What is Paralytic Shellfish Poisoning?

Paralytic shellfish poisoning (PSP) is a neurotoxin. It is called paralytic shellfish poisoning due to people's ingestion of shellfish containing such toxins. Its toxicology is similar to that of tetrodotoxin (TTX), which mainly inhibits nerve conduction by affecting sodium channels. Paralytic shellfish poisoning is the most serious of many different shellfish poisoning incidents. Because of its strong toxicity, it often causes consumer poisoning deaths, and has widespread and high incidence ^[1] .

Chinese name: Paralytic shellfish poisoning
Foreign name: paralytic shellfish poisoning

Poisoning range: 600 ~ 5000Mu
Chemical structure: Tetrahydropurine tricyclic compounds
Subject: Marine Technology and Fisheries

Source of paralytic shellfish toxin

Paralytic shellfish poisoning (PSP) is derived from toxic algae in red tide. Red tide is generally divided into toxic red tide and non-toxic red tide. There are more than 260 species that are known to cause red tide in the coastal waters of China. Among them, 78 species can produce red tide toxins. There are five major categories of paralytic shellfish poisoning (PSP), diarrheal shellfish poisoning (DSP), neurogenic shellfish poisoning (NSP), memory loss shellfish poisoning (ASP) and ciguatera toxin. Among them, PSP is recognized as the most serious harm to public health. ^[2] Paralytic shellfish poisoning (PSP) is mainly composed of Alexandrium tamarensis, A.minutum, A. catenella, A.fraterculus, A Fundyense, A. cohorticula, Pyrodinium bahamense, Gymnodinium catenatum and other secretions are produced. In addition, some species of bacteria, cyanobacteria, and red algae (Janiasp.) Can also produce PSP toxins. PSP toxins were also found in freshwater cyanobacteria Cylindrospermopsis raciborskii, Lyngbya wollei (Onodera et al., 1997) in 1999. ^[3]

Mean change of PSP content in shellfish ^[4] When toxic red tide occurs, shellfish consume a large amount of toxic algae, and the algal toxin accumulates in the shellfish. When the toxin content exceeds the human food safety standards, humans often eat such shellfish products and they will be in danger of poisoning. Exposed shellfish cannot be distinguished by the freshness of appearance and taste, and freezing and heating cannot completely inactivate them. ^[5-6]

Chemical structure of paralytic shellfish poisoning

Paralytic shellfish poisons are a class of tetrahydropurine tricyclic compounds. The toxins that have been discovered are classified according to the different substituents. There is an isomerism on C11. In shellfish, isomers can be converted into more stable Alpha isomers whose molar ratio ( / ) can be used to analyze the exposure time of shellfish. ^[7]

Chemical structure of paralytic shellfish poison ^[8] According to the similarity of the groups, paralytic shellfish poisons can be divided into four categories: Carbamate toxins, including saxitoxin (STX), neosaxitoxin (NEO), knee Gonyautoxin, including GTX1, GTX2, GTX3 and GTX4; N-sulfocarbamoyl toxins, including C1, C2, C3, C4, GTX5 (B1) and GTX6 (B2); Decarbamoyltoxins, including Decarbamoyl saxitoxin (dcSTX), Decarbamoyl neosaxitoxin (dcneoSTX), Decarbamoylgonyautoxins1-4 (dcGTX1-4); Deoxydecarbamoyl toxins. Research on PSP has focused on studies of saxitoxin. ^[9-10]

HPLC profile of paralytic shellfish standard ^[11]

^[12]

