Post on 13-Oct-2020
Mass-spectrometry based survey of BMAAsources, distribution and transferNadezda Zguna
Academic dissertation for the Degree of Doctor of Philosophy in Analytical Chemistry atStockholm University to be publicly defended on Thursday 28 February 2019 at 10.00 inNordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 12.
Abstractβ-methylaminoalanine (BMAA) is a neurotoxic non-protein amino acid first isolated from cycad seeds in 1967. It is believed to be connected to neurodegenerative diseases such as Parkinson’s, Alzheimer’s and amyotrophic lateral sclerosis (ALS) and is a ubiquitous compound produced by cyanobacteria, diatoms and dinoflagellates. Consequently, elucidating natural production, distribution and routes for human exposure of BMAA are of particular importance. However, the natural function of BMAA and its mechanisms of toxicity have not been fully established yet. The contradictory results about BMAA presence in cyanobacterial cultures and food webs have been reported by different scientific groups, which required the development of more sensitive and reliable analytical methods.
This thesis is focused on the analytical chemistry dimension of BMAA research: covering both new method development and novel applications. New analytical methods for BMAA detection and quantification were developed, focusing on improving sensitivity, since BMAA is normally found in natural samples at low concentrations. In Paper I, a new derivatization technique was implemented, which increased sensitivity and selectivity in the analysis of BMAA and its isomers. Subsequently, this developed method was applied to determine the presence of BMAA in fat and oil matrices in Paper II, which is a step towards discovering BMAA forms other than the documented free and protein-bound BMAA species. In Paper III, a method for separation and quantification of L- and D-BMAA stereoisomers in complex biological matrix was developed and applied to determine the enantiomeric composition of BMAA in cycad seed. Studying environmental distribution of BMAA is important to evaluate potential exposure routes and health risks for humans. Part of the work was devoted to broaden assessment on environmental occurrence of BMAA by applying existing robust methodology to new samples, such as commercial seafood in Paper IV and Baltic Sea biota in Paper V. Some of the “overlooked” aspects in the existing BMAA literature (i.e., BMAA chiral analysis, possible BMAA presence in dietary oil supplements and defined food webs) were successfully addressed.
Overall, the thesis presents important analytical developments, which can help to further elucidate sources, distribution and transfer of BMAA.
Keywords: BMAA, LC-MS/MS, chiral analysis, seafood, toxins, Baltic Sea, derivatization.
Stockholm 2019http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-164088
ISBN 978-91-7797-544-1ISBN 978-91-7797-545-8
Department of Environmental Science and AnalyticalChemistry
Stockholm University, 106 91 Stockholm
MASS-SPECTROMETRY BASED SURVEY OF BMAA SOURCES,DISTRIBUTION AND TRANSFER
Nadezda Zguna
Mass-spectrometry basedsurvey of BMAA sources,distribution and transfer
Nadezda Zguna
©Nadezda Zguna, Stockholm University 2019 ISBN print 978-91-7797-544-1ISBN PDF 978-91-7797-545-8 Cover image author: Tanja Russita Printed in Sweden by Universitetsservice US-AB, Stockholm 2019
To my dear parents, Victor and Lyubov
Моим дорогим родителям, Виктору и Любови
Populärvetenskaplig sammanfattning
Aminosyror kallas ofta för livets byggstenar. Av de hundratals naturliga
aminosyrorna som man känner till är det dock endast 21 stycken som bygger
upp proteinerna i människokroppen. De övriga, så kallade icke-proteinogena
aminosyror, uppfyller andra viktiga biologiska funktioner i olika typer av
levande organismer.
På 1960-talet upptäcktes en hittills okänd icke-proteinogen aminosyra på ön
Guam och som misstänks ha ett samband med en förhöjd frekvens av
neurologiska sjukdomar, som amyotrofisk lateral skleros (ALS) och
Parkinsons sjukdom. Föreningen isolerades från fruktkärnorna från en
kottepalm och identifierades som β-metylamino-L-alanin (BMAA). Senare
studier har visat att BMAA också produceras av vanliga marina organismer,
som cyanobakterier. Dessutom har hypotesen om föreningens verkan som
nervgift fått ytterligare vetenskapligt stöd. Sammantaget har intresset för
BMAA ökat markant under de senaste åren.
I arbetet bakom avhandlingen har nya kemiska analysmetoder utvecklats för
att studera förekomst och spridning av BMAA i miljön, möjliga källor, som
mat och kosttillskott, samt eventuell transport inom näringskedjan.
Högupplösande masspektrometri har varit viktig för att kunna detektera
riktigt låga halter av BMAA. Alla analysmetoder som beskrivs i
avhandlingen har dessutom involverat kemisk omvandling, derivatisering,
med olika reagens innan analys, dels för att förbättra separationen från andra
ämnen, dels för en känsligare detektion. För BMAA har därför lägre
koncentrationer än tidigare kunnat uppmätas i exempelvis blåmusslor, fisk-
och algolja.
I ett av arbetena har två isomerer av BMAA, de optiskt aktiva D- och L-
formerna som är varandras molekylära spegelbilder, påvisats för första
gången i fruktkärnor från kottepalm. Dessa så kallade enantiomerer är
molekyler som har identiska fysikaliska och kemiska egenskaper och därför
inte kan åtskiljas genom vanlig kromatografi eller masspektrometri. Däremot
är det vanligt att enantiomerer har olika biologiska funktioner och det är
därför viktigt att kunna mäta dem separat. Genom att derivatisera
blandningen av BMAA-enantiomerer med en optiskt aktivt reagens erhålls
isomerer av BMAA-derivat som inte längre är spegelbilder och därför kan
separeras kromatografiskt och mätas var för sig.
Eftersom BMAA har kopplats till neurodegenerativa sjukdomar är det viktigt
att identifiera eventuella livsmedel som kan vara potentiella
exponeringskällor. Därför undersöktes förekomst av BMAA i fisk och
skaldjur från olika matbutiker i Stockholm. Låga halter observerades i
ostron, blåmusslor, räkor och rödspätta. Däremot kunde BMAA inte
detekteras i lax, torsk, abborre eller kräftor.
Möjliga transportvägar för BMAA inom näringskedjor från Östersjömiljö
undersöktes som modell för ett naturligt ekosystem. Endast växtplankton,
djurplankton och pungräkor visade detekterbara mängder av BMAA, medan
inga halter alls kunde uppmätas i sediment, bottenlevande ryggradslösa djur,
skarpsill eller skrubbskädda. Enbart prover tagna under sommarsäsongen
analyserades, men för en fullständigare bild skulle en längre, exempelvis
ettårig mätperiod behövas. Dessutom vore det intressant att undersöka fler
områden i Östersjön.
Det finns en risk för att personer som bor nära vattendrag skulle kunna
exponeras för BMAA genom inandning av naturligt bildade aerosoler.
Därför initierades en undersökning kring detta. Ytterligare studier krävs
dock för att fullt ut kunna bedöma riskens omfattning.
1
Table of Contents
List of publications ........................................................................................ 2
Abbreviations ................................................................................................ 5
Introduction ................................................................................................... 7
BMAA chemical properties ........................................................................ 7
BMAA natural production, distribution and human exposure .................... 8
Forms of BMAA in natural matrices ........................................................ 11
L-BMAA toxicity and neurodegenerative diseases ................................... 12
Toxicity of BMAA structural isomers and D-BMAA ............................... 16
Analytical strategies for BMAA ............................................................... 17
Aims of the thesis ........................................................................................ 21
Methods ........................................................................................................ 23
Sample pre-treatment and derivatization .................................................. 23
UHPLC separation .................................................................................... 24
MS/MS detection ...................................................................................... 25
Results and discussion ................................................................................ 29
Developing novel derivatization methods................................................. 29
Screening studies ...................................................................................... 37
Other analytical strategies and applications .............................................. 39
Conclusions .................................................................................................. 45
Further method development & application ............................................ 47
Acknowledgements ..................................................................................... 49
References .................................................................................................... 52
2
List of publications
I. Improved detection of β-N-methylamino-L-alanine using N-
hydroxysuccinimide ester of N-butylnicotinic acid for the
localization of BMAA in blue mussels (Mytilus edulis)
Rudolf Andrýs *, Javier Zurita
*, Nadezda Zguna, Leopold L. Ilag,
Klaas Verschueren and Wim M. De Borggraeve
Anal Bioanal Chem. 407(13) (2015)
II. Analysis of BMAA in fish oil and its vegan alternatives
Nadezda Zguna, Jan Holmback and Leopold L. Ilag
[Manuscript]
III. Chiral separation of β-Methylamino-L-alanine (BMAA)
enantiomers after (+)-1-(9-fluorenyl)-ethyl chloroformate (FLEC)
derivatization and LC-MS/MS
Javier Zurita*, Nadezda Zguna
*, Rudolf Andrýs, Anna Strzelczak,
Liying Jiang, Gunnar Thorsen and Leopold L. Ilag
Analytical Methods, 2019, DOI: 10.1039/C8AY02287A
IV. Quantification of neurotoxin BMAA (β-N-methylamino-L-
alanine) in seafood from Swedish markets
Liying Jiang, Nadezda Kiselova, Leopold L. Ilag and Johan Rosen
Scientific Reports, 4 (2014)
V. Is there a BMAA transfer in the pelagic and benthic food webs in
the Baltic Sea?
