'#
© Anupam Chandra, 2020
Series of dissertations submitted to the Faculty of Medicine, University of Oslo
ISBN 978-82-8377-729-1
All rights reserved. No part of this publication may be
reproduced or transmitted, in any form or by any means, without permission.
Cover: Hanne Baadsgaard Utigard.
Print production: Reprosentralen, University of Oslo.
Table of content
1. Acknowledgments 2. Abbreviations 3. List of papers 4. Thesis summary 5. Introduction
5.1 Fatty acids
5.2 Structure and sources
5.3 Historical aspects
5.4 Fatty acids and associations with cardiovascular disease and risk factors
5.4.1 Marine n-3 polyunsaturated fatty acids$
5.4.2 Linoleic acid&
5.4.3 Trans fatty acids
5.5 Intake of fatty acids world wide
5.6 Intake of fatty acids in Norway
6. Rationale and aims
6.1 Rationale and general aim
6.2 Paper specific aims
7. Material and methods
7.1 Study population!
7.2 Data collection"
7.3 Fatty acid analysis"
7.4 Statistics
7.5 Ethical considerations
8. Summary of results
8.1 Descriptive data of the ACE 1950 Study Cohort:
8.2 Paper I
8.3 Paper II!
8.4 Paper III#
9. Discussion
9.1 Methodological considerations
9.1.1 Study cohort"
9.1.2 Study design"
9.1.3 Bias and validity"
9.1.4 Fatty acid analysis""
9.1.5 Statistical considerations""
9.2 Marine n-3 polyunsaturated fatty acids, linoleic acid and cardiovascular risk factors
9.2.1 Serum lipids"$
9.2.2 Fasting plasma glucose and glycated hemoglobin"%
9.2.3 Body mass index"&
9.2.4 Renal function and blood pressure"&
9.2.5 Carotid intima-media thickness#
9.2.6 C-reactive protein#
9.2.7 General health and lifestyle#
9.3 Trans fatty acids and cardiovascular risk factors
9.3.1 Industrial trans fatty acids#!
9.3.2 Ruminant trans fatty acids#"
10. Conclusion and implications 11. Future perspectives 12. References
!
1. Acknowledgments
I found myself walking in the corridors of Akershus University Hospital, when I was
suddenly asked, in a mixed Swedish-Danish-Norwegian accent, whether I would like to join a research project. At that moment, Professor My Svensson made the neurons between my speech center and mouth fire without me even knowing. I had said yes to research, something I had never imagined doing, neither as a medical student nor as a clinician. My, in this way you probably handed me the biggest challenge of my life, and as my senior supervisor you also made sure I got all the way through. I am sincerely grateful for your enthusiasm for my project, your support, encouragement and patience. And for teaching me the art of
multitasking… which I did not think was possible.
As expected, these three years have been filled with ups and downs. And especially during the 2nd year, I faced some though times. Making it through had been difficult, if my supervisor Ivar Anders Eide, had not shown me the path. At that time, the famous lyrics by Mariah Carry “And then a hero comes along, with the strength to carry on. And you cast your fears aside, and you know you can survive” gave me a completely new meaning. You have been a mentor and a friend, and thanks to you I have been introduced to the storyteller hidden inside me somewhere. You have gone the extra mile for me, and that will never be forgotten.
Joe Chan; I have been lucky to have you around during this period. The perfect break from exhausting work was when you came barging in to inform me that Apple had released a new product. You never used more than 12 minutes to solve my recurrent computer problems; 2 minutes to solve the actual problem, after laughing at me for 10 minutes for my lacking computer skills. Your sense of humor has been an antagonist to work related stress and frustration. Thanks for being there for me, 24/7.
I am immensely thankful to my co-supervisor, Torbjørn Omland, for stepping in every time I faced challenges in my project. You always came up with a fitting solution to every problem and made sure that I could go back to my desk to continue my work. Thank you for sharing your knowledge.
A wholehearted thanks to Erik Berg Schmidt for treating me as one of your own PhD students. Your expertise in the field of fatty acids and cardiometabolic health has been invaluable to me. My stays in Denmark were educational as well as adventurous, and thanks to you, I can say, like every other Norwegian: “Det er dejlig å være norsk i Danmark”.
A special thanks to Rikke Bulow Eschen at The Lipid Research Center, Aalborg University Hospital.
Helge Røsjø, I thank you for your support and constructive feedback, and for introducing me to the ACE 1950 Study group in 2012. I was warmly welcomed when I joined this group in 2017, and especially Magnus Nakrem Lyngbakken made sure I did not feel like a stranger.
Magnus, you have been extremely helpful and guided me towards increasing the quality of
"
my work. Lunch together with the ACE 1950 Study group at “hytta” are among the better memories of this period, although my coffee never passed the “Thea-test”.
I am grateful to Arnljot Tveit for making me a part of the ACE 1950 Study group. Your advice and guidance have been essential for my project. Also, a special thanks to my colleges at Bærum Hospital; Trygve Berge and Håkon Ihle-Hansen.
I wish to show my gratitude to the entire ACE 1950 Study group. I cannot even imagine the time and effort you all have put into executing this project and collecting the data. It has not been taken for granted. Finally, I wish to thank the study participants of the ACE 1950 Study.
I have been honored to work with Karsten Midtvedt and Anders Åsberg from the Dept. of Renal medicine, Rikshospitalet. Thank you for giving me the opportunity to write with you on immunosuppression and reproductive health. It has for sure opened new doors for me.
It is at The Dept. of Renal Medicine, Akershus University Hospital, where I grew as a clinician. Although my days of research were filled with everything but renal medicine, I never felt fare away from home. Willy, Bastian, Carl Erik, Rüdiger, Toril, Trude, Geir, Helga, Marte, Krystina and Christian, you have all been very supportive and positive towards my work, and encouraged me with the words: “You will get there”.
I will forever be grateful to the University of Oslo and Dept. of Research, Akershus
University Hospital for accepting me as a PhD student and helping me each step on the way.
It has been a privilege to be connected to these institutes. I also wish to thank Ludvig André Munthe for your recommendations on how to write a thesis.
To the members of the doctoral thesis evaluation committee, I am immensely thankful for the time you have spent on my work.
My family has been like a sports team for me. My gorgeous wife, Beate, has been my biggest cheerleader, and my children, Amrita (8) and Arun (4), have been the mascots of Team Anupam. Beate, I thank you for bearing with me during these three years. There have been a lot of tears, however, we both know they have mostly been mine. You believed in me at times I did not, and for that I can never thank you enough. And my mascots Amrita and Arun, you are the energy drink every athlete dream about.
I thank my sister, Richa, for keeping reminding me that things could have been worse.
And finally, to my parents, Dinesh and Shikha Chandra, all I am today is because of you!
#
2. Abbreviations
ACE Akershus Cardiac Examination ALA Alpha linoleic acid
BMI Body mass index
CKD Chronic kidney disease
cIMT Carotid intima-media thickness
CRP C-reactive protein
CV Cardiovascular
DHA Docosahexaenoic acid
DM Diabetes mellitus
DPA Docosapentaenoic acid
EPA Eicosapentaenoic acid
FAs Fatty acids
FFQ Food frequency questionnaires FPG Fasting plasma glucose
HbA1c Glycated hemoglobin HDL High-density lipoprotein iTFAs Industrial trans fatty acids
IQR Interquartile range
LA Linoleic acid
LDL Low-density lipoprotein PUFAs Polyunsaturated fatty acids RCTs Randomized controlled trials rTFAs Ruminant trans fatty acids
SD Standard deviation
$ Std. β-coeff. Standardized regression coefficient
TFAs Trans fatty acids
Unstd. β-coeff. Unstandardized regression coefficient
wt% Weight percentage
%
3. List of papers
Paper I:
Anupam Chandra, Helge Røsjø, Ivar Anders Eide, Thea Vigen, Håkon Ihle-Hansen, Eivind Bjørkan Orstad, Ole Morten Rønning, Magnus Nakrem Lyngbakken, Trygve Berge, Erik Berg Schmidt, Torbjørn Omland, Arnljot Tveit, My Svensson. Plasma marine n-3
polyunsaturated fatty acids and cardiovascular risk factors: data from the ACE 1950 study.
