The regulation of tissue n-acylethanolamine and arachidonoylglycerol concentrations by

diet, ischemia, n-acyl phosphatidylethanolamine-specific phospholipase D and

fatty acid amide hydrolase

By

Lin Lin

A thesis submitted in conformity with the requirements

for the degree of Doctorate of Philosophy

Department of Nutritional Sciences

University of Toronto

© Copyright by Lin Lin, 2018

II

The regulation of tissue n-acylethanolamine and arachidonoylglycerol levels by diet, ischemia,

n-acyl phosphatidylethanolamine-specific phospholipase D and fatty acid amide hydrolase

Lin Lin

Doctor of Philosophy

Department of Nutritional Sciences

University of Toronto

2018

Abstract

Dietary fatty acids (DFAs) can modulate levels of n-acylethanolamines (NAEs) and

arachidonoylglycerols (AGs) via the synthetic enzyme, n-acylphosphatidylethanolamine-

specific phospholipase D (NAPE-PLD) and degradation enzyme, fatty acid amide hydrolase

(FAAH). However, other parallel pathways maybe also involved. This research hypothesizes

that the absence of NAPE-PLD will lower tissue NAEs; while the absence of FAAH will

increase NAEs. Also, basal NAE and AG levels are lower than ischemia-induced levels, while

DFA can modulate basal NAE levels. In study 1, wild-type (C57BL/6), NAPE-PLD (-/+) and

NAPE-PLD (-/-) mice were fed AIN-93G diets enriched with beef tallow, canola oil, corn oil

or fish oil for nine weeks then killed by microwave fixation. Results showed that NAPE-PLD

(-/-) had lower levels of plasma and jejunum oleoylethanolamide (OEA), lower food intake,

body weight, and body fat composition than wild-type. Also, corn oil diet elevated

arachidonoylethanolamide (AEA) and AGs; while fish oil elevated

docosahexaenoylethanolamide (DHEA) in all genotypes. Therefore, NAPE-PLD is specific to

OEA, but not other NAE, which may play a role in regulating body composition and weight

management. Also, DFA can still elevate NAE levels without NAPE-PLD suggesting it is not

necessary for dietary increased NAEs. In study 2, wild-type and FAAH-KO mice were fed a

standard chow diet for 9 weeks; then killed by control (microwave fixation) or partial CO2-

induced ischemia or CO2-induced ischemia. Brain NAE levels were higher in the FAAH-KO

than the wild-type. Interestingly, CO2-induced ischemia increased all NAEs and AGs in the

wild-type mice, but only DHEA was increased in the FAAH-KO mice. Thus, FAAH may play

III

an essential role in regulating lipid metabolism in response to ischemia. In conclusion, this

work provides evidence that diet, NAPE-PLD, FAAH and ischemia are independently

involved in maintaining tissue NAE levels.

IV “Few are those who see with their own eyes and feel with their own hearts” – Albert Einstein

Acknowledgment

Beyond the persistent, cogent and passionate scientific research, this thesis is also fulfilled with

my inner growth and the spirit of precious intellectual, psychological and emotional support

provided by many people. I would like to share many thanks and many moments, which built

the backbone of my strength to accomplish my Ph.D. degree.

Dr. Richard Bazinet: I feel honored to share my sincere gratitude to my supervisor, Dr. Richard

Bazinet, for the opportunity you provided me to learn and grow under your guidance, advice,

and support. I have been fortunate to have you as my supervisor who genuinely cared about my

research and who taught me how to think critically and write effectively to become a better

scientist. I also inspired by your research ethics and philosophy: “If you followed every step

carefully to test your hypothesis, then don’t worry about what the results might say. A result is

a result; this is science.”

Dr. Peter Jones: I sincerely thank my co-supervisor, Dr. Peter Jones, for your continuous

support and the guidance you gave to me during my Ph.D. Your countless encouragement and

mentorship since my Master degree at the University of Manitoba. Your support and belief in

my ability on researching the field of nutrition and endocannabinoids for the past ten years

have been invaluable. I become a better researcher since your first advice: “Be on time; use

more English, and get your work published.”

Dr. Harvey Anderson: I sincerely thank my advisory committee member, Dr. Harvey Anderson

for the knowledge and experience you guided to me and the tough questions you challenged

me to understand my project and the general concept of science in an exceptional training.

I thank Dr. Elena Comelli, my advisory committee member, for generously given your time

and insights to guide me on my graduate studies and thesis writing during the past five years.

I thank Dr. Deborah O’Connor and Dr. Mary R. L’Abbé for acting as my Departmental

Appraiser. Dr. Thomas Wolever for serving as my Departmental Chair; and Dr. Harold

Aukema for acting as my External Appraiser from the University of Manitoba. I feel honored

to have been among such a distinguished group of scientists.

V “When I walk along with two others, from at least one I will be able to learn”-Confucius

My lab family: Everyone in Dr. Bazinet lab lent their helping hands to my research. I

especially would like to thank Shoug Alashmali, who has been a fantastic supportive lab mate

and a wonderful friend during my research journey. I would like to thank Vanessa Guiliano,

Scott Lacombe, Adam Metherel, Raphael Chounard-Watkins and Maha Irfan Sarah Orr, Chuck

Chen, Marco Trepanier, Kathyrn Hopperton Alex Kitson, Lauren Lin, Kayla Hildebrand, for

the fun time, supportive research environment and advice to help me become a confident

speaker and researcher.

I also gave my special thanks to Louisa Kung and Emelia D’Souza, who has given the best

administrative support in such to ensure my research journey on time and well-performed.

I want to thank my teaching mentor Debbie Gurfinkel, for guiding me to be a better teaching

assistant and an instructor to deliver research in the past four years. I want to thank Fiona

Wallace, who is a definite hard-working role model for me as my mentor.

I would like to thank Kate Banks, Tracy McCook, Nancy Tomas, AJ Wang Warren Foltz,

Michael Leadley and Ashley St.Pierre for ensuring my experiments went well. Also, I want to

thank many collaborators, including Sophie Laye, Cobol Su, Laura Best, Mathieu Di-Miceli,

Mandy H, Ruslan Kubant, Ryan Bradley, Cigdem Sahin, Ivana Prce who shared their

knowledge and expertise in the different area of research, which opened my view on

conducting research containing diverse programs.

In addition, I want to thank Shirley Vien, Diana Sanchez Hernandez, Amel Taibi, Paraskevi

Masssara, Kit-Yi Yam and many others who are my colleagues and friends going through this

fantastic post-graduate journey together.

I want to thank Daniela D’Aniello and my friends, Aleese Smith, Melanie A, Marianthe

Maroulis, Tad Ferreira, Gursimran Sethi, Anamika Ray Elie Lee Livia Li, Dobrachina Zubek

and others who lived in Knox College and New College. We shared so many wonderful

conversations, meals, events, and parties which made our experience in the University of

Toronto enjoyable.

I want to thank my piano teacher Arsha Nersessian and Ed Jesus and my piano students for

sharing our joyful weekly music sections in the past five years

VI “There is always a deeper emotion which triggers all the motives seeing in people's ostensible needs.” -

Agatha Christie, 《Five Little Pigs》

I want to thank my dear friends Alex (Chen) Wang, Yanlin Zhao, Xin Yi, Ray Zhang,

Dom Kowk, Linda (Dan) Li, Dongfang Chao, and many others for sharing your wise words,

life and work experience with me through our numerous conversations. Life is a continuous

line filled with different colors of flowers that we observe together.

To my amazing parents, thank you so much for providing mental, emotional and material

support to cherish my life. You taught me to how to work hard while balanced, how to respect

people and keep smiling even in the face of adversity. Mother, you have a kind heart and a

strong spirit to lift me up in any circumstance. Whenever I had doubt, I remember you said:

“Don’t be afraid.” Father, you taught me how to be a hard worker with gratitude. You are a

great listener and a patient achiever. I learn from you and make you proud.

Through my fascinating Ph.D. journey, I inspired by some famous scholars and many ordinary

but fantastic people by their perception of life through their experience. I also came up with my

own understanding of science and life. These invaluable inspiration quotations are shared in

the footnotes of the acknowledgment and reference sections, that opened my mind and heart.

VII “Pleasure in the job puts perfection in the work”-Aristotle

Financial Disclosure

This research was funded by the Natural Sciences and Engineering Research Council of

Canada (NSERC). Lin Lin also received scholarships from Ontario Graduate Scholarship,

Ontario Student Opportunity Trust Fund and Doctoral Completion Award.

VIII “A successful relationship is an efficient relationship, which aims to a mutual direction and

motivates each other through the journey.”-Alex (Chen) Wang (Friend)

Contributions

Lin Lin contributed to and/or performed all experimental designs, method development,

procedures, data collection, analyses, and wrote the first draft, with the exception of the

following contributions:

Co-authors for submitted / published manuscripts

Dr. Adam Metherel - Contributed to statistical analysis and provided feedback on manuscript

editions (Chapter 4 & 5).

Dr. Alex Kitson - Contributed to database extraction and confirmation (Chapter 2); Data

validation (Chapter 4)

Dr. Kathryn E Hopperton and Dr. Marc-Olivier Trépanier - Contributed to animal tissue

collections and provided feedback on manuscript editions (Chapter 4).

