Circulating fatty acids : associations with diet, genetic variations, low-grade inflammation and type 2 diabetes

(1)

DISSERTATIONS | MARKUS TAKKUNEN | CIRCULATING FATTY ACIDS – ASSOCIATIONS WITH DIET... | No 350

uef.fi

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences

ISBN 978-952-61-2106-2 ISSN 1798-5706

Dissertations in Health Sciences

MARKUS TAKKUNEN

CIRCULATING FATTY ACIDS – ASSOCIATIONS WITH DIET, GENETIC VARIATIONS, LOW-GRADE INFLAMMATION AND TYPE 2 DIABETES

Fatty acids in erythrocyte membranes and plasma are used as objective biomarkers of dietary fat intake. These circulating fatty acids are particularly good in reflecting fish

oil intake. In this thesis, circulating marine n-3 fatty acids were associated with lower incidence of type 2 diabetes. N-6 fatty acids,

except for linoleic acid, were associated with higher low-grade inflammation. Novel evidence was found suggesting that gene-diet

interactions modulate circulating fatty acids.

MARKUS TAKKUNEN

(2)

(3)

Circulating fatty acids – associations with diet, genetic variations, low-grade

inflammation and type 2 diabetes

(4)

(5)

(6)

(7)

MARKUS TAKKUNEN

Circulating fatty acids – associations with diet, genetic variations, low-grade

inflammation and type 2 diabetes

To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in CA102, Kuopio, on Friday, 10^th of June, at 12 o’clock noon

Publications of the University of Eastern Finland Dissertations in Health Sciences

Number 350

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

Kuopio 2016

(8)

Printing Office: Grano Oy Jyväskylä, 2016

Series Editors:

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

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

Professor Hannele Turunen, 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. (pharmacy) School of Pharmacy

Faculty of Health Sciences Distributor:

University of Eastern Finland Kuopio Campus Library

P.O.Box 1627 FI-70211 Kuopio, Finland http://www.uef.fi/kirjasto

ISBN (print): 978-952-61-2106-2 ISBN (pdf): 978-952-61-2107-9

ISSN (print): 1798-5706 ISSN (pdf): 1798-5714

ISSN-L: 1798-5706

(9)

(10)

(11)

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

Kuopio Finland

Email: markust@student.uef.fi

Supervisors: Associate professor Ursula Schwab, Ph.D.

Institute of Public Health and Clinical Nutrition University of Eastern Finland

Kuopio Finland

Email: ursula.schwab@uef.fi

Professor Matti Uusitupa, M.D., Ph.D.

Kuopio Finland

Email: matti.uusitupa@uef.fi

Docent Vanessa de Mello Laaksonen, Ph.D.

Kuopio Finland

Email: vanessa.laaksonen@uef.fi

Reviewers: Professor Suvi Virtanen, M.sc., M.D., Ph.D.

Nutrition Unit

National Institute for Health and Welfare Helsinki

Finland

& School of Health Sciences University of Tampere Tampere

Finland

Docent Kirsi Laitinen, Ph.D.

Institute of Biomedicine University of Turku Turku

Finland

Opponent: Professor Harri Niinikoski, M.D., Ph.D.

Institute of Biomedicine University of Turku Turku

Finland

(12)

(13)

Takkunen, Markus

Circulating fatty acids – associations with diet, genetic variations, low-grade inflammation and type 2 diabetes

University of Eastern Finland, Faculty of Health Sciences

Publications of the University of Eastern Finland. Dissertations in Health Sciences 350. 2016. 78 p.

ISBN (print): 978-952-61-2106-2 ISBN (pdf): 978-952-61-2107-9 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706

ABSTRACT:

Proportions of fatty acids measured in erythrocyte membranes (EM) and plasma are commonly used as objective biomarkers of dietary fat quality. These circulating fatty acids also provide insight into fatty acid metabolism in the human body, and activities of certain desaturase enzymes are often estimated using circulating fatty acids. In this thesis the use of circulating fatty acids as biomarkers of dietary fat intake and their associations with genetic variation, low-grade inflammation and type 2 diabetes (T2D) were examined.

The associations of major sources of dietary fat, estimated by a qualitative food frequency questionnaire, with EM fatty acid composition were investigated in a subpopulation (n=1033) of Metabolic Syndrome in Men study (METSIM). All major sources of dietary fat studied (fish, meat, dairy fat, spreads and cooking fat) were associated with EM fatty acid composition. As expected, the strongest associations were found between fish products and marine n-3 fatty acids in EM.

The associations between EM fatty acids and markers of low-grade inflammation were examined in another subpopulation (n=1373) of METSIM. All n-6 fatty acids, except for linoleic acid (18:2n-6), were associated with higher low-grade inflammation, whereas the anti-inflammatory associations of n-3 fatty acids were modest. Palmitoleic acid (16:1n-7) was associated with a higher concentration of C-reactive protein, whereas its elongation product, vaccenic acid (18:1n-7), was associated with higher adiponectin concentration.

The associations of serum fatty acid composition with T2D incidence, insulin secretion and insulin sensitivity were analyzed in a prospective cohort study (n=407) using repeated measurements based on the randomized Finnish Diabetes Prevention Study. Higher marine n-3 fatty acids and Δ5 desaturase (D5D) activity predicted lower T2D incidence during the long follow-up (median 11 y). These same n-3 fatty acids and D5D also tended to be associated with higher insulin sensitivity.

We hypothesized that a polymorphism in the FADS1 gene (rs174550) could modulate the observed association between intake of marine fatty acids and circulating polyunsaturated fatty acids in EM and plasma. In the participants of the METSIM study, who were homozygous for minor alleles (C/C) of rs174550 (n=168), the association between diet and circulating long-chain n-3 fatty acids was stronger than in those men who were carries of major alleles (T, n=794). Polymorphisms in the same locus were associated with hepatic expression of FADS1 mRNA in the separate Kuopio Obesity Surgery study.

This thesis provides more evidence that endogenous and dietary fatty acids play a role in the development of chronic diseases. Novel evidence was found indicating that gene-diet interactions modulate circulating fatty acid concentrations, which should be considered in future studies that examine the role of dietary and circulating fatty acids in the development of chronic diseases.

National Library of Medicine Classification: QU 90, QU 145, QZ 150, QU 500, WK 810

Medical Subject Headings: Fatty Acids; Biomarkers; Humans; Diet; Diabetes Mellitus, Type 2; Inflammation;

Fatty Acid Desaturases; Polymorphism, Genetic; Dietary Fats; Erythrocyte Membrane;

(14)

(15)

Takkunen, Markus

Veren rasvahappokoostumus – yhteydet ruokavalioon, geneettisiin variaatioihin, lievään tulehdukseen ja tyypin 2 diabetekseen

Itä-Suomen yliopisto, terveystieteiden tiedekunta

Publications of the University of Eastern Finland. Dissertations in Health Sciences 350. 2016. 78 p.

