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Healthy dietary patterns, lipids and inflammation in human randomized controlled trials Lena Leder

Dissertation for the degree of Philosophiae Doctor (Ph.D.)

Department of Nutrition Institute of Basic Medical Sciences University of Oslo 2016

© Lena Leder, 2017

Series of dissertations submitted to the Faculty of Medicine, University of Oslo

ISBN 978-82-8377-000-1 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard. Print production: Reprosentralen, University of Oslo.

Acknowledgements This work was carried out at the Department of Nutrition, Institute of Basic Medical Sciences at the University of Oslo, Norway from 2012 until 2016. The primary financial support was a four-year doctoral fellowship from the Institute of Basic Medical Sciences, University of Oslo, Norway. Prof. Dr. Kirsten Holven was my principal supervisor. Thank you for providing an incredible environment for conducting my PhD in your expert supervision. The scientific guidance, critical questions, support, sharing extensive knowledge and never-ending optimism were greatly appreciated. Your positive and kind attitude was inspiring and always helped me to get back on track. With the same gratitude I want to thank my co-supervisor Prof. Dr. Stine Ulven for sharing your invaluable knowledge, your patience, and encouragement means a lot to me. Thank you for taking this role and supporting me wherever and whenever you could. Thank you to the SYSDIET consortium for allowing access to the study material and to all the people participating in the SYSDIET study. My special gratitude I want to express to Marjukka Kolehmainen for her tremendous help with the SYSDIET paper. Thank you for your inspiring suggestions, great ideas and very fruit-full discussions. Thank you to the NoMa study team at UiO, HiOA and Mills DA for making this study possible. I express my gratitude to all people participating in the NoMa study. All my colleagues formed an extremely friendly work environment that allowed to discuss ideas as well as critical issues. I am very grateful to Inger Ottestad, Gyrd Omholt Gjevestad, Ingunn Narverud, Jacob Juel Christensen, Patrik Hansson, Amanda Rundblad, Mari Myhrstad and Vibeke Telle-Hansen for the i

good and productive times in seminars, in our “Kollokvie”, in the lunch breaks, at conferences, at dinners, while running and cycling. A great thank you to Kristin Eckardt, Christin Zwafink and Rikke Nørgaard. Our discussions about lab methods, teaching and life in general were highly appreciated. Thank you to all who proofread my thesis and gave invaluable comments. Thank you to Marit Sandvik, Navida Akhter Sheikh and Ellen Raael for their expertise and technical support in the lab. Thank you to Anine Medin and Susanne Strohmaier for our master-mind group. It was extremely inspiring, funny, crazy, critical and knowledgeable. I hope we keep in touch and keep it running in one way or the other! I want to express my gratitude to Carina Knudsen for her creative support with the figures in my thesis and to Magne Thoresen for all the biostatistical support during my PhD. Deep gratitude goes to my dear family. My parents-in-law Beatrix and Reinhard Leder always made it possible to help out even though there are 1500km between us. You are incredible. A huge thank you for in-depth revision of the language in my thesis. My parents Beate and Oskar Lückel have always encouraged and believed in me whatever I have been up to; no matter if it was crazy, well-considered or just for fun. Thank you for your unfailing love and your never-ending support. My deepest thank you I want to express to my beloved husband, Felix Leder, and my children, Finus and Nuka. I am deeply thankful for your motivation and inspiration, and that you always believed in me. Finus and Nuka are my sunshines showing me what really matters in life. I would not have made it as far without the three of you. You guys rock!