Dr. Peter J Jones: contributed to the study designs, assisted in the data collection, provided

technical support and edited manuscript revisions (Chapter 2, 4, 5)

Dr. Richard P Bazinet: supervised the development of research ideas, provided laboratory

resources, and comprehensively edited manuscripts (Chapter 2, 4, 5)

Ms. Shoug Alashmali - Contributed to the risk of biases assessment and data validation

(Chapter 2); helped with sample preparation and provided feedback on manuscript editions

(Chapter 2 & 4)

IX “"There's a way to do it better - find it."-Thomas Edison

Table of Contents

Acknowledgement ..................................................................................................................... IV

Financial Disclosure ................................................................................................................. VII

Contributions ...........................................................................................................................VIII

Table of Contents ...................................................................................................................... IX

List of Tables ......................................................................................................................... XIV

List of Figures ........................................................................................................................... XV

List of Abbreviations .............................................................................................................. XVI

CHAPTER 1. Introduction ......................................................................................................... 1

CHAPTER 2. Literature review ................................................................................................. 6

2.1. Purpose of this literature review........................................................................................ 7

2.2. Background ....................................................................................................................... 7

2.3. Identification and quantification of n-acylethanolamines (NAEs) and

monoacylglycerols (MAGs) ............................................................................................. 8

2.4. The enzymatic synthesis of NAEs and MAGs .................................................................. 9

2.4.1.The biosynthesis and degradation of NAEs ................................................................ 9

2.4.2. The biosynthesis and degradation of MAGs (e.g., 2-arachidonoylglycerol) ......... ..12

2.5. Research gap ................................................................................................................... 14

2.6. Study design .................................................................................................................... 14

2.6.1. Study eligibility criteria ....................................................................................... 14

2.6.2. Literature search strategy ..................................................................................... 15

2.6.3. Data extraction ..................................................................................................... 15

2.6.4. Data synthesis and statistical analysis.................................................................. 18

2.7. Results ............................................................................................................................. 18

X ““Changes will only make people stronger, that’s how adaptation works.”-Dongfang Chao (Friend)

2.7.1. Risk of bias assessments ........................................................................................... 21

2.7.2. Study background / designs ...................................................................................... 21

2.7.3. Clinical trials: subject characteristic ......................................................................... 21

2.7.4. Animal studies: strains, types ................................................................................... 23

2.7.5. The impact of dietary fatty acids on human NAE and MAG levels ........................ 26

2.7.6. The impact of dietary fatty acids on animal NAE and MAG levels ........................ 29

2.7.6.1. Blood NAE and MAG levels ............................................................................. 29

2.7.6.2. Brain NAE and MAG levels .............................................................................. 34

2.7.6.3. Other organs NAE and MAG levels ................................................................. 43

2. 8. Summary and discussion ................................................................................................ 49

2.8.1. Impact of dietary fatty acids on NAE and MAG levels ........................................... 49

2.8.2. Other factors may affect on NAE and MAG levels in biological samples .............. 60

2.9. Strengths and limitations................................................................................................. 60

2.10. Conclusion .................................................................................................................... 62

CHAPTER 3. Rationale, Hypothesis and objectives ................................................................ 64

3.1. Rational and research gap ............................................................................................... 65

3.2. Overall hypothesis ........................................................................................................... 67

3.3. Specific hypothesis .......................................................................................................... 67

3.4. Overall objectives ............................................................................................................ 67

3.5. Specific objectives........................................................................................................... 67

CHAPTER 4. (Study 1) Dietary fatty acids augment tissue levels of n-acylethanolamines in

n-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) knockout mice .................... 68

4.1. Abstract ........................................................................................................................... 69

4.2. Introduction ..................................................................................................................... 69

XI “A question that sometimes drives me hazy: am I or others crazy?”-Albert Einstein

4.3. Methods ........................................................................................................................... 71

4.3.1 Diets .......................................................................................................................... 71

4.3.2 Animals ..................................................................................................................... 75

4.3.3. The percentage of fat oxidation ................................................................................ 75

4.3.4. Body fat composition ............................................................................................... 76

4.3.5. Euthanasia and sample collection ............................................................................. 76

4.3.6. Tissue lipid extraction and gas chromatography-mass spectrometry ....................... 77

4.3.7. Extractions of NAEs and arachidonoylglycerols (AGs) .......................................... 77

4.3.8. Identification and separation using high-performance liquid chromatography-mass

spectrometry ...................................................................................................................... 78

4.4. Statistics .......................................................................................................................... 79

4.5. Results ............................................................................................................................. 79

4.5.1. Tissue fatty acid composition ................................................................................... 79

4.5.2. Tissue NAEs, 1-AG and 2-AG levels ...................................................................... 83

4.5.2.1. Plasma.................................................................................................................... 83

4.5.2.2. Liver ..................................................................................................................... 85

4.5.2.3. Jejunum.................................................................................................................. 87

4.5.2.4. Brain ...................................................................................................................... 89

4.5.3. Food intake and body weight ................................................................................... 91

4.5.4. Fat oxidation and fat composition ............................................................................ 93

4.6. Discussion ...................................................................................................................... 95

CHAPTER 5. Study 2 Fatty acid amide hydrolase (FAAH) regulates hypercapnia/ischemia-

induced increases in n-acylethanolamines in mouse brain ...................................................... 100

5.1. Abstract ......................................................................................................................... 101

XII “I don’t give up, unless walking away is a better solution in that situation”-Lin Lin

5.2. Introduction ................................................................................................................... 102

5.3. Methods ......................................................................................................................... 103

5.3.1. Diets ........................................................................................................................ 103

5.3.2. Animals................................................................................................................... 103

5.3.3. Kill methods ........................................................................................................... 104

5.3.4. Whole brain lipid extraction and gas chromatography-mass spectrometry ........... 106

5.3.5. NAEs and 1-AG and 2-AG sample extractions ...................................................... 107

5.3.6. Identification and separation using high-performance liquid chromatography mass

spectrometry ..................................................................................................................... 107

5.4. Statistics ........................................................................................................................ 110

5.5. Results ........................................................................................................................... 110

5.5.1. Whole brain total lipids are not altered by CO2-induced hypercapnia/ischemia or

FAAH-KO ........................................................................................................................ 110

5.5.2. Unesterified lipids are elevated upon CO2-induced hypercapnia/ischemia ........... 112

5.5.3. OEA, AEA and DHEA are elevated in the FAAH-KO mice ................................. 114

5.5.4. OEA, AEA, and DHEA are elevated with CO2-induced hypercapnia/ischemia in

wild-type, but only DHEA is elevated in FAAH-K mice ................................................ 114

5.5.5. 1-AG and 2-AG are elevated upon CO2-induced hypercapnia/ischemia ............... 116

5.6. Discussion ......................................................................................................................... 118

CHAPTER 6. General discussion ........................................................................................... 122

6.1. Summary of research findings....................................................................................... 123

6.2. Regulatory pathway associated with NAE biosynthesis and degradation .................... 124

6.3. The effect of dietary fatty acids on NAE synthesis with/without NAPE-PLD ............. 125

.4. The effect of ischemia on NAE degradation with/without FAAH.................................. 125

XIII “Only one who loves can remember so well.” –Anton Chekhov

6.5. Strengths and Limitations.............................................................................................. 126

6.6. Significance and implications ....................................................................................... 127

6.7. Specific conclusion ....................................................................................................... 128

6.8. Overall conclusion......................................................................................................... 129

CHAPTER 7. Future directions .............................................................................................. 130

CHAPTER 8. Reference ......................................................................................................... 135

CHAPTER 9. Appendices ...................................................................................................... 149

Appendix 1. Search term for Medline 1946-October week1 2017 and Medline in-progress

and non-indexed citations. (Chapter 2) ................................................................................ 150

Appendix 2. Search Term for Embase October 14th, 2017 (Chapter 2) ............................... 153

Appendix 3. PRISM checklist 2009 ..................................................................................... 158

Appendix 4. The risk of bias assessment for human studies (Chapter 2) ............................ 161

Appendix 5. The risk of bias assessment for animal studies (Chapter 2) ............................ 163

XIV “Be happy in the moment, that’s enough. Each moment is all we need, not more”-Mother Teresa

List of Tables

Table 2.1. Description of the PICOS criteria used to perform this systematic review ............. 17

Table 2.2. Human study design and subject characteristics of individual studies .................... 22

Table 2.3. Animal studies .......................................................................................................... 24

Table 2.4. Endpoint plasma NAEs and MAGs concentrations in human studies ..................... 28

Table 2.5. Endpoint blood NAEs and MAGs concentrations in animal studies ....................... 32

Table 2.6. NAEs and MAGs in brain or brain regions .............................................................. 38

Table 2.7. NAEs and MAGs in the body .................................................................................. 46

Table 2.8. The association of dietary fat-induced NAEs and MAGs on the change of

physiological outcomes .............................................................................................................. 54

Supplemental Table 2.1. Baseline NAEs and MAGs concentration in clinical studies .......... 63

Table 4.1. Macronutrient composition of diets ......................................................................... 73

Table 4.2. Dietary fatty acid composition ................................................................................ 74

XV “There are and will be a thousand princes; there is only one Beethoven.”-Ludwig Van Beethoven

List of Figures

Figure 1.1. Internal and external factors influence n-acylethanolamine levels ....................... 5

Figure 2.1. Biosynthesis and degradation of NAE pathways ............................................... 11

Figure 2.2. Biosynthesis and degradation of 2-AG pathways .............................................. 13

Figure 2.3. Flow diagram of the systematic review .............................................................. 20

Figure 3.1. Rational and research gap .................................................................................. 66

Figure 4.1. Fatty acid composition of liver, duodenum, and whole brain............................. 81

Figure 4.2. Plasma NAE, 1-AG, and 2-AG levels ................................................................ 84

Figure 4.3. Liver NAE, 1-AG, and 2-AG levels 2.7 Results ................................................ 86

Figure 4.4. Jejunum NAE, 1-AG, and 2-AG levels ............................................................. 88

Figure 4.5. Brain NAE, 1-AG, and 2-AG levels ................................................................... 90

Figure 4.6. The progression of accumulated food intake and weekly body weight .............. 92

Figure 4.7. Ratio of fat volume to body weight .................................................................... 94

Supplemental Figure 4.1. Example of one magnetic resonance imaging slice of total body

composition ........................................................................................................................... 97

Figure 5.1. Study design ...................................................................................................... 105

Figure 5.2. The multiple reaction monitoring chromatography of NAEs and AGs ............ 109

Figure 5.3. Whole brain total lipids ..................................................................................... 111

Figure 5.4. Whole brain unesterified fatty acid concentrations .......................................... 113

Figure 5.5. Whole brain NAE concentrations ..................................................................... 115

