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Trial registered on ANZCTR


Registration number
ACTRN12625000174482
Ethics application status
Approved
Date submitted
20/01/2025
Date registered
14/02/2025
Date last updated
14/02/2025
Date data sharing statement initially provided
14/02/2025
Type of registration
Prospectively registered

Titles & IDs
Public title
Does Combining Dietary Protein Sources Improve Amino-Acid Digestibility and Net Protein Balance in Older Adults? The DIPO Study
Scientific title
How Does the Digestible Indispensable Amino Acid Score (DIAAS) Influence Protein Net Balance in Older Adults? The Efficacy Potential of Combinatorial Proteins in Humans
Secondary ID [1] 313016 0
Nil known
Universal Trial Number (UTN)
Trial acronym
The DIPO Study (the DIAAS Influence on Protein net balance in Older adults)
Linked study record

Health condition
Health condition(s) or problem(s) studied:
Protein malnourishment 335213 0
Condition category
Condition code
Metabolic and Endocrine 331765 331765 0 0
Normal metabolism and endocrine development and function
Diet and Nutrition 333013 333013 0 0
Other diet and nutrition disorders

Intervention/exposure
Study type
Interventional
Description of intervention(s) / exposure
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.
Intervention code [1] 329561 0
Lifestyle
Comparator / control treatment
Vegetarian protein meal (Quorn, chickpeas, quinoa, and bread) which will have a digestible indispensable amino acid score equal to or above 100.
Control group
Active

Outcomes
Primary outcome [1] 339417 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] 339417 0
Postprandial changes in protein net balance responses over 4 hours (pre to post ingestion of intervention meal). Timepoints: -90, -20, -10, 0, 15, 30, 45, 60, 90, 120, 150, 180, 210, and 240-min post-intervention meal consumption on intervention visits 1, 2, and 3.
Primary outcome [2] 339418 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] 339418 0
Postprandial changes in protein synthesis responses over 4 hours (pre to post ingestion of intervention meal). Timepoints: -90, -20, -10, 0, 15, 30, 45, 60, 90, 120, 150, 180, 210, and 240-min post-intervention meal consumption on intervention visits 1, 2, and 3.
Primary outcome [3] 339419 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] 339419 0
Post-prandial changes in protein degradation responses over 4 hours (pre to post ingestion of interventional meal). Timepoints: -90, -20, -10, 0, 15, 30, 45, 60, 90, 120, 150, 180, 210, and 240-min post-intervention meal consumption on intervention visits 1, 2, and 3.
Secondary outcome [1] 439881 0
Post-prandial plasma Area Under the Curve (AUC) for indispensable amino acids (IAA) concentration of total IAA.
Timepoint [1] 439881 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, and 3.
Secondary outcome [2] 439882 0
Postprandial plasma Area Under the Curve (AUC) for insulin
Timepoint [2] 439882 0
Postprandial changes in plasma insulin responses over 4 hours (pre to post ingestion of intervention meal). Timepoints: 0-, 30-, 60-, 120-, 180-, and 240-min post-intervention meal consumption on intervention visits 1, 2, and 3.
Secondary outcome [3] 439883 0
Post-prandial gastrointestinal comfort scale in response to the interventional meal.
Timepoint [3] 439883 0
Postprandial gastrointestinal comfort over 4 hours (post ingestion of intervention meal) designed specifically for this study. This will be measured at 30-, 60, and 240 min.
Secondary outcome [4] 439884 0
Post-prandial plasma Area Under the Curve (AUC) for total leucine
Timepoint [4] 439884 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, and 3.
Secondary outcome [5] 439885 0
Post-prandial plasma Area Under the Curve (AUC) for total lysine
Timepoint [5] 439885 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, and 3.
Secondary outcome [6] 439886 0
Postprandial plasma Area Under the Curve (AUC) for glucose
Timepoint [6] 439886 0
Postprandial changes in plasma insulin responses over 4 hours (pre to post ingestion of intervention meal). Timepoints: 0-, 30-, 60-, 120-, 180-, and 240-min post-intervention meal consumption on intervention visits 1, 2, and 3.

Eligibility
Key inclusion criteria
- Men and eumenorrheic women aged 67 to 77.
- BMI: 23 to 30 kg/m2
- 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 informed consent).
Minimum age
67 Years
Maximum age
77 Years
Sex
Both males and females
Can healthy volunteers participate?
Yes
Key exclusion criteria
- 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.
- Unwilling to ingest animal-based protein.
- 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.

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
Not yet recruiting
Date of first participant enrolment
Anticipated
3/03/2025
Actual
Date of last participant enrolment
Anticipated
31/05/2025
Actual
Date of last data collection
Anticipated
31/05/2025
Actual
Sample size
Target
8
Accrual to date
Final
Recruitment outside Australia
Country [1] 26568 0
New Zealand
State/province [1] 26568 0
Palmerston North

Funding & Sponsors
Funding source category [1] 317457 0
University
Name [1] 317457 0
Riddet Institute, Massey University
Country [1] 317457 0
New Zealand
Primary sponsor type
University
Name
Riddet Institute, Massey University
Address
Country
New Zealand
Secondary sponsor category [1] 319745 0
None
Name [1] 319745 0
Address [1] 319745 0
Country [1] 319745 0

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 316172 0
Central Health and Disability Ethics Committee
Ethics committee address [1] 316172 0
https://ethics.health.govt.nz/about/central-health-and-disability-ethics-committee/
Ethics committee country [1] 316172 0
New Zealand
Date submitted for ethics approval [1] 316172 0
06/08/2024
Approval date [1] 316172 0
23/08/2024
Ethics approval number [1] 316172 0
19566

Summary
Brief summary
The design is a 3-arm (3 visits) randomised crossover conducted in healthy 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: Combinatorial Protein 1. Chickpeas, Quinoa, Quorn, and Bread (DIAAS equal to or above 100), Combinatorial Protein 2. Chickpeas, Quinoa, Tofu, and Bread (DIAAS 75), Combinatorial Protein 3. Chickpeas, Quinoa, and Bread (DIAAS 50). The meals will comprise 22g protein and 9g indispensable amino acids, each with different digestibility, as defined by the DIAAS. Blood sampling and infusion catheters will be placed in veins at the top of the non-dominant hand and forearm on the other side. The hand will be placed into a heated-hand box to arterialise 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. After 1.5 h, participants will ingest one of three test meals comprising whole foods of combinatorial protein and L-[15N]-Phenylalanine (splanchnic extraction).

Prior to the first visit, participants will fill out questionnaires including: habitual protein intake, sustainability index, and thoughts about meat and alternative meats. There will be a minimum 1-week washout between test days to eliminate any carry-over effects.

This study aims to determine how dietary protein quality, as defined by the DIAAS, affects whole-body protein net balance (synthesis and breakdown) in older adults. We hypothesise that a meal with a DIAAS equal to or above 100 will have the greatest positive impact on whole-body protein net balance, followed by DIAAS 75, with DIAAS 50 having the lowest effect. Results will inform how dietary protein quality is related to whole-body protein net balance, as defined by the DIAAS of the meal and provide the first data on how dietary protein quality affects whole-body protein metabolism in older adults by combining dietary protein.
Trial website
Trial related presentations / publications
Public notes
- The intervention is altering dietary protein quality by way of food source in combinations to establish an excellent-quality protein meal (animal protein, DIAAS greater than or equal to 100), high-quality meal (DIAAS 75), a no-quality protein (DIAAS 50).

- The second objective is to see if formulating a complementary protein blend within in a lower quality whole-foods matrix to reach a DIAAS greater than or equal to 100 will elicit similar responses in whole-body protein balance to that of animal protein.

Contacts
Principal investigator
Name 137022 0
Prof David S. Rowlands
Address 137022 0
Massey University, SNW Extension Level 3, Albany Expressway (SH17), Albany, Auckland 0632, New Zealand
Country 137022 0
New Zealand
Phone 137022 0
+64 272099383
Fax 137022 0
Email 137022 0
[email protected]
Contact person for public queries
Name 137023 0
Robin D. Nielsen
Address 137023 0
Massey University, SNW Extension Level 3, Albany Expressway (SH17), Albany, Auckland 0632, New Zealand
Country 137023 0
New Zealand
Phone 137023 0
+64 212874100
Fax 137023 0
Email 137023 0
[email protected]
Contact person for scientific queries
Name 137024 0
Robin D. Nielsen
Address 137024 0
Massey University, SNW Extension Level 3, Albany Expressway (SH17), Albany, Auckland 0632, New Zealand
Country 137024 0
New Zealand
Phone 137024 0
+64 212874100
Fax 137024 0
Email 137024 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?

Doc. No.TypeCitationLinkEmailOther DetailsAttachment
24469Study protocol    Contains the full detailed study protocol. From th... [More Details] 388491-(Uploaded-20-09-2024-10-39-57)-Study Protocol_Study 2_01112023_1.12.pdf
24470Informed consent form    Contains both the participant sheet and consent fo... [More Details] 388491-(Uploaded-20-09-2024-10-39-57)-Participant Information Sheet - DIPO Study 2024.pdf
24471Other    Contains the questionnaire on habitual protein int... [More Details] 388491-(Uploaded-20-01-2025-11-17-53)-Questionnaire about Habitual Protein Intake.docx
24472Other    Contains the questionnaire on views towards meat a... [More Details] 388491-(Uploaded-13-02-2025-08-28-52)-Attitudes_towrads_meat_and_meat_alternatives.docx
24473Other    Contains the questionnaire on dietary patterns and... [More Details] 388491-(Uploaded-20-01-2025-11-18-27)-Environmental impact and diet patterns.docx



Results publications and other study-related documents

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