| Elevated Serum Lipid Peroxidation and Reduced Vitamin C and Trace Element Concentrations Are Correlated With Epilepsy
Abhijit Das 1, Md Shahid Sarwar 1, Md Shohel Hossain 2, Palash Karmakar 1, Mohammad Safiqul Islam 1, Mohammad Enayet Hussain 3, Sujan Banik 1
Abstract
Background: Epilepsy is one of the chronic and heterogeneous epidemic neurological disorders leading to substantial mortality. The aim of the present study was to investigate the serum levels of malondialdehyde (MDA), vitamin C, and trace elements namely zinc (Zn), copper (Cu), and manganese (Mn) in epileptic patients of Bangladesh and to establish if there are any pathophysiological correlations.
Methods: This was a case-control study with 40 generalized epileptic patients and 40 healthy subjects as controls. Epilepsy was determined by the presence of seizure events with an abnormal electroencephalography and magnetic resonance imaging report of brain.
Results: Anthropometric parameters highlighted that age is a major risk factor of epilepsy and men are more prone to epilepsy than women. Blood serum analysis demonstrated significantly ( P < .001) higher values of MDA and lower level of vitamin C in the patient group (4.41 ± 0.76 μmol/mL and 18.31 ± 0.84 μmol/L, respectively) compared with control (1.81 ± 0.70 μmol/mL and 29.72 ± 1.06 μmol/L, respectively). Pearson's correlation analysis revealed a negative correlation between the serum level of MDA and vitamin C for both patient ( r = -0.023, P = .887) and control group ( r = -0.142, P = .383). This study also revealed that the trace elements (Zn, Cu) were significantly ( P < .05) lower in epileptics (68.32 ± 4.59 and 50.81 ± 2.54 μg/dL, respectively) where the level of Mn in patients (187.71 ± 9.04 μg/dL) was almost similar to that of the control group ( P > .05). The univariate analysis demonstrated that zinc <70 μg/dL (odds ratio = 3.56, P < .05) and copper <50 μg/dL were associated (odds ratio = 14.73, P < .001) with an increased risk of epilepsy. Establishment of interelement relationship strongly supported that there was a disturbance in the element homeostasis of epileptic patients.
Conclusions: The study results strengthen the role of lipid peroxidation, antioxidants and trace elements in the pathogenesis and warrant larger studies to investigate the association of these biochemical parameters with epilepsy.https://pubmed.ncbi.nlm.nih.gov/29788779/ https://journals.sagepub.com/doi/10.1177/1550059418772755 Elevated Serum Lipid Peroxidation and Reduced Vitamin C and Trace Element Concentrations Are Correlated With Epilepsy
Abstract
Background. Epilepsy is one of the chronic and heterogeneous epidemic neurological disorders leading to substantial mortality. The aim of the present study was to investigate the serum levels of malondialdehyde (MDA), vitamin C, and trace elements namely zinc (Zn), copper (Cu), and manganese (Mn) in epileptic patients of Bangladesh and to establish if there are any pathophysiological correlations. Methods. This was a case-control study with 40 generalized epileptic patients and 40 healthy subjects as controls. Epilepsy was determined by the presence of seizure events with an abnormal electroencephalography and magnetic resonance imaging report of brain. Results. Anthropometric parameters highlighted that age is a major risk factor of epilepsy and men are more prone to epilepsy than women. Blood serum analysis demonstrated significantly (P < .001) higher values of MDA and lower level of vitamin C in the patient group (4.41 ± 0.76 μmol/mL and 18.31 ± 0.84 μmol/L, respectively) compared with control (1.81 ± 0.70 μmol/mL and 29.72 ± 1.06 μmol/L, respectively). Pearson’s correlation analysis revealed a negative correlation between the serum level of MDA and vitamin C for both patient (r = −0.023, P = .887) and control group (r = −0.142, P = .383). This study also revealed that the trace elements (Zn, Cu) were significantly (P < .05) lower in epileptics (68.32 ± 4.59 and 50.81 ± 2.54 μg/dL, respectively) where the level of Mn in patients (187.71 ± 9.04 μg/dL) was almost similar to that of the control group (P > .05). The univariate analysis demonstrated that zinc <70 μg/dL (odds ratio = 3.56, P < .05) and copper <50 μg/dL were associated (odds ratio = 14.73, P < .001) with an increased risk of epilepsy. Establishment of interelement relationship strongly supported that there was a disturbance in the element homeostasis of epileptic patients. Conclusions. The study results strengthen the role of lipid peroxidation, antioxidants and trace elements in the pathogenesis and warrant larger studies to investigate the association of these biochemical parameters with epilepsy.
