Seizure 21 (2012) 780–784

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, Indiab 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

Article history:

Received 3 April 2012

Received in revised form 31 August 2012

Accepted 2 September 2012

Keywords:

Lipid peroxidation

Protein carbonylation

Nitric oxide

Malondialdehyde

Seizures

Epilepsy

Antiepileptic drugs

A B S T R A C T

Purpose: Oxidative stress has been implicated in many pathological processes. The brain is highly

susceptible to oxidative stress because of its high lipid content. In this study, the oxidative parameters in

patients with epilepsy and those of a control group were measured. The study’s aim was to determine

whether there are differences between the oxidative levels of treated and untreated patients and

whether there are differences between individual antiepileptic drugs.

Methods: Oxidative parameters such as Malondialdehyde (MDA), Protein Carbonylation (PC) and Nitric

Oxide (NO) levels were evaluated in 100 patients with epilepsy and compared to an equal number of age

and sex matched healthy subjects. In the study group, the oxidative parameters of 25 untreated patients

were compared to treated patients and the control group. The treated patients were divided into 2

groups based on their antiepileptic drugs (AEDs). Group 1 consisted of patients on a single AED, and

group 2 was composed of patients taking more than one AED. A comparative study was performed on the

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.

� 2012 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

Contents lists available at SciVerse ScienceDirect

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1. Introduction

Epilepsy is a chronic neurological disorder that requires longterm treatment. Various studies have demonstrated the role ofoxidative stress in the pathogenesis of atherosclerosis, psychiatricand neurodegenerative disorders.1–3 The brain is generally in abalanced redox state between oxidative and reactive conditions.However, reactive oxygen species have damaging effects when theyare produced in excessive amounts and are unable to be eliminatedby endogenous antioxidants. The brain remains particularlyvulnerable to oxidative stress because of its high demand foroxygen. The oxygen is needed because the brain derives energy fromoxidative phosphorylation in mitochondrial respiratory chains.4–6 Adisproportionate production of free radicals compared to antioxi-dant levels is thought to be an important contributory factor to thepathogenesis 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).

1059-1311/$ – see front matter � 2012 British Epilepsy Association. Published by Else

http://dx.doi.org/10.1016/j.seizure.2012.09.003

can also exacerbate some forms of seizure. The end products of lipidperoxidation, protein carbonyl groups and nitric oxide have been themost studied markers of oxidative damage. Animal studies asseizure models have provided evidence of increased oxidative stress(increased lipid peroxidation and protein carbonylation) followingseizures.7–9 However, the role of nitric oxide is still debatable. Somestudies have shown increased levels of nitric oxide in patients,13

while other studies have demonstrated that nitric oxide has abeneficial role as an anticonvulsant11,12 or have had inconclusiveresults.10 AEDs may also modulate oxidative stress in epilepticpatients.14 Animal studies (in which antioxidants were used inaddition to anticonvulsants) showed decreased oxidative stress andreduced frequency of seizures.6,15,16 The complex mechanism ofepileptogenesis remains largely unclear. However, neuronal death isone of the important events, and factors altering or influencing thisevent may play an important role. Oxidative stress by free radicalgeneration does indeed play a role in mitochondrial dysfunction andcell death.

The aim of this study is to determine the effects of epilepsy onnitric oxide levels, lipid peroxidation and protein carbonylationlevels in patients with epilepsy and a healthy control group. The

vier Ltd. All rights reserved.

Table 1Demographic and treatment details of patients and control group.

Entire

group

Control Treated

patients

Untreated

patients

Age (yrs) 26.23�10.6 26.55�8.5 26.50�10.78 24.76�9.96

Sex (M:F) 56: 44 52:48 38:37 18:7

Duration of

illness (yrs)

7�6 – 6�5 4�2

Duration of

treatment (yrs)

7�5 – 5�4 –

Waist/hip ratio 0.95� 0.12 0.63–1.2 59.15�22.35 57.79�13.69

Body mass index 22.01�4.30 12–32.4 13.25�4.27 21.09�4.21

Table 2Biochemical and oxidative parameters in patients and control group.

