Seizure 25 (2015) 187–190
Contents lists available at ScienceDirect
Seizure journal homepage: www.elsevier.com/locate/yseiz
Short communication
Correlation of saliva and serum free valproic acid concentrations in persons with epilepsy Rekha Dwivedi a, Yogendra Kumar Gupta b, Meenakshi Singh a, Rupa Joshi b, Prabhakar Tiwari a, Thomas Kaleekal b, Manjari Tripathi a,* a b
Department of Neurology, All India Institute of Medical Sciences (AIIMS), New Delhi, India Department of Pharmacology, AIIMS, New Delhi, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 August 2014 Received in revised form 29 September 2014 Accepted 18 October 2014
Purpose: Therapeutic drug monitoring (TDM) of antiepileptic drugs (AEDs) in serum is frequently used in clinical settings however saliva could be an alternative to measure free concentration of drugs. In the present study, we observed the possible correlation of VPA concentration between serum and saliva in persons with epilepsy (PWE). Methods: A total of 59 paired serum and saliva samples were assayed from 65 consecutive PWE (51 males and 14 females; age range 9–65 years). Patients were subjected to either VPA monotherapy or its combination with other AEDs for at least three months. Steady state trough concentration of unbound VPA drug was quantified using HPLC. The correlation between serum and saliva free VPA concentration was evaluated. Results: Out of 65 patients, 27 were on monotherapy of VPA and 38 were on VPA with other antiepileptic drugs. Saliva VPA concentration significantly correlated with serum free VPA concentration (p < 0.05). Poor correlation was observed between serum and saliva VPA concentration with the daily dose (p > 0.05) respectively. Conclusions: Our study reveals that serum and saliva VPA concentrations are significantly associated in PWE. These associations may facilitate monitoring and evaluation of VPA levels non-invasively for PWE. ß 2014 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.
Keywords: Valproic acid (VPA) Saliva Therapeutic drug monitoring (TDM)
1. Introduction Valproic acid (2-propyl pentanoic acid, VPA) is a broad spectrum AED and is frequently prescribed by physicians to control various forms of seizures and syndromes. It is also indicated for the treatment of diseases like schizophrenia, bipolar disorder, depression, neuropathic pain and prophylactic treatment of migraine.1 TDM of VPA plays an important role in understanding its efficacy as well as toxicity. As clinical effects of therapy are more closely related to the levels of drug than dose, TDM may be helpful to optimize the dose in order to have enhanced efficacy and to minimize toxicity.2,3 In TDM, blood samples are commonly used for monitoring the concentrations of various AEDs in a routine practice. As of now, there in evidence supporting saliva being used as an alternative to serum for monitoring the free concentration of AEDs and few studies have shown a significant association between serum and saliva concentration in PWE.3,4 In different clinical settings, several pharmacokinetic and pharmacodynamic
studies were reported which showed either strong or weak association for the VPA levels in both serum and saliva. However these associations were affected by either variability of pH, interpatient variability, precision of different assays, drug–drug interactions or genetic variations in diverse ethnic population.2,3,8 In some reports, it has been shown that a significant correlation of saliva levels with serum levels of VPA is there using different approaches,5,7 whereas others have observed no association.6,8,9 These inconsistent reports give an opportunity to investigate this aspect in other populations. In the Indian population, serum samples for VPA estimation were observed however to the best of our knowledge this is a first report in Indian ethnicity where we used saliva for VPA estimation in PWE. We studied the association of free VPA concentrations to ascertain the correlation between serum and saliva in PWE. 2. Materials and methods 2.1. Ethical approval and study subjects
* Corresponding author. Tel.: +91 011 26594494; fax: +91 011 2658848. E-mail address: [email protected] (M. Tripathi).
All PWE were prospectively recruited from 2010 to 2012 at the out-patient epilepsy clinic, Department of Neurology, All India
http://dx.doi.org/10.1016/j.seizure.2014.10.010 1059-1311/ß 2014 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.
200 Level of VPA 100 150 0
Institute of Medical Sciences (AIIMS), New Delhi, India. This study was approved by Institute Ethics Committee and written informed consent was obtained from all subjects or guardians prior to sample collection. PWE, who were taking either VPA alone or in combination with other AEDs for a minimum of 3 months; able to produce saliva and age 65 years were included in our study. PWE who were either unable to give consent or had history of illness like stroke, tuberculosis, diabetes, endocrinal disorders, AIDS etc. or had adverse effect of AEDs were excluded. Diagnosis of epilepsy was made according to International League Against Epilepsy (ILAE) guidelines; 2010. Clinical and demographic details were recorded during recruitment.
