1
ASSOCIATIONS BETWEEN SERUM LONG-CHAIN OMEGA-3
POLYUNSATURATED FATTY ACIDS, HAIR MERCURY AND RISK OF
STROKE IN MIDDLE-AGED AND OLDER MEN
Roya Daneshmand
Master’s thesis
Public Health Nutrition
School of Medicine
Faculty of Health Sciences
University of Eastern Finland
April 2014
2
Contents
1.
Introduction ......................................................................................................................... 6
2.
Theoretical background ....................................................................................................... 7
2.1.
Definition of stroke and types of stroke ........................................................................ 7
2.2.
Risk factors for ischemic stroke.................................................................................... 8
2.3.
Age ................................................................................................................................ 8
2.4.
Blood pressure .............................................................................................................. 8
2.5.
Cigarette smoking ......................................................................................................... 9
2.6.
Serum Cholesterol....................................................................................................... 10
2.7.
Alcohol........................................................................................................................ 10
2.8.
Diabetes ...................................................................................................................... 11
2.9.
Atrial fibrillation ......................................................................................................... 11
2.10.
Dietary factors ......................................................................................................... 12
2.10.1
Fat intake ................................................................................................................. 12
2.10.2
Fish Intake ............................................................................................................... 13
2.11.
Pollutants and stroke ............................................................................................... 24
3.
Aims .................................................................................................................................. 27
4.
Subjects, participants and methods.................................................................................... 27
4.1
Study population ..................................................................................................... 27
4.2
Serum fatty acids measurements ............................................................................. 27
4.3
Other Measurements ............................................................................................... 28
4.4
Ascertainment of follow-up events ......................................................................... 29
4.5
Statistical analysis ................................................................................................... 29
3
5.
Results ............................................................................................................................... 30
6.
Discussion.......................................................................................................................... 39
7.
Conclusions ....................................................................................................................... 42
4
ABBREVIATIONS
BMI
Body Mass Index
CNS
Central Nervous System
CHD
Chronic Heart Disease
DHA
Docosahexaenoic
EPA
Eicosapentaenoic acid
ICH
Intracerebral hemorrhage
KIHD
Kuopio Ischemic Heart Disease
LDL
Low Density Lipoprotein
PUFA
Polyunsaturated Fatty Acids
RR
Relative Risk
5
UNIVERSITY OF EASTERN FINLAND, Faculty of Health Sciences
Public health
Daneshmand R.: Associations between serum long-chain omega-3 polyunsaturated fatty acids,
hair mercury and risk of stroke in middle-aged and older men
Master's thesis, 50 pages.
Instructors: Jyrki Virtanen, PhD; Sudhir Kurl, MD, PhD
April 2014
Key words: Stroke; Fatty acids, omega-3; Mercury
ASSOCIATIONS BETWEEN SERUM LONG-CHAIN OMEGA-3 POLYUNSATURATED
FATTY ACIDS, HAIR MERCURY AND RISK OF STROKE IN MIDDLE-AGED AND
OLDER MEN
ABSTRACT
Stroke is an important chronic disease that leads to morbidity and mortality worldwide. Stroke
has different subtypes; the most important are ischemic stroke and hemorrhagic stroke. Diet
may influence the risk of stroke. Fish consumption and omega-3 polyunsaturated fatty acids
(PUFA) from fish have been suggested to have a protective effect on stroke. However, fish
may also contain some harmful components, like methylmercury. Most of the previous studies
focus on the association between fish consumption and stroke risk. In this study serum omega3 PUFA, an objective biomarker for intake, was used as exposure. Hair mercury content was
used as a biomarker for mercury exposure. The associations between serum long-chain
omega-3 PUFA, hair mercury and risk of stroke were investigated in middle-aged and older
men. The study cohort included 1871 Finnish men aged 42-60 years from the Kuopio
Ischemic Heart Disease Risk factor Study (KIHD), who were free of coronary heart disease
(CHD) and stroke at baseline. During an average follow-up time of 16.1 years, 141 cases of
stroke occurred, of which 107 were ischemic strokes. In a Cox regression model, there was no
significant association between serum long-chain omega-3 PUFA concentration and risk of
stroke. In contrast, the relative risks of stroke in the quartiles of hair methyl-mercury were 1,
0.95 (95% CI: 0.57-1.59), 1.00 (95% CI: 0.60-1.66) and 1.59 (95% CI: 1.00-2.54) (P for trend
across quartiles=0.04) after adjusting for age and examination years. Further multivariate
adjustments only slightly attenuated the associations. No association was found with ischemic
stroke. In conclusion: this study could not find any association between serum long-chain
omega-3 PUFA and risk of stroke in middle-aged and older Finnish men free of prior CHD
and stroke. Mercury exposure was associated with higher risk of stroke.
6
1. Introduction
Stroke is the leading cause of disability and mortality in the world (Sacco et al. 2013). Since
four decades ago, the rate of stroke has been higher in economically robust countries rather
than in low and middle income countries (Norrving and Kissela 2013). For instance, stroke is
the third-leading cause of death and one of the main factors for disability in the United States
with more than 140,000 people dying of stroke each year. Stroke is responsible for
approximately 5.5 million deaths annually, and 44 million disability-adjusted life-years lost. In
the United States, 795,000 cases suffer from at least one kind of stroke annually, 600,000 of
these strokes cases are the first incident. First incident stroke cases in the United States are
similar to those in European countries with annual stroke incidence ranges between 94.6 per
100,000 population for women and 141.3 per 100,000 population for men (Mukherjee and
Patil 2011).
In Finland the incident of stroke has been declined. Stroke is responsible for 8.9% of all deaths
in Finland. Stroke incident is age dependent disease, Finland with rapid aging population has a
highest incident of stroke between European countries. The estimation pointed to the 50%
increase in stroke incident till 2030 due to the increase in the elder population (Meretoja et al.
2011). According to the cohort studies done by Sivenius et al., in Kuopio and Turku in 1980s
and 1990s the incident of stroke has been declined which is mostly due to decreases in
ischemic stroke both in men and women (Sivenius et al. 2009). Although the number of
elderly people will increase, by improving healthcare and controlling risk factors the burden of
stroke in 2030 will remain the same as year 2000 in Finland (Sivenius et al. 2009).
There are different kinds of stroke. Ischemic stroke is the main reason for morbidity and
mortality in the developed countries with huge financial burden for both family and society
(Fung et al. 2004, Lucke-Wold et al. 2012). Also, ischemic stroke is the most important stroke
with long-term disability (Lucke-Wold et al. 2012). Different studies have been carried out to
investigate associations between risk factors and stroke. Arterial hypertension is the most
important risk factor for ischemic stroke in the Western countries (Droste et al. 2003). In 78%
of ischemic stroke patients, with previous known hypertension history, high blood pressure
has not been controlled (Droste et al. 2003). Also, cigarette smoking is a preventable risk
7
factor for ischemic stroke (Hawkins et al. 2002). Hypercholesterolemia is a risk factor for
ischemic stroke while the role of cholesterol on hemorrhagic stroke is still inconsistent (Wang
et al. 2013). A study done in Finland and US showed that controlling smoking, cholesterol and
diastolic pressure in population level leads to decrease in stroke incidence (Stegmayr et al.
1997).
Different studies on the effect of fish consumption and coronary heart disease pointed to the
dose response association between fish intake and reduced risk factor for cardiovascular
disease (Kris-Etherton et al. 2003). Effect of fish consumption on stroke is still inconsistent
(Larsson et al. 2012); this inconsistency may be partly due to the different components in fish
which can have negative (environmental contaminants, food preparation methods) or positive
effect (EPA+DHA) on stroke.
Although fish is an important source of nutrients, there are environmental contaminants such
as polychlorinated biphenyls, dioxins and methylmercury in the fish. Level of these
environmental contaminants will be higher in old predatory fish and marine mammals. There
are different studies pointing to the harmful effects of methylmercury and risk of chronic
diseases (Virtanen et al. 2007).
2.
Theoretical background
2.1.
Definition of stroke and types of stroke
Any distortion and deficit in neurological system with rapidly focal clinical signs lasting for
24 hours with damaging effect on central nervous system (CNS) or leading to death with
vascular origin is defined as stroke. Stroke occurs due to blood flow restriction into the brain,
which may also be called brain attack. Two prominent types of stroke are hemorrhagic and
ischemic stroke (Sacco et al. 2013). Ischemic stroke is responsible for 80-85% of all stroke
cases while hemorrhagic stroke is responsible for only 15-20% of all stroke cases.
Thrombotic, embolic and lacunar are different types of ischemic stroke. Intracerebral and
subarachnoid are two kinds of hemorrhagic stroke. The most well-known cause of thrombotic
8
stroke is failure in perfusion due to stenosis in major vessels, while embolic stroke is mostly
due to cardiac failure. Arterial obstruction or small artery disease are the main causes of
lacunar stroke. Hypertension has main effect as a risk factor on intracerebral hemorrhage and
vascular malformations is the main etiology of subarachnoid hemorrhage (Zorowitz et al.
2004). Rupture of blood vessels due to their weakness and bleeding around the brain tissue is
the major cause of hemorrhagic stroke (Bouzan et al. 2005).
2.2.
Risk factors for ischemic stroke
High blood pressure, high blood cholesterol level, diabetes, smoking, alcohol use, carotid
stenosis, and atrial fibrillation are the major modifiable risk factors for ischemic stroke
(Medline Plus 2013). The most important non-modifiable risk factors for ischemic stroke are
gender, ethnicity, family history, and age (Sacco 1997, Medline Plus 2013).
2.3.
Age
Age is an important non-modifiable risk factor for all kinds of stroke, but especially for
ischemic stroke (Lucke-Wold et al. 2012). Risk of stroke will be doubled for each decades
after age 55 in both the genders (Sacco et al. 1997). 75% to 89% of strokes occur at the age
older than 65 years old (Chen et al. 2010). Aging also causes increase in the risk of atrial
fibrillation, which increases the risk of stroke (Chen et al. 2010). Aging is a process that leads
to the large number of inflammatory changes. Cytokines are secreted from different cells and
can have an effect on central nervous system (CNS). Cytokine secretion changes with
advancing age. Aging causes an increase in secretion of necrosis factor-alpha (TNF- α) - a
pro-inflammatory cytokine, and decreased interleukin-10 (IL-10) - an anti-inflammatory
cytokine. Some of the experimental studies done on animals concluded that aging leads to
increase in some cytokines like TNF-α, interleukin-1α (IL-1 α), IL-6, interleukin-17 (IL-17),
and interleukin-6R α (IL-6R α) secreted from endothelial cells with damaging effect on
vascular function and CNS which may have role on stroke incident (Lucke-Wold et al. 2012).
2.4.
Blood pressure
According to the previous meta-analysis, there is contradictory findings in regard to the
association between blood pressure and different kinds of stroke (Song et al. 2004). By
9
decreasing every 10 mmHg in systolic blood pressure there should be 30 to 40% reduction in
stroke incident (Sivenius et al. 2009). A study based on Asian population, found strong
association between hemorrhagic stroke and blood pressure (Eastern Stroke and Coronary
Heart Disease Collaborative Research Group 1998) while another study found association
between both ischemic and hemorrhagic strokes (Lewington et al. 2002). In a cohort study
there was a strong association between blood pressure and hemorrhagic stroke compared to
the ischemic stroke (Song et al. 2004).
The effect of sodium on blood pressure has a dose-response relation (Droste et al. 2003).
Arterial stiffness may be another factor causing stroke in people with high sodium intake
(Droste et al. 2003). Sodium may also increase adenosine diphosphate (ADP)-stimulated
platelet aggregation (Droste et al. 2003). Sodium chloride increases cell protein synthesis and
cell growth which may lead to increase in collagen synthesis with deposition in heart and renal
cortex (Droste et al. 2003). Hypertension leads to the major disorders like stroke, myocardial
infarction and hypertensive nephropathy, vascular remodeling, increase in collagen deposition,
and atherogenesis effect, hypertension and LDL activated angiotensin-I-receptor (Droste et al.
2003). In a cohort study done in Kuopio, hypertensive men (blood pressure > 140/90 mmHg)
without alcohol intake had a 1.72-fold relative risk for any stroke and 1.90-fold relative risk
for ischemic stroke. Among men with high blood pressure and alcohol intake the relative risk
for any stroke was 1.86-fold and for ischemic stroke 2.02-fold (Rantakomi et al. 2013).
2.5.
Cigarette smoking
Cigarette smoking has more than 4000 chemical components with harmful effects on human
body (Hawkins et al. 2002). According to the review article done on the association between
smoking and stroke, association between smoking and ischemic stroke is undeniable while the
exact mechanism of cigarette smoking and nicotine on ischemic stroke is inconsistent
(Hawkins et al. 2002). According to the previous studies smoking has association with
ischemic stroke and subarachnoid hemorrhage (US CDC 2004), while the association between
smoking and hemorrhagic stroke is not clear (Shah and Cole 2010). A meta-analysis of more
than 42000 stroke cases from 81 cohort studies found risk of stroke in men and women who
smoke will be 67% and 83% higher than nonsmokers. Results pointed that cigarette smoking
is a prominent and modifiable risk factor of stroke with same hazard of stroke in both men and
10
women, however results showed greater relative hazard of hemorrhagic stroke in women
rather than men (Peters et al. 2013). Smoking leads to the changes in cerebral blood flow,
damages blood brain barrier and changes in cerebrovascular endothelium (Hawkins et al.