Parameter Mean� SD P value

Patient Control

Haemoglobin, g/dl 13.40�2.11 13.36�1.81 0.88

Fasting blood sugar, mg/dl 84.18�14.63 83.11�7.45 0.51

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

HDL, mg/dl 46.49�11.63 45.4�10.06 0.48

LDL, mg/dl 112.70�27.71 102.5�29.08 0.01

Triglyceride, mg/dl 95.29�34.73 106.3�45.55 0.07

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

SGOT, IU/L 36.22�21.11 37.21�18.7 0.72

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

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

NO, nmol/L 50.41�18.29 51.95�9.56 0.45

PC, nmol/mg of protein 0.71�0.19 0.46�0.16 <0.0001

HDL: high density lipoprotein; LDL: low density lipoprotein; VLDL: very low density

lipoprotein; SGOT: serum glutamic oxaloacetic transaminase; SGPT: serum

glutamic pyruvate transaminase; MDA: malondialdehyde; PC: protein carbonyla-

tion; NO: nitric oxide.

B. Menon et al. / Seizure 21 (2012) 780–784 781

study also intends to assess the oxidative stress in patients withepilepsy who are not taking antiepileptic drugs and the effects ofAEDs, if any, in patients receiving monodrug or multidrug therapy.

2. Materials and methods

The study was composed of 100 patients with epilepsy whowere diagnosed with idiopathic generalised epilepsy on antiepi-leptic therapy. One hundred age and sex matched healthyvolunteers served as the control group.

Patients included in the study had been seizure free for morethan 3 days. All patients underwent a detailed examination whichincluded demographics, seizure frequency, duration of illness andtreatment. Patients with chronic medical illnesses such ashypertension, diabetes, rheumatoid arthritis, collagen vasculardisease, chronic neurological illness or localisation related epilep-sy; those who were smokers, alcoholics, tobacco chewers; andthose taking any other medications were excluded from the study.EEG was performed in all patients, and several patients underwentneuroimaging for definitive diagnoses.

Five millilitres of venous blood was collected after an overnightfast, and 2 ml was transferred to EDTA-coated vacutainers forhaemoglobin estimation. The remaining blood was transferred toclot activator coated vacutainers, allowed to clot and centrifugedfor 5 min at 3000 rpm to obtain serum that was stored at�20 8C forfurther analysis. The serum samples were used to analyse routinebiochemical investigation and oxidative biomarkers. The oxidativeparameters tested were lipid peroxidation, protein carbonylationand nitric oxide levels. Lipid peroxidation was estimated bymeasuring the Malondialdehyde levels using a UV-visible spec-trophotometer.17 We used the TBARS Assay (thiobarbituric acidreactive substances assay) to determine the Malondialdehyde(MDA) levels, the end product of lipid peroxidation.18 Proteincarbonylation (PC) was estimated with the Resnic and Packermethod.19 We measured the oxidation of the protein by addingcarbonyl groups to the spectrophotometric assay using 2,4-DNPH(2,4-dinitrophenylhydrazine) for detecting carbonyls.20 Carbony-lated plasma protein reacts with 2,4-DNPH, and the complex wasmeasured at 360 nm.21 Nitric oxide (NO) levels were determinedusing the Griess reagent in which NO reacts with 1% sulphani-lamide in 5% phosphoric acid, and 10% naphthalene ethylenedia-mine dihydrochloride forming chromospheres, which can be readat 543 nm.22

The oxidative levels were compared between patients and thecontrol group. The 25 patients who were not on AEDs werecompared with both patients on AEDs and the control group toeliminate the effects of the antiepileptic drugs. Depending upontreatment, the patients were divided into group 1 or group 2,composed of Monotherapy and Polytherapy recipients, respec-tively. Those patients on Monotherapy were treated with one ofthe following antiepileptic drugs: Carbamazepine, Phenytoin,Valproate, Clobazam, Phenobarbital, Lamotrigine, Topiramate orLevetiracetam. Group 2 was composed of patients on more thanone drug (Polytherapy). The oxidative parameters were comparedamong patients on Monotherapy. All patients provided writteninformed consent. The study was approved by the InstitutionalEthics Committee.

2.1. Statistical analysis

The data were analysed with the statistical package SPSS forWindows, version 11.5. Descriptive analyses are expressed asmean � S.D. Paired t-tests were performed between patients andcontrols, before treatment and controls, and before and aftertreatment. One way analysis of variance (ANOVA) was used toestimate the differences between the study groups. Correlation

coefficients were determined by Pearson’s correlation test. P < 0.05was considered to be statistically significant.

