OMEGA-3 FATTY ACIDS AND CARDIOVASCULAR DISEASE

BY

MUHAMMAD AISHATU IBRAHIM

PG23/MSC/BCHM/1001

A SEMINAR REPORT

SUBMITTED TO DEPARTMENT OF BIOCHEMISTRY

SCHOOL OF POSTGRADUATE STUDIES

GOMBE STATE UNIVERSITY

SUPERVISOR: PROF. HAJJA GANA HAMZA

JUNE, 2024
 DECLARATION

I hereby declare that this seminar was written by me and it is a record of my own research work.
It has not been presented before in any previous application for a higher degree. All references
cited have been duly acknowledged.

………………………………… ………………………………..

Student Signature/date

………………………………. …………………………………

Supervisor Signature/Date

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 CERTIFICATION
This is to certify that this seminar report was written by MUHAMMAD AISHATU IBRAHIM
with the registration number PG23/MSC/BCHM/1001 under the supervision of Prof.
HajjaGanaHamza.

PROF. HAJJAGANA HAMZA __________________________

(SUPERVISOR) SINATURE/DATE

Dr. ABUBAKAR AISAMI __________________________

(PG COORDINATOR) SINATURE/DATE

ASST. PROF. LAZARUS JOSEPH GOJE __________________________

(HEAD OF DEPARTMENT) SINATURE/DATE

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 DEDICATION
I dedicate this work to my parent for their support.

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 ACKNOWLEDGEMENTS

I express my gratitude to Almighty Allah (SWT) for His mercies upon me, guidance and
blessings, by making it possible to complete this seminar report successfully.

I would like to express my profound appreciation to my supervisor, Prof. HajjGanaHamza for

her support and guidance, the PG coordinator, Dr. AbubakarAisami, the HOD Associate Prof.
Lazarus Goje. My sincere gratitude to my beloved parents for their unwavering support and
prayers, my appreciation also goes to my husband for his support. Special appreciation goes to
Mubarak Saidu, Madam Patum, Maryam UsmanAbdulkadir, BilkisuAbubakar Mayo,
MalamaNusaiba, my sister Amina and other siblings and friends, who helped me during my
seminar paper.

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 ABSTRACT
Cardiovascular disease (CVD) is the world’s most recognized and notorious cause of death. It is
known that increased triglyceride-rich lipoproteins (TRLs) and remnants of triglyceride-rich
lipoproteins (RLP) are the major risk factor for CVD. Furthermore, hypertriglyceridemia
commonly leads to a reduction in HDL and an increase in atherogenic small dense lowdensity
lipoprotein (sdLDL or LDL-III) levels. The long-chain omega-3 fatty acids, eicosapentaenoic
acid (EPA) and docosahexaenoic acid (DHA), are found in seafood, supplements, and
concentrated pharmaceutical preparations. Thus, the evidence shows that omega-3 fatty acids
(eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have a beneficial effect on CVD
through reprogramming of TRL metabolism, reducing inflammatory mediators (cytokines and
leukotrienes), and modulation of cell adhesion molecules.

Prospective cohort studies demonstrate an association between higher intakes of EPA+DHA or

higher levels of EPA and DHA in the body and lower risk of developing cardiovascular disease
(CVD), especially coronary heart disease and myocardial infarction, and of cardiovascular
mortality in the general population. The cardioprotective effect of EPA and DHA is due to the
beneficial modulation of a number of risk factors for CVD. Some large trials support the use of
EPA+DHA (or EPA alone) in high-risk patients, although the evidence is inconsistent. This
review presents key studies of EPA and DHA in the primary and secondary prevention of CVD,
briefly describes potential mechanisms of action, and discusses recently published RCTs, cohort
studies and meta-analyses.

Key words: omega-3 fatty acid, fish oil, eicosapentaenoic acid, docosahexaenoic acid,
cardiovascular diseases.

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 TABLE OF CONTENTS

Contents
DECLARATION........................................................................................................................................i
CERTIFICATION.....................................................................................................................................ii
DEDICATION..........................................................................................................................................iii
ACKNOWLEDGEMENTS......................................................................................................................iv
ABSTRACT...............................................................................................................................................v
TABLE OF CONTENTS.........................................................................................................................vi
CHAPTER ONE........................................................................................................................................1
1.0 INTRODUCTION.............................................................................................................................1
CHAPTER TWO.......................................................................................................................................4
2.0 Omega-3 PUFAs...............................................................................................................................4
2.1 Structure and classification of omega-3 fatty acids............................................................................5
2.2 Sources of omega-3 fatty acids..........................................................................................................5
2.3 History of fish oil and lifestyle-related diseases................................................................................6
2.4 History of Fish Oil Preparation Development: Highly Purified EPA and EPA/DHA Combination. .7
2.5 Absorption of omega-3 PUFAS and metabolism in vivo...................................................................8
2.6 Molecular mechanisms of PUFAs in cardiovascular health...............................................................8
2.7 Recommended Intake of DHA and EPA.........................................................................................10
2.8 Cardiovascular health and omega-3 fatty acids................................................................................11
CHAPTER THREE.................................................................................................................................16
3.0 Roles of Omega-3 PUFAs in heart health........................................................................................16
3.1 Mechanisms by which EPA and DHA reduce the risk of cardiovascular disease............................16
3.2Risk factors for CVD that are targeted by EPA and DHA................................................................18
3.3 Therapeutic roles and mechanisms of omega-3 PUFA in lowering of triglycerides........................19
3.4 Anti-inflammatory mechanism of omega-3 PUFA in cardiovascular disease..................................19
3.5Omega-3 polyunsaturated fatty acids and hypertension....................................................................22
3.6 Controversies Surrounding omega-3 fatty acids..............................................................................27
CHAPTER FOUR...................................................................................................................................29
4.0 Summary, conclusion and recommendation....................................................................................29
4.1 Summary.........................................................................................................................................29

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 4.2 Conclusion.......................................................................................................................................29
4.3 Recommendations...........................................................................................................................30
RFERENCES...........................................................................................................................................31

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 CHAPTER ONE

1.0 INTRODUCTION
Fatty acids are classified into saturated fatty acids that have no double bonds and unsaturated
fatty acids possessing double bonds. Fatty acids with multiple double bonds are called
polyunsaturated fatty acids (PUFA) (Yasuhiro and Ichiro, 2020).

Omega-3 fatty acids, also known as ω-3 fatty acids, are polyunsaturated fatty acids (PUFA) of
plant and animal or marine-origin. They are biologically essential macromolecule that involves
in many metabolic processes and cannot be synthesized by mammals. The term “ω-3” fatty acids
is distinguished by the presence of a double bond in their chemical structure at threecarbon atom
away from the terminal (the omega) methyl group. Omega-3 fatty acids consist of longchain and
short-chain fatty acids. Plants synthesize shortchain ω -3 fatty acids such as alpha-linolenic acids
(ALA; 18:3 n-3) and linoleic acid (18:2n-6) that are found in many seeds, nuts, and seed oils.
Neither linoleic nor α-linolenic acid can be synthesized in animals. Long-chain fatty acids such
as docosahexaenoic (DHA);22:6 n-3 and eicosapentaenoic acid (EPA); 20:5 n-3 are mainly
found in fatty fish like mackerel, halibut, herring, salmon, tuna, and they may be collectively
referred to as marine n-3 fatty acid. Thus, omega-3 FAs are essential fatty acids that cannot be
synthesized de novo pathway by mammalian cell because they lack the necessary enzymes to
place a double bond in the ω-3 and ω-6 position (Shibabaw,2020).

As early as 1944, Sinclair described the rarity of coronary heart disease (CHD) amongst
Greenland Eskimos, who consumed a diet rich in fish, seal and whale. More than 40 years ago,
Bang and Dyerberg reported that despite low consumption of fruit, vegetables and complex
carbohydrates in exchange for a diet high in saturated fat and cholesterol, serum cholesterol and
triglyceride (TG) levels were lower in Greenland Inuit than in agematched residents of Denmark,
who also demonstrated lower risk of myocardial infarction (MI). These and other similar
observations sparked interest in the potential benefits of increased dietary fish intake, particularly
the benefits of omega-3 polyunsaturated fatty acids, for cardiovascular (CV) health (Elagiziet al.,
2021).
The 2013 Global Burden of Disease study estimating that cardiovascular diseases (CVD) caused
17.3 million deaths globally. It accounted for 31.5% of all deaths and 45% of all non-
communicable disease deaths, more than twice that caused by cancer, as well as more than all
communicable, maternal, neonatal, and nutritional disorders combined (Shramkoet al.,2020).

