Extracellular Vesicles; Generation and Function
Conditions
Keywords
Fish oil; N-3 polyunsaturated fatty acids
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.
Interventions
DIETARY_SUPPLEMENTFish oil capsules
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
DIETARY_SUPPLEMENTHigh-oleic safflower oil capsules
High-oleic safflower oil capsules for 12 weeks
Sponsors
Biotechnology and Biological Sciences Research Council
University of Reading
Study design
Allocation
RANDOMIZED
Intervention model
CROSSOVER
Primary purpose
PREVENTION
Masking
TRIPLE (Subject, Investigator, Outcomes Assessor)
Eligibility
Sex/Gender
ALL
Age
40 Years to 70 Years
Healthy volunteers
Yes
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
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| Numbers of Circulating Total EVs in Platelet-free Plasma (PFP) Detected by Nanoparticle Tracking Analysis (NTA) | 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) | 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 | 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. |
Secondary
| Measure | Time frame | Description |
|---|---|---|
| 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 | 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) |
| 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 | 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) |
| Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay | Change in ex vivo platelet activation after intake period of 12 weeks | 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). |
| Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay (CRP-XL Log EC50) | Change in ex vivo platelet activation after intake period of 12 weeks | 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). |
| Pro-thrombotic Activities of Platelet-derived Extracellular Vesicles (PDEVs) Prepared From the Supernatants of Stimulated Platelets (Endpoint and Maximum of Thrombus Formation) | 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 | 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. |
| 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 | 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) |
| Circulating EV Total Lipids Analysis | Change in total lipids of EVs after intake period of 12 weeks | 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. |
| Plasma Total Phospholipids Analysis | Change of plasma total phospholipids after intake period of 12 weeks | 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. |
| Concentrations of Lipid Profile in Plasma | Change in concentrations of plasma lipid profile after intake period of 12 weeks | 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. |
| Concentrations of TC/HDL-C Ratio in Plasma | Change in concentrations of plasma TC/HDL-C ratio after intake period of 12 weeks | 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. |
| Blood Pressure | Change in blood pressure after intake period of 12 weeks | 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. |
| Pro-thrombotic Activities of Platelet-derived Extracellular Vesicles (PDEVs) Prepared From the Supernatants of Stimulated Platelets (Area Under Curve) | Change in pro-thrombotic activities (area under curve) of PEVs prepared from the supernatants of stimulated platelets after intake period of 12 weeks | 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. |
| 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 | 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) |
| 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 | 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) |
Countries
United Kingdom
Participant flow
Recruitment details
Participants were recruited by using the Hugh Sinclair Unit of Human Nutrition volunteer database, emailing advertisements to staff and students of the University of Reading, to members of local community groups and to staff members in large local organisations and companies between Oct 2017 and March 2019. The first participant was enrolled on 16th Feb 2018 and the last participant was enrolled in March 2019.
Pre-assignment details
Of the 416 participants contacted, a total of 58 participants were screened, of which 14 subjects failed to meet inclusion criteria and 2 declined to participate; 42 participants were enrolled on the trial and 40 completed the study with 2 dropped out before starting.
Participants by arm
| Arm | Count |
|---|---|
| Fish Oil Capsules, Then Placebo Capsules Participants received fish oil capsules three times per day with meals for in the first 12-week period and after 12-week wash-out, they received placebo capsules for a further 12 weeks
Fish oil capsules: each serving contains 360mg eicosapentaenoic acid (EPA), 270mg docosahexaenoic acid (DHA) and total supplement is 1.8 g per day n-3 PUFA
Placebo capsules: high-oleic safflower oil capsules | 20 |
| Placebo Capsules, Then Fish Oil Capsules Participants received placebo capsules three times per day with meals for in the first 12-week period and after 12-week wash-out, they received fish oil capsules for a further 12 weeks.