3. Results

The study included 100 patients with epilepsy (56 males, 44females) and 100 control subjects (52 males, 47 females). Themean ages of the patients and control group were 26 � 10 and26 � 8 years, respectively. The mean duration of illness in the patientswas 7 � 6 yrs. Twenty-five per cent of the patients were not ontreatment, 66% were on monotherapy (Carbamazepine 16%, Phenyt-oin 20%, Valproate 17%, others 13%), and 34% were on Polytherapy.The demographics and epilepsy profiles are shown in Table 1. Thebiochemical and oxidative parameters are shown in Table 2. The totalcholesterol, LDL and alkaline phosphatase levels were higher inpatients than in the control group with a significant p value (Table 2).The remainder of the biochemical parameters were normal.

In the entire group, the MDA levels in patients(6.80 � 2.84 mmol/ml) were higher than those in the control group(1.64 � 0.82 mmol/ml) with a significant P < 0.001 (Graph 1). The PClevels (0.71 � 0.19 mg of protein/ml) in patients were higher thanthose of the controls (0.46 � 0.16 mg of protein/ml) with a significantP < 0.001 (Graph 2). However, the NO levels were not significantlydifferent between the patients (50.41 � 18.29) and control group(51.95 � 9.56) (P = 0.45) (Graph 3).

The mean ages of untreated patients and control group were24.76 � 9.96 and 25.72 � 7.77, respectively. Twenty-five patientswere not on AEDs. Hence, it was possible to evaluate the oxidativestress in these patients by eliminating the effects of AEDs.

[(Graph_1)TD$FIG]

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

[(Graph_2)TD$FIG]

Graph 2. Protein carbonylation levels in patients and the control group.

[(Graph_3)TD$FIG]

Graph 3. Nitric oxide levels in patients and the control group.

B. Menon et al. / Seizure 21 (2012) 780–784782

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 mgof protein/ml) of untreated patients were higher than those of thecontrol group (0.46 � 0.13 mg of protein/ml) with a significantP < 0.001 (Graph 2). However, the NO levels did not show anysignificant difference between untreated patients (47.96 � 18.00)and the control group (55.44 � 9.57) (P = 0.07) (Graph 3).

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

Parameter Mean� SD

Carbamazepine (n = 16) Phenytoi

Age 20.75�7.05 27.35�Total cholesterol 181.50�33.59 194.50�

HDL 48.00�7.81 52.45�

LDL 115.06�26.46 124.10�

Triglyceride 89.13�25.01 93.75�VLDL 17.63�5.06 18.55�MDA 6.52�2.01 6.66�NO 46.13�12.04 53.85�PC 0.77� 0.28 0.65�

HDL: high density lipoprotein; LDL: low density lipoprotein; VLDL: very low density li

There were no differences between the MDA levels in untreatedpatients (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 untreatedpatients (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 levelsin 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. Theoxidative 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); NO (49.69 � 18.67, monotherapy) (53.25 � 19.54, poly-therapy) (P = 0.43) (Graph 3), respectively.

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 didnot show any differences in oxidative levels between the groups.However, there was a difference in lipid parameters; the totalcholesterol, HDL and LDL levels were highest in patients takingPhenytoin and lowest in patients taking Carbamazepine.

P-value

n (n = 20) Valproate (n = 17)

8.86 25.76�9.87

45.77 156.59�25.63 CBZ/VPA P<0.02

PHT/VPA P<0.00

12.60 38.82�9.16 CBZ/VPA P<0.00

PHT/VPA P<0.00

30.05 98.29�18.01 CBZ/VPA P<0.04

PHT/VPA P<0.00

46.50 93.94�17.46 NS

9.26 19.71�4.21 NS

2.12 7.64�3.72 NS

21.73 49.47�16.78 NS

0.15 0.75� 0.25 NS

poprotein; MDA: malondialdehyde; PC: protein carbonylation; NO: nitric oxide.

