Trial Review

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Trial registered on ANZCTR


Registration number
ACTRN12623000834651
Ethics application status
Approved
Date submitted
7/07/2023
Date registered
3/08/2023
Date last updated
18/09/2023
Date data sharing statement initially provided
3/08/2023
Type of registration
Prospectively registered

Titles & IDs
Public title
Combining Dietary Protein Sources to Improve Amino-Acid Digestibility and Net Protein Balance - The EPiC Study
Scientific title
How Does the Digestible Indispensable Amino Acid Score (DIAAS) Influence Protein Turnover? The Efficacy Potential of Combinatorial Proteins in Humans
Secondary ID [1] 309843 0
Nil known
Universal Trial Number (UTN)
Trial acronym
The EPiC study (the Efficacy Potential of Combinatorial Proteins)
Linked study record

Health condition
Health condition(s) or problem(s) studied:
Protein malnourishment 330275 0
Condition category
Condition code
Metabolic and Endocrine 327142 327142 0 0
Normal metabolism and endocrine development and function
Diet and Nutrition 327534 327534 0 0
Other diet and nutrition disorders

Intervention/exposure
Study type
Interventional
Description of intervention(s) / exposure
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.
Intervention code [1] 326277 0
Lifestyle
Intervention code [2] 326536 0
Treatment: Other
Comparator / control treatment
Animal protein meal (beef and bread; DIAAS equal to or above 100).
Control group
Active

Outcomes
Primary outcome [1] 335014 0
Postprandial whole-body net protein balance responses to protein quality measured via infusion of stable isotope tracer amino acids: L-[Ring-2H5]-Phenylalanine, L-[Ring-2H2]-Tyrosine], and L-[Ring-2H4]-Tyrosine and ingestion of L-[15N]-Phenylalanine (splanchnic extraction), These data provide information to enable calculation of postprandial changes in protein net balance over 4 hours (pre to post ingestion of intervention meal).
Timepoint [1] 335014 0
Postprandial changes in protein net balance responses over 4 hours (pre to post ingestion of intervention meal). Timepoints: -90, -60, -30, 0, 15, 30, 45, 60, 90, 120, 150, 180, 210, and 240-min post-intervention meal consumption on intervention visits 1, 2, 3, 4, and 5.
Primary outcome [2] 335391 0
Postprandial whole-body protein synthesis responses to protein quality measured via infusion of stable isotope tracer amino acids: L-[Ring-2H5]-Phenylalanine, L-[Ring-2H2]-Tyrosine], and L-[Ring-2H4]-Tyrosine and ingestion of L-[15N]-Phenylalanine (splanchnic extraction), These data provide information to enable calculation of postprandial protein synthesis over 4 hours (pre to post ingestion of intervention meal).
Timepoint [2] 335391 0
Postprandial changes in protein synthesis responses over 4 hours (pre to post ingestion of intervention meal). Timepoints: -90, -60, -30, 0, 15, 30, 45, 60, 90, 120, 150, 180, 210, and 240-min post-intervention meal consumption on intervention visits 1, 2, 3, 4, and 5.
Primary outcome [3] 335392 0
Postprandial protein degradation responses to protein quality measured via infusion of stable isotope tracer amino acids: L-[Ring-2H5]-Phenylalanine, L-[Ring-2H2]-Tyrosine], and L-[Ring-2H4]-Tyrosine and ingestion of L-[15N]-Phenylalanine (splanchnic extraction), These data provide information to enable calculation of postprandial protein synthesis over 4 hours (pre to post ingestion of intervention meal).
Timepoint [3] 335392 0
Post-prandial changes in protein degradation responses over 4 hours (pre to post ingestion of interventional meal). Timepoints: -90, -60, -30, 0, 15, 30, 45, 60, 90, 120, 150, 180, 210, and 240-min post-intervention meal consumption on intervention visits 1, 2, 3, 4, and 5.
Secondary outcome [1] 422712 0
Post-prandial plasma Area Under the Curve (AUC) for indispensable amino acids (IAA) concentration of total IAA.
Timepoint [1] 422712 0
Postprandial changes in plasma IAA responses over 4 hours (pre to post ingestion of intervention meal). Timepoints: 0-, 15-, 30-, 45-, 60-, 90-, 120-, 150-, 180-, 210-, and 240-min post-intervention meal consumption on intervention visits 1, 2, 3, 4, and 5.
Secondary outcome [2] 424276 0
Postprandial plasma Area Under the Curve (AUC) for insulin.
Timepoint [2] 424276 0
Postprandial changes in plasma insulin responses over 4 hours (pre to post ingestion of intervention meal). Timepoints: 0-, 15-, 30-, 45-, 60-, 90-, 120-, 150-, 180-, 210-, and 240-min post-intervention meal consumption on intervention visits 1, 2, 3, 4, and 5.
Secondary outcome [3] 424280 0
Post-prandial gastrointestinal comfort scale in response to the interventional meal.
Timepoint [3] 424280 0
Postprandial gastrointestinal comfort over 4 hours (pre to post ingestion of intervention meal) designed specifically for this study. This will be measured every 15 min the first hour and every 30 min the last three hours.
Secondary outcome [4] 424747 0
Post-prandial plasma Area Under the Curve (AUC) for total leucine
Timepoint [4] 424747 0
Postprandial changes in plasma leucine responses over 4 hours (pre to post ingestion of intervention meal). Timepoints: 0-, 15-, 30-, 45-, 60-, 90-, 120-, 150-, 180-, 210-, and 240-min post-intervention meal consumption on intervention visits 1, 2, 3, 4, and 5.
Secondary outcome [5] 424748 0
Post-prandial plasma Area Under the Curve (AUC) for total lysine.
Timepoint [5] 424748 0
Postprandial changes in plasma lysine responses over 4 hours (pre to post ingestion of intervention meal). Timepoints: 0-, 15-, 30-, 45-, 60-, 90-, 120-, 150-, 180-, 210-, and 240-min post-intervention meal consumption on intervention visits 1, 2, 3, 4, and 5.
Secondary outcome [6] 424749 0
Postprandial plasma Area Under the Curve (AUC) for glucose
Timepoint [6] 424749 0
Postprandial changes in plasma glucose responses over 4 hours (pre to post ingestion of intervention meal). Timepoints: 0-, 15-, 30-, 45-, 60-, 90-, 120-, 150-, 180-, 210-, and 240-min post-intervention meal consumption on intervention visits 1, 2, 3, 4, and 5.
Secondary outcome [7] 424750 0
Postprandial plasma Area Under the Curve (AUC) for incretin
Timepoint [7] 424750 0
Postprandial changes in plasma incretin responses over 4 hours (pre to post ingestion of intervention meal). Timepoints: 0-, 15-, 30-, 45-, 60-, 90-, 120-, 150-, 180-, 210-, and 240-min post-intervention meal consumption on intervention visits 1, 2, 3, 4, and 5.

Eligibility
Key inclusion criteria
- Men and eumenorrheic women aged 18 to 45.
- BMI: 18 to 25
- HbA1c within the non-diabetic range of <40 mmol/mol.
- Physical activity level (PAL) is within the range of 1.60-1.99, defined as light to moderate by the FAO (FAO, 2001).
- Obtained his/her (or his/her legal representative's informed consent).
Minimum age
18 Years
Maximum age
45 Years
Sex
Both males and females
Can healthy volunteers participate?
Yes
Key exclusion criteria
- Planning on leaving the city or proximity that would affect the ability or willingness (we may be able to support travel costs) to participate in all 5 study arms for the entire study duration.
- Other foreseen factors that may prevent the completion of the study.
- Criteria-defined sedentary due to a precluding disability
- Missing hands (for arterialized-venous blood sampling)
- Active malignancy (cancer) within the past six months.
- Current pregnancy.
- Unwilling to ingest animal proteins.
- Allergy to experimental foods (i.e., gluten, lectin, and allergens).
- Any gastrointestinal disease or disorder that may affect the study outcomes.
- Gastrointestinal bypass surgery or congenital gastrointestinal issues.
- Chronic inflammatory disease (rheumatoid arthritis, psoriasis, psoriatic arthritis, Crohn's disease, ulcerative colitis, and ankylosing
spondylitis).
- Taking medications that may interfere with the study outcomes.
- Currently participating or having participated in another clinical study during the last four weeks prior to the beginning of this study that may affect results.

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)
Sealed opaque envelopes
Methods used to generate the sequence in which subjects will be randomised (sequence generation)
Simple randomisation to one of the Latin square sequences by randomisation function in Excel. Participants will be allocated to sequences based on the Latin Square design.
Masking / blinding
Blinded (masking used)
Who is / are masked / blinded?
The people receiving the treatment/s


Intervention assignment
Crossover
Other design features
Phase
Not Applicable
Type of endpoint/s
Bio-availability
Statistical methods / analysis
The sample size was determined from the only relevant available protein net balance data of Kim et al., 2018 where the egg vs. cereal difference (expected effect size between no and excellent quality) was 7 g net protein accretion in 165 min of infusion time, and the standard error derived from the p-value of 1.53 g (Kim et al., 2018). With the smallest important change of 2.2 g protein net balance, using traditional null hypothesis significance testing with 5% type-1 and 20% type-2 error rates, a sample size of 8 was required (Hopkins, 2006), which we felt provided sufficient precision for the 30% of the anticipated difference between high and excellent protein quality, relative to the no-excellent contrast. The meet the full Latin Square matrix for 5 treatment arms, a final sample size of 10 is required.

Primary analysis:
Outcomes will be analyzed using a mixed model analysis of variance. Fixed effects will be meal condition and time (where relevant). The random effect will be subject to an unstructured covariance matrix to account for correlated data within the crossover. Data will be log-transformed to improve linearity and model fit and to express outcomes as percent differences. Primary outcomes (protein synthesis, protein degradation, and protein net balance) will be referenced to our estimate of the smallest important clinical changes. Data will be presented as the least-squares mean and uncertainty (95% confidence interval) and the effect size as a standardized mean difference.

Secondary analysis:
The statistical analysis on secondary outcomes will be as for the primary, except psychometric scale data will not be log-transformed.

Recruitment
Recruitment status
Recruiting
Date of first participant enrolment
Anticipated
2/10/2023
Actual
Date of last participant enrolment
Anticipated
1/04/2024
Actual
Date of last data collection
Anticipated
30/08/2024
Actual
Sample size
Target
10
Accrual to date
0
Final
Recruitment outside Australia
Country [1] 25582 0
New Zealand
State/province [1] 25582 0
Auckland

Funding & Sponsors
Funding source category [1] 314029 0
University
Name [1] 314029 0
Riddet Institute, Massey University
Country [1] 314029 0
New Zealand
Funding source category [2] 314030 0
University
Name [2] 314030 0
Massey University
Country [2] 314030 0
New Zealand
Primary sponsor type
University
Name
Riddet Institute, Massey University
Address
Riddet Institute, Massey University
Private Bag 11 222, Palmerston North 4442
New Zealand
Country
New Zealand
Secondary sponsor category [1] 315920 0
None
Name [1] 315920 0
Address [1] 315920 0
Country [1] 315920 0

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 313164 0
Central Health and disability Ethics Commitee
Ethics committee address [1] 313164 0
Ethics committee country [1] 313164 0
New Zealand
Date submitted for ethics approval [1] 313164 0
04/07/2023
Approval date [1] 313164 0
18/07/2023
Ethics approval number [1] 313164 0
18248

Summary
Brief summary
Trial website
Trial related presentations / publications
Public notes

Contacts
Principal investigator
Name 127214 0
Prof David S. Rowlands
Address 127214 0
Massey University, SNW Extension Level 3, Albany Expressway (SH17), Albany, Auckland 0632, New Zealand
Country 127214 0
New Zealand
Phone 127214 0
+64 272099383
Fax 127214 0
Email 127214 0
[email protected]
Contact person for public queries
Name 127215 0
Robin D. Nielsen
Address 127215 0
Massey University, SNW Extension Level 3, Albany Expressway (SH17), Albany, Auckland 0632, New Zealand
Country 127215 0
New Zealand
Phone 127215 0
+64 212874100
Fax 127215 0
Email 127215 0
[email protected]
Contact person for scientific queries
Name 127216 0
Robin Dreymann Nielsen
Address 127216 0
Massey University, SNW Extension Level 3, Albany Expressway (SH17), Albany, Auckland 0632, New Zealand
Country 127216 0
New Zealand
Phone 127216 0
+64 212874100
Fax 127216 0
Email 127216 0
[email protected]

Data sharing statement
Will individual participant data (IPD) for this trial be available (including data dictionaries)?
No
No/undecided IPD sharing reason/comment


What supporting documents are/will be available?




Results publications and other study-related documents

Documents added manually
No documents have been uploaded by study researchers.

Documents added automatically
No additional documents have been identified.