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Abstract Healthy dietary patterns have been subject of considerable attention in recent years. A healthy Nordic diet may improve cardiovascular risk factors and thereby prevent cardiovascular diseases. In order to reduce plasma cholesterol and disease risk, one central aspect of the healthy Nordic diet is the combination of reduced dietary intake of saturated fatty acids and increased dietary intake of polyunsaturated fatty acids. However, although the effect of dietary fat quality on plasma cholesterol concentration is well established, the effects and mechanisms of whole diets on plasma lipids and inflammation are less examined. Therefore, we aimed to investigate the role of healthy dietary patterns on lipids and inflammation with special focus on fat quality in populations with cardiometabolic risk. Two randomized controlled dietary intervention studies were included in this thesis. In an eight-week double-blinded study, healthy adults aged 25-70 years with moderate hypercholesterolemia were assigned to an experimental diet or a control diet. The experimental diet group received commercially available food items in which saturated fat was replaced by vegetable sunflower and rapeseed oil. The control diet group received similar commercial food items with a higher content of saturated fat and lower content of polyunsaturated fat. In an 18-24 week, Nordic multi-center study, subjects between 30-65 years with features of metabolic syndrome were assigned to follow a healthy Nordic diet or an isocaloric control diet. An oral glucose tolerance test was performed at baseline and at the end of the study. Exchanging food products with improved fat quality reduced total- and LDLcholesterol by 9% and 11%, respectively, and increased the serum levels of bile acid, but we did not detect an effect on circulating inflammatory markers. iii

The cholesterol-lowering effect observed seemed to be induced by a change in mRNA expression of the LDL receptor, potentially leading to increased cholesterol in the cell, increasing mRNA expression of liver X receptor alpha (LXRA) and LXRA target genes in peripheral blood mononuclear cells (PBMCs). The increase in serum bile acid may reflect increased LXRA activity in liver, and thus our data confirm that changes in gene expression in PBMCs reflect changes in hepatic lipid metabolism, as has been shown by others. A long-term healthy Nordic diet modified the expression of genes involved in inflammation and lipid metabolism in PBMCs after a 2h oral glucose tolerance test (OGTT) in individuals at risk of metabolic diseases. In conclusion, a healthy Nordic diet and an improvement of the fat quality, as part of a healthy dietary pattern, has positive effects on a variety of markers of cardiovascular diseases and on the transcription of genes involved in lipid metabolism and inflammation.

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List of papers Paper I Ulven SM, Leder L, Elind E, Ottestad I, Christensen JJ, Telle-Hansen VH, Skjetne AJ, Raael E, Sheikh NA, Holck M, Torvik K, Lamglait A, Thyholt K, Byfuglien MG, Granlund L, Andersen LF and Holven KB: Exchanging few commercially regular-consumed food items with improved fat quality reduces total and LDL cholesterol– a double-blind randomized controlled trial. British Journal of Nutrition. In press. Paper II Leder L, Ulven SM,Ottestad I, Christensen JJ, Telle-Hansen VH, Granlund L, Andersen LF and Holven KB: Replacement of SFAs with PUFAs increases the excretion of bile acids and up-regulates the mRNA expression level of the LDL receptor and LXR alpha in peripheral blood mononuclear cells: a double-blind randomized controlled trial. Submitted. Paper III Leder L, Kolehmainen M, Narverud I, Dahlman I, Myhrstad MCW, de Mello VD, Paananen J, Carlberg C, Schwab U, Herzig K-H, Cloetens L, Ulmius Storm M, Hukkanen J, Savolainen MJ, Rosqvist F, Hermansen K, Dragsted LO, Gunnarsdottir I, Thorsdottir I, Risérus U, Åkesson B, Thoresen M, Arner P, Poutanen KS, Uusitupa M, Holven KB and Ulven SM: Effects of a healthy Nordic diet on gene expression changes in peripheral blood mononuclear cells in response to an oral glucose tolerance test in subjects with metabolic syndrome: a SYSDIET substudy. Genes & Nutrition 2016 Mar 17;11:3.

