Risk factors for sodium valproate-induced renal tubular dysfunction.

OBJECTIVE: To explore the risk factors for the development of sodium valproate (VPA)-induced renal tubular dysfunction for early diagnosis and treatment. STUDY DESIGN: The subjects were selected from patients who were diagnosed with epilepsy and administered VPA. Blood and spot urine samples were collected and measured the concentration of VPA, the level of serum phosphorus, serum uric acid, serum free carnitine, serum cystatin-c, and urine ß2-microglobulin (BMG). Patients with urine BMG/creatinine levels above 219.2 were treated as renal proximal tubular dysfunction (RTD), with all others treated as non-RTD. RESULTS: Eighty-seven patients, 4-48 years, 53 men and 34 women, were studied. RTD group is 17 patients and non-RTD group is 70 patients. Univariate analyses revealed that the RTD patients were more likely to be bedridden, receiving enteral tube feeding, taking more anticonvulsants, and demonstrating significantly lower serum levels of free carnitine, uric acid, and phosphorus. Among them, bedridden, free serum carnitine, and phosphorus levels were associated with the development of RTD by multivariate analysis. CONCLUSIONS: Bedridden patients receiving VPA are susceptible to hypocarnitinemia, which can cause RTD and may lead to FS. Therefore, urinary BMG should be measured regularly in all patients receiving VPA to assess renal tubular function. An additional measurement of serum free carnitine level should be considered in patients who developed RTD. Supplementation of carnitine for those patients to prevent such complication deserves for further study.

Challenges for Detecting Valproic Acid in a Nontargeted Urine Drug Screening Method.

BACKGROUND: Valproic acid (VPA) is a widely prescribed medicine, and acute toxicity is possible. As such, it should be included in any nontargeted urine drug screening method. In many published liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) methods, VPA is usually measured using a pseudo-multiple reaction monitoring (MRM) transition. We investigate a simple ultra-high-performance liquid chromatography-quadrupole time-of-flight (QTof) approach to detect the presence of VPA with more confidence. METHODS: Three commercially sourced VPA metabolites were characterized and added to a nontargeted high-resolution MS urine drug screening method. All analyses were performed on a Waters Xevo G2-XS LC-QTof in negative electrospray ionization mode. The mass detector was operated in MS mode, and data were processed with UNIFI software. Sixty-eight patient urine samples, which were previously identified by a well-established gas chromatography-MS method as containing VPA, were analyzed on the Waters Xevo G2-XS LC-QTof, to validate this approach. RESULTS: VPA metabolite standards were characterized, and their detection data were added to the broad drug screening library. VPA metabolites were readily detectable in the urine of patients taking VPA. CONCLUSIONS: The inclusion of characterized VPA metabolites provides a simple and reliable method enabling the detection of VPA in nontargeted urine drug screening.

In vivo inhibition of acylpeptide hydrolase by carbapenem antibiotics causes the decrease of plasma concentration of valproic acid in dogs.

1. Our previous in vitro studies suggest that inhibition of the acylpeptide hydrolase (APEH) activity as valproic acid glucuronide (VPA-G) hydrolase by carbapenems in human liver cytosol is a key process for clinical drug-drug interaction (DDI) of valproic acid (VPA) with carbapenems. Here, we investigated whether in vivo DDI of VPA with meropenem (MEPM) was caused via inhibition of APEH in dogs. 2. More rapid decrease of plasma VPA levels and increased urinary excretion of VPA-G were observed after co-administration with MEPM compared with those after without co-administration, whereas the plasma level and bile excretion of VPA-G showed no change. 3. Dog VPA-G hydrolase activity, inhibited by carbapenems, was mainly located in cytosol from both the liver and kidney. APEH-immunodepleted cytosols lacked VPA-G hydrolase activity. Hepatic and renal APEH activity was negligible even at 24 h after dosing of MEPM to a dog. 4. In conclusion, DDI of VPA with carbapenems in dogs is caused by long-lasting inhibition of APEH-mediated VPA-G hydrolysis by carbapenems, which could explain the delayed recovery of plasma VPA levels to the therapeutic window even after discontinuation of carbapenems in humans.