ISBN (print): 978-952-61-2106-2 ISBN (pdf): 978-952-61-2107-9 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706

TIIVISTELMÄ:

Rasvahappojen suhteellisia osuuksia punasoluissa ja plasmassa käytetään usein objektiivisina biomarkkereina ruokavalion rasvan laadulle. Verestä mitatut rasvahapot kertovat samalla elimistön rasvahappometaboliasta ja niiden perusteella voidaan esimerkiksi arvioida desaturaasientsyymien aktiivisuutta. Väitöskirjatyössä tutkittiin veren rasvahappokoostumuksen yhteyksiä ruokavalioon, geneettisiin variaatioihin sekä yhteyksiä lievään tulehdukseen ja tyypin 2 diabeteksen (T2D) ilmaantuvuuteen.

Metabolinen oireyhtymä miehillä (MOM) –tutkimuksen osajoukossa (n=1033) tarkasteltiin rasvaa sisältävien ruokien, joiden määrää arvioitiin laadullisella frekvenssikyselyllä, yhteyksiä punasolujen rasvahappokoostumukseen. Kaikki merkittävät rasvanlähteet (kala, liha, maitotuotteet, levitteet ja ruokaöljyt) olivat yhteydessä punasolujen rasvahappokoostumukseen. Yhteydet olivat voimakkaimmat kalarasvojen ja punasoluista mitattujen pitkäketjuisten n-3 rasvahappojen välillä.

Toisessa MOM-tutkimuksen osajoukossa (n=1373) tutkittiin punasolujen rasvahappokoostumuksen yhteyttä lievään tulehdukseen. N-6 rasvahapot, paitsi linolihappo (18:2n-6), olivat yhteydessä korkeampiin tulehdusmerkkiainetasoihin. N-3 rasvahappojen anti-inflammatoriset yhteydet olivat melko vaatimattomat. Endogeeninen palmitoleiinihappo (16:1n-7) oli yhteydessä suurempaan CRP:hen ja tämän rasvahapon elongaatiotuote, vakseenihappo (18:1n-7), oli puolestaan yhteydessä anti-inflammatorisen adiponektiinin pitoisuuteen.

Kohorttitukimuksessa (n=407), joka perustui randomoituun suomalaiseen diabeteksen ehkäisytutkimukseen (DPS), analysoitiin toistetusti mitattujen seerumin rasvahappopitoisuuksien yhteyksiä T2D:een, insuliiniherkkyyteen ja insuliinin eritykseen.

Pitkäketjuiset n-3 rasvahapot ja Δ5-desaturaasientsyymin aktiivisuus olivat yhteydessä matalampaan T2D:n ilmaantuvuuteen. Nämä kyseiset löydökset vaikuttivat selittyvän paremmalla insuliiniherkkyydellä.

Hypoteesina oli myös, että FADS1-geenin pistevaihtelu (rs174550) muuntaisi havaittua yhteyttä ruokavalion kalarasvojen ja verestä mitattujen monityydyttymättömien rasvahappojen välillä. MOM-tutkimuksessa (n=962) heillä, joilla oli molemmat harvinaisemmat alleelit (C/C) rs174550:sta, oli voimakkaampi yhteys kalarasvojen ja punasoluista ja plasmasta mitattujen pitkäketjuisten n-3 rasvahappojen välillä kuin heillä, joilla oli yleisempiä alleeleja (T). FADS1:n pistevaihtelut olivat yhteydessä maksan FADS1:n mRNA-ilmentymiseen erillisessä kuopiolaisten lihavuusleikattujen aineistossa.

Tämä väitöskirjatyö esittää lisää uuttaa näyttöä siitä, että rasvahapoilla on merkitystä eri kroonisten sairauksien kehityksessä. Uutta tietoa saatiin siitä, että geeni-ruokavalio yhdysvaikutukset voivat muuntaa veren rasvahappokoostumusta, mikä on huomioitava tulevissa tutkimuksissa, jotka käsittelevät rasvahappojen yhteyksiä sairauksiin.

Luokitus: QU 90, QU 145, QZ 150, QU 500, WK 810

Yleinen suomalainen asiasanasto: rasvahapot; markkerit; ihminen; ravitsemus; aikuistyypin diabetes;

tulehdus; entsyymit; geneettinen monimuotoisuus; ravintorasvat; punasolut;

(16)

(17)

Acknowledgements

I am grateful for the instructions and support by the supervisors of my thesis, associate professor Ursula Schwab, professor Matti Uusitupa and docent Vanessa de Mello Laaksonen. This dissertation would have not been possible without their guidance. I am especially indebted to professor Matti Uusitupa because of his enthusiasm and planning related to my dissertation. I give my thanks to Jyrki Ågren, whose special insight in biochemistry was of high value. I am grateful for the support of Jaana Lindström and professor Jaakko Tuomilehto from the DPS study. I also want to thank all other co-authors who participated to the substudies of this dissertation. I am also thankful for all the other members in the study groups of the METSIM, DPS and KOBS studies.

I want to express my gratitude to professor Markku Laakso and professor Johanna Kuusisto for allowing me to use data from the METSIM study in my thesis. I am thankful to professor Jussi Pihlajamäki for allowing me to use data from the KOBS study for one of the substudies. I thank laboratory technician Sirkku Karhunen for her excellent technical assistance. I thank Teemu Kuulasmaa for the outstanding data management in the METSIM study.

I thank docent Kirsi Laitinen and professor Suvi Virtanen for reviewing the thesis and providing comments that helped me significantly to improve the clarity of the thesis. I express my thanks to docent David Laaksonen for proofreading my thesis.

I thank my family and friends for their support during the past years. I am eternally grateful to Maria for her love and support. Without her, this thesis would have not been possible.

(18)

(19)

List of the original publications

This dissertation is based on the following original publications:

I Takkunen M, Ågren J, Kuusisto J, Laakso M, Uusitupa M, Schwab U. Dietary fat in relation to erythrocyte fatty acid composition in men. Lipids 48: 1093-102, 2013.

II Takkunen MJ, de Mello VD, Schwab US, Ågren JJ, Kuusisto J, Uusitupa MI.

Associations of erythrocyte membrane fatty acids with the concentrations of C- reactive protein, interleukin 1 receptor antagonist and adiponectin in 1373 men.

Prostaglandins Leukot Essent Fatty Acids 91: 169-74, 2014.

