Background: Epilepsy is a chronic medical condition that requires long-term therapy with antiepileptic drugs (AEDs). However, long-term employment of AEDs may lead to the onset of hyperhomocysteinemia, which has been found to modulate imperative metabolic mechanisms and induce cardiovascular disorders (CVDs). Therefore, adolescent population that have been diagnosed with epilepsy and utilize AEDs are among the most vulnerable, exhibiting higher risks of developing CVDs. Purpose: The present study was designed to explore the effects of folic acid (FA) supplementation on AED-induced hyperhomocysteinemia and CVD risk factors in adolescent epileptics. Methods: The randomized clinical trial included adolescent epileptics (i.e., 10–19 years of age) of either sex, on antiepileptic therapy for > 6 months with high homocysteine levels (i.e., >10.9 µmol/L). At the time of enrolment, their baseline BP, lipid and homocysteine levels were recorded. Participants were randomly assigned to either treatment or placebo groups and received the respective treatments. At the end of the first month, BP, lipid and homocysteine levels were recorded and compared to determine the effect of FA on these parameters. Results and conclusion: A significant fall in homocysteine levels was observed with FA supplementation (P < 0.05). However, this fall was significantly high in valproic acid treated epileptic patients. In addition, we observed an improvement in high-density lipoprotein levels, a risk factor for CVDs, but the change was statistically insignificant (P > 0.05). The study results suggest that FA supplementation in epileptic patients receiving AED therapy may minimize AED-induced hyperhomocysteinemia and other CVD risk factors. Keywords Adolescent, AEDs, hyperhomocysteinemia, CVD, folic acid an increased risk for cardiovascular disorders (CVDs). Since Introduction the epileptic adolescents are bound to consume AEDs for a Epilepsy is a group of CNS disorders, a chronic medical longer period of time due to their young age, in comparison to condition that requires long-term therapy with antiepileptic adult populations, this warrants an early intervention to abate drugs (AEDs). However, long-term employment of AEDs hyperhomocysteinemia and its potential to induce CVDs. may lead to the onset of hyperhomocysteinemia. Thiol- The re-methylation pathway recycles homocysteine back containing amino acid homocysteine is an intermediate to methionine and requires vitamin B12 and folic acid (FA) as product formed during methionine metabolism. With age, cofactors (Figure 1). the average concentration of homocysteine increases and Deptartment of Pharmacology, SKNMC, Narhe (Ambegaon), Pune, the range of blood homocysteine concentrations within Maharashtra, India. adolescents range from 4.3 μmol/L to 9.9 μmol/L. Blood Corresponding author: homocysteine concentration of greater than 10.9 μmol/L Uma A. Bhosale, Professor and Head Department of Pharmacology, 49/1, is defined as a hyperhomocysteinemia. Elevated blood Narhe, Off Mumbai–Pune Bypass, Pune, Maharashtra 411041, India. homocysteine concentrations, however, are associated with E-mail: email@example.com Creative Commons CC BY: This article is distributed under the terms of the Creative Commons Attribution 4.0 License (https:// creativecommons.org/licenses/by/4.0/) which permits any use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). Bhosale et al. 51 Figure 1. Homocysteine Metabolism Pathway of patients with high homocysteine levels, i.e., > 10.9 mmol/L (normal homocysteine levels are 4.3–9.9 mmol/L for male and 3.3–7.2 mmol/L for female adolescent and a high homocysteine concentration is defined as at least 11.4 mmol/L for male and at least 10.4 mmol/L for female; gender mean of high homocysteine concentration is 10.9 mmol/L), were randomly assigned to placebo and test groups by using a table of random numbers obtained from OpenEpi statistical software. Patients with diabetes, IHD, stroke, malignancy, psychiatric diseases, pregnancy-lactation renal dysfunction, thyroid dysfunction, chronic inflammatory diseases, inborn errors of homocysteine, cobalamin or folate metabolism, or any other condition known to interfere with homocysteine metabolism, receiving vitamin supplements were excluded. Source: The author. Homocysteine Assay Method Asian Indian adolescents are genetically more exposed to A fasting blood sample was collected by a standard- CVD risks; AED therapy is an additional risk for developing ized procedure. Blood was collected by venipunc- future CVDs due to folate deficiency leading to homocysteine elevation. It has been observed that homocysteine itself has ture into SST tubes. The serum was separated by got epileptogenic potential and can cause the risk developing centrifugation at 2000 × g for 15 min at 4 °C within refractory epilepsy. 