Amnesic Shellfish Poisoning toxin-Domoic acid
Dao Viet Ha Institute of Oceanography, VAST
*Email: [email protected]
ContentASP and domoic acid: PoisoningResponsible toxin Producing organismsCurrent study in region
Detection of domoic acid: Invitro assay: MBA In vivo assayChemical assay: HPLC
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Toxin
Shellfish vector: origin of toxins are unicellular microalgae
Paralytic shellfish poison (PSP) Diarrhetic shellfish poison (DSP) Amnesic shellfish poison (ASP)Neurotoxic shellfish poison (NSP) Azaspiracid (AZA)
Bloom of toxic phytoplankton Toxic shellfish Food poisoning
Shellfish poisoning
Amnesic shellfish poisoning(ASP)
Characters after ingestion of contaminated seafood
Poisoning cases was reported only in Canada with 108 patients including 3 mortality by softening of the brain.
Symptom: Typical: vomiting, diarrhea, stomachache, headache, and diminution of appetite.Sever case: lost memory (amnesia), confusion, and lost of sense of balance and paralysis. In heavy case: lose conscious and die.
Recover is slow. Amnesia is obvious.
No antidote
domoic acid
N H
COOH
Me
MeCOOH
COOH3
53'
2
2' 1'
44'
5'
6
H2N COOH
COOH
Responsible toxins of ASP
N H
COOH
MeCOOH
2
36
kainic acid
Main toxin - domoic acid (water soluble amino acid). Domoic acid and kainic acid compete with glutamic acid
to react to a receptor of nerve, and connect to it more than 10 times stronger than glutamic acid.
Glutamic acid is stimulant transmitter in the central nervous system. By domoic acid, glutamic acid cannot work.
Domic acid breaks memory center of brain irrecoverably.
glutamic acid
Domoic acid producing organisms
Pseudo-nitzschia spp.Pseudo-nitzschia produces domoic acid only late-log to stationary growth phases
18 diatoms are confirmed its toxin productivity: 16 Pseudo-nitzschia, 1 Nitzschia (N. navis-varingica) and 1 Amphora species (A. coffaeiformis)
Toxin producing species -6 (ASP toxin)
ASP toxin (domoic acid) accumulation in a bivalve Spondylus versicolor
Study on the toxin producing organisms
Pseudo-nitzschia cf. cacianth
(Photos from Drs. Dao and Omura)
Tohoku district, Japan n=14
DA 94% IB 6%
Okinawa, Japan n=56
DA 72% IB 28%
Bangkok, Thailand n=18
DA 95% IB 5%
Haiphong, Vietnam n=84
DA 65% IB 35%
Bulacan, Iba, Philippines n=29
IA 39% IB 61% South Luzon, Philippines n=31
DA 64% IB 36%Alaminos, Philippines n=10
IB 100%
DA 34% IA 12% IB 54%
Cavite, Philippines n=1
South Sulawesi, Indonesia n=15
DA 98% IB 2%
South ChinaSea
Pacific Ocean
Indian Ocean
North
Nitzschianavis-varingica
Domoic acid and its derivative composition
(from Dr. Kotaki)
Detection of ASP toxin (DA)
Monitoring (seafood safety): in vivo, in vitro assay (MBA, ELISA): Screening of net toxicity.
Scientific research: toxin chemical features, origin, mechanism to accumulate in organisms: Chemical method (HPLC, LC-MS, LC-MS/MS…).
1. In vivo Assays: Mouse Bioassay
Principle: An extract of a sample containing toxins is injected intraperitoneally (i.p.) into a mouse, then observe for symptoms caused by toxins in doubt.
The PSP toxins: AOAC, 1990/APHA, 1987. DA: The characteristic neurological effects
on the mouse.
A rapid screening method for total toxicity
2
Problems of MBA for DA detection
1) Infrastructures: Huge stock of mice (ddY or ICR strain, 17-21g, male) and supply systems are necessary.
2) Calibration: Only certain authorized labs can use official toxin std.
3) Sequential analysis:Have to analyze samples one by one, to observe symptoms and measure death time, if there is.
4) Ethics:Life of mouse is consumed.
5) Low sensitivity:Detection limit: 150 µg/g (regulation level: 20 µg/g).
2. In vitro Assays: 2.1. Receptor Binding Assays
Van Dolah et al. (1997): using a cloned rat GLUR6glutamate receptor.
No inter-laboratory study of this method has been carried out.
2. In vitro Assays: 2.2. Structure assays (ELISA = Enzyme linked immunosorbent assay):
The conformational interaction of the analyte (toxin) with the assay recognition factor (e.g. epitopic binding sites in immunoassays).
Cross-reactivity in such structural immunoassays is limited to components with compatible epitopic sites (not always reflect relative biological activity or specific toxicity).
