nutrients

Review
Beyond Fish Oil Supplementation: The Effects of
Alternative Plant Sources of Omega-3
Polyunsaturated Fatty Acids upon Lipid Indexes and
Cardiometabolic Biomarkers—An Overview
Heitor O. Santos 1, *, James C. Price 2 and Allain A. Bueno 2
1 School of Medicine, Federal University of Uberlandia (UFU), Uberlandia 38408-100, Brazil
2 College of Health, Life and Environmental Sciences, University of Worcester, Worcester WR2 6AJ, UK;
[email protected] (J.C.P.); [email protected] (A.A.B.)
* Correspondence: [email protected]




Received: 27 September 2020; Accepted: 14 October 2020; Published: 16 October 2020


Abstract: Cardiovascular diseases remain a global challenge, and lipid-associated biomarkers

can predict cardiovascular events. Extensive research on cardiovascular benefits of omega-3
polyunsaturated fatty acids (n3-PUFAs) is geared towards fish oil supplementation and fish-rich
diets. Nevertheless, vegetarianism and veganism are becoming more popular across all segments
of society, due to reasons as varied as personal, ethical and religious values, individual preferences
and environment-related principles, amongst others. Due to the essentiality of PUFAs, plant sources
of n3-PUFAs warrant further consideration. In this review, we have critically appraised the
efficacy of plant-derived n3-PUFAs from foodstuffs and supplements upon lipid profile and selected
cardiometabolic markers. Walnuts and flaxseed are the most common plant sources of n3-PUFAs,
mainly alpha-linolenic acid (ALA), and feature the strongest scientific rationale for applicability into
clinical practice. Furthermore, walnuts and flaxseed are sources of fibre, potassium, magnesium,
and non-essential substances, including polyphenols and sterols, which in conjunction are known
to ameliorate cardiovascular metabolism. ALA levels in rapeseed and soybean oils are only
slight when compared to flaxseed oil. Spirulina and Chlorella, biomasses of cyanobacteria and
green algae, are important sources of n3-PUFAs; however, their benefits upon cardiometabolic
markers are plausibly driven by their antioxidant potential combined with their n3-PUFA content.
In humans, ALA is not sufficiently bioconverted into eicosapentaenoic and docosahexaenoic acids.
However, evidence suggests that plant sources of ALA are associated with favourable cardiometabolic
status. ALA supplementation, or increased consumption of ALA-rich foodstuffs, combined with
reduced omega-6 (n6) PUFAs intake, could improve the n3/n6 ratio and improve cardiometabolic and
lipid profile.

Keywords: alpha-linolenic acid; flaxseed; lipids; omega-3; walnuts

1. Introduction
Cardiovascular diseases remain a major Public Health concern, with lipid-associated biomarkers
being trusted predictors of major cardiovascular events [1,2]. Added to the pharmacological strategies
and conscious effort to improve the patients’ lifestyle by adequate nutrition and physical activity,
nutraceutical strategies which include certain supplements, herbal extracts and functional foods, may be
a helpful complementary approach for individuals with cardiovascular disease and dyslipidaemia [3–9].
The dietary replacement of saturated fats for polyunsaturated fatty acids (PUFAs) has
shown beneficial effects upon lipid profile [10], as well as long-term protective benefits against

Nutrients 2020, 12, 3159; doi:10.3390/nu12103159 www.mdpi.com/journal/nutrients

Nutrients 2020, 12, 3159 2 of 19

major cardiovascular events and associated clinical complications [11,12]. For decades, omega-3
polyunsaturated fatty acids (n3-PUFAs) from either marine sources or fish oil (FO) supplementation
were broadly referred to in cardiology guidelines [13–15]. For example, amongst several clinical
investigations, Sagara et al. in 2011 showed that 2 g of DHA daily for five weeks improved blood
pressure and lipid profile in a sample population of 38 middle-age men with hypertension and
hypercholesterolaemia [16].
On the other hand, however, Manger et al. in 2010 did not find a significant trend in reduced
risk of coronary events with increased consumption of n3-PUFAs from fish and fish supplements.
Nonetheless, Manger argues that their sample population had a high intake of n3-PUA to begin with,
and possibly the lack of association could have been attributed to a ceiling effect of n3-PUFAs [17].
Interestingly, a robust meta-analysis published in 2018, which included 79 Randomized Control
Trials (RCTs) and a total of 112,059 participants, showed that n3-PUFAs supplementation actually
did not show significantly greater efficacy on reducing the occurrence of cardiovascular events [18].
Such results must be interpreted carefully due to factors such as methodology employed, varied sample
population investigated, other factors beyond the scope of the study, and a potential risk for bias.
At the same time, it has also been demonstrated that plant sources of n3-PUFAs have shown some
potential against cardiovascular events and dyslipidaemia [18].
The findings on fish consumption and FO supplementation upon cardiovascular health do not
disregard the merit of investigating the usefulness of plant-derived n3-PUFAs in clinical practice.
Studies on the potential efficacy of plant-derived n3-PUFAs become further justified as dramatic
reductions of fish stocks have been reported in the North Atlantic Ocean and Mediterranean Sea [19,20].
In the present review, we have critically appraised the efficacy of plant-derived n3-PUFAs from
foodstuffs, as well as its supplementation, upon the modulation of lipid profile and selected
cardiometabolic markers. More specifically, we searched for the effects of chia seeds, flaxseeds, walnuts,
as well as Spirulina and Chlorella. As those foodstuffs are gaining more popularity, a phenomenon
possibly attributed to the increased awareness of environmental issues related to overfishing, combined
with the increasing trend towards veganism, vegetarianism and flexitarianism across all segments of
society, it is of greatest interest to clarify the clinical potential of plant sources of n3-PUFAs.

