Fish and camelina sativa oil : effects on fatty acid composition of blood lipid fractions, lipoprotein subclasses and pro- and anti-atherogenic lipoprotein functions

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DISSERTATIONS | SUVI MANNINEN | FISH AND CAMELINA SATIVA OIL ON FATTY ACID ... | No 538

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PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences

ISBN 978-952-61-3219-8 ISSN 1798-5706

Dissertations in Health Sciences

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND

SUVI MANNINEN

FISH AND CAMELINA SATIVA OIL – EFFECTS ON FATTY ACID COMPOSITION OF BLOOD LIPID FRACTIONS, LIPOPROTEIN SUBCLASSES AND PRO- AND ANTIATHEROGENIC

LIPOPROTEIN FUNCTIONS

Fish and vegetable oils are considered important components of a cardioprotective

diet. Fish is a main source for long-chain omega-3 polyunsaturated fatty acids, whereas camelina sativa oil contains plant- derived omega-3 fatty acid,

α

-linolenic acid.

In this doctoral thesis, intakes of fatty fish and camelina sativa oil were found to have beneficial effects on the lipoprotein subclass profile and one of the key mechanisms in early

atherosclerosis. These findings highlight the importance of omega-3 polyunsaturated fatty

acids of both marine and plant origin for the cardiovascular health.

SUVI MANNINEN

31253624_UEF_Vaitoskirja_NO_538_Suvi_Manninen_Terveystiede_kansi.indd 1 17.10.2019 9.08

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FISH AND CAMELINA SATIVA OIL – EFFECTS ON FATTY ACID COMPOSITION OF BLOOD

LIPID FRACTIONS, LIPOPROTEIN

SUBCLASSES AND PRO- AND ANTI-

ATHEROGENIC LIPOPROTEIN FUNCTIONS

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Suvi Manninen

FISH AND CAMELINA SATIVA OIL –

EFFECTS ON FATTY ACID COMPOSITION OF BLOOD LIPID FRACTIONS, LIPOPROTEIN

SUBCLASSES AND PRO- AND ANTI- ATHEROGENIC LIPOPROTEIN FUNCTIONS

To be presented by permission of the

Faculty of Health Sciences, University of Eastern Finland for public examination in Medistudia auditorium MS302, Kuopio,

on Friday, November 29^th 2019, at 12 o’clock noon Publications of the University of Eastern Finland

Dissertations in Health Sciences No 538

Institute of Public Health and Clinical Nutrition, School of Medicine, Faculty of Health Sciences

University of Eastern Finland Kuopio

2019

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Series Editors

Professor Tomi Laitinen, M.D., Ph.D.

Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences

Associate professor (Tenure Track) Tarja Kvist, Ph.D.

Department of Nursing Science Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D.

Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences

Associate Professor (Tenure Track) Tarja Malm, Ph.D.

A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences

Lecturer Veli-Pekka Ranta, Ph.D.

School of Pharmacy Faculty of Health Sciences

Distributor:

University of Eastern Finland Kuopio Campus Library

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Grano Oy 2019

ISBN: 978-952-61-3219-8 (print/nid.) ISBN: 978-952-61-3220-4 (PDF)

ISSNL: 1798-5706 ISSN: 1798-5706 ISSN: 1798-5714 (PDF)

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Author’s address: Institute of Public Health and Clinical Nutrition University of Eastern Finland

KUOPIO FINLAND

Doctoral programme: Doctoral Programme in Health Sciences Supervisors: Docent Arja Erkkilä, Ph.D.

Institute of Public Health and Clinical Nutrition University of Eastern Finland

KUOPIO FINLAND

Professor Ursula Schwab, Ph.D.

KUOPIO FINLAND

Docent Maria Lankinen, Ph.D.

KUOPIO FINLAND

Reviewers: Professor Rolf Kristian Berge, Ph.D.

Department of Clinical Science University of Bergen

BERGEN NORWAY

Docent Tiina Solakivi, Ph.D.

Department of Medical Biochemistry University of Tampere

TAMPERE FINLAND

Opponent: Associate professor Kirsi Laitinen, Ph.D.

Institute of Biomedicine University of Turku TURKU

FINLAND

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“If you don’t go after what you want, you’ll never have it. If you don’t ask, the answer is always no. If you don’t step forward, you’re always in the same place.”

Nora Roberts

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7 Manninen, Suvi

Fish and camelina sativa oil – effects on fatty acid composition of blood lipid fractions, lipoprotein subclasses and pro- and anti-atherogenic lipoprotein functions Kuopio: University of Eastern Finland

Publications of the University of Eastern Finland Dissertations in Health Sciences 538. 2019, 109 p.

ISBN: 978-952-61-3219-8 (print) ISSNL: 1798-5706

ISSN: 1798-5706

ISBN: 978-952-61-3220-4 (PDF) ISSN: 1798-5714 (PDF)

ABSTRACT

The main long-chain n-3 polyunsaturated fatty acids (n-3 PUFAs) are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) which are primarily derived from fish. Intake of these fatty acids is considered cardioprotective. In contrast, the effects of n-3 PUFA of plant origin, -linolenic acid (ALA), on cardiovascular health are far less understood. Lipoprotein subclass profile and lipoprotein functions play a central role in the development and progression of cardiovascular diseases. Despite the extensive evidence regarding the beneficial health effects ofn-3 PUFAs, the effects of differentn-3 PUFAs from dietary sources on lipoprotein subclasses or lipoprotein functions have been less studied. Precise measures of dietary fatty acid intakes are required to investigate the health effects of these fatty acids. Fatty acid compositions of several blood lipid fractions can serve as objective biomarkers of dietary fat intake. However, it is unclear which fatty acid pool most accurately reflects the dietary intakes of different n-3 PUFAs. In this doctoral thesis, the effects of fish and camelina sativa oil (CSO) intakes on fatty acid composition of blood lipid fractions, lipoprotein subclasses and pro- and anti- atherogenic lipoprotein functions were examined.

The effects of fish and CSO intakes were investigated in a randomized controlled trial. Altogether 88 volunteers participated in the study, of which 79 subjects completed the study. The subjects were men and women aged 40–75 with impaired glucose metabolism. They were randomly assigned to the CSO, fatty fish, lean fish or control group for 12 weeks. Subjects in the CSO group ingested 30 ml of CSO per day, and subjects in the fish groups consumed 4 fish meals per week. Control and CSO groups were allowed to eat 1 fish meal per week. Compliance was monitored with consumption records regarding the intakes of fish and CSO and with 4-day food records at baseline and three times during the intervention. Furthermore, as an objective measure of compliance, the proportions of fatty acids in erythrocyte membranes (EM), phospholipids (PL), cholesteryl esters (CE) and triglycerides (TG) were measured by gas chromatography. Nuclear magnetic resonance spectroscopy

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was used to determine lipoprotein subclasses and their lipid components. In addition, the binding of lipoproteins to aortic proteoglycans, LDL aggregation and activation of endothelial cells by LDL and cholesterol efflux capacity of HDL were determinedin vitro.

