Cyanobacterial Blooms and the Occurrence of the neurotoxin beta-N-methylamino-L-alanine (BMAA) in South Florida Aquatic Food Webs

Recent studies demonstrate that most cyanobacteria produce the neurotoxin beta-N-methylamino-L-alanine (BMAA) and that it can biomagnify in at least one terrestrial food chain. BMAA has been implicated as a significant environmental risk in the development of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis (ALS). We examined several blooms of cyanobacteria in South Florida, and the BMAA content of resident animals, including species used as human food. A wide range of BMAA concentrations were found, ranging from below assay detection limits to approximately 7000 μg/g, a concentration associated with a potential long-term human health hazard.     

Microcystins,BMAA and BMAA isomers in 100-year-old Antarctic cyanobacterial mats collected during Captain R.F. Scott’s Discovery Expedition

Microcystins (MCN), β-N-methylamino-L-alanine (BMAA) and anatoxin-a were investigated in Antarctic cyanobacterial mats collected from Ross Island and the McMurdo Ice Shelf, East Antarctica during Captain Scott’s ‘Discovery’ National Antarctic Expedition (1901–1904). Ultra-performance liquid chromatography-photodiode array detection (UPLC-PDA) and tandem mass spectrometry (MS/MS) analysis were used to quantify the cyanotoxins in seven cyanobacterial mat samples. MCNs were identified in six of the mat samples at concentrations from 0.5 to 16.1 µg?g^–1 dry weight. BMAA was found in one sample (528 ng?g^–1 dry weight, total BMAA), as well as two BMAA isomers, 2,4-diaminobutyric acid (DAB) and N-(2-aminoethyl) glycine (AEG) in six samples up to 6.56 and 6.79 μg?g^–1 dry weight, respectively. No anatoxin-a was detected. The findings confirm that MCNs, BMAA and BMAA isomers are preserved under dry herbarium conditions. The ‘Discovery’ cyanobacterial mat samples represent the oldest polar cyanobacterial samples found to contain cyanotoxins to date and provide new baseline data for cyanotoxins in Antarctic freshwater cyanobacterial mats from prior to human activity in Antarctica, the development of the ozone hole and current levels of climatic change.     

Evaluation of a Commercial Enzyme Linked Immunosorbent Assay (ELISA) for the Determination of the Neurotoxin BMAA in Surface Waters

The neurotoxin β-N-methylamino-L-alanine (BMAA) is suspected to play a role in Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Because BMAA seems to be produced by cyanobacteria, surface waters are screened for BMAA. However, reliable analysis of BMAA requires specialized and expensive equipment. In 2012, a commercial enzyme-linked immunosorbent assay (ELISA) for determination of BMAA in surface waters was released. This kit could enable fast and relatively cheap screening of surface waters for BMAA. The objective of this study was to determine whether the BMAA ELISA kit was suitable for the determination of BMAA concentrations in surface waters. We hypothesised that the recovery of spiked samples was close to 100% and that the results of unspiked sample analysis were comparable between ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. However, we found that recovery was higher than 100% in most spiked samples, highest determined recovery was over 400%. Furthermore, the ELISA gave a positive signal for nearly each tested sample while no BMAA could be detected by LC-MS/MS. We therefore conclude that in its current state, the kit is not suitable for screening surface waters for BMAA.     

The non-protein amino acid β-N-methylamino-l-alanine in Portuguese cyanobacterial isolates

The tailor made amino acid β-N-methyl-amino-L-alanine (BMAA) is a neurotoxin produced by cyanobacteria. It has been associated with certain forms of progressive neurodegenerative disease, including sporadic Amyotrophic Lateral Sclerosis and Alzheimer's disease. Some different reports of BMAA in cyanobacterial blooms from lakes, reservoirs, and other water resources have been made by different investigators. We here report the detection of BMAA of both free and protein-bound produced by cyanobacteria, belonging to the Chroococcales, Oscillatoriales and Nostocales ordered. We use a rapid and sensitive HPLC-FD method that utilizes methanol elution and the Waters AQC Tag chemistry. On other hand, we have used three different assay procedures for BMAA extraction from cyanobacteria: Trichloroacetic acid (TCA), Methanol/Acetone and hydrochloric acid (HCl). All assays let successfully detect BMAA in all cyanobacteria samples analyzed. Nevertheless, with TCA and HCl extraction procedures the highest BMAA values, for free as well as protein-bound BMAA were detected. BMAA content could not be related to the taxonomy of the isolates or to their geographical origin, and no correlation between free and protein-bound BMAA concentrations were observed within or between taxonomic groups. These data offer confirmation of the taxonomic and geographic ubiquity of BMAA from naturally occurring populations of cyanobacteria, for the first time reported for estuaries.     