III Takkunen MJ, Schwab US, de Mello VD, Eriksson JG, Lindström J, Tuomilehto J, Uusitupa MI, DPS Study Group. Longitudinal associations of serum fatty acid composition with type 2 diabetes risk and markers of insulin secretion and sensitivity in the Finnish Diabetes Prevention Study. Eur J Nutr, 2015. (In press, published online)

IV Takkunen MJ, de Mello VD, Schwab US, Kuusisto J, Vaittinen M, Ågren JJ, Laakso M, Pihlajamäki J, Uusitupa MI. Gene-diet interaction of a common FADS1 variant with marine polyunsaturated fatty acids for fatty acid

composition in plasma and erythrocytes among men. Mol Nutr Food Res, 60: 381- 9, 2016.

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

(20)

(21)

5.3 Interactions between marine fatty acid intake and a FADS1 variant for circulating fatty acids (Study IV) ... 40 5.4 Erythrocyte membrane fatty acids and low-grade inflammation (Study II) ... 43 5.5 Prospective associations of serum fatty acids with type 2 diabetes, insulin secretion and insulin sensitivity (Study III) ... 45 6 DISCUSSION ... 51 6.1 Main sources of fat estimated by a FFQ are related to erythrocyte membrane fatty acid composition ... 51 6.1.1 Principal findings ... 51 6.1.2 Meat and fish intake in relation to n-6 fatty acids in erythrocyte membranes ... 51 6.1.3 Dairy fat and erythrocyte membrane fatty acids ... 51 6.1.4 Vegetable oil based fats and erythrocyte membrane fatty acids ... 52 6.2 A common FADS1 variant may modify the relationship between marine fatty acids and circulating fatty acids ... 53 6.2.1 Principal findings ... 53 6.2.2 Gene-diet interactions for circulating fatty acids ... 53 6.2.3 Mechanism for the gene-diet interactions? ... 53 6.3 N-6 fatty acids but not linoleic acid in erythrocyte membranes associate with low- grade inflammation ... 54

6.3.1 Principal findings ... 54 6.3.2 N-6 fatty acids and low-grade inflammation ... 54 6.3.3 N-3 fatty acids and low-grade inflammation ... 55 6.3.4 Estimated enzyme activities and low-grade inflammation ... 55 6.4 Marine n-3 fatty acids and Δ5 desaturase activity predict lower type 2 diabetes incidence and higher insulin sensitivity ... 56

6.4.1 Principal findings ... 56 6.4.2 Marine n-3 fatty acids and the risk of type 2 diabetes ... 56 6.4.3 Other serum fatty acids and the risk of type 2 diabetes ... 57 6.4.4 Desaturase enzymes and the risk of type 2 diabetes ... 57 6.5 Strengths and limitations ... 58 7 CONCLUSIONS AND FUTURE IMPLICATIONS ... 61 8 REFERENCES ... 63 ORIGINAL PUBLICATIONS (I-IV)

(23)

Abbreviations

ALA Alpha-linolenic acid (18:3n-3) ARA Arachidonic acid (20:4n-6) CE Cholesteryl esters

CoA Coenzyme A

CRP C-reactive protein CVD Cardiovascular disease D5D Δ5 desaturase

D6D Δ6 desaturase

DGLA Dihomo-gamma-linolenic acid (20:3n-6)

DHA Docosahexaenoic acid (22:6n-3)

DPA Docosapentaenoic acid (22:5n-3)

DPS Finnish Diabetes Prevention Study

EM Erythrocyte membranes EPA Eicosapentaenoic acid

(20:5n-3)

FFQ Food frequency questionnaire IL-1Ra Interleukin-1 receptor

antagonist IL-6 Interleukin-6

KOBS Kuopio Obesity Surgery Study

LA Linoleic acid (18:2n-6) Marine EPA (20:5n-3), DPA (22:5n-3) fatty acids and DHA (22:6n-3)

METSIM Metabolic syndrome in men study

MUFA Monounsaturated fatty acids OGTT Oral glucose tolerance test PC Phosphatidylcholine PL Phospholipids

PUFA Polyunsaturated fatty acids SCD1 Stearoyl-CoA desaturase-1 SFA Saturated fatty acids SNP Single nucleotide

polymorphism T2D Type 2 diabetes TG Triacylglycerides

WHO World Health Organization

(24)

(25)

1 Introduction

Fatty acids are a major source of energy in human diet due to their high energy content (9 kcal/g). However, their biological impact is much wider. Fatty acids are essential parts of cellular membranes, bind to various types of receptors and transcription factors (1,2), act as precursors to paracrine mediators (e.g., prostaglandins) and may even exhibit lipotoxic effects (2). Fatty acids are also thought to have an important contribution to human disease and health, and it is considered that certain types of fatty acids are more beneficial in the prevention of common chronic diseases, such as cardiovascular diseases (CVD).

Replacing unsaturated fatty acids with saturated fatty acids (SFA) in the diet increases plasma LDL cholesterol concentration, which in turns predisposes to the development of CVD (3,4). Similarly, trans-fatty acids increase LDL cholesterol concentrations and the risk of CVD, probably even more than SFA (5,6). N-3 fatty acids are generally thought to protect from CVD and lower plasma triglyceride concentrations (3,7), but the latest intervention studies supplementing n-3 fatty acids in the form of fish oil have not found any effect on CVD (8,9). Fatty acids may also play a part in the development of type 2 diabetes (T2D). In T2D and its prevention preference of unsaturated over saturated fatty acids is promoted (10), because this tends to improve insulin sensitivity (3,11). The role of n-3 fatty acids in the etiology of T2D, however, is unclear because in cohort studies n-3 fatty acids have been associated with both increased and decreased T2D risk, and supplementation has not improved insulin sensitivity in human intervention studies (3,12,13).

Furthermore, n-3 fatty acids are often referred as anti-inflammatory and n-6 fatty acids as proinflammatory fatty acids due to their eicosanoids products. This might be of high importance when considering the effects of these fatty acids on chronic diseases, as low- grade inflammation is one main pathogenic mechanism behind chronic diseases such as CVD, cancer and T2D (14-16). Yet, the evidence from intervention studies that n-3 and n-6 fatty acids modulate low-grade inflammation in the general population is insufficient (17- 19).

Fatty acid composition in plasma and various tissues have been successfully used as biomarkers of certain dietary fatty acids in a number of studies (20). These biomarkers are more objective estimates of dietary fat intake than dietary questionnaires, and they are more feasible to measure in large scale studies. They also provide insight in the endogenous metabolism of fatty acids. Thus, biomarkers of dietary fat intake offer a valuable tool to study the health effects of different types of fatty acids.

The aim of this thesis was to investigate the use of circulating fatty acids measured in erythrocytes and plasma as measures of fat intake and their relationship with genetic variation, low-grade inflammation and the risk of T2D in observational settings.

