Amnesic Shellfish Poisoning toxin-Domoic acid

Dao Viet Ha Institute of Oceanography, VAST

*Email: daovietha69@gmail.com

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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