Introduction
Epilepsy, one of the most common and serious brain disorders,1 is characterized by spontaneous and stereotyped disturbance of sensation, behavior, emotion, or motor function resulting from the rapid and local discharges of gray matter.2 This disorder is heterogenic in nature3 with idiopathic (genetic basis), symptomatic (identified brain injury), and cryptogenic (unknown) etiologies.4 It affects more than 50 million people worldwide and half of them are children.3 However, the incidence of epilepsy is more common in the developing countries than in the developed ones with a prevalence rate of 24 to 53 per 100 000 population in developed countries.5 Although the exact prevalence of epilepsy is unknown in Bangladesh, some hospital-based studies estimated that the number of epilepsy patients in the country is high (1.2%-3.5%) compared with other countries of the world.6,7 Ignorance of treatment or diagnosis and superstitious belief about epilepsy are the most common causes of such high prevalence rate of epilepsy in Bangladesh.6
There are comprehensive evidences demonstrating that epilepsy has a strong correlation with oxidative stress and lipid peroxidation.8-11 Oxidative stress and lipid peroxidation lead to a substantial decrease in cholinergic markers (eg, acetylcholinesterase)8 and muscarinic receptors of the brain resulting several brain disorders, including epilepsy.8,9 Moreover, lipid peroxidation produces free radicals, which attack the large lipid content of myelin sheaths causing cerebral oxidative metabolism and finally initiating the nerve tissue injury process.11,12 Lipid peroxidation process also involves in the production of several lipid byproducts, some of which exert adverse effects.13 Serum malondialdehyde (MDA) is an endogenous genotoxic product, generated by oxidation of arachidonic acid and larger polyunsaturated fatty acids (PUFAs). MDA is used as a marker to investigate the oxidative damage of lipids in many degenerative human diseases.14 However, it is an antioxidant which prevents or alleviates the free radical induced lipid peroxidation15 and acts as body’s defense system against oxidative damage.16 Several enzymatic and nonenzymatic antioxidants are available in human body, which scavenges these reactive oxygen species (ROS). Vitamin C (a water-soluble, nonenzymatic antioxidant) is a fundamental neurological antioxidant that plays an inevitable neuroprotective function.17 Vitamin C is also important for the proper functioning of the central nervous system. Reduction of optimum concentration causes structural and functional damage to the cells and imbalance in neurotransmission system.18,19
Microminerals and trace elements act as crucial components for the development of immunity and provide antioxidant defense in human body.20 For maintaining a healthy nervous system and neuronal susceptibility, it is very important to keep these elements in a balanced state.21 Several reports have suggested that altered level of some trace elements and body electrolytes play an important role in the pathophysiology and recurrence of seizures.21-23 Thus, the complex balance of trace elements is essential for proper functioning of human health, as well as preventing health problems; otherwise, imbalances may adversely affect biological processes and may lead to many neurological diseases.23-26 For example, zinc (Zn) is abundant in the hippocampus region of the brain which has been implicated as a causative factor of epilepsy. It is a fundamental element for the normal neuronal communication as well as proper functioning and synthesis of the inhibitory neurotransmitter, γ-amino butyric acid (GABA). Altered zinc metabolism and low levels of serum zinc may play an important role in the induction epileptic seizures via activation of N-methyl-d-aspartate receptors.27 Copper (Cu) is also an important micromineral that acts as a cofactor for different enzymes, including tyrosinase and dopamine hydroxylase.26 Release of Cu during synaptical transmission helps modulate neuronal excitability under normal conditions and depleted serum Cu level leads to epileptiform discharges.28 Manganese (Mn) is a fundamental micronutrient for the proper neuronal development and function. Either deficiency or excess Mn concentration in the body can severely affect the pathological conditions in the central nervous system. Alterations in Mn concentrations, whether excessive or deficient, can be accompanied by convulsions due to the alterations in dopamine, glutamate, and GABA regulatory systems.29
Considering the above issues together, the current study was undertaken to evaluate the association of serum MDA, vitamin C, and essential micronutrients (Zn, Cu, and Mn) with epilepsy in the Bangladeshi population.
