Skip to main content
SpringerLink
Log in

BMAA extraction of cyanobacteria samples: which method to choose?

  • Research Article
  • Published:
Environmental Science and Pollution Research Aims and scope Submit manuscript

Abstract

β-N-Methylamino-l-alanine (BMAA), a neurotoxin reportedly produced by cyanobacteria, diatoms and dinoflagellates, is proposed to be linked to the development of neurological diseases. BMAA has been found in aquatic and terrestrial ecosystems worldwide, both in its phytoplankton producers and in several invertebrate and vertebrate organisms that bioaccumulate it. LC-MS/MS is the most frequently used analytical technique in BMAA research due to its high selectivity, though consensus is lacking as to the best extraction method to apply. This study accordingly surveys the efficiency of three extraction methods regularly used in BMAA research to extract BMAA from cyanobacteria samples. The results obtained provide insights into possible reasons for the BMAA concentration discrepancies in previous publications. In addition and according to the method validation guidelines for analysing cyanotoxins, the TCA protein precipitation method, followed by AQC derivatization and LC-MS/MS analysis, is now validated for extracting protein-bound (after protein hydrolysis) and free BMAA from cyanobacteria matrix. BMAA biological variability was also tested through the extraction of diatom and cyanobacteria species, revealing a high variance in BMAA levels (0.0080–2.5797 μg g−1 DW).

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Al-Sammak M, Hoagland K, Cassada D, Snow D (2014) Co-occurrence of the cyanotoxins BMAA, DABA and Anatoxin-a in Nebraska reservoirs, fish, and aquatic plants. Toxins 6:488–508

    Article  CAS  Google Scholar 

  • Assmy P, Hernandez-Becerril DU, Montresor M (2008) Morphological variability and life cycle traits of the type species of the diatom genus Chaetoceros, C-dichaeta. J Phycol 44:152–163. doi:10.1111/j.1529-8817.2007.00430.x

    Article  Google Scholar 

  • Banack SA, Cox PA (2003) Distribution of the neurotoxic nonprotein amino acid BMAA in Cycas micronesica. Bot J Linn Soc 143:165–168

    Article  Google Scholar 

  • Banack SA, Johnson HE, Cheng R, Cox PA (2007) Production of the neurotoxin BMAA by a marine cyanobacterium. Mar Drugs 5:180–196

    Article  CAS  Google Scholar 

  • Banack SA et al (2011) Distinguishing the cyanobacterial neurotoxin beta-N-methylamino-L-alanine (BMAA) from other diamino acids. Toxicon 57:730–738. doi:10.1016/j.toxicon.2011.02.005

    Article  CAS  Google Scholar 

  • Baptista MS, Cianca RC, Lopes VR, Almeida CM, Vasconcelos VM (2011) Determination of the non protein amino acid beta-N-methylamino-l-alanine in estuarine cyanobacteria by capillary electrophoresis. Toxicon 58:410–414. doi:10.1016/j.toxicon.2011.08.007

    Article  CAS  Google Scholar 

  • Berntzon L, Erasmie S, Celepli N, Eriksson J, Rasmussen U, Bergman B (2013) BMAA inhibits nitrogen fixation in the cyanobacterium Nostoc sp. PCC 7120. Mar Drugs 11:3091–3108

    Article  CAS  Google Scholar 

  • Booth BC, Larouche P, Bélanger S, Klein B, Amiel D, Mei ZP (2002) Dynamics of Chaetoceros socialis blooms in the north water. Deep-Sea Res Pt II 49:5003–5025. doi:10.1016/S0967-0645(02)00175-3

    Article  CAS  Google Scholar 

  • Bowler C et al (2008) The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456:239–244, http://www.nature.com/nature/journal/v456/n7219/suppinfo/nature07410_S1.html

    Article  CAS  Google Scholar 

  • Cohen SA (2012) Analytical techniques for the detection of alpha-amino-beta-methylaminopropionic acid. Analyst 137:1991–2005. doi:10.1039/c2an16250d

