Fish Oil-derived N-3 Polyunsaturated Fatty Acids and Extracellular Vesicles

NCT03203512 | Completed Results DMC

• 1 other identifier: UREC 17/18
• Study Type: interventional
• Target: 42
• Locations: 1 country
• Sites: 1

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.

Conditions

Extracellular Vesicles; Generation and Function

Keywords

Fish oil; N-3 polyunsaturated fatty acids

Outcome Measures

Primary Outcomes (3)

• 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.

  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.

  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.

  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)

  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)

  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)

  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)

  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)

  Change of pro-thrombotic activities (endogenous thrombin potential) of circulating EVs in PFP after intake period of 12 weeks

Study Arms (2)

Intervention

ACTIVE COMPARATOR

Fish oil capsules

Dietary Supplement: Fish oil capsules

Placebo

PLACEBO COMPARATOR

High-oleic safflower oil capsules

Dietary Supplement: High-oleic safflower oil capsules

Interventions

Fish oil capsules DIETARY_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

Intervention

High-oleic safflower oil capsules DIETARY_SUPPLEMENT

High-oleic safflower oil capsules for 12 weeks

Placebo

Eligibility Criteria

• Age: 40 Years - 70 Years
• Sex: all
• Healthy Volunteers: Yes
• Age Groups: Adult (18-64), Older Adult (65+)

You may qualify if:

• 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

You may not qualify if:

• 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

Sponsors & Collaborators

• University of Reading: lead
• Biotechnology and Biological Sciences Research Council: collaborator

Study Sites (1)

University of Reading

Reading, RG6 6AP, United Kingdom

Location

Related Publications (1)

• 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.

  PMID: 38484976 DERIVED

MeSH Terms

Interventions

Fish Oils

Intervention Hierarchy (Ancestors)

Oils; Lipids

Limitations and Caveats

The collection, isolation and characterization of EVs are still undergoing standardization, so although the protocol for the characterization of EVs applied in this study was refined by combining NTA and fluorescence FCM, circulating EVs measured in this study did not represent all types of EVs as neither NTA nor FCM was able to provide a full picture of the EV population.

Results Point of Contact

• Title: Professor Parveen Yaqoob
• Organization: University of Reading

p.yaqoob@reading.ac.uk

+443787113

Study Officials

• Parveen Yaqoob, MA, DPhil, RNutr

  University of Reading

  PRINCIPAL INVESTIGATOR

Publication Agreements

• PI is Sponsor Employee: No
• Restrictive Agreement: No

Study Design

• Study Type: interventional
• Phase: not applicable
• Allocation: RANDOMIZED
• Masking: TRIPLE
• Who Masked: PARTICIPANT, INVESTIGATOR, OUTCOMES ASSESSOR
• Purpose: PREVENTION
• Intervention Model: CROSSOVER
• Sponsor Type: OTHER
• Responsible Party: PRINCIPAL INVESTIGATOR
• PI Title: Professor Parveen Yaqoob

Study Record Dates

• First Submitted: June 23, 2017
• First Posted: June 29, 2017
• Study Start: February 16, 2018
• Primary Completion: November 30, 2019
• Study Completion: March 30, 2021
• Last Updated: November 7, 2022
• Results First Posted: November 7, 2022

Record last verified: 2022-11

Locations

GB (1)