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Serum nitrite and nitrate levels in epileptic children using valproic acid or carbamazepine
Brain & Development 26 (2004) 15–18 www.elsevier.com/locate/braindev
Original article
Serum nitrite and nitrate levels in epileptic children using valproic acid or carbamazepine Hamza Karabibera,*, Cengiz Yakincib, Yasar Durmazc, Ismail Temeld, Nihayet Mehmetd a
KSU Medical School, Department of Pediatrics, 46050 Kahramanmaras, Turkey b Inonu University Medical School Department of Pediatrics, Malatya, Turkey c Kutahya SSK Hospital Department of Pediatrics, Kutahya, Turkey d Inonu University Medical School Department of Biochemistry, Malatya, Turkey
Received 22 January 2002; received in revised form 10 March 2003; accepted 2 April 2003
Abstract In experimental epilepsy studies, nitric oxide was found to act as both proconvulsant and anticonvulsant. The objective of this study was to investigate the effects of valproic acid and carbamazepine on serum levels of nitrite and nitrate, which are the metabolites of nitric oxide. To achieve this goal, serum nitrite and nitrate levels were determined in active epileptic 34 children using valproic acid and 23 children using carbamazepine and in non-active epileptic 38 children (control group) not using any antiepileptic drug. In the valproic acid group serum nitrite and nitrate levels were 2.66 ^ 2.11 mmol/l and 69.35 ^ 23.20 mmol/l, 1.89 ^ 1.01 mmol/l and 49.39 ^ 10.61 mmol/l in the carbamazepine group, and 1.22 ^ 0.55 mmol/l, 29.53 ^ 10.05 mmol in the control group, respectively. Nitrite and nitrate levels were significantly high in both valproic acid and carbamazepine groups compared to the control group (P , 0:01). When valproic acid and carbamazepine groups were compared to each other, level of nitrate was found statistically higher in the valproic acid group in relation to the carbamazepine group (P , 0:01), however, there was no statistically significant difference in the levels of nitrite (P . 0:05). No relation could be found between serum drug levels and nitrite and nitrate levels. According to these results, it can be suggested that valproic acid and carbamazepine might have antiepileptic effects through nitric oxide. q 2003 Elsevier B.V. All rights reserved. Keywords: Nitric oxide,; Epileptic children,; Valproic acid,; Carbamazepine
1. Introduction Nitric oxide (NO) is a short-lived free radical gas synthesized from L -arginine by activation of nitric oxide synthetase (NOS). It acts as an intra/intercellular mediator in many physiological and pathological processes. To date, three NOS isoforms, NOS-I, NOS-II and NOS-III have been identified in different tissues of various species. These are neuronal (nNOS), inducible (iNOS) and endothelial (eNOS) genetic isoforms [1,2]. Since NO is a very unstable and reactive substance, it shows its effect just after being synthesized and is degraded (transformed) into stable end products of nitrite and nitrate. Therefore, measurements of nitrite and nitrate levels are * Corresponding author. Tel.: þ90-344-215-24-65; fax: þ 90-344-22123-71. E-mail address: [email protected] (H. Karabiber). 0387-7604/03/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0387-7604(03)00076-7
used as a criterion of NO levels or NOS activity in body fluids [2 – 7]. Since NO can diffuse to all parts of CNS and provides neurosynaptic communication among neurons, it is regarded as a new class neuromodulator, which affects the basic neuronal functions such as excitatory and/or inhibitory of neuronal activities significantly [8]. Recent investigations related to NO elucidate the role of this molecule in the nervous system was attempted and its functional relation with epilepsy has been demonstrated. However, the nature of the inhibitory or excitatory effects of NO, in which it mediates the formation and spread of the epileptic phenomenon, is still complicated and conflicting [9,10]. In this study, we aimed to investigate the effects of valproic acid (VPA) and carbamazepine (CBZ) on the levels of nitrite and nitrate as an indicator of NO level in epileptic children.
16
H. Karabiber et al. / Brain & Development 26 (2004) 15–18
2. Patients and methods Ninety-five children who had been diagnosed as having epilepsy according to the clinical and EEG findings and had been followed at least for 6 months by the Pediatric Neurology Unit of Turgut Ozal Medical Center, were evaluated. Fifty-seven children were still taking CBZ or VPA and 38 children had taken antiepileptic drugs previously but were followed up without any medication due to not having active epilepsy. Of the children taking antiepileptic medication, 23 were taking CBZ (two tonic, three atonic, four complex partial, 14 generalized tonic –clonic seizures) and 34 were taking VPA (three tonic, one atonic, eight complex partial, 22 generalized tonic – clonic seizures). Ages of children in all groups ranged between 2 and 17, and both average age and its distribution according to sexes are given in Table 1. Treatment group included children taking CBZ or VPA regularly at least for the last 6 months and the control group comprised of children who had taken antiepileptic medication previously and did not get any treatment at least for the last 6 months and had no convulsion as well. All children were evaluated in the aspects of mental – motor retardation, renal or hepatic disorders, heart diseases and hypertension. After getting consent of the families, systemic complaints of the children were inquired prior to the study and physical examinations, complete blood count, hepatic and renal function tests were carried out and CBZ or VPA levels in the blood were measured as well. Children having active infection or accompanying diseases, abnormalities in hepatic or renal function tests in the laboratory, taking drugs irregularly and experiencing convulsions during the last 6 months were excluded from the study. Eight milliliters of blood sample was drawn from each patient in the morning prior to the morning dose of the antiepileptic drug before eating anything. Two milliliters of blood sample was transferred into tubes with EDTA for complete blood count and the rest was collected in sterile dry tubes for separation of serum. After waiting for 30 min for the coagulation, blood sample was centrifuged at 3000 £ g for 10 min. Some of the serum obtained was used in the same day for testing drug levels and routine biochemical analyses. Other serum samples were kept in deep freeze at 2 70 8C for 30 days in order to be used for the measurement of nitrite and nitrate levels. Fasting blood glucose, alanine aminotransferase (ALT),
aspartate aminotransferase (AST), alkaline phosphatase (ALP), urea, creatinine, Naþ, Kþ, Cl 2 , Ca2þ and other routine biochemical analyses were carried out in Olympus AU 600 autoanalyzer by utilizing Olympus brand kits with enzymatic, colorimetric and ion-selective electrodes (ISE) methods. Following the deproteinization of all samples by adding NaOH/ZnSO4, nitrite and nitrate levels were analyzed together. Nitrite was analyzed directly by the method of Griess, however, nitrate was first transformed to nitrite by cadmium reduction and then analyzed by the same method [11,12]. The data obtained were evaluated in the computer medium by SPSS for Windows 6.0 statistical package program. Serum nitrite and nitrate levels were compared among VPA, CBZ and control groups by one-way analysis of variance (ANOVA).
3. Results Serum nitrite and nitrate levels of groups taking VPA, CBZ and control are shown in Table 2. VPA and CBZ groups were compared to the control group separately by means of serum nitrite and nitrate levels. Serum nitrite and nitrate levels of both VPA and CBZ groups were found to be significantly higher than the control group statistically (P , 0:01). When VPA and CBZ groups were compared to each other, no statistically significant difference could be found in the nitrite levels (P . 0:05), however, nitrate levels of VPA group were found to be statistically significantly higher than the CBZ group (P , 0:01). Routine biochemical parameters (fasting glucose level, urea, creatinine, AST, ALT, ALP) and complete blood count values were in normal ranges both in antiepileptic users and the control group and no difference could be found between the groups statistically. When NO and drug levels in the blood were compared, there was no statistically significant correlation between them.
