Influence of conventional antiepileptic drugs on glutathione-S-transferase and lipid peroxidation in children with idiopathic epilepsy

Epilepsy is one of the most common neurological disorders; the WHO’s 2010 Global Burden of Disease study ranks epilepsy as the second most burdensome neurologic disorder worldwide in terms of disability-adjusted life years (Murray et al. 2012). The prevalence of active epilepsy was estimated to be 6.38 per 1000 persons (95% CI 5.57–7.30) (Fiest et al. 2017). The prevalence of epilepsy did not differ by age group, sex, or study quality, and incidence rate of epilepsy was higher in low- to middle-income countries (Fiest et al. 2014). Epilepsy is characterized by recurrent unprovoked seizures (Melcon et al. 2007). Epilepsy is usually controlled with conventional antiepileptic drugs (AEDs) but cannot be cured; however, more than 30% of people with epilepsy do not achieve seizure control, even with the best available medications (George and Krucik 2014).

Evidence suggests that conventional AEDs change the oxidative-antioxidative balance in epileptic children (Cárdenas-R et al. 2013; Aycicek and Iscan 2007).

Conventional AEDs greatly increase lipid peroxidation at the expense of protective antioxidants, leading to an increase in seizure recurrence and idiosyncratic drug effects (Tan et al. 2009). Peroxidation of lipid membranes caused by increased generation of free radicals or decreased activity of antioxidant defense systems has been suggested to critically affect seizure control (Shin et al. 2011). Malondialdehyde (MDA), an end product of lipid peroxidation, is an indicator of free radical metabolism and oxidative stress in humans and other organisms and is altered during administration of AEDs (Ebru et al. 2011). Glutathione is an important biomolecule that protects the cell against chemical-induced cytotoxicity by direct or enzymatic glutathione-S-transferase (GST) conjugation with electrophilic compounds and reactive oxygen species (ROS), such as lipid hydroperoxides and hydrogen peroxide, and is affected by anticonvulsant treatments (Cengiz et al. 2000).

Therefore, we aimed to evaluate the influence of conventional AEDs (valproic acid (VPA), carbamazepine (CBZ), or both) on antioxidant enzyme activity and lipid peroxidation in children with idiopathic epilepsy by measuring GST activity and the MDA concentration in both epileptic and healthy children.

The present study was a case control study conducted on 50 children and adolescents with idiopathic epilepsy recruited from the Paediatric Neurology Clinic, Children’s Hospital, Ain Shams University. Their ages ranged from 2 to 14 years, and the patients included 27 males and 23 females. The patients were classified into three groups according to the type of AED received:

Group A: It included 20 epileptic patients (14 males and 6 females) treated with VPA monotherapy.

Group B: It included 20 epileptic patients (8 males and 12 females) treated with CBZ monotherapy.

Group C: It included 10 epileptic patients (5 males and 5 females) treated with both VPA and CBZ.

Exclusion criteria were any patients with progressive neurological disease, with confirmed neurodegenerative disorders, or with a disease affecting the serum levels of GST and MDA, such as chronic liver disease. Twenty-five healthy children age- and sex-matched were recruited as control group.

A full history was taken for each patient with an emphasis on epilepsy type and age of onset of seizures and frequency. Seizure control was considered good if the seizure frequency was ≤ 2 times/month. However, a seizure frequency > 4 times/month was considered uncontrolled. The severity of seizures was rated according to the Chalfont seizure severity scale (Duncan and Sander 1991). AEDs (number, type, duration and dosage of each drug, and its effect on seizure control) were recorded. A thorough clinical examination was performed for all patients.

The CBC and liver and kidney functions were measured to exclude any associated illness or drug side effects. Electroencephalography (EEG), computed tomography (CT), and/or magnetic resonance imaging (MRI) were performed to reveal subtle structural abnormalities and identify lesions.

