Does Combining Dietary Protein Sources Improve Amino-Acid Digestibility and Net Protein Balance in Older Adults? The DIPO Study

The design is a 3-arm (3 visits) randomized crossover conducted in healthy older men and women aged 67 to 77. 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 4 times: 1 for introduction/screening and signed consent, and 3 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 1-3. After coming to the lab at an agreed time between 6:00 am and 9:00 am, catheters for blood sampling and stable isotope infusion (non-radioactive) 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 (L-[Ring-2H5]-Phenylalanine and L-[Ring-2H2]-Tyrosine), 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). 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, participants will ingest a pre-heated intervention meal (within 15 minutes under supervision of the research staff) comprising whole foods of combinatorial protein (commercially bought) and 10% of total meal phenylalanine of L-[15N]-Phenylalanine (to measure splanchnic extraction). The composition of the meals will be approximately 810 kcal, 22g protein (9g indispensable amino acids), 132g carbohydrates, 22g fats. The meal will comprise the following protein: Combinatorial Protein 1. Chickpeas, Quinoa, Quorn (micoprotein), and Bread (DIAAS 100), Combinatorial Protein 2. Chickpeas, Quinoa, Tofu, and Bread (DIAAS 75), Combinatorial Protein 3. Chickpeas, Quinoa, and Bread (DIAAS 50). We add maltodextrin and olive oil to make up the isocaloric composition of 810 kcal. The lead researcher or PhD candidate will deliver the meals. Participants will be directly supervised during the meals to ensure adherence. The Gastrointestinal scale will be measured at 30, 60, and 240 min postprandial. Prior to the first visit, participants will fill out questionnaires including: habitual protein intake, sustainability index, and thoughts about meat and alternative meats. 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. Blood plasma be stored and later analyzed for amino acids and tracer/tracee concentration using mass spectrometry at AgResearch, Palmerston North. Data will be analyzed to determine how much and how fast the combinatorial protein has affected whole-body protein net balance. Results will inform how protein quality is related to whole-body protein net balance determined by the DIAAS of the meal and provide the first data on how dietary protein quality defined by DIAAS affects protein metabolism in older adults.