A method for the estimation of amino acid radioactivity in biological samples

A method for quantitative estimation of total radioactivity present in the free amino acid fraction of tissue samples has been described. Samples deproteinized with cold acetone were extracted in acidic medium, with ethyl ether (peroxide free); after centrifugation, the aqueous phase was used for amino acid derivatization at 400C for 15 h with 1-fluoro-2,4-dinitrobenzene in bicarbonate-buffered medium. Aliquots of the derivatized samples were acidified and extracted twice again with ethyl ether. The combined organic phases were placed in glass scintillation vials, dried, and used for the determination of its radioactivity, corresponding to the radioactivity present in the free amino acid fraction of the sample. Deproteinized samples of rat blood plasma, as well as hen egg white and yolk were tested after addition of known quantities of 14C-labeled amino acids or glucose, for validation of the method. No glucose radioactivity was found in any of the extracted sample. All radioactivity added to the samples in the form 14C-labelled alanine, glutamic acid, leucine and phenylalanine was quantitatively recovered in the derivatized fraction; only a fraction of arginine radioactivity was recovered. (Auth.)