The impact of different proline and glycine containing test foods and supplements on serum amino acid availability, and engineered ligaments

The amino acid bio-availability of proline and glycine containing foods will be measured in 15 participants (part a) as per the protocol outlined below. These 15 participants will be separated further into 2 sub-groups of 8 for the assessment of urinary hydroxyproline (HYP) - (part b) and the assessment of food sources on In vitro collagen synthesis and ligament mechanical properties (part c). The second group of 8 participants included in part c will undertake the research protocol once the timing of appearance and peaks of amino acids are determined: derived from the 1st group and will inform the timing of part 3: serum sample collection for the engineered ligament. Research protocol: Part a: Healthy male participants will complete a single-blinded cross-over design. A total of 7 trials will be undertaken in fasted, rested conditions with a 72 hour wash-out period between trials. Participants will arrive on testing days after an overnight fast. Participants will be cannulated by a trained phlebotomist and a baseline (BL) blood sample collected (2 ml) for analysis of amino acids. The participants will then consume the test food (as described below) and blood samples (2ml) will be obtained every 20 minutes over the proceeding 3 hours for plasma amino acid assessment. Participants will ingest 7 different proline and glycine containing test foods (specifically whey protein powder, skim milk powder, casein protein powder, hydrolysed collagen powder, liquid gelatin, solid gelatin powder and a bone broth). Foods will be sent to an external lab for analysis of food amino acid concentrations (Australian Proteome Analysis Facilty). Participants will ingest 20 g of each supplements, and 300 ml of bone broth (to provide a practical amount). Commencement of intake of test food will be considered t = 0 and encouragement will be provided to finish consumption of test food within 5 min. Fluid volume associated with product intake will be standardised to 300ml. (this will consist of either the test food being used if applicable i.e. bone broth or a matched amount of fluid will be given in the form of water). No further food/fluid intake will be allowed for 60 min after ingestion of test food to standardise gastric emptying rates. Once the final blood sample is collected, the cannula will then be removed and pressure applied to the insertion site, the participant will be free to go and will return to the lab for subsequent testing after a minimum 48 hr washout. Part b: For the first 8 participants completing the study, participants will attend the lab the day prior to day 1 testing, and provide a urine samples for assessment of baseline HYP assessment (first morning, 12 hours after first morning (i.e. before bed) and 24h excretion) and 24 hour food/fluid intake records. On arrival to the lab of the 1st testing day, these 8 participants will be required to give a first morning urine sample and will then undergo testing as per part a. To assess for hydration status (USG) and urinary excretion of hydroxyproline (HYP), first morning urine will be collected on each trial day (BL) (USG), 12 hours after 1st morning urine (i.e. before bed) and for the 24 hour period following the ingestion of the test food. A 24hr food diary will also be required to be completed by participants to assess dietary intake of HYP containing foods. Part c: For the second 8 participants completing the trials, an additional 55ml of blood will be collected at BL and at the time point associated with the proline and glycine plasma peak post ingestion. This blood collection will occur for 3 of the 7 foods described above. Two collagenous protein sources (hydrolysed collagen, Gelatin) and one non collagenous protein source (hydrolysed casein powder). The serum obtained from this blood pull (~ 24ml) will be utilised for the treated constructs and serum will be sent to the University of California, Davis Proteomics Core facility. The second 8 participants will complete the trials after amino acid analysis has occurred (~3 months post part a, to ensure timing of blood pulls correspond to measured amino acid peaks. Once collected the 55ml blood will be allowed to clot and spun at 1, 500 x g for 10 min with the resultant serum separated into 2 x ~12ml samples and stored at -80C for further analysis. NB. . The research protocol, and any relevant information on dietary intake (i.e. 24 food diaries) will be completed by an Accredited Practicing Dietitian with a minimum of 3 years experience.