Figure 5.6. Whole brain 1, 2-AG concentrations ................................................................ 117

Supplemental Figure 5.2. The schematic diagram of this study ........................................ 121

Figure 7.1. Overview of thesis for future research directions .............................................. 13

XVI “Humans are undefined.”-Nan Zou (PhD. in statistics)

List of Abbreviations

ABH4, or 6 or 12, alpha/beta domain-containing hydrolase 4, or 6 or 12;

ACC1, Acetyl-CoA carboxylase1;

ACT-1, alpha serine/threonine protein kinase-1;

AEA, arachidonoylethanolamide;

ALA, α-linoleic acid;

ALEA, linolenoylethanolamide;

AMPKa1,2: AMP-activated protein kinase catalytic subunit alpha-1,2;

ARA, arachidonic acid;

AT, acyltransferase;

BDNF, brain derived neurotrophic factor;

BMI, body mass index;

CAO, canola oil;

CA2+-NAT, Ca-depedent-n-acyl-transacylase;

Camkk2: Ca2+/calmodulin-dependent protein kinase;

CB1 or 2, cannabinoid receptor 1 or 2;

CNS, central nervous system

CLA, conjugated linoleic acid;

CREBH, cyclic AMB-responsive element-binding protein 3-like3 hepatocyte specific;

CRP, C-reactive protein;

CO, corn oil;

COX-2, cyclooxygenase-2;

DAG, diacylglycerol;

DAGL, diacylglycerol lipase

DAGLαβ, diacylglycerol lipase-alpha, beta;

DGLEA, dihomo-γ-linolenoylethanolamide;

DHA, docosahexaenoic acid;

DHEA, docosahexaenoylethanolamide;

Di-acyl-PE, diacyl-phosphatidylethanolamine;

DTEA, docosatetraenoyletaenoylethanolamide;

E, Energy;

XVII “To put everything in balance is good, to put everything in harmony is better.”-Victor Hugo

EEA, eicosanoylethanolamine

EPA, eicosapentaenoic acid;

EPEA, eicosapentaenoylethanolamide;

FFA, free fatty acids;

FAAH, fatty acid amide hydrolase;

FlaxO, flaxseed oil;

FO, fish oil;

GC, gas chromatography;

GCK, glucokinase;

GDE1, glycerophosphodiester phosphodiesterase 1;

GLUT1,, insulin-regulated glucose transporter 1, 4;

G6P, glycose 6-phosphate;

GTPgS, guanosine tri phosphate gamma sulfur;

Gp-NAE, glycerophospho-NAE;

GPR, orphan-G protein-coupled receptor;

HDL, high density lipoprotein;

HFD, high-fat diet;

HOCAO, high-oleic canola oil;

HOMA-IR: homeostatic model assessment of insulin resistance;

IL-6, interleukin-6;

iSCAT, interpheromatric scattering microscopy;

ITT, insulin tolerance test;

KO, knockout;

KrillO, krill oil;

LC, liquid chromatography;

LDL, low density lipoprotein;

Lyso, lyso-phosphatidylinositol;

LEA, linoleoylethanolamide;

LFD, low-fat diet;

LNA, linolenic acid;

LPA, lysophosphatidic acid;

LPS, lipopolysaccharide;

LPL, lipoprotein lipase;

XVIII “What is the meaning of a sustained note in music?”

“It means to hold it until you feel like to let it go.”- Piano student (6 years old)

MAG, monoacylglycerol;

MAGL, monoacylglyceride lipase;

MFD, medium-fat diet;

MS, mass spectrometry;

MUFA, monounsaturated fatty acids;

NAAA, n-acylethanolamine-hydrolyzing acid amidase;

NAE, n-acylethanolamine;

NAPE, n-acylated ethanolamine phospholipid;

NAPE-PLD, n-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D;

NAT, n-acyl-translase

N-3 PUFA, omega-3 fatty acids;

N-6 PUFA, omega-6 fatty acids;

O-AEA, virodhamine

OA, oleic acid;

OEA, oleoylethanolamide;

OGTT, oral glucose tolerance test;

OO, olive oil;

PA, palmitic acid;

PC, phosphatidycholine;

PE, phosphatidylethanolamine;

PEA, palmitoylethanolamide;

PGE2-GE, prostaglandin E2 glycerol ester;

PIP2, phosphatidylinositol bisphosphate;

PLA1/2, phospholipase A1 or 2;

PLC, phospholipase C;

PLCβ, phospholipase Cβ;

PI, phosphatidyl inositol;

PPAR-, proliferator activated receptor-alpha;

PTPN22, protein tyrosine phosphatase, non-receptor type 22;

PUFA, polyunsaturated fatty acids;

SaffO, Safflower oil;

SCAT, subcutaneous adipose tissue;

SEA, stearoylethanolmide;

XIX “Feelings are as important as logic.” –Melanie Adamsons

SEM, standard error

SHIP1, Src homology 2 domain-containing inositol-5-phosphatase;

SIRT1, sirtuin1

sn-1-PC, sn-1 position phosphatidycholine

SO, soya-bean oil;

SREBP-1c, sterol regulatory element-binding protein 1c;

SunfO, sunflower oil;

TC: total cholesterol;

TG, triglycerides;

THC, delta-9-tetrahydrocannabinol;

TNF-α, tumour necrosis factor-alpha;

TRPV1, transient receptor potential vanilloid type 1;

VA, vaccenic acid;

VAT, viscous adipose tissue;

WC, waist circumference

1 or 2-AG, 1- or 2-arachidonoylglycerol;

2-EG, 2-eicosanoylglycerol;

2-EPG, 2-eicosapentanoylglycerol;

2-DHG, 2-docosahexenoylglycerol;

2-DTG, 2-docosatetraenoic acid glycerol

2-LG, 2-linoleoylglycerol;

1 or 2-OG, 1- or 2-oleoylglycerol;

2-PG, 2-palmitoylglycerol;

12-HPETE-G, 12- hydroxyperoxyeicosa-5,8,10,14-tetraenoic acid glycerol ester;

1

CHAPTER 1

Introduction

2

Dietary oils vary substantially in fatty acid composition, for example, animal fats are rich in

saturated fatty acids, canola or olive oils are high in omega-9 monounsaturated fatty acids; corn

oil is high in omega-6 polyunsaturated fatty acids and fish oil high in omega-3 polyunsaturated

fatty acids. Depending on their carbon chain length, chemical structure and number of double

bonds, dietary fatty acids have the ability to regulate lipoprotein metabolism, fat oxidation,

cholesterol metabolism and gene expression (Katan et al. 1994, Salter & Tarling 2007).

However, their exact mechanisms of action in regulating the processes discussed above are not

clearly understood. One of the possible mechanism is that dietary fatty acids can be converted to

n-acylethanolamines (NAEs) (e.g., arachidonoylethanolamide, AEA) and monoacylglycerols

(e.g., 2-arachidonoylglycerols,2-AG), which regulate lipid metabolism, fat oxidation and appetite

control (Sugiura et al. 2006, DiPatrizio et al. 2011b, Fu et al. 2005, Yang et al. 2011).

NAEs and MAGs are lipid-signalling molecules. In 1957, the first NAE compound,

palmitoylethanolamide (PEA) was discovered, which has anti-inflammatory properties (Coburn

& Moore 1943, Kuehl et al. 1957). Although a variety of clinical trials explored the safety, and

efficacy of PEA in the 1970s (Hesselink 2013b, Hesselink 2013a), interest on endocannabinoid

only surged after the discovery of AEA in 1990s (Devane et al. 1992). Improvement of NAE and

MAG analysis by using liquid chromatography-mass spectrometry (LC-MS) (Schmidt et al.

2006) opened the opportunity to study multiple NAE simultaneously in biological samples

(Richardson et al. 2007, Lin et al. 2012, Balvers et al. 2013).

An emerging body of evidence suggests that changes in dietary fatty acid compositions can

modulate NAE and MAG levels in blood and tissues (Lin et al. 2013a, Mennella et al. 2015b, Pu

et al. 2016a). For example, levels of NAEs and MAGs reflect their precursor fatty acids in the

diet (Artmann et al. 2008b). Other fatty acids can also alter NAE or MAG levels (Pintus et al.

2013b).

Early studies (Gillum et al. 2008b, Schwartz et al. 2008b) demonstrated the synthesis of NAEs

from fatty acids using labelled ethanolamine-C14 (Colodzin et al. 1963) upon fasting and

refeeding (Rodriguez de Fonseca et al. 2001, Petersen et al. 2006b), as well as sham feeding and

fatty acid infusion (DiPatrizio et al. 2013a, Schwartz et al. 2008b). It is now generally accepted

3

that NAE can be synthesized from membrane phospholipids via multiple redundant steps. One

well-studied pathway of NAE biosynthesis and degradation involves NAPE-specific

phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH) (LoVerme et al. 2005,

Okamoto et al. 2007). MAGs are mainly synthesized using other enzymes including

diacylglycerol lipase (DAGL) and monoacylglycerol lipase (MAGL). Other parallel pathways

are spontaneously involved for both NAE and MAG formations. However, the specific role of

NAPE-PLD and FAAH in regulating levels of NAEs upon dietary fat manipulation has not been

studied. It is hypothesized that NAPE-PLD are necessary for the conversion of fatty acids to

NAEs, but not MAGs (e.g. 1-AG and 2-AG) while FAAH is necessary for the catabolism of

NAEs to fatty acids.

Chapter 2 of this thesis is a systematic literature review, which is adapted from a submitted study

titled N-acylethanolamine and monoacylglycerol levels are regulated by dietary fatty acids in

diets with isocaloric fat content: a systematic review. This literature review systematically

summarizes the literature regarding the effect of dietary fatty acids on NAE and MAG levels

upon consumption of a diet containing isocaloric fat in humans and animals. Additionally,

Chapter 2 addresses other factors (eg., ischemia, health status, species) that can contribute to the

variation of NAE and MAG levels, which may be independently related to the effect of dietary

fatty acid modulation.

