[Progress in the analysis of amino acids with special reference to the determination of the intracellular amino acid pattern].

Abstract

Intracellular amino acid patterns are nowadays considered as suitable criteria to evaluate the therapeutical effects of newly developed nutritional techniques. Owing to the low sensitivity and resolution of conventional amino acid analysis (ion exchange chromatography with ninhydrin-detection), reproducible and reliable studies to assess alterations in intracellular amino acid concentrations are, however, limited. In this study, for the first time a routinely manageable HPLC method allowing fully automated determination of free amino acids by employing on-line precolumn derivatization with ortho-phthaldialdehyde is presented. The highly sensitive fluorescence detection (less than 10 pMol/amino and injection) enables the measurement of intracellular/extracellular amino acid levels in various tissues (blood, muscle, liver, kidney, leukocytes) by using minimum amounts to sample material. Furthermore, the high resolution of the HPLC method facilitates the simultaneous determination of amino acid derivatives as well as short chain peptides. The error and the reproducibility of the method (expressed as the coefficient of variation) ranged between 1.0-4.7% and 0.4-2.2%, respectively. In addition to ortho-phthaldialdehyde, alternative precolumn-derivatization reagents are described and evaluated with special regard to their applicability in the routinely manageable determination of intracellular amino acids in biological material.