2. Methods
We employed the electronic databases Pubmed/Medline and Google Scholar to identify relevant
publications. Randomised Controlled Trials (RCTs) written in English were the chosen sources of
results as a means of translating current research findings into clinical practice. Preferred Reporting
Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [21] were used to evaluate and
select the RCTs.
Limited to RCTs, we searched for plant sources of ALA by using the following combinations
of Medical Subject Heading (MeSH) keywords: (“Chia Seeds” OR “Flaxseeds” OR “Hemp Seeds”
OR “Walnuts” OR “Seaweeds” OR “Spirulina” OR “Chlorella”) AND (“LDL-C” OR “Low-Density
Lipoprotein Cholesterol” OR “Total Cholesterol” OR “Triglycerides” OR “HDL-C” OR “Low-Density
Lipoprotein Cholesterol” OR “Blood Pressure” OR “Cardiovascular Disease” OR “Cardiometabolic
Risk” OR “Inflammatory Biomarkers” OR “Proinflammatory Cytokines”). The period covered in the
search included inception to August 2020. Summary findings from the selected papers are presented in
Supplementary Tables S1–S3. After perusal of such findings, we discussed key aspects and manually
expanded the review by selecting further articles that have investigated nutrition facts, mechanisms of
action and clinical applications.
As the number and robustness of clinical studies that have investigated the metabolic effects of
plant-derived n3-PUFAs are only a fraction of those that employed FO, we have also summarized in
Supplementary Table S4 the outcomes of 38 selected FO supplementation studies identified using the
key terms listed above and published in the last five years, with a combined sample population of
Nutrients 2020, 12, 3159 3 of 19

4136 individuals. The evidence gathered confirms that whilst some studies did show improvements in
metabolic biomarkers after FO supplementation, some others did not.

3. Metabolic Pathways

3.1. Conversion of ALA in Humans

Not only the insufficient intake of dietary n3-PUFAs, but also the inefficient elongation and
desaturation of ALA to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in humans,
result in low n3 long chain-PUFA content in blood and other peripheral tissues [22,23]. Accordingly,
North America, Central and South America, and Africa, are geographical examples where EPA and
DHA concentrations are endemically low [24]. More specifically, the intake of n3-PUFAs in the average
USA population is low or very low [14].
Approximately only 5% of ALA is converted to EPA whilst less than 0.5% is converted to
DHA in humans, although mammals have the essential enzymes used in this pathway [25–27].
A proof-of-concept study published in 2010 showed in newborn infants that only approximately
0.04% of ALA administered was elongated and desaturated to circulating EPA, whilst the subsequent
conversion of EPA to DHA was comparatively more efficient [28]. The very low ALA to EPA
bioconversion ratio identified in that study does not account for the EPA that was actually incorporated
into solid tissues, but it does confirm the suggestion that even large amounts of dietary ALA will
probably have negligible effects on plasma DHA levels [27].
Moreover, the conversion of ALA to EPA and subsequently DHA appears to be more efficient in
women than in men, a phenomenon that could probably be explained by a possibly advantageous
action of oestrogens in protecting the mother and the lactating child. Gender difference is a factor to be
considered before making dietary recommendations for n3-PUFAs intake [25].

3.2. n3-PUFAs Versus n6-PUFAs

The inefficient bioconversion of ALA to EPA and DHA becomes more evident when considering
that n3 and omega-6 (n6) PUFAs, although in much different concentrations in the typical westernized
diet, compete almost equally for the same enzymatic pathway that elongates and desaturates the
precursors ALA and linoleic acid (LA) to EPA and arachidonic acid (AA), respectively [29]. On the other
hand however, potential roles for ALA in human health that may be independent of its bioconversion
onto DHA have been proposed [27].
Experimental evidence alludes to a slower enzymatic metabolism of n3-PUFAs in relation to
n6-PUFAs [30]. Therefore, focus on ALA intake is paramount for human health, but similarly important
is the adequate intake of LA, as not to imbalance the n6/n3 ratio. Typical westernized diets show
abundance of meats and poultry alongside deep-fried foodstuffs, an important dietary characteristic
that favours high LA intake.
As seen in Figure 1, dietary sources of n6-PUFAs lead to raised levels of AA, culminating
in increased synthesis of pro-inflammatory eicosanoids. Dietary sources of n3-PUFAs, in turn,
allow the synthesis of anti-inflammatory eicosanoids. Increased n3-PUFA concomitant to decreased
n6-PUFA decreases the AA content in platelets, vascular endothelial cells and vascular wall
macrophages, thus reducing AA-derived pro-inflammatory mediators [31]. Cyclooxygenases (COX)
and lipoxygenases (LOX) convert AA to prostaglandin E2, thromboxane A2, prostaglandin I2 and
leukotriene B4, amongst other pro-inflammatory eicosanoids, whilst the same enzymatic pathways
convert EPA to prostaglandin E3, thromboxane A3, prostaglandin I3 and leukotriene B5, amongst
other anti-inflammatory eicosanoids [32–34].
Nutrients 2020, 12, 3159 4 of 19
Nutrients 2020, 12, x FOR PEER REVIEW 4 of 19

Figure
Figure Whilst
1. 1. Whilstthe themain
maindietary
dietarysources
sources of the n3-PUFAs
n3-PUFAsEPA EPAand
andDHA
DHAare arefatty
fattyfish
fish (e.g.,
(e.g., salmon
salmon
and andsardines)
sardines)and andalgae,
algae, the n3
n3precursor
precursorALAALA is found
is found mainly
mainly in walnuts,
in walnuts, chia seeds,
chia seeds, flaxseeds,
flaxseeds, and
and rapeseed
rapeseed oil.oil.
Likewise, meat,
Likewise, poultry
meat, andand
poultry eggseggs
are important sources
are important of theofn6-PUFAs
sources the n6-PUFAs AA, andAA,its n6 its
and
n6 precursor
precursorLA LAisisfound
foundmainly
mainlyininsafflower,
safflower,sunflower,
sunflower,soybean
soybeanand
andcorn
corn oils
oils[35–37].
[35–37]. ALAALA and LALA
and
areare converted
converted by desaturases
by desaturases and and elongases
elongases to EPAto and
EPAAA,
andrespectively,
AA, respectively, and subsequently
and subsequently converted
in aconverted in a series
series of complex of complex
enzymatic reactionsenzymatic reactions
to the longer to the
forms DHA andlonger forms DHA and
n6-docosapentaenoic n6-
(n6-DPA),
docosapentaenoic
respectively [38,39]. (n6-DPA), respectively [38,39].

