary pathogenic event in ickle cell anaemia. Polymeriation can tranform a normal red blood cell into a dene, inflexible blood cell (Uzunova et al., 1). Thi phenomenon in ickle cell dieae caue harmful pathophyical effect, including ickling, vao-occluion, and ichemia-reperfuion injury. Thee complication have a cyclic nature which involve the generation and impairment of oxidative tre (Morri et al., 8). In addition to the oxygen pecie that are formed in ickle cell dieae, the protective mechanim uch a antioxidant are decreaed. Thoe that provide enzymatic defence, including uperoxide dimutae (SOD), Glutathione peroxidie (GPX), Catalae (CAT) and hemeoxygenae-i a well a thoe that cavenge free radical uch a glutathione, vitamin A,C and E are mot affected (Natta et al., 1). Antioxidant capacity i an important caue of tiue injury, particularly in patient with elevated oxidative tre (Walter et al., ). Thi neceitated the need to evaluate the antioxidant tatu of patient with the ickle cell dieae in the Niger Delta region of Nigeria. Thi i with a view to determine the effect of the ickling phenomenon on the antioxidant particularly the enzyme o a to etablih it uniquene in ickle cell anaemia with repect to thoe ubject that are carrier (HbAS) and thoe with normal haemoglobin (HbAA). Study Area The ample for thi tudy were collected from ubject in Bayela, and River State, South-South region of Nigeria. Sample were collected pecifically from the ickle cell centre, Federal Medical Centre in Yenegoa, the Niger Delta Teaching Hopital, Okolobiri and the Univerity of Port Harcourt Teaching Hopital, Port Harcourt. Study Subject A total of 13 participant contituted the ubject for thi tudy. Subject included 3 ickle cell dieae patient (4 male and female) in a teady tate, (8 male, 3 female) with HbAS and (3 male, 3 female) with normal haemoglobin HbAA. Subject were of varying age and gender, but are age- matched for the different haemoglobin electrophoretic pattern. All the ample were collected from the ame environment. For all ubject, ocio-demographic data and clinical examination finding were recorded uing a tandard form. Excluded from the tudy were ickle cell anaemia patient with acute febrile illne, vaooccluion cae and patient who have had a recent blood tranfuion. Method The haemoglobin electrophoretic pattern of each ubject wa confirmed uing haemoglobin electrophorei. The enzyme linked immunoorbent aay (ELISA) wa ued to etimate the antioxidant enzyme (Superoxide dimutae, Glutathione peroxidie and Catalae) and the vitamin A, C, and E. The Elabciece Biotechnology Company Ltd (ELISA) kit (Include Country of manufacture) wa pecifically ued for the tudy. The component of the ELISA kit were pecifically deigned to analye the antioxidant enzyme and the vitamin. It applie to in-vitro quantitative determination of the enzyme and vitamin concentration in plama (Uotila et al., 181; Peter et al., 1). Sample Collection About ten millilitre (1ml) of venou blood wa obtained from the ubject. A portion of the blood wa added to the EDTA anticoagulant tube for haemoglobin electrophorei. Another portion wa added to the lithium heparin tube for the variou enzyme and vitamin aay. Thi lithium heparin tube wa eparated by centrifugation at 3 rpm for 1minute at room temperature and the plama ued for the variou vitamin and enzyme etimation. The eence and detail of the tudy were explained to the ubject and their guardian and verbal informed conent wa obtained from them after approval from the management of the hopital. Statitical Analyi The data are expreed a mean ± tandard deviation. The paired-tet (tet of ignificance) wa done uing the tudent t-tet to compare the group. Difference were conidered ignificant at p<. (% confidence level). Correlation between the group tudied wa teted uing the regreion Inaugural Edition: Volume 1: Number 1 July 1 Page 3