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Abbreviations AA

arachidonic acid

cDNA

complementary DNA

ABCA1

ATP binding cassette subfamily A member 1

CE

cholesteryl ester

ABCG1

ATP binding cassette subfamily G member 1

CHD

coronary heart disease

ChREBP

ACAT

acyl CoA cholesterol acyltransferase

carbohydrate regulatory element-binding protein

CPT1A

carnitine palmitoyltransferase 1A

CPT1B

carnitine palmitoyltransferase 1B

CRAT

carnitine O-acetyltransferase

CRP

C-reactive protein

CVD

cardiovascular disease

CXCR2

C-X-C motif chemokine receptor 2

CYP27A1

cytochrome P450 family 27 subfamily A member 1

ADRB2

adrenoceptor beta 2

ADRB2

adrenoceptor beta 2

ALA

α-linolenic acid

ALOX5AP arachidonate 5-lipoxygenase activating protein apoB100

apolipoprotein B100

ARHGAP15 Rho GTPase activating protein 15 BMI

body mass index

Cyp7a1

cholesterol 7 alpha-hydroxylase

CCL

C-C motif chemokine ligand

DGLA

dihomo-c-linolenic acid

CCR2

C-C motif chemokine receptor 2

DHA

docosahexaenoic acid

CD14

CD14 molecule

DNA

deoxyribonucleic acid

CD19

CD19 molecule

DPA

docosapentaenoic acid

CD36

CD36 molecule

E%

percent of energy

CD40LG

CD40 ligand

EPA

eicosapentaenoic acid

CD72

CD72 molecule

ER

endoplasmatic reticulum

vii

ERK

extracellular signal-regulated kinase

FASN

fatty acid synthase

FXR

farnesoid X receptor

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

GPR

G protein-coupled receptor

HBEGF

heparin binding EGF like growth factor

HDL-C

IL23A

interleukin 23 subunit alpha

IL23R

interleukin 23 receptor

IL7R

interleukin 7 receptor

INSIG

insulin induced gene

L2HGDH L-2-hydroxyglutarate dehydrogenase LA

linoleic acid

LACTB

lactamase beta

HDL cholesterol

LDL

low density lipoprotein

HETE

hydroxyeicosatetraenoic acid

LDL-C

LDL cholesterol

HIF1A

hypoxia inducible factor 1 alpha subunit

LDLR

LDL receptor

LPAR2

lysophosphatidic acid receptor 2

HMGCR

HMG-CoA reductase hydroxyoctadecadienoic acid

LPS

lipopolysaccharide

HODE

hypoxanthine phosphoribosyltransferase 1

LTA4H

leukotriene A4 hydrolase

HPRT1

LTB4

leukotriene B4

hs-CRP

high-sensitive CRP

LXR

liver X receptor

HSPA5

heat shock protein family A (Hsp70) member 5

LXRA

liver X receptor alpha

LXRE

LXR response element

LY96

lymphocyte antigen 96

MAPK8

mitogen-activated protein kinase 8

ICAM1

intercellular adhesion molecule 1

IFN

interferon

IFNG

interferon gamma

MetS

metabolic syndrome

IGHD

immunoglobulin heavy constant delta

MIF

macrophage migration inhibitory factor

IKBKB

inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta

MLX

max-like factor X

MMD

IL

interleukin

monocyte to macrophage differentiation associated

IL1B

interleukin 1 beta

MMP

matrix metalloproteinase

IL1RN

interleukin 1 receptor antagonist

mRNA

messenger RNA

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MUFA

monounsaturated fatty acid

PPARG

peroxisome proliferator activated receptor gamma

NAGPA

N-acetylglucosamine-1phosphodiester alpha-N-acetylglucosaminidase

PPRE

PPAR response element

NFkB

nuclear factor kappa B

PREDIMED Primary Prevention of Cardiovascular Disease

NFKBIA

NFkB inhibitor alpha

PUFA

polyunsaturated fatty acid

NK

natural killer

qPCR

real-time quantitative polymerase chain reaction

OGTT

oral glucose tolerance test

RCT

randomized controlled trial

OLR1

oxidized low density lipoprotein receptor

RELA

RELA proto-oncogene, NF-kB subunit

OLTT

oral lipid tolerance test

RIN

RNA integrity number

PBMC

peripheral blood mononuclear cell

RIPK1

receptor interacting serine/threonine kinase 1