Bestimmung von Zearalenon in Speiseölen mit GPC und LC-ESI-MS/MS

A new method for the determination of zearalenone in edible oils with size exclusion chromatography (SEC) followed by LC-MS/MS as well as HPLC-FLD was developed and validated. By using the LC-MS/MS determination no further clean up step is necessary after the SEC. The correlation coefficient of 0.999 for the two detection systems is acceptable. In this research 77 edible oils were analyzed. The mean average value of 38 corn germ oils was 169 μg/kg, the maximum value amounted up to 921 μg/kg.     

The natural non-protein amino acid N-β-methylamino-l-alanine (BMAA) is incorporated into protein during synthesis

N-β-methylamino-l-alanine (BMAA) is an amino acid produced by cyanobacteria and accumulated through trophic levels in the environment and natural food webs. Human exposure to BMAA has been linked to progressive neurodegenerative diseases, potentially due to incorporation of BMAA into protein. The insertion of BMAA and other non-protein amino acids into proteins may trigger protein misfunction, misfolding and/or aggregation. However, the specific mechanism by which BMAA is associated with proteins remained unidentified. Such studies are challenging because of the complexity of biological systems and samples. A cell-free in vitro protein synthesis system offers an excellent approach for investigation of changing amino acid composition in protein. In this study, we report that BMAA incorporates into protein as an error in synthesis when a template DNA sequence is used. Bicinchoninic acid assay of total protein synthesis determined that BMAA effectively substituted for alanine and serine in protein product. LC–MS/MS confirmed that BMAA was selectively inserted into proteins in place of other amino acids, but isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) did not share this characteristic. Incorporation of BMAA into proteins was significantly higher when genomic DNA from post-mortem brain was the template. About half of BMAA in the synthetic proteins was released with denaturation with sodium dodecylsulfonate and dithiothreitol, but the remaining BMAA could only be released by acid hydrolysis. Together these data demonstrate that BMAA is incorporated into the amino acid backbone of proteins during synthesis and also associated with proteins through non-covalent bonding.     

Co-occurrence of beta-N-methylamino-L-alanine, a neurotoxic amino acid with other cyanobacterial toxins in British waterbodies, 1990-2004

The neurotoxic amino acid, β- N -methylamino- l -alanine, was found to be present in all of 12 analysed samples of cyanobacterial blooms, scums and mats, which had been collected in seven years between 1990 and 2004 inclusive and stored at −20°C. BMAA identification was by high performance liquid chromatography with fluorescence detection and by triple quadrapole mass spectrometry after derivatization. The samples originated from 11 freshwater lakes and 1 brackish waterbody, used either for drinking water, recreation, or both. BMAA was present at between 8 and 287 μg g⁻¹ cyanobacterial dry weight and was present as both the free amino acid and associated with precipitated proteins. Ten of the samples contained additional cyanotoxins (including microcystins, anatoxin-a, nodularin and saxitoxin) at the time of sample collection. Five of the samples were associated with animal deaths, attributable at the time of sample collection, to microcystins, nodularin or anatoxin-a. The data demonstrate the presence of BMAA by high performance liquid chromatography and mass spectrometry in a diverse range of cyanobacterial bloom samples from high resource waterbodies. Furthermore, samples collected over several years shows that BMAA can co-occur with other known cyanotoxins in such waterbodies. Health risk assessment of cyanobacterial BMAA in waterbodies is suggested.     