We found that EM and plasma PL, CE and TG seem to respond similarly to intake of long-chainn-3 fatty acids from fish and ALA from CSO. Dietary intakes of ALA, EPA and DHA also correlate with their respective proportions in a similar manner in different lipid fractions. These results together suggest that there is no difference in the ability of blood lipid fractions to reflect then-3 PUFA intake. Our study also suggests that fatty fish intake causes a shift toward larger and lipid-rich HDL particles. Furthermore, CSO intake decreases intermediate-desity lipoprotein (IDL) particle concentration and lipoprotein-proteoglycan interactions by decreasing serum LDL cholesterol concentration. This highlights the importance of serum lipoprotein levels in the accumulation of cholesterol within arterial wall. Lean fish intake did not significantly alter the fatty acid profile of blood lipid fractions or affect subclasses or functions of lipoproteins. Together these results suggest thatn-3 fatty acids from marine and plant origin differ in their impact on lipoprotein metabolism.

Therefore, these fatty acids may have complementary effects on the cardiovascular health.

In conclucion, in this doctoral thesis we have shown that with easily achieved dietary modifications it is possible to beneficially affect the lipoprotein subclass profile and one of the key mechanisms in the development of atherosclerosis. These results emphasize the importance of fish and food items rich in ALA in a healthy, cardioprotective diet.

National Library of Medicine Classification: QU 85, QU 90, QY 465, WB 425

Medical Subject Headings: Fatty Acids, Omega-3; alpha-Linolenic Acid; Eicosapentaenoic Acid; Docosahexaenoic Acids; Fish Oils; Lipids/blood; Lipoproteins/blood; Lipoproteins, HDL; Lipoproteins, IDL; Lipoproteins, LDL; Cholesterol/blood; Biomarkers; Randomized Controlled Trial

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9 Manninen, Suvi

Kala ja camelinaöljy – vaikutukset veren lipidifraktioiden rasvahappokoostumukseen, lipoproteiinien alaluokkiin ja aterogeenisiin sekä anti- aterogeenisiin lipoproteiinifunktioihin

Kuopio: Itä-Suomen yliopisto

Publications of the University of Eastern Finland Dissertations in Health Sciences 538. 2019, 109 s.

ISBN: 978-952-61-3219-8 (nid.) ISSNL: 1798-5706

ISSN: 1798-5706

ISBN: 978-952-61-3220-4 (PDF) ISSN: 1798-5714 (PDF)

TIIVISTELMÄ

Eikosapentaeenihappo (EPA) ja dokosaheksaeenihappo (DHA) ovat pitkäketjuisia omega-3-sarjan rasvahappoja, joiden pääasiallinen lähde ruoasta on kala. Näiden rasvahappojen saannilla tiedetään olevan sydänterveyden kannalta edullisia vaikutuksia. Kasviperäisen omega-3 rasvahapon, alfalinoleenihapon, vaikutuksista sydänterveyteen tiedetään sen sijaan merkittävästi vähemmän. Lipoproteiinien alaluokkien jakauma ja lipoproteiinien funktiot ovat keskeisessä roolissa sydän- ja verisuonitautien kehittymisessä ja etenemisessä. Laajasta omega-3-sarjan rasvahappojen hyötyihin liittyvästä tieteellisestä näytöstä huolimatta eri omega-3- sarjan rasvahappojen vaikutuksia lipoproteiinien alaluokkiin tai lipoproteiinien funktioihin on toistaiseksi tutkittu vähän. Jotta rasvahappojen terveysvaikutuksia voidaan tutkia, tarvitaan tarkkoja menetelmiä ruokavalion rasvahappojen saannin mittaamiseen. Ruokavalion rasvahappojen saannin objektiivisena biomarkkerina voidaan käyttää veren lipidifraktioiden rasvahappokoostumusta. Toistaiseksi kuitenkin on epäselvää, mikä näistä lipidifraktoista kuvastaa tarkimmin eri omega- 3-sarjan rasvahappojen saantia ruokavaliosta. Tässä väitöskirjatyössä tutkittiin kalan ja camelinaöljyn vaikutuksia veren lipidifraktioiden rasvahappokoostumukseen, lipoproteiinien alaluokkiin ja aterogeenisiin sekä anti-aterogeenisiin lipoproteiinifunktioihin.

Kalan ja camelinaöljyn käytön vaikutuksia selvitettiin kontrolloidussa, satunnaistetussa interventiotutkimuksessa. Tutkimukseen osallistui yhteensä 88 vapaaehtoista, joista 79 oli mukana tutkimuksen loppuun saakka. Tutkittavat olivat 40–75-vuotiaita miehiä ja naisia, joilla oli heikentynyt glukoosiaineenvaihdunta.

Tutkittavat jaettiin satunnaisesti camelinaöljyryhmään, rasvaisen tai vähärasvaisen kalan ryhmään tai kontrolliryhmään. Camelinaöljyryhmässä käytettiin päivittäin 30 ml camelinaöljyä, kun taas kalaryhmissä syötiin kalaa neljästi viikossa. Camelinaöljy- ja kontrolliryhmissä kalaa sai syödä kerran viikossa. Annettujen ruokavalio-ohjeiden noudattamista arvioitiin kalan ja camelinaöljyn käyttöön liittyvien kyselyiden sekä neljäpäiväisten ruokapäiväkirjojen avulla tutkimuksen alussa sekä kolmesti

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tutkimuksen aikana. Lisäksi kaasukromatografilla mitattiin punasolumembraanien sekä plasman fosfolipidien, kolesteryyliestereiden ja triglyseridien rasvahappokoostumukset, joita voidaan pitää objektiivisina mittareina rasvahappojen saannille. Lipoproteiinien alaluokkien jakauma ja näiden sisältämien lipidikomponenttien määrä määritettiin ydinmagneettisen resonanssispektroskopian avulla. Lisäksi lipoproteiinien kiinnittymistä aortan proteoglykaaneihin, LDL:n aggregaatiota, endoteelisolujen aktivoitumista LDL:n avulla sekä kolesterolin ulosvirtausta HDL-partikkeleihin tutkittiin in vitro- analyysien avulla.

Punasolumembraanien, fosfolipidien, kolesteryyliestereiden ja triglyseridien havaittiin reagoivan yhtäläisesti pitkäketjuisten omega-3 rasvahappojen saantiin kalasta sekä alfalinoleenihapon saantiin camelinaöljystä. Ruokavalion alfalinoleenihappo, EPA ja DHA myös korreloivat eri lipidifraktioista mitattujen vastaavien rasvahappojen kanssa. Näiden havaintojen perusteella eri lipidifraktioiden kyvyssä kuvastaa omega-3-sarjan rasvahappojen saantia ei ole merkittäviä eroja. Lisäksi rasvaisen kalan käytön havaittiin kasvattavan HDL- partikkeleiden kokoa ja lisäävän niiden sisältämien lipidien määrää. Camelinaöljyn käytön havaittiin vähentävän IDL-partikkeleiden pitoisuutta sekä lipoproteiinien kiinnittymistä proteoglykaaneihin vähentämällä seerumin LDL-kolesterolin pitoisuutta. Tämä havainto korostaa veren lipoproteiinitasojen merkitystä kolesterolin kertymisessä valtimon seinämän sisälle. Vähärasvaisen kalan käytöllä ei havaittu merkittäviä vaikutuksia veren lipidifraktioiden rasvahappokoostumukseen eikä lipoproteiinien alaluokkiin tai niiden funktioihin. Tämän tutkimuksen perusteella kala- ja kasviperäiset omega-3-sarjan rasvahapot näyttävät eroavan vaikutuksiltaan lipoproteiinien aineenvaihduntaan. Näiden rasvahappojen vaikutukset sydänterveyteen voivat siis olla toisiaan täydentäviä.