Omega-3 confer CV benefits through TG reduction, anti-inflammatory and anti-arrhythmic

effects, vasodilation, reduced blood pressure, improved arterial and endothelial function,
favorable autonomic tone, and reduced platelet aggregation. In particular, TG levels are a
historically well-studied, independent risk factor for CHD. Omega-3 or fish oil diet
supplementation is evidenced to lower TG levels in a dose-dependent fashion, whereby 3-4 g/day
of eicosapentaenoic acid (EPA) or a combined EPA and docosahexaenoic acid (DHA) reduces
blood levels by 20–50% in those with high TGs (Elagiziet al., 2021).

According to the World Health Organization (WHO), atherothrombotic cardiovascular (CV)

disease has become the leading cause of global mortality with a disproportionate impact on low
and middle income countries. Despite effective control of low-density lipoproteins (LDL) with
oral and non-oral interventions, CV risk persists, likely due in part to elevated triglyceride (TG)-
rich lipoproteins (TGRLs), a dyslipidemia particularly prevalent in patients with diabetes and
metabolic disease. Genetic and epidemiologic analyses support an independent role for TGRLs
as a risk factor and strong contributor to overall mortality (Sherrattet al., 2022).

Early interest in the cardiovascular effects of polyunsaturated fatty acids (PUFA) emerged from
observational studies conducted between the 1950s and 1970s indicating that populations with a
high intake of omega-3 PUFA had lower rates of mortality from cardiovascular diseases (CVDs).
Numerous subsequent randomized controlled trials (RCTs) indicated that dietary substitution of
carbohydrates or saturated fatty acids by PUFA had a protective effect on intermediate outcomes,
such as a reduction in low-density lipoprotein (LDL)-cholesterol and triglycerides(Borges et al.,
2022).Cardiovascular disease (CVD) remains a major health problem worldwide. Even after
implementing all current treatments, residual CVD risk remains high. During the past years,
epidemiological, genetic, and biological data on the potential contributors to residual risk
strongly suggested that elevated triglyceride (TG) levels (fasting and nonfasting) may represent
causal risk factors for atherosclerotic CVD and all-cause mortality. This led to a renewed clinical
and scientific interest in hypertriglyceridemia and elevated non-high-density lipoprotein
cholesterol (non-HDL-C) levels.

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The latest (2016) European Cardiology Society/European Atherosclerosis Society guidelines
recommend that TG-lowering drugs should be used in high-risk patients with TG levels >2.3
mmol/L (200 mg/dL) when lifestyle measures fail to lower them. Furthermore, non-HDL-C is
regarded as a strong independent CVD risk factor, especially in the presence of
hypertriglyceridemia, thus being a secondary (after low-density lipoprotein cholesterol [LDL-C])
treatment target. Among lipid-lowering drugs, n-3 fatty acids (ie, eicosapentaenoic acid [EPA]
and docosahexaenoic acid [DHA]) can be used at doses of 2 to 4 g/d to lower TG levels. It has
been shown that n-3 fatty acids affect serum lipids and lipoproteins, especially very-low-density
lipoprotein concentrations. A recent scientific advisory from the American Heart Association
(AHA) stated that n-3 (EPA plus DHA) supplementation may be reasonable in patients at high
CVD risk, as well as in those with prevalent coronary heart disease (CHD), especially after
amyocardial infarction (MI) (Perez-Martinez et al., 2020).

In November 2018, the long-awaited results of the Reduction of Cardiovascular Events with
EPA–Intervention Trial (REDUCE-IT) trial, a phase III multicenter, double-blind, placebo-
controlled, randomized trial, were published. The study met its primary end point demonstrating
that among patients with elevated TG levels on a statin, the risk of a major CVD event
(combined end point including CVD death, nonfatal MI, nonfatal stroke, unstable angina
requiring hospitalization, or coronary revascularization) was significantly lower (by 25%) in
patients receiving 2 g of icosapent ethyl (IPE), a highly purified EPA, twice daily (ie, total daily
dose of 4 g), compared with those on placebo.Briefly, patients enrolled (n ¼ 8179; mean age ¼
64 years; 71.2% men) had baseline LDL-C between 41 and 100 mg/dL (median LDL-C ¼ 75
mg/dL) and fasting TGs between 135 and 499 mg/dL (median TGs ¼ 216 mg/dL) with either
established CVD (70.7%, secondary prevention cohort) or diabetes mellitus (DM) and 1 other
CVD risk factor (primary prevention cohort). Patients were followed up for a median of 4.9
years(Perez-Martinez et al., 2020).

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 CHAPTER TWO

2.0 Omega-3 PUFAs

Poly unsaturated fatty acid has emerged as a subject of scientific research and public interest
over the past 30 years. In the 1970s, epidemiological studies of Greenland Eskimos connected
their diet rich in ω -3 PUFA of fish and fish oil to a low incidence of cardiovascular mortality
and hypertriglyceridemia (HTG) treatment. Even though 40 per cent of the total caloric intake is
high in fat, cardiovascular incidents such as stroke and myocardial infarction in Greenland
Eskimos were low or nearly nonexistent when compared with the Danish population. Then, in
the late 1970s scholars pointed to these fatty acids as potential anti-atherogenic nutrients.
Omega-3 fatty acids are used as structural components of phospholipid membranes in body
tissues and maintain cell membrane fluidity. In addition, it is also able to overcome other forms
of inflammatory disorder of atherosclerotic CVD. These fatty acids can partly inhibit many
aspects of inflammation, including leukocyte chemotaxis, adhesion molecule expression and
leukocyte–endothelial adhesive interactions, production of eicosanoids like prostaglandins and
leukotrienes from the n-6 fatty acid (arachidonic acid), and production of pro-inflammatory
cytokines (Shibabaw,2020).

In Japan, an epidemiological survey of the anti-arteriosclerotic effect of fish oil was conducted
for the first time by Chiba University in 1980. In this study, fishermen who had high fish intake
were compared with rural residents who had a relatively low fish intake. Despite the high calorie
intake and high degree of obesity in the fishermen, the mortality rate due to ischemic heart
disease and cerebrovascular disease was significantly lower than that in rural residents. The
mean intake of EPA as 2600 mg/day in fishermen, approximately three times that of rural
residents. This diet was associated with low platelet aggregation capacity and low blood
consistency, and the triglyceride level of the fishermen was approximately 40% lower than that
of the rural residents (Yasuhiro and Ichiro, 2020).

Multiple trials continue to use an ω -3 intervention dose of 1 g/day of EPA + DHA, which
demonstrated significant CVD benefits in the landmark GISSI-P trial. However, trials
demonstrating a benefit with this low dose, such as GISSI-P, GISSI-HF and JELIS, were
performed in Italian and Japanese populations with higher baseline ω -3 intake in their regular
diet, which may account for their ability to reach a therapeutic level of omega-3 which confers
CVD benefits.

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The efficacy of modern medical therapy for CVD can further confound the benefits of ω-3
supplementation due to reduced overall CVD events. Patients in Western nations or nations with
lower ω -3 intake in general may require higher-dose interventions (e.g., 2–4 g/day of EPA +
DHA) to reach a therapeutic effect of ω -3. Several decades and countless dollars have been
spent studying the relationship between ω -3 and CVD without reaching a consensus among
clinicians. There is, however, clear evidence from multiple studies that higher doses of ω -3 (2–4
g/day of EPA + DHA) appear to be safe and to reduce CVD events in multiple CVD populations,
which warrants further study to conclusively determine the potential benefits of this safe,
inexpensive, and well-tolerated therapy (Elagiziet al., 2021).

2.1 Structure and classification of omega-3 fatty acids

Omega-3 PUFAs are found in food in three forms: alpha linolenic acid (ALA), eicosapentaenoic
acid (EPA) and docosahexaenoic (DHA). ALA is mainly found in plant foods, such as
vegetables, fruits, nuts, soybeans, and vegetable oils. EPA mainly comes from marine animals,
such as fish, and shrimp, especially deep-sea fish. Like EPA, the main sources of DHA are fish,
shrimp, and deep-sea fish. The best dietary source of EPA and DHA (and also docosapentaenoic
acid) is seafood, especially fatty fish (also called ‘oily fish’). The blubber and tissues of sea
mammals, such as whales and seals, also contain EPA and DHA in significant amounts. Various
supplements, including fish oils, cod liver oil, krill oil and some algal oils, contain EPA and
DHA. Finally, concentrated pharmaceutical-grade preparations of EPA and DHA, or EPA alone,
are available (Innes et al., 2020).