B. Menon et al. / Seizure 21 (2012) 780–784 783

4. Discussion

This study included 100 patients with epilepsy and 100 healthycontrol subjects. Our study demonstrated that MDA and PC levelswere significantly higher in patients with epilepsy than in thecontrol group (P < 0.001), which is a finding that is consistent withprevious studies.23–25 However, the NO levels did not varybetween the 2 groups. The brain has a high lipid content, andits oxygen consumption and oxidative metabolism make itsusceptible to oxidative stress. Lipid peroxidation involvesoxidative degradation of lipids. Measuring Malondialdehyde, theend product of lipid peroxidation, is a relatively easy method forestimating lipid peroxidation.18 Protein oxidation is irreversibleoxidative damage, and protein damage is considered to be amarker for severe oxidative stress.19,26

Nitric oxide is a free radical that is formed biologically throughthe oxidation of L-arginine by nitric oxide synthase. The exact roleof NO in the pathophysiology of epilepsy is still unclear. Severalstudies have shown that NO may act as an endogenousanticonvulsant.27–29 Although some studies have shown that NOacts as a proconvulsant,30–32 our study did not find any differencein NO levels between the patients and control group.

In our study, 75 out of the 100 patients with epilepsy were ontreatment for durations of 5 � 4 years. We report significantly highlevels of MDA (P < 0.001) and PC (P < 0.001) in 75 patients whencompared to those of the healthy control group, but with nodifference between NO levels. Our study showed that patients whowere 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 thehealthy control group (P < 0.07). Because we found that patients whonot on AEDs also had high oxidative levels, we compared this groupwith patients on AEDs. We did not find any significant differencesbetween 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 differencein oxidative parameters in patients on monotherapy and polytherapy.Altogether, we conclude that oxidative stress is present in patientswith epilepsy and that AEDs do not appear to play any role. Similarresults have been documented in previous studies.23,24 Severalexperimental models of seizures have demonstrated increased levelsof oxidative stress markers in various regions of the brain afterseizures. Increased lipid peroxidation has been demonstrated inelectroshock-induced seizures in mice in the entire brain,33

Pentylenetetrazol (PTZ)-induced seizures in the entire cortex inrats,4 mice hippocampus6 and pilocarpine-induced status epilepticusin rat hippocampus.33 These experimental studies have suggestedthat seizures induce oxidative stress, which is confirmed in our studyas well as others.23,24 Recurrent seizures and prolonged seizures havebeen found to result in both necrosis and apoptosis of a specific area ofhippocampus, CA3, in experimentally induced seizures.34 Highoxidative stress and free radical generation are possible reasons forseizure-induced apoptosis and neuronal death.

Patients were also found to have high levels of cholesterol, LDLand 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 inthe evolution of hyperlipidemia.37 In our study, correlationanalysis was not significant between lipid parameters andoxidative levels. Hence, we can only comment that patients withepilepsy had high oxidative stress with dyslipidemia, but furtherstudies are needed to confirm this correlation.

Various studies have shown that AEDs appear to modulateoxidative stress. Studies have reported increased NO levels inpatients treated with VPA.38,39 Patients with epilepsy treated withPhenytoin, Carbamazepine and Valproate have increased MDAlevels.23,40,41 In our study, patients were on Carbamazepine,

Phenytoin and Valproate or Polytherapy regimens. Increased MDAand PC levels were found in patients on AEDs. There was a variationin 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 ineach group. Studies with larger sample sizes are needed to confirmthese results.

These results have therapeutic implications. Whether freeradicals released after seizure episodes lead to further seizures orrefractory epilepsy needs to be addressed. However, oxidativestress has indeed been implicated in several other diseasesincluding epilepsy. Few studies in animal models show the effectsof antioxidants in decreasing the oxidative stress. Antioxidantmelatonin has been found to decrease seizure activity or delay thedevelopment of epileptiform discharges in ferric chloride inducedseizure models in rodents. This phenomenon was also found toaccompany decreased levels of lipid peroxidation.38,39

Most of the previous studies were performed on patients whowere already on treatment and could not conclude that theoxidative stress is a result of epilepsy or AEDs. Some studies havecompared 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 weresignificantly increased levels of oxidative markers in patientswith epilepsy. AEDs did not influence oxidative markers, whichsuggests that seizures induce oxidative stress. The mechanism ofepileptogenesis is still unclear. Even with the advent of newerAEDs, a significant proportion of patients are refractory totreatment. Hence, it is important to have novel methods oftherapy, which include AEDs with possible adjunctive antioxidanttherapy.

This study adds information to the existing literature andnecessitates further detailed research to understand the mecha-nism of epilepsy to promote newer modalities of diseasemodification.

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 InternationalEpilepsy Trust.

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