Useful for detection of almost algae toxins such as PSP, DSP, NSP and CFP
Functional assay: ELISA: Antibody against toxin is necessary
Toxin (PSP, ASP) = Low molecule hapten(impossible to immunize directly)
Couple to a carrier carrier protein
PSP
PSP
PSP
PSP
PSP-Protein conjugate (antigen)
Polyclonalantibody
ELISAB-cell
Cell-fusion
Monoclonalantibody
3. Chemical Assays
N H
COOH
Me
MeCOOH
COOH3
53'
2
2' 1'
44'
5'
6
Domoic acid: C15H21NO6
Molecular weight: 311.14
Melting point: 215-216 ºC
UV (ethanol) absorption spectrum max: 242 nm
Decomposition: high temperature (>50)/pH 12, light or oxygen
3. Chemical Assays3.1. Thin layer chromatography (TLC): Quilliamet al. 1998: Principal: a weak UV-quenching spot that stains yellow
after spraying with a 1% solution of ninhydrin. Detection limit: 0.5 µg The routine screening of shellfish tissues in those
laboratories not equipped with an LC system. Useful as a chemical confirmation method for DA in
samples tested positive by assay methods such as immunoassay.
No in-depth quantitative studies have been reported for this method.
3.2. High Performance Liquid Chromatography-UV detection (HPLC-UV)
• Quilliam et al, 1989: Acidic mobile phase (0.1% TFA and 10% MeCN in MeOH).
Flow rate: 1.0 -1.5 mL/min Injected volume: 20 µL Column: 250 x 46 mm C18. Limitation: fault positives (tryptophan, the same RT to
iso-E).
• Quilliam et al. 1991, Quilliam et al. 1995:Clean up by SAX-SPE procedure (Strong Anion Exchange and Solid Phase Extraction cartridges).
Summary of Analytical Techniques for the detection of DA
Technique Detection limit Key features References
Thin-layerchromatography
10 µg/g Semi-quantitative Applicable to a variety of
matrices Inexpensive
Johannessen,2000; Lawrence et al., 1989
High Performance Liquid Chromatography
20-30 ng/g (UV)15 pg/g (FD)
Quantitative Sample cleanup usually
required Can detect isomers Derivatization required for
fluorescence AOAC approval method (UV)
Johannessen,2000; Quilliam et al., 1989; AOAC, 2000; Pocklingtonet al., 1990
Capillaryelectrophoresis
3 pg/injection150 ng/g
Quantitative Minimal cleanup required High resolution Small volume required
Johannessen,2000; Zhao et al., 1997
Mass Spectrometry
1 µg/g Quantitative and qualitativehigh resolution
Usually requires prior separation
Expensive equipment
Johannessen,2000; Hadley et al., 1997,
3.2. High Performance Liquid Chromatography-UVdetectionKotaki et al. 2005:
Mobiphase: 10% MeCN in Phosphate buffer (pH 2.5)NaH2PO4 : 3.12 gD.W. : 900 mLMeCN : 100 mLAdjust pH 2.5 by 50% H3PO4
(Filtered by Cellulose membrane, keep in cool room (2-4 ºC), before use: degas)
Analysis condition: Injected volume: 10 µLMobile phase pump flow: 0.8 mL/minTemp.: 32 oCAbsorbance length: 242 nmColumn: 4.5 mm x 250 mm, 5C8 5µmEnd time: 30 min
(After use: Wash mobile phase pump by 25% MeCN)
NRC-CNRC. 2012 (Takata et al. 2009)
Mobile phase: 0.2% formic acid and 9% MeCN in D.W. (Acetonitrile/Formic acid/Water/ (9/0.2/90.8)
Column: Wakosil C18-II 2.0x150mm HG 3µm Wako Japan Temp: 30 oC (~ room temperature) End time: 20 mins Injection volume: 5 µL Flow: 0.2 mL/min Absorbance length: 242 nm (After use: Wash mobile phase pump by 25% MeCN)
3.2. High Performance Liquid Chromatography-UVdetection (LC-UV)
Analysis condition Kotaki’s method Canadian method
Mobile phase Phosphate buffer (pH 2.5)
0.2% Formic acid
End time 30 mins 20 mins
Temperature 32 oC 30 oC
Column 4.5 x 250 mm 5C8 2.0 x 150 mm 3C18
Injected volume 10 µL 5 µL
Detection limit of HPLC for DA
UV-HPLC: 10-80 ng/ml, depending on the sensitivity of the UV detector.
Depending on extraction, cleanup: Crude extract: practical limit detection: 1 µg/g (ppm)SAX-SPE clean-up: 20-30 ng/g (ppb).
FD-HPLC, FMOC: 15 pg DA/ml in seawater.
