Relationship between hTERT levels and oxidative stress in epilepsy patients

Abstract

Objective

Human telomerase reverse transcriptase (hTERT) is a key determinant of telomere maintenance and cellular aging. Oxidative stress plays a role in neurodegenerative processes by causing cellular damage through increased reactive oxygen species. Our study aimed to reveal the relationship between hTERT and oxidative stress in the pathophysiology of epilepsy.

Methods

The study included 45 individuals diagnosed with epilepsy and 55 healthy controls. hTERT concentration, total oxidant status (TOS), total antioxidant status (TAS), superoxide dismutase (SOD), and thiol‐disulfide homeostasis were measured in serum samples.

Results

A significant decrease in hTERT levels, an increase in oxidative stress markers (TOS, OSI), and a decrease in antioxidant levels (TAS, SOD), and total and native thiol levels were determined in epilepsy patients. In addition, a significant and negative correlation was found between hTERT and native thiol.

Significance

Our study suggested that the interaction between oxidative stress and hTERT levels in the pathophysiology of epilepsy may play an important role in seizure generation and progressive neural damage. This study will form the basis for further research and guide the identification of potential biomarkers for treating epilepsy.

Plain Language Summary

We measured the levels of a protective protein that helps preserve chromosomes and several markers of cell stress in people with epilepsy and healthy volunteers. People with epilepsy showed lower levels of this protective protein, weaker antioxidant defenses, and higher oxidative stress. We also found that lower protein levels were linked to fewer molecules that guard cells from damage. These changes may help explain why seizures occur and why the brain can suffer ongoing injury in epilepsy.

Key points.

• Epilepsy patients show significantly lower serum hTERT levels than healthy controls.

• Total oxidant status (TOS) and oxidative stress index (OSI) are elevated in epilepsy.

• Antioxidant defenses (TAS, SOD) and thiol levels are decreased in epilepsy patients.

• Oxidative imbalance and reduced hTERT may contribute to seizure generation and neural damage.

1. INTRODUCTION

Epilepsy is a long‐term neurological condition characterized by frequent, unexpected seizures and impacts approximately 50 million individuals worldwide. ¹ The pathophysiologic basis of epilepsy is highly complex, and neuroinflammation, mitochondrial dysfunction, and oxidative stress play an important role in seizure progression. ² , ³ However, failure to fully elucidate the underlying molecular mechanisms leads to clinical problems such as treatment resistance and progressive neurodegeneration. ⁴

Oxidative stress contributes to epilepsy through multiple mechanisms, including direct neuronal damage, disruption of neurotransmitter homeostasis, and increased neuroinflammatory responses. ⁵ Between oxidative stress and seizure occurrence, it has been suggested that oxidative damage triggered by seizures increases neuronal excitability, leading to additional seizure activity, and this process has the potential to turn into a cycle in itself. ⁶ Thiol‐containing molecules play an important role in maintaining cellular redox balance by scavenging ROS and in antioxidant defense systems. ⁷ The balance between reduced thiols (natural thiols) and their oxidized counterparts (disulfides) is an indicator of oxidative stress. ⁸ Superoxide dismutase (SOD) functions as the first line of defense against oxidative stress by regulating superoxide radical accumulation. Epileptic patients frequently exhibit decreased SOD activity, which may predispose neurons to oxidative damage. ⁹

Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of the telomerase ribonucleoprotein complex, best known for maintaining chromosomal stability by elongating telomeres. ¹⁰ Beyond its classical telomeric functions, hTERT also influences cellular stress responses, including redox homeostasis and apoptosis regulation. ¹¹ Studies in cancer and aging models suggest that hTERT may modulate oxidative stress resistance by affecting mitochondrial function and facilitating the repair of oxidative damage. ¹² , ¹³ Research on the role of hTERT functions in oxidative stress responses in epilepsy is quite limited. Considering that epilepsy causes oxidative stress‐mediated neuronal loss and hTERT plays a role in cellular stress adaptation, we hypothesized that reduced hTERT expression may contribute to the pathophysiology of epilepsy. For this purpose, serum hTERT levels and oxidative stress markers in epilepsy patients were compared with those of healthy controls.

2. METHODS

2.1. Study population and ethical approval

Individuals aged 18–65 years with epilepsy were selected from Kahramanmaraş Sütçü İmam University Research Hospital Neurology Outpatient Clinic, and the diagnosis was confirmed by clinical criteria and EEG findings. The control group consisted of healthy volunteers without neurologic or chronic diseases. The study was approved by Kahramanmaraş Sütçü İmam University Medical Research Ethics Committee (Protocol No: 2024/06‐53), and written informed consent was obtained from all participants.

We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

2.2. Collection and processing of blood samples

A volume of 8 mL of venous blood was drawn from the antecubital vein of all subjects. The samples were placed into biochemistry tubes containing gel, allowed to sit at room temperature for 30 min, and then centrifuged at 4000 rpm for 7 min to separate the serum. The serum was then transferred into 1.5 mL storage tubes and stored at −80°C, being homogenized by slow thawing at 4°C prior to analysis.

