Ilse Smolders

Validation of bioanalytical LC-MS/MS assays: evaluation of matrix effects.

Liquid chromatography coupled to atmospheric pressure ionization tandem mass spectrometry is currently the method of choice for the quantitative determination of drugs in biological matrices. The advantages of this technique include high specificity, sensitivity and throughput. However, co-eluting matrix components, which are not observed in the chromatogram, can have a detrimental effect on the analysis, since they can cause ion suppression or enhancement of the analyte. The evaluation of matrix effects on the quantitative analysis of drugs in biological fluids is an important and sometimes overlooked aspect of assay validation. In this review, the influence of matrix effects on bioanalytical LC-MS/MS methods is discussed and illustrated with some examples. In addition, possible solutions to reduce or eliminate matrix effects are highlighted. A literature overview of validated LC-MS/MS methods published from January till June 2008 is also included. Although matrix effects are investigated in most papers, there is no consensus on how matrix effects should be evaluated during method validation. In addition, the definition of specificity should be changed for LC-MS/MS based methods.

The Cystine/Glutamate Antiporter System xc− in Health and Disease: From Molecular Mechanisms to Novel Therapeutic Opportunities

The antiporter system x(c)(-) imports the amino acid cystine, the oxidized form of cysteine, into cells with a 1:1 counter-transport of glutamate. It is composed of a light chain, xCT, and a heavy chain, 4F2 heavy chain (4F2hc), and, thus, belongs to the family of heterodimeric amino acid transporters. Cysteine is the rate-limiting substrate for the important antioxidant glutathione (GSH) and, along with cystine, it also forms a key redox couple on its own. Glutamate is a major neurotransmitter in the central nervous system (CNS). By phylogenetic analysis, we show that system x(c)(-) is a rather evolutionarily new amino acid transport system. In addition, we summarize the current knowledge regarding the molecular mechanisms that regulate system x(c)(-), including the transcriptional regulation of the xCT light chain, posttranscriptional mechanisms, and pharmacological inhibitors of system x(c)(-). Moreover, the roles of system x(c)(-) in regulating GSH levels, the redox state of the extracellular cystine/cysteine redox couple, and extracellular glutamate levels are discussed. In vitro, glutamate-mediated system x(c)(-) inhibition leads to neuronal cell death, a paradigm called oxidative glutamate toxicity, which has successfully been used to identify neuroprotective compounds. In vivo, xCT has a rather restricted expression pattern with the highest levels in the CNS and parts of the immune system. System x(c)(-) is also present in the eye. Moreover, an elevated expression of xCT has been reported in cancer. We highlight the diverse roles of system x(c)(-) in the regulation of the immune response, in various aspects of cancer and in the eye and the CNS.

Antagonists of GLUK5-containing kainate receptors prevent pilocarpine-induced limbic seizures

Developments in the molecular biology and pharmacology of GLUK5, a subtype of the kainate class of ionotropic glutamate receptors, have enabled insights into the roles of this subunit in synaptic transmission and plasticity. However, little is known about the possible functions of GLUK5-containing kainate receptors in pathological conditions. We report here that, in hippocampal slices, selective antagonists of GLUK5-containing kainate receptors prevented development of epileptiform activity—evoked by the muscarinic agonist, pilocarpine—and inhibited the activity when it was pre-established. In conscious rats, these GLUK5 antagonists prevented and interrupted limbic seizures induced by intra-hippocampal pilocarpine perfusion, and attenuated accompanying rises in extracellular L-glutamate and GABA. This anticonvulsant activity occurred without overt side effects. GLUK5 antagonism also prevented epileptiform activity induced by electrical stimulation, both in vitro and in vivo. Therefore, we propose that subtype-selective GLUK5 kainate receptor antagonists offer a potential new therapy for epilepsy.

NMDA receptor-mediated pilocarpine-induced seizures: characterization in freely moving rats by microdialysis.