30 min of collection, and samples were immediate- Current scientific literature though highlights the role of ly stored at －80 °C until analysis. The Diazyme vitamin B12 in the regulation of blood homocysteine levels; Enzymatic Homocysteine (Hcy) Assay reagent kits there has been very little research on the implication of FA supplementation on hyperhomocysteinemia, along with were used for the estimation of homocysteine lev- 5,6 AED supplementation for the treatment of epilepsy. A els by an enzymatic method. few studies have reported that there is negative correlation between hyperhomocysteinemia and low FA levels in Statistical Analysis patients on AEDs. At the same time, few others have reported effectiveness of FA supplementation to normalize The data were analysed using OpenEpi (version 2.3); the homocysteine levels. Therefore, the current study was Student’s t-test/ANOVA was used for comparison of the conducted to study the effects of FA supplementation on means of continuous variables and normally distributed data. homocysteine levels and hyperhomocysteinemia-induced P < 0.05 was considered significant. CVD risk factors, including BP and blood lipid levels in adolescent epileptics taking AEDs. Materials and Methods Study Design This single-blind two-arm parallel-group randomized controlled clinical trial included adolescent epileptics (n = 42) of either sex with an age range between 10 years and 10,11 19 years taking AEDs for > 6 months. Sample size was calculated by using OpenEpi statistical software expecting a standard deviation (SD) of 2 at an α-error of 5%, power of 80 and mean difference of homocysteine value of 2 µmol/L. In experimental and control groups, patients were allocated in a 2:1 ratio; thus, the minimum required sample size was 24:12. After getting approval from the Institutional Ethics Committee (Ref. SKNMC No/Ethics/App/229/2014) and requisite informed consent/assent from parents/relatives 52 Annals of Neurosciences 26(3–4) Table 1. Baseline Demographic Profile of the Epileptic Patients Characteristics Placebo Group (N = 14:28) (Oral Saccharine) Test Group (Folic Acid 5 mg) Age (mean ± SD) 16.2 ± 16.8 17.6 ± 12.6 Sex: male (female) 09(05) 16(12) BMI (mean ± SD) 23.3 ± 5.3 22.8 ± 2.8 Source: The author. Table 2. Effect of Folic Acid Supplementation on Homocysteine Levels of Epileptics Characteristics Baseline (mmol/L) At 1 Month (mmol/L) N = 14(28) (Mean ± SD) (Mean ± SD) Placebo group (oral saccharine 10 mg) 27.8 ± 12.9 26.1 ± 12.8 Test group (folic acid 5 mg) 24.6 ± 6.8 21.7 ± 10.2* Source: The author. Note: N = 42. *P < 0.05, when compared to baseline levels by Student’s t-test. Table 3. Effect of Folic Acid Supplementation on CVD Risk Factors of Epileptics Placebo Group Test Group (Oral Saccharine) (Folic Acid 5 mg) P Value Parameters N = 14(28) Baseline 1 Month Baseline 1 Month SBP (mm of Hg) 123.9 ± 11.3 124.2 ± 10.8 114.3 ± 4.5 116.3 ± 5.2 > 0.05 DBP (mm of Hg) 80.1 ± 9 80.4 ± 9.1 76.8 ± 6.4 76.2 ± 5.6 Random BSL (mg/dL) 111.9 ± 40.3 114 ± 32.6 117.8 ± 10.6 117.2 ± 12.2 HDL (mg/dL) 38.4 ± 7.6 37.3 ± 8.6 39.8 ± 12.1 42.7 ± 16.2 TGs (mg/dL) 137.2 ± 38.8 137.8 ± 32.5 139.3 ± 34 138.1 ± 36 Cholesterol (mg/dL) 186.3 ± 42.8 186.8 ± 38.2 184.2 ± 22.1 183.2 ± 25.3 Source: The author. Abbreviations: = ± > SBP—systolic blood pressure; DBP—diastolic blood pressure; BSL—blood sugar level; HDL—high-density lipoproteins; TGs—triglycerides. Note: N = Placebo (test); values are mean ± SD; P > 0.05 by ANOVA. in the test group that was administered FA (Figure 3). Amongst Results the CVD risk factors that were tested in this study, there was an improvement in test group’s high-density lipoprotein (HDL) In this randomized, single-blind and clinical study, we levels, but the change was statistically insignificant (Table 3). have assessed the effects of FA supplementation on AED- induced hyperhomocysteinemia and other CVD risk factors in adolescent epileptic patients. The demographic profiles of Discussion these patients were also studied and are presented in Table 1. There were 25 males and 17 females (mean, 16.2 and 17.6 FA is required for DNA formation where it serves as a carrier years of age, respectively) enrolled in this study. The effect of of hydroxymethyl and formyl groups. As a derivative from FA supplementation on AED-induced hyperhomocysteinemia this group, methylterahydrofolate converts homocysteine showed a significant decrease (P < 0.05) in the test group, to methionine, which maintains the blood homocysteine presented in Table 2. In the present