4. Discussion In many types of childhood epilepsy, CBZ and VPA are Table 2 Serum nitrite and nitrate levels in control, VPA and CBZ groups Control group (n ¼ 38)
Table 1 Distribution of groups according to sex and mean ages
Control group VPA group CBZ group
n
Girl
Boy
Mean age ^ S.D. (years)
38 34 23
16 17 10
22 17 13
9.01 ^ 3.57 7.90 ^ 3.93 9.45 ^ 3.73
Serum drug level (mg/ml) Nitrite level (mmol/l) Nitrate level (mmol/l)
VPA group (n ¼ 34)
CBZ group (n ¼ 23)
70.56 þ 38.27
6.65 ^ 3.71
1.22 ^ 0.55
2.66 ^ 2.11
1.89 ^ 1.01
29.53 ^ 10.05
69.35 ^ 23.20
49.39 ^ 10.61
–
H. Karabiber et al. / Brain & Development 26 (2004) 15–18
being used effectively. CBZ decreases consecutive neuronal stimuli and its frequency, inhibits neuronal impulse conduction and produces these effects by delaying the recovery of voltage-dependent sodium channels. VPA was suggested to conduct its effect by increasing the concentration (synthesis and secretion) of an inhibitory neurotransmitter GABA in the central nervous system. In some other studies, VPA was reported to show its effect by inhibiting excitatory neurotransmitters or sodium and calcium channels [11,12]. Even though in all of those reports, the mechanism of effect of both VPA and CBZ could not be understood thoroughly. Boshkatova and Rayevsky [13] detected an increase in the production of NO in the brain cortex of rats in which they caused convulsions by various methods. Smith et al. [14] found that convulsions occur in a later stage in rodents sensitive to stimulators in the case of inhibition. In conclusion, they explained the anticonvulsive effect of nNOS inhibitors in reflex epilepsy models by the accumulation of L -arginine or the decrease of NOS products NO and L -citrulline. In an experimental study, Rundfeldt et al. [15] concluded that two NOS inhibitors (NG-nitro-L -arginine and NG-nitro-L -arginine methyl ester) had been as effective as an antiepileptic drug clinically. Sandirasegarane et al. [16] measured NOS activity in the optic lobe, cerebellum and forebrains of chickens (hens) having hereditary primary generalized convulsive disorders. When compared with the non-epileptic species, they found an almost twofold increase of NOS activity in the forebrains of epileptic chickens. They evaluated this lack of change as if it showed the increase of NO as an adaptive response to hereditary epileptogenesis in some regions of the brain. In many of the studies related to NO and convulsion, it was also shown that NO had an anticonvulsive effect and when NOS was inhibited by different methods, seizures were initiated more easily with inducing agents [17 – 20]. Boda and Szente [17] formed in vivo focal seizures in the neocortex of rats by 3-aminopyridine. After inhibiting NOS by N-nitro-L -arginine, they found that seizures started more readily and lasted for a longer time. They commented that NO had an inhibitor effect on initiation and continuation of seizure activity. Yokoi et al. [18] studied cats and they stated that the decrease in the synthesis of NO could take part in the pathogenesis of convulsions induced by a-guanidinoglutaric acid. Kabuto et al. [19] found a decrease both in NOS activity and in the level of NO in the epileptic focus induced by Fe2þ. They commented that NO has an anticonvulsive role and when its level decreases then convulsions occur. Although CBZ and VPA are effective in different ways, nitrite and nitrate levels were found to be higher in both groups in our study when compared to the control group. Since liver, kidney and heart diseases that could elevate nitrite and nitrate levels were excluded previously, the results obtained reflect the data of epileptic patients. With reference to the fact that high levels of nitric oxide have an
17
anticonvulsive effect, the antiepileptic effect of VPA and CBZ might be through elevating the level of NO directly or indirectly. When VPA and CBZ groups were compared to each other, there were no statistical differences between nitrite levels, however, nitrate levels in the VPA group were found to be significantly higher. VPA may have a stronger effect on NO synthesis than CBZ and may use NO synthesis pathway more in demonstrating its anticonvulsive effect. Nagatomo et al. [20] reported that zonisamide with VPA or phenytoin did not change brain and serum NO metabolites and Tominaga et al. [21] showed a higher dose of zonisamide decreased brain NO metabolites in the EL mouse. These two studies exhibiting diverse results differed from our study by means of lack of chronic use of drugs, combined use of drugs, and provokation of convulsions by a ‘tossing-up’ stimulation method. On the other hand, another study including these authors [22] determined that the levels of nitrite plus nitrate in the whole brain were significantly lower in EL mouse than those of control mice, although levels of nitrite plus nitrate in the serum did not differ between groups. These results suggest that lower NO production in the brain may be related to the susceptibility of the EL mouse to convulsive seizures. Suzuki et al. [23] determined that zonisamide, carbamazepine and diazepam also increased the gene expression of NOS mRNA in the rat brain and they concluded that psychotropics modulate the gene expression of NOS in the brain. Those studies showed that lower levels of NO might cause convulsions and AEDs increased the level of NO [22,23] similar to our study. Faradji et al. [24] reported that VPA and ethosuximide produced an increase in the height of the cortical NO signal (VPA þ 35%, ethosuximide þ 20%) in GAERS rats (Genetic Absence Epilepsy Rat from Strasbourg), an experimental model of ‘petit mal’ human disease. They claimed in the study that they demonstrated VPA and ethosuximide were NO releasers for the first time. Thompson et al. [25] attempted barbiturate-induced expression of nNOS in the rat cerebellum and their data showed a threefold increase in nNOS mRNA expression. These two studies suggest that AEDs are NO releasers and nNOS producers rather than their effect on metabolism of nitrate in the liver, which supports our hypothesis. Even though the cause of increase in the level of NO cannot be explained clearly in all those studies, it can be suggested that VPA and CBZ might have antiepileptic effects through nitric oxide. Further studies dealing with antiepileptic drugs and NO levels and a more clear demonstration of the relation may lead to new treatment methods in refractory epileptics.