2.3. Biochemical analyses

hTERT was assessed using commercial ELISA kits (Elabscience hTERT [E‐EL‐H0706]) following the protocols provided by the manufacturer. The levels of total thiol (TT) and native thiol (NT) were evaluated spectrophotometrically with commercial kits from Rel Assay Diagnostics (Gaziantep, Turkey). Disulfide (DS) levels were obtained by taking half of the difference between TT and NT; subsequently, the ratios of DS/NT, DS/TT, and NT/TT were calculated. The Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) were measured using commercial kits from Rel Assay Diagnostics (Gaziantep, Turkey). Following the TOS and TAS levels measurements, the oxidative stress index (OSI), which serves as a reliable indicator of oxidative stress, was computed as the ratio of TOS to TAS. Malondialdehyde (MDA) levels, serving as an indicator of lipid peroxidation, were assessed using the method developed by Ohkawa et al. ¹⁴

2.4. Statistical analysis

Data distribution was assessed with the Shapiro–Wilk test. Seizure frequency was recorded in five clinical categories (1–2/month, >2/month, 1–4/year, >4/year, <1/year) and dichotomized for subgroup analysis into high frequency (≥1/month, n = 16) and low frequency (<1/month, n = 29) to ensure adequate subgroup sizes. For group comparisons, the Independent‐Samples t‐test was used for normally distributed variables and the Mann–Whitney U test for non‐normal variables. Associations between continuous variables were evaluated with Spearman's rank correlation. To control for multiple comparisons in correlation analyses, p‐values were adjusted using the Benjamini–Hochberg false discovery rate procedure (Q = 0.05); Bonferroni correction was calculated as a sensitivity analysis. Statistical analyses were performed with GraphPad Prism 10, and both raw and adjusted p‐values <0.05 were considered statistically significant.

3. RESULTS

This research involved 45 patients diagnosed with epilepsy who fulfilled the inclusion criteria and 55 healthy control participants. Among the patient group, 82% were female (n = 37) and 18% were male (n = 8). The average age of the patient group was 29.93 ± 8.19 years. In the group of healthy individuals, 51% were female (n = 28) and 49% were male (n = 27). The average age of the control group was 34.02 ± 12.80 years. The demographic and clinical characteristics of the study participants are presented in Table 1.

TABLE 1.

Demographic characteristics of epilepsy and control group individuals and clinical characteristics of epilepsy patients.

Parameters	Number of patients (n = 45)	Number of controls (n = 55)
Gender (Female/Male)	37 (%82)/8 (%18)	28 (%51)/27 (%49)
Age (years)	29.93 ± 8.19 (18–49)	34.02 ± 12.80 (19–55)
Age at diagnosis (years)	17.20 ± 8.51 (1–35)
Seizure type
Focal	21 (%46)
Generalized	24 (%54)
Seizure frequency
1–2/month	14
>2/month	2
1–4/year	5
>4/year	1
<1/year	23
Last seizure
Last 1 month	14
Last 3 months	6
Last 1 year	8
Last >1 year	17
Number of treatments received
1	18
2	12
3	13
>3	2

Note: Epileptic drugs used by patients: Lamotrigine, Levetiracetam, Carbamazepine, Lacosamide, Valproate, Ethosuximide, Topiramate, Pregabalin, Clobazam.

3.1. Biochemical analyses

As a result of the analyses performed in the study, it was determined that hTERT levels were significantly lower in the epilepsy group compared to the control group (p < 0.001). When oxidative stress markers were analyzed, TOS was significantly higher in the epilepsy group (p < 0.001), while TAS was significantly decreased (p < 0.001). Accordingly, Oxidative Stress Index (OSI) increased in the epilepsy group (p < 0.001). SOD activity, one of the antioxidant enzymes, was also significantly decreased in epilepsy compared to the control group (p < 0.001). MDA, an indicator of lipid peroxidation, was found to be significantly higher in the epilepsy group (p = 0.001).

When the thiol‐disulfide balance was analyzed, Total Thiol and Native Thiol levels were significantly lower in epilepsy patients (p < 0.001), whereas disulfide levels were significantly increased (p < 0.01). In parallel, the disulfide/Native Thiol and disulfide/Total Thiol ratios increased in the epilepsy group (p < 0.01), while the Native Thiol/Total Thiol ratio decreased (p < 0.01). These findings indicate that in addition to increased oxidative‐nitrosative stress and decreased hTERT levels, the thiol‐disulfide balance is also impaired in epilepsy. Thus, cellular redox homeostasis is severely affected. Statistical data for all parameters measured and calculated in the study are given in Figure 1 and Table 2.

FIGURE 1.