study, the majority of concentration at an appropriate level. However, AEDs the patients (i.e., 22 participants) were taking valproic acid have been found to inhibit the conversion of homocysteine 13,14 (VPA), 12 were taking carbamazepine (CBZ) and 8 patients to methionine. VPA—though a cytochrome P450 were taking phenytoin (PHN). Homocysteine levels were enzyme inhibitor, in conjunction with other cytochrome found to be significantly higher in patients on VPA (Figure 2). P450 enzyme-inducing AEDs such as CBZ and PHN, However, the greatest reduction in homocysteine levels was has high potential to cause folate deficiency and raised 15,16 found in the patients taking VPA. Overall, the results of the homocysteine levels. VPA inhibits methionine synthase present study revealed a significant fall in homocysteine levels while other AEDs target methylterahydrofolate reductase Bhosale et al. 53 inhibiting re-methylation of homocysteine (Figure 1). This Figure 4. Folate Synthetic Pathway leads to hyperhomocysteinemia, which is associated with an increased risk of CVDs in individuals diagnosed with epilepsy. It is believed that homocysteine and its related compounds may have a role as an excitatory agonist on the NMDA subtype of glutamate receptors; epileptic relapse can be discussed on this ground. Hyperhomocysteinemia leads to endothelial cell damage, reduction in the flexibility of vessels and atherosclerosis, and alters the process of haemostasis due to oxidative stress resulting from hypomethylation of 17,18 homocysteine. Although prior literature elucidates the effects of FA on homocysteine levels within adults, the results achieved in this study are comparable. In the present study, FA supplementation showed a significant decrease in blood homocysteine levels within adolescent epileptic patients on various AEDs. FA supplementation replenish levels of folate which facilitate re-methylation of homocysteine into methionine and hence DNA methylation (Figure 4). Source: The author. Figure 2. Homocysteine Levels in Epileptics Receiving Various In addition, a fall in homocysteine levels was greatest in AEDs VPA-treated patients which is an enzyme inhibitor and this is in contrast with the results of studies which mention that it was significant in enzyme-inducing AEDs such as CBZ and PHN since these AEDs have high potential to cause 15,16 folate deficiency and raised homocysteine levels. In these studies, however, only adult epileptic patients were enrolled, while in our study we have enrolled adolescent epileptic patients and majority of them were treated with VPA. Our results are comparable with earlier studies which mention that in children even VPA leads to low folate 15,16 and high homocysteine levels. Regarding CVD risk modulation, there were no changes in the measured CVD Source: The author. risk factors following FA administration, not including a Abbreviation: VPA—valproic acid; PHN—phenytoin; CBZ— minor improvement of HDL within the test group. Although carbamazepine; PHN: phenytoin. statistically insignificant, more improvement in HDL Note: A number of patients receiving AED mentioned in parenthesis. levels and other CVD risk factors could be induced if the parameters for a study allowed for extended periods of FA Figure 3. Effect of Folic Acid Supplementation and Placebo on Ho- supplementation. Therefore, long-term follows-up studies mocysteine Levels in Various AEDs are required to confirm the effects of FA supplementation on CVD risk factors. Conclusions FA supplementation in adolescent epileptics may aid in preventing AED-induced hyperhomocysteinemia, CVDs and epileptic relapse. However, further long-term follow- up studies are required to conﬁrm the effects of FA supplementation on CVD risk factors and prevent epileptic relapse. Acknowledgements Source: The author. Note: N = Pplacebo (Ttest); values are Mean±SD. VPA: Valproic Acid, PHN: The authors are thankful to Dr A. V. Bhore, Director, Dr R. S. Bangal, Phenytoin, CBZ: Carbamazepine. Dean SKNMC, and Dr S. M. Bhat, HOD Medicine, for providing *P < 0.05, **P < 0.001 when compared to placebo by sStudent’s’- t-test. facilities to carry out of the experiments of this work. 54 Annals of Neurosciences 26(3–4) Authors Contributions 8. Linnebank M, Moskau S, Semmler A, et al. Antiepileptic drugs interact with folate and vitamin B12 serum levels. Ann Neurol Uma A. Bhosale contributed in study design, literature research, data 2011; 69: 352–359. analysis and manuscript writing. Radha Yegnanarayan had edited the manuscript. Akhil Agrawal and Ashwini Patil contributed in data 9. ClinicalTrials.gov. 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Published: Jul 1, 2019