Pilocarpine administration has been used as an animal model for temporal lobe epilepsy since it produces several morphological and synaptic features in common with human complex partial seizures. Little is known about changes in extracellular neurotransmitter concentrations during the seizures provoked by pilocarpine, a non‐selective muscarinic agonist. Focally evoked pilocarpine‐induced seizures in freely moving rats were provoked by intrahippocampal pilocarpine (10mm for 40min at a flow rate of 2μl min−1) administration via a microdialysis probe. Concomitant changes in extracellular hippocampal glutamate, γ‐aminobutyric acid (GABA) and dopamine levels were monitored and simultaneous electrocorticography was performed. The animal model was characterized by intrahippocampal perfusion with the muscarinic receptor antagonist atropine (20mm), the sodium channel blocker tetrodotoxin (1μm) and the N‐methyl‐d‐aspartate (NMDA) receptor antagonist MK‐801 (dizocilpine maleate, 100μm). The effectiveness of locally (600μm) or systemically (10mg kg−1 day−1) applied lamotrigine against the pilocarpine‐induced convulsions was evaluated. Pilocarpine initially decreased extracellular hippocampal glutamate and GABA levels. During the subsequent pilocarpine‐induced limbic convulsions extracellular glutamate, GABA and dopamine concentrations in hippocampus were significantly increased. Atropine blocked all changes in extracellular transmitter levels during and after co‐administration of pilocarpine. All pilocarpine‐induced increases were completely prevented by simultaneous tetrodotoxin perfusion. Intrahippocampal administration of MK‐801 and lamotrigine resulted in an elevation of hippocampal dopamine levels and protected the rats from the pilocarpine‐induced seizures. Pilocarpine‐induced convulsions developed in the rats which received lamotrigine perorally. Pilocarpine‐induced seizures are initiated via muscarinic receptors and further mediated via NMDA receptors. Sustained increases in extracellular glutamate levels after pilocarpine perfusion are related to the limbic seizures. These are arguments in favour of earlier described NMDA receptor‐mediated excitotoxicity. Hippocampal dopamine release may be functionally important in epileptogenesis and may participate in the anticonvulsant effects of MK‐801 and lamotrigine. The pilocarpine‐stimulated hippocampal GABA, glutamate and dopamine levels reflect neuronal vesicular release.

Tonic GABA-ergic modulation of striatal dopamine release studied by in vivo microdialysis in the freely moving rat.

GABAA and GABAB receptor agonists and antagonists were administered locally in the striatum of intact and kainic acid lesioned rats. (+/-)-Baclofen, a GABAB receptor agonist, significantly decreased the level of extracellular dopamine in the striatum of intact rats. (+/-)-Phaclofen, a GABAB receptor antagonist, increased the level of extracellular dopamine in the striatum of intact rats and to a lesser extent in the striatum after kainic acid lesion. Pregnanolone (5 beta-pregnan-3 alpha-ol-20-one), a positive allosteric modulator of the GABAA receptor, significantly decreased the level of extracellular dopamine in intact rats. (-)-Bicuculline, a GABAB receptor antagonist, increased the level of extracellular dopamine in the striatum of intact rats, but failed to increase the level of extracellular dopamine after kainic acid lesion. The release of extracellular dopamine, due to infusion of phaclofen or bicuculline, was totally suppressed by tetrodotoxin. These results support a direct influence of GABA on the dopaminergic terminals via presynaptic GABAB receptors, while the effects via the GABAA receptor seem to be postsynaptic and mediated by striatal interneurons or the striatonigral feedback loop.

Anticonvulsant action of hippocampal dopamine and serotonin is independently mediated by D2 and 5-HT1A receptors

The present microdialysis study evaluated the anticonvulsant activity of extracellular hippocampal dopamine (DA) and serotonin (5‐HT) with concomitant assessment of the possible mutual interactions between these monoamines. The anticonvulsant effects of intrahippocampally applied DA and 5‐HT concentrations were evaluated against pilocarpine‐induced seizures in conscious rats. DA or 5‐HT perfusions protected the rats from limbic seizures as long as extracellular DA or 5‐HT concentrations ranged, respectively, between 70–400% and 80–350% increases compared with the baseline levels. Co‐perfusion with the selective D2 blocker remoxipride or the selective 5‐HT1A blocker WAY‐100635 clearly abolished all anticonvulsant effects. These anticonvulsant effects were mediated independently since no mutual 5‐HT and DA interactions were observed as long as extracellular DA and 5‐HT levels remained within these protective ranges. Simultaneous D2 and 5‐HT1A receptor blockade significantly aggravated pilocarpine‐induced seizures. High extracellular DA (> 1000% increases) or 5‐HT (> 900% increases) concentrations also worsened seizure outcome. The latter proconvulsive effects were associated with significant increases in extracellular glutamate (Glu) and mutual increases in extracellular monoamines. Our results suggest that, within a certain concentration range, DA and 5‐HT contribute independently to the prevention of hippocampal epileptogenesis via, respectively, D2 and 5‐HT1A receptor activation.