Serum and calculated parameter levels in control and epilepsy groups. Panels show (A) hTERT, (B) total oxidant status (TOS), (C) total antioxidant status (TAS), (D) oxidative stress index (OSI), (E) superoxide dismutase (SOD), (F) malondialdehyde (MDA), (G) total thiol, (H) native thiol, (I) disulfide, (J) disulfide/native thiol (DS/NT), (K) disulfide/total thiol (DS/TT), and (L) native thiol/total thiol (NT/TT). Control (n = 55); epilepsy (n = 45). Data are presented as mean ± SD; individual data points are overlaid on each bar. Data distribution was assessed with the Shapiro–Wilk test. Between‐group comparisons were performed using independent‐samples t‐tests for variables with normal distribution and Mann–Whitney U tests for non‐normally distributed variables; the specific test used for each panel and exact p‐values are reported in Table 2. Statistical significance is indicated as: **p < 0.01, ***p < 0.001. Error bars represent standard deviation.

TABLE 2.

Descriptive statistics of measured parameters in control and epilepsy groups.

Parameter	Control (mean ± SD)	Epilepsy (mean ± SD)	p‐value
hTERT	0.87 ± 0.33	0.40 ± 0.21	<0.001
TOS	10.5 ± 2.00	11.6 ± 2.65	0.006
TAS	0.95 ± 0.13	0.74 ± 0.16	<0.001
OSI	11.2 ± 2.32	16.2 ± 4.92	<0.001
SOD	1.33 ± 0.21	1.16 ± 0.25	<0.001
MDA	17.1 ± 1.77	18.4 ± 2.20	0.001
Total thiol	329 ± 23	313 ± 26	<0.001
Native thiol	287 ± 23	265 ± 21	<0.001
Disulphide	20.9 ± 5.46	24.2 ± 5.88	0.004
DS/NT	0.0737 ± 0.021	0.0924 ± 0.025	<0.001
DS/TT	0.0637 ± 0.016	0.0773 ± 0.017	<0.001
NT/TT	0.873 ± 0.032	0.845 ± 0.034	<0.001

Note: Values are expressed as Mean ± Standard Deviation (SD). p‐values were obtained using an independent samples t‐test for normally distributed data and the Mann–Whitney U test for non‐normally distributed data. A p‐value <0.05 was considered statistically significant.

Abbreviations: DS/NT, disulphide/native thiol; DS/TT, disulphide/total thiol; hTERT, human telomerase reverse transcriptase; MDA, malondialdehyde; NT/TT, native thiol/total thiol; OSI, oxidative stress index; SOD, superoxide dismutase; TAS, total antioxidant status; TOS, total oxidant status.

Given the sex imbalance between groups, we performed stratified comparisons. Among females (female controls n = 28; female patients n = 37), the differences reported above persisted: hTERT, TOS, TAS, OSI, SOD, MDA, Total Thiol, Native Thiol, Disulfide, and the calculated thiol ratios remained significantly different between female patients and female controls (all p < 0.05). In males (male controls n = 27; male patients n = 8), only serum hTERT (p < 0.001), OSI (p = 0.02), and Native Thiol (p = 0.004) reached statistical significance; the other parameters showed no significant differences (p > 0.05). The male epilepsy subgroup is small (n = 8); therefore, these male‐specific findings should be interpreted cautiously and considered exploratory (Tables 3).

TABLE 3.

Comparison of serum biomarkers between epilepsy and control groups stratified by gender.

Variable	Female control (n = 28)	Female epilepsy (n = 37)	p‐value	Male control (n = 27)	Male epilepsy (n = 8)	p‐value
hTERT	0.87 ± 0.36	0.40 ± 0.20	<0.001	0.86 ± 0.30	0.40 ± 0.22	<0.001
TOS	10.7 ± 2.10	11.6 ± 2.69	0.04	10.3 ± 1.92	11.6 ± 2.62	0.13
TAS	0.94 ± 0.14	0.72 ± 0.13	<0.001	0.96 ± 0.13	0.88 ± 0.26	0.26
OSI	11.4 ± 2.25	16.6 ± 4.67	<0.001	10.9 ± 2.40	14.5 ± 5.99	0.02
SOD	1.33 ± 0.21	1.15 ± 0.28	0.006	1.33 ± 0.22	1.21 ± 0.09	0.17
MDA	17.0 ± 1.72	18.4 ± 2.32	0.01	17.2 ± 1.85	18.5 ± 1.68	0.1
Total Thiol	327 ± 27	312 ± 24	0.02	330 ± 17	317 ± 19	0.1
Native Thiol	285 ± 27	264 ± 23	0.001	289 ± 18	268 ± 14	0.004
Disulphide	21.2 ± 3.23	24.2 ± 5.61	0.01	20.7 ± 7.14	24.4 ± 7.44	0.21
DS/NT	0.0750 ± 0.014	0.0926 ± 0.024	0.001	0.0724 ± 0.026	0.0915 ± 0.029	0.09
DS/TT	0.0649 ± 0.011	0.0774 ± 0.017	0.001	0.0623 ± 0.020	0.0764 ± 0.020	0.1
NT/TT	0.870 ± 0.021	0.845 ± 0.034	0.001	0.875 ± 0.041	0.847 ± 0.041	0.1

Note: Values are expressed as Mean ± Standard Deviation (SD). p‐values were obtained using an independent samples t‐test for normally distributed data and the Mann–Whitney U test for non‐normally distributed data. A p‐value <0.05 was considered statistically significant.

Abbreviations: DS/NT, disulphide/native thiol; DS/TT, disulphide/total thiol; hTERT, human telomerase reverse transcriptase; MDA, malondialdehyde; NT/TT, native thiol/total thiol; OSI, oxidative stress index; SOD, superoxide dismutase; TAS, total antioxidant status; TOS, total oxidant status.

When patients were dichotomized by seizure frequency (high vs. low), significant between‐group differences were observed for hTERT, SOD, MDA, Total Thiol, Native Thiol, DS/NT, DS/TT, and NT/TT (all p < 0.05) (Table 4). Specifically, the high‐frequency group showed higher hTERT, SOD, Total Thiol, and Native Thiol levels, together with higher MDA and elevated DS/NT and DS/TT ratios, compared with the low‐frequency group (Table 5).

TABLE 4.

Comparison of serum biomarkers between high‐frequency and low‐frequency epilepsy patients.

Parameter	High frequency (n = 16)	Low frequency (n = 29)	p‐value
hTERT	0.75 ± 0.35	0.41 ± 0.20	<0.001
TOS	11.6 ± 2.50	10.4 ± 2.42	0.28
TAS	0.76 ± 0.18	0.88 ± 0.19	0.22
OSI	14.0 ± 5.21	13.5 ± 4.74	0.84
SOD	1.42 ± 0.27	1.19 ± 0.23	0.006
MDA	18.9 ± 1.94	17.5 ± 2.13	0.03
Total Thiol	340 ± 16.9	312 ± 22.9	<0.001
Native Thiol	297 ± 14.0	263 ± 21.6	<0.001
Disulphide	24.5 ± 6.85	23.0 ± 4.14	0.9
DS/NT	0.0719 ± 0.008	0.0952 ± 0.025	0.001
DS/TT	0.0627 ± 0.006	0.0793 ± 0.017	<0.001
NT/TT	0.875 ± 0.013	0.841 ± 0.035	<0.001

Note: Values are expressed as Mean ± Standard Deviation (SD). Seizure‐frequency groups were defined from detailed clinical categories as follows: High frequency (≥1/month; n = 16) comprising patients with 1–2/month (n = 14) and >2/month (n = 2); Low frequency (<1/month; n = 29) comprising patients with 1–4/year (n = 5), >4/year (n = 1) and <1/year (n = 23). p‐values were obtained using an independent samples t‐test for normally distributed data and the Mann–Whitney U test for non‐normally distributed data. A p‐value <0.05 was considered statistically significant.

Abbreviations: DS/NT, disulphide/native thiol; DS/TT, disulphide/total thiol; hTERT, human telomerase reverse transcriptase; MDA, malondialdehyde; NT/TT, native thiol/total thiol; OSI, oxidative stress index; SOD, superoxide dismutase; TAS, total antioxidant status; TOS, total oxidant status.

TABLE 5.

Spearman's correlation coefficients among measured parameters.

	hTERT	TOS	TAS	OSI	SOD	MDA	Total thiol	Native thiol	Disulphide	DS/NT	DS/TT	NT/TT
hTERT	1	−0.136	−0.127	0.055	0.270	0.252	−0.150	−0.365	0.247	0.325	0.325	−0.325
TOS	−0.136	1.000	−0.073	0.679	0.054	−0.243	0.259	0.332	0.112	0.028	0.028	−0.028
TAS	−0.127	−0.073	1.000	−0.720	−0.144	−0.260	−0.077	−0.073	0.022	0.020	0.020	−0.020
OSI	0.055	0.679	−0.720	1.000	0.209	0.072	0.257	0.272	0.089	0.021	0.021	−0.021
SOD	0.270	0.054	−0.144	0.209	1.000	0.070	−0.093	−0.141	0.105	0.180	0.180	−0.180
MDA	0.252	−0.243	−0.260	0.072	0.070	1.000	−0.176	−0.297	−0.095	−0.062	−0.062	−0.062
Total thiol	−0.150	0.259	−0.077	0.257	−0.093	−0.176	1.000	0.803	0.399	0.137	0.137	−0.137
Native thiol	−0.365	0.332	−0.073	0.272	−0.141	−0.297	0.803	1.000	−0.003	−0.237	−0.237	0.237
Disulphide	0.247	0.112	0.022	0.089	0.105	−0.095	0.399	−0.003	1.000	0.951	0.951	−0.951
DS/NT	0.325	0.028	0.020	0.021	0.180	−0.062	0.137	−0.237	0.951	1	1	−1
DS/TT	0.325	0.028	0.020	0.021	0.180	−0.062	0.137	−0.237	0.951	1	1	−1
NT/TT	−0.325	−0.028	−0.020	−0.021	−0.180	−0.062	−0.137	0.237	−0.951	−1	−1	1