The core of this Ph.D. thesis involved both internal and external factors (Figure 1.1). Internal

factors, such as n-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) and fatty acid

amide hydrolase (FAAH) are key contributors in the endocannabinoid metabolic pathways.

External factors, such as dietary fatty acids and ischemia can also influence endocannabinoid

levels. However, the interaction between internal factors and external factors on NAE levels has

not been evaluated. In this thesis, Chapter 3 states the rationale, research gap, the hypotheses and

objectives. The overall objective of this thesis is to examine whether external factors (dietary

fatty acids or ischemia) can further influence levels of NAEs in the absence of internal factors

(NAPE-PLD or FAAH).

4

Chapter 4 examines the biosynthesis of NAEs via the interaction between internal factor, NAPE-

PLD and external factor, dietary fatty acids. This chapter is adapted from an study titled Dietary

fatty acids augment tissue levels of n-acylethanolamines in n-acylphosphatidylethanolamine

phospholipase D (NAPE-PLD) knockout mice, revision/publication to the Journal of Nutritional

Biochemistry. Chapter 4 contains the bulk of my experimental work, which focused on the effect

of different dietary fatty acids on NAE levels while NAPE-PLD was absent. This experiment

was carefully designed to quantify basal brain NAE and AG levels without ischemic stress.

Chapter 5 examined the interaction of internal factor, FAAH and external factor, ischemia on

levels of NAEs. This chapter is adapted from a published study titled Fatty acid amide hydrolase

(FAAH) regulates hypercapnia/ischemia-induced increases in n-acylethanolamines in mouse

brain, published in the Journal of Neurochemistry. Chapter 5 tested if the absence of FAAH will

result in lower levels of NAEs. Also, this experiment verified if ischemic stress could elevate

NAE and AG levels. Chapter 6 and 7 summarizes my thesis and suggest future directions.

5

Figure 1.1. Internal and external factors influence n-acylethanolamine levels

6

CHAPTER 2

Literature review

N-acylethanolamine and monoacylglycerol levels are regulated by dietary fatty acids in

diets with isocaloric fat content: a systematic review

Adapted from: Lin Lin, Alex Kiston, Shoug M Alashmali, Peter J Jones and Richard P Bazinet.

“N-acylethanolamine and monoacylglycerol levels are regulated by dietary fatty acids in

diets with isocaloric fat content: a systematic review”.

Under revision

7

2.1 Purpose of this literature review

The overarching objective of this review was to determine if the isocaloric fat content in the diet

explains the alternation of NAE and MAG levels in various types of human and animal samples.

2.2 Background

Dietary fatty acids can be converted to a family of lipid signaling mediators including n-

acylethanolamines (NAEs) and monoacylglycerols (MAGs). Previous studies demonstrate that

modulating dietary fatty acids can alter levels of NAEs and MAGs in tissues and blood, which in

turn can impact homeostatic signals and physiological outcomes (DiPatrizio et al. 2011b, Engeli

et al. 2014, Diep et al. 2011). Although NAEs and MAGs are both synthesized from fatty acids,

they have different structures. For example, NAEs are nitrogen-containing lipids, where the acyl

group(s) is linked to the nitrogen atom of ethanolamine. However, MAGs contain glycerol,

which links to fatty acids through ester bonds.

Importantly, NAEs and MAGs are further divided into two categories. First,

arachidonoylethanolamide (AEA, also called anandamide) is an endocannabinoid (Devane et al.

1992). AEA works similar to tetrahydrocannabinol (the active compound in marijuana), which

binds to cannabinoid receptor 1 (CB1) and partially to CB2 (Pertwee 2015), stimulating fear,

feeding, anxiety (Alger 2004), pain sensation (Cravatt & Lichtman 2003), energy balance,

appetite, memory (Blancaflor et al. 2014), sedation, euphoria (LoVerme et al. 2005) and has

psychotropic effects in humans. Additionally, other polyunsaturated NAEs, such as dihomo-γ-

linolenoylethanolamide (DGLEA), eicosapentaenoylethanolamide (EPEA) and

docosatetraenoyletaenoylethanolamide (DHEA), as well as polyunsaturated MAGs, such as 2-

AG, also act as CB1 agonists (Bradshaw & Walker 2005, Meijerink et al. 2013).

Second, some NAEs and MAGs are described as “endocannabinoid-like” compounds, because

they do not bind to CB1 receptors (Cravatt & Lichtman 2003, Esposito & Cuzzocrea 2013,

Lambert et al. 2002). These compounds include saturated NAEs and MAGs (e.g.,

palmitoylethanolamide, PEA and 2-palmitoylglycerol, 2-PG), monounsaturated NAEs and

MAGs (e,g., oleoylethanolamide, OEA and 2-oleoylglycerol, 2-OG) and some polyunsaturated

NAEs and MAGs (e.g., linoleoylethanolamide, LEA and 2-linoleoylglycerol, 2-LG) (Bradshaw

8

& Walker 2005, Zoerner et al. 2011). These compounds bind to proliferator-activated receptor-

alpha (PPAR-), transient receptor potential vanilloid type 1 (TRPV1), orphan G protein-

coupled receptors (GPR55 and GPR119) (Wang & Ueda 2009), which can result in anti-

inflammation, satiety and anti-pain (Fu et al. 2003, Hansen et al. 1999, Kuehl et al. 1957,

LoVerme et al. 2005, Ueda et al. 1993a, Schmid et al. 1990).

2.3. Identification and quantification of n-acylethanolamines (NAEs) and

monoacylglycerols (MAGs)

Since NAEs and MAGs have many synonyms, we used the naming system from Pubchem

(https://pubchem.ncbi.nlm.nih.gov/docs/subcmpd_summary_page_help.html) and the

recommendation from the International Union of Pure and Applied Chemistry (IUPAC)

(Rodriguez de Fonseca et al. 2001). Thus, we refer to these compounds as

arachidonoylethanolamide (AEA) and 2-archidonoylglycerol (2-AG), as examples of NAE and

MAGs, respectively (Berdyshev et al. 1996, Schmid et al. 2000).

The quantification of NAEs and MAGs in biological samples is most commonly done using gas

or liquid chromatography with tandem mass spectrometry (GC-MS or LC-MS). Improvements in

the analysis using LC-MS (Schmidt et al. 2006) opened the opportunity to study multiple NAEs

and their isomers simultaneously in various types of biological samples (Richardson et al. 2007,

Lin et al. 2012, Balvers et al. 2013). In general, levels of NAE in tissues, cells or plants are in the

pmol/g range (Hansen et al. 1999, Ueda et al. 2010, Blancaflor et al. 2014, Hansen & Diep

2009). Among all identified NAE, the most abundant NAEs in animal tissues are PEA, SEA,

OEA, and LEA (Ueda et al. 2010, Blancaflor et al. 2014, Hansen & Diep 2009), while in human

follicular fluid or plasma, the most abundant are PEA and OEA (Schuel et al. 2002). AEA

generally makes up less than 10% of the total NAE concentration (Lin et al. 2013a, Ueda et al.

2010, Maccarrone et al. 2001, Jones et al. 2014). Although tissue NAE levels are low under

normal physiological conditions, NAEs can accumulate during ischemia or upon cell injury

(Brose et al. 2016, Lin et al. 2017, Bazinet et al. 2005, Schmid et al. 1990). Also, it has been

shown that levels of AEA increase with EDTA at room temperature (Zoerner et al. 2011) or

when whole blood is stored at 4°C for one to two hours, likely due to ex vivo AEA release from

https://pubchem.ncbi.nlm.nih.gov/docs/subcmpd_summary_page_help.html

9

erythrocytes or leukocytes (Vogeser et al. 2006, Zoerner et al. 2011, Wood et al. 2008, Jain et al.

2017).

2.4. The enzymatic synthesis of NAEs and MAGs

2.4.1. The biosynthesis and degradation of NAEs

For the biosynthesis of NAEs (Figure 2.1), the first reaction is through a calcium-dependent-N-

acyl-transacylase (CA2+-NAT), which transfers a (Hansen et al. 1999)(PC) onto the amine of

diacyl-phosphatidylethanolamine (PE) to generate n-acylated ethanolamine phospholipid

(NAPE) (Hansen et al. 1999, Leung et al. 2006). In parallel, sn-1 fatty acids from PC and PE can

form NAPE via an enzyme phospholipase A1 (PLA1) or acyltransferase (AT) as alternative

pathways. It is worth noting that NAPE is a generic term for all of the three forms: “n-acylated

ethanolamine phospholipid species comprising the diacyl type”, “n-acyl-plasmenyl-

ethanolamine” and “n-acyl-plasmanyl-ethanolamine” (Ueda et al. 2013). NAPE can be

synthesized in small intestinal gut segments after fat feeding (Gillum et al. 2008a), and is present

in many other tissues (Rodriguez de Fonseca et al. 2001, Petersen et al. 2006a, Hansen et al.

1999). As well, NAPE is able to enter the central nervous system (CNS) and accumulates in the

hypothalamus (Gillum et al. 2008a); and it has been suggested that NAE can be synthesized in

the CNS from circulating NAPE (Gillum et al. 2008a).

After NAPE is synthesized, a group of specific hydrolases or a combination of acyltransferases

and hydrolases convert it into NAE (Wang & Ueda 2009, Ueda et al. 2010, Ueda et al. 2013).

The most studied pathway is the conversion of NAPE to NAE via n-acyl-

phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) (Okamoto et al. 2007,

LoVerme et al. 2005, Fu et al. 2007). However, NAPE can be converted to phosphate-NAE via

phospholipase C (PLC), then phosphate-NAE is further converted into NAE via protein tyrosine

phosphatase, non-receptor type 22 (PTPN22) or Src homology 2 domain-containing inositol-5-

phosphatase (SHIP1) (Ueda et al. 2010). Furthermore, NAPE can be converted into lyso-NAPE

via alpha/beta-hydrolase 4 (ABH4) or sPLA1/A2, then lyso-NAPE is further converted into NAE

via lyso-PLD (Ueda et al. 2010, Sun et al. 2004). As well, lyso-NAPE can also be converted into

glycerophospho-NAE (Gp-NAE) through ABH4, then the Gp-NAE is converted to NAE via

GDE1 (Wang & Ueda 2009). Because GDE1 is highly expressed in the brain, where it can

10

regulated NAE synthesis (Wang & Ueda 2009). Moreover, free fatty acids and ethanolamine can

be converted into NAE via fatty acid amide hydrolases (FAAH), when levels of free fatty acids

and ethanolamine are at high concentrations (Katayama et al. 1999, Ueda et al. 2000). NAE can

be degraded into free fatty acids and ethanolamine by hydrolases (Ueda et al. 2013). The most

well-studied hydrolase for NAE degradation is FAAH, which catabolizes numerous NAEs.

Another lysosomal enzyme, n-acylethanolamine-hydrolyzing acid amidase (NAAA), is also

involved in NAE degradation to fatty acid and ethanolamine, which selectively hydrolyzes PEA

over other NAEs (Ueda et al. 2013, Piomelli 2013, Cravatt et al. 1996, Izzo et al. 2010).

Moreover, NAE can also be converted to either prostamide E2 via coyclooxygenase-2 (COX-2)

or to 12-hydroperoxyeicosa-5,8,10,14-tetraenoylethanolamide via lipoxygenases (Ueda et al.

2010).

11

Figure 2.1. Biosynthesis and degradation of NAE pathways

Ingested dietary fat/oil is broken down into phospholipids (e.g., PC and PE), thereby converting to NAEs in tissues. The 1st step is to

synthesize NAPE, which is catalyzed by enzymes (e.g., CA2+-NAT or PLA1 and/or AT-1-5) in the gut. Then NAPE is able to enter

other tissues including the CNS. The 2nd step involves multiple pathways. The most well-known pathway is via the enzyme NAPE-

PLD. Also, in the CNS, NAPE can first be converted to phosphate-NAE via enzyme PLC, then phosphate-NAE is further converted

into NAE via enzyme PTPN22 or SHIP1. In parallel, NAPE can be converted into lyso-NAPE via enzymes (e,g. ABH4 or sPLA1/A2).

Then the lyso-NAPE can be further converted into NAE in multiple pathways 1) lyso-PLD in the gut or 2) ABH4 to synthesize Gp-

NAE then via GDE1 to synthesize NAE. In addition, FFA and ethanolamine can be converted into NAE via FAAH at high

concentrations. Spontaneously, NAE can be broken down into FFA and ethanolamine via FAAH or NAAA (target to PEA only).

Moreover, NAE can also be broken down into prostamide E2 via COX2 or 12-hydroperoxyeicosa-5,8,10,14-tetraenoylethanolamide

via lipoxygenases. Source from (Hansen et al. 1999, Gillum et al. 2008a, Ueda et al. 2013, Wang & Ueda 2009, Okamoto et al. 2007,

Lo Verme et al. 2005, Sun et al. 2004, Katayama et al. 1999, Izzo et al. 2010, Ueda et al. 2010)

12

2.4.2. The biosynthesis and degradation of MAGs (e.g., 2-arachidonoylglycerol)

After ingestion, dietary fatty acids are converted to phospholipids in tissues, which can be further

synthesized to MAGs. 2-AG is the most studied compounds in this family. The most accepted

route of biosynthesis pathways of 2-AG is illustrated in Figure 2.2. Specifically, PI can be

converted to 2-AG through the formation of lyso-PI by the enzyme PLA1 (Tsutsumi et al. 1994,

Ueda et al. 1993a). PC also serves as a precursor of 2-AG after being converted to 2-

arachidonoyl-DAG (Oka et al. 2005). As well, 2-arachidonoyl-lysophosphatidic acid (LPA) can

be converted to 2-AG (Nakane et al. 2002, Ueda et al. 1993a). PLC-diacylglycerol lipase

(DAGL) is used for 2-AG synthesis, which is in postsynaptic neurons in response to

depolarization and stimulation of G q/11-coupled receptors (Ahn et al. 2008). For instance, 2-

arachidonoyl phosphatidyl inositol-arachidonoyl-containing phosphatidylinositol bisphosphate

(PIP2) is hydrolyzed by PLCβ to ARA-containing diacylglycerol (DAG) or β (Ueda et al.

2013). So far, DAG is thought to be the isoform responsible for 2-AG production in the central

nervous system of adult mice (Lu & Mackie 2016, Pacher et al. 2006, Tanimura et al. 2010, Gao

et al. 2010).

2-AG hydrolysis to glycerol and arachidonic acid occurs via one of four hydrolytic enzymes: 1)

monoacyl glycerol lipase (MAGL), 2) ABH6, 3) ABH12 or 4) FAAH. Additionally, 2-AG can be

oxidized by COX-2 to prostaglandin E2 glycerol ester (PGE2-GE) or by lipoxygenases to 12-

hepete-G (Lu & Mackie 2016, Pacher et al. 2006). FAAH can hydrolyze 2-AG into ARA and

glycerol in vitro conditions (Goparaju et al. 1998), but in vivo studies suggest that 2-AG is

mainly hydrolyzed by MAGL and that 2-AG is not a substrate for FAAH (Pacher et al. 2006,

Osei-Hyiaman et al. 2005a, Sugiura et al. 1995). Furthermore, 2-AG can be oxidized to PGE2-

GE or 12-hepete-G via lipoxygenases (Lu & Mackie 2016, Pacher et al. 2006) (Figure 2).

13

Figure 2.2. Biosynthesis and degradation of 2-AG pathways

In postsynaptic neurons, arachidonoyl-containing PIP2 can first convert to DAG via PLCβ, then

this synthesized DAG is further converted to 2-AG via DAGL. Spontaneously, this PIP2 can be

converted to lyso-PI via PLA1, then the lyso-PI is further converted to 2-AG via lyso-PI-PLC.

Not only PIP2, but also PC can also form DAG, in which DAG is converted to 2-AG via DAGL.

In presynaptic neurons, 2-AG can be broken down to ARA and glycerol via 4 different enzymes

(e,g., MAGL, FAAH, ABH6 and ABH12). Additionally, in postsynaptic neurons, 2-AG can also

be broken down into PGE2-GE via COX-2 or degraded into 12-HPETE-G via lipoxygenases.

Source from (Oka et al. 2005, Nakane et al. 2002, Ahn et al. 2008, Gao et al. 2010, Ueda et al.

2013, Pacher et al. 2006, Goparaju et al. 1998, Lu & Mackie 2016, Osei-Hyiaman et al. 2005a,

Ueda et al. 1993a)

14

2.5. Research gap

Many studies reviewed the biosynthesis and degradation pathways of NAEs and MAGs. Dietary

modification is one significant component in regulating lipid metabolism and physiological

functions. Therefore, dietary modulation can thrive through multiple constantly competitive

pathways and unsustainable enzyme activities. It is still unknown how dietary fat/oil modulation

can impact levels of NAEs and MAGs.

In order to provide evidence-based information in this area, the present report aims to conduct a

systematic review of human and animal studies which have examined the effects of modulating

the composition of dietary fatty acids under isocaloric conditions on levels of NAEs and MAGs.

Also, this review aims to provide information on NAE and MAG concentrations from human

and animal biological samples, which will map their range in concentrations after dietary

interventions, which may further explain the feasibility of adjusting NAE and MAG levels via

dietary fat modulation.

2.6. Study design

Although there is no standard methodology to systematically review animal experiments, this

current paper adapted the tools used for clinical systematic reviews. This review followed the

Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) (Moher et al.

2009) and The Cochrane Handbook for Systematic Reviews of Interventions for planning and

conducting systematic search strategies for both clinical and animal studies (Higgins et al. 2008).

2.6.1 Study eligibility criteria

The study eligibility criteria are based on the PICOS (population, intervention, comparison,

outcome, setting) aspects of the Cochrane review format (Higgins et al. 2008), in which the

specification of this review inclusion and exclusion criteria are shown in Table 2.1. Briefly,

human and animal dietary intervention studies were included in this review, which reported the

effect of isocaloric dietary fats intake on NAE and MAG levels in biological samples (e.g. brain,

brain regions, liver, intestine, kidney, blood, etc.). Non-peer-reviewed articles, reviews/meta-

analyses and in vitro studies were excluded. In addition, this study excluded articles with 1) no

dietary fat treatments or comparison of different dietary fats, oils or fatty acids, or 2) non-

15

isocaloric fat diets (e.g. high fat vs low fat). Furthermore, this review also excluded studies

comparing the effect of dietary fat from whole foods or dietary oils (e.g. olive oil or fish oil)

with pure fatty acids (e.g. OA or DHA) (Diep et al. 2014). This review also excluded studies

comparing dietary fat to other dietary nutrients (e.g. proteins, carbohydrates) under isocaloric

diets (DiPatrizio et al. 2011b, Schwartz et al. 2008a). In addition, this article excluded studies

examining the effect of liquid diets (DiPatrizio et al. 2013b) and the effect of fatty acid position

in triacylglycerol (Carta et al. 2015b, Rossmeisl et al. 2012).

2.6.2. Literature search strategy

The literature search was conducted using three online sources: 1) MEDLINE (Ovid)-1946 to

October week 1 2017; 2) Embase-1947 to October 14th, 2017 and 3) MEDLINE-not-indexed

citation articles until October 14th, 2017. The electronic search strategy in Medline and -not-

indexed citation articles included both Medical Subject Headings (MeSH) and keywords in

search fields including abstract (ab), title (ti), keyword heading (kw) and Medline Subject

Heading (sh) following a three-section combination, step-by-step approach with ((“diet*”

[MeSH]) or (“diet*.ab.ti.kw.sh.”)) AND ((“fat” [MeSH]) or (“fat*.ab.ti.kw.sh”)) AND

((ethanolamine” [MeSH]) or (“ethanolamine*.ab.ti.kw.sh”)) as an example. Similar terms in

Embase included EMTREE and keywords in the same search fields ((“lipid diet” [Embase]) or

(“diet*.ab.ti.kw.sh.”)) AND ((“fats” [Embase]) or (“fat*.ab.ti.kw.sh.”)) AND ((ethanolamine”

[Embase]) or (“ethanolamine*.ab.ti.kw.sh”)) as an example. Overall, all the synonyms

commonly used were searched in this review. For more details, the complete search terms are

listed in Appendix 1,2. Microsoft Excel, 2007 was used to eliminate duplicate reports based on

title and abstract, and to manage the included complete reference. Also, manual searches were

performed by checking reference lists and related reviews to identify further relevant papers.

