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Tetrodotoxin and Its Analogues in the Pufferfish Arothron hispidus and A. nigropunctatus from the Solomon Islands: A Comparison of Their Toxin Profiles with the Same Species from Okinawa, Japan.

Puilingi CG, Kudo Y, Cho Y, Konoki K, Yotsu-Yamashita M - Toxins (Basel) (2015)

Bottom Line: TTXs concentrations were higher in the skin of both species from both regions, and relatively lower in the liver, ovary, testis, stomach, intestine, and flesh.Similar results were obtained from fish collected in Okinawa, Japan: TTX in the skin of A. hispidus and A. nigropunctatus were 12.7 and 255 µg/g, respectively, at highest, and saxitoxin was also detected in the skin (2.80 µg/g at highest) and ovary of A. hispidus.TTX, 5,6,11-trideoxyTTX (with its 4-epi form), and its anhydro forms were the most abundant, and 11-oxoTTX was commonly detected in the skin.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, Miyagi 981-8555, Japan. clyde.puilingi@yahoo.com.

ABSTRACT
Pufferfish poisoning has not been well documented in the South Pacific, although fish and other seafood are sources of protein in these island nations. In this study, tetrodotoxin (TTX) and its analogues in each organ of the pufferfish Arothron hispidus and A. nigropunctatus collected in the Solomon Islands were investigated using high resolution LC-MS. The toxin profiles of the same two species of pufferfish from Okinawa, Japan were also examined for comparison. TTXs concentrations were higher in the skin of both species from both regions, and relatively lower in the liver, ovary, testis, stomach, intestine, and flesh. Due to higher TTX concentrations (51.0 and 28.7 µg/g at highest) detected in the skin of the two species from the Solomon Islands (saxitoxin was <0.02 µg/g), these species should be banned from consumption. Similar results were obtained from fish collected in Okinawa, Japan: TTX in the skin of A. hispidus and A. nigropunctatus were 12.7 and 255 µg/g, respectively, at highest, and saxitoxin was also detected in the skin (2.80 µg/g at highest) and ovary of A. hispidus. TTX, 5,6,11-trideoxyTTX (with its 4-epi form), and its anhydro forms were the most abundant, and 11-oxoTTX was commonly detected in the skin.

No MeSH data available.


Related in: MedlinePlus

HR-LC-MS chromatograms (extracted ion chromatograms: EICs) of charcoal treated sample solution (2 µL) prepared from the skin of A. hispidus No.3 specimen (25 mL/g) from the Solomon Islands soaked in ethanolic solution. The sample solution contained TTX 1.4 µg/mL, 4-epiTTX 0.30 µg/mL, 4,9-anhydroTTX 0.80 µg/mL, 5-deoxyTTX 0.13 µg/mL, 6,11-dideoxyTTX 0.03 µg/mL, 5,11-dideoxyTTX 0.09 µg/mL, 5,6,11-trideoxyTTX and 4-epi-5,6,11-trideoxyTTX (total of two analogues 1.8 µg/mL), 4,4a-anhydro-5,6,11-trideoxyTTX 2.7 µg/mL, 11-oxoTTX 0.50 µg/mL, 4-epi-11-oxoTTX 0.50 µg/mL, and 4,9-anhydro-11-oxoTTX 0.60 µg/mL.
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toxins-07-03436-f003: HR-LC-MS chromatograms (extracted ion chromatograms: EICs) of charcoal treated sample solution (2 µL) prepared from the skin of A. hispidus No.3 specimen (25 mL/g) from the Solomon Islands soaked in ethanolic solution. The sample solution contained TTX 1.4 µg/mL, 4-epiTTX 0.30 µg/mL, 4,9-anhydroTTX 0.80 µg/mL, 5-deoxyTTX 0.13 µg/mL, 6,11-dideoxyTTX 0.03 µg/mL, 5,11-dideoxyTTX 0.09 µg/mL, 5,6,11-trideoxyTTX and 4-epi-5,6,11-trideoxyTTX (total of two analogues 1.8 µg/mL), 4,4a-anhydro-5,6,11-trideoxyTTX 2.7 µg/mL, 11-oxoTTX 0.50 µg/mL, 4-epi-11-oxoTTX 0.50 µg/mL, and 4,9-anhydro-11-oxoTTX 0.60 µg/mL.