GST activity was measured using 1-chloro-2,4-dinitrobenzene (CDNB), which is a synthetic GST substrate. Briefly, CDNB was added to a buffer containing GSH and an aliquot of sample to be tested. Upon the addition of CDNB, the change in absorbance at 340 nm was measured as a function of time. The results are expressed in units per liter (Habig and Jakoby 1981).

MDA level was determined by the reaction of MDA with thiobarbituric acid (TBA) at 95 °C. In the TBA test reaction, MDA and TBA react to form a pink pigment with an absorption maximum at 532 nm. The reaction was performed at pH 2–3 at 95 °C for 15 min. The sample was mixed with 2.5 volumes of 10% (w/v) trichloroacetic acid to precipitate the protein. The precipitate was pelleted by centrifugation, and an aliquot of the supernatant was allowed to react with an equal volume of 0.67% TBA in a boiling water bath for 15 min. After cooling, the absorbance was read at 532 nm. The arbitrary values obtained were compared with a series of standard solutions (1,1,3,3-tetramethoxypropane). The results are expressed as nanomoles per milliliter (Draper and Hadley 1990). None of the patients had received a blood transfusion within 2 weeks before blood sampling, as this may interfere with the serum levels of GST and MDA.

The study included 50 patients (27 males and 23 females) with a mean age of 5.6 ± 3.3 years as well as 25 healthy controls (12 males and 13 females) with a mean age of 6.9 ± 2.9 years. There were no significant differences in sex distribution among the patient subgroup and controls (χ² = 1.7 and p = 0.3).

The demographic and laboratory data of the patients and controls are presented in Table 1. The GST level was significantly lower in the patients than in the controls (p < 0.001), while the MDA level was significantly higher in the patients than in the controls (p < 0.001) (Table 1). No significant difference was noted in GST and MDA levels among the patient subgroups (Fig. 1).

	Patients	Controls	p value
Age (years)	5.6 ± 3.3	6.9 ± 2.9	0.9
Sex (males/females)	27/23	12/13	0.6
BMI (kg/m2)	20.7 ± 4.16	19.5 ± 3.5	0.2
Age of onset (years)	5.5 ± 3.6
Duration of the therapy (years)	2.87 ± 1.87
Frequency of seizure/month	2.22 ± 1.037
Type of seizure:
Focal
Simple partial	5(10%)
Complex partial	14(28%)
Generalized
Tonic clonic	29(58%)
Myoclonic	2(4%)
Uncontrolled/controlled seizure	18/32
Chalfont severity scale	28.38 ± 26.06
MDA (nmol/ml)	2.3 ± 0.3	1.6 ± 0.22	< 0.001
GST (U/l)	835.2 ± 200.6	1537.6 ± 298.7	< 0.001

Evidence suggests that glutathione homeostasis may be altered due to reactive metabolites and/or ROS produced during treatment with anticonvulsants (Cárdenas-R et al. 2013). Glutathione is an important biomolecule that protects the cell against chemical-induced cytotoxicity by direct or enzymatic (GST) conjugation with electrophilic compounds while demonstrating reactive oxygen teratogenic potential in patients with epilepsy (Ebru et al. 2011).

In the current study, the GST level was significantly decreased in the epileptic patients compared to that in the control group; however, no significant difference was found among the different patient subgroups. These results are consistent with the findings of studies by Michoulas et al. (2006) and Solowiej and Sobaniec (2003). Solowiej and Sobaniec (2003) reported that the intermediate metabolites of VPA and CBZ are detoxified via conjugation with glutathione, leading to the efflux of GST in bile. Thus, depletion of hepatic GST stores as a result of this detoxification might account for the changes observed in the serum GST level. However, Adnan et al. (2001) recorded a decrease in the GST level in patients treated with VPA, while it was not changed in patients receiving CBZ. They stated that CBZ may be a better drug for management of epilepsy, as its metabolic intermediates were more stable and thus resulted in less free radical injury than VPA.