Eur J Nutr. 2019 May 23.
Paper II:
Anupam Chandra, Helge Røsjø, My Svensson, Thea Vigen, Håkon Ihle-Hansen, Eivind Bjørkan Orstad, Ole Morten Rønning, Magnus Nakrem Lyngbakken, Ståle Nygård, Trygve Berge, Erik Berg Schmidt, Torbjørn Omland, Arnljot Tveit, Ivar Anders Eide. Plasma linoleic acid levels and cardiovascular risk factors: Results from the Norwegian ACE 1950 Study.
Submitted.
Paper III:
Anupam Chandra, Magnus Nakrem Lyngbakken, Ivar Anders Eide, Helge Røsjø, Thea Vigen, Håkon Ihle-Hansen, Eivind Bjørkan Orstad, Ole Morten Rønning, Trygve Berge, Erik Berg Schmidt, Arnljot Tveit, Torbjørn Omland, My Svensson. Plasma trans fatty acid levels and cardiovascular risk factors: Results from the Akershus Cardiac Examination 1950 Study.
Submitted.
&
4. Thesis summary
Background: A high intake of marine n-3 polyunsaturated fatty acids (PUFAs) and the major n-6 PUFA linoleic acid (LA), has been associated with reduced risk of cardiovascular (CV) morbidity and mortality in large epidemiological studies. In contrast, a high consumption of trans fatty acids (TFAs), in particular industrially produced TFAs (iTFAs), is reported to be harmful for CV health.
Intake of fatty fish, the major source of marine n-3 PUFAs, has decreased over the last decades in Norway, while the consumption of LA has been relatively low compared to other European countries. After 2004, the consumption of iTFAs was reduced to <1% of total daily energy intake. However, to avoid adverse effects form iTFAs, a total daily energy intake of
<0.5% might be necessary.
The relationship between the current intake of marine n-3 PUFAs, LA and iTFAs and CV health in Norway is unclear. Accordingly, the aim of this study was to examine the
associations between plasma levels of these fatty acids, reflecting their dietary intake, and multiple CV risk factors in a middle-aged Norwegian community-acquired cohort.
Methods: A total of 3,706 participants, born in 1950 and residing in Akershus County, were enrolled in this cross-sectional study. All participants underwent structural baseline
interviews, clinical examination, blood sampling and advanced imaging. Plasma phospholipid levels of marine n-3 PUFAs, LA and iTFAs were analyzed by gas chromatography and expressed as weight percentage (wt%) of total plasma phospholipid fatty acids. The main statistical approach was multivariable linear regression analysis.
Results: Plasma marine n-3 PUFA levels ranged from 2.7 to 20.3 wt%, with a median level of 7.7 wt% (interquartile range [IQR] 4.3 to 11.1 wt%). Plasma LA levels ranged from 11.4 to 32.0 wt%, with a median level of 20.8 wt% (IQR 16.8-24.8 wt%). Plasma iTFA levels ranged from 0.09 to 0.62 wt%, with a median level of 0.20 wt% (IQR 0.13 to 0.27 wt%).
' High plasma marine n-3 PUFA levels were associated with lower serum triglycerides
(Standardized regression coefficient [Std. β-coeff.] -0.14, p<0.001), C-reactive protein (CRP) levels (Std. β-coeff. -0.03, p=0.04) and higher levels of high-density lipoprotein cholesterol (Std. β-coeff. 0.08, p<0.001). Furthermore, we found weak but statistically significant inverse associations between plasma marine n-3 PUFA levels and glycated hemoglobin, body mass index (BMI) and serum creatinine.
High plasma levels of LA were associated with lower fasting plasma glucose (Std. β-coeff.
-0.10, p<0.001) and serum triglycerides (Std. β-coeff. -0.10, p<0.001). In addition, weak inverse associations were found between plasma LA levels and BMI, low-density lipoprotein cholesterol levels and systolic and diastolic blood pressure.
Plasma iTFA levels were inversely associated with serum triglycerides, fasting plasma glucose levels, BMI, systolic and diastolic blood pressure and CRP levels. Participants with high compared to low plasma iTFA levels were more educated, had a lower prevalence of smoking and consumed alcohol less often.
Conclusion: High plasma levels of marine n-3 PUFAs and LA were favorably associated with CV risk factors. Our findings signal that a high intake of marine n-3 PUFAs and LA might benefit CV health.
Plasma iTFA levels were low and inversely associated with CV risk factors, findings which were likely confounded by lifestyle related variables. Overall, our findings related to iTFAs suggest that the current low iTFA intake in Norway does no longer pose a threat to CV health.
5. Introduction
Cardiovascular (CV) disease is a common group of diseases affecting the heart or blood vessels, and include conditions such as coronary artery disease, cerebrovascular disease, peripheral artery disease and venous thromboembolism (1). Annually, more people die from CV disease than any other medical condition (1). Major CV risk factor, such as blood pressure and serum cholesterol (Figure 1), can be modified by dietary interventions. A link between various dietary patterns and different CV disease risks was described in 1960s by dr. Keys in The Seven Countries Study (2). His work laid the foundation for the well-
recognized diet-heart-hypothesis, which postulates a connection between dietary fats, serum cholesterol levels and occurrence of heart disease. A typically Western diet, containing a high amount of trans fatty acids (TFAs) and saturated fats, has been associated with increased risk of CV morbidity and mortality (3). In contrast, a Mediterranean diet, recognized for its high content of polyunsaturated fatty acids (PUFAs), is suggested to improve CV health (4).
According to The Global Burden of Disease Study 2016, a high TFA and a low PUFA intake are among the 25 most important risk factors for CV death (Figure 1).
Norwegians are recognized for their traditional Nordic diet, characterized by a high intake of fish and seafood, root vegetables, rapeseed oil and whole grain bread (5). A traditional Nordic diet is suggested to have a favorably influence on serum cholesterol and inflammation,
findings mainly attributed to the high content of marine n-3 PUFAs (5).
Figure 1. Cardiovascular death attributed to the 25 most important risk factors in 2016.
Includes both genders and all ages. Data is presented as number of deaths per 100 000 total deaths. Available at https://vizhub.healthdata.org/gbd-compare/
5.1 Fatty acids
Fatty acids (FAs) are found as components of complex lipid molecules, such as fats and phospholipids, in microorganisms, plants and animals. They serve as energy storage, are essential components of cell membranes, and play important roles in signal transduction pathways and genetic regulations (6). Most FAs, termed non-essential FAs, are synthesized in the body in sufficient quantity to satisfy our needs. Essential FAs, however, cannot be
produced by the body and must be consumed through food or dietary supplementation (6).
Marine n-3 PUFAs and linoleic acid (LA), the major n-6 PUFA, are examples of such essential FAs. They will, together with TFAs, be the focus of this theses.
