Correlation between selenium levels and selenoproteins expression in idiopathic generalized epilepsy: a study from Karachi

Abstract

Background

Oxidative damage has been implicated in multiple neurodegenerative diseases, including epilepsy. Selenium, in the form of selenoproteins is an integral part of the human antioxidant defense system. Though a relationship between the altered selenium levels and epilepsy has been reported, limited evidence is available about the expression pattern of selenoproteins in epileptic patients.

Objective

This study aimed to determine the serum selenium levels in idiopathic epileptic and healthy individuals. Expression profiling of selenoproteins (GPx1, TRxR1 and SEPW1) both at mRNA and protein levels was also evaluated.

Methods

Serum selenium levels of 30 patients with idiopathic generalized epilepsy and their age and gender matched 30 healthy controls were measured. Protein levels of Serum Glutathione Peroxidase 1 (GPx1), Thioredoxin Reductase 1 (TRxR1) and Selenoprotein W (SEPW1) were estimated using ELISA. mRNA expression of GPx1, TRxR1 and SEPW1 were determined using qRT-PCR.

Results

The mean values for serum selenium levels in cases and controls were 37.6 ± 2.0 µmol/ml and 38.9 ± 2.7 µmol/ml, respectively. Selenium levels in cases were significantly lower as compared to controls (p = 0.031). No statistically significant differences were observed between the serum levels of selenoproteins GPx1, TRxR1 and SEPW1 in epileptic patients and the healthy group. GPx1 and TRxR1 expression was found to be down regulated (0.34 and 0.13 folds respectively) whereas SEPW 1 was found to be 0.04 folds up regulated in epileptic patients compared to the healthy subjects.

Conclusion

Selenium deficiency observed in epileptic patients suggests the association between serum selenium levels and epilepsy. This study provides the information about the selenium status in Pakistani population and helps in understanding the role of selenium in the prevention of epilepsy.

Graphical Abstract

Introduction

Epilepsy is a complex and occasionally idiopathic neurological condition characterized by unprovoked, uncontrollable, and recurrent seizures of varying types and intensities [1, 2]. It is global condition that transcends ethnic, geographic, and social boundaries. Its occurrence is universal, reflecting its basis in fundamental neurological processes that are common to all humans [3].This aliment affects individuals of all genders and age groups [4]. Around 70 million people over the world are estimated to be affected by epilepsy and approximately 90% of them are residing in developing regions of the world [4]. Though appropriate epidemiological data of epilepsy is scarce for Pakistan however an estimated prevalence of epilepsy is reported to be 9.99 per 1000 that highlighted the presence of about 1/10th world epileptic burden is in Pakistan [5]. Idiopathic generalized epilepsy is a group of epileptic disorders with no apparent cause, no structural brain abnormalities but believed to have a strong underlying molecular basis [6].

Epileptic seizures alter cellular activities of the brain including disturbed blood circulation, increased cerebrospinal fluid pressure, brain edema, and hypoxia that lead in a reduction of energy carriers such as ADP, ATP, phosphocreatine (PCr) and brain pH [7]. During epileptic seizures, the release of arachidic acid in the postsynaptic membranes stimulates an excessive release of glutamate. This activity leads to increased production and accumulation of reactive oxygen species (ROS) and a decline in the brain's antioxidant defenses, resulting in oxidative stress that promotes neuronal death and brain damage [8, 9]. Oxidative stress is known to be directly involved in pathways leading to neurodegeneration and in the progressive pathogenesis of neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and Epilepsy [10]. In order to minimize the cellular damages caused by ROS; mammalian cells have evolved with a well-established antioxidant defense systems to neutralize and block harmful effects of those ROS [11]. The human body is fortified with both enzymatic and non-enzymatic antioxidant mechanisms [12]. The shift of balance between the increased formation of reactive species and the activity of antioxidant systems towards decreased antioxidant activity not only leads to different forms of epilepsy but also increases the chances of recurrent epileptic seizures [13]. Combination antioxidant therapy during seizure activity has been found to insert neuroprotective effect [14].