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R. Dwivedi et al. / Seizure 25 (2015) 187–190
50
188
2.2. Sample collection and determination of free VPA concentration by HPLC Venous blood samples (3 mL) were collected at trough level (just before the next dose) in plain vials (BD Vaccutainer tube, BD Bioscience, USA), at the same time, saliva samples were also collected by placing commercially available salivette cotton swab with citric acid (Sarstedt, Nu¨mbrecht, Germany). Blood and saliva samples were centrifuged within 2 h at 3000 rpm for 10 min at 4 8C. Clear supernatant of serum and saliva fractions were stored at 80 8C until further analysis. The free concentration of VPA in serum and saliva was quantified by HPLC System (Agilent 1200 series, Agilent Technologies, USA) using Chromolith RP, 18e (100 mm 4.6 mm) packed column (Merck, Germany). Further analysis was carried out as described method by Kishore et al.10 with slightl alterations (flow rate 1 mL/min, injection volume 25 mL, column oven temperature 35 8C, run time 10 min and wave length 210 nm). The standard curve of VPA for saliva and serum was plotted in the different concentrations range (5–100 mg/mL). Less than 5% intra and inter day variability for VPA was observed. 2.3. Statistical analysis All data were analyzed using STATA [v. 11.0 (Statacorp, College Station, TX, USA)] and summarized as mean SD. The Pearson correlation coefficient was used when data had a normal distribution. Otherwise, the spearman rank-correlation coefficient was performed to elucidate the correlation between serum and saliva VPA concentration. The p < 0.05 was considered as statistically significant. 3. Results 3.1. Demographic and clinical details A total of 65 PWE were recruited with the age range between 9 and 62 years (22.70 9.96, mean SD) and their body weight ranged 26.0–98.4 kg (53.55 12.92, mean SD). The percentage of male (78.5%) was higher than female (21.5%). The mean SD for duration of epilepsy, age at onset of seizures, frequency of seizures is presented in (years) and duration of treatment in (months) were 7.68 6.93, 15.09 9.92, 76.12 260.47 and 52.92 56.01 respectively. Patients with generalized seizures (61.5%) were higher as compared to focal seizures (38.5%) and seizures control was achieved in 18 (27.7%) patients with one or two AEDs regimen whereas 47 (72.3%) patients had drug refractory epilepsy (DRE) with more than two drugs. Out of 65 PWE, higher percentage of patients (58.5%) were taking VPA in combination with other AEDs, i.e. phenytoin, carbamazepine, phenobarbital, levetiracetam, lamotrigine and clobazam etc. at a daily dose of (996.05 418.23 mg/day mean SD) as compared to VPA (41.5%) alone (807.40 281.37 mg/day mean SD). Drug doses were adjusted
Monotherapy Duo-therapy Polytherapy
Serum
Monotherapy
Duo-therapy Polytherapy
Saliva
Fig. 1. Comparative graphical presentation for mono-, duo- and poly-therapy of PWE with VPA level in serum and saliva.