    Article  CAS  Google Scholar 

  • Combes A, El Abdellaoui S, Sarazin C, Vial J, Mejean A, Ploux O, Pichon V (2013) Validation of the analytical procedure for the determination of the neurotoxin beta-N-methylamino-L-alanine in complex environmental samples. Anal Chim Acta 771:42–49. doi:10.1016/j.aca.2013.02.016

    Article  CAS  Google Scholar 

  • Council Directive 98/83/EC on the quality of water intended for human consumption (1998) vol OJ L 330. European Commission

  • Cox PA, Banack SA, Murch SJ (2003) Biomagnification of cyanobacterial neurotoxins and neurodegenerative disease among the Chamorro people of Guam. Proc Natl Acad Sci U S A 100:13380–13383

    Article  CAS  Google Scholar 

  • Cox PA et al (2005) Diverse taxa of cyanobacteria produce beta-N-methylamino-L-alanine, a neurotoxic amino acid. Proc Natl Acad Sci U S A 102:5074–5078

    Article  CAS  Google Scholar 

  • Degerlund M, Eilertsen H (2010) Main species characteristics of phytoplankton spring blooms in NE Atlantic and Arctic waters (68–80° N). Estuar Coast 33:242–269. doi:10.1007/s12237-009-9167-7

    Article  CAS  Google Scholar 

  • Downing S, Contardo-Jara V, Pflugmacher S, Downing TG (2014) The fate of the cyanobacterial toxin β-N-methylamino-l-alanine in freshwater mussels. Ecotox Environ Safe 101:51–58. doi:10.1016/j.ecoenv.2013.11.028

    Article  CAS  Google Scholar 

  • Duncan MW (1991) Role of the cycad neurotoxin BMAA in the amyotrophic lateral sclerosis-parkinsonism dementia complex of the western Pacific. Adv Neurol 56:301–310

    CAS  Google Scholar 

  • Eriksson J, Jonasson S, Papaefthimiou D, Rasmussen U, Bergman B (2009) Improving derivatization efficiency of BMAA utilizing AccQ-Tag in a complex cyanobacterial matrix. Amino Acids 36:43–48. doi:10.1007/s00726-007-0023-4

    Article  CAS  Google Scholar 

  • Esterhuizen M, Downing TG (2008) Beta-N-methylamino-L-alanine (BMAA) in novel South African cyanobacterial isolates. Ecotox Environ Saf 71:309–313

    Article  CAS  Google Scholar 

  • Esterhuizen-Londt M, Downing S, Downing T (2011) Improved sensitivity using liquid chromatography mass spectrometry (LC-MS) for detection of propyl chloroformate derivatised 2-N-methylamino-L-alanine (BMAA) in cyanobacteria. Water SA 37:133–138

    Article  CAS  Google Scholar 

  • Faassen E (2014) Presence of the neurotoxin BMAA in aquatic ecosystems: what do we really know? Toxins 6:1109–1138

    Article  CAS  Google Scholar 

  • Faassen EJ, Gillissen F, Zweers HAJ, Lürling M (2009) Determination of the neurotoxins BMAA (β-N-methylamino-L-alanine) and DAB (α-,γ-diaminobutyric acid) by LC-MSMS in Dutch urban waters with cyanobacterial blooms. Amyotroph Lateral Scler 10:79–84. doi:10.3109/17482960903272967

    Article  CAS  Google Scholar 

  • Faassen EJ, Gillissen F, Lurling M (2012) A comparative study on three analytical methods for the determination of the neurotoxin BMAA in cyanobacteria. PLoS One 7, e36667. doi:10.1371/journal.pone.0036667

    Article  CAS  Google Scholar 

  • Glover WB, Liberto CM, McNeil WS, Banack SA, Shipley PR, Murch SJ (2012) Reactivity of beta-Methylamino-l-alanine in complex sample matrixes complicating detection and quantification by mass spectrometry. Anall Chem. doi:10.1021/ac301691r

