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Human fatality due to ingestion of the crab Demania reynaudii that contained a palytoxin-like toxin
Toxicon, Vol. 26, No. 1, pp. 105-107, 1988. Printed in Great Britain.
0041-0101/88 $3.00+ .00 © 1988 Pergamon Journals Ltd.
SHORT COMMUNICATION H U M A N F A T A L I T Y D U E TO I N G E S T I O N OF THE C R A B D E M A N I A R E Y N A UDII T H A T C O N T A I N E D A PALYTOXIN-LIKE TOXIN ANGEL C. ALCALA, 1 LAWTON C. ALCALA, 1 JOHN S. GARTH,2 DAISUKE YASUMURA3 and TAKESHI YASUMOTO3. ~Marine Laboratory, Silliman University, Dumaguete City, Philippines, 2Allan Hancock Foundation, University of Southern California, Los Angeles, California 90007, U.S.A., and 3Faculty of Agriculture, Tohoku University, Tsutsumidori-Amamiya, Sendal 980, Japan
(Accepted for publication 22 July 1987) A. C. ALCALA,L. C. ALCALA, J. S. GARTH, D. YASUMURAand T. YASUMOTO.Human fatality due to ingestion of the crab Demania reynaudiithat contained a palytoxin-liketoxin. Toxicon 26, 105- 107, 1988.--Clinical accounts of a human fatality resulting from ingestion of the crab Demania reynaudii are documented. The causative toxin was suggested to be palytoxin on the basis of dose-death time relationships and chromatographic properties.
OCCASIONAL outbreaks o f fatal crab poisoning have been known to occur in the Philippines (ALCALA, 1983; ALCALA and HALSTEAD, 1970; CARUMBANA et al., 1976; GONZALES and ALCALA, 1977). The causative toxin or toxins were identified by us to be either saxitoxin analogs or tetrodotoxin in Zosimus aeneus (YASUMURA et al., 1986) and palytoxin in Demania alcalai and L o p h o z o z y m u s pictor (YASUMOTO et al., 1986). We now report Demania reynaudii as a confirmed toxic species and the occurrence o f a palytoxin-like toxin in this species. At about noon on 5 N o v e m b e r 1984, a 49-year-old m a n (A.R.) ate a hairy crab cooked over live charcoal. The crab had been caught earlier in the morning in a fish net o f f the village o f T a b u k Tubig, in Tanjay Town, province o f Negros Oriental, Philippines. Within a few minutes after eating about one-fourth o f the crab, he felt dizzy, nauseated and tired. He also produced a cold sweat. The crab left a metallic taste in his mouth. To remove this taste, he drank Coca Cola and 7-Up soft drinks. Later he experienced diarrhoea. Sighting a dog which had died 1 hr after eating the remainder of the crab, he realized that he had eaten a toxic crab and requested his relatives to take him to a hospital in Dumaguete City, about 30 km away. Along the way he complained of tiredness and numbness o f the hands and the lower extremities. He also showed restlessness and vomited. On admission to the hospital at 5.45 p.m., he complained o f restlessness, muscle cramps and vomiting. H e died the following day at 3.14 a.m. Clinical records in the hospital showed that the patient suffered f r o m alternating periods o f normal heart rate and severe bradycardia (30 beats/min), rapid and shallow *To whom correspondence and reprint requests should be addressed. 105
106
Short Communication
Two l e g s
of
the
crab
(10.4
g)
I
Methanol extract I
MU)
(800
Partition between 1-butanol and water
l-Butanol extract (750 MU) I
DEAE Sephadex A-25 chromatography
Toxic eluates (20-35 ml) J
Desalting on a TSK G3000S column
75% ethanol eluate (800 MU) I
HPLC on ERC-ODS with methanol-0.05N acetic acid (8:2)
Toxic eluates (Rt 13-18 min, 400 MU) J
HPLC on ERC~ODS with acetonitrile-0.05N acetic acid (I:I)
Purified toxin (Rt 7.5-9.0 min, 300 MU) FIG. 1. PURIFICATIONSCHEME FOR THE TOXIN IN THE CRAB Demania reynaudii. H P L C and Rt denote high performance liquid c hroma t ogra phy and retention time, respectively.
E
8
¢M
o" 8 I
o
\
Demania toxin
lb
Retention
2'o
time
(rain.)