Effect of antiepileptic drug polytherapy on urinary pH in children and young adults.

OBJECTS: The relationship between antiepileptic drugs (AEDs) polytherapy and urinary pH was studied to demonstrate the effect and difference of AED polytherapy compared to monotherapy. MATERIALS AND METHODS: A total of 271 urine samples from patients receiving AED polytherapy aged from 7 months to 35 years were enrolled. Two AEDs were co-administered to 215 patients, three AEDs to 45 patients, four AEDs to ten patients, and five AEDs to one patient. RESULTS: The distribution of urinary pH shifted to the alkaline range with increasing numbers of co-administered AEDs (p < 0.0001). The distribution of urinary pH shifted to the alkaline side with AED polytherapy that included valproate (p < 0.05) or acetazolamide (p < 0.03). The distribution of urinary pH did not change with or without zonisamide, carbamazepine, phenobarbital, phenytoin, or clonazepam. CONCLUSIONS: Urinary pH should be monitored in patients receiving AED polytherapy, particularly those receiving valproate, acetazolamide, or various AEDs in combination.

The relationship between glucuronide conjugate levels and hepatotoxicity after oral administration of valproic acid.

The aim of this study was to investigate the relationship between hepatotoxicity, levels of glucuronide conjugates of valproic acid (VPA), and the toxic metabolites of VPA (4-ene VPA and 2,4-diene VPA). We also examined whether hepatotoxicity could be predicted by the urinary excretion levels of VPA and its toxic metabolites. VPA was administrated orally in rats in amounts ranging from 20 mg/kg to 500 mg/kg. Free and total (free plus glucuronide conjugated) VPA, 4-ene VPA, and 2,4-diene VPA were quantified in urine and liver using gas chromatography-mass spectrometry. Serum levels of aspartate aminotransferase, alanine aminotransferase, and alpha-glutathione S-transferase (alpha-GST) were also determined to measure the level of hepatotoxicity. The serum alpha-GST level increased slightly at the 20 mg/kg dose, and substantially increased at the 100 and 500 mg/kg dose; aspartate aminotransferase and alanine aminotransferase levels did not change with the administration of increasing doses of VPA. The liver concentration of free 4-ene VPA and the urinary excretion of total 4-ene VPA were the only measures that correlated with the increase in the serum alpha-GST level (p < 0.094 and p < 0.023 respectively). From these results, we conclude that hepatotoxicity of VPA correlates with liver concentration of 4-ene VPA and can be predicted by the urinary excretion of total 4-ene VPA.

Determination of DP-VPA and its active metabolite, VPA, in human plasma, urine, and feces by UPLC-MS/MS: A clinical pharmacokinetics and excretion study.

DP-VPA is a phospholipid prodrug of valproic acid (VPA) that is developed as a potential treatment for epilepsy. To characterize the pharmacokinetics and excretion of DP-VPA, four reliable ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods were validated for quantitation of DP-VPA and its metabolite, VPA, in human plasma, urine, and feces. Protein precipitation and solid-phase extraction (SPE) were used for extraction of C16, C18 homologs of DP-VPA and VPA, respectively, from plasma. Urine and fecal homogenate involving the three analytes were efficiently prepared by methanol precipitation. The determinations of C16 DP-VPA, C18 DP-VPA, and VPA were performed using the positive multiple reaction monitoring (MRM) mode and the negative single ion monitoring (SIM) mode, respectively. The analytes were separated using gradient elution on C8 or phenyl column. Satisfactory results pertaining to selectivity, linearity, matrix effect, accuracy and precision, recovery, stability, dilution integrity, carryover, and incurred sample analysis (ISR) were obtained. The calibration ranges in human plasma were as follows: 0.00200-1.00 µg/mL for C16 DP-VPA, 0.0100-5.00 µg/mL for C18 DP-VPA, and 0.0500-20.0 µg/mL for VPA. The linear ranges in urine and fecal homogenate were 0.00500-2.00 µg/mL and 0.00200-0.800 µg/mL for C16 DP-VPA, 0.00500-2.00 µg/mL and 0.0100-4.00 µg/mL for C18 DP-VPA, and 0.200-80.0 µg/mL for VPA, respectively. The intra- and inter-batch coefficients of variation in three matrices ranged from 1.7% to 12.4% while the accuracy values ranged from 85.4% to 111.7%. The developed methods were successfully applied to determine pharmacokinetics of DP-VPA tablet after a single oral dose of 1200 mg in 12 healthy Chinese subjects under fed condition.

