Yessotoxin

Yessotoxin

Clinical data
Pregnancy cat.	?
Legal status	?
Identifiers
ATC code	?
ChEMBL	CHEMBL1269577
Chemical data
Formula	?

Yessotoxins (YTXs) are a group of lipophilic sulphated polyester) compounds that are structurally related to brevetoxins and ciguatoxins.^[1] They are produced by a number of planktonic algal species particularly the dinoflagellates, Lingulodinium polyedrum, Gonylaulax spiniferia and Protoceratium reticulatum.^[2]

When the environmental conditions encourage the growth of these algal species YTXs can accumulate in the water supply and within the edible tissues of bivalve molluscs including mussels, scallops and clams, therefore enabling the entry of YTX into the food chain.^[3]

History

The first YTX analogue discovered was yessotoxin and it was initially found in the scallop species Patinopecten yessoensis in the 1960s.^[4] Since then numerous yessotoxin analogues have been isolated from shellfish and marine algea (including 45-Hydroxyyessotoxin and carboxyyessotoxin).^[1]

Initially, scientists wrongly classified YTXs with the other lipophilic phycotoxins that are found in shellfish. This includes the algal toxins oxadiac acid (OA), azaspiracid (AZA) and pectenotoxins (PTX). All of these toxins cause adverse health effects when present in humans. OA causes diarretic shellfish poisoning (DSP), where symptoms of poisoning include diarrhea and tumor growth and PTX can cause liver damage. Once scientists realised that YTXs did not have the same toxicological mechanism of action as the other toxins (via the inhibition of protein phosphatise) they were given their own classification.^[5]

Toxicity

A large number of studies have been conducted to assess the potential toxicity of YTX’S.

To date none of these studies have highlighted any toxic effects of YTXs when they are present in humans. They have however found that YTX’s have toxic effects in mice when the YTX had been administered by an intraperitoneal injection into the animal. The toxicicological effects encountered are similar to those seen for paralytic shellfish toxins and include hepatotoxicity, cardiotoxicity and neurotoxicity, with a YTX level of 100 µg/Kg causing toxic effects. Limited toxic effects have been seen after oral administration of the toxin to animals. The mechanism by which YTX exert a toxic effect is unknown and is currently being studied by a number of research groups. However some recent studies suggest the mode of action may have something to do with altering calcium homeostasis.^[6]

Although there is no data illustrating the direct association of YTX’s and toxicity in humans, issues with regards to the potential health risks of YTXs still stand due to the significant animal toxicity observed and as like other algal toxins present within shellfish YTK’s are not destroyed by heating or freezing.^[3] As a result, several countries including New Zealand, Japan and those in Europe regulate the levels of YTX’s in shellfish. In 2002 the European commission placed the regulatory level of 1 µg of YTXs per g-1 or 1g/kg of shellfish meat intended for human consumption (Directive 20012/225/EC).^[2]

Analysis

The analysis of YTX’s is necessary because of the possible health risks and the limits put in place by the European Commission directive.

The analysis of YTXs is complex due to the large number of YTX analogues that can be present in the sample. Analysis is also problematic because YTXs have similar properties to other lipophilic toxins present in the samples therefore methods can be subject to experiencing false negative or false positive results due to sample interferences.

Several experimental techniques have been developed to detect YTX’s each offering varying levels of selectivity and sensitivity whilst having numerous advantages and disadvantages.

