Metabolic Availability of Methionine in Tempeh Chips Among Reproductive-age Women Using Indicator Amino Acid Oxidation (IAAO) Method

For the pre-study assessment, participants will be required to measure their body weight, height, and resting energy expenditure (REE) using indirect calorimetry.

The study protocol is a 3-day experimental protocol consisting of 2 adaptation days and 1 indicator amino acid oxidation (IAAO) study day. For each 3-day experimental protocol, participants will be provided with either a reference diet for 3 days or a tempeh diet for 3 days. To begin another 3-day experimental protocol for the same participant, at least a 1-week interval will be required to ensure complete washout of the tracer.

During each adaptation period, every participant will be given 4 equal meals per day (breakfast, lunch, dinner, and snack) that fulfill 100% of their daily energy and protein requirements (Energy requirement = Resting Energy Expenditure (REE) × 1.7; Protein requirement = 1.0 g/kg/day × body weight). Meals provided will include a protein-free formula (required), protein-free butterscotch cookies (required), a crystalline amino acid mixture, or a combination of the crystalline amino acid mixture with tempeh chips. The meals should be finished by participants within 15 minutes. A daily multivitamin supplement and a 500 mg choline supplement will also be provided. In addition to plain water, only one cup of tea or coffee without sugar or cream is allowed during the adaptation days.

During the IAAO study period, after an overnight fast of 12 hours, participants will attend the metabolic laboratory at Kolej Tun Syed Nasir (KTSN) for 8 hours. A total of 9 isonitrogenous and isocaloric hourly meals will be provided, with each meal contributing 1/10 of the total energy requirement (REE × 1.5) and protein requirement (1.0 g/kg/day). Meals will consist of a protein-free formula (required), protein-free butterscotch cookies (required), and either a crystalline amino acid mixture or a combination of the crystalline amino acid mixture with tempeh chips. Each meal should be consumed within 15 minutes. The total meals provided will meet 90% of the participants' energy and protein requirements. During the experimental day, a daily multivitamin supplement and 500 mg of choline will also be provided.

During the first to fourth meals, participants will only be required to consume the provided meals. Starting from the fifth meal, participants will receive a constant oral isotope incorporation. Phenylalanine, an indispensable amino acid whose final metabolism produces carbon dioxide, will be used as the tracer to measure oxidation. In addition, NaH13CO3 (¹³C-sodium bicarbonate) will be administered as a priming dose to allow 13CO2(carbon-13 labeled CO₂) in the breath to reach a plateau within 2-3 hours. A dose of 0.176 mg/kg of NaH13CO3 and 0.66 mg/kg of L-\[1-13C\]phenylalanine will be given as the priming isotope during the fifth meal. From the fifth meal until the last meal, 1.2 mg/kg of L-\[1-13C\]phenylalanine will be provided continuously. The amount of phenylalanine provided as the tracer (excluding the priming isotope) will be subtracted from the dietary provision to maintain a total phenylalanine intake of 30 mg/kg/day. A total of 40 mg/kg/day (160% of the total aromatic amino acid requirement) of tyrosine will also be provided to ensure that no labeled phenylalanine is hydroxylated to meet the tyrosine requirement. After the fifth meal, the expired carbon dioxide production rate (VCO2) will be measured using indirect calorimetry for 15 minutes.

For breath sample collection, samples will be collected every 15 minutes before the tracer protocol is initiated (i.e., before the fifth meal) and every 15 minutes after isotopic steady state is reached (approximately 2.5 hours after isotope administration) until the end of the study. Additional breath samples will be collected 30 minutes after the end of the study. Each breath sample will be collected in triplicate using 10 mL non-silicon-coated tubes. The enrichment of 13CO2 in the breath will be analyzed using a continuous-flow isotope ratio mass spectrometer and expressed as atom percent excess (APE) over baseline. The fraction of 13CO2 (F13CO2) from L-\[1-13C\]phenylalanine oxidation relative to the amount administered will be calculated.

Using a linear mixed-effects regression model, the reference and test IAAO response slopes will be generated from F13CO2 measurements and their corresponding methionine intakes, with a common origin of the F13CO2 response at the base methionine intake. The metabolic availability (MA) of methionine in the test protein will be determined by the slope-ratio method using the following equation:

MA (%) = (Test protein / Reference protein) × 100.