Mechanism of paralytic shellfish poisoning

Process of paralytic shellfish toxin

One of the strong toxins of paralytic shellfish toxin, its toxicity is equivalent to that of tetrodotoxin. Before 1999, 24 people had died of paralytic shellfish poisoning in China. The Chinese government has stipulated that paralytic shellfish poisoning for marketed shellfish must be lower than 4 Mu / g. The toxicities of PSP in various tissues and organs of Scallop are: digestive gland> skirt> gill> gonad> shellfish. PSP toxins are very toxic and can cause death if ingested at 1 mg. ^{[13] It} is highly specific to the nervous system and cardiovascular system of animals, and the range of human poisoning is between 600 ~ 5000Mu (Mu is a virulence unit, 1Mu refers to the virulence that causes 18-22g mice to die within 15min. ), The lethal dose is 3000 ~ 30 000 Mu. It consists of more than 20 toxins from different dinoflagellates, which can grow in both tropical and temperate waters. This toxin is soluble in water and stable to acids. It is easy to decompose and inactivate under alkaline conditions. It is also stable to heat. Generally, heating does not make its toxicity ineffective. PSP toxin belongs to the guanidine toxin. Its active site is the guanidino group at positions 7, 8, and 9, and it has a high affinity for the amino acid residue at voltage-gated Na + channel position 1 on the excitable cell membrane. Current, hindering the formation of action potentials. Due to differences in various sodium channels such as neural sodium channels, brain sodium channels, cardiac sodium channels, and skeletal muscle sodium channels, the binding of the guanidino group at positions 7, 8, and 9 of the PSP toxin to the amino acid residues of the sodium channel are different, but all It binds to amino acid residues closer to the outer mouth of the sodium channel. In the adult rabbit skeletal muscle sodium channel, STX has a high affinity for Asp400 and Glu755 on the channel through the guanidino groups at positions 7, 8, and 9. In addition, the hydroxyl group at the C-12 position of the PSP toxin (as a hydrated ketone) and the carbamoyl side chain functional group also play a role in channel blocking, but they are not critical. In the skeletal muscle sodium channel of adult rabbits, the guanidino groups at positions 1, 2, 3 of STX have affinity with Asp1532 on the channel, which also increases its blocking effect on sodium channels. NeoSTX has one more hydroxyl group than STX at N1 position. This hydroxyl group can interact with Asp400 and Tyr401, and may form hydrogen bonds with Tyr401, so it has a higher affinity than STX. By studying lobster giant axons, calamari giant axons, and frog Langfie knots, it was found that PSP toxins can inhibit the transient sodium conduction in the membrane produced by depolarization stimulation. Each of the PSP toxin derivatives has a similar mechanism of action on the membrane, all blocking sodium influx in a dose-dependent manner, but has no effect on resting membrane potential or potassium channels. ^[14]

Sodium channel alpha subunit structure ^[15] PSP is in a bound state in shellfish, so ingestion of this toxin by shellfish will not cause harm to itself; when a person ingests food containing paralytic shellfish toxin, the toxin will be quickly released and exhibit toxic effects, with a latency period of only a few minutes Or for several hours, symptoms include muscle paralysis of the extremities, headache, nausea, salivation fever, rash, etc. In severe cases, muscle paralysis, dyspnea, and even suffocation die. ^[16]

Clinical characterization of paralytic shellfish poisoning

The initial symptoms of PSP toxin poisoning are paresthesia and numbness in the lips, mouth, and tongue, which is due to local absorption of PSP toxins in the oral mucosa. These sensations then spread to the parts near the face and neck. The fingertips and toes often feel acupuncture-like pain, accompanied by slight headaches and dizziness. Nausea and vomiting sometimes occur at an early stage. The poisoning is slightly severe, with paralysis of the arms and legs, voluntary movement disorders, and often dizziness. When the poisoning is severe, breathing difficulties and throat tension will occur; as the muscle paralysis continues to increase, it will eventually lead to death. The prominent feature of poisoning death is that the patient's consciousness is always clear before death. The dangerous period is 12 ~ 14h. Those who pass this period are expected to recover. The severity of PSP toxin poisoning is determined by the specific toxicity of the ingested PSP toxin, the amount ingested, and the rate of excretion. The toxicity of each derivative of PSP toxin is closely related to the firmness of binding to Na + channel site 1. ^[17]

Pathological photo of PSP exposure in mice ^[18]

First aid measures for paralytic shellfish poisoning

PSP food poisoning is difficult to diagnose. The general diagnosis is based on the history of eating, that is, the aquatic products that may contain PSP have been taken before the onset of the disease. The clinical symptoms are mainly the nervous system and neurological disorders, and there are obvious cardiac blood vessels. Comprehensive judgment of the performance of the drug, and need to be distinguished from anti-cholinesterase (such as toxic lentiline) insecticide poisoning, puffer fish poisoning and so on. There are no specific laboratory tests to assist in the diagnosis. However, water samples from fishing grounds where poisoned aquatic products are collected can be tested for toxic dinoflagellate and quantitative qualitative testing of suspected food PSP to determine paralytic shellfish poisoning. In the early stage of poisoning, 5 mg of apomorphine hydrochloride can be injected subcutaneously for emetic, or 1L of 2% sodium bicarbonate can be injected after gastric lavage. Oral activated carbon adsorbent can make the toxin adsorbed and discharged. For patients with muscle palsy, Shi Ning can be administered subcutaneously or intramuscularly at a dose of 2 to 3 mg after the acute phase. If breathing is found to be difficult, artificial respiration, tracheal intubation, or mechanical respiration must be performed immediately and oxygen should be provided in a timely manner. There is no effective antidote for PSP toxin poisoning. ^[19]

