Bioavailability of Omega-3 Long-chain Polyunsaturated Fatty Acids (LCPUFA) From Foods Enriched With Vegetable-encapsulated Omega-3 Oils
Phase: PHASE2
Conditions: Lifestyle
Sponsor: Commonwealth Scientific and Industrial Research Organisation, Australia

 
Protocol Number: OBP001 
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Version 3.0, 20th October 2020 
 
 
 
 
 
 
Clinical Study Protocol 
 
Omega-3 bioavailability from vegetable-
omega-3 enriched products 
Full Project Title: Bioavailability of long-chain omega-3 polyunsaturated fatty acids from 
foods enriched with vegetable-encapsulated omega-3 oils 
 
 
 
 
 
 
 
Protocol Number: 
 
OBP001 
Date of Protocol: 
20th October 2020 
 
Version No: 
3.0 
 
 
Sponsor/Grant:  
Singapore-Australia Bilateral Program on Innovations in Food for Precision 
Health 
 
 
 
 
 
 
 

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Table of Contents 
CLINICAL STUDY PROTOCOL ............................................................................................................................. 1 
OMEGA-3 BIOAVAILABILITY FROM VEGETABLE-OMEGA-3 ENRICHED PRODUCTS ............................................ 1 
FULL PROJECT TITLE: BIOAVAILABILITY OF LONG-CHAIN OMEGA-3 POLYUNSATURATED FATTY ACIDS FROM 
FOODS ENRICHED WITH VEGETABLE-ENCAPSULTED OMEGA-3 OILS ................................................................ 1 
TABLE OF CONTENTS ........................................................................................................................................ 2 
PROJECT MANAGERS AND KEY RESEARCH STAFF ............................................................................................. 3 
SITE SIGNATURES ............................................................................................................................................. 4 
STUDY SYNOPSIS ............................................................................................................................................. 5 
LIST OF ABBREVIATIONS .................................................................................................................................. 7 
1. INTRODUCTION ............................................................................................................................................ 8 
2. AIM, OBJECTIVES AND HYPOTHESES ............................................................................................................ 9 
3. STUDY POPULATION .................................................................................................................................... 9 
4. STUDY DESIGN ........................................................................................................................................... 10 
5. TEST MEALS ............................................................................................................................................... 14 
7. ASSESSMENTS ............................................................................................................................................ 14 
8. STATISTICAL ANALYSIS ............................................................................................................................... 16 
9. ASSESSMENT OF ADVERSE EVENTS ............................................................................................................ 16 
10. ETHICAL CONSIDERATIONS ...................................................................................................................... 18 
11. DATA HANDLING, RECORD KEEPING & RETENTION .................................................................................. 18 
13. QUALITY CONTROL AND QUALITY ASSURANCE ........................................................................................ 19 
14. DIRECT ACCESS TO SOURCE DATA/DOCUMENTS ...................................................................................... 19 
15. PUBLICATION POLICY ............................................................................................................................... 19 
16. REFERENCES ............................................................................................................................................. 19 
APPENDIX ...................................................................................................................................................... 21 
 
 
 
 
 
 
 
 

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Project Managers and Key Research Staff 
 
Principal Investigator (Australia-CSIRO):  
Dr Welma Stonehouse 
Email address: Welma.Stonehouse@csiro.au 
Telephone number: +61 8 83038919 
 
Principal Investigator (Singapore-A*STAR): 
A/Prof Ciaran Forde 
Email address: Ciaran_Forde@sifbi.a-star.edu.sg 
Telephone number: +65 64070104 
 
Co-Investigator: 
Dr Netsanet Shiferaw Terefe 
Email address:  Netsanet.Shiferawterefe@csiro.au 
Telephone number: +61 397313293 
 
CSIRO Clinic Manager: 
Dr Bianca Benassi-Evans  
Email address:  Bianca.Benassi@csiro.au  
Telephone number:  +61 8 8303 8982 
 
CSIRO Clinical Trial Coordinator: 
Bradley Klingner  
Email address: Bradley.Klingner@csiro.au 
Telephone number: +61 8 8303 8818 
 
CSIRO Clinical Research Data Manager 
Himanshu Tandon 
Email address: Himanshu.Tandon@csiro.au 
Telephone number: +61 8 8305 0673 
 
CSIRO Clinical Research Data Adviser 
Kathryn Bastiaans 
Email address: Kathryn.Bastiaans@csiro.au 
Telephone number: +61 8 8303 8906 
 
A*STAR Project Manager: 
Dr. Pey Sze Teo 
Email address: Teo_Pey_Sze@sifbi.a-star.edu.sg 
Telephone number: +65 64070497 
 
A*STAR Clinical Trial Coordinator: 
Vicki Tan Wei Kee 
Email address: Vicki_Tan@sifbi.a-star.edu.sg 
Telephone number: +65 64074361 
 
A*STAR Clinical Research Data Manager: 
Rachel Tso 
Email address: Rachel_Tso@sifbi.a-star.edu.sg  
Telephone number: +65 64070423 

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Site Signatures 
 
PROTOCOL TITLE: 
 
PROTOCOL NUMBER: OBP001 
DATE OF PROTOCOL: 13 October 2020 
 
Confidentiality and Good Clinical Practice (GCP) Compliance Statement: 
 
I, the undersigned, have reviewed this protocol, including the appendices and I will conduct the study 
as described in compliance with this protocol, GCP and relevant International Council on 
Harmonisation (ICH) guidelines. 
 
Once the protocol has been approved by the Human Research Ethics Committee (HREC), I will not 
modify this protocol without obtaining prior agreement and approval from CSIRO/A*STAR Principal 
Investigator (PI) and the HREC. I will submit the protocol modification and/or any Informed Consent 
Form (ICF) modifications to the CSIRO/A*STAR PI and HREC and approval will be obtained before any 
modifications are implemented. 
 
I understand that all the information obtained during the conduct of the study with regard to the 
participants’ state of health will be regarded as confidential. No participants’ names will be disclosed. 
All Participants will be identified by assigned numbers on all case report forms (CRF), laboratory 
samples or source documents. Clinical information may be reviewed by regulatory agencies. 
Agreement must be obtained from the participant before disclosure of participant information to a 
third party. 
 
 
INVESTIGATOR SIGNATURE: 
 
PRINTED NAME: Welma Stonehouse 
 
COMPANY/POSITION: CSIRO 
 
SIGNATURE: 
 
 
 
 
INVESTIGATOR SIGNATURE: 
 
PRINTED NAME: Ciarán Forde 
 
COMPANY/POSITION: A*STAR 
 
SIGNATURE: 
 
 
 
 

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Study Synopsis  
 
 
 
Protocol Title: 
Bioavailability of omega-3 long-chain polyunsaturated fatty acids (LCPUFA) 
from foods enriched with vegetable-encapsulated omega-3 oils 
Aim: 
To compare the bioavailability of omega-3 LCPUFA from two test food 
products containing vegetable-encapsulated omega-3 algal oil against a 
control test product (algal oil gel capsules) across two ethnicities (Australian 
European vs. Chinese Singaporean).  
Study Design: 
Multi-centre, randomised, controlled, acute, 3-way cross-over study design. 
Participants will consume in random order, at least 1-week apart, three test 
products after an overnight fast. Blood samples will be collected at 0, 2, 4, 6, 
8 and 24 hours after consuming the products. Plasma fatty acid 
concentrations (µg/mL) will be analysed by gas chromatography and 
bioavailability of omega-3 LCPUFA determined by calculating the 
incremental area under the curve (iAUC0-24h), time to maximal value (Tmax) 
and maximal value (Cmax). 
 
Study sites: 
CSIRO Nutrition and Health Research Clinic, Adelaide 
A*STAR Clinical Nutrition Research Centre, Singapore 
Number of 
Participants: 
N=24 (n=12 in Australia and n=12 in Singapore) 
Accounts for 20% attrition. 
Inclusion/Exclusion 
Criteria: 
INCLUSION CRITERIA: 
1. Healthy men, 21-50-year old 
2. BMI 18-27.5 kg/m2 
3. Consuming <2 meals of fatty fish/week and no fish oil supplements 
4. Ethnicity 
o If Australian site: Australian European 
OR  
o If Singapore site: Chinese Singaporean 
 
EXCLUSION CRITERIA: 
1. Self-reported history of chronic disease - cancer, type 2 diabetes, 
cardiovascular disease, liver disease or any condition that may, in the 
opinion of the principle investigator, influence the study outcomes. 
2. Self-reported history of gastrointestinal disease, pancreatic insufficiency, 
conditions resulting in fat malabsorption - chronic pancreatitis, cystic 
fibrosis, coeliac disease, Crohns disease, gastric bypass surgery, small 
bowel resection, abnormal thyroid function 
3. Bleeding disorders, currently taking anticoagulants or has received 
anticoagulants within 28 days of Day 1 of the trial, with the exception of 
low dose aspirin up to 150 mg daily* 
4. Any medical procedures deemed by the principal investigator to affect 
study outcomes 

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5. Known food allergies, hypersensitivity, dietary avoidance or intolerance 
to the study foods 
6. Taking medications/supplements known to influence lipid metabolism 
and gastric emptying  
7. On any weight-loss program 
8. History of smoking during the 6 months prior to the study 
9. Persons considered by the investigator to be unwilling, unlikely or 
unable to comprehend or comply with the study protocol. 
10. Self-reported history of drug abuse or alcoholism 
11. Participation in another research study within 30 days preceding the 
start of this study 
Treatment: 
Standardised breakfast plus: 
Test food 1: Semi-solid food – Soup (commercially available packet-soup) 
containing cauliflower encapsulated HiDHA® algal oil providing 400 mg DHA 
Test food 2: Solid food – Extruded snack (savory snack) containing 
cauliflower encapsulated HiDHA® algal oil providing 400 mg DHA 
Control: 2x Algal oil gel capsules containing HiDHA® algal oil providing 400 
mg DHA 
Study Duration: 
~1 month (3 test products at least 1-week apart) 
Study Population: 
Healthy men 
Primary Outcome: 
Plasma DHA and total omega-3 LCPUFA iAUC0-24h 

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List of Abbreviations 
 
Abbreviation 
Description  
AE 
Adverse Event 
AUC 
Area under the curve 
BMI 
Body mass index 
CHD 
Coronary heart disease 
CRF 
Case report form 
CVD 
Cardiovascular disease 
DHA 
Docosahexaenoic acid 
EPA 
Eicosapentaenoic acid 
EW 
Early Withdraw 
FRAT 
Food risk assessment team 
HREC 
Human Research Ethics Committee 
ICF 
Informed Consent Form 
ICH-GCP 
International Council for Harmonisation Good Clinical Practice 
LCPUFA 
Long-chain polyunsaturated fatty acids 
NCD 
Non-communicable disease 
SAE 
Serious adverse events 

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1. Introduction 
 
Non-communicable chronic diseases (NCD) in both Australia and Singapore account for significant 
proportions of deaths, disease burden and hospitalizations, with poor quality diet as the major 
contributor to the NCD burden (1, 2). Marine derived omega-3 long-chain polyunsaturated fatty acids 
(LCPUFA) (particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are associated 
with numerous health benefits throughout the life cycle. Omega-3 Index (O3I) (EPA+DHA as % of red 
blood cell fatty acids – a biomarker of EPA+DHA status (3)) levels of 8% vs. 4% were related to a 30% 
reduced risk of fatal coronary heart disease (CHD) (4). More recently O3I levels at 8% have also been 
linked to increased longevity, reduced all-cause mortality, improved depressive symptoms, improved 
arthritis symptoms (5) and improved cognitive function (6). Despite the well-established health 
benefits of omega-3 LCPUFA, intakes in Western and most Asian diets are below recommendations 
(7, 8), and low levels of consumption have been associated with an increased risk of cardiovascular 
mortality in a Chinese population (7). Consumption of ~2-3 serves/week of fish, including oily fish, is 
recommended to achieve ~250 – 500 mg/day of combined EPA+DHA, and up to 1g/day for those with 
CHD (9-11). Achieving these intakes through dietary means is challenging, particularly for individuals 
who do not consume fish or seafood (major dietary sources of omega-3 LCPUFA) (5). Vegans and 
vegetarians are particularly at risk. Several studies have shown that vegan diets are devoid of DHA and 
vegetarian diets that include dairy food and eggs only provide about 0.02 g DHA/day. These low 
intakes were accompanied by substantially lower levels of DHA in plasma, serum, red blood cells (RBC) 
and plasma phospholipids (PL) in vegans and vegetarians compared to omnivores (12). Furthermore, 
a high degree of inter-individual variability exists in how omega-3 intake affects O3I levels (5). Dose, 
baseline omega-3 status and chemical form of supplements have been shown to affect O3I levels in 
response to intake (13). Other factors such as characteristics of food products and individuals’ 
ethnicity have potential to impact O3I but have not been formally investigated previously. A better 
understanding of factors affecting response to increased omega-3 LCPUFA intakes will assist in the 
development of personalized approaches to assist consumers in achieving omega-3 status that is 
optimal for health. Furthermore, despite convincing evidence that diets rich in vegetables are 
associated with reduced risk of all-cause mortality and particularly cardiovascular mortality (14), 
populations globally fail to meet recommended intakes (15). Hence, strategies empowering 
consumers to achieve omega-3 LCPUFA and vegetable intake targets have potential to significantly 
impact health outcomes. 
 
Some of the challenges in the production of foods fortified with omega-3 fatty acids include their 
undesirable odour and taste and their susceptibility to oxidative degradation (16). CSIRO has 
developed a novel vegetable-based encapsulation system for delivery of omega-3 LCPUFA which 
provides superior protection against oxidation of omega-3 fatty acids compared to other commercially 
available microencapsulation techniques (17), while at the same time delivering the nutritional 
benefits of vegetable consumption. Vegetable encapsulated omega-3 fortified food has the potential 
to deliver daily recommended dosage of omega-3 fatty acids (~250-500 mg/serve) in addition to a 
serving of vegetables. In addition, if a vegetarian omega-3 LCPUFA oil is used (e.g. algal oil), these 
products will also be suitable for vegans and vegetarians. One of the challenges in incorporating 
vegetable encapsulated omega-3 LCPUFA into fortified foods is the potential effect of the vegetable 
carrier matrix (e.g. fibre and other bioactives) on release and bioavailability of omega-3 LCPUFA in 
vivo. Furthermore, there is a possibility of inter-ethnic differences in omega-3 bioavailability. 
Heritability and anthropometric phenotypes, previously associated with omega-3 status (18), differ 
between Asian and Caucasian populations. The current project aims to address these challenges and 
to use the data generated as the basis for development of personalised formulations tailored to 
improve omega-3 status and vegetable intake and consequently health outcomes of consumers in 
Australia and Singapore. 
 

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2. Aim, Objectives and Hypotheses 
Aim:   
To evaluate the bioavailability of omega-3 LCPUFA from foods enriched with vegetable-encapsulated 
omega-3 oils across two ethnicities. 
 
Objective/s: 
• 
To compare the bioavailability of omega-3 LCPUFA from two test food products containing 
vegetable-encapsulated omega-3 algal oil against a control test product (algal oil gel capsules). 
• 
To evaluate whether ethnic background (Australian European vs. Chinese Singaporean) affects 
the bioavailability of omega-3 LCPUFA when consuming vegetable-encapsulated omega-3 
products relative to a control test product. 
 
Hypotheses: 
H0: Delivery of omega-3 LCPUFA with vegetables as carriers does not impact bioavailability.   
 
H0:  Bioavailability of omega-3 LCPUFA is not influenced by inter-ethnic differences. 
 
Outcomes: 
• Primary outcome: Incremental area under the plasma DHA and total omega-3 LCPUFA 
concentration (µg/mL) curves (iAUC0-24h) 
• Secondary outcomes: Time to maximal value (Tmax) and maximal value (Cmax) on the plasma 
DHA and total omega-3 LCPUFA concentration (µg/mL) curves.  
   
 
3. Study Population 
 
N=24 (N=12 in Australia; N=12 in Singapore) healthy men. Men are chosen as study population to 
ensure a homogenous population.  
 
4.1 Inclusion and Exclusion criteria 
Inclusion Criteria 
1. Healthy men*, 21-50-year old**  
2. BMI 18-27.5 kg/m2 
3. Consume <2 meals of fatty fish/week 
4. Not Consume fish oil supplements over the past 3 months 
5. Ethnicity 
a. 
If Australian site: Australian European  
OR  
b. 
If Singapore site: Chinese Singaporean 
 
* Only males will be recruited to ensure a metabolically homogenous sample 

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** The lower-end age criteria has been increased from the typical 18 to 21 to provide consistency 
with Singaporean recruitment criteria (Participants under the age of 21 require guardian approval in 
Singapore). 
Exclusion Criteria 
1. History of chronic disease - cancer, type 2 diabetes, cardiovascular disease, liver disease or 
any condition that may, in the opinion of the principle investigator, influence the study 
outcomes* 
2. History of gastrointestinal disease, pancreatic insufficiency, conditions resulting in fat 
malabsorption - chronic pancreatitis, cystic fibrosis, coeliac disease, Crohns disease, gastric 
bypass surgery, small bowel resection, abnormal thyroid function* 
3. Bleeding disorders, currently taking anticoagulants or has received anticoagulants within 28 
days of Day 1 of the trial, with the exception of low dose aspirin up to150 mg daily* 
4. Any medical procedures deemed by the principal investigator to affect study outcomes 
5. Known food allergies, hypersensitivity, dietary avoidance or intolerance to the study foods 
6. Taking medications/supplements known to influence lipid metabolism and gastric emptying  
7. On any weight-loss program 
8. History of smoking during the 6 months prior to the study* 
9. Persons considered by the investigator to be unwilling, unlikely or unable to comprehend or 
comply with the study protocol 
10. History of drug abuse or alcoholism* 
11. Participation in another research study within 30 days preceding the start of this study 
*Self-reported, no clinical testing will be performed. 
 
4. Study Design and procedures 
 
A multi-centre, randomised, controlled, acute, 3-way cross-over single-blind study design will be used. 
The trial will be performed at CSIRO’s Nutrition and Health Research Clinic, Adelaide (n=12) and 
A*STAR’s Clinical Nutrition Research Centre in Singapore (n=12).  
 
Participants will attend a screening visit to confirm their eligibility for inclusion into the trial (Figure 1). 
If eligible, participants will remain at the clinic where they will be enrolled into the study and 
participate in the first intervention on that day (Screening will be conducted at V1). After enrolment 
participants will attend the research clinic for test procedures on 3 occasions at least 1-week apart 
during which they will consume one of three test products at each occasion in random order. 
Participants will attend the clinic on 6 occasions in total (to include returning for 24hr blood sample) 
– visits 2, 4 and 6 (Figure 1).   A 1-week washout period is sufficient for plasma sample to return to 
baseline (19). Participants will be randomly assigned by computer sequence generation 
(http://www.randomisation.com) to one of three treatment orders (Figure 1). The random allocation 
sequence will be generated by a staff member not involved with entering participants into the trial to 
ensure allocation concealment.   

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Figure 1. Study design 
 
Participants will visit the research clinic on six occasions (Figure 1).  
• 
Occasion 1: Screening + 1st intervention (randomised) involving in-clinic assessments (~9 
hours). 
• 
Occasion 2: Participants will return to the clinic for a short visit (30 minutes) where a 24-hour 
blood sample will be collected (Figure 2).  
• 
Occasion 3: (1 week following Occasion 1) 2nd intervention (randomised) involving in-clinic 
assessments (~8.5 hours). 
• 
Occasion 4: Participants return to the clinic for a short visit (30 minutes) where a 24-hour 
blood sample will be collected. 
• 
Occasion 5: (1 week following Occasion 3) 3rd intervention (randomised) involving in-clinic 
assessments (~8.5 hours). 
• 
Occasion 6: Participants return to the clinic for a short visit (30 minutes) where a 24-hour 
blood sample will be collected. 
 
Screening occasion: Participants will be pre-screened using a screening questionnaire and those 
determined to be potentially eligible will be invited to attend an in-clinic screening visit. After they 
have given signed informed consent (see details below) their eligibility will be confirmed by reviewing 
their medical history, demographic information and fatty fish intake obtained from the screening 
questionnaire, measuring height and weight and calculating BMI, and measuring vital signs (blood 
pressure and temperature). Eligible participants will be enrolled into the study and remain at the clinic 
for further 8.5 hours and complete their first intervention  
 
Intervention occasions (Figure 2): Participants will arrive at the research clinic after an overnight fast 
(>10h)). Weight, percentage body fat mass and vital signs (blood pressure and temperature) will be 
measured, an intravenous cannula will be inserted into the participants arm for a duration of 8 hours 
during which the participant will remain at the research clinic.  
 
A fasting blood sample (4 mL) will be collected after which participants will consume a standardised 
breakfast with their test product (see section 5 for more detail) within 15 minutes. Subsequent blood 
samples will be collected after 2, 4, 6 and 8 hours. The cannula will be removed, and participants will 
leave the research clinic. Participants will return to the research clinic the next morning after an 
overnight fast for collection of the 24-hour sample. The period of 24 hours was chosen as it represents 
an intake interval of daily intake of omega-3 supplements (19). Throughout the 9-hour day at the clinic 
participants will have access to low-fat snacks and a lunch meal devoid of omega-3 LCPUFA that they 
can consume ad libitum (see section 5 for more detail). Participants will leave the clinic with a take-
home low-fat dinner meal.  
 

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Apart from the study specific test products, participants will be requested to maintain their habitual 
lifestyle patterns throughout the duration of the study and to avoid fatty fish and omega-3 LCPUFA 
containing supplements. Participants will be provided with a checklist to record any non-compliance 
or accidental consumption of these foods and return the checklist at their subsequent visit.  
 
Staff responsible for sample and statistical analysis will be blinded to which treatment was consumed 
when, but participants and staff administrating treatments will not be blinded.  
 
Plasma fatty acid concentrations (µg/mL) will be analysed by gas chromatography and bioavailability 
determined by calculating the incremental area under the curve (iAUC0-24h) for plasma DHA and total 
omega-3 LCPUFA (Primary outcome), time to maximal value (Tmax) and maximal value (Cmax).  
 
 
 
Figure 2. Test day procedures 
 
Informed Consent 
 
The Principal Investigator or designee will ensure that all participants must sign freely given written 
informed consent, approved by HREC, before undergoing any in-clinic screening activity or procedure. 
 
The Principal Investigator or designee will inform the individual of the objectives, methods, anticipated 
benefits, and potential risks and inconveniences of the study. The individual will be given every 
opportunity to ask for clarification of any points he or she does not understand and, if necessary, to 
ask for more information. All questions about the study should be answered to the satisfaction of the 
individual. The participant will be given time to consider participation in the study. 
 
Participants will be required to sign and personally date the informed consent form (ICF) in the 
presence of the designee and the designee who conducted the informed consent discussion must also 
sign and personally date the ICF. After signatures are obtained a copy of the signed ICF will be provided 
to the participant for their reference and the original document filed in the Investigator Site File.  
 
Withdrawal of Participants 
 
Participants are free to withdraw from the study at any time without providing reason(s) for 
withdrawal and without prejudice or penalty/loss of benefits to which they are otherwise entitled. 
Participant can be withdrawn for any of the following reasons: 
• 
Participant request; withdrawal of informed consent 
• 
Any adverse event (AE), intercurrent illness, medical condition or any situation where in 
continued participation in the study Is not in the best interest of participant as decided by PI 
• 
Participant non-compliance with protocol at the discretion of the Principal Investigator or 
designee or study is terminated 

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The reason/s for withdrawal will be documented in the source documents. Participants who refuse to 
give their consent will not be included in the study, and participants who withdraw their consent will 
not continue in the study. 
 
The Principal investigator or designee may withdraw a participant from the study at any time. In this 
event, the clinic will ensure the reasons have been clearly explained to the individual. Adverse event 
and concomitant medications will be reviewed at the time of withdrawal. A two-week follow-up will 
be performed for any unresolved AE at the time of withdrawal. 
 

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5. Test meals 
 
Test meals: 
1. Semi-solid food – 200 g serve Soup (commercially available packet-soup) containing 
cauliflower encapsulated HiDHA® algal oil providing 400 mg DHA + standardised breakfast 
2. Solid food – 50 g serve extruded snack (savoury snack) containing cauliflower encapsulated 
HiDHA® algal oil providing 400 mg DHA + standardised breakfast 
3. Control: 2 x algal oil gel capsule containing HiDHA® algal oil providing 400 mg DHA + 
standardised breakfast 
 
HiDHA® algal oil will be supplied by NuMega Ingredients Pty Ltd.  
 
The standardised breakfast will consist of a small carton of Milo (Nestle: 200 ml), an apple (200 g), one 
muesli bar (Uncle Toby’s: 24 g) (total energy = ~1600 kJ) and the test products/control.  
 
Throughout the 8-hour day at the clinic participants will have access to low-fat snacks and a lunch 
meal devoid of omega-3 LCPUFA that they can consume ad libitum. Low-fat is defined as ≤3 g fat/100 
g solid food or ≤1.5g fat/100 ml for liquid food (20, 21). Snacks may include rice crackers, fruit, low-fat 
dairy products (yoghurt, chocolate milk), fruit juice, water, etc. Lunch and dinner meals will be low-fat 
frozen meals selected from the Lean Cuisine Steam range available in both Australia and Singapore 
https://www.vescofoods.com.au/brands/lean-cuisine). Breakfast (selection of cereal) will be provided 
to participants after collection of the 24-hour sample.  
 
The test products will be manufactured in facilities certified for production of food for human 
consumption in accordance with an approved HACCP (Hazard Analysis Critical Control Point) plan. Only 
food grade ingredients will be used in the production process. All the equipment that will be used in 
the production process will be sanitised, swabbed for microbial contamination and recleaned until it 
becomes suitable for food contact.   All the processing steps, from the purchase of the ingredients to 
the processing and handling conditions at each processing stage will be recorded in a process log 
sheet.  The microbial and physicochemical quality of the final products will be tested in accordance 
with the approved HACCP plan. The products will be released as fit for human consumption after the 
Food Risk Assessment Team (FRAT) team examined the process log sheet and the results of the 
microbial and physicochemical analyses.  
 
7. Assessments 
 
Medical history and demographic information: 
 
Medical and surgical history, medication and supplement use and demographic information will be 
obtained by questionnaire and interview of participants. This information will be used to screen 
participants.  
 
 

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Assessment of fatty fish intake: 
 
In order to assess participant’s eligibility to inclusion criteria 3 - “Consuming <2 meals of fatty 
fish/week over the past 3 months”, potential participants will be asked to record the frequency and 
type of fish they habitually consume. E.g. “How often do you consume fish?” Frequency options will 
include <1x/month, 1-3x/month, 1x/week, 2x/week, 3-4x/week, 1x/day, >1x/day. “What type of fish 
to you usually consume”. The categories of fish according to fatty fish / medium- or low-fat fish sources 
are summarised in the Appendix and will be used to determine participant’s eligibility accordingly.  
 
 
Anthropometric measurements: 
 
Height and weight will be measured at the screening visit and BMI calculated using the formula: weight 
(kg)/height (m) 2. Weight will also be collected at each clinic visit. Height will be measured using a 
stadiometer and body weight will be measured using calibrated electronic digital scales.  
 
Bioelectrical Impedance Analysis (BIA) will be used to assess % body fat mass using a multi-frequency 
BIA with 8 tactile electrodes InBody 230, Biospace Co. Ltd, Seoul) in Australia and Tanita BC-418, Tokyo 
in Singapore. Measurements will be obtained after voiding. Participants will stand upright, positioning 
their bare feet on the footpads and their hands on the handles. A small electrical current is passed 
through the body, resistance is measured, and total body water and the corresponding body 
composition measures are calculated by the inbuilt software. 
 
 
Vital signs: 
 
Resting blood pressure will be measured using an automated blood pressure monitor (InBody 
BPBIO750 Blood Pressure Monitor). In a seated position, an initial mock measurement will be 
performed at the start of the visit (measurement not recorded). Following 5 minutes of rest in a quiet 
room, a further measurement will be performed. If measurement is out of clinical range, a subsequent 
measurement will be performed after 5 to 15 minutes rest. 
 
. Body temperature will be measured using a digital tympanic thermometer. 
 
Vital signs will be assessed at each clinic visit.  
 
 
Blood sample collection and preparation: 
 
Fasted venous blood samples will be collected into vacutainers containing EDTA (purple top, 4mL) for 
the preparation of plasma. Plasma will be prepared by centrifugation at 2851g for 15 min at 4°C. The 
resulting plasma will be aliquoted (500 µL) and stored at -70°C until analysis. Samples from each 
subject will be analysed within the same analytic run to reduce variation. 
 
 
Fatty acid analysis of plasma and test products 
 
Plasma fatty acid concentrations (µg/mL) will be analysed using a gas chromatography–mass 
spectrometry (GCMS) technique. A 100 µL plasma sample will be treated with 1 mL 0.2M KOH/MeOH 
(0.005% BHT) and 50 µL of a 1 mg/mL solution of C17:0 (internal standard; 26.44 mg triheptadecanoin 
[C17:0 triglyceride, Sigma No. T-2151 99] in 25ml chloroform). This will be followed by 10 min of 

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heating at 90 C; addition of 2 mL methanol; acidification with 200 µL of acetyl chloride and heating 
at 90oC for 1 hr while vortexing approximately every 10min after which the samples will be returned 
to room temperature. Methyl esters will be extracted into 1.5 mL hexane and 1.0 µl aliquot injected 
onto a gas chromatographic column (DB – FastFAME, 20m x 0.18 mm from Agilent Technologies), 
using a PerkinElmer Clarus 690 gas chromatograph / Clarus SQ 8 T Mass Spectrometer with a split 50:1 
injector. Fatty acids will be identified by comparison with authentic Sepelco 37 component FAME mix 
(Sigma-Aldrich, Australia) and verified using the NIST MS search database software.  Peaks will be 
measured using TurboMass software and fatty acids will be calculated against the internal standard. 
 
8. Statistical analysis 
 
A sample size of n=10 per site provides >80% power to detect a difference of 10% in AUC (22). N=12 
per site will be recruited to account for a 20% attrition rate.  
 
A statistical analysis plan will be developed before statistical analysis commences. In brief, data will 
be presented as means and SD/SEM. Plasma DHA and total omega-3 LCPUFA concentrations will be 
plotted into graphs. iAUC0-24h (primary outcome), will be calculated using the trapezoidal rule and 
corrected to baseline levels. Time to maximal value (Tmax) and the maximal value (Cmax) will be 
determined. Primary analysis will be pairwise comparisons between test foods vs. control using linear 
mixed models with fixed effects for treatment, time, ethnicity and assessing the interaction of 
treatment*time and treatment*time*ethnicity. Participant identifier will be fitted as a random effect, 
i.e. one intercept per participant. Potential covariates will be identified a priori and included in the 
model (e.g. BMI). Intention-to-treat analysis will be performed primarily and per protocol analysis as 
secondary analysis.  
 
Statistical analysis will be performed using appropriate statistical packages e.g. SPSS software (IBM 
Corporation, New York, USA). Statistical significance will be determined at a P-value of <0.05. 
Statistical analysis will be performed blinded and treatments revealed after completion of the 
statistical analysis.  
 
9. Assessment of Adverse Events 
 
Following Informed consent, adverse events (AE) reported by participants for the duration of their 
time in the trial will be documented and followed up in accordance with International Conference on 
Harmonisation Good Clinical Practice (ICH-GCP) guidelines. 
Adverse Event 
 
Definition: 
An AE is defined as any unfavourable and unintended change in the structure (signs), function 
(symptoms) or chemistry (laboratory data) of the body associated in time with the use of the study 
product whether or not considered related to the use of the product. Any worsening (e.g. any clinically 
significant adverse change in frequency or intensity) of a pre-existing condition which is temporally 
associated with the use of the product is also an AE. A serious adverse event (SAE) is defined as any 
AE that result in any of the following outcomes: death; a life-threatening AE; inpatient hospitalisation 
or prolongation of existing hospitalisation; a persistent or significant disability/incapacity; or a 
congenital anomaly/birth defect(23)(22)(22)(21)(22)(21)(21)(20)(20)(20). 
 

Protocol Number: OBP001 
17 
Version 3.0, 20th October 2020 
 
Suspected Unexpected Serious Adverse reactions (SUSARs) are SAEs that are believed to be related to 
a study treatment and are both unexpected (i.e. the nature or severity is not expected from the 
information known on the product) and serious. SUSARs require expedited reporting to applicable 
regulatory authorities. 
 
AE will be scored for its severity and likelihood of the symptoms being related to the trial product as 
follows:  
 
Assessment of Severity: 
“1” Mild: An event that is easily tolerated by the subject, causing minimal discomfort and not 
interfering with everyday activities  
“2” Moderate: An event that is sufficiently discomforting to interfere with normal activities 
“3” Severe: An event which is incapacitating and prevents normal everyday activities 
 
Assessment of Causality: 
The causality of an AE (i.e. their relationship to study product) will be assessed by the Principal 
Investigator or designee at the study site. Assessing causality requires considering whether there was 
a reasonable possibility that the event may have been caused by the study treatment. 
 
The relationship of the AE to the study product will be specified by the investigator using the following 
definitions: 
 
Not Related: The adverse event is clearly explained by another cause not related to the study product. 
Probably Not Related: A potential relationship between study product and the adverse event could 
exist (i.e. the possibility cannot be excluded), but the adverse event is most likely explained by causes 
other than the study agent. 
Possibly Related: The adverse event and administration of study product are reasonably related in 
time, and the adverse event can be explained equally well by causes other than the study product. 
Probably Related: The adverse event and use of study product are reasonably related in time, and the 
adverse event is more likely explained by study product than other causes. 
Definitely Related: The adverse event and use of study product are related in time, and a direct 
association can be demonstrated. 
 
Assessment of Outcome: 
For each recorded AE or SAE an assessment of outcome at the time of last observation. The outcome 
of AEs or SAEs will be documented as follows: 
• Fatal: The participant died 
• Resolved: The AE or SAE has ended 
• Resolved with Sequelae: The AE or SAE has ended but changes are noted from baseline 
• Unresolved: The AE has not ended. The AE outcome can only be categorised as unresolved if the AE 
is: 
o Ongoing at the final safety or early withdrawal visit and the Medical Investigator deems that no 
further follow-up is required 
o Lost to follow-up after repeated unsuccessful attempts to contact the participant 
o Ongoing and referred to the participant’s physician or specialist 
 
Reporting: 
Details of the AE are documented by the relevant clinic staff member in the participant’s Case Report 
Form (CRF) on the applicable form while the participant is present or at the time of contact. Once the 
AE has been documented, the information is reviewed by the trial coordinator. Follow up of the event 
should be conducted by the coordinator or the person conducting the next consecutive visit unless 

Protocol Number: OBP001 
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the AE is reported at the end of a study then the coordinator will follow up. All unresolved AEs at the 
completion of the study will be followed after two weeks by phone call. At this follow-up call, any 
stable unresolved AEs of mild intensity would stay unresolved and would not have further follow-ups. 
All other unstable, unresolved AEs with moderate to severe intensity will be actioned as per PI’s 
discretion.  If the participant has been lost to contact, it must be demonstrated that three phone calls 
were made on different days and times followed by a final email to contact the person to follow up 
the AE. 
 
All SAE, whether causally related to the treatment or not, and SUSAR must be immediately reported 
firstly to the Research Clinician, the relevant Human Research Ethics Committee (HREC) and to 
applicable regulatory authorities within 24 hrs.  
 
10. Ethical Considerations 
 
The study will be performed in accordance with the ethical principles that has their origin in the 
Declaration of Helsinki, and that are consistent with ICH-GCP and the applicable local ethical & 
regulatory requirements. 
 
The PI is responsible to ensure the protocol, informed consent and all other related documents have 
been reviewed and approved by the (HREC prior to the commencement of the study. The PI is 
responsible for informing the HREC of any amendment to the protocol and will report promptly to the 
HREC new information that may adversely affect the safety of the Participants or the conduct if the 
study. Any necessary extensions or renewals of HREC approval will be obtained for changes to the 
study such as modification of the protocol, the informed consent forms and any information provided 
to participants. The PI or designee will submit written summaries and any study reports to HREC 
annually or as requested. On completion of the study, the PI (or designee) will notify the HREC that 
the study has concluded. 
 
All personal data collected and processed for the purposes of this study will be managed by site staff 
with adequate precautions to ensure confidentiality of those data and in accordance with the 
applicable privacy regulations per the investigators’ country/jurisdiction. Study data will be stored in 
a computer database and confidentiality maintained in accordance with national data legislation. 
Participants in this database will be identified by Participant number. 
 
 
11. Data Handling, Record Keeping & Retention 
 
All data collected from a participant will be recorded in source documents (paper and electronic). 
Study site will enter data from the participant’s source documents into an electronic case report form 
(eCRF) designed in REDCap. Copies of all Source, eCRF data and study related documentation must be 
retained at the site for the duration of the study.  
 
Data collected during this study will be handled, processed and managed as per study specific data 
management plan. Final datasets will be uploaded to the CSIRO Data Access Portal and shared in de-
identified manner as per study specific data sharing plan. 
Following completion of the study and publication of results, all study related documents will be 
archived by study sites and retained at least for 15 years. 
 
 

Protocol Number: OBP001 
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Version 3.0, 20th October 2020 
 
13. Quality Control and Quality Assurance 
 
Internal quality management of study conduct, data and biological specimen collection, 
documentation and completion in compliance with the protocol, ICH GCP and applicable regulatory 
requirements will be performed by study sites.  
 
14. Direct Access to Source Data/Documents 
 
Upon request or notification, the Investigator will ensure and permit direct access to source data and 
documents for trial related monitoring, audits, HREC review and regulatory inspections. In case of 
electronic source, a read-only assess will be provided. 
 
 
15. Publication Policy 
 
The results will be submitted for scientific publications including publication in scientific journals, 
conference abstract, and verbal or poster presentations in accordance with the publication policy 
outlined in the agreement between CSIRO and A*STAR.  
 
 
16. References 
 
1. 
Australian Institute of Health and Welfare (AIHW). Australia’s health 2018: in brief. Cat. no. 
AUS 222. Canberra: AIHW; 2018. 
2. 
Melaku YA, Renzaho A, Gill TK, Taylor AW, Dal Grande E, de Courten B, et al. Burden and 
trend of diet-related non-communicable diseases in Australia and comparison with 34 OECD 
countries, 1990-2015: findings from the Global Burden of Disease Study 2015. European journal of 
nutrition. 2019;58(3):1299-313. 
3. 
Harris WS, Von Schacky C. The Omega-3 Index: a new risk factor for death from coronary 
heart disease? Preventive medicine. 2004;39(1):212-20. 
4. 
Harris WS, Del Gobbo L, Tintle NL. The Omega-3 Index and relative risk for coronary heart 
disease mortality: Estimation from 10 cohort studies. Atherosclerosis. 2017;262:51-4. 
5. 
Jackson KH, Polreis JM, Tintle NL, Kris-Etherton PM, Harris WS. Association of reported fish 
intake and supplementation status with the omega-3 index. Prostaglandins, leukotrienes, and 
essential fatty acids. 2019;142:4-10. 
6. 
Stonehouse W. Does consumption of LC omega-3 PUFA enhance cognitive performance in 
healthy school-aged children and throughout adulthood? Evidence from clinical trials. Nutrients. 
2014;6(7):2730-58. 
7. 
Koh AS, Pan A, Wang R, Odegaard AO, Pereira MA, Yuan JM, et al. The association between 
dietary omega-3 fatty acids and cardiovascular death: the Singapore Chinese Health Study. European 
journal of preventive cardiology. 2015;22(3):364-72. 
8. 
Meyer BJ. Australians are not Meeting the Recommended Intakes for Omega-3 Long Chain 
Polyunsaturated Fatty Acids: Results of an Analysis from the 2011-2012 National Nutrition and 
Physical Activity Survey. Nutrients. 2016;8(3):111. 
9. 
National Health and Medical Research Council (NHMRC). Nutrient Reference Values for 
Australia and New Zealand Including Recommended Dietary Intakes. Canberra, Australia; 2006. 
10. 
Food and Agriculture Organisation of the United Nations. Fats and fatty acids in human 
nutrition. Report of an expert consultation. Rome, Italy; 2010. 

Protocol Number: OBP001 
20 
Version 3.0, 20th October 2020 
 
11. 
Nestel P, Clifton P, Colquhoun D, Noakes M, Mori TA, Sullivan D, et al. Indications for Omega-
3 Long Chain Polyunsaturated Fatty Acid in the Prevention and Treatment of Cardiovascular Disease. 
Heart, lung & circulation. 2015;24(8):769-79. 
12. 
Sanders TA. DHA status of vegetarians. Prostaglandins, leukotrienes, and essential fatty 
acids. 2009;81(2-3):137-41. 
13. 
Walker RE, Jackson KH, Tintle NL, Shearer GC, Bernasconi A, Masson S, et al. Predicting the 
effects of supplemental EPA and DHA on the omega-3 index. The American journal of clinical 
nutrition. 2019;110(4):1034-40. 
14. 
Wang X, Ouyang Y, Liu J, Zhu M, Zhao G, Bao W, et al. Fruit and vegetable consumption and 
mortality from all causes, cardiovascular disease, and cancer: systematic review and dose-response 
meta-analysis of prospective cohort studies. BMJ (Clinical research ed). 2014;349:g4490. 
15. 
Hall JN, Moore S, Harper SB, Lynch JW. Global variability in fruit and vegetable consumption. 
American journal of preventive medicine. 2009;36(5):402-9.e5. 
16. 
Sanguansri L, Day L, Shen Z, Fagan P, Weerakkody R, Cheng LJ, et al. Encapsulation of 
mixtures of tuna oil, tributyrin and resveratrol in a spray dried powder formulation. Food & function. 
2013;4(12):1794-802. 
17. 
Augustin MA, Sanguansri L, inventorsProtecting a bioactive or precursor thereof. 
Australia2018. 
18. 
de Groot RHM, Emmett R, Meyer BJ. Non-dietary factors associated with n-3 long-chain 
PUFA levels in humans - a systematic literature review. The British journal of nutrition. 
2019;121(7):793-808. 
19. 
Qin Y, Nyheim H, Haram EM, Moritz JM, Hustvedt SO. A novel self-micro-emulsifying delivery 
system (SMEDS) formulation significantly improves the fasting absorption of EPA and DHA from a 
single dose of an omega-3 ethyl ester concentrate. Lipids Health Dis. 2017;16(1):204-. 
20. 
Food Standards Australia New Zealand (FSANZ). Standard 1.2.7 Nutrition, Health and Related 
Claims 2016 [Available from: 
https://www.foodstandards.gov.au/consumer/labelling/nutrition/Pages/default.aspx. 
21. 
Health Promotion Board. A handbook of Nutrition Labelling Singapore; 2015. 
22. 
Raatz SK, Johnson LK, Bukowski MR. Enhanced Bioavailability of EPA From Emulsified Fish Oil 
Preparations Versus Capsular Triacylglycerol. Lipids. 2016;51(5):643-51. 
23. 
Therapeutic Goods Administration. Note for guidance on good clinical practice 
(CPMP/ICH/135/95). Canberra; 2000. 
 
 

Protocol Number: OBP001 
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Appendix 
Categories of fish according to fat content 
 
Fatty fish sources (~>500 
mg LCPUFA/serve) 
(serve = 150 g fish / 100g 
canned fish) 
• 
Salmon (fresh or canned) 
• 
Mackerel 
• 
Sardines (fresh or canned) 
• 
Anchovies 
• 
Trout 
• 
Tuna (fresh or canned) 
• 
Mullet 
 
• 
Silver perch 
• 
Morwong 
• 
Bream 
• 
Herring 
• 
Smoked/green/blue mussels 
(canned) 
• 
Smoked oysters (canned) 
 
Medium- to low-fat fish 
sources (<500 mg 
LCPUFA/serve) 
(serve = 150 g fish / 100g 
canned fish) 
• 
Yellowtail kingfish 
• 
Trevally 
• 
Snapper 
• 
Flounder 
• 
Mirror dory 
• 
Jack mackerel 
• 
Blue-spotted goatfish 
• 
Small-spotted herring 
• 
Blue-eye trevalla 
• 
Australian bass 
• 
King Dory 
• 
Wrasse 
• 
John Dory 
• 
Blue morwong 
• 
Coral trout 
• 
Blue grenadier (Hoki) 
• 
Golden perch  
• 
Ocean jacket 
• 
Leather jacket 
• 
Murray cod 
• 
Hussar 
• 
Garfish 
• 
Barramundi 
• 
Dhufish 
• 
Mangrove Jack 
• 
King George whiting 
• 
Smoked cod 
• 
Flathead 
• 
Shark (Flake) 
• 
Jackass morwong 
• 
Squid/calamari (100g) 
• 
Oysters, raw (100g) 
• 
Scallops (100g) 
• 
Prawn (100g) 
• 
Octopus (10 
• 
Crab (100g) 
• 
Lobster/crayfish (100g)  
• 
Moreton bay bug (100g) 
• 
Whiting 
 
Source: Australian Heart Foundation