PALYTOXINS: BIOLOGICAL AND CHEMICAL DETERMINATION
Pilar Riobó* and José M. Franco
Instituto de Investigaciones Marinas. CSIC. Eduardo Cabello, 6. CP36208 Vigo,
Spain.
*Corresponding author. Tel.: 0034986231930; Fax: 0036986498626
E- mail address: pilar.riobo@vi.ieo.es
ABSTRACT:
Palytoxin (PLTX) is a marine polyether toxin with a very large and complex
molecule that has both lipophilic and hydrophilic areas. It presents the longest
continuous carbon atoms chain known to exist in a natural product second only
to maitotoxin.
This toxin was first isolated from Palythoa toxica and was subsequently
reported in dinoflagellates of the genus Ostreopsis. Although PLTX has so far
been associated with ciguateric fish poisoning (CFP), recent evidence suggests
that PLTXs should be excluded from CFP toxins. NMR and LC-MS/MS
techniques have enabled the isolation of 10-15 new analogues from
dinoflagellates ever since their first discovery.
Literature data on biological origin, poisonings and chemistry of certain naturally
occurring PLTX analogues, commonly known as ostreocins, are detailed herein.
This paper reviews all reported biological and chemical analysis methods to
date for this group of compounds.
KEYWORDS:
Ostreopsis, matrix, Palytoxin (PLTX), mouse bioassay (MBA), haemolytic
assay, cell cultures, immunoassays, liquid chromatography (LC), ultraviolet
detection (UV), fluorescence detection (FLD)
INTRODUCTION
PLTX is one of the most poisonous non-protein substances known to date. It
was first isolated and purified from corals belonging to the family Zoanthidae,
order Zoantharia and phylum Coelenterata (Moore et al. 1971)., This zoanthid
was subsequently identified as Palythoa toxica (Walsh et al. 1971). PLTX was
also found in other zoanthids such as P. tuberculosa, P. mammilosa and P.
caribeaorum (Kimura et al. 1973; Attaway et al. 1974; Beress et al. 1983) in
different locations. The structure of PLTX was elucidated 10 years later by two
independent groups, one led by Professor Hirata in Japan (Uemura et al. 1985)
and the other by Professor Moore at Honolulu in the United States (Moore
1985).
The PLTX-group toxins are complex polyhydroxylated compounds with both
lipophilic and hydrophilic areas. They are white, amorphous, hygroscopic solids,
which have not yet been crystallized. They are insoluble in non-polar solvents
such as chloroform, ether, and acetone, are sparingly soluble in methanol and
ethanol and soluble in pyridine, dimethyl sulfoxide and water.
At least 8 different PLTX analogues are known: PLTX, ostreocin D, ovatoxin-A,
homopalytoxin,
bishomopalyloxin,
neopalytoxin,
deoxypalytoxin
and
42-
hydroxypalytoxin. Chemical structure has only been characterised for PLTX (J.
K. Cha et al. 1982), ostreocin-D (Ukena et al. 2001) and 42-hydroxypalytoxin
(Ciminiello et al. 2009). The chemical formula of PLTX is C129H233N3O54, 115 of
the 129 carbons are in continuous chain. The basic molecule consists of a long,
partially unsaturated aliphatic backbone containing cyclic ethers, 64 chiral
centres, 40-42 hydroxyl and 2 amide groups Fig.1. The primary amino-group at
the C-115 end of the molecule accounts for the basicity of PLTX-group toxins.
The molecular formula and molecular weight of PLTX analogues differ
depending on the Palythoa species from which they are obtained and range
from 2659 to 2680 Da (Moore et al. 1981). It was found to exist as a dimer in
aqueous solution with a molecular weight of 5700 Da (Uemura 2006; Inuzuka et
al. 2008).
