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Serum Trace Elements in Children with Well-Controlled and Drug Refractory Epilepsy Compared to Controls: An Observational Study
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.359205
Keywords: Copper, drug refractory epilepsy, epilepsy, iron, selenium, trace elements, well-controlled epilepsy
Essential trace elements (zinc, copper, and selenium) by their effects on synaptogenesis, ion channels, oxidative stress, neurotransmitters, and immune system presumably have a role in the pathogenesis of epileptic seizures.[1],[2] Selenium is essential for synthesis and functioning of antioxidant proteins like glutathione peroxidase contributing to glutamate-mediated excitotoxicity, membrane lipid peroxidation, and neurotransmitter turnover rates, which have a role in pathogenesis of epilepsy.[3],[31],[32],[33] Zinc is involved as a cofactor in N-methyl-D-aspartate receptor activation and T-type calcium b channel regulation.[4] Zinc can inhibit glutamic acid decarboxylase activity causing low concentrations of gamma-aminobutyric acid or an increase in carbonic anhydrase activity. It is being speculated that zinc is concentrated in the mossy fiber axons of the dentate gyrus in the hippocampus contributing to pathogenesis of epilepsy.[5] Copper (Cu) and zinc (Zn) effect the removal of free radicals through their involvement in the structure of superoxide dismutase (SOD), an important antioxidant enzyme.[6] Copper independently inhibits Mg and Na K ATPase, causing altered Na and K levels, which in turn increases susceptibility to epileptic seizures.[7],[34],[34],[35],[36] Iron, involved in several brain processes such as neurometabolism, myelination, and neurotransmitter function and deficiency, has been implicated in causation of epilepsy and febrile seizures.[8],[9] Studies have shown altered levels of micronutrients in children with epilepsy and further difference have been observed between drug-controlled and refractory epilepsy.[10],[11],[12],[13],[14],[15],[37],[38] Studies till date are inconclusive in terms of the role of trace elements and metals in pathogenesis and therapeutics of children with epilepsy. Moreover, there are few studies that have compared their levels between well-controlled and drug refractory epilepsy. Till date no studies have been done evaluating serum trace elements in Indian children with epilepsy. The current study has been planned to compare serum levels of micronutrients (copper, selenium, zinc, and iron) between children with drug-controlled epilepsy, drug refractory epilepsy, and controls.
This cross sectional study was conducted in a tertiary care teaching hospital in North India over a 6 month period. The primary objective was to compare mean serum levels of selenium and zinc in children with epilepsy and normally developing age and gender matched controls. The secondary objectives were to compare mean blood levels of
Drug refractory epilepsy was defined as failure of two tolerated and appropriately chosen antiepileptic drugs (whether as monotherapies or in combination) to achieve sustained seizure freedom. Seizure freedom was defined as freedom from all types of seizures for 1 year or three times the pretherapy interseizure interval, whichever was longer of the two.[16] All consecutive children with epilepsy, aged 2 to 12 years, presenting to the Department of Pediatrics were evaluated for inclusion in the study. Patients with chronic systemic illnesses and those who were critically sick were excluded. Healthy age and gender matched children who visited the Pediatric outpatient department because of mild infectious diseases (like upper respiratory infection excluding diarrhea) or vaccination who were not on any vitamin and mineral supplementation were included as controls. A dietary history was taken for all subjects at enrolment. Controls and well-controlled epilepsy patients who were on a balanced diet and still on vitamin and mineral supplements were excluded from the study. It was planned to exclude drug refractory epilepsy cases on milk predominant enteral feeds who were not on vitamin and mineral supplements. The study was approved by the Institute Ethics Committee. Written informed consent was taken from parents and/or guardians of the subjects who participated in the study. With an α of 0.05 and power of 90%,
As it was a time-bound study, for feasibility purpose, it was proposed to enroll at least 50 cases each of intractable epilepsy, well-controlled epilepsy, and controls. Serum levels of selenium, copper, zinc, and iron 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. Statistical analysis Data was analyzed by stata 11.2. Continuous variables were compared in two groups by using t-test and/or Wilcoxon ranksum test. Categorical variables were compared by Chi-square test. A P value of less than 0.05 was considered as statistically significant.
During the study period, 153 patients presented with epilepsy. After applying inclusion and exclusion criteria, 106 patients with epilepsy and 52 controls were included in the study. Of the 106 children with epilepsy, 55 belonged to drug refractory epilepsy group and 51 were drug-controlled epilepsy. The study flow is shown in [Figure 1]. In the epilepsy group who were on balanced diet, 31 were excluded as they were on vitamin and mineral supplementation. None of the drug refractory epilepsy patients on milk-based enteral feeds were excluded as all were receiving vitamin and mineral supplementation.
