Metabolomics is the comprehensive study of small-molecule metabolites. Obtaining a wide coverage of the metabolome is challenging because of the broad range of physicochemical properties of the small ...molecules. To study the compounds of interest spectroscopic (NMR), spectrometric (MS) and separation techniques (LC, GC, supercritical fluid chromatography, CE) are used. The choice for a given technique is influenced by the sample matrix, the concentration and properties of the metabolites, and the amount of sample. This review discusses the most commonly used analytical techniques for metabolomic studies, including their advantages, drawbacks and some applications.
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 actual utility of capillary electrophoresis‐mass spectrometry (CE‐MS) for biomarker discovery using metabolomics still needs to be assessed. Therefore, a simulated comparative metabolic profiling ...study for biomarker discovery by CE‐MS was performed, using pooled human plasma samples with spiked biomarkers. Two studies have been carried out in this work. Focus of study I was on comparing two sets of plasma samples, in which one set (class I) was spiked with five isotope‐labeled compounds, whereas another set (class II) was spiked with six different isotope‐labeled compounds. In study II, focus was also on comparing two sets of plasma samples, however, the isotope‐labeled compounds were spiked to both class I and class II samples but with concentrations which differ by a factor two between both classes (with one compound absent in each class). The aim was to determine whether CEMS‐based metabolomics could reveal the spiked biomarkers as the main classifiers, applying two different data analysis software tools (MetaboAnalyst and Matlab). Unsupervised analysis of the recorded metabolic profiles revealed a clear distinction between class I and class II plasma samples in both studies. This classification was mainly attributed to the spiked isotope‐labeled compounds, thereby emphasizing the utility of CE‐MS for biomarker discovery.
Objective
In the management of epilepsy, there is an ongoing quest to discover new biomarkers to improve the diagnostic process, the monitoring of disease progression, and the evaluation of treatment ...responsiveness. In this regard, biochemical traceability in biofluids is notably absent in contrast to other diseases. In the present preclinical study, we investigated the potential of neurofilament light chain (NfL) as a possible diagnostic and response fluid biomarker for epilepsy.
Methods
We gained insights into NfL levels during the various phases of the intrahippocampal kainic acid mouse model of temporal lobe epilepsy—namely, the status epilepticus (SE) and the chronic phase with spontaneous seizures. To this end, NfL levels were determined directly in the cerebral interstitial fluid (ISF) with cerebral open flow microperfusion as sampling technique, as well as in cerebrospinal fluid (CSF) and plasma. Lastly, we assessed whether NfL levels diminished upon curtailing SE with diazepam and ketamine.
Results
NfL levels are higher during SE in both cerebral ISF and plasma in kainic acid‐treated mice compared to sham‐injected mice. Additionally, ISF and plasma NfL levels are lower in mice treated with diazepam and ketamine to stop SE compared with the vehicle‐treated mice. In the chronic phase with spontaneous seizures, higher NfL levels could only be detected in ISF and CSF samples, and not in plasma. No correlations could be found between NfL levels and seizure burden, nor with immunohistological markers for neurodegeneration/inflammation.
Significance
Our findings demonstrate the translational potential of NfL as a blood‐based fluid biomarker for SE. This is less evident for chronic epilepsy, as in this case higher NfL levels could only be detected in ISF and CSF, and not in plasma, acknowledging the invasive nature of CSF sampling in chronic epilepsy follow‐up.
Stress, such as social isolation, is a well-known risk factor for depression, most probably in combination with predisposing genetic factors. Physical exercise on the other hand, is depicted as a ...wonder-treatment that makes you healthier, happier and live longer. However, the published results on the effects of exercise are ambiguous, especially when it comes to neuropsychiatric disorders. Here we combine a paradigm of social isolation with a genetic rat model of depression, the Flinders Sensitive Line (FSL), already known to have glutamatergic synaptic alterations. Compared to group-housed FSL rats, we found that social isolation further affects synaptic plasticity and increases basal synaptic transmission in hippocampal CA1 pyramidal neurons. These functional synaptic alterations co-exist with changes in hippocampal protein expression levels: social isolation in FSL rats reduce expression of the glial glutamate transporter GLT-1, and increase expression of the GluA2 AMPA-receptor subunit. We further show that physical exercise in form of voluntary running prevents the stress-induced synaptic effects but do not restore the endogenous mechanisms of depression already present in the FSL rat.
•Proper solubilization reduces aspecific adsorption of peptides and proteins.•A systematic approach to select the most appropriate solvent for dissolution.•Strategies to reduce sticking of peptides ...and proteins during sample preparation.•Reduction of adsorption at parts of the LC–MS system (carryover).
In the drug-discovery setting, the development of new peptide and protein-based biopharmaceuticals attracts increased attention from the pharmaceutical industry and consequently demands the development of high-throughput LC–MS methods. Regulatory guidelines require bioanalytical methods to be validated not only in terms of linearity, sensitivity, accuracy, precision, selectivity and stability, but also in terms of carryover. Carryover results from the aspecific adsorption of analyte(s) to parts of the analytical system and thus introduces bias in both identification and quantification assays. Moreover, nonspecific binding occurs at the surface of materials used during sample preparation, such as pipette tips, sample tubes and LC-vials. Hence, linearity, sensitivity and repeatability of the analyses are negatively affected. Due to the great diversity in physicochemical properties of biomolecules, there is no general approach available to minimize adsorption phenomena. Therefore, we aim to present different strategies which can be generically applied to reduce nonspecific binding of peptides and proteins. In the first part of this review, a systematic approach is proposed to guide the reader through the different solvents which can be used to dissolve the analyte of interest. Indeed, proper solubilization is one of the most important factors for a successful analysis. In addition, alternative approaches are described to improve analyte recovery from the sample vial. The second part focuses on strategies to efficiently reduce adsorption at components of the autosampler, column and mass spectrometer. Thereby carryover is reduced while maintaining a sufficiently wide dynamic range of the assay.
