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Trial registered on ANZCTR


Registration number
ACTRN12619001553167
Ethics application status
Approved
Date submitted
17/07/2019
Date registered
12/11/2019
Date last updated
12/11/2019
Date data sharing statement initially provided
12/11/2019
Type of registration
Prospectively registered

Titles & IDs
Public title
Discovering markers of flavonoid intake: The Flavi-MARKER study
Scientific title
Validity of novel urinary biomarkers of flavan-3-ol intake in healthy participants: A randomised, 5-way cross-over trial
Secondary ID [1] 298705 0
None
Universal Trial Number (UTN)
U1111-1236-7988
Trial acronym
n.a.
Linked study record
n.a.

Health condition
Health condition(s) or problem(s) studied:
Dietary biomarkers 313611 0
Condition category
Condition code
Diet and Nutrition 312044 312044 0 0
Other diet and nutrition disorders

Intervention/exposure
Study type
Interventional
Description of intervention(s) / exposure
This is a randomised, controlled, 5-way cross-over designed acute intervention trial. Volunteers will be randomised to receive each of the four interventions, plus control, in a random order:

1. Black tea – 1 cup of black tea, steeped for two minutes in 250ml of freshly boiled water and served with the tea bag out (no milk), 3 x per day, for one day.
2. Green tea – 1 cup of green tea, steeped for two minutes in 250ml of freshly boiled water and served with the tea bag out (no milk), 3 x per day, for one day.
3. Apple – 1 Pink Lady apple (~200g), with the skin on and eaten to the core, 3 x per day, for one day.
4. Cocoa – 1 CocoaVia cocoa sachet (4.5g) in 200ml of hot water, 3 x per day, for one day.

Participants will be provided with the tea/apple/cocoa as well as instructions to prepare and consume their drinks/food during the intervention period. Volunteers will be asked to maintain a low-flavan-3-ol diet, during each data intervention period, as a way of reducing the ‘background noise’ from their usual diet. To achieve this, volunteers will be provided with all meals for the intervention days as well as the meal the night before. Meals will be prepared by the metabolic research kitchen at Edith Cowan University. These will be prepared using foods that contain low levels of flavan-3-ols (i.e., <1mg/serve) and all participants will consume the same meals before each visit, apart from selected available snacks to raise energy intake according to individual appetite. Breakfast will be a selection from three low-flavonoid meals: cornflakes with milk/rice krispies with milk/eggs on white toast. A low-flavonoid lunch will be provided consisting of white bread with either pumpkin soup or chicken and sweet corn soup. Dinner will be a selection of: meat pie/mince samosas/chicken parcels. Snacks will consist of the following options: custard cream biscuit/plain yoghut/babybel cheese/tinned pineapple/popcorn. The low (poly)phenolic diet contains less than three servings of fruit and vegetables per day while, water will be provided ad labium. Volunteers will also be provided with an example menu and a list of foods to limit intake of (i.e., high flavonoid foods), on the day preceding the intervention (List 1 - 3). To monitor adherence, participants will keep a diary detailing: the time the intervention was consumed, the preparation of the intervention and the maintenance of the low flavan-3-ol diet. These diaries will be cross checked when the participants return their urine samples. There will be a minimum 2 day wash out between each intervention.

List 1. Foods participants will be asked to avoid.
Coffee
Red wine
White wine
Rose´ wine
Sherry wine
All Beer
Black tea
Green tea
Oolong tea
Rooibos tea
Peppermint tea
Chamomile tea
Any other tea
All fruit juices
Apple cider
All chocolate
All chocolate containing foods
Cacao beans
Chocolate drink
All fruit juice
All jams
All dried fruit
Apple
Banana
Grape
Raisin
Current
Plum
Prune
Dates
Cranberry
Kiwi
Pear
Blackberry
Blueberry
Strawberry
Raspberry
Nectarine
Mango
Cherry
Apricot
Peach
Garlic
Red onion
White onion
Lettuce
Olives
Shallots
Capers
Broccoli
Tomato
Capsicum
Asparagus
Carrot
Spinach
Artichokes
Herbs and spices
Capers
Kale
Avocado
Potato
Sprouts
Green bean
Sorghum- flour
Brown rice
All whole- meal flour or rye
Green bean
Broad bean
Kidney bean
Black bean
Soybean
Soy products
Tempeh
Tofu
Buckwheat
Almond
Pistachio
Pecan
Peanut
Hazelnut
Walnut
Flaxseed meal
Chestnut

List 2. A list of foods suitable for consumption
Water
Soda water
Milk
Eggs
Red meat
White meat
All Seafood meats
All plain cheese
All plain yoghurt
Butter (use for cooking)
Plain ice-cream
Plain yoghurt
Lemon
Melon
Orange
Pineapple
Vegemite
Plain sugar
Salt
Cauliflower
Pumpkin
Peas
Corn
Button-Mushroom
Cucumber
Zucchini
Celery
White bread
White pastry
White rice
White pasta
Chickpea
Quick oats
Popcorn
Plain biscuits
Corn flakes
Plain Weetabix

List 3. A description of suitable meals
Breakfast
Choice of: cornflakes with milk / rice krispies with milk / bacon, eggs, mushroom & toast / plain yoghurt with watermelon or honey / porridge / toast with butter & vegemite + orange / pancake with honey

Lunch
Choice of: Toasted chicken or steak sandwich / cheese sandwich / chicken soup with bread / eggs and rice

Evening Meal
Choice of: chicken and gravy pie with pumpkin and butter side / macaroni cheese / steak and kidney pie with cauliflower / beef stew / ham burger and chips / salmon, white rice and zucchini

Meal Accompaniments
Selection of: plain rice / bread with butter / peas / corn

Snacks
Selection of: custard cream biscuits / cheese scone / toffee yoghurt / crumpets / Babybel cheese / mini cheddars / melon / corn chips / popcorn / tinned pineapple

Drinks
Selection of: Water / Soda Water / Milk

Dessert
Plain ice-cream
Intervention code [1] 314989 0
Lifestyle
Comparator / control treatment
The control will correspond to:
Control – 1 cup of water 3 x per day for one day
Control group
Active

Outcomes
Primary outcome [1] 320703 0
Change in urinary gamma-valerolactones
Timepoint [1] 320703 0
24-hour urine samples will be collected, starting the morning of each intervention day.
Secondary outcome [1] 372577 0
This is an exploratory outcome and a panel of urinary metabolites of phytochemical breakdown will be explored.
Timepoint [1] 372577 0
24-hour urine samples will be collected, starting the morning of each intervention day.

Eligibility
Key inclusion criteria
Inclusion criteria:
• Men and women between 18 and 75 years of age
• No prescription medications
• BMI 18.5 – 30 kg/m2
• Previously consumed cocoa, tea and apples with no adverse reactions
Minimum age
18 Years
Maximum age
75 Years
Gender
Both males and females
Can healthy volunteers participate?
Yes
Key exclusion criteria
Exclusion criteria:
• Adults unable to consent
• Body mass index less than 18.5 or greater than 30 kg/m2
• Systolic blood pressure less than 150 mmHg
• Diastolic BP less than 100 mmHg
• Diagnosed diabetes
• Current or recent (less than 12 months) smoking
• History of cardiovascular or peripheral vascular disease
• Use of blood pressure lowering or cholesterol lowering medication
• Psychiatric illness or other major illnesses such as cancer
• Alcohol intake greater than 210 g per week for women and greater than 280 g per week for men
• Women who are lactating, pregnant or wishing to become pregnant during the study.
• Inability to attend clinic/office visits
• Use of antibiotics (within previous 3 months)
• Inability or unwillingness to follow the study protocol
• Any dietary allergies to cocoa, tea and apples or any ingredients in the standardised
meals
• GI tract disorders, previous GI surgery (except appendectomy)
• Self-reported malabsorption (e.g. difficulty digesting or absorbing nutrients from food, potentially leading to bloating, cramping or gas)
• Diarrhoea within the last 3 months
• Vegetarian, Vegan, food faddists, individuals using non-traditional diets, on a weight loss diet or individuals following diets with significant deviations from the average diet
• Currently participating in another clinical or dietary intervention study

Study design
Purpose of the study
Diagnosis
Allocation to intervention
Randomised controlled trial
Procedure for enrolling a subject and allocating the treatment (allocation concealment procedures)
The person who determines if a subject is eligible for inclusion in the trial will be unaware, when this decision is made, to which group the subject will be allocated to. This is because participants will be allocated to the treatment sequences using a random number allocation program.

Methods used to generate the sequence in which subjects will be randomised (sequence generation)
Five treatment periods will be used to allocate each of five treatments. In order to ensure a balanced number of individuals receive each treatment in each follow up period, each individual will be randomly allocated a treatment sequence using Williams type latin squares. From this design a series of twenty sequences will be produced and each of the 20 individuals will be randomly allocated to one of these sequences using a random number allocation program.
Masking / blinding
Blinded (masking used)
Who is / are masked / blinded?



The people analysing the results/data
Intervention assignment
Crossover
Other design features

Phase
Not Applicable
Type of endpoint(s)
Bio-availability
Statistical methods / analysis
As this is a pilot study, no sample size has been calculated. However, we believe that 12 people will provide adequate power to detect a difference in urinary gVL levels. This was enough people to detect a difference in a previous and similarly designed study.
Multilevel Principle Components Analyses (PCA) will be used to investigate the experimental and biological differences between the subjects, while a multilevel Partial-Least Squares-Discriminant Analysis (PLS-DA) will be used to reveal the net treatment effect within the subjects. This will be supplemented with linear mixed models on each individual response to examine the effect of treatment over time. Results of the linear mixed models will be presented as treatment differences at each time point with 95% confidence intervals. A multiplicity adjustment will be made to allow for the issue of multiplicity.

Recruitment
Recruitment status
Not yet recruiting
Date of first participant enrolment
Anticipated
Actual
Date of last participant enrolment
Anticipated
Actual
Date of last data collection
Anticipated
Actual
Sample size
Target
Accrual to date
Final
Recruitment in Australia
Recruitment state(s)
WA
Recruitment hospital [1] 14221 0
Royal Perth Hospital - Perth
Recruitment postcode(s) [1] 27209 0
6000 - Perth

Funding & Sponsors
Funding source category [1] 303253 0
Charities/Societies/Foundations
Name [1] 303253 0
Royal Perth Hospital Research Foundation, ‘Springboard Grants 2019’, ‘West Family Cardiovascular Grant’
Address [1] 303253 0
PO BOX 2323, East Perth, Western Australia, 6892
Country [1] 303253 0
Australia
Primary sponsor type
University
Name
University of Western Australia
Address
35 Stirling Hwy, Crawley WA 6009
Country
Australia
Secondary sponsor category [1] 303330 0
None
Name [1] 303330 0
Address [1] 303330 0
Country [1] 303330 0

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 303794 0
HUMAN RESEARCH ETHICS - THE UNIVERSITY OF WESTERN AUSTRALIA
Ethics committee address [1] 303794 0
The University of Western Australia
M459, 35 Stirling Highway
Crawley WA 6009 Australia
Ethics committee country [1] 303794 0
Australia
Date submitted for ethics approval [1] 303794 0
23/04/2019
Approval date [1] 303794 0
11/06/2019
Ethics approval number [1] 303794 0
RA/4/20/5366

Summary
Brief summary
Recommendations to increase intakes of foods rich in flavonoids have the potential to improve population health and reduce cardiovascular disease-related mortality. Flavan-3-ols are the most abundant subclass of flavonoids in the diet and are found in abundance in cocoa, apples, and tea. Currently, flavan-3-ol intake is estimated from food frequency questionaires. There is a crucial need for quality biomarkers of intake as a means of overcoming food questionnaire and food database limitations. Recently, a microbiome-derived flavan-3-ol catabolite, gamma-valerolactone (gVL), has emerged as a promising candidate. The primary aim of the proposed project is to establish the suitability of gVLs as biomarkers of flavan-3-ol intake.
Trial website
Trial related presentations / publications
Public notes

Contacts
Principal investigator
Name 94830 0
Prof Kevin Croft
Address 94830 0
The University of Western Australia (M570), 35 Stirling Highway,

6009 Perth, Western Australia

Australia
Country 94830 0
Australia
Phone 94830 0
+61 422210414
Fax 94830 0
Email 94830 0
kevin.croft@uwa.edu.au
Contact person for public queries
Name 94831 0
Mr Ben parmenter
Address 94831 0
50 Murray street, Royal Perth Hospital, Medical Research Foundation, Level 4,
Perth, Western Australia, 6000
Country 94831 0
Australia
Phone 94831 0
+61 473440405
Fax 94831 0
Email 94831 0
benjamin.parmenter@research.uwa.edu.au
Contact person for scientific queries
Name 94832 0
Prof Kevin Croft
Address 94832 0
The University of Western Australia (M570), 35 Stirling Highway,

6009 Perth, Western Australia

Australia
Country 94832 0
Australia
Phone 94832 0
+61 422210414
Fax 94832 0
Email 94832 0
kevin.croft@uwa.edu.au

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?
No other documents available
Summary results
No Results