Note: This table presents the Spearman's rank correlation coefficients (r) among the following parameters: hTERT, TOS, OSI, SOD, Total Thiol, NO, Native Thiol, Disulphide, DS/NT, DS/TT, and NT/TT. Positive r values indicate a direct (positive) relationship, while negative r values indicate an inverse (negative) relationship. The magnitude of r reflects the strength of the relationship (|r| closer to 1.0 indicates a stronger correlation).

Abbreviations: DS/NT, disulphide/native thiol; DS/TT, disulphide/total thiol; hTERT, human telomerase reverse transcriptase; MDA, malondialdehyde; NT/TT, native thiol/total thiol; OSI, oxidative stress index; SOD, superoxide dismutase; TAS, total antioxidant status; TOS, total oxidant status.

Spearman correlation analyses (Tables 6, 7, 8) identified several associations that remained significant after Benjamini–Hochberg correction (Table 7). The strongest relationships were among the thiol–disulfide measures: the derived ratios DS/NT, DS/TT, and NT/TT show near‐complete mathematical dependence (ρ ≈ ±1.00, FDR‐p < 0.001). Disulfide was very strongly correlated with DS/NT and DS/TT (ρ = 0.951, FDR‐p < 0.001) and strongly negatively correlated with NT/TT (ρ = −0.951, FDR‐p < 0.001). Total thiol and native thiol were highly positively correlated (ρ = 0.80, FDR‐p < 0.001), and total thiol showed a moderate positive association with disulfide (ρ = 0.40, FDR‐p = 0.044). Oxidant–antioxidant indices were also interrelated: TOS correlated positively with OSI (ρ = 0.68, FDR‐p < 0.001), while TAS correlated negatively with OSI (ρ = −0.72, FDR‐p < 0.001). Notably, the previously reported unadjusted correlation between hTERT and native thiol (ρ = −0.365, p = 0.014) did not survive FDR correction (FDR‐p = 0.082). The full pairwise ρ and p_FDR matrix is provided in Table 7; disease–duration correlations are shown in Table 8. The heat map (Figure 2), which displays ρ values, visually supports this overall correlation structure and highlights the strong interrelationships among thiol–disulfide measures and between oxidant/antioxidant indices.

TABLE 6.

p‐values for Spearman's correlation analysis among measured parameters.

	hTERT	TOS	TAS	OSI	SOD	MDA	Total thiol	Native thiol	Disulphide	DS/NT	DS/TT	NT/TT
hTERT		0.372	0.407	0.719	0.073	0.095	0.062	0.014	0.833	0.495	0.495	0.495
TOS	0.372		0.635	<0.001	0.723	0.108	0.056	0.026	0.557	0.998	0.998	0.998
TAS	0.407	0.635		<0.001	0.344	0.085	0.658	0.633	0.856	0.781	0.781	0.781
OSI	0.719	<0.001	<0.001		0.169	0.638	0.114	0.071	0.588	0.822	0.822	0.822
SOD	0.073	0.723	0.344	0.169		0.650	0.225	0.356	0.457	0.850	0.850	0.850
MDA	0.095	0.108	0.085	0.638	0.650		0.248	0.047	0.535	0.684	0.684	0.684
Total thiol	0.062	0.056	0.658	0.114	0.225	0.248		<0.001	0.003	0.184	0.184	0.184
Native thiol	0.014	0.026	0.633	0.071	0.356	0.047	<0.001		0.730	0.172	0.172	0.172
Disulphide	0.833	0.557	0.856	0.588	0.457	0.535	0.003	0.730		<0.001	<0.001	<0.001
DS/NT	0.495	0.998	0.781	0.822	0.850	0.684	0.184	0.172	<0.001		<0.001	<0.001
DS/TT	0.495	0.998	0.781	0.822	0.850	0.684	0.184	0.172	<0.001	<0.001		<0.001
NT/TT	0.495	0.998	0.781	0.822	0.850	0.684	0.184	0.172	<0.001	<0.001	<0.001

Note: This table displays the p‐values corresponding to the correlation coefficients shown in Table 5. A p‐value of <0.05 was considered statistically significant.

Abbreviations: DS/NT, disulphide/native thiol; DS/TT, disulphide/total thiol; hTERT, human telomerase reverse transcriptase; MDA, malondialdehyde; NT/TT, native thiol/total thiol; OSI, oxidative stress index; SOD, superoxide dismutase; TAS, total antioxidant status; TOS, total oxidant status.

TABLE 7.