PCSK9

proprotein convertase subtilisin kexin type 9

RNA

ribonucleic acid

PDGFA

platelet derived growth factor subunit A

ROS

reactive oxygen species

RSAD2

platelet derived growth factor subunit B

radical S-adenosyl methionine domain containing 2

RXR

retinoid X receptor

PDK4

pyruvate dehydrogenase kinase 4

SELP

selectin P

PEAR1

platelet endothelial aggregation receptor 1

SFA

saturated fatty acid

PGJ2

prostaglandin J2

sICAM1

soluble intercellular adhesion molecule 1

PL

phospholipid

SLC22A5

solute carrier family 22 member 5

PLIN2

perilipin 2

POLK

DNA polymerase kappa

PPAR

peroxisome proliferator activated receptor

PDGFB

SLC25A20 solute carrier family 25 member 20 SR

scavenger receptor

SREBP

sterol regulatory binding protein

PPARA

peroxisome proliferator activated receptor alpha

sTNFR

soluble tumor necrosis factor receptor

PPARD

peroxisome proliferator activated receptor delta

sVCAM1

soluble vascular cell adhesion molecule 1

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T2DM

diabetes type 2

TAB2

TGF-beta activated kinase 1/MAP3K7 binding protein 2

TNFRSF12A tumor necrosis factor receptor superfamily member 12A TNFSF10

tumor necrosis factor superfamily member 10

total-C

total cholesterol

TBP

TATA box binding protein

TG

triglyceride

UCP2

uncoupling protein 2

TGFB2

transforming growth factor beta 2

VCAM1

vascular cell adhesion molecule 1

Th

T helper

VEGFB

TLR

Toll-like receptor

vascular endothelial growth factor B

XBP1

X-box binding protein 1

TNF

tumor necrosis factor

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Contents Acknowledgements

i

Abstract

iii

List of Papers

v

Abbreviations

vii

Contents

xi

1

1 1 4 5 7 10 10 12 12 13 16 17

2

Introduction 1.1 Dietary patterns and cardiometabolic risk . . . . . . 1.2 Fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Dietary fat, lipids and cardiovascular risk . . . . . . 1.4 LDL cholesterol metabolism . . . . . . . . . . . . . . 1.5 Atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . 1.5.1 Atherosclerotic process . . . . . . . . . . . . . 1.5.2 Inflammation . . . . . . . . . . . . . . . . . . . 1.6 Dietary fat, inflammation and cardiovascular risk . 1.7 Fatty acids and gene regulation . . . . . . . . . . . . . 1.8 Peripheral blood mononuclear cells . . . . . . . . . . 1.9 Use of challenge tests in interventions . . . . . . . . Aims

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CONTENTS 3

Subjects and methods 3.1 The NoMa study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 The SYSDIET study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Selection of candidate genes . . . . . . . . . . . . . . . . . . . . . . . .

23 23 24 27

4

Summary of results

31

5

Discussion 5.1 Methodological considerations . . . . . . . . . . . . . . . . . . . . . . 5.1.1 Study design of intervention studies . . . . . . . . . . . . . 5.1.2 Gene expression in peripheral blood mononuclear cells as a model system in intervention studies . . . . . . . . . . 5.1.3 Quantitative real-time polymerase chain reaction . . . . . 5.1.4 Statistical considerations . . . . . . . . . . . . . . . . . . . . . 5.2 Discussion of results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1 Healthy dietary patterns and lipids . . . . . . . . . . . . . . 5.2.2 Healthy dietary patterns and inflammation . . . . . . . . . 5.2.3 Oral glucose tolerance test as a tool in dietary intervention studies to detect effects on inflammation . . . . . . . 5.3 Implication for public health . . . . . . . . . . . . . . . . . . . . . . .