References [1] Lowenstein CJ, Dinerman JL, Snyder SH. Nitric oxide: a physiologic messenger. Ann Intern Med 1994;120:227–37. [2] Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology,
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H. Karabiber et al. / Brain & Development 26 (2004) 15–18 pathophysiology, and pharmacology. Pharmacol Rev 1991;43: 109–42. Kimura S, Watanabe K, Yajiri Y, Motegi T, Masuya Y, Shibuki K, et al. Cerebrospinal fluid nitric oxide metabolites in painful diseases. Neuroreport 1999;10:275–9. Ikeda M, Sato I, Yuasa T, Miyatake T, Murota S. Nitrite, nitrate and cGMP in the cerebrospinal fluid in degenerative neurologic diseases. J Neural Transm Gen Sect 1995;100:263 –7. Navarro JA, Molina JA, Jimenez-Jimenez FJ, Benito-Leon J, OrtiPareja M, Gasalla T, et al. Cerebrospinal fluid nitrate levels in patients with Alzheimer’s disease. Acta Neurol Scand 1996;94:411–4. Zecca L, Rosati M, Renella R, Galimberti M, Ambrosini A, Fariello RG. Nitrite and nitrate levels in cerebrospinal fluid normal subjects. J Neural Transm 1998;105:627–33. Kuiper MA, Visser JJ, Bergmans PL, Scheltens P, Wolters EC. Decreased cerebrospinal fluid nitrate levels in Parkinson’s disease. Alzheimer’s disease and multiple system atrophy patients. J Neurol Sci 1994;121:46– 9. Lipton SA, Choi YB, Pan ZH, Lei SZ, Chen HS, Sucher NJ, et al. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds. Nature 1993;364:626 –32. Szabo C. Physiological and pathophysiological roles of nitric oxide in the central nervous system. Brain Res Bull 1996;41:131–41. Kirkby DR, Carroll DM, Grossman AB, Subramaniam S. Factors determining proconvulsant and anticonvulsant effects of inhibitors of nitric oxide synthase in rodents. Epilepsy Res 1996;24:91–100. Cortas NK, Wakid NW. Determination of inorganic nitrate in serum and urine by kinetic cadmium-reduction method. Clin Chem 1990;36: 1440–3. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite and (15N(nitrate in biological fluids. Anal Biochem 1982;126:131– 8. Bashkatova VG, Rayevsky KS. Nitric oxide in mechanisms of brain damage induced by neurotoxic effect of glutamate. Biochemistry (Mosc) 1998;63:866–73. Smith SE, Man CM, Yip PK, Tang E, Chapman AG, Meldrum BS. Anticonvulsant effects of 7-nitroindazole in rodents with reflex epilepsy may result from L -arginine accumulation or a reduction in
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Original article
Serum nitrite and nitrate levels in epileptic children using valproic acid or carbamazepine Hamza Karabibera,*, Cengiz Yakincib, Yasar Durmazc, Ismail Temeld, Nihayet Mehmetd a
KSU Medical School, Department of Pediatrics, 46050 Kahramanmaras, Turkey b Inonu University Medical School Department of Pediatrics, Malatya, Turkey c Kutahya SSK Hospital Department of Pediatrics, Kutahya, Turkey d Inonu University Medical School Department of Biochemistry, Malatya, Turkey
Received 22 January 2002; received in revised form 10 March 2003; accepted 2 April 2003