Pairwise Spearman correlations between serum hTERT and oxidative‐stress/thiol parameters (ρ and FDR‐adjusted p).

	hTERT	TOS	TAS	OSI	SOD	MDA	Total Thiol	Native thiol	Disulphide	DS/NT	DS/TT	NT/TT
hTERT	—	−0.14 (0.630)	−0.13 (0.671)	0.06 (0.867)	0.27 (0.268)	0.25 (0.285)	−0.15 (0.630)	−0.37 (0.082)	0.25 (0.286)	0.33 (0.129)	0.33 (0.129)	−0.33 (0.129)
TOS	−0.14 (0.630)	—	−0.07 (0.851)	0.68 (<0.001)*	0.05 (0.867)	−0.24 (0.286)	0.26 (0.280)	0.33 (0.129)	0.11 (0.748)	0.03 (0.910)	0.03 (0.910)	−0.03 (0.910)
TAS	−0.13 (0.671)	−0.07 (0.851)	—	−0.72 (<0.001)*	−0.14 (0.630)	−0.26 (0.280)	−0.08 (0.851)	−0.07 (0.851)	0.02 (0.910)	0.02 (0.910)	0.02 (0.910)	−0.02 (0.910)
OSI	0.06 (0.867)	0.68 (<0.001)*	−0.72 (<0.001)*	—	0.21 (0.399)	0.07 (0.851)	0.26 (0.280)	0.27 (0.268)	0.09 (0.821)	0.02 (0.910)	0.02 (0.910)	−0.02 (0.910)
SOD	0.27 (0.268)	0.05 (0.867)	−0.14 (0.630)	0.21 (0.399)	—	0.07 (0.851)	−0.09 (0.816)	−0.14 (0.630)	0.10 (0.775)	0.18 (0.505)	0.18 (0.505)	−0.18 (0.505)
MDA	0.25 (0.285)	−0.24 (0.286)	−0.26 (0.280)	0.07 (0.851)	0.07 (0.851)	—	−0.18 (0.512)	−0.30 (0.195)	−0.09 (0.816)	−0.06 (0.851)	−0.06 (0.851)	0.06 (0.851)
Total Thiol	−0.15 (0.630)	0.26 (0.280)	−0.08 (0.851)	0.26 (0.280)	−0.09 (0.816)	−0.18 (0.512)	—	0.80 (<0.001)*	0.40 (0.044)*	0.14 (0.630)	0.14 (0.630)	−0.14 (0.630)
Native Thiol	−0.37 (0.082)	0.33 (0.129)	−0.07 (0.851)	0.27 (0.268)	−0.14 (0.630)	−0.30 (0.195)	0.80 (<0.001)*	—	−0.00 (0.983)	−0.24 (0.286)	−0.24 (0.286)	0.24 (0.286)
Disulphide	0.25 (0.286)	0.11 (0.748)	0.02 (0.910)	0.09 (0.821)	0.10 (0.775)	−0.09 (0.816)	0.40 (0.044)*	−0.00 (0.983)	—	0.95 (<0.001)*	0.95 (<0.001)*	−0.95 (<0.001)*
DS/NT	0.33 (0.129)	0.03 (0.910)	0.02 (0.910)	0.02 (0.910)	0.18 (0.505)	−0.06 (0.851)	0.14 (0.630)	−0.24 (0.286)	0.95 (<0.001)*	—	1.00 (<0.001)*	−1.00 (<0.001)*
DS/TT	0.33 (0.129)	0.03 (0.910)	0.02 (0.910)	0.02 (0.910)	0.18 (0.505)	−0.06 (0.851)	0.14 (0.630)	−0.24 (0.286)	0.95 (<0.001)*	1.00 (<0.001)*	—	−1.00 (<0.001)*
NT/TT	−0.33 (0.129)	−0.03 (0.910)	−0.02 (0.910)	−0.02 (0.910)	−0.18 (0.505)	0.06 (0.851)	−0.14 (0.630)	0.24 (0.286)	−0.95 (<0.001)*	−1.00 (<0.001)*	−1.00 (<0.001)*	—

Note: Spearman rank correlation coefficients (ρ) for pairwise associations between serum hTERT, oxidative‐stress markers and thiol–disulfide parameters in the combined study cohort (n = 100; 45 epilepsy patients, 55 healthy controls). Each cell shows ρ (p_FDR), where p_FDR is the p‐value adjusted for multiple testing using the Benjamini–Hochberg false discovery rate procedure (Q = 0.05). Correlations that remained significant after FDR correction are indicated with an asterisk (*). Spearman ρ indicates both direction and strength of monotonic association (range −1 to +1).

TABLE 8.

Associations between disease duration and serum biomarkers (Spearman correlations).

