VPA is one of the antiepileptic drugs and the metabolism of valproate may trigger oxygen dependent tissue injury and elevate the free radicals in the body⁶. An increase in the free radicals can trigger the epileptic conditions, leading to neuronal degeneration through lipid peroxidation and decreased glutathione concentration in the epileptic focus⁷. The long term use of AEDs may result in an increased production of free radicals and elevated oxidative damage in neuronal cells⁴. It has been suggested that AEDs have occasionally been associated with significant adverse effects on the antioxidant defense system^5,8.

There is abundant invivo evidence of oxidative injury in animal models of epilepsy and for efficacy of antioxidant therapy in reducing this injury in animal models of epileptogenesis. However, there is sparse direct clinical data on the use of antioxidants in human epilepsy. Hence the aim of our study was to evaluate the effects of antiepileptic drug, valproate monotherapy in the management of childhood epilepsy.

MATERIALS AND METHODS:

Patients and Controls: The study population consisted of 25 epileptic children of both sexes aged between 0-12 years and 25 healthy age-matched children as normal. The blood samples were collected from the Laboratory. The samples were collected through proper channel from the Department of Pediatric Neurology, Stanley Medical College and Hospital, Chennai-600 108, India. After the baseline evaluation, the samples were collected from the epileptic children and they were administered with valproate (15mg/kg/day). Again the samples were collected at the lab at 2 months interval of time of the same children.

The venous blood collected was divided into two parts: one part was allowed to clot at room temperature and centrifuged at 5000rpm for 10 min and the serum was collected. The second part was collected in sterile vials containing EDTA, centrifuged at 3000rpm for 15 min and plasma was carefully separated. Buffy coat was removed and packed cells were washed thrice with 0.89% saline. A known volume of erythrocytes was lyzed with deionized water. The hemolysate was separated by centrifugation at 2500rpm for 15 min at 4°C. Biochemical estimations were carried out immediately.

Experimental groups: To investigate the VPA monotherapy in normal, pre- and post- treated epileptic children, their samples were categorized into three groups respectively as Group I- Normal; Group II- Before treatment; Group III- After treatment.

Chemicals: The chemicals and reagent kits used for the estimations were purchased from Sigma Chemical, Loba Chemie, Qualigens, Fischer, SDS and they were of analytical grade.

Biochemical Measurements: MDA level was measured in serum by the method of Hunter et al.⁹. The pink colored chromogen produced by the reaction of Thiobarbituric acid with MDA was measured at 533nm. The lipid peroxide content in serum was expressed as mmoles of MDA/ml.

The GSH was assayed in RBC hemolysate by the method of Moron et al.¹⁰, using 5, 5’- dithiobisnitrobenzoicacid which forms a complex with GSH that absorbs at 412nm. The amount of GSH was expressed as µmoles/g of Hb.

GPx activity in hemolysate was assayed by the method of Rotruck et al.¹¹, which is based on the reaction of remaining GSH with 5, 5’- dithiobisnitrobenzoicacid to form a complex that absorbs at 412nm. Enzyme activity was expressed as U/g of Hb.

The method of Misra and Fridovich¹² was adopted for the assay of SOD in hemolysate. The changes in absorbance were monitored using epinephrine at 480nm for 30 seconds interval of time. One unit of SOD is the amount of enzyme required to be oxidized in 50% auto oxidation of epinephrine. Enzyme activity was reported as Units/min/g of Hb.

CAT activity in hemolysate was determined by the method of Aebi¹³. The decomposition of the substrate H₂O₂ was monitered spectrophotometrically at 240nm for 3 min. Activity of enzyme was expressed as U/g of Hb.

Heamoglobin in RBC hemolyzate was estimated using the method of Drabkin and Austin¹⁴.

Statistical analysis: Descriptive statistics were calculated for all the outcome variables and expressed as mean±s.d. The results were analyzed statistically according to the Student’s t-test. The p values <0.05 were considered as significant.

RESULTS:

Effect of VPA on Lipid peroxides:

The levels of MDA (an index of extracellular membrane lipid peroxidation) were significantly (p<0.001) higher in Group II and Group III when compared to Group I. This shows marked elevation of LPO in epilepsy. In contrast, it shows less significant (p<0.05) difference between Group II and Group III as portraited in Table 1.

Effect of VPA on antioxidants:

Datas obtained in this study demonstrated a statistically significant p(<0.001) decrease in GSH, an non-enzymic antioxidant in Group II and Group III comparing to Group I. Similarly, studies on enzymic antioxidants (SOD, CAT, GPx) showed significant (p<0.001) decrease between Group I and Group III. But in contrast, there was no significant difference observed in comparing Group II and Group III except for the enzyme GPx as depicted in Table 2.

Table 1: Serum LPO profile in normal and in the pre- and post- treated epileptic children.

GROUPS	MDA (mmol/ml)
Normal	1.28±0.15^a,*
Before treatment	1.57±0.13^b,ε
After treatment	1.69±0.18^c,*

Values were expressed as mean±s.d. *p<0.001, ^εp<0.05 by Student’s t-test, ns-not significant.

(a - Group II Vs Group I; b - Group III Vs Group II; c - Group III Vs Group I)

Table 2: Erythrocyte Antioxidant levels in normal and in the pre- and post- treated epileptic children.