3.3.3.3.
Cardiometabolic
CardiometabolicPathways
Pathways
It has
It has been
been observedininvitro
observed vitrothat
thatALAALA is associated
associated withwithdecreased
decreasedexpression
expression levels of of
levels vascular
vascular
cellcell adhesion
adhesion molecule-1(VCAM-1),
molecule-1 (VCAM-1), interleukin
interleukin 6 (IL-6),
(IL-6), proliferating
proliferatingcell cellnuclear
nuclear antigen
antigen(PCNA),
(PCNA),
macrophagemarker
macrophage markerM3/84 M3/84 (mac-3)
(mac-3) and and stearoyl-CoA
stearoyl-CoAdesaturase-1
desaturase-1 (SCD-1)
(SCD-1) [40].[40].
Indirect effects
Indirect of
effects
ALA upon cardiometabolic pathways have also been observed;
of ALA upon cardiometabolic pathways have also been observed; ALA bioconversion to DPA ALA bioconversion to DPA is is
associated with decreased expression levels of COX-1, COX-2 and
associated with decreased expression levels of COX-1, COX-2 and tumour necrosis factor-alpha tumour necrosis factor-alpha
(TNF-α),
(TNF-α), whilst
whilst itsitsbioconversion
bioconversionto toEPA
EPAandand DHADHA is is associated
associatedwithwithdecreased
decreasedexpression
expression levels of of
levels
peroxisome proliferator-activated receptor
peroxisome proliferator-activated receptor gamma (PPAR-γ) [40].gamma (PPAR-γ) [40].
Given that such results have also been identified in aortic tissue [41], it is reasonable to speculate
Given that such results have also been identified in aortic tissue [41], it is reasonable to speculate
that the positive effects of ALA are driven by attenuation of inflammation, cell proliferation and
that the positive effects of ALA are driven by attenuation of inflammation, cell proliferation and
oxidation [40]. In a more optimistic, long-term speculative scenario, such ALA-induced effects could
oxidation [40]. In a more optimistic, long-term speculative scenario, such ALA-induced effects could
retard the progression, or even promote an amelioration, of the atherosclerotic state.
retard the progression, or even promote an amelioration, of the atherosclerotic state.
ALA administration to primary cultured endothelial cells induced inhibitions of NAD-
ALA administration to primary cultured endothelial cells induced inhibitions of NAD-dependent
dependent deacetylase sirtuin-3 (SIRT3) reduction, superoxide dismutase 2 (SOD2) hyperacetylation,
deacetylase sirtuin-3 reactive
and mitochondrial (SIRT3) oxygen
reduction, species superoxide dismutase 2 alongside
(ROS) overproduction, (SOD2) hyperacetylation,
restoration of
and mitochondrial
autophagic reactive
flux under oxygeninduced
damage speciesby (ROS) overproduction,
treatment alongside
with angiotensin restoration
II plus of autophagic
TNFα [42,43]. Such
flux
effects, apparently attributed to ALA, are in line with mitigation of endothelial dysfunction effects,
under damage induced by treatment with angiotensin II plus TNFα [42,43]. Such and
apparently
experimental attributed to ALA,[42].
hypertension are in line with mitigation of endothelial dysfunction and experimental
hypertension [42]. that n3-PUFAs have the capacity to decrease liver triglyceride (TG) synthesis by
It is known
It is known
competitive that n3-PUFAs
inhibition have the acyltransferase
of 1,2 diglyceride capacity to decrease (DAT),liver
at thetriglyceride
same time (TG) synthesis
suppressing theby
competitive
activity ofinhibition of 1,2 diglyceride
sterol regulatory acyltransferase
element-binding protein (DAT), at the same time
1c (SREBP-1c)—A suppressing
protein the activity
that regulates the
of expression
sterol regulatoryof genes involved in fatty
element-binding acid 1c
protein and TG synthesis—and
(SREBP-1c)—A proteinalsothattoregulates
increase the β-oxidation
expression in of
adipose
genes tissuein[44,45].
involved fatty acidRegarding
and TGthe latter, the highalso
synthesis—and affinity of n3-PUFAs
to increase for peroxisome
β-oxidation in adiposeproliferator-
tissue [44,45].
activatedthe
Regarding receptor alpha
latter, the high(PPAR-α)
affinity ofleads
n3-PUFAsto a for
greater synthesis
peroxisome of enzymes involved
proliferator-activated in lipid
receptor alpha
(PPAR-α) leads to a greater synthesis of enzymes involved in lipid catabolism, thus favoring not of
catabolism, thus favoring not only fatty acid β-oxidation in peripheral tissues but also catabolism only
circulating
fatty TG in chylomicrons
acid β-oxidation in peripheral and very but
tissues low-density lipoprotein
also catabolism cholesterolTG
of circulating (VLDL-C) [44,46,47].
in chylomicrons and
Nutrients 2020, 12, 3159 5 of 19

very low-density lipoprotein cholesterol (VLDL-C) [44,46,47]. Moreover, substrates for TG synthesis
are also decreased by reduced transport of non-esterified fatty acids to hepatocytes, consequently
reducing VLDL-C synthesis [44,48]. Nonetheless, as the majority of the cardiometabolic pathways so
far elucidated are based on experimental data [40–43], a more thorough, critical and applied appraisal
of the effects of ALA is needed before any general conclusions can be made.

4. Alternative Plant Sources of n3-PUFAs

4.1. Nuts and Seeds

4.1.1. Nutrition Facts

Nuts and seeds are important sources of ALA and also micronutrients, polyphenolic compounds,
sterols and fibres, which are protective elements against the exacerbation of chronic diseases [49–51].
For instance, nuts and seeds that are sources of ALA also contain a considerable amount of calcium,
magnesium, and potassium (Table 1), which are fundamental macrominerals for cardiovascular health,
mainly in the management of hypertension [52–56]. Furthermore, nuts and seeds are a source of
protein. Although at lower levels as compared to animal-derived protein in terms of needs for muscle
hypertrophy, the consumption of nuts and seeds is inversely correlated with cardiovascular events and
mortality, as demonstrated by recent studies [57,58].

Table 1. Nutrition facts of ALA-containing seeds.

Food Item, ALA,

LA, 18:2, n-6
one Ounce Energy Protein Total Lipid Total Fiber CHO Ca Mg K 18:3,
n-6 /n-3
/≈28 g (kcal) (g) (g) (g) (g) (mg) (mg) (mg) n-3
(g) Ratio
[FDC ID] (g)
Chia seeds
136 4.63 8.60 9.75 11.79 176.68 93.8 113.96 1.63 4.99 0.32
[170554]
Hemp seed
155 8.83 13.65 1.12 2.42 19.6 196 366 7.68 2.80 2.74
[170148]
Flaxseed
150 5.12 11.80 7.64 8.08 71.4 109.76 227.64 1.65 6.38 0.25
[169414]
Walnuts
183.12 4.26 18.25 1.9 3.83 27.44 44.24 123.48 10.8 2.54 4.25
[784410]
Adapted from the United States Department of Agriculture (USDA) database [59]. ALA, alpha-linolenic acid; CA,
calcium; CHO, carbohydrate by difference; FDC, FoodData Central; K, potassium; LA, linoleic acid; Mg, Magnesium.