2.6.3. Data extraction, risk of bias

The selected abstracts were analyzed by the primary author (LL) to identify all relevant articles.

The studies were selected or excluded according to the PICOS eligibility criteria (Table 2.1).

Two investigators (LL and AK) independently reviewed all the included studies. A modified

study parameter form (PROFORMA) was used to extract relevant information. Also, this review

16

is prepared in accordance with the existing guidelines of PRISM. The PRISM 2009 Checklist

(Moher et al. 2009) was used and provided in Appendix 3.

The investigators (LL and SA) evaluated the risk of bias assessments. The Cochrane Risk of Bias

Tool was used for human studies (Higgins et al. 2008). We modified the Cochrane Handbook

(Higgins et al. 2008) and the SYRCLE’S risk of bias tool (Hooijmans et al. 2014) for animal

studies.

17

Table 2.1. Description of the PICOS criteria used to perform this systematic review

Components Inclusion criteria Exclusion criteria

P = Population

1) Human interventional studies (Not

restricted to randomized clinical trials

only, No restriction on health status,

age, gender, BMI and ethnic groups at

baseline); 2) Animals (No restriction on

species, sex, age); 3) English only

1) Cell-culture;

2) Reviews, case-reports, abstract-only, newspaper

articles, non-peer reviewed articles, letters

I = Intervention

1) Fat type in meals (vegetable oils, animal fats)

2) Dietary fat composition (saturated, monounsaturated, polyunsaturated

fatty acids,)

1) Not dietary fat/ oil studies

C = Comparison

The comparison focuses on the effect

of consumption of dietary fatty acids

on blood and tissue NAE and MAG

levels upon isocaloric fat diets.

(Regular dietary feeding only, no

substance, no restriction on the types of

isocaloric fat diets)

Comparison focused on NAE and MAG levels

upon 1) non-isocaloric fat diets or just standard

diets, 2) fasting vs. refeeding, 3) not-regular

feeding (e.g., gavage, sham, emulsions, oral

administration or injection), 4) taking substance

(e.g., alcohol, lipopolysaccharide, pharmacological

doses of NAEs or MAGs)

O = Outcomes

1) Levels of NAEs and MAGs in

biological samples upon isocaloric fat

diet interventions, 2) Associated tests

(e.g., physiological outcomes or

biochemical outcomes)

Outcomes did not include levels of NAE and

MAG, but focused on 1) behavior tests, 2)

biomarker tests, 3) enzyme activities

S = Setting

No restriction on settings (e.g., multi-

center, community, hospital, nursing

home, chronic care institution,

outpatients or geographical regions)

18

2.6.4. Data synthesis and statistical analysis

The specific values for NAEs and MAGs levels were extracted based on the article’s reports or

estimated from figures using the PlotDigitizer (verson 2.1) software. To keep the values of NAEs

and MAGs consistent between articles, this review converted all of the values into pmol/ml for

blood samples and pmol/g for tissues samples. The mean ± SEM values were reported for NAE

and MAG levels in Table 2.3-2.7. All of the NAE data were double checked by two investigators

(LL and AK). The risk of bias assessment for human and animal studies were analysed using

Review Manager (RevMan), version 5.3 (The Nordic Cocharne Centre, The Cochrane

Collaboration, Copenhagen, Denmark).

This review focuses on the modulation of dietary fats / oils upon consumption of diet containing

isocaloric fat content. We applied one-way ANOVA statistical analysis to the endpoint NAEs

and MAGs concentrations in isocaloric groups, if the original studies only compared the effect

between baseline and endpoint NAEs and MAGs (Banni et al. 2011). We also conducted one-

way ANOVA statistical analysis only between isocaloric groups, when the original paper mixed

with different fat content (e.g., low-fat vs high-fat) (Artmann et al. 2008a, Alvheim et al. 2012b,

Alvheim et al. 2014a). Moreover, we conducted one-way ANOVA or paired T-test to 100%

sufficient diet groups, if the original studies combined the 100% sufficient diet groups with diet

restricted groups (Avraham et al. 2011, Liisberg et al. 2016). In addition, we conducted paired T-

test in between the formula groups, but did not include the sow’s milk group in the study by

Berger et.al (Berger et al. 2001a) because it was not isocaloric.

2.7. Results

A flow diagram depicting the search and selection process is shown in Figure 2.3. The search

identified a total of 6696 articles. After removing articles which were reviews, not in English or

not journal articles (1669), there were 5027 studies left. Then, we removed 1243 duplicates. 3784

articles were screened by title and abstract. After excluding the irrelevant articles (3619), the

remaining 165 articles were retrieved and screened in full-text, of which 141 were excluded. A

total of 24 articles containing 6 human and 18 animal dietary interventional studies were

selected. It is worth mentioning that within the included 24 studies, one experiment from

Avraham et al. (2011) were excluded because the outcome may have been driven by energy

19

restriction rather than the fatty acid intervention. Also, an experiment that measured NAE levels

in human plasma from Balvers et al. (2013) was removed because it did not have any dietary fat

intervention. As well, this review only focuses on the early low-fat diet period in the study by

Demizieux et al. (2016) and excluded the high-fat diet period which does not have compared

group. In addition, we only report the value in the formula groups in the Berger et al. (2001a) as

not the sow’s milk group.

20

6494 studies identified (through October

14th, 2017): 2111 Medline; 4189 Embase;

194 Medline-not-indexed citation

4825 articles left

1669 removed articles:

-71 Not-journal articles (12 Medline,

1 Medline in-progress and not-indexed

citation, 58 Embase;);

-222 Not-English articles (71 Medline, 1

Medline in-progress and not-indexed citation,

150 Embase);

-1376 Review articles (396 Medline, 36

Medline in-progress and not-indexed citation,

944 Embase);

3784 articles for title and abstract

screening

1041 duplicated articles (Searched by title

/abstract using Excel)

165 articles left for full-text screening

3619 articles were excluded based on the

PICOS criteria

-cell-culture studies;

- case-reports, abstract-only, newspaper

articles, non-peer reviewed articles, letters-

study design: 1) non-isocaloric fat diets or just

standard diets, 2) fasting vs. refeeding, 3) not-

regular feeding (e.g., gavage, sham,

emulsions, oral administration or injection) 4)

taking substance (e.g., alcohol,

lipopolysaccharide, pharmacological doses of

NAEs or MAGs), 5) behavior test only; 6)

enzyme activities only;

24 articles were included in this review

141 articles were excluded based on PICOS

criteria

Electronic searching databases: Medline

1946-October week 1 2017; Medline in-

progress and not-indexed citation; Embase

Figure 2.3. Flow diagram of the systematic review

21

2.7.1. Risk of bias assessments

All of the human studies and the majority of animal studies were judged as having a “low” or

“unclear risk of bias” using the Cochrane Risk of Bias tool (Appendix 4,5).

2.7.2. Study background / designs

Six clinical studies and 18 animal studies are included in this review. The summary

characteristics of the 24 studies are presented in Table 2.2 and 2.3.

2.7.3. Clinical trials: subject characteristics

Subject characteristics are presented in Table 2.2. All study subjects had an overnight fast before

blood sample collection, except for one study which did not provide details of their fasting

duration (Ramsden et al. 2015a). Of the 6 human studies, all trials were randomized and

controlled. The sample size varied from 15 subjects (Mennella et al. 2015b) to 130 subjects (Pu

et al. 2016b). One study (Ramsden et al. 2015a) examined chronic headache patients. One study

(Mennella et al. 2015b) examined the acute effect of dietary fats. The duration of the long-term

studies was typically one month, except one study, which was 12 weeks (Ramsden et al. 2015a).

Four clinical studies reported their baseline NAE and MAG levels, which are summarized in

Supplemental Table 2.1.

22

Table 2.2. Human study designs and subject characteristics of individual studies

Authors, year

/ countries Subject characteristics

Duration /

Study design Sample size /group Fasting Fat content Experimental diets

Banni et al.

(2011) / Italy

63 subjects (♂: wc >

102cm; ♀: wc > 88cm; 35-

64yrs; ) normal: BMI 84cm and LDL <

1.3mmol/L; ♂&♀: TC ≥

1.7mmol/L or, BP

≥130mmHg systolic, ≥

85mmHg diastolic or

glucose ≥ 5.5mmol/L;

46.5yr; BMI ~29)

30 ds / R, C,

cross-over,

DB

130 12 hrs

35% E

(Senanayake et al.

2014)

1) CAO (60% OA, 20% LA, 10% ALA;

2) HOCAO (72% OA, 15% LNA, 2%ALA);

3) DHA+ CAO (63% OA, 13% LA, 6%DHA);

4) CO/SaffO (18% OA, 69% LA);

5) FlaxO/SaffO (18% OA, 38% LA, 32%ALA)

Ramsden et al.

(2015a) /

Unite States

55 chronic headache

patients (♂, ♀; >18 yr old)

12 wks / R, P,

SB

Low n-6 PUFA: 27;

Low n-6 PUFA +

high n-3 PUFA: 28

> 10

hrs(Ramsden

et al. 2013)

30.4-33.6%E

(MacIntosh et al.

