A study of serum level of trace elements in patients with childhood epilepsy

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1 International Journal of Contemporary Pediatrics Singh A et al. Int J Contemp Pediatr Nov;3(4):xxx-xxx pissn eissn Research Article DOI: A study of serum level of trace elements in patients with childhood epilepsy Ankush Singh*, Biswaroop Chakrabarty, Sheffali Gulati, Vinod Kumar Paul Department of Paediatrics, AIIMS, New Delhi, India Received: 28 July 2016 Accepted: 02 August 2016 *Correspondence: Dr. Ankush Singh, ankushsinghdogra@yahoo.co.in Copyright: the author(s), publisher and licensee Medip Academy. This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Background: Equilibrium of trace elements is essential for a healthy nervous system. They activate specific enzymes that act as anti-oxidants. Epileptic seizures result in free radical production and oxidative damage to cellular proteins, lipids, and DNA. Increased production of free radicals leads to seizures or increase the risk of their recurrence. So, oxidative stress and production of the free radicals are now known both as a cause and as a consequence of epileptic seizures. Aim of the study was to estimate the serum level of trace elements in patients 2-12 years of age with epilepsy and comparing these levels in age and gender matched controls. Methods: Serum levels of selenium, copper, zinc and iron were done in patients with epilepsy and compared with healthy controls. The trace elements were analyzed by Inductively Coupled Plasma Mass Spectrometer (ICP-MS, X Series 2, Thermofisher Scientific). An ICP-MS combines a high-temperature ICP source with a mass spectrometer. The ICP source converts the atoms of the elements in the sample to ions. These ions are then separated and detected by the mass spectrometer. Results: The mean serum zinc level in patients with epilepsy was ± µg/l and in controls was ± µg/l. The mean serum selenium level in patients with epilepsy was ± µg/l and in controls was ± µg/l. Serum zinc level was not significantly decreased in patients with epilepsy as compared to control group (p=0.13). Serum selenium level was significantly decreased in patients with epilepsy as compared to control group (p=0.0077). The mean serum copper levels in patients with epilepsy were ± µg/l and in controls was ± µg/l. The median serum iron level in patients with epilepsy was 44.2 µg/dl and in controls was µg/dl. Serum copper level was significantly decreased in patients with epilepsy as compared to control group (p = ). Serum iron level was not significantly decreased in patients with epilepsy as compared to control group (p = 0.44). Conclusions: Decreased serum levels of trace elements in patients with seizures paves way for newer research on trace element supplementation in epilepsy patients. Keywords: Childhood epilepsy, Trace elements INTRODUCTION Equilibrium of trace elements is essential for a healthy nervous system. They activate specific enzymes that act as anti-oxidants. Epileptic seizures result in free radical production and oxidative damage to cellular proteins, lipids, and DNA. Increased production of free radicals leads to seizures or increase the risk of their recurrence. So, oxidative stress and production of the free radicals are now known both as a cause and as a consequence of epileptic seizures. 1,2 In the present study it was evaluated whether epilepsy decreases the serum levels of trace elements in patients with childhood epilepsy. Patients with epilepsy were enrolled and serum levels of trace International Journal of Contemporary Pediatrics October-December 2016 Vol 3 Issue 4 Page 1