Nadezda Zguna, Agnes Karlson, Leopold L. Ilag, Andrius Garbaras
and Elena Gorokhova
[Submitted]
* authors contributed equally to the paper
3
The author’s contribution to the papers was as described below:
I. The author was responsible for part of the experimental work and
part of the data evaluation.
II. The author was responsible for designing and performing the
majority of the experiments, all data evaluation and writing the
paper.
III. The author was responsible for part of the experimental design;
significant parts of the experimental work, significant parts of data
evaluation and significant part of writing the paper.
IV. The author was responsible for all the experimental work, part of the
data evaluation and part of writing the paper.
V. The author was responsible for designing and performing the
analytical part of the work and associated sample preparation as well
as LC-MS/MS data evaluation and writing a significant part of the
paper.
4
Publications not included in this thesis
VI. Human Scalp Hair as an Indicator of Exposure to the
Environmental Toxin β-N-Methylamino-l-alanine
Simoné Downing, Laura Louise Scott, Nadezda Zguna and Timothy
Grant Downing
Toxins, Vol 10, Iss 1, p 14 (2017)
VII. Host cell-derived lactate functions as an effector molecule in
Neisseria meningitidis microcolony dispersal
Sara Sigurlásdóttir, Jakob Engman, Olaspers Sara Eriksson, Sunil D
Saro, Nadezda Zguna, Pilar Lloris-Garcerá, Leopold L Ilag and
Ann-Beth Jonsson
PLoS Pathogens, Vol 13, Iss 4, p e1006251 (2017)
VIII. A Collaborative Evaluation of LC-MS/MS Based Methods for
BMAA Analysis: Soluble Bound BMAA Found to Be an
Important Fraction
Elisabeth J. Faassen, Maria G. Antoniou, Wendy Beekman-
Lukassen, Lucie Blahova, Ekaterina Chernova, Christophoros
Christophoridis, Audrey Combes, Christine Edwards, Jutta Fastner,
Joop Harmsen, Anastasia Hiskia, Leopold L. Ilag, Triantafyllos
Kaloudis, Srdjan Lopicic, Miquel Lürling, Hanna Mazur-Marzec,
Jussi Meriluoto, Cristina Porojan, Yehudit Viner-Mozzini and
Nadezda Zguna
Marine Drugs, Vol 14, Iss 3, p 45 (2016)
IX. An automated mass spectrometry-based screening method for
analysis of sulfated glycosaminoglycans
Nadezda Kiselova, Tabea Dierker, Dorothe Spillmann and
Margareta Ramström
Biochemical and Biophysical Research
Communications 450(1):598-603 (2014)
5
Abbreviations
AEG N-(2-aminoethyl)glycine 1H-NMR Proton nuclear magnetic resonance spectroscopy
ALS Amyotrophic lateral sclerosis
AMPA α-amino-β-methylaminopropionic acid
AP7 2-amino-7-phosphonoheptanoic acid
AQC 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
BAMA β-amino-N-methylalanine
BMAA β-methylaminoalanine
BOAA α-amino-β-oxalylaminopropionic acid
C4-NA-NHS N-hydroxysuccinimide ester of N-butylnicotinic acid
CE Capillary electrophoresis
DAB 2,4-diaminobutanoic acid
ELISA Enzyme linked immunosorbent assay
ESI Electrospray ionization
FLD Fluorescence detector
FLEC (+)-1-(9-fluorenyl)-ethyl chloroformate
GC Gas chromatography
HILIC Hydrophilic interaction liquid chromatography
(U)HPLC (Ultra) High performance liquid chromatography
LC Liquid chromatography
LLE Liquid-liquid extraction
LOD Limit of detection
LOQ Limit of quantification
LPI Low pressure impactor
MRM Multiple reactions monitoring
MS Mass spectrometry
MS/MS Tandem mass spectrometry
N-butyl-AQ-N-NHS N-butyl-aminoquinolyl-N-hydroxysuccinimide
N-methyl-AQ-N-NHS N-methyl-aminoquinolyl-N-hydroxysuccinimide
NFT Neurofibrillary tangles
NMDA N-methyl-D-aspartate
PD Parkinsonism-dementia
QC Quality control
SPE Solid phase extraction
TCA Trichloroacetic acid
UV Ultraviolet
6
7
Introduction
In the 1960s, the anthropologist Marjorie Whiting was working on Guam
Island, studying the indigenous Chamorro population lifestyle and, in
particular, their diet1. She was interested in the fact that the Chamorros
suffered from an unknown neurodegenerative disease similar to amyotrophic
lateral sclerosis (ALS)-parkinsonism-dementia (PD) complex at unusually
high occurrence (50-100 times higher than worldwide)2. It was suspected
that a plant that was widely used in Chamorros’ traditional cuisine (Cycas
micronesica), might contain a toxic amino acid, α-amino-β-
oxalylaminopropionic acid (BOAA), which had been previously linked to
lathyrism by Arthur E. Bell3, or an analogue to BOAA in terms of its toxic
properties.
A couple of years later, in 1966, Vega and Bell isolated an amino acid from
the seeds of Cycas micronesica. They proved the compound to be β-
methylaminoalanine (BMAA), though at that time it was named α-amino-β-
methylaminopropionic acid (AMPA). Vega and Bell also mentioned the
acute toxicity of BMAA to chicks and thus BMAA started its winding road
in science4.
BMAA chemical properties
BMAA is a non-protein amino acid with molecular mass of 118.1 Da. It is a
polar basic amino acid with pKa values of 2.1, 6.48 and 9.70 for carboxyl
group, primary and secondary amino group, respectively5. BMAA is a chiral
compound, which means it can be present as L-BMAA and D-BMAA. Thus,
BMAA has structural isomers and this fact should be taken into
consideration during development of analytical methods. Isomers which
were observed in natural matrices6 are 2,4-diaminobutanoic acid (DAB), N-
(2-aminoethyl)glycine (AEG) and β-amino-N-methylalanine (BAMA)
(Figure 1). BMAA is also known as a chelator of divalent metals, such as
zinc, copper and nickel7.
8
Figure 1. BMAA and its structural isomers. Chiral centers of the molecules are denoted with
asterisk (*).
BMAA natural production, distribution and human
exposure
Even though the biological role of BMAA is still not clear, it has been
detected in various ecosystems all around the globe. Guam Island4, Lake
Taihu in China8, Baltic Sea
9, British
10, Portuguese
11 and South Florida
12
water bodies, Dutch urban waters13
, and even deserts of Qatar14
are examples
of where BMAA has been discovered. BMAA is believed to be naturally
produced by ubiquitous organisms: cyanobacteria in marine, freshwater and
terrestrial environments (such as soil and limestone caves)15
, diatoms16,17
and
dinoflagellates11,17,18
as reported for Swedish coast and French ecosystems.
Generally, cyanobacteria are well-known producers of bioactive compounds,
including toxins, which may cause adverse health effects19
. Several lines of
evidence support the hypothesis of BMAA production by cyanobacteria. It
has been widely demonstrated that both field samples and axenic laboratory
cultures of cyanobacteria contain BMAA in detectable amounts, which
would be possible only in case of endogenous BMAA production15,20
.
Similar experiments have been performed with diatom cultures that were
also found to contain BMAA in the low µg∙g-1
range17
.
The biological function of BMAA in cyanobacteria has also been addressed.
In the experiment with cyanobacteria and the isotope-labelled 15
NH4+ that
was used as a sole nitrogen source, nitrogen starvation induced an increase in
free 15
N BMAA, indicating synthesis of BMAA. Moreover, the addition of
nitrate or ammonia to the culture medium after starvation reduced the
concentration of free BMAA and did not cause the increase in the protein-
9
associated fraction. That finding suggests that BMAA may have an
important role in nitrogen metabolism in cyanobacteria21
. Further
investigations by the same scientific group proposed a possible pathway for
BMAA metabolism in cyanobacteria, which presumes that a natural
precursor of BMAA is a keto acid undergoing reversible amination reaction
to produce BMAA22
.
Other researchers supported the idea of BMAA’s role in cyanobacterial
nitrogen metabolism. For instance, Berntzon and co-authors have
demonstrated that BMAA severely inhibits nitrogenase (enzyme
participating in nitrogen fixation) activity, reduces growth, induces
pigmentation change of cells from blue-green to yellow-green, and leads to
the massive accumulation of glycogen23
. However, publications reporting no
detectable levels of BMAA in cyanobacteria are also common24–26
. In most
cases considered nowadays, reliable analytical methods were used, so the
controversy of the findings could not be attributed to poor scientific
approach and, therefore, cannot be ignored.
Currently, no large-scale surveys comparing field and laboratory samples of
cyanobacteria are available. Indeed a systematic investigation is needed to
reveal which specific cyanobacteria produce BMAA and under which
conditions. Analysis of 27 publications reporting the presence of BMAA in
cyanobacteria has been made by Berntzon, and the conclusion was drawn
that BMAA production by cyanobacteria depends neither on their taxonomic
group nor on the geographic origin, growth stage, and nutritional status27
.