3 analyi and analyi of variance (ANOVA). The reult were conidered tatitically ignificant at % confidence level (p<.). Reult The reult howed a ignificant decreae (p<.) in the antioxidant parameter in HbSS ubject, but howed no ignificant difference (p>.) between the value obtained for HbAA and HbSS ubject. The reult howed a reduction of 4.3%,.%, and 48.1% in the level of antioxidant compound, Vit. A, C and E repectively in HbSS ubject from HbAA ubject. Alo a reduction of 33.8%, 4.3% and 4.8% in the level of antioxidant enzyme, SOD, GPX and CAT repectively in the ickle cell anaemia with repect to the control group (HbAA). Table 1 how the mean ±S.D reult for the variou antioxidant compound in the three human haemoglobin electrophoretic pattern. The reult howed that the difference in the variou antioxidant compound baed on gender were not tatitically ignificant (p>.) in the different haemoglobin electrophoretic pattern. Table how the mean + S.D reult for the variou antioxidant compound in the three human haemoglobin electrophoretic pattern with repect to the gender of the ubject. The decreae in the level for HbSS ubject that are le than 14year were not tatically ignificant (p>.) from the HbSS ubject that are greater than 14year. Table 3 how the mean + S.D reult for the variou antioxidant compound for the HbSS ubject baed on age group tudied. The decreae in the level of the compound etimated howed a poitive correlation in ickle cell patient with repect to the control group. Table 4 how the Pearon correlation analyi of the relationhip between the antioxidant enzyme level and the elected antioxidant compound in ickle cell anaemia and control group. Table 1: Mean + S.D reult for the variou antioxidant compound in the three human haemoglobin electrophoretic pattern Study No. of VIT. A VIT.C VIT. E SOD (ng/ml) GPX CAT (pg/ml) Group Subject (ug/dl) (mg/dl) (mg/dl) (ug/ml) HbAA.1±1..4±.1.3±.1 1.8±.1 4.3±.1 4.±1.8 HbAS 3.±1.48.±.3.34±.14 8.± ±. 44.8±.84 HbSS 3 41.±1..3±.1.1±.1.±..± ±1.1 CAT: Catalae Table : Mean + S.D reult for the variou antioxidant compound in the three human haemoglobin electrophoretic pattern, with repect to the gender of the ubject. Study Gende No. of VIT. A VIT.C VIT. E SOD GPX CAT Group r Subject (ug/dl) (mg/dl) (mg/dl) (ng/ml) (ug/ml) (pg/ml) HbAA M 3 4.3±3.1.8±. 4.38±.1.±4. 3.8±..±1. HbAA F 3.18±..± ±. 3.4± ±. 4.8±. HbAS M 8 1.±18..±. 1.4±.1 3.1±. 3.88± ±1.1 HbAS F 3.4±..±..3±.1 8.±.1 4.±. 4.±1. HbSS M 4 4.1±1.1.31±.1 4.3± ±. 1.±..14±1. HbSS F 44.±1..3±.1 8.1±.1 1.±.8.±.4 1.±11.8 CAT: Catalae Page 4 Inaugural Edition: Volume 1: Number 1 July 1

4 Table 3: Mean + S.D reult for the variou antioxidant compound for the HbSS ubject baed on age group tudied. Study Age No. of VIT. A VIT.C VIT. E SOD GPX CAT Group Group(Year ) Subject (ug/dl) (mg/dl) (mg/dl) (ng/ml) (ug/ml) (pg/ml) HbSS ±1.4.31±..1±. 18.1±. 1.84± ±1.4 HbSS > ±13..3±.1.1±.3.8±..31±. 1.1± CAT: Catalae Table 4: Pearon correlation analyi of the relationhip between the antioxidant enzyme level and the elected antioxidant compound in ickle cell anaemia and control group. Antioxidant compound R-Value Vitamin SOD GPX CAT Vitamin A.1*.8*.3* Vitamin C.11*.*.34* Vitamin E.4*.314*.3* SCA Patient (N=3) control (N=) Value in the table are Pearon correlation coefficient (r) *p <., indicate poitive correlation CAT: Catalae Dicuion Sickle cell dieae i an inherited diorder in which the red blood cell change hape in a characteritic way. It i emerging a an important model of oxidative tre. Since red blood cell carry oxygen to the blood tiue, they are already rich in oxidative fuel. According to Singer and Singer, 13, the HbS bind oxygen normally when in dilute olution (oxyhaemoglobin) but in deoxyhaemoglobin form, it polymerie giving rie to gel formation. Chronic oxidative tre reulting from an imbalance between the production of reactive oxidant