Mentions: Each tissue soaked in ethanolic solution was extracted with hot 0.2 M acetic acid (v/v). The extract was purified with activated charcoal and analyzed by High Resolution (HR) HILIC-LC-MS (Q-TOF MS) as previously reported [17,43,44,45]. The typical mass chromatograms of major TTX analogues in the skin of A. hispidus No.3 specimen are shown in Figure 3. All TTX analogues were quantified by HR-LC-MS based on the standard curve for TTX. However, in the samples from the Solomon Islands, the peak areas of 11-oxoTTX and 4-epi-11-oxoTTX on the extracted ion chromatograms (EIC) at m/z 336.1014 were smaller than that predicted by the LC-FLD analysis. Probably ionization was suppressed by certain compounds eluted at the same retention time. Therefore, 11-oxoTTX and 4-epi-11-oxoTTX were quantified using LC-FLD. The limit of detection (LOD) of all TTX analogues, except 11-oxoTTX and 4-epi-11-oxoTTX in the samples from the Solomon Islands, was (S/N > 3, 0.01 µg/g), while the limit of quantitation (LOQ) was (S/N > 10, 0.03 µg/g). The LOD and LOQ of 11-oxoTTX and 4-epi-11-oxoTTX in the samples from the Solomon Islands were 0.04 µg/g and 0.14 µg/g, respectively. TTX, 4,9-anhydroTTX, 5,6,11-trideoxyTTX (with its 4-epi form), 4,4a-anhydro-5,6,11-trideoxyTTX [46], and 4,9-anhydro-5,6,11-trideoxyTTX were major TTX analogues in almost all the skin samples. In addition, 11-oxoTTX which is considered more potent than TTX by some authors [23,24], was observed with its 4-epi and 4,9-anhydro forms. The MS/MS spectrum of 11-oxoTTX showed a similar fragmentation pattern to that of TTX, detecting m/z 162.0660 (C8H8N3O) and 178.0614 (C8H8N3O2) ions, corresponding to 2-aminohydroquinazoline and 2-aminodihydroquinazone, respectively [17], as the major fragment ions (Figure 4). Regarding minor analogues, monodeoxyTTX (11-deoxyTTX and 5-deoxyTTX) and dideoxyTTX (5,11-dideoxyTTX and 6,11-dideoxyTTX) were also detected.


Tetrodotoxin and Its Analogues in the Pufferfish Arothron hispidus and A. nigropunctatus from the Solomon Islands: A Comparison of Their Toxin Profiles with the Same Species from Okinawa, Japan.

Puilingi CG, Kudo Y, Cho Y, Konoki K, Yotsu-Yamashita M - Toxins (Basel) (2015)

HR-LC-MS chromatograms (extracted ion chromatograms: EICs) of charcoal treated sample solution (2 µL) prepared from the skin of A. hispidus No.3 specimen (25 mL/g) from the Solomon Islands soaked in ethanolic solution. The sample solution contained TTX 1.4 µg/mL, 4-epiTTX 0.30 µg/mL, 4,9-anhydroTTX 0.80 µg/mL, 5-deoxyTTX 0.13 µg/mL, 6,11-dideoxyTTX 0.03 µg/mL, 5,11-dideoxyTTX 0.09 µg/mL, 5,6,11-trideoxyTTX and 4-epi-5,6,11-trideoxyTTX (total of two analogues 1.8 µg/mL), 4,4a-anhydro-5,6,11-trideoxyTTX 2.7 µg/mL, 11-oxoTTX 0.50 µg/mL, 4-epi-11-oxoTTX 0.50 µg/mL, and 4,9-anhydro-11-oxoTTX 0.60 µg/mL.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591647&req=5