Tämän väitöskirjan johtopäätöksenä voidaan todeta, että helposti saavutettavissa olevilla ruokavaliomuutoksilla voidaan vaikuttaa edullisesti lipoproteiinien alaluokkien jakaumaan sekä yhteen ateroskleroosin kehittymisen keskeisimmistä mekanismeista. Tutkimuksesta saatu näyttö myös korostaa kalan sekä alfalinoleenihapon lähteiden tärkeyttä sydänterveyttä edistävässä ruokavaliossa.

Luokitus: QU 85, QU 90, QY 465, WB 425

Yleinen suomalainen ontologia: rasvahapot; omegarasvahapot; kalaöljyt; lipidit; lipoproteiinit;

kolesteroli; HDL-kolesteroli; LDL-kolesteroli; markkerit; satunnaistetut vertailukokeet

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ACKNOWLEDGEMENTS

The present study was carried out in the Institute of Public Health and Clinical Nutrition, Faculty of Health Sciences, Kuopio campus, University of Eastern Finland.

I wish to thank several people for supporting me during this process and for contributing to this thesis.

I am deeply grateful to my principal supervisor, Docent Arja Erkkilä, for the excellent guidance and support. I have been privileged to learn from your scientific expertise and experience. Your thorough feedback has helped me to get the best out of myself during the doctoral studies. I also want to express my warm thanks to my other supervisor, Professor Ursula Schwab, for your endless support and interest in my work. Thank you for believing in me from the very beginning and taking me into your research group right after my Master’s degree. I would also like to extend my deepest gratitude to my third supervisor, Docent Maria Lankinen, for your encouragement and valuable input to my doctoral thesis. You always had the door open for me and my questions.

I sincerely thank the reviewers of the thesis, Professor Rolf Kristian Berge and Docent Tiina Solakivi, for your careful work and constructive comments that helped me improve my thesis.

I am grateful to all the co-authors and collaborators for their contribution to this study. Special thanks to Katariina Öörni for introducing me to the world ofin vitro studies and helping me to understand the pathogenesis of atherosclerosis. Many thanks also to David Laaksonen for the careful revision of the language of this thesis.

I would like to thank all my colleagues at the Institute of Public Health and Clinical Nutrition, above all Heli, Timo, Topi and especially team Tehotieteilijät: Ulla, Katriina and Susanna. Thank you for all the great conversations and sharing the joy and the occasional agony of PhD student’s life.

Sincere thanks also belong to all the participants in AlfaFish study without whom this thesis would not have been possible.

I would like to thank my agility coach, Sini, for all the fun moments in training at Pro Perro. Agility hall has often been the best place for my mind to rest and reset outside the research world. I also want to thank Deep in the Forest ry for all the great times playing disc golf.

My dear friend Leija, thank you for cheering me during these years despite the distance and encouraging me, especially in times of self-doubt. I want to thank Laura for lifelong friendship and all the fun times we always have together – team Lievestuore forever! Warm thanks also to Aki and Varpu for your friendship and for trusting me with the most amazing dog, Jali – my little pikkumusta that lifts my spirits every day.

The completion of my doctoral thesis would not have been possible without the support of my family. I want to thank my parents, Eija and Pentti, for your unconditional love and care. Thank you for always letting me choose my own path

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and helping me along the way. I would also like to thank my brother Petri and my brother-in-law Niklas. Special thanks belong to my dear sister and partner in crime, Outi. Thank you for your endless understanding, encouragement and unwavering support. You’re the best!

Finally, I want to thank Ville for standing by my side through all these years because “these stories don’t mean anything when you’ve got no one to tell them to”.

You have always listened to my “stories” and supported me no matter what. For that I am forever grateful.

In appreciation of their financial support for this study, I would like to thank Finnish Cultural Foundation, Juho Vainio Foundation and State Research Funding of Kuopio University Hospital.

Kuopio, October 2019 Suvi Manninen

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LIST OF ORIGINAL PUBLICATIONS

This dissertation is based on the following original publications:

I Manninen S, Lankinen M, de Mello V, Ågren J, Laaksonen D, Schwab U, Erkkilä A. The effect of Camelina sativa oil and fish intakes on fatty acid compositions of blood lipid fractions. Nutrition, Metabolism & Cardiovascular Diseases 29: 51-61, 2018.

II Manninen S, Lankinen M, de Mello V, Laaksonen D, Schwab U, Erkkilä A.

Intake of fatty fish alters the size and the concentration of lipid components of HDL particles and Camelina sativa oil decreases IDL particle concentration in subjects with impaired glucose metabolism. Molecular Nutrition & Food Research 62: e1701042, 2018.

III Manninen S, Lankinen M, Erkkilä A, Nguyen S, Ruuth M, de Mello V, Öörni K, Schwab U. The effect of intakes of fish and camelina sativa oil on atherogenic and anti-atherogenic functions of LDL and HDL particles – a randomized controlled trial. Atherosclerosis 281: 56-61, 2018.

The publications were adapted with the permission of the copyright owners.

In addition, some previously unpublished data are presented.

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ABBREVIATIONS

ABCA1 Adenosine triphosphate binding cassette transporter A1

ABCG1 Adenosine triphosphate binding cassette transporter G1

ALA -linolenic acid apoB Apolipoprotein B apoE Apolipoprotein E CE Cholesteryl ester

CETP Cholesteryl ester transfer protein

CSO Camelina sativa oil CVD Cardiovascular disease DHA Docosahexaenoic acid DPA Docosapentaenoic acid EM Erythrocyte membrane EPA Eicosapentaenoic acid HDL High-density lipoprotein

hs-CRP High-sensitivity C-reactive protein

IDL Intermediate-density lipoprotein

IL-8 Interleukin-8 LCAT Lecithin/cholesterol

acyltransferase

LDL Low-density lipoprotein MUFA Monounsaturated fatty acid PL Phospholipid

PUFA Polyunsaturated fatty acid RCT Reverse cholesterol transport SAA Serum amyloid A

SFA Saturated fatty acid SMC Smooth muscle cell SR-B1 Scavenger receptor-B1 TG Triglyceride

VLDL Very low-density lipoprotein

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1 INTRODUCTION

Although the prevention of cardiovascular diseases (CVD) has advanced over the past few decades, CVD are still the major cause of premature death and chronic disability worldwide accounting for one-third of all deaths annually (1). Most of this burden owes to suboptimal lifestyle and is therefore preventable. Despite the extensive evidence of the healthy lifestyle factors in promoting cardiovascular health, incorporating these habits to daily lives of individuals is often challenging (2).

Therefore, practical approaches are needed to effectively promote healthy lifestyle changes.

Diet is an important modifiable lifestyle factor in the prevention of CVD (3).

Among several other food items, fish and vegetable oils have been considered important components of a cardioprotective diet. Fish is a main source of long-chain omega-3 polyunsaturated fatty acids (n-3 PUFAs), whereas several vegetable oils contain plant-derivedn-3 PUFA, -linolenic acid. Intake of long-chainn-3 PUFAs of marine origin beneficially modify several risk factors of CVD, whereas the effects of

-linolenic acid on cardiovascular health are far less understood (4).

