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

• 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 Me COOH COOH

3 5 3' 2 2' 1' 4 4' 5'

6

H2N COOH COOH

Responsible toxins of ASP

N H COOH Me COOH 2 3 6

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 China Sea Pacific Ocean

Indian Ocean

North

Nitzschia navis-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

• n 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 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 PSP PSP PSP

PSP-Protein conjugate (antigen)

Polyclonal

antibody

ELISA

B-cell Cell-fusion

Monoclonal antibody

• 3. Chemical Assays

N H

COOH Me Me COOH COOH

3 5 3' 2 2' 1' 4 4' 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 <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 chromatography 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; Pocklington et al., 1990 Capillary electrophoresis 3 pg/injection 150 ng/g  Quantitative  Minimal cleanup required  High resolution  Small volume required Johannessen, 2000; Zhao et al., 1997 Mass Spectrometry 1 µg/g  Quantitative and qualitative high resolution  Usually requires prior separation  Expensive equipment Johannessen, 2000; Hadley et al., 1997,

3.2. High Performance Liquid Chromatography-UV detection

Kotaki et al. 2005:

Mobiphase: 10% MeCN in Phosphate buffer (pH 2.5) NaH2PO4 : 3.12 g D.W. : 900 mL MeCN : 100 mL Adjust 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 µL Mobile phase pump flow: 0.8 mL/min Temp.: 32 oC 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)

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-UV detection (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 with

4 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)

• centrifuge

10,000 g, 5mins

• centrifuge

(10,000 g, 5mins) Milipore NMWL 10,000 Milipore NMWL 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 study

Mechanism 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-nitzschia

under light microscope.

Spondylus sinensis Spondylus squamosus Spondylus versicolor

Japan Philippines 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)

2 4 6

Viet Nam: 23.88  11.59 (n=10)

25 50 75 100

20 40 60

0.0 0.4 0.8 1.2 1 2 3 4 5 6 7 8 9 10 11 12 13 14

• No. of specimens

1 2 3 4 5 6 7 8 9 10

• No. of specimens

1 2 3 4

• No. 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 Oct 50 100 150

( ) DA in S. versicolor (µg/g of whole tissue)

5.0 10.0

( ) DA in plankton net sample (ng/L of seawater)

2004 2005 29 23 6 Oct 13 15 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 Phu Bay, 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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