Abstract In experimental epilepsy studies, nitric oxide was found to act as both proconvulsant and anticonvulsant. The objective of this study was to investigate the effects of valproic acid and carbamazepine on serum levels of nitrite and nitrate, which are the metabolites of nitric oxide. To achieve this goal, serum nitrite and nitrate levels were determined in active epileptic 34 children using valproic acid and 23 children using carbamazepine and in non-active epileptic 38 children (control group) not using any antiepileptic drug. In the valproic acid group serum nitrite and nitrate levels were 2.66 ^ 2.11 mmol/l and 69.35 ^ 23.20 mmol/l, 1.89 ^ 1.01 mmol/l and 49.39 ^ 10.61 mmol/l in the carbamazepine group, and 1.22 ^ 0.55 mmol/l, 29.53 ^ 10.05 mmol in the control group, respectively. Nitrite and nitrate levels were significantly high in both valproic acid and carbamazepine groups compared to the control group (P , 0:01). When valproic acid and carbamazepine groups were compared to each other, level of nitrate was found statistically higher in the valproic acid group in relation to the carbamazepine group (P , 0:01), however, there was no statistically significant difference in the levels of nitrite (P . 0:05). No relation could be found between serum drug levels and nitrite and nitrate levels. According to these results, it can be suggested that valproic acid and carbamazepine might have antiepileptic effects through nitric oxide. q 2003 Elsevier B.V. All rights reserved. Keywords: Nitric oxide,; Epileptic children,; Valproic acid,; Carbamazepine
1. Introduction Nitric oxide (NO) is a short-lived free radical gas synthesized from L -arginine by activation of nitric oxide synthetase (NOS). It acts as an intra/intercellular mediator in many physiological and pathological processes. To date, three NOS isoforms, NOS-I, NOS-II and NOS-III have been identified in different tissues of various species. These are neuronal (nNOS), inducible (iNOS) and endothelial (eNOS) genetic isoforms [1,2]. Since NO is a very unstable and reactive substance, it shows its effect just after being synthesized and is degraded (transformed) into stable end products of nitrite and nitrate. Therefore, measurements of nitrite and nitrate levels are * Corresponding author. Tel.: þ90-344-215-24-65; fax: þ 90-344-22123-71. E-mail address: [email protected] (H. Karabiber). 0387-7604/03/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0387-7604(03)00076-7
used as a criterion of NO levels or NOS activity in body fluids [2 – 7]. Since NO can diffuse to all parts of CNS and provides neurosynaptic communication among neurons, it is regarded as a new class neuromodulator, which affects the basic neuronal functions such as excitatory and/or inhibitory of neuronal activities significantly [8]. Recent investigations related to NO elucidate the role of this molecule in the nervous system was attempted and its functional relation with epilepsy has been demonstrated. However, the nature of the inhibitory or excitatory effects of NO, in which it mediates the formation and spread of the epileptic phenomenon, is still complicated and conflicting [9,10]. In this study, we aimed to investigate the effects of valproic acid (VPA) and carbamazepine (CBZ) on the levels of nitrite and nitrate as an indicator of NO level in epileptic children.