Regarding the ALA content of nuts and seeds, one ounce (28 g) of flaxseed, chia seeds, hemp seed
or walnuts exceeds the Adequate Intake (AIs) for ALA, which is 1.1 g/day for women and 1.6 g/day for
men, and 1.4 g/day and 1.3 g/day during pregnancy and lactation, respectively [59,60]. In one ounce,
there are 6.38 g (398% and 580% of AIs for men and women) of ALA in flaxseed, 4.99 g (175% and
254% of AIs for men and women) in chia seeds, 2.80 g (312% and 453% of AIs for men and women) in
hemp seed, 2.54 g (159% and 230% of AIs for men and women) in walnuts (Table 1).

4.1.2. Walnuts
Walnuts are an important source of ALA [61,62]. A 2-years follow-up study recruited 236 elderly
subjects, segregated into two groups: a control group without nut consumption, and an intervention
group in which 15% of the approximate daily energy intake consisted of walnuts, at approximately
30–60 g/day of walnuts [63]. The researchers found a reduction of 8.5 mmHg in the systolic blood
pressure, whose baseline levels were >125 mmHg; however, no changes were observed in diastolic
blood pressure. In the same study, the participants who consumed walnuts required lower dosages of
antihypertensive drugs as compared to the control participants. The blood pressure of all participants
in that study was monitored by the 24-h ambulatory blood pressure monitoring, which is considered
the gold standard in the diagnosis of hypertension [64]. In contrast, a recent meta-analysis [65]
Nutrients 2020, 12, 3159 6 of 19

did not support walnut consumption per se as a blood pressure-lowering strategy. Despite being a
meta-analysis, that study [65] may have had a few limitations due to heterogeneity. The results of
Domènech et al. [63] provide what appears to be reliable evidence, based mainly on its long-term
duration and sample size.
Le et al. [66] recruited 213 overweight and obese women to a weight loss study, and offered one of
the three following dietary regimens: a walnut-rich diet which consisted of 35% energy from fat and
45% energy from carbohydrates, or a low-fat (20%) high-carbohydrate (65%) diet, or a high-fat (35%)
low-carbohydrate (45%) diet. After six months of intervention, high-density lipoprotein cholesterol
(HDL-C) levels were significantly increased (p < 0.05) in the walnut-rich group (from ≈60 to ≈63 mg/dL),
whilst a small decrease was observed in the low-fat (from ≈60 to ≈57 mg/dL) and low-carbohydrate
(from ≈58 to ≈57 mg/dL) groups.
Interestingly, Fatahi et al. [67] randomised 99 overweight and obese women into three low
energy-diet groups: the first group consisted of 300 g/week of fatty fish such as salmon, avoiding the
intake of plant sources of n3-PUFAs; the second group consisted of 18 walnuts/week, avoiding the
intake of fish and other plant sources of n3-PUFAs; the third group consisted of 150 g fatty fish and nine
walnuts/week, avoiding the intake of other sources of n3-PUFAs. After 12 weeks of dietary intervention,
as compared to the fish-only group and walnut-only group, the fish + walnut group showed better
metabolic profile overall, evidenced by greater increase in HDL-C (+3.6 mg/dL) levels, followed with
greater decrease in systolic blood pressure (−5 mmHg), fasting blood glucose (−12 mg/dL), low-density
lipoprotein cholesterol (LDL-C) (−6 mg/dL), high-sensitivity C-reactive protein (hs-CRP) (−0.51 mg/L),
D-dimer (−0.45 mg/dL), fibrinogen (−22 mg/dL), alanine aminotransferase (ALT) (−6 IU/L), aspartate
aminotransferase (AST) (−6 IU/L), TNF-α (−0.08 ng/mL) and IL-6 (−1.6 ng/mL).

4.1.3. Flaxseed
Flaxseed is an important source of ALA, and a few trials have identified beneficial effects of
flaxseed intake upon lipid indexes and cardiometabolic biomarkers. In a study recruiting 21 patients
with coronary artery disease [68], a pivotal population to ascertain the magnitude of a cardiometabolic
intervention, the daily consumption of 30 g flaxseed for 12 weeks promoted better outcomes as
compared to the control group in increasing flow-mediated dilation (5.1 vs. −0.55% change from
baseline for the flaxseed and control groups, respectively), whilst decreasing the inflammatory status by
reducing the levels of CRP (−1.18 mg/L), IL-6 (−7.65 pg/mL), and TNF-α (−34.73 pg/mL). Importantly,
no significant change in body weight was observed in either groups [68], which appears to be a very
relevant result as it suggests that flaxseed may improve cardiovascular parameters independently of
weight loss.
In a systematic review and meta-analysis of RCTs with 1502 patients across 32 studies, flaxseed or its
derivatives (whole or ground flaxseed, flaxseed oil, or lignan supplements) reduced the concentrations
of hs-CRP (weighted mean difference (WMD): −0.75; 95% CI −1.19, −0.31) and TNF-α (WMD: −0.38;
95% CI −0.75, −0.01) but did not change IL-6 levels. Flaxseed was tested in the form of whole flaxseed,
golden flaxseed, flaxseed oil, and lignan supplements at dosages ranging from 360 mg to 60 g, for 2 to
12 weeks, with an averaged intervention period of approximately 10 weeks [69].
A recently published clinical trial recruited 41 women suffering with polycystic ovary syndrome,
randomly segregated into two groups, group 1 subjected to lifestyle changes (American Heart
Association recommendations + >30 min moderate to intense activity 3x/week) plus 30 g/day brown
flaxseed flour in salad, yogurt or cold drinks, and group 2 subjected to the same lifestyle changes
only, for 12 weeks [70]. The authors found that the flaxseed group showed significant improvements
in insulin, homeostasis model assessment of insulin resistance (HOMA-IR), TG, hs-CRP, IL-6, leptin,
HDL-C and adiponectin, as compared to the non-flaxseed group [70].
In a RCT recruiting 100 eligible patients suffering with non-alcoholic fatty liver disease
(NAFLD), Yari et al. [71] found that 30 g flaxseed daily plus positive lifestyle interventions for
12 weeks decreased serum concentrations of total cholesterol (TC) (−31.71 mg/dL), TG (−61.33 mg/dL),
Nutrients 2020, 12, 3159 7 of 19