    Google Scholar 

  • Guidance for Industry - Bioanalytical Method Validation (2001). US Food and Drug Administration (FDA), Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM), Maryland, USA

  • Guillard RR, Ryther JH (1962) Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran. Can J Microbiol 8:229–239

    Article  CAS  Google Scholar 

  • Guo T, Geis S, Hedman C, Arndt M, Krick W, Sonzogni W (2007) Characterization of ethyl chloroformate derivative of beta-methylamino-L-alanine. J Am Soc Mass Spectr 18:817–825. doi:10.1016/j.jasms.2007.01.006

    Article  CAS  Google Scholar 

  • Hasle GR (1978) Phytoplankton Manual: the inverted microscope method. In: Sournia A (ed) Monographs on oceanic methodology. Unesco, Paris, pp 88–96

    Google Scholar 

  • Jiang L, Aigret B, De Borggraeve WM, Spacil Z, Ilag LL (2012) Selective LC-MS/MS method for the identification of BMAA from its isomers in biological samples. Anal Bioanal Chem 403:1719–1730. doi:10.1007/s00216-012-5966-y

    Article  CAS  Google Scholar 

  • Jiang L, Johnston E, Aberg KM, Nilsson U, Ilag LL (2013) Strategy for quantifying trace levels of BMAA in cyanobacteria by LC/MS/MS. Anal Bioanal Chem 405:1283–1292. doi:10.1007/s00216-012-6550-1

    Article  CAS  Google Scholar 

  • Jiang L et al (2014) Diatoms: a novel source for the neurotoxin BMAA in aquatic environments. Plos One 9, e84578. doi:10.1371/journal.pone.0084578

    Article  CAS  Google Scholar 

  • Jiao Y et al (2014) Occurrence and transfer of a cyanobacterial neurotoxin β-methylamino-l-alanine within the aquatic food webs of Gonghu Bay (Lake Taihu, China) to evaluate the potential human health risk. Sci Total Environ 468–469:457–463. doi:10.1016/j.scitotenv.2013.08.064

    Article  CAS  Google Scholar 

  • Johansen M, Mohlin, M., Skjevik, A.T., (2013) Algae report number 1–12 vol Algal situation in marine waters surrounding Sweden. Swedish Meteorological and Hydrological Institute

  • Jonasson S et al (2010) Transfer of a cyanobacterial neurotoxin within a temperate aquatic ecosystem suggests pathways for human exposure. Proc Natl Acad Sci U S A 107:9252–9257. doi:10.1073/pnas.0914417107

    Article  Google Scholar 

  • Kaloudis T, Triantis TM, Hiskia A (2014) Basic validation protocol for the analysis of cyanotoxins in environmemtal samples. Athens, Greece

    Google Scholar 

  • Kruger T, Monch B, Oppenhauser S, Luckas B (2010) LC-MS/MS determination of the isomeric neurotoxins BMAA (beta-N-methylamino-L-alanine) and DAB (2,4-diaminobutyric acid) in cyanobacteria and seeds of Cycas revoluta and Lathyrus latifolius. Toxicon 55:547–557. doi:10.1016/j.toxicon.2009.10.009

    Article  CAS  Google Scholar 

  • Lage S, Costa PR, Moita T, Eriksson J, Rasmussen U, Rydberg SJ (2014) BMAA in shellfish from two Portuguese transitional water bodies suggests the marine dinoflagellate Gymnodinium catenatum as a potential BMAA source. Aquat Toxicol 152C:131–138. doi:10.1016/j.aquatox.2014.03.029

    Article  CAS  Google Scholar 

  • Lage S, Annadotter H, Rasmussen U, Rydberg S (2015) Biotransfer of beta-N-Methylamino-l-alanine (BMAA) in a Eutrophicated Freshwater Lake. Mar Drugs 13:1185–1201. doi:10.3390/md13031185

    Article  CAS  Google Scholar 

  • Masseret E et al (2013) Dietary BMAA exposure in an amyotrophic lateral sclerosis cluster from southern France. PLoS One 8, e83406. doi:10.1371/journal.pone.0083406