Detection and Pharmacokinetics of Alginate Oligosaccharides in Mouse Plasma and Urine after Oral Administration by a Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS) Method

A sensitive and simple liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the detection of alginate oligosaccharides (AOs) in mouse plasma and urine after oral administration. In an AO mixture, dimer, trimer, and tetramer were detected by LC-MS/MS equipped with an anion-exchange column with extremely high sensitivity. By this method, we detected certain levels of AOs in samples prepared from mouse plasma and urine after a single oral administration of the AO mixture. Based on a calibration curve made with an AO trimer peak area as a standard, the maximum plasma and urine concentrations of AOs were estimated to be 24.5 μg/ml at 5 min and 425.5 μg/ml at 30 min, respectively. These results suggest that the LC-MS/MS method is well suited to pharmacokinetic analysis of AOs in an in vivo system, and that some of orally administered AOs, at least from dimer to tetramer, are absorbed by digestive organs promptly, and that unaltered, these oligomers were excreted into an urine after a single oral administration to a mouse.     

神经毒素BMAA在淡水池塘水体中的健康风险及调控技术

为掌握中国常见淡水养殖生态系统中神经毒素β-N-甲氨基-L-丙氨酸BMAA的污染水平,文章选取典型淡水养殖池塘的水体、底泥及6种水产品(河蚬、铜锈环棱螺、日本沼虾、中华绒螯蟹、青鱼和鲫)进行BMAA的含量检测,在此基础上开展BMAA对人体的健康风险评估.同时采用L-半胱氨酸修饰后的氧化石墨烯为载体,结合化感物质"没食子...     

Is there a role for naturally occurring cyanobacterial toxins in neurodegeneration?: The beta-N-methylamino-l-alanine (BMAA) paradigm

The naturally occurring, non-essential amino acid beta-N-methylamino-l-alanine (BMAA) has been recently found in high concentrations in brain tissues of patients with tauopathies such as the Amyotrophic Lateral Sclerosis-Parkinsonism-Dementia Complex (ALS/PDC) in the South Pacific island of Guam and in a small number of Caucasian, North American patients with sporadic Alzheimer's disease. BMAA is produced by cyanobacteria that are present in all conceivable aquatic and/or terrestrial ecosystems and may be accumulated in living tissues in free and protein-bound forms through the process of biomagnification. Although its role in human degenerative disease is highly debated, there is mounting evidence in support of the neurotoxic properties of BMAA that may be mediated via mechanisms involving among others the regulation of glutamate. Glutamate-related excitotoxicity is among the most prominent factors in the etiopathogenesis of human neurodegenerative diseases. Due to the wide geographical distribution of cyanobacteria and the possible implications of BMAA neurotoxic properties in public health more research towards this direction is warranted.     

Diatoms: A Novel Source for the Neurotoxin BMAA in Aquatic Environments

Amyotrophic lateral sclerosis (ALS) or Lou Gehrig’s disease is a neurological disorder linked to environmental exposure to a non-protein amino acid, β-N-methylamino-L-alanine (BMAA). The only organisms reported to be BMAA-producing, are cyanobacteria – prokaryotic organisms. In this study, we demonstrate that diatoms – eukaryotic organisms – also produce BMAA. Ultra-high-performance liquid chromatography coupled with tandem mass spectrometry revealed the occurrence of BMAA in six investigated axenic diatom cultures. BMAA was also detected in planktonic field samples collected on the Swedish west coast that display an overrepresentation of diatoms relative to cyanobacteria. Given the ubiquity of diatoms in aquatic environments and their central role as primary producers and the main food items of zooplankton, the use of filter and suspension feeders as livestock fodder dramatically increases the risk of human exposure to BMAA-contaminated food.     

Cyanotoxins in inland lakes of the United States: Occurrence and potential recreational health risks in the EPA National Lakes Assessment 2007

A large nation-wide survey of cyanotoxins (1161 lakes) in the United States (U.S.) was conducted during the EPA National Lakes Assessment 2007. Cyanotoxin data were compared with cyanobacteria abundance- and chlorophyll-based World Health Organization (WHO) thresholds and mouse toxicity data to evaluate potential recreational risks. Cylindrospermopsins, microcystins, and saxitoxins were detected (ELISA) in 4.0, 32, and 7.7% of samples with mean concentrations of 0.56, 3.0, and 0.061 μg/L, respectively (detections only). Co-occurrence of the three cyanotoxin classes was rare (0.32%) when at least one toxin was detected. Cyanobacteria were present and dominant in 98 and 76% of samples, respectively. Potential anatoxin-, cylindrospermopsin-, microcystin-, and saxitoxin-producing cyanobacteria occurred in 81, 67, 95, and 79% of samples, respectively. Anatoxin-a and nodularin-R were detected (LC/MS/MS) in 15 and 3.7% samples (n = 27). The WHO moderate and high risk thresholds for microcystins, cyanobacteria abundance, and total chlorophyll were exceeded in 1.1, 27, and 44% of samples, respectively. Complete agreement by all three WHO microcystin metrics occurred in 27% of samples. This suggests that WHO microcystin metrics based on total chlorophyll and cyanobacterial abundance can overestimate microcystin risk when compared to WHO microcystin thresholds. The lack of parity among the WHO thresholds was expected since chlorophyll is common amongst all phytoplankton and not all cyanobacteria produce microcystins.     

Demonstrated transfer of cyanobacteria and cyanotoxins along a freshwater-marine continuum in France

The frequency of cyanobacterial proliferations in fresh waters is increasing worldwide and the presence of associated cyanotoxins represent a threat for ecosystems and human health. While the occurrence of microcystin (MC), the most widespread cyanotoxin, is well documented in freshwaters, only few studies have examined its occurrence in estuarine waters. In this study we evaluated the transfer of cyanobacteria and cyanotoxins along a river continuum from a freshwater reservoir through an interconnecting estuary to the coastal area in Brittany, France. We sampled regularly over 2 years at 5 stations along the river continuum and analysed for phytoplankton and cyanotoxins, together with physico-chemical parameters. Results show that cyanobacteria dominated the phytoplanktonic community with high densities (up to 2 × 10⁶ cells mL⁻¹) at the freshwater sites during the summer and autumn periods of both years, with a cell transfer to estuarine (up to 10⁵ cells mL⁻¹) and marine (2 × 10³ cells mL⁻¹) sites. While the temporal variation in cyanobacterial densities was mainly associated with temperature, spatial variation was due to salinity while nutrients were non-limiting for cyanobacterial growth. Cyanobacterial biomass was dominated by several species of Microcystis that survived intermediate salinities. Intracellular MCs were detected in all the freshwater samples with concentrations up to 60 μg L⁻¹, and more intermittently with concentrations up to 1.15 μg L⁻¹, at the most upstream estuarine site. Intracellular MC was only sporadically detected and in low concentration at the most downstream estuarine site and at the marine outlet (respectively <0.14 μg L^-1 and <0.03 μg L⁻¹). Different MC variants were detected with dominance of MC-LR, RR and YR and that dominance was conserved along the salinity gradient. Extracellular MC contribution to total MC was higher at the downstream sites in accordance with the lysing of the cells at elevated salinities. No nodularin (NOD) was detected in the particulate samples or in the filtrates.     

An LC-MS/MS procedure for the quantification of naproxen in human plasma: development, validation, comparison with other methods, and application to a pharmacokinetic study

A sensitive, precise and accurate quantitative LC-MS/MS method for the measurement of naproxen in human plasma was developed and completely validated according to current FDA and EMA guidelines. The new method employs acetonitrile protein precipitation for sample preparation and uses ketoprofen as the internal standard. Suitability of the new assay was assessed in comparison with 36 reported bioanalytical assays and the pharmacokinetic results obtained by the new method were compared to 11 reported studies in humans. The principal advantage of this LC-MS/MS method is the simultaneous achievement of high absolute recovery (90.0±3.6%), acceptable sensitivity (lower limit of quantitation of 0.100 μg/mL), high inter-day precision (CV≤9.4%), high analytical recovery (between 94.4 and 103.1%), and excellent linearity over the concentration range 0.100-50.0 μg/mL (r(2)≥0.998) combined with a short run time of only 2 min.     

2种检测灯盏花素血药浓度方法的比较

目的：建立测定Beagle犬血浆中灯盏花素浓度的反相高效液相色谱法和液相-质谱联用法,并将其进行多参数的两两比较。方法：采用液-液萃取法处理血清,反相高效液相色谱法测定,流动相为甲醇-水（pH3．0）=42：58,EclipseXDB-C18为固定相;将同样的样品用液相-质谱法开展平行检测;将所测得的数据做两两对比,采用SPSS统计软件进行分析。结果：2种方法测定的数据具有良好的相关性（r=0．982,P〉0．05）;最低检测限前者为0．05μg／mL,后者为0．01μg／mL,反相高效液相色谱法的最低检测限和灵敏度不及液相-质谱联用法。结论：反相高效液相色谱法可用于灯盏花素毒代动力学研究,与液相-质谱联用法检测结果无显著差异,且费用较低,不失为-种低成本且行之有效的检测方法。     

Quantification of endocannabinoids in biological systems by chromatography and mass spectrometry: a comprehensive review from an analytical and biological perspective

The endocannabinoids anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2AG) are physiologically occurring, biologically active compounds on CB(1) and CB(2) receptors with multiple physiological functions. AEA and 2AG have been identified and quantified in many mammalian biological fluids and tissues, such as human plasma, adipocytes, tissues and tissue microdialysates, at concentrations in the picomolar-to-nanomolar range under basal conditions. In this article, recently published chromatographic and mass spectrometric analytical methods, i.e., HPLC with fluorescence or ultraviolet detection, LC-MS, LC-MS/MS, GC-MS and GC-MS/MS, are reviewed and discussed, notably from the quantitative point of view. We focus on and emphasize the particular importance of blood sampling, sample storage and work-up including solvent and solid-phase extraction and derivatization procedures, matrix-effects, and stability of analytes. As 2AG spontaneously isomerizes to its CB(1)/CB(2) receptors biologically inactive 1-arachidonoyl glycerol (1AG) by acyl migration, this phenomenon and its particular importance for accurate quantification of 2AG are discussed in detail. Due to the electrical neutrality of AEA and 2AG their solvent extraction by toluene offers the least matrix-effect and minimum isomerization. LC-MS/MS is the most frequently used analytical technique for AEA and 2AG. At present, the utility of the GC-MS/MS methodology seems to be limited to AEA measurement in human plasma, bronchoalveolar liquid (BAL) and microdialysate samples. Despite great instrumental advances in the LC-MS/MS methodology, sampling and sample treatment remains one of the most crucial analytical steps in 2AG analysis. Extension of the LC-MS/MS methodology, for instance to microdialysate and BAL samples from clinical studies, is a big analytical challenge in endocannabinoid analysis in clinical settings. Currently available LC-MS/MS and GC-MS/MS methods should be useful to investigate the metabolism of AEA and 2AG beyond hydrolysis, i.e., by β- and ω-oxidation pathways.     

Is there a role for naturally occurring cyanobacterial toxins in neurodegeneration?: The beta-N-methylamino-l-alanine (BMAA) paradigm

The naturally occurring, non-essential amino acid beta-N-methylamino-L-alanine (BMAA) has been recently found in high concentrations in brain tissues of patients with tauopathies such as the Amyotrophic Lateral Sclerosis-Parkinsonism-Dementia Complex (ALS/PDC) in the South Pacific island of Guam and in a small number of Caucasian, North American patients with sporadic Alzheimer's disease. BMAA is produced by cyanobacteria that are present in all conceivable aquatic and/or terrestrial ecosystems and may be accumulated in living tissues in free and protein-bound forms through the process of biomagnification. Although its role in human degenerative disease is highly debated, there is mounting evidence in support of the neurotoxic properties of BMAA that may be mediated via mechanisms involving among others the regulation of glutamate. Glutamate-related excitotoxicity is among the most prominent factors in the etiopathogenesis of human neurodegenerative diseases. Due to the wide geographical distribution of cyanobacteria and the possible implications of BMAA neurotoxic properties in public health more research towards this direction is warranted.     

A new strategy for ionization enhancement by derivatization for mass spectrometry

Liquid chromatography-mass spectrometry (LC-MS) using atmospheric pressure ionization is drastically different from hitherto available analytical methods used to detect polar analytes. The electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources of MS have contributed to the advancement of LC-MS and LC-MS/MS techniques for the analysis of biological samples. However, one major obstacle is the weak ionization of some analytes in the ESI and APCI techniques. In this review, we introduce high-sensitivity methods using several derivatization reagents for ionization enhancement. We also present an overview of chemical derivatization methods that have been applied to small molecules, such as amino acids and steroids, in biological samples.     

Development of a high performance liquid chromatography method and a liquid chromatography-tandem mass spectrometry method with the pressurized liquid extraction for the quantification and confirmation of sulfonamides in the foods of animal origin

The residues of sulfonamides (SAs) in the foods of animal origin are of the major concern because they are harmful to the consumer's health and could induce pathogens to develop resistance. Rapid and efficient determination methods are urgently in need. A quantitative high performance liquid chromatography method (HPLC) and a confirmative liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of 18 sulfonamides such as sulfamidinum, sulfanilamide, sulfisomidine, sulfadiazine, sulfapyridine, sulfathiazole, sulfamerazine, sulfadimidine, sulfamethoxypyridazine, sulfamethoxydiazine, sulfisoxazole, sulfachloropyridazine, sulfamethoxazole, sulfamonomethoxine, sulfadoxine, sulfaclozine, sulfadimethoxine, sulfaquinoxaline in the muscles, livers and kidneys of swine, bovine and chicken were developed and validated. The sample preparation procedures included a pressurized liquid extraction (PLE) with acetonitrile conducted at elevated temperature (70°C) and pressure (1400 psi). After clean-up with hydrophilic-lipophilic balance cartridge, the extraction solution was concentrated and analyzed by HPLC and LC-MS/MS analysis. 18 SAs were separated by the HPLC with a Zorbax SB-Aq-C18 column and the mobile phase of methanol/acetonitrile/1% acetic acid with a gradient system. The wavelength of UV for the HPLC detection was set at 285 nm. The LC-MS/MS analysis was achieved with a Hypersil Golden column and the mobile phase of acetonitrile and 0.1% formic acid aqueous solution with two gradient systems. The Limits of detection (LOD) and the limits of quantitation (LOQ) were 3 μg/kg and 10 μg/kg, respectively, for both of the HPLC and LC-MS/MS. Linearity was obtained with an average coefficient of determination (R) higher than 0.9980 over a dynamic range from the LOQ value up to 5000 μg/kg. The recoveries of the methods range from 71.1% to 118.3% with the relative standard derivation less than 13%. The peaks of interest with no interferences were observed throughout the chromatographic run. The sample pretreatment provided efficient extraction and cleanup that enables a sensitive and rugged determination of 18 SAs, the obtained results revealed that PLE, in comparison with other sample preparation methods applied, has significantly higher efficacy for SAs isolation from animal tissues.     

Improving derivatization efficiency of BMAA utilizing AccQ-Tag<Superscript>®</Superscript> in a complex cyanobacterial matrix

Two different assays have been developed and used in order to investigate the optimal conditions for derivatization and detection of acid beta-N-methyl-amino-L-alanine (BMAA) in a cyanobacterial sample. BMAA was extracted from cyanobacterial cultures both from the cytosolic ("free") fraction and in the precipitated ("protein") fraction using a newly developed extraction scheme and the sample matrix was standardized according to protein concentration to ensure the highest possible derivative yield. A rapid and sensitive HPLC method for fluorescence detection of the non-protein amino acid BMAA in cyanobacteria, utilizing the Waters AccQ-Tag chemistry and Chromolith Performance RP-18e columns was developed. Using this new method and utilizing a different buffer system and column than that recommended by Waters, we decreased the time between injections by 75%. The limit of quantification was determined to be 12 nmol and limit of detection as 120 fmol. The linear range was in the range of 8.5 nmol-84 pmol. Accuracy and precision were well within FDA guidelines for bioanalysis.