2002), which are prominent factors that increase the risk of stroke. According to a metaanalysis, smokers have higher possibility for stroke incidence when compared to nonsmokers
(Hawkins et al. 2002). Endothelin is a peptide secreted from endothelial cells with
vasoconstrictor effect. Endothelin is a mediator with effect on ischemic stroke (Hawkins et al.
2002).
2.6.
Serum Cholesterol
Hypercholesterolemia is a modifiable risk factor for ischemic stroke (Goldstein et al. 2011),
while there is inconsistency in association between serum cholesterol level and intracerebral
hemorrhage and subarachnoid hemorrhage (Wang et al. 2013). A Japanese study was the first
study which found an inverse association between serum cholesterol level and hemorrhagic
stroke (Puddey 1996). Collaborative analysis of 12 Asian cohort studies found that, 0.6
mmol/L decrease in cholesterol concentrations leads to 27% increase in risk of hemorrhagic
stroke (Eastern Stroke and Coronary Heart Disease Collaborative Research Group 1998) while
Korea Medical Insurance Corporation Study found no association between low total serum
cholesterol level and risk of hemorrhagic stroke (Suh et al. 2001). Wang et al. found an
inverse association between total cholesterol and hemorrhagic stroke, also they found inverse
association between LDL-C and hemorrhagic stroke while they could not find any significant
association between HDL-C and risk of hemorrhagic stroke (Wang et al. 2013).
2.7.
Alcohol
Association between alcohol consumption and ischemic stroke has been shown to be a J or U
shaped, while the association with hemorrhagic stroke has been shown to be linear. Effect of
high alcohol intake on the increased risk of stroke may occur because of inducement
hypertension, cardiomyopathy and atrial fibrillation through alcohol consumption while
moderate consumption increase high density lipoprotein (HDL) and decreasing platelet
aggregation (Reynolds et al. 2003). Recently, Rantakomi et al., (Rantakomi et al. 2014) found
an association between the frequency of alcohol intake and stroke mortality. They showed the
11
highest risk of stroke death among men who consumed alcohol >2.5 times/week. Among those
men who consumed alcohol 0.5 –2.5 times/ week, the risk of stroke death was only slightly
increased and the association was not statistically significant.
2.8.
Diabetes
Diabetes mellitus is a well-known risk factor for stroke. Most of the epidemiological studies
have found strong association between type 2 diabetes mellitus and stroke (Janghorbani et al.
2007). Association between diabetes and hemorrhagic stroke remains inconsistent while there
are reports that found positive association between diabetes and changes in cerebral vessels
(Jorgensen et al. 1994). Most of the studies found type 2 diabetes as an important risk factor
for ischemic stroke while there is not strong evidence in regard to the association between type
2 diabetes and hemorrhagic stroke (Hu et al. 2005). According to a cohort study, there was
association between stroke and both two types of diabetes mostly with type 1 diabetes
(Janghorbani et al. 2007). The higher incidence of stroke subtype in patients with type 1
diabetes may explain by younger age at onset, insulin deficiency, hypertension followed by
nephropathy (Hu et al. 2005). Reports from a meta-analysis of 26 prospective studies showed
an association between chronic hyperglycemia and increased risk of all-cause mortality and
cardiovascular outcomes in type 2 diabetes (Zhang et al. 2012). They found that every 1%
increase in glycohemoglobin, was associated with a 15% increase in risk of all-cause
mortality, 25% in CVD mortality, 17% in CVD, 15% in CHD, 17% in fatal CHD, 11% in
heart failure and 11% in stroke (Zhang et al. 2012). This result was consistent with results
from meta-analysis of 10 prospective studies. The only difference was the higher risk of stroke
(17%) (Selvin et al. 2004).
2.9.
Atrial fibrillation
Atrial fibrillation is one of the most common arrhythmias in adults (Guerra et al. 2013). Atrial
fibrillation is responsible for 1/6 of all stroke cases (Armaganijan et al. 2012). Atrial
fibrillation alone is associated with a 3- to 4-fold increased risk of stroke after adjustment for
other vascular risk factors (Goldstein et al. 2006). Fibrillatory activity may occur due to the
irregular atrial response to the reentry circuit. Omega 3 long chain PUFA, especially DHA and
12
EPA are constituents of biological membrane and reduce membrane fluorescence anisotropy
by increasing
cell
membrane
fluidity,
reduce
oxidative
stress
also
has
direct
electrophysiological effect on some of ion channels like sodium and ultra-rapid potassium
currents, and the sodium–calcium exchanger (Patti et al. 2006). Any sustainable changes in
arterial function defined as atrial remodeling. Atrial fibrillation or heart failure conditions,
myocardial infarction, and cardiomyopathy leads to the atrial remodeling.
The effect of dietary lipids on arrhythmia has been known from 25 years ago. In 1988 an
experimental study on rabbit reported the lowering effect of polyunsaturated alpha-linoleic
acid on arrhythmia threshold (McLennan et al. 1988). Experimental study pointed to the effect
of PUFA on reducing atrial electrical remodeling also reducing structural changes in the atria
(Savelieva et al. 2010).
2.10.
Dietary factors
2.10.1 Fat intake
Intake of fats has effects on blood lipids, blood pressure, endothelial function and
inflammation (Strazzullo et al. 2004). Most of the studies underline the importance of type of
fat intake rather than total fat intake (Strazzullo et al. 2004). Association between dietary fat
intake and stroke is still inconsistent (Strazzullo et al. 2004).
A cohort study done on 43732 men aged 40-75 years and followed up for 14 years did not find
an association between intakes of total fat, animal fat, vegetable fat, saturated fat,
monounsaturated fat, polyunsaturated fat, trans unsaturated fat intake and risk of ischemic and
hemorrhagic stroke (He et al. 2003). Previous studies found a positive association between
dietary saturated fatty acid intake and risk of stroke and carotid artery thickness (Tell et al.
1994, Sasaki et al. 1995). However Yamagashi et al., (Zorowitz et al. 2004) found an inverse
association. A meta-analysis of 21 cohort studies did not find an association between SFA
intake and stroke risk (Siri-Tarino et al. 2010). In a prospective nested case-control study, the
positive association between plasma SFA as a biomarker of dietary fatty acids intake and
stroke has been mentioned (Iso et al. 2002).
13
Association between serum monounsaturated fatty acids and stroke is inconsistent. Results
from multicenter case control study (Ricci et al. 1997) also Framingham Heart Study, a
population-based cohort study with 20 years follow up of men (Gillman et al. 1997) found an
association between high risk of stroke and low levels of oleic acid in blood cells membrane,
however cohort study done by He et al., did not find this association (He et al. 2003) . Cohort
study done on 87025 postmenopausal women with 7.6 years follow-up did not find any
association between intake of saturated, mono unsaturated and polyunsaturated fatty acids and
total stroke or ischemic stroke (Yaemsiri et al. 2012). Framingham study found adverse effect
between monounsaturated fatty acids and stroke (Gillman et al. 1997). Other studies could not
find association between intake of monounsaturated fatty acids and polyunsaturated fatty acids
and stroke (Iso et al. 2001, Iso et al. 2002, He et al. 2003, Iso et al. 2003, Sauvaget et al. 2004,
Leosdottir et al. 2007).
Linoleic acid is one of the major polyunsaturated fatty acids in human diet with defined effect
on total cholesterol and LDL cholesterol (Strazzullo et al. 2004). Two nested case-control
studies found inverse association between serum linoleic acid (Iso et al. 2002) and also serum
α-linolenic acid (Simon et al. 1995) and risk of stroke. In a US study there was no association
between ALA intake and risk of stroke (He et al. 2002). In a Dutch study low ALA intake was
associated with increased risk of stroke (de Goede et al. 2011). In a study done by Strazzullo
et al., (Strazzullo et al. 2004), serum α-linolenic acid was selected as a biomarker to study the
effect of omega-3 PUFA on stroke. Results showed inverse association between serum αlinolenic acid and risk of stroke through decreasing in plaque formation and carotid thickness.
2.10.2 Fish Intake
Fish is a good source of protein, selenium, vitamin D, other minerals and vitamins. Fish may
contain beneficial components that some of them do not exist in supplements so
recommendations are to use fish rather than supplements. Fish intake have not only protective
effect on the human body through omega 3 long chain polyunsaturated fatty acids but there
may be some damaging effect through some toxic pollutants in fish like methylmercury
(MeHg).
14
According to the American Heart Association 2020 report, consumption of at least 2-3.5-oz
servings/week of fish, especially oily fish, is good for ideal cardiovascular health. Fish,
especially fatty fish, are a prominent source of omega-3 polyunsaturated fatty acid (PUFA),
EPA (20:5n-3) and DHA (22:6n-3) (Mozaffarian and Wu 2011). Level of long chain omega-3
PUFA differs among the fish type. Mackerel, salmon, red mullet have the highest content of
omega-3 polyunsaturated fatty acids (Domingo et al. 2007). Table 1 shows first 10 fish species
with high amount of omega-3 PUFA. Circulating DPA level is poorly related to the fish intake
and diet but mostly as a metabolite of EPA. There are few studies found inverse association
between DHA and chronic diseases (Mozaffarian and Wu 2011).
Table 1. EPA, DPA, DHA concentration in different fish
Fish
EPA
DPA mg/100g
DHA mg/100g
mg/100g
EPA+DHA
mg/100g
Anchovy
763
41
1,292
2,055
Herring
909
71
1,105
2.014
Salmon, farmed
862
393
1,104
1,966
Salmon, wild
411
368
1,429
1,840
Mackerel, Atlantic
504
1106
699
1,203
Bluefish
323
79
665
988
Sardines, Atlantic
473
0
509
982
Trout
259
235
677
936
Golden bass (tilefish)
172
143
733
905
Swordfish
127
168
772
899
Data from the U.S. Department of Agriculture National Nutrition Database for Standard
Reference Release 23, 2010. (U.S. Department of Agriculture 2010)
The study in Greenland Eskimos was a pioneer study on the association between fish intake
and the low incident of stroke (Bang et al. 1976). Ecological studies found inverse association
between long-chain omega-3 polyunsaturated fatty acids and ischemic stroke while positive
association with hemorrhagic stroke (He et al. 2004). According to the reports from a metaanalysis done on 16 prospective cohort studies, consisting of 402127 individuals aged between
15
30 and 103 years with 10568 incident stroke cases (Xun et al. 2012), a nonlinear association
was found between fish intake and stroke. According to the meta-analysis of 15 prospective
studies (seven studies were conducted in the United States, 4 in Europe, 3 in Japan, and 1 in
China) with 9360 stroke events and 383838 participants, 9 studies reported RR of 0.90 in
those with 3 servings fish intake/week for both ischemic and hemorrhagic stroke. Results
found weak inverse association between fish intake and risk of stroke (Larsson and Orsini
2011).
Different cohort studies found association between long chain omega-3 PUFA intake and
lower risk of stroke (Iso et al. 2001, He et al. 2002, Larsson et al. 2012). Cohort study done by
Mozaffarian et al., in 65 years or older men and women found that tuna and other fish have
inverse association with ischemic stroke however there was not any association with
hemorrhagic stroke. Fried fish and fish sandwiches had positive association with total and
ischemic stroke also using baked and broiled fish reduces the risk of total and ischemic stroke
while fried fish, increased the risk of stroke (Mozaffarian et al. 2005). However a prospective
study found conflicting result regard to the association between fish intake and risk of stroke,
which may because of methodological differences (Larsson et al. 2011). Methodological
differences were mostly because of method of fish preparation (deep fried fish), also fish type
(salted fish), which may attenuate all healthy impact of fish consumption (Atkinson et al.
2011, Larsson and Orsini 2011). Cohort study done by Folsom et al, in 41,836 postmenopausal
women aged 55–69 years pointed to positive association between the greater fish intake and
younger age; greater education, physical activity, alcohol consumption, estrogen use, vitamin
use, body mass index, and hypertension. In this study there was not any association between
intake of marine omega-3 fatty acids and total mortality also with coronary heart disease and
stroke mortality (Folsom and Demissie 2004). Moreover, in a Chinese cohort study (Yuan et
al. 2001), prospective population cohort study in the UK (Myint et al. 2006) and in a Finnish
study (Montonen et al. 2009) no association were found (Table 2).
There are few studies done on the effect of eicosapentaenoic acid (EPA) and docosahexaenoic
(DHA) intake from fish. In a study done by de Goede et al., an inverse association was found
16
between EPA+DHA and fish intake with risk of all subtypes of stroke in women while these
associations were not significant in men (de Goede et al. 2012) (Table 2).