The first reference of PLTX in a marine organism other than zoanthids is a fish
named Alutera scripta, which caused death in pigs fed on the same, in
Okinawa, Japan (Hashimoto et al. 1969). The toxin was later identified in other
species of anemones, fishes, crabs and sea urchins (Gonzáles et al. 1977;
Fusetani et al. 1985; Yasumoto et al. 1986; Fukui et al. 1987; Alcala et al. 1988;
Kodama et al. 1989; Mahnir et al. 1992; Gleibs et al. 1995; Granéli et al. 2002;
Taniyama et al. 2002).
The presence of PLTX and its derivatives has been well documented in
invertebrates (Yasumoto et al. 1986; Alcala et al. 1988; Aligizaki et al. 2008), in
fish (Fukui et al. 1987; Gleibs et al. 1999; Taniyama et al. 2001; Taniyama et al.
2003) and also in benthic dinoflagellates belonging to the genus Ostreopsis
(Nakajima et al. 1981; Yasumoto et al. 1987; Quod 1994; Tognetto et al. 1995;
Usami et al. 1995; Onuma et al. 1999; Rhodes et al. 2000; Pearce et al. 2001;
Ukena et al. 2001; Vila et al. 2001; Granéli et al. 2002; Rhodes et al. 2002;
Ukena et al. 2002; Sansoni et al. 2003; Simoni et al. 2003; Taniyama et al.
2003; Turquet et al. 2003; Fraga et al. 2004; Lenoir et al. 2004; Riobó et al.
2004; Riobó et al. 2004; Penna et al. 2005; Ciminiello et al. 2006; Morton et al.
2006; Riobó et al. 2006; Zingone et al. 2006).
Six of the nine currently recognised species are toxic and produce PLTX related
compounds. Ostreocin-D, an analogue of PLTX, was identified and isolated in
Ostreopsis siamensis (Usami et al. 1995). In recent years, Ostreopsis ovata
blooms in the Mediterranean region were found to cause respiratory illnesses
due to inhalation of aerosols released during such blooms (Bottalico et al. 2002;
Sansoni et al. 2003; Simoni et al. 2003; Simoni et al. 2004; Ciminiello et al.
2006).
However, it is yet to be demonstrated that the dinoflagellate is the primary
producer, because toxin levels found in polyps are not related with the presence
of symbiotic microalgae (Gleibs et al. 1995). Bacterial origin of PLTX has been
suggested by Frolova and collaborators based on results from a PLTX-sensitive
immunoassay (Frolova et al. 2000). Recently, the group of Seeman suggested
that bacteria might be the original producers of PLTX (Seemann et al. 2009). In
their work, bacteria from two zoanthid corals (Palythoa caribaeorum, Zoanthus
pulchellus) and one sponge (Neofibularia nolitangere) were isolated in order to
investigate a possible microbial origin of PLTX. A newly developed PLTX blood
agar assay was applied to screen toxin production and potential PLTXproducing bacteria were identified by 16S r RNA gene sequence analysis and
phylogenetic tree construction.
PLTX shows remarkable biological activity even at very low concentration
(Moore et al. 1971). Lethal doses of PLTX through intravenous administration in
rats, mice, guinea pigs, rabbits, dogs and monkeys ranged between 0.03 and
0.45 µg/Kg (Wiles et al. 1974). By extrapolation, a toxic dose in a humans would
range between 2.3 and 31.5 µg (Uemura 1991).
This toxin and its analogues have become a global concern due to their effects
on animals and especially on humans. In this sense, human fatalities arising
from consumption of seafood suspected to be contaminated with PLTX have
been reported after consumption of crab (Alcala et al. 1988), sardine (Onuma et
al. 1999), smoked fish (Kodama et al. 1989), groupers (Taniyama et al. 2002)
and parrotfish (Okano et al. 1998). One of the most commonly reported
complications arising from consumption of these fishes appears to be
rhabdomyolsis (Deeds et al. 2010). Symptoms associated with palytoxin
exposure vary greatly depending upon the exposure route. Mortalities in
humans have only occurred due to ingestion but a variety of additional nonlethal symptoms have been observed upon ingestion: dermal, ocular and
inhalational exposure in humans. Systemic PLTX effects have been reported
through dermal contact with marine aquarium zoanthids (Hoffmann et al. 2008;
Deeds et al. 2010), including an unusual case of inhalation exposure (Majlesi et
al. 2008). Many other anecdotal evidences of intoxications related with
aquarium zoanthids can be found in online marine aquarium forums (Deeds et
al. 2010)
There is no recognized official method to date for the determination of this toxin.