with their body weight (53.55 12.92 kg; range, 26.0–98.4 kg). In 65 patients, 41.5% of PWE were on monotherapy, 40% of PWE on duotherapy whereas 18% of PWE on polytherapy. As shown in Fig. 1, the levels of VPA in mono-, and poly-therapy were not found statistically significant (p > 0.05) in free serum and saliva whereas in duotherapy PWE it showed a statistically significant association (p < 0.05). Implying that the interaction of two AEDs may alter the level of drugs in PWE. Interestingly, with increase in the number of drugs in poly-therapy increased, it remains unchanged as in monotherapy. 3.2. Correlation between saliva and serum unbound VPA concentration Out of 65 PWE, paired samples (saliva and serum) were analyzed in 59 subjects. The VPA levels of serum in 6 patients were below the limit of quantification. However, mean SD (11.5 8.5 mg/mL) of VPA levels of saliva in these subjects were observed. These patients were excluded for correlation analysis. The mean SD of VPA concentration in free serum and saliva were 47.35 39.02 mg/mL and 19.02 19.43 mg/mL respectively. The mean ratio of saliva to serum free concentration of VPA was 0.68 1.29%. Free serum VPA concentrations significantly correlated with that of Saliva VPA concentrations (r = 36; p < 0.004) while determination of correlation coefficient (r2), slope, and confidence interval (CI) were 0.13, 0.73 and 0.23–1.22 respectively. As shown in Fig. 2A, we observed a linear relationship between the saliva and serum free concentration of VPA. However, poor correlation coefficient was observed between serum and saliva VPA concentration with daily dose (r2 = 0.01 and r2 = 0.02) respectively (Fig. 2B – left and right). These associations were not statistically significant (p > 0.05) and showed non-linear relation in PWE. As expected, we found that drug dose significantly correlated with age and weight (r = 0.39 and p < 0.005), (r = 0.40 and p < 0.0001). 4. Discussion TDM is highly useful in determination of drug levels and identification of therapeutic failure either due to under or optimal dose or toxicity due to an inter individual variability. Monitoring of VPA using saliva as compared to serum is an essential approach for the determination of free drug concentrations.4 Through simple diffusion, free drug transports across the biological membrane and reaches the effector site according to the concentration gradient.2,3 In our study by comparing the VPA estimation in saliva and serum, we observed that 64.3% of
R. Dwivedi et al. / Seizure 25 (2015) 187–190
189
Fig. 2. A. Scatter plot for the correlation between serum and saliva free valproic acid in 65 persons with epilepsy (PWE). B. Scatter plot for the correlation between daily dose of VPA with serum and saliva concentration in 65 persons with epilepsy (PWE) (left and right respectively).
patients in sub therapeutic range had seizure control and this was similar to results in an Iranian population.11 Interestingly, we found a poor association between VPA dose and its concentration in serum and saliva because of rapid or slow metabolization of drug and a heterogeneous population, this is similar to previous published reports in a pediatric population.12 The mean ratio of saliva to serum free VPA concentration indicates that the saliva levels can predict the free drug concentrations in serum and it also shows the protein binding of VPA in both. In our study, A statistically significant (p < 0.004) correlation was found in serum free VPA with saliva VPA concentration in an Indian population. Using different analytical approaches with different populations, the saliva VPA levels had significant correlation with total and free level of VPA in the serum.5,7,13 In contrast, other studies have reported a weak correlation which was not significant.6,8,9 Our results indicate that saliva could be used as a good substitute for the monitoring of free VPA concentration because it correlated with the free drug concentrations in serum. The limitation of our study was that our population was heterogeneous, affected by either the variability of pH, genetic variability and dug drug interaction in duo- or polytherapy. This may result in either a weak or strong association with substantial variation that alters the correlation coefficient. In addition, it also limits that the analysis of free VPA (serum or saliva) requires specialized laboratory techniques (e.g., ultrafiltration followed by HPLC or LC/MS) that may not be available in many hospital laboratories in resource limited settings. Therefore, it necessitates referral and extra expenses to perform analysis in an equipped external reference laboratory. In conclusion, our results suggest a positive correlation and help understand TDM system in our population. Saliva could be an alternative and non-invasive method of sampling in TDM. The collection of saliva is painless, convenient, easy to process, less stressful and does not involve any risk of secondary infection or thrombosis as that of blood.