Naturally, the variability of BMAA occurrence and its possible connection
with neurodegenerative diseases raised concerns about human exposure to
the toxin through food consumption, inhalation, and other routes. Seafood
analysis from the Swedish supermarkets showed that BMAA might be
present in mollusks and crustaceans (in oyster, blue mussel and shrimp
BMAA was detected in 16 samples out of 16 tested) However, it is less
likely to be found in fish and crayfish (BMAA was detected in 5 samples out
of 27 tested)28
. The occurrence of BMAA in human scalp hair indicates that
consumption of shellfish, and, possibly, pork may be a dietary route of
exposure, whereas residential proximity to the water bodies and
10
consumption of vegetables irrigated with water from water reservoir with
algal blooms does not correlate with BMAA content in hair29
. Other human
exposure routes through food might not be as straightforward as seafood
consumption, but should also be explored. For instance, the demand for
algae-derived food supplements increases30
, which makes it more urgent to
investigate their toxicological safety. The process of algal oil (a vegan
alternative to fish oil) production includes extraction of lipid compounds
with supercritical CO231
, and the remaining algal debris can be dried and
further used as the food seasoning known as furikake32
, which should also be
analyzed for algal toxins, specifically BMAA. Furthermore, the fact that blue
mussels are used for feeding hens33
makes poultry an interesting subject to
analyze for the presence of BMAA.
Inhalation, which is another plausible exposure route, was studied using a rat
model, but no clinical signs of toxicity were observed in exposed rats both at
environmentally relevant levels of the aerosolized BMAA and higher
concentrations (0.9 µg – 1.4 g BMAA dose per rat corresponding to 65 days
– 267 years of chronic BMAA exposure through inhalation from
environmental sources)34
. Aerosols collected from a lake in an area with
elevated ALS frequency (25-fold the expected average) were positive for
BMAA35
. There are only a few studies on BMAA availability through
inhalation and its link to ALS, and we undoubtedly need stronger evidence
to make any conclusions on this exposure route relevance, so it is an
interesting path for further investigations.
Studies of developmental exposure to BMAA have suggested that mother’s
milk could be an important pathway of BMAA transfer from lactating
mothers to its offspring in mice36
. Moreover, a preferential enantiomer-
specific uptake of L-BMAA was found in a human mammary cell line. The
efficient absorption of BMAA into intestinal cells and its transport into
neuronal and glial cells suggest that in humans it might be transferred via the
milk from a mother to the brain of the nursed infant37
. BMAA
transplacentral transfer from exposed mothers to the brain of fetus has been
demonstrated in mice38
, suggesting that exposure to BMAA in utero may
influence further offspring development. Autoradiography analysis revealed
11
pronounced BMAA transfer and deposition in the eggs of exposed quails,
predominantly in the yolk39
. Thus, the developmental exposure to BMAA in
birds should also be studied in more detail, and the level of BMAA in eggs
consumed by humans should be evaluated.
Forms of BMAA in natural matrices
In natural matrices, BMAA can be present both as a free compound and/or in
bound form. Therefore the analytical method must be selected wisely to
determine the concentrations of those fractions separately or as total BMAA.
Initially, free BMAA was discovered in cycad seeds, being extracted with
50% ethanol4. Later, the bound form was discovered by the observation that
BMAA concentration in samples increased 10- to 240-fold after
hydrochloric acid hydrolysis (6M HCl, 110°C for 24 h)40
. This discovery led
to introducing strong acid hydrolysis as a routine step in sample preparation
and reporting BMAA in further studies as “total BMAA” or as two separate
fractions of “free BMAA” and “protein-bound BMAA”16
. Although strong
acid hydrolysis is an unquestionably efficient way to cleave peptide bonds, it
might be unsuitable in cases, where it is important to preserve the absolute
configuration of a chiral molecule, since it may cause racemization41,42
.
Overall, determination of total BMAA concentration in the samples does not
provide information regarding its original form, i.e. what specific chemical
species exist in the matrix before hydrolysis, but it gives a good estimation
of the amounts of BMAA one can be exposed to from certain samples.
It has been discovered that BMAA does not exist solely in the protein-bound
form, but also as soluble bound BMAA. That means that after hydrolyzing
trichloroacetic acid (TCA) extracts of free BMAA, the concentration of the
latter increased up to 40%43
, implying the existence of TCA-soluble BMAA-
containing compounds44,45
or what here is referred to as a conjugated form.
Similar observations were made in pioneering studies of BMAA many years
ago, but have not been studied extensively43,46
. Possible soluble-bound forms
12
of BMAA include metal complexes7, carbamate
47,48, imines
49 and low-
molecular weight peptides50
, such as galantine and γ-glutamyl peptide51
.
There also exist a number of lipid compounds conjugated to amino acids,
namely lipoaminoacids and lipopeptides and various forms of these
compounds were discovered in marine organisms52
. Moreover, serine (one of
20 common amino acids), is a part of the most abundant acidic phospholipid
in human cell membranes – phosphatidylserine. Phosphatidylserine also
constitutes up to 20% of the total phospholipids of human blood plasma and
plays a crucial role in the human brain functions by influencing the
metabolism of neurotransmitters53
. Lipid compounds, as possible conjugates
or natural precusors of BMAA, to the best of our knowledge, have not yet
been studied.
L-BMAA toxicity and neurodegenerative diseases
In the half-century of BMAA research, its toxicity has always been in sharp
focus. The idea of the toxicity of BMAA arose from the fact that cycad seeds
are toxic both to animals and humans1. It has been demonstrated that BMAA
solution in low micromolar (10 µM) concentrations can cause selective death
of motor neurons in cortical cultures and that the same damage to the
neurons can be caused by crude cycad extract54,55
. Cox et al. hypothesized
that BMAA in cycads may function as a toxin for herbivores56
. Early
experiments by Vega et al.57
with purified BMAA showed that BMAA-
administered chicks and rats exhibited acute weakness, convulsions and
troubled gait. Furthermore, BMAA exposure experiments with macaques,
showed that the animals developed parkinsonian features and behavioral
anomalies; also degenerative changes were registered in their motor neurons
in the cerebral cortex and spinal cord58
. In a study by Cox and co-workers,
vervet monkeys were fed with L-BMAA or L-BMAA plus L-serine-dosed
fruit for 140 days. Animals developed neurofibrillary tangles (NFT) and β-
amyloid deposits in the brain, however, the animals co-administered with L-
serine expressed a significantly reduced density of NFT59
. Rats and mice
have also been used as animal models for studying BMAA toxicity. In a
13
study by Karlsson et al.60
, neonatal rats were dosed with 14
C-L-BMAA and
the radiographic data was obtained followed by ultra-high performance
liquid chromatography tandem mass spectrometry (UHPLC-MS/MS)
analysis. A dose-dependent increase of protein-associated BMAA in
neonatal rat tissues was observed; and BMAA concentration in the rat liver
was 10-fold higher than in the brain. No BMAA was detected in the samples
from adult rats, which suggests that it had been metabolized and not re-used
in protein synthesis. In other studies by the same group, it has been shown
that the rats neonatally treated with BMAA expressed long-term changes,
such as decrease in learning ability61
, deficits in spatial learning and memory
impairments62
. Rats dosed neonatally with 600 mg/kg of BMAA also
expressed morphological changes in the brain, developing fibrils63
. Long-
term effects of treating rats with BMAA manifested on the molecular level
as the decrease in several proteins expression. Specifically, the expression of
proteins involved in energy metabolism and intracellular signaling has been
registered64
. Other research groups have noted that L-BMAA-treated rats
mimic symptoms presented by ALS patients such as muscular atrophy, brain
structural changes, motor neuronal death and other65,66
. Moreover,
Alzheimer’s and Parkinson’s disease-like symptoms, such as decreased
locomotor activity, body tremors, inability to identify a learned odor and
developmental delays, could simultaneously be observed in BMAA-exposed
rats67
. Finally, BMAA toxicity was demonstrated to be gender and age
dependent, affecting younger rats more than older ones and showing that
different neurodegenerative disruptions manifested in male and female rats;
for instance, male rats exhibited unilateral splay of hind limb while female
rats demonstrated whole body tremors67
.
Linking BMAA to neurodegenerative diseases calls for elucidating the
mechanisms behind its neurotoxicity. The first suggested mechanism of
BMAA toxicity was presented in 1987 and described a concentration-
dependent excitotoxicity through N-methyl-D-aspartate (NMDA) glutamate-
receptor overactivation, manifesting as increased frequency of postsynaptic
vacuoles and dark shrunken cells in mouse cortex explants exposed to
BMAA. The ability of BMAA to block glutamate receptors has been
attributed to structural similarity between glutamate and BMAA carbamate
14
adduct (Figure 2), which can readily form47
in the presence of bicarbonate,
which is considered a required co-factor for BMAA neurotoxicity5. It was
also noticed that the acute neuronal toxicity of BMAA was selectively
blocked by specific antagonists at separate glutamate receptor sites. For
instance, 2-amino-7-phosphonoheptanoic acid (AP7), NMDA antagonist,
was shown specifically to block the neuronal changes produced by BMAA68
.