High serum low-density lipoprotein (LDL) cholesterol and low serum high- density lipoprotein (HDL) cholesterol concentrations are important risk factors for CVD (5). However, lipoproteins are a heterogeneous group of particles which not only differ in their size, structure and composition but also in their biological functions (6). Lipoproteins have a central role in atherosclerosis, which is a complex, inflammatory disease of arterial wall (7). Atherosclerosis consists of several lipoprotein-related reactions that trigger a self-accelerating process leading to development and progression of the disease (7,8). Despite the vast amount of evidence of beneficial effects of n-3 PUFAs on several cardiovascular risk factors, very little is known about the effects of these fatty acids on different lipoprotein subtypes or pro- and anti-atherogenic lipoprotein functions.

Knowledge on the effects of easily achieved dietary modifications on lipoprotein metabolism and lipoprotein functions provide information for more effective prevention of CVD. In order to investigate these health effects of fatty acids, precise measures of dietary fatty acid intakes are needed (9). However, there is no consensus on which biomarker most accurately reflects the increased intake of n-3 PUFAs (10).

The aim of this doctoral thesis was to investigate the effects of fish and camelina sativa oil intakes on fatty acid composition of blood lipid fractions, lipoprotein subclasses and lipoprotein functions associated with atherosclerosis in middle-aged and elderly Finnish men and women with impaired glucose metabolism.

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2 REVIEW OF THE LITERATURE

2.1 N-3 FATTY ACIDS, FISH AND CAMELINA SATIVA OIL

2.1.1 Structure and functions ofn-3 fatty acids

Fatty acids have hydrocarbon chains with carboxyl and methyl groups (11). Fatty acid chains differ by length and saturationi.e. the number of double bonds. Chain length and degree of saturation, to a large extent, determine the physical properties of fatty acids and of the molecules that contain them. Short-chain fatty acids contain 6 or fewer, medium-chain fatty acids from 7 to 12 and long-chain 14 or more carbon atoms in the chain (12). Fatty acids with at least 20 carbon atoms are sometimes referred as very long-chain fatty acids. Saturated fatty acids (SFAs) have no double bonds whereas monounsaturated fatty acids (MUFAs) have one double bond in the chain. Polyunsaturated fatty acids (PUFAs) have at least two double bonds in the chain and can be classified inton-series ( - or omega-series). Inn-3 fatty acids, the first double bond is located between the third and fourth carbon from the methyl end of the chain (Figure 1). The shorthand nomenclature of PUFAs is based on the chain length, number of double bonds and the location of the first double bond from the terminal methyl group. The shortest n-3 PUFA is -linolenic acid (ALA, 18:3n-3), which is synthesized in plants from linoleic acid (18:2n-6) by the 15-desaturase enzyme (13). The human body lacks 15-desaturase and, therefore, is not able to synthesize ALA, which makes it an essential fatty acid together with linoleic acid.

Figure 1. Chemical structures of ALA, EPA and DHA.

ALA, -linolenic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.

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ALA is a precursor for long-chainn-3 fatty acids, such as eicosapentaenoic acid (EPA, 20:50n-3) and docosahexaenoic acid (DHA, 22:6n-3) (14). The conversion takes place primarily in the endoplasmic reticulum in the liver. The metabolic pathway includes series of elongations that add 2-carbon units and desaturations that add double bonds to the fatty acid chain (15) (Figure 2). The conversion ofn-6 PUFAs occurs via shared enzymatic pathway and, therefore,n-6 PUFAs compete withn-3 PUFAs for these same desaturase and elongase enzymes in the biosynthesis of longer-chain PUFAs (16). The rate of conversion is affected by an abundance of each substrate so that the abundance ofn-6 PUFAs reduces the conversion ofn-3 PUFAs. The first reaction in the pathway catalyzed by 6-desaturase has been considered the rate- limiting step (14). Although 6-desaturase has higher affinity for ALA (17), a Western diet, which is generally high in linoleic acid, favors the conversion n-6 PUFAs (18). The rate of ALA conversion to longer-chain n-3 PUFAs varies but is generally considered limited, which is most likely explained by the high oxidation rate of ALA (13,15). Studies have estimated that about 0.4–8% of ALA is converted to EPA and about 0–4% of ALA is converted to DHA (19). Furthermore, retroconversion of DHA to EPA is possible (20,21). However, recent evidence suggests that an increase in plasma EPA following DHA supplementation is due to slowed EPA metabolism and not retroconversion of DHA (22). Studies investigating the effects of ALA intake onn-3 PUFAs with different biomarkers have reported varying degrees of conversion of ALA to EPA and either no change or a decrease in the proportion of DHA (Table 1).

Variation in the rate of conversion may be explained by the analytical methods, by the polymorphisms in genes encoding desaturases or age and gender of the study subjects (14,23). For example, the conversion has been found to be more efficient in women than in men (24). Conversion is also affected by the background diet. A deficiency of long-chain n-3 PUFAs, such as in vegan diets, could enhance the conversion, whereas diets that contain larger amounts ofn-6 PUFAs thann-3 PUFAs favor the biosynthesis ofn-6 fatty acids at the expense ofn-3 fatty acids (15,25,26).

Furthermore, the availability of conversion products (EPA+DHA) in the diet suppresses the conversion by feedback regulation (27). An overall poor diet with insufficient energy or protein and deficiencies of certain vitamins (e.g. biotin) and minerals (e.g. zinc) or excessive alcohol consumption can decrease the activity of conversion enzymes (28).

The conversion pathway of ALA to EPA and DHA also forms several othern-3 fatty acid intermediates (Figure 2). Of these fatty acids, mainly stearidonic acid (SDA) and docosapentaenoic acid (DPA) are found as significant amounts in dietary sources. SDA is found, for example, in echium oil and black currant seed oil and as a minor constituent also in fish (29–31). Also DPA is found in fish (32), although circulating DPA levels have been shown to correlate only weakly with fish intake (33). However, endogenous DPA may be a significant contributor to long-chainn-3 PUFA metabolism as it can provide a source for EPA via retroconversion and possibly also for DHA via the biosynthetic pathway (34).

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23 Fatty acids have many essential roles in the human body: they serve as fuel in fat metabolism, provide an energy store, alter biophysical properties of membrane (e.g.

fluidity) and take part in cellular signaling pathways and transcriptional regulation (15,35). Furthermore,n-3 PUFAs contribute to normal neurodevelopment in children (36). PUFAs are incorporated especially in heart muscle, the nervous system and retina cells (15,37). In the human body, DHA is the principaln-3 fatty acid in tissues.

Nutritional deficiency ofn-3 PUFAs can cause visual dysfunction (38). Furthermore, several signaling pathways in the brain are modulated by DHA in neural membrane phospholipids. Therefore, long-term deficiency ofn-3 PUFAs may potentially have effects on cognition and memory. Bothn-6 and n-3 PUFAs are also precursors for lipid mediators (e.g. eicosanoids,) which have an essential role in the regulation of several important biological functions, such as blood pressure, sensation of pain and inflammation (13,39). Lipid mediators derived fromn-6 PUFAs have been commonly considered pro-inflammatory and n-3 PUFA-derived lipid mediators anti- inflammatory, but recent studies have shown that the role of these lipid mediators in inflammation is far more complex and not fully understood (40).