FIG. 2. COMPARISON BETWEENTHE TOXIN FROM THE CRAB Dermania reynaudii AND PALYTOXIN ON HIGH PERFORMANCE CHROMATOGRAPHY. C h r o m a t o g r a p h y was carried out on an ERC-ODS column (Erma Optical Work, 0.6 x 25 cm) using acetonitrile - 0.05 N acetic acid (1 :l) solution as a mobile phase at a flow rate of 0.9 m i / m i n . Eluates were monitored by u.v. absorption at 263 nm and mouse bioassays. Fractions causing mouse lethality are indicated by ~---~. About 30 MU each of reference palytoxin (top) and the Demania toxin (bottom) were used for chromatography.
Short Communication
107
breathing, cyanosis around the mouth and in the hands, and renal failure (no urine produced). Administration of atropine, benadryl, demerol and adrenaline apparently failed to provide relief to the patient. The carapace and legs of the crab specimen which caused the poisoning were examined by one of us (J.S.G.), who identified them as belonging to Demania reynaudii. Two legs of the crab weighing 10.4 g were extracted with methanol at room temperature. After evaporating the methanol, the residue was suspended in water, freed of lipids with diethyl ether, and extracted with 1-butanol. The butanol extract was evaporated and the residue chromatographed successively on columns of DEAE Sephadex A-25 (Pharmacia, 2 x 25 cm), TSK gel G3000S (Toyo Soda, 2 x 20 cm) and ERC-ODS (Erma Optical Work, 0.6 x 25 cm). The purification procedure, as shown in Fig. 1, is essentially the same as z previously described method (YASUMOTO et al., 1986). The toxin in the eluates wa., monitored by a mouse bioassay method proposed by TEn and GARDINER (1974) and employed by us for determination of palytoxin in crabs (YASUMOTO et al., 1986). Mouse lethality of the toxin was expressed in a mouse unit (MU), which was defined as the amount of the toxin to kill a mouse of 17 g body weight in 24 hr by i.p. injection. The methanol extract from the two legs provided 800 MU of the toxin. The toxin remained quantitatively in the organic layer in the subsequent 1-butanol - water partition, indicating that neither saxitoxin nor tetrodotoxin occurred in this species. The chromatographic properties of the crab toxin on different columns agreed well with those of palytoxin (Fig. 1). Comparison between the purified Demania toxin and palytoxin on a high performance column is shown in Fig. 2. The crab toxin still contained an undefined contaminant, which appeared as a shoulder of the main peak. Mouse bioassays of the eluates confirmed that the toxin was in the main peak, which was identical with palytoxin in the retention time. The dose-survival time relationship was the same as that of palytoxin (YASUMOTO et al., 1986) obeying the equation MU = 2255.19X -°99, where X denotes the survival time in min. D. reynaudii is reputed to be toxic (GARTH and ALCALA, 1977), but no substantial evidence had heretofore been available. This report is the first to confirm a human fatality due to the ingestion of D. reynaudii and to show a palytoxin-like toxin as the cause of the intoxication. Acknowledgements - - We thank the relatives of the victim for information on the circumstances leading to the intoxication and the hospital authorities for giving us access to the clinical records. We also thank Dr D. UEMURA of Shizuoka University for a generous gift of authentic palytoxin.
REFERENCES ALCALA, A. C. (1983) Recent cases of crab, cone shell, and fish intoxication on southern Negros Island, Philippines. Toxicon Suppl. 3, 1. ALCALA, A. C. and HALSTEAD, B. W. (1970) Human fatality due to ingestion of the crab Demania toxica in the Philippines. Clin. Toxic. 3, 609. CARUMBANA, E. E., ALCALA, A. C. and ORTEGA, E. P. (1976) Some toxic marine crabs in southern Negros Island, Philippines. Silliman J. 23, 265. GARTH, J. S. and ALCALA, A. C. (1977) Poisonous crabs of Indo-west Pacific coral reefs, with special reference to the genus Demania Laurie. In: Proc. 3rd Int. Coral ReefSymp., p. 645. Rosentiel School of Marine and Atmospheric Sciences, University of Miami, Miami, Florida. GONZALES, R. B. and ALCALA, A. C. (1977) Fatalities from crab poisoning on Negros Island, Philippines. Toxicon 15, 169. TEH, Y. F. and GARDINER, J. E. (1974) Partial purification of Lophozozymus pictor toxin. Toxicon 12, 603. YASUMURA, D., OSHIMA, Y., YASUMOTO, T., ALCALA, A. C. and ALCALA, L. C. (1986) Tetrodotoxin and paralytic shellfish toxins in Philippine crabs. Agric. biol. Chem. 50, 593. YASUMOTO, T., YASUMURA, D., OHIZUMi, Y., TAKAHASHI, M., ALCALA, A. C. and ALCALA, L. C. (1986) Palytoxin in two species of xanthid crab from the Philippines. Agric. biol. Chem. 50, 163.
0041-0101/88 $3.00+ .00 © 1988 Pergamon Journals Ltd.
SHORT COMMUNICATION H U M A N F A T A L I T Y D U E TO I N G E S T I O N OF THE C R A B D E M A N I A R E Y N A UDII T H A T C O N T A I N E D A PALYTOXIN-LIKE TOXIN ANGEL C. ALCALA, 1 LAWTON C. ALCALA, 1 JOHN S. GARTH,2 DAISUKE YASUMURA3 and TAKESHI YASUMOTO3. ~Marine Laboratory, Silliman University, Dumaguete City, Philippines, 2Allan Hancock Foundation, University of Southern California, Los Angeles, California 90007, U.S.A., and 3Faculty of Agriculture, Tohoku University, Tsutsumidori-Amamiya, Sendal 980, Japan
(Accepted for publication 22 July 1987) A. C. ALCALA,L. C. ALCALA, J. S. GARTH, D. YASUMURAand T. YASUMOTO.Human fatality due to ingestion of the crab Demania reynaudiithat contained a palytoxin-liketoxin. Toxicon 26, 105- 107, 1988.--Clinical accounts of a human fatality resulting from ingestion of the crab Demania reynaudii are documented. The causative toxin was suggested to be palytoxin on the basis of dose-death time relationships and chromatographic properties.
OCCASIONAL outbreaks o f fatal crab poisoning have been known to occur in the Philippines (ALCALA, 1983; ALCALA and HALSTEAD, 1970; CARUMBANA et al., 1976; GONZALES and ALCALA, 1977). The causative toxin or toxins were identified by us to be either saxitoxin analogs or tetrodotoxin in Zosimus aeneus (YASUMURA et al., 1986) and palytoxin in Demania alcalai and L o p h o z o z y m u s pictor (YASUMOTO et al., 1986). We now report Demania reynaudii as a confirmed toxic species and the occurrence o f a palytoxin-like toxin in this species. At about noon on 5 N o v e m b e r 1984, a 49-year-old m a n (A.R.) ate a hairy crab cooked over live charcoal. The crab had been caught earlier in the morning in a fish net o f f the village o f T a b u k Tubig, in Tanjay Town, province o f Negros Oriental, Philippines. Within a few minutes after eating about one-fourth o f the crab, he felt dizzy, nauseated and tired. He also produced a cold sweat. The crab left a metallic taste in his mouth. To remove this taste, he drank Coca Cola and 7-Up soft drinks. Later he experienced diarrhoea. Sighting a dog which had died 1 hr after eating the remainder of the crab, he realized that he had eaten a toxic crab and requested his relatives to take him to a hospital in Dumaguete City, about 30 km away. Along the way he complained of tiredness and numbness o f the hands and the lower extremities. He also showed restlessness and vomited. On admission to the hospital at 5.45 p.m., he complained o f restlessness, muscle cramps and vomiting. H e died the following day at 3.14 a.m. Clinical records in the hospital showed that the patient suffered f r o m alternating periods o f normal heart rate and severe bradycardia (30 beats/min), rapid and shallow *To whom correspondence and reprint requests should be addressed. 105
106
Short Communication
Two l e g s
of
the
crab
(10.4
g)
I
Methanol extract I
MU)
(800
Partition between 1-butanol and water
l-Butanol extract (750 MU) I
DEAE Sephadex A-25 chromatography
Toxic eluates (20-35 ml) J
Desalting on a TSK G3000S column
75% ethanol eluate (800 MU) I
HPLC on ERC-ODS with methanol-0.05N acetic acid (8:2)
Toxic eluates (Rt 13-18 min, 400 MU) J
HPLC on ERC~ODS with acetonitrile-0.05N acetic acid (I:I)
Purified toxin (Rt 7.5-9.0 min, 300 MU) FIG. 1. PURIFICATIONSCHEME FOR THE TOXIN IN THE CRAB Demania reynaudii. H P L C and Rt denote high performance liquid c hroma t ogra phy and retention time, respectively.
E
8
¢M
o" 8 I
o
\
Demania toxin
lb
Retention
2'o
time
(rain.)
FIG. 2. COMPARISON BETWEENTHE TOXIN FROM THE CRAB Dermania reynaudii AND PALYTOXIN ON HIGH PERFORMANCE CHROMATOGRAPHY. C h r o m a t o g r a p h y was carried out on an ERC-ODS column (Erma Optical Work, 0.6 x 25 cm) using acetonitrile - 0.05 N acetic acid (1 :l) solution as a mobile phase at a flow rate of 0.9 m i / m i n . Eluates were monitored by u.v. absorption at 263 nm and mouse bioassays. Fractions causing mouse lethality are indicated by ~---~. About 30 MU each of reference palytoxin (top) and the Demania toxin (bottom) were used for chromatography.