Another possible mechanism has been discovered in an aquatic plant,
Ceratophyllum demersum, where BMAA inhibits the activity of several
oxidative stress defense enzymes, such as superoxide dismutase, catalase,
guaiacol peroxidase, glutathione peroxidase and glutathione reductase,
therefore allowing reactive oxygen species to damage cell walls, DNA and
proteins69
.
Figure 2. Glutamate and BMAA carbamate adduct are structurally similar70.
Dunlop and co-workers suggested that BMAA may misincorporate into
newly synthesized proteins in human cell lines. This could potentially cause
protein misfolding and lead to the formation of protein plaques in the brain,
characteristic of many neurodegenerative diseases. The hypothesis of
15
BMAA misincorporation was based on the concentration-dependent increase
of the protein-associated radiolabel when exposed to 3H-BMAA, as well as
observing toxicity of BMAA to neuronal cell culture; the latter could also be
attributed to other toxicity mechanisms, not necessarily misincorporation.
Moreover, since radioactivity measurements were used for detection, there
might exist a possibility of radio-label migration giving false positive
results71
. Subsequently, the concept of BMAA misincorporation into proteins
has been criticized. For instance, BMAA and canavanine toxicity and their
possible misincorporation into proteins in various bacterial species was
compared by van Onselen et al. The authors concluded that growth reduction
was not observed for any of the tested bacterial species treated with BMAA
in contrast to those incubated in canavanine-containing media. The
following liquid chromatography mass-spectrometry (LC-MS) analysis
showed no detectable BMAA in proteins produced in an Escherichia coli
expression system72
.
Two abovementioned potential mechanisms of toxicity were further
compared by van Onselen et al.73
using rat pheochromocytoma cell line
PC12. It was concluded that excitotoxicity through activation of mGluR1
glutamate receptors was the main and possibly the only, mechanism of
BMAA toxicity in PC12 cells. Moreover, BMAA capability to
misincorporate into proteins was studied on the human cell lines and no
signs of misincorporation into proteins (i.e., no concentration-dependent
apoptosis or necrosis and no BMAA in purified protein extracts of BMAA-
exposed cells) were observed in contrast with the known misincorporating
analogues L-4-fluorophenylalanin and m-tyrosine in the same cell lines74
.
Nunn and Ponnusamy proposed that BMAA reacts non-enzymatically with
pyridoxal-5’-phosphate, forming 2,3-diaminopropanoic acid and
methylamine. This was observed in model experiments at physiological pH
as well as in rat liver and kidney homogenates incubated with BMAA.
Methylamine release is toxic, because in the organism it converts into
formaldehyde, which in turn participates in protein cross-linking processes
in a variety of tissues49
.
16
Recently, it has been shown that BMAA can inhibit the activity of certain
enzymes, such as β-amylase, catalase and glutathione S-transferase, as well
as associate in vitro with commercial proteins (becoming impossible to
remove by protein precipitation or denaturation) and interfere with protein
folding in the absence of de novo protein synthesis75
. These findings suggest
an additional novel mechanism of BMAA toxicity that needs to be
elucidated to possibly clarify the role of BMAA in the development of
neurodegenerative diseases.
Toxicity of BMAA structural isomers and D-BMAA
The toxicity of BMAA structural isomers, such as BAMA, DAB and AEG
has not been studied extensively, however the neurotoxicity of DAB has
been reported in the early 70s, when it was shown that L-DAB administered
to rats in low mmol/kg body weight doses resulted in hyperirritability,
tremors and convulsions, while D-DAB at even much larger doses did not
cause toxic symptoms76
. A more recent study compared individual and
combined toxicity of BMAA, DAB and AEG in vitro on human
neuroblastoma cells and showed that both BMAA and 2,4-DAB treatment in
µM concentration range resulted in reduction of cell viability, while AEG at
the same concentration was not cytotoxic and its toxicity was observed at
concentrations above 1 mM77
. Moreover, combined treatment with BMAA
and DAB resulted in increased apoptosis compared to the individual
treatment with each compound.
A crucial aspect in studying BMAA toxicity is to distinguish between
stereoisomers and to understand the differences in toxicity between D and L-
BMAA. Although it is known that L-amino acids are predominantly used in
the organisms to build proteins, it is important to give due consideration to
the investigation of D-amino acids and their biological role. This was
summarized in the recent review by Genchi, that contrary to the opinion that
D-amino acids have a minor function in biological processes in comparison
with L-enantiomers, there is a growing body of evidences that D-amino acids
are present in relatively high concentrations in various microorganisms,
17
plants and mammals and fulfil specific biological functions78
. Examples
include marine invertebrates, the cellular fluids of which contain D-amino
acids as a main component; marine shellfish in which D-amino acids can
exceed 1%; amphibian whose skin secretion contains active peptides with
incorporated D-amino acids. Also higher plants, such as pea seedlings,
tobacco leaves, wild rice and lentils contain D-amino acids in free or bound
form. D-amino acids are routinely found in the peptidoglycan cell wall of
bacteria. It has been shown that D-amino acids have regulatory roles in
diverse bacteria. They regulate cell wall remodeling and cause biofilm
dispersal in aging bacterial communities79
.
It was believed that L-BMAA is neurotoxic, while D-BMAA had less or no
harmful effect on the organisms46,57
. In a later study by Andersson et al.37
it
was demonstrated that the uptake of L-BMAA in rats was much higher
compared to D-BMAA. However, a more recent study by Metcalf et al.
assessed in vitro toxicity of D-BMAA using mixed cortical cell cultures from
mice and found it to be comparable to the toxicity of L-BMAA80
. In the same
study it was also reported that L-BMAA used for dosing was converted into
D-BMAA in mouse brain and vervet monkey cerebrospinal fluid but
remained in the form of L-BMAA in the peripheral nervous system of mice.
Moreover, D-BMAA was found in mice liver, suggesting that racemization
might occur during digestion. Altogether, the most recent research calls for
toxicological studies of the D-enantiomer, as well as analytical methods
development that would take BMAA chirality into consideration.
Analytical strategies for BMAA
Historically, BMAA detection and quantification has been performed with
various analytical methods, including different separation techniques, such
as capillary electrophoresis81
(CE), gas chromatography82
(GC) and liquid
chromatography (LC), in combination with a selection of detectors
(fluorescence (FLD), ultraviolet81
(UV), mass spectrometry82
(MS) and
tandem mass spectrometry (MS/MS)). Additionally, proton nuclear magnetic
resonance spectroscopy (1H-NMR) was used in one study
83 and
18
commercially available enzyme linked immunosorbent assay (ELISA) for
BMAA determination in environmental samples was evaluated.
Unfortunately, the latter gave unacceptable false positive results and was not
found to be useful for BMAA analysis84
.
One of the commonly used methods for a long time for BMAA analysis has
been high performance liquid chromatography (HPLC)-FLD10,15,85,86
preceded by pre-column derivatization with 6-aminoquinolyl-N-
hydroxysuccinimidyl carbamate (AQC). However, FLD detection is less
selective than MS/MS and thus tends to produce false positive results. For
instance, two publications reporting unusually high BMAA content in
sharks87
and flying foxes88
(10-250 µg/g range compared to normally
reported 0.1-1 µg/g range for wild fish and animals) used HPLC-FLD for
quantification. That puts the accuracy of the reported results in question.
Overall, in comparison with MS/MS, FLD detection has less stringent
identification criteria for the analyte and cannot be the method of choice for
accurate and precise BMAA analysis86
.
Currently, the method of choice for BMAA analysis is HPLC or UHPLC
coupled to tandem mass spectrometry. This approach may involve analysis
of either derivatized or non-derivatized BMAA. For the derivatization,
normally a commercial AccQ∙Tag Kit with AQC reagent from Waters is
used prior to chromatography on C18 column. Other derivatization
techniques rely on using propyl chloroformate72
and dansyl chloride89,90
as
derivatization agents.
In this thesis, novel strategies of BMAA derivatization were studied. For
instance, the successful use of N-hydroxysuccinimide ester of N-
butylnicotinic acid led to the efficient separation of BMAA from its
structural isomers and higher sensitivity in detecting BMAA compared to the
AQC derivatization (Papers I and II). Specific analytical strategies are
required for the enantiomeric analysis of BMAA. In some methods,
derivatization serves the purpose to create diastereomers and make
enantiomer separation possible. One methodology is a pre-column
derivatization with o-phthaldialdehyde followed by the detection by
fluorimetry91
. However, this approach is not currently recognized as a
19
method with sufficient selectivity. Another approach using (+)-1-(9-
fluorenyl)-ethyl chloroformate (FLEC) derivatization has been proposed in
this thesis (Paper III). In yet another method described by Metcalf et al., the
underivatized enantiomers of BMAA are separated by HPLC on the chiral
chromatographic column and collected as fractions80
. The advantage of
derivatization is the added molecular mass and, in certain cases, increased
hydrophobicity, which leads to a better separation on the reversed phase
chromatographic column and can generate more detectable fragments in
tandem mass spectrometry. For underivatized BMAA analysis, hydrophilic
interaction liquid chromatography (HILIC) columns are commonly used.
However, HILIC-MS/MS methods do not resolve the issue of BMAA
structural isomers except DAB26,92
and lack diagnostic fragmentation for
AEG26
.
