Epilepsy is a common neurological disease caused by a transient electrical brain disorder that leads to sudden and repeated attacks (various types of seizures), short-term, accompanied by changes in the state of consciousness or abnormal movements. It may sometimes be accompanied by complete seizures or local muscle spasms, and depending on the nature and type of attacks, it may be associated with loss of consciousness [1][2]. All aspects of this disease are not well known and cannot be cured, but epileptic attacks can be controlled with some treatment methods, including drug treatments with anti-epileptic drugs [3].

There is a wide range of antiepileptic drugs with different mechanism, pharmacology, pharmacokinetics and important side effects. One of these drugs is sodium valproate (SV), which is an 8-carbon branched-chain fatty acid and has been used as a broad-spectrum drug since 1970 as one of the first-line anticonvulsant drugs [4].

SV, like other anticonvulsant drugs, has side effects, such as, if prescribed for a long time, the metabolism of folate and vitamin B12 is affected, and by increasing the plasma level of homocysteine (Hcy), they lead to cerebrovascular disorders [5]. Hcy, as an amino acid containing sulfur, plays a role in methionine metabolism, and folic acid and vitamin B12 are necessary for the remethylation of Hcy to methionine, So that the reduction of each of them may lead to an increase in the level of Hcy and a decrease in methionine [6].

Epilepsy is prevalent in about half to one percent of the world's population, and almost 80% of its cases are reported in developing countries [7]. Its prevalence in Iran has been about 5% higher than the statistics of similar countries, while in terms of geographical region, the highest frequency has been reported in the center of the country, then the east and finally the north of the country [8].

SV is one of the most widely used and common drugs used by epilepsy patients and is reported to be one of the first common drugs used in these patients. Therefore, this study was conducted to investigate its effect on the plasma levels of Hcy, folate and vitamin B12, and as a result, controlling side effects and ultimately helping patients recover.

Objective – to investigate the effects of SV on plasma concentrations of homocysteine, folate and vitamin B12 levels in epileptic patients with long-standing tonic-clonic seizures compared to newly diagnosed epileptic patients and healthy controls.

This descriptive-analytical study was conducted in the population of epilepsy patients referred to Ayatollah Rouhani Hospital in Babol between November 2015 and January 2018. A total of 90 people were studied.

The inclusion criteria were defined as patients who were suffering from epilepsy at the time of the study, and the exclusion criteria were defined as age less than 20 years, pregnancy, heart, liver and kidney disorders, suffering from cancer, taking supplements containing folate and vitamin B12 and hyperlipidemia.

The control group (Group 1) consisted of 30 healthy people without epilepsy and without diagnosis of neurological disorder. According to the available sampling method and the inclusion criteria, 30 patients with newly diagnosed epilepsy who did not take SV (Group 2) and 30 patients with long-term epilepsy with tonic-colonic seizures under SV treatment (Group 3) were selected and included in the study.

Healthy people (Group 1) included 12 men and 18 women with an average age of 36.30±12.80 years. The newly diagnosed group (Group 2) included 13 men and 17 women with an average age of 36.76±12.91 years. The group of patients with long-term epilepsy (Group 3) included 12 men and 18 women with an average age of 35.56±12.77 years and an average duration of the disease of 6 months (Table 1).

The researchers complied with the requirements of the Helsinki Declaration of the World Medical Association (Fortaleza, Brazil, 2013). The study was approved by the local Ethics Committee (MUBABOL.REC.1395.179-3570). The samples were collected with the consciously written consent of patients.

Differences in plasma Hcy, folate and vitamin B12 concentrations in studied patients after 6 months of follow-up were analyzed. Blood (5 ml) was collected from the veins of fasting and resting patients, and plasma was isolated. Standard biochemical kits were used to detect plasma concentrations of Hcy, folate and vitamin B12 according to the manufacturer's instructions.

SPSS Statistics 18 (IBM Inc., USA) was used for all statistical analyses. Data are presented as mean ± standard deviation. Parameters that were not normally distributed, including folate, vitamin B12, and Hcy, were log-transformed before analysis. Comparisons between groups were performed using analysis of variance (ANOVA). The Pearson’s test was used to assess the correlation of the parameters.