Conflict of interest All authors declare no conflict of interest. Acknowledgements We would like to thank Indian Council Medical Research (ICMR) for their funding support (Grant No. 5/4-5/Neuro/2008NCD-I). We acknowledge to INTAS pharmaceutical Ltd. (Hyderabad, India) for providing VPA (drug) standard. We are thankful to Dr. Mani Kalaiwani (Scientist), Department of Biostatistics, AIIMS for analyzing of the data. Also, we express our gratitude to the patients and their families for their co-operation consent in this study. References 1. Chateauvieux S, Morceau F, Dicato M, Diederich M. Molecular and therapeutic potential and toxicity of valproic acid. J Biomed Biotechnol 2010;2010: 1–18. 2. Gorodischer R, Burtin P, Verjee Z, et al. Is saliva suitable for therapeutic monitoring of anticonvulsants in children: an evaluation in the routine clinical setting? Ther Drug Monit 1997;19:637–42. 3. Al-Za’abi M, Deleu D, Batchelar C. Salivary free concentrations of anti-epileptic drugs:an evaluation in a routine clinical setting. Acta Neurol Belg 2003;103:19– 23. 4. Patsalos PN, Berry DJ. Therapeutic drug monitoring of antiepileptic drugs by use of saliva. Ther Drug Monit 2013;35:4–29. 5. Moemi S, Kiyoto E, Hiromichi I, et al. TDM using salivary VPA levels in saliva samples taken at patients’ homes. Jpn J Pharm Health Care Sci 2005;31:217– 22. 6. Monaco F, Piredda S, Mutani R, et al. The free fraction of valproic acid in tears, saliva, and cerebrospinal fluid. Epilepsia 1982;23:23–6. 7. Acheampong AA, Abbott FS, Orr JM, et al. Use of hexadeuterated valproic acid and gas chromatography–mass spectrometry to determine the pharmacokinetics of valproic acid. J Pharm Sci 1984;73:489–94. 8. Tokugawa K, Ueda K. Correlation between saliva and serum valproic acid concentrations in epileptic children. Pediatric International 1983;25:285–7. 9. Nitsche V, MascherF H.. The pharmacokinetics of valproic acid after oral and parenteral administration in healthy volunteers. Epilepsia 1982;23:153–62.
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10. Kishore P, Rajani Kumar V, Satyanarayana V, et al. HPLC determination of valproic acid in human serum. Pharmazie 2003;58:378–80. 11. Forooghipour M, Mohammadpour AH, Mashhadian NV, et al. Therapeutic drug monitoring of valproic acid in patients with monotherapy at steady state. Iran J Basic Med Sci 2009;12:146–9.
12. Kumar SV, Radhika Y, Kumar GV, et al. Therapeutic drug monitoring of valproic acid in pediatric epileptic patients. Int Bull Drug Res 2011;1:11–8. 13. Abbott FS, Burton R, Orr J, et al. Valproic acid analysis in saliva and serum using selected ion monitoring (electron ionization) of the tert.-butyldimethylsilyl derivatives. J Chromatogr 1982;227:433–44.
Contents lists available at ScienceDirect
Seizure journal homepage: www.elsevier.com/locate/yseiz
Short communication
Correlation of saliva and serum free valproic acid concentrations in persons with epilepsy Rekha Dwivedi a, Yogendra Kumar Gupta b, Meenakshi Singh a, Rupa Joshi b, Prabhakar Tiwari a, Thomas Kaleekal b, Manjari Tripathi a,* a b
Department of Neurology, All India Institute of Medical Sciences (AIIMS), New Delhi, India Department of Pharmacology, AIIMS, New Delhi, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 August 2014 Received in revised form 29 September 2014 Accepted 18 October 2014
Purpose: Therapeutic drug monitoring (TDM) of antiepileptic drugs (AEDs) in serum is frequently used in clinical settings however saliva could be an alternative to measure free concentration of drugs. In the present study, we observed the possible correlation of VPA concentration between serum and saliva in persons with epilepsy (PWE). Methods: A total of 59 paired serum and saliva samples were assayed from 65 consecutive PWE (51 males and 14 females; age range 9–65 years). Patients were subjected to either VPA monotherapy or its combination with other AEDs for at least three months. Steady state trough concentration of unbound VPA drug was quantified using HPLC. The correlation between serum and saliva free VPA concentration was evaluated. Results: Out of 65 patients, 27 were on monotherapy of VPA and 38 were on VPA with other antiepileptic drugs. Saliva VPA concentration significantly correlated with serum free VPA concentration (p < 0.05). Poor correlation was observed between serum and saliva VPA concentration with the daily dose (p > 0.05) respectively. Conclusions: Our study reveals that serum and saliva VPA concentrations are significantly associated in PWE. These associations may facilitate monitoring and evaluation of VPA levels non-invasively for PWE. ß 2014 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.