Extraction methods

Prior to analysis it is necessary to isolate YTXs from the sample medium whether this is the digestive gland of a shellfish, a water sample or a grown culture medium. This can be achieved by several methods:

Liquid–liquid or solvent extraction

Liquid–liquid extraction or solvent extraction can be used to isolate YTX’s from the sample medium. Methanol is normally the solvent of choice but other solvents can also be used including acetone and chloroform. The drawback of using the solvent extraction method is that the levels of analyte recovery can be poor therefore any results obtained from the quantification processes may not be representative of the sample.^[6][7]

Solid phase extraction

Solid phase extraction (SPE) is another method that can be used to isolate YTXs from the sample medium. This technique separates the components of a mixture by using their different chemical and physical properties. This method is robust and extremely useful when small sample volumes are being analysed. This method is advantageous over solvent extraction as it concentrates (can give sample enrichment up to the power of 10) and can purify the sample by the removal of salts and non-polar substances which can interfere with the final analysis. This technique is also beneficial because it gives good levels of YTX recovery ranging from 40–50%.^[6][7]

Analytical techniques

A range of analytical methods can be used to identify and quantify YTXs.

Mouse bioassay (MBA)

The MBA was developed by Yasumoto et al. and is the official reference method used to analyse for YTX and lipophilic toxins including okadiac acid (OA), dinophysistoxins (DTXs), azaspiracids (AZAs) and pectenotoxins (PTXs).

The MBA involves injecting the extracted toxin into a mouse and by monitoring the mouse survival rate, the toxicity of the sample can be subsequently deduced and the analyte concentration determined. This calculation is made on the basis that one mouse unit (MU) is the minimum quantity of toxin needed to kill a mouse in 24hours. The MU is set by regulating bodies at 0.05 MU/g of animal.

The original Yasumoto MBA is subject to interferences from paralytic shellfish toxins (PSP) and free fatty acids in solution which causes false positive results. Several modifications to the MBA can be made to allow the test to be performed without these areas for error.

The MBA however still has many drawbacks;

• The method is a non specific assay- it is unable to differentiate between YTX and other sample components including DSP toxins
• The method has economic and social issues with regards to testing on animals
• The results produced are not very reproducible
• The method has insufficient detection capabilities

Advantages:-

• The method is quick
• The method is cheap

Due to these factors the other more recently developed techniques are being preferred for analysis of YTX.^[8]

Enzyme-linked immunosorbent assay

The enzyme-linked immunosorbent assay (ELISA) technique used for the analysis of YTXs is a recently developed method by Briggs et al.^[6] It is a competitive, indirect immunoassay that uses polyclonal antibodies against YTX to determine its concentration in the sample. The assay is commercially available and is a rapid technique that can be used for the analysis of YTXs in shellfish, algal cells and culture samples.

This technique has several advantages: it is very sensitive, has a limit of quantification (LOQ) of 75 µg/Kg,^[9] is relatively cheap and it is easy to carry out. The major disadvantage to this method is that it cannot differentiate between the different YTX analogues and it takes a long time to generate results.^[6]

Chromatographic methods

A variety of chromatographic methods can be used to analyse YTX’s. This includes chromatographic techniques coupled to mass spectrometry and fluorescence detectors. All of the chromatographic techniques require a calibration step prior to sample analysis.

Chromatographic methods with fluorescence detection. Liquid chromatography with Fluorescence detection (LC-FLD) provides a selective, relatively cheap, reproducible method for the qualitative and quantitative analysis of YTX for shellfish and algae samples.^[6] This method requires an additional sample preparation step after the analyte extraction procedure has been completed (in this case SPE is preferentially used so that common interferences can be removed from the sample). This additional step involves the derivatization of the YTXs with a fluorescent dienophile reagent dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQ-TAD) which facilitates analyte detection. This additional sample preparation step can make LC-FLD analysis extremely time consuming and is a major advantage of the technique.^[5]

Chromatographic methods coupled to mass spectrometry. This technique is extremely useful for the analysis of multiple toxins. It has numerous advantages over the other techniques used. It is a sensitive and selective analytical method making it ideal for the analysis of complex samples and those with low analyte concentrations. The method is also beneficial in that it provides important structural information on the analyte which is helpful for aiding analyte identification and when unknown analytes are present in the sample. The technique has benefits over LC-FLD as the derivatisation and purification extraction steps are not necessary. For YTX analysis limits of detection of 30 mg/g of shellfish tissue for chromatographic methods coupled to mass spectrometry have been recorded.^[10]