2.2 Sources of omega-3 fatty acids

Naturally occurring fatty acids (FAs) can be classified according to their carbon-chain length and
the number of double bonds. Long-chain (LC) FAs contain more than 12 carbon atoms, and FAs
containing 22 or more carbon atoms are sometimes referred to as very long-chain (VLC) FAs.
Based on the number of double bonds, FAs can be classified into saturated FAs (no double
bonds), monounsaturated FAs (MUFAs, a single double bond), and polyunsaturated FAs
(PUFAs, ≥2 double bonds). In the diet, palmitic acid (C16:0) and stearic acid (C18:0) are the
SFAs, and oleic acid (C18:1) is the main MUFA.

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The PUFAs can be further classified into two groups, omega−3 (ω−3 or n−3) and omega-6 (ω−6
or n−6), based on the position of the first double bond on the methyl terminal end. For instance,
α-linolenic acid (ALA, C18:3 cis-9,12,15), with the first double bond at the third position from
the methyl terminal end, and linoleic acid (LA, C18:2 cis-9,12), with the first double bond at the
sixth position from the methyl terminal end, are termed n−3 and n−6 FA, respectively (Sainiet
al., 2021).
The intake of EPA and DHA from diet is strongly influenced by fish consumption because fish,
in general, and fatty fish, in particular, are the richest dietary source of these fatty acids. The
intake of fish and fatty fish is high in some countries, such as Japan, but it is low in many
Western countries, including the USA and the United Kingdom. As a result, the intake of EPA +
DHA among adults varies among different populations and is low in most Western countries; it
is generally considered that in non-fatty fish eaters.

2.3 History of fish oil and lifestyle-related diseases

Mortality rate from atherosclerotic cardiovascular diseases (CVD), particularly myocardial
infarction (MI), is high in Western countries. Epidemiological studies have thus focused on the
differences in life- style, in particular dietary habit, among countries that differ in the incidence
of atherosclerosis-associated MI. A study conducted in seven countries" reported that the
mortality rate from ischemic heart disease is lower in Japan and Mediterranean countries than
that in the United States and Northern European countries and highlighted the role of unsaturated
fatty acids that are abundant in Japanese and Mediterranean diets. An epidemiological study of
the Greenlandic Inuit suggests that fish oil (omega-3 fatty acids) is important in preventing
atherosclerotic diseases (Yasuhiro and Ichiro, 2020).
Consumption and high biomarker concentrations of omega3 (n-3) fatty acids, show convincing
cardioprotective benefits and offer additional mental and physical health benefits in humans
including decreased risk of chronic diseases and cognitive decline. The most beneficial n-3 fatty
acids, eicosapentaenoic acid (20:5n-3 (EPA) and docosahexaenoic acid (22:6n-3 (DHA)) are
mainly obtained from marine sources in the diet. Although recently debated, there is now
convincing evidence for the reduction of cardiovascular events with high dose intake of the n-3
fatty acid, EPA. DHA is also important especially with respect to mental and cognitive effects
(Lane et al., 2022).

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After that landmark study, the health benefits of omega-3 fatty acids as part of a fatty acid rich
diet have been extensively researched in large-scale epidemiological studies, clinical outcome
trials, and meta- analyses, the results of which show a statistically significant reduction in the
relative risk of CVD in persons consuming omega-3 fatty acids. In Japan, a study of fishermen at
Kawazu, a village in Katsuura city in Chiba Prefecture, also demonstrated the contribution of an
omega-3-rich diet in the prevention of CVD. As a result, the first highly purified EPA
preparation for human use was developed in Japan. Omega-3 fatty acids are now widely
recognized to have an important role in preventing atherosclerotic CVD, carcinogenesis, and a
wide range of other diseases and conditions, including those of the central nervous system (such
as dementia), CV system (such as arrhythmia and chronic heart failure [CHF]), and immune
system (including rheumatoid arthritis and psoriasis), and in the defense against infections
(Yasuhiro and Ichiro, 2020).

2.4 History of Fish Oil Preparation Development: Highly Purified EPA and EPA/DHA
Combination
In Japan, an epidemiological survey of the anti-arteriosclerotic effect of fish oil was conducted
for the first time by Chiba University in 1980. In this study, fishermen who had high fish intake
were compared with rural residents who had a relatively low fish intake. Despite the high calorie
intake and high degree of obesity in the fishermen, the mortality rate due to ischemic heart
disease and cerebrovascular disease was significantly lower than that in rural residents. The
mean intake of EPA as 2600 mg/day in fishermen, approximately three times that of rural
residents. This diet was associated with low platelet aggregation capacity and low blood
consistency, and the triglyceride level of the fishermen was approximately 40% lower than that
of the rural residents (Yasuhiro and Ichiro, 2020). On the basis of these studies, Chiba University
and Nippon Suisan Co., Ltd. developed a high-purity EPA preparation from fish oil and
administered it to healthy people for the first time in the world, reporting a decrease in platelet
aggregation ability and an improvement in erythrocyte deformability.
This highly purified EPA (Epadel ®) was clinically used with an indication for obstructive
arteriosclerosis in 1990. Thereafter, the indication for hyperlipidemia, especially for the
improvement of hypertriglyceridemia, has been added. While the highly purified EPA
preparation has been used in Japan as Epadel ® and the United States as Vascepa ®, a different fish
oil preparation containing a combination of EPA and DHA has been used in Europe and the
United States for more than 20 years and has been evaluated in large-scale clinical trials.

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This EPA/DHA preparation (Lotriga®) was launched in Japan in January 2013. A randomized
study comparing the highly purified EPA preparation developed in Japan with the EPA and DHA
product developed in Europe and America indicated higher triglyceride- lowering efficacy of the
EPA and DHA product when used at a high dose (Yasuhiro and Ichiro, 2020).

2.5 Absorption of omega-3 PUFAS and metabolism in vivo

In the body, omega-3 fatty acids are primarily available as EPA and DHA and less abundantly
available as docosapentaenoic acids (DPA). Omega-3 fatty acids are incorporated into
chylomicron triglycerides in the gastrointestinal tract and transported to the liver, where EPA and
DHA are incorporated into triglycerides as very-low-density lipoprotein cholesterol (VLDL-C)
and released into the blood stream.
Only a small proportion of omega-3 fatty acids are available as free fatty acids, most of which
are bound to albumin. In the liver, ALA and EPA, which are omega-3 PUFA, are converted to
DPA and DHA through the action of desaturase and fatty acid chain elongase. Conversely, the
administration of DHA increases DPA and EPA. Changes of omega-3 PUFA in platelet
membranes have been reported after fish oil administration. The administration of highly
purified EPA increases EPA and DPA, but does not change DHA in the platelet membrane. On
the contrary, the administration of highly purified DHA increases DHA, DPA, and EPA in the
platelet membrane. These results suggest that the conversion from DPA to DHA is tightly
controlled by the content of DHA in the platelet membrane. Thus, the incorporation of DHA into
the membrane may be necessary for the increment of DHA in the membrane and DHA may be
converted to DPA and EPA. The above findings in the human body have also been found in the
arteries of arteriosclerosis model mice and the kidneys of metabolic syndrome model rats. These
findings confirm that the administration of DHA is effective in increasing the amount of DHA in
membrane phospholipids (Yasuhiro and Ichiro, 2020).

2.6 Molecular mechanisms of PUFAs in cardiovascular health

Molecular and interventional studies have demonstrated the benefits of omega-3 fatty acids in
the primary and secondary prevention of cardiovascular disease. These benefits arise from
multiple, complex interactions, including inflammatory mediators, lipid metabolism, alteration of
cardiac ion channels, cell membrane chemistry and physiology, down-stream cell signaling
pathways, and anti-thrombotic pathways, to name a few.

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The ratio of EPA/AA has been found to correlate with cardiovascular risk and atherosclerotic
progression. A lower serum EPA/AA ratio is associated with an increased risk of cardiovascular
disease and coronary events. In the Japan EPA Lipid Intervention Study, a significant decrease in
cardiac risk in patients with an EPA/AA ratio of >0.75 was found. Interestingly, the study also
found that increased EPA, but not DHA, levels are associated with decreased major coronary
events. Two families of proteins derived from EPA and DHA called protectins and resolvins
were discovered to play a pivotal role in resolving inflammation. Resolvins have two structural
forms: resolvin E and D. Resolvin E is formed by the action of 5-lipoxygenase on EPA-derived
mediators and inhibits neutrophil chemotaxis, specifically transendothelial migration
(Sheikh et al., 2019).