5.2 Structure and sources
Most naturally occurring FAs consist of a straight chain of carbon atoms, with hydrogen atoms along the length of the chain. They have a methyl group at one end of the molecule, designated omega (ω), and terminate with a carboxyl group, also known as the “acid group”, at the other end (7). Saturated FAs are characterized by the presence of only single bonds between carbon atoms, while monounsaturated FAs have one double bond and PUFAs have two or more double bonds along the carbon atom chain (7). PUFAs are named after the
location of the first double bond, counted from the omega end. (7). In case of n-3 PUFAs, also known as omega-3 FAs, the first double bond is located after the third carbon atom. The most recognized n-3 PUFAs are the marine based ones, namely docosahexaenoic acid (DHA, C22:6n-3) and eicosapentaenoic acid (EPA, C20:5n-3). DHA consist of 22 carbon atoms with 6 double bonds, while EPA has 20 carbon atoms and 5 double bonds (Figure 2).
Figure 2. Chemical structures of docosahexaenoic acid and eicosapentaenoic acid.
! A third marine n-3 PUFA, docosapentaenoic acid (DPA, 22:5n-3) exists in smaller amounts in humans and acts mainly as an intermediate between EPA and DHA (8). A plant-derived n-3 PUFA, alpha linoleic acid (ALA), can be converted to EPA and DHA (Figure 3), however, suchendogenous conversion in humans is low (<5%) (9). Fatty fish and fish oil are the major sources of marine n-3 PUFAs.
Figure 3. The n-3 PUFA and n-6 PUFA metabolism pathways.
Reprinted with permission from ©InTechOpen 2017. Importance of Fatty Acids in Physiopathology of Human Body Nagy K, Tiuca ID. Published under CC BY 3.0 license. Available from: htpp://dx.doi.org/10.5772/67407
LA is the major n-6 PUFA, mainly derived from vegetable oils, nuts, eggs and meats (10). Its backbone consists of 18 carbon atoms, with the first double bond located after the sixth carbon atom from the omega terminal. LA is considered the parent compound in the n-6 PUFA metabolism, and can be elongated and desaturated to other active n-6 PUFAs (Figure 3). Although an essential FA, LA is the most consumed PUFA in human diet and deficiencies are therefore highly uncommon (10).
"
For both marine n-3 PUFAs and LA, it is the length of the carbon atom chain and the kinked structure, created by the double bonds, that forms basis for their favorable biological
properties (6).
TFAs are unsaturated FAs with at least one double bond in trans configuration, meaning that the attached hydrogen atoms are located on opposite sides of the carbon atoms (6) (Figure 4).
Such trans double bonds straightens the FA molecule, in contrast to isomers with cis double bonds, creating a bend in the FA chain (Figure 4). The shape and physiological properties of TFAs are more like those of saturated than unsaturated FAs (6).
Figure 4. Fatty acid chains with cis and trans double bonds.
TFAs occur naturally in ruminant meat and dairy products, and are produced by
biohydrogenation in ruminant gut (11). In modern diet however, most TFAs are found in industrial processed food, created by partial hydrogenation of vegetable and fish oils (11).
While ruminant TFAs (rTFAs) are found in very small amounts (<5% of total FAs) in ruminant products, the concentration of industrial produced TFAs (iTFAs) in partial
# hydrogenated oils may range up to 60% of total FAs (12). In addition to being cheap in
production, iTFAs have desirable physical properties like long shelf life and temperature stability, and have therefore been widely used in products like margarines, bakery products, crackers and deep-fried food (12).
5.3 Historical aspects
In early 19th century, dietary fat was simply considered a source of calories. This perception was changed when dr. George Oswald Burr (Figure 5) discovered LA as the first essential FA, through a simple experiment on rats in 1929 (13). His findings were initially met with skepticism, as the idea of fats being essential in diet was too revolutionary for many (14).
However, within a few years, findings of dr. Burr were confirmed by other scientists and generally accepted (14). The work of dr. Burr and colleagues reached new heights in 1931, when they discovered ALA, the omega-3 analog of LA (15). The essentiality of ALA, and its derivatives EPA and DHA, were determined over the following decades (14). These
discoveries are now considered landmarks in lipid research.
Figure 5. Dr. George Oswald Burr (left). Originally published in the Journal of Lipid Research. Spector A., Kim H. Discovery of essential fatty acids. J. Lipid Res. 2015. 56; 11-21. © the American Society for Biochemistry and Molecular Biology. Bang and Dyerberg on one of their expeditions in Greenland (right). Privat photograph, reprinted with permission from Prof. Jørn Dyerberg.
$ Except from being essential FAs, n-3 PUFAs were not believed to have any other important biological functions. However, this point of view changed dramatically when Jørn Dyerberg and Hans Olaf Bang (Figure 5), researchers from Denmark, reported a low incidence of myocardial infarction in Greenland Intuits, whose diet consisted of marine fat rich in EPA and DHA (16). In addition to higher plasma marine n-3 PUFAs, Greenland Intuits had lower serum cholesterol and triglyceride levels compared to the average Danish control, findings that could possibly explain lower incidence of ischemic heart disease (16). Their findings were published in Lancet in 1971, and are still regarded as the first connection between marine n-3 PUFAs and a healthy heart (16).
The story of iTFAs also started in early 19th century, when the German chemist Wilhelm Normann discovered that treating liquid fat with hydrogen gas would give fat a semi-solid consistency (12). He called this process for fat hardening and received a patent for it in 1903.
Hydrogenation of oils was later adapted by the food industry, and by 1950s iTFAs were used in margarines and bakery-products (12). Despite high content of iTFAs, margarines were often advertised as a healthier alternative to butter, which was rich in saturated FAs.
5.4 Fatty acids and associations with cardiovascular disease and risk factors 5.4.1 Marine n-3 polyunsaturated fatty acids
Since the time of Dyerberg and Bang, numerous studies have investigated the association between marine n-3 PUFA consumption and CV outcomes. Results from epidemiological studies support an overall protective role of marine n-3 PUFAs against CV morbidity and mortality (17-19). In a meta-analysis of prospective studies published between 1947 and 2015, a high intake of marine n-3 PUFAs was associated with an 18% risk reduction for CV events (20). A number of large randomized controlled trials (RCTs), such as the Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico (GISSI) – Prevenzione trial,
% the GISSI – Heart Failure trial and the Japan EPA Lipid Intervention Study (JELIS), reported reduced mortality in patients receiving marine n-3 PUFA supplementation compared to controls (21-23). However, the role of marine n-3 PUFAs in prevention and treatment of CV disease was challenged by later published meta-analyses of RCTs, indicating no effect of marine n-3 PUFA supplementation on CV outcomes (24-26). Although these meta-analyses were criticized for excluding relevant trials and applying conservative statistical approaches (27), uncertainties were created regarding the cardioprotective effects of marine n-3 PUFAs.
Late 2018 saw the publication of two RCTs, A Study of Cardiovascular Events in Diabetes (ASCEND) and Vitamin D and Omega-3 Trial (VITAL), aimed to examine whether marine n-3 PUFAs could reduce the risk for major CV events (28, 29). In ASCEND, patients with diabetes but without evidence of CV disease were randomized to approximately 900 mg/day of EPA and DHA or to placebo. Similarly, participants without prior history of CV disease were randomized to approximately 900 mg/day of EPA and DHA, vitamin D or placebo in VITAL. Although certain prespecified secondary outcomes were reduced by marine n-3 PUFA supplements, both ASCEND and VITAL were considered “null” studies as the primary end point of composite major CV events was not significantly reduced. They were both primary prevention trials with the use of low-dose marine n-3 PUFA supplementation. The recently published Reduction of Cardiovascular Events with Icosapent Ethyl – Intervention Trial (REDUCE-IT), brought marine n-3 PUFAs back into the limelight (30). This was a combined primary and secondary prevention trial, in which patients with established CV disease or diabetes mellitus (DM) were enrolled. It showed that use of EPA 4 grams daily was superior to placebo in reducing triglycerides, CV events, and CV death among patients with high triglycerides whom were already on statin therapy with well-controlled cholesterol levels. These findings were supported by a recent meta-analysis of RCTs, in which marine n-3 PUFA supplementation reduced risk of CV outcomes, even afterexclusion of REDUCE-IT
&
(31). The risk reduction in this meta-analysis was linearly related to the marine n-3 PUFA dose.