Variable	r	p	p_FDR
hTERT	−0.05	0.726	0.807
TOS	0.01	0.955	0.955
TAS	−0.06	0.704	0.807
OSI	0.08	0.610	0.807
SOD	0.08	0.604	0.807
MDA	0.1	0.529	0.807
Total Thiol	0.13	0.387	0.807
Native Thiol	0.05	0.740	0.807
Disulphide	0.26	0.082	0.246
DS/NT	0.28	0.065	0.246
DS/TT	0.28	0.065	0.246
NT/TT	−0.28	0.065	0.246

Note: Spearman rank correlation coefficients (ρ) and corresponding p‐values for associations between disease duration (years) and serum biomarkers/oxidative‐stress parameters. p‐values were adjusted for multiple testing using the Benjamini–Hochberg false discovery rate (FDR, Q = 0.05); FDR‐adjusted p‐values are reported in the p_FDR column. No associations reached significance after FDR correction in our cohort.

FIGURE 2.

Heatmap of Spearman's correlation coefficients. This heatmap visualizes the correlation coefficients (r) from Table 5. The color scale (shown on the right) ranges from −1.0 (strong negative correlation) to +1.0 (strong positive correlation). Shades of red represent negative correlations, whereas shades of blue represent positive correlations. The intensity of the color corresponds to the magnitude of the correlation, allowing for a quick visual assessment of the relationships among the measured parameters.

4. DISCUSSION

In this study, we demonstrated that epilepsy patients exhibited prominent alterations in oxidative stress and serum hTERT levels compared to healthy controls. In particular, decreased serum hTERT was observed in epilepsy patients. In contrast, increased total oxidant status (TOS), elevated oxidative stress index (OSI), decreased total antioxidant status (TAS), superoxide dismutase (SOD) activity, and marked decreases in both total and natural thiol levels were recorded. These findings suggest that increased oxidative stress and reduced hTERT expression may play critical roles in the pathophysiology of epilepsy.

Oxidative stress happens when there is an imbalance between the generation of reactive oxygen species (ROS) and the effective elimination of these species; thus, various cellular damage mechanisms, such as protein oxidation, are activated. ¹⁵ Enzymatic and non‐enzymatic antioxidant systems in the cell maintain this balance. ¹⁶ Our findings on oxidative stress are consistent with previous studies reporting increased oxidative stress markers in epilepsy patients. For example, increased TOS and decreased TAS values have been reported in both treatment‐naïve and antiepileptic‐treated pediatric and adult epilepsy patients. ¹⁷ , ¹⁸ Increased TOS indicates the presence of higher ROS levels, whereas decreased TAS suggests the depletion of endogenous antioxidant defense systems. In parallel, increased OSI, which reflects the balance between oxidants and antioxidants, better emphasizes the pathological state, and these results agree with previous studies. ¹⁹ In addition, superoxide dismutase (SOD) activity, which is the first‐line antioxidant enzyme that provides defense against superoxide radicals, one of the ROS that cause oxidative stress, was also decreased in the epilepsy group, and these findings are consistent with previous studies. ²⁰ , ²¹ It should be noted, however, that our SOD measurement reflects total/systemic SOD activity in serum and is not isoform‐ or compartment‐specific; therefore, these data do not directly report mitochondrial SOD2 levels or intramitochondrial O₂•⁻/H₂O₂ dynamics. Reduced total SOD activity may indicate a diminished capacity to dismutase superoxide to hydrogen peroxide at a systemic level. However, the local balance between superoxide and H₂O₂ depends on tissue compartment, relative expression of SOD isoforms, and the activity of downstream peroxide‐removing enzymes (e.g., catalase, glutathione peroxidases), which may lower seizure threshold and exacerbate neuronal hyperexcitability. Accordingly, although serum SOD is not compartment‐specific, the observed decrease in total SOD activity is nevertheless compatible with an increased cellular—potentially including mitochondrial—superoxide burden and impaired dismutation capacity. We also measured malondialdehyde (MDA), a well‐established marker of lipid peroxidation, and found significantly higher MDA levels in the epilepsy group. Elevated MDA indicates increased lipid peroxidation that can compromise neuronal membrane integrity and ion‐channel function, thereby promoting neuronal hyperexcitability and providing a plausible mechanistic link between oxidative stress and seizure generation. ²²

In our study, native and total thiol levels decreased, whereas disulfide levels increased in epilepsy patients. Thiol groups, especially glutathione, play a central role in preventing cellular damage by neutralizing ROS and reactive nitrogen species. ²³ Thiol‐disulfide homeostasis is an important indicator of oxidative stress and disruption of redox balance in epilepsy. ²⁴ , ²⁵ In the literature, decreased native and total thiol concentrations and elevated disulfide levels have been reported in patients with epilepsy. ²⁶ , ²⁷ During recurrent seizures, mitochondrial dysfunction and overproduction of reactive species rapidly deplete thiol groups and disrupt the thiol‐disulfide balance. In this process, the conversion of thiols to oxidized forms (disulfide) leads to a reduction in the total thiol pool and a decrease in functional native thiol capacity. Consequently, this depletion of thiol reserves can trigger epileptic activity in a neural environment susceptible to redox imbalance.

