Trial Review

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Trial registered on ANZCTR


Registration number
ACTRN12617000874314
Ethics application status
Approved
Date submitted
12/06/2017
Date registered
15/06/2017
Date last updated
15/06/2017
Type of registration
Retrospectively registered

Titles & IDs
Public title
The effects of dietary protein supplementation and energy restriction on whole body protein turnover, substrate oxidation and appetite in lean to obese healthy adults
Scientific title
The effects of dietary protein supplementation and energy restriction on whole body protein turnover, substrate oxidation and appetite in lean to obese healthy adults
Secondary ID [1] 292074 0
None
Universal Trial Number (UTN)
U1111-1197-0394
Trial acronym
Linked study record

Health condition
Health condition(s) or problem(s) studied:
Obesity 303490 0
Condition category
Condition code
Diet and Nutrition 302905 302905 0 0
Obesity

Intervention/exposure
Study type
Interventional
Description of intervention(s) / exposure
This study will consist of 4 phases, with each phase lasting 2 weeks; habitual diet 1 (HD1), habitual diet 2 (HD2), high protein energy balance (HPEbal) and high protein energy restriction (HPER). During the habitual diet phases (HD1 and HD2), participants will be instructed to continue to consume their normal diet, which based on being weight stable for greater than or equal to 6 months, should be equal to their current energy requirements. During HPEbal, the participants will be provided with a whey based protein powder supplement (0.5 g/kg/day) which will replace food items in their diet in order to maintain energy balance and a protein intake 0.5 g/kg/day greater than HD1 and HD2. During HPER subjects will continue to be provided with the protein supplement (0.5 g/kg/day) and will have further food excluded from their diet to create an energy restriction of 33% below their HD1 energy intake. The energy restriction will come entirely from carbohydrates and fats in order to maintain protein intake at the level of HPEbal. Energy and macronutrient intake will be recorded using a daily weighed food diary and recording app (myfitnesspal) throughout all study phases. These will be logged into a dietetics database (Foodworks Xyris software) for accuracy. An additional diet recall per week will be undertaken to monitor recording adherence and clarify any items unclear from the weighed records. During HPEbal and HPER, individualised energy and macronutrient goals will be set for all participants based on their energy and protein intake during HD1. Participants will be able to track their own progress in meeting these dietary goals on a daily basis through the app as well as regular feedback from the research team. The research team have over 20 years experience in dietary interventions and the study will be reviewed by a registered clinical dietitian.

Participants will attend 5 test days at the laboratory. The initial test session will be prior to starting diet recording to determine baseline values of body composition (Dual Energy X-ray Absorptiometry (DXA) and Air Displacement Plethysmography (BODPOD) and resting metabolic rate. Tests days 2-5 will occur at the end of each 2 week diet block and will include measuring whole body protein turnover (WBPT), resting and postprandial energy expenditure, body composition (bodpod only to monitor changes in body composition), appetite and food hedoncis (liking and wanting).

Participants will arrive at the laboratory between 6:00 am and 8:30 am on the morning of their test days after a minimum 10-hour fast. Participants will be required to minimise activity on the test day mornings and to abstain from all exercise for 24 hours prior to test days and abstain from high intensity exercise for greater than 48 hours prior to test days. On test days 2-5, following arrival at the laboratory, participants will undergo measures of their body weight and body composition (Bodpod) followed by a measurement of resting energy expenditure (indirect calorimetry). Participants will then be provided with their first test meal of the day. Postprandial energy expenditure and substrate oxidation will be measured twice, between 20-40 and 70-90 minutes following the breakfast meal. WBPT will be measured using the end products method as described by Picou and Taylor Roberts [1] and modified by Fern et al. [2], with a single dose of 200 mg of 15N Glycine (98% atom 15N) and ammonia as the end product. One hour following a breakfast meal, participants will provide a baseline urine sample for the background abundance of 15N ammonia, followed by ingestion of the 200 mg of 15N labelled glycine (98% atom 15N, Sigma-Aldrich) mixed in water. All urine will be collected for the following 24 hours in three separate urine containers between 0-9 hours and 9-12 hours and 12-24 hours. After 9 hours at the laboratory, participants will be sent home and instructed to continue to collect up to and at 24 hours. Participants will receive 6 isoenergetic and isoproteogenic meals; 1 every 2 hours during the study day to maintain a postprandial state. The total energy and protein intake in the provided meals will be based on each individual’s habitual intake recorded with their food diary during HD1 and modified according to the appropriate study phase (HPEbal or HPER). Appetite and liking and wanting will be assessed fasting and postprandial for 10 hours following the first test meal.