In a randomized control trial 11,324 patients surviving recent myocardial infarction, results
could not find any association between long-chain omega-3 PUFA supplementation and stroke
(Gissi 1999). Randomized, double-blind intervention trial done in patients from 11 different
dialyses centers in Denmark could not find association between omega 3 supplementation and
risk of stroke (Svensson et al. 2006). Randomized control trial done in men and women with
18 years and older in Italy could not find association between stroke and long-chain omega-3
PUFA (Gissi et al. 2008). In a RCT done in France, result could not find any significant
association between long chain omega-3 PUFA supplementation and risk of stroke (Galan et
al. 2010) (Table 3).
Only few studies have used objective biomarkers of fish and long-chain omega-3 PUFA intake
as exposure. According to the study done by Mozaffarian et al., among different n3-PUFA
biomarkers, DHA and Total n3-PUFA strongly associated to ischemic stroke incident without
any association with total and hemorrhagic stroke (Mozaffarian et al. 2013).
Cell lipids has effect on cellular functions. Omega 3 PUFA as one of the cell lipids, has effect
on molecular and Ion channels also on phospholipids of cell membrane with changing in
physiochemical properties of the membrane rafts and caveolae. It resulted to the membrane
protein function which may describe anti- inflammatory and anti-arrhythmic effect of omega 3
PUFA. Omega 3 PUFA can effect on the ion channels directly or through the effect on lipid
membrane. Animal experimental study showed omega 3 PUFA directly affects myocyte
electrophysiology (like changing the function of membrane sodium channel, L-type calcium
channel, and sodium–calcium exchanger) and effect on myocyte excitability especially in
ischemic cells and trigger arrhythmia (Mozaffarian and Wu 2011).
There are different mechanisms that describe the beneficial effect of omega 3 PUFA on
chronic diseases like CVD (Mozaffarian and Wu 2011). There is linear relation between
dietary intake of omega 3 PUFA for less than 750 mg/day and decreasing in heart rate, blood
pressure and arrhythmia. Reports from population-based observational studies showed inverse
associations between high intake of fish or polyunsaturated fatty acids through fish
17
consumption and blood pressure in general population and in healthy, non-hypertensive
participants (Pauletto et al. 1996, Mozaffarian et al. 2006, Virtanen et al. 2012).
Randomized control trials could not find the association between omega-3 supplementation
and all causes mortality, cardiac death and stroke (Rizos et al. 2012). Also, meta-analysis of 8
prospective studies could not find any significant association between omega-3 supplements
and stroke (Larsson et al. 2012).
Possible mechanism for decreasing HR and arrhythmia is increase in myocardial efficiency,
left ventricular diastolic filling and autonomic function. Decrease in systematic vascular
resistance and endothelial dysfunction and increase in vasodilatory response and arterial wall
compliance and decrease in endothelial dysfunction and systemic vascular resistance are the
possible mechanism to reduce blood pressure. The possible mechanism for decrease in
thrombosis are decrease in production of arachidonic acid derived eicosanoids (Mozaffarian
and Wu 2011). Intake of higher dose (at least up to 7g/d) has linear relation with decreasing
triglyceride production. The beneficial effect of omega 3 PUFA on thrombosis is just in the
format of high supplementary intake (more than 4 g/d) (Goldstein et al. 2011).
There are consistent results in association between fish consumption and coronary heart
disease in different studies while there is inconsistency between results of randomized control
trials and meta-analysis of cohort studies in regard to the association between fish
consumption and risk of stroke (Mozaffarian and Wu 2011). This inconsistency may occur
because of some residual confounding in the observational studies, inadequate statistical
power in trials or insufficient follow up time in trials, or because of beneficial components
exist in fish and not in fish oil (Mozaffarian and Wu 2011).
18
Table 2. Characteristics of prospective cohort studies of dietary fish or long-chain omega-3 PUFA intake, circulating long-chain
omega-3 polyunsaturated fatty acid levels and risk of stroke
study
Study name
No of
Age
participants
Follow
Exposure Categories
up, y
Outcome (No.
RR (95 %
RR (95 %
RR (95 % CI)
of Events)
CI)
CI)
for highest vs.
for
for highest
lowest category
highest
vs.
for
vs.
lowest
hemorrhagic
lowest
category for
stroke
category
Ischemic
for total
stroke
stroke
Fish intake
Larsson et al 2011
Fish consumption and
Swedish
Mammography
34670
49-83
10.4
< 1serving/week
Total stroke =
0.72
0.87 (0.73-
0.67 (0.42-
risk of stroke in Swedish
1-1.4 Serving/week
1680
(0.57-
1.04)
1.08)
women
1.5-2 Serving/week
Ischemic
0.92)
Cohort
2.1-3 Serving/week
stroke = 1310
(Larsson and
>3 Serving/week
Hemorrhagic
Orsini 2011)
stroke = 137
Unclassified
stroke = 125
Montonen et al
2009
Finland
(Montonen et al.
2009)
Fish consumption and
the incidence of
cerebrovascular disease
3958
40-79
28
6 (median g/day)
Total stroke =
1.01
0.99 (0.73-
1.23 (0.63-
18 (median g/day)
659
(0.81-
1.35)
2.42)
32 (median g/day)
Ischemic
1.27)
72 (median g/day)
stroke = 364
Hemorrhagic
19
stroke = 120
Unclassified
stroke = 175
Myint et al, 2006
Habitual fish
Norfolk, UK
<1 portion/week
Total stroke =
Women:
consumption and risk of
1-2 portion/week
421
0.86
cohort of the
incident stroke: the
>2 portion/week
European
European Prospective
Prospective
Investigation into Cancer
Investigation into
(EPIC)-Norfolk
Men: 1.34
Cancer
prospective population
(0.93-
study
2.93)
Mozaffarian et al.,
Fish Consumption and
2005
(Mozaffarian et al.
24312
40-79
8.5
-
-
-
(0.601.24)
4775
65-98
12
Once/mo
Total stroke =
0.72
0.68 (0.48-
Stroke Risk in Elderly
1-3 times/mo
626
(0.53-
0.95)
Individuals:
1-4 times/week
Ischemic
0.98)
≥ 5times/week
stroke = 529
2005)
Hemorrhagic
stroke = 65
Unclassified
stroke = 32
Folsom et al.,
Fish intake, marine
2004, The Iowa
41836
55-69
14
< 0.5 serving/week
Total stroke =
1.06
omega-3 fatty acids, and
0.5-1.0
313
(0.67-
Women’s Health
mortality in a cohort of
serving/week
Study cohort
postmenopausal women
1.0-1.5
French
serving/week
(Folsom and
1.5-2.5
Demissie 2004)
serving/week
1.67)
-
-
20
>2.5 serving/week
He et al 2002
Fish consumption and
Health
risk of stroke in men
43671
40-75
12
< once/month
Total stroke =
0.83
0.54(0.31-
1.55 (0.45-
1-3 times/month
608
(0.53-
0.94)
5.35)
Professional
once/week
Ischemic
1.29)
Follow-Up Study
2-4 times/week
stroke = 377
USA
> 5 times/week
Hemorrhagic
(He et al. 2002)
stroke = 106
Unclassified
stroke = 125
Iso et al 2001
Intake of fish and
Nurses’ Health
Study
79 839
34-59
14
< once/mo
Total stroke =
0.72
0.67 (0.42-
Could not find
omega-3 fatty acids and
1-3 times/mo
574
(0.53-
1.07)
association
risk of stroke in women
once/week
Ischemic
0.99)
USA
2-4 times/week
stroke = 303
(Iso et al. 2002)
> 5 times/week
Hemorrhagic
-
-
stroke = 181
Yuan et al 2001
Fish and shellfish
Shanghai, China
(Yuan et al. 2001)
18244
45-64
12
<30 (g/week)
Total stroke =
1.05
consumption in relation
30<60 (g/week)
480
(0.77-
to death from myocardial
60<100 (g/week)
infarction among men in
100<150(g/week)
Shanghai, China
≥150(g/week)
1.43)
EPA+DHA
Women 1
Total stroke =
Women:
Women:
Women: 0.45
Associations of Marine
Q1 < 57 mg/d
221
0.49
0.62 (0.29-
(0.14–1.42)
n-3 Fatty Acids
Q2 57–106 mg/day
Ischemic
(0.27-
1.35)
de Goede et al.,
Gender-Specific
20121
(de Goede et al.
20069
20-65
8-13
21
2012)
and Fish Consumption
Q3 107–188 mg/day
stroke = 142
0.91)
with 10-Year Incidence
Q4 > 188 mg/day
Hemorrhagic
of Stroke
Men
stroke = 47
Men: 0.87
Q1< 66 mg/day
Unclassified
(0.51-
Q2 66-118 mg/day
stroke = 32
1.48)
Men: 0.28
Men: 0.85
(0.05–1.46)
(0.45-1.60)
Q3 119-198 mg/day
Q4 > 199 mg/day
He et al 2002
Fish consumption and
Health
risk of stroke in men
43671
40-75
12
< 0.05 g/day
Total stroke =
0.73(0.43-
1.14 (0.34-
0.05 < 0.2 g/day
608
1.25)
3.84)
Professional
0.2 < 0.4 g/day
Ischemic
Follow-Up Study
0.4 < 0.6 g/day
stroke = 377
USA
≥ 0.6 g/day
Hemorrhagic
-
-
-
-
(He et al. 2002)
stroke = 106
Unclassified
stroke = 125
Yuan et al 2001
Fish and shellfish
<0.27 g/week
Total stroke =
1.01
Shanghai, China
consumption in relation
18244
45-64
12
0.27–0.43 g/week
480
(0.75-
(Yuan et al. 2001)
to death from myocardial
0.44–0.72 g/week
infarction among men in
0.73–1.09 g/week
Shanghai, China
≥ 1.10 g/week
1.33)
circulating long-chain omega-3 polyunsaturated fatty acid levels
Mozaffarian et al.,
Plasma phospholipid
2013
(Mozaffarian et al.
2,692
75±5
1 to 5
Total stroke = 406
EPA: 1.09
EPA: 0.70
long-chain ω-3 fatty
Ischemic stroke =
(0.76–1.57)
(0.30–
acids and total and
319
DHA: 0.74
1.67)
22
2013)
cause-specific mortality
Hemorrhagic stroke
(0.50–1.10)
DHA:
in older adults: a cohort
= 65
DPA: 0.78
1.24
study.
Unclassified stroke
(0.55–1.10)
(0.52–
= 22
2.94)
DPA:
0.66
(0.32–
1.35)
1
According to the EPA, DHA intake
23
Table 3. Characteristics of RCT studies of dietary long-chain omega-3 PUFA intake and risk of total stroke
Study, country
Intervention
Galan et al.,
omega-3
EPA,
DHA,
Vitamin E,
dose, g/d
g/d
g/d
mg/d
0.6
0.4
0.2
-
Control
Placebo
Duration,
No of participants,
HR (95
y
Treatment/Control
% CI)
4.7
1253/1248
1.04
(SU.FOL.OM3)
(0.62-
2010, (France)
1.75)
(Galan et al. 2010)
Gissi et al., , 2008,
850-882 mg
1
(Italy)
EPA+ DHA
(0.89–
(Gissi et al. 2008)
(ratio: 1:1.2)
1.51)
Svensson et al.,
Omacor (45%
2006, (Denmark)
EPA, 37.5%
(Svensson et al.
DHA)
1.7
-
0.77
-
0.64
-
-
Placebo
Placebo (olive
3.9
2
3494/3481
103/103
oil)
1.16
2.23
(0.588.46)
2006)
Gissi et al., 1999,
850-882 mg
(Italy)
EPA+ DHA
(Gissi 1999)
(ratio: 1:1.2)
1
-
-
300
Placebo
3.5
PUFA intake 2836
1.30
/2828
(0.871.96)
Vitamin E intake:
2830/2828
24
2.11.
Pollutants and stroke
Mercury (Hg) is a heavy metal. Elemental or metallic Hg, inorganic and organic Hg are three
forms of Hg. Hg is produced by burning fossil fuels, industrial waste, volcano, municipal
waste combustion and gold mining. It is a volatile metal and can remain in the atmosphere for
about one year (Virtanen et al. 2007). Methylmercury (MeHg) is a highly toxic form of Hg
which bioaccumulates in the fish and marine mammals (100% of Hg in fish is in the format of
MeHg) (Roman et al. 2011). MeHg level will be higher in older and predatory fish like sharks,
tuna, swordfish and pike (Virtanen et al. 2007) (Table 4).