Protectins and resolvins derived from DHA both play a role in blocking t-cell and neutrophil
migration, respectively. PUFAs can influence gene transcription by binding to nuclear receptors,
such as peroxisome proliferatoractivated receptor (PPAR). PPARs are ligand-activated
transcriptors that regulate lipid and carbohydrate metabolism, cellular proliferation, and
inflammation. They are the first identified transcription factors that are regulated by fatty acids.
Tree PPAR isoforms are PPAR-α, PPAR-β/δ, and PPAR-γ. PPAR-α is mostly involved in fatty
acid metabolism, including β-oxidation of fatty acids, decreased hepatic triglyceride secretion,
increased lipoprotein lipase activity with very low-density lipoprotein (VLDL) clearance, and
increased production of high-density lipoprotein cholesterol, thereby promoting a favorable
hypo-lipemic effect . Anti-diabetic agents known as fibrates have been designed to specifically
target PPAR-α. PPAR-γ modulates fatty acid storage and glucose metabolism; specifically, they
reduce insulin resistance, thereby stimulating lipid uptake and adipogenesis. The anti-diabetic
thiazolidinediones (glitazones) target the PPAR-γ receptor (Sheikh et al., 2019).

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2.7 Recommended Intake of DHA and EPA
The potential health benefits associated with consumption of omega-3 fatty acids have led to a
substantial increase in the number of fish oil supplements and fortified food products available
on the market. Based on the evidence from clinical trials and epidemiology studies, many
governments and scientific organizations have set dietary guidelines that specify the
recommended daily intake of EPA and DHA. For instance, the Institute of Medicine at the
National Institute of Health (NIH) in the USA recommends an adequate intake of 1.1 to 1.6 g per
day of omega-3 oils for adults.
The European Academy of Nutritional Sciences (EANS) recommends an average intake of 0.2 g
of omega-3 oil per day. The International Society for the Study of Fatty Acids and Lipids
(ISSFAL) and the American Heart Association recommends consumption of adequate fatty
fishes through daily diet. There should be a suitable balance between the omega-6 to omega-3
fatty acids in the food.

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They recommended that the ratio of omega-6 to omega-3 PUFAs should not exceed 4:1 for
enhancing their bioavailability and metabolism. However, this ratio has risen to about 10:1
because of the elevated consumption of vegetable fats and oils rich in omega-6 PUFAs. The joint
FAO/WHO (Food and Agriculture Organization/World health Organization) Expert Consultation
on Fats and Oils in Human Nutrition suggested that individuals with linoleic to linolenic acid
ratios in excess of 10:1 should consume more foods rich in omega-3 PUFAs, mainly sea foods
(Venugopalaet al. 2021).

Table showing the recommended intake of DHA and EPA

2.8 Cardiovascular health and omega-3 fatty acids

Cardiovascular disease (CVD) represents the leading cause of death worldwide, accounting for
about one-third of all global deaths with significant healthcare-related costs. Previous
observational studies reported beneficial effects in lowering CVD risk with an adequate dietary
intake or dietary supplementation of marine-derived omega 3 polyunsaturated fatty acids
(O3FA), including eicosapentaenoic acid (EPA) and docosahexaenoic acid. The exact
cardiovascular protective mechanism of O3FA remains largely unknown but may be related to
their multiple favorable effects on triglycerides and blood pressure, as well as their anti-
inflammatory, anti-arrhythmic, and anti-platelet effects (Marco et al., 2020).

11
American and European authorities have made recommendations for the clinical use of EPA and
DHA with respect to CVD, as detailed elsewhere. In recognition of the ability of long-chain
omega-3 PUFAs to lower blood triglycerides, in 2019, the American Heart Association (AHA)
updated its earlier recommendation for the use of 2–4 g/day EPA+DHA for triglyceride
lowering, “prescription n-3 fatty acids, whether EPA+DHA or EPA-only, at a dose of 4 g/d, are
clinically useful for reducing triglycerides, after any underlying causes are addressed and diet
and lifestyle strategies are implemented, either as monotherapy or as an adjunct to other
triglyceride-lowering therapies” (Djuricic and Calder, 2023).

With the critical functions of EPA and DHA in cellular protection, several clinical and
epidemiological studies have witnessed the protective role of n−3 PUFAs in chronic and
metabolic disorders, including cardiovascular diseases (CVD), obesity, bipolar disorder,
rheumatoid arthritis, non-alcoholic fatty-liver disease, cognitive impairment, and type II diabetes.
In view of cardioprotection, most studies have supported that higher body status of n−3 FAs
(especially EPA and DHA) can minimize the risk of CVD.

However, some studies have also reported a marginal reduction in coronary heart disease (CHD)
deaths (RR 0.90, 95% CI 0.81 to 1.00; 127,378 participants) and CHD events (RR 0.91, 95% CI
0.85 to 0.97; 134,116 participants) by increased intake of EPA and DHA. Moreover, a slightly
reduced risk of CVD events (RR 0.95, 95% CI 0.83 to 1.07; 19,327 participants) and arrhythmia
(RR 0.73, 95% CI 0.55 to 0.97; 4912 participants) were reported with increased intake of ALA.
Overall, the prospective and observational studies suggest that higher dietary intakes of EPA and
DHA are more beneficial in decreasing the risk of CVD and stroke than are higher intakes of
ALA (Sainiet al., 2021).

The potential for EPA and DHA to have a role in reducing the risk of cardiovascular disease
(CVD) was first identified by studies in the Greenland Inuit, where the low rate of mortality from
myocardial infarction (MI) and ischaemic heart disease was linked to the very high dietary intake
of EPA and DHA. These observations were replicated in other native Arctic populations and the
Japanese population. Subsequently, substantial evidence accumulated from epidemiological and
case-control studies in Western populations indicating that consumption of fish, fatty fish, or
EPA and DHA is associated with reduced risk of mortality from CVD, especially coronary heart
disease (CHD) (Innes et al., 2020).

12
For example, the United Kingdom’s Scientific Advisory Committee on Nutrition/Committee on
Toxicity established dietary recommendations for adults in the general population to intake at
least two portions of fish per week, at least one of which is fatty fish, equated to 450 mg
EPA+DHA per day. The International Society for the Study of Fatty Acids and Lipids set the
target of 500–650 mg EPA+DHA per day for the general population. Similarly, 400–500 mg
EPA+DHA per day with at least 100–120 mg DHA per day was set by the French Agency for
Food, Environmental, and Occupational Health and Safety, while a minimum of 250 mg
EPA+DHA per day is the appropriate daily intake recommended by the Food and Agricultural
Organization of the United Nations (Djuricic and Calder, 2023).

There is a large body of evidence gathered from long-term prospective cohort studies that
consistently demonstrates an association between higher intakes of fish, fatty fish and marine n-3
fatty acids (EPA+DHA) or higher levels of EPA and DHA in the body and lower risk of
developing CVD, especially CHD, having an MI and cardiovascular mortality in the general
population.This cardioprotective effect of EPA and DHA is plausible considering the robust
identification of mechanisms that show that EPA and DHA beneficially modulate a number of
known risk factors for CVD, such as blood lipids, blood pressure, heart rate and heart rate
variability, platelet aggregation, endothelial function and inflammation (Innes et al., 2020).

The omega-3 fatty acid also stimulates the anti-inflammatory transcription factor peroxisome
proliferator activated receptor γ (PPAR γ), which is involved in the prevention and treatment of
CVD and atherosclerosis. PPAR-γ is biologically active after heterodimers with the retinoid X
receptor (RXR); thus, omega−3 fatty acids affects not only PPAR-γ expression but also RXR.
Furthermore, marine n−3 PUFAs can affect RXR activation itself. In addition, omega-3 fatty
acid-activated PPAR-γ will interfere physically with the translocation of NF-kB to the nucleus.
Thus, supplementing the healthy individual with omega-3 fatty acid showed the suppression of
the synthesis of tumor necrosis factor (TNF α) and IL1. It also suppresses IL1 mRNA expression
at the transcription level. Similarly, supplementing healthy humans with omega-3 fatty acids
providing approximately 1.5 g EPA + DHA per day resulted in lower level of expression of
intercellular adhesion molecule (ICAM)-1 on the surface of blood monocytes stimulated ex vivo
with interferon-γ (Shibabaw,2020).

13
Cohort studies associating the intake of fish or marine n-3 fatty acids with cardiovascular or
coronary outcomes have been subject to a number of meta-analyses. These include a 2012
aggregation of seven prospective cohort studies, including 176,441 participants, which
investigated the association between dietary fish, EPA+DHA intake or plasma EPA + DHA
concentrations and heart failure. The investigators found a 15% risk reduction of heart failure
associated with the highest versus lowest fish intake and a 14% lower risk of heart failure for
those with the highest intake compared to those with the lowest dietary intake or plasma
concentrations of EPA + DHA. A comprehensive meta-analysis, published in 2014, investigated
the association between dietary intakes or blood levels of different classes of fatty acids
(including n-3 fatty acids) and combined coronary disease outcomes. The aggregation of data
from 16 studies involving over 422,000 individuals showed a risk reduction of 13% for those in
the top tertile of dietary EPA + DHA intake compared with those in the lower tertile of intake.
Furthermore, the aggregation of data from 13 studies involving over 20,000 individuals showed
risk reductions of 22%, 21% and 25% for those in the top tertile of circulating EPA, DHA and
EPA + DHA, respectively, compared with those in the lower tertile (Innes et al., 2020).

A 2012 meta-analysis of seven prospective cohort studies involving over 176,000 participants
confirmed consistency between long-chain omega-3 fatty acid consumption and a reduced
likelihood of heart failure. The investigators reported a 15% risk reduction of heart failure
associated with the highest intake compared to lowest intake of fish and a 14% lower risk of
heart failure for those with the highest versus lowest dietary intake or plasma concentrations of
EPA+DHA. The nature of these cohort studies does not enable the identified lower risk of heart
failure to be ascribed as a secondary effect of reducing CHD risk or as an effect on cardiac
contractile function independent of CHD. Both are possible. However, the GruppoItaliano per lo
Studio dellaSopravvivenzanell’Infartomiocardico heart failure (GISSI-HF) trial randomized
patients with prevalent heart failure to long-chain omega-3 fatty acids or placebo and reported
significantly reduced mortality in the omega-3 fatty acid group; this would suggest that omega-3
fatty acids act to improve cardiac function in patients with heart failure (Djuricic and Calder,
2023).

14
A summary of the impacts of omega-3 PUFAs on cardiovascular diseases is shown in the Figure
below.

(Venugopalaet al., 2021).

15
 CHAPTER THREE

3.0 Roles of Omega-3 PUFAs in heart health

3.1 Mechanisms by which EPA and DHA reduce the risk of cardiovascular disease
Alpha linolenic acid (ALA) promotes cholesterol conversion into bile acids by the cholesterol
7α-hydroxylase (CYP7). As a result of removing hepatic cholesterol from the circulation by the
synthesis of bile acids, ALA promotes SREBP activation (via SCAP activity), upregulating
LDLR expression and favoring the clearance of LDL cholesterol from the body. Omega 3FAs
such as EPA and DHA reduce TG levels by direct inhibiting liver diacylglycerol acetyl-
transferase, which catalyzes the formation of TG from diacylglycerol, essential for TG intestinal
absorption and fatty acyl-CoA, and the inhibition of the phosphatidic acid phosphohydrolase
required for triacylglycerol (TAG) synthesis from glycerol 3-phosphate (Rodriguezet al., 2022).
Other molecular effects are exerted by inhibiting the acyl-CoA:1,2-diacylglycerol
acyltransferase, increasing mitochondrial and peroxisomal-beta-oxidation in the liver, decreasing
lipogenesis, and increasing plasma lipoprotein lipase activity. EPA’s ability to stabilize cell
membranes, along with its ability to lower cholesterol, may contribute to the 30% and 40%
reductions seen in deaths from CVD and sudden cardiac death (SCD) and the 56% reduction in
cardiac arrest (Rodriguezetal., 2022).

It has been debated since the Inuits study whether omega 3FA consumption is solely responsible
for the CV benefits observed in this population, whether fish intake is beneficial on its own, or
whether an overall healthier diet resulting from a higher fish intake is beneficial in lowering
CVD risk. The Diet and Reinfarction Trial (DART) was the first RCT to show a reduction in
mortality during the two years after myocardial infarction (MI) among men who were advised to
eat about 300 g of FO per week or who took an equivalent amount of n-3 fatty acids in the form
of FO supplements. Later, these findings were confirmed by the GISSI– Prevenzione trial, the
Lyon Diet Heart Study, and various cohort studies Omega 3FAs lower TG-rich lipoproteins and
increase anti-aggregatory and vasodilatoryprostanoids such as prostacyclin, combating
thrombosis and vasospasm. It can incorporate into the mitochondria and plasma membranes,
stabilizing them and preventing them from oxidation, which is believed to have a role in
preventing arrhythmias(Rodriguez et al., 2022).

16
(Cavoet al., 2017)

Fig 1: Mechanisms of action of EPA & DHA

Additionally, omega 3 fatty acids are precursors to the synthesis of specialized mediators capable
of combating inflammation and have been demonstrated to decrease proinflammatory cytokines
such as interleukine-6 and tumor necrosis factor-α and inhibit the activation of the ikappaB
kinase and nuclear factor-κB, as well as several other transcription factors that inhibit reactive
oxygen species. These anti-inflammatory properties are believed to interfere less with self-
defense than direct anti-inflammatory treatments. A combination of these mechanisms is
believed to contribute to the CVD protection associated with omega-3FA consumption and the
added benefit on multiple other systems and pathologies (Rodriguez et al., 2022).

17
3.2 Risk factors for CVD that are targeted by EPA and DHA
Beneficial modification of a broad range of risk factors probably explains the protective effect of
long-chain omega-3 fatty acids toward CVD. These risk factors include blood triglyceride
concentrations, blood pressure, thrombosis, cardiac function, vascular function, and
inflammation, which are all improved by long-chain omega-3 fatty acids. Concerning the
modulation of blood lipid concentrations, there is overwhelming evidence that both EPA and
DHA have a triglyceride-lowering effect, apparently with a slightly higher impact for DHA.
Although EPA and DHA do not show a significant total cholesterol-lowering effect in humans,
they do have an independent effect on different lipid subfractions, with EPA lowering high-
density lipoprotein (HDL)3-cholesterol and DHA increasing the more cardioprotective HDL2-
cholesterol (Djuricic and Calder, 2023).

It has also been suggested that DHA increases low-density lipoprotein (LDL)-cholesterol (LDL-
C) more than does EPA and increases LDL particle size, an effect that was not seen for EPA.
DHA is reported to reduce mitochondrial oxidative stress and cytochrome c oxidase activity
while increasing manganese-dependent superoxide dismutase activity. The anti-inflammatory
properties of long-chain omega-3 fatty acids may also be important in this regard, since
inflammation induces oxidative stress. Recent meta-analyses have found a significant reduction
in platelet aggregation with long-chain omega-3 fatty acids, with a greater impact observed in
nonhealthy participants and considerable improvement of vascular endothelial function through
increasing flow-mediated dilatation. EPA and DHA have been reported to lower systolic and
diastolic blood pressure and heart rate (Djuricic and Calder, 2023).

Interestingly, DHA is reported to be more effective than EPA at lowering blood pressure and
heart rate in normotensive individuals, while neither EPA nor DHA had any effect in
hypertensive diabetic patients. Overall, there is strong evidence that both EPA and DHA
beneficially modify a range of risk factors for CVD,this most likely accounts for the reduced risk
of developing and mortality from CVD reported in cohort studies.

18
3.3 Therapeutic roles and mechanisms of omega-3 PUFA in lowering of triglycerides
Omega-3 fatty acids are well known for lowering TAG and VLDL-cholesterol levels in the
blood. However, the mechanisms by which EPA and DHA minimize serum TAGs are still not
fully understood. Numerous preclinical and clinical studies provide compelling proof that EPA
and DHA minimize synthesis and secretion of the hepatic VLDL-TAG. In addition, it
also increases LPL mediated TAG clearance from circulating TRL particles in pheripheral
tissues. As regards hyperchylomicronemia, by upregulating peripheral LPL function, both EPA
and DHA will accelerate the clearance of TAG chylomicrons equally. ω-3 PUFA reduces serum
TAG via several mechanisms such as by reduces hepatic lipogenesis, increases β-oxidation,
reduces VLDL-TAGs hepatic output or its secretion via facilitating apoB-100 degradation.
Furthermore, by decreasing the endoplasmic reticulum membrane-bound transcription factor
SREBP (sterol regulatory element-binding protein)-1c all genes involved in the de novo
lipogenesis pathway are decreased by interfering with the function of LXR/RXR heterodimer.
(Shibabaw,2020).
Taken together, TAG-lowering mechanism of ω-3 PUFAs are by either targeting lipogenic
enzymes at the proteins level or via modulating different gene encoding involved in the
homeostasis of lipid metabolism at transcription level.