(26)

(27)

2 Review of the literature

2.1 FATTY ACIDS 2.1.1 Classification

Fatty acids consist of a functional group, carboxylic acid (-COOH), and of a hydrocarbon chain with a variable length. Fatty acids are classified as saturated if the hydrocarbon chain does not contain double bonds. Unsaturated fatty acids contain one (monounsaturated) or more (polyunsaturated) double bonds in the hydrocarbon chain. The unsaturated fatty acids are further classified into different groups depending on which carbon atom, counting from the non-functional end (i.e. the methyl terminal end), has the first double bond. These fatty acid groups are referred e.g. as n-3, n-6 or n-9 fatty acids, or respectively, as omega-3, omega-6 and omega-9 fatty acids. This grouping is important because these fatty acid groups have different biological properties, and n-3 and n-6 fatty acids are not de novo synthesized in the human body (21). Consequently, two important n-3 and n-6 fatty acids, alpha-linolenic acid (ALA, 18:3n-3) and linoleic acid (LA, 18:2n-6), are referred as essential fatty acids. N-3 fatty acids with longer chain length than ALA, i.e.

eicosapentaenoic (EPA, 20:5n-3), docosapentaenoic (DPA, 22:5n-3) and docosahexaenoic (DHA, 22:6n-3) acids are often referred as marine fatty acids, indicating that their source in diet is largely of marine orgin, e.g. fish. Furthermore, the unsaturated fatty acids can be divided into trans- or cis-fatty acids depending on the isomer structure of the double bonds. Cis-fatty acids are the more commonly occurring fatty acid type in the nature.

Fatty acids can be divided according to their chain length. Fatty acids with an odd- numbered chain length are thought not to be de novo synthesized in the human body, but instead are produced by bacteria in the gut of ruminant animals (22). Fatty acids are also sometimes referred as short-chain fatty acids (< 7 carbon atoms), medium-chain fatty acids (>6 carbon atoms), long-chain fatty acids (>12 carbon atoms) and very-long-chain fatty acids (>20 carbons). Most fatty acids are straight chain fatty acids, but fatty acids with branched chains also exist in humans (23).

Fatty acids occur as free fatty acids in the body, but they are more often incorporated into other molecules. The most common of these are glycerol, making up triacylglycerides (TG); phosphate, making up very diverse types of phospholipids (PL); and cholesterol, forming cholesteryl esters (CE). Fatty acids bind also to other organic molecules, such as carbohydrates.

(28)

Figure 1. An example of a fatty acid. The trivial name is oleic acid (18:1n-9). Systematic name is (cis-9-)octadecenoic acid. Sometimes the abbreviation OA is used.

The nomenclature of fatty acids is diverse. Both trivial names and systematic names are used. Besides, some fatty acids are often referred to by their abbreviations, such as EPA.

The structure of fatty acid is expressed as number of carbon atoms (e.g. 22), number of double bonds in the hydrocarbon chain (:5) and the first double bond from the methyl end (n-3). Figure 1 shows another example of a common unsaturated cis-fatty acid, oleic acid (18:1n-9) that has a chain length of 18 carbons and one double bond at the n-9 position in the cis conformation.

2.1.2 Dietary sources and absorption

Men consume on average roughly 90 g and women 70 g of fat daily in Finland, totaling slightly over a third of daily energy intake (24). Some common sources of dietary fat are listed in Table 1. Dairy products and red meat are examples of food products that contain mostly SFA and monounsaturated fatty acids (MUFA) and only small amounts of polyunsaturated fatty acids (PUFA). Most of the trans-fatty acids are derived from dairy and meat products in Finland (25). Fish and chicken products have somewhat different fatty acid composition compared with beef and pork (Table 1). Eggs and fish contain mainly unsaturated fatty acids. The marine products, such as fish and fish oil, are the main sources of EPA and DHA. Nuts and seeds are also good sources of unsaturated fatty acids, and for example peanuts and sunflower seeds are high in essential fatty acid, LA. Rapeseed oil (Canola oil) is a commonly used cooking oil in Finland and in addition to LA, it has an especially high content of the other essential fatty acid, ALA, unlike other commonly used vegetable oils. Olive oil is a good source of LA and MUFA, especially oleic acid (18:1n-9).

Fat absorption takes place in the small intestine as reviewed in (26) and summarized here. First, fat is emulsified by bile acids and lecithin secreted by the liver. As the surface area of the fat increases, triglycerides and other fat molecules are hydrolyzed by enzymes called lipases, which are mainly secreted by the pancreas. Thus, free fatty acids and monoglycerides are produced. Then the free fatty acids and monoglycerides in combination with bile acids form very small micelles, which transport the fatty acids to epithelial cells.

Fatty acids with short chain lengths (≤12 carbon atoms), which are more water soluble, are then absorbed directly into the portal blood and transported to the liver. Monoglycerides and long-chain fatty acids are instead taken up by endoplasmic reticulum of enterocytes and recombined into triglycerides. Triglycerides are then packed into lipoproteins called chylomicrons and secreted into lymphatic vessels. These vessels combine into the thoracic

(29)

duct that ends at the subclavian vein and finally releases the chylomicrons to circulating blood.

2.1.3 Fatty acid metabolism

The main events in fatty acid metabolism after absorption are summarized in this paragraph as reviewed in (28). After the chylomicrons have entered the circulating blood, an enzyme called lipoprotein lipase, located in the endothelial cells of capillaries, hydrolyzes triglycerides into free fatty acids. Fatty acids are also transported from the liver by another type of lipoprotein, very low-density lipoprotein (VLDL). In adipocytes, the free fatty acids are again combined into triglycerides and stored as lipid droplets. In muscle cells, however, the fatty acids are primarily oxidized for energy. Fatty acids are activated by coenzyme A (CoA) and are then β-oxidized inside mitochondria. Each round of β-oxidation shortens the fatty acids by two carbons, and acetyl-CoA, NADH and FADH2 are produced.

These are used in the citric acid cycle and finally in the respiratory chain to produce energy, i.e. ATP and heat. Unlike other fatty acids, very-long chain fatty acids are β-oxidized and branched fatty acids α-oxidized inside peroxisomes. While a person is fasting and fatty

Table 1. Examples of dietary sources of fatty acids.

