Total Antioxidant Status and other Antioxidant Agent Levels in Children with P. Falciparum Infection in Calabar, Nigeria

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1 Original Aricle Toal Anioxidan Saus and oher Anioxidan Agen Levels in Children wih P. Falciparum Infecion in abar, Nigeria Akpouzor J. O, Udoh A. E, Eukudo M. H Deparmen of Medical Laboraory Science, Universiy of abar, abar, Cross River Sae, Nigeria. Malaria infecion and is reamen wih animalaria drugs have been repored o generae oxygen free radicals. These free radicals generaed in he cause of infecion and is reamen need o be removed from he body by reducing agens called anioxidans. This sudy is aimed a evaluaing he impac of malaria infecion on oal anioxidan saus of children. By using sandard procedures and kis purchased from Randox laboraories, USA and Quimica Clinica Aplicada Spain, oal anioxidan level, viamin C, Toal and HDL choleserol and albumin were assessed in 122 malaria infeced children. The haemoglobin and parasie densiy saus of he children were also measured. Sixy healhy children were used as conrols. I was observed ha all parameers measured excep he oal choleserol (which was similar in boh malaria infeced children and he conrol subjecs), were significanly lower in malaria infeced children when compared wih he respecive conrol values. Malaria parasiemia correlaed srongly and negaively wih oal anioxidan levels (r= ).Reducion in he level of oal anioxidan was dependen on he severiy of malaria hence he more severe he malaria, he lower he level of oal anioxidan. From his sudy i is observed ha here is a general depression in oal anioxidan levels as well as HDL Choleserol, albumin and viamin C levels suggesing increased consumpion of anioxidans and is agens during malaria infecion. Therefore, anioxidan inervenion may be crucial in he reamen of malaria infecion. Key words: Anioxidan saus, P. Falciparum infecion, malaria, Viamin C Inroducion Malaria is one of he mos devasaing global public healh problems. Abou 107 counries and erriories involving abou 3.2 billion people are sill a risk of malaria aack as a 2004 (World Healh Organizaion (1).Presen esimaes sugges ha around million clinical disease episodes occur annually (2) and around 60% of clinical cases and over 80% of he deahs due o malaria occur in Africa souh of he Sahara (3). Malaria is responsible for an esimaed average annual reducion of 1.3% in economic growh for hose counries wih he highes burden, Nigeria inclusive. Malaria also conribues o anaemia in children and undermines heir growh and developmen. An esimaed one million faaliies per year has been repored in which 85 % of deahs are children (4). Reacive oxygen species (ROS) are generaed by inflammaory cells recruied during infecion.the poenially damaging effecs of oxidaive sress are normally limied by anioxidans ha scavenge ROS in he body (5). However, low dieary inakes of anioxidan viamins or reduced synhesis of nondieary anioxidans such as albumin, bilirubin, gluahione peroxidase choleserol and uric acid are likely o Received: Sepember 30, 2010 Revised: December 29, 2010 Acceped: December 26, 2011 Corresponding auhor : Josephne Akpouzor, Deparmen of medical laboraory science, universiy of abar, abar, Cross-river sae, Nigeria, Tel: josephineakpouzor@ yahoo.com Inernaional Journal of Biomedical Laboraory Science (IJBLS) 2012 Vo1. 1, No. 2:

2 resul in an oxidan-anioxidan imbalance ha exacerbaes inflammaion and issue damage (6). Toal anioxidan saus is defined as he sum oal of endogenous and food derived anioxidans of he exra cellular fluid of an individual (7). Cooperaion of all he differen anioxidans provides greaer proecion agains aack by reacive oxygen or nirogen radicals han any single compound alone. Measuremen of serum oal anioxidan capaciy (TAC) level was repored o provide an inegraed index, as opposed o one based on simple summaion of measurable anioxidans (8). I possibly could be used o assess he real change in anioxidan saus in paiens wih severe infecion and migh lead o universally useful reamen (8). Albumin represens a very abundan and imporan circulaing anioxidan. Albumin has several imporan physiological and pharmacological funcions. I ranspors meals, fay acids, choleserol, bile pigmens, and drugs. I is a key elemen in he regulaion of osmoic pressure and disribuion of fluid beween differen comparmens and is plasma concenraion represens equilibrium no only beween is synhesis in he liver and is caabolism, bu also is ranscapillary escape. I represens he major and predominan anioxidan in plasma, which is he body comparmen known o be exposed o coninuous oxidaive sress. Previous work has shown ha more han 70% of he free radical-rapping aciviy of serum was due o human serum albumin (HSA) as assayed using he free radical-induced hemolysis es (9). Choleserol is carried around in he blood on carrier molecules called lipoproeins. HDL choleserol s main funcion is o ranspor fa from he cell o he liver. When he HDL choleserol level is high, i is more cardio proecive (10). One heory advanced by some scieniss is ha he oal serum choleserol is really an indicaor of he amoun of free radical damage in he body (10). The higher he free radical level, he higher he body needs o produce choleserol inernally from he liver o ac as an anioxidan and free radical scavenger. Plasma viamin C is a good anioxidan (11). Viamin C has also been repored o rejuvenae viamin E, making i an indirec conribuor o he figh agains free radical damage in he lipids (12). The relaionship in humans as regards he role of anioxidans and oxidaive sress in he pahogenesis of malaria is no well undersood hough repors have been made in murine models. On he oher hand, repors have shown ha anioxidans and anioxidan agens such as choleserol, albumin and viamin C would provide proecion agains he oxidaive sress induced by malaria infecion(11,13). To gain insigh ino he relaionship beween P. Falciparum malaria and hese anioxidan agens, a hospial based sudy among children wih uncomplicaed Falciparum malaria infecion was conduced in abar, Cross-River Sae, Nigeria and he associaion of malaria parasiemia and viamin C, oal and HDL choleserol, and oal anioxidan levels, was examined Maerials and Mehods The sudy Subjecs consised of one hundred and weny wo (122) children beween he ages of 1-10 years, who were seen in emergency uni of Universiy of abar Teaching Hospial abar, Nigeria. The sudy subjecs were children infeced wih P. Falciparum malaria parasie, who repored ill wih fever in he hospial. The children who did no mee hese crieria were excluded from he sudy.apparenly healhy children, consising of sixy (60) subjecs who were found o be negaive for P. Falciparum in heir peripheral blood, were used as conrols. Boh groups of subjecs mus have resided in he ciy of abar for a leas one year before he sudy. Five milliliers of venous blood was obained from paien and conrol subjecs by venepuncure. Two milliliers of he blood was placed ino an EDTA bole for he deerminaion of haemoglobin using Avid 60 CT auomaed haemaoloyg analyzer, and parasie coun WHO, 1991 mehod (14) and he res was discharged ino a clean plain ube and allowed o clo a room emperaure. The plasma or serum (as appropriae) was obained by cenrifugaion for 10 minues a 3000rpm. Toal anioxidan was deermined immediaely by he mehod of Miller e al 1993 (7) and he remaining samples were sored a -200C for viamin C assay by he mehod of Roe and Kueher, 1943( 15). Serum oal choleserol and high densiy choleserol was deermined by ki mehods purchased from Quimica Clinica Aplicada Spain, while serum albumin was also deermined by ki mehod purchased from Randox laboraories, USA. The daa were analyzed by suden s es and Pearson s correlaion. Unless oherwise saed he daa were expressed as means +/- sandard deviaion. P<0.05 was considered significan in all saisical comparisons Resuls Comparison of oal anioxidan level, oal Choleserol, HDL-Choleserol, viamin C, albumin, haemoglo- 36 Inernaional Journal of Biomedical Laboraory Science (IJBLS) 2012 Vo1. 1, No. 2:35-39