2 elements were done and compared with normal age and gender matched controls. METHODS The study was conducted in the Department of Pediatrics, All India Institute of Medical Sciences, New Delhi from October 2014-January All consecutive children with epilepsy 2-12 years of age presenting to the Paediatric Neurology OPD, Paediatric ward, Neurocysticercosis clinic at AIIMS, New Delhi were included in the study. Patients with chronic medical illness of other systems or with history of surgery, patients on chronic medications besides anti-epileptic drugs which can affect the level of these trace elements e.g. penicillamine, deferasirox, desferoxamine, dimercaprol, patients taking supplementations containing these trace elements in last 3 months e.g. multivitamin supplements, patients who were critically sick as defined by severe respiratory distress, circulatory insufficiency, altered sensorium or on ventilator were excluded from the study. Healthy age and gender matched children who visited our paediatric outpatient department because of mild infectious diseases (like upper respiratory infection excluding diarrhoea) or vaccination. In the study by Ashrafi et al serum selenium level in patients with epilepsy was 72.90±22.20 and in healthy controls was 86.00± In another study by Ashrafi et al serum selenium level in patients with intractable epilepsy was 68.88±17.58 and in healthy controls was 85.93± In the study by Seven et al serum zinc level in patients with intractable epilepsy was 176±46 and in healthy controls was 203±31. 5 In the study by Wojciak et al serum zinc level in patients with epilepsy was 114.4±23.90 and in healthy controls was 94.63± Using the above data the minimum and maximum sample sizes came out to be 19 and 576. For feasibility purposes, it was proposed to enroll at least 100 patients of epilepsy and 50 individuals as controls. Detailed information on the study was given to each patient with epilepsy and controls and written consent was taken from all subjects to attend this study. Approval from the Ethics committee was taken. Serum levels of selenium, copper, zinc and iron were done in patients with epilepsy and compared with age and gender matched healthy controls. The control group comprised of healthy age and gender matched children who visited our paediatric outpatient clinic because of mild infectious diseases (excluding diarrhoea) or vaccination. The trace elements were analyzed by Inductively Coupled Plasma Mass Spectrometer (X Series 2, Thermofisher Scientific). An ICP-MS combines a high-temperature ICP source with a mass spectrometer. The ICP source converts the atoms of the elements in the sample to ions. These ions are then separated and detected by the mass spectrometer. Data was analysed by stata 11.2 presented in frequency (%), mean (S.D.)/median (minimum-maximum). Continous variables were compared in two groups by using t-test (following normal distribution) and wilcoxon ranksum test (non-parametric data). Categorical variables were compared by chi-square test. P value less than 0.05 was accepted as statistically significant. RESULTS Table 1: Demographic profile of patients. One hundred and six patients with epilepsy and 52 healthy children who served as controls were included in the study (Table 1). Patient population Number of patients Average age (years) Sex distribution Males Females Controls ± (57.69%) 22 (42.31%) Epileptic patients ± (70.75%) 31 (29.25%) p=0.11 p=0.10 Aetiological diagnosis: In patients with epilepsy the aetiology was hypoxic-ischaemic encephalopathy (HIE, 22 cases), neurocysticercosis (18 cases), HIE/ Lennox- Gastaut syndrome (17 cases), idiopathic epilepsy (17 cases), stroke (6 cases), HIE/West syndrome (4 cases), global developmental delay/intellectual disability(4 cases), tuberous sclerosis (4 cases), cryptogenic Lennox- Gastaut syndrome (3 cases), post encephalitic Lennox- Gastaut syndrome (2 cases) and Neurotuberculosis, febrile seizure, head injury, intellectual disability, neonatal hypoglycaemic brain injury(nhbi), corpus callosal(cc) agenesis, childhood absence epilepsy(cae), Doose syndrome and Rasmussen syndrome (1 case each) (Table 2). Serum zinc level was not significantly decreased in patients with epilepsy as compared to control group (p = 0.13). Serum selenium level was significantly decreased in patients with epilepsy as compared to control group(p = ) (Table 3). Serum copper level was significantly decreased in patients with epilepsy as compared to control group(p = ). Serum iron level was not significantly decreased in patients with epilepsy as compared to control group(p = 0.44) (Table 4). International Journal of Contemporary Pediatrics October-December 2016 Vol 3 Issue 4 Page 2

