Measurement of 15N/14N 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 15N/14N isotopic composition of 13 plasma free amino acids out of 500 microliters plasma, measured as N-pivaloyl-i-propyl amino acid esters by gas chromatography-isotope ratio mass spectrometry. It is now possible to measure 15N isotopic natural abundances in plasma free amino acids, which has not been the case previously because of the minimum requirement of 60 ml plasma to supply, e.g., 1 mg free leucine, for example, when performing a conventional preparative isolation. Data from seven healthy American and German subjects reveal a similar delta 15N pattern in plasma free amino acids, although there are small differences in individual amino acids. Phenylalanine and threonine showed the lowest abundance (-12 and -5/1000 delta 15N, respectively), whereas alanine, leucine, proline, and ornithine ranged between +10 and +15/1000 delta 15N. Lysine ranged between +1 and +4/1000 delta 15N and showed the lowest variation of all measured amino acids. The metabolically related aromatic amino acids, phenylalanine and tyrosine, differ by 15/1000 in their 15N abundance. The isolation of amino acids by cation-exchange chromatography resulted in a small alteration of 15N/14N isotopic composition in the amino acids glycine, lysine, and glutamic acid of maximal 1.2, 0.4, and, 0.4/1000, respectively, but the remaining amino acids were unchanged. The method described offers new possibilities for the investigation of the amino acid metabolism in vivo.