EFFECT OF DIFFERENT CONCENTRATIONS OF N-3 AND N-9 FATTY ACIDS ON FATTY ACID ETHANOLAMIDE LEVELS IN RATS by Anthonia O. Olatinsu A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirements of the degree of MASTER OF SCIENCE Department of Human Nutritional Sciences University of Manitoba Winnipeg Copyright © 2017 by Anthonia O. Olatinsu ABSTRACT Dietary fatty acids are precursors of the lipid mediator group of compounds termed fatty acid ethanolamides (FAE). Prolonged intake of specific types of dietary fats has been shown to increase FAE levels. However, the short term effects of qualitative dietary fat intake on FAE levels remain understudied. Hence, the objective of this study was to identify the effect of diets containing varying concentrations of n-9 from canola oil (CO) and n-3 fatty acids from DHA rich oil (DRO) on plasma and organ FAE levels after different time points in male Sprague Dawley rats. Sixty-four rats were randomly assigned into four groups and were fed diets containing 40% as energy of either safflower, 95% CO: 5% DRO, 50% CO: 50% DRO and 5% CO: 95% DRO. These diets were consumed within a 2hr window in all groups. Circulating fatty acid and FAE levels were measured at 3, 6, 12 and 24hr within each group. At 3hr, significant differences. At 3hr, significant differences (p<0.05) in plasma oleoylethanolamide (OEA) levels were seen in the 95% CO group: 5% DRO group and 5% CO group: 95% DRO group as well as between 50% canola oil group: 50% DRO and 5% CO group: 95% DRO. In all dietary groups, palmitoylethanolamide (PEA) levels were not significantly different at 3, 6 and 24hr compared to 0hr, but did at 12hr where the 50% CO:50% DRO group showed significantly lower levels than seen in the 95% CO group, but PEA levels were not different from the 5% canola oil group. Linoleoylethanolamide (LEA) also failed to show any differences in plasma concentrations at all time points in the three dietary groups. Although plasma FAE levels were generally multiple times lower than observed in small intestine, liver or brain, arachidonoylethanolamide (AEA) levels were significantly lower in the 95% DRO group than in the remaining two groups. Plasma docosahexanoylethanolamide (DHEA) showed no difference across all time points except at 24hr where levels were higher (p<0.05) in the 95% DRO group than in the remaining two groups. No differences were seen in the small intestine across time points in any groups. In i liver at 3hr, OEA levels were higher (p<0.05) in the 95% CO group than the groups with lesser concentrations of oleic acid, while liver OEA levels showed no difference at any other time points across dietary groups. LEA levels were higher in 95% CO: 5% DRO group compared to the 5% CO group: 95% DRO group after 3hr of feeding. Liver DHEA levels were observed to be highest in the 5% CO group: 95% DRO group at 3 and 12, but not at 6 or 24hr. The dietary fatty acid composition affects plasma and organ fatty acid profiles in a time dependent manner and also produces time shifts in plasma and organ FAE levels. These dietary induced changes according to time points in the levels of FAEs may translate into discernible changes in energy expenditure and lipid levels which may in turn influence the risk of obesity. ii ACKNOWLEDGMENTS First, I want to deeply appreciate my supervisor, Dr. Peter Jones, for his counsel, supervision and support throughout my research program. His acceptance of me as his research student initiated my journey to Winnipeg. Thanks for being patient and giving me room to grow and develop. It has really been a wonderful and unforgettable experience working with you. I would like to thank my committee members, Dr. Mohammed Moghadasian and Dr. Trust Beta for their suggestions, recommendations and review of my thesis. A big thank you to my lab members for their support and help throughout my program. It has been a pleasure working with all of you. I want to specially thank Jyoti Sihag, Dr. Vanu Ramprasath, and Khatima Khaloufi for their support of my animal study and laboratory work. I would also like to acknowledge the staff at the Richardson Centre for Functional Foods and Nutraceuticals and also my friends, Anjalika, Bridget and Elizabeth. Thanks for creating beautiful memories with me outside the laboratory environment. I would like to acknowledge the financial support I received from the Natural Sciences and Engineering Research Council of Canada throughout my entire graduate program and also my scholarship from the Faculty of Graduate Studies. These sets of funding provided lots of support. Without them, this project would not have been possible. I am saying a big thank you to my parents and siblings for their undying support and encouragement throughout my program. I cannot trade you all for anything. May the good Lord bless you. I love you so much!!! Also, I would like to send thanks to my friends at home and around the world including, Gbemisola Akomolafe, Anyaduba Arthur, Boluwatife Obembe, Bori Atunwa, Olayemi Remi Oni, Adetolani Elegbede, Ayooluwa Jolaosho, and others for their support and assistance as well as for keeping my life outside of Richardson Center entertaining and positive. Omolayomi, thank you for your support and encouragement at times when I thought iii I could not continue. Your patience during times of stress helped make this journey a rewarding and interesting one. I could not have asked for a better companion. iv DEDICATION I dedicate this thesis to God Almighty for giving me wisdom, understanding and most of all the strength to study and successfully complete this program. v TABLE OF CONTENTS ABSTRACT............................................................................................................................... 1 ACKNOWLEDGMENTS .........................................................................................................iii DEDICATION .......................................................................................................................... iv TABLE OF CONTENTS.......................................................................................................... vi LIST OF ABBREVIATIONS ................................................................................................... ix LIST OF FIGURES .................................................................................................................. xi LIST OF TABLES ....................................................................................................................xii CHAPTER 1 .............................................................................................................................. 1 LITERATURE REVIEW....................................................................................................... 1 1.1 Introduction ...................................................................................................................... 