membrane lipidomics for personalized health

Membrane Lipidomics for Personalized Health

Membrane Lipidomics for Personalized Health

Carla FerreriConsiglio Nazionale delle Ricerche,

Institute of Organic Synthesis and Photoreactivity, Italy

Chryssostomos ChatgilialogluNational Center for Scientific Research “Demokritos”,

Institute of Nanoscience and Nanotechnology, Greece

This edition first published 2015© 2015 John Wiley & Sons, Ltd.

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Library of Congress Cataloging‐in‐Publication Data

Ferreri, Carla, author. Membrane lipidomics for personalized health / Carla Ferreri and Chryssostomos Chatgilialoglu. p. ; cm. Includes bibliographical references and index. ISBN 978-1-118-54041-1 (cloth) – ISBN 978-1-118-54032-9 (pbk.)I. Chatgilialoglu, Chryssostomos, author. II. Title.[DNLM: 1. Fatty Acids–metabolism. 2. Membrane Lipids–metabolism. 3. Individualized Medicine. 4. Metabolomics–methods. 5. Nutritional Physiological Phenomena. QU 85.6] QP752.F35 612.3′97–dc23 2015016361

A catalogue record for this book is available from the British Library.

Set in 10.5/13pt Sabon by SPi Global, Pondicherry, India

1 2015

http://www.wiley.com

Dedicated to our son Alexandros and daughter Raffaella, who made our lives

complete with love

Contents

About the Authors xiPreface xiiiAcknowledgments xviiAbbreviations xix

Part I Molecular and Nutritional Basis of Cell Membranes and Lipidomics 1

1 Membranes for Life and Life for Membranes 31.1 Cell Membranes: The Role of Fatty

Acids and the Exclusion of Trans Isomers 31.2 Organization and Homeostasis 11

1a In Depth: The Formation of a Cell Membrane 161b In Depth: Cholesterol and Membranes 171c In Depth: Lipid Rafts 19

2 Fatty Acid Families: Metabolism and Nutrition 212.1 Saturated Fatty Acids: Biosynthesis and

Dietary Regulation 232.2 Monounsaturated Fatty Acids:

The Importance to be cis 282a In Depth: The key Steps of Phospholipid

Synthesis 312b In Depth: Biosynthesis of the Double

Bond and Desaturase Features 342.3 Polyunsaturated Fatty Acids: The Essentiality

for Human Cells 37

viii COnTEnTS

Concepts’ Summary 38S1 Beware of the nutritional Label! 38S2 The Optimal Values of Fatty Acids in Tissues 38S3 Structural Role of Fatty Acids 40

3 Essential Fatty Acids 413.1 The Omega‐6 and Omega‐3 Families:

Cascades and Regulation 423a In Depth: The Definition of

Omega‐6 and Omega‐3 483b The Polyunsaturated Fatty Acids in

Cell Membrane Remodeling 503c In Depth: How do you Define an

Inflammatory Pathway? 553.2 The Balance Between Omega‐6 and Omega‐3

Pathways: nutritional and Metabolic Considerations 563.3 Food and Membranes: A Virtuous Cycle 60

4 Free Radicals and Lipids: Trans and Oxidized Fatty Acids 654.1 Trans Fatty Acids for Humans:

The nutritional Intake 664.2 Endogenous Sources of Trans Fatty

Acids by Free Radical Stress 714.3 Free Radicals and Lipid Oxidation:

The Threshold for Health 734.4 Lipoproteins and Development of

Markers for Lipid Reactivity 794a In Depth: Oleic versus Linoleic Acid

Reactivity with Free Radicals 83

Concepts’ Summary 84S1 Fatty Acid Geometry: A “Radical” Change 84S2 Antioxidants for Membranes 85

Part II Membrane Lipidomics for Personalized Health 87

5 What Is Lipidomics for Health 895.1 The Birth of the Postgenomics Era 895.2 Lipidomics in the Postgenomic Era 925.3 Fatty Acids Involved in Membrane and

Mediator Lipidomics 93

COnTEnTS ix

5.4 Membrane Lipidomics: Cellular Stress, Turnover, and Opportunities 955.4.1 How Does the Stress Involve Membranes? 97

5.5 Phospholipids From Dietary Intakes to Biological Functions 100

6 Lipidomics of Erythrocyte Membranes 1056.1 Erythrocyte as a Comprehensive Health Biomarker 1076.2 The Optimal Value Intervals and The Membrane

Unbalance Index 1156.3 Lipid Biosynthesis and Related Indices 1206.4 The Individuation of Molecular Indicators 122

7 Nutrilipidomics 1277.1 When Fatty Acids Become nutraceuticals:

Membrane Therapy With nutrilipidomics 1287.2 Fatty Acid–Based Membrane Lipidomics and

nutrilipidomics: The Personalized Approach for nutrition and nutraceuticals in Health and Diseases 131

8 Lipidomic Profiles and Intervention Strategies in Prevention and Diseases 1358.1 Lipidomics and Sport 1378.2 Lipidomics and Pregnancy 1408.3 Lipidomics and Aging 1438.4 Lipidomics and Cardiovascular Health 1458.5 Lipidomics and Overweight 1488.6 Lipidomics and Dermatology 1508.7 Lipidomics and neurology 1518.8 Lipidomics and Ophtalmology 1538.9 Conclusive Remarks 154

9 Lipidomics and Tutorials 1579.1 First Steps for the Lipidomic Analysis 159

9.1.1 Saturated Fatty Acid Excess 1609.1.2 Monounsaturated Fatty Acid Excess 1609.1.3 Omega‐6 PUFA Excess 1609.1.4 Omega‐3 PUFA Deficit 161

9.2 Learning Verification 162

References and Notes 167Index 181

About the Authors

Carla Ferreri was born in Napoli, graduated in Pharmacy in 1979 and postgraduated in Hospital Pharmacy in 1981. She started her studies in organic synthesis and medicinal chemistry as permanent research fellow at the University of Napoli. From 1990 she was involved in free radical research, and in 2001 she moved to the Consiglio Nazionale delle Ricerche, where she is now Senior Researcher, responsible for the project “Biomarkers of Free Radical Stress” at the Research Area of Bologna. She is interested in multidisciplinary research, involving free radicals, chemical transformations under biomimetic conditions (liposomes), bio-marker discovery related to free radical stress, and lipid remodeling caused to cell membranes by various stress types. She is also consultant to companies for lipidomic profiles and nutraceutical formulations. Her activity is described in more than 160 scientific contributions. From this research the innovation project “Lipidomic Profile of Cell Membranes: A Molecular Approach Applied to Human Health” started, with a wide applicability to medicine, prevention, and quality of life. For this project Carla Ferreri was awarded in 2010 with the ITWIIN award as the Best Innovator Woman in Italy and received a special mention at the EUWIIN award 2011. Carla Ferreri is cofounder and R&D director of the company Lipinutragen, a spin‐off officially recognized by CNR, and is cofounder of Lipinutramed, a start-up at of the NCSR “Demokritos” in Athens (Greece).

Chryssostomos Chatgilialoglu was appointed Director of the Institute of Nanoscience and Nanotechnology (INN) in the NCSR “Demokritos,” Athens, in March 2014. He is also the Honorary President and Cofounder of spin‐off companies Lipinutragen (Italy) and Lipinutramed (Greece). He chaired the COST Action CM0603 on Free Radicals in

xii ABOUT THE AUTHORS

Chemical Biology, from 2007 to 2011, and is now the Chairman of the COST Action CM1201 on Biomimetic Radical Chemistry, running from 2012 to 2016.

In 1976, he received his doctorate degree in Industrial Chemistry from the University of Bologna and completed his postdoctoral studies at York University (UK) and National Research Council of Canada, Ottawa. From 1983, he worked for the Consiglio Nazionale delle Ricerche (Bologna), and was Research Director from 1991 to 2014. He has received many honors and awards including the Fluka Prize “Reagent of the Year 1990,” and is a world expert on free radicals. His research interests lie in free radical reactions increasingly address-ing in the last decade applications in biomimetic chemistry and bio-marker discovery, with fundamental acquisitions in DNA, lipid, and protein transformations.

He has published over 240 papers in peer‐reviewed international jour-nals, 33 book chapters, 6 patents, and 6 books (2 as author and 4 edited); he is Coeditor of the Encyclopedia of Radical in Chemistry, Biology and Materials (4 volumes), 2012 John Wiley & Sons, Ltd. Over 100 invited lectures at international conferences and over 120 invited research sem-inars at institutions.

Preface

The idea of this textbook is to offer a multi‐ and interdisciplinary treatment on lipidomics, which lies at the interface of several life science disciplines, from chemistry, biochemistry, biology, pharmacology, to medicine and health care as the final application. In particular, for health applications lipidomics must be treated in a “functional” way, building connections of lipid structures with their metabolic and nutritional origins, and with biological, pharmacological, and medical functions.

The book focuses on cell membrane lipidomics for the important structural and functional roles played by lipid molecules, in particular phospholipids, whose influence goes beyond the membrane compartment itself, expanding from the start of cell signaling to the regulation of gene expression. Nowadays, the central role played by the structure and functionality of the cell membrane has been recognized in many processes, such as the start of cascades for lipid‐mediated signaling, hav-ing strong influence on the quality and sustainability of life. Phospholipids are evaluated in detail for their composition made of fatty acids, the hydrophobic part that forms the interior of the membrane bilayer. We will describe why and how the fatty acid residues of membrane phospholipids represent the result of a precise and successful balance between biosynthesis and diet, which can be realized in each individual. This is an important topic of molecular medicine.

In fact, considering that cell membranes display characteristic fatty acid compositions for each type of tissue, these compositions represent the “lipid code” necessary for the tissue functioning and to realize a normal tissue metabolism. Any change in the tissue fatty acid compo-sition corresponds not only to a “molecular change,” but also to the start of possible tissue malfunction or degeneration. Stress, such as an increased oxidative status or a decrease in protective elements for

xiv PrefAce

metabolism, can cause an initial change in the fatty acid composition and its consequent healthy balance. In this book, membrane lipidomics will be focused as a powerful diagnostic tool of “molecular health,” starting from cell membranes.

The importance of the concept of “unbalanced membrane fatty acid composition” for the application in molecular medicine will be treated, which cannot be in principle a pathological status in itself, but can indi-cate an initial failure of the healthy status when present under physiological conditions. At this stage, lipidomics will also be shown as an important preventive and problem‐solving tool, by which molecular unbalances can be addressed in a personalized way, applying the most appropriate strategy for the subject. It also contributes to the choice of diet, nutritional supplements, or functional foods for the restoration of the individual optimal balance.

In this context, lipidomic analysis can be part of the decisional activity of health operators for the formulation of the most adequate therapeutic strategy also based on nutritional lipid elements.

In fact, health operators cannot disregard the molecular aspect of the membrane since the lipid composition derives not only from the biosyn-thetic abilities of the body, but also from the dietary habits. As nutrition affords essential fatty acid, vitamins, and micronutrients strictly related with the enzyme and metabolic functioning to generate cells and cell membranes, the membrane status can be taken as a global health biomarker, interpreting the resulting balance among different fatty acid families.

from the patients’ point of view, a strategy suggested by health operators, which includes attention to nutritional guidelines and the use of an integrated medical approach, can have a positive impact on their lifestyle and relationship with the trusted doctor, as opposed to the dangerous and diffuse habit of “self‐prescription.”

from the societal point of view, the myriad of nutritional supplements present in the market clearly indicates the need of criteria for prescription. Indeed, this confused market started to warn the decision makers in the health care sectors for setting a rationalization policy based on scientifi-cally recognized claims. On the other hand, comprehensive indicators of health conditions, which have been science‐driven and fully validated, are still far from being used in clinical practice by health operators, mainly due to the lack of knowledge in molecular medicine and related biomarkers.

Starting from the basic knowledge of chemistry and biology in Part 1, this book is a user‐friendly manual of fatty acid–based functional

PrefAce xv

lipidomics, in particular membrane lipidomics, in order to familiarize with one of the most successful tools in molecular diagnostics. The ratio-nale of membrane lipidomics for human health is offered to the readers, refreshing basic concepts of biochemistry and pharmacology achieved during the academic formation of health operators.

In Part 2 membrane lipidomics will be discussed in the context of some metabolic and health conditions, introducing the concept of lipidomic profiles in different physiological and pathological situations. for a facilitate use in preventive medicine, membrane lipidomics will be explained in the format of molecular indicators, grouping fatty acids according to their main health indications.

Health operators are the main readers to whom this book is addressed to update their academic and clinical experience. The book can be also useful for those involved in life sciences and the health care market in various roles, from research to business, since the topics and their descriptions can help innovation of ideas and products of “pharma‐nutra” companies.

At the end of this journey through the various aspects of membrane lipidomics, we do hope that it will be much easier to combine the molec-ular status of the cell membrane with the clinical evaluation of the subject for assignment of personalized nutritional and nutraceutical strategies.

carla ferreri chryssostomos chatgilialogluBologna, November 2014 Athens, November 2014

Acknowledgments

We wish to thank all colleagues and coworkers that contributed to the research in this field and to the affirmation of membrane lipidomics and nutrilipidomics as tools in molecular medicine. Lipidomic profiles were developed thanks to the spin‐off company Lipinutragen, with special thanks to the long‐standing collaboration with Simone Deplano, Anna Rosaria Maranini, Michele Melchiorre, Valeria Minelli, and Valentina Sunda. The collaboration of Dr. Anna Sansone and Dr. Annalisa Masi of the ISOF-CNR group for the discovery of new stress biomarkers is gratefully acknowledged. With the enthusiasm and devotion of all these extraordinary people, the process to bring science innovation to the health care market has been an extraordinary journey.

We also wish to thank the COST organization (COST CM1201 Action: Biomimetic Radical Chemistry) for the scientific context provided by fruitful meetings and scientific exchanges with many research groups in European and extra‐European countries.

Abbreviations

AA Arachidonic acidADP Adenosine diphosphateAI Adequate intakeALA Alpha‐linolenic acidATP Adenosine triphosphateCAC Critical aggregation concentrationcAMP Cyclic adenosine monophosphateCLA Conjugated linolenic acidCoA Coenzyme ACOX CyclooxygenaseCTP Cytidine triphosphateCyt CytochromeDAG DiacylglycerolDGK Diacylglycerol kinaseDGLA Dihomogammalinolenic acidDHA Docosahexaenoic acidDNA Deoxyribonucleic acidDRV Daily (dietary) reference valueEFA Essential fatty acidsEFSA European Food Safety AgencyELISA Enzyme‐linked immunosorbent assayELOVL Fatty acid elongaseEPA Eicosapentaenoic acidER Endoplasmic reticulumFABP Fatty acid binding proteinFAD Fatty acid desaturaseFAS Fatty acid synthaseFATP Fatty acid transport protein

xx ABBREVIATIONS

FFA Free fatty acidGC Gas chromatographyGLA Gamma‐linolenic acidHDL High‐density lipoprotein4‐HNE 4‐HydroxynonenalLA Linoleic acidLCAT Lecithin cholesterol acyl transferaseLDL Low‐density lipoproteinLOX LipoxygenaseLPA Lysophosphatidic acidLT LeukotrieneLTC (A) Leukotriene C (A)MDA MalondialdehydeMg MagnesiumMUFAs Monounsaturated fatty acidsNADP Nicotinaminadenine dinucleotide phosphatePAF Platelet‐activating factorPG ProstaglandinPGE (F) Prostaglandin E (F)PKA Protein kinase APL PhospholipidPLA2 Phospholipase A2PPAR α (γ) Peroxisome proliferator‐activated receptor α (γ)PUFA Polyunsaturated fatty acidsSCD Stearoyl coenzyme A desaturaseSFA Saturated fatty acidsSNP Single nuclear polymorphismSREBP Sterol response element binding proteinTBA Thiobarbituric acidTBARS Thiobarbituric acid reactive substancesTFA Trans fatty acidTX ThromboxaneUSF Upstream stimulatory factorVLCFA Very long chain fatty acidVLDL Very low‐density lipoproteinWHO World Health OrganizationZn Zinc

Membrane Lipidomics for Personalized Health, First Edition. Carla Ferreri and Chryssostomos Chatgilialoglu. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

In Part 1 the basic concepts of chemistry and biology related to the cell membrane structure and functions will be described, starting from the relationship between phospholipid structures and membrane properties. The structural driving force leading to phospholipid organization with the different contributions of the hydrophobic fatty acid tails affords the fundamental properties of membrane permeability and fluidity, which regulate in their turn the accommodation of protein entities that consti-tute receptors and channel functionality. Therefore, the combinations of saturated and unsaturated fatty acid residues that form membrane phospholipids can be considered as the arrangement palette that charac-terizes the various tissues, being the basis of their optimal performance.

In this part the connection between membrane and nutrition will also be explained, based on two main aspects of the essentiality of fatty acid

Molecular and Nutritional Basis

of Cell Membranes and Lipidomics

Part I

2 Molecular and nuTrITIonal basIs of cell MeMbranes

structures and the nutritional influence of fatty acids on cellular fate and signaling.

an emphasis will be given to the consequences of free radical stress on membrane components, focusing on the reactivity of the double bonds in natural unsaturated fatty acids and the transformation of cis isomers into their corresponding trans isomers, which are not natural for eukaryotes. In the last decade or so the importance of the cis double bonds present in mono‐ and polyunsaturated fatty acids has become clearer and clearer, due to the studies on membrane models, such as liposomes, and the medical research on trans fatty acids. Indeed, the trans isomers can occur not only as result of chemical processes in the food industry, entering the human body by food consumption, but also as a consequence of free radical stress in the body. The formation of trans isomers interpreted in a metabolic way envisaged their role as biomarkers and early indicators of cellular stress. This pathway has been studied in different organisms, and involves mainly the chemical reactivity of sulfur‐centered radical species, which are very effective lipid isomerization agents.

The molecular structures of fatty acid residues in phospholipids are deeply involved in the regulation of the cell membrane as sensors of stressful conditions, and these stimuli bring to the cell response called lipid remodeling. This is a fascinating mechanism, whose potentiality has been evidenced in the last decade, but is still awaiting its full appli-cation in medicine. Indeed, the membrane response by a rapid change of fatty acid composition and release of active fatty acid signaling mole-cules constitute important molecular information to be interpreted in different metabolic conditions. The principles of membrane formation and remodeling in combination with that of nutritional requirements will be described in this part as the main biological path to get successful cell adaptation, which is strongly connected with nutritional habits and lifestyles of individuals.


Membranes for Life and Life for Membranes

1.1 CELL MEMBRANES: THE ROLE OF FATTY ACIDS AND THE EXCLUSION OF TRANS ISOMERS

The cell membrane represents the fundamental structure and organizational element in the cells of living organisms. In fact, no cell can exist without the membrane; actually, cell reproduction and multiplication, such as in cancerogenesis, implies formation of membranes [1]. The complex mixture of lipids in an overall fluid state, where proteins and other molecules such as cholesterol are immersed, identifies the cell space and its boundary with the extracellular environment, but its behavior is not like that of a wall. Instead, this is the structure through which all communi-cations and exchanges useful to cell life occur, and in the twenty‐first century it represents the most direct and innovative site for correlation with the health condition.

The fundamental unit of the membrane assembly is the phospholipid molecule, with a characteristic structure that is defined as amphipatic. This means that in the same molecule two different parts coexist: the hydrophilic and the hydrophobic parts. The hydrophobic part cannot stay in contact with water, the biological solvent, since it is impossible to estab-lish any type of interaction (the so‐called hydrogen bonding). Therefore, the hydrophobic effect occurs, which leads to the perfect separation of the water molecules and the hydrophobic components in two phases, as is observed between oil and water. In phospholipids the hydrophilic part

1

4 MEMBRANES FOR LIFE AND LIFE FOR MEMBRANES

is called the “head” and the hydrophobic part is called the “tail”; as the structure shown in Figure 1.1 indicates, the hydrophobic part is made of long fatty acid chains (generally with hydrocarbon chains containing from 12 up to 26 carbon atoms), with and without double bonds, whereas the hydrophilic part is a polar residue, sometimes charged (e.g., in phosphatidyl choline). The coexistence of these two parts with opposite interactivity with water drives the specific organization called double layer, as represented in Figure 1.1: the arrangement is obtained by two molecules that are placed one in front of the other, and their polar parts are disposed outward facing water.

The double layer can expand until a critical number of molecules are assembled, at the so‐called critical aggregation concentration (CAC) that causes the two extremities of the double layer to become close to each other and form a round sphere, with water in its interior. In this way “compartmentalization” occurs, which allows the organization of cellular life to be exploited. In natural membranes cholesterol is the other important lipid component forming part of the layer, with the general effect of modulating the fluidity property of this aggregation. This is not the place to go into a deeper description of the numerous factors influencing membrane formation and its properties, which are better described elsewhere [2–6]. However, it is worth recalling that, as water is the most important element for life, hydrophobicity is the complementary property needed for life organization, which in fact

Water

WaterInsidePhospholipidpolar heads

Outside

Hydrophobictails

Hydrophilichead

Lipidbilayer

Figure 1.1 The membrane structure made of a double layer of phospholipids, and the fatty acid chains that form the hydrophobic layer

CELL MEMBRANES: THE ROLE OF FATTY ACIDS 5

induces compartimentalization. Indeed, the presence of the aqueous and lipid compartments plays a fundamental role in the distribution of the various biological elements, from small molecules to macromolecules, according to their partition coefficient, thus determining their different concentrations, inside and outside cells, by which physical and chemical interactions are established.

The primary function of membranes is to compartimentalize mole-cules but not to separate them; therefore the regulation of membrane permeability and fluidity properties is studied for understanding the subsequent events of diffusion, exchange, and signaling [7].

In this book we will not study the contribution from the “head” in depth, which is not insignificant, in explaining lipid diversity. In Figure 1.2 the variation of the phospholipid molecules is shown as different tails and heads. As an example, it is worth citing the effects of inositol lipids, which are present in small quantities in membranes; however, they participate in cell signaling associated with growth and immune processes, as well as in programmed cell death, and the transport of chemicals into and out of cells. The protein receptors, after activation, can induce the breakage of inositol lipids into pieces and the phosphate‐containing head group (phosphatidylinositol 3‐phosphate, PtdIns3P or PI3P) released into the cells’ interior binds to other proteins, propagating the signal, while the remaining lipid tail is involved in other kinds of binding to proteins, completing the activation process. Glycolipids are also involved in other important signaling processes, such as insulin response, and help the docking of viral proteins (such as HIV virus) or toxins (e.g., cholerae and tetanus toxins) to membranes. They are found in the outward‐facing part of the membrane bilayer, and in red blood cells their presence determines the combination of the AB0 blood group a person has. Obviously the dis-tinction of properties and functions of lipids by the polar heads can be deepened by reading several papers on this topic [8, 9].

In this book we focus readers’ attention on the hydrophobic tails of the phospholipids composed of fatty acids. This subject will be developed to demonstrate how important these constituents are for health, specifi-cally connecting molecular and nutritional contributions. As shown in Figure 1.2, the fatty acid structures are linked with their carboxylic acid function to the positions C1 and C2 of the l‐glycerol moiety of phos-pholipids. l‐glycerol is one of the isomeric forms; therefore it is worth mentioning that nature chose one enantiomer in a similar way as it chose the l‐form of amino acids.

The fatty acid chains display a high degree of diversity concerning the carbon atom number (chain length) and the presence of unsaturations

Fatty acids

Ceramide

O

O

O

O

Fatty acyl chains

Phosphatidic acid Phosphatidylcholine

Phosphatidylserine Phosphatidylinositol Phosphatidylglycerol

Phosphatidylethanolamine

Glycerol Phosphate

O X

X=

X=

H

Head group

O

P

O

C NH CH

CH

CH2

CH2

CH2

CH2 CH2 CH2OHCHNH3CH HOOH OH

OH

OH OHCOO

CH2 CH2 CH2 NH3N(CH3)3

H2C

CH

R1 C

O

O

R2 C

OH

Sphingosine+

+

–

+

CH3

CH3 CH3

CH2CH2N+P

Choline

Figure 1.2 Details of the phospholipid molecule with variation of fatty acid tails and polar heads. In the box the structure of sphyngomielin is displayed


(double bonds): (i) the chain can contain from 11 to 25 CH2 groups plus the carboxylic group (COOH), which is numbered as Carbon‐1, and (ii) some of these CH2 groups can be substituted by CH groups for double bond function in unsaturated lipids. These could appear as small variations, but it is not so. The variability of physical, chemical, and biochemical properties due to chain length and number of unsaturations can be relevant for the effects on membrane fluidity and permeability, as well on its functions.

In Figure 1.3 the main structures and names of the naturally occurring fatty acids in eukaryotic membranes are shown. The trivial names indicate the natural sources where they were first discovered. For the nomenclature, the numbering of the carbon atom chain and indication of the double bonds represent a useful way, together with the specifica-tion of the position and geometry of the double bonds, when present.

For example, the nomenclature of 12 : 0 or C12 : 0 indicates a fatty acid with carbon atom chain of 12 and no (0) unsaturation, which belongs to the family of saturated fatty acids (SFA, lauric acid). Conversely, 9cis‐18 : 1

Number of C atom:Number of unsaturation

Common name Melting point ( °C)

12:0 Lauric acid 4414:0 Miristic acid 5816:0 Palmitic acid 6318:0 Stearic acid 7020:0 Arachidic acid 77

16:1 Palmitoleic acid 3218:1 Oleic acid 1618:2 Linoleic acid – 518:3 Alpha-linolenic acid – 1120:4 Arachidonic acid – 49

Saturatedfattyacids

Unsaturatedfatty acids

1

Numbering

Numbering

1

9

Oleic acid 18:1Stearic acid 18:0

H

H

H

C

HH

H

CH

H

CH

H

CH

H

CH

H

CH

H

CH

H

CH

H

CH

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C C

O

OHC

OH

O

Figure 1.3 List of main saturated and unsaturated fatty acids, with their melting points, trivial nomenclature, and numerical annotation (number of C atoms : number of double bonds)


indicates an 18‐carbon atom chain with one double bond in the C9 position with the cis geometrical configuration (starting the numbering from the C1 of the chain), belonging to the family of monounsaturated fatty acids (MUFAs). The positions of the double bond are also described with the notation delta Δ followed by the number of the double bond along the chain. For example, in Figure 1.3 oleic acid has the double bond in the Δ. The 18 : 2 notation corresponds to molecules with two double bonds, and the various structures with more than one double bond belong to the family of polyunsaturated fatty acids (PUFAs). A unifying nomen-clature has also been proposed, but the trivial names are still very preva-lent. The carboxylic group can be found in the form of carboxylic ester, such as in triglycerides (the C(O)OH group is connected with the OH group of l‐glycerol, forming an ester function: C(O)O‐glycerol). The carbon atom chain with only CH2 groups (i.e., numeric notation C12 : 0) is present in the saturated fatty acid family. The most abundant SFA in the eukaryotic cell membranes is palmitic acid C16 : 0. In unsaturated fatty acids a carbon atom is connected with another carbon atom by two bonds instead of one, so that in place of two CH2 groups there is a >CH=CH< functionality, which is in fact the carbon–carbon double bond (Figure 1.4).

As shown in Figure 1.3, the numbering indication is accompanied by the trivial names, which are very much in use despite the efforts of scientists to have a common and unequivocal nomenclature for fatty acids [10], to avoid misunderstanding that are very frequent (i.e., linoleic acid vs. linolenic acid or alpha‐linolenic acid vs. gamma‐linolenic acid). In Figure 1.3 the melting points are given, which can also be useful for envisaging the wide variety of temperatures realized by the different fatty acid structures. It can be seen that only saturated fatty acids can reach values over the physiological temperature of 37°C, and this can be intuitively extrapolated to the “hardening” effect of saturated fatty acids in the hydrophobic membrane layer. Conversely, the presence of

H H HH

H H

H

H

H

H

H

H

H

H H

C

C C

C

C

C C

CH

H H

Figure 1.4 A representative region of the carbon atom chain in the saturated fatty acids (left, –CH2–CH2– groups), in the cis unsaturated fatty acids (center, with the cis >CH=CH< functionality), and in the trans unsaturated fatty acids


double bonds reduces the melting temperature, which has the effect of “ softening” the membrane containing unsaturated fatty acids.

Indeed, the double bond is a very important element of the hydro-phobic layer, on which most of the cell membrane characteristics depend. Most of the fatty acids in living organisms (and in eukaryotes, in general) have the double bonds in a precise geometrical configura-tion, which is called cis (middle structure in Figure 1.4). This corre-sponds to the position of the two substituents connected to the >CH=CH< group that are in the same direction as the plane involving the double bond. As a consequence, the cis geometry creates a characteristic bent of about 30° in the unsaturated fatty acid chain, compared to saturated fatty acids, which have a typical linear molec-ular structure. The other possible disposition of substituents is the trans configuration, where the two substituents are in an opposite direction to the double bond plane. As shown in Figure 1.4, the fatty acid structure consequently looses the kink, becoming more similar to the saturated fatty acids. Considering the two types of double bond configurations and the fact that the trans geometry is the most thermodynamically stable one, it is remarkable that the most stable unsaturated isomer is excluded from the natural lipid structures of eukaryotes. The reasons for this exclusion have been considered only recently, based on its evo-lutionary meaning, since both cis and trans geometries are present in prokaryotes, and the geometrical interconversion via enzymatic activity is the basis of bacterial resistance to stress [11]. Interestingly, the trans lipid structure has a profound effect on membrane fluidity and perme-ability, as well as on protein and channel functioning, as can be intui-tively extrapolated from the sharp difference in the melting points of the corresponding free fatty acids (13.4°C for the 9cis‐18 : 1 isomer and 44°C for the 9trans‐18 : 1, whereas the corresponding saturated fatty acid, stearic acid 18 : 0, melts at 72°C) [12]. Indeed, the trans configura-tion has a completely different effect on the fluidity of the phospholipid bilayer, compared to the cis or saturated one at physiological tempera-ture, and also on its overall sensor functions [11, 13, 14]. The replacement of one cis acyl chain by a trans fatty acid in phosphatidylethanol amine increases the transition temperature in the range of 18–31°C, depend-ing on the structure of the other acyl chain of the lipid molecule. Therefore, the conversion of cis unsaturated fatty acids into their trans configuration results in a significant reduction in membrane fluidity, which is, however, intermediate with the replacement of cis by saturated fatty acids.

The trans geometry has an important role in fat dietary consumption, which became a hot topic in the nineties after the discovery that trans isomers


are found in oils chemically manipulated by the food industry, in particular deodorized and partially hydrogenated oils. In the following years, due to the strong involvement of consumers’ organizations especially in the United States, and to scientific research on the health effects of the trans fatty acids (increasing cardiovascular risk, in primis), the US laws became very strict with the obligatory indication of trans fatty acids cited as nutri-tional facts and a limit of 0.5% of these fats in foods [15]. Nowadays, the food industry seeks different ways to abandon the process of partial hydrogenation and the use of partially hydrogenated oils. In Europe, the control of trans fatty acids in foods and the need for limiting the dietary intake are considered as important issues for the protection of consumers, but disclosure of nutritional information indicating trans fatty acid content is not yet required by legislation. Therefore, their presence in foods of European countries remains unknown (see also Section 4.1). This can be a problem especially because the deodorization process is largely applied when the natural sources of omega‐3 fish oils, for example, have to lose their unpleasant smell for entering the functional food chain, such as in milk or margarines. This process employs high temperatures and under these conditions a certain percentage of the cis omega‐3 fats are trans-formed into their corresponding trans isomers, which can also be metab-olized to membrane lipids, thus reaching the level of mitochondria and causing functional impairment [16]. From the data obtained so far on the effects of trans fatty acids, it is clear that it is much safer to use only natural, unprocessed oils for food. Moreover, in the market for “healthy” foods, omega‐3 fats are perceived by consumers as useful compounds; therefore the use of deodorized fish oils containing trans modifications can belie expectations. The issue of omega‐3‐ containing products, including nutraceuticals, is still only at the level of research, but it is hoped the interest of producers regarding consumer safety can be kindled.

It is worth noting at this point that in recent years the presence of trans isomers has been evidenced not only in connection with diet and oil manipulation, but also with the process of endogenous transforma-tion of natural lipids, due to free radical production during cellular stress [11]. In Figure 1.5 the reaction of sulfur‐centered radicals is shown, which are able to enter the hydrophobic layer of the membrane and react with the double bond of unsaturated lipids, thus effecting the reac-tion of cis–trans isomerization. In Chapter 4, this reactivity will be more detailed, and the role of trans lipids as markers of endogenous stress will be explained. The source of trans lipids as a result of an endogenous transformation of the naturally occurring cis lipids has a different implication compared to nutritional (exogenous) sources, connected

ORGANIZATION AND HOMEOSTASIS 11

with oxidative pathways as a signaling activity and with a threshold for damage consequences.

1.2 ORGANIZATION AND HOMEOSTASIS

The membrane hydrophobic bilayer has a precise distribution of the fatty acid molecular functionalities, which can be evaluated by computer simulation [4, 17]. As shown in Figure 1.6 the double bond of monounsat-urated fatty acids is calculated to occupy the core region of the membrane at ±10 Å from the center in a membrane model whose thickness is about 60 Å. These models bring attention to the important feature of organization that the phospholipid molecules achieve in natural membranes, connected with other facts such as the insertion of protein structures, resulting inter-actions, and the overall functioning of the membrane, which acts more as a passage of nutritional and signaling substances than as a wall separating the internal and external cellular compartments.

To understand in depth this organization the fundamental unit of the membrane structure has to be described, which is the phospholipid shown in Figure 1.7. It has an amphipatic character, with a polar portion (the “head”) and an apolar portion (the “tail” with two hydrophobic fatty acid chains) in the same molecule. The molecular shape can be assimilated to a cylinder, and in aqueous systems a certain number of these molecules spontaneously organize themselves in order to expose only the polar heads toward water, whereas the hydrophobic tails are one in front of the other preventing water contact. In this way the double layer is formed and at a critical aggregation concentration (CAC),

X.

9

129

12

X.

transcis

Figure 1.5 Free radical attack (X•) in the membrane bilayer and formation of trans phospholipids


calculated as the number of molecules per liter of solvent (molarity), the double layer folds on itself forming a spherical form, enclosing an internal volume of water. This effect, called “compartimentalization” in biology, is considered the basis for all life, since the cells are formed only when the membrane is formed, and without the definition of internal/external compartments life cannot start.

–30

3020100–10–20–30

–20 –10 0Distance from bilayer center (Å)

10 20 30

PW

PGLYC

PCH2

PCHOLPCH3PCOO

DHH

PPO4

CHOL GLYC C4 C9 C15α

β

Figure 1.6 Model of the distribution of the structure of phospholipid chain within the membrane bilayer; P= position, D= distance, GLYC= glycerol, COO= ester group, Cn= various position of the fatty acid chain, PO4= phosphate group, CHOL= cholesterol, W= waterAdapted from Ref. [4]

Phosphate group

Cholinegroup

Polar head

L-glycerol

Figure 1.7 A representative phospholipid, with the polar head of phosphatidylcho-line and the two fatty acid hydrophobic tails attached to l‐glycerol


In the life of any organism, cells are formed, stay alive for a certain period of time, and then die, whereas new cells are formed during cell turnover in a continuous flow. In the turnover the assembly of phospho-lipids in a double layer to form the cell membrane is a fundamental aspect. What kind of phospholipids and fatty acids do form the cell membrane? When an in vitro experiment on membrane formation is carried out, phospholipids with all types of structures (saturated, cis or trans fatty acids) can form membranes, with the fatty acid residues having a particular influence on vesicle shapes and the resulting volumes, expressed as diameter of the vesicle [13]. In model vesicles, the cell size follows the fatty acid order of saturated < trans < cis, indicating that the latter geometry provides the largest cell volume. This is an important property of cis fatty acids necessary for the eukaryotic cells, which are larger cells than other organisms.

A different consideration must be done in case of in vivo formation of natural cell membranes, when the compartmentalization by the aggregation of phospholipids is crucial not only for the formation of the compartment, but also to produce the functionalization of the double layer with a series of proteins, which allow signaling and exchanges to occur. It is now known that the selection of different fatty acids of the membrane phospholipids is connected with the nature of the proteins that are formed in the cell according to the genetics of the cell line. In other words, tissues are composed of cell membranes containing var-iable quantities of fatty acids depending on the type of tissue and its function. In Chapter 2 the fatty acid families and the levels of different tissues will be treated in depth.

Here we would like to remark that when cells are formed or replicate, there is an enlargement of the cell volume and a duplication of the number of phospholipids, parallel to DNA duplication; therefore the recruitment of fatty acids for new phospholipids is needed (Figure 1.8). The availability of fatty acids from the lipid pool of each individual becomes a crucial point during cell turnover (see also Section 3.3).

In fact, the selection of fatty acids for cell membranes is effected on the basis of the available fatty acid pool, which in turn is connected to the metabolism and the diet of the individual. As will be described in the next chapter, fatty acids come from an integrated contribution of biosynthesis and nutrition, to achieve an adequate diversity in the fatty acid pool. For eukaryotes this availability is crucial, especially because some fatty acids like omega‐6 and omega‐3 fatty acids cannot be biosynthesized, and in fact are called essential fatty acids. During the fatty acid recruitment for the formation of cell membranes, the more complete and balanced the


fatty acid pool, the better the resulting membrane composition. Membranes can become an important observational point for personalized evalua-tion. In case of incompleteness of the fatty acid pool, membranes of the tissues are formed with an improper fatty acid balance compared to the standard values, and this corresponds to a wrong assembly from a biological point of view with impairment of membrane functionality.

However, the event of membrane impairment is not so dramatic initially to be noticed, because the membrane composition is very dynamic and undergoes continuous modifications, which have been the subject of intense research in the last two decades. The model of fluid mosaic envisaged by Singer and Nicholson in 1978 has been integrated with new knowledge on the processes of membrane remodeling and signaling, which imply continuous lipid changes. These processes will be considered in detail in Chapter 5. Here it must be mentioned that once the composition of the membrane compartment is established, the process of phospholipid remodeling takes place every moment as a response to any stimulus and the changes in the membrane composition are part of cell adaptation. The fatty acid selection for the membrane phospholipids after each stimulus represents the “molecular” response combined with the quality of the lipid pool. It is now well known that the regulation of this process is based on the principle of “homeostasis” (or homeoviscous adaptation), by which membranes after perturbation restore, or at least try to restore, their balance keeping all functions as constant as possible. This includes also the functioning of proteins embedded in the membrane structure; therefore, successful homeostatic control is reflected by the efficacy of the metabolic response to stimulus.

At this point it is worth underlining the fundamental link between the molecular aspect and the clinical outcome, which is of interest to health operators for improving the diagnostic interpretation of the individual

Phospholipid recruitmentand membrane duplication

DNA duplication

Cell duplication

Figure 1.8 Duplication of membranes during cell replication


status. Indeed, if the phospholipid selection during the earlier‐mentioned remodeling cannot occur in the proper way, the resulting homeostatic control is lost, and this can be reflected by a wrong cellular response with several symptoms, without representing a real pathological status. Membrane lipidomics provides information on the role of membranes for the homeostatic control as a fundamental molecular aspect for health. Based on the membrane asset and its homeostatic control, the cell adaptation to physical (pH, ionic changes, temperature, etc.) and biochemical (signal cascades, inflammation, stress, etc.) solicitations takes place, that has to occur instantaneously, with the final goal to harmonically follow up cellular events.

Therefore, cell homeostasis is based on “molecular” homeostasis obtained not only by retaining the properties of fluidity and perme-ability, but mainly by allowing the rapid exchange of its lipid elements as a short‐term response. On the basis of recent discoveries, it can be concluded that membrane organization has a key role in the regulation and balance of the whole cellular metabolism, which can deeply influence life. The definition of “membranes as metabolic pacemaker” from Professor Antony Hulbert of the University of Wollongong, Australia, indicates that membranes are not a passive spectator but an active element of cell life and fate [18].


1a In Depth: The Formation of a Cell Membrane

The formation of the membrane double layer is a fundamental biological phenomenon, which has inspired an important applica-tion in biotechnology, which is called liposome. As per Danilo Lasic, one of the scientists that first developed this field [19], “every cell on the planet is surrounded by a membrane that has the same basic lipid bilayer structure as that exhibited by liposomes.” The property of spontaneous aggregation exhibited by phospholipids was carefully studied, observing the typical behavior of these amphiphilic molecules, as shown in Figure 1.9. The mode of aggregation in an aqueous medium is the same for all types of phospholipids, whose molecular shape can be assimilated into a cylinder, therefore forcing the organization in a layer growing on the lateral sides. Since the molecules are amphipatic, the hydro-phobic tails cannot face the water and one layer becomes closer to another layer, in such a way that the hydrophobic parts are in the inner part of the layer, avoiding water contact, whereas the polar heads can be exposed to water. The so‐formed double layer continues to grow until the molecules reach a “critical” number (CAC, critical aggregation concentration) such that the bilayer folds on itself, thus creating a vesicle with a cavity full of water surrounded by water. In biology this is the cell prototype (proto‐cell); in biotechnology this is called liposome (Figure 1.9).

The diversity of fatty acids composing phospholipids and the role of cholesterol as the “additive” of this lipid assembly attracted considerable interest from researchers, since they certainly take part in the efficiency and regulation of membranes. An investiga-tion of liposome behavior provided numerous insights on mem-brane behavior and biological effects.

It is also clear that the membrane lipid library (about 1000 molecules up to now) combined with the membrane protein struc-tures discovered so far (among about 71 000 protein structures available in the RSCB Protein Data bank, 1 095 structures are those of membrane proteins) define a larger field of lipid–protein inter-actions still to be thoroughly explored [3], in order to figure out what are the important elements needed during membrane formation and reduce the possibility of functional impairment.

Definitely, if the DNA organization and its four bases describing the genetic code involved incredible efforts in terms of financial


and human resources, it is not easy to figure out how much involve-ment of money and researchers would be needed to unveil the “membrane code.”

As will be seen in Chapter 2, the simplest principle to be followed for optimal membrane performance is the completeness of the “fatty acid pool,” in quantity and quality of the fatty acid mole-cules, thus allowing the membrane formation process to occur with the spontaneity and self‐regulation used during millions of years of life experience!

1b In Depth: Cholesterol and Membranes

Cholesterol is a lipid molecule that can occupy the upper region of the bilayer, intercalating the phospholipids and increasing the space between them. Actually, recent research highlighted that the position and orientation of phospholipid/cholesterol membranes

Formation of the double layer

Planar double layerwith polar head exposed to water

Sealed compartmentformed by the double layer

Figure 1.9 Aggregation process of the phospholipid into a double layer, with folding and creation of the proto‐cell or a liposome


follows precise rules driven by the polar (OH group) and apolar (steroid ring and alkyl chain) molecular portions, avoiding the cholesterol–cholesterol direct contact, but mostly intercalating cholesterol between two phospholipids as in Figure 1.10 [20, 21].

The cholesterol effect is the increase of membrane fluidity; therefore the lower the fluidity, the higher the quantity of choles-terol required. Reduced fluidity occurs when the saturated fatty acid proportion exceeds the optimal values of the overall content of membrane phospholipids. This condition is in general due to an activation of biosynthesis (FAS, fatty acid synthase), as an endoge-nous process, or an increased intake of food rich in saturated fatty acids, as an exogenous source. The homeostatic regulation of fluidity with the increase of saturated fats brings to the biosyn-thesis of cholesterol as feedback. Since the value of cholesterol in the plasma is monitored as a preventive indicator of metabolic unbalance, it is very important for health operators to acquire a comprehensive scenario of the “molecular” factors influencing this biomarker, where the membrane properties must be taken into account due to the contiguity between lipids and cholesterol (Figure 1.10). On the other hand, the feedback of cholesterol bio-synthesis can also be induced by an excess of membrane fluidity, when stabilization of the membrane assembly is needed. This can occur when an excess of PUFA is incorporated in the bilayer and destabilizes the membrane asset. This aspect will be discussed in the section dedicated to nutraceuticals in Part 2.

CH3

CH3

H3CH3C

H3C

HO

Cholesterolmolecule

Polar portion

Apolar portion

Figure 1.10 Cholesterol and its position in membranes


Obviously, dietary factors have to be carefully considered and all aspects can vary due to the individual situation. As a general rule, among the factors influencing cholesterol levels, saturated fatty acids have a relevant effect and this cannot be dissociated from the previously examined effect on membrane properties. Indeed, it is well known that the diminution of saturated fatty acids from the diet lowers plasma cholesterol levels [22]. The correlation between cholesterol and saturated fatty acid levels should represent a seminal example of the application of “molecular” principles to the definition of a comprehensive “health biomarker,” giving to health operator an important piece of information about the dietary and metabolic status of the subject. In the medical practice discussed in Part 2, a panel of fatty acid bioindicators will be included in order to set up nutritional and nutraceutical strategies directed toward the correct membrane balance.

1c In Depth: Lipid Rafts

In membrane organization an important role was discovered for the so‐called lipid rafts, which are small aggregates of phospho-lipids and cholesterol, containing another type of phospholipid called sphingolipid (see Figure 1.2). A mixture of these three types of lipids creates a rigid packing with less lateral movement or dif-fusion, corresponding to microdomains existing in a liquid‐ordered phase that is significantly more fluid than a gel phase. This is quite different from the phospholipid liquid phase; therefore such a lipid mixture separates as “raft,” generally of small size (26–70 nm in diameter) floating in the double layer. Lipid rafts can be iso-lated from the rest of plasma membrane since they constitute a “ detergent‐resistant” section of the lipid bilayer. The name describes this phenomenon, which is very important for the accommodation of specific macromolecules, that is, proteins of specific size, and this makes the compartimentalization of the membrane even more precise, regarding separation of places and roles [23]. Lipid rafts can be isolated from the rest of plasma membrane since they con-stitute a “detergent‐resistant” section of the lipid bilayer; however, research is still ongoing on the existence and role of lipid rafts. It emerged that rafts are involved in the movement of cholesterol and


other proteins, thus furnishing organized places for signaling, or as the points for molecules to leave and to enter cells, even in the viral or microorganism attack (such as HIV or malaria) through also the attachment of glycolipids to rafts. This subject can be considered “in progress” and more knowledge will be gathered in the coming years for explaining completely the meaning of the membrane organization and the effective contribution of lipid rafts.


Fatty Acid Families: Metabolism and Nutrition

The main structural characteristics of fatty acid families have been introduced in Chapter 1, describing the saturated and unsaturated fatty acid molecules. Here more details will be provided on those fatty acids that are found in natural membranes, and the reader will be introduced to the specific role of each structure, from its biosynthetic origin to its biological role.

This chapter contains basic information on lipids, which have mostly been studied in the last two decades, bringing a revised interpretation of their biological and metabolic importance. For health operators it is worth noting that in the food pyramid of the twenty‐first century the position of fats is revolutionized compared to that in the pyramid of the nineties, moving them from the top to the bottom, due to increasing and strong evidences of the fundamental importance of lipid quantity and quality (Figure 2.1).

In particular, research pointed out that the fatty acid diversity of the lipid pool plays a crucial role in the regulation of the cell membrane compartment, from where all other metabolic and signaling functions derive. Membranes and their fatty acids are key factors in whole cell metabolism for reproduction and maintenance, and this book focuses on this subject in order to facilitate its comprehension and use in clinical practice. On the other hand, we cannot dismiss the other important role of fatty acids as energy provider, through the so‐called beta‐oxidation of fatty acids, gener-

2

22 FaTTy aCId FaMIlIes: MeTabOlIsM and nuTrITIOn

ating cellular fuel in the form of aTP. The changed requirements, due to the mainly sedentary life of most people in industrialized countries, will be treated in Part 2, examining fat accumulation, the functional role of adipose tissue, and membrane lipidomics of overweight and obesity.

Redmeat:butter

Whiterice,whitebread,potatoes,and pastasweets

Dairy or calciumsupplement, 1–2 times/day

Use sparingly

Fish, poultry, eggs0–2 times/day

Nuts, legumes, 1–3 times/day

Vegetables(in abundance)

Daily exercise and weight control

Multiple vitaminsfor most

Alcohol inmoderation

(unlesscontraindicated)

Fruits, 2–3 times/day

Whole grain foods(at most meals)

Plant oils, including olivecanola, soy, corn, sunflower,

peanut and other vegetable oils

Fats, oils and sweetsUse sparingly

Milk, yogurtand cheese group2–3 servings

Vegetablegroup3–5 servings

Meat, poultry, fishdry deans, eggs,and nuts group2–3 servings

Fruit group2–4 servings

Bread, cereal,rice, and pasta

group6–11 servings1992

2001

Figure 2.1 The food pyramid evolution in the turn of the twenty-first century

saTuraTed FaTTy aCIds 23

nowadays, the fatty acid–containing lipids cannot be considered syn-onymous with the unspecific word “fats”; knowledge of their structural and functional characteristics is needed for health operators to drive nutritional and therapeutic choices, and use their immense potentiality for the benefit of patients.

The fatty acid families will be described in this chapter, together with the main biosynthetic and nutritional pathways, introducing the reader to the concepts of the natural balance among these families, in connection with the homeostatic control described in Chapter 1, and the need of determination of the individual status for a personalized approach.

2.1 saTuraTed FaTTy aCIds: bIOsynTHesIs and dIeTary reGulaTIOn

The family of saturated fatty acids (sFas) has a typical linear molecular structure (Figure 2.2) with an even number of carbon atoms, deriving from the fact that the biosynthesis occurs with a 2 + 2 assembly of the carbon atom units.

The enzymatic system responsible for saturated fatty acid synthesis is called fatty acid synthase (Fas), a multienzymatic assembly that is based on the head‐to‐tail principle: the entrance of the first substrate from the head (the first two‐carbon units, named acetyl‐coenzyme a, with or without malonyl‐coenzyme a) and the transfer of the products from one to the other enzymatic subunit, until the exit of the assembled final product from the tail, that is, after the last enzymatic step. The final product of Fas is palmitic acid, the saturated fatty acid with a 16‐carbon

H

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C C

OH

O

Figure 2.2 The linear structure of sFa (18 carbon atom, 18:0, stearic acid): top, the molecular space-filling model; bottom, the molecular structure displaying the atoms and bonds.


atom chain. The total reaction occurring in fatty acid biosynthesis is represented by equation 2.1:

Acetyl-CoA ma nyl-CoA NADPH Hpalmitate CO NADP7 14 14

7 142

lo8 6 2CoA H O (2.1)

and independently from the malonate unit in equation 2.2:

8 14 714 8 7 7

Acetyl-CoA NADPH ATPpalmitate NADP CoA ADP Pi (2.2)

Fatty acid synthase (Fas) is localized in the cytoplasm of different cell types: hepatic, nervous, pulmonary, adipose cells, and generally in hormone‐sensitive cells (pituitary glands, breast, endometrial, prostate, seminal vesicle, and adrenocortex), as well as in gastrointestinal epithe-lial cells. It has also been evidenced that this enzyme is necessary for cell proliferation, and fatty acid biosynthesis is an indicator of cell growth due to the need for membrane fabrication. In some cases (breast and prostate cells) the Fas activity can be correlated with oncogene properties; therefore it can also be considered as a target for antitumoral activity [24, 25]. On the other hand, cell proliferation is also needed for tissue growth during pregnancy for fetal development and during neonatal and child life, and Fas is active in all these cases. The Fas activity depends on the presence of cofactors, such as nadPH, and thiols, such as phosphopantotein and cysteine. regulatory factors are also involved in the Fas activity, such as srebP (sterol response element binding protein) and usF (upstream stimulatory factor), which are correlated to cholesterol biosynthesis. The expression of Fas is favored by insulin, which indicates a strong relationship between carbohydrate intake and the synthesis of saturated fatty acids. since carbohydrate intake is a signal for lipid synthesis and accumulation, an important application of membrane lipidomics consists of the follow‐up of saturated/monounsaturated pathways for the design of nutritional inter-vention, as will be described in Part 2.

It is important to underline that membrane lipids also include choles-terol, as the second lipid type that has biosynthetic and nutritional origins, with an important role in overall membrane functioning. The copresence of cholesterol and fatty acids at the membrane level indicates a mutual regulation between these two elements. The “In depth” section in Chapter 1 has considered some details of this interaction.


Many other elements can regulate Fas directly acting on gene transcription, such as in the case of polyunsaturated fatty acids in hepatic cells, which influence srebP production and reduce steroid synthesis. In adipose cells srebP‐1 and fatty acid synthase expressions are inhibited by leptin, the hormone regulating food intake and fat metabolism pro-duced under the stimulus of exceeding fat depots. In this way, leptin inhibits fatty acid synthesis when the depots are high, regulating body weight, diminishing the food request and thus preventing other fats from being deposited. It is worth recalling that fat accumulation can also be indirectly inhibited by the hormones glucagon and epinephrine, which activate the beta‐oxidation process for fat burning. also, this process and the cofactors needed for the balance between fat accumulation and burning will be discussed in Part 2.

as previously mentioned, the first product of Fas is palmitic acid (C16 : 0), which is the most representative fatty acid in mammal tissues and membranes. another saturated fatty acid is stearic acid (C18 : 0, see Figure 2.2) obtained from palmitic acid by the activity of the elongase enzyme. This enzymatic step adds two carbon atoms to the fatty acid chains, and can work with both saturated, extending the carbon atom chains up to 26 carbon atoms, and unsaturated substrates [26]. This typ-ical biosynthetic scheme brings all fatty acids to have an even number of atoms in their molecular structures. The elongation steps occur mainly in the endoplasmic reticulum (er), specifically in the microsomes, which are a vesicle‐like formation containing pieces of the endoplasmic retic-ulum and possess many enzymatic activities. The family of the elonga-tion enzymes is called elOVl, categorized as elOVl1, 3, and 6, which preferentially elongate saturated and monounsaturated fatty acids, whereas elOVl2, 4, and 5 elongate polyunsaturated fatty acids [27]. The location of these enzymes is characteristic of the tissues (in humans elOVl3 in the brown adipose tissue, elOVl4 in the retina, and elOVl5 in many tissues). The elongation step has several uses during myelin development in the nervous system, as well as in tissues with a barrier function (stomach, lung, kidney, and skin), and this gene is well expressed. In nerve cells, sphingolipids containing very long saturated fatty acids (VlCFa) with chains up to 26 : 0 are important for the tight packaging of membrane structures called “raft,” which create the microenvironment for the functioning of ionic channel and receptors. However, in cell proliferation elOVl is also necessary, and the role of sphyngolipids and ceramides as markers is under evaluation [28]. The involvement of elOVl in metabolic and cardiovascular diseases still needs confirmation [29]. In Chapter 3 an overview of elongase and


desaturase genes will be given, although conclusive results are far from being obtained. as a matter of fact, elongase activity and its implications are important topics for undergoing research, which indicate that not only enzymatic activity but also the property and activity of each of the elongated fatty acids must be understood, putting them in the context of the overall balance of metabolic and nutritional contributions.

since saturated and polyunsaturated fatty acids are also substrates of desaturase enzymes, which are described in the next paragraphs, the interplay between these two enzymatic pathways is crucial for creating the lipid diversity and effecting the overall balance at the level of membrane composition [30]. Figure 2.3 summarizes the steps between saturated and monounsaturated fatty acids that will also be considered.

as the affinity of saturated fatty acids for desaturase enzymes is very high, long chain saturated fatty acids (>20 carbon atoms) cannot be formed in high amounts, and their increase can become an indicator of dismetabolic lipid pathways, in relationship with the desaturase activity measured by long chain polyunsaturated fatty acids. The plasma level measurements were applied to X‐linked adrenoleukodystrophy (the disease connected to the use of the so‐called lorenzo’s oil, rich in erucic acid, C22 : 1) and other peroxisomal disorders, and were found to be correlated to the severity of the disease conditions. The laborious meth-odology has not allowed for a wide use of this assay [30].

saturated fatty acids are also well‐known components of many foods, palmitic and stearic acids being the most frequent in animal fats, and are present in other vegetable sources, such as coconut, palm, cocoa, and

C16:0Palmitic acid

ElongaseElongase

(C18:0)Stearic acid

Long chain saturated fatty acids

20:022:024:026:0

(C18:1-∆9)Oleic acid

(C16:1-∆9)Palmitoleic acid

(C18:1-∆11)Vaccenic acid

∆9 desaturase

∆9 desaturase

Elongase

Figure 2.3 The main steps of the sFa–MuFa enzymatic interplay between elongase and desaturase


shea nut oils. The intake of saturated fatty acids should give a negative feedback to biosynthesis but, since Fas is stimulated by several other dietary factors as previously indicated, there is a tiny control that seems very easy to overcome, thus causing an excess of saturated fatty acids for the mixed contribution of diet and biosynthesis.

Considering the whole data on metabolism and food intake, the values given by the World Health Organization (WHO), the european Food safety agency (eFsa), and medical associations worldwide point toward maintaining the intake of saturated fatty acids at a minimum that is, in a total 25–30% caloric intake for an healthy adult; the sFa portion can reach a maximum of 33% of the total fats, but it is highly advisable to reduce this intake to the minimum possible [31, 32] Generally, the quantity of 10–15 g/day should be followed, and it is better to substitute saturated with unsaturated fatty acids to get better health effects.

It is worth underlining that a direct relationship between saturated fatty acids, plasma cholesterol, and lipoprotein levels emerges from several decades of research. However, the extrapolation that saturated fatty acids are directly correlated with risk of cardiovascular diseases is not yet proven [33]. In fact, a direct relationship is described for cardio-vascular diseases and lipoprotein/cholesterol metabolism, whereas the real harmful contribution of saturated fatty acids still remains to be determined.

saturated fatty acids and their health effects have been estimated using the measurement of these fatty acids in plasma lipids, generally without separating different lipid types, such as triglycerides, phospholipids, and cholesteryl esters. saturated fatty acids are mostly present in triglycer-ides as circulating lipids, whereas saturated fatty acids in membrane lipids play the most relevant role. In fact, from the membrane point of view, saturated fatty acids as phospholipid components have a very well‐known effect on the resulting properties, since their assembly gives rise to a compartment with a rigid structure, with the previously described “hardening” effect of the hydrophobic layers and interferences of trans-port and signaling phenomena. Therefore, perhaps the role of saturated fatty acids for health outcomes should be reconsidered by choosing which kind of parameters are used for estimating fatty acid levels, depending on the functional roles of these biomarkers. It is necessary for researchers in lipidomics to arrive at shared methodologies and interpre-tation tools, in order to offer clear indications for application to health, and avoid confusing scenarios that create a jungle‐type market with low guarantee for consumers.


Is the membrane compartment the best site to evaluate the effects of fatty acids? From a molecular point of view, it seems to be so and the basic information given in Part 1 is a premise for Part 2, where the readers will find several examples on membrane lipidomics of physiological and pathological conditions. Indeed, the fatty acid composition of membranes and the delicate balance between saturated and unsaturated fatty acids play a key role as “molecular” characteristics for satisfactory health improvement and outcome.

2.2 MOnOunsaTuraTed FaTTy aCIds: THe IMPOrTanCe TO be Cis

as previously said, the presence of the double bonds defines the sec-ond type of fatty acids as unsaturated fatty acids. Monounsaturated fatty acids (MuFas) have only one double bond. The most prevalent monounsaturated acid in cell membranes is oleic acid (9cis‐C18 : 1). For eukaryotic cells it can have two origins: (i) the biosynthetic origin, starting from stearic acid (C18 : 0) by the desaturase enzyme activity in position 9 of the carbon chain (see Figures 1.3 and 2.1); and (ii) the dietary origin with oleic acid as the main component of olive oil, in the form of triglyceride, which for some countries (Mediterranean area) is the most used source of fats. Oleic acid is also called omega‐9 fatty acid, applying the nomenclature that counts from the end of the carbon chain (C18) to the double bond position. Given that oleic acid may be supplied by the diet, the concept of the cell economy applies; that is, if the diet makes up for the needs, then the biosynthesis is spared, saving important coenzymes (i.e., nicotinamide dinucleotide nad, see section 2b) and the activity of desaturase enzymes. The effect of oleic acid on the properties of the cell membrane is enormously important, since the bending resulting from the presence of the cis double bond in position 9 gives the proper fluidity in the “heart” of the hydrophobic phospholipid packing. It must also be emphasized that the double bond of the monounsaturated fatty acids has the advantage of not being easily oxidized, or not to be so sensitive to the conditions of oxidative stress, either physical or chemical, which will instead be very consis-tent in transforming polyunsaturated fatty acids having more than one double bond. so, even in recent times, the role of oleic acid has been highlighted (also through the dietary intake) for its contribution

MOnOunsaTuraTed FaTTy aCIds: THe IMPOrTanCe TO be Cis 29

to membrane homeostasis, especially when the natural antioxidant defenses or enzyme is reduced, for example, as we shall see in Part 2, in aging or some pathological conditions.

another particularly important monounsaturated fatty acid is palmi-toleic acid (9cis‐C16 : 1), which is obtained by enzymatic desaturation of palmitic acid (Figure 2.3). This is also called omega‐7 for the number of carbon atoms counted from the end of the carbon atom chain (C16) up to the position of the double bond. The enzyme sCd (stearoyl‐Coa desaturase, sCd1 and sCd2, delta‐9 desaturase) works with the satu-rated fatty acid stearic acid, but also with palmitic acid, producing the double bond in position 9 with the cis configuration. Palmitoleic acid is not found in high amounts in normal foods (low percentages in butter, while the sources of macadamia nuts and sea buckthorn contain moderate amounts). This means that the level of this fatty acid detect-able in the fat tissues can give a direct measure of desaturase activity, definitely better than other monounsaturated fatty acids confusable with dietary intake. It is worth noting that palmitoleic acid can also be obtained from vaccenic acid (11cis‐18 : 1) by the process of beta‐oxidation, to eliminate two carbon atoms from the carboxylic head; however, this pathway is not predominant in human metabolism. From the latest research more and more details are being unraveled about the role of this fatty acid in very specific metabolic steps. For example, pal-mitoleic acid has been found to be particularly important in the balance of lipid metabolism, because it is able to act as a warning to the muscle and liver cells and prevent problems of lipid accumulation in experi-mental animal models. It was defined as a lipokine in 2008, and since then a lot of interest also arose for nutraceutical and pharmaceutical applications [34]. It is important to underline that, in case of palmi-toleic acid, the correct identification of this fatty acid as 9cis‐16 : 1 dur-ing the analytical experimental procedures must be carried out and the protocol must be double‐checked. Often, the use of mass spectrometry protocols to individuate palmitoleic acid may not be appropriate, because the molecular mass is the same for cis and trans geometrical and positional isomers of monounsaturated fatty acids with the same number of carbon atoms in the chain. Therefore, mass spectrometry cannot individuate the presence of a pure palmitoleic acid fraction. Instead, gas chromatography (GC) is the gold standard for the identification of all fatty acid isomers. a good example is represented by palmitoleic acid and its positional isomers 6cis‐16 : 1, named sapi-enic acid, which is obtained by the activity of delta‐6 desaturase on


palmitic acid [35]. In Figure 2.4 the two desaturase enzymes working on palmitic acid are shown. It is important to note that delta‐6 desatu-rase preferentially works on PuFa, specifically on linoleic acid (9cis,12cis‐18 : 2), and palmitoleic acid can also derive from metabolic degradation (beta‐oxidation) of vaccenic acid (11cis‐18 : 1). Metabolic interplay of these pathways can occur and have different outcomes in each individual; therefore the result of an unequivocal GC analysis is crucial for correct identification and clinical interpretation. The meaning of these different pathways for palmitic acid is still under investigation, and research on sapienic acid will unveil important roles of the mono-unsaturated fatty acid family.

Palmitoleic acid can be added with two carbon atoms by the elongase enzyme, to give vaccenic acid (11cis‐18 : 1) as shown in Figures 2.3 and 2.4. The latter can also originate from the diet with the intake of various foods, including milk and dairy products, and its degradation via beta‐oxidation can produce palmitoleic acid again.

at this point it is worth noting that the saturated and monounsatu-rated fatty acid pathways are very important for the life of bacterial cells since their lipids contain only these two fatty acid families. Multicellular organisms, such as eukaryotes, are developed by adding another class of fatty acids, the polyunsaturated fatty acids (PuFas), which will be introduced briefly below and treated more extensively in Chapter 3.

Palmitoleic acid (9cis-16:1)Elongase

Delta 9-desaturase (SCD1)

Palmitic acid (16:0)

Vaccenic acid (11cis-18:1)

Linoleic acid (9c,12c-18:2)

Gamma-linolenic acid (6c,9c,12c-18:3)

β-oxidation

Delta 6-desaturase (FADS)

Sapienic acid (6cis-16:1)

O

OH

O

OH

O

OH

O

OH

OH

O

OH

O

Figure 2.4 The two desaturase pathways on palmitic acid to give the two geomet-rical hexadecenoic isomers (9cis‐16 : 1, palmitoleic acid and 6cis‐16 : 1, sapienic acid) combined with other competitive pathways involving vaccenic acid (11cis‐18 : 1) and linoleic acid (9cis,12cis‐18 : 2)

2a In Depth: The key Steps of Phospholipid Synthesis

as previously indicated, the key molecule, bringing fatty acids to their highest functional role in forming cell membranes, is the phospholipid (Figure 1.7). From the combined fatty acid biosyn-thesis and dietary contribution, phospholipids are formed in the cytoplasm and then start to assemble spontaneously in vesicles or other organized structures (such as endoplasmic reticulum and microsomes, which host the protein synthetic apparatus and enzymes) to reach the largest organization with high structural complexity of plasma membranes. This spontaneous process cre-ates a continuous flow of molecules regulated by biophysics and by the homeostatic principle, as previously described.

The biosynthesis of phospholipids (Pl) occurs by several path-ways. The building blocks are fatty acids, l‐glycerol, phosphate and the polar groups, such as choline, for the most diffuse phospholipid in the eukaryotic membranes. Here we wish to indicate the influence of some factors, in order to understand how metabolic and nutri-tional conditions, which change for each individual, can influence the membrane status: starting from fatty acids obtained after the Fas activity, only saturated and monounsaturated pathways are formed (Figure 2.3). In order to enter in Pl synthesis they have to be transformed into acyl‐coenzyme a derivatives (Figure 2.5). The coenzyme a structure is made of adenosine with two phos-phate groups, pantotenic acid and cysteine, which has a free thiol (–sH) group connecting with fatty acids giving fatty acyl Coa. The enzyme acyl‐Coa synthetase catalyzes the formation of the thioester bond between a fatty acid and coenzyme a. Thioester

O

COH

+CoA-SH

O

CS CoA

Acyl-CoA

O

O O O O

OH OHCH2

CH2

CH3

HO CH

NH

C

C

CHN

OO

Coenzyme A(CoA-SH)

H3C

H2C CH2 CH2–CH2 SH

NH2

N

N N

N

P

O

O

P

O

ATP

Acyl-CoAsynthetase

AMP+PPi

Figure 2.5 structure of coenzyme a and reaction with the acyl group of a fatty acid



links are high‐energy bonds and the enzyme uses the energy from aTP to drive the formation of the thioester. It is worth recalling that thioesters are also intermediates for fatty acid catabolism, by enzy-matic conversion into the corresponding carnitine ester (carnitine acyl transferase, Figure 2.6). This is a crucial transfer of fatty acid into mitochondria to start the beta‐oxidation process (burning fats).

Concerning the coenzyme a structure, the vitamin component is pantotenic acid, which derives from a large variety of dietary sources, and plays an important role for the synthesis of androgens and sex hormones. There is scarce possibility of depletion except in cases of an increased requirement, such as during sexual development and the luteinic phase. The amino acid component is cysteine, which has a crucial involvement in the redox balance and in the synthesis of the tripeptide glutathione; therefore a depletion for coenzyme a synthesis can arise because multiple requirements of the organism are not adequately balanced.

a second Pl path uses the fatty acids and l‐glycerol from TaG, which derive also from the diet (Figure 2.7). In this case the types of fatty acids are more connected to dietary choices of each individual. This pathway is an important step to introduce the essential fatty acids (omega‐6 and omega‐3) into Pl, which cannot be biosynthesized in the eukaryotic cells (see Chapter 2). Hydrolysis of triglycerides occurs with intervention of lipase enzymes during digestion, whereas the recombination of fatty acyl chains (always as fatty acyl Coa) with l‐glycerol is obtained by the same lipase enzymes present in the bloodstream. during the rearrangement of TaG, 1,2‐diacylglycerol (daG) is formed, and at this stage the phosphorylation of the free OH group in position 3 is obtained by transferring the phosphate from glucose‐6‐phosphate by acyl transferase enzymes (dGK1, diacylglycerol kinase using cytidine triphosphate, CTP), to form phosphatidic

O

CS CoA

Carnitine Acylcarnitine

CoA-SHCarnitine

acyltransferase

OH

O

HO

OH

O

O

O

CN

N

Figure 2.6 Transformation of acyl‐Coa into acyl carnitine to pass into the mitochondria

acid, which can then take the choline molecule to become phos-phatidylcholine (PC) (Figure 2.7). From membrane phospholipids the change of the polar group can occur by action of phospholi-pase d, which cuts the lipid at the polar head to be substituted with other polar heads, or further transformed to daG.

another important point is that phospholipid synthesis must occur in all tissues, for ensuring cell growth and turnover; there-fore fatty acids must be available everywhere in the body. The fatty acid transport occurs endlessly from the bloodstream to tissues and vice versa, with fatty acids continuously passing through cell mem-branes to start their metabolic changes. How the transport of fatty acids occurs is a matter of current investigations. It is known that albumin is the most efficient transporter of fatty acids in the blood-stream, and that fatty acids enter the hydrophobic bilayer by sev-eral means: by the “flip‐flop” from the exterior to the interior layer or if there is a link with a membrane protein that makes the trans-port (fatty acid transport or binding proteins, FaTP or FabP). When the fatty acid is internalized, it is converted into a coenzyme a derivative, and is transformed as explained earlier. an important role for fatty acid transport is played by lipoproteins, with their various components that are treated in section 4.4.

Phosphatidic acid, PA

PC

PE

PS Membranephospholipids

PL D

TAG

OH

OH

OH

R CO

OH

R' CO

OH

R'' CO

OH

OCOR

OCOR'

OCOR''

Lipase

O

O

OO

RR

OO3 1

2

O

O

P

L-glycerol

Fatty acids

DAG

DGK1Choline

Nutrition

Fatty acyl CoAIn PA synthesis

Figure 2.7 Main transformations involved in phospholipid synthesis



an impairment of one of the stages or a depletion of one of the elements of Pl biosynthesis is reflected in the incorrect formation and balance of the membrane lipid assembly.

2b In Depth: Biosynthesis of the Double Bond and Desaturase Features

The formation of a double bond in the fatty acid chain occurs in the endoplasmic reticulum of the cell and involves the following proteins:

• nadH–Cyt b5 reductase, which is a flavoprotein with flavinadenin dinucleotide (Fad) as prosthetic group

• Cytochrome b5 (Cyt b5)• desaturase, which contains two iron atoms complexed to a

histidine residue in the active site

The transformation occurs as follows:Two electrons pass from nadH to the desaturase enzyme

via the Fad‐containing reductase enzyme and cytochrome b5: nadH → Fad → → cyt b5 desaturase. Two electrons are extracted when the double bond of the fatty acid is formed.

The formation of oleic acid from stearic acid (unbalanced equation 2.3) is:

Stearate NADH H O H O oleate NAD2 22 (2.3)

We will discuss in Part 2 several examples of the involvement of desaturase enzymes in the regulation of key fatty acid levels and disease onsets. Here it is worth underlining that desaturase enzymes play a big role in the control of the levels of saturated fatty acids, transforming them into monounsaturated fatty acids. Therefore, sFas are not harmful until a certain concentration threshold is reached in the membrane phospholipids causing the “hardening” of the cell membrane asset. Here it is worth mentioning the utility of a molecular diagnostic tool evidencing the impairment of this impor-tant enzymatic transformation by desaturase. The membrane com-position can be analyzed by lipidomics with a follow‐up along the years and the unbalance can be evidenced by an increase of the ratio between saturated and monounsaturated residues. It is intuitive to

understand how the two fatty acid families can affect membrane properties by seeing the melting point (mp) differences between corresponding saturated and unsaturated fatty acids reported in Figure 1.3. For example, stearic acid (mp 70°C) is transformed by the activity of delta‐9 desaturase into oleic acid with a melting point difference of more than 50° (mp. 16°C). When could the desaturase activity be impaired? Generally speaking, desaturase enzymes can vary along the human life span. They are absent until the sixth month of life; therefore maternal milk is an important source of unsaturated fatty acids in this period, provided that mothers take care of their diet during pregnancy and lactation. The absence of unsaturated fatty acids can influence the skin quality of the new-born baby, and is often correlated to the atopic disease at birth. desaturase activity can be critical in the elderly, and in these subjects unsaturated fatty acid supplementation by dietary habits is very important. On the other hand, it is also important to consider that cofactors of the desaturase enzymes are nucleotide molecules, such as nicotinadenine dinucleotide (nad) and flavinadenin dinucleotide (Fad), acting as electron‐transfer agents in many biological pro-cesses, as well as the iron metal in the active site of the enzyme, coadiuvated by other metal cofactors, such as Mg and Zn, and the prosthetic groups of group b vitamins. These compounds and cofac-tors are required in different processes related, for example, to infections, sportive activity, and the inadequate intake of essential components as well as of micronutrients, which can create critical conditions. Moreover, most of the microelements and vitamins are generated by intestinal microbiota, whereas the lipid absorption from intestine and turnover occur by intervention of hepatic and pancreatic enzymes and bile produced by the gallbladder. This also means that intestinal and liver functions are connected with the good functioning of fatty acid transformation. alcohol is also a well-known inhibitor of desaturase activity, together with trans fatty acids coming from the dietary intake of junk foods. a good anamnesis questionnaire highlighting the health and dietary factors of the patient must alert health operators of possible enzymatic impairment affecting the resulting fatty acid balance.

The behavior of the desaturase enzyme is regiospecific because it operates at a precise position of the fatty acid chain for removing two hydrogen atoms. Therefore, the position of double bonds is



determined by the types of desataurase enzymes existing in the species. In humans there are three main desaturase isoforms: delta‐9, delta‐5, and delta‐6 desaturases, as will be shown in the biosynthesis of mono‐ and polyunsaturated fatty acids. all of them work for the elimination of two hydrogen atoms in the stereospe-cific cis fashion, so that the double bond has its unique geometrical configuration. This configuration is in the same direction as the two substituent chains on the double bond, relative to the plane of the double bond.

In other words, the geometry of all lipids present in mammals is cis, because it comes from the action of the desaturase enzyme. The other possibility would be the trans geometry with substituent chains on opposite sides. However, in mammals this is not realized by an enzyme. Trans lipids will be treated in Chapter 4. Here it is worth mentioning that only in bacteria can the conversion of the cis to the trans geometry occur by specific enzymatic activity (cis–trans isomerase), which is part of the bacterial defense response in an unfavorable environment (high temperature, presence of toxic chemicals or disinfectants). It is also worth underlining that this well‐known resistance mechanism has not been considered for antibiotic resistance and pharmacological applications.

What is the advantage of having trans double bonds in membrane fatty acids? The shift from cis to trans of unsaturated phospholipids changes the molecular shape of the fatty acid, so that the corresponding membrane becomes more compact and less perme-able. In this way bacteria can reduce their exposure to an unpleasant environment (such as antibacterial compounds) and sustain their life until the danger has not elapsed. The eukaryotic cell, or mam-malian, does not have this defense mechanism, and the cis geometry is the only isomeric form of the double bond. The reasons for this biological choice are multiple: among others, the cis geometry has been studied for its contribution to the membrane arrangement, so that the dimension of the cellular compartment becomes the largest possible [13, 14]. For prokaryotes, which have smaller dimensions than eukaryotes, the geometry can still alternate between cis and trans. On this basis the cis configuration becomes an element of enormous significance because it demarcates a line of evolution in biological systems toward more complexity, such as in eukaryotes. The preservation of the cis geometry becomes a key element in the maintenance of the code related to membrane lipids in humans.

POlyunsaTuraTed FaTTy aCIds 37

2.3 POlyunsaTuraTed FaTTy aCIds: THe essenTIalITy FOr HuMan Cells

unsaturated fatty acids that have more than one double bond are called polyunsaturated fatty acids (PuFas) and are divided into two families—omega‐6 and omega‐3—which will be discussed in Chapter 3. These components are also called essential fatty acids (eFas) because they come to humans only through nutrition. The omega‐6 family has as its precursor linoleic acid, and alpha‐linolenic acid is the precursor of the omega‐3 family. both can be biosynthetically obtained from oleic acid, which has a double bond in position 9; in fact, from the structure of oleic acid a second double bond in position 12 may be formed, to afford linoleic acid, and a third double bond in position 15 produces linolenic acid (Figure 2.8).

It can be deduced that the notation “omega” derives from the number of carbon atoms in the “tail” from the last double bond in the chain to the end of the molecule (C18 is the “omega” carbon atom). For linoleic acid there are six carbon atoms from position 12 of the last double bond toward the C18 carbon atom. For alpha‐linolenic acid the remain-ing carbon atoms are three. so the two families have the names of omega‐6 and omega‐3 (also denoted as n-6 and n-3) derived from their structural features (see the “In depth” section for further details of the structures).

The two desaturase enzymes, delta‐12 and delta‐15, may intervene on oleic acid in succession and operate the two steps of dehydrogena-tion; however, these two enzymes are present only in plant cells. These enzymes are not present in eukaryotic cells, and this makes the two fatty acids, linoleic and alpha‐linolenic acids, essential, which means that they have to be taken from sources that instead naturally produce them, which are plants. life evolution from plants is there-fore an absolute fact, since the eukaryotic cell could develop only according the presence of plant cells from where assume the PuFa lipids as essential elements.

Omega-9

n-9

Omega-6

n -6

(C18:1-∆9)Oleic acid

(C18:2-∆9,12)Linoleic acid

(18:3-∆9,12,15)Alpha-linolenic acid

∆12 desaturase ∆15 desaturase

Omega-3

n -3

Figure 2.8 Formation of linoleic acid (omega-6) and alpha-linolenic acid (omega-3) from oleic acid occurring in plants


CoNCePTS’ SuMMAry

S1 Beware of the Nutritional Label!

The saturated fatty acid called palmitic acid (16 : 0) is prepared by the enzymatic complex of Fas (fatty acid synthase) but can also be intro-duced through the diet. Indeed, in this respect it must be said that the contribution from food is often not carefully considered and palmitic acid can be hidden in any of the baked foods bought at the supermarket. labels on the packaging of prepared foods or baked goods can help when they clearly show the content of fatty acids. The food label has to show the source (plant or animal) of the fat. The unspecific defini-tion of “vegetable oil” cannot be accepted anymore in the ingredient labels and from 2016 it will be not possible due to the new european legislation. Palm oil is frequently used as plant oil, and the presence of saturated fatty acids, namely, palmitic acid, is high. Palm oil should not be confused with coconut oil, which contains a high percentage of saturated fatty acids, but they are mostly medium chain fatty acids (<12 carbon atoms) and they have a completely different metabolic value, which is not discussed here in detail. Consumers that buy foods with whole grain flours have to be aware of the vegetable or animal fats (lard, always rich in saturated fatty acids) that are usually present as ingredients. The recommendation for consumers is to pay attention to the labels, and choose carefully to avoid creating the problem of overcoming the normal intake of saturated fatty acids. recall that under certain conditions, such as being overweight or in case of neurological disorders and degenerative diseases, the proportion of saturated fatty acids from the diet must drastically decrease, better dis-appear, or be <10 g/day together with a strong presence of mono‐ and polyunsaturated fats. The low intakes of saturated fatty acids and the health claim about substituting them with unsaturated fatty acids are well‐known data recommended by all health organizations [31, 32].

S2 The optimal Values of Fatty Acids in Tissues

saturated fatty acids are naturally occurring elements forming cell membranes, so they cannot be considered as harmful. The danger of such fats is in their accumulation, because beyond a certain

POlyunsaTuraTed FaTTy aCIds 39

threshold, they strongly influence the properties of cell membranes or boost metabolic transformations. The membrane effect is easily explained by considering the linear structure of saturated fatty acids (see Figure 2.2); therefore a high concentration of these elements as phospholipid components consequently leads to an extremely packed organization of the membrane. This does not allow spacing of the phospholipid units to accommodate between them the entire range of protein structures, receptors, and trans-port systems, which make functional the membrane assembly. It is clear that the increase of the saturated components and the relative impact may vary from tissue to tissue. In Table 2.1 it can be seen that saturated fatty acids sFa may vary by a percentage of 27–48% from the adipose tissue to the retina [36]. The different tissue com-position can be maintained with an adequate balance between the diet and biosynthesis and membrane lipidomics takes into account the different distribution of fatty acid types in the various body compartments and their constituent cells.

From the data in Table 2.1 it is easy to understand that is not possible to confuse adipose tissue with another tissue, for example, the red blood cell (rbC), the retina, or the brain cells. It is also clear that the cells of each tissue tend to retain much of the characteristic composition of the membrane, but in reality this depends on the availability and balance of all lipid components, especially those coming necessarily from nutrition, such as essential

Table 2.1 Percentages of fatty acids and families present in various human tissues

Fatty acids adipose tissue (%rel)

rbC(%rel)

liver(%rel)

retina(%rel)

brain(%rel)

18:2, omega-6, lIn 10.5 9.3 17.5 1.4 0.620:4 , omega-6, aa 0.3 15.2 7.7 9.6 7.720:3, omega-6, dGla 0.2 1.5 1.6 nd 1.220:5, omega-3, ePa Traces 0.7 0.4 0.1 Traces22:6, omega-3, dHa 0.3 3.2 3.4 19.7 7.2sFa 27.2 43.1 42.0 48.2 45.9MuFa 59.7 23.0 23.8 14.2 29.7PuFa 13.1 33.3 32.0 37.2 23.4Omega‐3/omega‐6 0.17 0.21 0.17 1.32 0.46

adapted from ref. [36].


fatty acids (omega‐6 and omega‐3). Therefore, the final composi-tion of the cell membrane will reflect the terms of metabolism and diet followed by the individual. as can be seen from Table 2.1, the erythrocyte membrane contains all the classes of fatty acids and, given its distribution and transport in all districts of the body, can be used as a “global reporter” of the fatty acid content in tissues, including those not withdrawable in a living organism (brain tissue).

S3 Structural role of Fatty Acids

evaluating the role of fatty acids in cell membranes we can distinguish:

• The general structural aspect, which is the role of maintaining an appropriate structure of the double layer to obtain favor-able characteristics of fluidity and permeability. This also forms part of the concept of homeostasis, that is, adaptation and flexibility that characterize the cell membrane, which responds faster than any other compartment to the stress coming from environmental changes through the precise modulation of fatty acid components.

• The intrinsic structural aspect, which relates to the ability of the cell membrane to maintain its identity (characteristic of each tissue) and return to it as closely as possible during cell replication, providing for the formation of a tissue with the proper composition and then functioning in specific activities for which it is responsible. Obviously, if this is not realizable, due to lack of the correct amount of individual fatty acids, the tissue will form anyway, but present less effective function-ality, thus contributing to the creation of a state of basal cell impairment. The effect of the availability of fatty acids during tissue development along the life of the individual deserves great attention from the medical point of view as a preventive tool (see Chapter 8, section 8.2).


Essential Fatty Acids

As we have briefly discussed in Chapter 2, the classes of polyunsatu-rated fatty acids (PUFAs) present in the membrane of eukaryotic cells are of two types: omega‐6 and omega‐3. It is important to note that the precursors, linoleic acid and alpha‐linolenic acid, originating only from dietary supply, are called essential fatty acids (EFAs); however, the other components of the two families obtained from these precursors can also be named semi-essential fatty acids. To learn more about these molecules is a necessary step in order to understand their importance and role in human health. The tissue composition shown in Figure 2.5 with the presence of omega‐6 and omega‐3 must be carefully consid-ered for their immediate application to health. In fact, tissue impair-ment and consequent organ malfunction can derive from incorrect fatty acid composition, causing a domino effect on signaling and meta-bolic processes. As will be shown in Part 2, dermatology and omega‐6 intakes were connected since early times by the experimental observa-tion of the Burr’s family of scientists [37].

It is worth underlining that the problems of incorrect tissue composi-tion can include several aspects not only derived from the dietary intake of essential fatty acids. In fact, after ingestion, lipids must be absorbed by the intervention of enzymes (lipase) and detergents (bile). The condition of the gastrointestinal tract and the functioning of the gall-bladder and liver play important roles in consequent fatty acid absorption. These concepts will be seen at work in Part 2; however, readers must be aware about them before going through the details of EFA pathways.

3

42 EssEnTIAl FATTy ACIds

3.1 THE OMEGA‐6 And OMEGA‐3 FAMIlIEs: CAsCAdEs And REGUlATIOn

The omega‐6 and omega‐3 pathways depart from precursors that can be subsequently processed by desaturase and elongase enzymes within human cells. These pathways are called “cascades,” since the enzymatic transformations concerning linoleic acid and alpha‐linolenic acid occur one after another in sequence, which are depicted in Figures 3.1 and 3.2, as well as in parallel, as shown in Figure 3.3.

The elongase enzymes expressed by the genes of the ElOVl series (ElOVls 1–6) work to elongate PUFA chains from 18 up to 24 carbon atoms (very long chain fatty acids, VlCFA), and some features have been discussed in section 2.1.

desaturase enzymes form the cis double bonds and for the PUFA structures there are specific isoforms that work preferentially with the different substrates. As seen in section 2.2, delta‐9 desaturase works

Diet

Diet

H3C

H3C

H3C

H3C

C

O

SCoA

C

O

SCoA

C

O

SCoA

C

O

SCoA

12

12

14

14

6ω

11 8 5

11 8

9 6

9

Linoleoyl-CoA 18:2

Gamma-linolenoyl-CoA (GLA) 18:3

Arachidonoyl-CoA (20:4)

Dihomo-gamma-linolenoyl-CoA (DGLA) 20:3(eicosatrienoyl-CoA)

O2+NADH+H+

(2)H2O+NAD+

O2+NADH+H+

(2)H2O+NAD+

Malonyl-CoA

CoASH

∆6-desaturase

∆5-desaturase

Elongation in ER

Figure 3.1 The enzymatic pathways of the omega‐6 family evidencing the fatty acids that can also be provided by dietary intake

THE OMEGA‐6 And OMEGA‐3 FAMIlIEs 43

with the saturated fatty acid family. In fact, stearoyl‐CoA desaturase (sCd1 and sCd2 isoforms are the most diffuse in humans) works with palmitic acid to form palmitoleic acid, and with stearic acid to form oleic acid, with the double bond in the delta‐9 position. On the other hand, desaturase enzymes called fatty acid desaturase FAds1 and FAds2 work preferentially with PUFA precursors recruited from the diet, and are encoded by the corresponding genes, which are a hot topic in the study of correlation between genotypes and diseases, addressed by the so‐called genome‐wide association studies (GWAs) initiated in 2006 (see Part 2).

The FAds1 FAds2 gene cluster can vary in different individuals and there are genetic variants that are associated with fatty acid changes [38, 39]. some of these variants could therefore be connected to the development of diseases, such as atopic disease or cognitive impairment; however, more studies are needed to draw conclusions.

Diet 18:3

20:318:4

FADS2

FADS2

FADS1

FADS2

p-βOx

Elovl5

Elovl5

Elovl2 or Elovl5

Elovl2

20:4

20:5

22:5

24:5

24:6

22:6

Diet

Diet

Retro

-con

version

Figure 3.2 The enzymatic pathways of the omega‐3 family evidencing the fatty acids with dietary intakes and the genes involved in enzyme formation


looking at Figures 3.1, 3.2, and 3.3 a note of attention goes to the nomenclature of fatty acids, underlining the difference between alpha‐linolenic acid of the omega‐3 series (18 : 3 omega‐3) and gamma‐ linolenic acid of the omega‐6 series (18 : 3 omega‐6). In this case the nomenclature can cause confusion whereas in biology the structures of these fatty acids are crucial for two subsequent enzymatic steps to occur. It is noted that alpha‐linolenic acid (AlA; 9cis,12cis,15cis‐18 : 3) is the substrate of the enzyme delta‐6 desaturase, while gamma‐linolenic acid (GlA; 6cis,9cis,12cis‐18 : 3, which has the double bonds moved of two units) is the substrate of the enzyme elongase.

To understand fatty acids, we have to be able to recognize them, as do the enzymes!

1. The examination of each pathway follows:• The OMEGA‐6 pathway (Figure 3.1) is initially involved in the

formation of gamma‐linolenic acid (GlA) by the action of the enzyme delta‐6 desaturase on the precursor linoleic acid; this first step is very important because it gives rise to GlA with

Omega-6 pathway

Delta-6 desaturation(FADS2)



Delta-6desaturation(FADS2)

Delta-6desaturation(FADS2)

(Delta-4 desaturation,probably does not existin humans)

Strong inflammatoryeicosanoids:LTs group 4 andPGs group 2

Light inflammatoryeicosanoids:LTs group 5 andPGs group 3

ElongationElongation

Elongation

Elongation

Elongation

Beta-oxidation Beta-oxidation

18:2 n–6 18:3 n–3

18:4 n–3

20:4 n–3

20:5 n–3

22:5 n–3

22:6 n–3 24:6 n–3

24:5 n–3

18:3 n–6

20:3 n–6

20:2 n–6

22:4 n–6

22:5 n–624:5 n–6

24:4 n–6

20:4 n–6Arachidonic acid)

Omega-3 pathway

Figure 3.3 Evidence of the enzymatic competition between the members of omega‐6, and omega‐3 families noted also as n-6 and n-3. Reproduced with permission from Ref. [36]. © 2001, Elsevier


important functions, such as, for example, the functionality of the epidermis and hydration (see Part 2). The functioning of the enzyme delta‐6 desaturase can be influenced by many conditions as previously described (section 2b). For example, it is inhibited by the absence of important cofactors such as Fe, Zn, Mg, vita-mins B2, B3, and B6, and, as in the case of sCd enzymes, nAdH. Also, the intake of alcohol and saturated or trans fatty acids leads to inhibition of enzyme function. The desaturase enzyme is also inhibited by catecholamines and the presence of diabetes and hepatitis. A feedback inhibition of the function of the enzyme comes from high levels of carbohydrates, and then from insulin response. As we will see in Part 2, dermatology is the first area where the deficiency of this enzyme was highlighted.subsequent to the formation of GlA is the intervention of elon-gase that leads to the synthesis of dihomo gamma‐linolenic acid (dGlA; eicosatrienoic acid, omega‐6; 8cis,11cis,14cis‐20 : 3). This fatty acid plays a key role in the balance of the omega‐6 cas-cade, since it performs various control functions [40]: (i) gives rise to series 1 prostaglandins (PGE1 with anti‐inflammatory activity, TXA1 and PGF1) and series 3 leukotrienes (lTA3, lTC3, and lTd3); (ii) regulates the enzyme phospholipase A2 (PlA2), which is responsible for the release of fatty acids from cell mem-brane phospholipids; and (iii) is an essential element for the mat-uration of lymphocytes, and then for the functioning of the immune system. Finally, dGlA is the substrate for the enzyme delta‐5 desaturase, which creates a fourth double bond leading to the synthesis of arachidonic acid (5cis,8cis,11cis,14cis‐20 : 4). Arachidonic acid in its turn is the precursor of prostanoids of group 2 (PGd2, PGE2, PGF2, PGI2, and TXA2), leukotrienes, and lipoxins of series 4. some of these mediators have well‐known inflammatory activity, so that arachidonic acid and the omega‐6 pathway are defined inflammatory track tout court. In the “In depth” section there are further considerations on this subject. The enzyme delta‐5 desaturase is regulated by the presence of cofactors, vitamins, and proteins, as we have seen for the enzyme delta‐6 desaturase, being activated by the presence of insulin and cortisol.

• The OMEGA‐3 pathway (Figure 3.2) starts from alpha‐linolenic acid (AlA; 9cis,12cis,15cis‐18 : 3), which is transformed by the enzyme delta‐6 desaturase in the corresponding derivative with four double bonds, stearidonic acid (6cis,9cis,12cis,15cis‐18 : 4).


The cascade provides for other subsequent steps of elonga-tion and desaturation (delta‐5), leading to the synthesis of eicosapentaenoic acid (EPA; 5cis,8cis,11cis,14cis,17cis‐20 : 5), an important precursor of the series 3 prostanoids with known anti‐inflammatory activity (PGd3, PGE3, PGF3, PGI3, and TXA3) and series 5 leukotrienes (lTA5, lTB5, and lTC5). And again from EPA the succession of elongation, desaturation (delta‐6), and finally beta‐oxidation gives doco-sahexaenoic acid (dHA; 4cis, 7cis, 10cis, 13cis, 16cis, 19cis‐22 : 6) as the final product. several observations have been made on the omega‐3 pathways and they have shown that: (i) alpha‐linolenic acid is not adequately present in the Western diet and there are a few food sources; also, in tissues it is not present in a high percentage as a component of mem-brane phospholipids (<0.1–0.2%). It is estimated at a wide range of 0.2–21% in this conversion. The conversion of alpha‐linolenic acid into EPA takes place with decent efficiency (about 8%), also depending on the competition given by the omega‐6 fatty acids, discussed later, together with the availability of cofactors. On the other hand, the conversion of alpha‐linolenic acid to dHA in humans occurs in low percentages (about 0.1%) [41, 42]. The role of AlA as a precursor of the omega‐3 fatty acids EPA and dHA is cou-pled with its metabolic role of balancing the omega‐6 pathway. Therefore, since both cascades start from the delta‐6 desaturase activity, AlA is the only omega‐3 to compete with linoleic acid for this enzyme (see Figure 3.3). In section 3.2, the nutritional and metabolic contributions of these two PUFA families and their importance in nutraceutical supple-mentation will be considered in detail.

2. The role of the fatty acids EPA and dHA is not only to balance the inflammatory effects of arachidonic acid, but they play several other roles, producing substances with protective activity: after release from cell membranes, they are transformed by oxidative enzymatic pathways into a series of substances, called neuroprotectins (from dHA) and resolvins (from EPA), which provide specific protective activity at picomolar concentration in tissues [43–45].In Figure 3.4 an example of the biosynthesis of resolvins of the E series from EPA is shown. Remarkably, the first step of this biosyn-thesis is triggered by acetyl salicylic acid via acetylation of the COX2 enzyme. This is a very interesting mechanism by which acetyl sali-cylic acid has the property of participating in the resolution of


inflammatory processes with the formation of mediators, and not only by inhibiting the enzymatic oxidative pathways (e.g., COX).

3. dHA is the last product of the omega‐3 pathway, and is the fatty acid most hardly transferred to membrane phospholipids with very important functions for many tissues, from adipose to intestinal and nervous systems, constantly unveiled by new discov-eries. This fatty acid has been more recently taken into consideration for the vegetarian and vegan dietary status, since fish is its main source and is not consumed by these subjects [46]. All these biological activities depart from dHA detached from membrane Pl; therefore it is crucial to have dHA embedded in membranes. It is also the “most” polyunsaturated fatty acid containing six double bonds. This molecular feature makes it more sensitive to oxidative degradation that has to be taken into account in the strategy of dHA supplementation. dHA-containing foods must be included in everyone’s diet. In particular, dHA content is good in algae, and hence also in fish, this intake being particularly important for bal-anced diets [46]. Research on the role of dHA intake is addressed by lipidomics. When dietary regimes, such as the vegan diet, do not include fish consumption, a very high risk of being deficient in dHA can occur, unless the algal source of dHA is supplied.

COOH

COOHAspirin/COX2

RvE2

Eicosapentaenoic acid 18R-hydro(per)oxy-EPE

5S-Hydroperoxy,18R-hydroxy-EPE

5,6-Epoxy,18R-hydroxy-EPERvE1

Reduction

5-LOX

LTA4H

COOH

OH

OH

OH

OHHO

OCOOH

COOHCOOH

OOH

OH

OH

O(O)H)

Figure 3.4 Transformation of EPA (omega‐3) into the corresponding hydroperoxy derivatives by aspirin‐triggered COX2 activity in the biosynthesis of resolvins of the E series


3a In Depth: The Definition of Omega‐6 and Omega‐3

Why are the two PUFA families called omega‐6 and omega‐3? This nomenclature can be understood by considering the end of the chain of carbon atoms, that is, the side opposite to the position of the carboxyl group, namely, position 1. Counting from this end, in Greek called “omega,” the number of carbon atoms before encoun-tering the first double bond is checked. In the omega‐3 series the first double bond is three carbon atoms from the end, while in the omega‐6 series it is spaced by six carbon atoms from the end (see structures in Figures 3.1 and 3.2). Another structural difference between omega‐6 and omega‐3 families concerns the precursors: in the family of the omega‐6 the precursor linoleic acid (9cis,12cis‐18 : 2) has only one methylene group CH2 between the two double bonds. The family of omega‐3 alpha‐linolenic acid (9cis,12cis,15cis‐18 : 3) has two CH2 groups between the three double bonds. As we will see in section 4.3, these methylenic sites may react under conditions of oxidative stress and the different number of reactive sites influences the omega‐6 and omega‐3 fatty acids reactivity.

As already underlined and shown in Figures 3.1 and 3.2, the families of omega‐6 and omega‐3 fatty acids depart from precur-sors that must be taken from foods. Once these molecules are assumed in the diet, the body is able to further modify them by lengthening them (adding two carbon atoms by elongase enzymes), as well as creating a double bond (subtracting two hydrogen atoms by desaturase enzymes). The essentiality of these precursors has been explained in Chapter 3 and, on this basis, three other impor-tant points must be further underlined:

1. The content of omega‐6 and omega‐3 varies depending on the type of plants, so it is advisable to consider the composi-tion of foods in terms of quality of fats, and choose appropri-ately. The content of omega‐3 and omega‐6 in animals also derives from diet. Therefore, fish contain omega‐3 because their food is mainly algae! An important concern arose some years ago due to the different diet that is used for fish breeding in aquacultures. In fact, some food was prepared with corn oil and animal fats, which are not natural in fish diet. Analyses


and inspections are necessary so that fish farms ensure a natural diet, which is then reflected by the appropriate quality of the food.

2. The essentiality of omega‐6 and omega‐3 fatty acid precur-sors is not the only example in biochemistry and biology. It is well known also for other components such as vitamins and amino acids, which are called essential because the body cannot form them and also we cannot live without them. It is surprising that the consequences of the lack of omega‐6 and omega‐3 fatty acids are not always taken into consideration by medical doctors, when they make a clinical observation, as in the case of well‐known deficiency of vitamins and amino acids. As a matter of fact, the deficiency of essential fatty acids (EFA syndrome) is present in the medical literature; however, attention to EFA values is not diffuse in daily practice. yet, just a question about the dietary habits of the patient is enough to realize if EFA deficiency can be present, and, consequently symptoms must be considered in the light of a possible lack of lipid components.

3. For EFA, lipid analysis can indeed shed light on the individual situation. The scenario offered by lipidomics has also clarified that the status of omega‐3 and omega‐6 fatty acids alone cannot satisfactorily represent the whole lipid status. some indexes such as the omega‐3 cardiovascular risk index, calcu-lated by the sum of EPA and dHA percentages in erythrocyte membranes, can be connected with the incidence of diseases, such as heart pathologies and other tissue impairments [47–50]. The work done on the omega‐3 index in the last years is impressive, a significant amount of data giving support to the measurement of omega‐3 index in the red blood cell mem-brane as a useful screening tool for preventive medicine. An important feedback can be obtained when the omega‐3 index is found <4%, due to a safety threshold of these fatty acids in maintaining membrane and tissue functioning. The use of fatty acid biomarkers, such as the omega‐3 index targeted to eryth-rocyte membranes (not in plasma!), can be used also for its low cost, as a first informative view on the patient status. However, taking into account the relevant roles of saturated and monounsaturated fatty acids and the regulation from the


omega‐6 dGlA, the full profile of membrane erythrocyte and the follow‐up of the lipidomic pathways can offer the best molecular information for clinical observation directed toward a therapeutic goal.

3b The Polyunsaturated Fatty Acids in Cell Membrane Remodeling

Why does the content of omega‐6 and omega‐3 fatty acids affect the cellular (and tissue) response so much? Omega‐6 and omega‐3 fatty acids present in the membrane are precursors of other molecules, called prostanoids, which act as “mediators” and “signals” for the cell. In Figure 3.5 a brief summary of the fate of four important PUFA components is provided. It is clear that the omega‐6 pathway produces prostaglandins that may have inflammatory action (e.g., starting from arachidonic acid, the series E2 prostaglandins) or anti‐inflammatory action (e.g., from dihomo gamma‐linolenic acid, the series E1 prostaglandins), whereas eicosapentaenoic acid pro-duces series 3 prostaglandins and series 5 leukotrienes with anti‐inflammatory activity.

We will not discuss in detail these mediators, which have been the subject of intense research, and reviews are available [51, 52]. Here we will describe the role of cell membrane remodeling, which can start signaling cascades by releasing active fatty acids. This is why the membrane is considered as a crucial site for the events that intervene in cell fate. How does cell membrane remodeling work? The scheme shown in Figure 3.6 describes the principal steps of this process called land’s cycle [53].

In brief, the enzyme phospholipase (in particular phospholipase PlA2, and we will not mention other phospholipases) is able to work on membrane phospholipids, detaching a fatty acid unit from the phospholipid molecule (Figure 3.7).

Generally, the position sn‐2 of the phospholipid is involved with the PlA2 and this is the position frequently occupied by unsatu-rated fatty acids. This means that the PlA2 activity on membranes can liberate omega‐6 or omega‐3, thus providing the precursors of eicosanoid compounds in the cells. The probability of fatty acid liberation, which results in the production of eicosanoids, increases

Vegetables, corn, seeds, seed oils

Flaxseeds, chia, hemp seeds/oils, walnuts

DESATURASEIncrease: sugar (insulin)

Reduce: alcohol,trans fats, omega-3

Marine algae

Red meat, egg yolk

Fish

PLA2

PGD2, PGE2, PGD2PGI2, TXA2, LTA4, LTB4,LTC4, LTD4, LTE4

Neuroprostaneresolvins

PLA2PGD3, PGE3, PGF3aPGI3, TXA3, LTA5, LTB5,LTC5, LTD5, LTE5

PLA2

OMEGA-6 PATHWAY

18:2 n-6

18:2 n-6

20:3 n-6

20:4 n-6(Arachidonic acid)

18:4 n-3

20:4 n-3

20:5 n-3

22:5 n-3

20:6 n-3

Delta6-Desaturation(FADS2)

Delta5-Desaturation(FADS1)

Elongation

18:3 n-3

OMEGA-3 PATHWAY

Figure 3.5 some of the mediators produced omega‐6 and omega‐3 fatty acids and the main food sources where these EFA can be found


according to the lipid composition of membranes, which is different for each individual. It is clear that membrane phospholipids rich in omega‐6 fatty acids, such as arachidonic acid, will consequently liberate more of this fatty acid in the cytoplasm, for example, precursor for the transformation to series 2 prostaglandins. On the

Fatty acid poolEicosanoids

Fatty acids

Lysophospholipids

Acyl-CoA

O

O

PAF,LPA, endocannabinoids

Phospholipase A2

PX

PX

O

O

O

HO

O

Phospholipids

Lands’ cycleLysophospholipidacyltransferases

Figure 3.6 The cell membrane remodeling (land’s cycle) starting from the phospholipase A2 activation and the intervention of the fatty acid pool. Reproduced with permission from Ref. [53]. © 2010, Wolters Kluwer Health

Phospholipase A2

Figure 3.7 detachment of a fatty acid tail by the action of phospholipase A2 to obtain a lysophospholipid


other hand, the “liberation” of a fatty acid molecule by the enzyme activity produces a lysophospholipid molecule that cannot stay in the membrane, due to its hydrophilicity, and moves to the cyto-plasm, being converted to another series of compounds, for example, platelet‐activating factor (PAF), endocannabinoids, lysophospha-tidic acid, and others. It is worth mentioning that the activation of phospholipase enzyme can be stimulated by different conditions, such as the accumulation of intracellular calcium ions for enabling phospholipase A2 activity.

The turnover of the detached phospholipid is coupled with the insertion of another phospholipid in the membrane. land’s cycle uses the molecules of acyl‐coenzyme A as a building block for the Pl synthesis via lysophospholipid acyl transferase and lysophos-pholipid coupling (Figure 3.6). In this replacement, the fatty acid moiety of the acyl‐coA comes from the fatty acid pool, which varies depending on biosynthetic availability and dietary habits of the individuals. As a consequence, the quality of the fatty acids involved in the cycle is not the same for everybody; instead, it is a personal-ized condition. The land’s cycle describes the overall phenomenon of membrane lipid remodeling, which is triggered by chemical and physical stimuli from the cell exterior. It is worth recalling that the fatty acid pool plays a fundamental role in the outcome of a stim-ulus, and that essential fatty acids (omega‐6 and omega‐3) must be provided by the diet in mammals. Indeed, the availability of fatty acids regulates the types of responses from the cells. The inflammatory response is a defense mechanism, and this stimulus must liberate an adequate amount of fatty acids, such as arachi-donic acid, for the response to be effective. However, if there is an excess of arachidonic acid in the fatty acid pool, the response to an inflammatory stimulus can induce a vicious cycle, with excess liberation of this fatty acid, whose concentration and transforma-tion to inflammatory mediators can be out of control, especially when the restoration period should start after the acute phase. This can trigger inflammation as a chronic status, due to the loss of balance, as will be considered in Part 2.

The molecular check‐up at the level of fatty acids incorporated in membrane phospholipids is important for their regulation by nutra-based strategy, and this aspect should always be consid-ered when inflammation is diagnosed.


The land’s cycle shown in Figure 3.6 and its role in membranes suggests that: (i) free fatty acids (FFA) cannot be present in large quantities, being liberated at nanomolar concentrations during stimulus; therefore FFA levels can inform on an obsessive occur-rence of remodeling, and (ii) the conversion of fatty acids into acyl‐coenzyme A to be incorporated into phospholipids (acyl‐CoA synthase) must be finely regulated, in parallel with phospholipase activity. This is particularly important in the case of arachidonic acid that, after its liberation in the cytoplasm, is partitioned between the formation of inflammatory mediators (PG, lT, TX) and, alternatively, the corresponding acyl‐CoA derivative (Figure 3.8).

As will also be seen hereinafter, the enzyme phospholipase A2 (PlA2) is involved in the replacement of membrane phospholipids, when the structure of the fatty acid has undergone a modification or an oxidation reaction, and thus cannot maintain its functional role any longer. Working for detachment from the phospholipid, phos-pholipase allows the damaged tail to be removed and therefore is an important enzyme for surveillance of bilayer integrity. In this view, the role of oxidative damage can be considered as a useful way to “reshape” the membrane composition, provided that the loss of functional fatty acids by oxidative and free radical pathways is not

O

OH

8 5

11 14

AA-C(O)SCoA Eicosanoid synthesis

Fatty acyl-coenzyme A PG, LT, TX

Arachidonic acid

Figure 3.8 Fate of arachidonic acid after detachment from membrane phos-pholipids by PlA2 partitioned between acyl‐CoA synthesis and eicosanoid synthesis


so massive to affect cell activities. This subject is especially important in preventive medicine, in order to use the molecular check‐up of the membrane fatty acids as a premonitory signal for health and nutri-tional interventions, and will be treated in Chapters 4 and 5.

3c In Depth: How do you Define an Inflammatory Pathway?

The omega‐6 pathway has the “bad” reputation of inflammatory activity, because it gives rise to arachidonic acid, which is then converted into prostaglandins and other inflammatory mediators (Figures 3.3 and 3.5). It is time to reflect more on the roles of omega‐6 to discover other aspects, showing that omega‐6 fatty acids have both inflammatory and anti‐inflammatory activities. In fact, in the omega‐6 pathway there is an active control and adjustment between these two activities where the fatty acid dGlA (20 : 3, omega‐6, eicosatrienoic

GLA

DGLA

PGE1

EP2/EP4 receptor

G protein

Adenylate cyclase

ATP

cAMP

PKA

Controlproliferation

Macrophage

Smooth muscle cells

Figure 3.9 The role of stimulation of macrophage proliferation by dGlA. Reproduced with permission from Ref. [54]. © 1998 Fan, Journal of nutrition


3.2 THE BAlAnCE BETWEEn OMEGA‐6 And OMEGA‐3 PATHWAys: nUTRITIOnAl And METABOlIC COnsIdERATIOns

At this point it should be clear that each subject has a personalized mem-brane composition that derives from the combination of a myriad of factors. Placing simple questions to the patient related to food intake and lifestyle, the specialist/doctor may form an idea of the contribution of the environment to cellular functions. The main food sources of fatty acids in the Western diet are known and Figure 3.10 gives a brief assay of foods containing different types of fatty acids.

detailed food compositions can be found in the food database provided by several institutions and research organizations [55]. It is worth noting that, depending on the origin and season of the food, the nutritional contents can vary.

Questions on dietary habits and lifestyle cannot be used for a precise strategy. In order to have a clear picture of the patient molecular status, the evaluation of fatty acid pathways is necessary. In particular information on the balance among saturated, monounsaturated, omega‐6 and omega‐3 intakes and enzymatic activities, and the effect of competition among these families, must be acquired. Examining Figure 2.2 it is evident that both EFA pathways use the same sequence of three enzymes: delta‐6 desaturase, elongase, and delta‐5 desaturase. Both omega‐6 and omega‐3 cascades start from delta‐6 desaturase, which converts linoleic acid for the omega‐6 track and alpha‐linolenic

acid) plays an important role. This fatty acid is the primary control element, by giving a feedback to the activity of cyclic AMP produc-tion and protein kinase A (PKA) activity, which solicits several path-ways. In fact, the dGlA transformation to PGE1 leads to the binding to G protein coupled surface binding receptors (EP family) and activation of the messenger cyclic AMP, as shown in Figure 3.9 [54]. This message can be driven by macrophages and can induce cell response such as proliferation. It is worth noting that the activity of PGE1 compounds can be up to 20‐fold higher than PGE2 series toward specific cellular functions. Therefore, a small transformation of dGlA into its metabolites can correspond to an efficient control of the activity of arachidonic acid mediators.

THE BAlAnCE BETWEEn OMEGA‐6 And OMEGA‐3 PATHWAys 57

acid for the omega‐3 track. These two substrates compete with each other for the same enzyme, which will be taken up by the most prevalent between the two compounds. The omega‐6‐rich diets efficiently help in the formation of arachidonic acid, reducing the possibility of omega‐3 competition, thus increasing inflammatory predisposition. The increase of the omega‐6/omega‐3 ratio is popularly known as “silent inflamma-tion,” which is a diet‐based condition. The contrast to this increase must be done through a careful choice of nutrients, to keep the membrane balance in an interval ratio omega‐6/omega‐3 up to 5 : 1, as will be further specified. The large number of nutritional supplements based on omega‐3, which do not replace a varied diet, have a pretty huge concentration of these fatty acids, and make market claims of reducing inflammation. This topic will be addressed in Part 2, giving also criteria for recognizing a good formulation and its wise utilization depending on the observed deficit. It is worth noting that imbalance can be created also as a result of supplementation, when it is not adequate to the subject’s needs. In recent years we have witnessed the growing interest of the public for dietary supplements based on fatty acids, such as omega‐3 from fish oils. supplements have become a best seller also based on the tam‐tam news on the various beneficial properties, from weight loss to cancer preven-tion, and the “do it yourself” is very diffuse. These are natural products so the general thinking is that they may not have contraindications. This is partially true and depends in principle on two factors: the real need for such a supplementation and the dosage of the supplementation. As explained before, the dietary intake can cover personal needs; however, if the intake is not sufficient or absent, supplementation

OIL 16:0 18:0 18:1 18:2 (ω6) 18:3 (ω3)

Olive 12 2 72 11 1

Palm 42 4 43 8 0

Sunflower 6 6 33 53 0

Butter 28 (+ other SFA) 16 26 1 2

OMEGA-3 rich oils ALA EPA DHA

Flaxseed 53 0 0

Linseed 56 0 0

Menhaden 1 11 11

Salmon 1 9 12

Figure 3.10 Examples of fatty acids contained in foods


can be recommended. As far as the dosage is concerned, it is worth recalling that fatty acids are hydrophobic molecules, which means that their excretion is slow. If polyunsaturated compounds are in excess and accumulate in lipid deposits and assembly, such as, for example, lipo-proteins, it must be also taken into account that they are chemically reactive to oxidation and free radicals. Therefore, such accumulation increases the tendency to oxidative transformations and can create a vicious circle with the formation of toxic metabolites (e.g., aldehydes, which are hepato‐ and neurotoxic). The decision to take supplements, especially when containing polyunsaturated fatty acids, has to be done on the basis of serious considerations of health, metabolic, and dietary status. such a decision is better taken by health professionals, using appropriate doses and quality of fatty acids. The main criterion is the adequacy based on individual need. The second criterion is the balance, so any supplementation should only be done after studying the status of the saturated, unsaturated, omega‐6 and omega‐3 pathways.

The dosage of supplementation of fatty acids must be calculated so as not to exceed the levels required. The self‐prescription of “harmless” capsules of omega‐3 can create the risk of unnatural conditions, with high dosages, then causing toxicity. There are other factors that must be taken into account in nutraceutical supplementation, but this will be discussed in Part 2.

The balance among the fatty acid families has been considered in nutritional studies. The examination of the Western diet food composi-tion evidenced that the intake of omega‐6 is much higher than that of omega‐3, reaching the omega‐6/omega‐3 ratio of 15–16 : 1 [56]. Investigations of the dietary patterns and health status of countries, taking into account also historical context, evidenced that a net difference can be drawn in the omega‐6/omega‐3 ratio as shown in Table 3.1 [57].

Table 3.1 Omega‐6/omega‐3 ratios in different population and historical context

Population Omega‐6/omega‐3

Paleolithic 0.79Greece prior to 1960 1.00–2.00Current Japan 4.00Current India, rural 5–6.1Current United Kingdom and northern Europe 15.00Current United states 16.74Current India, urban 38–50

Reproduced from the website reported in Ref. [57]

THE BAlAnCE BETWEEn OMEGA‐6 And OMEGA‐3 PATHWAys 59

The European Food safety Agency (EFsA) in 2010 gave an opinion on the daily Reference Values (dRV) of fatty acids [58]. The opinion says that total fats can have the lowest intake at 20%E (energy) and the highest intake at 35%E, with other specific requirements in infant and child ages. The quality of fatty acids was also evaluated giving the following directions:

• saturated and trans fatty acids must be consumed as little as possible• no dRV for cis monounsaturated fatty acids• no dRV for omega‐6/omega‐3 ratio and linoleic acid (omega‐6)• 5%E as adequate intake (AI) of alpha‐linolenic acid (omega‐3)• AI for EPA of 250 mg• AI for dHA in infants and children of 100 mg• AI for dHA in adults of about 100–200 mg

The omega‐6/omega‐3 ratio has profound biological consequences when considered in cell membranes, since it determines a strong contribu-tion to structural and functional properties as described earlier. Therefore, since this ratio has changed dramatically going from the agricultural to the industrial period (see Table 3.1), health professionals cannot disregard the idea that health consequences are connected to the quality of fatty acids. Foods shown in Figure 3.10 should be taken into consideration for inquiry into dietary habits. Even with a poor food questionnaire, restricted to EFA‐containing foods, health professionals can have an idea of their patients and individuate a possible imbalance condition. For example, alpha‐linolenic acid can be found satisfactorily in only a few sources (such as flax seeds and its oil) and is generally not part of the diet, as well as dHA is mainly found in fish and marine algae, food that is often refused because of taste‐related problems. Moreover, as previously explained, the efficiency of metabolic conversion can vary individually, so that based on the intake of precursors such as alpha‐linolenic acid and EPA, the resulting levels of dHA in humans can also vary.

Obviously, the evaluation of the diet does not exactly describe the metabolic transformations correlated to fatty acids, especially related to membrane formation and remodeling, which can be obtained only by fatty acid analysis. The correlations of fatty acids with health conditions will be explained in Part 2, showing that low levels of essential fatty acid intake are always connected with disease conditions, and can be manifest in newborn babies, as well as at various ages.

The importance of fatty acid analysis in order to assess precisely the individual situation is evident, determining the amount and the quality of the fatty acids present in the various compartments or tissues.


The analysis can be performed on plasma or blood samples: when the fatty acid analysis is used for lipidomics, the fatty acid values are used not only as nutritional information of the subject, but also to trace a lipidomic profile of the metabolic status, which is also related to the health condition. At this point the choice of the biological compartment to measure fatty acid levels is very crucial. As previously explained, cell membranes allows the fatty acids to be examined as structural and functional elements with very precise meaning and biological effects, and this is particularly true for polyunsaturated fatty acids, due to the previously detailed roles of omega‐6 and omega‐3 fatty acids in cell metabolism. In particular, red blood cell membranes are an important site for estimating the arachidonic acid values because they are the main storage for this fatty acid, being released by phospholipase A2, as previ-ously explained. In general, for their fatty acid composition, erythrocyte membranes express the efficiency of the metabolism in the distribution of fatty acids and can provide a good indication of what occurs in tissues much less easier to reach than blood [59]. later, we will enter into the details of erythrocyte fatty acid composition and how this cell can be used as a successful health biomarker.

3.3 FOOd And MEMBRAnEs: A VIRTUOUs CyClE

In this chapter dedicated to PUFAs it is very important to stress the concept of the fatty acid pool for tissue metabolism. Recalling the tissue compositions shown in Table 2.1, it is easy to understand that each tissue needs the appropriate fatty acid types in order to work properly. From the explanations given so far it is also clear that every subject builds up his/her own lipid pool, according to what is the nutritional choices and how his/her metabolism works. The study of the membrane fatty acid asset is an important diagnostic tool for evaluating many of these factors, and lipid research gave birth in the late nineties to the discipline of lipidomics, where a multidisciplinary dynamic and functional approach is proposed. In Part 2 the lipidomic approach and its relationship with health conditions and nutritional/nutraceutical strategies will be delineated. Indeed, the interchange of lipid molecules coming from either metabolism or diet, which occurs as natural processes to gain cellular homeostasis, indicates that lipid therapy can represent an important tool to be fully applied in preven-tion and disease treatment. It is now necessary that lipidomics enter the medical practice.

FOOd And MEMBRAnEs: A VIRTUOUs CyClE 61

For each subject, carefully assessing his/her food choices and lifestyle, the health professional can have a first indication whether the fatty acid pool contains all the necessary fatty acid elements. In Part 2 the food questionnaire will be the first step in the lipidomic approach, in order to gather essential information for deciding the second step, which is the personalized lipidomic profiling assessed by the examination of membrane fatty acids from the red blood cells.

The examination of the fatty acid asset can give other important information: (i) since dietary components, such as microelements and cofactors, are needed for lipid enzymatic pathways, some of the unbal-ances evidenced in the biosynthetic pathways of fatty acids can indicate the need of biotin, nAdH, vitamins, metals (Zn and Mg), which help enzyme functioning. On the other hand, the use of alcohol or trans fatty acids can be deleterious to enzyme functioning, especially desaturase enzymes. Therefore, in the investigation of possible causes of wrong lipid assets, these parameters have to be considered. (ii) The functioning of important organs that correlate directly with lipid absorption (gastroin-testinal tract, gallbladder) or lipid metabolization (liver and pancreas); the role of pancreatic lipase enzymes in the hydrolysis of triglycerides has already been mentioned and this is an important step for the fatty acid to enter in the circulation. Moreover, the role of bile, the gallbladder secretion, in the moment that fat emulsification is needed during digestion, can influence absorption and bioavailability. It is quite common that patients have gallbladder removal. This aspect cannot be disregarded in the set‐up of lipid therapy.

As already pointed out, it is highly advisable to choose carefully the type and dosage of fatty acids, which enter in cell compartments and play a plethora of biological activities, in order not to create undesired unbalance or effects after the supplementation. Here, we can underline that there is a striking difference between nutrition and supplementa-tion: in fact, in the case of PUFAs in foods, the presence of proteins, vitamins, and other molecules can exert a protective effect for the PUFA molecules, saving them from oxidative and free radical conditions. Instead, when PUFA natural sources such as fish oils are used, protection of the supplementation during biodistribution can occur with specific components added in the formulation. Only one type of vitamin (vitamin E) that can be found naturally in the oils is not an efficient protection; instead, an antioxidant mix is required. In the next chapter fatty acid reactivity will be discussed, showing several mechanisms of protec-tion given by biomolecules that can enhance PUFA resistance and bioavailability.


Why can the virtuous cycle between food and cell membranes work for improving health? looking at the correlation between nutrition and health, we touch a subject that was intuitively caught since early times. The Hippocrates’ quote “let food be thy medicine and medicine be thy food” makes part of the history of medicine, and we can say that this paradigm is ideal for fatty acids!

In fact, it is well known that cell turnover occurs at every moment in the life of a biological organism, which means the renewal of structural or functional elements of the cell, and consequently of the correspondent tissue and organ. Accompanying the replication of genetic material, membranes are absolutely needed for cells, and in fact in the telophase of the mitotic phase new phospholipids have to be synthesized for gen-erating daughter cells (Figure 3.11).

Only in 2014 did research report some information on this delicate phase, discovering that lipidome changes accompany cell division, which are not limited to phosphatidylinositol 4,5‐biphosphate or 3,4,5‐triphosphate (PIP2 and PIP3, respectively) as signaling molecules for polymerization and membrane trafficking. Indeed, the lipids of the s phase are very different from those of the cytokinesis phase (see Figure 3.11) where the two dividing cells are almost separate from each other. Eleven species of lipids were found to be different by at least four-fold in cultured cells [60].

Future research will provide more details on these important processes. Intuitively, it can be figured out that various factors have to be satisfied during the recruitment of phospholipids, such as the maintenance of the membrane protein functionality (e.g., channels, receptors) to be orga-nized within the lipid assembly, and the homeostasis, with maintenance of osmotic, pH, electric potential, and other parameters, able to follow variations in environmental conditions. Here, a role is also played by cholesterol as an important balancing factor. To give an idea of the number of cells that replicate during our life, some of the lifetimes of cells are shown in Table 3.2. This is an important piece of information for understanding how lipid therapy can influence cell turnover. Blood, intestinal, skin, and liver cells have short‐medium lifespans, meaning that changes after diet or supplementation can be followed up in a reasonable time range. Instead, for adipose tissues the length of the turnover suggests longer observation periods (1 year).

It is also important to take into account that all tissues, including the perennial tissues (muscle and nervous system), have a certain period of growth that goes from pregnancy to birth until adolescent age at least. Therefore, the constant availability of fatty acids for tissue formation is

INTERPHASE

Cytoplasm divides

© Clinical tools, Inc.

Cellular contents,excluding thechromosomes,are duplicated.

Cell cycle arrest

G0

G1

M

G2

SEach of the 46

chromosomes isduplicated by

the cell.

parent cell becomestwo daughter cells withidentical genetic information

Breakdown of nuclearmembrane

Spindle fibers appear

Chromosomes condense

Spindle fibers attachto chromosomes

Chromosomes condense

Chromosomes align

Centromeres divide

Cholesterol

Transmembraneproteins

Sister chromatids

Centromere

Loosely coiledreplicated chromosomes

Sister chromatids move toopposite poles

Spindle fibers disappear

Nuclear membrane reforms

Chromosomes decondese

Telophase

Cytokinesis

AnaphaseMetaphasePro

metaphas

e

Prop

hase

G2

The cell double checks˝ theduplicated chromosomes for

error, making any neededrepairs.

Figure 3.11 Cell replication phases and membrane formation


an important condition to avoid defiance and lipid therapy becomes crucial in such moments of life. Also, the organelles in the cells, such as mitochondria (key intracellular organelles for energy metabolism and aerobic respiration) are renewed, and have an average life span of a month. This happens in perennial tissues as well as in “labile” tissues.

To form the membranes, a flow of lipids from the endoplasmic retic-ulum within the cytoplasm, where the phospholipids are formed, moves to be part of the double layers as well as of other organelles. Phospholipids are provided during mitosis, as previously noted (Figure 3.11), and it is an ideal condition that at the time of mitosis the person can have all the types and amounts of fatty acids that must form the cells of that tissue, without restriction. What happens if the ideal condition is not met? The new cell will be born, although the fatty acids are not exactly the ones present in the original “lipid code” of that tissue, and at the same time this error of the code is incorporated, which means that it cannot operate correctly. However, since the cellular metabolism and the continuous formation of membranes accompany tissue renewal, in the turnover resides the opportunity to rebalance and settle again the ideal homeo-static conditions, even if they had been lost in a previous turnover.

Cell membranes, as a reliable reporter of information from internal and external factors, become an important basis for the innovative approach of molecular medicine, which has the purpose of connecting the molecular state with the metabolic state of the subject. Part 2 describes how lipidomics of the cell membrane can become an instru-ment not only for molecular diagnostics, extensively used as perfect complement to clinical observation, but also for the operative phase of decision making of an intervention to restore the balance of fatty acid pool and, consequently, influence positively the next cell generation.

Table 3.2 Mean life span of human cells in the body

White blood cell some hours-2 daysPlatelet 8–10 daysIntestinal cell 7 daysEpithelial cell 18–20 daysRed blood cell 120 daysliver cell 150 daysAdipose cell 6–15 months


Free Radicals and Lipids: Trans and Oxidized Fatty Acids

The last chapter of this part will be dedicated to more details on unsaturated fatty acid reactivity, mentioned in Chapter 2, attributable also to free radical stress conditions produced in living organisms. The most known reaction involving polyunsaturated fatty acids is the peroxidation process, which is certainly relevant in the life of aerobic organisms like humans. On the other hand, double bond reactivity was investigated almost a decade ago, evidencing that mono‐ and polyun-saturated fatty acid can be involved in the so‐called isomerization pro-cess catalyzed by free radicals. It is worth mentioning that both peroxidation and isomerization processes occurring in natural lipid structures cannot be considered only from a deleterious point of view, since they do not directly correspond to “biological damages.” Indeed, life is based on the reactivity of all molecules in the biological environ-ment, and the molecular modification of lipids by oxidative and free radical processes can have two different meanings: as “signal” or as “damage.” Often, the two roles are different only from a quantitative point of view, with a threshold that the transformations have to overcome for becoming harmful.

4

66 Free radICals and lIpIds: Trans and OxIdIzed FaTTy aCIds

4.1 Trans FaTTy aCIds FOr HUMans: THe nUTrITIOnal InTaKe

nutrition and medical research started to focus on fatty acid double bond structure–activity relationship only in the early nineties. Mono‐ and polyunsaturated fatty acids in eukaryotic cells always present the double bond (unsaturation) in the cis configuration (see Chapters 1 and 2); however, for many years the proper significance of this structural fea-ture was not considered. serendipitously, it was observed that the other configuration of fatty acids, which is called trans, is completely excluded for biological lipids of eukaryotes. Therefore, cis double bond was more appreciated for imparting precise biological properties (see Chapter 2 on membrane properties); consequently, the need for saving this structural feature and having good intakes of cis fatty acids in the diet was under-lined for its crucial importance. The discovery of how trans fatty acids (TFa) can enter the human body was first made by examining the com-position of oils manipulated by the food industry. In the fifties, the manipulation to render more solid the naturally oily fats was introduced with the process of partial hydrogenation. Then it was diffused in indus-trialized countries, since natural oils were not only more solid and spreadable (introducing “margarines” as vegetable semisolid fats in the daily dietary habits of most families, nOT naTUral!!), but also more stable for elongation of shelf lives of foods. partial hydrogenation is a chemical process, which adds hydrogen gas to the double bonds in the presence of a metal catalyst, and transforms the double into saturated bonds. However, in this reaction it can happen that the double bonds present in the lipid molecules are not all hydrogenated, but some of them are transformed into trans isomers. another process employed by the food industry involves temperature treatment of edible oils, carried out for the deodorization process (to eliminate unpleasant odors such as in fish oils) as well as the frying of vegetable oils containing double bonds, again at high temperature. In both cases the formation of trans fatty acids can occur.

The trans unsaturations after this processes can involve the same or the adjacent positions of the natural cis double bonds. In fact, geomet-rical and positional trans isomers can be formed, whose analytical dis-tinction becomes an urgent matter in food safety and toxicology. In Figure 4.1 some examples of positional and geometrical cis and trans fatty acid isomers are given.

Geometrical trans fatty acid isomers are the trans isomers maintain-ing the same position as the cis double bond, but with the opposite

Trans FaTTy aCIds FOr HUMans 67

disposition of the two chains with respect to the plane, whereas the positional trans isomers have changed position and geometry with respect to the original cis isomer. For pUFas, the number of geometrical isomers is 2n, where n is the number of the double bonds in the lipid molecule. For pUFas with two double bonds, such as linoleic acid (omega‐6), four geometrical isomers are possible. For arachidonic acid, epa and dHa with 4, 5, and 6 double bonds, the number of geomet-rical isomers obviously is high (16, 32, and 64 isomers, respectively).

as previously said, during industrial and heating processes of natural oils, transformations occur and destroy the natural cis fatty acid struc-ture, which is converted to trans fatty acids. due to the increasing use of these processes in the food industry, the trans lipid intake increased in industrialized countries and was found to be correlated with the curves of coronary heart disease mortality tracked over time. In the early 1900s, angina and myocardial infarction were unusual clinical events, but over the century they rapidly increased, becoming the major cause of death by midcentury [61]. among other factors, TFa consumption was discov-ered to play a crucial role in this increase, raising serious concerns mainly in north america and northern europe, where there is an estimated intake of 2–5 g/day. On the other hand, Mediterranean countries (Italy, portugal, Greece, and spain), which mainly use olive oil, reported lower TFa intake (1.4–2.1 g/day) and lower cardiovascular risk [62, 63]. as the result of a long debate, in 2003 the Food and drug administration

9 10

HO

O

9 10O

HO

7

8

O

HO

1110

O

HO

Figure 4.1 examples of positional and geometrical isomers of monounsaturated fatty acids


(Fda) in United states issued a regulation requiring manufacturers to list trans fats on the nutrition Facts of foods and some dietary supple-ments [64]. In europe, the label information of food products has been revised (eU regulation 1169/2011) active from december 2014 on; however, the trans content of foods has not been included, raising a debate over consumers’ safety [65].

It is worth noting that deep frying and wok cooking can produce geometric isomerization of the polyunsaturated fatty acids contained in the oils (linoleic and alpha‐linolenic acids) at temperatures above 200°C. The stability of the oil to oxidation and isomerization processes is also due to the content of different antioxidants that can delay the alteration. It is highly advisable to lower the frying temperatures and waste oils after the first uses.

research in this field is addressed to new processes and separation procedures to reduce the final trans content of the industrial chain, and to high‐performance analytical procedures in order to detect and characterize trans fatty acid isomers in the final products.

an important problem was highlighted in recent reports on the presence of trans omega‐3 fatty acids in oils after deodorization processes [11, 16]. In fact, the high temperatures used for distillation and elimination of fishy odor in the natural sources rich in omega‐3 (fish oils) were found to afford a certain percentage of epa and dHa isomers. These materials are used for nutraceuticals and functional foods claiming health benefits by supplementation [66]. particular attention has to be given to the quality of omega‐3‐containing oils, as the refining processes that involve thermal treatment can affect the geometrical integrity of these fatty acids. Indeed, commercially available deodorized fish oils have been found to contain trans epa isomers, and the follow‐up of supplementation in rats, fed a low (5%) and high (30%) fat content diet including fish oil, showed that trans epa isomers, prevalently the 17trans isomer, can be incorporated at the level of liver mitochondria [16, 67]. since omega‐3 pUFas are well known for their beneficial health effects, such as protection against car-diovascular diseases [68], anti‐inflammatory properties [69], ameliora-tion of physiological and cognitive functions [70], crucial role for brain and neurodevelopment [71], the interference of trans omega‐3 strongly affects the expected health benefits. Moreover, it is of great importance to establish the connection between the presence of TFas and pUFas; in fact, TFas can reduce the cis pUFa availability in human metabolism by direct inhibition of desaturase (Δ5 and Δ6) and elongase enzymes [72].

It is not within the scope of this book to focus more on the incorpora-tion and effects of trans fatty acids from the diet, which have been

described in several reviews and books [11, 63, 73]. However, it is worth underlining that two interesting points emerge from this research: (i) trans isomers can interact with most of the enzymes of the cis lipids, sometimes at the same rate; and (ii) the effects of the trans geometry cannot be generalized, but each isomer can have a specific activity that must be appropriately studied.

another important detail is that a few trans isomers can be considered “natural” and have gained importance for their biological effects. It is the case of conjugated linoleic acid isomers (Cla isomers), which are shown in Figure 4.2.

The term Cla (conjugated linoleic acid) can refer to the four isomers of linoleic acid with conjugated double bonds so, compared to linoleic acid, there is no methylene group to separate the double bonds. The major natural isomer (9cis,11trans‐18 : 2) was identified in 1977 and further named “rumenic acid,” whereas in minor amounts the 10trans,12cis‐isomer is present in dairy products or meat; their concen-trations can vary depending on the diet fed to cows or sheep. Interest in the biological activities played by these fatty acids arose for the

R2 R1

R2R2

R1

R2 R2R1 R1

R1

13

12 10

12 10

11

11

9

9

12

9c,12c-18:2

9c,11t-18:2

9t,11t-18:2

10t,12c-18:2

10t,12t-18:2

Linoleic acid: R1 = (CH2)7CO2H, R2 = (CH2)4CH3

10

13 12

11

1113

10

12 10

9

Figure 4.2 Conjugated linoleic acid (Cla) isomers and comparison with linoleic acid

Trans FaTTy aCIds FOr HUMans 69


antitumoral activity of 9cis,11trans isomer. On the other hand, the 10trans,12cis‐Cla isomer exerts specific effects on adipocytes, in particular reducing the uptake of lipids by inhibiting the activities of lipoprotein lipase and stearoyl‐Coa desaturase. Both the Cla isomers were then found to inhibit carcinogenesis in animal models [73]; there-fore it is also likely that some effects are induced and/or enhanced by these isomers acting synergistically. Further research is needed to clarify the activities of these isomers; however, in nutritional quantities they are already present when dairy products are present in the diet.

It is worth underlining that bacterial biohydrogenation occurring in the ruminant stomach is responsible for partial hydrogenation of the dietary lipids of ruminants. Therefore, milk and meat from these animals can contain a certain percentage of geometrical and positional trans iso-mers. The summary of the pathways leads to 11‐trans‐18 : 1 and by delta‐9 desaturase to Cla (9cis,11trans‐18 : 2) with beneficial effects, which have been explained in several reviews [11, 74].

To complete the scenario of “exogenous” sources of trans isomers, the bacterial process of cis to trans isomerization of fatty acids has to be mentioned. This process creates trans geometrical isomers, without changing the position along the fatty acid chain, but only the geometry. some Gram‐negative bacteria, in particular very resistant strains such as Vibrio cholerae, Pseudomonas putida and aeruginosa, were discovered to use this endogenous enzymatic isomerization as a short‐term response to environmental and temperature solicitations. In fact, transforming the cis to trans fatty acids gives different properties to the cell membranes, which become more resistant to survive under unpleasant conditions.

It is interesting to note that prokaryotes have the adaptation response by isomerase activity that was completely lost in the evolutionary steps to eukaryotes; therefore eukaryotic (so human) cells cannot prepare trans fatty acids themselves and, as previously discussed, this geometry is toxic for them. The enzymatic isomerization mechanism and bacterial resistance have been treated elsewhere [74, 75], where the reader can be addressed for deepening understanding on this subject. Two aspects of this process are underlined:

1. The isomerization mechanism is underevaluated so far as resis-tance phenomenon in bacteria is concerned, whereas it could be suggestive of novel pharmacological strategies for antibacterial activity.

2. The capability of bacteria to resist unfavorable conditions started to attract research for medical and biotechnological applications only recently, and important developments can be foreseen in this area.

endOGenOUs sOUrCes OF Trans FaTTy aCIds 71

4.2 endOGenOUs sOUrCes OF Trans FaTTy aCIds By Free radICal sTress

an interesting observation was made in early 2000, concerning the free radical reaction with double bonds of natural lipids, inducing the cis to trans isomerization process. This reaction tested first in biomimetic models of phospholipids in aqueous systems was found to be very effective [11]. The most reactive free radicals to induce isomerization were the sulfur‐centered radicals, according to the mechanism of addition and elimination depicted in Figure 4.3. In section a of the Figure the isomerisation of oleic acid is depicted, whereas in section B the isomerisation mechanism involving a double bond and the sulphur-centered radical is shown, with the formation of the beta-sulfur substituted intermediate radical.

The trans isomer due to its thermodynamic stability is the most favored geometry for the double bond. The discovery of this reaction highlighted also that the cis geometry chosen by nature is in principle very delicate, since it is the less stable isomeric configuration. However, it must be taken into account that this geometry is the result of a selection made by nature for the ideal fluidity and permeability of the cellular environment, especially for the cell membrane compartment. as explained in part 1, the optimal functionality of membranes can be obtained only by the bent structure of the cis lipids.

Free radical X∙

HO

HO

9 10

9

10

O(a)

(b)

O

RSH H

RS∙Radical source

RS∙ + RS∙ +

∙

Figure 4.3 Isomerisation of fatty acids (a) shown for oleic acid and addition- elimination mechanism (b) shown for s-centered radicals.


The lipid isomerization has been proven to occur under free radical stress conditions in cell, animal, and human studies [11], and here three main aspects are underlined:

1. The formation of trans isomers in a living organism can be consid-ered as a signal, especially at the level of membrane unsaturated phospholipids, since the effect of trans isomers is to increase the rigidity of the lipid assembly resembling that of saturated fatty acids (see Chapter 1). a threshold for the presence of trans isomers in erythrocyte membranes due to “natural” endogenous and exog-enous processes could be established, as 0.4% of the total fatty acids detected by lipid analysis [59], and this value also includes the trans isomers coming from a “healthy” diet.

2. The markers of the endogenous formation of trans isomers by free radical stress are arachidonic acid mono‐trans isomers in positions 5 and 8. In fact, as shown in Figure 4.4, during biosynthesis the two double bonds in the positions 5 and 8 of arachidonic acid are formed from linoleic acid by the desaturase enzymes; therefore their geometry is cis, unless there is an endogenous isomerization process to form the trans isomers.

Exogenous(diet)

Linoleate (Diet)9c,12c–18:2

Gamma-linolenate6c,9c,12c–18:3

Desaturation

Eicosatrienoate8c,11c,14c–20:3

Elongation

Arachidonate5c,8c,11c,14c–20:4

Desaturation

5 8

Endogenous

9 12

11 14

Figure 4.4 Biosynthesis of arachidonic acid and the importance of the position 5 and 8 of the double bonds, for indicating the trans isomers of arachidonic acid as marker of endogenous isomerization

Free radICals and lIpId OxIdaTIOn 73

The mono‐trans isomers of arachidonic acid are shown in Figure 4.5 and constitute a very important molecular library to determine the exposition of the cell membranes to free radical stress.

3. so far there is no enzymatic system in humans known to convert trans to cis fatty acids, or that recognizes specifically the presence of these unnatural isomers. Therefore, the trans fatty acids can make only part of the lipid remodeling and conversion pathways, such as for saturated fatty acids, and their washout from mem-branes is favored by using adequate quantities of the natural cis isomers.

4.3 Free radICals and lIpId OxIdaTIOn: THe THresHOld FOr HealTH

In the last decade or so, the roles of free radicals and oxidative processes have been linked to the occurrence of inflammation processes in many pathological conditions. Consequently, lipid transformations and dis-eases have been connected with each other, actually giving the idea that lipid oxidative and degradation processes are mostly damages that

R1

R2

R2

R2

R1 = (CH2)3CO2H, R2 = (CH2)3CH3

R2

5t

8t

14t

11t

R1

R1

R1

Figure 4.5 Mono-trans isomers of arachidonic acid


impair the cell response. This information is really incomplete, altering the importance of oxidative pathways that are instead naturally occur-ring processes and make part of the signaling cascades for activation of defense mechanisms. The case of resolvins obtained from epa has been previously cited (see Figure 3.4). In Figure 4.6 the oxidative pathways involving arachidonic acid are shown, which can be either enzymatic (cycloxygenase, lipoxygenase, and lTC synthase) or chemical (Fenton reaction with Fe+2 and H2O2) ‐driven processes. The products such as aldehydes (4‐hydroxynonenale, 4‐Hne), a2 isoprostane, and prosta-glandins, derive naturally from the cellular metabolism [76, 77]. It is worth noting that these very important transformations use the arachi-donic acid moiety that is present in the membrane phospholipids. Therefore, membranes assume the meaning of crucial site for the syn-thesis of mediators, with processes that start and are regulated by the phospholipase a2 enzyme, which detaches arachidonic acid from the membrane lipids (see land’s cycle treated in section 3b).

The release of arachidonic acid and the production of these com-pounds are very efficient during inflammatory events and any other cell

Non-enzymatic

Arachidonic acid

Cyclic endoperoxide LOOH 5-HPETE PGH2

Isoprostanes Aldehydes Leukotriene A4

Fe2+ H2O2O2

O2

5,12, 15-lipoxygenase(LOX)

Cyclooxygenase(COX)

Dehydrase Isomerases/synthases

LTCsynthase(GST)

Hydrolase Lipoxygenases

LTC4LTD4LTE4

TXA2PGD2PGE2PGF2PGl2

LTB4 Lipoxins

+OH

Heme

HemeCu2+

Enzymatic

Figure 4.6 enzymatic and nonenzymatic pathways of transformation of arachi-donic acid into oxidation products such as aldehydes (4‐Hne), isoprostanes, prosta-glandins, and others. reproduced with permission from ref. [76]. With kind permission from springer science and Business Media


response under stimulated and reactive conditions. In these cases, the levels of oxidation products can overcome a physiological threshold, and cause a transitory status of “oxidative stress.” In particular, reactive compounds such as those containing the aldehyde functionality (e.g., 4‐Hne) are electrophilic as regards their chemical nature, and can imme-diately react with compounds containing amino and thiol groups, such as proteins and peptides. so‐called aldehyde adducts are formed, thus producing covalent protein modifications, nowadays also interpreted as a signal belonging to the “lipid–protein interactions,” which are then metabolized by the proteasome. The accumulation of these irreversible modifications is observed in degenerative and chronic diseases, and care-ful analytical methodologies are under study to provide reliable mea-surements for clinical use. The formation of aldehyde compounds, such as 4‐Hne, is connected with the peroxidation process of polyunsatu-rated fatty acids (in particular omega‐6 such as linoleic and arachidonic acids) and has been described in detail in several reviews and papers [76–79].

In recent years, a link between the 4‐Hne production and the release of linoleic and arachidonic acids from the membranes has been pro-posed, since these fatty acids are detached from phospholipids after the stress signal and activation of phospholipase activity, as previously men-tioned (see the activation of land’s cycle in section 3b). experiments were carried out in cell cultures using the powerful tool of cell mem-brane lipidomics coupled with the measurement of the 4‐Hne formation. In this way, it was proven for the first time in beta‐pancreatic cells that increasing glucose concentration produces 4‐Hne through a cascade of events starting from a membrane‐stimulated process, involving the lipid remodeling pathway and release of omega‐6 from membranes, after which 4‐Hne is produced and interacts selectively with the ppar‐delta system for inducing nuclear transcription and insulin response [77, 80]. This simple experiment showed that lipid signaling is connected to glucose stimulation, highlighting membranes and their fatty acid compo-sition as very important sites of cell response and regulation. This also means that in order to understand the basis of cellular response, biological and molecular biology research must also include membrane lipidomic monitoring with special emphasis on phospholipid fate.

Coming back to the oxidative reactivity of pUFas, the entire mecha-nism was proposed by porter in the late 1970s [81], where hydroperox-ides deriving from the corresponding peroxyl radicals were individuated as the first formed products (Figure 4.7). The reactive site of pUFas in the peroxidation process is the methylenic group between two double


bonds, called bisallylic position, and the first step is the hydrogen atom abstraction from this position by a free radical.

The mechanism of lipid peroxidation (a radical chain reaction) starts with the abstraction of hydrogen atom producing the bisallylic (or pentadienyl) radical. Figure 4.7 illustrates the steps of this process, starting from: (i) hydrogen abstraction from bisallylic position; (ii) formation of the bisallylic radical; (iii) uptake of oxygen to give the conjugated hydroperoxy radical; and (iv) the products of lipid degrada-tion and decomposition commonly expressed as conjugated dienes, as well as the previously mentioned aldehyde end products, which are used to assess the occurrence of oxidative stress. Together with the earlier‐mentioned 4‐Hne, malondialdehyde (Mda) can be formed as the aldehydic product (Figure 4.8).

It is worth noting that the initiation step of hydrogen atom abstrac-tion occurs by free radicals, the hydroxyl radical OH• being the most effective species in this event. One of the most involved reactions in the production of HO• radicals is the Fenton reaction in the presence of hydroperoxides and metals in the reduced state (Cu+2, Fe+2, etc.). an increase of hydroperoxide production, also due to an incomplete control by the antioxidant enzymatic systems (e.g., catalase), or to an increase of free metal concentrations (facilitated by acidic conditions), can speed up the Fenton chemistry and the production of HO• radicals. The

Bisallylic position

R1 R2

O2

H HR1

R1 = (CH2)7C(O)OH; R2 = (CH2)4CH3

R2R1

HO•

OO∙

O2

R1 H

L∙LH

LOOH LOO•

+LHOOH

R1 R1R2 R2

R2H H

R2 R1 R2

OOH+

HOO

Figure 4.7 Hydroperoxides formed from linoleic acid by the peroxidation process, starting from the reactivity of the bisallylic position


effectiveness of enzymatic and microenvironmental control systems in maintaining the balance of the oxidative status is crucial, in order to keep pUFa peroxidation within physiological levels.

The chemical knowledge of peroxidation steps should be a priority toward further developments, such as:

1. Biomarker discovery to monitor the occurrence and the extent of this process in living organisms. Human fluids are the subject of intense research in this field and several methods must be seriously evaluated for their accuracy to detect free radical stress products [82, 83].

2. The research of inhibitors of this process, called “antioxidants,” among natural or artificial compounds, to be used also for treatment of degenerative processes of peroxidation in neurolog-ical, cardiovascular, or metabolic diseases.

as far as the first point is concerned, by spectrophotometric or chromatographic methods, the quantification of peroxides and alde-hydes can be carried out. The TBa (thiobarbituric acid) or TBars (thio-barbituric acid–reacting substances) test is the most famous, also the easiest and cheapest, checking the absorbance at or close to 532 nm or by fluorescence at 553 nm. Often the test is referred to micromolar Mda equivalents; however, this can be done in controlled systems,

R1R2

OO•

LOO•

Conjugatedhydroperoxides

LOOH CHO

HO

C5H11

CHO

CHOMDA

4-HNE

Figure 4.8 The main products from the peroxidation process: conjugated hydro-peroxides (lOOH), 4‐hydroxynonenal (4‐Hne) and malondialdehyde (Mda)


whereas in body fluids the measurement produces a host of problems. The TBa test reliability depends also on the antioxidants contained in the samples, as well as on the metal contaminants, and there are also many other criticisms, which suggest other methodologies such as a more precise separation of the aldehyde by HplC (high‐performance liquid chromatography), to avoid artifacts [84].

regarding the earlier‐mentioned aldehyde adducts, their capability to induce antibody is well known and became an important field of research for markers and bioassays of oxidative stress, with Hne or Mda anti-bodies able to visualize the adducts present in a tissue. depending on their mode of preparation, there are specific antibodies to the Hne–His epitope, whereas others may also recognize Hne bound to Cys and lys, and possibly even other amino acid residues like tyrosine, serine, argi-nine, and proline. Therefore, it is better to choose elIsa tests, such as the Hne–Hys–elIsa test and the Hne–protein adduct–elIsa test. These tests are promising to become crucial assays for determining the oxidative degenerative status in several pathological conditions.

On the other hand, the commercially available peroxide activity assays (pox‐act and d‐rOMs) are less specific on the measured compounds and can give several problems for the correlation with the specific clinical condition of subjects. It is reasonable to think that serum peroxide levels in patients is higher than in healthy subjects; however, problems of sensitivity and false positive can occur and the test response has to be considered with caution [85].

In general, the difficulty to envisage the contribution of the oxidative free radical pathway to the formation of peroxides and aldehydes, when the complex scenario of a living organism is involved, is well known. In fact, these two products can derive from many other metabolic path-ways, rather than being markers of pure free radical or oxidative stress.

For example, aldehydes can result from alcohol metabolism, via alcohol dehydrogenase activity, as well as peroxides, which are present in different enzymatic pathways, connected with antimicrobial defenses and favorable protective activities. Therefore, it is difficult so far to indicate these markers corresponding to oxidative processes exclu-sively attributable to pathological conditions. The common conclusion from thousands of research papers highlights that, although lipid per-oxidation products exert cytotoxicity, sublethal concentrations of such products induce cellular adaptive responses and enhance tolerance against subsequent oxidative stress through upregulation of antioxi-dant compounds and enzymes. The products obtained by enzymatic

lIpOprOTeIns and deVelOpMenT OF MarKers 79

oxidation are clearly more regulated than those formed by free rad-ical‐mediated processes. However, even such unregulated products may exert beneficial effects at low levels, whereas excessive produc-tion may lead to pathological disorders and diseases [85]. The only robust marker for free radical processes regarding pUFas so far is the isoprostane family, in particular F2‐Isops, currently considered the best available biomarker of oxidative stress status and lipid peroxidation in vivo [86, 87].

This marker has several advantages, such as: (i) the specificity of the assay itself for the product of lipid peroxidation being measured, (ii) the product being measured derives from lipid peroxidation, (iii) sufficient sensitivity to detect levels of the product being measured in normal sub-jects, thus allowing the definition of a normal range, (iv) levels of the product being measured not influenced by external factors, such as the lipid content of the diet, and (v) the assay is not invasive for human investigation. detailed description of the levels and the measurements is available in the literature, with liquid chromatography coupled with mass spectrometry (lC/Ms) being the gold standard, followed by immu-noassays with various methodologies for the purification of the sample before the reaction.

4.4 lIpOprOTeIns and deVelOpMenT OF MarKers FOr lIpId reaCTIVITy

Up to now the importance of phospholipids has focused on the mem-brane. another lipid assembly where phospholipids play an important role is represented by lipoproteins, present in human plasma as different types: very low‐density lipoproteins (Vldls), high‐density lipoproteins (Hdls), and low‐density lipoproteins (ldls), to cite the most impor-tant. They are generally formed by a protein part, called apolipoproteins (10–55%), and a lipid part made of variable proportions (in weight) of phospholipids (18–24%), triglycerides (5–50%), cholesteryl esters (15–37%), and free cholesterol (2–8%). Functionally speaking, the lipoprotein assembly is the carrier of the most complete mixture of lipid molecules; therefore its circulation in the body allows the tissues to receive an important lipid supply.

In Figure 4.9 the interconnection among all the different types of fatty acid–containing lipids is shown, where it is clear that lipoproteins contribute to the bioavailability of different lipid molecules, which are


insoluble in the aqueous plasma medium, and represent the richest fatty acid reservoir circulating all over the body (Figure 4.9).

In particular, the quality of the fatty acids transported by lipoproteins must be underlined. In fact, the abundant cholesteryl ester and phospho-lipid fractions, which sum up to 75% of the total lipoprotein lipids, contain high percentages of unsaturated fatty acids, with linoleic acid being the most abundant residue. another important point regarding lipoproteins is that cholesteryl esters derive from the enzymatic activity of lecithin cholesteryl ester transfer protein (lCaT) that moves one of the fatty acid tails (i.e., the pUFa residue in position 2) of phospholipids to a molecule of cholesterol. This step is not reversible, and occurs in chilomi-crons and Vldls (the smallest lipid emulsion formed during digestion), thus forming a cholesteryl ester core, which gives rise to Hdls and their maturation. From this scenario the importance of cholesteryl ester syn-thesis becomes clear, and novel research must be addressed toward fatty acid analysis of this lipid fraction, including comparison with the mem-brane phospholipids of the same patient, in order to understand better the metabolic transformations, thus excluding dietary contribution.

OH

OH

OH

R CO

OH

Rʹ CO

OH

Rʺ CO

OH

OCOR

OCORʹ

OCORʺ

Glycerol

Fatty acids

Triglycerides(diet)

Lipaseenzymes

Diglycerides

Phospholipids(PC, PS, PE)

+ Cholesterol(diet)

Cholesteryl esters

Choline+ polar heads

assembly

Lipoproteins

Assembly

Figure 4.9 The interconnections between the different pathways of fatty acid– containing lipids, and the formation of lipoproteins as lipid assembly (pC = phosphatidylcholine, ps = phosphatidylserine, pe = phosphatidylethanolamine)


With their high content of unsaturated moieties, lipoproteins are naturally exposed to the transformation of MUFas and pUFas by isomerization and oxidation, as previously discussed. excessive ldl oxidation has been recognized as an important contributor to the pathogenesis of atherosclerosis [88, 89]. The phospholipid part of ldls is the primary target of oxidation in an ldl particle. In vitro oxidation of ldls results in the isolation of several oxidized phospho-lipids, and, with the development of HplC methods coupled with ion‐spray mass spectrometry, primary products in the early stages of ldl oxidation have been identified [90]. The cholesterol ester component of lipoproteins is also involved in oxidation of the linoleic and arachi-donic acid moieties. These esters are incorporated into Vldls, and further transformed into ldls. When ldls is transferred to endothe-lial cells, toxic products are liberated and induce cell damage. The damage is accompanied by structural changes that influence neigh-boring cells and cause an influx of Ca+2 ions, with activation of, first, phospholipase, which is responsible for the fatty acid liberation from membranes, and, second, lipoxygenase enzymes, which produce lipid peroxides. The fatty acids liberated from the membranes, if exceeding the enzymatic capacity of metabolic transformations, remain “free” in the cytoplasm, and this corresponds to an important signal of toxicity against lipoxygenase, which commits suicide, liberating iron ions. These ions can react with lipid peroxides and produce peroxyl radicals via nonenzymatic reactions. The radical cascade is then activated with the attack on any biological compounds in the vicinity that can cause severe damage [91]. The formation of lipid peroxides is described to occur in cardiovascular, as well as in inflammatory and neurodegener-ative diseases, indicating a common ground for all these events. The formation of oxidized lipids induces a vicious cycle, since it can influence the overall membrane lipid balance with alteration of the channel functioning across the bilayer. To this change the influx of Ca+2 ions can be related, so that phospolipase enzymes are activated. The cleavage of phospholipids occurs and cause damage as described earlier [92].

another interesting, yet newly discovered, marker of free radical stress in lipoproteins is the level of geometrical trans fatty acid isomers. The total trans fatty acids (summing up the trans isomers of oleic, linoleic, arachidonic, and omega‐3 fatty acids) present in the cholesteryl ester fraction of human plasma and ldls have been determined and quanti-fied, and are expected to be developed for biomarkers in healthy and


unhealthy conditions [93]. The ldl fatty acid composition also results in hexadecenoic acid isomers, such as palmitoleic and sapienic acids, as explained in the section dedicated to MUFas (section 2.2). These trans MUFa isomers are found to be very interesting for development as markers of endogenous stress, due to the absence of dietary contribution of such components [35].


4a In Depth: Oleic versus Linoleic Acid Reactivity with Free Radicals

The unsaturated fatty acids possess double bonds that are suscep-tible to reactions under free radical stress. How different are the monounsaturated versus polyunsaturated fatty acids? We will focus our attention on the most important fatty acids of the two types, namely, oleic acid (MUFa) and linoleic acid (pUFa, omega‐6). The two structures are shown in Figure 4.10: both of them have two allylic positions, whereas MUFa has only one double bond and pUFa has two double bonds plus a bisallylic position.

For the peroxidation reaction both allylic and bisallylic positions can react, with the bisallylic position far most reactive than the allylic ones. This means that linoleic acid can be the substrate for fast consumption via the bisallylic reaction in the presence of oxygen and free radical conditions, provided that no protection or inhibition of the process is activated.

On the other hand, the double bonds of the two substrates have sim-ilar reactivity as regards the isomerization process with formation of the geometrical isomers depicted in Figure 4.11, being doubled reac-tive positions in linoleic acid compared to oleic acid. In Figure 4.11, the geometrical isomers of oleic and linoleic acids are shown, which can occur by exogenous and endogenous isomerisation as explained in section 4.2, and are recognized by gas chromatographic analyses.

9

9

1210

10

13

Bis-allylic group

HO

HO

O

O

Allylic groups

Figure 4.10 The two structures of oleic acid (MUFa) and linoleic acid (pUFa, omega‐6). reactive sites can be distinguished: linoleic acid shows the bisallylic groups, two allylic groups, and two double bonds (C9–C10 and C12–C13), whereas oleic acid has two allylic groups and one double bond (C9–C10)


CONCEPTS’ SUMMARY

S1 Fatty Acid Geometry: A “Radical” Change

The discovery of the isomerization reaction carried out by free radicals toward the double bonds of natural lipids was a scientific breakthrough, which is expected to gain more and more attention from life science researchers interested in understanding the fine‐tuning and molecular selectivity present in living organisms. The fact that in microbiology lipid isomerization is evaluated as a survival strategy of some resistant bacterial species whereas in the study of mammalian the change of lipid geometry is not even considered, is self‐explanatory about the separation among disciplines, indicating

O

HO

HO

HOOC

HOOC

HOOC

HOOC

O

9 10

9

9 12

c c

c

c

t

t

t t

10

Geometrical isomers of oleic acid

Geometrical isomers of linoleic acid

Figure 4.11 The structures of the geometrical isomers expected from oleic and linoleic acids


lack of a unitary vision regarding biological molecules and their functions. The “radical” change in favor of the most stable trans structure is dramatic for eukaryotes, since it produces the loss of the natural geometry and functions of lipid structures. Therefore, a scenario for the trans significance in eukaryote species can be figured out as follows: (i) first of all, the trans lipid formation can be one of the most sensitive signals to produce effects on the “all‐cis” organization of the membrane compartment, at the level of membrane biophysical response; (ii) second, it would be important to understand whether systems to repair or protect the geometry of natural lipids are operative in trans‐free systems like eukaryotes. research in this field can be pursued for expand-ing basic knowledge on the naturally occurring biomolecules. The extent of this transformation and the related signaling are still to be evaluated, whereas it is very important that molecular libraries and effective analytical methodologies are already avail-able in order to estimate the types and quantities of trans lipids in different organisms and conditions.

S2 Antioxidants for Membranes

at this point, it is worth considering another aspect related to the protection of membrane lipids. On the basis of the reactions described so far, the role of molecules able to interrupt or inhibit lipid transformations is very important. “antioxidants for mem-branes” means molecules able to interrupt the oxidative pathways of lipids in this important compartment. This effect can be obtained by different modalities depending on how the lipid transforma-tions occur. In Figure 4.12 a schematic distribution of several enzy-matic and molecular systems intervening in cell protection against free radicals and oxidation damages is shown: (i) with lipophilic compounds such as vitamin e and carotenoids (beta‐carotene) able to stay in the membrane layer, free radicals can be intercepted inside membranes (plasma or mitochondrial) prior to the reaction with bisallylic positions or double bonds; (ii) with more hydro-philic compounds (such as vitamin C, cysteine, and glutathione) as well as with enzymatic systems (glutathione peroxidase, catalase, and zn/Mn sOd), oxidative reagents (such as hydrogen peroxide)


and free radical species (diffusible HO• radicals, sulfur‐ centered radicals) can be intercepted inside the cytoplasm, reducing the pos-sibility of their diffusion in the membrane compartment according to their partition coefficient.

The “antioxidants for membranes” can be ideally formed by synergic cocktails of compounds, having either different inhibition mechanisms or different partition coefficients, in order to quench satisfactorily the various sources of reactive species that can perturb the natural lipid structures.

Vitamin E

Vitamin E

Vitamin C

Glutathioneperoxidase

GSHMitochondria

Lysosome

Peroxisome

Catalase

Endoplasmic reticulum

Cu/ZnSOD

Lipid membrane

Vitamin Eβ-carotene

MnSOD+Glutathione-peroxidase+GSH

DNANucleus

Vitamin C ed Eβ-carotene

β-carotene

Figure 4.12 different locations of enzymatic and molecular antioxidant systems


Membrane Lipidomics for

Personalized Health

Although lipid research has a long history, the last two decades or so have been crucial for the development of a comprehensive scenario of lipids in cell metabolism, especially concerning the importance of fatty acids and remodeling pathways in life sciences and nutrition. To place the functional roles of the fatty acid families in the wide scenario of homeostatic control and cell signaling, a new interdisciplinary field involving collecting and reinterpreting the data on lipids was born. Lipidomics is a newly proposed discipline that rationalizes the fatty acid transformations, partly explained in Part 1, aiming at furnishing a dynamic vision of lipid classes and their exchange during various meta-bolic conditions.

Lipidomics took the place of the “old” lipidology, with a suffix “‐omics,” which has a correlation with the other “‐omics,” such as genomics and proteomics.

Part II

88 MeMbrAne LiPidoMics for PersonALized HeALTH

Lipidomics can be defined as follows:

Lipidomics is a discipline of life sciences that studies lipid molecules in a “dynamic” context, which means not only understanding the relationship bet-ween structures and functions, but also following up the changes that occur to lipids in a cell compartment or in whole organism, under physiological or pathological conditions, and addressing the lipid diversity needed for life.

each organism and biological compartment has its own lipid compo-sition, defined as the lipidome, which can be monitored by lipidomics. The involvement of lipid changes in the functionality of the organism is named functional lipidomics. in particular, membrane lipidomics, treated in Part 2, is related to the compartment of cell membrane and includes phospholipids as well as fatty acids as their hydrophobic components.

since the start of lipidomics, it was clear that the understanding of the dynamic behavior of lipids under physiological and pathological conditions could open new frontiers to health prevention and disease treatment. in this context, lipidomics has become a mature field since its analytical methodologies have optimal performance level, and some of them, such as gas chromatography, are the gold standard for the qualitative and quantitative determinations of fatty acid. This is an important requisite for the application of a methodology to clinical practice. indeed, the combination between reliable analytical determina-tion and precise biological role or effect is needed in order that clinicians can use molecular diagnostics in their decisional process. Paradoxically, the majority of the oxidative stress tests available in the market (e.g., to measure lipid peroxides, see section 4.3) use imprecise or non‐optimized methodologies. nevertheless, the market of oxidative stress assays is well known and these tests are used more than that of fatty acid analysis or lipidome monitoring.

The fatty acid analysis is mature to translate the values found in humans into a valid molecular diagnostic tool; therefore, it is more appropriate now to include lipidomics, in particular membrane lipido-mics, in clinical practice for health prevention and intervention strat-egies. spreading the knowledge and educating health professionals on membrane lipidomics are highly recommended to help them to appreciate this tool, to learn how to interpret the fatty acid analysis, to envisage lipidomic profiles of their patients and, finally, to use lipidomics for deciding the appropriate intervention. The objective is to place membranes and their fatty acid components in the clinical protocols, recognizing the strength of lipidomics toward an integrated nutritional and metabolic vision for optimal health.


What Is Lipidomics for Health

5.1 THE BIRTH OF THE POSTGENOMICS ERA

Big efforts for human genome sequencing characterized the last century’s research. This brought about single nuclear polymorphism (SNP) to be individuated as a powerful molecular diagnostic tool, consisting of the variation in the DNA sequence, involving one of the four nucleotides substituted for another. For example, a SNP may replace the nucleotide cytosine (C) with the nucleotide thymine (T) or the nucleotide guanosine (G) with cytosine (C) in a certain DNA strand. This exchange occurs with an average frequency of 1 upon 300 sequences, so that more than 10 million SNPs can be present in the human genome. This diversity is responsible for the recognition of each individual largely adopted in forensic protocols. When occurring in regulatory regions near gene coding sequences, this trait difference can cause diseases or serious impairments (beta‐thalassemia, sickle cell anemia, cystic fibrosis). At most, SNPs do not affect health; however, they are still a very crucial determinant for the sensitivity to drugs, with application in pharmacogenomics assays, particularly useful to decide type and dosage of certain pharmacological treatments [94]. Another field of application, perhaps the most intriguing and discussed one, is genetic risk prediction, which is connected to a probabilistic factor that links the SNP or SNP group to the development of a disease. The predictive objective is very ambitious and since the beginning required a strong organizational effort with the Genome‐Wide Association Study (GWAS). This is a task force of

5

90 WHAT IS LIPIDOMICS FOR HEALTH

researchers collaborating to associate the frequency of a SNP with the probability of development of a disease.

The SNP test has already been put on the market with diagnostic products claiming to give a precise answer on the individual predisposition to develop a disease. For clinicians understanding the potential of this approach and the validity of this diagnostic tool is a serious matter, involving not only information, but also formation courses, in order to understand the correct use of this data. A relevant argument concerns how much is the probability of developing the disease once the subject discovers having a particular SNP or SNP combination. For example, for the predictive value of type 1 diabetes (T1D) the knowledge gained so far estimates that with a predictive value of 18% of the population above the risk threshold, those who will not develop T1D “would be ca. 60 times more numerous of the 80% of the future case among them” [95]. This statistical evaluation, that underlies a tremendous work done by the scientists in the field for this multifactorial disease, tells that screening are not recommended, except in very specific cases.

Knowing the higher risk population can be an indication for cost‐effective public health strategies focusing on disease prevention and intervention. For example, the prediction of the risk of neurodegenerative disease like Parkinson’ and Alzheimer’ diseases, which are associated with severe health implications and high costs, would be greatly helped by diagnostics and preventive tools. However, it is important to underline that the SNP tool is in development, still not in the mature stage for unequivocal identification of disease predisposition and onset probability. It is not the scope of this book to provide an in‐depth analysis of the role of genomics for health but only to define that, from the scenario available so far, it is advisable to treat this matter with caution.

A research advancement is the knowledge that the DNA sequence and the correlated predisposition read by the SNP tool are not the only driving force for ensuring a healthy life. In the twenty‐first century the approach to health includes molecular tools, which are not restricted to DNA analysis. Indeed, the role of each single molecule and process in a living organism has been revaluated for its capability of contrasting and even reversing the DNA predisposition, making part of the complex regulation that allows life. Humans are thought to have between 25 000 and 30 000 genes that encode proteins for all the parts of our bodies. On the other hand, the tiny roundworm Caenorhabditis elegans has nearly as many genes as humans do—approximately 20 000—but far fewer body parts. Organism complexity thus comes down to more

THE BIRTH OF THE POSTGENOMICS ERA 91

than just gene numbers. From recent research it was discovered that the regulation of gene expression is an important part of the game, the environment—external (such as chemicals, light, drugs) or internal (such as hormones and metabolism) to the organism—being a crucial factor to activate the final response [96]. All these driving forces have been collected in one word: epigenetics.

The postgenomic era is now at work with epigenetics that can offer an even larger and more complex scenario than genomics alone. Epigenetics includes a part dedicated to the regulation of the structure and organization of DNA. The fact that almost 1.7 m of DNA sequence is packed into about 5 µm of nuclei (nucleosomes) organized by chromatine (and histones as its important components) gave other hints to researchers, who discovered that at least three processes are involved in the DNA (and RNA) activation or deactivation to transcription: DNA methylation–demethylation, histone acetylation–deacetylation, and chromatin remodeling. The starting point and hierarchy of these transformations are not yet deeply understood, and a comprehensive scenario and know‐how are far from being available. These reactions and the epigenetics of each individual, the influence of nutritional elements, such as vitamins and cofactors, are also under investigation by many research groups [97]. The field of nutrigenomics is correlated to this enormous potential of nutrition in personalized medicine and already involves many research groups worldwide. After several years of research in this field, there are some concerns regarding the promise that nutrigenomics has made of “controlling health by foods” in the general population, and now opinions are gathered on avoiding too many expectations [98]. This field might bring important developments for personalized medicine; however, it depends also on the clarification of epigenetic pathways that will come in the future.

Despite the clear scientific indications of more work needed, commercial products for diagnostic and nutrition are proposed in the market, also from the web, and certainly this induces confusion. There is a serious possibility that health professionals, and also consumers, are more and more confused from these health promises. Knowledge is required to discriminate when and how to use diagnostic and nutritional tools as proposed by nutrigenomics. In order to do so, formative courses must be organized by academic institutions and professional organizations, and attended by health professionals for advancing and updating their knowledge in this promising sector of human health.


5.2 LIPIDOMICS IN THE POSTGENOMIC ERA

In the postgenomic era what is the place of lipids? Lipids cannot be underevaluated, starting from the simple observation that they, as well as DNA, are needed for starting life as cell membrane components. Lipidomic knowledge is essential for the complete understanding of the multiple roles of lipids and fatty acids. This scenario also includes fatty acid induction of gene expressions, which is a subject of nutrigenomics. As a matter of fact, PUFAs are studied for their interaction with two groups of transcription factors, such as sterol regulatory element binding proteins (SREBP) and peroxisome proliferator activated receptors (PPAR), which are critical for modulating the expression of genes controlling both systemic and tissue‐specific lipid homeostasis [99]. This is an important aspect of fatty acid biological activity, thus motivating a strong commitment in research for various medical applications. It can be foreseen that fatty acids or natural sources of fatty acids will be used in combination with pharmacological therapy, likely in multicomponent soft gel capsules, offering a synergic strategy also for the treatment of important diseases. In all this research transferred from bench to bedside, it is important to underline that the mode of action of fatty acids does not consist only of gene regulation, and lipidomic approach is needed to follow up fatty acid activity, starting from biodistribution to the metabolic involvement of different pathways, which are deeply connected with target gene interaction.

It is not trivial to recall that fatty acids come in abundant quantities from nutrition, and actually essential fatty acids, such as omega‐6 and omega‐3, are necessarily taken from the diet. Therefore, from scientific research on lipids and nutrition, considering the strong connections already existing between the nutritional and metabolic roles of lipids, including fatty acids in primis, the discipline of nutrilipidomics was defined [100]. As lipidomics offers the most effective integrated vision of the fatty acid fate, the corresponding nutrilipidomic approach examines the interplay between the food components—which can be not only lipids, but also cofactors and coadjuvants of lipid pathways—and the effects in the overall lipid activity and metabolism, addressing also health maintenance and improvement. Nutrilipidomics, treated in Part 2, has already a strong base in the immense work done by lipidologists and nutritionists in the last three decades. It will be very important to apply knowledge acquired so far to achieve health objectives. Nutrilipidomics can really ensure the stewardship of its expectations for personalized health improvement.

FATTY ACIDS INVOLVED IN MEMBRANE 93

From all the earlier considerations, we must step forward and toward an integrated vision, unifying the various “‐omics” approaches. The scientist’s duty will be to prepare platforms for the interdisciplinary evolution of molecular diagnostics, eliminating barriers, still present and too heavy, and facilitating the development of this integrated vision in clinical practice.

After presenting fatty acid structures and pathways in Part 1, with their biochemical and biological roles correlated to specific cell activities, in Part 2 the lipidome behavior and its transformations will be treated in a functional way, understanding how quality and quantities of fatty acids correlate to health and can be different in physiological and pathological conditions.

5.3 FATTY ACIDS INVOLVED IN MEMBRANE AND MEDIATOR LIPIDOMICS

Fatty acids with their different biological roles are described by the approach of functional lipidomics, which applies an additional distinction between mediator lipidomics and membrane lipidomics. Membrane lipidomics deals with the fatty acid cycle starting from the biosynthesis and nutritional contributions to constitute the fatty acid pool for the formation of membrane fatty acids. This formation occurs with a tissue‐specific selection, since each tissue requires its adequate fatty acid composition to function. Membrane lipidomics, as we have discussed in Part 1, studies the “lipid code” and its integrity correlated to protein, channel, and receptor activities embedded in the membrane phospholipid bilayer of each tissue. In membrane lipidomics also the role of specialized areas, called lipid rafts, is studied since it is crucial for the functioning of proteins. Moreover, the role of cholesterol, in making plasma membranes and lipid rafts with the appropriate composition and properties, is remarkable for its health consequences and the decision‐making process of pharmacological treatments with inhibitors of cholesterol biosynthesis. These topics are treated in the “In depth” sections of Part 1.

The virtuous cycle between balanced nutritional intakes and metabolic turnover provides membranes with their ideal fatty acid composition; however, this cycle can be altered due to different “environmental” causes, such as dietary habits, subjected to changes and industrialization, the stressful life of Western society with its metabolic consequences, the increased pollution and exposure to chemical agents, drug treatments, radiations, and others. Membrane lipidomics can also be used as an


indicator of the response to all these causes, as it will be seen in further sections, in order to find a very important application as a comprehensive biomarker in the screening of population health.

Mediator lipidomics has been proposed by Serham in 2005 to include the structural characterization and quantification of bioactive lipid species, in particular having low abundance, that create and propagate cell signaling [101]. In fact, mediator lipidomics is based on the follow‐up of lipid signaling with a module composed of a regulated enzyme, the bioactive lipid, and specific downstream targets. One of the most recognized pathways monitored by mediator lipidomics is inflammation [102]. Being a protective response, coming after an injury through physical damage or infection by microorganisms, acute inflammation is a cascade of highly coordinated events beginning with the production of soluble mediators by resident cells in the injured/infected tissue that promote the exudation of proteins and influx of granulocytes from the blood. Lipid mediators are mainly derived from eicosanoid precursors, such as arachidonic acid (AA), that are released from membrane phospholipids through the activity of phospholipase enzymes (predominantly PLA2). We have considered in Part 1 the lipid remodeling pathway, known as Land’s cycle, which is obviously active in inflammatory, as well as in other, stimuli received by the membrane.

In the eicosanoid production from the omega‐6 and omega‐3 fatty acid families (Figures 3.3 and 3.5), the dietary intake of the precursors LA and ALA, as well as the correspondent transformations by the Δ6, Δ5 desaturase, and elongase enzymes, are fundamental for the cascade outcomes. Therefore, dietary and life styles with increased intake of omega‐6 (see Section 3.2) have been related to the onset and progression of several diseases. This fact involves mediator lipidomics, following up the PUFA pathways, which have the roles of both pro‐ and anti‐inflammatory signaling, balancing each other’s effects. The discovery of resolvins from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) contributed to evidence that the anti‐inflammatory activity attributed to omega‐3 fatty acids could not be solely related to the simple production of the series 3 prostaglandins from EPA. In fact, omega‐3 PUFA‐derived products display a plethora of stereospecific and potent anti‐inflammatory and immunoregulatory activities, evidenced by in vitro and in vivo experiments and described in excellent reviews [50, 51]. A seminal example is offered by the omega‐6 dihomo‐gamma‐linolenic acid (DGLA), which corresponds to the crossroads of two transformations: mediators can be produced by cyclooxygenase and lipoxygenase enzymes from DGLA, but also DGLA is used by the Δ5 desaturase

MEMBRANE LIPIDOMICS 95

enzyme to yield arachidonic acid (see Figure 3.3). Mediator lipidomics helps to connect the membrane‐released fatty acids with the downstream cascade of signaling and gives important guidelines for the inflammatory process, highlighting that: (i) in the same omega‐6 pathway an accurate balance of DGLA and AA can realize the best physiological control of the pro‐inflammatory status, and (ii) PUFA fatty acids can produce both inflammatory and anti‐inflammatory mediators; therefore the balance between these pathways brings to a natural inflammatory response followed by resolution and restoration of the physiological activity. As already pointed out in Part 1, production of prostaglandin E1 from DGLA turns out to be crucial for the control of cAMP levels, thus indirectly regulating the activity of phospholipase and the release of fatty acids from membrane. Research actively investigates for new information on specific fatty acid signaling with health applications. Research progress in mediator lipidomics will comprehensively describe how the signaling network behaves in different situations. In the next chapters, mediators connected to health conditions will be described, and the reader will understand the importance of the unequivocal determination of chemical structure for a new active lipid compound to be used as biomarker in lipidomics.

5.4 MEMBRANE LIPIDOMICS: CELLULAR STRESS, TURNOVER, AND OPPORTUNITIES

In the functional vision of the cell membrane and lipidomic follow‐up, an important role is played by cellular stress, which can occur when the cell faces a higher metabolic involvement, such as in case of enhanced exposure to environmental factors (radiations, pollution, drugs, smoke) or specific health conditions (inflammation, as well as challenging conditions such as sport, pregnancy) requiring a high performance response. Such situations involve an immediate and substantial engagement of the endogenous defense systems, which are a combination of chemical and enzymatic processes controlling and neutralizing the effects of the exposure. The so‐called antioxidant network has an important role in this case, including specific molecules that are taken from the diet in an adequate quantity to prevent free radical overflow. The control systems must work at high performance and in a balanced way for ensuring support to metabolism and to sustain for the aging process. From scientific research it is clear that proper functions and satisfactory control of degradation and oxidation processes are in a delicate equilibrium, influenced


by the lifestyle and preventive measures taken by each subject. It is worth mentioning at this point that the word “stress” does not have a negative meaning in biology. Instead, as shown in Figure 5.1, depending on the dose of the solicitation and the time duration, as well as from the specific vulnerability of the organism, the stress induces an injury but at the same time an adaptation, that builds up the defense systems by which the injury becomes reversible [103]. However, the efficiency of the adaptive control takes into account many factors, including the availability and timing of the cell resources for adaptation; therefore, in case of impaired balance, the outcome could also be the progression of the injury until the cell death.

When an adequate response is obtained, stress is an opportunity for the cell, instead of being a drawback. We will not go into details of the many types of stress response, since we focus on the membrane fatty acid response. However, we must mention that the binomial protein–DNA response is an important part of the cell adaptation response, and there are many pathways involving protein activation with effects on transcription factors and gene expression [103]. One representative example is the p53 pathway, which is probably one of the most widely studied proteins for cell death and survival, including the tumorigenesis process that in many cases it is able to inhibit [104]. p53 is a transcription factor that mediates cell response to various detrimental stresses through a complex signaling network. When a cell endures a variety of insults including DNA damage, hypoxia, and oxidative stress, p53 becomes modified, which promotes both its stabilization and translocation into

Stress

Adaptation

Injury

Death

Defence

Reversible

Irreversible

Dose and duration Specific vulnerability

Figure 5.1 The cycle of stress, adaptation and the passage from injury to cell death. Reproduced with permission from Ref. [103]. © 2006 Goldring, Journal of Experimental Biology


the nucleus, where p53 activates the expression of genes that induce cell cycle regulation, DNA repair, senescence, and cell survival. It even enters in the sun exposition response. How p53 regulates metabolism is not understood, and is matter of intense research that will certainly clarify in the future this complicated scenario.

5.4.1 How Does the Stress Involve Membranes?

Cell membrane is a primary site to receive signals and stimuli from the environment, stress included. The main events of stress for membranes are:

• Biological stress, caused by the necessity of the cell to respond to a stimulus with a cascade of signaling. The most typical stress is the reactivity cascade called inflammation, with membranes involved in the Land’s cycle explained in Part 1. Here, we would like to remark that, together with lipid remodeling, which is connected with the enzymatic release of lipid mediators from phospholipids, there is an instantaneous change of the membrane properties since the lysophospholipids are hydrophilic, and leave immediately the lipid bilayer. This change is compensated with the remodeling, bringing again new phospholipids into the assembly, which should have exactly the same balance existing before the stimulus. If everything works well, the membrane properties (functioning of channels, receptors, etc.) are the same along the cell life, and homeostatic control is perfect. The problems can arise if the fatty acid pool, especially the essential fatty acid quantities and proportions among the four fatty acid families, is not adequately composed. This can derive from dietary unbalances, which are frequent in modern diets, or from impairment of the metabolic turnover of fats, at the level of absorption (involving the functioning of intestine, gallbladder, pancreas), metabolic transformations (involving mainly liver), and biodistribution (at the level of each tissue, also concerning the presence of environmental conditions such as pH, oxidative status, cofactor concentration, etc.). There are many different causes of malfunction of the lipid remodeling that can be silent for several years before manifesting a functional problem at the organ and organism levels. Therefore, health professionals must be aware of the molecular status of lipids, in order to have a complete overview of the patient’s status.


• Stress for microbial attack, which is also the natural stress our cells experience from sharing the environment with microorganisms. We cannot enter into the detail of this complex process, but an aspects must be underlined in connection with the presence of specific receptors in the surface of several cells connected with innate immunity. There receptors are able to recognize pathogen‐associated molecular patterns (PAMPs). Among them, the toll‐like receptor (TLR) family attracts a lot of interest, for its involvement in enterocyte activity to recognize the intestinal microbiota and regulate the response of inflammatory processes correlated to immune defenses [105]. TLRs are a family of receptors that recognize specific PAMPs and activate a response based on the nuclear factor NF‐kB pathway. As a consequence, the pro‐inflammatory cascade to induce cytokine and chemokine genes initiates. The family of TLR is specific for the molecules that they are able to recognize: Figure 5.2 shows

TLR1 TLR2 TLR5TLR6

TLR4

LipopeptidesLipopeptides

Flagellin LPSMannans

MyD

88

MyD

88 MyD

88 MyD

88

NF-kB activation

Multiple pathways

Figure 5.2 The TLR family at work


the different TLRs in the membranes and some events connected with NF‐kB activation. The TLR2 recognize peptidoglycan (PGN), which is a component of the cell walls of Gram+ bacteria; TLR3 recognizes viral double‐stranded RNA; TLR4 recognizes lipopolysaccaride (LPS), which is the major component of Gram− bacteria; TLR5 recognizes flagellin, a protein produced by Gram+ and Gram− bacteria. These receptors have different locations in the cells that compose a tissue, and are activated under different conditions of bacterial invasion. It is reported that the effects of fatty acids is decisive to orient TLR activity: saturated fatty acids induce activation of TLR2 and TLR4, whereas unsaturated fatty acids inhibit the TLR‐mediated signaling pathway, suppressing the NF‐kB cascade and inflammation [106]. The study of genetic variation in TLRs in various populations combined with information on infective agents suggested complex interaction between genetic variation in TLRs and environmental factors. This interaction can explain the differences in the effect of TLR polymorphisms on susceptibility to infection and autoimmune disease in various populations [107]. Work is in progress in order to have possibility of mapping these variations; however, an important role has been recognized about the regulatory effect of environmental and dietary factors. It can also be hypothesized that, with inappropriate membrane changes during remodeling, the position of receptors like TLR in the membrane bilayer could be not optimal, therefore producing an unbalance effect. It is known that a TLR‐mediated overresponse can occur in intestinal epithelial cells with the amplification in immune responses [105, 106]. A strategy based on a virtuous membrane turnover can be helpful to restore membrane balance at the molecular level, with restoration of receptor and signaling functions, taking into account that the mean half‐life of human cells like lymphocytes and intestinal cells is within few days (see Table 2.1).

• Physical or chemical stress is induced by different causes, such as radiations and free radical reactions. These causes induce changes in structural and macromolecular features, which generate the cell response. There is the tendency to attribute to this stress only a bad effect, but based on the discussions made on the oxidative pathways in Part 1, the overall evaluation of this stress must be revised again. For example, free radicals cannot be judged as “bad” after considering their involvement in oxidative transformations that generate signaling cascades of cell defense systems. Free radical


stress has become a “trendy” subject in different health conditions, from degenerative and metabolic diseases to aging, but this is also involved with some economical interests, for example, in the constantly growing market of “antioxidant” protection. The different radical reactive species can be found with acronyms like ROS (reactive oxygen species), RNS (reactive nitrogen species), RSS (reactive sulfur species), which means that the radical center (containing one unpaired electron) is located at the atom of oxygen, nitrogen, and sulfur, respectively. Radical species are very reactive, as described in Part 1, and generate products that, if not repaired or eliminated, can accumulate in cells and tissues. Actually, some of these products are measured in the body fluids by the antigen response that they are able to induce. Moreover, specific molecular modifications of the main biomolecules (lipids, DNA, proteins) are evaluated. We treated this subject in Part 1 in some detail especially concerning lipid reactivity and products.

5.5 PHOSPHOLIPIDS FROM DIETARY INTAKES TO BIOLOGICAL FUNCTIONS

In Part 1 we focused our attention on membrane phospholipids and the fundamental role they play in cell organization, signaling, and response. As shown in Figure 4.10 phospholipids can be formed from dietary triglycerides, which is actually the best and easiest way to provide these important cellular constituents. It is worth noting that a direct influence of the composition of dietary fatty acids on the final composition of membranes is observed, so that mistakes in the dietary intakes can have profound consequences on the functioning of this compartment. To complete this scenario, there are other delicate phases after food ingestion that must be taken into account for the successful incorporation of fatty acids into phospholipids. In a simplified scheme shown in Figure 5.3, lipids start to be “extracted” in the mouth and stomach that break down the food, so that small droplets of fats are formed in the chyme.

Then the droplets are attacked by lipase enzymes (mainly excreted from pancreas), which can work in two ways, creating free fatty acids and recombining them into triglycerides, as shown in Figure 5.4.

At the level of the small intestine the partially hydrolyzed lipids (also present as mono‐ and diglycerides) in the form of micelles arrive and emulsify with the bile excreted by the gallbladder, forming aggregates called chilomicrons. At this point, aggregates can be absorbed from the

PHOSPHOLIPIDS FROM DIETARY INTAKES 101

Dietary lipids(mostly triglycerides)

Physical break-up

Mouth

Stomach

Smallintestine

Intestinallining

Emulsification by bile saltsPartial hydrolysis by pancreatic lipase

Absorption

Lipid droplets in chyme

Mono-and diglyceridesin micelles

Synthesis of triglyceridespackaged into chylomicrons

for transport in blood

Figure 5.3 The fate of lipids from nutrition to digestion and absorption

R1

R2

R3

H2C

3H2O HC

OH

+

HO

HO

HOH2C OH

OH

O

O

OC

C

C

R1

R2

R3

H2C

H2C

HC

C

O

O

O

O

O

O

C

C

Figure 5.4 Hydrolysis from triglycerides to free fatty acids and glycerol and reversal triglyceride formation


intestinal cells and reach the blood vessels where they recombine to form lipoproteins, the largest lipid aggregation, as seen in Part 1. The formation of phospholipids from the dietary triglycerides is a crucial step accomplished mainly in the liver. In blood, phospholipids are present as free molecules as well as components of lipoproteins, and represent the circulating amount forming also vesicle aggregates, able to cross the cell membranes by endocytosis. It is worth mentioning that polyunsaturated fatty acids are the main component of lipoprotein lipids, so that the oxidation of these particles can give important information on the general status of the subject. The ready and the biosynthesized phospholipids present inside the cells are then recruited to form plasma membranes.

Maintaining the physiological condition of the gastrointestinal tract, including pancreas and the gallbladder, and taking care of the quality of food lipids, in order to create a balanced mixture of the fatty acid families, should be the best base for healthy status. As a consequence, when the patient manifests some problems of this tract, the physician must take into account the possibility of an unbalanced lipid management. This motivates a combined strategy for the success of pharmacological intervention, including specific molecular aid from nutritional elements to recover the lipid pool and the membrane functioning.

An important point to underline is the fates of different lipid types present in foods. In fact, triglycerides present in foods can undergo hydrolysis by lipase enzymes (Table 5.1) and start the cascade of events in

Table 5.1 Mean fatty acid percentages

Fatty acid Percent

Soybean lecithinsPalmitoyl 14.5Stearoyl 3.8Oleoyl 11.9Vaccenoyl 1.3Linoleoyl 63.5gamma‐linoleoyl 6.3

Egg lecithinPalmitoyl 32.0Stearoyl 14.1Oleoyl 27.0Vaccenoyl 1.2Linoleoyl 20.0Arachidonoyl 4.8

PHOSPHOLIPIDS FROM DIETARY INTAKES 103

digestion. On the other hand, when phospholipids are the source of fatty acids, lipase enzymes are not able to hydrolyse them, and phospholipids reach the intestinal tract intact, where they are adsorbed. Natural sources of phospholipids are lecithins, such as soybean or egg lecithins, which mainly possess the fatty acids from saturated, monounsaturated, and omega‐6 polyunsaturated families. Table 5.1 shows typical compositions of these two types of lecithins.

Lecithins are important since they can induce the consumption of the circulating triglycerides through their transformation into phospholipids. In fact, lecithins can furnish important building blocks for phospholipid synthesis, such as choline and phosphate (see Figure 4.10).

Phospholipids represent the most direct source providing precious elements for membrane assembly, overcoming possible impairments of metabolism, and favoring adequate exchange and remodeling in tissues. Recently, big industrial and pharmacological interests arose for omega‐3 phospholipid sources, and fish food is gaining more and more importance also for the contribution to this fatty acid category.

So far, we did not underline that the lipids coming from diet are very important to form membranes for another important cellular component called mitochondria, which contain the respiratory chain and are necessary for the energy production from fats. Besides phospholipids, cardiolipins are the main components of mitochondria, with a typical structure that allows the flexibility of the resulting bilayer, in order to form the numerous invaginations of these organelles. In Figure 5.5 representative mitochondria and cardiolipin structures are displayed [108].

The main fatty acid in the mitochondria cardiolipins is linoleic acid, which is the omega‐6 progenitor, also an essential fatty acid to be taken by the diet. The previously discussed problem of adequate intake, as well as the tendency to oxidative degradation in the absence of an appropriate balance with antioxidants, can affect the linoleic acid content of both plasma membranes and mitochondria; therefore the check‐up of the fatty acid status is indirectly connected to the verification of whole cell integrity. Health and disease conditions are nowadays seen also as mitochondrial dysfunctions, which are not treated in this book, and include the lipid unbalance and oxidation under cellular stress. It is worth keeping in mind the molecular requirements of mitochondria, since the pitfalls of this delicate cell compartment can also be explained by an inadequate membrane formation and organization [108].

Innermembrane

Outermembrane

Cristae

Matrix

O

OO

O

O

P

P

O

O

O

OO

O

O

O

O

O–

O–

H

H

H

OH

Figure 5.5 Mitochondria and the structure of cardiolipin containing linoleic acid as fatty acid moieties


Lipidomics of Erythrocyte Membranes

Analyzing the lipid or fatty acid composition of a biological sample cannot be considered to be lipidomic analysis, since this lipid analysis retains the “old” meaning attached to the quality and quantity of lipid components. For example, the analysis of fatty acids in plasma reports what derives from the last few weeks of dietary contribution and not an equilibration with all metabolic and functional needs of the organs and organism. Indeed, the plasma values are not stable, varying from week to week, and the data fluctuate in a very wide range. This means that it is possible to evidence only extreme changes, in excess or defect, but not representative of metabolic changes. On the other hand, lipid analysis can be directed to evidence the presence or formation of a biomarker, that is, a lipid molecule or metabolite whose level can refer to a specific health or metabolic condition, and this becomes interesting metabolic information when combined with other information regarding whole membrane fatty acid balance.

Therefore, we can define functional lipidomic analysis when referred to a cell compartment, having precise functions and properties, and to specific and stable ranges of lipids that refer to optimal conditions. The cell membrane has the proper characteristics to be the most relevant site for functional lipidomic analysis for the following reasons:

• For the cells belonging to each tissue the fatty acid values in membranes are known under physiological conditions and are established for large populations.

6

106 LIpIdOmIcs OF EryThrOcyTE mEmbrAnEs

• Altered values from normal/optimal ranges bring to consequences from the functional point of view (e.g., biophysical and biochemical changes), with a cause–effect relationship.

• The alteration also brings changes in other functions associated with membranes, such as protein functioning, which are involved in cell growth, channel activities distributing ions (i.e., calcium channels), receptor activities such as those intercepting the message from neurotransmitters, allergens, hormones, and also drugs.

The membrane compartment of a specific cell, such as erythrocyte, can be utilized to evaluate the subject status and examine homeostasis before and after a physiological or pathological event. For example, after drug administration, or stress condition, or a stabilized dietary regime, or in physiological changes such as those occurring in pregnancy, the quality and quantity of fatty acids can be affected and the analysis can reveal how they are involved in whole body management. Fatty acid composition is relevant for erythrocyte functioning, first of all, whose membrane is crucial for the exchange of oxygen or other nutrients within all human tissues. moreover, it is possible to utilize erythrocytes as reporter cells, since their fatty acid content can reveal more information on the body status: in particular, the status of essential fatty acids brought to tissues and organs through the bloodstream can be examined in the erythrocyte membranes to understand their metabolic availability. There are several papers dealing with the correlation of erythrocyte membranes with those of other tissues, such as nervous, muscle, and adipose tissues. For example, the levels of dhA in the cortex and erythrocyte [109], as well as those of muscle and erythrocyte membrane phospholipids [110], have been found to correlate in breast‐fed infants. Other fatty acid comparisons such as those of plasma, adipose tissue, and erythrocyte membranes have been reported to correlate quite well, especially in the case of pUFA omega‐3 and omega‐6, whereas no correlation was found for sex, and age was found to have some influence on pUFA levels [111]. Obviously, it must be taken into account that the uptake of different fatty acids can vary from tissue to tissue due to the differences of their characteristic composition as previously shown (see Table 2.1) and also to the nutritional and metabolic balance [112]. Actually, diet cannot be considered the only reason for a tissue to determine its fatty acid composition, whereas tissue activity is important to induce lipid remodeling. In this process the overall fatty acid balance is maintained by the intrinsic feedback because of membrane homeostasis, so that all components and signaling harmonize each other. As remarked several times, membrane

EryThrOcyTE As A cOmprEhEnsIVE hEALTh bIOmArKEr 107

functions include the activity of receptors and channels, with active and passive transports, as well as that of specialized cells—such as nervous and muscle cells—with the transmission effected by chemical compounds and electrical impulses, which are all “membrane phenomena”.

homeostasis, which can be evaluated by membrane lipidomics, is an important piece of an immense puzzle, which is cell metabolism. Information on the membrane assembly and its homeostatic maintenance provides an objective parameter, which is very useful for evaluating stress or the low quality of life and nutrition of individuals by a scientifically validated approach.

6.1 EryThrOcyTE As A cOmprEhEnsIVE hEALTh bIOmArKEr

Erythrocyte membranes are the crucial site for fatty acid evaluation as previously explained. blood is composed of cells (erythrocytes, lymphocytes, and platelets) suspended in a liquid (blood plasma or serum), and lipids are present in both parts. plasma is still an important site for lipid characterization, and a survey of the most relevant lipid species was published recently [113]. It is worth recalling that all lipid species present in plasma are “circulating,” which means that they must be transferred to the cells and their bioavailability in plasma does not mean that they are present in tissues directly. moreover, lipids taken from the diet, which are mainly triglycerides and phospholipids, circulate in blood plasma free or organized in transport vesicles (chilomicrons and lipoproteins), and the daily intake strongly influences the fatty acid levels in this part of the body. normal or optimal interval values are difficult to express for each fatty acid type, and plasma levels are still determined for scientific scopes, which are meaningful only if the dietary regime of the subjects is well known by researchers. plasma can be interesting for the evaluation of specific fatty acid markers, such as, for example, the cis and trans fatty acids present in plasma cholesteryl esters [35, 93], and also for comparative studies between membrane and plasma fatty acid compositions in order to map the metabolic capabilities of each subject. All these applications are under investigation and in future satisfactory mapping of the blood lipids and their exchanges is expected.

The erythrocyte cell membrane phospholipids are indeed the most interesting as readily available and very descriptive molecules for the


individual metabolism. As a matter of fact, the choice of the membrane, as an ideal observation point for fatty acids, depends on the following:

1. The composition must be representative of the general condition of the tissues and the whole organism, and of changes occurring under specific metabolic conditions, but not dependent on short‐term dietary changes.

2. noninvasive techniques and cheap methodologies should be utilized for isolating and analyzing these membranes.

3. The fatty acid types should be very representative of all families (saturated, monounsaturated, omega‐6 and omega‐3) in order to give more information on the nutritional and life status, as well as on the metabolic steps, influenced by the individual condition.

4. The evolution of the fatty acid status, following a stabilized diet or a therapeutic intervention, must be examinable in standard periods, not excessively long.

All these considerations point to blood as the body tissue to examine, and to erythrocytes as the most appropriate to apply the lipidomic approach. Among the blood cells, erythrocytes have the ideal lifetime of 120 days (see Table 3.1), compared to short‐lived lymphocytes (few hours–2 days) and platelets (8–10 days). due to the corresponding number of cells per milliliter of blood, erythrocytes represent without any doubt the high probability compartment for fatty acid distribution among blood cells, compared to the less numerous lymphocytes and platelets, which can be examined for the fatty acid composition regarding their specialized functions, but certainly are less representative of body functionality in the whole.

Another point of discussion is the possibility to use whole blood, without separating cells from plasma. some efforts examining this possibility are understandable, due to the easy collection of blood drops and fast workup without any need of centrifugation and separation steps. Therefore again the fatty acid percentages in plasma, erythrocyte membranes, and whole blood were examined in order to understand the differences in the information from these different sources [114]. Obviously, the fatty acid structures found in whole blood matches those composing membranes, and it was concluded that for estimating the immediate dietary intakes, whole blood can be used as plasma is used. In this case, a drop of plasma and a drop of blood are almost equivalent. On the other hand, different contents and proportions of fatty acid in membrane phospholipids were evident and, when whole blood is considered, the


content of the plasma “dilutes” the information contained in the membranes. Another important point to be taken into consideration is that erythrocyte membranes can change during cell life and, due to the lifetime of 120 days, in the blood the cells can be present with different ages. Therefore, membrane analysis must be realized with cells of approximately the same age, in order to have a homogeneous observational point of view for all individuals. In this respect, the mature erythrocyte can be chosen, which contains several pieces of information on the senescence progress, exposure to oxidative stress, together with the metabolic and nutritional effects produced in the individual during a reasonably long time window [115]. The senescent red blood cells have different parameters compared to younger cells, and are separable by density differentiation. In this way the information of membrane lipidomics becomes extremely precise and personalized, and the erythrocyte become a real global health marker. The methodology can be automated and, when applied to the Italian population, gave the possibility to determine the normal/optimal interval values, which will be detailed in the next sections.

blood withdrawal (0.5 mL) can be done by a syringe from the antecubital vein or by puncturing a finger followed by blood drop collection to reach the same volume (about 20 blood drops). The anticoagulant in blood is ethylenediamminotetracetate (EdTA), which acts by complexing the ions present in the sample. In this way some enzymes cannot function anymore, especially phospholipase enzymes that need calcium ions, and the membrane phospholipids cannot change after blood withdrawal. The stability of the blood sample is quite good, with the possibility of staying at room temperature for 24–48 h without suffering from any change, whereas by refrigeration at 4°c the sample can be stored for some weeks.

The erythrocyte membrane composition is the combined result of biosynthetic capabilities with the adequate presence of cofactors for enzymatic and hepatic activities (i.e., fatty acid and phospholipid synthesis and transformations), stabilized dietary regime, together with genetic factors, free radical balance, and cellular exchanges. The mature erythrocyte cannot biosynthesize lipids; therefore its membrane depends on the exchanges that occur in vivo with the circulating lipoproteins and tissues. moreover, being formed by all the families of fatty acids, the erythrocyte membrane contains fatty acids to become mediators and signaling molecules (for inflammatory and immune processes, etc.) and structural fatty acids, which allow the exchange activity of red blood cells. In particular, essential fatty acid moieties are very important in the


red blood cell membrane phospholipids. Arachidonic acid is naturally deposited in high quantities in the red blood cell membranes as a necessary mediator for all tissues, whereas brain tissues receive the polyunsaturated fatty acid components from the blood circulation, since they cannot biosynthesize double bond in the absence of desaturase enzymes.

The erythrocyte membrane has been thoroughly studied for its properties. starting from the individual composition of this membrane, changes can be observed on the mature erythrocyte fraction only if a stable solicitation or condition is applied. some states, such as starvation, traumatic events, drug treatment, can also produce membrane changes in the short term; however, in the majority of cases the changes are visible only if the condition is applied constantly for 3–4 months. The balance among the families of saturated, monounsaturated, and polyunsaturated fatty acids (sFA, mUFA, pUFA) is necessary for the functions of tissue oxygenation and nutrition. Therefore, selection of the best membrane composition is a natural process, which obviously depends on the bioavailability of the fatty acids in the individual.

since the number of red blood cells is much higher than the number of other blood cells, it is also a probabilistic factor, influencing the final membrane composition. Therefore, by examining red blood cells it is possible to have information on a representative fraction of cells, which become “reporter” of a general condition, instead of their own functions such as lymphocytes and platelets. Obviously, fatty acid levels in the erythrocyte membranes usually also reflect in other blood cell conditions.

For all these reasons, the mature erythrocyte is the best cell to observe and its selection was initially performed manually, after applying centrifugations steps to separate plasma and to stratify the cell layers. Applying selection criteria it was evident that a crucial amelioration of erythrocyte fatty acid analysis was obtained, since without selection the membrane fatty acid is isolated randomly from the mix of differently aged cells and the analysis of aliquots of the same blood sample can vary consistently. The methodology is now available on automated devices, and hopefully this approach will help in aligning the workup of blood and the results in all laboratories.

Let’s summarize the information on fatty acid families obtained from erythrocyte membrane lipidomics:

1. saturated and monounsaturated fatty acids and their ratio (sFA/mUFA): these fatty acids are both biosynthesized by the human body and introduced through the diet. Therefore their ratio can


indicate the equilibrium of these components with opposite effects: saturated components give a more rigid packing of the membrane, whereas monounsaturated fatty acids possess the bent structure with a fluid effect on the membrane assembly. The increase of saturated components can derive from an increase of biosynthesis or of dietary intake, and lipidomics can provide precise information of the individual situation. For example, in the activation of biosynthetic pathways, the role of palmitoleic acid (9cis‐16 : 1, see Figure 2.3) is crucial since it cannot derive principally from the diet, but its level is due to the activation of delta‐9 desaturase enzyme. In the monitoring of the individual management of the transformation of palmitic to palmitoleic acid, lipidomics can be of help to envisage the factors of the saturated fatty acid management of each subject. moreover, it can monitor which effects of a dietary regime or pharmacological intervention are connected with the sFA pathway, in terms of decreasing or increasing effects. The time frame of 4 months, given by the lifetime of the erythrocyte, is ideal to expect the metabolic change of the individual. It is worth mentioning that so far the lipid monitoring in routine checkup analysis concerns only the plasma levels of cholesterol and triglycerides, whereas no attention is given to the membranes and fatty acid composition. This is an unjustified limitation given the advancement in knowledge and techniques, which renders this information very easy to obtain. physicians and nutritionists must be aware that an important part of the metabolic outcome of the subjects depends on the type of fatty acids constituting their lipid pool and membranes, as detailed in part 1. moreover, taking into account the actual health recommendation on the saturated fatty acid risk for health, it appears really odd that this risk factor is not evaluated for each individual, also as prevention plan, whereas the estimation of palmitic acid, palmitoleic acid, and sFA/mUFA ratio would allow the metabolic pathway to be followed up.

2. polyunsaturated fatty acids omega‐6 and omega‐3 and their ratio (omega‐6/omega‐3) present in the mature erythrocyte membranes: as previously said, these essential fatty acids (EFA) must be taken from the diet as their corresponding precursors (linoleic and alpha‐linolenic acids); therefore any decrease in their levels can be due principally to inadequate dietary intake. This information is of crucial importance in the clinical evaluation of the patient’s symptoms, since any diagnosis cannot disregard the loss of essential factors for tissue functionality. As a comparison, although trivial,


we can mention the absence of vitamin c intake in humans. before discovering the vitamin c essentiality, the symptoms that include psychological and physical signs were taken as for other diseases. many times in medical reports over the centuries it is possible to envisage how the dietary requirements of essential vitamins were forgotten in the clinical evaluation. In twenty‐first‐century medical practice, especially considering the advancements in molecular medicine, such inattention cannot be allowed, as it affects therapeutic success and also the confidence of patients as to how up to date their health operators are, when compared with even simple information available on the Internet. The levels of omega‐6 and omega‐3 fatty acids in the individuals cannot be expected and predictable. The analysis of omega‐3 and omega‐6 fatty acids in the plasma can be taken as summary indication of the dietary intake, but not as a precise indication of metabolic use and transformation into membrane phospholipids for structural and signaling roles. In all cases, the analysis of pUFA and the evaluation of their lipidomic balance, according to their metabolic transformation (see Figures 3.1 and 3.2), can afford precious information for health evaluation, such as the effects of compromised tissues by EFA deprivation. Another important factor is the equilibrated intake between omega‐6 and omega‐3 fatty acids, which is needed for the balance of the metabolic effects and functions. In epidemiological investigations regarding the increased number of diseases, such as allergy, including dermatology and respiratory pathologies, the role of unbalanced omega‐6/omega‐3 ratio as the most probable cause has been evidenced. recently, also in the cardiovascular area, omega‐3 (percentages of EpA + dhA) in the erythrocyte membranes, denominated as “omega‐3 index,” has been proposed as a risk factor [47]. An overview of main health conditions and fatty acid relevance is provided in chapter 4. pUFA evaluation is therefore necessary to assess the clinical status of the patients, since the loss of these components can cause health problems in a wide range of tissues and functionalities.

3. The third element of lipidomic analysis, connected to the two previous ones, is the level of arachidonic acid, which is well represented in the erythrocyte membrane (about 15% of the total fatty acid composition), and is released from the red blood cell membranes on metabolic demand. The level of this important fatty acid is crucial to define the “silent inflammation” status of the patients. This concept concerns a silent condition of higher levels of the


mediator precursor arachidonic acid, which is consequently released in higher percentages upon cells being exposed to stimuli. In practice, if there is an exceeding level of arachidonic acid in the cells, upon stimulus the quantity of this fatty acid released in the cell compartment and producing the corresponding prostaglandins and mediators will also be high, causing an “amplification” of the response. On this basis it is clear that the functional levels of arachidonic acid established in membrane phospholipids provide a direct measure of the cell response capabilities by release and subsequent mediator production. The level of arachidonic acid in cell membranes, which can first affect the quality of life and then influence health condition in the subjects, was never assayed in large populations. It is worth noting that the level of arachidonic acid, determined either in plasma or in whole blood, is not an indicator of a silent inflammation or pro‐inflammatory status, being referred to the “circulating” fatty acid and not directly to the membrane‐bound fatty acid. Obviously, the individual condition of the patient must be taken into account: inflammation can be produced as a protective or metabolic response under several physiological conditions that require cell activation, such as in pregnancy and sportive activity. In other cases it can be influenced by drug consumption. Under these different situations, the arachidonic acid value is important to predict the role of mediator and signaling molecules. In part 1 also the role of its precursor, dihomogammalinolenic acid (dGLA), has been detailed, since it plays a relevant role as feedback in the omega‐6 cascade [40]. Finally, the transformation of arachidonic acid into its corresponding trans isomers (Figures 4.4 and 4.5) is also an important indicator of the exposure of this pUFA to free radicals, with loss of its natural geometry. All these issues can be addressed with a lipidomic analysis targeted toward erythrocyte membranes, which is then expected to become a necessary tool for clinical profiling.

The efficacy of the lipidomic analysis is also connected to a very good procedural and analytical methodology: the first step is to collect the patient’s anamnesis and food diary, for the data of the individual condition as a key to the metabolic and dietary interpretation of the found values. The individual history becomes connected to the clinical evaluation and correlated with the molecular asset of cell membranes. It must be underlined that lipidomic profiling does not have a diagnostic value yet, and large population studies must be undertaken to allow


such utilization, by collecting lipidomic profiles and elaborating the data using bioinformatics facilities. The correlation between the membrane asset and the metabolic functioning of an apparatus or organ started to be studied and reported in the literature of the last 20 years. As repeatedly underlined, the need for essential fatty acids and membrane balance of all fatty acid components must be taken into account in clinical evaluation. molecular medicine has to develop tools in order to use the molecular inventory of humans for a modern classification of the pathologies. It is worth underlining that the lipid inventory has an important and additional advantage compared to other biomolecules (dnA, proteins, carbohydrates) in living organisms, regarding the intervention strategy that can be performed mostly with the use of natural components from the diet and natural cofactors for the biosynthesis, chosen in a personalized way. It is now time to introduce this methodology in clinical practice. Also, in case of disease conditions, lipidomics can adjuvate therapies with fatty acids and cofactors, that ensure the equilibrium of the membrane assembly, and help the pharmacological treatment decided by the physician.

In various life stages, from birth to aging, lipidomics can indicate the outcome of metabolic and environmental conditions in each individual, correlating it with the quality of life. since all stimuli and signaling involve membranes as a crucial reactive site, the maintenance of homeostatic conditions according to lipidomics constitutes a useful resource for facing sudden events during life.

The cell membrane also exists when its fatty acid balance is imperfect, and at the stage of small unbalances the situation can be silent. For example, the membrane can accumulate arachidonic acid out of its optimal interval values, therefore determining an intrinsic “silent” pro‐inflammatory condition, as previously said. At the same time, membranes can be silently depleted of important constituents, for example, the retina be depleted of dhA, its most important constituent reaching 20% of the total fatty acid composition. how much loss and for how much time the fatty acid impairment can exist in a tissue without altering its basal activities have not been evaluated as yet. From lipidomic practice it can be said that the unbalance is associated with diseases, whereas it can also be associated with “silent” conditions without pathologies. With lipidomic monitoring, applied to large populations, it will be possible to establish the relationship between membrane fatty acid status and health conditions, going from bench to bedside with innovative nutritional strategies to improve health status. As a matter of fact, lipidome monitoring can be used for prevention, maintaining the efficiency and completeness of the fatty acid pools in the subject. In this case an annual checkup will be required. The

ThE OpTImAL VALUE InTErVALs And ThE mEmbrAnE 115

consequence of this approach to the market for supplements and functional foods would be to create a virtuous cycle, undoing the damage caused by the terrible confusion and simplification that are actually present. consumers’ needs are not addressed for their precise requirements and the majority of the health professionals are not able to use the innovative personalized tools at their best. Among others, lipidomics is the best method so far, since it is based on reliable and well defined basis, putting also nutrition at the center of the medical approach, as has also been historically proposed since hippocrates’ time, with a modern and personalized vision applying by the latest advancements in molecular medicine.

6.2 ThE OpTImAL VALUE InTErVALs And ThE mEmbrAnE UnbALAncE IndEX

Lipidomics of erythrocyte membranes attracts a lot of interest in research. The lipid and fatty acid organization of erythrocyte membranes reveals many applications spanning from nutritional to metabolic and immunological fields. Even the detailed fatty acid composition is a matter of further insight, as demonstrated by the research in the identification of geometrical trans arachidonic acid isomers and hexadecenoic positional and geometrical isomers [11, 35, 116]. starting from the consideration that membranes are compartments made of phospholipids at their critical aggregation concentration, we can estimate an almost fixed, or “close”, number of fatty acids as constituents. This is a basic consideration, since the morphology of each cell is kept by the organization of membrane lipid constituents, and the number of entities of this organization constitutes a well‐defined cluster. Therefore, some work was carried out to rationalize the basic fatty acid cluster in erythrocytes, i.e., the meaningful fatty acid cohort to satisfactory describe the membrane homeostasis and biological activities, which could be then expressed as percentage of each fatty acid composing the cluster (expressed as % rel). The examination of erythrocyte membrane fatty acid analyses reported in the literature gave a clear‐cut indication of this cluster, where all fatty acid families are represented. The total number of fatty acids is 12, including cis and trans fatty acids, the latter recognized by synthetic libraries of geometrical trans isomers as described in the last decade of research (see chapter 4). In Table 6.1 the optimal interval values of the 12 fatty acids are shown, as evaluated by a survey among literature values plus the values gathered by the Italian population database of about 5000 analyses [59]. It must also be said that these interval values have been determined also for the mature erythrocytes separated by an automated


procedure of cell selection, and so far the validity of these interval values has been tested satisfactorily as reported recently in the literature [59].

Table 6.1 presents the following fatty acid components:

• saturated fatty acids as palmitic and stearic acids.• monounsaturated fatty acids as palmitoleic, oleic, and vaccenic

acids.• Omega‐6 polyunsaturated fatty acids as linoleic acid, dihomogam

malinolenic acid, and arachidonic acid.• Omega‐3 fatty acids as eicosapentaenoic and docosahexaenoic

acids. The content of the dietary omega‐3 precursor alpha‐linolenic acid in the erythrocyte membranes is reported to be as low as <0.1%; therefore it was decided not to represent it in the cluster.

• The geometrical trans fatty acids as the values of elaidic acid (9trans‐18 : 1) and arachidonic acid mono‐trans isomers (trans‐20 : 4, see Figure 4.5), in latter case identified with appropriate synthetic libraries that are available only in a few laboratories [116]. In chapter 4 a thorough treatment of these isomers has been made.

Table 6.1 mature Erythrocyte membrane fatty acid interval values of the Italian population composing the reference fatty acid cluster for lipidomic analysis

FA residues Acronym normal values

c16:0 17–27c16:1‐∆9 0.2–0.5c18:0 13–20c18:1‐∆9 9–18c18:1‐∆11 0.7–1.3c18:2‐∆9, 12 LA 9–16c20:3‐∆8, 11, 14 dGLA 1.9–2.4c20:4‐∆5, 8, 11, 14 AA 13–17c20:5‐∆5, 8, 11, 14, 17 EpA 0.5–0.9c22:6‐∆4, 7, 10, 13, 16, 19 dhA 5–7Total saturated FA sFA 30–45Total mUFA mUFA 13–23Total pUFA pUFA 28–39sFA/mUFA 1.7–2omega‐6/omega‐3 3.5–5.5sum of mono‐trans trans ≤0.4

LA, linoleic acid; AA, arachidonic acid; dGLA, dihomo‐gamma‐linolenic acid; dhA, docosahexaenoic acid; EpA, eicosapentaenoic acid; FA, fatty acid; mUFA, monounsaturated fatty acid; pUFA, polyunsaturated fatty acid; sFA, saturated fatty acid.


A graphical representation of the optimal interval values and the linear profile of the found values can be used to communicate with the patient, showing at which points his/her condition is not aligned with the normality, thus motivating one to recover the personal situation (Figure 6.1).

based on the lipidomic profile, the correspondent nutrilipidomic strategy that targets the unbalance with a personalized nutritional suggestion and a cycle of nutraceutical supplementation can be designed. In fact, the use of nutraceuticals is defined in a cycle of 4 months, in particular executing only 3 weeks per month. It is worth noting that the individuation of specific nutritional elements and their adequate administration form as nutraceuticals are of crucial importance to induce the first important changes of the membrane asset, which can be much slower to obtain only with the diet. As explained in part 1, the target of the intervention is to recover the fatty acid pool of the individual, making available the needed fatty acid types and quantities, so that the natural lipid remodeling and choice for the new cells can be effected by the organisms, without restrictions due to dietary or metabolic conditions.

16:0

0 25 50 75 0 25 50 75 0 25 50 75

18:0 16:1 18:1(o) 18:1(v) 18:2

Membrane unbalance index (MUI)

Percentage and arrow show patient membrane unbalance

Slight unbalance Medium unbalance Strong unbalance

MUI:15.15%

20:4 20:3 20:5 22:6 18:1(t) 20:4(t)

Figure 6.1 Graphic representation of the lipidomic profile, compared to the optimal values (green area), and of the membrane Unbalance Index coloured scale


The supply of the fatty acid elements, together with appropriate cofactors and adjuvants, to protect their bioavailability and transformations, can induce the changes of membranes forming all tissues in the organisms. A profound health benefit can be expected by this strategy and the 4–6 months period after the blood test is important to determine the extent of the individual response. The protocol established for the lipidomic approach in health care was transferred as innovation some years ago in the Italian market and involved a group of health professionals as early adopters, also organizing formative courses about molecular medicine. The aim in the future is to involve other countries with a common methodological and laboratory platform, in order to unify the analytical workup condition, as well as to abandon odd methodologies, which are not correctly set up for obtaining good results. This effort will avoid discrepancies in the market for lipidomics, protecting customers for providing them the best scientific supported analysis and advancing science by gathering data from population.

It is worth noting that in the lipidomic approach the blood test allows for the personalization of the dietary and nutraceutical intervention for each individual. Gathering the data of patients and their analyses, another important achievement of this approach is the buildup of a database, where the results can be grouped by pathologies or physiological status, dietary habits, age, and so on, providing the possibility to map lipidomics characteristics and obtain significant indications leading to specific profiles connected with the health conditions. The preliminary results obtained from the database organized for the Italian subjects are very promising, and the expertise gained so far oriented the idea of this book to describe how useful lipidomics can be for profiling humans in various health conditions. As a general observation, lipidomic profiles represent a palette of lipid assets and can be obtained through a large population screening. moreover, membrane recovery can be addressed by the lipidomic approach, targeting the molecular compartment of the membrane for its optimal balance. Indeed, in the majority of the supplementation reported in the scientific literature no personalized need of the patients’ groups is taken into account, and often only one type of fatty acid at high dosage is used, without even taking into concern the type of pharmaceutical form (oil or capsule). As fully explained in this book, to achieve the membrane balance is a more complex task than the supplementation of a single fatty acid or family, and the lipidomic approach is the only one to show clearly which synergy has to be applied to recover functionality of this important cell compartment.

In 2008 the idea of utilizing the data of the above reported fatty acid cluster describing membrane composition to calculate a global factor of


“membrane balance” was born. In this calculation the percentages of the fatty acids in the cluster formed by 12 fatty acids present in the mature erythrocyte membrane analysis are used, and a factor is added to their “metabolic” significance. The mathematic algorithm calculates the individual membrane composition when it finds that the subject does not have the proper ranges, in comparison with the “ideal” membrane where all the found values are within the optimal range, and then obtains a score (in percentage) expressing the “distance” between the ideal membrane (having all normal fatty acid values) and the membrane of the subject. The algorithm is a European patent and the number is reported as the membrane Unbalance Index, mUI, which gives a comprehensive measurement of the erythrocyte membrane discomfort [117]. In this calculation fatty acids are “weighted” for their biological and metabolic importance, and the mUI is as higher as the significance of the fatty acids involved in the unbalance. For example, if the membrane unbalance of a subject concerns the percentage of arachidonic acid, the corresponding mUI will be higher compared to another individual with the unbalance of oleic acid, since the metabolic significance of arachidonic acid pertains biologically more relevant processes, such as inflammation.

The mUI number is a percentage from 0 to 100, which indicates the percentage of unbalance compared to the ideally balanced membrane. If the value is close to 0% it means that the fatty acid values have a small deviation from the optimal values, whereas if there is more than one metabolically significant fatty acid departing from the optimal values, the mUI will have a higher percentage values.

Figure 6.1 reports an example of the mUI output as a scale with three colors, indicating the mild, medium, and strong membrane unbalance regions, which are mainly connected to the quality of the fatty acids found in nonoptimal ranges. In fact, when saturated fatty acids, omega‐6 fatty acids, such as arachidonic acid, the sFA/mUFA and omega‐6/omega‐3 ratios, are found unbalanced, the position in the colored region takes into account that they are health risk factors and goes to the redcolored “strong” unbalance. Analogously, the yellow area indicates the unbalance of biologically important fatty acids, still not considered as health risk (e.g., excess or defects of omega‐3 fatty acids). Further cases of unbalanced values for fatty acids not included as risk factor (i.e., oleic acid) are located in the green area. The software is kept updated with the scientific advancements for the significance of fatty acids and for the optimal ranges of fatty acids in the population. In fact, using the normal distribution of the erythrocyte membrane values found in the population of individuals examined by lipidomic analysis, the optimal


interval values are constantly verified. In this respect, the enlargement of the lipidomic analysis to European and international cohorts will allow to expand the database, constantly referring to the normal distribution of fatty acid ranges.

It is worth underlining that when the mUI index is in the strong unbalance zone (red) this may not be only in case of a pathologic status, but it can also occur under physiological conditions. The saturated fatty acid excess present in a healthy subject is a preventive risk factor for cardiovascular, neurologic, and metabolic diseases; also, arachidonic acid is an indicator of an inflammatory predisposition, not only of an inflammatory process. For example, the frequency by which arachidonic acid is found in excess in a cohort of Italian population is lower in healthy people than in illaffected subjects (see next section, Figure 8.1). This underlines the necessity of a strategy to recover the optimal values, which means prevention in a healthy subject. The visualization of the strong unbalance as a red zone is useful for the patient to understand that he/she has to cooperate in order to recover his/her situation. In all cases, the membrane unbalance can be considered a “temporary” condition, in the sense that the unbalance can be recovered by a nutritional/nutraceutical intervention using the remodeling and turnover processes as natural cellular events to reshape the membrane composition.

The comparison of the mUI index before and after the intervention will show the progress of the individual.

6.3 LIpId bIOsynThEsIs And rELATEd IndIcEs

The values of the 12 fatty acids can be useful to evaluate the functioning of the corresponding lipid biosynthetic pathways of saturated, monounsaturated, omega‐6, and omega‐3 in each individual (see Figures 2.3, 3.1, and 3.2). In particular, as shown in the Table 6.2:

• The sFA/mUFA ratio indicates the balance between saturated and monounsaturated fatty acids, which indirectly expresses the increase of saturated fatty acids possibly due to dietary factors or to a variety of metabolic and enzymatic impairments (section 2.2). This index is connected to fatty acid synthase and elongase enzymatic activities to synthesize palmitic and stearic acids. It also gives an indirect indication of the ability of the body to convert the “rigid” structure of the sFA into a more flexible and fluid mUFA moiety, which is needed for membrane functionality. consequently, with this excess ratio the membranes tend to be more rigid. The increase of this

LIpId bIOsynThEsIs And rELATEd IndIcEs 121

ratio and the overpassing of the maximum threshold (>2) are in connection with the functioning of tissues, such as adipose and liver tissues, both of which suffer because of the accumulation of saturated fatty acids.

• The omega‐6/omega‐3 ratio represents the balance of these two pUFA families starting from the dietary contributions since both fatty acids are essential. Their ratio also gives a direct estimation of the metabolic transformations that lead to the incorporation of mediator precursors in the membrane phospholipids, such as inflammatory and anti‐inflammatory eicosanoids. The presence of values higher than the optimal range (3.5–5.5) indicates a pro‐inflammatory condition for the omega‐6 prevalence, which in our modern times can derive from the diet. Also, a diminution of omega‐3 can produce a higher omega‐6/omega‐3 ratio. It is worth mentioning that this index is often obtained from plasma analysis, for estimation of dietary contribution; however, the meaning of the ratio calculated in the membrane is not only connected to the dietary contribution, but it also gives a more interesting metabolic indication of how membranes are ready to produce the signaling after the stimulus. In fact, it should not be forgotten that pUFA transformations into their corresponding eicosanoids and other signaling molecules always occur after detachment from membrane phospholipids (chapter 3).

Table 6.2 The seven indices of lipid biosynthesis

Lipid biosynthesis index Optimal values*

sFA/mUFA 1.7–2.0

Omega‐6/omega‐3 3.5–5.5

Omega‐3 cardiovascular risk index0–4% high risk4–8% intermediate risk>8% minimal risk

delta‐9 desaturase index 18 : 0/9c‐18 : 1 <0.7 hyperactivity>1.3 hypoactivity

delta‐9 desaturase index 16 : 0/9c‐16 : 1 <45 hyperactivity>132 hypoactivity

delta‐6 desaturase index + elongase 18 : 2/20 : 3 <5 hyperactivity>8 hypoactivity

delta‐5 desaturase index 20 : 4/20 : 3 <6 hypoactivity>9 hyperactivity

*derived from the values in Table 6.1


• The omega‐3 risk index is obtained by the sum of EpA and dhA (omega‐3) percentages in the membrane, and this is an indicator of the presence of precious elements for the functioning of tissues, such as those of the cardiovascular system (see section 3c).

• Index of delta‐9 desaturase for the transformation of palmitic acid (16 : 0) to palmitoleic acid (9cis‐16 : 1) and index of delta‐9 desaturase for the transformation of stearic acid (18 : 0) to oleic acid (9cis‐18 : 1); these are important pathways regulated with the activity of the corresponding genes (scd) that can have different expressions and this subject has also been treated in chapter 2.

• Index of delta‐6 desaturase and elongase for the transformation of linoleic acid (9cis,12cis‐18 : 2) to dihomogammalinolenic acid (dGLA, 8cis,11cis,14cis‐20 : 3). This index can have also an influence on the omega3 pathway (EpA and dhA biosynthesis).

• Index of delta‐5 desaturase calculated for the transformation of dGLA into arachidonic acid (5cis, 8cis,11cis,14cis‐20 : 4).

consulting these indices together with the single levels of fatty acids, health professionals can have an overview of the individual status reflected in the membrane compartment, using erythrocytes as the global functional reporter. moreover, since membranes are in continuous lipid exchange and can be modified by environmental and stable dietary conditions, the findings of unbalances are not permanent and can be oriented to reach a better balance through natural and nutritional intervention.

6.4 ThE IndIVIdUATIOn OF mOLEcULAr IndIcATOrs

In using lipidomic analysis for prevention, fatty acid analysis can be expressed as molecular indicators, taking into account their main metabolic role or function. In this way six molecular indicators can be individuated (Table 6.3):

• Metabolic slowness, given by the sFA/mUFA ratio and palmitoleic acid level. Indeed, a signal of metabolic slowness is the increase of saturated fatty acids in membranes and their transformation to monounsaturated fatty acids. As previously explained, the sFA/mUFA ratio becomes a comprehensive and effective indicator of the sFA intakes and transformation, envisaging dietary mistakes or enzymatic (desaturase) impairment, associated also to the insulin response to foods and the capability of the body to make a

ThE IndIVIdUATIOn OF mOLEcULAr IndIcATOrs 123

satisfactory lipid turnover by the liver functions, without accumulation. Together with the sFA/mUFA ratio, in this indicator the level of palmitoleic acid is included, which derives from palmitic acid by the delta‐9 desaturase activity. This fatty acid does not come prevalently from the diet; therefore it is a direct indication of enzymatic activity, and is accentuated when there is a surplus of palmitic acid, either for the diet or for biosynthesis. The value of this fatty acid is important to evidence the feedback for “activation” of the desaturase pathway, which occurs as a response of the saturated fatty acid increase, or to evidence the absence of this feedback that is correlated to factors that have been recalled several times in the sFA pathway, among others: the absence of enzymatic cofactors, the inhibition of desaturase activity, liver impairment.

• Cellular defense and immunity indicator, given by two fatty acids values of the omega‐6 and omega‐3 pathways: dGLA and EpA. The involvement of these two molecules in cellular defense has been evidenced in part 1, also for the production of signaling molecules that triggers the response and helps the recovery of inflammatory processes. In general, if these values are defective, they call attention to both diet and impaired metabolism, which can also involve intestinal health. A crucial role has also been evidenced for dGLA in the control of proliferative processes of tumor cells; therefore the terms “defense” must be intended in a wide sense [118]. On the other hand, an excess of dGLA can indicate an increased production of fatty acids and mediators for inflammatory, immune, and defense processes, with loss of control from omega‐3 to balance omega‐6 excess.

• Silent inflammation, which involves arachidonic acid and the omega‐6/omega‐3 ratio. both values are necessary information to estimate the degree of cellular reactivity due to the individual environmental and metabolic situations. Indeed, as recalled several times, an omega‐6/omega‐3 ratio exceeding the optimal range (3.5–5.5) in membrane phospholipids can give information on the pro‐inflammatory balance mainly due to the diet, whereas the value of arachidonic acid is a more direct connection with cellular response. The increase of this fatty acid in membranes can indicate the existence of a cellular stimulation to produce proliferative mediators or the pro‐inflammatory effect of a diet rich in omega‐6 or poor in omega‐3 intakes.

• Cardio indicator is formed by the omega‐3 cardiovascular risk index and the total saturated fatty acid value (total sFA). In fact, a reduced content of omega‐3 coupled with the high sFA content in the membranes can really represent a risk for the health of the


cardiovascular system. It is obvious that the omega‐3 value does not have any significance if the subject is having an omega‐3 supplementation.

• Neuro indicator is given by the value of dhA, which is the principal component of the central nervous system, nerve, and retina tissues. This is an important value to consider when it is defective, since dhA governs important functionalities of the earlier‐mentioned tissues and a long‐term deficiency must be avoided.

