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Paralytic Shellfish Poison

Chuck Miller rellim at MAILHOST.TCS.TULANE.EDU
Mon Nov 16 10:21:08 EST 1998

Here are a few references from Medline:

Anal Biochem 1998 Jul 15;261(1):51-6
A high-throughput, microtiter plate assay for paralytic shellfish poisons
using the saxitoxin-specific receptor, saxiphilin.
Llewellyn LE, Doyle J, Negri AP
Australian Institute of Marine Science, Townsville, 4810, Australia.

An isoform of the paralytic shellfish poison (PSP)-specific receptor
saxiphilin, from the tropical centipede Ethmostigmus rubripes, was used as
the basis for a radiometric, high-throughput, microtiter plate assay for
this group of toxins. Characterization of the assay revealed that it was
able to detect several representatives from the various
structural PSP subgroups and yet was insensitive toward tetrodotoxin. To
test the utility of the assay as a seafood toxin-monitoring tool, the assay
was subjected to a variety of marine organism extracts, some of which were
known to contain PSPs, and whole extract toxicity expressed as STX
equivalents (STXeq) was measured by two methods: First, by comparison of
values from a screening assay with a standard STX inhibition curve and,
second, for highly active extracts, by calculation using the IC50 from a
full inhibition curve of the extract. For extracts which could be
by both methods, there was almost 100% correlation between the derived
values. STXeq derived by both methods from the bioassay highly correlated
with absolute toxin quantities from HPLC analysis. Copyright 1998 Academic

Toxicon 1998 Apr;36(4):627-30
Occurrence of a methyl derivative of saxitoxin in Bangladeshi freshwater
Zaman L, Arakawa O, Shimosu A, Shida Y, Onoue Y
Laboratory of Aquatic Resource Science, Faculty of Fisheries, Kagoshima
University, Shimoarata, Japan.

A new component of paralytic shellfish poison was isolated from a
Bangladeshi freshwater puffer Tetraodon cutcutia. Its structure was deduced
to be carbamoyl-N-methylsaxitoxin based on electrospray ionization mass
spectrometry, [1H] NMR, and conversion experiments.

Appl Environ Microbiol 1997 Aug;63(8):3104-10
Evidence for paralytic shellfish poisons in the freshwater cyanobacterium
Lyngbya wollei (Farlow ex Gomont) comb. nov.
Carmichael WW, Evans WR, Yin QQ, Bell P, Moczydlowski E
Department of Biological Sciences, Wright State University, Dayton, Ohio
45435, USA.

Lyngbya wollei (Farlow ex Gomont) comb. nov., a perennial mat-forming
filamentous cyanobacterium prevalent in lakes and reservoirs of the
southeastern United States, was found to produce a potent, acutely lethal
neurotoxin when tested in the mouse bioassay. Signs of poisoning were
similar to those of paralytic shellfish poisoning. As part of the Tennessee
Valley Authority master plan for Guntersville Reservoir, the mat-forming
filamentous cyanobacterium L. wollei, a species that had recently invaded
from other areas of the southern United States, was studied to determine if
it could produce any of the known cyanotoxins. Of the 91 field samples
collected at 10 locations at Guntersville Reservoir, Ala., on the Tennessee
River, over a 3-year period, 72.5% were toxic. The minimum 100% lethal
doses of the toxic samples ranged from 150 to 1,500 mg kg of lyophilized L.
wollei cells-1, with the majority of samples being toxic at 500 mg kg-1.
Samples bioassayed for paralytic shellfish toxins by the Association of
Official Analytical Chemists method exhibited saxitoxin equivalents ranging
from 0 to 58 micrograms g (dry weight)-1. Characteristics of the neurotoxic
compound(s), such as the lack of adsorption by C18 solid-phase extraction
columns, the short retention times on C18 high-performance liquid
chromatography (HPLC) columns, the interaction of the neurotoxins with
saxiphilin (a soluble saxitoxin-binding protein), and external blockage of
voltage-sensitive sodium channels, led to our discovery that this
neurotoxin(s) is related to the saxitoxins, the compounds responsible for
paralytic shellfish poisonings. The major saxitoxin compounds thus far
identified by comparison of HPLC fluorescence retention times are
decarbamoyl gonyautoxins 2 and 3. There was no evidence of paralytic
shellfish poison C toxins being produced by L. wollei. Fifty field samples
were placed in unialgal culture and grown under defined culture conditions.
Toxicity and signs of poisoning for these laboratory-grown strains of L.
wollei were similar to those of the field collection samples.