Direct determination of valproic acid in biological fluids by capillary electrophoresis with contactless conductivity detection.

Capacitively coupled contactless conductivity detection (C(4)D) is a new technique providing high sensitivity in capillary electrophoresis (CE) especially for small ions that can otherwise only be determined with indirect methods. In this work, direct determination and validation of valproic acid (VPA) in biological fluids was achieved using CE with C(4)D. VPA is of pharmacological interest because of its use in epilepsy and bipolar disorder. The running electrolyte solution used consisted of 10mM 2-(N-morpholino)ethane sulfonic acid (MES)/dl-histidine (His) and 50microM hexadecyltrimethylammonium bromide (HTAB) at pH 6.0. Caproic acid (CA) was selected as internal standard (IS). Analyses of VPA in serum, plasma and urine samples were performed in less than 3min. The interference of the sample matrix was reduced by deproteinization of the sample with acetonitrile (ACN). The effect of the solvent type and ratio on interference was investigated. The limits of detection (LOD) and quantitation (LOQ) of VPA in plasma samples were determined as 24 and 80ng/ml, respectively. The method is linear between the 2 and 150microg/ml, covering well the therapeutic range of VPA (50-100microg/ml).

Observation of Clinically Relevant Drug Interaction in Chimeric Mice with Humanized Livers: The Case of Valproic Acid and Carbapenem Antibiotics.

BACKGROUND AND OBJECTIVE: Human in vitro and dog in vitro/in vivo researches indicate that the drug-drug interaction (DDI) of decreased plasma valproic acid (VPA) concentration by co-administration of carbapenem antibiotics is caused by inhibition of acylpeptide hydrolase (APEH)-mediated VPA acylglucuronide (VPA-G) hydrolysis by carbapenems. In this study, we investigated VPA disposition and APEH activities in TK-NOG chimeric mice, whose livers were highly replaced with human hepatocytes, to evaluate the utility of this animal model and the clinical relevance of the DDI mechanism. METHODS: VPA and VPA-G concentrations in plasma, urinary excretion of VPA-G and APEH activity in humanized livers were measured after co-administration of VPA with meropenem (MEPM) to chimeric mice. RESULTS: After co-administration with MEPM to the chimeric mice, plasma VPA concentration more rapidly decreased than without the co-administration. An increase in plasma AUC and urinary excretion of VPA-G was also observed. APEH activity in humanized livers was strongly inhibited even at 24 h after co-administration of MEPM to the chimeric mice. CONCLUSION: The DDI of VPA with carbapenems was successfully observed in chimeric mice with humanized livers. The DDI was caused by long-lasting inhibition of hepatic APEH-mediated VPA-G hydrolysis by carbapenems, which strongly supports the APEH-mediated mechanism of the clinical DDI. This is the first example showing the usefulness of chimeric mice with humanized livers for evaluation of a DDI via non-cytochrome P450 enzyme.

Influence of acylpeptide hydrolase polymorphisms on valproic acid level in Chinese epilepsy patients.

BACKGROUND: Concomitant use of meropenem (MEPM) can dramatically decrease valproic acid (VPA) plasma level. It is accepted that inhibition in acylpeptide hydrolase (APEH) activity by MEPM coadministration was the trigger of this drug-drug interaction. AIM: To investigate the influence of APEH genetic polymorphisms on VPA plasma concentration in Chinese epilepsy patients. PATIENTS & METHODS: Urinary VPA-d6 ß-D-glucuronide concentration was determined in 19 patients with VPA treatment alone (n = 10) or concomitant use with MEPM (n = 9). A retrospective study was performed on 149 epilepsy patients to investigate the influence of APEH polymorphisms rs3816877 and rs1131095 on adjusted plasma VPA concentration (CVPA) at steady-state. RESULTS: Urinary VPA-d6 ß-D-glucuronide (VPA-G) concentration was increased significantly in patients with MEPM coadministration. The CVPA of patients carrying the APEH rs3816877 C/C genotype was significantly higher than that of C/T carriers, and the difference was still obvious when stratified by UGT2B7 rs7668258 polymorphism. CONCLUSION: APEH polymorphism has significant influence on VPA pharmacokinetics in Chinese population.