Each laboratory, according to its ability, develops its particular methodology,
through a combination of methods, analytical and/or biological testing in order to
confirm the presence/absence of PLTX in a sample.
Moreover there are no regulations on PLTX-group toxins in shellfish, either in
the EU, or in other regions of the world. During the first meeting of the working
group on toxicology of the national reference laboratories (NRLs) for Marine
Biotoxins (Cesenatico, Italy, 24-25 October 2005), a provisional limit of 250
μg/kg shellfish was proposed by the Community Reference Laboratory for
Marine Biotoxins. Several published methods exist for the determination of
PLTX, but none of them have been formally validated in inter-laboratory
validation studies, probably due to the lack of certified standards and certified
reference materials for this group of toxins.
Little is known about the real consequences that these toxins may have on
coastal communities despite the obvious acute biological impact of PLTXs.
There are ongoing attempts to develop a validated assay for rapid, sensitive
and specific detection of PLTXs because of human health risks. None of the
PLTX determination methods are able to meet all requirements per se, and so a
combination of fast and confirmatory methods still seems to be the most
appropriate approach for monitoring purposes. This special issue describes
analytical methods and biological assays that are currently available for
determination of PLTXs, with special emphasis on their main advantages and
disadvantages.
PALYTOXIN EXTRACTION.
Most of extraction and purification processes for biological and chemical
determinations of PLTXs follow a general procedure with slights modifications.
Ethanol and methanol are the most common solvents used to extract PLTX, but
the toxin is quite soluble in water or other water-miscible solvents too. The
following steps include partitions with hexane and butanol and finally SPE
cartridges or flash chromatography (Moore et al. 1971; Kimura et al. 1973; Teh
et al. 1974; Beress et al. 1983; Uemura et al. 1985; Yasumoto et al. 1986; Fukui
et al. 1987; Alcala et al. 1988; Hirata et al. 1988; Mahnir et al. 1992; Lau et al.
1993; Lau et al. 1995; Onuma et al. 1999; Oku et al. 2004).
Depending on the matrix, exhaustive extraction and purification processes may
be required. In this sense, polyps, molluscs, crabs and fish are the most
complex ones. Extraction method can be simplified when dinoflagellates
samples are obtained from cultures or seawater since the matrix is less
complex and the amount of lipophilic compounds is smaller than in molluscs
and fish. Thus, a simple extraction performed with methanol following by a later
partition with hexane is enough for their determination by LC FLD or LC - MS.
(Riobó unpublished).
PLTX dissolved in seawater has never been detected during Ostreopsis
blooms. Extraction from this matrix is complicated because of the presence of
low toxin levels in seawater and high concentration of salts. Recently, extraction
of PLTX from growth media of Ostreopsis ovata cultures (945 ml) after
removing Ostreopsis cells was performed using an equal volume of butanol for
three times. The butanol layer was evaporated to dryness, then dissolved in 5
ml of methanol/water (1:1, v/v) and analyzed directly by LC-MS (Guerrini et al.).
Likewise, the presence of PLTX in environmental aerosol samples has not yet
been demonstrated despite respiratory intoxications reported.
BIOLOGICAL DETECTION METHODS.
Detection of PLTX in biological samples can be accomplished by both
instrumental means and biological assays. While chemical determination
methods are needed to confirm their presence, biological tests allow detection
and sometimes quantification. These methods have the advantage of defining
characteristic symptoms of different complexity models (mice, cells, etc...)
because they use the functional properties or biological activities of the toxin.
However, a combination of methods is needed to confirm the presence of the
toxin. Methods listed in this section have been successfully used to detect
PLTX, and some of them are highly sensitive.
MOUSE BIOASSAY
Mouse bioassay (MBA) is one of the simplest ways to detect the presence of
PLTX and analogues in biological samples. Despite ethical and logistical
problems inherent in bioassays with mammals, such tests are widely used in
marine phycotoxins monitoring due to their reliability. The main advantage over
physical-chemical analysis or in vitro methods is that toxicity can be considered
directly proportional to the toxic effects in humans because these assays
provide a measure of total toxicity based on the biological response of animal to
these toxins. Knowledge of potential global toxicity is therefore a priority in
monitoring programs to protect human health. They are fast and do not require
expensive equipment or complex sample preparation processes. Furthermore,
determination of toxicity does not require availability of standards for all
analogues with toxicological concern as would normally be the case with
instrumental analytical methods. Bioassays can therefore check presence of
toxic compounds including poorly defined or unknown ones from a little-known
matrix which could produce an impact on public health.
The MBA for PLTXs is based on the neurotoxic effect caused by an organic
extract obtained from a biological sample, which is dried and re-suspended in
aqueous Tween 60 1% solution following the protocol described for lipophilic
toxins (Yasumoto et al. 1978). In this case, multiple interferences may occur
from domoic acid (DA), Saxitoxin (STX)-group toxins, yessotoxin (YTX)-group
toxins and cyclic imines (CI). Interferences can be reduced using the protocol
described by (Taniyama et al. 2002), but still include YTX- group toxins and the
water-soluble toxins. Another significant disadvantage of the MBA in general, is
the inherent variability in results between laboratories due to, for example,
specific animal characteristics (strain, sex, age, weight, general state of health,
diet, stress, etc)
Bibliography on MBA for PLTXs is not very clear in relation to definition of
relevant parameters such as mouse unit (MU), detection limit (LOD), LD 50 value
(which ranges between 150 and 720 ng/µL) and observation time in mice (from
4 to 48 h) (Ballantine et al. 1988; Onuma et al. 1999; Tan et al. 2000; Rhodes et
al. 2002; Taniyama et al. 2002; Taniyama et al. 2003; Riobó et al. 2007)
This method uses mouse lethality through intraperitoneal (i.p.) injection of the
sample. The initial distinguishing symptoms seen in mice with PLTX are really
important because these are always present regardless of whether mice die or
survive (Riobó et al. 2007). All mice show stretching of hind limbs, lower backs
and concave curvature of the spinal column within 15 minutes of i.p. injection of
PLTX. When survival time of mice is less than an hour, mice show sudden
jerking movements, stretching of hind limbs and lower back, weakening of
forelimbs, ataxia, decreased locomotion, convulsions, gasping for breath, and
finally death. However, if they survive for more than one hour, time of death
varies considerably and overlaps for the different concentrations (Riobó et al.
2007). In short, MBA provides a measure of total toxicity based on biological
response of the animal to the toxin(s) and it does not require complex analytical
equipment. These advantages are important for monitoring programs. However,
there are several reasons that make MBA undesirable. It cannot be automated,
it requires specialised animal facilities and expertise, the injection volume of one
mL exceeds good practice guidelines (<0.5 mL) which are intended to minimise
stress in mice, the inherent variability in results between laboratories due to
specific animal characteristics and ethical reasons.
HAEMOLYSIS ASSAY
PLTX is a potent but slow haemolysin in pigs, rats, mice, rabbits, guinea pigs,
and in human erythrocytes. Their haemolytic action was exploited to develop an
assay in order to detect the toxin.
Methods based on the haemolytic effect depend on the same molecular or
toxoforic architecture as in the case of neural failure in mice. Haemolysis due to
palytoxin produces degenerate sigmoid profiles over time due to the population
nature of the dose-response phenomenon. This is because affinity between
receptor and toxin are not constant in all erythrocytes but varies with probability
distribution within the assay erythrocyte population (Riobó et al. 2008).
Assays to detect the haemolytic activity of palytoxin are normally carried out
following Bignami's method (Bignami 1993). In the original method, whole blood
from a mouse was collected from the rail vein and diluted 1:9 in phosphate
buffered saline (pH 7.0), followed by red cells separation from plasma by
centrifugation. Such erythrocytes were washed once with buffer, and the cell
buffer was diluted in medium containing 5% (v/v) foetal bovine serum. For the
haemolysis neutralization assay, phosphate buffered saline (PBS) was
supplemented with 0.1% (w/v) bovine serum albumin (BSA), 1mM calcium
chloride, and 1mM sodium tetraborate (pH 7.2). Blood suspension was diluted
1:49 in this buffer with or without a PLTX monoclonal antibody. Samples of the
blood cell suspension with and without toxin-containing solutions were mixed
and incubated at 37°C for up to 24 h. After incubation, samples were
centrifuged and supernatants were used to measure the absorption at 540-595
nm. Haemolysis evolution over time needs to be evaluated by varying the
incubation time. The amount of haemoglobin released was found to be time and
concentration dependent. Toxin concentration in the sample can be determined
by incubating red cells with PLTX standards followed by measurement of the
amount of haemoglobin released after a fixed period of time. The haemolysis
effect of PLTX is specifically inhibited by ouabain (Habermann et al. 1982), a
glycoside poison that binds to and inhibits the action of the Na+/K+ pump in the
cell membrane. PLTX also binds to the sodium pump and converts the enzyme
into a channel. PTX and ouabain binding sites are not identical but share some
structural determinants for binding (Artigas et al. 2003; Artigas et al. 2006).
In this sense the specific presence of PLTX or its analogues in the sample can
be demonstrated by preventing haemolytic activity of the toxin with ouabain or
with a palytoxin monoclonal antibody.
PLTX toxicological dynamics inhibition with ouabain was studied by (Riobó et al.
2008). Results obtained were used to define a sensitive and reliable assay
which, under moderate temperature and partial inhibition of the PLTX with
ouabain, would permit one to obtain responses that give consistent and precise
parametric estimations. Blood is a critical element in this method. So, LOD
would vary depending on the origin of the erythrocytes (sheep, horse, rabbit,
pig, mouse, human, etc) and other parameters like age, sex… LOD value is
nevertheless around 0.5 pg, which is still below than in other methods (Riobó
unpublished).
CYTOTOXICITY ASSAYS
Cell toxicity assay is an alternative method to the use of animals in toxicity tests.
It is based on morphologic changes caused by the toxin and permits detection
of PLTX concentrations in the picomolar range. Use of microplate formats
enable multiple samples to be analysed in a single run. Cytotoxicity assays
however have some disadvantages: a) facilities are needed for maintenance
and handling of cell cultures, b) they do not provide any information on toxin
profile, and c) possible interference from other toxins (OA, AZA, YTXs…)
require confirmation of any positive results by chemical analysis.
A rapid release of K+ from cells appears to be the primary action of PLTX when
causing cytotoxicity. PLTX induces the release of potassium before the onset of
other secondary effects such as haemolysis or the inhibition of Na+-dependent
processes (Habermann et al. 1981). Measurement of K+ released from cells is
perhaps the simplest and most sensitive method for the assay of palytoxin and
can be carried out using a flame photometer. Such release is concentration
dependent and sensitivity of this method is approximately 1 pM for PLTXinduced release of K+ in rat and human erythrocytes (Habermann 1989).
Several types of cells including Hela cells (Lau et al. 1995), rat 3Y1 cells (Oku
et al. 2004) and the MCF-7 breast cancer cell line (Bellocci et al. 2008; Sala et
al. 2009) have been used to evaluate the presence of the toxin. Cells are
monitored for the characteristic morphological changes and cell damage in the
presence of toxin. Quantification of cell damage can be done by using dyes that
are absorbed by intact cells (e.g. neutral red) or by damaged cells (e.g. trypan
blue). These dyes can then be released and their intensity measured in a
spectrophotometer. Cytotoxicity
can be evaluated either through release of
lactate dehydrogenase (LDH) (Lau et al. 1995; Bellocci et al. 2008) or by using
the MTT-microculture tetrazolium assay (Oku et al. 2004).
