Thermostability of Alpha Amanitin in Water and Methanol

Ertuğrul Kaya 1 * , Mustafa Hancı 1, Selim Karahan 2, Sait Bayram 1, Kürşat Oğuz Yaykaşlı 3, Mustafa Gani Sürmen 1
More Detail
1 Düzce Üniversitesi, Tıp Fakültesi, Tıbbi Farmakoloji Anabilim Dalı, Düzce
2 Dicle Üniversitesi, Tıp Fakültesi, Tıbbi Farmakoloji Anabilim Dalı, Diyarbakır
3 Düzce Üniversitesi, Tıp Fakültesi, Tıbbi Genetik Anabilim Dalı, Düzce
* Corresponding Author
EUR J BASIC MED SCI, Volume 2, Issue 4, pp. 106-111. https://doi.org/10.21601/ejbms/9189
OPEN ACCESS
Download Full Text (PDF)

ABSTRACT

Alpha amanitin mostly found toxin in Amanita phalloides mushroom is responsible for toxicity. In this study, the chemical stability of the alpha amanitin in water and methanol at different temperatures was investigated. Alpha amanitin was purified by the method mentioned earlier from Amanita phalloides extract, and compared to the standard. Obtained toxin was dissolved in distilled water and methanol, and separated into 42 vials. Vials divided into 7 groups randomly. The stability of the toxin was measured at room temperature (25 °C), in the freezer (-20 °C), refrigerated (4 °C) and boiling (only water) (98 °C) using analytical HPLC system. In this method, 4.6 x50mm reverse phase C18 column, UV detector at a wavelength of 303nm, 1 mL/min flow rate of mobile phase (acetonitrile ammonium acetate + methanol + 80 +10 +10) were used. The initial amount of toxin was taken as100%, and toxin ratio was calculated according to the time, reduction in the amount of toxin. The remaining toxin level was only 5% after 6 hours in boiled water. The remaining toxin level was 86%, and 96% after 6 mounts in water, and methanol respectively at room temperature. The remaining toxin level was 91%, and 97% after 6 mounts in water, and methanol respectively at 4 °C. The remaining toxin level was 76%, and 97% after 6 mounts in water, and methanol respectively at -20 °C. Almost all of the toxin with boiling 6 hours changed to another chemical structure, but this does not imply toxicity completely disappeared. The most stability in the water was measured at 4 °C. However, the most stability in the methanol was measured at all temperatures. The chemical degradation does not mean loss of toxicity. To clarify it needs further invesitagation.
Keywords: Amanita phalloides, alpha amanitin, thermostability, HPLC

CITATION

Kaya E, Hancı M, Karahan S, Bayram S, Yaykaşlı KO, Sürmen MG. Thermostability of Alpha Amanitin in Water and Methanol. Eur J Basic Med Sci. 2012;2(4):106-11. https://doi.org/10.21601/ejbms/9189