Urinary excretion and metabolism of miglustat and valproate in patients with Niemann-Pick type C1 disease: One- and two-dimensional solution-state (1)H NMR studies.

Niemann-Pick type C1 (NP-C1) disease is a neurodegenerative lysosomal storage disease for which the only approved therapy is miglustat (MGS). In this study we explored the applications and value of both one- and two-dimensional high-resolution NMR analysis strategies to the detection and quantification of MGS and its potential metabolites in urine samples collected from NP-C1 disease patients (n=47), and also applied these techniques to the analysis of the anticonvulsant drug valproate and one of its major metabolites in ca. 30% of these samples (i.e. from those who were also receiving this agent for the control of epileptic seizures). A combination of high-resolution 1D and 2D TOCSY/NOESY techniques confirmed the identity of MGS in the urinary (1)H NMR profiles of NP-C1 patients treated with this agent (n=25), and its quantification was readily achievable via electronic integration of selected 1D resonance intensities. However, this analysis provided little or no evidence for its metabolism in vivo, observations consistent with those acquired in corresponding experiments performed involving an in vitro microsomal system. Contrastingly, the major valproate metabolite 1-O-valproyl-ß-glucuronide was readily detectable and quantifiable in 14/47 of the urine samples investigated, despite some resonance overlap problems (identification of this agent was confirmed by experiments involving equilibration of these samples with ß-glucuronidase, a process liberating free valproate). In order to facilitate and validate the detection of MGS in urine specimens, full assignments of the (1)H NMR spectra of MGS in both buffered aqueous (pH 7.10) and deuterated methanol solvent systems were also made. The pharmacological and bioanalytical significance of data acquired are discussed, with special reference to the advantages offered by high-resolution NMR analysis.

Ultrasound-assisted dispersive liquid-liquid microextraction followed by GC-MS/MS analysis for the determination of valproic acid in urine samples.

BACKGROUND: Valproic acid (VPA) is an anticonvulsant drug used for the treatment of epilepsy and bipolar disorder. A method based on simultaneous derivatization and dispersive liquid-liquid microextraction followed by GC-MS/MS analysis has been developed for the determination of VPA in urine samples. RESULTS: This optimized and validated method shows good linearity with R(2) value of 0.999. LOD and LOQ of VPA was found to be 0.4 ng ml(-1) and 1.4 ng ml(-1), respectively. Recovery of VPA was found to be in the range of 80 to 92%. CONCLUSION: The developed method can find its wide applicability for the routine analysis of VPA in toxicological and clinical laboratories.

The effect of aspirin on valproic acid metabolism.

Urinary valproic acid (VPA) and VPA metabolite profiles were determined before (day 1) and after (day 2) the administration of antipyretic doses of acetylsalicylic acid (ASA) to seven subjects with steady-state levels of VPA. Of the 13 metabolites assayed by GC/MS, levels of (E)-2-ene VPA and 3-keto VPA were significantly decreased on day 2, whereas those of the VPA conjugates (glucuronide) and 4-ene VPA were significantly increased. The beta-oxidation pathway consisting of (E)-2-ene VPA, 3-OH VPA, and 3-keto VPA was decreased from 24.5% +/- 10.3% of total metabolites excreted on day 1 to 8.3% +/- 4.2% on day 2, a decrease of 66% (P less than 0.05). VPA glucuronide content increased from 50.5% +/- 12.6% on day 1 to 65.5% +/- 14% of total excreted on day 2, an increase of 30% (P less than 0.05). The day 2/day 1 ratios of VPA glucuronide correlated significantly with the day 2/day 1 ratios of VPA mean free fraction (r = 0.9424; P = 0.005) in six of the seven subjects. Inhibition of VPA beta-oxidation by salicylate was sufficient to counterbalance the increased elimination of VPA as its conjugates and explains why total clearance of VPA after salicylate remains unchanged even though the free fraction of VPA is increased. Metabolic profiles indicate that salicylate likely inhibits VPA beta-oxidation by reducing valproyl-coenzyme A formation.

Effects of polytherapy with phenytoin, carbamazepine, and stiripentol on formation of 4-ene-valproate, a hepatotoxic metabolite of valproic acid.

The incidence of valproic acid hepatotoxicity has been reported to increase in patients who are receiving polytherapy. A minor valproic acid metabolite, 2-propyl-4-pentenoic acid (4-ene-VPA), formed by a cytochrome P450-mediated reaction, has been shown to be a potent inducer of microvesicular steatosis in rats. This study tested the hypothesis that formation of 4-ene-VPA would be increased in patients taking valproic acid with carbamazepine or with phenytoin but decreased with coadministration of an inhibitor of cytochrome P450 (the antiepileptic drug stiripentol in 300 to 1200 mg daily doses) in healthy subjects. Blood and urine samples in the studies were collected during a dosing interval at steady state. Valproic acid was assayed in plasma by capillary gas chromatography; valproic acid and 15 metabolites were measured in urine by gas chromatography/mass spectrometry. The formation clearance (CLf) of 4-ene-VPA was increased twofold in the valproic acid-carbamazepine and valproic acid-phenytoin groups. In the valproic acid/stiripentol studies, the CLf of 4-ene-VPA decreased by 32% in the 1200 mg/day stiripentol study. Similar findings were obtained at 600 and 300 mg/day stiripentol. These findings provide evidence supporting a role for cytochrome P450 in the formation of the hepatotoxic metabolite, 4-ene-VPA, in humans. The increased formation of 4-ene-VPA associated with carbamazepine and phenytoin is striking in relation to the epidemiologic finding of increased incidence of valproic acid-related hepatotoxicity during polytherapy with P450 inducers.

Valproic acid and plasma levels of phenobarbital.

During concurrent administration of phenobarbital and valproic acid, phenobarbital plasma concentrations often increase. This often requires a reduction of phenobarbital dosage. In normal cats and patients with epilepsy, we found no evidence of decreased renal excretion of phenobarbital. Metabolic studies in four patients revealed a decrease in the conversion of phenobarbital to hydroxyphenylphenobarbital and decreased urinary ratios of hydroxyphenylphenobarbital to phenobarbital. These data suggest that phenobarbital metabolism is inhibited by therapeutic plasma levels of valproic acid.

Gas chromatography/negative ion chemical ionization mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry quantitative profiling of N-acetylcysteine conjugates of valproic acid in urine: application in drug metabolism studies in humans.

We report a GC/NICI-MS assay and a LC/ESI-MS/MS assay for the analysis of N-acetylcysteine (NAC) conjugates of (E)-2,4-diene VPA (NAC I and NAC II) identified in humans. The assay also includes the analysis of the NAC conjugate of 4,5-epoxy VPA (NAC III), an identified metabolite in rats treated with 4-ene VPA for its use in metabolic studies in animals. The highly sensitive GC/MS assay was designed to monitor selectively the diagnostic and most abundant [M - 181](-) fragment anion of the di-PFB derivatives of NAC I, NAC II, and NAC IV, the internal standard (IS) and the PFB derivative of NAC III. The higher selectivity of LC/MS/MS methodology was the basis for an assay which could identify and quantitate the underivatized conjugates simultaneously using MRM of the diagnostic ions m/z 130 and 123 arising from the CID of their protonated molecular ions [MH](+). The GC/MS assay employed liquid-liquid extraction whereas the LC/MS/MS assay used a solid-phase extraction procedure. Linearity ranges of the calibration curves were 0.10-5.0microg ml(-1) by GC/MS and 0.10-1.0microg ml(-1) by LC/MS/MS for NAC I, NAC II and NAC III (r(2) = 0.999 or better). Both assays were validated for NAC I and NAC II and provided good inter- and intra-assay precision and accuracy for NAC I and NAC II. The LOQ by LC/MS/MS was 0.1microg ml(-1), representing 1 ng of NAC I and NAC II. The same LOQ (0.1microg ml(-1)) was observed by GC/MS and was equivalent to 100 pg of each metabolite. NAC III was detected at concentrations as low as 0.01 microg ml(-1) by both methods. The total urinary excretion of the NAC conjugates in four patients on VPA therapy was determined to be 0.004-0.088% of a VPA dose by GC/MS and 0.004-0. 109% of a VPA dose by LC/MS/MS.

