Analysis of the neurotoxin ß-N-methylamino-L-alanine (BMAA) and isomers in surface water by FMOC derivatization liquid chromatography high resolution mass spectrometry.

The neurotoxin ß-N-methylamino-L-alanine (BMAA), suspected to trigger neurodegenerative diseases, can be produced during cyanobacterial bloom events and subsequently affect ecosystems and water sources. Some of its isomers including ß-amino-N-methylalanine (BAMA), N-(2-aminoethyl) glycine (AEG), and 2,4-diaminobutyric acid (DAB) may show different toxicities than BMAA. Here, we set out to provide a fast and sensitive method for the monitoring of AEG, BAMA, DAB and BMAA in surface waters. A procedure based on aqueous derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) was investigated for this purpose. Under optimized conditions, a small aqueous sample aliquot (5 mL) was spiked with BMAA-d3 internal standard, subjected to FMOC-Cl derivatization, centrifuged, and analyzed. The high-throughput instrumental method (10 min per sample) involved on-line pre-concentration and desalting coupled to ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS). Chromatographic gradient and mobile phases were adjusted to obtain suitable separation of the 4 isomers. The method limits of detection were in the range of 2-5 ng L-1. In-matrix validation parameters including linearity range, accuracy, precision, and matrix effects were assessed. The method was applied to surface water samples (n = 82) collected at a large spatial scale in lakes and rivers in Canada. DAB was found in >70% of samples at variable concentrations (<3-1,900 ng L-1), the highest concentrations corresponding to lake samples in cyanobacterial bloom periods. BMAA was only reported (110 ng L-1) at one HAB-impacted location. This is one of the first studies to report on the profiles of AEG, BAMA, DAB, and BMAA in background and impacted surface waters.

Kainic acid in the seaweed Palmaria palmata (dulse).

Twenty samples of the seaweed Palmaria palmata (dulse) purchased mainly from commercial Internet shops on the European market were analysed by a liquid chromatograph coupled with a tandem mass spectrometer method for the content of kainic acid, a naturally occurring neurotoxic compound in P. palmata. Kainic acid levels in the samples ranged widely from trace levels to approximately 560 µg g-1 dry weight.

Transfer of developmental neurotoxin ß-N-methylamino-l-alanine (BMAA) via milk to nursed offspring: Studies by mass spectrometry and image analysis.

The cyanobacterial non-proteinogenic amino acid ß-N-methylamino-l-alanine (BMAA) is proposed to be involved in the etiology of amyotrophic lateral sclerosis/parkinsonism dementia complex. When administered as single doses to neonatal rats, BMAA gives rise to cognitive and neurodegenerative impairments in the adult animal. Here, we employed mass spectrometry (LC-MS/MS) and autoradiographic imaging to examine the mother-to-pup transfer of BMAA in rats. The results show that unchanged BMAA was secreted into the milk and distributed to the suckling pups. The concentration of BMAA in pup stomach milk and the neonatal liver peaked after 8h, while the concentration in the pup brain increased throughout the study period. About 1 and 6% of the BMAA recovered from adult liver and brain were released following hydrolysis, suggesting that this fraction was associated with protein. No association to milk protein was observed. Injection of rat pups with [methyl-(14)C]-l-BMAA or [carboxyl-(14)C]-l-BMAA resulted in highly similar distribution patterns, indicating no or low metabolic elimination of the methylamino- or carboxyl groups. In conclusion, BMAA is transported as a free amino acid to rat milk and suckling pups. The results strengthen the proposal that mothers' milk could be a source of exposure for BMAA in human infants.

Reduced microglial immunoreactivity for endogenous NMDA receptor agonist quinolinic acid in the hippocampus of schizophrenia patients.

Postmortem and positron emission tomography studies have indicated the pathophysiological involvement of microglial cells in schizophrenia. We hypothesized that the microglial production of quinolinic acid (QUIN), an endogenous N-methyl-d-aspartate receptor (NMDAR) agonist, may be linked to the previously described glutamatergic deficits in the hippocampus of schizophrenia patients. We performed a semi-quantitative assessment of QUIN-immunoreactive microglial cells in schizophrenia patients and matched controls in the CA1, CA2/3, and dentate gyrus (DG) area of the posterior hippocampal formation. Complementary immunostaining of the commonly used microglial surface marker HLA-DR was performed in adjacent histological sections. Fewer QUIN-immunoreactive microglial cells were observed in the CA1 hippocampal subregion of schizophrenia patients compared to controls (left p=0.028, right p=0.018). No significant diagnosis-dependent changes were observed in the CA2/3 and DG regions. These results were controlled for potential confounds by age, duration of disease, autolysis time, psychotropic medication, and hippocampal volume. No diagnosis-related differences were observed for the overall density of microglial cells (HLA-DR expression). Our findings suggest that reduced microglial QUIN content in the hippocampal CA1 region is associated with schizophrenia. We hypothesize that this association may contribute to impaired glutamatergic neurotransmission in the hippocampus of schizophrenia patients.

ß-N-methylamino-L-alanine induces neurological deficits and shortened life span in Drosophila.

The neurotoxic non-protein amino acid, ß-N-methylamino-L-alanine (BMAA), was first associated with the high incidence of Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC) in Guam. Recently, BMAA has been implicated as a fierce environmental factor that contributes to the etiology of Alzheimer's and Parkinson's diseases, in addition to ALS. However, the toxicity of BMAA in vivo has not been clearly demonstrated. Here we report our investigation of the neurotoxicity of BMAA in Drosophila. We found that dietary intake of BMAA reduced life span, locomotor functions, and learning and memory abilities in flies. The severity of the alterations in phenotype is correlated with the concentration of BMAA detected in flies. Interestingly, developmental exposure to BMAA had limited impact on survival rate, but reduced fertility in females, and caused delayed neurological impairment in aged adults. Our studies indicate that BMAA exposure causes chronic neurotoxicity, and that Drosophila serves as a useful model in dissecting the pathogenesis of ALS/PDC.

Detection of the neurotoxin BMAA within cyanobacteria isolated from freshwater in China.

The cyanobacterial neurotoxin, beta-N-methylamino-l-alanine (BMAA), has been suggested as an important environmental factor for neurodegenerative disease such as amyotrophic lateral sclerosis- Parkinsonism dementia complex (ALS/PDC) in Guam. BMAA was detected within the majority of cyanobacterial isolates surveyed in both free and symbiotic cyanobacteria, living in freshwater as well as marine environments. In this study, we report two methods using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) each coupled with a different type of hydrophilic interaction liquid chromatography (HILIC) column to detect BMAA. A third method using AQC-derivatized BMAA was also used for comparison. Axenic cultures of Microcystis aeruginosa and Nostoc sp. isolated from Chinese freshwater were analyzed for both free and protein-bound BMAA at the exponential growth stage. Cultures of two strains of M. aeruginosa collected at four growth stages were also analyzed for the presence of BMAA. BMAA was detected in the Nostoc sp. at very low concentrations (<0.07pmoles on column) only when precolumn AQC derivatization was used. No BMAA was detected in the Chinese derived axenic cultures of Microcystis; detection limits for the LC-ESI-MS and LC-ESI-MS/MS without precolumn derivatization were 10ng and 2pg BMAA on column, respectively. We suggest that cyanobacteria grown under some culture conditions may be relatively free of BMAA.

Intracellular domoic acid production in Pseudo-nitzschia multistriata isolated from the Gulf of Naples (Tyrrhenian Sea, Italy).

Twenty-six Pseudo-nitzschia multistriata cultures were tested for intracellular domoic acid production and fourteen were found to be toxic. Four suboptimal growth conditions were compared with conditions observed to be optimal to explore possible triggers for intracellular domoic acid production. Silica- and phosphate-limitation and low light treatment induced elevated toxin concentrations whereas high temperature appeared to suppress it. Inheritance of the toxin-production ability was investigated by measuring intracellular toxin content in a total of thirty-nine F(1) strains from two different crosses. Results showed radical differences in domoic acid levels among the F(1) offspring from the same parents.

Determination of the neurotoxin BMAA (beta-N-methylamino-L-alanine) in cycad seed and cyanobacteria by LC-MS/MS (liquid chromatography tandem mass spectrometry).