Accumulating evidence shows a key function for astrocytic connexin43 (Cx43) signaling in epilepsy. However, the lack of experimental distinction between Cx43 gap junction channels (GJCs) and ...hemichannels (HCs) has impeded the identification of the exact contribution of either channel configurations to epilepsy. We therefore investigated whether TAT‐Gap19, a Cx mimetic peptide that inhibits Cx43 HCs but not the corresponding Cx43 GJCs, influences experimentally induced seizures in rodents. Dye uptake experiments in acute hippocampal slices of mice demonstrated that astroglial Cx43 HCs open in response to the chemoconvulsant pilocarpine and this was inhibited by TAT‐Gap19. In vivo, pilocarpine‐induced seizures as well as the accompanying increase in D‐serine microdialysate levels were suppressed by Cx43 HC inhibition. Moreover, the anticonvulsant action of TAT‐Gap19 was reversed by exogenous D‐serine administration, suggesting that Cx43 HC inhibition protects against seizures by lowering extracellular D‐serine levels. The anticonvulsive properties of Cx43 HC inhibition were further confirmed in electrical seizure mouse models, i.e. an acute 6 Hertz (Hz) model of refractory seizures and a chronic 6 Hz corneal kindling model. Collectively, these results indicate that Cx43 HCs play a role in seizures and underscore their potential as a novel and druggable target in epilepsy treatment.
Main Points
Astrocytic Cx43 HCs open in response to pilocarpine.
Cx43 HC inhibition diminishes pilocarpine‐induced seizures and extracellular D‐serine levels in vivo.
Cx43 HC inhibition suppresses electrically‐induced refractory seizures.
The increased interest in the separation of peptides, proteins, immunoglobulins, and polynucleotides, led to an increased demand for appropriate analytical methodologies and instrumentation. CE, ...because of its unique separation mechanism and high efficiency, is frequently used in the analysis of those molecules. In this study, a CE method for the separation of six angiotensin analogues was developed in the first step. In the second step, the method was transferred to a CE instrument of another brand, taking into account guidelines defined earlier about the interinstrumental transfer of CE methods. Although previously successful, the application of these guidelines during this study was not able to maintain the baseline separation. Further research on the instrumental differences revealed that the electrical resistance on both instruments differed. At constant current, the electrical resistance, generated voltage, and separation efficiency on the Agilent instrument were lower than on the Beckman instrument. Increase of the electrical resistance, by reducing the capillary temperature, leads to an increased applied voltage and separation efficiency on the Agilent system. The guidelines developed earlier were, therefore, updated with an additional step equalizing the electrical resistances, which led to a successful interinstrumental analytical method transfer for the separation of six angiotensin derivatives.
Bidirectional communication between neurons and astrocytes shapes synaptic plasticity and behavior. D-serine is a necessary co-agonist of synaptic N-methyl-D-aspartate receptors (NMDARs), but the ...physiological factors regulating its impact on memory processes are scantly known. We show that astroglial CB1 receptors are key determinants of object recognition memory by determining the availability of D-serine at hippocampal synapses. Mutant mice lacking CB1 receptors from astroglial cells (GFAP-CB1-KO) displayed impaired object recognition memory and decreased in vivo and in vitro long-term potentiation (LTP) at CA3-CA1 hippocampal synapses. Activation of CB1 receptors increased intracellular astroglial Ca2+ levels and extracellular levels of D-serine in hippocampal slices. Accordingly, GFAP-CB1-KO displayed lower occupancy of the co-agonist binding site of synaptic hippocampal NMDARs. Finally, elevation of D-serine levels fully rescued LTP and memory impairments of GFAP-CB1-KO mice. These data reveal a novel mechanism of in vivo astroglial control of memory and synaptic plasticity via the D-serine-dependent control of NMDARs.
•Astrocytes are important for long-term object recognition memory•Astroglial CB1 receptors are coupled to D-serine availability at synapses•Appropriate D-serine levels are required for NMDAR activity and LTP induction
Robin et al. show that astroglial CB1 receptors in the hippocampus regulate D-serine supply to NMDA receptors, a process necessary for LTP induction and object recognition memory.
Temporal lobe epilepsy (TLE) is an acquired form of focal epilepsy, in which patients not only suffer from unprovoked, devastating seizures, but also from severe comorbidities, such as cognitive ...dysfunction. Correspondingly, several animal models of TLE exhibit memory dysfunction, especially spatial memory. The Morris water maze test is the most commonly used test for assessing spatial learning and memory in rodents. However, high stress and poor swimming abilities are common confounders and may contribute to misinterpretation. Particularly epileptic mice show altered behaviour during the test as they fail to understand the paradigm context. In the Barnes maze test, a dry-land maze test for spatial learning and memory that uses milder aversive stimuli, these drawbacks have not yet been reported. In the present study, we use this task to evaluate spatial learning and memory in the intrahippocampal kainic acid mouse model of TLE. We demonstrate that the epileptic mice understand the Barnes maze paradigm context, as they learn the location of the escape-chamber by using a serial search strategy but fail to develop the more efficient spatial search strategy. Our data indicate that the Barnes maze may be a better alternative to the Morris water maze for assessing search strategies and impairment of learning and memory in epileptic mice.
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