2013)

1) Low n-6 PUFA (< 2.5% E LNA, 60mg/d

ARA, 0.6% E ALA+EPA+DHA);

2) Low n-6 PUFA + High n-3 PUFA (< 2.5%E

LNA,1.5%E ALA,1000mg/d EPA+DHA)

23

2.7.4. Animal studies: strains, types

Of the 18 animal studies, more than half were conducted using mice (Table 2.3). Other animals

included salmon (Alvheim et al. 2013), hamsters (Lin et al. 2013a), pigs (Berger et al. 2001a)

and rats (Artmann et al. 2008a, Piras et al. 2015, Carta et al. 2015a). All animal studies

examined relatively long-term effects, in which the shortest duration was one week and the

longest was six months. Tissue samples of animal studies were collected with various euthanasia

methods including anesthesia, decapitation, cervical dislocation or combinations.

24

Table 2.3. Animal studies

Authors,

year

Type of animals

(sex, age) Duration

Sample size /

housing Fasting Euthanasia Fat content Experimental diets

Alvheim et

al. (2012b)

C57BL/6J mice

(♂, ~23 ds) 14 wks 9-10 / 2 Unknown Unknown

35% E for MFD;

60% E for HFD

1) 1% E LA; 2) 8% E LA;

3) 8% E LA + 1% E EPA/DHA;

Alvheim et

al. (2013)

Expt. 1: Atlantic

salmon (~340g) 6 mos 3 / 6 24 hrs

Sharp blow to the

head 51% E 1) SO diet; 2) FO diet

Expt. 2:

C57BL/6J mice

(♂; ~6 wks)

16 wks 8-9 / single Unknown Isoflurane, then

decapitation

32% E (SO diet);

35% E (FO diet);

1) SO-based fillet (8% E LA);

2) FO-based fillet (1% E LA + 2.7% E

EPA/DHA)

Alvheim et

al. (2014a)

C57BL/6J mice

(♂; ~ 6 wks) 16 wks 9 / single Unknown

Isoflurane, then

decapitation

LFD: 11% E;

MFD: 32% E; 1) 1% E LNA diet; 2) 8% E LNA diet

Artmann et

al. (2008a)

Sprague Dawley

rats (♂; ~250g) 1 wk 8 / single Unknown Decapitation

Experimental diets:

45% E (Piscitelli et al.

2011)

1) Palm oil diet; 2) OA diet; 3) Saf-O diet;

4) Pure AA + OO diet; 5) FO + OO diet,

Avraham et

al. (2011)

Sabra mice (♀;

~30g) 12 ds 10 / unknown Unknown Decapitation

100% sufficient energy:

12.4% E (calculated

from 5% fat wt/wt

Cat.901682)

1) 5% wt/wt CAO diet;

2) 4% wt/wt FO + 1% wt/wt CAO diet

Balvers et

al. (2013)

C57BL/6J mice

(♂; 6 wks)

(Verhoeckx et al.

2011)

6 wks 8 / 2-3 Unknown Anesthetized

9% E (Calculated from

4% wt/wt fat in AIN93-

M diet)

1) 1% SO + 3% HOSunfO;

2) 1% SO +2% HOSunfO + 1% FO;

3) 1% SO + 3% FO(Verhoeckx et al. 2011)

Batetta et

al. (2009)

Zucker rats (♂; 4

wks) 4 wks 6 / unknown Fasted

Sodium

pentobarbital

anesthesia,

kill unknown

11.2% E (calculated

from 7g/100g oil in

AIN-93 diet )

1) Blended oils (SunfO, rapeseed oil, coconut

oil, linseed oils (0.0% E EPA / DHA);

2) FO (0.8% E EPA+DHA); 3) KO (0.8% E

EPA+DHA)

Berger et

al. (2001a)

piglets (♂; < 1

kg,

25

Jacome-

Sosa et al.

(2016)

JCR:LA-cp rat

(♂; 8 wks) 8 wks 5 / unknown Unknown Not specific

34% E (calculated from

15% fat by wt/wt) and

1% cholesterol(Jacome-

Sosa et al. 2014)

1) Control (high SFA and OO); 2) VA (2% E

VA); 3) CLA (2% E cis-9, trans-11 CLA); 4)

VA/CLA (1% E VA, 0.5% E CLA)

Liisberg et

al. (2016)

C57BL/6J mice

(♂; 9 wks) 12 wks 9 / single Unknown

Isoflurane, then

cardiac puncture

39.1% E (calculated

from 19.8% fat by

wt/wt)

1) Cod in western diet;

2) Pork in western diet

Kim et al.

(2016)

C57BL/6J mice

(♂; 3 wks)

62 ds or

118 ds 9 / unknown 8 hrs

Isoflurane, then

cervical

dislocation

22.8% E (calculated

from 11% fat by wt/wt)

1) Control diet (SaffO);

2) DHA (~1100mg/kg DHA administered)

Lin et al.

(2013a)

Syrian Golden

Hamster (♂;

~80-100g, plus

1wk acclimation)

4 wks 12 / single 12 hrs Isofluorane, then

phlebotomy

23.4% E (calculated

from 10% fat by w/w in

AIN-93 diet)

1) CO diet; 2) HOCAO diet;

3) FO diet; 4) HOCAO+ DHA diet

Piras et al.

(2015)

Zucker rats (♂; 6

wks) 14 wks 8 / single Unknown

Isofluorane, then

Decapitation

33.1% E (Calculated

from 16.6% wt/wt fat)

1) Control (11.25% ovine fat, 3.75%SunfO, 2%

cholesterol wt/wt);

2) CLA (11.25% ovine fat, 2.53% SunfO,

1.22% of CLA, 2% of cholesterol wt/wt)

(Martins et al. 2012)

Tsuyama et

al. (2009)

Jcl: ICR mice (♂,

♀; 4 wks old) 4 wks

LNA: 8; CLA:

9 / unknown Unknown

Cervical

dislocation

13.2% E (Calculated

from 5.3% wt/wt in

diet)

1) LNA (3% wt/wt from SaffO /kg);

2) CLA (3% wt/wt from CLA triacylglycerol oil

/kg)

Watanabe

et al.

(2003)

Study1: ddy mice

(♂; starts at

utero)

Utero to

10 wks old

n-3 sufficient:

4;

n-3

deficient :3 /

unknown Unknown

Diethylether, then

decapitated

11.5% E (calculated

from AIN-76 diet by

wt)

1) n-3 PUFA sufficient (5% wt/wt SaffO +

DHA ethylester);

2) n-3 PUFA deficient (5% wt/wt SaffO)

Study 2: ddy

mice (♂;4wks) 4 wks 6 / unknown

23% E (calculated from

AIN-76 diet by wt)

1) 10% wt/wt LA from SaffO;

2) 10% wt/wt DHA from FO;

Wood et al.

(2010)

CD1 mice (♂;

~16-18g, plus 1

wk acclimation)

2 wks 10 / single Unknown

Cervical

dislocation, then

decapitated

13.7% E (Standard

Purina 5P00 prolab

RMH300 diet)

1) Pork fat (0.25% by wt n-3 PUFA);

2) DHA-rich FO diet (1.45% by wt n-3 PUFA:

8.9mg EPA, 59mg DHA/d)

Zamberletti

et al.

(2017)

Sprague-Dawley

rats (♂; starts at

utero)

Utero to

postnatal

75 days

8 / unknown Unknown Unknown

6% fat (unknown by wt

or by E; from Dr.

Piccioni Laboratory)

1) Control (3% peanut+ 3% rapeseed oils);

2) n-3 PUFA deficient (6% peanut oil);

3) n-3 PUFA enriched (6% rapeseed oil)

26

2.7.5. The impact of dietary fatty acids on human NAE and MAG levels

Six human studies (Mennella et al. 2015b, Pintus et al. 2013a, Banni et al. 2011, Ramsden et al.

2015a, Jones et al. 2014, Pu et al. 2016b) are included in this study (Table 2.4). The dietary fatty

acids interventions on NAEs and MAGs can generally be categorized into 1) dietary n-3

polyunsaturated (PUFA), 2) dietary n-6 to n-3 balance, 3) dietary n-9 monounsaturated fatty acid

(MUFA), 4) mixed dietary fatty acid composition (Western diet, Mediterranean diet), or 5)

dietary CLA consumption studies.

Dietary n-3 PUFA: Banni et al. (2011) compared plasma AEA and 2-AG levels after

consumption of diets enriched with long-chain n-3 PUFA (Menhaden oil or krill oil) or with

MUFA, olive oil, in normal weight, overweight, and obese subjects. Results showed that obese

subjects consuming krill, but not Menhaden, oil had significantly decreased plasma 2-AG levels

compared to baseline. In addition, we evaluated the impact of dietary fatty acids on the endpoint

plasma AEA and 2-AG levels (mean ± SEM, one-way ANOVA) across the three diets, but did

not find any statistical differences.

Dietary n-6 to n-3 balance: Ramsden et al. (2015a) examined the effect of modulating the

dietary n-6/n-3 PUFA ratio on plasma NAEs and MAGs in chronic daily headache patients.

Results showed that high n-3 /low n-6 PUFA diet had higher levels of DHEA, 2-

docosaheaenoylglycerol (2-DHG) and lower levels of 2-AG compared to a low n-6 PUFA diet.

However, plasma levels of AEA, OEA, PEA, and 2-OG were not different between diets.

Dietary n-9 MUFA: Mennella et al. (2015b) examined the acute dietary effects of subjects

consuming isocaloric diets containing either sunflower oil, high in n-6 PUFA, high-oleic

sunflower oil, high in n-9 MUFA or virgin olive oil, high in n-9 MUFA, in normal weight

subjects. Results showed that both n-9 MUFA groups had higher OEA than the n-6 MUFA group

at 120-mins post-meal, but plasma PEA, LEA, AEA, and 2-AG levels were not different across

all three groups at any time point.

Dietary conjugated linoleic acid (CLA): Pintus et al. (2013a) compared plasma AEA and 2-AG

levels upon consumption of cheese rich either in CLA, α-linolenic acid (ALA), vaccenic acid

27

(VA), or in saturated fat (control). After three weeks consumption of 90g cheese/ day, AEA was

lower in the CLA enriched-cheese group compared to high saturated fat control diet. Notably,

when subjects consumed 45g cheese/day for three weeks, there was no difference in AEA and 2-

AG levels between these two diets.

