Direct determination of seleno-amino acids in biological tissues by anion-exchange separation and electrochemical detection.
Several studies have described the determination of selenium in protein extracts from tissues of marine or terrestrial animals, but have not identified the different chemical forms of selenium that are present. Selenium may be present as seleno-amino acids. Selenocysteine, for example, is a normal component of glutathione peroxidase, an antioxidant enzyme which may behave like other antioxidants, such as vitamin E, protecting tissues against methylmercury toxicity. The present study illustrates a method for the characterization of seleno-amino acids, such as selenocysteine and selenomethionine, in proteins extracted from the liver of marine mammals. The mechanism of detoxification of methylmercury, which involves seleno-compounds, is identified. The analytical determination was carried out using high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection (HPAEC-IPAD). This method allows the direct determination of underivatized amino acids, eliminating the procedure of pre- or postcolumn derivatization. The chromatographic separation was carried out on an anion-exchange column using a quaternary gradient elution. In order to optimize this method, interferences of amino acids and the influence of pH and ionic strength on the separation and electrochemical detection were studied. The IPAD response for the direct detection of amino acids is optimum at pH > 11. The detection limit (S/N = 3) for selenocysteine was found to be 450 micrograms/l. The application of this method for the identification of seleno-amino acids in protein hydrolysates is also shown.
Cavalli
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Dionex S. r. l., Laboratorio Applicazioni, Pieve Emanuele MI, Italy.
Cardellicchio
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D000818:Animals; D000838:Anions; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004289:Dolphins; D004563:Electrochemistry; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000737:chemistry; D008628:Mercury / Q000032:analysis; D008767:Methylmercury Compounds / Q000032:analysis; D009994:Osmolar Concentration; D012643:Selenium / Q000032:analysis; D017279:Selenocysteine / Q000032:analysis* / Q000302:isolation & purification; D012645:Selenomethionine / Q000032:analysis* / Q000302:isolation & purification
D000838:Anions; D008767:Methylmercury Compounds; D017279:Selenocysteine; D012645:Selenomethionine; D008628:Mercury; D012643:Selenium