HRCE in Amanita poisoning: Analysis of cyclopeptides in fungi, urine and blood of intoxicated patients, and in enriched biological fluids. PP-A-55.

S. M. BATTISTA; A. B. POMILIO; A. A. VITALE

Buenos Aires (NH City & Tower)

Congreso; The 18th Latin-American Symposium on Biotechnology, Biomedical, Biopharmaceutical and Industrial Applications of Capillary Electrophoresis and Microchip Technology (LACE 2012); 2012

LACE

The deadly poisonous fungi of the genera Amanita, Lepiota and Galerina produce toxins that are cyclic oligopeptides. Poisoning is currently reported in our country by ingestion of Amanita phalloides, as the fruit body can be confused with edible mushrooms. The lethal dose is of only 0.1-0.3 mg/kg, and the poisoning is characterized by the late detection and treatment failure. Since only amatoxins are orally toxic, our attention was focused on these bicyclic octapeptides, which are known to induce a deficient protein synthesis due to the specific inhibition of RNA polymerase II in all eukaryotic cells.  Samples analyzed by HRCE in our labs: A) Basidiocarps of A. phalloides from Pereyra Iraola Park and other Argentina places (amatoxins and phalotoxins). B) Extraction, isolation, and quantification of alpha- and beta-amanitins. C) Blood and urine samples enriched with amanitins. D) Poisoined patients. E) Optimization of CE conditions. F) Validation of the CE mode. G) Statistical evaluation.   The identification was carried out by LC-ESI-MS/MS. Equipment: Capillary Electrophoresis BioFocus 3000 (Bio-Rad); conditions: borate buffer, pH 8.6; detection at 210 nm; injection: 20 psi * sec; 20 kV; 20°C; run duration: 10 min, 70 cm column length, 50 microm internal diameter.    The results showed that a rapid, reproducible and suitable method was developed for the detection and quantification of these cyclopeptidic toxins by high-resolution capillary zone electrophoresis in order to make an early assessment of the poisoning, and further adequacy of treatment for patients who have ingested toxic mushrooms. It is of value for toxicological research and forensic analysis of suspected poisoning cases, allowing to work with stored samples.REFERENCES:1. Pomilio AB, Battista ME, Vitale AA. Curr Org Chem 2006, 10, 2075-2121. 2. Pomilio AB, Battista SM, Vitale AA. QSAR studies on bioactive cyclopeptides. In: Castro EA, QSPR-QSAR Studies on Desired Properties for Drug Design, Research Signpost, ch. 1, 2010. 3. Pomilio AB, Battista M, Vitale AA, et al. Acta Bioquím Clín Latinoamer 2012, 46, 171-182. Acknowledgments:  To CONICET, MINCYT and University of Buenos Aires (Argentina). ABP and AAV are Research Members of CONICET.