Minced meat (pork

beef) and Butter Egg Salmon Fish oil

capsule* Peanuts

Rape- seed

oil Olive oil Typical portion size

(g) 80-230 3-10 60 100-215 1-3 20-100 10-40 10-40

/100 g of product

Energy (kcal) 221 727 143 195 760 568 884 884

Fatty acids (g) 13.5 76.7 6.6 11.9 72.6 35.1 98.4 93.3 Saturated fatty

acids (g) 5.6 52.8 2.1 2.5 5.1 6.3 5.7 14.0

Monounsaturated

fatty acids (g) 6.1 19.4 3.3 4.5 16.1 17.2 59.6 68.4 N-3 fatty acids (g) 0.2 0.4 0.2 4.2 50.3 <0.1 10.9 0.5 N-6 fatty acids (g) 0.9 1.3 1.0 0.7 1.1 11.6 22.1 10.4 Trans fatty acids

(g) 0.2 2.0 0 0 - 0 0 0

Linoleic acid,

18:2n-6 (g) 0.9 1.0 0.9 0.5 - 11.6 22.1 10.4

Alpha-linolenic

acid, 18:3n-3 (g) 0.1 0.4 0.1 0.3 - <0.1 10.9 0.5

EPA, 20:5n-3 (g) <0.1 0 0 0.8 26.3 0 0 0

DHA, 22:6n-3 (g) <0.1 0 0.1 2.3 18.2 0 0 0

Portion sizes and amounts of fatty acids and energy (/100 g) are based on Fineli® food composition database (27).

*Based on a label of a common commercial supplement available in Finnish grocery stores.

EPA, Eicosapentaenoic acid; DHA, docosahexaenoic acid.

(30)

acids are the primary source of energy, the liver converts the fatty acids into ketone bodies that are used as energy in extrahepatic tissues.

Fatty acids are also de novo synthesized in the human body, especially by the liver, as reviewed in (21) and summarized in the following. Acetyl-CoA is used to produce malonyl- CoA that is the substrate for fatty acid synthesis. The synthesis is catalyzed by an enzyme system called fatty acid synthase. This system is located in cytosol and it lengthens the fatty acid chain by two carbons in each cycle. The end product of fatty acid synthase is mainly palmitic acid (16:0). Palmitic acid in turn may be elongated or desaturated and used to produce phospholipids and triglycerides in the endoplasmic reticulum.

The processes related to modifying the fatty acids by elongation and desaturation are pivotal concerning this thesis. Figure 2 presents the outline of the main fatty acid modifications in human body adapted from several sources (29-34). Saturated and monounsaturated fatty acids can be de novo synthesized and modified in the human body (Figure 2A). N-3 and n-6 fatty acids can only be synthesized from the essential fatty acids ALA and LA, or they have to be acquired from the diet (Figure 2B).

After synthesis or absorption, fatty acids are elongated by a group of enzymes, of which the rate-limiting enzymes are called elongases (30). In humans seven different elongases are known with varying specificity for different fatty acid substrates (30). Addition of double bonds to fatty acids is catalyzed by three different types of desaturases: stearoyl-CoA desaturase-1 (SCD1), Δ5 desaturase (D5D) and Δ6 desaturase (D6D). SCD1 acts on saturated fatty acids and D5D and D6D on PUFA as shown in the Figure 2. Production of EPA, DPA and DHA from ALA is lower in men than in women and is also down-regulated by intake of EPA and DHA (35-37). The activity of SCD1 is also upregulated by a diet high in SFA in comparison with a diet high in MUFA (38). In addition to what is shown in the Figure 2, DHA can be retroconverted into EPA and DPA in the human body, even though this seems to happen in only minor quantities (39).

Besides being added to other organic molecules to produce e.g. triacylglycerides or phospholipids, certain PUFA are used as substrates by enzymes called cyclooxygenases and lipoxygenases to produce eicosanoids and docosanoids (Figure 2B). These products, such as prostaglandins and leukotrienes, act as paracrine mediators in processes such as inflammation and thrombosis (33).

(31)

Figure 2. An outline of the major modifications of fatty acids in the human body as adapted from several sources (29-34). Panel A presents the endogenous pathway, and panel B the pathways starting from the essential fatty acids, ALA and LA, i.e. the modification of n-3 and n- 6 fatty acids. FADS1 and FADS2 are the genes encoding D5D and D6D.

(32)

2.1.4 Impact of genetic variations on desaturation

D5D and D6D enzymes are encoded by the FADS1 and FADS2 genes, respectively, located in the FADS gene cluster in chromosome 11. In 2006, Schaeffer et al. showed that polymorphisms in FADS1 and FADS2 genes are strongly related to fatty acid composition in serum (34). There are several common (minor allele frequency > 5%) single nucleotide polymorphisms (SNP) in FADS gene cluster that associate with levels of circulating PUFA, and many of these SNP, located relatively close together, are in linkage disequilibrium, i.e.

they represent similar genetic variation of this genetic region. Most of the minor alleles of these SNP associate with increased levels of substrates of the desaturase enzymes and decreased levels of products, especially arachidonic acid (ARA, 20:4n-6) (34,40,41). These associations are indeed in accordance with the fatty acid metabolism pathway presented in Figure 2B and suggest especially decreased activity of D5D and also D6D caused by these variations. Although the exact mechanisms and functional SNP are still unknown, SNP in the FADS region associate with decreased hepatic expression of FADS1 and its protein product in human liver, which is expected to lead to decreased D5D activity (42). One example of a common SNP in FADS region is the intron variant of FADS1 gene, rs174550, which is strongly associated with fatty acid composition in erythrocytes, and is also, as explained above, associated with decreased activities of D5D and D6D (43).

SCD1 is encoded by the SCD gene on chromosome 10. There are also several SNP in the SCD gene, but their effect on SCD1 activity is unclear and probably low (44-46).

2.2 BIOMARKERS OF DIETARY FAT

2.2.1 Measurement and levels of fatty acids in different tissues

Gas chromatography is the prevailing method used to quantify fatty acid levels in tissues.

Besides gas chromatography, other methods such as nuclear magnetic resonance or mass spectroscopy are sometimes used (47,48). Fatty acids can and have been measured in a myriad of tissues, e.g. leukocytes, thrombocytes, erythrocytes, plasma, adipocytes, sperm, milk, skin, muscle and liver (20).

Lipid fractions are often, but not always, separated before the fatty acid quantification, for example in plasma, to separate PL, TG, CE and free fatty acids, chromatographic methods such as solid phase or thin layer chromatography are used (49). Before gas chromatography, the fatty acids need to be separated from the lipid molecules. This is done by a process called trans-esterification that produces fatty acid methyl esters. In gas chromatography, the fatty acids are separated by their retention times and can be quantified by their respective peak areas by flame ionization detectors. Although gas chromatography produces quantifiable results (mol/l) of each detected fatty acid when an internal standard is added, the common practice is to report fatty acids relative to all fatty acids, i.e. as weight or molar percentage.

The fatty acid composition of different tissues and lipid types greatly vary, and fatty acid composition of different tissues is extensively presented in (20). As an example of an important difference, CE contain abundantly LA (~50 mol%), due to the specificity of lecithin-cholesterol acyl transferase for this fatty acid, but contains very little of the n-3 fatty acids EPA, DPA and DHA (20). Other good examples are the very long-chain fatty acids, such as nervonic (24:1n-9) and lignoceric (24:0) acids, which are almost solely found in a type of PL called sphingolipids due to the action of enzymes called ceramide synthases (30).

