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Proton NMR spectroscopy has been used to identify the lethal toxic culprit in a spate of recent food poisoning incidents among Japanese people eating mushrooms including the species Russula subnigricans. The compound, cycloprop-2-ene carboxylic acid is well known to organic chemists as containing a highly strained, and so highly reactive, cyclopropene ring. R. subnigricans is a basidiomycete mushroom found in Asia it was named by Japanese mycologist Tsuguo Hongo in 1955 and shares characteristics of a North American fungus R. eccentrica. Ingestion of the mushroom has in recent years led to a spate of mushroom poisonings in Japan and elsewhere. Initial symptoms include nausea and diarrhoea, which can start within half an hour of eating the toxic mushrooms. Speech impairment, convulsions, pupil contraction, stiff shoulders, and backache, have also presented. However, it is the presence of myoglobin in urine that is the most worrying symptom. The mushroom apparently causes rhabdomyolysis, the breakdown of muscle fibres, which releases myoglobin into the bloodstream. Myoglobin, is a globular protein and has to be filtered out by the kidneys but it can cause blockages, kidney failure and fluid build up leading to shock, heart failure, and in extreme cases death. The potential toxicity of these mushrooms has been known in Japan since 1954 and Japanese researchers have previously isolated and identified several physiologically active agents in R. subnigricans. Russuphelin A was identified as a cytotoxic compound in 1992. Now, chemists Masanori Matsuura, Yoko Saikawa, and Masaya Nakata of Keio University, in Kohoku-ku, Yokohama, Kosei Inui of Ishihara Sangyo Kaisha Ltd., in Kusatsu, Shiga, Koichi Nakae and Masayuki Igarashi of the Bioactive Molecules Research Group, at the Microbial Chemistry Research Center, Shinagawa-ku, Tokyo, and Kimiko Hashimoto of the Kyoto Pharmaceutical University, in Japan, have homed in a single, small molecule. To uncover the lethal molecule, Hashimoto and colleagues collected fruiting bodies (the visible growing stem and cap of a fungus) R. subnigricans in the Kiyomizu and Fushimi areas of Kyoto during 2004 to 2007. They confirmed that this species is lethal to mice. They then cut up pieces of the fruiting bodies and soaked them in water at 4 Celsius overnight. They filtered the residue and concentrated it to about 100 millilitres under reduced pressure. Dialysis and chromatography were then used to obtain a final 1 millilitre sample containing just 15 milligrams of the suspected toxin. 1 H NMR analysis using 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TSP) as an internal standard led to the surprising identification of the toxic compound as cycloprop-2-ene carboxylic acid. "We estimated the amount of [the compound] contained in fruiting bodies from the 1H NMR spectrum of the crude extract," the researchers explain, "The content was very high: 360 mg per 500 g of fruiting body." The dose lethal to 100% of animals tested by oral injection in mice was 2.5 mg per kg of body mass, which is extrapolated to humans, would correspond to a mere two or three pieces of mushroom. Cycloprop-2-ene carboxylic acid is fairly well known to synthetic organic chemists, but has never before been observed in a biological system. Laboratory tests, however, showed that the compound causes the same severe symptoms as rhabdomyolysis in mushroom poisoning. "This compound is responsible for fatal rhabdomyolysis, a new type of mushroom poisoning that is indicated by an increase in serum creatine phosphokinase activity in mice," the team explains, "We found that polymerization of the compound at high concentrations via ene reaction abolishes its toxicity." They report details of their findings in the latest issue of Nature Chemical Biology as well as tests for antibiotic and anticancer activity for this compound results of which were fairly promising. The researchers explain that the toxin causes severe rhabdomyolysis, not by direct interaction with muscle cells, but probably by a trigger for some other biochemical reaction. They suggest that their discovery will allow biomedical researchers to determine precisely how these, and other mushrooms such as Tricholoma equestre, which has killed people in France and Poland, mushrooms cause fatalities and perhaps develop an antidote to the toxin.
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 NMR reveals toxic culprit in Asian mushroom |