    Article  CAS  Google Scholar 

  • Matuszewski BK, Constanzer ML, Chavez-Eng CM (2003) Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem 75:3019–3030

    Article  CAS  Google Scholar 

  • Metcalf JS, Banack SA, Lindsay J, Morrison LF, Cox PA, Codd GA (2008) Co-occurrence of beta-N-methylamino-L-alanine, a neurotoxic amino acid with other cyanobacterial toxins in British waterbodies, 1990–2004. Environ Microbiol 10:702–708

    Article  CAS  Google Scholar 

  • Mondo K, Broc Glover W, Murch SJ, Liu G, Cai Y, Davis DA, Mash DC (2014) Environmental neurotoxins beta-N-methylamino-l-alanine (BMAA) and mercury in shark cartilage dietary supplements. Food Chem Toxicol 70:26–32. doi:10.1016/j.fct.2014.04.015

    Article  CAS  Google Scholar 

  • Murch SJ, Cox PA, Banack SA (2004) A mechanism for slow release of biomagnified cyanobacterial neurotoxins and neurodegenerative disease in Guam. Proc Natl Acad Sci U S A 101:12228–12231

    Article  CAS  Google Scholar 

  • Pablo J et al (2009) Cyanobacterial neurotoxin BMAA in ALS and Alzheimer’s disease. Acta Neurol Scand 120:216–225

    Article  CAS  Google Scholar 

  • Pan M, Mabry TJ, Cao P, Moini M (1997) Identification of nonprotein amino acids from cycad seeds as N-ethoxycarbonyl ethyl ester derivatives by positive chemical-ionization gas chromatography–mass spectrometry. J Chromatogr A 787:288–294

    Article  CAS  Google Scholar 

  • Réveillon D et al (2014) Beta-N-Methylamino-l-Alanine: LC-MS/MS optimization, screening of cyanobacterial strains and occurrence in shellfish from Thau, a french mediterranean lagoon. Mar Drugs 12:5441–5467

    Article  CAS  Google Scholar 

  • Rines JEB, Hargraves PE (1988) The Chaetoceros Ehrenberg (Bacillariophyceae) Flora of Narragansett Bay, Rhode Island, U.S.A. J. Cramer

  • Rippka R, Deruelles J, Waterbury JB, Herdman M, Stainer RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1–61

    Google Scholar 

  • Rosen J, Hellenäs KE (2008) Determination of the neurotoxin BMAA (beta-N-methylamino-L-alanine) in cycad seed and cyanobacteria by LC-MS/MS (liquid chromatography tandem mass spectrometry). Analyst 133:1785–1789

    Article  CAS  Google Scholar 

  • Ross SM, Seelig M, Spencer PS (1987) Specific antagonism of excitotoxic action of ‘uncommon’ amino acids assayed in organotypic mouse cortical cultures. Brain Res 425:120–127

    Article  CAS  Google Scholar 

  • Salomonsson M, Hansson A, Bondesson U (2013) Development and in-house validation of a method for quantification of BMAA in mussels using dansyl chloride derivatization and ultra performance liquid chromatography tandem mass spectrometry. Anal Method 5:4865–4874. doi:10.1039/C3AY40657A

    Article  CAS  Google Scholar 

  • Sivonen K, Jones G (1999) Cyanobacterial toxins. In: Chorus I, Bartman J (eds) Toxic cyanobacteria in water: a guide to their public health consequences, monitoring and management. E & Fn Spon, London, pp 41–111

    Google Scholar 

  • Spacil Z, Eriksson J, Jonasson S, Rasmussen U, Ilag LL, Bergman B (2010) Analytical protocol for identification of BMAA and DAB in biological samples. Analyst 135:127–132

    Article  CAS  Google Scholar 

  • Spencer PS, Nunn PB, Hugon J, Ludolph A, Roy DN (1986) Motorneurone disease on Guam: possible role of a food neurotoxin. Lancet 1:965

