Iraqi J Pharm Sci, Vol.24(2) 2015 Trace elements in pediatric patients with otitis media effusion 72 Measurement of Serum Trace Elements (Zinc, Copper, Magnesium and Iron) Concentrations in Pediatric Patients with Otitis Media with Effusion in Iraq Najwan K. Fakree *,1 * Department of Clinical Laboratory Science, Collage of Pharmacy, University of Baghdad. Baghdad, Iraq. Abstract Otitis media with effusion (OME) is a common disease especially among young children (before school age) and it is one of the common causes of acquired hearing loss in childhood. Pediatric patients with OME are usually undernourished. The purpose of this study is to determine whether the serum levels of trace elements (zinc, copper, magnesium, iron) have a role in the development of OME in children. This study carried out on 55 children and subdivided them into two groups. Group 1 (patient group) consist of 30 children suffering from OME and group 2 (control group) included 25 apparently healthy children. Serum levels of zinc, copper, magnesium and iron were measured for both groups. Comparison the results between the two groups showed that group 1(patients) had significantly lower serum zinc, copper, and iron levels than group2 (P <0.05). Meanwhile the differences between the two groups in terms of serum values of magnesium were not statistically significant (p>0.05). This study postulated that serum levels of zinc, copper, and iron may have a role in the development of OME in children. While, serum magnesium may not have effect on the development of OME in children. Keyword: Pediatrics, Otitis media, Trace elements. قٍاس تراكٍز انعىاصر انىادرج فً مصم انذو ) انزوك، انىحاس ، انمغىسٍىو وانحذٌذ ( عىذ انمرضى تانتهاب االران انىسطى االوصثاتً فً انعراق االطفال انمصاتٍه فخريوجىان قٍصر ،*1 فشع انعهىو انًخرثشيح انسشيشيح، كهيح انصيذنح ، جايعح تغذاد ، تغذاد ، انعشاق .* الخالصة انرهاب االرٌ انىسطً االَصثاتي هى احذ االيشاض انشائعح خاصح عُذ االطفال ) قثم سٍ انًذسسح( وهى احذ االسثاب انًؤديح انً انطفىنح. وحيس اٌ االطفال انًصاتيٍ تانرهاب االرٌ انىسطً االَصثاتي هى عادج يعاَىٌ يٍ سىء انرغزيح . نهزا ضعف انسًع انًكرسة في يشحهح ذوز اصثح انغشض يٍ هزِ انذساسح هى نرحذيذ فيًا ارا كاٌ يسرىياخ انعُاصش انُادسج )صَك , َحاط , يغُسيىو ,حذيذ( في يصم انذو نها دوسفي ح طفم وذى ذقسيًهى انً يجًىعريٍ . انًجًىعح االونً )يجًىعح 55صثاتي نذي االطفال . هزِ انذساسح اجشيد عهً انرهاب االرٌ انىسطً االَ يٍ االطفال االصحاء . ذى 55طفم يعاَىٌ يٍ انرهاب االرٌ انىسطً االَصثاتي و انًجًىعح انثاَيح )انكىَرشول( وذشًم 03انًشضً( وذشًم انًغُسيىو وانحذيذ في يصم انذو نالطفال في كهرا انًجًىعريٍ. تعذ يقاسَح انُرائج تيٍ انًجًىعريٍ ذثيٍ اٌ في قياط يسرىياخ انضَك , انُحاط , . في حيٍ نى يكٍ انًجًىعح االونً كاَد يسرىياخ انضَك, انُحاط وانحذيذ في يصم انذو اقم تصىسج يهحىظح يقاسَح يع انًجًىعح انثاَيح غُسيىو في يصم انذو عُذ انًجًىعريٍ. هزِ انذساسح افرشضد اٌ يسرىياخ انضَك, انُحاط وانحذيذ في يصم االخرالف يهحىظ في يسرىياخ انً يهعة انذو يًكٍ اٌ يكىٌ نها دوس في طثيعح حذوز انرهاب االرٌ انىسطً االَصثاتي عُذ االطفال. تيًُا يسرىي انًغُسيىو في يصم انذو ستًا ال .لدوس في حذوز هزا انًشض عُذ االطفا . االطفالطة انعىاصر انىادرج ، انتهاب االرن انىسطى ، انكهماخ انمفتاحٍح : Introduction Otitis media with effusion (OME) is the most common ear disease in children; it is characterized by the accumulation of fluid in the middle ear space behind an intact tympanic membrane without symptoms of acute inflammation. It may lead to complications such as hearing loss, speech and language delay or poor balance (1, 2) . Approximately 90% of children experience OME at some time before school age, with peak incidence in 6 months to 4 years (3) . Pathogenesis of OME is not well understood, but a low-grade infection, especially with species similar to acute otitis media, eustachian tube dysfunction, inflammatory response following acute otitis media and complex interactions of biochemical, immunologic and inflammatory mediators in middle ear and adenoid hypertrophy have all been implicated (1, 4, 5) . The oxidative stress and the deficiency of antioxidants may be one of the factors leading to the pathogenesis of OME (6, 7) . 