Seasonal changes in paralytic shellfish toxicity

The accumulation of PSP in the shell has certain seasonal changes, and this change varies according to the type, sea area, year, and sampling point of the shell. Generally speaking, the content of PSP in shellfish is higher in spring and winter and lower in summer and autumn. The investigation showed that from 1996 to 1997, the PSP content in the digestive glands of mussels and scallops in Daya Bay and Dapeng Bay was highest in spring, decreased in summer and autumn, and recovered in winter. From 1997 to 1999, the peak infection period in Dongshan Bay of Daya Bay was in winter, but the peak infection period in Dayou Bay and Otou Bay was in spring. Gulf scallop PSP was most toxic in spring, and no PSP was detected in autumn. The most toxic PSP of Huagui scallop scallop occurred in winter, and the toxicity decreased in summer and autumn. PSP was detected throughout the year. The poisoning characteristics of shellfish in different sea areas are basically consistent, that is, spring and winter are the peak periods of poisoning, and the detection rate is higher than that in summer and autumn. ^[20]

Changes of PSP content in the digestive glands of scallops ^[21]

PSP Biochemical synthesis of paralytic shellfish poisoning PSP

Shimizu et al. Fed the stable isotope 13C and 15N labeled small molecule organic matter during the cultivation of toxin-producing cyanobacteria, and purified the toxin for NMR analysis after the culture. It was found that acetic acid, arginine, and adenosine methylsulfide S-adenosylmethionine and other unknown compounds first synthesize saxitoxin through an unknown pathway, and then convert it into other types of paralytic shellfish poison through the action of other modifying enzymes. ^[22] Shimizu et al. Also proposed a possible key step: CLAISEN polymerization of an acetic acid unit or derivative with arginine or its precursor on the alpha carbon. This step completes the theory of arginine precursors proposed by Chevolot, so that the structure of arginine or its precursors can be completely incorporated into the toxin molecule, while conforming to experimental observations. Shimizu et al. Found the source of all carbon molecules in the ring structure of toxin molecules. Kellmann et al. Conducted in vitro test on the extracted protein of the toxic cyanobacteria Cylindrospermopsis raciborskii T3 and found that the biosynthetic precursors of saxitoxin are acetic acid, arginine, adenosylmethionine, and carbamoyl phosphate. Synthetic enzymes are derived from cytoplasmic components, and an unknown coenzyme required in the experiment can be extracted from membrane components using polar solutions. On this basis, Moustafa et al. Used the sxt gene cluster (containing 26 putative genes) as a template or "bait" to study toxic blue-green algae through technologies such as whole genome sequencing, 16S rRNA analysis, rapid RAST labeling, and tree analysis. (STX +), non-toxic cyanobacteria (STX-), and other non-cyanobacteria toxin-producing organisms such as bacteria and dinoflagellates. It was found that 17 of the 26 target genes originated from cyanobacteria themselves, 4 of these 17 (Group I) belong to STX + and STX- in common, and 13 exist only in STX +. These 13 Genes include O-carbamoyltransferase, Sulfotransferase, Acyl-CoAN-acyltransferase, Saxitoxin-binding protein, etc .; 9 genes belong to non-cyanobacteria origin, 5 of which originate from Proteobacteria, they include Cytidine deaminase, Amidinotransferase, Phytanoyl -CoA dioxygenase, Chaperone-likeprotein, and 1 unknown gene; while the key sxtA gene, BLAST search found that it has 2 origins, the first 800 amino acid sequences and the Polyketide synthase gene of Myxococcus xanthus (a delta-proteobacterium) are Homologous, the last 390 amino acid sequences are identical to Frankia sp. The classI & IIaminotransferase gene (an actinomycete) is similar. Some ancestors of STX + acquired these genes through horizontal gene transfer (HGT) and gene fusion. ^{[twenty three]}

Extraction process of PSP from intestinal glands of scallops ^[24] Both cyanobacteria and dinoflagellates can produce saxitoxin, but they have evolved from multiple sources. Why does the saxitoxin biosynthesis mechanism occur independently in two different taxa, one hypothesis is that the dinoflagellates obtain these genes through HGT or other means from cyanobacteria or other bacteria, and the other hypothesis is that in the dinoflagellates Bacterial symbiotes are major contributors to saxitoxin. This may be an important research direction for scholars in this field in the future. ^[25]

Biochemical synthesis steps of paralytic shellfish poisoning ^[26]

PSP Distribution of PSP in the waters of paralytic shellfish in China

In the study of PSP toxins in the South China Sea, although the composition and content of the toxins are different between different regions, they are generally similar. The main components of the PSP toxins in the coastal areas of Guangdong are GTX5, C1 and C2 with low toxicity, and GTX1 with high toxicity. 4 also accounts for a certain proportion, and the STX toxoid is very low. Among the toxic shellfish in Daya Bay, Shenzhen, there are more -type toxins than -type toxins, and / is between 1.6 and 4.4 [12]. Of the 120 samples tested along the coast of Guangdong, only 13 were detected with PSP toxicity data, with a detection rate of 10.8%; PSP toxicity ranged from 152 to 198 MU · (100 g) -1. Among them, the PSP toxicity in the samples of Tossock pupa from Beijin Port (Dongping) and the river oysters of Shenquan Port (Shenquan) resulted in very high mortality of mice in 1 hour, but the toxicity was not strong. The value is much lower than China's tentative alert standard [about 423 MU · (100 g) -1 for KM mice]. ^[27]

Shellfish collection station map ^[28] In the spring of 1990s, the detection rate and toxicity value of PSP toxicity in the coastal waters of Guangdong were at a relatively high level. However, PSP toxicity in the coastal waters of Guangdong has been low since 2003. In the spring of 2003 and 2004, the South China Sea Environmental Monitoring Center of the State Oceanic Administration carried out shellfish toxin detection at multiple locations and samples in the South China Sea. Most of the cases did not detect shellfish toxin, and the highest value of the shellfish toxin detected was less than 200 MU. (100 g) -1. ^[29]

Distribution of PSP toxins in China's coast ^[30] In the north, the PSP toxin component of Dalian oyster scallops is also dominated by low-virulence C1 and C2, accounting for more than 60%, followed by GTX2 and GTX3, with the lowest STX and neoSTX content, about 5%; but no GTX5, GTX1, and GTX4; among the detected paralytic shellfish poisoning components, -type toxins include C1 and GTX2, -type toxins include C2 and GTX3, and / is close to 1, indicating that scallops have accumulated paralytic shellfish toxins. Time is short. ^[31] Paralytic shellfish poisoning found in the northern waters of the Yellow Sea exists in the scallop (Patinopecten yessoensis), confirming the presence of paralytic shellfish poisoning in northern China. Poisonous scallop scallop samples were found in May and June, of which the shellfish toxicity detected on June 25, 2003 was 445 MU / 100 g, and the shellfish toxicity detected on May 25, 2005 was 419 MU / 100 g, all have exceeded the food safety standards (400 MU / 100 g). It is worth noting that 9 of the 12 scallop samples were collected directly from the sea area near Dalian; the other 3 samples were purchased from Qingdao, and 2 of them were found to have paralytic shellfish toxicity, and their origin was also Dalian. It can be seen that the transportation and off-site sales of infected shellfish may endanger the health of consumers in other regions, and also cause difficulties in analyzing the source of toxins. At the same time that oyster scallop samples were found to contain paralytic shellfish poisoning, none of the purple stone house clams, purple mussels, and scallop scallops detected in Dalian waters were found to have paralytic shellfish poisoning. No surface seawater was found in the surface waters of Dalian during the study period. Poisonous red tide organisms. Poisonous red tide organisms may sink to the bottom of the sea in the form of cysts and accumulate toxins through the filtering action of scallops. The composition and toxicity of toxins in different species of Alexandrium algae are very different, and even for the same algae species, the composition and toxicity of toxins are different between algae strains from different sea areas. Toxins can also be complicated in shellfish. Conversion. Therefore, it is difficult to determine the source of toxins in shellfish samples only by referring to the analysis results of domestic toxic algae species, and the source of toxins in oyster scallop should be further studied.