Materials and Methods
Reagents
The reagents used for the study were of analytical grade and purchased from the commercially available companies. 2-Thiobarbituric acid (TBA), 2,4-dinitrophenylhydrazine (DNPH), n-butyl alcohol, and sulfuric acid (97%) were purchased from Merck (Darmstadt, Germany). Ascorbic acid was supplied by Globe Pharmaceuticals, Noakhali, Bangladesh as a gift sample. Trichloroacetic acid (TCA), nitric acid, and hydrochloric acid (37%) were procured from Merck (Darmstadt, Germany). Standards of Zn, Cu, and Mn were sourced from Buck Scientific (Norwalk, CT, USA).
Research Design and Study Population
For the study purpose, 40 diagnosed cases of generalized epilepsy (30 male and 10 female) with age between 8 and 55 years were randomly recruited by a specialist neurologist who was trained in Diagnostic and Statistical Manual of epilepsy. For comparison, 40 healthy volunteers who matched their age with patients were recruited as control. All subjects had to go through specific diagnostic procedures (eg, EEG, magnetic resonance imaging [MRI], computed tomography [CT] scan) to find out the existence of epilepsy. Patients with abnormal EEG and have been experiencing at least 1 seizure in the past 6 months were included in the study. Other inclusion criteria were no previous history of antiepileptic drug administration and not treated with any antioxidants. Subjects also had to undergo a routine physical checkup, including their organ activity, weight, nutritional condition, blood pressure, chest radiograph, and electrocardiogram. Subjects who were noncooperative, had severe general medical conditions (diabetes mellitus, renal, cardiovascular, liver disease, endocrine disorder, and any chronic illness) and those who denied donating blood sample were excluded from the study. Healthy human volunteers who had no previous history of any chronic diseases, no history of smoking and alcohol intake and had not taken any medications were selected as controls.
Ethical Considerations
This case-control study was carried out in the Department of Neurology, National Institute of Neurosciences and Hospital, Dhaka, Bangladesh. Ethical permission was taken from ethical committee of the respective hospital. In this study, it was necessary to ask and discuss about some sensitive issues regarding personal and family affairs. Precaution was maintained to ensure privacy and no financial involvement of the patient or respondents was encouraged. Subjects or key relatives were clearly informed about the scope and limitation of the study. Written or verbal consent was obtained from the subjects and /or their key relatives prior to study.
Data Collection
Detailed patients’ history was taken with a structured questionnaire which was arranged in accordance with the clinical experience and available information sources and which included gender, age, marital status, educational condition, socioeconomic status, and duration and severity of the illness. The questionnaires were distributed among patients with a written consent form that explained the purpose of the research and assured them of their confidentiality. The authors conducted a face-to-face interview of the participating patients for data collection.
Blood Sample Collection and Storage
Five-milliliter venous blood sample was drawn from each patient and control in a metal-free sterile tube. The blood sample was kept at room temperature for about 30 minutes to clot and centrifuged at 3000 rpm for 15 minutes to extract the serum. Then the serum was taken in an Eppendorf tube and was stored at −80°C until the study day. These samples were then used for analyzing the serum level of MDA, antioxidant (vitamin C), and trace elements (Zn, Cu, and Mn).