LDL-C (−22.64 mg/dL), ALT (−11.12 U/L), AST (−5.37 U/L) and gamma-glutamyltransferase
(−11.54 U/L), results that were not matched in the group submitted to positive lifestyle interventions
only. It should be noted however that both groups showed reductions in BMI (30.37 ± 4.42 to
28.05 ± 3.89 kg/m2 in the flaxseed plus lifestyle improvement group, and 33.37 ± 5.56 to 32.42 ± 5.98
in the lifestyle improvement only group) as well as the intensity of hepatic steatosis, a result most
likely attributed to decreased energy intake in both groups (2379.41 ± 473.74 to 2117.47 ± 378.46,
and 2424.45 ± 470.89 to 1966.39 ± 449.52 kcal, respectively). Such findings reinforce the hypothesis of
beneficial effects of flaxseed independently of changes in energy intake and body composition.
In a mirrored RCT [72], this time recruiting 98 patients suffering with metabolic syndrome,
the same researchers from the previously mentioned study [71] observed comparable results, in which
30 g flaxseed plus positive lifestyle interventions for 12 weeks reduced by 76% the prevalence of
metabolic syndrome, whilst the lifestyle intervention only group had this parameter reduced by 36.4%
(p = 0.013 for the difference between groups). Likewise, both groups reduced their calorie intake
before versus after, but without differences between them (2423.04 ± 468.98 to 2198.76 ± 455.47 and
2410.26 ± 451.87 to 2079.89 ± 465.46 for flaxseed and control groups, respectively).

4.2. Oils

4.2.1. Lipid Profile of Oils

Oils rich in ALA are a powerful tool to investigate the effects of ALA within a less complex
matrix. Despite the presence of other fatty acids and fat-soluble compounds in the oil, the removal of
fibre, vitamins, especially water-soluble ones, and water-soluble matter surely minimize the effects of
confounding variables. Flaxseed, walnut, and rapeseed oils are, respectively, the main sources of ALA.
Soybean oil is often considered a small to reasonable source of ALA, once research into its fatty acid
composition has shown ALA concentrations ranging from 2.7% to 7.8% [73,74]. The n6-PUFAs content
is nonetheless crucial when contemplating the n3 content of plant oils, as sunflower, corn, walnut,
cottonseed, soybean, and peanut oils are important sources of n6-PUFAs (Table 2).

Table 2. ALA content and principal nutrition facts of selected edible oils.

Oil Type, Total LA, ALA,

Energy Vitamin Saturated Monounsaturated Polyunsaturated n-6/n -3
1 Tablespoon/13.6 g Lipid 18:2/n-6 18:3 n-3
(kcal) E (mg) Fats (g) Fats (g) Fats (g) Ratio
[FDC ID] (g) (g) (g)
Flaxseed oil [789037] 120 13.6 0.064 1.22 2.51 9.23 1.95 7.26 0.26
Walnut oil [789048] 120 13.6 0.054 1.24 3.1 8.61 7.19 1.41 5.09
Rapeseed oil [172336] 120 13.6 2.38 1.01 8.64 3.82 2.58 1.24 2.08
Soybean oil [789045] 120 13.6 1.11 2.13 3.1 7.85 6.93 0.92 7.53
Corn oil [789035] 122 13.6 1.94 1.76 3.75 7.44 7.28 0.158 46.07
Olive oil [789038] 120 13.6 1.94 1.86 9.85 1.42 1.32 0.103 12.81
Cottonseed oil
120 13.6 4.8 3.52 2.42 7.06 7 0.027 259.25
[789036]
Coconut oil [789034] 121 13.5 0.015 11.2 0.861 0.231 0.229 0.003 76.33
Peanut oil [789039] 120 13.6 2.12 2.28 6.24 4.32 4.32 0 -
Almond oil [789033] 120 13.6 5.33 1.12 9.51 2.37 2.37 0 -
Sunflower oil [789047] 120 13.6 5.59 1.4 2.65 8.94 8.94 0 -
Highest to lowest sources of ALA among common oils used for cooking. In addition, the main sources of LA can be
noted, which are sunflower, corn, walnut, cottonseed, soybean, and peanut oils, respectively. Adapted from the
United States Department of Agriculture (USDA) database [59].

4.2.2. Flaxseed Oil

A RCT investigated the effects of either 25 mL/d flaxseed oil or 25 mL/d sunflower oil administered
for seven weeks to 60 patients suffering with metabolic syndrome [75]. Serum IL-6 levels decreased
significantly in both groups (9.37 to 7.90 pg/mL, p < 0.001 for flaxseed oil, and 9.22 to 8.48 pg/mL,
p < 0.006 for sunflower oil), but the flaxseed oil group presented a greater reduction (p = 0.017).
Nutrients 2020, 12, 3159 8 of 19