3 Table 2: Aetiological diagnosis in patients with epilepsy. Diagnosis Number of patients(n=106) Hypoxic-ischaemic encephalopathy(hie) 22 (20.75%) Neurocysticercosis 18 (16.98%) HIE/Lennox-Gastaut syndrome 17 (16.04%) Idiopathic epilepsy 17 (16.04%) Stroke 6 HIE/West syndrome 4 Global developmental delay/intellectual disability 4 Tuberous sclerosis 4 Cryptogenic Lennox-Gastaut syndrome 3 Post encephalitic Lennox-Gastaut syndrome 2 CNS tuberculosis 1 Febrile seizure 1 Intellectual disability 1 Head injury 1 Neonatal Hypoglycemic Brain Injury 1 Global developmental delay/corpus callosal agenesis 1 Childhood absence epilepsy 1 Doose syndrome 1 Rasmussen syndrome 1 Table 3: Serum zinc and selenium levels in controls and epileptic patients. Serum zinc levels mean± standard deviation (µg/l) Serum selenium levels mean± standard deviation (µg/l) Controls (n=52) Epileptic patients (n=106) ± ± p= ± ± p= Table 4: Serum copper and iron levels in controls and epileptic patients. Patient population Controls (n=52) Epileptic patients (n=106) Serum copper levels Mean±standard deviation (µg/l) ± ± p = Serum iron levels Median (range) (µg/dl) 43.01( ) 44.2( ) p=0.44 DISCUSSION The study shows that significant changes in serum selenium and copper concentrations are found in epileptic children as compared to the healthy children. However, there are no changes in serum zinc and iron concentration in epileptic patients as compared to control group. In the past many studies had suggested that epileptic patients have abnormal serum trace elements levels when compared with healthy controls. Verroti et al found no difference in serum levels of zinc between controls and epileptics. 7 Seven et al found that the patients with idiopathic intractable epilepsy had significantly decreased levels of serum zinc compared to those of the control group. 5 Wojciak et al found that there was increased levels of Zn concentration in all patients with epilepsy. 6 Since zinc and copper are co-factors for the same enzyme superoxide dismutase with the decrease in serum copper levels serum zinc levels may actually increase to compensate for the decrease in serum copper levels. In the present study no significant difference in serum levels of zinc was seen between patients with epilepsy and controls. Our findings are different as International Journal of Contemporary Pediatrics October-December 2016 Vol 3 Issue 4 Page 3

4 compared to that of Seven et al. Zinc being one of the cofactors for enzyme superoxide dismutase is expected to be low in cases of intractable epilepsy. But in the present study serum zinc levels were not decreased in patients with epilepsy as compared to healthy controls. Our findings are in line with those found by Verroti et al. The contradictory reports may be contributed also by the fact that zinc has a role in reducing levels of inhibitory neurotransmitter gammaaminobutyric acid. Verroti et al found no difference in serum levels of selenium between controls and epileptics. 7 Ashrafi et al found a significant lower mean of serum selenium in patients with intractable epilepsy group compared with that of the normal control group. 4 Ashrafi et al found a significant lower mean of serum selenium in patients with epilepsy compared to that of healthy control group. 3 Seven et al found that the patients with idiopathic intractable epilepsy had significantly decreased levels of serum selenium compared to those of the control group. 5 In the present study serum selenium levels were significantly decreased in patients with epilepsy as compared to normal controls. Selenium, being a co-factor of glutathione peroxidise which is an important antioxidant enzyme system in the human body, is expected to be low in patients with epilepsy, as it is a state of increased oxidative stress. Verroti et al found no difference in serum levels of copper between controls and epileptics. 7 Wojciak et al found that there was decreased level of copper in all patients with epilepsy. 6 In the present study serum copper levels were significantly decreased in patients with epilepsy as compared to normal controls. Our findings are similar to that of Wojciak et al. This can be explained by the fact that copper is a co-factor for superoxide dismutase which is the enzyme responsible for mitigation of injury due to free radicals. It is therefore understandable that in children with epilepsy, serum copper levels are low. Serum copper levels may have the potential of being a biomarker for severity of epilepsy. Ikeda et al found that serum iron was significantly higher in the epilepsy group than in the control group. 