DAP

=__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)**. Figure 1. Current domoic acid levels off the coast of Long Beach, WA. Black line indicates level at which shellfish are unsafe for human consumption (Source: WDFW).

__What is Domoic Acid?__



 * 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)**.

**__How can we detect DAP?__**

 * ===__Domoic Acid Receptor Binding Assay__===
 * [[image:http://www.nwfsc.noaa.gov/hab/habs_toxins/marine_biotoxins/detection/images/binding_assay_fig_1s.gif width="483" height="319" align="center" caption="Schematic diagram of receptor binding assay."]] ||
 * 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)**. Once a standard curve is established, unknown samples can be added to the radio-labeled kainic acid and receptor preparation. By comparing the amount of radioactivity in the samples to the standard curve, this allows you to estimate very low concentrations of domoic acid in a variety of sample types including sea water, shellfish, or phytoplankton cells **(4)**. 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)**.
 * [[image:http://www.nwfsc.noaa.gov/hab/habs_toxins/marine_biotoxins/detection/images/standardcurve.jpg width="341" height="288" align="center" caption="Image of receptor binding assay standard curve."]] ||
 * Image of receptor binding assay standard curve. ||
 * ===Surface Plasmon Resonance (SPR) Biosensor===
 * ===Enzyme Linked Immunosorbent Assay (ELISA)===

Other Links
[|ORHAB] [|WDFW -- Razor Clam Resource Affected By //Domoic Acid//] [|WA State DOH -- Shellfish Biotoxin Program] [|Domoic Acid and Pseudo-nitzschia References] [|NWFSC -- Harmful Algal Blooms Program]