Disaccharide compositional analysis of chondroitin sulphate using WAX HILIC-MS with pre-column procainamide labelling; application to the placenta in pre-eclampsia.

Chondroitin sulphate (CS) and dermatan sulphate are negatively charged linear heteropolysaccharides. These glycosaminoglycans (GAG) are involved in cellular signalling via binding to growth factors. CS is expressed in a range of tissue and biological fluids and is highly expressed in the placenta. There is evidence that decorin; a CS proteoglycan is significantly decreased in pre-eclampsia and fetal growth restriction. It is considered that GAG chain composition may influence cellular processes that are altered in pre-eclampsia. The goal of the present study was to develop an LC-MS method with precolumn procainamide labelling for the disaccharide compositional analysis of CS. The method was used to investigate whether the disaccharide composition of placenta-extracted CS is altered in pre-eclampsia. The study revealed differential disaccharide compositions of placental chondroitin sulphate between pre-eclampsia and other pregnancy conditions. This suggests that the method may have diagnostic potential for pregnancy disorders. Furthermore, the findings suggest that CS sulphation might play a significant role in maternal labour.

Antibacterial apple cider vinegar eradicates methicillin resistant Staphylococcus aureus and resistant Escherichia coli.

Methicillin-resistant Staphylococcus aureus (MRSA) and resistant Escherichia coli (rE.coli) infections can spread rapidly. Further they are associated with high morbidity and mortality from treatment failure. Therapy involves multiple rounds of ineffective antibiotics alongside unwanted side effects, alternative treatments are crucial. Apple cider vinegar (ACV) is a natural, vegan product that has been shown to have powerful antimicrobial activity hence we investigated whether ACV could ameliorate these resistant bacteria. The minimum dilution of ACV required for growth inhibition was comparable for both bacteria (1/25 dilution of ACV liquid and ACV tablets at 200 µg/ml were effective against rE. coli and MRSA). Monocyte co-culture with microbes alongside ACV resulted in an increase in monocyte phagocytosis by 21.2% and 33.5% compared to non-ACV treated but MRSA or rE. coli stimulated monocytes, respectively. Label free quantitative proteomic studies of microbial protein extracts demonstrated that ACV penetrated microbial cell membranes and organelles, altering the expression of key proteins. This resulted in significant reductions in total protein expression, moreover we could only detect ribosomal proteins; 50 s 30 s, enolase, phosphenol pyruvate and the ATP synthase subunit in rE. coli. Elongation factor iNOS and phosphoglycerate kinase OS were the only proteins present in MRSA samples following ACV treatment.

Detection of Extended-Spectrum ß-Lactamase and Carbapenemase Activity in Gram-Negative Bacilli Using Liquid Chromatography - Tandem Mass Spectrometry.

PURPOSE: Several mass spectrometry-based methods for antimicrobial sensitivity testing have been described in recent years. They offer an alternative to commercially available testing systems which were considered to have disadvantages in terms of cost- and time-efficiency. The aim of this study was to develop an LC-MS/MS-based antibiotic hydrolysis assay for evaluating antimicrobial resistance (AMR) of Gram-negative bacteria. MATERIALS AND METHODS: Four species of Gram-negative bacilli (Klebsiella pneumoniae, Escherichia coli, Providencia stuartii and Acinetobacter baumannii) were tested against six antibiotics from three different classes: ampicillin, meropenem, imipenem, ceftazidime, ceftriaxone and cefepime. Bacterial suspensions from each species were incubated with a mixture of the six antibiotics. Any remaining antibiotic following incubation was measured using LC-MS/MS. The results were interpreted using measurements obtained for an E. coli strain sensitive to all antibiotics and expressed as percentage of hydrolyzed antibiotic. These were subsequently compared to commercially-available system for the bacteria identification and susceptibility testing. RESULTS: Overall, LC-MS/MS assay and commercial antimicrobial susceptibility platform results showed good agreement in terms of an organism being resistant/sensitive to an antibiotic. The time required to complete the LC-MS/MS-based hydrolysis test was under 5 h, significantly shorter that commercially available susceptibility testing platforms. CONCLUSION: By using a sensitive strain for results interpretation and simultaneous use of multiple antibiotics, the proposed protocol offers improved robustness and multiplexing over previously described methods for antibiotic sensitivity testing. Nevertheless, further research is needed before routine assimilation of the method, especially for strains with intermediate resistance.

Effect of berberine on in vitro metabolism of sulfonylureas: A herb-drug interactions study.