Figure 2. Biosynthetic pathway ofn-3 fatty acids.

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24 Table 1. Overview of studies investigating the effects of ALA-treatment on the content of ALA, EPA and DHA in blood lipid pools in order of publication year 1Intakes excluding background diet unless otherwise specified.aTotal ALA intake,bALA intake from background diet 1.2 g/day.2Duration of the intervention period. 3Gas chromatography was used as the analytical method unless otherwise specified:cgas chromatograph–flame ionization detector,d gas-liquid chromatography,e high-resolution gas chromatography/mass spectrometry.4Statistically significant changes. ALA, -linolenic acid; CE, cholesteryl ester; DHA, docosahexaenoic acid; EL, erythrocyte lipids; EM, erythrocyte membranes; EPA, eicosapentaenoic acid; F, female; M, male; MNC, mononuclear cell; PL, phospholipids; TG, triglycerides.

ReferenceGender of subjectsThe form in which ALA was providedALA intake/day1Duration (weeks)2Biomarker3Results4 ALA EPA DHA Karvonen et al. 2002 (41)M + FCamelina oil Canola oil11.4 g 3 g6Total serum Finnegan et al. 2003 (42)M + FSpread4.5 ga 9.5 ga24PL Kew et al. 2003 (43)M + FSpread4.5 ga 9.5 ga24MNC PL Wallace et al. 2003 (44)MFlaxseed oil capsules3.5 gb12PL Wilkinson et al. 2005 (45)MFlaxseed oil and spread15 ga12EM PL Harper et al. 2006 (46)M + FFlaxseed oil capsules3 g26Total plasma Cao et al. 2006 (47)M + FFlaxseed oil capsules3510 mg8EM PL Schwab et al. 2006 (48)M + F30 ml flaxseed oil 30 ml hempseed oil

14 g 6 g

4CE TG CE TG Goyens et al. 2006 (49)M + FSpread and pastries1.1% of energya6PLc Zhao et al. 2007 (50)M + FWalnuts, walnut oil and flaxseed oil19.1 ga6MNC Total serum Barceló-Coblijn et al. 2008 (51)M + FFlaxseed oil capsules1197 mg 2393 mg 3590 mg

12EM PLd Dodin et al. 2008 (52)FFlaxseeds and flaxseed bread9 g52Total plasma Rajaram et al. 2009 (53)M + FWalnuts4.7 ga4EM Egert et al. 2012 (54)M + FSpread4.4 ga6Total ELe Zong et al. 2013 (55)M + FFlaxseed bread7 g12Total EL Chiang et al. 2012 (56)M + FWalnuts4.7 ga4PL Dittrich et al. 2015 (57)M + FFlaxseed oil7.4 g10Total plasma Total EL

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25 2.1.2 Biomarkers ofn-3 fatty acids

In order to elucidate the relation between fatty acids, health and disease or to assess compliance in clinical trials, precise methods to measure the intake of fatty acids are needed (9). The most common methods for assessment of dietary fatty acid intake, such as dietary records, are prone to bias due to memory, under- or overreporting, altered food intake behaviors during the recording period and limitations in food composition databases (9). Therefore, biological markers,i.e. biomarkers, are often used in studies together with common dietary assessment methods. A biomarker objectively measures the quantifiable characteristics of biological processes or responses to intake (58,59). Therefore, it can be any biological specimen that is an indicator of nutritional status with respect to intake or metabolism of dietary components. Analysis of fatty acids of biological samples generally consists of extraction of lipids, conversion of the extracted lipids to fatty acid methyl esters (FAMEs), and analysis of the FAMEs usually by gas chromatography (60). Isolation of lipid classes, such as plasma lipid fractions, is needed when the aim is to investigate the fatty acid composition of these lipid classes separately. In addition to commonly used methods, applying metabolomics to find new dietary biomarkers has been found promising (61).

Fatty acid composition of lipid pools as biomarkers of fatty acid intake

After consumption and absorption, fatty acids are distributed throughout the body via exchanges within and between lipid classes in tissues and blood (9).

Consequently, fatty acids are incorporated in different lipid pools in the human body and can be measured from different biological samples, such as adipose tissue, membranes and blood (9,15,59). These lipid pools can be categorized according to their role as a storage pool (adipose tissue), functional pools (cells and cell membranes) and transport pools (lipid pools in the bloodstream) (Figure 3). Each of these lipid pools have a unique pattern of fatty acid composition (9). Circulating fatty acids in the blood reflect a combination of intestinal absorption of fatty acids from dietary sources, endogenous synthesis and utilization from tissues and storage (62).

Consequently, it should be taken into account that blood fatty acid composition not only reflects the dietary fat quality but can be also a biomarker for other components in the food, such as high sugar intake (63).

Blood contains a mixture of fatty acid pools, such as phospholipids (PL), cholesteryl esters (CE), triglycerides (TG), non-esterified fatty acids (NEFA), erythrocyte membranes (EM), platelets and mononuclear cells (MNC). The fatty acid composition of these lipid pools with different turnover rates serve as biomarkers to reflect varying periods of fatty acid intake (9). Plasma TG reflects the short-term intake of fatty acids, from the preceding hours to days whereas plasma PL and CE reflect the intake from days to weeks (9,10,59). Plasma PL are the major lipid class in animal cell membranes (9,64). Therefore, they can also represent the fatty acid

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26

composition of biological membranes. Of individual PL classes, plasma phosphatidylcholine (PC) reflects rapid changes in fatty acids (65).

Blood cells, such as platelets and MNC, have been found to be more suitable for measuring habitual fatty acid intake (65). The fatty acid composition of erythrocytes has also been considered a biomarker of long-term intake of fatty acids due to its half- life of ~120 days in the circulation (9). However, transfer and exchange of fatty acids between plasma and erythrocytes occurs, for which reason the role of erythrocyte fatty acids as a long-term biomaker of fatty acid intake has been questioned. Due to the slow turnover rates, adipose tissue fatty acid composition reflects dietary fatty acid intake from several months to years in weight-stable individuals (9). Adipose tissue fatty acid composition has also been considered the gold standard biomarker for fatty acid status (9). Also, NEFA levels are predominantly determined by their release from adipose tissue (9). Although fatty acid compositions of NEFA and adipose tissue are correlated, NEFA content may not be a fully accurate surrogate for adipose tissue fatty acid content (66).

Biomarkers of n-3 PUFA intake

Becausen-3 PUFAs are mostly exogenously produced, the biomarkers of these fatty acids usually correlate well with the dietary intake (9,11). Various biomarkers have been used to assess the intake ofn-3 fatty acids, most often the fatty acid composition of different blood lipid pools (10,59) (Tables 1 and 2). Daily ALA intake ranging from

~2 g to almost 20 g either from supplements or dietary sources has been found to increase the proportion of ALA in total serum, total plasma, plasma lipid fractions and different blood cells (Table 1). Similarly, a variety of EPA+DHA doses from fish or supplements have been found to increase EPA and DHA content of different blood lipid pools (Table 2). Of note, a continuous regimen of moderate EPA and DHA intake has been found to result in higher EPA+DHA content in MNC and platelets than sporadic intake with high amounts (67).