Table 4. Mercury levels in selected fish
Species
Mercury concentration (mg/kg)
Mean
Range
Reference
Burbot
0.22–0.26
0.20–0.37
(E.-R. Venalainen)
Catfish
0.05
ND–0.31
(US Department of Health and Human
Services and US Environmental
Protection Agency)
Cod
0.10–0.11
ND–0.42
(US Department of Health and Human
Services and US Environmental
Protection Agency)
Herring
0.04
ND–0.14
(US Department of Health and Human
Services and US Environmental
Protection Agency)
Mackerel, king
0.73
0.23–1.67
(US Department of Health and Human
Services and US Environmental
Protection Agency)
Perch,
0.14
ND–0.31
freshwater
(E.-R. Venalainen) and (US Department
of Health and Human Services and US
Environmental Protection Agency)
Perch, ocean
ND
ND–0.03
(US Department of Health and Human
Services and US Environmental
25
Protection Agency)
Pike
0.38–0.40
0.15–0.85
(E.-R. Venalainen)
Pike–perch
0.11–0.30
0.06–0.37
(E.-R. Venalainen)
Pollock
0.04
ND–0.78
(US Department of Health and Human
Services and US Environmental
Protection Agency)
Salmon
0.01–0.07
ND–0.19
and (US Department of Health and
Human Services and US Environmental
Protection Agency)
Sardine
0.02
0.004–0.35
(US Department of Health and Human
Services and US Environmental
Protection Agency)
Shrimp
ND
ND–0.05
(US Department of Health and Human
Services and US Environmental
Protection Agency)
Shark
0.99
ND–4.54
(US Department of Health and Human
Services and US Environmental
Protection Agency)
Swordfish
0.98
ND–3.22
(US Department of Health and Human
Services and US Environmental
Protection Agency)
Tilefish
1.45
0.65–3.73
(US Department of Health and Human
Services and US Environmental
Protection Agency)
Trout,
0.07
ND–0.68
freshwater
(E.-R. Venalainen) and (US Department
of Health and Human Services and US
Environmental Protection Agency)
Tuna
0.38
ND–1.30
(US Department of Health and Human
Services and US Environmental
Protection Agency)
26
Whitefish
0.03–0.08
ND–0.31
(E.-R. Venalainen) and (US Department
of Health and Human Services and US
Environmental Protection Agency)
ND=mercury concentration below the detection level (0.01 mg/kg) (Virtanen et al. 2007)
MeHg is metabolized to inorganic Hg and is excreted via feces slowly, so the half-life of
MeHg in human body is 70-80 days (Virtanen et al. 2007). Hg is converted to MeHg in the
water-sources by microorganisms’ reaction. MeHg exposure leads to oxidative stress and
production of reactive oxygen species (ROS) which can lead to blockage of arteries and have a
negative effect on central nervous system (CNS) and also increase risk of CVD, arrhythmias,
hypertension and ischemia (Salonen et al. 1995, Virtanen et al. 2005, Virtanen et al. 2007,
Roman et al. 2011). Hg has high affinity to the sulfhydryl groups, which inactive enzymatic
reaction, sulfur containing antioxidants also sulfur amino acids and leads to decreasing
oxidative defense reaction and also endothelial dysfunction (Virtanen et al. 2005, Houston
2011). Some studies found association between MeHg and oxidized low density lipoprotein
(LDL) (Salonen et al. 1995). Hg exposure may induce hypertension through inactivation of
catecholamine-0-methyl transferase, which leads to elevating rate of epinephrine,
norepinephrine and dopamine in urine and serum (Houston 2011). However, recent
prospective cohort studies have not found an association between mercury exposure and blood
pressure (Mozaffarian et al. 2012, Virtanen et al. 2012).
Some studies suggest a protective effect of selenium in fish which can attenuate the negative
effect of MeHg by binding to Hg and making seleno-mercury complex which is less toxic
(Houston 2011).
KIHD was the pioneer study on the association between MeHg exposure and risk of CVD in
middle-aged men (Virtanen et al. 2005). MeHg can attenuate the positive effect of omega-3
polyunsaturated fatty acids (Virtanen et al. 2005, Wennberg et al. 2012). Result from
prospective population-based study found that high mercury will attenuate the beneficial effect
of fish on cardiovascular health (Virtanen et al. 2005) while cohort study done by Mozaffarian
et al., could not find any association (Mozaffarian et al. 2011). Also result from cohort study
27
done in US could not find any significant association between hair mercury and risk of stroke
(Mozaffarian et al. 2011). There is few information available regard to Hg and stroke risk.
3. Aims
The aim of this study is to investigate the association between serum long-chain omega-3
polyunsaturated fatty acids, hair Hg and risk of stroke in middle-aged and older men.
4. Subjects, participants and methods
4.1 Study population
KIHD is a population based, randomly selected sample of men from eastern Finland. It has
been designed to explore the associations between risk factors and risk of CVD,
atherosclerosis, stroke and other chronic diseases (Salonen 1988). The baseline examinations
were performed in 1984–1989 (Salonen 1988). A total of 2682 men who were 42, 48, 54 or 60
years old at baseline (82.9 % of those eligible) were recruited in two cohorts. The first cohort
consisted of 1166 men who were 54 years old, enrolled between 1984 and 1986, and the
second cohort included 1516 men who were 42, 48, 54 or 60 years old, enrolled between 1986
and 1989. During the years 1998-2001 all men from the second cohort were invited to the 11year re-examinations of the study, and 854 men (85.6%) participated. The KIHD study
protocol was approved by the Research Ethics Committee of the University of Kuopio and
complies with the Declaration of Helsinki. All subjects gave written informed consent for
participation.
Subjects with history of CHD at baseline (n= 677) and history of stroke at baseline (n=32) or
with missing data on dietary intakes (n=23), serum long-chain n-3 PUFA (n =122), hair
mercury (n= 14) were excluded, leaving 1814 men.
4.2 Serum fatty acids measurements
28
Serum esterified and non-esterified fatty acids were specified in one gas chromatographic run
without pre separation as described (Laaksonen et al. 2002). Chloroform-methanol used to
extract serum fatty acids. Quantification done with reference standards purchased from NuCheck Prep Inc. (MN, USA). Each analyte had specified reference standard and recovery of
analytes was confirmed with an internal standard eicosan (arachidic acid C20H40O2). Fatty
acids were chromatographed in an NB-351 capillary column (HNU-Nordion, Helsinki,
Finland) by a Hewlett-Packard 5890 Series II gas chromatograph (Hewlett-Packard Company,
Avondale, Pa, USA, since 1999 Agilent Technologies Inc., USA) with a flame ionization
detector. The coefficient of variation (CV) for repeated measurements of major esterified fatty
acids was about 5%. Because the relative degree of saturation of fatty acids varies among
esterified fatty acid types (i.e., cholesterol esters, phospholipids, and triglycerides), the
esterified fatty acid concentrations were adjusted for serum low-density lipoprotein
cholesterol, high-density lipoprotein cholesterol, and triglyceride concentrations. The CV for
major nonesterified fatty acids was about 15%. No adjustment was conducted for nonesterified
fatty acids. Results were obtained in mmol/L. Sum of EPA (20:5n-3), docosapentaenoic acid
(DPA, 22:5n-3) and DHA (22:6n-3) considered as total long-chain n-3 PUFA.
4.3 Other Measurements
The subjects gave fasting blood samples between 8 and 10AM on the baseline examinations in
1998-2001. They were instructed to abstain from ingesting alcohol for three days and from
smoking and eating for 12 hours prior to giving sample. Detailed descriptions of the
determination of blood glucose, (Salonen 1988) assessment of medical history and
medications, (Salonen 1988) family history of diseases, (Salonen 1988) smoking,(Salonen
1988) and alcohol consumption, (Salonen 1988) have been published. Diabetes was defined as
self-reported diabetes mellitus or fasting blood glucose of 6.7 mmol/L or more. Education was
assessed in years by using self-administrated questionnaire. Physical activity was assessed
using the KIHD 12-Month Leisure-Time Physical Activity Questionnaire (Lakka et al. 1994).
Body mass index (BMI) was computed as the ratio of weight in kilograms to the square of
height in meters. Dietary intake of foods and nutrients was assessed at the time of blood
sampling using 4-day food recording (Voutilainen et al. 2001). Mercury in hair was
29
determined by flow injection analysis-cold vapor atomic absorption spectrometry and
amalgamation, as described (Salonen et al. 1995).
4.4 Ascertainment of follow-up events
Incident strokes between years 1984-1992 were observed through FINMONICA stroke
register, Information regarding the stroke incident between years 1993 and 2004 was collected
through computerized linkage to the national hospital discharge registry. The diagnosis of
stroke was based on sudden onset of clinical signs or focal or global disturbance of cerebral
function lasting 24 hours (except in the case of sudden death or if interrupted by surgical
intervention) with no apparent cause other than a vascular origin. Each suspected stroke
(International Classification of Diseases [ICD]-9 codes 430_439 and ICD-10 codes I60–I68
and G45–G46) was classified into 1) a definite stroke, 2) no stroke, or 3) an unclassifiable
event. The FIN-MONICA stroke register data were annually rechecked with the data obtained
from the computerized national hospital discharge and death registers. Definite strokes and
unclassifiable events were included in the group of any stroke. Each definite stroke was
classified into 1) an ischemic stroke (ICD-9 codes 433 434; ICD-10 code I63) or 2) a
hemorrhagic stroke (ICD-9 codes 430 431; ICD-10 codes I60–I61). If the subject had multiple
nonfatal strokes during follow-up, the first stroke was considered as the end point. CT was
performed in 90% of the patients by 1993, and CT, MRI, and autopsy reached 100% by 1997.
Every resident of Finland has a unique personal identifier that is used in registers. There were
no losses to follow-up (Karppi et al. 2012).
4.5 Statistical analysis
Subjects were divided into quartiles according to the mean serum omega 3 PUFA (0-0.64,
0.65-1.26, 1.27-2.50, 2.51-15.67 % of all serum fatty acids) and the mean mercury content in
hair (1.70-3.63, 3.63-4.34, 4.34-5.34, 5.34-15.59 µg/g of hair). The univariate relationships
between serum EPA + DPA + DHA and hair mercury and baseline characteristics were
assessed by means and linear regression (for continuous variables) or χ2 tests (for categorical
variables). Three different models were used. First model was adjusted by age and
examination year. In the second model, body mass index; smoking; physical activity; alcohol
30
intake were also adjusted for. Further adjusted on systolic blood pressure; diabetes; HDL
cholesterol; LDL cholesterol; serum triglyceride; C - reactive protein was done in third model.
Three different models were conducted to find out about the effect of different variables which
may have the role as a confounders or modifier on the association between exposure and
outcome. Associations between serum long chain PUFA, hair mercury content and stroke were
analyzed using Cox regression models. Cohort mean was used to replace missing values in
covariates (< 0.5%). Tests of linear trend were conducted by assigning the median values for
each category of exposure variable and treating those as a single continuous variable. All P
values were two-tailed (α = 0.05). Data were analyzed using SPSS 21.0 for Windows (SPSS
Inc., Chicago, IL).
5. Results
5.1 Baseline characteristics
There were no significant differences between serum long chain PUFA concentrations in those
who had a stroke and those who did not (Table 5). In contrast, hair MeHg content was higher
in those who had a stroke in comparison to those who did not have a stroke.
141 cases of first strokes were identified, 107 cases out of 141 cases were ischemic stroke. At
baseline, men with higher serum long-chain omega-3 PUFA concentration had a higher
income, education, leisure-time physical activity, BMI, alcohol intake, serum LDL cholesterol,
serum HDL cholesterol and hair mercury concentration and lower serum triglyceride
concentration. They were also less likely to live in a rural area and smoke (Table 6).
Men with higher hair mercury concentration were older; had a lower education, physical
activity, income, and serum triglyceride concentrations; and had higher BMI, alcohol intake,
serum long-chain omega-3 PUFA, HDL and LDL concentrations, and higher systolic and
diastolic blood pressure. They were also more likely to live in a rural area and smoke (Table
7).
5.2 Association between serum long-chain omega-3 PUFA and risk of
stroke
31
During the average follow-up of 16.1 years, 141 men (7.7%) developed stroke. Of all strokes,
115 were ischemic strokes. After adjusted for age and year of examination (model 1 in Table
8), higher serum long chain polyunsaturated fatty acids concentration was not statistically
significantly associated with ischemic stroke and total stroke. Further multivariate adjustments
did not have an appreciable effect on the association (Models 2& 3 in Table 8). Further
adjustment for hair mercury slightly strengthened the association (RR in the highest quartile of
total serum long-chain omega-3 PUFA 0.84, 95% CI 0.51-1.34, P for trend=0.55). There were
no statistically significant associations when the individual fatty acids were evaluated
separately (Table 8).
The mean hair mercury concentration was 1.90 ug/g (SD 1.95). After adjusted for age and year
of examination the risk of stroke in the highest vs. the lowest hair Hg quartile was increased
by 59% [95% CI 0-154%, P for trend=0.01] (Model 1 in Table 9). Additional adjustments
slightly attenuated the associations (Models 2&3 in Table 9). The associations were attenuated
and not statistically significant, when only ischemic strokes were included in the analyses
(Table 9).
Table 5. Mean baseline concentrations of serum total long-chain omega-3 polyunsaturated
fatty acids and hair mercury in those who experienced stroke and those who did not during the
follow-up.
