Analysis Of The Hazardous Substan Cecausing Crayfish-Induced Rhabdomyolysis And The Allergens From Crayfish

During the fall of2010, rhabdomyolysis (23cases) caused by crayfish (Procambarus clarkii) was treated in Nanjing city. Domestic reseachers speculate that these cases of rhabdomyolysis may be associated with an unidentified toxin or allergen, but it has not yet been determined. Furthermore, allergy caused by shrimps is a common human allergic disease, but domestic reports on the crawfish allergens only shown in research of molecular biology. Thus, there is important and practical significance to analyze the hazardous substance causing rhabdomyolysis and allergens from crayfish.The health of consumers is seriously affected by shrimp allergy, we purify the allergen of crayfish and analyze its allergenic activity. In this study, the major allergen of crayfish was detected by Western-blotting using sera which from crustacean-allergic patients, purified through the procedure of ammonium sulfate grading precipitation, isoelectric precipitation and thermal process, and futher demonstrated. The sequence homology of the allergen was analyzed by DNAman software, and the cleavage sites were analyed by PeptideCutter software. The allergen was digested by simulated gastrointestinal fluid and simulated intestinal fluid in vitro, and the crude extracts of crayfish which boiled at different time were digested by two-phase of simulated gastrointestinal fluid in vitro, the digestive stability and immunoactivity of digestion products were analyzed by Western-blotting and inhibition ELISA. The results showed that a protein with molecular mass of36kDa reacted to all of13sera which from crustacean-allergic patients, specificly reacted to polyclonal antibody against tropomyosin (TM) from Scylla paramamosain, and the isoelectric point was about4.5. So it could be inferred that the major allergen of crayfish was TM. Crayfish TM shared high identities (>90%) with shrimp{Penaeus vannamei) and crab (Scylla paramamosairi), and contained49cleavage sites for trypsin and6cleavage sites for chymotrypsin (cleavage ratio>70%). TM was more resistant to pepsin degradation, but more susceptible to trypsin and chymotrypsin degradation. Furthermore, there was still a certain degree of immunoactivity in the digestion products. In addition, boiling could reduce the digestion stability and immunoactivity of TM, and the effect was more significant with the extension of cooking time. Thus, TM is the major allergen of crayfish, shares high identities with other crustacean TM, and can be digested by simulated gastrointestinal fluid but the digestion products are still containing a certain immunoactivity. Furthermore, boiling treating can reduce the digestion stability and immunoactivity of TM, but can not completely eliminate the allergenic activity.Crayfish-induced rhabdomyolysis has seriously threated health of consumers, and the etiology has not yet been confirmed. In order to analyze the hazardous substance in crayfish-induced rhabdomyolysis, we firstly mixed the sarcoplasmic and myofibril of mouse muscles with the extracts of Gills, Intestines, and Glands (GIG), and muscles from crayfish in vitro, respectively. Secondly, Mashed muscles and GIG from crayfish were administered by gavage to ICR mice, symptoms of mice were recorded during the observe time, sera were collected for clinical chemistry analysis, and tissues were collected for pathologic analysis. Finally, the methanol/water extracts of muscles or GIG from crayfish were analyzed by the cytotoxicity and hemolysis assay which was used for the detection of palytoxin. The results showed that mouse myofibril could only be degraded by salt-soluble and fat-soluble extracts of GIG. GIG gavage treatment made mice muscle weakness, elevated levels of serum (creatine kinase and lactate dehydrogenase and so on) and urine indicators, general changes in cardiac muscles, stomach, spleen, and skeletal muscle tissues. Furthermore, three GIG-treated mice suffered from rhabdomyolysis with paralyzed, five times or greater levels of creatine phosphokinase compared to the control, and pathological changes kidneys and skeletal muscles, it indicated that GIG contained hazardous substance causing rhabdomyolysis. Lower levels of serum IgE indicated that mice do not suffer from an allergic reaction. The methanol/water extract from GIG could induce hemolysis and cytotoxicity, but these effects were not specifically inhibited by ouabain, it indicated that crayfish was not containing palytoxin. Thus, these results suggest that crayfish GIG most likely contains rhabdomyolysis hazardous substance, which was neither a palytoxin nor an allergen.