Patients with type 2 diabetes may co-ingest herbal and prescription medicines to control their blood sugar levels. Competitive binding of drug and herb may mutually affect their metabolism. This can alter the level of drug and its kinetics in the body, potentially causing toxicities or loss of efficacy. Understanding how the metabolism of sulfonylureas like glyburide and gliclazide can be affected by the presence of berberine and vice versa can provide valuable information on the possible risk of toxicities caused by co-ingestion of drugs. METHODS: Berberine and sulfonylureas (glyburide and gliclazide) were co-incubated with rat liver microsomes in the presence of a NADPH-regenerating system. The metabolites of berberine and sulfonylureas were analysed using liquid chromatography with high-resolution mass spectrometry in the positive ion mode. The role of individual isozymes in the metabolism of berberine, glyburide and gliclazide was investigated by using specific inhibitors. RESULTS: In vitro metabolism of berberine led to the formation of demethyleneberberine (B1a) and its isomer B1b through demethylenation. Berberrubine (B2a) and its isomer B2b were formed through demethylation. The isozymes CYP3A and CYP2D were found to be involved in the metabolism of berberine. In vitro metabolism of glyburide and gliclazide led to the formation of hydroxylated metabolites. The isozymes CYP3A and CYP2C were found to be involved in the metabolism of glyburide. Gliclazide was metabolised by CYP2C. In vitro co-incubation of glyburide or gliclazide with berberine showed that each drug's metabolism was compromised as they share a common isozyme. A strong negative linear correlation of glyburide or gliclazide metabolite levels and the concentration of berberine confirmed the effect of berberine on the metabolism of sulfonylureas. CONCLUSIONS: The metabolism of sulfonylureas and berberine was affected when these compounds were co-incubated with each other. This may be attributable to competitive binding of the herb and drug to the catalytic sites of the same isozymes.

A method to assess bioavailability of antibiotics in anthropogenic polluted ecosystems by using a bacterial fitness test.

Antibiotics released in the environment exert a selective pressure on the resident microbiota. It is well accepted that the mere measurement of antibiotics does not reflect the actual bioavailability. In fact, antibiotics can be adsorbed or complexed to particles and/or chemicals in water and soil. Bioavailable concentrations of antibiotics in soil and water are subjected to great uncertainty, therefore biological assays are increasingly recognized as that allow an indirect determination of the residual antibiotic activity. Here we propose how a fitness test for bacteria can be used to qualitatively assess the bioavailability of a specific antibiotic in the environment. The findings show that by using a pair of resistant and sensitive bacterial strains, the resulting fitness can indirectly reflect antibiotic bioavailability. Hence, this test can be used as a complementary assay to other biological and chemical tests to assess bioavailability of antibiotics.

Analysis of procainamide-derivatised heparan sulphate disaccharides in biological samples using hydrophilic interaction liquid chromatography mass spectrometry.

Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6-4.9 ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated ∆UA-GlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biology. Graphical abstract Extraction and HILIC UPLC-MS analysis of procainamide-labelled heparan sulphate disaccharides.

Heparan sulfate disaccharide measurement from biological samples using pre-column derivatization, UPLC-MS and single ion monitoring.

Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 µg GAG. Limits of detection and quantitation were 0.02-0.15 and 0.07-0.31 µg/ml respectively. We have applied this method to the novel analysis of disaccharide levels extracted from heparan sulfate and human cancer cell lines. Heparan sulfate disaccharides extracted from biological samples following actinase and heparinase incubation and derivatized using reductive amination with 2-aminoacridone. Derivatized disaccharides were analyzed used UPLC-MS with single ion monitoring. Eight HS disaccharide subunits were separated and quantified from HS and cell lines in eleven minutes per sample. In all samples the most abundant subunits present were the unsulfated ΔUA-GlcNAc, ΔUA-GlcNAc,6S and ΔUA,2S-GlcNS,6S. There was considerable variation in the proportions and concentrations of disaccharides between different cell lines. Further studies are needed to examine the significance of these differences.

Detection of the tau protein in human serum by a sensitive four-electrode electrochemical biosensor.

This study presents a novel approach based on a four-electrode electrochemical biosensor for the detection of tau protein - one of the possible markers for the prediction of Alzheimer's disease (AD). The biosensor is based on the formation of stable antibody-antigen complexes on gold microband electrodes covered with a layer of a self-assembled monolayer and protein G. Antibodies were immobilized on the gold electrode surface in an optimal orientation by protein G interaction. Electrochemical impedance spectroscopy was used to analyze impedance change, which revealed a linear response with increasing tau concentrations. The assay is fast (<1h for incubation and measurement) and very sensitive. The limit of quantification for the full-length 2N4R tau protein is 0.03pM, a value unaltered when the assay was processed in bovine serum albumin or human serum. This technology could be adapted for the detection of other biomarkers to provide a multiple assay to identify AD progression in a point of care setting.