Marine n-3 PUFAs might improve CV health by beneficially influencing CV risk factors, such as lowering of triglycerides and inflammation (32, 33). In a recent meta-analysis of RCTs, marine n-3 PUFA supplements significantly reduced serum triglycerides, systolic and diastolic blood pressure and C-reactive protein (CRP) levels, while serum high-density lipoproteins (HDL) levels were increased (34). Effect of marine n-3 PUFA consumption on serum low-density lipoprotein (LDL) cholesterol has been controversial. While several interventional studies report an increase in serum LDL cholesterol levels after marine n-3 PUFA supplementation (35), a recent meta-analysis showed neutral effect (36). Marine n-3 PUFAs appear to have antiarrhythmic and platelet inhibiting effects (37, 38), and are also suggested to increase stability of atherosclerotic plaques (39). In a recent prospective analysis, erythrocyte marine n-3 PUFA levels were inversely associated with coronary artery
calcification (40). The ongoing Effect of Icosapent Ethyl on Progression of Coronary Atherosclerosis in Patients With Elevated Triglycerides on Statin Therapy (EVAPORATE) trial aims to examine whether high dose EPA (4 g/daily), as an add on to statin therapy, reduces progression of coronary atherosclerosis (41).
5.4.2 Linoleic acid
In contrast to marine n-3 PUFAs, a high intake of LA has generally been considered harmful for CV health. LA may act as a precursor for arachidonic acid with proinflammatory
properties, and is also suggested to decrease the conversion of ALA to EPA and DHA (42).
Based on these concerns, advice was given to maintain a low n-6/n-3 PUFA ratio (43). The attitude towards LA started to change in the late 1990s – early 2000, as epidemiological studies reported an inverse association between LA intake and risk of CV events (44, 45).
' However, LA once again became a matter of discussion when Ramsden et al. published results from their secondary analysis of the Sydney Diet Heart Study and Minnesota Coronary Experiment in 2013 and 2016, respectively (46, 47). In these studies, diets rich in LA were directly associated with an increase in overall and CV mortality. The findings have been questioned, as intervention group received more LA than generally recommended (48). In addition, the content of TFAs in diet was higher than recommended at the time of data collection, which might have confounded results (49). Two essential publications, a meta- analysis and a pooled analysis of cohort studies, support the role of LA in reduction of CV mortality risk (49, 50). In fact, Farvid et al. have demonstrated a linear inverse association between intake of LA and risk of coronary heart disease death (Figure 6) (50). This
relationship has been confirmed by a recent meta-analysis of 44 prospective cohort studies, including > 800,000 participants in which LA consumption was assessed by dietary surveys or biomarkers (51).
Figure 6. Dose-response analysis for curvilinear association between dietary intake of linoleic acid and risk of coronary heart disease deaths. Reprinted with permission from Wolters Kluwer Health, Inc.: Circulation; Farvid et al.43 (copyright 2014).
The favorable influence of a high LA intake on CV health is suggested to be mediated by lowering of serum triglycerides and LDL cholesterol levels (52, 53). In addition, high plasma LA levels have been associated with lower fasting plasma glucose (FPG) and a lower risk of incident type 2 DM (54, 55). Interestingly, LA does not seem to promote inflammation as previously presumed (43). In clinical trials, a high LA intake has not affected plasma or tissue concentration of arachidonic acid, nor increased markers of inflammation such as CRP, interleukin 6 or soluble tumor necrosis factor receptor (56, 57). This can be explained by the tight regulation of endogenous LA to arachidonic acid conversion, which in studies is reported to be less than 0.3% (58).
5.4.3 Trans fatty acids
In 1981, a group of Welsh researchers speculated that consumption of iTFAs might be linked to higher incident of heart disease (59). The first major study reporting a strong association between intake of iTFAs and risk of CV disease was published in 1993 (60). These findings were confirmed by several epidemiological studies over the next years (44, 61-63). A meta- analysis of prospective studies showed that a 2% increase in energy intake from iTFAs, increased the incidence of CV disease by 23% (64). In addition to dyslipidemia, inflammation and endothelial dysfunction, iTFAs are suggested to promote insulin resistance, adiposity and hypertension (52, 65-68). On the basis of these findings, legislative actions were taken in several countries to reduce iTFA consumption, with Denmark being the first country to do so in 2003 (69, 70). It is estimated that the Danish policy against iTFAs reduced CV disease mortality by 14.2 deaths per 100,000 person years on average during the period 2004-2006 (71).
In contrast to iTFAs, epidemiological studies have reported a nonsignificant inverse association between rTFA intake and risk of CV disease (60, 61). This might be related to
different biological properties of rTFAs compared to iTFAs, or presence of other dietary substances in ruminant products that might be cardioprotective (72). When consumed in moderate amounts, rTFAs have resulted in a favorable lipid profile, characterized by higher serum HDL and lower serum LDL cholesterol levels (73). Furthermore, an inverse association between rTFA intake and incident DM has been reported in epidemiological studies (74, 75).
5.5 Intake of fatty acids world wide
Intake of marine n-3 PUFAs is mainly dependent on consumption of fish and others seafood, and to some extend fish oil supplements. The European Food Safety Authority recommend a daily intake of 250 mg EPA and DHA, corresponding to 1-2 servings of fatty fish per week (76), while the American Heart Association recommend healthy individuals to eat two fish meals per week, with an emphasis on fatty fish (77).
Figure 7. Global and regional mean consumption levels of dietary seafood omega 3 fat in 2010 for adults ≥20 years of age. Reprinted with permission from BMJ Publishing Group Ltd..: BMJ;
Micha et al.74 (copyright 2014).
There are large variations in fish consumption between and within countries, which are
probably related to different dietary habits, income distribution and infrastructure (78). Mean daily intake of marine n-3 PUFAs is about 0.9 g in Japan (79), compared to 0.1 g in the US (80). Nearly 80% of the world’s adult population has a marine n-3 PUFA intake below 250 mg/day, with extremely low levels (often <100 mg/day) reported in Sub-Saharan Africa, South America and Asian mainland nations (Figure 7) (81).
There are large inconsistencies in global recommendations regarding LA intake. The
American Heart Association recommends daily consumption of at least 5% to 10% of energy from LA intake (82). Typical intake of LA in the US is approximately 6% of energy,
corresponding to 12-17 grams per day for individuals between 19 and 50 years of age (10). In comparison, the European Food Safety Authority, supported by the French national
guidelines, propose 4% of energy intake from LA per day, equivalent to 10 grams (76, 83).
The background for more restrictive European recommendations might be related to concerns for harmful LA effect, as previously mentioned. A recent systemic review, evaluating LA intake in various European nations, shows that the European Food Safety Authority recommendations are not met in half of the countries included in the study (84). In regions such as Southeast Asia and Sub-Saharan Africa, a great majority of nations consume LA below optimal levels, possibly due to limited focus on consumption of healthy vegetable oils (81).
Large variations in iTFA consumption are also seen across the world. After introduction of legislative bans, several nations in Europe have reduced their iTFA intake to <1% of energy consumption (81). While regions with limited or no iTFA regulations, such as North America and Middle East, still have 2% or more of their energy consumption from iTFAs (81).
Interestingly, the highest intake of iTFAs is seen among young adults, perhaps due to greater consumption of processed foods. A decrease in iTFA intake in Europe, accompanied by an
! increase in North Africa, Middle East and South Asia, resulted in a relatively unchanged global iTFA consumption between 1990 and 2010 (81).