3.4 Anti-inflammatory mechanism of omega-3 PUFA in cardiovascular disease

Inflammation, which is the body’s response to infection and cellular injuries, is mainly
manifested by the production of inflammatory mediators like cytokines and reactive oxygen
species (ROS), as well as the expression of adhesion molecules and arachidonicacid-derived
eicosanoids. Intake of PUFAs competitively inhibits the metabolism of arachidonic acid, thereby
increasing the production of omega-3-derived eicosanoids with anti-inflammatory effects.
Studies have reported that EPA and DHA consumption also has anti-inflammatory effects by
decreasing the expression of genes involved in inflammatory- and atherogenic-related pathways.
A recent meta-analysis of 31 randomized clinical trials reported that omega-3 PUFA
supplementation reduced serum inflammation markers (Venugopalaet al. 2021).

Several mechanisms have been suggested to explain the cardioprotective effects of EPA and
DHA. These include preventing arrhythmias, reducing plasma triacylglycerol, decreasing platelet
aggregation, arterial cholesterol delivery, and arterial inflammatory reaction, reducing the
expression of adhesion molecule, and the growth factor derived from platelets.

19
The underlining pathogenesis of atherosclerosis of CVD secondary to HTG is due to endothelial
dysfunction and inflammation. This is an opening window of a new avenue for the possible
impact of ω-3 PUFA on antiatherosclerotic. The most important mediators involved in the
homeostasis of the inflammatory response are generated from the metabolism of ω -3 PUFA
through the cyclooxygenase (COX) and lipoxygenase (LOX) pathways. These pathways are
synthesizing resolvins, resolution phase interaction products produced from both EPA (E-series,
RvE1-2) and DHA (D-series, RvD1-6), as well as protectins and maresins produced from DHA.

ω -3 PUFA can putatively exert anti-inlfammatory action by the production of distinct n-3
PUFA-derived metabolites, such as resolvins D (RvD) and E (RvE) series, maresins (MaR), and
protectins (PD), collectively referred to as specialized pro-resolving mediators (SPMs) and acing
as powerful anti-inflammatory agents(Shibabaw,2020).An epidemiological study of Greenland
Inuit found that autoimmune diseases, such as bronchial asthma and psoriasis, are extremely rare
among Inuit primarily subsisting on fish. Several subsequent animal and human studies provided
evidence that omega-3 PUFA, particularly EPA, have anti-inflammatory and immune
modulatory properties.

Omega-3 PUFA metabolites modulate eicosanoid and other immune pathways, leading to altered
inflammatory responses and modulation of molecules or enzymes associated with different
signaling pathways involving normal and pathological cell activity. Omega-3 fatty acids are
integrated into membrane phospholipids, having direct effects on gene expression.Its anti-
inflammatory effects are mediated by inhibiting the pathways of arachidonate 5-lipoxygenase or
5-lipoxygenase (5-LOX) in neutrophils and monocytes and by inhibiting the pro-inflammatory
leukotriene B4 (LTB4) (potent chemoattractant) PGE2 and TXB2-mediated activity of
leukotriene B5 (LTB5).Resolvins, in particular RvE1, partially antagonize LTB4 receptors on
polymorphonuclear (PMN) cells to inhibit NF-kB activation, suppress pro-inflammatory
cytokine (IL6, TNFα, IL1, and C-reactive protein (CRP)) gene production in aorta, and reduce
inflammatory cell (macrophage) infiltration into the intima, thereby attenuating atherosclerosis
and plaque formation (Yasuhiro and Ichiro, 2020).

It affects the expression of several key proteins as modulators of many genes involved in
inflammation, lipid metabolism (promoting LDL oxidation), and atherosclerosis mainly by
inhibiting the expression of inflammatory transcription factor nuclear factor-kB signaling by
blocking IkB phosphorylation, therefore reducing the transcriptional expression of inflammatory

20
genes such as several cytokines (TNF, IL1β, IL6, and IL8), adhesion molecules, and COX-2
(Shibabaw,2020).Omega-3 PUFA prevents the induction of arrhythmia caused by persistent
sodium overflow in myocardial cells after MI. In connection with such membrane-stabilizing
effects, the administration of omega-3 PUFA also decreases CVD events in patients with heart
failure, reduces hospitalization due to heart failure, and lowers mortality.

The use of omega-3 PUFA has been incorporated in the American College of Cardiology (ACC)
Foundation/American Heart Association (AHA) guidelines for the management of heart failure
in 2013. Indeed, according to the OMEGA-REMODEL study, the administration of EPA/DHA
preparation at a high dose of 4 g per day for 6 months inhibits myocardial fibrosis after acute MI,
thereby improving remodeling and protecting cardiac function (Yasuhiro and Ichiro, 2020).

The potential benefits of fish oil (omega-3 fatty acids) consumption to reduce CVD risk remains
controversial. On one hand, Food and Drug Administration (FDA) has approved that ω-3 PUFA
(Lovaza) as the primary option of HTG treatment. Supporting this, in the Japan EPA lipid
intervention study (JELIS) trial showed that the administration of 1.8 g/day of EPA was more
effective than statin monotherapy in minimizing the risk of coronary events in a patient present
with hypercholesterolemia and under statin treatment. The mechanisms for the effects of ω-3
PUFAs are either through multiple signaling pathways by incorporation into membrane
phospholipids or through acting as ligands for a variety of nuclear transcription factors.

Although not yet fully understood, some of the proposed mechanisms of action of omega-3
PUFA exhibit cardioprotective effects through increased mitochondrial role of β-oxidation
reduced endogenous TAG synthesis and increased plasma lipoprotein lipase (LPL) activity, in
turn, decreasing plasma TRL and its corresponding atherogenic TRL ruminants. Moreover,
mechanisms underlying the anti-inflammatory actions of EPA and DHA include altered cell
membrane phospholipid fatty acid composition and inhibition of activation of the pro-
inflammatory transcription factor nuclear factor κB. Taken together, ω -3 PUFA reduces the
expression of inflammatory genes and promotes the expression of anti-inflammatory
transcription factor like peroxisome proliferator-activated receptor ɣ (Shibabaw,2020).

Cardiovascular disease is exacerbated by combining HTG with high total cholesterol (TC) and
LDL-C. In addition, increased non-HDL-C, increased apolipoprotein B-100 (apoB-100) and
small dense LDL (sdLDL or LDL-III) particles, LPL gene mutation, and increased
apolipoprotein C-III (apo C-III) levels are responsible for the increased risk of CVD, all of which

21
may or may not be independent of each other. Very-low-density lipoprotein and its derivative
carry cholesterol thus form a non-HDL-C part. Non-HDL-C is the sum of atherogenic
lipoproteins bearing cholesterol such as VLDL, IDL, LDL, lipoprotein (a), and lipoprotein
remnants.
For CHD risk, it is a better predictive biomarker than LDL-C alone. A rise in nonHDL-C
atherogenic levels, therefore, improves the delivery of cholesterol to endothelial promotes
atherosclerotic progression and raises the risk of plaque rupture resulting in an increased risk of a
CVD event (Shibabaw,2020).
Apart from non-HDL-C, a test of apoB-100 is the second better predictor of CVD risk than LDL-
C alone. There is one apoB-100 molecule in every LDL, VLDL, and IDL particles. Thus, apoB-
100 represents the number of atherogenic lipoproteins in circulating, and this is a strong
predictor of CHD risk. Increasing TRL by increasing VLDL particles and their remnant increases
levels of apoB-100 and increases risk of CHD. Furthermore, the elevation of TAG levels is often
correlated with the formation of sdLDL particles. The sdLDL particle production also results
from the interplay of various enzymes, such as LPL, hepatic lipase, and cholesteryl ester transfer
protein. Although all LDL particles are atherogenic, sdLDL particles may be more atherogenic
and life-threatening risk factor for stimulation of atherosclerosis and CVD. As with HTG, not all
analyses support that LDL particle size is an independent predictor of CHD. A greater ability to
penetrate arterial walls, less oxidative stress resistance, and low affinity for LDL receptors are
the most likely atherogenic mechanisms of sdLDL particles (Shibabaw,2020).

3.5Omega-3 polyunsaturated fatty acids and hypertension

The characteristic manifestation of hypertension is a chronic increase in arterial blood pressure
(BP), generally defined as systolic (SBP) and diastolic (DBP) blood pressure values above 139
and 89 mmHg, respectively. 90%–95% of cases represent primary hypertension, which arises
independently of other conditions, and is associated with lifestyle and genetic factors.
Hypertension is divided into categories depending on severity and risk of complications, and
small reductions in blood pressure (such as 1 mmHg), even for those considered normotensive,
can represent a significant improvement in multiple predicted health outcomes such as all-cause
mortality or cardiovascular disease-related mortality. Hypertension is often characterised by
impaired vasodilation involving dysfunction of multiple vasodilatory mechanisms (Berceaet al.,
2020).

22
The first meta-analysis about omega-3 treating hypertension indicated that diet supplementation
with a relatively high dose of omega-3 could lead to clinically relevant blood pressure
reductions.
However, use of omega-3 as antihypertensive therapy will require demonstration of long-term
efficacy and patient acceptability of lower doses. Another systematic review suggested that
omega-3 is more effective in some cases than other lifestyle-related interventions (such as
increasing physical exercise, limiting alcohol or reducing sodium intake) for lowering blood
pressure among hypertensive populations without taking antihypertensive pill.
However, it could not provide a convincing proof that there is a strong association of increased
omega-3 consumption and reduced incidence of elevated blood pressure according to a review
published in 2016. Some recent clinical trials proposed that omega-3 had a hypotensive efficacy
in hypertensive patients. Therefore, this systematic review and meta-analysis might change the
conclusion about omega-3 treating hypertension patients (Tao et al., 2020).

Attention was first drawn to the potential benefits of seafood and “fish oils” when several
epidemiological studies reported a decreased incidence of cardiovascular disease, including
hypertension, in regions of the world with a high consumption of these foods. This led to a large
body of clinical, epidemiological, in vivo and in vitro data that identified ω-3 PUFAs derived
from marine sources as having cardiovascular effects. Numerous reviews and meta-analyses
have examined the evidence supporting cardiovascular effects of ω-3 PUFAs. Multiple proposed
mechanisms have emerged for these effects, including the lowering of blood pressure.

The blood pressure lowering and vasodilatory effects of ω-3 PUFAs have been investigated, as
will be discussed, using randomised controlled studies, epidemiological studies, in vivo animal
studies and in vitro studies. The beneficial effects of ω-3 PUFAs on blood pressure in
hypertensive or normotensive human subjects have been reviewed. Long-term cohort studies and
short-term randomised controlled trials have identified associations between increased
consumption of ω-3 PUFAs and lowered blood pressure in hypertensive individuals. Therefore,
most data point to the fact that individual ω-3 PUFAs have differential effects on blood pressure
and DHA has been suggested to be more potent in this respect (Berceaet al., 2020).

23
In JELIS trial, a lipid intervention trial shows daily treatment with 1.8 g EPA in combination
with statins proved to be more effective than statins alone in reducing cardiovascular events in
patients with high cholesterol.A meta-analysis in 2022, demonstrated that additional omega-3 FA
supplementation could decrease the incidence of major adverse cardiovascular events (MACEs),
cardiovascular death, and myocardial infarction (MI).The subgroup analysis found that omega-3
FA supplementation can reduce the risks of MACE and MI in people with acute MI, the risk of
MI and stroke in people with CHD, and the risk of MI in patients with high risks of CHD
(Critselis et al., 2023).

In addition, according to the used omega-3 FA dose in RCTs, study shows that moderate-dose
omega-3 FA ranging from 0.8 to 1.2 g attenuated the incidence of MACE, cardiovascular death,
and MI. A meta-analysis of 14 large-scale RCTs conducted to investigate the risk of
cardiovascular events after receiving omega-3 FA supplementation shows that omega-3 FA
supplementation can reduce the risk of MACE, cardiovascular death, and MI. Additionally, it
exhibits good clinical benefits for primary prevention and secondary prevention of CHD.
Omega3 FA supplementation application dose that ranges from 0.8 to 1.2 g exhibits more
superiority than other doses in reducing cardiovascular risks (Critseliset al., 2023).

A study by Critseliset al.,(2023) revealed that participants who regularly consumed seafood
exhibited a decreased risk of developing and/or dying from CVD. The study is the first of its
kind to evaluate the long-term effects of consuming seafood and/or small fish rich in n-3 fatty
acids on mainly 10-year, and less prominently on 20-year, CVD incidence and mortality in a
community dwelling Mediterranean population, participants with high consumption (>1
serving/week) of small fish rich in n-3 fatty acids had a significantly reduced risk of 10-year
CVD incidence and exhibited a 76% decreased 10-year risk of dying due to CVD, even among
normotensive individuals. When the analyses were focused on 20-year CVD incidence and
mortality, similar but not significant associations were detected. The above findings highlight the
importance for augmenting the consumption of seafood, and particularly small fish rich in n-3
fatty acids, for deterring particularly both 10-year CVD incidence and attributable mortality in
the general population, including among those without apparent excess CVD risk (i.e.,
normotensive individuals).

24
Findings of a 2019 meta-analysis of 13 RCTs and of a 2021 meta-analysis of 40 trials indicate a
dose-dependent therapeutic effect of EPA and DHA: a statistically significant inverse linear
dose–response relationship was found between EPA + DHA administration and risk of CVD
outcomes. It was estimated that every 1 g/day EPA + DHA corresponded to a 9% and 7% lower
risk of myocardial infarction and total coronary heart disease, respectively, and to a 5.8% lower
risk of CVD events.The Reduction of Cardiovascular Events with Icosapent Ethyl Intervention
Trial (REDUCE-IT) involved patients with established cardiovascular risk or with diabetes and
elevated triglyceride level (1.52 to 5.63 mmol/L), who were receiving statin therapy. In the trial,
they received 4 g/day of a formulation rich in EPA ethyl ester (providing 3.6 g of EPA daily).
(Djuricic and Calder,2023).

The decreased CVD mortality risk that was observed in participants consuming >2 servings of
marine fish and seafood on a weekly basis was notable in subpopulation groups at increased
CVD risk, such as women, the elderly, and those with low adherence to the Mediterranean Diet.
However, it is noteworthy that high intake of small fish rich in n-3 fatty acids was associated
with a reduction in CVD mortality even in otherwise healthy normotensive individuals. Previous
reports show that high intakes of fish rich in n3 fatty acids may decrease the risk of CVD
mortality. The beneficial health impacts of consuming small fish rich in n-3 fatty acids on CVD
health are primarily attributed to their rich EPA and DHA content, as well as their elevated
composition of minerals and vitamins known to enhance cardiometabolic health, such as
calcium, phosphorus, magnesium, zinc, and vitamin D (Critseliset al., 2023).

Another recent trial, the Effect of Vascepa on Improving Coronary Atherosclerosis in People
with High Triglycerides Taking Statin Therapy (EVAPORATE) trial used the same EPA ethyl
ester preparation and the same dose as used in REDUCE-IT. EVAPORATE demonstrated that
EPA might directly promote atherosclerotic plaque regression in hypertriglyceridemic
individuals. Thus, omega-3 PUFAs may directly target atherosclerotic plaques in patients who
already have advanced CVD, as suggested by earlier studies in mice and humans. The positive
findings of REDUCE-IT and EVAPORATE contrast with those of the more recently published
Long Term Outcomes Study to Assess Statin Residual Risk with Epanova in High
Cardiovascular Risk Patients with Hypertriglyceridemia (STRENGTH) trial.

In this trial, statin-treated patients with hypertriglyceridemia and high cardiovascular risk were
given 4 g/day of a formulation of highly purified EPA and DHA as free fatty acids (providing

25
approximately 2.2 g EPA and 0.8 g DHA daily) or corn oil as placebo. There was no significant
difference in a composite outcome of major adverse cardiovascular events between the groups
and the trial was stopped early (Djuricic and Calder,2023).

One explanation could be that EPA and DHA have differential effects on some cardiometabolic
risk factors, and therefore a formulation of pure EPA could have a different effect from a
formulation that combines EPA and DHA. Both EPA and DHA lower triglycerides, an effect
seen in both REDUCE-IT and STRENGTH to a similar extent (decrease of 18%). DHA is
reported to increase LDL-cholesterol more than EPA does. This could explain why LDL-
cholesterol was increased by 3% with EPA + DHA treatment in STRENGTH while LDL-
cholesterol was lowered by an average of 6.6% in REDUCE-IT which provided EPA but not
DHA.

Several biologic mechanisms have been proposed through which marine-derived omega-3 PUFA
can reduce biological pathways related to the occurrence of cardiometabolic diseases and cancer,
including anti-inflammatory, anti-oxidation, regulation of lipid metabolism, and amelioration of
insulin resistance. Chronic systematic inflammation is recognized as an important contributor to
endothelial dysfunction, phospholipid oxidation, insulin resistance, as well as tumor
development 4 and growth, all of which are believed to play a role in the subsequent
development of metabolic diseases, certain cancers and mortality (Cholewski, et al. 2018).

DHA increases HDL-cholesterol, especially the HDL2 subfraction which is cardioprotective, but
EPA does not affect HDL-cholesterol. However, both REDUCE-IT and STRENGTH reported
higher HDL-cholesterol with omega-3 PUFAs. One final difference between REDUCE-IT and
STRENGTH is the choice of placebo, mineral oil in REDUCE-IT and corn oil in STRENGTH.

It has been argued that mineral oil may have adverse effects on some cardiovascular risk factors,
therefore making omega-3 PUFAs appear beneficial, as in REDUCE-IT. A recent systematic
review of mineral oil when used as a placebo indicated inconsistent effects on blood lipids and
other biomarkers and reached the conclusion that mineral oil is not likely to be responsible for
effects seen in trials like REDUCE-IT and EVAPORATE. Thus, at this point in time, the exact
reasons for the different findings of REDUCE-IT and STRENGTH are not clear (Djuricic and
Calder,2023).

26
Recent AHA advisories support the use of marine n-3 fatty acids in the treatment of
hypertriglyceridemia and in a range of patients with CVD and the use of fish for primary
prevention of CVD. Both EPA and DHA beneficially modify a range of risk factors, although
DHA may be more effective. Nevertheless, the highly successful REDUCE-IT used pure EPA,
although at a high dose (Innes and Calder, 2020).

3.6 Controversies Surrounding omega-3 fatty acids

The initial landmark trial for omega-3 supplementation, GISSI-P, showed that 1 g/day Ω-3 (EPA
+ DHA) decreased the risk of death, non-fatal acute MI and stroke in patients with recent MI (<3
months). With approximately 11,000 subjects, almost 4 years of follow up with risk reduction of
14% in the combined primary endpoint and an overall 20% reduction in fatal events, there was
initial excitement. However, multiple subsequent studies have failed to corroborate those results.
Since GISSI-P(GruppoItaliano per lo studiodellasopraavvivenzanell’ micardico) was published,
however, there have been several fundamental developments in CV disease (CVD) research; for
example, how intervention study arms are treated with aggressive medical therapy. Only 5% of
GISSI-P patients received statin therapy at baseline, in contrast to the OMEGA trial, in which
81% of patients received statin therapy at baseline. The lack of an omega-3 benefit in the
OMEGA trial may have been due to the low rate of sudden cardiac death, total mortality and
major adverse CV events (MACE) within 1 year follow up after MI in those that received this
improved optimal medical therapy (Elagiziet al., 2021).

Nevertheless, recent meta-analyses confirm the benefit from long-chain omega-3 PUFAs and
that these benefits are dose dependent. Reasons for inconsistencies among RCT findings may
relate to the dose of long-chain omega-3. Despite the inconsistencies in the literature, there are
recommendations supporting the use of long-chain omega-3 PUFAs to treat hypertriglyceridemia
and patients with CVD (Djuricic and Calder,2023).

Further controversy resulted from articles in popular media which wrongly suggested that
omega-3 ingestion, including that from fish, increased prostate cancer risk, with many
commentaries thereafter advising against the use of supplemental fish oil. Habitual high intake of
fish and seafood in men who have prostate cancer, however, has been linked to significantly
improved survival.

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Accordingly, Japanese men, who consume approximately 8 times more fish than their American
counterparts, have a rate of prostate cancer mortality many-fold lower. Ultimately, a more
thorough review of the literature would suggest that increased omega-3 consumption does not
increase prostate cancer risk, but decreases prostate cancer mortality in addition to reducing
sudden cardiac death and CV events.

It is also important to consider the populations who show beneficial findings from omega-3
intake as well. Trials such as GISSI-P and JELIS were conducted in Italian and Japanese
populations, respectively, who have higher baseline omega-3 levels due to lifestyles composed of
increased fish and seafood intake as compared to the typical diet of Western populations.

One possibility is that there may exist a threshold effect regarding endogenous omega-3 levels
and CVD benefits, as some studies have shown that participants with lower baseline omega-3
levels have more impactful results after supplementation. Therefore, populations such as those in
GISSI-P and JELIS (Japan Eicosapentaenoic acid lipid intervention study) who may consume
more fish on a regular basis and therefore have higher baseline omega-3 levels may require a
lower dose of omega-3 to achieve a clinical benefit, as opposed to Western populations who on
average consume less fish and likely require a higher omega-3 dose to achieve the same
therapeutic benefit. Supporting this notion are the findings of the REDUCE-IT trial, in which
higher omega-3 doses (4 g/day of highly purified EPA) were associated with significant CVD
benefits, and, in particular, a more robust benefit in the US cohort (Elagiziet al., 2021).

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 CHAPTER FOUR

4.0 Summary, conclusion and recommendation

4.1 Summary
In summary, the cumulative results arising from observational studies regarding fish intake,
randomized clinical trials regarding fish oil supplementation, and related experimental studies
suggest that regular fish consumption may decrease the incidence of coronary heart disease or
acute coronary syndrome, albeit with uncertain impacts on total CVD events in the general
population.

In particular, a pooled analysis of four large-scale international cohort studies, including 191,558
people from 58 countries, indicated that minimal fish intake (approximately 2 servings or 175 g
weekly) is associated with lower risk of major CVD events and attributable mortality among
patients with prior CVD, but not in the general population. In contrast, the results suggest that
adequate intake of fish rich in n-3 fatty acids may induce 76% decreased risk in 10-year CVD
mortality in the general population, and respectively 69% diminished risk even among low risk
normotensive individuals (Critselis et al., 2023). From a clinical point of view and based on all
available evidence, n-3 fatty acids may be an effective treatment option for patients with high-
risk CVD to reduce the risk of major CVD events, as well as residual risk.

4.2 Conclusion
Regular intake of sufficiently high quantities of polyunsaturated fatty acids (PUFAs) has been
needed to reduce the incidences of innumerable types of ailments and chronic diseases, including
psoriasis, bowel diseases, mental illnesses, cancer, rheumatoid arthritis, cardiovascular diseases,
diabetes, pulmonary disorders, coordination disorders, movement illnesses, obesity, and weak
bone. As a result, the consumption of foods rich in PUFAs is strongly encouraged by health
agencies around the globe. Fish oil is one of the major dietary sources of PUFAs, especially
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are believed to have
particularly potent biological activities and health effects. The overall PUFA profile of fish oils
depends on fish species, sex, maturity, diet, and environment, which may impact their potential
health benefits (Venugopala et al. 2021).
Both omega-6 and omega-3 PUFAs lower CVD risk through multiple interacting mechanisms;
the omega-6 PUFA LA acts mainly through lowering LDL-cholesterol, while both the omega-3
PUFAs EPA and DHA lower triglycerides, promote blood flow and cardiac and vascular

29
function and control thrombosis and inflammation. The actions of pro-resolution mediators
produced from EPA and DHA are particularly important. The breadth of the effects of omega-3
PUFAs explains why they have roles in both the prevention and treatment of CVDs, especially of
CHD, and in the control of inflammatory conditions and various neurological disorders(Djuricic
and Calder, 2021). PUFAs display a plethora of biological activities at the molecular and cellular
levels that result in effects on cell and tissue function linked to health outcomes and disease risk.
Antioxidant and anti-inflammatory properties of omega-3 PUFAs are important and are
recognized as contributing to reduced disease risk and severity (i.e., better health). Furthermore,
these properties offer therapeutic opportunities in conditions marked by excessive inflammation
and oxidative stress.

4.3 Recommendations
Further clinical trials are necessary for elucidating the efficacy and appropriate fish intake levels
for securing optimal CVD health particularly in otherwise low risk normotensive individuals.
Additional investigations into the molecular mechanism of action for EPA and DHA may yield
new insights into the etiology and treatment of cardiovascular diseases and other diseases related
to heart. Identifying more clearly the dose-dependent effects of EPA and DHA, separately and
together, on cardiovascular risk factors and on clinical outcomes is important to further develop
long-chain omega-3 PUFAs as effective therapeutic agents for CVD. Furthermore, robust
primary prevention trials are still needed.

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