The clinicodemographic variables of cases and controls are enumerated in [Table 1]. Both the groups were matched for age and gender.
The serum trace element levels in cases and controls have been tabulated. Serum selenium and copper were significantly decreased in cases compared to controls. However, serum zinc and iron levels were not significantly different in the two groups [Table 2]. After classifying epilepsy into well-controlled and drug refractory cases, only in the latter the significant difference for serum selenium and copper levels remained compared to controls. Additionally, in the drug refractory cases, serum iron levels were significantly reduced compared to controls [Table 3].
Normal serum values for zinc, selenium, copper, and iron were defined as per age related reference values.[17],[18] Serum zinc was subnormal in 55.8% (29/52), 41.2% (21/51), and 52.7% (29/55) cases of controls, well-controlled, and drug refractory epilepsy, respectively, and this difference in distribution was not statistically significant. None of the cases in the 3 groups had values above normal range. Serum selenium was within normal limits in all cases in all the three groups. Serum copper levels were subnormal in 1 and 2 cases of well-controlled and drug refractory epilepsy, respectively, with none had subnormal value in the controls. It was above normal range in 1, 3, and 4 cases of drug refractory and well-controlled epilepsy, and controls respectively. Serum iron values were significantly (P = 0.04) more in drug refractory epilepsy (16.3%, 9/55) compared to well-controlled group (3.9%, 2/51). Although it was more than the control group as well (5.8%, 3/52), the difference was statistically not significant (P = 0.08).
Up to 10% of children experience one episode of seizure in first 16 years of life. The prevalence of epilepsy is 0.5% to 1% per year with cumulative lifetime incidence of 3%.[19] Up to 30% patients have medically intractable epilepsy.[20] Oxygen free radicals play a significant role in the neurotoxic cascade that leads to epileptic seizures. Trace elements are involved in antioxidant defense mechanism pathways. Increased production of oxygen free radicals and/or decreased functioning of antioxidant defense mechanism increase the recurrence risk of seizures.[21],[39],[40],[41],[42] Thus, it is worth investigating the role of trace elements in pathogenesis of seizure intractability which may have considerable therapeutic implications. In the current study, serum selenium levels were significantly reduced in the epilepsy group compared to controls and on further subgroup analysis, this difference was statistically significant only in the drug resistant group compared to well-controlled group and controls. In a study by Ashrafi et al.,[11] where they compared serum selenium levels in children with intractable epilepsy with age and sex matched healthy controls, they concluded that serum selenium was significantly low in the intractable epilepsy group. Similar results were also resonated by Seven et al.[12] A meta-analysis addressing role of trace elements in epilepsy and febrile seizures concluded that serum selenium levels are significantly reduced in febrile seizure patients compared to age matched controls.[15],[43],[44],[45],[46] There are anecdotal reports of reduction in seizure frequency in intractable epilepsy patients with selenium supplementation.[22],[23] The studies highlight that reduced selenium levels probably mediate pathogenesis of epilepsy by facilitating oxidative damage leading to glutamate-induced excitotoxicity and deranged neurotransmitter turnover rates.[1],[3] Relative reduction in serum selenium levels in intractable epilepsy cases compared to controls as seen in the current study may indicate an underlying vicious cycle of increased requirement of selenium to counter the ongoing oxidative stress leading to a state of relative deficiency. Thus, the role of selenium as a potential therapeutic option should be investigated further in intractable epilepsy patients in appropriately designed studies. Serum zinc levels were not significantly different in epilepsy group compared to controls in the present study. Moreover, there was no difference observed even after subclassifying epilepsy group into well-controlled and drug refractory epilepsy. In a study by Wojciak et al., serum zinc concentrations were significantly elevated in epileptic patients compared to controls.[13] Seven et al.[12] in their study concluded that serum zinc levels were significantly reduced in drug resistant epilepsy group compared to controls. In a large study with intractable epilepsy and controls, serum zinc levels were found significantly reduced in the former.[14] In a meta-analysis, it has been found that serum zinc concentrations are significantly low in both epilepsy and febrile seizure patients compared to controls.[15] Serum copper levels were significantly reduced in cases compared to controls in the current study. On further classifying epilepsy patients into well-controlled and drug refractory group, only the latter had significantly reduced levels compared to controls. Similar results have been echoed in a study by Wojciak et al.[13] with limited sample size. However, in a study with reasonable sample size by Seven et al.,[12] there was no significant difference between cases and controls. A meta-analysis and a large study have both shown significantly increased levels in epilepsy and febrile seizure patients compared to controls.