3 bin and parasie coun of malaria infeced children and normal subjecs or conrol are shown on able 1. Toal anioxidan saus, viamin C, albumin, haemoglobin and HDL-Choleserol were observed o be significanly lower (p<0.001) in malaria-infeced children when compared wih he normal subjecs. While oal Choleserol was observed o be similar in boh paiens of malaria infecion and conrol subjecs. Toal plasma anioxidan level decreased wih he severiy of malaria (able 2). Malaria paiens were divided ino wo groups, viz mild/moderae infecion and severe infecion. Paiens classified as mild/moderae infecion had parasie coun of <7000/ dl, moderae emperaure (<40 C) wih no oher severe sympoms. Those classified as having severe malaria had parasie couns of > 7000/dl, emperaure of 40oC and above, wih vomiing, and dehydraion. Toal plasma anioxidan, and haemoglobin levels were in severe malaria significanly lower han levels in Mild/moderae malaria. However no significan change was observed in he levels of viamin C, albumin, and oal choleserol and HDL-choleserol in boh severe and mild/moderae malaria infecion. Table 3 shows he relaionship beween age on one hand, and plasma oal anioxidan, viamin C, albumin, oal choleserol, HDL choleserol, haemoglobin and parasie couns on he oher hand in paiens. The able shows ha in conrol subjecs and wihin he age bracke (1 o 10 years) plasma oal anioxidan level, viamin C, albumin, oal choleserol, HDL choleserol, and haemoglobin do no change significanly (p>0.05) wih age. However, during malaria infecion, oal plasma anioxidan level and albumin were observed o be significanly lower in children beween 1 and 5 years when compared wih hose beween 6 and10 years Figure 1 shows he correlaion graph of parasie coun agains oal anioxidan level. The oal anioxidan level correlaed negaively wih parasie coun (r= , p <0.01). Table 1 Toal plasma anioxidan level, oal and HDL-choleserol, viamin C, albumin, haemoglobin and parasie couns in malaria infeced children. Parameers Conrol subjecs n=60 Paiens n=122 Criical Toal anioxidan Saus mmol/l 1.26 ± ± P<0.001 Viamin C µmol/l 59.0 ± ± P<0.001 Albumin g/l 39.6 ± ± P<0.001 Toal choleserol mmol /l 3.8 ± ± P>0.05 HDL-Choleserol mmol/l 0.89 ± ± P<0.001 Haemoglobin g/l ± ± P<0.001 Parasie coun dl 5,391.3 ± nc - n=number of subjecs sudied. nc No comparison Table 2 Toal plasma anioxidan level, oal and HDL-choleserol, viamin C, albumin, haemoglobin and parasie couns based on clinical and laboraory findings of malaria infeced children. Parameers Clinical/Laboraory Findings Mild/moderae parasie coun <7,000 n=79 Severe malaria parasie coun >7,000 n=43 Criical Toal anioxidan level mmol/l 0.74 ± ± <0.001 Viamin C µmol/l 39.7 ± ± >0.05 Albumin g/l 31.7 ± ± >0.05 Toal choleserol mmol L/L 3.1 ± ± >0.05 HDL-Choleserol mmol/l 0.6 ± ± >0.05 Haemoglobin g/l ± ± <0.001 Parasie coun dl ± ± <0.001 n=number of subjecs sudied. Inernaional Journal of Biomedical Laboraory Science (IJBLS) 2012 Vo1. 1, No. 2:

4 Table 3 Effec of age on plasma oal anioxidan, viamin C, albumin, oal choleserol, HDL choleserol, haemoglobin and parasie couns in malaria infeced children and Conrol Subjecs. Malaria infeced children (n = 122) Age1 5years n= years n=29 Cri T Conrol Subjec (n = 60) Age 1-5 n= years n=30 Cri T P-Value Toal anioxidan Saus mmol/l 0.59 ± ± < ± ± P>0.05 ViaminC µmol/l 37.9 ± ± > ± ± P>0.05 Albumin g/l 21.3 ± ± < ± ± P>0.05 Toal choleserol mno l/l 3.1 ± ± > ± ± P>0.05 HDL-Choleserol mnol/l 0.57 ± ± > ± ± P>0.05 Haemoglobin g/l 89.8 ± ± > ± ± P>0.05 Parasie coun dl 3,387.0± ,004.2± > Fig.1 scaer plo of parasie coun agains oal anioxidan. Discussion Toal anioxidan level in malaria paiens was lower han he level for conrol (p<0.001). The lower values observed in oal anioxidan levels in malaria may be aribued o increased uilizaion of he hos s plasma anioxidans by he malaria parasies o counerac oxidaive damages. Degradaion of anioxidan enzymes as well as haemoglobin by malaria parasie o produce is own proein has been repored (16). This migh hus be conribuing o he decline in oal anioxidan saus. Furhermore a negaive correlaion was obained beween parasie coun and oal anioxidan level in malaria paiens, which indicaed ha a higher parasiemia, here is increased consumpion of oal anioxidan hus lowering is level. The oal anioxidan level varies inversely wih he severiy of malaria. Paiens wih severe malaria had significanly lower oal anioxidan levels han hose wih mild/moderae malaria. Anioxidans are used up o counerac he effecs of free radicals generaed in he cause of malaria infecion. This explains why reducion in anioxidan level is dependen on he severiy of malaria. Furhermore, here was a significanly lower oal anioxidan level in children 5 years and below as compared wih hose of 6 years and above. Previous observaions (4, 17) on under fives have shown ha hey are he mos vulnerable age group for malaria. In his age bracke, i could be seen ha children had much reduced anioxidan level, which may be he consequence of frequen malaria infecion or of severiy of malaria. Toal choleserol in normal subjecs and paiens were similar. The mean oal choleserol for conrol and paiens were wihin our reference range. On he conrary, a significanly lower serum HDL-Choleserol ( mmol/l) was observed in malaria paiens when compared wih conrols ( mmol/l). This finding agrees wih he repor of Mezger e al 2001 (18), which made similar observaions in 273 children (aged 1-10 years), wih acue malaria bu differen from he findings of Das e al, 1996 (12), who repored significan decrease in oal choleserol. The low HDL-Choleserol observed in malaria infecion is aribued o increased caabolism seen during inflammaory processes (19). Low HDL-Choleserol in aduls is associaed wih increased risk of coronary hear disease (19). The implicaion of low plasma HDL-Choleserol seen in our paiens is ha hose in malaria endemic regions may be predisposed o fuure coronary diseases as a resul of frequen malaria infecion. In our presen work, viamin C level was also significanly lower (p<0.05) in malaria paiens when compared wih conrol subjecs. This was aribued o eiher, increased requiremen or increased desrucion during malaria infecion. Age had no independen effec on he 38 Inernaional Journal of Biomedical Laboraory Science (IJBLS) 2012 Vo1. 1, No. 2:35-39

5 levels of viamin C in children. Albumin was significanly lower (p<0.01) in malaria-infeced children paricularly among paiens 5years and below. Low albumin level in malaria may be due o a number of facors: hese include inhibiion of synhesis by increasing levels of cyokines such as TNF, ILI and IL6 (20), reduced food inake as a consequence of loss of appeie, and redisribuion ino exravascular spaces as a resul of inflammaion. The albumin in exravascular spaces enhances anioxidan aciviy a hese locaions (21). There was a reducion in haemoglobin levels in malaria paiens as compared o conrols. This is a sandard feaure in malaria infecion. The reducion in haemoglobin is undersandably due o haemolyic desrucion especially of parasiized red blood cells, suppression of bone marrow aciviy and ineffecive eryhropoiesis (17). Malaria undermines he healh and welfare of families. I endangers he survival of children. I