Contribution to clean-up procedures for serum amino acids

Abstract

Most studies dealing with gas chromatographic determinations of amino acids in biological samples involve proteinaceous starting material. PhysioIogical fluids requiring analysis for free amino acid are usually pretreated with a denaturating agent such as picric acid’*‘, suKosalicyclic acid=, ethanol’** or chloroformg. Following removal of the precipitated protein by centrifugation the amino acids are isolated by cation-exchange ciean-up. However, variable recoveries of the free plasma amino acids, caused mainly by co-precipitation of the basic amino acids omittine, lysine and arginine, found by other workers “‘-“, led to a search for an alternative procedure. A simple handy technique, involving dih~tion of the sample with acetic acid and, thus, elimination of the protein precipitation, was su_ggested by PelIizari et al.“, and in combination with cationexchange clean-up it was used for determination of free amino acids in physiological fluids ‘z-‘* Even when the results obtained with the _ acetic acid method proved to be more consistent with those obtained by the amino acid analyzer technique than those obtained by the picrate denaturation12, the yields of some plasma amino acids were higher with the picrate method, making the acetic acid procedure less attractive”. This fact ied us to investigate the acetic acid procedure more comprehensively, i.e. with respect to the ion-exchange material and the inherent isolation process. It was found that the rate of cross-linking of the ion-exchange polystyrene matrix with the diviuylbenzeue (DVB) iutlueuced yields of some amino acids markedly, and that by reversing the mechanism of uptake toward the elution a further improvement occurred. Moreover, pieces of polyethylene tubing were found to be a convenient substitute for the ordinary glass columns.