The pre-study assessments will include the measurement of Basal Metabolic Rate (BMR) / Resting Energy Expenditure (REE) by open-circuit indirect calorimetry, and body composition (fat and fat-free mass) by deuterium oxide dilution technique.
The study protocol is a 3-day experimental protocol (2 days of adaptation plus 1 study day), with an interval of ≥1 week to ensure complete tracer washout.
Each participant is adapted to the test diets for two days to provide them with adequate energy and protein (isonitrogenous diet), and to stabilize their metabolic pool. The energy during the adaptation period is provided at resting energy expenditure (REE/BMR) x 1.7 and protein 1.0 g.kg-1.d-1. During this period, each participant will be expected to consume 4 meals per day consisting of a protein liquid drink and protein free-cookies and/or cooked mung bean meal, which the investigators will provide.
On the study day, after a 10-12 h overnight fast, the participants will come to the research unit for 8-9 h, and receive 9 isonitrogenous, isocaloric hourly meals, each representing one-tenth of the participant's total daily protein (1 g.kg-1.d-1) and energy requirement (1.5 x REE/BMR).
The protein is supplied either as the reference amino acid mixture or as a combination of the reference amino acid mixture and the test proteins. The amount of each test protein provided at each level of selected IAA (Indispensable Amino Acid) intake should be calculated based on the concentration from all sources.
Indicator AA (Amino Acid), phenylalanine is provided at 30 mg.kg-1.d -1 in all study diets (from food protein and isotope). The amount of phenylalanine provided from crystalline amino acid should be deducted depending on the natural phenylalanine content in the test protein.
The non-protein energy in the test diets is provided through a flavored sugar-oil drink and protein-free wheat starch cookies.
The reference amino acid mixture, non-labelled crystalline phenylalanine, tyrosine, and selected IAA are added and mixed with the sugar-oil drink before administering the hourly mini-meals. The test proteins are separately administered to the participants with each mini-meal.
Breath samples are collected before starting the first mini meal, at baseline (before the tracer protocol; a stable fed state in breath 13CO2 background abundance occurs after 4 hourly mini-meals) and every 15-min during isotopic steady state (approximately 2.5 h after starting the isotope). Three breath samples should be collected in 10 mL non-silicon-coated tubes using a collection mechanism that permits the removal of dead-space air at each time point. When breath collection coincides with an hourly meal, the breath sample should be collected first, followed by the meal. Then, all breath samples are stored at room temperature, pending analysis.
Enrichment of 13C in breath is analyzed by continuous flow isotope ratio mass spectrometer. The 13CO2 enrichment in the breath samples due to isotopic administration during the fed state is expressed as atom % excess (APE) over baseline. Then, the fraction of 13CO2 (F13CO2) from the tracer phenylalanine oxidation in relation to the amount administered is calculated.
The reference and test IAAO response slopes are calculated from F13CO2 measurements and the respective IAA intakes using a linear mixed effect regression mode, with a common origin of F13CO2 response at base test IAA intake. The metabolic availability (MA) of test amino acid in the test meal is determined by the slope-ratio method using the following equation:
MA (%) = (Test protein slope × 100)/ Reference slope