As mentioned before, BMAA may be present in samples in different forms:
free BMAA, protein-bound BMAA and in conjugated forms other than to
protein. This defines the choice of appropriate analytical method for sample
pretreatment depending on which fraction is the focus of a certain study44,45
.
20
21
Aims of the thesis
Investigating BMAA requires sensitive, reliable, and validated analytical
methods. New instrumental and sample preparation strategies are to be
explored to provide an adequate toolbox for confident BMAA detection and
quantification in various biological matrices. Of particular interest is chiral
BMAA analysis, which is not extensively covered in current literature. Apart
from the analytical method development, it is crucial to understand the yet
unknown natural functions of BMAA and its possible dietary routes for
humans. To facilitate further elucidation of BMAA identification,
distribution and toxicity, the following aims were set:
To develop and analytically evaluate a more sensitive method for BMAA
quantification than the current method of choice, i.e., LC-MS/MS using
AQC derivatization.
To develop a quantitative method for the chiral analysis of BMAA.
To apply existing and/or developed methods to detect and quantify
BMAA in oil matrices, such as food supplements (fish and algal oil).
To conduct screening of BMAA presence in seafood from Swedish
supermarkets.
To explore BMAA distribution in pelagic and benthic Baltic Sea food
webs.
22
23
Methods
Sample pre-treatment and derivatization
Samples for the projects were either bought from commercial sources like
supermarkets (seafood for Papers III & IV, olive oil for Paper II) and
pharmacies (fish oil for Paper II) or received from collaborators (algae oil
for Paper II, cycad seeds for Paper III and Baltic Sea fauna for Paper V).
Main steps of sample preparation included homogenization, strong acid or
enzymatic hydrolysis, clean-up (filtering through spin filters, optional liquid-
liquid extraction with chloroform (LLE) and solid phase extraction (SPE))
followed by derivatization.
Rough homogenization was performed by mortar and pestle in liquid
nitrogen and was followed by sample homogenization by ultrasonication in
MilliQ water. Conditions of hydrolysis and the necessity of an LLE step
varied depending on the sample material and the detailed description can be
found in the corresponding papers (Papers I-V). SPE was always performed
according to the same protocol (column subsequent conditioning with
methanol and 0.1% formic acid in water, sample application, washing with
0.1% formic acid in water and methanol and elution with 5% NH4OH in
methanol). However, the derivatization step varied.
Different types of derivatization were studied and applied to address various
aspects of BMAA research. Briefly, a well-established derivatization
technique with commercial AccQ∙Tag Kit (AQC) from Waters was used in
Paper IV and Paper V to quantify BMAA in seafood and Baltic Sea food
webs respectively. Another derivatization technique with specially
synthesized N-hydroxysuccinimide ester of N-butylnicotinic acid (C4-NA-
NHS) was developed in Paper I and implemented in Paper II to quantify
BMAA extracted from spiked oil matrices. In Paper III, derivatization was
used to separate BMAA stereoisomers by adding FLEC reagent, which has
not been reported to be used previously with BMAA. The BMAA molecule
derivatized with each of three derivatization agents, used in the current
thesis, is shown in Figure 3.
24
Figure 3. BMAA (blue color) derivatized with AQC, C4-NA-NHS and FLEC reagents.
MS/MS fragmentation patterns shown for each compound.
UHPLC separation
In the framework of the current thesis, all chromatography was performed on
HPLC/UHPLC instruments equipped with different columns and using
different mobile phases according to the task. The following instruments
were used: Agilent G6410B Triple Quad LC/MS system (Santa Clara, CA,
USA); another system consisting of an Accela pump, a degasser and an
Accela auto-sampler coupled with TSQ Vantage triple quadrupole mass
spectrometer (Thermo Fisher Scientific, San Jose, USA) and another system
consisting of Acquity UPLC coupled with Xevo TQ-S micro mass
spectrometer (Waters, Milford, MA, USA).
For the separation of C4-NA-NHS and FLEC derivatized analytes a Hypersil
Gold C18 column (100×2.1 mm, 3 μm particle size; Thermo Scientific,
USA) was used. Elution was performed according to the linear gradient
25
elution program with a binary mobile phase (solvent A: 0.3 % or 0.075%
acetic acid in water; solvent B: 0.3 % or 0.075 % acetic acid in acetonitrile
for C4-NA-NHS or FLEC derivatives, respectively) at a flow rate of
300 μl/min. The first minute of elution was diverted to waste to avoid
introducing buffer salts into the mass spectrometer.
The AQC derivatized standards and analytes were separated on an ACCQ-
TAG ULTRA C18 column (100×2.1 mm, 1.7 µm particle size; Waters,
Ireland). Gradient elution with a binary mobile phase (solvent A: 0.3 %
acetic acid and 5% acetonitrile in water; solvent B: 0.3 % acetic acid in
acetonitrile) was run at a flow rate of 200 µl/min for 10 min and then 400
µl/min for 6 min. The post-column addition of solvent B at the flow rate of
600 µL/min was introduced to improve the detection. For the first two
minutes elution was diverted to waste to avoid introducing buffer salts into
the mass spectrometer.
MS/MS detection
Triple quadrupole mass analyzers consist of three four-rod quadrupoles
connected in series. The first and the third quadrupole are mass
spectrometers and the center quadrupole is a collision cell, where a collision
gas can be introduced and entering ions can undergo one or several
collisions93
.
In all studies, ion source was electrospray operating in positive ionization
mode (ESI+). Detection was performed in multiple reactions monitoring
(MRM) mode, where both first and third quadrupole were set to determine
ions with defined m/z. During the method development stage, product ion
scan mode was used to determine distinctive fragmentation patterns for each
compound derivatized with different tags, which means that the first
quadrupole selected the defined precursor ion and the third quadrupole
scanned m/z of all transmitted ions. In both modes, collision gas (argon) was
introduced to the second quadrupole (collision cell). For the consistency of
the results, collision energies and instrument parameters were tuned
26
specifically for each instrument. Schematic depiction of the triple
quadrupole instrument and its working modes can be seen in Figure 4.
A
B
Figure 4. Schematic diagram of a triple quadrupole instrument. A) Q1 and Q3 are mass
spectrometers, the center quadrupole, q2, is a collision cell (quadrupole using radio-frequency
(RF) only); B) Different operation modes of a tandem mass-spectrometer93.
To ensure that BMAA and each of its isomers were detected unambiguously,
four criteria were used for the identification: 1) compound retention time; 2)
presence of precursor ion; 3) presence of characteristic product ions (both
general and diagnostic); and 4) constant peak area ratio between two chosen
product ions. The complete workflow is summarized in Figure 5.
27
Figure 5. Diagram of sample preparation and analysis workflow, used in all Papers. Some
steps were excluded depending on the nature of the samples.
28
29
Results and discussion
Papers presented in the current thesis illustrate different aspects of analytical
chemistry: sampling; method development of sample treatment (Papers II
and IV) and instrumental analysis (Papers I, II, III and IV); method
application to matrices not previously analyzed for BMAA (Paper II);
method evaluation (Paper II, III and IV) and screening studies to deepen the
knowledge regarding BMAA natural distribution (Papers II, IV and V).
Developing novel derivatization methods
Paper I. Improved detection of BMAA using C4-NA-NHS for the localization
of BMAA in blue mussels.
In Paper I, chromatographic separation of BMAA and its structural isomers
was achieved after derivatization with C4-NA-NHS. Singly labelled species
of AEG and BAMA as well as doubly labelled species of BMAA and DAB
were predominant. For each compound, a characteristic fragmentation
pattern was obtained by MS/MS and diagnostic fragments were identified.
C4-NA-BMAA has three diagnostic product ions, at m/z 137.3, 192.9 and
249.1 (Figure 6), which allows more reliable determination of BMAA
compared to using only one diagnostic product ion at m/z 258.0 when AQC
derivatization is used6. It also allows quantification based on diagnostic ions,
which decreases the probability of false positive results, which can be rarely
done with AQC-derivatized BMAA due to the low abundance of diagnostic
product ion at m/z 258.0 compared to general product ion at m/z 119.0.
Calibration curves were produced for both C4-NA-NHS and AQC
derivatized BMAA standards in the range 100-1000 µg/L. These showed
that the curve for C4-NA-BMAA has a three-fold higher slope and thus
higher sensitivity (Figure 7). Instrumental limit of detection (LOD, defined
as S/N=3) of 40 μg/L was reached for C4-NA-BMAA.
30
Figure 6. Product ion spectra of doubly charged C4-NA-BMAA (m/z 221.2, CE=12, panel (a)
and singly charged AQC-BMAA (m/z 459.3, CE=20, panel (b).
Figure 7. Calibration curves for C4- NA-BMAA and AQC-derivatized BMAA. Higher
sensitivity is observed for C4-NA-BMAA.
31
The presence of BMAA in blue mussel tissues was detected using C4-NA-
NHS tag and two diagnostic ions were present for C4-NA-BMAA. For AQC
derivatized BMAA the diagnostic ion at m/z 258.0 was absent. This suggests
that signal enhancement through matrix effect is more pronounced for C4-
NA-NHS than for AQC derivatization probably due to the permanent
positive charge of the quaternary ammonium. In the electrospray ionization,
permanently ionized nicotine tag possibly facilitates charge repulsion, thus
favoring Coulomb explosion and faster desolvation. For AQC-derivatized
BMAA signal enhancement through matrix effect of around 10% was
observed and reported previously94
.
