Blood 15N:13C 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 N-15:C-13 enrichment ratio in the blood increases proportionally to the quantity ingested by applying different quantities of N-15 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 N-15-labeled milk protein concentrate simultaneous with 0.4 g of highly C-13-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 N-15- related to C-13-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 C-13 AUC for each of the measured amino acids in serum was observed when ingesting a low- or high-protein meal, whereas N-15 AUC of amino acids was similar to 2 times larger on the high-protein meal (P < 0.001). Doubling the intake of N-15-labeled amino acids increased the N-15:C-13 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 N-15:C-13 enrichment ratio in the peripheral circulation increased proportionally to the quantity of N-15-labeled milk protein ingested, especially for lysine, in healthy adults. However, when using N-15-labeled protein, correction for, e.g., alpha-carbon N-15 atom transamination is advised for determination of bioavailability of individual amino acids.