16
H. Karabiber et al. / Brain & Development 26 (2004) 15–18
2. Patients and methods Ninety-five children who had been diagnosed as having epilepsy according to the clinical and EEG findings and had been followed at least for 6 months by the Pediatric Neurology Unit of Turgut Ozal Medical Center, were evaluated. Fifty-seven children were still taking CBZ or VPA and 38 children had taken antiepileptic drugs previously but were followed up without any medication due to not having active epilepsy. Of the children taking antiepileptic medication, 23 were taking CBZ (two tonic, three atonic, four complex partial, 14 generalized tonic –clonic seizures) and 34 were taking VPA (three tonic, one atonic, eight complex partial, 22 generalized tonic – clonic seizures). Ages of children in all groups ranged between 2 and 17, and both average age and its distribution according to sexes are given in Table 1. Treatment group included children taking CBZ or VPA regularly at least for the last 6 months and the control group comprised of children who had taken antiepileptic medication previously and did not get any treatment at least for the last 6 months and had no convulsion as well. All children were evaluated in the aspects of mental – motor retardation, renal or hepatic disorders, heart diseases and hypertension. After getting consent of the families, systemic complaints of the children were inquired prior to the study and physical examinations, complete blood count, hepatic and renal function tests were carried out and CBZ or VPA levels in the blood were measured as well. Children having active infection or accompanying diseases, abnormalities in hepatic or renal function tests in the laboratory, taking drugs irregularly and experiencing convulsions during the last 6 months were excluded from the study. Eight milliliters of blood sample was drawn from each patient in the morning prior to the morning dose of the antiepileptic drug before eating anything. Two milliliters of blood sample was transferred into tubes with EDTA for complete blood count and the rest was collected in sterile dry tubes for separation of serum. After waiting for 30 min for the coagulation, blood sample was centrifuged at 3000 £ g for 10 min. Some of the serum obtained was used in the same day for testing drug levels and routine biochemical analyses. Other serum samples were kept in deep freeze at 2 70 8C for 30 days in order to be used for the measurement of nitrite and nitrate levels. Fasting blood glucose, alanine aminotransferase (ALT),
aspartate aminotransferase (AST), alkaline phosphatase (ALP), urea, creatinine, Naþ, Kþ, Cl 2 , Ca2þ and other routine biochemical analyses were carried out in Olympus AU 600 autoanalyzer by utilizing Olympus brand kits with enzymatic, colorimetric and ion-selective electrodes (ISE) methods. Following the deproteinization of all samples by adding NaOH/ZnSO4, nitrite and nitrate levels were analyzed together. Nitrite was analyzed directly by the method of Griess, however, nitrate was first transformed to nitrite by cadmium reduction and then analyzed by the same method [11,12]. The data obtained were evaluated in the computer medium by SPSS for Windows 6.0 statistical package program. Serum nitrite and nitrate levels were compared among VPA, CBZ and control groups by one-way analysis of variance (ANOVA).
3. Results Serum nitrite and nitrate levels of groups taking VPA, CBZ and control are shown in Table 2. VPA and CBZ groups were compared to the control group separately by means of serum nitrite and nitrate levels. Serum nitrite and nitrate levels of both VPA and CBZ groups were found to be significantly higher than the control group statistically (P , 0:01). When VPA and CBZ groups were compared to each other, no statistically significant difference could be found in the nitrite levels (P . 0:05), however, nitrate levels of VPA group were found to be statistically significantly higher than the CBZ group (P , 0:01). Routine biochemical parameters (fasting glucose level, urea, creatinine, AST, ALT, ALP) and complete blood count values were in normal ranges both in antiepileptic users and the control group and no difference could be found between the groups statistically. When NO and drug levels in the blood were compared, there was no statistically significant correlation between them.
4. Discussion In many types of childhood epilepsy, CBZ and VPA are Table 2 Serum nitrite and nitrate levels in control, VPA and CBZ groups Control group (n ¼ 38)
Table 1 Distribution of groups according to sex and mean ages
Control group VPA group CBZ group
n
Girl
Boy
Mean age ^ S.D. (years)
38 34 23
16 17 10
22 17 13
9.01 ^ 3.57 7.90 ^ 3.93 9.45 ^ 3.73
Serum drug level (mg/ml) Nitrite level (mmol/l) Nitrate level (mmol/l)
VPA group (n ¼ 34)
CBZ group (n ¼ 23)
70.56 þ 38.27
6.65 ^ 3.71
1.22 ^ 0.55
2.66 ^ 2.11
1.89 ^ 1.01
29.53 ^ 10.05
69.35 ^ 23.20
49.39 ^ 10.61
–
H. Karabiber et al. / Brain & Development 26 (2004) 15–18