Long-chainn-3 PUFA content of blood lipid fractions have been found to be good biomarkers for EPA and DHA intake (10). However, EPA and DHA have been found to differ in terms of incorporation and saturation into different lipid pools (47,65,68,69) (Figure 3). EPA tends to incorporate and clear from different lipid pools more rapidly than DHA. In contrast, DHA has been found to saturate in plasma PL with lower intakes of fish as compared with EPA (68). Furthermore, some specificity has been observed in the incorporation of EPA versus DHA in different lipid pools (20). It should be also considered that intake ofn-3 PUFAs from supplements or from fish may differ in their effectiveness to incorporaten-3 PUFAs to lipid pools due to the more complex composition of fish. Studies suggest that fish intake, at least in short-term (~4 weeks), results in more effective incorporation of EPA and DHA than fish oil (70,71).

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27 Factors influencing the choice of suitable biomarker

Each sample type has its own strengths and limitations as a biomarker for long-chain n-3 PUFA intake (72) (Figure 3). Whole blood contains all lipid fractions and blood cells, whereas total plasma contains all lipid fractions and free fatty acids. These pools are easily accessible and do not require analytical lipid class separation. The mixtures of different lipid pools in whole blood and whole plasma also give a wider view on fatty acid metabolism and could, therefore, be good biomarkers for overall changes inn-3 PUFA metabolism in the circulation. However, it is of note that EPA and DHA are not physiologically active in the circulation itself but in the membranes and tissues into which they are incorporated (37). Therefore, measuring long-chainn-3 PUFAs from whole blood allows only limited conclusions about their concentration at the site of physiological activity. Furthermore, the biological variability of fatty acids in whole blood and plasma is relatively large (72) and whole blood could be influenced by hematocrit or changes in white blood cell pools during infection (64).

Moreover, both whole blood and total plasma are also affected by the lipoprotein profile of individuals (9,64).

Lipid pools with rapid turnover, such as plasma lipid fractions, are more immediate markers of fatty acid intake showing faster incorporation of EPA and DHA than blood cells or whole blood (72) (Figure 3). Then-3 PUFA composition of blood cells (e.g. erythrocytes, platelets, MNC) are suitable biomarkers for habitualn- 3 PUFA intake (Figure 3) but are analytically more challenging. However, the fatty acid composition of erythrocytes has lower biological variability than whole blood or plasma (73,74). A significant part of the variation inn-3 PUFAs in erythrocytes have been found to be explained by genetic factors (75). Erythrocytes also contain a wider spectrum of PL classes than whole plasma that is concentrated in PC (9,64).

Therefore, fatty acids in erythrocytes may be a good indicator of the fatty acid composition of biological cell membranes. In fact, omega-3 index,i.e. EPA+DHA in EM expressed as a percent of total fatty acids, has been found to correlate with myocardial long-chainn-3 PUFA content in humans (76,77). Furthermore, omega-3 index 8% has been proposed optimal for cardiovascular health (77).

There are also several non-dietary factors that may influence the fatty acid profile and levels of biomarkers (11,78,79). Such factors are for example age, BMI, genetic factors, ethnicity, general nutritional status, tissue site of the sample, certain diseases and lifestyle factors, such as smoking and physical activity. Furthermore, gender differences have been observed in then-3 PUFA status. Especially plasma DHA tends to be higher in women than in men (35,79). Gender differences have been proposed to be due to differences in the rate of -oxidation, adipose tissue mobilization and fatty acid composition and the effects of sex hormones on elongase and desaturase activities.

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28 Table 2. Overview of studies investigating the effects of fish and marinen-3 PUFA supplements on the EPA, DPA and DHA content in blood lipid pools in order of publication year Continued on next page.ReferenceGender of subjectsTreatmentEPA+DHA1 intake/dayDuration2 Biomarker3Results5 Fish studiesEPA DPA DHA Vidgren et al. 1997 (20)M5 fatty fish meals/week1.05 g (0.38 g + 0.67 g)14 weeks PL CE TG Platelets EM

NA Elvevoll et al. 2006 (70)M + FSmoked salmon 400 g/week Cooked salmon 400 g/week Cooked cod 400 g/week

1.2 g (0.5 g + 0.7 g) 1.2 g (0.5 g + 0.7 g) 0.03 g (0.01 g + 0.02 g)

8 weeks Total serumNA NA NA Harris et al. 2007 (71)FTuna + salmon 2 meals/week 0.49 g (95 mg + 390 mg)16 weeks PL EMNA NA Erkkilä et al. 2008 (80)M + F4 fatty fish meals/week 4 lean fish meals/week

~1 g < 0.5 g

8 weeks PL CE TG PL CE TG

NA NA NA NA NA NA Telle-Hansen et al. 2012 (81)M + F150 g of salmon/day 150 g of cod/day3.1 g (1398 mg + 1710 mg) 0.13 g (48 mg + 86 mg)15 days PLcNA NA Chiang et al. 2012 (56)M + F2 fatty fish meals/week0.78 g (0.17g + 0.61 g)4 weeks PLNA Helland et al. 2017 (82)M + FFatty fish 750 g/week Lean fish 750 g/week~1.25 g ~0.3 g8 weeks Leucocytes Zacek et al. 2018 (83)M + F90 g salmon twice/week 180 g salmon twice/week0.31 g (158 mg + 149 mg) 0.62 g (316 mg + 299 mg)4 weeks PC4d CE4 TG4 Rundblad et al. 2018 (84)M + F3 fish meals/week (lean and fatty fish)0.54 g (195 mg + 343 mg)8 weeks Total plasma

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29

Table 2. (continued) ReferenceGender of subjectsTreatmentEPA+DHA intake/day1Duration2Biomarker3Results5 Supplementation studiesEPA DPA DHA Vidgren et al. 1997 (20)MDHA-rich capsules1.68 g (0 g + 1.68 g)14 weeks PL CE TG Platelets EM

NA Fish oil2.28 g (1.33 g + 0.95 g)PL CE TG Platelets EM

NA Wallace et al. 2003 (44)MFish oil capsules0.44 g (0.15 g + 0.29 g)a 0.94 g (0.27 g + 0.67 g) 1.9 g (0.49 g + 1.41 g)

12 weeks PL4NA Kew et al. 2003 (43)M + FSpread and fish oil capsules0.77 g (0.3 g + 0.47 g)b 1.68 g (0.66 g + 1.02 g)6 months MNC PL Cao et al. 2006 (47)M + FFish oil capsules2.16 g (1296 mg + 864 mg) 8 weeks EM Total plasma Elvevoll et al. 2006 (70)M + FCod liver oil3 g (1.4 g + 1.6 g)8 weeks Total serumNA Harris et al. 2007 (71)M + FFish oil capsules0.48 g (104 mg + 378 mg) 16 weeks PL EMNA NA Barceló-Coblijn et al. 2008 (51)M+ FFish oil capsules0.26 g (252 mg + 3 mg) 0.51 g (504 mg + 6 mg)12 weeks EM PL4c Metherel et al. 2009 (85)M + FFish oil4.8 g (3.2 g + 1.6 g)4 weeks EL Whole blood Plasma Rundblad et al. 2018 (84)M + FKrill oil0.65 g (445 mg + 209 mg)8 weeks Total plasma 1Intakes excluding background diet unless otherwise specified.aDaily EPA+DHA intakes from the diet 0.08 g + 0.14 g, 0.06 g + 0.08 g, 0.07 g + 0.1 g, respectively. bTotal EPA+DHA intake.2Duration of the intervention period.3Gas chromatography was used as analytical method unless otherwise specified:cgas liquid chromatography,dhybrid quadrupole ion-trap mass spectrometer.4Similar changes with all doses.5Statistically significant changes. CE, cholesteryl ester; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; EL, erythrocyte lipids; EM, erythrocyte membranes; EPA, eicosapentaenoic acid; F, female; M, male; MNC, mononuclear cell; NA, data not available; PC, phosphatidylcholine; PL, phospholipids; TG, triglycerides.