1 Corresponding author E-mail:najwankaisar@yahoo.com Received: 4/10/2015 Accepted: 15 /12/2015 Iraqi J Pharm Sci, Vol.24(2) 2015 Trace elements in pediatric patients with otitis media effusion 73 In OME (which is considering as inflammatory disease in the middle ear) Oxygen radicals are blamed in the pathogenesis of inflammation. Infections are one of the reasons of increased reactive oxygen species production (8) . Excessive production of reactive oxygen species which is known as oxidative stress may lead to increase the duration of middle ear inflammation by disturbing the immune function of leucocytes through damaging to the membrane lipids. Also DNA damage by free radicals decreases production of certain critical factors by leucocytes and lowered their proliferation capacity (6) . Moreover, oxidative stress and free radicals causes damage to the cellular DNA and proteins to the cilia in the middle ear (change the structure of cilia) leading to slow down the cilia activity and inhibit cellular regeneration (9) . Under normal physiological conditions enzymatic and non enzymatic antioxidant defenses protect aerobic organisms against the action of free radicals (7) .One of the most important enzymatic antioxidants is superoxide dismutase (SOD) (10) . The cytoplasmic copper/zinc-SOD, one of three forms of SOD, contains copper and zinc as cofactors and is believed to have a predominant role in the first step of antioxidant defense (7) . Ceruloplasmin, the copper-containing protein of extracellular fluids, has been shown to have important antioxidant properties towards peroxidising lipids (8) . This suggests that the risk of middle ear infection may potentially affect by factors (dietary or otherwise) which impair copper or zinc status (2) . Magnesium has an indirect antioxidant effect. it act as a cofactor on enzymatic reactions which have a role on energy production, thus activity of glutathione reductase (antioxidants) may decrease due to dietary deficiency of magnesium leading to free-radicals induced protein oxidation and lesion to tissues (11, 12) . Bacterial infections are considered as one of the etiological factors in OME (6) . Iron is an integral component of enzyme myeloperoxidase (MPO) activity which is used by neutrophil for killing process of bacteria by production of highly toxic reactive oxygen species intermediates (hydroxyl radicals) (13) . Poor iron status are associated with decreased neutrophil function and impaired their bactericidal activity, thymic atrophy with reduction of T-lymphocytes numbers, defective proliferation of T helper (Th)-1 and Th-2 lymphocytes, reduction in the activity of natural killer cell , reduction in the interleukin-2 levels produced by lymphocytes and decreased in the macrophage migration inhibition factors production (13) . In developing countries, studies on middle- ear diseases and their influence with micronutrient status may provide valuable information on the knowledge of the pathogenesis of middle-ear infection, prevention and treatment. Our hypothesis is that low serum values of trace elements (Zinc, Copper, Magnesium and Iron) may be a contributory factor in the development of OME in children. Patients and Methods This study was carried out at ear-nose – throat (ENT) department in Al Yarmook teaching hospital. The study consists of 55 children 29 male and 26 female with age range between 3 -6 years (with a mean age of 4.6± 1.06). They were divided into two groups: 1-Group 1(patients group): consisted of 30 patients diagnosed to have OME. 2-Group 2(control group): included 25 healthy children without any complaint in the ear-nose- throat region. No Infection or any other systemic diseases. Patients group were diagnosed to have OME by using otoscope, otomicroscopic and tympnometric evaluation by specialized physician. Venous blood samples of all children were taken. Serum zinc, copper, magnesium and iron measurements were obtained calorimetrically using commercial diagnostic kits (14- 17) . The reference ranges for the trace elements measured are listed in table 1. Table (1): The reference range for the trace elements measured. Trace element Reference ranges Zinc (umol/l) 10.7-17.6 Copper (umol/l) 4.72-23.6 Magnesium (mmol/l) 0.8-1.0 Iron (umol/l) 6.6-26 Results The serum trace elements values of both patient group (group 1) and control groups (group 2) are shown in table 2. Comparison between two groups showed that group 1(patients) had significantly lower serum zinc levels than group 2 (control) P < 0.05. (Serum levels of zinc in group 1 and group 2 were 8.7303 ±1.5503 and 16.8039± 2.4528(umol/l) respectively) Also patients group had significantly lower serum copper values as comparing with control group (P <0.05) (Serum copper levels were 14.2097± 4.3439 in group 1 and 18.1874± 3.01628 (umol/l) in group 2). As shown in table 2 and figure 1. According to the results in table 2 although the serum magnesium levels in group 1 was lower than in group 2 (0.8349± 0.08903 and 0.8645± 0.09598 (mmol/l)) respectively there was no significant difference between the group’s p > 0.05 (figure 1). The Serum iron levels of patients and control groups were 16.9850± 3.0323 and 19.4356± 3.1020 (umol/l) respectively. The patient http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3889347/#CR6 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3889347/#CR7 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3889347/#CR6 Iraqi J Pharm Sci, Vol.24(2) 2015 Trace elements in pediatric patients with otitis media effusion 74 group had significantly lower serum iron as comparing with control group (P <0.05) (table 2 and figure1). Table ( 2): Serum levels of Zinc, Copper, Magnesium and Iron in groups Group 1 (patients group). Group 2 (control). Data were expressed as mean± standard deviation (SD). N= number of children. The statistical significant differences in the concentrations of trace elements determined in group 1 and group 2 were investigated using student t-test) * P ≤ 0.05 Figure (1): Serum concentrations (mean serum concentrations) of trace elements in control group and patients group of children. Discussion Ear disease consider as one of the major health problem particularly in developing countries (2) . A close interaction among nutrition, infection, and health has been recognized since many decades. Adequate intake of micronutrients is essential for the proper function of the immune system (18) . Nutritional factors may play a contributory role in the middle ear diseases (2, 8) . Zinc is an essential trace element. It plays structural, regulatory, and catalytic roles in the body. Dietary zinc deficiency impairs immune function and resistance to infection by suppressing lymphocytes and neutrophils activity, decreased phagocytosis by macrophages, decreased functions of natural killer cell , and decreased antibody responses among other (19) . Some studies have found that children with vitamin A, Zinc and Iron deficiency were more susceptible to upper respiratory and ear infections (20, 21, 22) . In a previous study in Bangladeshi children, zinc supplementation significantly reduced the incidence of suppurative otitis media in those children (2) . Our results showed that patients in group 1 (OME) had significantly lower serum zinc values than those of group 2 (healthy control). Copper has a specific regulatory role in the development and expression of the immune reactions. It has been demonstrated that copper deficiency reduced numbers and functions of neutrophil, decrease function and proliferation of T- lymphocytes and reduced macrophages activity (18) . Also copper attends to important catalytic functions