Selenium (Se) is an essential element that is crucial for numerous metabolic functions, protecting the body against free radicals and preventing oxidative tissue damage [15]. Recent evidence have further emphasized the significance of selenium in the prevention of epilepsy because of its antioxidant potential [16]. This micronutrient exerts its protective effects mainly via selenium-dependent antioxidant enzymes and selenoproteins, which feature selenocysteine, the 21st amino acid, at their active sites [17]. The human proteome includes 25 identified selenoproteins, which are primarily classified into two groups based on the location of selenium within the protein structure [18]. In one group, selenium is located near the C-terminus, with examples including TrxRs, SelI, SelK, SelO, and Seps1 [19]. The other group has selenium positioned near the N-terminus, with examples such as GPXs, DIOs, SelH, SelM, Sepn1, SelT, Sepw1, SPS2, and Sep15 [19]. Additionally, selenium and selenoproteins play a crucial role in brain signaling pathways. Selenoproteins, besides working as antioxidant seems to exert diverse brain functions directly affecting GABAergic neurons and their signaling molecule GABA [20]. Studies have shown that selenium deficiency can cause degeneration of GABAergic neurons which can lead to impaired neuronal function seen in different neurological disorders including epilepsy [21]. The pathophysiological effects of selenoproteins are closely dependent on the status of selenium in the human body [22]. Previous literature has shown a relationship between the altered selenium levels and epileptic seizers [22]. Given that the micronutrients deficiency is a big public health problem in Pakistan and only few studies have evaluated the level of trace elements particular selenium in Pakistani population [23]. However, limited evidence are available about the expression pattern of selenoproteins in epileptic patients. This study is designed to understand the role of selenium and selenoproteins in the pathophysiology of epilepsy, by measuring both serum selenium levels and selenoproteins expression in epileptic and healthy individuals.

Materials and methods

Study site and participants

This case-control study recruited a total of 60 participants, divided into two study groups: drug naïve idiopathic generalized epileptic (IGE) patients (n = 30) and healthy individuals. The study was conducted over a one-year period, from July 2019 to June 2020.

Drug-naïve idiopathic generalized epileptic patients (n = 30) of either sex, aged 15 to 60 years, were enrolled after a confirmed diagnosis by a consultant neurologist from the Outpatient Department of Neurology, Dr. Ruth K. M. Pfau Civil Hospital Karachi (CHK), and the Outpatient Department of Neurology at Dow University Hospital Ojha Campus, Karachi. These two centers serve not only patients from Karachi but also from other parts of Pakistan. All patients recruited for this study were newly diagnosed with IGE and had not yet initiated antiepileptic drug therapy, ensuring that they were drug-naive at the time of sample collection. Age- and gender-matched healthy individuals (n = 30) were recruited from local colleges affiliated with DUHS and through personal contacts.

Informed consent was attained from all study participants aged 18 years and above. For participants under the age of 18, signed consent was obtained from parents or guardians. The anonymity of data was ensured to protect the privacy of the participants.

An approved questionnaire was completed through interviews with the study patients or their attendants. This questionnaire collected information regarding patient demographics and clinical characteristics.

Sample collection and processing

Blood samples (10 ml) were collected into three separate vacutainers: one containing a clot activator, one without anticoagulant, and one with EDTA (5ml in each tube). The collected samples were transported to the laboratory while maintaining the cold chain. Blood samples in the vacutainers containing the clot activator and without anticoagulant were centrifuged at 8000 rpm for 5 minutes to separate the serum. The serum was aliquoted into four polypropylene tubes and stored at −70°C until further analysis by ELISA for selenoproteins and selenium levels. The EDTA-containing vacutainers were processed immediately for RNA extraction.