A highly specific method for the analysis of beta-N-methylamino-L-alanine (BMAA) by LC-MS/MS (liquid chromatography tandem mass spectrometry) has been developed and applied for cycad seeds and cyanobacteria. BMAA was analysed as a free fraction or as total BMAA after acidic hydrolysis to release any protein-bound BMAA. Deuterium labelled BMAA was synthesised and used as internal standard. The method comprises HILIC (hydrophilic interaction chromatography) and positive electrospray ionisation of the native compound, i.e. no derivatisation was used. For safe identification five specific product ions (m/z 102, 88, 76, 73 and 44), all derived from a precursor ion of m/z 119 and originating from different parts of the molecule, were detected (typical relative abundance 100%, 16%, 14%, 12% and 22% respectively). Cyanobacteria or muscle tissue was spiked with BMAA (10 to 1000 microg g(-1)) to validate the method (accuracy 95% to 109%, relative standard deviation 1% to 6%). The detection limit for free and total BMAA in tissue was <1 microg g(-1) and <4 microg g(-1) respectively. BMAA was successfully identified and quantified in cycad seeds, whereas previously reported findings of BMAA in samples of cyanobacteria could not be confirmed. Instead, the presence of alpha-,gamma-diamino butyric acid (DAB), an isomer of BMAA, was confirmed in one sample. The possible implications of this finding are discussed.

Fate and availability of glyphosate and AMPA in agricultural soil.

The fate of glyphosate and its degradation product aminomethylphosphonic acid (AMPA) was studied in soil. Labeled glyphosate was used to be able to distinguish the measured quantities of glyphosate and AMPA from the background values since the soil was sampled in a field where glyphosate had been used formerly. After addition of labeled glyphosate, the disappearance of glyphosate and the formation and disappearance of AMPA were monitored. The resulting curves were fitted according to a new EU guideline. The best fit of the glyphosate degradation data was obtained using a first-order multi compartment (FOMC) model. DT(50) values of 9 days (glyphosate) and 32 days (AMPA) indicated relatively rapid degradation. After an aging period of 6 months, the leaching risk of each residue was determined by treating the soil with pure water or a phosphate solution (pH 6), to simulate rain over a non-fertilized or fertilized field, respectively. Significantly larger (p < 0.05) amounts of aged glyphosate and AMPA were extracted from the soil when phosphate solution was used as an extraction agent, compared with pure water. This indicates that the risk of leaching of aged glyphosate and AMPA residues from soil is greater in fertilized soil. The blank soil, to which 252 g glyphosate/ha was applied 21 months before this study, contained 0.81 ng glyphosate/g dry soil and 10.46 ng AMPA/g dry soil at the start of the study. Blank soil samples were used as controls without glyphosate addition. After incubation of the blank soil samples for 6 months, a significantly larger amount of AMPA was extracted from the soil treated with phosphate solution than from that treated with pure water. To determine the degree of uptake of aged glyphosate residues by crops growing in the soil, (14)C-labeled glyphosate was applied to soil 6.5 months prior to sowing rape and barley seeds. After 41 days, 0.006 +/- 0.002% and 0.005 +/- 0.001% of the applied radioactivity was measured in rape and barley, respectively.

Effective determination method for a cyanobacterial neurotoxin, beta-N-methylamino-L-alanine.

We developed a simple and effective analysis procedure that includes pretreatment and determination methods for beta-N-methylamino-l-alanine (BMAA), a cyanobacterial neurotoxin. BMAA may be produced by all known groups of cyanobacteria living in freshwater as well as marine environments. In this paper, we report a novel determination method for BMAA. A cation-exchange resin was effective for the selective concentration of BMAA from cyanobacterial extracts and yielded a high recovery rate. Moreover, liquid chromatography (LC)-electrospray ionization mass spectrometry with a hydrophilic LC column was effective for determining BMAA levels. The quantitation limit for BMAA based on selected ion monitoring (SIM) was determined as 0.5 ng at a signal/noise ratio of 5.

Effect of kindled seizures on rat behavior in water Morris maze test and amino acid concentrations in brain structures.