Participants with
Participants without
P value
stroke during follow-
stroke during follow-
up (n = 141), mean
up (n = 1673), mean
(SD)
(SD)
DHA, %
2.4 (0.7)
2.5 (0.7)
0.8
EPA, %
1.7 (0.8)
1.7 (0.9)
0.9
DPA, %
0.5 (0.1)
0.5 (0.1)
0.3
32
Total omega 3
4.7 (1.6)
4.7 (1.6)
0.9
2.2 (1.9)
1.9 (1.9)
0.05
PUFA, %
Hairhg, ug/g
33
Table 6. Baseline characteristics according to serum long-chain omega-3 PUFA concentration
Serum Omega 3 PUFA, %
P value
Q1
Q2
Q3
Q4
(0-0.64)
(0.65-1.26)
(1.27-
(2.51-
2.50)
15.67)
Number of subjects
453
454
454
453
-
Age (y)
52.2 (5.6)
52.1 (5.4)
52.6 (5.0)
52.7 (5.2)
0.05
Education (y)
8.9 (3.3)
8.7 (3.4)
8.8 (3.5)
9.6 (4.0)
< 0.001
Marital status, married (%)
85
87
85
91
0.06
Living in rural area (%)
30
28
28
25
0.40
Leisure-time physical activity (kcal/d)
134 (171)
132 (155)
132 (146)
153 (196)
0.07
Body mass index (kg/m2)
26.6 (3.5)
26.5 (3.3)
27 (3.5)
27 (3.5)
0.03
Current smoker (%)
31
28
28
25
0.27
Diabetes (%)
5
3
5
5
0.26
Family history of stroke (%)
21
20
22
18
0.32
Hypertension medication before stroke (%)
70
70
67
68
0.76
Alcohol intake (g/wk)
55 (90)
62 (93)
85 (136)
85 (127)
< 0.001
Serum LDL, mmol/l
3.8 (0.3)
4.0 (0.9)
4.1 (1.0)
4.1 (1.0)
< 0.001
Serum HDL, mmol/l
1.2 (0.3)
1.3 (0.3)
1.3 (0.3)
1.4 (0.3)
< 0.001
Serum TG, mmol/l
1.5 (0.9)
1.3 (0.8)
1.1 (0.6)
1.0 (0.5)
< 0.001
Systolic blood pressure (mmHg)
135 (17)
134 (16)
134 (16)
134 (17)
0.98
Diastolic blood pressure (mmHg)
90 (11)
89 (10)
89 (10)
89 (11)
0.97
Hairhg, ug/g
1.2 (1.3)
1.6 (1.7)
2.2 (2)
2.7 (2.3)
< 0.001
Income, Euros
13344
13833(9083
14162
15784
< 0.001
(7869)
)
(10070)
(10360)
34
Table 7. Baseline characteristics according to hair mercury concentration
Hair Hg, ug/g
P value
Q1
Q2
Q3
Q4
(1.70-3.63)
(3.63-4.34)
(4.34-5.34)
(5.3415.59)
Number of subjects
452
454
456
452
-
Age (y)
51.0 (5.7)
51.8 (5.8)
52.8 (4.9)
54.0 (4.4)
< 0.001
Education (y)
9.7 (3.6)
9.6 (3.9)
8.8 (3.5)
7.8 (3)
< 0.001
Marital status, married (%)
86
91
87
84
0.12
Living in rural area (%)
24
22
28
38
< 0.001
Leisure-time physical activity (kcal/d)
154 (186)
143 (190)
134 (149)
120 (141)
< 0.001
Body mass index (kg/m2)
26.3 (3.2)
26.6 (3.3)
27.0 (3.6)
27.0 (3.7)
< 0.001
Current smoker (%)
25
25
26
36
< 0.001
Diabetes (%)
4
4
5
5
0.58
Family history of stroke (%)
21
19
21
21
0.85
Hypertension medication before stroke
74
66
66
70
0.05
Alcohol intake (g/wk)
60 (112)
73 (106)
76 (129)
78 (106)
0.05
Serum LDL, mmol/l
3.8 (0.9)
3.9 (0.9)
4.0 (1)
4.3 (1)
< 0.001
Serum HDL, mmol/l
1.2 (0.9)
1.3 (0.3)
1.3 (0.3)
1.3 (0.3)
< 0.001
Serum TG, mmol/l
1.3 (0.8)
1.2 (0.7)
1.3 (0.9)
1.2 (0.6)
0.01
EPA, %
1.3 (0.6)
1.6 (0.8)
1.7 (0.8)
2.1 (1.1)
< 0.001
DHA, %
2.2 (0.6)
2.4 (0.6)
2.5 (0.7)
2.7 (0.8)
< 0.001
DPA, %
0.5 (0.1)
0.5 (0.1)
0.6 (0.1)
0.6 (0.1)
< 0.001
Total omega 3 PUFA, %
4.0 (1.2)
4.5 (1.4)
4.8 (1.4)
5.4 (1.9)
<0.001
Systolic blood pressure (mmHg)
134 (17)
134 (16)
133 (16)
136 (16)
0.04
Diastolic blood pressure (mmHg)
89.0 (10)
89.0 (10)
88.5 (11)
90.0 (10)
0.35
Income Euros
15267
15148
14649
12012
< 0.001
(10033)
(8617)
(10260)
(9428)
(%)
35
Table 8. Risk of incident total stroke and ischemic stroke in quartiles of serum long-chain
omega-3 polyunsaturated fatty acids
Serum long-chain Omega-3 PUFA, % 1
P value
for
trend
Q1
Q2
Q3
Q4
All stroke cases, (n=141)
Total serum long-chain omega-3 polyunsaturated fatty acids
< 3.22
3.22-3.98
3.99-4.77
> 4.77
No. of cases
36
29
38
38
Model 1
1(referent)
0.76(0.46-1.24) 1.03 (0.651.64)
Model 2
1(referent)
0.77(0.47-1.26) 1.02(0.641.61)
Model 3
1(referent)
0.78(0.48-1.28) 0.99(0.61-
0.99 (0.63-
0.70
1.56)
0.96(0.60-
0.86
1.52)
0.91(0.56-
1.60)
1.48)
0.93
EPA
< 0.92
0.92-1.27
1.28-1.67
> 1.67
No. of cases
32
31
35
43
Model 1
1(referent)
0.93 (0.56-
1.02 (0.63-
1.30(0.81-
1.52)
1.66)
2.07)
0.96 (0.58-
1.02 (0.63-
1.20 (0.77-
1.58)
1.66)
1.99)
0.93 (0.56-
0.99 (0.60-
1.16 (0.70-
1.55)
1.65)
1.93)
Model 2
Model 3
1(referent)
1(referent)
0.17
0.28
0.43
DHA
< 1.70
1.70-2.16
2.17-2.55
> 2.55
No. of cases
35
29
37
40
Model 1
1(referent)
0.80 (0.49-
1.05 (0.66-
1.08 (0.69-
1.31)
1.66)
1.71)
0.49
36
Model 2
Model 3
1(referent)
1(referent)
0.78 (0.48-
1.00 (0.63-
1.03 (0.65-
1.28)
1.60)
1.64)
0.71 (0.43-
0.94 (0.58-
0.95 (0.59-
1.17)
1.52)
1.55)
0.63
0.80
DPA
< 0.44
0.44-0.51
0.52-0.58
> 0.58
No. of cases
27
26
29
25
Model 1
1(referent)
0.92 (0.59-
0.96 (0.61-
0.70 (0.43-
1.45)
1.50)
1.14)
1.00 (0.63-
1.05 (0.67-
0.79 (0.48-
1.58)
1.65)
1.29)
1.05 (0.66-
1.12 (0.70-
0.81 (0.49-
1.66)
1.78)
1.34)
Model 2
Model 3
1(referent)
1(referent)
0.19
0.41
0.49
Ischemic stroke, (n = 107 )
Total serum long-chain omega-3 polyunsaturated fatty acids
< 3.22
3.22-3.98
3.99-4.77
> 4.77
No. of cases
29
21
26
31
Model 1
1(referent)
0.68(0.39-1.20) 0.87(0.511.47)
Model 2
1(referent)
0.70(0.40-1.22) 0.86(0.501.47)
Model 3
1(referent)
0.70(0.39-1.23) 0.82(0.47-
1.00 (0.60-
0.66
1.67)
0.99(0.59-
0.73
1.65)
0.91(0.53-
1.43)
1.58)
0.96
EPA
<0.92
0.92-1.27
1.28-1.67
> 1.67
No. of cases
24
23
27
33
Model 1
1(referent)
0.91 (0.52-
1.04 (0.60-
1.32 (0.77-
1.62)
1.82)
2.25)
0.97 (0.54-
1.06 (0.61-
1.30 (0.76-
1.72)
1.85)
2.40)
Model 2
1(referent)
0.20
0.26
37
Model 3
1(referent)
0.94 (0.53-
1.02 (0.57-
1.22 (0.69-
1.70)
1.82)
2.18)
0.39
DHA
< 1.70
1.70-2.16
2.17-2.55
> 2.55
No. of cases
28
20
29
30
Model 1
1(referent)
0.69 (0.39-
1.02 (0.61-
1.02 (0.61-
1.22)
1.72)
1.70)
0.68 (0.38-
0.98 (0.58-
0.97 (0.57-
1.21)
1.66)
1.64)
0.62 (0.35-
0.91 (0.53-
0.87 (0.50-
1.11)
1.56)
1.51)
Model 2
Model 3
1(referent)
1(referent)
0.63
0.77
0.99
DPA
< 0.44
0.44-0.51
0.52-0.58
> 0.58
No. of cases
27
26
29
25
Model 1
1(referent)
0.97 (0.57-
1.06 (0.63-
0.92 (0.53-
1.66)
1.80)
1.59)
1.05 (0.61-
1.16 (0.68-
1.05 (0.60-
1.82)
1.98)
1.83)
1.10 (0.64-
1.24 (0.72-
1.08 (0.61-
1.91)
2.14)
1.90)
Model 2
Model 3
1
1(referent)
1(referent)
0.85
0.78
0.72
Values in the models are relative risk (RRs) and 95% CIs (in parentheses).
Model 1: adjusted for age and examination year.
Model 2: adjusted for Model 1 plus body mass index; smoking; physical activity; alcohol
intake;
Model 3: adjusted for Model 2 plus systolic blood pressure; diabetes; HDL cholesterol; LDL
cholesterol; serum triglyceride; C-Reactive Protein (each in quintiles).
38
Table 9. Risk of incident total stroke and ischemic stroke in quartiles of hair mercury
HairHg, ug/g 1
P
value
for
trend
Q1
Q2
Q3
Q4
(< 3.63)
(3.63-4.34)
(4.35-5.34)
(> 5.34)
All stroke cases
(n=141)
No. of cases
25
24
21
37
Model 1
1(referent)
0.95(0.57-
1.00 (0.60-
1.59 (1.00-
1.59)
1.66)
2.54)
0.92 (0.55-
0.94 (0.56-
1.46 (0.91-
1.55)
1.57)
2.36)
0.94 (0.56-
1.00 (0.60-
1.47 (0.90-
1.57)
1.68)
2.41)
Model 2
Model 3
1(referent)
1(referent)
0.01
0.03
0.04
Ischemic stroke
(n = 107 )
No. of cases
29
21
26
31
Model 1
1 (referent)
0.91 (0.52-
0.74 (0.41-
1.29 (0.77-
1.59)
1.34)
2.18)
0.90 (0.51-
0.71 (0.40-
1.24 (0.73-
1.58)
1.29)
2.11)
0.92 (0.52-
0.76 (0.42-
1.24 (0.72-
1.62)
1.37)
2.14)
Model 2
Model 3
1
1 (referent)
1 (referent)
0.17
0.22
0.26
Values in the models are relative risk (RRs) and 95% CIs (in parentheses).
Model 1: adjusted for age and examination year.
Model 2: adjusted for Model 1 plus body mass index; smoking; physical activity; alcohol
intake;
39
Model 3: adjusted for Model 2 plus systolic blood pressure; diabetes; HDL cholesterol; LDL
cholesterol; serum triglyceride; C-Reactive Protein (each in quintiles).
6. Discussion
Results from this population based cohort study suggest that a higher serum concentration of
long-chain n-3 PUFA, mainly reflecting fatty fish consumption in this study population, does
not have any association with total stroke and ischemic stroke. The results from this study
suggest that exposure to methylmercury has a direct association with the risk of total stroke
but not with the risk of ischemic stroke. Although we did not evaluate the association between
methylmercury and hemorrhagic stroke separately due to the low number of participants with
hemorrhagic stroke, the lower risk of ischemic stroke compared to the risk of total stroke
suggests that methylmercury exposure increases especially the risk of hemorrhagic stroke.
Some studies have pointed to the long-chain omega-3 PUFA as a risk factor for hemorrhagic
stroke. Long-chain omega-3 PUFA have an effect on platelet aggregation (Park et al. 2009).