in a number of enzymes such as Cu,Zn superoxide dismutase (SOD), cytochrome oxidase , and ceruloplasmine. Thus impairing copper or zinc status (due to dietary or otherwise factors) markedly reduced tissue Cu, Zn-SOD (which consider as enzymatic antioxidant defenses important in ear disease) and cause peroxidative damage and mitochondrial dysfunction (12) . In a comprehensive review of literatures, no human research specifically examining the relation between copper status and middle ear diseases was identified. In our results, we found that group 1 children had significantly lower serum copper levels than those of group 2. Magnesium has a relationship with the immune system both in innate and acquired immune response (23) . In human, antibody production and susceptibility to infections have been associated with alack of magnesium (23) . Also magnesium has indirect antioxidant effect, and a dietary magnesium deficiency may reduce glutathion reductase activity (antioxidant defense) leading to radical-induced protein oxidation (11, 12) . Previous study showed that the levels of magnesium and iron were significantly depressed in children suffering from the exudative otitis media comparing with the control group (24) . In our study there were no differences in the level of Mg between patients and healthy children. Iron deficiency is the most widespread nutritional deficiency worldwide, especially in children living in poor resource countries (13) . Iron is essential for normal development of immune system and studies indicated that individuals with iron deficiency showed impairment of cell- mediated immunity, in addition phagocyte microbicidal function, natural killer cell activity and mucosal immunity are reduced leading to increase susceptibility to infections (13, 25) . In a previous study by Margareta B.et al children with frequent middle ear infections Trace element Group 1 N= 30 Group 2 N= 25 p value Zinc (umol/l) 8.7303 ± 1.5503 16.8039± 2.4528 0.000 Copper (umol/l) 14.2097± 4.3439 18.1874± 3.0162 0.000 Magnesium (mmol/l) 0.8349± 0.08903 0.8645± 0.09598 0.241 Iron (umol/l) 16.9850± 3.0323 19.4356± 3.1020 0.005 Iraqi J Pharm Sci, Vol.24(2) 2015 Trace elements in pediatric patients with otitis media effusion 75 seemed to account for most of the differences in the serum levels of Iron and Zinc between the patients and controls (26) . Other study by Golz A et al concluded that the children with iron-deficiency anemia have higher prevalence of episodes of acut otitis media in comparison to healthy (27) . These findings are in accordance with our results, we found that serum levels of iron in patients group were significantly lower than those in control group children. The antioxidant and micronutrient status of the patients group may be a significant factor in predisposition to OME. We found that serum levels of Zinc, copper, and iron in group 1 (OME) were significantly lower than group 2(control). However, there were not statistically significant differences between both groups regarding serum magnesium levels. We speculated that deficiency of serum Zinc, copper, and iron have a role in pathogenesis of OME in children. Conclusion This study postulated that deficiency of serum Zinc, copper, and iron levels may have a role in pathogenesis of OME in children. Further clinical studies needed to elucidate the effect of other micronutrient and vitamins status in pathogenesis of OME in children. References 1. Poopak Izadi, Mohammad-ebrahim, Nahid Kalid, et al. Effect of zinc on treatment of otitis media with effusion . Medical Journal of Islamic Republic of Iran. 2010; 24(3): 126- 132. 2. Elemraid MA, Makenzie IJ, Fraser WD. Nutritional factors in the pathogenesis of ear disease in children: a systemic review. Ann. Trop. Paediatr. 2009; 29: 85- 99. 