In Table 2, the comparison of mean plasma concen-trations of Hcy, folic acid and vitamin B12 of healthy controls, newly diagnosed patients and long-standing tonic-clonic seizures epileptic patients after SV therapy is shown. The plasma folate levels in Group 3 (following SV treat¬ment) were significantly higher than those in Group 2 (without SV treatment) and significantly lower than those Group 1 (p=0.001). Hcy level in Group 2 was significantly higher than in Group 3 (following SV treatment) and in the control group (p=0.001). The level of B12 was also higher in healthy people than in patients, and while it was reported higher in Group 2 (without SV therapy) than in Group 3 (with SV therapy), the difference between the groups was not significant (p=0.090).

In Table 3 and Figure 1, correlations between mean plasma concentrations of Hcy, folate, and vitamin B12 in healthy controls, newly diagnosed patients, and long-standing tonic-clonic seizures epileptic patients who have undergone SV therapy are demonstrated.

The current study determined SV impact on plasma levels of Hcy, folate and vitamin B12 B12 in long-standing tonic-clonic seizures epileptic patients following SV therapy compared to newly diagnosed epileptic patients and healthy controls. The main findings were a decrease in folate level, but Hcy levels elevated in patients with long-standing tonic-clonic seizures epilepsy (Group 3) compared to Groups 1 and 2. It is likely that SV could effect on the acidic properties of the digestive tract and the buffering system. These changes might effect on folate precursor enzymes and reduces folate absorption. Also, it may affect folate synthesizing pathway. Considering that vitamin B12 plays a role in the transport and storage mechanism of folate, by reducing vitamin B12 folate metabolism also fails. Vitamin B12 plays a role as a cofactor of methyl transferase [9–12].

But the reason for the elevation of Hcy level may be due to decreased folate levels. Because of the reduction of folate, the transfer of methyl group from Hcy to methionine is not done [10]. Folate is an important component in many biochemical and physiological reactions in the body. The amount of Hcy might remain high as folate level decreases. Also, with the reduction of vitamin B12, a disruption of the methyl group is caused.

On the other hand, elevated serum Hcy may be due to decreased vitamin B12 and folate, because vitamin B12 and folate are essential in the conversion of Hcy to the metabolite methionine. This may be the mechanism by which SV increases serum Hcy levels due to interference with feeding, absorption and renal excretion. These results indicate that SV interacts with folate and vitamin B12 metabolism.

Our results indicate that the plasma contents of Hcy, folic acid and vitamin B12 could be dependent risk factors for epilepsy, agreeing with previous studies [5][9]. Current findings demonstrated lower contents of sera folate and vitamin B12 contents in newly diagnosed epileptic patients and long-standing tonic-clonic seizures epileptic patients treated with SV, which was attributed to direct relationship between folate and vitamin B12 levels. But our results demonstrated higher levels of serum Hcy levels in newly diagnosed epileptic patients, which was attributed to inverse relationship between Hcy, folate and vitamin B12 contents.

Anyway, the mechanism underlying the effects of SV effect on folate and vitamin B12 content remains to be recognized. Therefore, additional basic and clinical studies should be managed to verify these results.

The present study demonstrated that taking SV is meaningfully connected with decreased serum levels of folate and tendency to decrease vitamin B12. This observation correlates with the finding of other researchers who had reported a significant decreased serum levels of vitamins B12 and folate in epileptic patients [5][13][14]. Our results agree with other researchers' reports [6]. But our findings indicate that receiving SV is meaningfully connected with increased serum levels of Hcy in epileptic patients. This findings agrees with most of clinical studies results [13][15][16], but is inconsistent with the findings of other researchers [17].

The study had certain limitations. Our sample size was small. Besides, we did not analyze plasma concentrations of other biochemical markers such as lipid profiles, because SV may cause alterations in these variables. Additionally, we did not take into account plasma hormone concentrations such as thyroid hormones.

The authors thank for support the Vice-Chancellor of Research and Technology of Babol University of Medical Sciences and the Development and Research Unit of Ayatollah Rouhani Hospital of Babol.

Administration of SV reduces the plasma contents of folate. It might disrupt the homeostatic level of Hcy and vitamin B12 and cause irregularity of their plasma contents in newly diagnosed and long-standing epileptic patients.