GROUPS	GSH (μmoles/g Hb)	GPx (U/g Hb)	SOD (U/min/g Hb)	CAT (U/g Hb)
Normal	2.98± 0.28^a,*	5.64± 0.31^a,δ	3180± 189^a,ε	258± 34^a,*
Before treatment	2.38± 0.17^b,*	6.08± 0.57^b,*	2961± 127^b,ns	240± 25^b,ns
After treatment	1.57± 0.13^c,*	5.12± 0.23^c,*	2902± 113^c,$	234± 29^c,*

Values were expressed as mean±s.d. *p<0.001, ^$p<0.02, ^δp<0.01, ^εp<0.05 by Student’s t-test, ns-not significant.

(a - Group II Vs Group I; b - Group III Vs Group II; c - Group III Vs Group I)

DISCUSSION:

Analysis of oxidant-antioxidant balance in epileptic children is difficult due to heterogeneity of epilepsy, its etiology, pathogenesis, symptomatology, therapy and many individual factors.

Oxidative stress exacerbates the etiology of epilepsy¹⁵. In amygdale kindled rats, the ROS are implicated in development of seizures under pathological conditions and are linked to seizure-induced neurodegeneration¹⁶. Due to destruction of membrane receptors the action of antiepileptic drug, VPA may be attenuated. Research findings concerning the effect of VPA monotherapy on LPO processes and endogenous antioxidant mechanisms exists to protect against the oxidative injury associated with normal metabolism are discordant.

MDA is one of the important aldehyde serves as an index of extracellular membrane LPO. Our results showed increased level of MDA in post- treated children with VPA than that of pre-treated epileptic children. The metabolism of valproate further lead to an increased free radical load of the body, which subsequently saturates and decreases the antioxidant enzymes.¹⁷

Maertens et al.⁴, Liu et al.¹⁸, Niketic et al.¹⁹ have also reported significant increased levels of MDA in untreated and treated patients in comparison to controls. We observed increased level of MDA in epileptic children compared to normal.

The results of Weber et al.²⁰ showed that serum LPO was increased in epileptic children receiving VPA for 13 months compared with control group and the results before treatment. Rettie et al.²¹, Rettenmeier et al.²² have suggested that the metabolism of VPA generates an increased body burden of free radicals and failure to remove this highly reactive species before they can inflict damage on various cellular constituents may be expressed by an increased susceptibility to the drug.

Glutathione- it’s reduced (GSH) and oxidized (GSSG) form is the major thiol redox system of the cell, providing protection against peroxidative damage. The results of our study shows a decline in the level of GSH in treated children compared to epileptic and normal children. This might be due to increased production of ROS and the reaction of 2,4-diene VPA with GSH in mitochondria could also produce a localized depletion of GSH that would result in oxidative stress, covalent binding and subsequent inactivation of enzymes²³. More recently, a glutathione in predisposed individuals by reactive metabolite can result in covalent binding of reactive metabolites to cellular macromolecules leading to toxicity and hypersensitivity.

Cengiz et al.⁶ have also shown that GSH level of epileptics were significantly changed as compared to those of healthy subjects. Tang and Abott²⁴, Kassahun et al.²⁵ have also suggested the hypothesis that the 2, 4-diene VPA forms a glutathione conjugate invivo as evidenced by NAC conjugate in the urine. Our study indicates that the conjugation of GSH with reactive of cellular GSH concentration that may exacerbate the oxidative damage.

GPx is the most important antioxidant enzyme which protects cells in the central nervous system. The oxygen radicals have been reported to inactivate GPx²⁶. GPx protects neurons from oxidative stress and subsequent damage²⁷. GPx deficiency has been connected to childhood seizures²⁰. We reported a reduced activity of GPx in pre- and post- treated children when compared to normal children.

An inherent low activity of GPx or possibly other antioxidant enzymes during VPA therapy, place these predisposed individuals at a higher risk of cellular damage by ROS. Pippenger et al.²⁸ reported that GSH-Px activity in erythrocytes of children with epilepsy receiving VPA was significantly reduced. Graf et al.¹⁷ have demonstrated that GPx can be depressed in VPA treated patients with clinically defined toxicity of the drug. On the contrary, those patients with good clinical tolerance of VPA showed normal GPx. They considered that GPx deficiency is a mediator of risk for toxicity in diverse applications of VPA. Indeed, young children have shown to be at a higher risk to develop undesired effects upon treatment with VPA. Hamed et al.²⁹ reported decreased level of GSH-Px in an untreated group of epileptics and an increase levels in treated patients but they did not reach the level of significance in comparison to control. This might be due to decreased level of hepatic synthesis of GPx and transport to blood.

CAT and SOD are the most important members of the antioxidant defense mechanism. We reported a decline in the level of these antioxidant enzymes in follow up cases of epileptic children than the normal. Kürekci et al.³⁰ have stated that reduced level of CAT and SOD was believed to cause the increased risk of an idiosyncratic drug reaction encountered in the management of epilepsy. Barbara Artemowicz et al.³¹ and Liao et al.³² have reported reduced SOD activity and also suggested that oxidants-antioxidants balance is disturbed in epileptic children and the antiepileptic therapy modifies this imbalance.

CONCLUSION:

The results of our study shows a strong evidence of oxidative stress in VPA treated children as evidenced by increased LPO and reduced antioxidant defense system. So the epileptic children under VPA treatment must be adequately supplemented with antioxidant vitamins to prevent further seizure recurrence.

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Received on 15.07.2011

Accepted on 31.10.2011

Research J. Pharmacology and Pharmacodynamics. 3(6): Nov.-Dec., 2011, 307-310