Blood (15)N:(13)C Enrichment Ratios Are Proportional to the Ingested Quantity of Protein with the Dual-Tracer Approach for Determining Amino Acid Bioavailability in Humans

BACKGROUND: Assessment of amino acid bioavailability is of key importance for the evaluation of protein quality; however, measuring ileal digestibility of dietary proteins in humans is challenging. Therefore, a less-invasive dual stable isotope tracer approach was developed. OBJECTIVE: We aimed to test the assumption that the (15)N:(13)C enrichment ratio in the blood increases proportionally to the quantity ingested by applying different quantities of (15)N test protein. METHODS: In a crossover design, 10 healthy adults were given a semi-liquid mixed meal containing 25 g (low protein) or 50 g (high protein) of (15)N-labeled milk protein concentrate simultaneous with 0.4 g of highly (13)C–enriched spirulina. The meal was distributed over multiple small portions, frequently provided every 20 min during a period of 160 min. For several amino acids, the blood (15)N- related to (13)C-isotopic enrichment ratio was determined at t = 0, 30, 60, 90, 120, 180, 240, 300, and 360 min and differences between the 2 meals were compared using paired analyses. RESULTS: No differences in (13)C AUC for each of the measured amino acids in serum was observed when ingesting a low- or high-protein meal, whereas (15)N AUC of amino acids was ∼2 times larger on the high-protein meal (P < 0.001). Doubling the intake of (15)N-labeled amino acids increased the (15)N:(13)C ratio by a factor of 2.04 ± 0.445 for lysine and a factor between 1.8 and 2.2 for other analyzed amino acids, with only phenylalanine (2.26), methionine (2.48), and tryptophan (3.02) outside this range. CONCLUSIONS: The amino acid (15)N:(13)C enrichment ratio in the peripheral circulation increased proportionally to the quantity of (15)N-labeled milk protein ingested, especially for lysine, in healthy adults. However, when using (15)N-labeled protein, correction for, e.g., α-carbon (15)N atom transamination is advised for determination of bioavailability of individual amino acids. This trial was registered at www.clinicaltrials.gov as NCT02966704.