Fish oil capsules: each serving contains 360mg eicosapentaenoic acid (EPA), 270mg docosahexaenoic acid (DHA) and total supplement is 1.8 g per day n-3 PUFA
Placebo capsules: high-oleic safflower oil capsules | 20 |
| Total | 40 |
Baseline characteristics
| Characteristic | Fish Oil Capsules, Then Placebo Capsules | Placebo Capsules, Then Fish Oil Capsules | Total |
|---|---|---|---|
| 10-yr CVD risk (QRISK2) | 13.0 percentage of risk STANDARD_DEVIATION 0.03 | 12.9 percentage of risk STANDARD_DEVIATION 0.03 | 12.9 percentage of risk STANDARD_DEVIATION 0.03 |
| Age, Continuous | 64.3 years | 65.0 years | 65.0 years |
| Blood Cholesterol Concentration | 5.8 mmol/L STANDARD_DEVIATION 0.9 | 6.2 mmol/L STANDARD_DEVIATION 1.1 | 6.0 mmol/L STANDARD_DEVIATION 1 |
| Blood Glucose Concentration | 5.9 mmol/L STANDARD_DEVIATION 0.8 | 5.6 mmol/L STANDARD_DEVIATION 0.4 | 5.8 mmol/L STANDARD_DEVIATION 0.6 |
| Blood High Density Lipoprotein Concentration | 1.6 mmol/L | 1.6 mmol/L | 1.6 mmol/L |
| Blood Triglyceride Concentration | 1.2 mmol/L | 1.0 mmol/L | 1.2 mmol/L |
| Body Mass Index | 25.0 kg/m^2 STANDARD_DEVIATION 2.8 | 25.7 kg/m^2 STANDARD_DEVIATION 3.1 | 25.4 kg/m^2 STANDARD_DEVIATION 2.9 |
| Diastolic Blood Pressure | 78.5 mmHg STANDARD_DEVIATION 7.3 | 79.1 mmHg STANDARD_DEVIATION 10.7 | 78.8 mmHg STANDARD_DEVIATION 9.1 |
| Race/Ethnicity, Customized White British | 20 participants | 20 participants | 40 participants |
| Region of Enrollment United Kingdom | 20 participants | 20 participants | 40 participants |
| Sex: Female, Male Female | 5 Participants | 11 Participants | 16 Participants |
| Sex: Female, Male Male | 15 Participants | 9 Participants | 24 Participants |
| Systolic Blood Pressure | 130.5 mmHg STANDARD_DEVIATION 11.5 | 137.1 mmHg STANDARD_DEVIATION 15.8 | 134.0 mmHg STANDARD_DEVIATION 14.2 |
Adverse events
| Event type | EG000 affected / at risk | EG001 affected / at risk |
|---|---|---|
| deaths Total, all-cause mortality | 0 / 40 | 0 / 40 |
| other Total, other adverse events | 1 / 40 | 0 / 40 |
| serious Total, serious adverse events | 0 / 40 | 0 / 40 |
Outcome results
Primary
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.
Time frame: Change in the numbers of circulating EVs subpopulation in PFP by fluorescence FCM after intake period of 12 weeks
| Arm | Measure | Group | Value (MEAN) |
|---|---|---|---|
| Fish Oil Capsules | Characterisation of Circulating EVs Subpopulation in PFP Detected by Fluorescence FCM | EDEV numbers | -5.5*10^5 vesicles per ml blood |
| Fish Oil Capsules | Characterisation of Circulating EVs Subpopulation in PFP Detected by Fluorescence FCM | PDEV numbers | -6.0*10^6 vesicles per ml blood |
| Placebo Capsules | Characterisation of Circulating EVs Subpopulation in PFP Detected by Fluorescence FCM | PDEV numbers | -3.0*10^5 vesicles per ml blood |
| Placebo Capsules | Characterisation of Circulating EVs Subpopulation in PFP Detected by Fluorescence FCM | EDEV numbers | 2.9*10^5 vesicles per ml blood |
Comparison: Null hypothesis is that there was no difference in change of PDEV numbers detected by FCM between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on PDEV numbers.p-value: =0.002ANOVA
p-value: <0.001ANOVA
Primary
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.
Time frame: Change of circulating total EV numbers in PFP detected by NTA after intake period of 12 weeks
| Arm | Measure | Value (MEAN) |
|---|---|---|
| Fish Oil Capsules | Numbers of Circulating Total EVs in Platelet-free Plasma (PFP) Detected by Nanoparticle Tracking Analysis (NTA) | -2.9*10^10 vesicles per ml blood |
| Placebo Capsules | Numbers of Circulating Total EVs in Platelet-free Plasma (PFP) Detected by Nanoparticle Tracking Analysis (NTA) | 7.5*10^9 vesicles per ml blood |
Comparison: Null hypothesis is that there was no difference in change of total EV numbers detected by NTA between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on total EV numbers.p-value: <0.001ANOVA
Primary
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.
Time frame: Change of total PS+EV numbers in PFP detected by FCM after intake period of 12 weeks
| Arm | Measure | Value (MEAN) |
|---|---|---|
| Fish Oil Capsules | Numbers of Total Phosphatidylserine Positive EVs (PS+EVs) in Platelet-free Plasma (PFP) Detected by Flow Cytometry (FCM) | -1.2*10^7 vesicles per ml blood |
| Placebo Capsules | Numbers of Total Phosphatidylserine Positive EVs (PS+EVs) in Platelet-free Plasma (PFP) Detected by Flow Cytometry (FCM) | -3.0*10^5 vesicles per ml blood |
Comparison: Null hypothesis is that there was no difference in change of total PS+EV numbers detected by FCM between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on total PS+EV numbers.p-value: =0.001ANOVA
Secondary
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.
Time frame: Change in blood pressure after intake period of 12 weeks
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Fish Oil Capsules | Blood Pressure | SBP | -6.7 mmHg | Standard Error 1 |
| Fish Oil Capsules | Blood Pressure | DBP | -2.9 mmHg | Standard Error 0.7 |
| Placebo Capsules | Blood Pressure | SBP | 3.4 mmHg | Standard Error 1.7 |
| Placebo Capsules | Blood Pressure | DBP | 0.9 mmHg | Standard Error 0.9 |
Comparison: Null hypothesis is that there was no difference in change of SBP between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on SBP.p-value: <0.001ANOVA
Comparison: Null hypothesis is that there was no difference in change of DBP between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on DBP.p-value: =0.002ANOVA
Secondary
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.
Time frame: Change in total lipids of EVs after intake period of 12 weeks
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Fish Oil Capsules | Circulating EV Total Lipids Analysis | DHA | 1.0 g/100g total fatty acids | Standard Error 0.1 |
| Fish Oil Capsules | Circulating EV Total Lipids Analysis | Total n-3 PUFA | 2.5 g/100g total fatty acids | Standard Error 0.3 |
| Fish Oil Capsules | Circulating EV Total Lipids Analysis | Oleic acid | -1.9 g/100g total fatty acids | Standard Error 0.4 |
| Fish Oil Capsules | Circulating EV Total Lipids Analysis | AA | -0.4 g/100g total fatty acids | Standard Error 0.1 |
| Fish Oil Capsules | Circulating EV Total Lipids Analysis | EPA | 0.9 g/100g total fatty acids | Standard Error 0.1 |
| Fish Oil Capsules | Circulating EV Total Lipids Analysis | Total MUFA | -2.2 g/100g total fatty acids | Standard Error 0.4 |
| Fish Oil Capsules | Circulating EV Total Lipids Analysis | DPA | 0.2 g/100g total fatty acids | Standard Error 0.02 |
| Placebo Capsules | Circulating EV Total Lipids Analysis | Total MUFA | 0.2 g/100g total fatty acids | Standard Error 0.8 |
| Placebo Capsules | Circulating EV Total Lipids Analysis | Oleic acid | 0.4 g/100g total fatty acids | Standard Error 0.7 |
| Placebo Capsules | Circulating EV Total Lipids Analysis | EPA | -0.1 g/100g total fatty acids | Standard Error 0.1 |
| Placebo Capsules | Circulating EV Total Lipids Analysis | DHA | -0.02 g/100g total fatty acids | Standard Error 0.1 |
| Placebo Capsules | Circulating EV Total Lipids Analysis | DPA | 0.04 g/100g total fatty acids | Standard Error 0.03 |
| Placebo Capsules | Circulating EV Total Lipids Analysis | AA | 0.1 g/100g total fatty acids | Standard Error 0.1 |
| Placebo Capsules | Circulating EV Total Lipids Analysis | Total n-3 PUFA | 0.2 g/100g total fatty acids | Standard Error 0.2 |
Comparison: Null hypothesis is that there was no difference in change of EPA in circulating EV total lipids between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on EPA in circulating EV total lipids.p-value: <0.001ANOVA
Comparison: Null hypothesis is that there was no difference in change of DHA in circulating EV total lipids between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on DHA in circulating EV total lipids.p-value: <0.001ANOVA
Comparison: Null hypothesis is that there was no difference in change of oleic acid in circulating EV total lipids between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on oleic acid in circulating EV total lipids.p-value: =0.011ANOVA
Comparison: Null hypothesis is that there was no difference in change of AA in circulating EV total lipids between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on AA in circulating EV total lipids.p-value: <0.001ANOVA
Comparison: Null hypothesis is that there was no difference in change of total n-3 PUFA in circulating EV total lipids between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on total n-3 PUFA in circulating EV total lipids.p-value: <0.001ANOVA
Comparison: Null hypothesis is that there was no difference in change of total MUFA in circulating EV total lipids between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on total MUFA in circulating EV total lipids.p-value: =0.013ANOVA
Secondary
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.
Time frame: Change in concentrations of plasma lipid profile after intake period of 12 weeks
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Fish Oil Capsules | Concentrations of Lipid Profile in Plasma | HDL-C | 0.06 mmol/L | Standard Error 0.02 |
| Fish Oil Capsules | Concentrations of Lipid Profile in Plasma | TAG | -0.1 mmol/L | Standard Error 0.04 |
| Fish Oil Capsules | Concentrations of Lipid Profile in Plasma | LDL-C | 0.1 mmol/L | Standard Error 0.06 |
| Fish Oil Capsules | Concentrations of Lipid Profile in Plasma | TC/HDL-C ratio | -0.04 mmol/L | Standard Error 0.05 |
| Fish Oil Capsules | Concentrations of Lipid Profile in Plasma | TC | 0.1 mmol/L | Standard Error 0.07 |
| Placebo Capsules | Concentrations of Lipid Profile in Plasma | TC/HDL-C ratio | -0.1 mmol/L | Standard Error 0.04 |
| Placebo Capsules | Concentrations of Lipid Profile in Plasma | TC | -0.02 mmol/L | Standard Error 0.05 |
| Placebo Capsules | Concentrations of Lipid Profile in Plasma | HDL-C | 0.04 mmol/L | Standard Error 0.02 |
| Placebo Capsules | Concentrations of Lipid Profile in Plasma | TAG | 0.03 mmol/L | Standard Error 0.04 |
| Placebo Capsules | Concentrations of Lipid Profile in Plasma | LDL-C | -0.1 mmol/L | Standard Error 0.04 |
Comparison: Null hypothesis is that there was no difference in change of plasma TAG between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on plasma TAG.p-value: =0.016ANOVA
Comparison: Null hypothesis is that there was no difference in change of plasma LDL-C between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on plasma LDL-C.p-value: =0.014ANOVA
Comparison: Null hypothesis is that there was no difference in change of plasma TC between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on plasma TC.p-value: =0.077ANOVA
Comparison: Null hypothesis is that there was no difference in change of plasma HDL-C between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on plasma HDL-C.p-value: =0.379ANOVA
Secondary
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.
Time frame: Change in concentrations of plasma TC/HDL-C ratio after intake period of 12 weeks
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Fish Oil Capsules | Concentrations of TC/HDL-C Ratio in Plasma | -0.04 ratio | Standard Error 0.05 |
| Placebo Capsules | Concentrations of TC/HDL-C Ratio in Plasma | -0.1 ratio | Standard Error 0.04 |
Comparison: Null hypothesis is that there was no difference in change of plasma TC/HDL-C ratio between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on plasma TC/HDL-C ratio.p-value: =0.285ANOVA
Secondary
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).
Time frame: Change in ex vivo platelet activation after intake period of 12 weeks
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Fish Oil Capsules | Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay | TRAP-6 logEC50 | 0.20 log(M) | Standard Error 0.18 |
| Fish Oil Capsules | Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay | Epinephrine logEC50 | -0.36 log(M) | Standard Error 0.24 |
| Fish Oil Capsules | Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay | U46619 logEC50 | 0.14 log(M) | Standard Error 0.3 |
| Fish Oil Capsules | Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay | ADP logEC50 | -0.17 log(M) | Standard Error 0.29 |
| Placebo Capsules | Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay | U46619 logEC50 | -0.37 log(M) | Standard Error 0.25 |
| Placebo Capsules | Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay | Epinephrine logEC50 | 0.16 log(M) | Standard Error 0.17 |
| Placebo Capsules | Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay | TRAP-6 logEC50 | 0.058 log(M) | Standard Error 0.16 |
| Placebo Capsules | Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay | ADP logEC50 | -0.078 log(M) | Standard Error 0.11 |
Comparison: Null hypothesis is that there was no difference in change of platelet function in affecting LogEC50 values in response to ADP between fish oil and placebo capsules. Comparisons of this parameter after each intervention were drawn using 2-way ANOVA with the Turkey multiple comparisons test.p-value: >0.05ANOVA
Comparison: Null hypothesis is that there was no difference in change of platelet function in affecting LogEC50 values in response to epinephrine between fish oil and placebo capsules. Comparisons of this parameter after each intervention were drawn using 2-way ANOVA with the Turkey multiple comparisons test.p-value: >0.05ANOVA
Comparison: Null hypothesis is that there was no difference in change of platelet function in affecting LogEC50 values in response to TRAP-6 between fish oil and placebo capsules. Comparisons of this parameter after each intervention were drawn using 2-way ANOVA with the Turkey multiple comparisons test.p-value: >0.05ANOVA
Comparison: Null hypothesis is that there was no difference in change of platelet function in affecting LogEC50 values in response to U46619 between fish oil and placebo capsules. Comparisons of this parameter after each intervention were drawn using 2-way ANOVA with the Turkey multiple comparisons test.p-value: >0.05ANOVA
Secondary
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).
Time frame: Change in ex vivo platelet activation after intake period of 12 weeks
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Fish Oil Capsules | Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay (CRP-XL Log EC50) | -0.39 log (mg/ml) | Standard Error 0.2 |
| Placebo Capsules | Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay (CRP-XL Log EC50) | 0.032 log (mg/ml) | Standard Error 0.27 |
Comparison: Null hypothesis is that there was no difference in change of platelet function in affecting LogEC50 values in response to CRP-XL between fish oil and placebo capsules. Comparisons of this parameter after each intervention were drawn using 2-way ANOVA with the Turkey multiple comparisons test.p-value: >0.05ANOVA
Secondary
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.
Time frame: Change of plasma total phospholipids after intake period of 12 weeks
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Fish Oil Capsules | Plasma Total Phospholipids Analysis | DPA | 0.2 g/100g total fatty acids | Standard Error 0.04 |
| Fish Oil Capsules | Plasma Total Phospholipids Analysis | Linoleic acid | -2.0 g/100g total fatty acids | Standard Error 0.4 |
| Fish Oil Capsules | Plasma Total Phospholipids Analysis | EPA | 2.9 g/100g total fatty acids | Standard Error 0.2 |
| Fish Oil Capsules | Plasma Total Phospholipids Analysis | DGLA | -0.8 g/100g total fatty acids | Standard Error 0.1 |
| Fish Oil Capsules | Plasma Total Phospholipids Analysis | Total n-3 PUFA | 5.6 g/100g total fatty acids | Standard Error 0.4 |
| Fish Oil Capsules | Plasma Total Phospholipids Analysis | Total MUFA | -1.0 g/100g total fatty acids | Standard Error 0.4 |
| Fish Oil Capsules | Plasma Total Phospholipids Analysis | DHA | 2.4 g/100g total fatty acids | Standard Error 0.2 |
| Placebo Capsules | Plasma Total Phospholipids Analysis | Total MUFA | -0.1 g/100g total fatty acids | Standard Error 0.4 |
| Placebo Capsules | Plasma Total Phospholipids Analysis | EPA | -0.1 g/100g total fatty acids | Standard Error 0.1 |
| Placebo Capsules | Plasma Total Phospholipids Analysis | DHA | -0.1 g/100g total fatty acids | Standard Error 0.1 |
| Placebo Capsules | Plasma Total Phospholipids Analysis | DPA | -0.1 g/100g total fatty acids | Standard Error 0.02 |
| Placebo Capsules | Plasma Total Phospholipids Analysis | Total n-3 PUFA | -0.2 g/100g total fatty acids | Standard Error 0.1 |
| Placebo Capsules | Plasma Total Phospholipids Analysis | Linoleic acid | 0.4 g/100g total fatty acids | Standard Error 0.2 |
| Placebo Capsules | Plasma Total Phospholipids Analysis | DGLA | 0.04 g/100g total fatty acids | Standard Error 0.1 |
Comparison: Null hypothesis is that there was no difference in change of EPA in plasma total phospholipids between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on EPA in plasma total phospholipids.p-value: <0.001ANOVA
Comparison: Null hypothesis is that there was no difference in change of DHA in plasma total phospholipids between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on DHA in plasma total phospholipids.p-value: <0.001ANOVA
Comparison: Null hypothesis is that there was no difference in change of DPA in plasma total phospholipids between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on DPA in plasma total phospholipids.p-value: <0.001ANOVA
Comparison: Null hypothesis is that there was no difference in change of linoleic acid in plasma total phospholipids between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on linoleic acid in plasma total phospholipids.p-value: <0.001ANOVA
Comparison: Null hypothesis is that there was no difference in change of dihomo-γ-linolenic acid (DGLA) in plasma total phospholipids between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on DGLA in plasma total phospholipids.p-value: <0.001ANOVA
Comparison: Null hypothesis is that there was no difference in change of AA in plasma total phospholipids between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on AA in plasma total phospholipids.p-value: <0.001ANOVA
Comparison: Null hypothesis is that there was no difference in change of total n-3 PUFA in plasma total phospholipids between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on total n-3 PUFA in plasma total phospholipids.p-value: <0.001ANOVA
Comparison: Null hypothesis is that there was no difference in change of total n-6 PUFA in plasma total phospholipids between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on total n-6 PUFA in plasma total phospholipids.p-value: <0.001ANOVA
Secondary
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)
Time frame: Change of pro-thrombotic activities (endogenous thrombin potential) of circulating EVs in PFP after intake period of 12 weeks
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Fish Oil Capsules | Pro-thrombotic Activities of Circulating EVs in PFP (Endogenous Thrombin Potential) | -437.2 nM thrombin × min | Standard Error 70.1 |
| Placebo Capsules | Pro-thrombotic Activities of Circulating EVs in PFP (Endogenous Thrombin Potential) | -60.8 nM thrombin × min | Standard Error 66.2 |
Comparison: Null hypothesis is that there was no difference in change of EV thrombogenicity in affecting TF-dependent endogenous thrombin potential (ETP) between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on EV thrombogenicity.p-value: <0.001ANOVA
Secondary
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)
Time frame: Change of pro-thrombotic activities (lag time for thrombin generation)of circulating EVs in PFP after intake period of 12 weeks
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Fish Oil Capsules | Pro-thrombotic Activities of Circulating EVs in PFP (Lag Time for Thrombin Generation) | 2.5 min | Standard Error 0.5 |
| Placebo Capsules | Pro-thrombotic Activities of Circulating EVs in PFP (Lag Time for Thrombin Generation) | 1.2 min | Standard Error 0.5 |
Comparison: Null hypothesis is that there was no difference in change of EV thrombogenicity in affecting lag time for TF-dependent thrombin generation between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on EV thrombogenicity.p-value: <0.001ANOVA
Secondary
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)
Time frame: Change of pro-thrombotic activities (peak thrombin concentration) of circulating EVs in PFP after intake period of 12 weeks
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Fish Oil Capsules | Pro-thrombotic Activities of Circulating EVs in PFP (Peak Thrombin Concentration) | -16.2 nM | Standard Error 2.5 |
| Placebo Capsules | Pro-thrombotic Activities of Circulating EVs in PFP (Peak Thrombin Concentration) | -1.6 nM | Standard Error 2 |
Comparison: Null hypothesis is that there was no difference in change of EV thrombogenicity in affecting TF-dependent thrombin peak concentration between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on EV thrombogenicity.p-value: <0.001ANOVA
Secondary
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)
Time frame: Change of pro-thrombotic activities (time to peak thrombin concentration) of circulating EVs in PFP after intake period of 12 weeks
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Fish Oil Capsules | Pro-thrombotic Activities of Circulating EVs in PFP (Time to Peak Thrombin Concentration) | 4.6 min | Standard Error 1 |
| Placebo Capsules | Pro-thrombotic Activities of Circulating EVs in PFP (Time to Peak Thrombin Concentration) | 0.3 min | Standard Error 0.7 |
Comparison: Null hypothesis is that there was no difference in change of EV thrombogenicity in affecting the time to reach peak TF-dependent thrombin concentration between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on EV thrombogenicity.p-value: <0.001ANOVA
Secondary
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)
Time frame: Change of pro-thrombotic activities (velocity index) of circulating EVs in PFP after intake period of 12 weeks
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Fish Oil Capsules | Pro-thrombotic Activities of Circulating EVs in PFP (Velocity Index) | -1.1 nM/min | Standard Error 0.4 |
| Placebo Capsules | Pro-thrombotic Activities of Circulating EVs in PFP (Velocity Index) | -0.2 nM/min | Standard Error 0.2 |
Comparison: Null hypothesis is that there was no difference in change of EV thrombogenicity in affecting TF-dependent velocity index between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on EV thrombogenicity.p-value: =0.015ANOVA
Secondary
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.
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
| Arm | Measure | Value (MEAN) | Dispersion |
|---|---|---|---|
| Fish Oil Capsules | Pro-thrombotic Activities of Platelet-derived Extracellular Vesicles (PDEVs) Prepared From the Supernatants of Stimulated Platelets (Area Under Curve) | -945 FU x min | Standard Error 4843 |
| Placebo Capsules | Pro-thrombotic Activities of Platelet-derived Extracellular Vesicles (PDEVs) Prepared From the Supernatants of Stimulated Platelets (Area Under Curve) | 2502 FU x min | Standard Error 3587 |
Comparison: Null hypothesis is that there was no difference in change of pro-thrombotic activity of PDEVs in affecting area under curve for ex vivo thrombus formation between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on the pro-thrombotic activity of PDEVs.p-value: >0.05ANOVA
Secondary
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.
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
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Fish Oil Capsules | Pro-thrombotic Activities of Platelet-derived Extracellular Vesicles (PDEVs) Prepared From the Supernatants of Stimulated Platelets (Endpoint and Maximum of Thrombus Formation) | Endpoint of thrombu formation | -14.3 FU | Standard Error 13.4 |
| Fish Oil Capsules | Pro-thrombotic Activities of Platelet-derived Extracellular Vesicles (PDEVs) Prepared From the Supernatants of Stimulated Platelets (Endpoint and Maximum of Thrombus Formation) | Maximum of thrombus formation | -1.3 FU | Standard Error 16.1 |
| Placebo Capsules | Pro-thrombotic Activities of Platelet-derived Extracellular Vesicles (PDEVs) Prepared From the Supernatants of Stimulated Platelets (Endpoint and Maximum of Thrombus Formation) | Endpoint of thrombu formation | 9.9 FU | Standard Error 9.8 |
| Placebo Capsules | Pro-thrombotic Activities of Platelet-derived Extracellular Vesicles (PDEVs) Prepared From the Supernatants of Stimulated Platelets (Endpoint and Maximum of Thrombus Formation) | Maximum of thrombus formation | 20.6 FU | Standard Error 11.1 |
Comparison: Null hypothesis is that there was no difference in change of pro-thrombotic activity of PDEVs in affecting endpoint for ex vivo thrombus formation between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on the pro-thrombotic activity of PDEVs.p-value: >0.05ANOVA
Comparison: Null hypothesis is that there was no difference in change of pro-thrombotic activity of PDEVs in affectingamximum for ex vivo thrombus formation between fish oil and placebo capsules. A general linear model with fixed factors of treatment and period was conducted to determine the differences in the effect of two treatments and two periods on the pro-thrombotic activity of PDEVs.p-value: >0.05ANOVA