Measurement of serum selenium levels

Serum Selenium levels were measured in both study groups by selenium assay kit provided by Abbexa Ltd., Cambridge, UK (catalog number: abx298910). Absorbance was recorded at 520 nm and serum selenium concentrations were calculated as µmoles/ml.

Estimation of serum selenoproteins (GPx1, TRxR1 and SEPW1) levels

Serum levels of Glutathione Peroxidase 1 (GPx1), Thioredoxin Reductase 1 (TRxR1), and Selenoprotein-W (SEPW1) were measured using commercially available enzyme linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. The kits based on sandwich ELISA method, were provided by USCN Bioassay Technology, China, with the following catalogue numbers: SEA295Hu (GPx1), E3953Hu (TRxR1), and SEE684Hu (SEPW1).

Briefly, standard, blanks, and samples were added to the ELISA plate wells precoated with antibodies specific to human GPx1, TrxR1, or SEPW1 and incubated for 1 hour at 37^oC. After incubation, the liquid was removed, and the plates were incubated with a secondary antibody. The plates were then washed, and a substrate solution was added to each well. The enzyme-substrate reaction was terminated by adding a stop solution. Absorbance was measured at 450 nm. Samples were run in duplicates, and the serum levels of Gpx1, TRxR1, and SEPW1 levels in each sample were calculated using their respective standard curves and regression equations.

RNA extraction and quantitative real time PCR (qRT-PCR)

The total RNA was extracted from 250µl whole blood sample using the TRIzol™ LS Reagent according to the protocol of Invitrogen. Briefly, 250µl whole blood was mixed with 750µl Trizol LS reagent and invert mixed to obtain a homogenous solution. Sample was incubated for 5 min and followed by adding 200µl chloroform. Samples were mixed, incubated for 2–3 minutes at room temperature and then centrifuged at 13000rpm for 15min at 4°C. Upper phase was transferred into a new tube and 500µl isopropanol was added to the tube containing the aqueous phase. Tube was incubated again at room temperature for 10min and centrifuged at 13000rpm for 10min at 4°C. Supernatant was discarded and pellet was washed in 75% ethanol. Pellet was centrifuged again at 4°C at maximum speed and was air dried before resuspending in 20µl nuclease free water. Purity and quality of RNA samples was tested using microvolume spectrophotometer.

DNase treatment was carried out using RQ1 RNase-Free DNase kit from Promega to ensure that the samples were not contaminated with genomic DNA. 500 ng RNA in a total volume of 8µl was mixed with RNase-free DNase-I and incubated at 37°C for 30 minutes. 1 µL stop solution added, reaction stopped by incubating at 65°C for 10 minutes.

cDNA was prepared using RevertAid First Strand cDNA Synthesis Kit. To synthesized cDNA, purified RNA was mixed with primers (Oligo dT18), dNTPs, RNase inhibitor, reaction buffer, and reverse transcriptase enzyme in a final 20 µL reaction. Samples were incubated at 42°C for 60 minutes, then at 70°C to terminate the reaction. cDNA stored at −80°C.

QuantStudio-7 Flex Real Time PCR Detection System (Applied biosciences) was used for the comparative expression analysis of selenoproteins (GPx1, TRxR1and SEPW1). All reactions were run in triplicates and the data was normalized using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control. Samples were amplified using PowerUp™ SYBR™ Green Master Mix (2X) and gene specific primers (10mM each) in a total reaction volume of 10µl. The relative expression values were calculated in comparison to the transcript levels quantified in healthy control to achieve a relative fold change of expression. The data was analyzed by comparative 2^−ΔΔCT method, MIQE guidelines were followed for the overall qPCR experimentation [24]. All the primers were designed through Primer3 Input software (v.0.4.0) and their sequences are presented in Table 1.