As a consequence of reduced iTFA consumption, rTFAs are now the major dietary TFAs in many countries (85). Intake of rTFAs has remained relatively unchanged over the past decades in countries such as the US and Denmark (86, 87), while a recent German study reported a parallel decrease in intake of rTFAs with iTFAs between 2008 and 2015 (88).
5.6 Intake of fatty acids in Norway
For centuries, fish and seafood have been central items in the Norwegian diet. In contrast to some European countries, where fish intake has been related to healthy lifestyle and high socioeconomical status (89, 90), fish consumption in Norway has mostly been driven by geographical factors, such as easy access to fresh fish along the coastline (91). Norway is one of the countries with the highest fish intake per capita worldwide (78), with a mean daily intake of marine n-3 PUFAs estimated to 0.7 grams (92). However, over the last decades, fish consumption in Norway has decreased following a shift towards a more Western type of diet (93).
Compared to other European countries, the overall LA intake in Norway is relatively low (81). Different type of vegetable oils are the primary LA sources (94). In a report by Nordic Nutrition Recommendations, the average energy intake from total PUFAs (the sum of n-3 PUFAs and n-6 PUFAs) was approximately 6% in Norway in 2012 (94). Hence the energy intake from LA alone was probably lower than the recommendations given by the American Heart Association.
Total TFAs (the sum of iTFAs and rTFAs) intake was approximately 5% of overall energy consumption during the late 1950s in Norway, gradually decreasing over the next decades, mainly due to reduction in the use of margarines (95). A large Norwegian cohort study
"
reported a mean intake of iTFAs to 0.9-1.6% of energy consumption between 1974 and 1988 (96). In this study, iTFA intake was positively associated with CV death. A considerable reduction in iTFA consumption took place shortly after the Danish legislation, mostly due to societal pressure and efforts by Regulatory Authorities (95). In addition, cooperation by food industries, voluntarily excluding iTFA-rich products from their assortment and providing better food labelling for consumers, resulted in further reduction in iTFA consumption (95).
Similar to Denmark, the Norwegian rTFA intake has remained unchanged despite reduction in iTFA consumption (97). According to the Nordic Nutrition Recommendations report, the dietary intake of total TFAs was <1% of total energy consumption in 2012 (94). 2 years later, in 2014, the Norwegian iTFA-legislation was passed (98).
#
6. Rationale and aims
6.1 Rationale and general aim
FAs vary in their source of origin and degree of unsaturation. They are often categorized as beneficial or harmful, depending on their effect on CV heath. Marine n-3 PUFAs are generally considered cardioprotective. As Norwegian dietary habits are changing, with a continuous decrease in fish consumption, the beneficial effects of marine n-3 PUFAs on CV health might be attenuated (99). Today, on a population level, effects of marine n-3 PUFA intake would likely differ from data obtained in previous era, where fish consumption was higher.
Similar to marine n-3 PUFAs, studies support the role of LA in reduction of CV morbidity and mortality (49, 50).As previously mentioned, a recent report shows that the European Food Safety Authority recommendations on LA consumption are not met in various European nations (84). Intake of LA has traditionally been low in Norway, and generally lower than other European countries. Thus, there might be a possibility that levels of LA intake are suboptimal also in Norway.
In contrast to PUFAs, iTFAs are recognized for exerting adverse effects on CV health (64).
Intake of iTFAs was associated with CV death in Norway during 1970s and 1980s (96). To our knowledge, the relationship between iTFA intake and CV health has not been evaluated in a Norwegian general population since. Although the dietary intake of total TFAs was <1% of total energy consumption in 2012 (94), an intake of <0.5% might be necessary to avoid adverse effects (64).
How the intake of marine n-3 PUFAs, LA and TFAs today, influences CV health in Norway, is unclear. This project was conducted to better understand the relationship between the current intake of these FAs, reflected by their plasma phospholipid levels, and established CV risk factors in a middle-aged Norwegian general population.
$ 6.2 Paper specific aims
To examine the associations between plasma levels of - marine n-3 PUFAs (paper I),
- LA (paper II), - TFAs (paper III),
and CV risk factors and lifestyle related variables in a Norwegian cohort of participants born in 1950, residing in Akershus County.
%
7. Material and methods
7.1 Study population
This thesis is based on data from the Akershus Cardiac Examination (ACE) 1950 Study, a population-based cohort study aimed to examine the cardio- and cerebrovascular health of individuals born in 1950 and residing in Akershus County, Norway. It is a collaborative project between The Cardiothoracic Research Group, Akershus University Hospital and The Department of Medical Research, Bærum Hospital, Vestre Viken Hospital Trust. From the Norwegian Population Registry, a list was obtained of all men and women born in 1950 and residing in Akershus County. A total of 5,827 eligible individuals were identified and invited for study participation by letters and subsequent phone calls. Among invited individuals, 3,706 agreed to participate (64%), while the remaining 2,121 did not respond to the study invitation letter or declined participation (Figure 8).
Figure 8. Flowchart for inclusion of study participants.
&
Enrollment was performed at Akershus University Hospital and Bærum Hospital, Vestre Viken, from September 2012 through May 2015. All participants underwent structural baseline interviews, clinical examination, blood sampling and advanced imaging. The study design is described in details elsewhere (100).
7.2 Data collection
All participants completed a study-specific ACE 1950 questionnaire, based on Cohort of Norway standards (101), regarding medical history, current medication and lifestyle variables.
They were asked to indicate frequency of fatty fish consumption in a study-specific food frequency questionnaire (FFQ), that was validated using plasma marine n-3 PUFA levels as reference. Study participants underwent a standardized physical examination, including measurement of blood pressure, weight and height. Blood samples were obtained after overnight fasting and stored in biobanks at each study site (storage at -80 °C). The local laboratory performed immediate standard analyses, including serum lipids, FPG, glycated hemoglobin (HbA1c) and CRP. Ultrasound examination of the right and left carotid arteries was performed for the assessment of carotid intima-media thickness (cIMT) (102).
Definition of diseases (hypertension, obesity, hypercholesterolemia, DM and chronic kidney disease [CKD]) and lifestyle variables (educational level, physical activity, smoking habits and alcohol consumption) are provided in paper I-III.
7.3 Fatty acid analysis
From stored blood samples, aliquots of plasma were sent to The Lipid Research Center, Aalborg University Hospital for analysis of FA composition by gas chromatography, a chemical analysis commonly used to separate and identify compounds in a complex sample.
Total lipids were first extracted from plasma using a modified Folch method, in which the
' optimal ratio of chloroform, methanol and water solution allowed separation of protein and organic phase after centrifugation (103). With a modified Burdge method, using chloroform and methanol, phospholipid FA fraction was separated from total lipids and dried under nitrogen for 1 hour under 40° C (103). FAs were methylated and transferred into gas chromatographic tubes that were placed at sample injector site (Figure 9) of a Varian 3900 gas chromatograph (Varian, Middleburg, The Netherlands).
Figure 9. Basic scheme of gas chromatograph.
We used helium as carrier gas, which together with methylated FAs constituted the mobile phase of the analysis. With a continuous flow of carrier gas, FAs were forced through narrow metal tubes known as columns. We used 60 m x 0.25 mm columns coated with a microscopic layer of liquid which formed the stationary phase. FAs were separated as they passed through the columns, mainly due to differences in carbon chain length and interactions with the stationary phase. Large FAs, compared to smaller, were retained for a longer time period in the columns. As individual FAs exited the columns, they were electronically detected and
! identified from their retention time. Where retention time matched perfectly with
commercially available standards, the individual FAs were named. Each FA created a graphical peak, with retention time along the x-axis and detector response on the y-axis. By calculating area under peak, individual FAs were quantitated and expressed as weight percentage (wt%) of total plasma phospholipid FAs.