Evaluation of expression and function of the H+/myo-inositol transporter HMIT.

BACKGROUND: The phosphoinositide (PIns) signalling pathway regulates a series of neuronal processes, such as neurotransmitter release, that are thought to be altered in mood disorders. Furthermore, mood-stabilising drugs have been shown to inhibit key enzymes that regulate PIns production and alter neuronal growth cone morphology in an inositol-reversible manner. Here, we describe analyses of expression and function of the recently identified H+/myo-inositol transporter (HMIT) investigated as a potential regulator of PIns signalling. RESULTS: We show that HMIT is primarily a neuronal transporter widely expressed in the rat and human brain, with particularly high levels in the hippocampus and cortex, as shown by immunohistochemistry. The transporter is localised at the Golgi apparatus in primary cultured neurones. No HMIT-mediated electrophysiological responses were detected in rat brain neurones or slices; in addition, inositol transport and homeostasis were unaffected in HMIT targeted null-mutant mice. CONCLUSION: Together, these data do not support a role for HMIT as a neuronal plasma membrane inositol transporter, as previously proposed. However, we observed that HMIT can transport inositol triphosphate, indicating unanticipated intracellular functions for this transporter that may be relevant to mood control.

Chronic fluoxetine differentially modulates the hippocampal microtubular and serotonergic system in grouped and isolation reared rats.

Social isolation from weaning in rats produces behavioural and hippocampal structural changes at adulthood. Here, rats were group or isolation reared for eight-weeks. Following the initial four-week period of rearing, fluoxetine (10 mg/kg i.p.) was administered for 28 days. Changes in recognition memory, hippocampal monoamines, and cytoskeletal microtubules were investigated. Isolation-rearing for four- or eight-weeks produced recognition memory deficits that were not reversed by fluoxetine. Eight-weeks of isolation decreased alpha-tubulin acetylation (Acet-Tub) and the tyrosinated/detyrosinated alpha-tubulin ratio (Tyr/Glu-Tub), suggesting major alterations in microtubule dynamics and neuronal plasticity. In grouped rats, fluoxetine decreased Acet-Tub without changes in Tyr/Glu-Tub. In isolates, fluoxetine did not affect Acet-Tub but increased Tyr/Glu-Tub. Finally, fluoxetine altered serotonin metabolism in grouped, but not in isolated animals. Therefore, isolation-rearing changes the hippocampal responses of the serotonergic and microtubular system to fluoxetine. These findings show that early-life experience induces behavioural changes paralleled by alterations in cytoskeletal and neurochemical functions.

Effects of N-acetylaspartylglutamate (NAAG) at group II mGluRs and NMDAR.

A group II metabotropic glutamate receptor (mGluR) agonist was recently reported to be clinically efficacious against symptoms of schizophrenia [Patil, S.T., Zhang, L., Martenyi, F., Lowe, S.L., Jackson, K.A., Andreev, B.V., Avedisova, A.S., Bardenstein, L.M., Gurovich, I.Y., Morozova, M.A., Mosolov, S.N., Neznanov, N.G., Reznik, A.M., Smulevich, A.B., Tochilov, V.A., Johnson, B.G., Monn, J.A., Schoepp, D.D., 2007. Activation of mGlu2/3 receptors as a new approach to treat schizophrenia: a randomized phase 2 clinical trial. Nature Med 13, 1102-1107]. The endogenous neuropeptide N-acetylaspartylglutamate (NAAG) has been described as an agonist at mGluR2 and mGluR3 [Wroblewska, B., Wroblewski, J.T., Pshenichkin, S., Surin, A., Sullivan, S.E., Neale, J.H., 1997. N-acetylaspartylglutamate selectively activates mGluR3 receptors in transfected cells. J. Neurochem. 69, 174-181; Cartmell, J., Adam, G., Chaboz, S., Henningsen, R., Kemp, J.A., Klingelschmidt, A., Metzler, V., Monsma, F., Schaffhauser, H., Wichmann, J., Mutel, V., 1998. Characterization of [3H]-(2S,2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl)glycine ([3H]-DCG IV) binding to metabotropic mGlu2 receptor-transfected cell membranes. Br. J. Pharmacol. 123, 497-504] and is degraded by the enzyme glutamate carboxypeptidase II (also known as N-acetyl-alpha-linked acidic dipeptidase or NAALADase). Hence, elevating the concentration of endogenous NAAG by inhibition of NAALADase represents a potential strategy for the treatment of schizophrenia via group II mGluR activation. We therefore investigated the activity of NAAG at both rat native and human recombinant mGluRs. We found that NAAG had no effect on synaptic transmission at the medial perforant pathway inputs to the rat dentate gyrus which is known to be sensitive to group II mGluR activation. We proceeded to examine the effects of NAAG at human recombinant mGluR2 and mGluR3 in a cellular G protein-activated K+ channel electrophysiology assay. Furthermore, due to discrepancies in the literature concerning the activity of NAAG at the N-methyl-d-aspartate receptor [NMDAR; Westbrook, G.L., Mayer, M.L., Namboodiri, M.A., Neale, J.H., 1986. High concentrations of N-acetylaspartylglutamate (NAAG) selectively activate NMDA receptors on mouse spinal cord neurons in cell culture. J. Neurosci. 6, 3385-3392; Losi, G., Vicini, S., Neale, J., 2004. NAAG fails to antagonize synaptic and extrasynaptic NMDA receptors in cerebellar granule neurons. Neuropharmacology 46, 490-496], we also tested NAAG at NMDARs in rat hippocampal neurons in culture. We found that a purified NAAG preparation had no effect at mGluR2, mGluR3 or NMDAR. Taken together, these findings do not support a rationale for targeting NAALADase and increasing extracellular NAAG levels as a therapeutic strategy for the treatment of schizophrenia.