Given that that was a dosage considered high, it is worth mentioning that no side effects were reported
in either group [75]. Interestingly, in a study recruiting 60 women with gestational diabetes [76], the daily
supplementation for 6 weeks with 2 g/day flaxseed oil capsules, which contained 800 mg/day ALA,
reduced the concentrations of TG (−40.5 mg/dL), TC (−22.7 mg/dL), insulin (−2.2 µIU/mL), and hs-CRP
(−1.3 mg/L), as compared to a matched group that received sunflower oil capsules. The flaxseed
oil-receiving group also showed upregulated LDL receptor, downregulated IL-1 and TNF-α gene
expression, decreased malondialdehyde levels and increased total nitrite and total glutathione levels.
In a recent study [77] recruiting 59 overweight and obese adults with stage I hypertension without
pharmacological treatment, 10 g of refined cold-pressed flaxseed oil (4.7 g ALA) for 12 weeks decreased
fasting free fatty acid (−58 µmol/L) and TNF-α (−0.14 pg/mL) plasma concentrations. In contrast,
no changes were found in other metabolic risk markers (e.g., serum glucose and TG levels) nor
vascular function markers (e.g., brachial artery flow-mediated vasodilation, carotid-to-femoral pulse
wave velocity, and retinal microvascular calibres) before versus after testing, both on fasting and
postprandially. We consider this result to be extremely relevant for our critical discussion, as the
ALA intake in that study was about three to five times higher than the recommended daily intake
and, even so, it failed to improve cardiovascular markers. Furthermore, the volunteers of that study
not only had obesity or overweight and stage I hypertension, their average age was 60 ± 8 years,
a finding that is positively associated with vascular ageing, which by definition poses a greater risk for
hypertension and atherosclerotic disease than the more traditional risk factors, including lipid and
glucose levels, smoking and sedentary lifestyle [78].
The Omega-3 Index (O3I) reflects the relative percentage amount of EPA and DHA in erythrocyte
membranes, and is considered a surrogate biomarker for cardiovascular events [79]. Cao et al.
investigated in individuals with low baseline n3-PUFA levels the effects of supplementation with
fish oil or flaxseed oil upon O3I [80]. Cao found that supplementing 2.1 g/day FO (1296 mg EPA
+ 864 mg DHA) for eight weeks increased the O3I from 4.3% to 7.8% (p < 0.001), followed by a
gradual decline to 5.7% and to 3.8% at 4 and 16 weeks after the end of the supplementation period,
respectively. On the other hand, supplementation with flaxseed oil (3510 mg ALA + 900 mg LA/d) for
the same period, in turn, did not significantly change the O3I, but it did increase not only EPA but also
n3-docosapentaenoic acid (DPA) [80], a fatty acid that sits in between EPA and DHA in the elongation
desaturation pathway.
It can be argued that the study of Cao et al. [80] did not cover a period of supplementation
that would allow maximum incorporation and saturation of supplemented PUFAs into erythrocyte
membranes, therefore not raising the O3I to its maximum achievable level. A O3I higher than 8%
has been proposed to be favourable against cardiovascular events, whilst ≤ 4% is interpreted as
low [24,79,81]. Accordingly, the benefits of ALA as a mitigator of cardiometabolic events can be
supported by its role as a substrate for EPA. The latter is a well-known element against pro-inflammatory
pathways in the cardiovascular system.

4.2.3. Soybean Oil

Despite having an obviously different fatty acid profile as compared to FO, soybean oil, unarguably,
is a source of some ALA, and a few studies have already demonstrated a mild but relatively positive
potential for soybean oil. For example, in treated hepatitis C patients (n = 52), 6000 mg/day FO or
a soybean oil control for 12 weeks significantly decreased serum insulin levels (17.1 to 10.9 µIU/mL
p = 0.001, 12.6 to 10.6 µIU/mL p = 0.011 for FO and soybean oil groups, respectively) and HOMA
values (3.8 to 2.4 p = 0.002, 3.1 to 3.0 p = 0.046 for FO and soybean oil groups, respectively) when
comparing baseline versus end-of-intervention. The FO group was clearly more efficient; however,
differences between both groups (p = 0.016 for insulin levels and p = 0.015 for HOMA-IR values) have
been observed [82].
In a small controlled clinical trial recruiting 16 hypercholesterolaemic women in a 10 weeks
intervention, participants received an amount of soybean oil that consisted of 20% of their energy
Nutrients 2020, 12, 3159 9 of 19

intake in a weight-maintaining diet with <300 mg/day of cholesterol [83]. The control group consisted
of the same participants in a weight-maintaining diet with <300 mg/day of cholesterol for eight weeks
but no soybean oil. As compared to the eight-week control period, TC, HDL-C, LDL-C and small dense
low-density lipoprotein-cholesterol (sdLDL-C) levels were reduced at the end of the 10 week-soybean
oil intervention period [83]. Furthermore, the sdLDL oxidation lag time was reduced after soybean oil
consumption. In addition to the unfavourable cardiovascular conditions of maintaining low HDL-C
levels, the former is linked to the pathophysiology of atherosclerotic disease due to the damage
potential of sdLDL-C subclasses, especially in their oxidised form, on arterial structures [6].

4.2.4. Rapeseed Oil

In a meta-analysis of 27 RCTs [84], consumption of rapeseed oil was associated with reductions of
approximately 7.24 mg/dL in TC (95% CI, −12.1 to −2.7) and of approximately 6.4 mg/dL in LDL-C
(95% CI, −10.8 to −2) serum levels, as compared to sunflower oil and saturated fat. No changes were
observed in TG nor HDL-C. Overall, the daily dose of rapeseed oil ranged from 12 to 50 g for 21 to
180 days. Most trials in that meta-analysis addressed individuals with lipid disorders and patients with
heart disease, type 2 diabetes mellitus, obesity, metabolic syndrome, or non-alcoholic fatty liver disease.
Importantly, the trials were under isocaloric conditions and, thus, partially avoided the bias of weight
loss and its relationship with amelioration of lipid indices [84]. A clinical trial study in well-controlled
conditions, recruiting 10 healthy men aged 25.3 ± 1 years, found that 24 h lipid oxidation was more
pronounced when the participants received rapeseed oil-enriched meals for the duration of the study,
as compared to a matched meal enriched with palm oil, a source of saturated fat [85].
It is well known that replacing dietary saturated fats with monounsaturated fatty acids (MUFAs)
and PUFAs can reduce overall mortality [86]. The United Kingdom Scientific Advisory Committee
on Nutrition in 2019 concluded that the recommendation for the population average contribution of
saturated fat to total calorie intake should remain at no more than 10% of total dietary intake, and that
reducing intakes of saturated fats reduces the risk of cardiovascular and heart disease events [87].
The alleged cholesterol-lowering properties of rapeseed oil could be attributed not only to its
ALA content, but also possibly combined with its MUFA composition. Approximately 56% of the total
fatty acids in rapeseed oil are MUFAs, with oleic acid as the most abundant one, at 54.5% of the total
fatty acids, approximately [88]. The abundance of oleic acid in rapeseed oil may support its beneficial
properties, as it has been shown that oleic acid elicits improvement on lipids and lipoproteins, as well
as reduced risk of cardiovascular disease in humans [89]. The ALA content in rapeseed oil, in turn,
is estimated at approximately 6 to 10% of total fatty acids [59,90,91]. Interestingly however, a study
found a much lower ALA content in rapeseed oil, ≈1.2% [92].
A critical interpretation of published studies will see that other plant sources of MUFAs and PUFAs
may show positive results in comparison to rapeseed oil. For instance, the consumption of sesame
oil was more favourable for glycaemic control markers when compared to rapeseed oil in a recent
RCT recruiting individuals with type 2 diabetes mellitus [91]. In that study, rapeseed oil increased
serum fasting blood glucose (+7.72 ± 3.15 mg/dL, p < 0.05), whilst sesame oil decreased serum insulin
(−6.00 ± 1.72 mIU/mL, p < 0.05) levels in a nine-week intervention period. The dietary recommendation
was based on 30–32% of the total energy intake from fats but, despite the predominance of oil intake in
each intervention, we noticed that the authors did not present in their study the exact or estimated
amount of oil consumed. Some of the strengths of the study include its design, a triple-blind, cross-over
clinical trial with 92 subjects completing all treatment periods, which were composed of four-week
run-in and four-week wash-out periods based on sunflower oil.
Nutrients 2020, 12, 3159 10 of 19

