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 is otopic composition of 13 plasma free amino acids out of 500 mu l plasm a, measured as N-pivaloyl-i-propyl amino acid esters by gas chromatogr aphy-isotope ratio mass spectrometry. It is now possible to measure N- 15 isotopic natural abundances in plasma free amino acids, which has n ot been the case previously because of the minimum requirement of 60 m i plasma to supply, e.g., 1 mg free leucine, for example, when perform ing a conventional preparative isolation. Data hom seven healthy Ameri can and German subjects reveal a similar delta(15)N pattern in plasma free amino acids, although there are small differences in individual a mino acids. Phenylalanine and threonine showed the lowest abundance (- 12 and -5 parts per thousand delta(15)N, respectively), whereas alanin e, leucine, proline, and ornithine ranged between +10 and +15 parts pe r thousand delta(15)N. Lysine ranged between +1 and +4 parts per thous and delta(15)N and showed the lowest variation of all measured amino a cids. The metabolically related aromatic amino acids, phenylalanine an d tyrosine, differ by 15 parts per thousand in their N-15 abundance. T he 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 inves tigation of the amino acid metabolism in vivo. (C) 1997 Academic Press .