Fluoxetine administration modulates the cytoskeletal microtubular system in the rat hippocampus.

A number of studies suggest that stressful conditions can induce structural alterations in the hippocampus and that antidepressant drugs may prevent such deficits. In particular, the selective serotonin reuptake inhibitor (SSRI) fluoxetine was more effective in modulating different neuronal plasticity phenomena and related molecules in rat hippocampus. Cytoskeletal microtubule dynamics are fundamental to dendrites and axons remodeling, leading to the hypothesis that fluoxetine may affect the microtubular system. However, despite reports of stress-induced alterations in microtubule dynamics by different stressors, only few studies investigated the in vivo effects of antidepressants on microtubules in specific rat brain regions. The present study investigated the dose-related (1, 5, or 10 mg/kg i.p.) effects of acute and chronic (21 days) treatments with fluoxetine on the ratio of hippocampal alpha-tubulin isoforms which is thought to reflect microtubule dynamics. Western Blot analysis was used to quantify alpha-tubulin isoforms, high-performance liquid chromatography and fluorescence detection was used to measure ex vivo monoamine metabolism. The results showed that acute fluoxetine increased the stable forms acetylated and detyrosinated alpha-tubulin. Conversely, chronic fluoxetine decreased acetylated alpha-tubulin, indicative of increased microtubule dynamics. The neuron-specific Delta2-Tubulin was increased by chronic fluoxetine indicating neuronal involvement in the observed cytoskeletal changes. Although acute and chronic fluoxetine similarly altered serotonin metabolism by inhibition of serotonin reuptake, this showed no apparent correlation to the cytoskeletal perturbations. Our findings demonstrate that fluoxetine administration modulates microtubule dynamics in rat hippocampus. The cytoskeletal effect exerted by fluoxetine may eventually culminate in promoting events of structural neuronal remodeling.

Development and application of a liquid chromatography/tandem mass spectrometric assay for measurement of N-acetylaspartate, N-acetylaspartylglutamate and glutamate in brain slice superfusates and tissue extracts.

A liquid chromatography-tandem mass spectrometric method has been developed for measurement of N-acetylaspartate, N-acetylaspartylglutamate and glutamate. The analytes were separated within 5 min using an anion exchange/reverse phase column. The lower limit of quantification for Glu, NAA and NAAG was found to be 5, 50 and 6 nM, respectively, with a signal-to-noise ratio of 5:1. Using this methodology the basal levels of Glu, NAA and NAAG could be measured consistently in in vitro superfusion samples from rat hippocampus. The assay was also used for measurement of the distribution of Glu, NAA and NAAG in different regions of the rat brain.

High-performance liquid chromatography/tandem mass spectrometric assay for the simultaneous measurement of dopamine, norepinephrine, 5-hydroxytryptamine and cocaine in biological samples.

A rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed for the measurement of dopamine (DA), 5-hydroxytryptamine (5HT) and norepinephrine (NE) in brain microdialysates. The assay has also been utilised for the simultaneous measurement of these neurotransmitters and cocaine in brain dialysates. The neurotransmitters and cocaine were resolved in a single 4-min run using a binary gradient elution profile. The analytes were detected using tandem mass spectrometry in the positive ion electrospray mode. The limits of detection for DA, NE, 5HT and cocaine were 200, 1000, 900 pM and 1 pg ml(-1), respectively.