4.3. Seaweed

4.3.1. Lipid Profile of Seaweed

The use of seaweed for cooking and as a food supplement is gaining more popularity
worldwide [93,94]. As a functional food, seaweed is a vegetarian source of n3-PUFAs, protein,
and micronutrients [95]. Spirulina and chlorella are commercially available biomass extracts of
cyanobacteria and green algae respectively, designed to attend a growing demand [96]. Both spirulina
and chlorella are often claimed to be valuable sources of n3-PUFAs; however, the accuracy of such
statement needs to be clarified in context, as to obtain approximately 2 to 3 g of total lipids from these
microalgae it is necessary to ingest approximately 28 g of it in its powdered form. The total lipid
content can be practically zero in supplemental dosages (≈3 g/day) [59].
Chlorella minutissima UTEX 2219 and UTEX 2341 feature 3.3% and 31.3% EPA, respectively, of the
total fatty acid content, but DHA was not detected in either strain [97]. In a fatty acid profile analysis
of Spirulina platensis from seven commercially available products, EPA and DHA were detected in only
two samples, contributing with 1.79% and 7.70%, and 2.28% and 2.88%, respectively, of the total fatty
acids [98]. Similarly to flaxseed and nuts, whether the effects of dietary intervention with seaweed are
attributed to its n3-PUFAs per se remain to be investigated, as the food matrix should be considered.
Spirulina and Chlorella contain not only macro and micronutrients but also other compounds with
antioxidant properties which may play a role in positive health outcomes [99–101].

4.3.2. Clinical Findings

A meta-analysis published in 2016 [102] found that in general the daily consumption of 1 to 10 g
Spirulina led to significant improvements in lipid profile by reducing TC in ≈47 mg/dL (95% CI: −67.31
to −26.22), LDL-C in ≈41 mg/dL (95% CI: −60.62 to −22.03), TG in ≈44 mg/dL (95% CI: −50.22 to −38.24),
whilst increasing HDL-C in ≈6 mg/dL (95% CI: 2.37–9.76). Seven placebo-controlled clinical trials with
duration of 2–4 months were included in that meta-analysis, and the population appraised consisted
of patients with diabetes, cardiac diseases, nephrotic syndrome and HIV infection, illnesses whose
pathophysiologies are related to dyslipidaemia [103–105]. Seemingly, an effective and practical dose
was about 4 g/day of Spirulina, which can be administered in capsules or powder [102]. Nevertheless,
as discussed above, we believe that the n3-PUFAs content in Spirulina was not the sole player in
yielding such outcomes.
Supplementation with 300 mg/day of Chlorella for 8 weeks decreased TNF-α levels, in comparison
with the placebo group, in a RCT of 70 patients with NAFLD [106]. An 8 week-long RCT investigating
44 women suffering with primary dysmenorrhea and supplemented with 1500 mg/day Chlorella found
a significant reduction in hs-CRP levels (from 2590.00 ± 1801.66 to 974.21 ± 292.85 ng/mL), as compared
to the control group [107].
In a Japanese sample population, 40 daily tablets of Chlorella were provided to 17 individuals with
borderline high fasting blood glucose, TC, and TG levels, as well as to 17 healthy individuals [108].
After 16 weeks of supplementation, the researchers found reductions in body fat percentage, serum TC,
and fasting blood glucose levels in both groups [108]. In our view however, that study appears to show
a few limitations that should be considered in the context of a broader clinical scenario. The researchers
did not measure food intake, there was no matched control group, and body fat percentage was
obtained through bioelectrical impedance, which as a method of body composition analysis has some
limitations [109]. Lastly, Chlorella supplementation was used in an impracticable dosage that, although
it may be tested, cannot be translated into a broader clinical recommendation. As 40 tablets were
ingested together with a considerable volume of fluid every day, probably this posology in itself with
fluid may have resulted in lower food intake due to stomach filling.
Nutrients 2020, 12, 3159 11 of 19

5. Population
Type 2 diabetes, obesity, dyslipidaemia and hypertension, conditions that we have attempted
to address in the present study, are related to a pro-inflammatory state [110–113]. The evidence
so far available suggests that the consumption of ALA-rich foodstuffs may attenuate the levels of
inflammation-associated biomarkers. More importantly however, the consumption of ALA food
sources appears to be associated with reduced incidence of cardiovascular events. Further long-term
RCTs are imperative to further elucidate such preliminary findings.
In the context of liver diseases, NAFLD particularly, lifestyle improvement is a cornerstone in
ameliorating the disease. We have identified studies that showed beneficial effects of ALA interventions
in patients with NAFLD [71,84]. Accordingly, a favourable nutritional support based on ALA food
sources could be considered in therapies for NAFLD patients in cases of low frequency of fatty fish
intake or absence of FO supplementation.
Food sources of ALA may also be relevant during pregnancy and lactation due not only to their
rich nutritional composition but also due to a thoroughly justified need to avoid complex herbal
supplement mixtures that may jeopardize the health of the mother and of the child. Additionally,
plant sources of n3-PUFAs could be seen as an option for pregnant women who do not tolerate fatty
fish and for those who suffer with nausea and with hyperemesis gravidarum, relatively common
manifestations during the gestational period [114,115]. The amount and profile of n3-PUFAs ingested
by the lactating mother is paramount for infant health, as the mother’s diet directly reflects upon her
milk fatty acid profile [116].
Exclusively vegan diets are to be examined carefully due to the risk of n3-PUFAs deficiency.
Apart from a lower intake of total and saturated fats, another characteristic of exclusively vegan
diets is a higher proportional intake of n6-PUFAs, when compared to omnivorous and vegetarian
diets [110,117]. For those reasons, recommendations for vegan diets that include appropriate amounts
of ALA, necessarily combined with a balanced n3/n6 ratio, are paramount for the maintenance of
long-term health.
It is widely accepted that peanut and peanut butter have been speculated by the layperson and by
some health practitioners as friendly components of the exclusively vegan diet. Careful consideration
however should be exercised regarding the amounts of peanut and peanut butter consumed, as a
pilot study observed in 14 exercise-trained healthy individuals with an average age of 30 years that
the daily consumption of approximately 103 g of peanut butter for 4 weeks led to changes in body
composition, markedly increased body fat content [118]. Such results only confirm that careful calorie
counting is pivotal when adding a new source of lipids to the diet plan, regardless of whether those
are considered healthy food items. Furthermore, peanut is particularly rich in LA and virtually absent
of ALA (Table 2).