Keywords: Valproic acid (VPA) Saliva Therapeutic drug monitoring (TDM)
1. Introduction Valproic acid (2-propyl pentanoic acid, VPA) is a broad spectrum AED and is frequently prescribed by physicians to control various forms of seizures and syndromes. It is also indicated for the treatment of diseases like schizophrenia, bipolar disorder, depression, neuropathic pain and prophylactic treatment of migraine.1 TDM of VPA plays an important role in understanding its efficacy as well as toxicity. As clinical effects of therapy are more closely related to the levels of drug than dose, TDM may be helpful to optimize the dose in order to have enhanced efficacy and to minimize toxicity.2,3 In TDM, blood samples are commonly used for monitoring the concentrations of various AEDs in a routine practice. As of now, there in evidence supporting saliva being used as an alternative to serum for monitoring the free concentration of AEDs and few studies have shown a significant association between serum and saliva concentration in PWE.3,4 In different clinical settings, several pharmacokinetic and pharmacodynamic
studies were reported which showed either strong or weak association for the VPA levels in both serum and saliva. However these associations were affected by either variability of pH, interpatient variability, precision of different assays, drug–drug interactions or genetic variations in diverse ethnic population.2,3,8 In some reports, it has been shown that a significant correlation of saliva levels with serum levels of VPA is there using different approaches,5,7 whereas others have observed no association.6,8,9 These inconsistent reports give an opportunity to investigate this aspect in other populations. In the Indian population, serum samples for VPA estimation were observed however to the best of our knowledge this is a first report in Indian ethnicity where we used saliva for VPA estimation in PWE. We studied the association of free VPA concentrations to ascertain the correlation between serum and saliva in PWE. 2. Materials and methods 2.1. Ethical approval and study subjects
* Corresponding author. Tel.: +91 011 26594494; fax: +91 011 2658848. E-mail address: [email protected] (M. Tripathi).
All PWE were prospectively recruited from 2010 to 2012 at the out-patient epilepsy clinic, Department of Neurology, All India
http://dx.doi.org/10.1016/j.seizure.2014.10.010 1059-1311/ß 2014 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.
200 Level of VPA 100 150 0
Institute of Medical Sciences (AIIMS), New Delhi, India. This study was approved by Institute Ethics Committee and written informed consent was obtained from all subjects or guardians prior to sample collection. PWE, who were taking either VPA alone or in combination with other AEDs for a minimum of 3 months; able to produce saliva and age 65 years were included in our study. PWE who were either unable to give consent or had history of illness like stroke, tuberculosis, diabetes, endocrinal disorders, AIDS etc. or had adverse effect of AEDs were excluded. Diagnosis of epilepsy was made according to International League Against Epilepsy (ILAE) guidelines; 2010. Clinical and demographic details were recorded during recruitment.
250
R. Dwivedi et al. / Seizure 25 (2015) 187–190
50
188
2.2. Sample collection and determination of free VPA concentration by HPLC Venous blood samples (3 mL) were collected at trough level (just before the next dose) in plain vials (BD Vaccutainer tube, BD Bioscience, USA), at the same time, saliva samples were also collected by placing commercially available salivette cotton swab with citric acid (Sarstedt, Nu¨mbrecht, Germany). Blood and saliva samples were centrifuged within 2 h at 3000 rpm for 10 min at 4 8C. Clear supernatant of serum and saliva fractions were stored at 80 8C until further analysis. The free concentration of VPA in serum and saliva was quantified by HPLC System (Agilent 1200 series, Agilent Technologies, USA) using Chromolith RP, 18e (100 mm 4.6 mm) packed column (Merck, Germany). Further analysis was carried out as described method by Kishore et al.10 with slightl alterations (flow rate 1 mL/min, injection volume 25 mL, column oven temperature 35 8C, run time 10 min and wave length 210 nm). The standard curve of VPA for saliva and serum was plotted in the different concentrations range (5–100 mg/mL). Less than 5% intra and inter day variability for VPA was observed. 2.3. Statistical analysis All data were analyzed using STATA [v. 11.0 (Statacorp, College Station, TX, USA)] and summarized as mean SD. The Pearson correlation coefficient was used when data had a normal distribution. Otherwise, the spearman rank-correlation coefficient was performed to elucidate the correlation between serum and saliva VPA concentration. The p < 0.05 was considered as statistically significant. 3. Results 3.1. Demographic and clinical details A total of 65 PWE were recruited with the age range between 9 and 62 years (22.70 9.96, mean SD) and their body weight ranged 26.0–98.4 kg (53.55 12.92, mean SD). The percentage of male (78.5%) was higher than female (21.5%). The mean SD for duration of epilepsy, age at onset of seizures, frequency of seizures is presented in (years) and duration of treatment in (months) were 7.68 6.93, 15.09 9.92, 76.12 260.47 and 52.92 56.01 respectively. Patients with generalized seizures (61.5%) were higher as compared to focal seizures (38.5%) and seizures control was achieved in 18 (27.7%) patients with one or two AEDs regimen whereas 47 (72.3%) patients had drug refractory epilepsy (DRE) with more than two drugs. Out of 65 PWE, higher percentage of patients (58.5%) were taking VPA in combination with other AEDs, i.e. phenytoin, carbamazepine, phenobarbital, levetiracetam, lamotrigine and clobazam etc. at a daily dose of (996.05 418.23 mg/day mean SD) as compared to VPA (41.5%) alone (807.40 281.37 mg/day mean SD). Drug doses were adjusted
Monotherapy Duo-therapy Polytherapy
Serum
Monotherapy
Duo-therapy Polytherapy
Saliva
Fig. 1. Comparative graphical presentation for mono-, duo- and poly-therapy of PWE with VPA level in serum and saliva.