Changes in PSP content of Scallop Scallop ^[32] ^[33]

Paralytic shellfish toxin test

Paralytic shellfish test standard

There are different regulations on the toxicity standards of red tide toxins, and the analysis methods also differ. More than 80% of countries and regions use the mouse bioassay to analyze PSP toxicity. Only the Netherlands, Denmark, and the United Kingdom (Scotland) require the simultaneous use of HPLC (high performance liquid chromatography) to analyze PSP toxicity. The alert (safety) PSP toxicity is generally specified as 80 g STXeq · (100 g) -1, which is equivalent to 400 MU · (100 g) -1, which is a government-defined standard throughout the European countries. The United States, Japan, South Korea, and Australia have adopted this standard, which is also a standard prescribed and recommended by the United Nations Health Organization and the Food and Agriculture Organization of the United Nations. The PSP toxicity alert standard in the Philippines and Norway is limited to 40 g STXeq · (100 g) -1 [equivalent to 200 MU · (100 g) -1]. Northern Ireland in the United Kingdom sets the PSP toxicity alert standard at 32 g STXeq · (100 g) -1. ^[34] Based on the above, China has tentatively set the warning standard for PSP toxicity to 80 g STXeq · (100 g) -1, that is, the STX equivalent value of PSP toxicity in 100 g of shellfish soft tissue must not be higher than 80 g [for KM series For mice, about 423 MU · (100 g) -1]. Detection methods include mouse bioassay, electrophoresis, high performance liquid chromatography, liquid chromatography-tandem mass spectrometry, and so on. Among these detection methods, mouse biological method is the most commonly used method. ^[35]

Paralytic shellfish bioassay

The biological method for mice is to use the property that paralytic shellfish poison is easily soluble in acidic solution and is thermally stable under acidic conditions. The paralytic shellfish toxin is extracted at pH 2 ~ 3 and boiled for 5 min. Produce special symptoms of convulsions and die, and judge the magnitude of toxicity based on the time of death of the mouse. This method is easy to master and does not require the use of special equipment, but it is cumbersome to operate and has low sensitivity. Heating in an acidic environment may cause the sulfonylcarbamoyl toxoid to convert to the corresponding carbamate toxoid, which increases the toxicity. In addition, it has high cost, poor reproducibility, low comparability, and requires a large number of mice, which is not suitable for large-scale field testing. Based on the characteristics of paralytic shellfish specific binding to sodium channels, cytotoxicity tests have also been used to detect paralytic shellfish poisoning. Enzyme-linked immunosorbent assay (ELISA) based on the principle of antigen-antibody reaction has the advantage of convenience and speed. Commercially available enzyme-linked immunoassay kits are available. Because antibodies may have defects such as cross-reactivity, they cannot replace mouse biological methods. ^[36]

Physicochemical analysis of paralytic shellfish

The physicochemical analysis method is to analyze the toxin molecules through basic oxidation into fluorescent derivatives or colored organisms through ultraviolet or fluorescence detection systems. High-performance liquid chromatography (HPLC) is the most commonly used physicochemical analysis method. HPLC method used different elution systems and elution methods for the detection of different PSP, and the derivatization process is also different. Compared with mouse bioassay, HPLC has many advantages such as fast detection speed, low detection limit and high sensitivity. However, the need for calibration based on mouse bioassays and the lack of standards for PSP analysis are the main disadvantages of the HPLC method. In addition, another reason that affects the general promotion of HPLC is that the toxicity types of PSP analogs are unique and the analogs are easily converted to each other. It is difficult to determine the original or potential total toxicity in the sample. ^[37]

Paralytic shellfish immunochemical method

From the 1960s, researchers began to study the application of immunoassay technology to detect PSP; the enzyme-linked immunosorbent technology and radioimmunity established in the late 1980s were low in cross-reactivity and could not detect all toxins and were not popularized. Enzyme-linked immunosorbent assay technology was successfully established in the early 1990s. The application of immunochemical technology to PSP detection is mainly based on monoclonal antibodies (MAb). Foreign scholars have tried to prepare PSPMAb antibodies and quantify PSP with satisfactory results. PSP is a small-molecule substance and does not have immunogenicity. It needs to be connected to a large-molecule carrier to make it a complete antigen and then used to immunize animals. Monoclonal antibodies are generally prepared using low-dose long-term immunization protocols. Immunochemical detection method has low detection limit and low interference, and has low requirements for sample preparation, simple, fast, specific and economical, so it has a very high promotion value. However, the cross-reactions of antibody production and the lack of a series of standard toxins in the detection of shellfish poisoning by immunotechnology limit the further application of this technology. Moreover, antibodies are often only established against a certain toxin, and often show a low cross-reaction to other shellfish poisons, and cannot detect all shellfish poisons. ^[38]