(33)

In TG oleic acid (18:1n-9) is the predominant fatty acid and usually totals over one third of all fatty acids in TG (20). Despite these differences, most individual circulating fatty acids correlate with between different sites of measurement moderately or strongly (20,50). Of circulating fatty acids, erythrocyte membrane (EM) fatty acids are especially interesting to study, since erythrocytes, unlike thrombocytes, white blood cells and lipoproteins, have a long three- to four-month lifespan, and mature erythrocytes also lack most of the capability for fatty acid metabolism (51,52). Some examples of fatty acid distributions for circulating fatty acids in total serum, EM and plasma CE, PL and TG can also be found in chapter 5.1.

2.2.2 Circulating biomarkers of fat intake

It was shown already over 50 years ago that circulating levels of fatty acids respond to dietary changes (53,54). Nowadays, circulating fatty acids in EM and plasma are commonly used in the validation of dietary questionnaires, as measures of adherence in intervention trials and in the assessment of dietary fat type, whereas platelets and white blood cells are less often used (20). Certain fatty acid concentrations in blood can be considered as objective estimates of dietary intake of fat. Dietary questionnaires can be biased by the behavior of the responder and investigator. Furthermore, dietary questionnaires are somewhat laborious to fill in, performing them in large scale studies is time consuming, and the accuracy of databases for calculating fatty acid composition in different foods may be limited. As peripheral blood is commonly drawn in studies, measurement of fatty acids in plasma or erythrocytes is a good alternative to gain objective information on fat intake.

However, the individual fatty acid levels in blood need to be carefully interpreted, because, in addition to the dietary intake, endogenous fatty acid metabolism contributes to circulating fatty acid composition (see 2.1).

Circulating biomarkers of fat intake are most appropriate when estimating intake of EPA and DHA, and the correlations between dietary questionnaires and circulating fatty acids are usually in the range r=~0.4-0.6 (55). The sum of EPA+DHA, also known as the omega-3 index, increases in response to n-3 supplementation, and a low omega-3 index has been proposed to act as a risk predictor for coronary heart disease (56). This index also correlates strongly (r=0.9) with levels of EPA, DPA and DHA in whole blood and plasma PL (56). A randomized controlled trial supplementing varying amounts of EPA+DHA in healthy men and women showed that the dose explained almost 70% of total variation in the change of erythrocyte EPA+DHA, and the second strongest predictor was the baseline omega-3 index (5% of variation) (57). Both fish oil and DPA supplementations increase levels of circulating DPA notably (58,59). At least concerning EPA and DHA, erythrocytes seem to be more stable in their fatty acid composition and reflect the intake of EPA and DHA over longer period of time (months) than plasma (days-weeks) (60-63). This difference is supposed to be related to the three- to four-month life cycle of erythrocytes and their lack of capability for fatty acid synthesis. Although this makes erythrocytes more useful in long-term studies, on the other hand, EM fatty acid composition cannot be used in very short-term studies. The half-life of EPA in the different plasma fractions PL, TG and CE is in the order of days (60,64).

As the only source of the essential fatty acids ALA and LA is diet, it is not surprising that their circulating levels can be used as biomarker. For LA, the correlations between dietary questionnaires range usually between r=~0.2-0.7 and for ALA r=~0.1-0.4, and their circulating levels increase after their intake is increased (20). Both plasma (free fatty acids,

(34)

PL, CE and TG) and erythrocytes (total PL and phosphatidyl choline (PC)) seem to reflect increased intake of LA over a similar time period (days-weeks) (65). Furthermore, it is known that circulating levels of ARA change significantly even after low-dose ARA supplementation (66).

Some of the circulating saturated fatty acids can also be used to reflect SFA intake.

Myristic (14:0), pentadecanoic (15:0) and heptadecanoic (17:0) acid seem to reflect saturated fat intake especially from dairy products (67,68). However, pentadecanoic and heptadecanoic acids are also found in many other food products such as fish and meat and concerns have been raised about their use as biomarkers of dairy fat (69). Palmitic acid also seems to correlate with its estimated intake to small extent (20), but it has to be remembered that this fatty acid is the main product of fatty acid synthase. In one study pentadecanoic acid in plasma and erythrocytes seemed to reflect changes in SFA intake over similar period of time (days-weeks) (65).

Circulating MUFA are not generally used as biomarker of dietary fat. However, in populations with high intake of olive oil, oleic acid (18:1n-9) acts as a biomarker for oleic acid intake (70).

Circulating trans-fatty acids, which are not produced in the human body, are sometimes used to reflect their intake. Trans fatty acids, such as trans-elaidic (trans-18:1n-9) and trans- palmitoleic (trans-16:1n-7), reflect their intake from partly hydrogenated fats, dairy products and beef that contain some (Table 1) trans-fatty acids (70,71).

2.2.3 Estimating enzyme activities by biomarkers of dietary fat

Product-to-precursor ratios are commonly used in studies to estimate enzyme activities related to endogenous synthesis and modification of fatty acids presented in Figure 2.

Particularly, estimated enzyme activities are used for the three desaturase enzymes, SCD1, D5D and D6D, e.g. (72,73). The validation evidence for desaturase indices, however, is still somewhat limited. SCD1 is estimated by the ratio of 16:1n-7/16:0 or 18:1n-9/18:0. In a study in which SCD mRNA expression and estimated activity were measured in adipose tissue, both ratios 16:1n-7/16:0 and 18:1n-9/18:0 correlated with the mRNA expression (74).

However, in another study in which SCD expression was measured in liver, plasma and liver, the ratio of 16:1n-7/16:0 reflected mRNA expression better than the ratio of 18:1n- 9/18:0. The 16:1n-7/16:0 ratio may therefore be a more suitable index, possibly because it is less affected by diet (75). D5D and D6D are usually estimated by ratios of 20:4n-6/20:3n-6 and 18:3n-6/18:2n-6, respectively. Sometimes in phospholipid fractions also the ratio of 20:3n-6/18:2n-6 is used for D6D, because the amount of 18:3n-6 in PL is low. However, there is currently little evidence that these indices are associated with liver or adipose tissue mRNA expression of FADS1 or FADS2 (74,75). Still one study found that in children D6D activity is associated with mRNA expression of both FADS1 and FADS2 in peripheral blood (76). In another study a common FADS1 SNP was clearly associated with both D5D and D6D indices measured in erythrocytes (77), providing some validation for the use of these indices.

