Potential Production Of Neurotoxins BMAA And DAB In Freshwater Cyanobacteria And Effects Of Environmental Factors

It has been suggested that the non-protein amino acid, β-N-methylamino-L-alanine (BMAA), is an important pathogenic factor for the neurodegenerativedisease amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS/PDC)because of its neurotoxicity. Another non-protein amino acid,2,4-diaminobutyric acid(DAB), as an isomer of BMAA, is harmful to liver and nervous system of animals. Inrecent years, cyanobacterial blooms frequently occurred in China, which potentiallythreatened the human health and safety of drinking water because freshwaterresources including rivers, lakes, and reservoirs, were contaminated. It is an importantscientific proposal for human health to figure out the relationship betweencyanobacterial blooms and rising incidence of neurodegenerative diseases.A liquid chromatography-triple quadrupoles’ mass spectrometry (LC-MS/MS)method was developed for neurotoxins BMAA and DAB using the multiple reactionmonitoring (MRM) scan mode. The targets were separated on a hydrophilicinteraction liquid chromatography (HILIC) column. The purification performances oftwo polymeric cation-exchange cartridges Strata-X-C and Oasis-MCX forcyanobacterial extracts were compared, and the matrix effects before and afterpurification were evaluated. Two strains of Microcystis aeruginosa and one strain ofNostoc sp. were firstly cultured using BG-11medium recipe altered with differentlevels of four factors including temperature, illumination intensity, nitrate andphosphate. Total19strains of cyanobacteria incubated under normal conditions, threefield samples of phytoplankton collected from different lakes in China, spirulina tabletand dry laver were also analyzed. It is the object of this paper to provide scientificbasis for objective evaluation of the ecological risk and food safety caused by BMAAand DAB in freshwater environment. The performances of Oasis-MCX and Strata-X-C cartridges for purifyingcyanobacterial samples were compared. Results showed that an unknown small peakwas eluted after DAB in extract purified by the Strata-X-C cartridge. Recoveries ofBMAA and DAB in mixed standards and cyanobacterial extracts spiked withstandards were69.3%~87.4%and72.5%~82.1%, respectively. In general, no co-elutecompounds were found in the extracts cleand by Oasis-MCX cartridge, and therecoveries of BMAA and DAB in these extracts were higher than those cleaned byStrata-X-C cartridge. The Oasis-MCX cartridge was selected to purify the biologicalsamples. No signifcant matrix effects for BMAA were found in the LC-MS/MSanalysis, but there was suppression for DAB in some degree because somecompounds with high concentration were co-eluted with DAB on the HILIC column.Some basic amino acids lysine, histidine, arginine were confirmed, but other threecompounds could not be identifed presently. The LC-MS/MS analysis methoddeveloped in this thesis is very sensitive for BMAA and DAB, and the limits ofdetection (S/N=3) are lower than10pg.No BMAA was detected in any cyanobacterial strains, spirulina tablet, dry laverand sharkfins, although some cyanobacteria were incubated under altered conditionsto simulate extreme environment. However, trace levels of free status DAB weredetected in most biological samples. Both free and protein-bound statuses of DABwere found in some cyanobacterial samples. The effects of environmental factors onproduction of DAB were demonstrated in the cyanobacterial samples cultured withaltered conditongs. The contents of DAB in cyanobacteria cultured under1000lx and9000lx were higher than those under the medial levels, which demonstrated that theextreme illumination intensity could improve the producing ability of DAB. Thetemperature factor also has a similar influence tendency. Totally, the contents of DABin the cyanobacteria increased with the rise of phosphate concentration, reversely theyreduced with the rise of nitrate concentration. It was supposed that the cyanobacteriaproduce much DAB with lower ratio of nitrate to phosphate concentration (N/P)taking both nutrition factors into consideration.The universal detection of neurotoxin DAB in biological samples collected from freshwater should be paid attention to by us. It is urgent to develop the standardizeddetection method and the safety limit in sources of drinking water in order to protecthuman health and safety of drinking water. Presently the knowledge of chronictoxicity, toxic mechanism, and environmental behavior of DAB is still limited, whichwill be discovered deeply in the future.