Table 1 Primers used in the study

Statistical analysis

The sample size for the study was calculated using OpenEpi.com. The estimated sample size for this case-control study was 60 participants, with 30 in each group. This calculation was based on a comparison of previously reported mean selenium levels in epileptic patients (73.37 ± 13.31 µg/L) and non-epileptic individuals (85.55 ± 19.39 µg/L) [25], with 80% power and a 95% confidence interval.

The collected data were analyzed using SPSS version 24. The normality of the data was tested using the Shapiro-Wilk test. Descriptive statistics were reported as means ± standard deviations (SD) for continuous variables with a normal distribution, and as medians with interquartile ranges (IQR) for variables without a normal distribution. Categorical variables were reported as numbers and percentages (%). The Chi-square test was used to compare categorical variables such as gender, marital status, employment status, and ethnicity between the groups.

The independent t-test was applied to analyze serum selenium levels between cases and controls, as well as among gender, age groups, and clinical variables. Variables without a normal distribution (e.g., age and selenoprotein levels) were analyzed using the Mann-Whitney test. Mann-Whitney U test was also used to determine association between the mRNA expression profile of selected biomarkers in respective study groups. A p-value of < 0.05 was considered statistically significant.

Results

This study has evaluated 60 participants including 30 epileptic patients and 30 controls. The median age of participants was 20.0 years and most of them were males (65.0%). The majority of the participants were unmarried (63.3%), employed (48.3%) and from Urdu speaking ethnicity (65.0%). There was no significant difference found in age, gender, martial and employment status of healthy individuals and epileptic patients. Significant difference was found in the ethnicity of patients and controls (p = 0.003). Demographic data of the study participants is presented in Table 2.

Table 2 Demographic Data of Study Participants (n=60)

Baseline, clinical and biochemical characteristics of idiopathic generalized epileptic patients

Baseline, clinical and biochemical characteristics of the idiopathic generalized epileptic patients is shown in Table 3. The median duration of seizures in epileptic patients was 5.0 min. Epileptic patients had a median of 5.0 episodes in the past. The frequency of epileptic episodes was reported to be 26.7% once a week, and 56.7% for once a month. Most of the patients had reported that their frequencies of seizures were becoming more frequent (53.3%). Most of the epileptic patients were idiopathic (76.7%), while 23.3% had reported that seizures were triggered due to headache or stress. Most epileptic patients had no family history of seizures (66.7%). Comparison of epileptic episodes in male and female patients showed no significant difference and the frequency of epileptic episode was mostly reported once a month by both genders (Fig. 1). Most of the male patients in contrast to females have reported that their seizures are usually triggered by stress or headache. Majority of male patients have reported no family history of seizures (Fig. 1).

Table 3 Baseline, Clinical and Biochemical Characteristics of Idiopathic generalized epileptic patients (n=30)

Fig. 1

Family history of epilepsy in male and female epileptic patients

Comparison of serum selenium levels between epileptic patients and healthy individuals

This study analyzed and compared the serum selenium levels in patients with Idiopathic generalized epilepsy (cases) and healthy individuals (controls). Mean selenium levels in idiopathic generalized epileptic patients (37.6 ± 2.0 µmol/ml) were found to be lower as compared to the levels in healthy individuals (38.9 ± 2.7 µmol/ml). The difference in selenium levels between the cases and control was statistically significant (p = 0.031). Comparison of Selenium levels between cases and controls is shown in Table 4 and Fig. 2.

Table 4 Comparison of serum Selenium Levels between Epileptic patients (Cases) and normal healthy individuals (Controls), n=60

Fig. 2

Selenium Levels between Cases and Controls. ^*p<0.05, statistically significant and calculated using independent t-test

Comparison of selenoproteins levels between epileptic patients and healthy individuals