Study done by Park et al. pointed to the positive association between EPA + DHA and
hemorrhagic stroke in rat brains (Park et al. 2009). This effect has not been confirmed in
humans. Few studies have evaluated the positive association between long-chain omega-3
PUFA and risk of hemorrhage and bleeding disorders. However, none of them have
considered the effect of high intake of long-chain omega-3 PUFA on increased bleeding
through oxidative stress (Park et al. 2009). Although we did not have enough cases to study
the association between serum long-chain omega-3 PUFA and risk of hemorrhagic strokes
separately, it seems unlikely that there is an association because the associations with total and
ischemic strokes were similar. Previously in this study population higher serum concentration
of total long-chain omega-3 PUFA has been associated with lower systolic blood pressure and
pulse pressure (Virtanen et al. 2012), CRP (Reinders et al. 2012), SCD (Virtanen et al. 2012),
CHD and CVD (Virtanen et al. 2005) and also with the risk of atrial fibrillation (Virtanen et
al. 2009). Atrial fibrillation is a common cardiac arrhythmia, and it is considered as one of the
most important risk factors for stroke. Atrial fibrillation reduces the cardiac output and reduces
40
the cerebral blood flow, which has effect on stroke incident (Wolf et al. 1991). Atrial
fibrillation can also significantly increase the frequency of blood clots that are transported into
the carotid arteries by alteration in cardiac hemodynamics (Choi et al. 2013). However, inspite
of the inverse association with the risk of atrial fibrillation, serum long-chain omega-3 PUFA
do not seem to decrease the risk of stroke in this study population.
The results of this study are consistent with the result from previous meta-analysis (Larsson
and Orsini 2011, Xun et al. 2012) and with other cohort studies (Yuan et al. 2001, Folsom and
Demissie 2004, Myint et al. 2006, Montonen et al. 2009), which could not find any inverse
association between omega-3 PUFA and risk of stroke. However, opposite results have also
been found (Iso et al. 2001, He et al. 2002, Larsson et al. 2011). The possible mechanism of
the effect of fish consumption on stroke, especially ischemic stroke, could be explained by the
effect of long-chain omega-3 PUFA from fatty fish. Omega-3 fatty acids from fatty fish
influences blood pressure, lipid levels, inflammatory responses, red blood cell deformability,
endothelial cell function, and cerebral arteriolar reactivity (Agren et al. 1990, Ellis et al. 1992,
Knapp 1996, Nestel 2000). Each of these effects, separately or in combination, may plausibly
reduce risk of ischemic stroke (Mozaffarian et al. 2005). Although each of these effects can
have a role on reducing risk of ischemic stroke, this population cohort study could not find any
beneficial effect of total serum long-chain n-3 PUFA or individual fatty acids separately with
risk of stroke. Unlike with the other cardiovascular outcomes in this study cohort, high
exposure to Hg does not seem to be the explanation, because adjustment for Hg only slightly
strengthened the associations.
Mozafarrian et al., explained the physiological effects and molecular pathways of n-3 PUFA
that might influence CVD risk (Wu and Mozaffarian 2014). Omega 3 PUFA has effect on
different physiological functions in different tissues, including heart, liver, vasculature, and
circulating cells. Experimental studies show that n-3 PUFA has effect on cardiac
electrophysiology, which could contribute to decrease in heart rate and arrhythmic risk (Wu
and Mozaffarian 2014). n-3 PUFA reduce plasma triglyceride through decreasing very lowdensity lipoprotein production in liver and increasing fatty acid beta-oxidation (Wu and
Mozaffarian 2014). Blood pressure-lowering effect of n-3 PUFA is a result of reduced
41
systemic vascular resistance, which improves endothelial dysfunction, wall compliance, and
vasodilatory responses. Physicochemical properties of cellular membranes are influenced by
their lipid composition. n-3 PUFA have protective effect on CVD (Wu and Mozaffarian 2014)
through its role on membrane fluidity, ion channels function and gene expression via nuclear
receptors and transcription factors. There are no comprehensive studies in regard to the effects
on risk factors, molecular pathways, of protective effect of omega-3 PUFA and stroke.
Although most of the risk factors for CVD and stroke are similar, we could not find any
association between omega 3 PUFA and stroke.
We used hair mercury as a reliable marker of long-term exposure (World Health Organization
1990) . This study found a direct association between hair methylmercury content and risk of
total stroke, which contradicts the null findings from the only previous study done by
Mozaffarian et al (Mozaffarian et al. 2011). Several explanations may explain the conflicting
results. Higher mercury level in the KIHD population could explain the different study
findings. In the US cohort study the mean toenail mercury concentration was 0.44 µg/g in the
male controls, corresponding to about 1.2 µg/g in hair (Mozaffarian et al. 2012). In our study
the mean hair mercury concentration is 1.9 µg/g. Thus, in lower concentrations mercury may
not have any adverse effects and higher levels of exposure are needed for the adverse effects.
Also the effects of other factors, like dietary antioxidants and selenium, or differences in the
genetic-based defenses, may explain the conflicting study findings.
Hair methylmercury could also be a marker of other factors with possible effect on stroke risk.
As seen in the Table 7, high hair mercury concentration has associated with stroke risk factors,
such as systolic blood pressure, serum TG, body mass index. Other factors or residual
confounding possibly can explain at least partly the higher risk in the highest hair mercury
tertile. There were no significant differences in baseline serum long-chain omega-3 PUFA in
those who experienced stroke during the follow-up and in those who did not. However, hair
methylmercury concentration was higher in those who had a stroke (Table 5). Higher mercury
content of selected fish or genetical differences in the elimination of mercury (Schlawicke
Engstrom et al. 2008) could explain this.
42
Few studies have focused on the association between different risk factors and hemorrhagic
stroke. Study done by Wang et al., pointed to the positive association between low total
cholesterol and LDL-cholesterol levels and hemorrhagic stroke, especially intracerebral
hemorrhage (ICH) (Wang et al. 2013). Also, there is an association between low intake of
saturated fat and animal protein and increased risk of intraparenchymal hemorrhage (Iso et al.
2001). Previous studies did not focus on the association between mercury exposure and
different stroke types separately. Oxidative stress effect of methylmercury, which can decrease
anti-oxidative capacity in plasma and cells and increase lipid peroxidation in cell membranes,
could at least partly explain the association between mercury exposure and risk of stroke
(Virtanen et al. 2005). Mercury also has high affinity to sulfhydryl groups, which results in
inactivation of anti-oxidative thiolic compounds (Flora et al. 2008). In addition, mercury binds
with selenium and forms an inactive form that cannot act as a catalytic center of glutathione
peroxidase, a main scavenger of H2O2 and lipid peroxides (Virtanen et al. 2005). However, we
do not have an explanation for why Hg would increase the risk of hemorrhagic stroke but not
the risk of ischemic stroke. Further research is needed to elucidate the associations.
Strength of the present study is the use of serum long-chain n-3 PUFA measurements instead
of dietary intakes. Few prior studies have used circulating long-chain n-3 PUFA concentration
as an exposure, which is important to reduce bias by misclassification that attenuates the
associations in studies using dietary intakes. Serum long-chain n-3 PUFA concentration is a
good biomarker of n3 fatty acid exposure (Hunter 1998). The other strength is the populationbased recruitment and high participation rate. Limitation of the study is the low number of
stroke cases, especially hemorrhagic strokes and the participants were only men.
7. Conclusions
In summary, our findings suggest that circulating n3-PUFA levels are not associated with
stroke. However, methylmercury exposure was associated with higher risk of total stroke.
Although some studies have found an inverse association between serum omega 3 PUFA and
stroke, there is not enough evidence about the specific amount of long-chain omega-3 PUFAs
that is needed to reduce risk of stroke. Also, the amount of long-chain omega-3 PUFAs in
43
supplements is higher than what is commonly obtained from diet, so recommendation to take
fish oil as a supplement instead of dietary fish intake should be done cautiously (He et al.
2004).
References
Agren, J. J., O. Hanninen, A. Hanninen and K. Seppanen (1990). "Dose responses in platelet fatty acid
composition, aggregation and prostanoid metabolism during moderate freshwater fish diet." Thromb
Res 57(4): 565-575.
Armaganijan, L., D. Patel, C. Dietrich and C. A. Morillo (2012). "Stroke Prevention in Atrial Fibrillation:
Latest Clinical Trials and Guidelines." Pharmaceuticals (Basel) 5(4): 384-397.
Atkinson, C., E. Whitley, A. Ness and I. Baker (2011). "Associations between types of dietary fat and
fish intake and risk of stroke in the Caerphilly Prospective Study (CaPS)." Public Health 125(6): 345348.
Bang, H. O., J. Dyerberg and N. Hjoorne (1976). "The composition of food consumed by Greenland
Eskimos." Acta Med Scand 200(1-2): 69-73.
Bouzan, C., J. T. Cohen, W. E. Connor, P. M. Kris-Etherton, G. M. Gray, A. Konig, R. S. Lawrence, D. A.
Savitz and S. M. Teutsch (2005). "A quantitative analysis of fish consumption and stroke risk." Am J
Prev Med 29(4): 347-352.
Chen, R. L., J. S. Balami, M. M. Esiri, L. K. Chen and A. M. Buchan (2010). "Ischemic stroke in the
elderly: an overview of evidence." Nat Rev Neurol 6(5): 256-265.
Choi, H. W., J. A. Navia and G. S. Kassab (2013). "Stroke propensity is increased under atrial fibrillation
hemodynamics: a simulation study." PLoS One 8(9): e73485.
de Goede, J., W. M. Verschuren, J. M. Boer, D. Kromhout and J. M. Geleijnse (2011). "Alpha-linolenic
acid intake and 10-year incidence of coronary heart disease and stroke in 20,000 middle-aged men
and women in the Netherlands." PLoS One 6(3): e17967.
de Goede, J., W. M. Verschuren, J. M. Boer, D. Kromhout and J. M. Geleijnse (2012). "Gender-specific
associations of marine n-3 fatty acids and fish consumption with 10-year incidence of stroke." PLoS
One 7(4): e33866.
Domingo, J. L., A. Bocio, G. Falco and J. M. Llobet (2007). "Benefits and risks of fish consumption Part
I. A quantitative analysis of the intake of omega-3 fatty acids and chemical contaminants." Toxicology
230(2-3): 219-226.
Droste, D. W., M. A. Ritter, R. Dittrich, S. Heidenreich, T. Wichter, M. Freund and E. B. Ringelstein
(2003). "Arterial hypertension and ischaemic stroke." Acta Neurol Scand 107(4): 241-251.
E.-R. Venalainen, A. H., R. Parmanne, P.J. Vuorinen "[Heavy metal contents in Finnish sea and water
fish. National Food Agency of Finland]."
Eastern Stroke and Coronary Heart Disease Collaborative Research Group (1998). "Blood pressure,
cholesterol, and stroke in eastern Asia." Lancet 352(9143): 1801-1807.
44
Ellis, E. F., R. J. Police, L. Y. Dodson, J. S. McKinney and S. A. Holt (1992). "Effect of dietary n-3 fatty
acids on cerebral microcirculation." Am J Physiol 262(5 Pt 2): H1379-1386.
Flora, S. J., M. Mittal and A. Mehta (2008). "Heavy metal induced oxidative stress & its possible
reversal by chelation therapy." Indian J Med Res 128(4): 501-523.
Folsom, A. R. and Z. Demissie (2004). "Fish intake, marine omega-3 fatty acids, and mortality in a
cohort of postmenopausal women." Am J Epidemiol 160(10): 1005-1010.
Fung, T. T., M. J. Stampfer, J. E. Manson, K. M. Rexrode, W. C. Willett and F. B. Hu (2004). "Prospective
study of major dietary patterns and stroke risk in women." Stroke 35(9): 2014-2019.
Galan, P., E. Kesse-Guyot, S. Czernichow, S. Briancon, J. Blacher, S. Hercberg and S. F. O. C. Group
(2010). "Effects of B vitamins and omega 3 fatty acids on cardiovascular diseases: a randomised
placebo controlled trial." BMJ 341: c6273.
Gillman, M. W., L. A. Cupples, B. E. Millen, R. C. Ellison and P. A. Wolf (1997). "Inverse association of
dietary fat with development of ischemic stroke in men." JAMA 278(24): 2145-2150.
Gissi, H. F. I. (1999). "Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E
after myocardial infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della
Sopravvivenza nell'Infarto miocardico." Lancet 354(9177): 447-455.
Gissi, H. F. I., L. Tavazzi, A. P. Maggioni, R. Marchioli, S. Barlera, M. G. Franzosi, R. Latini, D. Lucci, G. L.
Nicolosi, M. Porcu and G. Tognoni (2008). "Effect of n-3 polyunsaturated fatty acids in patients with
chronic heart failure (the GISSI-HF trial): a randomised, double-blind, placebo-controlled trial." Lancet
372(9645): 1223-1230.
Goldstein, L. B., R. Adams, M. J. Alberts, L. J. Appel, L. M. Brass, C. D. Bushnell, A. Culebras, T. J.