toxins-07-03436-f003: HR-LC-MS chromatograms (extracted ion chromatograms: EICs) of charcoal treated sample solution (2 µL) prepared from the skin of A. hispidus No.3 specimen (25 mL/g) from the Solomon Islands soaked in ethanolic solution. The sample solution contained TTX 1.4 µg/mL, 4-epiTTX 0.30 µg/mL, 4,9-anhydroTTX 0.80 µg/mL, 5-deoxyTTX 0.13 µg/mL, 6,11-dideoxyTTX 0.03 µg/mL, 5,11-dideoxyTTX 0.09 µg/mL, 5,6,11-trideoxyTTX and 4-epi-5,6,11-trideoxyTTX (total of two analogues 1.8 µg/mL), 4,4a-anhydro-5,6,11-trideoxyTTX 2.7 µg/mL, 11-oxoTTX 0.50 µg/mL, 4-epi-11-oxoTTX 0.50 µg/mL, and 4,9-anhydro-11-oxoTTX 0.60 µg/mL.
Mentions: Each tissue soaked in ethanolic solution was extracted with hot 0.2 M acetic acid (v/v). The extract was purified with activated charcoal and analyzed by High Resolution (HR) HILIC-LC-MS (Q-TOF MS) as previously reported [17,43,44,45]. The typical mass chromatograms of major TTX analogues in the skin of A. hispidus No.3 specimen are shown in Figure 3. All TTX analogues were quantified by HR-LC-MS based on the standard curve for TTX. However, in the samples from the Solomon Islands, the peak areas of 11-oxoTTX and 4-epi-11-oxoTTX on the extracted ion chromatograms (EIC) at m/z 336.1014 were smaller than that predicted by the LC-FLD analysis. Probably ionization was suppressed by certain compounds eluted at the same retention time. Therefore, 11-oxoTTX and 4-epi-11-oxoTTX were quantified using LC-FLD. The limit of detection (LOD) of all TTX analogues, except 11-oxoTTX and 4-epi-11-oxoTTX in the samples from the Solomon Islands, was (S/N > 3, 0.01 µg/g), while the limit of quantitation (LOQ) was (S/N > 10, 0.03 µg/g). The LOD and LOQ of 11-oxoTTX and 4-epi-11-oxoTTX in the samples from the Solomon Islands were 0.04 µg/g and 0.14 µg/g, respectively. TTX, 4,9-anhydroTTX, 5,6,11-trideoxyTTX (with its 4-epi form), 4,4a-anhydro-5,6,11-trideoxyTTX [46], and 4,9-anhydro-5,6,11-trideoxyTTX were major TTX analogues in almost all the skin samples. In addition, 11-oxoTTX which is considered more potent than TTX by some authors [23,24], was observed with its 4-epi and 4,9-anhydro forms. The MS/MS spectrum of 11-oxoTTX showed a similar fragmentation pattern to that of TTX, detecting m/z 162.0660 (C8H8N3O) and 178.0614 (C8H8N3O2) ions, corresponding to 2-aminohydroquinazoline and 2-aminodihydroquinazone, respectively [17], as the major fragment ions (Figure 4). Regarding minor analogues, monodeoxyTTX (11-deoxyTTX and 5-deoxyTTX) and dideoxyTTX (5,11-dideoxyTTX and 6,11-dideoxyTTX) were also detected.

Bottom Line: TTXs concentrations were higher in the skin of both species from both regions, and relatively lower in the liver, ovary, testis, stomach, intestine, and flesh.Similar results were obtained from fish collected in Okinawa, Japan: TTX in the skin of A. hispidus and A. nigropunctatus were 12.7 and 255 µg/g, respectively, at highest, and saxitoxin was also detected in the skin (2.80 µg/g at highest) and ovary of A. hispidus.TTX, 5,6,11-trideoxyTTX (with its 4-epi form), and its anhydro forms were the most abundant, and 11-oxoTTX was commonly detected in the skin.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, Miyagi 981-8555, Japan. clyde.puilingi@yahoo.com.

ABSTRACT
Pufferfish poisoning has not been well documented in the South Pacific, although fish and other seafood are sources of protein in these island nations. In this study, tetrodotoxin (TTX) and its analogues in each organ of the pufferfish Arothron hispidus and A. nigropunctatus collected in the Solomon Islands were investigated using high resolution LC-MS. The toxin profiles of the same two species of pufferfish from Okinawa, Japan were also examined for comparison. TTXs concentrations were higher in the skin of both species from both regions, and relatively lower in the liver, ovary, testis, stomach, intestine, and flesh. Due to higher TTX concentrations (51.0 and 28.7 µg/g at highest) detected in the skin of the two species from the Solomon Islands (saxitoxin was <0.02 µg/g), these species should be banned from consumption. Similar results were obtained from fish collected in Okinawa, Japan: TTX in the skin of A. hispidus and A. nigropunctatus were 12.7 and 255 µg/g, respectively, at highest, and saxitoxin was also detected in the skin (2.80 µg/g at highest) and ovary of A. hispidus. TTX, 5,6,11-trideoxyTTX (with its 4-epi form), and its anhydro forms were the most abundant, and 11-oxoTTX was commonly detected in the skin.

No MeSH data available.


Related in: MedlinePlus