Altered metabolic profiles of valproic acid in a patient with Reye's syndrome.

Urinary valproate metabolite and endogenous organic acid profiles in a 6-yr-old girl with Reye's syndrome were investigated by means of gas chromatography-mass spectrometry. 2-n-Propyl-3-oxovaleric acid, normally the major metabolite of valproate in man, was undetectable, while 2-n-propylglutaric acid, the end product via omega-oxidation was markedly increased. Polyunsaturated valproate was not found. Valproate-glucuronide was still found as the major metabolite. The clinical findings coupled with a greatly increased excretion of lactate and adipate was compatible with Reye's syndrome. Ketone bodies were not detectable. This case study shows that Reye's syndrome causes altered valproate metabolism, consistent with the defective mitochondrial beta-oxidation of medium chain fatty acids, and suggests that valproic acid should not be used in the treatment of seizures in patients with this syndrome.

Identification of urinary metabolites of sodium dipropylacetate in man; potential sources of interference in organic acid screening procedures.

Organic acid screening of urine samples from two children with neurological disease demonstrated the presence of two unknown metabolites. The children were receiving an antiepileptic drug, sodium dipropylacetate. The major abnormal compound has been shown by gas chromatography-mass spectrometry to be 3-oxodipropylacetic acid, a previously unidentified metabolite of dipropylacetate in man, while the minor metabolite was indentified as 2-(n-propyl)-glutaric acid.

Direct observation of 3-keto-valproate in urine by 2D-NMR spectroscopy.

BACKGROUND: It is known that valproate and its metabolites cause hepatotoxicity. The drug monitoring of valproate is important to determine an effective dose to keep an appropriate concentration in blood. METHODS: In a 2-dimensional (2D)-NMR spectrum of double quantum filtered correlation spectroscopy (DQF-COSY), clear correlation peaks were ascertained to be due to 3-keto-valproate, which was a beta-oxidation product of valproate. RESULTS: A predominant metabolite of valproate was observed by proton NMR spectroscopy in the crude urine of a particular patient with metabolic disorder. The assignment of the signals was determined by synthesized 3-keto-valproic acid ethyl ester. The concentration of 3-keto-valproate in the urine was calculated to be 631 microg/mg creatinine by the integration of the peak of the isolated triplet methyl protons of C(5) at 1.016 ppm. CONCLUSION: Although the NMR spectra of crude urine of the patients who took valproate were usually complicated with many metabolites, the signals of 3-keto-valproate in a DQF-COSY spectrum of the urine of patients were easily connected according to the present assignment. The NMR analysis of the urine of patients who are prescribed valproate is useful for therapeutic drug monitoring and for checking the compliance of the patients.

Valproylcarnitine: a novel drug metabolite identified by fast atom bombardment and thermospray liquid chromatography-mass spectrometry.

Urine samples from three children at different stages of chronic valproate therapy were partially purified using a cation exchange column. A signal consistent with either valproylcarnitine or octanoylcarnitine was observed in one of these extracts by direct fast atom bombardment-mass spectrometry analysis. These isomeric acylcarnitines were synthesized, separated and characterized by thermospray high performance liquid chromatography-mass spectrometry. This new technique was then employed to positively identify intact valproyl-carnitine in the patients' urine samples. The implications of this finding with regard to a mechanism to account for carnitine deficiency in patients receiving valproate are discussed.