35 35 35

Conclusions

53

6

Bibliography

38 39 40 41 41 47 49 51

55

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Chapter 1 Introduction 1.1 Dietary patterns and cardiometabolic risk The four major noncommunicable diseases cardiovascular diseases (CVDs), cancer, chronic respiratory diseases and diabetes type 2 (T2DM) are responsible for 82% of noncommunicable disease deaths. CVDs are the leading cause of deaths worldwide. In 2012, 17.5 million people died from CVDs representing 31% of all global deaths [1, 2]. Lifestyle strategies are important for the reduction of noncommunicable diseases, and especially diet plays a crucial role [3, 4, 5]. The term "cardiometabolic risk" may be considered to represent the comprehensive catalogue of factors that contribute to the development of both CVDs and T2DM [6]. According to the World Health Organization, a risk factor is "any attribute, characteristic or exposure of an individual that increases the likelihood of developing a disease or injury". In general, several factors contribute to cardiometabolic risk such as tobacco use, unhealthy diet and obesity, physical inactivity and alcohol abuse, hypertension and dyslipidemia. However, diet-related cardiometabolic risk factors are insulin resistance, obesity, dyslipidemia, hypertension and inflammation [7]. Nutrition research has traditionally focused on nutrients to identify the specific mechanisms and health impact of diet. However, associations between single factors as nutrients as well as foods and chronic diseases can be difficult to identify and to interpret. In contrast, studies of dietary patterns or whole diets examine the association between the combinations of many foods and nutrients 1

CHAPTER 1. INTRODUCTION and health. Therefore, more emphasis should be placed on the role of dietary patterns in contributing to the prevention of the major diet-related chronic diseases [8]. A very well-described dietary pattern is the Mediterranean-type of diet. As early as mid last century, Ancel Keys started to investigate the role of a diet and CVDs in the Seven Countries Study. The study has shown that populations in different countries have widely diverse incidence and mortality rates from coronary heart disease (CHD) as well as from other CVDs and overall mortality [9]. Higher rates were found in North America and Northern Europe, and lower rates in Southern Europe - i.e. the Mediterranean countries - and Japan. These differences in CHD rates were strongly associated with different levels of saturated fatty acid (SFA) consumption and average serum cholesterol levels, with lowest rates in Greece and Japan where the total fat intake was very different [10]. There is no standard definition of the term "Mediterranean Diet", but the characteristics of healthy Mediterranean Diet are high intake of fruits, vegetables, legumes, fish, whole grains, nuts, and olive oil. Dairy products and wine are of moderate intake, and red and processed meats as well as foods that contain high amounts of added sugar are of low intake [11, 12]. There is very strong evidence from the Primary Prevention of Cardiovascular Disease (PREDIMED) [13] and the Lyon Diet Heart Study [14] showing that a Mediterranean-type diet is effective in primary and secondary prevention of CVDs, respectively. In observational studies, the association between the Mediterranean diet and inflammatory markers in healthy persons has been examined [15, 16, 17], and overall inverse correlations have been reported. Moreover, intervention studies have been shown that consumption of a Mediterranean diet resulted in a decline of inflammatory markers in healthy subjects [18, 19] as well as in those with metabolic syndrome (MetS) [20] or high risk of CVDs in the PREDIMED study [21]. Particularly, the results from large intervention studies strongly suggest that a Mediterranean diet can lead to reductions in chronic low-grade inflammation and improvements in endothelial function, and thereby offering cardioprotective effects [22]. The idea of a "healthy Nordic diet" conceived of the Mediterranean diet, which has long been related to improved health, but the acceptance of the Mediterranean diet in the Nordic countries is challenging, probably because of different food preferences and eating habits in these countries [23, 24]. Thus, a 2

CHAPTER 1. INTRODUCTION

Figure 1.1: Overview of a healthy Nordic diet and cardiometabolic health. A Healthy Nordic dietary pattern or foods present in healthy Nordic diets may improve cardiometabolic risk factors, such as blood lipids, endothelial function, inflammation, glucose metabolism, insulin sensitivity, blood pressure and obesity. Abbreviations: DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; LDL-C, low density lipoprotein cholesterol; MetS, metabolic syndrome; T2DM, diabetes type 2.