REFERENCES

  • Vetter J. Toxins of Amanita phalloides. Toxicon. 1998;36:13-24.
  • McPartland JM, Vilgalys RJ, Cubeta MA. Mushroom poisoning. Am Fam Physician. 1997; 55(5):1797-800, 1805-9, 1811-2.
  • Kaya E, Karahan S, Hancı M, Yaykaşlı KO, Sarıtaş A, Bayram R, Yılmaz İ, Arslan SO. Düzce yöresinde yetişen Amanita Phalloides mantarındaki Alfa Amanitin düzeyinin HPLC Yöntemiyle Ölçümü. Düzce Tıp Dergisi 2012; 14(2):15-7.
  • Faulstich H, Bloching M, Zobeley S, Wieland T. Conformation and toxicity of amanitins. Experientia. 1973; 15;29(10):1230-2.
  • Wieland T. Structure and mode of action the Amatoxins. Naturwissenschaften 1972; 59(6):225-31.
  • Wieland T, Faulstich H. Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms, CRC Crit Rev Biochem 1978; 5(3): 185-260.
  • Wieland T, Götzendörfer C, Zanotti G, Vaisius AC. The effect of the chemical nature of the side chains of amatoxins in the inhibition of eukaryotic RNA polymerase B. Eur J Biochem, 1981; 117(1): 161-4.
  • Buku A, Campadelli-Fiume G, Fiume L, Wieland T. Inhibitory effect of naturally occurring and chemically modified amatoxins on RNA polymerase of rat liver nuclei, FEBS Lett 1971; 12,14(1); 42-44.
  • Bensaude O. Inhibiting eukaryotic transcription: Which compound to choose? How to evaluate its activity? Transcription 2011; 2(3):103-8.
  • Bushnell DA, Cramer P, Kornberg RD. Structural basis of transcription: alpha-amanitin-RNA polymerase II cocrystal at 2.8 A resolution. Proc Natl Acad Sci 2002; 5,99(3): 1218-22.
  • Kröncke KD, Fricker G, Meier PJ, Gerok W, Wieland T, Kurz G. Journal Article alpha-Amanitin uptake into hepatocytes. Identification of hepatic membrane transport systems used by amatoxins. Journal of Biological Chemistry 1986; 261(27):12562-7.
  • Yamaura Y. Mushroom poisoning in Japan--recent trends and future considerations. Shokuhin Eiseigaku Zasshi. 2010;51(6):319-24.
  • Ward J, Kapadia K, Brush E, Salhanick SD. Amatoxin poisoning: case reports and review of current therapies. J Emerg Med 2013;44(1):116-21.
  • Lima AD, Costa Fortes R, Carvalho Garbi Novaes MR, Percário S. Poisonous mushrooms: a review of the most common intoxications. Nutr Hosp. 2012;27(2):402-8.
  • Gong XQ, Nedialkov YA, Burton ZF. Alpha-amanitin blocks translocation by human RNA polymerase II. J Biol Chem. 2004; 25,279(26):27422-7.
  • Christians FC, Hanawalt PC. Inhibition of transcription and strand-specific DNA repair by alpha-amanitin in Chinese hamster ovary cells. Mutat Res. 1992;274(2):93-101.
  • Faulstich H, Talas A, Wellhöner HH. Toxicokinetics of labeled amatoxins in the dog. Arch Toxicol 1985; 56:190-4.
  • Thiel C, Thiel K, Klingert W, Diewold A, Scheuermann K, Hawerkamp E, Lauber J, Scheppach J, Morgalla MH, Königsrainer A, Schenk M. The enterohepatic circulation of amanitin: kinetics and therapeutical implications. Toxicol Lett 2011; 10,203(2):142-6.
  • Cao M, Yan L, Nie YY, Yang ZR, Zhao. Toxicity of alphaamanitin on mice in vivo. J. Sichuan Da Xue Xue Bao Yi Xue Ban. 2009;40(5):901-4.
  • Rosenthal P. Auxiliary liver transplantation for Amanita phalloides poisoning. J Pediatr Gastroenterol Nutr 2012;54(3):438.
  • Karlson-Stiber C, Persson H. Cytotoxic fungi--an overview. Toxicon. 2003; 15,42(4):339-49.
  • Tata JR, Hamilton MJO, Shields D. Effects of -amanitin a in vivo on RNA polymerase and nuclear RNA synthesis. Nat New Biol 1972; 238:161–4.
  • Magdalan J, Ostrowska A, Piotrowska A, Izykowska I, Nowak M, Gomułkiewicz A, Podhorska-Okołów M, Szelag A, Dziegiel P. Alpha-Amanitin induced apoptosis in primary cultured dog hepatocytes. Folia Histochem Cytobiol 2010; 1,48(1):58-62.
  • Kaya E, Karahan S, Yaykaşlı KO, Bayram R, Sarıtaş A. Purification of High Purity Alpha Amanitin Using Preparative HPLC Method. Konuralp Med J 2012;4(3):35-41.
  • Kaya E, Yaykaşlı KO, Karahan S, Bayram R, Saraıtaş A, Yaykaşlı E: Yüksek Saflıkta Beta Amanitin Üretimi. Düzce Tıp Dergisi, 2012; 14 (3) (Baskı aşamasında).
  • Bonnet MS, Basson PW. The toxicology of Amanita phalloides. Homeopathy 2002;91(4):249-54.
  • Işıloğlu M, Gücin F, Mat A. Kasım 1994’de İstanbul’da meydana gelen mantar zehirlenmeleri. Ekoloji Çevre Dergisi 1995;14:22-8.
  • Defendenti C, Bonacina E, Mauroni M, Gelosa L. Validation of a high performance liquid chromatographic method for alpha amanitin determination in urine. Forensic Sci Int 1998;92(1):59-68.
  • Broussard CN, Aggarwal A, Lacey SR, Post AB, Gramlich T, Henderson JM, Younossi ZM. Mushroom poisoning--from diarrhea to liver transplantation. Am J Gastroenterol 2001;96(11):3195-8.
  • Mengs U, Trost W. Acute phalloidin poisoning in dogs. Arch Toxicol 1981;48(1):61-7.
  • Himmelmann A, Mang G, Schnorf-Huber S. Lethal ingestion of stored Amanita phalloides mushrooms. Swiss Med Wkly 2001; 20;131(41-42):616-7.