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

Abstract

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 15N:13C enrichment ratio in the blood increases proportionally to the quantity ingested by applying different quantities of 15N 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 15N-labeled milk protein concentrate simultaneous with 0.4 g of highly 13C–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 15N- related to 13C-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 13C AUC for each of the measured amino acids in serum was observed when ingesting a low- or high-protein meal, whereas 15N AUC of amino acids was ∼2 times larger on the high-protein meal (P < 0.001). Doubling the intake of 15N-labeled amino acids increased the 15N:13C 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 15N:13C enrichment ratio in the peripheral circulation increased proportionally to the quantity of 15N-labeled milk protein ingested, especially for lysine, in healthy adults. However, when using 15N-labeled protein, correction for, e.g., α-carbon 15N atom transamination is advised for determination of bioavailability of individual amino acids. This trial was registered at www.clinicaltrials.gov as NCT02966704.

Protein Quality in Perspective: A Review of Protein Quality Metrics and Their Applications

“…Transamination correction f actor [61,62] Abbreviations: TP = Test Protein; RP = Reference Protein; AA = amino acids; N = Nitrogen, IAA = indispensable amino acid IAA lim = first limiting indispensable amino acid.…”

Determinants of amino acid bioavailability from ingested protein in relation to gut health

“…in in the meal together with the test protein. The latter is labelled with one isotope, preferably 2 H [ 9 ▪ ], or alternately 15 N [ 10 ] and the reference protein (spirulina or free amino acids) with 13 C. The amino acid absorption from the test protein is determined by analyzing the relative ratio of these two tracers in the meal and the plasma amino acids. The low invasiveness is a major strength of this method, but it is analytically complicated.…”

Evaluation of Protein Quality in Humans and Insights on Stable Isotope Approaches to Measure Digestibility – A Review

“…Intrinsically labeled dietary proteins with stable isotopes have been used to measure true or real digestibility for the last 25 y. Intravenous, oral, or ruminal administration of single/multiple labeled free AAs or 15 N ammonium sulphate have been used for labeling milk ( 25 , 26 ), eggs ( 27 ), and meat ( 28 ), whereas deuterium oxide ( 2 H 2 O) and 15 N fertilizers ( 29–31 ), and more rarely 13 CO 2 ( 32 ), have been used to intrinsically label plant proteins ( Table 1 ). For milk proteins, the intravenous administration of labeled AAs is more efficient compared with oral administration as it avoids losses due to fermentation in the rumen, feed refusals, and impaired absorption due to the feed matrix.…”

“…For instance, 15 N-labeled milk, bovine meat, and plant proteins (lupin, soybean, wheat, rapeseed, and pea) have been widely used to measure ileal nitrogen and AA digestibility using direct ileal balance methods in humans ( 23 , 28 , 30 , 31 , 35 , 36 ). However, due to the exchange and loss of 15 N during transamination of AAs ( Supplementary Material 1 ), the use of this label has major limits especially for the indirect methods which measure plasma appearances of labeled AAs ( 25 , 37 ).…”

“…The transamination losses of the 2 H label can be addressed by using correction factors determined by controlled experiments ( 37 ) ( Table 2 , Supplementary Material 1 ). Transamination correction factors (F TCF ) have also been proposed for 15 N but experimental validation is necessary due to their high intraindividual variability and the estimates differed with the amount of protein in test meals ( 25 ). This could be attributed to the higher potential for exchange of 15 N, owing to a smaller pool of amino nitrogen compared with hydrogen in the body water ( 25 , 37 ).…”