Amino acids are constituents of protein that is the major functional and structural component of all the cells of the body, and are required for health and reproduction. In the nutritional classification, the amino acids are categorized into two groups, essential (indispensable) and non-essential (dispensable). The essential amino acids (L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, and L-valine) have carbon skeletons that cannot be synthesized from simpler molecules in animals, and therefore must be provided in the diet. The earlier methodologies for determining amino acid requirements, which was based on nitrogen balance, were criticized because of their experimental design, but now essential amino acid requirements for adult humans are estimated by isotopic tracer methods and linear regression analysis. This article reviews recent advances in studies on amino acid requirements estimated by using the
13C-labeled amino acid oxidation method, isotope ratio mass spectrometry (IRMS) for the measurement of
13CO
2-
12CO
2 ratio in expired breath CO
2 and gas chromatography-mass spectrometry (GC/MS) for the measurement of isotopic enrichment of the precursor in
13CO
2 production.
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