being used effectively. CBZ decreases consecutive neuronal stimuli and its frequency, inhibits neuronal impulse conduction and produces these effects by delaying the recovery of voltage-dependent sodium channels. VPA was suggested to conduct its effect by increasing the concentration (synthesis and secretion) of an inhibitory neurotransmitter GABA in the central nervous system. In some other studies, VPA was reported to show its effect by inhibiting excitatory neurotransmitters or sodium and calcium channels [11,12]. Even though in all of those reports, the mechanism of effect of both VPA and CBZ could not be understood thoroughly. Boshkatova and Rayevsky [13] detected an increase in the production of NO in the brain cortex of rats in which they caused convulsions by various methods. Smith et al. [14] found that convulsions occur in a later stage in rodents sensitive to stimulators in the case of inhibition. In conclusion, they explained the anticonvulsive effect of nNOS inhibitors in reflex epilepsy models by the accumulation of L -arginine or the decrease of NOS products NO and L -citrulline. In an experimental study, Rundfeldt et al. [15] concluded that two NOS inhibitors (NG-nitro-L -arginine and NG-nitro-L -arginine methyl ester) had been as effective as an antiepileptic drug clinically. Sandirasegarane et al. [16] measured NOS activity in the optic lobe, cerebellum and forebrains of chickens (hens) having hereditary primary generalized convulsive disorders. When compared with the non-epileptic species, they found an almost twofold increase of NOS activity in the forebrains of epileptic chickens. They evaluated this lack of change as if it showed the increase of NO as an adaptive response to hereditary epileptogenesis in some regions of the brain. In many of the studies related to NO and convulsion, it was also shown that NO had an anticonvulsive effect and when NOS was inhibited by different methods, seizures were initiated more easily with inducing agents [17 – 20]. Boda and Szente [17] formed in vivo focal seizures in the neocortex of rats by 3-aminopyridine. After inhibiting NOS by N-nitro-L -arginine, they found that seizures started more readily and lasted for a longer time. They commented that NO had an inhibitor effect on initiation and continuation of seizure activity. Yokoi et al. [18] studied cats and they stated that the decrease in the synthesis of NO could take part in the pathogenesis of convulsions induced by a-guanidinoglutaric acid. Kabuto et al. [19] found a decrease both in NOS activity and in the level of NO in the epileptic focus induced by Fe2þ. They commented that NO has an anticonvulsive role and when its level decreases then convulsions occur. Although CBZ and VPA are effective in different ways, nitrite and nitrate levels were found to be higher in both groups in our study when compared to the control group. Since liver, kidney and heart diseases that could elevate nitrite and nitrate levels were excluded previously, the results obtained reflect the data of epileptic patients. With reference to the fact that high levels of nitric oxide have an
17
anticonvulsive effect, the antiepileptic effect of VPA and CBZ might be through elevating the level of NO directly or indirectly. When VPA and CBZ groups were compared to each other, there were no statistical differences between nitrite levels, however, nitrate levels in the VPA group were found to be significantly higher. VPA may have a stronger effect on NO synthesis than CBZ and may use NO synthesis pathway more in demonstrating its anticonvulsive effect. Nagatomo et al. [20] reported that zonisamide with VPA or phenytoin did not change brain and serum NO metabolites and Tominaga et al. [21] showed a higher dose of zonisamide decreased brain NO metabolites in the EL mouse. These two studies exhibiting diverse results differed from our study by means of lack of chronic use of drugs, combined use of drugs, and provokation of convulsions by a ‘tossing-up’ stimulation method. On the other hand, another study including these authors [22] determined that the levels of nitrite plus nitrate in the whole brain were significantly lower in EL mouse than those of control mice, although levels of nitrite plus nitrate in the serum did not differ between groups. These results suggest that lower NO production in the brain may be related to the susceptibility of the EL mouse to convulsive seizures. Suzuki et al. [23] determined that zonisamide, carbamazepine and diazepam also increased the gene expression of NOS mRNA in the rat brain and they concluded that psychotropics modulate the gene expression of NOS in the brain. Those studies showed that lower levels of NO might cause convulsions and AEDs increased the level of NO [22,23] similar to our study. Faradji et al. [24] reported that VPA and ethosuximide produced an increase in the height of the cortical NO signal (VPA þ 35%, ethosuximide þ 20%) in GAERS rats (Genetic Absence Epilepsy Rat from Strasbourg), an experimental model of ‘petit mal’ human disease. They claimed in the study that they demonstrated VPA and ethosuximide were NO releasers for the first time. Thompson et al. [25] attempted barbiturate-induced expression of nNOS in the rat cerebellum and their data showed a threefold increase in nNOS mRNA expression. These two studies suggest that AEDs are NO releasers and nNOS producers rather than their effect on metabolism of nitrate in the liver, which supports our hypothesis. Even though the cause of increase in the level of NO cannot be explained clearly in all those studies, it can be suggested that VPA and CBZ might have antiepileptic effects through nitric oxide. Further studies dealing with antiepileptic drugs and NO levels and a more clear demonstration of the relation may lead to new treatment methods in refractory epileptics.
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