The Effect of Ethyl Eicosapentaenoic Acid on Circulating Low-density Lipoproteins and Plasma Lipid Metabolism in Healthy Volunteers
Phase: NA
Conditions: Atherosclerosis, Low-density Lipoproteins Aggregation Susceptibility, Low-density Lipoprotein Lipid Composition, Cardiovascular Diseases
Sponsor: Wihuri Research Institute

E-EPA and LDL 1 /15 
 
Translated from Finnish 4.2.2025 
Document last updated 17.6.2025 
CLINICAL RESEARCH STUDY PLAN Version 3/21.10.2019 
The Effect of E-EPA on Circulating LDL Particles and Plasma Lipid Metabolism-Regulating 
Proteins 
Abbreviated Title: E-EPA and LDL 
Clinicaltrials.gov registration: NCT04152291 
Partner Organizations: 
1. Wihuri Research Institute, Helsinki, Finland 
2. University of Helsinki, HiLipid Lipidomics Unit, HiLife, Helsinki, Finland 
3. Minerva Foundation Institute for Medical Research, Helsinki, Finland 
4. Karolinska Institute, Stockholm, Sweden  
Research Team Members: 
• 
Professor Petri T. Kovanen, MD, Principal Investigator1 
• 
Professor Katariina Öörni1,2 
• 
MSc Lauri Äikäs1,2 
• 
Associate Professor, PhD, Reijo Käkelä2 
• 
Associate Professor, PhD, Matti Jauhiainen3 
• 
Bioanalyst Maija Atuegwu1 
• 
Professor Magnus Bäck4  
• 
PhD Hildur Arnardottir4  
Contact Person: 
Professor Katariina Öörni 
Wihuri Research Institute, Biomedicum Helsinki, Haartmaninkatu 8, 00290 Helsinki 
kati.oorni@wri.fi 
Phone: +358 40 702 3711, +358 9 681 411 
 
 
 
 
 
 
 
 
 

E-EPA and LDL 2 /15 
 
I. BACKGROUND 
Atherosclerosis is a condition in which cholesterol and other lipids transported by low-density 
lipoprotein (LDL) particles accumulate in the inner layer (intima) of the arterial wall (Boren & 
Williams, 2016). Cells within the intima secrete various proteases, lipases, and oxidative agents, 
which can modify LDL particles. These modifications make LDL particles unstable, leading to 
aggregation (figure 1). Aggregated LDL particles bind tightly to the extracellular matrix of the 
arterial wall (Öörni et al, 1997), further promoting LDL accumulation. Macrophages within the 
intima can internalize modified LDL particles, triggering an inflammatory response that drives 
atherosclerosis progression and results in a chronic inflammatory state in the arterial wall (Boren & 
Williams, 2016).  
 
 
Figure 1. Aggregated LDL-particles in human atherosclerotic plaque.  
3D-reconstruction of a human atherosclerotic plaque lipid accumulation  based on electron 
microscopy (Lehti et al, 2018). 
At the Wihuri Research Institute, a method has been developed to measure LDL particle 
aggregation propensity (Ruuth et al., 2018). Previous studies demonstrated significant inter-
individual variability in LDL aggregation susceptibility, with findings indicating that aggregation-
prone LDL predicts future cardiovascular deaths (Ruuth et al., 2018). Our research has also 
established that the lipid composition of LDL particles regulates their aggregation susceptibility, 
with LDL particles rich in sphingomyelin being particularly prone to aggregation. 
The REDUCE-IT study (Bhatt et al, 2019) investigated the effect of eicosapentaenoic acid (EPA; 
20:5 n-3) ethyl ester (E-EPA) on blood lipoproteins and cardiovascular disease outcomes. This 
groundbreaking study demonstrated that a daily dose of 4 g E-EPA significantly reduced 
cardiovascular events over a 4.9-year follow-up period. 
Previous research at Wihuri Research Institute has shown that dietary modifications affecting LDL 
lipid composition can influence its aggregation propensity (Ruuth et al., 2018). The introduction of 
plant oils into the diet has been associated with decreased sphingomyelin levels in LDL particles 
and a significant reduction in aggregation susceptibility. Additionally, intake of alpha-linolenic acid 
(ALA, 20:3 n-3) (10g/day) or fatty fish (containing approximately 1g of n-3 fatty acids, including 

E-EPA and LDL 3 /15 
 
EPA and docosahexaenoic acid (DHA, 22:6 n-3)) led to changes in LDL lipid composition. 
However, as these modifications were relatively minor, no significant changes in aggregation 
susceptibility were observed ((Manninen et al, 2018) and unpublished results).
 
II. OBJECTIVES 
This research plan builds upon our previous studies investigating LDL particle modifications in the 
early stages of atherosclerosis and plaque formation. The primary objective of this study is to 
determine whether dietary supplementation with E-EPA influences LDL aggregation propensity, 
particle binding to proteoglycans, or LDL composition. Additionally, plasma or serum samples will 
be analysed for factors associated with lipid metabolism. 
 
III. STUDY IMPLEMENTATION AND TIMELINE 
Study Population 
The study aims to recruit 40-70 normolipidemic healthy individuals aged 18-65 years. The study 
will last for 35 days. The E-EPA-supplementation will last for 28 days, followed by a seven-day 
washout period. Blood samples are taken before (day 0), during day (day 7), after (day 28) the E-
EPA-supplementation and after the washout period (day 35). At day 0 visit, participants will 
undergo a health examination (inclusive an interview, health questionnaire, anthropometric 
measurements, blood pressure measurement) and blood withdrawal. The blood will undergo 
laboratory analyses including complete blood count and key metabolic biomarkers (total 
cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, apoB-100, apoA-I, and Lp(a), vitamin 
D3, and blood glucose). 
Individuals with exceptionally low LDL aggregation susceptibility will be excluded, as further 
investigation of their LDL particles would not be meaningful in the present context. Additional 
exclusion criteria include pregnancy, known blood coagulation disorders, LDL cholesterol >5 
mmol/L, triglycerides >3 mmol/L, regular use of anti-inflammatory drugs, and fish allergy. 
Supplementation 
We will follow the dosing model used in the REDUCE-IT (Bhatt et al., 2019) study, administering 
2 grams of pure E-EPA twice daily with meals. Pure E-EPA is not commercially available in 
Finland, and thus a supplement containing 650 mg of E-EPA and 12.5 µg of vitamin D3 per capsule 
will be employed. The supplement will be provided as three capsules to be consumed in the 
morning and three in the evening, providing a daily total of 3.9 g of E-EPA and 75 µg of vitamin 
D3. 
 
Based on previous studies, vitamin D3 is not expected to influence the primary outcome variables. 
The internationally accepted maximum tolerable daily intake of vitamin D3 for long-term use is 100 
µg. In Finland, the estimated daily dietary intake of vitamin D3 ranges between 10-20 µg, 
suggesting that overall vitamin D3-dosage will remain well within safe limits even with the 
increased intake provided by the supplement. Notably, the participants will be instructed not to use 
other (commercial) vitamin D supplements during the study. 

E-EPA and LDL 4 /15 
 
Research Methods 
Blood samples will be analyzed for key parameters related to lipid metabolism and cardiovascular 
health, including total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, apoB-100, 
apoA-I, and Lp(a). Additionally, vitamin D3 and blood glucose levels will be measured. All blood 
samples will be processed under anonymized identifiers. 
 
Plasma lipoproteins will be isolated via ultracentrifugation. The LDL particle aggregation 
propensity will be determined using a method developed in our laboratory (Figure 2), employing a 
dynamic light scattering-based microplate reader (Dyna Pro Plate Reader II). Furthermore, the lipid 
composition of lipoprotein samples will be analyzed using a method optimized for LDL particle 
analysis at the University of Helsinki’s HiLipid lipidomics unit in collaboration with Associate 
Professor Reijo Käkelä. The ability of LDL particles to bind to proteoglycans will be assessed by 
measuring plasma lipoprotein binding to immobilized proteoglycans isolated from the human aorta, 
a method developed in our laboratory (Ahmed et al, 2018). 
 
 
Phospholipid transfer protein (PLTP) and cholesterol ester transfer protein (CETP) activities will be 
assessed using radiometric assays (Groener et al, 1986; Jauhiainen & Ehnholm, 2005) and 
Paraoxonase 1 (PON-1) activity by a chromogenic method (Kleemola et al, 2002). Lecithin-
cholesterol acyltransferase (LCAT) activity will be measured by following formation of 
radiolabeled cholesterol esters (Jauhiainen & Dolphin, 1986). 
 
Angiopoietin-like proteins (ANGPTL) 3, 4, and 8 will be analyzed using specific ELISA assays 
(Robciuc et al, 2010; Tikka et al, 2017). 
 
Resolvin levels will be measured in collaboration with the Karolinska Institute in Stockholm. The 
analysis will be conducted using mass spectrometry optimized for lipid mediators.  
 
 
 
 
LDL-modifikaatio 
LDL-aggregation 
Plasma  
LDL eristetään 
ultrasentrifuugilla 
Aika 
 
Aggregaatioanalyysi 
Aggregaatin koko 
Figure 2. LDL Particle Aggregation Analysis. 
LDL is isolated from blood samples using ultracentrifugation. The LDL particles are modified under 
precisely controlled conditions, with modification induced by enzymatic treatment. This modification 
leads to strong LDL aggregation, which is monitored by measuring aggregate size using a dynamic light 
scattering-based method. The method has been developed, optimized, and standardized in our laboratory 
(Ruuth et al, 2018). 

E-EPA and LDL 5 /15 
 
Implementation  
 
The responsible physician of the study is Professor Petri T. Kovanen (MD, PhD) from the Wihuri 
Research Institute. Study participants will visit the Wihuri Research Institute, where blood samples 
will be collected under his supervision. In addition, Professor Kovanen will personally conduct the 
baseline health checkups. During these visits, participants will have the opportunity to discuss the 
study with the physician or the Principal Investigator, Dr. Katariina Öörni. Breakfast will be 
provided for the participants after each visit. 
 
Blood samples accrued during the study will be processed and analysed at the Wihuri Research 
Institute, the HiLipid Lipidomics Unit, the Minerva Foundation Institute for Medical Research, and 
the Karolinska Institutet in Stockholm. 
 
Professor Petri Kovanen, Associate Professor Katariina Öörni, and BSc Lauri Äikäs will inform 
participants about the study and collect signed consent forms prior to enrollment to the study. 
Bioanalyst Maija Atuegwu will execute blood withdrawals. BSc Lauri Äikäs will carry out LDL 
particle isolation, lipidomic analyses, aggregation analyses, and plasma enzyme measurements 
under the supervision of Associate Professors Katariina Öörni, Matti Jauhiainen, and Reijo Käkelä 
and Dr. Hildur Arnardottir at the Karolinska Institutet. All senior team members will participate in 
data analysis. 
 
Timeline and Reporting 
 
Participant recruitment will begin once ethical approval and research permits have been granted. 
Recruitment will be conducted via internal mailing lists at the University of Helsinki and 
Biomedicum Helsinki. Laboratory analyses will take place between 2019 and 2020, with data 
analysis and reporting occurring within the same timeframe. The results will be published in 
international scientific journals, with a target of 1-2 original research articles. Findings will also be 
presented at international conferences. BSc Lauri Äikäs will include part of the results in his 
master’s thesis. All research team members will contribute to study planning, data evaluation, and 
reporting. 
 
Funding 
 
The study is funded through research grants to Associate Professors Katariina Öörni and Matti 
Jauhiainen. 
 
Table 1. Cost Estimates 
Expenses 
 
Combined 
Salaries  
 
     Master’s thesis worker’s salary – including overheads  
15 600 € 
 
 
Analytics: 
 
      LDL aggregation: 10 €/sample, 200 samples  
2 000 € 
      Lipoprotein isolation: 12.5 €/sample, 200 samples  
2 500 € 
      Lipid extraction and mass spectrometry analyses 
5 000 € 
      Enzymatic analyses 
2 000 € 
      ELISA-analyses 
2 500 € 

E-EPA and LDL 6 /15 
 
Other costs: 
 
      E-EPA capsules 
2 000 € 
      Breakfast for the participants 
   500 € 
Combined 
32 100€ 
 
 
 
 
IV. ETHICAL CONSIDERATIONS 
The study adheres to Good Clinical Practice (GCP) guidelines, the study plan, and applicable 
regulations. The study plan aligns with the research objectives and provides sufficient guidance for 
the research staff to conduct the study appropriately. The research methods used are well-
established. 
Participants will provide four fasting blood samples over a five-week period. These samples will be 
analysed for LDL cholesterol, HDL cholesterol, apoB-100, apoA-I, Lp(a), and triglyceride levels. 
Blood pressure measurements will also be taken during study visits. Participants will receive a 
summary of these measurements after analyses have been concluded. Prior to each blood sample 
collection, participants will complete a 24-hour dietary record. As the results related to lipoprotein 
aggregation and lipidomics remain at an experimental and theoretical level, their clinical 
significance cannot be assessed at an individual level, and therefore, individual reports on these 
results will not be provided to participants. However, participants may access study findings after 
the study has concluded and the results have been published. 
 
E-EPA has not been associated with harmful side effects. Since fish oil-based E-EPA does not 
contain fish protein, it is generally safe for use; however, due to the high dosage, individuals with 
fish allergies will not be included in the study. EPA has blood-thinning effects, making 
anticoagulant medication a contraindication for participation. In the REDUCE-IT trial, 2.3% of E-
EPA users reported joint pain, compared to 1% in the placebo group. The amount of vitamin D3 
administered in the study exceeds Finland’s daily recommended intake but remains well below the 
EU's upper safe limit of 100 µg, which has been shown to have no adverse effects. Participants 
taking daily vitamin D3 supplements will be requested to discontinue their use minimum one week 
prior to commencing the study. Vitamin D3 levels in the blood will be monitored for potential side 
effects. 
 
Participants may withdraw from the study or revoke their consent at any point prior to its 
conclusion without any repercussions. Any data collected before consent withdrawal will be used if 
deemed necessary for the accuracy and reliability of overall study results. 
Personal data will be collected in accordance with current data protection regulations. Participants 
will provide standard personal information, such as name, date of birth, and health-related details, 
via the consent form. The risk associated with processing this data is assessed as low, and risk 
assessments will continue throughout the maintenance of the registry. Data protection rights will be 
upheld during data storage. 
 
Participant samples will be blinded so that only the responsible researcher can link them to personal 
information. Results or data containing personal identifiers will never be made publicly available. 
Personal data and study-related documents will be securely stored at the Wihuri Research Institute 
in locked archive cabinets and password-protected computers. All members of the research team 

E-EPA and LDL 7 /15 
 
have been trained in proper data handling. The Principal Investigator will maintain a data registry in 
compliance with applicable privacy laws. 
 
Participant-specific data will remain confidential and will not be shared with third parties or 
included in medical records. Sample material containing DNA will not be stored; instead, it will be 
properly disposed of after sample processing. 
 
V. SCIENTIFIC SIGNIFICANCE  
This is an exploratory clinical study utilizing advanced lipid analysis techniques to investigate how 
E-EPA affects the properties of low-density lipoproteins. Specifically, the study examines changes 
in LDL lipid composition, its ability to bind to proteoglycans, and its susceptibility to aggregate. 
Additionally, the study provides insights into how E-EPA influences key plasma enzymes and lipid 
transfer proteins that regulate lipid metabolism. These findings are significant for assessing the risk 
of atherosclerosis and coronary artery disease. 
VI. REFERENCES 
Ahmed O, Littmann K, Gustafsson U, Pramfalk C, Öörni K, Larsson L, Minniti ME, Sahlin S, Camejo G, 
Parini P et al (2018) Ezetimibe in Combination With Simvastatin Reduces Remnant Cholesterol Without 
Affecting Biliary Lipid Concentrations in Gallstone Patients. J Am Heart Assoc 7: e009876 
Bhatt DL, Steg PG, Miller M, Brinton EA, Jacobson TA, Ketchum SB, Doyle RT, Juliano RA, Jiao L, 
Granowitz C et al (2019) Cardiovascular Risk Reduction with Icosapent Ethyl for Hypertriglyceridemia. 
New England Journal of Medicine 380: 11-22 
Boren J, Williams KJ (2016) The central role of arterial retention of cholesterol-rich apolipoprotein-B-
containing lipoproteins in the pathogenesis of atherosclerosis: a triumph of simplicity. Curr Opin Lipidol 27: 
473-483 
Groener JE, Pelton RW, Kostner GM (1986) Improved estimation of cholesteryl ester transfer/exchange 
activity in serum or plasma. Clin Chem 32: 283-286 
Jauhiainen M, Dolphin PJ (1986) Human plasma lecithin-cholesterol acyltransferase. An elucidation of the 
catalytic mechanism. J Biol Chem 261: 7032-7043 
Jauhiainen M, Ehnholm C (2005) Determination of human plasma phospholipid transfer protein mass and 
activity. Methods 36: 97-101 
Kleemola P, Freese R, Jauhiainen M, Pahlman R, Alfthan G, Mutanen M (2002) Dietary determinants of 
serum paraoxonase activity in healthy humans. Atherosclerosis 160: 425-432 
Lehti S, Nguyen D, Belevich I, Vihinen H, Soliymani R, Käkelä R, Heikkilä HM, Jauhiainen M, Grabowski 
GA, Kummu O et al (2018) Extracellular lipid accumulates in human carotid arteries as distinct three-
dimensional structures with proinflammatory properties. Am J Pathol 188: 525-538 
Manninen S, Lankinen M, Erkkila A, Nguyen SD, Ruuth M, de Mello V, Oorni K, Schwab U (2018) The 
effect of intakes of fish and Camelina sativa oil on atherogenic and anti-atherogenic functions of LDL and 
HDL particles: A randomized controlled trial. Atherosclerosis 281: 56-61 
Öörni K, Pentikäinen MO, Annila A, Kovanen PT (1997) Oxidation of Low Density Lipoprotein Particles 
Decreases Their Ability to Bind to Human Aortic Proteoglycans. Journal of Biological Chemistry 272: 
21303-21311 
Robciuc MR, Tahvanainen E, Jauhiainen M, Ehnholm C (2010) Quantitation of serum angiopoietin-like 
proteins 3 and 4 in a Finnish population sample. J Lipid Res 51: 824-831 
Ruuth M, Nguyen SD, Vihervaara T, Hilvo M, Laajala TD, Kondadi PK, Gistera A, Lahteenmaki H, Kittila 
T, Huusko J et al (2018) Susceptibility of low-density lipoprotein particles to aggregate depends on particle 
lipidome, is modifiable, and associates with future cardiovascular deaths. Eur Heart J 39: 2562-2573 
Tikka A, Metso J, Jauhiainen M (2017) ANGPTL3 serum concentration and rare genetic variants in Finnish 
population. Scand J Clin Lab Invest 77: 601-609 

E-EPA and LDL 8 /15 
 
Deviations from the original study plan: 
Statistical analysis plan 
This study is a single-arm, longitudinal nutritional intervention study with a repeated-measures 
design, investigating the effects of icosapent ethyl on blood composition and functional parameters 
in healthy, normolipidemic volunteers. A total of 38 participants were recruited and screened for 
inclusion and exclusion criteria based on an initial blood draw. Eligible participants provided a 
baseline blood sample at Day 0 before receiving the intervention. Participants consumed the 
supplement daily for four weeks (Days 1–28), with blood samples collected at Days 7 and 28. 
Following the intervention period, participants underwent a 7-day washout phase, after which a final 
blood sample was collected at Day 35. No control or placebo group was included in the study, as each 
participant served as their own control through repeated within-subject comparisons over time. 
1. Statistical Methods 
The statistical methods employed in this study include tests for normality, group differences, analysis of 
variance, correlation analysis, time series clustering, principal component analysis (PCA), linear mixed 
modeling, hierarchical clustering, distance calculations, sparse partial least squares-discriminant analysis 
(sPLS-DA), uniform manifold approximation projection (UMAP), and machine learning models. Specific 
methods include: 
• Normality Testing: D’Agostino-Pearson omnibus K2 test. 
• Group Differences: Paired multiple t-tests (LIMMA) on log2-transformed data, with false discovery rate 
(FDR) correction. 
• Analysis of Variance: One-way ANOVA with post hoc analysis (Benjamini, Krieger, and Yekutieli FDR 
correction). 
• Correlation Analysis: Spearman’s correlation with exact p-values for small datasets and t-ratio derived p-
values for larger datasets. 
• Time Series Clustering: VSClust fuzzy c-means clustering with k-nearest neighbors (kNN) imputation 
for missing values. 
• PCA: Performed on log2-normalized lipid abundances, using centering and scaling. 
• Linear Mixed Modeling: Implemented using lmer in the lme4 package, with time as a fixed effect and 
subject as a random effect. 
• Hierarchical Clustering: Ward’s method applied to Euclidean distance matrices from PCA scores. 
• Distance Calculations: Euclidean distances calculated from PCA scores to assess intra- and inter-subject 
variability. 
• sPLS-DA: Conducted via MetaboAnalyst 6.0 with nested cross-validation. 
• UMAP: Dimensionality reduction using the umap package with predefined parameters. 
• Machine Learning: XGBoost regression models trained on lipidomic and clinical data using nested cross-
validation. 

E-EPA and LDL 9 /15 
 
No formal interim analysis was planned. 
2. Sample Size and Power Justification 
The study initially planned to enrol 40–70 participants, with a total of 72 recruited and 68 enrolled. Due to 
COVID-19 restrictions, only 38 participants completed the study. A formal power calculation was not 
conducted, and sample size selection was based on feasibility and the exploratory nature of the study. The 
final analysis will include only the 38 participants who completed the study. 
A sample size assessment was conducted post hoc to evaluate whether the study had sufficient statistical 
power to detect changes in LDL aggregation, triglycerides, and EPA incorporation. This assessment was 
based on observed variability in the data and repeated-measures ANOVA. The observed effect size was 
computed using partial eta-squared (η²p). The effect size was then converted to Cohen's f and used to 
estimate the required sample size for 80% power. The results indicated that for the primary outcomes, the 
study had sufficient power to detect moderate-to-large effects, with a required sample size of approximately 
16 participants for the observed effect sizes.  
3. Level of Significance 
A two-tailed significance level of 0.05 was used throughout the study. Multiple hypothesis testing 
corrections were applied using the false discovery rate (FDR) method where appropriate. 
4. Criteria for Termination of the Trial 
The trial would be terminated if more than 10% of the participants encountered adverse effects related to the 
intervention. 
5. Handling of Missing Data 
A complete case analysis approach was used for analyses requiring full datasets, such as UMAP, 
PCA, and machine learning analyses. Within these complete cases, missing values were handled 
using k-nearest neighbors (kNN) imputation where required. However, for single-variable analyses, 
all available data were used, and missing values were omitted rather than imputed. 
6. Deviations from the Original Statistical Plan 
The study deviated from the original protocol in the following ways: 
• Sample Size Reduction: The original plan aimed for 40–70 participants, but only 38 completed the study 
due to COVID-19 restrictions. The final analysis includes only these 38 participants under a per-
protocol approach rather than an intention-to-treat model. 
• Changes in Outcome Assessments: Some planned secondary outcomes, including enzyme activity assays 
(PLTP, CETP, PON-1, LCAT) and ELISA assays for ANGPTLs and resolvins, were not conducted 
due to COVID-19-related constraints. 
• 
Changes in study collaborators: Due to Covid-19 travel restrictions and delays, the international 
collaboration with Karolinska Institute was halted and Professor Magnus Bäck and PhD Hildur 
Arnardottir removed from the research team. 

E-EPA and LDL 10 /15 
 
• Expanded Statistical Methods: The study incorporated additional statistical methods, including machine 
learning (XGBoost), time series clustering (VSClust), hierarchical clustering, UMAP, and sPLS-DA, 
which were not in the original protocol. 
• Data Handling Approach: The original plan allowed for analysis of partially completed cases, but the 
final study adopted a complete case analysis approach, excluding participants with missing time 
points. 
• Stopping Criteria Introduced: The original plan did not specify stopping rules. The final study 
introduced a stopping rule stating that if >10% of participants experienced adverse effects, the study 
would be terminated. 
These modifications were made to accommodate study constraints while maintaining scientific rigor. 
7. Selection of Subjects for Analysis 
The final analysis will include only the 38 participants who completed the study, following a per-protocol 
approach. Participants with missing time points will not be included in the analysis. 
8. Direct Access to Source Data/Documents 
The study sponsor will ensure that the investigator(s)/institution(s) permit trial-related monitoring, audits, 
IRB/IEC review, and regulatory inspection(s), providing direct access to source data/documents. The source 
data will not be made publicly available in any repository due to legislation on anonymity. Data is stored on 
secure backup media and deposited on a secure university server for long-term storage and controlled access.  
9. Quality Control and Quality Assurance 
The study adhered to standardized protocols for data collection, processing, and statistical analysis 
to ensure data integrity and reliability. Quality control measures were implemented to maintain 
consistency and accuracy in data handling. 
10. Ethical Considerations 
Written informed consent was obtained from all participants. The study adhered to the ethical guidelines of 
the Declaration of Helsinki and was approved by an appropriate ethics committee. 
11. Data Handling and Record Keeping 
All study data were securely stored in a regulatory-compliant manner, with participant identifiers removed to 
maintain confidentiality. Data processing was performed using GraphPad Prism v10.1.2 and RStudio 
(Version 2023.12.1, build 402, Posit Software, PBC). 
12. Financing and Insurance 
The study is sponsored by the Wihuri Research Institute, a private, foundation-funded academic institution. 
Wihuri Research Institute is supported by the Jenny and Antti Wihuri Foundation. Additional funding for 
this study was received through grants from private foundations and the Research Council of Finland. 
Funding for this study was solely for academic research, with no commercial interests involved. All study 
participants were insured by the Wihuri Research Institute.  

E-EPA and LDL 11 /15 
 
13. Publication Policy 
The publication policy follows the predefined guidelines agreed upon by study investigators. The study 
findings will be published in gold/green open-access peer-reviewed journals, ensuring transparency and 
reproducibility. 
14. Supplements 
Further details regarding statistical methodologies, exploratory analyses, and supplementary findings can be 
found in the Supplementary Methods section of the final study report. 
 
 
 

E-EPA and LDL 12 /15 
 
Information for participants of E-EPA study 
Name of the study 
The effect of E-EPA on circulating lipoproteins and plasma proteins related to lipid metabolism  
We kindly ask you to participate in our study in which we study eicosapentaenoic acid (EPA), an 
omega-3 fatty acid, and its effect on circulating LDL-particle composition and other cardiovascular 
disease factors. 
Participation is completely voluntary 
Participation in this study is fully voluntary. You may stop the study or withdraw your approval at 
any point without further notice. This will not impact your future access to any medical treatment. 
Please read this document carefully! If you have any questions, you can contact the research doctor 
or the principal investigator, whose contact details are found at the end of this document, on page 4. 
If you decide to participate in the study, we will ask you to sign the agreement on the last page of 
the document.  
Executor of the study 
This study is carried out by Wihuri Research Institute and the medical doctor in charge is Professor 
Petri Kovanen. Docent Katariina Öörni is head of personal data registry of this study and is 
responsible that personal data is handled according to local laws and regulation. 
The goal of the study 
Cardiovascular diseases cause the number one killer both in Finland and globally. The most 
important factor causing the disease is cholesterol, which is carried in the LDL particles 
accumulating in the artery walls. Therefore, LDL-cholesterol is called the “bad cholesterol”. In our 
recent studies we have discovered that in addition to the amount of LDL particles in circulation, 
also the composition and quality of the particles have an impact on the development of 
cardiovascular diseases. 
LDL particle composition is affected by dietary fats. Polyunsaturated fatty acids, so called “soft 
fats” are beneficial to your health and especially omega-3 fatty acid EPA has been found to lower 
the risk of fatal cardiovascular diseases. To further understand the health benefits of EPA we are in 
this study focusing on the effects of EPA on LDL particle composition and other cardiovascular 
disease factors. 
Background 
EPA is a polyunsaturated omega-3 fatty acid that is necessary for normal health and derived from a 
diet containing fatty fish and food oils. In this study we use clinically manufactured ethyl esterified 
EPA capsules. Capsules contain EPA fatty acids and D3-vitamin. In an international study 
REDUCE-IT, was found that consumption of 4 grams of EPA daily reduced cases of fatal 
cardiovascular diseases by 25% in 5-year median follow up. In this study we use the same dosage. 
EPA is an important precursor from which several signal molecules that reduce inflammation, 
resolvins, are formed. Resolvins have been shown to reduce the development of cardiovascular 
diseases. We will measure resolvins from the blood samples. In addition, we will also investigate if 

E-EPA and LDL 13 /15 
 
EPA affects lipid metabolism enzymes such as LCAT, CETP, PLTP and PON-1 which all affect the 
LDL particle composition. 
Aggregating LDL in the artery walls is crucial for the development of atherosclerosis. Previous 
studies show that the LDL particles bind to proteoglycans and other components of the extracellular 
matrix in the inner layer of the artery wall where LDL particles start to bind to each other and 
aggregate. LDL particle composition impacts the aggregation susceptibility and hence the 
development of atherosclerosis. Dietary fats again affect the composition of LDL particles. In this 
study we examine, how impactful the consumption of EPA is at modifying LDL particle 
composition and the possible reduction in the LDL particle aggregation susceptibility or binding to 
proteoglycans extracted from human aorta.  
Participation in the study 
40-70 voluntary healthy participants, who agree to consume 4 grams daily of E-EPA are recruited to 
the study. Capsules are taken twice a day (3 in the morning and 3 in the evening) for 4 weeks. Each 
capsule contains 650mg of E-EPA which will add up to a daily dosage of 3,9 grams. Capsules 
should be ingested with fat-containing food. During the study period participants are expected to 
keep up their usual lifestyle and diet. Supplements or the usage of a medication, like cholesterol 
lowering statins, should remain the same during the study period. If you are pregnant, breastfeeding 
or planning pregnancy during the study period, you cannot participate in the study. Also, 
substantially high amount of LDL-cholesterol (>5 mmol/l) or triglycerides (> 3 mmol/l) that we test 
in our laboratory, will lead to exclusion from participation.  
Participation in the study requires 4 visits to Wihuri Research Institute’s laboratory. During each 
visit we will collect a blood sample, which is similar to one taken during doctor’s appointment 
(approximately 20 ml). Before giving the sample, an overnight (8h) fasting is required. Blood 
samples are collected before the study and after weeks 1 and 4 and 1 week after the end of the diet 
at week 5. After the 4th blood sample at week 5, your participation in the study ends. All blood 
sampled are coded and handled anonymously. We also ask you to fill a food dairy from the previous 
day before each blood sampling. Breakfast will be provided after each visit. At the first visit you 
will meet with a medical doctor and you blood pressure, blood glucose, weight, height and waist are 
measured, and you are asked to fill a questionnaire regarding some common factors regarding 
cardiovascular disease. We measure plasma LDL-cholesterol, HDL-cholesterol, triglycerides and 
D-vitamin and each participant will get an evaluation of these results after the end of the study.   
LDL particles are isolated from the blood samples and their composition, aggregation susceptibility 
and binding affinity to proteoglycans is measured. Also, previously mentioned enzymes and 
resolvins are measured. Insights from these results can be given to you after the study is concluded.  
Possible side effects from E-EPA 
Healthy participants have very low risk for any side effects. A possible joint pain has been reported 
during longer periods of E-EPA diet. 
Exclusion criteria 
1. Because E-EPA causes blood thinning, one should not normally take E-EPA without 
consulting a doctor beforehand. Therefore, the usage of blood thinning medicine or 

E-EPA and LDL 14 /15 
 
increased risk (e.g. family history) of problems with blood coagulation prohibits from 
participating in the study.  
2. The capsules are manufactured from fish oils; hence they should not contain fish 
proteins, but there may be small amounts of fish protein as an impurity and people with 
fish allergy should be careful regarding fish oil products in general and only start with 
small doses. Therefore, having a fish allergy prohibits participation in the study. 
Financial expenses and clarifications 
E-EPA capsules and any procedures regarding the study are free of charge. You will not be paid to 
participate in the study, but breakfast is provided after each visit. All participants are insured.  
The study is financed by Wihuri Research Institute. Principal doctor and other personnel are not 
given extra payment for conducting the research. Docent Katariina Öörni and Professor Petri 
Kovanen have applied for a patent regarding LDL particle aggregation susceptibility measurement 
protocol.  
Personal data handling  
Personal data is handled according to European Union legislation (GDPR). All samples are handled 
with code numbers from which a code registry is formed. Code registry is protected by a password 
and the personal details of the participants are only known by the persons conducting the research. 
Coded blood samples are preserved in the research facility’s freezers, and they are mainly analysed 
by researcher Lauri Äikäs. Your identity will not be revealed when the results of the study are 
presented.  
This study and the handling of the personal data is based on following decrees. Also, an approval 
from ethical committee of Helsinki and Uudenmaan hospital district has been given for conducting 
this study.  
- 
General Data Protection Regulation (GDPR) 2016/679, 6th article sections 1a), b), c) and e) 
and 9th article sections 3 a), g), i) and j) 
- 
Law regarding scientific research 1999/488 
Additional information 
If you have any questions regarding the study, we encourage you to be in touch with the principal 
investigator or rest of the research personnel. With them you may discuss at any time regarding 
possible concerns related to the study. 
Contact: 
Principal investigator 
Docent Katariina Öörni, Ph.D., Wihuri Research Institute Biomedicum I, Haartmaninkatu 8, 00290 
Helsinki. Phone. 09 681 411, email kati.oorni@wri.fi 
Principal Doctor 
Professor Petri T. Kovanen, M.D., Ph.D., Wihuri Research Institute Biomedicum I, Haartmaninkatu 
8, 00290 Helsinki.  
Phone. 09 681 411, email petri.kovanen@wri.fi 
 
 

E-EPA and LDL 15 /15 
 
Approval for the study 
I have been asked to participate in the study “The effect of E-EPA on circulating lipoproteins and 
plasma proteins related to lipid metabolism”. 
I have read the document meant for the participants of the study and have discussed with the principal 
investigator or other responsible personnel. I have been given enough information about the study and the 
included personal data collection and handling in the study. I have also been informed verbally and have 
been given answers to all my questions related to the study. Explanation was given to me by 
______________________________ (name of the person). I have had enough time to consider participation 
in the study. 
I understand that participating in the study is completely voluntary. I am aware that I may withdraw from the 
study at any time without further notice and that it won’t affect my doctor-patient relationship or future 
healthcare. I am also aware that personal and other data gathered from me up to this point may still be used 
in the study if it is necessary for method validation, quality or safety. 
I have read the “personal data handling in the code registry”- document and I understand what my personal 
data is being used for. I understand that the data I give is being coded into a blinded study registry and a 
separate code registry is formed. Code registry may be accessed by only the key personnel in the Wihuri 
Research Institute’s research group. Responsible person of the code registry is Docent Katariina Öörni. 
I give my consent to gather my personal data. I am aware of the personal data gathered and of my rights 
related to my personal data, such as being able to withdraw my consent at any point. I have been informed on 
how I may apply my rights and given contact information required for this purpose. I have been given 
contact information to supervising authority, whom I may contact if I feel that my personal data has been 
misused or my rights violated.  
I give my consent to be contacted in the future, if validating the research results requires it. 
With my signature I confirm that I am 18-65-year-old and that I take voluntarily part in the study described in the 
document. I am aware that my personal data may be handled during an inspection by a national or international 
supervision agency or by an independent agent providing quality control on behalf of the study provider. 
__________________________  
 
____________________________________ 
Signature  
 
 
 
Date 
 
__________________________ 
  
____________________________________    
Print name    
 
 
Date of birth or social security number 
 
 
 
 
 
 
 
 
_______________________________________________________  
Address  
 
Phone number________________________ Email _____________________________  
  
Acceptor of the written approval 
 
_____________________________   
 
_____________________________  
Acceptors signature  
 
 
Date 
 
_________________________  
Print name