Seizure 21 (2012) 780–784

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Oxidative stress in patients with epilepsy is independent of antiepileptic drugs

Bindu Menon a,*, Krishnan Ramalingam b, Rajendiran Vinoth Kumar b
a
Department of Neurology, Narayana Medical College and Superspeciality Hospital, Chintareddypalem, Nellore-2, Andhra Pradesh, India
b
Department of Biochemistry, Narayana Medical College and Superspeciality Hospital, Chintareddypalem, Nellore-2, Andhra Pradesh, India

A R T I C L E I N F O A B S T R A C T

Article history: Purpose: Oxidative stress has been implicated in many pathological processes. The brain is highly
Received 3 April 2012 susceptible to oxidative stress because of its high lipid content. In this study, the oxidative parameters in
Received in revised form 31 August 2012 patients with epilepsy and those of a control group were measured. The study’s aim was to determine
Accepted 2 September 2012
whether there are differences between the oxidative levels of treated and untreated patients and
whether there are differences between individual antiepileptic drugs.
Keywords: Methods: Oxidative parameters such as Malondialdehyde (MDA), Protein Carbonylation (PC) and Nitric
Lipid peroxidation
Oxide (NO) levels were evaluated in 100 patients with epilepsy and compared to an equal number of age
Protein carbonylation
and sex matched healthy subjects. In the study group, the oxidative parameters of 25 untreated patients
Nitric oxide
Malondialdehyde were compared to treated patients and the control group. The treated patients were divided into 2
Seizures groups based on their antiepileptic drugs (AEDs). Group 1 consisted of patients on a single AED, and
Epilepsy group 2 was composed of patients taking more than one AED. A comparative study was performed on the
Antiepileptic drugs oxidative parameters of medicated patients on single drug regimens and multidrug regimens.
Results: The MDA and PC levels were significantly higher in patients than in the control group
(P < 0.0001). However, there was no significant difference in the NO levels of patients and those of the
control group. The 25 untreated patients had higher MDA and PC levels compared to both the control
group (P < 0.0001) and treated patients (P < 0.0001). There was no difference in the oxidative levels
between untreated patients and treated patients, group 1 and group 2, and individual AEDs.
Conclusions: In this study, we demonstrated the presence of significantly increased levels of oxidative
markers in patients with epilepsy as compared to the control group. AEDs did not influence the oxidative
markers, suggesting the presence of seizure-induced oxidative stress.
ß 2012 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

1. Introduction
Epilepsy is a chronic neurological disorder that requires long
term treatment. Various studies have demonstrated the role of
oxidative stress in the pathogenesis of atherosclerosis, psychiatric
and neurodegenerative disorders.1–3 The brain is generally in a
balanced redox state between oxidative and reactive conditions.
However, reactive oxygen species have damaging effects when they
are produced in excessive amounts and are unable to be eliminated
by endogenous antioxidants. The brain remains particularly
vulnerable to oxidative stress because of its high demand for
oxygen. The oxygen is needed because the brain derives energy from
oxidative phosphorylation in mitochondrial respiratory chains.4–6 A
disproportionate production of free radicals compared to antioxi-
dant levels is thought to be an important contributory factor to the
pathogenesis of several diseases by oxidative injury. Free radicals
* Corresponding author. Tel.: +91 861 2317963/64/68x2358;
mobile: +91 9866223905.
E-mail address: bneuro_5@rediffmail.com (B. Menon).
can also exacerbate some forms of seizure. The end products of lipid
peroxidation, protein carbonyl groups and nitric oxide have been the
most studied markers of oxidative damage. Animal studies as
seizure models have provided evidence of increased oxidative stress
(increased lipid peroxidation and protein carbonylation) following
seizures.7–9 However, the role of nitric oxide is still debatable. Some
studies have shown increased levels of nitric oxide in patients,13
while other studies have demonstrated that nitric oxide has a
beneficial role as an anticonvulsant11,12 or have had inconclusive
results.10 AEDs may also modulate oxidative stress in epileptic
patients.14 Animal studies (in which antioxidants were used in
addition to anticonvulsants) showed decreased oxidative stress and
reduced frequency of seizures.6,15,16 The complex mechanism of
epileptogenesis remains largely unclear. However, neuronal death is
one of the important events, and factors altering or influencing this
event may play an important role. Oxidative stress by free radical
generation does indeed play a role in mitochondrial dysfunction and
cell death.
The aim of this study is to determine the effects of epilepsy on
nitric oxide levels, lipid peroxidation and protein carbonylation
levels in patients with epilepsy and a healthy control group. The

1059-1311/$ – see front matter ß 2012 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.seizure.2012.09.003
 B. Menon et al. / Seizure 21 (2012) 780–784 781

study also intends to assess the oxidative stress in patients with coefficients were determined by Pearson’s correlation test. P < 0.05
epilepsy who are not taking antiepileptic drugs and the effects of was considered to be statistically significant.
AEDs, if any, in patients receiving monodrug or multidrug therapy.

2. Materials and methods 3. Results

The study was composed of 100 patients with epilepsy who The study included 100 patients with epilepsy (56 males, 44
were diagnosed with idiopathic generalised epilepsy on antiepi- females) and 100 control subjects (52 males, 47 females). The
leptic therapy. One hundred age and sex matched healthy mean ages of the patients and control group were 26  10 and
volunteers served as the control group. 26  8 years, respectively. The mean duration of illness in the patients
Patients included in the study had been seizure free for more was 7  6 yrs. Twenty-five per cent of the patients were not on
than 3 days. All patients underwent a detailed examination which treatment, 66% were on monotherapy (Carbamazepine 16%, Phenyt-
included demographics, seizure frequency, duration of illness and oin 20%, Valproate 17%, others 13%), and 34% were on Polytherapy.
treatment. Patients with chronic medical illnesses such as The demographics and epilepsy profiles are shown in Table 1. The
hypertension, diabetes, rheumatoid arthritis, collagen vascular biochemical and oxidative parameters are shown in Table 2. The total
disease, chronic neurological illness or localisation related epilep- cholesterol, LDL and alkaline phosphatase levels were higher in
sy; those who were smokers, alcoholics, tobacco chewers; and patients than in the control group with a significant p value (Table 2).
those taking any other medications were excluded from the study. The remainder of the biochemical parameters were normal.
EEG was performed in all patients, and several patients underwent In the entire group, the MDA levels in patients
neuroimaging for definitive diagnoses. (6.80  2.84 mmol/ml) were higher than those in the control group
Five millilitres of venous blood was collected after an overnight (1.64  0.82 mmol/ml) with a significant P < 0.001 (Graph 1). The PC
fast, and 2 ml was transferred to EDTA-coated vacutainers for levels (0.71  0.19 mg of protein/ml) in patients were higher than
haemoglobin estimation. The remaining blood was transferred to those of the controls (0.46  0.16 mg of protein/ml) with a significant
clot activator coated vacutainers, allowed to clot and centrifuged P < 0.001 (Graph 2). However, the NO levels were not significantly
for 5 min at 3000 rpm to obtain serum that was stored at 20 8C for different between the patients (50.41  18.29) and control group
further analysis. The serum samples were used to analyse routine (51.95  9.56) (P = 0.45) (Graph 3).
biochemical investigation and oxidative biomarkers. The oxidative The mean ages of untreated patients and control group were
parameters tested were lipid peroxidation, protein carbonylation 24.76  9.96 and 25.72  7.77, respectively. Twenty-five patients
and nitric oxide levels. Lipid peroxidation was estimated by were not on AEDs. Hence, it was possible to evaluate the oxidative
measuring the Malondialdehyde levels using a UV-visible spec- stress in these patients by eliminating the effects of AEDs.
trophotometer.17 We used the TBARS Assay (thiobarbituric acid
reactive substances assay) to determine the Malondialdehyde Table 1
Demographic and treatment details of patients and control group.
(MDA) levels, the end product of lipid peroxidation.18 Protein
carbonylation (PC) was estimated with the Resnic and Packer Entire Control Treated Untreated