3

CHAPTER 1. INTRODUCTION healthy Nordic diet takes food culture, palatability and the environment into account [25, 26]. Healthy Nordic diets are characterized by fatty fish (e.g. salmon and herring), whole grain cereals including rye, barley and oats, berries (e.g. blueberries) and fruits (e.g. apples), vegetables, root vegetables and legumes and rapeseed oil [27, 28]. In randomized controlled intervention studies conducted in various Nordic populations it has been shown that healthy Nordic diets or foods present in healthy Nordic diets and in accordance to the Nordic Nutrition Recommendations improve key CVD risk factors (Figure 1.1), such as blood lipid profiles [29, 28, 30, 31], endothelial function [32], inflammation [32, 28, 33], glucose metabolism [34], insulin sensitivity [35, 36], and blood pressure [37, 38]. Ad libitum consumption of a healthy Nordic diet in overweight and obese subjects resulted in a weight reduction [29, 37], which may have an effect on cardiometabolic health [39]. In conclusion, improving fat quality, as part of a healthy dietary pattern such as the Mediterranean diet or the healthy Nordic diet, has shown to improve blood lipid profile and inflammation and subsequently decreasing cardiometabolic risk.

1.2

Fatty acids

Fatty acids occur freely or as part of complex lipids such as triglycerides (TGs), phospholipids (PLs) and cholesteryl esters (CEs), and play a central role in energy metabolism, membrane formation, cell signaling, and as regulators of gene expression (see section 1.7) [40]. TGs are the main contributors to dietary fat in humans and are composed of one molecule of glycerol esterified with three fatty acid molecules. Typical dietary fatty acids have between 6 and 24 carbons and are either saturated, monounsaturated or polyunsaturated according to the number of double bonds between the carbon atoms. SFAs, monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) have none, one or two or more double bonds, respectively [8, 40] (Figure 1.2). Fatty acids can be obtained from the diet and several can also be produced endogenously, either from glucose or protein sources by de novo lipogenesis or from other fatty acids due to the activity of desaturases (addition of double bonds) and elongases (addition of two carbon atoms) [41]. In the liver and 4

CHAPTER 1. INTRODUCTION

Figure 1.2: Structures of some common dietary fatty acids. Fatty acids consist of a chain of carbon atoms with a carboxyl group (-COOH) at one end and a methyl group (-CH3 ) at the other end. Dietary unsaturated fatty acids are classified as n-3, n-6 and n-9 specifying the first position of the double bond by counting from the methyl end of the carbon chain.

in adipose tissue, even-numbered SFAs can be synthesized endogenously by de novo lipogenesis with the main product being palmitic acid (16:0), which can further elongated and/or desaturated into palmitoleic acid (16:1n-7), stearic acid (18:0) and oleic acid (18:1n-9). The majority of dietary SFAs in a western diet is palmitic acid (16:0), stearic acid (18:0) and myristic acid (14:0) [42]. In humans, linoleic acid (LA) (18:2n-6) and α-linolenic acid (ALA) (18:3n-3) cannot be synthesized due to the lack of enzymes and therefore are called essential fatty acids which require adequate dietary intake. The main sources for these fatty acids are vegetable oils. Sunflower, rapeseed, soybean and corn oil are rich in LA. The main sources for ALA are rapeseed and soybean oil, nuts as well as flaxseed and flaxseed oil.

