Plant-based and Animal-based Protein Diets on Muscle Growth and Functional Health in Overweight Elderly - Pilot (Phase 1)

This study includes a pilot study, designed to assess the equipment at Deakin, using deuterium oxide (D2O) enrichment and LC-MS/MS and GC-MS techniques, to detect fractional protein synthesis rate (FSR) comparable to existing literature (FSR ~0.0542%·h-1) for future use in a larger RCT. Study aims: 1. The primary objective of the pilot is to assess D2O enrichment and LC-MS/MS and GC-MS techniques, to detect protein synthesis comparable to existing literature (FSR ~0.0542%·h-1) in both the plant-based (Intervention group) and animal-based (Control group) protein-source diets. Hypothesis: 1. We hypothesise that the pilot shows D2O enrichments and LC-MS/MS and GC-MS techniques can detect MPS comparable to existing literature, in both the intervention and control group, using current dosing protocol. The pilot study is a 2-week study including a pre-screening, a 1-week run-in period and a 2-week intervention, with a total of 4 visits to Deakin University. 1-week run-in period: At visit 1, participants will be councelled to follow a dietitian-guided isocaloric diet providing 1-1.2g of protein per kg of body weight. This diet will be based on their habitual intake, which is assessed by a 3-day dietary record performed prior to the first visit. This diet standardises protein intake and ensures a controlled baseline. Dietary intervention: At visit 2, all participants will have been allocated to either the intervention or control group. The participants will receive dietary councelling on how to follow an isocaloric diet providing 1-1.2g of protein per kg of body weight. The intervention group will be consuming 70% plant-based and 30% animal-based protein sources from their total protein intake every day of the 14-day dietary intervention, The intervention group will be supervised by dieticians who will tailor the diet to comply with the recommended daily nutrient intake. In addition, the diet will be aligned with the sensory needs and close to dietary habits, allowing a smooth behavioural transition. Examples of protein sources being used as plant protein are: legumes, nuts & seeds, vegetables, cereals and bread etc. Examples of protein souces being used as animal protein are: eggs, meat, poultry, fish etc. Study setting: Data collection, including anthropometric, physiological, objective and performance data, will occur at the Burwood Campus in the Physiology Laboratory, Deakin University. The muscle biopsies and blood samples will be conducted in our research labs in SENS at the Burwood Campus Deakin University. Muscle protein synthesis: For the primary outcome to detect MPS using D2O enrichment, the pilot will be collecting objective measurements including saliva and blood samples, as well as muscle biopsies. Participants will get an intital dosing of 140.28 ml 99.8% D2O spread out in intervals (35.07ml x 4 consumed 1 hour apart) on visit 2, whereafter they will continue to consume a maintenance dose of 14.03 ml D2O daily at home. A skeletal muscle biopsy and will be obtained the day after D2O dosing, on visit 3 on the left leg, and again on visit 4 (14 days later), on the right leg. Biopsies will be obtained from the middle region of the vastus lateralis muscle (~15 cm above the patella) and approximately 4 cm below entry through the fascia using the percutaneous needle biopsy technique (Holwerda et al., 2024). Participants will further collect a daily saliva sample at the same time every day, preferably in the morning, and body water enrichment is analysed using these saliva samples collected throughout intervention period (Holwerda et al., 2024). To assess plasma AA deuterated-alanine enrichments, blood samples are collected at visit 2 prior to the D2O dosing, as well as at visit 3 and visit 4. Plasma AA deuterated-alanine enrichments will be determined by gas chromatography-mass spectrometry (GC-MS) analysis (Holwerda et al., 2024). The FSR is determined by calculating the change in muscle protein-bound enrichment relative to the mean precursor enrichment over the experimental period. It is expressed as a percentage of the entire protein pool (i.e., mixed muscle, myofibrillar, sarcoplasmic, and/or mitochondrial muscle protein) that is synthesised during the study period. FSR is calculated using following 2 equations 1. Equation 1: FSR(% x d-1)=k =-ln (1 - f)tX 100 where 1 and k represents the turnover rate of the protein pool. 2. Equation 2: f=EPRO2 - EPRO1Ebw X 3.7 where EPRO2 and EPRO1 represent the protein-bound enrichments at two different timepoints, and Ebw represents the body water enrichments, and t is the duration between the two biopsies taken (Holwerda et al., 2024). Dietary recording: To assess the participants dietary intake, the participants will keep a detailed food record including all food, drinks and snacks they have consumed in the app EasyDietDiary. The first 3-day dietary record is made prior to visit 1 and will be used to assess and create their individualised diet for both the run-in period and the intervention. The data from the second 3-day dietary recording is made on days 6, 7 and 8 of the intervention, and will be used to analyse adherence to the diet. Data from the EasyDietDietary app will be uploaded to FoodWorks for analysis. Feasibility and acceptability: To assess the participants experience of the dietary intervention, they will complete questionnaires in REDCap with the assistance of one of the researchers. The questionnaires include a validated questionnaire provided by Gie Liem, assessing the sensory experience of the diet. To assess the overall feasibility and acceptability a new questionnaire was developed, as no existing validated tool included all the elements required for this study.