Several groups are currently working with cytotoxicity assays that involve the
use of neuroblastoma cells and include ouabain pre-treatment. They were able
to detect PLTX-group toxins at concentrations of around 50 µg PLTX/Kg
shellfish tissue (Cañete et al. 2008; Espiña et al. 2009; Ledreux et al. 2009).
Cañete and Diogène (2008) and Ledreux et al (2009) used Neuro-2a cell-based
bioassays and estimated the cell number by using the MTT assay for
mitochondrial oxido-reductase activity. Espiña et al (2009) used BE(2)-M17
human neuroblastoma cells and added alamar blue to measure mitochondrial
oxido-reductase.
IMMUNOASSAYS
Several antibody-based methods have been used for detecting the PLTX-group
toxins. A radioimmunoassay for the detection of the PLTX-group toxins was
developed by Levine et al. in 1988,. They labelled PLTX with
I25I-Bolton-Hunter
reagent on its terminal amino group. The method was very sensitive and
permitted detection of PLTX in the picomolar range. However, it was not
possible to distinguish between biologically active and inactive palytoxins. Four
years later, Professor Bignami’s group developed a sandwich enzyme-linked
immunosorbent assay (ELISA) (Bignami et al. 1992) where select antibodies
were used to develop five PLTX-specific ELISA formats for PLTX quantification
in crude extracts of P. tuberculosa, wherein they detected 10 pg PLTX per test.
This immunoassay was further improved by Frolova (Frolova et al. 2000), who
developed a competitive ELISA using the intact toxin as a coating antigen for
detecting PLTX in the range of 6-250 ng·mL-1. Recently, a rapid isolation of
single-chain antibodies by phage display technology directed against PLTX has
been developed (Garet et al. 2010),. Results obtained with an immunoassay
competitive ELISA using this PLTX8 phage antibody showed that this phage is
able to specifically detect PLTX with a LOD of 0.5 pg·mL -1. These methods are
fast, easy to use, and can be applied to screen many samples for possible
further confirmatory tests. However, antibodies are not readily available, they do
not provide any information on the toxin profile and the accuracy is questionable
since cross-reactivity does not necessarily reflect toxic activity.
PALYTOXIN CHEMICAL ANALYSIS
Chemical determinations are mainly based on a previous separation and later
detection of the analytes. Liquid chromatography (LC) with ultraviolet (UV) or
fluorescence (FLD) detection is widely used in laboratories. However, thin layer
chromatography (TLC) is not widely used at present, and is restricted to
isolation and purification processes.
HPCE-UV and HPLC-UV
UV detection has been applied in separations by means of capillary
electrophoresis (CE) (Mereish et al. 1991) and LC. When diode array detection
(DAD) is used, the PLTX UV spectrum shows two absorption peaks contributed
by the two chromophores at 233 and 263 nm Fig.1. This characteristic UV
absorption profile is a parameter that can be used to verify the presence of toxin
in samples.
(Riobó et al. 2006) used HPLC-UV and detected the presence of PLTX -like
substances with the same UV-spectra and retention times as found in literature
reference PLTX, in cultures of O. ovata and O. cf. siamensis isolated from Brazil
and the western Mediterranean Sea. The LOD for PLTX standard was about 1–
2 μg/injection, however, they were unable to detect a peak and obtain a
spectrum for confirmation with some toxic Ostreopsis extracts. There are no
reports on HPLC-UV methods for quantitative determination of PLTX and
analogues in shellfish samples probably due to interferences presented in the
biological matrix that notably diminish method sensitivity (Noguchi et al. 1987).
It is unlikely that they could be routinely used for regulatory monitoring of PLTX
and its analogues in shellfish tissues.
On the other hand, (Lenoir et al. 2004) analyzed the toxically active n-butanol
soluble extract from cells of O. mascarenensis from a natural bloom in the
Southwestern Indian Ocean. Comparison of both, retention time and UV
spectra, obtained with sample and reference PLTX by HPLC-UV coupled with
DAD, enabled verification of the presence of two different PLTX analogues
called mascarenotoxin-A and -B. However, the methodology based on UV
detection is unsuitable for shellfish analysis because of the strong matrix effect.