1.3

Dietary fat, lipids and cardiovascular risk

Dyslipidemia is a major risk factor for CVDs and is defined as elevated blood total cholesterol (total-C), LDL cholesterol (LDL-C) or TGs, or low levels of HDL cholesterol (HDL-C). In Norway, the national guidelines for primary prevention of CVD recommend total-C < 5.0 mmol/L, LDL-C < 3.0 mmol/L, TGs ≤ 1.7 mmol/L and HDL-C ≥ 1.0 mmol/L (men) and ≥ 1.3 5

CHAPTER 1. INTRODUCTION

mg/dL

mmol/L

2,4

0,06

2,0

0,05

1,6

0,04

1,2

0,03

0,8

0,02

0,4

0,01

0

0,00

-0,4

-0,01 -0,02

-0,8 12:0

14:0

16:0

18:0

18:1 n-9

18:2 n-6

Figure 1.3: Effects of dietary fatty acids on serum total-C (white bars), LDL-C (grey bars) and HDL-C (black bars) when 1 E% from carbohydrates in the diet is replaced by 1 E% from the fatty acid in question The values for lauric acid (12:0) are based on [48], for myristic acid (14:0) on [49], for palmitic acid (16:0) on [48, 49, 50, 51], for stearic acid (18:0) on [51, 52], and for oleic acid (18:1n-9) and LA (18:2n-6) on [46]. Abbreviations: HDL-C, high density lipoprotein cholesterol; LA, linoleic acid; LDL-C, low density lipoprotein cholesterol; total-C, total cholesterol. Adapted from [45] with permission.

mmol/L (women) [43]. Dietary fatty acid composition regulates lipoprotein metabolism, which may affect plasma lipids and thereby potentially CVD risk [44]. Several dietary SFAs such as lauric acid (12:0), myristic acid (14:0) and palmitic acid (16:0) have an total-C and LDL-C raising effect. Stearic acid (18:0) has a more neutral effect on LDL-C levels [45, 42, 41]. Human intervention studies that replace carbohydrates by SFAs, MUFAs or PUFAs have shown that SFAs increase LDL-C and HDL-C level, but do not change the total-C to HDL-C ratio compared with carbohydrates. The replacement of carbohydrates by MUFAs or PUFAs resulted in a decrease in the total-C to HDL-C ratio, a raise in total-C and LDL-C level and a slightly increase in HDL-C. The replacement of SFAs by PUFAs may lead to an even more favorable lipid profile [46, 45, 47] (Figure 1.3). A pooled analysis of 11 cohort studies found that replacing 5 percent of energy (E%) of SFAs with PUFAs was associated with a 13% lower risk of coronary events and a 26% lower risk of coronary deaths [53]. In a prospective cohort study, high circulating LA was inversely associated with total and CHD 6

CHAPTER 1. INTRODUCTION mortality [54]. A meta-analysis of prospective cohort studies showed that dietary LA was inversely associated with CHD risk in a dose–response manner [55]. The typical modern human diets contain more LA than ALA most likely due to the increased use of vegetable oils rich in LA [56, 41]. LA accounts for approximately 90% of the total dietary n-6 PUFA intake [57]. In 2012, a metaanalysis of seven randomized controlled trials (RCTs) confirmed this beneficial effect, with an estimated 10% reduction in CHD risk for each 5 E% increase in PUFA consumption [58]. Another meta-analysis with RCTs concluded that interventions with n-3 and n-6 PUFAs reduce the CHD risk whereas interventions with n-6 PUFAs alone tend to increase the CHD risk [59]. However, the overall evidence indicates that higher n-6 PUFA intake lowers the CHD risk [60, 57]. It has been well demonstrated in RCTs that LA lowers serum total-C and LDL-C concentrations, particularly when it replaces SFAs in the diet [46]. Also in a more recent study it has been shown that replacing 9.5E% from SFAs with MUFAs or n-6 PUFAs leads to a significant lower total-C and LDL-C and total-C to HDL-C ratio after 4 months [61]. Schwab and co-workers included 45 RCTs in a systematic review investigating the effect of different fatty acids on serum lipids and evaluated the reduction of total-C and LDL-C when SFAs are replaced by cis-MUFAs or PUFAs as convincing [62]. These results have been incorporated into the Nordic Nutrition Recommendations from 2012, in which it is recommended to limit the intake of SFAs to