Besides estimated desaturase activities, indices for de novo lipogenesis and elongase activity are used. Because LA is not synthetized in the human body and palmitic acid (16:0) is the main product of de novo lipogenesis, the ratio of these two, 16:0/18:2n-6 is used to reflect de novo lipogenesis. In plasma TG this index is increased when hepatic de novo lipogenesis is induced by a high-carbohydrate diet, and this ratio correlates with hepatic

(35)

mRNA expression of genes related to de novo lipogenesis (75,78,79). Still, this index does not distinguish between de novo lipogenesis and diets high in palmitic acid and low in LA.

Elongase activities can be estimated for example by ratios of 18:0/16:0, 18:1n-7/16:1n-7 and 22:4n-6/20:4n-6, but there is little evidence for validity of these indices (75).

2.2.4 Gene-diet interactions when studying biomarkers of dietary fat

It is possible that besides the direct effects of individual genes and diet on circulating fatty acid composition, their combined effect may modulate fatty acid composition differently, i.e. a genotype may increase or decrease the effect of diet on circulating fatty acids. A number of recent studies have investigated gene-diet interactions of fat intake and FADS1 and FADS2 polymorphisms for circulating biomarkers of dietary fat both in intervention and observational settings (77,80-87). The data, however, is still quite limited to draw firm conclusions about the gene-diet interactions. The studies tend to suggest that intake of ALA interacts with variants in FADS cluster for circulating EPA (82,84,86). Secondly, it is possible that intake of PUFA from marine sources, may interact with FADS SNP for D5D and D6D activities and related fatty acid products (80,81,83,85,87). Interestingly, the direction of interaction may differ depending in which tissue fatty acids are measured (80,83,84), which may suggest that the nature of these interactions is very complex or that some of the observed interactions have arisen by chance.

Two studies have also investigated if the relationship between diet and circulating SCD1 indices is modified by SNP in SCD. One study found that the type of dietary oil (olive oil vs. sunflower oil) and SCD variants may have an interaction with the SCD1 index based on 18:1n-9/18:0, but no interaction with 16:1n-7/16:0 was found (46). Another study, in which fish oil was supplemented, found no gene-diet interaction for either of SCD1 indices (88).

In light of the aforementioned, it is clear that more research is needed to understand the nature of gene-diet interactions, since these interactions may play a part in diet-disease and gene-disease relationships and may also affect how the biomarkers of dietary fat should be applied in further studies.

2.3 CIRCULATING BIOMARKERS OF DIETARY FAT AND LOW-GRADE INFLAMMATION

Low-grade inflammation is a phenomenon in which concentrations of inflammatory markers, such as C-reactive protein (CRP), are mildly increased without existence of actual infectious or inflammatory disease. Low-grade inflammation is also linked to progression of atherosclerosis and T2D (14,16). Higher low-grade inflammation is related to other common risk factors of chronic diseases, such as aging, smoking, obesity, high waist circumference and low physical activity (89).

An extensive literature review has indicated that many dietary components, including fatty acids, could play a role in low-grade inflammation and suggested that trans- and saturated fatty acids are proinflammatory and especially marine n-3 fatty acids are anti- inflammatory (90). Furthermore, n-6 fatty acids are often referred to as pro-inflammatory fatty acids because the eicosanoid products of ARA have often stronger effects on inflammation than the eicosanoid products of EPA (33). In contrast, n-3 fatty acids are commonly considered to be anti-inflammatory fatty acids because they replace and

(36)

compete for enzymatic pathways with ARA in cells and give rise to anti-inflammatory products such as resolvins (33).

There is no clear view which of the inflammatory markers should be used to describe low-grade inflammation, and a myriad number of markers of various types have been investigated (90). CRP is one of the most commonly reported markers of inflammation. It is an acute-phase protein secreted from liver and acts in the complement system. It seems to have no causal role, however, and thus serves solely a marker of low-grade inflammation (91). Interleukin-6 (IL-6) and adiponectin concentrations are also very commonly reported (Table 2). IL-6 is a pro-inflammatory cytokine secreted by leukocytes and adipocytes and probably has a causal role at least in CVD (92,93). Adiponectin on the other hand, is an anti- inflammatory adipokine secreted by adipocytes and also, for example, decreases glucose production in liver and is associated with decreased risk of T2D (94,95). Interleukin-1 receptor antagonist (IL-1Ra) has so far been used less often (Table 2), but was included for this thesis. IL-1Ra is an anti-inflammatory cytokine that blocks interleukin-1 signaling.

However, it has proinflammatory associations and is increased for example in T2D and non-alcoholic steatohepatitis probably since it is secreted by liver and adipose tissue in pro- inflammatory situations to impede the effect of interleukin-1β (90,96,97).

Many observational studies in humans have investigated the associations of biomarkers of dietary fat with a number of circulating markers of inflammation. Due to high number of studies and inflammatory markers, the overview of studies in Table 2 is focused only on circulating biomarkers of dietary fat and their associations with selected important markers of low-grade inflammation, i.e. with concentrations of pro-inflammatory CRP, IL1-Ra and IL-6 and with concentrations of anti-inflammatory adiponectin. The associations of circulating fatty acids with markers of low-grade inflammation were generally weak in these studies, and the studies often reported only some of the measured fatty acids (Table 2).

Saturated fatty acids were generally associated with higher concentrations of inflammatory markers (Table 2). Of individual saturated fatty acids, myristic acid (14:0) tends to be associated with higher low-grade inflammation, whereas for longer individual SFA the associations are not clear and associations to both directions have been observed.

For total circulating MUFA the associations were equivocal (Table 2). Palmitoleic acid (16:1n-7) associates somewhat consistently with high low-grade inflammation. This may be explained by the fact that SCD1 activity also tends to associate with higher inflammation.

Accordingly, also oleic acid (18:1n-9) tended to associate with increased low-grade inflammation. For longer MUFA the data are insufficient. In contrast, one large-scale study found that trans-16:1n-7 was associated with lower CRP and IL-6 (71).

Total PUFA, n-3 and n-6 fatty acids were mainly associated with lower low-grade inflammation, even though the n-6/n-3-ratio tended to be associated with higher inflammation (Table 2). Of n-3 fatty acids, ALA does not show consistent association with low-grade inflammation, whereas EPA, DPA and DHA tend to be associated with lower inflammation. Of the n-6 fatty acids, LA seems to be associated with lower inflammation, and 18:3n-6 and dihomo-gamma-linolenic acid (20:3n-6, DGLA) seem to be pro- inflammatory. Neither ARA nor 22:4n-6 is consistently associated with low-grade inflammation. Only a few studies have investigated associations with estimated D5D and D6D. In those studies, it seems that associations with D5D are anti-inflammatory and with

(37)

D6D pro-inflammatory, which is in accordance with the associations of the individual n-6 fatty acids, i.e. LA, 18:3n-6, DGLA and ARA.