method.19 We measured the oxidation of the protein by adding group patients patients
carbonyl groups to the spectrophotometric assay using 2,4-DNPH Age (yrs) 26.23  10.6 26.55  8.5 26.50  10.78 24.76  9.96
(2,4-dinitrophenylhydrazine) for detecting carbonyls.20 Carbony- Sex (M:F) 56: 44 52:48 38:37 18:7
lated plasma protein reacts with 2,4-DNPH, and the complex was Duration of 76 – 65 42
illness (yrs)
measured at 360 nm.21 Nitric oxide (NO) levels were determined
Duration of 75 – 54 –
using the Griess reagent in which NO reacts with 1% sulphani- treatment (yrs)
lamide in 5% phosphoric acid, and 10% naphthalene ethylenedia- Waist/hip ratio 0.95  0.12 0.63–1.2 59.15  22.35 57.79  13.69
mine dihydrochloride forming chromospheres, which can be read Body mass index 22.01  4.30 12–32.4 13.25  4.27 21.09  4.21
at 543 nm.22
The oxidative levels were compared between patients and the
Table 2
control group. The 25 patients who were not on AEDs were Biochemical and oxidative parameters in patients and control group.
compared with both patients on AEDs and the control group to
Parameter Mean  SD P value
eliminate the effects of the antiepileptic drugs. Depending upon
treatment, the patients were divided into group 1 or group 2, Patient Control
composed of Monotherapy and Polytherapy recipients, respec- Haemoglobin, g/dl 13.40  2.11 13.36  1.81 0.88
tively. Those patients on Monotherapy were treated with one of Fasting blood sugar, mg/dl 84.18  14.63 83.11  7.45 0.51
the following antiepileptic drugs: Carbamazepine, Phenytoin, Serum creatinine, mg/dl 0.95  0.16 1.04  0.24 0.002
Total cholesterol, mg/dl 178.40  40.04 167.5  37.78 0.05
Valproate, Clobazam, Phenobarbital, Lamotrigine, Topiramate or
HDL, mg/dl 46.49  11.63 45.4  10.06 0.48
Levetiracetam. Group 2 was composed of patients on more than LDL, mg/dl 112.70  27.71 102.5  29.08 0.01
one drug (Polytherapy). The oxidative parameters were compared Triglyceride, mg/dl 95.29  34.73 106.3  45.55 0.07
among patients on Monotherapy. All patients provided written VLDL, mg/dl 19.23  7.04 20.97  9.047 0.13
Total bilirubin, mg/dl 0.82  0.05 0.82  0.072 0.33
informed consent. The study was approved by the Institutional
SGOT, IU/L 36.22  21.11 37.21  18.7 0.72
Ethics Committee. SGPT, IU/L 30.31  20.79 27.05  19.56 0.25
Alkaline phosphatase, IU/L 223.40  72.55 181.6  59.44 <0.0001
2.1. Statistical analysis Total proteins, g/dL 7.83  0.62 7.81  0.46 0.85
Albumin, g/dL 3.95  0.35 3.8  0.37 0.27
MDA, mmol/L 6.80  2.84 1.64  0.82 <0.0001
The data were analysed with the statistical package SPSS for
NO, nmol/L 50.41  18.29 51.95  9.56 0.45
Windows, version 11.5. Descriptive analyses are expressed as PC, nmol/mg of protein 0.71  0.19 0.46  0.16 <0.0001
mean  S.D. Paired t-tests were performed between patients and
HDL: high density lipoprotein; LDL: low density lipoprotein; VLDL: very low density
controls, before treatment and controls, and before and after lipoprotein; SGOT: serum glutamic oxaloacetic transaminase; SGPT: serum
treatment. One way analysis of variance (ANOVA) was used to glutamic pyruvate transaminase; MDA: malondialdehyde; PC: protein carbonyla-
estimate the differences between the study groups. Correlation tion; NO: nitric oxide.
[(Graph_1)TD$FI]
782 [(Graph_3)TD$FI]
B. Menon et al. / Seizure 21 (2012) 780–784

Graph 1. Malondialdehyde levels in patients and the control group.

[(Graph_2)TD$FI] Graph 3. Nitric oxide levels in patients and the control group.

There were no differences between the MDA levels in untreated

patients (6.81  2.94 mmol/ml) versus the treated group
(6.76  2.82 mmol/ml) (P = 0.94) (Graph 1), the PC levels in untreated
patients (0.68  0.17 mg of protein/ml) and the treated group
(0.71  0.19 mg of protein/ml) (P = 0.63) (Graph 2) or the NO levels
in untreated patients (47.96  18) and the treated group
(51.08  15.26) (P = 0.44) (Graph 3).