hTERT is critical for neuronal stem cell proliferation and DNA repair. ²⁸ Beyond these roles, hTERT performs multiple non‐canonical functions in mature neurons, including protection against Aβ toxicity, preservation of cognitive function, enhancement of autophagy, and control of mRNA translation in RNA granules. ²⁹ , ³⁰ , ³¹ , ³² However, chronic oxidative stress and mitochondrial dysfunction in epileptic neurons may decrease hTERT expression by suppressing nuclear promoter activity. ³³ This mechanism may accelerate neuronal senescence, leading to loss of synaptic plasticity and decreased seizure threshold. In an in vitro study by Spilsbury et al., it was reported that TERT deficiency increased ROS production and caused oxidative damage in neurons induced by pathological tau. ¹³ Similarly, the inhibition of telomerase by Aβ aggregates in Alzheimer's disease ³⁴ and seizure‐induced tau hyperphosphorylation or excitotoxic protein accumulations (e.g., HMGB1) in the epileptic brain may accelerate neuronal aging by directly suppressing hTERT. This process creates a synergistic effect with DNA damage caused by chronic seizures. ³⁵ Although telomere shortening was not observed in coronary artery disease, chronic oxidative stress and inflammation have been shown to suppress hTERT expression, ³⁶ , ³⁷ which supports that decreased telomerase activity may be associated with neurodegenerative processes. Notably, all hTERT measurements in our study were performed in serum and therefore may not directly reflect intracellular hTERT regulation in neurons or other brain cells. hTERT is typically an intracellular protein component of the telomerase complex, and its presence in circulation may result from cell turnover or apoptosis, limiting the generalizability of peripheral measurements to central nervous system dynamics. ³⁸

A key limitation of this study is that only serum hTERT levels were assessed; telomerase enzymatic activity was not evaluated, and brain or cerebrospinal fluid samples were not analyzed. Therefore, findings related to neuronal hTERT regulation and its non‐canonical mitochondrial functions may differ. Future studies should include direct telomerase activity assays (e.g., TRAP) and, where feasible, examine both hTERT expression and telomerase enzymatic activity in neural tissues or CSF to better elucidate telomerase's roles in epilepsy.

However, although the changes in hTERT and oxidative stress biomarkers are significant, the cross‐sectional design of the study, as well as the limited sample size and the gender imbalance observed in the patient group (82% of the epilepsy group were female, whereas the healthy controls had approximately equal gender distribution) are limiting factors in generalizing the results. Furthermore, the difference in mean age between the patient and control groups may also create a potential confounding effect. In addition, the male epilepsy subgroup in our cohort is small (n = 8); gender‐specific analyses showed that the reported differences persisted in females, but several parameters did not reach statistical significance in males—this may reflect the absence of effect rather than limited statistical power in the male subgroup. Furthermore, while antiepileptic drugs (nine different agents in our cohort, with patients receiving mono‐ and polytherapy) may influence oxidative stress and related markers, the heterogeneity of drug types and combinations, along with the limited sample size per drug, precluded reliable drug‐specific subgroup analyses in this dataset. Further research is needed to determine whether the suppression of telomerase and hTERT precedes, accompanies, or follows the exacerbation of oxidative stress during epilepsy progression. Moreover, drug‐specific effects of antiepileptic medications on oxidative stress and hTERT should be evaluated in larger, prospective cohorts to disentangle medication effects from disease‐related changes.

In addition to the known literature, our study is among the first to report a simultaneous decrease in serum hTERT expression with oxidative stress in epilepsy patients. Furthermore, the parallel decrease in antioxidant defense systems (TAS, Total and Native Thiols, SOD) and serum hTERT with increasing oxidative stress indicates a mutually reinforcing relationship in the pathophysiological mechanisms of epilepsy. Reduced serum hTERT increases neuronal vulnerability, while ongoing oxidative stress may further suppress intracellular hTERT, deepening the pathological process.

AUTHOR CONTRIBUTIONS

MMÜ conceived and designed the study, performed biochemical analyses, interpreted the data, and drafted the manuscript. DTB handled patient recruitment, clinical evaluation, data collection, and interpretation. NÜ performed experimental procedures, contributed to data interpretation, and critically revised the manuscript. FİT contributed to data interpretation and manuscript revision.

FUNDING INFORMATION

The study was supported by the Kahramanmaraş Sütçü İmam University Scientific Research Projects Coordination Unit (Project No: 2024/3‐27M).

CONFLICT OF INTEREST STATEMENT

The authors state that there is no financial or other conflict of interest. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

ETHICAL APPROVAL

This study was approved by the Kahramanmaraş Sütçü İmam University Medical Research Ethics Committee (Protocol No: 2024/06‐53).

INFORMED CONSENT

Written informed consent was obtained from all participants.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.