1 1.2 Fatty Acid Nomenclature ............................................................................................ 3 1.3 Obesity and Diet ............................................................................................................... 3 1.4 Saturated Fats .............................................................................................................. 5 1.5 Monounsaturated Fats ................................................................................................. 6 1.6 Polyunsaturated Fats ................................................................................................... 7 1.6.1 N-6 PUFA ............................................................................................................ 8 1.6.2 N-3 PUFA ............................................................................................................ 8 1.7 Fatty Acid Ethanolamides ........................................................................................... 9 1.7.1 Arachidonoylethanolamide (AEA) ...................................................................... 9 1.7.2 Oleoylethanolamide (OEA) ............................................................................... 10 vi 1.7.3 Palmitoylethanolamide (PEA) ................................................................................. 11 1.7.4 Docosahexanoylethanolamide ................................................................................. 11 1.8 Study Rationale .............................................................................................................. 11 1.9 Objectives of the Research ........................................................................................ 12 BRIDGE TO CHAPTER 2 .................................................................................................. 13 CHAPTER 2 ............................................................................................................................ 14 Time dependent effects of different concentrations of n-3 and n-9 fatty acids on plasma, liver, small intestine and brain fatty acid levels in rats ........................................................ 14 Abstract ................................................................................................................................ 14 Background .......................................................................................................................... 15 Materials and Methods ......................................................................................................... 16 Animals and Diets ................................................................................................................ 16 Sample Collection and Analysis .......................................................................................... 17 Fatty Acid Extraction and Methylation ................................................................................ 18 Statistical Analysis ............................................................................................................... 18 Results and Discussion......................................................................................................... 18 Food Intake and Body Weight .......................................................................................... 18 Fatty Acid Profiles ............................................................................................................ 18 Discussion ............................................................................................................................ 20 Conclusion............................................................................................................................ 21 BRIDGE TO CHAPTER 3 .................................................................................................. 35 CHAPTER 3 ............................................................................................................................ 36 vii Relationship between circulating fatty acids and fatty acid ethanolamide levels after a single 2hr dietary fat feeding in male Sprague Dawley rats............................................................... 36 Abstract ................................................................................................................................ 36 Introduction .......................................................................................................................... 37 Materials and Methods ......................................................................................................... 39 Fatty Acid Ethanolamide Extraction .................................................................................... 39 Statistical Analysis ............................................................................................................... 40 Results .................................................................................................................................. 40 Fatty Acid Ethanolamide Levels in Plasma, Small Intestine, Liver and Brain at Different Time Points ....................................................................................................................... 40 Correlations between Fatty Acid Ethanolamide Levels and Their Precursor Fatty Acids in Plasma, Small Intestine, Liver and Brain ..................................................................... 42 Discussion ............................................................................................................................ 42 Figure Legend ...................................................................................................................... 46 CHAPTER 4 ............................................................................................................................ 55 Discussion and Conclusion .................................................................................................. 55 Future Directions .................................................................................................................. 56 Limitations and Strengths..................................................................................................... 56 References ................................................................................................................................ 58 viii LIST OF ABBREVIATIONS AA Arachidonic acid AEA Arachidonoylethanolamide ALA α-linolenic acid ANOVA Analysis of variance BF3 Boron trifluoride BM Body mass index BW Body weight CO Canola oil D Days DFA Dietary fatty acids DHA Docosahexaenoic acid DHEA Docosahexanoylethanolamide DRO DHA rich oil EE Energy expenditure EPA Eicosapentanoic acid FAE Fatty acid ethanolamide FA Fatty acid FFM Fat-free mass FFA Free fatty acids GC Gas chromatography GC-FID Gas chromatography-flame ionization detector Hr Hours LA Linoleic acid LCFA Long chain fatty acids ix