3.1. Comparison between patients on monotherapy and polytherapy

No differences were found between groups 1 (monotherapy)

and 2 (Polytherapy) with regard to the oxidative parameters. The
oxidative levels in monotherapy versus. Polytherapy were MDA
(7.28  2.93 mmol/ml, monotherapy) (6.63  3.25 mmol/ml, poly-
therapy) (P = 0.37) (Graph 1); PC (0.69  0.22 mg of protein/ml,
monotherapy) (0.70  0.12 mg of protein/ml, polytherapy) (P = 0.89)
Graph 2. Protein carbonylation levels in patients and the control group.
(Graph 2); NO (49.69  18.67, monotherapy) (53.25  19.54, poly-
therapy) (P = 0.43) (Graph 3), respectively.

The MDA levels of untreated patients (6.81  2.94 mmol/ml)
were higher than those of the control group (1.75  0.94 mmol/ml),
with a significant P < 0.001 (Graph 1). The PC levels (0.68  0.17 mg
of protein/ml) of untreated patients were higher than those of the
control group (0.46  0.13 mg of protein/ml) with a significant
P < 0.001 (Graph 2). However, the NO levels did not show any
significant difference between untreated patients (47.96  18.00)
and the control group (55.44  9.57) (P = 0.07) (Graph 3).
3.2. Comparison between individual AEDs
The MDA, PC and NO levels of patients on Carbamazepine,
Phenytoin and Valproate are shown in Table 3. ANOVA analysis did
not show any differences in oxidative levels between the groups.
However, there was a difference in lipid parameters; the total
cholesterol, HDL and LDL levels were highest in patients taking
Phenytoin and lowest in patients taking Carbamazepine.

Table 3
Demographics, lipid profiles and oxidative parameters in patients on various AED’s.

Parameter Mean  SD P-value

Carbamazepine (n = 16) Phenytoin (n = 20) Valproate (n = 17)

Age 20.75  7.05 27.35  8.86 25.76  9.87

Total cholesterol 181.50  33.59 194.50  45.77 156.59  25.63 CBZ/VPA P < 0.02
PHT/VPA P < 0.00
HDL 48.00  7.81 52.45  12.60 38.82  9.16 CBZ/VPA P < 0.00
PHT/VPA P < 0.00
LDL 115.06  26.46 124.10  30.05 98.29  18.01 CBZ/VPA P < 0.04
PHT/VPA P < 0.00
Triglyceride 89.13  25.01 93.75  46.50 93.94  17.46 NS
VLDL 17.63  5.06 18.55  9.26 19.71  4.21 NS
MDA 6.52  2.01 6.66  2.12 7.64  3.72 NS
NO 46.13  12.04 53.85  21.73 49.47  16.78 NS
PC 0.77  0.28 0.65  0.15 0.75  0.25 NS

HDL: high density lipoprotein; LDL: low density lipoprotein; VLDL: very low density lipoprotein; MDA: malondialdehyde; PC: protein carbonylation; NO: nitric oxide.
 B. Menon et al. / Seizure 21 (2012) 780–784
4. Discussion
This study included 100 patients with epilepsy and 100 healthy
control subjects. Our study demonstrated that MDA and PC levels
were significantly higher in patients with epilepsy than in the
control group (P < 0.001), which is a finding that is consistent with
previous studies.23–25 However, the NO levels did not vary
between the 2 groups. The brain has a high lipid content, and
its oxygen consumption and oxidative metabolism make it
susceptible to oxidative stress. Lipid peroxidation involves
oxidative degradation of lipids. Measuring Malondialdehyde, the
end product of lipid peroxidation, is a relatively easy method for
estimating lipid peroxidation.18 Protein oxidation is irreversible
oxidative damage, and protein damage is considered to be a
marker for severe oxidative stress.19,26
Nitric oxide is a free radical that is formed biologically through
the oxidation of L-arginine by nitric oxide synthase. The exact role
of NO in the pathophysiology of epilepsy is still unclear. Several
studies have shown that NO may act as an endogenous
anticonvulsant.27–29 Although some studies have shown that NO
acts as a proconvulsant,30–32 our study did not find any difference
in NO levels between the patients and control group.
In our study, 75 out of the 100 patients with epilepsy were on
treatment for durations of 5  4 years. We report significantly high
levels of MDA (P < 0.001) and PC (P < 0.001) in 75 patients when
compared to those of the healthy control group, but with no
difference between NO levels. Our study showed that patients who
were not on AEDs also had high MDA (P < 0.001) and PC levels
(P < 0.001) with no difference in NO levels when compared to the
healthy control group (P < 0.07). Because we found that patients who
not on AEDs also had high oxidative levels, we compared this group
with patients on AEDs. We did not find any significant differences
between the 2 groups with regard to MDA (P = 0.54), NO (P = 0.51)
and PC levels (P = 0.373). Furthermore, we did not find any difference
in oxidative parameters in patients on monotherapy and polytherapy.
Altogether, we conclude that oxidative stress is present in patients
with epilepsy and that AEDs do not appear to play any role. Similar
results have been documented in previous studies.23,24 Several
experimental models of seizures have demonstrated increased levels
of oxidative stress markers in various regions of the brain after
seizures. Increased lipid peroxidation has been demonstrated in
electroshock-induced seizures in mice in the entire brain,33
Pentylenetetrazol (PTZ)-induced seizures in the entire cortex in
rats,4 mice hippocampus6 and pilocarpine-induced status epilepticus
in rat hippocampus.33 These experimental studies have suggested
that seizures induce oxidative stress, which is confirmed in our study
as well as others.23,24 Recurrent seizures and prolonged seizures have
been found to result in both necrosis and apoptosis of a specific area of
hippocampus, CA3, in experimentally induced seizures.34 High
oxidative stress and free radical generation are possible reasons for
seizure-induced apoptosis and neuronal death.
Patients were also found to have high levels of cholesterol, LDL
and serum alkaline phosphatase as compared to the control group.
There are reports that show correlations between high LDL levels,
oxidative stress and endothelial dysfunction.35,36 Furthermore,
studies have shown that oxidative stress could be an early event in
the evolution of hyperlipidemia.37 In our study, correlation
analysis was not significant between lipid parameters and
oxidative levels. Hence, we can only comment that patients with
epilepsy had high oxidative stress with dyslipidemia, but further
studies are needed to confirm this correlation.
Various studies have shown that AEDs appear to modulate
oxidative stress. Studies have reported increased NO levels in
patients treated with VPA.38,39 Patients with epilepsy treated with
Phenytoin, Carbamazepine and Valproate have increased MDA
levels.23,40,41 In our study, patients were on Carbamazepine,
783
Phenytoin and Valproate or Polytherapy regimens. Increased MDA
and PC levels were found in patients on AEDs. There was a variation
in the Cholesterol, HDL and LDL levels between individual AEDs,
but there were no differences in the oxidative parameters.
However, our study had a relatively small number of subjects in
each group. Studies with larger sample sizes are needed to confirm
these results.
These results have therapeutic implications. Whether free
radicals released after seizure episodes lead to further seizures or
refractory epilepsy needs to be addressed. However, oxidative
stress has indeed been implicated in several other diseases
including epilepsy. Few studies in animal models show the effects
of antioxidants in decreasing the oxidative stress. Antioxidant
melatonin has been found to decrease seizure activity or delay the
development of epileptiform discharges in ferric chloride induced
seizure models in rodents. This phenomenon was also found to
accompany decreased levels of lipid peroxidation.38,39
Most of the previous studies were performed on patients who
were already on treatment and could not conclude that the
oxidative stress is a result of epilepsy or AEDs. Some studies have
compared treated and untreated patients and found no differ-
ence.20,38 Considering the results of these studies, including ours,
the role of AEDs in increasing oxidative stress is negligible.
In conclusion, our study demonstrated that there were
significantly increased levels of oxidative markers in patients
with epilepsy. AEDs did not influence oxidative markers, which
suggests that seizures induce oxidative stress. The mechanism of
epileptogenesis is still unclear. Even with the advent of newer
AEDs, a significant proportion of patients are refractory to
treatment. Hence, it is important to have novel methods of
therapy, which include AEDs with possible adjunctive antioxidant
therapy.
This study adds information to the existing literature and
necessitates further detailed research to understand the mecha-
nism of epilepsy to promote newer modalities of disease
modification.
Conflict of interest statement
None of the authors have any conflicts of interest to disclose.
Acknowledgement
The study was supported by a grant from the International
Epilepsy Trust.
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