Children with idiopathic epilepsy treated with VPA and/or CBZ have a disrupted oxidant-antioxidant balance and thus require an adequate supply of antioxidants for brain protection and prevention of neurological disturbances. In addition, long-term use of valproate could lead to a lack of selenium, copper, and zinc, which are cofactors of GST activity. Thus, long-term administration of AEDs in epileptic children leads to decreased GST levels (Adnan et al. 2001).

Graf et al. (1998) suggested that deficits in antioxidant activity enhance susceptibility to the adverse effects of VPA therapy. Moreover, another study reported no change in the glutathione peroxidase level in erythrocytes during treatment with VPA (Yiş et al. 2009).

In contrast to our results, Kürekçi et al. (1995) found that serum GST levels were increased in patients on VPA therapy. They reported that this increase might be due to an induction of hepatic synthesis of glutathione.

Lipid peroxidation is an indicator of free radical metabolism and oxidative stress in humans and other organisms. Free radicals have a short half-life, and the estimation of MDA, which is an end product of lipid peroxidation formed during the non-enzymatic oxidation of polyunsaturated fatty acids, is both simple and convenient for determining lipid peroxidation and thereby oxidative stress in human subjects (Waldbaum and Patel 2010).

Excess oxidative stress may be a final common pathway through which AEDs exert their teratogenic potential in epilepsy patients (Ebru et al. 2011).

Our results showed that the MDA level was significantly higher in the patients with idiopathic epilepsy than in the controls. This finding is consistent with that of Michoulas et al. (2006). No significant difference in the MDA level was detected among our patient subgroups. Zhang et al. (2011) and Bindu et al. (2012) reported an increased MDA level in epileptic patients receiving VPA compared to that in the control group. Solowiej and Sobaniec (2003) reported a decreased MDA level in patients treated with CBZ monotherapy, while the MDA level was elevated in patients treated with VPA. This might be due to the decreased activities of the antioxidant enzymes associated with VPA therapy, whereas these enzymes showed normal activity during CBZ therapy (Ebru et al. 2011). Yiş et al. (2009) reported that the MDA levels were significantly increased but did not reach pathological levels in epileptic patients receiving VPA.

In contrast to our results, Yüksel et al. (2000) detected no significant difference in serum MDA levels between epileptic children treated with VPA and healthy controls. Changes in the antioxidant defense mechanisms and the resulting lipid peroxidation are involved in the pathogenesis of epilepsy. However, research findings concerning these processes are very conflicting (Solowiej and Sobaniec 2003).

In the present study, the serum GST level was significantly lower in the poor seizure control group (for at least 6 months) than in the controlled seizure group. However, the serum MDA level was significantly higher in the patients with poor seizure control than in the patients who were controlled. Our results were consistent with those of Graf et al. (1998) who reported a normal GST level in children showing good clinical tolerance and a decreased level in patients with repeated seizures. Their study showed that antioxidant enzymes including GST were saturated when the free radical load of the body increased. They also demonstrated that when these enzymes failed to compensate for the excessive repeated free radical load due to repeated seizures, the antioxidant enzyme level started to decrease, while damage occurred within the cell membranes. They added that VPA especially increased the oxygen-associated tissue damage due to its toxic metabolites in the body.

The duration of drug intake was negatively correlated with the GST level and positively correlated with the MDA level in the three groups of epileptic patients. Zhang et al. (2011) reported a positive correlation between the plasma MDA level and the duration of VPA treatment. Additionally, Martinc et al. (2012) found that erythrocyte GST activity was markedly decreased after long-term administration of VPA. Liu et al. (1998) correlated long-term use of various AEDs to deficiencies in magnesium and serum zinc, which play a critical role in neuronal excitability, in red blood cells.

A limitation of this study is the small size of the patients.

The oxidant-antioxidant balance is disturbed in epileptic patients treated with VPA, CBZ, or both. These imbalances are affected by both the duration of the drug treatment and epilepsy control.