Soft tissue of Shellfish
Plankton sample (net haulings)
- homogenize with4 vol. of 50% MeOH
- filtered by GF/C- boiled in D.W. in 5 mins
Extract Extract
HPLC analysis(DA in µg/g tissue)
HPLC analysis(DA in ng/L of seawater)
- centrifuge10,000 g, 5mins
- centrifuge(10,000 g, 5mins)
MiliporeNMWL 10,000
MiliporeNMWL 10,000
Procedure of DA analysis by UV-HPLC(Canadian method)
Study on ASP toxin in the region
ASP (Amnesic shellfish poisoning) occurrences have been reported from several areas in the world. But no report from SEA.
Local peoples in Viet Nam sometimes told about sickness showing symptoms similar to ASP, but no medical report.
Interest in study on ASP, especially its causative agent (domoic acid).
Reference studyMechanism of DA accumulation in bivalve was unclear:Reasons:
No sufficient data showing clear correlation between bivalve toxicity and abundance of toxic Pseudo-nitzschia in the field.
No significant DA accumulation in bivalve fed by cultured toxic Pseudo-nitzschia.
Few reports on DA accumulation in bivalves in tropical waters.
Difficulty in species quantification of Pseudo-nitzschiaunder light microscope.
Spondylus sinensisSpondylus squamosus Spondylus versicolor
JapanPhilippines Viet Nam & Thailand
Reference study: Takata, 2005
DA
epi-DA
Iso-B
Iso-A
Iso-D
Iso-E
g/g (tissue)
DA level in Spondylus spp. collected from different areas (Takata, 2005)
The Philippines: 34.42 16.35 (n=14) Thailand: 2.48 0.75 (n=10)
Japan: 0.67 0.28 (n=4)
g/g (tissue)
0
2
4
6
Viet Nam: 23.88 11.59 (n=10)
0
25
50
75
100
0
20
40
60
0.0
0.4
0.8
1.2
1 2 3 4 5 6 7 8 9 10 11 12 13 14No. of specimens
1 2 3 4 5 6 7 8 9 10 No. of specimens
1 2 3 4No. of specimens
1 2 3 4 5 6 7 8 9 10 No. of specimens
Spondylus spp. are specific species to accumulate DA: appropriate for DA study.
DA producing organisms distributed widely in tropical areas.
Learning from reference review
More than 10 Spondylus species endemic including 3-4 common species
Some cases of food poisoning (ASP-like symptoms) after ingestion of Spondylus spp. were reported.
Spondylus spp. have been listed in the menu at some local seafood restaurants, recently.
Reference study: In Viet Nam
DA
Fig. 2. HPLC chromatogram of DA in Spondylus versicolor ‘s extract(Analytical conditions: Kodama and Kotaki, 2005)
Fig.1. Spondylus versicolor(Photo by Dao VH)
Fig. 3. HPLC chromatogram of DA in Spondylus versicolor ‘s extract (Analytical conditions: Canadian method)
DA
Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct0
50
100
150
( ) D
A in
S. v
ersi
colo
r(µg
/g o
f w
hole
tiss
ue)
0
5.0
10.0
( )
DA
in p
lank
ton
net s
ampl
e (n
g/L
of se
awat
er)
2004 2005
29
23
6 Oct
1315 Sep
Figure 7 Seasonal variation of domoic acid in S. versicolor and plankton net samples in Nha Phu Bay
• Dao Viet Ha, Po Teen Lim, Pham Xuan Ky, Yoshinobu Takata, Sing Tung Teng, Takuo Omura, Yasuwo Fukuyo and Masaaki Kodama. 2014. Diatom Pseudo-nitzschia cf. caciantha (Bacillariophyceae), the Most Likely Source of Domoic Acid Contamination in the Thorny Oyster Spondylus versicolor Schreibers 1793 in Nha PhuBay, Khanh Hoa Province, Vietnam. Asian Fisheries Science 27:16-29.
• Dao Viet Ha, Phan Bao Vy, Sing Tung Teng, Hajime Uchida, Chui Pin Leaw, Po Teen Lim, Toshiyuki Suzuki, Pham Xuan Ky. 2015. Pseudo-nitzschia fukuyoi (Bacillariophyceae), a domoic acid-producing species from Nha Phu Bay, Khanh Hoa Province, Vietnam. Fisheries Science. 81(3): 533-539.
• Teng, S.T., S.N. Tan, H.C. Lim, V.H. Dao, S.S. Bates, C.P. Leaw. 2016. High diversity of Pseudo-nitzschia along the northern coast of sarawak (malaysian borneo), with descriptions of P. Bipertita sp. Nov. And P. Limii sp. Nov. (Bacillariophyceae). J. Phycol. *, ***–*** (2016)DOI: 10.1111/jpy.12448
Publications
1. Technical guidance for DA studies in tropical regions, and
2. Development of IOC/WESTPAC Regional Research and Training center/Keylab in Institute of Oceanography, Vietnam for ASP toxin analysanalysis
Activity plan in next phase (TMO project in collaboration with HAB)
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