Toxicon 1997 Jul;35(7):1081-7
Occurrence of paralytic shellfish poison in the starfish, Asterias
amurensis in Kure Bay, Hiroshima Prefecture, Japan.
Asakawa M, Nishimura F, Miyazawa K, Noguchi T
Faculty of Applied Biological Science, Hiroshima University, Japan.

In May 1996, during surveillance on the toxicity of invertebrates such as
bivalves inhabiting the coasts of Hiroshima Bay, the starfish Asterias
amurensis collected in the estuary of the Nikoh River was found to contain
toxins which showed strong paralytic action in mice; the maximum toxicity
(as paralytic shellfish poison, PSP) was 8.0 MU/g for whole body and 28.7
MU/g for viscera throughout the monitoring period, March to July 1996.
Attempts were made to identify the paralytic toxins in the starfish. They
were extracted with 80% ethanol acidified with acetic acid, followed by
defatting with dichloromethane. The aqueous layer obtained was treated with
activated charcoal and then applied to a Sep-Pak C18 cartridge. The unbound
toxic fraction was analyzed by high-performance liquid chromatography
techniques. The starfish toxin was rather unexpectedly identified as PSP.
It was comprised of high toxic components (gonyautoxin-1; GTX1, GTX2, GTX3,
GTX4, decarbamoyl-GTX3; dcGTX3 and dcSTX) as the major components, which
accounted for approximately 77 mole% of all components, along with
protogonyautoxin-1, 2, 3 and 4 (PX1-4), which are N-sulfocarbamoyl
derivatives. Of the high toxic components, GTX1 was present in the largest
amounts. It was concluded that the toxin of starfish collected in the
estuary of Nikoh River in May 1996 consisted of PSP, which supposedly came
via the food chain from toxic bivalves living in the same area. To our
knowledge, this is the first report of the occurrence of PSP in starfish.

Toxicon 1997 Mar;35(3):423-31
Occurrence of paralytic shellfish poison in Bangladeshi freshwater puffers.
Zaman L, Arakawa O, Shimosu A, Onoue Y
Laboratory of Marine Botany and Environmental Science, Faculty of
Fisheries, Kagoshima University, Japan.

Two species of freshwater puffer fish, Tetraodon cutcutia and Chelonodon
patoca, collected from several locations in Bangladesh, showed lethal
potency in mice ranging from 2.0 to 40.0 MU/g tissue as paralytic shellfish
poison. In both species, toxicity of the skin was generally higher than the
other tissues examined (muscle, liver and ovary). Water-soluble toxins from
T. cutcutia were partially purified by activated charcoal treatment
followed by column chromatographies using Bio-Gel P-2 and Bio-Rex 70.
Analyses by cellulose acetate membrane electrophoresis and high-performance
liquid chromatography with fluorometric detection demonstrated that the
toxins were composed of saxitoxin, decarbamoylsaxitoxin, gonyautoxins 2 and
3, decarbamoylgonyautoxins 2 and 3, and three unidentified components which
are possibly related to paralytic shellfish poison.

J AOAC Int 1996 Sep-Oct;79(5):1111-5
Determination of decarbamoyl saxitoxin and its analogues in
shellfish by prechromatographic oxidation and liquid
chromatography with fluorescence detection.
Lawrence JF, Wong B, Menard C
Health Canada, Health Protection Branch, Banting Research Centre, Ottawa,
ON, Canada.