The median levels of the three selenoproteins (Glutathione Peroxidase 1, Thioredoxin Reductase 1, and Selenoprotein-W) are given in Table 5. Comparison of selenoprotein levels in epileptic patients and controls showed no significant difference for any of the three proteins studied (p > 0.005). The levels of Thioredoxin Reductase 1 were found similar in both study groups. Biomarker Selenoprotein W showed slight difference, with epileptic patients having higher levels as compared to controls; however the difference was not significant. The study subjects were categorized into two age groups, i.e., Group 1: comprised of subjects less than 18 years of age. Group 2: Comprised of subjects more than 18 years of age. The levels of selenium in both age groups were found to be the same (Table 6, Fig. 3). Similarly, no major differences were found in the levels of the biomarker Glutathione Peroxidase 1, Thioredoxin Reductase and Selenoprotein-W in subjects more and less than 18 years of age in cases and controls. Levels of selenoproteins were also evaluated in male and female gender. Slight difference was noted in the levels of the biomarker Selenoprotein W where the levels for Selenoprotein-W were higher in females as compared to males. No significant differences were found in biomarker Glutathione Peroxidase 1, Thioredoxin reductase 1 and Selenoprotein-W. The levels of the biomarkers in male and female genders are shown in Table 7 and Fig. 4.

Table 5 Comparison of Selenoproteins (Glutathione Peroxidase-I, Thioredoxin Reductase and Selenoprotein W) levels in epileptic patients and healthy control group, n=60

Table 6 Selenium and Selenoproteins levels among different age groups

Fig. 3

Serum selenium and selenoproteins levels in different age groups. A Selenium levels, B Levels of Glutathione Peroxidase (GPx1), C Levels of 1 Thioredoxin reductase (TRxR1), D Levels of Selenoprotein W (SEPW1)

Table 7 Selenium and Selenoproteins levels among genders

Fig. 4

Serum selenium and selenoproteins levels in both genders. A Selenium levels, B Levels of Glutathione Peroxidase (GPx1), C Levels of Thioredoxin Reductase (TRxR1), D Levels of Selenoprotein W (SEPW1)

Association between selenium and selenoprotein levels with clinical spectrum of idiopathic epilepsy

The association between selenium and selenoproteins levels with clinical data of epileptic patients was determined and presented in Table 8. No significant association was found between the selenium levels and any of the variables of clinical presentation of epilepsy which included duration of seizers, total number of attacks, frequency of epileptic, family history of seizure and reason for triggering of seizures. Similarly, no significant association was found between selenoprotein (GPx1, TRxR1 and SEPW1) levels and any of the variables related to the clinical presentation of epilepsy, except for total number of epileptic attacks category in which GPx1 levels were significantly lower in a group of patients that were having more than 5 attacks.

Table 8 Association between selenium and selenoproteins levels with clinical data of epileptic patients

Gene expression profiling of selenoproteins

The expression profiling of selenoproteins was analyzed and compared with healthy individuals. The data represents that the expression level of Selenoprotein W (SEPW1) is up regulated (1.5 fold up regulation) in epileptic patients whereas the gene expressions of GPx1 and TRxR1 are down regulated (RQ = 0.13 and 0.5 respectively) in epileptic patients (Fig. 5). Expression profiling was further compared in relation to gender of study participants. As deduced in Fig. 6, the comparison between males and females suggests that transcription levels of SEPW-1 are higher (1.3 fold in males and 2.8 fold in females) as compared to healthy individuals. The data further suggests that SEPW1 is comparatively up regulated in females as compared to males (2.8 fold vs 1.3 fold) The data is however non-significant. Contrary to SEPW1, GPx1 and TRxR1 are significantly downregulated in both male and female patients (RQ of GPx1 = 0.7 and 0.6 in males and females respectively and RQ of TRxR1 = 0.5 and 0.3 in males and females respectively). The data further reported that female patients displayed further reduced levels of GPx1 and TRxR1 as compared to male patients.