The effects of kindled seizures elicited by repeated pentetetrazole (PTZ) injections, on learning and memory in the Morris water maze test and on concentration of brain amino acids, were examined in rats. It was found that kindled seizures (a model of temporal lobe epilepsy) produced a profound decrease in learning and memory accompanied by a selective and long-lasting decrease in hippocampal and striatal concentration of glutamate, glycine and alanine in the striatum (ex vivo measurement). The concentrations of histamine, serine and gamma-aminobutyric acid (GABA) were not selectively affected by kindling. Alower concentration of glutamate and N-methyl-D-aspartate (NMDA) receptor co-agonists in the striatum (glycine and alanine) indicates the general malfunction of the brain glutamatergic system. It is suggested that a selective decrease in hippocampal glutamate concentration may account for deterioration in learning and memory processes in kindled rats, considering the important role of this neurotransmitter in the cognitive processes (e.g. in the long-term potentiation), and the key contribution of the hippocampus to the spatial memory. The intrinsic mechanisms of the reported behavioral effects may involve neuronal damage in the brain limbic structures, secondary to seizure-induced ischemia and hypoxia.

Analysis of hallucinogenic constituents in Amanita mushrooms circulated in Japan.

The constituents of seven mushrooms sold as Amanita muscaria or Amanita pantherina (five A. muscaria and two A. pantherina) and four "extracts purported to contain A. muscaria" products that are currently circulated in Japan were determined. All mushroom samples were identified as A. muscaria or A. pantherina by macroscopic and microscopic observation. The dissociative constituents, ibotenic acid (IBO) and muscimol (MUS), were extracted with 70% methanol twice and determined by gas chromatography/mass spectrometry. The IBO (as the hydrate)/MUS contents were in the range of <10-2845ppm/46-1052ppm in the cap of A. muscaria and 188-269ppm/1554-1880ppm in the cap of A. pantherina. In the caps, these compounds had a tendency to be more concentrated in the flesh than in the cuticle. On the other hand, the IBO/MUS contents in the stem were far lower than in the caps. In the "extracts purported to contain A. muscaria" products, IBO/MUS were detected below the lower limit of calibration curve (<10ppm/<25ppm) or not detected. However, these samples contained other psychoactive compounds, such as psychoactive tryptamines (5-methoxy-N,N-diisopropyltryptamine and 5-methoxy-N,N-dimethyltryptamine), reversible monoamine oxidase inhibitors (harmine and harmaline) and tropane alkaloids (atropine and scopolamine), which were not quantified. This is the first report of the chemical analysis of Amanita mushrooms that are circulated in the drug market.

Glutamate receptor incorporated in a mixed hybrid bilayer lipid membrane array, as a sensing element of a biosensor working under flowing conditions.

The realization of a reliable receptor biosensor requires stable, long-lasting, reconstituted biomembranes able to supply a suitable biomimetic environment where the receptor can properly work after incorporation. To this end, we developed a new method for preparing stable biological membranes that couple the biomimetic properties of BLMs (bilayer lipid membranes) with the high stability of HBMs (hybrid bilayer membranes); this gives rise to an innovative assembly, named MHBLM (mixed hybrid bilayer lipid membrane). The present work deals with the characterization of biosensors achieved by embedding an ionotropic glutamate receptor (GluR) on MHBLM. Thanks to signal (transmembrane current) amplification, which is typical of natural receptors, the biosensor here produced detects glutamate at a level of nmol L(-1). The transmembrane current changes linearly vs glutamate up to 100 nmol L(-1), while the limit of detection is 1 nmol L(-1). In addition, the biosensor response can be modulated both by receptor agonists (glycine) and antagonists (Mg(2+)) as well, and by exploiting the biosensor response, the distribution of different kinds of ionotropic GluR present in the purified sample, and embedded in MHBLM, was also evaluated. Finally, one of the most important aspects of this investigation is represented by the high stability of the biomimetic system, which allows the use of biosensor under flowing conditions, where the solutions flow on both biomembrane faces.

Low sensitivity of retina to AMPA-induced calcification.