Mixed dietary fatty acid composition: Two studies (Jones et al. 2014, Pu et al. 2016b) examined

the modulation of plasma NAEs and MAGs by comparing a Western diet, Mediterranean diet,

and habitual diet. First, Jones et al. (2014) examined 36 subjects on a cross-over study, which

showed that 4 weeks consumption of a Mediterranean-like diet, containing high-oleic canola oil,

led to significantly higher plasma OEA levels compared with a Western diet, enriched with LA,

or with a diet enriched with blended oils, containing blended high-oleic canola oil and flaxseed

oil. Also, this study showed that the blended diet had higher levels of ALEA compared to the

Western diet. Diets had no effect on PEA, LEA, AEA or DHEA levels.

Second, Pu et al. (2016b) examined 130 subjects on a cross-over study, which included five diets

containing different combinations of conventional oils. The results showed that OEA levels were

higher in diets enriched with n-9 MUFA, such as canola oil, high-oleic canola oil and high-oleic

canola/DHA oil than diets enriched with n-3 or n-6 PUFA, such as corn/safflower oil and

flax/safflower oil, respectively. Particularly, the diets enriched with n-9 PUFA, high-oleic canola

oil increased OEA levels more than the high-oleic canola/DHA diet. Both LEA and AEA were

higher in the corn/safflower oil and flax/safflower oil diets. DHEA was higher in DHA enriched

n-3 PUFA diet, high-oleic canola/DHA oil; while ALEA was higher in the ALA enriched

flax/safflower oil and canola oil diets.

28

Table 2.4. Endpoint plasma NAEs and MAGs concentrations in human studies Author /

year Diets

Characters/

Duration

NAEs (pmol/ml) MAGs (pmol/ml)

PEA OEA LEA AEA ALEA DHEA 2-AG Other MAGs

Banni et

al. (2011)*δ

KO Normal weight /

4 wks

4.8 ± 0.5 67.4 ± 13.1

Menhaden oil 4.5 ± 0.8 45.4 ± 15.1

OO 6.2 ± 0.8 49.2 ± 17.6

KO Over-weight / 4

wks

6.0 ± 1.0 96.0 ± 25.7

Menhaden oil 5.8 ± 0.5 143.8 ± 35.8

OO 8.2 ± 1.3 97.3 ± 14.9

KO

Obese / 4 wks

6.3 ± 0.7 61.3 ± 10.7

Menhaden oil 6.2 ± 0.5 84.8 ± 11.4

OO 5.5 ± 0.7 72.4 ± 13.2

Jones et

al. (2014)δ

Western diet Hyper-

cholesterolemia /

4 wks

7 ± 0.7 6.5 ± 0.3b 2.9 ± 0.1 2.3 ± 0.1 0.1 ± 0.0a 1.7 ± 0.1

HOCAO/FlaxO 6.3 ± 0.7 6.5 ± 0.6 b 3 ± 0.2 2.2 ± 0.1 0.3 ± 0.1b 1.8 ± 0.1

HOCAO 7.7 ± 2.0 8.3 ± 0.6 a 3.2 ± 0.2 2.5 ± 0.1 0.1 ± 0.0a 2.2 ± 0.3

Mennella

et al.

(2015b)

SunfO Normal weight /

Post-meal 30

mins

15.3 ± 2.0 6.8 ± 0.8 4.2 ± 0.6 3.0 ± 0.1 10.6 ± 2.4

HOSunfO 16.0 ± 2.1 7.8 ± 0.8 4.6 ± 0.9 3.2 ± 0.2 11.4 ± 3.0

Virgin OO 16.6 ± 1.9 8.1 ± 1.0 4.5 ± 0.8 3.1 ± 0.2 12.1 ± 3.4

SunfO Normal weight /

Post-meal 60

mins

15.1 ± 2.2 6.1 ± 0.3 4.2 ± 0.5 2.6 ± 0.1 9.0 ± 3.0

HOSunfO 15.1 ± 1.3 7.9 ± 0.6 4.4 ± 0.5 3.2 ± 0.2 11.2 ± 3.7

Virgin OO 15.1 ± 1.0 7.2 ± 1.1 4.0 ± 0.7 3.0 ± 0.3 11.7 ± 2.9

SunfO Normal weight /

Post-meal 120

mins

12.6 ± 2.2 4.2 ± 0.9a 4.1 ± 0.5 2.1 ± 0.3 9.1 ± 2.7

HOSunfO 12.9 ± 1.8 7.7 ± 0.8b 3.8 ± 0.5 2.6 ± 0.3 8.5 ± 2.4

Virgin OO 13.6 ± 1.4 7.1 ± 0.9b 4.0 ± 0.6 2.5 ± 0.2 8.9 ± 2.5

Pintus et

al. (2013a)

control-cheese Hyper-

cholesterolaemia

/ 3 wks

84.9 ± 7.9a 49.4 ± 7.8

CLA cheese 56.8 ± 6.8b 40.9 ± 6.7

Pu et al.

(2016b)δ

CAO

Healthy / 4 wks

10.7 ± 0.3 6.5 ± 0.1ab 2.5 ± 0.2ab 1.4 ± 0.0ab 0.2 ± 0.0a 3.0 ± 0.1a

HOCAO 11.0 ± 0.3 6.8 ± 0.1a 2.5 ± 0.2ab 1.5 ± 0.1a 0.1 ± 0.0ab 3.0 ± 0.1a

HOCAO-DHA 10.7 ± 0.3 6.1 ± 0.2b 2.2 ± 0.2c 1.3 ± 0.1b 0.1 ± 0.0b 5.1 ± 0.1b

FlaxO/SaffO 11.0 ± 0.3 5.5 ± 0.1c 2.8 ± 0.1bd 1.3 ± 0.1b 0.3 ± 0.0c 2.7 ± 0.2a

CO/SaffO 10.7 ± 0.3 5.5 ± 0.2c 3.0 ± 0.9d 1.3 ± 0.0b 0.1 ± 0.0b 2.7 ± 0.1a

Ramsden

et al.

(2015a)δ

Low n-6 PUFA Chronic

headache patients

/ 12 wks

11.8 ± 2.8 8.3 ± 2.9 1.4 ± 0.3 1.2 ± 0.3b 1,857 ± 687b 6511 ± 1034 (2-OG);

428 ± 134b (2-DHG);

High n-3 / Low

n-6 PUFA 10.8 ± 2.2 8.3 ± 2.2 1.3 ± 0.2 2.2 ± 1.1a 1,471 ± 536a

5179 ± 2851 (2-OG);

656 ± 338a (2-DHG);

The difference of statistical significance was indicated with different alphabet letters. * indicates the reanalysed articles.

29

2.7.6. The impact of dietary fatty acids on animal NAE and MAG levels

The following sections will discuss the effect of dietary fat intake on NAE and MAG levels in

different biological samples.

2.7.6.1 Blood NAE and MAG levels

Six animal studies (Balvers et al. 2013, Lin et al. 2013a, Wood et al. 2010, Jacome-Sosa et al.

2016, Kim et al. 2016, Liisberg et al. 2016) measured the effects of dietary fats on NAE and

MAG levels in the blood, in which four studies (Balvers et al. 2013, Wood et al. 2010, Kim et al.

2016, Liisberg et al. 2016) examined the effect of diets enriched with long-chain n-3 PUFA;

while two studies (Lin et al. 2013a, Jacome-Sosa et al. 2016) focused on the effects of mixed

dietary fatty acids (Table 2.5).

Dietary long-chain n-3 PUFA: Kim et al. (2016) aimed to test the effect of a diet-enriched with

DHA on modulating NAE and MAG levels in plasma of mice after 62 and 118 days of feeding.

The results showed that the diet-enriched with DHA significantly increased plasma DHEA levels

compared to the control diet rich in safflower oil. This DHA diet also increased n-3 PUFA-

derived DHEA. The diet-enriched with DHA reduced levels of n-6 PUFA-derived NAEs and

MAGs including LEA, AEA, DGLEA, DTEA, 1-AG and 2-AG, 1-LG only at day 62 and 2-LG

only at day 118. The diet-enriched with DHA had lower n-9 MUFA-derived OEA and 1-OG.

Interestingly, the diet-enriched with DHA had significantly higher 2-OG levels than the control

diet after 62 days of feeding, while levels of 2-OG were lower on the DHA diet after 118 days of

feeding. As well, the diet-enriched with DHA increased PEA levels only at day 62 and decreased

SEA levels.

Liisberg et al. (2016) investigated the effect of replacing lean pork meat with lean cod meat (rich

in DHA) on NAE and MAG levels. Additionally, this study also included a third group which

was calorie restricted, that is not included in this review. The results demonstrated that the diet-

enriched with DHA increased plasma DHEA and reduced AEA and 2-AG levels after 12 weeks

of feeding.

30

Wood et al. (2010) showed that a DHA-enriched diet (1.25% DHA) increased DHEA and 2-

eicosapentanoylglycerol (2-EPG), but decreased OEA, 2-AG, and 2-OG, compared with a pork-

fat diet (0.25% DHA) in mice. There were no differences in PEA, AEA, eicosanoylethanolamine

(EEA), as well as 2-PG, 2-eicosanoylglycerol (2-EG) or 2-DHG levels.

Balvers et al. (2013) showed that in the free plasma pool, both fish oil diets, containing 1 % or

3%, by weight significantly increased DHEA, but decreased AEA, and OEA compared to the

control, 3% high-oleic sunflower oil diet. However, only the 3% fish oil diet reduced SEA

compared to the control. This study did not report the difference between the two fish oil diets,

so we evaluated NAE and MAG levels across all three diets (mean ± SEM, n, one-way ANOVA

followed by Tukey’s test). The analysis revealed that the 1% of fish oil diet had higher levels of

free pool OEA and SEA than the 3% of fish oil diet. Levels of PEA, AEA, and DHEA were not

statistically different across three diets.

In the free + esterified NAE plasma pool, both the 1% and 3%