with their body weight (53.55 12.92 kg; range, 26.0–98.4 kg). In 65 patients, 41.5% of PWE were on monotherapy, 40% of PWE on duotherapy whereas 18% of PWE on polytherapy. As shown in Fig. 1, the levels of VPA in mono-, and poly-therapy were not found statistically significant (p > 0.05) in free serum and saliva whereas in duotherapy PWE it showed a statistically significant association (p < 0.05). Implying that the interaction of two AEDs may alter the level of drugs in PWE. Interestingly, with increase in the number of drugs in poly-therapy increased, it remains unchanged as in monotherapy. 3.2. Correlation between saliva and serum unbound VPA concentration Out of 65 PWE, paired samples (saliva and serum) were analyzed in 59 subjects. The VPA levels of serum in 6 patients were below the limit of quantification. However, mean SD (11.5 8.5 mg/mL) of VPA levels of saliva in these subjects were observed. These patients were excluded for correlation analysis. The mean SD of VPA concentration in free serum and saliva were 47.35 39.02 mg/mL and 19.02 19.43 mg/mL respectively. The mean ratio of saliva to serum free concentration of VPA was 0.68 1.29%. Free serum VPA concentrations significantly correlated with that of Saliva VPA concentrations (r = 36; p < 0.004) while determination of correlation coefficient (r2), slope, and confidence interval (CI) were 0.13, 0.73 and 0.23–1.22 respectively. As shown in Fig. 2A, we observed a linear relationship between the saliva and serum free concentration of VPA. However, poor correlation coefficient was observed between serum and saliva VPA concentration with daily dose (r2 = 0.01 and r2 = 0.02) respectively (Fig. 2B – left and right). These associations were not statistically significant (p > 0.05) and showed non-linear relation in PWE. As expected, we found that drug dose significantly correlated with age and weight (r = 0.39 and p < 0.005), (r = 0.40 and p < 0.0001). 4. Discussion TDM is highly useful in determination of drug levels and identification of therapeutic failure either due to under or optimal dose or toxicity due to an inter individual variability. Monitoring of VPA using saliva as compared to serum is an essential approach for the determination of free drug concentrations.4 Through simple diffusion, free drug transports across the biological membrane and reaches the effector site according to the concentration gradient.2,3 In our study by comparing the VPA estimation in saliva and serum, we observed that 64.3% of
R. Dwivedi et al. / Seizure 25 (2015) 187–190
189
Fig. 2. A. Scatter plot for the correlation between serum and saliva free valproic acid in 65 persons with epilepsy (PWE). B. Scatter plot for the correlation between daily dose of VPA with serum and saliva concentration in 65 persons with epilepsy (PWE) (left and right respectively).