HPLC-FLD
It is widely recognized that HPLC-FLD methods are much more sensitive than
HPLC-UV methods. Therefore, by taking advantage of the presence of one
amino terminal group in the PLTX molecule Fig.1, a pre-column derivatization
method for the separation and quantification of PLTX has been established by
Riobó and collaborators (Riobó et al. 2006). They derivatized the molecule
using
the
derivatization
reagent
6-aminoquinolyl-N-
hyroxysuccinimidylcarbamate (AccQ) followed by reverse-phase HPLC analysis
with FLD detection (ex 250 nm and em 395 nm). This method is suitable for
determining and quantifying PLTX toxins in samples of benthic dinoflagellates of
the genus Ostreopsis (Riobó et al. 2006) and also in shellfish samples (Riobó
unpublished).
Results correlated well with those obtained through haemolysis assay.
Instrumental LOD for derivatized PLTX was 0.75 ng standard injected.
The principal advantages of the HPLC-FLD method are: that it is simple, low
cost and it can be automated . However, LOD and limit of quantification (LOQ)
information in shellfish tissues is therefore unavailable.
LC-MS
Different LC-MS methods have been used for identifying and quantifying the
PlTX-group toxins in seawater and phytoplankton (Lenoir et al. 2004; Penna et
al. 2005; Ciminiello et al. 2006; Riobó et al. 2006; Ciminiello et al. 2008)
Presence of the PLTX-group toxins in samples is confirmed by the presence of
the m/z 327 fragment ion, the bicharged ion m/z 1340 and/or the tricharged ion
m/z 912. Another characteristic in the MS spectrum of PLTXs is the cluster
generated by multiple losses of water molecules.
The main advantages of the LC-MS methods are: they are fast; they can be
sensitive if high resolution MS instruments are used; they can screen and
measure PLTX-group toxins individually, giving information on the profile of
PLTX-group toxins and they can be automated. However, they require costly
equipment and highly trained personnel. Further research into the development
of such methods is essential for future application in routine testing of shellfish
tissues for the PLTX-group toxins.
This topic has been discussed in-depth by Ciminiello et al within another
contribution of this special issue on palytoxins.
CONCLUSIONS
The PLTX-group toxins are complex polyhydroxylated compounds with both
lipophilic and hydrophilic areas. At least 8 different PLTX analogues are known
but chemical structure has been characterised only for PLTX, ostreocin-D and
42-OH PLTX.
Due to the high acute toxicity of PLTX-group toxins and their increasing
occurrence, appropriate strategies to protect human health need to be
developed.
One should bear in mind that although there are extraction methods for several
marine biotoxins in shellfish there is nevertheless no information available on
their efficiency for the PLTX -group toxins.
MBA has been used to detect PLTX-group toxins in fish and shellfish tissues,
but there is a growing concern about its use arising from animal welfare and
poor specificity points of view.
Although cell based assays, which take advantage of certain PLTX functional
properties, appear to have the lowest LOD for PLTX-group toxins, some assays
showed interference with other toxins and any positive results should be
confirmed by chemical analysis.
HPLC-FLD and LC-MS/MS methods can be valuable tools for the determination
of the PLTX-group toxins. There is therefore a need a) to optimise these
methods for application to shellfish extracts, b) to perform inter-laboratory
validation and c) to develop the necessary standards and reference materials.
AKNOWLEDGEMENTS
EBITOX (Study of the biological and toxicological aspects of benthic
dinoflagellates assoiated with risks to human health) CTQ 2008-06754-C04-04
and CCVIEO (Culture Collection at the Instituto Español de Oceanografía in
Vigo, Spain)
CONFLICTS OF INTEREST
There are no conflict of interest
LEGENDS
Fig. 1
Structure of palytoxin. The boxes show double bonds responsible of
characteristic UV absorption profile. The circles show the amino terminal group
where fluorescence reagent AccQ reacts and the fragment m/z 327 obtained by
mass spectrometry.
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