Increased hippocampal noradrenaline is a biomarker for efficacy of vagus nerve stimulation in a limbic seizure model

J. Neurochem. (2011) 117, 461–469.

The analysis of excitatory, inhibitory and other amino acids in rat brain microdialysates using microbore liquid chromatography

Three microbore liquid chromatography (LC) assays for determination of amino acids in rat brain dialysates are described: one for separation of amino acids by gradient elution and electrochemical detection, one for analysis of GABA by isocratic elution and electrochemical detection, and one for fast measurement of glutamate and aspartate by gradient elution and fluorescence detection. The assays are reliable, reproducible and sensitive. In comparison with conventional LC, a 5-fold increase in sensitivity was obtained for GABA. Optimization of the derivatization chemistry and the microbore LC system are discussed, as well as important practical aspects.

Loss of system x(c)- does not induce oxidative stress but decreases extracellular glutamate in hippocampus and influences spatial working memory and limbic seizure susceptibility.

System xc− exchanges intracellular glutamate for extracellular cystine, giving it a potential role in intracellular glutathione synthesis and nonvesicular glutamate release. We report that mice lacking the specific xCT subunit of system xc− (xCT−/−) do not have a lower hippocampal glutathione content, increased oxidative stress or brain atrophy, nor exacerbated spatial reference memory deficits with aging. Together these results indicate that loss of system xc− does not induce oxidative stress in vivo. Young xCT−/− mice did however display a spatial working memory deficit. Interestingly, we observed significantly lower extracellular hippocampal glutamate concentrations in xCT−/− mice compared to wild-type littermates. Moreover, intrahippocampal perfusion with system xc− inhibitors lowered extracellular glutamate, whereas the system xc− activator N-acetylcysteine elevated extracellular glutamate in the rat hippocampus. This indicates that system xc− may be an interesting target for pathologies associated with excessive extracellular glutamate release in the hippocampus. Correspondingly, xCT deletion in mice elevated the threshold for limbic seizures and abolished the proconvulsive effects of N-acetylcysteine. These novel findings sustain that system xc− is an important source of extracellular glutamate in the hippocampus. System xc− is required for optimal spatial working memory, but its inactivation is clearly beneficial to decrease susceptibility for limbic epileptic seizures.

Muscarinic Modulation of Striatal Dopamine, Glutamate, and GABA Release, as Measured with In Vivo Microdialysis

Abstract: Intrastriatal microdialysis was used to administer muscarinic drugs in freely moving rats for 40 min at a flow rate of 2 µl/min. Administration of the nonselective agonist pilocarpine at 10 mM increased striatal dopamine release and decreased extracellular GABA and glutamate overflow. Perfusion with the muscarinic M2 antagonist methoctramine at 75 µM increased extracellular dopamine and glutamate concentrations but exerted no changes on extracellular GABA levels. Intrastriatal administration of the M1 antagonist pirenzepine at 0.05 µM decreased extracellular dopamine overflow. Application of pirenzepine (0.05 and 5 µM) exerted no effects on the measured GABA or glutamate levels. There are thus important differences in applied doses of muscarinic drugs needed to obtain modulatory effects. High doses of agonists are probably needed to superimpose on the background of tonic influences of striatal acetylcholine, whereas antagonists can block the receptors in small doses. We further suggest that M1 receptors might tonically facilitate striatal dopamine release, that M2 receptors might tonically inhibit striatal glutamate efflux, and that acetylcholine does not exert tonic effects on striatal GABA release. The link with the pilocarpine animal model for temporal lobe epilepsy will be discussed.