Using high-resolution Twin-Ion Metabolite Extraction (HiTIME) mass spectrometry with stable isotope labelling to investigate the metabolism of valproic acid in vivo
Graphical abstract
Introduction
Epilepsy is one of the most common neurological diseases and affects over 2 million people in the United States [1,2]. Valproic acid (2-propylpentanoic acid, VPA) is a broad-spectrum anti-epileptic drug (AED) that was serendipitously discovered in the 1960s and has become the first-line treatment for a range of seizure types [3,4]. Despite its place as a mainstay of epilepsy treatment, VPA is associated with numerous severe side effects [5]. VPA is a known teratogen, producing a 10 to 20-fold increase in the incidence of neural tube defects (NTDs) relative to the population-at-large if taken by pregnant women in the first trimester [[6], [7], [8], [9]]. As a result of these effects, VPA currently carries so-called ‘black-box’ warnings issued by the United States Food and Drug Administration for hepatotoxicity, teratogenicity and pancreatitis. Clinical observations in Australia and the United Kingdom of women who carried multiple pregnancies with sustained VPA treatment indicate that giving birth to one malformed child significantly increases the risk of teratogenic effects in subsequent pregnancies [10,11]. This suggests that maternal factors such as genetics and metabolism may result in a predisposition to birth abnormalities following VPA treatment. Identification of biomarkers that could differentiate between women who reside in high or low-risk groups for VPA-induced teratogenicity would be a useful prognostic tool for counselling women regarding epilepsy treatment during pregnancies. Attention has thus turned toward investigating potential differences in the metabolism of VPA between individuals.
VPA is known to undergo extensive metabolism through both phase I and phase II processes [12]. While transformations are thought to be primarily affected by CYP and UGT enzymes [13,14], the β-oxidation pathway that is typically involved in processing of endogenous fatty acids also plays a role [15]. Interestingly, multiple metabolites of VPA have been found to be effective anticonvulsants [[16], [17], [18]] while others are thought to be hepatotoxic [19,20]. Given that the abundances of metabolites formed can vary widely between individuals [21] and numerous AEDs are often used in combination therapy with VPA, which can affect changes to metabolic enzyme expression [22,23], it is hypothesised that differential metabolism may contribute to the differential onset of human teratogenicity.
The combined use of stable isotope labels, chromatography and mass spectrometry has proven invaluable in drug studies [[24], [25], [26]]. For example, the simultaneously administration of a 1:1 mixture of isotopically unlabelled (“light”) and labelled (“heavy”) drug provides a characteristic ‘twin-ions’ signature that can be used in the non-targeted detection of metabolic products of the precursor drug, ranging from small molecules [[27], [28], [29]] to protein adducts of reactive metabolites [30]. Other stable isotope mass spectrometry based experiments include pharmacokinetic parameters, including bioavailability studies [31,32], drug interaction studies, establishment of steady state kinetics and mechanistic studies that utilize kinetic isotope effects to probe biochemical pathways. Many of these stable isotope labelling approaches have been used in early GC/MS based metabolism studies on VPA, which required hydrolysis of glucuronide conjugates followed by chemical derivatization [[33], [34], [35], [36]].
Since the advent of LC/MS based methods which require little sample preparation and no chemical derivatization, there has been renewed interest in the application of the twin ion method. Due to the large datasets generated from these experiments, several computer algorithms have been developed to automate twin-ion metabolite detection [37]. Here we report on the results of a pilot study investigating VPA and metabolism in the rat that is designed to demonstrate the feasibility of using twin-ion methodologies combined with our recently developed automated HiTIME (High-resolution Twin-Ion Metabolite Extraction) data-mining software for: i) identifying metabolites of valproate; and ii) tracking the kinetics of metabolite formation.
Section snippets
Experimental
Valproic acid and 13C4-valproic acid (1,2,3,3′-13C4, 99.2% isotope enrichment) were purchased from Sigma-Aldrich and Cambridge Isotope Laboratories respectively. Acetonitrile, oxalyl chloride, methylene chloride, dimethylformamide, trifluoroacetic acid, formic acid and ascorbic acid were obtained from commercial sources and used without further purification.
- (i)
Synthesis of putative VPA-ascorbate metabolite
Oxallyl chloride (0.88 g, 7 mmol) was added to a stirred solution of valproic acid (0.5 g,
Detection of valproic acid metabolites
VPA and 13C4-VPA were simultaneously administered to rats in a 1:1 ratio and blood samples were drawn at various time points thereafter. LC-MS analysis of plasma extracts performed in the negative ion mode resulted in the detection of a vast number of signals, most of which did not correspond to the twin-ion signature. To selectively detect data regions that fit the twin-ion signature, HiTIME analysis was performed using a Δm/z value of 4.01341 and heat maps of the results are shown in Fig. 1.
Discussion
Given its low molecular weight and remarkably simple structure, the metabolism of valproic acid is clearly quite complicated. Following administration of VPA and 13C4-VPA to rats, 14 twin-ions were identified by HiTIME searching of LC-MS data. These were assigned as the unmodified drug VPA, 12 metabolites of VPA and a putative VPA-ascorbate adduct. The metabolites of valproate that were detected in rat plasma here (Scheme 1) are consistent with those detected previously [35,38]. These
Conclusions
The metabolism and kinetics of the epilepsy medication valproic acid have been investigated in rats. The twin-ion method was used to assess VPA metabolism under both acute and steady state conditions at different doses. By simultaneously administering VPA and 13C4-VPA in equal proportions, 14 twin-ions were detected in LCMS data from blood extracts by HiTIME searching that could be assigned to: VPA, 12 known VPA metabolites and a putative VPA-ascorbate adduct. These included numerous phase I
Acknowledgments
We thank the University of Melbourne Interdisciplinary Seed Grant program for funding. MGL thanks the Elizabeth and Vernon Puzey foundation for a PhD scholarship and The University of Melbourne Faculty of Science of the award of the Norma Hilda Schuster scholarship. We thank Andrew P. Isaac and Bernard J. Pope from Victorian Life Sciences Computation Initiative (VLSCI) for developing the HiTIME program. We gratefully acknowledge Linda Cornthwaite-Duncan for performing rat surgeries, drug
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