It is noteworthy that most of the reviewed studies are cross-sectional, and therefore provide little insight into whether the fatty acids affect inflammation or the other way around. Only study by Peterson et al. has investigated longitudinal associations (over 20 years) of circulating fatty acids with CRP in a population setting (98). This study found pro- inflammatory associations with palmitoleic and oleic acid and with SCD1 and D6D activities, but interestingly, also with EPA. Moreover, LA was associated with lower CRP.

Furthermore, three dietary intervention studies have investigated correlations between change in circulating n-6 and n-3 fatty acids and inflammatory markers, but found no associations (99-101).

Table 2. Overview of studies on the association of circulating fatty acids with markers of low-grade inflammation in adults in the order of publication year.

Ref Subjects

and setting* n (male%) Mean age,

years Fatty acid CRP IL-6 IL-1Ra Adipo- nectin (102) Coronary

angiography patients

269 (63.6%) 60 Granulocyte

EPA 0

Granulocyte

DPA 0

Granulocyte

DHA ↓

Granulocyte

LA 0

Granulocyte

18:3n-6 0

Granulocyte

ARA 0

(103) Spaniards 232 (58%) 40 Serum 14:0 0 0/↑

Serum 16:0 0 0/↑

Serum 18:0 0 0/↑

Serum 24:0 0 0/↑

Serum LA 0/↓ 0/↓

Serum EPA 0/↓ 0 Serum DHA 0/↓ 0 Serum SFA 0 0/↑

Serum Σn-6 0 0/↓

Serum Σn-3 0/↓ 0 (104) Healthy

Caucasians 116 (66%) 39 Plasma 14:0 0/↓

Plasma 16:0 ↓

Plasma

20:1n-9 ↓

Plasma SFA 0/↓

Plasma EPA 0

Plasma DHA 0

Plasma ALA 0

Plasma LA 0

Plasma

Others 0

(105) Italians 1,123 (45%) 68 Plasma LA 0 0 0/↓

Plasma ARA 0 ↓ ↓

Plasma ALA ↓ 0 ↓

Plasma EPA 0 ↓ 0/↓

Plasma DHA 0 ↓ ↓

Plasma Σn-3 0 ↓ ↓

Plasma Σn-6 0 0 ↓

Plasma Σn-

6/Σn-3 0 ↑ ↑ Continued on the next page.

(38)

Table 2. Continued.

years Fatty acid CRP IL-6 IL-1Ra Adipo- nectin (98) Swedish 50y

men, 20y longitudinal study

767 (100%) 50 until

70 CE 16:1n-7 ↑ CE 18:1n-9 ↑ CE 18:3n-6 0/↑

CE LA ↓

CE SCD1(-

16) ↑

CE D6D ↑

CE D5D 0

CE EPA 0/↑

CE Others 0 Cross-

sectional 320 (100%) 70

CE LA 0/↑

CE DGLA ↑

CE D5D 0/↓

CE D6D 0/↑

CE Others 0 (106) Female

nurses ~270 (0%) 60 EM EPA ↑ 0

EM DPA 0 0

EM DHA 0 0

Plasma EPA 0 0 Plasma DPA 0 ↓ Plasma DHA 0 0 (101) 12-wk

intervention (low

carbohydrate vs. low fat diet)

40 (50%) 18-55 PL ΔARA 0 0

(107) Coronary angiography patients

876 (77%) 60 EM ARA/LA ↑

(108) Patients with stable coronary heart disease

992 (82%) 67 EM EPA+DHA ↓ ↓

(109) 70 y old

Swedes 264 (56%) 70 CE 14:0 0 0

CE 16:0 0 0

CE 16:1n-7 ↑ 0

CE 18:0 0/↓ 0

CE 18:1n-9 0/↑ 0

CE LA ↓ 0

CE 18:3n-6 ↑ 0

CE ALA 0 0

CE DGLA 0/↑ 0

CE ARA 0 0

CE EPA 0 0

CE DHA 0 0

CE SCD1(-

16) ↑ 0

(110) English civil

servants 348 (100%) 79 PL SFA ↑

PL MUFA 0

PL PUFA ↓

Continued on the next page.

(39)

Table 2. Continued.

years Fatty acid CRP IL-6 IL-1Ra Adipo- nectin (111) Coronary

angiography patients

291 (63.6%) 60 Thrombocyte

EPA 0/↑

Thrombocyte

DHA 0

Granulocyte

EPA 0/↑

Granulocyte

DHA 0/↑

(112) Healthy

Australians 124 (37.1%) 48 Plasma Σn-3 ↓ Plasma EPA ↓ Plasma DPA ↓ (113) With stable

coronary heart disease

956 (82%) 67 Whole blood

EPA+DHA ↓ ↓

(114) Healthy

Greeks 374 (51%) 42 Plasma SFA ↑ ↑

Plasma MUFA ↓ 0/↓

Plasma PUFA ↓ ↓ Plasma Σn-3 ↓ ↓ Plasma Σn-6 ↓ ↓ Plasma Σn-

6/Σn-3 0/↑ 0/↑

Plasma LA ↓ 0/↓

Plasma ARA 0/↓ 0/↓

Plasma ALA ↓ 0/↓

Plasma EPA ↓ 0 Plasma DPA ↓ ↓ Plasma DHA ↓ 0/↓

Plasma

EPA+DHA 0/↓ 0 (71,115) US citizens 3,630 (45%) 75 PL 16:1n-7 0/↑

3,736 (45%) 75 PL

trans16:1n-7 0/↓

Female

nurses 327 (0%) 60 EM

trans16:1n-7 ↓ ↓ (116) Yup'ik

Eskimos 357 (41%) 45 EM EPA 0/↓ 0 0

EM DHA 0/↓ 0 0

(117) Middle-aged

Germans 1,980 (38%) 50 EM LA 0/↓ 0/↑

EM 18:3n-6 0/↑ 0/↓

EM DGLA ↑ 0/↓

EM ARA 0 0

EM 22:4n-6 0 0/↓

EM ALA 0 0/↑

EM EPA 0 0

EM DPA 0/↓ 0

EM DHA 0 0

Continued on the next page.

Circulating fatty acids : associations with diet, genetic variations, low-grade inflammation and type 2 diabetes

uef.fi

Dissertations in Health Sciences

MARKUS TAKKUNEN

CIRCULATING FATTY ACIDS – ASSOCIATIONS WITH DIET, GENETIC VARIATIONS, LOW-GRADE INFLAMMATION AND TYPE 2 DIABETES

MARKUS TAKKUNEN

Circulating fatty acids – associations with diet, genetic variations, low-grade

inflammation and type 2 diabetes

Circulating fatty acids – associations with diet, genetic variations, low-grade

inflammation and type 2 diabetes

Acknowledgements

List of the original publications

Contents

Abbreviations

1 Introduction

2 Review of the literature