DeGraba, P. B. Gorelick, J. R. Guyton, R. G. Hart, G. Howard, M. Kelly-Hayes, J. V. Nixon, R. L. Sacco, A.
American Heart and C. American Stroke Association Stroke (2006). "Primary prevention of ischemic
stroke: a guideline from the American Heart Association/American Stroke Association Stroke Council:
cosponsored by the Atherosclerotic Peripheral Vascular Disease Interdisciplinary Working Group;
Cardiovascular Nursing Council; Clinical Cardiology Council; Nutrition, Physical Activity, and
Metabolism Council; and the Quality of Care and Outcomes Research Interdisciplinary Working
Group." Circulation 113(24): e873-923.
Goldstein, L. B., C. D. Bushnell, R. J. Adams, L. J. Appel, L. T. Braun, S. Chaturvedi, M. A. Creager, A.
Culebras, R. H. Eckel, R. G. Hart, J. A. Hinchey, V. J. Howard, E. C. Jauch, S. R. Levine, J. F. Meschia, W.
S. Moore, J. V. Nixon, T. A. Pearson, C. American Heart Association Stroke, N. Council on
Cardiovascular, E. Council on, Prevention, R. Council for High Blood Pressure, D. Council on Peripheral
Vascular, C. Interdisciplinary Council on Quality of and R. Outcomes (2011). "Guidelines for the
primary prevention of stroke: a guideline for healthcare professionals from the American Heart
Association/American Stroke Association." Stroke 42(2): 517-584.
Guerra, F., M. Shkoza, L. Scappini, L. Roberti and A. Capucci (2013). "Omega-3 PUFAs and atrial
fibrillation: have we made up our mind yet?" Ann Noninvasive Electrocardiol 18(1): 12-20.
Hawkins, B. T., R. C. Brown and T. P. Davis (2002). "Smoking and ischemic stroke: a role for nicotine?"
Trends Pharmacol Sci 23(2): 78-82.
He, K., A. Merchant, E. B. Rimm, B. A. Rosner, M. J. Stampfer, W. C. Willett and A. Ascherio (2003).
"Dietary fat intake and risk of stroke in male US healthcare professionals: 14 year prospective cohort
study." BMJ 327(7418): 777-782.
45
He, K., E. B. Rimm, A. Merchant, B. A. Rosner, M. J. Stampfer, W. C. Willett and A. Ascherio (2002).
"Fish consumption and risk of stroke in men." JAMA 288(24): 3130-3136.
He, K., Y. Song, M. L. Daviglus, K. Liu, L. Van Horn, A. R. Dyer, U. Goldbourt and P. Greenland (2004).
"Fish consumption and incidence of stroke: a meta-analysis of cohort studies." Stroke 35(7): 15381542.
Houston, M. C. (2011). "Role of mercury toxicity in hypertension, cardiovascular disease, and stroke."
J Clin Hypertens (Greenwich) 13(8): 621-627.
Hu, G., C. Sarti, P. Jousilahti, M. Peltonen, Q. Qiao, R. Antikainen and J. Tuomilehto (2005). "The
impact of history of hypertension and type 2 diabetes at baseline on the incidence of stroke and
stroke mortality." Stroke 36(12): 2538-2543.
Hunter, D. (1998). Biochemical indicators of dietary intake. Nutritional Epidemiology. W. Willett. New
York, NY, Oxford University Press: 174–243.
Iso, H., K. M. Rexrode, M. J. Stampfer, J. E. Manson, G. A. Colditz, F. E. Speizer, C. H. Hennekens and
W. C. Willett (2001). "Intake of fish and omega-3 fatty acids and risk of stroke in women." JAMA
285(3): 304-312.
Iso, H., S. Sato, A. Kitamura, Y. Naito, T. Shimamoto and Y. Komachi (2003). "Fat and protein intakes
and risk of intraparenchymal hemorrhage among middle-aged Japanese." Am J Epidemiol 157(1): 3239.
Iso, H., S. Sato, U. Umemura, M. Kudo, K. Koike, A. Kitamura, H. Imano, T. Okamura, Y. Naito and T.
Shimamoto (2002). "Linoleic acid, other fatty acids, and the risk of stroke." Stroke 33(8): 2086-2093.
Iso, H., M. J. Stampfer, J. E. Manson, K. Rexrode, F. Hu, C. H. Hennekens, G. A. Colditz, F. E. Speizer
and W. C. Willett (2001). "Prospective study of fat and protein intake and risk of intraparenchymal
hemorrhage in women." Circulation 103(6): 856-863.
Janghorbani, M., F. B. Hu, W. C. Willett, T. Y. Li, J. E. Manson, G. Logroscino and K. M. Rexrode (2007).
"Prospective study of type 1 and type 2 diabetes and risk of stroke subtypes: the Nurses' Health
Study." Diabetes Care 30(7): 1730-1735.
Jorgensen, H., H. Nakayama, H. O. Raaschou and T. S. Olsen (1994). "Stroke in patients with diabetes.
The Copenhagen Stroke Study." Stroke 25(10): 1977-1984.
Karppi, J., J. A. Laukkanen, J. Sivenius, K. Ronkainen and S. Kurl (2012). "Serum lycopene decreases the
risk of stroke in men: a population-based follow-up study." Neurology 79(15): 1540-1547.
Knapp, H. R. (1996). "n-3 fatty acids and human hypertension." Curr Opin Lipidol 7(1): 30-33.
Kris-Etherton, P. M., W. S. Harris, L. J. Appel and C. Nutrition (2003). "Fish consumption, fish oil,
omega-3 fatty acids, and cardiovascular disease." Arterioscler Thromb Vasc Biol 23(2): e20-30.
Laaksonen, D. E., T. A. Lakka, H. M. Lakka, K. Nyyssonen, T. Rissanen, L. K. Niskanen and J. T. Salonen
(2002). "Serum fatty acid composition predicts development of impaired fasting glycaemia and
diabetes in middle-aged men." Diabet Med 19(6): 456-464.
Lakka, T. A., J. M. Venalainen, R. Rauramaa, R. Salonen, J. Tuomilehto and J. T. Salonen (1994).
"Relation of leisure-time physical activity and cardiorespiratory fitness to the risk of acute myocardial
infarction." N Engl J Med 330(22): 1549-1554.
46
Larsson, S. C. and N. Orsini (2011). "Fish consumption and the risk of stroke: a dose-response metaanalysis." Stroke 42(12): 3621-3623.
Larsson, S. C., N. Orsini and A. Wolk (2012). "Long-chain omega-3 polyunsaturated fatty acids and risk
of stroke: a meta-analysis." Eur J Epidemiol 27(12): 895-901.
Larsson, S. C., J. Virtamo and A. Wolk (2011). "Fish consumption and risk of stroke in Swedish
women." Am J Clin Nutr 93(3): 487-493.
Larsson, S. C., J. Virtamo and A. Wolk (2012). "Dietary fats and dietary cholesterol and risk of stroke in
women." Atherosclerosis 221(1): 282-286.
Leosdottir, M., P. M. Nilsson, J. A. Nilsson and G. Berglund (2007). "Cardiovascular event risk in
relation to dietary fat intake in middle-aged individuals: data from The Malmo Diet and Cancer
Study." Eur J Cardiovasc Prev Rehabil 14(5): 701-706.
Lewington, S., R. Clarke, N. Qizilbash, R. Peto, R. Collins and C. Prospective Studies (2002). "Agespecific relevance of usual blood pressure to vascular mortality: a meta-analysis of individual data for
one million adults in 61 prospective studies." Lancet 360(9349): 1903-1913.
Lucke-Wold, B. P., R. C. Turner, A. N. Lucke-Wold, C. L. Rosen and J. D. Huber (2012). "Age and the
metabolic syndrome as risk factors for ischemic stroke: improving preclinical models of ischemic
stroke." Yale J Biol Med 85(4): 523-539.
McLennan, P. L., M. Y. Abeywardena and J. S. Charnock (1988). "Dietary fish oil prevents ventricular
fibrillation following coronary artery occlusion and reperfusion." Am Heart J 116(3): 709-717.
Medline Plus. (2013). "Stroke." from http://www.nlm.nih.gov/medlineplus/ency/article/000726.htm.
Meretoja, A., M. Kaste, R. O. Roine, M. Juntunen, M. Linna, M. Hillbom, R. Marttila, T. Erila, A.
Rissanen, J. Sivenius and U. Hakkinen (2011). "Trends in treatment and outcome of stroke patients in
Finland from 1999 to 2007. PERFECT Stroke, a nationwide register study." Ann Med 43 Suppl 1: S2230.
Montonen, J., R. Jarvinen, A. Reunanen and P. Knekt (2009). "Fish consumption and the incidence of
cerebrovascular disease." Br J Nutr 102(5): 750-756.
Mozaffarian, D., J. S. Gottdiener and D. S. Siscovick (2006). "Intake of tuna or other broiled or baked
fish versus fried fish and cardiac structure, function, and hemodynamics." Am J Cardiol 97(2): 216222.
Mozaffarian, D., R. N. Lemaitre, I. B. King, X. Song, H. Huang, F. M. Sacks, E. B. Rimm, M. Wang and D.
S. Siscovick (2013). "Plasma phospholipid long-chain omega-3 fatty acids and total and cause-specific
mortality in older adults: a cohort study." Ann Intern Med 158(7): 515-525.
Mozaffarian, D., W. T. Longstreth, Jr., R. N. Lemaitre, T. A. Manolio, L. H. Kuller, G. L. Burke and D. S.
Siscovick (2005). "Fish consumption and stroke risk in elderly individuals: the cardiovascular health
study." Arch Intern Med 165(2): 200-206.
Mozaffarian, D., P. Shi, J. S. Morris, P. Grandjean, D. S. Siscovick, D. Spiegelman, W. C. Willett, E. B.
Rimm, G. C. Curhan and J. P. Forman (2012). "Mercury exposure and risk of hypertension in US men
and women in 2 prospective cohorts." Hypertension 60(3): 645-652.
Mozaffarian, D., P. Shi, J. S. Morris, D. Spiegelman, P. Grandjean, D. S. Siscovick, W. C. Willett and E. B.
Rimm (2011). "Mercury exposure and risk of cardiovascular disease in two U.S. cohorts." N Engl J Med
364(12): 1116-1125.
47
Mozaffarian, D. and J. H. Wu (2011). "Omega-3 fatty acids and cardiovascular disease: effects on risk
factors, molecular pathways, and clinical events." J Am Coll Cardiol 58(20): 2047-2067.
Mukherjee, D. and C. G. Patil (2011). "Epidemiology and the global burden of stroke." World
Neurosurg 76(6 Suppl): S85-90.
Myint, P. K., A. A. Welch, S. A. Bingham, R. N. Luben, N. J. Wareham, N. E. Day and K. T. Khaw (2006).
"Habitual fish consumption and risk of incident stroke: the European Prospective Investigation into
Cancer (EPIC)-Norfolk prospective population study." Public Health Nutr 9(7): 882-888.
Nestel, P. J. (2000). "Fish oil and cardiovascular disease: lipids and arterial function." Am J Clin Nutr
71(1 Suppl): 228S-231S.
Norrving, B. and B. Kissela (2013). "The global burden of stroke and need for a continuum of care."
Neurology 80(3 Suppl 2): S5-12.
Park, Y., S. Nam, H. J. Yi, H. J. Hong and M. Lee (2009). "Dietary n-3 polyunsaturated fatty acids
increase oxidative stress in rats with intracerebral hemorrhagic stroke." Nutr Res 29(11): 812-818.
Patti, G., M. Chello, D. Candura, V. Pasceri, A. D'Ambrosio, E. Covino and G. Di Sciascio (2006).
"Randomized trial of atorvastatin for reduction of postoperative atrial fibrillation in patients
undergoing cardiac surgery: results of the ARMYDA-3 (Atorvastatin for Reduction of MYocardial
Dysrhythmia After cardiac surgery) study." Circulation 114(14): 1455-1461.
Pauletto, P., M. Puato, M. G. Caroli, E. Casiglia, A. E. Munhambo, G. Cazzolato, G. Bittolo Bon, M. T.
Angeli, C. Galli and A. C. Pessina (1996). "Blood pressure and atherogenic lipoprotein profiles of fishdiet and vegetarian villagers in Tanzania: the Lugalawa study." Lancet 348(9030): 784-788.
Peters, S. A., R. R. Huxley and M. Woodward (2013). "Smoking as a risk factor for stroke in women
compared with men: a systematic review and meta-analysis of 81 cohorts, including 3,980,359
individuals and 42,401 strokes." Stroke 44(10): 2821-2828.
Puddey, I. B. (1996). "Low serum cholesterol and the risk of cerebral haemorrhage." Atherosclerosis
119(1): 1-6.
Rantakomi, S. H., S. Kurl, J. Sivenius, J. Kauhanen and J. A. Laukkanen (2014). "The frequency of
alcohol consumption is associated with the stroke mortality." Acta Neurol Scand.
