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Domoic Acid Poisoning (DAP) in Pacific Razor Clams (Siliqua patula)
The presence of domoic acid in aquatic species was reported for the first time in the United States in the late summer of 1991 in Monterey Bay, California. By October of 1991, domoic acid was found in razor clams (Siliqua patula) and in the viscera of Dungeness crab (Cancer magister) along the coasts of Washington and Oregon. In response to this outbreak, the National Marine Fisheries Service, in cooperation with the Washington State Department of Fish and Wildlife began analysis of Washington State razor clams. Surveys conducted from November 1991 to June 1993 indicated that domoic acid levels in the edible portion of the razor clams were unsafe for human consumption (1). Of the 392 razor clam samples investigated, 42% were found to contain the toxin in excess of 20 ppm, a level considered to be unsafe for human consumption by the Food and Drug Administration (FDA). In sharp contrast to the razor clam results, neither mussels nor oysters gathered from the same sampling sites showed any significant sign of domoic acid contamination (2). Unlike mussels (Mytilus edulis), where the toxin is found only in the viscera, domoic acid distributes itself throughout the various body parts of the razor clam. The highest concentration occurs in the foot and the lowest in the siphon. Concentrations of domoic acid in the razor clam foot have been recorded as high as 230 ppm (1).| longbeach.gif |
What is Domoic Acid?
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Domoic acid is an amino acid associated with certain harmful algal blooms (HABs) particularly those related to the proliferation of diatoms in the genus Pseudo-nitzschia. During HABs domoic acid can accumulate in marine organisms that feed on phytoplankton, such as shellfish, anchovies, and sardines. In mammals, including humans, domoic acid acts as a neurotoxin and can lead to a condition called amnesic shellfish poisoning (ASP) whose symptoms include short-term memory loss, brain damage and, in severe cases, death. In marine mammals, domoic acid typically causes seizures and tremors (3).
Physiological Impact of DAP
Domoic acid binds very tightly to a glutamate receptor in the brain. Under normal circumstances, these cells use glutamic acid (a common amino acid) as a neurotransmitter. This receptor also binds two other compounds even more tightly than glutamic acid; kainic acid and domoic acid. Kainic acid and domoic acid are similar to each other but not very similar to glutamic acid. Of the three compounds, domoic acid binds to the glutamate receptor most tightly and will displace both kainic acid and glutamic acid from the binding sites (4).In the brain, domoic acid especially damages the hippocampus and amygdaloid nucleus. It damages the neurons by activating AMPA and kainate receptors, causing an influx of calcium. Although calcium flowing into cells is a normal event, the uncontrolled increase of calcium causes the cell to degenerate. Because the hippocampus may be severely damaged, memory loss occurs (3).
AMPA & kainate receptors
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How can we detect DAP?
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| Schematic diagram of receptor binding assay. |
Kainic acid has a structure very similar to domoic acid, is relatively easy to synthesize, and is readily available from commercial sources. Kainic acid can be easily marked using tritium (a radioactive isotope of hydrogen). Domoic acid can be used to displace the radioactive kainic acid from binding to the receptor. By measuring the radioactivity in counts per minute (CPM) of samples with known amounts of domoic acid, you can create a standard curve like the one below which relates added amounts of domoic acid and counts of radioactivity (4).
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| Image of receptor binding assay standard curve. |
A portable surface plasmon resonance (SPR) biosensor system for the detection of domoic acid has been developed because of the need for rapid field quantification of toxin levels in both shellfish and seawater. Antibodies were raised against domoic acid and affinity purified. These antibodies were used to develop competition- and displacement-based assays using a portable six-channel SPR system. Standard curves for detection of domoic acid in phosphate buffered saline and in diluted clam extracts analyzed by the competition-based SPR assay demonstrated a limit of detection of 3 ppb (10 nM) and a quantifiable range from 4 to 60 ppb (13-200 nM). Comparison of analyses for domoic acid levels in Pacific razor clams, Siliqua patula, containing moderate to high levels of domoic acid by the standard high performance liquid chromatography (HPLC) analysis protocol and the SPR-based assay gave an excellent correlation. This same technology functions for detection of domoic acid in concentrated algal extracts or high dissolved levels in seawater (5).
An ELISA test kit was developed for detecting domoic acid using a monoclonal antibody. The assay gives equivalent results to those obtained using standard HPLC methods. It has a linear range from 1.1 to 3 ppb and was used successfully to measure domoic acid in razor clams, mussels, scallops, and phytoplankton. The assay requires approximately 1.5 h to complete and has a standard 96-well format where each strip of eight wells is removable and can be stored at 4 degrees C until needed. The first two wells of each strip serve as an internal control eliminating the need to run a standard curve. This allows as few as 3 or as many as 36 duplicate samples to be run at a time enabling real-time sample processing and limiting degradation of domoic acid which can occur during storage. There is minimal cross-reactivity with glutamine, glutamic acid. kainic acid. epi- or iso-domoic acid. It is an accurate,rapid, cost-effective assay offering environmental managers and public health officials an effective tool for monitoring domoic acid concentrations in environmental samples (6).
Other Links
ORHABWDFW -- Razor Clam Resource Affected By //Domoic Acid//
WA State DOH -- Shellfish Biotoxin Program
Domoic Acid and Pseudo-nitzschia References
NWFSC -- Harmful Algal Blooms Program
References
(1) Wekell et al. 1994. Occurrence of domoic acid in washington state razor clams (Siliqua patula) during 1991-1993.(2) Altwein et al. 1995. The detection and distribution of the marine neurotoxin domoic acid on the Pacific coast of the United-States 1991-1993.
(3) http://en.wikipedia.org/wiki/Domoic_acid
(4) Trainer et al. 2004. Characterization of a domoic acid binding site from Pacific razor clam.
(5) Stevens et al. 2007. Detection of the toxin domoic acid from clam extracts using a portable surface plasmon resonance biosensor.
(6) Litaker et al. 2008. Rapid enzyme-linked immunosorbent assay for detection of the algal toxin domoic acid.