    Article  CAS  Google Scholar 

  • Spencer PS, Hugon J, Ludolph A, Nunn PB, Ross SM, Roy DN, Schaumburg HH (1987) Discovery and partial characterization of primate motor-system toxins. Ciba Found Symp 126:221–238

  • Trufelli H, Palma P, Famiglini G, Cappiello A (2011) An overview of matrix effects in liquid chromatography-mass spectrometry. Mass Spectrom Rev 30:491–509. doi:10.1002/mas.20298

    Article  CAS  Google Scholar 

  • Van de Waal DB, Verspagen JMH, Lürling M, Van Donk E, Visser PM, Huisman J (2009) The ecological stoichiometry of toxins produced by harmful cyanobacteria: an experimental test of the carbon-nutrient balance hypothesis. Ecol Lett 12:1326–1335. doi:10.1111/j.1461-0248.2009.01383.x

    Article  Google Scholar 

  • Van de Waal DB et al (2011) Reversal in competitive dominance of a toxic versus non-toxic cyanobacterium in response to rising CO2. ISME J 5:1438–1450. doi:10.1038/ismej.2011.28

    Article  CAS  Google Scholar 

  • Vega A, Bell A (1967) α-Amino- β- methylaminopropionic acid, a new amino acid from seeds of Cycas circinalis. Phytochem 6:759–762

  • Viswanathan CT et al (2007) Quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays. Pharm Res 24:1962–1973. doi:10.1007/s11095-007-9291-7

    Article  CAS  Google Scholar 

  • Wasmund N, Pollehne F, Postel L, Siegel H, Zettler ML (2003) Biological state assessment of the Baltic Sea in 2002 vol 78. Institut fuer Ostseeforschung, Warnemuende

    Google Scholar 

Download references

Acknowledgments

We wish to thank the Swedish Research Council Formas for its financial support and the Baltic 2020 and Olle Engkvist Byggmästare foundations for funding the Acquity UPLC coupled to the Xevo-TQ-MS system (Waters) via the MiMeBS grant awarded to Birgitta Bergman. We are grateful to Nicola Wannicke of the Leibniz Institute for Baltic Sea Research, Germany and to Katja Pasdzierny of GEOMAR, Germany for isolating the diatom species from the Baltic Sea and North Sea samples.

Conflict of Interest

The authors declare no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Ecology, Environment and Plant Sciences, Stockholm University, 10654, Stockholm, Sweden

    Sandra Lage, Alfred Burian, Ulla Rasmussen & Sara Rydberg

  2. IPMA – Instituto Português do Mar e da Atmosfera, 1449 006, Lisbon, Portugal

    Pedro Reis Costa

  3. Regito AB, 28022, Vittsjö, Sweden

    Heléne Annadotter

  4. Department of Biological and Environmental Sciences, University of Gothenburg, 40530, Gothenburg, Sweden

    Anna Godhe

Authors
  1. Sandra Lage
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alfred Burian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ulla Rasmussen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pedro Reis Costa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Heléne Annadotter
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anna Godhe
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sara Rydberg
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sara Rydberg.

Additional information

Responsible editor: Philippe Garrigues

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

UPLC-MS/MS chromatograms (459.1 > 119.08 and 459.1 > 258.09 fragments) of protein fractions extracted with Method C (TCA protein precipitation method) followed by HCl hydrolysis and AQC derivatization (a) BMAA spiked (100 ng mL–1) in cyanobacteria blank matrix, i.e. Spirulina powder (Arthrospira fusiformis); BMAA detected in cultures of diatoms (b) Chaetoceros socialis SCCAP K-0550; (c) Coscinodiscus granii SCCAP K-1831 and (d) Phaeodactylum tricornutum SCCAP K-1280. (PPTX 452 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lage, S., Burian, A., Rasmussen, U. et al. BMAA extraction of cyanobacteria samples: which method to choose?. Environ Sci Pollut Res 23, 338–350 (2016). https://doi.org/10.1007/s11356-015-5266-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11356-015-5266-0

Keywords

Search

Navigation