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Trial registered on ANZCTR


Registration number
ACTRN12617000923369
Ethics application status
Approved
Date submitted
20/06/2017
Date registered
26/06/2017
Date last updated
12/06/2018
Type of registration
Prospectively registered

Titles & IDs
Public title
The impact of different proline and glycine containing test foods and supplements on serum amino acid availability, and engineered ligaments
Scientific title
The impact of different proline and glycine containing test foods and supplements on serum amino acid availability in healthy active males, and engineered ligaments
Secondary ID [1] 292001 0
Nil known
Universal Trial Number (UTN)
Trial acronym
Linked study record

Health condition
Health condition(s) or problem(s) studied:
Connective tissue health 303367 0
Collagen synthesis 303368 0
Condition category
Condition code
Musculoskeletal 302792 302792 0 0
Normal musculoskeletal and cartilage development and function

Intervention/exposure
Study type
Interventional
Description of intervention(s) / exposure
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.
Intervention code [1] 298125 0
Treatment: Other
Comparator / control treatment
Cross-comparison study between different collagen containing and non-collagen containing foods. Matched to amount of protein and volume. All subjects will complete identical consumption protocols, with the only difference being the type of food consumed.
Control group
Active

Outcomes
Primary outcome [1] 302461 0
Change in plasma amino acid after ingestion of treatment food - assessed within all participants
Timepoint [1] 302461 0
Pre - treatment (baseline), and then every 20 minutes for a total of 3 hours post ingestion of treatment
Secondary outcome [1] 336256 0
Urinary hydroxyproline excretion - this outcome is assessed in part b only.
Timepoint [1] 336256 0
Baseline (1 day before testing commences)
After the intake of 2 collagen containing and 2 non-collagen containing food sources/supplements
Both of the above at the following time points:
First morning (upon waking)
12 hours after first morning urine (before bed)
24 hours
Secondary outcome [2] 348001 0
Collagen content of engineered ligaments. Blood pulled at time points corresponding to plasma peaks of amino acids will be used to "feed" engineered ligaments that have been developed at University of California, Davis. Collagen content will be assessed by measuring hydroxyproline content of the ligaments (which makes up ~14% of the collagen content) The collagen fraction will then be determined by dividing the collagen content by the mass of the tissue.
Timepoint [2] 348001 0
BL and at timing of peaks after the consumption of 3 test supplements
Secondary outcome [3] 348002 0
Tensile properties of engineered ligaments . Ligaments will be developed as outlined above, tensile properties will be tested using digital calipers.
Timepoint [3] 348002 0
BL and at timing of peaks after the consumption of 3 test supplements

Eligibility
Key inclusion criteria
Active individuals exercising at least 2-3 times per week
Minimum age
18 Years
Maximum age
40 Years
Sex
Males
Can healthy volunteers participate?
Yes
Key exclusion criteria
Females: Variations in serum estrogen concentrations associated with female reproductive function has been shown to influence the mechanical properties of engineered ligaments via its action on lysyl oxidase. This would add confounding to the study and therefore we have decided to control for this by excluding female subjects.
2. Individuals with allergy/intolerance to porcine and/or bovine gelatin, milk and/or milk products and/or chicken/beef/seafood
3. Individuals with diabetes: Increase blood glucose has been shown to influence non-enzymatic cross linking in collagen.
4. Individuals with a history of recent (< 1 month) or current tendinopathies: Inflammatory cytokines have been shown to influence fibroblast activity and collagen synthesis and subsequent cross-linking.

Study design
Purpose of the study
Treatment
Allocation to intervention
Randomised controlled trial
Procedure for enrolling a subject and allocating the treatment (allocation concealment procedures)
Allocation will be controlled by a primary researcher. Samples will be coded so as to ensure blinding from assessors.
Methods used to generate the sequence in which subjects will be randomised (sequence generation)
Simple randomisation using a randomisation table created by computer software (i.e. computerised sequence generation)
Masking / blinding
Blinded (masking used)
Who is / are masked / blinded?


The people assessing the outcomes
The people analysing the results/data
Intervention assignment
Crossover
Other design features
Phase
Not Applicable
Type of endpoint/s
Bio-availability
Statistical methods / analysis
Sample size calculations
Part a. A power estimation for detecting differences in AUC of blood amino acid concentrations following intake of protein-rich food has previously been undertaken by Van Loon et al. 2000. The suitability of the derived sample size (n = 15) was further confirmed by other research undertaken by our team in which clear differences in post-meal concentrations of blood amino acid were detected in a cohort of athletes of this size (Burke et al. 2012).
Part b. The suitability of a sample size of 8 for detecting changes in urinary HYP was confirmed by a power calculation using a 5mg change assuming a SD of 2.5mg providing an estimation of 80% power at the 0.05 level of significance.
Part c. According to calculations undertaken in a similar study by our group (Shaw et al. 2016), a 2.4 ug change in engineered ligament collagen content, assuming a SD of 1 ug with 80% power at the 0.05 level of significance, will be detected with a sample size of 8 subjects.

Statistics
Descriptive statistics will be used to summarise amino acid content within each food sample, serum levels of amino acids and differences in engineered ligament properties between and within groups. The differences between amino acid data and engineered ligament properties between and within groups will be analysed using a repeated measures two way ANOVA with either LSD or Tukey HSD post hoc analysis. To determine the association between urine Hyp and intake of Hyp, Pearson’s correlation will be used. Statistical significance will be set at p <0.05. Statistical analysis will be undertaken using SPSS (Version 15 for Windows, SPSS Inc, Chicago, IL).

Recruitment
Recruitment status
Completed
Date of first participant enrolment
Anticipated
15/07/2017
Actual
17/07/2017
Date of last participant enrolment
Anticipated
1/01/2018
Actual
5/02/2018
Date of last data collection
Anticipated
28/03/2018
Actual
27/03/2018
Sample size
Target
16
Accrual to date
Final
16
Recruitment in Australia
Recruitment state(s)
ACT
Recruitment postcode(s) [1] 16454 0
2617 - Bruce

Funding & Sponsors
Funding source category [1] 296515 0
University
Name [1] 296515 0
Australian Catholic University
Country [1] 296515 0
Australia
Funding source category [2] 296755 0
Other
Name [2] 296755 0
ACT Brumbies
Country [2] 296755 0
Australia
Primary sponsor type
Government body
Name
Australian Institute of Sport
Address
Leverrier street, Bruce, Canberra ACT 2616
Country
Australia
Secondary sponsor category [1] 295731 0
University
Name [1] 295731 0
University of California Davis
Address [1] 295731 0
Hutchison Dr.
Davis, CA, United States 95616
Country [1] 295731 0
United States of America
Secondary sponsor category [2] 295736 0
University
Name [2] 295736 0
Maastricht University
Address [2] 295736 0
Minderbroedersberg 4-6, 6211 LK Maastricht, Netherlands
Country [2] 295736 0
Netherlands
Secondary sponsor category [3] 295767 0
Other
Name [3] 295767 0
Collagen Research Institute
Address [3] 295767 0
Schauenburgerstraße 116, 24118 Kiel
Country [3] 295767 0
Germany

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 297736 0
Australian Institute of Sport ethics committee
Ethics committee address [1] 297736 0
Ethics committee country [1] 297736 0
Australia
Date submitted for ethics approval [1] 297736 0
22/05/2017
Approval date [1] 297736 0
07/07/2017
Ethics approval number [1] 297736 0

Summary
Brief summary
Trial website
Trial related presentations / publications
Public notes
Attachments [1] 2771 2771 0 0
/AnzctrAttachments/372982-20170607 - Collagen and non-collagen contraining food courses - Rebekah Alcock - Approval.pdf (Ethics approval)

Contacts
Principal investigator
Name 74986 0
Miss Rebekah Alcock
Address 74986 0
Australian Institute of Sport
Leverrier St, Bruce ACT 2617
Country 74986 0
Australia
Phone 74986 0
+61476291511
Fax 74986 0
Email 74986 0
Rebekah.Alcock@ausport.gov.au
Contact person for public queries
Name 74987 0
Rebekah Alcock
Address 74987 0
Australian Institute of Sport
Leverrier St, Bruce ACT 2617
Country 74987 0
Australia
Phone 74987 0
+61476291511
Fax 74987 0
Email 74987 0
Rebekah.Alcock@ausport.gov.au
Contact person for scientific queries
Name 74988 0
Rebekah Alcock
Address 74988 0
Australian Institute of Sport
Leverrier St, Bruce ACT 2617
Country 74988 0
Australia
Phone 74988 0
+61476291511
Fax 74988 0
Email 74988 0
Rebekah.Alcock@ausport.gov.au

No information has been provided regarding IPD availability


What supporting documents are/will be available?

No Supporting Document Provided



Results publications and other study-related documents

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