High-performance liquid chromatography/tandem mass spectrometry assay for the determination of 1-methyl-4-phenyl pyridinium (MPP+) in brain tissue homogenates.

A high-throughput liquid chromatography/tandem mass spectrometry method has been developed for the quantitative assessment of 1-methyl-4-phenylpyridinium (MPP+) in brain tissue samples. This separation is based on reversed phase chromatography using formic acid and acetonitrile as the mobile phase. Using gradient separation conditions, MPP+ was resolved within 5 min and detected using tandem mass spectrometry in the positive ion electrospray mode. The limit of detection for MPP+ was found to be 1 fmol on column with a signal to noise ratio of 3:1. The assay has been used routinely in our laboratory for the measurement of MPP+ levels in brain tissue from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, and can be used to distinguish neuroprotective efficacy and monoamine oxidase inhibition.

Selective antagonism at dopamine D3 receptors enhances monoaminergic and cholinergic neurotransmission in the rat anterior cingulate cortex.

Recent neuroanatomical and functional investigations focusing on dopamine (DA) D(3) receptors have suggested a potential role of this receptor in psychiatric diseases such as schizophrenia and drug dependence. In line with the key role of the prefrontal cortex in psychiatric disorders, the present study aimed at assessing the effects of the acute systemic administration of the selective DA D(3) receptor antagonist SB-277011-A on the in vivo extracellular levels of monoamines (DA, norepinephrine (NE), and serotonin (5-HT)) and acetylcholine (ACh) in the anterior cingulate subregion of the medial prefrontal cortex. The in vivo neurochemical profile of SB-277011-A (10 mg/kg, i.p.) in the anterior cingulate cortex was compared with both typical and atypical antipsychotics including clozapine (10 mg/kg, s.c.), olanzapine (10 mg/kg, s.c.), sulpiride (10 mg/kg, s.c.), and haloperidol (0.5 mg/kg, s.c.). The acute administration of SB-277011-A, clozapine, and olanzapine produced a significant increase in extracellular levels of DA, NE, and ACh without affecting levels of 5-HT. Sulpiride also significantly increased extracellular DA, but with a delayed onset over SB-277011-A, clozapine, and olanzapine. In contrast, haloperidol failed to alter any of the three monoamines and ACh in the anterior cingulate cortex. These findings add to a growing body of evidence suggesting a differentiation between typical and atypical antipsychotic drugs (APDs) in the anterior cingulate cortex and a role of DA D(3) receptors in desired antipsychotic drug profile. Similar to their effects on DA and NE, SB-277011-A, clozapine, and olanzapine increased extracellular levels of ACh, whereas haloperidol and sulpiride did not alter ACh. The results obtained in the present study provide evidence of the important role of DA D(3) receptors in the effect of pharmacotherapeutic agents that are used for the treatment of psychiatric disorders such as schizophrenia and drug dependence.

Amino acid neurotransmitters: separation approaches and diagnostic value.

Amino acids in the central nervous system can be divided into non-neurotransmitter or neurotransmitter depending on their function. The measurement of these small molecules in brain tissue and extracellular fluid has been used to develop effective treatment strategies for neuropsychiatric and neurodegenerative diseases and for the diagnosis of such pathologies. Here we describe the separation and detection techniques that have been used for the measurement of amino acids at trace levels in brain tissue and dialysates. An overview of the function of amino acid transmitters in the brain is given. In addition, the type of sampling techniques that are used for the determination of amino acid levels in the brain is described.

High-performance liquid chromatography/tandem mass spectrometry assay for the rapid high sensitivity measurement of basal acetylcholine from microdialysates.

A high-throughput liquid chromatography tandem mass spectrometry (LC/MS/MS) method has been developed for the analysis of acetylcholine (ACh) in brain dialysates. This separation of ACh is based on cation exchange chromatography with elution buffer consisting of a mixture of ammonium acetate, ammonium formate and acetonitrile. Using isocratic separation conditions, ACh was resolved within a minute and detected using tandem mass spectrometry in the positive ion electrospray mode. The limit of detection for ACh was found to be 1 fmol on column with a S/N ratio of 3:1. The assay has been used routinely for the measurement of ACh in brain dialysates from awake freely moving rats. Furthermore, separation conditions were modified to allow simultaneous measurement of ACh and the acetylcholine esterase inhibitor, neostigmine.