Measurement of N-15/N-14 isotopic composition in individual plasma free amino acids of human adults at natural abundance by gas chromatography combustion isotope ratio mass spectrometry

We describe a high-precision methodology to determine the N-15/N-14 isotopic composition of 13 plasma free amino acids out of 500 mu l plasma, measured as N-pivaloyl-i-propyl amino acid esters by gas chromatography-isotope ratio mass spectrometry. It is now possible to measure N-15 isotopic natural abundances in plasma free amino acids, which has not been the case previously because of the minimum requirement of 60 mi plasma to supply, e.g., 1 mg free leucine, for example, when performing a conventional preparative isolation. Data hom seven healthy American and German subjects reveal a similar delta(15)N pattern in plasma free amino acids, although there are small differences in individual amino acids. Phenylalanine and threonine showed the lowest abundance (-12 and -5 parts per thousand delta(15)N, respectively), whereas alanine, leucine, proline, and ornithine ranged between +10 and +15 parts per thousand delta(15)N. Lysine ranged between +1 and +4 parts per thousand delta(15)N and showed the lowest variation of all measured amino acids. The metabolically related aromatic amino acids, phenylalanine and tyrosine, differ by 15 parts per thousand in their N-15 abundance. The isolation of amino acids by cation-exchange chromatography resulted in a small alteration of N-15/N-14 isotopic composition in the amino acids glycine, lysine, and glutamic acid of maximal 1.2, 0.4, and, 0.4 parts per thousand, respectively, but the remaining amino acids were unchanged. The method described offers new possibilities for the investigation of the amino acid metabolism in vivo. (C) 1997 Academic Press.