The major drawback to LC-MS is that the equipment is very expensive.^[6]

Capillary electrophoresis

Capillary electrophoresis CE is emerging as the preferred analytical method for YTX analysis as it has significant advantages over the other analytical techniques used including; high efficiency, it has a fast and simple separation procedure, a small sample volume required and minimal reagent is required.[12]

CE techniques that can be used for YTX analysis include: CE with ultraviolet (UV) detection and CE coupled to mass spectrometry (MS). CEUV is a good method for YTX analysis as it is a very selective technique that can easily differentiate between YTXs and DSP toxins. The sensitivity of these techniques can however be poor due to the low molar absorptivity of the analytes. The technique gives a limit of detection (LOD) of 0.3 µg/ml and a LOQ of 0.9 µg/ml. The sensitivity of conventional CEUV can be improved by using micellar electrokinetic chromatography (MEKC).

CEMS has the added advantage over CEUV of being able to give molecular weight and/or structural information about the analyte. This enables the user to carry out unequivocal confirmations of the analytes present in the sample. The LOD and the LOQ have been calculated as 0.02 µg/ml and 0.08 µg/ml respectively again meeting the European Commission Directive.^[5]

References

1. ^ ^{a b} A. Tubaro, V. Del’Ova, S. Sosa and C. Florio, Yessotoxins: A Toxicological Overview, Toxicon (2009), dio:10.1016/j.toxicon.2009.07.038
2. ^ ^{a b} M.D.A Howard, M. Silver and R.M Kudela, Yessotoxin detected in mussel (Mytilus californicus) and phytoplankton samples from the U.S. west coast, Harmful Algae, 7 (2008) 646–652
3. ^ ^{a b} http://www.efsa.europa.eu/cs/BlobServer/Scientific_Opinion/contam_op_ej1306_summaryopinionregulatedmarinebiotoxins_summary_en.pdf?ssbinary=true
4. ^ M. Murata, M.Kumagi, J.S. Lee and T. Yasumoto, Isolation and structure of yessotoxin, a novel polyether compound implicated in diarehetic shellfish poisoning, Tetraherdon Letters, 28 (1987) 5869–5872
5. ^ ^{a b c} P. de la Iglesia, ana Gago-Martinez and t. Yasumoto, Advanced studies for the application of high-performance capillary electrophoresis for the analysis of yessotoxin and 45-hydroxyyessotoxin, Journal of Chromatography A, 1156 (2007) 160–166
6. ^ ^{a b c d e f g} B. Paz, A.H. Daranas, M.Norte, p. Riobo, J.M Franco and J.J. Fernandez, Yessotoxins, a group of marine polyethet toxins; an overview, Marine drugs, 6 (2008) 73–102
7. ^ ^{a b} A. These, J. Scholz and A. Preiss-weigert, sensitive method for the determination if lipophilic marine biotoxins in extracts of mussels and processed shellfish by high-performance liquid chromatography-tandem mass spectrometry based on enrichment by solid-phase extraction, Journal of Chromatography A, 1216 (2009) 4529–4538
8. ^ http://www.fda.org/docrep/007/y468e/y54860g.htm
9. ^ L.R. Briggs, C.O. Miles, J.M. Fitzgerald, K.M. Ross, I. Garthwaite and N.R. Towers, J. Agric. Food Chem. 52 (2004), p. 5836
10. ^ M.F. Amandi, A. Furey, M. Lehane, H Ramstad and K.J. James, Liquid chromatography with electrospray ion-trap mass spectrometry for the determination of yessotoxins in shellfish, Journal of Chromatography A, 976 (2002) 329–334

External sources

J.Aasen, I.A. Samdal, C.O. Miles, E. Dahl, L.R. Briggs and T. Aune, Yessotoxins in Norwegian blue mussels (Mytilus edulis): uptake from Protoceraltum, metabolism and depuration, Toxicon, 45 (2005) 265–272