Combining Dietary Protein Sources to Improve Amino-Acid Digestibility and Net Protein Balance - The EPiC Study

The design is a 5-arm (5 visits) randomized crossover conducted in healthy men and women aged 18 to 45. In each arm of the study, the participant will ingest a meal comprising a different combination of proteins (22g protein, 9g indispensable amino acids), each with different digestibility defined by DIAAS (equal to or above 100 percent, 75 percent, 50 percent). The participant will visit the lab 6 times: 1 for introduction/screening and signed consent, and 5 times for each arm of the study, representing the test days. The participant will be asked to refrain from alcohol, caffeinated foods, and drinks the day prior from 6 pm the night before. Upon waking, the participant will be asked to toilet, drink 300 ml of water, and have no food before entering the research lab. Other requirements will be to refrain from hard exercise (equal to or below 45 min light aerobic only; no hard exercise, weight-lifting, eccentric exercise such as running downhill) two days before visits 1-5. After coming to the lab at an agreed time between 6:00 am and 9:00 am, catheters for blood sampling and infusion will be placed in veins at the top of your non-dominant hand and forearm on the other side. The hand with the line will be placed into a heated-hand box to arterialize the blood flow through the hand. Administration of stable isotope amino acids will occur continuously, and blood sampling will occur at regular time points over 5.5h (1.5h prior to and 4 hours after ingestion of the test meal) (L-[Ring-2H5]-Phenylalanine and L-[Ring-2H2]-Tyrosine. Infusion rates for L-[Ring-2H5]-Phenylalanine and L-[Ring-2H2]-Tyrosine will be 270 µmol/h-1 and 85.5 µmol/h-1, respectively, with priming doses of 270 µmol and 85.5 µmol. To prime the phenylalanine-derived plasma tyrosine pool, a bolus dose of L-[Ring-2H4]-tyrosine will be administered (priming dose of 23.25 µmol). After 1.5 h, you will ingest the pre-heated test meal (within 15 minutes under supervision of the research staff) comprising whole foods of combinatorial protein (commercially bought) and 83.9 mg/meal of L-[15N]-Phenylalanine (to measure splanchnic extraction). The composition of the meals will be 810 kcal, 22g protein (9g indispensable amino acids), 132g carbohydrates, 22g fats. The meal will comprise the following protein: CP1. Topside steak and Bread (DIAAS 105) (reference protein), CP2. Chickpeas, Quinoa, Quorn, and Bread (DIAAS 105) (matched to meet the per-meal bioavailability of the reference protein, CP1), CP3. Chickpeas, Quinoa, Tofu, and Bread (DIAAS 75), CP4. Chickpeas, Quinoa, and Bread (DIAAS 50), CP5. Chickpeas, Quinoa, Bread, and L-Lysine supplement (DIAAS 100) (matched to meet the per-meal IAA requirement, in digestible/bioavailable values, for the first limiting amino acid: lysine to increase the quality to match CP1). To make up the isocaloric composition of 810 kcal, we add juice (mango and passionfruit), fruit (banana), and oil (olive oil). The meals will be delivered by Professor David Rowlands or Mr. Robin D. Nielsen (Ph.d. candidate). The Gastrointestional scale will be measured every 15 min the first hour and every 30 min for the last three hours. Blood will be collected from a hand vein at specified intervals for another 4 hours after meal ingestion. During this time, the participant will remain rested either fully or semi-reclined on a research bed or chair during sampling and may do (1-handed) computer work, watch TV, or relax. There will be a minimum 1-week washout between test days to eliminate any carry-over effects; for women, to control for the effects of the menstrual cycle on gastric emptying and metabolism, we will test between 3-11 days after the first day of the start of the menstrual cycle, meaning most testing in women will occur approximately monthly. Blood plasma be stored and later analyzed for amino acids and tracer/tracee concentration using mass spectrometry at Massey University. Data will be analyzed to determine how much and how fast the combinatorial protein has affected whole-body protein turnover. Results will inform how protein quality is related to whole-body protein turnover determined by the DIAAS of the meal and provide the first data on how dietary protein quality defined by DIAAS affects protein metabolism in humans.