Amnesic Shellfish Poisoning toxin-Domoic acid Dao Viet Ha - PowerPoint PPT Presentation

Amnesic Shellfish Poisoning toxin-Domoic acid Dao Viet Ha Institute of Oceanography, VAST *Email: daovietha69@gmail.com

Content ASP and domoic acid:  Poisoning  Responsible toxin  Producing organisms  Current study in region Detection of domoic acid:  Invitro assay: MBA  In vivo assay  Chemical assay: HPLC

Shellfish vector: origin of toxins are unicellular microalgae ● ● ● ● ● Toxin ● ● ● ● ● ● ● ● ● ● ● ● ● ● Paralytic shellfish poison (PSP) Diarrhetic shellfish poison (DSP) Amnesic shellfish poison (ASP) Neurotoxic shellfish poison (NSP) Azaspiracid (AZA)

Shellfish poisoning Bloom of toxic Food poisoning Toxic shellfish phytoplankton

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

Responsible toxins of ASP Me Me 6 6 2' 1' COOH COOH COOH 4' 3 4 3 3' Me 5 2 2 COOH 5' N COOH COOH COOH H 2 N N H H kainic acid glutamic acid domoic 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. 

Domoic acid producing organisms 18 diatoms are confirmed its toxin productivity: 16 Pseudo-nitzschia , 1 Nitzschia ( N. navis-varingica) and 1 Amphora species ( A. coffaeiformis) Pseudo-nitzschia produces domoic acid only Pseudo-nitzschia spp. late-log to stationary growth phases

Toxin producing species -6 (ASP toxin) Study on the toxin producing organisms Pseudo-nitzschia cf. cacianth ASP toxin (domoic acid) accumulation in a bivalve Spondylus versicolor (Photos from Drs. Dao and Omura)

Domoic acid and its derivative composition Haiphong, Vietnam n=84 North Tohoku district, Japan n=14 DA 65% IB 35% DA 94% IB 6% Bangkok, Thailand n=18 Pacific Ocean Okinawa, Japan n=56 DA 95% IB 5% DA 72% IB 28% Bulacan, Iba, Philippines n=29 South China Sea IA 39% IB 61% Indian Ocean South Luzon, Philippines n=31 South Sulawesi, Indonesia n=15 DA 64% IB 36% Alaminos, Philippines n=10 DA 98% IB 2% Cavite, Philippines n=1 IB 100% Nitzschia navis-varingica DA 34% IA 12% IB 54% (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

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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 GLUR6 glutamate 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 Polyclonal PSP PSP antibody PSP PSP-Protein conjugate (antigen) B-cell Monoclonal ELISA antibody Cell-fusion

3. Chemical Assays Me 6 2' 1' COOH 4' 4 3 3' Me 5 2 COOH 5' N COOH H Domoic acid: C 15 H 21 NO 6 Molecular weight: 311.14 Melting point: 215-216 ºC UV (ethanol) absorption spectrum max: 242 nm Decomposition: high temperature (>50)/pH <2 or > 12, light or oxygen

3. Chemical Assays 3.1. Thin layer chromatography (TLC): Quilliam et 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-layer 10 µg/g  Semi-quantitative Johannessen, chromatography  Applicable to a variety of 2000; Lawrence et matrices al., 1989  Inexpensive High Performance 20-30 ng/g (UV)  Quantitative Johannessen, Liquid 15 pg/g (FD)  Sample cleanup usually 2000; Quilliam et Chromatography required al., 1989; AOAC,  Can detect isomers 2000; Pocklington  Derivatization required for et al., 1990 fluorescence  AOAC approval method (UV) Capillary 3 pg/injection  Quantitative Johannessen, electrophoresis 150 ng/g  Minimal cleanup required 2000; Zhao et al.,  High resolution 1997  Small volume required Mass 1 µg/g  Quantitative and qualitative Johannessen, Spectrometry high resolution 2000; Hadley et  Usually requires prior al., 1997, separation  Expensive equipment

3.2. High Performance Liquid Chromatography-UV detection Kotaki et al. 2005: Mobiphase: 10% MeCN in Phosphate buffer (pH 2.5) NaH 2 PO 4 : 3.12 g D.W. : 900 mL MeCN : 100 mL Adjust pH 2.5 by 50% H 3 PO 4 (Filtered by Cellulose membrane, keep in cool room (2-4 ºC) , before use: degas) Analysis condition: Injected volume: 10 µL Mobile phase pump flow: 0.8 mL/min Temp.: 32 o C Absorbance length: 242 nm Column: 4.5 mm x 250 mm, 5C8 5µm End time: 30 min (After use: Wash mobile phase pump by 25% MeCN)

3.2. High Performance Liquid Chromatography-UV detection (LC-UV) 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 o C (~ 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)

Analysis condition Kotaki’s method Canadian method Mobile phase Phosphate buffer (pH 0.2% Formic acid 2.5) End time 30 mins 20 mins Temperature 32 o C 30 o C 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.

Procedure of DA analysis by UV-HPLC (Canadian method) Plankton sample Soft tissue of (net haulings) Shellfish - homogenize with - filtered by GF/C 4 vol. of 50% MeOH - boiled in D.W. in 5 mins - centrifuge - centrifuge 10,000 g , 5mins (10,000 g , 5mins) Extract Extract Milipore Milipore NMWL 10,000 NMWL 10,000 HPLC analysis HPLC analysis (DA in µg/g tissue) (DA in ng/L of seawater)

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).