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30 Figure 3. Characteristics of biomarkers used for EPA and DHA intakes (9,10,15,59,65,72–74,86–89). AT, adipose tissue; CE, cholesteryl esters; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; MNC, mononuclear cells; PC, phosphatidylcholine; PL, phospholipids; TG, triglycerides.

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31 2.1.3 Nutrients in fish and camelina sativa oil

Fish can be classified as fatty fish (oily fish) and lean fish according to the fat content (13,90). Fatty fish contains 4–25% of fat whereas lean fish typically contains < 2% fat (Table 3). Fish and seafood are the primary dietary sources of long-chainn-3 PUFAs which are, therefore, often called marinen-3 PUFAs (13). In lean fish, long-chainn-3 PUFAs are mostly incorporated in the liver, whereas in fatty fish they are incorporated in the flesh. The amount of long-chainn-3 fatty acids in fish varies for example according to their diet, location, water temperature and season (13,91). In addition to fatty acids, fish contains also several minerals (e.g. selenium, iodine and magnesium) as well as protein with high biological value and complete amino acid profile (90,92). Furthermore, fish is a good dietary source of vitamin D. However, the content of vitamin D in fish varies (Table 3) and is not correlated with the fat content of the fish (93). Fish contains also several B vitamins as well as vitamins A and E.

Consequently, in addition to fatty acids, beneficial effects of fish intake may be mediated by combinatory effects betweenn-3 PUFAs and other compounds in the fish (94). Furthermore, fish may replace other, potentially more unhealthy, foods in the diet, such as meat (95).

Concerns about the potential risks of fish consumption due to methylmercury (MeHg) and other contaminants found in fish exist (96). Consumption of fish and seafood is the primary source of exposure to MeHg, which can have adverse effects of neurodevelopment and cardiovascular health (90). In addition to contamination of the environment, the concentrations of methylmercury in different fish depend on the life span and predatory nature of the species (95). Of the Finnish fish species, pike has the highest levels of methylmercury (97).

For minimizing potential harmful effects, consumption of a variety of seafood is generally recommended (95). Specific restrictions for consumption of certain fish species also exist for vulnerable groups, such as infants, children and pregnant and lactating women (98). Collectively, studies suggest that benefits of moderate fish consumption outweigh the potential harmful effects (95).

Camelina sativa oil (CSO) is extracted from the seeds ofCamelina sativa, an oilseed crop which is also known as false flax or gold of pleasure (99). CSO contains about 10% of total SFA, 33% of total MUFA and 57% of total PUFA (Table 4). It is also a good dietary source for ALA, containing about 38% of ALA (Table 4). Other good dietary sources of ALA are vegetable oils, such as canola oil, and nuts, especially walnuts (18). CSO is also a source for phenolic compounds and tocopherols, of which

-tocopherol is predominant (100).

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32 Table 3. Fat and vitamin D content of fish species commonly consumed in Finland1 DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids. 1 Data from National Institute for Health and Welfare: Finnish food composition database (fineli.fi). 2 Data from reference (101). 3 Data from reference (102).

Total fat (g/100 g)SFA (g/100 g)MUFA (g/100 g)PUFA (g/100 g)EPA (mg/100g)DHA (mg/100g)Vitamin D (µg/100g) Mackerel (smoked)25.15.39.67.0148033505.2 Canned sardine (in oil)14.32.63.96.47889655.2 Farmed salmon212.31.443.522.24106206.7 Anchovy11.43.04.33.076814985.7 Canned tuna (in oil)9.01.31.94.5231901.7 Arctic char37.91.43.12.54808305.8 Wild salmon26.30.91.81.02605205.2 Rainbow trout6.21.22.71.72377545.1 Baltic herring6.11.62.02.040260615.6 Farmed white fish5.91.02.61.620439414.4 Fresh tuna4.91.31.61.42838907.2 Vendace3.90.90.61.32883009.4 Bream3.20.51.00.719927314.0 Trout3.00.50.80.81702909.0 Roach2.40.30.80.511228710.0 Flounder1.80.40.40.52841330.7 Saithe1.00.20.10.41022801.5 Canned tuna (in water)0.90.20.20.2201404.0 Cod0.80.1<0.10.3871957.0 Burbot0.80.10.20.3631350.5 Perch0.70.10.20.2408015.2 Pike0.40.1<0.1<0.118302.1 Pike-perch0.4<0.1<0.10.126506.9

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33 Table 4. Fatty acid composition of camelina sativa oil¹

Fatty acid mol%

Myristic acid 14:0 0.09

Pentadecanoic acid 15:0 0.02

Palmitic acid 16:0 5.74

Margaric acid 17:0 0.05

Stearic acid 18:0 2.48

Arachidic acid 20:0 1.49

Behenic acid 22:0 0.29

Lignoceric acid 24:0 0.15

Total SFA 10.31

Palmitoleic acid 16:1n-7 0.09

Oleic acid 18:1n-9 13.18

Cis-vaccenic acid 18:1n-7 0.70

Eicosenoic acid 20:1n-9+11 14.71

Erucic acid 22:1n-9 3.41

Nervonic acid 24:1n-9 0.59

Total MUFA 32.67

Linoleic acid 18:2n-6 16.43

-linolenic acid 18:3n-3 38.37

Eicosadienoic acid 20:2n-6 2.22

Total PUFA 57.02

1Analyzed at the Institute of Public Health and Clinical Nutrition, University of Eastern Finland (103).

Fatty acids are expressed as mol% of total fatty acids.

MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids.

2.1.4 Recommendations for fish andn-3 fatty acid intakes and current consumption

At least two servings (or 8 ounces i.e. ~227 g) of fish per week is generally recommended in different guidelines (104–106). More specifically, global recommendations for totaln-3 fatty acid intake are between 0.5% and 2% of daily energy intake, for ALA at least 0.5% of daily energy intake and for EPA+DHA 200–

250 mg/day (107,108). Similar recommendations are found also in Nordic Nutrition Recommendations (109). However, there is some variation in the recommendations of different national and international authorities, especially regarding EPA and DHA intakes (110). Furthermore, specific recommendations exist for certain populations groups, such as infants, elderly, and pregnant and lactating women.

The annual global consumption of fish products per capita has increased significantly over the past 50 years (111). However, annual fish consumption varies considerably between countries and national regions from less than 1 kg per capita in some countries to over 100 kg per capita in others. Consumption has been found to be greatest in the island nations and coastal regions. In Europe, annual consumption of fish and seafood is highest in Portugal and Spain (57 kg and 46 kg per capita, respectively) whereas the average in the region of European Union is 24 kg per capita and in Finland 20 kg per capita (112).