Short Communication
107
breathing, cyanosis around the mouth and in the hands, and renal failure (no urine produced). Administration of atropine, benadryl, demerol and adrenaline apparently failed to provide relief to the patient. The carapace and legs of the crab specimen which caused the poisoning were examined by one of us (J.S.G.), who identified them as belonging to Demania reynaudii. Two legs of the crab weighing 10.4 g were extracted with methanol at room temperature. After evaporating the methanol, the residue was suspended in water, freed of lipids with diethyl ether, and extracted with 1-butanol. The butanol extract was evaporated and the residue chromatographed successively on columns of DEAE Sephadex A-25 (Pharmacia, 2 x 25 cm), TSK gel G3000S (Toyo Soda, 2 x 20 cm) and ERC-ODS (Erma Optical Work, 0.6 x 25 cm). The purification procedure, as shown in Fig. 1, is essentially the same as z previously described method (YASUMOTO et al., 1986). The toxin in the eluates wa., monitored by a mouse bioassay method proposed by TEn and GARDINER (1974) and employed by us for determination of palytoxin in crabs (YASUMOTO et al., 1986). Mouse lethality of the toxin was expressed in a mouse unit (MU), which was defined as the amount of the toxin to kill a mouse of 17 g body weight in 24 hr by i.p. injection. The methanol extract from the two legs provided 800 MU of the toxin. The toxin remained quantitatively in the organic layer in the subsequent 1-butanol - water partition, indicating that neither saxitoxin nor tetrodotoxin occurred in this species. The chromatographic properties of the crab toxin on different columns agreed well with those of palytoxin (Fig. 1). Comparison between the purified Demania toxin and palytoxin on a high performance column is shown in Fig. 2. The crab toxin still contained an undefined contaminant, which appeared as a shoulder of the main peak. Mouse bioassays of the eluates confirmed that the toxin was in the main peak, which was identical with palytoxin in the retention time. The dose-survival time relationship was the same as that of palytoxin (YASUMOTO et al., 1986) obeying the equation MU = 2255.19X -°99, where X denotes the survival time in min. D. reynaudii is reputed to be toxic (GARTH and ALCALA, 1977), but no substantial evidence had heretofore been available. This report is the first to confirm a human fatality due to the ingestion of D. reynaudii and to show a palytoxin-like toxin as the cause of the intoxication. Acknowledgements - - We thank the relatives of the victim for information on the circumstances leading to the intoxication and the hospital authorities for giving us access to the clinical records. We also thank Dr D. UEMURA of Shizuoka University for a generous gift of authentic palytoxin.
REFERENCES ALCALA, A. C. (1983) Recent cases of crab, cone shell, and fish intoxication on southern Negros Island, Philippines. Toxicon Suppl. 3, 1. ALCALA, A. C. and HALSTEAD, B. W. (1970) Human fatality due to ingestion of the crab Demania toxica in the Philippines. Clin. Toxic. 3, 609. CARUMBANA, E. E., ALCALA, A. C. and ORTEGA, E. P. (1976) Some toxic marine crabs in southern Negros Island, Philippines. Silliman J. 23, 265. GARTH, J. S. and ALCALA, A. C. (1977) Poisonous crabs of Indo-west Pacific coral reefs, with special reference to the genus Demania Laurie. In: Proc. 3rd Int. Coral ReefSymp., p. 645. Rosentiel School of Marine and Atmospheric Sciences, University of Miami, Miami, Florida. GONZALES, R. B. and ALCALA, A. C. (1977) Fatalities from crab poisoning on Negros Island, Philippines. Toxicon 15, 169. TEH, Y. F. and GARDINER, J. E. (1974) Partial purification of Lophozozymus pictor toxin. Toxicon 12, 603. YASUMURA, D., OSHIMA, Y., YASUMOTO, T., ALCALA, A. C. and ALCALA, L. C. (1986) Tetrodotoxin and paralytic shellfish toxins in Philippine crabs. Agric. biol. Chem. 50, 593. YASUMOTO, T., YASUMURA, D., OHIZUMi, Y., TAKAHASHI, M., ALCALA, A. C. and ALCALA, L. C. (1986) Palytoxin in two species of xanthid crab from the Philippines. Agric. biol. Chem. 50, 163.