7.4 Statistics
Demographic and clinical data were presented according to tertiles of plasma marine n-3 PUFA levels (paper I) and quartiles of plasma LA and iTFA levels (paper II and III).
Differences between groups were evaluated using Chi square test for dichotomous data, ANOVA for normally distributed continuous variables and Kruskal-Wallis test for skewed variables.
We investigated the associations between plasma levels of marine n-3 PUFAs, LA and TFAs and CV risk factors by univariable and multivariable linear regression analysis after testing assumptions for linearity. Linear regression is commonly used to analyze the influence of one or more independent variables on a continuous dependent variable. The relationship is
described with a straight line, also known as regression line, which can be used to make predictions about one variable based on particular values of other variables. Unstandardized regression coefficient (Unstd. β-coeff.) indicates slope of the line, whereas standardized regression coefficient (Std. β-coeff.) compares strength of the independent variable to the dependent variable. R-squared (R2) is the proportion of variance for a dependent variable that is explained by one or more independent variables in the regression model. We presented both univariable and multivariable models with Unstd. β-coeff., Std. β-coeff. and R2.
In paper II, assumptions of linearity between plasma LA levels and CV risk factors were investigated with restricted cubic spline modeling. It is an extension of basic linear predictor
! models which can relax the linearity assumption, while minimizing residual confounding when adjusting for a continuous exposure. Restricted cubic spline modeling was performed by subdividing the range of values of plasma LA levels by use of specific points, known as knots. We used three knots, placed at 10th, 50th and 90th percentile of the exposure
distribution. In this way, the original independent variable, plasma LA level, was augmented by introducing a set of new variables. This transformed version of plasma LA levels was used to check linearity with CV risk factors.
Adjustments were made with predefined candidate variables, using stepwise forward procedure with p<0.10 for selection of variables in paper I. In paper II, adjustments were made with covariates chosen from a set of predefined candidate variables by least absolute shrinkage and selection operator (LASSO) regression. This method preforms regularization, also known as shrinkage, in addition to variable selection. First highly inflated Std. β-coeff.
were penalized towards zero. Strength of penalty was controlled by a tuning parameter lambda, which was chosen for each CV risk factor after cross validation. During variable selection, those candidate variables that still had non-zero coefficients after the shrinkage process, were included in the final model. Candidate variables included for adjustment in paper III were predefined. Models in paper II and III were constructed using simultaneous entry.
In paper I and II, non-normally distributed variables, such as serum triglycerides, fasting plasma glucose and HbA1c, were truncated by replacing 15 outliers with mean +/- standard deviation (SD). CRP was severely skewed, and therefore logarithmically transformed to obtain normal distribution. In paper III, both non-normally distributed and skewed variables were logarithmically transformed.
All statistical calculations presented in papers of this thesis were performed using SPSS®
version 25.0 (IBM, NY, US), RStudio® version 1.1.419, R version 3.4.4 (RStudio Inc.,
! Boston, MA, US and R Foundation for Statistical Computing, Vienna, Austria) and STATA 16 (StataCorp LP, TX, USA).
7.5 Ethical considerations
The ACE 1950 Study was approved by the Regional Ethics Committee for Medical and Health Research Ethics in Norway (Ref. number 2011/1475). The approval includes repeated follow-up until December 31st 2050. Written informed consent, in accordance with the Declaration of Helsinki, was obtained from all participants before enrolment.
In 2016, to ensure high level of security, de-identified study data were transferred to TSD (Services for Sensitive Data, hosted by the University of Oslo), a national IT infrastructure for handling and storage of scientific data.
The ACE 1950 Study was registered at www.clinicaltrials.gov; NCT01555411.
!!
8. Summary of results
8.1 Descriptive data of the ACE 1950 Study Cohort:
Table 1. Descriptive data on cardiovascular risk factors.
CV risk factor n Mean/median SD/IQR
HDL cholesterol, mmol/L 3678 1.55 0.48
LDL cholesterol, mmol/L 3678 3.30 0.96
Triglycerides, mmol/L 3678 1.17 0.38-1.96
FPG, mmol/L 3683 5.30 4.40-6.20
HbA1c, % 3683 5.79 5.39-6.19
BMI, kg/m2 3645 27.2 4.39
SBP, mmHg 3645 137.9 18.7
DBP, mmHg 3645 77.0 10.1
Creatinine, μmol/L 3645 75.9 14.4
eGFR, ml/min x 1.73m2 3645 83.1 11.9
cIMT, mm 3645 0.73 0.11
CRP, mg/L 3645 1.5 1.5-1.5
Abbreviations: CV: Cardiovascular. SD: Standard deviation. IQR: Interquartile range. HDL: High-density lipoprotein. LDL: Low-density lipoprotein. FPG: Fasting plasma glucose. HbA1c: Hemoglobin A1c. BMI: Body mass index. SBP: Systolic blood pressure. DBP: Diastolic blood pressure. eGFR: Estimated glomerular filtration rate. cIMT: Carotid intima-media thickness. CRP: C-reactive protein.
Table 2. Descriptive data on medical conditions.
Medical condition n Cases Percentage (%)
Hypertension 3705 2297 62.0
Hypercholesterolemia 3696 1945 52.5
Coronary artery disease 3706 263 7.1
Cerebrovascular disease 3706 140 3.8
Diabetes mellitus 3704 317 8.6
Obesity 3706 839 22.6
Chronic kidney disease 3685 143 3.9
!"
Table 3. Descriptive data on lifestyle related variables.
Lifestyle related variable n Cases Percentage (%)
Higher education, (>12 years of formal education)
3695 1713 46.2
Physical activity, (≥ 2 times weekly)
3650 2255 60.8
Current smoking 3676 532 14.4
Alcohol consumption, (≥ 2 times weekly)
3694 1749 47.2
!#
8.2 Paper I
Plasma marine n-3 polyunsaturated fatty acids and cardiovascular risk factors: data from the ACE 1950 study
Plasma marine n-3 PUFA levels ranged from 2.7 to 20.3 wt%, with a median level of 7.7 wt%
(interquartile range [IQR] 4.3 to 11.1 wt%). High levels of plasma marine n-3 PUFAs were associated with lower serum triglycerides (Standardized regression coefficients [Std. β-coeff.]
-0.14, p<0.001), body mass index (BMI) (Std. β-coeff. -0.08, p<0.001), serum creatinine (Std.
β-coeff. -0.03, p=0.05), CRP levels (Std. β-coeff. -0.03, p=0.04) and higher levels of serum
HDL cholesterol (Std. β-coeff. 0.08, p<0.001). Plasma marine n-3 PUFA levels were also positively associated with LDL cholesterol (Std. β-coeff. 0.04, p=0.003), however, only in males with hypercholesterolemia. Furthermore, plasma marine n-3 PUFA levels were inversely associated with HbA1c (Std. β-coeff. -0.04, p=0.01), but only in participants without DM.
We found no associations between plasma marine n-3 PUFA levels and FPG or cIMT.
Participants with high levels of plasma marine n-3 PUFAs had lower prevalence of comorbidities, higher educational level, were more physically activity and smoked less.
Self-reported fatty fish consumption frequency was moderately correlated with plasma marine n-3 PUFA levels, with the highest levels seen among individuals with daily fish consumption (Pearson correlation coefficient 0.30, p<0.001, Figure 10).