pecie (ROS) and antioxidant enzyme contitute a critical factor in the different organ dyfunction. Thi tudy evaluated the plama level of vitamin A, C, E and the antioxidant enzyme, SOD, GPX and CAT a indicator of antioxidant tatu. The reult of thi tudy howed a ignificantly lower level of the antioxidant compound. Our finding i in agreement with everal tudie (Haanato, ; Ray et al., ; Foluke et al., 8; Emokpae et al., 1) both in Nigeria and other countrie. Some previou tudie reported conflicting reult regarding the level of thee antioxidant enzyme in ubject with ickle cell anaemia. Adelakun and colleague (14) reported that there wa no ignificant difference in SOD and CAT level in ickle cell patient with repect to control ubject. Alo, Hundekar and colleague (11) reported an increaed level of SOD in patient with ickle cell anaemia. A contrating tudy (Schacter et al., 188) howed that SOD level decreaed in proportion to dieae everity in HbS patient compared with healthy volunteer. Reduction in the plama antioxidant enzyme may be due to the everity of the enzyme being degraded by oxidant during ickling and from oxygen radical. In thi tudy, the vitamin were ignificantly lower in the ickle cell ubject compared to control group (HBAA). Thi finding i conitent with the finding of previou tudie (Wali et al., 13; Wright et al., 14). It ha been uggeted that the low level may be a a reult of diturbed metabolim of thee vitamin. Hydrogen peroxide (H O ) i produced in ickle cell dieae either from electron tranfer or due to ickling. Thi equally i removed by two method, either GPX or catalae. Due to the Inaugural Edition: Volume 1: Number 1 July 1 Page

5 decreaed level of thoe enzyme in ickle cell patient, the endogenouly produced H cannot be ufficiently cleared (Alan et al., ). The reduction in the level of thee antioxidant enzyme in relation to the antioxidant vitamin tudied howed a poitive correlation at % confidence level (p<.). Thi i a a reult of both the antioxidant enzyme and the vitamin been ignificantly (p<.) reduced in ickle cell anaemia. There wa no ignificant difference in the reult obtained for the HbAA and HbAS ubject. Thi howed that the heterozygote nature in HbAS did not impact any ickling phenomenon or oxidative tre that can lead to a reduction in the antioxidant compound tudied. Alo gender and age did not indicate any ignificant difference in the parameter tudied, both in the control group and the homozygote haemoglobin S ubject. Concluion The reult of thi tudy in the Niger Delta Region of Nigeria indicated that the ickle cell ubject exhibited an alteration in the level of variou antioxidant compound tudied. It howed that the antioxidant vitamin were ignificantly reduced by 4.3%,.% and 48.8% repectively for vitamin A, C and E in the ickle cell anaemia patient with repect to the control group HbAA. The antioxidant enzyme, SOD, GPX and CAT were reduced by 33.8%, 4.3%, and 4.8% repectively in comparion to the control ubject. Sex and age had no effect on the parameter tudied. The dieae i aociated with different pathophyiological condition with altered oxidative tre and thi could account for the lower level of thee antioxidant vitamin and enzyme. Thi tudy indicate that the antioxidant enzyme and vitamin can be effective therapeutic target or trategie for the management of ickle cell dieae. Funding Source Thi tudy wa upported in part by De-integrated Medical Diagnotic and Reearch Centre Port- Harcourt. Reference 1. Adelakun, A., Ajani, O., Ogunleye, T., Diu, E., Kooko, A. and Arinola, G. (14) Repiratory burt enzyme and oxidant antioxidant tatu in Nigeria children with ickle cell dieae. Britih Biotechnology Journal; 4 (3): -8.. Alan, M., Thornley Brown, D. and Freeman, B. A. () Reactive pecie in ickle cell dieae. Annal New York Academy of Science; 8: Beutler, E. (). Diorder of haemoglobin tructure. Sickle