Fig. 5

Expression profile of selenoproteins in epileptic patients. The expression profile in epileptic patients was represented as relative quantification (RQ) in comparison to healthy volunteers. p-value was calculated using Mann-Whitney U test with respect to healthy volunteers for every gene transcript respectively (NS-data not significant; ^** p<0.01; ^*** p<0.001)

Fig. 6

Comparison of expression profiling based on gender. The expression profile in epileptic patients was represented as relative quantification (RQ) in comparison to healthy volunteers. p-value was calculated using Mann-Whitney U test with respect to healthy volunteers for every gene transcript respectively and comparison was made with reference to genders. The data is represented with respect to every gene transcript mentioned as HM-healthy male; HF-healthy female; PM-patient male; PF-patient female. p-value were represented as NS-data not significant; ^* p<0.05; ^**p<0.01; ^*** p<0.001

Both healthy and epileptic patient groups were further sub-grouped as above (≥ 18) or below (< 18) years of age and compared in between different groups (Fig. 7). Since age plays a significant role in the metabolic ROS production, it was speculated that age dependent metabolic status might have an impact on the respective expression levels of selenoproteins. Furthermore, since epilepsy is a progressive neurological disorder, age is a significant factor when compared between adults and adolescents. A difference in expression profile was observed between healthy and patient’s age groups (Fig. 7). While the overall data aligns with the previous analysis as shown in Figs. 5 and 6 with GPx1 and TRxR1 being under expressed in patients as compared to healthy, while SEPW1 slightly over expressed. Comparing the expression levels between adults and minors suggests that subjects of more than 18 years of age have a more pronounced varied effect in the expression levels as compared to younger volunteers of less than 18 years of age. Strikingly, SEPW1 is downregulated in patients less than 18 years as compared to patients of bigger age group (RQ = 0.74 vs 1.75). GPx1 is significantly under expressed in groups containing individuals of more than 18 years old as compared to under 18 years age group (RQ = 0.19 vs 0.7). In contrast to SEPW1, expression of TRxR1 significantly downregulated in patients of more than 18 years of age (RQ = 0.6) while the transcript levels are 1.3 fold upregulated in epileptic patients of age less than 18 years which is contrary to the means data mentioned in Fig. 7.

Fig. 7

Expression profile of selenoproteins in epileptic patients with respect to age group. The expression profile in epileptic patients was represented as relative quantification (RQ) in comparison to healthy volunteers. p-value was calculated using Mann-Whitney U test with respect to healthy volunteers for every gene transcript respectively and comparison was made with reference to age groups being <18 and >18. P value were represented as NS- data not significant; ^* p<0.5; ^** p<0.01; ^*** p<0.001

Discussion

The current study was designed to understand the role of selenium and selenoproteins in the pathophysiology of epilepsy, by measuring both serum selenium levels and selenoproteins expression in epileptic patients and healthy individuals.

This study was conducted on a total of 60 subjects including 30 idiopathic generalized epileptic patients and 30 healthy individuals. Healthy participants were both age and gender matched to the epileptic patients. Median age of the study participants was 20.0 (15.3–27.0) years with a preponderance of male population. The ratio of male to female epileptic patients enrolled in this study was observed to be 2:1. These results with high incidence of epilepsy among males are in line with other studies reported from Pakistan [25]. A meta-analysis on Prevalence, Incidence and Etiology of Epilepsy however reported inverse data to our results with a higher prevalence of epilepsy in females [26]. The disparity in data can be due to geographical, economical, and climatic differences that influence the causes, management and outcome of epilepsy in the region [27]. Male predominance in epilepsy may be due to their frequent exposure to the environmental factors or trauma since they are being more active in social life [28].