!$
Figure 10. Relationship between plasma marine n-3 PUFA levels and self-reported fatty fish consumption frequency
!%
8.3 Paper II
Plasma linoleic acid levels and cardiovascular risk factors: Results from the Norwegian ACE 1950 Study
Plasma LA levels ranged from 11.4 to 32.0 wt%, with a median level of 20.8 wt% (IQR 16.8 to 24.8 wt%). The associations between plasma LA levels and CV risk factors were linear, with exceptions for serum LDL-cholesterol levels and HbA1c (Figure 11).
Figure 11. Cubic spline models of associations between plasma LA levels and cardiovascular risk factors.
High levels of plasma LA were associated with lower serum LDL cholesterol levels (Std. β- coeff. -0.04, p=0.02), serum triglycerides (Std. β-coeff. -0.10, p<0.001), BMI (Std. β-coeff.
-0.13, p<0.001) and systolic and diastolic blood pressure (Std. β-coeff. -0.04, p=0.03 and Std.
β-coeff. -0.02, p=0.02, respectively). Furthermore, high plasma LA levels were associated with lower FPG (Std. β-coeff. -0.10, p<0.001), however, only in participant without DM.
!&
Finally, high levels of plasma LA were associated with lower eGFR, but not in participants with eGFR <60 ml/min/1.73m2. Plasma LA levels were not associated with HDL cholesterol levels, HbA1c, cIMT or CRP levels.
Participants with high compared to low levels of plasma LA had less comorbidities, higher educational level and consumed less alcohol, whereas no differences were observed for smoking or physical activity. When stratifying participants according to educational level and alcohol consumption, associations between plasma LA levels and CV risk factors mainly remained unchanged.
!' 8.4 Paper III
Plasma trans fatty acid levels and cardiovascular risk factors: Results from the Akershus Cardiac Examination 1950 Study
Plasma iTFA levels ranged from 0.09 to 0.62 wt%, with a median level of 0.20 wt% (IQR 0.13 to 0.27 wt%) (Figure 12). Plasma iTFA levels were inversely associated with serum triglycerides (Std. β-coeff. -0.15, p<0.001), FPG levels (Std. β-coeff. -0.05, p<0.001), BMI (Std. β-coeff. -0.13, p<0.001), systolic and diastolic blood pressure (Std. β-coeff. -0.06, p=0.001 and Std. β-coeff. -0.03, p=0.03, respectively) and CRP levels (Std. β-coeff. -0.06, p=0.001). We found no associations between plasma iTFA levels and HDL or LDL cholesterol levels, HbA1c, eGFR or cIMT.
Participants with high plasma iTFA levels were more educated, had lower prevalence of comorbidities, and consumed tobacco and alcohol less often.
Plasma rTFA levels ranged from 0.14 to 2.87 wt%, with a median level of 1.60 wt% (IQR 1.30 to 1.90 wt%) (Figure 12). Plasma rTFA levels were positively associated with serum HDL cholesterol levels (Std. β-coeff. 0.08, p<0.001), and inversely associated with serum LDL cholesterol levels (Std. β-coeff. -0.10, p<0.001), serum triglycerides (Std. β-coeff. -0.15, p<0.001), FPG levels (Std. β-coeff. -0.08, p<0.001), HbA1c (Std. β-coeff.
-0.13, p<0.001), BMI (Std. β-coeff. -0.10, p<0.001), systolic and diastolic blood pressure (Std. β-coeff. -0.04, p=0.01 and Std. β-coeff. -0.07, p<0.001, respectively). Plasma rTFA levels were not associated with eGFR, cIMT or CRP levels.
Participants with high plasma rTFA levels had a lower prevalence of DM, but higher prevalence of coronary artery disease and used more lipid-lowering drugs. In addition, they had a higher consumption of margarine and butter, a higher prevalence of smoking and consumed alcohol more often.
"
Figure 12. Distribution of industrial and ruminant trans fatty acids measured in weight percentage (wt%) of total plasma phospholipid fatty acids.
"
9. Discussion
9.1 Methodological considerations 9.1.1 Study cohort
Indicators for socioeconomical status and general health are better for Akershus County compared to the national average, with higher income and educational level, lower unemployment, and lower prevalence of daily tobacco use and CV disease (104). These features were seen in the ACE 1950 Study population, for example nearly 50% of participants had higher education and <15% were current smokers, findings supportive of a representative selection of the Akershus population. Furthermore, a high proportion of the study cohort were on antihypertensive, glucose-lowering or lipid-lowering medication, suggestive for a health- conscious behavior with good access to healthcare services. Overall, the ACE 1950 Study cohort can be considered a rather healthy and well-educated population, which have been kept in mind when interpreting results.
9.1.2 Study design
Although the ACE 1950 Study was designed as a prospective cohort study, the studies
included in this thesis are cross-sectional. In a cross-sectional study, exposure and outcome of interest are measured at a single time-point (105). This study design can be used to assess prevalence of diseases in a population, or to examine relationship between exposure and outcome. Cross-sectional studies are also considered hypothesis generating, as findings indicative of associations between certain exposures and diseases, can be further investigated with clinical studies. This methodology allows study of multiple exposures and outcomes, and is usually more feasible and less time consuming than longitudinal cohort studies and RCTs.
However, cross-sectional studies cannot help to determine cause and effect relationship, which is the major limitation of this study design (105).
"
9.1.3 Bias and validity
In epidemiological studies, there are several potential sources of bias, and are commonly classified into three categories: selection bias, information bias and confounding (106).
Selection bias occurs when study population is not representative of target population. In such case, study findings may not accurately apply to target population to which conclusions are being extended. Low participation rates increases risk of selection bias, as non-responders often tend to have lower socioeconomical status and higher prevalences of chronic diseases (107). The willingness of responders to participate is often related to a health-conscious behavior and the wish to contribute to a good cause. The participation rate of 64% in the ACE 1950 Study is relatively high and comparable with other large epidemiological studies
conducted in Norway (108, 109).
Information bias may arise from collection of wrong data or measurement error. Recall bias is a type of information bias that occurs when participants are not able to correctly recall information, making self-reported data especially vulnerable for such bias. Previous reports suggest greater accuracy between self-reported data and medical records for diagnosis easily understood by the patient, such as hypertension and myocardial infarction, while not for more complicated conditions such as heart failure (110). Consciously or subconsciously,
participants might under- or overreport information related to social behavior, such as smoking, alcohol consumption, physical activity or fish intake. In the ACE 1950 Study, data on medical history and lifestyle, including intake of fatty fish, were self-reported, and potentially exposed to recall bias. Having plasma marine n-3 PUFA measurements, as an objective marker of marine n-3 PUFA intake, we were able to validate self-reported fatty fish intake.
Both selection and information bias can be reduced with careful planning and execution of a study, but cannot be corrected once the study is completed.
"!
Confounding occurs when effect of an exposure on a given outcome is mixed in with effects of an additional factor, resulting in a distortion of the true relationship. This is usually caused by one or more variables that are related to both exposure and outcome. The major advantage of RCTs is the investigation of cause–effect relationships with minimal confounding. In observational studies, confounding cannot be removed, but reduced in data analysis. Age and gender are examples of common confounding factors, and stratification of participants according to these variables can reduce their influence on the results (106). All the
participants in the ACE 1950 Study were born in 1950, thereby removing age as a possible confounding factor. Another method is to adjust for possible confounding variables in the statistical analysis, which we have done in paper I-III. However, variables not included in the regression models might still have distorted the measured associations, a phenomenon known as residual confounding (106).
Observational studies are susceptible to another type of bias, known as reverse causality bias. The term describes the situation where the association between exposure and outcome is not due to direct causality from exposure to outcome, but rather because the outcome results in a change in the exposure. For example, high plasma LA levels have been associated with lower fasting plasma glucose (54). On the other hand, individuals with high fasting plasma glucose are reported to have low plasma LA levels (111).