Glutamate is involved in most CNS neurodegenerative diseases. In particular, retinal diseases such as retinal ischemia, retinitis pigmentosa, and diabetic retinopathy are associated with an excessive synaptic concentration of this neurotransmitter. To gain more insight into retinal excitotoxicity, we carried out a dose-response study in adult rats using alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), a glutamate analogue. AMPA intraocular injections (between 0.27 and 10.8 nmol) caused no morphologic modification, but a 10.8 + 21 nmol double injection in a 10-day interval produced a lesion characterized by discrete neuronal loss, astroglial and microglial reactions, and calcium precipitation. Abundant calcium deposits similar to those present in rat and human brain excitotoxicity or hypoxia-ischemia neurodegeneration were detected by alizarin red staining within the retinal surface and the optic nerve. Glial reactivity, associated normally with astrocytes in the nerve fiber, was assessed in Müller cells. GABA immunoreactivity was detected not only in neuronal elements but also in Müller cells. In contrast to the high vulnerability of the brain to excitotoxin microinjection, AMPA-induced retinal neurodegeneration may provide a useful model of low central nervous system sensitivity to excitotoxicity.

A biosensing system based on extracellular potential recording of ligand-gated ion channel function overexpressed in insect cells.

We have used outer cell potential measurement to record agonist-dependent cellular responses in cells engineered to express ligand-gated ion channels and grown on a microelectrode surface. Application of glutamate, a natural agonist, induced a complex and robust potentiometric response in cells expressing homomeric GluR-D glutamate receptor, but not in nonexpressing control cells. The response consisted of an initial decrease in outer potential followed by a transient increase and was not obtained for other amino acids devoid of agonist activity at glutamate receptors. Furthermore, the pharmacological agonist of the GluR-D receptor, kainate, also produced the potentiometric response whereas 6-cyano-7-nitroquinoxaline-2,3-dione, a competitive antagonist, was not active in itself but attenuated the responses to glutamate. The time course of the measured changes was slow, which may be partially due to the ligand being applied by free diffusion but may also reflect a contribution by secondary changes in the behavior of the cells. This novel approach should be applicable to other ligand-gated ion channels and holds promise as a cell-based biosensor for high-throughput drug screening and other applications.

Screening of receptor antagonists using agonist-activated patch clamp detection in chemical separations.

We present a capillary electrophoresis-patch clamp detection system optimized for screening of antagonists and inhibitors of ligand-gated ion channels. In this system, highly selective receptor agonists are delivered through the electrophoresis capillary to the cell surface where they continuously activate a receptor, resulting in increased steady-state transmembrane currents. Thus, receptor selection and biosensor functionality is simply achieved by selection of an appropriate agonist. The antagonists are fractionated in the same electrophoresis capillary and inhibit the agonist-evoked response, resulting in transiently decreased steady-state transmembrane currents. Specifically, a mixture containing 6-cyano-7-nitroquinoxaline-2,3-dione, that reversibly blocks alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and kainate receptors, and 6,7-dichloro-3-hydroxy-2-quinoxaline-carboxylate, a broad-spectrum glutamate receptor antagonist, were separated and detected by kainate-activated patch-clamped interneurons freshly dissociated from rat brain olfactory bulb. In addition, Mg2+ that reversibly blocks the N-methyl-D-aspartate receptor in a voltage-dependent way was detected using the same cell detector system when activated by N-methyl-D-aspartate and the co-agonist glycine. The presented method offers new possibilities for drug screening and for identifying endogenous receptor antagonists and to determine their mode of action on any ionotropic receptor system of interest.

In vivo inhibition of veratridine-evoked release of striatal excitatory amino acids by the group II metabotropic glutamate receptor agonist LY354740 in rats.

In vivo microdialysis in freely moving rats was used to investigate the presynaptic mechanisms by which LY354740, a novel, potent, selective, and systemically active agonist for group II metabotropic glutamate receptors (mGluRs), alters glutamate neuronal transmission. Basal levels of glutamate and aspartate in striatal dialysates of LY354740 (10 mg/kg i.p.)-treated animals were not significantly different from the saline-treated control animals. In the saline treated controls, veratridine (100 microM) induced a 6-fold increase in glutamate and 9-fold increase in aspartate. However, following LY354740 administration the veratridine-evoked release of glutamate and aspartate was completely prevented. These data demonstrate that LY354740 blocks the evoked release of endogenous excitatory amino acids, and indicate a role for group II mGluRs in presynaptic modulation of glutamate neuronal transmission in vivo. Ireland Ltd.