6. Decision-Making Practice
A diet poor in n3-PUFAs and rich in n6-PUFAs in the long term leads to inflammation and
increased risk of diseases, including cardiovascular diseases [119]. If the patient does not comply with
a diet that includes fatty fish or FO supplements, ALA-rich foods are important to at least partially
supply some of the n3 requirement, at the same time reducing the LA bioavailability. Furthermore,
some individuals in society may choose not to adhere to food supplementation, be it because of added
costs, dietary choices such as veganism and vegetarianism, the wish to avoid polypharmacy if they are
already taking medicine tablets, or due to personal values. Such lifestyle choices and circumstances
further emphasize the need for a careful nutritional planning that includes sources of n3-PUFAs.
Along those lines, some period supplementing FO and followed by some period incrementing
plant sources of n3-PUFAs may be an example of what would be a ”periodized” loading of n3
status. Such hypothesis can be sustained due to the incorporation of n3-PUFAs into cell membranes,
which occurs over a few months after supplementation, whilst the period appears to be longer for
decreasing n3 status than the progress of storage. In the case of the study of Cao et al. [80] for example,
Nutrients 2020, 12, 3159 12 of 19

two months of FO supplementation was sufficient to almost achieve a reasonable n3 state, which was
maintained by one month afterwards but returning to the same baseline level after 16 weeks of
interruption. We believe that a ”periodized” FO supplementation regimen could be conceivable two to
three times a year for one to four months, followed with nutritional counselling and observing the
intake of foodstuffs rich in ALA in the period without the FO supplementation, hoping to provide
a better n3-DPA level. Nutritional advice however will always be tailored to the individual’s needs
and preferences, such as frequency of consumption of fatty fish and food sources of ALA, patient’s
income, and any coexisting morbidity. As nutritional strategy for individuals who, for one reason or
another, do not eat fish nor take FO supplementation, adding in ALA-rich foods, e.g., flaxseed and chia
seeds, or adding their oils within a dietary plan, alongside a careful consideration to dietary sources of
n6-PUFAs, in itself seems to be paramount for long term overall health.

7. Perspectives
In the present study we focused on the effects of ALA found naturally in foodstuffs, seeds,
nuts and oils, instead of isolated ALA supplementation. Further clinical research is urgently required
to broaden the current knowledge of the potential of ALA upon cardiometabolic dysregulations and
cardiometabolic protection. Well-designed RCTs could certainly minimize the residual confounding
variables caused by other nutrients.
The limitations of our study, as well as of other studies that have investigated ALA cardiometabolic
effects specifically, are many. Ultimately however, although the current study provides some insights
in a real-world prescription-based intervention, we also encourage the execution of meta-analyses to
expand the current knowledge of specific nutrients in specific populations, in an attempt to find ideal
dose-responses, as well as to continuously update guidelines in nutrition and cardiology.

8. Conclusions
In case of suspected insufficient n3 status, such as in individuals with low intake of fatty fish,
those who do not take FO supplement, and in vegan individuals with very narrow dietary habits,
alternative plant sources of n3-PUFAs may be candidates for partially attending the n3 metabolic
demands. Although plant sources of n3-PUFAs are less impactful on EPA and DHA levels, evidence
suggests that those are foodstuffs positively associated with favourable cardiometabolic outcomes,
which could be triggered by other plant components, in synergy with ALA. Not only ALA in isolation,
but its proposed effect in combination with other plant fatty acids and other plant components such as
fibre, potassium, magnesium, and non-essential substances, e.g., polyphenols and sterols, may be the
players in yielding benefits in cardiovascular metabolism.
Consumption of walnuts and flaxseed seems to be the main plant sources of n3-PUFAs with strong
scientific basis for translation into clinical practice. Regarding oil intake, we believe that flaxseed oil is
more advantageous than walnut oil, because the former’s ALA content is five times greater than that
of the latter, which in turn, can be considered the second principal source of ALA. Although several
studies have alluded to rapeseed and soybean oils as ALA sources, their ALA amount is slight when
compared to flaxseed oil, so that a usual oil serving must be considered in order not to exceed the daily
energy requirement in an attempt to achieve an optimal level of ALA. Regarding seaweed, Spirulina and
Chlorella have gained attention but there are no discernible studies corroborating a relevant amount of
n3-PUFAs in usual doses of supplementation. Seemingly, the benefits of seaweed over cardiometabolic
markers appear to be driven by their antioxidant content.
The introduction of ALA-rich foods is a cornerstone for individuals looking for n3 sources beyond
fish and fish oil. It is nevertheless of greatest concern that the proposal to increase the consumption of
ALA ought to be integrated with a controlled calorie intake and controlled n6-PUFAs intake, since both
of them can be raised concomitantly, thus ensuing in untoward effects such as increased fat mass and
cardiometabolic dysregulations.
Nutrients 2020, 12, 3159 13 of 19

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/12/10/3159/s1,

Table S1: Identification of RCTs that investigated the effects of nuts and seeds rich in ALA through the database
search, Table S2: Identification of RCTs that investigated the effects of ALA-rich oils through the database search,
Table S3: Identification of RCTs that investigated the effects of seaweed through the database search, Table S4:
Identification of recent RCTs that investigated the effects of fish oil supplementation through the database search.
Author Contributions: Conceptualization, H.O.S.; methodology, H.O.S., J.C.P.; writing—original draft preparation,
H.O.S.; funding acquisition, A.A.B.; supervision, A.A.B. All authors have read and agreed to the published version
of the manuscript.
Funding: The authors are grateful to the University of Worcester for funding the publication fees.
Conflicts of Interest: The authors declare no conflict of interest.

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affiliations.

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