Oxidation and chromatographic conditions for detecting the decarbamoyl
analogues of several paralytic shellfish poison (PSP) toxins were studied.
Prechromatographic oxidation with periodate or hydrogen peroxide under
slightly alkaline conditions was used as previously reported for the parent
PSP toxins. Both periodate and hydrogen peroxide oxidations produced 2
fluorescent products separable by liquid chromatography for each
decarbamoyl (dc) toxin (dc-saxitoxin, dc-neosaxatoxin and dc-gonyautoxins 2
and 3). Decarbamoyl saxitoxin produced the same 2 products as did
dc-neosaxitoxin but in different ratios. One of these products was the same
as the one obtained with neosaxitoxin after periodate oxidation.
Decarbamoyl gonyautoxins 2 and 3 (together) produced 2 products, one of
which was the same as the major product obtained with gonyautoxins 1 and 4
(together) after periodate oxidation. Decarbamoyl gonyautoxins 1 and 4 were
not available for study. The method was used to detect dc-saxitoxin and
dc-gonyautoxins 2 and 3 in shellfish extracts.

Toxicon 1996 Apr;34(4):467-74
Occurrence of paralytic toxin in Taiwanese crab Atergatopsis germaini.
Tsai YH, Hwang DF, Chai TJ, Jeng SS
Department of Marine Food Science, National Taiwan Ocean University,
Keelung, Republic of China.

Paralytic toxicity was detected by paralytic shellfish poison bioassay for
all 17 specimens of the xanthid crab A. germaini collected from northern
Taiwan in November 1993. The average toxicity of crab specimens was 3809
+/- 2591 mouse units (mean +/- S.D.). The toxin was partially purified from
ethanolic extract of the crab by ultrafiltration and Bio-Gel P-2 column
chromatography. Electrophoresis, TLC, HPLC, ultraviolet spectrum and GC-MS
analyses indicated that the crab toxin was composed of gonyautoxin 3 (50%),
neosaxitoxin and saxitoxin (7%), a novel paralytic shellfish poison-like
toxin (40%) and tetrodotoxin (3%).

Alaska Med 1996 Apr-Jun;38(2):54-8, 68
A population-based study of paralytic shell fish poisoning in Alaska.
Gessner BD, Schloss M
Alaska Division of Public Health, Anchorage 99501, USA.

During May and June 1994, the authors interviewed and requested a shellfish
sample from a population-based sample of 170 residents from Kodiak and Old
Harbor, Alaska. Of 51 Old Harbor and 68 Kodiak residents who had eaten
shellfish gathered from Kodiak Island, 18 and 6 percent, respectively, had
a history of PSP. We calculated the incidence of paralytic shellfish
poisoning in Old Harbor and Kodiak as 15 and 1.5 per 1000 persons per year,
respectively. Of 12 butter clam batches collected from residents, 6 had a
paralytic shellfish poison toxin level greater than the regulatory limit of
80 micrograms saxitoxin equivalent per 100 g of tissue; one of the 29
people who ate these shellfish developed illness. People who eat shellfish
collected from non-commercial beaches have a high rate of paralytic
shellfish poisoning. It may be possible to raise the regulatory level for
paralytic shellfish poison toxin without affecting the public health.

Toxicon 1995 Dec;33(12):1669-73
Occurrence of tetrodotoxin and paralytic shellfish poison in the
Taiwanese crab Lophozozymus pictor.
Tsai YH, Hwang DF, Chai TJ, Jeng SS
Department of Marine Food Science, National Taiwan Ocean University, Keelung.

Paralytic toxicity was detected by tetrodotoxin (TTX) bioassay in all 15
specimens of the xanthid crab Lophozozymus pictor collected from northern
Taiwan in 1993. The average toxicity of crab specimens was 921 +/- 231
(mean +/- S.E.) mouse units. The toxin of crab was partially purified and
then identified. It was found that the crab toxin contained TTX and
gonyautoxin. The ratio of TTX to gonyautoxin for crab toxin was about 9:1.

Toxicon 1995 May;33(5):691-7
Dinoflagellate Alexandrium tamarense as the source of paralytic shellfish
poison (PSP) contained in bivalves from Hiroshima Bay, Hiroshima
Prefecture, Japan.
Asakawa M, Miyazawa K, Takayama H, Noguchi T
Faculty of Applied Biological Science, Hiroshima University, Japan.

