19

1)

Food Poisoning and Consumer Complaint Cases Caused by Chemicals and Natural Toxins in Okinawa 2007

Naomasa OSHIRO, Natsuko TERUYA1), Satsuki SAKUGAWA, Ayuko KOJA

19 11 1

2 Abstract Food poisoning and consumer complaint cases caused by natural toxins or chemical substance in Okinawa

prefecture in the fiscal year 2007 are summarized as below. A pufferfish poisoning due to consumption of a fish caught off Kunigami occurred. The patients took a soup containing flesh, liver and ovary of the puffer. The left over sample of the ovary showed toxicity level at 130 MU/g. The puffer was identified as based on mtDNA analysis. Bitter tasted fruit of gourd was brought to our institute for investigation of principal substance. One of the spots developed on the Silicagel 70 FM-plate WAKO (CHCl3/EtOAc (1:1)), shows same Rf value and color as those of cucurbitacin B. A woman had burning sensation in her throat, cough, heartburn, nauseousness after ingestion of coffee contaminated with dish surfactant served at restaurant. HPLC chromatogram (linear alkylbenzene sulfonate) of the coffee was identical to that of surfactant used at the restaurant. Two ciguatera fish poisoning occurred. In the first case, a man purchased two individual of the grouper

from a fish dealer. The man cooked miso-soup and sashimi from a bigger specimen and consumed at lunch. He suffered backache, vomiting, thermoesthesia (dry-ice sensation), and fatigue. Although leftover food showed mouse lethality level at 0.1 MU/g, the smaller one had notoxicity. In the second case, six family members ingest miso-soup and sashimi cooked from a yellow-edged lyretail (lyretail grouper, coral trout) purchased from fish dealer. Of which, three people consumed miso-soup showed symptoms of thermoesthesia (dry-ice sensation), paresthesia of mouth and fatigue. Other three people who ate only sashimi had not any abnormality. The leftover of sashimi showed toxicity level at 0.025MU/g.

Key words

food poisoning pufferfish, , , surfactant, linear alkylbenzene sulfonate (LAS), ciguatera,

Variola louti

19

1 1

1 11

1

1.

19 4 14 2

2 0

19 4 16

9 10 60 14

60 cm 20

1

7

2.

3. (1) 1)

DNA mtDNA 16S RNA rRNA

5mg DNeasy Tissue Kit QIAGEN DNA

16Sar L (CGCCTGTTTATCAAAAACAT)16Sbr H (CCGGTCTGAACTCAGATCACGT)

16S rRNA PCR

III II I3%

(PCR-RFLP ) 1) PCR Wizard SV Gel and PCR

Clean-Up system Promega BigDye Terminator v3.1 Applied Biosystems

2)

2)

5g 5g

(ddY )3MU

2) 3) (TTX)

TTX Horie .3) LC/MS

( 1) OASIS HLB

TTX 0 0.5 1.0 µg/ml (n=3)TTX

(2) 1)

16S rRNA PCR 600 bp( )

III

(149 211 254 bp) ( 1) II

1. LC/MS

Agilent 1100 Series LC/MS (Agilent Technologies) Shodex Rspak NN-414 (150×4.6 mm id) 20mM ammonium acetate/methanol(75:25)

0.5ml/min 40

5 µl ESI, Positive

145 V N2 (60psi)

N2 (10 L/min, 350 V-cap 5,000V

/ 320 [M+H]+

Dde I( II

119 496 bp I 14 209 302 bp 1) PCR (614 bp)

(AB194240)1)

2)

LC/MS 2LC/MS

TTX 3.

4)

18 TTX5) 130 MU/g TTX

( 2) 10,000 MU80 g

TTX

1. 19 7 5

B

2

1( )

2.

1( ) 190.2 g 2( ) 192.3 g

3( ) 124.7 g 4( ) 329.9 g 5( ) 424.1 g

3. (1)

1. PCR 3%

PCR III

II I

100

200

400500600

300

1,0001,500

bp

M 1 2 3 4 M

2. *

LC/MS **

22 23 20 21 126 135

42 47

* MU/g

** 1 MU = 0.22 µg2)

1)

2) (TLC)

TLC 6) 10g10ml 10 (2,000 rpm 15 )

5ml 0.2 ml

B 7) (70FM )

(1:1) RfB

(2) 2 3

32

TLCB Rf

3.

1. 19 9 11

1 1

0 ( )

19 9 11

2.

( ) ( )

3.

L-1000 (16%

)2005 7)

(1) LAS

( ( ))

(SPE) 1) HPLC

LAS 10 µg/ml (total) HPLC

Inertsil ODS-3, 5 µm, 4.6 x 150 mm

65:35 0.1M 1 ml/min 10 µl

Ex. 221 nm Em. 284 nm 2)

100 mg 10 ml 50 ml

MeOH 5ml 5ml5 ml MeOH

3 ml 10 ml

InertSep (GL ) 60mg/3ml 250mg/6ml OASIS HLB 3cc (Waters)

InertSep 60mg/3mOASIS HLB 3cc (31.951.7 ) C10 C11

InertSep 250mg/6ml 90.4 94.0

3)

1ml 50ml 1ml10ml 100

1ml 0.5 ml 50mlMeOH 5ml 5ml

InertSep 250mg/6ml 5ml MeOH 3ml

10ml (2)

1,000LAS C10 C11 C12 C13

C14 SPE

3.

1,000

20 1 1,0001 50

1.

19 9 22 1

1 0 ( )

9 22 2 ( 50 cm

40 cm)

12:40 1520

2023 22:20

2:45 5:30

6:207

9

2. ( ) 1000 ( ) SPE ( ) ( )

HPLC

2429

30 (9 )3 km

2.

220 g 704 g

598 g 3. (1)

101 g

9)

(2)

( ) 0.1 MU/g 0.025 MU/g

4. 7

53 (1998 ) 3 (1991)

mtDNA

1.

19 10 22 6

3 0

10 20(19 ) 21

22( )

2222

235

3 2.

304 g 3. (1)

9)

(2)

0.025 MU/g 4.

0.025 MU/g

33

mtDNA

mtDNA

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Nagashima, Y., Shiomi, K., Watabe, S. (2006) Molecular identification of pufferfish species using

PCR amplification and restriction analysis of a segment of the 16S rRNA gene. Comp. Biochem. Physiol. D, 1: 139-144.

2) (2005)2005

pp. 661-666

3) Horie, M., Kobayashi, S., Shimizu, N., Nakazawa, H., (2002) Determination of tetrodotoxin in puffer-fish by liquid chromatography-electrospray ionization mass spectrometry. Analyst, 127, 755-759.

4) (2004) 13 15

pp. 9-23 5) (2007)

41 93-97 6) (2006)

1740 139-143

7) (1996)

30 53-56 8) (2005)

2005 ( ) pp. 752-753 9) 2005

2005pp. 691-695