Cell Anemia and related abnormalitie. Overview in Lichtman M.A. Beautler E, Kipp TJ eligohn U,Kauhanky,K, Prechal,J. editor william Haematology th Ed. Mc Craw-hill medical Education: New Tork New York: Emokpae, A.M., Ojiefo, U.P. and Aiha, K. G (1). Anti-oxidant enzyme and acute phae protein. Correlate with marker of lipid peroxide in adult Nigeria ickle cell dieae patient.. Iran Journal of Baic Science; 13: Faola, F., Adedapo, K., Anetor, J. and Kuti, M. () Total anitioxidant tatu and ome haematological value in ickle cell dieae patient in teady tate. Journal National Medical Aociation; (8): Foluke, F., Kayode, A., Johan, A and Modupe, K. (8) Antioxidant tatu in ickle cell dieae patient in teady tate. Journal National Medical Aociation; 1: Haanato, R.M. () Zinc and antioxidant vitamin deficiency in patient with evere ickle cell anemia. Annal of Saudi Medical; : Hundekar, P., Suryakar, A.N., Karnik, A.C., Valvi, R., Ghone., R.A and Bhagat, S.S. (11). The effect of antioxidant upplementation on the oxidant and antioxidant tatu in ickle cell anemia. Journal of Clinical Diagnotic Reearch; (): Huiman, J. H. J. (181). Sickle Cell anemia a a yndrome. A review of diagnotic feature. American Journal of Haematology; : Lamia, M, A., Mae äd, K.H and Jawad, K.M. (1). Aociation of erythrocyte antioxidant enzyme and their cofactor with marker of oxidative tre in patient with ickle cell dieae. Qatar Medical Journal; : Morri, C.R., Suh, J.H., Hagar,W., Lankin, S., Bland, D.A., et al (8). Erythrocyte glutamine depletion, altered redox environment and pulmonary hypertenion in ickle cell dieae. Blood; III: Natta, C.I., Chen, L.C. and Chow, C.K (1). Selenium and glutathione peroxidae level in ickle cell anemia. Acta Haematology; 83: Nur, E., Biemond, B.J., Otten, U.M., Brandje, D.P. and Schnong, J.J. (11). CURAMA tudy group, oxidative tre in ickle cell dieae; Pathophyology and potential implication for dieae management. American Journal of Haematology; 8(): Odievre, M.H., Vergar,E., Silver- Pinto, A.C and Elion J. (11). Pathophyiological inight in ickle Page Inaugural Edition: Volume 1: Number 1 July 1

6 cell dieae. Indian Journal of Medicine and Reearch 134 (4): Peter, H., Scott, E.W. and Steven, A.T (1). Enzyme Linked immune orbent aay (ELISA). current protocol in molecular Biology II. 1. Ray, D., Dehmukh, P., Gowami, K. and Garg, N. (). Antioxidant vitamin level in ickle cell diorder. National Medical Journal of India; (1) Schacter, L., Warth, J.A., Gordon, E.M., Praad, A. and Klein, B. L. (188) Altered amount and activity of uperoxide dimutae in ickle cell anaemia. FASEB Journal; : Singer, K. and Singer, L. (13). Studie on abnormal haemoglobin. The gelling phenomenon of ickle haemoglobin it biologic and diagnotic ignificance. Blood; 8: Uotila, M., Ruoiahti, E. and Engrall, E. (181). Enzyme Linked immunoaay. Journal of Immunological Method; 4: Uzunova, V.V., Pan, W., Galkin, O. and Vekilov, P.G. (1). Free heme and the polymeriation of ickle cell haemoglobin. Biophyical Journal; : Walter, P.B., Fung, E.B., Killilea, D.W., Jiang, Q., Hude, M., Madlen, J., Evan, P.,Vichinky, E. and Harmatz, P. () Oxidative tre and inflammation in iron-overloaded patient with beta-thalaaemia or ickle cell dieae. Britih Journal of Haematology; 13() Wali, U., Yeldu, M.H., Samaila, B. and Jiya, N. M. (13) Serum antioxidant vitamin level in children with ickle cell anaemia in Sokoto. Bayero Journal of Pure and Applied Science (1): World Health Organiation report (). Secretariat report on ickle cell anaemia. Geneva.. Wright, D., Reid, M, and Stenneth, R. (14) Antioxidant intake in ickle cell dieae. International Journal of Clinical Nutrition; (3):3-.. Yohikawa, T., Toyokuni, S., Yamamoto, Y.and Naito, Y. (). Free radical in chemitry, Biology and Medicine. London, UK: OICA International: 8. Inaugural Edition: Volume 1: Number 1 July 1 Page