BMAA localization in blue mussels was investigated. Fresh mussels were
dissected in visceral (digestion organs, dark color) and non-visceral (flesh,
light color) parts and analyzed. BMAA was detected in both parts, which
indicates that depuration (method used to get rid of toxins from shellfish) is
unsuitable for BMAA removal.
Paper II. Analysis of BMAA in fish oil and its vegan alternatives
Although the method developed in Paper I showed promising results for
increased sensitivity in BMAA detection, it was not a quantitative study
because the tag used was not yet so well characterized regarding its stability.
Thus, the method did not employ the use of internal standard, which is
crucial for BMAA quantification in native samples, since it compensates for
analyte loss during sample preparation. Internal standard (d3-BMAA) was
introduced during further method development. The mass transitions for
deuterated BMAA have been determined at m/z 222.73>195.90 and
222.73>140.11, and the internal standard was successfully incorporated in
the method, which increased its reliability for quantification purposes. The
developed quantitative method accuracy was determined from quality
control (QC) samples as 94-99% and precision was 0.9-1.8% RSD. Method
limit of quantification (LOQ) was determined from low concentration (1-10
µg/L) standards S/N ratio. Standards of 10 µg/L demonstrated stable S/N>10
and thus 10 µg/L was determined to be LOQ concentration for the method.
32
Method performance was tested on food supplement samples. Since there are
evidences that BMAA might be present in the natural matrices in conjugated
forms, which are not protein-bound BMAA44,45
, determination of the
possible forms is an interesting challenge. In the current paper the focus was
on the oil matrices, which have not been studied for possible BMAA
presence before. Fish oil, algal oil and olive oil samples were subjected to
hydrolysis, which required the development of a new approach compared to
previous studies on BMAA quantification to hydrolyze lipids. The new
hydrolysis method was successfully applied and its efficiency was proven by 1H-NMR analysis. BMAA presence was not detected in any of the analyzed
samples.
Cycad seed samples (n=3) were analysed as a positive control and BMAA
presence was quantified as 180±11 µg/g wet weight, which is in agreement
with another BMAA quantification in the same material by another method
(Paper III). To further evaluate method reliability, all analysed food
supplements and olive oil (n=3 for each sample) were spiked with standard
L-BMAA solution (1000 µg/L) and subjected to the same sample preparation
protocol as the native samples. Subsequently, BMAA concentration was
quantified in the spiked samples, and the following recoveries were
obtained: 87% for fish oil; 95% for olive oil; 83% for pigment free algal oil
and 91% for algal oil.
Although it was clear that BMAA was not detectable in all analyzed
samples, a peak was observed on the AQC-derivatized algal oil sample
chromatogram corresponding to the diagnostic mass transition for BMAA
(459>258) at the retention time of BMAA (Figure 8). No peak was observed
for the general MRM transition of AQC-derivatized BMAA (459>119) as
well as no peaks of BMAA were observed in C4-NA-NHS-derivatized
samples. However, the presence of the potentially confounding interfering
peak for 459>258 transition introduces the risk of data misinterpretation and
potential false negative or false positive results. If BMAA were present in
the sample, the interfering peak would skew the peak area ratio between
general and diagnostic transitions of BMAA by contributing to the peak area
of diagnostic BMAA transition. This would lead to not meeting one of the
33
peak identification criteria (constant peak area ratio between two chosen
product ions) and therefore to the erroneous interpretation of data. In turn,
C4-NA-NHS derivatization does not share the described issue and could be
recommended as a complementary parallel method to the routine AQC
derivatization of BMAA, serving to resolve cases of having false negatives
or false positives.
Figure 8. Chromatograms of transitions
characteristic for BMAA, DAB and d3-
BMAA (IS) monitored in AQC-derivatized
algal oil sample. A peak for the general
transition of BMAA (459>119) is absent,
while a peak for diagnostic transition
(459>258) is present. This may introduce
false negative or false positive results in cases
when the sample indeed contains BMAA by
skewing the peak area ratio.
Figure 9. Chromatograms of transitions
characteristic for BMAA, DAB and d3-
BMAA (IS) in C4-NA-NHS-derivatized
algal oil sample. An unidentified compound
gives rise to the peaks around 7-7.5 min with
m/z transitions 221>193 and 221>123.
34
Although BMAA and its isomers were not detected in the C4-NA-NHS-
derivatized samples based on peak-identification criteria, the intense peaks
for the transitions 221>193 and 221>123 similar to BMAA and DAB,
respectively, were observed at the retention time around 7.0-7.5 min (Figure
9). These are actually suggestive indicators of possible unknown isomers.
Speculating on their identities, one can hypothesize that it can be doubly-
labelled BAMA. In a previous study (Paper I) BAMA was detected only as
singly-labelled species, but theoretical fragmentation pattern of doubly-
labelled BAMA (Figure 10) shows that the precursor ion at m/z=221 can
potentially fragment into products with m/z=193 and 123. According to
elution order of singly-labelled compounds determined in Paper I, BAMA
elutes after BMAA close to DAB. If doubly-labelled compounds follow the
same elution order, observed retention time is consistent for BAMA.
However, further studies should be carried out to clarify the identity of the
observed peaks and the identity of potential yet-unidentified BMAA
isomers.
Figure 10. Suggested fragmentation pattern for C4-NA-NHS-derivatized doubly-labelled
BAMA species. Parent ion mass m/z=221; fragment masses m/z=193, 179 and 57. A fragment
with m/z=123 can be obtained from m/z=179 by loss of alkyl group with m/z=57.
35
Paper III. Chiral separation of BMAA enantiomers after FLEC
derivatization and LC-MS/MS
In Paper III, we focused on establishing a simple and accurate method for
BMAA enantiomer analysis, since the existing methodologies do not address
resolving enantiomers to determine accurately the true amount of either L- or
D-BMAA. A key aspect in this study was the recognition that racemization
of BMAA occurs during strong acid hydrolysis, which is a widely used step
for measuring bound BMAA from complex matrices. Thus, it might be
recommended to use enzymatic hydrolysis with Pronase to avoid
racemization, which was done in the current study (Figure 11).
Standards and samples were derivatized with FLEC prior to UHPLC-ESI-
MS/MS to produce chromatographically separable derivatives of D- and L-
BMAA. FLEC derivatization converts enantiomers into diastereomers†, thus
resulting in different physico-chemical properties and allowing separation on
the common achiral chromatography column. We established quick and
clean separation of BMAA from its structural isomers as well as of BMAA
enantiomers. Useful fragment ions for confident identification and
quantification were determined. D-BMAA presence in cycad seeds was
discovered, which is an important novel finding. The enantiomeric
composition of BMAA in cycad seed was 50.13 ± 0.05 and 4.08 ± 0.04 µg
for L- and D-BMAA/g wet weight, respectively (n=3).
Although it was claimed that enzymatic hydrolysis should be preferred for
enantiomer quantification, it must be noted that it is less efficient than strong
acid hydrolysis. We quantified total BMAA in the Cycad sample after HCl
hydrolysis to be 133 µg BMAA/g wet weight and after Pronase hydrolysis it
was 54 µg BMAA/g wet weight. Our findings are in accordance with reports
on albumin hydrolysis, where the evaluated hydrolysis efficiency was 95%
and 25% for 6M HCl and Pronase, respectively95
. These findings indicate the
need for further development of the hydrolysis protocol, which will yield
higher outcome while simultaneously preserving the absolute configuration
† Diastereomers of FLEC-derivatized BMAA have different configurations at one of
the equivalent stereocenters and, in contrast with enantiomers, are not mirror images
of each other.
36
of enantiomers for unambiguous determination of those. Probably, other
enzymatic cocktails may be tested to find a more efficient one. Another
hydrolysis improvement option may include protein denaturation followed
by internal disulfide bonds reduction and subsequent alkylation to expose
more sites to proteolytical digestion95
.
Moreover, an experiment with cycad seed samples, incubated with the same
buffer, but without Pronase has been performed. Surprisingly, BMAA was
detected in those samples as well and was quantified to be 50 µg BMAA/g
wet weight. That can be interpreted as the fact that BMAA in the cycad seed
occurs in conjugated form, which not necessarily protein-bound, but rather a
fraction called soluble-bound in the literature44,45
.
Figure 11. MRM chromatograms of L-and D standard solutions (1 mg/L). A) L-BMAA and D-
BMAA, no treatment; B) L-BMAA and D-BMAA after acid hydrolysis demonstrate that
racemization occurs; C) L-BMAA and D-BMAA after enzymatic hydrolysis do not racemize.
The method evaluation parameters were evaluated from QC samples (n=5)
and were determined as follows: accuracy 98-108%, intraday precision 1.0-
37
2.7 RSD % and interday precision 1.3-2.4 RSD %. LOQ was estimated to be
0.1 mg/L for both L- and D-BMAA.