Biochemical Analysis
Determination of Serum MDA
Assessment of lipid peroxidation was conducted by measuring serum MDA level according to the modified method described by Nahar et al.26 A volume of 0.5 mL of serum was treated with 20 mg/dL TCA and after 10 minutes the mixture was centrifuged at 3500 rpm for 10 minutes. Later, the supernatant was decanted whereas 2.5 mL of 0.05 M sulfuric acid was added to the precipitate followed by 3.0 mL of 0.2 mg/dL TBA (in 2 M sodium sulfate). Then the mixture was heated for 30 minutes in boiling water. After cooling, the resulting chromogen was extracted with 4.0 mL of n-butyl alcohol by vigorous shaking. Finally, separation of the organic phase was facilitated by centrifugation at 3000 rpm for 10 minutes and absorbance of the supernatant was measured spectrophotometrically (UV-1800 Spectrophotometer, Shimadzu, Kyoto, Japan) at 530 nm using 1,1,3,3-tetraethoxy-propane as standard.
Measurement of Vitamin C
The analysis of serum ascorbic acid (vitamin C) was carried out by treating the extracted serum properly with 5% TCA in a test tube. The mixture was centrifuged at 3000 rpm for 10 minutes and the clear supernatant thus obtained was kept at −80°C for further study. The concentration of ascorbic acid was measured by UV-spectrophotometer (UV-1201, Shimadzu, Kyoto, Japan) by using phenyl hydrazine as indicator according to the previously mentioned method by Sarwar et al.16
Determination of Serum Trace Elements Status
Flame atomic absorption spectrometry (Varian Spectra AA 220) with the graphite analysis technique was used to determine serum concentration of Zn, Cu, and Mn following the method of Sarwar et al.30 The samples of patients and controls were diluted with deionized water by a dilution factor of 10. Different concentrations (0.5, 1.0, 2.0, 5.0, and 10.0 mg/L) of trace elements were used for calibration of standard graphs. Absorbances were taken at 213.9, 224.8, and 279.8 nm for Zn, Cu, and Mn, respectively, in the atomic absorption spectrometer. To maintain quality and to verify the assay accuracy, the standard solutions were applied for every 10 test samples.
Statistical Analysis
All data were expressed as mean ± standard error mean (mean ± SEM) with their corresponding P values. Statistical analysis was performed using the statistical software package SPSS, version 16.0 (SPSS Inc, Chicago, IL). Comparison of the investigated parameters between patient and control groups was performed using independent-sample t test. Pearson’s correlation analysis was used to find the correlation among the various study parameters.
Results
Sociodemography of Subjects
The total study population of this research comprised of 80 samples (40 epileptic patients and 40 healthy volunteers). Sociodemographic data and clinical features of both epileptic patients and control subjects are presented in Table 1. It was observed that the mean age of patient and control group was 21.61 ± 12.69 and 22.57 ± 12.83 years, respectively. The study result demonstrated that men are more prevalent to epilepsy than women (75% vs 25%). In this study, it was also observed that patients from the urban area (52.50%) had higher prevalence of epilepsy than those from the rural area (47.50%). Furthermore, it was found that about 40% patients completed education up to primary level while 12.50% patients were illiterate. Assessment of clinical parameters showed that the mean difference of body mass index (BMI) between the patients (21.48 ± 4.17 kg/m2) and controls (23.53 ± 3.14 kg/m2) was significant (P < .05). The level of hemoglobin differed insignificantly (P > .05) between the patient (12.84 ± 0.96 g/dL) and control (14.13 ± 0.67 g/dL) group. The values of systolic and diastolic blood pressure (SBP and DBP, respectively) were 126.02 ± 8.84 and 82.72 ± 8.67 mm Hg for the patient group and 122.70 ± 14.28 and 79.57 ± 5.15 mm Hg for the control group, respectively.
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