[14],[15] Zinc and copper have both pro and anticonvulsant properties. Zinc has both facilitatory and inhibitory actions toward gamma amino butyric acid, the inhibitory neurotransmitter. Copper by its inhibitory action on Mg and Na K ATPases increases neuronal excitability thereby increasing epileptic potential. Zinc and copper are integral part of body's defense mechanism against oxidative stress by mediating action of various antioxidant enzymes like glutathione peroxidase and SOD. However, at higher concentrations both can cause free radical induced oxidative damage.[14] The current study saw no significant difference in serum iron levels between cases and controls. However, on classifying epilepsy group further, iron levels were significantly reduced in drug refractory group compared to controls. The proconvulsant effect of iron deficiency has already been demonstrated in humans and animal models of epilepsy.[8],[9],[24] Studies have also shown that other trace elements like calcium, magnesium, and chromium are also reduced in children with epilepsy.[13],[14],[25],[26],[27] The effect of antiepileptic drugs on trace elements is still uncertain. Verrotti et al.[10] showed that one year of antiepileptic drug therapy does not alter the serum levels of trace elements in epileptic patients. Talat et al.[28] documented significantly altered serum levels of zinc and copper in epileptic patients on antiepileptic drug therapy; however, the levels were not significantly different in patients on mono or polytherapy. A recent meta-analysis on role of trace elements in patients with epilepsy and febrile seizures has shown that serum levels of copper, zinc, and magnesium were significantly altered in epileptic children on antiepileptic drug therapy compared to treatment naive patients.[15] It has been hypothesized that the effect of antiepileptics on hepatic enzyme system and plasma proteins may lead to alteration in the serum trace element levels, particularly zinc and copper.[28] In the present study, the majority of the patients in the well-controlled epilepsy group were on low-dose phenytoin or valproate and there was no statistically significant difference between the serum levels of micronutrients between them. In the drug refractory epilepsy group, all the patients were on high-dose multidrug therapy, the most common combination being that of valproate, levetiracetam, and benzodiazepine. Different combinations of drugs were not available in sufficient number in the drug refractory group for appropriate statistical analysis. All the patients in the current study were included with at least 1 year of epilepsy duration as it is mentioned in the definition for sustained seizure freedom. The difference in duration of epilepsy between both the study groups was statistically not significant. The current study has certain strengths and limitations. For the first time, a number of trace elements were evaluated in Indian children with epilepsy in an adequate sample size. In the current study, in the well-controlled epilepsy group and controls, all included study subjects were on balanced diet with meat and/or dairy products, which have better dietary diversification of all nutrients.[29] In the drug refractory epilepsy group, included patients were either on milk-based enteral feeds with necessary vitamin and mineral supplementation or balanced diet.[30] However, quantitative estimation of micronutrients in the diet was not done. Both the groups had representation of wide variety of etiology and hence sufficient number was not available for looking at association of individual etiology with serum levels of micronutrients. However, there was no statistically significant difference between well-controlled and drug refractory groups in distribution of two broad etiological categories (primary generalized and structural). Appropriately designed longitudinal studies evaluating trace elements from treatment naïve state through the disease course can help in understanding the effect of duration and etiology of epilepsy, anti-epileptic drugs, and nutritional status on serum trace element levels in patients with epilepsy. Epilepsy is not a prominent feature in deficiency syndromes of these trace elements; however, the current study in consonance with previous studies show that serum levels of micronutrients are significantly altered in drug refractory epilepsy compared to controls. This may be related to impaired antioxidant defense mechanism and altered functional status of ion channels and neurotransmitters, which underlie drug intractability in epilepsy. Hence, their potential as a biomarker and therapeutic option in drug refractory epilepsy should be investigated further. Ethical approval The study was approved by Institute Ethics Committee (IESC/T-339). Author contributions: BC and ASD conducted the study and wrote the manuscript under the supervision of SG and VKP. The trace element levels were estimated under the supervision of GST. The study design, methodology, and statistical analysis were done under the supervision of RMP. The final manuscript draft was approved by all the authors. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.
[Figure 1]
[Table 1], [Table 2], [Table 3]
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