1. Picou, D.F. and T. Taylor-Roberts, The measurement of total protein synthesis and catabolism and nitrogen turnover in infants in different nutritional states and receiving different amounts of dietary protein. Clinical Science, 1969. 36(2): p. 283-296.
2. Fern, E.B., et al., The excretion of isotope in urea and ammonia for estimating protein turnover in man with [15N]glycine. Clinical Science, 1981. 61(2): p. 217-228.
Intervention code [1] 298214 0
Treatment: Other
Comparator / control treatment
Participants will act as their own control. The control is the habitual diet phases.
Control group
Active

Outcomes
Primary outcome [1] 302292 0
Whole body protein turnover will be measured using the end products method as described by Picou and Taylor Roberts [1] and modified by Fern et al. [2], with a single dose of 200 mg of 15N Glycine (98% atom 15N) and ammonia as the end product. One hour following a breakfast meal, participants will provide a baseline urine sample for the background abundance of 15N ammonia, followed by ingestion of 200 mg of 15N labelled glycine (98% atom 15N, Sigma-Aldrich) mixed in water. All urine will be collected for the following 12 hours in two separate urine containers between 0-9 hours and 9-12 hours.

Samples will be analysed using the Conway micro diffusion method as provided by Preston [3,4} to separate ammonia from the urine samples. 100 microlitres of the prepared samples will be pipetted into tin combustion containers for analysis on a mass spectrometer.
Protein flux will be calculated from the enrichment of 15N ammonia estimated using the end product equation provided by Waterlow [5] Flux Q = d x Ex/ex where d = dose, Ex = enrichment of end product x total amount of end product and ex is the total amount of end product.
Protein synthesis (S) and breakdown (B) will be calculated from the equations S = Q – E and B = Q – I, where I is total protein intake derived from participants’ food diaries and their protein intake on the test day, and E is the total urinary nitrogen excretion..

1. Picou, D.F. and T. Taylor-Roberts, The measurement of total protein synthesis and catabolism and nitrogen turnover in infants in different nutritional states and receiving different amounts of dietary protein. Clinical Science, 1969. 36(2): p. 283-296.
2. Fern, E.B., et al., The excretion of isotope in urea and ammonia for estimating protein turnover in man with [15N]glycine. Clinical Science, 1981. 61(2): p. 217-228.
3. Preston, T. and D.C. McMillan, Rapid sample throughput for biomedical stable isotope tracer studies. Biomedical and Environmental Mass Spectrometry, 1988. 16(1-12): p. 229-35.
4. Preston, T. and D.C. McMillan, 15N Sample preperatoin for protein turnover measurements in large populations. Analytrca Chrmtca Acta, 1990. 241: p. 255-260.
5. Waterlow, J.C., M.H. Golden, and P.J. Garlick, Protein turnover in man measured with 15N: comparision of end products and dose regimes. American Journal of Physiology - Endocrinology And Metabolism, 1978. 235(2): p. E165-E174.
Timepoint [1] 302292 0
Measured over 9 and 12 hours starting 1 hour post the first test meal on test days 2-5 (End of each study phase HD1, HD2, HPEbal, HPER). All urine will be collected for 0-9 and 9-12 hours in separate containers to measure 9hr and 12 hr protein turnover.
Primary outcome [2] 302293 0
Body weight using laboratory digitial scales and scales attached to the Bodpod
Timepoint [2] 302293 0
Initial test session and on test days 2-5 (End of each study phase HD1, HD2, HPEbal, HPER)
Primary outcome [3] 302294 0
Resting energy expenditure (Indirect calorimetry with a ventilated hood)
Timepoint [3] 302294 0
Initial test session and on test days 2-5 (End of each study phase HD1, HD2, HPEbal, HPER)
Secondary outcome [1] 335395 0
24 hour nitrogen balance. Calculated as Nitrogen intake minus nitrogen excretion.
Intake will be determined based on protein intake on the test day from weighing of all provided food items and weighing crockery pre and post food consumption and determined based on manufacturer details of food product protein content. Pro = N x 6.25
Nitrogen excretion will be based on total 24 hour urine collections collected into separate containers to identify 0-12 hour feeding and 12-24 hour fasting Nitrogen losses. Sub-samples of 0-12, 12-24 and 0-24 hour urine samples will be analysed for total nitrogen using a mass spectrometer.
Timepoint [1] 335395 0
Measured over 24 hours following the consumption of the first test meal on test days 2-5 (End of each study phase HD1, HD2, HPEbal, HPER).
Secondary outcome [2] 335396 0
Appetite using Visual Analogue scales to assess subjective appetite
Timepoint [2] 335396 0
Fasting and intermittently for 10 hours following the first test meal on test days 2-5 (End of each study phase HD1, HD2, HPEbal, HPER)
Secondary outcome [3] 335424 0
Food hedonics using the Leeds food preference questionnaire (LFPQ) to assess food liking and wanting.
Timepoint [3] 335424 0
Fasting, immediately post meal 1, 2-hours post meal 1 and post meal 6 (end of the day) on test days 2-5 (End of each study phase HD1, HD2, HPEbal, HPER)
Secondary outcome [4] 335425 0
Fasting substrate oxidation with indirect calorimetry and corrected for fasting urinary nitrogen excretion.
Timepoint [4] 335425 0
Measured on test days 2-5 (End of each study phase HD1, HD2, HPEbal, HPER)
Secondary outcome [5] 335426 0
Whole body protein turnover method reproducibility .

Determined with:
Calculation of mean and 95% confidence intervals of between day differences, paired t-tests, ICCs (two way random, single measures (2,1)), CV and the coeffecient of reliability.
Timepoint [5] 335426 0
Determined from 9 and 12 hour urine collections during HD1 and HD2 test days.
Secondary outcome [6] 335427 0
The effects of urine collection duration on the calculation of whole body protein turnover with the end products method with ammonia as end product. Comparison of 9 hour versus 12-hour urine collection times.

Agreement and differences between 9 and 12-hour urine collections will be assessed with
Bland Altman plots, paired t-tests, and Pearson's correlations.
Timepoint [6] 335427 0
0-9 and 0-12 hour urine collections from both HD1 and HD2.
Secondary outcome [7] 335428 0
Reproducibility of appetite measurements with visual analogue scales. Reproducibility will be assessed on fasting, intra-meal change scores, post-meal averages and area under the curve scores.

Determined with:
Calculation of mean and 95% confidence intervals of between day differences, paired t-tests, ICCs (two way random, single measures (2,1)), CV and the coeffecient of reliability.
Timepoint [7] 335428 0
Based on fasting, and 10 hour postprandial appetite scores during HD1 and HD2 test days.
Secondary outcome [8] 335429 0
Reproducibility of the Leeds food preference questionnaire (LFPQ) to assess liking and wanting
Reproducibility assessed on fasting, intra-meal change scores, daily averages across 4 time points.

Determined with:
Calculation of mean and 95% confidence intervals of between day differences, paired t-tests, ICCs (two way random, single measures (2,1)), CV and the coeffecient of reliability.
Timepoint [8] 335429 0
Fasting, immediately post meal 1, 2-hour post meal 1 , post meal 6 (final test meal of the day) measured on HD1 and HD2 test days.
Secondary outcome [9] 335955 0
Body Composition assessed with air displacement plethysmography with the bodpod
Timepoint [9] 335955 0
Initial test session and on test days 2-5 (End of each study phase HD1, HD2, HPEbal, HPER)
Secondary outcome [10] 335956 0
Postprandial energy expenditure measured with indirect calorimetry and a ventilated hood.
Timepoint [10] 335956 0
Measured at 20-40 and 70-90 minutes post the breakfast meal.
Measured on test days 2-5 (End of each study phase HD1, HD2, HPEbal, HPER)
Secondary outcome [11] 335957 0
Postprandial substrate oxidation with indirect calorimetry and corrected for feeding urinary nitrogen excretion..
Timepoint [11] 335957 0
Measured at 20-40 and 70-90 minutes post the breakfast meal.
Measured on test days 2-5 (End of each study phase HD1, HD2, HPEbal, HPER)

Eligibility
Key inclusion criteria
BMI between 18.5 - 40 kg/m2
Euthyroid, non-diabetic and weight stable for at least 6 months (+/-2kg)
Owning a mobile device capable of downloading Apps
Minimum age
18 Years
Maximum age
50 Years
Sex
Both males and females
Can healthy volunteers participate?
Yes
Key exclusion criteria
Smokers (current or recent: quit less than 6 months ago)
Currently dieting
Currently taking protein supplements or intentionally following a high protein diet
Any metabolic, liver, kidney or gastrointestinal disease
Food allergies or intolerances
Females: Irregular menstrual cycle or pregnant
Any medications known to a ect metabolic rate or neuroendocrine function

Study design
Purpose of the study
Treatment
Allocation to intervention
Non-randomised trial
Procedure for enrolling a subject and allocating the treatment (allocation concealment procedures)
Allocation was not concealed.
Methods used to generate the sequence in which subjects will be randomised (sequence generation)
All participants received the same intervention and in the same order.
Masking / blinding
Open (masking not used)
Who is / are masked / blinded?



Intervention assignment
Crossover
Other design features
Phase
Not Applicable
Type of endpoint/s
Efficacy
Statistical methods / analysis
Linear mixed models with study phase and BMI as factors were used to determine the effect of the HPEbal and HPER interventions, and any BMI x study phase interactions for all outcome measures.

Sample sizes were determined based on the primary measure of WBPT. Calculations for determining changes in whole body protein synthesis and breakdown were calculated using a power of 0.8 and alpha error of 0.05. In previous research, implementing a similar increase in dietary protein of ~0.6 g/kg/d and using similar methodology (end products approach with 15N glycine and ammonia as end product), increased protein intake resulted in increased whole body protein synthesis (low PRO: 2.1 ± 1.0 g PRO/kg/d, high PRO: 3.2 ± 1.7 g PRO/kg/d) and protein breakdown (low PRO: 2.7 ± 1.1 g PRO/kg/d, high protein diet: 4.0 ± 1.7 g PRO/kg/d) [1]. Cohen’s D effect sizes based on these results were 0.81 and 0.93 for synthesis and breakdown respectively corresponding with a minimum of 12 and 9 participants required to find significant changes in these variables [2].

While the data of studies comparing lean and obese individuals with the 15N method was not highly prevalent, studies with the precursor method have found significant differences between moderately and severely obese and between lean and obese groups with just 6 participants per group [3,4]. An attrition rate of 25% over the study was anticipated and 24 participants sought to obtain a minimum of 8 per group.
1. Pannemans, D.L., et al., Effect of variable protein intake on whole-body protein turnover in young men and women. The American Journal of Clinical Nutrition, 1995. 61(1): p. 69-74.
2. Cohen, J., Statistical power analysis for behavioural sciences. (revised edition) ed. 1977, New York: Academic Press.
3. Henderson, G.C., et al., Effects of Adiposity and 30 Days of Caloric Restriction Upon Protein Metabolism in Moderately vs. Severely Obese Women. Obesity, 2010. 18(6): p. 1135-1142.
4. Bruce, A.C., et al., Nutrient oxidation patterns and protein metabolism in lean and obese subjects. International Journal of Obesity, 1990. 14(7): p. 631-46.

Recruitment
Recruitment status
Completed
Date of first participant enrolment
Anticipated
Actual
3/10/2014
Date of last participant enrolment
Anticipated
Actual
18/03/2015
Date of last data collection
Anticipated
Actual
29/06/2015
Sample size
Target
24
Accrual to date
Final
23
Recruitment in Australia
Recruitment state(s)
QLD

Funding & Sponsors
Funding source category [1] 296604 0
University
Name [1] 296604 0
Queensland University of technology
Country [1] 296604 0
Australia
Primary sponsor type
University
Name
Queensland University of technology
Address
Institute of Health and Biomedical Innovation
60 Musk Avenue
Kelvin Grove, QLD 4059
Country
Australia
Secondary sponsor category [1] 295562 0
None
Name [1] 295562 0
Address [1] 295562 0
Country [1] 295562 0

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 297833 0
Queensland University of Technology Human Research Ethics Committee
Ethics committee address [1] 297833 0
Ethics committee country [1] 297833 0
Australia
Date submitted for ethics approval [1] 297833 0
05/08/2014
Approval date [1] 297833 0
01/10/2014
Ethics approval number [1] 297833 0
1400000608

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

Contacts
Principal investigator
Name 75222 0
Mrs Leonie Ruddick-Collins
Address 75222 0
Queensland University of Technology, Institute of Health and Biomedical Innovation
60 Musk Avenue,
Kelvin Grove, QLD
4059
Country 75222 0
Australia
Phone 75222 0
+61 7 3138 6397
Fax 75222 0
Email 75222 0
[email protected]
Contact person for public queries
Name 75223 0
Leonie Ruddick-Collins
Address 75223 0
Queensland University of Technology, Institute of Health and Biomedical Innovation
60 Musk Avenue,
Kelvin Grove, QLD
4059
Country 75223 0
Australia
Phone 75223 0
+61 7 3138 6397
Fax 75223 0
Email 75223 0
[email protected]
Contact person for scientific queries
Name 75224 0
Leonie Ruddick-Collins
Address 75224 0
Queensland University of Technology, Institute of Health and Biomedical Innovation
60 Musk Avenue,
Kelvin Grove, QLD
4059
Country 75224 0
Australia
Phone 75224 0
+61 7 3138 6397
Fax 75224 0
Email 75224 0
[email protected]

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