In April 1993, a phytoplankton dinoflagellate was isolated from Hiroshima
Bay, Hiroshima Prefecture, Japan, and unambiguously identified as
Alexandrium tamarense on the basis of the morphological characteristics.
The dinoflagellates, cultures in modified SW-2 medium at 15 degrees C for
15 days, showed a specific toxicity of 30.7 x 10-6 MU/cell. HPLC analysis
demonstrated that the toxin was composed mainly of gonyautoxin-4 (GTX4) and
protogonyautoxin-2 (PX2 or GTX8)(27.6 and 37.0 mole%, respectively). Total
toxin concentration of this strain was 39.5 fmole/cell. Short-necked clams,
mussels, and oysters contaminated by the dinoflagellate showed a more
complicated composition, with GTX1 as the major component (61.8 mole% for
short-necked clams, 60.5 mole% for mussels, 42.5 mole% for oysters), and
PX2 was only present in trace amounts.

Am J Epidemiol 1995 Apr 15;141(8):766-70
Paralytic shellfish poisoning in Alaska: a 20-year retrospective analysis.
Gessner BD, Middaugh JP
Epidemic Intelligence Service, US Centers for Disease Control and
Prevention, Atlanta, GA.

Outbreaks of paralytic shellfish poisoning have occurred worldwide. The
authors reviewed records at the Alaska Division of Public Health to
determine the epidemiologic characteristics of this disease. To assess risk
factors for illness, the authors conducted a case-control study. A case was
defined as illness compatible with paralytic shellfish poisoning within 12
hours of the consumption of shellfish, and a control was defined as a
non-ill participant at a meal in which at least one case occurred. The
authors documented 54 outbreaks of paralytic shellfish poisoning involving
117 ill persons from 1973 to 1992. One person died, four (3%) required
intubation, and 29 (25%) required an emergency flight to a hospital.
Outbreaks occurred with multiple shellfish species, during all four
seasons, and at many locations. During the case-control study, illness was
not associated with the shellfish toxin level, method of preparation, dose,
race, sex, or age; alcohol consumption was associated with a reduced risk
of illness (odds ratio =0.05; p = 0.03). Although paralytic shellfish
poisoning causes significant illness, the authors could not identify risk
factors with clear implications for prevention strategies. This suggests
that shellfish from uncertified beaches should not be eaten. Alcohol may
protect against the adverse effects of paralytic shellfish poison.

J AOAC Int 1995 Mar-Apr;78(2):514-20
Evaluation of prechromatographic oxidation for liquid chromatographic
determination of paralytic shellfish poisons in shellfish.
Lawrence JF, Menard C, Cleroux C
Health Protection Branch, Food Research Division, Ottawa, ON, Canada.

A liquid chromatographic (LC) method employing prechromatographic oxidation
for the determination of paralytic shellfish poison (PSP) toxins was
evaluated. A number of changes to an earlier method resulted in improved
separation and quantitation of most PSP analogues. Modification of the
periodate oxidation reaction for the N-hydroxy-containing toxins led to
improved sensitivity and stability of the products, enabling automated
overnight analyses. These changes also enabled quantitation of gonyautoxins
1 and 4 (together) in the presence of gonyautoxins 2 and 3.
Decarbamoylsaxitoxin can be identified and quantitated after peroxide
oxidation. A cleanup step using a strong-anion-exchange column removed the
C toxins and B-2 from the extracts and enabled a more accurate quantitation
of gonyautoxins 1 and 4 and neosaxitoxin. Chiral chromatography, employing
a reversed-phase column and chiral mobile-phase additives (copper-proline
complex), was briefly evaluated for the separation of the oxidation
products of the isomer pairs, gonyautoxins 1 and 4 and gonyautoxins 2 and
3. A comparison of the method with the mouse bioassay for the determination
of PSP in lobster hepatopancreas (58 samples) showed a reasonable
correlation (0.90) over a concentration range of 40-500 micrograms/100 g
(saxitoxin equivalents), although the LC results were consistently higher
than the mouse bioassay values by about 40%.

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