Clinical trial NCT03203512: Fish Oil-derived N-3 Polyunsaturated Fatty Acids and Extracellular Vesicles
Official title: Effects of Fish Oil-derived N-3 Polyunsaturated Fatty Acids on the Generation and Functional Activities of Extracellular Vesicles
Study type: INTERVENTIONAL
Overall status: COMPLETED
Phase: NA
Conditions: Extracellular Vesicles; Generation and Function
Keywords: Fish oil; N-3 polyunsaturated fatty acids
Lead sponsor: University of Reading
Sponsor class: OTHER
Collaborators: name: Biotechnology and Biological Sciences Research Council
First posted: 2017-06-29
Enrollment: 42
Sex: ALL
Minimum age (years): 40
Maximum age (years): 70
Standard ages: ADULT
OLDER_ADULT
Accepts healthy volunteers: yes
Allocation: RANDOMIZED
Intervention model: CROSSOVER
Primary purpose: PREVENTION

Brief 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. 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.

Eligibility criteria: Inclusion Criteria:

* 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

Exclusion Criteria:

* 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
* 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

Primary outcomes:
measure: Numbers of Circulating Total EVs in Platelet-free Plasma (PFP) Detected by Nanoparticle Tracking Analysis (NTA)
timeFrame: Change of circulating total EV numbers in PFP detected by NTA after intake period of 12 weeks
measure: Numbers of Total Phosphatidylserine Positive EVs (PS+EVs) in Platelet-free Plasma (PFP) Detected by Flow Cytometry (FCM)
timeFrame: Change of total PS+EV numbers in PFP detected by FCM after intake period of 12 weeks
measure: Characterisation of Circulating EVs Subpopulation in PFP Detected by Fluorescence FCM
timeFrame: Change in the numbers of circulating EVs subpopulation in PFP by fluorescence FCM after intake period of 12 weeks
Secondary outcomes:
measure: Pro-thrombotic Activities of Circulating EVs in PFP (Lag Time for Thrombin Generation)
timeFrame: Change of pro-thrombotic activities (lag time for thrombin generation)of circulating EVs in PFP after intake period of 12 weeks
measure: Pro-thrombotic Activities of Circulating EVs in PFP (Peak Thrombin Concentration)
timeFrame: Change of pro-thrombotic activities (peak thrombin concentration) of circulating EVs in PFP after intake period of 12 weeks
measure: Pro-thrombotic Activities of Circulating EVs in PFP (Time to Peak Thrombin Concentration)
timeFrame: Change of pro-thrombotic activities (time to peak thrombin concentration) of circulating EVs in PFP after intake period of 12 weeks
measure: Pro-thrombotic Activities of Circulating EVs in PFP (Velocity Index)
timeFrame: Change of pro-thrombotic activities (velocity index) of circulating EVs in PFP after intake period of 12 weeks
measure: Pro-thrombotic Activities of Circulating EVs in PFP (Endogenous Thrombin Potential)
timeFrame: Change of pro-thrombotic activities (endogenous thrombin potential) of circulating EVs in PFP after intake period of 12 weeks
measure: Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay
timeFrame: Change in ex vivo platelet activation after intake period of 12 weeks
measure: Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay (CRP-XL Log EC50)
timeFrame: Change in ex vivo platelet activation after intake period of 12 weeks
measure: Pro-thrombotic Activities of Platelet-derived Extracellular Vesicles (PDEVs) Prepared From the Supernatants of Stimulated Platelets (Endpoint and Maximum of Thrombus Formation)
timeFrame: 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
measure: Pro-thrombotic Activities of Platelet-derived Extracellular Vesicles (PDEVs) Prepared From the Supernatants of Stimulated Platelets (Area Under Curve)
timeFrame: Change in pro-thrombotic activities (area under curve) of PEVs prepared from the supernatants of stimulated platelets after intake period of 12 weeks
measure: Circulating EV Total Lipids Analysis
timeFrame: Change in total lipids of EVs after intake period of 12 weeks
measure: Plasma Total Phospholipids Analysis
timeFrame: Change of plasma total phospholipids after intake period of 12 weeks
measure: Concentrations of Lipid Profile in Plasma
timeFrame: Change in concentrations of plasma lipid profile after intake period of 12 weeks
measure: Concentrations of TC/HDL-C Ratio in Plasma
timeFrame: Change in concentrations of plasma TC/HDL-C ratio after intake period of 12 weeks
measure: Blood Pressure
timeFrame: Change in blood pressure after intake period of 12 weeks
Other outcomes:
Locations:
city: Reading
country: United Kingdom
facility: University of Reading
Study officials:
affiliation: University of Reading
name: Parveen Yaqoob, MA, DPhil, RNutr
role: PRINCIPAL_INVESTIGATOR
References: