Omega-3 Fatty Acids in Brain and Neurological Health 9780124105270, 1865843830, 0124105270

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OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

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OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH RONALD ROSS WATSON University of Arizona, Arizona Health Sciences Center, Tucson, AZ, USA

FABIEN DE MEESTER DMF Ltd Co., Belgium

AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD PARIS • SAN DIEGO • SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 32 Jamestown Road, London NW1 7BY, UK 225 Wyman Street, Waltham, MA 02451, USA 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA Copyright r 2014 Elsevier Inc. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (144) (0) 1865 843830; fax (144) (0) 1865 853333; email: [email protected]. Alternatively, visit the Science and Technology Books website at www.elsevierdirect.com/rights for further information. Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-410527-0 For information on all Academic Press publications visit our website at elsevierdirect.com Typeset by MPS Limited, Chennai, India www.adi-mps.com Printed and bound in United States of America 14 15 16 17 18 10 9 8 7 6 5 4 3 2 1

Contents Preface xi List of Contributors xiii Acknowledgments xvii

4. Unregulated Lipid Peroxidation in Neurological Dysfunction MOTOTADA SHICHIRI, YASUKAZU YOSHIDA AND ETSUO NIKI

Introduction 31 Lipid Oxidation Biomarkers for Neurological Dysfunction 32 Lipid Peroxidation Products from Linoleic Acid 32 Lipid Peroxidation Products from AA 32 Neurological Dysfunction Associated with Lipid Peroxidation 37 Mechanisms of Free Radical Production in Neurological Disorders 38 References 44 Additional References 51

1. Enhanced Longevity and Role of Omega-3 Fatty Acids VIJAY KARAM SINGH AND RONALD ROSS WATSON

Introduction 1 Longevity 1 Food Restriction for Enhanced Longevity 2 Calorie Restriction for Longevity 2 Smoking and Reduced Longevity 2 Genetics, a Key Modifier of Longevity 3 Genetic Diseases and Longevity 3 Genomics 4 Environmental Factors and Longevity 4 Animal Tests and Longevity 5 Omega-3 Fatty Acids and Longevity 5 References 6 Further Reading 7

5. Obesity, Western Diet Intake, and Cognitive Impairment SCOTT E. KANOSKI, TED M. HSU AND STEVEN PENNELL

Obesity and Cognitive Impairment 57 Western Diet Intake and Cognitive Impairment Summary 61 References 61

58

2. Molecular Gerontology: Principles and Perspectives for Interventions

6. Genetic Risk Factors for Diabetic Neuropathy

SURESH I.S. RATTAN

CARMINE GAZZARUSO AND ADRIANA COPPOLA

Introduction 9 Molecular Basis of Aging 10 Genetics, Post-Genetics, and Epigenetics of Aging Aging Interventions 12 Hormetics, Hormesis, and Hormetins 14 References 15

Diabetes Mellitus and Its Complications 63 Diabetic Neuropathy: General Characteristics 63 Risk Factors for Diabetic Neuropathy and pathophysiological Mechanisms 63 Genetic Risk Factors for Diabetic Neuropathy 64 Potential Use of Genetic Risk Factors in Clinical Practice 66 References 67

11

3. Peroxisomal Pathways, their Role in Neurodegenerative Disorders and Therapeutic Strategies

7. n-3 Fatty Acid-Derived Lipid Mediators against Neurological Oxidative Stress and Neuroinflammation

PATRIZIA RISE´, RITA PARONI AND ANNA PETRONI

Peroxisomes 19 Peroxisomal Pathologies 23 Leukodystrophies 24 Therapeutic Strategies 25 Demyelination and Other Leukodystrophies Conclusion 28 References 28

AKHLAQ A. FAROOQUI

Overview 69 DHA in the Brain 70 EPA-Derived Lipid Mediators in the Brain Conclusion 78 References 78

27

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71

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CONTENTS

8. The Impact of Omega-3 Fatty Acids on Quality of Life ONDINE VAN DE REST AND LISETTE CPGM DE GROOT

Introduction 81 Assessment of QoL 81 Current Evidence on Omega-3 Fatty Acids and QoL 82 Discussion 83 Conclusion and Recommendations 84 References 84

Involvement of Lipids, Fatty Acids, and Their Metabolites in Pain Regulation 134 Future Prospects 141 References 142

13. Fish Oil Supplementation Prevents Age-Related Memory Decline: Involvement of Nuclear Hormone Receptors SERGE ALFOS

9. Mammalian Fatty Acid Amides of the Brain and CNS DOMINIK P. WALUK, MATTHEW R. BATTISTINI, DANIEL R. DEMPSEY, EMMA K. FARRELL, KRISTEN A. JEFFRIES, PERRY MITCHELL, LUCAS W. HERNANDEZ, JOSHUA C. MCBRIDE, DAVID J. MERKLER AND MARY C. HUNT

Introduction 87 The Relevance of the Fatty Acid Amides to Neurological Disease 99 Acknowledgements 102 References 102

Introduction 147 Effects of Aging on Incorporation of Docosahexaenoic Acid in Brain Phospholipids 147 Effects of Aging on DHA Biosynthesis 148 Dietary Fish Oil and Prevention of Age-Related Memory Decline 150 DHA Improves Synaptic Plasticity During Aging: Involvement of Retinoid X Receptors and Peroxisome Proliferator-Activated Receptors 153 Summary and Concluding Remarks 155 References 157

10. Low Omega-3 Fatty Acids Diet and the Impact on the Development of Visual Connections and Critical Periods of Plasticity

14. Role of Omega-3 Fatty Acids in Brain and Neurological Health with Special Reference to Clinical Depression

CLAUDIO ALBERTO SERFATY AND PATRICIA COELHO DE VELASCO

H.M. CHANDOLA AND ILA TANNA

Introduction 109 Nutrition and the Impact of Omega-3 Fatty Acids on Brain Development 109 The Development of Visual Topographical Maps 112 Critical Periods for Brain Development 113 Role of Omega-3 on Development of Central Visual Connections 114 References 117

11. The Effects of Omega-3 Polyunsaturated Fatty Acids on Maternal and Child Mental Health MICHELLE PRICE JUDGE, ANA FRANCISCA DIALLO AND CHERYL TATANO BECK

Introduction 121 The Role of Omega-3 Fatty Acids in Neurotransmission 121 DHA and Maternal Mental Health 122 Omega-3 Fatty Acids and Child Mental Health 123 Conclusion 126 References 127

12. Pain as Modified by Polyunsaturated Fatty Acids

Introduction 163 Omega-3 Fatty Acids 163 Status of Omega-3 Fatty Acids in Clinical Depression 165 Neurological Alterations in Depression 167 Possible Mechanisms for Links Between Omega-3 Fatty Acids and Depression 169 Impact of Diet on AHN 170 Clinical Trials Supporting the Role of Omega-3 Fatty Acids in MDD 171 Conversion of ALA to EPA and DHA from Flax Seed Oil 173 Probable Mode of Action of Flax Seed Oil in Depression 173 Conclusion 175 References 176 Further Reading 179

15. Omega-3 Fatty Acid Supplementation for Major Depression with Chronic Disease LAUREN E. LAWSON AND RONALD ROSS WATSON

SHOGO TOKUYAMA AND KAZUO NAKAMOTO

Introduction 131 Factors Involved in the Supply and Physiological Function of Fatty Acids in the Brain 132

Introduction 181 Omega-3 Fatty Acids Summary 185 References 185

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16. The Effectiveness of Fish Oil as a Treatment for ADHD MODHI ALI S. ALSHAMMARI AND RONALD ROSS WATSON

A review of the Literature Conclusion 193 References 198 Further Reading 199

187

17. Fatty Acids and the Aging Brain

21. The Role of Omega-3 Fatty Acids in Hippocampal Neurogenesis

ALYSSA BIANCA VELASCO AND ZALDY S. TAN

Introduction 201 Physiologic Brain Aging 202 Fatty Acids and Brain Aging 205 Function of Omega-3 Fatty Acids in the Brain 206 Sources of Omega Fatty Acids 208 Omega Fatty Acid Metabolism 208 Pathological Brain Aging 209 Protective Effect of Omega-3 Fatty Acids against Dementia Conclusion 213 References 213 Further Reading 219

Substantia Nigra Dopamine Cell Populations Display Differential Vulnerability to Lesions 239 Repercussion of EFA Deficiency or Supplementation on Midbrain Dopaminergic Systems 240 Potential Mechanisms Involved in Substantia Nigra Dopamine Cell Loss Induced by EFA Dietary Restriction 241 Acknowledgments 245 References 245 Further Reading 249

SIMON C. DYALL

Introduction 251 Measurement of Neurogenesis (Markers of Proliferation) Developmental Neurogenesis 253 Adult Hippocampal Neurogenesis 254 References 259

252

212

22. Imaging Brain DHA Metabolism in Vivo, in Animals, and Humans STANLEY I. RAPOPORT AND AMEER TAHA

18. Cerebrovascular Changes: The Role of Fat and Obesity LINNEA R. FREEMAN

Introduction 221 Vascularization of the Brain 221 Effects of a High Fat Diet and Obesity on Overall Health and Proposed Mechanisms 222 Conclusion 226 References 227

19. Effects of Omega-3 Fatty Acids on Alzheimer’s Disease GAURAV PAUL AND RONALD ROSS WATSON

Introduction 231 Alzheimer’s Disease 231 Omega-3 Fatty Acid: a Role in Alzheimer’s Disease? References 235

232

20. Substantia Nigra Modulation by Essential Fatty Acids BELMIRA LARA DA SILVEIRA ANDRADE DA COSTA, PRISCILA PEREIRA PASSOS, HENRIQUETA DIAS CARDOSO, CATARINA GONC ¸ ALVES-PIMENTEL, ERALDO FONSECA DOS SANTOS JUNIOR, JULIANA MARIA CARRAZZONE ´ JO GUEDES BORBA AND RUBEM CARLOS ARAU

Importance of Essential Fatty Acids as Neuroprotectors During Brain Development and Aging 237 Substantia Nigra Vulnerability to Neurodegeneration 238

Introduction 265 Quantitative Imaging of Brain DHA Metabolism in Rodents Summary and Conclusions 272 Acknowledgments 273 References 273

265

23. Obesity and Migraine in Children PASQUALE PARISI, ALBERTO VERROTTI, MARIA CHIARA PAOLINO, ALESSANDRO FERRETTI AND FABIANA DI SABATINO

Introduction 277 Epidemiological Relationship Between Migraine and Obesity 277 Proposed Mechanism for the Relationship Between Obesity and Migraine 279 Influence of Weight Loss on Chronic Headache in Obese People and Effects of the Preventive Treatment of Migraine on Weight Change 281 Conclusions 283 References 283

24. Dietary Omega-3 Sources during Pregnancy and the Developing Brain: Lessons from Studies in Rats CAROLINE E. CHILDS AND PHILIP C. CALDER

Introduction 287 ALA Supplementation and Brain Fatty Acid Composition 288 Long-chain Omega-3 PUFA Supplementation and Brain Fatty Acid Composition 291 Direct Comparison of ALA and Long-chain Omega-3 PUFA Supplementation on Brain Fatty Acid Composition 297 Conclusions 297 References 301

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25. Omega-3 Fatty Acids and Cognitive Behavior

29. Effect of Omega-3 Fatty Acids on Aggression

GRACE E. GILES, CAROLINE R. MAHONEY AND ROBIN B. KANAREK

KEI HAMAZAKI, TOMOHITO HAMAZAKI AND HIDEKUNI INADERA

Introduction 303 Epidemiological Studies: The Association Between Omega-3 PUFA Intake, Omega-3 PUFA Levels, and Cognitive Decline 314 Conclusion 322 References 322

Introduction 359 First Trial of Omega-3 PUFAs and Aggression in Young Adults 359 Conclusion 362 References 364

30. Multiple Sclerosis: Modification by Fish Oil 26. Lipids and Lipid Signaling in Drosophila Models of Neurodegenerative Diseases KYOUNG SANG CHO, SE MIN BANG AND AMANDA TOH

Introduction 327 Drosophila as a Model System of Neurodegenerative Diseases 327 Effects of Lipids and Lipid Signaling on Drosophila Models of Neurodegenerative Diseases 330 Points to Consider When Drosophila Models are Used for Studying the Role of Lipids 334 Perspective 334 References 335

27. Polyunsaturated Fatty Acids in Relation to Sleep Quality and Depression in Obstructive Sleep Apnea Hypopnea Syndrome CHRISTOPHER PAPANDREOU

Introduction 337 OSAHS 337 Conclusion 345 References 345 Further Reading 347

28. Omega-3 Fatty Acids in Intellectual Disability, Schizophrenia, Depression, Autism, and Attention-Deficit Hyperactivity Disorder BASANT K. PURI AND DINA GAZIZOVA

Introduction 349 Background 349 Plasma Lipids in Adults with Intellectual Disability and the Efficacy of Omega-3 Fatty Acids 350 Plasma Lipids in Schizophrenia and the Efficacy of Omega-3 Fatty Acids 352 Plasma Lipids in Depressive Illness and Efficacy of Omega-3 Fatty Acids 353 Plasma Lipids in Autism and Efficacy of Omega-3 Fatty Acids 354 Plasma Lipids in ADHD and Efficacy of Omega-3 Fatty Acids 355 Conclusions 355 References 356

GILBERT LUJAN RIVERA JR. AND RONALD ROSS WATSON

Introduction 367 Overview 367 Cellular Level of Multiple Sclerosis 368 Omega-3 Supplementation of MS Patients 369 Varying Results Amongst Researchers 369 Summary 370 Acknowledgement 371 References 371

31. Deuterium Protection of Polyunsaturated Fatty Acids against Lipid Peroxidation: A Novel Approach to Mitigating Mitochondrial Neurological Diseases MIKHAIL S. SHCHEPINOV, VITALY A. ROGINSKY, J. THOMAS BRENNA, ROBERT J. MOLINARI, RANDY TO, HUI TSUI, CATHERINE F. CLARKE AND AMY B. MANNING-BO˘g

Introduction 373 PUFAs in Mitochondrial Membranes and Oxidative Stress 373 Mitochondrial Dysfunction, Oxidative Stress, and PD 375 Isotope Protection of PUFAS Against autoxidation 376 Yeast Models Confirm the Non-Linear Protective Effect of D-PUFAs in vivo 378 Isotope Reinforcement of PUFA in Pre-Clinical PD Modeling 378 Conclusion 380 References 381

32. Obesity, Cognitive Functioning, and Dementia: A Lifespan Prospective MERRILL F. ELIAS, GEORGINA E. CRICHTON AND AMANDA L. GOODELL

Introduction 385 Weight-Related Variables and Dementia 385 Weight and Cognitive Function: Role of CVD Factors 389 Initial Summary 391 MetS 391 Mechanisms: General 392 Early Influences on Relations between Obesity and Cognition 393 Morbid Obesity and Clinically Important Cognitive Deficit 394 Treatment of Overweight and Obesity 395 Treatment of Obesity with Omega-3 Fatty Acids 396

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General Discussion References 441

Omega-3 Fatty Acid Mechanisms for Reducing Weight Loss 397 Methodologies: General Issues 397 Final Summary and Conclusions 398 References 399

36. Fish Oil Supplements, Contaminants, and Excessive Doses

33. Dairy Products and Cognitive Functions GEORGINA E. CRICHTON AND MERRILL F. ELIAS

Introduction 403 Review of the Literature 403 Early Cross-Sectional and Prospective Research Findings in Associations between Dairy and Cognition 405 Discussion 410 Summary 413 Directions for Future Research 413 References 413 Further Reading 427

34. Obesity and Chronic Low Back Pain: A Kinematic Approach VERONICA CIMOLIN, LUCA VISMARA, MANUELA GALLI, NICOLA CAU AND PAOLO CAPODAGLIO

Introduction 417 Quantitative Movement Analysis Conclusions 425 References 425

418

35. Fatty Acids and the Hippocampus HEATHER M. FRANCIS AND RICHARD STEVENSON

Introduction 429 Fatty Acids and Memory/Hippocampus Fatty Acids and AD 435

440

429

NICOLE BURCA AND RONALD ROSS WATSON

Introduction 447 Mercury 447 Lead 449 Selenium 449 Arsenic 450 Cadmium 451 PCBs 451 Dichlorodiphenyltrichloroethane Dieldrin 452 Conclusion 453 Acknowledgment 453 References 453

452

37. Introduction to Fish Oil Oxidation, Oxidation Prevention, and Oxidation Correction PETER LEMBKE AND ANETT SCHUBERT

Introduction 455 Oxidation Process 455 Oxidation Indicators 455 Oxidation Correction 458 Summary 459 References 459 Further Reading 460

Index 461

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Preface

Brain disorders resulting from omega-3 fatty acid deficiencies or inadequacies are one of the major preventative health opportunities, as treatment by dietary food and supplementation show great benefits, are safe, and relatively economic. As reviewed by Singh and Watson, the dietary long-chain omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are showing benefits in longevity as well as in molecular gerontology applications, as defined by Rattan. Their mechanisms of action in neurological tissues include unregulated lipid peroxidation, as described by Yoshida and Shichiri, with peroxisomal pathways in neurodegenerative disorders and therapeutic strategies, as summarized by Rise, Paroni, and Petroni. Not surprisingly, mental health issues are significantly affected due to low intakes of omega-3 fatty acids in much of the world on a similar magnitude to that of the expanding obesity epidemic. Kanoski describes the role of traditional Western diets, with their promotion of obesity, on cognitive impairment. For example, the Japanese, through a high consumption of fish, have an adequate intake of 1000 mg/day, while typical European diets provide only 100
125 mg/day. This yields inadequacy or deficiency within the brain and neurological effects, so that adequate levels of omega-3 fatty acids impact the quality of life, according to van de Rest. Farooqui defines the fatty acids’ lipid mediators which reduce neurological oxidative stress as key mechanisms of action. Gazzaruso and coworkers note the importance of genetic risk factors as targets in the major chronic disease diabetic neuropathy.

on neurological diseases is still emerging. Waluk and coauthors describe the actions of mammalian fatty amides in the brain and central nervous system to help understand their roles in health and disease. Similarly, Serfaty and coworkers describe the extensive data showing that low omega-3 fatty acids adversely affect development of visual connections. With industrialization, societies have changed their traditional dietary consumption of fats, frequently with adverse consequences to health and mental function. A sedentary lifestyle with increased availability of high energy foods has occurred. There has been a dramatic increase in the use of corn oil with its omega-6 polyunsaturated fatty acids, trans fatty acids, with more animal products modified by diets high in fats. Simultaneously, the use of omega-3 fatty acids, primarily from fish, has been reduced. In the past several decades there has been approximately a 10-fold increase in the ratio of omega-6 compared to omega-3 consumption. As expected, Tokuyama found that omega-3 fatty acids modulated other aspects of neurological dysfunction, especially pain. In addition, Judge and coworkers found significant confirmatory research on the role of fatty acids in maternal and child mental health. At the other end of the age spectrum, Alfos defines the role of fish oil containing omega-3 fatty acids in preventing age-related memory loss via hormone receptors, while Tan reviews blood levels of fatty acids in aging brain function to understand their mechanisms of action. Paul also found limited evidence that omega-3 fatty acids acted on Alzheimer’s disease. Clearly the companion omega-6 fatty acids have adverse effects in high or disproportionate levels. Unfortunately, omega6 fatty acids are the primary fatty acids consumed in most Western diets. Freeman described the association between the accumulation of other fats and fatty acids and obesity and changed cerebrovascular functions. The actions of such fatty acids in clinical depression are outlined by Chandola and coworkers, and their effects on major depression are outlined by Lawson. In children, their potential functions incorporated in fish oil in the growing epidemic of attention deficit hyperactivity disorder (ADHD) are summarized and reviewed by Ali.

FATTY ACIDS AND THE BRAIN It is critical that more scientists research and evaluate the effects of long-chain omega-3 fatty acids in brain health and neurological function. This will increase understanding at government, scientific, and importantly, general population levels. While it is increasingly accepted that omega-3 fatty acids in high levels benefit a variety of human diseases, their effects

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PREFACE

OMEGA-3 FATTY ACIDS IN STRUCTURAL AND FUNCTIONAL NEUROLOGICAL CHANGES Actual brain and neuron structural changes are important in brain dysfunction. Reviews by da Costa showed that substantia nigra was modulated by omega-3 fatty acids, while Dyall found that they modulated hippocampal neurogenesis. Francis also described the effects of dietary fatty acids on the hippocampus. As might be expected, Parisi’s review found that obese children with high levels of fat and non-omega-3 fatty acids were at more risk of migraine headaches. Lower back pain was similarly susceptible to a kinematic approach in the obese, as summarized by Cimonlin and coauthors.

FATTY ACIDS AND NEUROLOGICAL OUTCOMES Fly models are routinely used as efficient systems to look at neurological signaling. Cho summarizes the role of lipids and their signaling on insect models of neurological diseases. Human diseases with a neurological component, such as cognitive behavior, are reviewed by Giles, while Papandreou summarized their role in sleep quality with its depression in obstructive sleep apnea. Puri described other neurological dysfunctions as being modified by omega-3 fatty acids including intellectual disability, schizophrenia, and autism, while Hamazaki and coworkers discussed their actions on aggression. Rivera described the autoimmune disease, multiple sclerosis, looking for a role of therapy with fish oil. The review by Elias provides a life-span prospective of the role of fatty acids in obesity, and thus, cognitive function and dysfunction yielding dementia. In addition to the standard fish oil as neuromodulators, Elias describes dairy products and their actions on the brain via cognitive function.

CONTAMINANTS AND THEIR REMOVAL FROM FISH OILS Fish oils are a major source of dietary and supplementary omega-3 fatty acids. They do, however, have the potential for contamination. Burca reviewed the

accumulation of pesticides, mercury, and other such materials that can be taken up by plants which are a food source for fish. Finally, Lembke reviews the risks of oxidation to fatty acids, its prevention, and correction to keep them useful and beneficial as well as safe. Shchepinov and coworkers describe the isotope deuterium as a novel approach to reducing mitochondrial lipid peroxidation, and thus, neurological diseases. In summary, fish oils provide some protection of cognitive function during dementia with senile decline in Alzheimer’s disease, normal aging of the brain, some peroxisomal biogenesis disorders, and multiple sclerosis. The book experts review the role and effect of various intakes of omega-3 fatty acids on cognitive function in normal aging, dementia and its treatment, multiple other neurological diseases, and the progression of multiple sclerosis and related disorders, citing research that supports the use of dietary and food sources to increase omega-3 fatty acid intake to promote health. The second section of the book focuses on the wide variety of neurological effects of high and low omega-3 and/or omega-6 fatty acids. The third section aims at defining the role of omega-3 fatty acids in some of the various structural changes in neurons and neurological tissues. The fourth section relates to special roles during the time neurological tissues are developing, primarily in infants and children and the importance of omega-3 fatty acid supplementation. The fifth section focuses on a diffuse area of diseases that have a neurological component, but are not primarily neurological diseases. The final section aims to define neurological diseases due to immune dysfunction, particularly multiple sclerosis. Clearly, fatty acids fit the modern paradigm of being dangerous in the wrong amounts and types, while being health promoting otherwise. This information, as it is analyzed and defined by experts in this book, will promote research as well as logical, health promoting choices by patients and their physicians. These are increasingly science-based dietary choices to prevent and treat neurological diseases that are increasingly prevalent in the 21st century, due to an increasingly aged population living with lifestyle changes, moving us away from the traditional diets and foods of our ancestors. Ronald Ross Watson

List of Contributors

Serge Alfos University of Bordeaux and INRA UMR 1286 Laboratory of Nutrition and Integrative Neurobiology Bordeaux, France Modhi Ali S. Alshammari Mel and Enid Zuckerman College of Public Health, University of Arizona, Arizona Belmira Lara da Silveira Andrade da Costa Departamento de Fisiologia e Farmacologia, Centro de Cie`ncias Biolo´gicas, Universidade Federal de Pernambuco, Recife (PE), Brasil Se Min Bang Department of Biological Sciences, Konkuk University, Seoul, Republic of Korea Matthew R. Battistini Department of University of South Florida, Tampa, Florida

Chemistry,

Cheryl Tatano Beck University of Connecticut School of Nursing, Connecticut

Catherine F. Clarke Department of Chemistry and Biochemistry, UCLA, Los Angeles, California Adriana Coppola Internal Medicine, Diabetes, Vascular and Endocrine-metabolic Diseases Unit and the Centre for Applied Clinical Research, Clinical Institute Beato Matteo, Vigevano, Italy, and Department of Internal Medicine, San Donato Milanese, Italy Georgina E. Crichton Nutritional Physiology Research Centre, University of South Australia, Adelaide, Australia Lisette C.P.G.M. de Groot Wageningen University, Division of Human Nutrition, the Netherlands Daniel R. Dempsey Department of Chemistry, University of South Florida, Tampa, Florida

Juliana Maria Carrazone Borba Departamento de Nutric¸a˜o, Centro de Cie`ncias da Sau´de, Universidade Federal de Pernambuco, Recife (PE), Brasil

Patricia Coelho de Velasco Laborato´rio de Plasticidade Neural, Departamento de Neurobiologia, Programa de Po´s-Graduac¸a˜o em Neurocieˆncias, Instituto de Biologia, Universidade Federal Fluminense, Nitero´i, Brazil

J. Thomas Brenna Division of Nutritional Sciences, Cornell University, Ithaca, NY, USA

Ana Francisca Diallo University of Connecticut School of Nursing, Connecticut

Nicole Burca University of Arizona Mel and Enid Zuckerman College of Public Health, and School of Medicine, University of Arizona

Fabiana Di Sabatino Division of Child Neurology, Faculty of Medicine & Psychology, Sapienza University, Rome, Italy

Philip C. Calder Human Development & Health Academic Unit, Faculty of Medicine, University of Southampton, Southampton, United Kingdom

Simon C. Dyall Department of Life Sciences, University of Roehampton, Whitelands College, London

Paolo Capodaglio Rehabilitation Unit and Research Laboratory in Biomechanics and Rehabilitation, San Giuseppe Hospital, Istituto Auxologico Italiano IRCCS, Piancavallo (VB), Italy Henriqueta Dias Cardoso Departamento de Fisiologia e Farmacologia, Centro de Cie`ncias Biolo´gicas, Universidade Federal de Pernambuco, Recife (PE), Brasil Nicola Cau Department of Electronics, Information and Bioengineering, Politecnico di Milano, Italy H.M. Chandola Ch. Brahm Prakash Ayurved Charak Sansthan, Khera Dabar, Najafgarh, New Delhi, India Caroline E. Childs Human Development & Health Academic Unit, Faculty of Medicine, University of Southampton, Southampton, United Kingdom Kyoung Sang Cho Department of Biological Sciences, Konkuk University, Seoul, Republic of Korea Veronica Cimolin Department of Electronics, Information and Bioengineering, Politecnico di Milano, Italy and Rehabilitation Unit and Research Laboratory in Biomechanics and Rehabilitation, San Giuseppe Hospital, Istituto Auxologico Italiano IRCCS, Piancavallo (VB), Italy

Merrill F. Elias Department of Psychology, University of Maine, Orono, Maine, USA and Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, Maine, USA Akhlaq A. Farooqui Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio Emma K. Farrell Department of Chemistry, University of South Florida, Tampa, Florida Alessandro Ferretti Division of Child Neurology, Faculty of Medicine & Psychology, Sapienza University, Rome, Italy Heather M. Francis Department of Macquarie University, Sydney, Australia

Psychology,

Linnea R. Freeman Medical University of South Carolina, Charleston, South Carolina Dina Gazizova Central and North West London NHS Foundation Trust, London Manuela Galli Department of Electronics, Information and Bioengineering, Politecnico di Milano, Italy and IRCCS San Raffaele Pisana Tosinvest Sanita`, Roma, Italy

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LIST OF CONTRIBUTORS

Carmine Gazzaruso Internal Medicine, Diabetes, Vascular and Endocrine-metabolic Diseases Unit and the Centre for Applied Clinical Research, Clinical Institute Beato Matteo, Vigevano, Italy, and Department of Internal Medicine, San Donato Milanese, Italy

Robert J. Molinari

Retrotope Inc., Los Altos, California

Kazuo Nakamoto Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan

Tufts

Etsuo Niki Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Osaka, Japan

Catarina Gonc¸alves-Pimentel Departamento de Fisiologia e Farmacologia, Centro de Cieˆncias Biolo´gicas, Universidade Federal de Pernambuco, Recife (PE), Brasil

Maria Chiara Paolino Division of Child Neurology, Faculty of Medicine & Psychology, Sapienza University, Rome, Italy

Amanda L. Goodell Department of University of Maine, Orono, Maine, USA

Psychology,

Christopher Papandreou Department of Nutrition & Dietetics, Harokopio University of Athens, Athens, Greece

Rubem Carlos Arau´jo Guedes Departamento de Nutric¸a˜o, Centro de Cieˆncias da Sau´de, Universidade Federal de Pernambuco, Recife (PE), Brasil

Pasquale Parisi Division of Child Neurology, Faculty of Medicine & Psychology, Sapienza University, Rome, Italy

Kei Hamazaki Department of Public Health, Faculty of Medicine, University of Toyama, Toyama, Japan

Rita Paroni Department of Health Science, University of Milan, Milan, Italy

Tomohito Hamazaki Laboratory for Molecular Psychiatry, RIKEN Brain Science Institute, Saitama, Japan

Priscila Pereira Passos Departamento de Fisiologia e Farmacologia, Centro de Cieˆncias Biolo´gicas, Universidade Federal de Pernambuco, Recife (PE), Brasil

Grace E. Giles Department University, Medford, MA

of

Psychology,

Lucas W. Hernandez Department of University of South Florida, Tampa, Florida

Chemistry,

Ted M. Hsu Neuroscience Graduate Program, University of Southern California, California Mary C. Hunt School of Biological Science, Dublin Institute of Technology, Dublin, Ireland Hidekuni Inadera Department of Public Health, Faculty of Medicine, University of Toyama, Toyama, Japan Kristen A. Jeffries Department of Chemistry, University of South Florida, Tampa, Florida Michelle Price Judge University of Connecticut School of Nursing, Connecticut Robin B. Kanarek Department of Psychology, Tufts University, Medford, MA Scott E. Kanoski Department of Biological Sciences, University of Southern California, and Neuroscience Graduate Program, University of Southern California Lauren E. Lawson Mel and Enid Zuckerman College of Public Health, University of Arizona, Arizona

Gaurav Paul Mel and Enid Zuckerman College of Public Health, University of Arizona, Arizona Steven Pennell Neuroscience University of Southern California

Graduate

Program,

Anna Petroni Department of Pharmacological and Biomolecular Sciences, University of Milan, Milan, Italy Basant K. Puri

Imperial College London, London

Stanley I. Rapoport Brain Physiology and Metabolism Section, Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, MD Suresh I.S. Rattan Laboratory of Cellular Ageing, Department of Molecular Biology and Genetics, Aarhus University, Denmark Patrizia Rise´ Department of Pharmacological and Biomolecular Sciences, University of Milan, Milan, Italy Gilbert Lujan Rivera Jr. Mel and Enid Zuckerman College of Public Health Undergraduate Student, University of Arizona, Arizona

KD Pharma Bexbach GmbH, Bexbach,

Vitaly A. Roginsky Institute of Chemical Physics, Russian Academy of Sciences, Moscow, Russia

Caroline R. Mahoney Department of Psychology, Tufts University, Medford, MA

Eraldo Fonseca Santos Junior Departamento de Fisiologia e Farmacologia, Centro de Cieˆncias Biolo´gicas, Universidade Federal de Pernambuco, Recife (PE), Brasil

Peter Lembke Germany

Amy B. Manning-Bog˘ Center for Health Sciences, SRI International, Menlo Park, CA, USA Joshua C. McBride Department of Chemistry, University of South Florida, Tampa, Florida David J. Merkler Department of Chemistry, University of South Florida, Tampa, Florida Perry Mitchell Department of Chemistry, University of South Florida, Tampa, Florida

Anett Schubert Germany

KD Pharma Bexbach GmbH, Bexbach,

Claudio Alberto Serfaty Laborato´rio de Plasticidade Neural, Departamento de Neurobiologia Programa de Po´s-Graduac¸a˜o em Neurocieˆncias, Instituto de Biologia, Universidade Federal, Fluminense, Nitero´i, Brazil Mikhail S. Shchepinov USA

Retrotope, Inc., Los Altos, CA,

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LIST OF CONTRIBUTORS

Mototada Shichiri Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Kagawa, Japan Vijay Karam Singh Mel and Enid Zuckerman College of Public Health, University of Arizona R.J. Stevenson Department of Psychology, Macquarie University, Sydney, Australia Ameer Taha Brain Physiology and Metabolism Section, Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, MD Zaldy S. Tan Department of Medicine, Division of Geriatric Medicine, David Geffen School of Medicine, University of California at Los Angeles, Easton Center for Alzheimer’s Disease Research and the UCLA Alzheimer’s and Dementia Care Program, Los Angeles Ila Tanna Mahatma Gandhi Ayurved College, Hospital & Research Centre, Datta Meghe Institute of Medical Sciences, Sawangi, Wardha (Maharashtra), India Randy To Department of Chemistry and Biochemistry, UCLA, Los Angeles, California Amanda Toh School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, Singapore Shogo Tokuyama Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan

Hui Tsui Department of Chemistry and Biochemistry, UCLA, Los Angeles, California Ondine van de Rest Wageningen University, Division of Human Nutrition, the Netherlands Alyssa Bianca Velasco, Department Boston Children’s Hospital, Boston

of

Cardiology,

Alberto Verrotti Division of Child Neurology, Faculty of Medicine & Psychology, Sapienza University, Rome, Italy Luca Vismara Rehabilitation Unit and Research Laboratory in Biomechanics and Rehabilitation, San Giuseppe Hospital, Istituto Auxologico Italiano IRCCS, Piancavallo (VB), Italy Dominik P. Waluk Department of Biochemistry and Biophysics (DBB), Stockholm University, Stockholm, Sweden. Ronald Ross Watson Mel and Enid Zuckerman College of Public Health, University of Arizona, Arizona Yasukazu Yoshida Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Osaka, Japan

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Acknowledgments

The work of Dr. Watson’s editorial assistant, Bethany L. Stevens and the project manager, Kristi Anderson, in communicating with authors and working on the manuscripts was critical to the successful completion of the book. It is very much appreciated. Support for Ms. Stevens’ and Dr. Watson’s work was graciously provided by Southwest Scientific Editing & Consulting

LLD, DMF Ltd, the TsimTsoum Institute, and the Natural Health Research Institute. Finally, the work of librarian Mari Stoddard, of the Arizona Health Science Library, was vital and very helpful in identifying key researchers who participated in the book.

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Robert Ross Watson and Fabian De Meester

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C H A P T E R

1 Enhanced Longevity and Role of Omega-3 Fatty Acids Vijay Karam Singh and Ronald Ross Watson INTRODUCTION

life expectancy of 82 years of age, whereas Nigeria has a life expectancy of 50 years of age. A variety of different studies describe how longevity has changed over the past 200 years. The mysteries of longevity have always been looked into as something that is both interesting and fascinating. Ultimately, longevity has been shown to be the result of a combination of many contributory factors. Trends also differ depending on the time and situation within different regions. As the baby boomer generation of the 1960s ages, increasing attention is being given to the study of longevity (Myers and Ryu, 2008). The increase in the worldwide proportion of the population that is elderly is a major economic and healthcare issue. There is more information available now to help us define how longevity has come about. How are centenarians able to escape the ailments present in aging? The elderly populace in the United States is growing in size, unsettling the declining death rates, growing life expectancy, and the aging of baby boomers (Rice and Fineman, 2004). Although the prevalence of chronic illnesses and disabilities has now increased with age, successful aging in the elderly population is widespread, and the elderly populace is generally healthy (Rice and Fineman, 2004). Deciphering the reasons for longevity uncovers a variety of different perspectives. Some of the primary factors affecting longevity include: staying smoke-free, exercising, eating healthily, getting a healthy amount of sleep, staying mentally and physically active, along with consumption of healthy foods and dietary supplements (Rice and Fineman, 2004). This review will investigate a food and dietary supplement known as omega-3 fatty acids and will look at how this modifies longevity. The intake of omega-3 fatty acids may play a key role in assessing an individual’s life span. How can longevity be enhanced by fish oils, and what has

The average life span for a human being is 78 years of age. Longevity is generally defined as duration of life, an individual’s life span. There are many different contributory factors that develop someone’s longevity including behavior, diet, exercise, and overall health. Further extending the average human life expectancy will be a very profound advance for science as well as the world. Longevity in humans may also be enhanced by omega-3 fatty acids. Omega-3 fatty acids are found in the oils of fish, algae, squid, and a few diverse plants. They have many health benefits, are considered to be ‘essential’ fatty acids to the body, and are depressed by omega-6 fatty acids. This review will investigate the key factors that affect longevity, focusing on omega-3 fatty acids.

LONGEVITY Life expectancy is a major factor in human progression. Historically, the human population has had a very low life expectancy. The increasingly aging nature of populations is a current phenomenon in most western societies. Evidence for this is given by the large increases in the number of older (85 years or older) humans (Waite, 2004), the increase in the number of centenarians (Robine and Paccaud, 2005), along with steady recordings of maximum-recorded life span (Wilmoth, 2000). These facts have contributed to an increased interest in the question of what really causes us to live longer. Longevity is defined as the capability to survive past the average age of death (De Benedictis and Franceschi, 2006). Developed countries such as Japan have the highest life expectancy rate, much higher than many African nations. Japan has an average Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00001-6

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© 2014 Elsevier Inc. All rights reserved.

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been discovered from recent studies about it? And do fatty acids really have a key role in health and longevity enhancement?

FOOD RESTRICTION FOR ENHANCED LONGEVITY Experimental data has indicated that oxidative stress contributes to processes related to aging and to the pathogenesis of many age-related diseases (Mecocci et al., 2000). Vitamins along with antioxidant enzymes have an important role in protecting the body from oxidative stress (Mecocci et al., 2000). Records show that most centenarians have lived very healthy lifestyles (Longo and Finch, 2003). Restriction of the number of calories consumed is associated with extending longevity for many organisms (Longo and Finch, 2003). This association is quite clear due to how they are able to exercise daily, and along with being able to live a smoke- and alcoholfree lifestyle. With improvements in nutritional choices available, there may well be a great increase in the numbers of centenarians in the near future. A major problem facing many western societies today is obesity (Van Itallie, 1979). The body’s fat content, as well as its proportion of fat (triglyceride levels), continues to increase with age (Van Itallie, 1979). Levels of dietary omega-6 fatty acids are also increasing and these are a risk factor for premature death. Obesity is developed by diets that are calorically dense (Van Itallie, 1979), and which thus increase overall morbidity in an individual’s system. With increasing rates of obesity, many people diet in an attempt to lose weight. However, nutrition offers the most effective means to improve health and overall well-being for successful aging and longevity (Van Itallie, 1979). Nutrition is a major determinant for longevity in centenarians and potential centenarians. Adequate nutrition is a major factor in determining how an individual progresses through their day mentally and physically (Van Itallie, 1979). In contrast, it is also the case that inadequate nutrition on a daily basis impairs an individual’s ability to be productive. Insufficient functioning has also been associated with progressive onset of diseases over time. Increased obesity and caloric intake has also been associated with exacerbation of diseases and disorders such as cardiovascular disease, diabetes, and cancer (Van Itallie, 1979).

CALORIE RESTRICTION FOR LONGEVITY Caloric restriction remains the most highly researched, non-genetic intervention in order to

improve health and also increase life span in a wide variety of organisms ranging from single-celled yeast organisms all the way to non-human primate models (Wilson et al., 2008). The benefits that are shown to come from having good health and longevity are directly proportional to the amount of caloric restriction an organism undergoes. Caloric restriction can also be considered a very dangerous issue, however, especially since it can sometimes lead to malnutrition (Kaiser et al., 2012). A major phenotype that is noticed among all organisms undergoing caloric restriction is the reduction of overall body weight as well as body fat (Sullivan and Cameron, 2010). When rodents are studied for longevity tests, there are many that go through a great deal of age-associated obesity, and this is even true when they are fed with a low-fat diet. When the rodents are fed a high-fat diet, they are shown to have a diet-induced obesity response similar to that expected of human beings who over-consume a calorie-rich diet (Kaiser et al., 2012). With switching from a low-fat to a calorie-rich diet, caloric restriction is shown to have an induced, rapid weight loss effect as well. The transition from a ‘negative energy balance’ is shown to continually equalize to the point that the reduced body weights are now entered into a normal energy balance (Kaiser et al., 2012). The body weight, as well as the body composition changes that are correlated with that of caloric restriction, are shown to be more long term overall, rather than a temporary effect that is experienced with the initial beginnings of caloric restriction (Kaiser et al., 2012). In rodent studies, caloric restriction was found to decrease the levels of overall plasma glucose and insulin-like growth factors (Longo and Finch, 2003). This is also associated with postponing cancer, inflammation, and immunosenescence without side effects occurring in the body (Longo and Finch, 2003). Organisms from yeast to mice have had mutations developing due to the insulin-like growth factors signaling pathways throughout their system (Longo and Finch, 2003). Results of the tests found that this signaling from the growth factors was associated with extending the life span but had also been known to cause fat accumulation throughout the body (Longo and Finch, 2003).

SMOKING AND REDUCED LONGEVITY Smoking cessation is a very important step in living a healthy lifestyle and in avoiding major issues later in life. Smoking still remains the leading cause of preventable death in the United States (McGinnis and Foege, 1993). Roughly 45 million Americans and 1.2 billion people worldwide use tobacco (Taylor et al., 2002).

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

GENETIC DISEASES AND LONGEVITY

Smoking cessation has been shown to have welldocumented health benefits (Taylor et al., 2002). Once someone quits smoking, the risk of lung cancer will decrease dramatically (Taylor et al., 2002). Recent studies from the UK have found that 90% of excess mortality attributable to cigarette smoking can be avoided if people quit before middle age is reached (Darby et al., 2000). With the decrease in the risk of lung cancer and overall mortality, overall longevity will increase significantly for individuals who undertake smoking cessation. Smoking cessation is shown to substantially reduce mortality risk and therefore increase longevity (Taylor et al., 2002). With the many risks of death that are associated with smoking, there are many different ways in which the mortality risk can decrease with smoking cessation (Freeman et al., 2006). Longevity improves due to the fact that smoking cessation gradually increases an individual’s life expectancy. Dietary restrictions persist as cessation begins, and omega-3 fatty acids thus play a key role in this process. The health benefits of omega-3 fatty acids are very important for patients that have psychiatric disorders due to high rates of smoking and obesity (Freeman et al., 2006). The levels of omega-3 fatty acids are lower in smokers than non-smokers, partly due to the severity of the psychiatric symptoms adversely affecting the smokers’ diets, self-care, and overall life span (Freeman et al., 2006).

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concerned with how the genetic variations are associated with each other (Sebastiani et al., 2012). Depending on the average genetic variations for a human being, most people can live up to their eighties or nineties. It is due to the significant impact of such genetic factors that people are not able to become more adaptive with their age (Sebastiani et al., 2012). Human society has shown considerable variation in mortality and longevity characteristics, and yet it has also shown a common increase in the average life expectancy in the past two centuries (Oeppen and Vaupel, 2002). There are many factors that could have lead to this: better hygiene, medicine, nutrition, and healthcare. Although there is a large variation of healthy life span, exceptional longevity can be reached with a low degree of age-related disabilities (Christensen et al., 2008). For life span extensions though, the most prominent genetic influence is through families (Perls et al., 2000). Unlike most genetic variations present with age, human longevity is presumed to be a complex trait (Finch and Tanzi, 1997). Western diets are deficient when it comes to omega-3 and omega-6 fatty acid intake compared to diets where humans evolved and their genetic patterns were established (Simopoulos, 1991). Omega-3 fatty acids play a role in maintaining the overall structure of the body, by providing neurological function that passes down through families (Simopoulos, 1991).

GENETIC DISEASES AND LONGEVITY GENETICS, A KEY MODIFIER OF LONGEVITY Longevity can be present amongst genetic factors through lifestyle alternatives, especially through individual and heritable genetics (Sebastiani et al., 2012). Considerable life span extensions have been found from organisms as diverse as yeast, worms, fish, flies, and even rodents (Slagboom et al., 2011). All of these models have shown life span extension mainly due to control through dietary restriction and genetic manipulation (Kenyon, 2010). There are numerous genetic pathways that can indicate longevity is stimulated through the influence of metabolism and the opposition of oxidative stress (Slagboom et al., 2011). A major challenge with this is how genetic variations have a great deal of range between each organism, and the complexity here is how each phenotype can contain the information as well. Genetics has also been shown to have a major effect on longevity. There has been a substantial difference identified between the genetics of aging and the genetics of longevity (Sebastiani et al., 2012). There have been a large number of studies into longevity

The majority of centenarians have distinctly delayed high mortality risk-associated diseases such as cancer towards the end of their lives (Sebastiani et al., 2012). The evidence of families living with long life spans (Barker, 2007), and children of centenarians experiencing delays in age-related diseases (Finch and Tanzi, 1997), and the similarity of centenarians’ lifestyles in the general population (Westendorp et al., 2009) all support the argument that genetic factors do play a very strong role in longevity in people living beyond their mid-eighties (Skytthe et al., 2003). With these results, centenarians have had a major history of enduring more chronic age-related diseases for many years, which are more prevalent in women than men (Sebastiani et al., 2012). Even with these chronic diseases present, centenarians usually do not feel the effects until well into their nineties (Sebastiani et al., 2012). Unlike most centenarians, people who live to more extreme ages, 107 years and more, generally support the morbidity hypothesis (Sebastiani et al., 2012). This means that they reduce morbidity and compress most of the disabilities present when they reach the elder years of their lives (Passtoors et al., 2008).

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There is a strong familial component to extreme longevity and findings are now available from the New England Centenarian Study, revealing that familial genetics is a very significant survival component for people over the age of 100 years (Bae et al., 2013). The genetic component also has a great deal of genetic modifiers present as well. These modifiers all have modest effects, but as a group, they are also shown to have a strong influence among individuals who are or will soon become centenarians.

GENOMICS Animal studies have shown that diets with reduced glucose, along with reduced fat and protein uptake, are associated with a delay in the risk of cancer and other metabolic diseases (Slagboom et al., 2011). The significance here is that these animal tests allow us to further develop what the definition of longevity is in humans (Slagboom et al., 2011). Additionally, studies have also related the genetics within humans and investigated how these genomic factors can become closely related to that of longevity (Slagboom et al., 2011). Genomic studies have shown that human longevity in animal models has a remarkably plastic life span, and major pathways of life-span regulation have now been discovered through these models (Kenyon, 2010). Genomics is usually very much influenced by the degree to which the focus of the study is population-based and the nature of the phenotype for the cohort being studied (Kenyon, 2010). An alternative to this hypothesis on longevity is that of genetic variation in humans (Slagboom et al., 2011). Human twin studies suggest that only 20
30% of life-span extension is determined through genetic variation up to the age of about 85 years (Fontana et al., 2010). In order to better understand genetic studies, families that have longevity genetics are being studied for their age-related phenotypes and for the way in which these are actually associated with familial longevity. Results have demonstrated that there is a lower prevalence of cancer in the families being tested (Westendorp et al., 2009). Less medication was also needed for family members that had cardiovascular disease, especially if they had spent more time in a common environment (Westendorp et al., 2009). Longevity is considered a major dynamic in the whole equation of these diseases. Studies of long-lived individuals, on how their families’ provide care, will allow us a better understanding as to the role of physiological behavior (Slagboom et al., 2011). Further studies have been analyzing what types of behavior can inhibit longevity (Johnson et al., 2001). Studies have been carried out upon the family members’ alleles to

investigate how they are able to protect them from age-related phenotypes (Barker, 2007). A low prevalence of cancer has also been noted for families of centenarians (Terry et al., 2004). The absence of certain alleles can promote diseases that are related to old age (Steegers-Theunissen et al., 2009). Other gene and genomic-wide studies have been conducted in order to analyze the role of these alleles (Rozing et al., 2010). The occurrence of genetic variants of highly aged individuals has been habitually compared with that of younger control individuals (Slagboom et al., 2011). This comparison revealed which alleles give a better survival rate when people reach an old age. This study only analyzed animal and mammal models. Observations from the animal studies have shown that longevity genes for animals exist but they in no way affect the mortality of the organism at all ages (Slagboom et al., 2011). Genome-wide expression has been studied through the lymphocytes, brain, and kidneys, along with the skeletal muscle tissues (Erraji-Benchekroun et al., 2005). The genomic expression of some of these body parts is able to reflect the functionality of some of these specific organs (Passtoors et al., 2008). The genes are also able to analyze and mark the chronological ages of the subjects being tested along with the biological age of the tissues. This can show a more accurate and optimal means of acquiring the age of these genes and ultimately track an individual’s longevity (Slagboom et al., 2011). Gene expression changes occur as a function of age (Slagboom et al., 2011). These reports have shown that the comparison between the offspring of long-lived subjects and their partners is able to provide a step further towards the identification of early and possibly fundamental contributions to the overall aging process of any organism.

ENVIRONMENTAL FACTORS AND LONGEVITY Many animal and human studies have focused on the negative effects of environmental influences on energy balance and life span. A low social and/or economic status seems to be associated with a greater production of adiposity. Studies have begun to show that caloric restriction of foods can lead to longevity for humans (Kaiser et al., 2012). Environmental factors can provide further information as to how longevity occurs with humans. There seems to be a correlation between this and body weight. Socioeconomic status is shown to be a very important factor related to longevity since it correlates with obesity (Kaiser et al., 2012). Animal tests have also indicated that substantially reducing

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OMEGA-3 FATTY ACIDS AND LONGEVITY

caloric intake while maintaining health standards can increase life span (Kaiser et al., 2012). Social and environmental stress by caloric amount and timing of intake was also studied in non-human primates. Rodents were studied under conditions of having their food supply restricted in stressed environments (Kaiser et al., 2012). Monkeys of different hierarchies were tested over a 3-week period for both dietary and caloric restriction. The tests were focused mainly on their overall exposure to low- and high-fat dieting. It was noted that monkeys of lower social status within the hierarchy consumed more calories with increased day and night-time feeding than the dominant monkeys (Kaiser et al., 2012). As for a model comparison for humans based on these studies, it can be noted that longevity is affected by social and environmental implications, due to the fact that high caloric intake can result in obesity, and thus also has implications for diseases such as diabetes and cancer (Kaiser et al., 2012). Environmental conditions play a key role in longevity. The genetic studies carried out in humans have not yet been able to explain the genetic components that make up many physiological health conditions (Gluckman and Hanson, 2004). Rare variants in relevant genes have been used to apply novel and wholegenome sequencing technologies (Bjornsson et al., 2008). However, most human genetic studies have not yet focused on these kinds of interactions, since most studies and evaluations need to be conducted on the genome itself. Clearly a larger study sample will also be necessary in order to study the environmental effects presented with longevity. Like most complex phenotypes present in a variety of different organisms, exceptional longevity is shown to reflect a combined influence of both environmental and genetic factors (Slagboom et al., 2011). In order to contribute to healthy aging, it is important to develop a model that takes into account all general life factors. These life factors include a proper diet, nutrient intake, exercise, and other factors that contribute to healthy living. Maintenance of a balance of daily activities, such as exercise and nutrition, can influence genetic factors. The environmental factors are defined as lifestyle choices such as how we go about our daily activities. In order to explore this genetic contribution, a study (Sebastiani et al., 2012) was undertaken with 801 centenarians, along with 914 genetically matched healthy controls. Nearly one third of the subjects being tested possessed exceptional longevity, and this also helped increase the overall power of the experiment (Tan et al., 2008). Within this study, the median age of death for the centenarians was 104 years of age. With this data, a genetic model was built to present 281 single nucleotide polymorphisms. The model was able to

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differentiate the cohorts among the centenarians that were studied, and it was able to distinctly present the individual’s own population base.

ANIMAL TESTS AND LONGEVITY Tests to explain how genetics correlates with longevity have been analyzed with the naked mole rat (Kim, 2011). The naked mole rat is strictly subterranean, and an extraordinarily long-lived mammal. The size of the naked mole rat is similar to that of a mouse, and its maximum life span is known to be around 30 years of age (Edrey et al., 2011). With this life span, the naked mole rat is the longest living rodent (Kim, 2011). These animals are unique in terms of their life span due to the fact that they have no age-related connections towards their mortality and they also have a very high fertility rate until death. The naked mole rats are also good test subjects for longevity studies due to the fact that they do not go through tumor genesis (Liang et al., 2010). Additionally, they are resistant towards spontaneous cancers (Kim, 2011), which may pose a challenge to the theories of aging and longevity. The naked mole rats have a single breeding female. The queen plays a special role in the naked mole rat colony by mobilizing the reproduction processes (Kim, 2011). She also suppresses the sexual maturity of her subordinates. Naked mole rats also live in full darkness, and with relatively low oxygen and high carbon dioxide concentrations. Analyzing the naked mole rat’s genome is important in understanding its consistent cancer resistance, hairlessness, and taste sensing, to define its longevity (Kim, 2011). These extreme traits of the naked mole rat are very significant in further advancing human understanding of how longevity works (Kim, 2011). Additional studies in the future will allow us to further develop our understanding of longevity in the naked mole rat and relate these findings to humans.

OMEGA-3 FATTY ACIDS AND LONGEVITY A correlation may exist between longevity and omega-3 fatty acids. They help control multiple issues such as oxidative stress (Kantha, 1987), which should enhance longevity. Omega-3 fatty acids are considered fundamental fatty acids for the human body. The body is not able to make them; instead, one must acquire them through consuming food (Ameur et al., 2012). Omega-3 fatty acids occur in certain fish such as salmon, tuna, and halibut. Additionally, other seafood is known to contain omega-3 fatty acids such as algae,

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some plants, and nut oils. Omega-3 fatty acids play a very important role in brain development, and also in lowering the risk of cardiovascular disease (KrisEtherton et al., 2003). Moreover, omega-3 fatty acids lower blood pressure in people with hypertension (Kris-Etherton et al., 2003). Ultimately, the question here is how are omega-3 fatty acids connected with an individual’s longevity? The use of omega-3 fatty acid as an aid to improving longevity is a developing area of research. The proportionally large human brain is very unique among primates, and most of the energybudget that remains within the body is distributed to the brain in order to support it (Leonard et al., 2010). In order to fully maintain the brain’s neurological functions, humans are very dependent on high amounts of polyunsaturated fatty acids: omega-3 and omega-6 (Darios and Davletov, 2006). These fatty acids are essential to humans in the sense that they cannot be synthesized within the body, but rather, through dietary intake. There are known to be a great deal of beneficial effects from consuming fish oils. Reports have found that consuming a diet rich in omega-3 fatty acids considerably reduces many different health risks including plasma cholesterol and triglyceride levels (Ameur et al., 2012). Oxidative stress as well as inflammation affects nitric oxide production in the human body by directly causing insulin resistance. A decrease in nitric oxide, along with high intakes of fat and sugar, leads to problems such as obesity. Oxidative stress as well as inflammation is increased in men that have erectile dysfunction, and these factors all increase with age (Chedraui and Perez-Lopez, 2013). Omega-3 fatty acids are able to reduce inflammatory markers, as well as decrease cardiac death, and increase nitric oxide production. Omega-3 fatty acids are vital for men under 60 years old who have erectile dysfunction, diabetes, hypertension, and coronary artery disease. Taking a comprehensive approach to lifestyle modifications including exercise, omega-3 fatty acid supplements, reduced fat and sugars, and improved antioxidant status should be of benefit to men with erectile dysfunction in improving their vascular health as well as their longevity (Chedraui and Perez-Lopez, 2013). Omega-3 fatty acids have been studied as a major supplement for brain and neurological development. There are many other direct correlations between omega-3 fatty acids and major diseases such as cardiovascular disease (Kris-Etherton et al., 2003). New information has become available concerning the way in which fish consumption affects cardiovascular disease, especially in patients that have the disease. Men who consumed more than 35 grams of fish daily were found to have a relatively lower risk of death from

coronary heart disease compared to those who consumed no fish at all (Kris-Etherton, 2013). Study findings have not yet provided conclusive evidence as to whether omega-3 fatty acid intake really is beneficial for cardiovascular disease mortality. Likewise, further studies are also needed on the association between omega-3 fatty acid intake and coronary heart disease in relation to sudden mortality and coronary artery bypass (Kris-Etherton, 2013). Omega-3 and chronic diseases have also been studied in relation to longevity (Chedraui and PerezLopez, 2013). A diet highly concentrated in omega-3 containing foods has been associated with low mortality rates and increased longevity frequencies. Having a diet rich in Mediterranean nutrients can also lower incidences of chronic diseases including cancer and depression, along with metabolic syndrome and various neurodegenerative diseases (Chedraui and Perez-Lopez, 2013). Reports have also indicated that consumption of dietary components such as olive oil, antioxidants, and omega-3 and -6 polyunsaturated acid facilitate anti-aging effects (Chedraui and Perez-Lopez, 2013). Currently, these studies support the argument that omega-3 fatty acids do play a role in longevity.

References Ameur, A., Enroth, S., Johansson, A., Zaboli, G., Igl, W., Johansson, A.C., et al., 2012. Genetic adaptation of fatty-acid metabolism: A human-specific haplotype increasing the biosynthesis of longchain omega-3 and omega-6 fatty acids. Am. J. Hum. Genet. 90 (5), 809
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FURTHER READING

Finch, C.E., Tanzi, R.E., 1997. Genetics of aging. Science 278, 407
411. Fontana, L., Partridge, L., Longo, V.D., 2010. Extending healthy life span—from yeast to humans. Science 328, 321
326. Freeman, M., Hibbeln, J., Wisner, K., Davis, J., Mischoulon, D., Peet, M., et al., 2006. Omega-3 fatty acids: Evidence basis for treatment and future research in psychiatry. J. Clin. Psychiatry 67, 1954
1967. (accessed 22.06.13). Gluckman, P.D., Hanson, M.A., 2004. Developmental origins of disease paradigm: a mechanistic and evolutionary perspective. Pediatr. Res. 56, 311
317. Johnson, T.E., de Castro, E., Hegi de Castro, S., Cypser, J., Henderson, S., Tedesco, P., 2001. Relationship between increased longevity and stress resistance as assessed through gerontogene mutations in Caenorhabditis elegans. Exp. Gerontol. 36, 1609
1617. Kaiser, K., Smith, D., Allison, D., 2012. Conjectures on some curious connections among social status, calorie restriction, hunger, fatness, and longevity. Ann. N. Y. Acad. Sci. 1264 (1), 1
12. (accessed 22.06.13). Kantha, S., 1987. Dietary effects of fish oils on human health: a review of recent studies. Yale. J. Biol. Med. 60 (1), 37
44. (accessed 22.06.13). Kenyon, C.J., 2010. The genetics of ageing. Nature 464, 504
512. Kim, E., 2011. Genome sequencing reveals insights into physiology and longevity of the naked mole rat. NIH Public Access 223
227. (accessed 22.06.13). Kris-Etherton, P., Harris, W., Appel, L., 2003. Fish Consumption, Fish Oil, Omega-3 Fatty Acids, and Cardiovascular Disease. AHA Scientific Statement 20
30. (accessed 22.06.13). Leonard, W.R., Snodgrass, J.J., Robertson, M.L., 2010. Evolutionary Perspectives on Fat Ingestion and Metabolism in Humans. In: Montmighteur, J.P., le Coutre, J. (Eds.), Fat Detection: Taste, Texture, and Post Ingestive Effects. CRC Press, Boca Raton, FL. Liang, S., Mele, J., Wu, Y., Buffenstein, R., Hornsby, P.J., 2010. Resistance to experimental tumorigenesis in cells of a long-lived mammal, the naked mole-rat (Heterocephalus glaber). Aging Cell. 9, 626
635. Longo, V., Finch, C., 2003. Evolutionary medicine: From dwarf model systems to healthy centenarians. Science 299 (5611), 1342
1346. (accessed 22.06.13). McGinnis, J.M., Foege, W.H., 1993. Actual causes of death in the United States. JAMA. 270, 2207
2212. Mecocci, P., Polidori, C., Troiano, L., Cherubini, A., Cecchetti, R., Pini, G., et al., 2000. Plasma antioxidants and longevity: a study on healthy centenarians. Free Radic. Biol. Med. 28 (8), 1243
1248. (accessed 22.06.13). Myers, D., Ryu, S., 2008. Aging baby boomers and the generational housing bubble: Foresight and mitigation of an epic transition. J. Am. Plann. Assoc. 74 (1), 17
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1031. Passtoors, W.M., Beekman, M., Gunn, D., Boer, J.M., Heijmans, B.T., Westendorp, R.G., et al., 2008. Genomic studies in ageing research: the need to integrate genetic and gene expression approaches. J. Intern. Med. 263, 153
166. Perls, T., Shea-Drinkwater, M., Bowen-Flynn, J., Ridge, S.B., Kang, S., Joyce, E., et al., 2000. Exceptional familial clustering for extreme longevity in humans. J. Am. Geriatr. Soc. 48, 1483
1485. Rice, D., Fineman, N., 2004. Economic implications of increased longevity in the United States. Annu. Rev. Public Health 25, 457
473. (accessed 22.06.13).

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Robine, J.M., Paccaud, F., 2005. Nonagenarians and centenarians in Switzerland, 1860
2001: A demographic analysis. J. Epidemiol. Community Health 59, 31
37. Rozing, M.P., Westendorp, R.G., de Craen, A.J., Fro¨lich, M., Heijmans, B.T., Beekman, M., et al., Leiden Longevity Study (LLS) Group 2010. Low serum free triiodothyronine levels mark familial longevity: the Leiden Longevity Study. J. Gerontol. A Biol. Sci. Med. Sci. 65, 365
368. Sebastiani, P., Solovieff, N., Dewan, A., Walsh, K., Puca, A., Hartley, S., et al., 2012. Genetic signatures of exceptional longevity in humans. Public Library of Science 1
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463. (accessed 22.06.13). Skytthe, A., Pedersen, N.L., Kaprio, J., Stazi, M.A., Hjelmborg, J.V., Iachine, I., et al., 2003. Longevity studies in GenomEUtwin. Twin Res. 6, 448
454. Slagboom, P., Beekman, M., Passtoors, W., Deelen, J., Vaarhorst, A., Boer, J., et al., 2011. Genomics of human longevity. Philos. Trans. R. Soc. B Biol. Sci. 35
42. (accessed 22.06.13). Steegers-Theunissen, R.P., Obermann-Borst, S.A., Kremer, D., Lindemans, J., Siebel, C., Steegers, E.A., et al., 2009. Periconceptional maternal folic acid use of 400 μg per day is related to increased methylation of the IGF2 gene in the very young child. PLoS ONE 4, e7845. Sullivan, E.L., Cameron, J.L., 2010. A rapidly occurring compensatory decrease in physical activity counteracts diet-induced weight loss in female monkeys. Am. J. Physiol. Regul. Integr. Comp. Physiol. 298, R1068
R1074. Tan, Q., Zhao, J.H., Zhang, D., Kruse, T.A., Christensen, K., 2008. Power for genetic association study of human longevity using the case-control design. Am. J. Epidemiol. 168, 890
896. Terry, D.F., Wilcox, M.A., McCormick, M.A., Pennington, J.Y., Schoenhofen, E.A., Andersen, S.L., et al., 2004. Lower all-cause, cardiovascular, and cancer mortality in centenarians’ offspring. J. Am. Geriatr. Soc. 52, 2074
2076. Van Itallie, T., 1979. Obesity: Adverse effects on health and longevity. Am. J. Clin. Nutr. 32 (12), 2723
2733. (accessed 22.06.13). Waite, L.J., 2004. Introduction: The demographic faces of the elderly. Popul. Dev. Rev. 30 (Suppl), 3
16. Westendorp, R.G., van Heemst, D., Rozing, M.P., Fro¨lich, M., Mooijaart, S.P., Blauw, G.J., et al., Leiden Longevity Study Group 2009. Nonagenarian siblings and their offspring display lower risk of mortality and morbidity than sporadic nonagenarians: the Leiden Longevity Study. J. Am. Geriatr. Soc. 57, 1634
1637. Wilmoth, J.R., 2000. Demography of longevity: Past, present and future trends. Exp. Gerontol. 35, 1111
1129. Wilson, M.E., Fisher, J., Fischer, A., Lee, V., Harris, R.B., Bartness, T.J., 2008. Quantifying food intake in socially housed monkeys: social status effects on caloric consumption. Physiol. Behav. 94, 586
594.

Further Reading Peto, R., Darby, S., Deo, H., Silcocks, P., Whitley, E., Doll, R., 2000. Smoking, smoking cessation, and lung cancer in the UK since 1950: combination of national statistics with two case-control studies. BMJ. 321, 323
329. Taylos, D., Hasselblad, V., Henley, J., Thun, M., Sloan, F., 2002. Benefits of smoking cessation for longevity. Am. J. Public Health 92 (6), 990
996. (accessed 22.06.13).

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C H A P T E R

2 Molecular Gerontology: Principles and Perspectives for Interventions Suresh I.S. Rattan INTRODUCTION

mechanism. Although individually no tissue, organ or system becomes functionally exhausted even in very old organisms, it is their combined interaction and interdependence that determines the survival of the whole. The evidence that genes have a limited (about 25%) influence upon lifespan in human beings has mainly come from the studies performed on centenarians and their siblings, twins, and long living families. The value of the genetic contribution to individual lifespan was calculated from longevity studies on mono- and dizygotic twins, and it was shown that the heritability of longevity in men and women was 0.26 and 0.23, respectively (Herskind et al., 1996). This implies that the environment and the lifestyle (milieu) have more than 75% contribution in determining the lifespan of an individual. Thus a combination of genes, milieu, and chance determine the course and consequences of aging and the duration of survival of an individual, which could be modifiable (Rattan, 2007b; Rattan, 2012b). The aim of this article is to give a brief overview of the present state of knowledge with respect to the biological and molecular basis of aging, and the ongoing lines of research and strategies for slowing down aging, maintaining health, and extending health span and longevity.

Aging research has made tremendous advances and major breakthroughs have been achieved in the understanding of aging at various levels. It is now generally believed that the biological basis of aging is well understood and a distinctive framework has been established which can facilitate formulating some general principles of aging and longevity. There are at least three main biological principles of aging and longevity which can be derived from more than fifty years of biogerontological research: 1. Aging starts after essential lifespan: Biological aging occurs during the period of survival beyond the natural lifespan of a species, termed ‘essential lifespan’ (ELS), in accordance with the theory of evolution of biological traits and requirements for successful reproduction (Rattan, 2000a,b; Rattan and Clark, 2005). 2. Aging is a post-genetic emergent phenomenon: Biological aging is an emergent phenomenon seen primarily in protected environments, which allow survival beyond ELS. There is no fixed and rigid genetic programme which determines the exact duration of survival of an organism, and there are no real gerontogenes whose sole function is to cause aging and to determine precisely the lifespan of an organism. 3. Aging phenotype is highly heterogeneous: The progression, rate and phenotype of aging is different in different species, organisms within a species, organs and tissues within an organism, cell types within a tissue, sub-cellular compartments within a cell type, and macromolecules within a cell.

Homeostasis Versus Homeodynamics Survival and longevity are the result of various maintenance and repair mechanisms. All living systems have the intrinsic ability to respond, to counteract, and to adapt to the external and internal sources of disturbance. The traditional and dominating conceptual model to describe this property is homeostasis. However, recent enhanced understanding of the processes of biological growth, development, maturation, reproduction, and aging have led to the realization that

Thus, aging is an emergent, epigenetic, and a metaphenomenon, which is not controlled by a single Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00002-8

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the homeostasis model as an explanation is seriously incomplete. The main reason for the incompleteness of the homeostasis model is its defining principle of ‘stability through constancy’, which does not take into account the new themes, such as cybernetics, control theory, catastrophe theory, chaos theory, information and interaction networks, which comprise and underline the modern biology of complexity (Rattan, 2007a; Rattan, 2012b). Instead, the term homeodynamics is being increasingly used to account for the fact that, unlike machines, the internal milieu of complex biological systems is not permanently fixed, is not at equilibrium, and is a dynamic regulation and interaction among various levels of organization (Yates, 1994). Aging, age-related diseases, and death are the final manifestations of unsuccessful homeostasis or failure of homeodynamics (Rattan, 2006; Rattan, 2012b). A wide range of molecular, cellular and physiological pathways of repair are well known, and these range from multiple pathways of nuclear and mitochondrial DNA repair to free radical counteracting mechanisms, protein turnover and repair, detoxification mechanisms, and other processes including immune and stress responses. All these processes involve numerous genes whose products and their interactions give rise to a ‘homeodynamic space’ or the ‘buffering capacity’, which is the ultimate determinant of an individual’s chance and ability to survive and maintain a healthy state (Holliday, 2007; Rattan, 2006; Rattan, 2012b). In a normal, healthy, and young individual, the complex network of stress responses, damage control, and continuous remodeling constitute a functional homeodynamic space (Demirovic and Rattan, 2013). Since no biological system can be 100% perfect 100% of the time, there is a probability of imperfect homeodynamics giving rise to a zone of vulnerability, manifested in ageindependent diseases and mortality. However, a progressive accumulation of molecular damage and its effects on the interacting molecular networks leads to the reduction in the functional homeodynamic space, and effectively increases the vulnerability zone, thus allowing for the occurrence and emergence of age-related diseases. Alzheimer’s disease, cancer, cataracts, type 2 diabetes, osteoporosis, Parkinson’s disease, sarcopenia, and other age-related diseases are the result of reduced homeodynamic space of the individual (Demirovic and Rattan, 2013). Thus, a progressive shrinkage of the homeodynamic space is the hallmark of aging and the cause of origin of all age-related diseases.

MOLECULAR BASIS OF AGING At the molecular level, the theories of the mechanisms of aging are mostly centered on the accumulation

of molecular damage (Rattan, 2006; Rattan, 2008c), although recently some other views, such as continuous growth leading to a kind of quasi-program of aging, have also been put forward (Blagosklonny, 2012). However, at the mechanistic level, occurrence and accumulation of damage and its consequences are the most well studied aspects of molecular gerontology. The origin of molecular damage is mainly from three sources: (1) various chemical species such as reactive oxygen species (ROS) and free radicals (FR) formed due to external inducers of damage (for example ultra-violet rays), and as a consequence of cellular metabolism involving oxygen, metals, and other metabolites; (2) nutritional glucose and its metabolites, and their biochemical interactions with ROS and FR; and (3) spontaneous errors in biochemical processes, such as DNA duplication, transcription, post-transcriptional processing, translation, and post-translational modifications. An age-related increase in the levels of damage in various macromolecules, including DNA, RNA, proteins, carbohydrates, and lipids is well established. The biological consequences of increased levels of molecular damage can be wide ranging, including altered gene expression, genomic instability, mutations, loss of cell division potential, cell death, impaired inter-cellular communication, tissue disorganization, organ dysfunctions, and increased vulnerability to stress and other sources of disturbance (Holliday, 2007; Rattan, 2006; Rattan, 2012b). The so-called mechanistic theories of biological aging have often focused on a single category of inducers of molecular damage as an explanation for possible mechanisms of aging (Rattan, 2006). Of these, the free radical theory and the protein error theory of aging have been the basis of most of the experimental biogerontology research. Although neither of them can be considered to be the complete theory of biological aging, their contributions in providing a solid scientific footing to experimental aging research and anti-aging interventions are highly significant.

Free Radical Theory of Aging The free radical theory of aging (FRTA), proposed in 1954, arose from a consideration of the aging phenomenon from the premise that a single common biochemical process may be responsible for the aging and death of all living beings (for an update, see Harman, 2006). There is abundant evidence to show that a variety of ROS and other FR are indeed involved in the occurrence of molecular damage, which can lead to structural and functional disorders, diseases, and death. The chemistry and biochemistry of FR is very well worked out, and the cellular and organismic

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GENETICS, POST-GENETICS, AND EPIGENETICS OF AGING

consequences are well documented. However, the main criticisms raised against this theory are with respect to its lack of incorporation of the essential and beneficial role of FR in the normal functioning and survival of biological systems (Gruber et al., 2008; Halliwell, 2009). Additionally, FRTA presents FR as the universal cause of damage without taking into account the differences in the wide range of FR-counteracting mechanisms in different species. Furthermore, a large body of data showing the contrary and/or lack of predictable and expected beneficial results of antioxidant and FR-scavenging therapies have restricted the FRTA to being only a partial explanation of some of the observed changes during aging (Le Bourg and Fournier, 2004; Le Bourg, 2005; Howes, 2006).

Protein Error Theory of Aging Since the spontaneous error frequency in protein translation is generally several orders of magnitude higher than that in DNA replication and RNA transcription, the role of protein errors and their feedback in biochemical pathways has been considered to be a crucial one with respect to aging. Several attempts have been made to determine the accuracy of translation in cell-free extracts, and most of the studies show that there is an age-related increase in the misincorporation of nucleotides and amino acids. It has also been shown that there is an age-related accumulation of aberrant DNA polymerases and other components of the transcriptional and translational machinery (Rattan, 2008c; Rattan, 2010). Further evidence in support of the protein error theory of aging (PETA) comes from experiments which showed that an induction and increase in protein errors can accelerate aging in human cells and bacteria (Holliday, 1996; Rattan, 1996; Rattan, 2003; Nystro¨m, 2002). Similarly, an increase in the accuracy of protein synthesis can slow aging and increase the lifespan in fungi (Rattan, 2008c). Therefore, it is not ruled out that several kinds of errors in various components of protein synthetic machinery, including tRNA charging, and in mitochondria do have long-term effects on cellular stability and survival (Kowald and Kirkwood, 1993a,b; Hipkiss, 2003; Holliday, 2005). However, almost all these methods have relied on indirect in vitro assays, and so far direct, realistic, and accurate estimates of age-related changes in errors in cytoplasmic and mitochondrial proteins, and their biological relevance, have not been made. Similarly, applying methods such as two-dimensional gel electrophoresis, which can resolve only some kinds of mis-incorporations, have so far remained insensitive

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and inconclusive (Rattan, 2008c). It will be necessary to combine several methods, such as electrophoresis, mass-spectrometry, protein
protein interactions and antibody-based detection of molecular heterogeneity to find out the extent of protein errors and their biological role in aging.

From FRTA and PETA to Higher Order Theories Both the FRTA and PETA provide molecular mechanisms for the occurrence of molecular damage. Additionally, nutritional components, especially the sugars and metal-based micronutrients can induce, enhance, and amplify the molecular damage either independently or in combination with other inducers of damage. The biological consequences of increased levels of molecular damage are wide ranging and include altered gene expression, genomic instability, mutations, molecular heterogeneity, loss of cell division potential, cell death, impaired intercellular communication, tissue disorganization, organ dysfunctions, and increased vulnerability to stress and other sources of disturbance. Historically, each of these biological consequences has been used as the basis of putting forward other theories of aging, such as replicative senescence theory, neuroendocrine theory, pineal gland theory, immunological theory and many more (Rattan, 2006; Rattan, 2008c).

GENETICS, POST-GENETICS, AND EPIGENETICS OF AGING Since all molecular processes in a living system are based in and regulated by genes, an attractive research strategy has been to discover genes for aging, termed gerontogenes (Rattan, 1985; Rattan, 1995; Johnson, 2002). However, the evolutionary explanation for the origins of aging and limited lifespan, as discussed above, have generally ruled out the notion of any specific genetic program involving specific gerontogenes. But a lack of specific gerontogenes, with the sole purpose of causing aging and terminating the lifespan of an individual, does not imply that genes do not or cannot influence survival, longevity, and the rate of aging. There is ample evidence from studies performed on yeast and other fungi, nematodes, insects, rodents, and humans that mutations in various genes can either prolong or shorten the lifespan, and some of these are also the cause of premature aging syndromes in human beings (Martin, 2005; Kenyon, 2005; Kenyon, 2010; Christensen et al., 2006). Genetic linkage studies for longevity and several other studies showing an

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association between human longevity and single nucleotide polymorphisms (SNPs) in a variety of genes in various biological pathways, including heat shock response, mitochondrial functions, immune response, cholesterol metabolism, and others (Singh et al., 2004; Rattan and Singh, 2009; Yashin et al., 2012; Yashin et al., 2013). An analysis of the various functions of the genes associated with aging and longevity shows that these genes cover a wide range of biochemical pathways, such as energy metabolism, kinases, kinase receptors, transcription factors, DNA helicases, membrane glucosidases, GTP-binding protein coupled receptors, chaperones, and cell cycle check point pathways. What is clear from the identification of the genes influencing aging and longevity is that whatever their normal function and mechanism of action may be, these gerontogenes did not evolve to cause and accumulate molecular damage, to cause functional disorders, and to terminate the life of the organism. Most of these genes have well defined roles in normal metabolism, in intra- and inter-cellular signaling, and in maintenance and repair functions including stress response. It is the damage-induced changes in the regulation, structure, and/or activity of their gene products which result in their altered biological role with age. Therefore, such genes have been termed ‘virtual gerontogenes’ (Rattan, 1995; Rattan, 1998). Furthermore, a lack of evolutionary selection of virtual gerontogenes has given rise to the notion of post-genetics or ‘postreproductive genetics’ as an explanation for different biological roles played at different ages by the same genetic variants (Franceschi et al., 2005).

Epigenetics of Aging Although genes are the foundation of life, genes in themselves are non-functional entities. It is the wide variety of gene products, including coding and noncoding RNAs, proteins, and other macromolecules which constitute the biochemical and biophysical milieu for life to exist. ‘Epigenetics’ is the most commonly used term to account for and to explain the consequences of the intracellular and extracellular milieu, which establish and influence the structural and functional stability of genes. These epigenetic effects and alterations are generally not passed down from one generation to the next, but have strong deterministic effects on the health, survival, and aging of the individual. So far, there is only scant information available about the involvement in aging of various intracellular epigenetic markers such as methylated cytosines, oxidatively modified nucleotides, alternatively spliced RNAs, and post-translationally modified proteins,

including protein folding (Lund and van Lohuizen, 2004). The full spectrum of epigenetics of aging is yet to be unraveled and at present is one of the most attractive and challenging areas of research in biogerontology (Johnson et al., 2012; Heyn et al., 2012; Hannum et al., 2013). A major reason for apparent difficulties in fully understanding the epigenetics of aging is the existence of several orders higher complexity and diversity of the constituent components such as physical, chemical, biological, and environmental factors, including psychological factors in human beings. Furthermore, in order to understand how various conditions influence, regulate, and modulate the actions, interactions, and networks of gene products during aging will require new intellectual and technical tools, such as systems analysis, bioinformatics, and functional genomics involving simultaneous multiple analyses.

AGING INTERVENTIONS The biological process of aging underlies all major human diseases. Although the optimal treatment of each and every disease, irrespective of age, is a social and moral necessity, preventing the onset of agerelated diseases by intervening in the basic process of aging is the best solution for improving the quality of human life in old age. According to the three principles of aging and longevity described above, having the bodies that we have developed after millions of years of evolution, occurrence of aging in the period beyond ELS, and the onset of one or more diseases before eventual death, appear to be the normal sequence of events. This viewpoint makes modulation of aging by prevention very much different from the treatment of a specific disease. Scientific and rational anti-aging strategies aim to slow down aging, to prevent or delay the physiological decline, and to regain lost functional abilities. In order to modulate aging for achieving healthy old age and for extending lifespan, three main conditions need to be fulfilled, as represented by the equation E 5 GMC2, where G genes and M milieu are the critical factors amenable to intervention (Rattan, 2007b).

Gene Therapy One of the earliest experimental studies which demonstrated that an induced mutation in a single gene can increase the lifespan of an organism was the discovery of the so-called age-1 mutant in the nematode Caenorhabditis elegans (Friedman and Johnson, 1988a,b). Since then hundreds of putative gerontogenes or

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AGING INTERVENTIONS

longevity genes have been reported in C. elegans, Drosophila and rodents, which when mutated result in the extension of average and maximum lifespan of the organism. The methods used for the identification of such genes include induction of mutations and deletions by irradiation and chemical mutagens, alterations in gene expression by knockout, homologous recombination, or by gene addition, and reduction in gene expression by RNAi-induced abrogation of translation. It is important to realize that in almost all such cases longevity extension had occurred when one or multiple interventions resulted in the reduction or total inhibition of the activity of one or more genes (Rattan and Singh, 2009). Some of the main pathways whose ‘loss of function’ is shown to associate with extended period of survival are: (i) energy generation and utilization in mitochondrial respiratory chain; (ii) nutrition and hormonal sensing and signaling including insulin/ insulin-like growth factor-1 and its target forkhead transcription factor FOXO, transcriptional silencing by sirtuin-mediated histone deacetylase; and (iii) translational interference through target of rapamycin (TOR). Similarly, there are other examples which show that several mutant mice strains with defects in growth hormone (GH) pathways including deficiencies of GH levels and GH receptor have extended lifespans. Application of RNAi technology will further identify numerous genes whose normal levels of activities are lifespan restricting (Rattan and Singh, 2009; Kenyon, 2010; Holliday and Rattan, 2010). Studies have also been performed in which the effects of adding one or multiple copies of various genes, leading to the increased expression of their gene products, has resulted in the extension of lifespan. Some such transgenic manipulations in model systems include the addition of gene(s) for one of the protein elongation factors, antioxidant genes superoxide dismutase and catalase, sirtuin, forkhead trascription factor FOXO, heat shock proteins (HSP), heat shock factor (HSF), protein repair methyltransferase and klotho, which is an inhibitor of insulin and IGF1 signaling (Rattan and Singh, 2009). Although these studies have demonstrated longevity-extending effects of various genes in controlled laboratory conditions, there is very little information available on the basic process of aging in terms of the rate and extent of occurrence and accumulation of macromolecular damage and its physiological consequences in these animals. There is also almost no information available as to what is the physiological price paid for inactivating such genes whose normal function is a part of the general metabolism and signaling. There is some evidence that laboratory-protected longevity mutants in C. elegans have reduced Darwinian fitness when competing with the wild type worms under nutritionally challenging conditions.

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Similarly, klotho-induced insulin resistance and the paradox of the insulin/IGF-1 signaling pathways in longevity extension seriously question the practicality of such gene manipulations in humans (Rincon et al., 2004; Van Voorhies et al., 2006; Unger, 2006). Another system in which genetic interventions have been tried as potential anti-aging therapies is the Hayflick system of limited proliferative lifespan of normal diploid differentiated cells in culture (Rattan, 2008a). Almost all the genetic interventions by transient or permanent transfection and ectopic expression of various genes on this model system have focused on extending the replicative lifespan of cells by bypassing the cell cycle check-points (Campisi and d’Adda di Fagagna, 2007; Collado et al., 2007). One of the most widely used genetic interventions in indefinitely extending the replicative lifespan of normal cells has been the ectopic expression of telomerase in a wide variety of cells (Simonsen et al., 2002; Davis and Kipling, 2005). However, continuous proliferation by such genetically modified non-aging cells often leads to their genomic instability, transformation, and cancer-forming activity (Wang et al., 2000; Serakinci et al., 2004). In the case of animals, whereas telomerase negative mice show reduced lifespan and some other abnormalities after six-generations (Lansdorp, 1997), overexpression of telomerase in the skin increases myc-induced hyperplasia (Flores et al., 2006) without any extension of lifespan. Considering that the molecular cause of aging is the progressive accumulation of macromolecular damage and increased molecular heterogeneity, there are at least three major targets for anti-aging genetic interventions: (1) increasing the repair of damaged macromolecules, for example DNA repair pathways; (2) increasing the removal of damaged macromolecules, for example proteasomal and lysosomal pathways; and (3) decreasing the source of damaging agents, for example ROS, other FR, and reactive sugar metabolites. Whereas the first two targets basically imply achieving genetic enhancement or genetic improvement, the third target requires resetting the metabolic pathways. All of the above targets for anti-aging interventions involve hundreds of genes and gene products, whose expression and action are evolutionarily highly regulated in a cell-type-specific manner. Although there are several approaches in development for gene-based enhancement of physical strength, endurance, appearance, and memory, there are serious technical limitations and ethical and safety concerns that remain to be resolved. Preventing or treating one or more age-related diseases by gene therapy, including stem cells, are at best piecemeal treatments which are often temporary or become unsuccessful since these are

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overshadowed by the systemic aging of the whole body. Ideally, gene therapy for the process of aging requires a significant and ‘intelligent’ redesigning already at the level of the zygote for better maintenance and survival of the body without having to trade-off with growth, development, and reproduction. The chances of such an ‘intelligently redesigned’ and directed evolution to succeed in competition with the Darwinian natural selection from much larger random variations and combinations are practically none.

Manipulating the Milieu The second parameter M in the equation E 5 GMC2 represents milieu
the environment in which living systems operate and survive. The milieu in which genes and gene products function ranges from the intracellular molecular and ionic milieu to all other levels of organization including cellular, physiological, psychological, and societal. Almost all the ongoing work on aging modulation and intervention at present is aimed at modifying the milieu by either replenishing those enzymes, hormones and other molecules, such as antioxidants and micronutrients, whose levels are reported to decrease during aging. Although some of these approaches have been shown to have some clinical benefits in the treatment of some diseases in the elderly, none of these really modulate the aging process itself. However, another approach for aging intervention that has been drawing a lot of attention and has significant potential is that of mild stress-induced hormesis, discussed below.

HORMETICS, HORMESIS, AND HORMETINS A promising strategy to slow down aging and prevent or delay the onset of age-related diseases is that of mild stress-induced hormesis. The consequences of stress can be both harmful and beneficial depending on the intensity, duration, and frequency of the stress, and on the price paid in terms of energy utilization and other metabolic disturbances. However, the most important aspect of biological stress response (SR) is that it is not monotonic with respect to the dose of the stressor. SR is almost always characterized by a nonlinear biphasic relationship. Several meta-analyses performed on a large number of papers published in the fields of toxicology, pharmacology, medicine, and radiation biology have led to the conclusion that the most fundamental shape of the dose response is neither threshold nor linear, but is U- or inverted U-shaped, depending on the endpoint being measured. This

phenomenon of biphasic dose response is termed hormesis (Calabrese et al., 2007), and the study and science of hormesis is termed hormetics (Rattan, 2012a). The key conceptual features of hormesis are the disruption, the modest overcompensation, and the re-establishment of homeodynamics. Hormesis in aging is characterized by the life-supporting beneficial effects resulting from the cellular responses to single or multiple rounds of mild stress. It is important to note that although the hormetic zone is usually small, both with respect to the dose and the effect, its biological consequences are cumulative, amplified, and physiologically significant (Rattan, 2008b,d; Demirovic and Rattan, 2013). All such conditions which bring about biologically beneficial effects by initially causing low level molecular damage, and then lead to the activation of one or more SR pathways and thereby strengthens the homeodynamics, are termed hormetins (Rattan, 2008b,d). Hormetins may be further categorized as: (1) Hormetin-P, physical hormetins, such as exercise, thermal shock, and irradiation; (2) Hormetin-M, mental hormetins, such as mental challenge and focused attention or meditation; and (3) Hormetin-N, nutritional and biological hormetins, such as infections, micronutrients, spices, and some oils and fatty acids. An example of stress-induced hormesis is the welldocumented beneficial effects of moderate exercise as a hormetin, which initially increases the production of FR, acids, and aldehydes. Another frequent observation in studies of hormesis is that a single hormetic agent, such as heat shock (HS) or exercise, can strengthen the overall homeodynamics of cells and enhance other abilities, such as tolerance to other stresses, by initiating a cascade of processes resulting in a biological amplification and eventual beneficial effects (Rattan, 2008b,d; Demirovic and Rattan, 2013). Various mild stresses that have been reported to delay aging and prolong longevity in cells and animals include temperature shock, irradiation, heavy metals, pro-oxidants, acetaldehyde, alcohols, hypergravity, exercise, and food restriction (Le Bourg and Rattan, 2008). Aging modulatory and other effects of hormesis have also been reported for human cells. For example, using a regimen of repeated mild HS given to cultured normal human skin fibroblasts, keratinocytes, endothelial cells, and telomeraseimmortalized bone marrow mesenchymal stem cells, a variety of hormetic effects have been reported. These effects include slowing down of cellular aging, extension of cellular replicative lifespan, maintenance of youthful morphology, reduction in molecular damage, and improvement in differentiation, wound healing, and angiogenesis. Other hormetic conditions, which

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REFERENCES

have been shown to have anti-aging effects in human cells are irradiation, mechanical stretching, and electromagnetic field shock (Rattan, 2008b,d; Demirovic and Rattan, 2013). Nutritional hormetins, especially those derived from plant and animal sources, including oils and fatty acids, have generated much scientific interest for their beneficial health effects (Canuelo et al., 2012; Hamel et al., 2008; Niki et al., 2005). This is because of the realization that not all chemicals found in plants are beneficial for animals in a simple and straightforward manner. Instead, they cause molecular damage by virtue of their electrochemical properties and have a typical biphasic hormetic dose response. Some examples of nutritional hormetins involving heat shock response (HSR) are phenolic acids, polyphenols, flavanoids, ferulic acid geranylgeranyl, rosmarinic acid, kinetin, zinc, and the extracts of tea, dark chocolate, saffron, and spinach, components of olive oil, and other fatty acids (Rattan, 2008b,d; Canuelo et al., 2012; Hamel et al., 2008; Niki et al., 2005). Hormesis may also provide an explanation for the health beneficial effects of numerous other foods and food components such as garlic, Gingko, and other fruits and vegetables (Everitt et al., 2006; Hayes, 2005; Hayes, 2007; Ferrari, 2004; Gurib-Fakim, 2006). Understanding the hormetic and interactive mode of action of natural and processed foods is a challenging field of research, and has great potential in developing nutritional and other lifestyle modifications for aging intervention and therapies. However, not all pathways of SR respond to every stressor, and although there may be some overlap, generally these pathways are quite specific. The specificity of the response is mostly determined by the nature of the damage induced by the stressor and the variety of downstream effectors involved. Yet, the major pathways of SR can be used as the screening platform for discovering, testing, and monitoring the effects of novel hormetins. For example, it may be possible to develop multi-hormetin formulations as drugs and nutriceuticals whose mode of action is through hormetic pathways by mild stressinduced stimulation of homeodynamic processes. Finally, while the G and M components of the E 5 GMC2 formula for eternal life are being taken care of by various experimental approaches, the third factor C represents chance, which is the probability of stochastic events leading to a cascade of error-catastrophe in complex interacting systems. Recent developments in our understanding of complex networks at all levels of organization from molecular to societal and global networks have highlighted the vulnerability of all strong and weak links, and have reasserted the significance of chance events which are not amenable to regulation and manipulation. In the context of

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modulating aging, repeated mild stress-induced hormesis increases the boundaries of the homeodynamic space thus giving cells and organisms wider margins for metabolic fluctuation and adaptation. Slowing down the shrinkage of the homeodynamic space hormetically will reduce the probability of occurrence and emergence of various diseases in old age, and thus extend the health span.

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Niki, E., Yoshida, Y., Saito, Y., Noguchi, N., 2005. Lipid peroxidation: mechansims, inhibition, and biological effects. Biochem. Biophys. Res. Commun. 338, 668
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modulate aging changes: lessons from analysis of longitudinal data. Front Genet. 4, 3. Yashin, A.I., Wu, D., Arbeev, K.G., Stallard, E., Land, K.C., Ukraintseva, S.V., 2012. How genes influence life span: the biodemography of human survival. Rejuvenation Res. 15, 374
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74.

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C H A P T E R

3 Peroxisomal Pathways, their Role in Neurodegenerative Disorders and Therapeutic Strategies Patrizia Rise´, Rita Paroni and Anna Petroni PEROXISOMES

of matrix proteins contain the C-terminal PTS1 and the minority the N-terminal PTS2. In the cytosol, PEX5p bind the newly synthesized matrix proteins at the PTS1 site; after translocation of this receptor-cargo complex into the peroxisome there is a dissociation and the cargo is released in the matrix while the receptor is recycled back to the cytosol (Lanyon-Hogg et al., 2010, Girzalsky et al., 2010, Nagotu et al., 2012). More than 50 enzymes have been identified in mammalian peroxisomes and about half of them are involved in lipid metabolism. The number of peroxisomes and their enzymatic content depend on the need of a specific tissue or cell type. The most common enzymes are the oxidases which generate peroxides broken down by antioxidant enzymes such as catalase, glutathione peroxidase, peroxiredoxin I (Schrader and Fahimi, 2004), contributing to cell detoxification. Peroxisomes are also involved in degradation of purines, polyamines and eicosanoids (Wanders and Waterham, 2006). Other reactions include anabolic and catabolic conversions of substrates in lipid metabolism; in particular, ether lipid synthesis (i.e. the synthesis of plasmalogens), α-oxidation, and β-oxidation take place (Figure 3.1) (Wanders et al., 2010). In addition, particularly important is the role of specific synthetases in lipid homeostasis (Watkins and Ellis, 2012).

Peroxisomes were discovered in the 1950s in mouse renal cells and were classified simply as microbodies due to the lack of any further specific information at that time; they later became known as ‘biochemical entities’ with the studies of DeDuve and colleagues (DeDuve and Baudhuin, 1966). Peroxisomes are DNAfree organelles present in almost all eukaryotic cells (with the exception of erythrocytes), including unicellular eukaryotes and higher plant cells. Their shape is round or oval with a single limiting membrane, a finely granular matrix, and a size ranging from 0.1 to 1.0 micron in diameter, being larger in kidney and liver (0.3
1 μm) and smaller (0.05
0.2 μm) in other mammalian tissues. The biogenesis of peroxisomes is not well understood. The first hypothesis was that peroxisomes were derived from the endoplasmic reticulum by gemmation; however, it now seems that peroxisomes grow by transport of post-translational proteins and they generate new peroxisomes via fission (Lazarow and Fujiki, 1985, Borst, 1989, Girzalsky et al., 2010). The proteins involved in these processes are called peroxins (PEX); the 34 known peroxins, important for peroxisomal biogenesis, are well conserved from yeast to man, and 13 orthologous human PEX genes have been described (Girzalsky et al., 2010, Nagotu et al., 2012). As peroxisomes are DNA-free, they require specific pathways to import matrix proteins. These pathways include two classes of matrix-targeting signals for peroxisomal proteins, namely peroxisomal targeting signal 1 and 2 (PTS1 and PTS2), and their cytosolic receptors, peroxin 5 and 7 (PEX5p, PEX7p), respectively. The majority

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00003-X

Ether Lipid Synthesis Ether lipids, or better, phospholipids, can be of two types, plasmanyl-phospholipids or plasmenylphospholipids (plasmalogens), characterized by an ether bond of an alkyl or alkenyl chain with the OH group in position one of the glycerol-phosphate. Usually these lipids contain ethanolamine or choline as

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© 2014 Elsevier Inc. All rights reserved.

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3. PEROXISOMAL PATHWAYS, THEIR ROLE IN NEURODEGENERATIVE DISORDERS AND THERAPEUTIC STRATEGIES

VLCFA Eiscosanoids Dicarboxylic acids Bile acid intermediates

+CoA

Phytanic acid + CoA

DHAP

Acyl-CoA Ether lipid synthesis

β-oxidation α-oxidation

H 2O

Cata

Alkyl-DHAP

lase

O2 H2O2

Pristanic acid

Acyl-CoA

ER

CAT

Per

oxis

ome

Pristanic acid

Plasmalogens Acyl-Carnitine /Acetyl-Carnitine Mitochondria Oxidation

FIGURE 3.1 Metabolic pathways characteristic of peroxisomes. CAT, carnitine acetyltransferase; CoA, coenzyme A; DHAP, dihydroxyacetone phosphate; ER, endoplasmic reticulum; VLCFA, very long chain fatty acids.

head group. The first steps of this biosynthetic pathway occur in peroxisomes, as the enzymes alkydihydroxyacetone phosphate (alkyl-DHAP) synthase and dihydroxyacetone phosphate acyltransferase (DHAPAT) are only located in peroxisomes. All subsequent steps take place in the endoplasmic reticulum.

α-oxidation The 3-methyl-branched chain fatty acids cannot be β-oxidized because of the presence of the methyl group in position 3; they are α- or ω-oxidized as a first step and then β-oxidized. Phytanic acid, a typical 3-methylbranched chain fatty acid, is α-oxidized in peroxisomes after its activation to CoA-ester outside peroxisomes and transport across the membrane.

β-oxidation Substrates of β-oxidation are the very long chain fatty acids (VLCFA), such as C24:0 and C26:0, eicosanoids, dicarboxylic fatty acids, the 2-methyl-branched

fatty acid pristanic acid and the bile acid intermediates (di-hydrocholestanoic acid, (DHCA), and trihydrocholestanoic acid, (THCA)). The fatty acid (FA) metabolism occurs in both mitochondria and peroxisomes with some differences (Table 3.1); short and medium chain length FA are oxidized in mitochondria, whereas VLCFA are metabolized in peroxisomes, after which the metabolites (shorter in chain length) are transported into mitochondria for subsequent oxidation to CO2 and H2O. VLCFA enter peroxisomes as CoA-esters via specific half-ABCtransporters (Kemp et al., 2011) but they cannot enter mitochondria; medium and long chain length FA pass across the mitochondrial membrane as a complex with carnitine. The peroxisomal β-oxidation is comparable to that in mitochondria and consists of the following steps: oxidation, hydration, dehydrogenation and thiolitic cleavage (Figure 3.2). In mitochondria the first step of dehydrogenation is catalyzed by acyl-CoA dehydrogenase whereas in peroxisomes it is catalyzed by acyl-CoA oxidase (ACOX), a flavoprotein that reacts with oxygen to generate H2O2,

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PEROXISOMES

TABLE 3.1 Differences Between Fatty Acid Mitochondrial and Peroxisomal Oxidation

n-6

Peroxisomal Oxidation

18:2 LA

Short, medium long chain FA

Long and very long chain FA

18:3

Carnitine involvement

Yes

No

20:3

First enzyme in oxidative cycle

Dehydrogenase

Oxidase

20:4 AA

Final products

Acetate

FA shorter in chain length

22:4

Inducers

Dietary FA

Dietary FA, lipidlowering drugs

Mitochondrial Oxidation Substrates

n-3 18:3 ALA Δ6 desaturase 18:4 Elongase 20:4 Δ5 desaturase 20:5 EPA Elongase 22:5 Elongase

24:4

24:5 Δ6 desaturase

FA, fatty acids; MUFA, monounsaturated fatty acids.

24:5 O R–CH2–CH2–CH2–C–SCoA O2 Oxidation (ACOX)

H 2O Catalase

H 2O

O

R–CH2–CH CH–C–SCoA H 2O

Hydration (DBP)

O OH R CH2 C CH2 C SCoA H NAD+

Oxidation (DBP)

O O R CH2 C CH2 C SCoA HS-CoA O R CH2 C SCoA

Thiolysis (Thiolase) O CH2 C SCoA

FIGURE 3.2 Peroxisomal β-oxidation of fatty acids. ACOX, acylCoA oxidase; DBP, D-bifunctional enzyme; FAD, flavine adenine dinucleotide; NAD, nicotinamide adenine dinucleotide.

the substrate of catalase. In humans, there are two oxidases: ACOX 1, specific for VLCFA, and ACOX 2, which metabolizes branched chain FA and bile acid intermediates. The next two β-oxidation steps are catalyzed by a bifunctional enzyme (DBP, the dominant form in humans, or LBP) and the last step by thiolases. It is not known how many β-oxidation cycles take place for VLCFA. In any case the end products of the oxidation (small and more hydrophilic molecules) are transferred to mitochondria as carnitine esters or free acids for the final oxidation.

24:6 Peroxisomal β-oxidation

22:5

22:6 DHA

FIGURE 3.3 Metabolism of n-6 and n-3 series fatty acids. The elongation and desaturation reactions take place in microsomes, whereas the last step of β-oxidation takes place in peroxisomes. ALA, α-linolenic acid; AA, arachidonic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid.

Peroxisomal enzymes also play a role in docosahexaenoic acid (22:6 n-3, DHA) formation from the precursor 24:6 n-3. DHA, the major FA of the n-3 series, has different biological actions such as modulation of cell functions and metabolic effects on lipid metabolism (Galli and Rise´, 2009). In humans, DHA levels depend largely on dietary intake, but it can be synthesized, albeit not very efficiently (Hussein et al., 2005), from the precursor, i.e. the essential fatty acid α-linolenic acid (ALA). ALA is converted, via elongation and desaturation reactions in microsomes, to 24:6 n-3 which is then β-oxidized in peroxisomes to give the final product DHA (Figure 3.3) (Moore et al., 1995). DHA has played a unique role during evolution of the modern hominid brain (Crawford et al., 1999) and adequate intakes of n-3 polyunsaturated fatty acids (PUFA), especially of DHA, are very important throughout life, especially in the perinatal period, when it is supplied by the mother to the fetus through the placenta, and then to the newborn through lactation. Maintenance of adequate DHA intake into old age is crucial to prevent the onset of brain dysfunctions (Innis, 2007). A continuous supply of FA to the CNS and their replacement in CNS membranes take place throughout life, a major role being played by the liver in the processes involving FA metabolism and delivery to peripheral organs, including the brain (Rapoport et al., 2007). In addition, the production of anti-inflammatory and protective compounds (resolvins and neuroprotectins) derived from DHA has been described (Serhan, 2005).

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3. PEROXISOMAL PATHWAYS, THEIR ROLE IN NEURODEGENERATIVE DISORDERS AND THERAPEUTIC STRATEGIES

A metabolic pathway similar to that of ALA has also been described for linoleic acid (18:2 n-6, LA), the essential fatty acid of the n-6 series, with the first steps taking place in microsomes and the last step of β-oxidation in peroxisomes (Caruso et al., 1994). Conjugated linoleic acid (CLA) is also catabolized in peroxisomes; the term conjugated linoleic acid refers to a mixture of positional and geometric isomers of linoleic acid characterized by conjugated double bonds not separated by a methylene group as in linoleic acid. These double bonds are located at position 8 and 10, 9 and 11, 10 and 12, 11 and 13 in cis or trans configuration. CLA is naturally produced in the rumen, for example of the cow, by bio-hydrogenation, and thus it is present in milk, dairy products, and meat; in this case, the TABLE 3.2 Biological Effects of the Two Major Conjugated Linoleic Acid Isomers Effect

cis-9, trans-11 CLA

trans-10, cis-12 CLA

Anti-carcinogenic

11

1

Anti-atherogenic

1

1

Anti-obesity

2

1

Normalization of glucose tolerance

1

1

Immune modulation

11

1

CLA, conjugated linoleic acid; 1 is a positive effect; 11 greater positive effect; 2 no effect.

Peroxisomal β-oxidation

Peroxisomal β-oxidation

16:2 8t, 10c

predominant isomer is the cis-9, trans-11 also known as rumenic acid (Banni, 2002, Banni et al., 2004). The CLA isomers have some different biological effects, especially the cis-9, trans-11 and trans-10, cis-12, and some of these effects are induced by both these isomers (Table 3.2). Both CLA isomers have anti-carcinogenic and antiatherogenic effects, but only the trans-10, cis-12 CLA has an effect on body fat, i.e. an anti-obesity action. CLA metabolism is similar to that of LA, whereas the pattern of incorporation in tissues is different. While LA is mainly incorporated in phospholipids (PL), CLA and its metabolites (with the exception of CD 20:4) are incorporated in neutral lipids (in adipose and mammary tissues, rich in triglycerides). Like LA, CLA can be metabolized by desaturases and elongases, with maintenance of the conjugated diene (CD) structure; in addition, partial β-oxidation of CLA and its metabolites can take place (Figure 3.4). The different biological activities of CLA isomers have been explained by small differences in the metabolism of the two isomers. In fact, cis-9, trans-11 CLA seems to be metabolized up to CD 20:4, whereas trans-10, cis-12 CLA is desaturated to CD 18:3 without formation of CD 20:3 and CD 20:4. CLA can also be β-oxidized in peroxisomes to CD 16:2, and its metabolite CD 20:4 can be β-oxidized to CD 16:3, like arachidonic acid (20:4 n-6). Thus CLA and its metabolites are the substrates of the same enzymes responsible for the metabolism of n-6 and n-3 FA. The perturbation of PUFA metabolism and competition with LA caused by CLA could affect

CLA, 18:2 10t, 12c

CLA, 18:2 9c, 11t

16:2 7c, 9t

Δ6 desaturase

18:3 6c, 10t, 12c

FIGURE 3.4 Conjugated linoleic acid metabolism. The elongation and desaturation reactions take place in microsomes, the enzymes being the same as those of the n-3 and n-6 series fatty acids. The two isomers of CLA, 18:2 10t, 12c, and 18:2 9c, 11t, can also be β-oxidized in peroxisomes to the corresponding 16:2; the 20:4 isomers produced are also possible substrates for peroxisomal β-oxidation.

18:3 6c, 9c, 11t

Elongase

20:3 8c, 11c, 13t

20:3 8c, 12t, 14c Δ5 desaturase 16:3 4c, 8t, 10c

20:4 5c, 8c, 12t, 14c

Peroxisomal β-oxidation

20:4 5c, 8c, 11c, 13t

16:3 4c, 7c, 9t

Peroxisomal β-oxidation

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PEROXISOMAL PATHOLOGIES

eicosanoid formation and metabolism, explaining in part the biological effects of CLA.

PEROXISOMAL PATHOLOGIES The crucial role of peroxisomal metabolism in humans became evident when peroxisomal abnormalities were found in a number of severe neurodegenerative and demyelinating diseases; the abnormalities in these metabolic diseases involved either the whole peroxisome or a single metabolic pathway (Steinberg et al., 2006, Wanders and Waterham, 2006). Although different organs can be affected (liver, kidney, eye, bone) the nervous system is always involved. In the CNS, peroxisomes are more abundant in differentiating neurons than in mature ones, and they are found in axon terminals and dendrites, suggesting that peroxisomes are important for neuronal cellular enlargement and formation of cellular processes (Faust et al., 2005). On the other hand, peroxisomal functions decrease with age, favoring neurodegenerative diseases such as Alzheimer’s disease (AD) and dementia (Lizard et al., 2012); moreover, peroxisomal alterations are reported in studies on AD postmortem brain tissues (Kou et al., 2011). Peroxisomal disorders are summarized in Table 3.3 and include disorders of β-oxidation, α-oxidation, and ether lipid synthesis.

Peroxisomal disorders, or Zellweger spectrum diseases, occur when at least one of the genes encoding for PEXs, involved in the importation of peroxisomal proteins, is mutated. As a consequence the encoded enzymes could be unstable or inactive, and the severity of the mutation will determine the clinical manifestation of the diseases, from mild to severe phenotypes. The first described and most severe disease is Zellweger, or cerebro-hepato-renal, syndrome, characterized by a lack of peroxisomes and their functions in all cells, leading to death before the first year of life. In Zellweger patients there is an accumulation of VLCFA, plasmalogens, and phytanic acid; in the past these particular observations led to the association between peroxisomes and lipid metabolism (Brown et al., 1982, Heymans et al., 1983, Poulos et al., 1985). A peroxisomal disorder involving only PEX7 is rhizomelic chondrodysplasia punctata (RCDP type I), characterized by decreased levels of plasmalogens and in some cases also by an increase of phytanic acid. At present, five different genetic diseases involving peroxisomal β-oxidation are known. The most frequent is X-linked adrenoleukodystrophy (X-ALD) with an incidence of 1 in 17,000 births; the disease is due to a mutation in the ABCD1 gene (Mosser et al., 1993) encoding for a protein (adrenoleukodystrophy protein, ALDP) located in the peroxisomal membrane. ALDP belongs to the ATP-binding cassette (ABC) transporter proteins and it is involved in the transport of acyl-CoA

TABLE 3.3 Characteristics of Different Peroxisomal Disorders and the Lipid Metabolites Affected Pathway Affected

Peroxisome biogenesis

Peroxisomal α-oxidation

Ether lipid synthesis

Peroxisomal α-oxidation

Disorder

Gene

Protein Function

Lipid Abnormalities VLCFA

Pris

Phyt

D/THCA

PLGN

Zellweger spectrum

12 PEXs

Import of peroxisomal proteins (PTS1/PTS2 signal)

m

_

m

_

m

RCDP type I

PEX7

Import of peroxisomal proteins (PTS2 signal)

_

_

2/m

_

k

X-ALD

ABCD1

Transport of acyl-CoA

m

_

_

_

_

DBP deficiency

HSD17B4

β-ox of straight/branched chain substrates

m

m

2/m

m

_

ACOX1 deficiency

ACOX1

β-ox of straight chain substrates

m

_

_

_

_

AMACR deficiency

AMACR

β-ox of branched chain substrates

_

m

2/m

m

_

SCPx deficiency

SCP2

Sterol carrier protein X

_

m

2/m

m

_

RCDP type II

GNPAT

DHAP acyltransferase

_

_

_

_

k

RCDP type III

AGPS

Alkyl-DHAP synthase

_

_

_

_

k

Refsum disease

PHYH/PAHX

Degradation of phytanic acid

_

_

m

_

_

X-ALD, X linked adrenoleukodystrophy; RCDP, rhizomelic chondrodysplasia punctata; ACOX, acyl-CoA oxidase; DBP, D bifunctional protein; AMACR, 2-methylacyl-CoA racemase; SCPx, sterol carrier protein X; PEX, peroxins; VLCFA, very long chain fatty acids; Pris, pristanic acid; Phyt, phytanic acid; D/THCA, di/tri-hydrocholestanoic acids; PLGN, plasmalogens. The arrows indicate increased or decreased lipid levels.

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3. PEROXISOMAL PATHWAYS, THEIR ROLE IN NEURODEGENERATIVE DISORDERS AND THERAPEUTIC STRATEGIES

esters across the membrane for oxidation. ALDP deficiency reduces this transport with consequent accumulation of VLCFA (in particular of 26:0) in plasma and in all tissues, although the nervous system, adrenal cortex, and testis are particularly affected (Moser et al., 1999). Other peroxisomal proteins of the ABCD family are also involved in the lipid metabolic pathways and their activation can be induced by a variety of compounds (McGuiness et al., 2001). More than 500 mutations of the ABCD1 gene have been found and different clinical phenotypes for X-ALD have been described (see below). The second most frequent disorder involves the D-bifunctional protein (DBP). Because of its two catalytic units, hydratase and dehydrogenase, there are three different types of DBP deficiency involving one, the other or both units, respectively. These disorders are characterized by accumulation of 26:0, pristanic acid, DHCA, and THCA. Third in frequency is ACOX deficiency, in particular that of ACOX1, leading to an accumulation of VLCFA in body fluids and tissues, as also found for X-ALD. The ACOX deficiency phenotype is more severe than that of X-ALD; the clinical symptoms appear at birth and patient survival is 5
10 years (Ferdinandusse et al., 2007). A few cases of 2-methylacyl-CoA racemase (AMACR) deficiency have been described, grouped under two phenotypes (Wanders and Waterham, 2006, Ferdinandusse et al., 2000, Setchell et al., 2003). AMACR play a role in the metabolism of pristanic acid and bile acid intermediates, and their deficiency is characterized by increased levels of pristanic acid, DHCA, and THCA. Finally, only one case of sterol carrier protein X (SCPx) deficiency has been reported, characterized by elevated levels of pristanic acid and bile acid intermediates (Wanders and Waterham, 2006). Peroxisomal α-oxidation disorders include Refsum disease, which at the moment is the only one well described. In this disease there is a defect in the enzyme phytanoyl-CoA hydroxylase, leading to an increase of phytanic acid levels. Disorders of ether lipid synthesis are also described in a few patients. These disorders include RCDP type II and RCDP type III with DHAPAT and alkyl-DHAP synthase deficiencies, respectively, both characterized by decreased synthesis of plasmalogens.

LEUKODYSTROPHIES Leukodystrophies are a group of inherited diseases including X-linked leukodystrophy (X-ALD), metachromatic leukodystrophy, Krabbe’s disease, PelizaeusMerzbacher disease, and Alexander’s disease (Mar and Noetzel, 2010).

X-linked Adrenoleukodystrophy As mentioned above, X-linked adrenoleukodystrophy (X-ALD) is caused by a mutation in the ABCD1 gene encoding for the ATP-binding transport protein ALDP located in peroxisomal membranes. As a consequence, β-oxidation is impaired and VLCFA accumulate in plasma and in all tissues, although brain, adrenal cortex, and testis are particularly affected. X-ALD has distinct clinical phenotypes in males, ranging from the more severe cerebral forms to adrenomyeloneuropathy (AMN) and finally to asymptomatic individuals or isolated adrenal insufficiency (Addison’s form) without CNS involvement. The two main phenotypes are adrenomyeloneuropathy (AMN) and the cerebral demyelinating form (CALD), most common in childhood (CCALD) and adolescence (Kemp et al., 2012, Wanders et al., 2010). In AMN, the most frequent phenotype of X-ALD, the first symptoms appear at 28 6 9 years. The progression of the disease, initially slow, involves the spinal cord and peripheral nerves. CALD is the most severe phenotype of X-ALD, with onset of the disease between the ages of 3 and 12 years. In addition, about 20% of AMN males are also at risk of developing this cerebral form. There is a correlation between age at onset and progression of the disease: the earlier the cerebral demyelination appears, the more rapid the progression. Initially, the demyelination does not have an inflammatory component and progression of the disease is slow; at this stage, patients have no neurological symptoms, although mild cognitive dysfunctions could be present. The inflammatory component appears suddenly and the disease progresses rapidly, with neurological and cognitive deterioration. A certain percentage of CALD patients do not enter into the inflammatory stage of the disease; this phenotype is known as ‘chronic or arrested cerebral X-ALD’. Finally, in most patients there is adrenocortical insufficiency, as in AMN, which can precede the onset of neurological symptoms. The mean age at onset of symptoms in heterozygous females (37 6 14.6 years, range 32
73 years) is greater than in men with AMN, clinical symptoms are milder, and progression is slower. Cerebral and adrenal involvement are rare in females. Moser et al. have also proposed the subdivision of the nervous system manifestations of ALD into two pathologically distinct categories: inflammatory and non-inflammatory forms. AMN is the non-inflammatory form, characterized by distal axonopathy, mainly involving the spinal cord long tracts and to a lesser extent peripheral nerves. All the cerebral forms are inflammatory, associated with a rapidly progressive, intensely inflammatory myelinopathy that may involve autoimmune mechanisms (Ito et al., 2001).

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THERAPEUTIC STRATEGIES

Lipid and Steroid Hormone Modifications The impairment of the adrenal cortex and testis clearly indicates a role of steroid hormones in this disorder (Assies et al., 1998). Since the 1970s, several clinical reports have described adrenocortical atrophy and cerebral sclerosis in young patients with adrenoleukodystrophy; post-mortem analyses revealed an increase of cholesterol esters in the white matter and cerebral cortex. In addition, male X-ALD patients may show testicular insufficiency. In the adrenal and testis, cholesterol is esterified with VLCFA and particular lamellar inclusions have been identified in these organs (Powers et al., 1980). Cholesterol is the precursor of steroid hormones and its entrapment in these particular esters as lamellar inclusions contributes to abnormalities of both lipid and steroid hormone homeostasis (Powers et al., 1980). Testosterone metabolites have an effect on VLCFA metabolism in X-ALD fibroblasts (Petroni et al., 2000). Moreover, specific enzymatic activities in androgen metabolism are altered in X-ALD fibroblasts (Petroni et al., 2004). Lipid inclusions have also been found in the nervous system of X-ALD patients, for example in Schwann cells and in brain macrophages; these inclusions contained cholesterol, phospholipids (PL) and gangliosides esterified with saturated VLCFA. VLCFA are also increased in different lipid classes, especially in gangliosides, sphingolipids, phosphatidylcholine, cerebrosides, and sulfatides; these VLCFA do not accumulate in phosphatidylinositol and the VLCFA nervonic acid (24:1 n-9) is reduced in brain sphingolipids. The use of HPLC-MS/MS has also demonstrated the accumulation of FA with 28, 30, 32 carbon atoms (Kemp et al., 2005), in particular in lysophosphatidylcholine (lysoPC), providing a useful method of neonatal screening for X-ALD (Hubbard et al., 2006, 2009).

Biological Markers of X-ALD VLCFA are increased in different samples such as blood cells, in particular erythrocytes (RBC), plasma, and chorion villus cells. Plasma VLCFA are a good marker for the diagnosis of X-ALD, in particular hexacosanoic acid (26:0), and the 24:0/22:0, 26:0/22:0 ratios. In male patients the analysis of VLCFA is recommended when there is spastic paraparesis, Addison’s disease or partial demyelination presenting at brain MRI; increased plasma levels of these FA allow the diagnosis of X-ALD. For heterozygous women the diagnosis is complicated by possible negative results in approximately 15
20% of cases (Kemp et al., 2012). We know that in X-ALD there is an abnormal accumulation of VLCFA due to the enhancement of FA

25

elongation and the impairment of VLCFA catabolism, which take place initially in the peroxisomes. These events contribute to a broader perturbation of lipid homeostasis, cholesterol esterification processes, and consequently, modified steroidogenesis. Secondary cascades of inflammatory pathways are also activated. All these events contribute differently to the progression of the disorder. The indicated lipid metabolic pathways have been studied for many years. However it seems that something is escaping our comprehension and we should consider viewing the information that we have in a different manner. For example, impaired peroxisomal metabolism could be related to reduced energy, which might be useful for cellular maintenance. Peroxisomes do not produce ATP like mitochondria but produce heat which is considered to be dispersed in the cell. However heat is a form of energy that could be useful for cellular maintenance and it might have a role in the pathogenesis of peroxisomal disorders (Petroni, 2013).

THERAPEUTIC STRATEGIES In both X-ALD and ACOX1 deficient patients, there is an accumulation of VLCFA in fluids and tissues, whereas other parameters are not affected. These two diseases have different clinical symptoms and progression. The residual FA β-oxidation activity is lower in ACOX deficient patients than in X-ALD ones and this influences a possible pharmacological therapy. One of the consequences of VLCFA accumulation is altered physical characteristics of cell membranes. Saturated VLCFA, especially 26:0, are highly insoluble in water due to the long aliphatic chain; their affinity binding with albumin is very low and desorption from membrane PL is about 10,000 times slower than that of shorter chain length FA. 26:0 perturbs cell membranes increasing their rigidity. This results in an increase of RBC viscosity in X-ALD patients and also impaired stability of axon and myelin membranes. The high concentration of saturated VLCFA alters membrane structure and function, such as the availability of the adrenocorticotropic hormone (ACTH) receptor. The toxic effects of VLCFA were studied in cultured cells (neurons, astrocytes, oligodendrocytes) treated with different FA and cell death was observed at relatively low saturated VLCFA concentrations due to impaired calcium homeostasis and mitochondrial dysfunction (Hein et al., 2008). In addition, induction of reactive oxygen species (ROS) production by 26:0 was found in both in vitro and in vivo experiments (Fourcade et al., 2008). The oxidative stress contributes to the pathogenesis of X-ALD and to neurodegeneration; in fact, in

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3. PEROXISOMAL PATHWAYS, THEIR ROLE IN NEURODEGENERATIVE DISORDERS AND THERAPEUTIC STRATEGIES

X-ALD, higher brain levels of VLCFA were correlated with the expression of several markers of oxidative stress (Paintlia et al., 2003). El-Bassyouni et al. (2012) reported elevated levels of malondialdehyde and nitric oxide and decreased levels of superoxide dismutase in the plasma of patients with peroxisomal disorders, including X-ALD, suggesting that oxidative stress plays a role in the pathogenesis of these diseases (El-Bassyouni et al., 2012). Another aspect concerns the inflammation characteristic of some leukodystrophy phenotypes. In the cerebral form of X-ALD, in which the inflammatory component is important, a greater accumulation of VLCFA has been found in those brain regions showing inflammatory demyelination, with increased levels of cytokines, chemokines, and tumor necrosis factor (TNF-α) (Paintlia et al., 2003). An involvement of the immune system has also been proposed since the inflammatory response is associated with autoimmune mechanisms involving an as yet undefined CD1 lipid self-antigen. It seems that gangliosides containing saturated VLCFA could be the trigger of antigens (Ito et al., 2001). On the basis of these findings, different approaches could be used in clinical therapy, ranging from dietary interventions to gene therapy.

Fatty Acids and Dietary Intervention Lorenzo’s oil (LO) is a 4:1 mixture of glyceryl trioleate (GTO) and glyceryl trierucate in which glycerol is esterified with oleic acid (18:1 n-9) and erucic acid (22:1), respectively. 22:1 is present in large amounts in Cruciferae seed oils such as rape, mustard, etc. The use of LO in X-ALD therapy is based on the observation that LO lowers saturated VLCFA levels in plasma and fibroblasts of patients. In the past, restricted dietary intakes of saturated VLCFA failed to reduce saturated VLCFA levels in X-ALD patients (Brown et al., 1982) whereas supplementation with GTO reduced the plasma levels of saturated VLCFA by reducing their synthesis (Rizzo et al., 1986, 1987). Glyceryl trierucate was added to GTO by Augusto and Michaela Odone, the parents of Lorenzo Odone (from whom the name LO is derived), a patient affected by X-ALD. LO is more effective than GTO alone in decreasing plasma levels of saturated VLCFA in X-ALD patients. Saturated FA are elongated by the same enzyme and it seems that 22:1 reduces the saturated VLCFA by negative feedback, a competitive inhibition (Bourre et al., 1976). The use of 22:1 was initially controversial due to possible cardiac side effects. Oils with a high erucic acid content produce cardiac lipidosis in rodents but not in primates, and negative cardiac effects are not found in

humans after LO treatment. The only side effect is a moderately reduced number of platelets in 30
40% of patients and thus monitoring of platelet counts is required. Several studies have confirmed that LO decreases the saturated VLCFA concentration in plasma of X-ALD patients and the reduction is negatively correlated with the plasma concentration of 22:1 (Moser et al., 2005). LO is generally administered as 20% of total calories; if the amount exceeds 30
35% of total calories, its effect is reduced or nullified. LO also has an effect on plasma levels of other FA. After administration it lowers the very long chain PUFA, in particular DHA, and this can be counteracted by DHA supplementation of patients (Moser et al., 1999). In contrast, LO increases very long chain MUFA such as 24:1, 26:1, 28:1, 30:1, and 32:1. The effects of this increase have not been well studied but since LO administration normalizes RBC viscosity it does not lead to a distortion of cell membrane structure, as instead does 26:0. The previous observations concerning LO and FA levels were made in plasma of X-ALD patients and only limited information is available on LO therapy and FA levels in brain tissue. A few post-mortem studies demonstrated that 22:1 accumulated in different tissues (liver, adipose tissue) after treatment of X-ALD patients, whereas its brain levels were similar in treated and untreated patients, leading to the hypothesis that 22:1 does not cross the blood
brain barrier (Poulos et al., 1994, Rasmussen et al., 1994). However, using 14C 22:1 in comparison with 14C AA, Golovko and Murphy (2006) demonstrated that 22:1 is able to cross the blood
brain barrier, albeit more slowly than AA, and it is rapidly metabolized in the brain, mainly via β-oxidation. An indirect demonstration of this is the normalization of brain 26:0 levels after LO treatment, observed in the two post-mortem studies cited. Regarding the effect of LO on the clinical symptoms and course of leukodystrophy, two different studies have shown a benefit: a preventive effect in asymptomatic boys with normal brain MRI and a slowing of the disease’s progression in ‘pure’ AMN patients. In the first study, there was a significant negative correlation between the reduction of saturated VLCFA and the development of MRI and neurological abnormalities; the reduction of 26:0 levels led to a decreased risk of developing the childhood cerebral form, with retention of normal cognitive functions and physical growth. In the second study, LO administration to patients with AMN normalized the saturated VLCFA levels, with no disease progression or a slower rate of progression. Cappa et al. (2012) published preliminary data on five female X-ALD patients treated with LO 1 CLA for 2 months. CLA is incorporated in brain tissue (Fa et al.,

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DEMYELINATION AND OTHER LEUKODYSTROPHIES

2005, Hunt et al., 2010) and after treatment its level is increased in cerebrospinal fluid (CSF). The synergy with LO could be explained as follows: like other FA (PUFA), CLA is a ligand of PPARα and it has been demonstrated that it up-regulates ACOX (Reddy and Hashimoto, 2001, Belury et al., 1997), thus increasing peroxisomal β-oxidation. CLA should also increase the catabolism of pro-inflammatory molecules. After LO 1 CLA administration, IL-6 levels were decreased in three out of five patients, remaining unchanged in the other two. Neurophysiologic findings after treatment are an improvement of somatosensory evoked potentials (SEPs), a sign of neurological amelioration. As reported previously, LO decreases plasma DHA levels and this can be counteracted by DHA supplementation of patients. In a case report a male patient with the typical AMN phenotype was treated with LO for 7 months and then with DHA for 8 months. DHA supplementation (600 mg/day) consisted of a mixture of triglycerides (medium chain length FA), fish oil (40% DHA and 5% EPA) and vitamin E as an antioxidant. After supplementation, DHA and also EPA levels increased in plasma and RBC, possibly leading to an anti-inflammatory effect. Although no neurological improvement was found, there was no progression of demyelination, suggesting that DHA can prevent progression of the disease (Terre’Blanche et al., 2011). Treatment with DHA alone has been used in other generalized peroxisomal diseases, such as Zellweger syndrome, in which a real DHA deficiency is reported (unlike in X-ALD), with different clinical effects (Petroni et al., 1998, Martinez et al., 2000, Paker et al., 2010). LO supplementation has also been associated with immunomodulatory strategies, i.e. treatment with interferon β or immunoglobulin. Unfortunately, in both cases, the patients showed a progression of neurological and MRI symptoms (Eichler and Van Haren, 2007).

Hormone Replacement Therapy Since 70% of male X-ALD patients present a primary adrenocortical insufficiency, adrenal hormone replacement therapy is needed and all male patients should be monitored for this problem. However the hormone replacement therapy improves the endocrine status, as expected, but not the neurological one, even though some moderate positive effects were reported in AMN (Zang et al., 2003).

Gene Therapy Gene therapy for the treatment of leukodystrophy patients has evolved rapidly in recent years. Initially

27

hematopoietic cell transplantation (HCT) was developed and used to treat some lysosomal diseases, including MLD and GLD. HCT was also used in X-ALD and then replaced by hematopoietic stem cell transplantation (HSCT). Various clinical trials have shown a halt of disease progression in treated patients (Biffi et al., 2011, Aubourg, 2013).

DEMYELINATION AND OTHER LEUKODYSTROPHIES In these disorders, abnormalities of glial cells can be present, which are responsible for defective formation or maintenance of the myelin sheath in the brain, spinal cord, and peripheral nerves. Inflammation, demyelination, and axonal degeneration are evident to different degrees in these leukodystrophies. The white matter is a complex structure composed of a large number of axons covered by a rich lipid membrane, the myelin; white matter also contains glial cells (astrocytes, microglial cells, and oligodendrocytes) with an important role in structural and trophic support of myelin and axons. Various molecular defects, associated with impaired enzymatic activities in lysosomes and/or peroxisomes, characterize a group of leukodystrophies in which demyelination plays a crucial role. In fact, acute or chronic demyelination impairs axonal function and integrity (Nave and Trapp, 2008, Nave, 2010). However, axonal function, and not only the integrity of myelin, also depends on oligodendrocytes. The concentration of N-acetylaspartate is a marker of mitochondrial metabolism in neurons and it also provides acetyl groups for myelin synthesis (Chakraborty et al., 2001). Elevated levels of N-acetyl aspartylglutamate (NAAG) in cerebrospinal fluid (CSF) were recently proposed as a marker of white matter diseases (Mochel et al., 2010). NAAG metabolism involves three different cell types: it is synthesized in neurons from N-acetylaspartate and glutamate, hydrolyzed again to N-acetylaspartate and glutamate in astrocytes and then hydrolyzed to aspartate and acetate in oligodendrocytes (Baslow, 2000). The elevated levels of NAAG in CSF could reflect a compensatory mechanism in altered oligodendrocytes to enhance the synthesis of myelin. Increased CSF levels of NAAG are found in PelizaeusMerzbacher disease (with differences in genotypes), whereas decreased levels of N-acetylaspartate are seen in cerebral white matter of X-linked leukodystrophy patients (Mochel et al., 2010, Ratai et al., 2008). Metachromatic leukodystrophy (MLD) is a rare and fatal autosomal recessive disorder with an incidence of 1:40,000; the disease is characterized by a deficiency of the lysosomal enzyme arylsulfatase-A, involved in sulfatide metabolism, or its activator protein (Austin,

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3. PEROXISOMAL PATHWAYS, THEIR ROLE IN NEURODEGENERATIVE DISORDERS AND THERAPEUTIC STRATEGIES

1959). Four variants according to the age at onset are described, with different severity of clinical symptoms involving the central and peripheral nervous systems. The diagnosis of MLD is based on the measurement of arylsulfatase-A activity in peripheral cells and the increased secretion of sulfatides in urine. Unmetabolized sulfatides accumulate in neurons leading to axonal degeneration; demyelination and a reduced number of oligodendrocytes occur without the presence of inflammatory cells. In Krabbe’s disease (known as globoid-cell leukodystrophy (GLD)), the inflammatory component is evident. GLD, another autosomal recessive disease, is caused by a deficiency of the lysosomal enzyme galactocerebrosidase, which catabolizes the galactosylceramides (GalCer), important lipids in myelin. GalCer are involved in the transduction signal for oligodendrocyte differentiation and in axon-glia interactions. An accumulation of cerebrosides and of psychosine (a toxic molecule with apoptotic effects) leads to demyelination and oligodendrocyte death, with their replacement by gliotic tissue and infiltration of macrophages, often multinucleated ones (globoid cells). GLD has an incidence of 1:100,000 and affects the central and peripheral nervous systems; three different levels of the pathology are possible according to the age at onset. Pelizaeus-Merzbacher disease (PMD) and the type 2 of X-linked spastic paraplegia are due to mutations in the X-linked gene encoding for the proteolipid protein (PLP). PLP is the most abundant protein in CNS myelin embedded in the lipid bilayer of the cell membrane. The severity of myelin loss depends on the particular PLP mutation. Three forms are described according to the age at onset and severity of the disease, with partial or complete absence of myelin, reduced number of oligodendrocytes, and axonal loss (Mar and Noetzel, 2010, Garbern, 2007). Alexander’s disease is classified as one of the leukodystrophies characterized by diffuse accumulation in the brain of Rosenthal fibers containing glial fibrillary acidic protein (GFAP). GFAP is expressed in neuroglial cells and it is a member of the intermediate filament superfamily. GFAP has structural functions in astrocytes but is also involved in astrocyte interactions important during regeneration, synaptic plasticity, and reactive gliosis. Mutations in the GFAP gene are the basis of Alexander’s disease and detection of a heterozygous mutation in the GFAP gene is currently sufficient for diagnosis. The mechanisms of Rosenthal fiber formation remain unclear. However both the quality and quantity of GFAP are important. Except for a few mutations, no clear phenotype-genotype correlation has been established for Alexander’s disease (Brenner et al., 2001, Yoshida and Nakagawa, 2012, Middeldorp and Hol, 2011).

CONCLUSION Inflammation, demyelination, and axonal degeneration are evident, albeit with differences, in X-ALD phenotypes (AMN and CALD) and in leukodystrophies in general. Singh and Pujol (2010) proposed a ‘three-hit hypothesis’ for the pathogenesis of CALD, starting from the common loss of ABCD1 function. A defect in ABCD1 leads to the accumulation of VLCFA and to a decrease in plasmalogen levels, characteristic of both AMN and CALD. This contributes to an increase in oxidative stress (first hit). The oxidative stress results in axonal degeneration in AMN, whereas in CALD it (together with genetic/environmental factors) promotes neuroinflammation (second hit). The pro-inflammatory molecules, such as cytokines and chemokines, impair peroxisomal functions (third hit), resulting in cell loss and demyelination (Singh and Pujol, 2010). Elucidation of the molecular mechanisms associated with the three linked events should aid in developing new strategies and pharmacological therapies for X-ALD.

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4 Unregulated Lipid Peroxidation in Neurological Dysfunction Mototada Shichiri, Yasukazu Yoshida and Etsuo Niki INTRODUCTION

the chain carrying peroxyl radicals, to produce a pentadienyl carbon-centered lipid radical; (2) reaction of the lipid radical with molecular oxygen to produce a lipid peroxyl radical; (3) fragmentation of the lipid peroxyl radical to produce oxygen and a lipid radical [a reverse reaction of the above reaction (2)]; (4) rearrangement of the peroxyl radical; and (5) cyclization of the peroxyl radical (Porter et al., 1995). Cyclization of the peroxyl radical is important for PUFA when it has more than three double bonds, and it does not take place during the oxidation of linoleates. On the other hand, the role of antioxidants has also received extensive attention. Antioxidant defenses may be divided into four categories: (1) prevention of the formation of active oxidants; (2) scavenging, quenching, and removal of active oxidants; (3) repair of damage and excretion of toxic oxidation products; and (4) adaptive responses. Free radical-mediated lipid peroxidation is inhibited by preventing chain initiation and propagation and by accelerating chain termination. The inhibition of enzymatic lipid peroxidation can be achieved by the suppression or deactivation of an enzyme. Lipid peroxidation induced by singlet oxygen is inhibited by preventing the formation and quenching of the singlet oxygen. Many natural and synthetic supplements that possess radical scavenging activity have been explored and proposed. The antioxidant activity in vivo is determined by several factors, such as reactivity towards radicals, fate of antioxidant derived radicals, absorption, distribution, localization, and mobility of the antioxidant. Here we review the use of lipid peroxidation products in vivo as biomarkers and their applications to neurological dysfunctions. Furthermore, the therapeutic effect of antioxidants will be discussed.

Lipid peroxidation has been the subject of extensive studies for several decades and has received renewed attention from the viewpoint of nutrition and medicine. Lipid peroxidation is implicated in the underlying mechanisms of several disorders and diseases, such as cardiovascular disease, cancer, neurodegenerative diseases, and aging, with increasing evidence showing the involvement of in vivo oxidation in these conditions (Gershman et al., 1954; Harman, 1956; Halliwell and Gutteridge, 2007). More importantly, it has been reported that specific lipid peroxidation products exert various biological functions in vivo such as regulating gene expression, signaling, activating receptors, and adaptive responses (Forman et al., 2008; Poli et al., 2008; Noguchi, 2008; Zmijewski et al., 2005). Researchers have focused their attention on lipid peroxidation products to elucidate the mechanism of lipid oxidation, its involvement in disease pathogenesis, and to develop specific and practical biomarkers for diagnosing diseases and evaluating therapies. Lipid peroxidation proceeds by three distinct mechanisms: (1) free radical-mediated oxidation; (2) free radical independent non-enzymatic oxidation; and (3) enzymatic oxidation. Lipids such as polyunsaturated fatty acids (PUFAs) and cholesterol are oxidized by enzymatic and non-enzymatic pathways. For example, the oxidation of linoleic acid (LA) by lipoxygenase proceeds catalytically to produce regio-, stereo-, and enantio-specific hydroperoxyoctadecadienoates (HPODEs). The specificity depends on the types of enzymes, substrates, and the reaction milieu. The free radical-mediated peroxidation of PUFA proceeds by five elementary reactions: (1) hydrogen atom transfer from PUFA to the chain initiating the radical or

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LIPID OXIDATION BIOMARKERS FOR NEUROLOGICAL DYSFUNCTION The role of oxidative stress in neurodegenerative diseases has received much attention recently, whereby elevated levels of lipid oxidation products have been observed in patients with neurological diseases (Roberts et al., 2004; Montine et al., 2002a; Su et al., 2008). A number of studies have been performed to measure the level of lipid oxidation products in humans. The biomarkers measuring the degree of lipid peroxidation are discussed below.

LIPID PEROXIDATION PRODUCTS FROM LINOLEIC ACID Linoleic acid (LA) is the most abundant PUFA in vivo, and its oxidation proceeds by a straightforward mechanism that yields much simpler products than arachidonic acid (AA) and docosahexaenoic acid (DHA). As shown in Figure 4.1, hydroperxyoctadecadienoic acids (HPODEs) that are formed by a radicalmediated oxidation mechanism consist of four isomers: 13-hydroperoxy-9(Z),11(E)-octadecadienoic acid (13-(Z,E)-HPODE); 13-hydroperoxy-9(E), 11(E)-octadecadienoic acid (13-(E,E)-HPODE); 9-hydroperoxy-10 (E), 12(Z)-octadecadienoic acid (9-(E,Z)-HPODE); and 9-hydroperoxy-10(E), 12(E)-octadecadienoic acid. 9- and 13-(Z,E)-HPODE are also formed by enzymatic oxidation via lipoxygenase as enantio-, regio-, and stereo-specific products. Thus, 9- and 13-(E,E)-HPODE are specific products of radical-mediated oxidation. The absolute concentrations of lipid hydroperoxides in vivo are considered to be minimal since they are substrates of many enzymes such as glutathione peroxidases. In such cases, the stable oxidation products are HODEs. LA is less susceptible than AA and DHA to free radicalmediated oxidation.

Singlet oxygen oxidizes linoleic acid by non-radical oxidation to form 13-hydroperoxy-9(Z), 11(E)-octadecadienoic acid (13-(Z, E)-HPODE), 10-hydroperoxy-8 (E), 12(Z) -octadecadienoic acid (10-(E, Z)-HPODE), 12-hydroperoxy-9(Z), 13(E)-octadecadienoic acid (12-(Z, E)-HPODE), 9-hydroperoxy-10(E), and 12(Z)octadecadienoic acid (9-(E, Z)-HPODE). In this case, 10- and 12-(Z, E)-HPODEs are specific oxidation products of singlet oxygen. We recently proposed the measurement of total hydroxyoctadecanoic acid (tHODE) as a biomarker of oxidative stress in vivo (Yoshida and Shichiri, 2011). In this method, biological samples such as plasma, erythrocytes, urine, and tissues are reduced followed by saponification. The hydroperoxides, as well as hydroxides of both free and ester forms of linoleic acid, are measured as tHODE.

LIPID PEROXIDATION PRODUCTS FROM AA Hydroxyeicosatetraenoic Acids AA is susceptible to oxidation because it has three bisallylic positions that are possible sites for initial hydrogen atom abstraction. In the presence of good hydrogen atom donors, six major hydroperoxide products are obtained, such as hydroperoxyeicosatetraenoic acid (HPETE) (Figure 4.2a) (Yin et al., 2011). Furthermore, lipoxygenases oxidize AA to give 5-, 8-, 12-, and 15-HPETE (Yamamoto, 1992). Recently, elevated levels of 20-hydroxyeicosatetraenoic acid (HETE) in cerebrospinal fluid (CSF) and plasma have been reported in a small number of subarachnoid hemorrhage (SAH) patients with documented evidence of cerebral vasospasm and neurological deficits (Roman et al., 2006; Ward et al., 2011). Cytochrome P450 oxidoreductase catalyzes AA to 20-HETE. It has been reported FIGURE 4.1 Mechanisms of HODE formation.

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LIPID PEROXIDATION PRODUCTS FROM AA

FIGURE 4.2A Mechanisms of HETE formation.

that an inhibitor of 20-HETE synthesis reduced infarct size in rats after cerebral ischemia (Renic et al., 2009).

FIGURE 4.2B

Mechanisms of isoprostane (IsoP) and isofuran

(IsoF) formation.

F2-Isoprostanes F2-isoprostanes (F2-IsoPs), a group of prostaglandin F2α-like compounds produced by a noncyclooxygenase free radical-catalyzed mechanism, are products of free radical-induced peroxidation of AA and are currently thought to be the most reliable markers of oxidative damage in humans (Morrow et al., 1990; Halliwell and Lee, 2010; Roberts et al., 2004; Cracowski, 2004). Prostaglandin H2-like bicyclic endoperoxide (H2-isoprostanes) intermediates, which are formed by autoxidation of AA, are reduced to form F-ring IsoPs (Figure 4.2b). Three arachidonyl radicals give rise to the formation of four F2-IsoP regioisomers, 5-, 12-, 8-, and 15-series, each of which comprises eight racemic diastereomers for a total of 64 compounds (Morrow et al., 1990). F2-IsoPs are present in phospholipids in an esterified form and are released to free form by the activity of phospholipase A2 (PLA2) and platelet-activating factor-acetylhydrolase (PAF-AH) (Stafforini et al., 2006). Once they are released from cell membranes by phospholipases, IsoPs circulate in the plasma. IsoPs have been measured in biological fluids such as urine, plasma, exhaled breath condensate, bile, cerebrospinal

fluids, and normal tissues (for example, see Crago et al., 2011 for cardiovascular diseases). Recently, a large body of clinical data has been reported in terms of assessing IsoP levels in patients. However, as shown in Figure 4.2b, F2-IsoPs are minor oxidation products of AA as there are many kinds of isomers through various reactions. Furthermore, AA is not abundant in vivo, especially in human plasma. Thus, the absolute concentrations that are measured in vivo are considered to be quite low. Furthermore, artificial oxidation during sample processing, storage, and analysis is always a potential concern. These compounds are shown to increase in the hippocampus of patients with Alzheimer’s disease (AD) (Reich et al., 2001), as well as in the CSF of patients with AD (Montine et al., 1999a) and Huntington’s disease (Montine et al., 1999b).

Isofurans Similar products generated from the peroxidation of AA, that are characterized by a substituted tetrahydrofuran ring structure and are termed isofurans (IsoFs), have also been measured and found to increase with

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increasing oxygen tension, in contrast to IsoPs (Fessel et al., 2002; Roberts et al., 2004; Cracowski, 2004). The mechanisms of formation of F2-IsoPs and IsoFs occur via similar mechanisms and share a carbon-centered radical as an intermediate (Figure 4.2b). To generate IsoPs, the intermediate-carbon-centered radical undergoes 5-exo cyclization to form a cyclopentane ring. However, IsoFs are made by reaction with molecular oxygen. The levels of IsoFs in the substantia nigra (SN) were reported to be significantly elevated in Parkinson’s disease (PD) patients compared with controls, whereas levels of F2-IsoPs were not (Fessel et al., 2003). Milne et al. (2012) reported on methods of measuring F2-IsoPs and IsoFs using gas chromatography-mass spectrometry (GC-MS), and showed that the normal levels of F2-IsoPs (15-F2t-IsoP) in human plasma quantified using their methodology are 0.035 6 0.006 ng/mL while normal levels in human urine are 1.6 6 0.6 ng/mg Cr. Normal levels of IsoFs in human plasma are 0.071 6 0.010 ng/mL and in human urine are 5.8 6 1.0 ng/mg Cr, although it is unclear what kind of IsoF they measured.

Lipid Peroxidation Products from Docosahexaenoic Acid Owing to the fact that docosahexaenoic acid (DHA) has a higher number of double bonds compared with AA, DHA is more susceptible to free radical-mediated oxidation. Neuroprostanes The oxidized products of docosahexaenoic acid (DHA), neuroprostanes (NPs), are highly concentrated in neuronal membranes (Roberts et al., 1998). Endoperoxide intermediates, which are formed by autoxidation of DHA, are reduced to form F-ring NPs (F4-NPs). However, because of the higher number of double bonds in DHA, eight regioisomers can be formed. Abstraction of a bis-allylic hydrogen from positions C6, C9, C12, C15, and C18 gives rise to eight regioisomers representing 4-, 7-, 10-, 11-, 13-, 14-, 17-, and 20-series F4-NPs, each of which comprises eight racemic diastereomers, for a total of 128 compounds (Figure 4.3). It has been noted that measurement of F4NPs provides a more sensitive indicator of oxidative neuronal injury compared with measuring F2-IsoPs in AD studies (Reich et al., 2001). It has been reported that F4-NP levels are higher in CSF of patients with aneurysmal subarachnoid hemorrhage (SAH) (Hsieh et al., 2009). Signorini et al. (2011) reported that plasma F4-NP concentrations are higher in Rett syndrome, a pervasive developmental disorder.

Neurofurans Recently it was shown that a novel class of IsoFslike compounds, neurofurans (NFs), is formed in vivo and in vitro from the free radical-mediated peroxidation of DHA (Song et al., 2008) (Figure 4.3). Analyzing both NPs and NFs could more accurately reflect the levels of lipid peroxidation in DHA-rich tissues such as the brain. Both NPs and NFs are sensitive and specific markers of neuronal oxidative damage. Arneson and Roberts (2007) reported on a method of measuring NPs, and NFs using GC-MS. Solberg et al. (2012) reported on the levels of IsoPs, IsoFs, NPs, and NFs in brain tissues of newborn piglets. They showed that the levels of IsoPs, IsoFs, NPs, and NFs were increased with the supplementary oxygen concentration used for resuscitation, but the kind of isomers they measured was unclear. Their data indicated that the concentrations of NPs and NFs were similar to those of IsoPs and IsoFs, at about 10 ng/g brain tissue. Lipid Peroxidation-Derived Short-Chain Aldehydes Lipid peroxidation breaks down PUFA to reactive short-chain aldehydes (Uchida, 2003). These shortchain aldehydes are mainly classified into three families: 2-alkenals, 4-hydroxy-2-alkenals and ketoaldehydes (Figure 4.4A). These products are generated from oxidation of free fatty acids including AA and LA. Figure 4.4B shows one of the mechanisms of formation of 4-hydroxy-2-nonenal (HNE) and 4-oxo-2nonenal (ONE) from LA. 2-Alkenals represent a group of highly reactive aldehydes containing two electrophilic reaction centers and they can attack nucleophiles such as protein. Acrolein and its methyl derivative, crotonaldehyde, represent the most potent electrophilic 2-alkenals commonly detected in automobile emissions, cigarette smoke, and other products of thermal degradation (Ghilarducci and Tjeerdema, 1995). 4-Hydroxy-2-alkenals represent the most prominent lipid peroxidation-specific aldehydes. HNE is known to be a major aldehyde produced during peroxidation of ω6-PUFA. Peroxidation of ω3-PUFA generates 4-hydroxy-2-hexenal (HHE). Other important reactive aldehydes originating from lipid peroxidation include ketoaldehydes, such as malondialdehyde (MDA), glyoxal, and ONE. Glyoxal is generated by lipid peroxidation and glycation. MDA is the most abundant lipid peroxidation-specific aldehyde. The thiobarbituric acid (TBA) assay has been widely used as an indicator of lipid peroxidation (Kohn and Liversedge, 1944). Heating samples with TBA in an acidic medium gives rise to the formation of a red 1:2 MDA:TBA adduct with an absorption maximum at

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FIGURE 4.3 Structures of neuroprostanes (NPs) and neurofurans (NFs).

532 nm (Figure 4.4C). Kikugawa et al. (1992) reported that in addition to MDA, alkenals and alkadienals, TBA produces a red pigment. While the pigment formation from MDA is not affected by reaction conditions, the pigment formation from alkenals and alkadienals is dependent on reaction conditions, such as dissolved oxygen, ferric or ferrous ion, and pH. These aldehydes are highly reactive with proteins, DNA, and phospholipids, and cause deleterious effects. Modification of amino acids of proteins and peptides by these aldehydes occurs mainly at cysteine, lysine, and histidine. HNE- and acrolein-protein adducts are considered to be good biomarkers of lipid peroxidation in vivo and they have been applied

widely by using antibodies directed against these adducts (Toyokuni et al., 1995). In many clinical reports, these antibodies were used for not only measuring biological samples (Lovell et al., 2001; Markesbery and Lovell, 1998) but also for immunohistochemical staining of brain sections from individuals with neurodegenerative diseases (Sayre et al., 1997).

Lipid Peroxidation Products from Cholesterol Cholesterol is present in all cells and regulates the fluidity of lipid bilayers. Cholesterol oxidation products, which are commonly referred to as oxysterols, have received

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4. UNREGULATED LIPID PEROXIDATION IN NEUROLOGICAL DYSFUNCTION

FIGURE 4.4 General structures of short-chain aldehydes (A), one of the mechanisms of HNE and ONE formations (B), and mechanism of red pigment formation from malondialdehyde, alkenals, and alkadienals in the thiobarbituric acid (TBA) reaction (C).

increasing attention as diagnostic biomarkers of oxidative stress, as intermediates in bile acid biosynthesis and as messengers for cell signaling and cholesterol transport (Diczfalusy, 2004). Cholesterol is also oxidized by both enzymatic and non-enzymatic mechanisms (Figure 4.5). The free radical-mediated oxidation of cholesterol yields 7α- and 7β-hydroperoxycholesterol (7α-OOHCh

and 7β-OOHCh), 7α- and 7β-hydroxycholesterol (7α-OHCh, 7β-OHCh), 5α,6α- and 5β, 6β-epoxycholesterol, and 7-ketocholesterol (7-KCh) as major products (Diczfalusy, 2004). The conversion of 7-KCh into 7β-OHCh in vivo has been previously reported (Erickson et al., 1977; Larsson et al., 2007). The oxidation of 7-OHCh by either 7α-hydroxycholesterol

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NEUROLOGICAL DYSFUNCTION ASSOCIATED WITH LIPID PEROXIDATION

FIGURE 4.5 Mechanisms of the oxidation of cholesterol.

dehydrogenase (CYP7A1) (Song et al., 1996) or by nonenzymatic autoxidation yields 7-KCh. 7β-OHCh may be regarded as a marker of free radical-mediated oxidation. Oxysterols are present in vivo in different forms, namely the esterified, sulfated, and conjugated forms, as well as free oxysterols (Brown and Jessup, 1999). Singlet oxygen oxidizes cholesterol to give 5α-OOHCh as a major primary product (Smith et al., 1989). Various enzymes oxidize cholesterol to give specific hydroxycholesterols (Niki, 2009). Many of the enzymes belong to the cytochrome P450 family and are present in hepatocytes; however, the 24-hydroxylase, CYP46A1, is found exclusively in neuronal cells in the brain and retina and gives 24(S)-hydroxycholesterol (24(S)-OHCh) as a specific product (Lund et al., 1999; Pikuleva, 2006). 24 (S)-OHCh is formed exclusively in the brain in humans and there is a continuous flux of 24(S)-OHCh from the brain to the circulation. The mitochondrial enzyme CYP27A1 gives 27-hydoroxycholesterol (27-OHCh). 24(S)-OHCh is an endogenous regulator of the nuclear receptor liver X receptor (LXR) and potentially regulates cholesterol and fatty acid synthesis pathways in the brain. In the adult brain, almost all the neuronal requirements for cholesterol are supplied by ApoEcontaining lipoproteins released from astrocytes (Pfrieger, 2003). 24(S)-OHCh regulates the expression of the sterol transporters’ ATP-binding cassette transporter-A1, -G1, and -G4 on astrocytic membranes via LXR, and is involved in the transport of cholesterol from glia to ApoE particles (Leoni and Caccia, 2011, 2013). It is likely that 24(S)-OHCh may play an important role in affecting cholesterol metabolism in the brain.

37

Because almost all the plasma 24(S)-OHCh seems to have a cerebral origin, the plasma levels of 24(S)-OHCh are likely to reflect the number of metabolically active neuronal cells in the brain (Bjo¨rkhem, 2006). Leoni and Caccia (2013) observed that in neurodegenerative diseases (such as AD, PD, multiple sclerosis, Huntington’s disease, and vascular dementia) there were reduced levels of 24(S)-OHCh, presumably because of the loss of metabolically active neurons. Such reduction was related to the degree of atrophy. However, slightly increased 24(S)-OHCh was observed in mild cognitive impairment and AD patients (Lu¨tjohann et al., 2000). Almost all cells in the body contain CYP27A1 located in the inner membrane of the mitochondria. CYP27A1 converts cholesterol to 27-OHCh. 27-OHCh is able to pass the blood
brain barrier (BBB) and its level in the CSF is closely correlated with its corresponding level in the circulation. Damage to the BBB results in higher efflux of 27-OHCh from the circulation into the brain (Heverin et al., 2005).

NEUROLOGICAL DYSFUNCTION ASSOCIATED WITH LIPID PEROXIDATION There are four main reasons why the central nervous system is vulnerable to reactive oxygen species (ROS)-mediated injury: 1. Neurons consume a large amount of oxygen (Halliwell, 2006). The brain accounts for only 2% of body weight, but accounts for about 20% of basal O2 consumption. A major reason for the high O2 uptake is the vast amounts of ATP needed to maintain neuronal intracellular ion homeostasis in the face of ion channels that are associated with action potentials and neurosecretion. 2. Neuronal membranes are rich in PUFA, especially those of AA, eicosapentaenoic acid (EPA) and DHA (Chen et al., 2008). These PUFAs are especially vulnerable to oxidative stress because of the unsaturated double bonds. 3. Several brain areas (e.g. SN, caudate nucleus, putamen, globus pallidus) have high iron content (Zecca et al., 2004). It is generally accepted that iron accumulates in the brain in older people. Iron ions that are released by brain damage catalyze free radical reactions. 4. Neuronal mitochondria generate O22. Complex I-dependent hydrogen peroxide generation in brain mitochondria is greater than in skeletal muscle mitochondria (Malinska et al., 2009).

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MECHANISMS OF FREE RADICAL PRODUCTION IN NEUROLOGICAL DISORDERS Although neurodegenerative disorders have various causes, accumulating evidence does show that oxidative stress is involved in their pathologies. Moreover, the mechanism of ROS production differs in each disease. Here, the mechanisms in each neurological disease are described. Table 4.1 summarizes changes in lipid oxidation biomarkers in AD, PD, amyotrophic lateral sclerosis (ALS), stroke, and Down syndrome (DS).

Alzheimer’s Disease AD is the most common form of dementia. Increased markers of oxidative stress in AD and mild cognitive impairment have been shown to be associated with amyloid β-peptide (Aβ), a 40 to 42 amino acid residue neurotoxic peptide derived from the proteolytic cleavage of amyloid precursor protein (APP), by the action of β- and γ-secretase (Varadarajan et al., 2000). Aβ(1-42) has been shown to aggregate more quickly than Aβ(1-40) and is proposed to play a central role in AD pathogenesis. Aβ(1-42) as small oligomers can insert into the lipid bilayer and initiate lipid peroxidation, and can consequently cause oxidative damage to proteins and other biomolecules (Butterfield et al., 2001). It was reported that Aβ (1-42 . 1-40) can spontaneously generate hydrogen peroxide (Huang et al., 1999). In the presence of redox-active transition metal ions, such as Fe and Cu, hydrogen peroxide leads to the formation of hydroxyl radicals via the Fenton reaction. Oligomeric Aβ, which is considered to be the highly toxic form, has been reported to be localized in mitochondria (Caspersen et al., 2005; Manczak et al., 2006), the main centers of free radical generation. It has been suggested that oxidative stress might occur during the progression of AD as the consequence of, and as a cause of amyloid formation. Oxidative stress and lipid peroxidation seem to be able to induce Aβ accumulation; studies in a mouse model of AD demonstrated that brain lipid peroxidation increases Aβ levels (Pratico et al., 2001). Many studies have demonstrated increased levels of oxidation-associated metabolites and decline of antioxidant levels in the biological fluids of AD patients. For example, it has been observed in several studies that F2-IsoPs, a group of lipid peroxidation products derived from AA, are increased in AD patients (Markesbery et al., 2005; Montine et al., 2005). Increased levels of HNE have also been observed in AD patients (Reed et al., 2009; Vo¨lkel et al., 2006; Williams et al., 2006). Through Michael addition and Schiff base formation, HNE binds to

proteins, and covalent modification of proteins by HNE leads to changes in protein conformation and enzyme activities (Reed et al., 2009). It was found that HNE promotes major conformational changes in tau (τ), which is associated with neurofibrillary tangles (Liu et al., 2005). We previously reported that tHODE oxidatively modified peroxiredoxin (oxPrx)-2 and oxPrx-6 in plasma and erythrocytes in AD patients (Yoshida et al., 2009). We showed that these levels in AD patients were significantly higher than those in healthy controls. Furthermore, the tHODE levels increased with increasing clinical dementia ratings. Two enzymatically formed metabolites of cholesterol, 24(S)-OHCh, a brain-derived metabolite, and 27-OHCh, a peripherally derived metabolite, cross the BBB directly by diffusion and can be measured in the blood. 24(S)-OHCh is a primary metabolite of cholesterol in the brain, and is associated with both AD and brain volume (Leoni, 2009; Solomon et al., 2009). The levels of plasma 24(S)-OHCh are higher in the early stage of AD and vascular dementia (Lu¨tjohann et al., 2000), and lower in long-term cases of AD (Bretillon et al., 2000; Papassotiropoulos et al., 2000). As shown in Table 4.1, associations between plasma 24(S)-OHCh levels and AD have been inconsistent in many reports. In addition to lipids, the oxidation products of DNA (Lovell and Markesbery, 2001) and proteins (Sultana et al., 2001) have also received attention.

Parkinson’s Disease It is known that genetics, metabolism, and environmental factors contribute to the pathogenesis of PD. PD is clinically manifested by resting tremor, slowness of movements, rigidity, and postural instability. It is pathologically defined by the loss of neurons in the substantia nigra (SN), the striatum body, and brain cortex, and by the presence of cytoplasmic protein inclusions named Lewy bodies and neuritis (Jellinger, 2002). SN cell loss is closely related to striatal dopamine (DA) deficiency and to both the duration and clinical severity of the disease. An immunohistological study found that the small synaptic protein α-synuclein is the main component of Lewy bodies (Spillantini et al., 1997). Strong proof of the involvement of α-synuclein in neurodegeneration came from studies showing that three independent mutations in this protein, including A53T, A30P and E46K, lead to the development of familial PD. Recombinant synuclein is reported to produce hydrogen peroxide by ESR (Turnbull et al., 2001). Hydrogen peroxide exposure induces α-synuclein fragmentation and accumulation in the nucleus (Xu et al., 2006). α-Synuclein also modulates the expression of DA synthesis enzymes (Baptista et al., 2003) and physically

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MECHANISMS OF FREE RADICAL PRODUCTION IN NEUROLOGICAL DISORDERS

TABLE 4.1 Summary of the Changes in Lipid Peroxidation Biomarkers for Alzheimer’s Disease, Parkinson’s Disease, Amyotrophic Lateral Sclerosis, Stroke, and Down Syndrome Plasma or Serum F2-IsoPs

Alzheimer’s Disease

Parkinson’s Disease

Amyotrophic Lateral Sclerosis

Stroke

Down Syndrome

m -68,

m -50)




m




183, 232, 272) 108, 154, 158,

155)

209)

91, 113, 210, 260)

IsoFs
















HODE

m













HETE




m

209)




m

7-OHCh

m

m

209)




-




7-ketosholesterol




m

209)










24-hydroxycholesterol

m -272, 248,

k11,




m91)




m209) k11, 191, 248)




-99, 127)







272)

272)

109, 22, 125, 280) 191, 208,

104)

k11, 27-hydroxycholesterol

209, 248) 191)




91, 210, 260) 99)

27, 99, 127)

27, 75, 127)

m197) -27) k11, 104, 127,

191,

248)

neuroprostane

-154)

m209)




m91,

malondialdehyde

m

m -147)

m

m

m214,

m25,

40, 89, 142, 169, 181,

215,232)

-143) TBARS

m79,

4-HNE

m143,

acrolein




Urine F2-IsoPs

191, 214) 212)

12, 47, 206, 215, 219)

12, 66)




277)

273)

79)

m3,45, 81, 77,

213)

m80,

144)




m231)

m195, 122)

-278)







m251)




Stroke

Down Syndrome

m -256)

m184) -37, 250)

Parkinson’s Disease

Amyotrophic Lateral Sclerosis

m - 24,

-

m

154, 155,

10, 18, 38, 49, 61,

168, 180, 195, 220,

Alzheimer’s Disease 183, 253)

210)

50, 209)

146)

159)

91, 121, 210)

IsoFs
















HETE




m




m




7-OHCh
















7-ketocholesterol
















24-hydroxycholesterol










m




27-hydroxycholesterol
















neuroprostane

-










malondialdehyde













m111)

4-HNE
















acrolein
















TBARS













-37)

121)

154)

91, 210)

210)

(Continued)

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4. UNREGULATED LIPID PEROXIDATION IN NEUROLOGICAL DYSFUNCTION

TABLE 4.1 (Continued) CSF F2-IsoPs

Alzheimer’s Disease

Parkinson’s Disease

Amyotrophic Lateral Sclerosis

Stroke

m56, 87,




-150)







148, 150,

152, 156, 183, 185,

Down Syndrome

188)

-114) IsoFs
















HETE

m







m




7-OHCh
















7-ketocholesterol
















24-hydroxycholesterol

m

126, 171, 208, 217)













27-hydroxycholesterol

m

126, 217)













neuroprostane
















malondialdehyde




m -226)

-







4-HNE

m132, 212)




-228, 231,







acrolein
















Alzheimer’s Disease

Parkinson’s Disease

Amyotrophic Lateral Sclerosis

m185, 197,

-70, 185,

Brain Autopsy or Spinal Cord F2-IsoPs

268)

106)

233,

269)

66)

235)

265)

53)

Down Syndrome Stroke





IsoFs

m148)

m148, 232)










HETE

m













7-OHCh
















7-ketocholesterol
















24-hydroxycholesterol

m

95)













27-hydroxycholesterol

m k95)













neuroprostane

m197, 233)










malondialdehyde

m

m










m43, 165)

187)

216)

57, 112, 170,

199)

4-HNE and HNE bound protein

m4, 7, 26,

33,

44, 78, 84, 141,

m

54, 55, 58)

35, 69, 222)

m55)

m173,

m218)

m222)










m166)




m165)

222, 223, 224)

167, 207, 243, 276)

acrolein

m7, 26,

36, 134,

199, 233)

TBARS

m7, 112, 245)

131,

-247) The numbers adjacent to the arrows correspond to the reference numbers.

interacts with both the DA transporter (Wersinger et al., 2003) and the DA synthesis enzyme tyrosine hydroxylase (Castellani et al., 2002). These observations can explain the selective vulnerability of the SN.

It has been reported that the lipid peroxidation adduct HNE and Nε-(carboxymethyl)lysine is localized in Lewy bodies in post-mortem brain tissue from PD patients (Castellani et al., 2002). It has been

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suggested that HNE modification of α-synuclein results in conformational changes and oligomerization, and HNE-modified oligomers are potentially toxic and could contribute to the demise of neurons subjected to oxidative damage (Qin et al., 2007). Recently, it was reported that IsoFs are increased in the SN of patients with PD (Fessel et al., 2003). In addition to lipid peroxidation products, the levels of protein carbonyl (Alam et al., 1997), DNA oxidation products (8-hydroxy-2’-deoxyguanosine; 8-OHdG) (Nakabeppu et al., 2007) and glucose oxidation products (advanced glycation end products) (Mu¨nch et al., 2000) are increased in the SN of PD patients. Oxidative damage biomarkers were reported to be elevated in plasma and urine samples of PD patients (Seet et al., 2010). The levels of plasma F2-IsoPs, HETEs, 7β-and 27-OHCh, 7-KCh, NPs, and urinary 8-OHdG were elevated, whereas the levels of PLA2 and PAF-AH activities were lower in PD patients compared with controls. The levels of plasma F2-IsoPs, HETEs, and urinary 8-OHdG were higher in the early stages of PD. Recently, the finding of a negative correlation between plasma and CSF levels of urate and disease progression in PD has raised the possibility of altered antioxidant activity (Ascherio et al., 2009). It was also previously reported that glutathione, catalase, and glutathione peroxidase are reduced in PD (Ambani et al., 1975; Sian et al., 1994). Investigation of familial PD has so far revealed at least 17 autosomal dominant and autosomal recessive gene mutations responsible for variants of the disease (Dexter and Jenner, 2013). These include α-synuclein mutations and triplication, parkin, ubiquitin carboxylterminal hydrolase L1 (UCH-L1), DJ-1, phosphatase and tensin homolog-inducible kinase 1 (PINK1), leucine-rich repeat kinase 2 (LRRK2), and glucocerebrosidase (GBA). We recently developed specific antibodies against DJ-1, which was oxidized to sulfonic acid on cysteine-106 (Saito et al., 2009). The oxidation of DJ1 protein is thought to be critical for regulating its function. We reported that the levels of oxidized DJ-1 in the erythrocytes of unmedicated PD patients are markedly higher than those in healthy subjects and medicated PD patients. Immunohistochemical analysis also revealed that the number of oxidized DJ-1 antibody-positive cells in the SN of MPTP-treated mice increased in a dose-dependent manner (Akazawa et al., 2010).

Amyotrophic Lateral Sclerosis ALS is a neurodegenerative disease characterized by progressive muscular atrophy and weakness resulting from loss of both upper and lower motor neurons. The disease generally progresses rapidly and is inevitably

41

fatal. The cause of death is typically respiratory failure, on average about 3 years after onset of symptoms. A characteristic feature of degenerating neurons is the presence of cytoplasmic inclusions positive for ubiquitin (Mackenzie et al., 2010). Approximately 5% of patients with ALS have a family history of ALS. The first pathological mutations identified in ALS were in Cu/Zn-superoxide dismutase 1 (SOD1) (Rosen, 1993), and account for approximately 20% of familial ALS cases. Misfolded SOD1 exists in the cytoplasmic inclusion of patients with ALS due to SOD1 mutation, but is not observed in other ALS forms (Bruijn et al., 1998). This finding indicates that other proteins are involved in the pathogenesis of ALS. In 2006, TAR DNA-binding protein 43 (TDP-43) was identified as the major component of ubiquitin-positive neuronal and glial inclusions in SOD1-negative ALS and frontotemporal lobar degeneration (FTLD) (Neumann et al., 2006). TDP-43 binds to a large number of RNA targets and is involved in the regulation of transcription, splicing, and trafficking (Tollervey et al., 2011). Mutations in another RNA binding protein, fused in sarcoma/translated in liposarcoma (FUS/TLS), have been identified in approximately 4% of autosomal dominant familial ALS (FALS) (Kwiatkowski et al., 2009). FUS/TLS is ubiquitously expressed and plays an important role in the regulation of RNA transcription, splicing and transport (Fujii and Takumi, 2005). Previous pathological studies have reported evidence of increased oxidative stress in ALS post-mortem tissue compared with control samples. Lipid oxidation markers, including HNE-histidine and crotonaldehydelysine as markers of lipid peroxidation, N(ε)-(carboxymethyl)lysine, were detected in spinal cord from sporadic ALS (SALS) patients (Shibata et al., 2001), and levels of 8-OHdG, a marker of oxidized DNA, were elevated in whole cervical spinal cord from ALS patients (Fitzmaurice et al., 1996). Elevated protein carbonyl levels have been shown in the spinal cord (Shaw et al., 1995) and motor cortex (Ferrante et al., 1997) from SALS patients, and increased 3-nitrotyrosine levels, a marker for peroxynitrite- and NO2-mediated damage, were found within spinal cord motor neurons in both FALS with SOD1 mutation and SALS patients (Beal et al., 1997). Studies using CSF from ALS patients have reported elevated levels of HNE (Smith et al., 1998), 8-OHdG (Bogdanov et al., 2000; Ihara et al., 2005) and ascorbate free radical (Ihara et al., 2005). The pathology of ALS is complex, multifactorial, and not completely understood. Motor neuron death in ALS does not occur because of a single insult, but rather through a combination of mechanisms including oxidative stress, excitotoxicity, mitochondrial dysfunction, endoplasmic reticulum stress, protein aggregation, cytoskeletal dysfunction, and defects in RNA processing and trafficking. Oxidative stress, whether

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as a primary cause or a secondary consequence of disease, has been implicated in many of these processes. Barber and Shaw reviewed six hypotheses of how oxidative stress can exacerbate other neurodegenerative mechanisms in ALS (Barber and Shaw, 2010): (1) Elevated glutamate levels increase intracellular calcium levels, which are buffered by mitochondria, leading to increased ROS production. ROS inhibit glutamate uptake by glial cells, thereby increasing extracellular glutamate levels. This is a vicious cycle of increasing oxidative stress and excitotoxicity that leads to motor neuron degeneration (Rao and Weiss, 2004). (2) Pathological studies have revealed the presence of morphologically abnormal mitochondria in motor neurons from ALS patients (Siklo´s et al., 1996), and the increased uptake of calcium into mitochondria as a result of glutamate receptor-mediated toxicity has also been reported to trigger production of ROS (Carriedo et al., 2000). (3) Prolonged activation of the unfolded protein response in the endoplasmic reticulum increases ROS production (Ilieva et al., 2007). (4) Aberrant oxidative reactions catalyzed by mutant SOD1 increase the production of the highly reactive peroxynitrite and hydroxyl radicals, causing aggregation of proteins, including SOD1 itself (Rakhit et al., 2004). (5) Abnormal neurofilament accumulation is a pathological feature observed in spinal cord motor neurons in human ALS cases (Hirano et al., 1984). ROS attack neurofilament subunits, causing dityrosine cross-link formation and aggregation of neurofilament subunits (Kim et al., 2004). (6) ROS released from damaged motor neurons disrupt glutamate uptake into neighboring astrocytes (Rao et al., 2003). ROS can also activate glial cells, resulting in release of more ROS and proinflammatory cytokines that activate neighboring glial cells (Banati et al., 1993).

Stroke Acute ischemic cerebrovascular syndrome, or stroke, causes 9% of all deaths around the world and is the second most common cause of death after ischemic heart disease (Murray and Lopez, 1997). Stroke is characterized by a blockage of blood flow to the brain. There are two types of stroke: that induced by a total loss of blood flow to the brain such as during a cardiac arrest, and cerebral ischemia arising from a focal loss of blood flow to the brain due to an artery blockage (Hou and MacManus, 2002). Important risk factors for stroke are hypertension, diabetes, hypercholesterolemia, smoking, and old age (Feigin, 2005). Endothelial dysfunction associated with these risk factors underlies pathological processes leading to atherogenesis and cerebral ischemic injury.

Cerebral ischemia resulting from vascular disorders induces several biochemical and cellular reactions such as inflammation, increased ROS production, impairment of BBB, and calcium overload. With cerebral reperfusion by thrombolytic therapy, ROS production is further stimulated, which causes cytotoxicity through oxidation of lipids, proteins, and DNA (Crack and Taylor, 2005; Gu¨rsoy-Ozdemir et al., 2004). Thrombolytic agent tissue plasminogen activator (tPA), used commonly to restore cerebral blood flow before brain injury becomes irreversible, can increase ROS production through different pathways and cause further damage (Green, 2008). Endothelial cells in cerebral vessels are the basis for the BBB, which prevents the free flow of ions and polar molecules from the blood into brain tissue. Disruption of the BBB during stroke is important because it can lead to local edema because of increased vascular permeability, which in turn can decrease the perfusion of the area (Bektas et al., 2010). Recently, we reported changes in lipid peroxidation products in the CSF of patients with or without symptomatic vasospasm (SVS) after SAH (Hirashima et al., 2012). One of the most feared complications of SAH is ischemia, which occurs in about 30% of patients surviving the initial hemorrhage, mostly between days 4 and 10 after SAH. Decreased cerebral perfusion induced by vasospasm after SAH is thought to be a cause of ischemia. We found that the levels of free IsoPs (8-iso-PGF2α) and HODE in CSF and plasma PAF-AH activity are higher in patients without SVS than with SVS. We speculate that the plasma PAF-AH can hydrolyze oxidized phospholipids and attenuate the spreading of lipid peroxidation.

Down Syndrome DS is caused by a chromosomal aberration involving total or partial trisomy of chromosome 21. DS is considered to be the most common genetic cause of mental retardation. Mental retardation associated with DS is accompanied by learning and memory deficits, and impairments in adaptive behavior (Chapman and Hesketh, 2000; Evans and Gray, 2000). The brain abnormalities observed in DS have been reported to be related to inherent oxidative stress. Busciglio and Yankner (1995) reported that neurons of patients with DS exhibited a three- to four-fold increase in intracellular ROS and elevated levels of lipid peroxidation products. It has been suggested that one source of ROS in DS is the excessive production of hydrogen peroxide through the action of Cu, Zn-superoxide dismutase (Cu,Zn-SOD) (Groner et al., 1994). As a result of the overexpression of Cu,Zn-SOD in DS, there may be an imbalance between Cu,Zn-SOD and other antioxidant

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enzymes, such as catalase and glutathione peroxidase, which may induce oxidative damage (Tanabe et al., 1994). Perrone et al. (2007) reported that the concentration of IsoPs in the amniotic fluid of mothers who were pregnant with a DS fetus was nine-fold greater than in mothers with a normal fetus. The levels of lipid peroxidation products (thiobarbituric acid (TBA) reactive substances and HNE), protein carbonyl (2,4-dinitrophenylhydrazine), and advanced glycation endproducts (pyrraline and pentosidine) were found to be significantly increased in the brains of DS fetuses (18
20 weeks of gestation) in comparison with matched controls, providing evidence that accelerated brain oxidation occurs very early in the life of DS subjects (Odetti et al., 1998).

Other Neurological Dysfunctions in Childhood Similar to adult-onset neurodegenerative diseases, oxidative stress may be involved in various childhoodonset neurological disorders. Periventricular leukomalacia (PVL), the major neuropathological cause of the motor deficits in cerebral palsy, is characterized by focal periventricular necrosis and diffuse gliosis in the surrounding immature white matter. Immunostaining for MDA protein adduct was high in PVL cortical neurons from autopsy samples at postconceptional ages of 34 to 109 weeks (Folkerth et al., 2008). Spinal muscular atrophy (SMA), which is progressive loss of motor neurons in the spinal cord, leads to weakness and atrophy in the leg and respiratory muscle. HNE has been shown to exist in the motor neurons of the spinal anterior horn of SMA patients (Hayashi, 2009). Subacute sclerosing panencephalitis (SSPE), a persistent mutated measles virus infection of the central nervous system, shows slowly progressive brain atrophy and demyelination of white matter. In SSPE autopsy brains, immunoreactivity to 8-OHdG and HNE was observed. Therapeutic Intervention with Antioxidants for Neurological Dysfunction As shown above, oxidative damage is observed in neurological dysfunction, including AD, PD, ALS, stroke, and DS. Disease-specific origins of oxidative stress exist in these diseases. Oxidative stress induces neuronal death via mitochondrial dysfunction, proteasome-inhibition, or endoplasmic reticulum stress. Although there is increasing evidence that oxidative stress is involved in the pathogenesis of neurological diseases, there are few reports that antioxidants are effective in these neurological diseases. Investigations that showed effects of antioxidants, mainly vitamin E, against AD, PD, ALS, stroke, and DS are discussed below.

43

Petersen et al. (2005) reported on a double-blind study of high doses of vitamin E (2000 units per day) given to 769 patients with mild cognitive impairment. However, the results indicated that vitamin E treatment for 3 years did not affect the progression to AD. Significant effect of vitamin E was not corroborated in other studies on vitamin E treatment for AD (Sano et al., 1997; Lloret et al., 2009). Zhang et al. (2002) reported that vitamin E reduces the risk of developing PD. Other studies have shown contradictory results about dietary intake of vitamin E, vitamin C, and carotenoids, and their efficacy for preventing PD progression (Etminan et al., 2005; Pham and Plakogiannis, 2005; Weber and Ernst, 2006). Wang et al. (2011) reported on a large investigation including data from 1,055,546 participants from five prospective cohort studies. ALS risks were similar in users and nonusers of vitamin E supplement. However, among participants in cohorts with information on years of vitamin E supplement use, ALS risk declined with increasing duration of vitamin E use. Since stroke is an acute disease, the effect of vitamin E is difficult to assess. It has been suggested that combining a thrombolytic plus a neuroprotectant, including an antioxidant, might offer advantages, such as improving the clinical outcome after cerebral ischemia, or extending the treatment window for tPA (Green, 2008). Edaravone has been used as a neuroprotective agent for the treatment of acute cerebral infarction in Japan. Five controlled clinical trials have been conducted with children and young adults with DS, testing various doses and combinations of antioxidant vitamins and mineral supplements (Ellis et al., 2008; Bennett et al., 1983; Bidder et al., 1989; Smith et al., 1984; Ani et al., 2000). However, none of these trials reported a significant effect of antioxidants on cognitive function. In these trials, antioxidant vitamin supplementation was begun at 7.5 months of age or later, possibly accounting for the lack of efficacy observed. We performed an experiment to test whether chronic administration of vitamin E could reverse the cognitive deficit found in Ts65Dn mice, a mouse model of DS (Shichiri et al., 2011). Vitamin E was administered to pregnant Ts65Dn females from the day of conception throughout the pregnancy, and to Ts65Dn and control pups over their entire life, from birth until the end of the behavioral testing period. Supplementation with vitamin E attenuated cognitive impairment and decreased the levels of lipid peroxidation products in Ts65Dn mice. The results of animal experiments cannot be easily extrapolated to humans, but this study implies a potential benefit of vitamin E supplementation to DS patients at an early stage. In conclusion, it may be stated that, although not fully demonstrated, oxidative stress, notably lipid

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4. UNREGULATED LIPID PEROXIDATION IN NEUROLOGICAL DYSFUNCTION

peroxidation, is involved in the onset and progression of various diseases including neurodegenerative diseases and that the antioxidants should be beneficial for maintenance of health and prevention of diseases when given to the right subjects at the right time for the right duration and in the right amount.

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C H A P T E R

5 Obesity, Western Diet Intake, and Cognitive Impairment Scott E. Kanoski, Ted M. Hsu and Steven Pennell OBESITY AND COGNITIVE IMPAIRMENT

accuracy in the go/no-go task in children (aged 7
9), where higher BMI was associated with lower response accuracy (Kamijo et al., 2012). Other measures of inhibitory control have yielded similar results in adults
obese individuals show worse performance on the Stroop Task (a test requiring response inhibition for incongruent stimuli), as well as increased impulsivity ratings on psychometric tests (Maayan et al., 2011, Jasinska et al., 2012). These types of inhibitory control deficits may be causally related to the unhealthy eating habits that contribute to the development and maintenance of obesity (Maayan et al., 2011, Jasinska et al., 2012). This notion has been previously described as a ‘vicious circle’ in which cognitive deficits associated with obesity (i.e., inhibitory control deficits) contribute to further impulsive intake of unhealthy energy dense foods, which perpetuates further excessive eating and weight gain (Davidson et al., 2007, Kanoski and Davidson, 2011, Kanoski, 2012). While mental flexibility and inhibitory control are often considered to be separate domains of cognitive function, these psychological constructs may, in fact, involve overlapping cognitive and central nervous system (CNS) processing (Reinert et al., 2013). Growing evidence has begun to show an inability for obese individuals to disengage from previously relevant responses and then actively engage in a new set of responses. For example, administration of a test of cognitive flexibility called the Wisconsin Card Sorting Test revealed increased perseverative errors in 12-year-old obese subjects compared to lean individuals (Cserjesi et al., 2007). Similar results were also observed in adult subjects, where greater perseverative errors were seen in obese adults compared to normal weight controls (Cohen et al., 2011). Further support for these results has been seen in other measures of set shifting; Verdejo-Garcia and colleagues showed that obese

The increasing prevalence of obesity and its clear associations with several negative health outcomes has become a serious concern in modern Westernized societies. While research has revealed an apparent relationship between obesity and health outcomes like cardiovascular disease and Type 2 diabetes mellitus (T2DM), recent findings have also begun to unveil a relationship between obesity and cognitive decline, as well as various types of neurodegenerative dementias (Sellbom and Gunstad, 2012, Gustafson, 2008, Hassing et al., 2009). Furthermore, while accumulating evidence links obesity to increased risk of Alzheimer’s and other types of dementia in later life, the relationship between obesity and cognitive decline has been shown across a range of age groups, from childhood to adulthood (Cohen et al., 2011, Benito-Leon et al., 2013, Reinert et al., 2013). Multiple domains of cognitive and executive function have been examined in relation to obesity, especially inhibitory control, attention and mental flexibility, and learning and memory (Sellbom and Gunstad, 2012, Reinert et al., 2013). One domain that is particularly disrupted in obese individuals is impulsivity/inhibitory control. Several pieces of evidence have demonstrated that obese individuals adopt an impulsive strategy of favoring immediate rewards despite negative consequences in the future (Davis et al., 2004, Davis et al., 2010). Similarly, several studies have demonstrated reduced inhibitory control in obese individuals across various age groups. For example, a common behavioral measure for inhibitory control is the go/no-go task that requires subjects to respond during ‘go’ trials and inhibit the same response during ‘no-go’ trials. A recent study revealed a negative relationship between body mass index (BMI) and response Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00005-3

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5. OBESITY, WESTERN DIET INTAKE, AND COGNITIVE IMPAIRMENT

individuals had poorer performance during set shifting during an inhibition test and trail making test (Verdejo-Garcia et al., 2010). Taken together, results associating these cognitive deficits with obesity have strong implications with regards to the development of unhealthy lifestyle choices that, as discussed above, may perpetuate the development and maintenance of obesity. There is also a wealth of evidence showing that obesity is associated with learning and memory impairments. Gunstad and colleagues demonstrated that in a population of young and middle aged adults, obese participants demonstrated impaired delayed recall and recognition in a verbal list learning task (Gunstad et al., 2006). Similar results have been reported in a comprehensive study of obese and overweight elderly participants, where obese participants, independent of confounding factors that might influence cognition, had poorer performance on several learning and memory indexes such as free recall (Benito-Leon et al., 2013). Some studies have also shown obesity-associated impairments in working memory (the ability to actively store and manipulate information) (Cohen et al., 2011), although some conflicting findings (Cserjesi et al., 2007, Verdejo-Garcia et al., 2010) suggest that working memory may be less susceptible to disruption by obesity compared to other types of memory such as delayed recall and recognition. Neurological measures provide further evidence for the association between cognitive impairment and obesity. Batterink et al. (2010) utilized functional magnetic resonance imaging (fMRI) in conjunction with measures of cognitive functioning to demonstrate parallel deficits in both behavior and brain activation. More specifically, their results showed a correlation between high BMI and reduced activation of frontal executive regions involved with inhibitory control, such as the orbitalfrontal cortex and the ventrolateral prefrontal cortex. Similarly, structural magnetic resonance imaging (sMRI) has revealed correlations between reduced orbitalfrontal cortical volume, increased disinhibition, and high BMI (Maayan et al., 2011). This pattern has also been shown in an elderly female sample, where obese subjects had decreased orbitalfrontal cortical grey matter volume and poorer performance on tests of executive functioning (Walther et al., 2010). Overall, the recent neurological and behavioral evidence that associates obesity with cognitive deficits reveals that the harmful health impact of obesity is not limited to physiology, but also extends to CNS function. While research has highlighted a relationship between obesity and cognitive impairment, understanding the causality within this connection is critical for developing intervention and prevention strategies. In other words, it may be that cognitive impairment

(e.g., poor learning and memory function and/or inhibitory control) is what’s driving hyperphagia and the development of obesity rather than the other way around. Sorting out this direction of causality is difficult given that systematic controlled prospective studies evaluating whether cognitive impairments are causally related to obesity development (and vice versa) are thus far lacking. Another consideration discussed below is disentangling the relative contributions of Western diet intake versus obesity on cognitive impairment.

WESTERN DIET INTAKE AND COGNITIVE IMPAIRMENT Consuming dietary components that are commonly found in Western diets, such as saturated fatty acids (SFAs) and simple sugars (e.g., glucose, sucrose, high fructose corn syrup) is associated with cognitive impairment in both humans and experimental animal models. However, evidence was discussed above describing strong associations between obesity and cognitive decline. Given that consumption of SFAs and simple sugars (i.e., a typical ‘Western diet’) is linked with increased adiposity and body weight gain, many of the cognitive deficits attributed to consumption of these dietary components may be a secondary result of weight gain and related comorbidities (e.g., increased adiposity, metabolic syndrome, peripheral insulin resistance) and not diet, per se. This section will focus on whether diet-induced cognitive decline can occur independently of increases in body weight and adiposity. We will review studies, primarily those using controlled animal models, which show cognitive differences in behavioral tasks between different dietary groups that did not differ significantly in body weight. Two recent studies report that memory deficits can arise in rodents following a short period of Western diet consumption, well before significant increases in body weight and adiposity occur. First, Kanoski and Davidson observed spatial memory impairments in male rats after only 72 hours on a Western diet. The memory deficits persisted throughout the study and became more pronounced after longer periods of Western diet maintenance (Kanoski and Davidson, 2010). A similar result was obtained in rats by Murray et al. (2009) after only 9 days on a Western diet. These findings suggest that Western diet consumption can rapidly change the metabolic profile in a manner that contributes to CNS dysfunction well before the development of metabolic syndrome and obesity. Most animal model studies employ longer periods of Western diet maintenance feeding (e.g., 1
3 months) that eventually produce increased body weight and adiposity relative to

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control groups maintained on low-fat diets (see Kanoski and Davidson, 2011 for a review). However, some studies have reported working memory impairments in rodents following longer periods of Western diet intake that did not produce differential body weights relative to controls (e.g., Granholm et al., 2008). Privitera and colleagues recently assessed the role of a Western diet during development on cognitive abilities later in life (Privitera et al., 2011). Male rats were fed either a Western diet (WD) or a low-fat control diet (LF) during postnatal days (PD) 21
40; afterward, both groups of rats were fed standard lab chow. This developmental stage (PD days 21
40) in rats is analogous to pre-adolescence and adolescence in humans, in which significant brain development occurs. Cognition was measured using a conditioned-place preference (CPP) paradigm in which rats learned to associate a context (e.g., a location) with a reward (e.g., food). CPP training began on postnatal day 41 (immediately after the end of the diet manipulation phase) or on postnatal day 81 (delayed training). For comparison, another group of rats were fed either a WD or LF diet during postnatal days 61
81 with CPP trials beginning immediately afterwards on postnatal day 81. Both the immediate and delayed CPP testing in the adolescent LF groups learned the CPP. Interestingly, the adult WD group also learned the CPP; however, the rats that were fed the WD during adolescence did not, regardless of whether CPP training began immediately or following a delay after the dietary manipulation. Therefore, a WD during adolescence impaired food reward learning and memory later in life when the animals were consuming a healthy LF standard lab chow diet. Importantly, there were no significant differences in body weight between the LF and WD adolescent diet groups that were subsequently fed LF and had the delayed test (they were put on an LF diet after the diet manipulation for 40 days before the CPP training and testing). This means that the differences in reward learning and memory were not due to increased adiposity and obesity, per se, but rather were attributable to previous consumption of an obesigenic WD. Francis and Stevenson investigated whether a Western diet affected human memory performance. In the first part of the study, participants answered a dietary questionnaire (DFS-SQ) that measured the amount of saturated fat and refined sugar consumed in the past year. Participants then completed two computer tasks to assess delayed verbal memory performance. Participants that reported a greater consumption of Western diet-related foods in the past year performed worse on the memory tasks after controlling for other variables such as BMI. This further corroborates the idea that consuming a Western diet impairs cognitive function, independent of obesity. The second part of

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the study examined whether Western diet consumption affected memory of recent meal size. Results showed that participants who consumed less saturated fatty acids and simple sugars were significantly better at estimating the amount of food recently consumed than those consuming higher levels of these dietary components (Francis and Stevenson, 2011). This latter finding by Francis and Stevenson is important with regards to overeating in humans, as recent studies show that the strength of a memory for a recent eating episode is negatively correlated with the amount of food consumed at a subsequent meal (Higgs, 2002, Higgs, 2008, Brunstrom et al., 2012). In other words, poorer memory of a recent eating episode leads to excessive feeding at a subsequent meal. Thus, Western diet consumption appears to disrupt memory for a recent eating episode (independent of, or prior to, obesity development), which may lead to overeating at subsequent meals and potentially contribute to the development of obesity later in life. This notion is similar to the vicious circle model discussed above proposed by Davidson, Kanoski, and colleagues (Davidson et al., 2007, Davidson et al., 2005, Kanoski, 2012, Kanoski and Davidson, 2011) in which shortterm consumption of Western diets can lead to impaired hippocampal-dependent inhibitory memory function (prior to obesity onset), which can contribute to hyperphagia and eventually obesity development.

Western Diet Intake and Cognitive Impairment: Underlying Neuroendocrine Mechanisms Several interrelated neurobiological mechanisms underlie Western diet-induced cognitive impairment, including impaired peripheral glucose regulation, increased circulating triglycerides, reductions in brainderived neurotrophic factor (BDNF), and impairments in various forms of neural plasticity. These mechanisms have been reviewed elsewhere (Kanoski and Davidson, 2011) and are beyond the scope of this section. Here we focus on the novel hypothesis that Western diet consumption contributes to cognitive dysfunction by disrupting neuroendocrine signaling in the hippocampus, a brain region that is strongly tied to learning and memory function. Learning and memory ability is regulated, in part, by various neuroendocrine signals (hormones) that are produced in the periphery and act on specific protein receptors in the brain. These hormonal signals have the capacity to influence behavioral measures of learning and memory and alter dynamic structural changes in the hippocampus that are purported to contribute to the formation and maintenance of new memories, including changes in the strength of the connections

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between neurons (i.e., ‘synaptic plasticity’), as well as the generation of new neurons (i.e., ‘neurogenesis’). An accumulating body of data shows that these neuroendocrine signaling systems (and subsequently the memory functions that they support) can become disrupted by the intake of Western diets. We will focus on a few of these hormonal signaling systems that not only modulate memory function, but are also intimately linked with energy status (i.e., body weight, hunger, satiety, ongoing food intake) and potently influence food intake and energy balance regulation via communication between the peripheral organs and the brain. Leptin is a hormone produced from fat tissue that signals in multiple brain regions to reduce food intake and body weight, as evident from the extreme obesity and hyerphagia in both humans and rodents with genetic mutations to either the gene that encodes leptin or its receptor (LepRb) (see Leshan et al., 2006 for a review). Leptin action in the hippocampus promotes various types of neural plasticity that facilitate memory formation, including synaptic plasticity (glutamate receptor-mediated long-term potentiation and long-term depression) (Moult et al., 2009, Shanley et al., 2001) and neurogenesis (Garza et al., 2008). Unfortunately, leptin’s ability to act in the brain to reduce food intake (and potentially to improve memory function) appears to be disrupted with Western diet intake and obesity, a phenomenon known as ‘leptin resistance’. Leptin resistance is characterized by a

disrupted ability of leptin to engage intracellular and secondary signaling pathways after binding to its receptor on neurons (Munzberg et al., 2004). While leptin resistance has been established in the hypothalamus, the traditional ‘feeding center’ in the brain, the notion that leptin resistance occurs in the hippocampus has not been fully explored. However, our unpublished preliminary data show that leptin administration increases hippocampal expression of BDNF, a neurotrophin that promotes memory formation and neural plasticity, in control rats fed a low-fat diet but not in rats fed a Western diet for two weeks (Figure 5.1). Thus, these preliminary findings suggest that Western diet consumption may promote memory dysfunction by blunting the ability of leptin to promote neural plasticity. Insulin is a hormone produced in the pancreas that helps to regulate blood glucose metabolism. Like leptin, insulin signals in the brain to decrease food intake and body weight (Bruning et al., 2000, Obici et al., 2002). Insulin also acts on hippocampal neurons to facilitate memory function, an effect demonstrated following central administration of insulin to rodents (Park et al., 2000) and following intravenous (Craft et al., 2003) or intranasal (Reger et al., 2008) administration in humans. Insulin signaling is compromised, however, by Western diet consumption and obesity. T2DM is a comorbidity of obesity that is characterized by impaired ability of insulin to regulate blood glucose levels in the periphery. In addition to disrupting peripheral insulin signaling, Western diet intake and

BDNF protein expression in the hippocampus following CNS leptin delivery

BDNF/B-actin protein (% of chow-vehicle)

1.4

*

1.2

1 0.8 0.6 0.4 0.2

0 Low-fat Low-fat Western diet Western diet (vehicle injection) (leptin injection) (vehicle injection) (leptin injection)

FIGURE 5.1 Leptin administration (4 μg in 1 μl volume; intra-lateral cerebral ventricle) increases protein expression of brain-derived neurotrophic factor (BDNF), assessed via immunoblot analysis, in the hippocampus in rats fed a low-fat control diet, but not in rats maintained on a high-fat Western diet for two weeks.

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REFERENCES

obesity are also linked with CNS insulin resistance. When insulin accesses the brain and activates its specific receptor in neurons, it engages an intracellular signaling cascade that promotes neurogenesis (activation of protein kinase B, also known as Akt) (Shioda et al., 2009). Recent data show that insulin’s capacity to engage this intracellular signaling cascade in the hippocampus and hypothalamus is impaired with high fat diet intake (Clegg et al., 2011, Pratchayasakul et al., 2011), as is insulin’s ability to promote neural plasticity (Pratchayasakul et al., 2011). In one instance, neuronal insulin resistance occurred following Western diet intake independently of (prior to) the development of obesity (Clegg et al., 2011). Thus, disrupted CNS insulin signaling by Western diet intake is one mechanism that may underlie cognitive impairment associated with these dietary factors. Unlike the other neuroendocrine signals discussed above, ghrelin, a hormone discovered in 1999 (Kojima et al., 1999), communicates from the gut to the brain to increase (rather than suppress) food intake and foodmotivated behavior. In addition to effects on feeding behavior, ghrelin promotes neural plasticity in the hippocampus (long-term potentiation and increased spine density in neuronal dendrites) and promotes memory consolidation in a passive avoidance task (remembering that a location is associated with aversive reinforcement) (Diano et al., 2006). Ghrelin also signals in the ventral subregion of the hippocampus to stimulate meal initiation in free-feeding rats in response to stimuli (e.g., auditory tones) that previously signaled food availability when energy restricted (Kanoski et al., 2013). This study also demonstrated that ghrelin resistance occurs in the hippocampus. Rats that were maintained on a Western diet for several weeks showed reduced activation of the intracellular PI3KAkt signaling pathway, which promotes neural plasticity in ventral hippocampal neurons in response to ghrelin administration (Shioda et al., 2009). It is not yet established whether ghrelin’s diminished impact at the intracellular signaling level following Western diet consumption translates to an effect on memory function at the behavioral level.

SUMMARY Obesity and Western diet intake are clearly linked with cognitive decline, and in some cases, dementia onset. This is particularly concerning given that obesity rates have risen dramatically since the late 1970s, such that over one-third of U.S. adults are classified as obese, with another third classified as overweight. Perhaps of even more concern is that the nature of the cognitive impairment associated with Western diets and obesity

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(e.g., impaired inhibitory control and memory for recent eating episodes) may perhaps exacerbate the problem by contributing to a ‘vicious circle’ of further excessive food intake and weight gain. Efforts to understand the underlying neurobiological mechanisms that contribute to dietary and metabolic effects on cognition can hopefully lead to effective behavioral and pharmacological treatments to alleviate the harmful effects of both obesity and cognitive impairment.

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1703. Benito-Leon, J., Mitchell, A.J., Hernandez-Gallego, J., Bermejo-Pareja, F., 2013. Obesity and impaired cognitive functioning in the elderly: a population-based cross-sectional study (NEDICES). Eur. J. Neurol. 20, 899-e77. Bruning, J.C., Gautam, D., Burks, D.J., Gillette, J., Schubert, M., Orban, P.C., et al., 2000. Role of brain insulin receptor in control of body weight and reproduction. Science 289, 2122
2125. Brunstrom, J.M., Burn, J.F., Sell, N.R., Collingwood, J.M., Rogers, P.J., Wilkinson, L.L., et al., 2012. Episodic memory and appetite regulation in humans. PLoS One 7, e50707. Clegg, D.J., Gotoh, K., Kemp, C., Wortman, M.D., Benoit, S.C., Brown, L.M., et al., 2011. Consumption of a high-fat diet induces central insulin resistance independent of adiposity. Physiol. Behav. 103, 10
16. Cohen, J.I., Yates, K.F., Duong, M., Convit, A., 2011. Obesity, orbitofrontal structure and function are associated with food choice: a cross-sectional study. BMJ Open. 1, e000175. Craft, S., Asthana, S., Cook, D.G., Baker, L.D., Cherrier, M., Purganan, K., et al., 2003. Insulin dose-response effects on memory and plasma amyloid precursor protein in Alzheimer’s disease: Interactions with apolipoprotein E genotype. Psychoneuroendocrinology 28, 809
822. Cserjesi, R., Molnar, D., Luminet, O., Lenard, L., 2007. Is there any relationship between obesity and mental flexibility in children? Appetite 49, 675
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746. Davidson, T.L., Kanoski, S.E., Schier, L.A., Clegg, D.J., Benoit, S.C., 2007. A potential role for the hippocampus in energy intake and body weight regulation. Curr. Opin. Pharmacol. 7, 613
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747. Kamijo, K., Khan, N.A., Pontifex, M.B., Scudder, M.R., Drollette, E.S., Raine, L.B., et al., 2012. The relation of adiposity to cognitive control and scholastic achievement in preadolescent children. Obesity (Silver Spring). 20, 2406
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Moult, P.R., Milojkovic, B., Harvey, J., 2009. Leptin reverses longterm potentiation at hippocampal CA1 synapses. J. Neurochem. 108, 685
696. Munzberg, H., Flier, J.S., Bjorbaek, C., 2004. Region-specific leptin resistance within the hypothalamus of diet-induced obese mice. Endocrinology 145, 4880
4889. Murray, A.J., Knight, N.S., Cochlin, L.E., McAleese, S., Deacon, R.M., Rawlins, J.N., Clarke, K., 2009. Deterioration of physical performance and cognitive function in rats with short-term high-fat feeding. FASEB J, 23, 4353
4360. Obici, S., Feng, Z., Karkanias, G., Baskin, D.G., Rossetti, L., 2002. Decreasing hypothalamic insulin receptors cause hyperphagia and insulin resistance in rats. Nat. Neurosci. 5, 566
572. Park, C.R., Seeley, R.J., Craft, S., Woods, S.C., 2000. Intracerebroventricular insulin enhances memory in a passiveavoidance task. Physiol. Behav. 68, 509
514. Pratchayasakul, W., Kerdphoo, S., Petsophonsakul, P., Pongchaidecha, A., Chattipakorn, N., Chattipakorn, S.C., 2011. Effects of high-fat diet on insulin receptor function in rat hippocampus and the level of neuronal corticosterone. Life Sci. 88, 619
627. Privitera, G.J., Zavala, A.R., Sanabria, F., Sotak, K.L., 2011. High fat diet intake during pre and periadolescence impairs learning of a conditioned place preference in adulthood. Behav. Brain Funct. 7, 21. Reger, M., Watson, G., Green, P., Baker, L., Cholerton, B., Fishel, M., et al., 2008. Intranasal insulin administration dose-dependently modulates verbal memory and plasma amyloid-beta in memoryimpaired older adults. J. Alzheimer’s Dis. 13, 323
331. Reinert, K.R., Po’e, E.K., Barkin, S.L., 2013. The relationship between executive function and obesity in children and adolescents: a systematic literature review. J. Obes. 2013, 820956. Sellbom, K.S., Gunstad, J., 2012. Cognitive function and decline in obesity. J Alzheimer’s Dis. 30 (Suppl. 2), S89
S95. Shanley, L.J., Irving, A.J., Harvey, J., 2001. Leptin enhances NMDA receptor function and modulates hippocampal synaptic plasticity. J. Neurosci. 21, RC186, 1
6. Shioda, N., Han, F., Fukunaga, K., 2009. Role of Akt and ERK signaling in the neurogenesis following brain ischemia. Int. Rev. Neurobiol. 85, 375
387. Verdejo-Garcia, A., Perez-Exposito, M., Schmidt-Rio-Valle, J., Fernandez-Serrano, M.J., Cruz, F., Perez-Garcia, M., et al., 2010. Selective alterations within executive functions in adolescents with excess weight. Obesity (Silver Spring). 18, 1572
1578. Walther, K., Birdsill, A.C., Glisky, E.L., Ryan, L., 2010. Structural brain differences and cognitive functioning related to body mass index in older females. Hum. Brain Mapp. 31, 1052
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C H A P T E R

6 Genetic Risk Factors for Diabetic Neuropathy Carmine Gazzaruso and Adriana Coppola DIABETES MELLITUS AND ITS COMPLICATIONS

unmyelinated fibers can be damaged. There are attempts at axon regeneration, but they usually fail. DN often worsens because there is a reduced blood flow due to the involvement of the autonomic nervous system that affects microvessels (Vincent et al., 2011).

Diabetes mellitus is an epidemic condition that is increasing in prevalence worldwide due to unbalanced diet, physical inactivity, obesity, and aging (Wild et al., 2004, Danaei et al., 2011). Subjects with diabetes have a high prevalence of chronic complications such as cardiovascular and cerebrovascular disease, peripheral artery disease, retinopathy, kidney disease, and neuropathy.

RISK FACTORS FOR DIABETIC NEUROPATHY AND PATHOPHYSIOLOGICAL MECHANISMS Many risk factors seem to be associated with the development and progression of DN in diabetic patients. Table 6.1 reports the most important risk factors identified in several studies (Monastiriotis et al., 2012, Smith and Singleton, 2013, Lu et al., 2013). These risk factors can act by activating one or more of the following pathophysiological mechanisms: increased polyol pathway activity, oxidative stress, nonenzymatic glycation of nerve proteins and their receptors, mitochondrial damage, inflammation, activation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK), endoneural hypoxia, deficiency of long chain fatty acids, increased levels of homocysteine, and deficiency of folate and vitamin B12 levels (Wada and Yagihashi, 2005, Mata et al., 2008, Vincent et al., 2011, Chan et al., 2011, Miranda-Massari et al., 2011, Kamenov, 2012). Hyperglycemia certainly represents the most important activator of the pathophysiological mechanisms of DN, in particular of the polyol pathway. This is an alternative catabolic pathway that is activated when there is an increased intracellular glucose level. As is well-known, in the majority of the cells, action of insulin is needed to permit the glucose entry into them. For the cells of the retina, kidney, and nervous tissue this process is insulin-independent. So, in the hyperglycemic state, the important increase in glucose into the cell causes the activation of the enzyme aldose

DIABETIC NEUROPATHY: GENERAL CHARACTERISTICS The most common complication among diabetic patients is certainly diabetic neuropathy (DN): indeed, more that 50% of subjects with diabetes suffer from DN over the course of their disease (Tesfaye and Selvarajah, 2012). In addition, DN can represent the main source of morbidity and mortality, since it increases the risk for foot ulcers, foot and ankle fractures, depression, chronic pain, infections, and lower-limb amputations (Tesfaye and Selvarajah, 2012, Athans and Stephens, 2008, Veves et al., 2008). DN can affect sensory, motor, and autonomic neurons of the peripheral nervous system (Kamenov, 2012a; Kamenov, 2012b): virtually every type of nerve fiber can be damaged by diabetes and all organ systems may be involved, including cardiovascular, gastrointestinal, ophthalmologic, and genitourinary systems. Nevertheless, the most common type of DN is a symmetric distal sensory/sensorimotor polyneuropathy that begins at foot level and progresses in a proximal direction (Kamenov, 2012b). The symptoms generally develop over years. DN is usually an axonopathy that initially involves the longest axons (Vincent et al., 2011). DN is usually a dynamic flux between neuronal degeneration and regeneration. Both myelinated and Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00006-5

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© 2014 Elsevier Inc. All rights reserved.

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6. GENETIC RISK FACTORS FOR DIABETIC NEUROPATHY

TABLE 6.1 Main Risk Factors for Diabetic Neuropathy

TABLE 6.2 Potential Mechanisms and Pathways Implicated in the Development/Progression of Diabetic Neuropathy

Main Risk Factors for Diabetic Neuropathy Glycemic control

Potential Mechanisms and Pathways Implicated in the Development/Progression of Diabetic Neuropathy

Diabetes duration

Polyol pathway activity

Age

Oxidative stress

Socio-economic status

Inflammation

Insulin treatment

Insulin resistance

Dyslypidemia

Formation of AGEs

Hypertension

Action of AGEs on RAGE

Obesity

Mithocondrial damage

Height

Activation of PKC

Smoking

Activation of MAPK

Alcohol consumption

Endoneural hypoxia

Impairment in renal function

Deficiency of long chain fatty acids

Genetic factors

High homocysteine levels Deficiency of folate and vitamin B12

reductase, with an accumulation of sorbitol, which does not cross the cell membrane. The reduction of glucose to sorbitol due to aldose reductase is mainly obtained by the oxidation of NADPH1 to NADP1. Sorbitol accumulation results in osmotic stress, cellular edema, and cell lysis. Another important mechanism leading to peripheral nerves may be represented by an increase in oxidative stress, since a decreased availability of NADPH reduces reduction and regeneration of glutathione. This causes an increase in pro-oxidative substances, called reactive oxygen species (ROS). Interestingly, in the cell, elevated glucose levels are also able to increase diacylglycerol, which can activate PKC. This enzyme seems to be involved in several pathophysiological mechanisms of the DN, in particular in impairing blood flow that negatively affects metabolic pathways of the nerve. Glucose can non-enzymatically form covalent bonds with proteins, lipids, and nucleic acids. In the hyperglycemic state there is an increased formation of advanced glycation end products (AGEs). AGEs can favor DN both by decreasing the biological functions of some proteins and by binding specific receptors, including receptors for AGE (RAGE). The decreased action of some proteins inhibits neuronal activity, while the activation of RAGE results in increases in both oxidative stress and neuronal vascular dysfunction, leading to microangiopathy of the nerve. Oxidative stress in diabetes can lead to increased oxidation of lipoproteins, in particular of low-density lipoprotein (LDL). So oxidized LDL has a particularly high affinity for some scavenger receptors, called

Lipoprotein oxidation AGEs: advanced glycation end products. RAGE: receptors for AGEs. PKS: protein kinase C. MAPK: mitogen-activated protein kinase.

oxLDLs. The internalization of oxidized LDL increases intracellular ROS. Oxidative stress is also able to boost RAGE expression, via the enzyme MAPK, which is often activated under conditions of insulin resistance. The most important pathophysiological mechanisms and pathways involved in the development and progression of DN are summarized in Table 6.2.

GENETIC RISK FACTORS FOR DIABETIC NEUROPATHY All the mechanisms reported in Table 6.2 may have a role in promoting the development and progression of DN. Hyperglycemia is certainly the main trigger in activating these pathophysiological mechanisms, but many other environmental risk factors can be implicated (Table 6.1). Nevertheless, in clinical practice it is evident that diabetic patients sharing the same risk factors often show significant differences in the occurrence of diabetic complications. This suggests that a genetic predisposition towards the development of complications exists. Many studies have identified several candidate genes, in particular for the development of retinopathy, nephropathy, and macrovascular complications. There are less data available in the literature on the genetic risk factors involved in the

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GENETIC RISK FACTORS FOR DIABETIC NEUROPATHY

occurrence of DN. Studies particularly regard gene polymorphisms as being potentially involved in the pathophysiological mechanisms of the condition. The most studied gene is probably the aldose reductase gene, which can act on the polyol pathway activity. In a population of 85 Finnish type 2 diabetic patients and 126 nondiabetic controls, Sivenius and colleagues (Sivenius et al., 2004) found that diabetic patients with some polymorphisms of the aldose reductase gene had a lower sensory response amplitude and, during a 10-year follow-up period, a greater decrease in the conduction velocity. Nevertheless, no association was found with overt DN. In a cohort of 262 young type 1 diabetic patients, a decline in autonomic and quantitative sensory nerve testings linked to aldose reductase gene polymorphism was observed during a 7-year follow-up period (Thamotharampillai et al., 2006). However, a small study failed to find any association between aldose reductase gene polymorphism and microvascular complications, including neuropathy, in Caucasian type 1 diabetic patients (Ng et al., 2001). Several genetic polymorphisms can potentially influence the development of DN by increasing the vulnerability to oxidative stress. Suzuki and colleagues have hypothesized that a defect in aldehyde dehydrogenase 2 (ALDH2) could increase oxidative stress after alcohol ingestion (Suzuki et al., 2003). In any case, the authors speculated that increased tissue levels of toxic aldehyde could result from inactive ALDH2 expression, which results in the increased level of reactive aldehyde in sensory neuron pathways, thereby causing DN (Suzuki et al., 2004). In a group of 216 type 1 diabetic patients with DN and 250 without DN it was observed that the 262 T allele of the catalase gene seems to have a protective effect against the rapid development of DN (Chistiakov et al., 2006). In another Russian population of type 1 diabetic patients, an association of the genes manganese superoxide dismutase (SOD2) and extracellular superoxide dismutase (SOD3) with the presence of DN was observed (Strokov et al., 2003). The Na/K ATPase gene polymorphism has been seen associated with the presence of DN in type 1 diabetic patients with at least 10 years of diabetes duration (Vague et al., 1997). It has been hypothesized that this could promote the development of ROS. A similar action may be produced by the gene of the uncoupling protein 2, as suggested by two studies (Yamasaki et al., 2006, Rudofsky et al., 2006). This protein may mediate mitochondrial function. A Japanese study of 197 type 2 diabetic patients suggested that the uncoupling protein 2 gene seems to negatively affect nerve conduction and vasomotor sympathetic functions (Yamasaki et al., 2006), but in type 1 diabetic subjects, it was associated with a reduced prevalence of DN (Rudofsky et al., 2006).

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Therefore, the real role of uncoupling protein 2 remains to be elucidated. In the neuronal cells there are some of the so-called metabotropic glutamate receptors (mGluRs) that, if adequately activated, can have neuroprotective effects, as shown in vivo and in vitro (Anjaneyulu et al., 2008). The activation of these receptors may antagonize glucose-induced oxidative damage that may cause cell injury by the production of ROS and mitochondrial dysfunction. The neuroprotective effects can be obtained by an increased synthesis and release of glutathione (GSH) that is able to counter the increased ROS production. The use of specific agonists of these receptors has been suggested for the prevention and treatment of DN. Inflammation seems to play a major role in the development and progression of neuropathy. Some genetic polymorphisms have been proposed as possible risk factors for DN by acting on inflammatory mechanisms. In a group of type 1 diabetic patients, Barzilay and colleagues observed that the HLA DR3/4 phenotype may significantly increase the risk for autonomic neuropathy by approximately 6.2 times (Barzilay et al., 1992)). A genetic variation in or near the tumor necrosis factor (TNF) receptor 2 gene (TNFRSF1B) has been associated with an increased risk for DN (odds ratio: 2.1) in a group of 357 type 2 diabetic patients (Benjafield et al., 2001). Kolla and colleagues evaluated TNF-alpha, interferon gamma and interleukin 10 gene polymorphisms in relation to the presence of DN in type 2 diabetic patients (Kolla et al., 2009). These authors did not find an association between TNF-alpha and DN, but they observed that some genotypes associated with high production of interleukin 10 and low production of interferon gamma may have a role in DN development. Facer and colleagues have hypothesized that the gene for capsaicin (or vanilloid) receptors, such as TRPV1, could have a role in DN (Facer et al., 2007). To understand the possible action of TRPV1 on DN, it is important to remember that this receptor is involved in inflammatory pain and hyperalgesia and may modulate vasodilatation and natriuretic/diuretic action. Other inflammation genes may be involved in the onset and progression of DN, such as bradykinin receptor B2 (BDKRB2) (Kakoki et al., 2010) and membrane associated adenosine A3 receptor (ADORA3) (Hur et al., 2011). Another gene that could increase the risk for DN is represented by the alpha 2B adrenoreceptor gene that is characterized by a deletion/insertion polymorphism (Papanas et al., 2007). In type 2 diabetic patients allele D seems to be associated with a greater prevalence of DN. Interestingly, alpha 2B adrenoreceptor mediates a wide variety of functions, including regulation of blood pressure, sympathetic tone, insulin sensitivity, and lipolysis. Therefore, it

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6. GENETIC RISK FACTORS FOR DIABETIC NEUROPATHY

could favor DN both by hypoxia and by insulin resistance. On the other hand, it is interesting to remember that this gene may also be associated with the development of type 2 diabetes. Other genetic risk factors can act by affecting lipid metabolism. Among these the role of apolipoprotein E (ApoE) polymorphism may be particularly important. Recently, Monastiriotis and colleagues reviewed the studies that investigated the possible role of this polymorphism (Monastiriotis et al., 2012). They found four relevant studies but the results on the association between ApoE and DN were conflicting, probably because of methodological differences. However, the possible role of ApoE polymorphism in DN onset and progression seems to be due to actions on oxidative stress and inflammation. It is of interest to remember the potential role of the peroxisome proliferatoractivated receptor (PPAR) gamma, a nuclear receptor involved in lipid and glucose metabolism, in the development and progression of DN (Hur et al., 2011). For the endothelial function to perform correctly, it is important to have adequate production of nitric oxide (NO). In NO production the enzyme endothelial nitric oxide synthase (eNOS) plays a pivotal role. On the contrary, vascular endothelial growth factor (VEGF) levels are usually increased under conditions of endothelial dysfunction. Some researchers have found that eNOS polymorphism can be associated with DN in type 2 diabetic patients (Mehrab-Mohseni et al., 2011) and VEGF polymorphism may favor DN in type 1 diabetic patients (Bazzaz et al., 2010). The possible role of the homocysteine (Hcy) may be of some interest in the development and progression of DN. Indeed, it has been hypothesized that high Hcy levels can play a role in the development not only of cardiovascular complications, but also of DN (Miranda-Massari et al., 2011). The enzyme methylenetetrahydrofolate reductase (MTHFR), folate, and vitamin B12 are crucial in the synthesis of the amino acid methionine. Hcy is an intermediate in the biosynthesis of methionine and when this biosynthetic mechanism is reduced, Hcy levels increase. Interestingly, long-term therapy with metformin, an important first-line treatment for type 2 diabetes, can cause malabsorption of vitamin B12 with a potential increase in Hcy. In addition, a genetic variant in the MTHFR gene can determine an MTHFR deficiency with an increase in Hcy. This suggests that subjects with the genetic variant of the MTHFR gene and long-term treatment with metformin can be more prone to DN. Therefore a supplementation with folate and vitamin B12 may be useful. Table 6.3 summarizes genetic risk factors potentially involved in the development and progression of DN and their hypothetical role in the pathophysiological mechanisms implicated in neural injury. It is evident that the classification on the basis of the mechanisms is

TABLE 6.3 Potential Genes Involved in the Development/ Progression of Diabetic Neuropathy Genes

Hypothetical Main Mechanism

Aldose reductase

Polyol pathway activity

Aldehyde dehydrogenase 2 (ALDH2)

Oxidative stress

Catalase Manganese superoxide dismutase (SOD2) Extracellular superoxide dismutase (SOD3) Na/K ATPase Uncoupling Protein 2 Metabotropic Glutamate Receptors HLA DR3/4

Oxidative stress
mitochondrial dysfunction Inflammation

Tumor necrosis factor receptor 2 gene (TNFRSF1B) Interferon gamma Interleukin 10 Capsaicin (or vanilloid) receptors Bradykinin receptor B2 (BDKRB2) Membrane associated adenosine A3 receptor (ADORA3) Alpha 2B adrenoreceptor

Hypoxia
insulin resistance

Apolipoprotein E (ApoE)

Lipid metabolism (inflammation
oxidative stess)

Perixosome proliferator-activated receptor (PPAR) gamma

Lipid and glucose metabolism

Nitric oxide syntase (eNOS)

Endothelial dysfunction

Vascular endothelial growth factor (VEGF) Methylene-tetrahydrofolate reductase (MTHFR)

Homocysteine levels

absolutely arbitrary, since each gene can be potentially implicated in more than one mechanism and some mechanisms are only hypothetical. Nevertheless, a classification can be useful in providing an initial framework in an otherwise unstructured field.

POTENTIAL USE OF GENETIC RISK FACTORS IN CLINICAL PRACTICE There are a relatively large number of studies on the possible association between specific genetic risk factors (genetic phenotypes or genotypes) and the

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REFERENCES

presence of DN. Nevertheless no clinical implication can be obtained from these associations. This is mainly due to the large number of genes potentially implicated. Therefore it may be very difficult to stratify the personal genetic risk for DN in a specific diabetic patient. In addition, data available in the literature have to be considered as preliminary: indeed they have usually been drawn from small and case-control studies. This implies that large and longitudinal studies are needed to confirm a possible association with the aim of using these risk factors in clinical practice. In addition, these studies should clarify not only whether an association exists but also whether it is valid for both type 1 and type 2 diabetes, whether it is affected by drugs and other environmental factors (it can be useful to remember metformin or alcohol for some genes), and whether it promotes the onset and/ or progression of DN. Therefore it is evident that the correct identification of each gene involved in the development of DN and its real ‘weight’ in determining neural damage can be important in establishing a panel of genes in order to be able to assign a specific score to each patient reflecting their personal genetic risk for DN. This can assist in the prevention and/or early diagnosis and treatment of DN by allowing informed decision-making in adopting the most appropriate specific programs. In addition, the identification of reliable genetic pathways in DN may be useful in helping to identify specific treatments, including possible gene therapies. In conclusion, the current knowledge of genetic risk factors involved in DN cannot yet be used in clinical practice, and further research is necessary in order to more fully establish their exact role. This will then allow the identification of subjects at particularly high risk for DN, along with the adoption of reliable and specific protocols of prevention and care.

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93. Athans, W., Stephens, H., 2008. Open calcaneal fractures in diabetic patients with neuropathy: a report of three cases and literature review. Foot Ankle Int. 29, 10
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3630. Benjafield, A.V., Glenn, C.L., Wang, X.L., Colagiuri, S., Morris, B.J., 2001. TNFRSF1B in genetic predisposition to clinical neuropathy and effect on HDL cholesterol and glycosylated hemoglobin in type 2 diabetes. Diabetes Care 24 (4), 753
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Chan, L., Terashima, T., Urabe, H., Lin, F., Kojima, H., 2011. Pathogenesis of diabetic neuropathy: bad to the bone. Ann. N.Y. Acad. Sci. 1240, 70
76. Chistiakov, D.A., Zotova, E.V., Savost’anov, K.V., Bursa, T.R., Galeev, I.V., Strokov, I.A., et al., 2006. The 262T . C promoter polymorphism of the catalase gene is associated with diabetic neuropathy in type 1 diabetic Russian patients. Diabetes Metab. 32 (1), 63
68. Danaei, G., Finucane, M.M., Lu, Y., Singh, G.M., Cowan, M.J., Paciorek, C.J., Global Burden of Metabolic Risk Factors of Chronic Diseases Collaborating Group (Blood Glucose), et al., 2011. National, regional, and global trends in fasting plasma glucose and diabetes prevalence since 1980: systematic analysis of health examination surveys and epidemiological studies with 370 country-years and 2.7 million participants. Lancet 378, 31
40. Facer, P., Casula, M.A., Smith, G.D., Benham, C.D., Chessell, I.P., Bountra, C., et al., 2007. Differential expression of the capsaicin receptor TRPV1 and related novel receptors TRPV3, TRPV4 and TRPM8 in normal human tissues and changes in traumatic and diabetic neuropathy. BMC Neurol. 7, 11. Hur, J., Sullivan, K.A., Pande, M., Hong, Y., Sima, A.A., Jagadish, H.V., et al., 2011. The identification of gene expression profiles associated with progression of human diabetic neuropathy. Brain 134 (Pt 11), 3222
3235. Kakoki, M., Sullivan, K.A., Backus, C., Hayes, J.M., Oh, S.S., Hua, K., et al., 2010. Lack of both bradykinin B1 and B2 receptors enhances nephropathy, neuropathy, and bone mineral loss in Akita diabetic mice. Proc. Natl. Acad. Sci. USA 107 (22), 10190
10195. Kamenov, Z.A., Traykov, L.D., 2012a. Diabetic somatic neuropathy. Adv. Exp. Med. Biol. 771, 155
175. Kamenov, Z.A., Traykov, L.D., 2012b. Diabetic autonomic neuropathy. Adv. Exp. Med. Biol. 771, 176
193. Kolla, V.K., Madhavi, G., Pulla Reddy, B., Srikanth Babu, B.M., Yashovanthi, J., Valluri, V.L., et al., 2009. Association of tumor necrosis factor alpha, interferon gamma and interleukin 10 gene polymorphisms with peripheral neuropathy in South Indian patients with type 2 diabetes. Cytokine 47 (3), 173
177. Lu, B., Hu, J., Wen, J., Zhang, Z., Zhou, L., Li, Y., et al., 2013. Determination of peripheral neuropathy prevalence and associated factors in Chinese subjects with diabetes and pre-diabetes
ShangHai Diabetic neuRopathy Epidemiology and Molecular Genetics Study (SH-DREAMS). PLoS One 8 (4), e61053. Mata, M., Chattopadhyay, M., Fink, D.J., 2008. Gene therapy for the treatment of diabetic neuropathy. Curr. Diab. Rep. 8, 431
436. Mehrab-Mohseni, M., Tabatabaei-Malazy, O., Hasani-Ranjbar, S., Amiri, P., Kouroshnia, A., Bazzaz, J.T., et al., 2011. Endothelial nitric oxide synthase VNTR (intron 4 a/b) polymorphism association with type 2 diabetes and its chronic complications. Diabetes Res. Clin. Pract. 91 (3), 348
352. Miranda-Massari, J.R., Gonzalez, M.J., Jimenez, F.J., Allende-Vigo, M.Z., Duconge, J., 2011. Metabolic correction in the management of diabetic peripheral neuropathy: improving clinical results beyond symptom control. Curr. Clin. Pharmacol. 6, 260
273. Monastiriotis, C., Papanas, N., Veletza, S., Maltezos, E., 2012. ApoE gene polymorphisms and diabetic peripheral neuropathy. Arc. Med. Sci. 8, 583
588. Ng, D.P., Conn, J., Chung, S.S., Larkins, R.G., 2001. Aldose reductase (AC)(n) microsatellite polymorphism and diabetic microvascular complications in Caucasian Type 1 diabetes mellitus. Diabetes Res. Clin. Pract. 52 (1), 21
27. Papanas, N., Papatheodorou, K., Papazoglou, D., Kotsiou, S., Christakidis, D., Maltezos, E., 2007. An insertion/deletion polymorphism in the alpha2B adrenoceptor gene is associated with peripheral neuropathy in patients with type 2 diabetes mellitus. Exp. Clin. Endocrinol. Diabetes 115 (5), 327
330. Rudofsky Jr., G., Schroedter, A., Schlotterer, A., Voron’ko, O.E., Schlimme, M., Tafel, J., et al., 2006. Functional polymorphisms of

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UCP2 and UCP3 are associated with a reduced prevalence of diabetic neuropathy in patients with type 1 diabetes. Diabetes Care 29 (1), 89
94. Sivenius, K., Pihlajama¨ki, J., Partanen, J., Niskanen, L., Laakso, M., Uusitupa, M., 2004. Aldose reductase gene polymorphisms and peripheral nerve function in patients with type 2 diabetes. Diabetes Care 27 (8), 2021
2026. Smith, A.G., Singleton, J.R., 2013. Obesity and hyperlipidemia are risk factors for early diabetic neuropathy. J. Diabetes Complications (Epub ahead of print). Strokov, I.A., Bursa, T.R., Drepa, O.I., Zotova, E.V., Nosikov, V.V., Ametov, A.S., 2003. Predisposing genetic factors for diabetic polyneuropathy in patients with type 1 diabetes: a population-based case-control study. Acta. Diabetol. 40 (Suppl. 2), S375
S379. Suzuki, Y., Taniyama, M., Muramatsu, T., Higuchi, S., Ohta, S., Atsumi, Y., et al., 2004. ALDH2/ADH2 polymorphism associated with vasculopathy and neuropathy in type 2 diabetes. Alcohol. Clin. Exp. Res. 28 (8 Suppl. Proceedings), 111S
116S. Suzuki, Y., Taniyama, M., Muramatsu, T., Ohta, S., Atsumi, Y., Matsuoka, K., 2003. Influence of alcohol intake and aldehyde dehydrogenase 2 phenotype on peripheral neuropathy of diabetes. Diabetes Care 26 (1), 249. Tesfaye, S., Selvarajah, D., 2012. Advances in the epidemiology, pathogenesis and management of diabetic peripheral neuropathy. Diabetes Metab. Res. Rev. 28 (Suppl. 1), 8
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Thamotharampillai, K., Chan, A.K., Bennetts, B., Craig, M.E., Cusumano, J., Silink, M., et al., 2006. Decline in neurophysiological function after 7 years in an adolescent diabetic cohort and the role of aldose reductase gene polymorphisms. Diabetes Care 29 (9), 2053
2057. Vague, P., Dufayet, D., Coste, T., Moriscot, C., Jannot, M.F., Raccah, D., 1997. Association of diabetic neuropathy with Na/K ATPase gene polymorphism. Diabetologia. 40 (5), 506
511. Veves, A., Backonja, M., Malik, R.A., 2008. Painful diabetic neuropathy: epidemiology, natural history, early diagnosis, and treatment options. Pain. Med. 9, 660
674. Vincent, A.M., Callaghan, B.C., Smith, A.L., Feldman, E.L., 2011. Diabetic neuropathy: cellular mechanisms as therapeutic targets. Nat. Rev. Neurol. 7, 573
583. Wada, R., Yagihashi, S., 2005. Role of advanced glycation end products and their receptors in development of diabetic neuropathy. Ann. N. Y. Acad. Sci. 1043, 598
604. Wild, S., Roglic, G., Green, A., Sicree, R., King, H., 2004. Global prevalence of diabetes. Estimates for the year 2000 and projections for 2030. Diabetes Care. 27, 1047
1053. Yamasaki, H., Sasaki, H., Ogawa, K., Shono, T., Tamura, S., Doi, A., et al., 2006. Uncoupling protein 2 promoter polymorphism
866G/A affects peripheral nerve dysfunction in Japanese type 2 diabetic patients. Diabetes Care 29 (4), 888
894.

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7 n-3 Fatty Acid-Derived Lipid Mediators against Neurological Oxidative Stress and Neuroinflammation Akhlaq A. Farooqui OVERVIEW

neuroketals (NKs), and neurofurans (NFs). Enzymicallyderived lipid mediators of EPA and DHA metabolism not only down-regulate pro-inflammatory cytokines but also produce antioxidant, anti-inflammatory, antithrombotic, antiarrhythmic, hypolipidemic, and vasodilatory effects (Hong et al., 2003; Hong et al., 2008; Marcheselli et al., 2003; Serhan, 2005; Serhan et al., 2008; Serhan et al., 2009; Marcheselli et al., 2010; Farooqui, 2009; Farooqui, 2012a). The nonenzymic lipid mediators of EPA and DHA metabolism also produce prooxidant and proinflammatory effects (Farooqui, 2011). Accumulating evidence suggests that ARA- and DHA-derived lipid mediators compete with each other and modulate induction and regulation of neuroinflammation by controlling the duration and magnitude of acute inflammation and oxidative stress, as well as the return of the injury site to homeostasis in the process of catabasis (the decline of the disease state) (Serhan et al., 2008; Farooqui, 2009; Farooqui, 2012a). Another important function of ARA, EPA, and DHA-derived lipid mediators is their involvement in the signal transduction network, which conveys the message of extracellular signals from the cell surface to the nucleus to induce a biological response at the gene level (Fahrenkrog, 2006). It is also reported that levels of ARA-, EPA-, and DHA-derived lipid mediators in neural and non-neural tissues are partly regulated by diet. Accumulating evidence supports the view that levels of ARA, EPA, and DHA, and their lipid mediators, not only orchestrate and control the onset of neuroinflammation and oxidative stress by coupling lipid metabolism with neural membrane lipid organization, but also cooperate with the action of lipid-dependent enzymes to execute appropriate downstream actions and responses. The present day Western diet is deficient in EPA and

In neural membrane phospholipids, essential fatty acids, namely arachidonic acid (ARA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3), are exclusively located at the sn-2 position of glycerol moiety of phospholipids. A small amount of eicosapentaenoic acid (EPA, 20:5n-3) is also present at the sn-2 position of neural membrane phospholipids. ARA belongs to the n-6 and EPA and DHA belong to the n-3 family of essential fatty acids. A high intake of food enriched in vegetable oils elevates levels of enzymic and nonenzymic mediators of ARA metabolism. Enzymic lipid mediators of ARA metabolism include prostaglandins (PGs), leukotrienes (LTs), thromboxanes (TXs), lipoxins (LXs), 2-arachidonylglycerol (2-AG), and arachidonylethanolamide (AEA) (Farooqui, 2009) (Figure 7.1). Nonenzymically, ARA is metabolized to 4-hydroxynonenal (4-HNE), isoprostanes (IsoPs), isoketals (IsoKs), and isofurans (IsoFs). ARA-derived lipid mediators produce prooxidant, prothrombotic, proaggregatory, and pro-inflammatory effects. In contrast, diets enriched in EPA and DHA (fish and fish oil) generate different enzymic and nonenzymic lipid mediators (Farooqui, 2011). The enzymic lipid mediators of EPA metabolism include E-series resolvins, 3-series PGs, 5-series LTs, and 17, 18-epoxyeicosatetraenoic acid (17, 18-EEQ). Nonenzymic mediators of EPA metabolism are cyclopentenone-isoprostanes (A3/J3-IsoPs) (Brooks et al., 2008). Similarly, the enzymic lipid mediators of DHA metabolism include D-series resolvins, neuroprotectins (NTPs), and maresins (MaRs) (Figure 7.1). The nonenzymic lipid mediators of DHA metabolism include 4-hydroxyhexanal (4-HHE), neuroprostanes (NPs),

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FIGURE 7.1 Phospholipid-derived enzymic and non-enzymic lipid mediators in the brain. Arachidonic acid (ARA); eicosapentaenoic acid (EPA); docosahexaenoic acid (DHA); prostaglandins (PG); leukotrienes (LT); 17, 18-epoxyeicosatetraenoic acid (1, 18-EEQ); 4-hydroxynonenal (4-HNE); isoprostanes (IsoP); isoketal (IsoK); isofuran (IsoF); 4-hydroxyhexanal (4-HHE); neuroprostane (NP); Neuroketal (NK); neurofuran (NF); 2-arachidonylglycerol (2-AG); N-arachidonoylethanolamine (AEA); N-arachidonyl-dopamine (NAD); and virodhamine (VDA). Modified from Farooqui, 2009.

DHA, but has high amounts of ARA (Farooqui, 2009; Farooqui, 2012a). The purpose of this article is to describe the antioxidant and anti-inflammatory effects of EPA- and DHA-derived lipid mediators in the brain.

DHA IN THE BRAIN DHA is an absolute requirement for the development of the human central nervous system (CNS) and the continuous maintenance of brain cell function. EPA and DHA play an important role throughout life, as critical modulators of neuronal function and regulation of neuroinflammation and oxidative stressmediated mechanisms in the normal brain during aging and chronic neurological diseases. Inadequate levels of DHA in the brain during development and old age induce cognitive deficits such as memory loss and learning disability in experimental animals (Farooqui, 2009). Thus, inadequacy of EPA and DHA

in neural membranes may contribute to cholinergic, dopaminergic, and glutamatergic receptor dysfunction in synapses associated with the hippocampal neurons, and growing evidence suggests that low levels of DHA in the brain are associated not only with neurotraumatic, neurodegenerative, and neuropsychiatric diseases, but also with peroxisomal disorders (Farooqui, 2009). Dietary intake of EPA antagonizes the synthesis of PGE2 from ARA and reduces the IL-1β-mediated increase in levels of PGE2 (Song et al., 2004; Song et al., 2009). Furthermore, expression of IL-10 is also blocked by ethyl-EPA treatment. Based on these results, it is proposed that ethyl-EPA treatment produces beneficial effects in those neuropsychiatric disorders in which inflammation and oxidative stress play a critical role (Song et al., 2004; Song et al., 2009; Farooqui, 2009). DHA acts as a ligand for the PPAR-γ and the RXR receptors. In conjunction with these receptors, which act as transcription factors, DHA modulates various

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neurochemical processes that maintain cellular homeostasis by modulating lipid metabolism, neural cell differentiation, and apoptosis (Farooqui, 2009). DHA not only downregulates protein kinase C, Ras, and NF-κB, but also activates the Jak/Stat pathway, and sustains phosphorylation of EGFR. DHA attenuates the transcription of NF-κB-dependent genes. Thereby, the COX-2/PGE2-dependent generation of pro-angiogenic vascular endothelial growth factor and levels of antiapoptotic bcl-2 and bcl-X(L) are reduced. Eicosanoidindependent proapoptotic pathways include enhanced lipid peroxidation, modulation of mitochondrial calcium homeostasis, and enhanced production of reactive oxygen species (ROS), as well as activation of p53. In the brain, DHA also restores levels of cerebellar phospho (p)-AKT, phospho-extracellular regulated kinase (p-ERK) and phospho-c-Jun N-terminal kinase (p-JNK), supporting their role in downregulation of neuronal apoptosis (Sinha et al., 2009). DHA also quenches gene expression of cyclooxygenase-2 and other enzymes, thereby diminishing the formation of pro-inflammatory eicosanoids. Pre-administration of DHA increases the corticohippocampal glutathione levels and glutathione reductase activity and suppresses the increase in lipid peroxide and ROS levels in the cerebral cortex and hippocampus of Alzheimer’s disease (AD) (Dan, 2009). In addition, DHA scavenges for free radicals, which diminish inflammatory response and oxidation of lipoprotein particles, notably low density lipoproteins (LDLs). DHA also suppresses insulin/neurotrophic factor signaling deficits, neuroinflammation, and oxidative damage that contribute to synaptic loss and neuronal dysfunction in old age and demented subjects (Cole and Frautschy, 2010). Finally, DHA increases brain levels of neuroprotective brain-derived neurotrophic factor and reduces the ARA and its oxidative metabolites. The cross-talk among these molecular processes has distinct neuroprotective effects not only through the stabilization of neural membranes, modulation of ion channels, and receptors, but also through inhibition of inflammatory processes and generation of anti-inflammatory lipid mediators (Farooqui et al., 2007; Farooqui, 2009). Dietary intake of DHA results in incorporation of this fatty acid into ethanolamine and serine glycerophospholipids. Among ethanolamine glycerophospholipids, ethanolamine and choline plasmalogens are closely associated not only with the stability of synapse and functioning of various receptors but also with membrane fluidity and permeability, and maintenance of electrophysiological characteristics (Farooqui, 2009). DHA-enriched phosphatidylserine (PtdSer) is an essential cofactor for the activation of several proteins including protein kinase C, Raf-1 kinase, and AKT, which translocate from cytoplasm to the membrane for

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their activation, supporting the view that translocation of these kinases may be target signaling events modulated by the DHA-mediated neuronal specific increase of PtdSer (Kim et al., 2010). PtdSer also modulates activities of diacylglycerol kinase, nitric oxide synthase, and Na1, K1-ATPase (Ikemoto et al., 2000). Collective evidence suggests that EPA- and DHAinduced changes in membrane properties may further affect the ability of membrane receptors to interact with their ligands or intracellular signaling molecules as well as modulate the effect of membrane bound enzymes (Farooqui, 2009; Farooqui, 2011). As stated above, compared to DHA, levels of EPA in brain tissue are quite low. This may be due to its rapid β-oxidation of EPA following its uptake by the brain tissue (Chen et al., 2009a). In rat hepatocytes L-carnitine, a long chain fatty acid mitochondrial matrix transporter, not only increases β-oxidation of EPA, but only marginally elevates the oxidation of ARA and alleviates competitive inhibition of ARA-dependent PGE2 synthesis and COX-2 expression by EPA. It is suggested that L-carnitine modulates the competition between ARA and EPA in PG synthesis in liver cells by enhancing oxidation of EPA. This suggests that the beneficial effects of n-3 PUFA, especially EPA, are modulated by cellular oxidation capacity (Du et al., 2010).

EPA-DERIVED LIPID MEDIATORS IN THE BRAIN EPA-derived lipid mediators include the 3-series PGs and TXs, 5-series LTs, and E-series resolvins (Resolvin E1 and E2 or RvE1 and RvE2). The oxidized metabolites of EPA mediate anti-inflammatory and antiproliferative effects. The oxidation of EPA by COX and LOX enzymes results in the production of 3-series PGs and TXs, and 5-series of LTs. These lipid mediators have different biological properties from the corresponding analogs generated by COXs and LOXsmediated oxidation of ARA. For example, TXA3 is less active than TXA2 in aggregating platelets and constricting blood vessels (Calder, 2009). In addition to generating less active lipid mediators, EPA exerts its effects on other aspects of inflammation such as leukocyte chemotaxis and inflammatory cytokine production. Some of these effects are likely due to changes in nuclear factor-κB-mediated gene expression (e.g. adhesion molecule) in microglia, astrocytes, and in visceral inflammatory and immune cells. In contrast, recent studies on the effects of prostaglandins (PGE2 and PGE3) and leukotrienes (LTB4 and LTB5) on endothelium permeability and mononuclear adhesion and migration across endothelial cell cultures indicate that PGE3 produces more pronounced effects on

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trans-endothelial Evans blue-albumin (EBA) permeability than PGE2, and these effects are antagonized by EP1 and EP2 antagonists (Moreno, 2009). LTB4 and LTB5 produce a slight effect on EBA extravasation (Moreno, 2009; Yin et al., 2007). It is suggested that EPA and ARA compete for the same COX enzymes (Zhao et al., 2004; Phillis et al., 2006), but the rate of oxidation of EPA is only 10% of the ARA. However, EPA significantly inhibits COX-1-mediated oxidation of ARA (Wada et al., 2007; Schmitz and Ecker, 2008), but the oxidation of ARA by COX-2 is only modestly inhibited by EPA. Metabolism of EPA by 15-LOX-like enzyme results in the synthesis of resolvins of the E-series (Arita et al., 2006; Arita et al., 2007), including resolvin E1 (RvE1; (5S,12R,18R)-trihydroxy-6Z,8E,10E,14Z,16Eeicosapentaenoic acid) and resolvin E2 (15S,18R-dihydroxy-EPE) (Figure 7.2). EPA is oxidized to 18R-hydroxyeicosapentaenoic acid (18R-HEPE) by endothelial cell cyclooxygenase-2 (COX-2). Aspirin acetylates COX-2 and the acetylated enzyme no longer catalyzes the synthesis of PGs, but can still convert EPA

to 18R-HEPE. During cell
cell interactions, 18R-HEPE is released to neighboring leukocytes, which through the action of 5-LOX converts it to RvE1 via a 5(6) epoxide containing intermediate. RvE1 is present in human whole blood, and its levels can be increased by ingestion of aspirin (Arita et al., 2006; Arita et al., 2007). RvE1 is transformed into several metabolic products, including 20-hydroxy-RvE1, 20-carboxy-RvE1, 19-hydroxy-RvE1, 18-oxo-RvE1, and 10,11-dihydro-RvE1 by human PMNs and whole blood as well as in murine inflammatory exudates, lungs, spleen, kidney, and liver (Seki et al., 2010). Among these products, 20-carboxy-RvE1, 18-oxo-RvE1, and 10,11-dihydro-RvE1 are essentially biologically inactive and may serve as inactive biomarkers of RvE1 metabolism in vivo. In contrast, 20-hydroxylated product of RvE1 has some of the activity of RvE1, suggesting that more metabolites of RvE1 are generated during inflammatory response. In non-neural tissues, RvE1 and RvE2 induce potent anti-inflammation/pro-resolution effect in vivo (Arita et al., 2006) via specific G protein-coupled receptors

FIGURE 7.2 Synthesis and roles of resolvins E1 and E2 in neural and non-neural tissues.

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(ChemR23 receptors) (Ohira et al., 2010). RvE1 suppresses the activation of NF-κB by tumor necrosis factor-α (TNF-α) through binding to human polymorphonuclear leukocyte (PMN) (Arita et al., 2007). Intrathecal RvE1 injection blocks spontaneous pain and heat and mechanical hypersensitivity evoked by intrathecal capsaicin and TNF-α. RvE1 mediates its anti-inflammatory activity not only by decreasing neutrophil infiltration, paw edema, but also by inhibiting the expression of pro-inflammatory cytokines (Xu et al., 2010). In addition, RvE1 also inhibits TNFα-mediated N-methyl-D-aspartic acid (NMDA) receptor hyperactivity in spinal dorsal horn neurons via inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway (Xu et al., 2010). Based on these results, it is suggested that resolvins may normalize the spinal synaptic plasticity associated with pain hypersensitivity. In heart tissue, EPA is also oxidized by cytochrome P450 enzyme (CYP2C/2J), which converts it into 17,18-epoxyeicosatetraenoic (17,18-EEQ) acid. These n-3 epoxides are highly active as antiarrhythmic agents. They suppress Ca21-induced increased rate of spontaneous beating of neonatal rat cardiomyocytes at low nanomolar concentrations (Arnold et al., 2010). Thus, dietary EPA/DHA supplementation in rats is accompanied by concomitant changes in endogenous CYP metabolites, which also mediate beneficial cardiovascular effects of dietary n-3 fatty acids (Arnold et al., 2010).

DHA-derived Lipid Mediators in the Brain Metabolism of DHA by 15-LOX-like enzyme results in the synthesis of D-series resolvins. This enzyme converts DHA into 17S-hydroperoxy-DHA (17S-H(p) DHA), which is converted into several bioactive compounds, including resolvin D1-D6 (RvD1, RvD2, RvD3, RvD4, RvD5, and RvD6). In addition, interactions with aspirin result in the formation of aspirin-triggered D-series resolvins (AT-Rv) through sequential oxygenation initiated by aspirin-acetylated COX-2. These lipid mediators not only antagonize the effects of PGs, LTs, and TXs, but also modulate leukocyte trafficking and down-regulate the expression of cytokines in glial cells. They possess potent anti-inflammatory, neuroprotective, and pro-resolving properties (Hong et al., 2003; Marcheselli et al., 2003; Serhan, 2005).

DHA-derived D-series Resolvins In neural and non-neural cells, D-series resolvins act through resolvin D receptors (resoDR1) (Serhan et al., 2008). ResoDR1 modulate potent anti-inflammatory and immunoregulatory activities. D-series resolvins not only

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block the production of pro-inflammatory mediators, but also regulate the trafficking of leukocyte cells at the sites of inflammation (Serhan et al., 2008; Hong et al., 2003) and inhibit the expression of cytokines leading to modulation of neuroinflammation. Studies on characterization of ResoDR1 are urgently needed to progress understanding of this area of DHA-derived lipid mediators.

DHA-derived Protectins and Neuroprotectins Synapses are specialized anatomical sites (structures) that facilitate communication between neurons. They are essential for transmitting, processing, and storing information from one cell to another in the brain. They are enriched in DHA-containing plasmalogens, a unique class of phospholipids characterized by the presence of a vinyl ether bond at the sn-1 position of the glycerol moiety. It is well known that loss of synapses in the hippocampal and cortical regions is accompanied by a decrease in plasmalogens in Alzheimer’s disease (AD) patients (Selkoe, 2002; Selkoe 2008; Ginsberg et al., 1995; Ginsberg et al., 1998; Han et al., 2001; Guan et al., 1999). The molecular mechanism associated with loss of synapse and decrease in plasmalogens is not yet fully understood. However, it is proposed that Aβ oligomers induce stimulation of plasmalogen-selective PLA2 (PlsEtn-PLA2) and may be responsible for the decrease in plasmalogen levels and synaptic loss in AD (Farooqui, 2010a; Farooqui, 2010b; Farooqui, 2012a). Since plasmalogens are essential structural phospholipids of synapses and neurons, decreases in plasmalogen pools may likely be responsible for the loss of synapse and the shrinkage of neurons that precedes neurodegeneration in AD. Synaptic loss not only correlates with cognitive impairment in AD, but also with insufficient mitochondrial ATP (Terry et al., 1991; Selkoe, 2002; Dong et al., 2007). The biosynthetic conversion of DHA to NPD1/PD1 involves the action of 15-LOX to form an epoxide intermediate followed by enzymic hydrolysis (Figure 7.3). Investigations into the stereochemistry of PD1 synthesis have confirmed that PD1 is a 10R,17S- dihydroxydocosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid (Hong et al., 2003; Marcheselli et al., 2003; Serhan et al., 2008). The reaction sequence of biosynthesis for PD1 via the epoxide intermediate distinguishes it from the formation of the double dioxygenation product 10S, 17S-dihydroxy-DHA. PD1 is more potent than DHA in neuroprotective action. In sharp contrast, PD1 positional isomers, including 4S,17S-diHDHA or 7S,17S-diHDHA, display less potent and non-selective actions. The occurrence of PD1 has also been reported in brain, where it is called neuroprotectin D1 (10R, 17S-dihydroxydocosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid, NPD1)

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FIGURE 7.3 Generation and roles of neuroprotection D1 from DHA in the brain. Modified from Farooqui, 2012a.

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EPA-DERIVED LIPID MEDIATORS IN THE BRAIN

(Hong et al., 2003). Tritium-labeled NPD1 (3H]-NPD1) binds to ARPE-19 cells with high affinity (Kd 31.3 6 13.1 pmol/mg of cell protein). The stereo-specific NPD1 interactions with these cells provide potent protection against oxidative stress-mediated apoptosis. 3 H-NPD1/PD1 also shows specific and selective high affinity binding with isolated human neutrophils (Kd approximately 25 nM). Neither resolvin E1 nor lipoxin A4 compete for 3H-NPD1/PD1-specific binding with human neutrophils. Collectively, these results indicate that stereo-selective specific binding of NPD1/PD1 with retinal pigment epithelial cells as well as human neutrophils may occur through specific receptors in both the immune and visual systems (Marcheselli et al., 2010). The isolation and characterization of 10(S),17(S)-dihydroxydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid, a main dihydroxy conjugated triene derived from the lipoxygenation of DHA has also been reported, but nothing has been published on its interactions in neural and non-neural cells. It is an isomer of protectin/neuroprotectin D1 (PD1/NPD1) and has been named PDX (Chen et al., 2009b). This metabolite inhibits human blood platelet aggregation at submicromolar concentrations. Like resolvins, the neuroprotectins not only block the infiltration of PMN (Serhan et al., 2006), but also down-regulate the expression of cytokines in the glial cells (Hong et al., 2003; Serhan et al., 2008). NPD1 reduces retinal and corneal injury (Mukherjee et al., 2004) and produces neuroprotective effects in ischemic injury (Marcheselli et al., 2003). Similarly, NPD1 promotes neural cell survival via the induction of antiapoptotic and neuroprotective gene expression programs that suppress Aβ42-mediated neurotoxicity in AD (Lukiw et al., 2005; Bazan, 2009a,b). DHA and NPD1 protect synapses and decrease the number of activated microglia in the hippocampal system (Pomponi et al., 2008). The molecular mechanisms associated with the above processes are not fully understood. However, it is becoming increasingly evident that NPD1 not only inhibits IL-1β-stimulated expression of COX-2, but also regulates apoptotic signaling at the level of the mitochondria, inducing the

release of cytochrome c and activating effector enzyme, caspase-3. In addition, in rats infused with Aβ, DHA and its oxidative metabolites attenuate elevation in levels of lipid peroxides and ROS in the cerebral cortex and the hippocampus, indicating that DHA and its metabolites facilitate neuroprotection by downregulating γ-secretase activity, an enzyme that liberates Aβ from soluble amyloid precursor protein-β (Lukiw et al., 2005). Furthermore, soluble amyloid precursor protein-β stimulates the synthesis of NPD1 (Lukiw et al., 2005; Bazan, 2009a,b). It is also reported that DHA increases protein levels of a genetically implicated risk factor, SorLA/LR11, a neuronal sorting protein that regulates APP processing to decrease Aβ production in a dose-dependent manner (Ma et al., 2007). This observation anchors the growing connection between LR11 and causal mechanisms of AD pathogenesis (Dodson et al., 2008). Recently, variants of the LR11 gene (SORL1) have been shown to correlate with risk of sporadic AD in several populations, providing direct genetic evidence for a proximal role of LR11 in AD (Lee et al., 2007). Collective evidence suggests that DHA and its oxidative metabolites limit the generation and accumulation of the Aβ peptide, which is closely associated with the pathogenesis of AD. DHA and its metabolites also suppress several signal transduction pathways induced by Aβ, including two major kinases that phosphorylate the microtubule-associated protein tau and promote neurofibrillary tangle pathology (Farooqui, 2009; Farooqui, 2010a) (Table 7.1). It has recently been shown that in macrophages, DHA is also metabolized through a 14-LOX pathway resulting in generation of 7,14-dihydroxy-docosa4Z,8,10,12,16Z,19Z-hexaenoic acid. This metabolite is called maresin (MaR1). This metabolite not only terminates PMN infiltration, but also stimulates macrophage phagocytosis. An isomer of MaR1, 7S,14S-diHDHA acts less potently than MaR1. This suggests that in macrophages MaR1 and other DHA-derived metabolites may stereo-selectively regulate catabasis and facilitate arrival of tissues to homeostasis through the inhibition of leukotriene A4 hydrolase, a bifunctional

TABLE 7.1 Effects of Resolvins and Neuroprotectins in Animal Models of Neural and Non-Neural Diseases Animal Model

Effect of Resolvin/ Neuroprotectin

Stroke

Alzheimer’s disease

Molecular Mechanism

Reference

Beneficial

Decrease in proapoptotic Bcl-2 expression and inhibition of caspase-3; induction of neuroprotective gene expression

Mukherjee et al., 2007; Bazan, 2009a,b

Beneficial

Decrease in proapoptotic Bcl-2 expression and inhibition of caspase-3; induction of neuroprotective gene expression

Mukherjee et al., 2007; Bazan, 2009a,b

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zinc containing enzyme that contains epoxide hydrolase and aminopeptidase activities (Serhan et al., 2009; Farooqui, 2011). In retina, epithelium-derived factor acts as an agonist and induces the synthesis of NPD1, thus promoting NPD1-mediated paracrine and autocrine signal transduction processes. Also, DHA and epitheliumderived factor not only synergistically activate NPD1 generation and antiapoptotic protein expression, but also down-regulate proapoptotic Bcl-2 protein expression and activation of caspase 3 during oxidative stress (Mukherjee et al., 2007). NPD1 also promotes AKT translocation and activation and interacts with the PPAR-gamma family of ligand-activated nuclear receptors, which may be involved in various aspects of neuroinflammation and neurodegeneration (PalaciosPelaez et al., 2010; Niemoller and Bazan, 2010; Farooqui, 2010a,b). Receptors for NPD1 have not been characterized in brain tissue, but their occurrence has been suggested (Hong et al., 2003; Marcheselli et al., 2003; Mukherjee et al., 2004). Thus, NPD1-mediated regulation targets upstream events of brain cell apoptosis and modulation of neuroinflammatory signaling promotes the cellular homeostasis and restoration of brain damage through the above mentioned mechanisms. It is tempting to speculate that the generation of DHA-derived Rvs, NPD1, MaR, and synthesis of ARAderived lipoxins may be internal neuroprotective mechanisms that block neuroinflammation and apoptosis-mediated brain damage caused by neurotraumatic and neurodegenerative diseases (Serhan, 2005; Bazan, 2009a,b; Farooqui, 2010a). Lipidomics, proteomics, and genomics techniques have been used to identify and determine levels of ARA, EPA, and DHA-derived lipid mediators (PGs, LTs, TXs, LXs, RvEs, RvD, and NPD1) (Serhan et al., 2006; Ariel and Serhan, 2007; Serhan et al., 2008; Bazan, 2009a,b). This information can be used for developing diagnostic tests in cerebrospinal fluid (CSF) for acute neural trauma in neurodegenerative and neuropsychiatric patients. The use of proteomics techniques for characterizing EPA and DHA metabolizing enzymes in subcellular organelles of the human brain and CSF may provide new information on properties and therapeutic targets of neurological disorders. Rvs and RvDs synthesizing and catabolizing enzymes may not only modulate the levels of these lipid mediators in the normal and diseased brain, but may also regulate the onset and progression of chronic, acute, and psychiatric diseases. Collectively, these studies suggest that combining lipidomics, proteomics, and genomics techniques may greatly enhance the existing knowledge of molecular homeostasis that occurs between inflammation and oxidative stress inducing mediators (PGs, LTs, TXs) and inflammation and

oxidative stress blocking lipid mediators (LXs, RvEs, RvD, and NPD1) in neural trauma, neurodegenerative and neuropsychiatric diseases (Serhan et al., 2008; Bazan, 2009a,b; Farooqui, 2010a).

Effect of EPA and DHA in Neurological Disorders As stated above, diet influences and modulates brain function. Diets high in saturated fat and n-6 fatty acids not only increase insulin resistance (Tschop and Thomas, 2006), but also negatively affect cognitive processing and increase the risk of neurological disorders (Luchsinger et al., 2002; Greenwood and Winocur, 2005; Convit et al., 2003). Consumption of EPA and DHA modulates neural membrane fluidity and permeability and improves spatial learning by regulating synaptic and cognitive function (Farooqui, 2009; Gomez-Pinilla, 2008). High intake of fruits, vegetables, fish, and whole grains (such as is typically found in a Mediterranean-type diet) along with phytochemicals (resveratrol and other polyphenols) produces beneficial effects on the human brain (Engelhart et al., 2002; Scarmeas et al., 2009; Farooqui, 2012b). The Western diet has extremely high levels of ARA, with an ARA to DHA ratio of about 20:1. The Paleolithic diet, on which human beings have evolved and lived for most of their existence, had a ratio of 2
1:1, and was high in fiber, rich in fruits, vegetables, lean meat, and fish (Simopoulos, 2002; Simopoulos, 2008; Cordain et al., 2005). The high intake of ARA-enriched food in the modern Western diet not only elevates levels of PGs, LTs, and TXs, but also upregulates the expression of pro-inflammatory genes including genes for cytokines (TNF-α, and IL-1β) enzymes (secretory phospholipase A2, cyclooxygenase-2, and nitric oxide synthase). These genes and enzymes initiate and maintain neuroinflammation. In contrast, consumption of an EPA- and DHA-enriched diet produces anti-inflammatory effects that are partly supported by repression of genes that code for pro-inflammatory cytokines. Since the Western diet is low in DHA and high in ARA, it is linked to many chronic visceral diseases as well as neurodegenerative and neuropsychiatric disorders (Simopoulos, 2002; Simopoulos, 2008; Cordain et al., 2005; Farooqui, 2009). It is reported that intake of EPA and DHA is associated with reduced risk of agerelated cognitive decline (Farooqui, 2009; Gorby et al., 2010). DHA is highly concentrated in the brain and enhances synaptic activities in neuronal cells. DHA attenuates neuronal cell death after ischemic injury not only by modulating neural membrane biophysical properties, but also by maintaining integrity in functions between presynaptic and postsynaptic areas.

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EPA-DERIVED LIPID MEDIATORS IN THE BRAIN

These processes result in better stabilization of intracellular ion balance in ischemic injury. Additionally, EPA and DHA prevent apoptotic cell death in the brain by generating resolvins and neuroprotectins and inducing antiapoptotic activities such as decreasing responses to ROS, upregulating antiapoptotic protein expression, down-regulating apoptotic protein expression, and maintaining mitochondrial integrity and function (Farooqui, 2009; Mayurasakorn et al., 2011) (Figure 7.4). DHA is enriched in synaptic fractions and there is a good correlation between low tissue levels of EPA and DHA and increased risk of depression, schizophrenia, memory loss, and a higher chance of developing AD (Farooqui, 2009). Furthermore, low dietary intake of

77

EPA and DHA produces cognitive decline in AD patients. Based on these observations, it is suggested that enrichment of EPA and DHA in the diet may improve inflammation and oxidative stress in neurotraumatic and neurodegenerative diseases, not only through the effects of EPA and DHA on physicochemical properties of neural cell membranes, but also through modulation of genes and generation of RvDs and NPD1 (Farooqui, 2009; Farooqui, 2011). It should be noted that, to date, studies on the dietary intervention of DHA and EPA have so far failed (Quinn et al., 2010). The cause of failure is not fully understood. It is likely that short-term use of dietary DHA alone may not stop cognitive decline in AD, but long-term

FIGURE 7.4 Regulation of inflammation, oxidative stress, and apoptotic cell death by neuroprotections under pathological conditions (ischemic injury and Alzheimer’s disease) in the brain. NMDA-R, N-methyl-D-aspartate receptor; Glu, glutamate; PtdCho, phosphatidylcholines; lyso-PtdCho, lyso-phosphatidylcholine; cPLA2, cytosolic phospholipase A2; ARA, arachidonic acid; PAF, platelet activating factor; COX-2, cyclooxygenase2; 5-LOX, 5-lipoxygenase; 15-LOX, 15-lipoxygenase; PlsEtn, plasmalogen; PlsEtn-PLA2, plasmalogen-selective phospholipase A2; sPLA2, secretory phospholipase A2; iNOS, inducible nitric oxide synthase; ROS, reactive oxygen species; TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin 1beta; IL-6, interleukin-6; NF- κB, nuclear factor- κB; βAPP, β-amyloid precursor protein; sAPP, soluble amyloid precursor protein; ADAM10, alpha-secretase; BACE1or beta-site APP cleaving enzyme, β-secretase (). Adapted from Farooqui, 2012a.

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supplementation of EPA and DHA from childhood through to old age may not only restore signal transduction processes associated with behavioral deficits and learning activity, but may also produce several neuroendocrinological and immunological effects on brain tissue, which may be beneficial in ischemia and AD (Farooqui, 2010a). Thus, double blind, large AD patient, and multicenter studies are needed to test the efficacy of EPA and DHA.

CONCLUSION Phospholipid-derived lipid mediators are important endogenous regulators of neural cell proliferation, differentiation, oxidative stress, inflammation, and apoptosis. They originate from enzymic and nonenzymic degradation of glycerophospholipids, ARA-derived lipid mediators including PGs, LTs, TXs, and LXs. These mediators induce and modulate proliferation, differentiation, oxidative stress, and neuroinflammation. LXs produce anti-inflammatory effects. The lipid mediators of EPA and DHA metabolism are RvEs, RvDs, NPD1, and MaR. These mediators produce antioxidant, anti-inflammatory, and antiapoptotic effects in the brain tissue. Regular intake of EPA and DHA may produce beneficial effects in ischemic injury and in AD.

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C H A P T E R

8 The Impact of Omega-3 Fatty Acids on Quality of Life Ondine van de Rest and Lisette CPGM de Groot INTRODUCTION

fact that we are living longer is a good thing for many people, particularly if these years can be lived well. QoL is hampered, however, by inevitable age-related deterioration of physical and mental health and by an increased risk of developing chronic diseases. These age-related problems are likely to affect the QoL of individuals in later years, as well as adding pressure to infrastructures such as health and social care services. For studies in health promotion, generic outcomes are even more relevant than disorder-specific outcomes. Such data are particularly needed since caregivers and professionals may wish to improve the patient’s QoL, especially for depression, dementia or cognitive impairment for which pharmacological treatment is often ineffective. In the current chapter, first the assessment of QoL will be addressed, and subsequently the chapter provides an overview of the current observational and clinical trial evidence with respect to the impact of omega-3 fatty acids on QoL.

Current evidence indicates that a low dietary intake as well as low levels of omega-3 fatty acids, such as eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3), may be associated with mental health problems, which are among the leading causes of impaired quality of life (QoL) in old age. A possible role for omega-3 fatty acids in mental health is supported by multiple plausible biochemical pathways, which are described by Parletta et al. (2013) and include the role of omega-3 fatty acids as structural components of the brain cell membranes, in increased brain phospholipid synthesis, increased neurite growth, improved membrane fluidity, more effective neurotransmission, its effects on eicosanoid synthesis, its anti-inflammatory and vasodilatory properties, and its role in protection against neuronal loss and neurodegeneration (Parletta et al., 2013). QoL has been defined by the World Health Organization (WHO) as an individual’s perception of their position in life in the context of culture and value systems in which they live and in relation to their goals, expectations, standards, and concerns (The WHOQOL Group, 1995). It is a broad ranging concept affected in a complex way by a person’s physical health, psychological state, level of independence, social relationships, and relationship to salient features of their environment (WHO, 1993). Depression, anxiety, and the presence of diseases such as cancer, cardiac disease, and diabetes are highly associated with a reduced QoL (Ali et al., 2010; Baumeister et al., 2011). Therefore, studies investigating these associations have mostly been performed in diseased populations and much less so in the general population. However, improving QoL is becoming an increasingly important outcome in research into the elderly population. The Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00008-9

ASSESSMENT OF QOL There are several valid and reliable questionnaires that can be used to assess QoL. The most commonly used questionnaire is the Short-Form 36 Health Survey (SF-36) and another generic QoL questionnaire is the WHO QoL questionnaire (WHOQOL). These questionnaires are generic health-related QoL surveys, because they can be used across age ($18 years), disease, and treatment groups, as opposed to disease-specific health surveys, which focus on a particular condition or disease. Disease-specific QoL measures are not included in the current chapter because this would introduce too much heterogeneity to the results and detract from the focus, which is on the general, particularly the elderly population.

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8. OMEGA-3 FATTY ACIDS AND QUALITY OF LIFE

The SF-36 consists of 36 items, grouped into eight scales; each 0
100 scale measuring a different aspect of health (Ware and Sherbourne, 1992). Two summary scores can be calculated: a psychometrically-based physical component summary score (PCS) and a mental component summary score (MCS). Physical health is assessed with scales on physical functioning, role limitations due to physical health, bodily pain, and general health. The mental health score is calculated with scales on vitality (energy/fatigue), role limitations due to emotional well-being, social functioning, and mental health. The reference period is the past four weeks for the scales that have a recall period. Higher scale scores represent better self-reported health and the scales have a good validity for measuring physical and mental health constructs (McHorney et al., 1993). There are also two shortened versions; the SF-12 and the SF-8, which measure the same eight health domains (Ware et al., 1996; Ware et al., 2001). The WHOQOL is a 100-item QoL instrument developed by the WHO to facilitate cross-cultural comparisons in QoL research (The WHOQOL Group, 1998b). The WHOQOL instrument focuses on the individual’s own views of their well-being. The WHOQOL-BREF was developed to enable a brief, but accurate, assessment of QoL in routine clinical work, epidemiological studies, and clinical trials (The WHOQOL Group 1998a). The questionnaire comprises 26 items, including two general items and 24 items covering four domains: (1) physical health; (2) psychological health; (3) social relationships; and (4) environment. The scores of the two general items range from 1 to 5 and the domain scores range from 7 to 35, 6 to 30, 3 to 15

and 8 to 40 for the domains 1 through 4 consecutively. The total score range of the WHOQOL-BREF is 26
130 with higher scores indicating a favorable condition. The reference period is the previous two weeks and questions have a 5-point Likert scale. Studies have shown good content validity, discriminant validity and test
retest reliability (Nelson and Lotfy 1999; Trompenaars et al., 2005).

CURRENT EVIDENCE ON OMEGA-3 FATTY ACIDS AND QOL Evidence from Observational Studies Two observational studies have assessed the association between omega-3 polyunsaturated fatty acids (PUFAs) and QoL (Table 8.1) (Crowe et al., 2007; Silvers and Scott, 2002). Silvers and Scott (2002) observed a significant positive association between fish intake and self-reported scores on the mental health part of the SF-36 in 4,644 New Zealand adults (Silvers and Scott, 2002). Conversely, the association between fish consumption and physical health was negative (P for trend 5 0.045). The second study was also crosssectional and based on data from the same populationbased survey performed in New Zealand. This time fatty acid composition of serum phospholipids was used as an indicator of omega-3 status, which resulted in a smaller sample size (n 5 2,416) than in the first analysis. A significant positive trend (P for trend 5 0.009) was observed across quintiles of the proportion of EPA in relation to self-reported physical well-being of the

TABLE 8.1 Summary of Observational Studies Assessing the Association Between Omega-3 Fatty Acids and QoL Author, Year

Study Population (n)

Crowe et al., 2007

Population-based survey in New Zealanders, aged $ 15 years (n 5 2,416)

CrossFatty acid SF-36 sectional composition in serum phospholipids

Age category, sex, BMI category, season of blood collection, urban or rural dwelling, socioeconomic status, marital status, employment, smoking status, diabetes, asthma, use of blood pressure medication, AUDIT category

Positive association between the proportion of EPA and the ratio of EPA:AA and self-reported physical well-being, P for trend 5 0.009 and 0.012, respectively. No associations between the proportion of EPA or DHA with mental health. However, positive association between the EPA:AA ratio and self-reported mental health (P for trend 5 0.044).

Silvers and Scott, 2001

Population-based survey in New Zealanders, aged $ 15 years (n 5 4,644)

CrossFish sectional consumption assessed with FFQ

Age, annual household income, smoking status, AUDIT category, eating patterns

Significant association between more fish intake and better selfreported mental health status (P for trend 5 0.005) and between more fish intake and impaired physical functioning (P 5 0.045).

Design

Nutritional Status

Measure (s) of QoL Covariate(s)

SF-36

Results

QoL: Quality of life; SF-36: Short Form-36 Health Survey; BMI: Body mass index; AUDIT: Alcohol Use Disorders Identification Test; EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid; AA: arachidonic acid; FFQ: Food Frequency Questionnaire.

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DISCUSSION

TABLE 8.2 Summary of Intervention Studies Assessing the Effect of Omega-3 Fatty Acid Supplementation on QoL Author, Year

Measure(s) of QoL Results

Study Population (n)

Intervention and Design

Sinn et al., 2012

Population-based prospective cohort of elderly, 70
89 years (n 5 937)

DHA-rich oil (1.55 g DHA 1 0.40 g EPA); EPA-rich oil (1.67 g EPA 1 0.16 g DHA), or 2.2 g LA for 6 months

SF-36

No treatment effects on QoL. Increased erythrocyte DHA was associated with improved self-reported physical health.

Rondanelli et al., 2010

Depressed women living in a nursing home, aged 66
95 years (n 5 46)

2.5 g omega-3 fatty acids (1.67 g DHA 1 0.83 g EPA) or placebo for 8 weeks

SF-36

Significant difference between the omega-3 and the placebo group on the physical function score (difference (95% CI) 21.1 (10.8, 31.3), P ,0.001) and on the mental function score (difference (95% CI) 25.2 (16.3, 34.1), P ,0.001).

Van de Rest et al., 2009

Individuals without depression or dementia, aged .65 years (n 5 302)

1,800 mg EPA 1 DHA (EPA:DHA ratio 5 3:2); 400 mg EPA 1 DHA, or placebo for 26 weeks

WHOQOL- The mean difference (95% CI) compared to BREF placebo was 21.42 (2 3.40
0.57) for the 1,800 mg EPA 1 DHA and 0.02 (2 1.95
1.99) for the 400 mg EPA 1 DHA group, which was not significantly different.

QoL: Quality of life; SF-36: Short Form-36 Health Survey; WHOQOL-BREF: World Health Organization Quality of Life questionnaire, short version.

SF-36. The ratio of EPA to arachidonic acid (AA) was also positively associated with self-reported physical well-being (P for trend 5 0.012). Neither the proportion of EPA nor that of DHA was associated with the mental health component, however, there was a positive association between the EPA:AA ratio and self-reported mental well-being (P for trend 5 0.044) (Crowe et al., 2007).

Evidence from Clinical Trials Three intervention studies have been performed and these are summarized in Table 8.2. The first randomized trial was performed in healthy individuals (n 5 302, mean age 70 years), who were supplemented with either 1,800 mg EPA 1 DHA (EPA:DHA ratio 5 3:2;  1093 mg EPA 1 847 mg DHA), 400 mg EPA 1 DHA (226 mg EPA 1 176 mg DHA) or placebo capsules for a period of 6 months (van de Rest et al., 2009). No treatment effects were found on total QoL or any of the separate domains as measured with the WHOQOL-BREF. Another trial was performed in 46 depressed women aged 66
96 years and living in a nursing home in Italy (Rondanelli et al., 2010). These women were supplemented with 2.5 g omega-3 fatty acids (1.67 g DHA 1 0.83 g EPA) or placebo over 8 weeks. A significant difference between the omega-3 and the placebo group was found on the physical function score (difference (95% CI) 21.1 (10.8, 31.3), P , 0.001) and also on the mental function score (difference (95% CI) 25.2 (16.3, 34.1), P , 0.001) of the SF36. The results on the total QoL score were not reported. A third trial was performed in 50 elderly subjects ( . 50 years) with mild cognitive impairment who were randomly allocated to daily supplements

containing DHA-rich oil (1.55 g DHA 1 0.40 g EPA), EPA-rich oil (1.67 g EPA 1 0.16 g DHA) or a control oil (safflower oil providing 2.2 g linoneic acid (LA), an n-6 fatty acid) (Sinn et al. 2012). After six months of supplementation there were no treatment effects on QoL parameters. However, increased erythrocyte DHA levels were significantly associated with improved self-reported physical functioning on the SF-36 scale. With respect to depressive symptoms, a beneficial effect was shown in both the EPA and the DHA group and these improved scores correlated with increased EPA and DHA erythrocyte levels. Another published trial was performed in 100 patients with Alzheimer’s disease (AD) who received SR-3, a compound comprising a 1:4 ratio of n-3 to n-6 fatty acids, for a period of 4 weeks. Twelve areas of behavior were rated and beneficially improved in this study. The authors state that based on improvements in these areas, a beneficial effect on QoL was observed, however, QoL had not been assessed with a validated QoL questionnaire and therefore this study is not included in Table 8.2 or the conclusions of the present chapter (Yehuda et al., 1996).

DISCUSSION With only two observational and three intervention trials, the number of studies that investigated omega-3 fatty acids in relation to QoL is very limited. Some results from the observational studies are favorable, but not consistently so, for mental and physical health components. The results only showed an effect of omega-3 fatty acid supplementation in the trial that included depressed individuals.

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Dementia, depression, and coronary heart disease constitute the main causes of impaired QoL in the Netherlands (RIVM, 2006). It could well be that QoL in general, non-diseased populations is already close to optimal and that further improvement could not be achieved with a higher omega-3 fatty acid intake or supplementation. The total body of evidence from observational, as well as clinical studies on omega-3 and depression, one of the strongest determinants of QoL, is much larger and will be addressed in another chapter. However, the results observed in these studies support the current results for QoL, i.e. the evidence provides some support of a beneficial effect of omega3 fatty acids in depressed individuals, but not in individuals without a diagnosis of depression (Appleton et al., 2010). If true, it can be concluded that extra omega-3 fatty acids are of little added value to mental health in a generally healthy population, but they could have a role as an adjuvant treatment for depression or dementia (Yehuda et al., 1996) and as such, also improve QoL. In the field of omega-3 fatty acids and QoL, four studies used the SF-36 and one study used the WHOQOL-BREF questionnaire. Both questionnaires have shown good internal consistency, excellent discriminant validity, and good sensitivity (McHorney et al., 1993; Nelson and Lotfy, 1999; Trompenaars et al., 2005). Although the SF-36 is generally the most widely used QoL questionnaire, the WHOQOL includes a strong mental health component and emphasizes the perception of the individual. Because of the implications for an association between omega-3 PUFAs and mental well-being, the WHOQOL questionnaire is also a very suitable questionnaire to be used in future studies in the field. Despite the availability of several valid and reliable QoL instruments, it is a pity that they have seldomly been applied in efficacy trials that address mental well-being (Scholzel-Dorenbos et al., 2007). The aspects included in QoL questionnaires have been generated by systematic qualitative and quantitative research among different cultures and populations and the psychometric properties have been validated in several studies. However, because mood state, physical pain or disease, and QoL are strongly correlated, QoL is a subjective concept to measure as it depends on self-reporting and on an individual’s perception of status. This possible lack of objectivity has to be taken into account. Dietary DHA can be incorporated into the brain not only during development, but also later in life. The half-life of DHA in the brain is about 21 days, so studies should last at least 3
4 weeks before the additional DHA is incorporated into the brain (Connor et al., 1990). Furthermore, the underlying mechanism that is targeted should also be taken into account, since

different plausible mechanisms have been proposed and could be classified as either short term (i.e. weeks to months) or long term (i.e. months to years). The three intervention studies had a study duration of 8 weeks or 26 weeks and a beneficial effect was only found in the shortest 8-week study. However, this result probably has more to do with the sensitive nature of the subjects studied in that particular study, i.e. depressed patients, rather than with the study duration.

CONCLUSION AND RECOMMENDATIONS Based on the current evidence we conclude that the total body of evidence is too limited to draw a firm conclusion with respect to the impact of a higher intake of omega-3 fatty acids, either via diet or supplementation, on QoL. The results from the few available observational studies are partly favorable, but the results from the trials are not, except in depressed participants. Because causality cannot be inferred from crosssectional studies, which comprised two of the five studies performed, at least some prospective studies should be performed before a valid conclusion about the role of omega-3 fatty acids in QoL can be drawn. Moreover, intervention studies of sufficient study duration are warranted, and particularly in subjects with an impaired QoL and/or impaired omega-3 fatty acid status, in order to further develop our understanding of the effects of omega-3 fatty acids on QoL.

References Ali, S., Stone, M., Skinner, T.C., Robertson, N., Davies, M., Khunti, K., 2010. The association between depression and health-related quality of life in people with type 2 diabetes: a systematic literature review. Diabetes Metab. Res. Rev. 26 (2), 75
89. Appleton, K.M., Rogers, P.J., Ness, A.R., 2010. Updated systematic review and meta-analysis of the effects of n-3 long-chain polyunsaturated fatty acids on depressed mood. Am. J. Clin. Nutr. 91 (3), 757
770. Baumeister, H., Hutter, N., Bengel, J., Harter, M., 2011. Quality of life in medically ill persons with comorbid mental disorders: a systematic review and meta-analysis. Psychother. Psychosom. 80 (5), 275
286. Connor, W.E., Neuringer, M., Lin, D.S., 1990. Dietary effects on brain fatty acid composition: the reversibility of n-3 fatty acid deficiency and turnover of docosahexaenoic acid in the brain, erythrocytes, and plasma of rhesus monkeys. J. Lipid. Res. 31 (2), 237
247. Crowe, F.L., Skeaff, C.M., Green, T.J., Gray, A.R., 2007. Serum phospholipid n 3 long-chain polyunsaturated fatty acids and physical and mental health in a population-based survey of New Zealand adolescents and adults. Am. J. Clin. Nutr. 86 (5), 1278
1285.

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REFERENCES

McHorney, C.A., Ware Jr., J.E., Raczek, A.E., 1993. The MOS 36-Item Short-Form Health Survey (SF-36): II. Psychometric and clinical tests of validity in measuring physical and mental health constructs. Med. Care. 31 (3), 247
263. Nelson, C.B., Lotfy, M., 1999. The World Health Organization’s WHOQOL-Bref quality of life assessment of psychometric qualities. Results of field trial. WHO. Parletta, N., Milte, C.M., Meyer, B.J., 2013. Nutritional modulation of cognitive function and mental health. J. Nutr. Biochem. 24 (5), 725
743. RIVM, 2006. Mortality, Morbidity and Quality of Life. RIVM, Bilthoven. Rondanelli, M., Giacosa, A., Opizzi, A., Pelucchi, C., La Vecchia, C., Montorfano, G., Negroni, M., Berra, B., Politi, P., Rizzo, A.M., 2010. Effect of omega-3 fatty acids supplementation on depressive symptoms and on health-related quality of life in the treatment of elderly women with depression: a double-blind, placebo-controlled, randomized clinical trial. J. Am. Coll. Nutr. 29 (1), 55
64. Scholzel-Dorenbos, C.J., van der Steen, M.J., Engels, L.K., Olde Rikkert, M.G., 2007. Assessment of quality of life as outcome in dementia and MCI intervention trials: a systematic review. Alzheimer. Dis. Assoc. Disord. 21 (2), 172
178. Silvers, K.M., Scott, K.M., 2002. Fish consumption and self-reported physical and mental health status. Public. Health. Nutr. 5 (3), 427
431. Sinn, N., Milte, C.M., Street, S.J., Buckley, J.D., Coates, A.M., Petkov, J., Howe, P.R., 2012. Effects of n-3 fatty acids, EPA v. DHA, on depressive symptoms, quality of life, memory and executive function in older adults with mild cognitive impairment: a 6-month randomised controlled trial. Br. J. Nutr. 107 (11), 1682
1693. The WHOQOL Group, 1995. The World Health Organization Quality of Life assessment (WHOQOL): position paper from the World Health Organization. Soc. Sci. Med. 41, 1403.

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The WHOQOL Group, 1998a. Development of the World Health Organization WHOQOL-BREF quality of life assessment. The WHOQOL Group. Psychol. Med. 28 (3), 551
558. The WHOQOL Group, 1998b. The World Health Organization Quality of Life Assessment (WHOQOL): development and general psychometric properties. Soc. Sci. Med. 46 (12), 1569
1585. Trompenaars, F.J., Masthoff, E.D., Van Heck, G.L., Hodiamont, P.P., De Vries, J., 2005. Content validity, construct validity, and reliability of the WHOQOL-Bref in a population of Dutch adult psychiatric outpatients. Qual. Life. Res. 14 (1), 151
160. van de Rest, O., Geleijnse, J.M., Kok, F.J., van Staveren, W.A., Olderikkert, M.G., Beekman, A.T., de Groot, L.C., 2009. Effect of fish oil supplementation on quality of life in a general population of older Dutch subjects: a randomized, double-blind, placebocontrolled trial. J. Am. Geriatr. Soc. 57 (8), 1481
1486. WHO, 1993. Study protocol for the World Health Organization project to develop a Quality of Life assessment instrument (WHOQOL). Qual. Life. Res. 2 (2), 153
159. Ware Jr., J., Kosinski, M., Keller, S.D.A., 1996. 12-Item Short-Form Health Survey: construction of scales and preliminary tests of reliability and validity. Med. Care. 34 (3), 220
233. Ware Jr., J.E., Sherbourne, C.D., 1992. The MOS 36-item short-form health survey (SF-36). I. Conceptual framework and item selection. Med. Care. 30 (6), 473
483. Ware, J.E., Kosinski, M., Dewey, J., Gandek, B., 2001. How to Score and Interpret Single-Item Health Status Measures: a Manual for Users of the SF-8 Health Survey: (with a Supplement on the SF-6 Health Survey). Quality Metric, Inc, Boston, MA: Lincoln, RI. Yehuda, S., Rabinovtz, S., Carasso, R.L., Mostofsky, D.I., 1996. Essential fatty acids preparation (SR-3) improves Alzheimer’s patients quality of life. Int. J. Neurosci. 87 (3-4), 141
149.

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C H A P T E R

9 Mammalian Fatty Acid Amides of the Brain and CNS Dominik P. Waluk, Matthew R. Battistini, Daniel R. Dempsey, Emma K. Farrell, Kristen A. Jeffries, Perry Mitchell, Lucas W. Hernandez, Joshua C. McBride, David J. Merkler and Mary C. Hunt INTRODUCTION

cerebrospinal fluid of the cat, rat, and man (Cravatt et al., 1995). Work since these discoveries in the mid1990s clearly shows that many different fatty acid amides are found in the mammalian brain and CNS (Bradshaw et al., 2009b; Han et al., 2013; Saghatelian and Cravatt, 2005; Schmid et al., 1995; Tan et al., 2010), but challenges remain. The neurological functions for many of the fatty acid amides are unclear, the receptors for the fatty acid amides are often undefined, and the chemistry used in vivo for their biosynthesis and degradation is not fully elucidated. The goal of this review is to present our current understanding of the fatty acid amides and to foster research into this intriguing family of neurologically important lipids.

The fatty acid amides are a family of relatively simple biomolecules, R1aCOaNHaR2. Simplicity does not preclude diversity as R1 can be CH3, but is usually a chain of three or more carbon atoms, including straight chains, branched chains, saturated chains, monounsaturated chains, and polyunsaturated chains. The R1aCO moiety of the fatty acid amides discussed within this review will represent well known long-chain fatty acids like arachidonic acid and oleic acid. Similarly, simplicity in R2 does not preclude diversity as R2 can range from a hydrogen atom, as found in the primary fatty acid amides (R1aCOaNH2), to a plethora of more complex structures. Diversity leads to complexity as the different fatty acid amides bind to different receptors and elicit different biological responses. Not surprisingly, fatty acid amides are important cell signaling molecules in the brain, the central nervous system (CNS), and throughout the body. As reviewed by Conti and Bickel (1977), fatty acid amides have long been known from biological systems, dating back to the late 1820s with the identification of hippurate as a urinary metabolite of benzoic acid. The importance of the fatty acid amides in the brain was established in the early twentieth century in the early work to characterize brain sphingolipids (Breathnach, 2001; Levene, 1914). Interest in the fatty acid amides increased dramatically with the discovery of N-arachidonoylethanolamine (anandamide) as an endogenous brain cannabinoid receptor ligand (Devane et al., 1992) with the field further expanding with the identification of oleamide, a sleep inducing fatty acid amide, in

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00009-0

Primary Fatty Acid Amides The primary fatty acid amides (PFAMs) were first characterized from a biological source (human plasma) by Arafat et al. (1989) (see Figure 9.1). This class of fatty acid amides was underappreciated until one PFAM, oleamide, was identified as an endogenous sleep inducer in the mammalian brain (Cravatt et al., 1995). This discovery sparked considerable interest in the PFAMs and led to advances in our understanding of their functions and metabolism in biological systems. PFAMs are now recognized as important signaling lipids and have been identified in the mammalian CNS and serum (Table 9.1). As found with other members of the fatty acid amide family, the functions exhibited by the PFAMs are dependent on the nature of their respective acyl group with respect to length and degree of unsaturation.

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9. MAMMALIAN FATTY ACID AMIDES OF THE BRAIN AND CNS

FIGURE 9.1 The structure of the primary fatty acid amides. R is a long-chain acyl group, which can be saturated, unsaturated, or polyunsaturated.

Oleamide is the best studied long chain PFAM due to an early understanding of its neurological functions. Oleamide was found to accumulate in the cerebrospinal fluid (CSF) of sleep-deprived cats and to induce physiological sleep when administered to experimental animals (Cravatt et al., 1995). Other biological effects that have been attributed to oleamide are summarized in Table 9.2. The effects exerted by oleamide are mediated

TABLE 9.1 Identification of Primary Fatty Acid Amides in Mammalian CNS and Seruma Primary Fatty Acid Amide

Organism

Location in the Organism

Endogenous Level

Reference

Myristamide (C14:0)

Human

Serum

Not reported

Lian et al., 2011

Palmitamide (C16:0)

Human

Plasma

13.8 nmol/mL

Arafat et al., 1989

Rabbit

Brain

11.4 2 25.6 nmol/g

N18TG2 cells

930 6 250 pmol/10 cells

Farrell et al., 2012

Sheep

Choroid plexus cells

1200 6 210 pmol/10 cells

Farrell et al., 2012

Human

Plasma

15.6 nmol/mL

Arafat et al., 1989

N18TG2 cells

340 6 110 pmol/10 cells

Farrell et al., 2012

Sheep

Choroid plexus cells

410 6 50 pmol/10 cells

Farrell et al., 2012

Human

Plasma

Not reported

Tao et al., 2008

Rabbit

Serum

Not reported

Lian et al., 2011

Brain

12.4 2 15.6 nmol/g

Sultana, 2005

Human

Plasma

13.1 nmol/mL

Arafat et al., 1989

Cat

CSF

10 2 50 nmol/mL

Cravatt et al., 1995

Human

Plasma

112.6 nmol/mL

Arafat et al., 1989

Mouse

Serum

Not reported

Qiu et al., 2009

Rabbit

N18TG2 Cells

Not reported

Li et al., 2013

Rat

Brain

Not reported

Tao et al., 2008

Squirrel

CSF

Not reported

Lian et al., 2011

Mouse

9

Palmitoleamide (C16:1 cis-Δ )

Mouse

Stearamide (C18:0)

9

Elaidamide (C18:1 trans-Δ ) 9

Oleamide (C18:1 cis-Δ )

Sheep

Sultana, 2005

7

7

7

7

10

Plasma

55 nmol/10

cells

Bisogno et al., 1997

Brain

120 6 50 pmol/10 cells

Farrell et al., 2012

Choroid plexus cells

82 2 99 nmol/g

Sultana, 2005

0.16 nmol/mL

Hanuˇs et al., 1999

0.035 nmol/mL

Hanuˇs et al., 1999

7

0.91 2 2.3 nmol/g

Stewart et al., 2002

6.4 6 2.3 nmol/10 cells

Farrell et al., 2012

Plasma

7.8 nmol/mL

Arafat et al., 1989

N18TG2 cells

100 6 60 pmol/10 cells

Farrell et al., 2012

Sheep

Choroid plexus cells

250 6 180 pmol/10 cells

Farrell et al., 2012

Rabbit

Brain

Not reported

Sultana, 2005

Pig

Plasma

0.011 nmol/g

Hamberger and Stenhagen, 2003

Brain

0.0018 nmol/g

Hamberger and Stenhagen, 2003

7

9,12

Linoleamide (C18:2 cis,cis-Δ

)

Human Mouse

13

Eicosenoamide (C20:1 cis-Δ ) 13

Erucamide (C22:1 cis-Δ )

7

7

a

This table is modified from Farrell (2010).

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INTRODUCTION

TABLE 9.2 Functions of Mammalian Primary Fatty Acid Amidesa Primary Fatty Acid Amide

Biological Effect

Receptor Reference

Palmitamide (C16:0)

Modestly attenuates seizures in mice, modest FAAH inhibitor

Palmitoleamide (C16:1 cis-Δ9) Gap junction communication

Lambert et al., 2001; Vandevoorde et al., 2003; Sultana, 2005 5-HT1A

Boger et al., 1998

5-HT2A Elaidamide (C18:1 trans-Δ9)

Induces sleep, inhibits epoxide hydrolase, and phospholipase A2

5-HT1A 5-HT2C 5-HT7

Oleamide (C18:1 cis-Δ9)

Sleep, memory, thermal and locomotor regulation, gap junction communication, Ca21 flux, vasodilatation, hunger, analgesia, anxiety, erg current inhibition, epileptic seizure, antiinflammatory, vasorelaxation

GABAA 5-HT1A 5-HT2A 5-HT2C 5-HT7 PPARγ CB1

Linoleamide (C18:2 cis,cis-Δ9,12)

Sleep and Ca21 flux regulation, erg current inhibition, epoxide hydrolase and phospholipase A2 inhibition, gap junction communication

5-HT2A

Erucamide (C22:1 cis-Δ13)

Fluid balance, angiogenesis

5-HT2A

5-HT1A

Arafat et al., 1989; Boger et al., 1998; Cravatt et al., 1995; Hedlund et al., 2003; Huidobro-Toro and Harris, 1996; Jain et al., 1992; Morisseau et al., 2001 Akanmu et al., 2007; Basile et al., 1999; Boger et al., 1998; Cravatt et al., 1995; Di Marzo et al., 2007; Dionisi et al., 2012; Guan et al., 1997; Hedlund et al., 2003; Herrera-Solis et al., 2010; Hopps et al., 2012; Huidobro-Toro and Harris, 1996; Maurelli et al., 1995; Mueller and Driscoll, 2009; Oh et al., 2010; Prieto et al., 2012; Solomonia et al., 2008; Soria-Gomez et al., 2010a; Stewart et al., 2002; Tao et al., 2008; Thomas et al., 1997; Verdon et al., 2000; Wei et al., 2007 Boger et al., 1998; Huang and Jan, 2001; Jain et al., 1992; Liu and Wu, 2003; Maurelli et al., 1995; Morisseau et al., 2001; Ueda et al., 2000 Boger et al., 1998; Hamberger and Stenhagen, 2003; Mitchell et al., 1996; Wakamatsu et al., 1990

a

This table is modified from Farrell (2010).

by well-established receptors found in the brain and CNS, including the CB1 receptor (Leggett et al., 2004), the GABAA receptor (Verdon et al., 2000) and serotonin receptor subtypes 5-HT1A, 5-HT2A, 5-HT2C, and 5-HT7 (Boger et al., 1998; Hedlund et al., 2003; Huidobro-Toro and Harris, 1996). In vitro evidence suggests that oleamide also activates peroxisome proliferator-activated receptor gamma (PPARγ) (Dionisi et al., 2012). The neurological functions of oleamide and the other endogenous mammalian PFAMs are summarized in Table 9.2. The cellular concentration of PFAMs is a balance between the pathways for biosynthesis and degradation. PFAM degradation involves their hydrolysis by fatty acid amide hydrolase (FAAH) to the corresponding fatty acid and ammonia: RaCOaNH2RaCOOH 1 NH3. Rat and human FAAH have similar substrate specificities; linoleamide is the best substrate (highest V/K value) followed by oleamide, myristamide, palmitamide, and stearamide (Giang and Cravatt, 1997; Maurelli et al., 1995). In FAAH knockout mice, the levels of oleamide were B10-fold higher throughout the CNS and the rates of oleamide hydrolysis in brain extracts were decreased B60-fold relative to the values measured for wildtype (McKinney and

Cravatt, 2005), providing strong evidence that FAAH is responsible for PFAM hydrolysis in vivo. Metabolic studies in the mouse neuroblastoma N18TG2 cells and sheep choroid plexus cells show that the relative amounts of PFAMs produced by these cells were inversely proportional to FAAH specificity (Farrell, 2010). The pathway(s) responsible for the in vivo production of the PFAMs are not as well understood. One postulated route is the oxidative cleavage of N-fatty acylglycines (NAGs) to the PFAM and glyoxylate by peptidylglycine α-amidating monooxygenase (PAM) (Merkler et al., 1996). Support for this chemistry comes from in vitro studies demonstrating that the NAGs are good PAM substrates (Wilcox et al., 1999) and in cellulo PAM knockout studies showing a decrease in the production of the PFAMs coupled to an increase in the cellular levels of the NAGs (see Figure 9.2) (Farrell, 2010; Merkler et al., 2004). The second proposed route is the nucleophilic attack of acyl-CoA thioesters by ammonia to yield a PFAM and free CoA-SH. Driscoll et al. (2007) report that this reaction is catalyzed by cytochrome c (cyto-c) in vitro and is stimulated by H2O2. Knockdown of somatic and testicular cyto-c expression in the oleamide-producing

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FIGURE 9.2 PFAM metabolism. The enzymes catalyzing the individual reactions are in the shaded boxes. The role for PAM and cyto-c in PFAM for biosynthesis is largely based on in vitro data. The in vivo significance of these reactions has yet to be established. ASC 5 ascorbate and SDA 5 semidehydroascorbate.

N18TG2 neuroblastoma cells did not decrease the levels of oleamide when these cells were cultured in the presence of exogenously added oleic acid, suggesting that cyto-c is not responsible for PFAM formation in these cells (Table 9.3) (Farrell, 2010). Note that Bisogno et al. (1997) found that oleamide production in the N18TG2 cells could not be attributed to the direct condensation of ammonia with oleic acid: oleic acid 1 ammonia-oleamide. There is still work remaining to be completed to define exactly how the PFAMs are produced in vivo and, as suggested by Muller and Driscoll (2009), there are likely to be multiple pathways for PFAM biosynthesis as has been demonstrated for other members of the fatty acid amide family.

TABLE 9.3 Oleamide Production in Mouse N18TG2 cells with and without siRNA Knockdown of Cytochrome c in the N18TG2 Cellsa Oleamide (nmol/107 cells) Incubation Time (hours)

Cytochrome c (2) Knockdown

Cytochrome c (1) no Knockdown

0

0.11 6 0.05

0.27 6 0.1

48

2.3 6 0.4

4.7 6 1.5

a Table adapted from Farrell (2010). No decrease in oleamide was detected after siRNA knockdown of cytochrome c, suggesting that cytochrome c does not play a role in oleamide biosynthesis in these cells.

N-Acylethanolamines (NAEs) Cannabis has been used medically for at least 2000 years, dating back to descriptions in the Chinese pharmacopoeia in the 2nd century (Di Marzo, 2006) (Figure 9.3). Seminal discoveries related to the medicinal uses of Cannabis were (a) the identification, synthesis, and structural elucidation of Δ9-tetrahydrocannabinol, the major psychoactive ingredient (Mechoulam and Hanuˇs, 2000), (b) the identification of the cannabinoid receptors, CB1 (Matsuda et al., 1990) and CB2 (Munro et al., 1993), and (c) the identification and characterization of N-arachidonoylethanolamine (anandamide) as an endogenous ligand in the mammalian brain for the CB1 receptor (Devane et al., 1992). Anandamide and other endogenous ligands to the CB1 and CB2 receptors are called the endocannabinoids. We now know that anandamide is simply one member of a family of N-acylethanolamines found in the brain, CNS, and peripheral tissues (Fontana et al., 1995; Koga et al., 1997). The most abundant NAE of the mammalian brain is N-palmitoylethanolamine, with anandamide actually representing only ,10% of total brain NAEs (Table 9.4). Clearly,

FIGURE 9.3 The structure of the N-acylethanolamines. R is a long-chain fatty acid, which can be saturated, unsaturated, or polyunsaturated.

more research into the NAEs of the brain and CNS is necessary as thousands of published works revolve around anandamide, one of the least abundant brain NAEs. Anandamide is known to modulate a number of biological and behavior processes, including body temperature (Cravatt et al., 2001), locomotion (Cravatt et al., 2001), appetite (Williams and Kirkham, 1999), pain perception (Guindon and Hohmann, 2009; Walker et al., 1999a,b, 2002, 2005), as well as anxiety and fear (Kathuria et al., 2003; Marsicano et al., 2002). The activities elicited by anandamide are the result of its binding to receptors found in the brain and CNS. In addition to binding to the CB1 receptor, anandamide will bind to the CB2 receptor (Lin et al., 1998), the transient receptor potential vanilloid type 1 (TRPV1) (Ross et al., 2001),

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INTRODUCTION

TABLE 9.4 Relative Amounts of Mammalian N-acylethanolaminesa Rat Cortical Neurons

Human Astrocytes

Rat Astrocytes

Rat Brain

%

%

%

%

N-Docosatetraenoylethanolamine

N/Ab

4.1

4.8

N/Ab

N-Dihomo-γ-linolenoylethanolamine

N/Ab

3.5

5.2

N/Ab

Anandamide

4.9 6 1.1

28

N-Linolenoylethanolamine

10.6 6 1.1

N-Linoleoylethanolamine

N-Acylethanolamine

Composition

8.1 6 5

19.3 b

N/A

b

N/A

N/Ab

1.1 6 0.3

N/Ab

N/Ab

N/Ab

N-Oleoylethanolamine

31.3 6 1.7

2.6

6.8

7.6 6 5

N-Stearoylethanolamine

21.7 6 2.7

N/A

N/A

19.7 6 4

N-Palmitoylethanolamine

25.7 6 2.2

62.3

63.9

65.5 6 6

b

b

a

Data in this table were taken from Fontana et al. (1995), Cadas et al. (1997), and Walter et al. (2002). The indicated NAE was not quantified in the published study.

b

TABLE 9.5 Affinity of Rat Receptors for Anandamide Receptor

Kd (nM)

Reference

CB1 (Human)

61 (90)

Lin et al., 1998 (Showalter et al., 1996)

CB2 (Human)

1930 (370)

Lin et al., 1998 (Showalter et al., 1996)

TRPV1

1660

Ross et al., 2001

150
3,000

De Petrocellis et al., 2007

TRPM8 Mouse

PPARγa

8,000
11,000 Rockwell and Kaminski, 2004; Bouaboula et al., 2005

a

Kd value for the binding of anandamide to rat PPARγ is not available.

the transient receptor potential of melastatin type 8 (TRPM8) (De Petrocellis et al., 2007), the peroxisome proliferator-activated receptor alpha (PPARα) (Sun et al., 2007), and the peroxisome proliferator-activated receptor gamma (PPARγ) (Rockwell and Kaminski, 2004). While there is a considerable range in the reported Kd values for the binding of anandamide to the receptors listed in Table 9.5, due to differences in experimental conditions and the source for the receptors (species and tissue), the general trend of the data in Table 9.5 is valid. Anandamide binds with the highest affinity to the CB1 receptors, with low affinity to PPARα and PPARγ, and the ratio of Kd,CB2/Kd,CB1 is usually 3
5 (McPartland et al., 2007). The majority of the biological effects of anandamide most likely result from its high affinity binding to the CB1 receptor. The functions of the NAEs other than anandamide, and the receptors to which these bind, are not as well understood. N-palmitoylethanolamine (PEA), a ligand for the PPARα and GPR55 receptors (Lo Verme et al., 2005b; Ryberg et al., 2007), is a neuroprotective lipid

participating in the regulation of the inflammatory response, pain perception, neuronal excitability, and pruritus (Esposito and Cuzzocrea, 2013; Sheerin et al., 2004). PEA accumulates after injury to the brain or spinal cord (Berger et al., 2004) and its neuroprotective functions likely result from its binding to PPARα (Lo Verme et al., 2005a). N-Oleoylethanolamine (OEA) is another brain NAE shown to bind to a number of receptors including the PPARα, PPARβ, PPARγ, TRPV1, and GPR119 (Ahern, 2003; Fu et al., 2003; Hansen, 2010). Most likely, the major role served by OEA is in the regulation of feeding behavior and body weight by activation of PPARα (Fu et al., 2003), with other neurologically relevant functions ascribed to OEA being memory enhancement (Campolongo et al., 2009), the regulation of alertness and the sleep-wake cycle (Murillo-Rodrı´guez et al., 2007; Soria-Go´mez et al., 2010b), and pain perception (Suardı´az et al., 2007). N-Stearoylethanolamine (SEA) exhibits cannabimimetic activity even though this NAE does not bind to the cannabinoid receptors (Maccarrone et al., 2002). Like PEA, recent studies show that N-lauroylethanolamine (LEA) and N-linoleoylethanolamine (LOEA) are neuroprotective in a rat model for stroke (Garg et al., 2011). The activities attributed to PEA, OEA, and the other non-anandamide NAEs may be derived, to some extent, from the ‘entourage effect’: the stabilization of the cellular concentrations of anandamide by serving as alternative substrates for the anandamidedegrading enzymes (Lambert and Di Marzo, 1999; Smart et al., 2002). The biosynthesis of anandamide and other NAEs is well described in a series of reviews (Farrell and Merkler, 2008; Liu et al., 2008; Okamoto et al., 2009; Tsuboi et al., 2013; Ueda et al., 2010a) (see Figure 9.4).

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FIGURE 9.4 N-acylethanolamine metabolism. The enzymes proposed to catalyze the individual reactions are in the shaded boxes and the numbers that refer to specific reactions in the text are above the arrows.

One widely accepted biosynthetic route to the NAEs is the cleavage of an N-acylphosphatidylethanolamine (NAPE) to the corresponding NAE and phosphatidic acid, as catalyzed by NAPE-specific phospholipase D (NAPE-PLD) (Figure 9.4, reaction 1) (Schmid and Berdyshev, 2002; Sugiura et al., 1996). Another pathway

for the production of the NAEs is the phospholipase C (PLC) catalyzed cleavage of NAPE to O-phosphorylNAE (pNAE) and diacylglycerol (Figure 9.4, reaction 2) followed by hydrolysis of the pNAE to the NAE and inorganic phosphate (Figure 9.4, reaction 3) (Liu et al., 2006, 2008; Ueda et al., 2010a). Hydrolysis of pNAE to

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the NAE is catalyzed by a phosphatase, either protein tyrosine phosphatase (PTPN22) or inositol-5’-phosphatase (SHIP1). The NAEs can be derived from the NAPEs by another set of reactions: two sequential O-deacylations to yield glycerophospho-NAE (GPNAE) and two free fatty acids. The first O-deacylation step, hydrolysis of NAPE to lysoNAPE (Figure 9.4, reaction 4), is catalyzed by phospholipase A2 (PLA2) or α/β-hydrolase 4 (Abh4) (Simon and Cravatt, 2006) while the second O-deacylation step, hydrolysis of lysoNAPE to GP-NAE (Figure 9.4, reaction 5), is catalyzed by Abh4. GP-NAE is either directly metabolized to the NAE and glycerol 3-phosphate by glycerophosphodiesterase (GDE1) (Figure 9.4, reaction 6) or hydrolyzed to pNAE and glycerol by lysophospholipase C (lysoPLC) (Figure 9.4, reaction 7) (Liu et al., 2008). As discussed, the pNAEs can then serve as a precursor to the NAEs. Finally, the lysoNAPEs can serve as substrates for lysophospholipase D (lysoPLD) to yield the NAEs and lysophosphatic acid (LPA) (Figure 9.4, reaction 8) (Tsuboi et al., 2013). The NAPEs, key precursors to the NAEs, are produced in vivo by a reaction between phosphatidylethanolamine (PE) and phosphatidylcholine (PC) (Figure 9.4, reaction 9), as catalyzed by either a calcium-activated PE N-acyltransferase (CaNAT) (Natarajan et al., 1983; Natarajan et al., 1986) or a calcium-independent PE N-acyltransferase (iNAT) (Jin et al., 2007). The NAPEs only constitute 0.01% of all the phospholipids found in mammalian membranes (Coulon et al., 2012), are present at low levels in the mammalian brain (low nmoles/gram of tissue), and, like the NAEs, are cell signaling lipids. The NAPEs are cytoprotective, exhibit anti-inflammatory properties, and regulate food intake. NAE catabolism involves two sets of reactions, hydrolysis or oxidation. NAE hydrolysis of the fatty acid and ethanolamine is catalyzed by either FAAH (two forms known, FAAH-1 and FAAH-2) (Giang and Cravatt, 1997; McKinney and Cravatt, 2005; Ueda et al., 2000) or N-acylethanolamine-hydrolyzing acid amidase (NAAA) (Ueda et al., 2010b) (Figure 9.4, reaction 10). NAE oxidation occurs either at the hydroxyl group or unsaturated acyl groups of the NAEs. Sequential oxidation of the NAE hydroxyl by a fatty aldehyde dehydrogenase (fAldDH) and a fatty alcohol dehydrogenase (fADH) first yields an N-acylglycinal and ultimately an N-acylglycine (Figure 9.4, reactions 11 and 12) (Burstein et al., 2000; Ivkovic et al., 2011). Oxidation of the acyl chain of anandamide by cyclooxygenase-2 (COX-2), lipoxygenase (LOX), or cytochrome P450 (CYP450) yields different N-(oxidized arachidonoyl)-ethanolamine products (Kozak and Marnett, 2002) (see Figure 9.5). Oxidation by COX-2 produces prostamide H2, which can be further metabolized to a set of prostanoid

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ethanolamides: prostamides D2, E2, and/or F2α (Weber et al., 2004; Yang et al., 2005 by LOX yields N-hydroxyeicosatetraenoyl-ethanolamines (HETE-EAs), primarily 15-HETE-EA and 12-HETE-EA (Ueda et al., 1995), which can be conjugated to glutathione to generate novel cysteinyl-containing HETA-EAs (Forsell et al., 2012); and by cytochrome P450 produces 20hydroxyanandamide (20-HETE-EA), N-[5,6-epoxy8,11,14-eicosatrienoyl]-ethanolamine (5,6-EET-EA), N-[11,12-epoxy-5,8,14-eicosatrienoyl]-ethanolamine (11,12-EET-EA), and other epoxylated/hydroxylated products (Snider et al., 2010). N-Linoleoylethanolamine and N-linolenoylethanolamine are also reported to serve as lipoxygenase substrates to generate N-(hydroxylated acyl)-ethanolamine products (van der Stelt et al., 1997, 2000). Recent evidence suggests that many of the oxidized anandamide products are bioactive suggesting that anandamide catabolism might represent a novel strategy to regulate the functions of these fatty acid amides (Glaser and Kaczocha, 2010; Piscitelli and Di Marzo, 2012).

N-Acyl Amino Acids (NAAs) N-Acyl amino acids are important ‘new players’ in many physiological processes throughout the body, especially in the CNS (Figure 9.6). These lipids are structurally related to the endocannabinoids, although they do not activate cannabinoid receptors. They contain one fatty acid and one amino acid linked via an amide bond. A nomenclature system has been proposed, naming these as elmiric acids and numbering them based on the amino acid e.g. EMA1 (20:4) is N-arachidonoylglycine (NAGly), as glycine is EMA1, alanine EMA2, valine EMA3, etc. (Burstein, 2008). Burstein and co-workers first suggested that NAGly was a putative endogenous compound in 1997 (Burstein et al., 1997). In 2001, NAGly was the first signaling lipid in this group of fatty acid amides to be identified in bovine and rat brain (Huang et al., 2001) and since then, approximately 50 novel N-acyl amino acids have been identified in mammalian systems (Tan et al., 2010). These N-acyl amino acids are involved in regulating pain processes, are anti-inflammatory and regulate body temperature. Recent studies have significantly contributed to a better understanding of the pathways, function, and metabolism of N-acyl amino acids as novel signaling molecules. The physiological roles of N-acyl amino acids are only now emerging and modulatory functions in the nervous system, vasculature, and immune system have been identified (Bradshaw et al., 2006; Conner et al., 2010). N-Acyl amino acids appear to have a variety of pharmacological effects and it is likely that their function requires

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FIGURE 9.5 The oxidative metabolism of anandamide as catalyzed by lipoxygenase (LOX), cyclooxygenase (COX), or cytochrome P450. The oxidation of the alcohol moiety of anandamide and the other NAEs to corresponding aldehyde and a carboxylic acid is shown in Figure 9.4.

particular receptors. This section of the chapter focuses on the physiological functions and the identification of pathways for production and regulation of N-acyl amino acids, in particular N-acyl glycines (NAGs, RaCOaNHaCH2aCOOH) and N-acyl taurines (NATs, RaCOaNHaCH2aCH2aSO3H). NAGs, a Specific Class of the NAAs The first annotation of glycine conjugates was 171 years ago, when Wilhelm Keller carried out an experiment on himself by injecting thirty-two grains of pure benzoic acid in syrup, and the next morning collecting his urine (Keller, 1842). The results from Keller were the first known reaction in the human body describing the conjugation of benzoic acid with glycine, to produce hippuric acid (N-benzoylglycine).

The conjugation of xenobiotics and endogenous carboxylic acids, particularly acyl-CoA molecules of chain lengths C6:0
C8:0 to amino acids such as glycine, is one of the major metabolic pathways in Phase II liver detoxification as it enhances the water solubility of these compounds, thereby promoting their excretion in substances such as urine and bile (Schachter and Taggart, 1954; Knights et al., 2007). More recently, longer chain NAGs have been identified in vivo and the various long-chain and very long-chain NAGs identified to date will be discussed herein. Glycine conjugates of medium- and long-chain saturated and unsaturated fatty acids have been detected in the CNS (Tan et al., 2010). N-Arachidonoyl glycine (NAGly) was one of the first long-chain N-acyl glycines to be identified in vivo in rat brain, spinal cord, small intestine, kidney and skin, at concentrations of

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(c) sequential oxidation by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AlDH) (Aneetha et al., 2009; Ivkovic et al., 2011). These three pathways are described below and are shown in Figure 9.7.

FIGURE 9.6 The structure of the N-acyl L-amino acids. R is a long-chain fatty acid, which can be saturated, unsaturated, or polyunsaturated, and X represents the functional groups that define the different amino acids.

approximately 50
140 pmol/g of dry tissue weight (Huang et al., 2001). Huang et al. (2001) and Succar et al. (2007) have reported anti-nociceptive and antiinflammatory effects by NAGly in rat models of pain. Moreover, Wiles et al. (2006) showed inhibition of the glycine transporter (GLYT2a) through direct, noncompetitive interactions by NAGly. NAGly has been described in the literature as a ligand for a number of G-protein coupled receptors: GPR18 (Kohno et al., 2006), GPR72 (Roy and Hannedouche, 2007), and GPR92 (Oh et al., 2008). A number of processes like migration of mouse microglia cells (McHugh et al., 2010), macrophage apoptosis in mouse (Takenouchi et al., 2012), or regulation of the intraocular pressure of the murine eye (Caldwell et al., 2013), all involve activation of GPR18 by NAGly. While no detectable levels of the GPR18 transcript were found in the human brain (Gantz et al., 1997), strong expression was found in hypothalamus, thyroid, peripheral blood leucocytes, cerebellum, and brain stem of the mouse (Vassilatis et al., 2003). There is controversy regarding the NAGly-mediated activation of GPR18. Yin et al. (2009) found that NAGly is not a GPR18 ligand and, more recently, Lu et al. (2013) found that NAGly was not an agonist for GPR18 in rat superior cervical ganglion neurons. These results suggest that GPR18 is not directly activated by NAGly and that another NAGly-dependent signaling system must exist. Three pathways have been suggested for the formation of the NAGs, in particular NAGly and N-oleoyl glycine (OLGly): (a) enzymatic synthesis by glycine N-acyltransferases (GLYATL2) (Waluk et al., 2010) or by bile acid-CoA:amino acid N-acyltransferase (O’Byrne et al., 2003), (b) synthesis via cytochrome c (Mueller and Driscoll, 2007; McCue et al., 2008), and

a. Enzymatic synthesis of the NAGs: Waluk et al. (2010) showed that human GLYATL2 produces the glycine conjugates of medium- and long-chain saturated and unsaturated acyl-CoA esters, in particular OLGly, N-stearoyl glycine, N-palmitoleoyl glycine, and NAGly. The steadystate kinetic values for the hGLYATL2 catalyzed production of OLGly were Km,oleyl-CoA of 4.4 μM and a Vmax of 933 nmol/min/mg. The activity of hGLYATL2 is regulated by acetylation/ deacetylation of Lys-19, which is likely carried out by members of the acetyltransferase family (HATs) and deacetylase enzymes (SIRTs). The acetylation of Lys-19 in hGLYATL2 links the posttranslational modification of proteins with the production of N-acyl glycines, thus allowing a rapid regulation of hGLYATL2 in response to lipid signaling requirements (Waluk et al., 2012). hGLYATL2 is localized in the endoplasmic reticulum and its mRNA is mainly expressed in the salivary gland, trachea, and spinal cord (Waluk et al., 2010), although it is unclear whether NAGs are produced in the CNS or whether other peripheral tissues are also involved in their production. The second enzyme shown to produce long-chain N-acyl glycines is the peroxisomal enzyme, bile acid-CoA: amino acid N-acyltransferase (BAAT). In vivo, BAAT is responsible for the production of glycine and taurine conjugates of bile acids. In addition, BAAT will utilize the fatty acyl-CoA thioesters as substrates at about 5
20% of its bile acid conjugating activity (O’Byrne et al., 2003). Thus, BAAT can conjugate long-chain and very longchain saturated and unsaturated acyl-CoA esters (from C16 carbons up to C26 carbons) to taurine or glycine (Hunt et al., 2012; O’Byrne et al., 2003). The BAAT catalyzed production of the N-acyl glycines is probably not of neurological significance as BAAT is expressed mainly in the liver (Pellicoro et al., 2007) and its mRNA has been identified in human gallbladder mucosa and in mouse proximal and distal intestine (O’Byrne et al., 2003). However, it is unclear as yet whether the NAGs can be transported to the CNS from peripheral tissues. b. Production of the NAGs by cytochrome c: Recent in vitro work has shown that cytochrome c can act on an acyl-CoA (specifically arachidonoyl-CoA or oleoyl-CoA) and glycine in the presence of hydrogen peroxide, leading to the production of N-acyl glycine (McCue et al., 2008; Mueller and Driscoll, 2007).

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FIGURE 9.7 N-acylglycine metabolism (A) and N-arachidonoyl oxidation (B). The enzymes proposed to catalyze the reactions of NAG metabolism in panel A are enclosed by the ovals: ACS, acyl-CoA synthetase; ADH, alcohol dehydrogenase; AlDH, aldehyde dehydrogenase; BAAT, bile acid-CoA:amino acid N-acyltransferase; FAAH, fatty acid amide hydrolase; GLYATL2, glycine N-acyltransferase like-2; and PAM, peptidylglycine α-amidating monooxygenase. The details of the PAM-catalyzed oxidation of the NAGs to the PFAMs are shown in Figure 9.2. The oxidation of arachidonoylglycine by COX is based on the report from Prusakiewicz et al. (2002). Other NAGs possessing an unsaturated acyl chain might undergo similar chemistry.

The contribution that cytochrome c may make towards the NAG biosynthesis in vivo is unclear. c. Oxidation of the NAEs to the NAGs: Burstein et al. (2000) first demonstrated that anandamide could be oxidized to NAGly in cultured Chang liver cells. Later work demonstrated that the NAEs would serve as substrates for a purified ADH (ADH7, see Aneetha et al., 2009 or ADH3, see Ivkovic et al., 2011) for the NAD1-dependent generation of either the N-acyl glycinals or the N-acyl glycines (Figure 9.7).

RaCOaNHaCH2 aCH2 aOH-RaCOaNHa CH2 aCHO-RaCOaNHaCH2 aCOOH As discussed by Ivkovic et al. (2011), N-acyl glycinal oxidation to the N-acyl glycine could be catalyzed by an alcohol dehydrogenase or an aldehydrogenase. The in vitro experiments with the purified ADHs are supported by a set of in cellulo experiments carried out with cultured mammalian cells. Addition of deuterated anandamide to RAW

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INTRODUCTION

264.7 cells yielded deuterated NAGly (Bradshaw et al., 2009a) and the growth of mouse neuroblastoma N18TG2 cells in the presence of [13C,15N]-oleoylethanolamine yielded approximately equal levels of both [13C,15N]oleamide and [13C]-oleamide (Farrell, 2010). These studies not only provide evidence for the cellular oxidation of NAEs to the NAGs (and the PFAMs), but also demonstrate that routes from the NAEs and acyl-CoAs to the NAGs are both operative in the cell. Future work is necessary to define exactly which isozymes of alcohol- and/or aldehydedehydrogenase catalyze NAE oxidation in vivo and the respective contribution of each pathway to total cellular NAG biosynthesis. As stated above, it is unclear whether the NAGs are produced in CNS or whether other peripheral tissues are also involved in production of these signaling lipids. The wide distribution of the NAGs throughout the body (Bradshaw et al., 2009b), including NAGly, likely implies its involvement in many physiological functions other than those described in the CNS and may suggest a different localization for production of these signaling molecules or the potential for transport through the blood
brain barrier. Levels of NAGly in vivo can be regulated by the action of FAAH, which hydrolyzes NAGly to free arachidonic acid and glycine (Bradshaw et al., 2009a). NAGly is also a substrate for selective oxygenation via cyclooxygenase-2 (Prusakiewicz et al., 2002), or finally NAGly can be oxidized by peptidylglycine α-amidating monooxygenase (PAM) to yield the PFAM, arachidonamide (Wilcox et al., 1999). The available data suggests that the in vivo processes used to produce NAGly are also operative to generate the other known NAGs (Waluk et al., 2010; Wilcox et al., 1999; Farrell et al., 2012). N-Oleoyl glycine (OLGly) formation has been detected in partially purified rat brain lipid extracts. Other results show the presence of OLGly in skin, lung, spinal cord, ovaries, kidney, liver, and spleen at concentrations from B150 to B750 pmol/gram of dry tissue weight (Bradshaw et al., 2009b). OLGly is a signaling lipid that regulates body temperature and locomotion in rats (Chaturvedi et al., 2006), and also may serve as a precursor to oleamide (Merkler et al., 2004; Farrell, 2010). To date, there is no reference in the literature to receptors that are activated by OLGly. N-Palmitoyl glycine (PAGly) is a further N-acyl amino acid that has been detected at high levels in rat skin, lung, spinal cord, kidney and liver, at concentrations from 388 to 1612 pmol/g of dry tissue (Bradshaw et al., 2009b). PAGly is an endogenous lipid and acts as a modulator of calcium influx and nitric oxide production in sensory neurons (Rimmerman et al., 2008). In

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addition, PAGly potently inhibited heat-evoked firing of nociceptive neurons in rat dorsal horn and occurs at high levels in FAAH knockout mice, implying a pathway for enzymatic degradation (Rimmerman et al., 2008). Bradshaw et al. (2013) noted that the activity profiles of PAGly and the transient receptor potential channel 5 (TRPC5) are remarkably similar, hinting that PAGly might be the endogenous ligand for TRPC5. A number of other NAGs have been detected in the CNS, including N-stearoyl glycine (STRGly) and N-docosahexaenoyl glycine (DOCGly). STRGly is most abundant in the skin (B1600 pmol/gram of dry tissue weight), with significantly lower levels in the brain and CNS (#100 1600 pmol/gram of dry tissue weight). The highest levels for DOCGly are found in skin, lung, spinal cord, kidney, and liver (B100 pmol/g to B200 pmol/g of dry tissue weight) (Bradshaw et al., 2009b; Tan et al., 2009, 2010). N-Linoleoyl glycine has also been found in mammals, but is only found in the peripheral tissues (Bradshaw et al., 2009b). The physiological roles for STRGly, DOCGly, and LINGly have yet to be elucidated.

N-Acyl Taurines, a Specific Class of the NAAs The N-acyl taurines (NATS) are members of the fatty acid amide family that consist of a fatty acid conjugated to taurine, RaCOaNHa(CH2)2aSO3H. NATs were first identified as a novel class of brain and spinal cord metabolites in FAAH(2 / 2 ) knockout mice (Saghatelian et al., 2004, 2006). MS/MS analysis of the brain and spinal cord NATs of the FAAH(2 / 2 ) mice identified a range of saturated and unsaturated long-chain acyl chains, C16aC24, with N-lignoceroyl (C24:0) taurine being the most abundant. Interestingly, there was a striking difference between the acyl chain distribution of FAAHregulated NATs between the CNS and periphery with saturated acyl chains in the CNS and polyunsaturated acyl chains in the periphery. More recent analysis of the NATs (and the NAEs) in the CNS and periphery in FAAH(2 / 2 ) mice reveals a complex temporal and anatomical relationship between amounts and acyl chain distribution of these fatty acid amides upon FAAH disruption (Long et al., 2011). Long et al. (2011) suggest that these data argue for multiple in vivo routes for the biosynthesis of NATs and NAEs. Multiple biosynthetic routes for the NAEs are known, as has been reviewed herein (Figure 9.4). The identification of NATs in vivo in mouse models, as outlined above, coincided with the characterization of an enzyme called acyl-CoA:amino acid N-acyltransferase 1 (ACNAT1), which was identified in mouse peroxisomes (Reilly et al., 2007). ACNAT1 is mainly expressed in the liver and the kidney and efficiently

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conjugates very long-chain and long-chain saturated fatty acids (C12aC24aCoA) to taurine (Km 5 11 μM and Vmax 160 nmol/min/mg for N-palmitoyl taurine production). ACNAT1 catalyzes the conjugation of fatty acids to taurine. Interestingly, ACNAT1 is not active on unsaturated fatty acids and appears to be a specific taurine-conjugating enzyme (Reilly et al., 2007). A second enzyme, which is 95% identical to ACNAT1 at amino acid level, was also identified in mouse peroxisomes, and was named ACNAT2. ACNAT2 is also a peroxisomal enzyme (Hunt et al., 2012), expressed mainly in kidney (mRNA), but, to date, the substrate specificity for ACNAT2 has not been defined. We have speculated that ACNAT2 conjugates unsaturated fatty acids to taurine (or another amino acid); however, this needs to be confirmed experimentally. The other enzyme identified to date which conjugates fatty acids to taurine is the bile acid-CoA:amino acid N-acyltransferase (BAAT), which shows approximately 45% sequence identity to ACNAT1 and ACNAT2 (for a review, see Hunt et al., 2012). Experiments reported by O’Byrne et al. (2003) showed that recombinant human BAAT can conjugate long- and very long-chain fatty acids to taurine, although the activity is low, 5
10% of the activity identified with the bile acid-CoAs (choloylCoA and chenodeoxycholoyl-CoA). The bile acid-CoAs are the common characterized substrates for BAAT (Falany et al., 1994; He et al., 2003; Hunt et al., 2012; O’Byrne et al., 2003). So far, data show that NATs are produced enzymatically in vitro by ACNAT1 or BAAT, with the latter enzyme localized in the peroxisomes. To date, no further pathways have been identified. Inhibition of FAAH in mice resulted in accumulation of micromolar levels of NATs in the liver, the kidney and the CNS, suggesting that micromolar levels can be reached in vivo (Saghatelian et al., 2004, 2006). FAAH has been shown to hydrolyze both N-arachidonoyl taurine and N-oleoyl taurine to the free fatty acid and taurine (McKinney and Cravatt, 2005). In addition to FAAH hydrolysis, a second pathway to degrade and/or regulate NAT levels in vivo is via oxidative metabolism, resulting in the generation or termination of novel signaling molecules (Turman et al., 2008). Although N-arachidonoyl taurine was a poor substrate for cyclooxygenases, two mammalian lipoxygenases (LOXs), 12S-LOX and 15S-LOX, oxygenated N-arachidonoyl taurine efficiently, generating 12- and 15-hydroxyeicosatetraenoyltaurines (HETE-Ts) and dihydroxyeicosatetraenoyltaurines (diHETE-Ts). The functional roles of the NATs have not been thoroughly investigated. N-Arachidonoyl taurine does not activate cannabinoid receptors, CB1 and CB2, or the peroxisome proliferator-activated receptors. N-Arachidonoyl taurine does activate TRPV1 and TRPV4 at relatively high concentrations (20
30 μM),

hinting at a signaling role for NATs at cell surface receptors (Saghatelian et al., 2006). The TRPV1 and TRPV4 are cation channels, activated by a number of endogenous substances, resulting in an influx of calcium into cells. They have roles in pain perception, regulation of blood pressure, osmotic sensation, and in respiratory diseases (Vriens et al., 2009). Peripherally, N-arachidonoyl taurine and N-oleoyl taurine are antiproliferative at concentrations as low as 1 μM (Chatzakos et al., 2012). More recently, Waluk et al. (2013) found that N-arachidonoyl taurine and N-oleoyl taurine regulate insulin secretion in pancreatic β-cells via TRPV1 channels, by increasing calcium flux (Waluk et al., 2013). Both of these NATs induced a high frequency of calcium oscillations in β-cell and rat islet cell lines. However, the data also indicated that receptors other than TRPV1 are involved in the insulin secretion response to N-oleoyl taurine. The only other report of an N-arachidonoyl taurine receptor was its binding to GPR72 at concentrations of B1 μM (Roy and Hannedouche, 2007). Other N-Acyl Amino Acids In addition to the NAGs and NATs, a large diversity of other endogenous N-acyl amino acids has been identified in rat brain and bovine spinal cord (Tan et al., 2009, 2010). Han et al. (2013) identified three other N-arachidonoyl amino acids in mouse brain (N-arachidonoyl-alanine, serine, and γ-aminobutyric acid) and N-arachidonoyl serine has also been characterized from bovine brain (Milman et al., 2006). N-Arachidonoyl serine (NASer) binds weakly to CB1, CB2, and TRPV1 (Kd . 10 μM) and is activated in the vascular system as a vasodilator and a vasoprotective agent that suppresses the formation of TNF-α, NO, and other reactive oxygen species (Milman et al., 2006). Limited work on the bioactivity of N-arachidonoyl γ-aminobutyrate (NAGABA) and N-arachidonoyl alanine (NAAla) has demonstrated that NAGABA was antinociceptive but that NAAla was not, in rat models for pain perception (Huang et al., 2001; Succar et al., 2007). The in vivo reaction for the synthesis and degradation of the N-acyl amino acids is not known and has been little studied. McCue et al. (2008) reported that cytochrome c will catalyze the H2O2-dependent formation of NASer, NAGABA, and NAAla in vitro and from arachidonoyl-CoA and the appropriate amino acid. Treatment of mice with an FAAH inhibitor led to an increase in the brain levels of NASer (Han et al., 2013). In contrast, FAAH inhibition in the mouse had no effect on the levels of mouse brain NAAla and decreased NAGABA (Han et al., 2013). Other work had shown that NAAla only weakly inhibited the FAAHmediated hydrolysis of anandamide (Ki 5 20
50 μM) (Cascio et al., 2004). In summary, these data suggest that NAAla is not an FAAH substrate, that NASer

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THE RELEVANCE OF THE FATTY ACID AMIDES TO NEUROLOGICAL DISEASE

an FAAH substrate for hydrolysis (NASer 1 H2Oarachidonate 1 L-Ser), and that NAGABA is a product of FAAH synthesis (arachidonate 1 GABANAGABA 1 H2O)
intriguing, if true! Clearly, much remains to be elucidated to fully understand the many N-acyl amino acids found in the mammalian brain and CNS.

is

N-Acyldopamines Only a small number of N-acyldopamines (NDAs) are known from the brain and CNS (see Figure 9.8), including N-arachidonoyl- (NADA), N-oleoyl- (OLDA), N-palmitoyl- (PALDA), and N-stearoyldopamine (STEARDA) (Huang et al., 2002; Chu et al., 2003). In bovine, the highest levels of NADA are found in the striatum, with lower amounts found distributed throughout the central and peripheral nervous system (Huang et al., 2002). There is a single report of other NDAs, such as N-myristoyldopamine and N-nonadecanoyldopamine, in rat striata at levels significantly lower than PALDA and STEARDA (Walker et al., 2005). NADA is an endogenous ligand for the CB1 and TRPV1 receptors and, as a consequence, has a role in the perception of pain, locomotion, and the regulation of body temperature (Bisogno et al., 2000; Huang et al., 2002). OLDA also binds to TRPV1 and CB1 (weakly) and, thus, exhibits effects similar to NADA (Chu et al., 2003). In addition, NADA binds to PPARγ (O’Sullivan et al., 2009) and OLDA may bind to GPR119 (Chu et al., 2010), but neither is relevant to the neurological functions of these NDAs. The functions of PALDA, STEARDA, and the other NDAs described by Walker et al. (2005) are unclear, but there is evidence suggesting that PALDA and STEARDA function by enhancing the effect of NADA on TRPV1, ‘the entourage effect’ (De Petrocellis et al., 2004). The most straightforward biosynthetic route to the NDAs would be the conjugation of dopamine to a fatty acid or fatty acyl-CoA thioester: RaCOOH (or RaCOaSaCoA) 1 H2Na(CH2)2aC6H4(OH)2-RaCO aNHa(CH2)2aC6H4(OH)2 1 H2O (or CoAaSH) (Hu et al., 2009). The direct conjugation of the free fatty acid with dopamine seems unlikely; an activated fatty acid susceptible to nucleophilic attack by dopamine is a chemically more attractive reaction. Alternative strategies for the in vivo production of the NDAs would

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first involve the N-acyltransferase catalyzed formation of the N-acyltyramines, N-acyl-L-DOPAs, or the N-acyltyrosines. Hydroxylation of the N-acyltyramines or decarboxylation of N-acyl-L-DOPAs would yield the NDAs. Also, the NDAs could be produced from the N-acyltyrosines by either (a) decarboxylation to the N-acyltyramines followed by N-acyltyramine hydroxylation or (b) hydroxylation to the N-acyl-DOPAs followed by N-acyl-DOPA decarboxylation (Figure 9.9). Despite the ‘unattractiveness’ of the chemistry, current data suggest that the direct conjugation of a fatty acid to dopamine is the major biosynthetic route for the NADAs (Akimov et al., 2007; Hu et al., 2009). The enzyme catalyzing fatty acid/dopamine conjugation could be FAAH working in reverse of its welldocumented hydrolysis reaction. A secondary route to NDAs involving N-acyltyrosine hydroxylation/decarboxylation is likely. Support for this secondary route comes from the identification of the N-acyltyrosines in rat brain, the lack of N-acyltyramines in tissues producing the NDAs, the stimulation of NDA formation by the N-acyltyrosines, and the dependence of NDA biosynthesis in dopaminergic termini on tyrosine hydroxylase (Akimov et al., 2007; Hu et al., 2009). Catabolism of the NDAs is relatively complex. The NDAs are relatively poor FAAH substrates indicating that hydrolysis is not the major route for NADA degradation (Chu et al., 2003). Other catabolic fates for the NDAs include methylation or sulfation at the 3’-hydroxyl group of the catechol ring or oxidation of the catechol ring (Akimov et al., 2007, 2009). Hydroxylation of the arachidonoyl moiety of NADA by rat liver microsomes (P450) has been reported and it was suggested that this chemistry could also occur in the brain (Rimmerman et al., 2009). Akimov et al. (2007, 2009) show that methylation or sulfation of the 3’-hydroxyl group is, most likely, the major catabolic fate of the NDAs in the brain and CNS. Based on the report of N-acetylnorepinephrine in a patient suffering from brain cancer (Herrlich and Sekeris, 1964), it is possible that the NDAs are hydroxylated in a reaction catalyzed by dopamine β-monooxygenase (DβM). Some of the NDA-derived products may be biologically active (Akimov et al., 2007; Rimmerman et al., 2009), but additional research is required to address this question (see Figure 9.10).

THE RELEVANCE OF THE FATTY ACID AMIDES TO NEUROLOGICAL DISEASE FIGURE 9.8 The structure of the N-acyldopamines. R is a longchain fatty acid, polyunsaturated.

which

can

be

saturated,

unsaturated,

or

The discovery of anandamide and other endogenously produced ligands for the CB1 and CB2 receptors (the endocannabinoids) coupled with an understanding of their roles in neuroinflammation, neurodegeneration,

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FIGURE 9.9 N-acyldopamine (NDA) biosynthesis. The enzymes proposed to catalyze the individual reactions are indicated by numbers within the ovals: 1 5 a decarboxylase, 2 5 a hydroxylase, and 3 5 an N-acyltransferase. Enzyme 1 is likely aromatic L-amino acid decarboxylase and enzyme 2 is likely tyrosine hydroxylase or tyrosinase. Enzyme 3 would be an unidentified N-acyltransferase and more than one N-acyltransferase could be involved in NDA biosynthesis depending on the substrate specificity of the particular enzyme.

and neurobehavioral activities like pain, anxiety, addiction, and the stress response, points towards exciting new strategies to treat and/or diagnose diseases related to dysfunction in these processes. The discovery of other neuroactive fatty acid amides, with different functions within the brain and CNS that bind to receptors other than CB1 and CB2, only expends the potential medical relevance of this family of lipids.

Earlier excellent reviews on the therapeutic potential of the fatty acid amides were from Conner et al. (2010); Gowran et al. (2011); Heal et al. (2009); Ligresti et al. (2009); Micale et al. (2013); and Piscitelli and Di Marzo (2012). Unfortunately, the complexities of fatty acid amidemediated signaling present significant challenges in developing novel therapeutics based on their

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FIGURE 9.10 N-acyldopamine (NDA) degradation. The pathways for NDA degradation as based largely on the work of Akimov et al. (2007). The enzymes proposed to catalyze the individual reactions are in the ovals: FAAH, fatty acid amide hydrolase; COMT, catecholO-methyltransferase; ST, sulfotransferase; OX, oxidoreductase. COMT is known to exist in the brain and CNS (Rivett et al., 1982) and the one possible ST involved in NDA degradation is SULT4A1, which is found in the mammalian brain (Liyou et al., 2003). Other abbrevations used in this figure are: PAPS, 3’-phosphoadenosine 5’-phosphate; PAP, 3’-phosphoadenosine 5’-phosphate; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine.

neuroactivities. Fatty acid amide-based agonists pose the threat of undesirable psychotropic and immunosuppressive side effects. One solution to this concern would be the development of fatty acid-amide based antagonists and/or inverse agonists. Rimonabant, a CB1 reverse agonist (Rinaldi-Carmona et al., 1994), was

approved in approximately 60 countries for the treatment of obesity (Ligresti et al., 2009), based on the orexigenic effects of CB1 agonists (Cota et al., 2003) and successful studies demonstrating that rimonabant reduced food intake in rodents (Tallett et al., 2007). However, severe psychiatric side effects of rimonabant

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treatment resulted in the removal of this drug from the market and a cessation of development of other CB1 antagonists/inverse agonists as drugs (Heal et al., 2009; Ligresti et al., 2009). The lessons from rimonabant treatment have pointed to alternative strategies for fatty acid amide-based therapies to treat neurological dysfunction. One approach is the inhibition of FAAH to increase the cellular concentration of anandamide and the other fatty acid amides degraded by this hydrolase (Bisogno and Maccarrone, 2013; Piscitelli and DiMarzo, 2012). The coadministration of an FAAH inhibitor with (a) an inhibitor of other enzymes involved in fatty acid amide/ endocannabinoid degradation, (b) an inhibitor of fatty acid amide uptake, or (c) an agonist/antagonist/reverse agonist of a specific fatty acid amide could prove more efficacious than the use of a selective FAAH inhibitor alone (Micale et al., 2013; Piscitelli and Di Marzo, 2012). Conner et al. (2010) point out that the rich chemical diversity of the fatty acid amide family provides many opportunities to development analogs as drugs, imaging agents, and research tools targeted at the fatty acid amide receptors and enzymes of fatty acid amide metabolism. Continued fatty acid amide research, including a better understanding of their biosynthesis, will foster the ultimate development of fatty acid amide-based therapies to treat neurological disease.

Acknowledgements This work was supported, in part, by grants from The Swedish Research Council to M.C.H, the National Institutes of Health (R15GM059050, R15-GM073659, and R03-DA03432), the Florida Center for Excellence for Biomolecular Identification and Targeted Therapeutics (FCoE-BITT grant no. GALS020), the Gustavus and Louise Pfieffer Research Foundation to D.J.M., a Graduate Student Success Fellowship from the University of South Florida to P.M., and a Graduate Multidisciplinary Scholars Award from the University of South Florida (USF Thrust Life Sciences Program) to E.K.F.

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3245. Wiles, A.L., Pearlman, R.-J., Rosvall, M., Aubrey, K.R., Vandenberg, R.J., 2006. N-Arachidonyl-glycine inhibits the glycine transporter, GLYT2a. J. Neurochem. 99, 781
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2751. Yin, H., Chu, A., Li, W., Wang, B., Shelton, F., Otero, F., et al., 2009. Lipid G protein-coupled receptor ligand identification using β-arrestin PathHuntert assay. J. Biol. Chem. 284, 12328
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C H A P T E R

10 Low Omega-3 Fatty Acids Diet and the Impact on the Development of Visual Connections and Critical Periods of Plasticity Claudio Alberto Serfaty and Patricia Coelho de Velasco INTRODUCTION Appropriate neural circuits develop through the selective elimination of misplaced axons and the maintenance of correct ones and their synapses (Chandrasekaran et al., 2005). These processes will determine the specificity of brain connections responsible for sensory perception and coordination of motor and cognitive systems that characterize a mature nervous system (Serfaty and Linden, 1994, Serfaty et al., 2005). Nutrition is a major factor that affects central nervous system functioning, especially in light of the existence of critical periods for the formation and differentiation of neural circuitry since its effects comprise two limiting phases: embryonic and neonatal life (Morgane et al., 1993, McIlvain et al., 2003).

NUTRITION AND THE IMPACT OF OMEGA-3 FATTY ACIDS ON BRAIN DEVELOPMENT Nutritional deficiencies are related to structural changes in the brain, such as alterations in overall size, decrease in hippocampal volume, modifications in developmental processes such as neurogenesis and gliogenesis, synthesis and release of neurotransmitters, the onset of neural activity, including excitatory and inhibitory circuits, and finally behavioral and cognitive aspects (Ranade et al., 2008, Dauncey, 2009). The consequences of malnutrition will depend on the nature and severity of disabilities, including the type of nutritional deficiency, level of malnutrition and exposure time (Joseph et al., 2009).

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00010-7

Essential nutrients are exclusively acquired through diet (Smith, 2008). Therefore, social and cultural factors involved in nutritional needs could lead to functional changes in neurochemical aspects of synaptic organization, with serious consequences for neural circuitry maturation (Ballabriga, 1990, Gonzalez et al., 2008, Serfaty, 2011). Lipids are responsible for about 50
60% by brain dry weight of an adult, of which approximately 35% are as long-chain polyunsaturated fatty acids (LCPUFAs). α-Linolenic acid (omega-3 fatty acid) and linoleic acid (omega-6 fatty acid) are considered essential fatty acids (EFAs) as they cannot be endogenously synthesized (Wainwright, 2002, Heird and Lapillonne, 2005). Docosahexaenoic acid (DHA/ omega-3) and arachidonic acid (AA/omega-6) derived from their respective precursors, are the main LCPUFAs found in the brain (Innis, 2000, Innis, 2008, Yehuda et al., 2005) (Figure 10.1). These lipids, highly sensitive to dietary changes, participate in the formation and physiology of the neural membrane and may thus regulate events which depend on its functional integrity (Marszalek and Lodish, 2005). Adequate supplies of EFAs are required during development and in adulthood, in order to ensure appropriate brain function (Innis, 2011, Bhatia et al., 2011, Luchtman and Song, 2012). Numerous studies on the deficiency of omega-3 fatty acids mention the initial stages of development as a critical period of brain modeling (Chen and Su, 2012, de Velasco et al., 2012, Moreira et al., 2010). During pre-natal development, DHA is transported from mother to the offspring through the placenta. After birth, omega-3 acquisition is achieved by breast milk consumption (Koletzko et al., 2008, Uauy et al., 2000, Innis, 2011). Thus, the

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Omega-3

Omega-6

α-linolenic acid ALA (18:3ω3)

linoleic acid LA (18:2ω6) Δ6 desaturase γ-linolenic acid GLA (18:3ω6)

octadecatetraenoic acid (18:4ω3) elongase

dihomo-γ-linolenic acid DGLA (20:3ω6)

eicosatetraenoic acid (20:4ω3)

Δ5 desaturase eicosapentaenoic acid EPA (20:5ω3)

arachidonic acid AA (20:4ω6) elongase

docosapentaenoic acid DPA (22:5ω3)

docosatetraenoic acid (22:4ω6) elongase

(24:5ω3)

(24:4ω6) Δ6 desaturase

(24:6ω3)

(24:5ω6) β-Oxidation

docosahexaenoic acid DHA (22:6ω3)

docosapentaenoic acid (22:5ω6)

FIGURE 10.1 Essential fatty acids pathway (adapted from Le et al., 2009).

fetus and newborn is completely dependent on maternal supply of EFAs. Several factors can interfere with the conversion of EFAs into their specific compounds. These factors include: intake of saturated fatty acids and hydrogenated lipids; deficiency of vitamins and minerals that act as cofactors (mainly zinc deficiency) (Bourre, 2006) and; excessive alcohol consumption and stress-related hormones (Pawlosky et al., 2001, Tajuddin et al., 2013). These conditions indicate that even with an adequate dietary intake of EFAs, deficiency of AA and DHA may still occur. Therefore, differences in the metabolism of constitutional EFAs have been recognized among possible risk factors of neurodevelopmental disorders (Richardson and Puri, 2000). The balance of omega-3/omega-6 fatty acids is an important determinant in maintaining homeostasis and normal brain development (Gomez-Pinilla, 2008,

Simopoulos, 2011). An adequate ratio of omega-3/ omega-6 has changed in the modern Western human diets (Singh, 2005, Simopoulos, 2006). This change has been attributed to an increase in omega-6 intake due to high protein diets and also to an increased intake of saturated fat found in industrialized food. Both habits have been associated with DHA deficiencies even in breast-fed infants (Chen and Su, 2011, Innis, 2008). The main food sources of α-Linolenic acid (ALA) are flaxseed and some nuts. DHA is also primarily available from fish and seafood (Youdim et al., 2000, Marszalek and Lodish, 2005, Siriwardhana et al., 2012). Findings from randomized clinical studies in children highlighted the importance of the introduction of omega-3 fatty acids into infant formulas as a method of satisfying children’s growing and developing needs (Uauy and Castillo, 2003). A study reported a persistent effect of DHA in visual acuity in the first year of life in children fed with breast milk as opposed to infants fed with DHA-supplemented formula, indicating that blood levels of DHA are directly related to changes in visual acuity (Uauy et al., 2000, Carlson, 2001). It has been demonstrated in a clinical trial study that infants at 12 months of age who received a formula supplemented with different concentrations of DHA demonstrated increased visual potential and acuity when compared with control subjects fed with a formula lacking DHA (Birch et al., 2010). Thus, an adequate DHA supply is necessary for the correct shaping of brain circuitry. DHA levels in the brain seem to be strictly controlled, since any disturbance leads to severe impairment in brain development and maturation (Fedorova and Salem, 2006, Kim et al., 2010, Kitajka et al., 2002). Most of the incorporation of DHA in the brain and retina occurs throughout the last trimester of gestation and continues up to the first four years in humans (Carlson, 2001, DijckBrouwer et al., 2005). This time-course overlaps with major landmarks of visual system development, from neurogenesis to axonal elimination and the critical period of use-dependent plasticity, when visual circuits acquire their ultimate functionality. Incorporation of DHA into neuronal membranes occurs primarily by the addition of DHA in circulating plasma or by biosynthesis that occurs in the liver. Local synthesis may still occur in the brain tissue (Kim, 2007). The omega-3 fatty acids are found esterified in membrane glycerophospholipids containing phosphatidylserine and phosphatidylethanolamine and are released to participate directly or indirectly in the regulation of physiological and metabolic processes (Innis, 2011). About 20% of the fatty acids found in the human retina is DHA, which is enriched in membrane disks at the specialized outer segment of photoreceptors (Innis, 2011) and DHA is extensively retained

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NUTRITION AND THE IMPACT OF OMEGA-3 FATTY ACIDS ON BRAIN DEVELOPMENT

through a recycling mechanism between the outer membranes of rods and cones and the retinal pigmented epithelium (RPE) (Sangiovanni and Chew, 2005). The importance of these fatty acids as integral membrane components includes organization and activity of membrane proteins, facilitation of neurotransmission release and cell signaling (Bazan, 2005). DHA deficiency results in reduced amplitude of waves of spontaneous electrical activity. Those waves are essential for the development of topographical matching between the retina and visual target nuclei (Jeffrey et al., 2001). In a biological model of the human retinitis pigmentosa, mice with mutations in rhodopsin present reduced levels of DHA in photoreceptors (Anderson et al., 2002). Moreover, DHA was shown to promote survival and inhibition of apoptosis in photoreceptors in vitro (Rotstein et al., 1997, German et al., 2006). Omega-3 fatty acids have been shown to modulate the levels of neuronal synaptic proteins (de Velasco et al., 2012, Chytrova et al., 2010, Mazelova et al., 2009). It has been reported that rats that received a supplementation with DHA and uridine during the gestational period until postnatal day 21 (PND21) showed enhanced synapsin-1, mGluR1, and PSD-95, suggesting that DHA is involved in synaptic stabilization (Cansev et al., 2009). DHA also increases the number of dendritic spines and synapses probably in hippocampal neurons, particularly at excitatory synapses (Wurtman, 2008). DHA is highly concentrated in synaptic membranes that facilitate exocytosis of neurotransmitter-containing vesicles, indicating an important role in regulating neurotransmitter release (Pongrac et al., 2007). Moreover, it not only modulates the physical properties of the neuronal membranes (Tanabe et al., 2004), but also promotes the formation of second messengers that can function in signaling processes (Phillis et al., 2006). As a free fatty acid, DHA can regulate the activity and insertion of membrane receptors such as NMDA and AMPA glutamate receptors (Moreira et al., 2010), as well as being able to modulate dopaminergic (Zimmer et al., 2000, Kuperstein et al., 2008) and serotonergic neurotransmission (Chalon, 2006). One of the potential mechanisms of DHA function is that it can directly modulate NMDA receptors or alter the lipid microenvironment of neuronal membranes enhancing the receptor-mediated response (Nishikawa et al., 1994). DHA can also restore long-term potentiation (LTP) attenuated by blocking the activity of phospholipase A2 (PLA2) (Fujita et al., 2001). Furthermore, increased PLA2 activity is related to the insertion of AMPA receptors (AMPAr) in postsynaptic densities (Martel et al., 2006). DHA as a free form, or by way of bioactive derivatives such as neuroprotectin D1 (NPD1), can be

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released by calcium-independent phospholipase A2 (iPLA2) activity in neurons, glial cells, endothelial cells, and even of cerebral blood vessels by stimulation of neurotransmitters, neurotrophic factors, cytokines, membrane depolarization, and activation of ion channels (Bazan, 2009, Palacios-Pelaez et al., 2010). Those lipid messengers can regulate and interact with multiple signaling cascades, contributing to the development, differentiation, synaptic function, protection, and repair of cells in the nervous system (Bazan, 2003). Also, it has been shown that the biotransformation of DHA into bioactive lipids, such as resolvins and maresins (Farooqui, 2012), protectins (neuroprotectin D1), and docosatriens (Mukherjee et al., 2007) can also be engaged in synaptic mechanisms and act together with DHA in facilitating brain development. Recent studies demonstrated a synaptogenic function of a specific DHA amide derivative, called N-docosahexaenoylethanolamide (DEA). Kim and collaborators showed that DEA-treated hippocampal neurons increased synaptic machinery expression (Kim et al., 2011). The group observed enhanced synapsin labeling, which indicated an increase in synaptic puncta, as well as an increase in glutamate receptor subunits. The study also revealed that a DHA nutritional restriction can decrease DEA levels in mouse fetal hippocampi, indicating that an imbalance in omega-3 food consumption can affect lipid mediators involved in neuronal development. DHA has been described as a trophic molecule since it is able to influence a broad spectrum of mechanisms such as the expression of genes related to signal transduction, synaptic plasticity, energy metabolism and traffic through membrane receptors (Kitajka et al., 2004). DHA plays a role as an endogenous ligand for retinoid X receptors (RXR) (De Urquiza et al., 2000). The receptors RXRα and RXRβ together as peroxisome proliferatoractivated receptor (PPAR) gamma can be activated by DHA, leading to dimerization of these receptors and their insertion into the nucleus, where they can act as transcription factors inducing differentiation and synaptic stability (Kim, 2007). Studies have shown that DHA can regulate expression of BDNF mRNA, neurotrophic factors directly related to processes of neuronal survival and synaptic strengthening (Bousquet et al., 2009, Katsuki et al., 2009, Venna et al., 2009). During aging, the concentration of polyunsaturated fatty acids (PUFAs) in neuronal membranes decreases. There are at least two possible reasons for this decline. The first is a decrease in the ability of fatty acids acquired from diet to cross the blood
brain barrier, since it is known that the function of the barrier becomes less effective during the aging process (Yehuda et al., 2005). Another factor is influenced by a diminished level and activity of enzymes involved in

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dietary precursors of EFAs. Thus, desaturase enzyme activity declines during aging (Youdim et al., 2000, Haag, 2003). This modifies cerebral membrane turnover which could indicate omega-3 relevance in neurodegenerative disorders such as Parkinson (Bousquet et al., 2009) and Alzheimer diseases (Oster and Pillot, 2010, Cunnane et al., 2012). Moreover, accumulating evidence points out that both omega-3 and omega-6 are deficient in schizophrenic patients, particularly DHA levels (Horrobin, 2001).

THE DEVELOPMENT OF VISUAL TOPOGRAPHICAL MAPS Synaptic specification and the importance of topographical maps has been recognized as a major feature of brain organization and neural processing since the pioneering studies of Roger Sperry (Sperry, 1963). Since then, the concept that sensory, as well as motor and cognitive abilities depend, in a fundamental way, on the correct patterning of connections, has become part of our current knowledge about brain function (Meyer, 1998). In this way, a keystone feature of sensory visual processing relies on the correct patterning of visual connections that allow the proper development of various visual attributes of form, color, and motion, that directly influence visual acuity, and the same is true for all other sensory systems (Graven, 2004). The development of organized and specific connections found among mammalian species depends on two general strategies: an initial over-production of SUPERIOR COLLICULUS PND 0

neurons and synaptic contacts followed by the death of excess neurons and elimination of misplaced ineffective axons/synapses (Linden and Perry, 1982, Serfaty and Linden, 1994). During the last trimester of human gestation and the first two postnatal weeks of the rodent development, about the time that neuronal contacts begin to emerge, nearly 50% of the immature neuronal population undergoes a process of natural neuronal death. However, natural neuronal death seems to play only a marginal role in topographical maps development (Serfaty et al., 1990, Reese, 2011). The connections between retinal ganglion cells and their target nuclei, the superior colliculus (SC) and the dorsal lateral geniculate nucleus (dLGN)
the so called retinofugal connections
have been extensively studied in rodents as a biological model for the study of organized neuronal circuits, (de Velasco et al., 2012, Kano and Hashimoto, 2009, Huberman et al., 2008). It has been shown that the rodent retinocollicular pathway develops within the first three postnatal weeks, producing highly specific patterns of axonal connections. This form of developmental plasticity occurs mainly through axonal elimination and synaptic growth at appropriate territories (Serfaty et al., 2005) (Figure 10.2). Thus, the initial development of retinocollicular topography is strongly influenced by repulsive/attractive molecules between retinal axons and target neurons. Retinal ganglion cell axons and target cells in the SC express Ephrins and Eph receptors in complementary gradients that vary along the main retinal axis (dorsal to ventral / temporal to nasal) (Cang et al., 2008). A later and complementary step of topographical refinement is achieved by activity-dependent SUPERIOR COLLICULUS PND 21

Contralateral pathway

lpsilateral Pathway

RETINA

FIGURE 10.2 Development of the ipsilateral retinocollicular pathway as a model of topographical specification. At postnatal Day 0, ipsilateral axons labeled by the anterograde transport of neuronal tracers are found dispersed throughout the collicular visual layers, both in the medio-lateral and rostro-caudal axis. By the end of the second and third postnatal weeks, an adult topography develops in which specific groups of neighboring retinal ganglion cells project their axons to adjacent post-synaptic sites.

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CRITICAL PERIODS FOR BRAIN DEVELOPMENT

mechanisms that are required to ensure the fine tuning of the correct representation of retinal axons over their targets, and thus circuitry maturation (O’Leary and McLaughlin, 2005). This includes both spontaneous and evoked activity of retinal ganglion cells (Torborg and Feller, 2005).

CRITICAL PERIODS FOR BRAIN DEVELOPMENT Neuronal connections in the mammalian brain become highly specified during a time window known as the critical period. The duration of the critical period is highly variable between mammalian species and is inversely related to the longevity of the species: rodents display a three-week critical period, while humans develop protracted critical periods that extend up to 5
12 years, and possibly beyond (Berardi et al., 2000, Hensch, 2005). The critical period corresponds to a developmental stage in which environmental cues provide rapid plasticity of neuronal circuits, necessary to the acquisition of appropriate sensory, motor, as well as cognitive skills. Indeed critical periods have been described in many brain systems and in a large variety of species: song learning in birds, auditory localization in barn owls and, in humans, the development of sensory acuity, motor, and language skills (Levelt and Hubener, 2012). The end of the critical period considerably affects plasticity of primary sensory areas of the brain and, as a result, a considerable slow-down in use-dependent modifications have been described in visual, auditory, and somatosensory cortical (Erzurumlu and Gaspar, 2012, Hensch, 2005) and subcortical areas such as the superior colliculus (Serfaty et al., 2005). Although the end of the critical period affects plasticity in cortical and subcortical primary sensory areas, it does not limit plasticity in other associative regions, which are still able to undergo use-dependent modifications even in adulthood (Majewska et al., 2006, Tailby et al., 2005, Barnes and Finnerty, 2010). One important question is how use-dependent activation of primary sensory areas during early postnatal development impacts development and plasticity of other brain areas later on. It has been shown that early visual experience directly influences plasticity in the adult visual cortex (Hofer et al., 2006) and early musical training (before the age of 7) influences sensory motor integration and, thus, performance of musicians (Penhune, 2011). Therefore, the experiences acquired during this developmental window may directly affect the emergence of several other brain abilities and influence the outcome of our individuality. Importantly, those use-dependent modifications in neuronal

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connectivity ultimately result in certain behaviors or capabilities, which would not be revealed/developed otherwise (Berardi et al., 2000, Penhune, 2011, Serfaty et al., 2008, Levelt and Hubener, 2012). Furthermore, it has been suggested that various forms of mental retardation/autism are related to errors in the selective elimination of synapses that takes place during the initial stages of postnatal development (Pfeiffer et al., 2010, Baudouin et al., 2012). It is worth mentioning that critical periods have different time courses in different sensory, motor, and cognitive systems, and the correct timing of such overlapping periods may be of fundamental importance for the progressive gain in complexity that is found in neural processing from primary to associative areas of the CNS (Serfaty and De Velasco, 2013, Berardi et al., 2000, Penhune, 2011). Therefore it has been proposed that pathological conditions of brain development such as those found in Autism Spectrum Disorders and Fetal Alcohol Spectrum Disorder may result from disturbances in duration and/or timing of critical periods (LeBlanc and Fagiolini, 2011, Geschwind and Levitt, 2007, Medina, 2011). The influence of critical periods on the development of sensory brain connections was originally defined after the experiments made by Wiesel & Hubel (1963) in kittens. Those investigators showed that a visual deprivation of one eye causes a dramatic change in the ocular dominance distribution, in favor of the open eye, between the fourth and the eighth postnatal weeks (Wiesel and Hubel, 1963, Hubel and Wiesel, 1970). In humans, neonatal strabismus can also result in similar loss of visual acuity: eye misalignment, if not appropriately treated by the age of 5, produces a permanent loss of visual acuity, a condition known as amblyopia (Kanonidou, 2011). This acuity loss results from the weakening of synapses originating from the nonaligned eye. Furthermore, cats raised under visually biased environments (e.g. exposed to visually stereotyped patterns of horizontal or vertical lines) do not develop accurate discrimination of visual stimuli (except for those horizontal or vertical stimuli), as well as the proper binocular representation of the visual field (Crair et al., 1998). In the visual cortex, the critical period has been correlated with mechanisms involving neurotrophin signaling, especially brain-derived neurotrophic factor (BDNF), which aids the differentiation of inhibitory gamma-aminobutyric acid (GABA) circuits (Levelt and Hubener, 2012). The development of GABAergic innervation seems to be crucial for the onset of the critical period (Hensch et al., 1998) and inhibitory circuits are under the control of both visual experience and BDNF (Hanover et al., 1999). In addition, insulin-like growth

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factor 1 (IGF-1) has been shown to facilitate the development of inhibitory innervation and increase visual acuity (Ciucci et al., 2007). The closure of the critical period in the primary visual cortex involves extracellular matrix (ECM) molecules that develop an environment that inhibits axon and dendritic remodeling. Such molecules include chondroitin sulphate proteoglycans (CSPGs) (Pizzorusso et al., 2002), tissue plasminogen activator (tPA) (Mataga et al., 2002) and growth inhibitory proteins like Nogo, MAG, and OMgp (McGee et al., 2005). In visual subcortical nuclei such as the superior colliculus, the critical period overlaps with the period of fine tuning of topographical maps (Serfaty and Linden, 1994, Simon and O’Leary, 1990). Lesion studies, either monocular enucleation or restricted retinal lesions, have been used to induce reorganization of axons originating from the intact eye (Serfaty et al., 2005, Mendonca et al., 2010, Thompson et al., 1995). Those experiments revealed that the plastic capacity of retinocollicular connections during the critical period is characterized by a rapid reactive growth of axons from the non-lesioned eye in response to lesions during the first three postnatal weeks. After the third postnatal week, a single retinal lesion was still able to elicit a certain amount of reorganization of the intact pathway. However, this took several weeks to develop. Therefore, a second slow stage of plasticity does occur even after the end of the critical period (Serfaty et al., 2005).

ROLE OF OMEGA-3 ON DEVELOPMENT OF CENTRAL VISUAL CONNECTIONS DHA has been shown to exert several roles in the visual system from photoreceptor differentiation to synaptic plasticity in a series of events leading to a direct influence on visual acuity (Hoffman et al., 2009, Jeffrey et al., 2001). In order to address a more specific role for omega-3 in the developing visual system, we used a nutritional approach in which female rats were given an isocaloric diet containing coconut oil as a lipid source (de Velasco et al., 2012). This diet protocol started 5 weeks before mating in order to deplete omega-3 fatty acids. Females were then kept under this nutritional restriction during mating, pregnancy, and after delivery until the litters reached postnatal day (PND) 42. Lipid levels were measured in samples from the collicular visual layers of rats at PND28. Those samples revealed a 53% reduction in the levels of DHA without any changes in AA content (Table 10.1) (de Velasco et al., 2012). The topographical distribution of uncrossed retinocollicular terminal fields was determined by the

TABLE 10.1 Fatty Acid Composition (% of Total) in Samples from the Visual Layers of the Superior Colliculus Fatty Acid

Control

ω-3 Deficient

16:0

17.78 6 0.76

24.09 6 0.23

16:1

0.68 6 0.01

0.84 6 0.02

17:0

Nd

0.20 6 0.01

18:0

24.96 6 0.44

29.09 6 0.32

18:1n9t

15.09 6 0.22

16.01 6 0.17

18:1n9c

2.56 6 0.08

3.19 6 0.02

18:2n6t

0.79 6 0.06

0.65 6 0.15

20:1

0.69 6 0.02

0.60 6 0.04

20:2

Nd

20:3n6

0.41 6 0.01

20:4n6

14.79 6 0.31

22:6n3

21.22 6 0.34

22:6n3 / 20:4n6

1.43

0.57 6 0.02 nd 14.92 6 0.41 9.99 6 0.35 0.67

The values represent mean 6 SEM.  vs control. p , 0.01; nd 5 not detected. (Adapted from De Velasco et al., 2012.)

anterograde transport of horseradish peroxidase (HRP). It was shown that this chronic form of malnutrition was able to disrupt the topographical development as early as the second postnatal week (PND13) when terminal fields displayed a two-fold increase in label density when compared to control, soy oil fed animals (Figure 10.3). Coconut fed litters also revealed topographically expanded terminal fields at PND28 and PND42, strongly suggesting that an omega-3 restriction, and the subsequent reduction of DHA levels, produced abnormal connections in the rodent visual system (de Velasco et al., 2012). This could be due either to a slowdown in axonal elimination of transitory synapses or to unspecific sprouting as a result of a decrease in DHA-induced synaptic stabilization mechanisms. The latter mechanism was in part confirmed by a decrease in phopho-GAP43 (pGAP-43) content observed in the visual layers of the SC (de Velasco et al., 2012). The phosphorylated form of GAP-43 (pGAP-43) protein has been involved in hippocampal synaptic plasticity and in the stabilization of developing synapses (Schaechter and Benowitz, 1993, Mendonca et al., 2010). The disturbance in visual system development induced by omega-3/DHA reduction was not confined to the ipsilateral retinocollicular pathway. During normal development, most of the development of retinogeniculate segregation has finished by PND28 and terminal zones from each become restricted to eyespecific layers of the dorsal lateral geniculate nucleus

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ROLE OF OMEGA-3 ON DEVELOPMENT OF CENTRAL VISUAL CONNECTIONS

115

Normal omega-3 @ PND 13

Retinocollicular connections @ PND 0

Restricted omega-3 /DHA @ PND 13

FIGURE 10.3 Topographical specification is impaired by an omega-3/DHA nutritional restriction. Control groups display a normal topographical restriction at postnatal Day 13, in which specific groups of retinal ganglion cells axons converge to non-overlapping post-synaptic sites. However, omega-3/DHA nutritional restriction resulted in expanded retinal projections (de Velasco et al., 2012).

(Guido, 2008). However, under omega-3/DHA restriction, the ipsilateral and contralateral eye specific zones were still expanded at PND 28 in relation to control animals, suggesting that the disturbances in the fine tuning of the visual system topography are a common finding in visual system development (de Velasco et al., 2012) (Figure 10.4). Since DHA is related to several mechanisms that influence neurogenesis, apoptosis, and cell differentiation, we asked whether the disturbances in retinal connectivity in visual nuclei would be reversed by a DHA supplementation protocol. This was achieved by the oral administration of fish oil, a source of DHA, and eicosapentaenoic acid (EPA). Fish oil supplementation during three weeks (from PND 7 to 28) was able to completely reverse the effects of a gestational/neonatal DHA deprivation, resulting in normal densities of terminal fields across the superior colliculus (de Velasco et al., 2012). Thus, the results clearly show that disturbances in visual connectivity can be restored by an adequate amount of DHA intake during the critical period of development.

The results described by de Velasco and colleagues are consistent with a general delay in development which results in errors in topographical fine tuning of retinal connections (de Velasco et al., 2012). To directly address whether other aspects of development could also exhibit a similar delay, we made a series of retinal lesion experiments which are suitable to access the critical period limits. As described earlier (Serfaty et al., 2005), restricted retinal lesions to one eye induce a rapid sprouting of axons from the intact eye that converge to the same aspect of the superior colliculus contralateral to the lesioned eye. It has been shown that after the third postnatal week, a slow plasticity is observed only within weeks or months (Serfaty et al., 2005). Under normal conditions virtually no sprouting of intact axons can be detected one week after a retinal lesion made at PND21, which characterizes the end of the collicular critical period (Campello-Costa et al., 2000, Campello-Costa et al., 2006). However, animals depleted of DHA still displayed a vigorous plastic response to a retinal lesion (Figure 10.5) suggesting that among other things, DHA restriction altered the

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10. LOW OMEGA-3 FATTY ACIDS DIET AND THE IMPACT ON THE DEVELOPMENT OF VISUAL CONNECTIONS

FIGURE 10.5 Nutritional restriction of omega-3/DHA increases

FIGURE 10.4 Omega-3/DHA nutritional restriction expands retinogeniculate terminal fields. Animals at PND28 from control (omega-31 ) and restricted (omega-32 ) groups received a unilateral injection of HRP. In the omega-31 control group, terminal labeling revealed an equal distribution of the ipsilateral terminal field and the corresponding gap in the contralateral projection. In the omega 32 group, however, ipsilateral labeling is expanded in relation to the contralateral gap. Quantitative measurements corroborated the qualitative findings, revealing significant differences (p , 0.01; n 5 4 for omega-31 and n 5 5 for omega-32 group). Scale bar 5 100 μm (adapted from De Velasco et al., 2012).

duration of the critical period (de Velasco et al., 2012). Since DHA is involved in transcription of neurotrophic factors such as BDNF (Wu et al., 2004) and that neurotrophin reduction, as well as dark rearing (Fagiolini et al., 1994, Viegi et al., 2002), delays the cortical critical period, it seems reasonable to suppose that a reduction

lesion-induced plasticity revealing an extended critical period in retinocollicular development. Dark field photomicrographs of coronal sections through the visual layers of the SC of control and omega-3 restricted diets. A restricted lesion to the temporal retina of the left eye was performed at PND21. The anterograde labeling of the intact uncrossed retinocollicular projection from the right eye was made at PND 28. (A) Omega-31 group revealed virtually no detectable labeling at the subpial aspect of the lateral SC, since the critical period closes by PND21. On the other hand, the omega-32 group. (B) Displayed an increased innervation density at the same subpial aspect of the lateral SC (arrows), in register with the retinal lesion in the opposite eye. (C) Optical density analyses confirmed a pronounced sprouting reaching the lateral surface of the SC in response to a temporal retinal lesion in the contralateral eye. (p , 0.01 and 0.05; n 5 4 for omega-31 group; n 5 6 for omega-32 group). Scale bar 5 100 μm (adapted from De Velasco et al., 2012).

in BDNF content might explain the developmental delay of retinofugal connections found in this study. In conclusion, omega-3 nutritional restriction directly impacts DHA availability within visual nuclei and dramatically alters the time course of topographical refinement and critical period windows. The consequences of those influences on such a precisely regulated time-course may explain the dysfunctions observed in DHA deficient children, who display reduced visual acuity (Birch et al., 1998) and impaired cognitive performance (Wurtman, 2008, Hoffman et al., 2009). Thus, an

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

REFERENCES

improved understanding of the role of essential fatty acids in visual system development is, thus, mandatory for the establishment of adequate dietary requirements for these essential lipids during early postnatal life.

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11 The Effects of Omega-3 Polyunsaturated Fatty Acids on Maternal and Child Mental Health Michelle Price Judge, Ana Francisca Diallo and Cheryl Tatano Beck INTRODUCTION

THE ROLE OF OMEGA-3 FATTY ACIDS IN NEUROTRANSMISSION

Nutrition plays an integral and complex role in the brain. Nutrients provide structural substrates and serve as cofactors in many biological reactions. Docosahexaenoic acid (DHA, 22:6, n-3) and eicosapentaenoic acid (EPA, 20:5, n-3) are omega-3 fatty acids that are the most prominent fatty acids in the brain and retinal tissue. Therefore, these nutrients are of great interest in the area of cognitive and mental health research. The polyunsaturated nature of DHA makes it highly fluid. As new growth and synaptic communications are being formed in the brain, fatty acids with fluidity are highly desirable to ensure adequate neurotransmission and growth. The incorporation of DHA into biological tissue, however, is dependent upon a complex chain of events involving other nutrients, and these interactions are not completely understood. Maternal and child mental health problems represent a significant public health issue worldwide (Ramakrishnan et al., 2009). Supplemental nutrition with polyunsaturated fatty acids such as omega-3 fatty acids has received recent attention as an alternative intervention for the prevention and management of mental health problems in women and children. The first part of this chapter will discuss the role of omega-3 fatty acids in neurotransmission as it relates to mood regulation and maternal postpartum depression. The second part of the chapter will discuss the role of omega-3 fatty acids on child mental health, including cognition, behavioral, and mood disorders like attention deficit hyperactivity disorder (ADHD), childhood depression, and autistic spectrum disorders (ASDs).

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00011-9

The omega-3 fatty acid DHA (22:6 omega-3) has a central role in regulating the biophysical properties of neural membranes (Wisner et al., 2006). Based upon animal studies, specific regions of the brain, including the cerebral cortex, synapses, and retinal rod photoreceptors, have a particularly high DHA concentration (Bowen and Clandinin, 2002; Sarkadi-Nagy et al., 2003). Studies conducted in animals provide evidence for disturbances in brain development of offspring relating to DHA deficiency induced during the gestational period (Aı¨d et al., 2003; Auestad and Innis, 2000; Hamano et al., 1996; Innis and De La Presa Owens, 2001; Levant et al., 2004). DHA deficiency during gestation in rats has been found to modify catecholamine biosynthesis in the brain, induce behavioral disturbances, and decrease learning ability in their offspring (Takeuchi et al., 2002). These findings are of particular interest because they link DHA deficiency to subsequent altered brain development and impaired functional status. Along the same line of research, Aı¨d et al. (2003) investigated the influence of an omega-3 deficient diet on cholinergic neurotransmission in the rat frontal cortex and hippocampus. The omega-3 deficient rats had significantly reduced DHA content in both brain regions. In the hippocampus, DHA deficient rats had 72% higher acetylcholine release than controls. An intact septo-hippocampal cholinergic system is crucial for learning and memory, and changes in acetylcholine would result in reduced performance. Takeuchi et al. (2002) investigated the impact of an omega-3 fatty acid deficient diet versus a diet rich in

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DHA during the gestational period in pregnant rats on the cerebral catecholamine content in the rat pups. The n-3 deficient rat pups had lower noradrenaline in the cerebral cortex, hippocampus and striatum compared to pups of DHA-fed dams. Additionally, dopamine contents in the omega-3 deficient rats were also significantly lower than in the DHA group. Learning tests conducted in the two rat groups indicated significantly lower learning scores in the omega-3 deficient group. Levant et al. (2004) investigated the impact of an omega-3 deficient diet versus a control diet during the gestational period on brain DHA content and dopamine-related behaviors in rat pups. Additionally, Levant et al. aimed to determine if the provision of fish oil in the diet would result in any changes in brain DHA content or behavior. The deficient pups had 80% less brain DHA at full maturity and demonstrated alterations in adult behavior compared to the control groups. The remediation diet resulted in the restoration of DHA levels and behaviors comparable to controls. The behaviors of note included catalepsy and amphetamine-stimulated locomotor activity, which have been previously associated with decreased brain dopamine, and increased dopamine receptors in the nucleus accumbens. In the U.S.A. and Canada, maternal DHA intake during pregnancy is far below the current recommended level of 200 mg/d (Denomme et al., 2005; Innis and Elias, 2003; Judge et al., 2003a,b; Lewis et al., 1996), which raises concerns for maternal health and infant neurodevelopment, because developmental advantages have been reported for infants of mothers who consume DHA during pregnancy (Colombo et al., 2004; Judge et al., 2007a,b). In fact, the last trimester of pregnancy is a critical interval for fetal neurological development. During this time, DHA accumulates in neural tissue at an accelerated rate (Fox, 1998). Reports of maternal DHA intake during pregnancy indicate that intakes are generally far below the recommended level, which suggests that many infants are at risk for associated impairments in development (Cheruku et al., 2002; Judge et al., 2003a; Olsen and Secher, 2002). Compounding the problem of reduced maternal intake of DHA, the overall intake of fatty acids in the omega-6 fatty acids (n-6) family is typically high in Western industrialized countries such as the U.S. Such high intakes of n-6 fatty acids are related to an abundance of processed and fried foods containing plant oils with linoleic acid (LA, 18:2, n-6). The omega-3 fatty acid linolenic acid (LNA, 18:3, n-3) and the omega-6 fatty LA both compete for the n-6 desaturase, and excessive intake of LA can inhibit the further desaturation and elongation of LNA toward DHA synthesis (Simopoulos 2002). Investigations carried out into high intakes of omega-6 fatty acids suggested that an

increased ratio of omega-6: omega-3 fatty acid intake might be linked to an increased risk of mental health issues for the mother and infant (Bodnar and Wisner, 2005; Kris-Etherton et al., 2000). In 1999, an expert panel including the International Society for the Study of Fatty Acids and Lipids (ISSFAL), the U.S. National Institute on Alcohol Abuse and Alcoholism, the U.S. Office of Dietary Supplements at the National Institutes of Health, and the Center for Genetics, Nutrition, and Health, was convened to formulate recommendations for dietary intakes of omega-3 and omega-6 fatty acids. The recommendation for DHA during pregnancy put forward by the expert panel was 200 mg/day (Koletzko et al., 2008). In addition, the panel recommended 2.22 g/day of LNA for all adults, and an upper limit was established for LA of 6.67 g/day.

DHA AND MATERNAL MENTAL HEALTH Postpartum Depression Postpartum depression is the most common complication of childbirth, affecting 13
15% of pregnant women in the U.S., and is a major public health problem (Gavin et al., 2005; Wisner et al., 2006). In a U.S. national survey, Listening to Mothers II, 63% of the women surveyed screened positive for elevated postpartum depressive symptoms with the Postpartum Depression Screening Scale (PDSS) (Beck et al., 2011). Furthermore, 25
50% of women diagnosed with postpartum depression have episodic events for six months or more (O’Hara and Swain, 1996). Postpartum depression is a universal phenomenon, affecting women in countries throughout the world (Oates et al., 2004). A striking characteristic of this mood disorder is how covertly it is suffered (Spinelli, 1998). Because postpartum depression is a term applied to a wide range of postpartum emotional disorders, women may be misdiagnosed. In the major depressive episodes of postpartum depression, which last at least two weeks, women experience either depressed mood or a loss of interest or pleasure in activities. Current DSM-IV-TR guidelines (DSM-IV-TR, 2000) outline that, in addition to depressed mood or loss of interest or pleasure in activities, women need to have three or more other symptoms including insomnia, psychomotor agitation or retardation, fatigue, feelings of worthlessness or excessive or inappropriate guilt, inability to concentrate or suicidal thoughts (American Psychiatric Association, 2000; First et al.,1997) The importance of preventing, diagnosing, and treating postpartum depression is underscored by the findings that postpartum

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depression has significant adverse effects on the cognitive and emotional development of children (Beck, 1998; Hay et al., 2001; Hay et al., 2003).

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of postpartum depression (Freeman et al., 2006; Peet and Stokes, 2005). Future research should focus on dose
response relationships in the treatment of postpartum depression and expand investigations to include baseline DHA status (Sontrop and Campbell, 2006).

Omega-3 Fatty Acids in Postpartum Depression Although multiple reports have provided evidence of an inverse relationship between DHA intake and depression (Sontrop and Campbell, 2006), few investigations have focused specifically on postpartum depression (De Vriese et al., 2003; Hibbeln, 2002; Miyake et al., 2006; Otto et al., 2003). Hibbeln et al. (2002) conducted a meta-analysis of 41 studies that used the Edinburgh Postpartum Depression Scale (EPDS). These investigations reported that the DHA content of mothers’ milk and seafood consumption rates were associated with a lower prevalence of postpartum depression. Otto and colleagues (2003) investigated plasma phospholipid DHA in 112 women at delivery and at 32 weeks postpartum. The EPDS was given to the women at the 32-week timepoint to assess postpartum depression. There was found to be an inverse relationship between DHA status and depressive symptoms. De Vriese et al. (2003) conducted a similar investigation of maternal DHA status immediately following delivery. DHA and total omega-3 fatty acids were significantly lower in the women who developed postpartum depression compared to the women who did not. Conversely, Miyake and colleagues (2006) investigated the risk of postpartum depression related to dietary fatty acid intake in 865 Japanese women. Again, the EPDS was used to evaluate postpartum depression and diet history questionnaires were self-administered to measure dietary fatty acid intake. No significant relationships were reported between dietary fish consumption or omega-3 fatty acid intake and postpartum depression. Likewise, Browne et al. (2006) investigated fish consumption and plasma DHA status after birth in relation to postpartum depression diagnosed using the Composite International Diagnostic Interview. No significant findings were reported between maternal fish consumption during pregnancy or maternal DHA status following delivery and depressive symptoms in the postpartum period. Evidence exists to support the notion that DHA status may have a protective effect in the prevention of postpartum depression (De Vriese et al., 2003; Hibbeln, 2002; Otto et al., 2003). However, further investigations are necessary to better define this relationship in light of some of the more recent conflicting reports (Browne et al., 2006; Miyake et al., 2006). To date, although DHA appears to help prevent postpartum depression, it has not been found to be beneficial in the treatment

OMEGA-3 FATTY ACIDS AND CHILD MENTAL HEALTH Neurodevelopmental Outcomes: Infancy through Childhood Starting in the last trimester of pregnancy, major neural development continues during the first five years of life. Omega-3 fatty acids and especially DHA are critical for the central nervous system and early brain development (Hallahan and Garland, 2005). In fact, omega-3 fatty acids are required for brain maturation due to their effect on the neuronal membrane structure and regulation of neurotransmission (Crawford, 1992; Fontani et al., 2005; Kidd, 2007). However, omega-3 fatty acids do not occur naturally in the body; they must be obtained from the diet. Adequate DHA intake is especially important during pregnancy, accumulating in fetal tissue at a high rate during the third trimester (Fox, 1998). This period corresponds to significant growth of the fetal brain and an increase in the amount of DHA. Infancy Based upon findings from animal studies, specific regions of the brain, including the cerebral cortex, synapses, and retinal rod photoreceptors have particularly high levels of DHA concentration (Bowen and Clandinin, 2002; Sarkadi-Nagy et al., 2003). These animal studies also provide evidence for disturbances in brain development of offspring relating to DHA deficiency during the gestational period (Aı¨d et al., 2003; Auestad and Innis, 2000; Hamano et al., 1996; Innis and De La Presa Owens, 2001; Levant et al., 2004). In infants, Farquharson et al. (1995) showed that infants who had received breast milk known to contain DHA had significantly higher levels of DHA in the cerebral cortex than those fed formula containing no DHA. Hence, maternal DHA levels during the gestational period are critical to fetal neurodevelopment as the process of cellular differentiation occurs during the fetal period and has been largely completed by birth (Kolb and Whishaw, 2003). As mentioned earlier in this chapter, poor maternal DHA intake compounds this problem. Given the concerns regarding DHA deficiency in pregnancy in the general population, increased clinical attention has focused on evaluating the efficacy of

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maternal DHA consumption during pregnancy in improving infant developmental outcomes with results supporting this notion (Colombo et al., 2004; Helland et al., 2001; Helland et al., 2003; Judge et al., 2007a,b; Makrides et al., 2010). Helland et al. (2001) and Colombo et al. (2004) have investigated infant cognitive function (recognition memory and habituation, respectively) relating to maternal DHA supplementation. Helland et al. (2001) investigated recognition memory at 6 and 9 months of age and reported no advantages for cod liver oil supplementation versus placebo. Judge et al. investigated maternal DHA consumption and infant problemsolving abilities at 9 months of age (Judge et al., 2007b) and visual acuity at 4 and 6 months of age (Judge et al., 2007a) and reported significantly better problem-solving and visual acuity in infants of mothers supplemented with DHA compared to the placebo group. In contrast, Makrides et al. (2010) evaluated infant outcomes using the Bayley Scales of Infant Development in women supplemented with DHA compared to a placebo group and reported no group differences. Although these study results are mixed (Colombo et al., 2004; Helland et al., 2003; Judge et al., 2007a,b), overall findings point to maternal DHA supplementation as being beneficial to infant information processing and attention in the general population. A recent meta-analysis of eleven randomized control trial studies assessed the effects of omega-3 fatty acids (Gould et al., 2013). Although the authors concluded that the existing clinical evidence on the effects of maternal omega-3 fatty acid supplementation in infant neurodevelopmental outcomes is inconclusive, they reported higher developmental standard scores of children between the ages of 2 and 5 years whose mothers received omega-3 supplementation, compared to the control group (mean difference: 3.92; P 5 0.01) (Gould et al., 2013). Childhood To date, a number of studies have investigated the effects of omega-3 fatty acid supplementation on cognitive functions of children whose mothers had received omega-3 supplements during pregnancy or of children who were taking omega-3 supplementation (Colombo et al., 2004; Helland et al., 2003; McNamara et al., 2010; Milte et al., 2011; Richardson and Montgomery, 2005; Sinn and Bryan, 2007; Sinn 2008). While some investigations have reported no significant effects of the use of omega-3 fatty acid supplementation on outcomes in childhood, there is increasing evidence in the literature that supports the notion of positive cognitive, learning, and behavioral outcomes in children who were exposed to maternal supplementation during pregnancy or who were supplemented with omega-3 fatty acids during childhood alone, compared to those who were not (Frensham et al., 2012).

The possible link between omega-3 fatty acids and cognitive function in children was investigated by Helland et al. (2003) as an outcome of omega-3 supplementation of the mothers during pregnancy. In a subset of a study cohort, Helland et al. (2003) showed improved IQ at 4 years of age in those children whose mothers consumed cod liver oil during pregnancy and lactation compared to controls. Likewise, Colombo et al. (2004) showed better infant performance on habituation and free-play attention tasks in infancy and less distractibility during the second year related to maternal DHA intake during pregnancy. Omega-3 supplementation during childhood has been reported as having a direct impact on developmental outcomes in childhood. In a double-blind, placebo-controlled trial, Richardson and Montgomery (2005) investigated the effects of omega-3 fatty acids on children between the ages of 5 and 12 years with developmental coordination disorder. The study findings reported a significant improvement in the intervention group compared to the control group in reading (z 5 2.87, p , 0.004) and spelling (z 5 3.36, p , 0.001) (Richardson and Montgomery, 2005). In a similar investigation, Richardson et al. (2012) studied the effects of DHA supplementation on 7 to 9 year old children in reading, working memory, and behavior. Although DHA supplementation had no significant effect on the reading skills of the entire sample, a significant improvement was noted in the reading skills of children who were underperforming in reading at baseline compared to the controls (p , 0.04 in the subgroup with initial reading ,20th centile) (Richardson et al., 2012). In summary, nutritional intakes, as well as other environmental factors, play a critical role in the structural and functional development of the brain (Levitt, 2003). Due to their effects on neurodevelopment, there is a growing body of evidence supporting a potential association between omega-3 fatty acid levels and brain development during pregnancy and childhood. Evidence points to better cognitive, learning, and behavioral outcomes from omega-3 supplementation given in the fetal period to their pregnant mothers or in childhood. However, because levels of maternal intake of omega-3 fatty acids during pregnancy are far below those of expert recommendations in the U.S., an increased effort is necessary to ensure adequate intake of omega-3 fatty acids during pregnancy and early childhood.

Childhood Developmental Disorders Pharmacological interventions for childhood developmental disorders are often associated with unwanted or unknown side effects. Given the

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disadvantages of pharmacological interventions, omega-3 fatty acids have been receiving increased attention for their potential role as adjunctive or alternative therapy in childhood mental health, especially for the treatment of ADHD, depression, and ASDs. Although the exact role of omega-3 fatty acids on children’s behavioral problems still remains unclear, findings from clinical trials point to levels of omega-3 fatty acid intake as a significant factor in the occurrence of these disorders. Attention Deficit Hyperactivity Disorder The DSM-V defines ADHD as a multi-faceted pattern of behaviors characterized by inattention, hyperactivity, and impulsivity (Burns et al., 2013). ADHD is the most common childhood developmental disorder that affects approximately 4% to 15% of school-age children in the U.S.A. (Richardson, 2006). A significant number of children diagnosed with ADHD are treated with complementary or alternative medicines. These alternative therapies, used mostly in conjunction with conventional treatments (pharmaceutical, behavioral, and psychosocial) include vitamins, minerals, and omega-3 fatty acids (Sarris et al., 2011). Multiple investigations have looked at the role of omega-3 fatty acids in ADHD and its comorbidities. Since omega-3 fatty acids are essential to the structural and functional development of the brain, it was hypothesized that deficiencies in omega-3 were linked to ADHD symptom domains and behavioral comorbidities (Antalis et al., 2006; Gow et al., 2013a,b). Associated symptoms include impaired affective and emotional behaviors such as impulsivity, as well as aggression, depression, anxiety, and conduct-disorder traits. There is increasing evidence linking behavioral and attention issues specific to ADHD with omega-3 fatty acid deficiency. Three longitudinal studies conducted by Llorente et al. (2003), Krabbendam et al. (2007) and Hibbeln et al. (2007) have reported that DHA does play a specific role in the disorder, and DHA concentration in early life has an inverse association with behavioral problems occurring subsequently in late childhood (Hibbeln et al., 2007; Krabbendam et al., 2007; Llorente et al., 2003). Investigations carried out by Gow and colleagues (2013), for instance, have reported that children with lower plasma and blood levels of omega-3 fatty acids, especially DHA and EPA, present with ADHD-related symptoms such as abnormal emotion processing, as well as anti-social traits (Gow et al., 2013a). A recent meta-analysis presented a compelling case that corroborated the findings of earlier investigations regarding the link between omega-3 fatty acids and ADHD symptoms (Frensham et al., 2012). These investigations evaluated supplementation with omega-3

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fatty acids, in particular DHA and EPA, in children with ADHD. The findings reported moderate to significant improvements in ADHD-related behavioral and cognitive problems such as inattention, restlessness/ impulsiveness, oppositional behavior, and hyperactivity (Johnson et al., 2009; Milte et al., 2011; Sinn and Bryan, 2007; Sinn, 2008; Stevens et al., 2003). In contrast, some studies have reported little to no effects of omega-3 supplementation on children with ADHD-related behaviors. One such investigation evaluated the efficacy of DHA and EPA supplementation in children diagnosed with symptoms of ADHD (n 5 40) and reported no significant differences between the treatment and control groups on ADHD symptoms (Hirayama et al., 2004). In a similar investigation, Voigt et al. (2001) supplemented children between 6 and 12 years of age with 345 mg/day of DHA over four months and reported no significant improvement in ADHD symptoms or parent-rating scales. In summary, the balance of the literature is supportive of a plausible link between omega-3 deficiency and the benefit of supplementation with respect to the frequency and severity of ADHD-related symptoms. However, experimental evidence also exists refuting the link between omega-3 fatty acids in the treatment of ADHD, making results inconclusive at this point (Gillies et al., 2012; Richardson, 2006; Sarris et al., 2011). Future studies are therefore warranted to expand knowledge on the mechanism by which omega-3 fatty acids affect behavioral disorders such as ADHD in children. Childhood Depression Evidence for a possible causal effect of omega-3 in behavioral disorders, including mood disorders, appears to have become stronger over the years. Investigations utilizing adjunctive treatment with omega-3 have yielded results supporting the efficacy of omega-3 in reducing symptoms of unipolar depression in children (Nemets et al., 2002; Nemets et al., 2006; Peet and Stokes, 2005; Su et al., 2003), bipolar disorder (Stoll et al., 1999), and borderline personality disorders (Zanarini and Frankenburg, 2003). Major depressive disorder in children is a rising public health issue in the U.S., especially since these disorders are intimately associated with poor psychological outcomes such as increased risk of suicide and substance use (Nemets et al., 2006). It is estimated that approximately 2
4% of children in the U.S. are diagnosed with major depressive disorders (Nemets et al., 2002). Since a number of studies have suggested the positive effects of omega-3 fatty acids in depression in the adult population, Nemets and colleagues (2002) examined the potential treatment effects of EPA/DHA supplementation in children aged between 6 to 12 years of age

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suffering from major depressive disorder. Depressive symptoms were rated by the Children’s Depressive Rating Scale (CDRS), the Children’s Depression Inventory (CDI), and the Clinical Global Impression (CGI) scale. Seven out of ten children in the intervention group had a 50% decrease in CDRS scores. Also, the treatment group had significantly lower self-rating CDI scores compared to the control group (p , 0.001) (Nemets et al., 2002). Like many investigations assessing the effectiveness of omega-3 fatty acids as an adjunct therapy in major depressive disorders in adults, these studies support the efficacy of omega-3 supplementation in improving symptoms of childhood depression and mood disorders in general. Clearly, more studies are needed to explore the effects of omega-3 fatty acids on prepubertal childhood depression.

Autistic Spectrum Disorders Autistic spectrum disorders (ASDs) constitute a heterogonous continuum of neurodevelopment disorders characterized by impaired communication, social functioning, and repetitive behaviors (Bell et al., 2004). ASDs have become a public health issue in the U.S. with a prevalence of approximately 66 per 10,000 children in the U.S. being affected (James et al., 2011). The etiology of ASDs is still unclear; fewer than 25% of cases have a known cause. The use of omega-3 fatty acids as an adjunct therapy has also been explored in ASDs (Baker, 1985; Bell et al., 2000; Bell et al., 2004; Sinn et al., 2010). Since 2001, evidence has emerged supporting the use of DHA and EPA for ASDs. Bradstreet and Kartzinel (2001) found that nearly 100% of the infants with ASD in their study also presented with deficiencies in omega-3 fatty acids. Similarly, reports of a study conducted by Vancassel et al. (2001) indicated that children with autism had approximately 20% lower than normal DHA levels. In addition, evidence suggested that omega-3 fatty acids can reduce symptoms of inflammatory bowel disorders that are frequently found in children with autism and are even suspected be part of its etiology (Belluzzi, 2002). Amminger et al. (2007) conducted a double-blind, randomized controlled trial of 13 children with ASDs between the ages of 5 to 17 years. Participants in the study displayed symptoms of severe tantrums, aggression, and self-injurious behaviors. The study’s intervention consisted of DHA/EPA supplementation, and the results indicated significant improvement for hyperactivity (Amminger et al., 2007). A similar pilot study was conducted by Bent et al. (2011). In this randomized controlled trial, 27 children between the ages of 3 to 8 years, diagnosed with ASDs, received omega-

3 fatty acid supplementation for 12 weeks. Although the findings were not statistically significant, the expert researchers reported a lower hyperactivity in the intervention group compared to the control group that was meaningful from a clinical perspective (Bent et al., 2011). Since studies have reported that symptoms associated with ASDs are significantly related to omega-3 fatty acid deficiency, supplementation of these key nutrients may lead to improvements in ASDs-related symptoms. To date there is no strong clinical evidence supporting the efficacy of omega-3 fatty acid supplementation for improving symptoms of ASDs. Further investigations are currently underway to expand the understanding of the important role that omega-3 fatty acids might play in the etiology and as an adjunctive therapy in ASDs.

CONCLUSION Mental health disorders disproportionally affect women, children, and adolescents in the U.S. (Kessler, 2003). The most current reports emphasize the need for further research into the role of omega-3 in maternal and child mental health. There is growing evidence suggesting a possible link between deficiencies in omega-3 fatty acids and psychopathology affecting the mothers’ and infants’ health. Consumption of omega-3 fatty acids as a supplement has been shown to reduce depressive symptoms among women during the postpartum period as well as children and adolescents. Studies have also demonstrated that omega-3 fatty acid supplementation is associated with improvement in cognitive function in children, especially those with initial learning disabilities. Improvements of outcomes have also been suggested in children diagnosed with depression, ASD, and ADHD-related symptoms such as inattention, aggressive and violent behaviors, and unemotional traits. While promising, findings of clinical trials using omega-3 supplementation have been inconclusive, thus highlighting the need for further investigation. Differences in experimental methodologies, supplementation dosages, fatty acid composition/source, and specific outcome measures vary between investigations. Additionally, larger scale investigations are warranted (Sinn et al., 2010). Essential fatty acids play a key role in maintaining a healthy central nervous system: serving as structural components of brain tissue; altering neurochemical properties of membranes involved in neurotransmission; functioning as precursors in the formation of neurotransmitters; and serving directly as neurotransmitters. A well-balanced diet comprised of adequate

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

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omega-3 fatty acids will help ensure optimal brain development and function. Therefore, efforts must be made to expand our current knowledge of the effects of omega-3 fatty acids on mental health and to ensure adequate intake of these nutrients for women during pregnancy, in the postpartum period, and during child development.

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C H A P T E R

12 Pain as Modified by Polyunsaturated Fatty Acids Shogo Tokuyama and Kazuo Nakamoto INTRODUCTION Nutrients provide an energy source to carry out and maintain physiological activities in the body. Lipids are currently generating lots of attention as a major energy source in the brain, in addition to glucose and protein. In particular, a continuous supply of lipids is required for the metabolism, differentiation, and development of cells (Park and Vasko, 2005). Fatty acids produced by the hydrolysis of lipids are not only a nutrient for cells and a component of cell membranes, but also a lipid-soluble signal molecule that plays a vital role in a wide variety of physiological functions (Horrocks and Farooqui, 2004). Fatty acids are classified as saturated fatty acids with no double bonds or as unsaturated fatty acids with double or triple bonds. The latter are divided into monounsaturated fatty acids with only one double bond and polyunsaturated fatty acids (PUFA) with two or more double bonds. PUFA are further divided into omega-3 fatty acids represented by α-linolenic acid (C18:3), eicosapentaenoic acid (EPA, C20:5), and docosahexaenoic acid (DHA, C22:6) and omega-6 fatty acids represented by linoleic acid (C18:2), γ-linolenic acid (C18:3), and arachidonic acid (C20:4) (O’Sullivan, 2009; Hashimoto and Hossain, 2011) (Figure 12.1). In the human body, the majority of saturated and monounsaturated fatty acids are synthesized from carbohydrates and proteins in adipose tissue. These fatty acids are the major components of animal fat and are used for energy production and for the construction of cell membranes (Hwang and Rhee, 1999). On the other hand, PUFA function as a cellular mediator and intrinsic ligand for nuclear receptors in addition to being core components of cell membranes (Dyall and Michael-Titus, 2008; Jump and Clarke, 1999). A deficiency of linoleic acid and α-linolenic acid, both of Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00012-0

which are PUFA, causes various disorders including skin disorders, infertility, and reduced immunity. In particular, omega-3 fatty acids are major phospholipids of cell membranes in the brain (Wall et al., 2010), retina, and sperm, and they are essential for the development, structure, and function of the brain (Hashimoto and Hossain, 2011). Other physiological actions of omega-3 fatty acids are anti-oxidation (Kim and Chung, 2007), anti-inflammation (Wall et al., 2010), and the protection of the cardiovascular (Saravanan et al., 2010) and nervous systems (Lauritzen et al., 2000). In addition, clinical studies have reported the prevention of cardiovascular events by omega-3 fatty acids and the efficacy of omega-3 fatty acid in patients with attention-deficit hyperactivity disorder (Richardson and Puri, 2002), neurodegenerative disease such as Alzheimer’s disease (Jicha and Markesbery, 2002), and psychiatric disorders such as depression (Logan, 2003). These studies have revealed various roles of omega-3 fatty acids in the regulation of biological function (Saravanan et al., 2010). Only a trace amount of omega-3 fatty acid is produced from α-linolenic acid in the body, with the rest coming from food. EPA and DHA are particularly popular supplements often found in health foods, and because of their safety, they are currently attracting lots of attention as a functional lipid. The effects of daily consumption of DHA and arachidonic acid in the human brain were assessed in a clinical study. The study investigated 14 healthy volunteers using synthetic [1-11C] arachidonic acid or [1-11C] DHA as a positron emission tomography (PET) tracer and showed that the daily consumption of arachidonic acid and DHA was 17.8 and 4.6 mg/1500 g brain, respectively (Umhau et al., 2009). The amount of DHA consumption in this study corresponds to 2.5
5% of the combined average daily intake of EPA

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Fatty acids

Unsaturated fatty acids

Saturated fatty acids

Polyunsaturated fatty acids

Monounsaturated fatty acids

Omega-6 fatty acids

Omega-3 fatty acids

Linoleic acid C18:2ω-6

Alpha-linolenic acid C18:3 ω-3 Δ 6-Desaturase

Gamma-linolenic acid Elongese Dihomo-gamma-linolenic acid C20:3 ω-6

Eicosatetraenoic acid Δ 5-Desaturase

Arachidonic acid C20:4 ω-6

Eicosapentaenoic acid (EPA, C20:5 ω-3)

COX 5-LOX

Docosapentaenoic acid (DPA) Prostaglandins, thromboxanes, leukotrienes

Elongese Docosahexaenoic acid (DHA, C22:6 ω-3)

FIGURE 12.1 The classification of fatty acids.

and DHA in the United States. Interestingly, the consumption of arachidonic acid was reportedly increased, while that of DHA was reduced in the brain of patients with Alzheimer’s disease compared with healthy individuals (Umhau et al., 2009). Because healthy brain regularly consumes DHA and arachidonic acid and because these fatty acids become imbalanced in a disease state, it is important to balance the levels of these fatty acids through daily meals.

FACTORS INVOLVED IN THE SUPPLY AND PHYSIOLOGICAL FUNCTION OF FATTY ACIDS IN THE BRAIN Blood
Brain Barrier Lipids constitute 55% and 30% (dry weight) of the cerebral medulla and cortex, respectively (Figure 12.2). In particular, phospholipids constitute approximately 25% of these brain structures (Freeman et al., 2006). Approximately 35% of fatty acids in the phospholipid population are PUFA, containing a large amount of

DHA (17%) and arachidonic acid (12%) (Wainwright, 2002). Because the blood
brain barrier (BBB) is not permeable to triglycerides and phospholipids, fatty acids are transported into the brain as nonesterified fatty acids, or free fatty acids, bound to albumin owing to the competition between the hydrophobic region of albumin and the hydrophobic region of the BBB endothelial cell (Dhopeshwarkar and Mead, 1973). The BBB is made of endothelial cells, pericytes, and astrocytes, and the microvascular endothelial cells (Hawkins and Davis, 2005) in the brain are tightly held together by a tight junction (Benarroch, 2011) which blocks drugs from entering the brain while maintaining a stable chemical environment. On the other hand, the BBB is equipped with unique transportation systems that assist important molecules with moving into the brain. For example, these unique transportation systems enable low molecular weight molecules, such as glucose, and certain amino acids can enter the brain, despite being non-lipid soluble. In addition, fatty acids are effectively transported through the BBB into the brain by fatty acid transport protein (FATP), fatty acid transporter (FAT), and fatty acid

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Saturated acids or unsaturated fatty acids

Blood vessel FA alb

133

FA alb

FA alb FA

Fatty acid transporter protein (FATP, CD36)

FA

alb

FA

Plasma membrane

GPCR

TLRs

(GPR40)

(TLR4)

FA

FA FA

FA Fatty acid binding protein (FABP)

cPLA2

iPLA2

↓ Arachidonic acid ↓ PGs, LTs ↓ Inflammatory response

↓ DHA ↓ Resolvins, NPD1 ↓ Anti-inflammatory

Endoplasmic reticulum Peroxisome Mitochondoria

Nucler Nucler

FA

Signal pathways

FA FA Gene expression

PPARs PPARs RXRs RXRs β-oxidation

Gq

GPCRs : G-protein coupled receptors;

TLRs: toll like receptors

FIGURE 12.2 Schematic representation of fatty acid uptake into the brain.

binding protein (FABP), which are present in brain microvascular endothelial cells (Mitchell et al., 2011).

Fatty Acid Transporter Protein The fatty acid transporter protein (FATP, CD36) family of transmembrane proteins is composed of 6 isoforms, and they have an extracellular binding site for fatty acid, intracellular acyl-CoA synthase activity site, and an ATP binding domain. Fatty acid translocase/cluster of differentiation 36 (FAT/CD36), a member of FATP, is an integral membrane glycoprotein isolated from rat adipose tissue in 1993 (Hirsch et al., 1998). The protein has an extracellular glycosylation site, and the C- and N-terminal ends are located inside the cell. FAT/CD36 is not only a major fatty acid transporter in the BBB, but is also present in other tissues (Abumrad et al., 2005). The affinity of FAT/CD36 for fatty acids is in the nanomolar range (Baillie et al., 1996), and oxidized low-density lipoprotein (Puente Navazo et al., 1996) and thrombospondin. Dawson et al. (1997) also serve as ligands for FAT/CD36. After being transported into cells via FATP, fatty acids are converted into metabolites such as ceramide, diacylglycerol, and inositol phospholipid derivatives, which

participate in a variety of cellular regulatory mechanisms as second messengers. It has also been proposed that FAT/CD36 functions as a receptor of long-chain fatty acids rather than as a transporter. In fact, the binding of linoleic acid to CD36-positive gustatory cells was shown to inhibit the release of serotonin and dopamine via the phosphorylation of Src kinase and a subsequent change in the intracellular calcium concentration (El-Yassimi et al., 2008).

Fatty Acid Binding Protein Fatty acid binding protein (FABP), low molecular weight proteins of 14
15 kDa, transport long-chain fatty acids through cell membranes and mediate intracellular transport as a chaperone. Nine FABP isoforms have been identified (Veerkamp and Zimmerman, 2001), of which FABP3, 5, and 7 are expressed in the brain (Owada, 2008; Kurtz et al., 1994). FABP7 binds specifically to DHA, FABP3 binds to arachidonic acid, and FABP5 binds to both DHA and arachidonic acid. FABP bound to PUFA enters the nucleus and hands PUFA to a nuclear receptor as a ligand, consequently regulating the expression of target genes. In addition to transcriptional activity and peroxidase-like activity,

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FABP is involved in various physiological phenomena in cells, such as energy production via the facilitation of β-oxidation in mitochondria, the production of the cell membrane in the endoplasmic reticulum, and various enzymatic activities in the cytosol (Sprecher, 2000). According to Kim et al. (2001), FABP mRNA was upregulated in the dorsal root ganglion (DRG) 7 days after chronic constriction injury (CCI), one of the neuropathic pain models, suggesting that FABP play a role as a signal transduction molecule in neuropathic pain.

the development of more potent analgesics. In recent years, an increasing number of physiological and pharmacological studies have shown that functional properties of fatty acids are modulated by factors such as the amount of individual fatty acid intake and their distribution in the body. For example, several studies have reported in the last few years that pain is regulated by fatty acids, particularly unsaturated fatty acids, their metabolites, and other lipid mediators. Below, we summarize studies which have investigated the relationship of fatty acids and their related molecules with pain.

Long-Chain Fatty Acid Receptor GPR40 Long-chain fatty acid receptor GPR40 is a seventransmembrane G-protein-coupled receptor (GPCR) and has a high affinity for PUFA (Briscoe et al., 2003). GPR40 is activated upon binding to PUFA, which in turn activates phospholipase C and phosphatidylinositol pathways, resulting in an increase in the intracellular calcium concentration (Briscoe et al., 2003). In peripheral tissues, GPR40 is expressed highly in the pancreas and controls blood glucose levels via the regulation of insulin secretion (Itoh et al., 2003). Studies using humans and primates have shown that GPR40 is widely expressed in the central nervous system as well and that the PUFA-GPR40-cAMP-response element binding protein (CREB) signaling pathway is involved in neurogenesis and memory (Kaplamadzhiev et al., 2010). We have also reported the presence of GPR40 throughout mouse brain and its involvement in DHA-mediated pain regulation (Nakamoto et al., 2012).

Toll Like Receptor 4 Saturated fatty acids activate toll like receptor 4 (TLR4) (Lee et al., 2012, Huang et al., 2011). They also serve as an agonist for a complex of TLR4 and the lipidbinding accessory protein MD-2, or TLR4/MD2 complex, and they induce the production of inflammatory cytokines and interferons. It was recently reported that saturated fatty acids, such as palmitic acid and stearic acid, induce the production of the inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin (IL)-6 (Huang et al., 2011). Because such induction is not observed with PUFA, saturated fatty acids may be a cause of astrocyte activation and inflammation in the brain. In addition, saturated fatty acids may be associated with neuropathy caused by central inflammation in metabolic disease such as obesity (Kleinridders et al., 2009). Conventionally, non-opioid analgesics such as nonsteroidal anti-inflammatory drugs (NSAIDs), opioid analgesics including morphine, and other supplementary analgesics have been used to treat pain. However, some cases are refractory to these drugs, necessitating

INVOLVEMENT OF LIPIDS, FATTY ACIDS, AND THEIR METABOLITES IN PAIN REGULATION Dietary Lipids Lipids in the diet are digested and absorbed by the small intestine and enter the circulatory system via the lacteal vessels. Fatty acids are stored as triglycerides in adipose tissue and are transported to the liver and skeletal muscle when needed, where they undergo oxidation and degradation. Basic research studies using animals have shown that fatty acids taken into cells modulate cellular reactions to acute and chronic noxious stimuli (Shir et al., 1998; Shir et al., 2001). For example, the dietary fatty acids corn and soy oil suppress mechanical allodynia (Perez et al., 2004) and thermal hyperalgesia (Perez et al., 2005; Yehuda and Carasso, 1993) in the animal model of partial sciatic nerve ligation (PSL), presumably via lipid
protein interactions. In a study using diets with different compositions of linoleic acid, an omega-6 fatty acid, and α-linolenic acid, an omega-3 fatty acid, hyperalgesia was suppressed more in a group of animals fed on a diet rich in α-linolenic acid than in a group of animals fed on a diet rich in linoleic acid, suggesting the close association of omega-3 fatty acids with pain regulation. Similarly, Perez et al. (2005) fed rats on diets containing different lipids prior to the induction of neuropathic pain and showed that the symptoms of mechanical allodynia and thermal hyperalgesia varied by dietary lipid content. This suggests that dietary lipids, particularly omega-3 fatty acids, play an important role in the development of neuropathic pain. In addition, rats were fed on diets low in omega-3 fatty acids for 2 weeks prior to chronic constriction injury (CCI), followed by evaluation with mechanical and heat stimuli. These rats started to exhibit oversensitivity toward mechanical stimuli soon after the induction of CCI, suggesting that pain behavior is modulated by a prolonged deficiency in omega-3 fatty acids (Martin and Avendano, 2009).

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Omega-3 Fatty Acids Omega-3 fatty acids, abundant in fish oil, engage in various physiological actions as an essential fatty acid and play an important role in homeostatic maintenance in the body. Recently, the functional role of omega-3 fatty acids in pain regulation has been the focus of many studies including those investigating the inhibitory effect of omega-3 fatty acids on inflammatory pain associated with rheumatoid arthritis (Berbert et al., 2005, Calder, 2008), dysmenorrhea (Harel et al., 1996), and inflammatory bowel disease (Belluzzi et al., 2000). Because omega-3 fatty acids inhibit the production of inflammatory cytokines and inflammatory eicosanoids (Bagga et al., 2003), they may regulate pain via an anti-inflammatory mechanism. It is also possible that intake of omega-3 fatty acids inhibits pain by blocking the activation of mitogen-activated protein kinase (MAPK) involved in central sensitization associated with neuropathic pain and inflammatory pain (Woolf and Salter, 2000; Mirnikjoo et al., 2001). Interestingly, intake of α-linolenic acid, an omega-3 fatty acid, reduced the production of lysophosphatidic acid, a known factor in the progression of neuropathic pain (Miyazawa et al., 2003). With regard to clinical studies, a meta-analysis of 17 randomized controlled trials by Goldberg and Katz, (2007) showed the relationship between the level of omega-3 fatty acids and the degree of inflammatory pain associated with rheumatoid arthritis, inflammatory bowel disease, and dysmenorrhea. Moreover, long-term intake of omega-3 fatty acids in a range of 2,400
7,200 mg/day alleviated pain associated with radiculopathy, thoracic outlet syndrome, cervical radiculopathy, carpal tunnel syndrome, and neuropathy due to febrile disease (Ko et al., 2010). Ozgocmen et al. (2006) similarly reported the efficacy of omega-3 fatty acids in a large quantity on fibromyalgia. These studies therefore strongly indicate that intake of omega-3 fatty acids is highly effective on inflammatory and neuropathic pain. Although no guidelines currently stipulate dietary intake of omega-3 fatty acids for pain control, the Omega-3 Fatty Acids Subcommittee assembled by the American Psychiatric Association recommends that all adults consume fish more than twice a week. In Japan, the Dietary Reference Intakes issued by the Ministry of Health, Labour and Welfare, recommends intake of 1 g/day of DHA and EPA. Therefore, a higher dose than that recommended of omega-3 fatty acids may be necessary in order to observe the effects of omega-3 fatty acids. According to Ko et al. (2010), up to 7,500 mg/ day of EPA and DHA can be administered to patients with severe pain, even though this requires routine blood testing under the supervision of a physician.

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Potential roles of fatty acid metabolites in pain regulation have also been proposed. DHA DHA, an omega-3 fatty acid, consists of 22 carbons with 6 unsaturated bonds. The level of DHA in the human body reflects the level of DHA in dietary food because of a minute amount of DHA production and the limited interconversion between omega-3 and omega-6 fatty acids in the human body. DHA is present mainly in the central nervous system, and the membrane phospholipids phosphatidylethanolamine and phosphatidylserine (Tinoco, 1982) are particularly rich in cerebral cortex synaptic membranes, retina, and axons. The heart and sperm also contain DHA (Marszalek and Lodish, 2005). Anti-inflammatory activity via blockade of the arachidonic acid cascade (Gaudette and Holub, 1990), inhibition of the voltage-dependent sodium channel (Xiao et al., 1995), and inhibition of the calcium channel (Vreugdenhil et al., 1996) are known pain regulatory mechanisms of DHA. In our previous study using different types of pain, we showed that DHA exhibits an anti-nociceptive activity in a dose-dependent manner via the release of β-endorphin, an intrinsic opioid peptide (Nakamoto et al., 2010, Nakamoto et al., 2011). Moreover, according to Lu et al. (2013), the intrathecal administration of DHA was effective for inflammatory pain and neuroinflammation induced by carrageenan, and this mechanism was mediated by the inhibition of inflammatory cytokines originating from microglial cells via the activation of p38 MAPK. A recent study investigating ligands for orphan GPCRs revealed that certain fatty acids bind to these receptors (Hirasawa et al., 2008). In particular, DHA and EPA activate GPR40 and GPR120 (Hirasawa et al., 2005; Briscoe et al., 2003). GPR40 is expressed mainly in the brain and spleen and is involved in the secretion of insulin and cholecystokinin (Liou et al., 2010), while GPR120 is expressed in the small intestine and involved in the secretion of glucagon-like peptide 1 (GLP-1) and insulin (Hirasawa et al., 2005). Our study showed that the intracerebroventricular administration of DHA and GW9508, a selective agonist of GPR40 and GPR120, suppressed pain behavior induced by formalin (Nakamoto et al., 2012). The antinociceptive action of DHA and the GW compound was abolished by pretreatment with different opioid receptor antagonists and an anti-β-endorphin antibody. Furthermore, immunohistochemical analysis showed that 10 min after the intracerebroventricular administration of DHA and GW9508, the number of β-endorphin-positive cells started to increase in the hypothalamic arcuate nucleus, suggesting that the

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Docosahexaenoic acid (DHA)

DHA Long chain fatty acid receptor GPR40

Gq β-endorphin

Opioid receptor

Pain relief Pain

FIGURE 12.3 Hypothetical scheme for β-endorphin release on DHA-induced antinociception. The scheme outlines the potential mechanism of β-endorphin release on DHA-induced antinociception in this system. From Nakamoto et al., 2011, 2012.

anti-nociceptive property of DHA requires the GPR40mediated release of β-endorphin in the brain and therefore that GPR40 plays an important role in pain regulation (Figure 12.3). In our recent study, GPR40 protein was upregulated transiently in the midbrain, hypothalamus, and medulla oblongata 7 days after the administration of complete Freund’s adjuvant (CFA) to generate a mouse model of inflammatory pain, suggesting that GPR40 mediates the development of chronic pain. It is possible that fatty acids are involved in the transient upregulation of GPR40 as an internal ligand. In double-fluorescence immunohistochemistry, GPR40 was co-localized with NeuN, but not the astrocytespecific marker glial fibrillary acidic protein (GFAP) or the microglial cell marker Iba-1, demonstrating that GPR40 is expressed in neurons. In the brain, fatty acids are regulated by astrocytes (Moore et al., 1991; Moore, 1993; Moore, 2001). Consistent with this finding, increased expression of fatty acids in astrocytes was observed one day after the administration of CFA. Moreover, treatment with the cell growth inhibitor flavopiridol abolished the upregulation of astrocytes, suppressed mechanical allodynia and thermal hyperalgesia induced by CFA, and inhibited the upregulation of GPR40 on Day 7. Furthermore, mechanical allodynia and thermal hyperalgesia were significantly blocked by the intracerebroventricular administration of the GPR40 agonist GW9508 on Day 7, but not Day 1, after

CFA injection, suggesting that the expression of GPR40 protein is required for this action. Currently, investigations are underway to examine anti-allodynic mechanisms that are mediated by GPR40. Although preliminary, observations have been made of the expression of GPR40 in proopiomelanocortin (POMC)-positive neurons, as well as in serotonergic neurons in the nucleus raphe magnus, where the descending inhibition pathway originates, and in noradrenergic neurons in the locus coeruleus. This suggests that GPR40 regulates the functions of neurons associated with the intrinsic regulatory mechanism of pain. However, further studies are needed to clarify the actual mechanism by which DHA regulates pain. Metabolites Derived from Omega-3 Fatty Acids Omega-3 fatty acids improve various inflammatory diseases, and their mechanism of action is thought to center around antagonistic action toward the arachidonic acid cascade (Gaudette and Holub, 1990). However, recent studies have revealed that EPA and DHA are metabolized by lipoxygenase and cyclooxygenase into resolvin and protectin, each with a powerful anti-inflammatory action (Serhan, 2005). Schwab et al. (2007) reported that resolvin E1 (RvE1) and protectin D1, the oxidized metabolites of EPA and DHA, facilitate the inflammation-resolution programs. The study also showed that the intrathecal administration of RvE1 derived from EPA and protectin D1

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FIGURE 12.4 The involvement of metabolites derived from omega-3 polyunsaturated fatty acid in pain.

Omega-3 fatty acids Alpha-linolenic acid C18:3 ω-3

Eicosatetraenoic acid CYP450

Epoxy-eicosatetra enoic acid (EpETE)

Eicosapentaenoic acid (EPA, C20:5 ω-3)

COX-2, aspirin and 5-LOX

Resolvin E (RvE)

Docosapentaenoic acid (DPA)

Antinociception CYP450

Epoxy-docosapenta enoic acid (EpDPE)

Docosahexaenoic acid (DHA, C22:6 ω-3)

15-LOX, and 5-LOX

ResolvinD (RvD)

derived from DHA (0.3
20.0 ng) alleviated inflammatory pain. This is presumably because RvE1 and resolvin D1 (RvD1) act on chem R23, a known resolvin receptor, in the spinal cord, inhibiting the expression of extracellular signal-regulated kinase (Veerkamp and Zimmerman, 2001) as well as excitation mediated by N-methyl-Daspartate (NMDA) (Xu et al., 2010). It was reported that 17 (R)-hydroxy-docosahexaenoic acid (17(R) HDoHE), a precursor of resolvin D, and aspirin-triggered resolvin D1 (AT-RvD1) suppress inflammatory pain in a mouse model of arthritis at a concentration as low as 1 μg/kg (Bang et al., 2010; Lima-Garcia et al., 2011). In addition, the subcutaneous administration of RvD1 into the paw alleviated inflammatory pain by blocking the activation of transient receptor potential (TRP) channels A1, TRPV3, and TRPV4 (Lima-Garcia et al., 2011; Huang et al., 2011). Moreover, intrathecal administration of RvD1 effectively prevented postoperative pain and alleviated subsequent symptoms (Huang et al., 2011). Interestingly, the anti-nociceptive action of 17(R) HDoHE, RvD1, and AT-RvD1 are more effective on mechanical allodynia than thermal hyperalgesia, and this appears to involve the activation of signaling pathways mediated by GPCR, such as GPR32 and ALX/ FPR2 (Krishnamoorthy et al., 2010). The involvement of other metabolites such as AT-RvD2, 3, 4, and 5 has also been proposed (Serhan et al., 2002; Serhan, 2007). Furthermore, neuroprotectin/protectin D1 has recently been shown to inhibit the activation of glial cells in

neuropathy due to traumatic brain injury and the accompanying inflammatory response (Xu et al., 2013). These lipid mediators are expected to serve as novel therapeutic agents for inflammatory pain associated with arthritis and inflammatory bowel disease. Linoleic acid, arachidonic acid, EPA, and DHA released from membrane phospholipids are converted to epoxygenated fatty acids (EpFAs) by cytochrome P450 (Liu et al., 2010). EpFAs, which are present throughout the central and peripheral tissues (Chacos et al., 1983; Bernstrom et al., 1992; Spector and Norris, 2007), are known as bioactive lipids with an extremely short half-life due to hydrolysis by soluble epoxide hydroxylase (sEH) (Jones et al., 2005). When stabilized by the use of an sEH inhibitor, EpFAs display various physiological activities, including the promotion of cell proliferation, the regulation of blood vessel tones, and anti-inflammatory action, and are also important for certain pathophysiological processes. Morisseau et al. (2010) reported that the intrathecal administration of epoxy-docosapentaenoic acid (EpDPE), an epoxygenated omega-3 fatty acid, reduced inflammatory pain induced by carrageenan. Inceoglu et al. (2012) also reported that the subcutaneous administration of sEH inhibitor in an animal model of type-1 diabetes inhibited peripheral neuropathy dosedependently by acutely increasing the plasma level of EpFAs without affecting blood glucose levels, the secretion of insulin, or insulin sensitivity (Figure 12.4).

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Omega-6 Fatty Acids and Their Metabolites Inflammatory eicosanoids, such as leukotriene and prostaglandin, and inflammatory cytokines, such as IL-1 and IL-6, are synthesized from omega-6 fatty acids. Consequently, omega-6 fatty acids are regarded as a negative factor in many diseases. For example, the levels of dihomo-γ-linolenic acid (C20:3) and docosatetraenoic acid (C22:4) were significantly increased in patients with complex regional pain syndrome which is a neuropathic pain syndrome with abnormal immune and autonomic nervous systems (Ramsden et al., 2010). Interestingly, pain was evoked by metabolites from the major plant oil component linoleic acid (Patwardhan et al., 2009). Pain was also evoked via the activation of transient vanilloid receptor 1 following heat-induced production of linoleic acid metabolites in the spinal dorsal horn (Patwardhan et al., 2010). Consequently, omega-6 fatty acids have been focused on as novel factors in the molecular mechanism that evokes pain. Arachidonic acid of the omega-6 fatty acids has been studied intensely because it is a major component of the cell membrane and a precursor of prostaglandins and leukotrienes, both of which are involved in blood pressure, inflammatory reaction, and coagulation. Ca-dependent cytosolic phospholipase A2 (cPLA2) hydrolyzes and releases arachidonic acid from the cell membrane (Shimizu et al., 2006). The intrathecal administration of cPLA2 inhibitor suppresses mechanical allodynia associated with neuropathy (Tsuda et al., 2007a). Furthermore, the phosphorylation of cPLA2 at Ser505 and that of cPLA2 at the cell membrane were increased in neuropathic DRG neurons. Time-dependent activation of cPLA2 in the neuropathic DRG neurons coincided with the development of mechanical allodynia, which was not observed in the CFA-induced inflammatory pain model, suggesting that the activation of cPLA2 in DRG cells is specific to neuropathy. MAPKs, Ca21/calmodulin-dependent protein kinase II (CaMKII), MAPK-interacting kinase I (MNK1), and other isoforms closely related to the above kinases activate cPLA2 by phosphorylating a serine residue in the catalytic domain (Hirabayashi and Shimizu, 2000). This suggests that CaMKII may be an intracellular regulator of cPLA2 in peripheral neuropathy. P2X2 or P2X3 receptor was also reportedly involved in the activation of the CaMKII-cPLA2 pathway (Tsuda et al., 2007b). Cytochrome P450s CYP2J and CYP2C convert arachidonic acid into epoxyeicosatrienoic acid, which is known to reduce blood pressure and inhibit antiplatelet activity (Terashvili et al., 2008). However, it was recently shown that epoxyeicosatrienoic acid, as well

as the EET hydrolase she, also has an anti-nociceptive property (Inceoglu et al., 2008). On the other hand, the CYP metabolite 5,6-EET derived from arachidonic acid evokes mechanical hyperalgesia via TRPA1 (Sisignano et al., 2012). These results suggest that omega-6 fatty acids, represented by arachidonic acid, play important roles in both the development and suppression of pain (Figure 12.5).

Prostaglandins Prostanoids, which are metabolites of arachidonic acid in the cyclooxygenase pathway, are lipid mediators associated with inflammatory pain. Of the many prostanoids, PGE2 is an important signaling molecule in pain pathways. Currently, most commonly used NSAIDs block the production of PGE2 and other prostanoids by inhibiting COX-1 and COX-2 and consequently suppress pain caused, for example, by arthritis, osteoarthritis, and migraine. However, these drugs have a risk of gastrointestinal disorders, cardiovascular disorders, renal failure, and other adverse reactions (Bresalier et al., 2005). This is presumably because these drugs block the protective action of prostacyclin (PGI2) in the renal and cardiovascular systems (Zeilhofer and Brune, 2006). PGE2 is synthesized from PGH2 by three isomerases (cytosolic PGE synthase (cPGES) and membranebound PGE synthases 1 and 2 (mPGES1, 2). Of these, cPGES and mPGES-2 are constitutively expressed in many tissues (Zeilhofer and Brune, 2006). Similarly to COX-2, the expression of mPGES-1 is upregulated in response to various inflammatory stimuli. At present, mPGES1 inhibitors MF63, PF-9184, and AF3442 inhibit the production of PGE2 without affecting other prostanoids (Mbalaviele et al., 2010, Xu et al., 2008; Bruno et al., 2010). Moreover, unlike NSAIDs, MF63 suppresses inflammatory pain without causing gastrointestinal disorders, and selective mPGES1 inhibitors are beneficial for the cardiovascular system via a compensatory increase in the production of PGI2 (Wang et al., 2008). Four subtypes of prostaglandin E receptors (EPs) EP1, EP2, EP3, and EP4 have been identified to date. EP1 is coupled with Gq, EP2 and EP4 with Gs, and EP3 with Gi (Wang et al., 2007). These GPCRs are expressed in sensory neurons, suggesting their involvement in pain regulation in the periphery and at the spinal cord level (Southall and Vasko, 2001; Narumiya, 2009). A recent study using various inflammatory pain models has revealed the usefulness of selective EP4 inhibitors. AH23848, MF766, CJ-023423, and CJ-042794 have a potent analgesic action toward CFA-induced inflammatory pain (Lin et al., 2006;

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Omega -6 fatty acids Linoleic acid C18:2ω-6

Heat

9-Hydroxyoctadecadienoic acid (9-HODE), 13-HODE

Gamma-linolenic acid

Dihomo-gamma-linolenic acid C20:3 ω-6

Arachidonic acid C20:4 ω-6

Nociception

COX, 5-LOX EP1 EP2 EP3 EP4

CYP450 Prostaglandins (PG) 14, 15-Epoxyeicosa enoic acid (14, 15-EET)

CYP450

PGE2

Thromboxanes Leukotrienes

LTB4

5, 6-Epoxyeicosa enoic acid (5, 6-EET)

Antinociception

Omega-6 fatty acids Linoleic acid C18䠖2 ω-6

Omega-3 fatty acids

Heat

9-Hydroxyoctadecadienoic acid (9-HODE), 13-HODE

Gamma-linolenic acid

Alpha-linolenic acid C18:3 ω-3

Eicosatetraenoic acid Epoxy-eicosatetra enoic acid (EpETE)

Dihomo-gamma-linolenic acid C20:3 ω-6

CYP450

Eicosapentaenoic acid (EPA, C20:5 ω-3)

Resolvin E COX-2, aspirin (RvE) and 5-LOX

Arachidonic acid C20:4 ω-6

Docosapentaenoic acid (DPA)

CYP450

14, 15-Epoxyeicosa enoic acid (14, 15-EET)

CYP450

Epoxy-docosapenta enoic acid (EpDPE)

COX 5-LOX Docosahexaenoic acid (DHA, C22:6 ω-3)

5, 6-Epoxyeicosa enoic acid (5, 6-EET)

Prostaglandins, thromboxanes, leukotrienes

CYP450

15-LOX, and 5-LOX

FIGURE 12.5 The involvement of metabolites derived from omega-6 polyunsaturated fatty acid in pain.

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ResolvinD (RvD)

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Nakao et al., 2007), while MF498 alleviates joint pain associated with osteoarthritis and rheumatism. On the other hand, EP1 and EP4 did not reveal clear antinociceptive properties in the knockout mice. For example, while the analgesic action of EP1 toward an acute noxious heat stimulus was augmented in the knockout mice, the action was suppressed in the EP4 knockout mice. Furthermore, the knockout of neither gene responded to formalin-induced pain or acute mechanical stimulus. It is possible that each EP receptor knockout mouse had an alternative adaptation mechanism to compensate for the knockout or that each receptor had a site-specific role. However, further studies are needed to address these questions (Minami et al., 2001; Popp et al., 2009; Zamponi et al., 2009). Protein kinases C (PKC) and A (PKA) have been shown to mediate hyperalgesia induced by a subcutaneous injection of PGE2 into an animal footpad (Sachs et al., 2009). These kinases are downstream of EP1 and EP4, and the activation of the PKC and PKA pathways by PGE2 is thought to induce hyperalgesia via the sensitization and activation of many molecules including receptors TRPV1 and P2X3 in nociception, Ca channel protein Cav3.2, and Na channel proteins Nav1.8 and Nav1.9 (Amaya et al., 2006). Interestingly, PGE2 plays a major role in sensitization and in the development of allodynia in the spinal cord, in addition to the periphery, but through different mechanisms (Nakayama et al., 2004). EP1 in the spinal cord was reported to play a more important role in nociception than EP1 in the periphery. Mechanical allodynia and thermal hyperalgesia induced by intrathecal injection of PGE2 were blocked in EP1 knockout mice and also by an EP1 receptor inhibitor (Nakayama et al., 2004). PGE2 is also reported to be involved in neuropathic pain. Following PSNL to ligate one-third to one-half of the mouse sciatic nerve, the expression of EP1 and EP4 was increased in the injured nerve and in the surrounding tissue, and the administration of an EP1 receptor inhibitor was effective (Ma and Eisenach, 2002). Furthermore, EP1 receptor is present in the injured brachial plexus nerve and in the DRG of patients with avulsion injury (Durrenberger et al., 2006). PGE2 synthesized by membrane-associated PGE synthase-1 (mPGES-1) was also shown to be involved in the maintenance period of neuropathy via the activation of EP1 and E2 in the primary sensory neurons in the central side (Kunori et al., 2011). The EP1 receptor inhibitor ONO-8130 suppresses animal behavior associated with bladder pain as well as hyperalgesia associated with cystitis induced by cyclophosphamide, indicating that the inhibitor is useful for visceral pain (Miki et al., 2011). These studies showed that PGE2 greatly influences pain signaling, and

therefore, a pharmacological intervention study targeting the downstream and upstream activity of PGE2 may be a useful treatment approach for intractable pain. These results suggest that the inhibition of microsomal PGE synthase 1 and the use of subtype-selective inhibitors of PGE2 receptors, in particular EP1 and EP4, may serve as an effective analgesic with minor, if any, adverse effects.

Leukotrienes Leukotrienes (LTs) are lipid mediators derived from arachidonic acid via the 5-lipoxygenase (5-LO) pathway. Arachidonic acid is converted to leukotriene A4 (LTA4), then enzymatically to bioactive LTB4, LTC4, LTD4, and LTE4. LTs are a major factor in allergic disorders such as asthma and atopy, spinal cord injury, and systemic inflammatory diseases such as rheumatoid arthritis and cancer (Henderson, 1994). LTC4, LTD4, and LTE4 are collectively called cysteinyl leukotrienes (CysLts) (Lynch et al., 1999; Yokomizo et al., 1997; Heise et al., 2000). LTs exert their effects by binding to specific receptors on the outer membrane of inflammatory cells and other cells. LT B4 receptor 1 (BLT1) has a high affinity to LTB4, but BLT2 has a low affinity to LTB4 as well as to other LTs (Peters-Golden and Henderson, 2007). CysLT1 and CysLT2 recognize CysLT with different affinities. LTs and the synthases are present in the central nervous system including the spinal cord. Recent studies showed that LTs are involved in peripheral inflammatory pain and that lipoxygenase metabolites are involved in hyperalgesia in the spinal cord (Shimizu and Wolfe, 1990; Shimada et al., 2005; Chiba et al., 2006). Following peripheral nerve damage, the synthesis of LTs was increased in the spinal microglia due to the secretion of LTs by neutrophils and other infiltrating cells. The inhibition of the synthase suppressed pain behavior during the developmental phase of neuropathic pain. The inhibitors of the 5-LO and CysLT1 receptors suppressed mechanical allodynia due to SNI, suggesting that CysLT1 expressed in microglia and BLT1 in neurons are involved in the early phase of neuropathic pain (Okubo et al., 2010).

Platelet-Activating Factor Platelet-activating factor (PAF) is a phospholipid that was discovered as a platelet-agglutinating factor derived from rabbit basophils and as a depressor substance derived from the renal medulla. To generate PAF, phospholipase A2 removes fatty acid in the sn-2 position of the ester-type membrane phospholipid, followed by acetylation. It is now clear that PAF is

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synthesized in keratinocytes and various other cells including inflammatory cells. Moreover, PAF activates inflammatory and immune cells, enhances vascular permeability, and has bronchoconstrictor activity. PAF is also involved in acute pulmonary edema and bronchial asthma as an important mediator. Furthermore, PAF is associated with pregnancy, childbirth, circulation, and ischemic tissue damage, showing that PAF participates in a wide variety of physiological and pathophysiological mechanisms (Ishii and Shimizu, 2000; Prescott et al., 2000). Because the administration of PAF to a peripheral site, such as skin, augments pain sensitivity, PAF is thought to function as an important factor in the development of pain (Dallob et al., 1987; Bonnet et al., 1981). In addition, the PAF receptor (PAFR) antagonist BN52021 has been shown to inhibit inflammatory pain in animal models (Teather et al., 2002). This suggests that endogenous PAF is involved in pain behavior associated with inflammatory pain induced by tissue damage. In the study conducted by Tsuda et al. (2007b), PAFR knockout mice and control mice responded similarly to heat and mechanical stimuli; however, formalin- and capsaicin-induced pain and acetic acid-induced writhing were suppressed in the knockout mice. Moreover, in the PAFR knockout mice, pain behavior due to neuropathy was suppressed via the inhibition of microphagederived inflammatory cytokines in the DRG (Hasegawa et al., 2010; Doi et al., 2006). These results therefore indicate that the PAF/PAFR system is deeply associated with the development of visceral pain and pain associated with tissue damage. Morita et al. (2004) reported that the intrathecal administration of PAF induced hyperalgesia and allodynia. In fact, PAF is synthesized in spinal glial cells and inflammatory cells and facilitates the release of ATP, glutamate, and other neurotransmitters from the nerve terminals of the primary sensory neurons. The maintenance of PAF-induced allodynia was deeply correlated with the disinhibition of glycinergic inhibition by targeting the spinal glycine receptor GlyRα3 subunit by cGMP/PKG, and this process required ATP and the activation of microglial cells. Furthermore, glycine transporter inhibitors appear to increase the concentration of glycine in synaptic cleft and consequently inhibit heat-induced sensitization and allodynia mediated by PAF.

Tokumura et al., 2002; Sonoda et al., 2002; Oka et al., 2010). In mammals, LPA is constitutively synthesized and degraded in multiple metabolic pathways inside and outside of the cells, and the metabolic abnormality is thought to induce diseases such as cancer, arteriosclerosis, and myofibrosis (Hecht et al., 1996; Nishimasu et al., 2011; van Meeteren et al., 2006). Nagai et al. (2010) revealed that LPA was a lipid mediator produced at the time of sciatic nerve injury and was a primary causal factor of neuropathic pain. They also reported that the molecular mechanism involved demyelination mediated by LPA1 receptor and the upregulation of pain-related genes in the primary sensory neurons and spinal cord dorsal horn cells. Furthermore, an increase in LPA production in the spinal cord and DRG neurons was inhibited by the removal of pain stimuli, suggesting the involvement of LPA in pain regulation (Inoue et al., 2004; Inoue et al., 2006). The mechanism that increases LPA in neuropathy has been elucidated in recent years, showing that LPA synthesis is induced by noxious inputs of substance P and glutamate arriving at the spinal dorsal horn from neuropathic primary sensory neurons (Inoue et al., 2008). In humans, the LPA receptor GPR35 is highly expressed in the small and large intestines and stomach in the gastrointestinal system, spleen, and peripheral white blood cells, where the molecule regulates neural excitation and pain (Cosi et al., 2011). Because pain behavior associated with cerebral ischemia is suppressed in LPA1 knockout mice, LPA1-mediated signaling may be required for the development of central pain after cerebral ischemia (Halder et al., 2013; Ueda et al., 2013). Also in LPA5 knockout mice, PSL-induced neuropathic pain was alleviated by inhibiting the phosphorylation of EPAC and the upregulation of Caα2δ1 expression (Lin et al., 2012; Callaerts-Vegh et al., 2012). This suggests that the downstream signaling pathway in LPA5-mediated pain regulation differs from that in LPA1-mediated regulation (CallaertsVegh et al., 2012). In addition, emotional behavior such as anxiety was also inhibited in LPA5 knockout nice. In recent years, inhibitors of autotaxin (ATX), lysophospholipase D that hydrolyzes lysophosphatidyl choline to LPA and choline, have attracted attention as a drug target for pain and cancer. However, research is still in the early stages, and no clinical trials using ATX inhibitors have been reported. We are keenly anticipating further developments in this area (Barbayianni et al., 2013).

Lysophosphatidic Acid Lysophosphatidic acid (LPA) is another major lipid mediator that exhibits various physiological activities via specific receptors such as LPA1
6, GPR35, and GPR87 (Noguchi et al., 2001; Umezu-Goto et al., 2002;

FUTURE PROSPECTS It has become increasingly clear that fatty acids play a major role in the induction and regulation of pain.

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In particular, omega-3 fatty acids have been the focus of many physiological and pharmacological studies that have investigated these fatty acids from various viewpoints. Because of their well-established safety profiles, omega-3 fatty acids are likely to be used in a wide range of clinical trials in the future. The concept of ‘Eco-Pharma’ was recently proposed to screen existing drugs with a known safety record to discover a new drug seed. However, omega-3 fatty acids may attract attention as candidate drugs for the treatment of pain because of the safety from the perspective of nutritional value and preventive medicine. In addition, omega-3 fatty acids may be effective if used in combination with existing drugs. For example, combination therapy with omega-3 fatty acids and NSAIDs, such as celecoxib, may reduce adverse drug reactions. With continuous and vigorous investigation of the relationship between fatty acids and pain, fatty acids may serve as novel regulators of pain to elucidate the pathogenesis of intractable pain and contribute to the discovery of new analgesics.

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13 Fish Oil Supplementation Prevents Age-Related Memory Decline: Involvement of Nuclear Hormone Receptors Serge Alfos INTRODUCTION Public health, economic development, and medicine gradually extend average life expectancy and the number of individuals older than 60 in the world is projected to exceed 1 billion in the year 2030, which will represent 16.5% of the total population (Kowal et al., 2010). Concern arises regarding the quality of life available to the elderly, not only in terms of physical well-being, but also in terms of mental well-being. Indeed, beyond Alzheimer’s disease, the prevalence of which increases with age, most of the elderly will exhibit subtle deficits in memory that are unrelated to neuropathologies. These mild memory deficits associated with normative aging are nonetheless disturbing for those affected (Erickson and Barnes, 2003). Therefore, cognitive decline in the elderly is a major socio-economic and healthcare concern. In order to reduce the effects of normal aging on memory performance and hence increase the mental well-being of the elderly, closer attention should be paid to the possible impact of modifiable lifestyle factors such as the quality of diet. Indeed, nutrition is one of the major determinants of successful aging and may affect mental health (Dauncey, 2009). Among the dietary nutrients most closely associated with optimal brain function, long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFAs) are particularly important (Gomez-Pinilla, 2008; Parletta et al., 2013). Increasing the intake of n-3 PUFAs, particularly the LC n-3 PUFAs, may be a strategy to delay the onset of memory decline in the elderly.

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00013-2

EFFECTS OF AGING ON INCORPORATION OF DOCOSAHEXAENOIC ACID IN BRAIN PHOSPHOLIPIDS The principal LC n-3 PUFA found in the adult mammalian brain is docosahexaenoic acid (22:6n-3, DHA) comprising 10
20% of total fatty acid composition, whereas eicosapentaenoic acid (EPA, 20:5n-3) and docosapentaenoic acid (DPA, 22:5n-3) represent less than 1% of total fatty acid composition (McNamara and Carlson, 2006). Studies in adult rodents and primates demonstrate that DHA concentrations differ between brain regions (Carrie´ et al., 2000a; Diau et al., 2005). In rodents, DHA concentration is higher in the frontal cortex and hippocampus (16
22% of total fatty acids) and lower in other brain regions such as the striatum (14% of total fatty acids) (Xiao et al., 2005). Most DHA accumulates in brain structures during prenatal development and the early post-natal period (Green et al., 1999; Innis, 2007; Martinez, 1992; McNamara and Carlson, 2006). In brain membranes, DHA is preferentially incorporated into the stereospecifically numbered-2 (sn-2) position of the phospholipids phosphatidylethanolamine (PE) and phosphatidylserine (PS), and to a smaller proportion, into phosphatidylcholine (PC) (Rapoport, 1999). Fish oil supplementation can rapidly modify brain phospholipid composition as shown by a mass spectroscopy analysis. In this study, an oral gavage of fish oil (38% DHA 1 48% EPA) in two-month-old rats, for a period of one month only, significantly increased the proportion of DHA-containing

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PC and PE species in the striatum, hippocampus, and cortex (Lamaziere et al., 2011). Thus membrane phospholipids in the adult brain are highly enriched in DHA, suggesting specific functional roles of this fatty acid in neural processes. Brain aging is associated with significant changes in the levels of major phospholipid classes. Depending on the animals’ age and diet and also on the brain region studied, the results sometimes differ from one study to another. Globally, aging is associated with a significant progressive decrease in PE and PS in the prefrontal cortex and the hippocampus in rats between 18 to 32 months of age compared to 2
3-month-old rats (Babenko and Semenova, 2010; Dyall et al., 2007; Favrelie`re et al., 2000; 2003). In rodents, most of the studies also show a decrease in the levels of DHA in the brain during aging (Giusto et al., 2002; Labrousse et al., 2012; Little et al., 2007). More specifically, aging alters DHA concentrations in specific brain phospholipid classes. Therefore, during aging, a reduction in DHA levels in PE, PS, and PC is observed in the cortex, the hippocampus, and the cerebellum, associated with an increase in monounsaturated fatty acids (Barcelo-Coblijn et al., 2003a; Dyall et al., 2007; Favrelie`re et al., 2000; Latour et al., 2013; Little et al., 2007; Lopez et al., 1995). This age-related DHA decrease can be reversed by 2
3 months of fish oil supplementation (Barcelo-Coblijn et al., 2003a; Dyall et al., 2007; Labrousse et al., 2012; Little et al., 2007). Moreover, the accumulation in the brain, mostly in the PC, of orally administered radiolabeled DHA is reduced with increasing age from 2- to 10-week-old rats (Graf et al., 2010). These results suggest a reduced capacity of the aging brain to incorporate DHA in phospholipids. The situation in humans seems more complex with limited alterations in brain DHA levels (Carver et al., 2001; So¨derberg et al., 1991) or a slight decrease in the orbitofrontal cortex (McNamara et al., 2008) during normal aging. However, several studies have clearly shown a decrease in DHA amount in the PE in the hippocampus and the cortex of patients with Alzheimer’s disease (review in Cunnane et al., 2009). The reduced DHA level in brain phospholipids may be due to a reduction in the activity of the enzymes allowing the incorporation of DHA into brain phospholipids (Andre et al., 2006; Giusto et al., 2002), but other changes in DHA metabolism during aging may contribute to a decrease in brain DHA level.

EFFECTS OF AGING ON DHA BIOSYNTHESIS In mammals, DHA can be synthesized from its nutritionally essential plant-derived precursor, α-linolenic

acid (α-LNA, 18:3n-3), through a series of elongation and desaturation steps in the endoplasmic reticulum followed by peroxisomal β-oxidation (Sprecher, 2000). Although the liver is the major site of DHA synthesis, it can also be synthesized locally in the brain but with much less efficiency (Rapoport et al., 2010). Indeed, primary cultures of rat hippocampal neurons and astrocytes were able to synthesize and incorporate DHA from radiolabeled α-LNA, but the amounts produced were considerably less than those incorporated when the culture was incubated with radiolabeled DHA (Kaduce et al., 2008; Williard et al., 2001). Human and rodent studies using stable isotopes have concluded that less than 1% of dietary α-LNA is used to form longer chain n-3 fatty acids (Plourde and Cunnane, 2007). However, it now seems clear that in rats on an adequate α-LNA containing diet, the liver is capable of synthesizing sufficient DHA from circulating α-LNA to maintain a normal brain DHA level in the absence of dietary EPA or DHA (Rapoport and Igarashi, 2009). Nevertheless, DHA may also be obtained directly from dietary sources, particularly from fatty fish, but also from n-3 PUFA enriched foods or from fish oil containing both EPA and DHA in the form of nutritional supplements (Gebauer et al., 2006; Whelan and Rust, 2006). Different fish oils provide different ratios of EPA/DHA but also differ in their digestibility and oxidation properties (Tou et al., 2011). In humans, intravenously injected radiolabeled DHA rapidly entered the brain and accumulated preferentially in the gray matter in the neocortex (Umhau et al., 2009). Studies in mice using radiolabeled DHA and EPA suggest that these compounds cross the blood
brain barrier to accumulate in the brain by a simple diffusion process (Ouellet et al., 2009). Most reports comparing DHA versus α-LNA intake suggest that the maximal concentration in the brain can only be achieved by including DHA in the diet (Arsenault et al., 2012; Brenna et al., 2009; Talahalli et al., 2010). Thus, it is questionable whether α-LNA can be as efficient as DHA in maintaining brain DHA levels and neurobiological functions (BarceloCoblijn and Murphy, 2009). Several studies suggest that aging alters n-3 PUFA metabolism. It has been shown that hepatic delta-6desaturase activity, the first and rate limiting enzyme in the conversion of α-LNA to LC n-3 PUFAs, decreases during aging in rat and mouse (Bourre and Piciotti, 1992; Bourre et al., 1990; Dinh et al., 1993; Hrelia et al., 1989). The brain obtains DHA synthesized in the liver directly from the plasma unesterified fatty acid pool (Chen et al., 2008) and an age-related decrease in hepatic DHA biosynthesis may change plasma fatty acid concentrations. A recent study shows that the liver synthesis secretion rate of DHA is reduced in 20- and 30-month-old rats compared with

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10-month-old rats, leading to reduced unesterified n-3 PUFA concentrations, mainly DHA and α-LNA, in the plasma, whereas the esterified PUFA concentration is unchanged (Gao et al., 2013). These data suggest that the reduced circulating concentrations of unesterified n-3 PUFAs resulting from an altered liver metabolism of PUFAs may contribute, in association with the reduced activity of the enzymes allowing the incorporation of DHA into brain phospholipids, to a decline in brain DHA levels observed during aging. In humans, Astarita et al. (2010) have reported a decrease in DHA level in the PE in the cerebral cortex, the cerebellum, and the liver in Alzheimer’s disease patients. This was associated with a reduced mRNA expression in the liver of the D-bifunctional protein, a peroxisomal enzyme which catalyzes the conversion of tetracosahexaenoic acid to DHA. These results indicate that alterations in DHA metabolism in the liver may contribute to reduced brain DHA levels during aging in humans. Moreover, when the plasma DHA response to a 3-week fish oil supplement was compared between 74-year-old healthy elderly and 24-year-old young adults, results show that plasma DHA increased to significantly higher levels and more rapidly in the elderly, reaching a plateau after 2 weeks of supplementation, whereas in young adults, plasma DHA was still increasing after 3 weeks (Vandal et al., 2008). More recently, a comparison of 13C-DHA metabolism into plasma lipids in young (26-year-old) and healthy elderly (77-year-old) subjects reveals that, 4 hours after ingestion of an oral dose of 50 mg radiolabeled DHA, 13C-DHA was significantly higher in plasma triglycerides and free fatty acids in the elderly than in the young subjects (Plourde et al., 2011). Moreover 13C-DHA β-oxidation was 1.9 times higher at 4 hours post-dose in the elderly. These authors conclude that LC n-3 PUFA metabolism is subtly altered during healthy aging.

DHA is Involved in Learning and Memory One of the classical approaches to studying the role of a nutrient in cognitive functions and more specifically in memory is to induce a dietary deficiency of this nutrient in animals. However this is not easy to achieve for the DHA, since the adult mammalian brain tissue is predominantly composed of lipids and the brain has homeostatic mechanisms which delay pathological effects of reduced n-3 PUFA intake by increasing DHA half-life from 33 to 90 days in post-weaning n-3 PUFA deficient rats (DeMar et al., 2004). For these reasons, in general, brain DHA deficiency is induced by feeding an n-3 fatty acid deficient diet in utero (via the maternal intake) and during one to three generations, thus even leading to an 80% decrease in brain

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DHA levels (Moriguchi et al., 2000). In order to study the role of n-3 PUFAs in memory, several studies have investigated the effects of n-3 PUFA deficiency on animal behavior using different tasks. Most of these studies demonstrate that n-3 PUFA deficiency impairs cognitive functions and mainly learning and memory capacities (review in Fedorova and Salem, 2006). For example, using a multi-generational deficiency model, it has been shown that n-3 PUFA deprivation in rats results in reduced performance in learning and reference memory tasks evaluated in the Morris water maze (Moriguchi et al., 2000). Moreover, these authors have also shown that in n-3 PUFA deficient rats repleted with α-LNA and DHA, the degree of brain DHA level and spatial performance recovery in the Morris water maze depended upon the duration of dietary repletion (Moriguchi and Salem, 2003). These results demonstrate a positive correlation between brain DHA content and spatial memory performance in deficient or repleted animals. Other studies have addressed the effects of dietary DHA supplementation in young animals without n-3 PUFA deprivation. Young (2-month-old) adult rats receiving fish oil by gavage during brain development and for 80 days of adulthood had enhanced performance in reference and working memory, as evaluated in the Morris water maze, and displayed an increase in DHA content in the hippocampus (Chung et al., 2008b). This study indicates that DHA may modulate memory capacities in animals without n-3 deprivation and that a link exists between DHA levels in the hippocampus and memory performance. The relationship between the duration of DHA intake and mazelearning ability was also studied. Dietary intake of DHA (2 g DHA-ethyl ester/100 g diet) improved maze-learning ability in mice after 1 month of feeding and was maintained for up to 3 months, whereas the increased DHA level in the brain was apparent after 2 weeks of feeding but not after 1 week (Lim and Suzuki, 2001). These results suggest that a sufficient level of brain DHA must be reached to induce an improvement in learning abilities. Tanabe et al. (2004) reported that chronic administration by gavage of fish oil in 5-week-old rats for 12 weeks improved reference and working memory using an eight-arm radial maze paradigm. The beneficial effect of fish oil supplementation on memory was associated with an increase in c-Fos positive neurons in the hippocampal CA1 region (Tanabe et al., 2004). Recent studies have also investigated the effect of DHA specifically in the adult hippocampus by using an electrophysiological approach. DHA supplementation (1% DHA from algal source in the diet) in mice from 6 to 12 months of age increased DHA content in total phospholipids by 29% in the hippocampus and enhanced hippocampal synaptic

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transmission following brief high-frequency stimulation (Connor et al., 2012). Another study compared the effect of α-LNA or DHA on the electrophysiological properties of the entorhinal cortex neurons, a brain region linked with the hippocampus and involved in memory function. Mice exposed from 4 to 14.5 months of age to a diet enriched in DHA (1.14 mmol/kg/day) displayed an increase in DHA concentration in the cortex (1 34%), whereas the DHA-increasing effect of an α-LNA diet (1.5 mmol/kg/day) was lower (1 23%), when compared with control animals (Arsenault et al., 2012). Moreover, only the DHA diet was able to increase the passive electrical properties of entorhinal cortex neurons. These authors suggest that α-LNA is partly converted to DHA, as reflected by the increased DHA concentrations in the cortex, but insufficiently to modulate the electrophysiological properties of entorhinal cortex neurons. A study in humans has investigated the potential association between cognitive performance and serum phospholipid levels of DHA, EPA, and ALA in healthy middle-aged adults (Muldoon et al., 2010). The results indicated that DHA was associated with cognitive functioning, as well as with better scores on tests of nonverbal reasoning, mental flexibility, working memory, and vocabulary. In contrast, EPA and ALA were unrelated to any measure of cognitive performance. In a recent randomized controlled trial, 176 healthy adults aged between 18
45 years of age with a low intake of DHA were supplemented with 1.16 g DHA per day over a 6-month period and cognitive performance was assessed using a computerized cognitive test battery (Stonehouse et al., 2013). Reaction time of working memory in men and episodic memory in women were improved in the DHA supplemented group when compared with the placebo group. Altogether these data suggest that brain DHA plays an important role in the maintenance of learning and memory performance in adults and that DHA may improve memory performance by enhancing synaptic plasticity in the hippocampus.

DIETARY FISH OIL AND PREVENTION OF AGE-RELATED MEMORY DECLINE Since DHA levels decrease with age and as this nutrient plays a major role in brain function, mainly in learning and memory, several studies, both in animals and humans, have investigated the effects of LC n-3 PUFA supplementation, mainly in the form of DHA or fish oil, on memory performance during aging. Old age is the main risk factor for Alzheimer’s disease since its prevalence increases with age. Numerous studies have investigated the effects of LC n-3 PUFA

supplementation in Alzheimer’s disease patients or in animal models of this pathology (review in Boudrault et al., 2009; Cole et al., 2009; Cunnane et al., 2009; Huang, 2010). A recent meta-analysis showed that in experimental animal models of Alzheimer’s disease, an LC n-3 PUFA supplementation improved cognitive function and reduced the amount of brain amyloid-β (Hooijmans et al., 2012). However, in humans, randomized controlled trials (RCTs) have failed to demonstrate a benefit of LC n-3 PUFA supplementation in patients with Alzheimer’s disease, except in subjects with the mild form of the disease or mild cognitive impairments (MCI), supporting the idea of a preventive effect of LC n-3 PUFAs rather than a potential treatment modality (Solfrizzi et al., 2010). Thus, in a goal of nutritional prevention of Alzheimer’s disease, the LC n-3 PUFA supplementation must be given before irreversible damage has accumulated in the brain of Alzheimer’s disease patients. For this reason, in this review we only reported studies investigating the effects of LC n-3 PUFA supplementation during normal aging in humans, i.e. subjects with MCI or healthy elderly, or in animals, excluding experimental Alzheimer’s disease models.

Animal Studies Several studies have investigated the effects of LC n-3 PUFA supplementation on learning and memory in rodents during aging. They mostly differ depending on the animal species, the route of administration, the dose and source of the LC n-3 PUFAs, the duration of the supplementation, and the behavioral test used to evaluate memory. The great strength in animal models compared to human clinical trials or epidemiological studies is that they afford the opportunity to control experimental variables such as the composition of the diet and the environment. The few studies that have investigated the effects of fish oil containing both EPA and DHA, in various ratios, in very old animals (20
25 months) when compared with young ones (1
5 months) on memory performance during aging give conflicting results. Two studies conducted in 2-year-old rats have not evidenced any effect of a fish oil supplementation over periods of either 1 or 4 months on learning and memory capacities (Barcelo-Coblijn et al., 2003a; Sergeant et al., 2011). Barcelo-Coblijn et al. (2003a) have shown that 2-year-old rats receiving a fish oil enriched diet (11% DHA 1 3% EPA) for one month did not perform better in the Morris water maze learning test than the 2-month-old control rats. However, these authors only evaluated the learning ability of the rats to find the hidden platform in the Morris water maze over a

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5-day period, but the memory performance using the classical probe test without platform was not evaluated. More recently, when using rats of the same age as in the Barcelo-Coblijn study but supplemented with fish oil (12% DHA 1 18% EPA) for a longer period (4 months), Sergeant et al. (2011) did not observe any beneficial effect of fish oil supplementation on spatial learning or reference memory performance in very old rats (28 months) when compared with 6-month-old rats. Altogether these results suggest that dietary fish oil supplementation given at a late stage of life is not effective in the prevention of age-related memory decline. In a recent study, Labrousse et al. (2012) investigated the preventive effects of a two-month fish oil enriched diet on age-related memory decline in 20-month-old mice. The results of this study have shown that spatial working memory deficits evaluated in the Y-maze were reduced in aged mice fed with the fish oil enriched diet (10% EPA and 7% DHA) when compared with aged mice receiving a DHA-free diet (Labrousse et al., 2012). The effect of life-long consumption of fish oil was studied by Carrie´ et al. (2000b). Pregnant OF1 mice were fed on diets enriched with or without fish oil (7% DHA 1 12% EPA), offspring were maintained on these diets after weaning, and behavioral tests were performed at 7
10 weeks, 9
10 months, and 17
19 months of age. A beneficial effect of fish oil supplementation on spatial reference memory performance, evaluated by the retention of platform location in the Morris water maze, was only observed in the group of mice aged 9
10 months, but not in younger or older mice. In an active avoidance paradigm, mice aged 7
10 weeks that were supplemented with fish oil performed better during the first training session than age-matched controls, whereas the opposite effect was observed in the oldest supplemented mice. This group displayed worse avoidance behavior, suggesting a negative effect of fish oil supplementation on learning performance on this task. Since PUFAs are readily oxidizable, the authors hypothesized that long-term fish oil supplementation may result in the formation of oxidation products in the brain leading to decreased memory performance (Carrie´ et al., 2000b). In an as yet unpublished study we have investigated the potential preventive effect of fish oil on memory decline during aging when the supplementation is given at middle-age. Our results show that fish oil supplementation (18% EPA and 12% DHA) in the diet for 5 months in middle-aged rats (from 13 to 18 months of age) improved spatial working memory, but not reference memory, when evaluated by a delayed matching-to-place paradigm in the Morris water maze (Alfos et al., unpublished data). Two other studies have recently been conducted on a non-human

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primate, the gray mouse lemur (Languille et al., 2012; Vinot et al., 2011). Languille et al. (2012) observed that 14 weeks of fish oil supplementation (6 mg EPA and 30 mg DHA per day) in 6- to 9-year-old female mouse lemurs did not significantly improve spatial memory performance in the Barnes maze task, even if the fish oil diet supplemented animals tended to find the target compartment more frequently than the control animals when tested after a 24-hour delay. These results are in contrast to those by Vinot et al. (2011), whose study findings showed that the same fish oil supplementation (6 mg EPA and 30 mg DHA per day) for 5 months in 2-year-old mouse lemurs improved spatial memory performance in the Barnes maze task. It should be noted that for mouse lemurs, the median survival time is 4.9 years and the maximum lifespan is 8
10 years. Thus, some of the animals used in the study of Languille et al. were old and others were middle-aged. This difference in age may contribute to the great variability of the results obtained, leading to a nonsignificant effect of fish oil in this study, whereas in the study by Vinot et al. the animals were younger. These results suggest that fish oil supplementation in aged animals may have beneficial effects on the prevention of memory decline if it is given at middle-age rather than later in life. As fish oil is composed of both EPA and DHA at various concentrations, it is difficult to distinguish the specific effects of DHA or EPA in supplementation studies. For this reason and because DHA is the main LC n-3 PUFA in the brain, some authors have investigated the specific effect of DHA alone, and more rarely of EPA alone, on learning and memory performance in aged animals. Gamoh et al. (2001) demonstrated, in 25-month-old rats fed a fish oil deficient diet for three generations, that peroral administration of DHA 300 mg/kg/day, in the form of ethyl ester for 10 weeks, improved working and reference memory, as evaluated in a partially baited eight-arm radial maze. This effect of DHA supplementation on memory performance was associated with an increase in DHA levels in the cortex but not in the hippocampus and a decrease in lipid peroxide levels in the hippocampus (Gamoh et al., 2001). The effect of a DHA-fortified oil fraction extracted from a green alga, Chlorella vulgaris, containing 20% DHA and only 3% EPA of total fatty acids, was also investigated in middle-aged mice (Sugimoto et al., 2002). The authors showed that DHAfortified oil fraction supplementation (2 g/100 g diet) for 2 months in 9-month-old mice increased brain levels of DHA and EPA, and improved working memory, but not reference memory, in a partially baited eight-arm radial maze, compared to control mice receiving a standard diet. Two studies compared the dose effect of DHA supplementation on memory in

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aged animals. In the first study, Lim and Suzuki (2000) investigated the effect of different doses of DHA on maze-learning ability in young (3-week-old) and middle-aged (14-month-old) mice. Mice were fed for 5 months with diets enriched with different sources of DHA, ethyl ester, or/and PC from eggs. Thus, the experimental diets contained different amounts of DHA: 0.9 g/100 g fatty acids in the egg-PC group, 13.8 g/100 g in the egg-PC 1 DHA-ethyl ester group, and 23.7 g/100 g in the DHA-ethyl ester group. Globally, old mice supplemented with DHA, regardless of form or dose, took less time to reach the maze exit and strayed fewer times in the blind alleys of the maze, showing an increased learning ability. Jiang et al. (2009) also investigated the effects of two oral gavage doses of DHA, 50 mg or 100 mg DHA/kg/day, for 7 weeks, on the cognitive ability of 15-month-old female mice assessed by step-through and passageway water maze tests. The results showed that 7-week DHA supplementation in middle-aged mice significantly improved learning and memory compared to control mice and also demonstrated a dose effect of the DHA on memory performance. More recently, Kelly et al. (2011) studied the effect on memory performance in aged rats of two specific LC n-3 PUFAs, EPA and DPA, which is an intermediate product in the biosynthesis of DHA from EPA (Sprecher, 2000). Compared with aged control animals, 20
22-monthold rats supplemented with EPA or DPA (200 mg/kg/ day) for 56 days performed better in a spatial learning task using the Morris water maze (Kelly et al., 2011). Moreover, the EPA supplementation, but not the DPA, induced an increase in DHA level in the cortical tissue of aged rats. The animal studies described above globally demonstrate that fish oil supplementation in aged animals has beneficial effects on the prevention of memory decline if it is given at middle-age rather than later in life. Moreover this result could be more specifically attributed to DHA since a DHA supplementation alone leads to an improvement of memory in aged animals. However, in the fish oil containing both EPA and DHA, the EPA can be efficiently converted to DHA in the liver (Gao et al., 2009). The beneficial effect of fish oil supplementation on memory observed in animals during aging must be confirmed in humans by RCTs.

Human Studies Several epidemiological studies have investigated the link between n-3 PUFA intake or fish consumption and cognitive decline in the elderly (Fotuhi et al., 2009). Most of these studies suggest that an EPA 1 DHA intake in the form of fatty fish can reduce the

risk of cognitive decline in elderly subjects with cognitive impairment no dementia (CIND) i.e. subjects with MCI or memory complaint without a dementia diagnosis of Alzheimer’s disease. In the Etude du Vieillissement Arte´riel (EVA) study, erythrocyte membrane lipid composition was used as a measure of omega n-3 fatty acid intake in 246 healthy men and women whose cognitive function was assessed by the Mini-Mental State Examination (MMSE). The results showed that the proportion of total n-3 PUFAs in the erythrocyte membrane was inversely associated with the risk of cognitive decline (Heude et al., 2003). In the Atherosclerosis Risk in Communities study based on 2,251 subjects, the risk of global cognitive decline increased with elevated plasma palmitic acid, and arachidonic acid (AA) and higher n-3 PUFAs in the plasma reduced the risk of decline in verbal fluency (Beydoun et al., 2007). Moreover, lower red blood cell DHA levels were associated with smaller brain volumes and reduced performance on tests of visual memory, executive function, and abstract thinking in persons free of clinical dementia (Tan et al., 2012). Two other studies also showed that cognitive test scores in elderly subjects were higher in fish oil supplement users than in non-users and this was associated, in one study, with an increase in erythrocyte membrane n-3 PUFA content in fish oil supplement users (Gao et al., 2011; Whalley et al., 2004). Other cross-sectional or longitudinal population-based studies have shown that fatty fish intake was associated with a reduced risk of impaired cognitive function in non-demented subjects (Kalmijn et al., 2004; Kesse-Guyot et al., 2011; Morris et al., 2005; Roberts et al., 2010; van Gelder et al., 2007). In the Zutphen Elderly Study, the authors estimated that an average difference in daily DHA 1 EPA intake of approximately 380 mg is associated with a 1.1 point reduction in cognitive decline over 5 years (van Gelder et al., 2007). These beneficial effects of fatty fish consumption observed in epidemiological studies, associated with the results obtained in animals studies, have encouraged the set-up of RCTs to investigate the protective properties of DHA and other n-3 PUFAs on cognitive performance in the elderly. A recent meta-analysis has examined the neuropsychological benefits of n-3 PUFAs in randomized double-blind placebo-controlled studies in healthy, CIND or Alzheimer’s disease subjects (Mazereeuw et al., 2012). This meta-analysis of studies published up to September 2011 combined 10 RCTs in subjects over 65 years: among them two RCTs were specifically conducted on Alzheimer’s disease subjects, three in healthy elderly, four in subjects with MCI or memory complaint and one in both Alzheimer’s disease and MCI subjects. The authors of this meta-analysis concluded that n-3 PUFA

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supplementation has no benefit on global cognitive performance in Alzheimer’s disease patients as measured by the MMSE or the Alzheimer’s Disease Assessment Scale-Cognitive Subscale (ADAS-cog). There was no global effect of n-3 PUFAs on composite memory but when examined by domain, the metaanalysis indicated that the effects of n-3 PUFAs were limited to CIND subjects for immediate recall, attention, and processing speed. No effect was observed in healthy elderly subjects or those with Alzheimer’s disease even when cognitive subdomains were assessed. In the five studies including CIND subjects only, DHA supplementation had a beneficial effect mostly in verbal fluency (Johnson et al., 2008; Sinn et al., 2012), immediate and delayed verbal recognition memory, but not on working memory (Yurko-Mauro et al., 2010), immediate memory and attention (Kotani et al., 2006), or immediate and delayed verbal recall and learning abilities (Vakhapova et al., 2010). It should be noted that these studies differ greatly in duration (from 12 weeks to 108 weeks), in the dose of n-3 PUFA intake (from 240 to 1550 mg per day), and in the form of n-3 PUFA supplementation (DHA alone, DHA-PS or DHA 1 EPA). From this previous metaanalysis new RCTs have been published partly confirming its conclusions. In a small crossover placebo-controlled trial, 40 healthy subjects aged 51
72 years receiving 3 g per day of fish oil for 5 weeks (EPA 1500 mg and DHA 1050 mg) displayed better performance in a working memory test (Nilsson et al., 2012). In another small RCT conducted on seventy 45- to 80-year-old healthy subjects who received 252 mg DHA per day over a 90-day period, no beneficial effect of the supplementation on the cognitive function was observed (Stough et al., 2012). Geleijnse et al. (2012) conducted a large double-blind placebocontrolled trial (Alpha Omega Trial) including 2,911 coronary patients aged 60 to 80 years old consuming margarines (400 mg/day of EPA-DHA and/or 2 g/day of α-LNA) but observed no effect on global cognitive functions measured by the MMSE after 40 months of n-3 PUFA consumption. These two negative studies are in agreement with the meta-analysis of Mazereeuw et al. (2012) showing no effect of n-3 PUFA supplementation either in Alzheimer’s disease or CIND subjects when cognitive functions were only evaluated by the MMSE test, nor in elderly healthy subjects. More recently, an RCT conducted on 36 low socioeconomic-status elderly subjects between 61 to 71 years of age with MCI receiving a daily capsule of 430 mg of DHA and 150 mg of EPA for 12 months showed an improvement in shortterm and working memory, immediate verbal memory and delayed recall capability in the fish oil group (Lee et al., 2013). This last study confirms the beneficial effect of fish oil supplementation in MCI subjects.

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There are now nine RCTs that have assessed the effect of long-chain n-3 PUFA supplementation on cognitive function in elderly subjects with MCI. Results of these studies indicate that a high LC n-3 PUFA intake ( .200 mg/day of DHA) slows down cognitive decline among elderly subjects with MCI without dementia. Such an effect was not observed in patients with Alzheimer’s disease, suggesting that LC n-3 PUFAs are less efficient on memory performance in the later course of the disease. Thus increasing the intake of LC n-3 PUFAs may be a strategy to maintain memory performance in the elderly. Other RCTs that are still in progress should finally ascertain the efficacy of LC n-3 PUFA supplementation alone or in combination with other nutrients in the prevention of cognitive decline in the elderly (Fotuhi et al., 2009). The results obtained on animals, together with epidemiological and RCT evidence, support the idea that LC n-3 PUFAs given in the form of fish oil may have a role in the prevention of age-related memory decline in subjects with mild cognitive impairment. The question still to be addressed is how LC n-3 PUFAs, and mainly DHA, act on neurobiological processes to maintain cognitive function during aging.

DHA IMPROVES SYNAPTIC PLASTICITY DURING AGING: INVOLVEMENT OF RETINOID X RECEPTORS AND PEROXISOME PROLIFERATORACTIVATED RECEPTORS LC n-3 PUFAs may have multiple and complex mechanisms of action in the cells. Among them: 1) they could modulate the structure and the function of neuronal membranes by modifying the composition of phospholipids and/or by acting on membraneincorporated proteins such as G-protein coupled surface receptors; 2) they can also be metabolized into lipid mediators which are involved in the regulation of many tissue functions (Calder, 2012; Schmitz and Ecker, 2008). Moreover LC n-3 PUFAs can act as direct ligands of transcription factors that modulate gene expression (Afman and Muller, 2012). Indeed, animal experiments and human studies have shown that LC n-3 PUFAs regulated gene expression in various tissues. Some studies on the effect of fish oil supplementation on gene expression have been conducted in humans by using microarray technology in peripheral blood mononuclear cells (PBMC) that are easily obtainable. In elderly subjects aged between 66 and 80 years, fish oil supplementation for 26 weeks changed the expression of 1,040 genes in PBMC leading to a more anti-inflammatory and anti-atherogenic gene expression profile (Bouwens et al., 2009). More recently, it

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has been shown that 6-month fish oil supplementation in elderly subjects with Alzheimer’s disease induced up- or down-regulation of several genes in PBMC (Vedin et al., 2012). Further studies in animals have demonstrated that fish oil supplementation in mice and rats modulated the expression of several genes in the liver and the brain, and more specifically in the hippocampus, both in adult (Barcelo-Coblijn et al., 2003b; Berger et al., 2002) and old animals (Barcelo-Coblijn et al., 2003a; Kitajka et al., 2002; Puskas et al., 2003). Through these effects on gene expression, LC n-3 PUFAs may modulate the expression level of synaptic plasticity-related genes that sustain learning and memory processes impaired during aging (Blalock et al., 2003; Lee and Silva, 2009). Indeed, EPA or DHA supplementation in aged rats (20
22 months old) for 2 months reversed the age-related decrease in long-term potentiation (LTP) in the hippocampus (Martin et al., 2002; McGahon et al., 1999). Moreover, Kelly et al. (2011) have shown that the beneficial effect of EPA or DPA (200 mg/kg/day) supplementation for 56 days on a spatial learning task in rats aged 20
22 months old was associated with an improvement of synaptic plasticity since DPA and EPA supplementation can attenuate the age-related decrease in LTP in the hippocampus. In adult (12-week-old) fat-1 transgenic mice with high endogenous DHA levels compared to wildtype mice, expression of synaptic plasticity-related genes such as synapsin-1, glutamate receptor subunit GluR1, neuromodulin (GAP-43) and post-synaptic density protein-95 (PSD-95) were up-regulated in the hippocampus and water maze memory performance was improved (He et al., 2009). In the aged (24-month-old) rat brain, the decrease in the levels of GluR2 and NR2B glutamate receptor subunits involved in synaptic plasticity was reversed after a 12-week fish oil supplementation (160 mg EPA and 110 mg DHA/kg per day) (Dyall et al., 2007). In an as yet unpublished study we have shown that a 5-month fish oil supplementation (18% EPA and 12% DHA) in the diet of middle-aged rats (from 13 to 18 months old) restored GAP-43 mRNA levels in the hippocampus to that of adult rats and induced an increase in hippocampal RC3 mRNA expression (Alfos et al., unpublished data). This effect of fish oil supplementation on synaptic plasticity was associated with an improvement of spatial working memory in aged rats (Alfos et al., unpublished data). Altogether, these results suggest that LC n-3 PUFAs might improve or maintain memory performance by, partly, modulating the expression of synaptic plasticityassociated genes in the aging brain, thus reinforcing the strength of connections between neurons. LC n-3 PUFAs regulate gene expression by binding to a variety of nuclear receptors including those belonging to the steroid/thyroid hormone nuclear

receptor superfamily (Vanden Heuvel, 2012). Indeed, it was initially reported in the 1990s that EPA, DHA, and some of their derivatives act as endogenous ligands of the peroxisome proliferator-activated receptors (PPARs) (Krey et al., 1997). More recently, DHA has also been shown to bind the retinoid X receptors (RXRs), primarily involved in the transduction of the retinoid signaling pathway (de Urquiza et al., 2000; Lengqvist et al., 2004). RXRs can also be activated by 9-cis retinoic acid (9-cis RA), one of the active metabolites of vitamin A, and this receptor is the obligate common dimerization partner of numerous nuclear receptors, including PPARs, retinoic acid receptors (RARs, activated by all-trans retinoic acid), the vitamin D receptor (VDR), thyroid hormone receptors (TRs) (Desvergne, 2007; Lefebvre et al., 2010). Therefore, RXRs are master regulators of multiple distinct biological pathways (Dawson and Xia, 2012). Nevertheless, there is a debate regarding the physiological ligands of RXRs (Wolf, 2006), as 9-cis RA is very difficult to detect in vivo because of its instability and its very low concentration (Kane, 2012). The RXRs are a converging point in mediating the physiological effects of retinoids and LC n-3 PUFAs either by directly binding retinoids and DHA or by acting as a heterodimerization partner of RARs and PPARs. It is now well established that these nuclear hormone receptors act as transcription factors which, upon activation by their ligands, modulate the transcription of several target genes by binding on specific sequences on DNA (Chambon, 1996; Gronemeyer et al., 2004). Several isotypes of these receptors are widely expressed in various regions of the adult brain and notably in the hippocampus, both in rodents and humans, suggesting a role of these receptors in brain functions (Krezel et al., 1999; Moreno et al., 2004; Rioux and Arnold, 2005; Zetterstrom et al., 1999). In the adult brain, these nuclear receptors modulate the transcription of target genes involved in synaptic plasticity processes, suggesting that these nuclear hormone receptors play major roles in learning and memory (Lane and Bailey, 2005; McCaffery et al., 2006; van Neerven et al., 2008). Fatty acids can modulate the expression of some nuclear receptors such as PPARs, RARs, and RXRs. Indeed, Hajjar et al. (2012) have shown that 10-week fish oil consumption in young rats improved spatial learning and memory in the Morris water maze and increased the expression of PPARβ and PPARγ in the hippocampus. Moreover, we have also shown that a high-fat diet in rats induced a decreased expression of RARβ and PPARγ mRNA and an increased expression of RXRβ/γ, associated with changes in the expression level of RC3 and GAP-43, both in the striatum and hippocampus (Buaud et al., 2010).

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Using a gene microarray approach Blalock at al. (2003) have shown that RXRγ gene expression was one of the several genes down-regulated in the CA1 region of the hippocampus in aged (24-month-old) and middle-aged (14-month-old) rats. This age-related down-regulation of RXRγ gene expression was positively correlated with impaired memory performance evaluated in the Morris water maze task and the object memory task. Moreover, it has recently been demonstrated in mice that the reduced despair behavior and improved working memory in mice treated with DHA implicated more specifically the RXRγ isotype (Wietrzych-Schindler et al., 2011). These results highlight the major role of RXRγ in the maintenance of memory processes. We have also previously shown that aging in rodents induced a decrease in the expression of some nuclear hormone receptors, mainly RARβ and RXRβ/γ in the brain and more particularly in the hippocampus, which was associated with a decreased expression of some of their target genes involved in synaptic plasticity such as neurogranin (RC3) and GAP-43 (Boucheron et al., 2006; Enderlin et al., 1997; Feart et al., 2005). This hypo-expression of nuclear receptors and target genes involved in synaptic plasticity in the aging brain can be reversed by RA administration or vitamin A supplementation which also reverses the age-related memory deficit (Etchamendy et al., 2001; Mingaud et al., 2008; Touyarot et al., 2013). As for the retinoids, we can postulate that an age-related decrease in the brain DHA level leads to a hypo-activation of fatty acid nuclear receptors and of their target genes which can be reversed by LC n-3 PUFA supplementation. In fact, we have recently demonstrated that a 5-month fish oil enriched diet in middle-aged rats (13 to 18 months old) suppressed the age-related decrease in RXRγ mRNA expression and increased RXRβ mRNA expression in the hippocampus (Alfos et al., unpublished data). This effect of fish oil supplementation on RXR expression was concomitantly associated with an increase in GAP-43 and RC3 mRNA, together with an improvement of spatial working memory in aged rats (Alfos et al., unpublished data). Dyall et al. (2007) also observed that the age-related decrease in RXRα, RXRβ and PPARγ protein levels in the rat forebrain were reversed by 12 weeks of fish oil supplementation. In order to activate nuclear receptors or to be converted in biologically active derivatives such as neuroprotectin D1 (NPD1) in the cells, DHA must either be directly captured from the plasma in the unesterified form or released from membrane phospholipids by Ca21-independent phospholipase A2 (iPLA2) (Green et al., 2008; Rapoport et al., 2011). As reported above, aging in rats is associated with a reduced unesterified DHA concentration in the plasma (Gao et al., 2013).

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Moreover, the iPLA2 mRNA expression decreased in the rat hippocampus during aging whereas the mRNA expression of cPLA2, involved in AA release from membrane phospholipids, was unchanged (Aid and Bosetti, 2007). These data, in combination with those showing a decrease of DHA in brain phospholipids, suggest that the brain intracellular level of DHA may be reduced during aging, thus leading to a hypoactivation of nuclear receptors which can be reversed by fish oil supplementation. Taken together, these data suggest that one of the mechanisms supporting the effects of LC n-3 PUFAs on synaptic plasticity that underlies memory processes may be the regulation of gene expression in the brain via nuclear hormone receptors and mainly PPARs and RXRs which are able to bind LC n-3 PUFAs and their derivatives. Aging is associated with a decrease in some of these nuclear receptors in the brain, which may reflect a reduction in intracellular DHA level, leading potentially to impaired gene expression. Therefore, LC n-3 PUFAs contained in fish oil may act as positive nutritional modulators that activate RXRs and PPARs and therefore restore normal gene expression and signaling pathways in the brain.

SUMMARY AND CONCLUDING REMARKS An analysis of the data from animal studies indicates that aging is associated with a reduction in DHA levels in specific classes of phospholipids in brain regions involved in memory such as the hippocampus and the cortex. These animal study findings are in contrast to those from studies in humans, which found a slight or no decrease in brain DHA contents during normal aging and a clear decrease in DHA levels in the hippocampus and the cortex in Alzheimer’s disease. These contrasting results in human post-mortem studies may be linked to variations in the age and cognitive status of the subjects studied, but also to variations in life-long consumption of fatty acids among subjects that may influence brain fatty acid content. In animal studies, environmental factors and, in particular the diet, are easily controllable and standardized, thus allowing for fewer variations in the results. The reduced DHA content in brain phospholipids during aging could be the result of a reduction in the activity of the enzymes specifically incorporating DHA in these phospholipids. But an altered metabolism of LC n-3 PUFAs in the liver, as brain DHA is obtained from the unesterified fatty acid pool of the plasma which is maintained by liver synthesis, could also contribute to the decrease in brain DHA content. In order to confirm that intracellular DHA bioavailability is reduced in the

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brain during aging, further studies are needed to measure free (unesterified) DHA and its derivatives in brain structures involved in memory. Considering the major role of DHA and, to a greater extent, LC n-3 PUFAs in the maintenance of synaptic plasticity processes that underlie learning and memory, a reduction in brain DHA level during aging may contribute to memory impairment in the elderly. Therefore LC n-3 PUFA supplementation in the form of fish oil could be one of several valuable strategies to prevent age-related memory decline. Indeed, an analysis of the data presented in this review shows that, in most animal and human studies, a fish oil supplementation has a beneficial effect on the prevention of memory decline during aging. However, in humans, RCTs demonstrate that fish oil supplementation was only able to slow down memory decline in subjects with middle cognitive impairments but not in patients with Alzheimer’s disease. These results indicate that LC n-3 PUFAs act as preventive nutrients on cognitive decline during aging but are less efficient when pathophysiological changes may have accumulated to an irreversible degree. Although LC n-3 PUFAs are major constituents of the brain and play a major role in its functioning, other nutrients that are also involved in the maintenance of normal brain functions (review in Bourre, 2006a; 2006b) could also be effective in the improvement of memory during aging. Indeed, studies in rodents have demonstrated that food supplements containing DHA and EPA, in association with other nutrients such as B vitamins, vitamin C, vitamin E, choline, uridine, and phospholipids, increased the number of dendritic spines in the hippocampus, increased the levels of synaptic proteins, enhanced synaptogenesis and improved memory (de Wilde et al., 2011; Wurtman et al., 2010). However, in humans, as for the single LC n-3 PUFA supplementation, the combined supplementation of LC n-3 PUFAs with other nutrients for 12 to 24 weeks improved memory performance (delayed verbal recall) only in the patients with very mild Alzheimer’s disease (Scheltens et al., 2010; 2012). Several other RCT studies using LC n-3 PUFAs with a combination of other nutrients that are ongoing or in preparation will broaden our understanding of the nutritional prevention of memory decline in elderly healthy or MCI subjects (Mi et al., 2013). Several mechanisms of action could be involved in explaining the beneficial effect of EPA and DHA on the prevention of memory decline during aging. Here we postulate that LC n-3 PUFAs act on synaptic plasticity via nuclear hormone receptors that regulate gene expression. Indeed LC n-3 PUFAs and their derivatives are ligands of PPARs and RXRs that are involved in the maintenance of learning and memory by regulating

genes involved in synaptic plasticity processes. Animal studies suggest that LC n-3 PUFA supplementation during aging could restore hippocampal RXR levels, and mainly RXRγ, that in turn up-regulates synaptic plasticity-associated target genes leading to a reduction in age-related memory decline. Recent study results highlight the major role of RXRγ in the maintenance of adult brain function. Indeed it has been shown that RXRγ is involved in learning and memory processes (Wietrzych-Schindler et al., 2011) and in remyelination processes after brain injury (Huang et al., 2011). It is now well accepted that RXRs play unique modulatory and integrative roles as a converging point in mediating the physiological effects of retinoids and LC n-3 PUFAs either by directly binding retinoids and DHA or by acting as a heterodimerization partner of RARs and PPARs. These data suggest that the beneficial effect of LC n-3 PUFAs on gene expression and memory could be in part mediated by the RXRs, and more particularly RXRγ, by acting as a lipid sensor in the cell or by integrating other nutritional signals. Besides their action on the transcriptional regulation of synaptic plasticity-related genes, nuclear hormone receptors are known to regulate the expression of genes involved in numerous functions in the adult brain that can also be modulated by LC n-3 PUFAs such as inflammation and neurogenesis. Indeed, LC n-3 PUFAs have anti-inflammatory properties by modulating the expression of cytokines and are able to reduce the naturally occurring neuro-inflammation in the aging brain (Calder, 2011; Laye, 2010). PPAR and RXR agonists modulate neuro-inflammation processes mainly by inhibiting pro-inflammatory gene expression (Chung et al., 2008a; Moraes et al., 2006; Xu et al., 2005). LC n-3 PUFAs have also been shown to promote hippocampal neurogenesis both in adult and aged animals (Dyall et al., 2010; He et al., 2009; Kawakita et al., 2006). Several results suggest that nuclear hormone receptors such as PPARs and RARs/RXRs play major roles in neurogenesis by modulating the survival and differentiation of newly proliferated cells (Bonnet et al., 2008; Yu et al., 2012; Touyarot et al., 2013). Other studies are needed to determine the complex mechanisms of action of LC n-3 PUFAs on memory processes during aging. As previously discussed, it is now well recognized that vitamin A and its derivatives, the retinoids, play major roles in the synaptic plasticity process and in learning and memory in the adult brain. More specifically, results from our laboratory have demonstrated that pharmacological activation of retinoid signaling by short-term treatment with RA, the active metabolite of vitamin A, or life-long nutritional vitamin A supplementation, have beneficial effects on hippocampal plasticity and restore impaired memory performance in aged mice (Etchamendy et al., 2001;

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

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Mingaud et al., 2008; Touyarot et al., 2013). In the same way our recent results in middle-aged rats supplemented for 5 months with fish oil show a beneficial effect of LC n-3 PUFAs on synaptic plasticity markers associated with an improvement of spatial working memory (Alfos et al., unpublished data). Moreover, RXRs can both bind DHA and 9-cis retinoic acid and are the obligate heterodimerization partner of RARs. These data suggest that the potential synergistic effects of a combined DHA and retinoid supplementation on nuclear receptor signaling pathways, synaptic plasticity, and memory should be investigated in aged animals.

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13. FISH OIL SUPPLEMENTATION PREVENTS AGE-RELATED MEMORY DECLINE: INVOLVEMENT OF NUCLEAR HORMONE RECEPTORS

McNamara, R.K., Carlson, S.E., 2006. Role of omega-3 fatty acids in brain development and function: potential implications for the pathogenesis and prevention of psychopathology. Prostaglandins Leukot. Essent. Fatty Acids 75, 329
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C H A P T E R

14 Role of Omega-3 Fatty Acids in Brain and Neurological Health with Special Reference to Clinical Depression H.M. Chandola and Ila Tanna INTRODUCTION Mood disorders, including major depression, are recurrent, debilitating and potentially life-threatening diseases. Depression is the most common psychiatric disorder affecting about 121 million people worldwide. More than 90% of people who commit suicide have a diagnosable mental disorder, commonly a depressive disorder. In 2000, it was the third leading cause of death among 15 to 24 year olds. The World Health Organization (WHO) estimates that major depressive disorder will become the second leading cause of disability worldwide by 2020, which is second only to ischemic heart disease. The earlier age of onset and increased frequency of depression over the last 100 years are likely related to neurobiology, physiology, genetics, stress, and environmental factors, but a role for nutrition in depressive symptoms has been underestimated. Despite advances in pharmacotherapy, a significant proportion of depressed patients are considered treatment-resistant (Kornstein and Schneider, 2001). Poor compliance, side effects, or lack of desired effects are not uncommon with anti-depressant medications. Lack of desired results encourages the continued search for improvements in current pharmacotherapy and novel treatments. Omega-3 fatty acids in particular represent an exciting area of research as a new potential agent in the treatment of depression. Omega-3 fatty acids promote transmission of the chemical messengers that facilitate communication between nerve cells and are associated with emotional stability (e.g., serotonin) and positive emotions (e.g., dopamine). Omega-3 fatty acids have been linked to depressive conditions such as bipolar disorder,

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00014-4

unipolar depression, borderline personality disorder, premenstrual syndrome, and perinatal depression. Omega-3 fatty acids affect brain-derived neurotrophic factor, which encourages synaptic plasticity, provides neuroprotection, enhances neurotransmission and has anti-depressant effects (Ikemoto et al., 2000).

OMEGA-3 FATTY ACIDS Brain development is a complex interactive process in which early disruptive events can have longlasting effects on later functional adaptation. It is a process that is dependent on the timely orchestration of external and internal inputs through sophisticated intra- and intercellular signaling pathways (Wainwright, 2002). Nerve tissue possesses one of the highest concentrations of fatty acids in the body, with approximately 50
60% of the dry weight of the adult brain comprised of lipids, of which approximately 35% are in the form of long chain polyunsaturated fatty acids (LCPUFAs), mainly arachidonic acid (AA) (20: 4n-6) and docosahexaenoic acid (DHA) (22: 6n-3). These LCPUFAs are derived through biosynthesis from their respective dietary essential fatty acid precursors, linoleic acid (LA) (18: 2n-6) and α-linolenic acid (ALA) (18: 3n-3), or they can be obtained directly from dietary sources such as eggs, fish, and meat or, more recently, from single-cell oils (Wainwright, 2002). These long chain fatty acids provided in food are essential for both the structure and function of nerve cells. The three most nutritionally important omega-3 fatty acids are ALA, eicosapentaenoic acid (EPA)

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14. ROLE OF OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH WITH SPECIAL REFERENCE TO CLINICAL DEPRESSION

Omega-6 series

Metabolic enzymes

Linolenic acid

Omega-3 series Alpha-linolenic acid

(18:2 n-5)

(18:3 n-3) Delta-6-desaturase

Gamma-linolenic acid

Steridonic acid

(18:3 n-6)

(18:4 n-3) Elongase

Dihomo-gamma-linolenic acid

Eicosatetraenoic acid

(20:3 n-6)

(20:4 n-3) Delta-5-desaturase

Arachidonic acid

Retroconversion

Eicosapentaenoic acid

(20:4 n-6)

(EPA) (20:5 n-3) Elongase

Adrenic acid

Docosapentaenoic acid

(22:4 n-6)

(22:5 n-3) Delta-4-desaturase

Docosapenteanoic acid (22:5 n-6)

Docosahexaenoic acid (DHA) (22:6 n-3)

FIGURE 14.1 Metabolic pathways for polyunsaturated fatty acids. Source: Parker et al., 2006.

(20: 5n-3), and DHA. Omega-3 fatty acids are LCPFAs found in various plant and marine life (Leaf and Weber, 1987). The marine-based omega-3 fatty acids primarily consist of EPA and DHA and appear to be highly biologically active. In contrast, those from plants (flax seed, walnuts, and canola oil) are usually in the form of the parent omega-3 fatty acid, ALA. This is considered to be an essential fatty acid since it cannot be synthesized by the body and hence must be supplied by dietary sources. Although dietary ALA can be endogenously converted to EPA and DHA (see the metabolic pathways in Figure 14.1), research suggests that this occurs inefficiently at a rate of only 10
15% (Eaton and Konner, 1985). There are many factors that interfere with the conversion of ALA to long chain fatty acid synthesis in the body.

Factors that Interfere with Conversion of ALA There are essentially four things that influence the conversion of ALA and hence which influence long chain fatty acid synthesis in the body. Omega-3 to Omega-6 Ratio A factor which is relatively consistent among individuals is related to another polyunsaturated fatty acid, LA, an omega-6 fatty acid. This is also an essential fatty acid and must be consumed through the diet. The

issue is that the same enzymes that convert ALA, also convert LA into its long chain metabolite AA (20: 4n6). Given this scenario it is understandable that diets high in omega-6 fatty acids can influence, and in reality reduce, the conversion of short chain omega-3 fatty acid into its long chain metabolites. Widespread use of cotton seed oil, vegetable oil (corn, safflower, and soyabean) leads to much greater intake of omega-6 to omega-3 fats. Since the conversion enzyme for omega-3 and omega-6 to their long chain metabolites is common (Figure 14.1), high intake of omega-6 preoccupies these enzymes and they are then no longer available for converting omega-3 to their long chain fatty acids i.e., ALA to EPA and DHA. This imbalance can lead to the conversion enzymes getting used up for omega-6, restricting omega-3 conversion. This has resulted in a high proportion of the common omega-6 fatty acid AA, rather than EPA (a well documented inhibitor of pro-inflammatory cytokines such as IL-1 β and TNFα), in the cell membranes of most tissues, leading in turn to a high proportion of inflammatory eicosanoids (Endres, 1993). An increase in AA also affects the production of EPA and DHA, owing to competition for metabolizing enzymes. Such dietary changes in fatty acid intake have been held to have numerous pathological consequences. The sharp rises in rates of depression and other neurological disorders in the 20th century are being fueled by increased consumption of vegetable oils rich in

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

STATUS OF OMEGA-3 FATTY ACIDS IN CLINICAL DEPRESSION

omega-6 fatty acids (Smith, 1991; Hibbeln and Salem, 1995). Indirect support for that hypothesis emerges from data indicating high levels of inflammatory eicosanoids derived from AA in both patients with unipolar depression and those with bipolar depression (Lieb et al., 1983). Genetic Factors The second factor is genetic variations in gene encoding for delta-5 desaturase and delta-6 desaturase enzymes. These variations have been reflected in changes in desaturase expression or activity, and have been found to influence long chain fatty acid levels in the blood. (Glaser et al., 2010). Dietary and Lifestyle Factors It has been shown that high intakes of saturated fats, trans-fat, alcohol, and caffeine can have a detrimental effect on the role of delta-6 desaturase (Horrobin, 1993). The result is impaired synthesis of long chain fatty acids. SATURATED FAT
TRANS-FAT

Fats compete for the conversion pathways. Consumption of saturated and hydrogenated fats causes a double whammy to the body’s ability to convert good fats to DHA and EPA. Trans-fatty acids (found in hydrogenated vegetable oils in shortenings, deep fat fried and processed foods) cripple the conversion enzymes irreparably. Tissues have to manufacture brand new enzymes to replace those damaged by trans-fats. CAFFEINE, NICOTINE, ALCOHOL, SUGAR

Too much caffeine from coffee and tea, nicotine from smoking cigarettes, and even sugar can set the body into a downward spiral of artificial stimulus that just results in an ever-increasing psychological and eventual physiological addiction. The result is just more stress on the body and a reduced chance of optimal conversion of good fats to DHA and EPA. They can lead to higher levels of insulin in the body, which presents another potential stumbling block for conversion. Even alcohol abuse poisons conversion enzymes. STRESS

Stress is the constant state of emergency for energy, which diverts the body’s focus from digestion, absorption, and complex nutrient conversion. The result is that, no matter how much flax one eats, the body could be too busy keeping one awake, maintaining increased levels of heart beat and blood flow, to be able to efficiently convert omega-3 fatty acids to DHA and EPA.

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Co-nutrients A final influencing factor is the effect that other components of the diet have on conversion. Zinc, magnesium, selenium, vitamin B complex, and vitamin C are all necessary co-factors for elongase and desaturase enzymes. Deficiencies in these nutrients can have significant effects on long chain fatty acid synthesis (Pawlosky et al., 2001; Burdge and Calder, 2005).

STATUS OF OMEGA-3 FATTY ACIDS IN CLINICAL DEPRESSION A number of epidemiological studies support a connection between dietary fish/seafood consumption and a lower prevalence of depression. A significant negative correlation has been reported between worldwide fish consumption and rates of depression (Hibbeln, 1998). Separate research involving a random sample within a nation confirms the global findings, as frequent fish consumption in the general population is associated with a decreased risk of depression and suicidal ideation (Tanskanen et al., 2001). A number of investigations have found a decreased omega-3 content in the blood of depressed patients (Adams et al., 1996; Peet et al., 1998; Maes et al., 1999; Tiemeier et al., 2003). In fact, EPA content in RBC phospholipids is negatively correlated with the severity of depression, while the omega-6 fatty acid AA to EPA ratio positively correlates with the clinical symptoms of depression (Adams et al., 1996). In addition, a negative correlation between adipose tissue DHA and depression has been observed. Mildly depressed subjects had 34.6% less DHA in adipose tissue than non-depressed subjects (Mamalakis et al., 2002). Overall, the reports show that the severity of depression increases as the concentrations of EPA, DHA, ALA, and total omega-3 fatty acids fell. Very recently, a survey study was carried out to evaluate the omega-3 fatty acid deficiency in depressed subjects. For that, depressed subjects (n 5 130) and non-depressed healthy control subjects (n 5 100) were evaluated for their consumption of dietary articles containing omega-3 fatty acids. The results of the survey study proved that the depressed subjects ingested significantly lower amounts of omega-3 fatty acids and the nutrient co-factors that support the function of conversion enzymes (p , 0.001) and higher amounts of dietary articles which interfere with the conversion of ALA to EPA and DHA (p , 0.001). This makes them omega-3 deficient and makes them prone to depression. Depressed subjects were found to be more deficient in omega-3 fatty acids provided by eggs, seeds, and nuts (sesame

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Egg

Source of selenium, has moderate serotonin concentration

Almond

Selenium, zinc, tryptophan

Walnut, cashew nuts

Selenium, tryptophan, rich in omega-3 fatty acid

Figs

Zinc, tryptophan

Sesame seeds

High in zinc, magnesium, calcium, which helps in serotonin production

Fennel seeds

Tryptophan

Cabbage

Selenium, zinc, vit BC, folic acid

Green leafy veg

vit B12, vit B6, vit C, folic acid, magnesium

Tomatoes

Serotonin, glutathione, folic acid

Citrus fruits

Magnesium, selenium, vit B12, folic acid

Kiwi

Serotonin, folic acid

seeds, walnuts, almonds, figs), spices (basil, cloves, pepper, fennel seeds), vegetables (cabbage, pumpkin, tomatoes, green leafy vegetables), and fruits (mangoes, citrus fruits, kiwis, strawberries) compared to non-depressed healthy subjects (p , 0.001). Moreover, they were consuming larger amounts of trans-fat offered by packaged snack foods, chips, deep fried foods, and sweet articles (p , 0.001), which hampers the conversion of ALA to EPA and DHA. Lastly, the authors concluded that omega-3 fatty acid deficiencies combined with adverse life events, childhood adversities, disharmony in marital life, psychological stress, sleep disturbances, lack of exercise, addictions such as tobacco and alcohol are the contributing factors or risk factors for depression (Tanna et al., 2013).

FIGURE14.2 Deficiency of omega-3 fatty acids and other nutrient co-factors affecting omega-3 status in depression.

Selenium Deficiency Lowered levels of selenium have been associated with negative mood scores in at least five studies. Selenium plays a significant role in the human antioxidant defense system. In addition, selenium deficiency can interfere with the normal conversion of ALA into EPA and DHA, resulting in an increase in the omega-6 to omega-3 ratio (Logan, 2004).

While analyzing these articles, it became apparent that, as well as being sources of omega-3 fatty acids, they are also sources of selenium, zinc, tryptophan, antioxidants, folic acid, serotonin, and vitamin B12 (Figure 14.2). Deficiency in these nutrients is also linked with depression as well as with the status of omega-3 fatty acids.

Folic Acid Deficiency A growing body of research has documented the low levels of folic acid among patients with depression (Paul et al., 2004). In addition, there are small clinical trials showing a beneficial effect of folic acid in depression, and its ability to enhance the effectiveness of anti-depressant medications at just 500 mcg (Paul et al., 2004; Coppen and Bailey, 2000). Folic acid has been shown to increase omega-3 status when supplemented, and to decrease omega-3 status when it is deficient in animal models (Pita and Delgado, 2000). In addition, a folic acid deficient diet can enhance lipid peroxidation (Durand et al., 1996). Higher levels of serum folate have been linked to fewer mood swings and negative moods. High folate levels can also improve other depression treatments.

Zinc Deficiency

Antioxidants

A number of studies have shown that zinc levels are lower among patients with depression and a recent study found that a 25 mg zinc supplement may improve depressive symptoms (Logan, 2004).

In patients with major depressive disorder (MDD) there are, in fact, signs of oxidative stress and lipid peroxidation. A recent human study found that depressive symptoms are independently correlated

Nutrient Co-Factor Deficiencies

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NEUROLOGICAL ALTERATIONS IN DEPRESSION

with lipid peroxidation (Tsuboi et al., 2004). Dietary antioxidants are known to influence the antioxidant defense system, and new research suggests that dietary antioxidants can influence omega-3 status (Watson et al., 2004). Omega-3 fatty acids have been shown to decrease lipid peroxidation in vivo (Erdogan et al., 2004), and antioxidant supplementation can prevent the negative influence of saturated fat on brainderived neurotrophic factor (BDNF) levels and cognitive function in animals (Wu et al., 2004b). Vitamin B6, Tryptophan, and Serotonin Tryptophan is a precursor of serotonin. Tryptophanderived food is transported to the brain to make the neurotransmitter serotonin. At the appropriate place inside a brain cell, two enzymes and vitamin B6 transform tryptophan to serotonin (Nutrition Wonderland, 2009). Omega-3 Fatty Acids Essential fatty acids necessary for serotonin production are the omega-3 fatty acids (Nutrition Wonderland, 2009). Thus, omega-3 fatty acid deficiency may lead to depression by hampering serotonin production. Deficiency of nutrient co-factors that support the function of conversion enzymes are vitamins B3, B6, and C, plus the minerals zinc and magnesium, so deficiency in these nutrient co-factors hampers the conversion of ALA to EPA and DHA and, ultimately, leads to depression (Omega Science Institute, 2013).

NEUROLOGICAL ALTERATIONS IN DEPRESSION In order to appreciate the potential role of omega-3 fatty acids in mental health, some of the neurobiological alterations that exist in depression must first be examined. Among the various brain changes induced by stress, the neurohistological changes may be the most fundamental as an explanatory model for depression. The hippocampus is a key structure involved in learning, mood, and memory, especially explicit (consciously acquired) memory. It also regulates the autonomic nervous system (ANS) and neuro-endocrine system (NES). Perhaps as a result of hippocampal impairment, stressed animals and depressed humans show impaired learning and memory (Sapolsky, 2000a; Diamond et al., 2004). It may contribute to dysthymic mood, impaired memory and disturbance in ANS and NES in depression. The ventral tegmental area projects to the nucleus accumbens. Mesolimbic circuitry regulates the response to novelty and the experience of reward. Stressed animals show impaired response to

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novelty. The parallel in depressed humans could be anhedonia. Thus, hippocampal impairment may explain anhedonia as a downstream effect (Pittenger and Duman, 2008). The prefrontal cortex regulates cognitive functions such as attention, concentration, learning and memory. The prefrontal cortex also regulates higher mental functions such as motivation and judgment. All these functions are impaired in depression and contribute to difficulty in concentration, attention deficit, and impairment in motivation and judgment, perhaps as a result of the neurohistological prefrontal changes associated with stress and depression. The amygdala is involved with social and emotional learning and especially with emotions such as anxiety and fear (Sheline, 2004). Depressed humans are often anxious and afraid and show an upregulated anxiety response to minor provocations. Thus, functional changes in different parts of the brain give rise to neuroendocrine, neurovegetative, neurocognitive, neurobehavioral and other deficits, which, put together, form the depressive syndrome (Figure 14.3). There is a growing body of research indicating involvement of the frontal cortex and limbic system, including the hippocampus and the nucleus accumbens (NA) in pathogenesis of depression (Nestler et al., 2002). Research shows decreased levels of dopamine turnover in the prefrontal cortex and dopamine levels that are up to six-fold higher in the NA in animal models of depression (Heidbreder et al., 2000; Zangen et al. 1999). The NA is involved in learning, reward, and motivation, and abnormalities in this area have been linked to major depression (Di Chiara et al., 1999; Brown and Gershon, 1993; Jentsch et al. 2000). Modern brain imaging technology has allowed investigators to examine cerebral blood flow and glucose utilization in patients with major depression. There are blood flow abnormalities in depressed patients, including hypoperfusion in the limbic system and prefrontal cortex. In addition, depressed patients have decreased glucose metabolism in a number of brain regions and this hypometabolic state correlates with severity of depression (Ito et al., 1996; Kimbrell et al., 2002). Reduction in omega-3 intake (in the form of ALA) results in a reduction of omega-3 content throughout the brain cells and organelles along with a compensatory rise in omega-6 fatty acid content. A number of studies have specifically examined the effect of an omega-3 deficient diet on dopamine and serotonin levels in animals. Animals on such a diet have a reduction in the dopaminergic vesicle pool (Zimmer et al., 2000b), along with a 40
60% decrease in the amount of dopamine in the frontal cortex, and an increase in the NA (Delion et al., 1996; Zimmer et al., 2000a), alterations strikingly similar to the animal models of depression described above. Although overall dopamine

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FIGURE 14.3 Neuro-histological changes: The most fundamental model for depression.

levels in the NA are higher in an omega-3 deficient and the animal model of depression, the function of the NA-dopaminergic system appears to be abnormal in both. In an omega-3 deficient model, the release of dopamine from the vesicular storage pool under tyramine stimulation is 90% lower than in rats receiving an adequate omega-3 intake (Zimmer et al., 2000b). In the

animal model of depression, although overall NAdopamine levels are higher, the extracellular levels of dopamine in the NA are lower than normal controls and do not respond to normal serotonin stimulation (Zangen et al., 2001). The increase in dopamine in the NA of omega-3 deficient rats is thought to be a result of loss of normal

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POSSIBLE MECHANISMS FOR LINKS BETWEEN OMEGA-3 FATTY ACIDS AND DEPRESSION

FIGURE 14.4 Potential mechanism of action of inflammatory cytokines in depression and its counteraction by omega-3 fatty acids. Source: Logan, 2003.

Omega-3 fatty acids

Activating HPA axis causes resitance to glucocorticoid hormones

–

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Lower serum tryptophan precursor to serotonin

Pro-inflammatory cytokines

+ Alter neurotransmitter mRNA

Alter neurotransmitter metabolism

Stress depression

inhibitory control by reductions in frontal cortex dopamine input (Chalon et al., 2001). A number of investigators have examined the role of pro-inflammatory cytokines in the patho-physiology of depression. A growing body of research indicates that depression is associated with excessive production of pro-inflammatory cytokines. These cytokines, including interleukin-1beta (IL-1β), -2, and -6, interferon-gamma, and tumor necrosis factor-alpha (TNF-α), can have direct and indirect effects on the CNS. For example, they may lower neurotransmitter precursor availability, activate the hypothalamic pituitary axis, and alter the metabolism of neurotransmitters and neurotransmitter transporter mRNA (Figure 14.4) (Maes and Smith, 1998). Researchers have found elevations in IL-1β and TNF-α are associated with severity of depression (Suarez et al., 2003). Chronic, but not acute, administration of antidepressants can cause an increase in nerve growth factors, particularly BDNF, and these nerve growth factors can play a role in the plasticity and survival of the developed adult nervous system. Serum BDNF has been found to be negatively correlated with the severity of depressive symptoms (Shimizu et al., 2003).

POSSIBLE MECHANISMS FOR LINKS BETWEEN OMEGA-3 FATTY ACIDS AND DEPRESSION Several neurophysiological mechanisms have been proposed to explain the relationship between omega-3 polyunsaturated fatty acids and depression.

Omega-3 Fatty Acids Affect Cell Membrane Integrity and Fluidity One possible mechanism relates to the abundance of DHA in CNS membrane phospholipids, where it plays a vital role in maintaining membrane integrity and fluidity (Yehuda et al., 1998a). The hypothesis that omega-3 polyunsaturated fatty acids can affect cell membrane fluidity is supported by a recent study using magnetic resonance imaging (Hirashima et al., 2004). Twelve women with bipolar disorder received omega-3 fatty acids for 4 weeks and were contrasted with two non-treatment groups. The bipolar subjects receiving omega-3 fatty acids had significant decreases in T2 values, with a dose-dependent effect shown by subdividing the treatment group into patients receiving a high dose (10 g/day) and those receiving a low dose (2 g/day). Another postulated, more direct mechanism involves gene expression and the binding of fatty acids to specific nuclear receptors early in life, leading to genetic transcription (Sampath and Ntambi, 2004) and predisposing subjects to a range of diseases later in life related to DHA and EPA depletion such as Alzheimer’s disease, cardiac disease, and depression. Omega-3 fatty acids are an essential component of CNS membrane phospholipid-acyl chains and, as such, are critical to the dynamic structure of neuronal membranes (Bourre et al., 1991). DHA is continuously secreted by astrocytes, bathing the neuron in omega-3 fatty acid (Williard et al., 2001). An optimal fluidity is required for neurotransmitter binding and the signaling within the cell (Heron et al., 1980). Omega-3 fatty acids can alter neuronal fluidity by displacing cholesterol from the membrane (Yehuda et al., 1998b). By varying

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lipid concentrations in cell membranes, changes in fluidity can affect either the structure or functioning of proteins embedded in the membrane, including enzymes, receptors, and ion channels, leading to changes in cellular signaling. Support for the involvement of omega-3 fatty acids in receptor functioning, neurotransmitter levels, and the metabolism of monoamines implicated in depression has been provided by animal studies (Logan, 2003; Hibbeln and Salem, 1995).

Omega-3 Fatty Acids Decrease the Production of Pro-Inflammatory Cytokines Patients with major depression have been found to exhibit increased peripheral blood inflammatory biomarkers, including inflammatory cytokines, which have been shown to access the brain and interact with virtually every pathophysiological domain known to be involved in depression, including neurotransmitter metabolism, neuroendocrine function, and neural plasticity. Indeed, activation of inflammatory pathways within the brain is believed to contribute to a confluence of decreased neurotrophic support and altered glutamate release/reuptake, as well as oxidative stress, leading to excitotoxicity and loss of glial elements, consistent with neuropathologic findings that characterize depressive disorders. Two omega-3 fatty acids, EPA and DHA, appear to decrease the production of inflammatory eicosanoids from AA by means of two mechanisms (Boissonneault, 2000). First, they compete with AA for incorporation into membrane phospholipids, decreasing both cellular and plasma levels of arachidonic acid. Second, EPA competes with AA for the cyclo-oxygenase enzyme system, inhibiting the production of pro-inflammatory eicosanoids derived from AA (e.g., prostaglandins, leukotrienes, and thromboxanes), and prostaglandin E2 and thromboxane B2 have been linked to depression. DHA and EPA also inhibit the release of pro-inflammatory cytokines, such as interleukin-1 beta, interleukin-2, interleukin-6, interferon-gamma, and TNF-α, which depend on eicosanoid release and are also associated with depression (Logan, 2003). Further, omega-3 fatty acids affect BDNF, which encourages synaptic plasticity, provides neuroprotection, enhances neurotransmission, and has antidepressant effects (Ikemoto et al., 2000).

Omega-3 Fatty Acids and Hippocampal Neurogenesis One of the brain structures associated with learning and memory, as well as mood, is the hippocampus. Interestingly, the hippocampus is one of the two structures in the adult brain where the formation of new

neurons, or neurogenesis, persists. Adult hippocampal neurogenesis (AHN) has been linked directly to cognition and mood (Zhao et al., 2008); therefore, modulation of AHN by diet could emerge as a possible mechanism by which nutrition impacts on mental health. Research shows major depression is associated with neuronal atrophy in the hippocampus (Sapolsky, 2000b) and prefrontal cortex (Rajkowska et al., 1999). Evidence has accumulated that omega-3 fatty acids have an influence on hippocampal neurogenesis by increasing BDNF. Animal studies have revealed that short-term augmentation of dietary omega-3 fatty acids relative to omega-6 fatty acids upregulated adult neurogenesis (Beltz et al., 2007), and that dietary omega-3 fatty acids elevate levels of BDNF, which promotes neuronal survival and growth (Wu et al., 2004a; Wu et al., 2008). BDNF influences the survival of existing neurons and the growth and differentiation of new neurons, and is also involved in the regulation of various neurotransmitter systems (Katoh-Semba et al., 1997; Hashimoto, 2010). Moreover, BDNF infused directly into the dorsal hippocampus of rats significantly increased the granule cell layer, indicating neurogenesis (Scharfman et al., 2005). Further, DHA promotes the development of hippocampal neurons in vitro by increasing neurite extension and branching (Calderon F. et al., 2004), as well as the maturation of neurons and hippocampal neurogenesis in adult rats (Kawakita et al., 2006). DHA dietary supplementation also enhances the effects of exercise on cognition and BDNF-related synaptic plasticity (Wu et al., 2008). A significant correlation was found between omega-3 fatty acid consumption and gray matter volume of the amygdala, hippocampus, and anterior cingulate gyrus in healthy adults (Conklin et al., 2007). Conversely, a selective deficit of DHA was reported in the postmortem frontal cortex of patients with depressive disorder (McNamara et al., 2007).

IMPACT OF DIET ON AHN Diet can impact on AHN from four different parameters (Stangl andThuret, 2009): calorie intake, meal frequency, meal texture, and meal content. Not only do these four parameters modulate AHN in rodents, but independent rodent studies and intervention or epidemiological studies in humans have shown that they also modulate cognitive performance and mood. A reduction in calorie intake of 30
40% increases AHN in rodents, and this effect is partly mediated by BDNF (Lee et al., 2002a; Lee et al., 2002b). Independent of calorie intake, meal frequency is a key player in modulating AHN, so without reducing calorie intake, extending the time between meals increases AHN. It

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CLINICAL TRIALS SUPPORTING THE ROLE OF OMEGA-3 FATTY ACIDS IN MDD

TABLE 14.1

Modulation of Depressive Behavior by Diet

Diet

Author

Effect on Depressive Behavior

Study Model

Omega-3 fatty acids

Jazayeri et al., 2008

Improved EPA

Human

Stoll et al., 1999

Delayed onset of depressive periods

Human (bipolar)

Osher et al., 2005

Decreased

Human (bipolar)

Keck et al., 2006

No benefit 6 g/day EPA

Human (bipolar)

Frangou et al., 2006

Improvement with 1 g/day EPA

Human (bipolar)

Appleton et al., 2006

Various effects with various concentrations of various fatty acids

Human

Flavonoids

Dimpfel, 2009

Improved

Rat

Zinc

Szewczyk et al., 2008

Improved

Rodents

Szewczyk et al., 2008

Improved

Human

Nowak et al., 2003

Improved

Human

Smith AP, 2009

Reduced risk

Human

Caffeine

also changes hippocampal gene expression and correlates with performance in hippocampus-dependent tasks and mood (S. Thuret, unpublished data). Interestingly, food texture also has an impact on AHN; rats fed with a soft diet, as opposed to a solid/hard diet, exhibit decreased hippocampal progenitor cell proliferation. The authors hypothesize that chewing resulting in cell proliferation is related to corticosterone levels (Aoki et al., 2005). Interestingly, independent studies have shown impairment in learning and memory abilities with similar soft diets (Kushida et al., 2008; Tsutsui et al., 2007). Meal content offers the most flexibility in regulating AHN, as a variety of bioactives/nutrients have been identified as potential modulators. For example, flavonoids, which are enriched in foods such as cocoa and blueberries, have been shown to increase AHN in chronically stressed rats (An et al., 2008), and treatment with flavonoids improves symptoms of depression (Dimpfel, 2009). The authors hypothesized that this effect might be mediated by BDNF. Deficiency in zinc inhibits AHN (Corniola et al., 2008) and induces depression in rodents (Thompson et al., 2008), whereas independent intervention studies have shown the efficacy of zinc supplements in improving symptoms of depression (Szewczyk et al., 2008). Caffeine is a dose-dependent bioactive. It decreases AHN and performance in hippocampus-dependent learning tasks in rodents at low doses, whereas at supraphysiological doses, there is an increase in proliferation of neuronal precursors (Han et al., 2007). However, neurons induced in response to supra-physiological levels of caffeine have a lower survival rate than control cells and

increased proliferation does not yield an increase in AHN (Wentz and Magavi, 2009). Curcumin is a natural phenolic component of yellow curry spice that increases AHN in rodents (Kim et al., 2008). It activates extracellular signal-regulated kinases (ERKs) and p38 kinases, cellular signal transduction pathways known to be involved in the regulation of neuronal plasticity and stress responses (Kim et al., 2008). Finally, it is important to note that independent of calorie intake, diets with high fat content impair AHN in male rats. The authors hypothesize that high dietary fat intake disrupts AHN through an increase in serum corticosterone levels (Lindqvist et al., 2006). There is increasing evidence for the role of diet in modulating depressive behavior, suggesting that omega-3 fatty acids, zinc, flavonoids, caffeine etc. improve depressive behavior (Table 14.1). It is now getting clearer that AHN affects cognition and mood. It is also firmly established that nutrition has an impact on cognition and mood. Therefore, AHN is emerging as a possible mediator of the effect of certain foods on cognition and mood. Consequently, modulating AHN by diet could be a target of choice to prevent cognitive decline during aging, as well as to counteract the effect of stress and prevent depression.

CLINICAL TRIALS SUPPORTING THE ROLE OF OMEGA-3 FATTY ACIDS IN MDD While there is evidence that essential fatty acids may play a biological role in depression, there is very little

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published clinical data available. One well-designed trial demonstrated the effect of omega-3 fatty acids in bipolar disorder by lengthening remission and showed a highly significant effect in treating depression (p , 0.001 Hamilton Rating Scale for Depression) with four months of treatment with 9.6 g EPA (Stoll et al., 1999). A treatment-resistant case of major depression, when placed on a daily dose of 4 g purified EPA, reported a significant improvement in one month and was symptom free within nine months. The EPA treatment was also associated with structural brain changes that may be the result of increased phospholipid biosynthesis and decreased phospholipid breakdown (observed through MRI) (Puri et al., 2001). This may be an indication that omega-3 fatty acids can influence depressionrelated brain atrophy. These observations are important in light of recent MRI studies indicating a decrease in volume of various areas of the brain in depressed patients (Bremner et al., 2002; Steingard et al., 2002). There is some evidence that adding fish oil to standard anti-depressant medication may enhance the therapy efficacy. One double-blind, placebo-controlled study (n 5 22) showed that the addition of 2 g EPA to standard anti-depressant medication enhanced the effectiveness of that medication compared to the medication plus placebo after three weeks of treatment. EPA had an effect on insomnia, depressed mood, and feelings of guilt and worthlessness. Importantly, no clinically relevant side effects were noted. Similarly, an eight-week, doubleblind placebo-controlled trial comparing high doses of fish oil (9.6 g/day) to placebo in addition to standard anti-depressant therapy in 28 patients with MDD significantly decreased scores on the Hamilton Rating Scale for Depression compared to those on placebo (p , 0.001). This relatively high dose of fish oil was well tolerated and no adverse events were reported during the twomonth trial (Su et al., 2003). The most impressive clinical study to date on omega-3 fatty acids and unipolar depression (n 5 70) demonstrated a 50% reduction on Hamilton depression scores, when added to 1 g ethyl-EPA for 12 weeks, in subjects experiencing persistent depression, despite ongoing standard pharmacotherapy at adequate doses. The 1 g EPA dose led to improvements in depression, anxiety, sleep, lassitude, libido, and suicidal ideation. To date, the published clinical data on the effect of marine-derived omega-3 fatty acids (combination EPA/ DHA or pure EPA alone) on major depression have been positive. But there is no convincing clinical evidence on the effect of plant-derived omega-3 fatty acids (ALA) on major depression except for one case study which indicates that flax seed oil, a source of ALA, may be of benefit in the treatment of bipolar depression and agoraphobia (Rudin 1981). So the authors (Tanna et al., 2013) carried out research to evaluate the clinical

efficacy of flax seed oil as monotherapy and as an adjuvant to Ashwagandharishta in the management of MDD. An administration of 10 ml flax seed oil twice a day mixed with a meal for 2 months (n 5 20) demonstrated a 48.97% reduction in Hamilton Rating Scale for Depression (HRSD) score and a 50.31% reduction in Montgomery Asberg Depression Rating Scale (MADRS) score with highly significant improvement in almost all the depressive symptoms. Adding the same amount of flax seed oil to Ashwagandharishta 25 ml twice a day mixed with an equal amount of water after a meal (n 5 45) demonstrated a 50.38% reduction in HRSD score and a 53.39% reduction in MADRS score, demonstrating the efficacy of plant-based omega-3 fatty acid, flax seed oil, in MDD as monotherapy and also as an adjuvant to an Ayurvedic formulation, Ashwagandharishta (Tanna et al., 2013). Moreover, , 9.51% reduction was observed in serum cortisol levels in the Ashwagandharishta treated group but when flax seed oil was added to the treatment, it showed a 15.02% decrease which was statistically significant. Though statistically insignificant, flax seed oil, as a monotherapy, decreased highly sensitive C-reactive protein (HsCRP) by 93.18% but when it was added to Ashwagandharishta, a highly significant decrease of 37.40% was reported, demonstrating the role of flax oil as an adjuvant in the treatment of depression. These reports suggest that omega-3 fatty acids can alleviate depression by entirely different means than standard antidepressants; they improve the functionality of antidepressants, or both. Many ingredients of Ashwagandharishta such as Ashwagandha, Mushali, Manjishtha, Yashtimadhu, Arjuna, Trivritta, SwetaChandana, Vacha, Chitraka, Shunthi, Twaka, Ela, and Tamalapatra are rich in antioxidants, which are a known factor for influencing omega-3 status. Antioxidant supplementation can prevent the negative influence of saturated fat on BDNF levels. This may be the reason for better alleviation of depression and biological markers in the combined group. In experimental models, flax seed oil exhibited an excellent antidepressant activity, with mild to moderate anxiolytic and antipsychotic properties (Tanna et al., 2013). Additionally, the authors also observed that the patients with major depression who were also known cases of epilepsy (n 5 4), benefited, in terms of their epilepsy, from combined therapy of flax seed oil and Ashwagandharishta, since in these patients the interval of two epileptic fits was increased, demonstrating the antiepileptic activity of flax seed oil. This clinical observation was further confirmed by animal experiments in which flax seed oil exhibited significant anticonvulsant activity by decreasing tonic extensor phase (p , 0.05), tonic flexion phase (p , 0.001) and recovery time (p , 0.05) with 76.44% protection against maximal

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electroshock induced seizures (Tanna et al., 2012). This observation confirms the protective effect of omega-3 fatty acids against the seizures as was reported earlier (Cysneiros et al., 2010). It has also been reported that chronic treatment with omega-3 promotes neuroprotection and positive plastic changes in the brains of rats with epilepsy (Ferrari et al., 2008), with a decrease in neuronal death in CA1 and CA3 subfields of the hippocampus. This could be attributed to ion channel modulation of n-3 PUFAs (Young et al., 2000; Xiao et al., 1995; Xiao and Li, 1999) and anti-inflammatory action. In in vitro studies, DHA has been reported to inhibit epileptiform activity and synaptic transmission mainly through the frequency-dependent blockade of Na 1 channels in the rat hippocampus (Young et al., 2000), and to stabilize neuronal membranes by suppressing voltage-gated Ca21 currents (Xiao et al., 1997) and Na1 channels.

CONVERSION OF ALA TO EPA AND DHA FROM FLAX SEED OIL In February 2010, the European Parliament passed a regulation permitting the labeling of nutrition claims for omega-3 fatty acids, allowing food products to claim they are either a ‘source of omega-3 fatty acids’ where they contain at least 300 mg ALA per 100 g (and per 100 kcal) or at least 40 mg of the sum of the EPA plus DHA per 100 g (and per 100 kcal) or a ‘high source of omega-3 fatty acids’ where they contain at least 600 mg ALA per 100 g (and per 100 kcal) or at least 80 mg of the sum of EPA plus DHA per 100 g (and per 100 kcal) (EU, 2010). According to this regulation, flax seed oil is a high source of omega-3 fatty acids as it contains 53.3 g ALA/100 g flax seed oil. Though human conversion of ALA to EPA and DHA is very limited, there is some evidence demonstrating the effective conversion of ALA to EPA and DHA from flax oil. Due to physiologic and lifestyle factors, human conversion of ALA to EPA ranges from 8% to 21% and conversion of ALA to DHA ranges from 1% to 9%. Based on superior conversion ability, two tablespoons of flax seed oil (28 g), which contains 15 g ALA, provides 3.15 g EPA (21% conversion) and 600 mg DHA (4% conversion). A multiple-dosing trial comparing two sources of n-3 fatty acids, flax seed oil and fish oil, demonstrated that fish oil produces a rapid increase in erythrocyte DHA and total omega-3 fatty acids and the consumption of either 2.4 g or 3.6 g flax oil/day is sufficient to significantly increase erythrocyte total phospholipid ALA, EPA, and DHA fatty acid content. There were no differences among groups in plasma inflammatory

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markers or lipid profiles. These results show the effectiveness of ALA conversion and accretion into erythrocytes. Thus, the amounts of ALA required to obtain these effects are easily achievable in the general population by dietary modification (Barcelo´Coblijn et al., 2008). Bioavailability from flax seed diet showed that the subjects who received flax seed oil exhibited a significant increase in plasma ALA and EPA concentrations over a period of 4 weeks on a course of 6 g ALA (Patenaude et al., 2009). Another piece of research evaluating the influence of omega-3 fatty acids from flax seed on the brain development of newborn rats showed that a maternal diet of flax seed during pregnancy influences the incorporation of omega-3 fatty acids in the composition of brain tissue since it significantly increased DHA by 38% and total omega-3 fatty acids by 62% assuring a good development of this organ in newborn rats (Lenzi Almeida et al., 2011).

PROBABLE MODE OF ACTION OF FLAX SEED OIL IN DEPRESSION Flax seed oil is a rich source of ALA which is endogenously converted into DHA. Recent research has demonstrated that the conversion of ALA to DHA in the liver is upregulated if a direct source of DHA is unavailable and, thereby, the normal level of DHA in the brain is maintained (Dermar et al., 2005). DHA, by maintaining membrane fluidity (by displacing cholesterol from the membrane) corrects receptor functioning, regulates neurotransmitter levels, neurotransmission, and signaling within the cells. It acts as a second messenger source within the cells (Figure 14.5). It helps in metabolism of monoamines implicated in the etiopathogenesis of depression. Flax seed oil is the richest source of ALA which can be rapidly diffused from plasma to brain. So, even if the conversion is ineffective, recent research (Blondeau et al., 2009) has shown that sub-chronic treatment with ALA induces an increase in BDNF levels, improves neurogenesis and synaptic plasticity in specific brain regions, properties well known for the efficiency of anti-depressant drugs (Castren et al., 2007). Thus, neurogenesis and synaptogenesis associated with subchronic ALA treatment are strong arguments in favor of such a mechanism being involved in the ALA anti-depressant effect, which can also be related to additive/or synergic interaction with serotonin, norepinephrine, or dopamine pathways (Delion et al., 1994; Delion et al., 1996) (Figure 14.6). BDNF encourages synaptic plasticity, provides neuroprotection, enhances neurotransmission, and has antidepressant effects (Ikemoto et al., 2000).

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Flax seed oil omega-3 fatty acids

ALA α-Linolenic acid

Conversion of ALA to DHA is upregulated in the liver

Receptor functioning

Maintains Membrane integrity & fluidity by displacing cholesterol from the membrane

Neurotransmitter levels, neurotransmission & signaling within the cells Metabolism of monoamines implicated in depression

DHA

It acts as a source for second messenger within the cells Thus, anti-depressant activity of flax seed oil may be due to the physiologic roles of omega-3 PUFA in regulating cell membrane fluidity, dopaminergic and serotonergic transmission, membrane bound enzymes, and cellular signal transduction and also by increasing BDNF and thereby increasing neurogenesis and synaptic plasticity.

FIGURE 14.5 Mode of action of flax seed oil through action of DHA after conversion from ALA.

FIGURE 14.6 Mode of action of flax seed oil through action of ALA.

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CONCLUSION

FIGURE 14.7 hippocampal BDNF.

Evidence has accumulated that omega-3 fatty acids have an influence on hippocampal neurogenesis by increasing BDNF. Adult hippocampal neurogenesis has been linked directly to cognition and mood (Zhao et al., 2008) (Figure 14.7), therefore, modulation of AHN by flax seed oil might be a possible mechanism by which nutrition impacts on mental health. A growing body of research indicates that depression is associated with excessive production of cytokines which may lower neurotransmitter precursor availability, activate the hypothalamic pituitary axis, and alter the metabolism of neurotransmitters and neurotransmitter transporter mRNA (Maes and Smith, 1998). Flax seed oil being a source of omega-3 fatty acids may play an influential role in treating depression via inhibition of these pro-inflammatory cytokines and thereby regulating neurotransmitter production, metabolism, transportation, and regulating the hypothalamic pituitary axis. Thus, the anti-depressant activity of flax seed oil may be due to the physiologic roles of omega-3 PUFAs in regulating cell membrane fluidity, dopaminergic and serotonergic transmission, membrane bound enzymes, and cellular signal transduction and also by increasing BDNF and thereby increasing neurogenesis and synaptic plasticity.

CONCLUSION Depressed subjects ingest significantly lower amounts of omega-3 fatty acids and nutrient cofactors such as zinc, selenium, antioxidants, folic acid, and magnesium, which influence the status of omega-3 fatty acids, than non-depressed subjects.

Influence of omega-3 fatty acids on neurogenesis through increasing

Moreover, they ingest significantly higher amounts of dietary articles which interfere with the conversion rate of ALA to EPA and DHA compared to nondepressed healthy control subjects, which makes them omega-3 deficient and makes them prone to depression. There is enough clinical data, combined with the rapidly growing support of experimental, laboratory, and epidemiological studies, to suggest that omega-3 fatty acids may play a role in the prevention and management of depression. There is experimental and clinical evidence demonstrating the efficacy of omega-3 fatty acids in the treatment of depression even when administered via flax seed oil, a plant-derived omega-3 source. For now, the bulk of clinical evidence indicates that EPA may be most important in mood stability. Further research is necessary before firm conclusions can be drawn regarding which omega-3 fatty acid, EPA or DHA, is likely to provide the greatest benefit and to more accurately ascertain the neurobiological influences of flax seed oil and its clinical value in the treatment of depression. It is anticipated that additional research will shed further light on the exact mode of action of flax seed oil in depression. This will help to identify whether ALA itself works as an anti-depressant or whether its effect occurs after bioconversion to EPA and DHA in contributing to correcting the pathophysiology of depression. Essential fatty acid supplements rich in omega-3 fatty acids are also generally inexpensive, making them attractive as an adjuvant or alternative to standard pharmacotherapy. Thus, deficits in omega-3 fatty acids have been identified as a contributing factor to mood disorders and offer a potential rational treatment approach.

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FURTHER READING

Zimmer, L., Delion-Vancassel, S., Durand, G., 2000a. Modification of dopamine neurotransmission in the nucleus accumbens of rats deficient in n-3 polyunsaturated fatty acids. J. Lipid. Res. 41, 32
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Further Reading Calderon, F., Kim, H.Y., 2004. Docosahexaenoic acid promotes neurite growth in hippocampal neurons. J. Neurochem. 90, 979
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8.

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C H A P T E R

15 Omega-3 Fatty Acid Supplementation for Major Depression with Chronic Disease Lauren E. Lawson and Ronald Ross Watson INTRODUCTION Omega-3 fatty acids are essential in that the body cannot produce them. Low levels of omega-3 fatty acids occur in individuals suffering from various depressive disorders (Andreeva et al., 2012; Giltay et al., 2011; Prior and Galduro´z, 2012; Appleton et al., 2010; Hegarty and Parker, 2012; Lucas et al., 2011). While some studies suggest that there is a positive correlation between a high intake of fish oil supplements and improvement of mood disorders, recent studies offer complicating evidence (Appleton et al., 2010; Hegarty and Parker, 2012; Freeman et al., 2006).

OMEGA-3 FATTY ACIDS Omega-3 fatty acids are long chain unsaturated molecules only found within certain foods such as flax seed, walnuts, and some types of fish (Prior and Galduro´z, 2012). Almost 20% of the brain’s dry weight is attributed to omega-3 fatty acids. These polyunsaturated fatty acids help maintain brain function by playing a role in the structure and composition of receptors within the central nervous system. Omega-3 fatty acids also help in maintenance of membrane potentials by promoting depolarization and repolarization of neurons and by promoting signal transmission throughout the cortex. As a result, the depletion of omega-3 fatty acids can interfere with neurotransmitter production and neuron firing (Prior and Galduro´z, 2012). There are different varieties of omega-3 fatty acids, including α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). ALA and linoleic acid (LA) are parent molecules that can be converted to form both EPA and DHA (Hegarty and Parker, 2012). Fish oil supplements can vary in their Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00015-6

composition, with one formulation being richer in either EPA or DHA, while others maintain almost even ratios of the two types of omega-3 fatty acid (Grenyer et al., 2007). EPA may have a larger impact on mood by utilizing eicosanoid mechanisms to increase cerebral blood flow (Grenyer et al., 2007). DHA is crucial to brain development as it is the primary component of brain phospholipids and necessary for neurotransmission of serotonin and dopamine (Hegarty and Parker, 2012). Additionally, DHA is the chief omega-3 fatty acid gained through consumption of fish. Therefore it may be responsible for the negative correlation between fish consumption and depression (Grenyer et al., 2007). In addition, an increase in levels of omega-3 fatty acids may impact on depression symptoms due to the anti-inflammatory effects that EPH and DHA both have in addition to their ability to suppress cytokine production (Hegarty and Parker, 2012). The lack of fish and fish products in the diet of the average individual living in industrialized countries has led to a decrease in consumption of omega-3 fatty acids of 80% in the last 100 years (Saldeen and Saldeen, 2004). These changes to the diet of industrialized nations over time occur concomitantly with an increase in the numbers of individuals affected with depressive disorders, possibly pointing to the larger role of diet in mental illnesses. (Hegarty and Parker, 2012). More specifically, as omega-3 fatty acids are vital in maintaining neural functions, decreased consumption may be linked to mental disorders. Foods with omega-3 fatty acids have varying amounts of omega-6. Unlike the tendency for omega-3 fatty acids to reduce inflammation, omega-6 fatty acid has a tendency to encourage inflammation. However, there are multiple types of omega-6 fatty acids, and not all have been associated with promoting inflammation (Hegarty and Parker, 2012). Individuals usually

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obtain sufficient amounts of omega-6 fatty acids without a need for supplementation. Omega-6 fatty acids are commonly found in vegetable oils, seeds and nuts, as well as meats and soy (Loef and Walach, 2013).

Major Depressive Disorder Major depression is a mental illness affecting over 350 million individuals worldwide (World Health Organization, 2012). Approximately 6.6% of adults living in the United States experience or have experienced major depression within the past year (Centers for Disease Control and Prevention (CDC), 2010). There are many types of mental depression, with major depressive disorder (MDD) being among one of the most severe and chronic forms. In order to be diagnosed with MDD, an individual must exhibit five symptoms from a list of common symptoms associated with the disorder and have experienced these symptoms consecutively for a minimum of two weeks. The most common symptoms associated with MDD are a depressed mood, loss of interest in most activities, significant weight loss or gain, insomnia, fatigue, inability to concentrate, and thoughts of death or suicide (Cahoon, 2012). Other types of depressive disorders include many of the same symptoms; however, individuals usually experience fewer symptoms with less severity (Alexopoulos et al., 2001). While the physiology of depression is not totally understood, depression may occur as the result of a chemical imbalance within the brain involving the regulation of neurotransmission. The CDC reports that depressive disorders are more common among those with chronic conditions (e.g., obesity, cardiovascular disease, diabetes, asthma, arthritis, and cancer) and among those with unhealthy behaviors (e.g., smoking, physical inactivity, and binge drinking). Depression can lead to chronic diseases, and chronic diseases can exacerbate depressive conditions (Champman et al., 2005). Although pharmaceutical medications are routinely prescribed for depressive disorders, other non-pharmaceutical treatments do exist. Some treatments are as simple as exercise and socializing with other people, while other treatments include therapies that focus on having an individual identify the problems with their own pattern of thinking or identify areas of conflict in their own life (Cahoon, 2012). The World Health Organization identified depression as the third leading cause of disease burden worldwide and estimates that it will be second only to cardiovascular disease by 2020 (CDC, 2010).

Omega-3 Fatty Acids and Depression Inflammation may be the culprit for the evolution of mental illness, which can be caused by increased

cytokine production, migration of inflammatory cells, and activation of glial cells (Prior and Galduro´z, 2012). Omega-3 fatty acids are essential nutrients that have been shown to reduce inflammation. Additionally, lower levels of omega-3 fatty acids have been reported in individuals suffering from psychiatric disorders. Due to these trends, recent studies have examined the relationship between an increased intake of omega-3 fatty acids and symptoms in individuals with mental disorders. In most individual countries where populations have higher fish consumption, depressive symptoms are less prevalent (Freeman et al., 2006). Positive results were reported in conjunction with three doubleblind placebo-controlled studies in which participants were either given an omega-3 fatty acid supplement or a placebo capsule in addition to their depression medication. Participants were assessed using the Hamilton Rating Scale for Depression. The participants who were given 1 g of EPA each day were more likely than participants who were given the placebo to show a dramatic reduction in their depression symptoms (Freeman et al., 2006). Another study provided participants with 2 g EPA each day and noted a dramatic improvement in depression symptoms in the experimental group in comparison to the control group (Freeman et al., 2006). A third study which used a supplement with a combination of EPA and DHA seemed to produce even lower scores than the preceding two trials on the Hamilton Rating Scale for Depression (Freeman et al., 2006). The dramatic decrease in the depressive symptoms of the participants suggests the efficacy of omega-3 fatty acid supplements, containing both EPA and DHA, for improvement of depression symptoms in individuals with a diagnosis of depression. In a later study, the efficacy of tuna fish oil supplements as a treatment for major depression was analyzed using a randomized, double-blind placebocontrolled trial. In this study, a group of participants with a primary diagnosis of major depression with no other major medical conditions were given either an olive oil placebo capsule or a tuna fish oil capsule (2.2 g DHA and 0.6 g EPA). Patients were assessed weekly for sixteen weeks. Results from this study showed no benefit of fish oil compared to placebo when added to a standard outpatient treatment (Grenyer et al., 2007). While no benefit was shown, it was noted that because DHA, as a component of neuronal membrane phospholipids, affects neurotransmission, and EPA acts through eicosanoid mechanisms to increase cerebral blood flow, EPA may have a greater effect on mood than DHA. Thus a higher concentration of EPA than DHA in the supplement may have a greater influence on mood (Grenyer et al., 2007). More recently, further research has been conducted to define the relationship between omega-3 fatty acids

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OMEGA-3 FATTY ACIDS

and depression symptoms. One meta-analysis attempted to identify and solve the potential sources of disparity among studies exploring this area of interest (Appleton et al., 2010). Thirty-five randomized control studies were analyzed and reviewed. Among these studies, the severity of patient symptoms and diagnosis contributed to varying results among studies. Greater effects of omega-3 fatty acids were found in individuals with more severe depressive symptoms. Trials that used participants with depressive symptoms but without diagnosis of depression showed little improvement (Appleton et al., 2010).

Effects of Omega-3 Fatty Acids on Depression with Cardiovascular Disorders Depression often affects coronary heart disease patients, especially those who have suffered from myocardial infarction (MI). Not only does depression affect the mood of the individual burdened with coronary heart disease, but it interferes with the quality and time of recovery of the individual, often worsening the already existing heart disorder. On average, 20% of individuals having suffered from an MI and suffering from major depression are not receiving adequate treatment for their depressive symptoms (Giltay et al., 2011). Patients suffering from coronary heart disease and exhibiting depressive symptoms were found with lower levels of DHA in their systems than those patients suffering from coronary heart disease and exhibiting no depressive symptoms (Giltay et al., 2011; Andreeva et al., 2012). Recently, low doses of both EPA-DHA (3:1) and ALA supplements were compared with placebo in individuals who had experienced an MI. Because coronary heart disorders are associated with lower intakes of fish oil and patients with coronary heart disorders expressing depressive symptoms have lower levels of DHA in their blood, the depressive state of these individuals may be improved by increasing their consumption of omega-3 fatty acids. The purpose of this study, lasting approximately four years, was to evaluate whether omega-3 fatty acid supplementation would improve the affective states of individuals suffering from coronary disorders (Giltay et al., 2011). After evaluation from cardiologists, 4,116 patients having experienced an MI were randomly assigned to one of four different types of supplementation: EPA-DHA and ALA, EPA-DHA (3:2), ALA, and placebo. The supplements were supplied in the form of a margarine that the patients would use instead of fish oil capsules. The patients had no differences in demographic, lifestyle or biological factors or in their use of psychotropic medications. The patients’ depressive symptoms were assessed by giving the patients a questionnaire to complete at each evaluation.

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Although no effect on depressive symptoms was found in these individuals, these patients had no psychiatric diagnosis of depression; they showed only depressive symptoms. In a small group of individuals who were prescribed anti-depressants at baseline, individuals using the EPA-DHA supplemented margarine had greater improvement of their symptoms in comparison to their counterparts who only received a placebo (Giltay et al., 2011). More recently, another study examined omega-3 fatty acids and their effect on depressive symptoms in individuals previously suffering from an MI, unstable angina, or ischemic stroke within the preceding year. With a similar goal in mind, the objective of this study was to assess both the effects of B vitamins and the effects of omega-3 fatty acid supplementation on depressive symptoms in individuals who survived cardiovascular disease. Patients in this study were between 40
80 years of age and had previously suffered an MI, unstable angina, or ischemic stroke within the previous year. The patients were divided into one of four groups: B vitamin with omega-3 fatty acid; omega-3 fatty acid; B vitamins; and placebo. The supplements were provided as two capsules. Each year the patients were reexamined, their depression symptoms were evaluated using the Geriatric Depression Scale, and they were provided with additional supplements. After approximately four years of taking omega-3 fatty acid supplements, (600 mg, EPA:DHA / 2:1), no positive change was observed in these individuals, contrary to the expected outcome that omega-3 fatty acid supplements are effective in the prevention of depression in cardiovascular disease survivors. Gender-specific effects of omega-3 fatty acid supplementation were, however, observed in men. In men who received omega-3 fatty acid supplementation, compared to men who did not receive supplementation, a 28% higher risk of presenting depressive symptoms was exhibited; however, there was no such risk exhibited in women (Andreeva et al., 2012).

Effects of Omega-3 Fatty Acids on Depression with Diabetes Depression is common in individuals suffering from both Type 1 and Type 2 diabetes. Low levels of omega-3 fatty acids are common in both diabetic patients and depressed patients (Decsi et al., 2007). In order to measure the effects of omega-3 fatty acids on the depressed moods of individuals with diabetes, participants with MDD between 18
75 years of age and taking antidepressant medication for two months or more were recruited and given either 1 g EPA per day or a rapeseed oil placebo (Bot et al., 2010). After 12 weeks from baseline, the average level of EPA in the erythrocyte membrane tripled in the group receiving the EPA

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supplement daily, while the level of EPA in the erythrocyte membrane remained the same in individuals receiving the rapeseed placebo supplement; however, no therapeutic effect was noted in the participants receiving the EPA supplement (Bot et al., 2010). While a recent meta-analysis showed that omega-3 fatty acids possessed anti-depressant efficacy in patients without diabetes (Appleton et al., 2010) it was noted that the lack of a positive correlation between omega-3 fatty acids and anti-depressant efficacy in patients with diabetes could be the result of a number of factors. One factor could be due to the ratio of EPA to DHA in the omega-3 fatty acid supplement. Because EPA has shown more promising effects on depressive behaviors than DHA (Pouwer et al., 2005), a pure EPA supplement was used in the diabetic study; however, it may be that a specific ratio of EPA to DHA is more effective (Bot et al., 2010). Additionally, the sample size from this trial was smaller than those of most other randomized controlled trials and the effects of rapeseed oil on mood cannot be fully excluded from having some beneficial effect on mood (Bot et al., 2010).

Effects of Omega-3 Fatty Acids on Depression and Pregnancy MDD affects twice as many women as men (Lucas et al., 2011). While evidence from randomized control trials have shown results indicating that omega-3 fatty acids obtained from fish oil have a positive effect on symptoms of depression (Appleton et al., 2010; Freeman et al., 2006), other research has found that higher intake of the omega-3 fatty acid ALA from vegetable sources showed an association with lower risk of clinical depression. However, no associations were shown between a lowered risk of clinical depression and higher consumption of omega-3 fatty acids from fish or from EPA and DHA supplements (Lucas et al., 2011). In the case of pregnant women, DHA levels may drop by 50% during pregnancy and levels may not return to normal until six or more months after birth (Saldeen and Saldeen, 2004). Many mothers who experience postpartum depression note having consumed little fish and have lower levels of DHA in breast milk than mothers who are not suffering from postpartum depression (Saldeen and Saldeen, 2004; Hibbeln, 2001). Observational trials and clinical studies assessing the effects of higher consumption of omega-3 fatty acids and postpartum depression show an association between omega-3 fatty acids and the effects of depression following pregnancy (Freeman et al., 2006, 2008; Golding et al., 2009). In a recent study, omega-3 fatty acids alone showed no association with postpartum depression; however, it was noted that the median

ratio of omega-6 to omega-3 fatty acids consumed in the first three months of pregnancy was higher in women with postpartum depression (da Rocha and Kac, 2012), These results indicate an association between the ratio of omega-6 to omega-3 fatty acids within an individual and depression symptoms rather than the level of omega-3 fatty acids alone.

Effects of Omega-3 Fatty Acids on Depression and Old Age Major depression is not uncommon in older individuals. Approximately 15
19% of Americans over the age of 65 are affected by depression (Cahoon, 2012). Depression is reported in 13% of community dwelling seniors, in 24% of medical outpatients, and in 43% of those living in nursing homes (Rizzo et al., 2012). Associations have been noted between the levels of polyunsaturated fatty acids and older adults suffering from major depression. Lower levels of omega-3 fatty acids have been found in older people with depression (Jadoon et al., 2012). In an effort to investigate the association between the levels of polyunsaturated fatty acid in the plasma of erythrocyte membrane and residual depressive or anxiety symptoms in older individuals with remitted major depression, participants over the age of 60 having a history of a previous diagnosis of major depression were observed. This cross-sectional study observed patients that were drawn from outpatient psychiatric services from hospitals in Taipei, Taiwan. No noteworthy relationship was observed between the levels of omega-3 fatty acid and an individual’s depression (Jadoon et al., 2012). While this crosssectional study showed no association between polyunsaturated fatty acids and depression symptoms, another recent study offers conflicting results. Females between 66 and 95 years of age with a diagnosis of depression were provided with either 2.5 g of omega-3 fatty acid each day or with a placebo for eight weeks. Using the Geriatric Depression Scale, the participants were asked to report on their depression symptoms throughout the study. Additionally, blood samples were taken and analyzed at baseline and at the study’s end. Results from the study showed an overall drop in depression symptoms by 35% in the participants who had been given omega-3 fatty acid supplements and no change was noted in the participants who had been given placebo (Rizzo et al., 2012).

Effect of Omega-3 Fatty Acids on Anxiety and Depression in Students Anxiety disorders are very similar to comorbid depressive disorders in that they are both commonly

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REFERENCES

diagnosed disorders and that they share some common symptoms. Additionally, individuals with some anxiety disorders, like social anxiety disorder, also have low levels of omega-3 fatty acids and both depression and anxiety disorders enhance the production of proinflammatory cytokines (Kiecolt-Glasser et al., 2011). For this reason, omega-3 supplementation should lower the production of pro-inflammatory cytokines, but its supplementation should also lessen symptoms of anxiety and depression. To test this hypothesis, 68 first and second year medical students between the ages of 21 and 29 were given either an omega-3 fatty acid supplement (2085 mg EPA and 348 mg DHA) or a placebo (mixture of palm, olive, soy, canola, and cocoa butter oils). Blood samples were collected when students had no exams (baseline) and the day before an important exam, continuing this pattern for three months. Results showed both decreases in inflammation and a 20% decrease in anxiety symptoms compared to the control group (Kiecolt-Glasser et al., 2011). Evidence from this study suggests that omega-3 fatty acids may have a diminishing effect on anxiety symptoms.

SUMMARY The anti-inflammatory effects of omega-3 fatty acids have some influence on the depressive symptoms of individuals and are dependent on multiple factors. Omega-3 fatty acid supplements containing both EPA and DHA, having a higher concentration of EPA, seem to be most effective on depressive symptoms. While a higher concentration of EPA has been more effective, EPA alone has shown to have no positive impact on depressive symptoms. Omega-3 supplementation alone has been ineffective, but in individuals who have been clinically diagnosed with depression and who are currently taking depression medications, omega-3 supplementation is effective in reducing depressive symptoms. The results of studies in which omega-3 fatty acid supplements were used to treat depression symptoms in individuals with chronic diseases are varied. It seems that supplementation may have no effect on depression symptoms of individuals with cardiovascular disorders, diabetes, and old age. However, the differences in participant criteria, EPADHA ratios, duration, and assessment procedures among the varying studies may have affected the overall results.

References Alexopoulos, G., Katz, I.R., Reynolds III, C.F., Carpenter, D., Docherty, J.P., Ross, R.W., 2001. Pharmocotherapy of depression

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in older patients: a summary of the expert consensus guidelines. J. Psychiatr. Pract. 7 (6), 361
376. Andreeva, V.A., Galan, P., Torre`s, M., Julia, C., Hercberg, S., KesseGuyot, E., 2012. Supplementation with B vitamins or n-3 fatty acids and depressive symptoms in cardiovascular disease survivors: ancillary findings from the SUpplementation with FOLate, vitamins B-6 and B-12 and/or OMega-3 fatty acids (SU.FOL. OM3) randomized trial. Am. J. Clin. Nutr. 96, 208
214. Appleton, M.K., Rogers, J.P., Ness, R.A., 2010. Updated systemic review and meta-analysis of the effects of n-3 long-chain polyunsaturated fatty acids on depressed mood. Am. J. Clin. Nutr. 91, 757
770. Bot, M., Pouwer, F., Assies, J., Jansen, E.H., Diamant, M., Snoek, F.J., et al., 2010. Eicosapentaenoic acid as an add on to antidepressant medication for co-morbid major depression in patients with diabetes mellitus: a randomized, double-blind placebo-controlled study. J. Affect. Disord. 126, 282
286. Cahoon, C.G., 2012. Depression in older adults. Am. J. Nurs. 112 (11), 22
30. Centers for Disease Control and Prevention, 2010. MMWR: Current Depression Among Adults- United States, 2006 and 2008. U.S. Department of Health and Human Services, Atlanta, Georgia. Champman, D., Perry, G., Strine, T., 2005. The vital link between chronic disease and depressive disorders. Prev. Chronic Dis. 2, A14. da Rocha, C., Kac, G., 2012. High dietary ratio of omega-6 to omega-3 polyunsaturated fatty acids during pregnancy and prevalence of post-partum depression. Matern. Child. Nutr. 8, 36
48. Decsi, T., Szabo´, E., Burus, I., Marosvo¨lgyi, T., Koza´ri, A., Erhardt, E., et al., 2007. Low contribution of n-3 polyunsaturated fatty acids to plasma erythrocyte membrane lipids in diabetic young adults. Prostaglandins Leukot. Essent. Fatty Acids. 76 (3), 159
164. Freeman, M.P., Hibbeln, J.R., Wisner, K.L., Davis, J.M., Mischoulon, D., Peet, M., et al., 2006. Omega-3 fatty acids: evidence basis for treatment and future research in psychiatry. J. Clin. Psychiatry. 67 (12), 1954
1967. Freeman, M.P., Davis, M., Sinha, P., Wisner, K.L., Hibbeln, J.R., Gelenberg, A.J., 2008. Omega-3 fatty acids and supportive psychotherapy for perinatal depression: a randomized placebocontrolled study. J. Affect. Disord. 110, 142
148. Giltay, E., Geleijnse, J., Kromhout, D., 2011. Effects of n-3 fatty acids on depressive symptoms and dispositional optimism after myocardial infarction. Am. J. Clin. Nutr. 94, 1442
1450. Golding, J., Steer, C., Emmett, P., Davis, J.M., Hibbeln, J.R., 2009. High levels of depressive symptoms in pregnancy with low omega-3 fatty acid intake from fish. Epidemiology. 20 (4), 598
603. Grenyer, B.F., Crowe, T., Meyer, B., Owen, A.J., Grigonis-Deane, E. M., Caputi, P., et al., 2007. Fish oil supplementation in the treatment of major depression: a randomized double-blind placebocontrolled trial. Prog. Neuropsychopharmacol. Biol. Psychiatry. 31, 1393
1396. Hegarty, B., Parker, G., 2012. Fish oil as a management component for mood disorders
an evolving signal. Curr. Opin. Psychiatry. 26, 33
40. Hibbeln, J.R., 2001. Seafood consumption and homicide mortality. A cross-national ecological analysis. World. Rev. Nutr. Diet. 88, 41
46. Jadoon, A., Chiu, C.C., McDermott, L., Cunningham, P., Frangou, S., Chang, C.J., et al., 2012. Associations of polyunsaturated fatty acids with residual depression or anxiety in older people with major depression. J. Affect. Disord. 136, 918
925. Kiecolt-Glasser, J., Belury, M., Andridge, R., Malarkey, W., 2011. Omega-3 supplementation lowers inflammation and anxiety in medical students: a randomized controlled trial. Brain. Behav. Immun. 25, 1725
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Loef, M., Walach, H., 2013. The omega 6/omega 3 ratio and dementia or cognitive decline: a systematic review on human studies and biological evidence. J. Nurt. Gerontol. Geriatr. 32 (1), 1
23. Lucas, M., Mirzaei, F., O’Reilly, E.J., Pan, A., Willett, W.C., Kawachi, I., et al., 2011. Dietary intake of n-3 and n-6 fatty acids and the risk of clinical depression in women: a 10-y prospective followup study. Am. J. Clin. Nutr. 93, 1337
1343. Pouwer, F., Nijpels, G., Beekman, A.T., Dekker, J.M., van Dam, R.M., Heine, R.J., et al., 2005. Fat food for a bad mood. Could we eat and prevent depression in Type 2 diabetes by means of omega-3 polyunsaturated fatty acids? A review of the evidence. Diabet. Med. 22, 1465
1475.

Prior, P.L., Galduro´z, J.C., 2012. (N-3) Fatty acids: molecular role and clinical uses in psychiatric disorders. Adv. Nutr. 3, 257
265. Rizzo, A.M., Corsetto, P.A., Montorfano, G., Opizzi, A., Faliva, M., Giacosa, A., et al., 2012. Comparision between the AA/EPA ratio in depressed and non depressed elderly females: omega-3 fatty acid supplementation correlates with improved symptoms but does not change immunological parameters. Nutr. J. 11 (82), 10.1186/1475-2891-11-82. Saldeen, P., Saldeen, T., 2004. Women and Omega-3 Fatty Acids. CME Rev. Artic. 59 (10), 722
730. World Health Organization, 2012. World Health Organization. [Online] Available at: ,http://www.who.int/mediacentre/factsheets/fs369/en/index.html. (accessed 20.03.13).

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C H A P T E R

16 The Effectiveness of Fish Oil as a Treatment for ADHD Modhi Ali S. Alshammari and Ronald Ross Watson A REVIEW OF THE LITERATURE The history of attention deficit hyperactivity disorder (ADHD) began in 1902 when a British doctor named George Frederick Still diagnosed cases in the United Kingdom of impulsive behavior in some young patients who were exhibiting challenging behaviors. For the past 50 years, researchers in many different fields (e.g., education, psychology, medicine, and nutrition) have investigated the nature of ADHD in attempts to find an effective treatment for it. Recently, ADHD has been defined as a psychiatric disorder in the American Psychiatric Association’s Diagnostic and Statistical Manual-IV (DSM-IV, 2000). There are two indices of symptoms by which a diagnosis of ADHD can be made: First, ADHD can be diagnosed using a minimum of six of the following inattention symptoms: (a) not paying close attention to detail, (b) having trouble maintaining attention on tasks, (c) not listening when spoken to directly, (d) not following through on instructions and failing to finish schoolwork, (e) having trouble organizing activities, (f) avoiding activities that require a high amount of sustained mental effort, (g) losing things needed for tasks and activities, (h) becoming distracted easily, and/or (i) being forgetful in daily activities. Second, ADHD can also be identified if six of the following hyperactivity-impulsivity symptoms are displayed: (a) fidgeting with hands or feet or squirming in seat, (b) getting up from seat when remaining in seat is expected, (c) running about or climbing when and where it is not appropriate, (d) having trouble playing or doing free time activities quietly, (e) acting as if driven by a motor, (f) talking excessively, (g) answering questions before they have been finished, (h) having trouble waiting one’s turn, and/or (i) interrupting or intruding upon others (DSM-IV, 2000). Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00016-8

Upon reviewing both sets of symptoms (i.e., inattention and hyperactivity-impulsivity) found in the DSM-IV (2000), the authors of the current study identified two facts about how researchers have been dealing with ADHD. First, this disorder is an educational concern (i.e., identified and treated in schools), and second, its diagnostic criteria describe childhood or adolescent (i.e., school age) behavioral problems. Reviewing the educational literature showed that ADHD is one of the recognized learning disabilities, and researchers in the field of special education (SE) have conducted an enormous amount of research into assisting young people with ADHD in achieving better success in school. However, in the field of SE educators have been highly influenced by the medical model, in which a physician identifies a problem in their patient with the goal of fixing it. Following this model, teachers have been struggling to identify the problems or weaknesses in young people with ADHD and then trying to fix these deficiencies. The exact causes of ADHD are still unknown, yet both physicians and teachers have been working to design treatments that might help reduce its symptoms. In the past two decades, ADHD has been a real issue for families, teachers, physicians, and researchers. For instance, Schwing (2009) stated that ADHD has been the most prevalent childhood disorder, with an estimated incidence as high as 18% (Schwing, 2009). Furman (2005) described ADHD as the most common neurobehavioral condition of childhood (Furman, 2005). Glo¨ckner-Rist et al. (2013) stated that ADHD is characterized by symptoms of inattention, hyperactivity, and impulsivity. As shown in the DSM-IV (2000) criteria, ADHD is not a disease per se, but rather a group of symptoms representing a final common behavioral pathway for a gamut of emotional, psychological, and/or

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learning problems (Furman, 2005). Although the exact cause of ADHD remains unknown, many risk factors have been implicated. In a review conducted by the State University of New York Upstate Medical University, Banerjee et al. (2007) concluded that in addition to genetic predispositions, there were many environmental factors that could contribute to a person’s development of ADHD, such as exposure to alcohol before birth, the mother smoking during pregnancy, pregnancy and delivery complications, and low birth weight. One critique that could be made about the conclusion of Banerjee et al. is that those environmental factors could also lead to a number of other problems and therefore might not be an adequate causal explanation for the development of ADHD. Making bold statements about the cause and effect relationship between those types of factors and ADHD may not be appropriate because it may mislead researchers. The authors of the current study concluded that all of the current treatments for ADHD (educational, pharmaceutical, and nutritional) have been geared towards reducing the symptoms, as there is no known cure for the disorder. Consequently, we believed that after a patient stopped their medication, symptoms would return. However, prolonged exposure to the medications used for treating ADHD comes with side effects. These include, but are not limited to, decreased appetite, sleeping difficulties, and behavioral or verbal tics, although these are not common. In addition, different patients react differently to the same medication. This means that there is no one-size-fits-all dosage, which creates problems for the healthcare professional writing the prescription. Varying doses must be tried until the optimum dosage is found that reduces symptoms in the patient without producing substantial side effects. As a result, researchers in all fields have been using different approaches to find a treatment for the symptoms of ADHD that minimizes side effects and maximizes the positive effects of the treatment. The various approaches include social, educational, pharmaceutical, and psychotherapeutic interventions. Although the educational approach to understanding ADHD and its treatment is the most researchable one in the literature, since so many people receiving ADHD diagnoses have been students, the authors of the current study will focus on the medical approach. The rationale for this is that the medical approach reflects the research interests, clarifies the problem, and answers the research questions relating to the current study. It is of interest to the authors of the current study that researchers in the nutrition and SE fields have found that, although there are no specific foods that have been found to cause ADHD, some nutritional deficiencies may worsen ADHD symptoms. Two

nutritional deficiencies that have been the subject of research linked to ADHD symptoms relate to essential fatty acids and iron (Yehuda et al., 2011). One of the most promising findings was emphasized by Colter et al. (2008), who found that patients with ADHD had significantly lower levels of docosahexaenoic acid (DHA, 22:6n-3), which is an omega-3 fatty acid that is a primary structural component of the human brain (Guesnet and Alessandri, 2011).

Purpose The purpose of this study was to investigate the effect of fish oil (i.e., omega-3 and omega-6 fatty acids) in patients with ADHD by exploring two major areas: (a) the effect of the substances in fish oil on the level of fatty acid in the blood of patients with ADHD, and (b) the effect of consuming fish oil on the behavioral/physical symptoms experienced by patients diagnosed with ADHD.

Analyzing the Studies A total of 15 studies were selected: Antalis et al. (2006); Bulut et al. (2007); Colter et al. (2008); Germano et al. (2007); Gustafsson et al. (2010); Huss et al. (2010); Johnson et al. (2009); Kirby et al. (2010a, b); Manor et al. (2012); Perera et al. (2012); Raz et al. (2009); Sinn, 2007; Vaisman et al. (2008) and; Yehuda et al. (2011). Table 16.1 outlines the purpose, sample, design, and results of each of these 15 studies. All of the studies are arranged alphabetically by the researchers’ surnames and numbered from 1 to 15 for ease of cross referencing to the other tables (i.e., Tables 16.2
16.5).

Findings Based on the 15 selected studies, the authors of the current study calculated the number of studies conducted during each year (i.e., from 2006 to March 2013) to observe how researchers’ interests might have varied from year to year. We demonstrated those trends with a simple chronological graph included in this review (see Figure 16.1). The graph shows that researchers were the most interested in investigating this research problem in 2010. The results that were found in the 15 studies ranged from significant improvements in patients after administering the treatments to some positive outcomes. Also, because the 15 studies were conducted using different research problems, sampling procedures, research designs, and measurement systems, drawing an overall conclusion regarding results would have

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A REVIEW OF THE LITERATURE

TABLE 16.1

Summary of the 15 Studies in Which Fish Oil was Used in Patients with ADHD

Study

Purpose

Participants

Design

Results

1. Antalis et al. (2006)

To assess the frequency of skin/thirst symptoms in patients with ADHD To compare the level of blood fatty acids to those of patients in the control group

35 Patients with ADHD 112 Patients without ADHD

Case-control

Patients with ADHD had higher skin/thirst symptoms Saturated fatty acids were higher in patients with ADHD

2. Bulut et al. (2007)

To examine the association between malondialdehyde and ADHD

20 Patients

Case-control

Malondialdehyde was significantly higher in patients with ADHD

3. Colter et al. (2008)

To determine the relationships between EFA and specific ADHD behaviors

11 Patients with ADHD 12 Patients without ADHD

Case-control

Lower omega-3 correlated with higher score on several Conners’ behavioral scales

4. Germano et al. (2007)

To investigate the effects of EPA and DHA on patients’ attention levels To investigate the correlation between omega-3 supplement and AA/EPA ratio

31 Patients with ADHD 36 Patients without ADHD

Regional pilot study

Decrease in the hyperactivity score mean values Decrease in AA/EPA ratio

5. Gustafsson et al. (2010)

To measure efficacy of EPA in patients with ADHD

92 Patients with ADHD

Randomized controlled trial

Improvement in Conners’ Parent/ Teacher Rating Scales

6. Huss et al. (2010)

To evaluate the nutritional effects of polyunsaturated fatty acids in combination with magnesium and zinc in patients with ADHD

810 Patients

Observational longitudinal study

Reduction in symptoms of ADHD

7. Johnson et al. (2009)

To assess whether supplementation with omega-3/6 fatty acids was effective in reducing ADHD symptoms

75 Patients with ADHD

Randomized placebocontrolled trial

Improvement in Clinical Global Impression results No significant difference in Attention Deficit Hyperactivity Disorder Rating Scale-IV Scores In the end 47% showed improvement in ADHD symptoms

8. Kirby et al. (2010a)

To examine the relationship between polyunsaturated fatty acid status in cheek cells with behavior reports and cognitive performance

411 Patients

Randomized controlled trial

Showed correlation between fatty acid levels and parents’ and teachers’ scores

9. Kirby et al. (2010b)

To assess the effects of omega-3 supplementation on cognitive test performance and behavior ratings in a typically developing population

348 Patients

Randomized controlled trial

Reduction in hyperactivity/ impulsivity using SNAP and teacher SDQ Placebo showed better results in overall SDQ teachers’ scores

10. Manor et al. (2012)

To study the efficacy of phosphatidylserine omega-3 in reducing ADHD symptoms

150 Patients with ADHD

Randomized controlled trial

Improvement in the Conners’ Parents Scale and CHQ, but not in SDQ scale

11. Perera et al. (2012)

To assess the effectiveness of combined omega-3 and omega-6 supplementation in patients with ADHD

98 Patients with ADHD

Double-blind placebocontrolled study

No change after 3 months Showed reduction in symptoms after 6 months

12. Raz et al. (2009)

To test the influence of short term essential fatty acids on ADHD

78 Patients

Double-blind placebocontrolled study

Significant improvement was shown in the experimental group.

13. Sinn (2007)

To determine fatty acid deficiency symptom levels and their relationship with items on Conners’ ADHD index, and whether FADS predicted degree of response to polyunsaturated fatty acid supplementation

Study 1, in a Crossgeneral population sectional (N 5 347) correlational Study 2, in patients with ADHD (N 5 104)

No improvement was shown in FADS in the polyunsaturated fatty acids group compared to the placebo group

(Continued)

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

190

16. EFFECTIVENESS OF FISH OIL IN TREATING ADHD

TABLE 16.1

(Continued)

Study

Purpose

Participants

Design

Results

14. Vaisman et al. To investigate the incorporation of long-chain (2008) omega-3 fatty acids provided as phospholipids or fish oils into blood and compare it with placebo

83 Patients with ADHD

Randomized controlled trial

The TOVA score showed an increase in the phospholipid and fish oil groups, but not in the placebo group

15. Yehuda et al. (2011)

78 Patients with ADHD

Controlled clinical trial

Showed improvement in quality of life and quality of sleep

To address the effects of a mixture of EFA on sleep-deprived ADHD patients

Note: Studies were arranged alphabetically by the researchers’ surnames and numbered from 1 to 15; the purpose, participants, design, and intervention were provided as a summary of each study.

Number of reviewed studies

5 4 3 2 1 0 2006

2007

2008

2009

2010

2011

2012

Years

FIGURE 16.1 The number of studies into the use of fish oil in patients with ADHD over time.

been impossible, and categorizing similar studies together in sub-groups was required. The authors of the current study identified two major sub-groups: (a) nine studies investigated the effect of fish oil on ADHD behavioral/physical symptoms and (b) seven studies investigated the level of fatty acid in the blood of patients with ADHD. The Effect of Fish Oil on the Behavioral/Physical Symptoms of ADHD OMEGA 3

Three out of the nine researchers in this category (Manor et al., 2012; Germano et al., 2007; Kirby et al., 2010b) used an omega-3 supplement with the participants. Manor et al. (2012) studied the effect of omega-3 in reducing the symptoms of ADHD. The participants were 150 patients, whose ages ranged from 6 to 13 years old. All participants had been diagnosed using the criteria for ADHD as presented in the DSM-1V (2000). The patients were divided into two groups, with one receiving phosphatidylserine (PS) omega-3, and the other receiving a placebo. Changes in participants’ ADHD symptoms were measured using three measurements (i.e., Conners’ Teacher/Parent Rating Scale [CRS-T], the Strengths and Difficulties Questionnaire

[SDQ], and the Patient Health Questionnaire [CHQ]). The findings of Manor et al. were as follows: (a) in the CRS-T, there was an improvement in the PS omega-3 group’s score; however, there were no differences when comparing the PS omega-3 group to the placebo group; (b) results of CRS-T and CHQ showed that the PS omega-3 group performed better than the placebo group; and (c) there were no observed differences between the two groups for any of the SDQ subscales. The authors of the current study criticized the study by Manor et al. regarding one aspect, which was that switching the study from a double-blind study to openlabel in the middle of the treatment reduced the time for comparing the two groups to 15 weeks, and might have negatively affected the results. In contrast to the study by Manor et al. (2012), Kirby et al. (2010b) included more participants (i.e., 348 patients) in their study, whose ages ranged from 8 to 10 years old. However, the Kirby et al. study was similar to that of Manor et al. in that they were both investigating the effect of omega-3 supplementation on children with ADHD. In the study by Kirby et al., the participants were divided into two groups, with 171 participants receiving omega-3 and 177 receiving placebo. For 16 weeks, the study was a comparison between the two groups; however, the researchers included an extra 8 weeks as an open-label study, in which all patients in both groups were given omega-3. To measure the effects of the supplement, the researchers used parent-teacher questionnaires (i.e., Swanson Nolan and Pelham [SNAP] and SDQ). The researchers found a reduction in hyperactivity/impulsivity when comparing SNAP results and the teachers’ SDQ results to baseline after 16 weeks. However, the overall SDQ teachers’ scores were better in the placebo group. The authors of the current study criticized Kirby et al. regarding two aspects of the study: (a) the researchers reported a variety of outcomes in tables without further explanation; and (b) the researchers focused on the results found after 16 weeks but generalized the data for 24 weeks, which might mislead the reader.

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

A REVIEW OF THE LITERATURE

In contrast to the studies of Manor et al. (2012) and Kirby et al. (2010b), Germano et al. (2007) conducted their study with a very small sample size (i.e., only 16 patients with ADHD) as a one-group pretest-posttest design. Also, in the Germano et al. study, the treatment period lasted only 8 weeks. However, similar to the Manor et al. study, Germano et al. used Conners’ scale to measure the differences between the pre- and posttests. Germano et al. included two parts in their study: first, they investigated the effect of eicosapentaenoic acid (EPA) and DHA on patients’ attention levels. The second part of their study will be discussed in the section below that examines findings on fatty acid levels in the blood of patients with ADHD. All patients in the Germano et al. study received omega-3 supplements for 8 weeks, as the study included only one group. The effects of the supplement were measured using Conners’ scale, and the researchers found that hyperactivity and inattention scores decreased compared to baseline. The small sample size of 16 patients, using only one group, and implementing the treatment for only 8 weeks might have negatively influenced the results from the Germano et al. study. In conclusion, after analyzing the three studies (Manor et al., 2012; Germano et al., 2007; Kirby et al., 2010b), the authors of the current study believed that some evidence had been found across the three studies showing promising effects of using omega-3 to treat patients with ADHD. However, more investigations in this area are needed. Future researchers may want to consider using direct observation as a method of measuring patients’ behaviors, as we believe this might be a superior method to questionnaires, which rely to some degree on the feelings and opinions of teachers and parents. Also, treatment periods in future studies need to be longer so that clear results about the effectiveness of omega-3 can be obtained. OMEGA-3 AND OMEGA-6

Four out of the nine researchers in this category (Johnson et al., 2009; Perera et al., 2012; Raz et al., 2009; Yehuda et al., 2011) used omega-3 and omega-6 as a supplement with the participants. In the Perera et al., 2012 study, the researchers investigated the effect of omega-3 and omega-6 as a treatment for patients with ADHD in behavioral and educational settings. The researchers included 94 patients aged between 6 and 12 years, who had been treated with a methylphenidate and standard behavioral therapy for 6 months or more (Perera et al., 2012). The total sample was divided into two groups (i.e., 48 patients were allocated to the experimental group and 46 patients were allocated to the control group). The researchers did not find any differences between the two groups after 3 months of treatment. Parera et al. measured the

191

patients’ behaviors by using an 11-item checklist, which was completed by the patients’ parents. After 6 months of treatment, Parera et al. found that the combination of omega-3 and omega-6 was effective in reducing most of the symptoms of ADHD compared to patients who took the placebo (i.e., the control group). The authors of the current study criticized the Parera et al. study regarding one critical aspect, which was that all patients included in the study had been taking medication and attending behavioral therapy sessions before, and continued to do so during, and after the study was conducted. Therefore, we believed that these uncontrolled variables might have affected the validity of the treatment results. The 2009 study by Johnson et al. included 75 patients aged between 8 and 18 years of age, none of whom received any outside treatment for ADHD during the study. The researchers in this study investigated the effects of using omega-3 and omega-6 in adolescents with ADHD. In this study, 78% of the patients had at least one comorbid diagnosis such as anxiety, autism or a learning disability (Johnson et al., 2009). The participants were divided into two groups, with one group receiving an active supplement and the other receiving a placebo for the first period of the study (i.e., 3 months), and then both groups receiving an active supplement for another 3 months (i.e., period 2). Two measurements were used to assess outcomes: the ADHD Rating Scale-IV and the Clinical Global Impression (CGI). Therefore, the Johnson et al. study was more reliable than that of Parera et al. because it used two measurement systems. Johnson et al. found that the results of the ADHD Rating Scale-IV were not significant for the group who took an active supplement compared to the placebo group. However, when using the CGI measurement results, the researchers found that the active group showed a significant improvement compared to the placebo group. The improvement in CGI results was to a near-normal range. The authors of the current study criticized the Johnson et al. study for failing to mention whether the patients received any other type of medication or school intervention to treat comorbid diagnoses. If so, this might have affected the results of the study, and may have explained the positive findings. Approaching the effects of using omega-3 and omega-6 in patients with ADHD from a different direction, Yehuda et al. (2011) investigated the effect of an essential fatty acid (EFA) mixture in sleep-deprived ADHD children (Yehuda et al., 2011). Similar to the studies by Parera et al. and Johnson et al., Yehuda et al. included a reasonable sample size of 78 patients diagnosed with ADHD, who ranged from 9 to 12 years of age. In the study, there were 40 patients who received omega-3 and omega-6 (i.e., the experimental

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

192

16. EFFECTIVENESS OF FISH OIL IN TREATING ADHD

group) and 38 who received a placebo (i.e., the control group). One of the strengths of this study was that the patients in the sample had been diagnosed with ADHD by two different psychologists. The findings were measured using a questionnaire, which was completed only by the patients. Asking the patients to selfcomplete the questionnaire was unique within this study category, and was not used in either the Parera et al. or the Johnson et al. studies. The researchers found a significant improvement in the quality of life of the participants who took the supplement, specifically in the quality of participants’ sleep. The authors of the current study criticized the Yehuda et al. study for using only a self-reported questionnaire, as this might have biased the results. One of the most interesting studies in this category was the investigation by Raz et al. (2009), because of its well-developed measurement systems. The researchers used Test of Variables of Attention (TOVA), which is a computer-based test. However, the Raz et al. study was similar to those of Perera et al. (2012), Johnson et al. (2009), and Yehuda et al. (2011) in investigating the effects of omega-3 and omega-6 in patients with ADHD. Raz et al. included 78 patients who were diagnosed with ADHD and who had not taken any medication or EFAs for the past 3 months. The patients were aged between 7 and 13 years of age. To measure the effects of the supplement, the researchers used parent and teacher questionnaires. They used TOVA to measure the attentiveness of the patients. The researchers found a significant difference in the active (i.e., experimental) group compared to the placebo (i.e., control) group. The authors of the current study criticized Raz et al. for the short treatment period used in the study, which only lasted 7 weeks. In conclusion, after this portion of the analysis, the authors of the current study believed that major evidence had been found across the four studies, which could be seen as showing fairly effective results of using omega-3 and omega-6 to reduce ADHD symptoms. However, as found in our review of the omega-3 research, more investigations are needed in this area. Future researchers might use direct observation or TOVA as methods of measuring patients’ behaviors, as we believe that relying solely on questionnaires is not always effective. The treatment periods in this category also need to be longer so that clear results can be found. Overall, and on the basis of the selected studies, the authors of the current study believed that the effectiveness of using omega-3 and omega-6 was clearer than using only omega-3 in patients with ADHD. COMBINATION OF ESSENTIAL FATTY ACIDS AND OTHER SUPPLEMENTS

Two out of the nine researchers in this category (Huss et al., 2010; Sinn, 2007) used this supplement

with participants. In Sinn’s 2007 study, the goal was to find the relationship between fatty acid deficiency symptoms (FADS) in patients and the patients’ scores on Conners’ ADHD index. Sinn’s first study analyzed the results of 347 patients from the general population. Using Conners’ ADHD index and a background questionnaire completed by the parents as a measurement scale in this study, Sinn found that Conners’ index scores were positively correlated with the total FADS (Sinn, 2007). This means that an increase in the Conners’ index scores of the participants was accompanied by an increase in participants’ FADS. Sinn’s second study was a randomized, controlled, doubleblind study that investigated the effect of a polyunsaturated fatty acid (PUFA) supplement, including 400 g fish oil, on the FADS of a group with ADHD after 15 weeks. In this study, 451 patients with ADHD aged 7 to 12 were included. After supplementation with PUFA or placebo oil, the researcher used Conners’ rating scale to measure the outcomes. The researcher found that after 15 and 30 weeks of taking the supplement, there were no improvements in the FADS of the control group compared to the placebo group. However, there were overall improvements in all groups (i.e., active and placebo). The authors of the current study criticized Sinn’s study for using patients who were not officially diagnosed with ADHD. While Sinn (2007) used Conners’ scales to measure the outcomes, Huss et al. (2010) used different instruments (SNAP and SDQ) to investigate the effect of an omega-3/-6-zinc-magnesium combination on the ADHD symptoms of participants. Huss et al.’s study was the largest in terms of participants with 810 patients, 94.7% of whom received the recommended dose of four capsules of the supplement per day. The researchers found that the four items related to emotional problems from the SDQ scale decreased in participants compared to baseline. Sleep problems also decreased. The authors of the current study criticized Huss et al. for not using a control group, which made the effectiveness of the treatment unclear. In conclusion, after analyzing these two studies, the authors of the current study believed that some evidence had been found in support of the effectiveness of the supplements. However, when we compared the findings in this category with those from the previous categories, we noted that the findings from the studies that used omega-3 and omega-6 showed greater improvements in outcomes. One of the limitations that prevented us from making any bold statements about the findings from this category was the fact that there were only two studies investigating this research problem. Therefore, there is a need for additional research in this area.

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

193

A REVIEW OF THE LITERATURE

Fatty Acid Levels in Blood of Patients with ADHD OMEGA 3

Four out of the seven researchers in this category (Germano et al., 2007; Gustafsson et al., 2010; Kirby et al., 2010a; Vaisman et al., 2008) used omega-3 as a supplement with participants. Vaisman et al. investigated the incorporation of omega-3 and fish oil into the blood. The researchers included 83 patients aged between 8 and 13 years. The researchers tested fatty acid levels in the plasma of the participants after receiving a placebo, omega-3 or fish oil. They found that TOVA scores differed between the three groups, with the highest scores being found in the omega-3 group, followed by the fish oil group, and the lowest scores being found in the placebo group. Using a different measurement system, Kirby et al. (2010a) investigated levels of fatty acid in patients and compared it to patients’ learning and behavior styles. The researchers included 411 patients in the study, aged between 8 and 10 years, with participants being selected from the general population. The fatty acid results were collected from a cheek cell sample and compared to results from a variety of questionnaires. These questionnaires were designed to identify symptoms of ADHD in the participants. The researchers found a correlation between fatty acid levels and parent-teacher questionnaire results. They also found a positive association between omega-3 fatty acid levels and pro-social behavior (Kirby et al., 2010a). Gustafsson et al. (2010) measured the level of fatty acid directly from patients’ blood samples. In this study, 92 patients aged between 7 and 12 years old were included. The patients received omega-3 or a placebo for 15 weeks. The researchers analyzed the fatty acid in the serum phospholipid and red blood cell membranes. They found that EPA improved Conners’ Teacher Rating Scales (CTRS), but not Conners’ total score (Gustafsson et al., 2010). Similarly, Germano et al. (2007) investigated the correlation between an omega-3 supplement and arachidonic acid (AA)/EPA ratios to measure the change in AA/EPA. In this study, there were 31 patients with ADHD and 36 non-ADHD diagnosed controls. All participants received an omega-3 supplement for 8 weeks. The results were obtained using the fatty acid content and AA/EPA ratio in the blood. The researchers found that before the supplementation, participants with ADHD had higher AA/EPA ratios, which decreased in this group after supplementation. In conclusion, after analyzing the studies on fatty acid levels, the authors of the current study believed that all the studies were consistent in that they all attempted to measure/monitor the fatty acid in patients; however, each one used a different approach. While some evidence was found across the four

studies, we believe more research is needed in this area and that it should be conducted using similar methods. NO SUPPLEMENTS WERE GIVEN

Three out of the seven researchers in this category (Antalis et al., 2006; Bulut et al., 2007; Colter et al., 2008) did not use supplements with participants. Antalis et al. (2006) investigated the frequency of skin/thirst symptoms in patients with ADHD, and compared the level of blood fatty acid to the control group. The researchers included 35 patients with ADHD and 112 controls, all of whom were college students. The researchers also used Conners’ Adult ADHD Rating Scale (CAARS) and blood analysis on both groups. In the first analysis they found that patients with ADHD had higher results on the skin/ thirst symptoms and on all CAARS subscales. In the second analysis, they found that the omega-3 level was lower in patients with ADHD; however, omega-6 was higher in those with ADHD. With a very small sample size of 23, Colter et al. (2008) conducted a study in which they investigated the difference in fatty acid levels between patients with ADHD and a control group. The researchers wanted to examine the differences in dietary intake between 11 patients with ADHD and 12 controls (aged 10 to 16 years). The researchers used blood samples to measure fatty acid levels. They also asked participants to record their diets for 7 days. They found that the ADHD group had higher dietary intake of protein and saturated fatty acid, but not of omega-3, EPA or DHA. The ADHD group also had lower DHA and omega-3 blood levels compared to the control group. Bulut et al. (2007) examined the levels of malondialdehyde (MDA) in ADHD patients. In this study, the researchers included 20 patients with ADHD and 21 healthy volunteers. The measurement in this study was different from those used in other blood studies, because the researchers measured the level of MDA in the plasma. The researchers’ rationale was that MDA is the end product of fatty acid oxidation. They found that patients with ADHD had higher levels of MDA than the control group, which means that the oxidation level in ADHD patients is higher. In conclusion, after analyzing these three studies, the authors of the current study believed that enough evidence had been found across the three studies to indicate promising findings. However, more research is needed to confirm those findings and to arrive at a clear conclusion.

CONCLUSION The purpose of this study was to investigate the effects of fish oil (i.e., omega-3 and omega-6 fatty acid)

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

TABLE 16.2

Analyses of the Patients and Geographical Regions Across All 15 Studies Participants Age as Categorized by School Levels

S

P

E

M

H

C

To.

AM

O

O

O

35

O

O

O

O

O

20

O

O

O

O

23

O

O

O

67

O

O

O

O

92

O

O

O

O

810

O

O

75

O

411

O O

O O

3. O

5. O

O

O

O

O

O

O

7.

O

8. 9.

O

O

O

O

O

O

O

348

O

O

O

150

O

O

O

98

O

O

O

78

O

O

O

O

O

O

O

11.

O

O

12.

O

13.

O

14.

O

15.

O

T

O

Un.

O

O

O

G

O

10.

O

B

Geographical Regions

Nor.

2.

6.

Un.

Patients Included AD.

1.

4.

Gender

O

O

O

O

O

O

3

1

1

12

11

3

14

7

26%

86%

40%

20%

6%

6%

80%

73%

20%

93%

46%

O O O O

O

2819

6

O

O

78

AU

O

83 O

13

ME

451

O

4

EU

2

7

5

1

13%

46%

33%

6%

Note: S 5 Study (i.e., studies were numbered from 1 to 15 alphabetically by the researchers’ surnames [See Table 16.1]); T 5 Total; P 5 Pre-school (i.e., from 3 to 6 years old); E 5 Elementary (i.e., from 7 to 12 years old); M 5 Middle school (i.e., from 13 to 15 years old); H 5 High school (i.e., from 16 to 18 years old); C 5 College (i.e., from 19 to 25 years old); Un. 5 Unreported; B 5 Boys; G 5 Girls; AD. 5 ADHDdiagnosed; Nor. 5 Normal; To. 5 Total included patients; AM 5 America (i.e., the United States of America and Canada); EU 5 Europe (i.e., the United Kingdom, Sweden, Germany, and Italy); ME 5 Middle East (i.e., Israel and Turkey); AU 5 Australia; “O” 5 Yes.

TABLE 16.3 Analyses of the Overall Examined Variables and Research Designs Across All 15 Studies Behavioral/Physical ADHD Symptoms After Using S

n-3

n- 3/ n-6

FA 1

Level of Fatty Acid in Blood of Patients with ADHD After Using n-3

n- 3/ n-6

FA 1

No

Duration of Treatment (Weeks)

General Research Design CT

OL

CC

CS

RP

0
15

1.

O

O

O

2.

O

O

O

3.

O

O

O

4.

O

O

O

O

5.

O

O

6.

O O

O

O

7.

O

O O

8.

15
30

O

O

O

9.

O

O

O

10.

O

O

O O

11.

O

O

12.

O

O O

13.

O O

14. O

15. T

O O

O

O

O

O

3

4

2

4

0

0

3

9

1

3

1

1

10

5

20%

26%

13%

26%

0%

0%

20%

60%

6%

20%

6%

6%

66%

30%

Note: S 5 Study (i.e., studies were arranged alphabetically by the researchers’ surnames and numbered from 1 to 15 [See Table 16.1]); T 5 Total; n-3 5 Omega 3; n-6 5 Omega 6; FA 1 5 Fatty acid with other supplements; No 5 Nothing has been given; CT 5 Controlled trial (i.e., Randomized controlled trial, Randomized placebo-controlled trial, Controlled clinical trial, Double-blind placebo-controlled study); OL 5 Observational and longitudinal study; CC 5 Case-control; CS 5 Cross-sectional correlation; RP 5 Regional pilot study; “O” 5 Yes.

TABLE 16.4

Analyses of the Measurement Systems and the Overall Outcomes Across the Nine Studies About Behavioral Symptoms of ADHD Measuring Behavioral/Physical Symptoms of ADHD Using: SNAP

SDQ

Conners’

CL/QU

Overall Outcomes

TOVA

CGI

RS

Sub.

Dr.

Dr.

PO

SPO

S

Sub.

P. / T.

Sub.

P. / T.

P. / T.

Sub.

P. / T.

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

O

4. ----

----

----

----

----

6.

O

O

O

O

----

O ----

----

----

----

----

----

O

9.

----

----

----

O

O

12.

O

13.

O ----

----

----

----

O

----

----

O ----

----

O

O

O

O O

O O

----

----

----

----

O

15. T

----

O

11.

----

----

O

O

10.

----

---O

7. ----

----

----

----

O

1

2

1

3

4

1

2

1

1

1

2

7

11%

22%

11%

33%

44%

11%

22%

11%

11%

11%

22%

77%

Note: S 5 Study (i.e., studies were arranged alphabetically by the researchers’ surnames [See Table 16.1]); T 5 Total; SNAP 5 Swanson Nolan and Pelham; SDQ 5 Strengths and Difficulties Questionnaire; Conners’ 5 (Conners’ Adult ADHD Rating Scales, Conners’ ADHD index, Conners’ Teacher Rating Scale, and Conners’ Abbreviated Parent-Teacher); CL/QU 5 Checklist/Questionnaire; CGI 5 Clinical Global Impression; RS 5 Attention Deficit Hyperactivity Disorder Rating Scale; Sub. 5 Subject/participants (i.e., adults and children); P. / T. 5 Parents and/or teachers; TOVA 5 Test of Variables of Attention; Dr. 5 trained doctors (i.e., pediatricians or patients’ psychiatrists); PO 5 positive outcomes have been found; SPO 5 some positive outcomes have been found; “O” 5 Yes; “----” 5 Not included (i.e., those are the studies that focus on the blood of the ADHD patients [See Table 16.5]).

TABLE 16.5 Analyses of the Measurement Systems and the Overall Outcomes Across the Seven Studies About the Fatty Acid Levels in the Blood of Patients with ADHD Measuring the Fatty Acid Levels in the Blood of the ADHD Patients Using: Blood Test S

Fatty Acid

1.

O

AA/EPA Ratio

Cheek Cell Malondialdehyde

Fatty Acid

Overall Outcomes Dietary Intake

PO

SPO O

O

2.

O

3.

O

O

4.

O

5.

O

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

O

O O O

O

8.

O

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

14.

O

----

----

----

----

----

----

----

----

T

5

1

1

1

1

3

4

71%

14%

14%

14%

14%

42%

57%

O

Note: S 5 Study (i.e., studies were arranged alphabetically by the researchers’ surnames [See Table 16.1]); T 5 Total; AA/EPA 5 Arachidonic acid/eicosapentaenoic acid; PO 5 positive outcomes have been found; SPO 5 some positive outcomes have been found; “O” 5 Yes; “----” 5 Not included (i.e., those are the studies that focus on the behavioral/physical symptoms [See Table 16.4]).

198

16. EFFECTIVENESS OF FISH OIL IN TREATING ADHD

in patients with ADHD by exploring two major areas: (a) the effects of fish oil supplements on the levels of fatty acid in ADHD patients’ blood; and (b) the effects of using fish oil on the behavioral/physical symptoms of ADHD. The major limitations of the current study were that we were restricted only to literature that was written in the English language, and to that which was available online. Therefore, we might have missed out on other potentially significant research and findings that were written in different languages or published only in print. However, with respect to the existing limitations, the authors of the current study identified gaps in the literature as follows. For the 7-year span of research that we analyzed (i.e., from 2006 to March 2013), only 15 studies were chosen for the current study. The 15 studies were conducted in four geographical regions: (a) two in America (i.e., the United States of America and Canada); (b) seven in Europe (i.e., the United Kingdom, Sweden, Germany, and Italy); (c) five in the Middle East (i.e., Israel and Turkey); and (d) one in Australia. The total sample size across all 15 studies was 2819, of which 86% were elementary school students. Therefore, we encourage future researchers to include pre-school children as well as older students and adults in their studies (for more information about gaps in the literature regarding sampling procedures, see Table 16.2). In the treatments procedures, in most of the studies we reviewed (i.e., 26%), the researchers used omega-3/ omega-6 as a supplement to affect the behavioral/ physical symptoms of patients with ADHD. However, in most reviewed studies (i.e., 26%), the researchers used omega-3 as a supplement to affect fatty acid levels in the blood of patients with ADHD. Therefore, the authors of the current study encourage future researchers to be consistent in examining all supplements (for more information about gaps in the literature regarding treatment procedures, see Table 16.3) One of the most challenging issues we found across all 15 studies was the variation of measurement systems. For instance, the measurements that the researchers used to record behavioral/physical symptoms of ADHD varied from study to study. Therefore, the authors of the current study encourage future researchers to use the most reliable and valid tests so that future reviewers can draw clear and complete conclusions (for more information about gaps in the literature regarding measurement systems, see Tables 16.4 and 16.5).

Final Thought ADHD presents a very challenging and complicated problem. However, by organizing cooperative research

teams in which researchers from different fields can work together, we might find a solution for this problem and a way to help individuals diagnosed with ADHD to experience higher levels of academic/vocational success and a better quality of life.

References American Psychiatric Association, 2000. Diagnostic and Statistical Manual of Mental Disorders, fourth ed., text rev., Washington, DC. Antalis, C.J., Stevens, L.J., Campbell, M., Pazdro, R., Ericson, K., Burgess, J.R., 2006. Omega-3 fatty acid status in attention-deficit/ hyperactivity disorder. Prostaglandins Leukot. Essent. Fatty Acids. 75 (4), 299
308. Banerjee, T.D., Middleton, F., Faraone, S.V., 2007. Environmental risk factors for attention-deficit hyperactivity disorder. Acta. Paediatr. 96 (9), 1269
1274. Bulut, M., Selek, S., Gergerlioglu, H.S., Savas, H.A., Yilmaz, H.R., Yuce, M., et al., 2007. Malondialdehyde levels in adult attentiondeficit hyperactivity disorder. J Psychiatry. Neurosci. 32 (6), 435
438. Colter, A.L., Cutler, C., Meckling, K.A., 2008. Fatty acid status and behavioural symptoms of attention deficit hyperactivity disorder in adolescents: a case-control study. Nutr. J. 7 (8), 79
85. Furman, L., 2005. What is attention-deficit hyperactivity disorder (ADHD)?. J. Patient Neurol. 20 (12), 994
1002. Germano, M., Meleleo, D., Montorfano, G., Adorni, L., Negroni, M., Berra, B., et al., 2007. Plasma, red blood cells phospholipids and clinical evaluation after long chain omega-3 supplementation in children with attention deficit hyperactivity disorder (ADHD). Nutr. Neurosci. 10 (1-2), 1
2. Glo¨ckner-Rist, A., Pedersen, A., Rist, F., 2013. Conceptual structure of the symptoms of adult ADHD according to the DSM-IV and retrospective Wender-Utah criteria. J. Atten. Disord. 17 (2), 114
127. Guesnet, P., Alessandri, J.M., 2011. Docosahexaenoic acid (DHA) and the developing central nervous system (CNS)—Implications for dietary recommendations. Biochimie. 93 (1), 7
12. Gustafsson, P.A., Birberg-Thornberg, U., Duche´n, K., Landgren, M., Malmberg, K., Pelling, H., et al., 2010. EPA supplementation improves teacher-rated behaviour and oppositional symptoms in children with ADHD. Acta. Paediatr. 99 (10), 1540
1549. Huss, M., Vo¨lp, A., Stauss-Grabo, M., 2010. Supplementation of polyunsaturated fatty acids, magnesium and zinc in children seeking medical advice for attention-deficit/hyperactivity problems—an observational cohort study. Lipids. Health. Dis. 9 (1), 105. ¨ stlund, S., Fransson, G., Kadesjo¨, B., Gillberg, C., 2009. Johnson, M., O Omega-3/Omega-6 fatty acids for attention deficit hyperactivity disorder. A randomized placebo-controlled trial in children and adolescents. J. Atten. Disord. 12 (5), 394
401. Kirby, A., Woodward, A., Jackson, S., Wang, Y., Crawford, M.A., 2010a. Childrens’ learning and behaviour and the association with cheek cell polyunsaturated fatty acid levels. Res. Dev. Disabil. 31 (3), 731
742. Kirby, A., Woodward, A., Jackson, S., Wang, Y., Crawford, M.A., 2010b. A double-blind, placebo-controlled study investigating the effects of omega-3 supplementation in children aged 8
10 years from a mainstream school population. Res. Dev. Disabil. 31 (3), 718
730. Manor, I., Magen, A., Keidar, D., Rosen, S., Tasker, H., Cohen, T., et al., 2012. The effect of phosphatidylserine containing omega 3 fatty-acids on attention-deficit hyperactivity disorder symptoms

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FURTHER READING

in children: A double-blind placebo-controlled trial, followed by an open-label extension. Eur. Psychiatry. 27 (5), 335
342. Perera, H., Jeewandara, K.C., Seneviratne, S., Guruge, C., 2012. Combined ω3 and ω6 supplementation in children with attentiondeficit hyperactivity disorder (ADHD) refractory to methylphenidate treatment—a double-blind, placebo-controlled study. J. Patient Neurol. 27 (6), 747
753. Raz, R., Carasso, R.L., Yehuda, S., 2009. The influence of short-chain essential fatty acids on children with attention-deficit/hyperactivity disorder: A double-blind placebo-controlled study. J. Patient Adolesc. Psychopharmacol. 19 (2), 167
177. Schwing, L.J., 2009. Attention deficit hyperactivity disorder (ADHD): Has diet therapy taken a place at the table? J. Consum. Health Internet. 13 (1), 93
102. Sinn, N., 2007. Physical fatty acid deficiency signs in children with ADHD symptoms. Prostaglandins Leukot. Essent. Fatty Acids. 77 (2), 109
115.

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Vaisman, N., Kaysar, N., Zaruk-Adasha, Y., Pelled, D., Brichon, G., Zwingelstein, G., et al., 2008. Correlation between changes in blood fatty acid composition and visual sustained attention performance in children with inattention: Effect of dietary n 2 3 fatty acids containing phospholipids. Am. J. Clin. Nutr. 87 (5), 1170
1180. Yehuda, S., Rabinovitz-Shenkar, S., Carasso, R.L., 2011. Effects of essential fatty acids in iron deficient and sleep-disturbed attention deficit hyperactivity disorder (ADHD) children. Eur. J. Clin. Nutr. 65 (10), 1167
1169.

Further Reading Brookes, K.J., Chen, W., Xu, X., Taylor, E., Asherson, P., 2006. Association of fatty acid desaturase genes with attention-deficit/ hyperactivity disorder. Biol. Psychiatry. 60 (10), 1053
1061.

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C H A P T E R

17 Fatty Acids and the Aging Brain Alyssa Bianca Velasco and Zaldy S. Tan INTRODUCTION The past few decades have presented a significant shift in the age demographic of the world’s population. For the first time in history, the 65 and older age bracket will represent the largest proportion of the population, increasing from an estimated 11% in 2000, to 22% in 2050 (World Health Organization [WHO], 2013). This demographic change brings with it the challenge of understanding how to care for a population of patients with a distinct set of disease characteristics. While modern medicine has lowered the mortality rate for many acute diseases and thus allowed people to live longer, aging and its associated physiological decline have increased the chronic disease burden and the need for more research on age-related diseases. Among the most prevalent diseases associated with aging is dementia, a neurological condition characterized by a decline in global cognitive function beyond that expected with normal aging. The cognitive decline associated with dementia can be extremely debilitating; it often interferes with activities of daily living, and results in both emotional and financial stress for those affected and their families. As of 2010, the WHO reports 35.6 million cases of dementia worldwide, and projects that this number will double approximately every twenty years up to a staggering 115.4 million cases in 2050 (WHO, 2012). Though dementia is not a normal part of the physiologic aging process, age is the single greatest risk factor for the condition (Corrada and Brookmeyer, 2008; Evans, 1996). As a growing proportion of the population reaches old age, dementia will become an increasing burden to our society. Understanding the mechanisms that contribute to pathological brain aging and identifying potential prevention and treatment measures have been a focus of current research in the field of neuroscience. In particular, preventing premature neurological decline and

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00017-X

delaying the onset of age-related brain diseases for even a few years can yield significant benefits at a population level (Viswanathan and Rocca, 2009). Recently, several studies have identified diet as a potentially modifiable risk factor by demonstrating an association between the consumption of omega-3 fatty acids and a lower risk for dementia and cognitive decline (Kalmijn and Launer, 1997; Morris and Evans, 2003). However, other investigations in this area did not confirm this association, and the true relationship remains an important area of research (Laurin and Verreault, 2003). Comprehending the effect of fatty acid consumption on brain aging is especially pertinent because of the important role of fatty acids in the brain. Fatty acids are vital to the structure of biological membranes and the integrity of membrane properties including fluidity and ion transport (Florent-Be´chard et al., 2007). Of all the organs of the human body, the brain has the greatest concentration of fatty acids, particularly long-chain polyunsaturated fatty acids (PUFAs), omega-3 docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), and the omega-6 PUFA arachidonic acid (AA) (Brenna, 2002). Over the past century, the consumption of dietary fats has increased substantially in modern Westernized diets (Muskiet and Fokkema, 2004). In fact, fat has become the single greatest source of energy for most Americans (Arab, 2003)
a statistic that is representative of the typical diet for Westernized countries. Further, the proportion of different types of fat in the human diet has also changed significantly, with an increase in saturated fats and trans-fats (Simopoulos, 1999). In addition, of the unsaturated fatty acids, consumption of the PUFA (n-6) family of fatty acids such as linoleic acid (LA) and AA has increased, while the consumption of the PUFA (n-3) family, including DHA and EPA, has decreased drastically (Muskiet and Fokkema, 2004).

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Though the precise effects on brain aging of these dietary modifications are not yet fully understood, support for the neuroprotective nature of the omega-3 fatty acids continues to accumulate (Cole and Ma, 2009). This leads some to surmise that increasing consumption of omega-3 fatty acids, either by dietary modification or supplementation, may be a viable method for preventing pathological cognitive decline. Nutritional interventions are attractive because they are more cost effective and are likely to be safer than novel drug treatments (Cole and Frautschy, 2010). Though the questions of ideal time of intervention and recommended dosage remain unanswered, a nutritional intervention with omega-3 fatty acids offers a promising prevention-based solution to rising healthcare costs associated with diseases of the aging brain.

PHYSIOLOGIC BRAIN AGING Due to the unprecedented growth that is expected in the aging population, a plethora of today’s research is focused on understanding disease processes related to aging. One of the biggest challenges facing investigators in this area is that the definition of what constitutes ‘normal aging’ is subjective and dynamic. In the past, cognitive decline was often viewed as an inevitable component of aging, but with time it has become clear that there is significant individual variability in the aging process, i.e. some people can retain their cognitive functions until late in life, while others have declines beyond what can be accounted for by a natural physiologic cause. Another challenge specific to studies on normative cognitive aging is that they must be representative of contemporary lifestyles (Au and Seshadri, 2004). For example, people born today typically have more years of formal education when compared to people born in the previous generations. Because education has been shown to be a protective factor against cognitive decline, there is a need for updating the standards for ‘normal’ (Birren and Morrison, 1961; Cagney and Lauderdale, 2002; Christensen et al., 1997). Despite these challenges, understanding the differences between physiologic and pathological brain aging is essential because knowledge of the natural processes promotes our understanding of disease processes, and provides insight on how to intervene at an early stage. The impact of aging on the brain can be observed at many different levels, from a cellular level to a functional level. Chemical changes influence structural changes, which in turn, result in differences in cognitive capacity, and are thus interrelated. The structural, chemical, and cognitive alterations that take place during physiologic aging are reviewed in the following sections.

Structural Changes Structural brain changes associated with physiologic aging occur at both macro- and micro-levels. Macrostructural changes include decreases in regional brain volumes (Allen, 2005; Fotenos, 2005; Piperhoff, 2008), cortical thinning (Magnotta, 1999; Salat, 2004.), and ventricular system expansion. Micro-structural changes include loss of neural circuits and brain plasticity, and accumulation of neurofibrillary tangles. These two levels of changes will be discussed individually. Macro-Structural Changes One of the most notable neuro-anatomical changes in the aging process is a general decrease in brain volume. Studies have shown that even in a population of healthy adults, older adults experience more than double the rate of brain shrinkage experienced by younger adults (Raz and Lindenberger, 2005) and specifically, that age accounted for 50% of the total volume differences (DeCarli and Masaro, 2005). A study by Fjell (2010) quantified the aging effect further by describing volume variance in particular regions of the brain. The study showed that more than a third of the volume variances in the thalamus, nucleus accumbens, and hippocampus can be accounted for by aging (Fjell, 2010). Brain shrinkage occurs throughout all regions, but certain regions are more susceptible to shrinkage than others. Some brain regions shrink significantly, while others retain their structure until late in life. A study by Raz and Rodrigue (2006) demonstrated that the greatest volume decreases were seen in the frontal and temporal lobe regions including the caudate, cerebellum, hippocampus, and tertiary association cortices. In contrast, changes in the fusiform (secondary association cortex), entorhinal, and primary visual cortex were negligible. Further, the study found that volume reduction in the hippocampus, cerebellum, and prefrontal white matter accelerates with advancing age, which suggests that brain volume decreases are not only region-specific, but also nonlinear (Raz and Rodrigue, 2006). Differential volume declines can also be observed between different types of brain tissue, which can be broadly classified as either grey matter or white matter (Craik and Salthouse, 2000). Grey matter consists of neuronal cell bodies, while white matter consists of myelinated axons. Though grey matter volume loss is linear throughout adulthood (Giedd et al., 1999), white matter volume loss is nonlinear (Bartzokis et al., 2001; Fjell et al., 2005; Jernigan et al., 2001; Raz and Lindenberger, 2005), seeing abrupt decline later in life. It has been surmised that regions that contain more myelin, such as white matter, are more likely to experience age-related volume declines (Raz and Lindenberger, 2005).

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The brain also shows indications of aging through thinning of the cortex, the cerebrum’s outer layer of neural tissue. Cortical thinning, which can be observed throughout cortical regions as early as middle age (Salat, 2004), is associated with small-scale structural changes. Because the thickness of the cortex roughly correlates to the number of neurons, thinning can be a proxy for the level of deterioration in the brain. As the cortex becomes thinner there is a corresponding expansion of the ventricular system, a network of cavities filled with cerebrospinal fluid. A review of studies showed that cerebral ventricles increase in size at an average rate of 0.43% per year for middle-aged adults, and 4.25% for older adults (Raz and Rodrigue, 2006), suggesting that the rate of expansion increases significantly with age. Micro-Structural Changes The macro-level structural changes in the aging brain can be better understood by looking into the changes that occur on a cellular level. The brain undergoes a number of alterations on a micro-structural level including neuron loss and reduction of synaptic plasticity. As noted previously, changes in regional brain volume and cortical thinning can be partially attributed to neuron loss. However, this does not account for all age-induced neural changes; differences in the morphology of surviving neurons are also observed (Barnes and Burke, 2006). The membranes of neurons and mitochondria, which are rich in phospholipids, experience significant degradation, resulting in impaired function (Sato and Endo, 2010; SolsonaSancho and Blasi-Cabus, 1999). Further, neuronal damage is evident through neuron shrinkage and death, which has been shown to contribute directly to 0.5% and 1.0% of cortical thinning (Fjell, 2010). The aging brain also experiences synaptic alterations. A study by Duan and Wearne (2003) has shown that aging results in the decrease of dendritic spine number and density in the cortex. Because dendrites are essential in transmitting signals from the synapse to the neuron’s cell body, this leads to a decrease in the brain’s ability to process information. In addition, a decrease in as much as 50% of the length of myelinated axons in neurons (Rabbitt, 2001) leads to slower signal transmission times, and less adaptability. In fact, many studies have suggested a link between neuron plasticity and the amount of myelination in the brain region (Kapfhammer and Schwab, 1994). Together, these micro-structural changes result in an overall loss in brain plasticity (Chen and Hillman, 1999; Sametsky, 2010).

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Chemical Changes Neurotransmitters are chemical messengers that transmit signals between neurons in the brain. In addition to structural differences associated with brain aging, the brain undergoes several changes in neurotransmitter activity, including impairments in synthesis, uptake, and signaling (Hoekzema, 2010; Ba¨ckman, 2010). Studies have demonstrated that dopamine, serotonin, and glutamate are neurotransmitters for which chemical signaling is the most disrupted by the process of aging. Dopamine Dopamine, a member of the catecholamine family, has important roles in regulating cognitive functions such as attention and memory. Dopamine is also involved in motor control and coordinating movement and balance. During aging, biochemical changes in dopaminergic activity are observed, and posited to be related to reduced brain plasticity (Wang and Snyder, 1998). A study by Ota et al. (2006), used positron emission tomography (PET) on live human subjects and demonstrated that there was an association between age and decreased dopamine synthesis. In addition to decreased blood levels of circulating dopamine to transmit the chemical signal, there is also a significant age-related decrease in dopamine receptors to receive and propagate the message. Of the five types of receptors within the dopamine family of receptors, several studies have supported marked decreases in D1, D2, and D3 (Kaasinen et al., 2000; Wang et al., 1998). The decline in D1 and D2 is especially apparent in the caudate nucleus and putamen (Wang et al., 1998; Wong, 1984), while that of D2 and D3 was noted in the anterior cingulate cortex, frontal cortex, lateral temporal cortex, hippocampus, medial temporal cortex, amygdala, medial thalamus, and lateral thalamus (Kaasinen et al., 2000). Serotonin The monoamine neurotransmitter, serotonin, regulates mood, appetite, and sleep, as well as cognitive functions such as memory, attention, and learning. Like dopamine, the density of serotonin receptors in the aging brain is markedly decreased (Marcusson et al., 1984). This decline has been shown to be preferential to the caudate nucleus, putamen, and frontal cerebral cortex for the S2 receptor (Wong, 1984), and in the frontal cortex and hippocampus for the S1 receptor (Marcusson et al., 1984). The effect of binding site decrease is aggravated by a concomitant loss of affinity for binding sites. A post-mortem study on humans conducted by Marcusson et al. (1984) showed that

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aging led to decreased affinity for serotonin receptors, especially in the putamen. In addition, 5-HTT, the serotonin transporter responsible for regulating circulating serotonin levels, has also been shown to be affected by aging. Studies have shown that serotonin’s ability to bind to the serotonin transporter is impaired in the thalamus and midbrain (Yamamoto et al., 2002), as well as in the frontal cortex (5-HT2) (Iyo and Yamasaki, 1993). Glutamate Glutamate is the major excitatory neurotransmitter in the central nervous system, and is involved with cognitive functions including memory and learning. More recent studies have focused on the role of changes in glutamate in aging. A significant age-related decline in glutamate content, especially in the cerebral cortex and hippocampus, has been noted (Segovia et al., 2001). However, most of the studies on glutamate release in the cerebral cortex, hippocampus, and striatum show that levels of glutamate release are unchanged during aging. This indicates that the decreased concentrations of glutamate are not attributable to differences in synthesis or release, but to differences in glutamate uptake processes (Segovia et al., 2001). Reduced glutamate uptake capacity has been demonstrated in aging rat models (Vatassery et al., 1998). Further, glutamergic N-methyl-D-aspartate (NMDA) receptor density has been shown to decrease with age by as much as 20
50% (Segovia et al., 2001).

Cognitive Changes The previous sections detail the structural and chemical modifications that occur during physiologic aging. These alterations are closely tied to alterations in cognitive function, though the exact associations remain largely speculative. As with structural brain aging, certain cognitive domains are more affected by the aging process than others. Some functions, including language, general knowledge, and numerical abilities show little change with aging, while others, such as memory and attention, can begin to decline as early as middle age (Hedden and Gabrieli, 2004; Park and Reuter-Lorenz, 2009). These changes can result in decreased ability to process new information and they account for much of the age-related cognitive function loss (Der and Deary, 2006). We will now discuss agerelated changes in the individual cognitive domains. Changes in Attention Studies have shown that aging results in diminished capacity to maintain and control attention in older adults. Though some investigations suggest that older

adults do not perform as well as younger adults in divided attention tasks (Light, 1991), others have suggested that distractions do not result in differential cognitive performance (McDowd and Shaw, 2000; Verhaeghen and Cerella, 2002). Similarly, sustained attention, or the ability to attend to and respond to stimuli for an extended period of time, remains relatively stable through the aging process (Carrier et al., 2010). It is possible that these age-related attention deficits are related to processes other than direct changes in attention capacity. Declines in information processing resources, and consequent decreased processing speed have been implicated (Glisky, 2007). In addition, it is possible that attention declines are due to the decline in sensory abilities, which makes performing tasks more challenging (Kensinger, 2009). Changes in Memory Memory can be broadly classified into long-term memory and short-term memory. Long-term memory, such as semantic memory
commonly held knowledge, and autobiographical memory
facts related to one’s personal history (Levine, 2002), remain relatively unchanged despite increasing age. On the other hand, significant declines in short-term memory, also called working memory, have been demonstrated (Peltz et al., 2011). Memory functions that are associated with the medial temporal lobe and the frontal lobes seem to be most susceptible to age-related changes (Craik and Salthouse, 2000). Theories on the reason for memory changes suggest that, like attention, much of the age-related decline can be attributed to impaired information processing. Studies suggest that a general slowing of information processing is at the root of cognitive changes related to memory (Salthouse, 1994), and account for most agerelated variance in performing cognitive tasks. With age, it is also increasingly difficult to recollect memories that have been stored (Jennings and Jacoby, 1997). The ability to retrieve relevant information is dependent on both initial encoding and retrieval processes. A study by Craik (1986) suggests there is minimal age variation when environmental support for retrieval processes is provided. Retrieval processes are dependent on the prefrontal cortex and the hippocampus (Davidson and Glisky, 2002; Nolde et al., 1998). Other studies suggest that memory can be viewed as a function of attention, and that a decrease in attentional resources causes memory impairment (Craik and Byrd, 1982). It is possible that the inability to suppress irrelevant information in the retrieval process for appropriate information, or the lack of inhibitory control, results in decreased cognitive capacity (Hasher et al., 1999). Further, according to a study by Engle

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(2002), the ability to sustain attention accounted for variation in working memory capacity. Changes in Orientation Though disorientation is a hallmark for neurodegenerative diseases (Alverzo, 2006), recent studies suggest that mild decreases in orientation ability are part of physiologic cognitive aging (Sweet et al., 1999). A study by Kok (2000) demonstrated decline in orientation with aging through a significant reduction in the brain’s response speed, as measured through the magnitude of deviance-related scalprecorded event-related potential (ERP) components. ERP measures brain activity in response to a specific cognitive event. Still, research on this topic is inconclusive as some studies suggest that orientation does not decline over the lifespan (Hopp et al., 1997; Benton et al., 1981). Changes in Perception Perception, or the process of interpreting sensory information, is another cognitive function that shows significant age-related declines. Most, but not all, of these changes may be accounted for by a decline in sensory capacities such as visual and auditory impairment. Several studies have shown that after controlling for differences in sensory capabilities, there are minimal age-related differences in cognitive function related to perception (Baltes and Lindenberger, 1997). The common cause hypothesis, proposed by Baltes and Lindenberger (1997) suggests that both the sensory and cognitive aspects of perception changes with aging can be accounted for by neural degeneration. Further, it has been suggested that there is a single pool of attentional resources dedicated to perceiving and processing external stimuli. The dulling of the senses that accompanies aging results in excess stress in the perception process, which results in a complementary negative effect in information processing (Schneider and Pichora-Fuller, 2000). Changes in Executive Control Executive control is the cognitive domain that is responsible for regulating other cognitive processes such as attention and memory. Impairments in executive control result in deficiencies in all aspects of cognition because it is involved in coordinating all aspects of cognitive function. This cognitive function is responsible for regulating inhibitory control, allotting attentional resources, and guiding encoding and retrieval processes (Baddeley, 2002). There has been an increased focus on the role of executive control in age-related cognitive decline. The decline in executive control in aging is associated with structural changes in the prefrontal brain regions (Raz, 2000), which are

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regions essential to executive control function. This supports the frontal lobe hypothesis of aging, which suggests that brain functions associated with the frontal lobes are preferentially affected by aging processes (West, 1996).

FATTY ACIDS AND BRAIN AGING Fatty Acid Basics: An Introduction to the Biochemistry of Fatty Acids Fatty acids, the building blocks of the hydrophobic class of molecules known as lipids, are carboxylic acids with long aliphatic tails. These hydrocarbon side-chains usually have an even number of carbon atoms, and range in length from 6 to 24 carbons (Arab, 2003). Fatty acids are derived from triglycerides and phospholipids, and are rarely found as free fatty acids in vivo. Fatty acids are important as storage units of energy, as their metabolism generates larger amounts of ATP than glucose or protein metabolism. In addition, fats play a vital role as structural units in cell membranes, and as precursors to eicosanoids (Arab, 2003). The biochemical properties of fatty acids are determined by differences in their aliphatic tails. Fatty acids can be classified by chain length, i.e. short-chain fatty acids have less than eight carbons, while long-chain fatty acids have more than sixteen carbons. The length of the chain determines the fatty acids’ physical properties such as melting point. More importantly, fatty acids can be classified based on the number and placement of double bonds. A fatty acid is considered saturated when it does not contain double bonds, i.e. all carbons in the side-chain are bonded to the maximum number of hydrogen atoms. This structure results in a straight chain that can be packed tightly, so saturated fats are usually solids at room temperature. Unsaturated fats, on the other hand, contain at least one double bond, which creates a kink in the chain and allows for more fluidity in the molecule. Unsaturated fatty acids can be further divided into monounsaturated and polyunsaturated families. Monounsaturated fats contain a single unit of unsaturation, while polyunsaturated fats have more than one. The configuration of the double bonds in unsaturated fats determines whether the fatty acid is a transfat or a cis-fat. Trans-fats rarely occur naturally, but are present in high quantities in Westernized diets due to the hydrogenation of oil common in producing processed foods (Kritchevsky, 1990). These differential characteristics of fatty acids have physiologic effects on the aging brain.

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Fatty Acid Composition of the Brain The lipid composition of the brain is unique from other tissues in the body. More than half of the solid matter in the brain is composed of membrane lipids (O’Brien, 1986), which in turn are predominantly composed of phospholipids (Crawford and Sinclair, 1972). Svennerholm (1968) studied human brain material from a range of developmental stages in order to understand the changes that occur in brain fatty acid composition throughout the lifespan. The results showed that the concentration of brain phosphoglycerides was age-dependent. The concentration of the linoleate family of fatty acids, which includes omega-6 PUFAs, decreased while the linolenic family of fatty acids, which includes omega-3 PUFAs, increased in aging brains (Svennerholm, 1968). The alteration of the specific pattern of fatty acid concentration with age likely contributes to the deterioration of central nervous system functions, but whether these changes are a natural component of physiologic aging is unresolved. Some studies have suggested that aging leads to decreased content of PUFAs in the frontal cortex, but others did not find significant age-related differences in fatty acid composition (So¨derberg et al, 1991). Further, it has been suggested that changes in brain lipid composition are indicative of pathological brain aging (So¨derberg et al., 1991).

Omega Fatty Acids A large proportion of phospholipids in the brain are PUFAs that belong to the (n-6) and (n-3) classes, collectively known as omega fatty acids (Crawford et al., 1976; O’Brien et al., 1964). Omega-3 and omega-6 fatty acids differ in the position of the double bonds within their hydrocarbon chain: the terminal double bond begins on carbon 3 for omega-3 fatty acids, and on carbon 6 for omega-6 fatty acids. Linoleic acid is the simplest n-6 fatty acid form, while α-linolenic acid (ALA) is the simplest n-3 fatty acid form (Calder, 2004). However, the most physiologically important PUFAs are AA for the n-6 fatty acid series, and EPA and DHA for the n-3 fatty acid series (Muskiet and Fokkema, 2004). AA is the most abundant n-6 fatty acid in the brain, comprising 8
11% of fatty acid phospholipids. The brain content of DHA is comparable at 12
15%, while EPA is not stored in significant quantities in the brain (Whelan, 2008). There appears to be a growing body of literature on the importance of PUFA in brain function (Uauy and Dangaur, 2006) due to recent changes in fatty acid consumption in the modern diet. Omega fatty acids have important roles in maintaining healthy brain functioning, which are described in the following section.

FUNCTION OF OMEGA-3 FATTY ACIDS IN THE BRAIN Omega-3 fatty acids have several important roles in brain function. As the major component of neural cell membrane phospholipids, omega-3 fatty acids regulate membrane properties such as fluidity, flexibility, permeability, and modulation of membrane-bound proteins (Suzuki et al., 1998). Through its control of the membrane, the n-3 PUFAs also regulate the speed of signal transduction by affecting neurotransmitter synthesis, release, and reuptake processes (de la Presa Owens and Innis, 2000; Chalon et al., 1998; Delion et al., 1996). DHA is also important in neurogenesis and phospholipid synthesis (Coti Bertrand et al., 2006, Kawakita et al., 2006). Further, free omega-3 fatty acids are precursors to a class of hormones called eicosanoids. Eicosanoids can be derived from both n-6 and n-3 PUFAs, but the omega-6 products are highly potent and pro-inflammatory, while omega-3 products have an anti-inflammatory effect (DeCaterina and Basta, 2001; Funk, 2001; Soberman and Christmas, 2003). As antiinflammatory agents, these eicosanoids exert important effects on the vasculature, including increased blood flow (Tsukada et al., 2000; Katayama et al., 1997), increased levels of antioxidants (Hossain et al., 1998), decreased levels of peroxides (Kubo et al., 1998), and decreased ischemic damage (Okada et al., 1996). The protective role of omega-3 fatty acids against brain aging can thus be broadly divided into two categories: neural and cerebrovascular.

Neural Mechanisms Omega-3 fatty acid consumption is involved in preventing neurodegenerative pathways in the brain through its various roles including neurogenesis, neurotransmission regulation, reduction of amyloid-β production, and increasing levels of brain-derived neurotrophic factor (Cole and Frautschy, 2010). Neurogenesis Because of the high DHA content in neural membranes, n-3 exerts control over the production of new neurons, or neurogenesis. Studies in animal models have shown that consumption of n-3 PUFA leads to increased hippocampal nerve growth (Ikemoto et al., 2000). Other studies have observed a decrease in the mean cell body size of neurons in the hippocampus, hypothalamus, and parietal cortex (Ahmad et al., 2002; Wainwright et al., 1998). Further, DHA has been implicated in promoting neural stem cell differentiation through regulation of phosphatidylserine synthesis,

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which plays an important role in apoptosis (Salem et al., 2001). As ligands for peroxisome proliferator-activated receptor (PPAR), a class of nuclear transcription factors, omega-3 fatty acids regulate the transcription of genes associated with stem cell proliferation (Wada et al., 2006). DHA is also a ligand for brain retinoic X receptor (RXR). RXR and its receptor, retinoic acid receptor (RAR) are highly expressed in the hippocampus (Lengqvist et al., 2004). RAR-RXR signaling pathways lead to several downstream effects including changes in synaptic plasticity, membrane assembly, signal transduction, ion channel formation, and neurogenesis (Lane and Bailey, 2005).

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which may have a protective role against amyloid-β production (Cole and Frautschy, 2010).

Vascular Mechanisms Omega-3 fatty acids promote cerebrovascular health through anti-inflammatory effects mediated through its derivative eicosanoids (Calder, 2004). Consumption of diets with higher omega-3 to omega-6 ratios have been linked to beneficial outcomes including lowering of thrombosis (Keli et al., 1994), lowering of blood pressure (Bonaa et al., 1990), and lowering serum triglyceride levels (Kelley et al., 2007). Reduction of Inflammation

Neurotransmission Omega-3 acids are involved in maintaining the balance of neurotransmitter systems in the brain. Multiple animal-based studies have confirmed that n-3 fatty acid deficiency during development results in disruptions of dopaminergic, serotonergic, and cholinergic signal transmission (Aid et al., 2003; Delion et al., 1996). The effect of n-3 PUFAs is pleiotropic, resulting in changes in neurotransmitter synthesis, release, and reuptake. In addition, it has been suggested that neurotransmission differences due to n-3 fatty acid deficiency contribute to disruptions observed in neurogenesis (Innis, 2007). Reduction of Amyloid-β Production Amyloid-β is a peptide derived from amyloid precursor protein (APP) and forms the amyloid plaques which are markers of Alzheimer’s disease in the brain and are believed to be the culprit in initiating the pathological cascade of the disease. DHA reduces amyloidβ through several mechanisms. Studies have shown that DHA supplementation in APPsw Tg2576 transgenic mice results in dose-dependent reductions in total amyloid in hippocampi and parietal cortices (Calon et al., 2004; Lim et al., 2005), amyloid-β pathology (Lim et al., 2005), and neuritic damage associated with amyloid-β plaques (Calon et al., 2004). In another study, DHA supplementation reduced amyloid-β and tau pathology (Green et al., 2007) in mice carrying three mutant transgenes (App, Ps1, Tau) associated with Alzheimer’s disease pathology. Increasing Brain-Derived Neurotrophic Factor Brain-derived neurotrophic factor (BDNF), is part of a family of growth factors that helps regulate neurogenesis (Benraiss et al., 2001; Pencea et al., 2001; Zigova et al., 1998). BDNF supports the survival of neurons and encourages new neuron growth and differentiation (Acheson et al., 1995; Huang and Reichardt, 2001). DHA increases brain levels of BDNF,

Chronic systemic inflammation has been linked with cognitive impairment. It stimulates the production of inflammatory cytokines, such as IL-1β, IL-6, and IL-18 (Whitney, 2009), which impair neurogenesis (Liu, 2005; Monje, 2003; Vallieres, 2002) and damage existing neurons (Marx, 2001; Jarskog, 1997). Excessive consumption of omega-6 fatty acids leads to increased production of highly potent pro-inflammatory eicosanoids. Omega-3 fatty acids reduce inflammation through competition with omega-6 fatty acids for phospholipid incorporation in the brain (Goodknight et al., 1981; Sanders et al., 1981) and access to cyclooxygenase and lipoxygenase enzymes required for synthesis of their own less potent eicosanoid products. The presence of EPA increases synthesis of significantly less inflammatory eicosanoids, and effectively lowers the output of AA’s eicosanoids. Recent studies have also suggested that DHA inhibits pro-inflammatory genes in the brain through antioxidant activity. Studies in animal models have shown that DHA protects against free radical peroxidative damage of lipids and proteins in the brain (Green et al., 2001). Lowering of Thrombosis The anti-thrombotic effect of n-3 PUFAs is also mediated by competition between omega-6 and omega-3 eicosanoid production (von Schacky et al., 1985). Omega6 fatty acids promote vasoconstriction by production of the eicosanoid, thromboxane A2 (TXA2), a potent stimulator of platelet aggregation (Kinsella et al., 1990). Experimental studies in vitro and in humans showed that n-3 PUFAs such as EPA and DHA reduce platelet aggregability (Dyerberg et al., 1978; Fischer and Weber, 1983), by reducing formation of TXA2. Omega-3 fatty acids also produce TXA3, which has a much weaker proaggregatory effect than its counterpart, TXA2. Further, omega-3 fatty acids can be metabolized to the anti-thrombotic eicosanoid, prostaglandin I3 (Dyerberg et al., 1978; Fischer and Weber, 1983; Kinsella et al., 1990).

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Blood Pressure Reduction Eicosanoids derived from omega-3 fatty acids promote vasodilation (Nasjletti, 1998), and inhibit vasoconstriction induced by omega-6 fatty acid eicosanoid products. Increased omega-3 levels have been associated with vasodilation and decreased blood pressure. Studies have shown that omega-3 fatty acid supplementation resulted in lower blood pressure in a population of people with normal blood pressure (Lorenz et al., 1983; Sanders et al., 1981; Singer et al., 1983), as well as in those with hypertension (Knapp and Fitzgerald, 1989; Norris et al., 1986), though not all study results are in agreement. Some investigations found no lowering of blood pressure (Demke et al., 1998; von Houwelingen et al., 1987). These disparities could be attributed to the differing efficacy of supplementation in populations with varying levels of hypertension. Bonaa et al. (1990) suggests that those with lower initial omega-3 intake were more likely to benefit from supplementation, while the effect was less significant in those with higher initial omega-3 intake. Further, the study suggests that EPA is the more active omega-3 in mediating effects on blood pressure, though the exact mechanism is not known. Lowering Triglyceride Levels Elevated levels of triacylglycerols (triglycerides) is a risk factor for the development of cardiovascular diseases (Schaefer, 2002; Kris-Etherton et al., 2002; Mora et al., 2007), but long-chain n-3 PUFAs have been shown to decrease blood triacylglycerol concentrations, by reducing triglyceride synthesis (Harris and Bulchandani, 2006). Consequently, decreased n-3 index (Harris, 2007; Harris and von Schacky, 2004; von Schacky and Harris, 2007) has been associated with an increased risk of cardiovascular diseases. The mechanism by which n-3 omega fatty acids effect a decrease in triglyceride synthesis is unclear, though there are several hypotheses. These include the reduction in availability of substrates for triglyceride synthesis, increase in phospholipid synthesis, or alteration of the activity of enzymes involved in triglyceride synthesis. A review by Harris and Bulchandani (2006) suggests that rat models have most consistently supported the first hypothesis that a decrease in fatty acid synthesis results in less substrate availability, though further clinical studies are required to confirm this effect in humans.

SOURCES OF OMEGA FATTY ACIDS Because mammals lack desaturase enzymes that allow conversion between n-9 and n-6 families, and

between n-6 and n-3 families, omega-6 and omega-3 fatty acid families must be obtained primarily from dietary sources. They are thus considered essential fatty acids because they have vital physiologic roles, but cannot be synthesized from precursors by the body (Brenna, 2002). There are several different dietary sources for essential fatty acids. Omega-6 fatty acids, which are consumed in large quantities by those following a typical Western diet, are most often consumed in the shorterchain form, linoleic acid, in vegetable oils, nuts, and seeds. Longer-chain variations, such as AA, are found in eggs and animal meat. Omega-3 fatty acids can be found as ALA in some plant-based foods, such as flaxseed oil and canola oil, but the longer-chain derivatives, DHA and EPA, are only found in significant portions in fatty fish (Innis, 2003). Though n-3 and n-6 fatty acids are both derived from dietary intake, some studies have found that the DHA and EPA concentrations are the most sensitive among the essential fatty acids to dietary modifications. Unlike the n-6 AA, for which circulating levels were relatively constant across individuals, DHA and EPA levels showed the greatest inter-individual variations (Fokkema et al., 2000).

OMEGA FATTY ACID METABOLISM Although mammals cannot produce linoleic acid or ALA directly, they are able to metabolize them further into more complex omega fatty acid products. Through processes of desaturation and elongation that occur primarily in the liver, linoleic acid can be converted into the longer-chain AA. Similarly, ALA can be converted into EPA, which can then be further metabolized to DHA (Calder, 2004). Because omega-6 and omega-3 fatty acids require the same enzymes for their metabolism, they effectively compete with one another for a limited resource of enzymes. More specifically, the metabolic pathways are rate-limited by the delta-6-desaturase enzyme (British Nutrition Foundation, 1992), which prefers ALA as a substrate. However, the high quantity of omega-6 consumption in the Western diet overwhelms the system, leading to preferential metabolism of linoleic acid (Calder, 2004). Moreover, stable isotope tracer studies suggest that conversion of ALA to DHA is minimal in humans, with less than 1% of dietary ALA being ultimately converted to DHA (Williams and Burdge, 2006). The conversion between EPA and DHA is particularly limited, with one study demonstrating that consumption of DHA in the diet was seven times more likely to result in uptake by the brain than DHA derived through conversion of ALA (Su et al., 1999).

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As a result of the limited inter-conversion between omega fatty acid types, humans need to consume a balanced diet that includes preformed sources of both short- and long-chain omega fatty acid derivatives. Recent studies have highlighted the significance to health of the omega-6 to omega-3 fatty acid ratio, also called the omega-3 index. A study by Connor et al. (1990), showed that omega-3 fatty acid dietary supplementation led to reciprocal changes in n-3 and n-6 fatty acids in the phospholipids of cerebral cortex, suggesting that the brain attempts to maintain a constant level of polyunsaturation. Optimal ratios of omega-6 to omega-3 range from 1:1 to 1:4 (Lands, 2005) but typical Western diets provide ratios of between 10:1 and 30:1 (Hibbeln et al., 2006). Several studies suggest that a lower ratio of omega-6 to omega-3 fatty acids is desirable in reducing the risk of many chronic diseases including cardiovascular diseases, cancer, and inflammatory diseases (Simopoulos, 2008).

PATHOLOGICAL BRAIN AGING Fatty acids are implicated in multiple neurologic diseases, some of which will be discussed in detail later in this volume. One of the most important agerelated conditions affected by fatty acids is dementia. Dementia is not a specific disease, but an umbrellaterm for diseases that result in severe gradual loss of global cognitive function beyond that which might be expected from normal aging (Burns et al., 1991). There are many types of age-related dementias, many of which target the central nervous system, including Parkinson’s disease, Huntington’s disease, and frontal temporal dementia. However, the most common form is Alzheimer’s disease, which accounts for 60
80% of dementia cases (Alzheimer’s Association, 2010). Recently, there has been a focus on dementias that are associated with cardiovascular disease, and their relationship to neurodegenerative forms of dementia. Vascular dementia is the second most prevalent type of dementia associated with aging (Drachman et al., 1991). Alzheimer’s disease and cardiovascular disease have many common risk factors, and it is common for people to exhibit a combination of Alzheimer’s disease and vascular dementia pathology, called mixed dementia. In fact, it is estimated that more than a third of dementia cases have mixed etiology (Lee, 2011). Because omega-3 fatty acids have important roles in maintaining both neural and cardiovascular health, dietary interventions of omega-3 fatty acid intake have the potential to protect against age-related dementia on multiple levels. Several studies have confirmed the benefits of omega-3 fatty acid on cardiovascular profiles, but the role of fatty acids on promoting cerebral

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health is only beginning to be understood. Though further investigations will be necessary to elucidate the role of fatty acids in preventing pathological brain aging, initial reports suggest that omega-3 fatty acid consumption is, in fact, protective against age-related dementia. Here we will review the pathological mechanisms associated with the two most prevalent types of dementia, and provide support for the importance of omega-3 fatty acids in preventing both.

Alzheimer’s Disease Alzheimer’s disease manifestation is heterogeneous, but a set of common characteristic symptoms have been identified in those diagnosed. As in physiologic aging, Alzheimer’s disease involves several structural and chemical changes in the brain, and decline of cognitive function. However, these changes are pathologic and are clinically distinct from those associated with healthy aging. Disease Characteristics STRUCTURAL DIFFERENCES FROM NORMAL AGING

Alzheimer’s disease is associated with a unique brain structure phenotype. As in aging, widespread neuron loss and decreases in synaptic density are observed, though Alzheimer’s disease results in a significant preferential effect in the neocortex, hippocampus, amygdala, and basal nucleus of Meynert (Wenk, 2003). In addition, extensive amyloid plaques and neurofibrillary tangles are indicators of Alzheimer’s disease pathology (Tiraboschi et al., 2004). Amyloid plaques are made up of fragments of the APP, called amyloidβ. In Alzheimer’s disease, these fragments clump together to form dense, insoluble plaques that adversely affect neuron signal transmission (Ohnishi and Takano, 2004; Tiraboschi et al., 2004). Similarly, neurofibrillary tangles are composed of the protein tau, which is altered by pathological processes. This results in the disintegration of microtubules and the formation of tau aggregates (Hernandez and Avila, 2007). Normal aging can result in the formation of plaques and tangles, but the amount and distribution does not compare to the brains of people with Alzheimer’s disease, in which greater quantities are common, especially in regions such as the temporal lobe (Bouras et al., 1994). Further, it has been suggested that Alzheimer’s disease can be identified by different patterns of fatty acid composition in the brain. A post-mortem study showed that the content of the n-3 PUFA DHA was decreased in the hippocampus and frontal grey matter of Alzheimer’s disease brains, though fatty acid

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composition remained unaltered in normal aging brains (So¨derberg et al., 1991). CHEMICAL DIFFERENCES

The most consistent chemical change in Alzheimer’s disease is observed in the cholinergic neurotransmitter system. Acetylcholine levels are decreased due to decreased activity of associated enzymes. Choline acetyltransferase, which is involved in acetylcholine synthesis, was shown to decrease as much as 50
85% in cerebral cortex and the hippocampus in Alzheimer’s disease patients (Reinikainen et al., 1990), particularly in areas of the brain that had amyloid depositions (Mullan, 2000). Acetyl cholinesterase, the enzyme that terminates the signal transmission at the post-synaptic terminal, is also affected by disease processes (Wenk, 2003). Further, there are changes in receptor density and selective loss of cholinergic neurons (Katzman, 1989). Degenerative changes have also been demonstrated in a variety of related neurotransmitter systems including norepinephrine, serotonin, dopamine, and glutamate (Boncristiano et al., 2002). A study by Reinikainen et al. (1990) showed that Alzheimer’s disease affected serotonergic, noradrenergic, and dopaminergic activity. Serotonin concentration was decreased by 21
37% in the hippocampal cortex, hippocampus, and striatum, noradrenaline was decreased by 18
36% in the frontal and temporal cortex and putamen, and dopamine was decreased by 18
27% in temporal and hippocampal cortex and hippocampus. These changes were significant, but not as severe as changes to cholinergic activity. Recent studies have also implicated glutamate in Alzheimer’s disease related neuron loss in the cerebral cortex and hippocampus (Katzman, 1989). Decreased glutamate concentrations result in excessive stimulation of glutamate receptors, in particular the NMDA receptor. The decreased glutamate is associated with the death of cholinergic neurons in the forebrain (Wenk et al., 2000). COGNITIVE DIFFERENCES

Memory loss is the most prominent symptom of Alzheimer’s disease. The ability to form new memories is particularly impaired, which leads to difficulties in learning and acquiring new information (Ba¨ckman et al., 2004). Memory loss associated with Alzheimer’s disease is caused by impairments in the initial encoding process, rather than differences in attentional resources that is observed in normal aging. Further, though long-term memory remains relatively unaltered in physiologic aging, it is often impaired in later stages of Alzheimer’s disease (Fo¨rstl and Kurz, 1999). Another major cognitive domain affected by Alzheimer’s disease is language. People with Alzheimer’s

disease have diminished naming, word fluency, comprehension, and associations (Hart et al., 1988; Murdoch et al., 1987; Nicholas et al., 1997). Language difficulties may be observed in the inability to recall words, which often leads to incorrect word substitution. Decreased language function also leads to impairments in reading and writing (Taler and Phillips, 2008). Alzheimer’s disease also affects other cognitive functions including executive function (Ba¨ckman et al., 2004), impaired reasoning ability including abstract thinking and judgement (Zec, 1993), and motor control and coordination (Fo¨rstl and Kurz, 1999). Though physiologic aging also involves diminished cognitive function, this impairment in Alzheimer’s disease is severe enough to interfere significantly with activities of daily living. As the disease progresses, those with Alzheimer’s disease increasingly lose their ability to live independently. During the final stage of Alzheimer’s disease, the person is completely dependent upon caregivers (Fo¨rstl and Kurz, 1999). Disease Mechanisms The cause and progression of Alzheimer’s disease are not well understood. However, there is increasing evidence that the neuronal cell death and synaptic impairments associated with the disease are related to abnormal amyloid-β peptide activity (Florent-Be´chard and Malapate-Armande, 2007). The amyloid cascade hypothesis suggests that amyloid-β accumulation and aggregation is the proximate cause of several downstream pathological events that occur in Alzheimer’s disease (Cummings and Cole, 2002). As described previously, Alzheimer’s disease is associated with the widespread formation of amyloid plaques in the brain. These plaques are accumulations of amyloid-β (Aβ) protein, which are derived from the APP (Selkoe, 1989; Sisodia and Price, 1995) through pathologic proteolysis processes. The plaques are primarily composed of amyloid-β oligomers (Selkoe, 1999) and fibrils. Though amyloid-β is present in nonpathologic brains, the concentrations of the peptide are elevated due to imbalanced levels of Aβ production and clearance, which leads to aggregation. Many theories have been proposed regarding how amyloid-β accumulation leads to Alzheimer’s disease. Though amyloid fibrils have been associated with disrupting calcium homeostasis and apoptosis (Yankner et al., 1990), it has been suggested that the oligomers are responsible for the Alzheimer’s disease related pathological effects (Klein et al., 2001). The neurotoxic and synaptotoxic Aβ oligomers cause decreases in synaptic plasticity and neurogenesis by impairing mitochondrial function, initiating apoptotic pathways, altering glutamate receptors (Pillot et al., 1999), and affecting signaling pathways.

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Amyloid-β also contributes to the formation of neurofibrillary tangles, by activating kinases that hyperphosphorylate the microtubule-associated protein, tau. The aggregation of hyperphosphorylated tau results in neurofibrillary tangles that disrupt microtubule structure (Goedert et al., 1991), the neuron’s transportation system (Iqbal, 2005), and cause biochemical communication dysfunction (Chun and Johnson, 2007). Further, amyloid plaques can cause oxidative damage and inflammation by microglial activation. The relative contributions of these different pathologies to neurodegeneration and cognitive dysfunction remain controversial.

Vascular Dementia Vascular dementia, more broadly referred to as vascular cognitive impairment (VCI), is cognitive decline associated with vascular factors (Hachinski and Bowler, 1993; Bowler and Hachinski, 2003). It is estimated to be the cause of (50
70%) of all dementia cases, but these numbers may underestimate its true prevalence because it is often seen concomitantly with other forms of dementia. Recent studies have focused on understanding the pathology of isolated vascular dementia, and have demonstrated that the disease is associated with unique physiological and cognitive changes.

Disease Characteristics Structural Changes Vascular dementia can be diagnosed through the presence of vascular lesions in the brain. The lesions are the result of cerebrovascular disease and can be diffuse or focal, though patients often demonstrate a combination of both. The lesions are associated with white matter hyperintensities (Longstreth et al., 1996), lacunar infarcts (Fisher, 1982), and microhemorrhages (Viswanathan and Chabriet, 2006), and can be found in various brain regions, though frontal structures are the most often affected. Though these lesions may be present in the grey matter, the white matter is preferentially affected, which is apparent through tissue loss, artery damage, and accumulation of lipid deposits and blood clots. The ischemic damage also results in significant myelin loss, which leads to neuronal dysfunction (Englund, 2000). Cognitive Changes Vascular dementia is related to the impairment of several cognitive functions, though it may vary in severity and type on an individual basis. Because vascular dementia is often related to cerebrovascular disease and a series of several small strokes, the cognitive

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domains affected depend on the area in which the infarcts occurred. In general, vascular dementia affects frontal lobe function the most, which is supported by the fact that most small vessel diseases occur in the frontal lobe. Patients with vascular dementia pathology have been shown to have better verbal fluency and free recall when compared to those with Alzheimer’s disease. A study by Yoon et al. (2013) showed that patients with vascular dementia had worse frontal function, but better memory function than patients with Alzheimer’s pathology. Vascular disease is associated with declines in cognitive domains that affect executive function including information processing, attention shifting, and abstract thinking (Snowdon et al., 1997). As in Alzheimer’s disease, vascular disease can interfere with the ability to perform activities of daily living independently. Chemical Changes As in Alzheimer’s disease, vascular dementia presents with significant cholinergic dysfunction. Many studies have shown that patients with vascular dementia have impairments in cholinergic function due to cerebrovascular injuries in brain regions with dense cholinergic fibers (Roman, 2005). There is a reduction in choline acetyltransferase activity in the hippocampus and temporal cortex, reduction of acetylcholine activity in the cortex, hippocampus, striatum, and cerebrospinal fluid, and significant cholinergic neuron loss (Court et al., 2002). Disease Mechanisms Vascular dementia can be manifested through many different clinical phenotypes and pathophysiologies. However, the most consistent finding in vascular dementia cases is an association with cardiovascular or cerebrovascular disease that leads to impaired blood flow to the brain, and consequent dementia (Sorrentino et al., 2008). Vascular dementia has also previously been called multi-infarct dementia because the cognitive impairments are often a result of a series of minor strokes, secondary to primary vessel disease, that contribute to brain damage by affecting the regulation of blood flow and disrupting the blood
brain barrier. Though the exact mechanism has not been determined, studies have shown that ischemia leads to cholinergic impairments and inflammation that contribute to brain damage (Kuang et al., 2008).

Mixed Dementia Conservative estimates suggest that as much as a third of dementia cases have mixed pathology, often a

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combination between the neurodegenerative Alzheimer’s disease, and vascular forms as in vascular dementia (Lee, 2011). Dementia is increasingly being viewed as a spectrum with Alzheimer’s disease patients at one end and vascular dementia patients at the other end. Most cases are somewhere in the middle and have varying contributions from both pathologies. Though Alzheimer’s disease is neurodegenerative in nature, several vascular risk factors have also been shown to be associated with it (Kivipelto et al., 2001). Both Alzheimer’s disease and vascular dementia are associated with the presence of atherosclerosis, atrial fibrillation, hypertension, and angina. Further, cardiovascular risk factors have been shown to accelerate cognitive decline in Alzheimer’s disease (Helzner et al., 2009). Several studies have suggested that patients who demonstrate both Alzheimer’s disease and vascular disease have more severe cognitive impairment than those with either pathology alone (Snowdon et al., 1997). Although many studies have tried to understand the pathology of the pure forms of each dementia, the current classification methods are not able to fully distinguish these entities, making classification and, thus, treatment and management difficult (McKhann et al., 1984; Mirra et al., 1991). However, because there are several cardiovascular risk factors for Alzheimer’s disease and vascular dementia that are shared (Staessen et al., 2007), targeting these can be an effective approach to decreasing the incidence and severity of dementia cases, whether Alzheimer’s disease, vascular dementia, or mixed dementia. Consumption of omega3 fatty acids is favorable in this respect because it has been shown to address cardiovascular risk factors, and is increasingly being linked to a protective role against dementia.

PROTECTIVE EFFECT OF OMEGA-3 FATTY ACIDS AGAINST DEMENTIA As discussed previously, omega-3 fatty acids have several important roles in maintaining neural and cardiovascular health. Further, accumulating evidence suggests that the consumption of omega-3 PUFAs, DHA and EPA, is protective against pathological cognitive decline. Because dietary intake of fatty fish is the main source of the omega-3 PUFAs DHA and EPA, many studies have investigated the effect of increased fish consumption or dietary supplementation. The results of population-based studies are not all in agreement, but most studies have shown that higher dietary fish intake leads to lower risk of dementia and cognitive decline (Cole and Ma, 2009).

Epidemiological studies have shown that omega-3 fatty acid consumption reduces the risk of dementia. A study by Kalmijn and Launer (1997) found that n-3 fatty acid consumption was associated with a reduced risk of dementia, especially Alzheimer’s disease. The results of this study also showed that high saturated fat intake, and high total fat intake led to an increased risk of mixed dementia. Similarly, a study by Heude et al. (2003) showed that consumption of a higher proportion of total omega-6 PUFAs was associated with greater risk of cognitive decline, while consumption of a higher proportion of total omega-3 PUFA intake was associated with a reduced risk. These dietary fat intake studies have been confirmed by studies of plasma fatty acid levels. Several studies have shown that people with high plasma omega-3 fatty acid levels were less likely to develop Alzheimer’s disease (Schaefer et al., 2006). Another case-control study reported that patients with Alzheimer’s disease had lower levels of omega-3 fatty acids in plasma phospholipids. Compared to an agematched cohort, Alzheimer’s disease patients had 60
70% of omega-3 fatty acid levels (Conquer et al., 2000). In addition, a longitudinal study following an elderly French cohort for 4 years showed that lower plasma concentrations of omega-3 PUFA were associated with a higher risk of cognitive decline (Heude et al., 2003). Furthermore, several studies have found that consumption of fish, which is the main dietary source of omega-3 fatty acids, is also associated with a reduced risk of cognitive decline or dementia (Kalmijn and Launer, 1997; Morris and Evans, 2003; BarbergerGateau et al., 2002; Lim et al., 2013; Fotuhi et al., 2009). The protective effects of dietary fish intake have been confirmed by a number of recent studies. A large prospective study conducted by Morris and Evans (2003) showed that eating at least one meal of fish per week resulted in a 60% reduced risk of Alzheimer’s disease compared to less frequent fish consumption. The Cardiovascular Health Study conducted by Virtanen et al. (2008) also found that the consumption of fish three or more times a week, based on dietary surveys, was associated with a lower risk of subclinical vascular brain abnormalities. In a prospective cohort study we conducted in the Framingham Heart Study, we related red blood cell (RBC) fatty acids composition
shown to be a reliable biological indicator of the dietary omega-3 PUFA intake
with structural brain and cognitive measures of aging (Tan et al., 2012). We found that lower levels of RBC DHA and EPA in late middle age were associated with accelerated brain aging in both structural and cognitive measures. The MRI data revealed that lower intake was correlated with lower brain volume,

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REFERENCES

with a representative change equivalent to approximately 2 years of structural brain aging. Though many large epidemiologic studies (Kalmijn and Launer, 1997; Morris and Evans, 2003) have demonstrated an association between the estimated intake of fatty fish and a lower risk for dementia, not all investigations have confirmed this protective association (Laurin and Verreault, 2003). A supplementation study with DHA in older adults for 6 months led to improved cognitive functions (Yurko-Mauro et al., 2010), but another supplementation study with DHA in subjects with mild to moderate Alzheimer’s disease did not observe benefits to cognitive functions (Quinn et al., 2010). One possible reason for the inconsistency in these results is related to the limitations of omega-3 fatty acid measurements. Many studies estimated fatty acid intake through dietary surveys or questionnaires, which can be unreliable. However, more recent studies have focused on using dependable biological indicators to quantify levels of fatty acids, such as RBC fatty acid composition (Hjartaker et al., 1997; Philibert et al., 2006). In addition, the inconsistencies among supplementation studies might be attributed to differences in the dosage of omega-3 PUFAs administered as well as the proportion of DHA to EPA. Some studies have suggested that consumption of DHA is protective against the development of dementia, while EPA was not associated with a reduced risk (Morris and Evans, 2003). Several studies have also demonstrated that the efficacy of omega-3 fatty acid supplementation can be increased by combining the treatment with antioxidants. DHA is highly susceptible to lipid peroxidation because of its double bonds (Cole and Frautschy, 2010). One study showed that cognitively normal elderly participants had higher cognitive outcome measures when they received DHA co-administered with the antioxidant lutein than when receiving DHA alone (Johnson et al., 2008). Further, some studies have suggested that only certain subgroups derived benefits from supplementation. A study with DHA and EPA supplementation in subjects with mild to moderate Alzheimer’s disease did not observe a delay in the rate of cognitive decline, but an analysis of participants with only very mild Alzheimer’s disease revealed a positive effect (FreundLevi et al., 2006). This suggests that omega-3 PUFAs most effectively exert their protective effects in people in a pre-clinical dementia state. Similarly, a study by Quinn et al. (2010) found that supplementation with DHA did not slow the rate of cognitive and functional decline in patients with mild to moderate Alzheimer’s disease. However, a subgroup of patients that were negative for the APOE-e4 gene did see improved

outcomes with supplementation, suggesting that certain genetic factors can also affect the efficacy of the dietary intervention.

CONCLUSION The omega-3 PUFAs are abundant in the brain and serve many important biological functions. As integral components of membranes, they influence membrane properties, signal transduction, and neurogenesis (Florent-Be´chard et al., 2007). Further, they are precursors for eicosanoids that promote vascular health through anti-inflammatory effects. Because omega-3 PUFAs are essential fatty acids that are not efficiently produced endogenously, including sufficient amounts of omega-3 PUFAs in the diet, through consumption of fatty fish, is important to sustain healthy physiologic processes (Brenna, 2002). Omega-3 PUFA intake has been associated with a reduced risk for age-related neurological diseases. Increased fish consumption, the main source of omega-3 fatty acids, has been shown by the majority of currently published studies to lower the risk of dementia and cognitive decline (Cole and Ma, 2009), though the mechanisms that explain this association are not yet fully understood. Omega-3 PUFAs may protect against cognitive decline by a combination of mechanisms that target the neurodegenerative pathogenesis of Alzheimer’s disease (Cole and Frautschy, 2010) directly and/or mechanisms that target cardiovascular risk factors that are associated with a higher risk of dementia. Additional investigation is necessary to understand these mechanisms and to identify the optimal target population for a nutrition-based intervention with omega-3 polyunsaturated fatty acid supplementation to promote brain health.

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Yamamoto, M., Suhara, T., Okubo, T., Ichimiya, T., Sudo, Y., Inoue, Y., et al., 2002. Age-related decline of serotonin transporters in living human brain of healthy males. Life Sci. 71, 751
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Further Reading Bermejo-Pareja, F., Benito-Leo´n, J., Vega, S., Medrano, M., Roma´n, G., 2008. Incidence and subtypes of dementia in three elderly populations of central Spain. J. Neurol. Sci. 264 (1
2), 63
72. Brookmeyer, R., Johnson, E., Ziegler-Graham, K., Arrighi, H., 2007. Forecasting the global burden of Alzheimer’s disease. Alzheimers Dement. 3 (3), 186
191. Dicarlo, A., 2002. Incidence of dementia, Alzheimer’s disease, and vascular dementia in Italy. The ILSA Study. J. Am. Geriatr. Soc. 50 (1), 41
48. Doraswamy, P., Steffens, D., Pitchumoni, S., Tabrizi, S., 1998. Early recognition of Alzheimer’s disease: what is consensual? What is controversial? What is practical? J. Clin. Psychiatry. 59 (Suppl. 13), 6
18. Gauthier, S., 1999. Managing expectations in the long-term treatment of Alzheimer’s Disease. Gerontology. 45 (Suppl. 1), 33
38. Wimo, A., Jonsson, L., Winblad, B., 2006. An estimate of the worldwide prevalence and direct costs of dementia in 2003. Dement. Geriatr. Cogn. Disor. 21 (3), 175
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C H A P T E R

18 Cerebrovascular Changes: The Role of Fat and Obesity Linnea R. Freeman INTRODUCTION Of great concern to the United States and worldwide is the epidemic proportion of overweight and obese people. In 1960, the United States reported 13.4% of the population as being obese. This number has now nearly tripled to 35.7% in 2010 (Flegal et al., 2010; Flegal et al., 2012). Furthermore, it is estimated there are one billion overweight or obese people worldwide (Runge, 2007). Unfortunately, these numbers continue to rise. The toll of a high fat diet and/or obesity on an individual’s health can be remarkable. In fact, healthcare costs attributable to obesity are projected to reach between $861 and $957 billion by 2030. Overweight and obese people pay significantly more in healthcare costs compared to normal weight individuals, about $1429 per year (Go et al., 2013). Obesity is a risk factor for many conditions including, but not limited to, diabetes, hypertension, metabolic syndrome, stroke, heart disease, certain cancers, sleep apnea, and arthritis (Malnick and Knobler, 2006; Flegal et al., 2010). A high fat diet impacts on health at various levels, particularly contributing to cardiovascular issues via inflammation and oxidative stress (Calder et al., 2011; Maachi et al., 2004; Uranga et al., 2010). While a large amount of research has helped us to understand the detrimental effects of a high fat diet and obesity to overall health, more research must be done. In particular, we must further understand the effects of a high fat diet and obesity upon the brain and cognition. The focus of the following chapter is the cerebrovascular changes that occur due to a high fat diet and/or obesity. First, vascularization of the brain and the blood
brain barrier (BBB) will briefly be reviewed. Then, proposed mechanisms, human studies, and rodent studies will be discussed. The effects of both a high fat diet and obesity will be presented. It is Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00018-1

possible to consume a high fat diet without attaining characteristic levels of obesity (such as a body mass index (BMI) between 25
30 kg/m2 (Wang and Beydoun, 2007)), however, detrimental effects to the cardiovascular system, changes to lipid profile, glucose tolerance, inflammation, and other measures can nevertheless still occur and cause cerebrovascular damage. Furthermore, the path to obesity can be complex and involve factors outside of a high fat diet such as genetic influence, carbohydrate intake, and lack of exercise. Again, these factors can also cause cerebrovascular damage. Therefore, this chapter will aim to explore cerebrovascular changes due to both obesity and a high fat diet.

VASCULARIZATION OF THE BRAIN The brain comprises about 2% of our body weight, yet it requires 20% of our total blood volume and 20% of our body’s oxygen (Shulman et al., 2004; Kalaria, 2010). Interestingly, the large volume of blood required by the brain depends on two sets of branches from the dorsal aorta. Briefly, these two sets of branches originate at the common carotid arteries and the subclavian arteries, which become the internal carotid arteries and vertebral arteries, respectively. At the base of the brain is the Circle of Willis, an important junction where the basilar artery joins the blood supply and from where the posterior cerebral arteries, anterior communicating arteries and posterior communicating arteries are derived. Feeding the cortex, basal ganglia, thalamus, and internal capsule is the anterior circulation. The posterior cortex, midbrain, and brainstem receive blood via the posterior circulation (Purves et al., 2012). Proper blood flow is essential to the health and activity of neurons, cells which have a high rate of metabolic

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activity (Cipolla, 2009). In fact, the brain exhibits autoregulation and functional hyperemia in order to maintain a relatively constant blood flow. Autoregulation is intrinsic to a number of organs allowing for local blood flow regulation. Functional hyperemia is essential for coordination of neuronal activity and proper blood flow (Cipolla, 2009). A well-known consequence of blood flow obstruction is stroke, which can lead to neurodegeneration and often physical and cognitive impairments. Stroke is the fourth leading cause of death in the United States with 795,000 Americans suffering from a stroke every year, of whom 130,000 die from the condition each year (Go et al., 2013).

The Blood
Brain Barrier Another important component of the cerebral vascular system is the blood-brain-barrier (BBB). At the foundation of the BBB are endothelial cells which become securely bound together via tight junctions (Nico and Ribatti, 2012; Weiss et al., 2009). The tight junctions are made up of transmembrane proteins such as occludin, claudins, and junctional adhesion molecules which are anchored to endothelial cells via scaffolding proteins such as zona-occludens-1 (ZO-1) and zona-occludens-2 (ZO-2) (Abbott et al., 2010). Further support for the BBB comes from astrocytic end feet which provide structural as well as biochemical support (Nico and Ribatti, 2012). The BBB is essential to brain health as it is only permeable to small hydrophobic molecules, but restricts the passage of hydrophilic molecules, toxins, and bacteria (Weiss et al., 2009). There is evidence that the BBB can weaken during the aging process, various illnesses, and due to inflammation (Nico and Ribatti, 2012; Weiss et al., 2009). Arguably, the damaging effects of a high fat diet and obesity could weaken the BBB leading to neurodegeneration.

EFFECTS OF A HIGH FAT DIET AND OBESITY ON OVERALL HEALTH AND PROPOSED MECHANISMS With the rising number of obese and overweight people in the world, a great amount of research has focused on understanding the damaging effects to overall health of obesity and an unhealthy, high fat or ‘Western’ diet. Primarily, research has focused on metabolic syndrome, type II diabetes, and cardiovascular disease. Therefore, most strides in research have been made in understanding the effects of a high fat diet and obesity from the neck down. In recent years, more effort has also been placed on understanding the

effects on the central nervous system: both directly and indirectly via the changes occurring in the periphery. With the rise in dementia, Alzheimer’s disease, and stroke in parallel to the epidemic rise in obesity, it is essential to determine how these disorders could be related. In the section below, a number of mechanisms will be introduced which have been evaluated in various systems such as the heart, kidney, and adipose tissue, but are also likely to play a role in high fat diet/obesity-induced cerebrovascular changes. First, elevated cholesterol levels are well known to occur due to an unhealthy diet/obesity (Grundy, 1997; Saggini et al., 2011; Go et al., 2013). A major component of the negative effects of a high fat diet is the high density lipoprotein (HDL)/low density lipoprotein (LDL) profile (Wang and Beydoun, 2007). For example, it has been found that consumption of mainly saturated fats leads to an increase in LDL. Saturated fat raises LDL levels by down-regulating its receptor, rLDL. On the other hand, it has been shown that polyunsaturated fats are protective against this phenomenon because they promote rLDL activity. Furthermore, atherosclerotic plaques have been shown to contain cholesterol and cholesterol esters built up from oxidized LDL, therefore revealing a mechanism by which high consumption of saturated fat can lead to endothelial dysfunction and cardiovascular issues (Chong et al., 2006). In addition to changes in cholesterol due to a high fat diet/obesity, circulating triglycerides are typically increased as well. Triglycerides consist of three fatty acids joined together by glycerol, which are primarily produced by the intestines and liver, and commonly enter the circulation within chylomicrons (Mansbach and Siddiqi, 2010). High serum triglyceride levels are a significant predictor of stroke (Miller et al., 2011). While the mechanism of action of triglycerides on vasculature is not as clear as that of cholesterol, primarily due to the fact that triglyceride levels are so variable and hard to characterize, one proposed mechanism for damage includes lipolysis of triglyceride-rich lipoproteins which can release toxic oxidized free fatty acids (FFAs) (Wang et al., 2009). Release of these oxidized FFAs can increase inflammation and oxidative stress, as well as altering endothelial function (Wang et al., 2009). Furthermore, lipolysis of triglyceride-rich lipoproteins is atherogenic, similar to the effects of oxidized LDL (Chung et al., 1989; Shin et al., 2004; Wang et al., 2009). Another proposed mechanism for the various disease processes caused by consumption of a high fat diet/obesity is inflammation. A positive correlation between saturated fat intake and plasma inflammatory markers has been shown (Fung et al., 2001). A high fat diet can act as an inflammatory insult to the body,

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triggering synthesis and secretion of pro-inflammatory cytokines, which will lead to even more cytokine production as well as increased reactive oxygen species (ROS) and nitrogen species (Bullo et al., 2007). A commonly used clinical marker for inflammation, especially for obesity issues and cardiovascular disease, is C-reactive protein (CRP), an acute phase reactant produced by the liver (Madsen et al., 2001). One of the pro-inflammatory cytokines, interleukin-6 (IL-6), controls hepatic CRP production (Lopez-Garcia et al., 2004). CRP then induces synthesis of many other cytokines leading to a cascade of inflammatory events (Basu et al., 2006). Correlations between CRP levels and a high fat diet as well as CRP levels and atherosclerosis have been found in humans (Giugliano et al., 2006; Bullo et al., 2007). Diets that provide a high glycemic load such as the ‘Western diet’ are associated with increased serum CRP (Liu et al., 2002). Diets with low levels of n-3 PUFAs also increase CRP levels (Madsen et al., 2001). Furthermore, increased levels of pro-inflammatory markers correlate with endothelial dysfunction and insulin resistance (Giugliano et al., 2006). The increased risk for cardiovascular disease from a ‘Western diet’ is not only due to increased cholesterol levels but also to a chronic inflammatory state which contributes to atherosclerotic plaques (Rifai and Ridker, 2002). Lastly, adipose tissue in the obese state has been shown to release pro-inflammatory molecules and is typically infiltrated by macrophages, contributing to the chronic, low-level of inflammation associated with obesity (Wellen and Hotamisligil, 2003). Although these macrophages reside in the adipose and cytokines are released from it, secondary effects on insulin resistance and cardiovascular disease have been reported (Heilbronn and Campbell, 2008; Lumeng et al., 2007; Berg and Scherer, 2005; Fantuzzi, 2005). Therefore, it is plausible that inflammatory molecules originating from increased adipose tissue could also reach the brain and affect the cerebrovascular system. A system that arguably works hand in hand with inflammation and is known to be altered due to a high fat diet/obesity is oxidative stress (Uranga et al., 2010). In the Framingham Offspring Cohort Study reported in 2003, oxidative stress was found to be independently associated with obesity as measured by BMI. In more than 2800 subjects, the level of F2-isoprostane 8-epiPGF2α (a biomarker of lipid peroxidation) in urine was significantly increased in those subjects with a high BMI (Keaney et al., 2003). An in vitro study using cultured adipocytes also revealed that when elevated levels of fatty acids were added, increased levels of oxidative stress were observed due to NADPH oxidase activation. This increase in oxidative stress can lead to dysregulated production of adipose inflammatory molecules, adipokines (Furukawa et al., 2004).

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Oxidative stress is proposed to be an early key event preceding inflammation as well as insulin resistance (Matsuzawa-Nagata et al., 2008). In terms of the role of oxidative stress in cardiovascular events, Dobrian et al. reported that obesity-prone rats fed a moderately high fat diet for 10
16 weeks (32% kcal/fat) developed hypertension, had increased oxidative stress in the aorta and kidneys, and also exhibited decreased NO bioavailability. On the other hand, obesity-resistant rats fed the same diet were normotensive and did not have elevated oxidative stress or decreased NO bioavailability (Dobrian et al., 2001). Therefore, it appears that in this case, the development of obesity and the secondary effect of hypertension are linked to oxidative stress and damage to the aorta and kidney, and not to the diet alone, since obesity-resistant rats were normotensive and did not have elevated oxidative stress. Vincent et al. (1999) performed their studies in the lean and obese Zucker rats and found obese Zucker rats exhibited increased myocardial oxidative stress as evidenced by increased lipid peroxidation and superoxide dismutase in the left ventricle. Again, those rats which developed obesity had elevated oxidative stress levels while the lean rats did not. A number of studies have linked increased oxidative stress to development of atherosclerosis as it can lead to increased lipoprotein oxidation and increased cytokine release into the bloodstream (Stone, 2005; Tibolla et al., 2010; Wang et al., 2009; Saggini et al., 2011). Lastly, in a recent study from our group, we demonstrated for the first time that diet-induced obesity (DIO) elevates levels of total ROS in the mouse brain, a result which was also supported by finding a significant decrease in cortical glutathione peroxidase activity (Freeman et al., 2013). Taken together, important mechanisms for damage to overall health by a high fat diet and obesity include hyperlipidemia, inflammation, and oxidative stress. These mechanisms are all likely to interact and play a key role in the cerebrovascular changes observed following a high fat diet/obesity.

Clinical Studies: Vascular Changes Due to a High Fat Diet and Obesity It has been well established that obesity is a risk factor for cardiovascular disease as well as stroke (Go et al., 2013). In addition to understanding the mechanisms involved in developing vascular disorders, more research has begun in order to understand what obesity can do to the cerebrovasculature. For example, researchers have shown a high BMI is associated with reduced cerebral blood flow velocity (CBFV) (Selim et al., 2008; Willeumier et al., 2011). As stated previously, the brain requires a large portion of our body’s

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blood volume (20%); therefore, when CBFV is reduced, vital functions in the brain will likely be impaired. In fact, other studies have shown increased adiposity can decrease brain metabolism and cognitive performance. Specific regions of the brain appear to be affected more than others including the frontal lobe, bilateral medial temporal lobes, anterior lobe of the cerebellum, and occipital lobe (Volkow et al., 2009; Gunstad et al., 2008; Walther et al., 2010). Current studies have also focused on metabolic syndrome, a collection of disorders including obesity, hypertension, hyperglycemia, and dyslipidemia and its effects on cerebrovasculature. These studies have provided evidence that metabolic syndrome is connected with silent ischemic brain lesions and early white matter deterioration (Bokura et al., 2008; Park et al., 2008; Segura et al., 2009). Koren-Morag et al. (2005) conducted a large cohort study in patients with atherosclerotic cardiovascular disease where all factors of the metabolic syndrome were found to be associated with increased risk for ischemic stroke or transient ischemic attack. However, impaired fasting glucose and hypertension were found to be the strongest predictors of risk. Previous studies identified metabolic syndrome to significantly increase risk of morbidity and mortality from cardiovascular disease (Koren-Morag et al., 2005). These subsequent studies are now determining cerebrovascular effects as well. In a study by Haley et al. (2012), an increased level of peripheral atherosclerosis in subjects was linked with an increased level of cerebral glutamate concentration. Glutamate is an excitatory neurotransmitter, however, it is also an endogenous neurotoxin (Lipton and Rosenberg, 1994). It has previously been shown that severe cerebral ischemia is associated with a large accumulation of extracellular glutamate (Hossmann, 1994). Additionally, in an in vitro study using cerebral vascular endothelial cells from newborn pigs and heme oxygenase-2-knockout mice, it was shown that glutamate’s neurotoxic properties can serve to break down endothelial cells (Parfenova et al., 2006). Therefore, there is accumulating evidence to support the hypothesis that obesity/metabolic syndrome not only affect the peripheral cardiovascular system, but the cerebrovasculature as well. Furthermore, there are high fat diet/obesity-related cerebrovascular changes that likely occur before or in the absence of stroke which must be further evaluated for their role in cognitive and longterm brain changes.

Animal Studies: Vascular Changes Due to a High Fat Diet and Obesity Studies in the peripheral circulation tell us hypercholesterolemia has effects on the large arteries

including the aorta and coronary arteries leading to atherosclerosis (Franciosi et al., 2009). Increased levels of cholesterol and oxidized LDL can deposit in vessels leading to occlusion and inflammation (Stone, 2005). But in terms of what occurs in the central nervous system (CNS), it is commonly argued that cholesterol does not cross the BBB, as the brain synthesizes cholesterol in situ to be incorporated into cell membranes and myelin sheaths (Vance et al., 2006). More research must be performed in order to determine the passage of cholesterol and other substances through the BBB when it is potentially compromised in a proinflammatory state. It is well known that the BBB can be disrupted under conditions such as stroke, traumatic brain injury, and multiple sclerosis (Bhat, 2010). There is also evidence that monocytes can enter the CNS from the periphery during acute or chronic peripheral inflammation (Bhat, 2010; Audoy-Remus et al., 2008; D’Mello et al., 2009). Therefore, it is possible that brain inflammation occurs following a cardiovascular insult in the periphery due to a high fat diet/ obesity. For example, Tibolla et al. have demonstrated that atherosclerosis in the aorta can lead to vascular inflammation in the brain (Tibolla et al., 2010). Obesity and/or a high fat diet are known to lead to a chronic, low-grade inflammation and could affect BBB integrity and infiltration of monocytes into the CNS. In fact, it has been shown that rats fed a high energy diet compared to control-fed rats display a leaky BBB in the hippocampus as shown by passage of sodium fluorescein through blood vessels. In this study, the authors also found decreased claudin-5 and claudin-12 mRNA levels, providing evidence for damage to the BBB via a high energy diet (Kanoski et al., 2010). In addition to the studies which have measured the effects of cholesterol on the peripheral vasculature, altered microvessel morphology in the CNS has also been examined (Franciosi et al., 2009). For example, in a study by Constantinescu et al., hamsters were fed a hyperlipidemic diet for 3 or 6 months. The hyperlipidemic diet was developed by adding 1% cholesterol and 15% butter to a standard chow diet consisting of 2.5% fat. Hamsters fed the hyperlipidemic diet for 3 months exhibited fatty streaks in the carotid artery and the hamsters fed the hyperlipidemic diet for 6 months developed atherosclerotic plaques. Not only did these animals reveal peripheral changes, the authors also found altered microvascular pathology in the cortex including vessels with an uncharacteristic shape, enlarged perivascular spaces, and enlarged endothelial cells. In some rare cases, endothelial cell lumen filled with lipoprotein particles and perivascular cells filled with lipid were observed (Constantinescu et al., 2011). In 2009, Franciosi et al. provided a detailed evaluation of microvessels in the hippocampus and entorhinal

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EFFECTS OF A HIGH FAT DIET AND OBESITY ON OVERALL HEALTH AND PROPOSED MECHANISMS

cortex of C57BL/6J mice and LDL receptor null mice (LDLR 2/2 ) fed a high cholesterol diet for 4 or 10 months. The abnormal pathology observed in high cholesterol fed mice included twisted and string vessels, thin and irregularly shaped microvessels mixed with enlarged microvessels, as well as degenerating microvessels. Furthermore, the authors observed changes at the endothelial cell including altered chromatin structure in the endothelial cell nucleus, luminal degeneration, and expanded perivascular space, sometimes filled with debris. The author’s proposed mechanisms for this cholesterol-induced damage included increased ROS and inflammatory molecules (Franciosi et al., 2009). In a manuscript from our previous work, we also proposed a role of inflammation in vascular changes to the hippocampus following longterm high fat, high cholesterol feeding. We observed reduced BBB integrity along with increased microgliosis; in fact, the number of microglia correlated with SMI-71 densitometry (a measure of BBB integrity, an antibody specific to rat endothelial barrier antigen) in the cornus ammonis 1 (CA1) region of the hippocampus (Freeman and Granholm, 2012). Therefore, increased inflammation from a high fat diet appears to have damaging effects on the BBB; interestingly, the hippocampus is particularly vulnerable to this damage. Unfortunately, few current studies have investigated the effects of a high fat diet/obesity on the brain’s vascular system in wild-type animals, but a number of studies have been performed on different transgenic and stroke models. For example, increased cerebral infarct size and poor outcomes of stroke have been observed in a number of genetic and DIO models subsequently induced with ischemia. These studies included models of DIO via administration of a high fat diet (36% fat; high in saturated fat) for 10 weeks (Deutsch et al., 2009), administration of a ‘Western Diet’ for 12 weeks (Langdon et al., 2011), administration of a high fat diet (45% fat) for 8 weeks (Li et al., 2013), TallyHo mice (a model of type II diabetes) (Didion et al., 2007), the ob/ob mouse (Mayanagi et al., 2008; Kumari et al., 2010), the diabetic db/db mouse (Vannucci et al., 2001), and the obese Zucker rat (Osmond et al., 2009; 2010). In the 2009 study by Deutsch et al., the authors found that the middle cerebral arteries (MCAs) from obese rats had smaller lumens and thicker walls. The authors also reported increased MMP-2 activity and collagen I expression and reduced MMP-13 expression in DIO rats, concluding that changes to MCA structure during obesity may be a primary cause of increased ischemic damage (Deutsch et al., 2009). The 2009 study by Osmond et al. also reported a smaller lumen diameter and increased myogenic vasoconstriction in the MCA. Furthermore, development of hypertension was correlated with

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these reported changes (Osmond et al., 2009). The 2010 and 2011 studies by Kumari et al. in diabetic ob/ob and db/db mice revealed increased matrix metalloprotease (MMP)-9 mRNA, protein and gelatinolytic activity, increased degradation of occludin and collagen IV, a subsequent breakdown of the BBB, and greater infiltration of macrophages into the brain parenchyma (Kumari et al., 2010; 2011). In the recent study by Li et al. (2013), the authors reported impaired dilation of small arterioles, altered contraction and dilation of basilar arteries after ischemic injury, and impaired functional hyperemia in the high fat diet-fed rats. Interestingly, these findings were observed in animals fed a fairly short-term high fat diet and, therefore, did not have overt obesity or altered plasma lipid profiles (Li et al., 2013). This study in particular suggests mechanisms involved in high fat diet-induced cerebrovascular changes outside of increased circulating cholesterol and triglyceride levels. Altogether, the proposed mechanisms for diet- or obesity-induced changes to cerebrovasculature in these stroke models include structural changes to the MCA, more specifically, reduction in lumen size and altered MMP expression, inflammation, and BBB breakdown. In many of these studies, regulation of cerebral perfusion appears to be a major factor in high fat diet-induced cerebrovascular changes. Another rodent model for the study of high fat dietmediated changes to the brain’s vascular system is the LDL-null mouse. In a study by Buga et al. (2006), it was shown that administration of a ‘Western diet’ (42% fat, 0.15% cholesterol) for 6
8 weeks resulted in increased inflammation in large arterial vessels as well as small arterioles in the brain. The authors reported increased number of microglia, particularly found on the adventitial side of the arteriole, leading to a thicker arteriole wall. Notably, the authors did not find a change in blood pressure, suggesting these inflammatory effects are dependent on mechanisms other than regulation of blood perfusion, however, cerebral blood flow was not directly measured in the study. As mentioned above in the ischemia models, regulation of cerebral perfusion was central to worsened stroke outcome by a high fat diet and, in many of the studies, hyperlipidemia was not observed. On the other hand, this LDL-null mouse model displayed hyperlipidemia, but no change in blood pressure and a resulting increase in inflammation in arterioles. Together, it could be hypothesized that these two independent events, inflammation and cerebral blood flow regulation, are contributors to high fat diet-induced cerebrovascular changes, and that they are due to hypertension and dyslipidemia, respectively. Both hypertension and dyslipidemia can occur due to high fat feeding/obesity but would depend on the degree and length of time under these conditions.

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18. CEREBROVASCULAR CHANGES: THE ROLE OF FAT AND OBESITY

Impact of Omega-3 Fatty Acids on Vascular Health Intake of omega-3 fatty acids through increased fish consumption as well as supplementation has been a large area of research for decreasing risk and improving outcomes of cardiovascular disease. In a review by Chowdhury et al. (2012), 38 unique studies were compared and it was determined that high fish consumption, but not omega-3 supplementation, is significantly associated with a reduced risk of cerebrovascular disease. Interestingly, circulating biomarkers were not significantly associated with risk of cerebrovascular disease, suggesting the beneficial effects of high fish consumption include a number of nutrients in addition to omega-3 fatty acids and their interaction. Nonetheless, a number of mechanisms have been described for the beneficial effects of omega-3 fatty acids on the cardiovascular system. As mentioned previously in this review, hyperlipidemia, inflammation, and oxidative stress are all possible mechanisms for damage to the cerebrovascular system following a high fat diet/obesity. Omega-3 fatty acids have been shown to reduce each of these factors (Chowdhury et al., 2012; Mozaffarian et al., 2011; James and Cleland, 1997; Adkins and Kelley, 2010; Jump, 2002; Harris and Bulchandani, 2006). In terms of the effects specific to the vascular system, n-3 polyunsaturated fatty acids can increase endothelium-dependent relaxation through increasing nitric oxide (NO) release, reduce resting heart rate, reduce systolic and diastolic blood pressure, and modify eicosanoid production to favor vasodilation and anti-thrombosis (Abeywardena and Head, 2001; Harris and Bulchandani, 2006; Kang, 2012). In a study by De Caterina et al., endothelial cells were challenged with pro-inflammatory molecules: IL-1, IL-4, TNF-α or LPS. However, endothelial cells that were first incubated with DHA revealed a reduced inflammatory response

as well as decreased expression of VCAM-1, ICAM-1, and E-selectin, providing a protective response to the endothelial cell (De Caterina et al., 1994). In vivo studies have also revealed protective effects of dietary omega-3 fatty acids. For example, rhesus monkeys fed an omega3 fatty acid-rich diet versus a coconut oil-rich diet presented a significant reduction in atherosclerotic lesion formation after 1 year (Davis et al., 1987). In a recent study, n-3 fatty acids were carried in n-3 rich triglyceride emulsions to neonatal mice after hypoxic-ischemic brain injury. Those animals which received the triglyceride emulsions containing DHA up to 2 hours after injury had a reduced total infarct volume, but no difference in cerebral blood flow (Williams et al., 2013). This finding suggests not only a protective role of dietary omega-3 fatty acid consumption, but a potential therapeutic effect as well, if given in a strict time window.

CONCLUSION In conclusion, a number of mechanisms involved in diet and obesity-induced cerebrovascular changes have been proposed and include hyperlipidemia, altered blood pressure, inflammation, oxidative stress, and structural changes to large vessels as well as microvessels (Figure 18.1). More research must be done in order to better understand the role and interaction of these mechanisms which have already been shown to be involved in peripheral vascular changes due to a high fat diet/obesity. Using the strides in research that have been made in heart disease, type II diabetes, and metabolic syndrome, these mechanisms are likely important starting points for measuring cerebrovascular changes. Further advancements in this field will certainly help us to better understand other diseases such as dementia, Alzheimer’s disease, and

High fat diet/obesity

Hyperlipidemia

Breakdown of the BBB

Inflammation

Oxidative stress

Cerebrovasculature Altered cerebral blood flow

Hypertension

FIGURE 18.1 A number of mechanisms involved in diet- and obesity-induced cerebrovascular changes have been proposed. Primary effects include hyperlipidemia, inflammation, oxidative stress, and hypertension; secondary effects include changes to the BBB and cerebral blood flow.

Obstructed blood flow

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REFERENCES

stroke. Lastly, improvements in diet and lifestyle modifications, such as including more omega-3-rich fish, may be important therapies for high fat diet/obesityinduced cerebrovascular changes.

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19 Effects of Omega-3 Fatty Acids on Alzheimer’s Disease Gaurav Paul and Ronald Ross Watson INTRODUCTION Omega-3 Fatty Acids Biology in Health Omega-3 fatty acids, polyunsaturated fatty acids with a double bond after the third carbon atom in the carbon chain, are fats that are commonly found in marine and plant oils. Although they are considered essential fatty acids, the exact health benefits are still being defined. Consuming omega-3 fatty acids versus other fatty acids as a supplement helps reduce the risk of cancer, cardiovascular disease, inflammation, and disorders. They are needed for a normal metabolism. A favorable omega-6 to omega-3 polyunsaturated fatty acids ratio and fish consumption have a protective effect against cancer (Cole et al., 2013). However, the studies that were conducted to support this hypothesis were only performed on adults, so the role of omega-3 in the diet of children for cancer prevention is not understood. It is possible that omega-3 has a greater positive effect in preventing cancer in children, but more tests would have to be performed in order to confirm this. Furthermore, omega-3 can play a role in preventing cardiovascular disease. Evidence does support a beneficial role for omega-3 fatty acid supplementation in preventing cardiovascular disease (including myocardial infarction and sudden cardiac death) or stroke. Eating a diet high in fish that contain longchain omega-3 fatty acids decreases the risk of stroke. Omega-3 fatty acids play a positive role in preventing cardiovascular disease when they are consumed through fish oils by eating fish. In related studies, a high intake of omega-3 fatty acids can increase low density lipoprotein (LDL) cholesterol, lower blood pressure, and reduce the risk of heart attacks.

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00019-3

Consumption of long-chain omega-3 fatty acids can change inflammation, which is one of the body’s responses to pathogens. One example of inflammation modification by omega-3 fatty acids reduces symptoms of rheumatoid arthritis. People with rheumatoid arthritis can have reduced pain by taking omega-3. Finally, omega-3 may be able to do something against developmental disorders, psychiatric disorders, and cognitive aging. Consuming omega-3 fatty acids may prevent some developmental disorders and even reduce symptoms in certain cases such as children with attention deficit hyperactivity disorder (ADHD). This is also true with psychiatric disorders, as omega-3 fatty acids may be able to reduce symptoms of bipolar disorder. With cognitive aging such as the degradation of neurogenesis, which is how neurons are generated, omega-3 may enhance the process of neurogenesis.

ALZHEIMER’S DISEASE Alzheimer’s disease, which is the most common form of dementia, has no cure. Over time, the disease simply gets worse until it leads to death. It can have varied development in different individuals, but common symptoms include confusion, irritability and aggression, memory loss, and mood swings. Over time, the Alzheimer’s disease patient will lose bodily function leading to death. The average life expectancy after diagnosis is around seven years and is associated with plaques and tangles in the brain. Alzheimer’s disease can have a variety of possible causes. Since it deals with plaque build-up and tangles in the brain, prevention is associated with brain

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activity. Mental stimulation, exercise, and a balanced diet are ways to delay cognitive symptoms. Once the patient has been diagnosed with Alzheimer’s, there is nothing that can be done for the person. Since the disease is degenerative, it will progress until death occurs (Cole et al., 2013). Since a person diagnosed with Alzheimer’s is not able to do many of the daily tasks that people perform, they will need a caregiver. Alzheimer’s disease places a great burden on caregivers; the pressures can be wide-ranging, involving social, psychological, physical, and economic elements of the caregiver’s life (Cole et al., 2013).

OMEGA-3 FATTY ACID: A ROLE IN ALZHEIMER’S DISEASE? Omega-3 fatty acids may play a huge role in Alzheimer’s disease. There are two ways in which omega-3 can be used against Alzheimer’s disease. Fish oils containing omega-3 fatty acids can be taken before onset of the disease, as a form of prevention, or they can be taken by someone who has been diagnosed with Alzheimer’s disease to fight off the progression of the disease. Alzheimer’s disease resembles cardiovascular disease since both show age-dependent accumulation of lipophilic material in multicellular plaque lesions, which involves a macrophage (microglial) inflammatory response, oxidative damage, and injury to surrounding cells (Cole et al., 2013). Therefore, is it possible that research done on cardiovascular disease could be connected to Alzheimer’s disease research? Two major advantages in the study of cardiovascular disease prevention have been the availability of adequate biomarkers (blood lipid profiles) and the opportunity to conduct prevention trials in at-risk patients with secondary cardiovascular ‘events’ as endpoints. Unfortunately, the lack of suitable biomarkers, ‘second events,’ and the cost and time involved in conducting prevention trials for Alzheimer’s disease have prohibited prevention testing in clinical trials. Most testing has been in Alzheimer’s disease patients (Cole et al., 2013). Initial clinical trials did not show many positive effects in reducing the progression of Alzheimer’s disease. More testing on animal models is needed in this area. One of the most important components of omega-3 fatty acid is docosahexaenoic acid (DHA). Neurons and synapses, which become enriched in unsaturated fatty acids with long chains, prefer DHA since it has six double bonds, making it highly unsaturated. Therefore, by stimulating the synapses, cognitive decline caused by Alzheimer’s disease, which is common with synapse loss, is prevented. DHA is oxidized in Alzheimer’s disease to produce elevated

F4-isoprostanes or neuroprostanes (Cole et al., 2013). By looking at the level of these oxidation products in the neurons, the level of neuronal oxidative damage can be determined. Lower levels of F4-isoprostanes or neuroprostanes means that the person is not getting enough DHA in their system (Cole et al., 2013). Fish are a major source of omega-3 fatty acids and they are also a main dietary source of DHA. An aged Tg2576 APPsw transgene-positive mice fed from 17 to 22 months on a safflower oil-rich, DHA-depleting diet exhibited increased oxidative damage and transgenedependent loss of central nervous system DHA and massive loss of post-synaptic proteins such as the actin-regulatory drebrin, a dendritic spine protein known to be 70
90% lost in Alzheimer’s disease (Sima & Li, 2006). Thus, when DHA was depleted from the system, there was much oxidative damage and loss of proteins that affects both the neurons and synapses (Sima & Li, 2006). Some of this damage is identical to that seen with Alzheimer’s disease patients (Sydenham et al., 2012). Not only is the dendritic spine protein drebrin lost with DHA deficiency as well as Alzheimer’s disease, but DHA depletion also causes transgene-dependent caspase activation and deficits in the neuroprotective insulin signaling pathway (Sydenham et al., 2012). Each of these components that is lost or damaged with Alzheimer’s disease is essential to proper functioning of the whole nervous system. Drebrin, a protein of the spine, is encoded by the DBN1 gene. The protein encoded by this gene is a cytoplasmic actin-binding protein thought to play a role in the process of normal growth (Sydenham et al., 2012). It is a member of the drebrin family of proteins, which are developmentally regulated in the brain. A decrease in the amount of this protein in the brain has been implicated as a possible contributory factor in the pathogenesis of memory disturbance in Alzheimer’s disease (Hooper et al., 2006).

DHA Deficiency and Neurological Function Affected by Diabetes in Alzheimer’s Disease Transgene-dependent caspase activation, caused by DHA deficiency, involves the random activations of caspases, which play essential roles in apoptosis (programmed cell death), necrosis, and inflammation (Li et al., 2002). Thus, DHA deficiency with apoptosis, necrosis, and inflammation all occur without any kind of programming. The insulin signaling pathway, another result of DHA deficiency, is very important because it affects homeostasis. When carbohydrates enter a system, the pancreas releases insulin so that more glucose is taken from the blood stream. Insulin will bind to an insulin receptor on the cells and will

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either give the signal to use the glucose or store it in the cell. This pathway is made up of different trigger mechanisms that are the different signals within a cell. Some of these signals are also there to stop the production of insulin when there is too much in the body and not very much glucose. DHA is involved in regulating the insulin signaling pathway and keeping the entire system functioning. Therefore, a deficiency of DHA can lead to dysfunctional glucose metabolism and could affect Alzheimer’s disease (Scheinder et al., 1999). The pancreas either may not make enough insulin or it may not store the glucose properly, which can lead to diabetes, hyperglycemia, and hypoglycemia. Therefore, DHA supplementation can help prevent diabetes, hyperglycemia, and hypoglycemia, as well as any other diseases or symptoms that are caused by either an excess or shortage of glucose in the system. In these circumstances, DHA also helps to regulate the neuroprotective pathway, which is there to make sure insulin does not cause any neurological damage, which may help prevent Alzheimer’s disease. Insulin-induced severe hypoglycemia was found to cause brain damage (Scheinder et al., 1999). The hypothesis to be tested was that diabetes is associated with more extensive brain tissue damage following an episode of severe hypoglycemia. Nine-week-old male streptozotocin-diabetic or vehicle-injected control Spraque-Dawley rats were subjected to hyperinsulinemic severe hypoglycemic clamps while awake and unrestrained (Scheinder et al., 1999). In both cases, the rats that had severe hypoglycemia were subject to long episodes of seizures due to damage in the hippocampus region of the brain, one of the major regions of the brain (Sima & Li, 2006). This region is a part of the limbic system of the brain and has an important role when it comes to both short-term and long-term memory. The hippocampus is the first and most severely affected part of the brain with Alzheimer’s disease patients (Tiraboschi et al., 2004). More specifically, although at first it begins with simple short-term and sometimes long-term memory loss, over time it can develop into severe amnesia, which is when the patient can no longer form or keep any new memories (Tiraboschi et al., 2004). This is most commonly seen in late stage Alzheimer’s disease, where the patients are nearing the end of their life (Tiraboschi et al., 2004). Insulin-induced hypoglycemia caused by diabetes and a deficiency of DHA leading to neurological damage has been linked to Alzheimer’s disease (Tiraboschi et al., 2004). What is the exact process behind the neurological damage and, are diabetes patients more likely to develop Alzheimer’s disease as a secondary disease? Neurological damage caused by insulin-induced hypoglycemia is slightly different than patients diagnosed

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only with Alzheimer’s. With hypoglycemia, the hippocampus can be damaged in a variety of ways. Lack of oxygen to the brain, inflammation of the brain (encephalitis), and the neurological condition known as epilepsy can account for some of the reasons why patients with severe hypoglycemia from either DHA deficiency or diabetes experience severe seizures and extensive memory loss. Are diabetes patients more prone to being diagnosed with Alzheimer’s disease? During the last decade, epidemiological data suggests a linkage between type 2 diabetes and Alzheimer’s disease (Shim & Drebrin, 2002). Insulin is a factor that plays a large role in the linkage of these two diseases. Impaired insulin signaling is involved in hyperphosphorylation of the tau protein, which is part of neurofibrillary tangles in Alzheimer’s disease (Shim & Drebrin, 2002). Here, the common linkage is the tau protein which is modified in both Alzheimer’s disease and type 2 diabetes. Cognitive impairments are more frequent in diabetic patients than in non-diabetic subjects, due to ischemic events resulting from cerebral microvascular and macrovascular disease, in addition to repeated episodes of severe hypoglycemia, and are causative for secondary diabetic encephalopathy. Accumulating evidence suggests that cognitive dysfunction is also caused by diabetic dysmetabolism, meaning that it could be primary diabetic encephalopathy (Shim & Drebrin, 2002). Although the higher risk of cognitive dysfunction appears in both type 1 and type 2 diabetes patients, it is more common with type 2 diabetes patients (Shim & Drebrin, 2002). Not only are hyperglycemia and altered insulin signaling transduction involved, but so too are other risk factors such as hypercholesterolemia, hyperlipidemia, and hypertension, which are connected to type 2 diabetes.

Animal Models, Diabetes, and Alzheimer’s Disease Experimental studies have demonstrated significantly more severe abnormalities in the expression of amyloid precursor protein, beta-secretase, amyloidbeta, and phosphorylated tau in the type 2 BBZDR/ Wor rat model as compared to its type 1 counterpart, the BB/Wor rat (Shim & Drebrin, 2002). These rat models can be applied to human patients who have type 2 diabetes and cognitive dysfunction. Together, the different risk factors outlined in the rat models are known as the metabolic syndrome, which has been identified as a way to predict complications such as cerebrovascular disease, accelerated cognitive dysfunction, and dementia. These complications comprise different features of Alzheimer’s disease and are also

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found in type 2 diabetes patients. Since similar factors lead to both diseases, there is a definite connection. Most of these studies that test those risk factors have been on rats with type 2 diabetes. With humans, sometimes aging alone is enough for the metabolic syndrome to start degrading, and this is because aging causes decreased glucose utilization as well as declining insulin and IGF-1 signal transduction due to receptor desensitization (Shim & Drebrin, 2002). With Alzheimer’s disease, these features are all magnified because the receptors are not just desensitized as with aging and type 2 diabetes, but there is degradation of both the insulin and IGF-1 receptor. Hyperphosphorylation of the tau protein, discussed before as one of the biggest links between the two diseases, is one of the most important pathobiological events in Alzheimer’s disease (Shim & Drebrin, 2002). Generally, it leads to a build-up of toxicity and degeneration of the neurites, which is known as tauopathy. Eventually, this leads to slower firing of the neurons and slow communication within the brain. In Alzheimer’s disease, it is much more accelerated than in type 2 diabetes. After looking at all of these different factors and examining the similarities of the two diseases, one group concludes that there is no doubt that mechanistic linkages exist between diabetes and Alzheimer’s disease (Shim & Drebrin, 2002). The major problems with both diseases deal with impaired actions by insulin, IGF-1, and NGF. This plus the accumulation of amyloid-beta products, and hyperphosphorylated tau, exert toxic effects on neuronal neurites with their subsequent degeneration and eventual neuronal death, which eventually leads to the death of the patient (Shim & Drebrin, 2002).

Omega-3 Fatty Acids in Prevention and Treatment of Alzheimer’s Disease The brain is particularly rich in fatty acids and several mechanisms have been postulated for the possible protective role of omega-3 fatty acids in dementia (Shim & Drebrin, 2002). First, DHA is a key component of membrane phospholipids in the brain; therefore, adequate omega-3 fatty acids status may help maintain integrity and neuronal function. Second, the oxidative products of polyunsaturated fatty acids act as key cellular mediators of inflammation, allergy and immunity, oxidative stress, bronchial constriction, vascular responses, and thrombosis and may thereby influence risk, especially of vascular dementia (Shim & Drebrin, 2002). Third, there is a suggestion that DHA may be directly involved in enhancing neuronal health in the aging brain through a range of potential mechanisms. And finally, there is a growing body of evidence that

DHA may modify the expression of genes that regulate a variety of biological functions important for cognitive health, including neurogenesis and neuronal function (Shim & Drebrin, 2002). In order to test all of this, a study was set up in which participants had to be aged 60 or over, and without dementia or cognitive impairment at the beginning of the study. There was extensive screening before the study to make sure that all the participants did not have any cognitive impairment. Once this was confirmed, the omega-3 intervention involved dietary supplementation or provided meals, versus placebo or usual diet (Van de Rest et al., 2008). Together, there were 4080 participants in the three studies, but each study had a different procedure. In the first study, there were 302 participants. After screening all participants with a Mini Mental State Examination (MMSE) and making sure their scores were 21 out of 30 or less, the intervention period began. It was 6 months long and consisted of six 900 mg soft gelatin capsules per day containing a total of 1940 mg omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) in the high-dose arm, 400 mg omega-3 LC-PUFAs in the low-dose arm, and high-oleic sunflower oil in the placebo arm. All of these capsules were identical and packed in foil strips containing the dose for each day. Out of all the participants, there is cognitive function outcome data available for 299 of these after six months. The primary outcome was cognitive function at six months. The average scores of all cognitive domains did not differ between the different treatment groups. After 13 weeks, there was a decline in the memory domain for the group that was supplemented with 400 mg EPA-DHA compared to the placebo group. However, after 26 weeks, at the end of the study, the differences were no longer there. Between 13 to 26 weeks of supplementation, there were not any significant changes for fish oil versus placebo in any of the cognitive function categories, meaning that the study did not show any positive results. The second study, (Van de Rest et al., 2008), involved 867 male and female participants between the ages of 70 and 79. They were first pre-screened using general practice recorded information for diabetes, dementia, and significant illness. Those who passed this and were potential participants then went through a cognitive function screen, the MMSE, in common with the previous study (Van de Rest et al., 2008). Any person who scored less than 24 out of 30 on the MMSE was excluded from the study. The intervention period was 24 months and the intervention consisted of two 650 mg soft gelatin capsules per day containing a total of 700 mg marine-source omega-3 LC-PUFAs in the active arm, as well as omega-9 rich olive oil in the placebo arm. These capsules looked identical and were usually packed in pots. Out of the 867 participants, there is cognitive function outcome data on 744 of

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REFERENCES

these after 2 years. The primary outcome was change in cognitive function at 24 months. There was no change in cognitive function over the 24 months in any of the different study arms, with the z scores remaining the same (Van de Rest et al., 2008). Finally, the third study was the largest of the three by far. It involved 2911 participants in total and only people who were between the ages of 60 and 80 and got a score of at least 21 out of 30 on the MMSE were allowed to take part in the trial. Participants were provided with margarine containing either 400 mg of eicosapentaenoic acid (EPA)-DHA (3:2 ratio), 2 g of α-linolenic acid (ALA), the EPA-DHA and ALA combined, or placebo margarine for 40 months. However, it is important to note that this was a study of people who had suffered from a heart attack up to 10 years before the trial, and the primary outcome from the trial was mortality from a subsequent heart attack during the trial period. There were actually 4837 participants in the whole study, but only 2911 of them participated in the omega-3 sub-study. After the trial period finished, there were 2493 participants who did not have cognitive impairment. The overall MMSE score decreased by 0.67 points after the study in most participants and the changes in MMSE score did not differ much between EPA-DHA and placebo groups. Each study had different outcomes depending on what was given to the participants (Toda et al., 1993). However, there is no evidence from these controlled trials that taking omega-3 fatty acid as a supplement can prevent against incident dementia (Toda et al., 1993). While none of the trials showed any benefit, there was also no harm. Participants in both the intervention and control groups experienced very little or no cognitive decline during the studies, and the main side effect experienced was gastrointestinal problems but that was only reported by a small fraction of all the participants. Those who performed the study said that for any conclusive evidence to be found, studies of longer duration are required because they may identify greater change in cognitive function in the study participants (Toda et al., 1993). Although this test did not show any conclusive evidence, there may be some good information here for

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further studies. Does not having any conclusive evidence make the study a failure? Not necessarily, because since there was no negative evidence for the trials, it is very possible that omega-3 fatty acid can in fact play a positive role in preventing Alzheimer’s disease. It could be that omega-3 fatty acids just need years of supplements to work in order to successfully prevent Alzheimer’s disease, and that if many more people started using them, there could be a natural study done to see how many live without Alzheimer’s disease after 10 years or so.

References Cole, G.M., Lim, G.P., Yang, F., Teter, B., Begum, A., Ma, Q., et al., 2013. Prevention of Alzheimer’s disease: omega-3 fatty acid and phenolic anti-oxidant interventions. National Library of Medicine. Hooper, L., Thompson, R.L., Harrison, R.A., Summerbell, C.D., Ness, A.R., Moore, H.J., 2006. Risks and benefits of omega 3 fats for mortality, cardiovascular disease, and cancer: systematic review. BMJ. 332, 752. Li, Z.G., Zhang, W., Grunberger, G., Sima, A.A., 2002. Hippocampal neuronal apoptosis in type 1 diabetes. Brain. Res. 946, 212
231. Scheinder, J., Murray, J., Banerjee, S., Mann, A., 1999. EUROCARE: a cross-national study of co-resident spouse carers for people with Alzheimer’s disease: Factors associated with carer burden. Int. J. Geriatr. Psychiatry. 14 (8), 651
661. Shim, K.S., Drebrin, L.G., 2002. A dendritic spine protein is manifold decreased in brains of patients with Alzheimer’s disease and Down syndrome. Neuroscience. 324, 209
212. Sima, A.A., Li, Z.G., 2006. Diabetes and Alzheimer’s disease
is there a connection? Rev. Diabet. Stud. 3, 161
168. Sydenham, E., Dangour, A.D., Lim, W.S., 2012. Omega-3 fatty acid for the prevention of cognitive decline and dementia. Cochrane Database of Systematic Reviews. 6. Tiraboschi, P., Hansen, L.A., Thal, L.J., Corey-Bloom, J., 2004. The importance of neuritic plaques and tangles to the development and evolution of AD. Neurology. 62, 1984
1989. Toda, M., Shirao, T., Minoshima, S., Shimizu, N., Toya, S., Uyemura, K., 1993. Molecular cloning of cDNA encoding human drebrin E and chromosomal mapping of its gene. Biochem. Biophys. Res. Commun. 196, 468
472. Van de Rest, O., Geleijnse, J., Kok, F.J., Van Staveren, W.A., Hoefnagels, W.H., Beekman, A.T.F., 2008. Effect of fish-oil supplementation on mental well-being in older subjects: A randomized, double-blind, placebo-controlled trial. Am. J. Clin. Nutr. 88, 706
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C H A P T E R

20 Substantia Nigra Modulation by Essential Fatty Acids Belmira Lara da Silveira Andrade da Costa, Priscila Pereira Passos, Henriqueta Dias Cardoso, Catarina Gonc¸alves-Pimentel, Eraldo Fonseca dos Santos Junior, Juliana Maria Carrazzone Borba and Rubem Carlos Arau´jo Guedes IMPORTANCE OF ESSENTIAL FATTY ACIDS AS NEUROPROTECTORS DURING BRAIN DEVELOPMENT AND AGING A growing body of experimental and clinical evidence has indicated the importance of long-chain polyunsaturated fatty acids (LCPUFA) docosahexaenoic acid (DHA) and arachidonic acid (AA), derived from the essential fatty acids (EFA) α-linolenic acid (ALA) and linoleic acid (LA), respectively, as critical modulators of brain function (reviewed in Uauy and Dangour, 2006; Innis, 2007; Zhang et al., 2011). From an evolutionary point of view, it has been discussed that the rapid expansion of gray matter in the cerebral cortex coincided with the inclusion of nutrients from coastal seafood and other sources from inland freshwater containing high levels of DHA in the human diet (Crawford et al., 1999, 2001; Broadhurst et al., 2002; Bradbury, 2011). As early as the 1920s some researchers had already described the importance of EFAs, observing signs of dermal changes in rats and neurological and visual disorders in humans subjected to fat restriction in their diets (Burr and Burr, 1929). Since then, several functional aspects related to EFAs have been studied. An expressive increase in the number of studies on the importance of their balanced levels in the diet has been published, considering that DHA and AA can exert opposite effects on brain metabolism (Schmitz and Ecker, 2008; Bradbury, 2011). During the growth spurt period, coincident with later stages of gestation and whole lactation period,

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00020-X

there is a significant accumulation of all fatty acids in the brain (Green and Yavin, 1993; Martinez and Mougan, 1998; Green et al., 1999). Saturated, monounsaturated, and polyunsaturated fatty acids are differently distributed between the gray and white matters and among brain regions (Xiao et al., 2005; Levant et al., 2006). Their accretion occurs at distinct stages of brain development (Burdge and Postle, 1995; Green and Yavin, 1998; Green et al., 1999; Uauy and Dangour 2006; Innis, 2007). During pregnancy, the need for the LCPUFA is higher (Koletzko et al., 2008). They need to be shared among the various maternal tissues and the fetus according to their availability in the diet and their metabolism in the liver (Frazer and Huggett, 1970; Rapoport et al., 2007). As ligands for the retinoid X receptor, AA and DHA participate in diverse neurodevelopmental steps, including neurogenesis, morphological differentiation of some neurons and activitydependent plasticity (Castro et al., 2001; Lengqvist et al., 2004). In vitro studies have also shown that DHA stimulates cell-cycle exit in retinal neuroprogenitor cells (Insua et al., 2003) and glial cell maturation (Joardar and Das, 2007; Joardar et al., 2006). Recently, it was also demonstrated that DHA promotes dopaminergic differentiation in induced pluripotent stem cells and inhibits teratoma formation in rats with Parkinson-like pathology (Chang et al., 2012). Early and recent studies using in situ hybridization or microarray analysis have shown that DHA is able to regulate the transcription of many genes related to cell metabolism, cell signaling including the oxidative stress response, cell division, outgrowth and apoptosis

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(Sessler and Ntambi, 1998; Berger et al., 2002; Kitajka et al., 2004; Lapillonne et al., 2004). In line with these findings, accumulating evidence has indicated that LCPUFA may act as neuroprotectors in the brain. DHA has been implicated in reducing apoptosis and necrosis in different classes of neurons (Lang-Lazdunski et al., 2003; Kim et al., 2010), and the inflammatory and antineurogenic functions of activated microglial cells (Ajmone-Cat et al., 2012) while AA can act as a neurotrophic factor on sensory neurons (Robson et al., 2010). Some of the neuroprotective effects of DHA involve sub-products of its metabolism called docosanoids, especially neuroprotectin D1 (NPD1). Under conditions of inflammation and/or oxidative stress, NPD1 is able to reduce pro-apoptotic agents such as caspase-3, Bad and Bax, as well as to inhibit cyclooxigenase-2 activation and the pro-inflammatory factor NF-kB (Marcheselli et al., 2003; Chen and Bazan, 2005; Bazan et al., 2011; Mukherjee et al., 2004). Recent evidence has also shown that an ethanolamide derivative of DHA, called Ndocosahexaenoylethanolamide (DEA) is a mediator of the DHA-induced increase in neurite outgrowth and synaptogenesis in hippocampal neurons (Kim et al., 2011a,b). A beneficial effect of the LA derivative 8-[2(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) on oxidative stress-induced neuronal death has been also reported (Yaguchi et al., 2010). Taken together, these findings suggest that imbalance in AA/DHA levels early in life, and especially DHA deficiency, induce neurodegeneration (Yavin, 2006; Schmitz and Ecker, 2008; Bazan, 2006; Bazan et al., 2011). However, under physiological conditions, LCPUFA levels in brain membrane phospholipids decrease with aging (Guisto et al., 2002; Uauy and Dangour, 2006) and for this reason their role in healthy brain aging has been widely debated in the literature (reviewed in Zhang et al., 2011). The extent and consequences of dietary omega-3 or omega-6 fatty acid deficiency on the brain are not yet completely understood. Nevertheless, it has been proposed that such deficiency can either contribute to the etiology of some neurodegenerative diseases (McNamara and Carlson, 2006; Heinrichs, 2010; Zhang et al., 2011) or worsens the age-induced modifications in neuron and glial cell reactivity that increases cell vulnerability to lesions (Latour et al., 2013). Therefore, dietary supplementation of LCPUFA, especially those of the omega-3 family, has been indicated as a potential therapeutic strategy as nutraceuticals, to reduce the risk of certain dopamine-associated neurological disorders (Chen et al., 2003; de Lau et al., 2005; Chao et al., 2012). This section focuses on experimental data which reinforces the modulatory effect of EFAs on midbrain

dopamine systems with special emphasis in the nigrostriatal system of basal ganglia circuitry.

SUBSTANTIA NIGRA VULNERABILITY TO NEURODEGENERATION The basal ganglia are important components of the forebrain circuitry which fulfill cognitive, limbic, motor and learning functions (Graybiel et al., 1994; Hikosaka et al., 2000; Yin and Knowlton, 2006; Kreitzer and Malenka, 2008; Surmeier et al., 2011a). In this circuitry, the corpus striatum is the largest and the primary input nucleus that integrates afferent information from several regions of the cerebral cortex, the thalamus and dopaminergic and GABAergic innervation from the midbrain (Surmeier et al., 2011b). Using interconnected closed or open neuronal loops with prefrontal, limbic, sensory and motor cortex, the basal ganglia network displays activity-dependent synaptic plasticity and coordinates action plans according to the motivation and motor information, and represents a neural substrate for procedural memory (revised in Graybiel, 2004; Kreitzer and Malenka, 2008; Haber and Calzavara, 2009; Pennartz et al., 2009). The dorsal striatum is especially involved in motor control while the ventral striatum, including nucleus accumbens (NAc), is mainly related to limbic and cognitive functions (Nicola, 2007). In this context, midbrain dopaminergic neurons from the substantia nigra and the ventral tegmental area (VTA) exert crucial roles modulating short-term cellular excitability as well as long-term changes in synaptic strength that shape network activity into the striatum and other basal ganglia nuclei (Joel and Weine, 1994; Surmeier et al., 2007; Kreitzer and Malenka, 2008). Dopamine neurons from the substantia nigra pars compacta project mainly to the dorsal striatum characterizing the nigrostriatal or mesostriatal system. The dopamine mesocorticolimbic system that originated from the VTA can be subdivided into two pathways: one starts in the medial posterior part of VTA and projects to the medial prefrontal cortex, the basolateral amygdala and the core and medial shell of NAc; the second one originates from lateral portions of the VTA and the medial part of the substantia nigra projecting to the lateral shell of the NAc (Lammel et al., 2008). Thus, modifications in these dopaminergic systems underlie changes in movement and thought in some neurological disorders such as Parkinson’s, Huntington’s and Gilles de la Tourette’s diseases (Albin et al., 1989; Graybiel, 2000; Wichmann et al., 2007) as well as in a number of psychiatric disorders such as schizophrenia

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SUBSTANTIA NIGRA DOPAMINE CELL POPULATIONS DISPLAY DIFFERENTIAL VULNERABILITY TO LESIONS

(van Kammen et al., 1989) and obsessive compulsive disorder (Aouizerate et al., 2004). The vulnerability of substantia nigra dopamine neurons to lesions has been widely debated in the literature especially considering that it is a multifactorial process, involving extrinsic risk factors related to the substantia nigra environment and interconnected brain regions, and intrinsic factors related to cell metabolism and neuron-glia interaction (Hassler, 1938; Duke et al., 2007; Gonza´lez-Herna´ndez et al., 2010; Bolam and Pissadaki, 2012; Sulzer and Surmeier, 2013; Shao et al., 2013). Compared to other midbrain dopaminergic nuclei such as VTA, retrorubral field and interfascicular nucleus or other basal ganglia nuclei, the substantia nigra has unique biochemical properties which render it particularly vulnerable to oxidative stress such as high iron content, low levels of endogenous antioxidant resource such as glutathione, catalase (CAT), and peroxidase enzymes (reviewed in Kidd, 2000; Gonza´lez-Herna´ndez et al., 2010). Studies on rat or human substantia nigra have also indicated a progressive decrease in the activity of some antioxidant enzymes including superoxide dismutase (SOD) and CAT during physiological brain aging (Kolosova et al., 2003; Venkateshappa et al., 2012) which worsen its selective vulnerability to oxidative stress. A high concentration of microglia has also been reported in the substantia nigra (Lawson et al., 1990). In conditions of mitochondrial dysfunction (Madathil et al., 2013) or lipopolysaccharide-induced insult (Arimoto and Bing, 2003; Arimoto et al., 2007), activated microglia release cytokines and free radicals such as superoxide radicals and nitric oxide (NO) (Minghetti et al., 1999; Duncan and Heales, 2005) which renders substantia nigra susceptible to develop neuroinflammation (reviewed in Gonza´lez-Herna´ndez et al., 2010). The presence of receptors for proinflammatory cytokines has been shown in substantia nigra dopaminergic cells and especially in Parkinson’s disease patients or experimental models of this disease, reinforcing their sensitivity to lesion under this stressful condition (Boka et al., 1994; Mogi et al., 2000; Ferger et al., 2004; Lofrumento et al., 2011). As cell intrinsic risk factors, it has been well established in the literature that the dopamine (DA) metabolism per se contributes to the selective vulnerability of the substantia nigra. Dopaminergic cells possess a distinct physiology intrinsically associated with increased production of reactive oxygen species via metabolization by monoamino oxidase or auto-oxidation (Meiser et al., 2013). In these metabolic pathways, dopamine either generates hydrogen peroxide or it is converted into reactive quinones and superoxide anion. The latter can react with nitrogen reactive species producing

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peroxynitrite (Meiser et al., 2013). Evidence in mouse has also indicated that lower mitochondria content is found in dopaminergic compared to other nondopaminergic cell populations present in the substantia nigra or in other midbrain dopaminergic nuclei such as VTA and interfascicular nucleus (Liang et al., 2007). Other studies have shown a redox modulation on the activity and expression of the tyrosine hydroxylase (TH), which represents the rate-limiting enzyme in the biosynthesis of DA (Di Giovanni et al., 2012). Structural risk factors contributing to dopamine cell vulnerability include unmyelinated or poorly myelinated long, highly branched axons and terminal fields. Physiological risk factors are pacemaker activity and broad action potentials. These features demand high energy and can be impaired under sustained conditions of oxidative stress or neuroinflammation (Bolam and Pissadaki, 2012; Sulzer and Surmeier, 2013). Besides the singular electrical properties, modifications induced by oxidative stress in some ion channels present in nigrostriatal dopaminergic neurons have been investigated as potential mechanisms involved in their selective demise in Parkinson’s disease (Michell et al, 2007; Liss and Roeper, 2010). Moreover, it has been shown that maintaining L-type Ca21 channels open in substantia nigra compacta DA neurons to keep pacemaker activity creates a basal mitochondrial oxidant stress. Epidemiological data also supports a linkage between L-type Ca21 channels and the risk of developing Parkinson’s disease (reviewed in Surmeier et al., 2011b).

SUBSTANTIA NIGRA DOPAMINE CELL POPULATIONS DISPLAY DIFFERENTIAL VULNERABILITY TO LESIONS Despite the general features related to the substantia nigra environment and cell metabolism described above, dopamine cell populations located in the rostrodorso-medial (SNrm) and caudo-ventro-lateral (SNcv) regions of this nucleus are not homogeneous. They rather differ in aspects related to their ontogeny, morphological and neurochemical profiles (Bayer et al., 1995; Gonza´lez-Herna´ndez et al., 2004; Duke et al., 2007). Their projections to the corpus striatum are segregated into distinct functional divisions (Joel and Weiner, 2000; Prensa and Parent, 2001) and they also differ on their susceptibility to degeneration in Parkinson’s disease in humans (Damier et al., 1999; Duke et al., 2007) and in rodent models (Rodrı´guez et al., 2001). DA cells located in the SNcv are usually more vulnerable to lesions compared to those of the SNrm. Several potential mechanisms involved in such

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differential vulnerability have been proposed: expression of calcium binding proteins (Yamada et al., 1990; Gaspar et al., 1994) associated or not with the homeodomain transcription factor Pitx3 (Luk et al., 2013); availability of the glial cell line-derived neurotrophic factor (GDNF) from the striatum (BarrosoChinea et al., 2005), levels of plasma membrane dopamine transporter (DAT) (Uhl et al., 1994; Gonza´lez-Herna´ndez et al., 2004) and the transient down-regulation of DAT glycosylated form after dopamine cell loss (Afonso-Oramas et al., 2010). Increased expression of genes encoding pro-inflammatory cytokines and subunits of the mitochondrial electron transport chain and decreased expression of several glutathione-related genes have been found in the SNcv, compared to SNrm (Duke et al., 2007). Considering that most of these genes are particularly expressed in glial cells, one hypothesis was also raised that the nature of the selective sensitivity of SNcv dopamine neurons to adverse conditions could involve a distinct relationship among these cells, their environment and glial cells (Duke et al., 2007).

REPERCUSSION OF EFA DEFICIENCY OR SUPPLEMENTATION ON MIDBRAIN DOPAMINERGIC SYSTEMS Several studies have indicated that serotonergic, mesostriatal and mesocorticolimbic dopamine systems can be particularly affected when brain DHA availability is reduced (review in Chalon, 2006). Using an animal model deficient in ALA over three generations, Delion et al. (1994) demonstrated that DHA deficiency was able to induce a significantly higher serotonergic 5-HT2 receptor density and a reduction in dopamine specific binding to receptor D2, associated with lower endogenous dopamine level in the frontal cortex of adult rats. However, in this study, none of these modifications were seen in the striatum. Deficiency of ALA for three generations also impaired dopamine vesicular release in the NAc and frontal cortex (Zimmer et al., 1998) and reduced the density of synaptic vesicles containing dopamine in this cortex (Zimmer et al., 2000a). In contrast, increased basal dopamine release and density of D2 dopamine receptors were observed in the NAc (Zimmer et al., 2000b). The vesicular monoamine transporter 2 (VMAT2) and D2 dopamine receptor mRNA levels were differentially altered in the dopamine mesolimbic and mesocortical systems of omega-3 deficient rats (Zimmer et al., 2002). In piglets, dietary EFA restriction for only 18 days from birth was also able to induce lower levels of dopamine, serotonin and norepinephrine in the frontal cortex (de la Presa Owens and Innis, 1999) indicating the sensitivity of these systems to a very short dietary treatment.

Studies on the reversibility of omega-3 LCPUFA deficiency-induced changes in dopaminergic neurotransmission demonstrated that even after restitution of an adequate diet after weaning, the stimulated release of dopamine in the NAc and frontal cortex, and the VMAT2 binding sites in the NAc did not recover completely (Kodas et al., 2002). Rats fed for 21 months with trans isomers of ALA showed reduced levels of endogenous dopamine in the frontal cortex, striatum and hippocampus. However, subsequent dietary supplementation with cis ALA was able to recover the dopamine concentration only in the frontal cortex (Acar et al., 2003). Other studies showed that, even when rats were fed from conception on a diet that produces a relatively modest decrease in brain DHA content (B20%), modifications in adult behavior indicative of dopaminergic dysfunction, such as basal and amphetamine-stimulated locomotor activity were found. Nonetheless, changes in catalepsy induced by haloperidol were reversed by supplementation of the diet at weaning (Levant et al., 2004). An extensive analysis using microarray, Western blot and immunohistochemistry was carried out by Kuperstein et al. (2005) in several brain regions of 2 week-old pups whose mothers were fed an α-linolenic deficient diet from conception. According to this study, a widespread increase in D1 and D2 dopamine receptors throughout the brain was found, including the substantia nigra and striatum, as well as several nuclei of the mesocorticolimbic system, such as VTA, NAc, amygdala, hippocampus, septum, thalamus, and frontal cortex. Taken together, these findings have been discussed as potential modifications related to a behavioral hypersensitivity caused by impairment in DA production, consistent with the large spectrum of behavioral and cognitive modifications observed in omega-3 deficient animals (Reisbick and Neuringer, 1997; Wainwright et al., 1997; Carlson and Neuringer, 1999; Wainwright, 2002; Fedorova and Salem, 2006). In line with the hypothesis of a deficient DA production, Ahmad et al. (2008), associating successive parity and ALA dietary restriction, reported fewer (B33%) TH-immunoreactive neurons in the substantia nigra pars compacta and VTA of omega-3 deficient adult animals as compared to animals fed an adequate diet. Moreover, a time-dependent decline was reported in TH mRNA and protein levels in the midbrain of lactating pups subjected to perinatal ALA deficiency (Kuperstein et al., 2008). The same authors also showed the time course of increasing dopamine receptor D1 and D2 mRNA expression in cerebral cortex and striatum, and D2 protein expression in the substantia nigra, septum, hippocampus, frontal cortex, amygdala and cerebellum, during the lactation period. In these same regions, no modification was detected in

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the expression of dopamine plasma membrane transport (DAT), while a time-dependent decline was observed in the VMAT-2 protein and transcript levels in several brain regions related to midbrain dopamine system (Kuperstein et al., 2008). Recently, Passos et al. (2012) investigated whether two functionally distinct dopamine cell populations of substantia nigra could be differentially affected by EFA dietary restriction over two generations. Considering their neurochemical profile and the distinct susceptibility to degeneration described above, it was hypothesized that dopaminergic cells located in the SNcv could be more vulnerable than those of SNrm to deleterious effects of an omega-3 deficiency. Using stereological assessment, the results did not corroborate the hypothesis, but paradoxically demonstrated for the first time a higher vulnerability of SNrm to the harmful effects induced by DHA depletion (B50%) in the midbrain of young animals. Although EFA dietary restriction affected the dopamine cell growth, assessed by body cell size in both SNrm and SNcv, significantly fewer TH-immunoreactive neurons (B20%) were found only in the SNrm compared to control conditions, consistent with reduced TH protein expression levels in the midbrain (Passos et al., 2012).

POTENTIAL MECHANISMS INVOLVED IN SUBSTANTIA NIGRA DOPAMINE CELL LOSS INDUCED BY EFA DIETARY RESTRICTION The mechanisms involved in the substantia nigra dopamine cell loss induced by EFA dietary restrictions are not yet completely understood, but it seems that during brain development such deficiency could impair multiple homeostatic mechanisms that usually confer resistance to the dorsal tier of substantia nigra and even to the VTA, modifying the degeneration profile of midbrain dopaminergic cells. Accumulating evidence has pointed to oxidative stress in the demise of dopamine cells as a relevant factor involved in the etiology and evolution of Parkinson’s disease as well as in other neurodegenerative disorders (Thomas and Beal, 2007; Hashimoto and Hossain, 2011; Melo et al., 2011). Accordingly, in experimental models of Parkinson’s disease, it has been recently shown that the dietary supplementation of DHA may partially restore dopaminergic neurotransmission after 6-hydroxidopamine (6-OHDA)- or 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)-induced striatal lesions which produce oxidative stress (Bousquet et al., 2008; Cansev et al., 2008). Moreover, DHA supplementation was able to increase the SOD activity in the corpus striatum

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(Sarsilmaz et al., 2003) as well as significantly decreased cyclooxigenase-2 activity and prostaglandin E2 levels in the substantia nigra, attenuating MPTP-induced dopaminergic cell death (Ozsoy et al., 2011). Considering these previous studies, Cardoso et al. (2012) investigated whether EFA dietary restriction for one (F1) or two (F2) generations could induce lipoperoxidation or modify the antioxidant activity of SOD or CAT in the substantia nigra and striatum of rats. This study demonstrated that in first generation adult animals, this nutritional deficiency caused a 28% DHA depletion. Nevertheless, increased SOD enzymatic activity was observed in both regions, protecting them against lipoperoxidation. In young animals subjected to EFA deficiency for two generations, signals of degeneration in dopaminergic and non-dopaminergic neurons were associated with a significant increase in lipoperoxidation and decreases in the CAT enzymatic activity were detected in the substantia nigra. In contrast, a strong resilience of the striatum to this oxidative insult was observed, in spite of a similar level of DHA depletion (B50%) in both regions. The results obtained by Cardoso et al. (2012) corroborated the hypothesis that oxidative stress in the substantia nigra could be one of the potential mechanisms involved in the dopamine cell loss induced by DHA deficiency (Ahmad et al., 2008; Passos et al., 2012). Moreover, they highlight the importance of DHA in maintaining the redox balance in the substantia nigra, reinforcing the protective action of DHA dietary supplementation on substantia nigra cell populations under oxidative stress conditions (Ozsoy et al., 2011). As previously described in this chapter, studies on rat or human substantia nigra have indicated a progressive decrease in the activity of several antioxidant enzymes including SOD and CAT during physiological brain aging (Kolosova et al., 2003; Venkateshappa et al., 2012). Thus, higher levels of oxidative stress induced by DHA deficiency in the substantia nigra of young animals may accelerate the degenerative profile of this nucleus increasing the risk of dopamine-related diseases such as Parkinson’s disease. Nevertheless, evidence from our laboratory has shown that the presence of oxidative stress depends on the magnitude of DHA depletion in the midbrain. Recent data from our group in F1 adult animals subjected to EFA dietary restriction from conception demonstrated that 28% DHA depletion in the midbrain is enough to induce dopamine cell loss in the SNrm, even in the absence of lipoperoxidation, previously demonstrated by Cardoso et al. (2012). Figure 20.1A shows results reported by Cardoso et al. (2012) demonstrating the absence of lipoperoxidation in F1 adult animals fed an EFA-deficient diet. Figure 20.1B shows representative images of brain parasaggital sections through substantia nigra in F1 control and

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TBARS (nmol MDA/mg prot.)

(A)

(B) c

b

d

1.5

1.0

#

#

C EF1 C + SNP

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0.0 SN

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2.0 SOD activity (U/mg prot.)

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(C) Number TH+ neurons 350 SNrm

300

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0.5

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250 200

0.0 SN

CS

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150 100 50 0

F1

F2

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FIGURE 20.1 Effect of EFA dietary restriction for one or two generations on oxidative stress markers in the nigrostriatal system and TH-immunoreactivity in the substantia nigra. A: Thiobarbituric acid-reactant substances (TBARS) levels (A) and total superoxide dismutase (t-SOD) activities (B) in the pool of substantia nigra and corpus striatum from first generation adult rats fed essential fatty acid-restricted diet and controls (n 5 12 per group). P , 0.05 compared to control group. Treatment of control homogenates with sodium nitroprusside (SNP) was used as a positive control in all the experiments. # P , 0.001 compared to control or EF1groups. Source: Cardoso et al. (2012). B: Representative photomicrographs of TH-immunoreactive parasagittal sections at the mid-level of substantia nigra from first generation adult rats fed control (C) or EFA-deficient diet (EF). Images of (A) and (B) show dopaminergic cells in the SNrm and SNcv from control group; (C) and (D) from EF1 group (bar 5 30 μm). C: Average number of TH positive neurons in the SNrm and SNcv of F1 adult and F2 young control (C) and EFA-deficient groups. Data were obtained in one parasagittal section at the mid-level of the substantia nigra and express mean 6 SD. p , 0.05 compared to control group.

experimental animals. Figure 20.1C compares the number of TH-immunoreactive cells, at the mid-level of substantia nigra, between control and omega-3 deficient animals in F1 adult animals and F2 young animals in the rostro-dorso-medial (SNrm) and caudo-ventrolateral (SNcv) regions. This bidimensional analysis in F2 young animals shows that the magnitude of SNrm dopamine cell loss (B20%) is similar to results previously reported by Passos et al. (2012) using stereological assessment. The apparent striatum resilience to oxidative insult in F2 young animals subjected to EFA deficiency (Cardoso et al., 2012) is also occurring in the presence of other reactive processes in this nucleus. We have examined stereotyped behaviors such as licking, head bobbing, turning and jumping effects induced by acute

administration of the dopaminergic agonist apomorphine (1 mg/Kg, i.p.) in F2 young animals (35
40 days; n 5 11 per group). Figure 20.2 shows the increased degree of apomorphine-induced stereotypy in the experimental (EF2Y) group when compared to controls (C). Such behavioral analysis reinforces previous evidence that the expression of dopamine receptors in the nigrostriatal system can be modified by EFA dietary restriction. It is worth noting that a relatively short-term feeding of an ALA-restricted diet was able to induce lower brain-derived neurotrophic factor (BDNF) levels in the mouse striatum (Miyazawa et al., 2010) and cerebral cortex (Rao et al., 2007). This sensitivity of striatum to changes in BDNF levels as a function of DHA concentration was also observed in other studies. Dietary

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50

Stereotypy degree

40 30 20 10 0 Control

EF2Y

FIGURE 20.2 Effect of EFA dietary restriction over two generations on apomorphine-induced stereotypy. Apomorphine-induced stereotypy in young rats (30
45 days) fed the control (C) and EFAdeficient diet (EF2Y). Stereotyped behaviors such as licking, head bobbing, turning and jumping were measured following apomorphine (1 mg/kg, i.p.).  p 5 0.043, Wilcoxon matched-pairs signed rank test.

DHA supplementation led to a strong reaction of the striatum under conditions of MPTP-induced oxidative stress, increasing BDNF content more than under control conditions (Bousquet et al., 2009). A stimulatory effect of DHA on BDNF expression has also been reported for other regions such as hippocampus, cerebral cortex and spinal cord (Vines et al., 2012; Ying et al., 2012) indicating a widespread effect that can occur in neurons with different neurochemical profiles. BDNF is a potent dopaminergic neurotrophin produced in the substantia nigra (Hyman et al., 1991; Stahl et al., 2011) and transported to the striatum (Altar and Distefano, 1998). It is also a direct target gene of the transcription factor Nurr1 (Volpicelli et al., 2007) which is involved in the genesis, development, and function of dopaminergic cells (Jankovic et al., 2005). Another point to be considered is the fact that DHA supplementation can reduce the loss of the transcription factor Nurr1 in the substantia nigra under conditions of MPTP-mediated oxidative stress (Bousquet et al., 2009). Early and recent studies support the hypothesis that reduced BDNF levels can be a potential mechanism involved in the DHA depletion-induced dopamine cell loss in the nigrostriatal system. Intrathecal infusion of BDNF reduced the loss of dopamine neurons and the severity of Parkinson’s disease in MPTP treated monkeys (Tsukahara et al., 1995). Patients with Parkinson’s disease showed reduced BDNF mRNA expression in the substantia nigra (Howells et al., 2000) and BDNF protein levels in the substantia nigra and striatum (Mogi et al., 1999). Furthermore, chronic deprivation of TrkB signaling led to selective late onset of nigrostriatal dopaminergic degeneration (Baydyuk et al., 2011)

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It was recently reported that there is a modulatory effect of DHA on other neurotrophins with action in the nigrostriatal system. DHA supplementation was able to increase GDNF and neurturin levels in the substantia nigra, reducing MPTP-induced dopaminergic cell death (Tanriover et al., 2010). As shown by Barroso-Chinea et al. (2005), most dopaminergic neurons of SNrm and VTA, but not those of SNcv, contain GDNF retrogradely transported from the striatum. Thus, the GDNF reduction caused by DHA depletion cannot be disregarded as a potential mechanism involved in the differential vulnerability of the SNrm dopamine cells previously reported (Passos et al., 2012). Future studies should be carried out in order to address this issue. Figure 20.3 shows several effects induced by EFA dietary restriction on midbrain dopamine systems. A modulatory effect of omega-3 LCPUFA upon neuroinflammation in the nigrostriatal system has recently been demonstrated. In the striatum of suckling pups subjected to perinatal ALA deficiency, microglia activation was reported by Kuperstein et al. (2008). Conversely, elevated dietary omega-3 LCPUFA levels were protective against dopaminergic damage associated with neuroinflammation in experimental models of Parkinson’s disease. For example, dietary supplementation with ethyl-EPA was able to protect mice against MPTP-induced hypokinesia and other behavioral deficits, and prevented the increase in TNF-α and interleukins (Luchtman et al., 2012). Another recent study showed that rats fed a 15% fish oil diet for 2 weeks prior to injection of lipopolysaccharide in the substantia nigra were protected against dopaminergic cell loss, microglia activation, TNF-α and interleukin 1 expression (Ji et al., 2012). Although these are promising results, the mechanisms involved in such effects still deserve future investigation. Glutamate excitotoxicity has also been implicated in the vulnerability of substantia nigra dopamine cells (reviewed in Gonza´lez-Herna´ndez et al., 2010). Under physiological conditions, LCPUFA can exert multiple effects on the glutamatergic system of the cerebral cortex or hippocampus: some of them favor hyperexcitability (Miller et al., 1992; Nishikawa et al., 1994) while others can decrease synaptic glutamate transmission and increase neuroprotection (Vreugdenhil et al., 1996; Lauritzen et al., 2000). In the substantia nigra, experimental evidence has indicated that DHA and AA can play an important role in the modulation of neuronal excitability by reducing GABA and glycine response, and potentializing glutamatergic transmission via NMDA receptors (Hamano et al., 1996). The substantia nigra receives glutamatergic afferent neurons from the pedunculopontine tegmental nucleus (PPTg) and subthalamic nucleus (Forster and Blaha, 2003). Stimulation of PPTg glutamatergic neurons with

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FIGURE 20.3 Schematic drawing illustrating neurochemical effects induced by EFA dietary restriction on midbrain dopamine systems. All data were obtained from literature cited in this chapter. [DA] 5 dopamine concentration; [D2] 5 dopamine receptor type 2 expression; [D1] 5 dopamine receptor type 1 expression [VMAT2] 5 vesicular monoamine transporter 2 expression; CAT 5 catalase enzymatic activity; SOD 5 superoxide dismutase enzymatic activity; LP 5 lipoperoxidation; BDNF 5 brain-derived neurotrophic factor expression.

kainic acid was able to induce neurodegeneration in substantia nigra dopamine cells (Gonzalez-Hernandez et al., 1997). Although some experimental data indicate that glutamate might contribute to substantia nigra dopamine cell degeneration, the impact of omega-3 dietary deficiency or supplementation on this glutamatergic action into the substantia nigra is not yet completely understood. However, the repercussion of such deficiency during aging in the hippocampus has recently been investigated (Latour et al., 2013). According to these authors, reduced levels of DHA were able to worsen the age-induced degradation of glutamatergic transmission, modifying its astroglial regulation and glial cell proliferation. Under some pathological conditions, increased levels of AA and DHA are synthesized and released from astrocytes, exerting modulatory actions on neuronal excitability and protection (Yoshida et al., 1980; Siesjo¨ et al., 1982). A number of studies have demonstrated the importance of DHA on astrocyte differentiation and viability (Champeil-Potokar et al., 2004; Joardar and Das, 2007), distribution of connexin-43 gap junctions (Champeil-Potokar et al.,

2006) and glutamatergic activity involving astrocytes (Berry et al., 2005; Grintal et al, 2009) in the cerebral cortex. Therefore, it remains to be shown how neuron
astrocyte cross-talk can be modulated by DHA deficiency or supplementation in the nigrostriatal system, considering the importance of astrocytes to this system (Shao et al., 2013). In conclusion, current experimental data reinforces the idea that adequate EFA levels can act as important players for the tonic activity of midbrain dopamine systems. Providing adequate molecular signaling, these fatty acids seem to be necessary to modulate key functions such as dopamine metabolism, release and uptake; receptor affinity; redox balance; anti-inflammatory response; and neurotrophin synthesis. Taken together, these actions may improve the competence of this neuronal system to maintain a suitable resilience during development and brain maturation. Such experimental studies seem to be in agreement with prospective studies in humans that have positively associated a dietary intake of omega-3 PUFA with a lower risk of developing Parkinson’s disease (Chen et al., 2003; de Lau et al., 2005).

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REFERENCES

Acknowledgments The authors are grateful to the Brazilian National Research Council (CNPq); CAPES (PROCAD # 0008052/2006 and PROCAD NF-2009); FACEPE (APQ 0036-2.07/11). We are also grateful to FACEPE which provided scholarships for Henriqueta Dias Cardoso, Eraldo Fonseca dos Santos Junior and Catarina Gonc¸alves-Pimentel (DCR 0079-2.07/10).

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262. Yin, H.H., Knowlton, B.J., 2006. The role of the basal ganglia in habit formation. Nat. Rev. Neurosci. 7, 464
476. Ying, Z., Feng, C., Agrawal, R., Zhuang, Y., Gomez-Pinilla, F., 2012. Dietary omega-3 deficiency from gestation increases spinal cord vulnerability to traumatic brain injury-induced damage. PLOS ONE.

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Yoshida, S., Inoh, S., Asano, T., Sano, K., Kubota, M., Shimazaki, H., et al., 1980. Effect of transient ischemia on free fatty acids and phospholipids in the gerbil brain. Lipid peroxidation as a possible cause of postischemic injury. J. Neurosurg. 53, 323
331. Zhang, W., Li, P., Hu, X., Zhang, F., Chen, J., Gao, Y., 2011. Omega-3 polyunsaturated fatty acids in the brain: metabolism and neuroprotection. Front. Biosci. 17, 2653
2670. Zimmer, L., Hembert, S., Durand, G., Breton, P., Guilloteau, D., Besnard, J.C., et al., 1998. Chronic n-3 polyunsaturated fatty acid diet-deficiency acts on dopamine metabolism in the rat frontal cortex: a microdialysis study. Neurosci. Lett. 240, 177
181. Zimmer, L., Delpal, S., Guilloteau, D., Aı¨oun, J., Durand, G., Chalon, S., 2000a. Chronic n-3 polyunsaturated fatty acid deficiency alters dopamine vesicle density in the rat frontal cortex. Neurosci. Lett. 284, 25
28. Zimmer, L., Vacanssel, S.D., Durand, G., Guilloteau, D., Bodard, S., Besnard, J.C., et al., 2000b. Modification of dopamine neurotransmission in the nucleus accumbens of rats deficient in n-3-polyunsaturated fatty acids. J. Lipid. Res. 41, 32
40. Zimmer, L., Vancassel, S., Cantagrel, S., Breton, P., Delamanche, S., Guilloteau, D., et al., 2002. The dopamine mesocorticolimbic pathway is affected by deficiency in n-3 polyunsaturated fatty acids. Am. J. Clin. Nutr. 75, 662
667.

Further Reading Barzilai, A., Melamed, E., Shirvan, A., 2001. Is there a rationale for neuroprotection against dopamine toxicity in Parkinson’s disease? Cell. Mol. Neurobiol. 21, 215
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21 The Role of Omega-3 Fatty Acids in Hippocampal Neurogenesis Simon C. Dyall INTRODUCTION Neurogenesis is the process of generation of new neurons from neuronal precursor cells, and was first described in adult mammals in 1965, where it was identified in rodents (Altman and Das, 1965). Adult neurogenesis has subsequently been shown to occur in two specific regions of the adult brain, the subventricular zone of the olfactory bulb and the subgranular layer of the hippocampal dentate gyrus, where it has been identified in all mammals studied to date, including man (Ehninger and Kempermann, 2008). Adult neurogenesis involves several distinct stages, beginning with the proliferation of resident neural stem and progenitor cells, followed by differentiation, migration, selection, and ultimately functional integration into the pre-existing circuitry (Ehninger and Kempermann, 2008). The rate of turnover of cells is very low. In the mouse there is approximately one new cell born per 200 cells in the granule cell layer per day (Kempermann et al., 1997), and in the macaque monkey it is one cell per 24,000 per day (Kornack and Rakic, 1999). The rate of neurogenesis and survival of new neurons in adults are enhanced by many factors such as estrogen (Tanapat et al., 1999), growth factors and neurotransmitters (Abrous et al., 2005), living in an enriched environment (Kempermann et al., 1998), voluntary exercise (van Praag et al., 1999) and antidepressant treatment (Malberg et al., 2000). Furthermore, associative learning tasks involving the hippocampus also increase neurogenesis (Gould et al., 1999), indicating a potential link between learning and adult neurogenesis. Other evidence also links neurogenesis with learning and memory. For example, new neurons display distinct electrophysiological properties and longterm potentiation (LTP), an experimental model of memory, can be induced more easily (Schmidt-Hieber Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00021-1

et al., 2004) and ablation of neurogenesis induces learning and memory impairments (Shors et al., 2001, 2002). However, it must be acknowledged that the methods used to inhibit neurogenesis in these studies also interfere with other cellular events. Overall, current evidence strongly indicates that neurogenesis plays an important role in learning, memory, and neural plasticity (Yirmiya and Goshen, 2011). The greatest negative regulator of neurogenesis is aging, where the rate of neurogenesis in the dentate gyrus declines significantly with age (Kuhn et al., 1996), and there is a strong correlation between memory impairment in hippocampal-dependent tasks, such as those based on spatial memory, and this age-related decline (Drapeau et al., 2003), suggesting a link between decreased neurogenesis and memory impairments. Although the age-related reduction in neurogenesis has been shown consistently, there is limited evidence of the effects of aging on other aspects, such as survival and migration. Rao and colleagues have shown that the reduction in new granule cells is due to reduced production of new cells rather than impairments in cell survival (Rao et al., 2005). Furthermore, they show that the aged dentate gyrus is able to support the survival of newly differentiated granule cells for up to five months, highlighting the importance and potential relevance of therapies that positively influence age-related deficits in neurogenesis. Interestingly, neurogenesis has been shown to be increased in the hippocampus of both patients with Alzheimer’s disease (Jin et al., 2004b), and in an animal model of Alzheimer’s disease, the transgenic (PDGFAPP Sw,Ind) mouse (Jin et al., 2004a). These mice express the Swedish and Indiana amyloid precursor protein mutations, and show an approximate two-fold increase in the number of BrdU-labeled cells as early as 3 months, which is prior to neuronal loss and

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amyloid deposition. BrdU (5’-bromo-2’-deoxyuridine) is a thymidine analog and neurogenic marker, and is discussed below. Brains from patients with Alzheimer’s disease show increased expression of the neurogenic immunological markers, doublecortin, PSA-NCAM, neurogenic differentiation factor, and TUC-4 (turned on after division/Ulip-1/CRMP-4, a protein expressed early in neuronal differentiation in the rat) compared to age-matched controls (Jin et al., 2004b). Expression of doublecortin and TUC-4 was associated with neurons in the neuroproliferative (subgranular) zone of the dentate gyrus. The physiological destination of these neurons (the granule cell layer), and the CA1 field, is the principal site of hippocampal pathology in Alzheimer’s disease. The findings of increased neurogenesis in the Alzheimer’s disease hippocampus may indicate an attempt at brain self-repair. This is consistent with similarly increased neurogenesis following certain acute neurological disorders, such as after ischemia (Liu et al., 1998), stroke (Darsalia et al., 2005), and seizures (Parent et al., 1997). However, increased neurogenesis has not consistently been shown in Alzheimer’s disease, and further studies are needed at different stages of the disease to establish the role of neurogenesis in Alzheimer’s disease (Mu and Gage, 2011) Overall, adult hippocampal neurogenesis has been shown to be important in learning and memory in a variety of paradigms, linking neurogenesis to both normal brain function and disease. This raises the intriguing possibility that treatments that enhance the rate of neurogenesis and survival of new neurons in adults may have a potential therapeutic role in the treatment of neurodegenerative and psychiatric disorders. This review provides an overview of the effects of omega-3 polyunsaturated fatty acids (PUFAs) in neurogenesis, particularly adult neurogenesis in the dentate gyrus region of the hippocampus, and explores some of the potential mechanisms of action which may underlie the observed effects.

Adult Neurogenesis Space limitations restrict a detailed review of adult hippocampal neurogenesis, and the interested reader is directed to some of the many excellent reviews on the topic, such as (Ehninger and Kempermann, 2008; Mu et al., 2010). Throughout adult life, granule neurons are generated from progenitor cells in the subgranular zone of the hippocampal dentate gyrus. Distinct proliferative cell types have been classified based on their morphology and expression of immunohistochemical markers (Mu et al., 2010). Adult hippocampal neurogenesis originates from type 1 precursor cells

resembling radial glial cells, both morphologically and functionally, and expressing the astrocytic marker, glial fibrillary acidic protein (GFAP), and intermediate filament, nestin. They have passive membrane properties, consistent with astrocytes, and give rise to fast proliferating intermediate type 2 and type 3 precursor cells. Type 2 cells are non-radial, and express the neuronal markers doublecortin (DCX) and polysialylated nerve cell adhesion molecule (PSA-NCAM), but not GFAP. Type 2a cells still express nestin, whereas type 2b cells express the neuronal markers, NeuroD and Prox1, and therefore show the first signs of neuronal differentiation. Type 3 cells are the transition to postmitotic immature neurons, and express DCX, PSANCAM, NeuroD and Prox1, but not nestin. The type 3 cell stage is where exit from the cell cycle typically occurs. Young adult rats generate around 9000 new neurons per day, although the survival rate is only around 50% (Cameron and McKay, 2001). The surviving cells integrate into the existing neuronal circuitry by extending dendrites to the molecular layer of the dentate gyrus and axonal projections to the CA3 region (van Praag et al., 2002).

MEASUREMENT OF NEUROGENESIS (MARKERS OF PROLIFERATION) Immunological markers of proliferation are normally thymidine analogs, which are incorporated into DNA of dividing cells in the S-phase of mitosis and their progeny are permanently labeled. Traditionally, tritiated thymidine was used, which could be measured by autoradiography (Altman and Das, 1965). This method has now been replaced and the most widely used current technique is based on the administration of 5’-bromo-2’-deoxyuridine (BrdU), also a thymidine analog. Newly-injected BrdU is available for a few hours for incorporation. This replacement of autoradiographic assays by immunological quantification allows for double or triple labeling with cell-specific markers and analysis by confocal microscopy (Kuhn et al., 1996). However, there are controversies with the BrdU technique, as subsequent cell divisions can dilute the nuclear BrdU content, and there has been debate over the optimum dose sufficient to assess neurogenesis, as BrdU is toxic at high doses and the use of lower doses can lead to inconsistent cellular labeling (Cameron and McKay, 2001; Gould and Gross, 2002). The standard dose used in rodent studies is 50
100 mg/kg body weight. However, it has been shown that a dose of 300 mg/kg BrdU may be needed to fully detect neurogenesis in adults. Doses of 50 or 100 mg/kg may label only 45
60%, respectively, of the neurogenesis events that can be detected by injection of 300 mg/kg BrdU

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(Cameron and McKay, 2001), suggesting that studies at the lower dose may have substantially underestimated neurogenesis. Although other studies indicate that doses of 50
100 mg/kg BrdU are sufficient to label the vast majority of S-phase proliferative populations (Burns and Kuan, 2005). For these reasons, DCX has been suggested as a potential primary neurogenic marker (CouillardDespres et al., 2005). DCX is a phosphoprotein expressed in migrating neuronal precursor cells in the developing central nervous system (CNS). Expression of DCX overlaps with PSA-NCAM, spanning the type 3 cell stage, exit from cell cycle, and the initial postmitotic neuronal development (Couillard-Despres et al., 2005; Rao and Shetty, 2004). As a marker of neurogenesis, DCX has the important advantage that it does not require prior in vivo labeling of proliferating cells. Importantly, only a small number of the proliferating progenitor cells are selected for long- term survival, as most of the new cells are eliminated by apoptosis after exit from cell cycle. Consequently, measurement of proliferation of precursor cells may not be a good predictor of overall neurogenesis, and ultimately, it may be most informative to use a variety of immunological markers in the assessment of neurogenesis.

Omega-3 PUFAs and Hippocampal Neurogenesis Omega-3 PUFAs have an essential role in brain development and function (Innis, 2007) and there is increasing evidence that increased intake of the longchain omega-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), may confer benefits in a variety of neurological and neurodegenerative conditions (Dyall and Michael-Titus, 2008). Importantly, the beneficial effects of omega-3 PUFA treatment have consistently been demonstrated in a variety of hippocampal-dependent tasks. For example, omega-3 PUFAs enhance spatial memory tasks in adult and old rats (Gamoh et al., 1999, 2001), enhance hippocampal LTP (Fujita et al., 2001; McGahon et al., 1999), and DHA deficiency impairs spatial learning (Greiner et al., 1999). They also possess antidepressant effects (Freeman, 2009), increase synaptogenesis (Cao et al., 2009), and enhance hippocampal neurite outgrowth (Calderon and Kim, 2004). However, the mechanisms underlying these effects are still unclear.

DEVELOPMENTAL NEUROGENESIS An adequate supply of the long-chain omega-3 PUFA, DHA, during the pre- and postnatal period is

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important for normal brain growth and development, and low DHA status results in impairments in cognitive and behavioral performance in infants (see Innis, 2007). During pre- and postnatal development, DHA rapidly accrues in brain gray matter membrane phospholipids, particularly phosphatidylethanolamine and phosphatidylserine (Martinez, 1992), and the prenatal DHA must be derived from placental transfer from the mother, either from preformed DHA or synthesized from α-linoleic acid (ALA). In animal models, maternal ALA deprivation before and during gestation has consistently been shown to decrease the DHA content of brain phospholipids in the offspring, and leads to impaired behavior and learning abilities, decreases in sensory and motor activities, and impaired vision (Innis, 2007), and dopaminergic, serotonergic, and cholinergic neurotransmitter production (Dyall and Michael-Titus, 2008). Importantly, maternal dietary ALA deprivation also negatively affects brain development. For example, following maternal ALA deprivation, neuron size is diminished in the hippocampus, hypothalamus, and parietal cortex (Ahmad et al., 2002) and there is reduced dendritic arborization complexity in omega-3 PUFA deficient rodents compared to controls (Wainwright et al., 1998). Whereas, DHA enrichment enhances neurite outgrowth in hippocampal (Calderon and Kim, 2004) and sensory (Robson et al., 2010) neurons in culture, and DHA supplementation increases dendritic spine density in the weanling rat (Cansev et al., 2009). Omega-3 PUFA deficiency has been shown to lead to decreases in neurogenesis in embryonic and newborn rats. For example, maternal ALA deficiency beginning two weeks prior to gestation results in altered neurogenesis in the embryonic rat brain (Coti Bertrand et al., 2006). In this study there were significant decreases in the mean thickness of the cortical plate and mean cross sectional area of the primordial dentate gyrus, and significant increases in the mean thickness of the cortical ventricular zone and primary gyrus dentate neuroepithelium in the ALA-deficient embryonic brains compared to controls, suggesting an inhibition or delay in neurogenesis. Furthermore, maternal ALA deprivation also decreases newborn neuronal cell migration in several regions such as cortical layers IV
VI, corpus collosum, the subventricular zone, and CA1 and dentate gyrus regions of the hippocampus (Yavin et al., 2009). A recent study looked at the effects of ALA deficiency and postnatal supplementation on cell proliferation, early neuronal differentiation and apoptosis in the hippocampal dentate gyrus (Niculescu et al., 2011). Control (Con) and ALA-deficient (Def) diets were fed to mice for 30 days prior, and during, gestation. Then, one day prior to delivery date, these groups were split

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into subgroups, either remaining on their original diet (Con-Con and Def-Def) or receiving an ALAsupplemented diet (Con-Sup and Def-Sup) until postnatal day 19. In this way the effects of ALA deficiency during gestation and subsequent postnatal ALA supplementation could be studied. Mitosis was assessed using phosphorylated histone H3 (pH3), a marker specific for the M-phase, and was significantly increased only in the control ALA-supplemented group. Similarly, early neuronal differentiation, assessed by calretinin levels, was also significantly increased only in this group. Apoptosis assessment of activated caspase-3 positive cells indicated increased apoptosis in the deficient-deficient compared to all other groups. Overall, this study showed that postnatal ALA supplementation enhances neurogenesis in the dentate gyrus of the offspring at postnatal day 19, but the beneficial effects are offset by maternal ALA deficiency during gestation, suggesting omega-3 PUFAs are required in both fetal and postnatal stages of brain development. In summary, omega-3 PUFA deficiency negatively influences developmental neurogenesis and delays or inhibits normal development, and is ultimately implicated in impaired functioning in the developing and adult brain. Interestingly, these alterations in neurogenesis have been demonstrated in the dentate gyrus region of the hippocampus, one of two regions capable of producing new neurons in the adult. The remainder of the chapter reviews the role of omega-3 PUFAs in adult neurogenesis.

ADULT HIPPOCAMPAL NEUROGENESIS The first published evidence of omega-3 PUFAs enhancing adult hippocampal neurogenesis was provided by Kawakita and co-workers (Kawakita et al., 2006). In this study, 18 month old rats were fed DHA at 300 mg/kg for 7 weeks. DHA treatment significantly increased the number of BrdU-positive and NeuNpositive newborn neurons in the dentate gyrus, indicating enhanced neuronal proliferation and maturation. In the second part of their study, neural stem cells were cultured under differential conditions with or without DHA (10 μM) for 4 and 7 days. DHA significantly increased the number of Tuj1-positive neurons compared with the control groups on both culture days, and the newborn neurons in the DHA group were morphologically more mature than in the control group. DHA also significantly decreased the incorporation ratio of BrdU during the first 24 hour period; it also significantly decreased the number of pyknotic (degenerating) cells on day 7, indicating that DHA promotes the differentiation of neural stem cells into neurons by promoting cell cycle exit and suppressing cell death.

The fat-1 transgenic mouse expresses the fat-1 gene from Caenorhabditis elegans, which encodes an omega-3 desaturase, and is therefore able to convert omega-6 to omega-3 PUFAs. It is thought to model the effects of dietary enrichment with omega-3 PUFAs (Kang et al., 2004). Fat-1 mice have increased brain DHA content compared to wild type litter mates. Importantly, they also have significantly enhanced hippocampal neurogenesis, with an increased number of proliferating neurons and neuritogenesis, as evidenced by increased density of dendritic spines of CA1 pyramidal neurons in the hippocampus (He et al., 2009). They also exhibit a better spatial learning performance in the Morris water maze compared with wild type litter mates, suggesting that the positive effects on neurogenesis by omega-3 PUFAs may contribute to improved cognitive performance. Positive effects of omega-3 PUFA treatment have also been reported in the lobster (Homarus americanus) brain, a model of adult neurogenesis, where short-term omega3 PUFA dietary enrichment significantly upregulates neurogenesis (Beltz et al., 2007). Lobsters were fed one of three diets differing in omega-3:omega-6 PUFA ratios (1:1, 2:1, and 2:1) and EPA and ALA levels, for 25 days. None of the diets had detectable DHA. It should be noted that the diets did not replicate the natural diet of young lobsters (rich in EPA and DHA), but rather investigated the influence of altering the omega-3 PUFA content on neurogenesis. In the projection neuron cluster, the omega-3 PUFA-enriched diets significantly increased the number of S-phase cells. These data show that 25 days of dietary omega-3 PUFA supplementation significantly increased the number of new neurons. Although there was a correlation between neurogenesis and both dietary omega-3:omega-6 PUFA ratio and ALA levels, the changes in cell proliferation did not correspond to the amount of EPA in the diet, suggesting the effects were as a result of an elevated omega-3:omega-6 PUFA ratio and increased ALA content of the diet. It should, however, be noted that in addition to omega-3 PUFA, the experimental diets also included additional components, such as the antioxidant astaxanthin, which may have further complicated the observations. Taken together, these observations strongly support the enhancing effects of omega-3 PUFAs on adult neurogenesis and neuritogenesis, and also suggest that this effect may be a potential mechanism underlying the beneficial effects observed on hippocampaldependent functions. A number of putative targets have begun to be suggested for the positive effects of omega-3 PUFAs on neurogenesis.

Mechanisms of Action Adult neurogenesis occurs in a complex microenvironment and the progression from neural stem cells to

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mature neurons is subject to tightly coordinated control by a multitude of cell- extrinsic and intrinsic factors (Johnson et al., 2009; Mu et al., 2010). Extrinsic factors which have previously been shown to be modifiable by omega-3 PUFA treatment include glutamatergic signaling (Dyall et al., 2007) and neurotrophic factors (Venna et al., 2009), whereas intrinsic factors include a variety of transcription factors (Dyall et al., 2010). The effects of omega-3 PUFAs on these regulatory factors in adult hippocampal neurogenesis have begun to be explored. Brain-derived neurotrophic factor (BDNF), a neurotrophin involved in spatial learning and memory, plays an important role in the regulation of dietary restriction-induced neurogenesis (Lee et al., 2002). Several studies have reported enhanced hippocampal neurogenesis in parallel with increased levels of BDNF levels following omega-3 PUFA treatment (Cysneiros et al., 2010; Venna et al., 2009; Blondeau et al., 2009). For example, three sequential injections of ALA (500 nmol/kg) significantly enhanced adult hippocampal neurogenesis in mice, and increased the expression of BDNF in hippocampal neurons and cortical and hippocampal tissue (Blondeau et al., 2009). In this study, the treatment also significantly increased the expression of proteins critical for synaptogenesis and synapse function, synatophysin-1, VAMP-2, and SNAP-25, and proteins supporting glutamatergic neurotransmission, V-GLUT1 and V-GLUT2. These studies therefore indicate that the positive effects of omega-3 PUFAs on neurogenesis may at least in part be mediated via effects on BDNF expression. Since calorific or dietary restriction have been shown to closely relate to hippocampal neurogenesis (Lee et al., 2000), it is important to monitor the food intake and weight of the animals throughout omega-3 PUFA treatment. In our studies, omega-3 PUFA-treated animals ate a similar amount of food to the control and untreated old animals, and indeed there was even a small nonsignificant increase in the average weight of the omega-3 PUFA-treated animals compared to the other groups, indicating dietary restriction was not a factor in the observed effects on neurogenesis (Dyall et al., 2010). There is strong evidence indicating the importance of the retinoic acid receptor family of transcription factors in regulating neural plasticity and neurogenesis in the hippocampus (McCaffery et al., 2006; Jacobs et al., 2006). Retinoids are metabolites of vitamin A (alltrans-retinol), and the actions of retinoids are predominantly mediated through regulation of gene expression by binding to their receptors, which function as transcription factors. These receptors are the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) and belong to the steroid/thyroid hormone/vitamin D nuclear receptor superfamily. There are three subtypes

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of each receptor, -α, -β, and -γ, and each subtype of receptor can undergo differential splicing to produce multiple receptor isoforms, e.g. RARβ1, RARβ2, RARβ3, and RARβ4. The RARs function as heterodimers with RXRs, whereas RXRs form either homo- or heterodimers with a variety of other nuclear receptors, such as liver X receptors (LXRs), farnesol X activated receptors (FXRs), and peroxisome proliferatoractivated receptors (PPARs) (Moreno et al., 2004). The dimers bind to the response elements in the promoter region of their cognate gene. Two retinoic acid (RA) stereoisomers can bind and activate RARs, all-trans RA (ATRA) and 9-cis RA, whereas RXRs are only activated by 9-cis RA (Heyman et al., 1992; Levin et al., 1992). The physiological relevance of 9-cis RA remains questionable since endogenous levels are difficult to detect in vivo (Kurlandsky et al., 1995). However, there is now mounting evidence indicating that DHA is an endogenous agonist of RXRs. DHA was first identified as an agonist ligand of RXRα in mouse brain (de Urquiza et al., 2000). The initial results suggested a high dose was required for activation. The dose
response curves for the activation showed an EC50 (median effective concentration) of 50
100 μM, whereas more recent evidence using more sensitive assays indicates lower doses are required, with EC50 values of 5
10 μM (Lengqvist et al., 2004). In this study the authors also show that DHA synergistically activates RXR-RAR heterodimers in combination with ATRA, a property shared with 9-cis RA. Further evidence has been provided by the crystal structure of DHA complexed to the ligandbinding domain of RXRα (Egea et al., 2002). Upon DHA binding RXR forms homodimers and exhibits the active conformation previously shown when complexed with 9-cis RA, and indeed DHA has a higher number of ligand-binding contacts than 9-cis RA, although DHA has a lower binding affinity. The role of RA in embryonic development is well known. However, the continued expression of RARs and RXRs in the adult CNS indicates important functions in the adult brain (Moreno et al., 2004; Zetterstro¨m et al., 1999). Indeed, retinoid receptors are involved in several forms of hippocampal-dependent learning and memory, such as LTP and spatial memory. For example, impaired LTP and long-term depression (LTD) have been reported in mice lacking RARα or RARβ and RXRγ (Chiang et al., 1998). In adult mice on a vitamin A-deficient diet for 12 weeks, both LTP and LTD are reduced, and after 15 weeks LTD is abolished (Misner et al., 2001). Vitamin A supplementation reverses these deficits, indicating that synaptic plasticity can be regulated by retinoids in adult mice. Furthermore, concomitant with age-related deficits in memory are decreases in RAR and RXR mRNA levels,

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and RA treatment not only restores age-related deficits in RAR and RXR, but also partially alleviates the agerelated deficits in LTP (Enderlin et al., 1997a,b; Etchamendy et al., 2001). Depletion of RA in adult mice leads to significantly decreased neuronal differentiation within the granular cell layer of the dentate gyrus, and a decrease in newborn cells expressing an immature neuronal marker, suggesting that retinoid signaling is an early event in neurogenesis (Jacobs et al., 2006). The authors also used microarray analysis to identify the retinoidinduced genes, and identified metabolic targets including the lipid transporters, CD-36 and ABCA-1, SREBP1c, and components of the Wnt signaling pathway, which are a principal regulator of adult hippocampal neurogenesis and have a role in adult hippocampal function (Lie et al., 2005). Overexpression of Wnt3 is sufficient to increase hippocampal neurogenesis and blockade of Wnt signaling abolishes neurogenesis almost completely. Furthermore, another Wnt protein, Wnt7b, is involved specifically in dendrite development and increases dendritic branching in cultured hippocampal neurons (Rosso et al., 2005), and this dendritic arborization is required for proper neuronal connectivity. Thus, the Wnt proteins may be downstream of retinoid signaling in the promotion of adult neurogenesis and dendrite development in the dentate gyrus. Furthermore, RXRs are expressed in differentiated neural stem cells and primary hippocampal cultures (Calderon and Kim, 2007; Cimini and Ceru, 2008), and although DHA-induced neurite outgrowth in neuroblastoma neuro 2A cells may not be mediated by direct activation of RXRα, it has been suggested that there may be involvement of other RXR-isoforms or DHA metabolites in neurite outgrowth (Calderon and Kim, 2007). RAR agonists also enhance BDNF expression (Katsuki et al., 2009), suggesting potential for a link between the effects of omega-3 PUFAs on these transcription factors and neurotrophins. RARα and RARβ agonists are also involved in the induction of neural progenitor cells to become DCX-expressing cells (Goncalves et al., 2009). It is therefore interesting to note that dietary supplementation of aged rats with an omega-3 PUFA-enriched diet (270 mg/kg/day, EPA: DHA ratio 1.5:1) for 12 weeks partially reverses the agerelated decline in neurogenesis in the dentate gyrus, as assessed by DCX expression, with a concomitant reversal of the age-related decreases in RARα and RXRβ expression in the dentate gyrus (Dyall et al., 2010). A further indirect line of evidence linking retinoid signaling and adult neurogenesis relates to 13-cis RA (isotretinoin). In one study a clinical dose of 1 mg/kg was injected intraperitoneally to mice for between 1 to 6 weeks (Crandall et al., 2004). By 3 weeks the

treatment had significantly decreased hippocampal neurogenesis and hippocampal-dependent memory, as measured by the spatial radial maze task, indicating that chronic exposure is detrimental to the process. It may be that the dose at this level is causing these adverse effects. Furthermore, the type of signal mediated by retinoids may depend on the retinoid receptor activated by endogenous ligands such as DHA. Interestingly, depression has been implicated as a side effect of chronic 13-cis RA treatment for severe acne (Lamberg, 1998), and it has been suggested that the etiology of depression may be related to a decline in hippocampal neurogenesis (Vogel, 2000). The positive influence of omega-3 PUFAs on neurogenesis may consequently be a mechanism for the beneficial effects of omega-3 PUFA treatment seen with depression (Sarris et al., 2012; Sublette et al., 2011). It remains to be established if the positive effects of omega-3 PUFAs on neurogenesis are directly mediated via retinoid signaling; however, it is likely that omega-3 PUFAs possess proneurogenic cell-intrinsic effects mediated by actions of transcription factors. A further member of the steroid/thyroid hormone/ vitamin D nuclear receptor superfamily of particular interest are the PPARs. There are also three isotypes of the PPAR, called PPARα, PPARβ/δ and PPARγ, which also undergo alternate splice variations comparable to retinoid receptors. PPARs only bind to RXRs (Desvergne and Wahli, 1999). PPARγ in particular has been reported to be neuroprotective in a variety of CNS injury paradigms (Collino et al., 2006; Costello et al., 2005; Zhao et al., 2006) and PPARγ agonists also suppress monocyte production of the pro-inflammatory cytokines TNF-α and IL-1β (Jiang et al., 1998). PPARγ has also been reported to regulate the development of the CNS during early embryogenesis via control of neural stem cell proliferation (Wada et al., 2006). In this study, moderate activation of the PPARγ pathway by the agonists, rosiglitazone and pioglitazone, stimulated proliferation and inhibited differentiation of murine neural stem cells into neurons. Whereas, agonist activation at higher concentrations caused cell death, suggesting a biphasic action of PPARγ and optimal concentrations of PPARγ agonists are required for neural stem cell survival and proliferation. Since EPA has been reported to regulate the gene expression of PPARγ by increasing mRNA levels in human adipose tissue (Chambrier et al., 2002) and both DHA and DHA derivatives are endogenous PPARγ agonists (Itoh et al., 2006; Yu et al., 1995), activation of PPARγ may also be involved in the neurogenic effects of omega-3 PUFA, although this remains to be explored. In neural stem cells, neurogenesis is regulated by activator-type and repressor-type basic helix-loop-helix

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(bHLH) transcription factors (Johnson et al., 2009; Mu et al., 2010). Neurogenesis is promoted by activatortype bHLH transcription factors such as neurogenin, NeuroD, and Ascl1 (also known as Mash1), whereas, hairy and enhancer of split 1 and 5 (Hes1 and Hes5) prevent terminal differentiation and preserve a pool of stem cells. An interesting recent study has investigated the effects of DHA treatment on the expression of these bHLH transcription factors in neural stem cells (Katakura et al., 2009). DHA treatment significantly decreased the expression of Hes1, an inhibitor of neuronal differentiation, and increased expression of the cyclin-dependent kinase (Cdk) inhibitor p27kip1, which causes cell cycle arrest in neural stem cells, in a dosedependent manner. Neurogenin1, NeuroD, and MAP2 (a neuron-specific protein) expression were also significantly increased. Since MAP2 is activated by NeuroD and repressed by Hes1 (Bhat et al., 2006), these results suggest that DHA stimulates neuronal differentiation by altering the balance of these bHLH transcription factors. However, the mechanisms by which DHA alters the expression of these transcription factors remain to be established. This group has also examined the effects EPA and the omega-6 PUFA, arachidonic acid (AA) on differentiation, expression of bHLH transcription factors (Hes1, Hes6, and NeuroD), and the cell cycle of cultured neural stem cells (Katakura et al., 2013). Consistent with DHA, EPA also increased mRNA levels of Hes1, Hes6, an inhibitor of Hes1, NeuroD, and MAP2 and Tuj-1-positive cells, indicating that EPA-induced neuronal differentiation. EPA also increased the levels of the Cdk inhibitors p21cip1 and p27kip1, indicating EPA-induced cell cycle arrest. Treatment with AA decreased Hes1, but did not change NeuroD or MAP2 levels, or the number of Tuj1-positive cells or cell cycle progression. These results indicated that EPA could be involved in neuronal differentiation by mechanisms other than those of DHA. Early studies indicated that inflammation and microglial activation were detrimental to neurogenesis; however, the situation may be far more complex (Ekdahl, 2012). Activation of microglia encompasses several diverse functional states, ranging from a proinflammatory antineurogenic phenotype, to a neurotrophic phenotype with pro-neurogenic properties (Kohman and Rhodes, 2013). The pro-inflammatory cytokines, IL-1β, IL-6, and TNF-α have been shown to reduce proliferation, survival and/or differentiation of neural stem cells and hippocampal progenitors; however, the mechanisms whereby inflammation reduces hippocampal neurogenesis are not completely understood (Kohman and Rhodes, 2013). Omega-3 PUFAs have well characterized antiinflammatory properties (Calder, 2009), and DHA and

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EPA are also the precursors of biologically active anti-inflammatory lipid mediators, such as resolvins and neuroprotectins. For example, DHA can be metabolized to oxygenated compounds, such as 10-17S-docosatriene (neuroprotectin-D1, NPD-1) with anti-inflammatory properties in a variety of conditions (Dyall and Michael-Titus, 2008). Recent work has begun to explore the relationship between the anti-inflammatory properties of omega-3 PUFAs and their pro-neurogenic effects. DHA treatment reverses the anti-neurogenic activities of conditioned media from activated microglia on neural stem progenitor cells. Moreover, DHA at 10 and 20 μM inhibits the synthesis of the proinflammatory products, IL-1β, IL-6 and TNF-α in a dose-dependent manner in lipopolysaccharide activated microglia (Ajmone-Cat et al., 2012). DHA was also shown to strongly reduce NO production, the expression of inducible NO synthase (iNOS), to inhibit p38 MAPK phosphorylation and the activation of PPARγ. Overall, this study suggests that the proneurogenic properties of DHA may be at least in part mediated via their immunomodulatory effects and modification of activated microglial functions. G-protein coupled receptors (GPRs) are a family of seven-transmembrane receptors, which play an important role in the response to peptide hormones, neurotransmitters, and free fatty acids (Yamashima, 2008). GPR40 (also known as Free Fatty Acid Receptor 1, FFAR1), is a putative target of unsaturated fatty acids, with DHA one of the most potent ligands with an EC50 of 1 μM (Briscoe et al., 2003), and is ubiquitously expressed in the human brain and pancreas, but is not detected in rat brains or rat neural stem cells (Ma et al., 2010). GPR40 is expressed throughout the primate brain, with abundant expression in the hippocampus (Ma et al., 2007). Furthermore, GPR40 signaling has been shown to play an important role in cell proliferation and survival in mammary epithelial cells (Yonezawa et al., 2008), and importantly, GPR40 expression in the dentate gyrus is closely related to neurogenesis following ischemia (Ma et al., 2008). To explore the relationship between DHA and GPR40 signaling in neurogenesis, the effects of DHA were investigated in rat neural stem cells transfected with the GPR40 gene (Ma et al., 2010). It was found that DHA-induced neuronal differentiation, neurite growth, and branching of adult rat stem cells potentially via GPR40. DHA-GPR40 binding led to activation of the phospholipase C (PLC) inositol trisphosphate (IP3) signaling pathway, and consequently increased intracellular Ca21. Although the further downstream events of GPR40 activation remain to be elucidated, increases in intracellular Ca21 have well established effects on the induction of signaling cascades involved

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in changes in neurotransmitter release, modulation of enzyme activity, and gene transcription (Boneva et al., 2011). Further studies are needed to identify the nature and extent of the DHA-GPR40 signaling; however, a putative signaling cascade integrating DHA-GPR40pCREB-BDNF pathway in cognition and adult neurogenesis has recently been suggested (Boneva and Yamashima, 2012). Phosphorylated cAMP response element-binding protein (pCREB) is a transcription factor involved in adult neurogenesis (Nakagawa et al., 2002), learning and memory (Sakamoto et al., 2011), and importantly a Ca21-dependent regulator of BDNF transcription (Tao et al., 1998). The endocannabinoid system has been shown to play a regulatory role in adult hippocampal neurogenesis (Jin et al., 2004c; Rueda et al., 2002). The two main endocannabinoids are N-arachidonylethanolamide (AEA, anandamide), and 2-arachidonylglycerol (2-AG), which are derivatives of the omega-6 PUFA, AA. They exert most of their biological effects by binding to the CB1 and CB2 cannabinoid receptors (Pertwee et al., 2010). The CB1 receptor is widely expressed in the brain, with higher levels in the hippocampus, cerebellum, and basal ganglia (Herkenham, 1991; Herkenham et al., 1991, Moldrich and Wenger, 2000), whereas expression of the CB2 receptor is restricted in the brain and more abundant in the immune system (Patel et al., 2010). Studies into the effects of AEA on promoting hippocampal neurogenesis via the CB1 signaling pathway have produced conflicting results. Adult rats treated with the AEA analog methanandamide show significantly decreased neurogenesis in the dentate gyrus, and this is increased by the CB1 antagonist SR14171 (Rueda et al., 2002). However, other evidence indicates that chronic treatment with the synthetic endocannabinoid agonist, HU210, increases hippocampal neurogenesis in adult rats (Jiang et al., 2005), and CB1 receptor knockout mice show significant reductions in BrdUlabeled cells in the dentate gyrus and subventricular zone (Jin et al., 2004c). The same group also treated either wild type mice or CB1 receptor knockout mice with the CB1 antagonist SR141716A. Both the wild type and CB1 receptor knockout mice showed increased neurogenesis, indicating involvement of a non-CB1 receptor pathway. The involvement of the transient receptor potential vanilloid 1 (TRPV1, VR1) was then assessed by administering SR141716A to VR1 knockout mice. The ability to enhance neurogenesis was abolished, suggesting a role for VR1 receptors in regulating adult neurogenesis. Since omega-3 PUFAs have been shown to modulate the VR1, where DHA acts as an agonist, and EPA an inhibitor (Matta et al., 2007), this may represent a further target responsible for the pro-neurogenic effects of DHA. Overall, chronic

stimulation of the CB1 receptor by methanandamide (Rueda et al., 2002) and the knockout of the CB1 receptor (Jin et al., 2004c) both negatively affect neurogenesis, suggesting that the effect of the endocannabinoid system on neurogenesis is a fine balance of receptor activation. There is an analogous endocannabinoid series derived from omega-3 PUFAs, including Ndocosahexanoylethanolamine (DEA) and 2eicosapentanoylglycerol (EPG), from DHA and EPA, respectively (Berger et al., 2001; Wood et al., 2010). DEA is formed from DHA in fetal mouse hippocampal neuron cultures and in a hippocampal homogenate, and importantly, DEA is formed endogenously in the mouse hippocampus, and the levels can be modified by dietary omega-3 PUFA status (Kim et al., 2011a). In this study, DEA stimulated neurite growth and synaptogenesis, at concentrations as low as 0.1 μM, substantially lower than those observed with DHA (1 μM). Similarly, both DEA and DHA increased synapsin and the NR2B glutamate receptor subunit expression in the hippocampal neurons. The effects of DHA on neurite growth and synaptogenesis, and the NR2B and GluR1 glutamate receptor subunit expression, were all substantially greater in hippocampal cultures co-treated with URB597, a fatty acid amide hydrolase (FAAH) inhibitor. The degradation of endocannabinoids is carried out via two specific enzymatic reactions: FAAH (Cravatt et al., 1996) and monoacylglyceride lipase (MAGL) (Dinh et al., 2002), suggesting that some of the effects may be mediated by its transformation to DEA (Kim et al., 2011a). In addition to DEA, further N-acylamide derivatives of DHA have been detected in the brain, with as yet unidentified biological activity (Kim et al., 2011b). It may therefore be that some of the effects of DHA on hippocampal neurogenesis may also be mediated at least in part via their conversion to bioactive lipid mediators, such as DEA and EPG, and/or by their interaction with endocannabinoid signaling pathways. Further targets of DHA are also being explored, such as the immediate early response genes. DHA supplementation (10 and 30 μM) of nerve growth factor induced differentiated PC12 (pheochromocytoma) cells significantly elevates the mRNA levels of the immediate early biomarkers, early growth response transcriptional regulator (Egr3) and PC3 (also known as Tis21 or BTG2) in parallel with enhanced neurite outgrowth (Dagai et al., 2009). Immediate early genes are a class of genes activated rapidly and transiently and constitute an important mechanism for specific early genomic responses to signaling cascades (Poirier et al., 2008). Egr3 has been shown to be involved in hippocampal-dependent memory tasks (Poirier et al., 2008), and has an important role in neurite outgrowth

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FIGURE 21.1 The pro-neurogenic effects of EPA and DHA potentially operate through a variety of overlapping mechanisms. For a detailed explanation refer to the text. a) Positive and negative regulators of neurogenesis potentially modified by omega-3 PUFA. Negative factors include neuroinflammation, which causes reactive microgliosis and contributes to impaired neurogenesis by releasing inflammatory and neurotoxic factors (IL-1β, IL-6, TNF-α, and NO), and aging. Positive factors include neurotrophins such as BDNF, pro-neurogenic transcription factors (TF), and EPA- and DHA-derived endocannabinoids (2-eicosapentanoylglycerol, EPG, and N-docosahexanoylethanolamine, DEA). b) Alterations in the neuroinflammatory response are mediated via competition between AA and EPA for eicosanoid biosynthetic enzymes, with a high EPA content favoring the production of EPA-derived anti-inflammatory mediators such as series 3 prostaglandins, prostacyclins and thromboxanes, and series 5 leukotrienes. Nonesterified EPA and DHA are also the precursors of anti-inflammatory lipid mediators, such as RvE1 and NPD1, respectively. Non-esterified EPA and DHA may also regulate gene expression via effects on transcription factors such as retinoid and PPAR signaling pathways and bHLH transcription factors. Binding of non-esterified DHA to GPR40 may also lead to activation of phospholipase C (PLC), IP3 production, mobilization of intracellular Ca21 from the endoplasmic reticulum, and activation of protein kinase C (PKC). Solid arrows indicate positive effects, flat arrow-heads inhibition, dotted arrows competition, and open arrows phospholipase A2-induced release from the cell membrane. Abbreviations: bHLH, basic helix-loop-helix; cPLA2, cytosolic PLA2; iPLA2, Ca21-independent PLA2; LT, leukotriene; PG, prostaglandin; PGI, prostacyclin; PPAR, peroxisomal proliferator-activated receptor; RXR, retinoid X receptor; TXA, thromboxane.

(Levkovitz et al., 2001). PC3 is normally expressed in neuronal precursor cells immediately before the last asymmetric division, and induces terminal differentiation in several areas of the CNS during neurogenesis

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(Farioli-Vecchioli et al., 2008). These results indicate that DHA-enhanced neurite outgrowth is coupled with a transient increase in the levels of immediate early genes, suggesting further pathways by which DHA enhances neurogenesis. In summary, omega-3 PUFA treatment has consistently been shown to enhance adult hippocampal neurogenesis in a variety of animal models. Elucidating the mechanisms for this effect has been complicated by the convergent pathways involved in regulating neurogenesis and the pleiotropic effects of omega-3 PUFAs. However, a number of potential targets have been identified. This chapter has briefly reviewed some of the evidence of the effects of omega-3 PUFAs on cellextrinsic and -intrinsic regulatory factors. These include influencing neurotrophin levels such as BDNF, modulation of transcription factors such as retinoid receptors and the bHLH transcription factors, modulation of pro-inflammatory cytokine levels, conversion to bioactive lipid mediators, and interactions with endocannabinoid signaling pathways. However, the effects of omega-3 PUFAs are undoubtedly mediated by further mechanisms, which may include biophysical effects in neuronal membranes. Furthermore, it is important to identify the individual roles of ALA, EPA, and DHA, as they cannot be considered functionally equivalent. In order for omega-3 PUFAs to achieve their therapeutic potential, it is imperative to understand the molecular mechanisms that mediate their observed effects, and understanding their regulatory effects in neurogenesis (Figure 21.1) is an important avenue for exploration.

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C H A P T E R

22 Imaging Brain DHA Metabolism in Vivo, in Animals, and Humans Stanley I. Rapoport and Ameer Taha INTRODUCTION The polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (AA, 20:4n-6), are major components of membrane phospholipids in the central nervous system (Bazan, 2009; Salem et al., 2001). Both help to maintain normal brain structure, metabolism, and function, and are second messengers during synaptic neurotransmission (Garcia & Kim, 1997; Ong et al., 2005; Ramadan et al., 2010; Rapoport, 2003). Neither can be synthesized de novo in vertebrate brain tissue where they are minimally subjected to oxidation (Holman, 1986). Thus, their brain concentrations depend on their dietary intake as well as on their hepatic synthesis from shorter-chain nutritionally essential precursors, α-linolenic acid (ALA, 18:3n-3) for DHA and linoleic acid (LA, 18:2n-6) for AA (Igarashi et al., 2007b; 2007c). In rodents, dietary n-3 PUFA deprivation for as short as 15 weeks or as long as 3 consecutive generations reduces brain DHA concentration while increasing the concentration of docosapentaenoic acid (DPA) (22:5n-6), an elongation product of AA formed in the liver (Figure 22.1) (Kim et al., 2011; Salem et al., 2001). Brain AA concentration is changed minimally. A graded dietary analysis showed that the changes in brain concentration initiated rather than followed decreased expression of DHA- selective Ca21-independent phospholipase A2 (iPLA2) VIA and increased expression of AA-preferring Ca21-dependent cytosolic cPLA2 β (IVA) (Kim et al., 2011). Rodent dietary deprivation studies usually involved terminal experiments and direct chemical analysis of postmortem brain. They have suggested clinically relevant mechanisms, as low dietary intake of DHAcontaining fish products are reported to contribute to

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00022-3

pathological human brain aging and disease, including primary depression and Alzheimer’s disease, whereas dietary DHA supplementation may be helpful in these conditions (Bloch & Hannestad, 2012; Conquer et al., 2000; Hibbeln, 1998; Martins, 2009; Quinn et al., 2010). Understanding brain PUFA metabolism in vivo is also relevant, since PUFA metabolism may be disturbed in patients with alcoholism (Taha et al., 2012), and mutations in PUFA-metabolizing enzymes can contribute to brain disease (Kurian et al., 2008; Morgan et al., 2006; Paisan-Ruiz et al., 2008). Thus, it is useful to have methods to study brain PUFA metabolism in unanesthetized animal models and in humans. With such methods, we can test the efficacy of dietary PUFA intervention, and elucidate the roles of PUFAs in neurotransmission, neuroplasticity, aging, and other processes under normal and pathological conditions. The present review describes newly available in vivo brain imaging methods, with a particular focus on DHA, and results obtained with them.

QUANTITATIVE IMAGING OF BRAIN DHA METABOLISM IN RODENTS Incorporation of Circulating PUFAs into Brain Membrane Lipids Within blood, PUFAs circulate when esterified within phospholipids, cholesteryl esters and triacylglycerols of lipoproteins, or in their unesterified (free) form when largely but reversibly bound to albumin. They enter brain largely in their unesterified form, after dissociating from albumin in brain capillary blood. Although receptors to lipoproteins are found at cerebral capillaries, studies in rodents show that

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FIGURE 22.1 Structures of major monounsaturated, saturated, and polyunsaturated n-3 and n-6 fatty acids. From ChemSpider Database. Available at: http://www.chemspider.com. (Moreno et al., 2012).

measurable PUFA uptake by brain is proportional to the unesterified plasma PUFA concentration (Purdon et al.,1997; Smith & Nagura, 2001). In addition, there is no difference in PUFA uptake between mice genetically lacking and having lipoprotein receptors (Chen et al., 2008). Brain uptake of unesterified long-chain fatty acids is unaffected by changes in regional cerebral blood flow (rCBF), thus it is independent of changes in functional or drug-induced activity that may influence flow (Chang et al., 1997; Pardridge & Mietus, 1980; Robinson et al., 1992; Smith et al., 2001). Having entered brain, an unesterified plasmaderived PUFA such as DHA (Figure 22.2) is rapidly (within 1 minute) and largely ( . 80%) esterified in membrane phospholipids, and to a lesser extent in cholesteryl esters and triacylglycerols (DeGeorge et al., 1989). Incorporation requires its activation by an acylCoA synthetase (Acsl) to its acyl-CoA, with consumption of two ATPs. An Acsl-4 is relatively selective for AA and an Acsl-6 is selective for DHA (Soupene &

Kuypers, 2008). While palmitic acid (PAM) is esterified preferentially into the stereospecifically numbered (sn)-1 position of phospholipids, AA and DHA are esterified preferentially into the sn-2 position, with DHA entering mainly ethanolamine glycerophospholipid and choline glycerophospholipid, and AA entering mainly choline glycerophospholipid and phosphatidyl inositol (DeGeorge et al., 1989). The shorter-chain circulating precursors of DHA, ALA or eicosapentaenoic acid (EPA, 20:5n-3), and of AA, namely LA, are largely β-oxidized within brain mitochondria via their acylCoA, rather than entering phospholipid (DeGeorge et al., 1989; 1991; DeMar et al., 2005; Gavino & Gavino, 1991; Washizaki et al., 1994), and their esterified brain concentrations are much lower than the DHA and AA concentrations. The brain has a very limited ability to convert the PUFA precursors to AA and DHA (Igarashi et al., 2007a). This ability largely resides in the liver, which has high activities of appropriate elongases and desaturases (Jump, 2002).

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FIGURE 22.2 Model for measuring incorporation and turnover of DHA into brain phospholipid. Intravenously injected unesterified radiolabeled DHA dissociates from albumin within the brain capillary and diffuses across the capillary endothelium (blood
brain barrier) into the intracellular unesterified brain fatty acid pool (endoplasmic reticulum, ER). DHA is activated with consumption of two ATPs to DHACoA by acyl-CoA synthetase, then esterified at the sn-2 position of a lysophospholipid (lower structure), which had been formed by receptormediated hydrolysis of unlabeled DHA (higher structure). Released DHA (top) is metabolized to bioactive docosanoids (oxo-DHAs, resolvins, docosatrienes, neuroprotectins, epoxydocosapentaenoic acids (EDPs)) or returned to ER by fatty acid binding protein (FABP).

These unique properties, rapidly-reversible albumin binding, preferential uptake of unesterified but not esterified PUFA from blood, flow independent uptake, and rapid (,1 min) incorporation (pulse labeling) into membrane phospholipids compared to oxidative metabolism, allow us to use measured incorporation coefficients and rates as explicit quantitative markers of regional brain DHA or AA metabolism at rest and during activation or disease states.

Methods and models for determining PUFA incorporation into brain In an unanesthetized rodent, we quantify incorporation parameters by infusing radiolabeled [1-14C]

DHA intravenously for 5 min, removing and freezing the brain, then measuring regional brain radioactivity CbrainðDHAÞ using quantitative autoradiography. An incorporation coefficient k is calculated as the ratio of measured brain radioactivity to integrated plasma radioactivity between time 0 and T 5 5 min of infusion, ÐT  C (it is the ‘input’ function to brain), plasmaðDHAÞ 0 where identifies labeling (Figure 22.2) (Robinson et al., 1992), k 5 Ð T 0

cbrainðDHAÞ CplasmaðDHAÞ

ð22:1Þ

As k applies both to labeled and unlabeled DHA (there is no significant isotope effect of 14C or of 3H), multiplying k by the unesterified plasma unlabeled

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DHA concentration CplasmaðDHAÞ gives the net rate of DHA incorporation into brain structural lipid, Jin(DHA), JinðDHAÞ 5 k CplasmaðDHAÞ

ð22:2Þ

In the equations, the incorporation coefficient k represents a number of different steps (it is a ‘lumped’ parameter) involving, among others, DHA dissociation from circulating albumin, unesterified DHA diffusion from plasma across the blood
brain barrier and into the unesterified intracellular compartment(s), activation to DHA-CoA by Acsl and subsequent esterification to membrane lipid by an acyltransferase (Figure 22.2) (Sun & MacQuarrie, 1989; Yamashita et al., 1973). Some of these steps are enzymatic and show saturation, competition or other non-linear properties that may change with precursor or product availability, genetics, enzyme level, or other factors. For example, k for DHA was increased when plasma and brain DHA were reduced by dietary n-3 PUFA deprivation in rats (Contreras et al., 2000), and in chronic alcoholics (Umhau et al., 2013), while k was decreased in mice genetically lacking iPLA2β VIA (see below) (Basselin et al., 2010). Additional kinetic parameters such as PUFA turnover (i.e. rate of acylation and reacylation) in phospholipids (Figure 22.2), can be calculated when the specific activity of the brain acylCoA pool is determined experimentally (Deutsch et al., 1994; Rapoport, 2003; Robinson et al., 1992). Experiments demonstrate that Jin(DHA) (Eq. 22.2) stoichiometrically replaces DHA that is consumed by brain in different metabolic pathways, since there is no de novo resynthesis and precursors are insignificantly elongated (see above). Integrating Jin(DHA) over whole brain gives a net DHA incorporation (consumption) rate of about 0.19 μmol/g brain/day in adult male rats (Contreras et al., 2000), while measuring half-life of injected radiolabeled DHA in brain gives a loss rate of 0.25 μmol/g/day (DeMar et al., 2004). This is equivalent to the single injection value, considering variance of the data in the measurements. Thus, the efficiency and simplicity of the single injection measurement, using quantitative autoradiography, makes it ideal for quantitatively imaging brain DHA consumption at rest or during functional or pharmacological activation.

Imaging Membrane Synthesis We have used quantitative imaging with radiolabeled fatty acids including palmitate, AA, and DHA, to follow changes in brain lipid membrane synthesis in animal models with implanted brain tumors, ocular enucleation, during neurodevelopment, and following denervation of the hypoglossal nerve (Greig et al., 1991; Tabata et al., 1986; Yamazaki et al., 1989). The

following subsections describe experimental and clinical conditions where DHA metabolism was shown to be disturbed (Table 22.1), and discuss the utility of imaging DHA incorporation in humans with positron emission tomography (PET). Neuroplasticity with Ocular Enucleation In the rat, visual input to one eye stimulates contralateral central visual areas; removal of one eye leads to multiple structural and functional changes in these contralateral areas over time. A role for DHA metabolism in these neuroplastic changes was shown in rats in which one eye was removed 3 months prior to injecting [1-14C]DHA (Figure 22.3). At 3 months, contralateral compared with ipsilateral k for DHA was lower in superficial gray of the superior colliculus (in the dark and during visual stimulation) and in the dorsal nucleus of lateral geniculate body (during stimulation), while glucose metabolism did not differ between hemispheres (Wakabayashi et al., 1995). These results indicate a role for DHA independently of glucose metabolism in neuroplastic membrane changes following chronic visual deprivation. Brain Tumor Imaging Tumor growth involves synthesis of new phospholipid-containing membrane, and this can be picked up in the brain with fatty acid neuroimaging. [1-14C]DHA, [1-14C]AA or [3H]PAM was injected intravenously in rats implanted with Walker 256 carcinosarcoma cells in brain 2 weeks earlier (Figure 22.4 left) (Nariai et al., 1993; 1994). In autoradiographs of coronal brain sections containing the tumor (Figure 22.4 TABLE 22.1 Incorporation of Coefficient and Plasma Unesterified DHA Changes in Different Experimental Conditions

Experimental Condition

k for DHA

Plasma Unesterified DHA

ANIMAL MODELS Ocular enucleation

l

3

iPLA2 knockout mice

l

l

Implanted Walker 256 carcinosarcoma

n

3

n-3 PUFA deficiency

n

l

DRUG-INDUCED NEURORECEPTOR ACTIVATION Glutamatergic ionotropic NMDA receptors 3 3 Dopaminergic D2-like G-protein coupled receptors

3

3

Muscarinic M1,3,5 G-protein coupled receptors

n

3

n

3

CLINICAL CONDITION Alcohol withdrawal

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QUANTITATIVE IMAGING OF BRAIN DHA METABOLISM IN RODENTS

[14C]Docosahexaenoate incorporation during visual stimulation, 3 months after unilateral enucleation

Te1

Oc2L Oc1M

DLG VLG

SuG InG DpG

k* 1.5 2.0 3.0 4.0

269

FIGURE 22.3 Color autoradiographs of coronal rat brain sections showing incorporation coefficients k of DHA 3 months after eye removal at 15 days of age. Intact eye was stimulated by black and white pattern at 1500 Lux. During stimulation, contralateral ipsilateral superior gray of superior geniculate nucleus (SuG) and dorsolateral geniculate body (DLG) and other regions showed decreased values of k relative to ipsilateral regions. From Wakabayashi et al., 1995.

6.5 11.0

MG Contralateral

Ipsilateral

Contralateral

ml/sec/g x 104

Ipsilateral

FIGURE 22.4 Left. Coronal rat brain section stained with cresyl violet, 7 days after implantation of Walker 256 carcinosarcoma. Right. Adjacent autoradiograph following i.v. injection of [1-14C]docosahexaenoic acid. N, necrotic tumor region. From Nariai et al. 1993.

right), tracer was not incorporated in necrotic tumor regions. Ratios of incorporation coefficients (k) into tumor compared with intact brain were 4.5, 3.4, and 1.7 for [3H]PAM, 1-14C]AA, and [1-14C]DHA, respectively; lipid radioactivity comprised more than 80% of total tumor or brain radioactivity for each probe. Thus, differences in relative increases with the different tracers might be used to image and to characterize lipid metabolism of different brain tumors, and to evaluate therapies that interfere with lipid synthesis (De Vries & van Noorden, 1992).

(Fagiolini et al., 2005; Segura et al., 2009; van den Berg et al., 2007). Rats fed a high-sucrose diet (60%) for 8 weeks are a model for the early-stage metabolic syndrome in humans. The rats develop peripheral insulin resistance and increased activity in brain of DHAselective iPLA2VIA and AA-selective cPLA2 IVA, reduced synaptic drebrin, and brain derived neurotrophic factor (BDNF) compared with controls (Taha et al., 2012). These results suggest disturbed brain AA and DHA metabolism as a consequence of the metabolic syndrome.

Upregulated DHA and AA Releasing Enzymes in an Animal Model of the Metabolic Syndrome

Neurotransmission

The metabolic syndrome is characterized by glucose intolerance and dyslipidemia (Eckel et al., 2005), and is a risk factor for cognitive decline and mood disorders

Esterified DHA and AA in the sn-2 position of brain phospholipids turn over rapidly, with half-lives in some molecular species of 1 hour or less in rat brain (Robinson et al., 1992; Shetty et al., 1996). As noted

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above, AA and DHA are hydrolyzed by selective PLA2 enzymes that have been characterized in the test tube (Six & Dennis, 2000; Yang et al., 1999). In the intact brain and in vitro, cPLA2 type IVA, which is selective for AA and has been shown to have a post-synaptic location, has also been shown to be coupled via G-proteins to serotonergic 5-HT2A/2C receptors (Berg et al., 1998; Ong et al., 2005; Qu, et al., 2005), cholinergic muscarinic M1,3,5 receptors (Bayon et al., 1997; DeGeorge et al., 1991) and dopaminergic D2-like receptors (Bhattacharjee et al., 2005; Vial & Piomelli, 1995), and by extracellular Ca21 entering the cell following glutamate binding to ionotropic N-methyl-D-aspartate (NMDA) receptors (Figure 22.5) (Basselin et al., 2006a). In the intact rat brain, a DHA signal was produced by stimulating muscarinic receptors by arecoline, but not by stimulating NMDA receptors that let calcium into the cell, confirming its in vivo calcium independence and mediation by iPLA2, which is calcium independent (Figure 22.5) (DeGeorge et al., 1991; Ramadan et al., 2010). As with cPLA2, iPLA2 has post-synaptic locations in the mammalian brain (Ong et al., 1999; 2005), but it is also distributed in the cytoplasm associated with the endoplasmic reticulum (ER), and has been shown in vitro to be activated in association with the release of calcium from the ER. Figure 22.6 provides a proposed model for such activation by arecoline (Cooper et al., 1996; Nowatzke et al., 1998; Rosa & Rapoport, 2009; Ueda, 2008). Briefly, arecoline activates phospholipase C (PLC) via a G-protein coupled mechanism, converting phosphatidyl inositol to diacylglycerol (DAG) and inositol trisphosphate (IP3). IP3 stimulates IP3 receptors on the ER/sarcoplasmic reticulum (SR) to release Ca21 and calcium efflux factor (CEF), which dissociates calmodulin (CaM) to activate Docosahexaenoic acid Saline

Arachidonic acid

NMDA

Saline

NMDA

iPLA2 and thereby hydrolyze DHA from membrane phospholipid. While muscarinic receptor activation stimulated both an AA and DHA signal in the awake rat, preliminary evidence indicates that stimulation of dopamine D2 receptors by the agonist quinpirole produced an AA signal but no DHA signal (Bhattacharjee et al., 2005; 2008) (Taha, unpublished). This demonstrates that DHA signal transduction does not follow the mechanism outlined in Figure 22.6 for muscarinic receptor activation. This difference may be related to limited calcium release following dopaminergic receptor stimulation, compared to significant release following cholinergic stimulation (Neve et al., 2004). Human Mutations and Mouse Knockouts of iPLA2β It is possible to use genetic knockout or inhibition rodent models to study mechanisms of signaling involving AA and DHA in vivo (Basselin et al., 2006b; 2007; Cheon et al., 2012). In this regard, there are two iPLA2 isoenzymes in rodent brain, iPLA2β (VIA) and iPLA2γ, both of which are considered selective for DHA hydrolysis. Mutations in the PLA2G6 gene encoding iPLA2β occur in patients with idiopathic neurodegeneration plus brain iron accumulation, dystonia-parkinsonism without iron accumulation, and other progressive neurological diseases (Kurian et al., 2008; Morgan et al., 2006; Paisan-Ruiz et al., 2008). Further, mice in which PLA2G6 is knocked out show neurological symptoms after about 1 year of age, with the appearance of significant neuropathology (Basselin et al., 2010; Cheon et al., 2012; Ma et al., 1998; Malik et al., 2008). We studied behaviorally normal 4-month-old iPLA2β mice, to address the functional metabolic defect in brain iPLA2β that leads to later pathology, by injecting arecoline or saline intravenously and measuring

Fr

CPu

DB Mot Hipp

k* (ml/s/g) x 10–4

5

25

FIGURE 22.5 NMDA (25 mg/kg i.p.) initiates arachidonic but not docosahexaenoic acid signal in rat brain. From Ramadan et al. 2010.

FIGURE 22.6 Proposed mechanism for receptor-mediated activation of iPLA2.

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QUANTITATIVE IMAGING OF BRAIN DHA METABOLISM IN RODENTS

iPLA2β+/+ Saline

iPLA2β+/+

Arecoline

Saline

Arecoline

iPLA2β–/– Saline

Mot

CPu Hipp

Arecoline

271

FIGURE 22.7 Reduced baseline and arecoline-initiated DHA signals in 4-month-old heterozygous (iPLA2β1/2) and homozygous (iPLA2β2/2) knockout mice, compared with wild types (iPLA2β1/1) mice. Coronal autoradiographs show brain DHA incorporation coefficients k (color scale) at rest and following cholinergic agonist, arecoline. From Basselin et al. 2010.

Hb Vis

SN

12 k* –4 (ml/s/g) x 10

60

[1-14C]DHA incorporation with quantitative autoradiography. Arecoline compared with saline increased k in homozygous iPLA2β (1 / 1 ) mice, but significantly less so than in wild type iPLA2β(2 / 2 ) or heterozygous iPLA2β (1 / 2 ) mice (Figure 22.7) (Basselin et al., 2010). The iPLA2β (1 / 1 ) mice also had a lower brain DHA concentration, but there was no evidence of compensation by a marked change in iPLA2γ expression (Cheon et al., 2012). Since significant neuropathology and motor dysfunction appear in the knockout mice, genetically defective DHA metabolism is likely a risk factor for neurological disease, and this might be identified with PET neuroimaging (see below) in at-risk subjects with PLA2G6 mutations (Kurian et al., 2008; Morgan et al., 2006; Paisan-Ruiz et al., 2008).

Quantitative Imaging of Brain DHA Metabolism in Human Subjects Baseline Brain DHA Incorporation To directly consider human brain DHA metabolism, we synthesized positron-labeled [1-11C]DHA and used PET to image DHA incorporation into the brain of healthy volunteers (Channing & Simpson, 1993; Umhau et al., 2009). As illustrated in Figure 22.8, DHA incorporation coefficients k were higher in gray than in white matter; the mean net rate of incorporation for the entire brain equaled 3.8 6 1.7 mg/day. This is the first direct estimate of human brain DHA daily requirements, and is one-fiftieth a recommended dietary DHA supplementation of 200 mg per day in the United States (Kris-Etherton et al., 2000).

FIGURE 22.8 PET scan of human brain. Horizontal image of scan following injection of [1-11C] DHA in a healthy volunteer, showing color coded incorporation coefficients and higher incorporation in gray than white matter regions. From Umhau et al., 2009.

Upregulated Incorporation Coefficients in Chronic Alcoholics Our PET method provides an opportunity to quantify human brain DHA metabolism in health and disease. As a first step in this regard, we imaged brain DHA metabolism with PET in 15 non-smoking chronic alcoholics within 7 days of their last drink, and 22 non-smoking controls (Umhau et al., 2013). Chronic alcoholics frequently have a low plasma concentration of DHA, particularly if they have liver disease, and show cognitive and behavioral abnormalities. We also measured rCBF using [15O]H2O in the same PET session, and made partial volume error (PVE) corrections for brain atrophy. Both mean brain k for DHA and rCBF were significantly elevated in the alcoholics compared with controls (Figure 22.9) (Umhau et al., 2013).

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FIGURE 22.9 Significantly higher global gray matter k for

[1-11C]DHA in alcoholics compared to controls (p # 0.05) (Umhau et al., 2013).

The elevated k suggests increased brain avidity for DHA in relation to plasma availability, whereas the higher rCBF indicates a hyperactive state perhaps expected following alcohol withdrawal (Victor, 1973). Earlier imaging studies that reported reduced rCBF or glucose metabolism in chronic alcoholics used nonPVE corrected measurements and did not exclude smokers (Volkow et al., 1992).

SUMMARY AND CONCLUSIONS In this critical review, we provide evidence that DHA incorporation coefficients k and rates Jin(DHA) are biomarkers of regional brain DHA metabolism in vivo. These kinetic parameters can be quantitatively imaged following a single intravenous injection of radiolabeled DHA, using autoradiography in unanesthetized animals or PET in humans. Our modeling and experiments show that k for DHA is a lumped value that involves diffusional, binding and enzymatic steps, and thus represents the brain’s affinity for DHA, whereas Jin(DHA) equals the net rate of DHA consumption by brain, representing downstream formation of bioactive DHA metabolites and other products, and membrane synthesis. In rodent models, in vivo imaging showed that k was increased in experimental brain tumors and in visual brain regions contralateral to chronic enucleation of one eye. These increases demonstrate that we can identify DHA’s regional involvement in membrane synthesis and remodeling. A PET study also showed

that k for DHA is increased within 7 days after alcohol withdrawal in chronic alcoholics, suggesting increased brain avidity for DHA compared with plasma availability. Interpreting in vivo images using our model depends on evidence that unesterified but not esterified radiolabeled DHA is taken up from plasma into brain in a blood flow independent manner; 80% is esterified within membrane lipids within 1 minute after injection, satisfying the condition of pulse labeling. The major site of labeling by DHA is the sn-2 position of brain membrane phospholipids. During neurotransmission that gives a DHA signal, DHA is likely hydrolyzed preferentially from the sn-2 position of phospholipids by Ca21-independent iPLA2, of which two isoforms exist in rodent brain, iPLA2β and iPLA2γ. Imaging shows profound reductions in DHA incorporation into brain, at rest, and during drug activation, in homozygous iPLA2β knockout compared with wild type or heterozygous knockout mice at 4 months of age, long before the knockouts develop Parkinson-like symptoms and profound neuropathology. This imaging study identifies a critical need for DHA metabolic integrity during normal brain development, and may inform human neurological diseases ascribed to a PLA2G6 mutation. DHA can act as a second messenger during neurotransmission, and this process can be imaged as well. In unanesthetized rats, a DHA signal reflecting DHA release from membrane phospholipid was shown to follow arecoline induced activation of G-protein coupled cholinergic muscarinic M1,3,5 receptors; this response was dampened in iPLA2β knockout mice. On the other hand, there was no DHA signal following activation by injected NMDA of ionotropic NMDA receptors that allow extracellular Ca21 into cells, or of G-protein coupled dopaminergic D2 receptors (Taha, unpublished). These observations, combined with in vitro information that iPLA2 activation is calciumindependent, confirm that iPLA2 activation in vivo is independent of extracellular Ca21 but may follow IP3-induced intracellular calcium release from the ER. As brain lipid metabolism involves a balance between the AA and DHA cascades, changes in the relations of these cascades by diet, drugs or disease should have profound effects; this area remains to be explored in detail, and brain imaging of DHA metabolism may help in this regard. One example of such a condition may be excitotoxicity, in which excess glutamate stimulates NMDA receptors that are coupled to AA but not DHA release (Chang et al., 2008; Rao et al., 2010). Some clinical conditions such as Alzheimer’s disease have been associated with reduced hippocampal DHA and DHA metabolite concentrations (Lukiw

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REFERENCES

et al., 2005), and this might be examined in patients with DHA PET. It is not known whether reductions in DHA or in DHA metabolites are related to reduced n-3 PUFA dietary intake. A reduction in plasma DHA concentration would be accompanied by a compensatory increase in k, as reported in n-3 deficient rats (Table 22.1) (Contreras et al., 2000). It is unlikely, however, that extreme n-3 deficiency modeled in rodent studies exists clinically, although human intake of the DHA precursor, ALA, may be 30% lower than the recommended amount (Blasbalg et al., 2011). Finally, given the challenges of PET imaging with [111C]DHA, which has a radioactive half-life of 20 minutes, it would be worthwhile to develop fluorinated, 18 F-DHA, which would extend the radioactive half-life of the tracer to 2 hours and allow for better resolution and shorter scanning time. We have synthesized a fluorinated analog for AA and validated it similarly to [1-14C]DHA in mice (Pichika et al., 2012). Developing a chemical synthetic method for 18F-DHA would allow the PET imaging method to be extended to centers that do not have the capability to synthesize 11C-fatty acid probes, thus enabling the widespread use of DHA as a probe for G-protein receptor neurotransmission and function in humans, and in disease, genetic or dietary conditions where its metabolism is altered.

Acknowledgments This research was supported entirely by the Intramural Program of the National Institute on Aging. Neither author has a conflict of interest.

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1268. Umhau, J.C., Zhou, W., Thada, S., Demar, J., Hussein, N., Bhattacharjee, A.K., et al., 2013. Brain docosahexaenoic acid [DHA] incorporation and blood flow are increased in chronic alcoholics: a positron emission tomography study corrected for cerebral atrophy. PloS One. 8, e75333. Vial, D., Piomelli, D., 1995. Dopamine D2 receptors potentiate arachidonate release via activation of cytosolic, arachidonic-specific phospholipase A2. J. Neurochem. 64, 2765
2772. Victor, M., 1973. Alcoholism. II. Withdrawal, neurological syndromes, and EEG. Ann. N. Y. Acad. Sci. 215, 210
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122. Washizaki, K., Smith, Q.R., Rapoport, S.I., Purdon, A.D., 1994. Brain arachidonic acid incorporation and precursor pool specific activity during intravenous infusion of unesterified [3H]arachidonate in the anesthetized rat. J. Neurochem. 63, 727
736. Yamashita, S., Hosaka, K., Numa, S., 1973. Acyl-donor specificities of partially purified 1-acylglycerophosphate acyltransferase, 2-acylglycerophosphate acyltransferase and 1-acylglycerophosphorylcholine acyltransferase from rat-liver microsomes. Eur. J. Biochem. 38, 25
31. Yamazaki, S., Noronha, J.G., Bell, J.M., Rapoport, S.I., 1989. Incorporation of plasma [14C]palmitate into the hypoglossal nucleus following unilateral axotomy of the hypoglossal nerve in adult rat, with and without regeneration. Brain Res. 477, 19
28. Yang, H.C., Mosior, M., Johnson, C.A., Chen, Y., Dennis, E.A., 1999. Group-specific assays that distinguish between the four major types of mammalian phospholipase A2. Anal. Biochem. 269, 278
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985.

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C H A P T E R

23 Obesity and Migraine in Children Pasquale Parisi, Alberto Verrotti, Maria Chiara Paolino, Alessandro Ferretti and Fabiana Di Sabatino INTRODUCTION Migraine and obesity are highly prevalent disorders that significantly affect quality of life, health, and individual well-being in adults and children. In particular, migraine is one of the most common chronic conditions in the pediatric population. Similarly, obesity has reached epidemic levels, with increases in rates of obesity having been observed among children and even infants. Both of these health issues together incur a significant financial burden on society (Abu-Arefeh and Russell, 1994; Kim et al., 2006; Kinik 2010). Recent data suggest that these two conditions may be directly related through biochemical and physiological pathways, however, their interaction is particularly complex and has been the subject of multiple large and sometimes conflicting studies. Both migraine and obesity seem to be influenced by genetic factors and are characterized by a local and systemic pro-inflammatory state.

EPIDEMIOLOGICAL RELATIONSHIP BETWEEN MIGRAINE AND OBESITY Migraine and obesity represent two major public health problems in adults and children. Migraine is defined by the International Headache Society (IHS) as a recurrent headache that occurs with or without aura and lasts between 4
72 hours (1
72 hours in children) and is often accompanied by nausea, vomiting or sensitivity to light, sound or movement (Oleson, 2004). It is among the most common chronic conditions with an estimated prevalence of 10
28% among children and adolescents (Abu-Arefeh and Russell, 1994). Similarly, obesity has reached epidemic levels, and increases in obesity rates have been observed among children of all ages (Kim et al., 2006). From 1963 to 1970, the

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00023-5

prevalence of obesity in children was near to 5%, while by 2003 to 2004, it had increased to 17% (Ogden et al., 2006). Particularly in the USA, 12% of individuals are migraineurs (Lipton et al., 2001) and one-third of people are obese (Bigal and Lipton., 2009; Flegal et al., 2010). During the past 30 years in the USA, the prevalence of obesity has substantially increased among the young (Odgen et al., 2006). Finally, both migraine and obesity create an economic liability in society. Healthcare costs attributed to migraine are predicted to surpass $11 billion annually (Hawkins et al., 2008), and obesity is projected to account for $147 billion in healthcare costs (Finkelstein et al., 2009), with indirect costs contributing an additional $65 billion to the total economic liability (Trogdon et al., 2008). Recent data suggest a possible relationship between chronic migraine and obesity in children, as well as in adult population studies. Bigal et al. (2006) conducted a large population-based cross-sectional study (30,215 participants with headache history, 3719 of whom had migraine, aged between 18 to 89 years, mean 38,7) to investigate the influence of BMI on the prevalence and clinical attributes of migraine. Their findings suggest that the prevalence of migraine in the population does not vary significantly as a function of the BMI group, but obesity is significantly associated with the number of headache days per month among migraineurs, particularly for very frequent headaches, even after adjusting for covariates (gender, age, use of headache medications, sleep problems, education status, and depression). Reporting of frequent headaches (10
14 days per month) increased from 4.4% in the normal weight group to 13.4% in the obese group, and 20.7% in the severely obese group. BMI is also significantly associated with the severity of attacks experienced by migraine sufferers, with the perception that attacks are worsened by physical activity.

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Bigal et al. (2009) subsequently reported findings that further supported the relationship between obesity and migraine frequency in the pediatric population. In this study, 18,968 of 162,576 individuals (11.7%) aged 12 years and older screened positive for migraine. Among these individuals, the occurrence of very frequent headaches was significantly higher in the obese (8.2%) and morbidly obese (10.4%) groups, compared to the normal weight group (6.5%). Additionally, the percentage of participants with some headache disability was higher in the obese (38.4%) and morbidly obese (40.9%) groups, relative to the normal weight group (32.0%). Hershey et al. (2009) examined the prevalence of obesity and the relationship between weight, headache frequency, and disability outcomes within a pediatric headache population (913 patients seen at seven pediatric headache specialty clinics. Subjects were aged from 3 to 18 years, mean 11.9 6 3.4). They found that the prevalence of overweight patients at initial visit did not significantly differ from that of the general pediatric population. They also found that BMI percentile was significantly correlated with headache frequency and disability at initial visit. In a study of Kinik et al. (2010) the influence of obesity on the severity of migraine was investigated in 124 children (mean age 12.9 6 2.8 years; range 4.0
17.0 years). The authors hypothesized that obesity might be a risk factor for the frequency and severity of migraine attacks during childhood and adolescence. In this study, although the severity and duration of migraine attacks and migraineassociated symptoms (presence of an aura, nausea, vomiting, phonophobia or photophobia accompanying pain) was not significantly different between obese and non-obese patients, there was an association between obesity and migraine attack frequency in children and adolescents with migraine. Obese patients had migraine attacks more frequently than non-obese patients. In another adult migraine study, BMI was shown to influence some migraine-associated symptoms such as phonophobia and photophobia, but not nausea or aura. These associations also held true after correcting for several potential confounders (Bigal et al., 2006). In contrast, vomiting was identified as occurring more frequently in obese migraineur than in non-obese migraineur women (Kaplan, 2006). Findings from research are more consistent in supporting the link between obesity and migraine frequency, severity, and clinical features than between obesity and migraine prevalence. Despite this, there is also some evidence to suggest that obesity is associated with migraine prevalence but only in the adult population (Ford et al., 2008; Peterlin et al., 2010b).

Previous studies in adults (Kaplan, 2006; Peres et al., 2005; Pinhas-Hamiel et al., 2008; Horev et al., 2005) found a high incidence of migraine, especially migraine with aura, among morbidly obese women. It was thought that migraine with aura in women might be associated with high estrogen levels produced by adipose tissue (McGregor, 2004). Kaplan has also shown that obese women with migraine had more frequent and severe attacks (Kaplan, 2006). PinhasHamiel et al. reported that overweight girls had fourfold increased risk of headache when compared with normal weight girls (Pinhas-Hamiel et al., 2008). Further evidence for the connection between obesity and headache can be found in studies examining the progression of headache over time. In a populationbased study, obesity was the strongest predictor of progression from episodic to chronic headaches (Scher et al., 2002). BMI was also associated with the development of chronic migraine, suggesting that individuals who are obese are at risk for developing chronic headache. The reasons for the inconsistencies in the findings regarding the relationship between BMI and migraine prevalence are not entirely clear, but several methodological differences may underlie this variation. There has been a variety of methods used to define and diagnose migraine and to measure height and weight, thus creating difficulty in interpreting and comparing findings across studies. Furthermore, few studies have been conducted in the pediatric population. Finally, while there is stronger evidence to suggest that obesity exacerbates migraine, it is less clear whether obesity increases risk for having migraine. Additional population studies are needed, which use direct measures of obesity and standardized IHS criteria to diagnose migraine, in order to better address this important clinical and research question.

Comorbidity Both chronic migraine and obesity have been linked to increased risk of numerous medical and psychological conditions. Pediatric chronic migraine has been associated with functional disability (Fichtel and Larsson, 2002) and some research has suggested an increased risk of psychopathology (Powers et al. 2006), while pediatric obesity has been linked with type II diabetes, high blood pressure, various psychosocial difficulties (Erickson et al., 2000; Storch et al., 2007), and chronic pain conditions such as fibromyalgia (Yunus et al., 2002), and back and neck pain (Webb et al., 2003). Both conditions are independent risk factors for cardiovascular disease (Pi-Sunyer, 2009; Bigal et al., 2010)

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PROPOSED MECHANISM FOR THE RELATIONSHIP BETWEEN OBESITY AND MIGRAINE

and they have also been associated with reduced quality of life compared with healthy peers (Powers et al., 2006; Schwimmer et al., 2003).

Cognitive Profile, Migraine, and Obesity It has frequently been hypothesized that migraine and obesity may be associated with an increased risk of cognitive impairment. However, few studies have so far been conducted to assess the impact of these issues on neurocognitive performance in the pediatric population and, therefore, the possible effects of migraine and obesity on executive abilities remain controversial. Significant differences were found between the headache and control groups in the mean total intelligence quotient and verbal intelligence quotient scores (Parisi et al., 2010a and Parisi et al., 2010b). Children with migraine had impairment in many cognitive functions such as attention, memory, information speed, and perceptual organization (Moutran et al., 2011) compared to the control group. At any rate, it is not clear why the verbal comprehension subscale appears to be more clearly involved in migraine patients than other verbal skills, or why the verbal subscales are all involved and the performance subscales are not (Esposito et al., 2012; Parisi et al., 2010b). Performance intelligence quotient scores (sequential motor planning, visual perceptive skills, and ability to adapt to sudden environmental changes) (Parisi et al., 2010b; Reinert et al., 2013; Kamijo et al., 2012) were found to be markedly lower in overweight and obese groups than in the normal weight group. Unfortunately, the impact of both conditions on the cognitive profile was never assessed within a single study. It would be intriguing to investigate, first, whether early treatment of obesity and a prompt antiheadache therapy can prevent a possible decline of cognitive profile and, second, whether the cognitive profile associated with headache and obesity is reversible at any time during the course of the disease. Additionally, some data show that both headache (Arruda and Bigal, 2012) and obesity (Krukowski et al., 2009) can affect the performance of children at school.

PROPOSED MECHANISM FOR THE RELATIONSHIP BETWEEN OBESITY AND MIGRAINE Several potential mechanisms have been proposed to explain the link between migraine and obesity. As mentioned earlier, data from the literature are more

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consistent in supporting the evidence that obesity is linked principally to headache frequency and severity rather than to headache prevalence. However, recent research shows multiple areas of overlap between migraine and the central and peripheral pathways regulating feeding, suggesting a direct biochemical link between these two health problems. Given the recently characterized metabolic activity of adipose tissue (Bigal and Lipton, 2006) and the multiple metabolic abnormalities identified in migraine patients, the relationship between these two disorders could be partially explained by common inflammatory mediators. Neurogenic inflammation resulting from activation of the trigeminovascular system figures predominantly in the pain of migraine, resulting in autonomic and nociceptive nervous system dysfunction, coupled with neurogenic inflammation and neuropeptide dysregulation. It has been hypothesized that a phenomenon known as cortical spreading depression (CSD) is the underlying factor that causes migraine attacks (Dalkara et al., 2006; Moskowitz, 2007; D’Andrea and Leon, 2010); it stimulates the release of glutamate and nitrous oxide from brain and blood that activates the trigeminal meningeal nociceptors to release several neuropeptides (calcitonin gene-related peptide (CGRP), substance P, and neurokinin A), inducing vasodilation and leading to neurogenic inflammation and employment of macrophages and mast cells. Simultaneously, modulation of both central pain processing pathways, in the thalamus, gray matter of the midbrain and hypothalamus, as well as vascular and autonomic function, occurs via serotonin and catecholamines. So multiple neurotransmitters and hormones, including CGRP, substance P, neuropeptide Y, adiponectin, leptin, orexin-A, interleukin (IL)-1, IL-6, and tumor necrosis factor alpha (TNF-α) have all been implicated in migraine pathogenesis (Valenzuela et al., 2007; Peterlin et al., 2007; Guldiken et al., 2008; Ray and Kumar, 2009; Yilmaz et al., 2010; Rozen and Swidan, 2007) and different literature data demonstrate their link to both migraine and obesity. It has been found that CGRP levels are raised in obese adults’ plasma and in the external jugular venous blood of animal models of obesity (Zelissen et al., 1991; Peterlin et al., 2010a), and that exogenous CGRP administration in rat increases fat intake. CGRP levels seem to be elevated prior to the onset of obesity and its receptors are a major focus for pharmacological treatment of migraine (Ho et al., 2008). Substance P has been identified in adipose tissue and may contribute to amplification of fat depots and of the pro-inflammatory milieu that occurs in obesity (Perini et al., 2005).

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Moreover, an increase in pro-inflammatory cytokines such as TNF-α and IL-6, which are characteristic of obesity, has been found to occur at the onset of migraine attacks (Vanmolkot and de Hoon, 2007; Jang et al., 2010). Inflammatory and immune mediators including TNF-α, IL-1, and IL-6 are also involved in migraine pathogenesis; TNF-α is shown to be elevated in obesity, produced by excess adipose tissue (Ro¨nnemaa et al., 2000), and in menstrual migraine (Mueller et al., 2001). From a genetic point of view, the gain of function TNF-α-308 A and IL-1β 1 3953 T allele gene polymorphisms have been demonstrated in patients with migraine without aura (Yilmaz et al., 2010), although the implications are unclear. Similarly, IL-6 is elevated in migraine patients and is thought to lower the pain thresholds. Thus, the inflammatory state of obesity may exacerbate the inflammatory response in migraine, probably contributing to increases in headache frequency or severity (Bigal et al., 2007a; Bond et al., 2010). Adipocytes and associated macrophages are known to be metabolically active (Bigal and Lipton, 2006), producing many mediators of migraine. Adiponectin and leptin, hormones involved in the regulation of appetite and metabolism, play a central role in adipose tissue development and in metabolic alterations of obesity (Arita et al., 1999; Stofkova, 2009). Serum adiponectin levels have been inversely correlated with BMI (Arita et al., 1999) and adiponectin may mediate many of the inflammatory metabolites associated with migraine (Peterlin et al., 2007). Some authors have suggested the role of leptin in the hypothalamus in blocking the effect of feeding stimulants, such as neuropeptide Y, and reducing appetite (Peterlin, 2007). Elevated concentrations of leptin in obesity are associated with increased susceptibility to chronic inflammatory and autoimmune disease (Ferna´ndez-Riejos et al., 2010). Moreover, leptin deficiency is believed to affect neurodevelopment through a deficit in migration of neuronal lineage cells to the cortical plate and in neural differentiation (Buettner et al., 2008). In one study of episodic migraine subjects, leptin was found to be decreased compared to controls with similar BMI (Guldiken et al., 2008). In patients with acute inflammation, leptin levels are increased, but they decrease with chronic exposure to inflammation, suggesting that patients with a long history of frequent migraine would be at greatest risk of weight gain (Peterlin, 2007). Other neurotransmitters and peptides directed by the hypothalamus in the regulation of eating behavior also had a role in migraine pathophysiology. For example, serotonin is involved in control of energy intake, signaling the satiety state when it binds to 1B and 2C receptors. Migraine has been shown to be a

condition characterized by brain serotonin deficiency, except for transient rises in serotonin levels during headache attacks. So, the chronically low serotonin activity of migraineurs could contribute to increased caloric intake and weight gain. Serotonin play its role in feeding via orexin-A, a neuropeptide also associated with reward, arousal, physical activity, and control of blood glucose level. Orexin-A and B receptors have been studied in migraine. Orexin-A is thought to depress CGRP release (Holland et al., 2005), and orexin activation is thought to confer resistance to dietary-related features of metabolic syndrome (Funato et al., 2009). An orexin deficiency is supposed to promote inflammation in the trigeminal system (Bigal et al., 2007a). However, orexin levels were found to be elevated in the cerebrospinal fluid of chronic migraine patients (Sarchielli et al., 2008) and, from animal models, it has been shown that orexin administration in rat reduces perception of painful stimuli (Watson et al., 2010) and inhibits CGRP release from trigeminal neurons and neurogenic vasodilatation (Holland et al., 2005). Therefore, it has been purported that the presence of high levels of orexin-A in the cerebrospinal fluid of migraineurs is a compensatory response to chronic pain. In conclusion, both migraine and obesity independently give rise to an altered inflammatory and immune marker milieu, and further research will be required to elucidate how these two diseases interact.

Lifestyle Factors Lifestyle factors such as dietary habits have also been associated with both migraine and obesity. For example, certain lifestyle habits associated with obesity and weight gain, such as irregular meal frequency, consumption of a high-fat diet, and low physical activity have also been shown to be associated with increased risk of migraine (Molarius et al., 2008). Psychological and behavioral diseases represent another area of overlap between migraine and obesity. Major depression and anxiety, for example, have been shown to be most common in the obese migraineurs and to be associated with higher headache frequency and disability, suggesting that these disorders may modify the relationship between obesity and migraine (Tietjen et al., 2007). Psychological stress, moreover, is hypothesized to promote migraine onset or precipitate and aggravate headache attacks by increasing the sensitivity of the trigeminal system and through its maladaptive response in reducing the pain threshold (De Benedittis et al., 1990). At the same time its association with increased risk of obesity and weight gain has also been shown (Block et al., 2009), probably due to a reduced

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INFLUENCE OF WEIGHT LOSS ON CHRONIC HEADACHE IN OBESE PEOPLE AND EFFECTS

control of food intake, mediated by dysregulation of the hypothalamic pituitary adrenal axis (Bose et al., 2009). Another factor that influences both metabolism and nervous system function is sleep. Sleep disorders have been connected to high frequency migraines, metabolic syndrome, and obesity. In particular, short sleep duration and sleep fragmentation are more frequent in migraineurs (Odegard et al., 2010) and they are also linked, together with obstructive sleep apnea syndrome, to metabolic disease and obesity (Nielsen et al., 2010). Moreover, chronic autonomic abnormalities during both sleep and wakefulness have been demonstrated in adults and children with sleep disorders, caused by sympathovagal balance alterations and characterized by an increase in sympathetic nerve activity and impaired reaction to several physiological stimuli (Montesano et al., 2010). Sympathetic dysregulation plays a role in both migraine and obesity (Lambert et al., 2010; Peroutka, 2004). In migraine patients a sympathetic hypofunction has been shown during asymptomatic periods and a sympathetic hyperfunction during headache attacks, meanwhile obesity is related to sympathetic hyperstimulation (Bigal et al., 2007a; Lambert et al., 2010). Some authors considered that, as a result of sympathetic hypofunction, obese migraineurs may have difficulty in adapting to the elevated sympathetic tone associated with obesity and sleep disorders and are therefore more susceptible to an increased number of attacks (Bigal et al., 2007b). Food and diet can also act as environmental trigger factors on the occurrence of migraine attacks (Diamond et al., 1986; Millichap and Yee, 2003; Peatfield et al., 1984). IgE-specific food allergy has been shown to be related to migraine, supported by the success of individualized diet in controlling migraine attacks (Diamond et al., 1986; Millichap and Yee, 2003). Non-IgE antibody-mediated mechanisms have also been proposed in food allergy (Halpern and Scott, 1987). Aljada et al. (2004) provided evidence for the pro-inflammatory effect of food intake. IgG antibodies against food antigens have been found to be correlated with inflammation and intima media thickness in obese juveniles (Wilders-Truschnig et al., 2008). IgG could be one of the markers to identify food which causes inflammation and could cause migraine attacks in predisposed individuals, and at the same time could increase the inflammatory state in obese individuals. All of this indicates that there is a need for an individualized approach to diet to relieve migraine, and that this could be guided by determination of specific IgG antibodies against food antigens for prevention and cure of food-induced migraine attacks, leading to lower drug consumption, fewer adverse drug reactions, and fewer days with migraine (Alpay et al., 2010).

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INFLUENCE OF WEIGHT LOSS ON CHRONIC HEADACHE IN OBESE PEOPLE AND EFFECTS OF THE PREVENTIVE TREATMENT OF MIGRAINE ON WEIGHT CHANGE Findings from the literature show a clear correlation between migraine and obesity, both in adult and pediatric populations; they also give explanations about epidemiology, mechanisms, and implications. Study results suggested that weight loss may well prove to be a means of reducing headache frequency and severity in obese migraineurs. It could prevent or decrease the severity of migraine attacks by altering central nervous signaling pathways via increases in orexin-A (Bronsky et al., 2007), and through inflammatory pathways mediated in part by increases in adiponectin and decreases in leptin (Esposito et al., 2003; Williams et al., 1999). Associated reductions in inflammatory cytokines (Forsythe et al., 2008), CRP (Selvin et al., 2007), and sympathetic tone (Straznicky et al., 2010) may also play a role in altering the central and peripheral inflammatory cascades that lead to the pain of migraines. Additionally, weight loss-related improvements in psychological factors including mood (Williamson et al., 2009) and stress-coping ability (Westenhoefer et al., 2004) could help to alleviate migraine (Bigal et al., 2007a). Similarly, enhancement of sleep quality and disturbances that may occur with weight loss could reduce headache frequency and severity (Chaput et al., 2005; Calhoun and Ford, 2007). Decreases in fat consumption and increases in physical activity are strongly correlated with weight loss (Raynor at al., 2004; Wadden et al., 2009), and both behaviors have been shown to be associated with reduction in headache frequency and severity (Bic et al., 1999; Narin et al., 2003). Finally, weight loss may impact migraine in obese individuals via improvements in conditions that are comorbid to both disorders, such as hypertension and hyperlipidemia (Scher et al., 2005; The Look AHEAD Research Group, 2007), depression (Tietjen et al., 2007; Williamson et al., 2009), diabetes (Guldiken et al., 2009; Albu et al., 2010) and sleep apnea (Scher et al., 2003; Foster et al., 2009). Despite these plausible mechanisms linking obesity and migraine, few studies have examined the impact of weight loss on headache until now. Regarding adults, the observation concerned only cases of surgical intervention; the results are briefly reported in the following lines. Surgerman et al. (1999) showed the effects of bariatric surgery on idiopathic intracranial hypertension, an obesity-related disease of which severe, persistent headaches is a symptom. Weight loss at 4 months post-surgery was associated with resolution of

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headaches in all but 1 of 19 female patients; in addition, headaches recurred in two patients who had subsequent weight regain. Similarly Bond et al. (2011) examined whether headache measures were improved in severely obese migraineurs at 6 months after bariatric surgery. A total of 29 patients proceeded to surgery. Twenty-four (83%) of these completed the 6-month postoperative follow-up. They found that headache frequency was markedly reduced from before to 6 months postoperatively, with nearly half (46%) of participants showing at least a 50% reduction, regarded as the gold standard for treatment outcome. The chances of experiencing a $ 50% improvement in headache frequency were higher in participants who had greater weight loss, regardless of the type of surgery that was performed. Remarkably, headache improvements occurred postoperatively despite the fact that 70% of participants remained obese, suggesting that weight loss can help alleviate migraine in the absence of resolution of obesity. At the same time a reduced frequency of migraine attacks was found with improvement of headacherelated disability post-bariatric surgery in premenopausal obese women with migraine (Novack et al., 2011). Post-bariatric surgery, the migraine-suffering women reported a lower frequency of migraine attacks (p , 0.001), shorter duration of the attacks (p 5 0.02), lower medication use during the attack (p 5 0.005), less non-migraine pain (44.8% vs. 33%, p 5 0.05), and postbariatric surgery reduction in headache-related disability assessed by the MIDAS and HIT-6 scores. There was a reduction in migraine frequency among both

TABLE 23.1

episodic (from four to one episodes a month) and chronic (from 16.8 to 8.5 episodes per month) migraine patient cohorts, separately and combined. For pediatric patients, the experimentation moved towards direct observation of changes in headache development, in relation to weight reduction achieved without surgical intervention. Hershey et al. (2009) examined the effect of weight change on various headache parameters in 913 pediatric headache sufferers (71% of whom who had migraine). BMI percentile was positively associated with headache frequency at the initial visit. Greater decreases in BMI were associated with greater reductions in headache frequency at 3- and 6-month followup for children who were initially overweight or obese, but not for those who were of normal weight. A further analysis by Verrotti and colleagues (2013) which organized a 12-month program consisting of the following three arms: balanced diet in agreement with the energy and nutrient dietary reference values; specific aerobic activity, based on the recommendations for treatment of adolescent obesity; and cognitivebehavioral training in groups of eight to 10 participants (4 monthly sessions). The program studied 135 patients (78 female and 57 male; mean age 6 SD, 15.9 6 0.9 years) were evaluated. Their data demonstrate significant improvements in both adiposity and headache variables 6 months after the intervention program that were maintained through 12 months (see Table 23.1). They confirmed that obesity is linked with migraine and that a decrease in BMI is associated with a reduction of migraine. In addition, they observed that

Adiposity and Headache Data Before and After Intervention Program

Variables

Baseline

After 6 Months

After 12 Months

85.2 6 8.2

78.1 6 7.3

76.9 6 9.1

BMI (kg/m2)

32.9 6 4.6

30.5 6 5.1

29.9 6 6.0

Waist circumference (cm)

103.4 6 15.4

98.6 6 18.7

97.8 6 19.9

5.3 6 2.1

2.4 6 1.1

2.2 6 0.9

6.7 6 3.8

6.3 6 4.6

5.9 6 3.4

ADIPOSITY VARIABLES Weight (kg)

HEADACHE VARIABLES Headache frequency (per month) Headache duration (h)



Headache intensity (10-point scale)

7.4 6 1.7

3.9 6 2.1

4.2 6 2.5

Migraine-associated symptoms (%)

84

79

82



Use of acute medication (%)

62

52

49

PedMIDAS score

27.5 6 12.7

21.3 6 13.9

19.7 6 10.8



P , 0.05. P , 0.01 versus baseline values. BMI, body mass index; PedMIDAS, Pediatric Migraine Disability Assessment Score. Values are expressed as means 6 SD and percentages. Adapted from Verrotti et al., 2013, with permission.



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REFERENCES

baseline BMI and change in BMI had a predictive impact on headache frequency, headache intensity, use of acute medications, and PedMIDAS 12 months after treatment. BMI-related changes in headache parameters were independent of acute headache medication use. These findings demonstrate that a weight management strategy should be incorporated within a migraine treatment plan for adolescents who are obese. To have a full picture of the situation, it is also useful to follow the development of studies into the effects of weight loss without surgery in adults too, such as the one started by Bond et al. (2013). However, although the prevailing achievements bring an affirmative answer to the question posed at the beginning of the paragraph, these results should be taken cautiously: further studies are needed to evaluate the possible specific effects of weight loss on migraine in obese patients. Finally, there is another interesting point related to whether the pharmacological treatment of migraine has some kind of influence on weight. Maggioni et al. (2005) found that drugs used for prophylactic therapy of primary headaches can cause undesirable effects with longterm treatment; weight gain is often observed in clinical practice, which in some cases can become a relevant problem. For 89 patients the study was extended to 6 months in monotherapy with the following drugs: amitriptyline (20 mg and 40 mg), pizotifen (1 mg), propranolol (80
160 mg), atenolol (50
100 mg), verapamil (160
240 mg), valproate (600 mg) and gabapentin (900
1200 mg). Weight variations of $ 1 kg were considered. After 6 months of therapy, the percentage of patients with weight increase was 86% with pizotifen (6/7; mean weight increase 4.4 6 2.5 kg), 60% with amitriptyline 20 mg (6/10; 3.1 6 1.6 kg), 47% with amitriptyline 40 mg (9/19; 5.4 6 2.7 kg), 25% with verapamil (1/4, 2.5 kg), 20% with valproate 600 mg (2/10, 3.0 6 2.8 kg), 20% with atenolol (3/15, 1.7 6 0.6 kg), 9% with gabapentin (1/11, 1.5 kg), and 8% with propranolol (1/13; 6 kg). Furthermore William and Young (2008) made an exhaustive classification of the effects of the various migraine prophylactics on weight variations. Those which give weight gain are methysergide, β-blockers, amitriptyline, fluoxetine, nortriptyline and paroxetine among antidepressants, divalproex sodium and gabapentin between antiepileptic drugs, and flunarizine as a calcium channel blocker. The ones that reduce weight are instead protriptyline and zonisamide. Venlafaxine, duloxetine, lamotrigine, and verapamil have no effects on weight. Different medications within each drug class have widely divergent effects on weight. Age and diagnosis may also influence the magnitude of a drug’s effect on weight. Thus the physician should take potential consequences into account when prescribing headache medication, particularly when weight changes may be

associated with the exacerbation (or improvement) of comorbid metabolic disorders.

CONCLUSIONS Obesity and migraine may be linked in several ways. Indeed both are influenced by genetic and inflammatory mediators that are increased in obese individuals. It is crucial to identify the numerous factors and mechanisms that contribute to the comorbidity of obesity and several types of headache. Moreover, a multi-faceted relationship between migraine, specific food intake-related cortico-subcortical neuronal networks, hormones, neuropeptides, inflammatory mediators, BMI, and cognitive profile does exist. So, multiple interventions such as, for example, modifying BMI to reduce headache frequency and severity in obese migraineurs, may provide useful therapeutic models. Accordingly, recent research has shown multiple areas of overlap between migraine and the central and peripheral pathways regulating feeding and behavior, suggesting a direct biochemical link between these two health problems.

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24 Dietary Omega-3 Sources during Pregnancy and the Developing Brain: Lessons from Studies in Rats Caroline E. Childs and Philip C. Calder INTRODUCTION The most common dietary omega-3 fatty acid is the essential fatty acid α-linolenic acid (ALA; 18:3n-3), with good dietary sources of this including plant seeds, seed oils (especially flaxseed oil, also known as linseed oil), and some nuts. ALA is a substrate for the production of long-chain omega-3 polyunsaturated fatty acids (PUFAs) including eicosapentaenoic acid (EPA; 20:5n-3), docosapentaenoic acid (DPA; 22:5n-3), and docosahexaenoic acid (DHA; 22:6n-3) (Leonard et al., 2004, Figure 24.1). Alternatively, these longchain omega-3 PUFAs can be consumed directly from food sources such as oily fish. Animal studies have demonstrated that consuming a diet deficient in omega-3 fatty acids during pregnancy and lactation results in neurological abnormalities in offspring, such as impairments of cognitive and visual function (Brenna, 2011; Lauritzen et al., 2001) and that these impairments are associated with a reduction in brain DHA content. DHA is a major component of the brain and retina, and it accumulates within the fetal rat brain during pregnancy and during the post-natal period (Green et al., 1999), peaking at around 6 weeks of age (Childs et al., 2011). In humans, transfer of DHA to the developing fetus occurs predominantly in the last 10 weeks of pregnancy, with the majority of this DHA accumulated within fetal adipose tissue in order to support brain and retinal development during the first months of post-natal life (Haggarty, 2004). There is significant interest in whether dietary provision of pre-formed DHA is advantageous in maximizing brain DHA content compared to a diet replete

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00024-7

in its precursor ALA. It has been hypothesized that direct provision of DHA will prevent any limitations in either the maternal or fetal endogenous synthesis pathways from adversely affecting brain development. The series of desaturase and elongase enzymes which generates DHA from ALA (Leonard et al., 2004; Figure 24.1) is also involved in the metabolism of the omega-6 PUFA linoleic acid (LA; 18:2n-6) into its longer-chain, more unsaturated derivatives (e.g. arachidonic acid, AA, 20:4n-6). In Western diets, consumption of LA is about ten times that of ALA (Blasbalg et al., 2011; Burdge and Calder, 2006), suggesting that synthesis of omega-6 PUFA will predominate over that of omega-3 PUFA, and that the ability to synthesize DHA from ALA may be limited. Indeed, studies with stable isotopes in humans have indicated that human infants convert just 1% of ALA into DHA, and that this capacity is significantly lower in adults (Brenna et al., 2009). If pre-formed dietary DHA is required for infant development, this has significant public health implications, as the current recommended intake of EPA 1 DHA for adults in the UK is a minimum of 450 mg/day, with oily fish as the major source of these two fatty acids, yet it is estimated that only 27% of UK adults and less than 10% of UK children and adolescents habitually eat oily fish (Scientific Advisory Committee on Nutrition, 2004). The use of studies of rat pregnancy to inform knowledge and understanding of human pregnancy has both limitations and advantages. The developmental maturity of rats and humans is significantly different at the time of birth. In terms of relative size, the rat reaches a proportion of adult size comparable to that of a newborn human at 12 days post-partum,

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24. DIETARY OMEGA-3 SOURCES DURING PREGNANCY AND THE DEVELOPING BRAIN: LESSONS FROM STUDIES IN RATS

18:3n-3

18:2n-6

α-linolenic acid (ALA)

linoleic acid (LA) Δ6 desaturase

18:4n-3

18:3n-6

stearidonic acid

γ-linolenic acid Elongase

20:4n-3

20:3n-6

eicosatetraenoic acid

di-homo-γ-linolenic acid Δ5 desaturase

20:5n-3

20:4n-6

eicosapentaenoic acid (EPA)

arachidonic acid (AA)

Elongase

22:5n-3

22:4n-6

docosapentaenoic acid (DPA)

docosatetraenoic acid

Elongase

24:5n-3

24:4n-6

tetracosapentaenoic acid

tetracosatetraenoic acid Δ6 desaturase

24:6n-3

24:5n-6

tetracosahexaenoic acid

tetracosapentaenoic acid β-oxidation

22:6n-3

22:5n-6

docosahexaenoic acid (DHA)

docosapentaenoic acid

FIGURE 24.1 Biosynthesis of long-chain polyunsaturated fatty acid from ALA and LA. ALA, α-linolenic acid; LA, linoleic acid.

indicating that the rat is significantly less developmentally mature at birth compared to a human infant (Quinn, 2005). In terms of brain development, comparative studies indicate that the rat cerebral cortex corresponds to that of a newborn infant at day 12
13 postpartum (Romijn et al., 1991). This suggests that rat models using interventions during pregnancy may be considered suitable models for early prenatal brain development in humans, and only studies which include interventions which continue to at least day 12 post-partum (i.e. during lactation) can be hypothesized as a model of interventions during full-term human gestation. While there are many differences between human and rat pregnancies, including the much larger litter size among rats, with a corresponding increased relative lactation burden, and shorter duration of lactation, rat models allow for far more stringent and/or extreme dietary manipulation than is possible in humans, which is essential for hypothesis testing and mechanistic studies. Use of a rat model, for example, allows for the possibility of timed interventions from

the moment of, or even before, conception, and greater access to developing tissues than would ever be possible in human studies. Here, we review available data from rat studies which have provided supplementary dietary ALA or long-chain omega-3 PUFA during pregnancy and/or lactation, which include measures of brain fatty acid composition. Five papers which provided supplementary ALA were identified, nineteen which provided long-chain omega-3 PUFA, and six which directly compared the efficacy of ALA and long-chain omega-3 PUFA. Data from studies where rats were provided with omega-3 PUFA-deficient diets were not included, as these have been reviewed in detail elsewhere (Brenna, 2011; Lauritzen et al., 2001).

ALA SUPPLEMENTATION AND BRAIN FATTY ACID COMPOSITION Based on the data from the five papers which provided dietary ALA in excess of the amount typically included in laboratory rat food, it was clear that the duration of the nutritional intervention was key to whether brain fatty acid composition was influenced (Table 24.1). In the studies where dietary interventions were provided throughout pregnancy, and brain samples were collected from offspring at birth, data indicate that increased dietary ALA can significantly increase brain long-chain omega-3 PUFA status, with a higher brain content of EPA (Cheon et al., 2000), DPA (Fernandes et al., 2011; Guesnet et al., 1997; Lenzi Almeida et al., 2011) and DHA (Cheon et al., 2000; Fernandes et al., 2011; Guesnet et al., 1997; Lenzi Almeida et al., 2011) compared to controls. An effect of diet on long-chain omega-6 PUFA status was also observed, with lower AA (Cheon et al., 2000; Fernandes et al., 2011; Lenzi Almeida et al., 2011), 22:4n-6 (Cheon et al., 2000; Lenzi Almeida et al., 2011), and 22:5n-6 (Guesnet et al., 1997) content (Table 24.1). Only one study provided information on the longevity of any effect by providing ALA for a limited period and taking samples after animals returned to a control diet (Rao et al., 2007). In this study, rats were provided with additional ALA only during pregnancy. In offspring post-weaning, no significant differences in brain fatty acid composition were apparent compared to controls, although it should be noted that only male pups were considered in the analysis. The study by Cheon et al. (2000) indicated that there were subtle sex differences in the offspring response to maternal dietary ALA, as female pups at birth did not display the reductions in AA and 22:4n-6 seen in males, and had a higher ALA, EPA, and DHA at weaning compared to controls.

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

TABLE 24.1 Rat Studies of ALA Supplementation Which Reported Effects Upon Brain Fatty Acid Composition Duration of Intervention

Brain Samples Analyzed

Fernandes Wistar Control (7% w/w soybean et al., 2011 oil) Flaxseed (10% w/w flaxseed oil) Modified control (10% w/ w soybean oil)

Diets to males and females from weaning, rats mated and diet continued throughout pregnancy, lactation, and post-weaning

Total fatty acids in Flaxseed vs. modified control hippocampus at (% total fatty acids): birth m18:1n-9, LA, DPA, DHA k18:0, ALA, AA, EPA

At day 30 post-partum: Flaxseed vs. control: kTime taken to complete Morris water maze test (spatial memory) on day 1
3 and day 5 Flaxseed vs. control/modified control: mTime spent in correct quadrant of Morris water maze when platform removed (spatial memory storage)

Lenzi Wistar 2.5% w/w Flaxseed Almeida 0.7% soybean oil 1 casein et al., 2011 1% Soybean oil 1 modified casein

Diets to males and females from weaning, rats mated and diet continued throughout pregnancy

2hours postpartum, whole brain

Flaxseed vs. casein (% total fatty acids): m11:0, 15:0, 17:1n-9, 18:1n-9, trans-LA, LA, 18:3n-6, 20:3n-6, 20:3n-3, 22:2n-9, 24:0, DPA, DHA k14:1n-9, 15:1n-9, 16:1n-9, 18:0, trans 18:1n-9, 22:1n-9, AA, EPA, 22:4n-6, 24:1n-9 Flaxseed vs. modified casein (% total fatty acids): m15:0, 17:1n-9, 18:1n-9, LA, 20:3n-6, 20:3n-3, 22:2n-9, DPA, DHA k12:0, 13:0, 16:1n-9, 18:0, trans 18:1n9, ALA, AA, EPA, 22:4n-6, 24:1n-9

Brain weight and relative brain weight in flaxseed diet significantly higher than casein or modified casein diet

Rao et al., 2007

During pregnancy, returned to control diet during lactation

Brain membranes of male pups at weaning

No significant differences between any group at weaning

Treatment II vs. control: kabsolute brain weight in male pups mrelative brain weight in male pups

Study

Rat Model Dietary Supplement

Wistar Control (18% protein, 7% soybean oil w/w) Treatment I (12% protein, 7% soybean oil w/w) Treatment II (3% flax oil, 4% soybean oil w/w)

Effects on Brain Fatty Acid Composition

Effects Reported on Brain Functional Outcome Measures

(Continued)

TABLE 24.1 (Continued) Study Cheon et al., 2000

Rat Model Dietary Supplement

Brain Samples Analyzed

Effects on Brain Fatty Acid Composition

Effects Reported on Brain Functional Outcome Measures

Liquid diets providing 35% One week prior to mating and throughout pregnancy, lactation and energy from fat, with weaning varying LA:ALA ratio 1.07 (LA1) 2.64 (LA2) 4.45 (LA3) 7.68 (LA4) 10.35 (LA5)

Male and female pups at 0, 3, 8, and 16 weeks old

Male pups, LA1 vs. LA5 (% total fatty acids): mLA (8, 16), ALA (0, 8, 16), EPA (0, 8, 16), DHA (0) kLA (3), AA (0, 3, 8, 16), 22:4n-6 (0, 3, 8, 16) Female pups, LA1 vs. LA5 (% total fatty acids): mLA (16), ALA (0,3,8,16), EPA (0,3,8,16), DHA (0,3) kLA (3,8), AA (3,8), 22:4n-6 (8,16)

No significant differences between LA1 and LA5 in Morris water maze or Open Field test

2 weeks prior to mating and throughout pregnancy and lactation

Brain phospholipids of pups at day 1, 3, 7, and 14 postpartum

In one-day-old pups, ALA vs. control (% total fatty acids): Dose dependent m in DHA content Dose dependent k in 22:5n-6 content. mDPA at 400 mg and 800 mg DHA content plateaued on day 7 and 14 post-partum among pups fed .200 mg / 100 g diet, while 22:5n-6 reached lowest levels

N/A

Guesnet Wistar 5% fat content w/w with et al., 1997 varying ALA content per 100 g diet: 5 mg 100 mg (0.22% energy) 200 mg (0.45% energy) 400 mg (0.9% energy) 800 mg (1.8% energy)

Duration of Intervention

LONG-CHAIN OMEGA-3 PUFA SUPPLEMENTATION AND BRAIN FATTY ACID COMPOSITION

In terms of neurological outcome measures, supplementary ALA had inconsistent effects. For example both higher absolute brain weight (Lenzi Almeida et al., 2011) and lower absolute brain weight (Rao et al., 2007) have been reported compared to controls. However, both these studies identified that there was increased brain weight relative to body weight, indicating an influence of dietary ALA on the pattern of fetal growth. No consistent effects on cognitive function with increased dietary ALA were observed, but available data are limited to just two studies. Fernandes et al. (2011) reported that performance in the Morris water maze was significantly greater among offspring of ALA-supplemented dams, while Cheon et al. (2000) reported no differences in Morris water maze or Open Field Test performance between offspring of dams fed a control or an ALA-rich diet.

LONG-CHAIN OMEGA-3 PUFA SUPPLEMENTATION AND BRAIN FATTY ACID COMPOSITION Twenty papers were identified where long-chain omega-3 PUFAs were provided to rats and brain fatty acid composition of offspring was assessed (Table 24.2). Of these, nine included interventions during pregnancy only (Childs et al., 2011; Glozman et al., 1999; Ikeno et al., 2009; Innis and de la Presa Owens, 2001; Joshi et al., 2004; Martin et al., 2004; Roy et al., 2012; Sable et al., 2012; Schiefermeier and Yavin, 2002), four during pregnancy and lactation (Amusquivar et al., 2000; Ho¨gyes et al., 2003; Sable et al., 2012; Suganuma et al., 2010), seven during pregnancy, lactation, and the post-weaning period (Haubner et al., 2002; 2007; Ozias et al., 2007; Saste et al., 1998; Stockard et al., 2000; Trevizol et al., 2013; Yonekubo et al., 1993) and two during lactation only (Amusquivar et al., 2000; Yeh et al., 1993). Providing additional dietary long-chain omega-3 PUFA during pregnancy significantly increased brain DHA content (Childs et al., 2011; Glozman et al., 1999; Ikeno et al., 2009; Innis and de la Presa Owens, 2001; Martin et al., 2004; Roy et al., 2012; Schiefermeier and Yavin, 2002; Trevizol et al., 2013), and in one study was also associated with increased brain EPA content (Ikeno et al., 2009). The long-chain omega-6 PUFA content of brain was significantly lower among rats fed a diet containing long-chain omega-3 PUFA, with lower AA (Ikeno et al., 2009; Innis and de la Presa Owens, 2001; Martin et al., 2004; Roy et al., 2012; Trevizol et al., 2013), 22:4n-6 (Innis and de la Presa Owens, 2001; Schiefermeier and Yavin, 2002; Trevizol et al., 2013) and 22:5n-6 (Innis and de la Presa Owens, 2001; Schiefermeier and Yavin, 2002; Trevizol et al., 2013)

291

reported. Three of these studies involved intervention during pregnancy only, with offspring followed up post-natally, enabling the duration of any effect arising from this discrete dietary intervention to be evaluated (Childs et al., 2011; Ikeno et al., 2009; Joshi et al., 2004). In the study by Ikeno et al. (2009), offspring at 1 week still had significantly higher DHA content and lower AA content compared to controls. Childs et al. (2011) found that a higher DHA content was only apparent until offspring were aged 3 weeks. Joshi et al. (2004) evaluated offspring at 6 months of age, and found no significant differences in brain long-chain omega-3 PUFA content, but did identify that water maze performance was significantly better among offspring from dams fed a long-chain omega-3 PUFA enriched diet. Studies which report additional brain biochemistry outcomes identified that a long-chain omega-3 PUFA rich diet significantly reduced brain monoamine concentrations, but only when compared to an omega-6 PUFA rich diet group (Innis and de la Presa Owens, 2001), and increased brain malondialdehyde content (Roy et al., 2012). All studies which provided supplementary longchain omega-3 PUFA during pregnancy and lactation reported significantly higher brain DHA and lower AA (Amusquivar et al., 2000; Ho¨gyes et al., 2003; Sable et al., 2012; Suganuma et al., 2010). In addition to the changes in brain fatty acid composition, three studies identified potential benefits and adverse effects of long-chain omega-3 supplementation upon outcome measures. Dietary long-chain omega-3 PUFA delayed the appearance of offspring developmental markers (Amusquivar et al., 2000), but reduced markers of hypoxic damage (Suganuma et al., 2010) and lowered levels of damage seen after toxic brain stimulation (Ho¨gyes et al., 2003). Two studies were designed to investigate the effect of supplementing the diet during pregnancy alone versus supplementing during pregnancy and/or lactation (Amusquivar et al., 2000; Sable et al., 2012). Neither of these studies reported significant differences in brain fatty acid composition among offspring from dams only provided with long-chain omega-3 PUFA during pregnancy, indicating that continued feeding during the lactation period results in greater changes to brain long-chain PUFA content. Those studies which provided a long-term dietary intervention throughout pregnancy, lactation, and post-weaning (Haubner et al., 2002; 2007; Ozias et al., 2007; Saste et al., 1998; Stockard et al., 2000; Trevizol et al., 2013; Yonekubo et al., 1993) found increased brain long-chain omega-3 PUFA content and lower long-chain omega-6 PUFA content in accordance with those studies conducted during pregnancy and lactation alone. Several of these longer duration studies

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

TABLE 24.2 Studies of Long-chain PUFA Supplementation Study

Rat Model

Dietary Supplement

Duration of Intervention

Brain Samples Analyzed

Effects on Brain Fatty Acid Composition

Effects Reported on Other Brain Outcome Measures

Trevizol et al., 2013

Wistar

3 g/kg p.o. soybean oil, fish oil, Six weeks prior to hydrogenated vegetable fat conception, throughout pregnancy and lactation, provided to male pups until 90 days old

Male pups aged 90 days: total lipid from cortex, hippocampus, and striatum

Fish oil vs. soybean oil (% total fatty acids): m17:1n-7 (hippocampus, striatum), LA (striatum), DPA (cortex, striatum), DHA (cortex, hippocampus, striatum). k18:0 (cortex), 18:1n-7 (cortex, striatum), 20:1n-9 (striatum), LA (hippocampus), AA (cortex, striatum), 22:4n-6 (cortex, hippocampus), 22:5n-6 (cortex)

Amphetamine (AMPH) -induced mania: mExploratory activities after AMPH in all groups except the fish oil treated rats. mRecognition memory in fish oil group. kReactive species generation in hippocampus and protein carbonyl levels in hippocampus and striatum among fish oil group

Sable et al., 2012

Wistar

7% w/w content: Control (NF/B) 7% w/w soybean oil, omega-3 supplemented (NF/BD/O) 2.5%, soybean oil, 4.5% MaxEPA fish oil (0.5% EPA, 0.3% DHA)

Throughout pregnancy; Throughout pregnancy and lactation

Total brain membranes, day 22 post-partum.

Omega-3 supplemented vs. control throughout pregnancy and lactation (g/100 g): mDHA kAA Omega-3 supplemented vs. control during pregnancy only: No significant differences in brain membrane composition

No effect of omega-3 supplementation on brain-derived nerve growth factor or nerve growth factor levels

Childs et al., 2011

Wistar

13% w/w fat content Soybean oil Sunflower oil Salmon oil

Pregnancy

Brain PE, day 20 gestation, 3, 6, 9, 12 weeks post-partum

Salmon vs. soybean/sunflower (% total fatty acids). mDHA at day 20 and week 3 only

N/A

Roy et al., 2012

Wistar

Omega-3 supplementation used Throughout pregnancy alongside B12 deficiency. 7% w/w content: Vit B12 deficient Vit B12 deficient 1 omega-3 High folate, B12 deficient High folate, B12 deficient 1 omega-3

Total brain membranes, day 22 gestation

Vit B12 deficient 1 omega-3 vs. Vit B12 deficient (g/100 g): mDHA kAA High folate, vit B12 deficient 1 omega-3 vs. High folate, vit B12 deficient (g/100 g): mDHA kAA

No effect on relative brain weight. Vit B12 deficient 1 omega-3 vs. Vit B12 deficient: mBrain malondialdehyde

Suganuma et al., 2010

Wistar

Ikeno et al., 2009

Total fat content not specified. Control (soybean) DHA-enriched (soybean 1 fish oil)

Day 7 gestation
post-natal day 14

Post-natal day 7, total brain phospholipids

DHA vs.control (% fatty acids): mDHA kAA

At post-natal day 7 all rats subjected to cerebral hypoxic-ischemia DHA vs. control: kTUNEL-positive cells (neurons) at day 10 kCapsase-3 immunoreactivity (marker of neurons undergoing apoptotic cell death) at day 8, 10, and 14. Confirmed with lower Western blot analysis. k8-OHdG immunoreactivity (marker of oxidative DNA injury) at day 10 and 14

Sprague- Soybean oil diet (5.8% w/w) Day 7 gestation
Dawley DHA-enriched diet (5.5% w/w) delivery. IUGR induced at day 14 gestation

Day 1 and 7 postdelivery, brain phospholipids

IUGR DHA-enriched vs. IUGR soybean oil (% phospholipid), day 1: mEPA, DHA kAA IUGR DHA-enriched vs. IUGR soybean oil (% phospholipid), mday 7: mDHA kAA

N/A

Haubner et al., 2007

Not 10% w/w fat content: specified 0% DHA 0.3% w/w DHA 0.7% w/w DHA 3% w/w DHA

Day 2 gestation, throughout pregnancy, lactation, and weaning

Myelin fatty acid composition of whole brain at post-natal day 24

3% DHA vs. 0% DHA (g/100 g): mLA, 20:3n-6, DPA, DHA kAA, 22:5n-6

Auditory startle latency at postnatal day 15: Significantly longer latency on 0.7% and 3% DHA diets vs. control

Ozias et al., 2007

LongEvans

7% w/w soybean oil 7% w/w sunflower oil 6.8% w/w soybean oil 1 0.2% w/w DHASCO

From mating and throughout weaning for four litters

Total brain phospholipids

N/A Soybean 1 DHASCO vs. Soybean (area %): No significant differences in DHA content at weaning of 1st, 3rd or 4th litter offspring. 2nd litter: m DHA Soybean 1 DHASCO vs. sunflower (area %) among 1st litter offspring: mDHA k22:5n-6

Joshi et al., 2004

Wistar

All 7% w/w fat content I: 18% protein II: 12% protein, folic acid deficient III: 12% protein, folic acid supplemented IV: 12% protein, fish oil supplemented

Day 1 gestation
delivery

Offspring at 6 months

Group 4 vs. Group 1 (g/100 g): Brain phospholipids: m18:3n-6 (among male offspring only)

Group 4 vs. Group 1: Significantly lower time spent in water maze at 6 months of age among male pups on days 2
5 of 5 consecutive days of testing

(Continued)

TABLE 24.2 (Continued) Study

Rat Model

Dietary Supplement

Duration of Intervention

Brain Samples Analyzed

Effects on Brain Fatty Acid Composition

Effects Reported on Other Brain Outcome Measures

Conception
Delivery

Forebrain phospholipids (PE, PC, PS/PI) at birth

Low protein 1 DHA vs. low protein (weight %): mDHA (PE, PC, PS/PI) kAA (PE, PC)

N/A

Phospholipids (PE, PS, PC, PI) of neural membranes at day 12 post-delivery

Supplement vs. placebo (% fatty acids) m18:1n-9 (PE), AA (PC), DHA (PE, PS, PC, PI) k16:0 (PE), 16:1n-9 (PC), 18:1n-9 (PC) AA (PE), 22:5n-6 (PE, PS)

At day 14 post-delivery pups were exposed to toxic brain stimulation. Long-chain PUFA supplemented rats lost fewer cholinergic cells and neurons compared to controls and had a lower degree of cholinergic fibre degeneration.

Cerebrum post-natal day 3 (g/100 g): 1% DHA vs. 0% DHA m20:3n-6, DHA k22:5n-6 3% DHA vs. 0% DHA m20:3n-6, EPA, DPA, DHA kAA, 22:4n-6, 22:5n-6 Brainstem post-natal day 31 (g/100 g) 1% DHA vs. 0% DHA mLA, DHA k16:0, 18:0, AA, 22:4n-6, 22:5n-6 3% DHA vs. 0% DHA mLA, DPA, DHA k16:0, AA, 22:4n-6, 22:5n-6

Significantly higher auditory brainstem conduction time in rat pups in 3% DHA group vs. 0% DHA at post- natal day 24 and 31. Auditory startle reflex appeared significantly later in the 3% DHA group vs. 0% DHA group

Martin et al., 2004

Wistar

10% w/w fat content low protein, 10% corn oil normal protein, 10% corn oil (control) low protein, 8.5% corn oil 1 1.5% DHASCO

Ho¨gyes et al., 2003

Wistar

Supplement formula (5% w/w 1 week prior to containing DHA, EPA and AA) conception
end of Placebo (5% w/w containing lactation LA and ALA) Control

Haubner et al., 2002

Sprague- 10% w/w soybean:olive oil Dawley blend diets with various DHA content 0% DHA 1% DHA 3% DHA

Day 2 gestation
end of lactation, diet maintained at weaning

Pup cerebrums at post-natal day 3, brainstem at postnatal day 31

Schiefermeier and Yavin, 2002

Wistar

All 5% w/w content: 5% sunflower oil 5% w/w soybean oil 2.5% soybean oil 1 2.5% DHATG

Day 15
20 gestation

Offspring at day 20, DHA supplemented vs. control (% brain phospholipids fatty acids): (PE, PE, PS) m18:1n-9 (PC), DHA (PC, PE, PS) kAA (PC, PE), 22:4n-6 (PC, PE, PS), 22:5n-6 (PC, PE, PS)

Innis and de la Presa Owens, 2001

Wistar

All 2% w/w content: Safflower oil Soybean oil High fish (tuna) oil

One week before mating Growth cone and throughout membranes from gestation brains collected within 12 hours after birth

High fish (tuna oil) vs. soybean oil (g/100 g): mDHA in growth cone PE, PS, PI kLA in growth cone PC kAA in growth cone PC, PE, and PS k22:4n-6, 22:5n-6 in growth cone PC, PE, PS, PI High fish (tuna oil) vs. safflower oil (g/100 g): mDHA in growth cone PC, PE, PS, PI kLA in growth cone PC, PE kAA in growth cone PC, PE, and PS k22:4n-6, 22:5n-6 in growth cone PC, PE, PS, PI

N/A

High fish (tuna oil) vs. soybean oil: No significant differences in brain monoamine concentrations. High fish (tuna oil) vs. safflower oil kdopamine and 3,4dihydroxyphenylacetic acid

Sprague- 10% w/w content: Dawley Fish oil Olive oil

Throughout pregnancy and/or lactation

Brain phospholipid, day 21 post-partum

Stockard et al., 2000

Sprague- DHA 2% total fatty acids Dawley DHA 4% total fatty acids DHA 6% total fatty acids

Day 2 gestation
end of lactation, pups weaned onto corresponding maternal diet

Total lipid extract of 6% DHA vs. 2% DHA (g/100 g): cerebrum at postDay 3: natal day 3 and 29 m18:1n-9, 20:3n-6, EPA, DPA, DHA kAA, 22:4n-6, 22:5n-6 Day 29: m18:1n-9, LA, 20:3n-6, EPA, DPA, DHA k16:0, 18:0, AA, 22:4n-6, 22:5n-6

Dose-dependent increase in auditory brainstem conduction time at day 31

Glozman et al., 1999

Wistar

Administered at day 17
20 gestation

Total lipid of fetal rat brain, brain PE at day 20

Et-DHA vs. control (% total fatty acids) mDHA in total lipid and PE

Et-DHA reduced TBARS production (indicator of oxidative stress) in fetal brain after stimulation with Fe21 up to 72 hr post injection. Et-DHA treatment reduced TBARS production following 20 minute ischemia episode (placental blood flow restriction)

Saste et al., 1998

Sprague- All 10% w/w content: Day 2 gestation
end Dawley Reference diet of lactation, pups Synthetic diet
corn oil weaned onto Synthetic diet
menhaden fish corresponding maternal oil diet

Cerebrum collected post-natal day 3

Fish oil vs. corn oil (g/100 g): m18:1n-9, EPA, DPA, DHA kLA, 20:3n-6, AA, 22:4n-6, 22:5n-6

Fish oil vs. corn oil: Significant effects upon brainstem auditory pathway. Auditory startle reflex appeared significantly later (mean day 12.5 vs. day 11.8). Significantly higher auditory brainstem conduction time at postnatal day 23 and 29

Intra-amniotic administration of ethyl-DHA

Fish oil vs. olive oil throughout pregnancy and lactation (g/100 g): mDPA, DHA k14:0, AA Fish oil during pregnancy only vs. olive oil: No significant differences in brain phospholipids. Fish oil during lactation only vs. olive oil: m16:0, EPA k18:1n-9, LA, AA

Fish oil vs. olive oil: Significant effects upon post-natal development indices. Significantly later eyelid and ear opening. Cross fostering indicates that diet during lactation period is most influential. Significantly later development of air righting and surface righting reflexes

Amusquivar et al., 2000

(Continued)

TABLE 24.2 (Continued) Study

Rat Model

Duration of Intervention

Brain Samples Analyzed

Effects on Brain Fatty Acid Composition

Effects Reported on Other Brain Outcome Measures

Day 2
12 post-partum

Brain phospholipids (PC, PI, PS and PE) in mitochondria, microsomes and synaptosomes

Menhaden oil vs. corn oil (weight %): Synaptosomes m16:0 (PS), EPA (PC, PS, PI, PE), DHA (PC, PS, PE) k18:0 (PS), LA (PI, PE), AA (PE) Microsomes m18:1n-9 (PC), EPA (PC), EPA (PS, PI, PE), DHA (PS, PE) k18:0 (PC, PI), 18:1n-9 (PS, PI), LA (PC, PE), AA (PE) Myelin m18:1n-9 (PC), LA (PS), EPA (PC, PS, PI, PE), DHA (PC, PI, PE) k16:0 (PI, PE), LA (PI), AA (PC, PS, PE) Mitochondria m18:1n-9 (PC), AA (PI), EPA (PC, PI, PE), DPA (PS, PE), DHA (PC, PS, PE) k16:0 (PI), 18:0 (PI), LA (PC, PS, PE), AA in (PS, PE)

N/A

10% fat w/w: Throughout pregnancy Palm oil, lard oil, soybean oil & and lactation/weaning coconut oil blend (control) 7% control oil 1 3% w/w sardine oil

Brain phospholipids (PE, PC, PS, PI) at day 17, 19, 21 gestation, 2- and 7-week- old pups

N/A Fish oil vs. control (mol % fatty acids): mDHA in PE at day 19, 21 gestation and 2 weeks post-delivery. mDHA in PS at day 19 and 21 gestation. mAA in PE at 2 weeks post-delivery. kAA in PE at day 19, 21 gestation and 7 weeks post-delivery. k AA at day 19 and 21 gestation and 2 weeks post-delivery

Dietary Supplement

Yeh et al., 1993

Sprague- 20% w/w fat content: Dawley Corn oil (20% w/w) Menhaden oil (20% w/w) 1 corn oil (1% w/w)

Yonekubo et al., 1993

Wistar

297

CONCLUSIONS

also assessed a wide range of other outcome measures. The study by Trevizol et al. (2013) used a rat model of amphetamine-induced mania, and identified that fishoil supplemented offspring had improved recognition memory, reduced brain reactive species production, and protein carbonyl levels, with an attenuated amphetamine-induced mania response, as measured by changes to exploratory activity. Four studies generated from one research group investigated the effect of long-chain omega-3 PUFA upon auditory development (Haubner et al., 2002; 2007; Saste et al., 1998; Stockard et al., 2000). It was identified that dietary long-chain omega-3 PUFA resulted in significantly longer auditory startle latency (Haubner et al., 2007), higher auditory conduction time (Haubner et al., 2002; Saste et al., 1998; Stockard et al., 2000) and later appearance of the auditory startle reflex (Haubner et al., 2002; Saste et al., 1998). These papers hypothesized that these impairments in auditory function were due to adverse effects of DHA upon myelination. Myelin is rich in cholesterol, saturated fatty acids, and sphingomyelin. Work by Yeh et al. (1993) specifically examined the fatty acid composition of brain myelin and, contrary to their expectations, found that dietary long-chain omega-3 PUFA could lead to a higher DHA content of myelin, with this change occurring during a critical phase of active myelination. Only two studies investigated the effect of dietary long-chain omega-3 PUFA during lactation alone (Amusquivar et al., 2000; Yeh et al., 1993). While the work of Yeh et al. (1993) identified significant changes to the long-chain omega-3 PUFA and long-chain omega-6 PUFA content of brain synaptosomes, microsomes, myelin, and mitochondria, including higher DHA and lower AA, Amusquivar et al. (2000) found that while supplementation during lactation could induce higher EPA and lower AA brain content, it did not significantly alter brain DHA content (Table 24.2).

DIRECT COMPARISON OF ALA AND LONG-CHAIN OMEGA-3 PUFA SUPPLEMENTATION ON BRAIN FATTY ACID COMPOSITION In the five papers where ALA and long-chain omega-3 PUFA are compared for their effect on brain fatty acid composition, the majority report no significant differences in brain DHA status (Alsted and Høy, 1992; Childs et al., 2010; Sommer Hartvigen et al., 2004; Valenzuela et al., 2004) between the two forms of dietary omega-3 PUFA, while one found that providing a diet rich in long-chain omega-3 PUFA resulted in significantly higher brain DHA status (Fernandes et al., 2012) compared to an ALA-rich diet (Table 24.3).

Several differences in brain EPA, DPA, and long-chain omega-6 PUFA content between the two diets were observed, but again with varied responses. A minority of studies reported that an ALA-rich diet resulted in significantly higher brain EPA (Fernandes et al., 2012) and DPA content (Sommer Hartvigen et al., 2004) compared to a long-chain omega-3 PUFA rich diet. The studies included here also describe mixed effects upon brain long-chain omega-6 PUFA status, with both lower (Fernandes et al., 2012; Sommer Hartvigen et al., 2004), and higher status (Alsted and Høy, 1992; Childs et al., 2010) reported with an ALA diet compared to a long-chain omega-3 PUFA diet. These differences between studies are likely to reflect differences in the mode of supplement delivery (i.e. direct gastric instillation was used in Valenzuela et al., 2004), the dietary dose of ALA (2
5% w/w) and long-chain omega-3 PUFA (0.2
1.84% w/w EPA 1 DHA) provided, and the relative dose of ALA:long-chain omega-3 PUFA selected for use in these studies for comparison (1.5
10:1). Only one paper assessed other brain outcome measures, with performance in the Morris water maze not found to significantly differ between ALA and longchain omega-3 PUFA supplemented rats (Sommer Hartvigen et al., 2004). Taken together the results indicate that direct provision of long-chain omega-3 PUFA has little advantage over dietary ALA in promoting brain DHA status in rats, but that differing effects upon other long-chain omega-3 PUFA and long-chain omega-6 PUFA are apparent. There is insufficient evidence to establish whether there are differences in functional outcomes between the two dietary interventions.

CONCLUSIONS There is clear and convincing evidence that providing additional ALA or long-chain omega-3 PUFA to rats during pregnancy and/or lactation and early life can significantly influence brain fatty acid composition, and that this has the potential to induce measurable changes in cognitive performance (Fernandes et al., 2011; Joshi et al., 2004; Trevizol et al., 2013) and brain chemistry (Innis and de la Presa Owens, 2001; Glozman et al., 1999; Roy et al., 2012; Trevizol et al., 2013) in rats. Data suggests that there are both potential risks and benefits of omega-3 PUFA supplementation during pregnancy upon offspring outcome measures. Altered patterns of fetal brain growth (Lenzi Almeida et al., 2011; Rao et al., 2007) and rates of neonatal maturation (Amusquivar et al., 2000) were observed, and evidence suggesting an adverse effect upon auditory development (Haubner et al., 2002;

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

TABLE 24.3 Studies which Directly Compare Long-chain PUFA and ALA Supplementation

Study

Rat Model

Dietary Supplement

Duration of Intervention

Brain Samples Analyzed

Effects on Brain Fatty Acid Composition

Effects Reported on Other Brain Outcome Measures

Fernandes et al., 2012

Sprague- 9% w/w fat content: Dawley Soybean oil (0.4% w/w ALA) Olive oil (0.04% w/w ALA) Fish oil (1.2% w/w EPA 1 DHA) Linseed oil (3.1% w/w ALA)

Day 1
12 gestation

Day 12 gestation, day 20 gestation, day 7 post-partum brain phospholipids

Linseed vs. fish oil (% fatty acids): m16:0, EPA k18:1n-9, AA, DHA Linseed oil vs. soybean oil (% fatty acids): m16:0, EPA, DPA k18:1n-9, AA, 22:5n-6 Fish oil vs. soybean oil (% fatty acids): mDPA, DHA k22:5n-6

N/A

Childs et al., 2010

Wistar

Low fat (LF, 3% w/w): Soybean oil (0.1% w/w ALA) High fat (HF, 13% w/w): Soybean oil (0.9% w/w ALA) Linseed oil (5% w/w ALA) Salmon oil (1.3% w/w EPA 1 DHA) Sunflower oil (0.02% w/w ALA)

Day 1
20 gestation

Day 20 gestation, brain phospholipids (PC, PE)

HF linseed oil vs. HF fish oil (g/100 g): mAA, 22:5n-6 in PE HF linseed oil vs. HF soybean oil (g/100 g): mDPA in PE k22:5n-6 in PE HF fish oil vs. HF soybean oil (g/100 g): mEPA, DPA in PE k22:5n-6 in PE

N/A

Sommer Hartvigsen et al., 2004

Wistar

Day 8 gestation
pups aged Brain phospholipids (PE, PS) of 20% fat w/w diets: 13 weeks pups at 1, 3, and 13 weeks of High structured oil age (2% w/w ALA) High linseed oil (2% w/w ALA) Low structured oil (0.4% w/w ALA) Low linseed oil (0.4% w/ w ALA) Low salmon oil (0.2% w/ w EPA 1 DHA) Control (1.26% w/w ALA, 0.5% w/w EPA 1 DHA)

High linseed vs. low salmon oil (weight %): mDPA in PS (1) k22:5n-6 in PE (1) and PS (1,3); 22:4n-6 in PE (3) and PS (13) High linseed vs. control (weight %): k22:4n-6 in PS (1); 22:5n-6 in PE (1) and PS (1,13) mDPA in PS (1) Low salmon oils vs. control (weight %): No significant differences

Morris water maze: No significant differences in time taken to complete

N/A

Valenzuela et al., 2004

Wistar

Habitual 8% w/w fat diet (0.26% ALA) Supplemented by gastric instillation with: 6 mg/kg DHA per day 60 mg/kg ALA

40 days prior to mating and throughout gestation

Day 16 and 19 of gestation, 2 and 21 days post-delivery. Total lipids from frontal cortex, hippocampus, and cerebellum

No significant differences reported between ALA and DHA groups. ALA and DHA vs. control (mg/g tissue): mDHA in frontal cortex at day 19 gestation and 2 and 21 days post-delivery. mDHA in cerebellum at day 16 and 19 gestation and day 2 and 21 days post-delivery. mDHA in hippocampus at 2 and 21 days postdelivery

Alsted and Høy (1992)

Wistar

20% w/w fat content: Fish oil diet (1.8% w/w EPA 1 DHA) Linseed oil diet (2.76% w/ w ALA) Control diet (0.14% w/w ALA)

Female rats fed diet from weaning and throughout pregnancy and lactation until offspring aged 18 weeks

Brain PE, PS, PE, PIP, and PIP2 of offspring aged 18 weeks

N/A Linseed oil vs. fish oil (weight %) of offspring: k16:0 (PIP2), 16:1n-7 (PIP, PIP2), 18:1n-9 (PIP), LA (PS, PIP, PIP2), 20:1n-9 (PI), 20:3n-6 (PE, PIP, PIP2), EPA (PI, PE, PIP, PIP2), 22:2(n-6) (PIP), DPA (PI, PS, PE) m18:0 (PIP), AA (PE, PIP, PIP2), 22:4n-6 (PS, PE), 22:5n-6 (PS) Linseed oil vs. control (weight %) of offspring: k20:1n-9 (PI), AA (PI, PE), 22:3n-6 (PI), 22:4n-6 (PS, PE), 22:5n-6 (PS, PE) m16:1n-7 (PE), 18:0 (PI), 18:1n-9 (PI), 20:3n-6 (PE, PIP2), EPA (PIP2) DPA (PS, PE). DHA (PS) Fish oil vs. control (weight %) of offspring: k18:0 (PS, PIP), 20:1n-9 (PI), AA (PI, PS, PE, PIP, PIP2), 22:4n-6 (PS, PE), 22:5n-6 (PS, PE) m16:0 (PIP2), 16:1n-7 (PE, PIP, PIP2), 18:1n-9 (PI, PIP), LA (PI, PS, PIP, PIP2), 22:1n-9 (PS), 20:3n-6 (PE, PIP, PIP2), EPA (PI, PE, PIP, PIP2), 22:2n-6 (PIP), DPA (PI, PS, PE), DHA (PS)

300

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TABLE 24.4 Summary of Studies which Identify Changes to Brain Long-chain omega-3 and omega-6 PUFA after omega-3 Supplementation During Pregnancy and/or the Post-Natal Period in Addition to the Effects Observed upon AA and DHA m EPA

m DPA

k 22:4n-6

k 22:5n-6

Alsted and Høy, 1992 Amusquivar et al., 2000 Cheon et al., 2000 Childs et al., 2010 Fernandes et al., 2012 Ikeno et al., 2009 Haubner et al., 2002 Saste et al., 1998 Stockard et al., 2000 Yeh et al., 1993

Alsted and Høy, 1992 Amusquivar et al., 2000 Childs et al., 2010 Fernandes et al., 2011 Guesnet et al., 1997 Haubner et al., 2002 Haubner et al., 2007 Lenzi Almeida et al., 2011 Saste et al., 1998 Stockard et al., 2000 Sommer Hartvigsen et al., 2004 Trevizol et al., 2013 Yeh et al., 1993

Alsted and Høy, 1992 Cheon et al., 2000 Fernandes et al., 2012 Haubner et al., 2002 Innis and de la Presa Owens, 2001 Lenzi Almeida et al., 2011 Saste et al., 1998 Schiefermeier and Yavin, 2002 Stockard et al., 2000 Sommer Hartvigsen et al., 2004 Trevizol et al., 2013

Alsted and Høy, 1992 Childs et al., 2010 Fernandes et al., 2012 Guesnet et al., 1997 Haubner et al., 2002 Haubner et al., 2007 Ho¨gyes et al., 2003 Innis and de la Presa Owens, 2001 Ozias et al., 2007 Saste et al., 1998 Schiefermeier and Yavin, 2002 Sommer Hartvigsen et al., 2004 Stockard et al., 2000 Trevizol et al., 2013

2007; Saste et al., 1998; Stockard et al., 2000). However, supplementary omega-3 PUFA was protective in models of toxic brain stimulation (Ho¨gyes et al., 2003), hypoxia (Suganuma et al., 2010), ischemia (Glozman et al., 1999), and amphetamine-induced mania (Trevizol et al., 2013) and improved performance in measures of spatial awareness (Fernandes et al., 2011; Joshi et al., 2004), spatial memory (Fernandes et al., 2011), and recognition memory (Trevizol et al., 2013). There are plausible mechanisms to support the pluripotent effects of omega-3 supplementation during pregnancy. AA status among premature infants has been demonstrated to be positively correlated to body weight, indicating that AA may have a growthpromoting effect (Koletzko and Braun, 1991). Provision of ALA or long-chain omega-3 PUFA has been demonstrated to significantly reduce circulating and tissue AA content (Childs et al., 2010) and may therefore be associated with a risk of impaired infant growth. The adverse effects observed upon neonatal auditory development in rats may be attributable to changes in the fatty acid composition of myelin at a critical phase of auditory development (Yeh et al., 1993). However, identifying whether these effects persist into an impaired auditory function in adulthood, or whether they reflect a transient delay in auditory development will require further research. The protective effect of omega-3 PUFA against acute or druginduced brain injury is consistent with the role of DHA as a substrate for neuroprotectins. Neuroprotectins are potent anti-inflammatory and anti-apoptotic molecules (Serhan et al., 2004). Research into brain injury and aging has identified potential benefits of neuroprotectins in Alzheimer’s disease by blocking neurotoxicity in in vitro models

(Lukiw et al., 2005), inhibiting white blood cell infiltration and inflammatory gene expression in a mouse model of stroke (Marcheselli et al., 2003), and improving neurological outcomes and reducing lesion size in a rat model of stroke (Bazan et al., 2012). Further research is required to confirm whether similar mechanisms underpin the protective effects observed in studies of acute brain injury during fetal development. Data consistently indicate an increase in brain DHA content and a reduction in AA content with both ALA and long-chain omega-3 PUFA supplementation. In many studies, only the brain content of these two fatty acids is reported, but data suggest that the brain content of other long-chain omega-3 PUFAs and longchain omega-6 PUFAs is also strongly influenced by diet, with significantly higher brain EPA and DPA and lower 22:4n-6 and 22:5n-6 reported in a large number of studies (Table 24.4). Further studies will be required before firm conclusions are drawn regarding the relative efficacy of ALA versus long-chain omega-3 PUFA supplementation upon functional brain outcomes, but available data indicate that ALA is as effective at inducing higher brain DHA content in rats as a diet rich in long-chain omega-3 PUFA (Alsted and Høy, 1992; Childs et al., 2010; Sommer Hartvigen et al., 2004; Valenzuela et al., 2004), but is also suggestive of a more modest effect in lowering brain long-chain omega-6 PUFA status. Further studies of whether omega-3 fatty acids may confer protection against hypoxia, ischemia, toxic brain injury, drug-induced mania or alter auditory function in humans, and whether ALA and long-chain omega-3 PUFA are equipotent in this regard, is worthy of further investigation.

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

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C H A P T E R

25 Omega-3 Fatty Acids and Cognitive Behavior Grace E. Giles, Caroline R. Mahoney and Robin B. Kanarek INTRODUCTION Omega-3 and omega-6 fatty acids are the main dietary components of the family of polyunsaturated fatty acids (PUFAs). Omega-3 fatty acids include α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), whereas omega-6 PUFAs include linoleic acid (LA) and arachidonic acid (AA). Since our bodies cannot synthesize these compounds, they must be obtained from our diet. The primary dietary sources of EPA and DHA are fatty fish such as salmon, herring, tuna, and halibut, while ALA comes mainly from plant sources such as canola oil, walnuts, and flaxseed. Omega-6 PUFAs are most commonly consumed as LA, which is found predominantly in plant oils (e.g. corn oil, sunflower oil, and soybean oil), as well as in products made from these oils (Kris-Etherton et al., 2000). Both omega-3 and omega-6 PUFAs are necessary for cells to maintain normal structure, function, and signal transduction. However, there is growing evidence to demonstrate that the ratio of omega-3 to omega-6 is more important than their absolute levels for these cellular processes (Loef and Walach, 2013; Simopoulos, 2011). For example, the ratio of omega-3 to omega-6 PUFA influences inflammation. Intake of omega-6 PUFA increases proinflammatory cytokine production, whereas omega-3 PUFAs reduce omega-6 PUFA activity, and thus decrease proinflammatory cytokine activity (Calder, 2009, 2010). In the nervous system, PUFAs are released via neurotransmitter stimulation and metabolized to active compounds including prostaglandins, thromboxanes, and leukotrienes. These compounds: 1) act as neuronal second messengers; 2) interact with G-protein coupled receptors on glial cells, thereby affecting neuromodulation and synaptic output; 3) affect cell migration; 4) moderate neurogenesis and synaptogenesis; and 5) increase adenylate cyclase and protein kinase A, which mediate serotonin, norepinephrine, and dopamine receptors (Fontani et al., 2005b; Innis, 2007). Additionally, the omega-3 fatty acid

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00025-9

DHA is a major component of the neuronal membrane, where it plays both a functional and a structural role. DHA helps to maintain the fluidity of the membrane at optimum levels for the transmission of neuronal information and moderates the characteristics of the hydrophobic core of the membrane to permit interactions with membrane proteins (for a review see Crawford, 2006). The importance of omega-3 PUFAs in brain function has been extensively studied in relation to psychological and neurological disorders. For instance, research indicates a beneficial influence of omega-3 PUFAs on depressive symptoms in individuals with major depressive disorder, although the relationship is far from understood (Giles et al., 2013; Lin et al., 2010; Martins, 2009; Parker et al., 2006; Sinclair et al., 2007). Omega-3 PUFAs may also benefit individuals with bipolar disorder (Turnbull et al., 2008), anxiety disorders (Ross, 2009) and attention deficit hyperactivity disorder (Frensham et al., 2012). Despite the reported benefits of omega-3 PUFAs when brain functions go awry, as in psychological and neurological disorders, less research has examined the influence of omega-3 PUFAs on cognitive function in healthy individuals. Recent research has begun to fill this gap by examining how omega-3 PUFAs influence cognitive development in infants and children, cognitive performance in young adults, and age-related cognitive impairments in older adults. Just as the brain changes throughout the lifespan, so too may nutritional influences on cognition (Benton, 2010; Luchtman and Song, 2013). Thus, this chapter will review recent research examining the cognitive effects of omega-3 intake across the lifespan.

Infants and Children Omega-3 PUFAs are critical for neurocognitive development from conception through early childhood

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304

25. OMEGA-3 FATTY ACIDS AND COGNITIVE BEHAVIOR

(Rogers et al., 2013). In the developing neonate, high levels of DHA are found in the cerebral cortex and photoreceptors in the retina (Innis, 2007). DHA accumulates in these areas during the last trimester of pregnancy and first months of life, which are periods of active neurogenesis, neuroblast migration, differentiation, and synaptogenesis (McNamara, 2010). As a result, pre-term infants who do not receive the thirdtrimester intrauterine supply of DHA may be particularly susceptible to the detrimental consequences of DHA deficiency, which include deficits in visual acuity, reductions in neural growth, and decreases in regional cortical gray matter volumes (Helland et al., 2003; McNamara, 2010). After birth, infants obtain DHA from breast milk or formula, as most infant formulas now contain DHA and AA (Consumer Reports, 2012). Initial epidemiological studies explored the relationship between maternal intake of fish and other foods containing omega-3 PUFAs and cognitive behavior in their offspring. In the majority of studies, intakes of fish by the mother during pregnancy and lactation were determined using food frequency questionnaires, while cognitive performance in the offspring was measured with standardized tests including the Fagan Test of Infant Intelligence, which measures novelty preference and is thought to predict IQ later in life (Fagan and Detterman, 1992), the MacArthur Communicative Development Battery, which assesses infant and toddler linguistic abilities (Fenson et al., 1993), the Peabody Picture Vocabulary Test, which measures listening comprehension and vocabulary (Dunn and Dunn, 1997), and the Bayley Scales of Infant Development, which evaluates cognitive and motor development (Bayley, 1969). Results of these studies revealed a positive relationship between mothers’ fish intake during pregnancy and lactation and cognitive development in their infants and young children (e.g. Boucher et al., 2011; Daniels et al., 2004; Hibbeln et al., 2007; Oken et al., 2008a,b). For example, Daniels and colleagues (2004) reported that maternal fish intake during pregnancy and infants’ fish intake during the first year of life were positively associated with scores on tests of language comprehension and social activity determined when the children were 15 and 18 months of age. Similarly, in an observational cohort study, Hibbeln and co-workers (2007) observed a direct relationship between maternal seafood intake measured at 32 weeks’ gestation and children’s cognitive performance between the ages of 6 months to 8 years. However, it should be noted that not all studies have observed a positive relationship between early exposure to omega-3 PUFAs and later cognitive functions (Keim et al., 2012). Although limited in number, epidemiological studies have also assessed the relationship between omega-3 PUFA intake and cognitive performance in adolescents.

In two studies conducted in Sweden, adolescents who regularly consumed fish performed better on a variety of cognitive measures including IQ, verbal performance, visuospatial performance, and academic achievement than adolescents who infrequently ate fish (Aberg et al., 2009; Kim et al., 2010). Similarly, in a study in Holland, adolescents who consumed the recommended levels of fish did better with respect to vocabulary and academic performance than those who consumed less than the recommended levels of seafood. However, caution must be exercised in reaching conclusions from this study, as exceeding the recommended level of fish intake actually decreased performance on a number of cognitive tasks (de Groot et al., 2012). While the results of these epidemiological studies suggest that dietary exposure to omega-3 PUFAs can have positive effects on cognitive behavior, their correlational design makes it impossible to assign causality. Moreover, a number of factors must be considered as possible confounds in such studies. For example, mothers who consume more fish may have a generally healthier diet, a greater level of education, and/or be older (or younger) than mothers who consume less fish. Another concern is that to quantify PUFA intake, these studies generally rely on food frequency questionnaires, which are subject to under-reporting of energy and food, including fats and fatty acids (Schaefer et al., 2000). To begin to address causality, randomized, doubleblind, placebo-controlled trials (RCTs) on the effects of omega-3 PUFAs on behavior are required. As a result, the following sections will concentrate on RCTs investigating the effects of omega-3 PUFAs on cognitive behavior. These trials can generally be divided into three categories, depending on the age at supplementation: (1) prenatally or infancy, via maternal supplementation; (2) infancy via formula supplementation; and (3) childhood.

Maternal Supplementation As shown in Table 25.1, results of experiments assessing the effects of maternal supplementation of omega-3 PUFA on cognitive development have not been consistent. On the negative side, Helland and colleagues (2001) found no differences in cognitive development of 6- and 9-month-old infants whose mothers had received supplements of either omega-3 PUFArich cod liver oil or omega-6 PUFA-rich corn oil during pregnancy and lactation. In contrast, the same researchers reported that children whose mothers had consumed cod liver oil during pregnancy and lactation did better on the mental processing subscale of the Kaufman Assessment Battery for Children at 4 years of

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

TABLE 25.1 Randomized Controlled Trials Assessing the Influence of Maternal Omega-3 PUFA Intake on Cognitive Development of Offspring

Authors

Mothers n

Maternal Age (Years)

Omega-3 PUFA Manipulation

Infants n Age at (Female) Testing

Childhood Cognitive Measures a

Results

(Helland 341 et al., 2001)

19
35

Cod liver oil (2632 mg/10 mL omega-3, 235 mg/ 10 mL omega-6) versus corn oil (350 mg/10 mL omega-3, 4747 mg/10 mL omega-6) 17
19 weeks pregnant until three months post-partum

288 (175)

27, 39 weeks

FTII

341 (Helland et al., 2003)

19
35 Cod liver oil: 28.5 6 3.3 Corn oil: 28.0 6 2.4 years

Cod liver oil (2494 mg/10 mL omega-3) versus corn oil (4747 mg/10 mL omega-6) 8 weeks pregnant until three months post-partum

84 (43)

4 years

K-ABCb

Cod liver . corn oil Mental Processing Composite Positive association plasma [DHA] and K-ABC subscales. Positive association between maternal DHA, EPA intake during pregnancy, and K-ABC subscales. Maternal DHA intake predicted Mental Processing Composite

(Lauritzen 150 et al., 2005)

X

FOc (1500 mg omega-3 PUFA: 900 mg DHA) versus OOd 9 6 3 days post-delivery until four months postpartum versus high habitual fish intake

148 (65)

9 months 1, 2 years

Motor function (9 months) Means-end problem solving (9 months) MCDIe (1, 2 years)

No differences in motor function. Higher problem-solving scores in FO than OO in girls only. Lower vocabulary production in FO than OO at 1 year (no differences at 2 years). Negative association between active vocabulary and RBC [DHA] at 1 year

98 (Dunstan et al., 2008)

X-X FO (2200 mg DHA, 1100 mg EPA) versus OO FO: 30.9 6 3.7 20 weeks pregnant until birth Control: 32.6 6 3.6

72 (39)

2.5 years

GMDSf PPVTg CBCLh

Higher hand eye coordination score on GMDS in FO than OO. Hand eye coordination positively associated with erythrocyte [EPA] and [DHA] and negatively correlated with [AA]. No differences between PPVT or CBCL

No differences

(Continued)

TABLE 25.1 (Continued)

Authors

Mothers n

Maternal Age (Years)

Omega-3 PUFA Manipulation

Infants n Age at (Female) Testing

Childhood Cognitive Measures

Results

(Makrides 2320 et al., 2010)

X-X Fish oil (800 mg DHA, 100 mg EPA) FO: 28.9 6 5.7, versus vegetable oil Control: 28.9 6 5.6

1196 (600)

18 months

BSID-IIIi

Mean scores did not differ by maternal treatment group. FO , control group for language score, language development, adaptive behavior (girls only)

(Cheatham 107 et al., 2011)

X

98 (44)

7 years

Woodcock Johnson Tests of Cognitive Abilities III Day/Night Stroop Task SDQj

No differences in speed of processing, Stroop Score, SDQ. Lower prosocial functioning in FO than OO (boys only). Total maternal omega-3 PUFA intake negatively associated with speed of processing scores

a

FO (1500 mg/day omega-3 PUFA: 790 mg DHA, 62 mg EPA) versus OO versus high habitual fish intake

Fagan Test of Infant Intelligence (FTII) Kaufman Assessment Battery for Children (K-ABC) c Fish oil (FO) d Olive oil (OO) e MacArthur Communicative Development Inventory (MCDI) f Griffiths Mental Development Scales (GMDS) g Peabody Picture Vocabulary Test (PPVT) IIIA h Child Behavior Checklist (CBCL) i Bayley Scales of Infant and Toddler Development Third Edition (BSID-III) j Strengths and Difficulties Questionnaire (SDQ) X Not Reported b

INTRODUCTION

age than children whose mothers had consumed corn oil. Additionally, the children’s mental processing scores correlated significantly with maternal intake of DHA and EPA during pregnancy (Helland et al., 2003). In more recent studies, the use of DHA-rich fish oil capsules compared with vegetable oil capsules during pregnancy did not result in improved cognitive and language development as measured by the Bayley Scale of Infant and Toddler Development (Makrides et al., 2010). Although the aforementioned studies suggest that maternal levels of omega-3 PUFAs may influence infant development, they are limited by the use of corn oil or vegetable oil which are high in omega-6 PUFAs as the control treatment. As a consequence, it is possible that rather than high levels of omega-3 PUFAs having a positive effect, high levels of omega-6 PUFAs, or a decreased ratio of omega-3 to omega-6 PUFAs, had a detrimental effect on cognitive behavior. To overcome this problem, recent studies have used olive oil, which contains monounsaturated fatty acids (MUFAs), and thus does not disrupt the balance between omega-3 and omega-6 PUFAs for the control condition (Cheatham et al., 2011; Dunstan et al., 2008; Lauritzen et al., 2005). A particularly well-designed RCT compared the effects of maternal fish oil and olive oil supplementation on infants’ behavior in women whose habitual fish intake fell below the population median (i.e. # 4 g/day omega-3 PUFA) to non-supplemented women whose habitual consumption of fish fell within the upper quartile of the population ($8 g/day omega-3 PUFA). When infants reached 9 months of age, better problemsolving skills were seen in girls whose mothers had been supplemented with fish oil relative to girls whose mothers had received olive oil. No differences emerged for boys or for other cognitive measures. At 1 year of age, however, vocabulary production was actually lower in children whose mothers had been supplemented with fish oil than in those whose mothers had received olive oil. Moreover, higher red blood cell DHA levels were associated with lower vocabulary scores. By 2 years of age, children did not differ in measures of cognitive behavior. Using a similar design, the same research group assessed executive function in children when they reached 7 years of age. Boys whose mothers had been supplemented with olive oil during the first four months of breastfeeding had lower prosocial behavior scores, as assessed by the Strengths and Difficulties Questionnaire, than boys whose mothers were given corn oil. In contrast, prosocial behavior in girls did not differ as a function of maternal supplementation. Moreover, no group effect of the intervention was evident in the speed of processing, inhibitory control or working memory abilities of 7 year olds whose mothers were or were not supplemented with

307

DHA during the first 4 months of breastfeeding (Cheatham et al., 2011). In a study in which mothers were supplemented with fish oil or olive oil from 20 weeks gestation through birth, hand eye coordination, as measured by the Griffiths Mental Development Scales, was more developed in 2.5 year old children whose mothers had received fish oil than in children whose mothers had been given olive oil. Moreover, hand eye coordination was positively associated with maternal erythrocyte EPA and DHA concentrations, and negatively associated with AA concentrations. However, performance on the MacArthur Communicative Development Inventory and the Child Behavior Checklist, which evaluates internalizing versus externalizing behavior, did not differ between treatment groups (Dunstan et al., 2008). Thus, while some studies found beneficial effects of maternal omega-3 PUFA supplementation on cognitive measures such as mental processing, problem solving, and hand eye coordination (Dunstan et al., 2008; Helland et al., 2003; Lauritzen et al., 2005), other studies have found few differences (Helland et al., 2001) and even negative effects (Cheatham et al., 2011; Makrides et al., 2010). Differences in the type, dose, and duration of supplementation with omega-3 PUFA, control conditions, cognitive measures which were employed, and age at assessment may contribute to inconsistent results among studies. Additionally, a recent review points out several methodological limitations that may influence results, including small sample size compounded with high attrition rates, as well as a lack of clear reporting of randomization and intent-to-treat analysis (Gould et al., 2013). Taken together, these factors make it premature to conclude that maternal intake of omega-3 PUFAs plays a significant role in cognitive development.

Supplementation During Infancy To assess the influence of omega-3 PUFA levels on cognitive development in a more direct manner than through maternal supplementation, researchers have supplemented the infant’s diet with omega-3 PUFAs (Table 25.2). Using this method, it was found that preterm infants fed human milk supplemented with DHA and AA for approximately 2.25 months displayed enhanced problem-solving skills and recognition memory at 6 months of age relative to infants fed milk without DHA and AA (Henriksen et al., 2008). Further support for a role for DHA in cognitive development comes from work investigating the effects of a formula containing either one of three doses of DHA or the same formula without DHA on cognitive performance.

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

308

25. OMEGA-3 FATTY ACIDS AND COGNITIVE BEHAVIOR

TABLE 25.2 Development

Authors

Randomized Controlled Trials Assessing the Influence of Infant Omega-3 PUFA Supplementation on Cognitive

Sample n (Female) Term

Gestational Age

Omega-3 PUFA Manipulation

Supplement Duration (Months)

Age at Cognitive Testing (Months) Measures

Results

(Scott et al., 274 (X) term 1998)

X

0.2% DHA formula versus 0.12% DHA 1 0.43% AA formula versus control formula versus BFa

14

12, 14

BSIDb MCDIc

No differences in BSID DHA , BF vocabulary comprehension. DHA , control vocabulary production

(Auestad et al., 2001)

404 (203) term

Egg-DTGd: 39.0 6 1.3 FOe
Fungal: 39.3 6 1.2 Control: 39.4 6 1.2 Egg-DTG BF: 39.6 6 1.3 Control BF: 39.2 6 1.2

FO 1 fungal oil formula (0.46 g/100 g AA, # 0.04 g/100 g EPA, 0.13 g/100 g DHA) versus Egg-DTG formula (0.45 g/100 g AA, 0.14 g/100 g DHA) versus control formula versus BF 1 Egg-DTG versus BF control

12

1, 2, 4, 6, 9, 12, 14

BSID MCDI FTIIf Infant Behavior Questionnaire

No differences in BSID, FTII. FO-Fungal . egg-DTG formula vocabulary expression. Egg-DTG formula , control formula smiling and laughter (Infant Behavior Questionnaire)

(O’Connor et al., 2001)

470 (214) Pre-term

Egg-DTG: 29.7 6 2.0 FO-Fungal: 29.8 6 2.1 Control: 29.6 6 1.9 BF: 29.7 6 2.1

FO 1 fungal oil formula (0.43 g/100 g AA, 0.08 g/100 g EPA, 0.27 g/100 g DHA) versus egg-DTG formula (0.41 g/100 g AA, 0.24 g/100 g DHA) versus control formula versus BF

12

2, 4, 6, 9, 14

BSID MCDI FTII

No differences in BSID, except FO-Fungal . control motor score in infants # 1250 g birth weight. Egg-DTG . fish-fungal, control groups novelty preference. No differences in MCDI, except egg-DTG, FO-fungal , control vocabulary comprehension after removing infants from Spanishspeaking families

(Auestad et al., 2003)

157 (71) term

All: 39 6 1

AA 1 DHA (0.43 g/100 g AA 1 0.12 g/100 g DHA) versus DHA (0.23 g/100 g DHA) versus control formula versus BF

52

14, 39

SBISg PPVT-Rh BVMi MLUj

No differences

(Henriksen et al., 2008)

141 (64) pre-term

48 mg/kg day Omega-3: DHA 1 48 mg/kg day AA 28.4 6 X Control: 28.9 6 X versus soybean

2.25 (median)

6

Ages and Omega-3 . control problem solving. Stages Omega-3 . control recognition Questionnaire memory Recognition memory via ERPk

oil 1 medium-chain triglyceride oil

(Drover et al., 2011)

131 (52) term

All: 37
42

0.32% DHA (17 mg/ 100 kcal), 0.64% DHA (34 mg/100 kcal) or 0.96% DHA (54 mg/100 kcal) 10.64% AA (54 mg/ 100 kcal) versus control formula

12

18

BSID BRSl

No differences between groups analyzed separately. Combined DHA groups . control mental development, language. Combined DHA groups . controls BRS emotion regulation

(Meldrum et al., 2012)

420 (139) term

FO: 39.1 6 1.1 Control: 29.4 6 1.3

FO (230 mg DHA, 110 mg EPA) versus OOm

6

12, 18

BSID MCDI CBCLn

FO . control MCDI later developing gestures, and total number gestures. No differences in BSID. FO . control CBCL anxious/ depressed behaviors

a

i

b

j

Breastfed (BF) Bayley Scales of Infant Development (BSID) c MacArthur Communicative Development Inventories (MCDI) d Egg-derived triglyceride (Egg-DTG) e Fish oil (FO) f Fagan Test of Infant Intelligence (FTII) g Stanford-Binet Intelligence Scale (SBIS) Form L-M h Peabody Picture Vocabulary Test-Revised (PPVT-R)

Beery Visual-Motor Index Test (BVM) Mean length of utterance (MLU) k Event Related Potential (ERP) l Behavior Rating Scale (BRS) m Olive oil (OO) n Achenbach Child Behavior Checklist (CBCL) X Not Reported

INTRODUCTION

While no differences were found among the four groups at 18 months of age, when the three DHA groups were combined, infants supplemented with DHA had enhanced language development on the Bayley Scales of Infant Development and emotion regulation behavior on the Behavior Rating Scale relative to those fed the control formula. In addition, higher red blood cell concentrations of the omega-6 PUFA, LA, were associated with lower language development and lower emotion regulation (Drover et al., 2011). More recently, Meldrum and co-workers (2012) reported no differences in global development at 12 and 18 months of age between infants supplemented with a high dose of fish oil and those supplemented with olive oil from birth to 6 months of age. However, infants supplemented with fish oil did display a higher number of gestures on the MacArthur Communicative Development Inventory than infants supplemented with olive oil. Moreover, erythrocyte DHA concentrations were directly associated with communication scores on the Bayley Scales of Infant and Toddler Development. In contrast to results of studies that suggest beneficial effects of omega-3 supplementation on infants’ cognition, other studies have found null effects or even detrimental effects. For instance, Auestad et al. (2003) observed no differences in IQ (Fagan Test of Infant Intelligence), language (MacArthur Communicative Development Inventory) or motor function (Bayley Scales of Infant and Toddler Development) at 1 through 14 months of age in infants supplemented with DHA with or without AA. Similarly, in a comparison between breast-fed infants and infants receiving DHA-supplemented, DHA and AA-supplemented or control formula for 14 months, no differences were found in mental or motor development at 12 or 14 months of age. Results from the MacArthur Communicative Development Inventory showed lower vocabulary comprehension in infants fed DHAsupplemented formula than in breast-fed infants, and lower vocabulary production than in infants fed the control formula. The potentially detrimental influence of DHA supplementation on language development was further evidenced by results showing that red blood cell DHA was negatively correlated with vocabulary production in breast-fed and formula-fed children, and with vocabulary comprehension in breast-fed children (Scott et al., 1998). Omega-3 PUFAs are found in a variety of sources which vary in terms of the relative DHA and EPA composition (Barcelo-Coblijn et al., 2008; Gebauer et al., 2006). Plant sources of PUFAs such as walnuts and flaxseed are relatively high in ALA, whereas marine sources such as salmon and fish oil contain a greater percentage of EPA and DHA. If the cognitive effects of omega-3 PUFAs are contingent upon the

309

relative constitution of EPA, DHA, and ALA, research into the differential cognitive effects of plant versus marine omega-3 PUFAs is essential (Auestad et al., 2001; O’Connor et al., 2001). To begin to examine this issue, infants were fed formulas containing fish oil plus fungal oil or egg-derived triglyceride, or breast fed for 12 months and tested on a range of cognitive measures including novelty preference, information processing, cognitive development, and motor development at 1, 2, 4, 6, 9, and 12 months of age. Few differences in cognitive behavior were observed among the groups, with the exception that infants fed the fish oil fungal oil formula scored higher in vocabulary expression on the MacArthur Communicative Development Inventory than those the fed egg-derived triglyceride formula (Auestad et al., 2001). However, in another study, infants fed either the fish oil fungal oil or the egg-derived triglyceride formulas scored lower in vocabulary comprehension than control groups, but only after removing infants from Spanish-speaking households (O’Connor et al., 2001). Although the influence of omega-3 PUFA supplementation on language remains poorly understood, the extant data suggest that higher DHA intake and levels are associated with poorer language development. In summary, while some studies have found a beneficial influence of omega-3 PUFAs on measures of problem solving and memory (Henriksen et al., 2008) and language (Drover et al., 2011; Meldrum et al., 2012), this has not universally been the case, as others have found detrimental effects of omega-3 PUFAs, particularly on language development (O’Connor et al., 2001; Scott et al., 1998). Like maternal omega-3 PUFA supplementation, infant omega-3 PUFA supplementation does not seem to influence global indices of cognitive development, but may have more circumscribed effects on certain cognitive domains.

Supplementation During Childhood As we move up the lifespan a few years, we will now examine RCTs assessing omega-3 PUFA supplementation in children from 4 to 12 years of age (Table 25.3). Relatively few researchers have looked at this age group, and from the few studies that do exist, there is no strong support for a positive effect of omega-3 PUFAs on cognitive performance. For example, no differences in episodic memory, working memory, psychomotor performance or mood measured at multiple time points, i.e. before and after breakfast, were found in 10 to 12 year old children who received either 400 mg/day DHA, 1000 mg/day DHA or a placebo. DHA did enhance self-reported feelings of relaxation, but this result was interpreted with caution, as

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

TABLE 25.3 Randomized Controlled Trials Assessing the Influence of Omega-3 PUFA Supplementation on Cognitive Development in Childhood

Authors

Sample n (Female)

Age Range (Mean 6 SD) Years

Omega-3 PUFA Manipulation

Duration (Weeks)

Cognitive Measures

Results

(Kennedy et al., 2009)

88 (42)

10
12 (400 mg DHA: 11.11 6 0.79 1000 mg DHA: 10.70 6 0.79 Control: 10.87 6 1.1)

400 mg/day DHA versus 1000 mg/day DHA versus vegetable oil

8

IB CDRb

Control . 400 mg, 1000 mg DHA relaxation ratings at baseline. Post . pre-DHA treatment relaxation ratings. 400 mg , placebo word recognition reaction time before and after breakfast. 1000 mg DHA . placebo word recognition reaction time before breakfast

(Ryan and Nelson, 2008)

175 (79)

4.0
4.67 (Omega-3: 4.3 6 0.2 Control: 4.3 6 0.2)

400 mg DHA versus sunflower oil

16

Leiter-Rc PPVTd Day-Night Stroop Test kCPTe

No differences

(Muthayya et al., 2009)

598 (204)

6
10 (All: 8.7 6 1.2)

Micronutrient (100% RDAf: high versus 15% RDA: low) x omega-3 PUFA (900 mg ALA 1 100 mg DHA: high versus 140 mg ALA: low)

52

KABC-IIg WISC-R, WISC-4h RAVLTi NEPSYj Number Cancellation

High . low micronutrient short-term memory (6 months only). Low . high micronutrient fluid reasoning (6 and 12 months). No differences in omega-3 groups

(Richardson et al., 2012)

362 (170) below 33rd centile in reading

6
10 (Omega-3: 8.6 6 0.8 Control: 8.7 6 0.8)

Algal oil (600 mg DHA) versus corn/ soybean oil

16

BAS-IIk Recall of Digits Forward Backward CTRS-Ll, CPRS-L

No changes in working memory or reading scores across entire sample. DHA . control reading score in children below 22nd centile in reading. DHA . control anxiety, restless-impulsive, emotional lability, global index from parent but not teacher ratings

a

a

Internet Battery (IB): Word Presentation, Picture Presentation, Arrow Reaction Time Test, Arrow Flanker Test, Paired Associate Learning, Sentence Verification, Delayed Word Recognition, Mood and Fatigue Visual Analogue Scales b Cognitive Drug Research (CDR) Battery: Picture Presentation, Word Presentation/Immediate Word Recall, Simple Reaction Time, Spatial Working Memory, Numeric Working Memory, Delayed Word Recall, Delayed Word Recognition, Delayed Picture Recognition c Leiter-R Test of Sustained Attention (Leiter-R) d Peabody Vocabulary Test (PPVT) e Conner’s Kiddie Continuous Performance Test (kCPT) f Recommended Dietary Allowance (RDA) g Kaufman Assessment Battery for Children, second edition (KABC-II) h Wechsler Intelligence Scales for Children (WISC-R and WISC-4) i Rey Auditory Verbal Learning Test (RAVLT) j Neuropsychological Assessment Tool (NEPSY) k British Ability Scales (BAS-II) l Conner’s Rating Scales (CTRS-L, CPRS-L)

INTRODUCTION

relaxation ratings were lower in the DHA groups than in the control group at baseline. Reaction time on a word recognition task was lower following 400 mg/day DHA intake both before and after breakfast and higher following 1000 mg/day DHA intake, before breakfast only (Kennedy et al., 2009). Similar results were found in a study using pre-school aged children in which DHA supplementation had no effect on cognitive measures including attention, vocabulary, and executive function. The only positive results showed that capillary whole blood DHA levels were positively associated with vocabulary scores (Ryan and Nelson, 2008). Recent work indicates that omega-3 PUFA supplementation not only affects behavior but also may modify brain activation. Using functional magnetic resonance imaging it was found that compared with placebo, DHA supplementation increased activation in the dorsolateral prefrontal cortex (DLPFC) during a sustained attention task in 8 to 10 year old boys. Lower activation was observed in low-dose (occipital cortex) and high-dose (cerebellar cortex) DHA groups compared with placebo. Despite similar performance on the behavioral task, erythrocyte DHA composition was positively correlated with DLPFC activation and inversely correlated with reaction time at baseline and endpoint, suggesting that omega-3 PUFAs, particularly DHA, may influence PFC-associated cognitive outcomes (McNamara et al., 2010). It is possible that omega-3 PUFAs may be more effective in improving mental functioning in children with cognitive or nutritional deficits than in healthy children. To investigate this possibility, the effects of 600 mg/day DHA versus control oil were examined in 6 to 10 year old children initially underperforming in reading, i.e. below the 33rd centile on reading ability, working memory, and behavior. Across the entire sample of children initially underperforming in reading, children did not differ in reading as a function of DHA supplementation. However, DHA improved reading scores relative to controls in children initially below the 20th centile. Parent ratings suggested that DHA elevated several behaviors including anxiety, restlessness, and emotional lability, but no such differences were found through teacher ratings (Richardson et al., 2012). Thus, DHA supplementation may aid reading performance in children with relatively low reading ability, but whether behavioral differences also emerge is questionable due to the mismatch in parent and teacher ratings. Another study looked at children with compromised nutrition, specifically marginally nourished children from low-income households in India. Although micronutrient supplementation improved memory and reasoning, omega-3 PUFA supplementation had no effects (Muthayya et al., 2009). Thus, little evidence exists for a beneficial influence of

311

omega-3 PUFA supplementation on cognitive performance in school-age children.

Young Adults To date only a handful of studies have assessed the cognitive effects of omega-3 PUFA supplementation in young adults (Table 25.4). Among the few studies that exist, Fontani and colleagues (2005a) found that relative to olive oil supplementation, five weeks of fish oil supplementation increased feelings of vigor and reduced feelings of anger, anxiety, fatigue, depression, and confusion. In a subsequent study, fish oil had similar effects on mood and reduced reaction time on tasks of response inhibition (Go/No-Go) and sustained attention (complex Go/No-Go). Measuring electromyography (EMG) activation showed that reduced reaction time was not due to increased muscle contraction but instead due to enhanced central processing (Fontani et al., 2005a). Thus, evidence from Fontani’s group suggests that omega-3 PUFAs have beneficial effects on mood, but does not present a clear picture on psychomotor performance, as fish oil did not influence straightforward tests of simple and choice reaction time. In contrast to the previous results, more recent data showed that, in comparison to olive oil supplements, four weeks of fish oil supplements only reduced ratings of fatigue, and had no effect on other mood scales measuring vigor, anger, anxiety, depression or confusion in healthy 18 to 27 year old men and women. Further, fish oil did not influence cognitive performance across a range of tasks measuring response inhibition, facial expression recognition, and memory. Fish oil did increase risk-seeking decision-making in gains only trials of a gambling task as well as reaction time in both gains-only and losses-only trials relative to placebo, together suggesting that omega-3 PUFA supplementation increased willingness to make calculated risks rather than increased impulsiveness (Antypa et al., 2009). However, the risk-based decision-making task was only administered after supplementation, making it impossible to determine whether fish oil and olive oil differentially changed risk taking pre- to post-supplementation. Another study found that fish oil improved verbal learning, but did not influence response inhibition or mood (Karr et al., 2012). A recent study found that DHA supplementation improved episodic memory in women and working memory in men, but not attention or processing speed in either sex (Stonehouse et al., 2013). Inconsistent results among studies may be due to differences in DHA content of fish oil (e.g. 0.8 g/day in Fontani et al. (2005a) compared to 0.25 g/day in Antypa et al. (2009) and 0.24 g/day in Karr et al. (2012)).

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

TABLE 25.4 Randomized Controlled Trials Assessing the Influence of Omega-3 PUFA Supplementation on Cognitive Performance in Young Adults

Authors

Sample n Age Mean Age (Female) Range 6 SD

Omega-3 PUFA Manipulation

Duration (Weeks)

Cognitive Measures

Results

(Fontani et al., 2005b)

33 (20)

22
51 33 6 7

Diet (40% carb, 30% pro, 30% fat versus 55% carb, 15% pro, 30% fat) x FOa (4 g/day FO: 0.8 g DHA, 1.6 g EPA versus OOb)

5

POMSc

FO . control vigor FO , control anger, anxiety, fatigue, depression, confusion

(Fontani et al., 2005a)

36 (32)

22
51 FO: 33 6 7 4 g/day FO (0.8 g DHA, 1.6 g Control: 33 6 3 EPA) versus OO

5

POMS, SRTd, CRTe, GNGf, Complex GNG (sustained attention)

FO . control vigor FO , control anger, anxiety, fatigue, depression, confusion FO , control GNG reaction time, number of errors No differences in SRT, CRT

(Antypa et al., 2009)

54 (44)

18
27 FO: 22.2 6 3.6 Control: 22.6 6 4.1

3 g/day FO (0.25 g DHA, 1.74 g EPA)

4

Affective GNG Attentional GNG Facial Expression Recognition Task Decisionmaking (gambling) task M.I.N.I.g BDI-IIh, POMS BIS/BASi LEIDS-Rj

FO , control fatigue, LEIDS-R Control/perfectionism, risk aversion and total score FO . control gambling task risk-seeking decision-making in gains-only trials No differences in GNG, facial expression recognition

(Jackson et al., 2012c)

140 (94)

18
35 DHA-rich FO: 21.96 6 0.54 EPA-rich FO: 22.74 6 0.61 Control: 21.94 6 0.50

1 g/day DHA-rich FO (0.45 g 12 DHA, 0.09 g EPA) versus EPArich FO (0.2 g DHA, 0.3 g EPA) versus OO

COMPASSk CDBl Bond-Lader VASm DASSn

DHA-rich FO , control Stroop reaction time EPA-, DHA-rich FO , control names-to-faces task (episodic memory) EPA-rich FO , control CBD self-reported fatigue No differences in mood or other cognitive measures

(Jackson et al., 2012a)

22 (13)

X

All: 21.96 6 X

1 g/day DHA-rich FO (0.45 g 12 DHA, 0.09 g EPA) versus EPArich FO (0.2 g DHA, 0.3 g EPA) versus OO

Stroop Task Peg-and-ball task 3-back task Wisconsin card sort task NIRSo

DHA-rich FO . control oxy-HB following DHA-rich FO during Stroop Task DHA-rich FO . control total-HB during Stroop Task, peg-and-ball, 3-back tasks

(Jackson et al., 2012b)

65 (49)

18
29 1 g FO: 20.5 6 0.43 2 g FO: 19.95 6 0.34 Control: 21.35 6 0.62

1 g/day FO (0.45 g DHA, 12 0.09 g EPA) versus 2 g/day FO (0.9 g DHA, 0.18 g EPA) versus OO

COMPASS

1, 2 g FO . control oxy-HB during all tasks 2 g FO . control total-HB during all tasks 1 g FO . FO control during Stroop, RVIPp tasks

(Karr et al., 2012)

41 (29)

1 g/day FO (240 mg DHA, 360 mg EPA) versus coconut oil

RAVLTq, Stroop Test, TMTr, PANASs

FO . control final stages (6 and 7) RAVLT FO , control TMT No differences in Stroop Test, PANAS

a

FO: 19.90 6 18.3 Control: 20.43 6 1.63

4

Fish oil (FO) Olive oil (OO) Profile of Mood States (POMS) d Simple Reaction Time (SRT) e Choice Reaction Time (CRT) f Go/No-Go (GNG) g Mini International Neuropsychiatric Interview (M.I.N.I) h Beck Depression Inventory-II (BDI-II) i Behavioral Inhibition/Behavioral Activation Scales (BIS/BAS) j Leiden Index of Depression Sensitivity
Revised (LEIDS-R) k Computerized Mental Performance Assessment System (COMPASS): episodic memory, psychomotor performance, attention, executive function, working memory l Cognitive Demand Battery (CDB) m Visual Analogue Scales (VAS) n Depression, Anxiety, and Stress Scales (DASS) o Near IR spectroscopy (NIRS) p Rapid Visual Information Processing (RVIP) q Rey Auditory Verbal Learning Test (RAVLT) r Trail Making Test (TMT), Parts A and B s Positive and Negative Affect Schedule (PANAS) X Not Reported b c

314

25. OMEGA-3 FATTY ACIDS AND COGNITIVE BEHAVIOR

Subsequent research has compared DHA-rich fish oil to EPA-rich fish oil to better understand whether the DHA or EPA content moderates differences in cognitive performance. DHA-rich fish oil lowered reaction time on the Stroop Task relative to olive oil and EPArich fish oil lowered self-reported fatigue during high cognitive demand. Both DHA- and EPA-rich fish oil impaired episodic memory on the Names-to-Faces task, but this task was one of five tasks measuring episodic memory, the other four of which did not generate differences, indicating that the influence of fish oil on episodic memory is not entirely reliable (Jackson et al., 2012c). Near-infrared spectroscopy (NIRS) is a brain imaging method that measures light absorbance to calculate oxy-hemoglobin (oxy-HB) and deoxy-hemoglobin (deoxy-HB), which provides an indirect measure of brain activity, particularly in the frontal cortex. Given that previous research had found increased prefrontal activation following DHA treatment (McNamara et al., 2010), this technique could shed further light on the relationship between omega-3 PUFA intake and prefrontal-related cognition. DHA- but not EPA-rich fish oil increased oxy-HB and total-HB in participants performing the Stroop Task as well as tasks measuring executive function, cognitive flexibility, and working memory (Jackson et al., 2012a). These effects were replicated in a subsequent study that compared two doses of DHA-rich fish oil (1 g/day and 2 g/day) to olive oil on a number of cognitive tasks, including those measuring episodic memory, psychomotor performance, executive function, and working memory. Both doses increased oxy-HB during all of the cognitive tasks relative to olive oil and whereas 2 g/day fish oil increased total-HB during all tasks, 1 g/day increased total-HB only during the Stroop Task and a rapid visual information processing task which measured sustained attention. Thus, two studies have found enhanced response inhibition following fish oil relatively high in DHA content (Fontani et al., 2005a; Jackson et al., 2012c). In addition, the influence of fish oil on the performance of the Stroop Task is further evidenced by increased oxy-HB and total-HB across multiple studies and doses (Jackson et al., 2012a
c). However, the effects on mood and other cognitive measures are less consistent.

Older Adults Older adulthood poses an additional critical period in cognitive development, as aging is associated with a number of cognitive changes, including decline in episodic and working memory (for a review, see Nyberg et al., 2012). Although cognitive decline often occurs

with normal aging, it may also serve as an early indicator of Alzheimer’s disease (Britton and Rao, 2011). Age-related cognitive impairments range from mild cognitive impairments to intermediate degrees of dementia to more severe cases, as in Alzheimer’s disease. In elderly individuals the estimated prevalence of mild cognitive impairment is 16% (Petersen et al., 2010), while the estimated prevalence of Alzheimer’s disease is 13% (Alzheimer’s Association, 2009) and all forms of dementia is 46% (Wimo et al., 2010). Rates of Alzheimer’s disease are higher in women than in men; however, it is important to note that the gender difference may owe to the fact that women have a longer lifespan than men (Alzheimer’s Association, 2009). Well-established risk factors for Alzheimer’s disease include advancing age, family history of Alzheimer’s disease, and carrying the apolipoprotein E ε4 (APOE ε4) gene as well as modifiable conditions such as hypertension, diabetes, and smoking (Reitz et al., 2010). A number of epidemiological studies as well as RCTs have assessed the relationship between omega-3 PUFA intake and age-related cognitive decline, dementia, and Alzheimer’s disease. We will begin with epidemiological studies and then move to RCTs in: (1) healthy older adults; and (2) individuals with mild cognitive impairment and Alzheimer’s disease.

EPIDEMIOLOGICAL STUDIES: THE ASSOCIATION BETWEEN OMEGA-3 PUFA INTAKE, OMEGA-3 PUFA LEVELS, AND COGNITIVE DECLINE Epidemiological studies assessing the influence of omega-3 PUFAs on cognitive decline in older adults generally measure dietary intake of fatty acids and/or plasma or erythrocyte levels of the fatty acids. Associations with cognitive performance are then determined at one or more time points (Table 25.5). The most common behavior measure utilized in such designs is the Mini Mental State Exam, which measures general cognitive impairment and is often used to characterize mild cognitive impairment or cognitive decline on a discrete yes-or-no basis. For example, older adults completed the Mini Mental State Exam at two time points four years apart, and were put into cognitive decline and no-decline categories. Individuals who had experienced cognitive decline had lower erythrocyte DHA, EPA, omega-3 to omega-6 ratio, DHA:AA ratio, and higher total omega-6 PUFA levels than those who had not experienced cognitive decline (Heude et al., 2003). Other researchers have reported that higher initial levels of plasma EPA and total omega-3 PUFAs are associated with reduced risk of dementia over a four-year follow-up period.

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

TABLE 25.5 Epidemiological Studies Assessing the Association Between Omega-3 PUFAs and Age-Related Cognitive Decline

Authors

Sample n (Female)

Age Range

FollowUp Duration (Years)

Cognitive and Physiological Measures a

b

Results

(Kalmijn et al., 1997)

476 (0)

69
89

3

MMSE , FA intake

Baseline: Higher intake of total fat, PUFAs, and LA and lower intake of total energy, fish, EPA, and DHA associated with cognitive impairment (no association with total omega3 PUFA). 3-year: No association between FA intake and cognitive decline in sample as a whole, but higher intake of LA and lower intake of fish associated with cognitive decline in individuals free of cognitive decline at baseline

(Heude et al., 2003)

246 (143)

63
74

4

MMSE, Erythrocyte FA (baseline only)

Lower DHA, omega-3:omega-6, DHA:AA and higher total omega-6 in individuals showing cognitive decline than no decline

(Laurin et al., 2003)

174 (117)

$65

5

MMSE, DSM-III-Rc dementia diagnosis, Plasma FA (baseline only), APOE ε4 genotype

No association with dementia risk and plasma FA. Lower omega-6 and total PUFAs in individuals with dementia than without (APOE ε4carriers only) Higher DHA in individuals with dementia than without (APOE ε4 non-carriers only) Prospective analysis (only participants free of dementia at baseline): Higher EPA in individuals who developed CIND than those who did not (free of dementia at baseline only). Higher DHA, omega-3 PUFAs, and total PUFAs in individuals who developed dementia than in those who did not (free of dementia at baseline only)

(Whalley et al., 2004)

350 (171)

B64

B53

MHTd (11 years old), MMSE, RPMe, RAVLTf, Uses of Common Objects Test, Digit Symbol Test, Block Design Test, FFQg, Erythrocyte FA, APOE ε4 genotype

No association between childhood IQ and supplement use. Higher IQ and digit symbol score for fish oil, vitamin, and other supplement users than non-users. Higher block design scores in fish oil users than non-users. Higher erythrocyte omega-3 PUFA, EPA, DHA, and omega3:omega-6 associated with higher IQ in childhood and 64 years. Higher erythrocyte AA:DHA associated with higher block design and RPM scores. Higher erythrocyte DHA and DHA:AA associated with higher digit symbol scores

(Huang et al., 2005)

2233 (1306)

$65

0.1
8.4 (mean 5.4)

MMSE, Digit Symbol Test, Benton Visual Retention Test, CES-Dh, ADLsi, TICSj, IQCoDEk, APOE ε4 genotype

No association between risk of dementia or AD and fried fish intake. Reduced risk of dementia and AD in individuals consuming $ 4 servings/week tuna or non-fried fish. (Continued)

TABLE 25.5 (Continued)

Authors

Sample n (Female)

Age Range

FollowUp Duration (Years)

Cognitive and Physiological Measures

Results Reduced risk dementia in $ 2 servings/week fatty fish in individuals (APOE ε4 non-carriers only)

(Morris et al., 2005)

3178 (2306)

$65

3, 6

MMSE, East Boston Tests of Immediate and Delayed Recall, Symbol Digit Modalities Test, Harvard FFQ

Less cognitive decline in individuals who consumed $ 1 serving fish/week. No association between cognitive decline and omega-3 PUFA intake

(Beydoun et al., 2008)

2251 (1141)

45
64

N/A

Delayed Word Recall, Digit Symbol Substitution, Word Fluency, Plasma FA

Higher plasma AA and lower LA associated with GCIl. No associations between plasma omega-3 and GCI. Higher omega-3 PUFAs associated with reduced decline in verbal fluency (individuals with higher hypertensive and dyslipidemic markers and lower depressive symptoms only)

(Dullemeijer et al., 2007)

807 (226)

50
70

3

Concept Shift Test, Stroop Test, Word Learning Test, Letter Reduced 3-year cognitive decline in participants with higher Digit Substitution Test, Verbal Fluency Test, Plasma FAs omega-3 PUFAs (placebo group in RCT). No differences in memory, information processing speed, word fluency. No associations between omega-3 PUFAs and cognitive performance (all participants in RCT)

(van Gelder et al., 2007)

210 (0)

70
89

5, 10

MMSE FFQ

Greater cognitive decline in men who consumed no fish than in men who consumed fish. Greater cognitive decline in lowest than highest tertile of EPA 1 DHA intake

(Samieri et al., 2008)

1214 (748)

$65

2, 4

Dementia diagnosis, CES-D, Plasma FA (baseline only), APOE ε4 genotype

Higher EPA and total omega-3 PUFA associated with reduced risk of dementia. Association between AA:DHA ratio and dementia risk higher in subjects with depression than without

(Whalley et al., 2008)

120 (68)

63.8
65.3

64, 66, 68

MHT (11 years old) Positive association between total omega-3 PUFA and RPM, RAVLT, Uses of Common Objects Test, Digit Symbol cognitive performance at 11 and 64 years (APOE ε4 nonTest, Block Design Test, APOE ε4 genotype carriers only) Erythrocyte FA (64 years only)

(Devore et al., 2009)

5395 (3185)

$55

10

MMSE, GMSm, DSM-III-R dementia diagnosis, Semi quantitative FFQ

No association between fish or omega-3 PUFA intake and risk of dementia

(Kroger et al., 2009)

663 (401)

$65

5, 10

MMSE, DSM-III-R dementia diagnosis, Plasma FA, APOE ε4 genotype

No association between FA and dementia

(van de Rest et al., 2009)

1025 (0)

68 6 X

3, 6

MMSE, Tests of memory, language, perceptual speed, and attentionn, 126-item Willet FFQ

No associations between cognitive performance and omega3 PUFA intake

(Gonzalez et al., 2010)

304 (177)

X(75.3 6 6.7)

N/A

MMSE FFQ

Higher cognitive score associated with higher intake of omega-3 PUFA, EPA, DHA, LNA, and lower omega-6: omega-3 ratio (no differences in total energy, lipids, SFA, MUFA, PUFA or omega-6 PUFA). In regression, higher intake of EPA and DHA and lower intake of omega-6:omega-3 PUFA predicted lower cognitive impairment

(Roberts et al., 2010)

1223 (592) free of 70
89 dementia

N/A

CDRo Scale, Neuropsychological test battery, DSM-IV dementia diagnosis, Modified Block 1995 Revision of the Health Habits History Questionnaire

MCI associated with lower intake of PUFA, omega-6 PUFA, omega-3 PUFA, fatty acids, LA, ALA, and (MUFA 1 PUFA): SFA ratio. Lower risk of MCI associated with higher intake of MUFA (men only)

(Gao et al., 2011)

1475 (969) free of dementia

$55

1.5

MMSE FFQ

Lower cognitive decline associated with omega-3 supplement use

(Milte et al., 2011)

79 (29) With MCI: 50 (16) HC: 27 (13)

$65

MMSE, Memory Functioning Questionnaire, SF-36 Health Survey, GDSp, RAVLT, Stroop Test, Boston Naming Test, Digits Forward, Digits Backward, Letter-Number Sequencing, Trail Making Task Erythrocyte FA

Lower EPA and higher omega-6 PUFAs in individuals with MCI than HC and associated with impaired cognitive performance. Higher DHA associated with impaired self-reported mental health

(Chiu et al., 2012)

132 (96) With history of MDD without cognitive impairment

$60

WAIS-IIIq CTTr, Semantic verbal fluency, Erythrocyte and plasma membrane FA, APOE ε4 genotype

Higher erythrocyte ALA, total omega-3 PUFA associated with greater immediate verbal memory

(Ronnemaa et al., 2012)

2009 (0)

50

Dementia diagnosis , Serum FA (baseline only), APOE ε4 genotype

Higher LA in individuals who developed dementia than in those did not (no difference with omega-3 PUFAs)

a

35

Mini Mental State Exam (MMSE) Fatty acid (FA) Diagnostic & Statistical Manual of Mental Disorders—3rd Edition Revised (DSM-III-R) d Moray House Test (MHT) e Raven’s Standard Progressive Matrices (RPM) f Rey’s Auditory Verbal Learning Test (RAVLT) g Food frequency questionnaire (FFQ) h Center for Epidemiological Studies of Depression Scale (CES-D) i Activities of Daily Living scale (ADLs) j Telephone Interview for Cognitive Status (TICS) k Informant Questionnaire for Cognitive Decline in the Elderly (IQCoDE) l Global cognitive decline (GCI) m Geriatric Mental State schedule (GMS) n Word list memory test, Backward digit span test, Pattern memory, Verbal fluency, Boston Naming Test-short-form, Vocabulary, Pattern comparison, Continuous performance test, Spatial copying task-constructional praxis o Clinical Dementia Rating (CDR) Scale p Geriatric Depression Scale (GDS) q Wechsler Memory Scale (III) (WAIS-III) r Color Trail Test (CTT) X Not Reported b c

318

25. OMEGA-3 FATTY ACIDS AND COGNITIVE BEHAVIOR

Additionally, in this study, depressive symptoms were used as a covariate, a factor which is often overlooked but nevertheless important to consider, given that history of major depressive disorder increases risk of Alzheimer’s disease (Samieri et al., 2008). Other studies have compared cognitive impairment at one time point rather than across multiple years, and have found similar results. Individuals with mild cognitive impairment had lower erythrocyte EPA and higher omega-6 PUFAs than those without mild cognitive impairment. Moreover, erythrocyte levels were associated with performance on cognitive tests (Milte et al., 2011). However, higher erythrocyte DHA levels were associated with lower self-reported mental health, suggesting that enhanced cognitive performance does not necessarily equate to enhanced subjective wellbeing. The previous studies point to a positive association between omega-3 PUFA levels and global cognitive function in older adults, although other studies have failed to correlate omega-3 PUFA levels with performance in specific cognitive domains. For instance, higher erythrocyte ALA and total omega-3 PUFAs were associated with enhanced verbal memory, but in the same study no associations were found between fatty acid levels and other cognitive measures including intelligence, attention, psychomotor speed, and executive function (Chiu et al., 2012). Higher plasma omega-3 PUFA levels were associated with reduced global cognitive decline over a three year period, though no differences were found in specific cognitive domains such as executive function, memory or visual information processing (Dullemeijer et al., 2007). The association between plasma fatty acid levels and cognitive decline may also depend on other health-related factors, including cardiovascular and psychological impairments. For instance, plasma omega-3 PUFAs were associated with reduced decline in verbal fluency in individuals with higher hypertensive and dyslipidemic markers, and in individuals with lower depressive symptoms (Beydoun et al., 2007). In an attempt to determine the relationship between fatty acids and cognitive decline, other cross-sectional and prospective studies have measured dietary intake of fatty acids. For instance, higher intake of total fat, PUFAs, and LA and lower intake of total energy, fish, EPA, and DHA were associated with lower cognitive scores at baseline, however, only individuals initially free of cognitive impairment showed associations between high LA and lower fish intake and cognitive decline (Kalmijn et al., 1997). Across six years, individuals who consumed at least one serving per week of fish showed less cognitive decline, but no association was found between total omega-3 PUFA intake and cognitive decline (Morris et al., 2005). Across ten years, individuals who consumed fish and those in the

highest tertile of EPA and DHA intake experienced less cognitive decline than those who consumed less fish, EPA, and DHA (van Gelder et al., 2007). Additionally, lower cognitive impairment was associated with higher intake of EPA and DHA and lower intake of omega-6 versus omega-3 PUFAs (Gonzalez et al., 2010) as well as the use of omega-3 PUFA supplements (Gao et al., 2011). Mild cognitive impairment was associated with lower intake of total omega-3 and omega-6 PUFAs, as well as LA and ALA (Roberts et al., 2010). Thus, although data from food frequency questionnaires give us some indication that increased omega-3 PUFA intake is associated with reduced cognitive decline, the reliability of such measures is questionable, given the inconsistencies in many studies, e.g. correlations between cognitive function and fish but not omega-3 PUFA intake (Morris et al., 2005). Using retrospective data, Whalley and colleagues (2004, 2008) correlated intelligence scores of individuals measured when they were 11 years of age with their intelligence scores, cognitive behavior, and erythrocyte fatty acid levels at 64 years of age. Higher erythrocyte total omega-3 PUFAs, EPA, DHA, and omega-3 to omega-6 ratio at 64 years of age were associated with higher IQ at both 11 and 64 years of age. Moreover, 1) a higher erythrocyte AA to DHA ratio was associated with higher constructional ability and nonverbal reasoning at 64 years of age and 2) older adults who consumed fish oil supplements had higher IQ, psychomotor performance, and constructional ability than those who did not use supplements (Whalley et al., 2004). In a subsequent study, the positive association between erythrocyte total omega-3 PUFA levels and cognitive performance at 11 and 64 years was found to be stronger for individuals who did not carry the APOE ε4 gene than for APOE ε4 carriers (Whalley et al., 2008). The results suggest that omega-3 PUFA intake and levels may have greater beneficial effects for individuals not already at increased risk for Alzheimer’s disease by carrying the APOE ε4 gene. Genetic variations located near the APOE ε4 gene are most consistently associated with Alzheimer’s disease, particularly the late onset form of the disease (Bertram and Tanzi, 2008). Indeed, another study found that APOE ε4 noncarriers who consumed at least two servings per week of fish were at reduced risk for dementia, but no such association was found for APOE ε4 carriers. Across all subjects, only those who consumed more than four servings per week of non-fried fish or tuna had a reduced risk of dementia and Alzheimer’s disease (Huang et al., 2005). Although these studies focus on the APOE gene associated with late-onset Alzheimer’s disease, future studies should also address correlations with genes associated with early-onset Alzheimer’s disease, including PSEN1, PSEN2, and APP (Rao et al., 2013).

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

EPIDEMIOLOGICAL STUDIES: THE ASSOCIATION BETWEEN OMEGA-3 PUFA INTAKE, OMEGA-3 PUFA LEVELS, AND COGNITIVE DECLINE

Despite the evidence above that omega-3 PUFA intake may confer protection against age-related cognitive decline, other studies have failed to find such effects. A number of studies found no association between the risk of dementia and fish or omega-3 PUFA intake (Devore et al., 2009; van de Rest et al., 2009) or serum or plasma fatty acid levels (Kroger et al., 2009; Ronnemaa et al., 2012). Some studies have even found negative associations between omega-3 PUFA intake and cognitive performance in older adults. In individuals initially free of dementia, plasma EPA levels were higher in those who developed cognitive impairment than those who did not, and plasma DHA, total omega-3 PUFA, and total PUFA levels were higher in those who developed dementia than those who did not. When all subjects beginning with and without dementia were analyzed together, plasma fatty acids were not associated with dementia. However, higher plasma DHA and lower total omega6 PUFA and total PUFA levels were associated with dementia in APOE ε4 carriers only (Laurin et al., 2003). Thus, although evidence from epidemiological studies is somewhat inconsistent, with some studies failing to find associations, a larger number of studies suggest that higher omega-3 PUFA intake and/or blood levels are associated with lower risk of age-related cognitive decline.

RCTs: Healthy Older Adults Conclusions drawn from epidemiological studies are limited to possible associations between omega-3 PUFAs and cognitive decline, whereas RCTs are necessary to determine whether increased omega-3 PUFA intake plays a causal role in reducing cognitive decline. A number of studies have assessed the influence of omega-3 PUFA supplementation on cognitive function in healthy older adults (Table 25.6). DHA improved aspects of verbal, visuospatial, and episodic memory (Johnson et al., 2008; Yurko-Mauro et al., 2010). These results were supported by physiological data showing associations between higher serum DHA and improved verbal fluency (Johnson et al., 2008), and between higher plasma DHA and improved visuospatial and episodic memory (Yurko-Mauro et al., 2010). Similar to results from epidemiological studies in which omega-3 PUFA levels were differentially associated with cognitive decline between APOE ε4 carriers and non-carriers (Huang et al., 2005; Whalley et al., 2008), low and high doses of EPA and DHA (i.e. 226 mg EPA 1 176 mg DHA and 2093 mg EPA and 847 mg DHA, respectively) enhanced attention relative to placebo only in APOE ε4 carriers (van de Rest et al., 2008). However, a lower dose of DHA

319

(i.e. 252 mg/day DHA compared to 800 mg/day and 900 mg/day in Johnson et al. (2008) and Yurko-Mauro et al. (2010), respectively) did not influence cognitive function (Stough et al., 2012). The discrepancy in results may be due to methodological differences in the Stough and colleagues (2012) study design, including differences in DHA dose, treatment duration (limited to 90 days), or wide age range (45
77 years). Nonetheless, the majority of research to date points to a beneficial influence of omega-3 PUFA, and particularly DHA, intake on cognitive function in older adults.

RCTs: Older Adults with Mild Cognitive Impairment or Alzheimer’s Disease Evidence suggests omega-6 PUFAs exacerbate β-amyloid deposition, a hallmark outcome of Alzheimer’s disease, and omega-3 PUFAs (or low omega-6 to omega-3 ratios) may reduce the effects (Corsinovi et al., 2011). Yet few RCTs have evaluated whether omega-3 PUFA supplementation results in behavioral changes in mild cognitive impairment and Alzheimer’s disease, and results are largely inconsistent (Table 25.7). Omega-3 PUFAs had few cognitive effects in individuals with a range of Alzheimer’s disease severity. However, omega-3 PUFAs prevented declines in memory in samples of individuals with either mild or severe Alzheimer’s disease (Freund-Levi et al., 2006). Additionally, omega-3 PUFAs reduced depressive symptoms in individuals with Alzheimer’s disease more in APOE ε4 non-carriers than carriers (Freund-Levi et al., 2008). DHA-rich fish oil has been shown to improve verbal fluency in individuals with mild cognitive impairment, and both EPA- and DHArich fish oil improved depressive symptoms, a result further supported by associations between higher erythrocyte DHA 1 EPA and lower AA:EPA levels and improved depressive symptoms (Sinn et al., 2012). In a small study of individuals with mild to moderate Alzheimer’s disease or mild cognitive impairment, omega-3 PUFAs improved global clinical Alzheimer’s disease status. Although no differences in cognitive impairment were found, higher red blood cell membrane EPA was associated with improved cognitive Alzheimer’s disease symptoms (Chiu et al., 2008). However, other researchers failed to find cognitive effects of omega-3 PUFA supplementation in individuals with mild to moderate Alzheimer’s disease (Quinn et al., 2010). A recent meta-analysis summarized the effects of RCTs evaluating the influence of omega-3 PUFAs on cognitive performance in healthy adults and those with cognitive impairment but no dementia and those with Alzheimer’s disease

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

TABLE 25.6 Randomized Controlled Trials Assessing the Influence of Omega-3 PUFA Supplementation on Cognitive Function in Healthy Older Adults

Authors

Sample n Age Range (Female) (Mean 6 SD)

Omega-3 PUFA Manipulation

Duration (Weeks)

Cognitive Measures

Results

Verbal Fluency, Digit Span Forward and Backward, Shopping Test Task, Word List Memory Test, Memory in Reality Apartment Test, Stroop Test, NES2 Mood Scales: selfreported mood

DHA, lutein, DHA 1 lutein . control verbal fluency. DHA 1 lutein , control response time on Shopping List Memory Test, delayed recall in Memory in Reality Apartment Test

(Johnson et al., 2008)

49 (49)

60
80 DHA: 68.5 6 1.3 Lutein: 66.7 6 1.9 DHA 1 lutein: 68.6 6 1.3 Control: 68.0 6 1.2

800 mg/day DHA versus 12 mg/day lutein versus DHA 1 lutein versus placebo

(van de Rest et al., 2008)

196 (88)

65 High EPA-DHA: 69.9 6 3.4 Low EPADHA: 69.5 6 3.2 Control: 70.1 6 3.7

900 mg/day fish oil high EPA-DHA 26 (226 6 3 mg EPA, 176 6 4 mg DHA) versus low EPA-DHA (2093 6 17 mg EPA, 847 6 23 mg DHA) versus sunflower oil

Verbal Fluency, Word Learning Test, Digit Span Forward and Backward, Trail Making Test version A and B, Stroop Test

Low EPA-DHA , control memory at 13 but not 26 weeks. Low and high EPADHA . control attention at 26 weeks in APOE ε4 carriers only. Low EPA-DHA . control attention at 26 weeks in men only

(YurkoMauro et al., 2010)

485 (282)

$ 55 DHA: 70 6 9.3 Control: 70 6 8.7

Algal triglyceride oil (900 mg/day DHA) versus corn 1 soybean oil

24

WAIS-IIIa logical memory, MMSEb, CANTABc Subtests: PALd, PRMe, VRMf, SOCg, SWMh, Frequency of Forgetting-10 Scale, ADCS-ADL PIi, GDSj

DHA . control CANTAB PAL, VRM

(Stough et al., 2012)

74 (43)

45
77 DHA: 55.08 6 8.70 Control: 57.66 6 8.67

1000 mg/day tuna oil (252 mg DHA, 60 mg EPA) versus soybean oil

90 days

STAIk, CDR assessmentl

No differences in CDR factors

a

Wechsler Memory Scale (III) (WAIS-III) Mini Mental State Exam (MMSE) c Cambridge Neuropsychological Test Automated Battery (CANTAB) d Paired Associative Learning (PAL) e Pattern Recognition Memory (PRM) f Verbal Recognition Memory (VRM) g Stockings of Cambridge (SOC) h Spatial Working Memory (SWM) i Alzheimer’s Disease Cooperative Study-Activities of Daily Living Prevention Instrument (ADCS-ADL PI) j Geriatric Depression Scale (GDS) k State Trait Anxiety Inventory (STAI) l Cognitive Drug Research (CDR) assessment b

16

TABLE 25.7 Randomized Controlled Trials Assessing the Influence of Omega-3 PUFA Supplementation on Cognitive Function in Older Adults with Mild Cognitive Impairment or Alzheimer’s Disease Sample n Authors (Female)

Age Range (Mean 6 SD)

Omega-3 PUFA Manipulation

Duration (Weeks)

Cognitive Measures b

c

Results d

(Freund- 204 (110) Levi ADa et al., 2006)

Omega-3 PUFA: 72.6 6 9.0 Placebo: 72.9 6 8.6

(Chiu et al., 2008)

Omega-3 Omega-3 PUFA (1080 mg EPA, PUFA: 720 mg DHA) versus olive oil 70.1
77.8 (74.0) Placebo: 71.8
81.1 (76.5)

24

(Freund- 204 (90) AD Levi et al., 2008)

Omega-3 PUFA: 72.6 6 9.0 Placebo: 72.9 6 8.6

Omega-3 PUFA (600 mg EPA, 1700 mg DHA) versus corn oil

24 (1 24 weeks NPIh, MADRSi, CGBj, DADk open label omega-3 for all participants)

(Quinn et al., 2010)

402 (210) mildmoderate AD

Omega-3 PUFA: 76 6 9.3 Placebo: 76 6 7.9

2 g Algal DHA (0.9
1.1 g DHA) versus corn or soy oil

72

MMSE (baseline only), ADAS-cog, CDR, ADCSADLl, Quality of Life of Alzheimer’s Disease Scale

No differences

(Sinn et al., 2012)

50 (16) MCI 65 EPA-rich FO: 74.88 6 5.06 DHA-rich FO: 74.22 6 7 Control: 73 6 3.96

EPA-rich fish oil (1.67 g EPA, 0.16 g DHA) versus DHA-rich fish oil (1.55 g DHA, 0.4 g EPA) versus safflower oil (2.2 g LA)

24

GDSm, SF-36 Health Survey: health and quality of life, RAVLTnWAIS-IIIo (Digits Forward, Boston Naming Task, Letter-Number Sequencing, Digits Backward), Trail-Making Task, Stroop Test, Verbal Fluency

EPA, DHA . LA depressive symptoms. DHA . LA verbal fluency. No differences in quality of life

a

43 (20) mildmoderate AD (23) or MCIe (23)

Omega-3 PUFA (150 mg EPA, 430 mg DHA) versus corn oil

Alzheimer’s disease (AD) Mini Mental State Exam (MMSE) c Cognitive portion of Alzheimer’s Disease Assessment Scale (ADAS-cog) d Clinical Dementia Rating (CDR) Scale e Mild cognitive impairment (MCI) f Clinician’s Interview-Based Impression of Change (CIBIC) Scale g Hamilton Depression Rating Scale (HDRS) h Neuropsychiatric Inventory (NPI) i Montgomery
A˚sberg Depression Rating Scale (MADRS) j Caregiver Burden Scale (CGB) k Disability Assessment for Dementia Scale (DAD) l ADCS Activities of Daily Living (ADCS-ADL) Scale m Geriatric Depression Scale (GDS) n Rey Auditory Verbal Learning Test (RAVLT) o Wechsler Memory Scale (III) (WAIS-III) b

24 (1 24 weeks MMSE , ADAS-cog , CDR open label omega-3 for all participants)

ADAS-cog, CIBICf, HDRSg

No differences in MMSE or ADAScog across all participants. Omega-3 . control MMSE delayed recall, attention (very mild AD). Omega-3 . control ADAS-cog delayed recall (severe AD) Omega-3 . control CIBIC. No differences in ADAS-cog, MMSE, HDRS

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(Mazereeuw et al., 2012), and found more support for a beneficial effect of omega-3 PUFAs in studies focusing on cognitive impairment without dementia than on healthy or Alzheimer’s populations. The majority of evidence of a beneficial effect of omega-3 supplementation on Alzheimer’s disease progression comes from epidemiological trials whereas RCTs are less conclusive. Thus, additional RCTs are necessary to determine whether omega-3 PUFA supplementation may prevent or reverse age-related cognitive decline, specifically Alzheimer’s disease (Barberger-Gateau et al., 2011).

CONCLUSION Multiple studies have found positive effects of maternal and formula supplementation on infant cognitive development, particularly in problem solving, memory, and language development (Drover et al., 2011; Helland et al., 2003; Henriksen et al., 2008; Lauritzen et al., 2005; Meldrum et al., 2012). However, others have found no differences, suggesting that although omega-3 PUFA supplementation may not influence global cognitive development among infants, it may aid particular cognitive functions. The opposite may be true in older adults, as prospective and crosssectional studies suggest that higher omega-3 PUFA intake and plasma levels are associated with reduced overall cognitive decline with less evidence for specific cognitive domains (e.g. Heude et al., 2003; Milte et al., 2011; Samieri et al., 2008). Epidemiological results are supported by some RCTs showing that omega-3 PUFA supplementation, particularly DHA, reverses agerelated cognitive decline in otherwise healthy individuals (Johnson et al., 2008; van de Rest et al., 2008; Yurko-Mauro et al., 2010), but there is less evidence to suggest such an effect in individuals with mild cognitive impairment and Alzheimer’s disease. Alzheimer’s disease risk factors including history of depression and carrying the APOE ε4 gene may influence the efficacy of omega-3 PUFAs in preventing cognitive decline in older adults (Heude et al., 2003; Huang et al., 2005; Whalley et al., 2008). Research in young adults remains fairly limited, and although some data suggest positive effects of omega-3 PUFA supplementation on mood and executive function (Fontani et al., 2005a,b), other studies have failed to replicate these effects. Thus, though the evidence to date points to a beneficial influence of omega-3 PUFAs on cognitive performance across the lifespan, the conclusions remain tenuous, and additional research is necessary to more fully tease apart which omega-3 PUFAs are most beneficial to which cognitive processes at which stage of life, as well as to more fully understand the mechanism by which omega-3 PUFAs may influence brain function.

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1657. Whalley, L.J., Deary, I.J., Starr, J.M., Wahle, K.W., Rance, K.A., Bourne, V.J., et al., 2008. n-3 Fatty acid erythrocyte membrane content, APOE varepsilon4, and cognitive variation: an observational follow-up study in late adulthood. Am. J. Clin. Nutr. 87 (2), 449
454. Wimo, A., Winblad, B., Jonsson, L., 2010. The worldwide societal costs of dementia: Estimates for 2009. Alzheimers Dement. 6 (2), 98
103. Yurko-Mauro, K., McCarthy, D., Rom, D., Nelson, E.B., Ryan, A.S., Blackwell, A., et al., 2010. Beneficial effects of docosahexaenoic acid on cognition in age-related cognitive decline. Alzheimers Dement. 6 (6), 456
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26 Lipids and Lipid Signaling in Drosophila Models of Neurodegenerative Diseases Kyoung Sang Cho, Se Min Bang and Amanda Toh INTRODUCTION The fruit fly Drosophila, has been used for approximately 100 years in the laboratory setting, making it one of the oldest animal models to be utilized for scientific research. It is considered to be the most useful animal genetic model, owing to several advantages: its high fecundity (800 eggs per female), short life cycle (eggs grow to become fertile adults within 2 weeks), and low maintenance (small body size and cheap cost to maintain). Moreover, more than 10,000 genetic mutants of Drosophila have been developed, which can be employed to discover the function of such mutated genes. In 2000, the full Drosophila genome sequence was released and compared with the human genome. A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster has revealed that about 75% of the human disease genes have a Drosophila ortholog. On the basis of its genetic similarity to humans, the use of Drosophila has since been extended from the study of development to the modeling of human diseases. One of the major objectives in the quest to find cures for human diseases is to understand the molecular and cellular mechanisms of the disease processes. Although frequently appropriate, cell culture systems and mouse models are time-consuming and expensive. In contrast, fruit flies can be generated quickly and are more cost-efficient. Additionally, Drosophila provides an impressive array of available genetic tools that facilitate the screening for interacting proteins, and allow foreign genes to be expressed in tissue-specific and temporally regulated patterns. Using the powerful genetic and cell biological tools available in Drosophila, much has been revealed about the pathophysiology of several neurodegenerative diseases. Furthermore, the Drosophila models can also be used to rapidly screen Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00026-0

for dietary components, drugs, and regimens of administration. In this chapter, the use of several Drosophila models for studying neurodegenerative diseases is introduced, and studies that have been carried out using these models that show the function of lipids and dietary oils in neurodegenerative diseases are presented.

DROSOPHILA AS A MODEL SYSTEM OF NEURODEGENERATIVE DISEASES Genetic Tools for Making Neurodegenerative Disease Models in Drosophila P-Element-Mediated Mutagenesis P-element-mediated mutagenesis is a powerful genetic method that allows for the creation of genetic mutants on a genome-wide scale. The P-element is a transposon that is found specifically in Drosophila (Figure 26.1A). Transposons are also known as ‘jumping genes’ owing to their ability to excise and insert themselves in various locations within the genome. The hallmark of a transposon lies in its 31-bp inverse terminal repeat ends—the enabling factor for transposition. Autonomous (complete) P-elements are 2.9 kb in size and contain 4 exons (Ryder and Russell, 2003). An important feature of these elements is the functional encoding of the transposase gene. Transposases are enzymes that identify the inverse terminal repeat sequences within the DNA and proceed to bind and excise the DNA transposons in between the terminals. Subsequently, the transposons can be re-inserted elsewhere through the identification of the same inverse terminal repeats, while the donor site in the DNA is then repaired. Insertions result in the generation of an 8-bp duplication at the target sites (50 end and 30 end).

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Experimentally, the P-element used is specially engineered, with the intron between the 3rd and 4th exons being spliced (P{W2
3}) so that the stop codon is removed and complete alternative splicing can occur, resulting in a somatically active functional mRNA from the P-element transcript. This functional P-element transcript does not occur naturally in the soma, as the stop codon in the intron between exon 2 and exon 3 prevents complete alternate splicing from happening. With this engineered P-element, transposition can occur in the somatic cells. In Drosophila, the non-autonomous (incomplete) transposon and transposase are kept within separate fly lines, which can then undergo crossing. The resultant F1 generation will then have an autonomous transposon, allowing for transposition to occur in their germ cells. In the F2 generation, a variety of the P-element insertion mutants can be obtained, in which the P-elements are inserted in various loci of their genome. On the basis of their insertion sites in the genome, the P-element can disrupt a specific gene (Figure 26.1B). For example, if the P-element is inserted in the regulatory regions or exons of a gene X, the expression of gene X will be affected. Several

genome-wide P-element insertion projects have been carried out in different laboratories. Therefore, currently, a large majority of the Drosophila genes are associated with at least one P-element mutant. The information relating to the mutants exists in the gene data bank Flybase (http://flybase.org), and the insertion mutants of genes of interest can be purchased from the fly stock centers. However, the insertion is often unable to disrupt gene expression completely; that is, they generate hypomorphic mutants. Therefore, to disrupt the gene expression completely (i.e., generate null mutants), further genetic crosses, termed imprecise excision, are needed. In this process, the fly with a P-element surrounding a gene X is again crossed with the fly with the transposase. Precise or imprecise excision of the P-element occurs, which can be reflected in the eyes of the Drosophila fly. Usually, this engineered P-element contains eye pigment genes such as miniwhite, and so the eyes of the flies in which transposition has occurred will be white, whereas the eye of the flies with the P-element will remain red. To determine the exact location of excision, a polymerase chain reaction is usually carried out. Imprecise excision occurs randomly in each transposition, and the genes involved are usually those that flank the target gene. Using these techniques, the mutants of most of the neurodegenerative disease-associated genes have been generated and characterized in Drosophila. UAS-GAL4-Based Gene Expression System The UAS-GAL4 system is a method of activating gene expression in Drosophila (Figure 26.2). The GAL4

FIGURE 26.1 The structure of P-element and P-element-mediated mutagenesis. A. P-element structure and its tissue-specific alternative splicing. B. A cartoon showing P-element-mediated mutagenesis. A, adapted with permission from Laski and Rubin 1989.

FIGURE 26.2 A schematic diagram of the UAS-GAL4 system. Induction of the expression of the gene of interest, X, at the specific tissue of interest is enabled. Adapted with permission from Brand and Perrimon 1993.

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protein, derived from yeast, serves as the transcriptional activator in this system. Its lack of endogenous targets within Drosophila, together with the ability to activate transcription within the fly, makes it a favorable tool. The upstream activation sequence (UAS) is an enhancer that is specific to the GAL4 protein. The UAS-GAL4 system is an efficient bipartite approach in the activation of gene expression (Duffy, 2002). UAS, together with a specific gene of interest, is kept in one fly line, and GAL4 with a tissue-specific promoter is kept in another. When flies of these two lines undergo crossing, the GAL4 protein will bind to the UAS and activate the gene at the tissue that the promoter is specific for. One of the advantages of this system is that toxic genes will only be expressed when bound to the GAL4 protein. This allows flies carrying the inactivated form of a toxic gene to survive normally. Another advantage of this system is that the effects of various genes can be studied through their overexpression or misexpression at various sites in the body using the array of tissue-specific promoters available. Reporters for Amyloid Precursor Protein γ-secretase Activity in Drosophila As the Drosophila genome contains genes for all of the γ-secretase complex components that show well conserved γ-secretase activity, it is considered to be a suitable model to study the regulation of amyloid precursor protein (APP) cleavage activity of γ-secretase. Several reporter systems were developed for measuring APP γ-secretase activity in vivo in Drosophila. Among these, a reporter system that can be applied as a powerful genetic screening for isolating the γ-secretase activity-regulating molecules is introduced here. Using the transgenic system expressing human APP-GAL4 under the eye-specific glass multimer reporter (GMR) promoter, Guo et al. (2003) generated a living reporter for APP γ-secretase activity in Drosophila (Figure 26.3). The transgene, known as APP-GAL4, encodes a fusion protein with a fragment of human APP, together with β- and γ-secretase cleavage sites as well as GAL4. The APP-GAL4 is expressed in the developing eye under the control of the eye-specific GMR promoter. The genome of the reporter fly also contains UAS-GRIM, which results in cell death when induced by GAL4. If γ-secretase activity is absent, GAL4 remains tethered at the membrane, unable to activate the transcription of GRIM. However, if γ-secretase activity is present, the intracellular domains of APP and GAL4 enter the nucleus and induce GRIM expression, which results in cell death in the eye. As the eye phenotype of the reporter fly is easily visible and quantifiable, this method can be used for in vivo genetic screening.

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FIGURE 26.3 A fly reporter system for gamma-secretase activity. N, amino terminus of protein. Adapted with permission from Guo et al. 2003.

Representative Drosophila Models for Neurodegenerative Diseases Using powerful genetic modification techniques, many human disease models have been generated in Drosophila. In particular, neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and polyglutamine diseases are considered suitable to be modeled with Drosophila, as its neuronal system is well conserved and extensive studies have been done. Indeed, a variety of neurodegenerative diseases have been modeled with Drosophila, and together with powerful genetic systems, Drosophila geneticists have successfully used these models to identify many novel disease-associated genes, shedding light on our understanding of the pathology of these diseases. AD is the most common neurodegenerative disease, which causes a deficiency in memory and other cognitive functions. As AD-associated genes are largely conserved in its genome, Drosophila is considered to be a relevant model system for determining the molecular mechanisms underlying AD. The Drosophila genome contains genes that encode orthologs of β-APP (APPL) and Tau, which are involved in the most important pathogenic event of AD. Moreover, the genes of 4 major protein components of γ-secretase (presenilin, Nicastrin, APH-1, and PEN-2) are also well conserved. As anticipated, transgenic flies expressing human Aβ42 or Tau using the UAS-GAL4 system, resulted in AD-like neuronal degeneration and early death (Iijima et al., 2004; Wittmann et al., 2001). To investigate the molecular mechanisms responsible for the neurodegeneration, several genetic modifier screenings have been conducted in the Drosophila models of AD. As a result, it was found that several biochemical processes

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such as secretion, cholesterol homeostasis, and regulation of chromatin structure and function were involved in mediating toxic Aβ42 effects (Cao et al., 2008). Moreover, kinases and phosphatases comprised the major class of modifiers of the tauopathy (Shulman and Feany, 2003). Interestingly, the modifiers of Tauexpressing models are largely distinct from those of polyglutamine toxicity, despite some clinical and pathological similarities among neurodegenerative disorders (Shulman and Feany, 2003). However, from the genetic screening study with Aβ42-expressing flies, a few candidate mutations of the genes involving vesicular trafficking, autophagy, and metal homeostasis have been identified to mediate common mechanisms of neurodegeneration by Tau, polyglutamine, and Aβ42 (Rival et al., 2009). Toward the end of the 1990s, Drosophila models of polyglutamine diseases including spinocerebellar ataxia type 3 (SCA3/MJD) and Huntington’s disease (Hashimoto et al., 2002) were developed (Jackson et al., 1998; Warrick et al., 1998). Targeted expression of a segment of the SCA3/MJD or human huntingtin protein with an expanded polyglutamine repeat induced neuronal degeneration, which suggested that cellular mechanisms of human polyglutamine disease are conserved in invertebrates. These models have been used to identify a variety of genetic modifiers of polyglutamine degeneration (Bilen and Bonini, 2007; Fernandez-Funez et al., 2000; Li et al., 2008), as well as some beneficial drugs, such as histone deacetylase inhibitors, the transglutaminase inhibitor cystamine, and mTOR inhibitor rapamycin (Karpuj et al., 2002; Lai et al., 2008; Ravikumar et al., 2004; Sang et al., 2005; Steffan et al., 2001). In addition to AD and HD, many other neurodegenerative disease models have been generated in Drosophila. Among them, X-linked adrenoleukodystrophy (X-ALD) is closely associated with lipid metabolism. A genetic screening with P-element-mediated mutagenesis identified a very long-chain acyl coenzyme A (CoA) synthetase mutant as being a brain degeneration mutant that showed a similar phenotype to that of human patients with X-ALD (Min and Benzer, 1999). This model provided a good opportunity to test the effect of dietary oil on the neurodegeneration in the metazoan. Mutations of the trp gene, which encodes a transient receptor potential (TRP) channel, are also good examples of the neurodegeneration induced by 1 gene deficiency in Drosophila. Interestingly, TRP channels have been implicated in several neurodegenerative diseases, such as AD, PD, stroke, and hypoxia (Leonelli et al., 2011). TRP channel activities have been extensively associated with fatty acid function in both mammalian and Drosophila studies (Chyb et al., 1999; Leonelli et al., 2011).

EFFECTS OF LIPIDS AND LIPID SIGNALING ON DROSOPHILA MODELS OF NEURODEGENERATIVE DISEASES Although Drosophila is one of the most studied animal models, surprisingly, only a small number of studies about the roles of lipids and lipid signaling in Drosophila models of neurodegenerative diseases have been performed to date. This might be due to the difficulty in the study of the lipid system in insects, as the biochemical knowledge of insect lipid metabolism is largely unknown. In addition, disparity in lipid metabolism between insects and mammals makes it challenging to study the effect of lipids in Drosophila. However, several recent studies that will be described in this chapter demonstrate the potential of Drosophila as a useful model for lipid biology in relation to neurodegenerative diseases.

Effects of PUFA and Cholesterol Levels on Drosophila AD Models Expressing Human Aβ42 The effects of hempseed meal (HSM) intake and linoleic acid on the human Aβ42-expressing Drosophila AD model were studied (Lee et al., 2011). Hempseed is a rich source of oil, composed of more than 80% polyunsaturated fatty acids (PUFAs). The fatty acids in hempseed oil include a variety of essential fatty acids, including linoleic acid (LA, 18:2n6) and α-linolenic acid (ALA, 18:3n3), as well as γ-linolenic acid (GLA, 18:3n6) (Callaway, 2004). HSM shows a strong antioxidant effect, which was tested by rearing flies on either HSM or cornmealsoybean standard media, together with H2O2, and then comparing their survival rates (Lee et al., 2011). Intriguingly, the survival rates of flies reared on HSM were much higher, indicating that HSM exerts a protective effect from the toxicity of H2O2, which suggests that HSM has antioxidant properties. LA, a major non-polar component of hempseed, also showed a protective effect against the toxicity of H2O2 when supplemented in standard medium containing H2O2. However, because the degree of increase in the survival rate of LA-fed flies was lower than that observed in the flies fed on HSM, the antioxidant activity of HSM is probably not caused solely by LA, but rather, a result of the complex effects of various HSM components, such as other PUFAs and phytosterols. HSM intake also showed a protective effect against the cytotoxicity of Aβ42. When Aβ42 was ectopically expressed in the fly eye, it induced profound eye degeneration. The defective eyes could be divided into 2 groups (mild and severe) according to their size

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(Lee et al., 2011). Eighty percent of the flies reared in standard medium showed severe phenotypes. However, feeding with HSM reduced the rate of occurrence of the severe defects to 50%, indicating that HSM intake suppressed Aβ42 cytotoxicity. In an effort to find the molecular components of hempseed that mediate the protective effect against eye degeneration, four major components of HSM
LA, ALA, GLA, and campesterol
were tested in the fly AD model. Interestingly, LA and ALA, but not GLA or campesterol, ameliorated the eye degeneration phenotypes in a dosedependent manner. At the moment, the molecular mechanism by which HSM, LA, and ALA exert their protective effects have yet to be clarified. As oxidative stress is an important mediator of Aβ42 toxicity, the antioxidant property of the HSM and fatty acids could be the main factor for the protective effects. Indeed, some studies have shown that the supplementation with PUFAs decreased oxidant parameters such as lipid peroxide and reactive oxygen species levels in mammalian animal models including the rat AD model (Hashimoto et al., 2002; Sarsilmaz et al., 2003). However, other studies reported that PUFA treatment does not prevent amyloid-β-mediated oxidative stress (Florent et al., 2006; Florent-Be´chard et al., 2009). Consistently, HSM feeding does not affect the disease-like phenotypes of the Drosophila model of PD and HD, two diseases that have been extensively associated with oxidative stress. Therefore, it is unlikely that the suppression of oxidative stress is a plausible way to explain the protective effect of HSM against Aβ42 cytotoxicity. Alternatively, HSM and PUFAs could exert their protective activity by regulating cholesterol level (Figure 26.4). Although its role in AD pathology is still not fully understood, cholesterol has been implicated in AD at various aspects of pathology. Aβ42 is produced from APP through the amyloidogenic pathway, which occurs in the cholesterol-enriched lipid rafts, while APP is alternatively cleaved by α-secretase in the non-raft region (Florent-Be´chard et al., 2009). Accordingly, cholesterol depletion has been found to suppress Aβ42 production in hippocampal neurons (Simons et al., 1998). In Drosophila, a screening of genetic modifiers of Aβ42 cytotoxicity identified loechrig mutants, in which the cholesterol homeostasis-associated protein AMP kinase γ is deficient (Cao et al., 2008). HSM and LA intake reduced the cholesterol uptake level in Drosophila. When flies were reared in high-cholesterol food with HSM or LA, the body cholesterol level was reduced to a nearly normal level; on the contrary, the body cholesterol level of control flies reared on high-cholesterol food without HSM and LA was greatly increased (Lee et al., 2011). Consistently, LA intake affects Drosophila development by decreasing the cholesterol level

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FIGURE 26.4 The molecular mechanisms of the lipid functions in Drosophila models of neurodegenerative diseases. C, carboxyl terminus; DAG, diacylglycerol; DGK ε, diacylglycerol kinase ε; Htt, huntingtin; IP3, inositol trisphosphate; N, amino terminus; OCB, open channel block; PA, phosphatidic acid; PE, phosphatidylethanolamine; PIP, phosphatidylinositol monophosphate; PIP2, phosphatidylinositol 4,5bisphosphate; Poly Q, polyglutamine; PUFA, polyunsaturated fatty acid; sAPPβ, soluble beta amyloid precursor protein.

FIGURE 26.5 The effect of linoleic acid on Drosophila development. Adapted with permission from Lee et al. 2011.

(Figure 26.5), which is crucial for metamorphosis, as cholesterol is a precursor of the molting hormone ecdysone. Drosophila larval growth is delayed by intake of a PUFA mixture or LA, and is enhanced by cholesterol. Interestingly, the delayed larval growth induced by LA feeding is almost completely rescued by an intake of cholesterol. These results suggest that linoleic

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acid can act antagonistically with cholesterol during Drosophila development. Therefore, PUFAs may exert their beneficial effects on Drosophila AD models by reducing the brain’s cholesterol level, which causes the deleterious effects of AD at high levels.

Effect of Phosphatidylethanolamine Depletion on γ-secretase-Mediated APP Processing in Transgenic Drosophila Recently, the role of membrane lipids on the generation of Aβ was studied in mammalian cells and Drosophila (Nesic et al., 2012). Brain phospholipid (PL) metabolism has been implicated in the pathogenesis of AD. The level of PLs was significantly reduced in some gray areas of patients with AD (Svennerholm and Gottfries, 1994). In particular, among the PLs, phosphatidylethanolamine (PE) was shown to be reduced in the plasma membranes of the synaptosome, glial, and neuronal cell bodies (Wells et al., 1995). APP-cleaving machinery such as β- and γ-secretases are present in detergent-resistant microdomains (DRMs), in which the level of cholesterol is important, despite that APP and α-secretase are localized in detergent-soluble (non-DRM) microdomains, which are PL-enriched membrane domains. Therefore, the lipidic components of the membrane microdomains, such as cholesterol and PL, are likely to be important in the pathology of AD, by influencing the production of Aβ. Interestingly, PE, the major membrane PL in both Drosophila (Jones et al., 1992) and mammalian cells, influences Aβ production (Figure 26.4). Using the fly reporter system for γ-secretase activity (Figure 26.3), Nesic et al. (2012) showed that a depletion of PE by the deficiency of easPC80, a Drosophila ethanolamine kinase (Etkn), decreased γ-secretase
mediated APP processing. The rough eye phenotype and reduced eye size in the GMR . C99-GAL4/UAS-GRIM reporter line were ameliorated by the EasPC80 homozygous mutant, suggesting that the PE level is important for γ-secretase activity in Drosophila. This is coincident with the data in mammalian cells, where the knockdown of Etkn by siRNA reduced the Aβ level, by both promoting the cleavage of α-secretase and decreasing the γ-secretase activity to process APP. It was suggested that the perturbed membrane PL content reduced the γ-secretase activity in DRM by inhibiting the assembling of its complex or the accessibility to its substrate APP in non-DRM. However, the altered PL content could increase the accessibility of α-secretase to APP. These results highlight the importance of the lipidic environment of the membrane in the pathology of AD.

Effects of Lipid Signaling Enzyme Diacylglycerol Kinase ε Inhibition on Mutant Huntingtin Toxicity Membrane lipids are also associated with huntingtin (Htt)-induced toxicity in Drosophila and mammalian cell models of HD. In Drosophila, HD models are well established and have been used for screening the genetic components that could be associated with HD (Jackson et al., 1998). When the N-terminal of Htt with a 128Q expansion was ectopically expressed in the fly nervous system using the elav-GAL4 driver, the flies showed a locomotive defect compared with the normal control. On the other hand, decreasing the diacylglycerol kinase ε (DGKε) level by shRNA reduced the toxic effects of the mutant Htt, which implicates DGKε-associated lipid signaling in the pathology of HD (Zhang et al., 2012) (Figure 26.4). DGK catalyzes the phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA), and is an important regulator of lipid metabolism. DGK-deficient mice showed a decreased polyphosphoinositide level, and treatment of mutant Htt-expressing cells with a DGK inhibitor reduces the level of phosphatidylinositol monophosphate (PIP) and phosphatidylinositol 4,5bisphosphate (PIP2) (Zhang et al., 2012). Therefore, altered lipid metabolism by DGK inhibition is supposed to be a critical mechanism for the rescue of HD cytotoxicity. The huntingtin peptide is associated with the membrane through its interactions with PLs. The region of huntingtin interaction with the membrane was predicted by structural analyses to be at the N-terminus (Kegel et al., 2009). Moreover, over-expressed and endogenous human huntingtin proteins in cells are associated with PLs. In particular, endogenous wild-type huntingtin from the mouse brain associates more with multivalent phosphoinositides such as PI(3,4)P2, PI(3,5)P2, PI(4,5)P2, and PI(3,4,5)P3, as compared to monovalent PLs or to PE and phosphatidylcholine (PC) (Kegel et al., 2009). Interestingly, an altered interaction of mutant huntingtin with phosphatidylinositols was revealed. That is, the interaction with PE and monovalent PLs such as PI(3)P and PI(4)P is greatly increased in the mutant huntingtin from the brain of mice with HD (Hdh140Q/140Q mouse). This suggests that the alteration of lipid metabolism could be a therapeutic treatment for HD, which is currently an incurable disease.

Drosophila Mutant of Very Long-Chain Acyl Coenzyme a Synthetase and Glyceryl Trioleate Oil X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disease that causes a rapidly progressive

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inflammatory demyelination within the brain, or noninflammatory distal axonopathy in the spinal cord (Kemp et al., 2012). In patients with X-ALD, various tissues, including adrenal and cerebral white matter and the testis, have lipid inclusions that contain cholesterol, phospholipids, and gangliosides esterified with saturated very long-chain fatty acids (VLCFAs), suggesting that the aberrant metabolism of VLCFAs is the key factor in the pathogenesis of X-ALD (Kemp et al., 2012). The ABCD1 gene has been identified as a causative gene of X-ALD and encodes adrenoleukodystrophy protein (ALDP), which transports VLCFA acyl-CoA into peroxisomes, after which beta-oxidation of VLCFAs occurs. Therefore, the lack of ALDP results in the abnormal accumulation of VLCFAs, mainly hexacosanoic acid (26:0), due to a defect in VLCFA peroxisomal beta-oxidation. Presently, the only available therapeutic treatment is Lorenzo’s oil, which is a dietary intake of a 4:1 mixture of glyceryl trioleate oil (GTO) and trierucin. Treatment with this mixture decreases plasma VLCFAs to nearly normal levels, probably through competitive inhibition of the elongase that forms VLCFAs. However, the clinical efficacy of this oil and the clinical indications for its use are still controversial. In the genetic screening for Drosophila brain degeneration mutants, bubblegum, a Drosophila VLCFA acyl CoA synthetase (VLCS) was isolated (Min and Benzer, 1999). The optic lobe of bubblegum mutant flies showed a bubbly appearance by degeneration on light microscope sections, and electron micrographs revealed that the mutant photoreceptor axons had become greatly expanded in the diameter. Because the β-oxidation of VLCFAs is catalyzed in the peroxisomes after activation to thioester derivatives by VLCS, the first enzyme in the beta-oxidation pathway, a lack of this enzyme may result in the accumulation of VLCFAs, as observed in the cells of patients with X-ALD. Indeed, mutant male flies showed increased levels of VLCFAs, and dietary treatment with GTO restored the accumulation of VLCFAs to a normal level. Moreover, the degeneration of the optic lobes and the phototactic behavior of the bubblegum homozygous mutant were rescued by feeding with GTO. Although VLCS-deficient flies showed X-ALD-like phenotypes, VLCS knock-out mice failed to mimic the X-ALD phenotype. Therefore, the implication of VLCS in X-ALD is not clear. However, the study with the bubblegum mutant provides a good example of how the efficacy of dietary lipids on neurodegenerative diseases can be tested using the Drosophila model.

Lipids, TRP Channels, and Neurodegeneration in Drosophila A number of human diseases including neurodegenerative diseases have been associated with the

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dysfunction of transient receptor potential (TRP) channels, most of which are non-selective Ca21-permeable cation channels. Lipids possibly affect neurodegeneration by modulating TRP channels. There are 13 family members of TRP in Drosophila, which are involved in various biological pathways including light sensing, pain transduction, and temperature preference. Among them, two members of the TRP protein family, proteins encoded by the trp gene and the closely related trpl gene, appear to account for all lightactivated channel activities in Drosophila photoreceptors. TRP channels are localized within microvilli of the Drosophila eye, forming a rhabdomere. The canonical G protein-coupled signaling cascade, involving rhodopsin, Gαq protein and phospholipase C, is responsible for the activation of the light-sensitive TRP channels. Both trp gain-of-function or loss-of-function mutants undergo retinal degeneration, which is closely related with Ca21 homeostasis. One of the trp mutants, TrpP365 showed an increase of spontaneous Ca21 entry into the eye, which resulted in a constitutive current development. Interestingly, the TrpP365 mutant showed extremely rapid and massive photoreceptor degeneration (Yoon et al., 2000). In TrpP365 heterozygotes at 2 days after eclosion, raised in light-dark cycles, the photoreceptors began to show evidence of degeneration. From the genetic complementation tests that examined the electroretinogram phenotypes of TrpP365 heterozygotes and several trp alleles, it was suggested that TrpP365 was an allele of the trp gene. This was confirmed by rescuing experiments in which the wild-type trp gene was reintroduced to the TrpP365 background. As TRP channels of TrpP365 are constitutive active, ensuing accumulation of Ca21 would lead to cell death. Interestingly, loss-of-function mutants of trp also showed retinal degeneration. As the cell death in these mutants is suppressed by a loss-of-function mutation of Na1/Ca21 exchanger, the retinal degeneration in the trp mutants is due to reduced light-dependent Ca21 influx caused by disruption of TRP channel activity (Wang et al., 2005). PI(4,5)P2 depletion followed by delocalization of phosphorylated Moesin, a PIP2-regulated membrane-cytoskeleton linker, was suggested to be an underlying mechanism of the retinal degeneration in Drosophila trp loss-of-function mutants. Consistently, a deficiency in phospholipase C, which is responsible for the hydrolysis of PIP2, completely rescued the trp degeneration under red light. Moreover, the depletion of PIP2 by PIP2 phosphatase was sufficient to induce retinal degeneration. As TRP channels are implicated in various biological processes in the nerve system, disruption of PIP2 homeostasis could be associated with the neurodegenerative diseases caused by TRP channel dysfunction.

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The TRP channel has been known to be regulated by PUFAs in Drosophila (Figure 26.4). PUFAs such as arachidonic acid and ALA have been shown to activate Drosophila light-sensitive channels, TRP and TRPL, in whole-cell recordings from a photoreceptor (Chyb et al., 1999). Moreover, recombinant TRPL channels were also activated by ALA. The activation of the TRP and TRPL channels may occur by the direct binding of PUFAs with the channels, rather than through the metabolites of PUFAs or through the increasing membrane fluidity by PUFAs. Indeed, TRP channels were also activated by monounsaturated fatty acids, which are not substrates for the major PUFA-metabolizing enzymes, or trans-isomers of ALA. Moreover, inhibitors of enzymes that metabolize PUFAs, such as lipoxygenase inhibitors nordihydroguaiaretic acid and cinnamyl-3,4-dihydroxy-α-cyanocinnamate, suggest that an increase in endogenous PUFAs results in excitation of the photoreceptors through TRP channel activation (Chyb et al., 1999). Consistently, DAG lipase, which generates PUFAs from DAG, and DAG kinase, have been shown to be involved in TRP channel regulation (Leung et al., 2008; Raghu et al., 2000). Recently, a molecular mechanism suggesting how PUFAs activate TRP has been proposed. PUFAs such as LA activate TRP channels by alleviating open channel block (OCB), which is a process that blocks the flow of ions through the channel by the binding of ions to the inside of a channel pore (Parnas et al., 2009a). Drosophila TRP and TRPL channels require OCB removal in order to be activated (Parnas et al., 2009a). On the contrary, LA inhibits TRP channels with intrinsic voltage sensitivity (Parnas et al., 2009b). This type of channel does not show OCB and includes the heat-activated TRPV1 and the cold temperatureactivated TRPM8 of mammals. As the proper level of TRP channel activity is very important in maintaining neuronal health, PUFAs could influence the maintenance of nervous system by modulating the activity of TRP channels.

POINTS TO CONSIDER WHEN DROSOPHILA MODELS ARE USED FOR STUDYING THE ROLE OF LIPIDS Many studies have shown that supplementation of PUFAs are beneficial in patients with neurodegenerative diseases, as well as in animal models of the diseases (Das, 2006). In particular, the majority of the studies have been focused on the effects of long-chain fatty acids, such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), since they are major PUFAs found in the human brain. However, unlike the human brain, the Drosophila brain lacks C20 and

C22 long-chain fatty acids. Moreover, genes encoding for Δ-6/Δ-5 desaturases, the key enzymes for the synthesis of C20/C22 PUFAs, do not exist in Drosophila (Shen et al., 2010). Furthermore, GLA is also absent in the head of Drosophila, including the brain and compound eyes (Yoshioka et al., 1985). This suggests that the lipid metabolism of insects is different from that of the mammalian system. Therefore, Drosophila should be cautiously employed for the study of lipid metabolism and related diseases. Another point that should be considered when studying lipid functions in the Drosophila model is the role of cholesterol in Drosophila development. As Drosophila cannot synthesize cholesterol in its body, it relies completely on a dietary intake of whole cholesterol for maintaining its proper development, especially since cholesterol is also an essential component of the molting hormone ecdysone. It is highly probable that most PUFAs, such as ALA and LA, delay fly development through the suppression of cholesterol uptake (Lee et al., 2010) (Figure 26.5). Supporting this hypothesis, a previous study showed that LA significantly reduced the cholesterol uptake in flies fed on both LA and cholesterol (Lee et al., 2011). Based on the crucial role of cholesterol in insect development, the effect of PUFAs on the developmental phenotype of a disease model should be carefully interpreted.

PERSPECTIVE An excellent genetic model system, Drosophila has been used extensively in studies on most of the biological processes, including the pathology of human diseases. Together with the availability of various powerful tools in both genetics and molecular biology, the fly system may be a useful alternative model for the investigation of the role of lipids in the pathology of neurodegenerative diseases. Yet surprisingly, only a limited number of studies in this field have been carried out using the Drosophila model. This may be due to the difficulty in studying the lipid biochemistry in Drosophila. In addition, the contrast in lipid metabolism between insects and mammals also makes it more complicated to study the effects of lipids in Drosophila. Despite these obstacles, a growing body of evidence supports the notion that a large portion of lipids and lipid signaling are well conserved and play a crucial role in the neuronal health of Drosophila. Furthermore, as Drosophila contains many lipid metabolism-associated genes in its genome, application of the powerful genetic tools together with Drosophila mutant models of the lipid metabolism-associated genes may provide invaluable insights into the roles of lipids and lipid signaling in the maintenance of neuronal health.

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C H A P T E R

27 Polyunsaturated Fatty Acids in Relation to Sleep Quality and Depression in Obstructive Sleep Apnea Hypopnea Syndrome Christopher Papandreou INTRODUCTION

OSAHS

Obstructive sleep apnea hypopnea syndrome (OSAHS) is considered to be one of the most prevalent sleep-related breathing disorders, with an enormous effect on public health. OSAHS is being increasingly recognized as an important cause of medical morbidity and mortality (Punjabi, 2008). It is a relatively common sleep disorder that is characterized by recurrent episodes of partial or complete collapse of the upper airway during sleep. Sleep quality is disturbed by frequent arousals, as well as reduced rapid eye movement and slow wave sleep (Guilleminault et al., 1991). Depression is also a common problem in patients with OSAHS (Saunama¨ki and Jehkonen, 2007). Characteristics of OSAHS including sleep fragmentation and hypoxia could be considered possible causes of depression (Ishman et al., 2010). Moreover, there is a suspicion for a possible effect of serotonin on depression although the true role of serotonin remains unclear (Mun˜oz et al., 2000). The implication of polyunsaturated fatty acids (PUFAs) in OSAHS has also been highlighted in a recent study (Ladesich et al., 2011). More specifically, the aforementioned study suggested that disordered membrane fatty acid patterns may play a causal role in OSAHS and that the assessment of red blood cell docosahexaenoic acid (DHA) levels might help in the diagnosis of OSAHS. In this chapter associations between PUFAs and OSAHS are considered, with a particular emphasis on issues related to sleep quality and depressive symptoms.

OSAHS is characterized by recurrent episodes of partial or complete upper airway obstructions during sleep (AASM, 1999). This manifests as a reduction in (hypopnea) or a complete cessation (apnea) of airflow despite ongoing inspiratory efforts resulting in oxygen desaturations and arousals. Daytime symptoms such as excessive sleepiness are thought to be related to sleep disruption due to repetitive arousals and possibly to recurrent hypoxemia. The severity of OSAHS is evaluated by taking into consideration both severity of daytime sleepiness and overnight monitoring. Full polysomnography (PSG) is traditionally regarded as the gold standard for the diagnosis of OSAHS (Figure 27.1). The apnea-hypopnea index (AHI), the frequency of apnea and hypopnea events per hour of sleep, is widely used to define OSAHS (many clinical and epidemiological studies use this metric) (Azagra-Calero et al., 2012). In turn, apnea is defined as the absence of airflow for $ 10 seconds; and hypopnea is defined as reduction in respiratory effort with $ 4% oxygen desaturation (HQO, 2006). Based on the AHI, the following cut-off points are widely used to describe sleep apnea, but it is important to note that the clinical importance of any particular cut-off point has not been adequately determined.

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00027-2

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Mild: 5 to 15 events per hour of sleep. Moderate: 15 to 30 events per hour of sleep. Severe: more than 30 events per hour of sleep.

© 2014 Elsevier Inc. All rights reserved.

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Full polysomnography Computer Patient unit (PU)

Bedside unit (BU)

Ethernet

up to 3m

Communication unit (CU) Power

Third party device

15
29, $ 30 events per hour), values on the Epworth Sleepiness Scale were found to be above normal ($11) for 21%, 28%, 28%, and 35%, respectively, in the four categories of AHI severity. Thus, even in individuals categorized as having severe OSAHS ($30 events per hour), excessive daytime sleepiness may be present in only one third of the population. Approximately 2
4% of adults experience some degree of OSAHS (Young et al., 1993). This percentage increases even more with obesity, up to 20
40%, especially in individuals with an excessive body mass index (BMI) ($30 kg/m2). Obesity affects upper airway anatomy because of fat deposition and metabolic activity of adipose tissue (de Sousa et al., 2008). Apart from obesity, age, male sex, family history, menopause, craniofacial abnormalities, and certain health behaviors such as cigarette smoking and alcohol use increase vulnerability for the syndrome (Punjabi, 2008).

OSAHS and Associated Complications FIGURE 27.1

Polysomnography. Adapted with permission from

Braley et al., 2012.

In the general adult population, the prevalence of OSAHS is defined by an AHI of $ 5 associated with excessive sleepiness (AASM, 1999).

Sleepiness Mild: unwanted sleepiness or involuntary sleep episodes occur during activities that require little attention. Moderate: unwanted sleepiness or involuntary sleep episodes occur during activities that require some attention. Severe: unwanted sleepiness or involuntary sleep episodes occur during activities that require active attention. This emphasizes that the diagnosis of OSAHS is not based solely on the detection of respiratory events, but equally includes clinical factors such as sleepiness and impairment of social or occupational functioning. The relationship between AHI level and excessive daytime sleepiness can be illustrated by the results of a cross-sectional cohort study of adults participating in the Sleep Heart Health Study (Gottlieb et al., 1999). The study sample consisted of 886 men and 938 women, with a mean age of 65 ( 6 11) years. Sleepiness was quantified using the Epworth Sleepiness Scale. Sleep-disordered breathing was quantified by AHI measured during in-home polysomnography (PSG). When AHI was categorized into 4 groups (,5, 5
14,

OSAHS can lead to a number of complications, ranging from daytime sleepiness to possible increased risk of death. The repetitive obstructions of the upper airways during sleep and nocturnal hypoxemia cause excessive daytime sleepiness resulting in road traffic accidents (Antonopoulos et al., 2011), neurocognitive conditions like loss of alertness, memory deficit, reduced vigilance, impaired executive function, increased risk for automobile and occupational accidents, and decreased quality of life (Vijayan, 2012), removal from the social environment due to depression (Lin and Winkelman, 2012), and sexual dysfunction (Khafagy and Khafagy, 2012). In addition, there is evidence for associations between the syndrome and adverse effects on health such as hypertension (Iftikhar et al., 2013), inflammation (Peled et al., 2007), oxidative stress (Yamauchi et al., 2005), ischemic heart disease-stroke (Levy et al., 2012), and diabetes mellitus (Reichmuth et al., 2005).

Polyunsaturated Fatty Acids Polyunsaturated fatty acids (PUFAs) are fatty acids that contain more than one double bond in their structure. These are widely known as essential fatty acids since they cannot be synthesized in the human body and must be obtained from diet (Nakamura and Nara, 2003). These fatty acids are essential for maintaining normal growth and development, including that of the brain, and are important components of all cell membranes in the body (Das, 2006). There are two types of PUFAs, omega-3 and omega-6. The main omega-3 fatty acid is α-linolenic acid (ALA), and the main omega-6

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FIGURE 27.2 The two major families of polyunsaturated fatty acids. Adapted with permission from Leonard et al., 2000.

fatty acid is linoleic acid (LA). Although the human body cannot synthesize either of these fatty acids, it can use them to synthesize other essential fatty acids. ALA is a precursor of the longer chain omega-3 fatty acids eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), and LA is a precursor of the longer chain omega-6 fatty acids gamma-linolenic acid (GLA), dihomogamma-linolenic acid (DGLA), and arachidonic acid (AA) (Figure 27.2). These longer chain fatty acids can also be provided directly from dietary sources. The two classes of PUFAs are metabolically and functionally separate, and often have important opposing physiological functions. AA (n-6), DGLA (n-6), and EPA (n-3) are used to synthesize eicosanoids in the body; these are signaling molecules that exert complex control over many bodily systems, mainly in inflammation or immunity. There are four families of eicosanoids: prostaglandins; prostacyclins; thromboxanes; and leukotrienes. The eicosanoids derived from AA tend to increase inflammation, blood clotting, and cell proliferation, while those derived from EPA and DGLA decrease those functions. The amounts and balance of omega-3 and omega-6 fatty acids in a person’s diet will therefore affect the body’s eicosanoid-controlled functions, and

are therefore important in maintaining optimum health. Research is needed to establish the optimal level of dietary PUFAs that maximally affects the greatest number of health risk factors.

PUFAs and Health As described in the previous paragraph, PUFAs have properties that affect in many ways a person’s health. This paragraph describes the role of these fatty acids in several health issues. The dietary omega-3 PUFAs have been associated with various favorable functions such as anti-inflammatory effects, improving vascular and cardiac hemodynamics, triglycerides, and possibly endothelial function, controlling the blood pressure, and reducing hypertriglyceridemia and insulin insensitivity (Jafari et al., 2013). The observed effects on the aforementioned cardiovascular risk factors are caused by the influence of omega-3 PUFAs on multiple relevant molecular pathways (Guttler et al., 2012). Moreover, the composition of PUFAs in diet seems to be of particular importance in relation to risk of cancer. Epidemiological studies have shown a reduced incidence of colorectal cancer in populations consuming

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high levels of fish. Also a variety of experimental studies and different clinical trials substantiated the beneficial role of omega-3 PUFAs. Such an anti-neoplastic activity has been related to the regulatory effects exhibited by omega-3 PUFAs on cell proliferation and apoptosis. Anti-angiogenic and anti-metastatic effects have been also reported for these fatty acids. Moreover the fatty acids may act as adjuvant therapeutic agents sensitizing tumors, including colon cancer, to different antineoplastic drugs (Calviello et al., 2007). There is evidence that diets high in omega-3 PUFAs may protect people from cognitive decline and dementia. A previous review indicated that an adequate amount of DHA, the main omega-3 PUFA in cell membranes in the brain, may limit the impact of stress, an important age-aggravating factor, and influences the neuronal and astroglial functions that govern and protect synaptic transmission. This transmission, particularly glutamatergic neurotransmission in the hippocampus, underlies memory formation. The brain DHA status also influences neurogenesis, nested in the hippocampus, which helps maintain cognitive function throughout life (Denis et al., 2013). On the other hand, excessive levels of certain omega-6 PUFAs and a very high omega-6/omega-3 ratio may increase the probability of a number of diseases, including cardiovascular disease, cancer, and inflammatory and autoimmune diseases (Simopoulos, 2008).

Depression Several studies have indicated a link between PUFAs and depression. Depression constitutes a public-health concern in modern societies, and is recognized as the most prevalent mental illness in adults (Zheng et al., 1997). Depression is a common mental disorder that presents with depressed mood, loss of interest or pleasure, decreased energy, feelings of guilt or low self-worth, disturbed sleep or appetite, and poor concentration. Moreover, depression often comes with symptoms of anxiety. These problems can become chronic or recurrent and lead to substantial impairments in an individual’s ability to take care of their everyday responsibilities. At its worst, depression can lead to suicide. Almost 1 million lives are lost annually due to suicide, which translates to 3000 suicide deaths every day. For every person who completes a suicide, 20 or more may attempt to do so (WHO, 2012). Several risk factors have been reported to mediate the development of depression (Department of Health and Human Services, 1999). In general, depression results from a complex interaction of social, psychological, and biological factors (Figure 27.3).

Risk Factors for the onset of Depressive Disorders •••••••••••••••••••••••••••••••••••••••••• • Having a parent or other close biological relative with a mood disorder. • Having a severe stressor such as a loss, divorce, marital separation, unemployment, job dissatisfaction, a physical disorder such as a chronic medical condition, a traumatic experience, or in children, a learning disorder. • Having low self-esteem, a sense of low self-efficacy, and a sense of helplessness and hopelessness. • Being female. • Living in poverty.

FIGURE 27.3

Risk factors for depression. Adapted with permission from Beardslee & Gladstone, 2001.

A full patient medical history, physical assessment, and thorough evaluation of symptoms may help determine the cause of the depression. Standardized depression rating scales can be helpful such as the Hamilton Rating Scale for Depression (Zimmerman et al., 2004), the Beck Depression Inventory (McPherson and Martin, 2010) and the Zung Self-Rating Depression Scale (Zung, 1965).

Link Between PUFAs and Depression in OSAHS Depression is often diagnosed in patients with OSAHS with a prevalence ranging from 24% to 45% (Banno and Kryger, 2007). The reason remains unclear, however, OSAHS results in excessive daytime sleepiness and fatigue, symptoms that are also characteristic of depression (Pillar and Lavie, 1998). In addition, the hypoxic events during sleep, a main feature of OSAHS, can affect mood by inducing cerebral metabolic impairment (Schro¨der and O’Hara, 2005). On the neurotransmitter level, the serotoninergic system is involved in the pathophysiology of both depression and OSAHS (Adrien, 2002). OSAHS is frequent in obese patients and there is evidence that obesity is present in 70% of adults with OSAHS (Malhotra and White, 2002). Obesity can result, in turn, in the development of depressive symptoms by inducing body image dissatisfaction, low self-esteem, and social discrimination (Roberts et al., 2003). Previous studies using biomarkers of short-term (plasma cholesteryl esters and phospholipids) (Glatzz et al., 1989; Katan et al., 1997) and/or long-term (gluteal adipose tissue) (Beynen et al., 1980; Dayton et al., 1966) dietary fat intake showed a correlation between omega-3 and omega-6 PUFAs and mood disorders. Specifically, PUFAs of the omega-3 family have been inversely associated with depressive symptoms (Hibbeln, 1998; Mamalakis et al., 2006), whereas

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341

OSAHS

omega-6 polyunsaturates and the ratio of omega-6 to omega-3 PUFAs have been reported to correlate positively (Hallahan and Garland, 2005; Maes et al., 1996). Biomarkers can provide a more accurate measure of long-term dietary intake than dietary questionnaires (Baylin et al., 2002), and especially gluteal adipose tissue, since it changes much more slowly in response to diet in comparison to adipose tissue in other sites (Bagga et al., 1997). The proposed mechanisms by which PUFAs are connected with depression may be through their role in the membrane fluidity; by influencing processes such as neurotransmission and ion channel flow and/or the formation of proinflammatory eicosanoids effecting neurotransmission (Pawels and Volterrani, 2008). Only one previous study has examined differences in the gluteal adipose tissue in relation to omega-3 and omega-6 PUFA profiles in OSAHS patients with and without symptoms of depression, and investigated possible correlations (Papandreou et al., 2011). For this purpose, consecutive patients diagnosed with OSAHS by overnight attended PSG in the Sleep Disorders Unit, Department of Thoracic Medicine, Medical School, University of Crete, over a one-year period, were included. Exclusion criteria were: a) diseases such as cardiac ischemic disease, diabetes mellitus, thyroid disorders, and malignancies b) upper airway surgery c) gestation d) alcoholism e) therapy with sleeping pills f) use of anti-depressive medication, and g) ages # 18 and .65 years. Sixty-three obese patients (54 males and 9 females) composed the sample. No statistically significant differences were observed in adipose tissue omega-3 and/or omega-6 fatty acid content between OSAHS patients with and without symptoms of depression (Table 27.1). The multiple linear regression analysis revealed significant associations between depressive symptoms and variables included in this model. 20:3n-6/18:3n-6 ratio (B 5 0.34) was related positively, whereas the omega-6/omega-3 ratio (B 5 20.36) negatively (Table 27.2). Based on the study results, there is a positive correlation between adipose tissue 20:3n-6/18:3n-6 ratio and depressive symptoms in this population. On the other hand, no notable differences in gluteal adipose tissue PUFAs between the studied patients with and without symptoms of depression were found. Obesity and OSAHS may mediate mechanisms of depression overlapping with other risk factors. Taking into account the grade of depression symptoms severity, one explanation could be that our sample consisted of patients with mild and not severe depressive symptoms, probably affecting the possibility of finding any significant difference in fatty acid content when they were compared with patients without symptoms. The

TABLE 27.1 Adipose Tissue Fatty Acid Measures in Subjects with Mild Symptoms of Depression Versus Subjects Without symptoms of Depression Without Symptoms of Depression (n 5 42)

With Mild Symptoms of Depression (n 5 17)

Fatty Acid

Mean 6 SD

Mean 6 SD

Sum n-6

11.96 6 2.47

12.42 6 1.87

0.425

18:2n-6

10.98 6 2.34

11.42 6 1.78

0.420

18:3n-6

0.02 6 0.01

0.02 6 0.00

0.133

20:2n-6

0.17 6 0.05

0.17 6 0.03

0.146

20:3n-6

0.27 6 0.08

0.31 6 0.11

0.134

20:4n-6

0.51 6 0.11

0.50 6 0.13

0.711

Sum n-3

0.81 6 0.16

0.85 6 0.19

0.173

18:3n-3

0.21 6 0.06

0.21 6 0.07

0.786

20:3n-3

0.05 6 0.03

0.04 6 0.02

0.632

20:5n-3

0.03 6 0.05

0.03 6 0.05

0.464

22:3n-3

0.20 6 0.05

0.22 6 0.08

0.090

22:5n-3

0.17 6 0.05

0.19 6 0.06

0.185

22:6n-3

0.15 6 0.08

0.17 6 0.07

0.626

P-Value

TABLE 27.2 Multiple Linear Regression Predictors of Depressive Symptoms Measured by ZSRDS Scale 95% Upper CI

P-Value

Predictor

Beta

95% Lower CI

Age

20.44

2 0.52

20.11

0.003

Gender

0.67

11.05

23.53

,0.001

Smoking

0.32

0.95

5.7

0.007

Educational level

20.19

2 3.18

0.12

0.069

ESS

0.31

0.15

0.83

0.005

n-6/n-3

20.36

2 1.13

20.13

0.015

20:3n-6/18:3n-6

0.34

0.15

1.15

0.012

ZSRDS, Zung Self-rating Depression Scale. ESS, Epworth Sleepiness Scale.

correlation between depressive symptoms and PUFAs remains controversial. Most of the already published studies showed no associations (Hakkarainen et al., 2004; Appleton et al., 2008; Mamalakis et al., 2008). On the other hand, other studies have reported significant correlations between symptoms of depression and PUFAs from gluteal adipose tissue (Mamalakis et al., 2002; 2006). A possible explanation for the different results might be related to the fact that the latter studies focused on lean individuals while our patients were obese. Taking into account the low metabolic

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342

27. POLYUNSATURATED FATTY ACIDS – SLEEP-DEPRESSION

function of gluteal adipose tissue generally, and especially under conditions where other fat stores are full (abdominal region) (Tan et al., 2004), the lack of any measurable relationship between adipose tissue PUFAs of the omega-6 and omega-3 families in our study may be explained in part from the inactive nature of the site, where the adipose tissue biopsy was undertaken. One unexpected finding of this study was the negative association between the ratio of omega-6 to omega-3 fatty acids and depressive symptoms. However, recent studies have shown that the above ratio may not be a useful marker in specific pathological situations like atherosclerosis (Willet, 2007) and it would be better if the omega-6 and omega-3 fatty acids were considered individually. The multifactor etiology of depression, especially in specific populations such as OSAHS patients, might be an explanation for this unexpected association. The omega-6/omega-3 ratio in the depression profile in our population might be of limited importance compared to other, still unresolved, factors. The 18:3n-6 obtained by Δ6 desaturation can be immediately converted into 20:3n-6 (Oulhaj et al., 1992) or can be incorporated into cells (Hasler et al., 1991). The 20:3n-6/18:3n-6 ratio (Oulhaj et al., 1992) indicates the elongase activity that turns 18:3n-6 to 20:3n-6, versus 18:3n-6 availability and actual incorporation into cells. DGLA (20:3n-6) (Robertson, 1987) is present in adipose tissue and, as a precursor for prostaglandin E1 (PGE1), it may exercise an inhibiting effect in the release of stress-related hormones (epinephrine, norepinephrine, dopamine, histamine, serotonin, and gastrin) (Mamalakis et al., 1998). On the other hand, the 20:3n-6 fatty acid is the immediate precursor of AA that is converted to the prostaglandins family. Whether the increase of the 20:3n-6/18:3n-6 ratio that we found is correlated with the PGD2, PGF2, and PEF2a prostaglandins increase reported in the saliva of major depressive patients (Ohishi et al., 1988) requires further research.

Sleep Quality Sleep is a naturally recurring state characterized by reduced or absent consciousness, relatively suspended sensory activity, and inactivity of nearly all voluntary muscles. Sleep has been recognized as an essential component for rejuvenating the body to keep good health and performance (National Sleep Foundation, 2006). Furthermore, several studies suggest an independent association between sleep deprivation and psychological, cognitive, and chronic conditions reducing and threatening quality of life (Pilcher and Huffcutt, 1996; Leonard et al., 1998; Naitoh et al., 1990). Research on sleep has traditionally examined

the effects of sleep quantity; however, a more recent distinction has been made between the amount of sleep people get and the quality of that sleep. Sleep quality has been associated with mental and physical health and is therefore a major public health issue (Augner, 2011). Humans, like most animals, have circadian rhythms, which are controlled by an endogenous biological clock that dictates sleep time and duration as a natural process (Gillin et al., 2003; Kryger et al., 2000). There are several factors such as physical, physiological, psychological, and environmental, that can greatly influence the onset and quality of sleep (Figure 27.4). In humans, sleep is characterized by the rapid eye movement (REM) and non-rapid eye movement (NREM or non-REM) sleep. Each type of sleep has a distinct set of associated physiological and neurological features. The American Academy of Sleep Medicine (AASM) further divides NREM into three stages: N1, N2, and N3, the last of which is also called delta sleep or slow-wave sleep (Iber et al., 2007). Generally, the course of a night’s sleep follows a pattern of alternating non-REM and REM phases, with the first REM phase occurring within 90 minutes of falling asleep. During REM blood flow to the brain increases by 50
200%. This is when dreaming occurs. Typically, after initially falling asleep, an individual will pass through non-REM stages before entering the first period of REM sleep. Sleep researchers term this initial transition period the ‘latency to REM sleep.’ After this, there is a transition through non-REM Stages 1 and 2, and into Stages 3 and 4, characterized by high-amplitude, slow frequency delta wave activity. The course of a night’s sleep follows a parabolic curve of repeating ‘ascents’ through the non-REM stages, into REM periods, and ‘descents’ back into deep sleep (Figure 27.5). The cycle speeds up toward morning, with more time spent in REM and Stages 1
2, and less in Stage 3
4 sleep. In a healthy sleep, roughly 25% of total sleep time is spent in REM activity, and 75% in nonREM phases (Gillin et al., 2003; Kryger et al., 2000). Sleep stages and other characteristics of sleep are commonly assessed by PSG in a specialized sleep laboratory.

Link Between PUFAs and Sleep Quality in OSAHS Until now, there have been two studies that have examined the association between PUFAs and sleep quality in obese adults with OSAHS (Papandreou, 2013a; 2013b). The main difference between these studies is the different statistical analysis approach. Both

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343

OSAHS

Macro sleep environment • Temperature • Noise • Light • Toxins • Safety

Micro sleep environment • Sleep surface • Bedding • Position

Sleep practices • Schedules • Feeding • Napping

Health issues • Illness • Medications • Nutrition

Developmental context • Sleep • Cognitive

Optimal sleep

Social/Emotional context • Attachment • Temperament • Maternal mental health/stress

Sociocultural context • Values • Parenting practices

FIGURE 27.4 Factors influencing sleep quality. Adapted with permission from Owens, 2008.

Hypnogram Brief awakening Awakening REM sleep Stage 1 Stage 2 Stage 3 Stage 4

Midnight

0130

0300

0500

0630

FIGURE 27.5 Hypnogram showing sleep cycles from midnight to 6.30 am, with deep sleep early on. There is more REM before waking.

obesity and moderate to severe OSAHS have independently been associated with poor sleep quality (Malhotra and White, 2002; Fernandez-Mendoza et al., 2012). Among the risk factors for weight gain is an excessive dietary fat intake that has been found to play a significant role in obesity (Bray and Popkin, 1998). On the other hand, there is evidence suggesting that dietary fatty acids as measured in serum could be involved in sleep regulation (Irmisch et al., 2007). The proposed mechanisms by which fatty acids are connected with sleep may be through their effect on the dynamics of biochemical compounds (complex lipids,

prostaglandins, neurotransmitters, amino acids, interleukins) necessary for the initiation and maintenance of sleep (Yehuda et al., 1998). Adipose tissue, mainly consisting of fatty acids, has in recent years been recognized as a biologically highly active organ that undertakes a range of metabolic and endocrine functions and affects other organs through signals (Trayhurn and Wood, 2005). According to a recent review, alterations in adipocyte function could lead to alterations in sleep. If sleep and the functional integrity of the adipocyte are related, even indirectly, perhaps changes in the cellular function of the adipocyte could manifest as changes in

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27. POLYUNSATURATED FATTY ACIDS – SLEEP-DEPRESSION

sleep physiology (Broussard and Brady, 2010). Compared to adipose tissue in other sites, gluteal adipose tissue is considered a reliable measure of longterm dietary fat intake, since it changes more slowly in response to diet (Bagga et al., 1997). The aim of the aforementioned studies was to examine the relationships between gluteal adipose tissue fatty acids and sleep quality in obese patients with OSAHS. In both studies, a series of consecutive patients, who were diagnosed with OSAHS by overnight attended PSG in the Sleep Disorders Unit, Department of Thoracic Medicine, Medical School, University of Crete, during a one-year period were evaluated and the study population was selected based on the following criteria. Inclusion criteria were as follows: a) age 18
65 years; b) body mass index $ 30 kg/m2; c) AHI .15 events/h. Exclusion criteria were: a) continuous positive airway pressure treatment b) diseases such as cardiac ischemic disease, diabetes mellitus, thyroid disorders, and malignancies c) upper airway surgery d) gestation e) alcoholism f) therapy with sleeping pills g) use of anti-depressive medication, and h) ages # 18 and .65 years. Sixty-three obese patients (54 males and 9 females) composed the sample. In the first study (Papandreou, 2013a) Pearson correlation was used to examine the associations between sleep quality parameters and gluteal adipose tissue fatty-acids. PUFAs (r 5 0.299) and omega-6 fatty acids (r 5 0.306) were found to be positively correlated with slow-wave sleep (Table 27.3). The rest of the measured fatty acids did not correlate significantly with the sleep quality parameters. The contribution of PUFAs to slow-wave sleep has also been indicated by a previous study that found a decrease of this sleep parameter in subjects with prolonged absence of PUFAs [17]. Generally, omega-6 fatty acids generate prostaglandins of the 2-series such as prostaglandin D2 that has been found to be important for the quality of sleep [8] and may affect slow-wave sleep. However, this study did not exclude the possible confounding effect of several factors in the aforementioned associations. Taking this into account, a second study (Papandreou, 2013b) aimed to examine the association TABLE 27.3 Pearson’s Correlation Values between Gluteal Adipose Tissue Fatty Acids (PUFAs and omega-6) and Sleep Quality Parameters

of sleep quality with gluteal adipose tissue fatty acids among obese patients with OSAHS after controlling for possible confounders. Thus, multiple linear regression analysis was performed with sleep quality parameters as the dependent variable and age, gender, waist circumference, BMI, AHI, arousal index, arterial oxygen saturation (% mean and lowest), desaturations per hour, Zung Self-rating Depression Scale scores, and adipose tissue fatty acids as the initial independent variables set. Significant positive associations were found between PUFAs and sleep efficiency (B 5 0.58) and REM (B 5 0.73). Moreover, omega-3 fatty acids were positively associated with sleep efficiency (B 5 0.47), slow-wave sleep (B 5 1.15), and REM (B 5 1.21) (Table 27.4). The meaning of this finding is not clear yet. However, it might be explained by the function of the fatty acids as precursors for sleep inducing substances. More specifically, a previous study that investigated the short-term effects of feeding rats a diet high in PUFAs concluded that tissue levels of endocannabinoids, which are considered as essential factors in sleep promotion, can be affected (Arias-Carrio´n et al., 2011). The generation of prostaglandins by omega-3 or omega-6 fatty acids, with prostaglandin D2 representing an important substance for the maintenance of physiological sleep (Urade and Hayaishi, 2011), would also explain the findings of this study. Apart from the generation of prostaglandins, omega-3 fatty acids have been related to serotonin production, a sleep-promoting substance that, in turn, helps the synthesis of melatonin, which is well known as a sleep agent (Kunz et al., 2004). Contrary to a previous study, concluding that the quiescent nature of gluteal adipose tissue possibly accounted TABLE 27.4 Multiple Linear Regression Analyses Demonstrating the Associations between Sleep Quality Parameters and Gluteal Adipose Tissue Fatty Acids (PUFAs and omega-3) Adjusted by Subject’s Age, Gender, Waist Circumference, BMI, AHI, Arousal Index, Arterial Oxygen Saturation (% mean and lowest), Desaturations Per Hour, and Zung Self-Rating Depression Scale Scores

Parameters

Standardized Regression Coefficient Beta

P-Value

SLEEP EFFICIENCY (%) Omega-3 fatty acids

0.47

0.023

Fatty Acid

TST (min)

Sleep Efficiency (%)

SWS (min)

PUFAs

NS

NS

0.299

PUFAs 0.58 SLOW-WAVE SLEEP (MIN) Omega-3 fatty acids 1.15 RAPID EYE MOVEMENT SLEEP (MIN) Omega-3 fatty acids 1.21

Omega-6 fatty acids

NS

NS

0.306

PUFAs

R-values given represent p , 0.05. NS, not significant; PUFAs, polyunsaturated fatty acids; TST, total sleep time; SWS, slow-wave sleep.

0.73

0.027 0.001

0.040

0.028

PUFAs, polyunsaturated fatty acids; BMI, body mass index; AHI, apnea-hypopnea index.

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

REFERENCES

for the absence of any significant relationship reported between omega-6 and/or omega-3 PUFAs and depressive symptoms (Papandreou et al., 2011), the present study highlighted the possible role of this adipose tissue site in the regulation of sleep. This is interesting because both studies consisted of the same patients. In the present study sleep quality was associated with fatty acids derived from an adipose tissue of low metabolic activity. The fatty acids in the gluteal adipose tissue may have signaled in the brain to impact sleep quality through the aforementioned mechanisms. However, the investigation of the role of fatty acids from other more metabolically active adipose tissue sites like abdomen (Tan et al., 2004) would shed more light on this issue.

CONCLUSION OSAHS plays a role in a number of medical conditions like low sleep quality and depression that affect a person’s quality of life. PUFAs may mediate mechanisms of sleep quality and depression overlapping with other risk factors. As we improve our understanding of the relationship between these conditions and PUFAs, we will be able to provide effective dietary interventions for sleep quality improvement and reduction in the incidence of depression.

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572. Papandreou, C., Schiza, S.E., Tsibinos, G., Mermigkis, C., Hatzis, C. M., Kafatos, A.G., et al., 2011. Gluteal adipose-tissue polyunsaturated fatty-acids profiles and depressive symptoms in obese adults with obstructive sleep apnea hypopnea syndrome: a crosssectional study. Pharmacol. Biochem. Behav. 98 (2), 316
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Robertson, R.P., 1987. Arachidonic acid metabolites relevant to medicine. In: Braunwald, E., Isselbacher, K.J., Petersdorf, R.G., Wilson, J.D., Martin, J.B., Fauci, A.S. (Eds.), Harrison’s Principles of Internal Medicine. McGraw-Hill, New York, pp. 374
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688. Tan, G.D., Goossens, G.H., Humphreys, S.M., Vidal, H., Karpe, F., 2004. Upper and lower body adipose tissue function: a direct comparison of fat mobilization in humans. Obes. Res. 12, 114
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1001.

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C H A P T E R

28 Omega-3 Fatty Acids in Intellectual Disability, Schizophrenia, Depression, Autism, and Attention-Deficit Hyperactivity Disorder Basant K. Puri and Dina Gazizova INTRODUCTION Lipids encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as other sterol-containing metabolites such as cholesterol. Changes in some lipid parameters are commonly reported in patients with all forms of intellectual disability and severe mental illness such as psychosis, depression, and bipolar disorder. These metabolic changes are probably related to a combination of genetic predisposition, lifestyle factors, and psychotropic treatments. There are also hypotheses relating to abnormalities of fatty acid and phospholipid metabolism in some mental disorders and they have given rise to potential new treatment approaches. In this chapter the authors focus on the lipid profile of major psychiatric disorders
intellectual disability, schizophrenia, depression, autism, and attentiondeficit hyperactivity disorder (ADHD), as well as the effects of omega-3 fatty acids on the treatment of mental illness.

BACKGROUND Fatty acids may be broadly defined as saturated, monounsaturated, and polyunsaturated. The polyunsaturated fatty acids (PUFAs) constitute two main types: the omega-6 and the omega-3, and have the most functional significance. Arachidonic acid (AA) and docosahexaenoic acid (DHA) are the most abundant fatty acids in the brain. AA, dihomo-γ-linolenic acid (DGLA), and eicosapentaenoic acid (EPA) are also important as cell-signaling and enzyme-regulating

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00028-4

molecules and as precursors of eicosanoids (prostaglandins, thromboxanes, and leukotrienes). The parent 18-carbon fatty acids, linoleic (omega-6) and α-linolenic (omega-3), have been called ‘essential’ fatty acids because they cannot be synthesized in humans. Although established metabolic pathways exist for the biosynthesis of the other omega-3 and omega-6 fatty acids, involving enzymes which elongate or desaturate their substrates, there is evidence that the first enzymatic step, common to both the omega-3 and the omega-6 biosynthetic pathways, involving the enzyme delta-6-desaturase, may be particularly vulnerable to inhibition by factors such as micronutrient deficiencies, viral infections, and the stress hormone cortisol (Puri, 2007). This potential block can be bypassed by obtaining omega-3 PUFAs from marine sources such as oily fish, which tend to be a rich source of the 20-carbon PUFA EPA and the 22-carbon PUFA DHA. Similarly, the block to the biosynthesis of omega-6 PUFAs may be bypassed through the use of γ-linolenic acidcontaining sources such as evening primrose oil and borage oil. In turn, the γ-linolenic acid can be converted by the body into DGLA, and this in turn can be converted into AA (Puri, 2007). PUFAs make up 15
30% of the dry mass of the brain, with AA and DHA constituting 80
90% of that total in some samples, of which AA may constitute about two-thirds and DHA one-third. Some PUFAs, including AA and EPA, together with their metabolites, have important functions as second messengers and neuromodulators. They have long been investigated for their cardioprotective and antiinflammatory roles, which has led to their increased use as dietary supplements and therapeutic agents.

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© 2014 Elsevier Inc. All rights reserved.

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A new application for omega-3 fatty acids has emerged recently, namely the treatment of certain forms of mental illness.

PLASMA LIPIDS IN ADULTS WITH INTELLECTUAL DISABILITY AND THE EFFICACY OF OMEGA-3 FATTY ACIDS Intellectual disability is a condition which includes deficits in cognitive abilities as well as behavior required for personal and social sufficiency, known as adaptive functioning. Both of these impairments should be present before the age of 18 years. The impairment in intellectual function manifests as an intelligence quotient (IQ) below 70. There are three categories of intellectual disability: mild, moderate, and severe. Intellectual disability may be caused by genetic and chromosomal abnormalities as well as environmental factors, for example, host subtle biological factors, including subclinical lead intoxication, prenatal exposure to drugs, alcohol, and other toxins. In this section, the authors will concentrate on inherited conditions associated with intellectual disability and caused by the defects in lipid metabolism as well as on the lipid status in people with intellectual disabilities. There is a group of inborn errors of metabolism with neurological and muscular presentations which are associated with intellectual disabilities. This group includes defects in phospholipids, sphingolipids, and long-chain fatty acid biosynthesis. Clinical presentations are diverse but can be divided into the following (Lamari et al., 2013): 1. Diseases of the central nervous system; 2. Peripheral neuropathies; 3. Muscular/cardiac presentations. Disorders of sphingolipid metabolism are associated with the class of lipid storage disorders known as sphingolipidoses. The main members of this group include Niemann-Pick disease, Fabry disease, Krabbe disease, Gaucher disease, Tay-Sachs disease, and metachromatic leukodystrophy. They are generally inherited in an autosomal recessive fashion, except for Fabry disease, which is X-linked. The incidence of sphingolipidoses is approximately 1 in 10,000 but substantially higher in certain populations such as Ashkenazi Jews. Enzyme replacement therapy is available mainly to treat Fabry disease and Gaucher disease, and people with these types of sphingolipidoses may live well into adulthood. The other types are generally fatal by age 1 to 5 years for infantile forms, but progression may be mild for juvenile- or adultonset forms.

Adrenoleukodystrophy and Adrenomyeloneuropathy This disorder affects 1 in 20,000 males either as cerebral adrenoleukodystrophy (ADL), with usual onset in childhood (Laan et al., 2000), although adult-onset cases can occur (Sutovsky et al., 2007) or as adrenomyeloneuropathy (AMN) in adults (Walterfang et al., 2007). The neonatal form is autosomal recessive, whereas the adult form is X-linked. Women are usually carriers and if affected have milder presentations. The defect lies in the gene ABCD1. Childhood adrenoleukodystrophy (cALD) is a metabolic disorder in which very long-chain fatty acids (VLCFAs) such as hexacosanoate accumulate in lipidcontaining tissue in the brain due to ALD protein gene defects, ultimately leading to lipotoxicity-induced neuroinflammatory demyelinating disease. cALD is a metabolic disorder of VLCFAs that eventually leads to inflammatory bilateral demyelination with marked activation of microglia and astrocytes and accumulation of proinflammatory cytokines (TNF-α and IL-1β) and extracellular matrix proteins (Khan et al., 2010). There are characteristic lamellar inclusions in the Schwann cells of the central nervous system and adrenal cortex, leading to non-inflammatory axonopathy involving the spinal cord. X-linked adrenoleukodystrophy (X-ALD), an inherited peroxisomal disorder, is characterized by progressive demyelination and adrenal insufficiency. It is the most common peroxisomal disorder affecting between 1 in 15,000 to 1 in 20,000 boys and manifests with different degrees of neurological disability. The onset of childhood X-ALD, the major form of X-ALD, occurs between the ages of 4 to 8 years and then death occurs within the next 2 to 3 years. As yet no proven therapy improves or changes the course of the disease process in X-ALD patients. All forms of X-ALD accumulate pathognomonic amounts of VLCFAs. VLCFA levels have been used as a tool for both prenatal and postnatal diagnosis. Similar to other genetic diseases affecting the central nervous system, gene therapy in X-ALD does not seem to be a realistic option in the near future and in the absence of such a treatment a number of therapeutic possibilities have been investigated (Moser, 1995; Skjeldal et al., 1994). Adrenal insufficiency associated with X-ALD responds readily to steroid hormone replacement therapy, however, there is as yet no proven therapy for neurological disability. Two forms of therapies are presently under current investigation. Dietary therapy with ‘Lorenzo’s oil’, which is a mixture of glycerol trioleate and glycerol trierucate, normalizes plasma VLCFA levels. However, it does not seem to improve the clinical status of X-ALD patients and, in particular, the normalization of

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VLCFA levels does not appear to be associated with improvement in or retardation of demyelination of visual pathways (Moser, 1993; Kaplan et al., 1993). These results, in part, may be due to the fact that the fatty acid composition of the brain is not normalized because of a failure of erucic acid to enter the brain in significant quantity. The use of unsaturated fatty acids to block the synthesis of VLCFA is based on the assumption that unsaturated fatty acids are non-toxic; it should be noted that exogenous unsaturated VLCFAs (and, indeed, also AA, EPA, and DHA) induce the production of superoxide ions by human neutrophils (Hardy et al., 1994). Bone marrow therapy also appears to be of limited value because of the complexity of the protocol and of insignificant efficacy in improving the clinical status of the patient. cALD patients die in early childhood whereas AMN patients may have a normal life span (Pahan et al., 1998). AMN is the milder form, with onset at 15
30 years of age and a progressive course. It affects the brain, spinal cord, adrenal glands, and testes. Clinical features include spastic paraparesis, ataxia, and sensory loss in the lower limbs, language difficulties, and adrenal insufficiency. Rarely, focal signs and memory problems may mimic Alzheimer’s disease. Adrenal insufficiency progresses to Addison’s disease (also known as chronic or primary adrenal insufficiency, hypocortisolism or hypoadrenalism). Biochemical assay of body fluids and biopsy of skin and conjunctival nerve terminals are useful in diagnosis. Treatment is symptomatic with adrenal hormone replacement and hematopoietic stem cell transplantation. Psychological support and physiotherapy are helpful.

Fatty Acid, Lipid, and Cholesterol Levels It has been hypothesized that alteration in plasma fatty acid composition may play a role in intellectual disabilities. One study has shown a significant inverse association between plasma DHA and mental retardation and suggests that the proportion of plasma total omega-3 fatty acids, particularly DHA, and the ratio of total omega-6 fatty acids to total omega-3 fatty acids is associated with mental retardation in children. For each unit increase in plasma DHA, the odds of mental retardation are reported to decrease by 74% (Neggers et al., 2009). Patients with intellectual disabilities have a variety of enhanced risk factors for cardiovascular disease. These include sedentary lifestyle, poor diet, smoking, and obesity. A higher prevalence of obesity in women with intellectual disability compared with men with intellectual disability and with women in the general population has been reported (Emerson, 2005).

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Moreover, although people with intellectual disability have lower levels of physical activity than participants from the general population, the risk of inactivity is higher in women with intellectual disability than in men. A recent study of adults with intellectual disability and Down’s syndrome found that of 37% of a sample of 38 young adults with Down’s syndrome were overweight and 37% were obese; the body mass index was significantly higher in the women than in the men (Soler Marin and Xandri Graupera, 2011). The average serum cholesterol level has been reported to have increased in people with intellectual disability with deinstitutionalization. In their survey of 1400 long-stay hospital intellectual disability adult inpatients, Eastham and Jancar reported that serum cholesterol levels were lower than in the general population (Eastham and Jancar, 1969). However, examination of cardiovascular risk factors in adults with mental retardation in 1994 indicated that they had cardiovascular risk profiles similar to those in the Framingham Offspring Study, including increased cholesterol levels (Rimmer et al., 1994). Health behaviors, such as high-fat dietary intakes and sedentary physical activity habits, likely play a major role in the development of high obesity rates and elevated cardiovascular disease risk factors for adults with mental retardation who reside in community settings. Given the association of such medication with metabolic syndrome, another possible explanation of increased body mass index and impaired lipid profile, including elevated triglycerides, in people with intellectual disabilities may be due to the frequent use of antipsychotic medication in such people (Holden and Gitlesen, 2004); however, in a recent study, the present authors have reported that serum triglyceride and cholesterol levels in such subjects did not appear to be unduly affected by first- or second-generation antipsychotic medication (Gazizova et al., 2012). The usual indication for use of antipsychotic medication is comorbid psychiatric disorders. The rate of psychiatric illness in people with intellectual disability is several times higher than in the general population. For example, about 3% of people with intellectual disabilities meet the criteria for schizophrenia. They are also four times more likely to suffer from depression or anxiety compared with people without intellectual disability. Another more frequent indication is challenging behavior, which is an umbrella term for culturally unacceptable behavior (e.g. self-injury, aggression, destruction of property, and screaming). About 25
30% of all individuals with intellectual disability using services in the UK regularly receive antipsychotics, rising to 48% of those with challenging behavior (Brylewski and Duggan, 2004). Recent concerns have focused on the inadequate definition of challenging behavior and the relative

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absence of controlled evidence for efficacy. People with intellectual disability are less likely to identify relevant medical symptoms accurately and are less amenable to routine blood testing. Hence such adverse effects carry a particular and enhanced threat to their health (Frighi et al., 2011).

PLASMA LIPIDS IN SCHIZOPHRENIA AND THE EFFICACY OF OMEGA-3 FATTY ACIDS Schizophrenia is a severe and enduring mental illness with multifactorial etiology. The prevalence of schizophrenia is between 1% and 2%. The course of the disease is chronic and is characterized by episodes of remission and relapses. During the relapses the patients displays two classes of symptoms: positive, which include hallucinations, delusions, disorganized thought and behavior; and negative (deficit), such as blunted affect and emotions, poverty of speech, and lack of motivation. The main treatment of the disorder includes antipsychotic medication, which may have significant side effects, such as extrapyramidal side effects and metabolic syndrome (Puri, 2012a). As with most other diseases, a combination of genetic and environmental factors is likely to play a role in the development of schizophrenia. Current research is focused on the role of neurobiology including biochemical and neurotransmitter factors (such as dopamine and glutamate), neural circuits, and brain metabolism. There is evidence relating to abnormal phospholipid and fatty acid metabolism in schizophrenia and abnormalities in the cell membranes; this was referred to as the membrane phospholipid hypothesis of schizophrenia by the late David Horrobin (Horrobin, 1998, Horrobin et al., 1994). A reduced level of phosphomonoesters and an increased level of phosphodiesters have been found in the brain in schizophrenia, using 31-phosphorus neurospectroscopy (Pettegrew et al., 1991), with the changes occurring pre-symptomatically (Keshavan et al., 1991). These phosphomonoesters index cerebral cell membrane phospholipid anabolism (Puri and Treasaden, 2009), while the phosphodiesters index cerebral cell membrane phospholipid catabolism (Puri, 2012b). Thus, schizophrenia may be associated with increased cerebral cell membrane phospholipid breakdown and reduced biosynthesis. These findings are consistent with reports of increased serum and plasma levels of phospholipase A2 enzyme activity in schizophrenia (Gattaz et al., 1990b; 1990a); this group of calciumindependent enzymes preferentially break down phospholipid molecules by cleaving the fatty acid (usually

a long-chain polyunsaturated fatty acid) attached to the middle carbon atom (the sn2 position) of the glycerol backbone of the phospholipid molecule. Indeed, such increased activity has also been found in platelets in schizophrenia patients (Gattaz et al., 1995) and may alter dopaminergic neurotransmission in the brain (Schaeffer et al., 2012). There are also changes in the metabolic pathway of AA in schizophrenia. Prostaglandin D2 is a cyclooxygenase metabolite of AA which mediates the skin flush response to topical niacin (nicotinic acid); this response is reduced in people with schizophrenia (Puri et al., 2001b; Ward et al., 1998; Wilson and Douglass, 1986; Vaddadi, 1981). A reduced inflammatory response, which depends in part on the same pathway, is also manifested by the relative rarity of rheumatoid arthritis in schizophrenia (Horrobin, 1977; Mellsop et al., 1974; Mellsop, 1972). Plasma and erythrocyte membrane levels of α-linolenic acid (ALA) have been reported to be elevated in schizophrenia (Obi and Nwanze, 1979). Linoleic acid (LA) levels have been reported to be reduced in non-lithiumresponsive patients with schizophrenia, while AA levels may be elevated and DGLA levels low (Horrobin and Huang, 1983). Moreover, schizophrenia is associated with changes in prostaglandin biosynthesis, with a relative deficit for series 1 (biosynthesized from DGLA) and an excess for the AA-derived series 2 (Horrobin, 1979b; 1979a; 1977; Horrobin et al., 1978; Abdulla and Hamadah, 1975; Mathe et al., 1980). Furthermore, schizophrenia is associated with increased exhaled ethane levels (Puri et al., 2008). Ethane levels in schizophrenia are not correlated with either symptom severity or erythrocyte omega-3 PUFA levels, so that the exhaled ethane is likely to be a biomarker of increased omega-3 lipid peroxidation in this disorder (Ross et al., 2011; Puri et al., 2008). Dietary fatty acid supplementation may have a direct, positive effect on the symptoms of schizophrenia and may enhance the efficacy of antipsychotic medication. There have been case reports and case series suggesting that omega-3 PUFAs may be useful in the treatment of schizophrenia. The first case report, by Puri and Richardson, described an essentially antipsychotic drug-naive schizophrenia patient who showed sustained improvement of positive and negative symptoms while being treated with ethyl-EPA (without any antipsychotic medication) (Puri and Richardson, 1998). Furthermore, the ethyl-EPA treatment was also associated with normalization of blood fatty acid levels, reversal of cerebral ventricular dilatation, and a reduction in neuronal membrane phospholipid turnover (Puri et al., 2000). The second case report, by Su and colleagues, described a patient with an acute relapse of schizophrenia during

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TABLE 28.1

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Prospective Efficacy Trials of Omega-3 PUFAs in Schizophrenia

Study

Methods

Participants

Interventions

(Berger et al., 2007)

Double-blind RCT Duration: 12 weeks. Setting: inpatient and outpatient.

n 5 80. Age: 15
29 y. Firstepisode psychosis, currently psychotic.

1.2 g purified ethyl-EPA Some evidence of response, dose daily 1 current of antipsychotic lower than in antipsychotic medication. placebo group.

(Emsley et al., 2002)

Double-blind RCT Duration: 12 weeks.

n 5 40. Age: 18
55 y, mean 46 y. Chronic schizophrenia, mean duration of illness 23 years.

3 g/day ethyl-EPA. Previous neuroleptic treatment remained constant throughout the trial.

EPA associated with reduction of symptoms and tardive dyskinesia.

(Emsley et al., 2006)

Double-blind RCT Duration: 12 weeks. Setting: inpatient and outpatient from single site.

n 5 84. Age: 18
60 y. Schizophrenia and schizoaffective disorder with tardive dyskinesia.

2 g/day ethyl-EPA. Allowed anticholinergic medication for treatmentemergent extrapyramidal symptoms.

Significant improvement in mental health, change in score on PANSS and significant improvement on CGI subscore for tardive dyskinesia.

(Fenton et al., 2001)

Double-blind RCT Duration: 16 weeks. Setting: outpatient clinic.

n 5 87. Schizophrenia or schizoaffective disorder with residual symptoms despite neuroleptic treatment. Age: 18
65 y, mean 40 y.

3 g/day ethyl-EPA. No difference between EPA and Previous neuroleptic placebo. treatment remained constant throughout trial.

n 5 81. Mean age 16.4 y. Adolescents at risk of firstepisode psychosis.

0.84 g/day EPA 1 0.7 g/ day DHA.

(Amminger et al., 2010) Double-blind RCT Follow-up: 12 months.

pregnancy who improved when treated with omega-3 fatty acids as monotherapy (Su et al., 2001). An open-label evaluation of fish oil (EPA and DHA) added to psychotropic medication, within a group of schizophrenia patients, demonstrated significant improvement in positive symptoms. There was a strong correlation between the level of intake of EPA/ DHA in the normal daily diet and the severity of symptoms: more omega-3 PUFAs were associated with less severe symptoms (Mellor et al., 1995). Some prospective trials point to the possible efficacy of fatty acids in the treatment of schizophrenia; these are shown in Table 28.1. In summary, evidence has shown that adding 2
3 g of omega-3 PUFAs daily to antipsychotic medication has beneficial effects on the residual symptoms of schizophrenia. Such treatment may also prevent the development of psychosis in high-risk groups. In the study by Amminger and colleagues, carried out in a high-risk group of adolescents and young adults with sub-threshold psychosis, approximately 5% of those receiving omega-3 PUFAs developed psychotic illness compared with 28% of the placebo group (Amminger et al., 2010). Patients with schizophrenia have a higher level of comorbidity than people not suffering from schizophrenia. They are more likely to be overweight, which may result from factors such as poor nutrition, decreased physical activity, and the effect of antipsychotic

Outcomes

Some evidence of benefits in preventing development of psychotic disorder.

medication. In turn, an increased body mass index contributes to an increased risk of cardiovascular morbidity, type 2 diabetes mellitus, and hyperlipidemia. Historically, schizophrenia blood lipid studies dating from the 1930s to around 1960 were concerned primarily with relating cholesterol levels to diagnosis and prognosis in mental disease. The consistent findings were that acutely disturbed and ‘excited’ patients had higher cholesterol levels than chronic anergic schizophrenia patients; and high cholesterol levels during acute psychiatric illnesses were associated with a good chance for recovery (Friedman and Rosenman, 1959). Thereafter, high cholesterol levels have been believed to contribute to cardiovascular disease, and studies became focused on the problem of cardiovascular disease prevention in schizophrenia (Sletten et al., 1964).

PLASMA LIPIDS IN DEPRESSIVE ILLNESS AND EFFICACY OF OMEGA-3 FATTY ACIDS Depressive illness is an affective disorder with a lifetime prevalence estimated to be between 5% and 11%. It is characterized by depressed mood, anhedonia, changes in sleep patterns and weight, and reduced cognitive abilities and concentration. The lifetime prevalence of mental disorders has been found to be lower

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28. OMEGA-3 FATTY ACIDS IN INTELLECTUAL DISABILITY

in Mediterranean countries than in Northern European countries. Age-standardized suicide rates, which may indirectly reflect the prevalence of severe depression, tend also to be lower in Mediterranean countries. One of the possible explanations is the abundant use of olive oil, which is rich in monounsaturated fatty acids (MUFAs). A beneficial effect of MUFA intake from olive oil with regard to depression has been hypothesized to be that it may improve the binding of serotonin to its receptors. In fact, an inverse association between olive oil consumption and a 15-point geriatric depression scale score has been reported; this inverse association was mainly present at the higher end of the range, suggesting that high olive oil consumption was associated with lower risk of more-severe depression (Kovess-Masfety et al., 2007). Ecological and cross-sectional studies provide epidemiological support. A strong negative association between apparent fish intake and the annual prevalence of major depression across nine countries has been reported (Hibbeln, 1998). A large, census-based, Japanese 17-year cohort study of over a quarter of a million people found that daily fish consumption was associated with a reduced risk of suicide compared with non-daily fish intake (Hirayama, 1990). In a much smaller cross-sectional study of 3004 adults (aged 25 to 64 years) in Kuopio, Finland, the risk of being depressed and the risk of having suicidal ideation were also found to be lower among frequent lake-fish consumers compared with more infrequent consumers (Tanskanen et al., 2001). Another, larger, cross-sectional study, this time of 4644 New Zealand adults aged at least 15 years, reported that fish consumption was significantly associated with higher self-reported mental health status (Silvers and Scott, 2002). A study of the Northern Finland 1966 Birth Cohort followed up prospectively from pregnancy up to the age of 31 years, including 2721 males and 2968 females, showed that, after adjusting for body mass index, serum total cholesterol level and socioeconomic situation, there was an increased risk of developing depression in females who rarely ate fish compared with regular fish eaters; no such association was found in the males (Timonen et al., 2004). In a population-based sample of 3317 African-American and Caucasian men and women from the Coronary Artery Risk Development in Young Adults study, the highest quintiles of intakes of EPA, DHA, and EPA plus DHA were associated with a lower risk of depressive symptoms at year 10, particularly in women. In view of the evidence of associations between various types of cardiovascular disease and depression, and that both disorders are associated with low blood EPA levels, ethyl-eicosapentaenoate may be of particular benefit in depressed patients who are also at risk for cardiovascular disease (Puri, 2008). EPA lowers

triglyceride levels, inhibits platelet aggregation, and inhibits cardiac arrhythmias (Horrobin and Bennett, 1999). The large Japan EPA Lipid Intervention Study, studying almost 19,000 patients, has provided good evidence that EPA may help prevent major coronary events and reduce the risk of recurrent stroke in hypercholesterolemic patients receiving low-dose statin therapy, even though the intake of fish in the Japanese is already high (Oikawa et al., 2009; Tanaka et al., 2008; Saito et al., 2008; Yokoyama et al., 2007). The first study of EPA in depression was a case report in which ethyl-EPA was added to the conventional antidepressant treatment of a treatment-resistant severely depressed and suicidal male patient with a seven-year history of unremitting depressive symptoms; not only did his symptomatology improve markedly, but nine months of treatment was associated with reduced lateral ventricular volume and reduced neuronal phospholipid turnover (Puri et al., 2002; 2001a). Since then, there have been many controlled trials of omega-3 PUFAs in adults suffering from depression. A comprehensive meta-analysis of such studies not only showed evidence of efficacy, but also provided evidence that EPA may be more efficacious than DHA in treating depression (Martins, 2009). One randomized, placebo-controlled trial in children aged between 6 and 12 years indicated that omega-3 PUFAs might also be of therapeutic value in childhood depression (Nemets et al., 2006).

PLASMA LIPIDS IN AUTISM AND EFFICACY OF OMEGA-3 FATTY ACIDS Autism (or autistic disorder) is a pervasive developmental disorder which is part of the autism spectrum disorders (ASDs) characterized by severe deficits and pervasive impairment in multiple areas of development including impairment in reciprocal social interaction, impaired communication, and the presence of stereotyped behaviors, interests, and activities (APA, 2000). A steady increase in prevalence has been reported; in the most recent Icelandic birth cohort study of children born during 1994 to 1998, the prevalence of ASD in boys was 172.4 per 10,000 and in girls 64.8 per 10,000, with the proportion of ASD children with an IQ of less than 70 being approximately 45% (Saemundsen et al., 2013; Manning-Courtney et al., 2013). In fact, the reported prevalence rates vary widely, as do the epidemiological methodologies employed, and the high rates reported recently may, in part, be a function of the latter (Isaksen et al., 2013). There is no strong evidence of any significant changes in plasma or erythrocyte membrane omega-3 PUFA levels in ASD, although there is a suggestion that

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CONCLUSIONS

the ratio of AA to EPA may be raised in children with autism (Bell et al., 2010; Bu et al., 2006). Furthermore, phospholipid-related signal transduction has been reported to be normal in autism (Puri and Singh, 2002). There is a preliminary report from a 16-week study (seven active versus six placebo) that large doses of AA added to DHA may improve impaired social interaction in individuals (mean age 14.6 years) with ASD (Yui et al., 2012; 2011). While some pilot studies suggest that the use of pure omega-3 PUFAs (without any AA) may be beneficial in ASD (Meguid et al., 2008; Amminger et al., 2007), others have failed to find any benefit (Bent et al., 2011; Politi et al., 2008). Unsurprisingly, therefore, recent reviews have concluded that there is no evidence at the present time to support the use of omega-3 PUFAs for ASD, although there is a need for large, well-conducted randomized controlled trials examining both high- and lowfunctioning individuals (Williams and Marraffa, 2012; James et al., 2011).

PLASMA LIPIDS IN ADHD AND EFFICACY OF OMEGA-3 FATTY ACIDS ADHD is usually first diagnosed before adulthood and is characterized by prominent symptoms of inattention and/or hyperactivity-impulsivity; inattention may manifest in academic, occupational, or social situations; hyperactivity may manifest in ways such as fidgeting, excessive inappropriate running or climbing, difficulty playing or engaging quietly in leisure activities, excessive talking, or often being ‘on the go’; impulsivity may manifest as impatience, difficulty with delayed gratification or frequently interrupting others inappropriately (APA, 2000). An increase in prevalence has been reported over many decades, with the prevalence in the US tending to be higher than that elsewhere. A recent American study reported a prevalence of ADHD in children aged between 5 and 13 years of between 8.7% and 10.6% (Wolraich et al., 2012). In comparison, the prevalence in a sample of Norwegian 7- to 9-year-olds was reported as 5.2% (Ullebo et al., 2012). It should be borne in mind that ADHD also affects adults; indeed, a recent Dutch study found that 2.8% of adults aged over 60 years are affected (Michielsen et al., 2012). In their seminal study, Stevens and colleagues reported that boys with ADHD had lower concentrations of certain plasma polar lipids (20:4n-6, 20:5n-3, and 22:6n-3) and of certain erythrocyte lipids (20:4n-6 and 22:4n-6) than controls (Stevens et al., 1995). Furthermore, subjects with lower compositions of total omega-3 fatty acids had more behavioral problems, temper tantrums, and learning, health, and sleep

problems than did those with high proportions of omega-3 fatty acids (Burgess et al., 2000; Stevens et al., 1995). Adults with ADHD symptoms have been reported to have lower erythrocyte total omega-3 fatty acids and DHA (Young et al., 2004). In a separate study by the Purdue group, young adults with ADHD have also been found to have a lower proportion of omega-3 fatty acids in plasma phospholipids and erythrocyte membranes (Antalis et al., 2006). Interestingly, in an event-related potential response to the presentation of facial expressions study in 20 adolescent boys with ADHD, erythrocyte membrane EPA was positively associated with a cognitive bias in orientation to overt expressions of happiness over both sad and fearful faces as indexed by midline frontal P300 amplitude, while the DHA level correlated with right temporal N170 amplitude in response to covert expressions of fear (Gow et al., 2009). Again, in a resting state electroencephalographic study in 46 adolescent boys with ADHD, erythrocyte DHA was positively associated with fast frequency activity (alpha during eyes-open and beta during eyes-closed), while frontal theta activity during both eyes-open and eyes-closed was positively associated with EPA (Sumich et al., 2009). In 2000, Richardson and Puri put forward the hypothesis that many of the clinical features of ADHD could be explained on the basis of an underlying abnormality of fatty acid metabolism, and further hypothesized that treatment with PUFAs may be of therapeutic value (Richardson and Puri, 2000). This hypothesis has been published again more recently, based on non-human mammalian research findings (Transler et al., 2013). This fatty acid model might also help explain the comorbidity of ADHD with disorders such as dyslexia, dyspraxia (developmental coordination disorder), and ASD (Richardson and Ross, 2000). There have been several PUFA trials in ADHD during this century. A recent Cochrane review concluded that ‘there is little evidence that PUFA supplementation provides any benefit for the symptoms of ADHD in children and adolescents. The majority of data showed no benefit of PUFA supplementation, although there were some limited data that did show an improvement with combined omega-3 and omega-6 supplementation. It is important that future research addresses current weaknesses in this area, which include small sample sizes, variability of selection criteria, variability of the type and dosage of supplementation, short follow-up times, and other methodological weaknesses.’ (Gillies et al., 2012).

CONCLUSIONS In this chapter evidence has been presented showing that omega-3 PUFAs play an important role in

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schizophrenia, depression, intellectual disability, ASD, and ADHD. Large, well-conducted clinical trials are now indicated to determine the efficacy (or otherwise) of PUFA therapy in these disorders.

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28. Ullebo, A.K., Posserud, M.B., Heiervang, E., Obel, C., Gillberg, C., 2012. Prevalence of the ADHD phenotype in 7- to 9-year-old children: effects of informant, gender and non-participation. Soc. Psychiatry Psychiatr. Epidemiol. 47, 763
769. Vaddadi, K.S., 1981. Niacin flushing and schizophrenia. Med. Hypotheses. 7, 599
600. Walterfang, M.A., O’Donovan, J., Fahey, M.C., Velakoulis, D., 2007. The neuropsychiatry of adrenomyeloneuropathy. CNS Spectr. 12, 696
701. Ward, P.E., Sutherland, J., Glen, E.M., Glen, A.I., 1998. Niacin skin flush in schizophrenia: a preliminary report. Schizophr. Res. 29, 269
274. Williams, K., Marraffa, C., 2012. No evidence yet to support omega-3 fatty acids as a treatment for autism. J. Paediatr. Child. Health. 48, 534
536. Wilson, D.W., Douglass, A.B., 1986. Niacin skin flush is not diagnostic of schizophrenia. Biol. Psychiatry. 21, 974
977. Wolraich, M.L., McKeown, R.E., Visser, S.N., Bard, D., Cuffe, S., Neas, B., et al., 2012. The prevalence of ADHD: Its diagnosis and treatment in four school districts across two states. J. Atten. Disord. [Epub ahead of print]. Yokoyama, M., Origasa, H., Matsuzaki, M., Matsuzawa, Y., Saito, Y., Ishikawa, Y., et al., 2007. Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis. Lancet. 369, 1090
1098. Young, G.S., Maharaj, N.J., Conquer, J.A., 2004. Blood phospholipid fatty acid analysis of adults with and without attention deficit/ hyperactivity disorder. Lipids. 39, 117
123. Yui, K., Koshiba, M., Nakamura, S., Onishi, M., 2011. Therapeutic effects of larger doses of arachidonic acid added to DHA on social impairment and its relation to alterations of polyunsaturated fatty acids in individuals with autism spectrum disorders. Nihon Shinkei Seishin Yakurigaku Zasshi. 31, 117
124. Yui, K., Koshiba, M., Nakamura, S., Kobayashi, Y., 2012. Effects of large doses of arachidonic acid added to docosahexaenoic acid on social impairment in individuals with autism spectrum disorders: a double-blind, placebo-controlled, randomized trial. J. Clin. Psychopharmacol. 32, 200
206.

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C H A P T E R

29 Effect of Omega-3 Fatty Acids on Aggression Kei Hamazaki, Tomohito Hamazaki and Hidekuni Inadera INTRODUCTION The basic membrane components of neuronal cells are phospholipids and cholesterol with small amounts of protein. Therefore, the brain consists mainly of lipids, which account for about 60% of its dry weight. Omega-6 polyunsaturated fatty acids (PUFAs), primarily arachidonic acid (AA), and omega-3 PUFAs, primarily docosahexaenoic acid (DHA), are esterified at the sn-2 position of phospholipids. The levels of these PUFAs are higher in the brain than in other organs. Clinical studies have revealed that omega-3 PUFAs ameliorate depression; in fact, a recent metaanalysis showed a significant benefit of omega-3 PUFAs in depression (Martins et al., 2012). However, few investigators have examined the effect of omega-3 PUFAs on aggression. Weidner et al. (1992) measured aggressive hostility in a 5-year study. Aggressive hostility did not significantly change at the end of the study in participants who ate a typical American diet, but was significantly reduced in those who consumed a low-fat, highcarbohydrate diet (including fish). This was probably the first cohort study relating fish oils and hostility, but the amount of fish consumed was not reported. Because the number of related studies is rather limited, the outcome measures differ, and the populations studied vary (i.e., children versus the elderly), here we perform a narrative review of the effects of fish oil on aggression.

FIRST TRIAL OF OMEGA-3 PUFAs AND AGGRESSION IN YOUNG ADULTS We initially performed a double-blind study investigating the effect of omega-3 PUFAs on aggression in medical students (Hamazaki et al., 1996). Forty-one students were allocated to the control (n 5 22,

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00029-6

soybean oil) or omega-3 PUFA group (n 5 19, DHA 1.5
1.8 g/day) for 3 months. At the start and end of the study, aggression was measured according to the Picture Frustration (PF) Study (Rosenzweig, 1976). A stressor component was applied at the end of the study. A few days after the second (last) PF Study, important exams started for all participants. Therefore, participants were likely to be stressed while preparing for exams around the time of the last PF Study. Aggression in the control group increased because of the presence of the stressor, but it remained unchanged in the omega-3 PUFA group (Figure 29.1). There were highly significant differences in the changes in aggression between the two groups (p 5 0.003). The results of this study indicated a possibility that stressor-enhanced aggression is controlled by prior administration of omega-3 PUFAs (Hamazaki et al., 1996). There were no aggression-controlling effects in the absence of a stressor, and this was later confirmed by another trial without stressors (Hamazaki et al., 1998). Forty-six university students were randomly divided into two groups; the same protocol was used as before, but without any stressor. As expected, aggression measured by the PF Study was not improved in the omega-3 group compared with the control group (Hamazaki et al., 1998). This effect of omega-3 PUFAs on aggression in young male adults was recently re-examined by another group in the United Kingdom (Long and Benton, 2013). University students (mean age 20.9 years) were allocated to four groups receiving placebo (n 5 42), multivitamins/minerals (n 5 43), omega-3 PUFAs (672 mg DHA 1 96 mg eicosapentaenoic acid (EPA) per day) (n 5 47), or both multivitamins/minerals and omega-3 PUFAs (n 5 41) for 3 months in a double-blind manner. Only omega-3 PUFAs decreased ‘extra aggression’ measured by the PF Study. No significant effects were observed in the vitamins/minerals group. Additionally, there was no evidence of a

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© 2014 Elsevier Inc. All rights reserved.

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29. EFFECT OF OMEGA-3 FATTY ACIDS ON AGGRESSION

50

Aggression

40

30

20

0 Start

End

Start

Control group

End

Omega 3

FIGURE 29.1 The effects of omega-3 PUFA-rich fish oil on aggression in students. Forty-one students were randomly allocated to the control and omega-3 PUFA groups in a double-blind fashion. After 3 months of intervention, aggression measured with the PF Study increased in the control group (p 5 0.002) because of the presence of a stressor (important exams), whereas it was stable in the omega-3 PUFA group (inter-group difference: p 5 0.003). Reproduced with permission from Hamazaki et al., 1996.

synergistic interaction between vitamins/minerals and omega-3 PUFAs. Of interest, the trial was conducted at the time of stressful end-of-year examinations as in our first study (Hamazaki et al., 1996).

Effect of Omega-3 PUFAs on Aggression in Schoolchildren We have also investigated the effect of omega-3 PUFAs on aggression in schoolchildren (Itomura et al., 2005). A placebo-controlled double-blind study was performed with 166 schoolchildren aged 9
12 years. The subjects in the omega-3 PUFA group (n 5 83) were given omega-3 PUFA-fortified foods (bread, sausage, and spaghetti) (3600 mg DHA 1 840 mg EPA per week) and the placebo group (n 5 83) received control foods (50% soybean and 50% rapeseed oil) for 3 months. In girls, physical aggression assessed by the Hostility-Aggression Questionnaire for Children (HAQ-C) increased significantly (median 13 to 15; n 5 42) in the control group and did not change (median 13 to 13, n 5 43) in the omega-3 PUFA group, with a significant intergroup difference (p 5 0.008). By contrast, there were no significant changes in physical aggression in boys. Impulsivity in girls assessed by parents/guardians using the diagnostic criteria for attention deficit hyperactivity disorder (ADHD) of the Diagnostic and Statistical Manual of Mental DisordersIV was significantly reduced in the fish oil group compared with the control group (p 5 0.008). In this study, we did not intend to set a stressor during the trial; however, blood sampling in front of the

schoolchildren while they were answering questionnaires unexpectedly acted as a stressor. Because we were also interested in whether omega-3 PUFAs worked in the same way in other countries, we performed a similar intervention trial in Lampung, Indonesia. Elementary schoolchildren in the fourth to sixth grade (mostly 9
12 years of age) were randomly allocated to either the omega-3 PUFA group (n 5 116) or the control group (n 5 117) in a doubleblind manner. The subjects in the omega-3 PUFA group were given 6 fish oil capsules per day (650 mg DHA 1 100 mg EPA) for 3 months. Subjects in the control group received soybean oil capsules. However, unlike in the previous study in Japan, behavior assessed using the HAQ-C did not show any differences between groups, although the administered amounts of DHA and EPA were similar in the two studies. When we interviewed a local teacher at the school, we found that the lifestyle of schoolchildren in Lampung was very different from that of children in Japan. First of all, there is no after-school private supplementary education in this area. When the Indonesian children go home after school, they often help their parents (usually farm work) instead of studying. At harvest time, farm work has priority over attending school. Secondly, despite the presence of bullying, it rarely becomes a serious social problem, as in Japan where bullying sometimes leads to suicide. Consequently, it seems that not only short-term stress but also chronic stress is important for the effect of omega-3 PUFAs on aggressiveness.

Effect of Omega-3 PUFAs on Aggression in the Elderly The effect of omega-3 PUFAs on aggressiveness in elderly subjects was also investigated (Hamazaki et al., 2002). Forty Thai subjects (50
60 years of age; 22 men and 18 women) were recruited from a university and from nearby villages in Thailand. They were allocated to either the control (mixed plant oils; 47% olive oil 1 25% rapeseed oil 1 25% soybean oil 1 3% fish oil) or omega-3 PUFA group (1500 mg DHA 1 200 mg EPA per day) for 2 months in a double-blind manner. Immediately prior to the PF Study at the end of the study, subjects were asked to watch a stressful videotape (containing real crimes and accidents caused by culpable negligence) as a stressor component. There was no significant difference in change in extraaggression. However, a sub-analysis showed that this difference reached significance only in subjects recruited from the university and not from local villages. The administration of omega-3 PUFAs favorably controlled extra-aggression at least in elderly

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white-collar workers. The average DHA intake as assessed using a food questionnaire was 150
160 mg/ day, indicating that this level of DHA was not enough to control extra-aggression. The reason for the significant difference only in subjects recruited from the university may be the presence of chronic stress. As discussed above, chronic stress, as well as short-term stress, is important, and subjects from the university might have been exposed to both types of stress.

without fish oil. Unfortunately we could not find any improvement in ADHD-related symptoms (Hirayama et al., 2004), but the sum of the hostility scores rated by parents and teachers was significantly reduced in the omega-3 PUFA group (Hamazaki and Hirayama, 2004).

Effect of Omega-3 PUFAs on Aggression in Prisoners

Acute symptoms of schizophrenia such as aggression, agitation, and psychosis can be a formidable clinical problem in psychiatric hospitals, and violence is currently one of the primary reasons for admission. In a cross-sectional study, we have investigated the relationship between hostility and omega-3 PUFAs in 75 acute drug-free patients with schizophrenia (Watari et al., 2010). Multiple regression analysis showed that the concentrations of EPA and DHA in red blood cells were inversely correlated with hostility after adjustment for age and sex.

Two clinical trials have been conducted in this field with prisoners. The first study was conducted by Gesch et al. (2002) with 231 young adult prisoners in the United Kingdom. The prisoners were allocated to a vitamins/minerals 1 omega-3 PUFA group (44 mg DHA 1 80 mg EPA per day) or a placebo group. Compared with the participants in the placebo group, those receiving the active capsules committed significantly fewer offences (decreased by 26.3%). However, the levels of fatty acids in this study were much lower than those often used when examining the effect of aggression. In fact, as previously shown in our study in the elderly (Hamazaki et al., 2002), the average DHA intake was 150
160 mg/day, which was not suitable for controlling extra-aggression. The cause of the effect in this trial is not clear. The second trial was conducted by Zaalberg et al. (2010) with 221 young adult prisoners (mean age 21.0, range 18
25 years) in the Netherlands. They essentially replicated the study of Gesch and co-workers except that they used much higher doses of fatty acids (400 mg DHA 1 400 mg EPA per day). They found that rule-breaking incidents (aggressive or disruptive behavior or incidents involving the possession or use of drugs) decreased by 34% in the omega3 PUFA group, whereas there was only a 14% decrease in the placebo group. No significant reductions in aggressiveness or psychiatric symptoms were found.

Effect of Omega-3 PUFAs on Aggression in Patients with ADHD A placebo-controlled double-blind study was performed in 40 children (6
12 years of age) with ADHD, most of whom were without medication. Subjects in the omega-3 PUFA group (n 5 20) received the same active foods as in our previous studies in schoolchildren (3600 mg DHA 1 840 mg EPA per week) for 2 months, whereas controls (n 5 20) received indistinguishable control foods

Association Between Omega-3 PUFAs and Hostility in Patients with Schizophrenia

Mechanism of Action of Omega-3 PUFAs Serotonin Modification of the serotonergic system by omega-3 fatty acids is probably the most important factor for controlling aggression. In several preclinical studies, serotonergic neurotransmission was shown to be influenced by omega-3 PUFAs. A diet low in omega-3 PUFAs has been shown to decrease serotonin and 5-hydroxyindoleacetic acid (5-HIAA; the main metabolite of serotonin) concentrations in several brain regions in rat (Olsson et al., 1998) and increase 5-HT2A receptor density in the frontal cortex by 44% (Delion et al., 1996). Moreover, deficits in fenfluramineinduced serotonin release in the rat hippocampus could be normalized when dietary omega-3 PUFA fortification was initiated (Kodas et al., 2004). The levels of serotonin were decreased by 40% to 65% in three brain regions (the frontal cortex, hippocampus, and striatum) by unpredictable chronic mild stress (Vancassel et al., 2008) in mice, and omega-3 PUFA supplementation reversed this stress-induced reduction in serotonin levels. The authors concluded that omega-3 PUFAs can improve resistance to stress through attenuation of the impact of stress on specific aspects of cerebral function; however, no impact on behavior such as aggressiveness was seen in this study (Vancassel et al., 2008). In observational studies, Hibbeln et al. (1998a) found that higher plasma concentrations of DHA and AA predicted higher concentrations of 5-HIAA in cerebrospinal fluid (CSF) among healthy volunteers, but

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29. EFFECT OF OMEGA-3 FATTY ACIDS ON AGGRESSION

plasma concentrations of DHA were inversely correlated with CSF 5-HIAA concentrations among early-onset alcoholics, who are at risk for aggressive behavior. It was also reported that violent subjects had significantly lower concentrations of 5-HIAA in the CSF than nonviolent subjects matched for severity of alcohol dependence and that plasma DHA concentrations were inversely correlated with CSF 5-HIAA among those violent subjects (Hibbeln et al., 1998b). Consequently, the relationship between plasma DHA and CSF 5-HIAA levels might depend on whether or not study subjects had early-onset alcoholism, or whether or not they were violent. Noradrenalin Sympathetic nervous system tone is known to be enhanced in subjects with high hostility scores (Williams, 1994). Singer et al (1990) showed for the first time that administration of omega-3 PUFAs (EPA 1.8 g 1 DHA 1.1 g per day) for 36 weeks reduced plasma noradrenaline (NA) levels in male patients with mild essential hypertension. We have previously investigated the effect of omega-3 PUFAs in two different studies with healthy volunteers (both university students) and found that administration of omega-3 PUFAs for 8
9 weeks reduced plasma NA concentrations (Sawazaki et al., 1999; Hamazaki et al., 2005). However, this was not supported by short-term (1 month) intervention studies with omega-3 PUFAs (Hughes et al., 1991; Mills et al., 1990). Changes in central NA levels by omega-3 PUFAs might explain the relationship between these fatty acids and aggression. Cortical-Hippocampal-Amygdala Pathway Activation of the cortical-hippocampal-amygdala pathway by elevated levels of corticotropin-releasing hormone (CRH) is associated with increased fear, depression, and violence (Ressler and Nemeroff, 2000). Prostaglandin E2, production of which is depressed by omega-3 fatty acids, increases the RNA expression of CRH (Bugajski, 1996). In this context, Hibbeln et al. (2004) assessed CSF and plasma for CRH and fatty acid compositions, respectively, among 21 perpetrators of domestic violence. They found that lower plasma DHA alone predicted greater CSF CRH levels. To our knowledge, no intervention studies have shown a reduction in CRH following omega-3 PUFA administration; therefore, further investigation is necessary. Endocannabinoids The two best characterized endocannabinoids, 2arachidonoyl glycerol (2-AG) and anandamide (AEA), are both metabolic derivatives of AA in phospholipids. From the findings of multiple preclinical studies, it seems that the combined effects of stressful situations

and endocannabinoids cause changes in serotonin levels leading to aggression (Mechoulam, 2002). Aggression may be modulated by omega-3 PUFAs through the endocannabinoid system. Watanabe et al. (2003) conducted two separate experiments in mice. An omega-3 PUFA-deficient diet for two generations markedly reduced phospholipid DHA levels in the brain compared with an omega-3 PUFA-sufficient group. The brain 2-AG level in the omega-3 PUFAdeficient group was significantly higher than in the omega-3 PUFA-sufficient group. In another experiment, short-term supplementation with DHA reduced the brain 2-AG level as compared with a diet containing a low level of omega-3 PUFAs. Because linoleic acid (LA) is a precursor of AA, increasing LA from 1 percent of energy (en%) to 8 en% elevated AA-phospholipids (PLs) in the liver and erythrocytes, which tripled 2-AG 1 1-AG and AEA as a result (Alvheim et al., 2012). Adding 1 en% of dietary omega-3 PUFAs to the 8 en% LA diet reversed the elevations of AA-PL, 2-AG 1 1-AG, and liver AEA in a pattern similar to lowering LA to 1 en% (Alvheim et al., 2012). However, it remains unclear how the reduction of endocannabinoids modulates aggressive behavior. Brain-Derived Neurotrophic Factor Brain-derived neurotrophic factor (BDNF) has trophic effects on serotonergic neurons in the central nervous system (Siuciak et al., 1996). BDNF also appears to be involved in the connection between omega-3 fatty acids and modulation of aggression. Deprivation of omega-3 PUFAs in rats increased their scores on the isolation-induced resident-intruder test for aggression (DeMar et al., 2006). This deprivation of omega-3 PUFAs decreased the frontal cortex DHA levels and reduced frontal cortex BDNF expression (Rao et al., 2007). On the other hand, dietary omega-3 PUFA supplementation induced an increase in the expression of BDNF mRNA and protein in the hippocampus in mice (Venna et al., 2009). Another group also found that an omega-3 PUFA-enriched diet increased levels of proBDNF and mature BDNF in the hippocampus in rat (Wu et al., 2008). Furthermore, we have found that administration of omega-3 PUFAs increased levels of serum BDNF significantly in humans (Matsuoka et al., 2011). Together, these findings suggest the possibility that BDNF might be one of the factors through which omega-3 PUFAs modulate aggression.

CONCLUSION Neither aggression nor hostility is considered a disease. However, both can lead to serious problems.

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363

CONCLUSION

TABLE 29.1

Overview of Intervention Studies on the Effects of Omega-3 Pufas on Aggression

Reference

Participants Sample n (Age, Years)

Daily Dose (mg)

Duration (Days)

Outcome

p value

Hamazaki et al., 1996

41

University students DHA 1500 (19
30) EPA 200

90

Extra-aggression was increased in 0.002 the control group at the time of exams but not in the omega-3 PUFA group

Hamazaki et al., 1998

46

University students DHA 1500 EPA (20
30) 200

91

Extra-aggression was slightly increased in the DHA group if there was no stressor

0.05

Hamazaki et al., 2002

40

University employees and farmers (50
60)

DHA 1500 EPA 200

60

Extra-aggression was decreased in university employees, whereas there was no change in farmers

0.04 in university employees; ns in farmers

Gesch et al., 2002

231

Young adult prisoners (18 1 )

DHA 44 EPA 80 ALA 1260 γ-LNA 160

142

26.3% fewer violent offences with 0.03 (0.001 if active capsules; 35.1% fewer if supplemented for supplemented for $ 2 weeks $ 2 weeks) (n 5 172)

Zanarini and Frankenburg, 2003

30

Female subjects with borderline personality disorder (18
40)

EPA 1000

56

EPA was superior to placebo in diminishing aggression and depressive symptoms

0.0001

Stevens et al., 2003

50

ADHD children (6
13)

DHA 480 EPA 80 AA 40 γ-LNA 96

120

Omega-3 PUFAs significantly reduced oppositional defiant behavior according to parents’ assessment

0.02

Bradbury et al., 2004

30

Moderately stressed DHA 1500 university staff EPA 360 (18
60)

42

Significant reduction was seen in Perceived Stress Scale compared with the no-treatment control group, but not with the placebo (olive oil) group

0.05 compared with no-treatment group

Hamazaki and Hirayama, 2004

40

ADHD children (6
12)

DHA 510 EPA 120

60

Aggression was significantly reduced in omega-3 PUFA in comparison to placebo group

0.001

Fontani et al., 2005

33

Healthy adults (22
51)

DHA 800 EPA 1600

35

Anger was significantly reduced in omega-3 PUFA group

0.001 for anger; ns for placebo

Richardson and Montgomery, 2005

117

Children with developmental coordination disorder (5
12)

DHA 174 EPA 558 γ-LNA 60

90 & 90 Omega-3 PUFAs decreased (crossover) opposition scores compared with control

0.02

Itomura et al., 2005

166

Elementary school children (9
12)

DHA 510 EPA 120

90

Physical aggression in girls was increased in the control group compared with the DHA group

0.008 in girls; ns in boys

Hallahan et al., 2007

49

Patients who repeatedly selfharm (16
64)

DHA 908 EPA 1220

90

Omega-3 PUFAs did not improve ns for aggression aggression but improved outcomes for depression, suicidal behavior, and daily stresses

Amminger et al., 2007

13

Children with autism (5
17)

DHA 700 EPA 840

42

There was a trend toward ns for irritability superiority of omega-3 PUFAs over placebo for hyperactivity but not for irritability

Sinn and Bryan, 2007

132

Children with ADHD-related symptoms (7
12)

DHA 174 EPA 558 γ-LNA 60

105 & 105 Omega-3 PUFAs for 15 weeks (crossover) decreased opposition scores compared with placebo

,0.01

(Continued)

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

364 TABLE 29.1

29. EFFECT OF OMEGA-3 FATTY ACIDS ON AGGRESSION

(Continued)

Reference

Participants Sample n (Age, Years)

Duration (Days)

Daily Dose (mg)

Outcome

p value

Buydens-Branchey and Branchey, 2008

24

Male substance abusers (mean age 51.2)

EPA 2250 DHA 500

90

Supplementation with omega-3 PUFAs significantly decreased anger compared with placebo

0.025

Hamazaki et al., 2008

233

School children (9
14)

DHA 650 EPA 100

90

No change in aggression was detected

ns

Zaalberg et al., 2010 221

Young adult prisoners (18
25)

DHA 400 EPA 400 γ-LNA 100

142

Supplementation with omega-3 PUFAs significantly reduced reported incidents; however, there were no significant reductions in aggressiveness or psychiatric symptoms

0.017 for reported incidents; ns for aggressiveness or psychiatric symptoms

Long and Benton, 2013

University students DHA 672 1 EPA (mean age 20.9) 96, vitamin 1 mineral, or both

90

Only supplementation with omega-3 PUFAs significantly reduced extra-aggression

,0.05

173

All studies were placebo-controlled double-blind trials. AA, arachidonic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; ALA, linolenic acid; γ-LNA, γ-linolenic acid.

Barefoot et al. (1983) followed 255 medical students for 25 years and examined the relationship between hostility scores and subsequent health status. Those with hostility scores above the median were nearly 7 times more likely to be dead by the age of 50 than those with hostility scores at or below the median. Many studies including animal experiments appear to suggest that supplementation with omega-3 PUFAs (or at least treating omega-3 fatty acid deficiencies) may modulate aggression. In addition, serotonergic neurons probably play a major role in the mechanism of action of omega3 fatty acids. Furthermore, it seems that the presence of a stressor is the key factor determining whether or not the effects of omega-3 PUFAs are observed. Because of the limited number of studies in this field and the heterogeneity of findings to date, as summarized in Table 29.1, further investigations are needed.

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30 Multiple Sclerosis: Modification by Fish Oil Gilbert Lujan Rivera Jr. and Ronald Ross Watson INTRODUCTION Multiple sclerosis (MS) patients have a limited number of treatment options available to reduce the debilitating symptoms of the disease (Gourraud et al., 2012). Diet is one environmental factor that may have a link in the development of the disease and possible alleviation of some symptoms (Riccio, 2011). Since the disease currently has no cure, physicians have tried different treatment therapies to lessen the various manifestations that can occur. Complementary and alternative medicine therapy has a prevalence rate ranging from 33
70% with an overall estimated average that around one-third of patients are using this form of treatment (Yadav et al., 2010). This type of treatment includes co-administration of medications; primarily INF-β, glatiramer acetate, natalizumab, and mitoxantrone with other supplements such as omega-3 fatty acids (Yadav et al., 2010). Patients have experienced benefits from dietary changes and supplements, specifically from omega-3 fatty acids and antioxidants, due to their immunomodulatory and neuroprotective properties (Yadav et al., 2010; Shinto et al., 2008). Two omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are found in fish and fish oils, act as anti-inflammatory agents and influence neuronal cell membrane health (Yadav et al., 2010). In this overview, we will explore the effects of fish oil on MS patients and the use of them as a dietary supplement coadministered with current pharmaceuticals.

OVERVIEW MS is the most common cause of neurological disability among youth and affects about 350,000
500,000 individuals in the United States (Yadav et al., 2010; Farinotti et al., 2012; Shinto et al., 2008). MS is a chronic neurological inflammatory disease that affects Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00030-2

the brain and spinal cord of the central nervous system (CNS) due to the formation of multiple foci inflammatory lesions (Gourraud et al., 2012; Unoda et al., 2013). In a healthy individual, when the CNS is stimulated, the brain provides a response via an action potential that is relayed through the cells of the CNS. Some of the neurons that send and receive the messages from the brain become compromised as the myelin sheaths are damaged or destroyed which, in turn, causes areas of the body to not function properly. The myelin sheath serves as insulation for the neuron and allows the signals to travel quickly and uninterrupted down the axon of the neuron to the next neuron or intended effector tissue, organ, etc. As the disease progresses, it becomes progressively more difficult for the body to send and receive these messages due to the demyelination of the neurons (Gourraud et al., 2012). More specifically, MS is characterized by blood
brain barrier breakdown, perivascular inflammation, axonal and oligodendrocyte injury, and the principal trait, demyelination, caused by T-lymphocytes, macrophages, and antibodies (Yadav et al., 2010; Riccio, 2011; Shinto et al., 2011). As the messages become increasingly problematic in delivery, symptoms of the disease manifest in the following forms: visual disturbances, muscle weakness, coordination and balancing trouble, ‘pins and needles’ sensations or numbness, thinking and memory problems, tremors, dizziness, and fatigue. Further complications can include muscle stiffness or spasms, paralysis, problems with the bladder, bowel, or sexual functioning, depression, and epilepsy (Yadav et al., 2010). Researchers have conducted worldwide studies on the development of the disease and have found that, in the Western World, the prevalence rates are around 0.5
1.5 per 1,000 inhabitants (Gourraud et al., 2012). Approximately 10
15% of patients with primary progressive MS experience deterioration upon the onset of the disease. However, most individuals

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with MS have relapsing-remitting MS, characterized by episodes of sporadic relapses followed by remission or vice versa (Yadav et al., 2010). In some instances, individuals may not completely recover from these relapses, making the disease characteristically progressive and debilitating. Of these individuals with relapsing-remitting MS, approximately 50% enter a secondary phase of the disease known as secondary progressive MS. It is characterized by the same sporadic episodes, however, remission is less likely to occur and further deterioration of the nervous system persists. In the primary forms of the disease, inflammation and demyelination are the primary factors influencing the pathology of the disease, whereas in secondary forms of MS, neuro-degeneration causing axonal degeneration is more dominant (Yadav et al., 2010). Some factors that can influence the development and progression of MS include multiple gene expressions and environmental factors that may simultaneously trigger the inflammation of the CNS causing destruction and other complications associated with the disease (Gourraud et al., 2012).

CELLULAR LEVEL OF MULTIPLE SCLEROSIS The immune system is compromised by various mechanisms naturally inhabiting the body that begin to break it down based on their influences on one another. Activated T-cells, B-cells, and macrophages, as well as soluble mediators of inflammation such as antibodies, cytokines, free radicals, and proteases are among the mechanisms compromising the body’s immunity (Yadav et al., 2010; Riccio, 2011). Regulatory T-cells are a heterogeneous T-cell sub-population that regulates the immune system in various ways, with the ability to suppress the activity of the immune system and regulate self-tolerance. The most common T-cell lineage is called CD41CD251FoxP31 regulatory T-cells. These T-cells have been found to suppress several immune cells such as CD81 T-cells, dendritic cells, monocytes/macrophages, B-cells, natural killer cells, and natural killer T-cells. It is speculated that natural Tregs (nTregs) can prevent autoimmunity and generally raise the activation threshold to initiate an immune response (Issazadeh-Navikas et al., 2012). High fat diets in mice studied by Issazadeh-Navikas et al. (2012) have shown a gradual decrease in hepatic Tregs due to hepatic steatosis, a model for nonalcoholic steatohepatitis. This decrease has been linked to an increased susceptibility to apoptosis in Tregs and an increased expression of the pro-inflammatory cytokine TNF-α (Issazadeh-Navikas et al., 2012). There

are correlations between the expression of proinflammatory cytokines, TNF-α, IFN-γ and IL-2, during periods of clinical worsening and of regulatory cytokines, IL-4 and IL-10, during periods of remission (Yadav et al., 2010). Transmigration of activated T-cells across the blood
brain barrier is crucial in developing new inflammatory lesions in MS, which explains the varying degrees of debilitation that individuals can experience. T-cell migration across the blood
brain barrier can be facilitated by the binding between integrins and their counter ligands, ICAM-1, onto endothelial cells, leukocyte function associated antigen 1 onto T-cells, and proteases produced by activated T-cells, matrix metalloproteinase-9 (MMP-9) (Yadav et al., 2010). MMP-9 aids in remodeling the extracellular matrix, basement membrane, and other tissues by digesting collagen components in tissues and also has a role in the transmigration of inflammatory cells into the CNS by aiding in the disruption of the blood
brain barrier (Shinto et al., 2011). It has been documented that patients with MS have higher levels of MMP-9 in blood serum samples compared to that of controls (Shinto et al., 2011). Oligodendrocytes are the myelin forming cells of the CNS and are the target cells in the pathogenesis of the disease that are accompanied by microglial activation upon apoptosis of these cells. The mechanisms involved in MS include immune-mediated inflammation, oxidative stress, and excitotoxicity. Each of these mechanisms may contribute to oligodendrocyte and neuronal damage and even to cell death, hence the varying degrees of progression and remission. The immune-mediated inflammation is mediated primarily by myelin specific CD4 1 T-cells, whereas CD8 1 T-cells and antibodies have been identified as causing axonal damage (Van Meeteren et al., 2005; Unoda et al., 2013). Other inflammatory cells and mediators such as reactive oxygen species (ROS), induce axonal loss as well as demyelination. In the case of oxidative stress, levels of ROS increase significantly under inflammatory conditions and exhaust the antioxidant defense ability within the lesions that form. However, excessive levels of ROS are generated by activated macrophages and microglia. ROS can damage cellular structures ultimately resulting in cell death. Oligodendrocytes and their extensive networks of myelin sheaths are highly susceptible to oxidative stress and can selectively result in cell death, demyelination, and damage to the myelin sheaths promoting attack by macrophages. The third possible cause for MS development is excitotoxicity in which activated immune cells release large amounts of glutamate, causing an overreaction of glutamate receptors. Since oligodendrocytes aid in maintaining the homeostasis of glutamate in the CNS, this over-activation causes subsequent excitotoxic

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death due to the cells’ high vulnerability to excitotoxic signals (Van Meeteren et al., 2005).

OMEGA-3 SUPPLEMENTATION OF MS PATIENTS Omega-3 fatty acids have become a focused ingredient in functional foods that are aimed at satisfactorily benefitting one or more target functions in the body with an emphasis on improvement in health and wellness and/or reduction in disease risk (IssazadehNavikas et al., 2012). For individuals utilizing contemporary and alternative treatment and pharmaceuticals to combat MS, omega-3 fatty acids have been used as a supplement. Two omega-3 fatty acids, EPA and DHA, are derived from α-linolenic acid (ALA). These two compounds cannot be synthesized by the body itself and, therefore, must be obtained in the form of food or dietary supplements. Sources of EPA and DHA are typically plant-based and include flaxseeds, flaxseed oil, soy, soybean oil, and canola oil, and are also found in high proportions in oily fish and fish oil. They show anti-inflammatory, anti-thrombotic, and immunomodulatory activities and are part of a class of fatty acids known as polyunsaturated fatty acids (PUFAs) (Yadav et al., 2010; Riccio, 2011). In the Western world, individuals with high fat and/or carbohydrate diets are known to have a high disproportion in the ratio of consumption of omega-3 and omega-6 fatty acids that contribute to higher rates of cardiovascular and inflammatory diseases (Riccio, 2011). The recommended consumption ratio of omega-6 to omega-3 acids is 3:2, whereas the actual Western diet is about 10
30:1 (Torkildsen et al., 2008). Linoleic acid (LA) is an unsaturated omega-6 fatty acid and should be consumed in a proportionate amount relative to omega-3 to maintain equilibrium. LA leads to the production of arachidonic acid, which is the precursor to the proinflammatory prostaglandins, leukotrienes, and thromboxanes. However, synthesis of these eicosanoids is favored by insulin and inhibited by EPA and DHA (Riccio, 2011). EPA and DHA both decrease the proinflammatory properties of cytokines and reduce the immune cell secretion levels of MMP-9 as well as its activity. While inhibiting MMP-9, DHA also increases the levels of tissue inhibitor of metalloproteinase-1 (TIMP-1). Together these modulatory effects on MMP-9 may impede cell migration across the basement membrane of the CNS that can activate further crossing of activated T-cells into the CNS (Shinto et al., 2011; Shinto et al., 2009). DHA reduces the suppressive and migratory functions of Tregs through its inhibitory capacity by affecting the negative T-cell signaling via upregulation of CTLA-4

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(Yadav et al., 2010). EPA can be converted to prostaglandin I3 and E3, thromboxane A3, and leukotriene B5, and therefore has immunomodulatory capacity acting as an anti-inflammatory agent (Yadav et al., 2010). Most profoundly, EPA and DHA inhibit the formation of IFN-γ, which is involved in the breakdown of myelin (Riccio, 2011). The mechanism by which PUFAs induce immunomodulation is through the modulation of gene expression (Riccio, 2011). Supplementation with omega-3 PUFAs reduced T-cell numbers and transcriptional levels of IL-6 in plaques of the human carotid artery and in mouse models of atherosclerosis. In ApoE-deficient mice, EPA supplementation suppressed the development of atherosclerosis by suppressing the expression of adhesion molecules in endothelial cells and of matrix metalloproteinase-2 and MMP-9 in macrophages through a peroxisome proliferatoractivated receptor PPARα-dependent pathway. These omega-3 PUFA augmented adiponectin, an anti-inflammatory adipokine, in a PPARγ-dependent manner, improved insulin resistance and gave rise to a family of anti-inflammatory mediators termed resolvins via trans-cellular biosynthesis (Unoda et al., 2013).

VARYING RESULTS AMONGST RESEARCHERS The scientific community has varying results of PUFA supplementation in humans and mice and has suggested more research to identify benefits associated with PUFAs. The studies outlined below have very limited data and make no claims as to whether CAM treatment is beneficial or not; their mention is used to demonstrate the results gained from various researching parameters. The Cochrane Collaboration (Farinotti et al., 2012) stated that six randomized controlled trials conducted, out of a total of 46, showed that omega-6 fatty acids (LA) 11 to 23 g/day showed no benefits in 144 MS patients; LA 2.9 to 3.4 g/day showed no benefits in 65 chronic progressive MS patients; and omega-3 fatty acids showed no benefits in 292 relapsing-remitting MS patients. Although PUFAs did not have a significant effect on disease progression after 24 months, during the two-year period, the relapse frequency appeared to be lower in patients treated with a PUFA spread. However, the data available are insufficient to assess a real benefit or harm associated with PUFA supplementation (Farinotti et al., 2012). The study conducted by Shinto et al. (2011) utilized 13 individuals for a blood draw of peripheral blood mononuclear cells (PBMC) to be used as controls. These cells were resuspended in X-Vivo 15 supplemented with 1% L-glutamine, 100 U/mL penicillin,

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100 ug/mL streptomycin, 1% sodium pyruvate, and 25 mM HEPES at 2 3 106 cells/mL. After 2 hours, the PBMC samples were pretreated with EPA, DHA, and oleic acid at the following concentrations: 10 μg/mL; 25 μg/mL; and 50 μg/mL followed by stimulation by canavalin A. The oleic acid PBMC sample was used as an oil control and is a monounsaturated fatty acid (Shinto et al., 2011). Cell supernatants were taken to measure MMP-9 activity following the exposure to EPA and DHA. Human CD4 1 T-cell lines (Jurkat cell cultures) were chosen for transmigration and exposed to 10 μg/mL, 30 μg/mL, and 100 μg/mL of DHA and EPA. The results of the study showed that DHA at 10 μg/mL decreased mean MMP-9 levels to 85.5 ng/mL, a decreased mean of 21.5%; at 25 μg/mL decreased mean MMP-9 levels to 41.5 ng/mL, a decreased mean of 29%; and at 50 μg/mL deceased MMP-9 levels to a non-detectable level, with a decreased mean of 51.4%. EPA at 10 μg/mL decreased mean MMP-9 levels to 50.9 ng/mL, a decreased mean of 9.6%; at 25 μg/mL decreased mean MMP-9 levels to 4.4 ng/mL, a decreased mean of 29.0%, and at 50 μg/mL decreased MMP-9 levels to a non-detectable level, a decreased mean of 69.0% (Shinto et al., 2011). This study shows that DHA and EPA, at low in vitro concentrations, have the ability to decrease MMP-9 production and activity in PBMC from healthy controls. Also, both DHA and EPA were able to inhibit T-cell migration via a fibronectin barrier in a concentration-dependent fashion. Together, DHA and EPA have the ability to decrease immune cell secretion of the protein MMP-9 and reduce its activity that will result in less transmigration into the CNS (Shinto et al., 2011). According to Torkildsen et al. (2008), omega-3 fatty acids are a group of essential fatty acids and could possibly have both an anti-inflammatory and a neuroprotective effect on MS; however, neither had an effect on disease activity either as monotherapy or in combination with INF-β (Torkildsen et al., 2008). In the study conducted by Torkildsen et al. (2008), the cuprizone model was used to create the same effects of demyelination experienced by MS patients. The mice used were given copper chelator cuprizone, which lead to demyelination in the corpus callosum and the cerebral cortex. Through the supplementation of PUFAs in their diets via cod liver or salmon fillets, researchers were able to determine the effects these had on the demyelination of the mice brains. Concluding the study, the mice that were given the salmon fillets in their diets had less demyelination and weight loss than those of the other two groups. Through the use of controlled areas for the mice to move around, the salmon fed mice explored the environment more frequently than those of the cod-liver and soybean oil groups indicating preserved locomotor activity. By monitoring the rate of

weight loss, a correlation was made between the degree of demyelination and weight loss. One interesting fact observed by researchers concerned the amount of omega-3 in both the salmon fillets and cod-liver. The cod-liver contained more omega-3, however, in laboratory testing, it showed less of an effect than its counterpart. Further analysis of the amount of omega-3 supplementation must be conducted to differentiate between the degrees of effectiveness achieved through supplementation with PUFAs (Torkildsen et al., 2008). In another study conducted by Torkildsen et al. (2012), researchers examined 92 patients who were given 1350 mg of EPA and 850 mg of DHA daily or a placebo. After the initial treatment of 6 months with DHA and EPA, patients were given an additional subcutaneous injection of 44 μg of interferon β-1a three times per week for an additional 18 months (Torkildsen et al., 2012). The primary measured outcome was an MRI of disease activity that measured the total number of new T-1 weighted gadoliniumenhancing lesions during the first 6 months of treatment. The secondary outcome was another MRI of disease activity taken after 9 months and 24 months with the inclusion of relapse rate, disability progression, fatigue, quality of life, and safety (Torkildsen et al., 2012). The primary outcome was observed to be the same in both placebo and non-placebo groups after the first 6 months following the MRI scan for disease activity. Additionally there was no difference in relapse rate detected after 6 or 24 months. The only difference among the groups detected was the increase in fatty acid serum levels in the patients treated with omega-3 fatty acids compared to those who were given the placebo. Therefore, researchers have determined that there are no beneficial effects on disease activity from omega-3 fatty acids when compared to the placebo as monotherapy or in combination with interferon β-1a. As expected, interferon β-1 did reduce disease activity based on MRIs (Torkildsen et al., 2012). Studies are still controversial concerning the effectiveness of omega-3 fatty acid supplementation in patients with MS. Researchers have changed their positions on the actual effectiveness of PUFAs in human trials due to a lack of substantial data to prove any benefits (Torkildsen et al., 2012; Shinto et al., 2011).

SUMMARY The pathogenesis of MS is centralized to complex cellular processes including but not limited to elevated MMP-9 protein expression, T-cell transmigration into the CNS, blood
brain barrier destruction, oligodendrocyte, and myelin sheath degradation which ultimately deteriorates the immune system. Some of these

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REFERENCES

processes at certain levels are appropriate and naturally occurring, however, it is when outside factors influence these processes to go beyond their naturally intended purposes that they begin to cause disease activity centers. Pharmaceuticals have already been created to address some of the cellular processes that have gone array, i.e., interferon β-1, however alternative methods have been sought by physicians and patients in an attempt to alleviate symptoms of MS. Due to the lack of research conducted on PUFA supplementation for use by MS patients, it is still difficult at this time to determine if the consumption of fish oil is effective in alleviating the debilitating symptoms associated with the disease. Of the current research carried out with omega-3 fatty acids, it is known that EPA and DHA both decrease the proinflammatory properties of cytokines and reduce the immune cell secretion levels of MMP-9 as well as its activity. EPA can be converted to prostaglandin I3 and E3, thromboxane A3, and leukotriene B5, and therefore has immunomodulatory capacity acting as an anti-inflammatory agent (Yadav et al., 2010). As research is continued on the potential for the use of PUFAs as a contemporary and alternative treatment option for MS patients, a point of interest should be actual dosing levels since some of the research already conducted does show potential. Researchers have shown DHA and EPA to exhibit beneficial effects, even though they were small. An important note to make of these new findings is that they have shown a decrease in disease activity based on MRIs. It was shown in the study conducted by Torkildsen et al. (2012) that combination therapy with interferon β-1a and omega-3 fatty acids showed a reduction in disease activity which may be a key concept in future studies. Although by themselves they show minimal benefits, co-administration of omega-3 fatty acids with current pharmaceutical regimens may be of importance in assisting patients to reduce the symptoms of MS.

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Acknowledgement Preparation of this review was part of the course CPH 499.

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31 Deuterium Protection of Polyunsaturated Fatty Acids against Lipid Peroxidation: A Novel Approach to Mitigating Mitochondrial Neurological Diseases Mikhail S. Shchepinov, Vitaly A. Roginsky, J. Thomas Brenna, Robert J. Molinari, Randy To, Hui Tsui, Catherine F. Clarke and Amy B. Manning-Bo˘g INTRODUCTION Increased life span firmly places neurological diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) at the forefront of the unmet needs category in modern pharmacology. It has been estimated that the worldwide economic impact of brain-related illness has reached $2 trillion a year (Neurotechnology Industry Organization, 2014).This is not for the lack of trying, for mitochondrial bioenergetics and its contribution to neurological damage and disease has been a focal point of research for over 30 years (Barlow et al., 1979; Jenner, 2003). Accordingly, growing evidence implicates mitochondria as a causative factor in many age-related neurodegenerative diseases (Lin and Beal, 2006), through an imbalance in pro-oxidant/ antioxidant homeostasis and linked phenomena such as regulation of cell death. The physiology of mitochondria clearly contributes to the onset of oxidative injury. During oxidative phosphorylation, as electrons pass from electron donors to an electron acceptor (oxygen), a substantial fraction of electrons ‘leaks’ away which leads to formation of reactive oxygen species (ROS). According to some estimates, in normal conditions 1
2% of all oxygen consumed is converted into ROS (Cadenas and Davies, 2000), a figure likely to be even higher in mitochondrial pathologies (Barnham et al., 2004). Failed attempts to use antioxidants to curtail this leak invite different approaches to reset the redox homeostasis in neurodegenerative disease. Here we

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00031-4

describe a novel approach to minimize the ROSinflicted damage, which focuses on the weakest link in the polyunsaturated fatty acid (PUFA)-laden mitochondrial membranes.

PUFAs IN MITOCHONDRIAL MEMBRANES AND OXIDATIVE STRESS PUFA content influences membrane fluidity, a vital parameter determining the efficiency of interactions between membrane proteins and membrane-bound small molecules, particularly in mitochondria. The more double bonds a PUFA contains, the more ‘fluid’ the membrane. However, this same feature makes PUFAs susceptible to autoxidation. The process of protein and DNA oxidation differs from that of PUFAs. For proteins and nucleic acid components, the ROS-inflicted oxidation is a stoichiometric reaction: one ROS moiety typically damages one amino acid of a protein, or a nucleic acid base. However, in the case of PUFA, ROS initiate the chain reaction. In other words, cone ROS moiety initiates a process where multiple PUFA residues are autoxidized, before the reaction is terminated by homologous recombination or interactions with chain-terminating antioxidants (Yin et al., 2011; Zimniak, 2011). Arguably, this makes PUFA peroxidation by far the most dangerous of all other types of oxidative damage.

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FIGURE 31.1 Chain reaction of PUFA autoxidation. Shown here is a linoleic acid chain reaction. More PUFAs are entering the cycle at the propagation step. The substitution at pos. 11 occurs at particular levels of hydrogen donors such as vitamin E (Yin et al., 2011). R, phosphoglyceryl moiety. Abstraction of a bis-allylic hydrogen is the rate-limiting step. For kinetic equations, see isotope protection of PUFAs against autooxidation.

The composition of lipids within the inner mitochondrial membrane enhances the propensity toward oxidative damage (Bindoli, 1988). Along with other phospholipids, cardiolipin encompasses a substantial portion of this mitochondrial PUFA fraction. Human cardiolipin normally contains 18-carbon unsaturated acyl chains, often linoleate residues, although this may vary depending on tissue and age (Chicco and Sparagna, 2007).The PUFA-rich mitochondrial membranes are highly relevant to the study of oxidative stress in neurodegenerative disease because PUFAs are one of the first targets of oxidation by ROS, and lipid peroxidation is one of the first and major outcomes of oxidative stress that characterize such disorders (Figure 31.1). PUFA peroxidation occurs at the bisallylic methylene groups (between double bonds) and proceeds through a chain reaction format. This deteriorates the membrane fluidity by increasing its rigidity, leading to reduced respiratory control (Dobretsov et al., 1977). Lipid peroxides undergo further transformations through variable pathways, leading to the subsequent liberation of α,β-unsaturated electrophilic carbonyl derivatives (reactive carbonyl species, RCS) such as acrolein and 4-hydroxynonenal (HNE) (Esterbauer et al., 1991) (Figure 31.2). These nonenzymatic lipid peroxidation processes also generate various regulatory molecules (such as isoprostanes and eicosanoids), hydrocarbons, and other species. Recent research suggests that the strongest detrimental effect on the etiology of oxidative stress-related diseases, including neurological disorders, is exercised

specifically by electrophilic toxicity of RCS on other biomolecules, most notably proteins and DNA (Lim et al., 2004), through RCS such as HNE, 4-hydroxyhexenal (HHE), and malondialdehyde (MDA) (Zimniak, 2011). Other destructive mechanisms are also at play. A ‘stub’ of the acyl chain formed upon cleavage of a tip (such as in the form of HNE or HHE) of a membranebound PUFA, for example in cardiolipin, is typically hydroxylated or peroxylated and so it is more hydrophilic than the surrounding membrane. It may flip over away from the hydrophobic interior of the membrane and emerge on its surface, protruding into the aqueous environment, according to a ‘lipid whisker’ model (Greenberg et al., 2008). These ‘whiskers’ can then recruit white blood cells and induce inflammation, or participate in an apoptotic signaling mechanism (Kagan et al., 2005; 2006; Gonzalez and Gottlieb, 2007) by reducing the binding of cytochrome c to the mitochondrial inner membrane and facilitating permeabilization of the outer membrane. Cytochrome c, in turn, activates a proteolytic cascade that culminates in apoptotic cell death (Schug et al., 2009). RCS have longer life spans compared to ROS, and are non-reactive towards many radical quenching antioxidants such as vitamin E. However, they react with thiolate residues on susceptible proteins, many of which are involved in redox signaling, as well as with glutathione, depleting this critical reserve (Gutierrez et al., 2006). Most RCS bear several reactive groups (Figure 31.2) and so can irreversibly cross-link

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FIGURE 31.2 PUFA peroxidation products (the series 8 IsoP shown here is formed from arachidonic acid initially peroxidized at pos. 12). Multiple pathways result in a large number of possible products, from isoprostanes (left) to reactive carbonyl compounds (right) like HNE (formed from omega-6 PUFAs) and HHE (formed from omega-3 PUFAs). A different pathway is responsible for ethane (omega-3) and pentane (omega-6) formation. The reactive carbonyl compounds are typically bi-functional and so are capable of cross-linking proteins. More hydrophobic ones like HNE tend to do damage to membrane proteins, while more hydrophilic HHE, MDA, etc can operate in an aqueous environment. PUFA monohydroperoxyls can also cross-react giving unstable dimers that cleave to give secondary electrophilic products (Schneider et al., 2008; Yin et al., 2011).

proteins. Until recently, detecting free RCS or their conjugates was challenging and depended mostly on immunochemical methods, although current advances in mass spectrometry are changing this. The lack of availability of antibodies for the majority of RCS, and the variable selectivity of the available ones, led to a somewhat biased body of published data on protein conjugates, with HNE and MDA featuring somewhat disproportionately, reflecting the availability of detection methods (Kitase et al., 2005), although the arrival of mass spectrometry-based methods is changing this (Carini et al., 2004). So while it is likely that other RCS also damage the neuronal proteome, HNE and MDA have been well documented to affect PD-related pathways. For instance, dopamine (DA) catabolism is distorted by elevated levels of HNE and MDA. Monoamine oxidase converts DA into a corresponding aldehyde, further oxidized to 3,4-dihydroxyphenylacetic acid by mitochondrial aldehyde dehydrogenase (ALDH). The aldehyde, which is toxic, can accumulate if increased lipid peroxidation produces elevated levels of HNE and MDA which preferentially react with ALDH (Rees et al., 2007). HNE has been shown to produce nerve terminal toxicity, through damage to striatal synaptosomes, inhibiting DA transport and vesicular storage (LoPachin et al., 2009). DA can also react with lipid peroxidation forming neurotoxic products. Particularly toxic species are formed when DA reacts with peroxidation products of arachidonic acid (AA) and docosahexaenoic acid (DHA) (Liu et al., 2008). α-Synuclein (αSyn), a protein that associates with lipid membranes and is localized in several organelles within the cell including mitochondria, forms neurotoxic aggregates which have been implicated in the etiology of PD and other α-synucleinopathies such as multiple system atrophy (MSA). There is evidence suggesting that the oligomerization and stabilization is, at least in part, driven by reactive carbonyls such as HNE and 4-oxo-2-nonenal (ONE) (Qin et al., 2006;

Selley et al., 1998; Shibata et al., 2010; Nasstrom et al., 2011). In a vicious circle of neurotoxicity, HNE-αSyn conjugates have been shown to further increase production of ROS (Xiang et al., 2013). Other RCS such as acrolein have also been shown to post-translationally modify αSyn with resulting inhibition in proteasome activity (Shamoto-Nagai et al., 2007). Some RCS are more hydrophobic (HNE) and tend to reside, and do the damage, inside of the lipid membranes. Others are more hydrophilic (HHE, MDA) and tend to diffuse into, and modify, biomolecules in the aqueous environment. It is therefore interesting to note that αSyn, having an affinity for lipid membranes, has an increased chance of reacting with more hydrophobic HNE species. There is data suggesting that protein modification by RCS is an early step and precedes the onset of clinical symptoms (Matveychuk et al., 2011).

MITOCHONDRIAL DYSFUNCTION, OXIDATIVE STRESS, AND PD Brain tissue is particularly sensitive to oxidative stress, given the enhanced rate of oxygen consumption compared to other tissues and high levels of PUFA. Mitochondrial pathology and lipid peroxidation are apparent in many progressive neurological diseases including AD, amyotrophic lateral sclerosis, Huntington’s disease, Friedreich’s ataxia, PD, and other neurodegenerative disorders related to PD such as dementia with Lewy bodies (DLB) and MSA (Dexter et al., 1989; Olanow, 1990; Schapira, 1999). There is substantial evidence from neuropathology, genetic analyses, and environmental exposure models implicating mitochondrial dysfunction and its ensuing oxidative stress as major factors in the pathogenesis of PD (Jenner 2003; Zhou et al., 2008). For example, markers of lipid peroxidation (i.e. 4-HNE, MDA, and ONE) are increased in post-mortem brain from PD

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patients, and not surprisingly, this damage is accompanied by a reduction in levels of PUFA in substantia nigra (Dalfo et al., 2005). Enhanced oxidative modification of DNA, the formation of 8-hydroxyguanine, is also apparent in PD brain (Bender et al., 2006), and mitochondrial complex I activity is dramatically reduced in substantia nigra (Schapira et al., 1990). Further, autosomal recessive parkinsonism is linked to mutations in genes involved in mitochondrial function such as DJ-1, PINK1, and PARKIN (Nussbaum, 1998; Moore et al., 2005; McCoy et al., 2012). DJ-1 deficiency promotes oxidative injury and enhances vulnerability to mitochondrial toxins (Lev et al., 2008; Li et al., 2005; Raman et al., 2013; Manning-Bog et al., 2007; Kim et al., 2005); loss of PINK1 function impairs mitochondrial calcium influx, promoting mitochondrial permeability transition pore opening and subsequent apoptosis (Gandhi et al., 2009). Decreased mitochondrial membrane potential signals Parkin-mediated ubiquitination of mitochondrial proteins, activating removal of the dysfunctional organelle by mitophagy; however, PARKIN or PINK1 mutation prevents clearance of the damaged mitochondria, thereby potentiating oxidative injury (Geisler et al., 2010; Narendra et al., 2010). Similarly, studies of environmental parkinsonism also support a definitive role for mitochondrial dysfunction. Exposure to complex I inhibitors such as 1-methyl-4phenyl-2,3,4,6-tetrahydropyridine (MPTP) and rotenone has been linked to parkinsonism in humans (Langston et al., 1983; Dhillon et al., 2008; Tanner et al., 2011); similarly, pesticides which cause oxidative damage through redox cycling agents, such as the herbicide paraquat, are implicated in disease development (Tanner et al., 2011). Furthermore, animal models of systemic challenge to these toxicants demonstrate robust nigrostriatal degeneration along with evidence for ROS generation including lipid peroxides (McCormack et al., 2005; Sherer et al., 2003). Work on these model systems, and others,

suggests that other important enzymatic and nonenzymatic intermediates relevant to the etiology of PD can be affected by lipid peroxidation. It is critical to note that antioxidants employed to diminish this oxidative stress were found to be not just inefficient, but, on occasion, outright harmful. There are several possible reasons for this phenomenon, most recently reviewed by Murphy (2013). They include the stochastic nature of ROS production; almost saturated amounts of antioxidants already present; involvement of ROS in hormesis as well as cell signaling; and toxicity of some antioxidants. Other, non-antioxidant based approaches are therefore required.

ISOTOPE PROTECTION OF PUFAS AGAINST AUTOXIDATION An isotope effect (IE) is an influence of substitution of a heavy atom for a light one (e.g. deuterium for hydrogen or 13C for 12C) on the strength of a chemical bond. The ground state vibrational energy of a bond is lower when the reduced mass (proportional to the masses of atoms forming the bond, m1m2/(m1 1 m2)) is higher. Thus bonds between heavier isotopes will have lower energy in the ground state, so the dissociation of these bonds will require more energy. The primary kinetic IE (KIE) describes the bond cleavage during or before the transition state (rate-limiting step). The rate-limiting step of PUFA autoxidation is hydrogen abstraction off a bis-allylic (between double bonds) site. Deuteration at this position (Figure 31.3) causes a dramatic reduction in the rate of abstraction. In stoichiometric (non-chain, monomeric, nonenzymatic reactions) the typical range of the KIE is 2
10 (Bigeleisen, 1949). However, the chain reaction format of PUFA autoxidation seems to lead to

FIGURE 31.3 Isotopic reinforcement of PUFAs inhibits the chain reaction of lipid peroxidation. As depicted in Figure 31.1, hydrogen abstraction from a bis-allylic site initiates the chain reaction of PUFA autoxidation, which generates numerous end-products. However, deuteration of the bis-allylic site slows the rate of initiation, through amplification of the KIE along the kinetic chain, thus halting downstream oxidative events. Linoleic (18:2,n-6) and α-linolenic (18:3,n-3) acids are considered the dietary essential PUFAs from which all longer chain and more unsaturated PUFAs are produced metabolically via elongation and desaturation. Acronyms shown in this figure are more accepted in the chemistry field, while in nutrition, LA (for linoleic acid) and α-LA (for α-linolenic acid) are often used.

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‘amplification’ of the KIE over many reaction steps, essentially resulting in the total inhibition of the reaction and the generation of toxic products (Shchepinov, 2007). Linoleic acid (LA) and D2-LA oxidation was compared in vitro using the Clark electrode technique (Figure 31.4). The autoxidation of LA can be represented by the following simplified kinetic scheme: k1

AMVN 1 2 ðLH1O2 Þ


! 2 LO2 1 products k2

LO2 1 LH


! LOOH1L L



ð1Þ

RIN

1O2 -LO2

ROX ðslowÞ

ð2Þ

ðfastÞ

k3

2 LO2


! products

ð2aÞ ð3Þ

RDEG

To monitor the process, a controlled chain reaction mode was used, with an azo-initiator (AMVN) that spontaneously yields free radicals that initiate (Eq. 1) lipid peroxidation at a constant rate RIN (proportional to the concentration of AMVN, and not changed by either [LH] alteration or by addition of an inhibitor). The slow reaction (Eq. 2) and fast reaction (Eq. 2a) correspond to chain propagation. The chain is terminated by reaction (Eq. pffiffiffiffi3). ffi The oxidizability of LH is characterized by k2 = k3 . The rate of chain oxidation (ROX), measured as the rate of oxygen consumption, is given by (Eq. 4): pffiffiffiffiffiffiffiffi k2 ½LH RIN pffiffiffiffiffi ROX 5 ð4Þ k3

RIN was measured with the inhibitor method (Roginskii, 1990). A typical kinetic run is shown in Figure 31.4, left, plot 1. ROX (0.71 M LA) was 6.1 3 1026 M/s. When the process was inhibited by 0.23 mM HPMC, induction period, tIND, increased to 48 min (RIN 5 0.16 3 1026 M;/s). The calculated length of p kinetic chain is: ν 5 ROX/RIN 5 38. The average of ffiffiffiffiffi k2 = k3 was found to be 0.0215 6 0.008 M20.5s20.5, similar to values reported previously for the oxidation of LA esters in PhCl (M20.5s20.5): 0.0203 and 0.022 for methyl linoleate; and 0.0268 for LA monoglyceride (Cosgrove et al., 1987; Roginskii, 1990). For 11,11-D2LA, we observed a dramatic decrease of ROX to 0.18 3 1026 M/s as compared to LA (plot 3). In contrast to LA, addition of HPMC does not result in the increase in ROX or the appearance of induction period, precluding a direct determination of RIN. Assuming that RIN during11,11-D2-LA oxidation does not change compared with LA, we conclude that 11,11-D2-LA oxidation is not a chain process (ν 5 0.18 3 1026/ 0.16 3 1026  1.1). Therefore, KIE may only be estimated from comparison of ROX for LA and 11,11D2-LA: KIE 5 6.1 3 1026/0.18 3 1026  35. A similar KIE was determined during the oxidation of LA and 11,11-D2-LA in Triton X-100 aqueous micelles (data not shown). This KIE is most likely associated with (Eq. 2). This method is based on oxygen consumption measurement over all steps of the process (initiation, propagation, and termination). Using the reaction product measurement-based competition and radical clock techniques, a KIE value of around 12 was obtained (Hill et al., 2012).

3.5

5.0

3.0 4.0 ROX, μM/sec

–(O2), mM

2.5 2.0 1.5 1.0 2

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20

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40

Time, min

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0.0 0.00

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FIGURE 31.4 Left, kinetic measurements of oxygen consumption using the Clark electrode. Kinetics of O2 consumption accompanying the oxidation of 0.71 M LA (plots 1 and 2), and 0.71 M 11,11-D2-LA (plot 3), in chlorobenzene initiated by 40 mM AMVN (2,2’-azobis(2,4-dimethylvaleronitrile, free radical initiator) at 37 C under air. HPMC (0.23 mM; 6-hydroxy-2,2,5,7,8-pentamethyl benzochroman, a chain-terminating antioxidant) was added to 0.71 M LA (plot 2). The induction period (tIND) is designated for plot 2. The rate of initiation, RIN, was determined by inhibitor with HPMC as a reference inhibitor. RIN was calculated from the induction period of inhibited oxidation, tIND: RIN 5 2 3 [HPMC]/tIND. Right, non-additivity effect. Dependence of the rate of oxidation of the mixture of LA and 11, 11-D2-LA in chlorobenzene on mixture composition. Conditions: [LA] 1 [11, 11-D2-LA] 5 0.775 M; [AMVN] 5 0.0217 M; 37 C. RIN 5 (1.10 6 0.08) 3 1027 M/sec.

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The HPMC results (plot 2) indicate that the slowed oxygen consumption rate observed for 11,11-D2-LA (plot 3) is not due to possible inhibitor contaminants. For short kinetic chain lengths, the rate of O2 uptake measurement (Eq. 2) is difficult, since oxygen uptake by the AMVN-derived radicals in the initiation reaction (Eq. 1) is significant and may affect the analysis. Therefore, for the short chain length in the case of 11,11-D2-LA, the KIE is likely to be even larger than the experimentally determined value of 35. Autoxidation of mixtures of LA with D2-LA suggests that the effect is markedly non-additive (Figure 31.4 Right). Indeed, inclusion of just 20% of D2-LA (D2-LA/LA 5 {1/4}) rendered the mixture as resistant to oxidation as pure 11,11-D2-LA. These in vitro assays suggest that D2-LA ‘protects’ the nondeuterated LA against autoxidation, although the mechanisms responsible for inhibition of autoxidation remain to be elucidated. The findings are in stark contrast to the enzyme-mediated soybean lipoxygenase oxidation of the 50/50 mixture of LA/11,11-D2-LA, where the rate of the process was shown to be fully additive (Glickman et al., 1994). However, in contrast to PUFA autoxidation, the enzymatic oxidation of PUFA induced by lipoxygenase is not a chain process. The dependence of oxygen consumption on the fraction of one of the two components need not be linear (Equations 5.1 and 5.2 in Denisov et al., 2005). This, in combination with the above mentioned issues associated with the oxygen uptake measurements and short kinetic chain, encouraged us to turn to a yeast model to further investigate the effects of isotope-reinforced deuterated PUFA in vivo.

YEAST MODELS CONFIRM THE NONLINEAR PROTECTIVE EFFECT OF D-PUFAS IN VIVO The non-additive effect described above, whereby a (relatively) small fraction of D-PUFA inhibits the chain reaction in non-deuterated PUFA, is potentially of great importance, because it makes it more feasible to reach necessary therapeutic levels of D-PUFA in mitochondrial membranes in vivo by dietary supplementation. To check if the effect can be observed in living systems, we used a well-established viability test based on coenzyme Q-deficient Saccharomyces cerevisiae coq mutants. These mutants are very sensitive to PUFA, however D-PUFAs are not toxic to them (Hill et al., 2011). Our extensive studies of different mixtures of synthetic D-PUFAs and non-deuterated linoleic and linolenic acids, as well as different deuteration positions, revealed that the non-additive effect operates in vivo (Hill et al., 2012). We then tested the ability of

11,11-D2-LA to inhibit the chain reaction of autoxidation in more biologically relevant lipids, including AA (20:4n-6), eicosapentaenoic acid (EPA; 20:5 n-3) and the exquisitely sensitive DHA (22:6 n-3). As is shown in Figure 31.5, a fraction of D2-LA can prevent these PUFAs from autoxidation, rescuing the viability of the yeast mutants.

ISOTOPE REINFORCEMENT OF PUFA IN PRE-CLINICAL PD MODELING In order to address whether isotopic reinforcement of PUFA could mitigate damage in PD, proof-ofprinciple testing in vivo was required with several steps including drug delivery, incorporation, potential toxicity profile, and PD paradigm to be considered. In terms of drug administration, PUFAs are essential nutrients (i.e. required in diet), and as such, must be delivered to the brain to replenish normal tissue PUFA turnover. Consequently, 11,11-D2-LA and 11,11,14,14D4-ALA were administered via diet as the only source of PUFA. Individually housed mice were given MP Bio Fat Free Mouse Diet AIN 76A (Cat. # 960321). For nutritional purposes, the following fat components were added to the diet: (i) saturated fatty acids containing a mixture of palmitate, stearate, and myristate, and (ii) the monounsaturated oleic acid, H-PUFA, or the deuterated form, (D-PUFA), at 0.8 g (0.8%) LA and 0.8 g (0.8%) ALA per 100 g diet, was given by daily application to food pellet to prevent extensive oxidation. In total, the combined fat composition (saturated, monounsaturated, and PUFA) was 9.7%. Animals were given this diet daily for 6 days prior to acute toxicant exposure (i.e. MPTP at 40 mg/kg, i.p.) or vehicle (i.e. saline) administration and continued on the diet for 6 days until euthanized. As stated previously, a critical first step in determining the utility of D-PUFA as a therapeutic for neurodegenerative disease is confirming delivery of the drugs to the brain. Since PUFA deuteration decreases autoxidation, yet does not alter PUFA as a substrate for enzymatic (courtesy of the non-additivity effect) or transporter (deuterium has natural abundance) processes, we expected incorporation of 11,11-D2-LA and 11,11,14,14-D4-ALA into brain tissue. Forebrain tissues were obtained from each group and lyophilized, and brain deuterium levels measured by isotope ratio mass spectrometry and standardized to the Vienna Standard Mean Ocean Water (VSMOW) scale. A dramatic 14-fold increase in deuterium brain incorporation in D-PUFA versus H-PUFA was noted with 12 days of drug administration (Figure 31.6, Shchepinov et al., 2011); it is important to point out these deuterium levels were consistent with literature references of

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0 hour

WT cor1Δ coq9Δ

10 hour

WT cor1Δ coq9Δ

No fatty acid

100% EPA

80% EPA 20% D2-Lin

50% EPA 50% D2-Lin

100% D2-Lin

No fatty acid

100% DHA

80% DHA 20% D2-Lin

50% DHA 50% D2-Lin

100% D2-Lin

No fatty acid

100% ARA

80% ARA 20% D2-Lin

50% ARA 50% D2-Lin

100% D2-Lin

10 hour

WT cor1Δ coq9Δ

10 hour

WT cor1Δ coq9Δ

FIGURE 31.5

Fore brain deuterium incorporation in %00 normalized to V-SMOW

Small amounts of D-PUFA inhibit peroxidation of non-deuterated PUFA. Q-less coq9, or respiratory deficient cor1 null mutants were harvested during mid-log phase growth (0.2
1.0 OD600). Yeast cells were washed twice with sterile water and diluted to 0.2 OD600 in phosphate buffer. Yeast cells were treated with 300 μM of the designated fatty acid or PUFA mixture for 10 h. Serial dilutions (1:5) starting at 0.2 OD/ml were spotted on YPD solid plate medium. A zero-time untreated control is shown in the top left. Pictures were taken after 3 days of growth at 30 C.

20000 *

16000

*

12000 8000 4000 0 –4000 H-PUFA Saline

H-PUFA MPTP

D-PUFA Saline

D-PUFA MPTP

FIGURE 31.6

Deuterium accumulation of mice administered dietary D-PUFA. C57BL/6 mice were fed a diet using either natural LA and ALA (H-PUFA) or 11,11-D2-LA and 11,11,14,14- D4-ALA for 12 days. Deuterium levels were measured by isotope ratio mass spectrometry and normalized to the Vienna Standard Mean Ocean Water (VSMOW). Data are expressed in per mille (m), and reflect a 14-fold increase in brain deuterium levels in brain from animals receiving the D-PUFA paradigm ( denotes significance; p , 0.001 versus HPUFA).

3
8% incorporation per day (Rapoport et al., 2001). Since PUFAs are elongated and extended enzymatically into higher level fatty acid compounds, as well as serving as substrates for numerous enzymes and pathways including β-oxidation, lipoxygenases, and

cyclooxygenases, another key question to address was whether these critical processes were altered with PUFA deuterated at specific sites. D-PUFAs have been used previously in tracer studies to examine these reactions in vivo (Emken et al., 1976); furthermore, in many instances the PUFAs were perdeuterated (i.e. total deuterium-for-hydrogen substitution) with no change in reaction products (Emken, 2001). Subsequent evaluation in our model system confirmed incorporation and, importantly, further revealed normal assimilation into higher-order fatty acid congeners with no alteration in distribution between H- and D-PUFA-treated groups (Figure 31.7). Given that ethyl esters of deuterated fatty acids have been administered in quantities of up to 9 g per day and in nursing mothers (Emken et al., 1989; Emken, 2001) to evaluate lipid metabolism in breast milk, we did not anticipate systemic toxicity in the drug-treated group. Indeed, serum markers of liver or renal function revealed no abnormalities between H-versus D-PUFA-fed animals, nor significant alteration in blood glucose or lipids (e.g., triglycerides and non-esterified fatty acid). Taken together, the absence of adverse side effects along with evidence of normal PUFA metabolism in these experiments indicates that supplementation with D-PUFA drugs represents a safe option.

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FIGURE 31.7 Tissue PUFA distribution is not affected in D-PUFA-fed animals. Mouse brain fat profile: 90 day dosing starting from the age of two months. Prior to that, mice were fed a regular chow. The diets contained either LA and ALA (1:1) (righthand black bars) or 11,11-D2-LA and 11,11,14,14-D4-ALA (left-hand gray and black bars) as the only source of PUFA. The analysis of PUFA composition reveals that the incorporation of deuterium into higher PUFAs was about 40% (gray bars). Importantly, the PUFA patterns (heights of bars in each pair) are very similar, suggesting that the enzymatic pathways are largely unchanged. Note the absence of ALA, due to full conversion into higher omega-3 PUFAs.

60

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40% of total fat deuterated

30

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0 18:2n–6 20:2n–6 20:3n–6 20:4n–6 22:2n–6 22:4n–6

18:n–3 20:5n–3 22:5n–3 22:6n–3 24:5n–3 24:6n–3

Pre-Clinical Efficacy Although no model fully replicates all of the characteristics of PD, a model that readily mimics the complex I inhibition, subsequent lipid peroxidation, cell death, and striatal DA depletion that underlies motor dysfunction in PD is the MPTP model. Although previous studies have indicated that non-deuterated PUFA supplementation provides some level of protection against degeneration in the central nervous system (CNS) (Simopoulos et al., 2000; Youdim et al., 2000; Carrie et al., 2009), our approach addresses a unique question: whether isotopic reinforcement of PUFA will prevent oxidative stress-related injury and thus modify outcome. As described above, mice were fed with either the D-PUFA drugs or H-PUFA and then challenged with MPTP or saline. Marked protection was revealed at the levels of the striatal dopaminergic terminals; MPTPchallenged mice administered D-PUFA revealed DA measurements virtually 3-fold greater than the toxinexposed H-PUFA cohort (Figure 31.8). Immunoblot analyses for tyrosine hydroxylase (TH), the rate-limiting enzyme in DA synthesis, confirmed preservation of striatal dopaminergic terminals with steady-state protein levels approximately 1.5-fold higher in the D- versus H-PUFA MPTP-exposed animals (Figure 31.8). Interestingly, in saline-treated mice, a trend in increased striatal DA (11%) was noted in the D-PUFA- versus H-PUFA-treated cohorts (p 5 0.053; Figure 31.8); however, this did not correlate with TH steady-state levels (Western blot). In our in vivo studies with the MPTP model, we evaluated DA turnover as an indicator of injury (i.e. intracellular ATP depletion) and subsequent secondary oxidative damage (from metabolism of dopamine

itself). Interestingly, in the H-PUFA MPTP group, striatal DA turnover is notably elevated, more than 3fold, over samples from saline-treated striatum. Remarkably, there was no significant difference in striatal DA turnover in mice administered D-PUFA and challenged with MPTP versus vehicle-treated animals (Figure 31.8). These data not only support a neuroprotective role against MPTP lesions in the model, but also indicate reduced secondary toxicity due to oxidative stress. These data are further supported by evaluation of a marker for mitochondrial dysfunction and oxidative stress, the mitochondrial chaperone HSP60. This is an inducible chaperone that is upregulated under conditions of oxidative injury within mitochondria to prevent abnormal protein aggregation (Lee et al., 2009; Bender et al., 2011). Following MPTP exposure, we detected a dramatic increase of this protein in striatal homogenates from H-PUFA-fed, MPTP-treated mice that was diminished in MPTP-treated mice given D-PUFA (Figure 31.8). Taken together, these data support the idea that mitochondrial injury may be partially attenuated by isotopic reinforcement through D-PUFA administration.

CONCLUSION The studies shown here demonstrate that deuterium-for-hydrogen substitution, even at low levels, can protect against oxidative challenge in the test tube, in yeast cells, and in an animal model of nigrostriatal cell death. This novel approach targets a pathway that contributes to oxidative injury and degeneration and exploits known properties in

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(A)

140

(C)

*

70

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100 80

*,†

60 40

Striatal DA tunrover (%)

Striatal DA in% II-PUFA-saline control

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(D)

TII Optical density in% II-PUFA-saline control

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H-PUFA D-PUFA H-PUFA D-PUFA Saline Saline MPTP MPTP

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— 60 kDa

β-actin

— 40 kDa

20 0 H-PUFA Saline

H-PUFA MPTP

D-PUFA Saline

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FIGURE 31.8 Dietary D-PUFA protects against MPTP-induced nigrostriatal injury. C57BL/6 mice were administered either hydrogenated Lin and Lnn (H-PUFA) or 11,11-D2-Lin and 11,11,14,14- D4-Lnn in diet for 6 days, exposed to MPTP or saline, continued on diet regimen and then sacrificed 6 days later. (A) Striatal DA was significantly reduced in mice administered MPTP vs. saline (; p , 0.05), but toxin-treated mice administered D-PUFA drugs revealed significantly elevated striatal DA compared to H-PUFA-MPTP (†; p , 0.05). (B) TH immunoreactivity declined in the H-PUFA-MPTP cohort (; p , 0.05), but was significantly higher in the D-PUFA-MPTP group (†; p , 0.05). (C) Striatal DA turnover is enhanced following MPTP treatment in the H-PUFA group (; p , 0.05), but the D-PUFA-MPTP cohort revealed DA turnover values similar to saline-treated mice (†; p , 0.05, D-PUFA-MPTP versus H-PUFA-MPTP). (D) Western blot analyses of HSP60 immunoreactivity revealed elevated levels of the chaperone HSP60 indicative of the mitochondrial response to oxidative stress.

biophysics and nutrition: the KIE and the dietary need for PUFA. Isotopic replacement of PUFA may represent a safe, easy, and effective method to dramatically slow the progression of injury caused by oxidative stress in PD and related neurodegenerative disorders.

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32 Obesity, Cognitive Functioning, and Dementia: A Lifespan Prospective Merrill F. Elias, Georgina E. Crichton and Amanda L. Goodell INTRODUCTION The prevalence of obesity (body mass index (BMI) $ 30 kg/m2) and overweight status (BMI of 25
29.9 kg/m2) is rising dramatically and, by 2015, will involve more than 2.3 million and 700 million people, respectively (World Health Organization, 2011). This is a major concern with regard to health in general, diabetes mellitus, and heart disease (Eckel, 1997; Sowers, 1998), but also because cardiovascular diseases (CVD) are associated with lower levels of cognitive performance and dementia (Waldstein and Elias, 2001) and lower cognitive performance earlier in life is a risk factor for dementia later in life (Elias et al., 2000). In this paper we review the literature on weight, including BMI, obesity, and central obesity, as it relates to dementia and to variations in cognitive ability in non-demented persons. We take a lifespan perspective, arguing that it is imperative to move backward in time from adult cognitive deficit and diagnosed dementia per se in order to examine its precursors. These precursors include weight and obesity, but also cognitive changes that occur prior to a diagnosis of mild cognitive impairment (MCI) or probable dementia. It is critical to consider the possible longitudinal co-variation of weight-related variables and cognitive outcomes across the lifespan. Because we cannot review all pertinent literature due to space limitations, we focus on prospective data and highlight selected studies that illustrate important issues of theory, design, methods, and the need for a lifespan perspective. For other perspectives and emphases see the following reviews (Anstey et al., 2011; Barrett-Connor, 2007; Beydoun et al., 2008a; Gustafson, 2008, 2012; Sellbom and Gunstad, 2012; Smith et al., 2011; Whitmer, 2007). There has been some amount of emphasis on which specific cognitive abilities are related to obesity and Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00032-6

overweight and in this chapter we comment on important methodological considerations with respect to this research goal. In addition, we discuss mechanisms underlying adiposity and cognitive decline. See also a comprehensive approach to the topic of mechanisms by Gustafson (2012). Where necessary, we note findings with respect to underweight and related conditions, but the focus is on overweight and obesity. Metabolic syndrome is an area of research which is an important and integrative extension of studies of obesity in relation to metabolically-related CVD risk factors. This topic is examined in a recent review by Crichton et al. (2012) and is thus briefly summarized in this chapter. Omega-3 fatty acids have shown promising results in the treatment of diabetes and may play a role in the mechanisms underlying the relation between overweight, obesity, and cognitive performance and dementia.

WEIGHT-RELATED VARIABLES AND DEMENTIA Results of prospective investigations into the relationship between BMI and incident dementia are decidedly mixed. Some studies report no association between BMI and dementia (Stewart et al., 2005). Others report that higher BMI in elderly persons is associated with increased risk of dementia (Gustafson et al., 2003), and still others report that higher BMI in middle age is associated with dementia three decades later (Kivipelto et al., 2005; Whitmer et al., 2005). Moreover, lower BMI has been related to increased risk for dementia, particularly Alzheimer’s disease (AD) (Nourhashemi et al., 2003). Nonlinear associations may exist between BMI and dementia. In that regard, authors of a recent meta-analysis concluded

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that being underweight, overweight or obese at midlife increases the risk for dementia later in life (Anstey et al., 2011). Among other factors, these conflicting findings may be related to the measures used to assess weight and to the surveillance periods for exposure to weight gain or loss and obesity. BMI has decreased in value as a measure of adiposity in the oldest old. Aging is characterized by a decline in lean body mass (i.e., sarcopenia) and an increase in adipose tissue without weight gain, a phenomenon that is not captured by BMI (Fitzpatrick et al., 2006). Waist circumference (WCir) and waist-hip ratio (WHR) appear to be better adiposity measures in elderly people as these indices of central obesity reflect the abdominal distribution of body fat (Allison et al., 1997; Janssen et al., 2004; Stevens et al., 1999; Whitmer et al., 2008). Whitmer et al. (2008) make the following points. Central obesity is a more potent risk factor for CVD and mortality than total body obesity assessed by BMI. This phenomenon may be related in part to the role of intra-abdominal fat (visceral adiposity) on metabolic abnormalities associated with increased risk of diabetes and CVD. Visceral fat is more active metabolically than subcutaneous fat and is thought to have a stronger influence on insulin resistance and adipocytokine (cell to cell signaling protein secreted b adipose tissue) production. In dementia and stroke-free individuals, WHR and WCir (indicators of central obesity and indirect indictors of visceral fat) were more strongly associated with cognitive performance levels than BMI (Wolf et al., 2007). In a study by Whitmer et al. (2008) in which participants were followed for 36 years from baseline, the highest quintile of sagittal abdominal diameter measured in midlife (40
45 years of age) was associated with a 3-fold increased risk of dementia. In a cross-sectional study comparing subcutaneous versus visceral viscosity measured by abdominal CT, Yoon et al. (2012) reported that visceral adiposity (highest tertile) was associated with increased risk of low MiniMental State Examination (MMSE) scores (2 1 SD), with adjustment for age, gender, education, and diabetes, but that subcutaneous adipose tissue and MMSE scores were not significantly related. Findings were significant only for participants under age 70 (n 5 188), but the over 70 groups had fewer subjects (n 5 62) and thus the negative finding may relate to relatively lower power. We need more studies with prospective and longitudinal designs that employ this type of quantification of visceral versus subcutaneous obesity. Positive associations between being overweight or obese at midlife and incident dementia may be at least partially attributed to the emergence of cardiovascular risk factors and events that correlate with obesity at midlife. These include components of the metabolic syndrome (MetS) such as central obesity, glucose

intolerance, dyslipidemia, hypertension, the development of diabetes mellitus, and clinical cardiovascular or cerebrovascular events (Fitzpatrick et al., 2006). However, at the end of the life-cycle the role of cardiovascular risk factors in cognitive performance may be diminished by attrition due to morbidity and mortality of subjects who are in the poorest health at midlife and who are likely the poorest performers on tests of cognitive ability. It is also important to consider the changes in protective reproductive hormones and weight gain experienced during midlife by women undergoing the menopausal transition (Will and Randolph, 2009). Data from the Cardiovascular Health Study (59% women) indicate that high BMI at midlife was related to increased risk of dementia in women, but after 65 years of age it was associated with decreased risk for dementia. The importance of this study is that weight variables were assessed both at midlife, age 50, and at 65 years or older (Fitzpatrick et al., 2006). Obesity at midlife (BMI . 30 kg/m2), as compared to normal weight (BMI 20
25 kg/m2), was associated with incident dementia [hazard ratio (HR) 5 1.38; confidence interval (CI) 5 1.03 to 1.95] when adjusted for demographic variables. These associations were attenuated, but not eliminated (HR 5 1.36; CI 5 0.94 to 1.95), with additional adjustments for CVD and risk factors, suggesting that they were among those mechanisms that mediated relations between obesity and cognition. However, in old age, risk estimates were reversed. Underweight study participants (BMI , 20 kg/m2) showed an increased risk of dementia (HR 5 1.62; CI 5 1.02 to 2.64) and presence of obesity was associated with reduced risk for dementia (HR 5 0.63; CI 5 0.44 to 0.91) as compared to normal weight status (Fitzpatrick et al., 2006). The same results were obtained for the condition of overweight (BMI 25
30 kg/m2) (HR 5 0.92; CI 5 0.72 to 1.18), in comparison with normal weight status. These curvilinear associations between weight and cognition in old age have been widely reported (Anstey et al., 2011; Brubacher et al., 2004; Fewlass et al., 2004; Sturman et al., 2008). Among older age cohorts, associations between weight loss and dementia may reflect the fact that weight loss is a common consequence of dementia rather than a cause. Importantly, however, weight loss often predates dementia onset by as many as 10 years (Knopman et al., 2007), and it is one of the principal manifestations of AD (Gillette Guyonnet et al., 2007). Prospective studies, in which risk factors are measured prior to onset of dementia, avoid confounding of precursors of dementia with the consequences of dementia such as weight loss and poor health. The length of the pre-dementia surveillance period used to assess weight, weight change, and obesity and its

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distance from the onset of dementia are both important design parameters. Clearly, more reliable assessments of risk factors are possible with more time to assess them and more time between risk exposure and dementia. The ideal study would start as early as possible in development with time points for measurement reasonably spaced, based on rate of development or decline, and would feature measurements of obesity, body weight, relevant comorbidity, confounders, and cognitive functioning at each of these points in time. It is difficult to achieve this ideal in a single study and these data will most likely evolve from an integration of data with surveillance periods beginning and ending at different points in the lifespan. The pre-clinical phase of dementia may begin decades before dementia is diagnosed (Elias et al., 2000; Sperling et al., 2011; Whalley et al., 2006). There is now a growing body of literature indicating that weight at midlife is a good predictor of cognition many years later and prospective studies are the design of choice. A detailed review of every study contributing to this growing literature is not possible, and thus in the next two sections we focus on studies in two categories based on these outcome measures: (1) dementia; and (2) cognitive performance, including studies that exclude persons with dementia and those that include them. We focus largely on studies with large sample sizes, and prospective designs with reasonably long surveillance periods.

Prospective Studies with a Focus on Dementia In the large sample (N 5 6,538) Kaiser-Permanente Study of Central Obesity (Whitmer et al., 2008), standardized anthropometric measures were obtained when participants were between the ages of 40 and 45 years and related to medical records of dementia three decades later. Compared to those in the lowest quintile of sagittal abdominal diameter, participants in the highest quintile showed nearly a 3-fold increase in risk of dementia (HR 5 2.72; CI 5 2.23 to 3.33). These associations were maintained when adjusted for demographic variables, diabetes, cardiovascular comorbidity, and BMI. The relation between increased sagittal abdominal diameter and risk for dementia was attenuated but not eliminated when adjusted for BMI (HR 5 1.92; CI 5 1.58 to 2.35). The number of years of follow-up and sample size are key strengths of the study, but the diagnosis of probable dementia was based on insurance records rather than comprehensive clinical and cognitive evaluation in a central laboratory or clinic setting. In a study with the Baltimore Longitudinal Study of Aging (Beydoun et al., 2008b) 2,322 participants were

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followed for a median of 23.4 years with AD as the outcome variable. BMI and WCir increased with age at an annual rate of 0.07 kg/m2 and 0.35 cm, respectively. For men, weight gain between ages 30 and 50 years for any 5-year longitudinal interval was associated with an approximate 3-fold increase in risk of AD. However, being an underweight male at ages 30, 40, or 45 years of age was associated with a more than 5-fold increased risk for AD (HR 5 5.76). Among older and midlife women, the joint presence of obesity and central obesity (WCir . 80 percentile) at 30, 35, or 50 years increased the risk of developing AD 6.6-fold. However, women who experienced weight loss between 30 and 35 years of age exhibited a 2-fold increase in AD. A notable longitudinal increase in WCir was found to be protective against AD in women, but not men. The follow-up period is exemplary, but data on possible mediators of the sex differences among weight variables and AD outcomes were not available, and thus a substantive elaboration on the reasons for these differences was not possible. Later in this paper we discuss the possibility that sex differences may be a product of events which occur many years prior to AD. A discussion of possible causes of sex differences for obesity and other risk factors for dementia may be found in Azad et al. (2007). In the Italian Longitudinal Study on Aging (ILSA) investigators (Solfrizzi et al., 2011) examined the progression from normal cognitive performance through two phases of clinically defined impairment, MCI, and dementia. The study is exemplary in terms of the prospective design, the population-based sample, the large number of participants (N 5 5,632, ages 65 to 84 years old), and adjustment for a large number of potential confounders including psychosocial variables such as depressed mood and under-nutrition. The presence of MetS and its major components, abdominal obesity, hypertension, and hypertriglyceridemia, were associated with a significantly higher incidence rate for progression to dementia in persons with MCI. However, obesity and the other MetS components did not predict progression from normalcy to MCI. Thus, abdominal obesity did not predict incident MCI, but MCI modified the prospective relation of abdominal (central) obesity to dementia. In an explanation of negative results, the authors noted that the MMSE was a major source of dementia case definition and diagnosis of impairment, that measures of executive function were not considered, and that amnestic versus non-amnestic subtypes were not defined because of missing data on critical indices of these subtypes. Study participants were at the lower end of formal educational attainment (mean 5 7.5 years; SD 5 4.9). This raises the possibility that cases of MCI and/or dementia may have been overestimated,

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albeit informant data and a neurologist’s diagnosis were employed to make these classifications. Regardless of these issues, MCI is an important diagnosis based on measures of clinical impairment and other evidence and there is a pressing need for prospective studies that differentiate between subtypes of MCI as a critical step in the progression from normal cognition to dementia. Sensitivity and specificity of cognitive measurement is best achieved by considering cognitive outcomes where the full range of test scores is employed. Accordingly, we now briefly review the literature on weight-related variables. In each of these studies a full range of cognitive test scores is the outcome measure or is used to create a binary outcome measure (good or poor performance).

Prospective Studies Focusing on Cognitive Functioning In the Swedish Adoption/Twin Study of Aging (SATSA) (Dahl et al., 2010) the association between BMI and cognitive decline was examined over 40 years of follow-up among participants initially aged 25 to 63 years (mean age 5 41.6 years; N 5 781). Without excluding persons with probable dementia, BMI and global cognitive ability (a continuously distributed variable) were measured five times over a 20-year period beginning at age 25. Global cognitive ability was measured by taking the first principal component of 11 tests at baseline and standardizing it across times of measurement (specific tests in the composite not reported). Latent growth curve analyses indicated that higher BMI was inversely related to the measure of global cognitive ability with longitudinal decline for both women and men. Results were similar when persons with probable dementia were excluded from the sample. Two study limitations were noteworthy: (1) BMI was calculated from self-report of height and weight at the earliest assessments (1963 and 1967) and no measures of WCir, WHR, and/or obesity were employed; (2) control for education was less than ideal, i.e. low education 5 # 6 years and high education 5 . 6 years. This leaves a wide range of years of education above 6 that can have a major effect on cognitive outcomes. Aside from these issues, the long follow-up period adds important lifespan information to the literature. In a later study with mixed self-report and assessed height and weight, these same investigators reported a negative effect on mean cognitive test performance across verbal and spatial abilities, memory, and perceptual speed for individuals who were overweight or obese in early midlife, late midlife, or across

midlife (Dahl et al., 2010). With adjustment for age, education, physical activity, and vascular risk factors, overweight (BMI between 25 and 30) in midlife for the OCTO-Twin study subjects was related to lower performance on long-term memory, short-term memory, speed, and verbal and spatial ability 30 years later (Hassing et al., 2010). Employing the Whitehall II study data, Sabia et al. (2009) examined the cumulative effect of BMI from early adulthood (25 years) to late midlife (mean age 61 years) with respect to cognitive outcomes. In a sample restricted to white participants (N 5 9,181), BMI was self-reported at 25 years (early adulthood), but was measured at midlife (mean age 5 43.8 years) and later life (mean age 5 60.8 years). Persons with dementia were not excluded. Associations between obesity and two measures of cognitive ability (MMSE and executive functioning indexed by semantic and verbal fluency) became more pronounced over the follow-up period (mean duration 36 years). Although attenuated, these associations were observed after adjustment for health behaviors, blood pressure, and cholesterol at mid and late life. An association between underweight and cognition, both at mid and late life, was also seen in the Sabia et al. (2009) investigation. The risk of cognitive deficit was enhanced with increasing levels of underweight in a dose
response manner, and this association was sustained with statistical adjustment for cardiovascular risk factors and events. Sabia et al. (2009) hypothesized that underweight may be a product of poor health (Truett et al., 1967) and/or dysregulation in hormone secretion corresponding to that seen in anorexia, a disease associated with cognitive disorder (Laitala et al., 2011). Limitations of the study were that measures of central adiposity were not employed, weight was selfreported for the 25-year-old cohort, and an opportunity to compare results with the inclusion and exclusion of African Americans was missed. However, the sample size was impressive, as was the duration of longitudinal follow-up. In the Basel Study, Brubacher et al. (2004) related weight change and cognition for a sample of 445 men and 86 women described as optimally healthy and non-optimally healthy community-dwelling persons. Baseline measurements of BMI were obtained in 1960, with follow-up examinations in 1965, 1971, 1990, and 2000, and multiple measures of cognitive performance were obtained at an average age of 69.4 years (SD 5 7.8 years). Based on the full continuous distribution of test scores for 11 key variables extracted from the Consortium to Establish a Registry for Alzheimer’s Disease-Neuropsychological Assessment Battery (CERAD-NAB) test scores, good and poor performance were defined as binary variables, i.e., two or more

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scores 1.5 SD below the mean was defined as poor performance. With zero annual weight change as the reference group, weight change in the positive direction was associated with progressively increasing odds for poor performance, but weight change in the negative direction from zero was also associated with increasing odds of poor performance, although risk did not reach the same level as change in the positive direction. The same curvilinear relation was seen for participants carrying the ApoE-ε4 allele but with modestly higher risk of poor performance at each point along the curve. The limitation in this study was that probable dementia was not defined by a dementia review, but was estimated from telephone test scores and identified as ‘likely dementia.’ Based on research with a large sample (2,606 twin individuals) from the Finnish Twin Cohort Study, Laitala et al. (2011) reported that midlife BMI (ages not specified) and other cardiovascular risk factors measured by self-report and medical registry records were related to cognitive functioning, as assessed via a validated phone interview technique. Height and weight estimates were based on self-report in 1975 and 1978 and related to cognition measured between 1999 and 2007. With adjustment for sex, education, birth year, and age, relations among other CVD risk factors and BMI at midlife were associated with a later-life cognition measure, based on the phone interview questions. Overweight classification was associated with an increased risk of likely dementia. Most importantly, weight gain in excess of 1.7 kg/m2 and weight loss of more than 2 kg/m2 within an average of 5.6 years prior to cognitive assessment was related to lower telephone cognitive performance scores despite adjustment for BMI. Additive genetic correlation (the proportion of variance that two traits share due to genetic causes) appeared to mediate between BMI and cognition, but this association was attenuated when adjusted for education. As the authors noted, telephone interviews cannot be definitive as a technique in assessing dementia, and in our view is less desirable than direct cognitive assessment. However, the study employed a very large sample, a necessity for genetic studies by making use of an emerging and important method of general cognitive assessment, e.g., telephone interview, where large samples are important. It should be noted that none of these studies examined weight in relation to multiple cognitive abilities, or multiple cognitive domains each indexed by one or more tests of ability, and several did not adjust for CVD risk factors other than weight. There are other prospective studies that we have not reviewed. To read further on these investigations and this topic, see the paper by Beydoun et al. (2008a) for a review and meta-analysis of ten relevant

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methodologically acceptable prospective studies employing adults between 40 and 80 year of age at baseline. This meta-analysis supports the general conclusion that obesity earlier in life is a risk factor for dementia and AD later in life. In the next section we focus on studies addressing two important questions: (1) do CVD risk factors and events that correlate highly with obesity account for relations between obesity and cognition; and (2) are there other variables that mediate and modify relations between weight-related variables and cognition?

WEIGHT AND COGNITIVE FUNCTION: ROLE OF CVD FACTORS The earliest study reporting an association between the classification of overweight and cognitive functioning did not adjust for CVD correlates of obesity (Sorensen et al., 1982). Extensive controls for CVD risk factors were employed for the first time in a prospective study employing data from the Framingham Heart Study (Elias et al., 2003). Persons with probable dementia and prevalent acute stroke were excluded from the study. The surveillance period for examination of multiple cardiovascular risk factors and events was 8 to 10 years prior to measurement of multiple cognitive abilities (Elias et al., 2005). With statistical adjustment for CVD factor variables and demographic variables, obesity was associated with test measures of attention, visual-spatial ability, memory, executive function, and global performance for both men and women. Relations between obesity and cognitive performance were not as strong for women. Stronger and more robust associations of obesity and cognitive performance for men than women (Elias et al., 2003) with control for CVD variables may be related to their generally higher risk for CVD-related mortality and morbidity. See Elias et al. (2003, 2005) for a more detailed elaboration of these hypotheses. Regardless of the reason for sex differences, this prospective study provides evidence for the conclusion that CVD risk factors play an important mediating role in relations between weight and cognitive functioning, but does not entirely account for these associations. Three years later, support for the generalization was provided by a cross-sectional study in which BMI and measures of central obesity were related to multiple measures of cognitive ability with extensive exclusions and adjustments for health and medical variables (Waldstein and Katzel, 2006). Study participants with renal, hepatic, pulmonary, and neurological disease, stroke, probable dementia, psychiatric disorder, heavy alcohol use, and low MMSE scores (,24) were excluded from the analyses. Despite these exclusions, both BMI

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and WCir were associated with lower levels of cognitive performance on measures of motor speed, manual dexterity, and executive functioning. Adjustment for demographic variables, fasting glucose, insulin, and specific lipid fractions attenuated, but did not eliminate, the associations between the measures of overweight and cognitive measures. Both the Elias et al. (2003) and the Waldstein and Katzel (2006) studies indicate that the correlations between overweight or diabetes and prevalence of other CVD risk factors does not explain why persons who are overweight or diabetic perform at a lower level on tests of cognitive functioning. These data are generally consistent with reports that obese adults who are otherwise healthy perform more poorly on measures of memory and learning (Gunstad et al., 2010, 2006), speed and attention (Cournot et al., 2006), and executive function (Gunstad et al., 2007). One may conclude from these studies that disease and risk factor correlates of overweight status and obesity partially mediate relations among weight and cognition, but they are insufficient to explain these associations. Other explanatory mechanisms are discussed following the next section on interactions.

Interactions of Obesity and CVD Risk Factors Associations between obesity and cognition are modified by the presence of other CVD risk factors. In the Framingham Heart Study (Elias et al., 2003), obesity and hypertension were independently associated with lower scores on multiple measures of cognitive functioning, but among men only, the adverse effects of obesity and hypertension were additive with respect to measures of episodic memory and visual-spatial constructive abilities. The highest level of performance was observed in the absence of obesity and hypertension, the second highest for those with either hypertension or obesity, and the lowest for those with both obesity and hypertension. Interactions between diabetes and obesity were also found in the progeny of the Framingham study participants. In a 12-year prospective study with 1,184 men and women participating in the Framingham Offspring Study population, the lowest levels of cognitive performance were seen for participants with both hypertension and central obesity as compared to those with either hypertension or obesity or neither of these risk factors (Wolf et al., 2007). The synergistic relation between diabetes and hypertension has been reported in at least two additional studies, one cross-sectional (Waldstein and Katzel, 2006), and one longitudinal (Solfrizzi et al., 2011), both featuring extensive controls for demographic variables, CVD, and CVD risk factor correlates of obesity.

Sakakura et al. (2008), employing the MMSE as an outcome variable, reported interactions between hypertension and weight in a sample of young-elder and elder adults with type 2 diabetes. There was no relation between obesity and performance, but the lean participants (BMI range 5 14.5
20.3 kg/m2) with hypertension were the poorest performers on the MMSE. This finding suggests that hypertension enhances the magnitude of associations between low weight and cognition, and this is consistent with reports of higher mortality rate in lean hypertensive subjects (Stamler et al., 1991). An interaction study with important implications for individuals experiencing heart failure (F) was performed by Alosco et al. (2012). Cerebral blood flow velocity (CBF-V), measured by transcranial doppler sonography in 99 study participants, was the index of cerebral perfusion and obesity and was defined by BMI. There were significant statistical main effects of CBF-V and BMI but a combination of hypoperfusion and higher BMI showed a stronger relation to measures of attention and executive function than did either hypoperfusion or BMI alone. Weight re-education would appear to be advantageous, particularly in the early stages of heart failure. Synergistic and additive relations among obesity and other CVD risk factors should come as no surprise. In general, risk factors are additive with respect to their impact on cognitive functioning. In a re-analysis of Framingham Heart Study data, obesity, diabetes, cigarette smoking, and hypertension were significantly related to lower cognitive performance; and, regardless of which risk factors were in the mix, risk for poor performance increased as the number of risk factors in the regression model increased (Elias et al., 2001, 1998). In a previous section we discussed the fact that obesity-related CVD comorbidity provides an incomplete explanation of relations between overweight or obesity and cognitive performance. In the next section we turn to the issue of needed additional controls and studies and variables that intervene (mediate) between, and partially explain, relations between weight and cognitive functioning, i.e. effect mediators.

Controls and Potential Mediators Failure to adjust for activity level, eating behaviors, and mood disorders is common in the weight and cognition literature we have reviewed, but the following studies indicate that these variables may mediate between weight and cognition. In a cross-sectional analysis using the Maine Syracuse Longitudinal data (N 5 917, ages 23
98 years, with stroke and

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METS

dementia excluded), Dore et al. (2008) found inverse associations between WHR and WCir for global cognition, scanning and tracking, and abstract reasoning domains with adjustment for age, other demographic variables, and multiple cardiovascular risk factors. But with further adjustment for self-reported physical ability expressed as metabolic energy expenditure, only the relation between central obesity and abstract reasoning was sustained, thus suggesting that activity and metabolic expenditure may be an important mediator between central adiposity and cognition. Dore et al. (2008) statistically adjusted for demographic variables and multiple cardiovascular risk factors, including depressed mood as an estimated metabolic energy expenditure, but important nutrition and dietary habit variables were not considered. Annesi and Gorjala (2010) found that exercise was associated with weight loss, but that this association was more related to improvements in mood state and improved eating and nutrition behaviors than by direct energy expenditure. Mood state may be important in explaining sex differences with respect to relations between weight and cognitive functioning. Obesity has a stronger relation to depressed mood in men than in women and obese cohorts exhibit more depression than non-obese cohorts (Heo et al., 2006). Daily, monthly or seasonal changes in mood are related to an excessive intake of carbohydrate-rich food and resistance to involvement in physical activity. Brain serotonin may also be involved in these mood and appetite disturbances (Wurtman, 1993). Clearly, co-control of these mood state, dietary change, and nutrition variables is important when relating activity level to cognitive performance; these controls are not widely employed; and few if any studies have examined them as effect mediators using formal statistical methods such as path analysis or structural equation modeling.

INITIAL SUMMARY Based on studies of weight and obesity where normal variation and a full range of cognitive test scores define the cognitive outcome variable, the following generalizations are possible: (1) cardiovascular risk factors, CVD, and CVD events play an important mediating role in relations between obesity and cognition, but are insufficient to fully explain these associations; (2) in large sample studies, central obesity shows a stronger relation to cognitive deficit than BMI; (3) effect sizes for weight and obesity are enhanced by the addition of other risk factors such as hypertension, diabetes, and ApoE-ε4 genotype; (4) adjustments for activity level and energy expenditure are associated with a

reduced magnitude and number of relations between central obesity and cognitive performance; and (5) it is important that studies in which weight is adjusted for activity separately include further adjustment or control for diet and mood state. A review of obesity and the overweight condition is incomplete unless tied into the MetS.

METS It is not possible to provide a comprehensive review of this rapidly growing area of literature in this chapter, but a brief summary is important because abdominal obesity is the key component of MetS. We refer readers to a recent and thorough review of this literature by Crichton et al. (2012) and identify a few major studies. The Crichton et al. (2012) study reviewed nine studies that assessed MetS in relation to cognitive performance, and ten studies which used a combination of screening measures, neuropsychological testing, clinical evaluations, and brain imaging to assess MetS in relation to dementia. The authors conclude that, despite the widespread variability in study design, statistical control of confounders, type of cognitive testing, and classification of tests, the presence of MetS increased the risk of poor cognitive test performance variously defined, and of cognitive impairment in an absolute sense (Crichton et al., 2012). For example, for cognitive function measured by neuropsychological testing, detriments in executive function, information processing speed, attention, and verbal memory were most frequently reported (Cavalier et al., 2010; Dik et al., 2007; Knopman et al., 2009; Komulainen et al., 2007; Segura et al., 2009; van den Berg et al., 2008; Yaffe et al., 2007). MetS was associated with increased risk of AD (Razay et al., 2007; Vanhanen et al., 2006), and with an increased risk for both the development of vascular dementia and the progression from MCI to dementia over a four-year period (Raffaitin et al., 2009; Solfrizzi et al., 2010, 2011). The Crichton et al. (2012) review highlights some of the major issues facing MetS investigations. While studies have demonstrated relationships between the number of MetS components present and cognitive function (Cavalier et al., 2010; Gatto et al., 2008; Komulainen et al., 2007; Yaffe et al., 2009), it is often difficult to determine which among the metabolic risk factors included in the syndrome is driving the relation between MetS and cognitive performance, and many studies have not addressed this issue. It is important in future research to distinguish if the MetS relationship to cognition is driven by one or more major risk factors such as obesity or diabetes.

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Stated differently, the role of major metabolic risk factors such as obesity and diabetes must be separated from the cumulative negative force of MetS. Secondly, it will be important to determine if MetS is having a diffuse influence on global cognition or on specific cognitive abilities, which may help identify patterns of cognitive deficit prior to cognitive decline. This goal, as we discuss later, is made difficult by the different definitions of various specific cognitive abilities and domains, especially where studies are examined in the context of meta-analysis. This is a very important area of future research because it is quite clear that risk factors do not act alone and that they cluster within the individual. We now turn to the consideration of biological mechanisms that may explain relations between weight, obesity, and cognition.

MECHANISMS: GENERAL As discussed previously, cardiovascular risk factors and CVD at least partially mediate relations between obesity and cognition. Yet, relations between overweight/obesity and cognition remain despite control for these health variables. There are other potentially important mediators between weight gain and cognitive performance. Insulin resistance has been used infrequently as a covariate in studies of obesity and cognitive performance, although it is a correlate of central obesity and it is associated with cognitive decline (Whitmer et al., 2008). Whitmer et al. (2008) found that there are toxic effects of visceral adiposity, a metabolically active endocrine tissue secreting several inflammatory cytokines and hormones, such as adiponectin and intereukin-6, which are both associated with cognitive deficit (Chaldakov et al., 2003; Kuller et al., 2003; Landin et al., 1990; Prins, 2002; Yaffe et al., 2003). Whitmer et al. (2008) also cite work indicating that leptin crosses the blood
brain barrier, plays a role in neurodegeneration, and is involved in the depositing of beta 42, the main ingredient in AD-associated plaques. There is evidence that total brain atrophy, and especially atrophy of the hippocampus, is related to obesity as increased white matter disease (Jagust, 2007; Jagust et al., 2005). Research by Verstynen et al. (2012) indicates that BMI is associated with a reduction of cerebral white matter throughout the brain. Walther et al. (2010) reported a negative association between increased body fat and gray and white matter in older women, aged between 52 to 92 years, who underwent extensive high-resolution magnetic resonance imaging (MRI) scanning of the brain. Compared to normal weight woman, these obese women performed more poorly on tests of executive function, and reduction in

gray matter in the left orbital frontal area was associated with executive functioning, but while gray matter predicted performance on memory and visual-motor performance, obesity did not. This phenomenon may, or may not, reflect the fact that executive functioning is more sensitive to gray matter deficits than other cognitive measures or the relatively small sample size for a cognitive study. There were only 53 normal weight women, 22 overweight women, and 20 obese women in the study. Thus we suggest that in studies combining MRI and cognitive measures, power should be determined on the basis of the cognitive measures. Driscoll et al. (2012) cite numerous cross-sectional studies in which obesity is related to whole brain, prefrontal regions, and the temporal lobe volumes, including the hippocampus across a variety of age groups in non-demented study participants. But this phenomenon, as they point out, could be related to the fact that some of the individuals with normal cognitive functioning in midlife may progress to dementia and the volume reduction may be related to pathological processes inherent to the dementing process. The need for longitudinal studies is emphasized in a longitudinal study of midlife obesity in relation to trajectories of brain volume change in older adults conducted with 152 Baltimore Longitudinal Study of Aging (BLSA) community-dwelling men and women, aged between 56 and 86 (Driscoll et al., 2012). The investigators predicted that the same brain regions that exhibit early pathological changes in association with AD would be related to obesity. There were no associations between central or global midlife obesity and brain volume changes when compared against a background of general age-related atrophy in their non-demented sample. They note that modest relations began to emerge when data up to the point of dementia were analyzed for those individuals who progressed to dementia. The BLSA study authors argue that cross-sectional studies, particularly those focusing on middle-aged individuals, may overstate the relation between obesity and brain volumes in non-demented individuals because they do not identify and/or do not exclude individuals who progress to dementia. Limitations of the BLSA study are that it is not population-based and most of the sample were Caucasians and very highly educated. But the study makes a major contribution to the literature, and it is clear that there is a need for further longitudinal work, especially work in the context of a background of pathological changes for those who will progress to dementia. A review by Gustafson (2012) has addressed the issues of underlying mechanisms with regard to adiposity-related cognitive decline in far more detail than is possible in this chapter. A brief summary of

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highlights is possible. Brain imaging studies have indicated that BMI is associated with multiple brain pathologies, including temporal lobe atrophy, white matter changes in the brain, and that blood
brain barrier disturbances are more pronounced in late life (Gustafson, 2012). Importantly, Gustafson (2012) concludes, based on the literature reviewed, that crosssectional studies indicate that BMI is related to cerebral spinal fluid markers and MRI markers in a way that is consistent with a relation between BMI and dementia. BMI is inversely correlated with cerebrospinal fluid (CSF) levels of total tau, is positively correlated with CSF αβ levels and inversely related to amyloid deposits in the brain. Gustafson (2012) points out that amyloid metabolism also occurs in peripheral adipose tissue and discusses the ‘potential of cross-talk between adipose tissue and the brain in relation to amyloid and perhaps in relation to dementia etiology.’ (p S107) and concludes that: ‘Irrespective of the direction and temporality of associations, vascular and neurodegenerative pathologies in the brain occur against the background of peripheral metabolic correlates and consequences of overweight and obesity and other vascular factors, and endocrine and neuroendocrine feedback loops. These alterations may lead to direct neuronal toxicity perhaps due to an altered blood
brain barrier (Gustafson, 2012. pS10).’

Obesity-related sleep disturbance, including obstructive sleep apnea, via tiredness, and hypoxia, among other mechanisms, may play a role in obesity-related lowered cognitive performance in children and adults (Amin et al., 2002; Bass et al., 2004; Carvalho et al., 2005; Halbower and Mahone, 2006; Li et al., 2008; Spruyt and Gozal, 2012). The study by Spruyt and Gozal (2012) is especially noteworthy because they employed structured equation modeling (an extension of path analysis) to test whether sleep disordered breathing mediates between overweight and cognition in 351 communityliving snoring and non-snoring children between 6 and 10 years of age. They found diminishing conceptualization and reasoning abilities with increasing BMI (direct association with weight), but they also found a modest (0.55%) increased risk of poorer cognitive ability that was mediated by sleep disorder Frisardi et al. (2010) and Gustafson (2012) have also provided a comprehensive and detailed integrative model and discussions of mechanism. See also a minireview by Kumari et al. (2000) on mechanisms relating obesity and overweight status to memory loss. It is clear that psychosocial variables such as activity level, dietary habits, and mood state disorders need to be included in theories relating obesity to cognitive ability and that sleep disturbance, and related physiological changes, play an important role as a mediating factor.

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Spruyt and Gozal (2012) note that the functional and structural differences in brains of children with sleep disorders (a possible mediator between weight and lower cognitive performance) have yet to be examined, but point out, with supporting literature, that children with congenital central hypoventilation syndrome, a genetic disorder associated with sleep disordered breathing, show evidence of localized injury to the hippocampus, and in adults, preliminary neuroimaging studies indicate that atrophy of the hippocampus and white matter lesions in the frontal lobes are seen, in association with neural differences in motor, sensory, and autonomic regions of the brain. Thus, imaging studies in non-obese, overweight, and obese children could advance our understanding of the role of sleep disorder in cognitive deficits observed as a result of these conditions of overweight.

EARLY INFLUENCES ON RELATIONS BETWEEN OBESITY AND COGNITION The pre-clinical period for dementia is at least 10 years, but its time course has not yet been defined. Pathophysiological processes related to dementia may begin decades prior to the diagnosis of clinical dementia. Further, there may be different pre-clinical stages depending on how the neuropathological processes involved are characterized (Beydoun et al., 2008a; Sperling et al., 2011). It is generally known that early experience and genotype alter brain structure and function (Dalton and Bergenn, 2007; Eleftheriou et al., 1975). Moreover, the early years of schooling are a critical period in acquisition of cognitive ability and motor development and these skills affect education and cognitive performance in adulthood (Strauss and Pollack, 2003). Lower levels of occupation, education, and income are risk factors for cognitive deficits and lowered cognitive performance (Scazufca et al., 2010). Lowered cognitive ability can lead to poorer health literacy including decisions about health practices, nutrition, and decreased activity level that increase the likelihood of sustaining or developing obesity later in life (Datar and Sturm, 2004, 2006; Datar et al., 2004; Reilly, 2005). Li et al. (2008) suggests a reverse causality model in which lower levels of cognition lead to being overweight and obese. It is very likely that multidirectional influences exist across the lifespan. More work with path analysis and structural equation modeling statistics will be helpful in modeling these influences and in the development of relevant theory. It is well established that obesity in childhood can affect tests of cognition, learning, and academic performance at a very early age (Li et al., 2008). For example, Mond et al. (2007) in a study of pre-school children in

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Germany, assessed a wide array of cognitive domains important to development between 4.4 and 8 years of age. Boys exhibited more impairment than girls in motor development, development of speech, memory, abstraction, visual perception, and arithmetic, as well as emotional behavior. After controlling for potential confounders, impairment in gross motor ability was greater in obese male children than normal weight male children, but impairment in attention skills was greater in obese female children than normal weight female children. These and other studies of children lead to the hypothesis that the basis of sex differences in relations between obesity, being overweight or underweight, and cognition in adults may relate to sex differences in psychosocial variables very early in life. A recent study by Guxens et al. (2009) with a U.S. population summarizes the literature on obesity and cognition in childhood and is pertinent to the reverse cognition hypothesis, i.e. that lowered cognition predicts obesity. Early-life cognitive function scores were related to obesity and overweight classifications at two important developmental periods, 4 and 6 years of age. Higher executive functioning ability, verbal, quantitative skills, and memory skills scores at baseline (age 4) were associated with lower likelihood of being overweight at age 6 with adjustment for a significant number of socioeconomic variables including maternal BMI, sex, birth weight and height, gestational age, maternal smoking during pregnancy, maternal education, maternal age, pregnancy height and BMI, breastfeeding, maternal smoking at baseline, number of siblings at baseline, and child’s consumption of sweetened beverages, sweets, and meat at baseline. As is true in the adult literature on obesity and cognition, results with respect to obesity effect modification are conflicting and the precise mechanisms are unresolved. However, the literature on childhood obesity indicates that the answer to why we see sex by weight interactions in adults may lie in the literature on school children and prospective studies that span many years. Of particular interest is the emerging evidence that attention-deficit symptoms are associated with higher prevalence of obesity and hypertension in early adulthood (Fuemmeler et al., 2011; Puder and Munsch, 2010); thus raising the possibility of causal paths from attention disorder to obesity and to cognitive performance, particularly with regard to attention deficits. A recent review by Smith et al. (2011) cites several prospective studies indicating that poor performance on measures of executive and motor performance predicts higher BMI in children from 6 to 11 and discusses obesity as a cause and consequence of cognitive deficits.

Associations between weight and cognition may relate to a chain of events from infancy to dementia. Whalley et al. (2006) emphasize the importance of a lifespan approach to the etiology of late-onset dementia, postulating that: ‘Possible pathways from adverse developmental exposure to dementia include a chain of events triggered by fetal malnutrition, leading to low birth weight, low average childhood intelligence, low educational attainments, low occupational status, and an unhealthy life style [p. 93].’

Whalley and colleagues (2006) hypothesize that malnutrition may trigger at least two pathways to lowered cognitive functioning, one adversely influencing neurodevelopment and the other affecting physiological regulation of blood pressure and glucose metabolism. Models with bi-directional relations between weight and cognitive performance make good sense but these hypotheses need to be tested further, especially using path analysis and structural equation modeling methods.

MORBID OBESITY AND CLINICALLY IMPORTANT COGNITIVE DEFICIT Morbid obesity (i.e., BMI . 40) provides a good model for the study of relations between cognition and obesity in those circumstances where cognitive deficits are of major clinical importance (Chelune et al., 1986). Morbidly obese patients often seek surgery for this condition, thus allowing comparisons of performance before and after substantial weight reduction. Further, although morbidly obese persons represent a small portion of the total US population (Stunkard, 1983), they have a disproportionate risk for obesity-related cardiovascular morbidity as well as psychosocial disability (Chelune et al., 1986). Morbidly obese individuals illustrate the wide range of biological mechanisms serving as mediators between obesity and cognition, including a greater prevalence of eating and psychosocial disorders. Given that the hypothalamicpituitary adrenal axis (HPAX) is excessively activated in morbid obesity, examination of the role of HPAX activation in the morbidly obese provides an opportunity to examine relations between alteration of the counter-regulatory response to insulin-induced hypoglycemia and cognitive function after dramatic weight reduction via banded gastroplasty. In a study of HPAX response and cognition, Guldstrand et al. (2003) evaluated visual-spatial problem solving and maze performance following weight loss from surgery. The mean weight loss was 40 kg

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(SD 5 9), and after approximately 12 months, insulin sensitivity improved to an average increase of 376% of initial values. Cognitive function in persons with both normoglycemia and hyperglycemia was modified by weight reduction and changed from an accuracyoriented approach to a speed-oriented approach with post-surgical improvements in processing rate, inspection rate, and correctly solved problems. More recently, Gunstad et al. (2011) have reported that bariatric surgery patients showed improved memory and concentration after 12 weeks of surgery-related weight loss, but adherence to post-operative procedures is essential to sustained improvement in cognition (Spitznagel et al., 2013). Additional work featuring clinical trials with randomization to surgical treatment is needed. We now turn to a necessary question in relation to the causal associations between weight and cognitive performance: are there randomized-to-treatment clinical trials indicating an improvement in cognitive performance or lessening of dementia symptoms with weight reduction?

TREATMENT OF OVERWEIGHT AND OBESITY The first question is whether therapies aimed at improving cognitive performance are potentially useful in the restoration of cognitive performance or slowing of cognitive deficit. Opinion varies but our view is that they are often ineffective or improvement is modest (Elias et al., 2012b). Barnes et al. (2013) point out that many of the intervention studies involving the goals of weight loss and improvement in cognitive performance in healthy persons with MCI report that resistance training and aerobic exercise achieve modest improvements in cognitive functioning, particularly with respect to attention, processing speed, and executive function (Bak et al., 2004; Colcombe and Kramer, 2003). Very often the specifically targeted cognitive abilities improve but the improvement does not generalize to other cognitive abilities or general cognitive functioning (Bak et al., 2004; Colcombe and Kramer, 2003). There have been few randomized controlled trials (RCTs) in which both weight reduction and cognitive training have been employed in the same trial. However, in a recent trial (Barnes et al., 2013) 126 inactive community-residing individuals were randomized to either mental activity (intensive computer work) or a mental activity control group (educational DVDs) plus exercise intervention (aerobic) or exercise control (stretching and toning). Thus there were four intervention

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groups and the outcome was global cognitive change based on a comprehensive neuropsychological test battery. Groups did not differ at baseline. The global composite cognitive score improved by 0.16 SD over time, but there were no differences among the intervention groups. As is very often seen in these types of studies, improvement due to the practice effect could not be separated from improvement due to specific interventions. Also, as noted in the editorial commentary on this paper by Lautenschlager and Cox (2013), the 12-week interval may have been too short in order for differences between the intervention strategies to be observed. It is clear that long-term intervention studies relating weight loss to cognitive performance are necessary. The Look AHEAD Ancillary Study is presently ongoing (ClinicalTrials.gov, 2013) with the purpose of determining effects of a lifetime intervention program designed to achieve and maintain weight loss and to determine whether this program will be related to better physical and cognitive functioning. This study involves 8 years of follow-up in 1000 participants and purports to be employing a comprehensive neuropsychological battery. No data have yet been reported, but findings will be of interest and importance because short-term interventions of this nature, in studies of hypertension and cognition, often yield trivial to modest or no positive results (Elias et al., 2012a). Again, practice effects will prove to be a problem and controls will be necessary. There is observational evidence that bariatric surgery improves cognitive functioning if post-operative treatment protocols are carefully followed (Spitznagel et al., 2013) but we are unaware of any RCTs. From a broader perspective, many interventions other than weight loss need to be examined. The best approach to reducing cognitive deficits associated with overweight is to prevent, manage, and treat obesity, and as early as possible in the lifespan. Obviously diet, exercise, and activity, prescription-weight medication, and weight loss surgery are all important in weight loss and this literature is too extensive to review here. Behavioral and psychiatric treatment can be important but behavioral disorders may not be the primary issue in many cases of overweight and obesity, or they may not be an issue at all, as obesity involves an interaction of genetic, environmental, neuro-endocrinological, psychosocial, and behavioral factors. Eckel (1997) reported that heredity is involved in 30
70% of cases of obesity, but as the prevalence and incidence of obesity is increasing, thus diet and exercise remain important factors in weight control. RCTs with other than weight reduction are important given reasonable evidence of longitudinal data indicating that higher BMI results in less physical

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activity, but that more physical activity did not result in a decline in BMI (Bak et al., 2004; Mortensen et al., 2006; Petersen et al., 2003) in the University of North Carolina Alumni Study. We now turn to a possible approach to the treatment of obesity which is particularly relevant to the central theme of this chapter, i.e. use of omega-3 polyunsaturated acids (PUFAs).

TREATMENT OF OBESITY WITH OMEGA-3 FATTY ACIDS Long-chain omega-3 PUFAs are essential nutrients that may potentially be of benefit to the management of several disease processes including obesity. Studies in animals have indicated promising early results in this area, in particular for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Omega-3 fatty acids are necessary for normal cognitive development, playing an important role in brain function (Schuchardt et al., 2010). Sources of EPA and DHA include fatty fish such as salmon and mackerel, as well as fish oil supplements. Despite considerable evidence from a number of studies that long-chain omega-3 PUFAs can attenuate weight gain and reduce body fat in rodents (Huang et al., 2004; Ruzickova et al., 2004), few studies have investigated such effects in humans. A small amount of research indicates that increasing longchain omega-3 PUFA intake by 0.3
3.0 g per day may have similar beneficial effects on overweight and obese people (Buckley and Howe, 2010). Our brief review of the literature in this chapter will consider the evidence from the few RCTs that have been conducted in this area. One of the earliest trials conducted in just six adults (Couet et al., 1997) evaluated the effects of a three-week control diet, followed by a wash-out period, and then the same diet in combination with 6 g/day of fish oil (1.1 g/day EPA and 0.7 g/ day DHA). Resting fat oxidation increased (22%) and body fat was reduced (0.88 kg) on the fish oil diets: however, there was no placebo group included in this study. In a double-blind parallel study, Kabir et al. (2007) examined the effects of long-chain omega-3 PUFA supplementation on body composition in 27 overweight and obese post-menopausal women with type 2 diabetes. The two-month study involved supplementation of 3 g/day of EPA-rich fish oil (1.08 g EPA, 0.72 g DHA) or 3 g/day of paraffin oil. Neither treatment group resulted in reduced body weight and there were no changes in subcutaneous or visceral fat assessed by computerized tomography. However, body fat mass was significantly reduced (B1.6 kg) in the longchain omega-3 PUFA group, primarily due to a loss of trunk fat.

In a similar study, Crochemore et al. (2012) evaluated the influence of long-chain omega-3 PUFA supplementation on body composition in women with type 2 diabetes and high blood pressure. Forty-one women were randomized to three groups: Group A (2.5 g/day fish oil, 547.5 mg EPA and 352.5 mg DHA, n 5 14); Group B (1.5 g/day fish oil, 328.5 mg EPA and 211.5 mg DHA, n 5 14); and Group C (placebo, n 5 13) for a 30-day intervention. Interestingly, the lower fish oil dose group had a greater loss of body mass and waist circumference (P , 0.05), and an increase of high-density lipoprotein cholesterol compared with the high dose group. Only a handful of studies have examined the effects of long-chain omega-3 PUFA supplementation combined with energy restriction on body composition (Fontani et al., 2005; Krebs et al., 2006; Munro and Garg, 2013; Thorsdottir et al., 2007). Results have been mixed; neither Fontani et al. (2005) nor Krebs et al. (2006) found any additional benefit from long-chain omega-3 PUFA supplementation on weight loss in addition to a controlled diet in athletes, or energy restriction in overweight women, respectively. However, Thorsdottir et al. (2007) showed a modestly greater increase in weight loss with either long-chain omega-3 PUFA supplementation (6 3 1 g capsules yielding 1.5 g/day long-chain omega-3 PUFA) or the inclusion of either lean or fatty fish, as part of an energy restricted diet over 8 weeks in males only. This differential with regard to gender is in contrast to the most recently conducted study, a double-blind RCT, investigating the effects of supplementation with longchain omega-3 PUFA alone, then consumed concomitantly with a very low energy diet on weight loss (Munro and Garg, 2013). In this study, the placebo group (n 5 19) consumed 6 3 1 g capsules per day of monounsaturated oil, and the treatment group (n 5 20) consumed 6 3 1 g capsules per day of long-chain omega-3 PUFA (fish oil, 70 mg EPA and 270 mg DHA), while consuming their usual diet for 4 weeks. Each group continued with their supplements for another 4 weeks while following a very low energy diet. Despite no change at 4 weeks, there was a greater percentage decrease for females in the supplementation group compared to the placebo for both body weight (2 7.21% vs. 25.82%) and BMI (2 7.43% vs. 25.91%) respectively (P , 0.05 for both) at 8 weeks. Much more work is necessary and, hopefully, will be inspired by the rapidly growing literature on overweight, obesity, and cognitive functioning. The majority of studies have been of short duration, used small samples, and shown only modest effects (Buckley and Howe, 2010). Differences in study design, and the dosage, timing, and duration of supplement administration may also contribute to the conflicting results to

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METHODOLOGIES: GENERAL ISSUES

date. Further human studies are necessary as physiological and behavioral changes in animals may not be equivalent in humans. In the longer term, large intervention studies in overweight and obese adults are needed to determine if long-chain omega-3 PUFA supplementation may be effective in reducing body fat, with the possibility of it being used as a public health strategy to combat the obesity epidemic.

OMEGA-3 FATTY ACID MECHANISMS FOR REDUCING WEIGHT LOSS A short summary of mechanisms by which the dietary intake of long-chain omega-3 PUFAs may facilitate body weight reduction may be helpful with respect to research hypotheses for further studies. Appetite reduction is an important possible mechanism. There is some evidence that long-chain omega-3 PUFA intake may increase postprandial satiety, thereby reducing subsequent food intake (Parra et al., 2008). A smaller sub-study of the weight loss trial by Thorsdottir et al. (2007) in overweight and obese volunteers, showed that individuals who consumed higher long-chain omega-3 PUFA content meals (fatty fish and fish oil) had lower hunger sensations immediately after the test dinner and two hours after the meal (Parra et al., 2008). In addition to influencing satiety, EPA and DHA may have a direct effect on specific brain structures and functions involved in appetite, food intake, and energy homeostasis (Golub et al., 2011). More specifically, animal studies have shown that EPA and DHA may reduce brain endocannabinoids involved in overeating, and effect dopaminergic neurotransmission implicated in the regulation of mood, motivation, and reward (e.g., in response to food intake) (Golub et al., 2011). Another mechanism for operation of long-chain omega-3 PUFA is possible alterations in the expression of genes involved in the regulation of fat oxidation in adipose, liver, cardiac, intestinal, and skeletal muscle tissue (Flachs et al., 2005; Mori et al., 2007). These mechanisms may contribute to body fat loss by increasing fat oxidation in these organs, reducing the capacity for deposition in adipose tissue and the storage of fat (Buckley and Howe, 2010). Finally, longchain omega-3 PUFA supplementation may improve vasodilator function, increasing blood flow and energy utilization (Hill et al., 2007). Further studies are needed to investigate all of these possible mechanisms, as they provide a guide to studies relating long-chain omega-3 PUFA to weight loss and cognitive performance and relations between these phenomena.

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METHODOLOGIES: GENERAL ISSUES Measurement of Cognitive Function A number of methodological issues deserve attention and it is important to define which cognitive abilities are adversely associated with the conditions of overweight and obesity because the pattern of cognitive deficits provides an indication as to whether low cognitive performance will more likely progress to AD or vascular dementia if conversion to dementia occurs. This goal is often frustrated by the fact that tests of different abilities may vary in difficulty level. Many of the clinical test measures employed do not represent pure measures of the constructs they purport to measure because they measure related and thus overlapping abilities. See thorough and thoughtful discussions of these issues in Rabbitt (1997) and Lyon and Krasnegor (1996). In our interpretation of the literature, global cognitive ability and a wide range of specific abilities are adversely affected by the conditions of underweight, overweight, and by changes in weight and obesity. Studies specifically concerned with the pattern of cognitive deficiencies associated with obesity (Cournot et al., 2006; Gunstad et al., 2010, 2007, 2006) indicate that memory, attention, psychomotor speed, and executive ability are among those cognitive skills particularly vulnerable to obesity. These conclusions mainly result from consideration of amalgamating results from multiple studies because the memory studies we reviewed met our criteria for a comprehensive test battery. This is perhaps due to issues of participant burden or because investigators are making use of available data sets. In our view, a comprehensive battery should include a global measure and/or a composite of the individual measures in the battery, which would be, at minimum, attention, verbal and nonverbal memory (immediate and delayed), speed of performance, abstract reasoning, and some form of visual-spatial organization ability. Ideally, it would also have a measure of pre-morbid ability such as reading abilities. Obviously test batteries with multiple neuropsychological tests measuring specific abilities are important. Ideally we should be examining cognitive domains and those domains should be indexed by one or more specific abilities measured by specific tests. For example, executive functioning is a popular construct but is often measured by one or a few test measures. More than one test purporting to measure executive function should be used to index the executive functioning domain. It needs to be distinguished from general fluid intelligence on the basis of an examination of results obtained for the constellation of all tests in the study. Under no circumstances should

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executive function be referred to as a test of ‘frontal lobe function’ in a specific study in the absence of evidence from neuroimaging or other brain function and structure indexing sources used in the same study.

Diagnosis of Dementia With respect to diagnosis of dementia and its employment as an outcome measure, differences in results may relate to differences in the way probable dementia is diagnosed in various studies, comprehensive clinical dementia reviews, medical records, insurance records, and inferences from MMSE scores, an instrument with a well-recognized low sensitivity and specificity. In our view, under no circumstances should a diagnosis of dementia be made solely on the basis of MMSE scores, although clearly the MMSE plays a role as an important component of the diagnostic process.

cognitive performance. But error of measurement with anthropomorphic measurements of body weight, waist circumference, and central obesity is substantial, especially in younger and elderly persons (Atlantis et al., 2006). Thus more reliable computer imaging methods and dual-energy, X-ray absorptiometry methods will, hopefully, be more frequently employed in future studies. Functional and structural neuroimaging can play a very important role in studies relating potential mediating brain mechanisms (gray and white matter hyperintensities and brain atrophy) to brain areas and specific cognitive abilities and major domains.

Trials RCTs with joint weight reducing methods and cognitive outcomes are in their infancy and hopefully will increase in number in future years.

Formal Definitions of Mild Cognitive Impairment From a clinical perspective, the important new focus on MCI and subtypes of MCI represented by the work of Sperling et al. (2011) on obesity and other risk factors represents an important step backward in time from dementia and should receive more emphasis in future studies.

Prospective Designs Given that predictors of and outcomes from dementia are confounded in studies conducted after dementia has been diagnosed, the ideal designs are prospective, involve long surveillance periods prior to the onset of dementia and, where possible, include periodic tracking of relations between weight and cognition over time. In the future, more lifespan longitudinal studies are needed, or at least the emergence of a connected body of literature that extends collectively over all periods of the life-cycle. There are three important reasons why this is true: (1) bi-directional relations between obesity and cognition may begin in childhood; (2) poorer cognitive performance in non-demented individuals is itself a risk factor for dementia; and (3) the direction of the relation between obesity and cognition is yet to be determined and further research is necessary to examine theoretical models suggesting that relations are bi-directional.

Neuroimaging Studies Self-report of height and weight really is not an acceptable option for future studies of weight and

FINAL SUMMARY AND CONCLUSIONS From an empirical perspective it is clear that overweight and obesity are conditions related to lowered cognitive performance and to the dementias, and that deficits in cognition are enhanced by the presence of obesity with other risk factors, especially a constellation of correlated risk factors that we refer to as MetS. Measures of central obesity appear to be more strongly associated with dementia and cognitive deficit than BMI, and results are less conflicting when these measures are used as predictors. Visceral obesity, reflected in central obesity, is more strongly related to cognitive performance than subcutaneous obesity, but more studies with objective measurement of these weight parameters are needed in the future. Less is not better than more with respect to risk of cognitive deficit or decline related to weight gain or loss. Especially with measures of central obesity as predictors, J- and U-shaped functions between weight and cognitive ability are seen. Where studies of weight loss are undertaken, it is particularly important to have a long prospective surveillance period as weight loss may be precipitated by the dementing processes. In the first controlled studies of weight and obesity, cardiovascular risk factors were viewed as mediators of relations between weight and cognition, but a rapidly evolving literature indicates that many other mediating mechanisms are possible. The next phase of research on weight and cognition will need to involve more studies of effect moderators and mediators. This phase of research could make much better use of path analyses and structural equation modeling statistical

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REFERENCES

methods for evaluating and modeling the role of mediating variables in a systematic manner. The mechanisms underlying sex modification of relations among overweight status, weight change, obesity, and cognition are not completely understood. It is our view that variables that will explain the role of sex in relations between obesity and cognition in later life are established at an early age. Fetal development, infancy, and childhood obesity is at the other end of the life-spectrum from dementia, but understanding the path to dementia requires an understanding of the role of psychosocial variables, cognition, and obesity in childhood. This is especially important if we are to better understand possible reciprocal relations between cognition and weight. As empirical research on dementia in general has moved backward in time from dementia diagnosis, we have achieved a better understanding of the predictors of dementia. The success achieved by moving backward in time from dementia reinforces arguments for lifespan approaches to dementia that appeared as early as 2001 (Breteler, 2001; Launer, 2005) and with the emphasis on the importance of studying relations between weight and cognition and dementia beginning with fetal development (Whalley et al., 2006). In the popular motion picture, Back to the Future, a young man journeys into the past and influences, for the better, his parents’ experiences and life in the future (Wikipedia, 2011). Our goal must be to influence the future of those who are at risk of dementia due to preventable and treatable risk factors, overweight, and obesity. Among many other treatment strategies, supplementation with long-chain omega-3 PUFAs should be studied. Certainly gaining a better understanding of the role of cognitive deficits in overweight and obesity is as important as understanding the role of overweight and obesity in cognitive deficits and the search for answers here leads us back to infancy, childhood, and possibly to neonatal development.

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Fitzpatrick, A.L., Lopez, O.L., Kuller, L.H., 2006. Mid- and late life obesity: risk of dementia in the Cardiovascular Health Cognition Study. Ann. Neurol. 60, S3
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C H A P T E R

33 Dairy Products and Cognitive Functions Georgina E. Crichton and Merrill F. Elias INTRODUCTION As the populations of developed nations around the world age (United Nations, 2009), cognitive decline is becoming a major public health problem. Cognitive decline may range from minimal decline, associated with healthy aging, to mild cognitive impairment (MCI), to very severe dementia, the clinical end-point of cognitive impairment. Brain dysfunction accompanied by cognitive impairment interferes with an individual’s ability to function independently, placing considerable burden on individuals, their families, and the community. Cognitive function in later life may be further reduced in the presence of cardiovascular disease risk factors, including obesity (Elias et al., 2012b, Waldstein and Elias, 2001). An increasing number of individuals around the world will be inflicted with some degree of cognitive impairment as the prevalence of obesity and chronic disease increase, coinciding with an aging population. The main challenge to combat the devastating effects of neuropsychological disease is to deliver early and effective interventions to prevent or slow cognitive decline. Although the precise etiology of cognitive decline is not completely understood, and there is no cure, there is, however, some evidence that drug interventions may slow this condition (Waldstein and Elias, 2001). Thus the search for effective interventions is of key importance. There is a substantial body of evidence indicating that nutrition may play an important role in the causation and prevention of age-related cognitive decline and dementia (Bryan, 2004; Solfrizzi et al., 2003). While positive associations have been shown between a number of nutrients and cognitive performance, including antioxidants, folate, and omega-3 and omega-6 fatty acids, little attention has been paid to the potential role of dairy foods in modulating neuropsychological parameters. This is despite recent epidemiological,

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00033-8

observational, and intervention studies providing evidence for the role of dairy foods in improving cardiovascular risk factors and reducing the prevalence of metabolic syndrome (Azadbakht et al., 2005a; Azadbakht et al., 2005b; Liu et al., 2006; Lutsey et al., 2008; Pereira et al., 2002; Steffen et al., 2005; Zemel et al., 2009). Dairy consumption may reduce the risk of cognitive decline either directly, or via mediating effects on cardiometabolic health.

REVIEW OF THE LITERATURE Early observational research largely designed to examine general dietary habits with health outcomes has produced some interesting findings with regard to potential relationships between intakes of dairy products and cognitive status. The few, but seemingly consistent findings, from this research have led to the instigation of a number of cross-sectional studies with the specific intention of examining this potential association in large data sets. However, no longitudinal studies have been conducted. Subsequent to this observational research, one randomized controlled clinical trial has been conducted with the aim of providing more definite evidence for the potential role of dairy foods impacting upon cognition. We will first present a brief overview of the early observational literature and then focus this chapter on a discussion of those studies designed to specifically examine the dairy
cognition relationship. For a more detailed summary and methodological appraisal of the early observational studies reporting on cognitive performance in relation to dairy, we refer interested readers to a review of this literature by Crichton et al. (2010a). A summary of all studies identified and discussed in this chapter are presented in Table 33.1. This table describes the study design, cohort, location, number, and ages of participants, and the exclusion criteria

403

© 2014 Elsevier Inc. All rights reserved.

404 TABLE 33.1

33. DAIRY PRODUCTS AND COGNITIVE FUNCTIONS

Characteristics of Studies Reporting Associations Between Dairy Consumption and Cognitiona

Study

Cohort FUD (Prospective Studies)

CROSS-SECTIONAL Lee et al. (2001) Korean elderly

Country

Age, Mean or Range (y)

N

Exclusion Criteria

Adjustments

Korea

60
83

210 M, 239 F

Major cognitive impairment

Age

Avila-Funes et al. (2006)

Study on Health, Welfare and Aging

Mexico

64.4

465 M, 1283 F

Not specified

None

Rahman et al. (2007)

Survey of Alabama’s Elderly

USA

55
94

345 M, 711 F

Not specified

Age, race, gender, education, dietary factors

Crichton et al. (2010b)

Survey of South Australian adults

Australia 39
65

432 M, 751 F

Incomplete or implausible dietary data (intakes too low or high)

Age, self-rated health, medical conditions, medications, BMI, total energy intake, alcohol

Crichton et al. (2012a)

MSLS

USA

23
98

399 M, 573 F

Dementia, acute stroke, current dialysis treatment, alcohol abuse, inability to read English

Age, education, gender, smoking, alcohol, depression, waist circumference, energy intake, folic acid, plasma homocysteine, missing data

Park and Fulgoni (2013)

NHANES

USA

20
59

4355 aged 20
59, 4282 aged $ 60

Pregnancy or currently lactating, missing data, missing cognitive test

Age, gender, smoking, ethnicity, energy intake, BMI, poverty income ratio, intakes of fat, protein, carbohydrate and cholesterol

601 M

Not specified

Analyses repeated excluding subjects with AD, dementia/memory problems, depression

baseline: 50.4 549 M, 900 F follow-up: 71.3

Not specified

Age, gender, education, FUD, other fat subtypes from milk products, ApoE4 status, vascular risk factors, history of vascular disorders

Not specified

Age, sex, education

PROSPECTIVE Almeida et al. (2006)

Australian elderly Australia baseline: men FUD: 3.3
6.8 y $ 75 followup: $ 80

Laitinen et al. (2006)

CAIDE study FUD: 21 y

Finland

Yamada et al. (2003)

Adult Health Japan Study, atomic bomb survivors FUD: 25
30 y

baseline: 35
60 follow-up: 60
90

Eskelinen et al. (2008)

CAIDE Study FUD: 21 y

Finland

baseline: 50.2 506 M, 835 F follow-up: 71.1

Dementia at follow-up

Age, gender, education, FUD, other fat subtypes from milk products, ApoE4 status, midlife vascular risk factors

Vercambre et al. (2009)

Cohort from the Epidemiological Study of Women of MGEN Study FUD: 13 y

France

baseline: 62
68 follow-up: 76
82

Energy intake deemed non- plausible

Age, education, physical activity, energy intake, smoking, supplement consumption, use of postmenopausal hormones, diabetes, hypertension, hypercholesterolemia, CHD, stroke, cancer, depression

475 M, 1299 F

4809 F

(Continued)

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

EARLY CROSS-SECTIONAL AND PROSPECTIVE RESEARCH FINDINGS IN ASSOCIATIONS BETWEEN DAIRY AND COGNITION

TABLE 33.1

Study

405

(Continued) Cohort FUD (Prospective Studies)

Country

RANDOMIZED CONTROLLED TRIALS Crichton et al. Dairy Health Study (2012b)

Age, Mean or Range (y)

Australia 18
75

N

Exclusion Criteria

Adjustments

11 M, 27 F

Weight .135 kg, dairy or lactose intolerance, smoking, diabetes, CVD, liver disease, renal disease, hypertension, pregnancy, use of weight loss medications, .1 g of fish oil/day, other medication that may influence study outcomes

Cross-over study, participants acted as own controls

a Modified from Crichton et al. (2010a, Table 2, p. 355). Copyright r 2010, Karger Publishers, Basel, Switzerland. Abbreviations: AD, Alzheimer’s disease; ApoE4, Apolipoprotein E4; BMI, body mass index; CAIDE, Cardiovascular risk factors, Aging and Incidence of Dementia; CHD, coronary heart disease; CVD, cardiovascular disease; F, female; FUD, follow-up duration; M, male; MGEN, Mutuelle Generale de l’Education Nationale; MSLS, Maine-Syracuse Longitudinal Study; NHANES, National Health and Nutrition Examination Survey.

for each study. The predictor and outcome measures used are detailed in Table 33.2, and the main findings of each study are presented in Table 33.3. All 12 papers were published after the year 2000. Studies were conducted in the United States, Europe, and Australasia.

EARLY CROSS-SECTIONAL AND PROSPECTIVE RESEARCH FINDINGS IN ASSOCIATIONS BETWEEN DAIRY AND COGNITION Cross-Sectional Studies Three cross-sectional studies were initially designed to assess the factors associated with dietary patterns and depressive symptoms in the elderly (Avila-Funes et al., 2006), and dietary intake and cognitive performance (Lee et al., 2001; Rahman et al., 2007), in elderly populations. These studies showed associations between a high intake of milk (Lee et al., 2001) and cheese (Rahman et al., 2007) with better cognitive function. In addition, milk intake was also associated with fewer depressive symptoms in men and women (Avila-Funes et al., 2006).

associated with impaired cognitive function and poorer mental health (Almeida et al., 2006). The Cardiovascular risk factors, Aging, and Incidence of Dementia (CAIDE) studies were instigated to evaluate associations between dietary fat intake in midlife with risk of MCI, dementia, Alzheimer’s disease (AD), and performance on a number of cognitive domains in later life (Eskelinen et al., 2008; Laitinen et al., 2006). These studies showed that high saturated or total fat intakes from milk products were associated with an increased risk for MCI, poorer global cognitive function, and poorer psychomotor speed (Eskelinen et al., 2008), but not with risk of dementia or AD (Laitinen et al., 2006). The final propective study assessed the long-term impact of dietary habits on age-related cognitive decline among 4809 elderly women in a large French epidemiological cohort (Vercambre et al., 2009), and similarly showed that higher consumption of dairy desserts and ice-cream was associated with an increased risk for cognitive decline (Vercambre et al., 2009). No significant associations were found between milk, yogurt or cheese consumption and cognitive decline (Vercambre et al., 2009).

Prospective Studies

Summary of These Study Findings

Two prospective studies were designed to examine associations between lifestyle and cardiovascular risk factors with mental health and dementia (Almeida et al., 2006; Yamada et al., 2003). Milk intake was associated with a significantly lower likelihood of vascular dementia in older age among both men and women (Yamada et al., 2003), while the Australian study of elderly men demonstrated that whole fat milk consumption was

These observational studies provided preliminary evidence for a beneficial association between some dairy products (milk and cheese) and cognitive function. Conversely, the findings also suggested that whole fat dairy products including milk, dairy desserts, and ice-cream, as well as total or saturated fat from milk products per se, may have the opposite effect on cognitive function.

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33. DAIRY PRODUCTS AND COGNITIVE FUNCTIONS

Outcome Measures of Studies Reporting Associations between Dairy Consumption and Cognitiona Outcome Measure: Cognition Function

Author

Domain Assessed

CROSS-SECTIONAL Lee et al. (2001) Dementia screening

Definition of Tool/ Criteria Used Impairment MMSE-K-12

Predictor: Dairy Intake Measures Individual Dairy Foods Measure Used Specified

Impaired Interview: 24 cognitive function: hour diet MMSE-K # 19 recall Inadequate cognitive function: MMSE-K 19
23

No

No

No

No

No

No

Avila-Funes et al. (2006)

Dementia screening (Depression)

Abbreviated MMSE Impaired GDS-15 cognitive function: MMSE # 12 Depression: GDS15 $ 6

Rahman et al. (2007)

Dementia screening

MSQ

Impaired Home cognitive function: interview MSQ ,9

Milk, cheese

No

No

Crichton et al. (2010b)

7 measures of selfreported cognitive function & psychological wellbeing

CFQ, MFQ, SF-36, STAI-Y, PSS, CESD, RSE-B

Not defined according to test scores

FFQ

Milk, cheese, yogurt, cream, ice-cream, dairy desserts

Yes

Yes

Crichton et al. (2012a)

Verbal episodic memory, visualspatial memory and organisation, scanning and tracking, working memory, executive function, global composite

MSLS neuropsychological test battery (19 tests)

Cognitive test scores

Nutrition and Milk, cheese, No Health yogurt and Question- naire dairy desserts, cream and icecream, total dairy

For milk only

Simple reaction Percentile rank on time task cognitive test Symbol-digit scores substitution test Serial digit learning task Story recall test Digit-symbol substitution test

NHANES 24hour dietary recall

Total dairy products (milk, cheese, yogurt), milk, cheese

Yes

No

MMSE GDS-15

At baseline: self-report questionnaire, including diet measures

Full cream milk

No

Yes

Park and Fulgoni Vasomotor speed (2013) Information processing speed Immediate learning and memory recall In those $ 60 years: Attention and delayed verbal recall Processing speed and attention PROSPECTIVE Almeida et al. (2006)

At follow-up: Dementia screening Depression

‘Preserved cognitive function’: MMSE $ 24 ‘Preserved mood’: GDS # 5 ‘Successful mental health aging’: MMSE $ 24 & GDS # 5

MiniNutritional Assessment questionnaire

Serving Size Fat Content Defined Specified

(Continued)

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EARLY CROSS-SECTIONAL AND PROSPECTIVE RESEARCH FINDINGS IN ASSOCIATIONS BETWEEN DAIRY AND COGNITION

TABLE 33.2

407

(Continued) Outcome Measure: Cognition Function

Predictor: Dairy Intake Measures Individual Dairy Foods Measure Used Specified

Serving Size Fat Content Defined Specified

Author

Domain Assessed

Definition of Tool/ Criteria Used Impairment

Laitinen et al. (2006)

At follow-up: Dementia screening Clinical dementia

MMSE Clinical diagnosis: DSM-IV criteria

Clinical diagnosis of dementia or AD

At baseline: FFQ

Milk, sour milk, spreads

No

Fat from milk products used in analyses

Yamada et al. (2003)

At follow-up: Clinical dementia:

CASI IQCDE (by the caregiver) Hachinski’s Ischemic Score Clinical Dementia Rating Clinical diagnosis: DSM-IV criteria

Clinical diagnosis of dementia

At baseline: FFQ: milk

Milk

No

No

Eskelinen et al. (2008)

At follow-up: Dementia screening Clinical MCI Episodic memory Semantic memory Psychomotor speed Executive function Prospective memory

MMSE Mayo Clinic AD Research Centre criteria Word recall tests Category Fluency test Purdue Peg Board task, Letter digit substitution Stroop test Einstein task

Clinical diagnosis of MCI

At baseline: FFQ

Milk, sour milk, spreads

No

Fat from milk products used in analyses

Vercambre et al. (2009)

At follow-up: Recent cognitive decline

DECO scale (by close relative or friend)

‘Recent cognitive decline’: DECO ,33

At baseline: diet history questionnaire

Milk and No yogurt, cheese, dairy desserts & ice-cream

Significant decrease in performance on psychological testing following specific dairy diet

FFQ Personal dairy intake records

Dairy foods provided in HD phase: milk, flavored milk, yogurt, custard; own dairy food permitted including cheese

RANDOMIZED CONTROLLED TRIALS Digit-Symbol Crichton et al. Processing speed Coding (2012b) Abstract reasoning Matrix Reasoning Working memory Letter Number Verbal fluency Sequencing Spatial Selective attention Span Verbal memory Verbal Initial Letter Processing speed Nonverbal executive Fluency task Stroop Colourfunctioning Word test Visual-spatial RAVLT scanning Inspection Time Computer task Design Fluency test Letter cancellation

Yes

No

Yes; all dairy food provided was reduced fat

a Modified from Crichton et al. (2010a, Table 3, p. 356). Copyright r 2010, Karger Publishers, Basel, Switzerland. Abbreviations: AD, Alzheimer’s disease; CASI, Cognitive Ability Screening Instrument; CES-D, Center for Epidemiological Studies-Depression Scale; CFQ, cognitive failures questionnaire; DECO, Observed Cognitive Deterioration scale; DSM-IV, Diagnostic and Statistical Manual, Fourth Edition; FFQ, food frequency questionnaire; GDS, Geriatric Depression Scale; HD, high dairy; IQCDE, Informative Questionnaire on Cognitive Decline in the Elderly; MCI, mild cognitive impairment; MFQ, Memory Functioning Questionnaire; MMSE, Mini-Mental State Examination; MMSE-K, Mini-Mental State Examination for Koreans; MSLS, Maine-Syracuse Longitudinal Study; MSQ, Mental Status Questionnaire; NHANES, National Health and Nutrition Examination Survey; PSS, Perceived Stress Scale, RAVLT, Rey Auditory Verbal Learning Test; SF-36, Medical Outcomes Study 36-Item Short Form Health Survey; RSE-B, Rosenberg Self-Esteem Scale; STAIY, Spielberger State-Trait Anxiety Inventory, Form Y.

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408 TABLE 33.3

Study

33. DAIRY PRODUCTS AND COGNITIVE FUNCTIONS

Main Findings of Studies Reporting Associations between Dairy Consumption and Cognitiona Statistical Analyses

CROSS-SECTIONAL Lee et al. (2001) ANOVA

% With Impairment

Range of Dairy Intake

Cognitive function: Poor: 22.3% Inadequate 28.3%

Milk and dairy products: Women with poor cognitive absolute amount function had significantly lower intakes of milk and dairy products, P ,0.05, than those with inadequate or normal cognitive function. No significant differences for men. Not specified

Avila-Funes et al. (2006)

Linear regression

Cognitive impairment: 8.2% (Depressive symptoms: 66%)

Rahman et al. (2007)

Logistic regression

Cognitive Cheese and milk: ,1 impairment: 16.6% serving/ week or never to $ 1 serving/ week

Crichton et al. (2010b)

ANOVA

Clinical impairment not assessed

Crichton et al. (2012a)

ANOVA

Park and Fulgoni (2013)

PROSPECTIVE Almeida et al. (2006)

Main Findings with Regard to Dairy and Cognitive Function

Secondary Findings

No reported association between cognitive impairment and milk product consumption.

Consumption of milk products significantly lower in those with depressive symptoms (P , 0.01).

Higher cheese intake associated with reduced likelihood of cognitive impairment (OR 0.68, 95% CI 0.47
0.99). Milk intake not associated with cognitive impairment.

Dose
response effect; as cheese intake increased, risk of cognitive impairment decreased (P ,0.01).

Low ( . 250 g/day) to high ( . 750 g/day)

Women: reduced fat cheese intake positively associated with social functioning, negatively associated with perceived stress. Men: reduced fat yogurt intake positively associated with memory recall and social functioning.

Full fat ice-cream, cream, milk, and cheese associated with a number of poorer self-reported psychological outcomes

Clinical impairment not assessed

Never to at least one time per day

Cognitive performance increased with increasing dairy food intake for 7/8 outcome measures, controlling for cardiovascular, lifestyle, and dietary factors. Highest scores observed from those who consumed dairy foods at least daily for all outcomes.

Individual dairy products (milk, cheese, yogurt, and dairy desserts, cream, and ice-cream) not significantly associated with cognitive performance.

ANOVA

Clinical impairment not assessed

None to highest quintile of intake for total dairy: .2.38 cup equivalents for those .60 years

In adults ,60 years, total dairy product and cheese consumption associated with higher information processing speed. In adults .60 years, total dairy product consumption and cheese associated with higher short-term memory compared to non-consumers and coding speed.

Milk consumption not associated with any outcome measure.

Cox proportional hazards regression

Impaired Full cream milk: ‘regular cognitive function: consumption’ (quantity 17.6% (Impaired not specified) mood: 8.8% Poor mental health: 24%)

Consumption of full cream milk associated with impaired cognitive function (OR 0.59, 95% CI 0.37
0.94).

Consumption of full cream milk associated with poor mental health (OR 0.57, 95% CI 0.37
0.86).

(Continued) OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

EARLY CROSS-SECTIONAL AND PROSPECTIVE RESEARCH FINDINGS IN ASSOCIATIONS BETWEEN DAIRY AND COGNITION

TABLE 33.3

409

(Continued) Main Findings with Regard to Dairy and Cognitive Function

Study

Statistical Analyses

% With Impairment

Range of Dairy Intake

Laitinen et al. (2006)

Logistic regression

Clinical dementia: 4.2%

Fat intake from milk products and spreads, g

Yamada et al. (2003)

Logistic regression

Clinical dementia: 6.4%

Milk: ,4 servings/ week Almost daily milk intake to almost daily associated with significantly lower likelihood of vascular dementia, compared to consuming milk less than four times per week (OR 0.35 95% CI 0.14
0.77).

Eskelinen et al. (2008)

Logistic regression ANCOVA

Clinical MCI: 5.7% Fat intake from milk products and spreads, g

Stratified by gender: High SFA intakes from milk products and spreads ( . 21.6 g) primarily significant in women. associated with increased risk for MCI compared to those with intakes less than this (OR 2.36, 95% CI 1.17
4.74). High SFA from milk products associated with poorer global cognitive function (MMSE) (P , 0.05). High total fat from milk products & spreads associated with poorer psychomotor speed (P , 0.05).

Cognitive decline: 12.4%

Mean intakes in g/day; no consumption compared with consumption # median, and . median intake

Consumption of dairy desserts and ice-cream associated with cognitive decline (OR 1.33, 95% CI 1.07
1.65).

HD phase: 4 servings of reduced fat dairy/day; LD phase: ,1 serving of reduced fat dairy/day (habitual intake)

8% increase in spatial working No significant change in memory performance cardiometabolic parameters following 6 months of between HD and LD. consuming 4 servings of reduced fat dairy/day.

Vercambre et al. Logistic (2009) regression

RANDOMIZED CONTROLLED TRIALS Crichton et al. Paired t-test Clinical (2012b) impairment not assessed

Secondary Findings

Fat intake from milk products (milk and sour milk) not significantly associated with risk of dementia or AD. Moderate intakes of PUFA from spreads associated with decreased risk of dementia (OR 0.40, 95% CI 0.17
0.94). Moderate intakes of SFA from spreads associated with increased risk of dementia (OR 2.45, 95% CI 1.10
5.47) & AD (OR 3.82, 95% CI 1.48
9.87).

No significant associations between milk and yogurt, and cheese consumption with cognitive decline.

a Modified from Crichton et al. (2010a, Table 4, p. 357). Copyright r 2010, Karger Publishers, Basel, Switzerland. Abbreviations: AD, Alzheimer’s disease; ANOVA, analysis of variance; ANCOVA, analysis of covariance; BMI, body mass index; CHD, coronary heart disease; CI: confidence interval; FUD, follow-up duration; HD, high dairy; LD, low dairy; MCI, mild cognitive impairment; MMSE, Mini-Mental State Examination; OR: odds ratio; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids.

Focused Dairy
Cognition Studies Cross-Sectional Studies These preliminary findings were supported by a large cross-sectional study conducted in over 1000 Australian middle-aged men and women, assessing

intakes of dairy foods and self-reported cognitive function and psychological well-being (Crichton et al., 2010b). The investigators reported that reduced fat cheese intake was positively associated with social functioning, and negatively associated with perceived stress, while in men, reduced fat yogurt intake was positively

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33. DAIRY PRODUCTS AND COGNITIVE FUNCTIONS

associated with memory recall and social functioning. While the measures of dairy intake in this study were assessed using a comprehensive food frequency questionnaire (FFQ), the assessment of cognitive function was only via self-report of perceived functioning. Subsequently, two large cross-sectional studies were designed to specifically examine the potential association between dairy food intake and cognitive performance assessed by an extensive battery of cognitive tests. These were both conducted in large U.S. samples (Crichton et al., 2012a; Park and Fulgoni, 2013). Data from 972 participants (mean age 62.0 6 12.8 years) in the Maine-Syracuse Longitudinal Study (MSLS) were used to examine intakes of: milk, cheese, and yogurt; dairy desserts, ice-cream, and cream; and total dairy food intake, in relation to cognitive performance as measured by a battery of 19 tests used to create domains of cognitive ability via factor analysis and previously used in numerous studies on health and cognition (Crichton et al., 2012a). Outcome measures (cognitive domains indexed by specific tests) included verbal episodic memory, visual-spatial memory and organization, scanning and tracking, working memory, executive function, and a global composite score derived from all tests. Those excluded had either missing data, probable dementia, suffered acute stroke, were undertaking dialysis treatment, a history of alcohol abuse, or an inability to read English. In this carefully controlled study, which adjusted for cardiovascular, lifestyle, and dietary factors, participants who consumed dairy products at least daily had significantly higher scores on multiple domains of cognitive function (seven out of eight measures) compared with those who never or rarely consumed dairy foods. More recently, Park and Fulgoni (2013) utilized the large National Health and Nutrition Examination Surveys (NHANES) database to examine relationships between intakes of milk, cheese, and yogurt (and total dairy) and cognitive measures of vasomotor speed, information processing speed, and learning and memory recall. Dairy intake in relation to performance on three measures of cognitive function, in addition to a calculated measure of overall cognitive function was assessed. Analyses were conducted to compare dairy consumers with non-consumers in two separate age groups, namely adults aged under 60 years and those aged 60 and over. The most notable findings were that in those over 60 years, those who consumed dairy products (total dairy) had significantly higher scores on verbal recall than those who did not, and this finding was also significant for cheese intake. In the younger age group, across the range of consumers of dairy, there was a positive association between total dairy and cheese intakes and information processing speed. Interestingly, milk intake was not associated with any

cognitive measure. Unfortunately, the study was unable to disassociate full fat and reduced fat dairy foods, which may have impacted upon these findings. Importantly, these three studies included communitybased individuals over a broad range of adult ages (20 to 98 years) and found statistically significant associations. Previous observational research has largely included elderly subjects only. They also controlled for cardiovascular risk factors and cardiovascular disease variables, suggesting that these associations found between dairy foods and cognition may be independent of vascular factors. Additionally, cognitive performance was assessed by neuropsychological tests in the large U.S. studies (Crichton et al., 2012a; Park and Fulgoni, 2013), as compared to the majority of previous studies using indicators of probable dementia, often obtained from global screening measures (e.g., Mini-Mental State Examination, MMSE) rather than neuroimaging and case review as in the Framingham studies of dementia (Elias et al., 2000).

Randomized Controlled Trials Very few studies have involved manipulation of the intake of dairy foods in order to examine any effect on cognitive health, with only one such trial having been reported recently in the literature. In this Australian study, overweight men and women (BMI $ 25 kg/m2) with typically low intakes of dairy foods (less than one serving per day) were randomized to either a low dairy diet (where they were required to consume no more than one serving of reduced fat dairy per day), or a high dairy diet (required to consume four servings of reduced fat dairy per day). Participants were instructed on how to incorporate the additional dairy into their diet so as not to increase their overall energy intake. Each participant crossed over to the alternate diet after 6 months. Cognitive function (consisting of nine aspects, refer to Table 33.3) was assessed at baseline and at the end of each dietary phase using a standardized clinical test battery. Spatial working memory performance was marginally better following the 6-month high dairy diet compared with the low dairy diet.

DISCUSSION Limitations of this Literature There are many limitations to the current research and unanswered questions remain. The evidence to suggest that a higher consumption of dairy foods may be associated with better cognition is based on a small number of observational studies, and one

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DISCUSSION

finding of borderline significance in a single clinical trial. Therefore, definitive conclusions regarding cause and effect between dairy intake and cognition cannot be made.

Assessment of Cognitive Functioning The majority of the early cross-sectional and prospective research used a dementia screening tool, such as the MMSE, as a measure of cognitive function. This widespread use of non-specific global screening tools to measure cognition limits the scope of information available. Many of these screening tools have demographic biases in score distributions, have not been suitably validated in community samples representative of the populations evaluated, and do not assess different cognitive domains (Cullen et al., 2007). These tools also do not typically provide a wide range of scores to assess variability in relatively healthy community-dwelling samples and are not sensitive to subtle cognitive effects that may occur. The later studies, using neuropsychological tests, have enabled some discrimination between effects on different cognitive functions to be determined.

Control of Confounding Variables Many of the early studies in this area did not adequately adjust for potential confounding variables. Incomplete adjustment might falsely indicate an association. It cannot be ruled out that people who have high dairy intakes might also have other lifestyle characteristics, e.g., regular exercise habits, which have not been controlled for, and which might account for the relationship with cognitive health. As with any health research, it is often those who are more health conscious that are interested in volunteering to participate in such studies. Similarly, the presence of illness such as vascular disease may influence any relationship between diet and cognition, and needs to be taken into account in any nutritional epidemiology research. Those with poor cognitive function or dementia may have poorer dietary patterns or awareness, which could result in lower dairy intakes. Therefore, the possibility of reverse causation also cannot be ruled out. It remains unknown as to how deteriorating cognition may impact upon food selection or dietary behavior. The quality of food intake by participants also needs to be addressed. Dietary Assessment The assessment methods and reporting of actual dairy food intake varied substantially in the studies reviewed in this chapter. Some studies assessed

411

intakes of individual dairy food only, e.g., milk or cheese, while others assessed total dairy food consumption. Some did not specify the particular foods included within ‘dairy products’ or ‘milk products’, which does not allow clear conclusions to be drawn. Frequency measures of dairy food intake were generally reported in terms of times per week or day, however, the actual quantity of milk (or dairy/milk product) consumed on these occasions, as a measure in grams or milliliters, was rarely specified. Similarly, the fat content of dairy foods consumed was infrequently reported. This lack of information makes it difficult to draw conclusions regarding the optimal intake and type of dairy that may be associated with greater cognitive health, to directly compare outcomes of studies, or to determine whether the fat content of dairy is important in mediating any relationship between diet and cognition. Finally, the measurement of diet at baseline only, as done in all included prospective studies, may not accurately reflect longterm consumption patterns, as dietary habits are unlikely to remain stable. Mechanisms of Action There is a paucity of evidence surrounding the way in which nutritional intake may impact upon cognitive functioning. Even less is understood about how dairy foods may impact upon cognitive function. As a large amount of epidemiological research has linked traditional cardiovascular risk factors with cognitive disease states, via their impact upon the vascular system, dietary habits that positively impact upon these cardiovascular factors may also work to reduce cognitive decline. For example, ‘healthy fats’ such as omega-3 and omega-6 polyunsaturated fatty acids, may work to reduce inflammation and low density lipids and raise high density lipids. There is some evidence that inflammation is related to deficits in cognitive performance (Teunissen et al., 2003; Trollor et al., 2012), although the results of effects of high density lipoproteins (HDL) and total cholesterol on cognition are mixed (Muldoon et al., 2001; Schreurs, 2010). Similarly, an antioxidant-rich diet to prevent oxidative damage and preserve cognitive function has been suggested (Devore et al., 2010; Engelhart et al., 2002; Li et al., 2012) but results are still conflicting. Vascular alterations such as white matter lesions have been associated with lower performance on abilities often associated with the front lobe function, including attention, memory, executive function, cognitive flexibility, sensorimotor ability, and processing speed (Akisaki et al., 2006; Breteler et al., 1994; Rabbitt et al., 2007; Soderlund et al., 2006; Tiehuis et al., 2008; Verdelho et al., 2007; Wright et al., 2008). However, one must be cautious about these conclusions because

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33. DAIRY PRODUCTS AND COGNITIVE FUNCTIONS

the frontal lobes have neuronal connections to all areas of the brain and performance deficits, while in the executive domain, my not be related to frontal lobe lesions and each of the cognitive abilities may index more fluid intellect (Lyon and Krasnegor, 1996; Rabbitt, 1997). This generalization that many risk factors are associated with deficits in multiple cognitive domains is supported by findings that diabetic individuals perform more poorly than non-diabetic individuals on tests of attention, verbal and non-verbal memory, and processing speed (Kodl and Seaquist, 2008; Roriz-Filho et al., 2009; van den Berg et al., 2009), psychomotor speed and complex motor function (Kodl and Seaquist, 2008, Roriz-Filho et al., 2009). A question might be asked as to what cognitive domains are not associated with diabetes, hypertension, and obesity and the answer seems to be, over-learned verbal abilities (e.g. vocabulary and general information) in dementia-free individuals (Elias et al., 2012a; Elias et al., 2012b). One of the important methodological issues here is that the relatively healthy brain tends to function as a whole and thus specific tests of ability are highly correlated with each other and global functioning (Rabbitt, 1997). Most clinical tests employed in studies of health and cognition lack test purity, i.e., they measure abilities in addition to the ability they were intended to measure (Rabbitt, 1997). In our own studies of dairy and cognition we see that poorer performance on multiple cognitive outcomes and global performance are associated with a lower consumption of dairy (Crichton et al., 2012a). Dairy may reduce the risk for cognitive impairment by modifying vascular factors linked to these detrimental brain changes, particularly via weight reduction. Epidemiological studies, animal studies, and a small number of randomized trials provide evidence for an anti-obesity effect of calcium and dairy foods (Teegarden, 2005; Zemel, 2004; 2005; Zemel et al., 2009). In addition to calcium, bioactive compounds derived from whey protein in dairy may contribute to accelerating weight and fat loss (Luhovyy et al., 2007; Teegarden, 2005; Zemel, 2004; 2005). Other components of dairy may play a role in reducing metabolic risk. Dairy products are the primary dietary source of vitamin D (Teegarden and Donkin, 2009), which through its role in regulating calcium homeostasis may improve insulin sensitivity and glucose homeostasis (Teegarden and Donkin, 2009; von Hurst et al., 2010). Phosphorus and magnesium are involved in blood pressure regulation (Alonso et al., 2010; Sontia and Touyz, 2007). Magnesium may also modulate vascular function through its antioxidant and anti-inflammatory properties (Weglicki et al., 1996). There is also evidence to suggest that increasing dietary intake of magnesium may also help reduce cholesterol and triglycerides (Singh et al., 1990), and

improve diabetes control, due to its role in glucose homeostasis (Ma et al., 2006; Saris et al., 2000). These components of dairy may help to prevent or decrease the vascular alterations and structural brain changes that occur with cognitive decline. The beneficial effects of these components of dairy on vascular disease may also be mediated by an improvement in inflammatory state. Individuals with greater adiposity have an increased inflammatory state in comparison with their leaner counterparts (Festa et al., 2001; Pannacciulli et al., 2001) which has been linked with adverse cognitive function (Schmidt et al., 2002; Yaffe et al., 2004; 2003). Any decrease in inflammation (via weight loss, improved regulation of glucose and blood pressure) may subsequently reduce the risk for cognitive decline. As the aim of this chapter is not to discuss possible mechanisms in detail, we refer interested readers to a paper by Camfield et al. (2011) who have thoroughly reviewed this topic. As discussed briefly here, these researchers also hypothesize that dairy foods may reduce the risk of cognitive decline via a beneficial impact on metabolic syndrome, a collection of cardiovascular risk factors. They also suggest that dairy foods may have a more direct effect on brain health through the action of bioactive peptides, vitamin B12, calcium, and end products of fermentation in dairy.

Epidemiological and Clinical Significance The worldwide prevalence of dementia is increasing and it is predicted to affect 81.1 million people by 2040 (Ferri et al., 2005). The economic burden is severe; the cost of dementia in the U.S. is estimated to reach US $200 billion in the near future (Alzheimer’s Association, 2012). The non-financial costs, namely the pain and suffering of those afflicted and their families, are immeasurable. Perhaps any dairy-derived benefit may be achieved by a small alteration in intake, from none or rarely to an average consumption of twice daily (Crichton et al., 2012a; Park and Fulgoni, 2013). This is important from a public health perspective, as it suggests that modest and achievable dietary changes may be enough to impact positively on health. It is a relevant point to consider, with recent studies suggesting that many people in both the U.S. and Australia are not meeting recommended intakes of dairy food (Crichton et al., 2012a; 2010b). This is particularly concerning given the heavy and increasing consumption of sugar-sweetened beverages among children and young adults in these countries (Clifton et al., 2011; Han and Powell, 2013). This represents a detrimental shift in dietary habits and is of public health concern. Evidence-based dietary strategies to prevent cognitive

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REFERENCES

decline, through the adoption of a healthier diet, have the potential to have a positive impact upon chronic disease at a population level, and improve the day-today well-being of individuals.

SUMMARY This chapter has summarized the current available literature on the relation between dairy consumption and cognitive performance. Despite considerable methodological variability and limitations in this small body of research, overall the literature supports the concept that high dairy consumption may be beneficial to cognitive functioning. However, high intakes of fullfat dairy and/or dairy fats may be associated with declines in cognitive performance. Research on this topic is in its infancy and many further studies are required in order to fully understand how dairy is related to cognitive functioning.

DIRECTIONS FOR FUTURE RESEARCH The quality of evidence from future observational studies would be improved if detailed assessments of both diet and cognition at multiple time periods (well before the onset of cognitive decline) were included. Regular assessment and recording of dairy intake is needed, distinguishing full fat from reduced fat dairy, and reporting dairy intake in grams per day. Future studies should include a very thorough assessment of cognition, including tests on a range of cognitive functions. It is important that any future research involves better controls for potential confounding variables, including not only socio-demographics, but also lifestyle factors, dietary variables, and vascular health. Longitudinal studies with detailed assessment of both diet and cognition over a sufficient time period are imperative if we are to determine whether dairy consumption over meaningful and critical segments of the life span reduces the risk for cognitive decline. Studies of dairy consumption patterns and methods of obtaining dairy (breast versus bottle feeding) in infancy are important. The best evidence for determining the effects of dairy intake on cognition will come from randomized, clinical intervention studies. There is presently only one such study. Finally, it will be important to examine dairy intake in relation to whole dietary patterns and in combination with other specific nutrients, such as antioxidants and healthy fats, as the importance of synergistic relationships between nutrients is being increasingly recognized.

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C H A P T E R

34 Obesity and Chronic Low Back Pain: A Kinematic Approach Veronica Cimolin, Luca Vismara, Manuela Galli, Nicola Cau and Paolo Capodaglio INTRODUCTION The problem of obesity has become a major health concern in the Western world. In the years 2003
2004 the prevalence of obesity was reported to be 17%, which is a significant increase from the previous two decades (Ogden et al., 2006). Obesity is known to lead to a wide array of medical comorbidities, and its physical and psychological impact on the affected individual cannot be understated. These complications include an increased risk of developing musculoskeletal problems, such as arthrosic degeneration, (Anandacoomarasmamy et al., 2009; Chan and Chen, 2009) and also as a risk factor for chronic lower back pain (cLBP). Low back pain (LBP) is among the most common chronic pain conditions (Janke et al., 2007). Symptoms of cLBP are varied. Indeed, LBP is often a symptom rather than a discrete diagnosis in and of itself, and pain may be experienced in one position or many positions and may vary in intensity and interference over time (Manek and MacGregor, 2005). LBP is typically defined as chronic when lasting more than 3 months. While many patients with LBP recover quickly, LBP commonly follows a recurrent course, with exacerbations occurring over time. Risk factors for the presence and severity of LBP include both socio-demographic factors such as age (Stranjalis et al., 2004), as well as lifestyle factors, such as smoking and physical conditioning (Kaila-Kangas et al., 2003; Wright et al., 1995). Additionally, a growing body of research questions whether excess body weight is a likely risk factor for LBP. A remarkable number of studies have been published that attempt to address the nature of the relationship between LBP and overweight/obesity, and equally remarkable given these attempts is the lack of

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00034-X

conclusive evidence elucidating the link between weight and LBP (Aro and Leino, 1985; Heliovaara et al., 1991; Han et al., 1997; Leboeuf-Yde et al., 1999; Lake et al., 2000; Andersen et al., 2003). A nationwide study of 15,974 patients with common spine diagnoses found that higher BMI was associated with increased disability, more severe pain symptoms, and more comorbidities than in the non-obese spine patient population (Fanuele et al., 2002). Combined, studies such as these that report a positive association between overweight/obesity and LBP suggest a possible dose
response relationship between the two, such that increasing prevalence of LBP occurs with increasing BMI. However, other studies have found more equivocal results. Several recent reviews have attempted to address the relationship between LBP and weight. Generally, these reviews have suggested that, while a relationship between overweight/obesity and LBP may indeed exist, the current state of science lacks evidence clearly establishing a direct relationship between the two (Garzillo and Garzillo, 1994; Leboeuf-Yde, 2000). Mirtz and Greene conducted a review on the relationship between overweight/obesity and LBP and similarly concluded that while current data do not provide a clear answer for untangling the relationship between overweight/ obesity and LBP, results suggest that individuals with BMI ,30 are at minimal risk of developing LBP, those between 30 and 40 are at moderate risk, and patients with BMI .40 are at high risk (Mirtz and Greene, 2005). One reason for these controversial results could be two-fold. Firstly, perhaps the relationship between the two is much weaker than previously hypothesized. A direct causal relationship between weight and LBP may or may not exist, or may be weak, as suggested by Leboeuf-Yde et al. (1999).

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Another reason may be methodological; for example, some of the difficulty may stem from the fact that LBP is often a non-specific disease and most cases of LBP have no identifiable cause and no reliable and valid classification system. Clinically, the joint overload, muscle weakness, and sedentary lifestyle are obesity-related factors that appear to be determinants for cLBP. The aim of this chapter is to illustrate and review the applications of quantitative movement analysis in obese individuals with cLBP and to review the experience of the motion analysis laboratory at the Istituto Auxologico Italiano, IRCCS, San Giuseppe Hospital, Piancavallo (VB), Italy, which routinely applies quantitative evaluation in obese patients for identifying specific patterns and optimizing rehabilitation interventions.

QUANTITATIVE MOVEMENT ANALYSIS Three-dimensional motion analysis has turned out to be a powerful tool for quantitative assessment of movement. It possesses several important features: it is non-invasive; it allows repeated examination within a short period of time; it provides quantitative and three-dimensional data for kinematics (trajectories, velocity, accelerations, angles), kinetics (forces, joint moments, joint powers); and it provides quantitative evaluation of muscle activity (electromyography). Due to these features, quantitative evaluation of motion represents a fundamental instrument in human movement analysis. Assessment of the characteristics of a patient’s movement and of significant deviations from normality can be extremely useful in clinical settings, both in the diagnosis of diseases with implications of one of the above systems, and in the planning and monitoring of specific rehabilitative treatments. In clinical practice, the low number of reliable, valid, and objective measures to monitor treatment outcomes is one of the most crucial problems in the management of pathological subjects. Clinical evaluations, such as passive range of motion, measures of muscle tone, and even video recording, which are frequently used by clinicians, have all shown poor reproducibility between observers and sessions, even when tested under standardized conditions. From these limitations, it is evident that there is a need to introduce a method of instrumental assessment that is able to supply the clinician with quantitative three-dimensional information relating both to kinematic and kinetic aspects of motion and to the pattern of muscle activation during the motor task. For this reason this method is largely applied in clinics to quantitatively assess patients with different pathologies with movement disorders, both in children and in adults.

In the rehabilitation of patients with obesity, quantitative motion analysis has an increasing importance. Obesity has, in fact, a profound effect on disability and quality of life (Bray, 2004). As the prevalence of obesity is increasing at an alarming rate worldwide, obesityrelated disabilities will eventually become a serious threat to national health systems. The excessive amount of fat modifies the body geometry by adding passive mass to different regions and causing impairment in skeletal statics and dynamics. Excess weight is able to influence the biomechanics of several activities of daily living such as walking, standing up, bending, and other movements (Saibene and Minetti, 2003; Sibella et al., 2003; de Souza et al., 2005; Vismara et al., 2007), causing functional limitations, and possibly predisposing to injury (Wearing et al., 2006). Quantitatively investigating these capacities appears necessary in order to define the functional profile in the obese population and to then be able to plan appropriate rehabilitation interventions.

Equipment The laboratory at the Istituto Auxologico Italiano, IRCCS, San Giuseppe Hospital is used to perform quantitative analysis of movement and in particular to contemporaneously acquire kinematic data (e.g., motion trajectories), kinetic data (e.g., ground reaction forces), and data relating to muscle activation (electromyographic data). In particular, the equipment is composed of: • Six-camera optoelectronic system (Vicon, UK): its cameras (Figure 34.1(a)) measure the threedimensional coordinates of reflective markers (Figure 34.1 (b)) positioned at specific points of reference on the patient’s body. From the threedimensional coordinates of the markers it is possible to calculate angles of flex-extension, abd-adduction and intra-extrarotation of the main joints, speed, and accelerations, and thus to know in detail the kinematics of the body segments on which the markers have been positioned. • Two piezoelectric force plates (Kistler, CH): they measure the system of ground reaction forces (Figure 34.2). From the system of ground reaction forces and the kinematic parameters, which have been acquired using the optoelectronic systems, the moments and powers of the different joints can be calculated. • One 8-channel telemetric electromyographic unit (Noraxon, USA): it records, via surface electrodes, the responses of muscles to electrical stimulation, i.e., electrical signals generated by muscle contraction (Figure 34.3).

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FIGURE 34.1 Optoelectronic camera system (a); markers (b).

FIGURE 34.2 On the left, a force plate (a); on the right, the vectogram during foot contact with the force plate during walking is represented (b).

• Two-camera system for the video recording of patients: they allow the clinician to observe a patient’s motor action from a qualitative point of view.

Obesity and LBP: Our Experience Obese patients often report musculoskeletal disorders with a high incidence and prevalence of nonspecific and cLBP which may influence daily life movements such as locomotion and trunk movement. Following our experience in the use of quantitative movement analysis to assess the functional limitation of obese patients with cLBP, we describe analysis of walking (or gait analysis) and trunk movement, reporting also the preliminary results of the effects of a rehabilitative treatment (osteopathic manipulative treatment) on trunk functionality.

Gait Analysis Walking represents the most common modality of physical activity, significantly contributing to mass reduction programs for obese subjects (Hill and Peters, 1998; Jakicic et al., 2003). Walking at a constant intensity for a prolonged lapse of time is a useful and frequently used strategy for body mass reduction in obese patients because it is a convenient type of physical activity that can be used to expend a significant amount of metabolic energy (Browing et al., 2009). Therefore walking abnormalities should be taken into account to avoid overload and possible musculoskeletal problems leading to a prolonged rehabilitation phase. Knowledge of biomechanical alterations induced by LBP can help tailoring specific treatment to recover gait pattern. The effect of obesity and LBP on gait could in fact generate a vicious circle hampering the rehabilitation process. In

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34. OBESITY AND CHRONIC LOW BACK PAIN: A KINEMATIC APPROACH

FIGURE 34.3 Electromyographic unit.

addition to spine flexibility and strengthening, range of motion exercises at knee level, strengthening of distal (ankle dorsal flexor), and proximal (knee flexor) muscles should be part of the rehabilitation program in obese patients with LBP. The analysis of gait pattern in obese subjects affected by LBP has received scant attention in the literature. Some studies have been performed using different techniques (clinical assessment, accelerometers, threedimensional movement analysis) and under different experimental conditions (activities of daily living, treadmill and ground walking) (Spenkelink et al., 2002; Lamoth et al., 2006a; 2006b). Spenkelink et al. (2002) investigated differences between LBP patients and healthy subjects in daily living activities and the dayto-day variability using an ambulant monitoring system based on accelerometers. LBP patients showed lower activity levels with lower gait velocity and standing time and higher resting time than the control group. Lamoth et al. (Lamoth et al., 2006a; 2006b; 2008) examined the capacity of LBP vs. healthy subjects to adapt their gait pattern to changes in velocity during treadmill walking. Their analysis was conducted in terms of trunk and pelvis rotations and erector spinae muscular activity to evaluate the trunk-pelvic coordination. They found that LBP patients had difficulties in modulating trunk-pelvis coordination flexibility, especially on the transversal plane and erector spinae activity following velocity perturbations. In a more recent study, the same authors (Lamoth et al., 2008) examined the relationship between attention and gait by varying conditions of attentional load during treadmill walking. In line with the previous study, they confirmed the reduced motility of the upper body in LBP

patients during gait, which was accentuated during an attention demanding task. Despite being frequently associated, the effects of LBP and obesity on biomechanical gait parameters have been investigated separately. None of the previous studies have investigated the combined effects of obesity and LBP and the quantitative differences in gait strategy in obese with LBP as compared to those without LBP. To our knowledge, the only study which quantified the gait pattern of obese women with and without LBP by using three-dimensional gait analysis was conducted by Cimolin et al. (2011). They quantitatively assessed 8 obese females (BMI $ 35 Kg/m2) with chronic LBP. The patients had chronic nonspecific pain and were not under any medication at the time of the experiment. Subjects were excluded who had secondary LBP, sciatica, osteoporosis, osteoarthritis, rheumatological, metabolic or hematological abnormality with potential to affect lower limb function and neurological diseases precluding physical exercise, cardio-respiratory conditions (diagnosed after treadmill stress tests) or acute illnesses. As shown in Table 34.1, the presence of LBP induces some further alterations of the gait pattern compared to obese subjects without LBP, who already yield some distinct biomechanical features from normal-mass controls. The coexistence of obesity and LBP seems therefore to affect gait more severely than obesity alone. Obese participants with LBP were characterized by a reduced stability during gait, as assessed by prolonged stance duration and lower velocity and step length compared to controls, and a less physiological knee and ankle strategy. These features can also affect pelvic biomechanics. It can be speculated that pain relief may represent the major underlying cause of this strategy: ankle dorsal flexion and knee flexion can potentially provoke traction on the sciatic nerve, inducing subjects to cautiously limit their range of motion. One important fact, however, is that subjects affected by clinically sound sciatic pain were excluded. In terms of kinetics, the low ankle power generation during push-off phase which characterized obese individuals with LBP resulted in greater power at hip level; this means that patients did not generate much walking power from their ankle plantar flexors, and as a result, they had to obtain additional power from their hips. The abnormal spatio-temporal parameters have been associated with an underlying instability in the obese, with a greater period of double support associated with a slower gait velocity in order to guarantee the maintenance of dynamic balance. The presented results showed a different motor strategy during gait among obese with and without LBP. From a clinical point of view, our results suggest that rehabilitation programs should include specific treatments to improve gait pattern in obese patients with and

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TABLE 34.1

Spatio-Temporal and Kinematic Parameters of the Study Groups OLG

OG

CG

% stance (% gait cycle)

63.44 (1.24) 1

61.99 (1.00)

59.45 (1.45)

Double stance phase (% gait cycle)

26.54 (2.44) 1

25.01 (2.14) 1

23.60 (3.65)

Step length

0.34 (0.06) 1

0.38 (0.06) 1

0.88 (0.21)

Cadence (step/min)

113.67 (5.99)

111.29 (8.28)

111.80 (4.80)

Velocity (1/s)

0.69 (0.07) 1

0.71 (0.09) 1

0.79 (0.07)

42.99 (5.84)

42.89 (4.35)

43.52 (4.76)

15.12 (2.60) 1

18.78 (1.81) 1

10.71 (3.06)

KIC

3.27 (3.87)

4.23 (3.89)

4.06 (6.63)

KmSt

22.63 (3.94)

20.13 (4.23)

0.12 (3.82)

KMSw

52.23 (6.72) 1

58.12 (3.22)

59.01 (6.18)

55.26 (6.45) 1

58.16 (5.41)

60.28 (6.31)

AIC

23.51 (3.03)

1.28 (2.15)

1.81 (4.87)

AMSt

10.51 (1.17) 1

14.52 (2.98)

13.04 (5.16)

AmSt

214.32 (4.81) 1

217.08 (5.06) 1

211.74 (9.40)

ADP-ROM

27.95 (4.21)

30.55 (5.89)

22.72 (6.56)

AMSw

2.81 (2.54) 1

5.05 (1.85)

5.63 (4.93)

2.41 (1.73) 1

1.05 (0.54) 1

0.67 (0.37)

APmin

21.05 (0.21) 1

20.98 (0.19) 1

20.50 (0.29)

APMax

2.84 (0.64) 1

3.05 (0.61) 1

3.75 (0.86)

SPATIO-TEMPORAL PARAMETERS



Hip Joint ( ) HFE-ROM HAA-ROM 

Knee Joint ( )

KFE-ROM 

Ankle Joint ( )

Ankle Joint ( ) HPMax Ankle Joint ( )

Data are expressed as mean (standard deviation).  5 p , 0.05, OLG compared to OG; 1 5 p , 0.05, OLG and OG compared to CG. OLG: Obese with LBP Group; OG: Obese Group; CG: normal weight Control Group; ROM: Range Of Motion; PT: Pelvic Tilt; PO: Pelvic Obliquity; HIC: Hip at IC; HFE: Hip Flex-Extension; HAA: Hip Ab-Adduction; KIC: Knee at IC; KFE: Knee Flex-Extension; AIC: Ankle at IC; ADP: Ankle Dorsi-Plantarflexion; AP: Ankle Power; HP: Hip Power; IC: Initial Contact; St: Stance; Sw: Swing; M and Max: maximum value; m and min: minimum value.

without LBP. In parallel with weight loss, gait retraining and selective muscle strengthening with attention to ankle, hip and pelvis strategies appear crucial therefore to prevent possible musculoskeletal disorders, improve the gait pattern, and the ability to sustain this key aerobic activity for managing mass reduction programs.

Trunk Movement Quantification of Functional Limitation The morphology of the spine accounts for its own mechanical behavior and changes in morphology often

have consequences for biomechanical performance. As consequence, the analysis of spinal mobility can play a central role in understanding the relationship between function and clinical conditions. Obese patients often report musculoskeletal disorders with a high incidence and prevalence of non-specific and specific LBP which may influence the trunk movements. Methods for the quantitative analysis of trunk movements in vivo need to take into account non-invasiveness together with accuracy and clinical feasibility. Some techniques based on optoelectronic systems have been proposed but most of these studies are focused on specific segments (for example lumbar and/or pelvis) and

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34. OBESITY AND CHRONIC LOW BACK PAIN: A KINEMATIC APPROACH

considering different movements (during walking, posturo-locomotor tests, forward and lateral flexion) (Sibella et al., 2003; Lamoth et al., 2006a; 2006b; SchonOhlsson et al., 2005). To our knowledge, only Menegoni et al. (2008) described and quantified the functional mobility of the spine during flexion, lateral bending, and rotation in obese women. They estimated potential errors introduced by different skin movement artifacts affecting obese and normal weight participants, without detecting any marker movements from the bony landmarks during rotation and bending. Only during forward flexion did the markers located on the spine and pelvis dislocate from the bony landmarks; the errors were similar in healthy and obese participants and only the error concerning the angle relative to the lumbar segment was greater in obese. Intra-subject and inter-subject variability was found relatively limited with a standard deviation lower than 6 . From the investigation of the relationship between obesity and spinal mobility and its clinical consequences, it appears that body weight influences standing posture in the sagittal plane: obese subjects are characterized by a forward flexed trunk and anteversion of the pelvis. Obese individuals seem to compensate for the forward translation of the center of mass with an increased pelvic tilt. Body weight is also a constraint for thoracic movement, demonstrated by the angle related to kyphosis, the thoracic movement in forward flexion, and by the limited range of motion in lateral bending. In addition dorsal stiffness with normal lumbar range of motion (ROM) appears to be a feature of obese subjects. These strategies could play an important role in the onset of non-specific and even more of specific LBP, which is known to affect many obese patients. In this field, few studies demonstrate a correlation between obesity and functional impairment of the spine secondary to weakness and stiffness of the lumbar muscles, possibly leading to LBP and disability (Larsson, 2004); moreover, there is a lack of quantitative data on spinal mobility in obese subjects who already suffer from LBP. Vismara et al. (2010) investigated the relationship between obesity and LBP during flexion and lateral bending. They quantitatively assessed 37 adult females, who were divided into three groups: 13 obese patients without LBP (age: 38.3 6 8.9 years, BMI: 39.2 6 3.6 kg/m2), 13 obese patients with non-specific cLBP (age: 42.8 6 11.9 years, BMI: 41.9 6 5.3 kg/m2), and 11 healthy women with no history of musculoskeletal complaints as the control group (age: 31.9 6 8.6 years, BMI: 20.1 6 1.2 kg/m2). The cLBP patients were not under any treatment. The marker set up used in this study was by Menegoni et al. (2008) (Figure 34.4). The authors showed biomechanical differences in spinal mobility between healthy and obese subjects

FIGURE 34.4 Marker set up. Markers were placed on superior posterior iliac spines (LPSI, RPSI), on superior anterior iliac spines (LASI, RASI not visible), on spine spinous processes (S1, L3, L1, T6, T1), and on acromions (LACR, RACR).

under static and dynamic conditions, with more pronounced differences when comparing obese patients to those without LBP (Table 34.2). They suggested that obesity may modify spinal posture and function favoring the onset of LBP. Significant differences were found at lumbar and pelvic level among the different groups. They confirmed previous results (Menegoni et al., 2008) showing an increased anterior pelvic tilt while maintaining a normal lumbar lordosis under static conditions in obese subjects. The increased anterior pelvic tilt induces a greater flexion of the sacroiliac joints, and therefore a higher torque on the L5-S1 joint and discs. This possibly increases the shear forces at this level and overloads the disc, thus increasing the risk of disc degeneration (Liuke et al., 2005; Hangai et al., 2008). In line with Gilleard and Smith (2007), they observed an increased lumbar lordosis in obese patients with LBP which may well represent a pain-related strategy. Abdominal circumference and gravity may influence the lumbar lordosis and its mobility during forward flexion or lateral bending. All these factors could impair the dynamic function of some muscles, in particular the erector spinal muscles, so that their counteraction to the anterior shear forces on the spine could be jeopardized (McGill et al., 2000). During forward flexion, they observed that thoracic ROM was significantly lower in obese and significantly lower in LBP as compared to controls, while lumbar ROM remained similar among the three groups. Due to thoracic stiffness, forward flexion in obese and particularly in LBP

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423

QUANTITATIVE MOVEMENT ANALYSIS

TABLE 34.2

Parameters of the Study Groups for the Forward Flexion and Lateral Bending Movement CG

OG

OLG

START 1

1.2 (2.7)

5.0 (2.5) 1

4.0 (3.5) 1

MAX 1

119.4 (9.2)

112.1 (7.5) 1

103.9 (14.8) 1

ROM 1

118.2 (9.3)

107.1 (7.5) 1

99.8 (14.6) 1

START 1

11.2 (2.4)

20.9 (7.8) 1

23.9 (8.6) 1

MAX

72.7 (6.5)

75.2 (13.7)

77.1 (12.4)

ROM

61.4 (6.2)

54.3 (10.4)

53.2 (9.5)

START

30.2 (5.2)

32.7 (8.6)

41.0 (12.9) 1 ,

MAX

221.3 (2.6)

214.6 (5.1) 1

25.5 (8.5) 1 ,

ROM

51.5 (5.0)

47.3 (5.9)

46.5 (15.9)

START

21.7 (5.1)

27.8 (13.5)

215.3 (14.2) 1

MAX

22.8 (5.2)

19.2 (11.0)

10.9 (11.3) 1 ,

ROM

24.5 (5.6)

27.0 (12.2)

26.1 (12.2)

START

23.7 (6.4)

25.5 (4.1)

24.9 (5.9)

MAX

34.6 (8.2)

27.2 (5.5) 1

29.0 (7.4)

ROM

10.9 (7.2)

1.8 (5.4) 1

4.1 (6.4) 1



FORWARD FLEXION ( ) Forward trunk inclination (αFTI)

Anterior pelvic tilt (α1)

Angle related to lordosis (αL)

Lumbar movement (α2)

Angle related to kyphosis (αK)

Thoracic movement (α3)

START

210.2 (6.7)

29.0 (14.6)

24.9 (9.6)

MAX (,)

33.9 (5.2)

25.5 (6.6) 1

23.4 (9.2) 1

ROM (,)

44.1 (8.5)

34.5 (10.0) 1

28.2 (9.6) 1

START

20.2 (1.0)

0.7 (1.5)

0.5 (1.7)

ROM

77.8 (13.7)

80.7 (8.0)

60.7 (21.3) 1 ,

START

20.5 (1.7)

0.0 (1.6)

20.2 (2.6)

ROM

12.1 (2.6)

15.2 (4.8)

11.7 (5.6)

START

1.9 (4.6)

2.1 (3.1)

1.5 (5.5)

ROM

46.0 (7.0)

43.9 (11.3)

29.4 (11.8) 1 ,

START

21.9 (1.7)

20.9 (3.0)

21.1 (4.2)

ROM

20.1 (8.2)

26.6 (9.3)

21.3 (16.8)

START

2.2 (2.3)

0.4 (3.1)

0.1 (3.2)

ROM

42.2 (9.0)

31.3 (9.0) 1

23.0 (8.9) 1 ,

START

2.7 (2.4)

2.8 (2.6)

1.4 (5.3)

ROM

59.2 (9.7)

50.5 (11.8)

35.5 (12.9) 1 ,

Lateral Bending Lateral trunk inclination (βLTI)

Pelvic obliquity (β1)

Lumbar curve (βDC)

Lumbar movement (β2)

Thoracic curve (βPC)

Thoracic movement (β3)

Data are expressed as mean (standard deviation).  5 p , 0.05, OLG compared to OG; 1 5 p , 0.05, OLG and OG compared to CG. OLG: Obese with LBP Group; OG: Obese Group; CG: normal weight Control Group. Trunk, pelvis, lumbar and thoracic positive values were indicative of forward flexion of the considered segment, negative values otherwise.

subjects appears to be performed mainly by the lumbar spine, which is most frequently involved in pain syndromes. Thoracic stiffness with normal lumbar ROM appears to be a feature of obesity and it appears

plausible that it might play a role in the onset of LBP in obese patients. According to these results, lateral bending is performed in a qualitatively different modality when LBP is present and it appears the most

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424

34. OBESITY AND CHRONIC LOW BACK PAIN: A KINEMATIC APPROACH

meaningful clinical test for detecting lower spinal impairments and monitoring functional consequences of obesity. Quantification of the Effects of Osteopathic Manipulative Treatment As concerns the reduction of pain in cLBP patients, manual therapies have rapidly become some of the most fashionable and diffuse approaches to cLBP (Moffett et al., 1999). These are considered to be particularly effective in the acute and sub-acute phase (Licciardone, 2004; Fritz et al., 2007). Osteopathic Manipulative Treatment (OMT), a conservative treatment using only manipulative techniques, is commonly used by osteopaths to integrate more conventional rehabilitation treatment for LBP. While a solid body of literature is available for the effectiveness of specific exercises in cLBP (Deyo and Tsui-Wu, 1987; Moffett et al., 1999; Heymans et al., 2004; Negrini et al., 2006; Fritz et al., 2007; Hough et al., 2007; McCarthy et al., 2007), no specific protocol has been developed to treat obese patients with cLBP. Obese patients with cLBP can be considered a specific LBP subgroup having particular features which may require a customized approach. In particular, the thoracic ROM limitation may play a role in the onset or maintenance of cLBP (Gilleard and Smith, 2007; Vismara et al., 2010) as the thoracic spine function has a relevant impact on

the functioning of the lower (lumbar) and upper (cervical) traits (Cleland et al., 2005). Thoracic spinal manipulation has been shown to be effective in reducing neck pain (Cleland et al., 2005; Lau et al., 2011), thus supporting the mechanical connection between cervical and thoracic spine (Cleland et al., 2005). The improvement of thoracic ROM of obese cLBP patients could therefore represent a key goal in rehabilitation and prevention of LBP. To verify this hypothesis, Vismara et al. (2012) designed a randomized controlled study to compare the biomechanical results of two rehabilitative interventions, OMT combined with Specific Exercise (SE) versus SE alone, in obese patients affected by cLBP. As for the SE group (specific exercises only) the protocol consisted of a combined back school (Heymans et al., 2004) and cognitive behavioral approach (Henschke et al., 2010) aimed at reinforcing and stretching the abdominal and back muscles, mobilizing the spine, and providing patients with the correct ergonomic knowledge for the safe use of the spine. All patients underwent ten 45-minute SE sessions. As for the OMT 1 SE group, in addition to specific exercises previously described, patients underwent an additional 45-minute individual session provided by an experienced and skilled osteopath. The OMT was targeted at the patient’s clinical picture. The techniques used were high-velocity low-amplitude thrust in

TABLE 34.3 Parameters Related to Forward Flexion Angles and Clinical Scales for the Two Considered Groups in PRE and POST Session Group SE 1 OMT Biomechanical Parameters

Group SE

PRE

POST

PRE

POST

START

7.21 (3.54)

6.39 (4.37)

6.32 (4.55)

4.76 (3.11)

ROM

101.74 (14.48)

104.69 (11.98)

92.02 (12.58)

92.14 (10.37)

START

21.33 (3.72)

20.30 (4.76)

22.19 (6.21)

23.85 (6.49)

ROM

52.11 (12.74)

55.23 (9.73)

48.03 (11.44)

49.17 (11.58)

START

28.53 (7.77)

26.65 (9.39)

213.96 (13.34)

216.75 (10.41)

ROM

27.86 (4.43)

24.80 (7.16)

23.44 (9.74)

22.68 (8.74)

START

26.35 (7.79)

210.45 (9.76)

22.29 (13.96)

23.43 (13.22)

ROM

30.24 (5.88)

35.99 (7.76)

29.25 (10.85)

28.99 (8.39)

VAS

55.00 (6.46)

14.12 (11.52)

54.36 (8.02)

29.64 (8.13)

RM

9.38 (2.13)

3.13 (2.85)

9.64 (2.54)

7.27 (2.19)

OQ

9.63 (2.45)

3.5 (1.69)

13.00 (4.47)

9.18 (3.34)



αFTI [ ]



α1 [ ]



α2 [ ]



α3 [ ]

Clinical scales

Data are expressed as mean (standard deviation). Trunk, pelvis, lumbar and thoracic positive values were indicative of forward flexion of the considered segment, negative values otherwise.  5 p , 0.05, PRE vs. POST session. Abbreviations: SE: Specific Exercises; OMT: Osteopathic Manipulative Treatment; forward trunk inclination (αFTI), anterior pelvic tilt (α1), lumbar movement (α2), and thoracic movement (α3); RM: Roland Morris Disability Questionnaire; OQ: Oswestry Low Back Pain Disability Questionnaire.

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

REFERENCES

thoracic spine (Downie et al., 2010); cranial techniques (Sutherland, 1939; Magoun, 1966; Kostopoulos and Keramidas, 1992); and myofascial release (Fryer et al., 2009). The application of such techniques was based on the methodological and conceptual theory of osteopathic dysfunction, i.e., ‘damaged or altered function of the somatic components: skeletal structure, joint, myofascial in relation to the vascular lymphatic and nervous system’ (Centers for Medicare and Medicaid Services, 2009). Each of the participants was quantitatively evaluated using a marker set up by Menegoni et al. (2008) before and after treatment during forward trunk flexion and the secondary outcome measures were clinical scales for LBP: Visual Analogue Scale (VAS 0-100) for measuring pain (Huskisson, 1974); the Roland and Morris Disability Questionnaire (RM) (Roland and Morris, 1983); and the Oswestry Low Back Pain Disability Questionnaire (OQ) (Fairbank et al., 1980) for the assessment of disability. The obtained data, displayed in Table 34.3, evidenced that significant effects on kinematics were reported only for OMT 1 SE with an improvement in thoracic ROM of nearly 20%. All scores of the clinical scales used improved significantly. The greatest improvements occurred in the OMT 1 SE group. Data results showed that combined rehabilitation treatment (OMT 1 SE) showed to be effective in improving biomechanical parameters of the thoracic spine in obese patients with cLBP. Such results are to be attributed to OMT, since they were not evident in the SE group. A reduction of disability and pain was observed, too. An approach combining specific exercises and OMT was found to be effective in reducing pain and disability in cLBP obese patients, similarly to SE alone, but unlike SE alone it was also associated with a significant improvement in kinematics of the thoracic spine flexion. ROM improvements seem to be specifically due to OMT, since the group treated with SE alone did not report the same results. OMT seems to provide an additional benefit when integrated within a multidisciplinary approach which includes active specific exercises.

425

analysis of static and dynamic posture, and they are also able to quantify the degree of improvement of a rehabilitative treatment. This is a crucial aspect in research into rehabilitation and related healthcare costs. However, although there is increasing evidence that obesity and cLBP affect lower extremity joint kinematics, kinetics, and trunk movements, these data must be viewed with a clear understanding of the challenges associated with quantifying motions of a skeletal system covered by extensive soft tissue. Almost all studies reporting lower extremity kinematics/kinetics in obese individuals during gait have used a standard gait marker set (Davis et al., 1991) which could be influenced by inaccurate marker placement and soft tissue artifact (Della Croce et al., 2005; Baker, 2006). Of particular concern especially in obese subjects is the reliance on Anterior Superior Iliac Spine (ASIS) and/or greater trochanter markers to establish the pelvis and hip joint centers leading to inaccurate estimates of joint centers and errors in the resultant kinematics/kinetics (hip and knee in particular) (Stagni et al., 2000). However, we know that some parameters are not so much affected by these errors (i.e. ROMs) (Kirtley, 2002) and so they could be considered reliable measures in these patients. As for trunk kinematics the proposed experimental set up can represent a non-invasive clinically useful technique for functional investigation in various spinal conditions and evaluation of effectiveness in rehabilitation. Potential biases of spinal motion measurement with surface markers are soft tissue artifacts which can affect the measure especially during forward flexion. However, these artifacts seemed to produce a similar systematic error between the obese and healthy. A limit of this approach is that it is unable to estimate the error introduced by the thickness of the skin, a layer interposed between the marker and the bone, which hinders the correct localization of the bony landmarks. Although there are limitations, the use of motion analysis appears necessary to define the functional profile in the obese population in presence of cLBP, to have indications for planning appropriate rehabilitation interventions and to quantify the effects of specific rehabilitative programs.

CONCLUSIONS In this chapter an overview was presented of the role of three-dimensional motion analysis to quantify functional tasks useful to assess the functional limitation of obese subjects with cLBP from a biomechanical point of view. These studies show how motion analysis could represent a useful tool to quantify the performance of these subjects giving crucial information that clinical evaluations and video recordings are not able to provide. The examination results are important for the

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FURTHER READING

McCarthy, C.J., Gittins, M., Roberts, C., Oldham, J.A., 2007. The reliability of the clinical tests and questions recommended in international guidelines for low back pain. Spine. 32 (8), 921
926. McGill, S.M., Hughson, R.L., Parks, K., 2000. Changes in lumbar lordosis modify the role of the extensor muscles. Clin. Biomech. 15 (10), 777
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1360. Sutherland W.G.1939. The Cranial Bowl. Mankato, Minnesota, reprinted 1994. Vismara, L., Romei, M., Galli, M., Montesano, A., Baccalaro, G., Crivellini, M., et al., 2007. Clinical implications of gait analysis in the rehabilitation of adult patients with ‘Prader-Willi’ Syndrome: a cross-sectional comparative study (‘Prader-Willi’ syndrome vs matched obese patients and healthy subjects). J. Neuroeng. Rehabil. 10 (4), 14. Vismara, L., Menegoni, F., Zaina, F., Galli, M., Negrini, S., Capodaglio, P., 2010. Effect of obesity and low back pain on spinal mobility: a cross sectional study in women. J. Neuroeng. Rehabil. 7, 3. Vismara, L., Cimolin, V., Menegoni, F., Zaina, F., Galli, M., Negrini, S., et al., 2012. Osteopathic manipulative treatment in obese patients with chronic low back pain: a pilot study. Man. Ther. 17 (5), 451
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C H A P T E R

35 Fatty Acids and the Hippocampus Heather M. Francis and Richard Stevenson INTRODUCTION

FATTY ACIDS AND MEMORY/HIPPOCAMPUS

The typical Western diet is high in saturated fatty acids (SFAs) and has reduced intake of polyunsaturated fatty acids (PUFAs). It is becoming recognized that the poor dietary practices typical of the modern world may have important consequences for cognition, and perhaps especially learning and memory, and may be a risk factor for development of neurodegenerative diseases such as Alzheimer’s disease (AD). Our focus here is on long-term memory function and its impairment, which in turn is closely tied to the integrity of the hippocampus. In particular, we explore two claims in this chapter. First, that higher levels of dietary SFAs can adversely impact learning and memory function by impairing synaptic plasticity, elevating oxidative stress, and causing neuroinflammation, which in the longer term may then contribute to the onset of AD. Second, that diets rich in omega-3 PUFAs such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may be able to remediate some of the harm caused by SFA consumption, as well as having a neuroprotective effect, particularly in respect of AD. In structuring this review we have tried to draw as generally as we can on the relevant animal and human literature. Because this is an emerging literature, especially in humans, we have chosen to include all of the studies that utilize measures likely to be sensitive to changes in hippocampal function. We have arranged the review into two basic sections, the first addressing the evidence for the claim that SFAs can impair memory function, and that omega-3 fatty acids may have a neuroprotective effect. The second section then addresses whether SFA and omega-3 fatty acid intake can affect the development of AD.

Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00035-1

This section explores whether the hippocampus, and hippocampal-dependent learning and memory, in both animals and humans, are affected by consumption of SFAs and omega-3 fatty acids. In addition, we also examine how these effects may be instantiated in the brain.

SFAs, Memory, and the Hippocampus
Evidence from Animal Models There is a substantial amount of animal research demonstrating an effect of high levels of dietary SFAs on performance in tests of hippocampal-dependent learning and memory function. In one of the earliest studies in this field, Greenwood and Winocur (1990) fed rats a diet consisting of 20% lard as a source of SFA. This was designed to represent the upper limits of typical Western consumption patterns. Rats maintained on this diet for 3 months were impaired on a series of learning and memory related tasks: Radial Arm Maze, Hebb-Williams Maze, and a variable interval delayed alternation task. Spatial memory tasks are a widely accepted model for assessing hippocampal dysfunction in rodents, with the most common being the Morris Water Maze (MWM). The MWM involves placing the rodent in a pool of opaque water that contains an escape platform hidden beneath the surface. Over several trials, the animal should learn and remember the location of the platform based on spatial cues. The task was originally developed by Morris (1981) who showed that memory for the platform location was impaired in rats

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with hippocampal lesions (Morris et al., 1982). Performance on this task has been compared after rats have been maintained on diets that are high in SFA or low fat standard chow. These studies typically use lard, corn oil, and vegetable oil as a source of saturated fat. Impairment on the MWM task has been reported following 2 months’ maintenance on a diet with 39% lard and corn oil as a source of SFA (Wu et al., 2004b) and after 4 months on a 60% lard diet (Morrison et al., 2010). MWM performance has been compared between 8-week-old rats that were maintained on a diet with 16% lard compared to standard chow, and whose mothers had been fed the same diet. Rats consuming the high SFA diet were poorer on the MWM task than rats consuming standard chow (Yu et al., 2010). Dietinduced obese mice that were maintained on a 10% fat diet from 8 weeks of age and were classified as obese when they reached 30% higher weight than the average control mouse, took significantly longer to find the platform in the MWM task than controls. In contrast, Jurdak et al. (2008) found no effect of 5 weeks consumption of a 55% fat diet on MWM performance. These discrepant results may be due to differences in diet, as the fat source in the latter experiment was hydrogenated vegetable oil, whereas the previously described studies used lard and corn oil. There are also a number of studies that demonstrate hippocampal-dependent memory impairment following maintenance on diets that are high in SFA but also high in refined sugars (Goldbart et al., 2006; Molteni et al., 2002; Stranahan et al., 2008). While these studies are more representative of the typical Western diet, it is difficult to attribute the effects to the separate different dietary components. Nevertheless, these results do lend weight to the general finding that diets high in SFAs have a detrimental impact on memory functioning. Neurological Impact of Saturated Fat Consumption There are several neurological changes that have been shown to occur as a result of high SFA consumption and a number of studies have linked these neurological changes to the dietary-induced memory impairments described in the previous section. Such changes include reductions in brain-derived neurotrophic factor (BDNF), oxidative stress, neuroinflammation, and impaired blood
brain barrier (BBB) integrity. BDNF

Learning and memory require synaptic plasticity: activity-dependent changes in the strength of synaptic connections. BDNF is a neurotrophin that is abundant in the hippocampus and cerebral cortex (Leibrock et al., 1989) and is a major player in the molecular mechanisms underlying synaptic plasticity (Lu and

Gottschalk, 2000). In particular, BDNF has been shown to facilitate synaptic transmission (Tyler and PozzoMiller, 2001) and is essential for long-term potentiation (Linnarsson et al., 1997). Animals with deletion in one copy of the BDNF gene show impaired performance on the MWM (Linnarsson et al., 1997). Additionally, the BDNF val66met polymorphism, which affects the release of BDNF, results in impaired fear extinction learning (Soliman et al., 2010) and episodic memory function (Egan et al., 2003), and reduces hippocampal activity during memory encoding and retrieval processes (Hariri et al., 2003). There are several studies that link diet-induced impaired memory function to reductions in BDNF in the hippocampus. Molteni et al. (2002) showed that rats maintained on a high SFA and refined sugar diet for 2 years had decreased levels of BDNF mRNA in the hippocampus after 2 months, and levels decreased further, with the lowest levels of BDNF mRNA observed after 2 years. Importantly, the results of MWM testing performed at 2 months significantly correlated with the hippocampal BDNF levels. Stranahan et al. (2008) found that rats maintained on a high SFA and refined sugar diet for 8 months had reduced concentrations of hippocampal BDNF, which was accompanied by impaired MWM learning, as well as reduced long-term potentiation and dendritic spine density. This research provides further support for the notion that diet-induced impairment of hippocampal function is due to BDNF-mediated effects on synaptic plasticity. In another study, decreased expression of BDNF and its receptor TrkB in diet-induced obese mice was normalized when mice were placed on a low fat or energy-restricted diet (Yu et al., 2009). Notably, in the above studies the effects of SFA compared to refined sugars cannot be separated. However, Wu et al. (2004b) demonstrated an effect of 2 months’ maintenance on a diet differing only in regards to higher SFA content. Rats on the high SFA diet showed impaired learning on MWM and this was accompanied by reductions of hippocampal BDNF, as well as its downstream effectors synapsin I and cyclic AMP-response element-binding protein (CREB). BDNF-dependent phosphorylation of these proteins can affect synaptic plasticity through promotion of synaptic vesicle exocytosis (Jovanovic et al., 2000), the establishment and maintenance of functional synapses (Melloni et al., 1994), and gene expression (Finkbeiner, 2000; Ying et al., 2002). OXIDATIVE STRESS

Oxidative phosphorylation is the process through which energy, in the form of adenosine triphosphate (ATP), is generated (Wallace, 1992). While oxidative phosphorylation is essential for energy metabolism, it

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produces reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (Wallace, 1992), which can interact with, and cause damage to, many cellular components including DNA, proteins, and lipids (Bayir, 2005). Typically, antioxidants such as superoxide dismutase, vitamin E, vitamin C, and glutathione reduce the levels of ROS to prevent free radical mediated damage (Bayir, 2005). An imbalance between levels of ROS and the rate at which the damage caused by ROS can be removed or repaired causes oxidative stress. Several studies have observed increased oxidative stress following high SFA diets. Rats fed a diet comprised of 18% lard as a source of SFA for a period of 5 months showed increased levels of ROS in the cerebral cortex (Zhang et al., 2005). NADPH oxidase is one of the predominant sources of ROS and the above study found high dietary SFA resulted in increased cerebral cortex levels of NADPH oxidase subunits. This suggests high levels of SFA might cause oxidative stress via the NADPH pathway. In support of this, endothelial cell dysfunction in obese Zucker rats resulted from oxidative stress due to overactivation of NADPH oxidase (Chinen et al., 2007). Diet-induced oxidative stress has also been associated with cognitive impairment. For example, markers of oxidative stress were increased in rats consuming a high fat diet and accompanied by impaired MWM performance, with this effect exacerbated in offspring of dams who had also consumed a high fat diet (White et al., 2009). Wu et al. (2004b) showed that consumption of a high SFA diet for 2 months caused an increase in two markers of oxidative stress, oxidized protein levels and nucleic acids, in the rat hippocampus. SFA-fed rats were impaired on the MWM task and a strong negative relationship (r 5 20.84) was observed between oxidized protein levels and MWM performance, suggesting oxidative stress contributed to cognitive impairment. Interestingly, oxidative stress in this study was associated with decreased levels of BDNF, synapsin I, and CREB in the hippocampus. The authors therefore suggested that oxidative stress mediates the effects of diet on cognition by reducing levels of BDNF and its downstream effectors, subsequently impairing synaptic plasticity and cognitive function. NEUROINFLAMMATION

The neurophysiological mechanisms underlying learning and memory, such as long-term potentiation, are susceptible to the effects of neuroinflammation (Bellinger et al., 1995; Jankowsky and Patterson, 1999). Obesity has been associated with chronic inflammation in the brain, with studies finding elevated levels of inflammatory markers in obese compared to lean individuals (Cazettes et al., 2011; Vachharajani and Granger, 2009). There is evidence from animal studies

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that consumption of dietary SFA plays a role in driving this difference. For example, mice consuming a high SFA diet showed microglial reactivity and elevated levels of cytokines (markers of inflammation), which were associated with reduced BDNF and impaired cognitive performance on the T-stone maze task (Pistell et al., 2010). High SFA-fed rats that were mothered by high SFA-fed dams showed increases in inflammatory signaling in the brain (White et al., 2009). Rats maintained on a high SFA diet for 5 months had increased cortical levels of markers of inflammation, and this was associated with impaired performance on the MWM. Importantly, the two aforementioned studies were also mentioned above as having observed increased oxidative stress. Thus, these two factors are likely to work together as mechanisms underlying the effects of a high SFA diet on learning and memory (Zhang et al., 2005).

SFAs, Memory, and the Hippocampus
Human Data Several studies in a human population have also provided evidence that high saturated fat intake is implicated in hippocampal impairment. These studies show impairment on tasks that assess long-term memory function and are therefore assumed to reflect hippocampal functioning. However, these studies also tended to assess functioning of other cognitive domains. While these are referred to where appropriate, they are not the focus of this chapter. Devore et al. (2009) found that in women with a mean age of 74 years and with type 2 diabetes, higher intake of saturated fat since midlife was associated with significantly worse performance on cognitive tests measuring immediate and delayed memory, working memory, and verbal fluency. In a population of men and women aged 45
70 years old, higher rates of saturated fat intake were associated with a trend towards greater impairment of memory (Kalmijn et al., 2004). In a longitudinal study, high saturated fat intake over a period of 6 years was associated with poorer performance on delayed memory tasks, as well as other aspects of cognitive functioning (Morris et al., 2004). Importantly, this relationship was present after adjusting for age, sex, race, education, and antioxidant intake. A study of women aged over 45 found that verbal memory performance assessed over the course of 4 years was associated with higher saturated fat intake, but not total fat or PUFA intake (Okereke et al., 2012). These results suggest that the effects on hippocampal-dependent memory functioning are specific to saturated fat and not fat intake as a whole. However, a general weakness in the human study literature is a difficulty in separating

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the effects of specific nutrients given that individuals who consume a high saturated fat diet will also tend to have an overall greater fat intake, as well as a greater sugar intake. Indeed, Francis and Stevenson (2011) showed that hippocampal-dependent memory function was impaired in individuals who self-reported high intake of saturated fat and sugar compared to individuals who self-reported low intake of these nutrients. Nevertheless, these human studies support the animal literature where there is greater ability to control dietary factors such as specific nutrient intake.

Omega-3 Fatty Acids, Memory, and the Hippocampus
Evidence from Animal Studies Omega-3 fatty acids are prominent in neural tissue and have profound influences in the central nervous system (CNS) (Chalon et al., 1998; Kaplan and Greenwood, 1998). For instance, DHA is the predominant structural omega-3 PUFA in the brain and is an important component of the phospholipid bilayer that surrounds all neurons (Salem et al., 2001). DHA is essential for normal CNS development and is vital to brain structure and function (Gomez-Pinilla, 2008). EPA is detected as a trace component in the brain and is used to synthesize DHA (Moore et al., 1991). The brain is unable to efficiently synthesize these PUFAs by itself, therefore, they must be obtained through dietary sources (Sinclair, 1975). Fish oil is a rich source of DHA and EPA and its use as a dietary supplement has therefore been the focus of much research in this field. Other good sources of omega-3 fatty acids include krill oil, nuts (notably walnuts), and plant seeds and oils such as perilla and flaxseed (Asif, 2011; Tou et al., 2007; Willis et al., 2009; Winther et al., 2011). There is a wealth of evidence that omega-3 enriched diets can enhance learning and memory function. Rats fed a diet with 1.25% DHA showed improved MWM performance compared to controls after only 7 days on the diet (Chytrova et al., 2010). Similarly, mice treated with 50 and 100 mg/kg/day for 7 weeks had significantly improved spatial memory on the passageway water maze test (Jiang et al., 2009). Improved spatial memory performance was also demonstrated in rats administered a combination of fish oil and soybean oil for 10 weeks and in rats fed diets rich in alternative sources of dietary omega-3 fatty acids such as walnuts (Haider et al., 2011) and perilla oil (Lee et al., 2012). Thus, diets with high levels of omega-3 fatty acids from a variety of sources that are particularly rich in DHA and EPA have consistently been shown to be beneficial to hippocampal-dependent memory function. The role of omega-3 fatty acids in learning and memory has been demonstrated through various

studies showing changes to performance on spatial memory tasks following manipulation of dietary omega-3 availability. Methods of inducing dietary DHA deficiency typically involve removing all omega3 fatty acids from the diet throughout gestation and lactation so that the offspring then has lower levels of DHA. This can be conducted over several generations. Employing this approach, Moriguchi et al. (2000) reduced DHA by over 80% in second and third generation DHA-deprived rats and that deficiency was accompanied by impairment in learning and memory performance on the MWM task. Findings of impaired memory performance due to DHA deficiency have been consistently replicated in subsequent studies in the same laboratory (Catalan et al., 2002; Lim et al., 2005a; Lim et al., 2005b). Adding weight to the argument that it is the deprivation of DHA specifically that is responsible for the observed effects, substitution of DHA with omega-6 PUFAs resulted in impairment of MWM performance (Lim et al., 2005b). Further, the effect of omega-3 fatty acid deficiency on MWM performance can be reversed by provision of an adequate diet containing DHA (Fedorova and Salem, 2006; Moriguchi and Salem, 2003). Given the evidence for a role of omega-3 fatty acids in hippocampal learning and memory, omega-3 supplementation has therapeutic potential in remediating memory deficits induced by various conditions. The conditions that will be the focus of this chapter are associated with neuroinflammation, synaptic plasticity, and oxidative stress. DHA and EPA have anti-inflammatory properties that have been extensively documented in non-neural tissues and more recently in neural tissues (Calder, 2007). In the hippocampus specifically, decrease in dietary DHA has been shown to cause increased production of the proinflammatory cytokine interleukin-6 (Mingam et al., 2008). Therefore, DHA and EPA may be relevant candidates for reversing impairment in learning and memory due to conditions associated with neuroinflammation. For instance, age-related impairments in learning and memory are associated with neuroinflammatory processes in the hippocampus (Kadish et al., 2009). Labrousse et al. (2012) demonstrated that 2 months’ supplementation with an omega-3 PUFA diet resulted in increased levels of DHA and EPA in the brain, as well as reduction in hippocampal levels of the proinflammatory cytokines interleukin-6 and tumor necrosis factor α. These hippocampal changes were accompanied by remediation of age-related deficits in spatial memory on the Y maze task. Research suggests that omega-3 fatty acids, particularly DHA, are implicated in synaptic plasticity: changes in the strength of synaptic connections that are necessary for memory formation and storage.

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Of particular relevance to this chapter is the role of omega-3 fatty acids on synaptic plasticity in the hippocampus specifically. Dietary supplementation with DHA causes enhanced processes related to synaptic plasticity in the hippocampus, such as increased glutamatergic synaptic activity, synaptogenesis, and expression of proteins known to be involved in synaptic plasticity (Cao et al., 2009; Chytrova et al., 2010; Lee et al., 2012). Similarly, supplementation with EPA improved long-term potentiation in the hippocampus (Kawashima et al., 2010). There is also evidence for involvement of omega-3 fatty acids in regulation of synaptic plasticity-related genes such as BDNF and CREB. For instance, omega-3 deficiency reduced the levels of BDNF, BDNF signaling through its TrkB receptor, and activation of its downstream effector CREB (Bhatia et al., 2011). Conversely, DHA supplementation and krill oil supplementation have both been shown to increase expression of BDNF (Jiang et al., 2009; Wibrand et al., 2013). In light of these findings, it has been suggested that omega-3 fatty acids have potential as strategies for preventing memory impairments related to disorders that reduce synaptic plasticity in the hippocampus. Indeed, fish oil supplementation ameliorated spatial learning deficits induced by diabetes by increasing neuronal excitability (Yang et al., 2012). Fish oil supplementation also normalized levels of BDNF, synapsin I, and cAMP response element-binding protein (CREB) following TBI in rats, and counteracted learning deficits on the MWM task (Wu et al., 2004a). Agrawal and GomezPinilla (2012) showed that high fructose consumption impacted on synaptic plasticity by decreasing phosphorylation of CREB, synapsin I, and synaptophysin levels. All of these parameters were attenuated by the presence of dietary omega-3 fatty acid. The antioxidant properties of omega-3 fatty acids render them an effective means of neuroprotection against the neuronal damage caused by ROS. For instance, fish oil supplementation has been shown to protect against oxidative stress and mitochondrial dysfunction induced by rotenone and 3-nitropropionic acid (Denny Joseph and Muralidhara, 2013; Denny Joseph and Muralidhara, 2012). In the hippocampus specifically, fish oil supplementation reduced oxidative stress and counteracted learning impairment in a rat model of traumatic brain injury (Wu et al., 2004a). Oxidative stress is also characteristic of aging and is associated with a deficit in cognitive function (O’Donnell et al., 2000). Dietary omega-3 fatty acid supplementation has the ability to modulate the agerelated increase in oxidative changes in the hippocampus, as well as the associated decrease in spatial learning (Kelly et al., 2011). In conclusion, there is evidence that omega-3 fatty acids are involved in hippocampal-dependent learning

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and memory function and that they are effective in remediating deficits caused by TBI, aging, high fructose intake, and diabetes. In light of the fact that the underlying mechanisms for their ability to remediate cognitive symptoms (i.e. neuroinflammation, synaptic plasticity, and oxidative stress) are the same mechanisms underlying deleterious effects of high SFA diets on learning and memory (see the section entitled Fatty acids and memory/hippocampus), it is possible that omega-3 fatty acids could have a therapeutic use in remediating damage to the hippocampus caused by SFAs.

Can Omega-3 Fatty Acids Remediate Damage to the Hippocampus in Humans? There is no direct evidence in humans at present that omega-3 fatty acids can prevent or remediate damage to the hippocampus caused by SFAs or other factors. Nonetheless, there are currently two lines of indirect evidence, both of which suggest a possible neuroprotective effect of omega-3 fatty acids on this structure. Omega-3 Fatty Acids and Depression Abnormalities of the hippocampus accompany depression. Several studies have found that a smaller hippocampus is associated with depression, although there is uncertainty as to whether this may precede the onset of depressive symptoms or may be a consequence of depression (e.g., den Heijer et al., 2011). Whatever the case, there is good evidence that anti-depressant medications act to increase neurogenesis in the hippocampus and that this happens over a time course that seems to parallel remission of symptoms in patients taking these drugs (Santarelli et al., 2003). Together, this would suggest that any agent that acts to reduce the likelihood of depression or to act in a curative manner, is likely to be affecting the hippocampus. Dietary omega-3 fatty acid consumption can be measured indirectly by assessing oily fish consumption using food frequency questionnaires or by measuring plasma levels of DHA, EPA, α-linolenic acid (ALA), and total omega-3 fatty acids. Using a food frequency approach has provided only weak evidence that a diet rich in omega-3 fatty acids is protective against depression (Sanhueza et al., 2012). More support comes from studies using plasma estimates. Conklin et al (2007a) found that plasma omega-3 fatty acid levels in otherwise healthy hypercholesterolemic individuals were associated with scores on a standardized depression inventory and on related measures of affect regulation. In particular, higher plasma levels of EPA, DHA, and overall omega-3 fatty acids were associated with better mood regulation. In a further study, Conklin et al (2007b) found that higher EPA levels

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were associated with better affect regulation in healthy non-hypercholesterolemic individuals. The significance of these observations in healthy people is that plasma levels of omega-3 fatty acids are depleted in people in the acute phase of a major depressive episode (Tiemeier et al., 2003), suggesting that low levels of omega-3 fatty acids may be a risk factor for the onset of depression. Finally, a recent meta-analysis of the use of omega-3 fatty acids to treat major depression indicates that they exert a significant curative effect (Martins, 2009). This appears to be mediated by EPA and it seems to work best when combined with antidepressant drugs (Martins, 2009). In sum, lower omega-3 fatty acid levels seem to be associated with a greater risk for depression, and elevating these levels seems to assist recovery. As the hippocampus is significantly involved in depression and the drugs which target depression seem to exert some of their therapeutic effect by up-regulating neurogenesis in this structure, we tentatively suggest that omega-3 fatty acids exert some of their beneficial effects by targeting this structure. Omega-3 Fatty Acids and their Effect on Memory and Cognition One form of evidence that would suggest a favorable effect of omega-3 fatty acids on the hippocampus would be to find that supplementation, or higher plasma levels, were associated with enhanced performance on neuropsychological tests known to tap hippocampal function. A few studies have now addressed this question in children and adults. In one study, Inuit children had plasma omega-3 levels established at birth from cord blood, and when aged 11 (Boucher et al., 2011). On the California Verbal Learning Test (CVLT), recognition scores from this test were found to correlate with DHA levels from cord blood and from the most recent plasma sample. These relationships withstood the use of various covariates, designed to control other plausible explanations for this result. Importantly, the CVLT recognition score has been shown in a number of studies to be sensitive to hippocampal integrity (Beyer et al., 2012; Libon et al., 1998). While this cross-sectional and longitudinal study suggests that higher plasma omega-3 fatty acids are beneficial for memory, not all studies support this conclusion. The NEMO study group (2007) found no beneficial effect of DHA/EPA supplementation in Australian or Indonesian children on tests of learning and memory. In contrast, a South African study using a much higher dosage of DHA/EPA did observe significant improvements in tests of verbal learning and memory in healthy children (Dalton et al., 2005). Three studies have looked for an association between hippocampal structure or function, and

questionnaire-based estimates of omega-3 intake. In the first, a sample of healthy middle-aged adults undertook two dietary recall interviews, from which their omega-3 fatty acid consumption was estimated (Conklin et al., 2007c). All of these participants then underwent a structural brain scan, so that gray matter volume in three structures (the amygdala, hippocampus, and anterior cingulate cortex) could be correlated with estimates of their omega-3 fatty acid intake. Even after controlling for confounding variables (e.g., sex, age, total gray matter volume) significant associations were observed between omega-3 intake and the three target structures. Larger omega-3 intake was associated with a larger gray matter volume in the hippocampus, amygdala, and anterior cingulate cortex. A second cross-sectional study in healthy middleaged adults examined the relationship between plasma estimates of omega-3 fatty acids and performance on a range of neuropsychological tests (Muldoon et al., 2010). Memory measures known to be associated with hippocampal integrity, i.e., logical memory and verbal paired associates (Saling et al., 1993; Sass et al., 1992) were found to correlate positively with plasma estimates of DHA, but not EPA or ALA. These relationships withstood control for confounding variables including intelligence, blood pressure, age, and gender. These controls are important, especially as higher omega-3 intake may reflect generally more healthful dietary choices as well as being a proxy for intelligence (i.e., following health-related guidelines). In a final cross-sectional study (Kalmijn et al., 2004), omega-3 intake was assessed from dietary recall in a large Dutch sample of 45
70 year-old participants, with memory performance obtained using a delayed verbal recall measure. In this case, no relationship was observed between performance on this memory test and omega-3 intakes, although general cognitive impairment was found to be inversely correlated with omega-3 intake. In sum, the child and adult data are generally supportive of the idea that higher levels of omega-3 fatty acids are associated with a healthier hippocampus. This is reflected in better performance on memory measures known to be sensitive to damage to this structure, and in one case on a more direct measure obtained by neuroimaging. Conclusion on Human Data There is currently no direct evidence that omega-3 fatty acids act to protect the human hippocampus or relatedly that they can offset or remediate the effects of consuming larger quantities of SFAs. Nonetheless, there is good indirect evidence that higher intakes of omega-3 fatty acids probably are neuroprotective to

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structures that may include the hippocampus, as well as being remedial.

General Conclusion: Fatty Acids and their Impact on the Hippocampus This section explored two claims. First, that SFAs have an adverse effect on hippocampal-dependent learning and memory. Second, that omega-3 fatty acids have a remedial or neuroprotective effect. Both claims are generally supported in the literature, especially by the animal data, where it is much easier to directly test causality. In addition, the animal literature has also provided a number of plausible candidate pathways by which SFAs and omega-3 fatty acids could exert their effects on the hippocampus.

FATTY ACIDS AND AD AD is a form of neurodegeneration characterized by memory loss due to neuronal abnormalities, with pathology predominantly seen in the hippocampus. The early onset form of the disease has a known genetic basis, linked to the Apoe ε4 allele and with abnormalities of β-amyloid precursor protein (APP) production and metabolism. However, late onset sporadic AD is the more common form and many theories regarding causality propose a contribution of environmental factors leading to neuronal dysfunction. This section will discuss evidence for the hypothesis that dietary fatty acid intake is an important environmental factor contributing to AD development. First, we discuss a role for high intake of SFAs and the potential neurological mechanisms underlying their detrimental effects. Second, we discuss the role of omega-3 fatty acids as a protective factor and their therapeutic potential to remediate memory deficits associated with AD.

SFAs and Risk of AD Several cross-cultural epidemiological studies have found higher risk of AD in Western countries compared to less developed countries (Havlik et al., 2000; Hendrie et al., 2001; Ogunniyi et al., 2000). Further, studies have reported a higher prevalence of AD in populations of elderly African-Americans and Japanese living in the United States compared to those living in their ethnic homelands. These studies suggest that AD development is strongly linked to environmental factors rather than genetic influence. This has led to the proposal that cultural differences in diet might account for the higher prevalence of AD in Western cultures. Indeed, Grant (1997) performed a

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regression analysis of 18 community studies in 11 different countries that assessed AD risk in individuals over 65 years of age. Results showed that the prevalence of AD significantly correlated with per capita daily fat consumption and that fish consumption was associated with a lower prevalence of AD. Human epidemiologic studies provide further evidence for a link between dietary SFA intake and development of AD. In a cross-sectional study, greater SFA intake was associated with increased incidence of mild cognitive impairment (MCI), a prodromal state preceding AD where individuals have impaired memory and other cognitive functions (Eskelinen et al., 2008). Incidence of dementia, including AD, was strongly associated with high SFA intake over an average of 2.1 years in individuals aged over 55 (Kalmijn et al., 1997). Similarly, high SFA intake assessed over a period of 4
6 years in adults over 65 years of age was associated with increased risk of AD (Morris et al., 2004). An analysis of dietary patterns found those consuming lower amounts of foods high in SFAs (including high fat dairy products, red meat, organ meat, and butter) over a 3.9 year period had the lowest risk for developing AD (Gu et al., 2010). A limitation of the epidemiologic studies described above is that causality cannot be determined and it is possible that pre-existing factors may contribute to both high SFA intake and AD. In an attempt to provide more causative evidence for SFA intake in the development of AD, Bayer-Carter et al (2011) conducted a randomized controlled trial where healthy participants and participants with MCI were maintained on either a high SFA/high glycemic index (HIGH) diet or a low SFA/low glycemic index (LOW) diet. For the MCI group, the LOW diet reduced Aβ42, a marker for AD, in the cerebrospinal fluid. Further, both groups exhibited an improvement in delayed visual memory performance. In summary, high SFA intake appears to be related to risk for developing AD, and there is some evidence from human studies that SFAs play a causative role. However, what remains to be elucidated are the mechanisms underlying this relationship and we describe some of the literature addressing this question in the following section.

Putative Causal Basis for Link between SFAs and AD One of the hallmark pathological features of AD is an increase in β-amyloid deposition, which is hypothesized to play a causative role in the disease (Joachim et al., 1988). In animal studies, including transgenic mice models of AD, maintenance on typical Western diets (high in SFA and cholesterol) increases

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accumulation of β-amyloid in the cortex (Refolo et al., 2000; Shie et al., 2002) and hippocampus (Oksman et al., 2006; Sparks et al., 1994). In this section, we present several lines of evidence for how SFAs contribute to increases in β-amyloid: (1) high SFA intake increases production and secretion of enterocytic β-amyloid; (2) amyloid plaques are a marker of neuronal injury due to SFA-induced oxidative stress; and (3) SFA-induced oxidative stress further causes BBB dysfunction. Recent research demonstrates that SFAs can cause a direct increase in β-amyloid levels. Galloway et al (2007) demonstrated that β-amyloid and its precursor protein are secreted by epithelial cells of the small intestine and that rats fed a high SFA diet showed a high epithelial cell β-amyloid/precursor protein concentration. In a subsequent study by the same laboratory, SFA-fed rats had a higher concentration of cells that expressed β-amyloid than rats fed a low fat diet (Galloway et al., 2008). These findings suggest that high SFA diets may contribute to β-amyloid accumulation in AD by modulating its production in the small intestine. Mitochondrial and nuclear oxidative damage occurs early in AD and precedes plaque pathology and β-amyloid deposition (Nunomura et al., 2001; Pratico et al., 2001). Further, enlarged and deformed mitochondria are one of the earliest changes observed in the development of AD, supporting the idea that oxidative stress occurs early and is a likely causative process (Hirai et al., 2001). As discussed in detail in the section entitled Neurological Impact of Saturated Fat Consumption high dietary intake of SFAs increases oxidative stress. In light of this, high SFA intake may be an environmental factor that induces or exacerbates oxidative stress, thus initiating the process that eventually leads to AD. The BBB is a system of tight junctions between endothelial cells that acts to restrict the diffusion of certain blood components into the cerebrospinal fluid (Persidsky et al., 2006). The BBB appears to be vulnerable to dietary factors, with rats maintained on high SFA diets showing changes to expression of tight-junction proteins and increased BBB permeability in the brain (Badaut et al., 2012; Freeman and Granholm, 2012) and hippocampus specifically (Davidson et al., 2012; Kanoski and Davidson, 2010). These impairments to BBB integrity have been suggested to result in increased blood
brain perfusion of peripheral β-amyloid of intestinal origin (Takechi et al., 2010). In summary, SFAs may contribute to β-amyloid accumulation in AD via a number of pathways that likely act in concert. There is evidence for increased production and secretion of β-amyloid resulting in greater amounts available for crossing the BBB. This may then be exacerbated by increased BBB permeability due to oxidative stress.

Omega-3 Fatty Acids, Cognitive Decline, and Dementia This section examines whether omega-3 fatty acids can exert a protective or curative effect in relation to cognitive decline, dementia, and in particular AD. Four classes of evidence are reviewed in this section: (1) global prevalence data to try and establish whether any regional variation in AD can be accounted for by dietary factors; (2) matched group studies that examine whether AD and other groups have or had different dietary or plasma omega-3 fatty acid profiles relative to matched controls; (3) longitudinal studies of omega3 cognitive decline, which constitute the largest set of findings, examining whether plasma fatty acid levels or diet-based estimates can predict cognitive decline or the onset of AD; and (4) treatment studies, which aim to maintain healthy aging by preventing the onset of AD or cognitive decline, by using omega-3 fatty acids. Global Prevalence If environmental factors such as diet are important in dictating the prevalence of diseases such as dementia, it might be possible to see these effects by examining countrywide differences. One country that initially attracted interest in this regard was Japan, where certain regions have a diet rich in fish. Early studies suggested differences in the proportion of cases of AD relative to vascular dementia, with the former being rarer than one would expect based on prevalence of these diseases in Western populations (Jorm, 1991). However, more recent work, both in Japan (e.g., Dodge et al., 2012), and in relation to other nationbased differences, have failed to support any moderate to large systematic effect (e.g., Prince et al., 2013). Matched Group Studies Six studies have examined whether actual or selfreported omega-3 fatty acid profiles differ between groups stratified by cognitive profile or more formally by dementia diagnosis. Titova et al. (2012) examined whether self-report dietary-based estimates of omega-3 fatty acid intake was related to cognitive function and brain volume (measured using magnetic resonance imaging), in a large sample of elderly people. They found that EPA and DHA levels were weakly correlated with total gray matter volume and cognitive function, but the former effect was only marginally significant once relevant control variables had been added to the model. The remaining five studies all compared groups diagnosed with dementia against various clinical groups and controls. Tully et al. (2003) compared plasma estimates of omega-3 fatty acids between controls and an AD group, finding higher levels of EPA and DHA in the plasma of healthy controls. Cherubini

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et al. (2007) obtained plasma estimates of omega-3 fatty acids. They found these to be significantly lower following adjustment for differences in various control variables in an MCI and dementia group relative to a healthy elderly group. SFA levels were also found to be significantly higher in the dementia group. In a conceptually similar study, Conquer et al. (2000) observed significantly lower levels of DHA and omega-3 fatty acids overall (adjusted for age and education), in an AD group. Two other groups were also tested, one with dementia and the other with an MCI diagnosis, and these fell in between the AD group and controls for plasma DHA and omega-3 fatty acid levels. Wang et al. (2008) used a different approach. They found a significant association, in a group of AD patients, between degree of global cognitive impairment on the Mini Mental State Exam (MMSE) and plasma DHA, with high DHA levels associated with better functioning. The final study in this set examined whether plasma- and brain-derived lipid profiles differed between AD, MCI, and elderly controls, at postmortem (Cunnane et al., 2012). Plasma phospholipid fraction was observed to differ across the three groups, with the AD patients having significantly lower levels of EPA and DHA relative to the MCI and elderly controls, who did not differ. For gray matter measures, they found that the proportion of DHA in the phosphatidylserine fraction was significantly lower in the AD group relative to the other groups. Thus, in common with the other studies described here, and after controlling for factors likely to co-vary with the dependent variable(s) of interest, there seems to be a fairly consistent pattern of abnormal lipid profiles in dementia patients, which is often reflected in lower levels of omega-3 fatty acids in the diet, plasma, and brain. Longitudinal Studies of Cognitive Decline Eighteen studies have now examined whether changes in cognitive ability over time are associated with self-reported or plasma-based estimates of omega-3 fatty acids. These studies can be further organized by whether the focus is on the emergence of AD or cognitive decline, which itself may be a precursor of AD. Thus, this section is organized by the focus on AD/cognitive decline, and whether the lipid measures are based on self-report or plasma. The highest standard of evidence is presumably the emergence of diagnosed AD and plasma-based markers of omega-3 fatty acids. COGNITIVE DECLINE AND SELF-REPORT MEASURES OF DIETARY INTAKE

Five studies have examined the relationship between cognitive decline and self-reported dietary-based estimates of omega-3 fatty acid intake. Morris et al. (2005) assessed whether cognitive decline, established using a broad battery of measures over a six-year

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period in initially healthy elderly Americans, was associated with fish intake. After controlling for confounding variables (principally age and education), only a weak relationship was observed, with less cognitive decline associated with greater overall fish intake. In another study, Kalmijn et al. (1997) used the MMSE as the principal measure of cognitive decline over a three-year period in a group of aging Dutch men. Fish consumption was found to be slightly lower in those with greater cognitive decline, after controlling for confounding factors. A further study also employed the MMSE to assess cognitive decline in a group of French men and women. Here, Kess-Guyot et al. (2011) found no correlation between dietarybased estimates of omega-3 fatty acid intake and cognitive decline. Two further studies, both using the MMSE as the principal measure of cognitive decline, have obtained more positive findings. van Gelder et al. (2007) described a five-year prospective cohort study based in the Netherlands, where greater cognitive decline, as measured by the MMSE, was associated with lower dietary-based estimates of omega-3 fatty acid intake. Nurk et al. (2007) examined a similar relationship in a large sample of elderly Norwegian men, finding that lower fish intake was predictive of cognitive decline on the MMSE. They also found a dose
response relationship between cognitive decline and fish intake. In sum, while not all published studies have found positive results, the general conclusion would seem to be that of a weak relationship between self-reports of omega-3 fatty acid intake and cognitive decline, with greater intake being protective. DEMENTIA DIAGNOSIS AND SELF-REPORT MEASURES OF DIETARY INTAKE

Six studies have explored whether dementia diagnosis at a later time point can be predicted by dietary-based self-report estimates of omega-3 fatty acid consumption at an earlier point in time. Morris et al. (2003) established food intake patterns in a group of elderly Americans around 3.5 years prior to an assessment for AD. Higher rates of AD were associated with lower reported levels of omega-3 fatty acid intake. A similar conclusion emerged from a French study (Barberger-Gateau et al., 2002) with dietary estimates of omega-3 fatty acid intake obtained between 2
7 years prior to AD assessment being predictive of who would go on to develop AD. A later study by the same group provided further confirmation of these findings (Bargerger-Gateau et al., 2007). Notably, in all three of these studies, the ability to predict who would go on to receive an AD diagnosis remained significant, even after controlling for confounding variables. Not all studies have reported significant relationships. Kalmijn et al. (1997) initially found a relationship between fish consumption and later diagnosis of AD,

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with this being more frequent in those consuming less fish. However, this relationship was not observed in a six-year follow-up of the same Dutch sample (Engelhart et al., 2002). Huang et al. (2005) found some evidence of a relationship between fish intake and a later AD diagnosis, but this relationship did not survive correction for confounding variables (age, education, and gender). In sum, these findings present a weakly supportive case for a relationship between self-reported omega-3 fatty acid intake and the later onset of AD. COGNITIVE DECLINE AND PLASMA LIPID ESTIMATES

Four studies have examined the link between cognitive decline and plasma lipid estimates in the elderly. Heude et al. (2003) examined cognitive decline based on the MMSE over a four-year period in a group of elderly French men and women. They found that higher levels of omega-3 fatty acids were associated with greater preservation of cognitive function, in contrast to saturated fat levels, which were associated with cognitive decline. These relationships withstood control for confounding factors of age, sex, education, and other medical and demographic variables. Beydoum et al. (2007) also found a significant relationship between higher plasma levels of omega-3 fatty acids and reduced risk for cognitive decline over a 4
6 year period in an elderly American cohort (this too survived statistical adjustment for confounding factors). A Dutch study by Dullemeijer et al. (2007) reported another similar observation, in this case using a general cognitive battery including the MMSE. Cognitive decline over a three-year period in elderly Americans could be predicted by plasma-based measures of omega-3 fatty acids obtained prior to the study, even after control variables were entered into the model. A final study by Whalley et al. (2008) is particularly interesting as intelligence data was available about each of the elderly Scottish participants when they were children. Cognitive assessment, conducted when they were elderly, and stratified by childhood intelligence, still revealed that cognitive decline over a four-year period could be predicted by initial plasma levels of omega-3 fatty acids. Notably, Whalley et al. (2008) also found that the relationship between plasma levels of omega-3 fatty acids and decline was moderated by certain genetic markers (APO status) and by age, but the most notable feature of this study is that the link between omega-3 fatty acids and cognition should remain significant for all levels of IQ. This would suggest that differences in intelligence
of which having a more healthy diet may be a proxy measure
does not account for the link between cognitive decline and omega-3 fatty acid intake. Together, these four studies provide a strong level of support for the notion that greater dietary intake

of omega-3 fatty acids, as reflected in plasma, is associated with slower cognitive decline. DEMENTIA DIAGNOSIS AND PLASMA LIPID ESTIMATES

Three studies have reported relationships between plasma markers of omega-3 fatty acid levels and a later diagnosis of dementia. Laurin et al. (2003) conducted a five-year prospective cohort study with a sample of elderly Canadians. They found that people who went on to develop dementia had higher levels of plasma omega3 fatty acids, a relationship that remained significant even after controlling for age, education, and gender. In contrast to this unexpected result, Schaefer et al. (2006) reported data from the Framingham study, which had an average 9.1 year follow-up from plasma sample to assessment. They found that plasma DHA was significantly, but only weakly, predictive of a later dementia diagnosis, after controlling for confounding variables. In this study, greater DHA levels at baseline predicted a lower likelihood of later developing dementia. Interestingly, a later dementia diagnosis could not be predicted by EPA levels or by fish consumption. Finally, Samieri et al. (2008) found that at four-year follow-up, plasma EPA levels, but not DHA, were predictive of who among a French elderly sample would go on to receive a dementia diagnosis. In sum, two out of the three published studies finds the expected relationship, although the Laurin et al. (2003) study suggests that much remains to be learned about the relationship between omega-3 fatty acids and dementia. Prevention and Treatment Studies Eight clinical trials have now been conducted to examine whether giving omega-3 fatty acids as dietary supplements can maintain healthy aging, prevent the onset of or treat AD and MCI. Yurko-Mauro et al. (2010) selected a large sample of elderly Americans who performed poorly on a test of memory (more than 1 SD below the mean on the logical memory subtest of the Wechsler Memory Scale). From this pool, participants were randomly allocated in a double-blind manner to receive either DHA supplementation or placebo for 24 weeks. Relative to the placebo group, DHA supplementation improved performance on two measures of memory, paired associate learning and immediate and delayed verbal recognition memory. Improvements in the paired associate learning score were found to correlate with the degree to which plasma DHA changed over the course of the intervention, further supporting the conclusion that these improvements in memory were driven by the consumption of this omega-3 fatty acid.

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FATTY ACIDS AND AD

Four studies have now examined whether supplementation with omega-3 fatty acids can delay or prevent the onset of dementia. Three of these studies were recently included in a Cochrane collaboration metaanalysis (Sydenham et al., 2012). These studies were: (1) Dangour et al. (2010), which involved DHA and EPA supplementation versus placebo with a 2 year duration in healthy elderly individuals; (2) Geleijnse et al. (2011), which compared DHA and EPA supplementation against other conditions, including a placebo, for 40 months, in healthy elderly people; and (3) van de Rest et al. (2008), which compared low and high dose DHA and EPA supplementation against placebo for 6 months. Overall, the Cochrane meta-analysis found no significant effect of omega-3 supplementation on reduction in dementia diagnosis relative to the placebo arms. A further study by Freund-Levi et al. (2006), where outcome was based on cognitive decline as indexed by the MMSE, also found no overall effect when comparing omega-3 fatty acid treatment (DHA and EPA supplements) versus placebo. These studies seem to suggest that supplementation with omega-3 fatty acids does not delay the onset of dementia, at least over the dose and time course studied. Three studies have now examined whether supplementation with omega-3 fatty acids can ameliorate the symptoms of MCI and AD. Terano et al. (1999) randomly assigned patients diagnosed with mild-tomoderate vascular dementia to receive DHA supplementation or placebo for one year. Although some evidence of slowed cognitive decline on the MMSE between the two groups was evident at 3 and 6 months, at 12 months there was no significant difference in the degree of decline between the placebo and treatment arms. Kotani et al. (2006) compared the effects of 3 month treatment with either DHA or placebo in patients with varying types of amnesia (AD or MCI). Overall, DHA supplementation did not slow cognitive decline as measured by a broad battery of neuropsychological measures. Finally, Quinn et al. (2010) randomly assigned patients with AD to receive either DHA supplementation or placebo, to examine whether this would slow or remediate cognitive decline. No overall effect of treatment was observed, but sub-group analysis did suggest that patients without the APO4E gene might demonstrate some therapeutic benefit. Overall, these results suggest that DHA supplementation does not offer a particularly useful treatment, but as many parameters remain to be studied, it is possible that higher doses, specific omega-3 fatty acids, or specific target groups might benefit. Human Data
Conclusions From the various human lines of evidence it would seem that dietary intake of omega-3 fatty acids

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probably exerts a small but positive effect on human cognition, slowing age-related cognitive decline. This seems to extend to slowing the rate at which AD appears in particular cohorts who presumably consume greater quantities of omega-3 fatty acids in their diet. However, the final step of determining whether addition of omega-3 fatty acids to elderly people’s diets has shown little benefit as yet.

Omega-3 Fatty Acids, AD, and Memory Impairment
Evidence from Animal Models There is some research using rodent models of AD to suggest that omega-3 fatty acids, particularly DHA, may have a protective effect against the formation of AD pathology. Transgenic mice models of AD include 3xTg-AD, APP/PS1, TgCRND8, and Tg2576. An additional rat model is produced by infusion of β-amyloid into the cerebral ventricle. All of these models mimic the β-amyloid pathology seen in AD (Chishti et al., 2001; Hashimoto et al., 2005; Higgins and Jacobsen, 2003), with the 3xTg-AD additionally developing neurofibrillary tangles (Oddo et al., 2003). The omega-3 fatty acids docosapentaenoic acid (DPA), docosatrienoic acid (DTA), and DHA significantly decreased in vitro β-amyloid peptide levels in an animal model of AD (Amtul et al., 2011). From this in vitro study, DHA stood out as the most potent omega-3 fatty acid for reducing β-amyloid levels and was therefore administered as a dietary supplement in TgCRND8 mice. DHA supplemented mice showed a significant decrease in plaque density in the hippocampus, frontal cortex, and amygdala when compared to mice fed standard chow. Similarly, 3xTg-AD mice fed a DHA supplemented diet for 6 months showed decreased levels of soluble β-amyloid (Green et al., 2007). DHA supplementation has also been shown to attenuate β-amyloid pathology in Tg2576 mice (Lim et al., 2005). In light of the fact that DHA supplementation can attenuate β-amyloid pathology and DHA enriched diets can enhance memory function (see the section entitled Omega-3 fatty acids, memory and the hippocampus
evidence from animal studies), DHA may have therapeutic potential to remediate memory impairments observed in AD
although the available human literature is not currently that supportive. For instance, Tg2576 mice showed increased dendritic and synaptic pathology following DHA depletion and mice fed a DHA replacement diet showed an improvement in MWM performance compared to those remaining on the DHA depleted diet (Calon et al., 2004). In another study, third generation dietary DHA depleted rats were divided into 4 groups; β-amyloid infusion,

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DHA supplement, β-amyloid 1 DHA, and a vehicle group. DHA supplementation in β-amyloid infused rats reduced the number of reference and working memory errors on the RAM task and suppressed the increases in markers of oxidative stress in the cortex and hippocampus (Hashimoto et al., 2005). In Tg2576 mice fed a DHA depleting safflower oil-based diet, DHA supplementation significantly reduced β-amyloid accumulation, oxidative stress, and dendritic pathology, as well as improving cognitive performance on the MWM task (Cole and Frautschy, 2006). These studies first involved DHA depletion, prior to supplementation, which is ecologically valid given that Western diets are typically deficient in DHA. The rationale for this is that standard rat chow contains sufficient levels of DHA and other omega-3 fatty acids, which is not representative of what the typical individual in a Western society would be consuming. Studies without prior depletion remain equivocal. While DHA supplementation in APP/PS1 mice decreased β-amyloid levels and activated microglia in the hippocampus, no improvement was seen in MWM performance (Oksman et al., 2006). Similarly, APP/PS1 mice showed no improvement on a battery of cognitive tasks following 7 months of DHA supplementation (Arendash et al., 2007). Conversely, Hooijmans et al. (2009) found that DHA-fed APP/PSI mice showed decreased β-amyloid deposition and improved memory during the MWM task. Possible differences that may explain these equivocal findings are the age of the animals and the duration of supplementation. Diets were administered from age 6 months through 10 months in the Oksman et al. (2006) study and from 2 months through 9 months in the Arendash et al. (2007) study. In contrast, in the Hooijmans et al. (2009) study, diet administration began at 2 months and was maintained until 15 months when the documented effects of DHA were observed. There is convincing evidence that DHA supplementation reduces β-amyloid pathology. The finding that DHA supplementation improves cognitive function, particularly spatial memory performance, has been consistently demonstrated in rodent models of AD, where DHA deprivation precedes supplementation. However, in cases where diet contains sufficient levels of DHA, the influence of DHA on AD pathology and cognition appears to require greater age (and therefore likely greater amyloid burden and cognitive deficits to remediate) and/or greater duration of supplementation.

GENERAL DISCUSSION This chapter explored two claims. The first was that SFAs impair hippocampal-dependent memory, and

that they may ultimately contribute to neurodegenerative diseases, in particular AD. The second claim was that omega-3 fatty acids may have a neuroprotective effect, and may possibly be able to remediate some of the adverse effects of SFAs on hippocampaldependent memory. The human and animal data generally concur on both these claims. In particular, animal data not only indicate a causal link between diets rich in SFAs and hippocampal-dependent memory impairments, but they also suggest likely causal pathways. Currently, these include oxidative stress, reduced levels of BDNF, and dietary-induced neuroinflammatory responses. While the human data is much less developed, there is clear epidemiological data linking elevated SFA intake to poorer hippocampal-dependent memory performance, and some albeit indirect evidence suggesting that omega-3 fatty acids may exert protective effects on cognition, including memory. Two conclusions emerge from this work. First, there is an important need to establish the causal effects of SFAs and omega-3 fatty acids on human learning and memory. This needs to employ both short-term dietary intervention studies, where diet can be carefully manipulated over several days, as well as longer-term dietary interventions lasting months or years. If the animal studies are correct there should be reasonably large and readily detectable effects on cognition following changes in SFA and omega-3 fatty acid consumption. Second, and again in humans, we need to establish whether any of the proposed causal pathways in animals are triggered by similar diets in people. This would help to establish the case in humans that SFAs and omega-3 fatty acids do exert causal effects on hippocampal-dependent memory. This is important, because as pointed out before, adverse changes to the hippocampus may themselves predispose people to over-consume the very foods that initiated this neural impairment, leading to weight gain and further damage to the hippocampus (see Francis and Stevenson, 2011). A further reason why it is important to know if SFAs and omega-3 fatty acids do cause changes in human hippocampal-dependent memory is because of the putative link with the development of AD. The second section of the review focused on this issue. In this case, the animal data again suggested that there is a causal link between SFA consumption and β-amyloid accumulation. Similarly, animal data also strongly suggest that omega-3 fatty acids are protective, and may even ameliorate some of the adverse effects of AD. Crucially, however, the human data are much weaker. There are again some epidemiological findings indicating that diets rich in SFAs are associated with higher rates of AD, but there is as yet no strong case that diet and SFA consumption, in particular, is a

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REFERENCES

causal agent. The human literature on omega-3 fatty acid and a remedial effect in AD is also weak at present, with no successful treatment outcome or prevention studies, and with only indirect correlational evidence available. This contrasts with the relative strength of the animal data, which must raise the obvious question over the validity of the animal models of AD in regard to their sensitivity to dietary manipulations. The key conclusion from these studies would seem to be that a better understanding of how the healthy human brain is affected by SFA and omega-3 fatty acid consumption might lead us to a more nuanced appraisal of the role of dietary fatty acids in neurodegenerative conditions such as AD.

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FURTHER READING

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1147. van de Rest, O., Geleijnse, J., Kok, F., van Staveren, W., Hoefnagels, W., Beekman, A., et al., 2008. Effect of fish oil supplementation on mental well-being in older subjects: a randomized, doubleblind, placebo controlled trial. Am. J. Clin. Nutr. 88, 706
713. Wallace, D.C., 1992. Diseases of the mitochondrial DNA. Annu. Rev. Biochem. 61, 1175
1212. Wang, W., Shinto, L., Connor, W., Quinn, J., 2008. Nutritional biomarkers in Alzheimer’s disease: The association between carotenoids, n-3 fatty acids and dementia severity. J. Alzheimer’s Dis. 13, 31
38. Whalley, L., Deary, I., Starr, J., Wahle, K., Rance, K., Bourne, V., et al., 2008. n-3 Fatty acid erythrocyte membrane content, APOE e4, and cognitive variation: an observational follow-up study in late adulthood. Am. J. Clin. Nutr. 87, 449
454. White, C.L., Pistell, P.J., Purpera, M.N., Gupta, S., Fernandez-Kim, S. O., Hise, T.L., et al., 2009. Effects of high fat diet on Morris maze performance, oxidative stress, and inflammation in rats: contributions of maternal diet. Neurobiol. Dis. 35, 3
13. Wibrand, K., Berge, K., Messaoudi, M., Duffaud, A., Panja, D., Bramham, C.R., et al., 2013. Enhanced cognitive function and antidepressant-like effects after krill oil supplementation in rats. Lipids Health Dis. 12, 6. Willis, L.M., Shukitt-Hale, B., Cheng, V., Joseph, J.A., 2009. Dosedependent effects of walnuts on motor and cognitive function in aged rats. Br. J. Nutr. 101, 1140
1144. Winther, B., Hoem, N., Berge, K., Reubsaet, L., 2011. Elucidation of phosphatidylcholine composition in krill oil extracted from Euphausia superba. Lipids. 46, 25
36. Wu, A., Ying, Z., Gomez-Pinilla, F., 2004a. Dietary omega-3 fatty acids normalize BDNF levels, reduce oxidative damage, and

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counteract learning disability after traumatic brain injury in rats. J. Neurotrauma. 21, 1457
1467. Wu, A., Ying, Z., Gomez-Pinilla, F., 2004b. The interplay between oxidative stress and brain-derived neurotrophic factor modulates the outcome of a saturated fat diet on synaptic plasticity and cognition. Eur. J. Neurosci. 19, 1699
1707. Yang, R.H., Wang, F., Hou, X.H., Cao, Z.P., Wang, B., Xu, X.N., et al., 2012. Dietary omega-3 polyunsaturated fatty acids improves learning performance of diabetic rats by regulating the neuron excitability. Neuroscience. 212, 93
103. Ying, S.W., Futter, M., Rosenblum, K., Webber, M.J., Hunt, S.P., Bliss, T.V., et al., 2002. Brain-derived neurotrophic factor induces longterm potentiation in intact adult hippocampus: requirement for ERK activation coupled to CREB and upregulation of arc synthesis. J. Neurosci. 22, 1532
1540. Yu, H., Bi, Y., Ma, W., He, L., Yuan, L., Feng, J., et al., 2010. Longterm effects of high lipid and high energy diet on serum lipid, brain fatty acid composition, and memory and learning ability in mice. Int. J. Dev. Neurosci. 28, 271
276. Yu, Y., Wang, Q., Huang, X.F., 2009. Energy-restricted pair-feeding normalizes low levels of brain-derived neurotrophic factor/tyrosine kinase B mRNA expression in the hippocampus, but not ventromedial hypothalamic nucleus, in diet-induced obese mice. Neuroscience. 160, 295
306. Yurko-Mauro, K., McCarthy, D., Rom, D., Nelson, E., Ryan, A., Blackwell, A., et al., 2010. Beneficial effects of docosahexaenoic acid on cognition in age-related cognitive decline. Alzheimer’s Dementia. 6, 456
464. Zhang, X., Dong, F., Ren, J., Driscoll, M.J., Culver, B., 2005. High dietary fat induces NADPH oxidase-associated oxidative stress and inflammation in rat cerebral cortex. Exp. Neurol. 191, 318
325.

Further Reading Granholm, A.C., Bimonte-Nelson, H.A., Moore, A.B., Nelson, M.E., Freeman, L.R., Sambamurti, K., 2008. Effects of a saturated fat and high cholesterol diet on memory and hippocampal morphology in the middle-aged rat. J. Alzheimer’s Dis. 14, 133
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36 Fish Oil Supplements, Contaminants, and Excessive Doses Nicole Burca and Ronald Ross Watson INTRODUCTION Omega-3 fatty acids are types of dietary polyunsaturated fatty acids (PUFAs) that humans cannot synthesize. Dietary consumption of fatty acids is essential for optimal health. Fatty acids are recommended for brain and neurological health for a number of reasons. Omega-3 fatty acids serve as a preventive measure against neurological disorders. Research has shown that people who consume high intakes of omega-3 fatty acids have a lower risk of developing dementia, which is a condition associated with a loss of brain functioning (Yashodhara et al., 2008). In addition, patients diagnosed with dementia have lower levels of the omega-3 fatty acid docosahexaenoic acid (DHA) in their brain and blood plasma (Yashodhara et al., 2008). Sources of omega-3 fatty acids such as fish and fish oil supplements may contain contaminants including metals, industrial products, and pesticides that may compromise their health benefits. Some fish oil supplements are concentrated in order to produce higher percentages of the beneficial omega-3 fatty acids eicosapentaenoic acid (EPA) and DHA (Mahaffey et al., 2008). Excessive doses of fish oil supplementation increase bleeding, cause excessive levels of vitamins A and D, loss of vitamin E, increase in low-density lipoprotein (LDL) cholesterol levels, heartburn, and diarrhea (Morton, 2012). However, the level of contaminant toxicity is determined by the species of fish in the supplement consumed, the amount of fish, and the frequency of fish oil supplement consumption. Heavy metals associated with fish oil supplementation include methylmercury, lead, selenium, arsenic, and cadmium. Mercury poisoning may be a key factor in the development of neuropsychiatric conditions (Bays, 2007). Lead poisoning affects the nervous system. In adults, it is primarily the peripheral nervous system, Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00036-3

while in children it is the central nervous system which becomes damaged. Although selenium is an essential mineral for humans, its intake level is infinitesimal compared to the levels that fish sources are capable of providing (Hamilton, 2004). Arsenic levels should be carefully monitored because it is the only element that the Environmental Protection Agency (EPA) has linked with cancer (Juma et al., 2002). The existence of cadmium in the body can last up to thirty years (Al-Busaidi et al., 2011). Despite the illegal status of polychlorinated biphenyls (PCBs), these organic toxins contaminate bodies of water as well as the fish living in them (Ashleya et al., 2010). Dichlorodiphenyltrichloroethane (DDT) is an organic pesticide that accumulates in fish and may result in neurotoxicity (Melanson et al., 2005). Another organic pesticide, dieldrin, has been found in fish oil supplement sources and has been linked with damaging the hypothalamus in the brain (Martyniuk et al., 2010a). Although these contaminants are potential dangers to the nervous system, the benefits of fish oil supplements outweigh the risks.

MERCURY Mercury is a toxic heavy metal commonly found in fish, the source of fish oil supplements. There are two forms of mercury, inorganic and organic. Organic mercury can be found in the fish used to make fish oil supplements. Atmospheric mercury is caused by coal burning, mining, and volcanic activity (Bernhoft, 2011). When the mercury particles settled down on Earth, they may have ended up in water. Both fresh and salt water sources have the possibility of being contaminated with mercury. In the United States, over 3000 lakes have been closed for fishing because of high

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levels of mercury contamination (Bernhoft, 2011). Microorganisms living in the water convert the atmospheric mercury into methyl, dimethyl, or ethyl mercury, which are then consumed by small organisms and ultimately by large fish (Bernhoft, 2011). These fish are often pressed for fish oil supplement production, but have accumulated a significant amount of mercury in their systems. As a result, mercury ends up in fish oil supplements and can cause toxic effects on the human body. Table 36.1 shows typical mercury concentrations found in different types of fish. Methylmercury is the most common type of organic mercury and is a neurotoxin. As a relatively stable molecule, methylmercury is a significant source of human mercury exposure. Any person who has consumed seafood has trace amounts of methylmercury in their system (Mahaffey, 2004). Ethyl mercury is another form of organic mercury, but has an excretory half-life of about one third of methylmercury’s (Bernhoft, 2011). Methylmercury is found as 75
90% of the total mercury found in fish muscle tissue and is easily absorbed through the intestinal tract and progresses to enter the bloodstream (Mahaffey, 2004). Since it is lipid soluble, mercury can pass through the blood
brain barrier (Zahir et al., 2005). Importantly, methylmercury cannot be removed from the fish source by cooking or cleaning methods (Mahaffey, 2004). Methylmercury binds to sulfhydryl groups, especially to ones in the amino acid cysteine (Bernhoft, 2011). After a short span of four days, methylmercury evenly distributes throughout the body but concentrates in the brain, liver, kidneys, placenta, and fetus (Bernhoft, 2011). Methylmercury’s half-life is roughly 70 days, with the majority released with feces. However, some methylmercury may end up in the enterohepatic circulation or may be excreted in breast milk (Bernhoft, 2011). Both inorganic and organic mercury interrupts normal cell function by binding with sulfhydryl and TABLE 36.1 Mercury concentration in fresh and salt water fish Fish

Mercury Concentration (ppm)

Sword fish

0.99

Mackerel king

0.73

Marlin

0.49

Tuna (fresh/frozen)

0.38

Lobster

0.31

Bass

0.27

Carp

0.14

Arctic krill

0.009

(Modified from Zahir et al., 2005.)

selenohydryl groups (Bernhoft, 2011). This action changes a protein’s tertiary and quaternary structure, and ultimately its biological function (Bernhoft, 2011). Studies have shown that methylmercury disturbs normal DNA transcription and protein synthesis (Bernhoft, 2011). In addition, the presence of methylmercury adversely affects the normal operations of the central nervous system. Damages include neurotransmitter disruption, stimulation of neural excitotoxins, and free radical accumulation (Bernhoft, 2011). Methylmercury negatively affects fine motor and sensory functions (Mahaffey, 2004). Studies have associated the presence of methylmercury in the body with a decline in natural killer cells, whose role includes the defense against cancer cells (Bernhoft, 2011). Methylmercury may also be responsible for lowering the effectiveness of autoimmune cells and damaging mitochondrial membranes, causing constant fatigue (Bernhoft, 2011). Pregnant women, as well as women who plan on becoming pregnant within one year, are advised to avoid fish oil supplements because of the risk of mercury toxins (Zahir et al., 2005). Fetuses exposed to large amounts of mercury have an increased risk for developing cerebral palsy, neurodevelopment retardation, and overall cognitive defects (Bernhoft, 2011). Excessive mercury exposure has the greatest effect on the nervous system. The amount of methylmercury exposure in an individual that shows toxicity is determined by the person’s weight (Zahir et al., 2005). Fish oil supplements may provide an accumulation of methylmercury toxicity, but the amount that determines poisoning in a smaller individual is significantly less than that in a heavier individual. Mercury toxicity can be diagnosed with several common symptoms. They include depression, fatigue, anxiety, weight loss, memory loss, and trouble concentrating on simple tasks (Bernhoft, 2011). Common modalities such as blood or urine tests do not determine whether the level of mercury toxins is overwhelming to the body (Bernhoft, 2011). Weight serves as an important measure in factoring the toxic effect methylmercury has on the body. However, blood and hair tests are used to determine concentration levels of exposed mercury from dietary sources in the body (Mahaffey, 2004). Research has shown that individuals who consume seafood on a more frequent basis have higher levels of methylmercury in their systems (Mahaffey, 2004). The United States Environmental Protection Agency and the National Academy of Science recommends an upper limit of whole blood mercury 5.0 μg/L (Zahir et al., 2005). However, if a person does develop mercury toxicity, there are steps to treat it. First, the individual should avoid continued consumption of foods that may be contaminated with

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mercury. Then, the individual may undergo chelation therapy in which chelating agents are administered to the patient (Hightower & Moore, 2003). The chelating agents bind to mercury and are then excreted from the body before mercury can bind to other molecules (Hightower & Moore, 2003).

LEAD Lead is a naturally occurring element. Organic lead has lipophilic properties and is more likely to bioaccumulate than inorganic lead (Meador et al., 2005). Lead leaches into the environment through soil erosion and atmospheric disposition (Castro-Gonzalez & Me´ndezArmenta, 2008). Lead is toxic to marine organisms (Nair et al., 2006). Since fish sit on top of the aquatic food chain, they are at a high risk of concentrating lead in their tissues (Nair et al., 2006). In addition, fish also store trace amounts of lead in their livers and gills (Nair et al., 2006). Individuals commonly consume fish as a part of their daily diet. After ingestion, lead is absorbed into the blood stream and transported to other tissues (Castro-Gonzalez & Me´ndez-Armenta, 2008). The metal accumulates in bone, teeth, liver, lungs, kidneys, and spleen (Castro-Gonzalez & Me´ndez-Armenta, 2008). In addition, the metal is able to cross the blood
brain barrier and the placenta (Castro-Gonzalez & Me´ndez-Armenta, 2008). Lead inhibits the biosynthesis of heme in the blood (Al-Busaidi et al., 2011). Lead is not necessary for any type of biological function in humans (Fallah et al., 2010). Instead, chronic ingestion of lead may lead to a number of human health conditions. Complications include increased blood pressure and risk of cardiovascular disease in adults and a reduction of cognitive development in children (Fallah et al., 2010). Symptoms of lead poisoning include headaches, irritability, stomach discomfort, and physiological issues related to the nervous system (Castro-Gonzalez & Me´ndez-Armenta, 2008). Chronic exposure to lead may result in the development of poor attention span, constipation, vomiting, seizures, comas, and even death (Castro-Gonzalez & Me´ndez-Armenta, 2008). Chronic lead toxicity most heavily impairs the gastrointestinal, neuromuscular, renal, and hematological systems (Castro-Gonzalez & Me´ndez-Armenta, 2008). Children are especially sensitive to lead exposure (Castro-Gonzalez & Me´ndez-Armenta, 2008). In children, lead exposure may result in encephalopathy, aggression, psychosis, and mental deficit (CastroGonzalez & Me´ndez-Armenta, 2008). In children, lead has a longer half-life in comparison to adults. In blood, lead has a half-life of thirty-five days (Castro-Gonzalez & Me´ndez-Armenta, 2008). Lead’s half-life in soft tissue lasts forty days, while its half-life in bones can persist

for up to thirty years (Castro-Gonzalez & Me´ndezArmenta, 2008). Humans are primarily exposed to lead through their diet (Castro-Gonzalez & Me´ndez-Armenta, 2008). When humans consume fish, they are at risk of accumulating metals in their own bodily tissue (Nair et al., 2006). The association between blood lead levels and the consumption of fish cannot be considered causal because the concentrations of lead in fish are relatively low (Nair et al., 2006). The European Union has established the maximum level for lead ingestion as 300 μg kg21 (Pastorelli et al., 2012).

SELENIUM Selenium is a naturally occurring essential trace element and is necessary for consumption in small amounts for optimal human functioning. Studies have shown that the average adult should consume at least 40 μg/day of selenium, with a recommended amount of 55 μg/day (Navarro-Alarcon & Cabrera-Vique, 2008). Consuming fish can serve as a source of selenium. A lack of selenium in the body may lead to a decrease in antioxidants in the system and a lack of energy (Navarro-Alarcon & Cabrera-Vique, 2008). Selenium deficiency may cause Keshan disease, a fatal form of cardiomyopathy that involves the degeneration of heart muscle tissue (Lenz & Lens, 2009). Trace amounts of this mineral are necessary for normal human growth and development, as well as metabolic function. In addition, selenium has an antioxidative property imperative to human health. Some studies suggest that consuming as much as 300 μg/day may help decrease the risk of cancer (Navarro-Alarcon & Cabrera-Vique, 2008). However, too much selenium can also cause toxicity in the body. Factors that determine selenium toxicity include the specific selenium chemical compound, the method of administration, exposure time, and interaction with other heavy metals (Navarro-Alarcon & Cabrera-Vique, 2008). Selenosis, characterized by hair loss, brittle finger nails, skin rash, and garlic breath, is a condition caused by chronic selenium toxicity (Navarro-Alarcon & Cabrera-Vique, 2008). Paralysis has been observed in some people who experience selenium toxicity (Lemly, 2004). Also, too much selenium has been associated with lower T3 levels, impairment of natural killer cells, and liver toxicity (Navarro-Alarcon & Cabrera-Vique, 2008). The hormone T3 is vital for thyroid functioning and natural killer cells target tumor cells. The established tolerable upper limit of selenium is set at 400 μg/day (Navarro-Alarcon & Cabrera-Vique, 2008). Unfortunately, there is a narrow range between selenium deficiency and toxicity (Miklavˇciˇc et al., 2012). Consumption of selenium should be considered with caution.

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Reported amounts of selenium in certain foods are not an accurate measure of how much is actually being absorbed by the body (Navarro-Alarcon & CabreraVique, 2008). A complete assessment of selenium’s bioavailability includes the total nutrient content of the food, its absorbable fraction, the amount actually absorbed, and the percent of the absorbed amount actually used by the person (Navarro-Alarcon & Cabrera-Vique, 2008). However, usually about 80% of consumed selenium is absorbed by the body (NavarroAlarcon & Cabrera-Vique, 2008). One major influence of the level of bioavailability in selenium is the form of micronutrient found in foods. Organic forms of selenium have a greater bioavailability compared with inorganic forms (Navarro-Alarcon & Cabrera-Vique, 2008). The frequency of selenium consumption also influences toxicity levels (Miklavˇciˇc et al., 2012). Fish is a protein-rich food that contains high levels of selenium (Navarro-Alarcon & Cabrera-Vique, 2008). Studies have shown that selenium helps decrease mercury toxicity (Navarro-Alarcon & Cabrera-Vique, 2008). Since fish contains both mercury and selenium, some fish have low concentrations of selenium because it binds with the mercury and other heavy metals and creates insoluble organic complexes (Navarro-Alarcon & Cabrera-Vique, 2008). Salmon typically contains high amounts of selenium (Navarro-Alarcon & Cabrera-Vique, 2008). Seafood is a primary source of selenium in the human diet (Miklavˇciˇc et al., 2012). There are various sources of selenium leaching into the waters and thus contaminating fish. Selenium is a waste product that stems from certain mining, agricultural, petrochemical, and manufacturing operations (Lemly, 2004). Selenium pollution affects aquatic environments on a broad scale. The mineral can contaminate areas of rural, urban, and suburban settings, as well as biomes including mountains, plains, deserts, rainforests, tropics, and even the Arctic (Lemly, 2004). Marine animals can absorb selenium through their gills or epidermis (Hamilton, 2004). However, the main source of selenium accumulation in marine animals comes from the biomagnification of selenium caused by the consumption of smaller marine animals (Navarro-Alarcon & Cabrera-Vique, 2008). Selenium found in fish can have concentrations of up to 2000 times more than that of the waters (Lenz & Lens, 2009). The larger fish commonly used for fish oil supplements eat phytoplankton and zooplankton, whose greatest selenium exposure comes from contaminated waters (Navarro-Alarcon & Cabrera-Vique, 2008). The levels of selenium in trophic levels of marine animals established that plankton have the lowest levels of selenium while fish had the highest concentrations (Kehrig et al., 2009). The study also found a high correlation between the size of the fish and the

concentration of selenium found. This research suggests that the older and larger the fish, the greater the biomagnification of selenium it accumulates (Kehrig et al., 2009). The larger the fish, the longer the half-life of selenium (Hamilton, 2004). Therefore, it will take a shorter amount of time for a small fish to naturally detoxify itself of selenium compared to a larger fish. Factors that influence the detoxification of selenium in fish include the cleanliness of the aquatic environment, the age of the fish, the size of the fish, season, and initial selenium concentrations in the fish (Hamilton, 2004). The type of fish that fish oil supplement manufacturers use depends on the company. Although selenium toxicity does exist, research concerning how to incorporate appropriate amounts of the mineral into fish oil supplement continues to be conducted.

ARSENIC Arsenic is a metalloid that is toxic to humans in trace doses (Juma et al., 2002). Arsenic has a natural tendency to chemically bind to iron and manganese oxides (Meador et al., 2005). These oxides are aquatic particles found in bed sediments (Casado-Martinez et al., 2010). Arsenic is a naturally occurring element found in the earth (Juma et al., 2002). However, other sources of arsenic found in the environment come from mining, agricultural, and industrial practices (Shah et al., 2009). Recently, water sources have been tested and found to be tainted with arsenic levels higher than expected (Zhang et al., 2011). Over twenty organic and inorganic chemical compounds containing arsenic have been found in marine water sources. Research has supported the argument that inorganic arsenic is more toxic than organic arsenic to organisms (Zhang et al., 2011). In addition, inorganic arsenic has been shown to be more carcinogenic than organic arsenic (CasadoMartinez et al., 2010). The type of chemical compound present in the water is influenced by several factors including redox conditions, temperature, salinity, and concentrations of phytoplankton and bacteria (Zhang et al., 2011). Marine bacteria can readily convert one form of arsenic into another (Zhang et al., 2011). Deposit feeding invertebrates introduce arsenic into the marine food chain (Shah et al., 2009). Fish prey on these invertebrates and ingest some of the arsenic particles (Shah et al., 2009). Research has suggested that fish is the most significant form of arsenic exposure in humans (Juma et al., 2002). Fish ingest suspended particles of arsenic in the water (Shah et al., 2009) and have a tendency to accumulate arsenic in their muscular tissues (Juma et al., 2002). Although it is known

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PCBs

that fish are contaminated with arsenic, the mechanisms concerning their bioaccumulation have not rendered significant scientific evidence. One study has shown that fish with higher concentrations of arsenic tend to come from areas with a higher prevalence of invertebrates (Meador et al., 2005). In addition, the study has suggested that arsenic levels in fish are directly influenced by dietary consumption (Meador et al., 2005). The amount of arsenic in the soil may also contribute to arsenic concentrations in fish (Meador et al., 2005). Research has shown that the higher the levels of arsenic in ground water, the higher the arsenic concentrations found in the local fish (Das et al., 2004). According to the EPA, it is recommended that 30% inorganic arsenic of the total arsenic concentrations in fish is to be considered in the calculations for human health criteria (Juma et al., 2002). In one study, arsenic concentrations in fish were three times that of the EPA’s recommendation of 1.0 ppm (Juma et al., 2002). Humans have no biological need for arsenic in their bodies (Juma et al., 2002). The majority of ingested organic arsenic is absorbed in the gastrointestinal tract and excreted within ten days (Juma et al., 2002). However, inorganic arsenic is more persistent and remains in the human body for a longer period of time (Juma et al., 2002). With frequent exposure, arsenic concentration can build up in the human body (Juma et al., 2002). According to the EPA, arsenic is the only metalloid that is directly related to cancer (Juma et al., 2002). The metalloid has been associated with lung, liver, skin, and bladder cancer (Shah et al., 2009). Trace concentrations of arsenic in the human body can lead to detrimental effects. Diseases associated with arsenic poisoning include skin conditions, nervous system complications, gastrointestinal system distress, and blood abnormalities (Juma et al., 2002). Additional health conditions that may ensue include melanosis, hyperkeratosis, lung disease, peripheral vascular disease, gangrene, diabetes mellitus, hypertension, and ischemic heart disease (Das et al., 2004).

CADMIUM Cadmium is a toxic metal that is naturally occurring in the environment through rocks and soil (Rashed, 2001). In addition, this metal is leached into the environment by industrial and agricultural practices (Rashed, 2001). The EPA has established an upper limit of 0.2 ppm for cadmium (Juma et al., 2002). Fish will acquire cadmium through their gills, skin, and mouth (Rashed, 2001). Cadmium selectively accumulates in the kidneys and livers of fish (Juma et al., 2002). Relatively little cadmium accrues in the muscle tissues of the fish (Juma et al., 2002). Lesser

concentrations of cadmium can be found in the fish’s gills and intestines (Rashed, 2001). In one study, ten fish species were collected and analyzed for cadmium concentrations (Al-Busaidi et al., 2011). According to the study’s findings, the highest cadmium concentrations were present in yellow fin tuna, which is a large species of fish toward the top of the food chain (Al-Busaidi et al., 2011). Research has shown that cadmium has no biological function in the human body (Juma et al., 2002). Cadmium has a long half-life. According to one study, approximately 5% of an ingested dose of cadmium is absorbed in the human body and remains in the tissue for up to thirty years (Juma et al., 2002). In long-term exposure in humans, cadmium accumulates in the kidneys for up to thirty years (Al-Busaidi et al., 2011). Cadmium toxicity is also associated with respiratory problems, liver conditions, reproductive issues, and bone disease (Al-Busaidi et al., 2011).

PCBS PCBs are organic pollutants with hydrophobic properties that resist the process of metabolism (Van den Berg et al., 1998). PCBs includes 209 chemically-related compounds with insulating and fire-retarding properties (Ross, 2004). Many forms of PCBs degrade slowly in nature, and thus build up in the food chain (Ross, 2004). PCBs affect the immune, reproductive, cardiovascular, nervous, and endocrine systems (Hopf et al., 2009). In addition, these organic pollutants are carcinogenic and overexposure to PCBs has been associated with vascular inflammation and atherosclerosis (Birnbaum & Staskal-Wikoff, 2010). PCB toxicity may lead to the inhibition of dopamine transportation (Birnbaum & Staskal-Wikoff, 2010). In humans, the most common health problems due to PCB toxicity are skin and eye problems (Ross, 2004). Multiple tests involving PCBs have been performed on various animal species. Health effects include reproductive issues, growth inhibition, immunotoxicity, liver toxicity, neurotoxicity, and dermal toxicity among other biochemical effects (Ross, 2004). For ethical purposes, similar testing has not been performed on humans. The effects of PCBs on humans have been collected through other means. One source of data collection is the history of PCBs in workers through occupational exposure (Ross, 2004). Another source includes large-scale or widespread accidental exposure in populations (Ross, 2004). A third source includes individuals with low-level environmental exposure in the form of consumption of fish known to contain PCBs (Ross, 2004). Low-level environmental exposure causes the most concern in the public. Approximately

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36. FISH OIL SUPPLEMENTS, CONTAMINANTS, AND EXCESSIVE DOSES

90% of PCB exposure in humans is because of supplements and the average diet (Fernandes et al., 2006). Humans may develop PCB toxicity by consuming fish that swim in contaminated waterways (Hopf et al., 2009). The waters are tainted by the inappropriate disposal of waste materials that contain PCBs (Hopf et al., 2009). The organic chemicals settle in the sediment rock and are accidentally consumed by bottom dwelling fish (Ross, 2004). Other fish eat the bottom dwellers and also accumulate PCBs in their systems (Ross, 2004). Although fish can metabolize some PCBs, the organic pollutants not metabolized or excreted instead accumulate in the fish’s fatty tissues (Ross, 2004). The half-life of PCBs are long in comparison to other chemical compounds (Kris-Etherton et al., 2002). The gradual accumulation of PCB ingestion in humans leads to toxicity (Hopf et al., 2009). In 2001, the European Union’s Scientific Community on Food established a weekly tolerable limit of PCBs of 14 pg WHO-TEQ kg21 body weight (Fernandes et al., 2006). One method of reducing PCB exposure is to remove the skin and fat from fish before cooking it (Kris-Etherton et al., 2002). Fortunately, enacted laws have reduced the overall exposure of PCBs in the general population (Ross, 2004). Research suggests that PCB exposure and concentrations in individuals have shown a significant decrease in the past 25 years (Ross, 2004). There are several techniques used to reduce the amount of PCBs in fish oil refining. Generally, PCBs are removed with high temperature deodorization (Maes et al., 2005). Another method of removing PCBs from fish oil is the adsorption of the PCBs on apolar adsorbents such as activated carbon. However, this method has not been proven to be very effective (Maes et al., 2005). One effective method of removing PCBs from fish oil is steam stripping the chemicals under optimized process conditions (Maes et al., 2005). The most effective process of removing PCBs is molecular distillation at an extremely hot and high pressured environment (Ross, 2004). Unfortunately, this process also removes some of the heart health fatty acids, such as DHA, found in fish products (Ross, 2004). Although these techniques are effective in reducing the amount of PCBs in fish, they do not completely remove the organic toxins. Research into ascertaining a safe level of PCBs in food is still being conducted. However, some international organizations, such as the European Union (EU), have established their own maximum levels of PCB intake. A study testing the levels of PCBs in thirty-three fish oil supplements found that twelve of them had levels that exceeded the EU’s recommended PCB limit of 14 pg WHO-TEQ kg21 body weight (Fernandes et al., 2006). Eleven of these fish oils were extracted from cod, while one of these fish oils came from salmon (Fernandes et al., 2006).

The data may show that cod fish have higher levels of PCBs compared to other fish used to produce fish oil. However, research is still being conducted to determine a universally safe level of PCBs.

DICHLORODIPHENYLTRICHLOROETHANE Dichlorodiphenyltrichloroethane (DDT) is an organochloride pesticide residue that can have toxic health risks (Carvalho et al., 2009). Although it was first introduced as an inexpensive yet effective pesticide for agricultural purposes and repels mosquitoes, DDT is toxic to the environment and potentially the human body (Beard, 2006). As a result, the pesticide has been banned in the United States since the early 1970s (Beard, 2006). However, the synthetic pesticide is chemically stable with lipophilic properties (Beard, 2006). The chemical composition of DDT makes it a persistent contaminant and it has a slow elimination rate in living organisms (Beard, 2006). A significant proportion of the population has detectable levels of the toxin in their blood or adipose tissue (Beard, 2006). Fish are commonly exposed to DDT in their marine environment (Stahl et al., 2009). Higher DDT concentrations were found in bottom dwelling fish as opposed to predator fish (Stahl et al., 2009). The EPA has not set a toxic upper limit on DDT in fish (Stahl et al., 2009). Overall, research has shown weak evidence between DDT exposure and negative health effects (Beard, 2006).

DIELDRIN Dieldrin is a synthetic pesticide commonly used between the 1950s to the 1970s for agricultural practices (Martyniuk et al., 2010b). Fish raised in salmon farms are more susceptible to dieldrin poisoning because of the contaminants in their feed (Jenkins et al., 2009). These chemicals tend to bioaccumulate and biomagnify due to their low volatile, chemically stable, and lipophilic properties (Hatcher et al., 2007). The pesticide’s highly lipophilic property causes significant accumulation in animal tissues, particularly in the brain, fat, muscle, liver, and gills (Martyniuk et al., 2010b). This lipophilic chemical crosses the blood
brain barrier and remains in the tissue of the brain (Kanthasamy et al., 2005). The liver is the primary target of acute dieldrin poisoning (Kanthasamy et al., 2005). Dieldrin in the environment has a half-life of approximately five years (Sava et al., 2007). The chemical’s persistent presence in the environment created concern and caused the United States government to ban them from the market (Hatcher et al., 2007). However, the intensive use of dieldrin in agriculture

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REFERENCES

has left sediment with heavy concentrations in some areas (Martyniuk et al., 2010b). High concentrations of dieldrin have toxic effects on dopaminergic and monoaminergic neurons (Sava et al., 2007). The pesticide affects the dopaminergic system by inducing apoptosis and oxidative stress (Martyniuk et al., 2010b). These two cellular processes have been linked to the developments of Parkinson’s disease and Alzheimer’s disease (Martyniuk et al., 2010b). The presence of dieldrin interferes with the mitochondrial oxidative phosphorylation process (Sava et al., 2007). Overall, dieldrin causes neurotoxicity and targets the central nervous systems of vertebrates (Martyniuk et al., 2010b). Dieldrin blocks the gamma-amino-butyric-acid (GABA) receptors, thus interfering with the synaptic transmission to the central nervous system (Martyniuk et al., 2010b). Physiological symptoms of dieldrin poisoning in humans include headache, nausea, vomiting, convulsion, and coma (Kanthasamy et al., 2005). The World Health Organization has established a maximum intake of 100 ng of dieldrin per kilogram of body weight (Kanthasamy et al., 2005). One United States research study found that approximately 100,000 adults consume an average of 0.5 μg of dieldrin per day. Dieldrin has a half-life of approximately three hundred days in the human body (Kanthasamy et al., 2005). As a result, dieldrin has a tendency to accumulate in the body and pose health risks. Exposure to certain pesticides, including dieldrin, has been linked to Parkinson’s disease (Hatcher et al., 2007). However, research has not been able to directly correlate this association with specific chemicals. In one research study, mice given acute and slow doses of dieldrin showed no changes in behavior (Sava et al., 2007). However, the slow infusion of dieldrin caused a dose-dependent increase in oxidative stress in all regions of the brain (Sava et al., 2007). Injecting the rats with dieldrin caused a brief depletion of dopamine, but normal levels were restored after 72 hours (Sava et al., 2007). Although this research shows that dieldrin affects dopamine levels in the brain, the doses administered to the rats were significantly higher than normal amounts to which humans are exposed on a daily basis (Sava et al., 2007). Research concerning the effects of chronic, low-dose dieldrin is still being conducted (Sava et al., 2007). Higher concentrations of dieldrin have been found in farm-raised salmon in comparison to wild salmon (Jenkins et al., 2009). Industrial contaminants play a large role in the increasing levels of toxins, including dieldrin, in water sources (Jenkins et al., 2009). The proximity of farm-raised fish locations to large manufacturing industries may play a role in this observation. In a 2010 New Zealand study, researchers found that the median concentration of dieldrin in eel

was 0.4 μg/kg (Stewart et al., 2011). However, within the 95th percentile range of dieldrin concentrations, 10.71 μg/kg of dieldrin were found in the eel (Sava et al., 2007). Although this worst case scenario is rare and highly unlikely, the study shows that there is still a nominal risk of extremely high concentrations of toxic chemicals to be found in fish.

CONCLUSION In conclusion, high concentrations of heavy metals and pesticides compromise the benefits of consuming fish oil supplements. Fish provide an excellent source of proteins, vitamins, and minerals to the human diet. In addition to vitamins A, D, and B12, fish is also a rich source of heart-healthy essential PUFAs (Pastorelli et al., 2012). However, waste products from agricultural, industrial, and municipal production processes have leached into water sources (Fallah et al., 2010). As a result, the pollutants bioaccumulate in the marine organisms that inhabit the waters. Consequently, humans who consume the fish are at risk of ingesting contaminants (Fallah et al., 2010). Although fish oil supplements supply beneficial nutrients, the heavy metal and pesticide contaminants present in fish may lead to health disorders and conditions. The benefits and risks of fish oil supplements may seem counterproductive. However, fish oil refineries are developing new technology in order to lower the contamination rates even further. The amount of contaminants in fish oil supplements is relatively minute. However, chronic exposure to these toxins may cause build-up and may result in health complications.

Acknowledgment Preparation of this review was part of course CPH 492.

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792. Yashodhara, B.M., Umakanth, S., Pappachan, J.M., Bhat, S.K., Kamath, R., Choo, B.H., 2008. Omega-3 fatty acids: a comprehensive review of their role in health and disease. Postgrad. Med. J. 85 (1000), 84
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C H A P T E R

37 Introduction to Fish Oil Oxidation, Oxidation Prevention, and Oxidation Correction Peter Lembke and Anett Schubert INTRODUCTION

within the lipid molecule. Initiators and accelerators for the formation of these radicals are:

The main source of dietary omega-3 fatty acids is cold water blue fish, in fact, fish oil. However, only approximately 30% of fish oil consists of omega-3 fatty acids. The rest consists of saturated fats, omega-6 fats, cholesterol, and other lipids (see Figure 37.1). These omega-3 fatty acids are characterized by a high degree of unsaturation (up to 6 C 5 C double bonds) and because of that they are very sensitive to oxidation. The result of the oxidation process is the breakdown of the polyunsaturated long chain fatty acid molecules to smaller, often very volatile components belonging to the ketones, aldehydes, and carboxylic acids, partly responsible for the characteristic ‘fishy’ smell and taste of a fish oil which has started to oxidize. Fresh fish and fresh fish oil do not smell fishy. For the addition of fish oil to food, beverages, supplements, etc., this fishy smell and taste is of huge disadvantage and has to be controlled. In this paper we will discuss very briefly the fish oil oxidation process, its consequences, and some important techniques to prevent it. The fish oil oxidation process is very complex and is not yet fully understood. We have therefore tried to keep it as simple as possible, introducing the reader to this interesting subject and giving helpful hints for the daily handling of these sensitive oils, but not wanting to confuse the reader with detailed chemical oxidation pathways of the polyunsaturated fatty acids. For more detailed information we suggest, for example, Frankel (1991), (2005), Choe (2006), and Velasco et al. (2003).

OXIDATION PROCESS The oxidation process is a very complex reaction which usually starts with the formation of free radicals Omega-3 Fatty Acids in Brain and Neurological Health. DOI: http://dx.doi.org/10.1016/B978-0-12-410527-0.00037-5

• • • •

Heat and already thermally oxidized compounds. Free fatty acids, mono- and diacylglycerols. Metal ions (mostly iron). Sunlight.

In the presence of one or more of these initiators, the omega-3 fatty acids, which belong to the longchain polyunsaturated fatty acids (LCPUFAs) may lose a hydrogen radical, thus forming a lipid free radical counterpart. The lipid free radicals are able to react with oxygen molecules under formation of hydroperoxides. The hydroperoxides tend to decompose to smaller molecules belonging to the group of saturated and unsaturated ketones or aldehydes and short chain carboxylic acids which, as mentioned above, contribute significantly to the fishy off flavor of ‘old fish’ and oxidized fish oil. Figure 37.2 shows a simplified oxidation pathway of a fish oil and Table 37.1 lists some of the main volatile compounds found in oxidized fish oil and their corresponding flavors. Due to the high degree of unsaturation of the LCPUFAs there are many different positions within the fatty acid molecule where the radical formation can take place. Hence numerous oxidation products can be expected.

OXIDATION INDICATORS The fact that especially unsaturated oils oxidize easily, become rancid, and finally spoil a food has been known for a long time. Therefore, the need for reliable measurement of the oxidative stability of polyunsaturated oils is not new and has been

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37. INTRODUCTION TO FISH OIL OXIDATION, OXIDATION PREVENTION, AND OXIDATION CORRECTION

Polyunsaturated fatty acid

7%

Sunlight, metal ions, heat –H* 43%

Omega-3 fats

Lipid radical

Other fats

+ O2

Cholesterol 50%

Hydroperoxide metal ions

FIGURE 37.1 Constituents of fish oil.

Aldehydes Ketones Carboxylic acid

Volatile compounds in rancid fish oils causing undesired fishy smell and taste

reviewed many times, for example, by Gray (1978) and Hamilton (1994). According to what has been discussed above the oxidation of an omega-3 oil containing LCPUFAs results in the formation of:

FIGURE 37.2 A simplified oxidation pathway of a fish oil.

• Hydroperoxides, also known as primary oxidation products. • Ketones, aldehydes, and carboxylic acids, also known as secondary oxidation products.

Compound

Odor

2,4-Heptadienal

Rancid hazelnuts

2-Hexanal

Green grass

2,4,7-Decatrienal

Oxidized fish oil

1-Octen-3-ol

Mushroom, melon

1,5-Octadien-3-ol

Mushroom, seaweed

2-Nonenal

Cucumber

As these two groups of compounds have been shown to be characteristic for fish oil oxidation, it makes sense that they were chosen as oxidation indicators for standard analytical methods used to characterize the state of oxidation of a fish oil or omega-3 concentrate: Peroxide value (POV): This analytical method detects and quantifies directly the generated hydroperoxides in oxidized oil. The POV is expressed in milli-equivalents of active oxygen (meq O2) contained in 1000 g of the substance or oil. As long as the POV is below 10 meq O2/Kg the oil is considered as suitable for human consumption. We recommend good quality oils and omega-3 supplements should have a POV below 5 or even 3 meq O2/Kg. However, such a low POV result is no guarantee for a fresh omega-3 oil. Unfortunately, the POV has a disadvantage in that the stability of the peroxides which are measured is very short. This is why they are referred to as primary oxidation products. Consequently, if you wait long enough, the hydroperoxides will decompose to the secondary oxidation products, which the POV method cannot detect. In other words, a low POV alone can be very misleading and is not a reliable guarantee for the freshness of fish oils or omega-3 concentrates. Therefore it makes sense to also analyze the secondary oxidation products in order to evaluate the freshness of fish oils. To analyze these secondary oxidation products the Anisidine value (AV) was developed. The AV is defined as the 100-fold optical density measured in a 1 cm optical cell containing a solution of 1 g

TABLE 37.1 Common Volatile Compounds Found in Oxidized Fish Oil and Omega-3 Concentrates

substance/oil to be examined in 100 mL of a defined solvent mixture. AVs of up to 20 are still considered as acceptable for omega-3 oils. In our view, good products should strive for an AV of 15 or even lower. In practice, the best way to describe the freshness of an omega-3 oil is to consider both the POV and AV. This is done by the total oxidation (TOTOX) value which is defined as: • TOTOX 5 (2 3 POV) 1 AV As long as the TOTOX value is below 26 an omega3 oil is still considered fresh and acceptable for human consumption. According to our experience a good quality fish oil or omega-3 oil should comply with: • TOTOX ,20 • AV ,15 • POV , 5 During the oxidation process the concentration of the individual oxidation indicators changes with time, as shown in Figure 37.2. In the first phase of the oxidation process the hydro-peroxides are formed and the

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OXIDATION INDICATORS

Relative intensity

200

150 POV AV TOTOX

100

50

0 0

2

4

6

8

Relative time

FIGURE 37.3

Changes in POV, AV, and TOTOX values

over time.

POV increases rapidly. The next step is the breakdown of the now oxidized LCPUFA molecules to smaller secondary oxidation compounds (ketones and aldehydes). This slows down and eventually even reverses the growth of the POV curve, while the Anisidine curve shows a steady growth. Analyzing at that point of time only the POV will falsely indicate a qualitatively good oil. At this point only the AV discloses its true state of oxidation. On the other hand, as shown in Figure 37.3 relying only on the AV, especially during the initial oxidation phase of the oxidation process, is also no solution. The best choice is the combination of both oxidation parameters, expressed as the TOTOX value. Figure 37.3 shows that the TOTOX value has the advantage that it will never return to a misleading zero value once the oxidation process has started. However, in the case of fish oils and their derivatives, one has to be aware of the fact that all the above methods to analyze the state of oxidation are far too insensitive to make any statement on the organoleptic properties of the oil. Typical volatile compounds identified in oxidized fish oil are, among many others, cis-4.heptenal, trans, cis-2,6-nonadienal and cis, cis-3,6-nonadienal, which have extremely low sensory threshold values, going as low as 600 ppb. These concentrations are so low that the chemical method of analysis described above will not be able to detect them, but our nose and tastebuds will do. This explains why a fish oil or omega-3 concentrate may show reasonably good POV and AV, but may already have an undesired ‘fishy’ taste and/or smell.

Prevention of Oxidation and Oxidative Stability The oxidative stability of an oil, the importance and mechanisms of antioxidants and the way to determine

its shelf-life and consumer acceptance has been discussed in the past many times in the literature, for example by Hamilton, (1994), Hamilton et al. (1998), and Frankel, (2007a). There are several possibilities to protect fish oil against oxidation. First of all, and most importantly, the fish oil should be fresh. To avoid oxidation the oil should be stored and handled under an inert gas atmosphere (nitrogen (N2), argon (Ar), carbon dioxide (CO2)). The oil should also be stored in an airtight container that protects it not only from oxidation but also from light. It is well known that light can initiate radical formation. Heat also should be avoided because of the possible thermo-dissociation of hydroperoxides (see below): Lipid
OOH-heat-Lipid-O 1  OH Metal ions should be avoided because they act as catalysts in the oxidation process, especially during the hydroperoxide decomposition step leading to the lipid radicals (Lipid-O ). In general the oxidation process can be described in three basic steps: 1. Initiation phase: triggered by light, metal ions, and existing peroxides. This leads to the formation of free radicals. 2. Propagation phase: requires the presence of oxygen and leads to the formation of hydroperoxides (primary oxidation products). 3. Termination phase: coupling of the radicals to nonreactive, often volatile compounds (secondary oxidation products). If we want to avoid or reduce oxidative decomposition of fish oil and thus prolong its shelf-life, we have different options as to how to do this. No doubt the best way is to avoid step 1, the initiation phase. As mentioned above, this can be achieved by choosing the freshest possible fish oil available, avoiding direct light, metal traces, and preformed peroxides. In order to interrupt and stop the propagation phase, the oil has to be protected from direct contact with oxygen. This is usually done by nitrogen flushing and blanketing. Instead of N2, other chemically inert gases can be used, for example, CO2 or Ar. In contrast to N2, these gases are a little more expensive but have the advantage that they are heavier than air and thus remain as a protection layer on the oil. If you open a fish oil drum flushed with N2, the N2 will ‘evaporate’ as it is lighter than air, and oxygen (O2) will get into contact with the oil. Using an inert gas which is heavier than air will maintain a protective layer on top of your oil, reducing the risk of forming hydroperoxides. Very important for the oxidative protection during this propagation and termination step is the addition

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TABLE 37.2 Commonly Used Antioxidants, their Mode of Protection, and their Typical Applied Concentration Range Oxygen- and Lipid Radicals

LightMetal Induced Catalysis Radicals

Typ. Conc. Range (ppm)

α-Tocopherol X (,100 ppm)



MixedTocopherol

X



Ascorbylpalmitate

X



Ascorbic acid

X



Citric acid

X



X



X



Lecithin

X

EDTA Rosemary extract

X



Sage extract

X



N2 or Ar blanket

X

Light-proof container

X

N/A

Micro(X) encapsulation

X

N/A



5 μM.

of suitable antioxidants belonging to the group of hydroperoxide inhibitors and metal chelators (see below) (Frankel, 2007b). Due to the fact that the oxidation process is triggered by several causes, it is recommended to utilize not only one type of antioxidant but a mixture of different antioxidants having distinctive protection characteristics as previously discussed, for example, by Drusch et al. (2008). Typical antioxidant mixes are: α-tocopherol in combination with citric acid and/or ethylene-diamine-tetra-acetic acid (EDTA). Another possibility is to combine phenolic compounds, for instance, in Rosemary oil, with lecithin, and tocopherol. Table 37.2 shows common antioxidants, their mode of action, and their typical applied concentration range. Table 37.2 distinguishes between three different categories of antioxidants. Hydroperoxide inhibitors like α-tocopherols or BHA and BHT, also known as hydroperoxide destroyers or chain-breaking antioxidants, deactivate the lipidand hydroperoxide radicals (‘Lipid-O ’ and ‘ROO ’ below) by rapidly donating hydrogen atoms and converting themselves into relatively stable antioxidant radicals before they react among each other forming stable unreactive compounds. ROO 1 A-H-ROOH 1 A

ROO 1 A -ROOA ðunreactiveÞ Lipid-O 1 AH-LOH 1 A Lipid-O 1 A -LOA ðunreactiveÞ A 1 A -A 2 A ðunreactiveÞ In the case of the different tocopherol isomers, it should be mentioned that α-tocopherol has much stronger antioxidant properties than the ‘vitamin active’ γ-isomer (Kula˚s & Ackman, 2001a). Trace metal chelators, also sometimes called ‘preventive antioxidants,’ like citric acid, ascorbic acid or EDTA and lecithin, effectively bind traces of metals, such as iron, in the oil and oil-in-water emulsions. These metals are essential catalysts for the formation of the lipid- and oxygen radicals (Frankel et al., 2002). The preventive antioxidants interfere in one of the initial stages of the oxidation process, reducing significantly the amount of free radicals in the oil and hence ensuring long-term shelf-life and stability. Synergistic compounds like lecithin and phenolic rosemary or sage extracts rich in carnosic acid, carnosol and rosmarinic acid, are characterized by the fact that both peroxide initiation and subsequent autoxidation reactions are effectively suppressed. Lecithin, for example, can act as a binding agent for free metal ions in the oil and, additionally, due to its emulsifying properties, improve the direct contact between the polar phenolic Rosemary extract and the lipophilic polyunsaturated fatty acids. Lecithin and ascorbic acid are also known for their synergistic properties (Yi et al., 1991).

OXIDATION CORRECTION After oxidation has occurred, technically it is possible to ‘correct’ this status and remove the undesired oxidation products from the oil, turning it back into an excellent product. Andersen (1962) discussed this topic more than 50 years ago. It has also received more recent comment by Brekke (1980) who discussed the correction of the oxidation status with vegetable oils. Table 37.3 shows a selection of commonly applied technical procedures and their suitability for correcting the oxidation status of a fish oil or omega-3 concentrate. The most popular process is that of deodorization. In this process, hot steam up to 210 C is pumped through the oil under a vacuum. The heat accelerates the decomposition of the hydroperoxides into volatile short-chain ketones, aldehydes, and carboxylic acids, which are then carried away together with the steam as it passes

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

459

REFERENCES

TABLE 37.3 Selection of Commonly Applied Technical Procedures and their Suitability to Correct the Oxidation Status of Fish Oil or Omega-3 Concentrates AnV

Color Fishy Taste TOTOX Reduction & Smell

Deodorization Yes

Yes

Yes

Yes

Yes

Mol. dest.

Yes

Yes

Yes

Yes

Process

POV

Yes

Active carbon Limited Limited Limited Yes

Limited

SFE & SFC

Yes

Yes

Yes

Yes

Yes

Urea fract.

No

No

No

No

No

Enzymes

No

No

No

No

No

HPLC

Limited Limited Limited Limited

No

through the oil (Cesarettin et al., 2011). Pigments, tocopherols, hydrocarbons, and alcohols are partly removed. The result of the deodorization process depends strongly on the quality of the incoming oil and the technical abilities of the deodorizing plant. Strongly oxidized fish oil will be very difficult to deodorize completely with this technology. Therefore it is always better to make sure that even the crude fish oil is protected against oxidation from the very beginning. The steam used during the deodorization process, which heats up the oil to between 160 C and 270 C has to be as much as possible free of oxygen, otherwise it would be counterproductive and even cause more oxidation problems. In order to avoid excessive process temperatures, typically a vacuum is applied. The stronger the applied vacuum, the lower the required temperature to deodorize the oils and the gentler is the process. The deodorization process can produce an oil which is almost free from primary oxidation products. The POV can be reduced to almost zero, providing that sufficiently high temperatures and extended process times are applied. However, high temperatures in combination with prolonged process times always bear the risk of undesired isomer formation and AVs cannot be reduced very effectively with this technology. Another popular process to remove primary and secondary oxidation products is Molecular Distillation (MD). With this technology volatile oxidation compounds are removed from the oil under a strong vacuum and at relatively low temperatures of approximately 140 C. This is why the MD process is generally considered as a more gentle process than the classical deodorization technology. An even milder method to remove oxidation products from a fish oil or omega-3 concentrate is the use of highly compressed CO2 at temperatures not exceeding 45 C. This technology, known as Supercritical

Fluid Extraction (SFE) and Supercritical Fluid Chromatography (SFC), makes use of the fact that the volatile oxidation compounds can be removed from the oil in a counter- or co-currant mode, resulting in qualitatively superior products which have suffered no thermal stress.

SUMMARY Only approximately 32% of fish oil consists of omega-3 fatty acids. Two of the most important omega-3 fatty acids are EPA and DHA, which are characterized by having five or even six double bonds. This high degree of unsaturation is the reason why the fish oils and especially the omega-3 concentrates are so sensitive towards oxidation and thermal decomposition. The primary oxidation products, known as hydroperoxides, decompose to the secondary oxidation products belonging to the group of ketones, aldehydes, and carboxylic acids. These small and often very volatile molecules contribute significantly to the undesired fishy off-flavor found in oxidized fish oils and omega-3 concentrates. The best way to characterize oxidized fish oil is to determine the POV, the AV and then use both of these values to calculate the TOTOX value. A good fish oil or omega-3 concentrate should have a TOTOX value of below 15. To protect the oils against oxidation the most important preventative step is to select the freshest crude fish oil available. Once chosen, this oil should be stored under an inert gas atmosphere (N2, Ar, CO2, etc.), in an airtight container protected from direct sunlight. During storage, a mixture of antioxidants is generally much more effective against oxidation than the use of only one antioxidant. The specific antioxidant mixture that should be used depends on the composition of the oil, its storage container, and storage conditions. Once oxidation has occurred, common processes like adsorption, deodorization, molecular distillation, and/or supercritical fluid technology can be applied to remove the undesired oxidation products from the oil and improve its organoleptic profile.

References Andersen, A.J., 1962. Refining of Oils and Fats, second ed. Oxford, Pergamon, p. 166. Brekke, O.L., 1980. Handbook of Soy Oil Processing and Utilization. American Soybean Association, St. Louis, MO, p. 166. Cesarettin, A., Kazou, M., Fereidoon, S., Udaya, W. (Eds.), 2011. Handbook of Seafood Quality. John Wiley & Sons. Drusch, S., Groß, N., Schwarz, K., 2008. Efficient stabilization of bulk oil rich in long-chain polyunsaturated fatty acids. Eur. J. Lipid Sci. Technol. 110, 351
359.

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

460

37. INTRODUCTION TO FISH OIL OXIDATION, OXIDATION PREVENTION, AND OXIDATION CORRECTION

Frankel, E.N., 1991. Recent advances in lipid oxidation. J. Sci. Food Agric. 54, 495
511. Frankel, E.N., 2005. , The Oily Press .second edition Lipid Oxidation, vol 18. The Oily Press Lipid Library, Bridgwater, UK, p. 1
98. Frankel, E.N., 2007a. Antioxidants in Food and Biology: Facts and Fiction. The Oily Press, Bridgwater, UK. Frankel, E.N., 2007b. May 12
13. Lipid Oxidation and Antioxidants AOCS Short Course, Quebec City, Canada. Frankel, E.N., Satue-Gracia, T., Meyer, A.S., German, J.B., 2002. Oxidative stability of fish and algae oils containing long-chain polyunsaturated fatty acids in bulk and in oil-in-water emulsions. J. Agric. Food Chem. 50 (7), 2094
2099. Gray, J.I., 1978. Measurement of lipid oxidation: a review. J. Am. Oil Chem. Soc. 55 (6), 539
546. Hamilton, R.J., 1994. The chemistry of rancidity in foods. In: Allen, J.C., Hamilton, R.J. (Eds.), Rancidity in Foods, third ed. Blackie Academic & Professional, London, pp. P1
21. Hamilton, R.J., Kalu, C., McNeill, G.P., Padley, F.B., Pierce, J.H., 1998. Effects of tocopherols, ascorbyl palmitate, and lecithin on autoxidation of fish oil. J. Am. Oil Chem. Soc. 75, 813
822. Kula˚s, E., Ackman, R.G., 2001a. Different tocopherols and the relationship between two methods for determination of primary oxidation products in fish oil. J. Agric. Food Chem. 49, 1724
1829.

Velasco, J., Andersen, M.L., Skibsted, L.H., 2003. Evaluation of oxidative stability of vegetable oils by monitoring the tendency to radical formation. A comparison of electron spin resonance spectroscopy with the rancimat method and differential scanning calorimetry. Food Chem. 77, 623
632. Yi, O.S., Han, D., Shin, H.K., 1991. Synergistic antioxidative effects of tocopherol and ascorbic acid in fish oil/lecithin/water systems. J. Am. Oil Chem. Soc. 68, 881
883.

Further Reading Chaiyasit, W., Elias, R.Y., McClements, D.J., Deckler, E., 2007. Role of physical structures in bulk oils on lipid oxidation. Crit. Rev. Food Sci. Nutr. 47, 299
317. Choe, E., Min, D.B., 2006. Mechanisms and factors for edible oil oxidation. Compr. Rev. Food Sci. Food Saf. 5, 169
186. Kula˚s, E., Ackman, R.G., 2001b. Properties of α-, γ- and δ- tocopherol in purified fish oil triacylglycerols. J. Am. Oil Chem. Soc. 78, 361
367. Serfer, Y., Drusch, S., Schwarz, K., 2009. Chemical stabilization of oils rich in long-chain polyunsaturated fatty acids during homogensisation, microencapsulation and storage. Food Chem. 113, 1106
1112.

OMEGA-3 FATTY ACIDS IN BRAIN AND NEUROLOGICAL HEALTH

Index Note: Page numbers followed by “f ” and “t”refers to figures and tables respectively.

A ABCD1 gene, 23
24 Acid-CoA:amino acid N-acyltransferase, 95
97 Acyl-CoA:amino acid N-acyltransferase 1 (ACNAT1), 97
98 Acyl-CoA dehydrogenase, 20
21 Acyl-CoA oxidase (ACOX), 20
21 deficiency, 24 Acyl-CoA thioesters, 89
90 ADHD Rating Scale-IV, 191 Adrenocortical atrophy, 25 Adrenocorticotropic hormone (ACTH) receptor, 25
26 Adrenoleukodystrophy (ADL), 25, 350
351 Adrenomyeloneuropathy (AMN), 24, 350
351 Adult hippocampal neurogenesis (AHN), 170, 175, 252, 254
259 impact of diet on, 170
171 link between learning and, 251 mechanism of action, 254
259 omega-3 fatty acids and, 170 survival of new neurons in, 251 Adult-onset neurodegenerative diseases, 43
44 therapeutic intervention with antioxidants, 43
44 Advanced glycation end products (AGEs), 64 AF3442, 138 Age-1 mutant, 12
13 Aging, 251 age-related diseases and, 10 beginning of, 9 biological principles of, 9 brain, 148 DHA concentrations and, 147
148 effects on biosynthesis, 148
150 free radical theory of aging (FRTA), 10
11 genes associated with, 11
12 genetic contribution to, 9 genetics, post-genetics, and epigenetics of aging, 11
12 homeostasis vs homeodynamics, 9
10 interventions, 12
14 gene therapy for, 12
14 manipulating the milieu, 14 modulatory and other effects of hormesis, 14
15 molecular basis of, 10
11 phenotype of, 9 as a post-genetic emergent phenomenon, 9 protein error theory of, 11 synaptic plasticity and, 153
155 AH23848, 138
140 Alcohol abuse, 165

Alcohol dehydrogenase (ADH), 95
97 Aldehyde dehydrogenase (AlDH), 95
97 Aldehyde dehydrogenase 2 (ALDH2), 65 Alexander’s disease, 24, 28 Alkydihydroxyacetone phosphate (alkylDHAP) synthase, 19
20 Alzheimer’s disease (AD), 10, 23, 73
75, 111
112, 131
132, 147, 149, 153
154, 231
232, 251
252, 314, 319
322, 329, 373, 405, 429 antioxidant levels in, 38 average life expectancy after diagnosis, 231 chemical changes, 210 cognitive changes, 210 diabetes in animal studies, 233
234 DHA deficiency and neurological function affected by, 232
233 disease characteristics, 209
210 fatty acids and, 435
440 saturated, 435
436 free radicals (FR) and, 38 lipid peroxidation biomarkers for, 39t omega-3 fatty acid supplementation and, 83, 232
235 oxidation-associated metabolites, 38 progression of, 210
211 structural changes, 209
210 Alzheimer’s Disease Assessment ScaleCognitive Subscale (ADAS-cog), 152
153 Amyotrophic lateral sclerosis, 41
42 lipid peroxidation biomarkers for, 39t neurodegenerative mechanisms in, 41
42 pathology of, 41
42 spinal cord from sporadic ALS (SALS), 41 Anandamide. see Narachidonoylethanolamine (anandamide) Animal tests and longevity, 5 monkeys, 5 Anti-aging therapies, 12
14 genetic interventions, 12
14 Antioxidant defenses, 31 Anxiety disorders, 184
185 Apnea-hypopnea index (AHI), 337 Apolipoprotein E (ApoE), 66 APP-GAL4, 329 Arachidonic acid (AA), 69
70, 87, 109, 140, 201, 257, 303, 338
339, 349, 359 lipid mediators of, 69
70 Arachidonylethanolamide (AEA), 69
70, 258 2-arachidonylglycerol (2-AG), 69
70 Arsenic, 450
451 Arylsulfatase-A, 27
28

461

Ashwagandharishta, 172
173 Aspirin, 72 ATP-binding cassette (ABC) transporter proteins, 23
24 ATP-binding transport protein (ALDP), 24 Attention deficit hyperactivity disorder (ADHD), 121, 125, 187
193, 231, 355 development of, 187
188 diagnosis of, 187 fatty acid levels in blood of patients, 193 omega-3, 193 in the field of special education (SE), 187 fish oil, study of effects analysis, 188 on behavioral/physical symptoms, 190
192 findings, 188
193, 189t purpose, 188 hyperactivity-impulsivity symptoms, 187 malondialdehyde (MDA) in, 193 symptoms of inattention, hyperactivity, and impulsivity, 187
188 treatments for, 188 Autism (or autistic disorder), 354
355 Autistic spectrum disorders (ASDs), 121, 126 Azo-initiator (AMVN), 377

B Baby boomer generation of 1960s, 1 Bayley Scales of Infant Development, 124 Bile acid-CoA:amino acid N-acyltransferase (BAAT), 97
98 Biochemical entities, 19 Biological aging, 9 Blood-brain-barrier (BBB), 222 BN52021, 141 Body mass index (BMI), 57
58. see also Obesity Brain aging, 148 effect of fatty acid consumption, 201 pathological, 209
212 physiologic, 202
205 chemical changes, 203
204 cognitive changes, 204
205 structural, 202
203 vascularization of brain, 221
222 Brain-derived neurotrophic factor (BDNF), 59, 113
116, 170, 175, 175f, 207, 242
243, 255, 269, 362 Brain development, 163, 181 catecholamine biosynthesis, 121 critical periods of, 113
114 essential fatty acids as neuroprotectors during, 237
238 omega-3 fatty acids, role of, 109
112 central visual connections, 114
117

462

INDEX

C Ca-dependent cytosolic phospholipase A2 (cPLA2), 138 Ca21-independent phospholipase A2 (iPLA2), 155 Cadmium, 451 Caenorhabditis elegans, 12
13 Caffeine, 165, 171 Calcitonin gene-related peptide (CGRP), 279 Calcium-activated PE N-acyltransferase (CaNAT), 91
93 Calcium-independent PE N-acyltransferase (iNAT), 91
93 Caloric restriction for longevity, 2 Cancer, 10 Cannabis, 90 Carbonyl (2,4-dinitrophenylhydrazine), 43 Cardiovascular risk factors, Aging, and Incidence of Dementia (CAIDE) studies, 405 Catalase (CAT), 239 Cataracts, 10 Cerebral demyelinating form (CALD), 24 Cerebral ischemia, 42 Childhood depression, 121, 125
126 Childhood developmental disorders, 124
126 Children’s Depressive Rating Scale (CDRS), 125
126 Cholesterol, 25, 31, 222 fatty acid composition and, 351
352 Chondroitin sulphate proteoglycans (CSPGs), 113
114 Chronic constriction injury (CCI), 133
134 Chronic or arrested cerebral X-ALD, 24 CJ-023423, 138
140 CJ-042794, 138
140 Clinical depression cell membrane integrity and fluidity, 169
170 neurological alterations in, 167
169 neuro-histological changes, 168f omega-3 fatty acids, role in, 165
167 in MDD, 171
173, 182 pro-inflammatory cytokines, role of, 169
170 PUFAs and, 340 Clinical Global Impression (CGI) Scale, 191 Cognitive impairment, 398 obesity and, 57
58 neurological measures, 58 Western diets and, 58
61 neuroendocrine mechanisms, 59
61 Composite International Diagnostic Interview, 123 Conditioned-place preference (CPP) paradigm, 59 Conjugated linoleic acid (CLA), 22, 22f, 22t metabolism, 22 Conners’ Adult ADHD Rating Scale (CAARS), 192
193 Conners’ Teacher/Parent Rating Scale (CRST), 190 Conners’ Teacher Rating Scales (CTRS), 193 C-reactive protein (CRP), 222
223

Cu,Zn-superoxide dismutase (Cu,Zn-SOD), 41
43 Curcumin, 171 Cyclization of peroxyl radical, 31 Cyclooxygenase-2 (COX-2), 72, 93 Cyclo-oxygenase enzyme system, 170 Cyclopentenone-isoprostanes (A3/J3-IsoPs), 69
70 CYP27A1, 37 CYP46A1, 37 CysLT1, 140 Cytochrome P450 (CYP450), 93, 138

D D-bifunctional protein (DBP), 24 Dementia with Lewy bodies (DLB), 375
376 Demyelination, 27
28 Deodorization process, 459 Depressive illness, 353
354 Developmental neurogenesis, 253
254 DHA/EPA supplementation, 125
126, 148, 183, 353 cognitive function outcome, 234 for myocardial infarction (MI), 183 DHA-GPR40-pCREB-BDNF pathway, 257
258 DHA-GPR40 signaling, 257
258 Diabetes mellitus, 63 Diabetic neuropathy (DN) ApoE polymorphism in, 66 general characteristics, 63 genetic polymorphisms, 65 genetic risk factors, 64
66 clinical implication, 66
67 genes in progression, 66t homocysteine (Hcy) in, 66 inflammation, 65
66 neuronal degeneration and regeneration, 63 NO production, 66 pathophysiological mechanisms, 63
64, 64t risk factors, 63
64, 64t studies, 64
65 symmetric distal sensory/sensorimotor polyneuropathy, 63 VEGF polymorphism, 66 Diacylglycerol (DAG), 332 Diary consumption and health outcomes cognition functions assessment of, 411 cross-sectional studies, 409
410 randomized controlled trials, 410 cross-sectional studies, 405 dairy food intake, assessment of, 411 discussion of studies, 410
413 epidemiological and clinical significance, 412
413 literature review, 403
405, 404t observational studies, 405
408 outcome measures of studies, 406t prospective studies, 405 Dichlorodiphenyltrichloroethane (DDT), 447, 452 Dieldrin, 452
453

Dietary omega-6 fatty acids, 2 Dietary questionnaire (DFS-SQ), 59 Diet-induced obesity (DIO), 2, 223 Dihomogamma-linolenic acid (DGLA), 338
339 Dihomo-γ-linolenic acid (DGLA), 349 Di-hydrocholestanoic acid (DHCA), 20 Dihydroxyacetone phosphate acyltransferase (DHAPAT), 19
20 Dihydroxyeicosatetraenoyltaurines (diHETETs), 98 DJ-1 protein, 41 D2-LA oxidation, 377 Docosahexaenoic acid (DHA), 69
70, 121, 131, 169
170, 181, 188, 201, 232, 237, 287, 338
339, 349, 359, 429, 447 aging and, 147
148 alterations, 148 effects on biosynthesis, 148
150 from ALA, 287 biotransformation of, 111 in brain, 70
71 circuitry, 110 derived lipid mediators in, 73 child mental health, 123
126 attention deficit hyperactivity disorder (ADHD), 125 autistic spectrum disorders (ASDs), 126 at childhood, 124 childhood depression, 125
126 childhood developmental disorders, 124
126 at infancy, 123
124 neurodevelopmental outcomes, 123
124 cognitive decline and dementia, curative effect for, 438
440 deficiency, impact of, 111, 121, 123
124 transgene-dependent caspase activation, 232
233 dendritic spines and synapses, effect on, 111 deprivation and effects of a gestational/ neonatal, 115 derived protectins and neuroprotectins, 73
76, 74f dietary intake of, 71 and QoL, 84 dietary provision of pre-formed, 287 D-series resolvins, 73 effects of daily consumption, 131
132 enzymic lipid mediators of, 69
70 from flax seed oil, 173 as a free form, 111 functions, 70
71 during gestational period, 123 in gestational period, 122 on hippocampal neurogenesis, 258
259 GPR40 signaling in, 257
258 incorporation and turnover into brain phospholipid, 267f incorporation into neuronal membranes, 110
111 learning and memory, role in, 149
150 animal studies, 149
150 human studies, 150

INDEX

maternal health and postpartum depression, 122
123 maternal intake during pregnancy, recommendations, 122 membrane integrity and fluidity, role in maintaining, 169 neurological disorders, role in, 76
78 and neurological function affected by diabetes in Alzheimer’s disease, 232
233 neuroplastic changes with ocular enucleation, 268 neuroprotective effects of, 238 omega-3 nutritional restriction and, 116
117, 116f pain regulatory mechanisms of, 135
136 Parkinson’s disease and, 241 in preventing age-related memory decline, 150
153 animal studies, 150
152 human studies, 152
153 quantitative imaging of metabolism, 265
272 and AA releasing enzymes in an animal model of metabolic syndrome, 269 baseline brain DHA incorporation, 271, 271f brain with fatty acid neuroimaging, 268
269 chronic alcoholics, 271
272 imaging membrane synthesis, 268
269 of iPLA2β and iPLA2γ, 270
271 mutations in the PLA2G6 gene, 270 neuroplastic changes, 268 neurotransmission, 269
271 N-methyl-D-aspartate (NMDA) glutamatergic receptors, 269
270, 270f partial volume error (PVE) corrections, 271
272 PET scan, 270
272, 271f regulation activities, 111 status and depressive symptoms, relation, 123 synaptic plasticity and, 153
155 in visual acuity, 110 Docosahexaenoic acid (DHA/omega-3), 109 Docosapentaenoic acid (DPA), 265, 287 Dopamine cell loss induced by EFA dietary restriction, 241
244 Doublecortin (DCX), 252
253 Down syndrome, 42
43 lipid peroxidation biomarkers for, 39t Drebrin, 232 Drosophila models genetic screening, 333 genetic tools for, 327
329 huntingtin (Htt)-induced toxicity in, 332 linoleic acid, effects of, 331f of neurodegenerative diseases, 327
330 effects of lipids and lipid signaling on, 330
334 perspective, 334 phosphatidylethanolamine depletion onγsecretase-mediated APP processing, 332

points to consider, 334 PUFA and cholesterol levels, effects of, 330
332 reporters for amyloid precursor protein γ-secretase activity in, 329 representative, 329
330 TRP channels and, 333
334

E Early growth response transcriptional regulator (Egr3), 258
259 Edinburgh Postpartum Depression Scale (EPDS), 123 Eicosanoid-independent proapoptotic pathways, 70
71 Eicosapentaenoic acid (EPA), 69
70, 82
83, 115, 131, 163
164, 170, 181, 287, 297, 303, 338
339, 349, 378, 447 as anti-depressant medication, 172 on attention levels, 191 in brain, 71
78 cognitive decline and dementia, curative effect for, 438
440 for depressed patients with cardiovascular disorders, 183 for depressed patients with diabetes, 183
184 enzymic lipid mediators of, 69
70 15-LOX-like enzyme and, 72 neurological disorders, role in, 76
78, 77f nonenzymic lipid mediators of, 69
70 pregnant depressed patients, 184 resolvins of the E-series, synthesis of, 72, 72f 2-Eicosapentanoylglycerol (EPG), 258 Elmiric acids, 93
94 Endocannabinoids, 258 Environmental factors affecting longevity, 4
5 EpFAs, 137 Epigenetics of aging, 12 Epoxy-docosapentaenoic acid (EpDPE), 137 E-selectin, 226 Essential fatty acids (EFAs), 109
110, 192. see also Omega-3 fatty acids; Omega-6 fatty acids balance of omega-3/omega-6 fatty acids, 110 conversion of, 109
110 dietary intake of, 109
110 dopamine cell loss induced by EFA dietary restriction, 241
244 metabolism of constitutional, 109
110 pathways, 110f Essential lifespan (ELS), 9 Ether lipids, 19
20 Ethylene-diamine-tetra-acetic acid (EDTA), 457
458 Ethyl-EPA treatment, 70, 352
353 Evans blue-albumin (EBA), 71
72

F F2-isoprostanes (F2-IsoPs), 33 F4-isoprostanes, 232 Fagan Test of Infant Intelligence, 309

463 Farnesol X activated receptors (FXRs), 255 Fatty acid amide hydrolase (FAAH), 89, 102, 258 Fatty acid amides, 87 neurological disease and, 99
102 primary, 87
90 biological effects, 88
89 cellular concentration of, 89 functions, 89t identification, 88t metabolism, 90f pathway(s) responsible for, 89 R1
CO
NH
R2, 87 Fatty acid binding protein (FABP), 133
134 Fatty acid deficiency symptoms (FADS), 192 Fatty acid (FA) metabolism, 20 Fatty acids AD and, 435
440 biochemistry of, 205 blood
brain barrier (BBB) and, 132
133, 133f classification of, 131, 132f composition of the brain, 206 function in brain, 132
134 memory and hippocampus, 429
435 animal studies, 429
433 human studies, 431
432 neuroinflammation, 431 neurological changes with SFA consumption, 430
431 omega, 206. see also Omega-3 fatty acids; Omega-6 fatty acids metabolism, 208
209 sources of, 208 Fatty acid translocase/cluster of differentiation 36 (FAT/CD36), 133 Fatty acid transporter protein (FATP), 133 Fatty alcohol dehydrogenase (fADH), 93 Fatty aldehyde dehydrogenase (fAldDH), 93 Fish oil supplementation, 115, 147
149, 181, 311
314, 447, 456f aggression and in schoolchildren, 360 in young adults, 360f beneficial effects of, 152
153, 226 brain phospholipid composition, role in modifying, 147
148 in preventing age-related memory decline, 150
153 animal studies, 150
152 human studies, 152
153 as a treatment for major depression, 182 Flavonoids, 171 Flax seed oil anti-depressant activity of, 173
175 antiepileptic activity of, 172
173 EPA plus DHA from, 173, 174f Food restriction for enhanced longevity, 2 Free radical-mediated lipid peroxidation, 31 Free radicals (FR), 10
11 neurodegenerative disorders and, 38
44 Free radical theory of aging (FRTA), 10
11 Frontotemporal lobar degeneration (FTLD), 41

464

INDEX

G

I

Galactosylceramides (GalCer), 28 GAL4 protein, 328
329 Gamma-aminobutyric acid (GABA) circuits, 113
114 Gamma-linolenic acid (GLA), 338
339 Gene therapy aging, 12
14 leukodystrophies, 27 Genetics longevity, role in, 3 Geriatric Depression Scale, 183 Ghrelin, 61 Gilles de la Tourette’s diseases, 238
239 Glial cell line-derived neurotrophic factor (GDNF), 239
240 Glial fibrillary acidic protein (GFAP), 28, 252 Globoid-cell leukodystrophy (GLD), 28 Glucocerebrosidase (GBA), 41 Glutamate receptor subunit GluR1, 154 Glycerophospho-NAE (GPNAE), 91
93 Glycine, 94
97 Glycine N-acyltransferases (GLYATL2), 95
97 Go/no-go task, 57 G-protein-coupled receptor (GPCR), 94
95, 134, 257

ICAM-1, 226 Imperfect homeodynamics, probability of, 10 Inducible NO synthase (iNOS), 257 Inhibitory control deficits, 57 Inositol-50 -phosphatase (SHIP1), 91
93 Insulin, 60
61 Insulin-like growth factor 1 (IGF-1), 113
114 Intellectual disability omega-3 fatty acids and, 350
352 ADHD, 355 autism (or autistic disorder), 354
355 depressive illness, 353
354 psychiatric illness, 351 schizophrenia, 353t risk factors for, 351 serum cholesterol level and, 351
352 International Society for the Study of Fatty Acids and Lipids (ISSFAL), 122 Isofurans (IsoFs), 33
34, 69
70 Isoketals (IsoKs), 69
70 Isoprostanes (IsoPs), 69
70

H Hamilton Rating Scale for Depression (HRSD), 172 Hayflick system, 13 Hempseed meal (HSM), 330
332 High fat diet/obesity-related cerebrovascular changes, 222
226 animal studies, 224
225 clinical studies, 223
224 Homeodynamics age-related diseases and, 10 vs homeostasis, 9
10 Homeostasis vs homeodynamics, 9
10 Homocysteine (Hcy), 66 Hormesis, 14
15 Hormetics, 14
15 Hormetins, 14
15 Hormone replacement therapy, 27 Horseradish peroxidase (HRP), 114 Huntingtin peptide, 332 Huntington’s disease, 238
239, 330 27-Hydoroxycholesterol (27-OHCh), 37 Hydroperoxide inhibitors, 458 Hydroperoxyeicosatetraenoic acid (HPETE), 32
33 Hydroperoxyoctadecadienoates (HPODEs), 31 20-Hydroxyanandamide (20-HETE-EA), 93 12- and 15-Hydroxyeicosatetraenoyltaurines (HETE-Ts), 98 4-Hydroxyhexanal (4-HHE), 69
70 4-Hydroxynonenal (4-HNE), 69
70, 373
374, 374
375 Hyperglycemia, 63
65, 233 Hyperlipidemia, 226 Hypoglycemia, 233

J Jak/Stat pathway, 70
71

K Krabbe’s disease, 24, 28

L Lead, 449 Leptin, 60 administration, 60f resistance, 60 Leucine-rich repeat kinase 2 (LRRK2), 41 Leukodystrophies, 24
25 fatty acids and dietary intervention, 26
27 gene therapy, 27 hormone replacement therapy, 27 Leukodystrophies, 24
25 Leukotriene A4 (LTA4), 140 Leukotrienes (LTs), 69
70, 140 Life expectancy, 1 Linoleic acid (LA), 31, 122, 131, 137, 163, 331
332, 338
339, 352, 377 α-Linolenic acid (ALA), 21, 163, 181, 233, 235, 287, 303, 331
332, 338
339, 352 conversion of, 164
165 dietary and lifestyle factors, 165 from flax seed oil, 173 genetic variations, 165 long-chain omega-3 pufa supplementation, comparison with, 297 supplementation and brain fatty acid composition, 288
291, 289t Lipid and steroid hormone homeostasis, 25 Lipid mediators, 69
70, 70f Lipid metabolic pathways, 25 Lipid peroxidation, 375
376 from arachidonic acid (AA), 32
33 F2-isoprostanes (F2-IsoPs), 33 hydroperoxyeicosatetraenoic acid (HPETE), 32
33 isofurans (IsoFs), 33
34

biomarkers for neurological dysfunction, 32, 39t from cholestrol, 35
37 distinct mechanisms, 31 from docosahexaenoic acid (DHA), 34
35 lipid peroxidation-derived short-chain aldehydes, 34
35 neurofurans (NFs), 34, 35f neuroprostanes (NPs), 34, 35f free radical-mediated, 31 induced by singlet oxygen, 31 from linoleic acid (LA), 32 neurological dysfunction associated with, 37 Lipoxins (LXs), 69
70 Lipoxygenases (LOXs), 93, 98 Liver X receptors (LXRs), 37, 255 Long-chain fatty acid receptor (GPR40), 134 Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFAs), 147, 153
155, 172
173 Long-chain omega-3 polyunsaturated fatty acids, 287, 288f and brain fatty acid composition, 291
297 comparison of ALA and, 297 studies, 292t Long-chain omega-6 PUFA, 297 Long-chain polyunsaturated fatty acids (LCPUFAs), 109, 163, 234, 237
238, 243, 455 Longevity, 1
2 animal tests and, 5 caloric restriction for, 2 defined, 1 in developed countries, 1 environmental factors of, 4
5 extending effects of various genes, 13 food restriction for enhanced, 2 genetic diseases and, 3
4 genetics, role of, 3 genomic factors of, 4 nutrition, influence of, 2 omega-3 fatty acids and, 1
2, 5
6 primary factors affecting, 1
2 principles of aging and, 9 smoking and, 2
3 studies on mono- and dizygotic twins, 9 Longevity genes, 12
13 Lorenzo’s oil (LO), 26
27, 333 Low back pain (LBP), 417 defined, 417 relationship between overweight/obesity and, 417 forward flexion and lateral bending movement, 423t gait analysis, 419
421 osteopathic manipulative treatment (OMT), 424
425 quantitative movement analysis, 418
425 spatio-temporal and kinematic parameters, 421t trunk movement, 421
425 severity, 417

465

INDEX

Low density lipoproteins (LDLs), 64, 70
71 LT B4 receptor 1 (BLT1), 140 Lysophosphatidic acid (LPA), 141 Lysophospholipase D (lysoPLD), 91
93

M MacArthur Communicative Development Inventory, 309 Macromolecular damage, 13 Major depressive disorder (MDD), 182 omega-3 fatty acids, role of, 171
173, 182 symptoms associated with, 182 Malondialdehyde (MDA), 193 Matrix metalloprotease (MMP)-9 mRNA, 225 Mediterranean nutrients, 6 Mercury, 447
449 Metachromatic leukodystrophy (MLD), 24, 27
28 Metal-based micronutrients, 11 2-Methylacyl-CoA racemase (AMACR), 24 Methylenetetrahydrofolate reductase (MTHFR), 66 Methylmercury, 448 1-Methyl-4-phenyl-2,3,4,6-tetrahydropyridine (MPTP), 376, 380 MetS, 391
392 MF766, 138
140 Migraine and obesity, epidemiological relationship between, 277
279 balanced diet therapy, 282
283, 282t BMI-related changes, 282
283 cognitive profile, 279 comorbidity condition, 278
279 food and diet as triggers for, 281 lifestyle factors, 280
281 potential mechanisms, 279
281 serotonin levels, 280 serum adiponectin levels, 280 sleep disorders, 281 weight loss, 281
283 Mini Mental State Examination (MMSE), 152, 234, 438 Mitochondrial dysfunction, 375
376 Mitochondrial proteins, 11 Mitogen-activated protein kinase (MAPK), 63 Mixed dementia, 211
212 Molecular basis of aging, 10
11 Molecular damage, 10 Monounsaturated fatty acids (MUFAs), 353
354 Montgomery Asberg Depression Rating Scale (MADRS), 172 Multiple sclerosis (MS) cellular level of, 368
369 omega-3 supplementation for, 369 scientific community on, 369
370 overview, 367
368 Multiple system atrophy (MSA), 375 Myocardial infarction (MI), 183

N N-[5,6-epoxy-8,11,14-eicosatrienoyl]ethanolamine (5,6-EET-EA), 93 N-[11,12-epoxy-5,8,14-eicosatrienoyl]ethanolamine (11,12-EET-EA), 93

N-acetylaspartate, 27 N-acetyl aspartylglutamate (NAAG), 27 N-acyl amino acids (NAAs), 93
99 N-acyl-DOPA decarboxylation, 99, 99f N-acyldopamines (NDAs), 99 biosynthesis, 100f catabolism, 99 degradation, 101f N-arachidonoyl- (NADA), 99 N-oleoyl- (OLDA), 99 N-palmitoyl- (PALDA), 99 N-stearoyldopamine (STEARDA), 99 in vivo production, 99 N-acylethanolamines (NAEs), 90
93, 91t biosynthesis of, 91
93 catabolism, 93 functions, 91 metabolism, 92f oxidation of, 96 N-acylglycines (NAGs), 94
97 from cytochrome c, 95
96 distribution of, 97 enzymatic synthesis of, 95 metabolism, 96f oxidation of, 96 pathways for formation of, 95
97 N-acyl-L-DOPAs, 99 N-acyl taurines (NATS), 97
99 FAAH-regulated, 97 functional roles of, 98 identification of, 97
98 TRPV1 and TRPV4, activation of, 98 N-acyltransferase, 99 Na/K ATPase gene polymorphism, 65 NAPE-specific phospholipase D (NAPEPLD), 91
93 N-arachidonoyl alanine (NAAla), 98 N-arachidonoyl amino acids, 98 N-arachidonoyl γ-aminobutyrate (NAGABA), 98 N-arachidonoylethanolamine (anandamide), 87, 90
91 affinity to rat receptors, 91t biosynthesis of, 91
93 oxidative metabolism, 94f N-arachidonoylglycine (NAGly), 93
94 N-arachidonoyl serine (NASer), 98 N-Arachidonoyl taurine, 98 N-docosahexaenoylethanolamide (DEA), 111, 238 N-docosahexaenoyl glycine (DOCGly), 97 Negative energy balance, 2 Neurodegeneration, 238
239 Neurofurans (NFs), 69
70 Neurogenesis by activator-type bHLH transcription factors, 256
257 definition, 251 developmental, 253
254 hippocampal, omega-3 PUFAs and, 253, 259 immunological markers of, 252
253 link between learning and, 251 survival of new neurons in, 251 Neurogranin (RC3), 155 Neuroketals (NKs), 69
70

Neurokinin A, 279 Neurological disease/disorders docosahexaenoic acid (DHA) and, 76
78 eicosapentaenoic acid (EPA), role of, 76
78, 77f fatty acid amide therapy for, 99
102 lipid peroxidation and, 32, 37 weight-related variables and dementia, 385
389 BMI and cognitive decline, 388
389 controls and potential mediators, 390
391 CVD correlates, 389
391 diagnosis, 398 prospective studies, 387
389 Neuromodulin (GAP-43), 154 Neuronal apoptosis, 70
71 Neuroprostanes (NPs), 69
70, 232 Neuroprotectin D1 (NPD1), 111, 155 N-fatty acylglycines (NAGs), 89 N-hydroxyeicosatetraenoyl-ethanolamines (HETE-EAs), 93 N-(hydroxylated acyl)-ethanolamine products, 93 Nicotine, 165 Nitric oxide production, 6 N-Linoleoylethanolamine, 93 N-Linoleoyl glycine, 97 N-Oleoylethanolamine (OEA), 91 N-Oleoyl glycine (OLGly), 97 Nonsteroidal anti-inflammatory drugs (NSAIDs), 134, 138 N-Palmitoyl glycine (PAGly), 97 N-Stearoylethanolamine (SEA), 91 N-stearoyl glycine (STRGly), 97 N18TG2 neuroblastoma cells, 89
90 Nucleus accumbens (NAc), 238 Nutrient co-factor deficiencies, 166
167 antioxidants, 166
167 folic acid, 166 omega-3 fatty acids, 167 selenium, 166 serotonin, 167 tryptophan, 167 vitamin B6, 167 zinc, 166 Nutrition, 121, 147 central nervous system functioning and, 109 deficiencies, changes in brain, 109 Nutritional hormetins, 15

O Obesity, 2, 134, 385 cognitive impairment and, 57
58 clinical perspective, 398 imaging studies, 398 morbid, 394
395 neurological measures, 58 epidemiological relationship between migraine and, 277
279 cognitive profile, 279 comorbidity condition, 278
279 lifestyle factors, 280
281 potential mechanisms, 279
281

466 Obesity (Continued) weight loss, 281
283 executive function and, 393
394 measurement, 397
398 inhibitory control, 57 learning and memory impairments, 58 morbid, 394
395 overall health, effect on, 222
226 relationship between LBP and, 417 forward flexion and lateral bending movement, 423t gait analysis, 419
421 osteopathic manipulative treatment (OMT), 424
425 quantitative movement analysis, 418
425 spatio-temporal and kinematic parameters, 421t trunk movement, 421
425 treatment of overweight, 395
396 omega-3 PUFAs, 396
397 Obstructive sleep apnea hypopnea syndrome (OSAHS), 337
345 characteristics of, 337 relationship between AHI level and excessive daytime sleepiness, 338 sleepiness, 338 complications associated with, 338 defined, 337 diagnosis of, 338 polyunsaturated fatty acids (PUFAs) and, 337
339, 339f sleep quality and, 342
345, 344t Oleamide, 88
89 Oleic acid, 87 Omega-3/DHA nutritional restriction, 114
117, 115f, 116f vs diet rich DHA, 121
122 Omega-3 fatty acid linolenic acid (LNA), 122 Omega fatty acids, 206. see also Omega-3 fatty acids; Omega-6 fatty acids athological cognitive decline, protective against, 212
213 metabolism, 208
209 sources of, 208 Omega-3 fatty acids, 1
3, 5
6, 131, 163
165, 167, 181
185 amyloid-β production, reduction of, 207 anti-thrombotic effects, 207 anxiety disorders and, 184
185 behavioral/physical symptoms, effects on, 190
192 blood pressure reduction, 208 brain-derived neurotrophic factor (BDNF) and, 207 brain development, role in, 109
112 brain function, role in, 206
208 cardiovascular disorders, role in preventing, 231 with depression, 183 central visual connections, role in, 114
117 chronic diseases and, 6 in clinical depression, 165
167, 182
183, 433
434

INDEX

with cardiovascular disorders, 183 with diabetes, 183
184 and old age, 184 and pregnancy, 184 in students, 184
185 cognitive decline and dementia, curative effect for, 436
439 animal studies, 439
440 group studies, 436
437 longitudinal studies, 437
438 plasma lipid estimates, 438 prevention and treatment studies, 438
439 self-report measures, 437
438 cognitive function in children and, 124 conversion to omega-6, 164
165 deficiency, impact of, 109
110, 166f foods with, 181
182 and hippocampal neurogenesis, 170 inflammation reduction, 207 intellectual disability and, 350
352 ADHD, 355 autism (or autistic disorder), 354
355 depressive illness, 353
354 schizophrenia, 352
353, 353t MDD, role in, 171
173, 182 memory and cognition, 434 metabolites derived from, 136
137 for multiple sclerosis (MS), 369 neurogenesis and, 206
207 neuronal synaptic proteins and, 111 neurotransmission and, 121
122 neurotransmission regulation, 207 nitric oxide (NO) release and, 226 pain regulation, role in, 135, 141
142 in postpartum depression, 123 production of inflammatory eicosanoids, 170 protective effect against seizures, 172
173 quality of life (QoL), impact on, 81 assessment, 81
82 clinical trials, evidence from, 83 conclusion and recommendations, 84 discussion, 83
84 observational studies, evidence from, 82
83, 82t SF-36, assessment using, 82, 84 WHOQOL-BREF questionnaire, assessment using, 82, 84 randomized clinical studies, findings, 110 in reducing inflammation, 182 supplementation during childhood, 124 triacylglycerols (triglycerides), lowering of, 208 vascular health, impact on, 226 Omega-3 PUFAs, 253
254, 287, 303, 349, 359. see also Polyunsaturated fatty acids (PUFAs) aggression and in elderly, 360
361 in patients with ADHD, 361 in patients with schizophrenia, 361 in prisoners, 361 in young adults, 359
362, 360f ASD and, 355

in brain function, 303 on cognitive development, 305t supplementation, effects of, 308t, 310t epidemiological studies, 314
322, 315t RCTs, 319
322, 320t, 321t in infants and children, 307 mental functioning, 311 supplementation, effects of, 307
311 in maternal supplementation, 304
307 mechanism of action, 361
362 brain-derived neurotrophic factor (BDNF), 362 cortical-hippocampal-amygdala pathway, 362 endocannabinoids, 362 noradrenaline (NA), 362 serotonin, 361
362 mechanisms for reducing weight loss, 397 in older adults, 314 schizophrenia and, 352
353, 353t treatment of obesity with, 396
397 in young adults, 311
314 cognitive development, 312t Omega-6 fatty acids, 1, 3, 122, 191
192 arachidonic acid of, 138 pain regulation, role in, 138, 139f ONO-8130, 140 Osteopathic manipulative treatment (OMT), 424
425 Osteoporosis, 10 α-Oxidation, peroxisomal, 20 disorders, 24 β-Oxidation, peroxisomal, 20
23 Oxidation correction, 458
459 Oxidation indicators, 455
458 Oxidation process, 455 Oxidative stability of an oil, 457
458 Oxidative stress, 2
3, 6, 41
42, 375
376, 430
431 vitamins, role in reducing, 2 4-Oxo-2-nonenal (ONE), 375

P PAF/PAFR system, 141 Pain regulation DHA-mediated, 134 docosahexaenoic acid (DHA), role in, 135
136 fatty acids, role in, 134 lipids, role in, 134
141 leukotrienes (LTs), 140 lysophosphatidic acid (LPA), 141 platelet-activating factor (PAF), 140
141 prostaglandins, 138
140 non-opioid analgesics for, 134 omega-6 polyunsaturated fatty acid in, 138, 139f Palmitic acid (PAM), 266 Parkinson’s disease, 10, 238
239, 243, 329, 373, 375
376 familial, 41 free radicals (FR) and, 38
41 isotopic reinforcement of PUFA, preclinical pd modeling, 378
380 Lewy bodies, 40
41

467

INDEX

lipid peroxidation biomarkers for, 39t Pathological brain aging, 209
212 Patient Health Questionnaire, 190 Pedunculopontine tegmental nucleus (PPTg), 243
244 P-element-mediated mutagenesis, 327
328, 328f Pelizaeus-Merzbacher disease (PMD), 24, 28 Pentadienyl carbon-centered lipid radical, 31 Pentosidine, 43 8-[2-(2-Pentyl-cyclopropylmethyl)cyclopropyl]-octanoic acid (DCP-LA), 238 Periventricular leukomalacia (PVL), 43 Peroxide value (POV), 456 Peroxins (PEX), 19 Peroxisomal disorders, 23, 23t Peroxisomal targeting signal 1 and 2 (PTS1 and PTS2), 19 Peroxisome proliferator-activated receptors (PPARs), 154, 255
256 Peroxisomes, 19
23 biogenesis of, 19 classification, 19 differences between fatty acid mitochondrial and, 21t docosahexaenoic acid (22:6 n-3, DHA) formation, 21 enzymes in, 19 ether lipid synthesis and, 19
20 functions of, 19 α-oxidation, 20 β-oxidation, 20
23 pathologies, 23
24 pathways, 19, 20f receptor-cargo complex, 19 PF-9184, 138 Phosphatase and tensin homolog-inducible kinase 1 (PINK1), 41 Phosphatidic acid (PA), 332 Phosphatidylcholine (PC), 91
93, 332 Phosphatidylethanolamine (PE), 91
93, 110
111, 147
149 Phosphatidylinositol pathways, 134 Phosphatidylserine (PtdSer), 71, 110
111 Phospholipase C (PLC), 91
93, 134 O-Phosphoryl-NAE (pNAE), 91
93 Phosphorylated cAMP response elementbinding protein (pCREB), 257
258 Physiologic brain aging, 202
205 chemical changes, 203
204 dopamine, 203 glutamate, 204 serotonin, 203
204 cognitive changes, 204
205 attention, 204 executive control, 205 memory, 204
205 orientation, 205 perception, 205 structural, 202
203 macro-level, 202
203 micro-level, 203 Phytanic acid, 20 PI3KAkt signaling pathway, 61

Platelet-activating factor (PAF), 140
141 Polychlorinated biphenyls (PCBs), 447, 451
452 Polyglutamine diseases, 329 Polysialylated nerve cell adhesion molecule (PSA-NCAM), 252 Polysomnography (PSG), 337, 338f Polyunsaturated fatty acids (PUFA), 21, 31, 111
112, 131, 133
134, 201, 222
223, 265, 303, 330
332, 349, 429. see also Omega-3 PUFAs autoxidation, 374f isotope protection, 376
378, 376f into brain membrane lipids, 265
267 depression and, 340
342 D-PUFAs, 378
379, 379f free radical-mediated peroxidation of, 31 function as a cellular mediator, 131 health and, 339
340 in mitochondrial membranes and oxidative stress, 373
375 OSAHS and, 339f PUFA-GPR40-CREB signaling pathway, 134 sleep quality, 342
345 sources of, 309 trials in ADHD, 355 Post-synaptic density protein-95 (PSD-95), 154 Primary fatty acid amides (PFAMs), 87
90 biological effects, 88
89 cellular concentration of, 89 functions, 89t identification, 88t metabolism, 90f pathway(s) responsible for, 89 Pro-inflammatory cytokines, 184
185 clinical depression, role in, 169
170 Pro-inflammatory eicosanoids, 70
71 Prostaglandin D2, 352 Prostaglandins, 69
70, 138
140 cytosolic PGE synthase (cPGES), 138 membrane-bound PGE synthases 1 and 2 (mPGES1, 2), 138 prostaglandin E receptors (EPs), 138
140 prostanoids (PGE2), 138 Protein error theory of aging, 11 Protein kinase A (PKA), 140 Protein kinase B, 60
61 Protein kinase C (PKC), 63, 140 Protein tyrosine phosphatase (PTPN22), 91
93 Proteolipid protein (PLP), 28 Pyrraline, 43

R Rapid eye movement (REM), 342, 343f Rate of chain oxidation (ROX), 377 Reactive oxygen species (ROS), 10
11, 63
64, 222
223, 373 Receptors for AGE (RAGE), 64 Refsum disease, 24 Replicative lifespan of cells, 13 Retinal pigmented epithelium (RPE), 110
111

Retinoic acid (RA) depletion, effect of, 256 in embryonic development, 255
256 stereoisomers, 255 Retinoid X receptors (RXR), 111 Rhizomelic chondrodysplasia punctata (RCDP type I), 23 18R-hydroxyeicosapentaenoic acid (18RHEPE), 72 Rodent dietary deprivation studies, 265 Rotenone, 376

S Sarcopenia, 10 Saturated fatty acids (SFAs), 58, 131, 165, 429 AD and, 435
436 Schizophrenia, 352
353 efficacy trials of omega-3 PUFAs in, 353t Selenium, 449
450 24(S)-hydroxycholesterol (24(S)-OHCh), 37 Single nucleotide polymorphisms (SNPs), 11
12 Smoking longevity and, 2
3 Smoking cessation, 2
3 24 (S)-OHCh, 37 Soluble epoxide hydroxylase (sEH), 137 Somatosensory evoked potentials (SEPs), 26
27 Spinal muscular atrophy (SMA), 43 Spinocerebellar ataxia type 3 (SCA3/MJD), 330 SR14171, 258 SR141716A, 258 ‘Stability through constancy,’ principle of, 9
10 Sterol carrier protein X (SCPx) deficiency, 24 Strengths and Difficulties Questionnaire, 190 Stress response (SR), 14, 165 Stroke, 42 lipid peroxidation biomarkers for, 39t Stroop Task, 57 Structural magnetic resonance imaging (sMRI), 58 Subacute sclerosing panencephalitis (SSPE), 43 Substance P, 279 Substantia nigra vulnerability, 238
239 dopamine cell populations, 239
240 cell loss, 241
244 effect of EFA dietary restriction, 242f Supercritical Fluid Chromatography (SFC), 459 Supercritical Fluid Extraction (SFE), 459 Superoxide dismutase (SOD), 65, 239 Symptomatic vasospasm (SVS), 42 Synapsin-1, 154 α-Synuclein (αSyn), 375

T TAR DNA-binding protein 43 (TDP-43), 41 Test of Variables of Attention (TOVA), 192 Thiobarbituric acid (TBA), 43 Thromboxanes (TXs), 69
70

468

INDEX

Tissue plasminogen activator (tPA), 42, 113
114 Toll like receptor 4 (TLR4), 134 TOTOX value, 456
457 Trans-fatty acids, 165 Transient receptor potential channel 5 (TRPC5), 97 Transient receptor potential of melastatin type 8 (TRPM8), 90
91 Transient receptor potential (TRP) channel, 330 Transient receptor potential vanilloid type 1 (TRPV1), 90
91 Trihydrocholestanoic acid (THCA), 20 TRNA charging, 11 Tumor necrosis factor (TNF) receptor 2 gene (TNFRSF1B), 65
66 Type 2 diabetes, 10

U UAS-GAL4 system, 328
329, 328f Ubiquitin carboxylterminal hydrolase L1 (UCH-L1), 41 Unsaturated fatty acids, 131 Upstream activation sequence (UAS), 328
329

V Vascular dementia, 211

disease characteristics, 211 chemical changes, 211 cognitive changes, 211 structural changes, 211 disease mechanism, 211 Vascular endothelial growth factor (VEGF), 66 Vascularization of brain, 221
222 blood-brain-barrier (BBB), 222 high fat diet and obesity, effect of, 223
224 animal studies, 224
225 VCAM-1, 226 Ventral tegmental area (VTA), 238
241 Verbal list learning task, 58 Very long chain fatty acids (VLCFA), 20, 23
25, 332
333, 350
351 brain levels of, 25
26 consequences of, 25
26 saturated, 25
26 toxic effects of, 25
26 Vesicular monoamine transporter 2 (VMAT2), 233 Vienna Standard Mean Ocean Water (VSMOW) scale, 378
379 Virtual gerontogenes, 12 Visual topographical maps, 112
113

critical periods of development of sensory brain connections, 113
114 ipsilateral retinocollicular pathway, 112f visual cortex, 113
114 visual subcortical nuclei, 114 Vitamin E, 43

W Western diet, 3, 69
70, 201, 222
223, 225 for cognitive impairment, 58
61 Wisconsin Card Sorting Test, 57
58

X X-linked adrenoleukodystrophy (X-ALD), 23
24, 330, 332
333, 350
351 X-linked leukodystrophy (X-ALD), 24 biological markers of, 25 lipid and steroid hormone modifications in, 25 RBC viscosity in, 25
26 use of LO in, 26

Z Zellweger spectrum diseases. see Peroxisomal disorders Zinc, 171