The results of this study showed a significantly lower serum selenium levels in epileptic patients compared to healthy controls (p = 0.031). However, serum selenium levels were found not to be affected by either age or gender within the patient group or when patient group was compared to the controls. These results are in agreement with numerous published studies that presented lower serum selenium concentrations in different types of epilepsy [24, 29,30,31]. The study reported by Per et al., 2012 stated statistically significant lower levels of selenium in children with resistant epilepsy in comparison to the healthy controls [32]. Another study showed lower levels of serum selenium in epileptic subjects as compared to healthy individuals [22, 33]. As a critical antioxidant, selenium primarily aids in reducing hydrogen peroxide and harmful lipid and phospholipid hydroperoxides in the brain to harmless byproducts. Growing evidence suggest that the depleted selenium levels in epileptic patients are due to its high consumption in quenching excessive free radicals generated in the initiation and progression of epilepsy particularly during seizures [13]. The inclusion of drug-naive patients eliminates the potential confounding effects of antiepileptic drugs (AEDs), which are known to influence selenium metabolism and oxidative stress pathways. This criterion strengthens the reliability of our findings by attributing changes in selenium levels and selenoprotein expression directly to IGE.

In biological systems, Selenium is essentially attributed to selenoproteins where selenium is incorporated as selenocysteine. Selenoproteins play important role in muscle regeneration, oxidative homeostasis, thyroid hormone metabolism, production of growth factors and immune responses [34]. The precise metabolic function of many selenoproteins is currently unknown however, thioredoxin reductases (TrxR), and glutathione peroxidases (GPx) and Selenoprotein W (SEPW 1) are well characterized selenoproteins involved in redox regulation and oxidative homeostasis. Their levels indirectly reflect the status of selenium level in the human body. Gene expression profile revealed downregulation of GPx1 gene in epileptic patients (RQ = 0.13) compared to the healthy group. Furthermore, lower serum levels of glutathione peroxidase 1 (GPx1) were also observed in epileptic patients compared to the healthy group. However, those results are statistically not significant. Similar observations have been reported in epileptic children with Febrile Seizures where serum glutathione peroxidase levels were higher in healthy group compared to the epileptic children however no significant difference was found [35]. Another study determined lower GPx activity in the serum of children with epilepsy than those of the control group with no significant relationship [29]. A study on five Progressive myoclonic epileptic patients aged 19–29 years and 12 healthy subjects demonstrated slightly lower levels of GPx in patients compared to the controls however their results were statistically insignificant [36]. Ristić et al., 2015 further reported an increased expression of GPx in hippocampi of patients with mesial temporal lobe epilepsy [37]. In contrast some studies have noted no difference in the serum GPx levels between epileptic patients and healthy controls [38]. Contrary to our results, Yüzbaşioğlu et al., 2009 reported 2.3-fold up regulation of GPx1 gene expression [39]. The anti-oxidative role of glutathione peroxidase has been well emphasized. Excessive generation of free radicals during the course of epilepsy and compromised ROS detoxification due to lower levels of GPx1 enzyme may increase the risk of severe oxidative damage in brain cells [40]. GPx1 is reported to be more sensitive to cellular Selenium levels compared to other selenoprotein and possibly works as an efficient storage device [41]. Thus, lower levels of GPx1 noted in our study may be a reflection of selenium depletion in epileptic patients.

In the present study, serum levels of Thioredoxin Reductase 1 (TRxR1) were observed to be the same in both epileptic patients and control groups however gene expression results showed reduced transcript levels of TRxR1 gene (RQ = 0.5). Various studies reported varied levels TRxR1. A study conducted on the surgically excised hippocampus samples of intractable mesial temporal lobe epilepsy (MTLE) patients reported low expression of TRxR1 in immunostained MTLE neurons compared to the controls [39]. Contrary to that, Yu et al., 2019 reported a high protein level of TRxR1 in the cortex of PTZ induced seizure model mice [42]. Variation in the protein level of TRxR1 might be due to the sample type used in different studies. Both aforementioned reference studies measured localized levels of TRxR1 in the excised brain tissues of either epileptic patients or drug induced seizures mice, however in our study serum levels of TRxR1 were estimated. TRxR1 levels are reported to be different at different brain parts. Increased levels of TRxR1 at both protein and mRNA are noted to be mainly occurred in the cortex and somewhat in the hippocampus of PTZ kindling seizure model mice. However, a decreased level of this selenoprotein is observed in in the cerebellum and diencephalon [42]. For these varied levels of TRxR1, authors of those studies believe that might be a reflection of less oxidative stress happened or absence of ant oxidative response in those brain regions.