debiliaes he acive populaion and srains he resources of he affeced persons. Anaemia is a common occurrence in malaria infecion paricularly in severe cases as seen in his work. Parasiemia of red cells appears o be he major facor in developmen of anaemia in malaria infecion. The major funcion of anioxidan is o comba oxidaive challenges. Our findings have revealed significan reducion in oal plasma anioxidan level, as well as Viamin C, HDL-Choleserol and albumin in malaria paiens. Reducion in he aciviy of hese analyes as anioxidan agens may lead o reducion in he body s capaciy o mop-up free radicals generaed. The accumulaion of hese free radicals in he body during malaria infecions, cause sress on he cellular vialiy ulimaely leading o desrucive effec on cells as well as exposure of he paien o free radical associaed disease. To ameliorae hese effecs, we sugges he incorporaion of anioxidan agens as par of he componen drugs in he reamen and managemen of malaria infecions paricularly in children. References 1. World Healh Organisaion. World Malaria Repor, Geneva Bawah, A.A and F.N.Binka. How many years of life could be saved if malaria were eliminaed from a hyperendemic are of Norhern Ghana, populaion Council Working PaperNo. 203, Alaba, A.O. Malaria in children: implicaions for he produciviy of female caregivers in Nigeria, seleced paper for he 2002 annual conference of he Nigerian Economic Sociey, Cabe J. Naional Insiue of Healh News release. Aug 2, Wood LG, Gibson PG, Garg ML. Biomarkers of lipid peroxidaion, airway inflammaion and ashma. Eur Respir J 2003; 21: Cross CE. The anioxidan milieu a ashmaic respiraory rac surfaces. Pediar Res 2003; 53: Miller,N, Rice Evans, C, Davies, MJ, A novel mehod for measuring anioxidan capaciy and is applicaion o monioring he anioxidan saus in premaure neonaes. Clin Sci, 1993, 84, Mooyama T, Okamoo K, Kukia I, Hamaguchi M, Kinoshia Y, Ogawa H: Possible role of increased oxidan sress in muliple. Care Med 2003, 31: Bourdon, E. and Blache, D. The imporance of proeins in defense agains oxidaion. Anioxid. Redox Signal.,2001 3, Lichensein, AA, Ausman, LM, Jalber, SM, Scheafer, EJ. Effecs of differen forms of dieary hydrogenaed fas on serum lipoproein choleserol levels. New Eng J Med, (25), Winklehofor Roob, BM, Ellemuner,H, Fruhwirh M, Schlege Haueer, SE, Kloschsorui, G, Van, MA Plasma Viamin C Concenraions in Paiens wih cysic fibrosis; evidence of associaions wih lung inflammaion. Am J Clinl Nur, , Das, BS., Thurnham, DI, Das D B, Plasma alpha- ocopherol, reinol and caroenoids in children wih falciparum malaria. Am J clin Nur, 1996, 64, Adelekan, DA, Adeodu OO, Thurnham, DI, Comparaive effec of malaria and malnuriion on plasma anioxidan viamins in children. Ann of Trop Paed, 1997, 17 (3), World Healh Organizaion,. Basic Laboraory Mehods. Medical Parasiology, Geneva, 1991, Roe, N. Kueher, J, Viamin C esimaion. In Teiz Texbook of Clinical Chemisry, 3 rd ediion, Philadelphia, W. B. Saunders Company. 1943, Koracevic, D; Koracenc,GD Djevic,V,. Mehod for he measuremen of anioxidan aciviy in human fluids. J of Clin Pah, 2001, 54, Adediran, K.I, Adejuigbe, EA, Oninla,SO. Haemaological profile and malaria parasiaemia in Nigeria. Nig J of Med, (3), Mezger A, Gelasius M, Schankar A, Ndeezi G, Melikiang G, Semba R, Anioxidan saus and acue malaria in children in Kampala, Uganda. Am J Trop Med and Hyg, (2), Caselli, WP, Doyle, JT, Gordon,T. HDL-choleserol and oher lipids in coronary hear disease. Circulaion, 1997, 55, PeersTJr., All abou serum albumin, biochemisry, geneics and medical applicaions. Am Asso of Clin chem press, Washingon DC, 1996, Galloway P, McMillan C, Scaar N, Effec of he inflammaory response on race elemens and viamin saus. Ann Clin Biochem 2000, 37, Inernaional Journal of Biomedical Laboraory Science (IJBLS) 2012 Vo1. 1, No. 2: