Extracellular Vesicles; Generation and FunctionHI-FIVENCT03203512
Fish Oil-derived N-3 Polyunsaturated Fatty Acids and Extracellular Vesicles
A study testing Fish oil capsules and High-oleic safflower oil capsules in people with Extracellular Vesicles; Generation and Function, run by University of Reading at 1 site in United Kingdom.
Trial summary
N-3 polyunsaturated fatty acids (n-3 PUFA), which are abundant in oily fish and fish oils, have been suggested to play a role in reducing the risk of cardiovascular diseases (CVDs) by modifying a wide range of risk factors, such as blood fats, blood clotting, blood vessel function and inflammation. Read more about this study
- Who is it for?
- Ages 40 to 70 yearsHealthy volunteers can take part
- Conditions the study is for
- Extracellular Vesicles; Generation and Function
- What is being tested?
- Fish oil capsules and High-oleic safflower oil capsules
- Type of treatment
- Pill or oral medicine
- Could I receive a placebo?
- Yes, some participants may receive a placebo
- How many people
- 42 participants
- Where
- 1 siteUnited Kingdom
- Study dates
- 16 Feb 2018to 30 Mar 2021
- Phase
- Interventional
- Are there results?
- Yes, posted in the registrySummary in the doctors' view
Key requirements
Who can take part, as listed by the sponsor. Only the study team can confirm whether you are eligible, so take this list to your doctor.
- Ages 40 to 70 years
- All sexes
- Healthy volunteers accepted
- Adult, Older adult
Who may be able to join
- Aged 40-70 years
- Non-smoker
- At moderate risk of cardiovascular diseases
- The risk will be evaluated by an online calculator called "QRISK2". This online calculator (https://qrisk.org/2016/), which use traditional risk factors (age, systolic blood pressure, smoking status and ratio of total serum cholesterol to high-density lipoprotein cholesterol) together with body mass index, ethnicity, measures of deprivation, family history, will provide a percentage of risk of having a heart attack or stroke within the next 10 years.
- Subjects with 10%-20% will be regarded as being at moderate risk
Who cannot join
- BMI: <18.5 kg/m2
- Anaemia (haemoglobin concentration <12.5 g/L in men and<11.5 g/L in women)
- Hyperlipidaemia (total cholesterol concentration >8 mmol/L)
- Diabetes (diagnosed or fasting glucose concentration >7 mmol/L) or other endocrine disorders
- Angina, stroke, or any vascular disease in the past 12 months
- Renal, gastrointestinal, respiratory, liver or bowel disease
Show all 18Show fewer
- Inflammatory disease
- Take drug treatment for hypertension, hyperlipidaemia, inflammation, depression or thyropathy.
- Take aspirin, ibuprofen or other nonsteroidal anti-inflammatory drugs (NSAIDs) > 4 times per month, or once in the week preceding the study
- Take any other anti-platelet or anti-coagulant drugs, like triflusal, clopidogrel and warfarin.
- Have allergies
- Smoking (including e-cigarettes and nicotine products)
- Alcohol misuse or intakes >21 units/wk for men and >15 units/wk for women or have a history of alcohol misuse
- Regularly consume oily fish and/or dietary supplements
- Planning to start or on a weight reducing regimen
- Intense aerobic exercise (>20 min, three times a week)
- Females who are pregnant, lactating, or if of reproductive age and not using a reliable form of contraception (including abstinence)
- Have participated in another clinical trial within the last three months
Trial locations and contacts
1 site in 1 country.
About this study
Official title: Effects of Fish Oil-derived N-3 Polyunsaturated Fatty Acids on the Generation and Functional Activities of Extracellular Vesicles
N-3 polyunsaturated fatty acids (n-3 PUFA), which are abundant in oily fish and fish oils, have been suggested to play a role in reducing the risk of cardiovascular diseases (CVDs) by modifying a wide range of risk factors, such as blood fats, blood clotting, blood vessel function and inflammation. Extracellular vesicles (EVs) are small particles released from various cells when they are activated or damaged. High numbers of EVs in the blood have been associated with a higher risk of CVDs, and it is thought that this is because they carry 'bioactive' components which can affect many processes involved in CVDs. However, very few clinical trials have investigated the relationships between the consumption of n-3 PUFA and circulating EVs. This study aims to investigate the effects of dietary n-3 PUFA on the generation and functional activities of EVs, which would provide new insight into the benefits of n-3 PUFA on cardiovascular health.
Read the detailed description
The proposed study will be a randomised, double-blind, placebo-controlled crossover intervention. Subjects (40-70y) at moderate CVDs risk will be supplemented with either fish oil (1.8 g/d n-3 PUFA) or placebo (high-oleic safflower oil) for 12 weeks. After a 12-week washout and then cross-over to the other intervention for another 12 weeks. Blood samples will be collected before and after each intervention. A food frequency questionnaire will be administered to assess the subject's habitual intake of n-3 PUFA. Subjects will also be expected to maintain a low consumption of n-3 fatty acids, refrain from the use of all supplements, and maintain their body weight during the study. The dose is based on our previous work, which demonstrated a reduction in numbers of endothelial-derived EVs (EEVs) and a trend for reduced numbers of platelet-derived EVs (PEVs), and a dose at which beneficial effects of n-3 PUFA on plaque stability are reported. The experimental work will follow two main strands. The first strand will examine the influence of n-3 PUFA supplementation on the characteristics and functional activities of total EVs from plasma. The second strand will examine the influence of n-3 PUFA on the generation of PEVs from platelets taken from subjects and stimulated in vitro; the PEVs generated will subsequently be assessed for their composition and functional activity. This experimental design will allow simultaneous investigation of both the composition and activity of total EVs taken directly from blood, and the generation and activity of PEVs. Based on our previous work, 27 subjects are required to detect a 10% reduction in numbers of EVs following fish oil supplementation with a two-sided significance level of 5% and a power of 90%, and 34 subjects are required for a power of 95%. Also based on previous data, 22 subjects would give 95% power to detect 10% differences in thrombus formation and 30 subjects are required to detect a significant effect of n-3 PUFA on platelet aggregation and phosphatidylserine (PS) exposure. Allowing for a 15% dropout rate, and aiming for 95% power based on a 10% reduction in EVs numbers, we will therefore recruit 40 subjects in total.
- Being tested
- Fish oil capsules, High-oleic safflower oil capsules
- Keywords
- Fish oil; N-3 polyunsaturated fatty acids
What participants receive
What participants receive. In a randomised study each person is assigned to one group by chance.
Fish oil capsulesDietary supplement
Each serving contains 360mg eicosapentaenoic acid (EPA), 270mg docosahexaenoic acid (DHA) and total supplement is 1.8 g per day n-3 PUFA for 12 weeks
High-oleic safflower oil capsulesDietary supplement
High-oleic safflower oil capsules for 12 weeks
| Study group | Role | What they receive |
|---|---|---|
| Intervention | Active comparator | Fish oil capsules Receives: Dietary Supplement: Fish oil capsules |
| Placebo | Placebo comparator | High-oleic safflower oil capsules Receives: Dietary Supplement: High-oleic safflower oil capsules |
What is being measured
The primary outcome is the main question the study is designed to answer.
Numbers of Circulating Total EVs in Platelet-free Plasma (PFP) Detected by Nanoparticle Tracking Analysis (NTA)
Circulating EVs were first isolated to obtain fractions 7~9 by size exclusion chromatography (SEC) using Izon qEV columns (Izon Science Ltd, Oxford, United Kingdom). Fractions were then diluted with PBS to maintain the recommended concentration range of particles (1~10*10^8 vesicles/ml) before being analysed on NanoSight 300 (Malvern, Amesbury, United Kingdom). For each analysis, five videos, each of 60 seconds duration, were captured with the camera level at 13. Data were analysed using the instrument software NTA 3.20, which can identify individual particles and estimate their sizes based on the Stokes-Einstein Equation. Finally, a threshold of 70nm was set for NTA to ensure minimal interference by small lipoproteins.
Measured: Change of circulating total EV numbers in PFP detected by NTA after intake period of 12 weeks
Numbers of Total Phosphatidylserine Positive EVs (PS+EVs) in Platelet-free Plasma (PFP) Detected by Flow Cytometry (FCM)
A 5μl of PFP was added into nonsticky microcentrifuge tubes (Alpha Laboratories Ltd, Hampshire, United Kingdom), which contained 5μl FcR blocking reagent (Miltenyi Biotec Ltd, Surrey, United Kingdom) and Annexin V buffer and incubated for 15 minutes in the dark at room temperature. Antibodies and isotype-matched controls were then added and samples incubated for another 15 minutes in the dark at room temperature. After incubation, samples were diluted with 200μl Annexin V buffer and transferred into FACS flow tubes (BD Biosciences, Wokingham, United Kingdom), ready to be analysed by FCM. PS+EVs were identified as Annexin V+EVs when triggering on APC fluorescence.
Measured: Change of total PS+EV numbers in PFP detected by FCM after intake period of 12 weeks
Characterisation of Circulating EVs Subpopulation in PFP Detected by Fluorescence FCM
A 5μl of PFP was added into nonsticky microcentrifuge tubes (Alpha Laboratories Ltd, Hampshire, United Kingdom), which contained 5μl FcR blocking reagent (Miltenyi Biotec Ltd, Surrey, United Kingdom) and Annexin V buffer and incubated for 15 minutes in the dark at room temperature. Antibodies and isotype-matched controls were then added and samples incubated for another 15 minutes in the dark at room temperature. After incubation, samples were diluted with 200μl Annexin V buffer and transferred into FACS flow tubes (BD Biosciences, Wokingham, United Kingdom), ready to be analysed by FCM. Platelet-derived EVs (PDEVs) were identified as Annexin V+EVs which also stained positive for CD41-PE in APC vs PE quadrant plot, and endothelial-derived EVs (EDEVs) were identified as Annexin V+EVs which also stained positive for CD105- eFluor450 in APC vs PB quadrant plot.
Measured: Change in the numbers of circulating EVs subpopulation in PFP by fluorescence FCM after intake period of 12 weeks
Secondary outcomes (14)
Pro-thrombotic Activities of Circulating EVs in PFP (Lag Time for Thrombin Generation)
A commercially available, plate-based thrombin generation assay was used to measure thrombin generation in either a standard, pooled vesicle and platelet-free plasma (termed vesicle-free plasma or VFP) or in the same VFP but with added circulating EVs from subjects in the intervention study. This enabled the assessment of TF-dependent thrombin generation specifically attributed to circulating EVs in samples from the intervention study. Results were presented as five variables: (i) lag-phase for initiation of thrombin generation after addition of the trigger (time to 1/6 of the peak height) (min); (ii) peak thrombin concentration (nM); (iii) time to reach the peak (min); (iv) velocity index, defined as = [peak height/(time to peak - lag time)] and (v) area under the curve, defined as endogenous thrombin potential (ETP) (expressed as nM thrombin × min)
Measured: Change of pro-thrombotic activities (lag time for thrombin generation)of circulating EVs in PFP after intake period of 12 weeks
Pro-thrombotic Activities of Circulating EVs in PFP (Peak Thrombin Concentration)
A commercially available, plate-based thrombin generation assay was used to measure thrombin generation in either a standard, pooled vesicle and platelet-free plasma (termed vesicle-free plasma or VFP) or in the same VFP but with added circulating EVs from subjects in the intervention study. This enabled the assessment of TF-dependent thrombin generation specifically attributed to circulating EVs in samples from the intervention study. Results were presented as five variables: (i) lag-phase for initiation of thrombin generation after addition of the trigger (time to 1/6 of the peak height) (min); (ii) peak thrombin concentration (nM); (iii) time to reach the peak (min); (iv) velocity index, defined as = [peak height/(time to peak - lag time)] and (v) area under the curve, defined as endogenous thrombin potential (ETP) (expressed as nM thrombin × min)
Measured: Change of pro-thrombotic activities (peak thrombin concentration) of circulating EVs in PFP after intake period of 12 weeks
Pro-thrombotic Activities of Circulating EVs in PFP (Time to Peak Thrombin Concentration)
A commercially available, plate-based thrombin generation assay was used to measure thrombin generation in either a standard, pooled vesicle and platelet-free plasma (termed vesicle-free plasma or VFP) or in the same VFP but with added circulating EVs from subjects in the intervention study. This enabled the assessment of TF-dependent thrombin generation specifically attributed to circulating EVs in samples from the intervention study. Results were presented as five variables: (i) lag-phase for initiation of thrombin generation after addition of the trigger (time to 1/6 of the peak height) (min); (ii) peak thrombin concentration (nM); (iii) time to reach the peak (min); (iv) velocity index, defined as = [peak height/(time to peak - lag time)] and (v) area under the curve, defined as endogenous thrombin potential (ETP) (expressed as nM thrombin × min)
Measured: Change of pro-thrombotic activities (time to peak thrombin concentration) of circulating EVs in PFP after intake period of 12 weeks
Pro-thrombotic Activities of Circulating EVs in PFP (Velocity Index)
A commercially available, plate-based thrombin generation assay was used to measure thrombin generation in either a standard, pooled vesicle and platelet-free plasma (termed vesicle-free plasma or VFP) or in the same VFP but with added circulating EVs from subjects in the intervention study. This enabled the assessment of TF-dependent thrombin generation specifically attributed to circulating EVs in samples from the intervention study. Results were presented as five variables: (i) lag-phase for initiation of thrombin generation after addition of the trigger (time to 1/6 of the peak height) (min); (ii) peak thrombin concentration (nM); (iii) time to reach the peak (min); (iv) velocity index, defined as = [peak height/(time to peak - lag time)] and (v) area under the curve, defined as endogenous thrombin potential (ETP) (expressed as nM thrombin × min)
Measured: Change of pro-thrombotic activities (velocity index) of circulating EVs in PFP after intake period of 12 weeks
Pro-thrombotic Activities of Circulating EVs in PFP (Endogenous Thrombin Potential)
A commercially available, plate-based thrombin generation assay was used to measure thrombin generation in either a standard, pooled vesicle and platelet-free plasma (termed vesicle-free plasma or VFP) or in the same VFP but with added circulating EVs from subjects in the intervention study. This enabled the assessment of TF-dependent thrombin generation specifically attributed to circulating EVs in samples from the intervention study. Results were presented as five variables: (i) lag-phase for initiation of thrombin generation after addition of the trigger (time to 1/6 of the peak height) (min); (ii) peak thrombin concentration (nM); (iii) time to reach the peak (min); (iv) velocity index, defined as = [peak height/(time to peak - lag time)] and (v) area under the curve, defined as endogenous thrombin potential (ETP) (expressed as nM thrombin × min)
Measured: Change of pro-thrombotic activities (endogenous thrombin potential) of circulating EVs in PFP after intake period of 12 weeks
Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay
96-well high-throughput aggregometry technique, allowing testing of a wide range of concentrations of different agonists, was used to examine the influence of n-3 PUFA supplementation on platelet function. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) from each study visit was used in the platelet aggregation assay using pre-prepared 96-well microplates, containing the agonists (ADP, EPI, TRAP-6 and U46619). Dose-response curves in response to each agonist were obtained and results were represented as a LogEC50 (log concentration of agonist, M, giving a response halfway between maximum and minimum aggregation).
Measured: Change in ex vivo platelet activation after intake period of 12 weeks
Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay (CRP-XL Log EC50)
96-well high-throughput aggregometry technique, allowing testing of a wide range of concentrations of different agonists, was used to examine the influence of n-3 PUFA supplementation on platelet function. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) from each study visit was used in the platelet aggregation assay using pre-prepared 96-well microplates, containing the agonists (CRP-XL). Dose-response curves in response to each agonist were obtained and results were represented as a LogEC50 (log concentration of agonist, mg/ml, giving a response halfway between maximum and minimum aggregation).
Measured: Change in ex vivo platelet activation after intake period of 12 weeks
Pro-thrombotic Activities of Platelet-derived Extracellular Vesicles (PDEVs) Prepared From the Supernatants of Stimulated Platelets (Endpoint and Maximum of Thrombus Formation)
Ex vivo thrombus formation was measured by the addition of in vitro-generated PDEVs from stimulated platelets into whole blood under flow. Results were presented as three variables: (i) endpoint for ex vivo thrombus formation (FU); (ii) endpoint for ex vivo thrombus formation (FU); (iii) area under curve.
Measured: Change in pro-thrombotic activities (endpoint and maximum of thrombus formation)of PEVs prepared from the supernatants of stimulated platelets after intake period of 12 weeks
Pro-thrombotic Activities of Platelet-derived Extracellular Vesicles (PDEVs) Prepared From the Supernatants of Stimulated Platelets (Area Under Curve)
Ex vivo thrombus formation was measured by the addition of in vitro-generated PDEVs from stimulated platelets into whole blood under flow. Results were presented as three variables: (i) endpoint for ex vivo thrombus formation (FU); (ii) endpoint for ex vivo thrombus formation (FU); (iii) area under curve.
Measured: Change in pro-thrombotic activities (area under curve) of PEVs prepared from the supernatants of stimulated platelets after intake period of 12 weeks
Circulating EV Total Lipids Analysis
A 500μl aliquot of frozen PFP was defrosted at room temperature using a roller mixer and subjected to SEC for the isolation and purification of EVs. The fractions 7~9 were pooled together, and 800μl of pooled fractions was prepared for total lipid extraction and methyl esterification. The EV total lipid methyl esters were then analysed by gas chromatography on a Hewlett-Packard 6890 series GC (Hewlett-Packard, California, United States), with the following protocol: split ratio was set as 30:1 for plasma and EV analysis. The injection volume was 1μl for plasma and 5μl for EVs, respectively. The temperature of both injector and detector were kept at 300°C and the temperature program was initial temperature 115°C for 2 minutes, increased at 10 °C/min to 200°C and hold at this temperature for 16 minutes, and finally increased at 60°C/min to 240°C for 2 minutes (total run time: 29.2 minutes). Samples were analysed by using ChemStation software and Microsoft Excel.
Measured: Change in total lipids of EVs after intake period of 12 weeks
Plasma Total Phospholipids Analysis
A 400μl aliquot of frozen PFP was defrosted and centrifuged to remove denatured protein. The 400μl of 0.9% NaCl was added to the PFP sample to make up 800μl in total, and 30μg of phosphatidylcholine (PC) and 15μg of phosphatidylethanolamine (PE) internal standards were then added for the quantitative analysis. After lipid extraction, separation of PC and PE, and methyl esterification of plasma phospholipid extracts, samples were analysed by GC.
Measured: Change of plasma total phospholipids after intake period of 12 weeks
Concentrations of Lipid Profile in Plasma
A 250μl aliquot of frozen PFP was defrosted at room temperature using a roller mixer and centrifuged at 500xg for 5 minutes at room temperature (Eppendorf Centrifuge 5415 R, DJBlabcare, United Kingdom). Then the sample was analysed by a RANDOX clinical analyser (RANDOX Daytona+ Analyser, Randox Laboratories Ltd, United Kingdom) for the concentration of TC, TAG, HDL-C, LDL-C and TC/HDL-C ratio.
Measured: Change in concentrations of plasma lipid profile after intake period of 12 weeks
Concentrations of TC/HDL-C Ratio in Plasma
A 250μl aliquot of frozen PFP was defrosted at room temperature using a roller mixer and centrifuged at 500xg for 5 minutes at room temperature (Eppendorf Centrifuge 5415 R, DJBlabcare, United Kingdom). Then the sample was analysed by a RANDOX clinical analyser (RANDOX Daytona+ Analyser, Randox Laboratories Ltd, United Kingdom) for TC/HDL-C ratio.
Measured: Change in concentrations of plasma TC/HDL-C ratio after intake period of 12 weeks
Blood Pressure
Subjects were asked to have a rest for 10 mins before blood pressure detection, and then blood pressure cuff was placed firmly on their upper left arms approximately 2 cm above the elbow with the indicator mark on the cuff over the brachial artery to start measurement. Subjects should put their arms at the level of the heart and should not speak and cross their legs during the measurement. Measurement was performed three times and waited for 2 mins between each reading ((Omron M2 Upper Arm Blood Pressure Monitor, OMRON Healthcare Europe BV, United Kingdom). The average of the three readings was taken to obtain the final result.
Measured: Change in blood pressure after intake period of 12 weeks
Timeline
From the start of the study to its completion; the mark is today. Estimated dates are the sponsor’s plan.
- First posted29 Jun 2017
- Study start16 Feb 2018
- Primary completion30 Nov 2019
- Study completion30 Mar 2021
Resources
- Official record on ClinicalTrials.govThe registry entry this page is built from, with its full change history.
- Posted resultsParticipant flow, outcome measures and adverse events reported by the sponsor.
- Publications (1)Papers the sponsor has linked to this study.
- Other trials for Extracellular Vesicles; Generation and FunctionStudies of the same condition, open and under way.
- What the trial phases meanHow a treatment moves from first tests in people to approval.
More details
For doctors and researchers: how the study is designed, who runs it, and its registry IDs.
Study design
- Study type
- Interventional
- Phase
- Phase N/A
- Primary purpose
- Prevention
- Allocation
- Randomised
- Intervention model
- Crossover
- Masking
- Triple blind
- Who is masked
- Participant, Investigator, Outcomes assessor
- Enrolment
- 42 participants (actual)
Sponsor and investigators
- Lead sponsor
- University of Reading
- Sponsor type
- University, hospital or other
- Collaborators
- Biotechnology and Biological Sciences Research Council
- Responsible party
- Principal investigator, Professor Parveen Yaqoob, MA, DPhil, RNutr, FAfN, University of Reading
- Investigators
- Parveen Yaqoob, MA, DPhil, RNutr, Principal investigator, University of Reading
IDs, dates and publications
- ClinicalTrials.gov ID
- NCT03203512
- Sponsor's protocol ID
- UREC 17/18
- Acronym
- HI-FIVE
- Status last verified
- Nov 2022
- First submitted
- 23 Jun 2017
- Last updated
- 7 Nov 2022
- Results posted
- Yes, on ClinicalTrials.gov
- Data monitoring committee
- Yes
- FDA-regulated drug
- No
- FDA-regulated device
- No
Publications
Bozbas E, Zhou R, Soyama S, Allen-Redpath K, Mitchell JL, Fisk HL, Calder PC, Jones C, Gibbins JM, Fischer R, Hester S, Yaqoob P. Dietary n-3 polyunsaturated fatty acids alter the number, fatty acid profile and coagulatory activity of circulating and platelet-derived extracellular vesicles: a randomized, controlled crossover trial. Am J Clin Nutr. 2024 May;119(5):1175-1186. doi: 10.1016/j.ajcnut.2024.03.008. Epub 2024 Mar 13.
PubMed 38484976
Study at a glance
From the registry record, last updated by the sponsor on 7 Nov 2022. Recruitment status is as the sponsor reported it: confirm with the study team.
- Official title
- Effects of Fish Oil-derived N-3 Polyunsaturated Fatty Acids on the Generation and Functional Activities of Extracellular Vesicles
- ClinicalTrials.gov ID
- NCT03203512
- Acronym / sponsor ID
- HI-FIVE / UREC 17/18
- Keywords
- Fish oil; N-3 polyunsaturated fatty acids
- Study type
- Interventional
- Recruitment status
- Completed
- Enrolment
- 42 participants (actual)
- Lead sponsor
- University of ReadingUniversity, hospital or other
- Collaborators
- Biotechnology and Biological Sciences Research Council
- Study start
- 16 Feb 2018 (actual)
- Primary completion
- 30 Nov 2019 (actual)
- Study completion
- 30 Mar 2021 (actual)
- First posted
- 29 Jun 2017 (actual)
- Results
- Posted 7 Nov 2022
Clinical summary
The research question as the sponsor registered it. This describes what the study plans to test, not what it found.
N-3 polyunsaturated fatty acids (n-3 PUFA), which are abundant in oily fish and fish oils, have been suggested to play a role in reducing the risk of cardiovascular diseases (CVDs) by modifying a wide range of risk factors, such as blood fats, blood clotting, blood vessel function and inflammation. Extracellular vesicles (EVs) are small particles released from various cells when they are activated or damaged. High numbers of EVs in the blood have been associated with a higher risk of CVDs, and it is thought that this is because they carry 'bioactive' components which can affect many processes involved in CVDs. However, very few clinical trials have investigated the relationships between the consumption of n-3 PUFA and circulating EVs. This study aims to investigate the effects of dietary n-3 PUFA on the generation and functional activities of EVs, which would provide new insight into the benefits of n-3 PUFA on cardiovascular health.
Detailed description
The proposed study will be a randomised, double-blind, placebo-controlled crossover intervention. Subjects (40-70y) at moderate CVDs risk will be supplemented with either fish oil (1.8 g/d n-3 PUFA) or placebo (high-oleic safflower oil) for 12 weeks. After a 12-week washout and then cross-over to the other intervention for another 12 weeks. Blood samples will be collected before and after each intervention. A food frequency questionnaire will be administered to assess the subject's habitual intake of n-3 PUFA. Subjects will also be expected to maintain a low consumption of n-3 fatty acids, refrain from the use of all supplements, and maintain their body weight during the study. The dose is based on our previous work, which demonstrated a reduction in numbers of endothelial-derived EVs (EEVs) and a trend for reduced numbers of platelet-derived EVs (PEVs), and a dose at which beneficial effects of n-3 PUFA on plaque stability are reported. The experimental work will follow two main strands. The first strand will examine the influence of n-3 PUFA supplementation on the characteristics and functional activities of total EVs from plasma. The second strand will examine the influence of n-3 PUFA on the generation of PEVs from platelets taken from subjects and stimulated in vitro; the PEVs generated will subsequently be assessed for their composition and functional activity. This experimental design will allow simultaneous investigation of both the composition and activity of total EVs taken directly from blood, and the generation and activity of PEVs. Based on our previous work, 27 subjects are required to detect a 10% reduction in numbers of EVs following fish oil supplementation with a two-sided significance level of 5% and a power of 90%, and 34 subjects are required for a power of 95%. Also based on previous data, 22 subjects would give 95% power to detect 10% differences in thrombus formation and 30 subjects are required to detect a significant effect of n-3 PUFA on platelet aggregation and phosphatidylserine (PS) exposure. Allowing for a 15% dropout rate, and aiming for 95% power based on a 10% reduction in EVs numbers, we will therefore recruit 40 subjects in total.
Study design
- Primary purpose
- Prevention
- Allocation
- Randomised
- Intervention model
- Crossover
- Masking
- Triple blind (participant, investigator, outcomes assessor)
- Number of arms
- 2
| Arm | Type | Receives |
|---|---|---|
| Intervention | Active comparator | Dietary Supplement: Fish oil capsulesFish oil capsules |
| Placebo | Placebo comparator | Dietary Supplement: High-oleic safflower oil capsulesHigh-oleic safflower oil capsules |
Who may qualify
- Ages
- 40 to 70 years
- Sex
- All
- Healthy volunteers
- Accepted
- Age groups
- Adult, Older adult
Key inclusion criteria (5 listed)
- Aged 40-70 years
- Non-smoker
- At moderate risk of cardiovascular diseases
Key exclusion criteria (18 listed)
- BMI: <18.5 kg/m2
- Anaemia (haemoglobin concentration <12.5 g/L in men and<11.5 g/L in women)
- Hyperlipidaemia (total cholesterol concentration >8 mmol/L)
- Diabetes (diagnosed or fasting glucose concentration >7 mmol/L) or other endocrine disorders
- Angina, stroke, or any vascular disease in the past 12 months
Eligibility is decided by the study team after screening; this overview does not establish that a patient qualifies. Full inclusion and exclusion criteria.
Interventions
| Intervention | Type | Arms |
|---|---|---|
| Fish oil capsulesEach serving contains 360mg eicosapentaenoic acid (EPA), 270mg docosahexaenoic acid (DHA) and total supplement is 1.8 g per day n-3 PUFA for 12 weeks | Dietary supplement | Intervention |
| High-oleic safflower oil capsulesHigh-oleic safflower oil capsules for 12 weeks | Dietary supplement | Placebo |
Primary outcome
Numbers of Circulating Total EVs in Platelet-free Plasma (PFP) Detected by Nanoparticle Tracking Analysis (NTA)
Time frame: Change of circulating total EV numbers in PFP detected by NTA after intake period of 12 weeks
Circulating EVs were first isolated to obtain fractions 7~9 by size exclusion chromatography (SEC) using Izon qEV columns (Izon Science Ltd, Oxford, United Kingdom). Fractions were then diluted with PBS to maintain the recommended concentration range of particles (1~10*10^8 vesicles/ml) before being analysed on NanoSight 300 (Malvern, Amesbury, United Kingdom). For each analysis, five videos, each of 60 seconds duration, were captured with the camera level at 13. Data were analysed using the instrument software NTA 3.20, which can identify individual particles and estimate their sizes based on the Stokes-Einstein Equation. Finally, a threshold of 70nm was set for NTA to ensure minimal interference by small lipoproteins.
Numbers of Total Phosphatidylserine Positive EVs (PS+EVs) in Platelet-free Plasma (PFP) Detected by Flow Cytometry (FCM)
Time frame: Change of total PS+EV numbers in PFP detected by FCM after intake period of 12 weeks
A 5μl of PFP was added into nonsticky microcentrifuge tubes (Alpha Laboratories Ltd, Hampshire, United Kingdom), which contained 5μl FcR blocking reagent (Miltenyi Biotec Ltd, Surrey, United Kingdom) and Annexin V buffer and incubated for 15 minutes in the dark at room temperature. Antibodies and isotype-matched controls were then added and samples incubated for another 15 minutes in the dark at room temperature. After incubation, samples were diluted with 200μl Annexin V buffer and transferred into FACS flow tubes (BD Biosciences, Wokingham, United Kingdom), ready to be analysed by FCM. PS+EVs were identified as Annexin V+EVs when triggering on APC fluorescence.
Characterisation of Circulating EVs Subpopulation in PFP Detected by Fluorescence FCM
Time frame: Change in the numbers of circulating EVs subpopulation in PFP by fluorescence FCM after intake period of 12 weeks
A 5μl of PFP was added into nonsticky microcentrifuge tubes (Alpha Laboratories Ltd, Hampshire, United Kingdom), which contained 5μl FcR blocking reagent (Miltenyi Biotec Ltd, Surrey, United Kingdom) and Annexin V buffer and incubated for 15 minutes in the dark at room temperature. Antibodies and isotype-matched controls were then added and samples incubated for another 15 minutes in the dark at room temperature. After incubation, samples were diluted with 200μl Annexin V buffer and transferred into FACS flow tubes (BD Biosciences, Wokingham, United Kingdom), ready to be analysed by FCM. Platelet-derived EVs (PDEVs) were identified as Annexin V+EVs which also stained positive for CD41-PE in APC vs PE quadrant plot, and endothelial-derived EVs (EDEVs) were identified as Annexin V+EVs which also stained positive for CD105- eFluor450 in APC vs PB quadrant plot.
14 secondary outcomes
Pro-thrombotic Activities of Circulating EVs in PFP (Lag Time for Thrombin Generation)
Time frame: Change of pro-thrombotic activities (lag time for thrombin generation)of circulating EVs in PFP after intake period of 12 weeks
Pro-thrombotic Activities of Circulating EVs in PFP (Peak Thrombin Concentration)
Time frame: Change of pro-thrombotic activities (peak thrombin concentration) of circulating EVs in PFP after intake period of 12 weeks
Pro-thrombotic Activities of Circulating EVs in PFP (Time to Peak Thrombin Concentration)
Time frame: Change of pro-thrombotic activities (time to peak thrombin concentration) of circulating EVs in PFP after intake period of 12 weeks
Pro-thrombotic Activities of Circulating EVs in PFP (Velocity Index)
Time frame: Change of pro-thrombotic activities (velocity index) of circulating EVs in PFP after intake period of 12 weeks
Pro-thrombotic Activities of Circulating EVs in PFP (Endogenous Thrombin Potential)
Time frame: Change of pro-thrombotic activities (endogenous thrombin potential) of circulating EVs in PFP after intake period of 12 weeks
Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay
Time frame: Change in ex vivo platelet activation after intake period of 12 weeks
Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay (CRP-XL Log EC50)
Time frame: Change in ex vivo platelet activation after intake period of 12 weeks
Pro-thrombotic Activities of Platelet-derived Extracellular Vesicles (PDEVs) Prepared From the Supernatants of Stimulated Platelets (Endpoint and Maximum of Thrombus Formation)
Time frame: Change in pro-thrombotic activities (endpoint and maximum of thrombus formation)of PEVs prepared from the supernatants of stimulated platelets after intake period of 12 weeks
Pro-thrombotic Activities of Platelet-derived Extracellular Vesicles (PDEVs) Prepared From the Supernatants of Stimulated Platelets (Area Under Curve)
Time frame: Change in pro-thrombotic activities (area under curve) of PEVs prepared from the supernatants of stimulated platelets after intake period of 12 weeks
Circulating EV Total Lipids Analysis
Time frame: Change in total lipids of EVs after intake period of 12 weeks
Plasma Total Phospholipids Analysis
Time frame: Change of plasma total phospholipids after intake period of 12 weeks
Concentrations of Lipid Profile in Plasma
Time frame: Change in concentrations of plasma lipid profile after intake period of 12 weeks
Concentrations of TC/HDL-C Ratio in Plasma
Time frame: Change in concentrations of plasma TC/HDL-C ratio after intake period of 12 weeks
Blood Pressure
Time frame: Change in blood pressure after intake period of 12 weeks
Results and safety
Loading the posted results from ClinicalTrials.gov…
Research links
- Official record on ClinicalTrials.govLast updated by the sponsor 7 Nov 2022.
- Posted resultsParticipant flow, baseline, all outcome measures and adverse events.
- Derived from this study: PubMed 38484976Bozbas E, Zhou R, Soyama S, Allen-Redpath K, Mitchell JL, Fisk HL, Calder PC, Jones C, Gibbins JM, Fischer R, Hester S, Yaqoob P. Dietary n-3 polyunsaturated fatty acids alter the number, fatty acid profile and coagulatory activity of circulating and platelet-derived extracellular vesicles: a randomized, controlled crossover trial. Am J Clin Nutr. 2024 May;119(5):1175-1186. doi: 10.1016/j.ajcnut.2024.03.008. Epub 2024 Mar 13.
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Referral and contact
AzTrials does not enrol participants or take referrals. Contact the study team or a site listed below; they decide who can take part. This study is not recruiting at the moment.
Parveen Yaqoob, MA, DPhil, RNutr principal investigator
University of Reading
1 site in 1 country: see every site and its status.
Questions about this trial
What is this clinical trial studying?
A study testing Fish oil capsules and High-oleic safflower oil capsules in people with Extracellular Vesicles; Generation and Function, run by University of Reading at 1 site in United Kingdom.
The full registered description is in the About this study section and the official record.
Who may be eligible to take part?
The sponsor lists 5 inclusion and 18 exclusion criteria. Eligibility is decided by the study team after screening, so read the full criteria and contact a listed site.
Is this clinical trial currently recruiting?
The record says: finished, as of the sponsor's last update on 7 Nov 2022. Status can change between updates, so confirm with the study contact before making plans.
Where is the study taking place?
The record lists 1 site in United Kingdom. Whether a site is taking part right now should be confirmed with that site. See every site.
What are the possible benefits and risks?
They depend on the study and the treatment, and no study can promise a benefit. Some participants in this study may receive a placebo. The study team explains the known risks during informed consent, before anyone joins; the record itself does not list them.
Are results available for this study?
Yes. Results are posted in the registry (first posted 7 Nov 2022). A summary is in the For doctors view, and the full results are on ClinicalTrials.gov.
How can a doctor refer a patient or contact the research team?
The record lists no contact. The official record names the sponsor, who can be asked directly. AzTrials does not enrol participants or take referrals.
What does the primary outcome measure?
Numbers of Circulating Total EVs in Platelet-free Plasma (PFP) Detected by Nanoparticle Tracking Analysis (NTA) (time frame: Change of circulating total EV numbers in PFP detected by NTA after intake period of 12 weeks). The study lists 3 primary outcomes in all. It is what the study was designed to measure, not a finding.
Where can I read the original study information?
On the ClinicalTrials.gov record for NCT03203512, which also lists 1 linked publication.
Does a listing mean the treatment is approved or proven to work?
No. A listing means the study is registered. It does not show that the treatment is safe, works, or is approved by a regulator.
How often is this information updated?
AzTrials copies changed records from ClinicalTrials.gov every night and reloads the whole registry every week. The sponsor last updated this record on 7 Nov 2022.