The proposed methodology can be used to determine the enantiomeric
composition of BMAA in other biological matrices and improve the
understanding of the contribution of enantiomers to the biological role of
BMAA and its toxicity. Despite that it has been believed that D-BMAA has
less pronounced to no toxic effect on the organisms46,57
, later studies
demonstrated that in vitro toxicity of D-BMAA on cell cultures is
comparable to the toxicity of L-BMAA80
. The same study reported that L-
BMAA conversion into D-BMAA occurs in vivo in mouse brain and liver as
well as in vervet monkey cerebrospinal fluid. That requires more
investigation of possible in vivo BMAA racemization and its toxicological
assessment.
Screening studies
Papers IV and V present screening studies, where BMAA was determined in
seafood from Swedish supermarkets as well as in pelagic and benthic food
webs of the Northern Baltic Proper.
Paper IV. Quantification of BMAA in seafood from Swedish markets
Taking into consideration that yearly seafood consumption in Sweden has
grown from 8.2 kg per capita in 2011 to 11.0 kg per capita in 2015 and is
still growing96
, it is crucial to determine potential dietary exposure to BMAA
through seafood. In Paper IV we present data on the occurrence of BMAA in
seafood available to the general public. Samples were obtained from popular
supermarkets in Stockholm. We detected BMAA in all blue mussel, oyster,
shrimp and plaice samples with little variation in concentrations between the
samples of the same species. We also detected BMAA in one out of three
samples of Baltic herring and char. Levels of BMAA in the positive-tested
seafood varied between 0.01 and 0.90 mg/kg seafood. However, no BMAA
38
was detected in crayfish and all other fish (salmon, cod and perch) samples
using the validated quantitative method with LOQ<0.01 mg BMAA/g wet
tissue.
Comparing with other studies containing quantitative data on BMAA in
environmental samples, our results are in the agreement with the studies
using LC-MS/MS for quantification8,97
and show lower values than those
obtained by quantification with LC-FLD method12,98
. This discrepancy adds
evidence for the tendency of LC-FLD method to give overestimated values
for BMAA concentration in the samples due to the method’s lower
selectivity. Nevertheless, the reason for the significant difference of several
magnitudes in the reported values could also be attributed, to a certain
extent, to the diverse nature of the samples (species, lifestyle, environmental
conditions).
Paper V. Is there BMAA transfer in the pelagic and benthic food webs in the
Baltic Sea?
In addition to BMAA screening in the seafood, it is important to elucidate
BMAA distribution according to trophic pathways. In Paper V, we analyzed
BMAA in defined pelagic and benthic food webs in the Baltic Sea (Northern
Baltic Proper) applying the same analytical method as in Paper IV. To
confirm that all animals in our study occupied the trophic positions as
expected and belonged to the same food web, we applied stable isotope
analysis (SIA). SIA uses elemental analyzer coupled to isotope ratio mass
spectrometer to detect ratios 14
N:15
N and 12
C:13
C, thus providing information
about trophic level of the analyzed organism to clearly distinguish producers
from consumers of various levels and define a food web99
.
In our study, only pelagic (phytoplankton, zooplankton and mysid shrimps)
organisms tested positive for BMAA, whereas none of the benthic
invertebrates and fish species as well as the sediment were found to contain
BMAA. In the BMAA-positive samples, the concentration varied between
7.5 to 18.1 µg/g dry weight, which is about 100-fold higher than previously
39
reported concentrations for BMAA in the Baltic Sea plankton by Jonasson et
al9. The conflicting results might be explained by the fact that BMAA
quantification in the Jonasson et al. study was based on a semi-quantitative
method, with no internal standard and no reported LOD. Their method
involved a clean-up procedure, which can introduce a significant (up to
90%44
) loss of BMAA. The absence of the internal standard that is subjected
to the same sample preparation procedures as the analyte can lead to the
underestimated concentrations of BMAA. For our study we used the
evaluated method28
with the reported LOQ<0.01 mg BMAA/g wet tissue.
Results suggest that BMAA was not present in the benthic food web, and
levels in the pelagic food web are below the risk level considering the effect
concentrations reported for invertebrates100–102
. However, to see the full
picture of BMAA distribution in various ecosystem compartments, a
systematic sampling throughout a year and in different parts of the Baltic
Sea should be conducted.
Other analytical strategies and applications
In addition to the studies described above, there are several on-going
projects that are important for BMAA quantification in the environmental
samples and understanding its natural pathways.
Investigation of novel derivatization possibilities
One of the studies addressed applicability of BMAA derivatization tags that
have not been used in the other studies of the thesis. These tags were N-
methyl-aminoquinolyl-N-hydroxysuccinimide (N-methyl-AQ-N-NHS) and
N-butyl-aminoquinolyl-N-hydroxysuccinimide (N-butyl-AQ-N-NHS)
(Figure 12). The idea behind designing these tags was to have permanent
positive charge and simultaneously introduce additional aromatic rings to
increase hydrophobicity of the molecule, which would be favorable for
ionization of the analyte in ESI source.
40
O
N+
N
O
O
O
CH3
N-methyl-aminoquinolyl-NHS N-butyl-aminoquinolyl-NHS
Figure 12. Alternative tags for BMAA derivatization. N-butyl-aminoquinolyl-NHS has
longer side chain, which increases the hydrophobicity of the molecule.
Using N-methyl-AQ-N-NHS for BMAA derivatization, we were not able to
detect any signal from BMAA or its isomers. However, analysis of N-butyl-
AQ-N-NHS BMAA-derivatives was far more promising. In Full Scan
positive ion mode, the molecular ion at m/z=271.0 was detected, which
corresponds to the BMAA molecule with the tag molecule attached to both
amino groups (doubly labelled and doubly charged species). Furthermore,
the product ion scan mode was applied to detect the fragmentation pattern of
the derivatized species. Two characteristic product ions at m/z 199.0 and
m/z 174.1 were determined at sufficient intensities to be qualified for using
in MRM mode.
Standard solutions of BMAA in concentrations ranging from 1 to 10 ppm
were derivatized with AQC, C4-NA-NHS and N-butyl-AQ-N-NHS and
analyzed in MRM mode to compare the performances of these tags. As seen
in Figure 13, BMAA derivatized with N-butyl-AQ-N-NHS demonstrated the
highest response at m/z 271.0>174.1, which is also higher than the response
from BMAA derivatized with AQC and C4-NA-NHS tags. More
development and evaluation is required to establish usable and robust
quantitative methods employing N-butyl-AQ-N-NHS for derivatization, but
this study shows that there is a potential for development of methods based
on novel derivatization techniques for BMAA.
O
N+
N
O
O
O
CH3
41
Figure 13. Calibration curves for BMAA (0.1-10 ppm) derivatized with AQC, C4-NA-NHS
and N-butyl-AQ-NHS tags with N-butyl-AQ-NHS demonstrating the highest signal.
Although novel derivatization strategies look promising, it must be stressed
that C4-NA-NHS and N-butyl-AQ-N-NHS tags are not yet commercially
available, but were custom synthesized for us. Thus, the stability and shelf-
life of the reagents have not been evaluated. During our studies involving
C4-NA-NHS derivatization agent, it was noted that proper storage conditions
(tube or vial with tight lock, covered in parafilm and aluminum foil, kept in a
dessicator) were crucial to preserve the reagent as stable as possible
preventing it from hydrolysis. The shelf-life of the reagent under proper
storage conditions was about 8-9 months and it cannot be recommended for
use after that time. The appearance of tag gradually changes with age from
dark-yellow coarse powder to dark-brown sticky liquid due to hydrolysis by
water vapor in the atmosphere, so the visual inspection of the tag is
suggested before use. Further detailed investigation should be performed to
determine storage time more precisely.
0
500000
1000000
1500000
2000000
2500000
0 2 4 6 8 10
Pe
ak a
rea
BMAA concentration, µg/L
AQC 459>258
C4-NA-NHS 221>193
N-butyl-AQ-NHS 271>174
N-butyl-AQ-NHS 271>199
42
Sea spray aerosol as possible exposure route to BMAA.
Potential aerosolisation of BMAA was studied using the Baltic
cyanobacterium Nodularia spumigena under controlled conditions. More
specifically, we aimed to evaluate whether these cyanobacteria and their
metabolites (such as BMAA) could be aerosolized in laboratory conditions.
The Baltic Sea is known to have extensive summer cyanobacterial blooms103
,
which are a growing environmental concern due to being harmful to a wide
range of organisms. The risks of marine toxins to humans have been
evaluated based on seafood consumption as the major route of human
exposure. However, inhalation of sea spray containing cyanobacteria or their
metabolites may be an alternative exposure pathway which has not yet been
studied.
Preliminary experiments, using a sea spray chamber (Figure 14) were
conducted.
Figure 14. Schematic picture of the sea spray chamber used to study BMAA aerosolisation104.
43
Figure 15. BMAA content on filters from the sea spray chamber. BMAA was detected on low
pressure impactor (LPI) stages 2, 7 and 14 (n=5) in different amounts. The medians are
marked with red horizontal lines, the ends of the box are the upper and lower quartiles and the
whiskers extend to the highest and lowest observations.
Briefly, artificial seawater was spiked with BMAA standard and d3-BMAA
was added as an internal standard. Sample was aerosolized in the sea spray
chamber with low pressure impactor (LPI) containing a set of filters with
different pore sizes (16 nm to 10 µm) and arranged with 14 levels of
decreasing pore size. BMAA was collected from several levels of filters,
namely 16 nm, 250 nm and 2.46 µm, and analyzed with UHPLC-MS/MS on
a system consisting of an Accela pump, a degasser and an Accela auto-
sampler coupled to TSQ Vantage (Thermo Fisher Scientific, San Jose, USA)
triple quadrupole mass spectrometer.
The results showed that different amounts of BMAA could be found on the
filters with the certain diameters (Figure 15). Highest BMAA concentration
was detected on stage 7 with pore size 250 nm, while filters on stage 2 (pore
size 16 nm) and stage 12 (pore size 2.46 µm) showed values close to blank.
44
These results point to the fact that filter’s size for BMAA analysis in
aerosols should be selected very carefully and suggest that 250 nm pore size
is suitable for BMAA collection from aerosols. Thus, previous studies that
report poor BMAA aerosolisation14,34
may have underestimated BMAA
concentration due to unsuitable pore size of the filters used (100 µm).
Next steps to be taken in this project include use of cyanobacterial culture at
different stages of the bloom in the sea spray chamber to determine the
BMAA transfer to aerosols as a function of the cyanobacterial abundance
and bloom stage.
45
Conclusions
The current thesis covered analytical aspects of BMAA research, more
specifically the development of new, more sensitive analytical methods for
BMAA quantification in biological matrices as well as the application of
developed and existing methods to natural samples to address issues on
BMAA distribution and possible public exposure.
A new method for BMAA quantification using C4-NA-NHS derivatization
agent was developed. It showed better sensitivity in comparison with
routinely used AQC derivatization, due to the permanent charge, which
facilitates compound detection in MS. The developed method was tested on
blue mussel samples and oil samples. Repeatability and accuracy of the
method were evaluated. The developed method can be recommended as a
complementary technique to the routine AQC analysis of BMAA to avoid
false negative or false positive results.
Another quantitative method was developed for the chiral analysis of
BMAA. BMAA optical isomers were successfully separated on a C18
column after derivatization with FLEC reagent. The study provided a crucial
observation that, to avoid racemization, enzymatic hydrolysis should be used
instead of acidic hydrolysis when chirality of the molecule is important. The
method was applied to quantify BMAA in cycad seeds, and the
concentrations were 50.13 ± 0.05 and 4.08 ± 0.04 µg of L- and D-BMAA/g
wet weight, respectively.
Studies for BMAA detection and quantification in seafood from Swedish
supermarkets and Baltic Sea food webs were also carried out. It was
demonstrated that BMAA is present in bivalves (mussels and oysters) and
shrimps as well as in several fish species (plaice, herring and char) in low
µg/g wet weight concentrations. In the Baltic Sea food webs, BMAA was
detected in the pelagic food chain (phytoplankton, zooplankton and mysids)
at 0.75-2.0 µg/g wet weight concentrations, but not in the benthic system;
moreover, no detectable levels were found in fish.
46
47
Further method development & application
Undoubtedly, the BMAA research field is fast-developing and challenging in
many ways. There are still more questions than answers, and due to possible
links to neurodegenerative diseases and ubiquity of BMAA, it remains a
highly relevant topic. Here are some suggestions about next steps to be taken
in analytical method development, application and evaluation in BMAA
studies.
Analytical method development
Further investigations regarding the identity of potential yet-unidentified
BMAA isomers should be carried out based on the observations made in
algal oil samples.
For the chiral analysis of BMAA, it would be beneficial to find the
appropriate enzyme cocktail or to develop an additional treatment method
for efficient hydrolysis without racemization.
Novel derivatization tags should be explored and tested on natural
samples to further improve selectivity and sensitivity of the quantification
methods.
Novel derivatization tags should be explored as a possible route to more
sensitive and robust analysis. Deuterated tags105
, such as isotope-coded
affinity tag (ICAT) and isobaric tags for relative and absolute quantitation
(iTRAQ), might be useful for labeling BMAA from different samples,
which are then pooled and analyzed by LC-MS/MS. This approach will
allow comparing BMAA concentrations in different sources while also
reducing the time required for the analysis.
Method application
For the production of food supplements, BMAA should be monitored at
every production step, including by-products, to get the full picture of
possible exposure routes.
48
Aerosolisation of BMAA should be studied as a possible exposure route
through inhalation. It is important to choose correct filter sizes for the
aerosol collection to perform accurate evaluation.
Chiral composition of BMAA in natural samples should be studied more
extensively, thus elucidating BMAA’s natural role.
Method evaluation
Collaborative efforts and interlaboratory projects should be directed
towards evaluation and validation of existing and newly developed
methods for BMAA quantification. This will reduce controversy about
the results and allow mapping BMAA distribution in various ecosystems
and relevant pathways.
49
Acknowledgements
My more-then-five-year PhD studies journey is coming to its end, and
before arrival to the PhD defense destination I would like to take the
opportunity and thank everyone who has been there on the road together
with me: during all time, part of the journey or just for a couple of stations.
First of all, I would like to thank the Department of Environmental Science
and Analytical Chemistry for the opportunity to study here and grow both
scientifically and personally in a friendly and inspiring atmosphere. I am
especially grateful to the Head of the Unit of Analytical Chemistry Cynthia
de Wit for her understanding, support and kindness.
I am deeply thankful to my supervisor Leopold L. Ilag for his guidance,
genuine support and scientific enthusiasm. It was an honor and a pleasure to
work with you during these years.
I was lucky to have three co-supervisors during the PhD studies and I thank
Ulrika Nilsson, Ingrid Granelli and Elena Gorokhova for their
involvement in my work at different stages and their readiness to help
whenever I needed that.
The PhD studies gave me a gift of meeting and collaborating with wonderful
people and I am thankful for the opportunity to know all of you: Anders
Colmsjö, Conny Östman, Magnus Åberg, Leopold L. Ilag, Roger
Westerholm and Gunnar Thorsén (thank you both for sharing teaching
experience in the department courses), Ulrika Nilsson, Ingrid Granelli, Jan
Holmbäck (thank you for introducing me to lipid chemistry), Vibhu Rinwa,
Karin Englund, Carlo Crescenci, Lena Elfver and Jonas Rutberg (thank
you both for your invaluable help with papers and documents), Emilia
Eklund, Rudolf Andrýs, Isabella Karlsson, Anna Sroka-Bartnicka,
Cynthia de Wit, Margareta Törnqvist, Lillemor Asplund, Hitesh
Motwani, Jenny Aasa, Henrik Carlsson, Ioannis Athanassiadis, Elena
Gorokhova, Oskar Sandblom and Matthew Salter.
50
I thank Oskar Karlsson, Hitesh Motwani, Elena Gorokhova, Cynthia de
Wit and Javier Zurita for the valuable comments on this thesis, which
certainly made it better.
I thank Tanja Russita for creating a very special cover image for my thesis.
I also thank all PhD students for being friendly, open and ready to help.
Most of you are already doctors and it was a pleasure to share our paths and
to witness your achievements: Aljona (thank you for sharing your
experience with instruments and being very supportive at the beginning of
my studies); Liying (thank you for the inspiration and support you gave me
and for our collaboration); Javier (thank you for all the work we did
together and for being so kind); Petter, Giovanna, Erik and Nicolò (thank
you all for sharing the room and having nice chats); Silvia, Rozanna, Jonas,
Hwanmi (thank you for nice conversations and help), Mohammad, Farshid
(thank you for sharing your knowledge and time so generously and for being
so helpful), Sara, Aziza, Trifa, Ioannis and Ramzy. I wish all the best to
the continuing PhD students and thank you for the time spent together:
Alessandro (thank you for the collaborations and always being ready to
help), Pedro (thank you for sharing a room and nice Lego-chats), Francesco
(thank you for sharing experience and helping me with Xevo), Hatem,
Josefine (thank you for nice health-walks and for the Swedish translation of
my abstract), Nikola (thank you for help with aerosols), Nicklas (thank you
for help with the sample preparation), Johan and Lorena.
Being teaching assistant was an important part of my PhD studies, so I want
to thank all the students I met in the courses as well as the exchange students
from France. I am particularly thankful to Patrik, Emil, Petra, Nargiza,
Maïwenn, Lea and Anthony. I learned a lot while teaching you.
I want to thank the external collaborators for their significant input in our
shared projects: Rudolf Andrýs, Klaas Verschueren, Wim M. De
Borggraeve, Johan Rosén, Agnes Karlson, Andrius Garbaras, Simoné
Downing, Laura L. Scott, Timothy G. Downing, Sara Sigurlásdóttir and
Elisabeth J. Faassen as well as organizers and participants of Cyanocost
Training School in Wageningen (2015).
51
My special thanks are for Zhongping Yao from Hong Kong Polytechnic
University who was supervising me during three-month internship in Hong
Kong and to the members of his research group who I was collaborating
with: Betty, Ho Yi and Tommy.
I am sincerely grateful to my friends who were always there for me and gave
me so much love and support: Aleksandr, Juta, Esther, Artur and Julia.
My very special thanks are to my family who always believes in me. I thank
my parents Victor and Lyubov for supporting me and being truly
understanding. I always feel your love no matter the distance.
I deeply thank my husband Petr for his strength, care and support. I am so
happy to share my life and my love with you.
52
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