• Radical stress is given by the values of trans isomers of 18 : 1 and 20 : 4. As previously described, these trans isomers are present in low quantities in membranes, and up to a total 0.4% they can be considered not harmful but derived from the natural response to stress. It can be expected that low levels of these fatty acids can induce a different membrane organization and trigger a defense response. On the other hand, when the isomers are present at borderline or higher percentages, their effect can be more substantial as described in part 1. Therefore, for prevention the value of trans isomers in membrane phospholipids must be kept under the threshold of 0.4%, a target that can be easily reached in three ways:

Table 6.3 The output of molecular indicators for preventive panels of membrane lipidomics

molecular indicators Optimal values

Metabolic fatiguesFA/mUFA 1.7–2.0palmitoleic acid 0.2–0.5

Cardio indicator

Total sFA 30–450–4% high risk

Omega‐3 cardiovascular risk index 4–8% intermediate risk>8% minimal risk

Cellular defense/immunitydGLA (omega‐6) 1.9–2.4EpA (omega‐3) 0.5–0.9

Silent inflammationArachidonic acid 13–17Omega‐6/omega‐3 3.5–5.5

Neuro indicatordhA 5–7

Radical stressTrans‐18 : 1 0.1–0.3Trans‐20 : 4 0.1–0.4

ThE IndIVIdUATIOn OF mOLEcULAr IndIcATOrs 125

the use of only cis fatty acids in the diet, a successful control of free radical activity, and no consumption of trans‐containing foods.

The output of molecular indicators is given in Table 6.3. It is clear that from the 12 fatty acid cohort, the total fatty acid families, and 7 indices of lipid biosynthesis of the previously shown erythrocyte membrane profile, the molecular indicators are reduced to 7 fatty acids, 3 indices, and 1 total fatty acid family (sFA). It is worth noting that this simplified panel of indicators can be useful as fast prevention screening in a population study. It allows also for the individuation of a “first aid” nutraceutical supplementation, which is personalized to the individual needs. such panel works well in the absence of pathological status, allowing important deficit or excess to be discovered before becoming a health issue.


Nutrilipidomics

The research in the postgenomic era and epigenetics have shown that cellular response is regulated by molecular processes and by the environ-ment, which are crucial to determine the whole organ and organism response, and also that there is an individual response based on the status of the subject. The individuation of membranes as the preferred point of evaluation of the lipid content and effects, as well as their rela-tionship with the other molecular components, opened the possibility of a membrane‐based strategy for personalization of foods and nutritional plans. Analogously, the use of nutritional supplements can be personal-ized through the evaluation of membrane status and need of specific fatty acid, cofactors, and microelements to overcome membrane impair-ment, as previously explained in the description of lipidomic profile and molecular indicators.

Taking the erythrocytes as a global reporter of the performance of membrane assembly in the individual, membranes can be used for a new approach, mapping their fatty acid content obtained by a simple anal-ysis. Membrane fatty acids are at the crossroads of metabolic and nutri-tional control processes, connecting the cell with its exterior environment, formed by other cells and the whole network of signaling coming from various parts of the body. Membrane homeostasis is so well regulated to balance the changes instantaneously and continuously during life, so that membrane composition acquires an important signature for quality of life, which can be useful for preventive strategies.

7

128 NuTrilipidoMics

7.1 WHEN FATTY Acids BEcoME NuTrAcEuTicAls: MEMBrANE THErApY WiTH NuTrilipidoMics

The rationalization of membrane behavior and the continuous discoveries of the role of fatty acids in cell metabolism and signaling contributed to the affirmation of the use of lipids as therapeutic agents. in particular, lipids are naturally directed toward membranes, as described previously for digestion and biodistribution; therefore, they can be used to “repair” unbalanced conditions using nutrilipidomics, thus combining nutritional and nutraceutical intervention. The membrane lipidomics of erythrocyte gives details of the individual condition and the health professional can build up the appropriate strategy for the recovery of the optimal balance, which is conditio sine qua non for cell functioning. in this way fatty acids can be defined as nutraceuticals, a word coined by dr. stephen l. defelice in 1989, which means a nutritional substance or cocktail with additional health benefits, such as disease treatment, health improve-ment, and prevention. Fatty acids have good characteristics to become nutraceutical compounds, which have been examined in several chapters of this book:

• well known for structures and functions, biochemical pathways, biology and interactions;

• present in all body districts and necessary for life;• found also in natural sources with wide availability;• well‐known composition and biodistribution in tissues, monitored

by simple methodologies.

As previously explained, all the major health organizations give the suggested intake values [31, 32, 119, 120]. recommended dosage by EFsA for omega‐6 linoleic acid is 10 g/day, whereas for omega‐3 AlA it is 2–3 g/day for energy intakes of 1800–2700 kcal/day, and for long chain omega‐3 puFA the dosage of 250 mg/day is recommended, although they are cited as EpA plus dHA intakes [120]. Nutrition can ensure these levels only for a balanced diet, also according to the necessary ratio between omega‐6 and omega‐3, as well as with the other two fatty acid families of saturated and monounsaturated fatty acids.

As noted in previous chapters of this book, it is common to find unbal-anced dietary conditions, and also the nutritional contribution can be impaired because of health problems and exposure to stress condi-tions. Therefore, nutraceuticals can be useful to recover fast and to keep

WHEN FATTY Acids BEcoME NuTrAcEuTicAls 129

the good levels of these fatty acid families. in this context, lipidomic monitoring becomes the exact method to assign fatty acids upon personal needs, thus making the correct use of these nutraceuticals.

in fact, other characteristics have to be mentioned when fatty acids are considered as nutraceuticals and medicaments, such as:

• types and quantities of fatty acids have specific biological meanings;• if not present in the diet, serious impairments can occur;• in several conditions such as stress and inflammation, their quantity

and integrity can be compromised;• their deficit can result in disease conditions;• fats and oils contain different quality and quantity of fatty acids.

These features underline that the choice of the ingredients and the for-mulations must be created with precise knowledge and know‐how, which ensure the efficacy of the products. How to formulate the fatty acid–based nutraceutical in the right way? using the lipidomic database, membrane profiles can be organized according to health conditions and subject char-acteristics, so that nutraceutical lines can be designed upon rationalization of molecular profiles. The formulas can include cofactors and antioxidants, which help the bioavailability and the activity of the lipid components to ameliorate membrane conditions. As in pharmaceutical interventions the molecular target and interaction of nutraceuticals has to be clearly defined; with the nutrilipidomic approach the cell membrane is identified as the target that can be reached and restored by nutraceutical intervention. using erythrocyte as reference, the length of the nutraceutical intervention can be kept in the period which is the lifetime of this cell type therefore the suggested supplementation period is of 4 months. Taking into account that the supplementation contains hydrophobic components such as lipids, it is advisable to interrupt the therapy at the end of each month for 1 week. in other words, there are 3 weeks of treatment per month for a total period of 4 months. This protocol comes from the clinical experience made with health professionals collaborating with the spin-off company lipinutragen founded by the authors of this book.

Another important aspect of this approach is the monitoring of the supplementation effects, by performing the analysis before and after the intervention. in this way, the nutraceutical approach is not only personalized, but also measurable as different metabolic conditions can generate different responses to the therapy.

it is also clear that nutrilipidomics can be used to assay new protocols and formulations with a science‐based and precise protocol referred to

130 NuTrilipidoMics

cell membranes. researchers in life science disciplines can collaborate with pharma industries in order to bring this innovation to market, tak-ing care of the consumers’ needs and therapeutical efficacy. The future development of molecular medicine and “omics” technologies can be the leading motif for the innovation of the productive chain of nutraceuti-cals and functional foods, where science is in the middle of the formula-tion, and not in the end to proof the activity of a marketed product, as it occurs nowadays. in Figure 7.1 an innovative vision of the productive chain of nutraceutical and functional food is shown: after the market analysis and the literature search for ingredients, there is an important r&i&d step for the consulting of data bases of molecular profiles which orient the formulations toward the specific needs of the customers. Moreover, connecting nutraceuticals and functional foods with the molecular profiling, the uncertainty and skepticism of the effects can be easily defeated, since the choice and personalization will be based upon the verification of the patients’ need with high precision. The existing confusion in the supplement and so‐called nutraceutical markets creates difficulty for the individuation of the best product, and often the spe-cialist even gives up to consider this nutritional contribution to the med-ical intervention. instead, the “nutraceutical shelf” can become a real opportunity when rationalization and personalization criteria are intro-duced, and the target is the patients’ need, not the market interest. This

Figure 7.1 science innovation in the center of the development of nutraceutical or food formulations oriented by the human molecular profiles

MARKET ANALYSIS

LITERATURE

R&D

DATA BASE MOLECULARPROFILES

Nutraceutical/food formulations

CONSUMERS’ NEEDSand

PERSONALIZATIONOPPORTUNITY

FATTY Acid–BAsEd MEMBrANE lipidoMics 131

is also interesting for an effective application of scientific advancements, instead of using science as a “justification” after the products are already on the market. Finally, this is the only way to introduce ethics in the market, since innovation contains not only a novel concept to apply to the productivity, but also improvement of the existing conditions for a better world.

7.2 FATTY Acid–BAsEd MEMBrANE lipidoMics ANd NuTrilipidoMics: THE pErsoNAliZEd ApproAcH For NuTriTioN ANd NuTrAcEuTicAls iN HEAlTH ANd disEAsEs

The opportunity given by nutrilipidomics is that, upon the analysis of membrane lipidomics, there is a personalized indication of the supple-mentation and nutrition to give to the patient, in order to recover the unbalance using the natural membrane turnover. As previously explained, the intervention consists of 4 months, with 3 weeks/month of supple-mentation, the last week of each month being useful to avoid accumulation of the lipid components which are hydrophobic molecules and their excretion is slow. indeed, fats tend to deposit, and with continuous sup-plementation there is the possibility to create accumulation of specific fats in the body, especially in more lipophilic tissues. using high dosages for long periods this can occur and the functionality of such tissues can be impaired due to the change of their natural composition of membranes; we recall from part 1, where we treated the membrane fatty acid characteristics, that the presence of polyunsaturated fatty acids increases the membrane fluidity. This is in principle a positive effect, unless it overcomes the natural content with unfavorable destabilization of membrane properties. Moreover, nutraceuticals with polyunsaturated molecules, which are components with tendency to oxidative reactions, must be taken without an excessive dosage or accumulation due to pos-sible increase of oxidizability during distribution in the body. one week of suspension per month is a safe measure to avoid the deposit and to keep the natural status of tissues. The dosages and formulas can also be created for combination with pharmacological therapies, in order to be synergic with the molecular interactions of the drugs. it is worth noting that the nutrilipidomic approach aims at realizing the molecular inter-vention with membrane as target, which is not “alternative” to the

132 NuTrilipidoMics

accepted therapeutics and protocols of medicine. in the nutrilipidomic approach, nutrition is also used taking into account its important contri-bution to everyday life. This is particularly important for the quality of the fats in the diet, and the dietary plan must consider not only the quantity of lipids but also the distribution among the four fatty acid families. Therefore, the new concept of “lipidomic diet” was put forward with the tuning of the fatty acid content of foods for a balanced diet personalized to the membrane lipidomic requirements.

The opportunity that comes directly from lipidomics, compared to the other “omics,” is the most direct relationship between nutrition and membranes, which can have a cascading effect on other relevant meta-bolic and signaling processes. Nutrilipidomics is based on this “natural” approach, personalized to the individual status, and aims at realizing a perfect combination between metabolism and diet. The approach of nutrilipidomics is based on the following considerations for fatty acid supplementation:

1. Metabolism and individual conditions mostly influence bioavail-ability and fate of the administered polyunsaturated fatty acids. in case of increased oxidative stress conditions or production of free radical species, unsaturated lipids can be altered, as shown in part 1, and toxic products can be generated, such as aldehydes and trans lipids. Free radicals are also involved in pathological condi-tions, when inflammatory and immune processes are active. in pathologies and aging a decrease of endogenous defense systems can occur together with lipid loss, especially essential elements; in all these cases the need for supplementation must be ascertained. in these conditions lipids must be associated with protection from free radicals and oxidations, in order to avoid the consumption of degradable fatty acid during biodistribution and absorption with failure of their activity. Fatty acid supplementation reported in most of the clinical studies are not associated with protective sub-stances, and even the dietary conditions of the individuals are not taken into due consideration. sometimes the association with an antioxidant vitamin, which is in general only one and represented by vitamin E, is used but this is not enough since vitamin E can also be a pro‐oxidant when taken alone. Moreover, as explained in the metabolic pathways of section 5.5, the intake of triglycerides from diet or supplementation does not directly mean that these fatty acids will be found incorporated in the membrane phospholipids. The overall metabolic transformations, connected

FATTY Acid–BAsEd MEMBrANE lipidoMics 133

with the status of organs, are crucial to the effectiveness of the lipid therapy.

2. A complete and balanced diet is the most important guarantee for health. However, unbalanced diets are also frequent and supple-mentation becomes important, especially as it must be targeted toward individual needs. This is valid for lipids, but also for anti-oxidants and other protective elements. The actual consumption of supplements without knowing the effective need causes an excess of some fatty acids, which alters the normal composition of tissues. At the same time, the inappropriate use of antioxidants can be deleterious toward many of the enzymatic mechanisms that in the body function through the generation of free radicals. indeed, it is necessary to mention that free radicals do not always mean danger, since many of our biological processes work through the free radical mechanism. The phenomenon called “antioxidant paradox” can occur, as described by prof. Barry Halliwell in 2000, losing the beneficial effect and shifting toward the detrimental role of an excess of antioxidants [121]. Therefore, it is advisable that the use of this type of molecules be motivated by a real and specific need.

3. Nutrilipidomics with membranes as targets relies also upon the pharmaceutical form used for supplementation, since it can influence biodistribution, as occurs for a drug. The soft‐gel capsule has favor-able adsorption and bioavailability characteristics for oral supple-mentation. indeed, it is worth mentioning that for nutraceutical supplementation the release form and the formula adjuvants are as important as they are for the activity of a drug.

To conclude, nutrilipidomics can be associated to the following criteria:

• study of the real needs of the individual and couple them with the dietary plan.

• choice of the type and dosage of lipids for an appropriate balance of metabolic pathways.

• control of degradative processes.• Help to the metabolic functions of intestine and liver.• Target the important compartment for the activity, which is the cell

membrane, using erythrocyte membranes as the comprehensive biomarker and monitoring after 4 months’ treatment.


Lipidomic Profiles and Intervention Strategies in Prevention and Diseases

Lipidomics and nutrilipidomics are tools at the disposal of health professionals involved in the diagnosis and care of patients, as well as in the management of optimal conditions for prevention. The impor-tance of the lipidomic approach is that it refers to a comprehensive condition of the cell membrane, which is a highly organized structure, whose homeostasis is basal for the correct functioning of the processes and recovery after any stimulus and stress.

The lipidomic approach has been introduced as innovation of med-ical practice in Italy as the core activity of the company Lipinutragen born as spin-off of the National Council of Research and founded by the authors of this book. The Lipidomic Laboratory of the company is officially recognized by the Health Minsitry and can execute the analyses which are prescribed by medical doctors to the patients. By this activity the lipidomic and clinical data are gathered in anonymous form and allow to form a data base which is organized by the different health conditions. Up to now around ten thousand of analyses have been organized and the picture of the importance of the lipidomic mon-itoring starts to be delineated.

Lipidomic monitoring allows the individual situation to be defined in detail, but also to group common characteristics for a specific dis-ease or health condition, in order to individuate the targeted fatty acid

8

136 LIpIdoMIC pRofILes aNd INTeRveNTIoN sTRaTegIes

metabolism of each situation. In fact, each tissue or organ has its own composition, from which it is also possible to understand what is the fatty acid crucial for the its activity as in the case of retina, or heart, or adipose tissues (see Table 2.1 with specific composition of some tissues). as an example of the powerfulness of the lipidomic database, figure 8.1 shows the value of the arachidonic acid percentage (optimal values 13–17%, see Table 3.1) present in Italian subjects under different health conditions, including healthy people. These data can be considered pre-liminary and make part of the collaboration between the authors and the company toward the definition of lipidomic profiles in the Italian population, which will be published in due time.

from the snapshot on arachidonic acid as relevant fatty acid and mediator for lipid signaling, it is evident that its level differs from healthy conditions and pathologies: 1 individual upon 4 (24%) has the arachidonic acid values >17% in healthy conditions, whereas in all other pathological conditions the frequency increases with 1 individual upon 2, or more.

In order to summarize the most relevant finding from the literature on fatty acid importance for lipidomic profiles, discussion of physiological and pathological conditions are given, underlining the importance of fatty acids in each case as described in literature and also confirmed by the, which satisfactorily matched the results of the database of human profiles gathered in the Italian population. It is worth underlining that

24.0

54.7

65.3 68.0 66.5 66.970.8

58.0

71.5 74.0

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40.0

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Ind

ivid

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s

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y con

trol (

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chole

stero

lemia

(N=20

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es (N

=144)

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y (N

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eight

(N=25

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(N=37

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Psoria

sis (N

=130)

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tens

ion (N

=200)

Allerg

ies (N

=200)

Neuro

logy (

N=15

0)

Figure 8.1 percentages of individuals having arachidonic acid percentage greater than 17% in their erythrocyte membranes, under different pathological conditions including healthy controls. data by courtesy of Lipinutragen srl from the data base of 10000 analyses available in the company.

LIpIdoMICs aNd spoRT 137

the information provided must be not considered exhaustive for describing health conditions, also in view of new knowledge coming from the ongoing research.

8.1 LIpIdoMICs aNd spoRT

sport is connected to the concept of maintaining optimal health. Indeed, sportive people are convinced that they are producing benefits to their life by exercising their body; therefore, it could be really dramatic for them to discover that they obtain the opposite result! on the other hand, the continuous solicitation that sportive activity produces at the levels of heart, muscle, and overall metabolism must be considered as a “stress” factor induced by exercise. at the level of muscle cells the ruptures caused by exercise must be promptly repaired, and this depends on the capability of each organism to intervene with repair systems and the right components after the stress event [122].

The role of fats has been considered in the metabolism at least for these two aspects:

1. energy providers, with substantial difference from carbohydrates and sugar components, for the balance between insulin response and fatty acid oxidation. for example, the hyperinsulinemia condition can derive from a decrease in pUfas of skeletal muscle membrane phospholipids, as well as from the increase in sfas in the diet, with several other metabolic consequences such as lipo-genesis (obesity), hypertension, type 2 diabetes [123].

2. Membrane components orient signals and the functioning of receptors and proteins present in the muscle cells, such as in the case of the transport protein of glucose (gLUT4) during exercise, which increases for high percentage of unsaturation in the muscle cell membrane phospholipids and decreases under higher satu-rated fat intake [124, 125].

These evaluations have been carried out in models, such as animals or cells; however, they can be extrapolated to humans and can vary according to the type of tissue that is under consideration. In a human study, regular physical exercise compared to sedentary lifestyle brought about several changes to the muscle membrane phospholipids, such as an increase in the content of oleic acid and dHa, an increase in vaccenic acid, a reduction in omega‐6/omega‐3, and an increase in the oleic/ palmitic


ratio, suggesting an increase in desaturase activity by training and a recruitment of oleic acid during exercise [126]. In the athlete world it has been estimated that the effects of exercise are opposite to the aging process [127], especially concerning the increased production of antiox-idant enzymes (catalase, glutathione peroxidase, and superoxide dis-mutase), increased insulin sensitivity, and prevention of hyperglycemia. on the other hand, it has also been demonstrated that aerobic sportive activity increases oxygen consumption, especially in certain tissues like muscles; therefore, if the endogenous production of antioxidant enzymes and presence of antioxidant vitamins and cofactors are not sustained, possible excess of oxidative and free radical processes can occur [128–130]. It is worth remarking that a balanced activity and adequate dietary styles ensure the training to be only positive; however, the individual conditions can vary or change along the years of life, and a preventive panel of molecular diagnostics could help to keep the situation under control. It is clear that the interruption of the agonist sportive activity can also produce consequences on the metabolism, trained to have a turnover from liver and muscle functions, especially concerning saturated fat accumulation. There is no knowledge as yet of the lipidomics of postsportive agonistic activity, which could even be involved in the development of degenerative diseases, as is more frequently observed.

The use of antioxidants and fatty acid supplementation became very common among sportive people in the last decade, guided by the idea that these natural substances are consumed during sportive activity and they are not harmful at any dosage, so that nutraceutical and food lines dedicated to sports are very successful on the market [131, 132]. Based on the concepts developed in the previous chapters on the nutraceutical formulas and the appropriateness of the supplementation, the use of these products needs to be regulated respect to individual needs. The future direction in the field of sportive health is to use diagnostic tools in order to personalize the intervention. In this regard, lipidomic anal-ysis is one of the most useful candidates as a reference point for the assignment of appropriate dietary and nutraceutical directions.

Lipidomic analysis can inform us on several aspects:

1. Molecular effects of the lifestyle and sportive activity in the impor-tant compartments of the membranes, taking also into account that favorable properties of permeability and fluidity of natural membranes favor the correct passage of oxygen and nutrients for tissue functioning.

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2. oxidative processes, which can affect the polyunsaturated content of natural membranes, either for the consumption of omega‐3 and omega‐6 fatty acids and for the formation of trans lipids due to production of free radicals; pUfas are used during the stress conditions to mediate signaling and cell responses, and can also be consumed during an increased oxidation state; therefore, an unbal-ance can occur in the remodeling mechanism and phospholipid exchange (see Chapter 3). Indeed, sportive activity is a natural stimulus for increasing metabolic turnover and the fatty acid pool composition is very crucial in order to keep the homeostatic control of the “repaired” membranes. The exaggerated use of antioxidants in this context can suppress the natural response; also, the erroneous balance among the different types of antioxi-dant activities can produce a paradoxical pro‐oxidant effect [121].

examining data available in the literature about this topic gives a very controversial situation, with a growing number of papers in which the deleterious effect of the antioxidant supplementation has been shown. Recently, a review summarized the basic knowledge on the effects of antioxidant supplementation on the physical performance of athletes [133]. The main features are as follows:

• In 1971 it was reported that vitamin e supplementation (400 IU/day for 6 weeks) did not present any beneficial effect in adolescent swimmers, but even appeared unfavorable for the duration of the sport performance.

• In 1996 and 1997, two papers showed the deleterious effect of ubiquinone‐10 supplementation in the athlete performances after an intensive training program.

• No effect was obtained from antioxidant supplementation in triathletes in the maximum oxygen uptake.

• It did not attenuate postexercise muscle cramps after intense physical activity, but rather it delayed the recovery time; a molec-ular explanation to all these data was put forward, showing that supplementation with vitamin C (0.5 g/day) and e (400 IU/day) inhibits the release of interleukin‐6 (IL‐6) from the contracted skeletal muscle. IL‐6 is a cytokine that facilitates lipid turnover and stimulates lipolysis as well as beta‐oxidation, and because it is pro-duced in the muscle it has been defined as “myokine.”

• The only antioxidant supplementation that seems to have a benefi-cial effect is the use of NaC, a cysteine donor that increases the


synthesis of endogenous glutathione. It has also been demonstrated to improve the human tolerance towards different types of physical exercise.

finally, the inflammatory effects connected with sportive activity have to be considered based on tissue resistance. These effects are expected on the basis of the release of arachidonic acid from membranes, upon exercise stimulus, and the consequent formation of eicosanoids, which activate the cytokine response and cell reactivity. However, this stimulus should occur under controlled endogenous conditions, as the membrane composition itself contributes to the required reactivity. The omega‐6 reactivity can be balanced with the anti‐inflammatory and compensative role of omega‐3 and their mediators [127]. since these fatty acids are essential, dietary habits of sportive people are crucially important to fur-nish the desired balance thus realizing an efficient anti‐inflammatory control. If this is not the case with good omega‐3 intakes, which can be checked by a simple food questionnaire, sportive people should then be controlled in order to evidence whether their sportive activity had induced a pro‐inflammatory condition.

To conclude, lipidomic analysis is necessary to obtain the maximum benefits from sportive activity, controlling the role of the exchange of lipids for an effective membrane homeostasis and informing on the impairment of the fatty acid composition derived from the incorrect composition of the individual pool.

8.2 LIpIdoMICs aNd pRegNaNCY

In pregnancy the role of fatty acids, and in particular of essential fatty acids that cannot be produced by the body, is evident. The exponential growth of new tissues in the fetus, whose functionality depends on the quality of the cell membranes and the related organization of cell functions, needs a continuous flow of fatty acids. The importance of both pUfa families and the appropriate balance is not only limited to the formation of functional membranes, but is extended to mediator production (prostaglandin, leukotriens, resolvins, etc.) and signaling, which provide day by day the maturation of the new organs and organism. as a consequence, in pregnancy either defect or excess of fatty acids can exert a negative effect on natural tissue formation; therefore it is very important that the fatty acid families be in correct proportion to avoid competitive effects between them. This condition is not easily

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realized in industrialized countries, since it is well known that omega‐6 and saturated fatty acids are present in excess, so that pregnancy becomes the time in a woman’s life that requires specific attention. Moreover, the so‐called long‐term effect extends from the mother to the fetus, even far from the pregnancy; therefore correct fatty acid intake protects the mother and the baby [134, 135].

The long‐term effects of food deprivation in pregnancy have been eval-uated along the years on a historical cohort of people (300 000 people) whose mothers were exposed for 6 months of 1944–1945 to the dutch famine, caused by the Nazi embargo in Holland during the second World War. In a study the hypothesis that prenatal and early postnatal nutrition determines subsequent obesity was examined. outcomes were opposite depending on the time of exposure. during the last trimester of pregnancy and the first months of life, exposure produced significantly lower obesity rates (P < 0.005). This result is consistent with the inference that nutri-tional deprivation affected a critical period of development for adipose tissue cellularity. during the first half of pregnancy, however, exposure resulted in significantly higher obesity rates (P < 0.0005). It could be observed that nutritional deprivation affected the differentiation of hypo-thalamic centers regulating food intake and growth, and that subsequent increased food availability produced an accumulation of excess fat in an organism growing to its predetermined maximum size [136]. Interestingly, from this large cohort of people metabolic considerations could also be carried out, putting forward in the early seventies a suggestive scenario that maternal malnutrition during gestation may permanently affect adult health, not evaluable from the size of the baby at birth. Indeed, the long‐term consequences of improved nutrition of pregnant women could be underestimated if the observations are solely based on the size of the baby at birth. The fetal origin hypothesis was advanced in a new dimension, suggesting that adaptations that enable the fetus to continue to grow may, nevertheless, have adverse consequences for health in later life.

another study was conducted regarding the gestational exposure to famine associated with health‐related quality of life. Nine hundred and twenty‐three individuals, including persons born in western Holland between January 1945 and March 1946, persons born in the same three institutions in 1943 and 1947, and same‐sex siblings of persons in series 1 or 2 were followed up between 2003 and 2005 (mean age: 59 years), and self‐reported quality of life and depressive symptoms were assessed. The conclusions were that a mother’s exposure to famine prior to conception of her offspring is associated with lower self‐reported mea-sures of mental health and quality of life in her adult offspring [137].


Considering other metabolic consequences on the babies of that period, like glucose tolerance, obesity, coronary heart disease, athero-genic lipid profile, hypertension, microalbuminuria, schizophrenia, anti-social personality, and affective disorders, exposure to famine during childhood resulted in changes in reproductive function, earlier meno-pause, changes in insulin‐like growth factor‐I, and increases in breast cancer [138]. This extreme example of loss of important components from the diet can alert medical doctors on the evaluation of the causes of disease onset in a variety of patients, confirming that an important anamnestic data should be dedicated to the nutrition.

during pregnancy the fatty acid concentration in plasma increases as triglycerides and the phospholipids increase in polyunsaturated fatty acid content, which is also due to the mobilization from tissues. In particular, several studies evidenced the strong mobilization of dHa and the LC‐pUfa transfer through placenta toward the fetus. at least 67–75 mg/day of dHa is transferred from mother to fetus in the last trimester of pregnancy, also to allow the development of neuronal and retina tissues, which are rich in this component [139, 140]. This fatty acid becomes an important nutritional supplement suggested in preg-nancy, especially if the food intake of fish is low (<3 times/week). It is worth underlining again that an excess of the intake of only one type of fatty acid can produce membrane unbalance; therefore it is appro-priate to use lipidomic monitoring to assess the need for the 9 months of pregnancy. This is recommended in the first months in order to start a personalized intervention for 3–4 months, and conclude the treatment within the first 6 months of pregnancy, since in the last 3 months the lipid changes need to be associated to the delivery process with pro-duction of mediators that must occur without any interference from supplementation.

The lipidomic monitoring in pregnancy must also include the evalua-tion of the trans fatty acid level. It is well known that, in the neuronal and cognitive development of the fetus, trans fatty acids interfere with the normal functioning of enzymes and metabolism [139], whereas the trans lipid intakes are discussed for their influence on the increase of weight in newborn babies [140], and for the availability of long‐chain pUfas [141]. These effects can also change in relationship with the diet followed by pregnant women; therefore, personalization of the status must be carried out to the right setup of related therapies.

Lipidomic analysis can provide important information on the lipids necessary to the fetus for correct membrane formation and functioning and to the mother to maintain homeostasis before and after the pregnancy.

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8.3 LIpIdoMICs aNd agINg

one of the most exciting subjects of medical and biological research is longevity, that is, the individuation of the molecular and functional char-acteristics, which provide a competitive advantage to prolong the life span to the best possible. aging is a complex multifactorial process still to be completely understood, where several molecules that have impor-tant biological roles start to be less efficient or to be degraded. The first observed aging phenomenon is known as Hayflick phenomenon, from the name of the researcher that in the sixties was reporting the impaired replication of in vitro cultured skin fibroblasts, loosing progressively their proliferative capability accompanied by degeneration of gene expressions and protein synthesis. These degradation processes were then connected to the free radical theory of aging, proposed by Harman in 1956 and more and more strengthened by several other experiments. The hypothesis is that one single common process is responsible for aging as well as death of living organisms, modifiable by genetic and environmental factors, and this is the event of free radical production, especially at the mitochondria level, which induce the transformation of biomolecules with consequent loss of their activities. This subject has been treated in part 1; therefore here it is pointed out that the delicate balance between functional radical reactions and free radical damage is the crucial factor that influences the fate of living organisms. Nowadays the effects of the so‐called antioxidant network, formed by enzymatic and molecular species, are hot research topics with a strong industrial interest. The delicate equilibrium between the biological functionalities and their progressive deterioration is influenced by the lifestyle, nutri-tion, and preventive habits of each individual, which help to maintain the level of components in the right proportion, avoiding harmful processes. for example, the balance of the redox state of metals (iron in primis) and the status of chelation by protein structures (hemoglobin in case of iron) offer a natural protection from metal reactivity, as, for example, occurs via the famous fenton reaction, with free fe+2 and per-oxides generating the reactive Ho• radical. The subsequent reactivity pathways occurring on nucleic acids, proteins, carbohydrates, and lipids creates oxidative and modified products, which can accumulate and influence the capability of cell adaptation. The formation of free radicals can also be induced upon exposure to radiations, including photoirra-diation, which is indeed one of the major causes of photoaging.

In the scenario of damage and repair, the cell membrane has a primary role in adaptation and survival. as described in the part dedicated to


stress (Chapter 3), the consequences of stress on membranes can be eval-uated by the oxidative loss of pUfas or by the formation of trans lipids. Indeed, pUfas are dual compounds, which are essential membrane com-ponents with incredibly important structural and biological roles and also display a delicate structure for the presence of reactive sites, attack-able by free radicals and oxidants. on the other hand the membrane compartment resisted millions of years; therefore pUfa reactivity should not be so critical! as a matter of fact, the studies carried out in the last two decades focused on the important role of this cell compartment in the regulation of whole cell metabolism and fate. professor antony Hulbert at the University of Wollongong is the reference author of studies on membrane composition of different species, coining the term “metabolic pacemaker,” due to the fine regulation of the lipid asset to make ionic channels and proteins to function in signaling and mediator activities [142]. Correlating the life span with the lipid species, Hulbert gave a possible explanation for the complexity of lipids as modulators of the life of living organisms. going from animals to humans he observed that the more the peroxidizability of the systems provided by pUfas the lesser the life span. In particular, membranes must be rich in compo-nents that provide fluidity but do not increase the susceptibility to peroxidation processes; therefore monounsaturated fatty acids are the most important elements to enrich cell membranes, especially when the defense systems decrease with aging. Monounsaturated components of olive oil are also connected to favorable aging observed in the Mediterranean area, affording the properties of stability and flexibility, which are necessary for structural compartments such as membranes. This is a dietary advantage as compared to other countries that do not regularly use this food. It is worth noting that the monounsaturated component can be prepared in the human body by desaturase enzymes, with the first step after the palmitic acid biosynthesis (16 : 0) to palmi-toleic acid (9cis‐16 : 1) (see figure 2.3).

an Italian research in 2008 examined centenarian offspring (41 indi-viduals) for the composition of their erythrocyte membranes, evidencing that these subjects have a significantly higher amount of palmitoleic acid than subjects living in the same geographical context and general Italian control population [143]. The calculation of two indexes can be done with the percentages of fatty acids in the membranes: unsaturation index and peroxidation or peroxidizability index. The first index can be calcu-lated from the percentages of the main unsaturated fatty acids found in membranes, using the equation: (monoenoic% × 1) + (% dienoic × 2) + (% trienoic × 3) + (% tetraenoic × 4) +(% pentaenoic × 5) + (% hexaenoic × 6)

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[144]. The peroxidation or peroxidizability index can be calculated as follows: (%Monoenoic × 0.025) + (%dienoic × 1) + (%Trienoic × 2) + (%Tetraenoic × 4) + (%pentaenoic × 6) + (%Hexaenoic × 8). It seems that centenarians give an advantage to their siblings in terms of favorable molecular composition of membranes and reduction of the peroxidiz-ability index [142, 143]. The biological advantage provided by an increased proportion of MUfas in membranes is fascinating, and population studies on lipidomic and genomic traits of desaturase activity can be foreseen to give further support to this hypothesis.

Together with the free radical and the membrane theories, the aging process has also been correlated to a progressive low‐grade inflammatory immune process, thus leading to the coining of the word “inflammag-ing” [145]. It is well known that chronic inflammation is associated with aging and plays a causative role in several age‐related diseases such as cancer, atherosclerosis, and osteoarthritis. This process puts the pro-gressive activation of immune cells over time into play. The scenario is very complicated, involving secretion of pro‐inflammatory proteins, dNa damage response, key transcription factors and kinases, and chromatin remodeling [145, 146]. Healthy aging and longevity are likely the result of the balance between inflammatory responses and efficient anti‐inflammatory networks, whereas the unbalance in normal aging is the major driving force for frailty and common age‐related pathologies.

Taking all these concepts into account, it can be concluded that mem-branes can be profitably used as an early marker of inflammaging and an important prevention tool, evidencing the consequent unbalance of the homeostasis and allowing for the planning of personalized nutri-tional‐based strategy for favorable aging.

8.4 LIpIdoMICs aNd CaRdIovasCULaR HeaLTH

It would be not enough to dedicate a whole book to the lipidomics of cardiovascular diseases, starting from the consideration of the main role played by intake of fats and health risk factors. The “diet–heart” paradigm, for the high intake of saturated fats and cholesterol correlated with ischemic heart disease and atherosclerosis, was individuated in the beginning of 2000 [147], and for several years the dietary indication of health organization in all the industrialized countries, especially the United states, promoted dietary programs with low if any importance given to the quality of fats.


The observations about fats and blood lipid levels are particularly referred to plasma cholesterol with the following correlations:

• sfas contained in foods have a powerful effect on the increased plasma cholesterol, LdL and HdL levels, with a superior effect on the former, which can be the major driver of Cvd risk. In particular, lauric, myristic, and palmitic acids have been found more effective [148].

• MUfas and pUfas have a positive effect in reducing LdL choles-terol levels, with both having the effects on the increase of HdL levels with the former being less effective toward LdL [149].

• The effect of sfas is stronger than the effect of pUfas.

The observations on saturated fats spurred the shift toward the elimination of fats from the diets, which induced a compensatory carbo-hydrate intake, nowadays thought to be responsible for the increase of overweight and obesity (see also the next section). It is worth mentioning that carbohydrates are obviously connected with insulin response and, as explained in part 1, fatty acid synthase activity with formation of palmitic acid is stimulated under these conditions, thus causing an accumulation of this saturated fatty acid at the level of several tissues (vascular and adipose tissues). With more and more data on the role of MUfas and pUfas, the reason for lipid diversity became more evident, and in particular the suspicion about fats is nowadays seen as completely wrong. as a matter of fact, the influence of new acquisitions can be clearly demonstrated by the change in the diet pyramids from 1990 to 2001 (see figure 2.1). Many nutritionists with the knowledge of the nineties are still at work and it is hoped that they updated their formation on fat intakes from the diets!

The third observation on the competitive effects between sfas and pUfas is also very relevant, and was important to motivate a reassess-ment of the quality of fats in cardiovascular diseases [150]. Indeed, among all fat categories, pUfas received the highest attention from the clinical research point of view and the number of studies on omega‐3 and omega‐6 effects increased exponentially each year. for omega‐3, starting from the historical epidemiological research on the eskimos, the effects are reported in so many papers that it would be impossible to give an overview in this small section. protective effects concern decrease in blood pressure, reduction in triglyceride levels, antithrombotic effects, anti‐inflammatory effects, antiarrhythmic effect, improvement in vascular function, and increment in plaque stability and in insulin sensi-tivity [151]. It is worth mentioning that generalization of the role of

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omega‐3 must be avoided, as demonstrated by a study of 53 patients affected by atrial flutter and atrial fibrillation, where the differences of the erythrocyte membrane profile compared to controls concerned mainly the strong decrease of MUfas and increased pUfa content with consequent increased peroxidation index, whereas omega‐3 levels were unimportant [152]. other issues regarding the role of fatty acids in the cardiovascular sector and nutritional recommendations have been treated in the sections on saturated fatty acids (section 2.1), and pUfa (Chapter 3), which are recommended to the reader in order to complete the information given in this specialty section.

Here we wish to add something more on the role of omega‐6 and trans fatty acids for the cardiovascular system.

The role of omega‐6 linoleic acid in this field and the benefits of dietary intake of vegetables that are rich in these components are well established. also, lecithins, which are mainly composed of omega‐6 fatty acids, are recommended for the lowering triglyceride contents, acting as providers of phosphate and choline building blocks, needed for the transformation of fatty acid derivatives into phospholipids (see figure 5.6). It is also evident that linoleic acid is the essential fatty acid starting the cascade in the biosynthesis of arachidonic acid, from which pro‐inflammatory molecules, such as prostaglandins and leukotrienes, can be prepared. Therefore, the benefits of omega‐6 were investigated, which also demonstrated that arachidonic acid generates not only pro‐inflammatory mediators but also potent protective and anti‐inflammatory mediators, whose balance is the key to omega‐6 control and beneficial activities. The discovery of lipoxins in 1984 by serhan was followed by other investigations, which designed a scenario of balanced activities for the eicosanoids and enzymatic oxidation products in the omega‐6 series fatty acids, arachidonic and dihomogammalinolenic acid [51, 153], as also discussed in previous sections.

The role of trans fatty acids as cardiovascular risk factors deserves some comments. Research have clarified the harmfulness of trans fats of industrial origin (see Chapter 4), which can be found in several foods, such as margarines, bakery products, pastries, cakes, french fries [154]. To summarize [155, 156]:

• an increment of LdL levels far higher than the sfa, without increasing HdL levels.

• an increment of lipoprotein a levels and reduction of LdL particle size, which amplifies the risk of Cvd, in particular myocardial infarction.


• promotion of systemic inflammation through the increment of C‐reactive protein, resulting in thinning of the arteries, diabetes, and sudden death due to cardiac arrest.

• endothelial dysfunction through the increment of soluble proteins, which are at the basis of atherosclerosis development.

• an increment of insulin resistance that has an important effect on the cardiometabolic level and on the onset of the metabolic syndrome.

The huge and negative impact on mortality associated to Cvd brought about the consumer associations to fight against food companies and food chains in order to ban trans fats. other interesting news and the progress of this campaign can be found on some websites [157].

8.5 LIpIdoMICs aNd oveRWeIgHT

The percentage population affected by overweight and obesity in the world is so high that it is hoped an immediate and effective solution can be obtained from research to see these numbers progressively diminishing [158]. overweight and obesity are risk factors for cardiovascular dis-eases, diabetes, and for several other diseases, thus significantly increasing costs for health care in the eU and worldwide; for example , an obese person incurs 25% higher health expenditures than a person of normal weight [159]. The connection between the problem of lipid accumulation and lipidomics is evident; however, the use of this diagnostic tool is far from being recognized by pathologists and nutritionists for checking the metabolic and dietary status of the patient.

adipocytes, which are the cells composing adipose tissues, have the role of fat depot and turnover, which has been clearly defined with the discovery of the two different cellular forms of white and brown adipo-cytes [160]. The nature of fatty acids is crucial to define lipid metabolism and the white adipocyte prevalence, with decisive roles of MUfa and pUfa families in limiting the increase of cell dimensions, whereas sfas are associated to hypertrophy and hyperplasia of adipose tissue. The membrane composition of adipocytes is also crucial in the functioning of the beta‐adrenergic receptor under adrenaline stimulation, which acti-vates the pathways of lipase enzymes and mobilization of triglyceride deposits, together with the simultaneous functioning of channels for glycerol, ion and free fatty acids. also, cold stimulus (for humans around 20°C) can induce activation of fat mobilization with change from white to brown tissue. The dietary habits associated with increased food intake

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cause adipocyte hypertrophy, which occurs with remodeling of their membrane composition; however, when this remodeling does not change the fatty acid composition dramatically, loss of weight is still possible, actuating a diet and exercise strategy. on the other hand, the unfavor-able change in fatty acid composition due to unbalanced fatty acid intakes, especially with the increase of saturated components, causes membrane malfunctioning, which is reflected in an impaired fat management. obviously, contemporary activation of the hypothalamus and liver and muscle production of signaling molecules have to occur for fat mobilization, and again the quality of the membrane receptor response plays a crucial role for the effectiveness of the overall control. Lipidomic monitoring of erythrocyte membranes and adipose tissues can identify the main fatty acid transformations, pointing attention to the increase of desaturase enzymatic activity and stress, as important players for the metabolic outcome:

1. The induction of the saturated–monounsaturated pathway, starting from palmitic acid as a component of the diet or derived from the insulin stimulus of the fatty acid synthase activity, with the level of palmitoleic acid as biomarker; indeed, an increased level of this nondietary fatty acid has been associated with obesity and the risk of metabolic syndrome [161–163]. an important warning for the identification of this fatty acid has been provided in section 2.2; therefore it is important that lipidomic monitoring be carried out appropriately, and mass spectrometry facilities are not able to pro-vide this absolute identification.

2. The induction of the omega‐6 pathway with the increase of arachi-donic acid as marker of cellular reactivity toward inflammation asso-ciated also to the adipokine level [164]; this is an important information obtained from lipidomic monitoring, which also controls the level of the arachidonic acid precursor, that is, dihomogammalin-olenic acid (dgLa), with balancing anti‐inflammatory properties. In recent times, the omega‐6 pathway has been highlighted as involved in early origins of obesity, sounding a note of caution on the recom-mendation of intake of these fatty acids [165].

3. omega‐3 fatty acids are also important in obesity for their balance with omega‐6, in order to avoid pro‐inflammatory outcome [164]. In metabolic and cardiovascular diseases the omega‐3 fatty acids are a crucial ingredient for diet and supplementation [166].

Membrane lipidomics can be very useful to determine the individual condition, which in obesity can vary enormously depending also on the


functions of several organs (liver, pancreas, intestine, muscle, brain) that exchange and correlate with the adipose one.

8.6 LIpIdoMICs aNd deRMaToLogY

The field of dermatology strictly correlates with cell membrane functions at the molecular level, along with phospholipid composition, which influ-ences permeability and fluidity, and consequently hydration, resistance, and aging of the skin. sfas, MUfas, and pUfas in dermal tissue are very important at least for three events:

1. The balance necessary for skin functionality due to fatty acids needed in this tissue, and metabolism at the individual level; this aspect includes the capability of skin to produce their own unsat-urated fatty acids by desaturase activity, since the presence of the enzymatic activity is still under evaluation. for example, delta‐9 desaturase activity (sCd‐1) has recently been evaluated in mice, as an important step for the transformation of palmitic and stearic acids into palmitoleic and oleic acids, with consequences on skin integrity; moreover, delta‐6 desaturase activity is known to occur in sebaceous glands and skin fibroblasts, with production of sapienic acid as discussed also in section 2.2 [167–169].

2. The oxidative and free radical transformations of fatty acids due to skin exposure, which can cause the accumulation of products responsible of the aging process, as explained in section 8.3.

3. The inflammatory process or better the unbalance between omega‐6 and omega‐3, which influences the production of media-tors (pge2) from arachidonic acid compared to those from epa (pge3), and includes omega‐6 gammalinolenic acid (gLa) and dihomogammalinolenic acid (dgLa), with the latter being the precursor of strong anti‐inflammatory prostaglandins (pge1). The individual situation can be examined by lipidomics, to establish the best strategy to revert to the natural balance.

It is not possible to address completely the importance of lipids in dermatological diseases, which is treated elsewhere [170–172]. dermatological diseases, such as psoriasis and atopic dermatitis, are strongly connected to diet and stress [173–175]. The increase of atopic dermatitis, as well as asthma and allergy, has been attributed to the unbalance of the dietary omega‐6 intakes compared to the omega‐3. The

LIpIdoMICs aNd NeURoLogY 151

two pathways use the same enzymes; therefore in case of omega‐6 excess, the omega‐3 series with its anti‐inflammatory metabolites is disfavored in substrate competition. It has been observed that a low‐calorie vege-tarian diet, as well as a gluten‐free diet together with elevated omega‐3 content, can have a positive influence on psoriasis evolution. such diets modify the polyunsaturated fatty acid metabolism and influence the eicosanoid profiles, reducing the inflammatory effects.

oxidative and free radical reactions have been associated to the inflammatory response of atopic disease and psoriasis [176]. also in skin aging, either chronological or induced by external factors, consumption of the natural antioxidant defenses and lipid reactivity are involved [177–179]. also the formation of trans isomers has been found to be involved in atopic diseases, with loss of the naturally fluid organization of cis lipids. The trans isomers found in children were significant in erythrocyte and lymphocyte membrane fatty acids, especially in Ige‐neg-ative subjects [180]. It is worth recalling that the identification of trans isomers requires appropriate molecular libraries to recognize each struc-ture and mass spectrometry cannot provide such identification.

The efficacy of supplementations with fatty acids in dermatological diseases reported in the literature is controversial [180–184]. This leads to confusion and skepticism among specialists in considering this strategy. on the other hand, an examination of the experimental setup of these studies raises doubts about the protocols followed, since in most cases supplementations of fatty acids are not performed in order to keep efficient and bioavailable the lipid molecules and to reach the target of the cell membranes, as described in the Chapter 3. Moreover, in all studies there is no preliminary examination of the subjects in order to understand their real needs; therefore the failure of the lipid therapy in dermatological problems could be more likely due to the fact that the supplemented fatty acids were unnecessary or inappropriate to these patients. The lipidomic approach can contribute to the most appropriate choice regarding fatty acid supplementation, not forgetting to add also the control of oxidative and free radical processes associated with the dermatological disease, as explained earlier.

8.7 LIpIdoMICs aNd NeURoLogY

The central nervous system is the area with the highest energy demand compared to all the other parts of the body, and with a lipid concentration second only to that of adipose tissue. The gray matter consists mainly of


neurons with 40% lipid components, whereas the white matter has about 50–70% of lipids. phospholipids form approximately 60% of the dry weight of the brain, mainly glycerophospholipids and sphingolipids, where monounsaturated fatty acids are prevalent in white matter and polyunsaturated fatty acids in the gray matter, in particular arachidonic acid and dHa as important constituents, with dHa being a key element during the development before birth and for subsequent neuronal growth. It is important to underline that desaturase activity is almost absent in the brain so that most of the unsaturated components must arrive from the liver biosynthetic activity through blood circulation [185, 186]. The composition of pUfas in the brain tissue is age‐specific. several clinical studies showed how the omega‐6/omega‐3 ratio was dependent on time: during the normal aging process, phospholipids of the cerebral cortex undergo a progressive increase in the levels of dHa with consequent decrease of aa, while in infants the dHa/aa ratio is close to 1. The La levels increase strongly with age suggesting a loss in the efficiency of desaturase enzymes, which are responsible for enzy-matic transformations [187]. an important observation on dHa during pregnancy has been made, evidencing that the transfer of this fatty acid from mother to fetus occurs by mobilization from the neuronal tissue and umbilical cord blood transfer [188–191] and is impaired by the health condition of the mother, such as gestational diabetes [192]. The nutritional status of the pregnant woman is crucial for the effectiveness of dHa supply, and this led to suggested intakes of this fatty acid during this specific condition [58].

omega‐3 pUfas are essential for homeostasis and the proper func-tioning of the central nervous system. In particular, dHa is the most abundant fatty acid in the brain tissue and retina, reaching in the latter almost 20% of the total fatty acid content. dHa has two important roles in the nervous structures: (i) providing the optimal composition of membranes, which accompany the functioning of proteins, such as in the case of photoreceptors in the retina; (ii) by the action of phos-pholipase a2 (pLa2) dHa is liberated from neuronal membrane and generates docosanoids, molecules with several protective roles such as anti‐inflammatory activity, regulation of the immune system, cytokine expression, neuroprotective action, which for this reason are called neuroprotectins [193].

deficiency of dHa and in general of omega‐3 is recognized as mainly responsible for several neurological diseases such as schizo-phrenia, dementia, as well as other central nervous system disorders (e.g., depression) [194].

LIpIdoMICs aNd opHTaLMoLogY 153

The importance of the optimal balance between the omega‐6 and omega‐3 pathways is confirmed also by studies conducted on alzheimer’s disease that showed a marked reduction of dHa levels in the hippo-campus, associated with the reduction of plasmalogens, which are an important class of neuronal phospholipids. Both dHa and plasmalo-gens are abundantly present in synaptosomes that are vesicles containing the neurotransmitters; thus this decrement can be associated to the loss of synapses and damage to cognitive function, which are typical events of alzheimer’s disease [194].

saturated fatty acids (sfas) represent another lipid class that is able to influence the integrity of the nervous tissue with their structure. It has already been demonstrated how sfa excess is connected to the onset of several pathologies, such as cardiovascular diseases, metabolic syn-drome, and dyslipidemia. several studies have also shown how these diseases are associated with an increased risk of developing neurodegen-erative diseases such as alzheimer’s disease [195]. furthermore, a diet rich in sfas, trans fatty acids (Tfa), and cholesterol may affect vascular integrity, thus creating a favorable condition for the onset of nervous system disorders [195, 196]. The presence of Tfa in neuronal mem-branes can trigger dopaminergic signaling because of the high produc-tion of dopamine, which results in the formation of pro‐oxidant metabolites [197]. In this respect, a proper lifestyle and the elimination/limitation of bad dietary habits certainly have a positive influence on the synapsis mechanisms; therefore they may offer a sort of protection against the onset of neurological disorders.

8.8 LIpIdoMICs aNd opHTaLMoLogY

The second leading cause of human blindness in the world is glaucoma, which shows a characteristic increase of intraocular pressure. early diag-nosis is important to give the chance of limiting degeneration and visual compromission. The diet is also very relevant in order to provide essential nutrients for the visual tissues. There is a strict correlation between pUfas and the levels of intraocular pressure, which connects with the deformability and functionality of membranes [198]. some essential pUfas such as arachidonic acid and dHa also produce metabolites, which regulate the homeostasis of uveoscleral outflow [199]. for example, pgf2a is a prostaglandin generated from arachidonic acid, which can modulate the outflow, and inspired the therapeutical use of an analogue for this disease, Latanoprost [200]. similar activities have been


demonstrated for the eicosanoids derived from epa, decreasing the intraocular pressure by increasing aqueous outflow [201]. analogously, the deprivation of omega‐3 leads to a downregulation of the ionic pumps that produce the aqueous humor, with the effect of reducing the intraoc-ular pressure, but then the reduced eicosanoid synthesis does not allow the aqueous outflow, with consequent pressure increase and predisposi-tion of glaucoma [202].

Interestingly, the analysis of erythrocyte membrane fatty acid levels in primary open‐angle glaucoma patients resulted with decreased levels of epa and dHa together with increased levels of arachidonic acid with significant differences with control healthy subjects [203]. dietary intake of omega‐3 is able to help the control of intraocular pressure and also degeneration with age.

another important group of ophthalmologic disorders involves retina, in particular macular degeneration, which is also connected with the aging after 50 years and is the main cause of blindness. The retina is the richest tissue in dHa, which is the fatty acid with a crucial role in pho-toreceptor development and functioning as well as their protection from apoptosis [204, 205]. However, it is also necessary to underline that the susceptibility of dHa to peroxidation is very high; therefore the levels of this fatty acid must be maintained in the right amount, thus avoiding excess that can increase stress‐induced degeneration [206].

The dietary intakes and the efficient incorporation of omega‐3 and omega‐6 in membrane phospholipids can be easily checked with a lipi-domic analysis of erythrocyte membranes, which become a very useful molecular diagnostic tool in ophthalmology, highlighting the individual situation and avoiding the progress of diseases that are strictly related to the functionality of the eye tissues.

8.9 CoNCLUsIve ReMaRKs

In many other health conditions, lipidomics can be important to get information on the molecular status of the most important cell compartment of the body, the cell membrane. The choice of cell mem-brane, in particular erythrocyte membrane, as a global health reporter has been discussed thoroughly in this book and is sustained by research, especially in the first decade or so of this new millennium. The knowledge on this topic allows us to be confident about this molecular diagnostics tool, and to expand its use in all medical applications. Besides the eight specialty fields indicated, we would like to remark that lipidomics is

CoNCLUsIve ReMaRKs 155

needed when nutrition and quality of life are the targets of medical intervention. In fact, the nutritional status of membranes inform on the metabolic and dietary outcomes of each individual, whereas the individ-uation of the membrane unbalance can highlight the underlying reason for a discomfort, which can be used successfully for prevention or for ameliorating the general conditions in unhealthy subjects. The interven-tion strategy decided after the analysis is personalized to the recovery of membrane balance and is created naturally, since the best drugs for membranes are lipids and cofactors coming from nutritional elements. Using lipidomics as the molecular version of the ancient saying “Let food be thy medicine and medicine be thy food” the health operators can speak also an “everyday” language to their patients, more comprehen-sible since it is closer to their habits and choices, orienting their daily efforts toward better health.


Lipidomics and Tutorials

In the conclusive chapter of this book lipidomics can assume practical importance after receiving the response.

When is the analysis indicated? First of all, on the basis of all the previous considerations and arguments, analysis can be recommended for prevention. In fact, the response is not an indication of pathology, but a way to discover a silent unbalance. The molecular profile with increased presence of fatty acids plays a role in inflammation (e.g., satu-rated fatty acid (SFA) and omega‐6 arachidonic acid excess), whereas with the decreased presence of protective fatty acids (omega‐6 DGLA, monounsaturated fatty acid excess (MUFA), EPA, and DHA) can induce a disadvantageous condition hardly distinguishable from pathological symptoms. Taking into account the obvious impairment that tissues without essential components such as EFA suffer, it is evident that lipi-domic analysis highlights health conditions substantiating the clinical diagnosis. Moreover, from the point of view of the patient, there are sev-eral practical advantages brought about by lipidomic analysis, since any strategy can be created without taking food habits into consideration. Indeed, one of the most diffuse criticisms in the medical prescription is that it does not include any information on the nutritional behavior, which instead is the daily concern of the patients, in acute and chronic diseases. The personalized indication of nutraceuticals as nutritional ele-ments of the lipidomic recovery strategy provides added value to patients and their families, who feel more proactive in overcoming their health problem.

9

158 LIPIDoMIcS AnD TUTorIALS

The approach that couples the clinical and the molecular (lipidomic) observations shown in Figure 9.1 represents a modern tool in the hands of health operators in order to define a personalized strategy to patients, combining therapeutics with nutrition and nutraceuticals.

The methodology of lipidomic analysis is very important in yielding another important contribution to the progress of molecular medicine. In fact, health operators can take an active part in the creation of data-base, coupling health conditions with molecular profiles, such as the membrane profile of fatty acids. As far as the lipidomic profiling the first two steps in the methodology are:

• gathering patient’s information through a questionnaire of anam-nesis, lifestyle, and health and dietary habits;

• providing a robust analytical protocol, which is on high- throughput device and a common platform for the organization of the analyses and the data.

Biotechnological and analytical skills are required to achieve the sec-ond task, and a first prototype of a high-throughput device is now at work in the spin-off company founded by the authors of this book in Italy. The spin-off company Lipinutragen born from the research of the authors of this book at the national council of research in Italy has worked in the last years in order to create the premises for the database of “health and membrane conditions” aiming at an interesting rational-ization of the membrane behavior in several health conditions. The

CLINICALPROFILE

Therapeutical strategy

LIPIDOMIC PROFILE

Nutrastrategy

PERSONALISATION

Figure 9.1 combined clinical–lipidomic observation for a personalized strategy

FIrST STEPS For THE LIPIDoMIc AnALYSIS 159

vision is that it is possible to examine health condition through the molecular membrane profiles as a fingerprint of the transformation of the membrane and a comprehensive descriptor of the metabolic changes induced by the health status. Using membrane as the target (in particular mature erythrocyte membranes) ensures that there is not a preponderant contribution of the short term dietary habits of the individual, as explained in details in Part 1. In collaboration with medical hospital units the possibility to draw profiles of human subjects started to be assayed as described in centenarian offspring [143], atrial flutter/atrial fibrillation [152], atopic dermatitis [180], autism [207], and celiac dis-ease [208]. obviously, a large number of analyses must be gathered in order to draw and assess the power of lipidomics profiles; however, it can be foreseen that the systematic collection of lipidomics analyses can reach an important mass of data in the next few years, opening many opportunities for interesting health applications.

9.1 FIrST STEPS For THE LIPIDoMIc AnALYSIS

The first steps to evaluate the data of a lipidomic analysis are the following:

1. Examination of the table with the fatty acid values found in the individual in comparison with the optimal ranges (see Figure 6.1).• See the values in excess or defect. The unbalance can be created

by both conditions.• Be careful to evaluate also the borderline values (close to the

upper or lower limit values).• Individuate also the behavior of the fatty acids in their specific

pathways (SFA, MUFA, oMEGA‐6, and oMEGA‐3), pointing attention to the trends of the pathway or in the pathways that are present for the individual: increase of all the MUFAs, the behavior of the omega-6 pathway, the decrease of the omega-3 pathways, etc.

2. Evaluate the biological and metabolic meaning of the unbalanced fatty acids.

Without being exhaustive, a brief guideline of the fatty acids is given here, which can be further personalized by considering the anamnestic and dietary information of the individual.


9.1.1 Saturated Fatty Acid Excess

The SFA values in excess influence the fluidity and permeability prop-erties of the membranes, reducing these properties and inducing an effect of metabolic slowness with possible malfunctions of channels, protein, and receptors of the membranes. This condition can occur as a result of stress situations stress situations, due to the remodeling of PUFA con-sumed by oxidative stress and replaced by saturated fatty acids which are more resistant to chemical and physical agents. Moreover, the increase in SFA can be due to increased biosynthesis, which is induced by proliferative and metabolic stimuli. The quantity and the quality of this family of fatty acids must be monitored in parallel with that of the monounsaturated family.

From the dietary point of view, several foods can increase SFA directly (cheese, meat) and also indirectly as explained with regard to carbohy-drate and the insulin stimulation of fatty acid synthase (chapter 2).

9.1.2 Monounsaturated Fatty Acid Excess

This family is formed from SFA by the activity of desaturase enzymes, and it is important for cis unsaturation. The increase in SFA accelerates the conversion to MUFA, which can occur in proliferative conditions, such as adiposity and tumor, but also in pregnancy for the new cells that are form-ing. It is known that dietary intake does not play a big influence in increasing the MUFA amount in membranes, whereas it provides feedback for reduced desaturase activity in cells.

9.1.3 omega‐6 PUFA Excess

In the omega-6 pathway, inflammatory mediators can be generated from arachidonic acid, and anti‐inflammatory mediators are generated from DGLA. The balance between these two fatty acids can provide information on the efficient regulation of this pathway. Linoleic acid must be taken from the diet (essential fatty acid) and is the most preva-lent PUFA in the body. Due to its structure and concentration in the body it is the most exposed to degradative and stress conditions. An examination of its level can inform of problems in the intake of appro-priate foods and in the oxidative conditions of the individual. In general, the intake of omega‐6 in the industrialized diet is higher than that of

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omega‐3; however, the analysis and the examination of the dietary habits are both necessary in order to evaluate the individual conditions and set up a personalised strategy in case of unbalanced values.

9.1.4 omega‐3 PUFA Deficit

Long chain fatty acids such as EPA and DHA are necessary for the func-tioning of tissues and for the production of important mediators with activity of protection from inflammatory processes. The involvement of these fatty acids in several tissues (nervous, muscle, intestine, etc.) ren-ders the deficit of omega‐3 PUFA very important to be discovered in prevention and disease. The dietary intake of some plant sources, fish, and algae is also very important for providing the appropriate levels of EPA and DHA. It is worth underlining, as described in chapter 3, that some plants or seeds (hemp, line seed, chia) contains the omega-3 pre-cursor alpha-linolenic acid, which is the essential fatty acid that humans cannot synthesize. From this precursor EPA and DHA are formed with variable efficiency depending on the individual metabolism. Therefore, the deficit can be associated to diet, but other possible causes are the incorrect formation from precursors, due to enzymatic malfunctions, as well as increased stress condition, which triggers the consumption of PUFAs, as it occurs also for omega-6 fatty acids. on the other hand, when omega‐3 supplementation is followed, it is also possible that an excess of these components is found. Patients sometimes forget to report the omega‐3 supplementation in the questionnaire. It has to be consid-ered that an excess of omega‐3 can be present even if the supplementa-tion has been stopped 2-3 months before the analysis.

The values of all fatty acids can also be affected because of enzyme impairment as evaluated on the basis of the seven lipid biosynthesis indi-cators (see Table 6.1). This information helps in establishing the need for cofactors and micronutrients, together with the specific fatty acid sup-plementation to cover the found unbalances.

An overall criterion for the strategy of nutritional/nutraceutical treatment is the restoration of the equilibrium among the four fatty acid families as evidenced by lipidomic analysis. Generally, the treatment lasts 4 months, three weeks per month as explained in chapter 7, connected to the mean lifetime of the erythrocyte and the metabolic turnover of the lipid therapy, and after this cycle a second analysis (within 6 months from the first analysis) highlights the effects of the strategy, which is also a measure of the individual response.


1. Phospholipids are:a. esters of l‐glycerol with three fatty acids.b. anhydrides of l‐glycerol with two fatty acids and one phosphate group.c. esters of l‐glycerol with two fatty acids and a phosphate group.

2. In the cell membranes of eukaryotes the unsaturated fatty acid geometry is:a. cis.b. trans.c. neither cis nor trans.

3. Phospholipids are:a. hydrophilic compounds.b. hydrophobic compounds.c. amphiphilic compounds.

4. Indicate the essential fatty acid:a. palmitic acid.b. linoleic acid.c. oleic acid.

5. Desaturase are enzymes able to:a. remove a double bond.b. insert a double bond.c. stabilize a double bond.

6. Membrane phospholipids contain:a. only saturated fatty acids.b. only unsaturated fatty acids.c. saturated and unsaturated fatty acids.

7. What is the fatty acid precursor of prostaglandin?a. arachidonic acid.b. alpha‐linolenic acid.c. oleic acid.

8. Which foods do fatty acids come from?a. from vegetable oils.b. from sugar.c. from pasta.

9.2 LEArnInG VErIFIcATIon

compiling the following questions, the reader can verify his/her learning of the basic concepts of membrane lipidomics and fatty acid significance. Answers are provided at the end of this chapter.

LEArnInG VErIFIcATIon 163

9. What are the essential fatty acids?a. saturated fatty acids.b. monounsaturated fatty acids.c. omega‐6 and omega‐3 fatty acids.

10. What is the richest source of alpha‐linolenic acid among these foods:a. olive oil.b. sunflower oil.c. linseed oil.

11. What are the foods that mostly contain omega‐6 fatty acids:a. fish.b. pasta and bread.c. seed oils.

12. Indicate the exact definition:a. Industrialized diets are rich in omega‐6 PUFAb. Industrialized diets are balanced in omega‐3 and omega‐6 PUFAs.c. Industrialized diets are rich in omega‐3 PUFA.

13. Which foods are a rich source of saturated fatty acids?a. fish.b. milk.c. cheese.

14. Which health condition is mostly known to influence the SFA pathway:a. dermatological health.b. cardiovascular health.c. neurologic health.

15. What is the tissue rich of DHA?a. nervous systemb. intestinec. retina

16. What is the “gold standard” for the analysis of fatty acids:a. spectrophotometry.b. gas chromatography (Gc).c. high‐performance liquid chromatography (HPLc).

17. What does the name linolenic acid omega‐3 mean:a. polyunsaturated fatty acid with two double bonds in positions

9 and 12 and an 18‐carbon atom chain.b. polyunsaturated fatty acid with three double bonds in positions

9, 12, and 15 and an 18‐carbon atom chain.


c. polyunsaturated fatty acid with three double bonds in positions 6, 9, and 12 and an 18‐carbon atom chain.

18. In the erythrocyte membrane what is the most representative MUFA and its structure?a. The most representative MUFA is oleic acid, with an 18‐carbon

atom chain and a double bond in position 9.b. The most representative MUFA is linoleic acid, with an 18‐carbon

atom chain and two double bonds in positions 9 and 12.c. The most representative MUFA is arachidonic acid, with a

20‐carbon atom chain and four double bonds in positions 5, 8, 11, and 14.

19. What is the enzyme producing stearic acid 18 : 0?a. stearic 18 : 0 comes from oleic acid by the activity of elongase.b. stearic 18 : 0 comes from vaccenic acid by the activity of

desaturase.c. stearic acid 18 : 0 comes from palmitic acid by the activity of

elongase.

20. Give an example of saturated and monounsaturated fatty acid:a. saturated fatty acid: palmitic acid; monounsaturated fatty acid:

stearic acid.b. saturated fatty acid: palmitic acid; monounsaturated fatty acid:

arachidonic acidc. saturated fatty acid: stearic acid; monounsaturated fatty acid:

oleic acid.

21. What is the precursor of omega‐3 fatty acids?a. linoleic acid.b. alpha‐linolenic acid.c. arachidonic acid.

22. What is the most representative saturated fatty acid in the erythro-cyte membrane?a. palmitic acid.b. stearic acid.c. miristic acid.

23. nutraceuticals contain:a. pharmacologically active compounds.b. nutritional elements that can have a positive effect on health and

diseases.c. components with an integrative function as regards dietary

habits.

LEArnInG VErIFIcATIon 165

24. The use of nutraceuticals occurs:a. all year long without specifying the needs.b. some cycles per year without specifying the needs.c. assigned by specific needs and verifying the effectiveness.

25. nutraceuticals represent:a. a nutritional tool.b. an alternative medicine tool.c. a tool that can complement pharmacological activity.

26. Indicate the right sentence:a. cells produce free radicals in their ordinary activity.b. cells do not produce free radicals in their ordinary activity.c. cells produce free radicals upon specific stimuli.

27. oxidative reactions occur in the following cell compartment:a. membrane.b. mitochondria.c. both compartments.

28. Peroxidation is a process that mainly concerns:a. saturated and monounsaturated fatty acids.b. mainly polyunsaturated fatty acids.c. all the fatty acid families.

29. How to distinguish saturated and monounsaturated fatty acids?a. they have different lengths.b. saturated fatty acids have more oxygen atoms.c. saturated fatty acids are linear whereas unsaturated fatty acids

are bent.

30. Is casual the choice of fatty acids forming a tissue?a. Yes, it is casual.b. no, it has to conform to a typical composition.c. The choice occurs depending on the availability of fatty acids.

AnSWErS

1c, 2A, 3c, 4B, 5B, 6c, 7A, 8A, 9c, 10c, 11c, 12A, 13c, 14B, 15c, 16B, 17B, 18A, 19c, 20c, 21B, 22A, 23B, 24c, 25c, 26A, 27c, 28B, 29c, 30B


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Index

adipocytes, 148aging, 143–5aldehyde adducts, 75, 78alpha‐linolenic acid, 37, 45, 48

biosynthesis, 43–4competition, 46

Alzheimer’s disease, 153antioxidants, 85–6

network, 143sportive activity, 140

arachidonic acidbiosynthesis, 44–5cardiovascular health, 147desaturase index, 122excess in Land’s cycle, 53–4mediator lipidomics, 94ophtalmology, 153oxidative pathways, 74–5prostaglandin pathways, 74RBC content, 112–13silent inflammation indicator, 123trans isomers, 72

atrial fibrillation, 147

blood lipids, 108–9blood withdrawal, 109

cardio indicator, 123–4cardiolipin, 103

cardiovascular health, 145–8carnitine

fatty acid transport, 32cellular defence and immunity indicator,

123cellular stress, 95–100cholesterol

biological role, 17–18PUFA excess, 18saturated fatty acid feedback, 19

cholesteryl ester formation, 80–81cis–trans isomerase, 36cis–trans isomerisation

bacterial, 70coenzyme A, 31conjugated dienes, 76conjugated linoleic acid (CLA) isomers

antitumoral activity, 70biological activity, 69–70

cysteine, 32

delta‐5 desaturase index see also FADS, 122

delta‐6 desaturase index see also FADS, 122, 150

delta‐9 desaturase/stearoyl CoA desaturase (SCD), 29–30

index, 122deodorization, 66–8

182 InDex

desaturase enzyme, 26, 30, 35alcohol inhibition, 35cofactors, 35, 45inhibition, 45maternal milk, 35trans fatty acid inhibition, 35

digestionlipids, 100–103

dihomo gamma linolenic acid (DGLA)cellular defence and immunity

indicator, 123desaturase index, 122functions, 45inflammation control, 55mediator lipidomics, 94–5proliferation control, 55

docosahexaenoic acid (DHA), 46–7foods, 47neuro indicator, 124neurology, 151–3ophtalmology, 153–4pregnancy, 142

double bondcis geometry, 9notation, 7trans geometry, 9

eicosanoids, 50eicosapentaenoic acid (ePA), 46

cellular defence and immunity indicator, 123

trans isomers, 68eicosatrienoic acid, 45–6, 56elongase enzyme (eLOVL), 25

PUFA biosynthesis, 42epigenetics, 91erythrocyte membrane, 107–9

conservability, 109fatty acid cluster, 115–16fatty acid interval values, 116

essential fatty acids (eFA), 41–4balance, 56–7RBC evaluation, 112–13syndrome, 49

fatty acid (FA)beta oxidation, 21–2, 30, 32cell volumes, 13

Dietary Reference Values, 59nomenclature, 7pool, 13, 17, 60–61

in membrane lipidomics, 93stress remodeling, 97–100

RBC composition, 110–114recommended intakes (eFSA), 128structural roles, 40–41structures, 7therapy, 131–3tissue composition, 39, 136

fatty acid‐based nutraceuticals, 128–31fatty acid desaturase enzyme (FADS), 43–4

delta‐5 and delta‐6 deasaturase index, 122fatty acid synthase (FAS), 23

insulin, 24leptin, 25

Fenton reaction, 74–6food deprivation, 141food pyramid, 21–2, 146free fatty acids (FFA), 54free radical theory of aging, 143functional lipidomics, 93

gallbladder, lipid metabolism, 61gamma‐linolenic acid, 44–5gas chromatography (GC), 29gene expression. 91glaucoma, 153glycolipids, 5

Hayflick phenomenon, 1434‐hydroxynonenal

formation, 75

inflammaging, 145inflammatory pathways, 44, 55, 74

mediator, 93–4skin, 150sportive activity, 140

isoprostanes, 79

Land’s cycle, 50–54lecithins, 103lecithin cholesterol transfer proteins

(LCAT), 80leukotrienes

synthesis, 45–6

InDex 183

L‐glycerol, 5, 11linoleic acid, 37, 48

biosynthesis, 42–4cardiolipins, 103–4reactivity, 83–4

lipid code, 93lipid hydroperoxides, 75–6lipid isomerisation

free radicals, 71–3oxidation, 73–7

lipidome, 88lipidomic analysis, 159–61lipidomic diet, 132lipidomic platform, 118lipidomic profiles, 118

aging, 143–5cardiovascular health, 145–8dermatology, 150–151neurology, 151–3ophtalmology, 153–4overweight, 148–50pregnancy, 140–142sport, 137–40

lipoproteins, 79–82oxidation, 79, 81trans fatty acids, 81

liposome, 16lipoxins, 45, 147lysophospholipid, 53

stress response, 97

macular degeneration, 154malnutrition in pregnancy, 141mature erythrocyte, 109–10mediator lipidomics, 93

inflammation signalling, 94membrane

adipocyte, 148compartimentalization, 4, 11critical aggregation concentration

(CAC), 4, 11, 16fluidity, 18homeostatic control, 14remodelling, 14, 50–54, 97–100, 148–9replication, 62–4

membrane unbalance index, (MUI), 117–19significance, 119–20

metabolic pacemaker, 144

metabolic slowness, molecular indicator, 122–3

microbial attack, 98molecular indicators, 122–5molecular profiles, 157mono‐trans isomers, 72monounsaturated fatty acids (MUFAs)

nomenclature, 8

natural phospholipids, 103neuro indicator, 124neuroprotectins, 46nuclear factor nF‐kB, 98nutraceutical supplementation, 117, 127–33nutrigenomics, 91nutrilipidomics, 92, 127–33

obesity and overweight, 148–50oils

deodorization, 10partial hydrogenation, 10

oleic acidbiosynthesis, 28reactivity, 83structure, 7, 28

omega‐3cardiovascular health, 146deodorization treatment, 10foods, 48health effects and trans geometry, 68intake for signalling, 94

omega‐3 risk index, 49, 123cardio indicator, 123–4

omega‐6/omega‐3balance, 56, 149, 150cardiovascular health, 147index of lipid biosynthesis, 121intake for signalling, 94neurology, 152ratio, 58RBC membranes, 112silent inflammation indicator, 123

p‐53 pathway, 96–7palmitic acid

biosynthesis, 23–4nutritional label, 38structure, 8

184 InDex

palmitoleic acidbiological roles, 29biosynthesis, 29–30desaturase index, 122metabolic slowness, 122obesity biomarker, 149RBC composition, 111structure, 8

partial hydrogenation, 66peroxidation/peroxidizability

detection methods, 77, 79index, 144life span, 144mechanism, 76–7

phosphatidylinositol 3‐phosphate (PI3P), 5phospholipase enzymes, 45, 50

Land’s cycle, 52–4phospholipid

biosynthesis, 31–3double layer, 4formation, 100–103structure, 11

plasma lipidsblood drop, 107–8

polyunsaturated fatty acids (PUFA)aging, 143biosynthesis, 37, 41–7in foods, 61nomenclature, 8nutraceuticals, 131peroxidation process, 76–7prostanoid precursors, 50reactivity, 75–6supplementation, 57–8

pregnancy, 140–142prostaglandins

series 1‐2‐3, 45–6synthesis, 45, 50, 74–5

radical stress indicator, 124resolvins, 46

sapienic acid, 29–30in lipoproteins, 81–2

saturated fatty acids (SFAs), 23–8biosynthesis, 23–8cardio indicator, 123–4desaturase enzyme, 34–5food sources, 26

hardening effect, 8, 27health effects, 27neurology, 153nomenclature, 8RBC composition, 111recommended intakes, 27structures, 23

SFA/MUFA ratioindex of lipid biosynthesis, 120–121metabolic slowness, 122RBC composition, 111

silent inflammation, 57, 123single nuclear polymorphism, 89

predictive power, 90skin functionality, 150sport, 137–40stearic acid

biosynthesis, 25stearidonic acid, 45stearoyl CoA‐desaturase, 29stress response, 95–6

toll‐like receptors (TLR), 98–9trans fatty acids (TFA)

cardiovascular risk, 147dermatology, 151dietary intakes, 10, 67endogenous formation, 10–11free radical isomerisation, 71–3, 83–4geometrical and positional

isomers, 66–7geometry, 9health problems, 67monounsaturated, 29–30neurology, 153nutrition, 66–8pregnancy, 142radical stress indicator, 124regulations, 67–8threshold in humans, 72

triglyceridesdigestion, 101–2

tromboxanes, 45

unsaturation index, 144

vaccenic acid, 30

whole blood lipids, 108

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