patients in sub therapeutic range had seizure control and this was similar to results in an Iranian population.11 Interestingly, we found a poor association between VPA dose and its concentration in serum and saliva because of rapid or slow metabolization of drug and a heterogeneous population, this is similar to previous published reports in a pediatric population.12 The mean ratio of saliva to serum free VPA concentration indicates that the saliva levels can predict the free drug concentrations in serum and it also shows the protein binding of VPA in both. In our study, A statistically significant (p < 0.004) correlation was found in serum free VPA with saliva VPA concentration in an Indian population. Using different analytical approaches with different populations, the saliva VPA levels had significant correlation with total and free level of VPA in the serum.5,7,13 In contrast, other studies have reported a weak correlation which was not significant.6,8,9 Our results indicate that saliva could be used as a good substitute for the monitoring of free VPA concentration because it correlated with the free drug concentrations in serum. The limitation of our study was that our population was heterogeneous, affected by either the variability of pH, genetic variability and dug drug interaction in duo- or polytherapy. This may result in either a weak or strong association with substantial variation that alters the correlation coefficient. In addition, it also limits that the analysis of free VPA (serum or saliva) requires specialized laboratory techniques (e.g., ultrafiltration followed by HPLC or LC/MS) that may not be available in many hospital laboratories in resource limited settings. Therefore, it necessitates referral and extra expenses to perform analysis in an equipped external reference laboratory. In conclusion, our results suggest a positive correlation and help understand TDM system in our population. Saliva could be an alternative and non-invasive method of sampling in TDM. The collection of saliva is painless, convenient, easy to process, less stressful and does not involve any risk of secondary infection or thrombosis as that of blood.
Conflict of interest All authors declare no conflict of interest. Acknowledgements We would like to thank Indian Council Medical Research (ICMR) for their funding support (Grant No. 5/4-5/Neuro/2008NCD-I). We acknowledge to INTAS pharmaceutical Ltd. (Hyderabad, India) for providing VPA (drug) standard. We are thankful to Dr. Mani Kalaiwani (Scientist), Department of Biostatistics, AIIMS for analyzing of the data. Also, we express our gratitude to the patients and their families for their co-operation consent in this study. References 1. Chateauvieux S, Morceau F, Dicato M, Diederich M. Molecular and therapeutic potential and toxicity of valproic acid. J Biomed Biotechnol 2010;2010: 1–18. 2. Gorodischer R, Burtin P, Verjee Z, et al. Is saliva suitable for therapeutic monitoring of anticonvulsants in children: an evaluation in the routine clinical setting? Ther Drug Monit 1997;19:637–42. 3. Al-Za’abi M, Deleu D, Batchelar C. Salivary free concentrations of anti-epileptic drugs:an evaluation in a routine clinical setting. Acta Neurol Belg 2003;103:19– 23. 4. Patsalos PN, Berry DJ. Therapeutic drug monitoring of antiepileptic drugs by use of saliva. Ther Drug Monit 2013;35:4–29. 5. Moemi S, Kiyoto E, Hiromichi I, et al. TDM using salivary VPA levels in saliva samples taken at patients’ homes. Jpn J Pharm Health Care Sci 2005;31:217– 22. 6. Monaco F, Piredda S, Mutani R, et al. The free fraction of valproic acid in tears, saliva, and cerebrospinal fluid. Epilepsia 1982;23:23–6. 7. Acheampong AA, Abbott FS, Orr JM, et al. Use of hexadeuterated valproic acid and gas chromatography–mass spectrometry to determine the pharmacokinetics of valproic acid. J Pharm Sci 1984;73:489–94. 8. Tokugawa K, Ueda K. Correlation between saliva and serum valproic acid concentrations in epileptic children. Pediatric International 1983;25:285–7. 9. Nitsche V, MascherF H.. The pharmacokinetics of valproic acid after oral and parenteral administration in healthy volunteers. Epilepsia 1982;23:153–62.
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R. Dwivedi et al. / Seizure 25 (2015) 187–190
10. Kishore P, Rajani Kumar V, Satyanarayana V, et al. HPLC determination of valproic acid in human serum. Pharmazie 2003;58:378–80. 11. Forooghipour M, Mohammadpour AH, Mashhadian NV, et al. Therapeutic drug monitoring of valproic acid in patients with monotherapy at steady state. Iran J Basic Med Sci 2009;12:146–9.
12. Kumar SV, Radhika Y, Kumar GV, et al. Therapeutic drug monitoring of valproic acid in pediatric epileptic patients. Int Bull Drug Res 2011;1:11–8. 13. Abbott FS, Burton R, Orr J, et al. Valproic acid analysis in saliva and serum using selected ion monitoring (electron ionization) of the tert.-butyldimethylsilyl derivatives. J Chromatogr 1982;227:433–44.