3. Tos M. Epidemiology and natural history of secretary otitis. Am J. Otol.1984; 5:459-462 4. Post JC, Preston RA, Aul JJ, et al. Molecular analysis of bacterial pathogens in otitis media with effusion. JAMA1995; 273(20): 1598- 1604 5. 5. Dadas M, Incessulu A, Cakmakci E, Erkam U, Akpz I, Unal G. Evaluation of serum interleukin-1 and zinc levels in children with otitis media with effusion. J. Trace Element Electrolytes health Disease 1989; 3(4): 203-208. 6. Yilmaz T, Koçan EG, Besler HT, Yilmaz G, Gürsel B. The role of oxidants and antioxidants in otitis media with effusion in children. Otolaryngol Head Neck Surg. 2004;131:797–803. 7. Unal M, Tamer L, Pata YS, Kilic S, Degirmenci U, Akbaş Y, Görür K, Atik U. Serum levels of antioxidant vitamins, copper, zinc and magnesium in children with chronic rhinosinusitis. J Trace Elem Med Biol. 2004;18:189–192. 8. Cemek M, Caksen H, Cemek F, Bayiroğlu F, Dede S, Dülger H, Ustün R. Investigation of antioxidant status in children with acute otitis media and tonsillitis. Int J Pediatr Otorhinolaryngol. 2004;68:1381–1385. 9. Testa B, Testa D, Mesolella M, et al. Management of chronic otitis media with effusion : the role of glutathione. Laryngoscope. 2001; 111:1486-9 10. Aladag I, Guven M, Eyibilen A, Sahin S, Köseoglu D. Efficacy of vitamin A in experimentally induced acute otitis media. Int. J. Pediatr. Otorhinolaryngol. 2007;71:623– 628. 11. Sancak. Serum levels of antioxidant vitamins (vitamin A,C, E) and magnesium in children with Allergic Rhinitis. Medical Journal of Trakya University. 2010;27(2):132-136. 12. Silvia Vertuani, Angela Angusti, Stefano M. The antioxidants and pro-antioxidants Network: an overview. Current pharmaceutical Design. 2004; 10:1677-1694. 13. Vishal Kumar and V. P.Choudhry. Iron deficiency and infection. Indian Journal of Pediatrics, 2010; 77(7):789-793. 14. Tetsuo Makino. A sensitive, direct colorimetric assay of serum zinc using nitro- PAPS and micro well plates. Clinica Chemica Acta. 1991;197(3):209-220. 15. Ciuti R., Galli A., Giorn IT. Determination of serum copper. Chim. Clin. 1987; 12(2):101- 111. 16. Bohoun C. Micro determination of magnesium in various biological media. Clin. Chim. Acta.1962; 7: 811-817. 17. Weippl G. et al., Serum iron and statistical distribution of single values in men and women. Blut 1973; 27(4):261-270. 18. Wintergerst ES, Maggini S, Hornig DH. Contribution of selected vitamins and trace elements to immune function. Ann. Nutr. Metab.. 2007;51:301–323. 19. Hasse H, Rink L. the immune system and the impact of zinc during aging. Immun Aging. 2009;12:6-9. 20. Prasad A, Fitzgerald B, Baof, et al. Duration of symptoms and plasma cytokine levels in patient with the common cold treated with zinc acetate. Ann. Intern. Med. 2000;133: 245-252. 21. Sazawal S, Jalla S, Mazamdar A. Zink supplementation reduces the incidence of acute lower respiratory infections in infants and preschool children. Adouble blind controlled trial. Pediatrics. 1998; 102:1-5. 22. Van Wouwe JP, Van Gelderen HH, Bos JH. Subclinical zinc deficiency in children with recurrent upper respiratory tract infection, Eur. J. Pediatr. 1987; 146(3): 293-5. Iraqi J Pharm Sci, Vol.24(2) 2015 Trace elements in pediatric patients with otitis media effusion 76 23. Tam M, Gomez S, Gonzalex M, et al. Possible role of magnesium on the immune system. Eur. J. Clin. Nutr. 2003; 57(10): 1193-7. 24. Grazyna N, Piotr S, Krystyna C. Magnesium, Calcium and Iron levels in blood serum of children with otitis media with effusion Journal of Elementologyy 2005; 18 (1): 53-62. 25. Bhaskaram C., Reddy V. Cell mediated immunity in iron-and vitamin-deficient children. Br. Med. J. 1975;48: 864-866. 26. Bondestam M., Foucard T., Gebre-Medhin M. Subclinical trace element deficiency in children with undue susceptibility to infections. Acta. Paediatr. Scand. 1985; 74(4):515-20. 27. Golz A., Netzer A., Goldenberge D., et al. The association between iron-deficiency anemia and recurrent acute otitis media. Am. J. Otolaryngol. 2001; 22(6): 391-4.