Rantakomi, S. H., J. A. Laukkanen, J. Sivenius, J. Kauhanen and S. Kurl (2013). "Alcohol consumption
and the risk of stroke among hypertensive and overweight men." J Neurol 260(2): 534-539.
Reinders, I., J. K. Virtanen, I. A. Brouwer and T. P. Tuomainen (2012). "Association of serum n-3
polyunsaturated fatty acids with C-reactive protein in men." Eur J Clin Nutr 66(6): 736-741.
Reynolds, K., B. Lewis, J. D. Nolen, G. L. Kinney, B. Sathya and J. He (2003). "Alcohol consumption and
risk of stroke: a meta-analysis." JAMA 289(5): 579-588.
Ricci, S., M. G. Celani, E. Righetti, A. Caruso, G. De Medio, G. Trovarelli, S. Romoli, E. Stragliotto and L.
Spizzichino (1997). "Fatty acid dietary intake and the risk of ischaemic stroke: a multicentre casecontrol study. UFA Study Group." J Neurol 244(6): 360-364.
Rizos, E. C., E. E. Ntzani, E. Bika, M. S. Kostapanos and M. S. Elisaf (2012). "Association between
omega-3 fatty acid supplementation and risk of major cardiovascular disease events: a systematic
review and meta-analysis." JAMA 308(10): 1024-1033.
48
Roman, H. A., T. L. Walsh, B. A. Coull, E. Dewailly, E. Guallar, D. Hattis, K. Marien, J. Schwartz, A. H.
Stern, J. K. Virtanen and G. Rice (2011). "Evaluation of the cardiovascular effects of methylmercury
exposures: current evidence supports development of a dose-response function for regulatory
benefits analysis." Environ Health Perspect 119(5): 607-614.
Sacco, R. L. (1997). "Risk factors, outcomes, and stroke subtypes for ischemic stroke." Neurology 49(5
Suppl 4): S39-44.
Sacco, R. L., E. J. Benjamin, J. P. Broderick, M. Dyken, J. D. Easton, W. M. Feinberg, L. B. Goldstein, P.
B. Gorelick, G. Howard and S. J. Kittner (1997). "Risk factors." Stroke 28(7): 1507-1517.
Sacco, R. L., S. E. Kasner, J. P. Broderick, L. R. Caplan, J. J. Connors, A. Culebras, M. S. Elkind, M. G.
George, A. D. Hamdan, R. T. Higashida, B. L. Hoh, L. S. Janis, C. S. Kase, D. O. Kleindorfer, J. M. Lee, M.
E. Moseley, E. D. Peterson, T. N. Turan, A. L. Valderrama, H. V. Vinters, C. o. C. S. American Heart
Association Stroke Council, Anesthesia, R. Council on Cardiovascular, Intervention, C. Council on, N.
Stroke, E. Council on, Prevention, D. Council on Peripheral Vascular, P. A. Council on Nutrition and
Metabolism (2013). "An updated definition of stroke for the 21st century: a statement for healthcare
professionals from the American Heart Association/American Stroke Association." Stroke 44(7): 20642089.
Salonen, J. T. (1988). "Is there a continuing need for longitudinal epidemiologic research? The Kuopio
Ischaemic Heart Disease Risk Factor Study." Ann Clin Res 20(1-2): 46-50.
Salonen, J. T., K. Seppanen, K. Nyyssonen, H. Korpela, J. Kauhanen, M. Kantola, J. Tuomilehto, H.
Esterbauer, F. Tatzber and R. Salonen (1995). "Intake of mercury from fish, lipid peroxidation, and the
risk of myocardial infarction and coronary, cardiovascular, and any death in eastern Finnish men."
Circulation 91(3): 645-655.
Sasaki, S., X. H. Zhang and H. Kesteloot (1995). "Dietary sodium, potassium, saturated fat, alcohol, and
stroke mortality." Stroke 26(5): 783-789.
Sauvaget, C., J. Nagano, M. Hayashi and M. Yamada (2004). "Animal protein, animal fat, and
cholesterol intakes and risk of cerebral infarction mortality in the adult health study." Stroke 35(7):
1531-1537.
Savelieva, I., A. Kourliouros and J. Camm (2010). "Primary and secondary prevention of atrial
fibrillation with statins and polyunsaturated fatty acids: review of evidence and clinical relevance."
Naunyn Schmiedebergs Arch Pharmacol 381(3): 1-13.
Schlawicke Engstrom, K., U. Stromberg, T. Lundh, I. Johansson, B. Vessby, G. Hallmans, S. Skerfving
and K. Broberg (2008). "Genetic variation in glutathione-related genes and body burden of
methylmercury." Environ Health Perspect 116(6): 734-739.
Selvin, E., S. Marinopoulos, G. Berkenblit, T. Rami, F. L. Brancati, N. R. Powe and S. H. Golden (2004).
"Meta-analysis: glycosylated hemoglobin and cardiovascular disease in diabetes mellitus." Ann Intern
Med 141(6): 421-431.
Shah, R. S. and J. W. Cole (2010). "Smoking and stroke: the more you smoke the more you stroke."
Expert Rev Cardiovasc Ther 8(7): 917-932.
Simon, J. A., J. Fong, J. T. Bernert, Jr. and W. S. Browner (1995). "Serum fatty acids and the risk of
stroke." Stroke 26(5): 778-782.
49
Siri-Tarino, P. W., Q. Sun, F. B. Hu and R. M. Krauss (2010). "Meta-analysis of prospective cohort
studies evaluating the association of saturated fat with cardiovascular disease." Am J Clin Nutr 91(3):
535-546.
Sivenius, J., J. Torppa, J. Tuomilehto, P. Immonen-Raiha, M. Kaarisalo, C. Sarti, K. Kuulasmaa, M.
Mahonen, A. Lehtonen and V. Salomaa (2009). "Modelling the burden of stroke in Finland until 2030."
Int J Stroke 4(5): 340-345.
Song, Y. M., J. Sung, D. A. Lawlor, G. Davey Smith, Y. Shin and S. Ebrahim (2004). "Blood pressure,
haemorrhagic stroke, and ischaemic stroke: the Korean national prospective occupational cohort
study." BMJ 328(7435): 324-325.
Stegmayr, B., K. Asplund, K. Kuulasmaa, A. M. Rajakangas, P. Thorvaldsen and J. Tuomilehto (1997).
"Stroke incidence and mortality correlated to stroke risk factors in the WHO MONICA Project. An
ecological study of 18 populations." Stroke 28(7): 1367-1374.
Strazzullo, P., L. Scalfi, F. Branca, G. Cairella, F. Garbagnati, A. Siani, G. Barba, P. Rubba and G. Mancia
(2004). "Nutrition and prevention of ischemic stroke: present knowledge, limitations and future
perspectives." Nutr Metab Cardiovasc Dis 14(2): 97-114.
Suh, I., S. H. Jee, H. C. Kim, C. M. Nam, I. S. Kim and L. J. Appel (2001). "Low serum cholesterol and
haemorrhagic stroke in men: Korea Medical Insurance Corporation Study." Lancet 357(9260): 922925.
Svensson, M., E. B. Schmidt, K. A. Jorgensen, J. H. Christensen and O. S. Group (2006). "N-3 fatty acids
as secondary prevention against cardiovascular events in patients who undergo chronic hemodialysis:
a randomized, placebo-controlled intervention trial." Clin J Am Soc Nephrol 1(4): 780-786.
Tell, G. S., G. W. Evans, A. R. Folsom, T. Shimakawa, M. A. Carpenter and G. Heiss (1994). "Dietary fat
intake and carotid artery wall thickness: the Atherosclerosis Risk in Communities (ARIC) Study." Am J
Epidemiol 139(10): 979-989.
U.S. Department of Agriculture. (2010). "Agricultural Research Service USDA National Nutrient ,
Database for Standard Reference, Release 23Nutrient Data Laboratory Home Page,." Retrieved
01.09.2014, from http://www.ars.usda.gov/ba/bhnrc/ndl
US CDC. (2004). "Surgeon General’s Report—The Health Consequences of Smoking, Chapter 3." from
http://www.google.fi/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CCgQFjAA&url=http%3A
%2F%2Fwww.cdc.gov%2Ftobacco%2Fdata_statistics%2Fsgr%2F2004%2Fpdfs%2Fchapter1.pdf&ei=S6
GPUpjMAYeS5AS42YDIAQ&usg=AFQjCNGfGjmgJnyINbSELXX6oFn6VmgaiA&bvm=bv.56988011,d.bGE
&cad=rja.
US Department of Health and Human Services and US Environmental Protection Agency. (February
2006). "Mercury levels in commercial fish and shellfish."
Retrieved December 2013, from
http://www.cfsan.fda.gov/~frf/sea-mehg.html.
Virtanen, J. K., J. A. Laukkanen, J. Mursu, S. Voutilainen and T. P. Tuomainen (2012). "Serum longchain n-3 polyunsaturated fatty acids, mercury, and risk of sudden cardiac death in men: a
prospective population-based study." PLoS One 7(7): e41046.
Virtanen, J. K., J. Mursu, S. Voutilainen and T. P. Tuomainen (2009). "Serum long-chain n-3
polyunsaturated fatty acids and risk of hospital diagnosis of atrial fibrillation in men." Circulation
120(23): 2315-2321.
50
Virtanen, J. K., A. N. Nyantika, J. Kauhanen, S. Voutilainen and T. P. Tuomainen (2012). "Serum longchain n-3 polyunsaturated fatty acids, methylmercury and blood pressure in an older population."
Hypertens Res 35(10): 1000-1004.
Virtanen, J. K., T. H. Rissanen, S. Voutilainen and T. P. Tuomainen (2007). "Mercury as a risk factor for
cardiovascular diseases." J Nutr Biochem 18(2): 75-85.
Virtanen, J. K., S. Voutilainen, T. H. Rissanen, J. Mursu, T. P. Tuomainen, M. J. Korhonen, V. P.
Valkonen, K. Seppanen, J. A. Laukkanen and J. T. Salonen (2005). "Mercury, fish oils, and risk of acute
coronary events and cardiovascular disease, coronary heart disease, and all-cause mortality in men in
eastern Finland." Arterioscler Thromb Vasc Biol 25(1): 228-233.
Voutilainen, S., T. H. Rissanen, J. Virtanen, T. A. Lakka, J. T. Salonen and S. Kuopio Ischemic Heart
Disease Risk Factor (2001). "Low dietary folate intake is associated with an excess incidence of acute
coronary events: The Kuopio Ischemic Heart Disease Risk Factor Study." Circulation 103(22): 26742680.
Wang, X., Y. Dong, X. Qi, C. Huang and L. Hou (2013). "Cholesterol levels and risk of hemorrhagic
stroke: a systematic review and meta-analysis." Stroke 44(7): 1833-1839.
Wennberg, M., U. Stromberg, I. A. Bergdahl, J. H. Jansson, J. Kauhanen, M. Norberg, J. T. Salonen, S.
Skerfving, T. P. Tuomainen, B. Vessby and J. K. Virtanen (2012). "Myocardial infarction in relation to
mercury and fatty acids from fish: a risk-benefit analysis based on pooled Finnish and Swedish data in
men." Am J Clin Nutr 96(4): 706-713.
Wolf, P. A., R. D. Abbott and W. B. Kannel (1991). "Atrial fibrillation as an independent risk factor for
stroke: the Framingham Study." Stroke 22(8): 983-988.
World Health Organization. (1990). "International Programme on Chemical Safety (IPCS),
Environmental Health Criteria 101: Methylmercury." from http://www.who.int/phe/news/Mercuryflyer.pdf.
Wu, J. H. and D. Mozaffarian (2014). "omega-3 Fatty acids, atherosclerosis progression and
cardiovascular outcomes in recent trials: new pieces in a complex puzzle." Heart 100(7): 530-533.
Xun, P., B. Qin, Y. Song, Y. Nakamura, T. Kurth, S. Yaemsiri, L. Djousse and K. He (2012). "Fish
consumption and risk of stroke and its subtypes: accumulative evidence from a meta-analysis of
prospective cohort studies." Eur J Clin Nutr 66(11): 1199-1207.
Yaemsiri, S., S. Sen, L. Tinker, W. Rosamond, S. Wassertheil-Smoller and K. He (2012). "Trans fat,
aspirin, and ischemic stroke in postmenopausal women." Ann Neurol 72(5): 704-715.
Yuan, J. M., R. K. Ross, Y. T. Gao and M. C. Yu (2001). "Fish and shellfish consumption in relation to
death from myocardial infarction among men in Shanghai, China." Am J Epidemiol 154(9): 809-816.
Zhang, Y., G. Hu, Z. Yuan and L. Chen (2012). "Glycosylated hemoglobin in relationship to
cardiovascular outcomes and death in patients with type 2 diabetes: a systematic review and metaanalysis." PLoS One 7(8): e42551.
Zorowitz, R., E. Baerga and S. Cuccurullo (2004). Types of stroke. Physical medicine and rehabilitation
board review. S. Cuccurullo. New York, Demos.