Internal validity describes how trustworthy the findings are for the population being studied, and relates directly to how well the study has been conducted. Factors improving internal validity of the ACE 1950 Study is recruitment of participants at only at two centers,
standardized procedures for data collection, and little missing data. For example, from 3,706 participants, only 23 FA analyses were missing.
External validity refers to how well the outcome of a study can be expected to apply to other populations in different setting or timepoints. As all participants of the ACE 1950 Study
""
cohort are born in 1950, our findings might not be relevant to other age groups. Furthermore, results from the present studies cannot be generalized to populations and regions with low background consumption of marine n-3 PUFAs, or a high LA or iTFA intake.
9.1.4 Fatty acid analysis
One of the major strengths of our studies was that plasma phospholipid FA levels were determined by gas chromatography, providing an objective measure of marine n-3 PUFA, LA and TFA consumption. We acknowledge that plasma FA levels do not reflect the long-term intake of FAs as accurate as erythrocyte membrane or adipose tissue levels (112). However, for most people dietary habits usually remain unchanged (113), and we therefore assume that plasma FA composition represents long-term average FA profiles for majority of participants in the present studies.
9.1.5 Statistical considerations
In paper I-III, we applied linear regression to investigate the associations between plasma levels of marine n-3 PUFAs, LA and TFAs and CV risk factors. This statistical approach allowed us to adjust for multiple candidate variables simultaneously. In paper I, adjustment variables were chosen from a set of predefined candidate variables with stepwise forward procedure. Although this selection technique has the ability to manage large amounts of potential variables, regression coefficients are often biased (114). To avoid biased regression coefficients, we applied the shrinkage method LASSO in paper II. In addition to penalizing inflated regression coefficients towards zero, LASSO performed variable selection, which reduced over-fitting of the final models.
We used restricted cubic spline modeling to test assumptions of linearity between plasma LA levels and CV risk factors in paper II. This method revealed non-linear relationships between
"#
plasma LA levels and LDL cholesterol levels and HbA1c. Thus, results obtained by linear regression in these cases did perhaps not reflect their true relationships.
Overall, our results were characterized by small but significant regression coefficients, findings which might have been driven by the large sample size. These are features of type 1 error, where the true null-hypothesis is incorrectly rejected (105). Thus, we cannot exclude the possibility that the associations between plasma FA levels and CV risk factors in our studies might have been overestimated.
"$
In section 9.2, results from paper I and II are discussed together.
9.2 Marine n-3 polyunsaturated fatty acids, linoleic acid and cardiovascular risk factors 9.2.1 Serum lipids
High plasma levels of both marine n-3 PUFAs and LA were associated with lower serum triglycerides in our cohort. Triglyceride lowering effect of marine n-3 PUFAs is well documented in clinical trials, with greater reduction achieved in individuals with higher triglyceride levels at baseline (115). In severe hypertriglyceridemia, lowering of triglycerides levels by 30% has been reported (32). Clinical trials also show that marine n-3 PUFAs can complement lipid-lowering drugs, such as statins, in patients with dyslipidemia (116). Marine n-3 PUFAs lower triglycerides by reduced hepatic very low-density lipoprotein synthesis and increased clearance of chylomicrons, lipoprotein particles rich in triglycerides (117). In addition, both marine n-3 PUFAs and LA are suggested to increase the activity of lipoprotein lipase (117, 118), an enzyme involved in hydrolysis of triglycerides.
In clinical trials, marine n-3 PUFA supplementation have shown to increase serum LDL cholesterol levels (35). A shift towards larger LDL particles has also been reported, which are suggested to be less atherogenic (119). We found a positive association between plasma marine n-3 PUFA levels and serum LDL cholesterol levels, however, only in participants with hypercholesterolemia. A possible explanation for this finding could be that participants with hypercholesterolemia might have received advice to increase their intake of marine n-3 PUFAs. In contrast, we found an inverse association between plasma LA and serum LDL cholesterol levels. However, this finding must be interpreted with caution as assumptions of linearity between plasma LA and serum LDL cholesterol were not met. In addition, plasma LA and serum LDL cholesterol levels exhibited an inverted U-shaped relationship (Figure 12), the biological plausibility for which can be questioned. Consumption of LA-rich diet has
"%
reduced serum LDL cholesterol levels by 10% in interventional studies (52, 53), however, substitution of LA for either carbohydrates or saturated FAs in these studies might have confounded results.
Plasma marine n-3 PUFA levels, but not plasma LA levels, were positively associated with serum HDL cholesterol levels in our cohort. Data from clinical trials indicate an increase in serum HDL cholesterol levels with marine n-3 PUFA supplementation (119). However, this effect is seen at high doses of marine n-3 PUFAs, not obtainable through regular diet. Recent meta-analyses conclude that neither marine n-3 PUFA supplementation nor LA-rich diet have effect on serum HDL cholesterol levels (36, 120).
9.2.2 Fasting plasma glucose and glycated hemoglobin
High levels of plasma marine n-3 PUFAs and LA were associated with lower prevalence of DM in the present cohort. Epidemiological studies on marine n-3 PUFA consumption and risk for incident type 2 DM have been divergent, showing both positive, neutral and negative associations (121). Similar reports on LA are more convincing, with a pooled analysis of 20 prospective studies, including >39,000 participants from 10 different countries, demonstrating a 43% reduced risk of incident type 2 DM associated with high plasma LA levels (55). The underlying mechanism by which these FAs might influence glucose-insulin metabolism is not fully understood. A possible mechanism might be increased glucose consumption related to upregulation of carnitine shuttle system required for mitochondrial β-oxidation of long-chain FAs (122). In addition, both marine n-3 PUFAs and LA are suggested to increase insulin sensitivity by altering insulin receptor binding capacity and by influencing ion permeability and cell signaling (123-125). High plasma marine n-3 PUFA levels were associated with lower HbA1c, while high plasma LA levels were associated with FPG in our cohort.
However, both findings were done in participants without DM, representing majority of the
"&
study population. Furthermore, we found no associations between plasma marine n-3 PUFA levels and FPG, or plasma LA levels and HbA1c. Thus, we cannot rule out the possibility that our findings, signaling a favorable influence of marine n-3 PUFAs and LA on glucose-insulin metabolism, might have been confounded by a healthy lifestyle.
9.2.3 Body mass index
Established CV risk factors such as dyslipidemia and insulin resistance are associated with obesity, and may improve with a modest weight loss of 5-10% of body weight (126). An inverse association between plasma long chain FA levels and adiposity has been reported in a meta-analysis of observational studies (127). A high intake of long chain FAs increases mitochondrial β-oxidation, a process suggested to induce weight loss (128). In addition, marine n-3 PUFAs are proposed to increase lipolysis and reduce lipogenesis (129), and
decrease circulatory leptin concentrations in non-obese adults (130). These mechanisms might partly explain lower prevalence of obesity and lower BMI associated with high plasma marine n-3 PUFA and LA levels in our cohort. In a clinical trial, consumption of LA-rich diet was associated with weight loss over a 4-year period (128). However, high LA intake in this study was obtained at the expense of energy from carbohydrates, and the results can therefore not be attributed solely to LA consumption. Furthermore, an elevated activity of delta-6-desaturase, an enzyme converting LA to longer chain metabolites, has been reported in obese subjects (131), which might be a potential source of reverse causality bias.
9.2.4 Renal function and blood pressure
A high intake of fish and marine n-3 PUFA supplements has been associated with lower prevalence of CKD (132). In an Italian prospective cohort study of adults aged 65 years, participants with high compared to low plasma marine n-3 PUFA and LA levels at baseline,