The present study further observed marginally elevated levels of Sel W (SEPW1) in the serum of epileptic patients when compared to the control group. Our data suggested that mRNA expression results of SEPW1 gene coincide with the protein data with an about 1.5 fold increase of SEPW1 gene expression in epileptic patient group compare to the healthy cohort. Higher expression of this gene is observed in female epileptic patients and those with > 18 years of age. These observations are in line with the results of Yüzbaşioğlu et al., 2009 which exhibited raised levels of SELW in neurons of in mesial temporal lobe epilepsy patients [39]. Contrary to these results, Özbas-Gerceker reported 11-fold reduced gene expression of Sel W 1 in the hippocampus of patients with temporal lobe epilepsy [43]. The up regulation of SELW 1 expression observed in our study among epileptic patients is likely to be caused by the high oxidative stress in epileptic patients and stimulation of the defense mechanism against this high oxidative trauma. Selenoprotein W is a glutathione dependent antioxidant that provides protection against oxidative stress and H₂O₂-induced cell apoptosis [44]. It is one of the selenoproteins that express at highest level in most of the human brain parts which reflects its central role in normal brain functions and altered levels at any levels (mRNA or protein) may imitate pathological status of the brain [45].

The present study has a few limitations, including a small sample size and the estimation of only selected selenoproteins. Furthermore, localized selenium and selenoprotein levels in the brain were not determined, which may limit the generalizability of the results.

Looking across the results of our study, we found that the selenium levels were significantly lower in epileptic patients compared to the healthy controls. That might be due to its extensive use as an antioxidant for removing free radicals during the course of epilepsy. Therefore, measuring serum selenium levels and addressing selenium deficiency could help in managing epilepsy. Selenium supplementation in epileptic patients may be beneficial in reducing the progression of seizures or the disease. The levels of selenoprotein GPx1 are also observed to be lowered among epileptic patients when compared to the control group which might suggest its sensitivity towards selenium depletion in epileptic patients. There were no significant differences in serum TRxR1 levels between the two study groups however slightly elevated levels of SEPW1 both at mRNA and protein levels were noted which suggesting a protective role of this selenoprotein during epilepsy. Due to serious obstruction in the availability of surgically excised brain tissue samples of healthy controls and idiopathic epileptic patients, analysis of selenium and selenoproteins was merely restricted to the whole blood samples of these individuals. Evidence supports a comparable mRNA of selenoprotein in whole blood to the major tissue in experimental animals. This study may provide a basis for using whole blood for assessing selenium and selenoprotein status as a molecular marker in generalized epileptic patients. While mRNA and ELISA results provide valuable insights, they are insufficient to fully elucidate the pathophysiology of IGE. Future studies integrating histological or pathological data will be critical in further unraveling the underlying mechanisms.

Conclusion

This study found significantly lower selenium levels in epileptic patients as compared to healthy controls. Selenium deficiency observed in epileptic patients suggests the association between selenium levels and epilepsy. It is concluded from the study that selenium affects the progression of neurological or neurodegenerative disorders in humans. Selenoprotein analysis showed non-significant variations in their expression both at mRNA and protein level, with GPx1 and TRxR1 expression was found down regulated whereas SEPW 1 was up regulated in epileptic patients. Furthermore, no significant differences were found in selenium levels and selenoprotein expression with respect to age and gender groups. SEPW was found to be up regulated both at mRNA and protein level in epileptic patients indicating a protective or antioxidant role of this protein. This study provides information about the selenium status in our population and helps in understanding the role of selenium in the prevention of epilepsy. However, further studies with larger sample sizes are recommended to further examine the correlation between selenoprotein levels and epilepsy clinical presentation.