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34

The regional variety in fish consumption is also seen in mean intake of marinen-3 PUFAs in the dietary surveys, ranging from less than 50 mg/day to over 700 mg/day globally (25). The lowest levels have been identified in Sub-Saharan Africa and the highest levels in Southeast Asia, Pacific Asia and Western Europe. This variation is reflected also in the blood levels of EPA and DHA in different populations (113).

Altogether, almost 80% of the world’s adult population has intake of marine n-3 PUFAs less than 250 mg/day (25).

In Europe, the recommendation of 0.5 E% of ALA per day is met relatively well (almost 80% of 17 included countries) whereas the recommendation for EPA+DHA (250 mg/day) intake is met only in 26% of the countries, as reviewed by Sioen et al.

(110). In Finland recommended intake for totaln-3 PUFAs (1 E%) is met for both men and women (1.7 E% for women and 1.6 E% for men) (114). Average daily intake of fish is 53 g for Finnish men and 37 g for women. In 2007, it was reported that less than 30% of Finnish population eat the recommended two fish meals per week (115).

Several factors can influence the choice of consuming fish, such as taste, cultural background, ethical views, cost and availability (92). Furthermore, knowledge of the health effects of fish consumption and, on the other hand, concerns about the contaminants in the fish and the sustainability of fishing and fish farming can be factors that affect these choices (92,96).

Long-chain n-3 fatty acids can also be obtained from fish oil supplements.

However, it has been shown that the bioavailability of long-chainn-3 fatty acids from fish is better than from fish oil supplements (70). The bioavailability ofn-3 fatty acids can be affected by concomitant intake of other food items (especially fat) and the presence of other compounds (e.g. calcium ions) that may alter the metabolism of these fatty acids (37). The chemical form in which EPA and DHA are bound may also influence their bioavailability (PL > TG > free fatty acids > ethyl esters). Moreover, existing evidence suggests that supplemental long-chainn-3 PUFAs are not useful for preventing CVD and are therefore not recommended for general population (116,117).

2.2 LIPOPROTEINS

2.2.1 Lipoprotein metabolism

Lipoproteins are complex particles that are essential for the transport of cholesterol and TG in the circulation (118,119). Lipoproteins consist of a hydrophobic core of CE and TG surrounded by a monolayer of PL, apolipoproteins and free cholesterol.

There are several apolipoproteins that have specific properties and distributions in the lipoprotein classes. Apolipoproteins, along with other protein components, have an essential role as structural elements, enzymes, enzyme cofactors and ligands for cell surface receptors. Lipoproteins can be divided into different lipoprotein classes:

chylomicrons, very low-density lipoproteins (VLDL), intermediate-density

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35 lipoproteins (IDL), LDL and HDL. These classes differ in size, lipid composition and apolipoprotein content (Table 5).

Table 5. Lipoprotein classes

Lipoprotein Diameter (nm) Major lipids Major apolipoproteins

Chylomicrons 75–1200 Triglycerides apo B-48, apo C, apo E,

apo A-I, A-II, A-IV Chylomicron remnants 30–80 Triglycerides,

cholesterol

apo B-48, apo E

VLDL 30–80 Triglycerides apo B-100, apo E, apo C

IDL 25–35 Triglycerides,

cholesterol

apo B-100, apo E, apo C

LDL 18–25 Cholesterol apo B-100

HDL 5–12 Cholesterol,

phospholipids

apo A-I, apo A-II, apo C, apo E

Adapted from reference (119). Apo, apolipoprotein; HDL, high-density lipoprotein; IDL, intermediate- density lipoprotein; LDL, low-density lipoprotein; VLDL, very low-density lipoprotein.

Chylomicrons are formed in the intestine, and they transport dietary TG and cholesterol from the intestine to other tissues to be metabolized during the postprandial state (119) (Figure 4). Fatty acids are released from the TG via hydrolysis and then taken up by muscle cells and adipocytes for either energy production or storage. The removal of TG from chylomicrons by the tissues results in the formation of chylomicron remnants, which are then rapidly taken up from the circulation by the liver where the cholesterol from the remnant particles is released.

This metabolic route of dietary TG and cholesterol to peripheral tissues and liver is referred to as the exogenous lipoprotein pathway (119) (Figure 4).

The endogenous lipoprotein pathway starts in the liver with the formation of VLDL particles (119) (Figure 4). VLDL transports the TG formed in the liver to the tissues. TG from VLDL are taken up by the muscle and adipose tissue, resulting in the formation of VLDL remnants, IDL particles. Further removal of TG from IDL leads to the formation of LDL particles, rich in cholesterol and CE. The role of LDL is to deliver cholesterol to the peripheral tissues (119).

Excess cholesterol from tissues is transported back to the liver by HDL particles in a multi-step process called reverse cholesterol transport (RCT) (120). This pathway is essential for the cholesterol homeostasis. HDL particles are remodeled throughout the transport of cholesterol between cells, lipoproteins and liver. Nascent HDL particles (i.e. pre- -HDL), which are protein-rich and contain low amounts of cholesterol, are produced in the liver and small intestine (Figure 4). Nascent HDL particles take up PL and cholesterol from peripheral cells gradually forming discoidal HDL particles. Lecithin/cholesterol acyltransferase (LCAT) enzymatically converts free cholesterol into CE which forms mature, spherical HDL particles (119).

Mature HDL particles then return to the liver to unload the cholesterol (Figure 4).

Another enzyme involved in HDL metabolism is cholesteryl ester transfer protein

Fish and camelina sativa oil : effects on fatty acid composition of blood lipid fractions, lipoprotein subclasses and pro- and anti-atherogenic lipoprotein functions

uef.fi

Dissertations in Health Sciences

SUVI MANNINEN

FISH AND CAMELINA SATIVA OIL – EFFECTS ON FATTY ACID COMPOSITION OF BLOOD LIPID FRACTIONS, LIPOPROTEIN SUBCLASSES AND PRO- AND ANTIATHEROGENIC

LIPOPROTEIN FUNCTIONS

α

SUVI MANNINEN

FISH AND CAMELINA SATIVA OIL – EFFECTS ON FATTY ACID COMPOSITION OF BLOOD

LIPID FRACTIONS, LIPOPROTEIN

SUBCLASSES AND PRO- AND ANTI-

ATHEROGENIC LIPOPROTEIN FUNCTIONS

Suvi Manninen

FISH AND CAMELINA SATIVA OIL –

EFFECTS ON FATTY ACID COMPOSITION OF BLOOD LIPID FRACTIONS, LIPOPROTEIN

SUBCLASSES AND PRO- AND ANTI- ATHEROGENIC LIPOPROTEIN FUNCTIONS

ABSTRACT

TIIVISTELMÄ

ACKNOWLEDGEMENTS

LIST OF ORIGINAL PUBLICATIONS

CONTENTS

ABBREVIATIONS

1 INTRODUCTION

2 REVIEW OF THE LITERATURE

2.1 N-3 FATTY ACIDS, FISH AND CAMELINA SATIVA OIL

2.2 LIPOPROTEINS