8 Idro et al concluded that iron deficiency is not associated with an increased risk of all acute seizures in children but of febrile seizures. 9 In the present study serum iron levels in patients with epilepsy were similar as compared to normal controls. Our findings are in contrast to that of Ikeda et al. Iron is part of various neurotransmitters and enzyme system in the brain, on the other hand it is also a trace element with pro oxidative stress properties. Thus the balance may tilt in either direction in a child with epilepsy. Reasonable sample sizes of epilepsy patients as well as controls were enrolled in the study. Serum levels of trace elements were done by using state of the art technology inductively coupled plasma mass spectrometer. The study included a wide spectrum of patients with epilepsy (well controlled to intractable). In the current study, serum levels of trace elements were estimated. However there are better indicators of body stores of these elements. Red blood cell (RBC) glutathione peroxidise level is a better marker of body stores of selenium. Similarly, RBC superoxide dismutase and metallothionein level reflect better the body stores of zinc. 10 Serum ceruloplasmin and RBC superoxide dismutase level indicate better the body stores of copper. Iron status is better assessed by serum ferritin concentration and serum transferring receptor concentration. Moreover brain level of these elements would be better reflected by levels in cerebrospinal fluid (CSF). Thus to what extent the serum levels represent actual body stores of these elements is a matter worth debating. Studies have shown that antiepileptic pharmacotherapy may adversely affect trace element status of the body; this factor has not been accounted for in the current study. Funding: No funding sources Conflict of interest: None declared Ethical approval: The study was approved by the Institutional Ethics Committee REFERENCES 1. Mizielinska SM. Ion channels in epilepsy. Biochem Soc Trans. 2007;35: Hayashi M. Oxidative stress in developmental brain disorders. Neuropathology. 2009;29: Ashrafi MR. A probable causative factor for an old problem: selenium and glutathione peroxidase appear to play important roles in epilepsy pathogenesis. Epilepsia. 2007;48(9): Ashrafi MR, Shabanian R, Abbaskhanian A, Nasirian A, Ghofrani M, Mohammadi M, et al. Selenium and Intractable Epilepsy: Is There Any Correlation? Pediatr Neurol. 2007;36: Seven M, Basaran SY, Cengiz M, Unal S, Yuksel A. Deficiency of selenium and zinc as a causative factor for idiopathic intractable epilepsy. Epilepsy Res. 2013;104: Wojciak RW1, Mojs E, Stanislawska-Kubiak M, Samborski W. The serum zinc, copper, iron, and chromium concentrations in epileptic children. Epilepsy Research. 2013;104: Verrotti A, Basciani F, Trotta D, Pomilio MP, Morgese G, Chiarelli F. Serum copper, zinc, selenium, glutathione peroxidase and superoxide dismutase levels in epileptic children before and after 1 year of sodium valproate and carbamazepine therapy. Epilepsy Res. 2002;48(1-2): Ikeda M. Iron overload without the C282Y mutation in patients with epilepsy. J Neurol Neurosurg Psych. 2001;70: Idro R, Gwer S, Williams TN, Otieno T, Uyoga S, Fegan G et al. Iron deficiency and acute seizures: International Journal of Contemporary Pediatrics October-December 2016 Vol 3 Issue 4 Page 4

5 results from children living in rural Kenya and a meta-analysis. PLoS One. 2010;5(11):e Wood RJ. Assessment of marginal zinc status in humans. Presented at the international workshop Zinc and Health: Current Status and Future Directions, held at the National Institutes of Health in Bethesda, MD, Cite this article as: Singh A, Chakrabarty B, Gulati S, Paul VK. A study of serum level of trace elements in patients with childhood epilepsy. Int J Contemp Pediatr 2016;3:xxx-xx. International Journal of Contemporary Pediatrics October-December 2016 Vol 3 Issue 4 Page 5